volume 4 number 2 2015 issn 2089-6514 biology, medicine, & natural product chemistry volume 4 – number 2 – 2015 issn 2089-6514 honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad ja’far luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia maizer said nahdi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: riyanto & m. iqbal a. t. publisher: sunan kalijaga state islamic university yogyakarta and the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@ yahoo.com guidance for authors biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international standards in their methodology. scientific feedback (short communicatiom) is only received for manuscript, which criticize published article before. manuscripts will be reviewed by managing editor and invited peer review according to their disciplines. the only articles written in english (u.s. english) are accepted for publication. this journal periodically publishes in may and november. in order to support reduction of global warming as a consequence of transportation vehicles emission and forest degradation for paper manufacturing, management of the journal prefer receiving manuscripts via e-mail rather than in hard copy. manuscripts and its communications can only be addressed to the managing editor; 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proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in java island. sebelas maret university, surakarta, 17-20 july 2000. [indonesian] thesis, dissertation: sugiyarto (2004) soil macro-invertebrates diversity and inter-cropping plants productivity in ag roforestry system based on sengon.[dissertation]. brawijaya university, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l (2008) a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com publication manuscript “in-press” can be cited and mentioned in reference (bibliography); “personal communications” can be cited, but cannot be mentioned in reference. research which not be published or “submitted” cannot be cited. some annotation. manuscript typed without sign link (-) (except repeated word in indonesian). usage of letter “l” (el) to “1” (one) or “o” (oh) to “0” (null) should be avoided. symbols of α, β, χ, etc. included through facility of insert, non altering letter type. no space between words and punctuation mark. progress of manuscript. notification of manuscript whether it is accepted or refused will be notified in about three months since the manuscript received. manuscript is refused if the content does not in line with the 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(photo: widodo) issn: 2089-6514 krokot (portulaca oleracea. l) as a natural sensitizer for tio dye-sensitized solar 2 cells: the effect of temperature extract reyza anni mufidah, khamidinal, endaruji sedyadi, didik krisdiyanto effect of lunasia amara blanco on sperm number, sperm motility, and testicular histology of male rats muhammad ja'far luthfi antioxidant potential of black, green and oolong tea methanol extracts wahyu widowati, tati herlina, hana ratnawati, gabriella constantia, i dewa gde sathya deva, maesaroh maesaroh medicinal plants: a prospect in developing male fertility enhancing agent muhammad ja'far luthfi, mahanem mat noor, jalifah latip tapak liman (elephantopus scaber l) as immunostimulant and its effect on lymphocyte differentiation in mice balb/c marmi kelik 25-29 31-33 35-39 41-47 49-51 issn 2089-6514 cover jurnal biomenaprochy vol 4 num 2 2015 dpn.pdf (p.1) editorial board 2015 v4 n2.pdf (p.2) guidance_v4n2.pdf (p.3) cover jurnal biomenaprochy vol 4 num 2 2015 blkg.pdf (p.4) untitled-1 volume 6 number 2 2017 issn 2089-6514 (paper) issn 2540-9328 (online) biology, medicine, & natural product chemistry volume 6 – number 2 – 2017 issn 2089-6514 (paper) | issn 2540-9328 (online) honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad jafar luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia widodo department of biology education, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: r i y a n t o publisher: state islamic university (uin) sunan kalijaga yogyakarta co-publisher: the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university (uin) sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@yahoo.com guidance for authors aims and scope biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international standards in their methodology. article types the journal seeks original full-length research papers, reviews, and short communication. manuscript of original research should be written in no more than 8,000 words (including tables and picture), or proportional with articles in this publication number. review articles will be accommodated, while, short communication should be written at least 2,000 words, except for pre-study. submission the journal accepts online submission, through ojs website system and/ or email to the editors at jafarluthfi@yahoo.com. submitted manuscripts should be the original works of the author(s). the manuscript must be accompanied by a cover letter containing the article title, the first name and last name 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2manuscript preparation manuscript is typed on a4 (210x297 mm ) paper size, in a single column, single space, 10-point (10 pt) times new roman font. the margin text is 3 cm from the top, 2 cm from the bottom, and 1.8 cm from the left and right. smaller lettering size can be applied in presenting table and figure (9 pt). word processing program or additional software can be used, however, it must be pc compatible and microsoft word based (.doc or .rtf or .docx). scientific names of species (incl. subspecies, variety, etc.) should be written in italic, except for italic sentence. scientific name (genera, species, author), and cultivar or strain should be mentioned completely for the first time mentioning it in the body text, especially for taxonomic manuscripts. name of genera can be shortened after first mentioning, except generating confusion. name of the author can be eliminated after first mentioning. for example, rhizopus oryzae l. uicc 524, hereinafter can be written as r. oryzae uicc 524. using trivial name should be avoided, otherwise generating confusion. biochemical and chemical nomenclature should follow the order of the iupac iub. for dna sequence, it is better used courier new font. symbols of standard chemical and abbreviation of chemistry name can be applied for common and clear used, for example, completely written butilic hydroxyl toluene (bht) to be bht hereinafter. metric measurement use is denomination, usage other system should follow the value of equivalent with the denomination of is first mentioning. abbreviations set of, like g, mg, ml, etc. do not follow by dot. -2 -1 -1minus index (m , l , h ) suggested to be used, except in things like "perplant" or "per-plot". equation of mathematics does not always can be written down in one column with text, in that case can be written separately. number one to ten are expressed with words, except if it relates to measurement, while values above them written in number, except in early sentence. the fraction should be expressed in decimal. in the text, it should be used "%" rather than "percent". avoid expressing ideas with complicated sentence and verbiage, and used efficient and effective sentence. title of the article should be written in compact, clear, and informative sentence, preferably not more than 20 words. name of author(s) should be completely written. name and institution address should also be completely written with street name and number (location), postal code, telephone number, facsimile number, and email address. manuscript written by a group, author for correspondence along with address is required. first page of the manuscript is used for writing above information. abstract should not be more than 200 words. keywords is about five words, covering scientific and local name (if any), research theme, and special methods which used; and sorted from a to z. all important abbreviations must be defined at their first mention. running title is about five words. introduction is about 400-600 words, covering the background and aims of the research. materials and methods should emphasize on the procedures and data analysis. results and discussion should be written as a series of connecting sentences, however, for manuscript with long discussion should be divided into subtitles. thorough discussion represents the causal effect mainly explains for why and how the results of the research were taken place, and do not only re-express the mentioned results in the form of sentences. concluding sentence should be given at the end of the discussion. acknowledgments are expressed in a brief; all sources of institutional, private and corporate financial support for the work must be fully acknowledged, and any potential conflicts of interest are noted. figures and tables of maximum of three pages should be clearly presented. title of a picture is written down below the picture, while title of a table is written above the table. colored figures can only be accepted if the information in the manuscript can lose without those images; chart is preferred to use black and white images. author could consign any picture or photo for the front cover, although it does not print in the manuscript. all images property of others should be mentioned source. there is no appendix, all data or data analysis are incorporated into results and discussions. for broad data, it can be displayed on the website as a supplement. references author-year citations are required. in the text give the authors name followed by the year of publication and arrange from oldest to newest and from a to z. in citing an article written by two authors, both of them should be mentioned, however, for three and more authors only the first author is mentioned followed by et al., for example: saharjo and nurhayati (2006) or (boonkerd 2003a, b, c; sugiyarto 2004; el-bana and nijs 2005; balagadde et al. 2008; webb et al. 2008). extent citation as shown with word "cit" should be avoided. reference to unpublished data and personal communication should not appear in the list but should be cited in the text only (e.g., rifai ma 2007, pers. com. (personal communication); setyawan ad 2007, unpublished data). in the reference list, the references should be listed in an alphabetical order (better, if only 20 for research papers). names of journals should be abbreviated. always use the standard abbreviation of a journal's name according to the issn list of title word abbreviations (www.issn.org/2 22661-ltwa-online.php). the following examples are for guidance. journal: saharjo bh, nurhayati ad. 2006. domination and composition structure change at hemic peat natural regeneration following burning; a case study in pelalawan, riau province. biodiversitas 7: 154-158. book: rai mk, carpinella c. 2006. naturally occurring bioactive compounds. elsevier, amsterdam. chapter in book: webb co, cannon ch, davies sj. 2008. ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. in: carson w, schnitzer s (eds) tropical forest community ecology. wiley-blackwell, new york. abstract: assaeed am. 2007. seed production and dispersal of rhazya stricta. 50 annual symposium of the international association for vegetation science, swansea, uk, 23-27 july 2007. proceeding: alikodra hs. 2000. biodiversity for development of local autonomous government. in: setyawan ad, sutarno (eds.) toward mount lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in java island. universitas sebelas maret, surakarta, 17-20 july 2000. [indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com the female population growth projection year 2021 in trenggalek regency by leslie matrix model on the birth rate and life expectancy dewi anggreini clustering of 18 local black rice base on total anthocyanin kristamtini, endang wisnu wiranti vitamin c and total sugar content characterization on 31 accessions of banana collection of banana germplasm plants of yogyakarta siti dewi indrasari, kristamtini, endang wisnu wiranti alliin as a natural bioactive from single bulb garlic (allium sativum) for nitric oxide (no) increasing in atherosclerotic process based on insilico screening riza rahayu ilmawati, ahya zhilalikbar amin, mohamad amin on designing interactive online atlas of reptile anatomy (mabouya multifacsiata) muhammad jafar luthfi, riyanto 37-45 47-51 53-57 59-62 63-68 volume 6 number 2 2017 published twice a year printed in indonesia front cover: commelina benghalensis (photo: widodo) issn 2089-6514 (paper) issn 2540-9328 (online) 3: font 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 5, number 2, 2016 | pages: 49-53 | doi: 10.14421/biomedich.2016.52.49-53 issn 2540-9328 (online) histological study of common house gecko (hemidactylus frenatus) regenerated tail rakhmiyati1, muhammad jafar luthfi2 1postgraduate program, universitas sebelas maret jalan ir. sutami 36 a, surakarta, 57126, tel. +62271-646994, fax. +62271-646655, indonesia 2biology department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: miarakhmiy@gmail.com1 abstract common house gecko (hemidactylus frenatus) belongs to suborder lacertilia that has capacity to shed its tail (autotomy) as a self-defense mechanism. after autotomy, tail regeneration occurs. axiale skeleton of an original tail is composed of bony vertebrae, whereas the the regenerate one is comprised of cartilaginous tube. the purpose of this study was to determine the histological difference between axial skeleton of the original tail and the regenerate one of the common house gecko. twenty four individuals consist of twelve common house gecko with original tail and twelve with regenerate tail were used. microanatomical observations were carried on histological slide of original and regenerated tail stained with hematoxylin-eosin and mallory acid fuchsin. the results showed that the original tail comprised of bony vertebrae whereas regenerated tail supported by cartilaginous tube. keywords: common house gecko (hemidactylus frenatus); autotomy; tail histology; cartilaginous tube of regenerated tail. introduction common house gecko belongs to the family lacertidae that has the ability to autotomized its tail as defense mechanism. caudal autotomy is the ability of an animal to shed off the tail to escape from predator (lutfi, 2002; soesilo, 1992). after autotomy, regeneration occurs (kimball, 1983; soesilo, 1992). the regeneration processes consist of three stages, namely the wound healing stage, blastema formation, differentiation and tail growth (soesilo, 1992). the original tail of lacertid is composed of vertebrae caudales, the spinal cord located within the vertebral canal, perivertebral fat tissue, muscle layers, blood vessels, nerve, skin and scales (white, 1925 in soesilo, 1999). the regenerated tail differs from the original one. according to soesilo (1999), the tail differs mainly in the structure of the vertebrae caudales and spinal cord. instead of vertebrae, regenerated tail is supported by elongated cartilaginous tube, whereas the spinal chord is replaced by ependymal cells, glia cells and nerve fibers without nerve cell body (soesilo, 1999). the muscles of a regenerated tail also have a difference with the original tail muscle. lacertid muscles arranged segmentally, consisted of 8 longitudinal bundles and each is limited by a septum composed by connective tissue. seosilo (1999), stated that tail regeneration can occur when there is ependymal layer on the remaining spinal cord. this study aimed to desribe histology of axial skeleton and muscle of regenerated tail of common house gecko (hemidactylus frenatus). materials and methods twenty four common house geckos (body length ranges from 6.5 to 7.5 cm, with intact tail) were used in this study. histological slides were made using paraffin method. histological observation were done using light microscopy on histological slides stained with hematoxylin-eosin (he) and mallory acid fuchsin. results and discussion from observation of original and regenerated tail of common house gecko it was found that there are difference in histological structure. http://dx.doi.org/10.14421/biomedich.2016.52.49-53 50 biology, medicine, & natural product chemistry 5 (2), 2016: 49-53 figure 1. longitudinal section of original tail (magnification 4x10). hematoxylin eosin (he). 1. skin; 2. muscles; 3. autotomy septum. figure 2. longitudinal section of original (magnification 4x10). mallory acid fuchsin. 1. epidermis; 2. perivertebral fat tissue; 3. muscle segment; 4. scales; 5. muscles; 6. scale joint. autotomy plane is lied transversal (figure 1). autotomy plane serve to facilitate tail sheding for minimalized excessive blooding. muscles pink colored due to eosin staining. the skin on the tail of common house gecko is thin. in the original tail, skin has scales and joints of the scales with thin epidermis (figure 2). muscles are segmental. the vertebral fracture is seen in figure 1. figure 3. cross section of original tail (10x10 magnification). hematoxylin eosin (he). 1. centrum vertebrae; 2. spinal cord; 3. transverse process; 4. muscles. figure 4. cross section of original tail (10x10 magnification). mallory acid fuchsin.1. muscles; 2. spinal chord; 3. centrum vertebrae; 4. caudal artery; 5. perivertebral fat tissue. the muscle in the original tail is divided into 8 bundles and between bundles separated by septum (figure 3). the transverses process is located at both side of vertebra. the original tail has a caudal artery. centrum vertebrae are lied between the caudal artery and the spinal cord (figure 4). the regenerated tail has a cartilaginous tube as a substitute for bony vertebrae and rakhmiyati & luthfi – histological study of common house gecko (hemidactylus frenatus) … 51 also has ependymocyti (figure 5). the regenerated tail does not have a tranverse process like the original one. the figure shows that the cartilaginous tube has chondrocytes. figure 5. longitudinal section of regenerated tail (20x10 magnification). hematoxylin eosin (he). 1. muscles; 2. perivertebral fat tissue; 3. cartilaginous tube; 4. meninx; 5. ependymal cell. figure 6. cross section of lizard tail that has undergone autotomy (4x10 magnification). coloring: mallory acid fuchsin. 1. skin; 2. muscles; 3. perivertebral fat tissue; 4. muscle segment; 5. cartilago pipe. figure 7. cross section regenerated tail (10x10 magnification). hematoxylin eosin (he). 1. perivertebral fat tissue; 2. cartilaginous tube; 3. nerves; 4. septum; 5. meninx; 6. ependymocyti; 7. muscles; 8. myotomes; 9. myoseptum; 10. myotube. figure 8. cross section of regenerated tail (10x10 magnification). mallory acid fuchsin. 1. perivertebral fat tissue; 2. nerves; 3. cartilago pipe; 4. meninx; 5. ependymocyti; 6. septum; 7. muscles; 8. myotomes; 9. myoseptum; 10. myotube. the cartilaginous tube is surrounded by perivertebral fat tissue in which there are nerve cells (figure 7). figure 8 shows that perivertebral fat tissue surrounds the cartilaginous tube and in the fatty tissue there are nerve cells. there are ependymocyti in regenerated tail which play a role in cartilage formation. observation of cross and longitudinal sections on the original tail of the common house gecko (hemidactylus frenatus) observation of cross and longitudinal sections on the original tail of the common house gecko (hemidactylus frenatus) stained with mallory acid fuchsin and hematoxylin-eosin showed autotomy plane and transverse process on vertebrae caudales. autotomy plane is located along the tail from the base of the tail to the tip of the tail. the autotomy pane lies transversely and divides the vertebra and fat tissue into two parts: the anterior and posterior. vertebrae caudales of the original tail is surrounded by muscles. the muscle on the tail is a striated muscle. according to irianto (2005), striated muscle is a muscle attached to the skeleton that can be moved. this muscle contracts quickly and strongly. in addition, striated muscles are also very sensitive to stimuli. the muscle of the regenerated tail is composed of multiple myotubes that merge into a myotomes. myotomes are separated by myoseptum. the muscles of a regenerated tail are divided into 16 bundles. according to research conducted by rachman & hadi (2008), the posterior part of original tail muscle of a lizard (mabouya multifasciata kuhl) has its origo on the chevron bone, spina neuralis, and transverse process, whereas the anterior part of the muscle inserted in the septum. muscle of original tail is divided into 8 bundles. 52 biology, medicine, & natural product chemistry 5 (2), 2016: 49-53 observation of cross and longitudinal sections on common house gecko regenerated tail (hemidactylus frenatus). the observation revealed that the regenerated tail is not supported by the bony vertebrae but cartilage or segmental cartilage (figures 5, 6, 7, and 8). inside the cartilaginous tube there is a layer of ependyma that is located parallel to the cartilage tube. according to soesilo (1999), ependymal layer is very important in the process of tail regeneration and in the formation of cartilaginous tube. rachman & luthfi (2004) stated that calcification in cartilaginous tube of regenerated tail of lizard (mabouya multifasciata) starting at 12 weeks of regeneration. this calcification process starts from the inner side and the outer side of the cartilaginous tube. cartilaginous tube of regenerated tail does not have an autotomy plane so the ability of autotomy in the regenerated tail is lower. autotomy capability on the original tail is higher because it has autotomy plane. conclusion based on the results of the study, we drew conclusion as follows: 1. the original tail is composed of bony vertebrae, while the regenerated tail is composed by cartilaginous tube. 2. the regenerated muscle tail is divided into 16 bundles, while the original tail muscle is divided into 8 bundles. references alibardi, l. 1994. muscle differentiation and morphogenesis in the regenerating tail of lizards. journal of anatomy. 186: 143-151. andayani, m.m.l. 2007. mikroanatomi ekor kadal (mabouya multifasciata kuhl) fase penyembuhan luka setelah autotomi dan irradiasi sinar gamma. thesis universitas gadjah mada (unpublished). anonim. 1985. biology of the reptilia (vol.15). new york: a wiley-interscience publication. bateman, p.w & fleming, p. a. 2009. to cut a long tail short: a reviewof lizard caudal autotomy studies carried out over the last 20 years. journal of zoology. london: the zoological society. bevelander, g & ramaley, j. a. 1988. dasar-dasar histologi. translated by wisnu gunarso. erlangga, jakarta. beresford, w.a. 1983. lecture notes on histology. black well scientific publications, oxford. carneiro, j & junqueira, l.c. 1992. histologi dasar (basic histology). translated by adji dharma. penerbit buku kedokteran egc, jakarta. elkarim, a. a. 1994. regeneration from different levels along the tail of the geckonid lizard, bunopus tuberculatus. zoology. 1 (14): 82-89. qatar: university of qatar. finerty, j.c and cowdry, e.v. 1962. a text book of histology (functional significance of cells and intercellular substances). lea and febiger, philadelphia. gardner, gray, dan o’rahilly. 1995 . anatomi (kajian ranah tubuh manusia). translated by z.s bustami. penerbit universitas indonesia, jakarta. geneser, f. 1994. buku teks histologi. binarupa aksara, jakarta. hadi, s & rachman, a. 2008. struktur makro dan mikroanatomi otot ekor pada ekor dan regenerat ekor kadal (mabouya multifasciata kuhl). berkala ilmiah biologi. 6 (2): 81-86. yogyakarta: universitas gadjah mada. irianto, k. 2005. struktur dan fungsi tubuh manusia untuk paramedis. bandung: cv. yrama widya. kardong, k. v. 2006. vertebrates: comparative anatomy, function, evolution. washington state university: mc graw hill higher education. kimball, j. w. 1983. biologi. fifth edition. jakarta: erlangga. luthfi, m. j. 2002. kalsifikasi skeleton aksial dan kemampuan autotomi regenerat ekor kadal (mabouya multifasciata kuhl). tesis. universitas gadjah mada (unpublished). luthfi, m. j, soesilo, n.p, & sagi, m. 2003. kalsifikasi skeleton aksial pada regenerat ekor kadal (mabouya multifasciata kuhl). jurnal berkala ilmiah biologi, 3 (1): 1-8. yogyakarta: universitas gadjah mada. leeson, c.r, leeson, t.s, and paparo, a. a. 1996. buku ajar histologi. translated by yan tambayong dkk. penerbit buku kedokteran egc, jakarta. maria, b. 1998. struktur vertebrae caudales pada 5 species anggota sub ordo lacertilia. skripsi. universitas gadjah mada (unpublished). nahdi, m. s. & solihah, j. 2007. buku ajar: biologi umum. uin sunan kalijaga yogyakarta. pratiwi, i.r. 2009. struktur organ reproduksi dan seksual dimorfisme pada hemidactylus frenatus dumeril & bibron, 1836; cosymbotus platyurus (schneider, 1792); & gekko gecko linnaeus, 1758. thesis universitas gadjah mada (unpublished). pratt, c.w.m. 1946. the plane of fracture of the caudal vertebrae of certain lacertilians. journal of anatomy. 80: 184-188. russell, a.p., bergmann, p.j. & barbadillo, l.j. 2001. maximal caudal autotomy in podarcis hispanica (lacertidae): the caudofemoralis muscle is not sundered. american society of ichthyologists and herpetologists. 1: 154-163. rachman, a & luthfi,m.j. 2004. studi histokimia kalsifikasi skeleton regenerat ekor cicak (hemidactylus sp), jurnal berkala ilmiah biologi. 3 (4): 223-230. yogyakarta: universitas gadjah mada. suntoro, s. h. 1983. metode pewarnaan (histologi dan histokimia). jakarta: bhratara karya aksara. soesilo, n. p. 2002. pengaruh regenerat ekor kadal (mabouya multifasciata kuhl) terhadap angiogenesis. biologi. 2 (14): 833-844. yogyakarta: universitas gadjah mada. soesilo, n. p. 1982. regenerasi ekor kadal (mabuya multifasciata kuhl) setelah mengalami autotomi. thesis. universitas gadjah mada (unpublished). soesilo, n. p. 1992. proses regenerasi ekor kadal (mabouya multifasciata kuhl). biologi. 1 (4): 169-175. soesilo, n.p. 1999. peranan lapisan ependima dalam regenerasi ekor kadal (mabouya multifasciata kuhl). biologi. 2 (8): 419-450. rakhmiyati & luthfi – histological study of common house gecko (hemidactylus frenatus) … 53 takahashi, h. 2009. preferensi pakan cicak rumah (hemidactylus frenatus, gray 1825) dan tokek (gekko gecko, linnaeus 1758) di gamping, sleman, daerah istimewa yogyakarta. thesis. universitas gadjah mada (unpublished). yatim, w. 1996. biologi modern: histologi. bandung: tarsito. zug, g. r. 1993. herpetology an introductory biology of amphibian and reptiles. academic press. london. histological study of common house gecko (hemidactylus frenatus) regenerated tail biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 783-786 | doi: 10.14421/biomedich.2025.142.783-786 issn 2540-9328 (online) optimization of genomic dna extraction method on nutmeg (myristica fragrans houtt.) tanti agustina1, abdul razaq chasani1*, budi setiadi daryono2 1laboratory of plant systematics; 2laboratory of genetics and breeding, department of biology, faculty of biology, universitas gadjah mada, sekip utara street, sleman, yogyakarta 55281, indonesia. corresponding author* ar.chasani@ugm.ac.id abstract nutmeg (myristica fragrans) is one of the commodities in the myristicaceae family that has numerous benefits and high phenolic compounds. elevated amounts of secondary metabolites in the myristica genus pose significant challenges for obtaining high-quality dna. this complexity often results in reduced dna recovery and suboptimal absorbance ratios, attributed to contamination by rna and proteins in dna extraction yields. the results of dna extraction that have been obtained through several variations of optimized methods are compared. the dna extraction method using a modified geneaid genomic dna mini kit was determined to produce the highest quality and quantity of dna. compared to other protocols such as doyle and doyle, modified doyle and doyle, and the basic protocol of the geneaid kit without improvement, the modified geneaid genomic dna mini kit had a high mean range purity of 1.83, and bands appeared. keywords: dna extraction; myristica fragrans; nutmeg; optimization. introduction nutmeg (myristica fragrans) is a commodity from the myristicaceae family that possesses numerous applications in culinary as food flavoring and medicinal as an essential oil and pharmaceutical ingredient (naeem, 2016; gupta, 2020). the study of the character of a plant is not only seen in its morphological character but can also be analyzed in the molecular area such as genetic material (dna, rna), proteins, and secondary metabolites (wijayanti, 2018). with advancements in molecular genetic techniques, dna analysis now enables the precise identification of genetic variations, offering deeper insights into plant biology (gupta et al., 2020). dna extraction is crucial for obtaining high-quality genomic dna. due to the differential concentrations of secondary metabolites in plant cells, each species requires a specific extraction protocol to produce genomic dna of sufficient quality for molecular analyses. impure dna can be caused by secondary compounds such as polysaccharides, phenolic compounds, or other contaminants (pharmawati, 2009). nutmeg has high levels of various types of phenolic compounds (periasamy et al., 2016). these metabolites negatively impact subsequent procedures such as dna extraction, restriction, amplification, and cloning (ashokkumar et al., 2022). the high levels of secondary metabolites present in leaves of the myristica genus pose significant challenges to obtaining high-quality dna (diyasree, 2014). the problems in dna extraction remain significant issues that need to be solved by optimization. procedure optimization can be attained by modifying the lysis buffer composition or employing specialized physical handling techniques to effectively separate genomic dna from other components. the primary aim of these optimization strategies is to prevent degradation of genomic dna caused by secondary compounds released during cellular lysis and subsequent handling (milligan, 1992). several techniques and procedures have been published, but these are not applicable because treatment for every genus or plant species is very specific. modification of the conventional dna extraction protocol is necessary for extracting dna from plant leaves with high concentrations of polysaccharides or secondary metabolites (sahu, 2012). therefore, it is necessary to select and optimize the extraction method that allows the genomic dna of the nutmeg plants to be obtained. studying plant dna isolation methods, especially nutmeg, is the first step that will be used as a reference for further research. this study aimed to obtain optimal dna extraction methods and produce highquality dna genomes so they could be used for genetic diversity analysis in nutmeg plants. manuscript received: 13 august, 2025. revision accepted: 30 september, 2025. published: 08 october, 2025. https://doi.org/10.14421/biomedich.2025.142.783-786 784 biology, medicine, & natural product chemistry 14 (2), 2025: 783-786 materials and methods sampling and preservation following sudhamayee’s (2010) recommendation, the third leaf from the shoot tip was collected from sangihe island. samples were cleaned using alcohol and quickly put into an envelope, then wrapped in a zip-lock plastic with silica gel. the samples were transported to the laboratory in ice-filled containers and stored at -20 °c until utilized for dna extraction. procedures doyle and doyle (1987) in accordance with the doyle and doyle (1987) protocol, 0.2 g of clean leaf tissue was homogenized with an extraction buffer consisting of 2% hexadecyltrimethylammonium bromide (ctab), 1.4 m nacl, 20 mm edta, 100 mm tris-hcl (ph 8.0), and 0.2% β-mercaptoethanol, as specified in the original method (doyle & doyle, 1987). modified doyle and doyle (1987) genomic dna was isolated using the original doyle and doyle (1987) protocols, with a modified protocol by adding pvp (polyvinylpyrrolidone). genomic dna mini kit (plant) tissue dissociation, a sample from 0.35 g of fresh leaf tissue was placed in a mortar, frozen with liquid nitrogen, and subsequently ground. the homogenized sample was transferred to a pre-labeled 1.5 ml microcentrifuge tube. during the lysis stage, 400 µl of gpx1 buffer and 5 µl of rnase were added to the sample, which was then vortexed and incubated at 60°c for 20 minutes. the tube was inverted every 5 minutes. following incubation, gp2 solution was added, the mixture was vortexed, and the sample was incubated on ice for 3 minutes. the sample was then transferred to a column filter placed in a 2 ml collection tube and centrifuged at 1000 × g for 1 minute. the filter column was discarded, and the supernatant was transferred from the 2 ml collection tube to a new, prelabeled 1.5 ml microcentrifuge tube. then, in the binding stage, the supernatant was added with 1.5 of the volume of gp3 (to which isopropanol has been added) and vortexed for 5 seconds, subsequently, 700 µl of the mixture was aliquoted into the gd column, which was then inserted into a 2 ml collection tube and centrifuged at 15,000 × g for 2 minutes. the eluate was discarded, and the gd column was returned to the 2 ml collection tube. the remaining mixture was added to the gd column and centrifuged again at 15,000 × g for 2 minutes. the resultant liquid was discarded, and the gd column was repositioned in the 2 ml collection tube. the next step is washing; this stage serves to remove the remnants of the extraction buffer and residual pigment. 400 µl of w1 buffer was added to the gd column, which was then centrifuged at 15,000 × g for 30 seconds. the eluate was discarded, and the gd column was returned to the 2 ml collection tube. next, 600 µl of wash buffer was added to the gd column, followed by centrifugation at 15,000 × g for 30 seconds. the liquid was discarded, and the gd column was placed back into the 2 ml collection tube. the column was then centrifuged at 15,000 × g for 3 minutes to ensure complete drying of the column matrix. in the last step, dna elution, the dried gd column was transferred to a new 1.5 ml microcentrifuge tube, and 80 µl of preheated elution buffer was carefully added to the center of the column matrix. the column was allowed to stand for 10 minutes to ensure complete absorption of the elution buffer. subsequently, the column was centrifuged at 15,000 × g for 30 seconds to elute the dna. modified genomic dna mini kit (plant) genomic dna was isolated using the geneaid brand extraction kit with a modified protocol by adding pvp at the tissue dissociation stage, incubation for 20 minutes at the original lysis stage (protocol: 10 minutes), and also incubation for 10 minutes in the dna elution stage after adding the elution buffer (protocol: 2-3 minutes), and the centrifugation temperature adjustment to 4 ˚c. dna concentration measurement the concentration of the extracted dna samples was determined by measuring 50 µl of the sample at each stage. the sample is pipetted onto a spectrophotometer plate to obtain od (optical density). the spectrophotometer used a single-wavelength method, and the od was measured at a wavelength of 260 nm. the results from each method were compared. agarose gel electrophoresis the extraction results were electrophoresed with a 100bp dna marker (dna ladder) on a 1% agarose gel with florosafe dna staining in tbe buffer (tris/borate/edta) for 40 minutes at 100 volts. then the gel was observed under a uv lamp using the gel documentation system. results and discussion based on the results of quantitative tests using a spectrophotometer, the average λ of dna produced in four methods was 1.17, 1.54, 0.00, and 1.83 (table 1). based on the comparison of the absorbance values of λ260/ λ280 from the four methods, the 4th method obtained a high level of purity, and the dna produced is quite clean. theoretically, dna samples that are considered quite pure have a ratio of λ260/ λ280= 1.80-2.0. according to watson et al. (2024), this range of numbers has met the requirements needed in molecular analysis. agustina et al. – nutmeg dna extraction method optimization 785 table 1. quantitative and qualitative test results from nutmeg dna extraction. no isolation method mean λ260/ λ280 remarks 1 doyle & doyle (1987) 1,17 low dna content. the pellet was brown in color and difficult to dissolve in te (tris-hcl and edta) buffer. high protein contamination, no bands appeared. 2 modified doyle & doyle (1987) 1,54 dna yield was higher when compared to the original protocol of doyle & doyle (1987), but the concentration was still low. the pellet was cleaner and still difficult to dissolve, and no bands appeared. 3 genomic dna mini kit (plant) 0,00 no evidence of dna, no bands appeared. 4 modified genomic dna mini kit (plant) 1,83 bands were obtained with the highest concentration of dna. the method using additional pvp resulted in a higher average dna concentration. phenolic compounds interact with proteins and dna through hydrogen bonding immediately after cell lysis, which can alter their properties and impede effective extraction. consequently, the incorporation of phenolic inhibitors is essential. polyvinylpyrrolidone (pvp), or polyvidone or povidone, is a water-soluble polymer synthesized from n-vinylpyrrolidone. pvp binds to polyphenols and is available in different molecular weights, with lower molecular weight forms particularly effective at binding proteins (li et al., 2021). using a modified kit also results in better dna quality because the kit is provided with a filter that is used specifically to filter macromolecules such as protein, so protein contaminants are lower than others. the extraction results in methods 1, 2, and 3 did not show any dna bands, respectively (figure 1). this shows that the three methods cannot produce dna extracts properly. good-quality dna extraction products are indicated by dna bands that look thick and clean and dna bands that glow. thus, the three methods are not suitable for use in the isolation of nutmeg genomic dna. figure 1. visualization of qualitative test results from nutmeg dna extraction. the addition of reducing compounds such as merchaptoethanol (ß-me) in the dna extraction process can prevent the oxidation of phenolic compounds, thereby inhibiting the activity of free radicals generated by the oxidation of phenol to nucleic acids (drygin, 2021). the use of ctab in the extraction buffer is helpful for eliminating polysaccharides. furthermore, valizadeh et al. (2021) and ali et al. (2019) stated that adding nacl with a concentration above 1 m can increase the solubility of polysaccharides so that they are easier to remove. thus, the ctab buffer is qualified to be used in dna extraction from plants containing high amounts of carbohydrates and phenols because it does not damage dna. ctab buffer with high salt content can separate polysaccharides from cell walls, while pvp can reduce browning due to phenol content in young leaves (gill et al., 2025; surzycki, 2000). conclusion based on the results of dna quantitative and qualitative tests on nutmeg samples, the best dna produced is using the modified genomic dna mini kit (plant). acknowledgment: the authors extend their heartfelt thanks to the lembaga pengelola dana pendidikan republik indonesia (lpdp ri) for their financial support, which was instrumental in conducting this research. authors’ contributions: t.a. designed the study, performed the experiments, analyzed the data, and drafted the manuscript. a.r.c. and b.s.d. supervised the research, contributed to methodology development, and revised the manuscript critically for important intellectual content. all authors read and approved the final manuscript. competing interests: the authors declare that there are no competing interests. 786 biology, medicine, & natural product chemistry 14 (2), 2025: 783-786 references ali, q., salisu, i. b., raza, a., shahid, a. a., rao, a. q., & husnain, t. (2019). a modified protocol for rapid dna isolation from cotton (gossypium spp.). methodsx, 6, 259264. ashokkumar, k., simal‐gandara, j., murugan, m., dhanya, m. k., & pandian, a. (2022). nutmeg (myristica fragrans houtt.) essential oil: a review on its composition, biological, and pharmacological activities. phytotherapy research, 36(7), 2839-2851. diyasree, p., lokesh, k. a., john, p. l., joy, t., ramachandran, v. t., muttakulath, a. t., ... & mathew, d. (2014). a simplified protocol for the recovery of high quality dna from nutmeg. journal of tropical agriculture, 52(1), 79-83. doyle, j. j. j. l. (1987). genomic plant dna preparation from fresh tissue‐ctab method. phytochemical bulletin, 19(11), 11. drygin, y. f., butenko, k. o., & gasanova, t. v. (2021). environmentally friendly method of rna isolation. analytical biochemistry, 620, 114113. gill, k., negi, s., kumar, p., & irfan, m. (2025). improved genomic dna extraction from citrus species using a modified ctab method. molecular biology reports, 52(1), 638. gupta, r., azhar, m., & kalam, m. a. (2020). an overview of myristica fragrans (nutmeg)-its benefits and adverse effects to humans. indian journal of integrative medicine, 45-50. https://mansapublishers.com/index.php/ijim/article/view/2948 hussein, m. a., eid, m., rahimi, m., filimban, f. z., & abd elmoneim, d. (2023). comparative assessment of ssr and rapd markers for genetic diversity in some mango cultivars. peerj, 11, e15722. li, m., ritzoulis, c., du, q., liu, y., ding, y., liu, w., & liu, j. (2021). recent progress on protein-polyphenol complexes: effect on stability and nutrients delivery of oil-in-water emulsion system. frontiers in nutrition, 8, 765589. milligan, b.g. (1992). plant dna isolation. in: a.r. hoelzel (ed). molecular genetic analysis of populations. a practical approach. new york: oxford university press naeem, n., rehman, r., mushtaq, a., & ghania, j. b. (2016). nutmeg: a review on uses and biological properties. international journal of chemical and biochemical sciences, 9, 107-110. https://www.iscientific.org/wpcontent/uploads/2019/10/13-ijcbs-16-09-13.pdf periasamy, g., karim, a., gibrelibanos, m., & gebremedhin, g., (2016). essential oils in food preservation flavor and safety: nutmeg (myristica fragrans houtt.) oils. cambridge: academic press. pharmawati, m. (2009). optimalisasi ekstraksi dna dan pcrrapd pada grevillea spp.(proteaceae). jurnal biologi, 13(1), 12-16. https://ojs.unud.ac.id/index.php/bio/article/view/577 sahu, s. k., thangaraj, m., & kathiresan, k. (2012). dna extraction protocol for plants with high levels of secondary metabolites and polysaccharides without using liquid nitrogen and phenol. international scholarly research notices, 2012. sudhamayee, m. (2010). sex determination in nutmeg (myristica fragrans houtt.) through molecular and biochemical markers. m. sc. (agri.) thesis, kerala agricultural university, india. surzycki, s. (2012). basic techniques in molecular biology. springer science & business media. valizadeh, n., holasou, h. a., mohammadi, s. a., & khawar, k. m. (2021). a comparison of genomic dna extraction protocols in artemisia annua l. for large scale genetic analyses studies. iranian journal of science and technology, transactions a: science, 45(5), 1587-1595. watson, j. f., arroyo-urea, s., & garcía-nafría, j. (2024). dna cloning. in handbook of molecular biotechnology (pp. 66-72). crc press. wijayanti, t., & setiawan, d. c. (2018). eksplorasi senyawa metabolit sekunder pada kulit batang tanaman duwet (syzygium cumini l.) dengan metode liquid chromatograph mass spectrometry (lcms). bioma: jurnal ilmiah biologi, 7(2), 196-210. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 2, 2017 | pages: 53-57 | doi: 10.14421/biomedich.2017.62.53-57 issn 2540-9328 (online) vitamin c and total sugar content characterization on 31 accessions of banana collection of banana germplasm plants of yogyakarta siti dewi indrasari1, kristamtini2, endang wisnu wiranti3 1,2,3assessment institute for agricultural technology (aiat) yogyakarta jln. stadion maguwoharjo no. 22, wedomartani, ngemplak, sleman, yogyakarta, indonesia author correspondency: dewindrasari@yahoo.com1 abstract banana is one of the tropical fruits that people like because it tastes good and contains good nutritional value that beneficial for health. the content of vitamin c and total sugar are an important character to complete the morphological characterization of banana accession that can be utilized by breeders in choosing accession as parent. the study aimed to determine the content of vitamin c and total sugar on 31 accessions of banana collection of banana germplasm plants of yogyakarta. the research was conducted at banana germplasm plantation and laboratory of agricultural technology production of gadjah mada university, yogyakarta from january to december 2016. the results indicated that 31 banana accessions showed their susceptibility to vitamin c content 60.42 + 39.22 mg / 100 g and total sugar 22.06 + 16.01%. high standard deviation values indicate the large diversity of banana accessions that were characterized, indicating that the accessions of each characterized banana were separate accessions of one another. keywords: characterization; banana; vitamin c; total sugar. introduction banana is one of the tropical fruits that people like because it tastes good and contains good nutritional value that beneficial for health. in addition to the relatively cheap price, banana was also one of the plants that have bright prospects because everyone in the whole world almost likes to consume bananas (komaryati & adi, 2012). indonesia is one of the centers of banana plant genes (sukartini, 2007). therefore, in 1988, the goverment of yogyakarta special region develop the banana germplasm garden and has been carry out the collection, maintenance and dissemination of various collections of bananas. with a land area of 19,525 square meters, this garden already has 346 varieties of bananas originating from indonesia and from abroad (kristamtini et al., 2016). the banana germplasm garden of yogyakarta was called as the most complete collection of banana conservation garden in indonesia by the ministry of agriculture of indonesia. indonesia has a high diversity of bananas, this condition was very helpful for breeding program in producing superior varieties. the superior varieties of bananas were expected to have high productivity, good quality, early age, resistant to certain pests and tolerance to environmental stress. on the other hand, in the banana diversity was not yet widely known for its characteristics. to support the assembly of superior varieties of bananas, it was necessary to characterize and evaluate existing germplasm. the information obtained from characterization and evaluation, can be used as a material for improving the character through a plant breeding program. banana plants have diverse morphological characters. each banana plant has its advantages and disadvantages based on its morphological appearance (wardiyati et al., 2004). as the first step of breeding program to get information about morphological character of a plant was needed characterization activity (subandiyah, 2002). as simmonds & shepherd (1955) have pointed out, one of the first steps to recognize the magnitude of such biodiversity was to identify genetic diversity. one approach to identifying a banana plant is to use morphological characters that include leaf, stem, fruit, and flower characters (wardiyati et al., 1997) plant breeders can utilize the plant after knowing the character thoroughly through exploration, collection, characterization, evaluation and selection (wardiyati et al., 1997). morphological characterization is the preliminary information required in seeking superior characters and the diversity that was still in need (santos et. al., 2003). morphological characters were considered still not enough to find a clear position so that other methods as complement to evaluate kinship. one method was to do chemical characterization by analyzing the content of vitamin c and total sugar. the content of vitamin c and http://dx.doi.org/10.14421/biomedich.2017.62.53-57 54 biology, medicine, & natural product chemistry 6 (2), 2017: 53-57 total sugar becomes the character that differentiates between accessions, because bananas contain high enough nutrients and high of vitamin c. in addition the content of vitamin c and total sugar was the required character in the description of banana plants the aim of this research was to know and get the diversity of 31 accessions of banana collection of banana germplasm garden of yogyakarta based on vitamin c and total sugar content so that the relation of various bananas based on vitamin c and total sugar content are known. materials and methods the experiment was conducted at banana germplasm plantation and agricultural production technology laboratory of gadjah mada university, yogyakarta from january to december 2016. thirty-one accessions of banana as research material are presented in table 1. banana tree ripe fruit was analyzed for vitamin c and total sugar content. the analysis of sugar content using nelson somogyi method, while analysis of vitamin c content using iodometric titration method. table 1. the banana accessions and its origin. no name origin 1 ambon jaran bantul 2 ambon sepet gunung kidul 3 ambon warangan kulon progo 4 anjasmoro sleman 5 australi kbh dongkelan 6 gajih putih kulon progo 7 kepok brentel bantul 8 kepok bung kulon progo 9 kepok hijau sleman 10 kepok kuning kbh dongkelan 11 kidang hijau sleman 12 kluthuk susu yogyakarta 13 koja pretel gunung kidul 14 koja sirimentak gunung kidul 15 lempereng kulon progo 16 mangsan sleman 17 mas raja sleman 18 mas sloka kulon progo 19 mas tropong sleman 20 morosebo yogyakarta 21 potho merah bantul 22 raja bagus yogyakarta 23 raja bandung bantul 24 raja lini gunung kidul 25 raja pulut yogyakarta 26 raja sabrang kulon progo 27 raja sawi sleman 28 raja sewu yogyakarta 29 rejang sleman 30 serawak kulon progo 31 triolin bantul the data of vitamin c content and total sugar were analyzed by cluster method using hierarchical cluster analysis to know the kinship relationship with the help of sas ver software 9.2. hierarchical cluster analysis was a common way to grouping objects in groups that share the same resemblance to each other. results and discussion the content of vitamin c and total sugar in 31 accessions of bananas is presented in table 2. the content of vitamin c and total sugar from 31 banana accessions varies according to their genetic potential, with an average vitamin c content of 60.42 + 39.22 mg/100 g, average of total sugar 22.06 + 16.01%. the highest vitamin c content is owned by the mangsan accessions (149.34 mg/100g) and the lowest is owned by the mas raja accessions (12.79 mg/100g). the content of vitamin c in 31 accessions of bananas is a hint of accession of bananas to be selected for consumption as fresh fruit that is beneficial to health. table 2. the content of vitamin c and total sugar of 31 bananas accessions. bananas accessions vit c (mg/100 g) total sugar (%) 1. ambon jaran 20.41 20.41 2. ambon sepet 128.20 18.54 3. ambon warangan 52.44 15.19 4. anjasmoro 34.38 17.01 5. australi 53.14 2.42 6. ceni 36.50 21.70 7. gajih putih 76.14 10.66 8. kapok brentel 30.85 20.31 9. kepok bung 135.69 17.25 10. kepok hijau 23.34 23.34 11. kepok kuning 93.48 18.99 12. kidang hijau 82.93 17.90 13. klutuk susu 61.70 21.36 14. koja pretel 27.39 22.73 15. koja sirimentak 50.38 13.20 16. lempereng 20.52 20.07 17. mangsan 149.34 14.36 18. mas jambe 75.53 16.38 19. mas raja 12.79 44.85 20. mas sloka 92.15 18.99 21. mas tropong 98.84 16.96 22. morosebo 85.78 14.31 23. potho merah 19.40 19.40 24. raja bandung 57.93 15.66 25. raja lini 17.79 22.97 26. raja pulut 122.29 20.94 27. raja sabrang 54.43 15.64 28. raja sewu 31.08 22.31 29. rejang 104.37 10.88 30. serawak 17.38 81.80 31. triolin 16.62 73.72 average + sd min max 60.92+39.22 12.79-149.34 22.06+16.01 2.42-81.80 indrasari et al. – vitamin c and total sugar content characterization on 31 accessions of banana … 55 vitamin c or ascorbic acid is needed for health. in human body vitamin c serves as an antioxidant that can protect the body, especially cellular dna from damage due to oxidation of free radicals. antioxidants prevent oxidative stress and play an important role in the development of chronic and degenerative diseases such as cancer, arthritis, autoimmune disease, cardiovascular and neurodegenerative diseases (lian et al., 2008 in harifah et al., 2017). daily requirement for vitamin c vary for each individual, in general experts estimate that 60 mg per day is enough but in certain conditions the body requires more than that (sitohang, 2006). based on widya karya nasional pangan dan gizi v (1993), the requirement of vitamin c per day for baby (7-12 months) is 35 mg, childrem (7-9 year) is 45 mg and women and men (20 59 year) is 60 mg. it appears in table 2 that accessions of bananas that have a high vitamin c content followed by a low total sugar content. this means that there is a negative correlation between vitamin c content and total sugar on 31 accessions of bananas observed. correlation analysis result showed negative correlation equal to 0.41. furthermore, from the chemical characteristic data of vitamin c content and total sugar was used for cluster analysis with the result of dendogram in figure 1. information: 1. ambon jaran : aj 12. kidang hijau : kih 22. morosebo : mro 2. ambon sepet : as 13. klutuk susu : ks 23. potho merah : pm 3. ambon warangan : aw 14. koja pretel : kp 24. raja bandung : rb 4. anjasmoro : am 15. koja sirimentak : ksi 25. raja lini : rl 5. australi : au 16. lempereng : lp 26. raja pulut : rp 6. ceni : ce 17. mangsan : ma 27. raja sabrang : rs 7. gajih putih : gp 18. mas jambe : mj 28. raja sewu : rse 8. kapok brentel : kb 19. mas raja : mr 29. rejang : rj 9. kepok bung : kbu 20. mas sloka : ms 30. serawak : sr 10. kepok hijau : kh 21. mas tropong : mt 31. triolin : to 11. kepok kuning : kk figure 1. dendogram of 31 banana accession of banana germplasm garden collection of based on vitamin c and total sugar content. dendogram of figure 1 shows the kinship relations of 31 banana accessions based on vitamin c and total sugar content. there appears to be some accessions that have very close kinship i.e. accession aj (ambon jaran) with accession lp (lempereng); and accession kk (kepok kuning) with ms accession (mas sloka). but among these accessions there is no equal accession to other accessions, meaning there is no duplication of names between the accessions. and each accession is a separate accession with one accession to another. this result is supported by the morphological character of the fruit, presented in figure 2. name of observation or cluster rj mt ms kk mro kih mj gp ma kbu rp as to sr au ks rb ksi rs aw mr kp rse kb ce am kh rl pm lp aj average distance between clusters 0 10 20 30 40 50 60 70 80 56 biology, medicine, & natural product chemistry 6 (2), 2017: 53-57 dendogram of figure 1 shows that 31 banana accessions based on vitamin c content and total sugar were divided into 5 groups. group i consists of 11 accessions of bananas ie: aj (ambon jaran); lp (lempereng); pm (potho merah); rl (raja lini); kh (kepok hijau); am (anjasmoro); ce (ceni); kb (kepok brentel); rse (raja sewu); kp (kojo pretel) and accession of mr (mas raja). group ii consists of 7 accessions of bananas ie: aw (ambon warangan); rs (raja sabrang); ksi (kojo sirimentak); rb (raja bandung); ks (kluthuk susu); au (australi);. group iii consists of 2 accessions of bananas ie: sr (serawak); and to (triolin). group iv consists of 4 accessions of bananas ie: as (ambon sepet); rp (raja pulut); kbu (kepok bung); and ma (mangsan); and group v consists of 8 accessions of bananas ie: gp (gajih putih); mj (mas jambe); kih (kidang hijau); mro (morosebo); kk (kepok kuning); ms (mas sloka); mt (mas tropong); and rj (rejang). figure 2. bananas fruit of 31 bananas accessions. (a). ambon jaran; (b). ambon sepet; (c). ambon warangan; (d). anjasmoro; (e). australi; (f). ceni; (g). gajah putih; (h). kepok brentel; (i). kepok bung; (j). kepok hijau; (k). kepok kuning; (l). kidang hijau; (m). kluthuk susu; (n). koja pretel; (o). koja sirimentak; (p). lempereng; (q). mangsan; (r). mas jambe; (s). mas raja; (t). mas sloka; (u). mas tropong; (v). morosebo; (w). potho merah; (x). raja bandung; (y). raja lini; (z). raja pulut; (aa). raja sabrang; (ab). raja sewu; (ac). rejang; (ad). serawak; (ae). triolin. indrasari et al. – vitamin c and total sugar content characterization on 31 accessions of banana … 57 conclusion from the result we draw conclusion as follows: 1. the content of vitamin c and total sugar from 31 accessions of bananas vary according to their genetic potential, with an average vitamin c content of 60.42 + 39.22 mg/ 100 g and average total sugar 22.06 + 16.01%. 2. each banana accession from 31 accession was a separate accession one with another accession and is divided into 5 groups based on vitamin c content and total sugar. references anonim. 1993. daftar angka kecukupan gizi yang dianjurkan. widya karya nasional pangan gizi ke v. jakarta komaryati & adi, s. 2012. analisis faktor-faktor yang mempengaruhi tingkat adopsi teknologi budidaya pisang kepok (musa paradisiaca) di desa sungai kunyit laut. kecamatan sungai. kabupaten pontianak. j. tiprekas: 53-61. kristamtini, wiranti, ew., indrasari, sd., wirasti, ch.a., & sutarno. 2016. deskripsi aksesi pisang koleksi kebun plasma nutfah pisang yogyakarta tahun 2016. bptp yogyakarta. harifah, i., mustofa, a., suhartatik, n., 2017. aktivitas antioksidan infuse water dengan variasi jenis jeruk (nipis, lemon, dan baby) dan buah tambahan (strawberry, anggur hitam, dan kiwi). ejurnal.unisri.ac.id/index.php/jtpr/article/download/1517/1335 . retrieved at 23 august 2017. santos, e.a., souza, m.m., viana, a.p., almeida, aaf., freitas, jco., & lawinsky, pr.. 2011. multivariate analysis of morphological charateristics of two species of passion flower with ornamental potential and of hybrids between them. gen. mol. res. 10 (4): 2457-2471. simmonds, n.w. and shepherd, k. 1955. the taxonomy and origins of the cultivated bananas. lennean soc bot. 55: 302312. sitohang, a. pengaruh konsentrasi gula dan suhu pengering terhadap mutu pada pembuatan sirup markisa. media unika tahun 26 no. 87. edisi 1. subandiyah. s, sumardiyono, christanti, rumahlewang & wilhelmina, w. 2002. pengimbasan ketahanan pisang terhadap penyakit layu bakteri (ralstonia solana cearum) dengan pseudomonas cepacia jurnal agrosains 15 (1): 9-16. sukartini. 2007. pengelompokan aksesi pisang menggunakan karakter morfologi ipgri. j. hort 17 (1). wardiyati. t, retnowati. a, & widaryanto, e. 1997. ketahanan empat kultivar pisang terhadap kekeringan. jurnal agrivita 20 (3): 167-170. wardiyati. t, sugianto. a, nugroho. a, lamadji. s, & mugiono. 2004. perbaikan sifat pisang kepok melalui mutasi buatan sinar gamma; keragaman fase genetik. jurnal ilmu hayati 16 (2): 90-98. http://portalgaruda.org/index.php?ref=browse&amp;mod=viewarticle&amp;article=17868 http://portalgaruda.org/index.php?ref=browse&amp;mod=viewarticle&amp;article=17868 http://portalgaruda.org/index.php?ref=browse&amp;mod=viewarticle&amp;article=17868 this page intentionally left blank vitamin c and total sugar content characterization on 31 accessions of banana collection of banana germplasm plants of yogyakarta biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 77-80 | doi: 10.14421/biomedich.2020.92.77-80 issn 2540-9328 (online) isolation and characterization of stigmasterol from fritillaria roylei gunpreet kaur1,*, vikas gupta1, r. g. singhal2, parveen bansal1 1baba farid university of health sciences, faridkot, india 2shobhit university, meerut, india corresponding author* reetjattana21@gmail.com manuscript received: 26 june, 2020. revision accepted: 06 november, 2020. published: 16 november, 2020. abstract fritillaria roylei (kshirakakoli) is the threatened species of “ashtwarga” group suffers lot of confusion for identification & authentification in ayurvedic system of medicine. due to lack of natural sources and insufficient availability of kshirakakoli, chances of adulteration and substitution increases which in turn leads to loss of faith of people in herbal drugs. thus for identification and differentiation, quality standardization and quality assurance of kshirakakoli containing herbal formulations there is a need to isolate chemical marker compound using advanced analytical techniques. the methanol extract of root samples of plant was prepared and phytochemical screening was performed. marker compound was isolated from the extract using column chromatography. single compound having rf value 0.31 was isolated with tlc by using mobile phase n-hexane: ethyl acetate: formic acid (8:2:0.1 v/v/v) and purified by re-crystallization with methanol. isolated compound was further characterized by using melting point and spectral analysis. the methanol extract was dark brown in color and showed the presence of steroids, amino acids and flavonoids. the isolated compound was found to be white crystalline powder with melting point range of 167-169°c. spectral analysis confirmed the presence of stigmasterol. in present study stigmasterol was isolated for the first time and can be used as chemical marker for identification and differentiation of the plant from its substitutes. keywords: isolation; kshirakakoli; marker; standardization; stigmasterol. introduction ayurveda is one of the most distinctive systems of medicine known to man (jaiswal and williams, 2017; sreena et al., 2011). it is regarded as the oldest divine knowledge in the humankind which is based on the principle of maintaining a balance between the interconnected relations within the body and mind. ayurvedic medicines include herbs, herbal materials, herbal preparations, minerals and finished herbal products. ayurveda is blessed with plentiful speculate medicinal plants including ashtawarga group of eight medicinal plants i.e. kakoli (roscoea purpurea), kshirakakoli (fritillaria roylei), jeevaka (microstylis muscifera), rishabbhaka (malaxis acuminatea), meda (polygonatum cirrhifolium), mahameda (polygonatum verticillatum), riddhi (habenaria edgeworthii) and vridhi (habenaria internedia) (balkrishna et al., 2012; saha d et al., 2015). in recent days, herbal medicines are getting more popular with the comprehensive movement of people towards natural therapies. this increasing demand of the population towards herbal medicines results in shortage of authentic raw materials leading to increase in chance of adulteration and substitution because the regulatory authorities lack the strict quality control measures of herbal medicines (shukla and dhanya, 2017). this same situation happens with plants of ashtawarga group. according to international union of conservation of nature (iucn) and conservation assessment and management plan (camp), fritillaria roylei (kshirakakoli) is considered as threatened medicinal plant (saha et al., 2015; iucn 2001; kuniyal et al., 2015). from various studies, it has been found that due to lack of natural sources and insufficient availability to meet the requirements of market for the raw material the department of ayush, govt. of india, permitted the use of available substitutes in place of original plant. the substitution of fritillaria roylei is done with ashwagandha (root) (withania somnifera) or safed musli root (chlorophytum arundinaceum boker) (balkrishan et al., 2012; sagar, 2014). however, literature survey reveals that ayurvedic parameters as well as pharmacological actions of the ashtawarga plants do not match with their substitutes. the substitute of f. roylei that is w. somnifera or c. arundinaceum shows 33% or 16% similarity which ultimately results in reduced efficacy of the drugs along with loss of faith of people towards use of herbal drugs (virk et al., 2015). thus, drug standardization and quality assurance is essential to assess the safety, efficacy and quality of herbal drugs. lack of chemical markers is a major problem for the https://doi.org/10.14421/biomedich.2020.92.77-80 78 biology, medicine, & natural product chemistry 9 (2), 2020: 77-80 regulatory authorities for enforcement of quality regulations. therefore, the study was designed to isolate chemical markers from fritillaria rolei (kshirakakoli) using advanced analytical techniques. materials and methods chemicals in the present study all the reagents and solvents used were of analytical grade. for isolation of marker compound, precoated aluminum-backed tlc plates with 0.2 mm layer of silica gel 60 f254 (20 cm × 10 cm) manufactured by e. merck (germany) were purchased from local authorized dealer. plant material the root samples of fritillaria roylei were procured from cultivar source of himachal pradesh and authenticated by national botanical research institute, lucknow wide authentication letter no. nbri/cif/535/2017 dated 04/01/2017. root samples of the plant were washed, shade dried and stored in air tight container. extract preparation the shade dried roots of fritillaria roylei (500gm) were coarsely powdered and defatted with petroleum ether followed by continuous hot extraction process with methanol. the methanol extract thus obtained was filtered and then evaporated to obtain a concentrated semisolid mass. the final extract thus obtained was then stored in vacuum desiccator for further use. phytochemical screening preliminary phytochemical screening of the extract were performed for the detection of phytoconstituents like alkaloids, flavonoids, steroids, proteins, tannins, saponins, phenolics, carbohydrates and amino acids (banu and cathrine, 2015; kokate et al., 2000; tiwari et al., 2011). isolation of chemical marker about 8.4g of methanol extract was mixed with methanol and then silica gel having pore size 60–120 mesh was added to form slurry which was then dried on water bath to form a free flowing powder. silica gel (675g) suspended in n-hexane was poured into the glass column having dimensions 1000mm x 50 mm to give rise to silica bed. saturated silica bed was allowed to stand overnight for uniform bed packing. after 12hrs, elution was started with non-polar solvent n-hexane followed by an increase in polarity of the solvents and fractions were collected with optimum flow rate of 10ml/min. thin layer chromatography (tlc) of collected fractions were performed using different solvents selected by hit and trial method and on the basis of tlc profile similar fractions were pooled together. elution with the solvent system n-hexane: chloroform: ethyl acetate 60:20:20 yielded a pool of two compounds with rf 0.31 and 0.42 on tlc plates by the use of mobile phase n-hexane: ethyl acetate: formic acid (8:2:0.1 v/v/v) along with mixture of other compounds on tlc plates. single compound was isolated by cutting and pooling of tlc plate of compound having rf value 0.31 and purified by re-crystallization with methanol. the fraction was kept in a refrigerator to get the crystallized compound (virk et al., 2016; cannell, 1998). characterization of isolated compound isolated crystallized compound was characterized by using different chemical test, melting point and spectral analysis (ir, nmr, mass, and uv spectroscopy). figure 1. nmr spectra of isolated compound stigmasterol from fritillaria roylei (kshirakakoli). kaur et al. – stigmasterol from kshirakakoli 79 result and discussion physical evaluation and percentage yield of extract the methanol extract of fritillaria roylei was dark brown in color, and the percentage yield of extract was 7.48%. phytochemical screening of extract preliminary phytochemical analysis confirmed the presence of steroids, amino acids, flavonoids and proteins characterization and identification of isolated compound isolated compound was found as white crystalline powder after crystallization from chcl3-meoh. the compound was shown positive test of steroids. melting point of the compound was found to be 167-169°c (lit. 164-171°c). spectroscopic data ir: the infra-red spectra of isolated compound, showed very intensely broad peak at 3428 cm-1 and moderately intense peak at 1192 and 699 cm-1 were observed for the o-h bond vibrations of hydroxyl group. the unsaturated part of c-h and c=c vibrations was observed at 881 cm1 and 1642 cm-1. the vibrations of -ch3 were observed at 2937 cm-1and at 1465 cm-1 and vibrations of =ch2 were observed at 2852 cm-1 and at 1460 cm-1 respectively. the c-c vibration peak was shown at 1053 cm-1which confirm the structure of stigmasterol. nmr: in the 1h-nmr spectrum of isolated compound showed two olefenic protons appeared downfield at δ 4.57 (m) and δ 4.14 (m) which were identical with the chemical shift of h-22 and h-23, respectively of stigmasterol. six methyl protons at δ 1.23(s, 3h), δ 1.19(s, 3h), δ 1.06(s, 3h), δ 1.00, δ 0.98(s, 3h) and δ 0.91 (s, 3h); [(3h each, s, ch3)] confirms the structure of stigmasterol. mass spectra: mass spectrum of isolated compound showed parent molecular ion [m+] peak at m/z 412 which was being in agreement with the proposed structure of stigmasterol. structure and molecular formula of isolated compound the molecular formula of isolated molecule stigmasterol is c29h48o that is confirmed by ir, mass spectra and nmr data. its iupac name is (3s,8s,9s,10r,13r,14s,17r)-17-[(e,2r,5s)-5-ethyl-6methylhept-3-en-2-yl]-10,13dimethyl,2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro1h-cyclopenta[a] phenanthren-3-ol. as phytosterols are not synthesized by humans and animals so they are needed to be included in the diet. stigmasterol is present in various naturally occurring items of plant origin like vegetables, nuts, seeds and unpasteurized milk. edible oils contain higher amount of stigmasterol than vegetables. it is widely used as food additives in food and beverage industries for production of sausages, yogurt, cold cuts, margarines, bakery products, milk and spreads (aboobucker et al., 2019; jun-hua et al., 2008; siddiqui et al., 1990). stigmasterol is also used as a precursor or intermediate for synthesis of various human steroid hormones like progesterone, androgen corticoids, estrogens etc (sundararaman and djerassi, 1977; hogg, 1992). stigmasterol is used as raw material in pharmaceutical industries for commercial synthesis of vitamin d3 (kametani and furuyama, 1987). in addition to this various studies showed that stigmasterol possesses various pharmacological properties like cytotoxic activity, antioxidant activity, anti-hypercholestrolemic, anti-osteoarthritic activity, anti-inflammatory activity, hypoglycemic activity etc. high therapeutic value and regulatory approval status as generally recognized as safe (gras) in the u.s., followed by an approval from the fda and by the eu scientific committee on food (sfc) increases the demand of phytosterol significantly (ghosh et al., 2011; chandler et al., 1979; batta et al., 2006; gabay et al., 2010; panda et al., 2009; navarro et al., 2001). as the market price of stigmasterol is 32,334/10g (approximately) and it will be difficult for commercial manufacturers to replace fritillaria roylei plant with stigmasterol just to claim the presence of fritillaria roylei (kshirakakoli). presence of stigmasterol has not been ever reported in its substitutes/adulterants. it has been reported first time from root of kshirakakoli plant. thus, for quality standardization of kshirakakoli containing formulations isolated compound i.e. stigmasterol may be used as chemical markers for identification and differentiation of authentic plants from its substitutes and common adulterants. conclusion in the present study, stigmasterol was isolated from roots of fritillaria roylei using column chromatography and tlc. as per the knowledge, this is first report on chromatographic method of isolation of stigmasterol from natural source that is fritillaria roylei plant. this compound can be used as a chemical marker by regulatory authorities for identification of kshirakakoli plant from other substitutes. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. source of funding: the authors gratefully acknowledge authorities of baba farid university of health sciences, faridkot for necessary permission and providing all research facilities. this work was supported by the national medicinal plants board, ministry of ayush, govt. of india, new delhi, india [grant numbers r&d/ch-01/2012]. 80 biology, medicine, & natural product chemistry 9 (2), 2020: 77-80 references aboobucker, s. i., suza, w. p. 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(2015). lack of pharmacological basis of substitution of an endangered plant group ashtawarga–a significant ingredient of polyherbal formulations. am j phytomed clin ther., 2 690712. cover jurnal biomenaprochy vol 13 num 2 2024.cdr biology, medicine, & natural product chemistry volume 13 – number 2 – october 2024 issn 2089-6514 (paper) | issn 2540-9328 (online) contents the effect of tapioca flour and bogor taro flour (colocasia esculenta l. schott) formulations on the chemical, physical, and sensory characteristics of catfish sausage (pangasius hypophtalmus) leona amelia, sussi astuti, esa ghanim fadhallah, dyah koesoemawardani 321-328 leaf extract of kentut (paederia foetida l.) as a preventive measure against interleukin-6 expression in the liver of mice in a sepsis model injected with escherichia coli lisa savitri, maria do carmo da costa freitas, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita 329-336 pharmacophore mapping of angiotensin converting enzymes (aces): insight to binding site of ace1 and ace2 navista sri octa ujiantari, cintya nurul apsari 337-341 potency of turmeric extract (curcuma domestica val.) with virgin coconut oil (vco) against incision wound healing yusni atifah, aifa kurnia 343-349 effect of justicia insularis leaf extract and fractions on oxidative stress markers, liver function parameters and liver histology of plasmodium berghei -infected mice veronica james enyiekere, martin osita anagboso, uduak peter ise, grace emmanuel essien, jude efiom okokon, nwakaego omonigho ebong 351-359 isolation and antibacterial activity of sembung (blumea balsamifera) leaf essential oil l., dc tika afriani, azimatur rahmi, mia darmita sari 361-365 the antibacterial and antibiofilm efficacy of citrullus lanatus rind ethanol extract against aggregatibacter actinomycetemcomitans wilbert alvin genesis cokro, priska natassya, sheila soesanto 367-372 antibacterial activity screening of bacillus sp. am12 associated with mangrove soil evellyne natania, anak agung gede indraningrat, ni wayan widhidewi 373-379 https://sciencebiology.org/index.php/biomedich/article/view/498 https://sciencebiology.org/index.php/biomedich/article/view/498 https://sciencebiology.org/index.php/biomedich/article/view/498 https://sciencebiology.org/index.php/biomedich/article/view/538 https://sciencebiology.org/index.php/biomedich/article/view/538 https://sciencebiology.org/index.php/biomedich/article/view/538 https://sciencebiology.org/index.php/biomedich/article/view/541 https://sciencebiology.org/index.php/biomedich/article/view/541 https://sciencebiology.org/index.php/biomedich/article/view/544 https://sciencebiology.org/index.php/biomedich/article/view/544 https://sciencebiology.org/index.php/biomedich/article/view/555 https://sciencebiology.org/index.php/biomedich/article/view/555 https://sciencebiology.org/index.php/biomedich/article/view/555 https://sciencebiology.org/index.php/biomedich/article/view/546 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397-406 effectiveness of ethanol extract clove leaves (syzygium aromaticum) in inhibiting biofilm of candida albicans atcc 14053 firdausi nuzula, masfufatun masfufatun 407-413 physicochemical characteristics of jelly candy cinnamon powder koerintji variation of red seaweed carrageenan concentration erwin setiawan, rizky muliani dwi ujianti, iffah muflihati, arief rakhman affandi 415-421 effectiveness test of methanol, ethyl acetate, and chloroform fractions of bidara leaf extract (zizyphus mauritiana l.) on wound healing in rabbits (oryctolagus cuniculus) novel umarella, wirasti wirasti, slamet slamet, urmatul waznah 423-431 prospecting marine natural products as the disease-modifying treatment of alzheimer’s diseases legis ocktaviana saputri, herpan syafii harahap, arina windri rivarti, fitriannisa faradina zubaidi 433-441 phytochemical and toxicity analysis of sonneratia alba mangrove leaf extract using the brine shrimp lethality test (bslt) method ni kadek dewi asri tiara arta, anak agung gede indraningrat, made dharmesti wijaya 443-448 antibacterial activity of sweet orange (citrus sinensis) peel tea against enterobacteriaceae isolated from a water depot yumiko yumiko, suhartomi suhartomi, sri wahyuni nasution, siti syarifah, ade pryta r. simaremare 449-458 the new technology for in vitro culture with induction of nanobubbles (nbsn2 and nbso2) in cattleya sp. moch mustakim, azis mawardi, lina purnama dewi, nela vede rhofa putri, tintrim rahayu, gatra ervi jayanti 459-465 https://sciencebiology.org/index.php/biomedich/article/view/466 https://sciencebiology.org/index.php/biomedich/article/view/466 https://sciencebiology.org/index.php/biomedich/article/view/565 https://sciencebiology.org/index.php/biomedich/article/view/565 https://sciencebiology.org/index.php/biomedich/article/view/565 https://sciencebiology.org/index.php/biomedich/article/view/566 https://sciencebiology.org/index.php/biomedich/article/view/566 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salmonella spp. in cured beef with different shelf-life expectancy mutia asri khairunisa, endah retnaningrum 467-473 effect of mixing time variation and framing time on the quality of coffee grounds-based solid soap insa dorra neisya, suratno suratno, kamalia fikri 475-483 histopathological description of mouse liver in a sepsis model infected with escherichia coli treated with paederia foetida l. leaf extract for sepsis prevention lisa savitri, ana retnowati, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita 485-492 effect of trichosanthes cucumerina methanol extract on crp and fibrinogen levels in diabetic ulcer rat models nazula rahma shafriani, tri dyah astuti, shofy farha aulia amri, riski aditya permana, zuvi fridhoriansyah, intan maharani 493-496 quantitative analysis, anti-inflammatory and analgesic effects of ethanol leaf extract and fractions of microsorium scolopendria (burm. f.) copel. in mice uwemedimo francis umoh, ekikere ezekiel ubengama, gina ramos, victor chubueze onyido 497-503 the effectiveness of fragrant pandan leaves ethanol extract in reducing cholesterol levels in high-fat diet-induced rats ni made wiasty sukanty, farida ariani, lina yunita 505-509 utilization of maggot larvae (hermetia illucens) for processing food waste at the uin raden intan lampung canteen ika listiana, marlina marlina, eko kuswanto, ade lenty hoya 511-516 formulation and evaluation of hard candy preparation as an innovative internal wound medicine from snakehead fish (channa striata) albumin extract ilvid diana, elfi abidah ardelia, nazilatun nadriani, dilna azizah, dea nova widhyati, mohammad rofik usman 517-522 preparation of ni/bentonite acid-activated using dragon fruit peel extract (hylocereus polyrhizus) as a reductor for one pot synthesis of menthol yuni marhayuni, priyagung dhemi widiakongko 523-530 https://sciencebiology.org/index.php/biomedich/article/view/586 https://sciencebiology.org/index.php/biomedich/article/view/586 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lombok with red blood cell membrane stability method ani fatin humaira, siti rahmatul aini, raisya hasina 555-558 the effect of paracetamol on the development of chicken embryos hendry tri sakti surya gunawan saragih, alfian primahesa, shintia oktaviana, agnessia asian budi setyo, alifia anisya, hayu swari allimi, laili mufli zusrina, ruth liananda citra dolok saribu, ardaning nuriliani, nur indah septriani 559-564 formulation and effectiveness test of red ginger (zingiber officinale var. rubrum) and starfruit (averrhoa bilimbi l.) combination shampoo against pediculus humanus capitis reza anindita, salwa zhafarina harahap, maya uzia beandrade, intan kurnia putri 565-573 identification of marbofloxacin-susceptible bacterial uropathogens in feline urinary tract infection of different age groups sekarani arifah udasmara, endah retnaningrum 575-584 the recent collection of climacodon roseomaculatus (hydnum roseomaculatum henn. & e. nyman) from the type locality (indonesia) ivan permana putra, oktan dwi nurhayat 585-589 antibacterial activity testing propionibacterium acnes combination of 70% ethanol extract of starfruit leaves (averrhoa bilimbi l.) and jarak pagar leaves (jatropha curcas l.) tarisya anilya dwi putri, agnes yuliana, krismayadi krismayadi 591-594 chemical constituents and evaluation of bioactivity of vernonia hymenolepis a. rich root extracts alemu gelata, mudin jemal, soressa gershe, negera abdissa 595-600 https://sciencebiology.org/index.php/biomedich/article/view/560 https://sciencebiology.org/index.php/biomedich/article/view/560 https://sciencebiology.org/index.php/biomedich/article/view/604 https://sciencebiology.org/index.php/biomedich/article/view/604 https://sciencebiology.org/index.php/biomedich/article/view/639 https://sciencebiology.org/index.php/biomedich/article/view/639 https://sciencebiology.org/index.php/biomedich/article/view/639 https://sciencebiology.org/index.php/biomedich/article/view/620 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polymorphism in schizophrenia of batak and minangkabau ethnicities in pematang siantar raysha awlia wijaya, edy fachrial, i nyoman ehrich lister, bayu ariatama 601-608 modulatory efficiency of vitamin c (ascorbic acid) on collagen-induced platelet aggregation and dysfunction osah martins onwuka, chukwukadibie adigwe, oghenefego michael adheke, chamberlin jamike elem, josiah soipiriala hart 609-615 exploring the therapeutic potential of kalanchoe pinnata: a comprehensive analysis of bioactive compounds and pharmacological activities dakshina bhusal, manila poudel, ashika tamang, sujan dhital, nirmal parajuli, timila shrestha, samjhana bharati, binita maharjan, jhashanath adhikari subin, bishnu prasad marasini, ram lal swagat shrestha 617-625 application of taguchi experimental design in optimization of levulinic acid production from cellulose derived from millet stalk shehu ibrahim, abdullahi sokoto muhammad, kabiru jega umar, ibrahim magami muhammad 627-633 https://sciencebiology.org/index.php/biomedich/article/view/569 https://sciencebiology.org/index.php/biomedich/article/view/569 https://sciencebiology.org/index.php/biomedich/article/view/575 https://sciencebiology.org/index.php/biomedich/article/view/575 https://sciencebiology.org/index.php/biomedich/article/view/609 https://sciencebiology.org/index.php/biomedich/article/view/609 https://sciencebiology.org/index.php/biomedich/article/view/609 https://sciencebiology.org/index.php/biomedich/article/view/642 https://sciencebiology.org/index.php/biomedich/article/view/642 this page intentionally left blank 3: front 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1325-1330 | doi: 10.14421/biomedich.2025.142.1325-1330 issn 2540-9328 (online) laboratory detection of toenail onychomycosis using malt extract agar in students lisa savitri1*, kharisul ihsan2, elfred rinaldo kasimo1 1department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia 2department of pharmacy, faculty of pharmacy, public health, hospital administration, radiology, universitas strada indonesia, kediri, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 04 october, 2025. revision accepted: 29 november, 2025. published: 14 december, 2025. abstract onychomycosis is a common nail disorder caused by dermatophytes, non-dermatophyte molds, and yeasts. despite being more prevalent in older adults, it is increasingly reported among younger populations, including university students, due to lifestyle factors and communal exposures. rapid and accurate diagnosis is critical for effective management, but conventional methods remain limited in sensitivity. this study aimed to evaluate the prevalence and etiological profile of toenail onychomycosis among university students using potassium hydroxide (koh) microscopy and malt extract agar (mea) culture. a total of 98 students with clinical signs of onychomycosis provided toenail specimens. direct microscopy examination with 20% koh was performed, followed by culture on mea at 28–30 °c for up to 21 days. fungal isolates were identified based on macroscopic and microscopic morphology. statistical analysis assessed associations between clinical features and culture results. koh microscopy detected fungal elements in 59.2% of specimens, while mea culture yielded growth in 63.3%. when combined, the overall prevalence of confirmed onychomycosis was 71.4%. trichophyton rubrum was the most frequent isolate (35.5% of positive cultures), followed by non-dermatophyte molds such as aspergillus and fusarium, and yeasts including candida albicans and candida parapsilosis. nail thickening and discoloration were significantly associated with culture positivity (p < 0.05). onychomycosis is prevalent among university students, with mea culture providing superior diagnostic yield compared to koh microscopy alone. the identification of non-dermatophyte molds and yeasts underscores the importance of comprehensive diagnostic strategies for guiding effective treatment. future studies should combine culture and molecular methods to enhance detection accuracy and clarify the clinical significance of emerging pathogens. keywords: onychomycosis; malt extract agar; potassium hydroxide; dermatophytes; non-dermatophyte molds. abbreviations: human immunodeficiency virus (hiv); potassium hydroxide (koh); malt extract agar (mea); non-dermatophyte molds (ndm); polymerase chain reaction (pcr); sabouraud dextrose agar (sda) introduction onychomycosis is a fungal infection of the nails that most commonly affects toenails rather than fingernails. the disease is characterized by nail discoloration, thickening, brittleness, and in advanced cases, pain and impaired function. while dermatophytes are the main etiological agents, non-dermatophyte molds and yeasts have increasingly been identified in recent years (nenoff et al., 2023). this condition represents an important public health concern due to its relatively high prevalence, particularly among adults and older populations. a recent global epidemiological analysis reported a prevalence of toenail onychomycosis of approximately 4% in the general population, with significantly higher rates in groups with specific risk factors such as diabetes or immunocompromised states (gupta et al., 2024). although often perceived as a cosmetic issue, onychomycosis has a substantial impact on quality of life, including reduced self-confidence and limitations in daily activities. clinical diagnosis alone is unreliable because several non-infectious conditions, such as psoriasis or trauma, may mimic the appearance of onychomycosis. therefore, laboratory confirmation remains essential to establish the presence of fungi and to determine the causative species (clinical diagnosis and laboratory testing study, 2024). updated international guidelines emphasize that direct microscopy and fungal culture remain the cornerstone of diagnosis, despite the growing use of molecular techniques in some centers (nenoff et al., 2023). the choice of culture medium is a critical factor influencing the success of fungal isolation. traditionally, sabouraud dextrose agar (sda) or dermatophyteselective media are used. however, other media such as malt extract agar (mea) have been shown to support https://doi.org/10.14421/biomedich.2025.142.1325-1330 mailto:lisasavitri@unik-kediri.ac.id 1326 biology, medicine, & natural product chemistry 14 (2), 2025: 1325-1330 the growth of a wide variety of fungi, including dermatophytes, non-dermatophyte molds, and yeasts. mea contains carbohydrates from malt extract that stimulate robust colony growth with clearer morphology, which may facilitate morphological identification (university of adelaide, 2024). while mea is widely employed in environmental and taxonomic mycology, its specific application for the clinical diagnosis of onychomycosis remains underexplored in recent literature. the urgency of this study lies in the need for more sensitive and representative culture methods to detect fungal agents of onychomycosis, particularly in young populations such as university students. students may be at risk due to shared facilities such as bathrooms, swimming pools, and communal footwear, along with lifestyle factors that increase nail humidity and fungal exposure (widaty et al., 2024). the novelty of this research is its focus on evaluating malt extract agar for toenail onychomycosis diagnosis in a student population — an area that has not been widely investigated in indonesia. findings from this study are expected to contribute valuable data for improving clinical mycology diagnostics and to inform preventive and educational strategies for nail health within academic environments. materials and methods study design and setting this was a descriptive cross-sectional study conducted at the microbiology laboratory of medical laboratory technology, faculty of health sciences, kadiri university. the study aimed to evaluate the use of malt extract agar (mea) for the detection of fungal pathogens in toenail samples of university students clinically suspected of having onychomycosis. study population the study population consisted of undergraduate students enrolled at laboratory of medical laboratory technology, faculty of health sciences, kadiri university. participants were recruited through an open call within the faculty premises. students who presented with clinical signs suggestive of toenail onychomycosis, such as nail discoloration, thickening, brittleness, or onycholysis, were invited to participate. inclusion criteria: 1. students aged 18–30 years. 2. presence of toenail changes clinically suspected as onychomycosis. 3. willingness to provide informed consent. exclusion criteria: 1. history of systemic or topical antifungal treatment in the past 3 months. 2. presence of nail abnormalities due to trauma or psoriasis confirmed by clinical examination. 3. students with comorbidities severely affecting immunity (e.g., hiv/aids, chemotherapy). sample collection toenail specimens were collected from affected nails under aseptic conditions. after cleaning the nail with 70% alcohol to remove contaminants, subungual debris and nail clippings were obtained using sterile surgical blades and nail clippers. samples were transported in sterile petri dishes or containers and processed immediately in the laboratory. direct microscopic examination a portion of each specimen was subjected to direct microscopic examination using a 20% potassium hydroxide (koh) preparation. samples were examined under light microscopy to identify fungal elements such as hyphae or yeast cells. culture procedure the remaining specimens were inoculated on malt extract agar (mea) plates. the inoculated plates were incubated at 28–30 °c for up to 21 days and examined periodically (every 3–4 days) for fungal growth. colony morphology (color, texture, topography) was documented. to avoid false-positive results due to contamination, isolates were only considered significant if they showed consistent growth in repeated inoculations or correlated with positive direct microscopy. identification was based on macroscopic colony features and microscopic morphology using lactophenol cotton blue mounts. quality control all culture media were prepared according to standard microbiological procedures and quality checked for sterility before use. positive control cultures of trichophyton rubrum and candida albicans were maintained to validate culture conditions. data analysis data were recorded in structured forms and analyzed using descriptive statistics. prevalence of onychomycosis was calculated, and fungal species distribution was presented in frequencies and percentages. concordance between direct microscopy and mea culture was assessed. statistical analysis was performed using spss version 26.0 and r software version 4.2.2. savitri et al. – laboratory detection of toenail onychomycosis using … 1327 results and discussion results study population a total of 120 university students were recruited for the study between march and june 2025. of these, 98 students met the inclusion criteria and provided toenail specimens. the mean age of participants was 21.4 ± 2.1 years, with a male-to-female ratio of 1.2:1. the most common clinical presentations included nail discoloration (76.5%), thickened nails (54.1%), and brittleness (32.6%). table 1. clinical characteristics and culture results. clinical characteristic culture positive (n) culture negative (n) p-value male students (n=54) 35 19 0.21 female students (n=44) 27 17 0.21 nail discoloration (n=75) 52 23 0.04* nail thickening (n=53) 39 14 0.03* brittleness (n=32) 21 11 0.12 *significant at p < 0.05 direct microscopy (koh examination) out of the 98 specimens examined by direct microscopy with 20% koh, 58 samples (59.2%) showed fungal elements, such as branching hyphae or yeast-like cells, while 40 samples (40.8%) were negative. culture on malt extract agar all 98 samples were inoculated onto malt extract agar (mea) and incubated at 28–30 °c for up to 21 days. fungal growth was observed in 62 samples (63.3%), while 36 samples (36.7%) showed no growth after the incubation period. concordance between koh and mea culture when comparing koh microscopy with mea culture: ▪ 45 specimens (45.9%) were positive by both koh and culture. ▪ 13 specimens (13.3%) were koh-negative but culture-positive. ▪ 12 specimens (12.2%) were koh-positive but culture-negative. ▪ 28 specimens (28.6%) were negative by both methods. this indicates that mea culture detected additional cases that were missed by direct microscopy, suggesting its potential to enhance diagnostic sensitivity. fungal isolates identified the distribution of fungal species isolated from mea cultures (figure 1) is summarized as follows: 1328 biology, medicine, & natural product chemistry 14 (2), 2025: 1325-1330 figure 1. distribution of fungal species isolated on mea. ▪ dermatophytes (41.9%) trichophyton rubrum (22 isolates, 35.5% of positives) trichophyton mentagrophytes (4 isolates, 6.4%) ▪ non-dermatophyte molds (27.4%) aspergillus spp. (7 isolates, 11.3%) fusarium spp. (6 isolates, 9.7%) scopulariopsis brevicaulis (4 isolates, 6.4%) ▪ yeasts (30.7%) candida albicans (11 isolates, 17.7%) candida parapsilosis (8 isolates, 12.9%) dermatophytes, particularly t. rubrum, remained the most common pathogen, but a considerable proportion of cases were caused by non-dermatophyte molds and yeasts (figure 2). figure 2. dermatophytes statistical analysis the overall prevalence of confirmed onychomycosis in this student population, defined as positive by either koh or mea culture, was 71.4% (70/98). the sensitivity of mea culture compared to koh microscopy was higher (63.3% vs. 59.2%), and the combined use of both methods increased diagnostic yield to 71.4%. chi-square analysis showed a statistically significant association between clinical signs of nail thickening and positive culture results (p < 0.05). no significant gender difference in prevalence was observed (p = 0.21). discussion this study demonstrated a relatively high prevalence of onychomycosis among university students, with 71.4% of participants confirmed positive by either koh or mea culture. mea culture yielded a higher positivity rate (63.3%) compared to direct microscopy (59.2%), and when combined, the diagnostic yield improved. trichophyton rubrum was the most frequently isolated species, but non-dermatophyte molds and yeasts also contributed substantially. these results highlight the importance of integrating mea culture in routine diagnostic workflows for nail infections (gupta et al., 2024; nenoff et al., 2023). diagnostic performance: koh versus culture (and the role of mea) koh microscopy remains the most commonly used initial test because it is rapid and inexpensive. however, its diagnostic accuracy is highly variable, with reported sensitivities ranging from 60% to 85% and specificities from 60% to 95% (hasan et al., 2023; gaol et al., 2024). the positivity rate in this study (59.2%) was consistent with the lower spectrum of published data, reinforcing concerns that koh alone may miss a considerable number of cases. savitri et al. – laboratory detection of toenail onychomycosis using … 1329 culture, despite being time-consuming, remains essential because it enables species-level identification and antifungal susceptibility testing. traditionally, sabouraud dextrose agar (sda) has been used; however, its performance is limited. malt extract agar (mea) has long been employed in environmental and taxonomic mycology due to its rich nutrient base, which promotes sporulation and colony morphology for a wide variety of fungi (university of adelaide, 2024; najafzadeh et al., 2014). this explains why mea culture identified more positive cases than microscopy alone in this study. species distribution and clinical implications dermatophytes remain the primary agents of onychomycosis globally, with t. rubrum as the predominant isolate (gupta et al., 2024; ha et al., 2025). however, non-dermatophyte molds such as aspergillus, fusarium, and scopulariopsis are increasingly reported (maskan bermudez, 2023; widaty et al., 2024). yeasts, particularly candida albicans and c. parapsilosis, were also frequent in this study, reflecting trends in recent epidemiological surveys (bersano et al., 2023). these findings are clinically significant because ndms and candida species often show different susceptibility patterns compared with dermatophytes, requiring tailored therapeutic strategies. concordance and added value of combined methods our findings confirmed that combining direct microscopy and mea culture improves diagnostic accuracy. previous work has similarly demonstrated that using more than one diagnostic approach increases sensitivity and reduces false negatives (nenoff et al., 2023; katsiaunis et al., 2024). while pcr-based molecular methods may provide even greater sensitivity, their high cost and limited accessibility in many regions restrict their widespread use (evrard et al., 2024). thus, culture with mea may represent a cost-effective alternative that still broadens pathogen detection. epidemiological considerations in university students young adults are often considered at lower risk for onychomycosis compared to older populations. however, communal living environments, shared bathrooms, and sports-related exposures increase fungal transmission in university settings (navarro-pérez et al., 2024). the prevalence observed in this study reinforces the need for awareness campaigns in younger populations, focusing on foot hygiene and preventive behaviors. limitations and future directions although mea culture improved detection, it also carries limitations. non-dermatophyte molds isolated on culture can represent contamination rather than true infection; repeated sampling and correlation with microscopy are necessary to confirm clinical relevance (dermnet nz, 2024). furthermore, while mea favors sporulation, it may also promote the growth of environmental fungi not implicated in disease. future multicenter studies should compare mea with sda and dermatophyte-selective media in parallel, incorporating molecular confirmation for species-level identification (katsiaunis et al., 2024). conclusions this study demonstrated a high prevalence of onychomycosis among university students, emphasizing that fungal nail infections are not limited to older populations. direct microscopy examination with koh remains a useful screening tool but has limitations in sensitivity. culture on malt extract agar proved valuable by detecting additional cases and enabling accurate identification of dermatophytes, non-dermatophyte molds, and yeasts. the predominance of trichophyton rubrum was consistent with global trends, but the notable presence of non-dermatophyte molds and candida species highlights the need for broader diagnostic approaches and tailored treatment strategies. integrating mea culture with routine koh examination can significantly improve diagnostic accuracy and provide clinicians with critical information for guiding therapy. future studies should validate these findings in larger, multicenter cohorts and explore the role of molecular methods alongside conventional mycology to further enhance diagnostic performance. acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors wrote the manuscript and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there are no funding. references bersano, j. m. q. b., litao, d. y. b., tin, m. s. a., gozum, i. e. g., & razon, m. n. 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(2024). clinical and microbiological characteristics of onychomycosis in a tertiary hospital: a cross-sectional study. medical journal of indonesia, 33(1), 17–23. https://doi.org/10.13181/mji.oa.247201 https://pubmed.ncbi.nlm.nih.gov/38392821/?utm_source=chatgpt.com https://dermnetnz.org/topics/non-dermatophyte-mould-onychomycosis?utm_source=chatgpt.com https://dermnetnz.org/topics/non-dermatophyte-mould-onychomycosis?utm_source=chatgpt.com https://www.adelaide.edu.au/mycology/laboratory-methods/malt-extract-agar?utm_source=chatgpt.com https://www.adelaide.edu.au/mycology/laboratory-methods/malt-extract-agar?utm_source=chatgpt.com biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 499-505 | doi: 10.14421/biomedich.2023.122.499-505 issn 2540-9328 (online) phytochemicals and larvicidal activity of sonneratia alba root extracts from ngurah rai mangrove forest, denpasar-bali made dharmesti wijaya1,*, anak agung gede indraningrat2, i gede yoga ayuning kirtanayasa3 1pharmacology department; 2microbiology department, faculty of medicine and health sciences; 3faculty of agriculture, warmadewa university, jl. terompong no 24 denpasar 80235, indonesia. tel. +62 361 240727. corresponding author* dharmestiwijaya@gmail.com abstract dengue is an endemic disease with a high incidence in almost all southeast asian countries, including indonesia. this infectious disease is caused by a virus transmitted by the bite of aedes aegypti mosquito as the main vector. effective mosquito vector control is a crucial step in stopping the spread of this virus. of the several methods available, the use of larvicides is considered one of the most successful treatments in reducing the number of mosquito vectors. however, widely used synthetic larvicides can have undesirable side effects on the environment and non-target organisms including human health. the aim of this study was to discover a new biolarvicide from natural materials that is relatively safer. in this study, the biolarvicidal activity of mangrove plant species that are commonly found in ngurah rai mangrove forest bali was tested, namely sonneratia alba. mangrove root simplicia was extracted by reflux method using three different solvents namely methanol, chloroform, and n-hexane. the content of compounds in the extract were analyzed using gc/ms method. the larvicidal activity of the extracts were tested on a. aegypti instar iii/iv larvae with concentrations of 0.1, 1, 10, 100, and 1000 ppm for 24 hours. subsequently, the average value of larval mortality was used to calculate the lc50 of each extract. the results showed that s. alba methanol extract provided the best larvicidal activity compared to chloroform and n-hexane extracts, with mortality of 69.33% at a concentration of 1000 ppm and lc50 of 1265 ppm. gc-ms analysis showed that the methanol extract of s. alba contained five dominant compounds namely methyl 2-hydroxyeicosanoate (19.55%); 4h-1-benzopyran-4-one, 3,5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy (16.48%); 4-((1e)-3-hydroxy-1propenyl)-2-methoxyphenol (10.06%); benzamide, n-[4-(2-naphtyl)-2-thiazolyl](9.40%); and 2,3-dihydro-3,5-bis(3-methoxyphenyl)-1hinden-1-one (6.52%). the results of this study provide a preliminary result on larvicide activity from mangrove s. alba in order to develop bio larvicides from nature which is safer for human health and environment. keywords: larvicidal activity; roots; sonneratia alba. introduction dengue is an infectious disease caused by the dengue virus (denv). this virus has four serotypes, namely denv 1, denv 2, denv 3, and denv 4 (gupta et al., 2021). dengue is a mosquito-borne virus with the main vector being the female aedes aegypti, which is also a vector of yellow fever, chikungunya, and zika (who, 2020). denv is estimated to infect 50-100 million people worldwide annually, causing an economic burden for both governments and individuals (guo et al., 2017). this disease has caused health burden to the community, mainly because limited diagnostic tool available for the early phase of infections, no specific therapy to cure the infections, and limited availability for effective and efficient vector control systems (cucunawangsih & lugito, 2017). in the last 50 years, indonesia has shown an increase in the incidence of dengue hemorrhagic fever, where bali and borneo (kalimantan) have been the areas with the highest incidence in recent years (harapan et al., 2019). to date, no antiviral therapy is available for dengue other than supportive therapy (who, 2020). cyd-tdv vaccine, the first and only dengue vaccine legally distributed in 2015 has failed to meet safety and efficacy aspects (wellekens et al., 2020). therefore, the current focus is to prevent transmission of the virus from mosquitoes to humans through vector control methods (jing & wang, 2019). one of the most common methods used to control dengue vectors is the use of the organophosphate compound temephos (abateⓡ) (george et al., 2015). unfortunately, long-term use of synthetic larvicides has been shown to cause resistance in vector organisms, as well as to cause unwanted effects on nontargets, including human health and the environment (pavela et al., 2019). this has led to intensive research to find natural larvicides that are safer and more effective since the last few decades (pavela et al., 2019). secondary metabolites derived from plants can be used as an alternative bio larvicides which are safer for humans and the environment, relatively inexpensive and manuscript received: 25 march, 2023. revision accepted: 30 august, 2023. published: 09 september, 2023. https://doi.org/10.14421/biomedich.2023.122.499-505 500 biology, medicine, & natural product chemistry 12 (2), 2023: 499-505 easy to obtain, biodegradable, as well as exhibit broad spectrum activity and specific targets against different species of mosquito vectors (ghosh et al., 2012). mangrove plants are a potential source that can be explored to find new bio larvicidal compounds. harsh environmental conditions such as high salinity and low oxygen levels make mangrove plants adapt, one of which is by synthesizing unique secondary metabolites (dahibhate et al, 2019). of the various parts of mangrove plants, roots are one of the important and interesting parts to study because they are directly exposed to fluctuating oxygen and salinity levels (reef & lovelock, 2014). a phytochemical study of mangrove plant sonneratia alba from musi river estuary, south sumatera, indonesia have shown that root extract of this species is rich in compounds such as flavonoids, triterpenoids, steroids, tannins, and phenols (rahmania et al, 2018). secondary metabolites such as flavonoids, triterpene, phenolic acid, and others were found to have larvicidal activity (de souza wuillda et al., 2019). therefore, in this study we aimed to screen for larvicidal activity of s. alba root extracts by using three different solvents: n-hexane, chloroform, and methanol, against aedes aegypti instar iii/iv larvae. this research is expected to accelerate the discovery and development of natural larvicides that are effective and safe for human health and the environment in order to create effective and sustainable vector control management. materials and methods materials sonneratia alba root was collected from simbar segara area of ngurah rai mangrove forest (8.72547397637542, 115.2002679693129) (figure 1), bali, indonesia, on november 2022 during low tide. solvents methanol, chloroform, and n-hexane were purchased from merck-supelco, germany. mosquito eggs were collected in west denpasar area using ovitraps. temephos (abateⓡ) was used as a positive control. figure 1. simbar segara area, ngurah rai mangrove forest, denpasar, bali. methods sample preparation root samples of s. alba (figure 2) were taken from five plants at different locations in simbar segara area of ngurah rai mangrove forest bali. sampling was carried out on thursday, 3 november 2022 at 11.00 to 13.00 wita during low tide. the collected roots were then washed under running water, cut into smaller sizes, and oven-dried at 50°c until the moisture content was <10%. the dry ingredients were chopped and blended into a coarse powder and then stored at room temperature until further processing. sample determination was carried out at characterization laboratory, “eka karya” botanical garden, baturiti, tabanan, bali-indonesia. figure 2. sonneratia alba tree (a), root part (b), and root samples (c). wijaya et al. – phytochemicals and larvicidal activity of sonneratia alba 501 extraction for each solvent (methanol, chloroform, or n-hexane), 100 grams of dry powder was refluxed 2x2.5 hours using 500 ml solvent at 50-55°c. the reflux results were filtered using whatmann no.1 filter paper and evaporated using rotary evaporator at 50°c. the extract was stored in a sealed and airtight vial at 4°c until used. larvae maintenance aedes aegypti mosquito eggs were collected using a simple ovitrap (djiappi-tchamen et al., 2022) in the west denpasar area. mosquito eggs were kept in plastic clips covered with silica gel until they were hatched. eggs were hatched by placing filter papers containing mosquito eggs in a tray filled with water to a depth of ± 2 cm. the room was maintained at an optimum temperature of 27 ± 2°c, humidity of 75 ± 10% and with dim lighting. the larvae of a. aegypti mosquitoes in instar iii/iv phase aged 3-6 days with a size of 4-6 mm were used as samples in this study. larvicidal activity testing larvicidal bioactivity testing was carried out based on the method described by world health organization who, 2005) with a slight modification. stock solutions (10,000 ppm) of each extract were prepared by dissolving 1000 mg of the extract in 100 ml of distilled water and 0.1% dimethyl sulfoxide (dmso). stock solutions were used to prepare working solutions with concentrations of 0.1, 1, 10, 100, and 1000 ppm. a total of 1 mg/l temephos (abateⓡ) was used as a positive control, while 0.01% dmso was used as a negative control. a total of 20 instar iii/iv larvae were transferred into 100 ml of each working and control solutions, then incubated for 24 hours. the number of dead larvae was counted after 24 hours of exposure. mortality rates were corrected using the abbott formula (abbott, 1925) and percentage mortality was calculated based on the average of three repetitions. phytochemical screening compounds identification of the extracts were carried out using gas chromatography coupled to mass spectrometry (gc-ms) at the forensic laboratory, bali regional police station. each component was identified by comparing between the mass spectrum and the retention time of the sample with a standard. data analysis the lc50 and lc90 values were calculated using probit analysis with statistical package for the social sciences (spss) software version 25. results and discussion larvicides are crucial in a. aegypti control program for killing mosquitoes’ larvae. however, long-term use of synthetic larvicides have serious negative impact on the human health, environment, as well as increase the risk of resistance in vector organism (pavela et al., 2019). in the contrary, some medicinal plants have larvicidal properties that have allowed them to be used as safer alternatives (al-rashidi et al., 2022; aljameeli, 2023). mangrove plants with their unique secondary metabolites are one of the most potential alternatives to synthetic larvicides. some studies have shown that mangrove plants such as rhizopora mucronata, avicennia marina, and excoecaria agallocha show the potential to be utilized as biolarvicides (ali et al., 2012; karthi et al., 2020; pradeepa et al., 2015). in this study, larvicidal activity of mangrove plant sonneratia alba was examined using three different solvents namely methanol, chloroform, and n-hexane. the larvicidal activity testing results with three repetitions showed that the methanol extract of s. alba showed the most prominent result compared to chloroform and n-hexane extracts with percentage of mortality of 69.33% at a concentration of 1000 ppm (table 1). this result indicated that different solvent polarity can affect the bioactivity of extracts from the same plant sample because of different active biochemicals extracted by each solvent. in addition, probit analysis results show that the lc50 of s. alba methanol extract is 1265 ppm. this result is lower than the positive control, which showed 100% mortality rate at only 1 ppm. a previous study conducted by karthi et al (2020) showed that acetone extract of r. mucronata leaves displayed an effective efficacy against a. aegypti larvae with lc50 of 0.11 ppm. a study by ali et al (2012) reported that ethanol-water mixture extract of a. marina demonstrates larvicidal activity against a. aegypti larvae with lc50 of 135.8 ppm. such large discrepancies that were obtained in this study compared to previous studies could possibly happened because the bioactive compounds in the methanol extract of s. alba present in low quantity. moreover, the larvicidal activity of plant extracts can varies greatly depends on the plant species, plant part used, age of plant part, solvent, and vector species (ghosh et al., 2012). to the best of our knowledge, this is the first study reported larvicide potential from s. alba root. previous study showed that methanol, chloroform, and n-hexane extracts of s. alba roots have no antibacterial activity against staphylococcus aureus, but n-hexane extract displayed activity against streptococcus mutans (wijaya & indraningrat, 2021). 502 biology, medicine, & natural product chemistry 12 (2), 2023: 499-505 table 1. larvicide activity screening of s. alba root extracts. solvents concentration (ppm) number of larvae (n) mortality average ± sd % mortality r1 r2 r3 methanol 0.1 25 0 0 0 0.00 0.00 1 25 0 0 0 0.00 0.00 10 25 0 0 0 0.00 0.00 100 25 4 1 5 3.33 ± 2.08 13.33 1000 25 18 15 19 17.33 ± 2.08 69.33 chloroform 0.1 25 0 0 0 0.00 0.00 1 25 0 0 0 0.00 0.00 10 25 0 0 0 0.00 0.00 100 25 0 0 0 0.00 0.00 1000 25 4 2 2 2.67 ± 1.15 10.67 n-hexane 0.1 25 0 0 0 0.00 0.00 1 25 0 0 0 0.00 0.00 10 25 0 0 0 0.00 0.00 100 25 0 1 0 0.33 ± 0.58 1.33 1000 25 2 5 3 3.33 ± 1.53 13.33 positive control 1 25 25 25 25 100 ± 0.00 100.00 negative control 25 0 0 0 0.00 0.00 note: r = repetition the results showed that there were five main compounds contained in the methanol extract of s. alba, namely methyl 2-hydroxy-eicosanoate (19.55%); 4h-1benzopyran-4-one, 3,5-dihydroxy-2-(4-hydroxy-3methoxyphenyl)-7-methoxy (16. 48%); 4-((1e)-3hydroxy-1-propenyl)-2-methoxyphenol (10.06%); benzamide, n-[4-(2-naphtyl)-2-thiazolyl](9.40%); and 2,3-dihydro-3,5-bis(3-methoxyphenyl)-1h-inden-1-one (6.52%) (table 2). meanwhile, the gc-ms analysis of chloroform extract showed the five dominant compounds were stigmasterol (57.91%); stigmasta-5,22-dien-3-ol, acetate, (3.beta.)(6.28%); 1h-indole, 5-methyl-2phenyl(4.66%); nonane, 2,2,4,4,6,8,8-heptamethyl (3.53%); and cyclic octaatomic sulfur (3.04%) (table 3). in addition, the five dominant compounds found in the nhexane extract were stigmasterol (32.61%); stigmast-4en-3-one (29.37%); campesterol (8.20%); 9,19cyclolanost-24-en-3-ol, (3.beta.)(5.70%); and 2,6,10dodecatrien-1-ol, 3,7,11-trimethyl-, acetate, (e,e) (4.80%) (table 4). table 2. gc-ms analysis results of methanol extract of s. alba. no compounds rt %area 1 1,2,3-benzenetriol 12.138 5.17 2 propanoic acid, 3-hydroxy13.519 2.00 3 3-hydroxy-4-methoxybenzoic acid 14.576 1.01 4 2,3,4,5-tetrafluoroanisole 14.678 1.62 5 4-((1e)-3-hydroxy-1-propenyl)-2-methoxyphenol 16.625 10.06 6 benzoic acid, 4-hydroxy-3,5-dimethoxy17.394 0.98 7 trans-sinapyl alcohol 19.203 2.92 8 (s,e)-4-hydroxy-3,5,5-trimethyl-4-(3-oxobut-1-en-1-yl) cyclohex-2-enone 24.200 1.79 9 orcinol, monoacetate 24.682 2.62 10 1,1’-biphenyl-3,4,4’-trimethoxy-6’-formylatau nitroscanate 24.740 3.43 11 1-octadecene 26.087 2.25 12 1-hexacosene 26.164 0.69 13 1-hexacosene 26.253 0.62 14 tricosane 26.425 1.87 15 9h-xanthen-9-one, 1-hydroxy-3,5,6-trimethoxy 26.581 1.50 16 1-hexacosene 27.087 0.90 17 methyl 2-hydroxy-eicosanoate 27.516 19.55 18 1-hexacosene 27.803 2.89 19 4h-1-benzopyran-4-one, 3,5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy 28.408 16.48 20 benzamide, n-[4-(2-naphtyl)-2-thiazolyl]28.458 9.40 21 2,3-dihydro-3,5-bis(3-methoxyphenyl)-1h-inden-1-one 28.793 6.52 22 1-hexacosene 29.043 5.73 note: rt = retention time wijaya et al. – phytochemicals and larvicidal activity of sonneratia alba 503 table 3. gc-ms analysis results of chloroform extract of s. alba. no compounds rt %area 1 fumaric acid, 2-dimethylaminoethyl hexadecyl ester 5.992 1.11 2 9,19-cyclolanost-6-ene-3,7-diol, diacetate 8.609 0.52 3 d-ribose, 2-deoxy-bis(thioheptyl)-dithioacetal 8.648 0.74 4 nonane, 2,2,4,4,6,8,8-heptamethyl11.400 3.53 5 4,25-secoobscurinervan-4-one-22-ethyl-15,16-dimethoxy-, (22.alpha.) 14.083 0.88 6 3(2h)-furanone, dihydro-2,2-dimethyl-5-phenyl16.797 0.80 7 octadecane 17.251 0.51 8 docosane 18.275 1.00 9 heptacosane 19.249 2.04 10 cyclic octaatomic sulfur 19.650 3.04 11 heptacosane 20.183 1.92 12 eicosane 21.073 0.89 13 17-pentatriacontene 21.923 0.73 14 17-pentatriacontene 22.754 0.46 15 octatriacontyl pentafluoropropionate 23.190 2.28 16 17-pentatriacontene 23.548 1.64 17 1h-indole, 5-methyl-2-phenyl23.938 4.04 18 1h-indole, 5-methyl-2-phenyl24.036 4.66 19 1-nonadecene 24.091 1.43 20 17-pentatriacontene 24.175 1.02 21 3,5,7-triazatricyclo[6.3.0.0(3,7)]undec-11-ene-4,6-dione, 2,2-diphenyl-5-methyl24.205 1.39 22 stigmasta-5,22-dien-3-ol, acetate, (3.beta.)29.026 6.28 23 stigmasterol 29.343 57.91 24 5-fluoro-3-trifluoromethylbenzoic acid, nonadecyl ester 29.652 1.17 note: rt = retention time table 4. gc-ms analysis results of n-hexane extract of s. alba. no compounds rt %area 1 2,4-di-tert-butylphenol 13.999 1.21 2 9,19-cyclolanost-24-en-3-ol, (3.beta.)23.611 5.70 3 2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-, acetate, (e,e)23.705 4.80 4 7,11-hexadecadienal 23.786 4.48 5 bis(2-ethylhexyl) phthalate 23.941 3.91 6 stigmast-4-en-3-one 25.483 29.37 7 21-hydroxyprogesterone, trifluoroacetate 25.602 2.36 8 17-pentatriacontene 27.827 1.21 9 campesterol 29.017 8.20 10 stigmasterol 29.342 32.61 11 stigmasterol 29.437 6.15 note: rt = retention time methyl 2-hydroxy-eicosanoate is a compound that majorly present in methanol extract of s. alba leaves. this compound is fatty acid methyl esters that previously found in oil palm roots, aerial parts of ipomoea eriocarpa, and terminalia bentzoe (alexander, dayou, abdullah, & chong, 2017; das & himaja, 2014; elrafie, mohammed, hamed, ibrahim, & abou zeid, 2016). however, its pharmacological activity data is still limited. other major compounds in methanol extract are still poorly studied as well. stigmasterol was abundant in both chloroform and nhexane extracts. stigmasterol is a phytosterol that belong to tetracyclic triterpenes class and is known to exhibit some pharmacological property such as anticancer, antiinflammation, antioxidant, antibacterial, antifungal, and antiparasitic (bakrim et al., 2022; morgan et al., 2021). in addition, a study conducted by gade et al (2017) indicates that acetylcholinesterase inhibitory activity of stigmasterol and 1-haxacosanol is accountable for larvicidal activity of chromolaena odorata (gade et al., 2017). however, percent mortality of chloroform and n-hexane extracts against a. aegypti larvae in this study are only 10.67% and 13.33% respectively at concentration of 1000 ppm. it could be that the amount of stigmasterol in both extracts was not sufficient to give strong larvicidal effect. 504 biology, medicine, & natural product chemistry 12 (2), 2023: 499-505 conclusions in conclusion, methanol extract of s. alba root displayed the highest larvicidal activity compared to chloroform and n-hexane extracts, with 69.33% mortality value at 1000 ppm and lc50 value of 1265 ppm. five main compounds found in the methanol extract were methyl 2hydroxy-eicosanoate; 4h-1-benzopyran-4-one, 3,5dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy; 4-((1e)-3-hydroxy-1-propenyl)-2-methoxyphenol; benzamide, n-[4-(2-naphtyl)-2-thiazolyl]-; and 2,3dihydro-3,5-bis(3-methoxyphenyl)-1h-inden-1-one. to the best of our knowledge, this is the first report on larvicidal activity from s. alba root extract. however, further studies are required to determine the exact larvicide compound(s) from s. alba root extract. acknowledgements: we would like to acknowledged financial support from research unit, faculty of medicines and health sciences, warmadewa university, under grant no. 259/unwar/fkik/unit-penelitian/pd13/ix/2022. authors’ contributions: made dharmesti wijaya & anak agung gede indraningrat designed the study. i gede yoga ayuning kirtanayasa carried out the 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(2021). antibacterial activity of mangrove root extracts from ngurah rai mangrove forest, denpasar-bali. 2021, 10(2), 5. doi:10.14421/biomedich.2021.102.117-121 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 443-448 | doi: 10.14421/biomedich.2024.132.443-448 issn 2540-9328 (online) phytochemical and toxicity analysis of sonneratia alba mangrove leaf extract using the brine shrimp lethality test (bslt) method ni kadek dewi asri tiara arta1, anak agung gede indraningrat2, made dharmesti wijaya3,* 1medical study program; 2department of microbiology and parasitology; 3department of pharmacology, faculty of medicine and health sciences, warmadewa university, jl. terompong no 24 denpasar 80235, tel. +62 361 240727, indonesia. corresponding author* dharmestiwijaya@warmadewa.ac.id manuscript received: 11 agustus, 2024. revision accepted: 08 october, 2024. published: 10 october, 2024. abstract mangroves are plants that contain various secondary metabolites with various potential pharmacological activities, one of which is cytotoxic activity. however, researchers have not extensively explored the evidence of secondary metabolite compounds and their toxicity effects in mangrove plants. this research aims to determine the content of secondary metabolite compounds and the toxicity of the leaf extract of the sonneratia alba mangrove plant in the ngurah rai grand forest park (tahura), bali. in this experimental research, compound extraction was carried out using a maceration technique for 2x24 hours using two types of solvents, namely methanol and nhexane. phytochemical tests were carried out using qualitative methods and gas chromatography-mass spectrometry (gc-ms), while the toxicity test used was the brine shrimp lethality test (bslt) method. potassium dichromate and sea water without extract were used as positive and negative controls, respectively. the lc50 value was calculated using probit analysis in microsoft excel. phytochemical test results showed that the methanol extract contains phenol, saponin, tannin, and steroid compounds. meanwhile, n-hexane extract only contains steroid compounds. gc-ms analysis showed that the compounds with the largest area percentages in the methanol and n-hexane extracts were 1,2,3-benzenetriol (3.94%) and n-hexadecenoic acid (2.41%), respectively. based on the lc50 value, the methanol extract of s. alba mangrove leaves was moderately toxic (178.17 ppm), while the n-hexane extract was weakly toxic (567.23 ppm). the results of this study provide information that the type of chemical solvent has a major influence on the level of toxicity of s. alba leaf extract, with polar methanol solvent providing the highest toxicity effect compared to nonpolar n-hexane solvent. keywords: bslt; mangrove; phytochemicals; sonneratia alba; toxicity. introduction cancer is a disease caused by the uncontrolled growth of abnormal cells in the body (suryaningrum, 2021). in 2020, the number of deaths recorded in the world due to cancer reached almost 10 million (pambudi, 2022). commonly used methods of treating cancer, such as surgery, radiotherapy, and chemotherapy, are considered to be less effective because they do not selectively destroy cancer cells and are also toxic to normal cells (latief et al., 2020). furthermore, chemotherapy drugs also cause mild to severe side effects. therefore, the discovery and development of safer and more selective anticancer compounds are still being conducted, especially in plants, considering that more than twothirds of anticancer drugs on the market currently come from plants (niksic et al., 2021). one of the natural resources that has the potential to be a new anticancer source is mangrove plants. several studies on the pharmacological activity of mangrove plants have shown that their compounds make them useful as traditional medicines (kadir et al., 2019; dotulong et al., 2020; mairing & ariantari, 2022). various secondary metabolite compounds are reported to be contained in mangrove plants, such as flavonoids, tannins, alkaloids, saponins, steroids, and triterpenoids (kadir et al., 2019). the content of these secondary metabolites makes mangrove plants have various pharmacological activities, namely antioxidants, anticancer, antibacterial, anti-inflammatory, antidiarrheal, antifungal, antinematode, antimalarial, and other pharmacological benefits (rahmania et al., 2018). among the mangrove plants, the sonneratia alba species shows significant potential for development as a new source of chemotherapy agents. leaves of the s. alba mangrove plant contain secondary metabolites that have toxic effects, namely tannins, which cause damage to biological structures at the organ, tissue, cell, and biomolecular levels (setiasih et al., 2016; mairing and ariantari, 2022). the toxic effect of a compound or extract can be determined from its lethal concentration 50 (lc50) value. the lc50 value indicates the ability of an extract to kill test larvae by 50% (fidyasari et al., 2020). an lc50 value of less than https://doi.org/10.14421/biomedich.2024.132.443-448 444 biology, medicine, & natural product chemistry 13 (2), 2024: 443-448 1000 ppm is considered toxic and has the potential to be developed as an anticancer drug (latief et al., 2020). the initial screening stage to map the potential toxicity before testing the anticancer activity of an extract is to carry out a toxicity test using the brine shrimp lethality test (bslt) method (latief et al., 2020). this study explored the potential of s. alba from ngurah rai mangrove forest, bali, indonesia, as a source of anticancer agents. two solvents, methanol, and n-hexane, were used to extract bioactive compounds from the leaves of s. alba, aiming to identify the most effective solvent for isolating compounds with cytotoxic properties. methanol, a polar solvent, is expected to extract a broader range of hydrophilic compounds, while n-hexane, a non-polar solvent, is likely to target lipophilic compounds. by comparing the phytochemicals, toxicity profiles, and lc50 values obtained from the bslt for both extracts, we aim to determine the most promising extract for further anticancer research. this investigation represents a significant step towards harnessing the therapeutic potential of s. alba, particularly in the development of novel, plant-derived anticancer agents. materials and methods sample preparation a total of two kilograms of s. alba leaves were collected from five different plants at the ngurah rai mangrove forests. taxonomy was identified at the plant conservation research center, botanical gardens and forestry, bedugul, bali. leave samples were cleaned using running water, cut into smaller sizes, and dried in an oven at 40°c. after drying, the sample was grounded using a blender until it became powder and then sieved (akasia et al., 2021). to make extracts from two different solvents, a minimum of 200 grams of sample powder was prepared. extraction the extraction stage was carried out using a maceration technique using two types of solvents with different polarities, namely methanol and n-hexane. 100 grams of simplicia were soaked in 500 ml of each solvent for 24 hours (v/v, 1:5) (wijaya & indraningrat, 2021). the filtrate from the first maceration was filtered using whatmann no.1 filter paper and the resulting residue was macerated again using 500 ml of new solvent for 24 hours. the first and second maceration results are combined and then stored in the refrigerator until the next process. the extract evaporation process was carried out using a vacuum rotary evaporator at a temperature of 50°c, then the thick extract resulting from the evaporation process was weighed. the resulting extract was put into a tightly closed vial and stored at 4°c until the next research stage (wijaya & indraningrat, 2021). phytochemical testing testing for the content of phytochemical compounds in extracts was carried out using qualitative methods and gas chromatography-mass spectrometry (gc-ms) to identify the specific compounds in the extracts. the gcms method compares the mass spectrum and retention time of the sample with a standard standard (suhaili et al., 2020). briefly, 0.1 grams of the crude extract was prepared and sent to the forensic laboratory polda bali for further analysis. the extract was injected to the gc/ms instrument (agilent technologies 7890b/agilent technologies 5977b) based on the following setting: hp-5ms ultra inert column 30 m x250 µm x 0.25 µm, oven temperature (-60oc to 325oc), mode (splitless), pressure (25.523 psi), total flow (20.9 ml/min), average velocity (62.662 cm/sec), purge flow to split vent (15 ml/min at 0.75 min), and gas (he). the chromatogram results were analyzed by matching the compound fragments from each chromatogram peak with literature to determine the type of content and function of the detected bioactive compounds. toxicity effect testing using the bslt method tests were conducted using 10 newly hatched artemia salina nauplii larvae (fidyasari et al., 2020). the extract consisted of 7 concentrations, namely 2000; 1000; 500; 250; 100; 50; and 25 ppm. at each extract concentration, 10 larvae were placed in the extract and left for 24 hours. after 24 hours, surviving a. salina was counted and the lethal concentration was analyzed. the test was carried out with 3 repetitions and used potassium dichromate as a positive control and sea water without extract as a negative control. the toxicity effect was determined by calculating the percent death of a. salina in each vial after 24 hours. the calculation is done by counting the number of dead a. salina divided by the initial number and then multiplying by 100%. the lc50 value was calculated using probit analysis with microsoft excel software. an extract is declared to have a toxicity effect if it has an lc50 of less than 1000 ppm (kurniawan & ropiqa, 2021). the toxicity category of an extract is based on the lc50 value, namely the very toxic category with an lc50 of less than 30 ppm, the strongly toxic category with an lc50 of 30-100 ppm, the moderate toxic category with an lc50 range of 100-250 ppm, the weakly toxic category of 250-1000 ppm, and the non-toxic with lc50 more than 1000 ppm (zulfiah et al., 2020). ethical clearance the research has been approved by ethical committees of the faculty of medicine and health sciences, warmadewa university, under ethical eligibility number: 338/unwar/fkik/ec-kepk/vii/2023. arta et al. – phytochemical and toxicity of sonneratia alba 445 results and discussion phytochemical screening the phytochemical test showed that the methanol extract contained four compounds, namely tannin, phenol, steroid, and saponin. meanwhile, only steroid was found in the n-hexane extract (table 1). these results showed that steroid compounds were found in both s. alba mangrove leaf extracts. table 1. phytochemical test results of methanol and n-hexane extracts. compounds reagent sonneratia alba leaf extracts methanol n-hexane tannins fecl3 1% + flavonoids mg+hcl alkaloids dragendorff phenolics fecl3 1% + saponins distilled water + steroids liebermann burchard + + triterpenoids liebermann burchard the gc-ms analysis showed that 251 compounds were found in the methanol extract. the five dominant compounds contained in the methanol extract can be seen in table 2, with 1,2,3-benzenetriol being the compound with the largest area (3.94%). on the other hand, the nhexane extract consisted of 338 compounds. the five dominant compounds in the n-hexane extract can be observed in table 2, with n-hexadecanoic acid being the compound with the largest area (2.41%). table 2. phytochemical analysis of s. alba mangrove leaf extracts using gc-ms. peak sonneratia alba leaf extracts retention time compounds %area 1 methanol 16.291 1,2,3-benzenetriol 3.94 2 27.832 linoelaidic acid 3.28 3 25.471 n-hexadecanoic acid 2.74 4 19.879 2,4-hexadiene,3,4-dimethyl-,(e,z)1.92 5 9.089 benzyl alcohol 1.56 1 n-hexane 25.499 n-hexadecanoic acid 2.41 2 19.511 dodecanoic acid 1.97 3 39.702 oxirane, hexadecyl1.45 4 38.685 dl-.alpha.-tocopherol 1.28 5 28.258 9,12,15-octadecatrienoic acid, (zzz)1.21 toxicity analysis the toxicity screening of methanol extract showed that the highest toxicity was obtained at a concentration of 2000 ppm. from the linear regression formula, an lc50 value of 178.17 ppm was obtained, which is included in the moderate toxic category. meanwhile, the toxicity test of n-hexane extract showed that the greatest toxicity effect with a death percentage of 70% was at a concentration of 2000 ppm. the probit analysis showed that the lc50 of n-hexane extract was 567.23 ppm, which is included in the weak toxic category. table 3. cytotoxic activities of s. alba mangrove leaf extracts with potassium dichromate as positive control sonneratia alba leaf extracts concentration (ppm) lc50 (ppm) toxicity category methanol (25-2000) 178.17 moderate n-hexane (25-2000) 567.23 weak potassium dichromate (25-2000) 58.59 strong discussion based on the research results, it is known that methanol extract has a moderate toxic effect with an lc50 of 178.17 ppm. this result differs from a study by eriani & usman (2017) that reported that methanol extract of s. alba leaves from the east kalimantan area yielded weak toxicity of 441.67 ppm (eriani and usman, 2017). differences in toxicity effects can be influenced by 446 biology, medicine, & natural product chemistry 13 (2), 2024: 443-448 variations in sample ages, as well as differences in the living environment and habitat of the s. alba mangrove plant such as nutrient and climate differences (supriatna et al., 2019). qualitative phytochemical test results show that the methanol extract contains four phytochemical compounds, namely tannins, phenolics, saponins, and steroids. the research results differ from those of muhaimin (2019), who found phytochemical content, namely alkaloids, flavonoids, terpenoids, phenolics, tannins, saponins, quinones, and glycosides. differences in phytochemical content are caused by the age of the test samples, differences in habitat, and environmental conditions of mangrove plants, such as light intensity, season, and water temperature.(supriatna et al., 2019). the high tannin content in s. alba leaves can be correlated with observed toxicity in plant extracts due to the nature of tannins themselves. tannins are a type of polyphenolic compound that can have astringent properties and can bind to proteins (alfarabi, 2018). phenolic is the largest class of natural compounds, including gallic acid, p-coumaric acid, ferulic acid, vanillic acid, and flavonoids, which have anticancer potential (fatimah et al., 2022). phenolic compounds are a diverse group of plant secondary metabolites that play essential roles in plant growth, development, reproduction, and defense against environmental stresses like herbivores, pathogens, and uv radiation (lin et al., 2016). while phenolic compounds generally benefit plants, they can also have toxic or inhibitory effects on other organisms. steroid compounds cause death in larvae because they work as antifeedant (to'bungan et al., 2021). this antifeedants effect leads to feeding disorders in larvae by triggering toxic effects on digestion and starvation (kurniawan & ropiqa, 2021). steroids also have anticancer and anti-inflammatory potential (to'bungan et al., 2021). steroid compounds can also inhibit tumor cells in the human cervix and lungs (putram et al., 2017). qualitative test results show that the methanol extract contains more phytochemical compounds than n-hexane extracts. this is in line with research by gazali (2020) showing that the methanol extract of s. alba contains more phytochemical compounds compared to ethyl acetate and n-hexane extracts (gazali et al., 2020). this can be caused by the difference in polarity between the three solvents. based on the principle of like dissolves like, polar compounds can be extracted perfectly in polar compounds and nonpolar compounds can be perfectly extracted in zero-polar compounds, therefore compounds with different polarities cannot be extracted properly (leksono et al., 2018). the methanol solvent is polar, while n-hexane is non-polar (leksono et al., 2018). consequently, methanol was able to extract the most active compounds, possibly because most of the compounds contained in s. alba leaves are polar compounds. the results of quantitative phytochemical tests using the gc-ms method show five compounds with the largest percentages area in the methanol extract of s. alba including1,2,3-benzenetriol, linoelaidic acid, nhexadecanoic acid, 2,4-hexadiene,3,4-dimethyl-,(e,z)-, and benzyl alcohol. compound1,2,3-benzenetriol is known to have anti-inflammatory, antioxidant, phytosterol, antifungal effects. however, there has been no research discussing the toxicity effects of this compound (cahyani et al., 2021). the compound with the second largest percentage area is linoelaidic acid, which is an unsaturated fatty acid and has anticancer activity by triggering the death of cancer cells and can increase necrosis in tumors (dutta et al., 2023). the nhexadecanoic acid compound has been associated with cytotoxic effects on human colorectal carcinoma cells and has potential as an anticancer drug (ravi & krishnan, 2016). the benzyl alcohol compound has been reported for antifungal and antibacterial activities, but there is no literature that mentions the toxicity activity of this compound (sulaiman et al., 2020). on the other hand, the pharmacological effects of the compound 2,4hexadiene,3,4-dimethyl-,(e,z) remain unknown. the n-hexane extract showed lower toxicity effects against brine shrimp larvae than the methanol extract. this may be because the n-hexane extract only contains steroids, compound known to have anti-inflammatory and anti-cancer activities (to’bungan et al., 2021). the gc-ms test results showed that the five compounds with the largest % area are n-hexadecanoic acid, dodecanoic acid, oxirane, hexadecyl-, dl-alpha.-tocopherol, and 9,12,15-octadecatrienoic acid, (zzz)-. of the five compounds, three compounds, namely n-hexadecanoic acid, 9,12,15-octadecatrienoic acid, (zzz)-, and dodecanoic acid, are known to have cytotoxic and anticancer effects (ravi & krishnan, 2016). apart from that, dodecanoic acid or also called lauric acid is a medium chain saturated fatty acid compound which is known to have antibacterial activity (renugadevi et al., 2021). another compound, namely dl-alpha-tocopherol or vitamin e, is a compound known to have antioxidant activity (mubarak et al., 2017), but its toxicity effects are not yet known. meanwhile, there has been no research regarding the pharmacological activity of the oxirane hexadecyl compound. conclusions in conclusion, the toxicity of methanol and n-hexane extracts of s. alba mangrove leaves obtained in the tahura ngurah rai bali area was moderately toxic (178.17 ppm) and weakly toxic (567 ppm), respectively. the secondary metabolites contained in the methanol extract of the leaves of the s. alba mangrove plant are tannins, phenolics, saponins, and steroids, with 1,2,3benzenetriol as the compound with the largest % area in arta et al. – phytochemical and toxicity of sonneratia alba 447 the results of the gc-ms test analysis. the secondary metabolite in the n-hexane extract is steroids, with nhexadecanoic acid being the compound with the largest % area in the gc-ms test analysis results. acknowledgements: the authors would like to acknowledged financial support from up2m of faculty of medicines and health sciences under grant no. 382/unwar/fkik/pd-13/ iv/2021. authors’ contributions: ni kadek dewi asri tiara arta & made dharmesti wijaya designed the study. ni kadek dewi asri tiara arta carried out the laboratory 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2021. antibacterial activity of mangrove root extracts from ngurah rai mangrove forest, denpasar-bali. biol. med. nat. prod. chem. 10, 117–121. https://doi.org/10.14421/biomedich.2021.102.117-121 zulfiah, z., megawati, m., herman, h., h. ambo lau, s., hasyim, m.f., murniati, m., roosevelt, a., kadang, y.k., ar, n.i., patandung, g., 2020. uji toksisitas ekstrak rimpang temu hitam (curcuma aeruginosa roxb.) terhadap larva udang (artemia salina leach) dengan metode brine shrimp lethality test (bslt). j. farm. sandi karsa 6, 44–49. https://doi.org/10.36060/jfs.v6i1.67 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1379-1384 | doi: 10.14421/biomedich.2025.142.1379-1384 issn 2540-9328 (online) formulation of an anti-acne cream containing ethanolic extract of purslane (portulaca oleracea) isna lailatusholihah*, dina alva prastiwi, afiatun ni’mah, yudhistira department of chemistry, sekolah tinggi analis kimia cilegon jalan lingkar selatan km 1.7, cilegon 42411, indonesia. corresponding author* isnalailatusholihah@gmail.com manuscript received: 18 september, 2025. revision accepted: 21 november, 2025. published: 15 december, 2025. abstract portulaca oleracea (purslane) is a medicinal plant known for its antibacterial potential due to the presence of bioactive compounds such as saponins, tannins, flavonoids, and alkaloids. this study aimed to formulate an anti-acne cream containing the ethanol extract of p. oleracea and to evaluate its antibacterial activity against staphylococcus aureus. the extract was obtained through maceration using 96% ethanol, and antibacterial testing was performed using the well diffusion method. the results showed that the ethanol extract demonstrated antibacterial activity, with the highest effect observed in formulation f3, which produced an inhibition zone of 13 mm (strong category). f3 also exhibited desirable physicochemical characteristics, including a spreading diameter of 6.5 cm, viscosity of 14,340 cp, and ph of 5, all of which complied with standard requirements. these findings indicate that p. oleracea ethanol extract has potential as an active ingredient in topical anti-acne formulations. keywords: cream anti-acne; portulaca oleracea; antibacterial activity; ethanolic extract. introduction the prevalence of acne in developing countries ranges from 40% to 80%, with 80–85% of adolescents affected and cases continuing to rise annually. a 2019 study involving 66 acne vulgaris patients at abdul moeloek hospital reported that 69.7% were female and 30.3% were male (sibero et al., 2020). acne has been shown to negatively affect quality of life, with 30–50% of patients experiencing psychological distress and reduced selfesteem due to its impact on appearance (pariury et al., 2021). acne vulgaris is a chronic inflammatory disorder of the pilosebaceous unit, associated with hyperkeratinization, excess sebum production, and microbial colonization, particularly by cutibacterium acnes (formerly propionibacterium acnes), staphylococcus epidermidis, and staphylococcus aureus (pariury et al., 2021). s. aureus is a gram-positive pathogenic bacterium commonly colonizing the respiratory tract, oral cavity, urinary tract, nasal mucosa, and skin, and is implicated in infections such as meningitis, abscesses, and acne (damayanti et al., 2022). while multiple endogenous and exogenous factors— including androgenic hormones, psychological stress, genetic predisposition, dietary habits, and climate— contribute to acne pathogenesis, colonization by s. aureus is recognized as an aggravating factor in the inflammatory process (sifatullah & zulkarnain, 2021). conventional acne management frequently involves the administration of antibiotics with anti-inflammatory and antibacterial activity. however, indiscriminate use of antibiotics has led to the emergence of antimicrobial resistance, highlighting the importance of exploring phytopharmaceutical alternatives with fewer adverse effects (wardania et al., 2020). purslane (portulaca oleracea) is a medicinal plant with reported antibacterial activity, attributed to its secondary metabolites such as saponins, tannins, flavonoids, alkaloids, and glycosides (purwanto et al., 2021; nabilah et al., 2023). despite its bioactive profile, purslane is often regarded as a weed, making it readily available and sustainable as a raw material for potential anti-acne formulations. previous investigations on ethanol extracts of medicinal plants, such as sungkai leaves, demonstrated concentration-dependent antibacterial activity against c. acnes and s. aureus, with higher extract concentrations producing larger inhibition zones. furthermore, maidawati (2022) confirmed the presence of phytochemical classes including phenolics, flavonoids, coumarins, alkaloids, saponins, and terpenoids in sicerek extracts using fourier transform infrared (ftir) spectroscopy. ftir spectroscopy provides a characteristic infrared absorption spectrum of the https://doi.org/10.14421/biomedich.2025.142.1379-1384 mailto:isnalailatusholihah@gmail.com 1380 biology, medicine, & natural product chemistry 14 (2), 2025: 1379-1384 purslane extract, enabling identification of chemical bonds and functional groups. these spectral fingerprints serve as a qualitative chemical profile, supporting the validation of phytoconstituents prior to formulation studies. therefore, this research investigates the formulation of an anti-acne cream containing ethanol extract of purslane (p. oleracea) against s. aureus, aiming to elucidate its antibacterial potential, provide supporting chemical evidence, and assess its prospective value as a phytopharmaceutical candidate. materials and methods study area this research was conducted at the laboratory of sekolah tinggi analis kimia cilegon. the materials used were purslane (portulaca oleracea), distilled water, benzoic acid, stearic acid, staphylococcus aureus culture, 96% ethanol, glycerin, tetracycline, nutrient agar, nutrient broth, propylene glycol, cetyl alcohol, triethanolamine (tea), 2n hcl, mgso₄, kbr, 1% h₂so₄, 1% bacl₂, and 0.9% nacl. the instruments used in this study included a rotary evaporator, fourier transform infrared (ftir) spectrophotometer, brookfield viscometer, incubator, analytical balance, hot plate, ph meter, micropipette, spatula, pipette, test tubes, watch glass, sterile absorbent cotton, petri dishes, vernier caliper, inoculating loop, graduated cylinder, beakers, and erlenmeyer flasks. procedures sample preparation of purslane (portulaca oleracea) a total of 5 kg of purslane was washed under running water to remove impurities. the whole plant was cut into small pieces and air-dried at room temperature until completely dry. the dried simplicia was then separated, pulverised into powder, and stored in a container protected from direct sunlight to prevent degradation of active compounds (sari et al., 2021). preparation of purslane ethanolic extract extraction was carried out using the maceration method. a total of 600 g of dried purslane powder was macerated with 3600 ml of 96% ethanol for five days, with stirring once daily. the extract was filtered, and the filtrate was concentrated using a rotary evaporator at 40 °c to obtain a viscous ethanolic extract (sari et al., 2021). flavonoid identification two millilitres of purslane extract were added into a test tube, followed by 0.5 g of mgso₄ powder and 2 ml of 2n hcl. the formation of an orange to red colour indicated the presence of flavonoids (johannes et al., 2023). fourier transform infrared (ftir) analysis functional group analysis of purslane extract was performed using ftir spectroscopy (shimadzu 8400s). the solvent was removed by freeze-drying prior to analysis. the process involved freezing the extract at –25 °c, followed by drying at –50 °c under 5 mpa vacuum. potassium bromide (kbr) was added before ftir measurement to obtain spectra representing the characteristic functional groups of the extract. preparation of purslane extract cream the cream was formulated using the fusion method. the oil phase was prepared by heating cetyl alcohol and stearic acid in a water bath, while the aqueous phase was prepared separately by dissolving benzoic acid in distilled water, followed by the addition of glycerin, propylene glycol, and triethanolamine (tea). both phases were heated to 70 °c to ensure complete melting and then combined gradually in a mortar with continuous trituration until a homogenous cream was obtained. purslane extract was incorporated into the base at different concentrations (0, 8, 12, and 16% v/v). the formulations are shown in table 1 (novianti & wirnawati, 2024). table 1. formulation of purslane extract cream. ingredients function formulation (ml) f0 f1 f2 f3 purslane extract active agent 0 8 12 16 stearic acid emulsifier 12 12 12 12 cetyl alcohol thickening agent 0.5 0.5 0.5 0.5 propylene glycol co-emulsifier 3 3 3 3 triethanolamine emulsifier 1.5 1.5 1.5 1.5 glycerin humectant 5 5 5 5 benzoic acid preservative 0.2 0.2 0.2 0.2 distilled water solvent 77.8 69.8 65.8 61.8 evaluation of purslane extract cream ph determination the ph of the cream was measured using a universal ph meter. according to sni 16-3499-1996, the acceptable ph range for topical preparations is 4.5–8 (chandra et al., 2022). lailatusholihah et al. – purslane extract anti-acne cream 1381 homogeneity test a small amount of cream was applied onto a glass slide. homogeneous cream was indicated by the absence of visible particles or bubbles (novianti & wirnawati, 2024). spreadability test about 0.5 g of cream was placed on graph paper covered with a glass plate. another glass plate was placed on top for 1 minute, followed by a 150 g weight. the spread diameter was measured, with the ideal spreadability for cream being 5–7 cm (isrul et al., 2023). viscosity measurement viscosity was determined using a brookfield viscometer. the spindle was immersed up to the marked level, and measurements were taken after ~10 seconds. according to sni 16-4399-1996, acceptable viscosity for cream formulations ranges from 2,000–50,000 cp (chandra et al., 2022). antibacterial activity assay against staphylococcus aureus sterilisation culture media were sterilised using an autoclave at 121 °c for 15 minutes. glassware was sterilised in a hot air oven at 160–170 °c for 1–2 hours, while forceps and inoculating loops were sterilised by flaming (wangloan et al., 2025). preparation of nutrient agar (na) medium a total of 28 g na was dissolved in 1 l distilled water, heated until fully dissolved, then distributed into test tubes (±7 ml) and petri dishes (±30 ml). the tubes were covered with cotton and aluminium foil, while the petri dishes were sealed with brown paper. media were sterilised in an autoclave at 121 °c and 1 atm for 15 minutes. test tubes were solidified in a slanted position (susanti et al., 2024). subculturing of staphylococcus aureus staphylococcus aureus was streaked on slanted na medium under aseptic conditions and incubated anaerobically at 35 °c for ~24 h (susanti et al., 2024). preparation of nutrient broth (nb) medium a total of 1.3 g nb was dissolved in 100 ml distilled water, heated until dissolved, and sterilised by autoclaving at 121 °c and 1 atm for 15 minutes (susanti et al., 2024). preparation of mcfarland standard solution a 1% h₂so₄ solution (9.5 ml) was mixed with 0.5 ml of 1% bacl₂ solution to obtain a turbid suspension equivalent to the mcfarland standard (setiawan et al., 2024). preparation of bacterial suspension colonies of s. aureus were collected using a sterile inoculating loop and suspended in 2 ml of 0.9% nacl solution. the turbidity was adjusted to match the mcfarland standard (setiawan et al., 2024). positive and negative controls aquadest was used as the negative control, while tetracycline served as the positive control. the cream formulations were tested at concentrations of 0, 8, 12, and 16% v/v. antibacterial assay procedure the antibacterial activity of the formulations was evaluated using the well diffusion method (6 mm diameter wells). each well was loaded with 20 µl of cream samples, along with positive and negative controls. plates were incubated at 37 °c for 18–24 h. the inhibition zones were measured as clear areas surrounding the wells, indicating antibacterial activity. results and discussion extraction of purslane (portulaca oleracea) the extraction process was carried out using maceration with 96% ethanol as the solvent. after maceration, a dark green extract was obtained (figure 4). the filtrate was concentrated using a rotary evaporator at 40 °c and 50 rpm, yielding an extract recovery of 9.04% from the initial sample weight. this yield was higher than that reported by sari et al. (2024), who obtained 7.04% using a similar extraction method. the variation in yield may be attributed to differences in evaporation temperature, as higher temperatures can increase the loss of volatile compounds. technical differences in the maceration procedure, such as multi-stage processing or variations in extraction time, may also contribute to discrepancies in yield. overall, the 9.04% recovery obtained in this study indicates that the extraction process was relatively efficient and suggests the potential presence of a higher concentration of active compounds compared to previous reports. figure 1. a dark green extract. 1382 biology, medicine, & natural product chemistry 14 (2), 2025: 1379-1384 phytochemical screening and ftir analysis of purslane ethanol extract the phytochemical test of the ethanol extract of purslane showed a colour change from orange to brick-red, indicating a positive result for flavonoids. this finding is consistent with the study of johannes et al. (2023), which also reported the presence of flavonoids and other secondary metabolites such as alkaloids, saponins, and tannins in purslane stem and leaf extracts. both studies highlight flavonoids as one of the dominant secondary metabolites in purslane. flavonoids are known to exhibit diverse biological activities, including antimicrobial effects through inhibition of nucleic acid synthesis in bacteria, as well as antioxidant, anti-inflammatory, and cytotoxic properties. these bioactivities support their potential application as active constituents in herbal-based therapeutic formulations. ftir spectral analysis of purslane ethanol extract the ftir spectrum of the ethanol extract of purslane, as presented in figure 6, confirmed the presence of flavonoid compounds, as indicated by several characteristic absorption bands. a broad and intense band observed at 3398.57 cm⁻¹ corresponds to hydroxyl (–oh) stretching vibrations. aromatic ring structures were further confirmed by absorption peaks at 1647.21 cm⁻¹ and 1516.05 cm⁻¹, attributed to c=c stretching in aromatic systems, along with c–h out-of-plane bending vibrations detected at 865.33 cm⁻¹ and 823.03 cm⁻¹. additionally, the peak at 1718.58 cm⁻¹ suggests the presence of a carbonyl (c=o) functional group, while an absorption band at 1246.02 cm⁻¹ is assigned to c–o stretching vibrations. these spectral features are consistent with the functional groups typically associated with flavonoids, supporting the results obtained from the phytochemical screening. figure 2. the ftir spectrum of the ethanol extract of purslane. this finding is consistent with the study of saxena et al. (2021) on portulaca oleracea, which reported similar absorption bands, namely at 3362.20 cm⁻¹ for the hydroxyl (–oh) group, 1640 cm⁻¹ for the carbonyl (c=o) group, 1300–1000 cm⁻¹ for the c–o group, 848.35 cm⁻¹ for aromatic c–h, and 1600 cm⁻¹ for aromatic c=c, which are characteristic of flavonoid compounds. these similarities support the validity of the present data and strengthen the assumption that flavonoids play a crucial role in the potential biological activities of purslane. cream preparation and characterization the purslane extract cream was formulated using the two-phase fusion method with a total volume of 100 ml for each preparation. the fusion method is a technique in which all or selected components are melted and then cooled while being continuously stirred until a homogeneous preparation is formed (nurrahman et al., 2022). in this formulation, the oil phase consisted of stearic acid and cetyl alcohol. stearic acid acted as a structure-forming agent and emulsifier, whereas cetyl alcohol served to increase viscosity and provide a smoother texture to the cream. the aqueous phase consisted of distilled water as the solvent, benzoic acid as a preservative to prevent microbial growth, and glycerin and propylene glycol as humectants to maintain skin moisture. in addition, triethanolamine (tea) was used to form and stabilise the oil-in-water emulsion system. ethanol extract of purslane was incorporated as the active ingredient into the cream base in three different volume-to-volume (v/v) concentrations: f1 (8 ml), f2 (12 ml), and f3 (16 ml) within a total 100 ml formulation. the final preparations produced homogeneous and stable creams that did not separate, with a smooth texture that spread easily when applied to the skin. the results of the cream preparation are shown in figure 3. figure 3. purslane (portulaca oleracea) ethanolic extract cream. ph evaluation the ph test was conducted to assess the compatibility of the formulation with the physiological ph of the skin. the results showed that f0 (cream base) had a ph of 6, whereas f1, f2, and f3 exhibited an identical ph of 5. these values fall within the range specified by the indonesian national standard (sni 16-4399-1996), namely 4.5–8.0, and are also consistent with the normal skin ph range of 4.5–7.5 (bsn, 1996). thus, the measured ph values (ph 5) indicate that all three cream formulations complied with the required acidity criteria for topical preparations. lailatusholihah et al. – purslane extract anti-acne cream 1383 homogeneity evaluation the homogeneity test was performed visually, and the results (figure 9) showed that f1, f2, and f3 exhibited a uniform physical appearance without coarse particles, clumps, or phase separation. in addition, no entrapped air bubbles were observed in the system. these findings indicate that all formulations possessed good homogeneity, in accordance with the criteria for topical preparations as described by novianti & wirnawati (2024). spreadability evaluation the spreadability test was conducted to assess the ability of the cream to spread upon application to the skin. the results presented in table 3 indicate that f0 exhibited a spread diameter of 3.0 cm, while f1, f2, and f3 showed spread diameters of 5.2 cm, 6.2 cm, and 6.5 cm, respectively. based on these results, formulations f1, f2, and f3 fulfilled the spreadability criteria for topical preparations, which range from 5–7 cm (isrul et al., 2023; setiawan et al., 2024). in contrast, f0 (the base cream without extract) did not meet the requirement due to its excessively high viscosity, which resulted in limited spreadability. this finding confirms the inverse correlation between viscosity and spreadability, as previously described by chandra et al. (2022). viscosity evaluation the viscosity test revealed a decreasing trend in viscosity with increasing concentrations of purslane extract. the average values from duplicate measurements are presented in table 2, showing that formulations f1–f3 were within the ideal viscosity range for topical preparations as specified by sni 16-4399-1996 (2,000– 50,000 cp). in contrast, f0 exceeded the upper limit due to the absence of extract, resulting in an overly dense cream base. this finding is consistent with the spreadability test results, which demonstrated the inverse relationship between viscosity and spreadability. higher viscosity reduces the ability of the cream to spread, requiring greater pressure to achieve adequate distribution on the skin (chandra et al., 2022). table 2. viscosity of purslane ethanolic extract creams. formulation viscosity (cp) f0 418.800 f1 29.220 f2 16.620 f3 14.340 antibacterial activity test of cream formulations the antibacterial activity of purslane ethanolic extract cream formulations was evaluated against staphylococcus aureus using the well diffusion method. antibacterial activity was determined by measuring the inhibition zone diameter (mm) formed around the wells (table 4). table 4. inhibition zone diameter of purslane ethanolic extract creams against staphylococcus aureus. no. sample inhibition zone (mm) category 1. f1 8,5 moderate 2. f2 12 strong 3. f3 13 strong 4. tertrasiklin (+) 29 very strong 5. akuades (-) 0 none according to yusrina et al. (2022), antibacterial activity is classified based on inhibition zone diameters as shown in table 5. table 5. antibacterial activity classification based on inhibition zone diameter. diameter (mm) activity strength ≤ 5 mm weak 6-10 mm moderate >11-20 mm strong >20-30 mm very strong based on table 4, formulation f1 demonstrated moderate antibacterial activity (8.5 mm), while f2 and f3 exhibited strong activity with inhibition zones of 12 mm and 13 mm, respectively. the positive control (tetracycline) produced a very strong inhibition zone of 29 mm, whereas the negative control (aquadest) showed no inhibitory effect (figure 4). figure 4. antibacterial activity of anti-acne cream formulations with ethanolic extract of portulaca oleracea. the positive control served as a reference standard to validate the assay system and confirm test accuracy, while the negative control verified that the solvent itself had no antibacterial effect. overall, the results indicate that purslane ethanolic extract cream possesses antibacterial activity against s. aureus, with activity increasing in line with extract concentration. these findings are consistent with yusrina et al. (2022), who reported that higher extract concentrations correlate with larger inhibition zones due to the presence of greater amounts of bioactive compounds. moreover, rasidah et al. (2019) noted that flavonoids exhibit antibacterial activity against s. aureus, supporting the role of 1384 biology, medicine, & natural product chemistry 14 (2), 2025: 1379-1384 flavonoids as active constituents in purslane extract creams. conclusions this study demonstrated that purslane ethanolic extract (portulaca oleracea) can be formulated into an anti-acne cream with effective antibacterial activity against staphylococcus aureus. the highest activity was observed in formulation f3, which showed a strong inhibition zone (13 mm), a spreadability value of 6.5 cm, a viscosity of 14,340 cp, and a ph of 5, all of which complied with topical formulation standards. acknowledgment: the authors would like to acknowledge kemenristekdikti for providing research funding. appreciation is also extended to the sekolah tinggi analis kimia cilegon for supporting this study with laboratory facilities, as well as assistance in data collection and processing. authors’ contributions: isna lailatusholihah designed the study. isna lailatusholihah, dina alva prastiwi, afiatun ni'mah and yudhistira carried out the laboratory work. dina alva prastiwi analyzed the data. isna lailatusholihah wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors would like to acknowledge kemenristekdikti for providing research funding. references chandra d, & rahmah r. 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(2022). uji efektivitas antibakteri ekstrak metanol daun ramania (bouea macrophylla griffith) terhadap bakteri staphylococcus aureus. pharmacoscript 5(2): 212–224. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 211-218 | doi: 10.14421/biomedich.2025.141.211-218 issn 2540-9328 (online) comparative assessment of nutritional, antioxidant and phytochemical properties of wild yams (dioscorea spp.) accessions in peninsular malaysia mohd zulkhairi azid1,*, mohd norfaizal ghazalli1, abdul muhaimin abdul kadir1, siti aisyah mohd noor1, saidatul aqilah mohamad yusof1, nur daliana yusof1, mohammad raiehan prayoga2, salmaniza salleh1 1agrobiodiversity and environment research centre, malaysian agricultural research and development institute (mardi), mardi headquarters, persiaran mardi-upm, 43400 serdang, selangor, tel. +603-8953 6325, fax. +603-8953 6330, malaysia. 2agrotechnology study program, faculty of agriculture, universitas muhammadiyah sumatera utara, north sumatra, jl. kap. mukhtar basri no. 3 medan, 20238, north sumatra, tel. +61-6619056, fax. +61-6625474, indonesia. corresponding author* zulkhairi@mardi.gov.my abstract yams (dioscorea spp.) are one of the important tuber crops for sustainability and food security in the world. however, dioscorea spp. is considered an underutilized crop and is not heavily consumed, especially by local people in malaysia, due to its limitations in its nutritional composition and beneficial studies. this study was carried out to evaluate various accessions of wild yams in peninsular malaysia. eight different accessions of yams (d. alata (da1-da5), d. alata var. purpurea (dvp), d. esculenta (de) and d. piscatorum (dp) were collected from various localities in farmer's farms in peninsular malaysia. all the samples were investigated for their proximate composition, polyphenols and flavonoid content and in vitro antioxidant activity. d. piscatorum (dp) collected in kuala koh, kelantan, showed the highest amount of carbohydrate content and energy compared to all accessions (p<0.05) with 90.8 g/100g and 374.33 kcal/100g, respectively. meanwhile, a variety of d. alata (da2) collected in machang, kelantan, showed a high amount of protein content (10.57 g/100g) (p<0.05). the flavonoid/phenolic ratio was highest in the methanolic extract of dvp (0.72), followed by de (0.64) and dp (0.61), respectively. however, the methanolic extract of dp showed the lowest inhibition concentration value (ic50 = 4.73 mg/ml), indicating its stronger scavenging activity towards dpph free radicals. the comparison and assessment of different accessions of dioscorea spp. on their nutritional composition and phytochemicals highlights the suitability of each sample to accommodate specific dietary or health-related applications. keywords: antioxidant; dioscoreaceae; polyphenols; ubi; underutilized. introduction dioscorea belongs to the dioscoreaceae family and is also known as yams, greater yam, "shanyao" in china, "umbi" or “uwi” in indonesia, “ube’ in the philippines or “ubi” in malaysia (mojiono et al., 2022; li et al., 2023; azahana et al., 2023; ferriol, 2023). the world production share of yams by regions from 2020-2022 in africa (98%), americas (1.2%), oceania (0.5%) and asia (0.2%), with the top producer countries being nigeria, ghana, côte d'ivoire and benin. the increase in yams production worldwide from 81 million tons (2020) to 88 million tons (2022) showed a significant demand for this crop (fao stat 2024). meanwhile, according to the statistics released by the department of agriculture of peninsular malaysia, the production of dioscorea alata or "ubi badak" in 2023 was estimated at around four metric tons (doa, 2023). limited knowledge of its nutritional value compared to other tuberous crops is one of the reasons this crop is not commercially cultivated and consumed in malaysia. the dioscorea spp. commonly consumed by local people on the east coast of peninsular malaysia and can be found abundantly in local markets, especially during the monsoon season from october to december each year (azahana et al., 2023). the tuber flesh of dioscorea spp. is rich in carbohydrates, making it one of the primary staple foods in certain countries, such as those in africa, and an alternative starchy food in some areas in malaysia. the carbohydrate content of several species of dioscorea (d. alata, d. esculenta, d.rotunda, d. cayenensis and d. bulbifera) was 50% 77% depending on their varieties and location of cultivation (oko & famurewa, 2015; ezeabara & anona, 2018; godfrey et al., 2023; sato et al., 2024). meanwhile, the protein content in the various manuscript received: 06 february, 2025. revision accepted: 20 may, 2025. published: 16 june, 2025. https://doi.org/10.14421/biomedich.2025.141.211-218 212 biology, medicine, & natural product chemistry 14 (1), 2025: 211-218 species of dioscorea was reported in the range of 1.13% 6.20% (d. hispida), 4.7% 8.99% (d. alata), 7.25% (d. esculenta) and 8.83% (d. bulbifera) (saleha et al., 2018; n'dri et al., 2018; yalindua et al., 2021; rayamajhi et al., 2024). recent studies on its biological activities from extracts of dioscorea spp. also revealed interesting activities in antioxidant (adomėnienė & venskutonis, 2022), antimicrobial (induar et al., 2024), antidiabetic (rayamajhi et al., 2024), antidiarrheal (islam et al., 2024), antihypertensive (logan et al., 2024), hypolipidemic (li et al., 2019; povydysh et al., 2023), antiproliferative and anticancer (mainasara et al., 2021; nurul makiyah et al., 2023). the astonishing pharmacological activities of dioscorea spp. mainly due to the presence of its secondary metabolites, such as phenolic compounds, including flavonoids and anthocyanins, steroidal saponin, and carotenoids (ren et al., 2015; price et al., 2018; ukom et al., 2020; safitri et al., 2021; kuagny et al., 2023; syahputra et al., 2024; islam et al., 2024). however, studies on proximate composition and phytochemicals of various dioscorea spp. accessions in malaysia remain limited. thus, this study aims to deliver and assess various accessions of dioscorea spp. collected in peninsular malaysia for its nutritional values, phenolics and flavonoid content, and antioxidant activity. materials and methods collection and preparation of the sample eight different accessions of dioscorea spp. were collected across peninsular malaysia (table 1 and figure 1). each species was identified by a botanist at the agrobiodiversity and environmental research centre, mardi, serdang. the flesh color and cross-section reviewed under a stereomicroscope are shown in figure 2. all the specimens were deposited in the herbarium, kompleks mygenebank, mardi, serdang. all the samples were cleaned, and the skin was peeled. the samples were cut into small cubes and dried in the industrial oven at 50°c for three days or until the moisture content remained constant. the samples were ground into a fine powder (18 mesh) using a mechanical grinder (ika werke mf 10 basic, germany). the sample powder was sealed and kept in the chiller at 4°c until further analysis was needed. table 1. various accessions of dioscorea spp. collected in peninsular malaysia. sample sample code location tuber flesh color d. alata da1 bukit payong, terengganu light white yellowish d. alata da2 machang, kelantan light white yellowish d. alata da3 pasar pasir putih, kelantan light white yellowish d. alata da4 marang, terengganu light white yellowish d. alata da5 wakaf bharu, kelantan light white yellowish d. alata var. purpurea dvp wakaf bharu, kelantan dark purple d. esculenta de teluk pelandok, negeri sembilan light yellow d. piscatorum dp kuala koh, kelantan dark yellow-orangish figure 1. the area of the sample (dioscorea spp.) was collected in peninsular malaysia. azid et al. – nutritional and antioxidant properties of wild yams 213 (a) (b) (c) (d) figure 2. flesh color and structure were reviewed after cross-section under a stereomicroscope of dioscorea spp. collected. a). dioscorea alata (white) b). dioscorea alata var purpurea c). dioscorea esculenta and d). dioscorea piscatorum. proximate and nutritional composition approximately 250g of dried samples were weighed and sent to the accredited laboratory for proximate composition analysis. each reading was done in triplicate. total phenolic content (tpc) the total phenolic contents of the sample were determined using the folin–ciocalteu method with some modifications (mirfat et al., 2013). a 2g dried powder sample was extracted in 20 ml of 70% methanol. crude extract (50µl) was mixed with 100µl of folin ciocalteau's phenol reagent (merck, germany). after 3 minutes, 100µl of 10% sodium carbonate (na2co3) (sigma aldrich, usa) was added to the reaction mixture and allowed to stand in the dark for 60 minutes. the absorbance was measured at 725 nm, and the total phenolic contents were calculated from a calibration curve using gallic acid (0-10 µg/ml) as a standard reference. the estimation of phenolic contents was conducted in triplicate. the results were presented as mean values ± standard deviations and expressed as mg gallic acid equivalent per 100 g of samples (mg gae/100g) in dry weight (dw). total flavonoid content (tfc) the total flavonoid content was measured using the previously published method with minor modifications (mirfat et al., 2013). approximately 100µl of extract was diluted with 400 µl of distilled water. subsequently, a 5% sodium nitrite (nano2) solution (30µl) was added and allowed to react for 5 min. afterwards, 10% 214 biology, medicine, & natural product chemistry 14 (1), 2025: 211-218 aluminium chloride (alcl3.6h20) (20µl) was added and left for 5 min. finally, 200µl of sodium hydroxide (naoh) was added, and the mixture was mixed using a vortex. all samples were analyzed in triplicate, and the absorbance was measured immediately at 510nm. quercetin was used to calculate the standard curve, and the results were expressed as mg quercetin per 100 g sample (mg qe/100g) in dry weight (dw). free radical scavenging assay all the powder samples underwent extraction and were assessed for their free radical scavenging ability, following the previously described procedure with minor modification (salahuddin et al., 2020; zulkhairi et al., 2021). the assay was conducted using a 96-well plate, with 2g of dried powder sample extracted in 20 ml of 70% methanol. the stock solution was diluted to the desired concentration for the working solution. the final volume obtained (7µl) was mixed with 280µl of a methanolic solution of 2,2-diphenyl-1-picrylhydrazyl (dpph) (sigma, usa). the plate was covered with aluminium foil to prevent exposure to sunlight and was kept in a dark place for 30 minutes. analysis was performed using a uv spectrophotometer (eon biotek instrument) at 517 nm. the results were expressed as the inhibition concentration (ic50) value in mg/ml, representing the concentration at which dpph radicals were scavenged by 50%. statistical analysis all triplicate samples were analyzed using duncan's multiple range test (dmrt) (mean ± sem, n = 3) with sas version 9.4. results and discussion proximate and nutritional composition proximate and nutritional composition of dioscorea spp. shown in table 2. accessions da1-da5, dvp and de showed a range of carbohydrate content <85 g/100g, while dp had significantly the highest carbohydrate content with 90.8 g/100g (p<0.05). additionally, dp also provides significantly (p<0.05) higher energy in 100g compared to other dioscorea accessions with 374.33 kcal/100g, while da1-da5 with 352.67 – 371.0 kcal/100g, dvp (368.67 kcal/100g) and de (366.0 kcal/100g), respectively. table 2 proximate composition of dioscorea spp. collected in peninsular malaysia. sample ash fat protein carbohydrate energy g / 100 g kcal / 100g da1 3.07b 0.10cd 7.43c 82.23d 358.67f da2 2.23d 0.13c 10.57a 80.17e 364.0e da3 2.73c 0.00d 8.0b 84.67b 371.0b da4 2.73c 0.40b 7.23c 80.03e 352.67g da5 2.87c 0.53a 6.0e 83.60c 363.33e dvp 1.90e 0.17c 8.03b 83.77c 368.67c de 3.83a 0.57a 6.50d 83.67c 366.0d dp 1.60f 0.00d 2.77f 90.80a 374.33a means followed by the same letter within a column are not significantly different at (p≤0.05) by duncan's multiple range test (dmrt) (mean sem, n=3). *, ** and *** significantly difference at p<0.05, 0.01 and 0.001 respectively and ns= not significant. the carbohydrate content in all accessions of dioscorea spp. does not vary from the previously published data. five different varieties of d. alata also showed a range of carbohydrate content ranging from 70.88 to 73.90 g/100g (oko & famurewa 2015), while research conducted by ezeabara & anona et al. (2018) showed that the carbohydrate content of d. alata in nigeria was 76.26 g/100g. meanwhile, the carbohydrate content in different varieties of dioscorea spp. showed carbohydrate content ranging from 20.46 g/100g to 92.50 g/100g dw (kulasinghe & ranaweera, 2019). additionally, recent research by godfrey et al. (2023) on comparative assessment of the proximate composition in d. alata, d. rotunda, and d.bulbifera revealed the carbohydrate composition in the range of 57.38 to 77. 51g/100g. meanwhile, da2 showed the highest protein content (10.57 g/100g) (p<0.05), while dp had the lowest protein content (2.77 g/ 100g). these findings and values agreed with the previous data on nutritional compositions of various landraces of dioscorea spp. twenty-five landraces of d.alata, d.opposita, d. fordii and d.persimilis in china were studied with a protein content range from 6.3% to 12.2%, while 36 yams landraces were studied in southwest ethiopia with protein content from 6.25% to 8.28% ( wu et al., 2016; mulualem et al., 2018). a study by padhan et al. (2020) on eight wild yams and one cultivated dioscorea revealed a high protein content in the wild yam of d. pubera with 10.3%. our findings were also aligned with the theory that different accessions of yams contribute to different nutritional compositions. for example, studies on five cultivars of d.alata flour fractions (yellow yam, orange azid et al. – nutritional and antioxidant properties of wild yams 215 yam, light purple yam, purple yam and dark purple yam) were compared, and the protein content in various flour fractions revealed light purple of d.alata had the highest of protein content (9.85%)(nadia et al. 2015). phytochemical analysis meanwhile, the total phenolic content from the methanolic extract of various accessions of dioscorea spp. is shown in table 3. d. piscatorum (dp) had the highest content of phenolics (300.55 mg gae/100g) (p<0.05) compared to other accessions. meanwhile, the phenolics content in all accessions of d. alata (light yellow variation, da1-da5) exhibited the amount of phenolics ranging from 38.9 mg gae/100g 74.1 mg gae/100g, with da3 showing the highest amount of phenolics content (74.1 mg gae/100g). meanwhile, the methanolic extracts of dvp and de displayed phenolic content of 60.92 mg gae/100g and 84.70 mg gae/100g, respectively. table 3. total phenolic, total flavonoid, flavonoid/phenolic ratio and inhibition concentration of dioscorea spp. sample total phenolic (mg gae/100 g) total flavonoid (mg qe/100 g) flavonoid/phenolic ratio inhibition concentration (ic50 mg/ml) da1 38.90d 11.97d 0.31 7.31b da2 39.52d 19.51d 0.49 15.61h da3 74.10b 38.53c 0.52 9.55d da4 49.85cd 20.05d 0.40 8.13c da5 43.44d 12.98d 0.30 15.07g dvp 60.92c 43.76bc 0.72 9.69e de 84.70b 54.52b 0.64 14.59f dp 300.55a 183.03a 0.61 4.73a means followed by the same letter within a column are not significantly different at (p≤0.05) by duncan's multiple range test (dmrt) (mean sem, n=3). *, ** and *** significantly difference at p<0.05, 0.01 and 0.001 respectively and ns= not significant. ic50 = inhibition concentration at which dpph radicals were scavenged by 50%. the methanolic extracts of dp, de and dvp demonstrated the highest flavonoid content with 183.03 mg qe/100g, 54.52 mg qe/100g and 43.76 mg qe/100g, respectively. the flavonoid/phenolic ratio in the dvp was high compared to other accessions, with 0.72, followed by de (0.64) and dp (0.61), while da1da5 had values ranging from 0.30 to 0.52. the variations of phytochemicals in different accessions of dioscorea spp. due to the many contributing factors. the dark purple hue of the flesh from dvp is due to the high amount of anthocyanin pigment, which is then proven to have the highest flavonoid/phenolic ratio across all the accessions. alatanin, a class of cyanidin in anthocyanins, was identified as a significant chemical compound present in d. alata (purple variety) in thailand and the philippines (moriya et al., 2015; srivichai & hongsprabhas 2020). meanwhile, the study of phenolic content from different landraces of dioscorea spp. including d. esculenta (highland and lowland) in the philippines was conducted, whereby the phenolics content in the highland variety showed an impeccable higher phenolics content as compared to the lowland with 156.5 mg gae/100g and 112. 4 mg gae/100g, respectively (cornago et al., 2011). the development of the hp-tlc method to quantify the secondary metabolites in the d. esculenta in vanuatu showed the presence of epicatechin, a flavonoid class of compound, along with saponin (lebot et al., 2019). additionally, the effects of pigment colours from the different landraces of d.alata in india and their correlation with the phenolics content showed that the yellow and purple flesh tuber share higher phenolics content and their antioxidant activity (jose et al., 2019). meanwhile, the in vitro antioxidant activity was measured using a free radical scavenging assay on eight extracts of dioscorea spp, and a significant difference in the inhibition concentration was revealed. the methanolic extracts of da1, da3, da4, dvp and dp showed ic50 values < 10 mg/ml, with dp having the lowest ic50 value of 4.73 mg/ml (p<0.05), indicating a more potent antioxidant activity compared to the others accessions. however, the flavonoid/phenolic ratio in dp is lower compared to dvp. still, it exhibits the most potent antioxidant activity (ic50 = 4.73 mg/ml), probably due to the presence of carotenoids, which is attributed to its dark yellow-orangish hue of the flesh tuber that is present in various accessions of dioscorea spp. (nadia et al., 2015; jose et al., 2019; ukom et al., 2020). conclusions the studies on different accessions of dioscorea spp. showed different proximate compositions, phenolics and flavonoid content. the free radical scavenging assay towards methanolic extracts also showed diverse 216 biology, medicine, & natural product chemistry 14 (1), 2025: 211-218 antioxidant activities depending on their varieties and localities. d. piscatorum (dp) showed the highest carbohydrate content and provided more energy than other accessions. this accession exhibits exceptional antioxidant activity that could benefit health applications emphasizing oxidative stress reduction. meanwhile, despite the high protein content in the variety of d. alata (da2), its antioxidant activity was weak compared to other accessions. in conclusion, the samples vary significantly in both nutritional and functional properties. these differences highlight the suitability of each sample for specific dietary or health-related applications. acknowledgements: the author wishes to extend their deepest gratitude to the herbarium (kompleks mygene bank) for identifying the samples and the alliance chemist laboratory sdn. bhd. for conducting the proximate analysis. authors’ contributions: all authors contributed to the manuscript. mohd zulkhairi azid designed and assisted with the experiments, visualized data, and drafted, compiled, and edited the manuscript. mohd norfaizal ghazalli and salmaniza salleh collected and identified all sample materials. abdul muhaimin abdul kadir assisted with data visualization. siti aisyah mohd noor, saidatul aqilah mohamad yusof, nur daliana yusof, and mohammad raiehan prayoga contributed to sample preparation and experimental design. all authors have read and approved the final manuscript. competing interests: all authors declared that they have no competing interests in this study. funding: the research was made possible by funding from the ministry of agriculture and food security (kpkm), malaysia, for the entire duration of the study. references azahana, a., dahlia, a.s., noor syaheera, m.y., noor haslinda, n.h., othman, h.s., & salleh, m.s. 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(2021). antioxidants capacity, phenolic and oxalate content from two varieties of solanum melongena at different maturity stages. asian journal of applied chemistry research, 8 (october 2020),54–63.63. doi:https://doi.org/10.9734/ajacr/2021/v8i430198 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1231-1240 | doi: 10.14421/biomedich.2025.142.1231-1240 issn 2540-9328 (online) the effectiveness of honey purity testing from several regions in central sulawesi province i made budiarsa*, manap trianto, akram, yulia windarsih, abdul ashari departement of biology education, faculty of teacher training and education, tadulako university, jl. soekarno hatta no.99, tondo, palu tim., palu, central sulawesi 94111, indonesia. corresponding author* budiarsa_imade@yahoo.com abstract honey is a valuable natural product with nutritional, medicinal, and economic importance, but its high commercial value makes it prone to adulteration, which threatens consumer trust and product quality. this study aimed to evaluate the effectiveness of simple purity tests in identifying adulteration in honey samples collected from several regions in central sulawesi province. a total of 12 honey samples from different bee species and floral sources were analyzed using seven tests, namely solubility, cloudiness and foam, heating, hexagonal pattern, seepage, raw fish, and onion tests. the results showed that most tests successfully distinguished pure honey from adulterated samples. specifically, the solubility, cloudiness and foam tests, as well as heating, and raw fish tests indicated that 66.67% of the samples were pure, while the hexagonal pattern, seepage, and onion tests confirmed purity in all samples (100%). these findings demonstrate that simple, low-cost methods can provide reliable initial screening of honey purity and are suitable for routine use in local communities where advanced laboratory facilities are limited. the application of these tests is expected to support honey quality assurance, protect consumer trust, and promote the sustainable development of the beekeeping industry in central sulawesi. keywords: adulteration detection; honey purity; regional quality; simple tests; sulawesi honey. introduction honey is a natural sweet substance produced by honey bees from the nectar of flowers or from secretions of living parts of plants (abeshu & geleta, 2016; tafere, 2021). it has been widely recognized for its nutritional, medicinal, and economic value. honey contains carbohydrates, amino acids, vitamins, minerals, organic acids, and various bioactive compounds with antioxidant, antimicrobial, and anti-inflammatory properties (manyiloh et al., 2011; ranneh et al., 2021; afroz et al., 2023). due to these benefits, honey is highly sought after in both local and international markets, making it a valuable commodity for beekeepers and traders (garcía, 2018). however, its high commercial value has also led to an increased risk of adulteration, which negatively impacts consumer trust, market stability, and public health (anagaw et al., 2024). adulteration in honey often involves the addition of sugar syrups, molasses, or other sweeteners to increase volume and reduce production costs (fakhlaei et al., 2020). such practices can compromise the nutritional quality and bioactive properties of honey, as well as mislead consumers (bose & padmavati, 2024). globally, honey adulteration has been identified as one of the most common forms of food fraud (siddiqui et al., 2022; morariu et al., 2024). in indonesia, including the province of central sulawesi, this issue remains a concern due to the diversity of honey production areas and the varying levels of quality control across regions. although honey from central sulawesi is known for its distinctive flavor and potential health benefits, systematic assessments of its purity are limited. central sulawesi is geographically diverse, with ecosystems ranging from lowland forests to mountainous regions, each offering different floral sources for nectar production (kahono et al., 2023). this environmental variation contributes to differences in honey characteristics, including color, aroma, viscosity, and chemical composition (oroian et al., 2016; mohammed, 2022). consequently, honey from different districts or regions may vary significantly in quality. identifying the purity of honey from these areas is not only important for consumer protection but also for maintaining the reputation and market value of local honey products (bose & padmavati, 2024). various methods have been developed to assess honey purity, including physicochemical tests (e.g., moisture content, ash content, acidity, electrical conductivity) (dobrinas et al., 2022), microscopic analysis of pollen content (melissopalynology) (nunes et al., 2024), and advanced instrumental techniques such as manuscript received: 03 september, 2025. revision accepted: 14 october, 2025. published: 12 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1231-1240 1232 biology, medicine, & natural product chemistry 14 (2), 2025: 1231-1240 fourier-transform infrared spectroscopy (ftir) or highperformance liquid chromatography (hplc) (halko et al., 2024). in indonesia, basic physicochemical analyses remain the most accessible and cost-effective approach for routine quality monitoring, particularly in rural beekeeping communities where advanced laboratory equipment may not be available (wakjira et al., 2021). these tests can detect abnormal values that may indicate adulteration or poor handling practices, such as excessive water content leading to fermentation or unusually high ash content, which suggests contamination (choudhary et al., 2020). previous studies have emphasized the importance of establishing regional honey quality profiles to support quality assurance and product certification (mădaş et al., 2020). for instance, moisture content is a critical parameter that affects honey stability and shelf life; the codex alimentarius standard sets a maximum of 20% moisture to prevent fermentation (correa-mosquera et al., 2022). similarly, the ash content reflects the mineral composition of honey, which is influenced by botanical and geographical origins but can also indicate adulteration if levels are abnormally high. additionally, parameters such as ph, electrical conductivity, and reducing sugar content provide valuable information for distinguishing pure honey from adulterated products (gela et al., 2023; inaudi et al., 2025). this research aims to evaluate the effectiveness of honey purity testing by applying standard physicochemical analyses to samples collected from several regions in central sulawesi province. the findings are expected to provide scientific evidence regarding the quality of honey in the region, identify potential cases of adulteration, and highlight differences attributable to geographic and floral variations. ultimately, this study will contribute to efforts in improving honey quality assurance, protecting consumer rights, and promoting the sustainable development of the local beekeeping industry. materials and methods time and location this research was conducted from july to august 2025. the experimental work took place at the biology laboratory, faculty of teacher training and education, tadulako university. tools and materials the tools used in this study included a stainless steel tablespoon, stainless steel teaspoon, glass beakers, thermometer, white ceramic plates (15 cm in diameter), matches, candles, jam jars, a camera for observation, and a stopwatch for recording observation time. the materials used consisted of honey samples, plain water, and warm water at 50 °c. the warm water was prepared at a constant temperature of 50 °c. the honey samples were obtained from four different bee species originating from various sources, totalling 12 samples. each honey sample was transferred into a 50 ml test tube. the sources of honey samples used in this research are presented in table 1 and figure 1. table 1. the sources of honey samples used in this research. sample code species types of feed addar apis dorsata multiflora acpmr apis cerana mango (mangifera indica) acbir apis cerana multiflora adbkr apis dorsata multiflora acblr apis cerana water apple (syzygium aqueum) tsmir tetragonula sapiens multiflora tsmur tetragonula sapiens multiflora wipor wallacetrigona incisa multiflora adsir apis dorsata rambutan (nephelium lappaceum) actur apis cerana multiflora actir apis cerana rambutan (nephelium lappaceum) acpur apis cerana multiflora note: information on bee species and forage type was obtained from the sellers. figure 1. the sources of honey samples used in this research. budiarsa et al – the effectiveness of honey purity testing from … 1233 the honey samples were obtained from different sources because the foraging sources of each bee species vary. the use of different samples was intentional, as this study aimed to evaluate the effectiveness of the tests. each test was expected to be effective in determining honey purity regardless of its origin (bee species and honey source). procedure each honey sample was stored in a 50 ml test tube and labelled according to its type. for each purity test, five replications were performed for every honey sample. the purity tests included the solubility test, turbidity and foam test, heating test, hexagonal pattern test, leakage test, raw fish test, and onion test. data were collected using the one-zero sampling method. solubility test a glass with a diameter of 10 cm was filled with 200 ml of warm water (50 °c) and placed on a white cardboard base to clearly observe the movement of the honey when poured. one tablespoon of honey was poured slowly into the glass from a vertical distance of 10 cm above the water surface at a 30° inclination. if the honey and water were mixed immediately, a score of 0 was assigned (indicating adulterated honey). conversely, if no immediate mixing occurred, a score of 1 was assigned (indicating pure honey). cloudiness and foam test a glass with a diameter of 10 cm was filled with 200 ml of warm water (50 °c) and placed on a white cardboard base to observe the color and foam of the honey when it was poured. one tablespoon of honey sample was added to the glass and stirred with a teaspoon approximately 100 times over 30 seconds until well mixed. if small bubbles formed, disappeared quickly, and the mixture appeared clear, a score of 0 was assigned (adulterated honey). conversely, if small bubbles formed, persisted, and the mixture became cloudy, a score of 1 was assigned (pure honey). heating test a 5 ml of a honey sample was placed in a tablespoon and heated over a candle flame with a 1 cm wick length, positioned 2 cm above the flame surface for 2 minutes. if the honey did not overflow (spill from the spoon) after 2 minutes, a score of 0 was assigned (adulterated honey). conversely, if foam formed and overflowed (spilt from the spoon) before 2 minutes, a score of 1 was assigned (pure honey). hexagonal pattern test a volume of 10 ml of a honey sample was poured onto a white ceramic plate with a diameter of 15 cm, and 100 ml of water was added along the plate’s edge until the honey was submerged. the plate was gently moved in a figure-eight motion three times. if the resulting hexagonal patterns were unclear, irregular, and disappeared within 10 seconds, a score of 0 was assigned (adulterated honey). conversely, if the hexagonal patterns were regular, distinct, and persisted for at least 10 seconds, a score of 1 was assigned (pure honey). seepage test a volume of 5 ml honey sample was dropped onto blotting paper placed on a flat surface and allowed to seep for 30 minutes. if the seepage reached 1–3 mm from the original drop, a score of 1 was assigned (pure honey). conversely, if the seepage extended more than 3 mm from the original drop, a score of 0 was assigned (adulterated honey). raw fish test a fresh, whole baby fish (bawal), 5 cm in length, was placed in a plastic cup with a diameter of 10 cm. a volume of 50 ml honey sample was poured into the cup to fully immerse the fish, which was secured with a bamboo skewer to keep it submerged. the cup was sealed tightly with plastic and stored in a cool, dark place for two weeks. after two weeks, the fish was examined. if the fish remained moist, did not shrink, and the honey did not liquefy (absorb water), a score of 0 was assigned (adulterated honey). conversely, if the fish became dry, odorless, and the honey liquefied (absorbed water), a score of 1 was assigned (pure honey). onion test a fresh, whole shallot without skin, 3 cm in length, was placed in a plastic cup with a diameter of 10 cm. a volume of 50 ml honey sample was poured into the cup, and the shallot was secured with a bamboo skewer to keep it submerged. the cup was sealed tightly with plastic and stored in a cool, dark place for two weeks. if the shallot remained intact and unspoiled, a score of 0 was assigned (adulterated honey). conversely, if the shallot shrank and changed color to dark purple, a score of 1 was assigned (pure honey). data analysis experimental design the experimental design employed was a completely randomized design (crd), with bee species as the treatment factor. the mathematical model used, as described by mattjik and sumertajaya (2002), is as follows: where: yij : observed value of the iii-th bee species in the jjj-th replication μ : overall mean pi : effect of the iii-th bee species on honey purity test effectiveness εij : experimental error 1234 biology, medicine, & natural product chemistry 14 (2), 2025: 1231-1240 the collected data were first tested for assumptions, followed by an analysis of variance (anova) to determine the effect of treatments on the observed variables, using sas software version 9.1.3. variables the observed variable in this study was the percentage effectiveness of honey purity tests. a honey sample was considered to have passed the test if it is effectiveness percentage reached 85%. the test effectiveness was calculated using the following formula: where: α : number of successful results in each test s : number of replications in each test results and discussion solubility test the solubility test was conducted on 12 honey samples to evaluate their authenticity. the results show that 8 out of 12 samples (66.67%) did not immediately mix with water, indicating purity, whereas 4 samples (33.33%) showed immediate mixing, suggesting adulteration (table 2 & figure 2). table 2. effectiveness of the solubility test on 12 honey samples. code*) repetition percentage 1 2 3 4 5 addar 1 1 1 1 1 100 acpmr 1 1 1 1 1 100 acbir 1 1 1 1 1 100 adbkr 0 0 0 0 0 0 acblr 1 1 1 1 1 100 tsmir 0 0 0 0 0 0 tsmur 0 0 0 0 0 0 wipor 1 1 1 1 1 100 adsir 1 1 1 1 1 100 actur 1 1 1 1 1 100 actir 1 1 1 1 1 100 acpur 0 0 0 0 0 0 note: the sources of honey vary figure 2. effectiveness of the solubility test on 12 honey samples. cloudiness and foam test the cloudiness and foam tests were performed on 12 honey samples to evaluate their authenticity. the results showed that 8 out of 12 samples (66.67%) produced small bubbles that persisted and formed a turbid mixture, indicating purity, whereas 4 samples (33.33%) produced small bubbles that quickly disappeared and resulted in a precise mixture, suggesting adulteration (table 3 and figure 3). table 3. effectiveness of the cloudiness and foam test on 12 honey samples. code*) repetition percentage 1 2 3 4 5 addar 1 1 1 1 1 100 acpmr 1 1 1 1 1 100 acbir 1 1 1 1 1 100 adbkr 0 0 0 0 0 0 acblr 1 1 1 1 1 100 tsmir 0 0 0 0 0 0 tsmur 0 0 0 0 0 0 wipor 1 1 1 1 1 100 adsir 1 1 1 1 1 100 actur 1 1 1 1 1 100 actir 1 1 1 1 1 100 acpur 0 0 0 0 0 0 note: the sources of honey vary budiarsa et al – the effectiveness of honey purity testing from … 1235 figure 3. effectiveness of the cloudiness and foam test on 12 honey samples. heating test the heating test was conducted on 12 honey samples to evaluate their authenticity. the results showed that 8 out of 12 samples (66.67%) produced foam that overflowed from the spoon within 2 minutes, indicating purity, whereas 4 samples (33.33%) did not overflow within 2 minutes, suggesting adulteration (table 4 and figure 4). table 4. effectiveness of the heating test on 12 honey samples. code*) repetition percentage 1 2 3 4 5 addar 1 1 1 1 1 100 acpmr 1 1 1 1 1 100 acbir 1 1 1 1 1 100 adbkr 0 0 0 0 0 0 acblr 1 1 1 1 1 100 tsmir 0 0 0 0 0 0 tsmur 0 0 0 0 0 0 wipor 1 1 1 1 1 100 adsir 1 1 1 1 1 100 actur 1 1 1 1 1 100 actir 1 1 1 1 1 100 acpur 0 0 0 0 0 0 note: the sources of honey vary figure 4. effectiveness of the heating test on 12 honey samples. 1236 biology, medicine, & natural product chemistry 14 (2), 2025: 1231-1240 table 5. time overflowed on 12 honey samples. code*) repetition average 1 2 3 4 5 addar 53.46 59.91 58.91 52.35 54.55 55.84 acpmr 58.86 55.66 58.74 57.75 59.05 58.01 acbir 57.26 58.16 57.65 56.15 57.20 57.28 adbkr acblr 56.61 58.31 52.74 55.50 57.80 56.19 tsmir tsmur wipor 57.66 56.91 54.57 56.55 57.79 56.70 adsir 54.86 56.76 57.75 53.95 56.64 55.99 actur 51.81 58.91 57.52 50.70 56.25 55.04 actir 58.82 55.16 54.55 57.90 56.63 56.61 acpur note: number in seconds. hexagonal pattern test the hexagonal pattern test was conducted on 12 honey samples to evaluate their authenticity. the results showed that all samples (100%) formed regular and distinct hexagonal shapes that remained visible for more than 10 seconds, indicating purity (table 6 and figure 5). table 6. effectiveness of the hexagonal pattern test on 12 honey samples. code*) repetition percentage 1 2 3 4 5 addar 1 1 1 1 1 100 acpmr 1 1 1 1 1 100 acbir 1 1 1 1 1 100 adbkr 1 1 1 1 1 100 acblr 1 1 1 1 1 100 tsmir 1 1 1 1 1 100 tsmur 1 1 1 1 1 100 wipor 1 1 1 1 1 100 adsir 1 1 1 1 1 100 actur 1 1 1 1 1 100 actir 1 1 1 1 1 100 acpur 1 1 1 1 1 100 note: the sources of honey vary figure 5. effectiveness of the hexagonal pattern test on 12 honey samples. seepage test the seepage test was conducted on 12 honey samples to evaluate their authenticity. the results showed that all samples (100%) exhibited leakage ranging from 1–3 mm from the initial drop, indicating purity (table 7 and figure 6). budiarsa et al – the effectiveness of honey purity testing from … 1237 table 7. effectiveness of the seepage test on 12 honey samples. code*) repetition percentage 1 2 3 4 5 addar 1 1 1 1 1 100 acpmr 1 1 1 1 1 100 acbir 1 1 1 1 1 100 adbkr 1 1 1 1 1 100 acblr 1 1 1 1 1 100 tsmir 1 1 1 1 1 100 tsmur 1 1 1 1 1 100 wipor 1 1 1 1 1 100 adsir 1 1 1 1 1 100 actur 1 1 1 1 1 100 actir 1 1 1 1 1 100 acpur 1 1 1 1 1 100 note: the sources of honey vary figure 6. effectiveness of the seepage test on 12 honey samples. raw fish test the raw fish test was conducted on 12 honey samples to evaluate their authenticity. the results showed that 8 out of 12 samples (66.67%) exhibited dry fish texture, absence of odor, and liquefied honey (absorbing water), indicating purity, whereas 4 samples (33.33%) exhibited wet fish texture, no shrinkage, and non-liquefied honey (not absorbing water), suggesting adulteration (table 8 and figure 7). table 8. effectiveness of the raw fish test on 12 honey samples. code*) repetition percentage 1 2 3 4 5 addar 1 1 1 1 1 100 acpmr 1 1 1 1 1 100 acbir 1 1 1 1 1 100 adbkr 0 0 0 0 0 0 acblr 1 1 1 1 1 100 tsmir 0 0 0 0 0 0 tsmur 0 0 0 0 0 0 wipor 1 1 1 1 1 100 adsir 1 1 1 1 1 100 actur 1 1 1 1 1 100 actir 1 1 1 1 1 100 acpur 0 0 0 0 0 0 note: the sources of honey vary 1238 biology, medicine, & natural product chemistry 14 (2), 2025: 1231-1240 figure 7. effectiveness of the raw fish test on 12 honey samples. onion test the onion test was conducted on 12 honey samples to evaluate their authenticity. the results showed that all samples (100%) caused the onion to shrink and change color to dark purplish (table 9 and figure 8). table 9. effectiveness of the onion test on 12 honey samples. code*) repetition percentage 1 2 3 4 5 addar 1 1 1 1 1 100 acpmr 1 1 1 1 1 100 acbir 1 1 1 1 1 100 adbkr 1 1 1 1 1 100 acblr 1 1 1 1 1 100 tsmir 1 1 1 1 1 100 tsmur 1 1 1 1 1 100 wipor 1 1 1 1 1 100 adsir 1 1 1 1 1 100 actur 1 1 1 1 1 100 actir 1 1 1 1 1 100 acpur 1 1 1 1 1 100 note: the sources of honey vary figure 8. effectiveness of the onion test on 12 honey samples. discussion 4 types of tests including the solubility, cloudiness and foam, heating, and raw fish tests obtained the same results, namely 66.67% of samples indicated pure honey and the remaining 33.3% were indicated as false while different results were shown by 3 types of tests, namely hexagonal pattern, seepage, and onion tests which indicated that 100% of honey samples were genuine. pure honey generally has a high viscosity, a greater density than water, and contains various natural components such as reducing sugars (fructose and glucose), enzymes, and phenolic compounds that make it not immediately dissolve when dripped into water (alkafaween et al., 2023; mello dos santos et al., 2025). pure honey also generally contains complex organic compounds that contribute to the formation of stable foams when homogenized with water (saxena et al., 2010; tyowua et al., 2023). stable foaming accompanied by solution turbidity occurs due to the interaction between complex sugars and natural protein and colloidal compounds in honey (brudzynski & sjaarda, 2021). in addition, turbidity can also be affected by the presence of budiarsa et al – the effectiveness of honey purity testing from … 1239 pollen (pollen) and minerals that are natural characteristics of pure honey (raweh et al., 2023). based on this data, 4 samples with the code adbkr, tsmir. tsmur and acpur are strongly indicated to include adulterated honey which is supported by the majority of test results. honey that has been adulterated dissolves more easily when dripped into water (damto, 2021; tomczyk et al., 2023). furthermore, adulterated honey is generally derived from sugar cane and sugar beet such as corn syrup, sucrose syrup, hfcs, inverted syrup, glucose syrup and high fructose inulin syrup which changes its physicochemical properties so that it can be known through honey authenticity detection tests (fakhlaei et al., 2020). although the 4 samples have been strongly indicated including fake honey, these results are not completely valid because they are limited to initial qualitative tests. to obtain valid results in proving the authenticity of honey, it is necessary to carry out physicochemical analysis and instrumental techniques that are more accurate and more measurable such as chromatographic and hyphenated techniques, spectroscopy, elemental techniques, bioanalytical techniques and electrochemical methods (mohamat et al., 2023; zhang et al., 2023). conclusions this study demonstrated that simple and low-cost methods are effective in detecting honey purity. among the seven tests applied, the solubility, cloudiness and foam, heating, and raw fish tests identified 66.67% of samples as pure, while the hexagonal pattern, seepage, and onion tests confirmed purity in all samples (100%). these results indicate that the combination of these traditional methods provides reliable preliminary screening to distinguish pure honey from adulterated products. the use of such techniques is highly beneficial for local communities in central sulawesi, where advanced laboratory facilities may be limited. overall, the application of these tests can strengthen honey quality assurance, safeguard consumer trust, and support the sustainable development of the regional beekeeping industry. acknowledgements: the authors would like to express their gratitude to tadulako university for providing financial support for the implementation of the 2025 research program. authors’ contributions: conceptualization, i made budiarsa and manap trianto; 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(2023). a comprehensive review of the current trends and recent advancements on the authenticity of honey. food chemistry: x, 19(july), 100850. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 817-824 | doi: 10.14421/biomedich.2025.142.817-824 issn 2540-9328 (online) predicting the anti-pulmonary fibrosis potential of physalis angulata compounds a computational study muhammad farid*, assa aulia kirana, nandita diah oktaviana, sofia rasyda, dwi anggraini, widya aryana ramadhania department of medicine, faculty of medicine, ahmad dahlan university, yogyakarta, indonesia jl. universitas ahmad dahlan kampus 4, kragilan, tamanan, kec. banguntapan, kabupaten bantul, daerah istimewa yogyakarta 55191, indonesia. corresponding author* muhammad2100034023@webmail.uad.ac.id abstract pulmonary fibrosis is a progressive lung disease characterized by tissue scarring and respiratory decline. existing treatments have limited efficacy and significant side effects. physalis angulata, a traditional medicinal plant, shows promise for antifibrotic therapy due to its bioactive compounds potentially targeting key fibrotic pathways. this study aims to predict the potential of physalis angulata compounds to pi3k/akt protein as candidates for antifibrotic therapy. ten active compounds from p. angulata were docked against the pi3k/akt protein (pdb id: 2uzt) using autodock vina. docking was validated by redocking the native ligand. binding affinities and molecular interactions were analyzed. admet properties were predicted via the pkcsm platform to assess pharmacokinetics and toxicity. myricetrin exhibited the strongest binding affinity (-9.6 kcal/mol), surpassing the native ligand (-9.1 kcal/mol). other flavonoids, including eriodictyol (-8.9 kcal/mol), naringin (-8.8 kcal/mol), and apigenin (-8.5 kcal/mol), also showed favorable affinities. critical amino acids involved were asp184 and glu121. the redocking rmsd value of 0.893 å confirmed methodological accuracy. admet predictions revealed high intestinal absorption for tangeretin and apigenin, with no mutagenic or hepatotoxic risks, indicating good pharmacokinetic profiles. physalis angulata flavonoids exhibit strong pi3k/akt binding and favorable pharmacokinetics, supporting their potential as antifibrotic agents. keywords: pulmonary fibrosis: physalis angulata; molecular docking; pi3k/akt. introduction pulmonary fibrosis is a progressive, chronic interstitial lung disease characterized by the excessive accumulation of fibrotic tissue in the lungs, leading to a steady decline in respiratory function (mazurek et al., 2025). among its various forms, idiopathic pulmonary fibrosis (ipf) is the most prevalent and severe, with a reported incidence of 58.7 cases per 100,000 individuals. the disease carries a high mortality rate, with an age-adjusted death rate of 5.4 per 100,000 population in 2017 (zheng et al., 2022). although several intrinsic risk factors have been identified, including genetic predisposition, age, sex, and alterations in the lung microbiome, as well as extrinsic factors such as cigarette smoke, environmental exposures, and air pollution, the exact etiology of ipf remains elusive (zaman & lee, 2018). comorbid conditions like gastroesophageal reflux disease, obstructive sleep apnea, diabetes mellitus, and viral infections have also been linked to the disease (alfaro & robalo cordeiro, 2020). clinically, patients with pulmonary fibrosis often present with non-specific symptoms such as exertional dyspnea and chronic cough, which can delay diagnosis (martinez et al., 2017). a detailed clinical history is essential and should include information on environmental exposures, smoking habits, prior treatments, illicit drug use, travel history, and familial cases of lung disease. to aid this process, the german respiratory society has developed a standardized patient questionnaire that enhances the accuracy and comprehensiveness of evaluations (kardos et al., 2020). diagnostic confirmation typically relies on an integrative assessment involving clinical evaluation, imaging such as high-resolution ct scans, and, in certain cases, histopathological examination (lee & song, 2024). currently, pirfenidone and nintedanib are the only antifibrotic agents approved for ipf management. although both drugs have demonstrated efficacy in slowing disease progression, their use is often limited by side effects, including gastrointestinal disturbances and hepatotoxicity, necessitating regular liver function monitoring (glass et al., 2022). in light of these limitations, interest in plant-based therapies has grown, with physalis angulata emerging as a promising natural candidate. commonly known as manuscript received: 13 july, 2025. revision accepted: 02 october, 2025. published: 17 october, 2025. https://doi.org/10.14421/biomedich.2025.142.817-824 mailto:muhammad2100034023@webmail.uad.ac.id 818 biology, medicine, & natural product chemistry 14 (2), 2025: 817-824 ciplukan in indonesia, this plant is traditionally used to treat various ailments, including diabetes, hepatitis, and asthma (prasetyo & purwanti, 2024). pharmacological studies have shown that p. angulata possesses diverse biological activities, such as antioxidant, antiinflammatory, anticancer, antidiabetic, and antibacterial effects (fadhli et al., 2023). the plant is well adapted to tropical climates and grows wild across much of indonesia, making it easily accessible for cultivation and medicinal use (pillai et al., 2022). furthermore, subacute toxicity studies have confirmed its safety profile, supporting its suitability for development as a herbal medicinal product. its wide geographic distribution across asia, the americas, australia, and the pacific further underscores its global therapeutic potential. the therapeutic properties of p. angulata are largely attributed to its rich content of withanolide compounds. recent investigations have identified eight novel withanolides designated withagulides a through h alongside twenty-eight known analogues (novitasari et al., 2024). among these, several compounds have shown potent antifibrotic activity, notably by inhibiting col1a1 gene expression by over 50% (zhou et al., 2024). one compound, physalin f, has been found to significantly suppress collagen i and α-sma expression induced by tgf-β1 in hepatic stellate cells, primarily through inhibition of the pi3k/akt/mtor signalling pathway (wang et al., 2022). this pathway is known to play a central role in cellular proliferation, fibrosis progression, and scar tissue formation. additionally, the plant exerts anti-inflammatory effects by reducing il-6 gene expression in tgf-β-induced fibrotic cells, suggesting its potential dual action as both an antifibrotic and anti-inflammatory agent (wiraswati et al., 2024). this study aims to explore the antifibrotic potential of physalis angulata through molecular docking analysis, targeting the pi3k/akt signalling pathway. this pathway is critically involved in the activation of fibrotic cells and the regulation of genes associated with extracellular matrix production and tissue scarring. through computationally screening the interaction between bioactive compounds from p. angulata and the pi3k/akt protein complex, the study seeks to identify candidate molecules with high binding affinity that may serve as novel therapeutic agents. the use of molecular docking offers a cost-effective, predictive method for assessing the pharmacological potential of natural compounds prior to in vitro or in vivo validation. ultimately, this research supports the advancement of plant-based antifibrotic therapies that are safer and potentially more effective than current pharmacological options. it also contributes to the broader scientific understanding of how natural compounds can be leveraged to modulate key fibrotic signalling pathways. materials and methods prediction of antifibrotic activity the potential antifibrotic activity of physalis angulata active compounds was assessed using the ddi-pred module of the way2drug software. this approach is grounded in the prediction of activity spectra for substances (pass) technology, which enables prediction of molecular interactions based on the structural and biological activity relationships of compounds. this method facilitates the evaluation of the likelihood that active compounds interact with molecular targets involved in pulmonary fibrosis, providing an initial indication of their antifibrotic potential (ananthathandavan & narayanasamy, 2024). receptor and ligand preparation this study utilized the pi3k/akt protein with pdb id: 2uzt, obtained from the rcsb protein data bank (https://www.rcsb.org/). this protein was selected based on previous research where it was employed to predict the anti-apoptotic potential of cinnamaldehyde (fukata et al., 2025). the retrieved protein structure was subsequently repaired to isolate the protein chains and native ligands essential for validation purposes (utami et al., 2025). this step was carried out using biovia discovery studio 2024 to determine the specific protein sequences accurately. the active compounds from physalis angulata served as test ligands in this study, with their chemical structures retrieved from the pubchem database (https://pubchem.ncbi.nlm.nih.gov/). a total of ten compounds were analyzed: rhoifolin (cid 5282150), tangeretin (cid 68077), eriodictyol (cid 440735), catechin (cid 9064), naringin (cid 442428), myricetrin (cid 5281673), kaempferol (cid 5280863), hesperidin (cid 10621), epigallocatechin (cid 72277), and apigenin (cid 5280443) (ekeke et al., 2019; nguyen et al., 2021). nintedanib was included as a reference standard due to its well documented clinical efficacy in slowing disease progression by reducing pulmonary function decline (kreuter et al., 2021). molecular docking simulation a crucial step in this study involved validating the docking methodology through a redocking procedure to confirm its accuracy. redocking evaluates whether the docking protocol can accurately reproduce the native ligand’s binding pose within the protein-ligand complex. this assessment is quantified by calculating the root mean square deviation (rmsd) between the docked and original ligand positions, with values below 2 å considered acceptable. lower rmsd values correspond to higher docking precision. besides validation, redocking aids in defining the grid box’s center coordinates (x, y, z) and dimensions for subsequent specific docking runs, referencing the native ligand’s position in the crystal structure (farid et al., 2025). docking simulations were performed using autodock https://www.rcsb.org/ https://pubchem.ncbi.nlm.nih.gov/ farid et al. – predicting the anti-pulmonary fibrosis potential … 819 vina integrated within pyrx software. prior to docking, ligand and receptor files were converted to the pdbqt format using open babel, which also facilitated energy minimization to ensure that both ligand and receptor conformations were energetically favorable. docking results were evaluated based on binding affinity values, with lower affinities indicating stronger ligand-protein interactions and potential biological activity (farid et al., 2025). visualization of docking results redocking outcomes were analyzed by calculating rmsd values to assess the congruence between docked ligand positions and their native conformations. additionally, three dimensional overlap visualization of ligands before and after docking was performed using pymol to observe positional deviations (farid et al., 2025). further interaction analysis between ligands and amino acid residues within the receptor binding site was conducted using biovia software, enabling detailed characterization of the types and strengths of molecular interactions formed (farid et al., 2025). pharmacokinetic and toxicity predictions pharmacokinetic and toxicity properties of the tested compounds were predicted using admet (absorption, distribution, metabolism, excretion, and toxicity) parameters via the pkcsm online platform (https://biosig.lab.uq.edu.au/pkcsm/prediction). for this analysis, each compound’s canonical smiles notation was obtained from the pubchem database. key parameters evaluated included intestinal absorption percentage, blood-brain barrier (bbb) permeability, central nervous system (cns) permeability, substrate specificity for cytochrome p450 enzymes cyp2d6 and cyp3a4, total clearance rate (log ml/min/kg), renal organic cation transporter 2 (oct2) substrate status, as well as toxicity profiles including ames mutagenicity and hepatotoxicity potential (utami et al., 2025). results and discussion results the pass online analysis revealed that several compounds from physalis angulata possess predicted pharmacological activities related to lung fibrosis and lung cancer. notably, myricetrin had the highest predicted activity (pa = 0.479) for antineoplastic effects in lung cancer, while also showing moderate potential for cystic fibrosis treatment (pa = 0.273). rhoifolin exhibited pa values of 0.478 and 0.306 for lung cancer and cystic fibrosis, respectively, indicating dual relevance in antifibrotic and antineoplastic contexts. apigenin and kaempferol both demonstrated moderate activity predictions for cystic fibrosis and small cell lung cancer, while hesperidin showed a pa of 0.405 for general lung cancer. tangeretin and naringin also displayed potential with pa values consistently near or above the 0.3 threshold across multiple lung cancer types. these findings suggest that multiple flavonoids from p. angulata may interact with targets relevant to fibrosis and neoplastic lung conditions (ananthathandavan & narayanasamy, 2024). redocking was conducted using the receptor file to validate the accuracy of the docking method. the grid box was defined with center coordinates at x = 21.580, y = 7.910, and z = 35.577, and dimensions of 14.153 å (x), 10.807 å (y), and 4.976 å (z). the evaluation yielded an rmsd value of 0.893 å based on the alignment of 27 atoms, confirming that the predicted ligand conformation closely resembled the original pose (farid et al., 2025). this indicates that the docking protocol was valid and reliable. the determined grid parameters were subsequently used for the main docking simulations. figure 1. native ligand before (green) and after (yellow) redocking. molecular docking simulations produced varied binding affinity values for the tested compounds against the pi3k/akt target. myricetrin exhibited the strongest binding affinity at -9.6 kcal/mol, surpassing even the native ligand at -9.1 kcal/mol. other compounds such as eriodictyol (-8.9 kcal/mol), naringin (-8.8 kcal/mol), epigallocatechin (-8.6kcal/mol), apigenin (-8.5 kcal/mol), and kaempferol (-8.3 kcal/mol) also demonstrated strong interactions. conversely, compounds like hesperidin (4.8 kcal/mol) and rhoifolin (-4.9 kcal/mol) showed the weakest binding affinities. the reference drug nintedanib showed a positive binding energy of +1.73 kcal/mol, which may suggest poor binding affinity under these docking parameters. https://biosig.lab.uq.edu.au/pkcsm/prediction 820 biology, medicine, & natural product chemistry 14 (2), 2025: 817-824 table 1. docking scores and amino acid interactions. compound δg (kcal/mol) hydrogen bonding amino acids non-hydrogen bonding amino acids native ligand -9.1 arg18, thr51 val57, ala70, leu173, asp184, met120, lys72 rhoifolin -4.9 asp184, lys72, asn171, gly50, ser53 val57, leu173, ala70, val123, leu49, met120, lys72 tangeretin -8.2 glu121, thr51, lys72 val57, leu173, ala70, val123, met120, asp184, arg18 eriodictyol -8.9 lys72, glu121, thr51 val57, ala70, leu173, val104, met120, thr183, asp184, arg18 catechin -8.2 — val57, leu173, ala70, asp184 naringin -8.8 glu127, val123, leu49, asp184, ser53 val57, ala70, leu173, lys72, asn171, leu49, asp184 myricetrin -9.6 glu121, glu170, asp184, asn171, gly50 val57, ala70, leu173, tyr330 kaempferol -8.3 glu121, asp184 val57, ala70, leu173, thr183, asp184 hesperidin -4.8 glu121, val123, lys72, asp184 val57, leu173, gly50, thr183 epigallocatechin -8.6 glu121, tyr330, leu49, glu127, arg18, thr51 val57, ala70, leu173, gly50 apigenin -8.5 glu121, gly50 val57, leu173, ala70, met120, val104, thr183 nintedanib +1.73 asp264, thr299 — the docking results also identified key amino acid residues involved in the interactions. non-hydrogen bonding interactions were most commonly observed with residues val57, leu173, and ala70 across nearly all ligands. in terms of hydrogen bonding, asp184, glu121, and lys72 were among the most frequently engaged residues. notably, asp184 formed hydrogen bonds with eight of the eleven tested ligands, highlighting its importance in ligand recognition. table 2. predicted admet profiles of selected compounds. compound absorption (%) bbb penetration cns penetration cyp3a4 substrate clearance ames toxicity hepatotoxicity rhoifolin 24.3 -1.702 -4.798 no -0.005 no no tangeretin 98.5 -1.026 -3.011 yes 0.780 no no eriodictyol 74.7 -0.827 -3.142 no -0.013 no no catechin 68.8 -1.054 -3.298 no 0.183 no no naringin 25.8 -1.600 -4.773 no 0.318 no no myricetrin 43.3 -1.811 -4.376 no 0.303 no no kaempferol 74.3 -0.939 -2.228 no 0.477 no no hesperidin 31.5 -1.715 -4.807 no 0.211 no no epigallocatechin 54.1 -1.377 -3.507 no 0.328 no no apigenin 93.3 -0.734 -2.061 no 0.566 no no based on admet prediction using the pkcms tool, most compounds from physalis angulata showed good intestinal absorption, except for rhoifolin (24.3%) and naringin (25.8%), which fell below the 30% threshold. tangeretin exhibited the highest absorption rate (98.5%), followed by apigenin (93.3%) and kaempferol (74.3%) (ramadhan et al., 2024). all ten compounds were predicted to have poor blood–brain barrier (bbb) permeability, with values below -1, particularly myricetrin (-1.811) and hesperidin (-1.715). only apigenin (-2.061) and kaempferol (-2.228) approached the cns permeability threshold. none of the compounds were substrates for cyp2d6 or oct2, while only tangeretin was predicted as a cyp3a4 substrate. all compounds were classified as non-mutagenic and nonhepatotoxic. in terms of clearance, tangeretin (0.78 log ml/min/kg) and apigenin (0.566) had the highest predicted elimination rates (utami et al., 2025). discussion the results of the docking analysis provide insight into the molecular interactions between active compounds from physalis angulata and the pi3k/akt protein, a critical signalling pathway implicated in fibrosis progression. the high binding affinity observed in compounds like myricetrin and eriodictyol suggests a strong and potentially biologically relevant interaction, comparable or even superior to the native ligand. these findings support the hypothesis that certain flavonoids may inhibit pi3k/akt activity and thus interfere with fibrotic signalling mechanisms. farid et al. – predicting the anti-pulmonary fibrosis potential … 821 figure 2. 3d structure of pi3k/akt protein (pdb id: 2uzt) with binding site. the frequent appearance of val57, leu173, and ala70 in non-polar interactions implies the presence of a conserved hydrophobic pocket that stabilizes the ligand through van der waals and hydrophobic interactions. this structural feature may contribute significantly to the overall binding strength of the tested ligands. moreover, the consistent involvement of polar residues such as asp184 and glu121 in hydrogen bonding highlights their central role in mediating strong and specific ligandreceptor interactions. asp184’s ability to act as both a donor and acceptor enhances its versatility and reactivity, potentially serving as a critical anchor point in the binding site. figure 3. binding pocket interactions of (a) myricetrin, (b) eriodictyol, (c) naringin, and (d) epigallocatechin with akt. 822 biology, medicine, & natural product chemistry 14 (2), 2025: 817-824 the results suggest that bioactive compounds from physalis angulata may have therapeutic potential for pulmonary fibrosis by targeting pathways shared with lung cancer, such as fibroblast proliferation and extracellular matrix accumulation through modulation of tgf-β/smad and nf-κb signalling (fan et al., 2023). compounds like myricetrin and rhoifolin showed high predictive activity values, indicating strong antifibrotic potential, while apigenin and kaempferol, despite slightly lower scores, have been shown to suppress key fibrosis markers such as α-sma and fibronectin (b. ke et al., 2018). hesperidin and tangeretin also demonstrated promising anti-inflammatory and antiproliferative effects that may slow fibrosis progression (fan et al., 2023). the overlap between antifibrotic and antineoplastic activities highlights the molecular parallels between fibrosis and cancer, especially involving emt and abnormal cell growth (vithalkar et al., 2025). this supports the use of network pharmacology as a useful approach to explore multi-target and multi-component therapies, which are better suited for complex diseases like pulmonary fibrosis where single-target drugs often fall short (liu et al., 2021; nogales et al., 2022). consequently, myricetrin, rhoifolin, and kaempferol emerge as promising candidates for further research and development as natural anti-fibrotic agents (h. l. ke et al., 2024). multiple in vivo and in vitro studies have consistently demonstrated the antifibrotic potential of physalis angulata (ciplukan), supporting the molecular docking results presented in this study. wiraswati et al. (2024) reported that ethanol extract of p. angulata significantly reduced fibrosis scores and serum levels of il-6, tgfβ1, and kl-6 in a bleomycin-induced pulmonary fibrosis mouse model. these biological outcomes are consistent with the docking findings, where flavonoid compounds such as myricetrin, eriodictyol, and hesperidin exhibited strong binding affinities and interacted with key residues including asp184, glu121, and lys72 associated with fibrotic and inflammatory pathways. a fibroblast model confirmed that p. angulata ethanol extract inhibited cell migration and downregulated il-6 and hif-1α expression in tgf-β-induced 3t3-l1 cells, which supports the proposed anti-inflammatory and antifibrogenic mechanism of the docked ligands (rohmawaty et al., 2021). further molecular validation was provided by imaduddin et al. (2024), who demonstrated that p. angulata extract significantly downregulated fibrosisrelated genes such as nox4, mmp8, and klf4 in a bleomycin-induced mouse model. these gene targets are central to oxidative stress response and extracellular matrix remodeling mechanisms possibly modulated via the observed ligand-residue interactions in silico. complementary liver fibrosis studies also support p. angulata's systemic antifibrotic activity. bestari et al. (2023) and rohmawaty et al. (2023) observed improvements in liver histopathology, alt, and cholesterol levels in nafld models, while rohmawaty et al. (2021) reported reduced fibrosis scores and serum transaminase levels in ccl₄-induced liver fibrosis rats treated with the plant extract. taken together, these findings demonstrate that the consistent interaction of docked flavonoids with polar and hydrophobic residues particularly asp184, glu121, and val57 may underlie the extract's observed antifibrotic efficacy, thus reinforcing p. angulata’s therapeutic promise across multiple fibrotic disease models. the admet profiles indicate that several physalis angulata compounds, particularly tangeretin and apigenin, possess favorable pharmacokinetic characteristics, including high intestinal absorption and relatively fast systemic clearance (farid et al., 2025). however, poor bbb and cns permeability across all compounds may limit their central nervous system effects, suggesting a more localized or peripheral antifibrotic action (utami et al., 2025). the absence of cyp2d6 and oct2 interactions reduces the risk of metabolic and renal toxicity, enhancing their safety profile. notably, the lack of ames toxicity and hepatotoxicity predictions across all compounds further supports their potential as safe therapeutic candidates. overall, the pharmacokinetic properties strengthen the viability of these bioactive agents especially tangeretin, apigenin, and kaempferol for further development as anti-fibrotic treatments with minimal systemic toxicity. this study is limited by its reliance on in silico predictions, which, while informative, do not fully replicate the complexity of biological systems. the docking results and admet simulations require further validation through experimental assays, such as in vitro tests on lung fibroblasts or in vivo models of pulmonary fibrosis. additionally, the pharmacokinetic predictions need to be confirmed by actual bioavailability and toxicity studies. future research should focus on elucidating the specific molecular mechanisms of promising compounds like myricetrin and apigenin, particularly their influence on the pathway and fibrotic markers. such investigations will be essential to assess their therapeutic relevance and advance them as potential candidates for natural anti-fibrotic drug development. conclusions several bioactive compounds from physalis angulata, particularly myricetrin (δg = -9.6 kcal/mol), apigenin (8.5 kcal/mol), and eriodictyol (-8.9 kcal/mol), demonstrated strong binding affinities to the pi3k/akt protein and interacted with key residues like asp184 and glu121, suggesting potential antifibrotic activity. admet predictions indicated favorable absorption and safety profiles, especially for tangeretin and apigenin. these findings highlight the therapeutic promise of p. angulata flavonoids in targeting fibrotic pathways. farid et al. – predicting the anti-pulmonary fibrosis potential … 823 further in vitro and in vivo studies are needed to validate their mechanisms and efficacy as multi target agents for pulmonary fibrosis treatment. acknowledgements: this study, and that the research was conducted without any external funding. all necessary details have been fully disclosed within this article. authors’ contributions: mf and aak designed the insilico study. ndo and sr conducted the computational analyses. da processed and analyzed the data. mf and war drafted the manuscript. all authors reviewed and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references alfaro, t. m., & robalo cordeiro, c. 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(2022). mortality and survival in idiopathic pulmonary fibrosis: a systematic review and meta-analysis. erj open research, 8(1). https://doi.org/10.1183/23120541.00591-2021 zhou, j., hu, j., liu, j., & zhang, w. (2024). elucidating the gastroprotective mechanisms of imperata cylindrica beauv.var. major (nees) c.e.hubb through uhplc-ms/ms and systems network pharmacology. scientific reports, 14(1). https://doi.org/10.1038/s41598-024-79483-z biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 151-160 | doi: 10.14421/biomedich.2022.112.151-160 issn 2540-9328 (online) anti-oxidant, anti-inflammatory and anti-atherosclerotic activity of bioactive peptide hpaedr isolated from catla catal muscle on lps induced inflammation on 246.7raw macrophage cells and hcf induced hyperlipidemic zebrafish larvae sabarinathan sethuramalingam, revathy leena ravi, janet rani rajiah* sadakathullah appa college, thirunelaveli – 627011, india. corresponding author* janetgnana@gmail.com manuscript received: 23 july, 2022. revision accepted: 13 august, 2022. published: 23 august, 2022. abstract a muscle wasdissected from the catla catlafish and enzyme hydrolysis was done using various digestive enzymes such as pepsisn, protease, papine, trypsin and alcalase at verity of time intervals (0th, 3rd, 6th, 9th and 12th) hour respectively. followed by, the amino acid composition was identified and the confirmative assays such as, the anti-oxidant assays (dpph and hydroxy radical scavenging activity) and anti-inflammatory assays (hrbc and ad) were done for various peptide hydrolysate. the active hr was identified as 9th hr alcalase hydrolysate which was purified through ultrafiltration (>30 kda, 30-10 kda, 10-3 kda and <3 kda). these fractions were again studied for its anti-oxidant and anti-inflammatory activity. based on the results obtained, the active fraction was identified as 10-3kda which was further purified and identified using gel filtration chromatography and lc-ms/ms as hpaedr (723.76 da). further, for in vitro and in vivo studies the peptide derived from ccm was synthetically designed with 98% purity (phtdpeptides co., ltd. zhemgzhou, china). additionally, the physiochemical properties (solubility, emulsifying properties and foaming properties) of these fraction was studied. finally, the purified fraction was tested for in vitro activity through cell viability, cox-2 production, no production and tnf-α production. moreover, the in vivo protective effect is tested on zebrafish larvae. the results suggest that the active purified peptide fraction isolated from catla catla muscle has a strong natural anti-oxidant, anti-inflammatory and cholesterol reduction activity which can be used in functional foods and pharmaceuticals. keywords: anti-inflammatory; antioxidant; peptide; zebrafish larvae; 246.7raw macrophage; inflammation. abbreviations: hcf-high cholesterol food; ad-albumin denaturation assay; hrbchuman red blood cell membrane stabilization assay; ccm-catla catla mussel introduction generally, fishes are considered as easily putrescible food hence, it starts to blemish as soon as it was harvested. to overcome these problems, processing and packaging methods such as drying and smoked fish that are required to increase its shelf life (majidiyan et al. 2022). moreover, the fish waste such as bones, muscles and viscera are not disposed in a proper way (hotel 2001). the fish muscles are rich in proteins and collagen which can be used in development of novel pharmaceutical and nutritious functional food (hadidi et al. 2021; zamorano-apodaca et al. 2020). these collagen and muscle proteins are rich in amino acids such as glycine and proline which can be hydrolyzed using digestive enzymes to develop a bioactive peptide (nurilmala et al. 2020). these enzymatic production of bioactive peptides will have various productive activities such as, anti-inflammatory, anti-oxidant and anti-microbial activities (qian et al. 2020). the peptide isolation via enzyme hydrolysis is a productive and non-toxic method because even when it comes to provide non-synthetic hydrolysed peptide it will be less toxic than chemical or solvent based extraction. mostly, digestive enzymes such as trypsin, pepsin, papine, protamex and alclase are used in enzyme hydrolysis, where alclase is considered as one of the active enzyme with ability to create bioactive peptide especially from fish species (qiu et al. 2019). salmon skin (grundtman 2012) and yellow tuna (ray et al. 2019) are some of the examples from which bioactive peptide is isolated using alclase enzyme. the reactive oxygen species (ros) is one of the major markers which caused verity of oxidative stress related diseases such as, cardiovascular diseases and cancer (huertas‐alonso et al. 2021). fish isolated bioactive peptide are a good source of anti-oxidant molecules which can reduce ros production. moreover, https://doi.org/10.14421/biomedich.2022.112.151-160 152 biology, medicine, & natural product chemistry 11 (2), 2022: 151-160 the research shows that the peptides with anti-oxidant activity will posses other important activities such as anti-inflammatory and anti-microbial activities (taheri et al. 2014). carpfish (catla catla) is one of the common fish that lives on fresh water (zamora-sillero et al. 2018). fresh water fish production highly depends on carp fish because it is available globally (franěk et al. 2021). many studies show that fishes can posses a high anti-oxidant activity when enzyme hydrolysis is done (chalamaiah et al. 2015). in this current study, we had investigated the anti-inflammatory and anti-oxidant activity of the bioactive peptide isolated from the carp fish. additionally, we have investigated the gene expression and toxicity studies on macrophage cells and zebrafish larvae. materials and methods sample collection the catla catla was obtained from coastal area thoothukudi (8° 45' 50.9976'' n 78° 8' 5.4024'' e) of tamil nadu, india and was verified from zoological surveyof india (zsi), chennai. chemicals used the chemicals used in this study were as follows: opa, dtt, dpph, pepsin, trypsin, papain, protamex, alcalse sephadex g-25, lps, phosphatase inhibitor (sigma). other chemicals were purchased commercially proximate analysis and amino acid quantification the catla catla muscle (ccm) dissected and the protein percentage is measured (covey et al., 1991). the other parameters of the proximate analysis were also measured using commercially available kits. the ccm was hydrolyzed using hcl at 100 degree celcius for whole day and sent for hplc to quantify the percentage of essential and non-essential amino acids present (waterborg and matthews 1984). enzyme hydrolysis the ccm was hydrolyzed using various digestive enzymes such as, pepsin, trypsin, papin, protamex and alcalase at respective ph and temperature (adler-nissen 1979). the extraction buffer was used to hydrolyze the ccm. the samples were taken for lyophilisation at different time intervals 0th hr, 3rd hr, 6th hr, 9th hr and 12th hr respectively. finally, the various lyophilized samples were used to determine the anti-inflammatory and anti-oxidant activity of the peptide hydrolysate. acid hydrolysis the degree of hydrolysis percentage (dh %) was calculate using opa method where, the various peptide hydrolysate were taken and dissolved in 1mg/ml concentration and 400 μl of sample was mixed with 300 μl of opa after 20 minutes incubation at 37 degree celsius the od was measured at 340nm (chandra et al. 2012). determination of anti-oxidant activity for dpph assay, 50 μl of each peptide hydrolysate (1mg/ml) was mixed with 500 μl of dpph (95% 1:1 v/v ethanol) and kept in 37 degree celcius for 30 minutes and the od was measured at 517nm. the ascorbic acid was used as a positive control (haghani et al. 2021). for hydroxyl radical scavenging assay, 200 μl of each peptide hydrolysate (1mg/ml) was mixed with 900 μl of pbs, 100 μl of edta, 100 μl of ferrous sulphate and 500 μl of deoxyribose followed by, 250 μl of h202 and incubated for 30 minutes at room temperature. finally, the od reading was taken at 532 nm. the ascorbic acid was used as a positive control (ghanbari et al. 2016). determination of anti-inflammatory activity in hrbc assay, the blood sample was collected from volunteries and mixed with equal volume of alsever solution and centrifuged at 1000 rpm for 15 min followed by, the addition of isosaline and incubated for 30 minutes at room temperature and finally, the absorbance is measured at 560 nm. the diclofenac was used as a positive control (mizushima 1966). in albumin denaturation (ad) assay, the sample is mixed with specimen 1% bsa and the mixture is incubated for 30 minutes at room temperature additionally, kept in water bath (60 degree celsius) for 5-6 minutes and the od reading was measured at 340nm. the diclofenac is used as a positive control (rajapakse et al. 2005). separation, purification and identification of peptide sequence ultracentrifugation was done for the active hour (9th) hydrolysate with different cutoff units (3, 10 and 30 kda) to separate peptide hydrolysate into various fractions based on molecular weight. then gel filtration coromatography was performed to purify the active fraction by maintain 1000 μl per minute speed at 280nm. the obtained fractions were again tested for inhibition activity and protein percentage (ranathungaet al., 2006). finally, the amino acid sequence of the purified fraction was identified using lc-ms/ms as hpaedr (723.76 da) and the results obtained were examined using bio tools software. physiochemical properties of peptide to determine the solubility of peptide the peptide was mixed with 1ml of water with a ph of around 3-9 and the mixture was centrifuged (8000 rpm) for 20 minutes. additionally, to determine the protein content, sodium hydroxide was used as solvent (ranathunga et al., 2006). for emulsifying property, 1ml of maize oil was mixed with 30ml of peptide solution followed by sethuramalingam et al. – effect of catla catla muscle derived peptide 153 adjusting the ph between 2-10 respectively and the mixture was centrifuged (20,000) for 1 minutes. then 0.5ml of sds was added and the od was measured at 500nm after 10 minutes incubation (coveyet al.,1991). to examine the foaming property the pure peptide was taken (200 μl) and centrifuge at 16000 rpm for 1 minutes. finally the entire volume of the solution was determined (pearce and kinsella 1978). in vitro assessment peptide activity on 246.7raw macrophage cells raw264.7 macrophage cells were cultured in dmem media along with 10 percent fbs and 1 percent (v/v) antibiotic at 36 °c in a 5 percent co2 incubator. cell viability raw264.7 macrophage cells (1 104 cells/well) are incubated for 3–4 h at 37 °c with 50–1000 g/ml of pure peptide concentrations. after 1x pbs washing, 20 l of mtt reagent was added afterwards. after 3 hours of incubation, 200 l of dmso was added to each well, and the od was measured at 570 nm. triton x-100 served as control substance (mosmann 1983). determination of nitrous oxide production raw264.7 macrophage cells (1 104 cells/well) were incubated for one hr at 37 °c with doses ranging from 60 to 260 g/ml, followed by a 24-hour incubation at 37°c with lps (1 g/ml). later, griess reagent (1:1) was added to the supernatant and maintained for 10 minutes. finally, od was measured at 540 nm was observed (nambiar et al., 2015). determination of pro-inflammatory cytokine synthesis the cells (1 104 cells/well) were treated with peptides (50–250 g/ml) and lps (1 g/ml) at 37 °c and 5% co2 for one day; diclofenac served as the reference. the quantitative analysis was performed by measuring the absorbance at 450 nm. in addition, standard concentrations versus absorbance values were used to produce standard curves. in vivo activity of purified ccm peptide on zebrafish larvae zebrafish maintenance and feeding zebrafish were purchased from local shops and grown and maintained in a cycling zebrafish aquaculture system at 28 °c with a 14:10 light-dark cycle. factors such as ph, ammonia and nitrite concentrations are maintained to ensure water purity. a cholesterol diet (hcd) for zebrafish was prepared from a cholesterol solution in diethyl ether (sigma) to obtain a cholesterol content of 4% (w/w) in artificial brine shrimp after evaporation of the ether. five-week-old zebrafish were fed a diet three times a day. the control group was given regular fish kernels and the experimental group was given purified ccm peptide. cholesterol measurement the control and experimental zebrafish larvae were euthanized using cold pbs and homogenized. by centrifugation at 1500 g at 4° c. for 5 minutes the supernatant was separated from 10 zebrafish larvae in each group. total cholesterol was measured using a commercially available test kit. in vivo microscopy for in vivo microscopy, sedated fish larvae were stored in droplets containing tricaine in a sealed, temperaturecontrolled environment. zebrafish larvae were observed under a microscope. pictures of zebrafish larvae were collected every 200 nm and all images were inspected and processed using software. gene expression and survival percentage the expression of tnf-α and nf-kβ is studied on zebrafish larvae and the survival percentage was calculated during the feeding and peptide treatment statistical analysis all the experiments were done in triplicate and the results were represented in the mean ± sd. the analysis was done using graphpad prism 8. results and discussion proximate analysis of ccm the muscle protein percentage was determined to be 33.67 ± 1.68% which has a similarity with other protein percentage (gökoolu and yerlikaya 2003). the percentage of other parameters of proximate analysis is shown (table 1). other studies reveals that higher protein percentage will eventivually have higher possibility of getting active bioactive peptide. table 1. protein percentage of baf. proximate analysis percentage (%) protein 35.77 ± 0.46 moisture 54.53 ± 0.03 (table 1) protein percentage and moisture of baf (mean ± sd) amino acid composition of ccm the anti-oxidant, anti-inflammatory and physiochemical properties of peptides are related to the amino acid composition of the samples (chen et al. 2017). the amino acid composition of the ccm is shown in the table 2. the results shows that, it has both essential and non-essential amino acids where it has higher percentage of amino acids such as, histidine and glutamine which is highly known amino acids for its anti-oxidant and antiinflammatory property (liu et al. 2010). it also has 154 biology, medicine, & natural product chemistry 11 (2), 2022: 151-160 arginine and lysine amino acids which are responsible for high anti-microbial activity (chi et al. 2014; liu et al. 2010). observed amino acids are known to posse’s anti-oxidant activity of peptides (alemán et al. 2011). table 2. amino acid composition of ccm. amino acids ccm (%) asparticacid 15.7 glutamic acid 25.7 serine 19.2 histidine* 7 glycine 0.9 threonine* 3.0 arginine 17.3 alanine 4.2 tyrosine 6.9 methionine* 0.9 valine* 0.9 phenylalanine* 5.2 isoleucine* 6.0 leucine* 9.5 *essential amino acids (table 2) it shows the amino acids composition present in the acid hydrolysis sample of oliva oliva visceral mass percentage of protein hydrolyzed (dh %) the percentage increases in the 9th hr hydrolysate as, 99.69 ± 1.79 %, 89.7 ± 1.54 %, 66.89 ± 0.85 %, 65.99 ± 0.77 % and 60.42 ± 0.39 % for trypsin, alcalase, papine, pepsin and protamex hydrolysate respectively (figure 1). similar studies show that the dh % can be calculate in the same method for marine organisms (ray et al. 2019). other studies shows that the pepsin and alclase has more dh% that other enzymes. in this study, we have used opa method of dh % estimation which is easy to perform and cheaply affordable. 0th 3rd 6th 9th 12th 0 20 40 60 80 100 trypsin alcalase papain pepsin protamex time (hr) d h ( % ) figure 1. it shows the percentage of degree of hydrolysis of oliva oliva visceral mass with five digestive enzymes (mean ± sd, n=3), *p>0.05. anti-inflammatory and anti-oxidant activity of ccm peptide the anti-inflammatory activity is quantified by albumin denaturation assay (ad) and hrbc membrane stabilization assay. current results show that in ad assay the alcalase 6th, 9th and 9th hr hydrolysate has higher inhibitory activity of 67.20 ± 0.88, 76.55 ± 0.37 and 69.84 ± 1.14 percentages respectively. when compared with other hydrolysate inhibitions the 12th hr alcalase shows higher activity (figure 2a). the diclofenac potassium is used as standard drug (95.97 ± 0.27 %). the inflammation may also leads to the lysosomal membrane rupturing hence hrbc membrane is performed because the rbc membrane are similar to lysosomal membrane (babu, pandikumar and ignacimuthu 2011). results shows that the 9th hr alcalase hydrolysate of ccm shows a higher hrbc membrane stabilization assay about 69.03 ± 1.04 % (figure 2b). the diclofenac potassium is used as standard drug (93.77 ± 0.99 %). figure 2. the ad and hrbc assay of different time variables during enzyme hydrolysis are shown in a and b respectively (mean ± sd, n=3), *p>0.005. the dpph is performed since the dpph free electron is paired off (taheri et al. 2014). the antioxidant capacity of ccm peptide shows that the 9th hr hrydrolysate has more activity when compared with sethuramalingam et al. – effect of catla catla muscle derived peptide 155 other hydrolysate (figure 3a). this can be due to the presence of alanine and glycine amnioacid (hajfathalian et al. 2018; taheri et al. 2014). hydroxyl radicals are the major reactive oxygen species (pavithra and vadivukkarasi 2015). the current results show the similar activity with higher anti-oxidant activity at 9th hr hydrolysate (figure 3b). some aminoacids detected also have the ability to chelate pro-oxidative transition metals, thus favoring the reduction and deactivation of oh˙ free radicals (hajfathalian et al. 2018). all these facts could explain the dependency between oh˙ scavenging activity and concentration. figure 3. the dpph and hydroxyl assay of different time variables during enzyme hydrolysis are shown in a and b respectively (mean ± sd, n=3), *p>0.005. purification and sequencing of active fraction the active hr sample was purified using the gel filtration chromatography (figure 4) and the fraction was subjected to amino acid sequencing and molecular weight analysis using lc-ms/ms. the sequence was obtained as hpaedr (723.76 da). further, for in vitro and in vivo studies the peptide derived from ccm was synthetically designed with 98% purity (phtdpeptides co., ltd. zhemgzhou, china) (figure 5). figure 4. the gel filtration chromatogram of the 9th hr alcalase hydrolysate. figure 5. the amino acid sequencing of purified fractions and its molecular weight. physiochemical properties the functional property relies on the structure and amino acid sequences of the protein. the results obtained show that, an increase in solubility was observed between ph (4-10) up to 84.37 ± 1.03% (figure 6). the observed increase in solubility is due to 156 biology, medicine, & natural product chemistry 11 (2), 2022: 151-160 amino acid serine which is reported earlier as an efficient amino acid regarding to solubility of protein (trevinoet al., 2007). similarly, the emulsifying ability also relies on the structure and amino acid sequences of the protein. another study shows that the foaming property are also responsible for the ph change and said that the ph (6-10) is steady in foaming property (majidiyan et al. 2022). additionally, the emulsification activity are active at ph 5 and its stability varies based on the ph (ma et al. 2018). figure 6. the functional properties of oliva oliva veseral mass derived peptide (mean ± sd, n=3), *p<0.05 cytotoxisity and pro-inflammatory cytokines expression the cell viability of the drug treated cells are generally evaluated using mtt assay whereas, the current results on mtt assay shows that the cell viability was more than 80% (figure 7) and hence the purified peptides were not toxic to the macrophage raw264.7 cells. similarly, on another study conducted using sargassum polycystum shows higher cell viability. additionally, a anti-inflammatory peptide derived from arca subcrenata shows lower cell toxicity on macrophage raw264.7 cells (chen et al. 2017; li et al. 2014). figure 7. cell viability (a) and anti-inflammatory activity of ccm peptide over (b) tnf-α, (c) il-1 β, (d) il-6, (e) cox2 and (f) no production inlps-induced raw264.7cells, where n and prepresent negative and positive control (mean ± sd) and there is significant difference, p< 0.05 the pro-inflammatory cytokines expression analyzed at a dosage of 50,100 and 200 μg/ml respectively. the results show that increase in the concentration of peptides increases the cox-2 inhibition (figure 7). it is found that comparatively, the 200μg/ml and higher concentration are effective in inhibition than the lower concentration. further, the studies carried out on oyster and yan-hou-qing shows the similar activity on cox-2 inhibition (hwang et al. 2012; ray et al. 2019). additionally, the expression of tnf-α and nf-kb of peptide treated zebrafish larvae is studied (figure 8). sethuramalingam et al. – effect of catla catla muscle derived peptide 157 figure 8. the peptide reduced the weight (a) of zebrafish. it has a considerable amount of mortality (b) throughout the experiment. the c and d shows the gene expression (hadhaa and hadhb) in the zebrafish liver, intestine and muscle of the atherosclerotic zebrafish against fish oil zebrafish group. ccm peptide downregulated the emergence of plaques in atherosclerotic zebrafish figure 9 shows that zebrafish of the ac group showed a huge amount of deposition of lipids near the vascular area, but there was no change in the control group. interestingly, the addition of 1 mg/l and 10 mg/l ccm peptide to the bath significantly reduced lipid deposition in zebrafish larvae compared to the ac group. there were no significant differences between the as (atherosclerotic) group and the 0.1 mg/l peptide group. figure 9. larvae induced with 4% red florescence attached high cholesterol food (hcf) for 10 days where, the dotted box represents the crucial area of accumulated lipids. ccm peptide improved liver metabolism and oxidative stress in atherosclerotic zebrafish compared to the manage organization, the as organization exhibited a enormous growth in lipid degrees, which have been considerably decreased with the aid of using 1mg/l and 10mg/l ccm peptide (figure 10(a)). figures 10(b) and 10(c) exhibit that tc and tg degrees have been additionally considerably reduced in as zebrafish dealt with with 1mg/l and 10mg/l peptide, however now no longer with 0.1mg/l peptide. in conclusion, peptide should successfully lessen lipid degrees in as zebrafish. 158 biology, medicine, & natural product chemistry 11 (2), 2022: 151-160 figure 10. nail red staining images of control anpeptide treated (a) at various concentration (exposed for a period of 10 days). total cholesterol (b) and triglycerides (c) of zebrafish. significance was calculated by one-way anova and unpaired t-test using graphpad prizom. ccm peptide protected against lesion in atherosclerotic zebrafish neutrophil is widely regarded to be the principal cell type responsible for tissue damage and acute lesion. to determine the characteristics of the lesion response in the early stages of atherosclerosis, we first created a genetically modified zebrafish line that upregulated the gfp of neutrophils in transgenic mpx: “egfp” zebrafish. we discovered a significant upregulation of green fluorescentlylabeled neutrophils in vascular locations in atherosclerotic zebrafish larvae group (figure 11). in contemplation to determine the benefit of fish oil as a therapeutic medication that inhibits gfpneutrophils transhumance and lesion response, we administered varying dosages of ccm peptide to zebrafish larvae fed an hcd diet. both the 1mg/l and 10mg/l concentrations of ccm peptide resulted in a reduction of neutrophils in the tails of zebrafish larvae, however the 0.1mg/l concentration was unaffected. in conclusion, ccm peptide protected zebrafish from oxidative damage and inflammation. figure 11. anti-inflammatory effect of peptidein hcf induced atherosclerosis on zebrafish (egfp) larvae. the green fluoresce represents the inflammation level of neutrophils. sethuramalingam et al. – effect of catla catla muscle derived peptide 159 conclusions the catla catla muscle(ccm) derived peptide is isolated and purified using ultrafiltration and gel filtration chromatography. by the anti-inflammatory assays (ad and hrbc) the active hour is identified as 9th hr alcalase hydrolysate. additionally, the fractions are purified and the sequence is found using lc/ms-ms as hpaedr with a molecular weight of 723.76 da. further, in vitro studies were carried out using lps induced inflammation on raw264.7 macrophage cells against synthetically designed peptide and found that the peptide is non-toxic and posses ability to suppress a proinflammatory cytokine in increased quantity. additionally, it exhibits reflex effects in hcd-induced hyper-lipidemia, lesion and oxidative stress and inhibits formation of atherosclerotic plaque. hence this study suggests that the ccm derived peptide may be work as a supplementary medicine to treat chronic inflammatory diseases and may posses cholesterol reducing effect. code availability: no code availability data availability: data sharing not applicable to this article as no datasets were generated or analysed during the current study. authors’ contributions: sabarinathan s and revathy lr have contributed in all aspects of research and manuscript preparation under the guidance of janet rani r. competing interests: the authors declare that there are no competing interests. funding: the authors declare that nofunding was received to assist this research references adler-nissen, 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indonesia. corresponding author* fachrial_edy@yahoo.co.id manuscript received: 15 july, 2023. revision accepted: 19 august, 2023. published: 05 october, 2023. abstract an analgesic-antipyretic drug widely used is paracetamol, which has various health benefits and several adverse effects. therefore, various natural products have been extensively studied as alternative analgesic-antipyretics, one of which is sweet orange peel. this study aimed to investigate sweet orange peel's analgesic and antipyretic activity by in vivo methods. this experimental study evaluated the analgesic and antipyretic effects of sweet orange peel extract extracted by the maceration method. the analgesic effect was evaluated by tail immersion (maximum possible analgesia) and acetic acid-induced writhing method (total abdominal writhing). meanwhile, the antipyretic effect was evaluated by the brewer yeast-induced hyperpyrexia (body temperature) method. this study showed that sweet orange peel methanol extract significantly increased the maximum possible analgesia value (132.79%) and reduced the number of abdominal writhing (44.05%) at the highest dose of 750 mg/kg bw. it indicated analgesic activity from sweet orange peels. meanwhile, the antipyretic effect of sweet orange peel methanol extract was observed from 1-4 hours after administration, and the highest percentage inhibition of body temperature 4 hours after administration was found in a moderate dose, that was 5.98% (p value: 0.042). therefore, it can be concluded that sweet orange peel methanol extract has analgesic and antipyretic effects with an optimal dose range of 500-750 mg/kg bw. keywords: analgesic; antipyretic; sweet orange; peel; methanol. introduction estimated 50 million adults in the united states suffer from chronic pain daily, with 19.6 million adults experiencing high-impact chronic pain that interferes with daily life or work activities (raja et al., 2020; treede, 2018; wang & meng, 2021). various injuries and illnesses are commonly occupied by pain and fever. nonsteroidal anti-inflammatory drugs (nsaids) are usually prescribed for the relief of pain and fever; however, some patients may express some adverse drug effects in the gastrointestinal tract, such as perforation, hemorrhage, ulceration, and obstructions (subedi et al., 2016). paracetamol or acetaminophen has been widely used since 1955 and that commonly used equally with other nsaids. these drugs are over-the-counter drugs with analgesic antipyretic activity. due to this reason, an irrational application of these drugs has widely occurred, including the recreational use of sports, to relieve pain from athletic activity or as prophylaxis before exercise (esh et al., 2017). the paracetamol utility was high in some countries, which may be due to the public having an opinion about paracetamol as a medicine for all diseases (pill for every illness). dorji et al. (2018) reported that 72.1% of 441 outpatients in phuentsholing general hospital, bhutan (india) was prescribed paracetamol in the last year (dorji et al., 2018). the paracetamol utility was also high in indonesia. surya et al. (2018) reported that 34 parents (69%) of 50 parents in laksana kumara kindergarten preferred paracetamol as an antipyretic for their children (surya et al., 2018). other than the benefits, paracetamol also has some adverse drug effects due to the metabolite of paracetamol, including acetanilide and phenacetin. paracetamol must be used judiciously; when prescribed with a higher dose (7.5-15 grams/24 hours), it may become hepatotoxic or ineffective when it is too low. more than 28 billion combination forms of paracetamol were distributed worldwide in 2005, and around 401 deaths were reported associated with paracetamol or paracetamol combination drugs in 2009 by the association of poison control centers. furthermore, paracetamol-induced liver failure is recently reported as the second leading cause of liver transplantation in the united states (khosravi et al., 2011). (bebenista dan nowak, 2014). due to the information above, looking for a natural product with similar efficacy with minimal adverse effects is essential. herbs have been widely used as an https://doi.org/10.14421/biomedich.2023.122.577-584 578 biology, medicine, & natural product chemistry 12 (2), 2023: 577-584 alternative treatment. however, direct used herbs were not effective and unadjusted. thus, it required a procedure to form the form of a pharmaceutical. the direct application of herbs was high in some undeveloped countries because of the cheaper cost than synthetic drugs (salmerón-manzano et al., 2020). some studies have investigated the various benefits of natural products in indonesia. sweet orange is one of the natural products widely utilized for various products such as beverages, pharmaceuticals, or cosmeceuticals. however, the peel becomes a wasted product for these industries. due to this reason, some studies have been performed to investigate the benefits of sweet orange peels. malleshappa et al. (2018) reported that some types of orange (citrus aurantifolia, citrus reticulata, citrus aurantium, citrus grandis, and citrus medica) had a significant anti-inflammatory effect by decreasing paw edema volume in carrageenan-induced rats. on the other hand, malleshappa also demonstrated the analgesic effect of these citruses by increasing reaction time in tail immersion assay and hot plate assay (120 and 150 minutes). another study by ahmed et al. (2019) reported a similar result whether hydroethanolic citrus extract, naringin, and naringenin had hepatoprotective effects in apap (n-acetyl-p-aminophenol)-induced liver injury. this protective effect was indicated by increased antioxidant defense systems and inflammation and apoptosis suppression. furthermore, schneider et al. (2020) also reported that citrus reticula ethanol extract had an analgesic effect by the formalin-induced hyperalgesia assay (control: 501.5 ± 40.0 seconds; c. reticulata 300 mg/kg: 161.8 ± 41.1 seconds), carrageenan model (control at hour 4th: 82.5 ± 9.6 %; c. reticulata 300 mg/kg at 4th hour: 47.5 ± 6.5%) and complete freund’s adjuvant model (control: 501.5 ± 40.0 s; c. reticulata 300 mg/kg: 161.8 ± 41.1 sec). however, none of these studies investigated analgesic and antipyretic activity from sweet orange peel. thus, this study aimed to investigate analgesic and antipyretic activity from sweet orange peel methanol extract by in vivo models. materials and methods study design this experimental study was performed between september 2022 to december 2022 in pharmacology laboratory, universitas prima indonesia. this study evaluated analgesic and antipyretic activity from sweet orange peel methanol extract by in vivo model. acetic acid-induced writhing and tail immersion methods evaluated the analgesic activity of sweet orange peel. meanwhile, the antipyretic activity was evaluated by brewer yeastinduced hyperpyrexia method. this study protocol has been approved by health research ethics committee universitas prima indonesia with letter no. 018/kepk/unpri/xii/2022. materials this study used some materials, including sweet orange peels, distilled water, magnesium powder, amyl alcohol, mayer, bouchard, and dragendorff reagent, iron (iii) chloride, hydrogen sulfate, 95% ethanol, lead (ii) acetate, isopropanol, chloroform, molisch reagent, liebermanburchard reagent, acetic acid, blood tube, paracetamol, sodium carboxyl methyl cellulose (scmc), acetic acid, brewer’s yeast, and ketamine. extraction process sweet orange peel was cleaned, cut, and dried without sunlight exposure to form a dry simplicial. after that, dry simplicia meshed into simplicial powder. this simplicial powder is then extracted by the maceration method. it was soaked into 95% methanol as the solvent in a ratio of 1:3 for three days and regularly stirred. after three days, it was filtered, and the residue was macerated again in the same way two times. meanwhile, all filtrate from each maceration was collected to evaporate by rotary evaporator, forming a concentrated sweet orange peel methanol extract. finally, the extract yield was determined by dividing the extract mass by fresh simplicial mass and multiplying it by 100% (chiuman et al., 2023; gulo et al., 2021; suhartomi et al., 2020). phytochemicals screening the obtained extract underwent phytochemical screening to identify the presence of some phytochemicals, including flavonoid, tannin, phenol, saponin, alkaloid, steroid/ triterpenoid, and glycoside (chiuman et al., 2023; girsang et al., 2019; widowati et al., 2018). oral suspension formulation a gram of concentrated extract and 150 milligrams of paracetamol were suspended into 0.5% scmc to form sweet orange peel extract and paracetamol suspensions. meanwhile, 0.5% scmc was formulated by dissolving 0.5% grams of scmc into ten millilitres of distilled water (chiuman et al., 2021; mutia et al., 2021). tail-immersion method analgesic activity was evaluated by tail immersion and abdominal writhing methods. firstly, the tail immersion method was performed among twenty-five male wistars rats, which were grouped into five groups: control, standard, sweet orange peel extract-1, 2, and 3. the control group received a milliliter of 0.5% scmc; the standard group received 10 ml/ kg bw of paracetamol suspension; sweet orange peel extract-1, 2, and 3 received 2.5 ml/ kg bw, 5.0 ml/ kg bw, and 7.5 ml/ kg bw of sweet orange peel extract suspension, respectively. an hour before and after the treatment, all rats were placed into a restrainer to fix the rat’s body, and the tail was pulled out from the restrainer. then, the rat tail was dipped 3 cm from the distal tail in heated distilled water at 55oc, which was heated by a hotplate. silalahi et al. – analgesic-antipyretic sweet orange peels 579 the maximum time for a dipped rat tail was 15 seconds. analgesic activity was described by maximum possible analgesia (mpa), which was obtained by the following formula (salim et al., 2021): 𝑀𝑃𝐴: 𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑔𝑟𝑜𝑢𝑝 − 𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐺𝑟𝑜𝑢𝑝 15 𝑑𝑒𝑡𝑖𝑘 − 𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐺𝑟𝑜𝑢𝑝 𝑥 100% acetic acid-induced abdominal writhing method besides the tail immersion method, the acetic acidinduced abdominal writhing method is also used to evaluate analgesic activity. it required a 0.7% acetic acid solution formulated by dissolving 0.7 ml of 100% glacial acetic acid into a hundred milliliters of distilled water in a hundred milliliters volumetric flask. after that, all rats were grouped into five groups, including control, standard, sweet orange peel extract-1, 2, and 3, that received some treatment as described in the tail immersion method. fifteen minutes after treatment, all rats were injected intraperitoneally with 0.7% acetic acid solution. five minutes after acetic acid injection, the number of abdominal writhing was counted for twenty minutes (saini & singha, 2012; salim et al., 2021). brewer yeast-induced hyperpyrexia this study evaluated the analgesic activity and antipyretic activity of sweet orange peel extract. this study used brewer yeast-induced hyperpyrexia model to investigate the antipyretic activity of sweet orange peel extract. this study used a 15% brewer’s yeast suspension formulated by dissolving 15 grams of brewer’s yeast in 100 ml of normal saline. then, 20 grams of the last suspension is dissolved with 100 ml of distilled water to form a 20% brewer’s yeast solution. this solution was subcutaneously injected at 10 ml/kg bw. however, the body temperature was initially measured by a rectal thermometer before the injection, and the body temperature was also measured 24 hours after the injection. after that, all rats were grouped into five groups control, standard, sweet orange peel extract-1, 2, and 3, which received some treatment as described in the analgesic activity assay. the antipyretic activity was analyzed from body temperature an hour to five hours after the treatment. finally, all rats were sacrificed for blood sampling intracardiac using three milliliters syringe with a 23 g needle. the blood sample obtained was then filled into the edta tube. before taking the rat blood, it was anesthetized using chloroform. the edta blood sample was investigated for routine blood count at the health laboratory, north sumatra provincial health office (saini & singha, 2012; salim et al., 2021; sivamurugan et al., 2016; veronica et al., 2017). data analysis phytochemicals screening of sweet orange peel extract, initial body weight, number of writhing, reaction time, and body temperature were analyzed by descriptive statistics. then, the analysis was continued based on data distribution by shapiro-wilk. if the data distribution is normal, then one-way anova analyzed it. if data distribution is not normal, and then kruskal-wallis analyzed it. results and discussion physical characteristics and phytochemicals screening this study used sweet oranges obtained from a traditional market in medan. this study used 836 grams of fresh sweet orange peel that was dried into 490 grams of dried sweet orange peel powder, and this dried powder was extracted into 75 grams of concentrated sweet orange peel extract. after that, the obtained sweet orange peel extract underwent phytochemicals screening, and it showed that sweet orange peel methanol extract has some phytochemicals, including alkaloid, saponin, flavonoid, and tannin. analgesic activity of extract tail immersion method the obtained sweet orange peel methanol extract underwent both analgesic and antipyretic assay. analgesic activity assay by tail immersion method in all groups was described in table 1. table 1. reaction time of all groups in the tail immersion method. group reaction time, seconds pvalue mean sd control 4.33 0.76 < 0.05 standard 12.97 2.19 sweet orange peel extract-1 7.80 1.26 sweet orange peel extract -2 8.50 1.99 sweet orange peel extract -3 10.08 3.06 based on table 1, all groups showed a significant difference in reaction time, which can be seen from pvalue < 0.05. the standard group expressed the highest reaction time, and the lowest reaction time was expressed by the control group. further analysis was performed to determine maximum possible analgesic (mpa), that was indicated the analgesic potency from the sample compared to a control group, and maximum possible analgesia in all groups was described in table 2. 580 biology, medicine, & natural product chemistry 12 (2), 2023: 577-584 table 2. maximum possible analgesia of all groups in tail immersion method. group maximum possible analgesia (%) control ref standard 199.54 sweet orange peel extract-1 80.14 sweet orange peel extract -2 96.30 sweet orange peel extract -3 132.79 table 2 above showed that the highest mpa value was found in the standard group, that was 199.54%, followed by sweet orange peel extract-3 (132.79%), 2 (96.30%), and the lowest one was found in sweet orange peel-1, which was 80.14%. acetic acid-induced abdominal writhing method on the other hand, this study also used the acetic acidinduced abdominal writhing method to investigate the analgesic activity from sweet orange peels, and the result of the acetic acid-induced abdominal writhing method was described in table 3. table 3. number of abdominal writhing of all groups in acetic acidinduced abdominal writhing method. group no. writhing pvalue mean sd control 16.80 1.92 < 0.05 standard 8.80 1.92 sweet orange peel extract-1 11.00 2.34 sweet orange peel extract -2 10.00 1.87 sweet orange peel extract -3 9.40 1.14 table 3 showed that all groups showed a significant number of abdominal writhing, which was shown by pvalue < 0.05. the lowest abdominal writhing was found in the standard group, and the highest in the control group. however, this study evaluated the analgesic potency in acetic acid-induced abdominal writhing by percent inhibition of abdominal writhing, described in table 4. table 4. percent inhibition of abdominal writhing of all groups in acetic acid-induced abdominal writhing method. group percent inhibition of abdominal writhing (%) control ref standard 47.62 sweet orange peel extract-1 34.52 sweet orange peel extract -2 40.48 sweet orange peel extract -3 44.05 table 4 showed that the highest percent inhibition of abdominal writhing was found in the standard group, that was 47.62%, followed by the sweet orange peel extract3 (44.05%), 2 (40.48%), and the lowest one was found in sweet orange peel extract-1, that was 34.52%. antipyretic activity other than analgesic activity, this study also investigated the antipyretic activity of sweet orange peel, which was used as evaluated outcome and body temperature in all groups described in table 5. table 5. body temperature of all groups in brewer’s yeast-induced hyperpyrexia method. group body temperature (oc) before induction after induction 1st hour 2nd hour 3rd hour 4th hour 5th hour control 36.84 ± 0.30 38.60 ± 0.68 36.58 ± 0.48 38.04 ± 0.78 37.12 ± 0.47 37.06 ± 0.30 36.88 ± 0.16 standard 36.70 ± 0.40 38.34 ± 0.44 37.20 ± 0.74 36.46 ± 0.44 36.68 ± 0.54 36.46 ± 0.24 36.64 ± 0.42 sweet orange peel extract-1 36.96 ± 0.43 38.50 ± 0.55 38.44 ± 0.71 37.78 ± 0.63 37.34 ± 0.46 36.94 ± 0.45 36.66 ± 0.26 sweet orange peel extract -2 36.98 ± 0.41 39.10 ± 0.23 38.42 ± 0.68 37.34 ± 0.30 36.98 ± 0.25 36.76 ± 0.30 36.66 ± 0.30 sweet orange peel extract -3 36.60 ± 0.39 38.16 ± 0.65 37.66 ± 0.87 36.68 ± 0.43 36.84 ± 0.57 36.46 ± 0.42 36.38 ± 0.40 p-value 0.601 0.084 0.025 0.001 0.252 0.042 0.230 table 5 above showed no significant difference in body temperature in all groups either before or after brewer yeast injection. it can be seen from the p-value of either before (p value: 0.601) or after (p value: 0.084) induction that was lower than 0.05. the mean body temperature before and after induction ranged between 36.60oc-36.98oc and 38.16oc-39.10oc, respectively. it indicated that the brewer yeast injection increased the body temperature. after the induction, all groups received some treatment based on their group. all groups reported significantly decreasing body temperature an hour to four hours after receiving the treatment. it can be seen from the p-value in 1st (p-value: 0.025), 2nd, and 4th (p-value: 0.043) hour after the treatment, which was silalahi et al. – analgesic-antipyretic sweet orange peels 581 lower than 0.05. however, the body temperature decrease was insignificant 5 hours after the treatment and can be seen from p-value > 0.05 (p value: 0.230). furthermore, the percent inhibition of body temperature is described in table 6. table 6. percent inhibition of body temperature of all groups in brewer’s yeast-induced hyperpyrexia method. group percent inhibition of body temperature (%) 1st hour 2nd hour 3rd hour 4th hour 5th hour control 5.23 1.45 3.83 3.99 4.46 standard 2.97 4.90 4.33 4.90 4.43 sweet orange peel extract-1 0.16 1.87 3.01 4.05 4.78 sweet orange peel extract -2 1.74 4.50 5.42 5.98 6.24 sweet orange peel extract -3 1.31 3.88 3.46 4.45 4.66 table 6 showed that the highest percent inhibition of body temperature 4 hours after treatment was found in the sweet orange peel extract-2, which was 5.98%, followed by the standard group (4.90%), 3 (4.45%), 1 (4.05), and the lowest one was the control group, that was 3.99%. after evaluating the rats’ body temperature, all rats in this study were sacrificed for intracardiac blood sampling. this blood sample is then used for routine blood counts, including rbc or red blood cell and wbc or white blood cell. the number of rbc and wbc from rat blood samples in all groups was described in table 7. table 7. rbc and wbc counts in all groups. group rbc wbc control 8.20 ± 0.35 7.86 ± 0.97 standard 4.27 ± 1.63 7.66 ± 0.36 sweet orange peel extract-1 7.68 ± 4.08 8.00 ± 0.29 sweet orange peel extract -2 5.89 ± 2.23 8.70 ± 0.34 sweet orange peel extract -3 3.97 ± 1.83 8.04 ± 0.85 p-value 0.030 0.149 table 7 showed no significant difference in the number of white blood cells or white blood cells in all groups and can be seen from p value > 0.05 (p value: 0.149). however, all groups showed significant differences in the number of red blood cells (rbc), which can be seen from p value <0.05. the highest rbc level was found in the control group, which was 8.20 ± 0.35 x 106/mm3, followed by the sweet orange peel extract-1 (7.68 ± 4.08 x 106/mm3), 2 (5.89 ± 2.23 x 106/mm3), standard (4.27 ± 1.63 x 106/mm3), and the lowest one was the sweet orange peel extract-3, that was 3.97 ± 1.83 x 106/mm3. discussion this study demonstrated the analgesic and antipyretic activity of sweet orange peel methanol extract. both analgesic and antipyretic assays showed that the sweet orange peel had analgesic and antipyretic activity. this analgesic and antipyretic activity from sweet orange peel was not better than the standard group that received paracetamol. however, this analgesic and antipyretic activity from sweet orange peel was better than the control group that received only scmc, a vehicle of extract, and another active compound. the international association for the study of pain (iasp) 2020 defined pain as an unpleasant sensory and emotional experience associated with or resembling that associated with actual or potential tissue damage. various organizations, including world health organization, have widely accepted and adopted this definition. this definition has also been revised many times; however, the meaning of this definition has remained the same from time to time. several painrelated terms include nociception, nociceptive system, and noxious stimulus. nociception is an activity that can be observed in response to an adequate stimulus to the nervous system, in contrast to pain, a subjective experience that can be said to only exist in people who feel it. the nociceptive system is a warning system for an adequate stimulus, while a noxious stimulus is a stimulus that can damage or threaten normal tissue (raja et al., 2020; treede, 2018). according to the pain definition, this study evaluated the analgesic effect of sweet orange peel methanol extract using acetic acid-induced abdominal writhing and tail immersion. the abdominal writhing method evaluated analgesic activity at either central or peripheral levels. meanwhile, tail immersion was used to evaluate 582 biology, medicine, & natural product chemistry 12 (2), 2023: 577-584 analgesic activity at the peripheral level via thermal stimulation of nociceptors (fan et al., 2014; moniruzzaman & imam, 2014; saha et al., 2013). acetic acid-induced abdominal writhing was performed by intraperitoneal injection of acetic acid, which induced an inflammatory response in the peritoneum. this injection induced local inflammation by activating arachidonic acid metabolism, initiating cyclooxygenase (pge2 and pge2α) or lipoxygenase pathways in the peritoneum tissue. both cyclooxygenase products such as pge2 or pge2α and lipoxygenase products accumulate in the peritoneal fluid and cause various inflammation cascades, including increased capillaries permeability, swelling, and the release of various endogenous mediators which stimulate pain in the nerve ends nociceptor (afsar et al., 2015). this study demonstrated analgesic activity by acetic acid-induced abdominal writhing and tail immersion methods from sweet orange peel. it indicated the analgesic activity from sweet orange peels at central and peripheral levels. this analgesic activity was reported from maximum possible analgesia and percent inhibition of abdominal writhing. an increase of sweet orange peel extract dose followed to increase maximum possible analgesia and percent inhibition of abdominal writhing. the highest maximum possible analgesia and percent inhibition of abdominal writhing at all tested sweet orange peel extract doses were found in the highest dose (750 mg/ kg bw), 132.79% and 44.05%, respectively. this study investigated analgesic activity from sweet orange peel and antipyretic activity. typically, human body temperature is ranged between 36.7°c to 37°c (98°f–98.6°f) for oral thermometer, and it can be 0.6°c (1°f) higher than oral thermometer for rectal thermometer or 0.6°c (1°f) lower than oral thermometer for axillar thermometer. when the human body can not keep this normal body temperature, it is defined as a fever and requires antipyretic drugs (estella et al., 2022). some methods can be used to evaluate the antipyretic activity of herbs. one of these methods is brewer’s yeastinduced hyperpyrexia models. brewer’s yeast is a lipopolysaccharide (exogenous pyrogen) which is a component of the cell wall of gram-negative bacteria. when pyrogens such as lipopolysaccharide (lps) or brewer’s yeast enter the body by escaping the natural barrier, this brewer’s yeast then binds to an immunological protein called lipopolysaccharide binding protein (lbp). it promotes the synthesis and release of various endogenous cytokines, such as il-1, il-6, and tnf-α, which easily cross the blood-brain barrier and act on the preoptic/anterior hypothalamus. thereby, it activates the arachidonic acid pathway for the synthesis and release of prostaglandin e2, which was synthase by the cyclooxygenase-2 pathway, leading to increased body temperature (eldahshan & abdel-daim, 2015; santra et al., 2014). brewer yeast-induced hyperpyrexia in this study indicated that the administration of brewer yeast suspension significantly increased rats’ body temperature in the range of 38.16oc-39.10oc. all doses of sweet orange peels revealed antipyretic activity within an hour after extract administration. it can be seen from the decrease in the rat’s body temperature in the first 1 hour and continued to show a decrease in body temperature for the next 4 hours. thus, the antipyretic activity reached the peak effect 4 hours after extract administration, and the highest antipyretic activity 4 hours after extract administration was found in the moderate dose of sweet orange peel extract, which was indicated by the highest percent inhibition of body temperature, 5.98%, among all tested extract doses. on the other hand, the line chart also clearly described the moderate dose line in line chart continued to increase and positioned above the other lines. in addition, increasing the sweet orange peel extract dose did not significantly increase the percent inhibition of body temperature. this study showed that the highest sweet orange peel extract did not reveal better antipyretic activity than the lower dose, which various factors can cause. mintarto and fattahillah (2019) reported an increased in body temperature affected by dehydration, blood flow velocity, and sweat secretion. this study did not control these factors, such as the beverages and food was not limited but was given freely (ad libitum), potentially affecting the rat body temperature. furthermore, both foods and beverages also potentially affect the rat’s body’s metabolic rate and hydration level (mintarto & fattahilah, 2019). the analgesic and antipyretic effects of sweet orange peel methanol extract are associated with the phytochemicals and the yield of the extract. phytochemical screening reported that sweet orange peels contained alkaloids, saponins, flavonoids, and tannins. limited previous studies investigated the antipyretic and analgesic effects of sweet orange peel methanol extract. however, several studies reported that alkaloids and flavonoids had analgesic activity. flavonoids inhibit prostaglandins’ biosynthesis, which is involved in the immunological response and product of cyclooxygenase and lipoxygenase pathways. in addition, flavonoids also affect protein kinase, one of the regulatory enzymes that can inhibit the inflammatory process (eldahshan & abdel-daim, 2015). meanwhile, alkaloids are also reported to inhibit the synthesis of prostaglandins, a product of the cyclooxygenase pathway (gaichu et al., 2017). hence, both flavonoids and alkaloids in sweet orange peel extract can inhibit prostaglandins’ biosynthesis, thereby preventing the cascade of inflammation and resulting in analgesic and antipyretic effects. analgesic and antipyretic activity from sweet orange peel methanol extract can be affected by either phytochemicals or yield from sweet orange peel silalahi et al. – analgesic-antipyretic sweet orange peels 583 methanol extract. some previous studies have reported the yield value of orange peel extract, which can be a reference value. the yield values of ethanol and ethyl acetate extracts were 19.49% and 5.45%, respectively. this study reported that the yield value of sweet orange peel methanol extract was 14.90%, which was not much different from that of sweet orange peel ethanol extract. nevertheless, the yield value of sweet orange peel methanol extract was higher than that of sweet orange peel ethyl acetate extract. this lower yield value indicates better extract quality, but this value is equal to the yield value of sweet orange peel ethyl acetate extract. the difference in yield values could be due to differences in the solvents and the duration of maceration in this study and these previous studies. pandey and tripathu (2014) reported that several factors affecting the extract’s quality included the plant part, the solvent used for extraction, and the extraction procedure (gulo et al., 2021; pandey & tripathi, 2014). conclusions overall, it can be concluded that sweet orange peel methanol extract has an antipyretic and analgesic activity with a range of dose 500-750 mg/ kg bw. the effective dose of analgesic activity of sweet orange peel methanol extract is 750 mg/ kg bw with central and peripheral acting. meanwhile, the effective dose of antipyretic activity is 500 mg/ kg bw, which has revealed antipyretic activity within one hour of administration and is persistent until 4 hours after extract administration. acknowledgements: this study was supported by faculty of medicine, universitas prima indonesia. authors’ contributions: conceptualization: elsa debora silalahi, i nyoman ehrich liester, and edy fachrial; methodology: elsa debora silalahi and edy fachrial; investigation: elsa debora silalahi; discussion of results: elsa debora silalahi; writing – original draft: elsa debora silalahi; writing – review and editing: i nyoman ehrich liester and edy fachrial; supervision: i nyoman ehrich liester and edy fachrial; approval of the final text: elsa debora silalahi, i nyoman ehrich liester, and edy fachrial. competing interests: the authors declare no competing interests. funding: this study did not receive any funding. references afsar, t., khan, m. r., razak, s., ullah, s., & mirza, b. 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(2018). antioxidant and antiaging activities of jasminum sambac extract, and its compounds. journal of reports in pharmaceutical sciences, 7(3), 270–285. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 489-498 | doi: 10.14421/biomedich.2025.141.489-498 issn 2540-9328 (online) diversity of anatomical morphological characters of leaves of mangrove plant types in panimbang banten amanda nabilah, suyamto, mu’jijah biology study program, faculty of health pharmacy sciences, universitas mathla’ul anwar banten, indonesia. corresponding author* suyamto35@yahoo.co.id manuscript received: 08 june, 2025. revision accepted: 27 july, 2025. published: 30 july, 2025. abstract mangroves are plants that produce seeds (spermatophyta), mangroves also have many benefits, namely as an economic or ecological function. mangrove plants have different types of leaf shapes. leaves have the task of absorbing nutrients and gases, as well as processing food through the process of photosynthesis. this study aims to determine the differences in leaf shape based on morphological and anatomical characters. the method used is exploration and preparation of preserved preparations. the samples used were some mangrove leaves found in panimbang district. the stages of the research are starting with taking the third leaf sample, observing the morphological characters of the leaves, taking samples. parameters used to observe morphological characters were leaf shape, base shape, leaf tip, leaf length, width and length of petiole. anatomical character of shape and number of stomata, shape and length of palidase. keywords: mangrove leaves; morphology; anatomy. introduction indonesia is one of the countries in the world that is blessed with abundant natural wealth and biodiversity. one of the biodiversity riches in indonesia is mangrove forests, which have irreplaceable functions and roles. mangroves have economic and ecological functions, such as reducing the impact of hurricanes and tsunamis, as well as being a buffer for the population and wildlife around them (kathiresan, 2011). indonesia has the largest mangrove forest in the world, covering around 21% of the total area of mangroves in the world. mangroves in indonesia are spread across islands such as java, sumatra, kalimantan, sulawesi, to papua, with varying areas depending on the physical conditions, substrate composition, and climate on the islands (spalding et al., 2010). banten is a province on the island of java that has various ecosystems, including mangrove ecosystems that provide high value and benefits to the surrounding community. the mangrove forest area in the pandeglang area, panimbang district, has various types of mangroves. pandeglang, as one of the regencies in banten province, is famous for its natural and culinary tourism. the pristine natural conditions make the mangrove forest in panimbang district a new tourist attraction that is starting to be known to many people, with beautiful views of various types of mangroves (pertiwi, 2021). the coast of pandeglang regency has a large potential for mangrove ecosystems. in 2015, the total number of mangroves along the coast of pandeglang regency reached 1,764 ha, but this area decreased by around 18% in 2020. the reduction in mangrove ecosystems was triggered by community activities that cut down mangroves to be used as animal feed, as well as the tsunami disaster on the west coast of banten in 2018 due to the eruption of anak krakatau (horstman et al., 2014). riyandani (2017) and melati (2020) stated that a good mangrove ecosystem can protect coastal areas from tsunami wave energy by up to 50%. the existence of mangrove ecosystems as coastal protectors from climate change mitigation in banten province has proven to be very important. areas protected by mangrove ecosystems experienced minimal damage compared to open areas during the tsunami disaster in 2018 (horstman et al., 2014). mangrove forests are a type of forest that grows in tidal areas, such as protected beaches, lagoons, and river estuaries, which are inundated at high tide and free from inundation at low tide. the mangrove forest area in panimbang district has a diversity of mangroves that is not yet fully known. the mangrove ecosystem consists of organisms (animals and plants) that interact with environmental factors in the mangrove habitat (sofian et al., 2012). the structure of mangrove roots that can deposit sediment protects the coast from the ebb and flow of sea water and strong waves, so that mangroves are https://doi.org/10.14421/biomedich.2025.141.489-498 490 biology, medicine, & natural product chemistry 14 (1), 2025: 489-498 useful for preventing coastal abrasion that can erode coastal land (susanto & etwin, 2011). mangroves have an important function as a habitat for biota, a stopover for migrant fauna, a place for spawning, nurturing, and foraging, a protector of the marine ecosystem, a protector of the coast from abrasion and tsunamis, a preventer of sea water intrusion, and the lungs of the earth. the potential and benefits of mangroves include forest products (timber and nontimber), fish, shellfish, molluscs, food, sources of medicine, and tourist areas (kordi & ghufran, 2012). the characteristics of mangrove forests usually grow in intertidal areas with muddy, clayey, and sandy soil, periodically inundated with water every high tide. waterlogging determines the composition of mangrove forest soil. mangrove plants have difficulty growing in areas with large waves and strong currents that allow mud to settle. physically, mangrove forests maintain a stable coastline, prevent sea erosion, and function as wave dampers and waste pollution (saputra, 2016). the characteristics of mangrove leaves vary, especially in thickness, surface, tip shape, and base of the leaf. various types of mangrove leaves can be found in various regions, including in panimbang district. the results of this study are expected to be useful for all parties, namely to increase insight into the diversity of morphological and anatomical characters of mangrove leaves, as well as additional information and comparison for further research, providing information on the diversity of morphological and anatomical characters of mangrove leaves. this study aims to determine the diversity of leaves of mangrove plant species in panimbang, pandeglang, banten; to determine the morphological characteristics of mangrove leaves in panimbang, pandeglang, banten; and to determine the anatomical characteristics of leaves of mangrove plant species found in panimbang district, pandeglang, banten. literature review mangrove plants description mangrove forests are complex ecosystems that significantly affect the surrounding area. this ecosystem is ecologically and economically productive, offering various benefits such as preventing coastal erosion, accelerating land expansion, regulating seawater intrusion, and protecting against waves and tsunamis (setiawan, 2013). mangroves also function as wave and storm wind absorbers, coastal protection from abrasion, sediment traps, preventing seawater intrusion, and are able to neutralize water pollution (wiharyanto & laga, 2010). mangroves act as buffer zones and shoreline protection (lungquist et al., 2017) and are found on the coast that is protected from large waves in tropical and subtropical regions (fao, 2007). mangrove forests support coastal fisheries, produce commercial forest products, and protect coastlines (rotich et al., 2016). these plants live between land and sea in tropical and subtropical latitudes with high salinity conditions, tides, strong winds, high temperatures, and anaerobic soil (kathires & bingham, 2001). mangroves have the ability to live in areas with high salinity and changing water conditions, with anaerobic soil (nugraha, 2011). this vegetation grows on muddy soil in tidal areas, beaches, and river estuaries (hotden, 2014). mangrove classification plant grouping depends on the variety of organs and plant parts that form a unit, and these structures are given binomial names to describe their complexity. higher taxon terms are broader and less concrete (aziz et al., 2021). figure 1. rhizophora mucronata rhizophora mucronata (noor, 2012) is included in the kingdom plantae, division magnoliophyta, class magnoliopsida, order malpighiales, family rhizophoraceae, and genera rhizophora. avicennia marina (rizky, 2019) is also in the kingdom plantae, division magnolyophyta, class magnoliopsida, order lamiales, family acanthaceae, and genera avicennia. avicennia is known as a mangrove pioneer that grows on the shores of the sea and rivers. this tree is useful as a medicine and soap ingredient. it grows in muddy places with upright breathing roots, smooth skin, and leaves with spots on the surface (noor et al., 2006). figure 2. bruguiera cylindrica. nabilah et al. – diversity of anatomical morphological characters of leaves … 491 bruguiera cylindrica (maghfiroh, 2010) is included in the kingdom plantae, division magnoliophyta, class magnoliopsida, order myrtales, family rhizophoraceae, and genera bruguiera. b. cylindrica grows on mature substrates with respiratory roots to obtain oxygen. the flowers are light and floating, and are used as firewood. the flowers are in groups of three with yellowish green petals (rosadi, 2013). figure 3. rhizophora apiculate. rhizophora apiculata (saptarini et al., 2012) is included in the kingdom plantae, division magnoliophyta, class magnoliopsida, order malpighiales, family rhizophoraceae, and genera rhizophora. r. apiculata grows in muddy and flooded soil, with elliptical leaves with small black spots. the flowers are paired with brownish yellow corollas, and hypocotyls that are green when young and red when old (saptarini et al., 2012). leaf morphological characteristics characteristics comes from the english word "character" which implies nature or character. in indonesian, character means a trait that shows the appearance or condition of an item. in science, characteristics are often associated with the morphological life system and quality of each plant. plant characteristics must be visible from the roots, stems, and leaves. figure 4. morfology leaves. leaves are important organs for plants, usually thin, wide, and green in color due to chlorophyll. leaves play a role in photosynthesis, gas exchange, and water evaporation. leaves have parts such as stalks (petiolus), blades (lamina), and sometimes sheaths (vagina). complete leaves have all three parts, while incomplete leaves only have blades and stalks. the morphological characteristics of leaves include the overall shape, base tip, surface, and leaf layout. leaf shape is important in taxonomic morphology because it is a characteristic of a species. in compound leaves, special attention is paid to the leaflets. morphological characters are also used in the classification and grouping of plants. leaf anatomy includes oxalate crystals, fibers in the vascular bundles, and five main tissue layers: adaxial epidermis, hypodermis, palisade, sponge, and abaxial epidermis. observations were made using the paraffin and leaf clearing methods to see the stomatal index and palisade ratio. the leaf epidermis consists of one or more layers of cells, often experiencing cuticle thickening. stomata in the epidermis function as a place for gas and water exchange. the compact structure of the epidermis and the presence of cuticles help in the reinforcing function of the leaf. mesophyll is a layer of basic tissue between the upper and lower epidermis, consisting of parenchyma rich in chloroplasts for photosynthesis. the columnar and spongy tissues in the mesophyll help in gas exchange and photosynthesis efficiency. the transport tissue in the leaf forms the leaf veins, consisting of xylem and phloem. in dicotyledonous plants, the leaf veins form a net, while in monocotyledons they form parallel rows. the reinforcing tissue in the form of collenchyma and sclerenchyma is located near the leaf veins and the edges of the leaf. collenchyma has cell walls coated with cellulose and pectin, helping in the mechanical strength of the leaf. secretory tissue in the form of glands that secrete secretions, found on the surface or inside of the leaf. these glands secrete various kinds of secretions and can function in defense against insect attacks. trichomes are epidermal derivatives in the form of hairs that grow on epidermal cells, functioning in defense against insects. trichomes are divided into non-glandular and glandular, with various shapes and structures. stomata are modifications of epidermal cells that play a role in gas and water exchange. stomata are surrounded by neighboring cells that regulate the width of the gap, helping in controlling evaporation and photosynthesis efficiency. 492 biology, medicine, & natural product chemistry 14 (1), 2025: 489-498 framework of thought figure 5. framework of thought hypothesis h1 = several leaves of mangrove plants in the panimbang banten area have different morphological forms. h2 = mangrove leaves in the panimbang sub-district have the same anatomical structure as the others relevant research hadi et al. (2016) studied the morpho-anatomical characteristics of the vegetative structure of the rhizophora apiculata species and found that the morphological leaves of rhizophora apiculata have leaf stalks 10-50 cm long and are whitish brown in color (jurnal pendidikan, 1(9): 166-1692). tihurua et al. (2020) found cuticles with different thicknesses and water storage tissue in mangroves in the form of subepidermis in the mangrove zonation area in the banggai islands, central sulawesi (jurnal kelautan tropical, 23(2): 255-264). tobing et al. (2021) observed avicennia marina leaf preparations consisting of upper (adaxial) and lower (abaxial) epidermis tissue (bulletin of anatomy and physiology, 6(1): 2541-0083). rizki et al. (2015) noted that the morphology of the bruguiera cylindria plant has elliptical-ovate leaves and green hypocotyl (jurnal sainstek, 9(1): 26-32). sabandar et al. found that the rhizophora apiculata mangrove has an anomastic stomata type with neighboring cells surrounding the stomata cells and various epidermal cell shapes, ranging from square to octagonal with irregular lengths (jurnal biologi science dan education, 10(1): 2252-858). research method this study uses a qualitative field research type. according to melong (2013), qualitative research involves direct observation in the field to understand phenomena in a particular context. the approach used is naturalistic, with exploration techniques in the field and in the mathla'ul anwar university laboratory. preparation of preparations was carried out at the structure and development laboratory of gadjah mada university (ugm). research on the morphological and anatomical characteristics of mangrove leaves in panimbang district was carried out from november 2023 to february 2024. research tools and materials the tools used in leaf morphology research include a digital camera, knife or scissors, pencil, description book, label, plastic bag, cardboard, and ruler. while the tools used in leaf anatomy research include a sliding microtome, petri dish, surgical blade, cutter, dropper pipette, flacon, small brush, hotplate, object glass, cover glass (18 mm x 18 mm), label paper, magnifying glass, light microscope, slide glass, micrometer object, micrometer eyepiece, and microphotography. the materials used in the leaf morphology study were several types of mangrove plants taken from panimbang district. meanwhile, the materials used for leaf anatomy observations were samples in the form of leaves from three types of mangrove plants originating from panimbang district, pandeglang regency. the chemicals used to make anatomical preparations included 70%, 80%, 95%, and absolute alcohol, xylol, a mixture of alcohol-xylol with various concentrations (3:1, 1:1, 1:3), canadian balsam, glycerin or albumin, chloral hydrate, distilled water, and nail polish. work procedure 1) sampling the sampling technique is carried out using two methods: first, through the field exploration method which includes direct observation of the morphology of various types of mangrove leaves for inventory, identification, and description such as shape, blade, tip, base, edge, and color of the upper and lower surfaces of the leaves. second, for research in the structure and development laboratory, the samples used are leaves from various types of mangroves found in panimbang district, pandeglang. the leaf morphology work stage includes observation and measurement of various characteristics such as leaf shape, base, tip, color of the upper and lower surfaces, texture, color of the leaf stalk, venation, phyllotaxis, and measurement of dimensions nabilah et al. – diversity of anatomical morphological characters of leaves … 493 such as leaf width, leaf length, stalk length, and stalk diameter. 2) leaf anatomy observation work procedure the initial stage includes cutting the leaves to a size of 1x1 cm, then soaking them in a plakon bottle containing 70% alcohol for fixation. after 24 hours, dehydration was carried out with graded alcohol solutions (70%, 80%, 95%, 100%) for two times with 30 minutes each. the dealcoholization process was carried out with alcohol/xylol solutions in a ratio of 3:1, 1:1, 1:3, followed by pure xylol twice for 30 minutes. furthermore, the samples were infiltrated with a mixture of xylol/paraffin 1:9 at a thermostat of 57°c for 24 hours, then pure paraffin was used for one hour before making paraffin blocks in a petri dish smeared with 15% glycerin. paraffin slices with a thickness of 12 microns were made using a rotary microtome, then the slices were placed on a glass object smeared with glycerin/albumin and distilled water, then heated on a hot plate at 45°c to stretch the paraffin. staining was carried out with pure xylol, a mixture of alcohol/xylol, and graded alcohol solutions, followed by 1% safranin staining in 70% alcohol. after staining, it was continued with graded alcohol and a mixture of alcohol/xylol before being covered with canadian balsam. the preparations were dried on a 45°c hot plate and labeled before being observed using a microscope with 4x, 10x, and 40x objective lenses. the parameters observed included the shape and type of stomata, as well as other characteristics such as leaf thickness, epidermis, trichomes, palisade, stomatal density, vascular tissue, and leaf veins. research flowchart observation of leaf morphological characters figure 6. observation of leaf morphological characters. observation of leaf anatomy figure 7. observation of leaf anatomy. data analysis morphological and anatomical characteristics of mangrove plant leaves were conducted by observing plants in the field as they are. no special planting was carried out on the plants. descriptive analysis methods will be used to examine the data collected for this investigation. the purpose of descriptive analysis is to provide a description of the topic of the variable data collected from the subjects studied. results and discussion types of mangroves found the types of mangroves found in panimbang sub-district are 5 types of mangrove species, and several families. the names of the types of mangroves found can be seen in the following table. 494 biology, medicine, & natural product chemistry 14 (1), 2025: 489-498 table 1. the names of the types of mangroves. no local name scientific name family name 1. red mangrove rhizophora stylosa rhizophoraceae 2. feather fire avicennia rumphiana verbenaceae 3. broadleaf fires avicennia officinalis acanthaceae 4. sea slug sonneratia alba lythraceae 5. white mangrove bruguiera cylindria rhizophoraceae from the data from the table above, 5 types of mangroves were found in the panimbang sub-district, namely r. stylosa, a. rumphiana, a. officinalis, s. alba, and b. cylindria. of the 5 types of mangroves, they have several different morphological characteristics of the leaves, which are presented in the table below: table 2. morphological characteristics of the leaves. no morfologis daun r. stylosa a. rumphiana a. officinalis s. alba b. cylindria 1 leaf shape oval, widened elips oval oval elips 2 length 6 cm 8-9 cm 10 cm 7 cm 10 cm 3 width 6,5 cm 6 cm 5 cm 3 cm 5 cm 4 tip sharp pointed rounded blunt tapered 5 base blunt pointed blunt rounded blunt 6 upper surface color green green dark green green yellowish green 7 lower surface color green yellowish green yellowish green green green 8 pa itd color green green green green green 9 pb itd color green green green green green 10 edge shape smooth wavy flat flat wavy 11 upper surface texture flat smooth smooth smooth smooth 12 stalk lenght 3,5 cm 4 cm 2,5 cm 5 cm 3,6 cm 13 stalk diameter 2 cm 2,5 cm 3 cm 3 cm 0,8 14 stalk color green yellow green green green 15 venation pinnate pinnate pinnate pinnate pinnate 16 phylotaxis alternate alternate alternate alternate alternate observation results in panimbang district show that bruguiera cylindria and rhizophora stylosa have the longest leaves, 10 cm and 9.5 cm respectively. the leaves with the shortest size are found in sonneratia alba. bruguiera cylindria has leaves with a length of about 15 cm. can be seen in the following table: table 3. leaves length of mangrove. no mangrove types long short leaf size 1. b. cylindria 10 cm 2. r. stylosa 9,5 cm 3. s. alba 7 cm anatomical the results of microscopic observations show that the cross-section of mangrove leaves in panimbang district generally have the same structure but with different sizes, especially in leaf thickness, palisade thickness, cuticle thickness, upper and lower epidermis thickness, and stomata position and type. the upper epidermis is usually thicker than the lower epidermis. the epidermis generally consists of one layer. the following are the results of leaf anatomy in the cross-section of mangrove leaves. figure 8. anatomical cross section of a. rumphiana leaf. nabilah et al. – diversity of anatomical morphological characters of leaves … 495 figure 9. glandular trichomes of a. rumphiana leaves. figure 9 above shows the presence of glandular trichomes on a. rumphiana leaves in the upper epidermis with multicellular glands that are round and adjacent to the cuticle. leaf anatomy research found differences in leaf thickness, palisade thickness, cuticle thickness, upper and lower epidermis thickness, stomata position and type, and stomata density. the following are the anatomical data of the leaves: table 4. anatomical data of the leaves. figure 10. mangrove leaf anatomy the leaf thickness ratio in a. officinalis is higher than other species with a value of 603.16 µm, indicating a greater water storage capacity to cope with drought. a. rumphiana and a. officinalis have the highest palisade thickness, which is 132.31 µm and 110.37 µm, respectively, which are important for photosynthesis. the cuticle in mangroves varies, with a. officinalis having a thin cuticle and s. alba being thick reaching 10.31 µm. the upper epidermis of a. officinalis is the thickest (22.63 µm) and b. cylindria the thinnest (10.82 µm). the lower epidermis is thickest in b. cylindria (17.24 µm). all species have hypostomatic leaves (stomata on the underside of the leaf) with anomostic, anisocytic, cyclocytic, and staurocytic stomata types. r. stylosa has cryptophorous stomata (on the underside of the leaf), while a. rumphiana, a. officinalis, s. alba, and b. cylindria have abaxial (underside) and panerophorous stomata (parallel to the epidermal cells). stomatal density varies, with a. rumphiana having the highest density (247.33 stomata/mm²) and s. alba the lowest (57.00 stomata/mm²). morfologycal leaf rhizophora stylosa rhizophora stylosa, or known as red mangrove, has distinctive morphological characteristics. the leaves are 496 biology, medicine, & natural product chemistry 14 (1), 2025: 489-498 dark green with spots on the underside, elliptical and wide with a tapered tip. the fruit is about 2 cm long, similar to a pear and brown in color, with a forked flower stalk that reaches 2-4 cm in length. the stem is quite tall, reaching 10 meters, with gray bark and supporting roots and aerial roots. the natural habitat of rhizophora stylosa is sandy and muddy areas. according to jalaludin et al. (2020), this plant can reach a height of 15 meters, with a blackish gray stem surface and leaves about 8 cm long. red mangrove flowers consist of 4-16 single flowers with ivory yellow petals, white crowns, and 8 stamens, while the fruit extends up to 20-60 cm. in indonesia, rhizophora stylosa thrives on sandy or muddy beaches, including on the coast of panimbang, banten, which has a mangrove forest potential of 2,214.45 ha. indonesia has 38 species of true mangroves, with rhizophora as a genus that has abundant seedlings and durable propagules. classification-wise, rhizophora stylosa is included in the kingdom plantae, division magnoliophyta, class magnoliopsida, order myratales, family rhizophoraceae, and genus rhizophora. avicennia rumphiana the a. rumphiana mangrove found in panimbang district has leaves with a green upper surface and a yellowish green lower surface. the leaves are oval with a pointed tip, smooth texture on the upper surface, and rough on the lower surface. the leaf stalks are about 1820 mm long. according to hardy et al. (2018), a. rumphiana leaves are elliptical with rounded tips and pointed bases. the flowers grow in clusters at the end of the bunch, are yellow to orange, with 4 brown stamens and 4 flower petals. the fruit is heart-shaped and usually appears at the end of the stalk with a pale green color. the leaf stems are dark brown with rough bark, and the tree can reach a height of 16 m. the respiratory roots are pointed like pencils and appear above the ground from shallow waters. the classification of avicennia rumphiana is as follows: kingdom plantae, division spermatophyta, class magnoliopsida, order lamiales, family verbenaceae, genera avicennia, species avicennia rumphiana. avicennia officinalis mangrove avicennia officinalis, known as one of the prominent species in panimbang sub-district, banten, is a fairly tall tree that can reach 15 to 30 meters. the trunk is relatively low compared to other mangroves, brown in color with distinctive white spots. the leaves are oval with rounded tips, about 8-10 cm long, and have a shiny green upper surface, while the underside has fine hairs. the fruit is oval-shaped like a capsule. anatomically, a. officinalis leaves have a dark green upper surface and a yellowish green underside, and concave glands that cover the upper part of the leaf. the leaves are simple, opposite each other, with a shape similar to a breech egg, flat edges, tapering base, and rounded tips. its scientific classification according to maghfirah (2010) is in the kingdom plantae, class magnoliopsida, order scrophulariales, family acanthaceae, genera avicennia, and species avicennia officinalis. this mangrove is also known as a compound flower that grows at the end of the stalk, forming 2-10 flowers per bunch. sonneratia alba the s. alba mangrove found in panimbang sub-district is a tall tree that grows around the coast. the leaves have a green upper surface with fine hairs on the underside, and round leaf tips with opposite positions. the trunk is dark white to brownish with a smooth and longitudinal peeling skin texture. the roots of the s. alba mangrove are cone-shaped and brown, forming respiratory roots that protrude above the ground. according to noor (2006), s. alba can reach a height of 15 meters, with eggshaped leaves and rounded tips, measuring around 5-12.5 x 3-9 cm. this mangrove grows in coastal ecosystems with high adaptation to environmental dynamics. in terms of scientific classification, s. alba is included in the kingdom plantae, division magnoliophyta, class magnoliopsida, order myrtales, family lythraceae, genera sonneratia, and species sonneratia alba. these mangroves also have the ability to produce secondary metabolites such as flavonoids, which act as antioxidants to protect plants from oxidative stress produced by their environment (hastuti et al., 2020). bruguiera cylindria l mangrove b. cylindria l is found in coastal areas with trees reaching 23 meters in height. the bark is brown mixed with white, has a spotted texture. the leaves are elliptical with pointed tips, wavy at the edges, and yellowish green. the roots are in the form of wide boards and spread at the base of the tree. mangrove b. cylindria flowers are grouped into three at the end of the stem, with a white crown, yellowish green petals, and are tubular about 3-4 mm. the hypocotyl is cylindrical or sometimes curved, 8-15 cm long and 5-10 mm in diameter, green in color. growing in areas with soil mixed with a little mud, this type of tree has slow growth and relies on respiratory roots to get enough oxygen, an adaptation that allows it to survive waterlogging. scientifically classified, bruguiera cylindria is included in the kingdom plantae, class magnoliopsida, order myrtales, family rhizophoraceae, genera bruguiera, and species bruguiera cylindria. leaf anatomical characters the anatomical components of the leaf from the upper surface to the abaxial surface are respectively the upper epidermis (ea), hypodermis tissue (h), palisade parenchyma tissue (pl), sponge parenchyma (sp), vascular bundles (vb), and stomata (st). the results of the analysis showed that r. stylosa leaves have high leaf thickness (604.67µm), indicating greater water nabilah et al. – diversity of anatomical morphological characters of leaves … 497 absorption capacity. bareja (2013) confirmed that increasing leaf thickness is related to high heat storage capacity, causing higher leaf temperatures, thereby increasing transpiration. the hypodermis tissue is located under the upper epidermis, with large and dense cells, functioning as a place to store water to prevent drought in high temperature habitats (jacoeb et al., 2011). the cuticle is located on the outermost layer of the leaf, thick in s. alba leaves to control the rate of transpiration and maintain water content. the epidermis consists of the upper epidermis (adaxial) and the lower epidermis (abaxial), both covered by the cuticle and composed of a single layer of thin cells. palisade parenchyma tissue is a mesophyll tissue located below the hypodermis tissue and above the sponge parenchyma. palisade cells are elongated, small, and numerous, important for co2 binding in photosynthesis, especially in drought conditions (peel et al., 2017). stomatal density affects transpiration and water balance in mangrove plants. all mangrove leaf species have mesophyll tissue consisting of palisade and sponge. the genus sonneratia has two layers of palisade tissue, which is an ecophysiological adaptation supported by high stomatal density. the transport bundle consists of xylem and phloem, playing an important role as a transportation system. xylem is on the inside, while phloem is on the outside, located in the leaf veins. phloem is a complex tissue with various elements and types. stomata are derivatives of the epidermis, in the mangrove leaves of a. rumphiana, a. officinalis, s. alba, and b. cylindria have panerophore stomata that are parallel to the epidermis, while r. stylosa has cryptophore stomata that are deeper than the leaf surface (samsuri, 2013). stomata types based on the thickening of the guard cells according to schwendener (in sutrian, 2011): a) amaryllidaceae type: kidney-shaped, thin dorsal wall, thick abdominal wall, with thickened cuticle. b) helleborus type: thick central guard cells, thin wall ends. c) thin wall type and ends. d) mnium type: kidney-shaped, thin abdominal wall, other walls are thin or thick (sarjani et al., 2017). conclusion in panimbang district, five types of mangrove plants were found, namely rhizophora stylosa, avicennia rumphiana, avicennia officinalis, sonneratia alba, and bruguiera cylindria. the morphology and anatomy of mangrove leaves vary depending on the type, with characteristics of thick leaves, thick cuticles, and stomata types. species that face the coast, such as sonneratia alba and bruguiera cylindria, have thicker cuticles. suggestions further research should deepen the diversity of morphological and 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however, its toxicological profile remains understudied. this study evaluated the metabolism, excretion and toxicity properties of gas chromatography-mass spectroscopy-identified phytochemicals from c. rostrata epicarp. in silico analysis and molecular docking of components were carried out using the admetlab2.0 platform and autodock4 tools. visualization of molecular binding interactions was done using discovery studio-2020. ten of the 48 compounds in the extract, including 1-(4-methoxyphenylazo)-2-phenoxynaphthalene, anthiaergostan-5,7,9-trien-14.alpha.,15.alpha.-diol and 2-hydroxychalcone, were predicted to have high probability of inducing liver injury, oxidative stress and inhibiting cytochrome-p450 enzymes. molecular docking revealed that 1-(4-methoxyphenylazo)-2-phenoxynaphthalene binds strongly to nadh dehydrogenase 1 (-7.78 kcal/mol) and cyp2c19 (-9.93 kcal/mol), with the compound interacting with thr301, leu361 and leu366 at the active site of cyp2c19. 2-hydroxychalcone binds strongly to cyp2c19 (-8.07 kcal/mol) and to na+/k+-atpase (-7.49 kcal/mol), while, anthiaergostan-5,7,9-trien14.alpha.,15.alpha.-diol binds strongly to cyp2c19 (-9.56 kcal/mol) and cyp1a2 (-8.71 kcal/mol). the extract showed strong potential to induce toxic outcomes. the abundance of antioxidant phytosterols in the extract may counterbalance the potential toxicity. while c. rostrata holds therapeutic potential, molecular interactions of its phytochemicals highlight risks of toxicity. keywords: cola rostrata; phytochemicals; metabolism; toxicity profile; gc-ms; molecular docking. introduction a large majority of people in developing areas of the world depend on natural products for the treatment and management of various diseases and illnesses (ekor, 2014; mintah et al., 2022). the current global hike in the prices of clinically used drugs, chemotherapeutic drugs in particular, is making more people choose plant-based remedies, owing to their affordability, accessibility, and availability (shin et al., 2022; serra-burriel et al., 2023; vasques et al., 2023). plants have proven to be a viable source of bioactive compounds that have been developed into drugs for treating several disease conditions (newman & cragg, 2020). the increase in the usage of plant remedies for healthcare has come with more research attention and warnings on the safety of these remedies and their potential to induce adverse reactions (okaiyeto & oguntibeju, 2021: başaran et al., 2022). the adverse effects reported for some plant extracts include, liver damage, injuries to tissues of the gastrointestinal tract, kidney failure, cerebral haemorrhage etc. (posadzki et al., 2013; frenzel & teschke, 2016). extracts of cola species are used in traditional medicine for the treatment of various diseases and ailments, with some of them used in the management of diabetes and cancer (ekalu & habila, 2020; ngoka et al, 2021). cola rostrata is a member of the genus that has limited information in the literature on the toxicity of its extracts. the extract of the root bark of c. rostrata, which contains flavonoids, phenols, steroids, tannins saponins, triterpenoids and alkaloids was found to be safe after the acute toxicity of the extract was assessed in mice (odion et al., 2013). dongmo et al (2019) found weak cytotoxic activity in the prenylated derivative of compounds isolated from the root of c. rostrata against human cervix carcinoma kb-3-1 cells. ajayi et al (2022, 2023) determined the components of the epicarp extract of c. rostrata and the protein targets of the drug-like compounds in it; they also found the extract to be significantly cytotoxic against mrc5-sv2 and hela cells with potential to be effective against diabetes, pain manuscript received: 27 february, 2025. revision accepted: 20 may, 2025. published: 11 july, 2025. https://doi.org/10.14421/biomedich.2025.141.369-379 370 biology, medicine, & natural product chemistry 14 (1), 2025: 369-379 and inflammatory diseases. the evaluation of absorption, distribution, metabolism, excretion and toxicity (admet) profile of the components of the crude extract of c. rostrata epicarp is yet to be done. generally, the toxicity of herbal preparations is a function of the contributions of each of the molecules in the extracts. the interactions among extract components could result in the elimination of the effect of some potentially harmful components or accentuation of the toxic effect of the few potentially harmful components. reports show that in some cases, the direct interactions of extract components could be synergistic, antagonistic or additive in nature to bring about the observed pharmacological response in living systems, while in some other cases, there is indirect interaction, products of the metabolism of the constituent of the extracts may interact with one another to produce the observed effect (matotoka & masoko, 2018; uduwana et al., 2023). to assess the safety and mechanisms of pharmacological activities of the crude extract of c. rostrata epicarp as a remedy for potential use in traditional medicine against diabetes, inflammatory diseases, pain and cancer, this study evaluated the metabolism, excretion, toxicological properties, mode of action, and binding affinities of the compounds present in ethanol extract of the plant using in silico analyses and molecular docking. molecular docking of most toxic components of the extract was carried out against selected proteins in the pathways perturbed by the extract fractions. materials and methods extract preparation, fractionation and gas chromatography-mass spectroscopy (gc-ms) analysis the collection of plant materials, extract preparation, and gc-ms analysis of the extract have been previously reported by ajayi et al (2022). briefly, the epicarp of fresh ripe c. rostrata fruit was collected, cut into small pieces, air-dried at room temperature under shade for four months, and pulverized with an electric blender. the pulverized material was soaked in absolute ethanol for six days with frequent rigorous shaking of the extraction vessel to disperse phytochemicals. the crude extract was obtained by filtration, followed by concentration under pressure, and then air-drying. the air-dried crude extract was fractionated via liquid-liquid partitioning with 70% methanol solution and n-hexane to obtain a polar fraction and a non-polar fraction. the constituents of the fractions were identified via gc-ms, the identified compounds were reported by ajayi et al (2022). in silico metabolism, excretion and toxicity analyses of the components of extract fractions the simplified molecular input line entry system (smiles) notation for each compound present in the two extract fractions was obtained from pubchem. the smiles were submitted to admetlab 2.0 online platform for systematic evaluation of the metabolism, excretion and toxicity properties of each constituent of the extract fractions. for each constituent of the extract fractions, the probabilities of having specific properties that are known to define the toxicity of known drug compounds were scored; these properties include, permeation of the blood-brain barrier, clearance (excretion), carcinogenicity, inhibition of members of cytochrome families, induction of liver injury, and inhibition of tox21 pathway proteins. the probabilities of the constituents binding to these proteins and inhibiting their actions (or inducing toxic responses) were reported as follows: probability 0.0–0.1 as no binding, 0.1–0.3 as very low inhibition potential, 0.3–0.5 as low inhibition potential, 0.5–0.7 as medium inhibition potential, 0.7–0.9 high inhibition potential and 0.9–1.0 as having very high inhibition potential; with the very high inhibition potential corresponding to the most profound toxic effect. molecular docking based on the toxicity potential of the extract constituents, the most toxic components were identified via crosstabulation of the scores of their toxicity properties. the identified compounds were docked against cyp1a2, cyp2c19, na+/k+–atpase, and nadh dehydrogenase 1. cytochrome p450 members are important in the metabolism of xenobiotics, their impairment have been reported to have profound toxicological implications in human cells (ogu & maxa, 2000; guengerich, 2022). na+/k+–atpase and nadh dehydrogenase 1 are important for the maintenance of cellular redox balance and mitochondrial membrane potential (pivovarov et al., 2019; herrmann & riemer, 2021). preparation of protein molecules the crystal structures of human cyp1a2 (pdb id: 2hi4), cyp2c19 (pdb id: 4ggs), na+/k+–atpase (pdb id: 7e1z), and nadh dehydrogenase 1 (pdb id: 5xtd) were downloaded from protein data bank (www.rcsb.org). using autodock4 molecular docking software, the protein subunits (polypeptide chains) of cyp2c19 and na+/k+–atpase were separated and treated individually; cyp1a2 comprises a single polypeptide chain. nadh dehydrogenase 1 comprises 44 subunits, the chains were divided into three clusters based on proximity to one another; cluster 1 contains 8 polypeptide chains: a, f, k, l, m, n, o and t; cluster 2 contains 16 polypeptide chains: b, c, e, g, h, i, j, j, m, p, q, s, s, u, w and w; while cluster 3 contains 20 polypeptide chains: a, b, c, d, e, f, g, h, i, k, l, n, o, p, r, v, v, x,y and z. water molecules, other heteroatoms, and non-standard amino acid molecules were deleted from the polypeptide chains. non-polar hydrogen atoms and kollman charges were added to the chains. the ajayi et al. – toxicity prediction of cola rostrata epicarp 371 polypeptide chains were checked for missing atoms, and repairs of missing atoms were done. the atoms of the polypeptide chains were formatted as autodock4 molecules, and the chains were saved in pdbqt format. preparation of ligand molecules using the “draw structure” provision on pubchem platform, the smiles notation of three components of the fractions were used to obtain their mdl molfile which were then converted to mol2 file with discovery studio 2021 software. the structures were converted to their pdbqt format on autodock4 after hydrogen atoms had been added to each compound and the gasteiger charges computed. docking experiments blind docking was done with the grid box coordinates covering the entire atoms of the polypeptide chains, but in few cases, due to the size of the polypeptide chains some atoms were not covered by the grid. the genetic algorithm (ga) parameters include, number of ga runs (30), and population size (300). for each docking experiment, the best pose i.e. the one with the highest binding energy, and the binding interactions between the ligands and the polypeptide chains were visualized on discovery studio 2021 software. results and discussion results chemical constituents of polar and non-polar fractions of c. rostrata epicarp were identified using gc-ms analysis. the inhibitory effects of these components on cyp450 family enzymes were evaluated as presented. gc-ms identification of constituents the constituents of the polar and non-polar fractions of the epicarp of c. rostrata identified via gc-ms and their proportion (area %) in the fractions are presented in table 1. a total of 51 compounds were identified in the fractions, 29 in the non-polar fraction and 22 in the polar fraction, with heptadecanoic acid methyl ester, nheptadecanoic acid and stigmasterol co-partitioning into both fractions. inhibitory effects of components of the extract fractions on cyp450 family members the detoxification of drug compounds in humans is chiefly done by members of the cytochrome p450 (cyp) families. the potential of the components of the polar and non-polar fractions of c. rostrata epicarp extract to bind to and inhibit members of cyp family is presented in table 2. a total of 10 compounds, five from each extract fraction, have very strong potential (probability between 0.9 and 1.0) to inhibit cyp1a2, these 10 compounds are (i) 3-cyclopentylpropionic acid, ethyl ester, (ii) cis-9-tetradecenoic acid, propyl ester, (iii) z,z10,12-hexadecadien-1-ol acetate, (iv) 7,10octadecadienoic acid, methyl ester, and (v) z-8-methyl9-tetradecen-1-ol formate from the non-polar fraction; and (vi) 7-pentadecyne, (vii) silicic acid, diethyl bis(trimethylsilyl) ester, (viii) 4-methoxy-6-methyl-5nitroisobenzofuran-1,3-dione, (ix) 2-hydroxychalcone, and (x) corynan-16-carboxylic acid, 16,17-didehydro9,17-dimethoxy-, methyl ester, (16e)from the polar fraction. these last three compounds listed above (viii, ix and x) also have strong potential to inhibit cyp2c19. 2hydroxychalcone, compound ix in the list above, could also inhibit cyp2c9 and cyp3a4 strongly, while anthiaergostan-5,7,9-trien-14.alpha.,15.alpha.-diol, which is also present in the polar fraction of the extract, has very strong potential to inhibit cyp2d6. table 1. components of the fractions of ethanol extract of cola rostrata epicarp sn non-polar fraction area% polar fraction area% 1 3-cyclopentylpropionic acid, ethyl ester 0.60 hexadecanoic acid, methyl ester 1.48 2 azelaic acid, monoethyl ester 1.20 n-hexadecanoic acid 8.57 3 tetradecanoic acid 0.67 bicyclo[2.2.2]octane, 2-methyl1.55 4 pentadecanoic acid 1.19 2-myristynoyl-glycinamide 1.29 5 hexadecanoic acid, methyl ester 0.80 oleic acid 2.89 6 n-hexadecanoic acid 27.45 methyl 9,12-heptadecadienoate 1.59 7 hexadecanoic acid, ethyl ester 6.59 7-pentadecyne 1.24 8 cis-9-tetradecenoic acid, propyl ester 0.80 2(1h)-naphthalenone, octahydro-4a-methyl-7-(1-methylethyl)-, (4a.alpha.,7.beta.,8a.beta.)6.93 9 heptadecanoic acid 1.69 (3r,4as,8as)-8a-methyl-5-methylene-3-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,8a-octahydronaphthalene 4.96 10 z,z-10,12-hexadecadien-1-ol acetate 0.66 13-tetradece-11-yn-1-ol 5.34 11 octadecanoic acid 7.09 silicic acid, diethyl bis(trimethylsilyl) ester 2.28 12 octadecanoic acid, ethyl ester 2.09 2h-3,9a-methano-1-benzoxepin, octahydro-2,2,5a,9-tetramethyl-, [3r(3.alpha.,5a.alpha.,9.alpha.,9a.alpha.)] 1.56 13 9,12-octadecadienoic acid (z,z)0.76 cyclododecanol, 1-aminomethyl1.38 14 cyclohexene, 4-(4-ethylcyclohexyl)-1-pentyl1.68 2-[1-(3,4-dimethyl-phenyl)-1h-tetrazol-5-ylsulfanyl]-n-phenethyl-acetamide 3.66 15 7,10-octadecadienoic acid, methyl ester 0.59 2-hydroxychalcone 1.76 16 z-8-methyl-9-tetradecen-1-ol formate 0.86 4-methoxy-6-methyl-5-nitroisobenzofuran-1,3-dione 2.55 17 9,12-octadecadienoic acid (z,z)5.32 corynan-16-carboxylic acid, 16,17-didehydro-9,17-dimethoxy-, methyl ester, (16e)13.33 18 ethyl 9.cis.,11.trans.-octadecadienoate 12.09 anthiaergostan-5,7,9-trien-14.alpha.,15.alpha.-diol 8.21 19 naphthalene, decahydro-2-methyl2.28 methanesulfonic acid, 17-cyano-10,13-dimethylhexadecahydrocyclopenta[a]phenanthren-17-yl ester 13.68 20 2-dodecen-1-yl(-)succinic anhydride 3.84 stigmasterol 9.62 21 9-tricosene, (z)0.88 [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid, 4,7-dihydro-7-imino-, ethyl ester 6.11 22 stigmasta-4,22-diene 1.59 23 β-sitosterol 2.69 24 docosanoic acid 2.75 25 3-(2-methoxymethoxyethylidene)-2,2 dimethylbicyclo[2.2.1]heptane 0.79 26 1-(4-methoxyphenylazo)-2-phenoxynaphthalene 0.92 27 γ-sitosterol 6.13 28 stigmasterol 3.68 29 stigmasta-4,22-diene 2.33 b io lo g y, m ed ic in e, & n atu ral pro d u c t c h em istry 1 4 (1 ), 2 0 2 5 : 369 -379 372 ajayi et al. – toxicity prediction of cola rostrata epicarp 373 table 2. numbers of cola rostrata extract components with various degrees of inhibitory binging to members of cytochrome p450 protein family. cyp family no binding very low low medium high very high np p np p np p np p np p np p cyp1a2 6 3 11 4 0 1 3 6 3 2 5 5 cyp2c19 7 3 9 9 5 1 5 3 2 2 0 3 cyp2c9 6 1 13 12 5 3 3 3 1 1 0 1 cyp2d6 17 13 8 4 1 2 2 0 0 1 0 1 cyp2a4 10 4 4 7 8 4 4 2 2 3 0 1 cyp: cytochrome p450; np: non-polar fraction; p: polar fraction. probability of inhibition: no binding (0–10%); very low (10–30%); low (30–50%); medium (50–70%); high (70–90%) and very high (90–100%). toxicity of the components of c. rostrata extract fractions the predicted toxic effects of the components of the polar and non-polar components of c. rostrata epicarp is mainly drug-induced liver injury, followed by hepatotoxicity and ability to block the herg channels (table 3). two compounds, one in each of the fractions, have 90 % to 100 % likelihood of causing liver damage, and these compounds are 2-hydroxychalcone and 1-(4methoxyphenylazo)-2-phenoxynaphthalene, in the polar and non-polar fractions, respectively. silicic acid, diethyl bis(trimethylsilyl) ester which is present in the polar fraction, also has 70 % to 90 % likelihood of causing injury to the liver. in the non-polar fraction, ethyl 9.cis.,11.trans.-octadecadienoate and β-sitosterol have high likelihood of blocking herg channels. hepatotoxicity could be caused by two compounds in the polar fraction: methanesulfonic acid, 17-cyano-10,13dimethylhexadecahydrocyclopenta[a]phenanthren-17-yl ester and [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid, 4,7-dihydro-7-imino-, ethyl ester. other predicted negative effects of the components include the likelihood of 3-(2-methoxymethoxyethylidene)-2,2 dimethylbicyclo[2.2.1]heptane and corynan-16carboxylic acid, 16,17-didehydro-9,17-dimethoxy-, methyl ester, (16e)-inducing carcinogenesis, and 1-(4methoxyphenylazo)-2-phenoxynaphthalene with a very high potential to induce mutation. table 3. numbers of cola rostrata extract components with various degrees of end-point toxicities. end-point toxicities nontoxic very low low medium high very high np p np p np p np p np p np p herg blockers 18 13 6 7 1 0 1 0 2 0 0 1 hepatotoxic 20 9 7 8 1 2 0 0 0 2 0 0 dili 17 11 6 3 2 2 2 2 0 2 1 1 mutagenicity 27 17 0 1 0 0 0 2 0 1 1 0 roat 26 14 2 2 0 2 0 1 0 1 0 1 fdamdd 18 9 1 6 4 1 4 1 1 1 0 3 carcinogenicity 14 10 9 3 2 0 1 5 1 2 1 1 herg: human ether-a-go-go-related gene; dili: drug-induced liver injury; roat: rat oral acute toxicity; fdamdd: federal drug administration maximum (recommended) daily dose; np: non-polar extract fraction; and p: polar extract fraction. probability of toxicity: nontoxic (0–10%); very low (10–30%); low (30–50%); medium (50–70%); high (70–90%) and very high (90–100%). the predicted binding (activatory or inhibitory) effect of the components of the extract fractions to proteins identified by the tox21 program as significant predictors of toxicity is shown in table 4. a total of 11 compounds in both extract fractions have from high to very high potential to bind to the nuclear receptor protein, peroxisome proliferator-activated receptor gamma (nr-ppar-gamma). out of these 11 compounds, eight are in the non-polar fraction, they are fatty acids and fatty acid derivatives, and they make up about 43% proportion by mass in the extract fraction. 2-hydroxychalcone is the only non-fatty acid component of the polar extract fraction that could bind strongly to nr-ppar-gamma. from the two extract fractions, a total of 10 compounds could strongly induce the stress response pathway leading to the disruption of mitochondrial membrane potential (sr-mmp). 2-hydroxychalcone, anthiaergostan-5,7,9-trien-14.alpha.,15.alpha.-diol, methanesulfonic acid, 17-cyano-10,13dimethylhexadecahydrocyclopenta[a]phenanthren-17-yl ester, and stigmasterol are compounds that could disrupt mitochondrial membrane potential in the polar fraction, and in the non-polar fraction of the extract, the same effect could be induced by 1-(4-methoxyphenylazo)-2phenoxynaphthalene, naphthalene, decahydro-2-methyl-, 2-dodecen-1-yl(-)succinic anhydride, γ-sitosterol, stigmasterol, and stigmasta-4,22-diene. 374 biology, medicine, & natural product chemistry 14 (1), 2025: 369-379 table 4. numbers of cola rostrata extract components with inhibitory binding potential against tox21 proteins. no binding very low low medium high very high np p np p np p np p np p np p nr-ar 14 17 4 0 8 3 1 0 1 1 0 0 nr-ar-lbd 28 20 0 0 0 0 0 1 0 0 0 0 nr-ahr 26 16 0 1 0 1 1 1 0 0 1 2 nr-aromatase 18 12 6 4 1 2 3 1 0 1 0 1 nr-er 3 7 12 8 12 4 0 1 0 0 1 1 nr-er-lbd 19 12 1 3 3 1 1 2 2 2 2 1 nr-ppar-gamma 14 12 4 3 2 2 0 1 0 1 8 2 sr-are 11 10 10 4 5 5 1 1 0 0 1 1 sr-atad5 26 20 1 0 0 0 0 0 0 1 1 0 sr-hse 8 9 11 4 3 5 3 1 1 1 2 1 sr-mmp 6 4 11 6 5 3 0 4 4 1 2 3 sr-p53 25 16 1 1 0 2 0 1 0 0 2 1 estrogen receptor-ligand-binding domain (nr-er-lbd peroxisome proliferator-activated receptor gamma (nr-ppar-gamma) mitochondrial membrane potential (sr-mmp) tumor suppressor protein (sr-p53), heat shock factor response element (sr-hse), antioxidant response element (sr-are), estrogen receptor (nr-er), aryl hydrocarbon receptor (nr-ahr) and atpase family aaa domain-containing protein 5 (sr-atad5). a total of seven compounds (mostly those with the gonane nucleus) have high to very high probability of binding to estrogen receptor-ligand-binding domain (nr-er-lbd) and activating series of reactions that result in endocrine disruption. in the non-polar fraction, γ-sitosterol, stigmasterol and stigmasta-4,22-diene have strong potential to bind to the ligand-binding domain of estrogen receptor; in the polar fraction of the extract, the same potential is seen in 2-hydroxychalcone, anthiaergostan-5,7,9-trien-14.alpha.,15.alpha.-diol and stigmasterol. among all the compounds identified in the two extract fractions, 2-hydroxychalcone is the most toxic component of the epicarp extract of c. rostrata since it could cause liver damage, disrupt mitochondrial membrane potential, modulate sugar metabolism through nr-ppar-gamma activation, and disrupt endocrine function through its effect on the estrogen receptor. in addition to 2-hydroxychalcone’s inhibitory effects on members of the cytochrome p450 family, it could also bind strongly to and modulate the functions of tumor suppressor protein (sr-p53), heat shock factor response element (sr-hse), antioxidant response element (srare), estrogen receptor (nr-er), aryl hydrocarbon receptor (nr-ahr) and atpase family aaa domaincontaining protein 5 (sr-atad5). next to 2hydroxychalcone is 1-(4-methoxyphenylazo)-2phenoxynaphthalene which also has strong potential to induce liver damage and mutation, and disrupt mitochondrial membrane potential. 1-(4methoxyphenylazo)-2-phenoxynaphthalene could also predict to bind strongly to and modulate the functions of sr-are, nr-ahr, nr-er, sr-atd5, and members of the cyp450 family, while also disrupting mitochondrial membrane potential. some of the compounds present in the extract that have been predicted to cause toxic outcomes are presented in figure 1. analysis of binding energies the docking experiment revealed that components of the fractions of c. rostrata epicarp extracts, 2hydroxychalcone, 1-(4-methoxyphenylazo)-2phenoxynaphthalene, and anthiaergostan-5,7,9-trien14.alpha.,15.alpha.-diol, are potent inhibitors of cyp2c19 and cyp1a2; the three compounds showed strong binding affinity for the four subunits of cyp2c19, having average free energy of -7.21, -9.65 and -8.62 kcal/mol, respectively. table 4 shows the binding energies of selected components of the extract fractions with na+/k+-atpase, nadh dehydrogenase and two members of the cyp450 family. out of the three components of the extract used in the molecular docking experiment, 1-(4-methoxyphenylazo)-2phenoxynaphthalene showed the strongest binding affinity for cyp2c19 and nadh dehydrogenase with the compound binding strongly to each of the four subunits of cyp2c19. 2-hydroxychalcone has the strongest affinity for na+/k+-atpase, binding to the b subunit with a free energy of –7.49 kcal/mol. ajayi et al. – toxicity prediction of cola rostrata epicarp 375 figure 1. some components of ethanol extract of c. rostrata epicarp predicted to have toxic effects. (a) 2-hydroxychalcone, (b) 1-(4-methoxyphenylazo)2-phenoxynaphthalene, (c) anthiaergostan-5,7,9-trien-14.alpha.,15.alpha.-diol, (d) 4-methoxy-6-methyl-5-nitroisobenzofuran-1,3-dione, (e) z-8-methyl-9tetradecen-1-ol formate, (f) corynan-16-carboxylic acid, 16,17-didehydro-9,17-dimethoxy-, methyl ester, (16e)-, (g) β-sitosterol, (h) methanesulfonic acid, 17-cyano-10,13-dimethylhexadecahydrocyclopenta[a]phenanthren-17-yl ester, and (i) [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid, 4,7dihydro-7-imino-, ethyl ester. examination of the binding interactions showed that the most predominant interaction of 1-(4methoxyphenylazo)-2-phenoxynaphthalene with the polypeptide chain d of cyp2c19 are pi-alkyl bonds with the interaction with conventional hydrogen bond having the shortest bond length of 2.93 å (figure 2 c). other interactions with the polypeptide chain include van der waals forces, and alkyl, pi-pi t-shaped, pi-sulfur, pisigma, and carbon-hydrogen bonds. the binding interaction of 1-(4-methoxyphenylazo)-2phenoxynaphthalene with cyp2c19 shows that the compound binds to residues: thr301, leu361 and leu366 at the active site of cyp2c19, with the free energy of -9.93 kcal/mol. also, the binding interactions of 1-(4-methoxyphenylazo)-2-phenoxynaphthalene to nadh dehydrogenase 1 has pi-alkyl bonds as the predominant interactions, nine bonds with residues on three subunits (r, i and v chains) in cluster 3 of the nadh dehydrogenase 1 structure (figure 2 a). the shortest bond distance (1.88 å) and the strongest bond seen in the interactions is the conventional hydrogen bonding between lone pairs of electrons on the hydroxyl group of 1-(4-methoxyphenylazo)-2phenoxynaphthalene and glu v:134. other bonds in the binding interaction include pi-pi stacked, pi-pi t-stacked and pi-donor hydrogen bonds. this binding of 1-(4methoxyphenylazo)-2-phenoxynaphthalene to cluster 3 chains has the lowest free energy (-7.78 kcal/mol) of the three compounds docked with the three chain clusters of nadh dehydrogenase 1 protein. the interaction of 1-(4methoxyphenylazo)-2-phenoxynaphthalene with nadh dehydrogenase (a critical enzyme in the mitochondrial electron transport chain that plays a central role in cellular energy production through oxidative phosphorylation) suggests the potential modulation of atp production. table 5. binding free energy (kcal/mol) of three components of cola rostrata extract to selected cellular proteins. components of cola rostrata epicarp extract na+k+-atpase nadh dehydrogenase 1 cyp2c19 cyp1a2 a b c cluster 1 cluster 2 cluster 3 a b c d 2-hydroxychalcone -5.41 -7.49 -4.97 -5.48 -6.82 -5.77 -8.07 -7.11 -6.89 -6.77 -7.31 1-(4-methoxyphenylazo)-2phenoxynaphthalene -5.76 -5.58 -5.13 -6.86 -6.46 -7.78 -9.68 -9.52 -9.49 -9.93 -7.51 anthiaergostan-5,7,9-trien14.alpha.,15.alpha.-diol -6.00 -5.17 -5.25 -6.54 -7.09 -7.17 -9.56 -8.16 -7.91 -8.83 -8.71 cluster 1 contains polypeptide chains: a, f, k, l, m, n, o and t; cluster 2 contains polypeptide chains: b, c, e, g, h, i, j, j, m, p, q, s, s, u, w and w; cluster 3 contains polypeptide chains: a, b, c, d, e, f, g, h, i, k, l, n, o, p, r, v, v, x,y and z. a, b, c and d: subunits of the indicated proteins. 376 biology, medicine, & natural product chemistry 14 (1), 2025: 369-379 for the binding interactions of 2-hydroxychalcone, its binding to chain b of na+/k+-atpase has the lowest free energy (-7.74 kcal/mol) of the dockings of the three compounds to the chains of na+/k+-atpase (table 5). the binding interaction of hydroxychalcone to chain b involves two conventional hydrogen bonds linking met b:202 with bond distances of 1.90 å and 2.25 å, and pi bonds: pi-alkyl, pi-anion, pi-sulfur and pi-pi t-shaped; the pi-sulfur bond is also linking met b:202 (figure 2 d). the binding of anthiaergostan-5,7,9-trien14.alpha.,15.alpha.-diol to cyp1a2 produced the best free energy value (-8.71 kcal/mol) of the three compounds docked with the protein (table 5). the binding of anthiaergostan-5,7,9-trien14.alpha.,15.alpha.-diol to cyp1a2 involves alkyl bonding (9 bonds) with hydrophobic aliphatic side chains of leu and ile residues; with the bond with the shortest distance (3.46 å) being the alkyl bond formed with cys 458 (figure 2 b). other bond interactions in the binding of anthiaergostan-5,7,9-trien-14.alpha.,15.alpha.-diol to cyp1a include, pi-sigma, amide-pi stacked and pidonor hydrogen bonds. figure 2. 2d images of the molecular binding interactions of selected components of cola rostrata extract with selected cellular proteins. (a) binding interactions of 1-(4-methoxyphenylazo)-2-phenoxynaphthalene with amino acid residues in 20 subunits (a, b, c, d, e, f, g, h, i, k, l, n, o, p, r, v, v, x,y and z) of nadh dehydrogenase 1 (b) binding interactions of anthiaergostan-5,7,9-trien-14.alpha.,15.alpha.-diol to cyp1a (c) binding interactions of 1-(4methoxyphenylazo)-2-phenoxynaphthalene to chain d of cyp2c19 (d) binding interactions of 2-hydroxychalcone to chain b of na+/k+-atpase. ajayi et al. – toxicity prediction of cola rostrata epicarp 377 discussion the ethanol extract of the fruit epicarp of cola rostrata has been found to possess strong anticancer activity with potential for usage against inflammation, pain and diabetes; the toxicity potential of this extract has not been evaluated. adverse side effects have been identified as one of the barriers to increased usage of herbal preparations (awodele et al., 2018; vaezi et al., 2021). though adverse reactions are associated with several herbal preparations, conventional orthodox drugs like carbamazepine, diclofenac and most antibiotics, including tetracycline, azithromycin andmoxifloxacin, have been identified as major causes of liver damage and adverse side effects (chalasani et al., 2015; mejia et al., 2020; osuntokun et al., 2020). the high potential for adverse reactions with the use of herbal preparations is attributable to the huge diversity and the numerical enormity of phytochemicals that are consumed at the same time, and in most cases, in unstandardized quantities. with 10 components of the extract of c. rostrata epicarp possessing the ability to induce oxidative stress and initiate processes leading to loss of mitochondrial membrane potential and massive apoptotic cell death; red blood cells and brain cells which require a high level of oxygen could be sites for adverse reactions after the extract is consumed. gc-ms and toxicity analyses the gc-ms analysis of components of ethanol extract of c. rostrata epicarp identified 48 different phytochemicals. this value represents the components of the extract that are already known, but possible novel components are not accounted for, and the possibility of having these unidentified components contributing significantly to toxic outcomes cannot be ascertained. the high potential of some of the identified components of the extract to cause liver damage relates well with the reported potential of the extract for the management of pain and inflammation as many non-steroidal antiinflammatory drugs have also been reported to cause damage to the liver. though the inhibition of members of cyp450 enzyme family has been identified as having therapeutic potential in some disease conditions, largely, their inhibition has been associated with dangerously toxic outcomes (alaei et al., 2023). a flavonoid component of the extract, 2hydroxychalcone, which binds strongly to members of cyp450 family, has been found to inflict damage to hepatocytes at concentrations above 20 µm through the generation of oxidative stress and down-regulation of the expression of antioxidant proteins in hepg2 cells (qian et al., 2019). 2-hydroxychalcone which also interferes with nr-ppar-gamma activity has been reported to reduce hyperglycemia through the nr-ppar-gamma pathway in diabetic rats (eissa et al., 2017). the strong binding of 2-hydroxychalcone to subunit b of na+/k+atpase, which involves hydrophobic interactions and conventional strong hydrogen bonding, shows that the compound could be a strong inhibitor of na+/k+-atpase. na+/k+-atpase inhibitors, like cardiac glycosides (e.g., digoxin), are clinically used to enhance cardiac contractility as they regulate ion balance across the plasma membrane especially in neurons and muscle cells (ren et al, 2024). 2-hydroxychalcone could alter ion transport, which has implications for conditions such as hypertension, heart failure, or neuronal excitability, thereby offering potential as a cardioprotective agent. the toxic effect of 1-(4-methoxyphenylazo)-2phenoxynaphthalene which is shown here to bind strongly to members of cyp450 family and also to subunits in nadh dehydrogenase 1 has not been reported in the literature. the inhibition of mitochondrial nadh dehydrogenase 1 has been reported to induce significant reactive oxygen species generation which have toxic outcomes in the liver and the brain (murali & shivanandappa, 2022). the inhibition of nadh dehydrogenase 1 in the mitochondria by 1-(4methoxyphenylazo)-2-phenoxynaphthalene may be one of the significant contributors to the cytotoxicity of c. rostrata epicarp extract against hela and mrc5-sv2 cells. this also suggests that cola rostrata extract might be able to modulate energy metabolism, potentially leading to therapeutic applications in cancer cells, or side effects if inhibition of this enzyme occurs in healthy cells. the inhibition of cyp2c19 by 1-(4methoxyphenylazo)-2-phenoxynaphthalene may invariably result in altered drug metabolism, potentially affecting the pharmacokinetics of drugs metabolized by this enzyme. the resulting modulation might be harnessed for controlling drug metabolism in individuals with cyp2c19-specific health issues. the pharmacological activity of anthiaergostan5,7,9-trien-14.alpha.,15.alpha.-diol, which is one of these 10 components, in living systems has not been fully elucidated. here, the molecular docking experiments revealed that the compound binds tightly to members of the cyp450 family and also to nadh dehydrogenase 1. the short hydrogen bond lengths (2.5–2.9 å) indicate strong binding affinity, while hydrophobic interactions stabilize the ligands within enzyme pockets. the molecular interactions of cola rostrata components with enzymes such as nadh dehydrogenase, cyp1a1, cyp2c19, and na+/k+-atpase demonstrate their potential to modulate key biological pathways which could result in toxic outcomes. the antioxidant effects of phytosterol constituents could counter toxicity the toxic effects of the identified components of c. rostrata epicarp extract could be ameliorated by the rich presence of phytosterols like stigmasterol, β-sitosterol, γ-sitosterol and stigmasta-4,22-diene in the extract. these phytosterols have been shown to possess strong oxidative stress-combating activities. koc et al (2021) reported that β-sitosterol showed anti-inflammatory and anti-oxidative stress activities in rats by increasing the 378 biology, medicine, & natural product chemistry 14 (1), 2025: 369-379 activities of glutathione and the antioxidant enzyme, superoxide dismutase, while it also downregulated the levels of inflammatory proteins, tumor necrosis factoralpha and interleukin-6. the analgesic and antiinflammatory activity of stigmasterol was reported by kariuki et al (2012), and the strong antioxidant potential of stigmasta-4,22-dien-3-one, a closely related compound to stigmasta-4,22-diene, was reported by sahidin et al (2014). the overall toxicity of the ethanol extract of c. rostrata epicarp could be reduced by the presence of these steroids. conclusion this study provides analyses of the pharmacological and toxicological profiles of components of the ethanol extract from the epicarp of cola rostrata fruit. the extract contains 48 identified phytochemicals, including 1-(4-methoxyphenylazo)-2-phenoxynaphthalene, anthiaergostan-5,7,9-trien-14.alpha,15.alpha.-diol, and 2-hydroxychalcone, which were evaluated for their metabolism, excretion and toxicity properties using insilico techniques and molecular docking. the binding of these compounds to key metabolic proteins suggests potential effects on ion transport and liver function. however, the predicted high potential for adverse reactions, including herb-drug interactions and mitochondrial toxicity, necessitates further in-vitro and in-vivo studies to validate these findings and ensure the safe use of cola rostrata in traditional medicine and drug formulations. authors’ contribution: abe: 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(2024) the use of herbal medicines among cancer patients. cureus 16(2): e53455. https://doi.org/10.7759/cureus.53455 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 457-461 | doi: 10.14421/biomedich.2023.122.457-461 issn 2540-9328 (online) proximate and mineral composition of atlantic mackerel (scomber scombrus) and atlantic horse mackerel (trachurus trachurus) joseph adaviruku sanni1,*, grace omayoza sanni2, rufus ranmilowo awoniyi2, remi osanyinlusi2, yvonne ego richards3 1department of biochemistry, faculty of science, adekunle ajasin university akungba-akoko, ondo state nigeria. 2science laboratory technology, faculty of applied science, rufus giwa polytechnic, owo, ondo state nigeria. 3department of crop science, faculty of agriculture, ladoke akintola university of technology, ogbomoso oyo state, nigeria. corresponding author* josephsanni59@gmail.com abstract atlantic mackerel (scomber scombrus) and atlantic horse mackerel (trachurus trachurus), locally known as kote, are fishery species consumed in nigeria due to their high nutritional values. this research determined the nutritional composition of the local dried fish, scomber scombrus and trachurus trachurus. results for scomber scombrus shows the mean value of the moisture, ash, crude fat, crude fiber, and crude protein contents in percent (%) as: 5.26±0.00, 5.20±0.10, 35.60±0.00, 2.90±0.10 and 46.30±0.01 respectively. for trachurus trachurus, the moisture, ash, crude fat, crude fiber, and crude protein contents in percent (%) were 9.52±0.00, 32.26±0.01, 24.14±0.00, 11.91±0.85 and 40.95±0.00 respectively. the mineral composition of scomber scombrus was as follows: sodium (na) had the highest mineral composition with 78.90 mg/100g, followed by magnesium (mg) with 15.90 mg/100g, manganese (mn) with 0.86 mg/100g, zinc (zn) with 0.282 mg/100g, and iron (fe) with 0.10 mg/100g. trachurus trachurus has magnesium (mg) at 27.00 mg/100g as its highest mineral composition, followed by sodium (na) at 22.50 mg/100g, zinc (zn), iron (fe) at 0.17 mg/100g, and manganese (mn) at 0.09 mg/100g. it is concluded from the study, that scomber scombrus and trachurus trachurus are good sources of essential nitrates, fat, proteins containing essential amino acids, and other micronutrients that are beneficial to human health. keywords: proximate composition; mineral content; scomber scombrus; trachurus trachurus. introduction fish is an essential food consumed in developing countries like nigeria because of its abundance and nutritional values, like its high protein, unsaturated fatty acids, carbohydrate, and mineral contents. it is cheap and available in urban and rural areas (bene and heck, 2005). it is acceptable worldwide because of its high nutritional value and low cholesterol content when compared with meat and is thus often recommended for consumption, especially among the adult population (eyo, 2001). several studies have recorded the importance of including fish in diets. fish contains essential nutrients, especially protein, vitamins, and minerals, which enhance the management of cardiovascular and other related diseases (damsgaard et al., 2006). according to (damsgaard, 2006), fish contains eicosapentaenoic and docosahexaenoic acids, long-chain n-3 polyunsaturated fatty acids that supply essential nutrients and promote good health. atlantic mackerel (scomber scombrus) and atlantic horse mackerel (trachurus trachurus), locally known as kote are the most predominant fishery species in various markets and are well consumed among the nigerian populace. trachurus trachurus originates from portugal and is consumed widely because of its availability and nutritional value (nadeisa et al., 2001). scomber scombrus is known for its high protein and fat content (ackman, 1990). proximate analysis or nutritional evaluation of fish indicates the percentage composition of essential nutrients or constituents such as proteins, crude fats, and other minerals in fish products. the chemical constituents are naturally used as a pointer to the nutritional value found in fish (moghaddam et al., 2007; aberoumand, 2011). fish generally differ from species to species. the differences can result from seasonal variation, feeding habits, or sex (islam et al., 2005). this study evaluates two samples of commercially available fish species obtained from a market in southwestern nigeria. therefore, this work determines the proximate and mineral compositions of scomber scombrus and trachurus trachurus to identify similarities and differences in their nutritional content. manuscript received: 14 may, 2023. revision accepted: 10 august, 2023. published: 15 august, 2023. https://doi.org/10.14421/biomedich.2023.122.457-461 458 biology, medicine, & natural product chemistry 12 (2), 2023: 457-461 materials and method materials powdered scomber scombrus and trachurus trachurus, hcl, h2so4, naoh, weighing balance, filter paper, heating mantle, crucible, thread, beaker, conical flask, distilled water, reagent bottle, chloroform, water bath, acetic acid, and pipette. figure 1. pictorial representation of scomber scombrus. figure 2. pictorial representation of trachurus trachurus. method sample preparation the materials were obtained from oja oba, owo local government area of ondo state. the fish was cut into small pieces, sundried for a month, and placed in an oven for further drying. the dried sample was crushed into powder using an el-850w blender and packed into a container. proximate analysis fat content determination a clean fat-free filter paper was weighed (w1). 5g of the sample was added to the filter and weighed (w2). the weighed sample was tied with a piece of thread and dropped into the thimble of the soxhlet apparatus. 250 ml of petroleum ether was poured into the round bottom flask of the apparatus. soxhlet was set up on the heating mantle, and the extraction process was done for four hours to extract the fat with the help of the solvent. petroleum ether was siphoned over the barrel, the condenser was detached, and the thimble was removed. the solvent extract (lipid) mixture was carefully poured into a clean, dried petri dish and transferred into a fume cupboard for two hours. the solvent evaporated, leaving behind the extracted fat. the filter paper containing the residue was dropped into a beaker and transferred into an oven at 50°c. it was then dried to a constant weight and cooled in a desiccator and reweighed (w3). the percentage of fat was calculated. % 𝐹𝑎𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊2 − 𝑊3 𝑊2 − 𝑊1 × 100 moisture content determination the moisture content was determined using a drying method based on weight loss. a clean and dry crucible was weighed using a weighing balance, and its weight was recorded (w1). samples were added to the empty crucible, and their weight was recorded (w2). the crucible containing the sample was transferred into the oven, maintained at 105°c, and dried for four hours. the dish was placed in a desiccator, cooled for one hour, and reweighed (w3). the percentage moisture content was calculated. 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊2 − 𝑊3 𝑊2 − 𝑊1 × 100 ash content an ash-free crucible was weighed, and the weight was recorded (w1). 2g of sample was weighed into the crucible (w2) and transferred into the muffle furnace. the muffle furnace was then ignited at 600 °c for about four hours until a grayish-white substance was obtained. the crucible was transferred into a desiccator, cooled, and reweighed (w3). the percentage ash content was calculated. % 𝑎𝑠ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊2 − 𝑊3 𝑊3 − 𝑊1 × 100 crude-fiber content determination 5g of the defatted sample was weighed (w1) into a 2500 ml conical flask. 200 ml of 1.25% h2so4 was added. the mixture was heated for 30 minutes, then cooled and filtered through poplin cloth by suction using a bunchier funnel. the residue was rinsed in hot, distilled water and scraped back into a flask. 200 ml of 1.25% naoh was added, and the mixture was heated for 30 minutes. it was cooled, filtered, and washed once with hot, distilled water, once with 10% hcl, four times with hot water, and twice with methylated spirit. the residue was drained, placed in a crucible, and dried in an oven at 105ºc. after drying in an oven, it was then cooled in a desiccator and weighed (w2). the crucible containing the residue was placed in a muffle furnace at 300°c for 30 minutes, placed in a desiccator to cool to room temperature, and weighed (w3). % 𝑐𝑟𝑢𝑑𝑒 𝑓𝑖𝑏𝑒𝑟 = 𝑊2 − 𝑊3 𝑊1 × 100 determination of protein content 2g of sample was weighed into a 50-ml kjeldahl flask, and 12.5 ml of concentrated h2so4 was added with one kjeldahl catalyst tablet. the flask was heated on low heat sanni et al. – proximate and mineral composition of atlantic mackerel … 459 for about 15 minutes, on medium heat for 30 minutes, and then on high heat until digested. the flask was rotated at intervals until the digest was clear, and the heating continued for a few minutes to ensure complete digestion. the flask was allowed to cool, and the sample residue was washed and filtered to make the digest up to 50 ml (v1). after the digestion was completed, 5 ml of 2% boric acid (h3bo3) was placed into a 100-ml conical flask, and 3 drops of the mixed indicator were added. the receiving flask was placed so that the tip of the condenser tube was below the surface of the boric acid. 5 ml of the digest (v2) was pipetted into the distillation tube, and 10 ml of 40% naoh was added. the heater was turned on, and the distillation continued until approximately 50 ml of distillate was collected into the receiving flask. the distillate was titrated with 0.01m hcl, and the blank was titrated with the acid. %𝑁 = 𝑀 × 𝑇 × 0.014 𝑊 × 𝑉1 𝑉2 × 100 % 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = % 𝑁 𝑥 6.25 results and discussion the result of proximate analysis of the nutritional composition of scomber scombrus and trachurus trachurus showed the presence of moisture, ash, crude fat, crude fiber, and crude protein in the following proportions, respectively: the result of the mineral analysis of the nutritional composition of scomber scombrus and trachurus trachurus showed the presence of sodium (na), zinc (zn), magnesium (mg), iron (fe), and manganese (mn), respectively: proximate composition table 1. shows the type and quantity of the nutritional composition of scomber scombrus and trachurus trachurus. s/n parameters measured scomber scombrus trachurus trachurus 1 moisture content (%) 5.26±0.00 9.52±0.00 2 ash content (%) 5.20±0.10 32.26±0.01 3 crude fat (%) 35.60±0.00 24.14±0.00 4 crude fibre (%) 2.90±0.10 11.91±0.85 5 crude protein (%) 46.30±0.01 40.95±0.00 table 2. showing the result of metal (mg/100g) analysis of scomber scombrus and trachurus trachurus. s/n parameters measured scomber scombrus trachurus trachurus 1 sodium(mg/100g) 78.90±0.02 22.50±0.01 2 zinc(mg/100g) 0.28±0.00 0.17±0.00 3 magnesium(mg/100g) 15.90±0.01 27.00±0.02 4 iron(mg/100g) 0.10±0.00 0.17±0.00 5 manganese(mg/100g) 0.09±0.00 0.09±0.00 discussion the proximate composition of both scomber scombrus and trachurus trachurus are in table 1. proteins and fats are known to be the major nutrients found in fish. considering the various species, there are variations depending on age, sex, and environment. the variations help to determine their nutritional status (aberoumad and pourshafi, 2010). the mean value of crude protein obtained for scomber scombrus was 46.29%. this was slightly higher than the value obtained from trachurus trachurus, which was 40.95%. this value was found to be lower when compared to 57.80% obtained from scomber scombrus reported when the sample was smoked with an electric oven (aremu et al., 2014); lower compared with the 70.24% for melon husk heat treatment reported (aremu et al., 2014); 62.14% for clarias gariepinus as observed by kumolu and ndimele, 2010; and higher than 26.25% reported for scomber scombrus (agu and bhandary, 2004). the value of the crude fat of scomber scombrus was observed to be 35.60%, while that of trachurus trachurus was recorded to be 24.14%. the mean value of crude fat for both scomber scombrus and trachurus trachurus was higher than the range of crude fat for scomber scombrus, ranging between 7.41 and 17.51% using different heat sources, as reported by (aremu et al., 2014). a moisture content of 5.26% was observed in scomber scombrus, while 9.52% was recorded for trachurus trachurus. the result shows that the moisture content found in trachurus trachurus was higher than that obtained in scomber scombrus. (olusola et al., 2011) reported the moisture content of scomber scombrus at 56.5% and roughear scad at 66.7%, which was seen to have a higher mean value. this can be a result of the type of the environment in which the 460 biology, medicine, & natural product chemistry 12 (2), 2023: 457-461 samples were collected or the season in which the fish was collected. the 5.26% of scomber scombrus is almost the same as the 5.28% reported by aremu et al. (2014) in scomber scombrus dried using an electric oven. another study reported by adeyi et al. (2010) gives a moisture content of 5.43% when coconut husk was used as a heat treatment. the result is almost similar, though it has about a 0.17% difference, which may imply that the heat generated from an electric oven dries the fish better and is stronger than the heat generated from the husk of a coconut. the ash content of scomber scombrus is 5.20%, and that of trachurus trachurus is 32.26%; this shows that trachurus trachurus has a higher ash content compared to scomber scombrus. the 5.20% of scomber scombrus obtained using an electric oven was compared to the ash content of 5.70% obtained for heat treatment using sawdust by (aremu et al., 2014) and 6.01% reported for mackerel fish (agu and bhandary, 2004). the mineral composition indicates certain organic matter that is not denatured by high heat intensity and is known for its low volatility compared to other food classes (remi, 2023). the mineral composition in mg/100g of scomber scombrus and trachurus trachurus is shown in table 2. from the result for scomber scombrus, sodium (na) was shown to be more abundant in the dried fish with 78.90 mg/100g, followed by magnesium (mg) with 15.90 mg/100g, manganese (mn) with 0.86 mg/100g, zinc (zn) with 0.282 mg/100 g, and iron (fe) with 0.10 mg/100g. for trachurus trachurus, magnesium is more abundant at 27.00 mg/100g, followed by sodium at 15.90 mg/100g. this value is higher than the 7.43 mg/100g reported by (aremu, 2014). magnesium is an important element in connection with the treatment of diseases of the circulatory system, such as ischaemic heart disease, and calcium metabolism in bones (ishida et al., 2000). iron (fe), zinc (zn), and manganese (mn) were low, albeit available for biological functions. conclusion it is concluded from this study that scomber scombrus and trachurus trachurus are high in fat, proteins, and some essential micronutrients, which are good sources of nutrients for the growth of the body. scomber scombrus was reported to have high protein and fat content, which implies that it would be a good source of protein for growing children and adults. authors’ contributions: rra, gos, and jas conceptualized the study, carried out the research, and wrote the manuscript draft. ro, gos, jas, and yer contributed to the study design and manuscript editing. all authors read and approved the final manuscript. competing interests: the authors declared that they have no competing interests. funding: not applicable. availability of data and materials: not applicable. ethics approval and consent to participate: not applicable. consent for publication: not applicable. references aberoumand, a. (2012). proximate composition of less known some processed and fresh fish species for determination of the nutritive values in iran. journal of agricultural technology, 8(3), 917-922. adeyi, o. (2010). proximate composition of some agricultural wastes in nigeria and their potential use in activated carbon production. journal of applied sciences and environmental management, 14(1). https://doi.org/10.4314/jasem.v14i1.56490 agu, h. o., & bhandary, c. s. (2005). effect of smoking time on keeping quality of smoked mackerel (scomboromorus scombrus). nigerian food journal, 23(1), 114-120. https://doi: 10.4314/nifoj.v23i1.33606 aremu, m. o., namo, b. s., oko, o. j., adelagun, r. o. a., & yebpella, g. g. (2014). compositional evaluation of local smoked nigerian mackerel (scomber scombrus). food science and quality management, 24, 42-50. aremu, m. o., olonisakin, a., otene, i. w., & atolaye, b. o. (2005). mineral content of some agricultural products grown in the middle belt of nigeria. oriental journal of chemistry, 21(3), 419. association of official analytical chemists-aoac (1984). official methods of analysis of the association of official analytical chemists. 14. ed. arlington, 1141. bandarra, n. m., batista, i., nunes, m. l., & empis, j. m. (2001). seasonal variation in the chemical composition of horsemackerel (trachurus trachurus). european food research and technology, 212, 535-539. https://doi.org/10.1007/s002170100299 béné, c., & heck, s. (2005). fish and food security in africa. http://hdl.handle.net/1834/25699 damsgaard, c. t., schack-nielsen, l., michaelsen, k. f., fruekilde, m. b., hels, o., & lauritzen, l. (2006). fish oil affects blood pressure and the plasma lipid profile in healthy danish infants. the journal of nutrition, 136(1), 94-99. https://doi.org/10.1093/jn/136.1.94 eyo, a. a. (2001). fish processing technology in the tropics, national instit. fresh water fish. res.(fifr) new bussa nigeria, 66-130. ishida, h., suzuno, h., sugiyama, n., innami, s., tadokoro, t., & maekawa, a. (2000). nutritive evaluation on chemical components of leaves, stalks and stems of sweet potatoes (ipomoea batatas poir). food chemistry, 68(3), 359367. https://doi.org/10.1016/s0308-8146(99)00206-x islam, m. s., & tanaka, m. (2004). impacts of pollution on coastal and marine ecosystems including coastal and marine fisheries and approach for management: a review and synthesis. marine pollution bulletin, 48(7-8), 624-649. https://doi.org/10.1016/j.marpolbul.2003.12.004 sanni et al. – proximate and mineral composition of atlantic mackerel … 461 kumolu-johnson, c. a., aladetohun, n. f., & ndimele, p. e. 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(2023). nutritional composition of processed and unprocessed samples of unripe plantain (musa paradisiaca). journal of advanced education and sciences, 3(1), 75-81. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 559-564 | doi: 10.14421/biomedich.2024.132.559-564 issn 2540-9328 (online) the effect of paracetamol on the development of chicken embryos hendry tri sakti surya gunawan saragih*, alfian primahesa, shintia oktaviana, agnessia asian budi setyo, alifia anisya, hayu swari allimi, laili mufli zusrina, ruth liananda citra dolok saribu, ardaning nuriliani, nur indah septriani faculty of biology, universitas gadjah mada, jl. teknika selatan, sleman, daerah istimewa yogyakarta 55281, indonesia. corresponding author* saragihendry@ugm.ac.id manuscript received: 27 august, 2024. revision accepted: 17 october, 2024. published: 07 november, 2024. abstract paracetamol (n-acetylaminophenol) is the most widely used over-the-counter drug in the world. although considered safe for pregnant mothers, there is a concern regarding the teratogenic effect of paracetamol. this study aims to observe the teratogenic effect of paracetamol developing embryos. using chicken embryos, the embryos were incubated for 48 hours and then injected with paracetamol in 3 concentrations, namely 10 ppm, 15 ppm, and 20 ppm. the control group and treatment group consisted of 3 replicates of fertile embryos. then, the embryos were incubated again for 48 hours in the incubator. data analysis was carried out descriptively by observing any developmental alterations. results showed that paracetamol caused enlargement of the head and heart edema. exposure to paracetamol at concentrations of 15 ppm and 20 ppm affects the morphology of chicken embryos, especially the formation of the head and disrupts the process of angiogenesis and normal heart formation, causing bleeding and edema at higher concentrations. keywords: abnormality; chicken embryo; development; paracetamol. introduction paracetamol (para-hydroxyacetanilide), the common name of acetaminophen (n-acetyl aminophenol), is an over-the-counter (otc) drug used as an analgesic and antipyretic (sharma and mehta, 2014). aside from pain and fever, it’s also commonly used to ease headaches, both by itself or in conjunction with other migraine medications such as caffeine and non-steroidal antiinflammatory drugs (nsaids) (pini et al. 2008), and it’s used to treat patent ductus arteriosus in newborns (ohlsson and shah, 2020). it is one of the most commonly used medications worldwide (moore and moore, 2016; becker, 2015). paracetamol is used by over 50% of pregnant women during their pregnancy (lupattelli et al. 2014; werler et al. 2005). existing guidelines recommend the use of paracetamol during pregnancy at the lowest possible dose in the shortest possible time, with health professional consultation recommended if prolonged use is necessary (european medicines agency, 2019; food and drug administration, 2019). over the last few decades, there has been a concern over the increasing prevalence of urogenital, neurodevelopmental, and reproductive disorders in children and adults alike (bauer et al., 2021). because paracetamol readily crosses the placental barrier rayburn et al. 1986), paracetamol was indicated as one cause of the rising incidence. thus, several researchers urge caution when administering paracetamol to pregnant mothers (nilsen et al., 2023; bauer et al., 2021), while some disagreed over the conclusion of the review due to faulty methodology and failure to consider confounding factors in studies used to support the notion that paracetamol is the leading cause of said birth disorders (alwan et al., 2022; damkier et al., 2022). even though several observational studies on paracetamol’s effect on human fetuses have been made (bauer et al., 2018; andrade, 2016), studies of the teratogenic effects of paracetamol in animal models are rather scarce. several studies conducted on zebrafish larvae have shown that paracetamol induces heart defects and affects the hatching rate of its larvae, as well as its motor function (rosas-ramírez et al., 2022; xia and zhou, 2017). however, an ex vivo study on rat embryos shows no prominent defect (moungmaithong et al., 2022). there is considerable difficulty in investigating the teratogenicity of paracetamol because, in mammals, maternal prostaglandin is often able to compensate for the disruption of fetal prostaglandin synthesis, thus rendering it difficult to assess the importance of prostaglandin and cyclooxygenase in embryogenesis (cha et al. 2006). chick embryo, with its similarity to the mammalian embryo and ease of labeling, is a great https://doi.org/10.14421/biomedich.2024.132.559-564 mailto:saragihendry@ugm.ac.id 560 biology, medicine, & natural product chemistry 13 (2), 2024: 559-564 alternative for embryology studies (ribatti and annese, 2023). research on the effect of paracetamol on chicken embryo has not been widely conducted. thus, in this study, we expose chick embryos to different doses of paracetamol to study its effect on embryo growth. materials and methods procedures incubation the eggs the eggs that were obtained are placed in an incubator at a temperature of 37 ± 1⁰c. incubation was carried out for 48 hours. total 12 eggs incubated and then divided into 4 groups (control/0 ppm, 10 ppm, 15 ppm and 20 ppm paracetamol). preparation paracetamol solution paracetamol solution was prepared by dissolving a paracetamol tablet and then diluting into 10 ppm, 15 ppm and 20 ppm. exposure of chicken embryo to paracetamol the eggs were injected with paracetamol in 3 concentration groups; 10 ppm, 15 ppm and 20 ppm. the eggs were hollowed out to inject 0.1 ml of paracetamol. after that, the eggs is closed with hypafix and returned to the incubator. the injection of the eggs were conducted in laf (laminar air flow) to minimize contamination. the eggs were incubated again for 48 hours. embryo collection and making whole mount preparations eggs that have been incubated for 96 hours are taken from the incubator. the eggshell was opened in a warm 0.9% nacl solution. the embryo was washed with 0.9% nacl solution. embryos were fixed using bouin’s solution for 24 hours. after fixation, the embryo was washed using 70% alcohol and stained using eosin for 30 seconds. the embryo was dehydrated using graded alcohol for 5 minutes each. after that, the embryo was cleared using xylol for 30 minutes. data analysis embryos were observed using a leica microscope and then documented. data analysis used a descriptive method by observing and explaining the visible abnormalities. results and discussion to check the possibility of abnormalities in chicken embryo growth caused by paracetamol, 3 treatments with different doses are needed, namely 10 ppm, 15 ppm, 20 ppm, and a control as a comparison. based on the results of microscopic observations, it can be seen that abnormalities can be seen in embryos treated with 10 ppm. the most visible abnormalities are the head (mesencephalon and telencephalon) and heart (figure 3). embryos treated with 15 ppm and 20 ppm experienced abnormal growth, and their size was huge, apart from that, embryos treated with 15 and 20 ppm experienced a very dark color change so that it was difficult to distinguish between the head and body (figure 4 and figure 5). untreated embryo embryos (control) did not experience abnormalities (figure 2). figure 1. control group. no abnormalities were seen. the sizes of (1) diencephalon, (2) mesencephalon, (3) eye, (4) conus arteriosus, (5) somite and (6) tail normal. magnification: x40. figure 2. embryos treated with 10 ppm paracetamol. abnormalities can be seen in the head and heart. (1) diencephalon, (2) mesencephalon, (3) telencephalon and (4) conus arteriosus look enlarged/swollen. magnification: x40. saragih et al. – effect of paracetamol on chicken embryo 561 figure 3. embryos treated with 15 ppm paracetamol. abnormalities can be seen in the head and heart. parts of the head such as (1) diencephalon, (2) mesencephalon, and (3) telencephalon look enlarged/swollen. magnification: x40. figure 4. embryos treated with 20 ppm paracetamol. abnormalities can still be seen in the head and heart. in some preparations, parts of the head such as (1) diencephalon, (2) mesencephalon, (3) telencephalon and (4) heart look enlarged/swollen. magnification: x40. discussion the results showed that several abnormalities were observed in embryos exposed to paracetamol. several abnormalities were larger head size and cardiac edema. the enlargement of the head is due to astrocyte swelling in the brain. napqi (n-acetyl-p-benzoquinone imine), a paracetamol metabolite, is known to be hepatotoxic (athersuch et al., 2018). damage to the liver thus impaired its capability of maintaining blood ammonia homeostasis, forcing other organ systems to compensate, including the nervous system via astrocytes (aldrige et al., 2015). this caused astrocyte swelling, which in turn caused fluid accumulation (edema) in the brain. however, the molecular mechanism of the swelling of astrocyte cells due to ammonia is still unknown (seperhrinezhad et al., 2020). traditionally, glutamine the product of ammonia detoxification accumulation in astrocytes was blamed as the sole cause of edema due to its nature as an osmolyte; however the emerging “trojan horse” hypothesis posits that excess glutamine converted by astrocytes was transported to astrocyte’s mitochondria and would later be converted to ammonia and glutamate, causing oxidative stress and mitochondrial damage that leads to swelling (scott et al., 2013). cardiac edema is caused by a buildup of fluid in the heart’s ventricles (edema). napqi is known to irreversibly bind to glutathione synthetase (gs) in vitro and cause glutathione depletion (walker et al. 2016). as glutathione plays a central role in antioxidant defense, depletion of glutathione causes oxidative stress (sekhar et al., 2011). ros and oxidative stress can act as signaling molecules that can stimulate 2 things: cardiomyocyte cell hypertrophy or apoptosis. cardiomyocyte apoptosis can cause cardiac dysfunction and congenital heart failure (chung and lin, 2018). one of the manifestations of heart failure is cardiac edema (abassi et al., 2022). mechanism of action of paracetamol in the body. paracetamol is mainly converted to soluble glucuronide and sulfonate by phase ii metabolizing enzymes, with a fraction of it converted to napqi in the cytochrome 450 system (roy et al., 2013). in therapeutic doses, approximately 10% of paracetamol is metabolized by cyps to form napqi, which is then conjugated by intracellular glutamate and ultimately excreted as conjugates with cysteine and mercapturic acid (mazaleuskaya et al., 2015). paracetamol has more potency when prostaglandins are synthesized from added arachidonic acid in low concentrations or after activating delayed pathways via cytokines (graham and scott, 2003). paracetamol has a major pharmacological effect as an inhibitor of prostaglandin synthesis in cells under low arachidonic acid conditions and is selective against cox-2, one of the cyclooxygenase (cox) enzymes (graham et al., 2013). cox enzymes, also known as prostaglandin h synthases (pghs), catalyze the first step of prostanoid synthesis by converting arachidonic acid to prostaglandins (pgs), including pgh2, pge2, 15-keto-pge2, pgd2, pgf2α, pgi2, and thromboxane a2 (txa2) (seo and oh, 2017). this enzyme has functions namely fatty acid cyclooxygenase by catalyzing the change of arachidonic acid to pgg2 and prostaglandin hydroperoxidase by catalyzing the change of pgg2 to pgh (mccrae et al., 2018). peroxidase function is the basis of cox-1 and cox-2 activity, but 562 biology, medicine, & natural product chemistry 13 (2), 2024: 559-564 they can function independently and allow them to oxidize various organic substances using hydrogen peroxide or other peroxides (yang et al., 2020). paracetamol is one of the cox-1 substrates that can be oxidized, and it is thought that the peroxidase function of cox-2 can also oxidize paracetamol (graham et al., 2013). cox-1 is constitutively expressed and present in the majority of healthy human tissues, also plays a role in normal physiological processes, while cox-2 is often upregulated by infection and inflammation, but can be constitutive although it is still upregulated by peripheral inflammation in the spinal cord (radi et al., 2010). cox1 is down-regulated in the oral mucosa after oral surgery, and it has been suggested that paracetamol may inhibit cox-1 in the early stages of inflammation, but with continued inflammation, cox-2 is induced (lee et al. 2007), and then subjected to inhibition by paracetamol. then, the following mechanism is cox-2 selective oxygenation inhibition of endocannabinoids, 2arachidonoylglycerol, and anandamide (arachidonoyl ethanolamide) resulting in active prostaglandin products (alhouayek and muccioli, 2014). selective inhibitors of paracetamol and cox-2 reduce the amount of endocannabinoids that are oxidized and cause inhibition of endocannabinoid metabolism so that it can establish interactions between endocannabinoids with paracetamol and some nsaids (roy et al., 2013). interestingly, risomers of ibuprofen and other related drugs also inhibit endocannabinoid-mediated oxidation of endocannabinoids (duggan et al., 2011). teratogenic effects of paracetamol the teratogenic effects of paracetamol can be caused by several mechanisms, namely the metabolic products of paracetamol n-apap (n-acetyl-p-aminophenol); napap will interfere with cell signaling pathways by increasing oxidative stress in the embryo which can then interfere with the organogenesis process (gutiérrez noya et al. 2021; rosasramírez et al. 2022). oxidative stress refers to increased levels of intracellular reactive oxygen species (ros) that cause damage to lipids, proteins, and dna (schieber and chandel, 2014). paracetamol will also inhibit the production of pge2 (prostaglandin) such as vegf (vascular endothelial growth factor) and bfgf (basic fibroblast growth factor) which play a role in the process of angiogenesis (cha et al. 2006). disruption of the angiogenic balance can increase vascular dysfunction, which is responsible for several systemic effects on blood pressure and organ function (waller et al. 2021). in addition to playing a role in the angiogenesis process, bfgf will also regulate muscle growth (pawlikowski et al. 2017), and regulate neuroepithelial proliferation, migration, and differentiation so that the combination of several mechanisms can interfere with the process of organogenesis by chicken embryos resulting in embryonic morphological abnormalities. conclusions paracetamol can cause abnormalities in chicken embryos. embryos that were not treated did not experience abnormalities, while embryos that were given 10 ppm paracetamol experienced abnormalities in the form of enlarged heads and hearts. exposure to paracetamol at concentrations of 15 ppm and 20 ppm affects the morphology of chicken embryos, especially the formation of the head and disrupts the process of angiogenesis and normal heart formation, causing bleeding and edema at higher concentrations. acknowledgements: the authors would like to thank the faculty of biology for their financial support to carry out this project. authors’ contributions: hendry t.s.s.g. saragih designed the research and wrote the manuscript. alfian primahesa, shintia oktaviana, agnessia a. b. setyo, alifia anisya conducted the experiments, performed analysis and data visualization and wrote the manuscript. hayu s. allimi designed, conducted the preliminary research, and wrote the manuscript. laili m. zusrina and ruth l. c. dolok saribu conducted the preliminary research. ardaning nuriliani and nur indah septriani wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors would like to thank the faculty biology for their financial support to carry out this research. references abassi, z., khoury, e. e., karram, t., & aronson, d. 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https://doi.org/10.1016/j.ajog.2005.02.100 https://doi.org/10.1016/j.chemosphere.2017.05.054 https://doi.org/10.1038/s41598-020-71237-x biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 1-7 | doi: 10.14421/biomedich.2025.141.1-7 issn 2540-9328 (online) mint leaf extract (mentha x piperita l.) and bay leaves (syzygium polyanthum (wight) walp.) as additional ingredients for making hand sanitizer ovi prasetya winandari*, shinta anisya, lili anisa carolin biology education study program, raden intan state islamic university lampung jl. let.kol endro suratmin, sukarame, bandar lampung, 35131, indonesia. corresponding author* oviprasetyawinandari@radenintan.ac.id manuscript received: 12 agustus, 2024. revision accepted: 11 november, 2024. published: 18 february, 2025. abstract spray hand sanitizer in this study is a product used as an alternative hand cleansing with the addition of mint leaf extract and bay leaves that contain secondary metabolitic compounds and can form an antibacterial inhibitory zone. this study aims to determine the effectiveness of the addition of mint leaf extract (mentha x piperita l.) and bay leaves (syzygium polyanthum (wight.) walp.) as a spray hand sanitizer formulation. the method used is quantitative with a complete random design research design (ral) consisting of 4 treatments, namely: p0 (30% bay leaf extract: control), p1 (3% mint leaf extract + 30% salam leaf extract), p2 (5 % mint leaf extract + 30% bay leaf extract) and p3 (7% mint leaf extract and 30% bay leaf extract). data analysis techniques in the form of anova tests and further dmrt tests. the results showed that the addition of mint leaf extract and bay leaves gave effectiveness to the hand sanitizer formulation with the ability to form an inhibitory zone for the staphylococcus aureus bacteria. the largest inhibitory zone is formed by the p3 formulation with an average diameter of 15.78 mm category of strong inhibition. the spray hand sanitizer formulation that is preferred by panelists is the p3 formulation (7% of mint leaf extract + 30% bay leaf extract). the average value of the p3 formulation in the color parameter of 3.57, the aroma parameter of 3.63, the parameter of the use of 3.4 and the absorption rate of 3.57 with a fairly like category. keywords: bay leaves; hand sanitizer; mint leaves. introduction health problems are one of the conversations that never end to be questioned, one of the factors that causes health problems in the body quickly is a dirty hand (holifah et al., 2020). therefore, one of the most important limbs to be kept clean is hands, because it is vulnerable to being used as a place for nesting bacteria and viruses. bacteria are typical and unicellular prokaryotic cells. bacteria are microorganisms that cannot be seen in plain view by humans (adinda fitri salsabila et al., 2022). types of bacteria that are often found in the hands are staphylococcus aureus bacteria. staphylococcus aureus is a bacterium that is easily found everywhere and is pathogenic for humans. staphylococcus aureus bacteria are round-shaped gram-positive bacteria with a diameter of 0.7-1.2 μm, arranged in irregular groups such as grapes, anaerobic facultative, not forming spores and immovables (yanto et al., 2021). efforts can be made to maintain a very simple hand health, namely by washing hands. hand washing is a method used to eliminate bacteria or germs that are on the surface of the hand through the process of rubbing both palms simultaneously using soap or cleaning substances that are appropriate and then rinsed using running water (sultan & zikri, 2021). the habit of washing hands using soap is an action that is still difficult to apply to the community, especially if you are traveling or in a journey and clean water is not available, then the dirty hand will be used to eat and touch other limbs (huliatunisa et al., 2020). hand sanitizer or hand cleansing spray is a handcleaning innovation that can be used without running water. hand sanitizer is a health product that can directly kill germs without rinsing with water (less et al., 2020). hand sanitizer began to bloom on the market because of its use which tends to be practical and its packaging is easy to carry everywhere (nakoe et al., 2020). the selection of materials in making hand sanitizers is very important to note, so that the hand sanitizer products used are skin-friendly products and do not irritate the skin. the addition of natural ingredients is an alternative that can be applied to overcome these problems (aprilia & yanti, 2019). examples of types of plants that have various properties besides being used as food sources are mint plants and greeting plants. https://doi.org/10.14421/biomedich.2025.141.1-7 2 biology, medicine, & natural product chemistry 14 (1), 2025: 1-7 mint plants (mentha x piperita l.) and salam (syzygium polyanthum (wight) walp.) can be processed as a natural ingredient to make hand sanitizers by utilizing the leaves. mint plants are plants that are included in the lamiaceae family with active ingredients and distinctive aroma, in addition to being antibacterial and antifungal mint leaves can also be used to increase skin moisture, treat acne, remove dead skin cells, smooth the skin and be able to control excess oil in skin (hasibuan maimunah aisah, 2022). mint leaves are widely used in the pharmaceutical industry, cigarettes, and food, among others, for the manufacture of toothpaste, wind oil, balm, confectionery and others. salam plants are one type of woody plant that is often found in everyday life. this plant can grow wild in the forests and mountains, or commonly planted in the case and the environment around the house. salam plants also have a diuretic, analgesic and antibacterial effect. the benefits of bay leaves are produced by the content of chemical compounds they have (fitri et al., 2020). both of these plants can be processed into hand sanitizer preparations because they contain antibacterial compounds that can kill germs. antibacterial compounds found in mint leaves and bay leaves are flavonoids, saponins, tannins and alkaloids (taba et al., 2019). this research is expected to provide information about the use of mint leaves and bay leaves that contain antibacterial compounds both for students and the community. so, this study aims to make natural spray hand sanitizer preparations using the additives of mint leaf extract (mentha x piperita l.) and bay leaves (syzygium polyanthum (wight) walp.) which contain antibacterial compounds as a solution that can be used to clean the hands practical after doing various activities. materials and methods materials this research material was conducted at the uin raden intan lampung biology education laboratory and lampung state polytechnic agricultural product technology laboratory in february to may 2024. equipment used in this study was analytic scales, spatel, stirring rods, dropper pipette, erlenmeyer, shaker, blender, vaporizer, rotary evaporator, glass object, watch glass, measuring cup, ph meter, sieve, flannel, spray hand sanitizer bottle. the ingredients used in this study are mint leaves, bay leaves, glycerin, methylparaben, propylparaben and ethanol 70%. methods the required sample is mint leaves and bay leaves. mint leaves are obtained from jl. abdul kadir, rajabasa district, bandar lampung city. salam leaves are obtained from jl. sukardi hamdani, labuhan ratu district, bandar lampung city. in this study, the type of research used is quantitative research with the experimental method. research using a complete randomized design (ral). provision of concentration treatment from mint leaf extract and bay leaves consists of 4 treatments and 3 repetitions. from this treatment, 12 experiments will be obtained with the following concentration: ▪ p0 = 0% mint leaf extract and 30% bay leaf extract ▪ p1 = 3% mint leaf extract and 30% bay leaf extract ▪ p2 = 5% mint leaf extract and 30% bay leaf extract ▪ p3 = 7% mint leaf extract and 30% bay leaf extract mint leaves are washed thoroughly with running water, and then separated with the leaf stalks. after that it is weighed and dry the mint leaves by aerating ± 1 week. after drying, the mint leaves are blended and sifted until it becomes a powder preparation. furthermore, the making of the extract is done by the maceration method. the extraction process is carried out by simplicia mint leaves inserted into a container and then soaked with 96% ethanol solvent for 1x24 hours. furthermore, the extract solution is inserted on the shaker and then covered with the lid of the container for 3x24 hours, every day stirred ± 5 minutes then filtered using a flannel. furthermore, it is concentrated using a rotary evaporator until 100 ml of thick extract is obtained. before entering the process of making hand sanitizer, some of each mint leaf extract and bay leaves are taken first for the process of phytochemical screening and testing antibacterial inhibition. the phytochemical screening test was carried out to find out what secondary metabolites compounds are contained in the plant leaf extract (sulistyarini et al., 2019). the antibacterial activity test was carried out to determine the potential for inhibition of antibacterial mint leaf extract and bay leaf extract which was added as a preparation in the spray hand sanitizer formulation (karmilah et al., 2023). this antibacterial activity test was carried out on staphylococcus aureus bacteria through the well method. the steps in testing antibacterial effectiveness are, sterilization of tools, manufacturing nutrient broth (nb) and nutrients agar (na), bacterial inoculation, making bacterial suspense, manufacturing test media, observation and measurement. observation and measurement were carried out after the incubation process for 24 hours (ishimora et al., 2023). observation and measurement of the diameter of the inhibitory zone (clear zone) is carried out using the calipers. given positive control treatment using clindamycin and negative control using aquadest. after all ingredients are weighed and recorded the weight, mint leaf extract and bay leaves are put into a test tube according to the concentration in each treatment, then dissolved with 70% ethanol until dissolved (l1) (zuhri & dona, 2021). propil paraben as much as 0.04 g and methyl paraben as much as 0.18 g are inserted into a test tube and dissolved with 70% ethanol until dissolved (l2). glycerin as much as 15 ml is put into a test tube winandari et al. – mint leaf extract (mentha x piperita l.) and bay leaves … 3 (l3). furthermore, l1, l2 and l3 are put into a spray hand sanitizer bottle and added with 70% ethanol as much as 100 ml, stirred until evenly distributed and evaluated the preparation. table 1. hand sanitizer formulation. component uses formulation p0 p1 p2 p3 mint leaf ekstract active substance 0 ml 0,3 ml 0,5 ml 0,7 ml bay leaf ekstract active substance 3 ml 3 ml 3 ml 3 ml metyl paraben preservatives 0,18 g 0,18 g 0,18 g 0,18 g propil paraben preservatives 0,04 g 0,04 g 0,04 g 0,04 g glycerin humectant 15 ml 15 ml 15 ml 15 ml ethanol 70% solvent 100 ml 100 ml 100 ml 100 ml results and discussion results the results of phytochemical screening test the results of phytochemical screening testing can be seen in table 2. table 2. the results of phytochemical screening test. no. materials compound results detected 1. mint leaf ekstract flavonoid + yellow tanin + dark green saponin + stable foam alkaloid + white precipitate 2. bay leaf ekstract flavonoid + orange tanin + dark blue black saponin + stable foam alkaloid + white precipitate the results of the antibacterial activity test the results for testing antibacterial activity can be seen in table 3. table 3. the results of the antibacterial activity test. deuteronomy test of the treatment k+ kp0 p1 p2 p3 1 32,27 0,00 14,93 15,07 15,47 15,83 2 32,27 0,00 14,73 15,20 15,53 15,73 3 32,37 0,00 14,63 15,23 15,37 15,78 average (mm) 32,27 0,00 14,76 15,17 15,45 15,78 one way anova test results one way anova test results can be seen in table 4. table 4. one-way anova test results. anova dh sum of squares df mean square f sig. between groups 1566.614 5 313.323 39165.344 .000 within groups .096 12 .008 total 1566.710 17 4 biology, medicine, & natural product chemistry 14 (1), 2025: 1-7 duncan further test results duncan's advanced test results can be seen in table 5. table 5. duncan further test results. dh duncana sample n subset for alpha = 0.05 1 2 3 4 5 6 kn 3 .00 p0 3 14.76 p1 3 15.14 p2 3 15.46 p3 3 15.78 kp 3 32.27 sig. 1.000 1.000 1.000 1.000 1.000 1.000 homogeneity test results homogeneity testing gets the results that hand sanitizer preparations have homogeneous variations. ph test results ph test results can be seen in table 6. table 6. ph test results. testing observation results p0 p1 p2 p3 ph 6,0 6,1 6,2 6,3 organoleptic test results organoleptic test results can be seen in table 7. tabel 7. organoleptic test results average. parameter treatment p0 p1 p2 p3 color 3,4 3,4 3,37 3,57 aroma 3,6 3,4 3,37 3,63 properties of use 3,3 3,4 3,4 3,47 absorption rates 3,27 3,2 3,13 3,57 discussion this research discussion was conducted from february to may 2024 by utilizing the uin raden intan lampung biology education laboratory and lampung state polytechnic agricultural product technology laboratory. the research design used is experimental using a complete random design (ral). provision of concentration treatment from mint leaf extract and bay leaves consists of 4 treatments and 3 repetitions. the resulting spray hand sanitizer product has a brownish color characteristic, has an aroma of mint leaves and bay leaves, gives a cold sensation to the skin after being sprayed and absorbs on the skin. the results of spray hand sanitizer products can be seen in figure 1. figure 1. spray hand sanitizer product. the results of phytochemical screening tests show that in mint leaf extract contained secondary metabolitic compounds in the form of flavonoids characterized by the detection of the yellow color in the extract after mixed with the reagent solution. tannins marked by the detection of dark green color on the extract after mixed with reagent solution. saponins were marked by the detection of stable foam after the extract was mixed with a reagent solution, as well as an alkaloid compound characterized by the detection of white deposits in the extract after mixed with the reagent solution. whereas in bay leaf extract it appears that the metabolitic compound contained in the form of flavonoids is characterized by the detection of orange colors in the extract after mixed with reagent solution. tannin is characterized by the detection of dark blue in the extract after being mixed with a reagent solution. saponins are marked by the winandari et al. – mint leaf extract (mentha x piperita l.) and bay leaves … 5 detection of stable foam after the extract is mixed with reagent solution, as well as alkaloid compounds are characterized by the detection of white deposits in the extract after mixed with reagent solution (darma & marpaung, 2020). phytochemical screening testing is carried out to detect secondary metabolitic compounds contained in a plant extract. the observations of the antibacterial activity test show the diameter of the inhibitory zone which means that the compounds contained in mint leaf extract and bay leaves can inhibitory power to the staphylococcus aureus bacteria. the biggest inhibition is made by extracts with p3 formulations, namely the concentration of bay leaves by 30% and mint leaf concentrations of 7% with an average inhibitory power of 15.78 mm strong category. while the smallest inhibition is made by extracts with p0 formulation, namely the concentration of bay leaves by 30% without the addition of the concentration of mint leaf extract with an average inhibitory power of 14.76 mm strong category. in the p1 formulation the average inhibition produced is 15.17 mm of the strong category, and in the p2 formulation the average inhibitory power produced is 15.45 mm of the strong category. the inhibition produced by the clindamycin as a positive control of 32.27 mm is very strong category and aquadest as a negative control does not indicate the presence of antibacterial activity with the formation of the inhibition zone. clindamycin as a positive control produces a diameter of the inhibitory zone which is classified as very strong because of clindamycin is a type of antibiotic that has antibacterial activity by inhibiting protein synthesis in bacteria (ardhany et al., 2023). while aquadest as a negative control does not produce an inhibitory zone because the compounds of aquadest are neutral which will not have an effect on bacterial growth or do not have antibacterial activity (pangestu & kusuma, 2023). the anova test results show a significant value of 0.000 which means that there is an effectiveness of the addition of mint leaf extract and bay leaves as a hand sanitizer formulation. then in the duncan follow -up test the results were obtained that all formulations experienced a significant difference, but the concentration of the extract formulation whose most effective inhibition was found in the p3 formulation with a mint leaf concentration of 7% and the concentration of bay leaves by 30%, this was due to the test duncan p3 formulation is right after the kp column (positive control). homogeneity testing aims to find out and observe the mixing of substance components in the hand sanitizer preparations that are made so that they don't seem to have rough granules. homogeneity testing of hand sanitizer solutions is done by means of a hand sanitizer solution, dropped on 1 ml of object glass using a measuring pipette to form a flat surface. the results show the absence of rough grains visible. these results indicate that the substances contained in the hand sanitizer formulation are evenly dissolved so that there are no coarse grains in the observation process. these results indicate that the formulation of each treatment has a homogeneous variation. the ph test was conducted to determine the sensitivity of the hand sanitizer preparation for the skin. the normal ph range of preparations that can be applied to the skin is 4.5-6.5 (arina et al., 2023). ph testing is measured by a ph meter to determine the degree of acidity or taste of the preparation and to ensure that spray hand sanitizer preparations do not cause irritation or damage to the surface of the skin. ph preparations are too acidic (low ph) can irritate the skin while the ph is too alkaline (high ph) and can make the skin dry (aprilianti et al., 2020). the hand sanitizer preparation shows that the higher the addition of the concentration of mint leaf extracts gives the influence the level of the hand sanitizer produced, namely the ph produced is increasing. the four hand sanitizer preparations formulations that are made meet the size of the skin ph ranging from 4,56.5. the p0 formulation has the lowest ph of 6.0, the p1 formulation has a ph of 6.1, the p2 formulation has a ph of 6.2 and the p3 formulation has the highest ph of 6.3. the degree of acidity (ph) is one of the important factors in bacterial growth, this affects the high and low density of the bacteria produced. the minimum and maximum ph value for bacterial growth in general is 4-9, but the most optimal ph ranges from 6.57.5. ph is very influential on the growth of bacteria because it is related to the activity of the enzymes needed by bacteria to catalyze reactions associated with bacterial growth. if the ph in a medium or the environment is not optimal, it will interfere with the work of these enzymes and eventually disrupt the growth of the bacteria itself (razmi et al., 2023). the ph of spray hand sanitizer products in this study is classified as acidic and not in the optimal range of bacterial growth ph. the study was followed by organoleptic testing using a questionnaire sheet involving 30 panelists in the panelist category not trained, to find out the response of community acceptance of the hand sanitizer products produced. this test includes 4 parameters, namely color, aroma, nature of use and absorption rate. the ability of the sensory device to form a response which will later become an assessment of the products tested in accordance with the stimuli received by the senses. based on the organoleptic test that has been carried out shows that in the color parameter the average organoleptic test results of the p0 formulation are 3.4. the p1 formulation is 3.4, the p2 formulation is 3.37 and the p3 formulation is 3.47. in the aroma parameter of the p0 formulation, the lowest was 3.6, the p1 formulation was a score of 3.4, the p2 formulation received a score of 3.37, and the p3 formulation received a score of 3.63. then in the parameter the nature of the use of the p0 formulation received a score of 3.3, the p1 formulation was a score of 3.4, the p2 formulation was a score of 3.4 and the p3 formulation was a score of 3.47. based on the 6 biology, medicine, & natural product chemistry 14 (1), 2025: 1-7 highest score absorption level parameter obtained by the p3 formulation with an average value of 3.57 while the lowest score is in the p2 formulation with an average value of 3.13, the p0 formulation is a score of 3.27 and the p1 formulation is a score of 3, 1. overall based on the results of the 4 parameters, each hand sanitizer formulation in this study can be accepted by panelists and is in the category quite liked by panelists. conclusion based on the results of the research that has been carried out, it can be concluded that there is the effectiveness of the addition of mint leaf extract (mentha x piperita l.) and bay leaf extract (syzygium polyanthum (wight) walp.) as a hand sanitizer formulation. optimal concentration on hand sanitizer products with the addition of mint leaf extract and bay leaves based on antibacterial activity tests and organoleptic tests, namely in the p3 formulation with a concentration of adding mint leaves as much as 7% and the addition of bay leaves as much as 30%. the inhibition zone formed by this concentration has an average of 15.78 mm and the average results of organoleptic testing results of a color parameter of 3.57, aroma parameter of 3.63, parameter of the use of 3.47 and absorption level parameter of 3.57 with whole parameter in the category quite like. competing interests: the authors declare that there are no competing interests. references adinda fitri salsabila, livia syafnir, & vinda maharani patricia. 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(2021). the use of alcohol for medical purposes istihsan review. journal of law, society, and islamic civilization, 9(1), 40. https://doi.org/10.20961/jolsic.v9i1.51849 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 635-640 | doi: 10.14421/biomedich.2025.142.635-640 issn 2540-9328 (online) antimicrobial properties of endophytic fungal culture filtrates from tinospora crispa oktira roka aji1,*, karomah indah fitria2, naresh wari mega vianingtyas2, adelyanty pangestuty wicaksono2 1biology department; 2microbiology laboratory, faculty of applied science and technology, universitas ahmad dahlan. jl. ahmad yani, banguntapan, bantul, yogyakarta 55166, tel. +62-274-563515, fax. +62-274-564604, indonesia. corresponding author* oktira.aji@bio.uad.ac.id manuscript received: 09 may, 2025. revision accepted: 13 august, 2025. published: 01 october, 2025. abstract endophytic fungi can produce bioactive compounds that are useful as antimicrobials. this study evaluates the antimicrobial potential of culture filtrate extracts derived from endophytic fungi isolated from the medicinal plant tinospora crispa. isolation was carried out from the roots, leaves, and stems of t. crispa, which were then identified based on the its gene. the culture filtrate was extracted using ethyl acetate and assessed for antimicrobial activity using the disc diffusion method against escherichia coli, staphylococcus aureus, and candida albicans. a total of 3 endophytic fungal isolates were isolated and identified as acrocalymma vagum, diaporthe tulliensis, and colletotrichum truncatum. the results showed that all culture filtrate extracts of the fungal endophyte isolates exhibited varying antimicrobial activity, with the highest antibacterial activity demonstrated by c. truncatum isolates against escherichia coli and staphylococcus aureus. the most significant anticandida activity was by d. tulliensis isolates. endophytic fungi of medicinal plant t. crispa can be developed as a source of antimicrobial agents, especially to overcome the increasing antibiotics resistance. keywords: antimicrobial; culture filtrate extract; endophytic fungi; tinospora crispa. introduction tinospora crispa (l.) miers ex hook.fil. & thomson is one of the most widely recognised medicinal plants in southeast asia. this medicinal plant has traditionally been used to treat of various diseases, such as fever, jaundice, malaria, arthritis, diabetes, urinary disorders, high blood pressure, and promoting wellness (ahmad et al., 2016). the plant is rich in phytochemical compounds such as alkaloids, flavonoids, glycosides, terpenoids, phenolics, as well as other compounds such as lactones, sterols, lignans, and nucleosides, which are known to have diverse biological activities (shree & krishnaveni, 2023). in addition to the extensively studied plant parts, microbial components residing within these plant tissues, particularly endophytic fungi, are increasingly recognised as a valuable source of bioactive compounds. endophytic fungi are fungi that live symbiotically in plant tissues without causing disease symptoms in the plants they inhabit (wen et al., 2022). endophytic fungi in all plants are known to play an important role in supporting plant growth and resistance to various environmental stresses (li et al., 2025). endophytic fungi facilitate nutrient absorption, promote plant growth by producing hormones like auxin, and assist plants in coping with stressors such as drought, pathogen attacks, or heavy metal exposure (baron & rigobelo, 2022; fite et al., 2023; morales-vargas et al., 2024). endophytic fungi benefit the host plant and are known for their ability to synthesize valuable bioactive compounds (hashem et al., 2023). these metabolites hold significant potential in the pharmaceutical industry, particularly as antimicrobial agents and antioxidants. this capability is especially crucial given the rise of microbial resistance to antibiotics. endophytic fungi from various medicinal plant species have been reported to produce compounds with antimicrobial activity. botryosphaeria mamane from the medicinal plant arrabidaea chica has potent antibacterial activity against various pathogenic bacteria, including staphylococcus aureus and candida parapsilosis (gurgel et al., 2023). additionally, isolates such as penicillium sclerotigenum and diaporthe kochmanii from ageratina adenophora have shown effectiveness against resistant bacteria like mrsa (wen et al., 2023). isolates of fusarium sp. and acremonium sp. obtained from paederia foetida produced metabolites that exhibited moderate to strong antibacterial activity against s. aureus and e. coli (widjajanti et al., 2021). endophytic fungi are reported to have great potential to inhibit both grampositive and negative bacteria (nguyen et al., 2025). https://doi.org/10.14421/biomedich.2025.142.635-640 636 biology, medicine, & natural product chemistry 14 (2), 2025: 635-640 previously, it has been reported that the endophytic fungus extract t. crispa has antibacterial activity against e. coli and s. aureus and anticandidal activity (fathoni et al., 2021, 2022, 2023). however, no studies exclusively utilise liquid culture filtrates, which contain secondary metabolites naturally secreted by fungi and may serve as a more practical source of bioactive compounds. this study aimed to evaluate the antimicrobial and antioxidant activities of the culture filtrate of endophytic fungi from t. crispa. this study is expected to provide new insights into the potential of endophytic fungal culture filtrates as promising therapeutic candidates derived from locally sourced biological resources. materials and methods materials the materials used in this study included healthy leaves, stems, and roots of tinospora crispa, pda, and pdb media with chloramphenicol, and solvents such as ethyl acetate and sterile distilled water. molecular work involved the wizard® genomic dna kit, its1f and its2 primers, gotaq green master mix, and a pcr thermocycler. antimicrobial assays used e. coli, s. aureus, and c. albicans with chloramphenicol and ketoconazole as positive controls. paper discs, cotton swabs, and standard microbiological tools were used, and data analysis employed mega-x, bioedit, and ncbi blast. preparation of plant samples plant samples were prepared using tinospora crispa plants from the berbah, yogyakarta special region. samples collected include healthy leaves, stems, and roots without any spots or defects. after collection, the samples were immediately taken to the microbiology laboratory, universitas ahmad dahlan, for the endophytic fungus isolation process. isolation of endophytic fungi the isolation process begins with sterilizing the sample surface, where fresh roots, stems, and leaves are washed using running water to remove dirt and soil. next, the samples were immersed in 70% (v/v) alcohol for one minute, then immersed in 0.5% (v/v) naclo solution for three minutes, and again immersed in 70% (v/v) alcohol for 30 seconds. after that, the samples were rinsed twice with sterile distilled water and dried in a sterile petri dish. samples were then aseptically cut using a sterile scalpel into 1-2 cm sizes and inserted into pda media supplemented with 100 μg/ml chloramphenicol aseptically. each organ sample was done in two repetitions and coded. to ensure the success of the sterilization process, 0.1 ml of sterile distilled water from the last rinse was poured onto pda media using the scatter method. the samples were then incubated at room temperature (25-28°c) for seven days. fungal isolates that successfully grew were then subcultured onto new pda media. the characteristics of endophytic fungi were observed microscopically by adding lactophenol cotton blue to the mycelial samples and then observing them under a microscope. preparation of culture filtrate extract of endophytic fungus of tinospora crispa plant the endophytic fungal culture filtrate was prepared using the method from baz et al. (2024). endophytic fungi that have been rejuvenated for 7 days on pda media in a petri dish are taken using a scalpel, as many as three snippets in the form of plates. every three plates of pure fungal hyphae from each strain were put into 2000 ml of pdb media placed in a dark bottle with a capacity of 2500 ml. the culture was then incubated at room temperature (25°c-27°c) for 14 days to allow the production of secondary metabolites. the fermented culture was divided into two components: fungal mycelia and fermentation media. this separation was achieved by filtering the culture through sterile filter paper. the fermentation media, also known as culture filtrate, was then mixed with ethyl acetate solvent in a 1:1 ratio and homogenized. afterwards, this mixture was evaporated using a vacuum rotary evaporator set at 44°c until a thick extract was obtained. antibacterial activity of culture filtrate extract of plant endophytic fungus tinospora crispa determination of the antibacterial activity of the culture filtrate extract of endophytic fungi isolated from tinospora crispa plants was carried out using the disk diffusion method. this test was performed on bacteria escherichia coli and staphylococcus aureus. in this test, sterile distilled water was used as the extract solvent as well as a negative control, while chloramphenicol was used as a positive control with a concentration of 30 μg. prior to testing, the bacterial suspension was standardised for turbidity using the 0.5 mcfarland standard, which represents a bacterial density of 1 × 10⁸ cells/ml. the bacterial suspension was then streaked evenly over mueller-hinton agar (mha) media using a sterile cotton swab. paper discs that have been soaked with fungal extracts are placed on the surface of the media. next, the entire medium was incubated at 30°c for approximately 24 hours to observe the antibacterial activity indicated by the zone of inhibition around the discs. anticandidal activity of culture filtrate extract of plant endophytic fungus tinospora crispa anticandida activity testing of filtrate culture extracts against candida albicans was carried out by the paper disc diffusion method. candida albicans stock that has been rejuvenated for 24 hours at 37°c was taken from potato dextrose agar (pda) media using an ose needle, then made a suspension in 0.85% nacl solution and standardized its turbidity using the 0.5 mcfarland aji et al. – antimicrobial properties of endophytic fungal culture … 637 standard. the fungal suspension that has met the standard is then taken using a sterile cotton swab and spread evenly on the surface of the pda media in a petri dish. paper discs were dipped for 5 minutes into the filtrate culture extract of endophytic fungi. the plate was then incubated for 24 hours at 37°c. to ensure the reliability of the results, the test was performed three times. after incubation, anticandidal activity was observed by measuring the diameter of the inhibition zone formed around the disk. the inhibition zone results of the extracts were compared with the positive control using ketoconazole (200 mg) and the negative control using sterile distilled water to assess the anticandida effectiveness of the endophytic fungal extracts. antibacterial activity of culture filtrate extract of plant endophytic fungus tinospora crispa the isolation and genetic identification of endophytic fungi begins with harvesting of four-day-old mycelium from potato dextrose broth (pdb) media. the mycelium was filtered, then frozen in a freezer until it froze. after that, the frozen mycelium was ground to destroy the cell tissue, and 40 mg was used for genomic dna isolation using the wizard® genomic kit method (promega corp). amplification of the its-1 rdna gene was performed using the polymerase chain reaction (pcr) method. the primer pairs were its1f (5'cttggtcatttagaggaagtaa-3') and its2 (5'gctgcgttcttcatcgatgc-3'). pcr reactions were prepared in a total volume of 50 µl, consisting of 6 µl of isolated dna, 25 µl of gotaq green master mix, 2 µl of each primer, and 15 µl of nuclease-free water. amplification was performed in a pcr thermocycler (bio-rad) with 35 cycles, including pre-denaturation at 95°c for 3 minutes, denaturation at 95°c for 10 seconds, annealing at 54°c for 30 seconds, elongation at 72°c for 45 seconds, and final elongation at 72°c for 8 minutes. pcr products were analyzed using 1.5% agarose gel electrophoresis with 0.5x tae buffer. electrophoresis was run for 60 minutes with a voltage of 50 v, and the gel results were observed using a uv transilluminator. the pcr amplification products were then sent for dna sequencing. after sequencing, the chromatogram images were analyzed to obtain the nucleotide base sequence. furthermore, the dna sequences were analyzed using ncbi's blast program. finally, based on the blast results and sequence analysis, a phylogenetic tree was constructed to determine the evolutionary relationship between the fungal isolates. phylogenetic tree construction was performed using the neighbor-joining method with 1000 bootstraps on mega-x software. data analysis the test was conducted three times to ensure accurate results. data on the percentage of inhibition was calculated by measuring the diameter of the inhibition zone of each culture filtrate extract of tinospora crispa endophytic fungus. statistical tests were conducted using analysis of variance (anova). if significantly different results were found, the duncan test was performed with a confidence level of α = 0.05. results and discussion fungal endophyte isolation and identification endophytic fungi were isolated using tinospora crispa plants in perfect, healthy, and non-deformed conditions. the isolation process uses pda media added with chloramphenicol, which inhibits the growth of contaminant and endophytic bacteria. isolation was carried out using the direct planting method with three repetitions. from the isolation results, three endophytic fungal isolates were obtained, each from the root (isolate a), stem (isolate b), and leaf organs (isolate d). endophytic fungal isolates obtained are observed based on the shape and colour of the colonies as an initial step in morphological characterisation to obtain pure isolates. based on its gene nucleotide similarity, it shows that isolate a is 98.09% similar to acrocalymma vagum (mh141292.1), isolate b is 99.65% similar to diaporthe tulliensis (kx688170.1), and isolate d shows 100% similarity to colletotrichum truncatum (kx621963.1). figures 1, 2, and 3 show the observation results for each isolate, including the top and bottom views of fungal colonies grown on pda medium, microscopic observations of hyphae, and the phylogenetic tree based on its gene sequences. figure 1. endophytic fungal isolate a (acrocalymma vagum). figure 2. endophytic fungal isolate b (dilaporthe tulliensis). 638 biology, medicine, & natural product chemistry 14 (2), 2025: 635-640 figure 3. endophytic fungal isolat d (colletotrichum truncatum). antibacterial activity of culture filtrate extract of plant endophytic fungus tinospora crispa culture filtrate extracts from endophytic fungi demonstrated antibacterial activity against escherichia coli and staphylococcus aureus, as observed in table 1 and figure 4. the culture filtrate extract from the three isolates (a, b, and d) demonstrated inhibition zones against e. coli and s. aureus. statistical analysis using one-way anova revealed significant differences between the treatment groups. the culture filtrate extract of isolate d showed the highest antibacterial activity compared to the other isolates against both bacteria. table 1. antibacterial activity of culture filtrate extract of fungal endophyte. categories inhibition zone (mm) e. coli s. aureus negative control 00,00 ± 0,00a 00,00 ± 0,00a positive control 27,33 ± 0,21e 30,00 ± 0.96d a 10,00 ± 0,20b 11,33 ± 0.76b b 10,66 ± 0,24c 13,33 ± 1.16c d 11,00 ± 0,14d 13,66 ± 0.41c note: data represent the average inhibition zone diameter (± standard deviation). statistical analysis using one-way anova showed significant difference among treatments (p > 0.05). anticandidal activity of culture filtrate extract of plant endophytic fungus tinospora crispa the results of the anticandidal activity test of the fungal endophytic culture filtrate extract, as shown in table 2 and figure 4, indicate that the culture filtrate extract can produce an inhibition zone against candida albicans. the highest activity was shown by the culture filtrate extract of endophytic fungal isolate b. statistical analysis using one-way anova showed significant differences between treatments (p > 0.05). table 2. anticandidal activity of culture filtrate extract of fungal endophyte. categories inhibition zone (mm) candida albicans negative control 00,00 ± 0,00a positive control 28,03 ± 0,24d a 6,50 ± 0,59b b 9,05 ± 0,81c d 8,21 ± 0,43c note: data represent the average inhibition zone diameter (± standard deviation). statistical analysis using one-way anova showed significant difference among treatments for candida albicans (p > 0.05). a. b. c. figure 4. the antibacterial and anticandidal activity of culture filtrate extracts from endophytic fungi. (a) escherichia coli, (b) staphylococcus aureus, and (c) candida albicans. each plate shows: negative control, positive control, and extracts from three fungal isolates (1: isolate a, 2: isolate b, and 3: isolate c). discussion this study showed that culture filtrate extract of endophytic fungi from tinospora crispa had varying antimicrobial activity against gram-negative escherichia coli, gram-positive staphylococcus aureus, and pathogenic candida albicans. the three isolates obtained, acrocalymma vagum (isolate a), diaporthe tulliensis (isolate b), and colletotrichum truncatum (isolate c), demonstrated the ability to produce inhibition zones against all test microbes, although at varying intensities. the highest antibacterial activity against e. coli and s. aureus was shown by isolate c. this study is in line with several previous studies that also reported the antimicrobial potential of this species. c. truncatum has been successfully isolated as an endophytic fungus from various plants, including chloranthus japonicus (an et al., 2020), gynura japonica (riga et al., 2025), and musa acuminata (yansombat et al., 2023). riga et al. (2025) reported that sydonic acid compounds isolated from endophytic c. truncatum showed antibacterial activity against staphylococcus aureus, escherichia coli, and streptococcus pyogenes. endophytic fungi are an important source of novel antimicrobial compounds with diverse chemical structures and the ability to inhibit human, plant and marine pathogens, thus contributing significantly to the global challenge of antimicrobial resistance (caruso et al., 2022). the difference in antibacterial potential between endophytic isolates could be due to variations in the secondary metabolites produced by each fungal species. the culture filtrate used in this study contains metabolite compounds secreted naturally during fermentation in liquid media. extraction with ethyl acetate solvent allows good separation of these active compounds. this suggests that using culture filtrate, without involving the fungal biomass directly, is potentially effective as a source of antimicrobial compounds. the higher level of inhibition against s. aureus than e. coli in most isolates was due to the difference in cell wall structure between gram-positive and gram-negative bacteria. gram-negative bacteria, such as e. coli, have a complex lipopolysaccharide outer membrane, which can inhibit the penetration of antimicrobial compounds (choi & lee, 2019). anticandidal activity testing revealed that the culture filtrate extract from isolate b (diaporthe tulliensis) aji et al. – antimicrobial properties of endophytic fungal culture … 639 showed the largest zone of inhibition against candida albicans. genus diaporthe have been successfully isolated from various plants, such as orthosiphon stamienus (tong et al., 2017), prunus domestica (abramczyk et al., 2022), etc. the anticandidiasis activity observed in this study is in line with the findings of yedukondalu et al. (2017), who reported that xylarolide compounds isolated from the endophytic fungus diaporthe terebinthifolii showed antifungal activity against candida albicans. in addition, diaporthe sp. ed2 isolate from o. stamienus produced a new ketone compound, 3-hydroxy-5-methoxyhex-5-ene-2,4dione, which showed fungicidal activity against candida albicans (tong et al., 2017). endophytic fungi of the genus diaporthe are also reported to produce various active compounds that have the potential to be used as biofertilizers and biopesticides (hilário & gonçalves, 2022). this finding align with previous reports on the antimicrobial activity of endophytic fungi from various medicinal plants. in addition, using culture filtrate extracts can be an alternative in exploring microbial resources as potential therapeutic agents. the results of this study indicate the potential utilisation of endophytic fungi from t. crispa. future research should focus on the purification and structural elucidation of the active metabolites from these endophytic fungi, as well as in vivo testing to assess their therapeutic efficacy and safety. conclusions the culture filtrate extract of endophytic fungal isolated from tinospora crispa has antimicrobial activity against escherichia coli, staphylococcus aureus and candida albicans. the results showed that secondary metabolites secreted into the culture medium were able to inhibit microbial growth, with colletotrichum truncatum isolate showing the highest antibacterial effect against escherichia coli and staphylococcus aureus while diaporthe tulliensis showed the highest anticandida activity. these findings support the potential application of fungal culture filtrates as an alternative source of bioactive compounds for antimicrobial therapy. acknowledgements: the authors gratefully acknowledge universitas ahmad dahlan for laboratory facilities. special thanks to the microbiology laboratory team for their assistance. authors’ contributions: oktira roka aji designed the study, analyzed the data, and finalized the manuscript. karomah indah fitria, naresh wari mega vianingtyas, and adelyanty pangestuty wicaksono carried out the data collection and wrote the initial draft. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that no funding was received for this study. references abramczyk, b., marzec-grządziel, a., grządziel, j., król, e., gałązka, a., & oleszek, w. 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(2017). diapolic acid a–b from an endophytic fungus, diaporthe terebinthifolii depicting antimicrobial and cytotoxic activity. the journal of antibiotics, 70(2), 212–215. https://doi.org/10.1038/ja.2016.109 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 225-232 | doi: 10.14421/biomedich.2023.121.225-232 issn 2540-9328 (online) chemical profile and in-silico docking studies on bioactives from essential oil of cymbopogan pendulus targeting penicillin binding proteins (pbps) in bacteria arun dev sharma*, inderjeet kaur pg department of biotechnology, lyallpur khalsa college jalandhar, india. corresponding author* arundevsharma47@gmail.com manuscript received: 25 january, 2023. revision accepted: 15 february, 2023. published: 17 february, 2023. abstract antibiotic resistance in bacteria is the major concern worldwide. pbp (penicillin binding proteins) have been cited as an appropriate target for therapeutic drug design. in the present study molecular docking followed by wet lab validation was designed to estimate the effect of potent bioactive molecules from cymbopogan pendulus essential oil against pbp5 protein. gc-fid (gas chromatography with flame-ionization detection) based composition profile, and in-silico docking study was conducted by using cb-dock 2 analysis followed by 2d and 3d interactions. gc-fid revealed limonene, neral, geranial, linalool, myrcene as major and minor compounds in cymbopogan pendulus essential oil. the docking score indicated effective binding of ligands to pbp5. interactions results indicated that, pbp5/ligand complexes form hydrogen and hydrophobic interactions. wet lab study validated the anti-bacterial potential of oil against gram-positive and gram-negative bacteria. therefore, essential oil from cymbopogan pendulus essential oil may represent potential herbal treatment to mitigate bacterial infections. keywords: bacteria; docking; lemon grass oil; geranial; herbal drug. introduction bacterial peptidoglycan, which is repeating disaccharide unit of n-acetylglucosamine (nag) and nacetylmuramic acid (nam), with nam bearing a peptide stem, provide resistance to bacteria not only by providing capability to resist against internal intracellular pressure but also helps to maintain well-defined cellshape (brockhurst et al., 2019). the final steps involved in cell wall bio synthesis and remodeling are mediated by penicillin-binding proteins (pbps), which comprise highmolecular-mass (hmm) and low-molecular-mass (lmm) pbp subgroups. the term ‘pbp’ has been cited in manuscripts to refer to any enzyme that identifies and/or metabolizes β-lactams, independently of its function in the cell (fair and tor, 2014). pbps are implicated in catalysis such as: in trans-glycosylation (polymerization of the glycan strands) and transpeptidation (the cross-linking between glycan chains), dd-carboxy-peptidation (hydrolyze the last d-alanine of stem pentapeptides) and endo-peptidation (hydrolyze the peptide bond connecting two glycan strands) (simoes et al., 2017). pbps represents excellent targets for β-lactam antibiotics such as penicillin’s, thus inhibiting penultimate physiological role of enzymes involved in bacterial cell wall synthesis and leading to cell death. however, worldwide uncontrolled use of β-lactam antibiotics has led to bacterial resistance to antibiotics which is a swiftly growing apprehension. this problem emerged due to the emergence spread, and persistence of multidrug-resistant (mdr) bacteria, which are frequently isolated in hospitals collectively known as “eskape”, which constitutes gram-positive and gram-negative species (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter spp.), also known as “superbugs”. “eskape” are also resistant to traditional and conventional treatments involved in majority of nosocomial infections (brockhurst et al., 2019). bacteria possess a variable number of pbps and among all pbp5, an lmm trans-peptidase, is the most abundant and a vital enzyme involved in peptidoglycan synthesis in cell wall of bacteria (welsh et al., 2017). in the cell wall, the major function of pbp5 is a ddcarboxypeptidase reaction, that regulates the degree of cross-linking by hydrolytically shortening the peptide stem of the nascent peptidoglycan. it was reported that blocking of either the carboxypeptidation, transpeptidation or reactions by β-lactams antibiotics, worsens the peptidoglycan and may cause cell death (chan et al., 2016). this process has widened the use of antibiotics and its analogs maximally but has been challenged by the transmission of drug-resistant strains, https://doi.org/10.14421/biomedich.2023.121.225-232 226 biology, medicine, & natural product chemistry 12 (1), 2023: 225-232 highlighting the inevitability for novel natural antibiotic therapies. in this regard, inhibition of the glycosyltransferase reaction by the natural product moenomycin also has been reported that weakens the peptidoglycan and kills bacterial cells (masters et al., 2020). pbp5, topology constitutes: a trans-membrane anchor, a cytoplasmic tail, and two domains joined by a beta-rich linker located on the outer surface of the cytoplasmic membrane where cell wall peptidoglycan synthesis takes place (lu et al., 2020). the antibacterial activity of β-lactams is arbitrated by covalent binding to pbps, thus inhibiting the transpeptidase (tpase) activity of pbp-mediated bacterial cell wall synthesis (da costa et al., 2018). since bacterial resistance to multiple drugs, including b-lactam antibiotics, is a main therapeutic problem thus development of new chemical entities as antibacterial agents are urgently needed (da costa et al., 2018). it was argued that gram-positive and gramnegative bacteria have mostly established resistance to all the available antibiotics and pose a grave problem not only in hospitals but also for the general population (contreras-martel et al. 2017). therefore, by virtue of its key role, pbps are considered as an appropriate objective for developing bacterial inhibitor. inhibition of pbps protein activity would block replication of bacteria. since in humans, not at all any pbps with comparable cleavage specific are recognized, so inhibitors are improbable to be considered as toxic. lemon grass essential oil (lgo) from cymbopogon species, also known as lemon grass, encompasses a number of bioactives. due to complex nature of essential oil, their anti-fungal mechanism of action is still not completely understood (bhatnagar, 2008, mancianti et al., 2020)]. lgo has long history to be used as complementary and traditional medicine in ancient times. in addition, various potent biological activities like antiamoebic, anti-inflammatory, anti-filarial, anti-diarrheal, anti-malarial, anti-fungal and anti-bacterial agent, antihiv have been attributed to lgo, hence playing a major role as therapeutics in the scientific community (oladeji et al., 2019). this study postulated that due to richness of geranial, essential oil from cymbopogon pendulus plants have potential to inhibit bacterial infections. hence as an objective this study was designed to study molecular docking of geranial and wet lab validation of antibacterial potential of lemon grass oil in relation with pbp5. the present study outcomes would offer scientists and doctors with prospects to identify the key antibacterial drugs to combat mdr. experimental gc-fid analysis lgo was extracted from fresh leaves of cymbopogan pendulus growing naturally at nearby areas of lyallpur khalsa college, jalandhar. the cymbopogan pendulus was authenticated by dr upma from botany dept and voucher with number bt103 was deposited in dept of biotechnology. hydro-distillation method was used for extraction of essential oil by using clevenger-type apparatus (borosil, india) (sharma and kaur 2021). to identify bioactive compounds in eo, gc-fid study was carried out (gc-fid, chemtron 2045). the specifications of column was: 2 m long, stainless steel having 10% ov-17 on 80-100% mesh chromosorb w (hp). nitrogen was used as carrier gas at flow rate of 35 ml/min. 0.2 µl leo sample was used. the temperatures for detector and injector were: 220 °c and 270 °c. oven ramping conditions were: 100°c (firstly maintained) ramped to 2100 °c at 3 °c/min. bioactive constituents in leo were identified by comparing relative retention times (rt) of gc-fid spectra of leo with authentic standards and literature data. ligand preparation for bacterial receptors (pbp5, pdb id: 3mzf), various bioactive compounds such as: limonene, neral, geraniaol, linalool, myrcene which are present in lemon grass essential oil as major and minor amounts were used as ligands for structures. to build 3d structure of ligand, smiles of ligands was recovered from ncbi-pubchem database. the structure was built by using ucsfchimera. molecular docking crystal structures of pbp5 bacterial penicillin binding protein recovered from pdb (https://www.rcsb.org/). before docking analysis, all target enzymes were cleaned from selected h2o molecules, cofactors, co-crystallized ligand, and energy minimized. then all protein target structures were prepared by means of the dock prep set up in ucsf-chimera. it is the process under optimization that bond length, charges anomalies and corrects atomic structure. for docking, cb-dock 2 tool was used for docking of ligands over pbp5 (https://cadd.labshare.cn/cb-dock2/php/index.php). to execute docking, both receptors and ligand molecules as “pdb files” were uploaded to the cb-dock 2 and docking was performed. for 2d and 3d interactions in docked complexes, biovia 2020, ucsf chimera and plip tools were used. active sites prediction in fungal receptors, identification and dimension of cavities on 3d active sites were computed by using castp web tool. for this all structures in “pdb” format were uploaded to server and prediction was executed with probe radius value of 1.4 angstroms. in-vitro anti-bacterial activity the in-vitro antimicrobial activity of lgo was determined through agar disc diffusion method against four test organisms, gram-negative escherichia coli (mtcc 40), pseudomonas aeruginosa (mtcc 424), and sharma & kaur – chemical profile and in-silico docking studies on … 227 gram-positive staphylococcus aureus (mtcc 3160) and bacillus subtilis (mtcc 121). pathogens were purchased from institute of microbial technology, chandigarh. sterile paper discs (10 mm in diameter) were impregnated with 100 µl lgo. 12-h cultures were used inoculums and od of suspensions was adjusted to 0.6. a swab of bacteria suspension was spread on to lb-agar plates and allowed to dry for 30 min. the discs with essential oil were then applied and plates were left for 20 min at room temperature to allow to diffusion of oil followed by incubation at 37oc for 24 hours. vancomycin antibiotic, (10mg) was taken as positive control. zone of inhibition was measured. result and discussion gc-fid analysis of bioactive molecules in lgo the gcfid chromatogram obtained was depicted in figure 3. the peaks observed and their respective retention time was also displayed. the gcfid analysis of lemon grass oil obtained from cymbopogon pendulus revealed 26 compounds for the total of 100%. in the present study, all identified compounds were micrene, limonene, linalool, geranial, neral, undececanone and geranial acetate. gc-fid chromatogram contained three major peaks along with many small peaks indicating the presence of minor compounds. the major constituents were geranial (27%), neral (31%), myrcene (6.7%), limonene (4.9%) and linalool (3.6%). the small peaks may be ascribed to the disintegrated major compounds bioactive compounds or present in small quantities. the literature studies also showed the presence and identification of mycrene, geranial and neral in lemongrass oil obtained from c. flexuous, (oladeji et al., 2019). during the course of time, use of lgo has become a major area of healthand medical-related research due to richness of bioactives. lgo also has been used as therapeutic agent in pharmaceutical preparations as anti-oxidative, antibacterial, antiviral, anti-diabetic, anti-tumor, antifungal, anti-obesity, antihypertensive, anti-histaminic, anti-cancer, anti-hiv and hepatoprotective agent (oladeji et al., 2019). in this study 2 major and 3 minor bioactive compounds as cited above were selected for 3d docking. peak no. retention time (min) biactive compound conc. 1 0.498 micrene 6.7075 2 5.582 limonene 4.9152 3 6.915 linalool 3.6378 4 18.998 geranial 27.0440 5 21.248 neral 31.5752 6 23.998 undececanone 1.5138 7 27.165 geranial acetate 1.3755 figure 1. gc-fid analysis of lemongrass essential oil (lgo). molecular docking structure-based drug design (sbdd) is most widely used in-silico technique in making drugs which is based on 3-d structures (srimai et al., 2013). in-silico docking has simplified investigators to screen conformations and affinities of an assembly of bioactive components against receptors (barcellos et al., 2019). present study aimed at docking of limonene, neral, geranial, linalool, and myrcene bioactive molecules from lgo as key antibacterial inhibitor candidates against pbp5. from docking analysis, it was apparent that ligands efficiently docked with pbp5 bacterial enzyme. 3d docking results illustrated that pbp5 depicted strong binding with ligand limonene (table 1) as apparent from its docking score of -5.3. based on docking score, the order to docking of ligand with pbp5 receptor was: limonene>, neral>geranial>linalool> myrcene. the best pose displaying 3d model of pbp5-ligand based on docking 228 biology, medicine, & natural product chemistry 12 (1), 2023: 225-232 score are displayed in figure 2. 2d/3d interaction of ligands with pbp5 is displayed in figure 3. with pbp5, it was revealed that ligands docked with penicillin binding domain of pbp5. the c-terminal module is responsible for the transpeptidase activity of pbps catalyzing peptide cross-linking between two adjacent glycan chains catalyzing peptide cross-linking between two adjacent glycan chains in peptidoglycan cell wall synthesis (contreras-martel et al., 2017). it was cited that blocking of either the transpeptidation or carboxypeptidation reactions by b-lactam antibiotics or therapeutic inhibitors, weakens the peptidoglycan and may engender cell death (straume et al., 2020). once a pbp is acylated by a by therapeutic inhibitors, it is unable to catalyze hydrolysis of the covalent acylenzyme intermediate and is inactivated; peptidoglycan transpeptidation cannot occur, and the cell wall is weakened (masters et al., 2020, moon et al., 2018). based on analysis, it was highlighted that lgo can be used as effective source of anti-bacterial compounds. table 1. docking score of ligands with pbp5. ligand vina score (kcal/mol) cavity volume (å3) center (x, y, z) docking size (x, y, z) interacting residues (4 a˚) h-bond interactions hydrophobic interactions limonene -5.3 1721 39, 4, 27 26, 17, 17 214athr,222atyr,222atyr,224aleu 245aphe, 248aarg, 248aarg neral -5.1 1721 39, 4, 27 26, 19, 19 216a his 198aarg,214athr,216ahis,222atyr, 222atyr,224aleu,248aarg, 248aarg,249aglu geraniaol -4.9 1721 39, 4, 27 26, 19, 19 216a his 198arg, 214athr 216ahis, 222atyr 222atyr, 224aleu 248aarg,249a,glu linalool -4.7 1721 39, 4, 27 26, 18, 18 216ahis 198aarg,214athr,222atyr,248aarg, 248aarg,249aglu myrcene -4.7 1721 39, 4, 27 26, 18, 18 214athr,216ahis,222a tyr,222atyr,222atyr, 248aarg,248aarg,249aglu figure 2. pictorial view of 3d model of pbp-ligand interactions. a: geranial; b: limonene; c: linalool; d: myrcene; e: neral; f: general pictorial view of pbp1 exhibiting domains. through 3d docking, with site residues of receptors, ligand could form h-bonds or hydrophobic bonds which designate affinity of ligand with receptor (lima et al., 2019). hence, docking interactions of limonene, neral, geranial, linalool, myrcene with pbp5 was further evaluated. it was observed that most effective ligand limonene forms hydrophobic interactions with pbp. with pbp5 receptors, hydrophobic interactions were detected a b c d fe sharma & kaur – chemical profile and in-silico docking studies on … 229 via 214thr, 222tyr, 224leu, 245phe, and 248arg active site residues at 3.38, 3.84 3.69, 3.68 and 3.56 å (figure 2). it was noted in addition to hydrophobic interactions that ligands neral, geranial, linalool exhibited h-bond interactions also by 216his. castp active sites prediction quantified interacting residues in the active site cavities of pbp1 receptors (data not shown). in pbp5 enzymes, a main pocket was documented with volume (sa) of 1721(å3) and area (sa) of 376 (å3) main pocket contained active site residues such as ser44 arg198 thr214 gly215 his216 tyr222 asn223 leu224 arg248 glu249. meanwhile, as all ligands shown good affinity to pbp5 enzyme via active site residues so it was conjectured that upon binding with ligand pbp5 becomes closed thus in-turn persuades change in conformation of bacterial enzymes and inhibit biosynthetic pathway involved in cell wall synthesis. earlier studies also documented that β-lactam antibiotics irreversibly acylate the active-site serine of pbps, which deprives bacteria of their biosynthetic functions and results in bacterial death (o’daniel et al., 2014). all these events halts bacterial viability thus mitigate infectivity of bacteria into the host cell. similar in-silico results citing antibacterial potential of polypharmacological natural agents like flavonoids, phenolics, steroids, and terpenoids, which have the ability to inhibit and kill bacteria strains have been, stated (soviati and widyarman, 2020, apriyanti and kurnia, 2020, kurnia and apriyanti, 2019, gartika and pramesti, 2018). anti-bacterial activity in the present study the in-vitro anti-bacterial activity of lgo was quantitatively assessed against drug resistant microbial strains of escherichia coli (mtcc-40), bacillus subtilis (mtcc-121), pseudomonas aeruginosa (mtcc-424) and staphylococcus aureus (mtcc-3160), the results of which are depicted in table-2 and figure 4. the present study shows that lgo exhibits substantial antimicrobial activity against gram negative escherichia coli (mtcc-40) while total inhibition was seen for gram positive bacillus subtilis (mtcc-121), pseudomonas aeruginosa (mtcc-424) and staphylococcus aureus (mtcc-3160) as indicated in figure 3. the variance action of lgo might be owing to the incidence of single target or multiple targets for their activity. the antimicrobial activity of lgo may arise due to the presence of major and minor bioactive component that affected hydrolytic enzyme inhibition (proteases) or inhibited partners like: cell wall envelop proteins, microbial adhesions, and non-specific interactions with carbohydrates (silva et al., 2003, siramon et al, 2007). earlier studies also have cited that anti-microbial activity was not always related to the high content of one major chemical compound, rather than to synergic effects between major and minor components (silva et al., 2003). siramon et al, (2007) also cited incidence of potent bioactive molecules like flavonoids, and terpenoids behind the antimicrobial activity. same authors cited that bioactive molecules have tendency to cross across the cell membranes and to induce biological reactions, thus upsetting electron flow, the proton motive force, active transport and coagulation of the cellular contents. lgo also exhibits high antifungal, insecticidal and bactericidal activity (chen et al, 2016). in present study high antimicrobial toxicity of lgo toward gram negative bacteria was observed which is a noteworthy observation as most studies suggests that the gram negative bacteria are more resistant than gram positive bacteria due to thick peptidoglycan layer, lipopolysaccharides, phospholipids, of cell wall that permit gram negative bacteria to be added resistant to most of the hydrophobic antibiotics and toxic drugs (su et al, 2006). table 2. antimicrobial analysis of lemon grass essential oil. strain strain type zone of inhibition (cm) c eo pseudomonas aeruginosa gram negative 3.0 fi escherichia coli gram negative 2.5 6.5 bacillus subtilis gram positive 2.6 fi staphylococcus aureus gram positive 2.5 fi here: c= positive control (vancomycin antibiotic, 10mg), eo= essential oil, fi: 100% inhibition, values are expressed as mean±sd (n=3), 230 biology, medicine, & natural product chemistry 12 (1), 2023: 225-232 figure 3. 3d interaction’s of pbp5 with ligands. a: geranial; b: limonene; c: linalool; d: myrcene; e: neral. figure 4. anti-bacterial activity of lgo against mtcc-121, mtcc-40, mtcc-424 and mtcc-3160. codenc, pc and lgo are for negative control (blank), positive control and lemongrass oil and codelgo-a, lgo-b, lgo-c and lgo-d represents mtcc-40, mtcc-121, mtcc-424, and mtcc3160 respectively. conclusions currently, antibiotic resistance against gram-positive and gram-negative bacteria has emerged in the human population, and is a potential threat to global health, worldwide. currently, the main target for bacterial infections is primarily pbps. the aim of this study was to examine bioactive molecules from lemon grass essential oil that may be used to inhibit the bacterial infection pathway. compositional analysis revealed the presence a b c d e nc-a pc-a lgo-a nc-b pc-b lgo-b nc-c pc-c lgo-c nc-d pc-d lgo-d sharma & kaur – chemical profile and in-silico docking studies on … 231 of bioactive compounds in lemon grass oil. in-silico docking depicted effective docking of all bioactive compounds. wet lab validation documented antibacterial role of lgo. therefore, we suggested that bioactives compounds from lemon grass essential oil may represent potential treatment options, and found in medicinal plants that may act as potential inhibitors of bacterial pbps. however, further studies should be conducted for the validation of these compounds using in vitro and in vivo models to pave a way for these compounds in drug discovery. conflict of interest: authors declares no conflict of interest. funding: dst govt of india. author contributions: ads: designed study, ijk: interpreted study. references apriyanti e, kurnia d. 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(2017). identification of a functionally unique family of penicillin-binding proteins. j am chem soc. 13:17727-17730 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 87-91 | doi: 10.14421/biomedich.2021.102.87-91 issn 2540-9328 (online) potential of tithonia diversifolia hemsley a. gray (kembang bulan) leaf extract as anti-cancer agents muflihah rizkawati department of pharmacology, faculty of medicine, universitas islam indonesia, indonesia. corresponding author dr.rizkawati@uii.ac.id manuscript received: 22 september 2021. revision accepted: 04 october, 2021. published: 07 october, 2021. abstract the main objective of this review is to explain the great potential of herbal plants as anticancer agents. cancer is a disease caused by abnormal cell growth in the body. the high number of cancer incidents still become a global concern because of the high mortality rate. the treatments of cancer such as chemotherapy can cause serious side effects by killing the normal cells. this is the reason why it is necessary to develop an alternative treatment of cancer. i discussed a plant that is believed to has health benefit. many studies have showed the positive effect of tithonia diversifolia plant for health. after 2000, the researchers discovered a new potential through its cytotoxicity to neoplastic cells. this plant needs to be developed sustainably. however, in the future this plant might become an effective alternative to treat cancer with lower side effects. keywords: tithonia diversifolia (hemsley) a. gray; kembang bulan; anticancer. introduction cancer is a disease with a multifactorial etiology which is initiated by an abnormal and uncontrolled expression of transcription factors. this can be affected by a gene mutation (babazadeh et al., 2018). cancer is one of the main causes of death in the world and still become a global concern. cancer has the second highest risk of death with a mortality ratio of 1:6 or around 9.6 million deaths in 2018. the most common types of cancer in men are lung, prostate, colorectal, stomach, and liver and the most common types of cancer in women are breast, colorectal, lung, cervical and thyroid cancers (who, 2021). cancer can develop in any part of the body and can spread (metastasize) to other parts of the body. although some types of cancer can be similar in some ways, how they develop, grow, spread, and respond to drugs can be very different (american cancer society, 2021). the research and development data in 2019 showed the incidence of cancer in indonesia reached 136.2/100,000 population and reach 8th place in southeast asia. the highest incidence rate of cancer for men is lung cancer, which is 19.4 per 100,000 population with an average death rate of 10.9 per 100,000 population, while the highest incidence rate of cancer for women is breast cancer, which is 42.1 per 100,000 population with an average 17 deaths per 100,000 population (kemenkes ri, 2019). cancer treatment methods are still being developed. there are several types of local treatment such as surgery and radiation therapy, and systemic treatment such as chemotherapy, immunotherapy, or targeted therapy that can affect not only cancer cells but also the entire body (american cancer society, 2021). understanding the causes, how they are formed, and especially the factors that promote or inhibit their growth required to solve the complex biological and medical problems. currently, there are many successful results from cancer treatment. for example, over the past half century, the survival rate for childhood leukemia has increased from 10% to more than 80%. but the healing method (or extended remission) is difficult, and is only available to those who living in affluent countries. there is nothing like "medicine" for cancer, and much remains for medical researchers to do (haussman, 2019). the cancer treatment ideally can stop the growth of cancer cells with a selective toxic effect on the targeted cancer cells without damaging other normal tissues. however, most of today's anticancer drugs are still potentially toxic to normal tissues. the condition can cause harmful side effects to the body. therefore, the discovery of anti-cancer drugs that have more positive effects with low toxicity is being developed. many potential phytopharmaceuticals have been investigated to find chemical elements in plants that are beneficial for treatment (wahyuningsih et al., 2013). the development of herbal studies using in vitro and in vivo methods has https://doi.org/10.14421/biomedich.2021.102.87-91 88 biology, medicine, & natural product chemistry 10 (2), 2021: 87-91 shown the beneficial effects of medicinal plants and their bioactive compounds. the most common anticancer mechanism is by inducing apoptosis in neoplastic cells. currently, more than 25% of drugs used over the last 20 years are derived directly from plants. according to who, 80 percent of the world's population has benefited from traditional medicine (aryan, 2018). nowadays, the efforts to develop new anti-cancer medicines and particularly novel drug delivery systems should be done. it is important to improve the pharmacodynamics and bioavailability of drugs and to deliver specific drug concentration to cancer cells so it can produce minimum cytotoxic effects on normal cells (babazadeh et al., 2018). tithonia diversifolia characterization the asteraceae family is known for its therapeutic effect such as antihelmintic, anti-inflammatory, astringent, antispasmodics, cholesteric, antioxidant, antihemorrhagic, antimicrobial, diuretics, and analgesics. one type of the species from this family is tithonia diversifolia (farias et al., 2019). t. diversifolia can be described as a flowering shrub-like species, growing up to over 2–3 m high. the flowers are 5–15 cm wide and shaped like a daisy. the leaves (15–30 cm long) can be described as sub-ovale, petiolate, 3to 7-lobed delicately hairy, and alternately to oppositely arranged (tagne at al., 2018). t. diversifolia (hemsl.) a. gray (mexican sunflower) originally from mexico and central america, but is now widely known in various parts of the world as an ornamental plant and a plant used for medicine. there are more than 150 types of secondary metabolites isolated from t. diversifolia, including the sesquiterpene lactones thyrotundin, tagitinin a, and tagitinin c (miranda et al., 2015). t. diversifolia can be found in tropical and subtropical climes. traditionally, all parts of the plant especially the leaves, are used by people to treat wounds, musculoskeletal disorders, abscesses, dermatological conditions, stomach pains, oral treatment for diabetes, malaria, fever, hepatitis and infectious diseases. some studies showed the heterogenous evidence from in vitro and in vivo research that supporting most of the traditional therapeutic claims. t. diversifolia can grow up for all year-round and used fresh or dry as traditional remedies in several cultures. all parts of the plant are useful include the leaves, flowers, stems and roots are mentioned in some ethnobotanical reports (tagne et al., 2018). the studies on phytochemical screening not only showed the presence of metabolites phenolics, flavonoids and tannins but also absence of alkaloids and saponins in t. diversifolia ethanolic flower extract. phenolics are well known for their antioxidants and antimicrobial properties. this plays a role in prevention activities of phenolic against infection and degenerative disease, inflammation and allergies through antioxidants, antimicrobial and protein or enzyme neutralization or modulation mechanism. the examples of phenolic compounds are flavonoids and tannins. flavonoids have many beneficial health effect and physiological properties. flavonoids have antioxidant effects, anti-inflammatory, anti-mutagenic, anticarcinogenic property and as a modulator to the function of cellular enzymes. it is also have a great potensial inhibitors of xanthine oxidase, cyclooxygenase, lipoxygenase and phosphoinositide 3-kinase. tannins has antioxidant, anticarcinogenic and antimutagenic potential. tannins are also known to accelerate blood clotting, lower blood pressure, lower serum lipid levels and modulation of immunoresponse. terpenoids has an antibiotic, chemopreventive and therapeutic effects on cancer. terpenoids have been developed in the prevention, inhibition and therapy of several diseases including cancer, antivirals, antimicrobial, anti-allergic, antispasmodic, antidiabetic, anti-inflammatory, anticancer and immuno-modulatory properties (gaule ang et al., 2019). farias et al, in 2019 also evaluate the composition, antioxidant, cytotoxic and microbiological activity of the essential oil of the leaves of the species t. diversifolia. the main constituent they was found are α-pinene (9.9%), limonene (5.40%), (z)-β-ocimene (4.02%), pcymen-8-ol (3.0%), piperitone (11.72%), (e)-nerolidol (3.78%) and spathulenol (10.8%). the activity of antioxidant from thee essential oil extracted from the leaves of t. diversifolia can be observed from the significant result with p < 0.0001 at the concentration of 5 mgml-1 with %aa 54.6 ± 0.06. the minimum inhibitory concentration (ic50) was 4.30 mgml-1 , with a strong correlation coeffificient (r2) of 0.9965. although t. diversifolia has good antioxidant activity, its ic value still lower than ascorbic acid (farias et al., 2019). anti-cancer potential activity-guided fractionation technique of an ethyl acetate extract from t. diversifolia, using an antiproliferative bioassay against human colon cancer cells (col2), resulted in the isolation of three new sesquiterpenoids, 2r-hydroxytirotundin (1), tithofolinolide (2), and 3r-acetoxydiversifolol. (3), together with eight known sesquiterpene lactones, 3'acetoxy-8'-isobutyryloxyreynosine (4), tagitinin c (5), 1',2repoxytagitinin c (6), 4r,10r-dihydroxy-3-oxo-8'isobutyryloxyguaia -11(13)-en-12,6r-olide (7), 3racetoxy4r-hydroxy-11(13)-eudesmen-12-oic acid methyl ester, 17,20-dihydroxygeranylnerol, tagitinin a, and thyrotundine. measurement of antiproliferative activity in col2 cells and induction of cellular differentiation in human promyelocytic leukemia (hl60) cells were carried out to determine the potential of these isolates as cancer chemopreventive agents. in this rizkawati – potential of tithonia diversifolia as anti-cancer agents 89 study, a culture test was used on rat breast organs that had been induced by 7,12-dimethylbenz[a]anthracene and the selected compound was tested for its ability to inhibit preneoplastic lesions. isolates tagitinin c and 1',2 repoxytagitinin c showed significant antiproliferative activity. isolat tithofolinolide, 3'-acetoxy-8'isobutyryloxyreynosine, and 4r,10r-dihydroxy-3-oxo8'-isobutyryloxyguaia -11(13)-en-12,6r-olide induced cellular differentiation of hl-60. the isolate 3'-acetoxy8'-isobutyryloxyreynosine significantly inhibited (63.0% at 10 μgml-1) lesion formation in rat breast organ culture assays. among these isolates there were several sesquiterpenoids and flavonoids that showed anti-human pro myelocytic leukemia (hl-60) activity and tagitinin c was the main sesquiterpenoid that had been identified (gu et al., 2002). the methanol extract of tithonia diversifolia showed antiproliferative activity against glioblastoma cells u373, with an ic50 value of 59.2 ±3.7 μgml-1, while tagitinin c showed an ic50 value of 6.1 ±0.1 μgml-1. based on flow cytometric analysis and pan-caspase inhibitory activity, the anti-glioblastoma effect was independent of apoptosis and it was concluded that tagitinin c induces u373 cell death through autophagy under certain conditions. autophagy is a transport pathway to all eukaryotic cells. cells degrade part of their own intracellular constituents, including cytoplasm and organelles. the first process for autophagi mechanism is the formation of a double membrane bound vacuole from endoplasmic reticulum. the autophagosomes receive hydrolases to form an autolysosome. this study also found that human glioblatoma u373 cell death induced by tagitinin c was apoptosis-independent and induced autophagosome (lee at al., 2011). another study showed the activity of the extract t. diversifolia in inhibiting the viability of keloid fibroblasts. antifibrotic activity of the ethanolic extract of t. diversifolia on keloid fibroblast cell proliferation and collagen accumulation was expressed by the ic50 value using probit regression analysis. the percentage of inhibition of keloid fibroblast proliferation increases gradually with the given dose extract of t. diversifolia with a very strong correlation (r = 0.838; p = 0.000 in 72 hours; r = 0.924; p = 0.000 in 120 hours of incubation) with the lowest ic50 value of 3.624 μgml-1 at an incubation time of 120 hours (wahyuningsih et al., 2015). furthermore, ranti et al, in 2018 tested the cytotoxic activity of tagitinin c on keloid fibroblast cells with ic50 values of 0.122 μgml-1 (72 hours incubation) and 0.039 μgml-1 (120 hours) which affected the deposition of 53.1% of keloid collagen (72 hours) and 44.3% (120h). the selectivity index of tagitinin c in normal fibroblasts (nf) was 287 (72 hours incubation) and 791 for (120 hours incubation). based on these results, it was concluded that the ability of tagitinin c to inhibit keloid fibrosis viability and reduce keloid collagen deposition was consistent with the concentration and incubation time. the process of collagen synthesis happened when the fibroblast got a stimulus from tgf-β that was bound to tgf-β receptors on the fibroblasts membrane. when an injury occured, the tgf-β will be separated from the binding protein give respond to mechanical trauma system. however, there have not been many studies on the inhibitory activity of tgf-β by t. diversifolia compound. but, another study have found that the sesquiterpene lactones is known to inhibit the keloid fibroblasts proliferation, inhibit tgf-β1, and inhibit collagen synthesis of keloid through the nf-κb (ranti et al., 2018). the next study was carried out by wahyuningsih, et al in 2019 to determine the antifibrocytic mechanism of t. diversifolia extract. the study showed that the ethanolic extract of t. diversifolia at concentrations of 20 gml-1, 10 gml-1, and 5 gml-1 for 24 hours in keloid fibroblast culture showed slower migration activity compared to untreated keloid fibroblast culture (p<0, 05). the study also showed that the expression of tgfβ1 and vegf in the treatment group given the ethanolic extract of t. diversifolia was significantly lower than untreated keloid fibroblasts (p<0.05). from those result, it can be concluded that the ethanolic extract of t. diversifolia can inhibit fibroblast migration activity and reduce the expression of vegf and tgf-β1 in keloid fibroblasts. this mechanism further strengthens that t. diversifolia has great potential to be used as a keloid therapy (wahyuningsih et al., 2019). traditionally, t. diversifolia have been used to treat chronic hepatitis and liver diseases in chinese herbal medicine. further researh must be done to explore whether t. diversifolia can be used to treat hepatoma. (lu et al., 2017). the study of the cytotoxic activity of the methanol extract of the leaves of t. diversifolia and tagitinin c against human hepatoma cells (hep-g2) showed positive results. the test results with the mtt assay showed ic50 values 40.0 ± 2.0 and 2.0 ± 0.1 μgml-1. this compound induces an increase population in the sub-g1, exhibited s phase arrest and results in the activation of caspase 3 and caspase 8 which confirms the data that the antiproliferative effect of this compound is through caspase-dependent apoptotic activity. tagitinin c caused membrane blebbing, cell shrinkage, nuclear fragmentation, and chromatin condensation in hepatoma cells. caspases, have an essential role in the regulation and the execution of apoptotic cell death. caspase 3 well known as a key effector caspase in the apoptosis pathway, amplifying the signal from initiator caspases (such as caspase 8). this study show that tagitinin c induced apoptosis done by the activation of the caspase cascade (liao at al., 2012). a follow-up study was conducted by lu in 2017 to showed another cytotoxic effect of t. diversifolia on hep g2 cells. the results of the study showed that the induction of apoptosis such as dna fragmentation could increase the level of apoptosis and apoptosis of the sub-g1 population in the treatment group that given the acetate extract leaves of t. 90 biology, medicine, & natural product chemistry 10 (2), 2021: 87-91 diversifolia. in addition, the acetate extract of the leaves of t. diversifolia was also increase the expression of the proapoptotic protein bax bcl-2. based on this study, it prove the cytotoxicity induced by ethyl acetate extracts of t. diversifolia leaves (td-l-ea) in human hepg2 hepatoma cells that strengthen ethnopharmocologic use of t. diversifolia (lu at., 2017). the cytotoxic effect of t. diversifolia leaf extract on raw264.7 cells and human mononuclear peripheral blood cells (pbmc) by mitochondrial respiration method was also studied. the results of the study showed that the ic50 concentration in pbmc proliferation was 4.42 μgml-1 and in raw264.7 cells the ic50 value was 11.63 μgml-1. the study demonstrated an immunomodulating effect by the leaf extract of t. diversifolia, which was produced the inhibition of phytohemagglutinin-m-induced pbmc proliferation and lps-induced nitric oxide production in raw264.7 macrophages. the hydrogen peroxide are able to induce cell death under stress conditions through apoptosis, but the anti-oxidant compound can protect this condition. the data showed that t. diversifolia aqueous extract showed an anti-oxidative capacity equivalent of (241.04 ± 11.93 mmol troloxg-1) from abts assay and (94.89 ± 2.69 mmol troloxg-1) from dpph assay, respectively. the anti-oxidative capacity of t. diversifolia aqueous extract was equivalent to (32.62 ± 1.87) and (20.99 ± 2.79) mg nacg-1 in the abts and dpph assays, when nac was used as the standard compound. however, the calculated concentration was out of the non-cytotoxic range, which should be lower than 30 mg/ml. thus, this calculated concentration based on the antioxidant capacity could not be used in practice in the hydrogen peroxide-induced cell death model. it could be conclude that the effects of the immunomodulation caused by the aqueous extract from t. diversifolia. the mechanism can desccribe such as anti-pha-m-induced pbmcs proliferation and antilpsinduced nitric oxide production from macrophages in the range of non-cytotoxic concentrations (hiransai et al., 2016). conclusion based on the literature study, it is found that some isolate from the extract of tithonia diversifolia (hemsley) a. gray) have the anticancer activity in several different types of cancer cells. the active substances contained in the leaves of the t. diversifolia. the compound material which has been known to become an anticancer alternative therapy is tagitinin c. the extract of the leaves t. diversifolia has several anticancer mechanisms related to 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of tithonia diversifolia (hemsley) a.gray) against hela cells. traditional medicine journal. vol. 18(1), p 2228. wahyuningsih, m., wirohadidjojo, y., hidayat, r., sadid, a. 2015. antifibrotic effect of standardized ethanol extract of tithonia diversifolia (hemsley) a. gray on keloid fibroblasts. international journal of pharmacognosy and phytochemical research. 7(4); 642-647. wahyuningsih, m., nugrahaningsih, d., budiyanto, a.2019. ethanolic extract of tithonia diversifolia (hemsley) a. gray inhibits migration activity and decrease the transforming growth factor-beta1, vegf expression on keloid fibroblasts. asian journal of pharmaceutical and clinical research. 12(1): 342-345. doi: http://dx.doi.org/10.22159/ajpcr.2019.v12i1.29850 world health organization, 2021. cancer. https://www.who.int/health-topics/cancer#tab=tab_1 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1033-1038 | doi: 10.14421/biomedich.2025.142.1033-1038 issn 2540-9328 (online) potential of fig fruit ethanol extract (ficus carica l.) against staphylococcus aureus mrsa and klebsiella pneumoniae esbl bacteria enmia septia padang, rini hafzari, endang sulistyarini gultom* department of biology, faculty of mathematics and natural sience, universitas negeri medan, william iskandar ps. v. medan, north sumatera, 20221 indonesia. corresponding author* endanggultom@unimed.ac.id manuscript received: 26 june, 2025. revision accepted: 04 november, 2025. published: 17 november, 2025. abstract ficus carica l. commonly known as figs originate from west asia and have bioactive components with antibacterial properties. this study aimed to determine the antibacterial activity of fig ethanol extract in inhibiting the growth of mdr staphylococcus aureus mrsa and klebsiella pneumoniae esbl bacteria, to determine the mic and mbc values, and the types of compounds contained in fig ethanol extract. the method began with fig extraction using concentrations of 20%, 40%, 60%, 80%, and 100% with 4 repetitions, testing mic and mbc using the liquid dilution method, tlc-bioautography test with ethyl acetate: methanol: aquades (7: 2: 1) eluent. the results showed that fig fruit extract had an inhibition zone with an average value of 3.30 mm at a concentration of 100% which was still in the weak category, the mic value of staphylococcus aureus mrsa bacteria was a concentration of 100% and klebsiella pneumoniae esbl bacteria was a concentration of 80%. the mbc value of mdr bacteria was not obtained because there was still bacterial growth. the results of the tlc-bioautography test of ethanol extract of fig fruit (ficus carica l.) can inhibit bacterial growth indicated by the presence of a clear zone with an rf value of 0.5 cm (unidentified), 0.31 cm (flavonoid), 0.2 cm (phenol). the compounds that act as antibacterials against staphylococcus aureus mrsa and klebsiella pneumoniae esbl bacteria are the flavonoid and phenol groups. keywords: fig fruit (ficus carica l.); antibacterial activity; mic; mbc; tlc-bioautography. introduction infectious diseases are diseases caused by pathogenic microorganisms such as bacteria. infectious diseases can also be caused by various microorganisms such as bacteria, viruses, and fungi are organisms that can attack the body (darsono et al., 2020). currently, many pathogenic bacteria are resistant to several antibiotics, known as mdr (multi drug resistant) bacteria. indonesia is ranked 8th out of 27 countries with the highest multi-drug resistant predicate in the world (estiningsih et al., 2020). one of the staphylococcus aureus mrsa bacteria is a grampositive bacterium that is pathogenic and is often found in contaminated milk (ramadhan et al., 2020). another bacteria that can cause infectious diseases is klebsiella pneumoniae esbl bacteria, which is a gram-negative bacterium from the enterobacteriaceae family that often causes lung infections (pneumonia), urinary tract infections (utis), and gastrointestinal infections (kurama, et al., 2020). mdr (multi drug resistant) bacteria is a condition where bacteria are resistant to more than one antibiotic. mdr (multi drug resistant) bacteria can be caused by several things, namely the use of antibiotics with inappropriate doses and diagnostics. the types of bacteria detected include staphylococcus aureus, streptococcus pneumoniae, neisseria sp., escherichia coli, shigella sp., salmonella sp., enterobacter aerogenes, pseudomonas aerogenosa, and klebsiella pneumoniae (kurniawan et al., 2024). one type of medicinal plant that has many benefits is figs (ficus carica l). figs have a high nutritional content and have the potential as a medicinal ingredient (nugraha, 2020). figs (ficus carica l.) are plants that have antibacterial activity against gram-positive and gram-negative bacteria, and have been shown to have health benefits. the benefits of figs in the scientific scope and pharmacological activity are used as antibacterials, antioxidants, antitumors, while for traditional medicine they are used to treat ulcers, digestive disorders, and diarrhea (ramadhanti, 2023). figs are an important source of bioactive components such as phenols, benzaldehyde, terpenoids, flavonoids, and alkaloids which have antioxidant properties (ramadhan et al., 2020). research on figs (ficus carica l) as an antibacterial is still minimal and there is still a lot of bacterial resistance to antibiotics, the author needs to develop an antibacterial compound as a basis for conducting https://doi.org/10.14421/biomedich.2025.142.1033-1038 1034 biology, medicine, & natural product chemistry 14 (2), 2025: 1033-1038 research on the antibacterial activity test of ethanol extract of figs (ficus carica l) against mdr bacteria using the tlc bioautography method. materials and methods place and time of research the research was conducted at the microbiology laboratory and chemistry laboratory of the faculty of mathematics and natural sciences (fmipa) state university of medan, the research was conducted in december 2023 march 2024. extraction of fruit the figs (ficus carica l.) used in this study were fresh and not rotten. the collected figs were cleaned with running water. the sampling technique used was random, namely simple random sampling method. figure 1. fig fruit (ficus carica l.). the study conducted to obtain fig extract using the squeezing method. the squeezing method was done by blending the figs. fig extraction was done by the maceration method. maceration was done 2 times. 2,500 grams of finely ground figs are put into a glass jar, then soaked in 2,500 ml of ethanol solvent p.a with a solvent ratio of 1: 1 (w / v). the first extraction process was carried out for 5 days and stirred every 24 hours. the extract was filtered using filter paper to separate it from the pulp. the second maceration is carried out with a ratio of 1: 0.5. maceration was carried out twice. the extract results were combined and concentrated using a rotary vacuum evaporator at a temperature of 40 ℃ until a thick extract is obtained. bacterial rejuvenation two grams of nutrient agar (na) media was taken, dissolved in 100 ml of distilled water and put into an erlenmeyer flask, heated on a hotplate until dissolved and homogeneous, then the dissolved na media was put into a sterile test tube. the na media that had been put into the test tube was sterilized by autoclaving at a temperature of 121℃ for approximately 15 minutes. the media was left at room temperature until it solidified in a tilted position, when scratching the bacteria, the tube was brought close to the bunsen, covered with cotton and incubated for 24 hours at a temperature of 37℃ all done aseptically in laminar airflow. antibacterial activity of ethanol extract of fig fruit this antibacterial activity test was carried out using the kirby-bauer method or disc diffusion method. a total of 10 ml of mueller hinton agar (mha) media was inserted into a petri dish, scratching the bacteria evenly into the petri dish using a sterilized cotton bud, placing a 6 mm diameter disc paper that had been soaked with fig extract with variations of 20%, 40%, 60%, 80%, 100%. in this study, the negative control used was the solvent used as a diluent for the extract used in the study, namely ethanol, the positive control used was chloramphenicol. the petri dish was incubated at 37℃ for 24 hours. minimum inhibitory concentration (mic) mic was conducted using the modified kirby and bauer liquid dilution method (fatisa, 2013). a total of 4 ml of sterile nb media was put into each test tube, and 0.5 ml of extract was added with each extract having 5 concentrations (20%, 40%, 60%, 80%, 100%), then the tubes were incubated for 24 hours at 37°c in an incubator. after incubation, the bacterial absorbance was measured again using a spectrophotometer. minimum bacterial concentration (mbc) mbc was further tested, namely on all tubes used in mic, by taking 0.2 ml of the suspension that showed the mic, then put into a test tube containing 5 ml of sterile nb media. the test tube was incubated for 24 hours at 37°c in an incubator, then the absorbance value was measured using a spectrophotometer. thin layer chromatography separation of fractions will occur when a solvent was added as a mobile phase, three solvents used are ethyl acetate, methanol and aquades with a ratio of (7: 2: 1). the mobile phase was put into a glass bottle (chamber). the chromatogram is made by cutting the tlc plate at a size of 8 x 2 cm, the upper and lower boundaries are marked using a pencil (0.5 cm upper limit and 1 cm lower limit). ethanol extract of figs was spotted using a capillary pipette on the tlc plate, spotted using a capillary pipette. the tlc plate that had been spotted was inserted into the chamber and eluted, after the solvent had reached the upper limit, the tlc plate was removed and dried, the spots that emerged were observed with uv light at a wavelength of 366nm (hidayat et al., 2017). padang et al. – potential of fig fruit ethanol extract (ficus carica l.) against 1035 tlc-bioautography testing the solidified mha media was swabbed with staphylococcus aureus mrsa and klebsiella pneumoniae esbl bacteria using a sterile ose needle, then the media was left for 10 minutes so that the bacterial suspension could be absorbed, after solidifying and absorbing, the eluted tlc plate was placed and pressed slightly on the surface of the media, after 30 minutes the tlc plate was lifted then the mha media was incubated at 37℃ for 24 hours, the inhibition zone formed on the surface of the media was observed. results and discussion antibacterial activity test the study revealed that fig fruit extract (ficus carica l.) has antibacterial activity against staphylococcus aureus mrsa and klebsiella pneumoniae esbl bacteria. the resulting thick extract is brown in color with a weight of 149.81 gr. the results of the observations can be seen in figure 2. figure 2 comparison of inhibition zone results formed from ethanol extract of fig fruit (ficus carica l.) description: positive control: chloramphenicol antibiotic, negative control: ethanol solvent p.a. based on the data obtained, it can be said that each ethanol extract of fig fruit (ficus carica l.) has the ability as an antibacterial against staphylococcus aureus mrsa and klebsiella pneumoniae esbl bacteria, indicated by the presence of an inhibition zone formed around the disc paper in the inhibition test that has been carried out, with an incubation time of 24 hours with an incubation temperature of 37℃. based on the data, the extract of fresh fruit juice has the potential to inhibit the growth of gram-positive and gram-negative bacteria which is still in the weak category. a b figure 3. antibacterial activity test of ethanol extract of figs against staphylococcus aureus mrsa bacteria. figure 4. antibacterial activity test of ethanol extract of figs against klebsiella pneumoniae esbl bacteria. the diameter of the inhibition zone formed does not always increase with the increase in the concentration made, this is due to differences in the diffusion rate of active antibacterial compounds in agar media (wahlanto et al., 2020). higher concentrations do not always provide a greater inhibitory effect but have a smaller inhibitory ability compared to other concentrations (zeniusa et al., 2019). several possibilities that cause this to happen, such as the lack of diffusion power of the extract into the media. the higher the concentration of the extract, the lower the solubility (thickens like a gel) so that it can slow down the diffusion of the active ingredients of the extract in the media so that it can reduce the ability of the extract. inhibition zones with small diameters have low antibacterial activity, while inhibition zones with large diameters have high antibacterial activity. the components of substances, quality and quantity contained in medicinal plants that can weaken, strengthen, improve, or change the inhibition zone cause a large increase and decrease (afriani et al., 2024). minimum inhibitory concentration (mic) mic value testing was carried out using a uv-vis spectrophotometer at a wavelength of 625 nm to measure the absorbance value (turbidity value) before and after incubation in the test tube. the results of the observations are seen in figure 5 1036 biology, medicine, & natural product chemistry 14 (2), 2025: 1033-1038 figure 5. graph of absorbance value of mic test. based on figure 5, the mic value in staphylococcus aureus mrsa bacteria is at a concentration of 100% with an average of -0.0476 while in klebsiella pneumoniae esbl bacteria at a concentration of 80% with an average of -0.0315. the amount of absorbance or absorption depends on the content of the substance in it. the more the content of the substance in the sample, the molecules that will absorb light with a certain wavelength which causes the resulting absorbance value to be greater, in other words the absorbance value is equivalent to the concentration (yanlinastuti, 2016). in the table, negative absorbance values indicate a decrease in the absorbance value, indicating a decrease in the number of cells that have been incubated, while positive absorbance values indicate no decrease in the absorbance value, indicating that the number of bacterial cells is still increasing after incubation. minimum bactericidal concentration (mbc) minimum bactericidal concentration (mbc) was carried out by taking the tube from the mic test results. based on the results of the research that has been carried out, the mbc value was not obtained because the absorption value after incubation showed a significant increase that there was still bacterial growth. the increase in absorbance value depends on the level of substance contained in the solution, the greater the level of substance contained, the more molecules will absorb light at a wavelength. at a concentration that experiences a decrease in the average absorbance value, because the molecules are in small quantities to absorb light coming at a certain wavelength (putri et al, 2023). determination of mic and mbc in this antibacterial test was carried out using the liquid dilution method. the advantage of using this method is the use of more efficient media. the parameters used are turbidity (there is bacterial growth) and clarity (no bacterial growth), which can be seen after incubation for 24 hours. however, this tends to be subjective, so the absorbance values before and after incubation are used to help determine the presence or absence of bacterial growth using a uv-vis spectrophotometer. the advantage of the spectrophotometric method is that this method provides a simple way to determine the quantity of a very small substance, and the results obtained are quite accurate, where the numbers read are directly recorded by the detector and printed in the form of digital numbers or graphs that have been regressed. the weakness of the uv-vis spectrophotometer is that it cannot distinguish samples from other particles that absorb light at the same wavelength, it can also be caused by the amount of light absorbed by the solution in the tube is not completely absorbed by the extract compound, but is also absorbed by dead bacterial cells (sekeon et al., 2018). the concentration of the sample affects the absorption of its turbidity so that the higher the concentration, the higher the absorption. thin layer chromatography (tlc) from the results of tlc-bioautography on ethanol extract of figs (ficus carica l.) there are secondary metabolite compounds that have the potential to be antibacterial in staphylococcus aureus mrsa and klebsiella pneumoniae esbl bacteria, namely the flavonoid and phenol compound groups. according to gultom 2020, compounds that act as antibacterials against klebsiella pneumoniae esbl bacteria are alkaloid, flavonoid, and phenol compounds. flavonoids are polyphenol compounds that function as antibacterial compounds by disrupting the integrity of bacterial cell membranes. phenol compounds are one of the compounds that are antibacterial, with their performance that can damage bacterial cell walls so that they have the potential to be natural antibacterials in pathogenic bacteria (anwariyah, 2011). a b figure 6. results of spots on uv light 366 after spraying reagents: a. after spraying 𝐴𝑙𝐶𝑙3 b. after spraying 𝐹𝑒𝐶𝑙3 according to research conducted by irwan (2017), spraying with the reagent 𝐴𝑙𝐶𝑙3 is marked by the presence of a yellow stain color, indicating that the compound is positive for flavonoids. flavonoids are polyphenol compounds produced by plants, various studies have been conducted that flavonoid compounds have been proven to have antibacterial activity. the activity of flavonoid compounds as antibacterials is stimulated through molecular mechanisms, namely they padang et al. – potential of fig fruit ethanol extract (ficus carica l.) against 1037 can damage the sheath of gram-negative and grampositive bacteria. flavonoid compounds are also thought to be able to damage bacterial cell components (cahyani et al., 2023). detection of phenol content in solution with color changes in the solution after being dripped into green, red, jet black, blue, or purple. spray reagent 𝐹𝑒𝐶𝑙3 will produce blue, red purple, dark brown, green and strong black to determine phenol compounds. spray reagent 𝐹𝑒𝐶𝑙3 will produce blue-black or green-blue to determine tannin compounds (mariana et al., 2013). tlc-bioautography the results of the tlc bioautography test of ethanol extract of figs (ficus carica l.) against staphylococcus aureus mrsa and klebsiella pneumoniae esbl bacteria can be seen in figure 7 and figure 8. figure 7. results of tlc bioautography test of ethanol extract of figs (ficus carica l.) on staphylococcus aureus mrsa bacteria. testing the antibacterial activity of fig fruit ethanol extract using the tlc bioautography method showed positive results against staphylococcus aureus mrsa bacteria. figure 7 shows spot 1 which has the potential to be an antibacterial with an rf value of 0.53 cm whose chemical compound was not identified and spot 2 has the potential to be an antibacterial with an rf value of 0.31 cm which is suspected of containing secondary flavonoid metabolite compounds. a b figure 8. results of tlc bioautography test of ethanol extract of fig fruit (ficus carica l.) on klebsiella pneumoniae esbl bacteria (a) 𝐴𝑙𝐶𝑙3 (b) 𝐹𝑒𝐶𝑙3 tlc bioautography showed positive results against klebsiella pneumoniae esbl bacteria. figure 8 shows that spots that have the potential to be antibacterial are spots 2 with an rf value of 0.31 cm which is suspected of containing secondary metabolite compounds of flavonoids, then sprayed with the reagent 𝐴𝑙𝐶𝑙3 showed positive results and the appearance of reddish yellow fluorescence spots under uv 366 nm, and spots 3 with an rf value of 0.2 cm are suspected of containing secondary metabolite compounds of phenol, sprayed with the reagent 𝐹𝑒𝐶𝑙3 and showed positive results with the formation of a green color. conclusion the ethanol extract of fig fruit (ficus carica l.) has antibacterial activity against staphylococcus aureus mrsa and klebsiella pneumoniae esbl bacteria with a weak category. the minimum inhibitory concentration (mic) in staphylococcus aureus mrsa bacteria is 0.0476 at a concentration of 100%, while the value in klebsiella pneumoniae esbl bacteria is -0.0315 at a concentration of 80%. the mbc value in staphylococcus aureus mrsa and klebsiella pneumoniae esbl bacteria cannot be found because it increases after incubation, which indicates that there is still bacterial growth so that the bacteria cannot be killed. secondary metabolite compounds that have the potential to be antibacterial found in fig fruit (ficus carica l.) are flavonoid and phenol compounds. competing interests: the 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(2016). pengaruh konsentrasi pelarut untuk menentukan kadar zirkonium dalam paduan uzr dengan menggunakan metode spektrofotometri uv-vis. pengelolaan instalasi nuklir. aces journal, 9(17) zeniusa, p., ramadhani, m. r., nasution, s. h., karima, n., (2019). uji daya hambat ekstrak etanol teh hijau terhadap escherichia coli secara in vitro. majority. 8(2): 136-143 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 143-150 | doi: 10.14421/biomedich.2023.121.143-150 issn 2540-9328 (online) phytochemical, antioxidant screening, antinociceptive, and anti-inflammatory activities of boswellia dalzielii hutch (burseraceae) root ethanol extract using animal model macdonald idu1, anthonia omoregbee1, benjamin ogunma gabriel1,2,* 1phytomedicine unit, department of plant biology and biotechnology; 2science laboratory technology, university of benin, pmb 1154, benin city, edo state. nigeria. corresponding author* alexthemain076@gmail.com manuscript received: 28 september, 2022. revision accepted: 05 december, 2022. published: 14 january, 2023. abstract this study investigated the biological activities and phytochemical screening of boswellia dalzielii root ethanol extract. standard procures were used to evaluate the phytochemicals and antioxidant capacity, antipyretic activity in baker’s yeast-induced pyrexia in mice, analgesic property (hotplate and acetic acid-induced in mice), acute anti–inflammation (carrageenan-induce in rats) and chronic arthritis (formalin– induced in rats) on boswellia dalzielii root ethanol extract. the phytochemical results revealed the presence of phenol, ascorbic acid, flavonoids, alkaloids, cardiac glycoside, tannin, saponin. the extract had a significant reduction in the body temperature in graded doses and 100 mg/kg paracetamol at 60 minutes when compared with the control, but 400 mg/kg was more effective (p<0.01). morphine and plant extract showed a slight significant analgesic property at 0 and 30 minute compared to the control. the extract at 100 mg/kg elicited a significant increase at 60 and 90 minutes compared with the control, and it is comparable to 5 mg/kg morphine. the plant extract (100, 200, and 400 mg/kg) and aspirin (100mg/kg) shows significant analgesic properties compared to control (p<0.01) but 200 mg/kg of extract revealed highest percentage inhibition. the extract produced no significant reduction on carrageenan induced inflammatory at all dose level compared to control (p>0.05). the plant extract (100, 200, 400 mg/kg) and indomethacin (1 mg/kg) reduced paw volume across the doses from day 4 compared to the control (p<0.01). the boswellia dalzielii root extract is a promising anti–inflammatory agent, it also possesses antipyretics, and analgesics effect validating the folklore claim. keywords: phytochemicals, ethanol; boswellia dalzielii; pyrexia; anti-inflammatory; mice. introduction phytochemicals with medicinal properties are also known as secondary metabolism. it ranges from cell wall substances through photosynthesis pigments, terpenes and terpenoids, the alkaloids, plants phenolics to plant hormones, plant non-proteins, amino acids and cyanogenic glycosides (harborne, 1973). many phytochemicals have been shown to be bioactive, that is they exhibit pronounced biological activity in other living organisms (harborne, 1973). many of the bioactive phytochemicals have found usefulness as chemotherapeutic agents, pesticides, food additives, and other biological (tripathi and tripathi, 2003; odesanmi et al., 2009). modern medicine recognizes herbal medicine as a form of alternative medicine, as the practice of herbal medicine is not strictly based on evidence gathered using the scientific method. modern medicine, however, make use of many plant-derived compounds as the basis for evidence-tested pharmaceutical drugs, and phytotherapy works to apply modern standards of effectiveness testing to herbs and medicines that are derived from natural sources. boswellia dalzielii hutch (burseraceae) commonly known as the frankincense tree. the bini called it ebanarukhoe, fulani called it januhi, while the hausa called it ararrabi, basamu and hanu. the stem bark secretes a fragrant white gum that is burnt to fumigate cloth and to drive out flies, mosquitoes, antiseptic, fever, rheumatism, gastrointestinal, antidotes for arrow poison, giddiness, palpation, cancers/fibrosis, inflammation, snake bite, arthritis, asthma, microbes, ulcer, syphilis and diarrhea (nwinyi et al., 2004; moses et al., 2005; abubakar et al., 2007; bello et al., 2013). bello et al., 2013 reported that the phytochemicals detected in ethanol and aqueous stem bark extracts include; flavonoids, terpenoids, alkaloids, tannins, saponins, quinones, and cardiac glycosides. this study investigated the phytochemical screening, antipyretic, analgesic, and anti-inflammatory activity of boswellia dalzielii ethanol extract. https://doi.org/10.14421/biomedich.2023.121.143-150 https://en.wikipedia.org/wiki/alternative_medicine https://en.wikipedia.org/wiki/evidence https://en.wikipedia.org/wiki/scientific_method https://en.wikipedia.org/wiki/pharmaceutical_drug https://en.wikipedia.org/wiki/phytotherapy 144 biology, medicine, & natural product chemistry 12 (1), 2023: 143-150 materials and methods collection of plant material the fresh root of boswellia dalzielii was collected from makaya forest, kibiya local government area, kano state in the mouth of may, 2015. the plant root was identified and authenticated by dr. timothy odaro in the herbarium unit of the department of plant biology and biotechnology, with the voucher number ughk257. the roots were chopped, air dried for four weeks and ground to course powder. the dried seeds were ground to powder using a mechanical grinder. preparation of plant extract the dried powder was weighed (100 g) and then macerated in 2.5 liters of ethanol for 72 hours. the solution of the root was decanted into another flask. the solution was filtered and concentrated at 40oc by means of a rotary evaporator. the evaporated extract was transferred into an oven at 40oc and evaporated to dryness for 24 hours. the concentrated extract was transferred into a sample bottle of known weight. the weight of the ethanol root extract was 111.5 g giving a yield of 15 %. phytochemical screening the preliminary qualitative and quantitative phytochemical test was screening and identification of bioactive chemical constituents in the ethanol root extract of boswellia dazielii under study that was carried out in extract. specimens using follows the standard procedures as described by sofowora (1993) and tiwari et al. (2011); naredra devanboyina et al. (2013). experimental animal adult swiss mice and wistar rats of male and female sexes weighed between (20 – 30 g) and (180-220 g) were obtained from the animal house, department of pharmacology, faculty of pharmacy, university of benin, benin city. they were acclimatization for 14 days. they were housed in a well-ventilated woody cages in a normal laboratory state (12 hours light/dark cycle: 23 ± 2°c) and fed using a standard diet. food and water were administered at free choice (ad libitum) to the animals designed for experiments. the animals were properly handled using the ethics of laboratory animals’ approval from the ethical committee of the faculty of life sciences with the ethical number ls21592. experimental design the animals were transferred into the laboratory 24 hours before the commencement of the experiment and were randomly grouped into five (n=5). this was done based on the design protocol of the study incudes: the groups for antipyretic study, rectal temperature were taken in order to accustom handling and the environment. the animals were exposed to natural lighting conditions and a room temperature of between 22-26oc and has free access to water and laboratory diet. the animals were carefully handled and the ethical committee of the faculty of life sciences issued an ethical number ls20619. antipyretics activity the animals had their rectal temperature measured, using a sfda lubricated probe digital thermometer (kft–04). a stock solution of 30% (w/v) of baker’s yeast in 0.9% nacl solution was prepared. the intended doses were 10ml/kg and the doses to be administered to individual mouse were calculated with respect to their weights. the different doses were administered intraperitoneally and the time of administration was noted. the different groups were observed for 6 – 7 hours (bafor et al., 2010). at the 7th hour, the rectal temperature was taken for the different groups and a calculated dose of 0.9% nacl solution (control), extract and paracetamol (standard). the control group received 10 ml/kg of nacl orally while the extract groups received 100, 200, 400 mg/kg orally and the standard group received 100 mg/kg of paracetamol intraperitoneally. the different groups were observed for change and sign and their rectal temperature were recorded for 2 hours at 30 minutes hour interval (bafor et al., 2010). analgesic property hot plate method the paws of mice are very sensitive to heat at temperature which is not damaging the skin. the responses are jumping, withdrawal of the paws and licking of the paws. the hot plate (ugo basile hot/cold plate 35100) consist of electrically heated surface, the temperature is controlled to 55.5oc. the animals were placed on the hot plate for 30 minutes before the experiment in order to accustom the environment, after which each animal was placed on the hotplate and the time until either licking or jumping is recorded (shanmugasundaram and venkataraman, 2005). the control group received 10 ml/kg 0.9% nacl orally, while extract groups received 100, 200, 400 mg/kg orally and the standard group received 5 mg/kg morphine intraperitoneally. after 30 minute the animals were placed on the hot plate and the observation were recorded and the time interval of 60, 90 and 120 minutes for all groups. the result of the hot plate method was tabulated. acetic acid induced writhing method the writhing model represents a chemical nociceptive test based on the induction of peritonitis like condition in animals by injecting irritant substances intraperitoneally. the control group received 10ml/kg 0.9% nacl orally, while extract groups received 100, 200, 400 mg/kg and the standard control received 100 mg/kg of aspirin orally and 30minute later all groups idu, et al. – phytochemistry, biological activity of boswellia dalzielii hutch (burseraceae) 145 received 10mg/ml of 1% acetic acid solution intraperitoneally (vogel, 2002). the mice were placed individually into glass beakers and immediately were observed for a period of 20 minutes and the numbers of writhes were recorded in each animal. for scoring purpose, a writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb percentage inhibition was calculated using the following formula: % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = {(𝑊𝑐 − 𝑊𝑡) × 100} 𝑊𝑐 where, 𝑊𝑐 : no. of writhes in control group, 𝑊𝑡 : no. of writhes in test group. carrageenan-paw induced edema in evaluating carrageenan-induce paw edema, 25 wistar rats were randomly divided into 5 groups (n=5). the control group was treated with distilled water, 10 ml/kg, the test groups and graded doses (100, 200 and 400 mg/kg) of the treatment groups, while the standard group received indomethacin (10 mg/kg) orally. after one hour, 0.1 ml/kg of 1% carrageenan was injected into the subplantar tissue of the right hind paw (vogel, 2002). paw edema was measured using a vernier caliper at 30, 60, 120, 180, and 240. formaldehyde induced arthritis arthritis was induced by injecting 0.1 ml of 1% of formaldehyde into the hind paw of albino rat using the model described by fatima and fatima (2016). twentyfive (25) wistar rats weighing 180-220 mg/kg were randomly divided into 5 groups (n=5). group 1 received the vehicle (distilled water) (1 ml/kg) without arthritis (normal control). group 2 received the vehicle (distilled water) with arthritis. group 3 received the standard drug dexamethasone (2 mg/kg). group 4, 5, 6 and 7 received the extract (100, 200, and 400 mg/kg). before administration of the standard and test drug, the rat was induced arthritis by injecting 0.1 ml of formaldehyde into the hind paw of the animal. at the third day, formaldehyde was re-injected again into the hind paw followed by treatment with the test agent. the thickness of the paw was measured at days 2, 3, 4, 5, 6, 7, 8, 9 and 10. statistical analysis data were expressed as the mean ± sem. the data were analyzed using one-way analysis of variance (anova) followed by tukey's post hoc test. statistical analysis was performed using graph pad prism version .6.01. p < 0.05 was considered significant results and discussion the qualitative and quantitative phytochemistry, antioxidant, identification and in vitro antioxidant property of boswellia dalzielii root ethanol extract. table 1 showed the presence of alkaloid, flavonoid and cardiac glycoside in abundantly while carbohydrate, saponin and tannin were moderately present. the total antioxidant capacity was shown in figure 1, which indicates that the total phenolic content was high compared to ascorbic acid that was very low. the phytochemicals screening of boswellia dalzielii root ethanol extract revealed the presence of alkaloid, flavonoid, saponins, tannins, cardiac glycoside and carbohydrate. alkaloids are naturally occurring chemical compounds with nitrogen atoms (andreas luch, 2009), and it is use as a starting points for drug discovery in traditional or modern medicine used for analgesic and anti-bacterial activities. the findings of raymond et al. (2010); cushnie et al. (2014), adhere to the analgesic and anti–inflammatory activities of this present study. study of raymond et al. (2010) showed the effect of flavonoids as a potent antioxidant, antiinflammatory, vasculopropertor, anti-hepatotoxic, antiallergic and antitumor properties (singh and gambhir, 1998). the presence of phenolic and ascorbic acid antioxidant contents of the extract may have contributed to the analgesic and anti–inflammatory properties. table 1. the qualitative and quantitative phytochemical screening of boswellia dalzielii root methanol extract. phytochemicals constituents observations concentration(mg/kg) mean ± sem alkaloid +++ 127 ± 0.01 flavonoid +++ 77.84 ±0.00 carbohydrate ++ – cardiac glycoside +++ – saponin ++ 124 ± 0.00 tannin ++ 145 ± 032 key: +++ = abundantly present, ++ – = moderately present n u m b e r o f r e p l ic a t e a n t i o x i d a n t v a l u e 0 1 2 3 4 0 5 0 1 0 0 1 5 0 t a c (m g v c e ) t p c (m g g a e 100 -1 ) t a c (m g /k g ) figure 1. total antioxidant capacity, phenolic content and ascorbic content of the ethanolic extract of boswellia dalzielii root. https://en.wikipedia.org/wiki/natural_product https://en.wikipedia.org/wiki/chemical_compound https://en.wikipedia.org/wiki/nitrogen https://en.wikipedia.org/wiki/drug_discovery https://en.wikipedia.org/wiki/drug_discovery https://en.wikipedia.org/wiki/traditional_medicine https://en.wikipedia.org/wiki/pharmaceutical_drug 146 biology, medicine, & natural product chemistry 12 (1), 2023: 143-150 the extract produced significant reduction (p < 0.05) in body temperature at all dose level and standard (100 mg/kg paracetamol) when compared with control. the antipyretics activities of the extract was in dose dependent and also comparable to the standard. the results also show that the antipyretics effect of the extract increase with time, in table 2. the root extract of boswellia dalzielii displayed a significant decrease in baker’s yeast-induced pyrexia with an increase in grade time, starting from 60 minutes and progresses a through the study when compared with the control, but at highest dose of 400 mg/kg, the extract showed more effect. antipyretic effect of the extract was comparable to that of the standard drug (paracetamol), with an underlying mechanism of action unknown. yeast is known to be an exogenic pyrogen responsible for an increased in the body temperature via the formation and synthesis of cytokines called as endogenic pyrogens, hence this endogenic pyrogen centrally promote thermosensitive neutrons in the preoptic area of the hypothalamus, increasing heat and decreasing heat loss. the body temperature increases to a set point that leads to fever (kelly et al., 2003). certain phytochemicals including tannins, flavonoids, and cardiac glycosides of the root extract as a potent antipyretic action observed. flavonoids have been reported in previous study carried out by germain et al. (2011) to inhibit prostaglandins and arachidonic acid peroxidation, which surmount to the lowering of the prostaglandin thus causes a reduction in fever occurrence (houghton and raman, 1998; germain et al., 2011). the main mechanism of action through which acetaminophen act is by the inhibition of cyclooxygenase (cox), or highly selective cox–2 receptors (hendrickson et al., 2006). paracetamol reduces the oxidized form of the cox enzyme, thereby reducing the release of prostaglandin e2 in the central nervous system to lower its binding from the hypothalamus set point of thermoregulatory center (hendrickson et al., 2006) which may be consider also as mechanism of action of the extract. table 2. the effect of boswellia dalzielii root ethanol extract on barker’s yeast-induced pyrexis in mice. groups dose mg/kg base line 0c 6 hrs 7 hrs control (nacl) 10 ml/kg 39.43±0.10a 39.65±0.17a 39.95±0.15a boswellia dalzielii 100 39.90±0.18a 37.15±0.16b 37.05±0.17b boswellia dalzielii 200 39.00±0.09a 37.28±0.16a 37.13±0.13b boswellia dalzielii 400 39.88±0.79a 37.30±0.15b 37.18±0.18b aspirin 100 39.08±0.14a 37.22±0.14b 37.20±0.19b the values were expressed in mean ± sem and the significant difference was spotted as p -value < a 0.05 boswellia dalzielii root ethanol extract exhibited a significant decrease in hot plate-induced analgesia when compared to the control. the analgesic effect of the extract was comparable to the morphine standard analgesic, at the lowest concentration. the presence of certain phytochemicals like alkaloids displayed a major role in the analgesic action observed from the extract. this agreed with sinatra et al. (2010) study on analgesic. the significant increase in pain threshold synthesized by hot plate model suggested the connection with the central pain pathways. pain is centrally modulated via series of multifaceted processes on the opiate, dopaminergic descending noradrenergic and serotonergic systems (headley and shaughnessy, 1985; wigdor and wilcox, 1987; yekkirala et al., 2012). the analgesic potential of the treatment groups act with central mechanisms of action connecting the receptor and the inhibitory effect of prostaglandins, leukotrenes, and other endogenous neurotransmitters that triggered pain. morphine being a phenanthrene opioid receptor agonist elicited its main effect is an requisite and activation of μ–opioid receptors in the central nervous system. the μ–δ–opioid receptor heteromer (yekkirala et al., 2010). the μ–binding sites are discretely spread across human brain, with a high densities in the posterior amygdala, hypothalamus, thalamus, nucleus caudatus, putamen, and certain cortical areas. they are also present in the terminal axons of the primary afferents within the laminae i and ii (substantia gelatinosa) of the spinal cord and in the spinal nucleus of the trigeminal nerve. morphine exerts its main action on the central nervous system and gastrointestinal tract to excite possible analgesic action. it also serve as an κ– opioid and δ–opioid receptor agonist, associated with spinal analgesia, meiosis (pinpoint pupils) and psychotomimetic effects. δ–opioid is thought to play a role in analgesia (chien and pasternak, 1995). https://en.wikipedia.org/wiki/phenanthrene https://en.wikipedia.org/wiki/opioid_receptor https://en.wikipedia.org/wiki/receptor_agonist https://en.wikipedia.org/wiki/mu_opioid_receptor https://en.wikipedia.org/wiki/central_nervous_system https://en.wikipedia.org/wiki/central_nervous_system https://en.wikipedia.org/wiki/gpcr_oligomer https://en.wikipedia.org/wiki/human_brain https://en.wikipedia.org/wiki/amygdala https://en.wikipedia.org/wiki/hypothalamus https://en.wikipedia.org/wiki/thalamus https://en.wikipedia.org/wiki/nucleus_caudatus https://en.wikipedia.org/wiki/nucleus_caudatus https://en.wikipedia.org/wiki/putamen https://en.wikipedia.org/wiki/chemical_synapse https://en.wikipedia.org/wiki/posteromarginal_nucleus https://en.wikipedia.org/wiki/substantia_gelatinosa_of_rolando https://en.wikipedia.org/wiki/substantia_gelatinosa_of_rolando https://en.wikipedia.org/wiki/trigeminal_nerve https://en.wikipedia.org/wiki/human_gastrointestinal_tract https://en.wikipedia.org/wiki/kappa_opioid_receptor https://en.wikipedia.org/wiki/kappa_opioid_receptor https://en.wikipedia.org/wiki/delta_opioid_receptor https://en.wikipedia.org/wiki/miosis https://en.wikipedia.org/wiki/psychotomimetic idu, et al. – phytochemistry, biological activity of boswellia dalzielii hutch (burseraceae) 147 g ro u p s r e a c ti n g t im e (s ) c o n tr o l 1 0 0 m g /k g 2 0 0 m g /k g 4 0 0 m g /k g ) m o rp h in (5 m g /k g ) 0 2 4 6 8 g ro u p s r e a c ti n g t im e (s ) c o n tr o l 1 0 0 m g /k g 2 0 0 m g /k g 4 0 0 m g /k g ) m o rp h in (5 m g /k g ) 0 2 4 6 8 g ro u p s r e a c ti n g t im e (s ) c o n tr o l 1 0 0 m g /k g `2 0 0 m g /k g 4 0 0 m g /k g ) m o rp h in (5 m g /k g 0 5 1 0 1 5 ** ** * g ro u p s r e a c ti n g t im e (s ) c o n tr o l 1 0 0 m g /k g 2 0 0 m g /k g 4 0 0 m g /k g m o rp h in (5 m g /k g ) 0 5 1 0 1 5 ** * * ** figure 2. (a, b, c, and d) effect of boswellia dalzielii root ethanol extract on hotplate-induced pain. plant extract at 100mg/kg, 400mg/kg and morphine increase the reaction time to pain induced by hotplate at 0, 30, 60 and 90 minutes exposure compared to control (**p < 0.01, *p < 0.05). the plant extract 100, 200, 400mg/kg and aspirin (100mg/kg) reduced the number of writhing compared to control (p<0.01), in table 2. the root ethanol extract of boswellia dalzielii exhibit a significant decrease in acetic acid induced analgesia when compared with the control. the analgesic effect of this present study was comparable to the standard analgesic, aspirin. abdominal constriction reaction induced by acetic acid serves as a sensitive procedure for the evaluation of peripherally pain acting analgesics (gene et al., 1989). acetic acid is a peripheral pain triggering factor capable of liberating the endogenous substances (histamine, prostaglandins (pgs), bradykinins and substance p, endings). the local peritoneal receptors are proposed to be complicated in the abdominal with a constrictive response (bentley et al., 1983). prostanoids is a general stimulatory factor that increases the levels of pge2 and pgf2α in the peritoneal fluids as well as lipoxygenase products (derardt et al., 1980). the analgesic effect instigated by the treatment via peripheral mechanisms could be involved in prostaglandins, leukotrenes, and other endogenous substances with inhibitory effect. the presence of certain phytochemicals (alkaloids) in the facilitated analgesic action observed from this study (sinatra et al., 2010). aspirin as a pure compound suppresses the production of prostaglandins and thromboxanes to trigger irretrievable inactivation of the cyclooxygenase (cox; known as prostaglandin– endoperoxide synthase, ptgs) enzyme needed for prostaglandin and thromboxane production. it also acts via acetylating agent where acetyl group is covalently bond to serine residue in the active site of ptgs enzyme. this makes aspirin different from other nsaids, which are reversible inhibitors (burke et al., 2006). table 3. the effect of boswellia dalzielii root ethanol extract on acetic acid-induced writhing in mice. group doses mg/kg numbers of writhing % inhibition control (nacl) 10 ml/kg 91.75±10.48a boswellia dalzielii 100 45.00±10.34b 50.95 boswellia dalzielii 200 34.00±7.22b 62.94 boswellia dalzielii 400 36.50±5.33b 60.22 aspirin 100 42.00±5.95b 54.22 the values were expressed in mean ± sem and the significant difference was spotted as p -value < a 0.05. the ethanol root extract of boswellia dalzielii exhibit a significant decrease in carrageenan-induced inflammatory when compared with inflammatory control. the report of cushnie et al. (2014) concurred to this present ant-inflammatory potential. various components leads to inflammatory response stimulating edema formation, leukocyte infiltration and granuloma formation inked with the components of inflammation (mitchell and cotron, 2010). edema formation in the paw is as a result of the synergistic action between inflammatory mediators that triggered an increase in the vascular permeability of blood flow (lalenti et al., 1995). the inhibitory effect of carrageenan-induced inflammation in rats was an established model for the evaluation of anti–inflammatory ingredient, with the frequency to evaluate the anti–inflammatory potential on natural remedies. the development of carrageenaninduced edema is consist of two phase system such as first phase (within one hour of carrageenan inflammation) and is usually intermediate with the release of cytoplasmic enzymes serotonin, discharged from mast cells. the second phase is (mediated release of prostaglandins) in most inflammatory area and linger between the two phases provided by kinins (goel et al., 2001). a b c d https://en.wikipedia.org/wiki/cyclooxygenase https://en.wikipedia.org/wiki/serine 148 biology, medicine, & natural product chemistry 12 (1), 2023: 143-150 table 4. the effect of boswellia dalzielii root ethanol extract on carrageenan induced inflammation in rats. paw volume (mm) treatment doses mg/kg t30 min t60min t120min t180min t240min control (nacl) 10 ml/kg 1.28±0.51a 1.46±0.55a 1.62±0.59a 1.08 ±0.29a 0.74±0.25a boswellia dalzielii 100 0.83±0.l14b 1.08 ±0.16b 2.06±0.27a 1.56±0.44a 1.12±0.47a boswellia dalzielii 200 0.69±0.62b 0.63±0.35b 0.72±0.37b 0.77±0.39b 0.49±0.37b boswellia dalzielii 400 0.99±0.80b 0.91±0.17b 1.70±0.44a 0.73±0.25b 0.56±0.20b indomethacin 10 0.79±0.34b 0.78±0.35b 0.18±0.17b 0.32±0.21b 0.09±0.05b the values were expressed in mean ± sem and the significant difference was spotted as p -value < a 0.05. the effect of ethanolic extract of boswellia dalzielii root on formalin–induced inflammation (arthritis) in rats (table 4). the plant extract 100, 200, 400 mg/kg and indomethacin (1mg/kg) reduced paw volume starting from day 4 and progresses through the study compared to control (p < 0.01). boswellia dalzielii root ethanol extract exhibit a significant decrease in formalin– induced arthritis across the graded doses when compared with the arthritic control. the anti-arthritic effect of the extract had a significant reduction when compared with indomethacin. the possible underlying mechanism of the extract has not be established rather it triggered an inhibitory effect of edema induced by formalin in rats, which is a most suitable test procedures to screen for anti–arthritic and anti–inflammatory agents (mitchell and cotron, 2010). table 5. the effect of boswellia dalzielii root ethanol extract on formalin–induced arthritis in rats. treatment doses mg/kg day 2 day3 day4 day5 day6 day7 day8 day9 day10 control (nacl) 10 ml/kg 3.18±0. 49a 1.54±0.38a 2.69±0.04a 2.36±0.64a 2.45±0.20a 2.40±0.26a 2.54±0.10a 3.28±0.20a 3.79±0.38a boswellia dalzielii 100 3.15±0.16a 1.06±0.23b 1.36±0.13b 0.72±0.19b 0.78±0.19b 0.66±0.19b 0.46±0.15b 0.56±0.15b 0.77±0.12b boswellia dalzielii 200 2.10±0.294b 0.79±0.10b 0.99±0.12b 0.79±0.09b 0.37±0.10b 0.51±0.16b 0.38±0.12b 0.56±0.15b 0.32±0.16b boswellia dalzielii 400 2.20±22.63b 0.55±0.18b 1.60±0.38b 0.99±0.33b 0.67±0.37b 0.49±0.27b 0.40±0.60b 0.39±0.14b 0.44±0.12b indomethacin 1 2.18±0.22b 0. 80±0.61b 0.80±0.30b 0.90±0.20b 1.10±1.00b 0.11±0.90b 1.13±0.10b 1.12±1.15b 0.64±.10b the results gotten from the formalin-induced tests is associated with cell and tissue damages instigated arthritic conditions. this is as a result of the changes in connective tissue metabolism, which is one of the major biochemical cascade events leading to inflammation. these changes occurred in the alteration of relative composition of the various constituents in the connective tissue incudes; mucopolysaccharides, glycoprotein, hexosamine and hydroxy proline, sialic acid (houck and jacob, 1969). hence the concentration of hexosamine and hydroxyproline had a significant increase in formalin-induced rats. boswellia dalzielii extract pretreatment elicited an inhibitory effect with the accumulation of hydroxyl proline and hexosamine in the edematous tissue of the formalin–induced rats. indomethacin is a nonselective inhibitor of cyclooxygenase (cox) i and ii, enzymes that triggered prostaglandin synthesis from arachidonic acid. prostaglandins are hormone–like molecules present in the body, with wide variety of effects, some of which it excite pain, fever, and inflammation (brayfield, 2014) conclusion the result from this investigation shows that the ethanolic extract of boswellia dalzieliiroot is a potent antipyretic analgesic and anti-inflammatory agent, as further study should be done to improve on it. conflict of interest: the authors declare that there is no conflict of interest. references abubakar, m. s., musa a. m., ahmed a. and hussaini, i. m. 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(2010). standard opioid agonists activate heteromeric opioid receptors: evidence for morphine and [d–ala(2)–mephe(4)– glyol(5)]enkephalin as selective μ–δ agonists. acs chemical and neuroscience, 1(2): 146–154. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 243-248 | doi: 10.14421/biomedich.2025.141.243-248 issn 2540-9328 (online) effect of robusta coffee extract (coffea canephora) on mice (mus musculus) on gastric histopathology and anxiety level difa hasna soebroto, harlita* department of biology education, faculty of teacher training and education, sebelas maret university, jl. ir. sutami 36 kentingan, jebres, surakarta, central java 57126, tel. 0271-646994, fax. 0271-646655, indonesia. corresponding author* harlita@staff.uns.ac.id manuscript received: 09 february, 2025. revision accepted: 20 may, 2025. published: 23 june, 2025. abstract robusta coffee (coffea canephora) contains bioactive compounds, such as caffeine that have the potential to affect the digestive system and anxiety levels. this study aims to evaluate the effects of robusta coffee extract on gastric histopathology and anxiety levels in mice (mus musculus). the method used involved 4 treatments, k1: negative control (without treatment), k2: positive control with aspirin 10.84 mg/20gr body weight of mice, k3: coffee extract 6.72 mg/20gr body weight of mice, k4: coffee extracts 13 mg/20gr body weight of mice. the results showed that robusta coffee extract impacted the histological structure of the stomach, with indications of changes in the gastric mucosa. in addition, there was a tendency for changes in the level of anxiety in mice analyzed through the behaviour test, the elevated plus maze test. this study provides insight into the potential physiological effects of robusta coffee consumption and its implications for gastric health and psychological conditions. keywords: coffea canephora; mus musculus; gastric; histopathology; anxiety level. introduction coffee is one of indonesia’s favorite drinks from time to time. this is supported by indonesia's ability to produce coffee as a leading commodity in the plantation sector (haryani et al., 2022). indonesia is ranked second in the asia and oceania region in coffee production. in 20222023, coffee production in indonesia increased by 2.4% or around 12 million bags (international coffee organization, 2023). one type of coffee widely circulated on the market and is often used is robusta coffee (coffea canephora) (lumaksono et al., 2021). the caffeine in robusta coffee is almost twice as high as that of arabica coffee, which is 2.4% compared to that of arabica coffee. arabica coffee contains 1.3% of caffeine in dry conditions. according to (sri & rubiyanti, 2020), excessive caffeine consumption can stimulate the heart and muscles, and increase gastric acid secretion. the caffeine in coffee can accelerate the process of stomach acid formation. the formation of gastric acid causes excessive gas production in the stomach, so people often complain of a bloating sensation. consumption of caffeine can damage the tissue that makes up the stomach, especially the mucosal tissue. if the gastric mucosa is damaged, there is diffusion of hcl to the gastric mucosa and hcl will damage the mucosa. the presence of hcl in the gastric mucosa stimulates the change of pepsinogen to pepsin which stimulates the release of histamine from mast cells. histamine will increase capillary permeability, resulting in fluid movement from the intracellular to the extracellular space, leading to edema and capillary damage, which in turn causes gastric bleeding. when the stomach continues to interact with irritants, the tissue will become inflamed, leading to the loss of the gastric mucosal layer and atrophy of gastric mucosal cells (azer et al., 2025). another effect of caffeine is that it can affect a person's anxiety levels. this compound target is adenosine receptor antagonists (1,3,7-trimethylxanthine) which can stimulate the central nervous system. it is said that caffeine has anxiogenic and panicogenic effects in both animals and humans (meurling, 2020). in some sensitive individuals, such as patients with panic disorder (pd), anxious behavior can be seen. the core symptoms of excessive anxiety are recurrent panic attacks, fear, or discomfort in some parts of the body, such as palpitations, shortness of breath, numbness, and dizziness. panic attacks can also occur in other anxiety disorders as well as depressive disorders, and/or even sometimes in healthy individuals (klevebrant & frick, 2022). identification of behavior in rodents can be seen using the elevated plus maze (epm), the most used test to measure anxiety-like behavior in rodents. the use of epm is based on the behavior of animals avoiding open https://doi.org/10.14421/biomedich.2025.141.243-248 244 biology, medicine, & natural product chemistry 14 (1), 2025: 243-248 areas and their tendency to move towards more protected places (florén lind et al., 2023) materials and methods study area this study used mice as experimental animals, with samples taken randomly using the completely randomized design (crd) method. the study lasted for 37 days, consisting of 7 days of acclimatization and 30 days of treatment. the sample criteria included male mice aged 3–4 months with a body weight of 25–35 grams, having a healthy physical condition, and showing no anatomical defects. this study was conducted at the biology education laboratory, fkip, sebelas maret university and lab pusat uns. this study requires various tools and materials to support its implementation. the tools used include elevator plus maze (epm), mouse cages, analytical scales, beakers, erlenmeyers, stirrers or spatulas, grinders, and sieves. in addition, filter paper, aluminum foil, black plastic, rotary evaporator, oven, mice drink bottle, mice food container, oral sonde, latex, paraffin surgical pad, scissors, scalpel, cutter, pins, and object glass are required. the materials used include 3–4-month-old male mice, robusta coffee bean extract, 96% ethanol, mouse feed, water, and wood dust as additional media. all these tools and materials are used systematically to ensure the research was conducted according to the designed method. procedures robusta coffee exctract extraction is obtained by the maceration method. the extraction process by the maceration method is most often used to prevent damage to the content of certain compounds in robusta coffee. coffee beans are roasted and ground to the same size. the coffee bean powder is stored in a container and tightly closed. coffee bean samples are ground by pounding and blending which aims to increase the surface area of the sample so that the interaction of caffeine withdrawal in coffee occurs optimally. robusta coffee bean extraction by the maceration method begins by inserting 120 g of coffee bean powder into an erlenmeyer flask. the powder is soaked in 225 ml of 96% ethanol and the container is covered with aluminum foil. this process is carried out for 5 days with occasional stirring. after that, the mixture is filtered using filter paper to obtain the first filtrate and dregs. the dregs left are then soaked again (remaceration) with 75 ml of 96% ethanol for 2 days while stirring occasionally. like the previous process, the mixture is filtered using filter paper to obtain the second filtrate. the two filtrates obtained were combined and evaporated using a rotary evaporator at 40-50°c to produce a thick extract (tanauma et al., 2016). preparation of animals the research sample criteria included male mice, aged 34 months, weighing 25-35 grams, in a healthy physical condition without anatomical defects. animals were adapted a week before treatment (1 week) excluding 30 days of test treatment. treatment of animals the treatment was conducted for 30 days, each group was given orally to 6 test animals which were divided into 4 groups: ▪ negative control (k1): no treatment ▪ positive control (k2): aspirin 10.84 mg/20gr body weight of mice to induce gastritis ▪ dose i (k3): 6.72 mg/20gr body weight of mice ▪ dose ii (k4): 13 mg/20gr body weight of mice data analysis anxiety level in the epm method, mice are placed in the center of the maze facing one of the closed compartments and are allowed to move freely to both sides. the duration and frequency spent by the experimental animals on the open and closed arms are recorded. anxious animals will tend to be on the closed arm, while animals affected by antianxiety compounds will spend more time on the open arm. this maze has two dimensions, the dimensions of the open arm: length 50 cm × width 10 cm. the dimensions of the closed arm: length 50 cm × width 10 cm with a wall height of 40 cm. the entire maze is elevated 50 cm above the floor. the anxiety level of the treatment effect was evaluated using the parameters (a) percentage of time spent on the open arm ([seconds on the open arm] / [300 s] × 100), (b) percentage of open arm entries ([open entries] / [total entries] × 100), (c) percentage of closed arm entries ([closed entries] / [total entries] × 100) (guillén-ruiz et al., 2021). histopathology after 30 days of treatment, the mice were prepared for histological observation by removing their abdominal organs. before removing the organs, the mice were euthanized by inhaling chloroform (avma, 2013)next, the lower abdomen of the mice was cut to the chest. next, the lower abdomen of the mice was cut to the chest, then the organs were removed from the mice's body. the abdominal organs must be separated from the attached intestines, liver, and gallbladder. after the mice died, a laparotomy was performed to remove the mice's stomach for microscopic preparations. histological examination of the rat stomach was carried out by observing the gastric mucosal layer using an optilab iris-4 microscope based on the wattimena score (lumaksono et al., 2021) which was then analyzed using the kruskall-wallis statistical test. soebroto & harlita – effect of robusta coffee extract (coffea canephora) … 245 wattimena score: 1 : no erosion found 2 : erosion only on the surface epithelium 3 : erosion reaches a depth of 1/3 of the upper gastric glands 4 : erosion reaches a depth of 1/3 of the middle gastric glands 5 : erosion reaches a depth of 1/3 of the lower gastric glands 6 : erosion reaches the depth of the lamina muscularis mucosa results and discussion body weight of mice this study was conducted using four different treatments on mice to observe their effects on changes in body weight, histopathology, and anxiety levels. the results of the observations showed variations in changes in body weight between treatment groups. the data obtained were analyzed using analysis of variance (anova) and tukey's advanced test in the study of the effect of robusta coffee extract on the body weight of mice shown in table 1. the results showed that the average between groups did not differ (p>0.05) with a significance level of 0.59 (body weight in week 1) and 0.29 (body weight in week iv). the treatments did not significantly affect the body weight of mice, despite a decrease observed in k2, k3, and k4 (table 1). there was no significant difference in body weight changes between treatment groups. the decrease that occurred showed the effect of the treatment, but the effect was too small to be detected by the statistical test used. table 1. average body weight (grams) of mice during treatment (30 days). treatments initial treatment final treatment body weight difference x±sd x±sd k1 (negative control) 31.13 ± 1.11 35.63 ± 1.11 +4.5 k2 (aspirin 10.84 mg/20g bw of mice) 32.13 ± 2.98 31.25 ± 2.25 -0.88 k3 (coffee extract 6.72 mg/20g bw of mice) 33.50 ± 3.72 31.00 ± 5.12 -2.5 k4 (coffee extract 13 mg/20g bw of mice) 34.50 ± 5.52 33.63 ± 4.80 -0.87 note: data presented as mean (x) ± standard deviation (sd) bw = body weight anxiety levels in mice the anxiety level of mice was analyzed using the elevated plus maze (epm) method. this method is one of the standard tests for evaluating anxiety behavior, which is based on the tendency of mice to choose open or closed areas. data were tested using anova and tukey's advanced test. the results of this observation are expected to provide an overview of the anxiety response that arises due to the treatment given. there is a significant difference (p <0.05) in each treatment group, which is interpreted as mice’s anxiety level. from the data presented k2, k3, and k4 significantly showed anxiety activity (figure 1). meanwhile, k1 (negative control) did not show anxiety activity. the average percentage of time spent in the open arm position was highest in the negative treatment group (k1) which showed a low level of anxiety. on the other hand, k4 showed the lowest frequency of entering the open area compared to other groups. this can indicate a high level of anxiety compared to other groups. this is due to k1 not receiving any treatment. giving coffee extract to mice affected the anxiety of mice, in the form of a low entry time and entry frequency when mice entered the open arm. figure 1. average duration (left) and frequency (right) of entries in open arms during elevated plus maze (epm) testing. data shown as mean values and standard deviation (sd). significant differences indicated by * and ** (one-way anova test with post hoc lsd). 246 biology, medicine, & natural product chemistry 14 (1), 2025: 243-248 table 2. data on the effect of robusta coffee extract on anxiety levels. treatment time in open arms entry to open arms entry to close arms x±sd x±sd x±sd k1 (negative control) 74.2b ± 13.93 17.8 a ± 12.8 15.5a ± 11.4 k2 (aspirin 10.84 mg/20g bw of mice) 9.89a ± 0.84 12.34a ± 7.7 20.98a ± 7.7 k3 (coffee extract 6.72 mg/20g bw of mice) 11.66a ± 6 12.5a ± 4.17 20.83a ± 8.3 k4 (coffee extract 13 mg/20g bw of mice) 17.22a ± 5.66 11.56a ± 3.12 21.7a ± 11.2 note: data presented as mean (x) ± standard deviation (sd) bw= body weight gastric histopathology table 3. kruskal-wallis test. histopatology kruskal-wallis 7.500 df 3 asymp. sig. 0.58 data analysis showed that the dose 2 group had the highest histopathology score, followed by the positive and dose 1 groups (with the same mean rank value), then the negative group with the lowest mean rank. the p value (0.058) exceeds the significance level (α = 0.05). thus, there was no significant difference in the gastric histopathology score between treatment groups. although there was a difference in mean rank between groups (especially at the higher dose 2), the kruskalwallis test results showed that the difference was not statistically significant. figure 2. microscopic histological picture of mice stomach. 40x magnification (left) and morphology of mice stomach (right). red arrows indicate erosion on the surface epithelium. note: (a) k1: negative control, wattimena score 1; (b) k2: aspirin 10.84 mg/20g bw mouse, wattimena score 2; (c) k3: coffee extract 6.72 mg/20g bw mouse, wattimena score 2; (d) k4: coffee extract 13 mg/20g bw mouse, wattimena score 2. in the negative group, the epithelium on the surface of the intact mucosal layer (score 1), without erosion. this is due to the absence of gastritis triggers such as caffeine in coffee drinks compared to other groups. compared to other groups, the morphological picture of the stomach in the negative group shows a normal and healthy stomach. in group k2: aspirin 10.84 mg/20gr of mouse body weight, k3: coffee extract 6.72 mg/20gr of mouse body weight, and k4: coffee extract 13 mg/20gr of mouse body weight showed erosion on the surface epithelial layer (score 2). morphologically, the negative group shows a healthy and normal stomach, shaped like red beans or j-shaped (chen et al., 2016), the color of the stomach shows an even red. group 2 shows changes in color and texture in the form of lumps indicating irritation due to aspirin administration. k3 and k4 show changes in color and shape of the stomach, in the form of shrinkage, where the normal shape of the stomach is like red beans or the letter j. discussion the results of the study showed that the treatment given did not have a significant effect on changes in the weight of mice. although there was a decrease in body weight in several treatment groups, the effect was too small to be detected by the statistical test. the insignificant difference was likely due to the relatively short duration of the study. according to wijayanti & kadita (2017), weight loss can be felt through long-term coffee consumption. at the level of anxiety, giving coffee extract to the mice affected the anxiety of mice. this is indicated by the low time and frequency of entry when mice entered soebroto & harlita – effect of robusta coffee extract (coffea canephora) … 247 with open arms. the caffeine content in coffee can have a significant negative impact on mood and work performance if consumed continuously. caffeine is known to trigger feelings of restlessness, anxiety, and anger which have the potential to hurt the body (purdiani, 2014). as a psychostimulant drink, coffee is considered to be able to reduce fatigue and drowsiness, as well as provide additional energy. the structure of caffeine is similar to that of adenosine, allowing it to bind to adenosine receptors in the brain, thereby increasing cortical activity through the aras (ascending reticular activating system) pathway and stimulating nerve activity (dewanti & tadjudin, 2023). in histopathological observations, although there was a difference in mean rank between groups (especially at a higher dose of 2), the results of the kruskal-wallis test showed that the difference was not statistically significant. this shows that the administration of coffee extract at the tested dose did not provide significant changes in the histological structure of the stomach of mice between treatment groups. under normal conditions, the defense mechanism of the preepithelial gastric mucosa, namely mucus, can protect the stomach from endogenous and exogenous damage. the normal stomach lining is due to the absence of gastritis triggers, such as caffeine and others. giving aspirin to mice aims to provide pain relief from gastritis. aspirin is a non-steroidal anti-inflammatory drug (nsaid) that can cause discomfort because it is induced by aspirin which causes nausea and vomiting (farhan et al., 2022). this drug has two mechanisms of action, namely irritating the gastric epithelium and/or inhibiting prostaglandins. then, gastric ulcers (inflammation of the gastric mucosa) are formed (oktrinorma & indriyanti, 2020). the effect of erosion on the mucosal layer is caused by the caffeine content in robusta coffee. in lumaksono et al (2021), 100 grams of coffea canephora seeds contain 6.1-11.3 grams of chlorogenic acid, 1.5-2.5 grams of caffeine, and 0.2-0.8 grams of diterpene. the caffeine content in coffee can increase stomach acid production if consumed continuously. excessive stomach acid can cause inflammation of the stomach wall, gastritis. in addition, caffeine can also weaken the esophageal sphincter (valve muscle between the esophagus and stomach) (haryani et al., 2022) in addition to internal factors, mouse epithelial erosion can also be influenced by uncontrolled external variables, such as psychological stress experienced by mice or initial stomach conditions not analyzed in this study. psychological stress can trigger the release of angiotensin ii which impacts decreasing blood flow to the gastric mucosa. as a result, reactive oxygen species are formed which trigger oxidative stress, ischemia, and damage to the gastric mucosal layer (lumaksono et al., 2021). conclusions the use of robusta coffee extract in this study had varying effects on several parameters. in the parameter of weight, there was no significant effect between treatments, although there was a tendency for weight loss in mice. in the histopathology of the stomach of mice, there was an effect of the treatment with robusta coffee extract. coffee extract caused damage in the form of erosion on the surface epithelium of the stomach. in addition, there was a significant effect too on the observation of anxiety levels. this is indicated by the frequency of mice in the open area being lower, compared to the closed area. acknowledgements: i would like to express my sincere gratitude to my advisor, dr. harlita, s.si., m.si, for her invaluable guidance, support, and encouragement throughout this research. also, lab pusat universitas sebelas maret provided the facilities to carry out this study. authors’ contributions: difa hasna soebroto designed the study, conducted laboratory work, analyzed the data, and wrote the manuscript. harlita as supervisor. all authors read and approved the final version of the manuscript competing interests: there are no competing interests. references avma. (2013). avma guidelines for the euthanasia of animals: 2013 edition. in american veterinary medical association. https://www.avma.org/kb/policies/documents/euthanasia.pdf azer, s., awosika, a., & akhondi, h. (2025). gastritis. statpearls publishing. https://www.ncbi.nlm.nih.gov/books/nbk544250/ chen, l., xu, y., fan, t., liao, z., wu, p., wu, x., & chen, x. d. (2016). gastric emptying and morphology of a ‘near real’ in vitro human stomach model (rd-iv-hsm). journal of food engineering, 183, 1–8. https://doi.org/https://doi.org/10.1016/j.jfoodeng.2016.02.025 dewanti, p., & tadjudin, n. s. (2023). hubungan minum kopi dan kecemasan pada mahasiswa fakultas kedokteran universitas tarumanagara angkatan 2019 pada bulan januari 2021. ebers papyrus, 28(2), 50–62. https://doi.org/10.24912/ep.v28i2.20930 farhan, m. r., oktora, m. z., & hasni, d. (2022). pengaruh pemberian aspirin terhadap gambaran histopatologi mukosa gaster mencit. jurnal kesehatan as-shiha, 79–86. florén lind, s., stam, f., zelleroth, s., meurling, e., frick, a., & grönbladh, a. (2023). acute caffeine differently affects risktaking and the expression of bdnf and of adenosine and opioid receptors in rats with high or low anxiety-like behavior. pharmacology, biochemistry, and behavior, 227–228, 173573. https://doi.org/10.1016/j.pbb.2023.173573 guillén-ruiz, g., cueto-escobedo, j., hernández-lópez, f., rivera-aburto, l. e., herrera-huerta, e. v, & rodríguez 248 biology, medicine, & natural product chemistry 14 (1), 2025: 243-248 landa, j. f. (2021). estrous cycle modulates the anxiogenic effects of caffeine in the elevated plus maze and light/dark box in female rats. behavioural brain research, 413, 113469. https://doi.org/10.1016/j.bbr.2021.113469 haryani, n. s., budiraharjo, k., & handayani, m. (2022). analisis faktor-faktor yang mempengaruhi permintaan kopi bubuk di umkm kopi kapal lampung. jurnal litbang provinsi jawa tengah, 20(1), 65–78. https://doi.org/10.36762/jurnaljateng.v20i1.946 international coffee organization. (2023). coffee report and outlook. international coffee organization ico, 1(1), 1–39. https://icocoffee.org/documents/cy202324/coffee_report_and_outlook_december_2023_ico.pdf klevebrant, l., & frick, a. (2022). effects of caffeine on anxiety and panic attacks in patients with panic disorder: a systematic review and meta-analysis. general hospital psychiatry, 74, 22–31. https://doi.org/10.1016/j.genhosppsych.2021.11.005 lumaksono, m. a., kusumo, p. d., & gultom, f. l. (2021). effect of coffea arabica and coffea canephora consumption on mus musculus gaster histopathological description. indonesian journal of biotechnology and biodiversity, 5(2), 53–60. https://doi.org/10.47007/ijobb.v5i2.79 meurling, e. (2020). t. effect of caffeine on anxiety-like behavior in rats. (2020). the effect of caffeine on anxiety-like behavior in rats. oktrinorma, w. v, & indriyanti, n. (2020). pengaruh induksi tukak lambung menggunakan aspirin terhadap kerusakan jaringan mukosa lambung mencit. proceeding of mulawarman pharmaceuticals conferences 11, 45–49. purdiani, m. (2014). hubungan penggunaan minuman berkafein terhadap pola tidur dan pengaruhnya pada tingkah laku mahasiswa/i universitas surabaya. calyptra: jurnal ilmiah mahasiswa universitas surabaya, 3(1), 1–15. sri, t., & rubiyanti, r. (2020). pengaruh pemberian ekstrak biji kopi arabika (coffea arabica l.) terhadap histopatologi lambung tikus putih galur wistar. fitofarmaka jurnal ilmiah farmasi, 10(1), 32–41. tanauma, h. a., citraningtyas, g., & lolo, w. a. (2016). aktivitas antibakteri ekstrak biji kopi robusta (coffea canephora) terhadap bakteri escherichia coli. pharmacon, 5(4), 243–251. wijayanti, h. s., & kadita, f. (2017). hubungan konsumsi kopi dan screen-time dengan lama tidur dan status gizi pada dewasa. journal of nutrition college, 6(4), 301–306. http://ejournal-s1.undip.ac.id/index.php/jnc biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 2, 2014 | pages: 65-67 | doi: 10.14421/biomedich.2014.32.65-67 the antidepressant effects of (arcangelisia flava (l.) merr) water-soluble extract in balb-c mice reviewed from immobility time by forced tiara a.1, arief r.h.2* and sudarsono3 1faculty of pharmacy; 2department of pharmacology; 3department of pharmaceutical biology, ugm, indonesia author correspondency*: ar_hakim2000@yahoo.com abstract this study was conducted to examine / determine the antidepressant effects of (arcangelisiaflava l.) on immobility time of the white male mice strain balb-c by the forced swim test method. the method of research using laboratory animals such as 25 micewas divided into 5 groups. as a negative control group was only given distilled water ad libitum. amitryptiline was used as the positive control group; the experiment group was a water-soluble extract of a.flava by multiple doses. the results showed that the best antidepressant effects were 312 mg/kg bw; it had a minimum of immobility time compared with the other groups. keywords: acangelisiaflava, berberine, immobility time, antidepressants introduction depression is usually as a public problem. world health organi-zation (who) in 2001 stated that depression was the fourth most commonbasic problem in the world. symptoms of depression that appears in the form of complaints related to mood (e.g depressed, sad, despair) to make a diagnosis of depression can be easily enforced, but when psychomotor and somatic complaints that appear frequently undiagnosed depression (amir, 2005). therapy for depression people are medications that can improve mood, better known as antidepressant drugs (grollman, 1972).antidepressant are drugs which can improve depressive symptoms. antidepressant can be classified as desipramine type; imipramine type; amytriptylin type. this antidepressant drug has the side effects of these drugs are : due to anticholinergic properties i.e dry mouth; accommodation disorders, constipation (mutschler, 1995), the development of local wisdom e.g. the people of west sulawesi were using hot water soluble extract of archangelisiaflava l. merr for their gastrointestinal problem (diarrhoea). according to their experience in using hot water soluble of a.flavastem, could be work for the treatment of their problem. according to some references, one of the constituent of a.flava stem are a group of berberine derivative alkaloid (siwon, 1982). this alkaloid berberine chloride is as one of the specific active constituent that active against shigella sp. in vitro and in vivo (larisu, 2010). in this research of interest was to examine, that berberine chloride constituent of the hot water soluble extract of a.flava, had an anti-depressant effect in vivo in mice. the active constituent of the a.flavais composed of alkaloid berberine, jatrorrhizin, palmatin and kolumbamin (keawpradub et al., 2005). from the research, it was known that berberinechloride was an isoquinolinealkaloid group found in a.flava. the group of isoquinolin alkaloid had many pharmacological activities, namely as an antihypertensive, anti-inflammatory, antidepressant, anticancer, antimicrobial, hypolipidemic, hepatoprotective and antidiabetic (singh et al., 2010). n a b c d + 1 2 3 4 5 6 8 9 10 11 12 13 r1 r2 r3 r4 r5 r1 r2 r3 r4 r5 berberine o-ch2-o och3 och3 h jatrorrhizine och3 och3 och3 hoh palmatine och3 och3 och3 och3 h figure 1. berberinederivative (siwon, 1982). materials and method plant material a.flava (“kayukuning”) was obtained from sorong, west papua, irian jaya. this experimental plants was identified by mr.jokosantosom.si and voucher specimen was found at the department of pharmaceutical biology faculty of pharmacy ugm. 66 biology, medicine, & natural product chemistry 3 (2), 2014: 65-67 figure 2. i. a.flava stem; ii. a.flava powder. animals male white mice balb-c strain 2-3 months old, weighing 30-45 grams were used in the present study. the minimum number of animals and duration of observations required to obtain consistent the data were employed. amitriptiline tablets was obtained from “kosudgama” drugstore ugm, was used as a positive control. the dose of the water-soluble extract containing berberine chloride of a.flava based on empirical dose by the local people, while the administration of one dose amitriptiline based on usage which was then converted to the test animals dose. qualitative and quantitative analize of plant material qualitative – quantitative analyze of berberine chloride were done by thin layer chromato-graphy 5 μl.extractand reference standard berberine chloride was spotted on silica gel 60 f254 each. mobile phase was a mixture of nbutanol-acetic acid and water (3:1:1 v/v/v). the spot visualization were uv254 and uv366 separated spots was scanned between 200 – 700 nm. the quantification was done by the use basic stock for 5 mg/ml.stock solution of test solution was 5 mg/ml and the several concentration of berberine chloride in ethanol with in 250; 200; 100; 50; dan 25 μg/ml. preparation of water soluble extract the extraction was done by hot water extraction method (infundation) 3,12%. 3,12gram stem powderand 100 ml aquadest for 15 minutes at 90oc.there was 100 ml filtrate. figure 3. preparation of water soluble extract. qualitative and quantitative analyze thin layer chromatography method, was used to analyze the constituents of this extract. quantitative analyze of the alkaloids constiturnts has done by tlc-scanner 5 μlextract was spotted on the tlc plate silica gel 60 f254 ; mobile phase was n-butanol-acetic acid-water (3:1:1 v/v/v) for 8 cm;visible light, uv254 nm, uv366 nm were used for spot detection. scanningwas done by 200-700 nm. pharmacological treatments forced swim test (fst) can be used as a sign of antidepressants effect. mice were divided into 5 groups, with the control group 2 positive control (+) and negative control (-) and 3 treatment groups (p1, p2, and p3). mice were adapted to feeding ad libitum for 1 week.after the mice were created stress by forcible allow to swim as in the forced swim test procedures on the first day up until the 14th day. then from day 15 to day 28, every day is oral, positive control group (+) was given po at a dose of amitriptyline were 3.25 mg / kg, negative control group (-) were given a placebo in the form of distilled water, groups p1, p2 and p3 were each given a stock solution in test p.o. at a dose of 78, 156, and 312. on day 29, all groups treated with the forced swim (forced swim test) for 6 minutes and measured the immobility time. 100 75 67 50 25 0 tiara a., et al. – the antidepressant effects of (arcangelisia flava (l.) merr) water-soluble extract … 67 im m ob ili ty ti m e (d et ik ) ekstrak (mg/kgbb) statistical analysis all results were expressed as mean ± sd. data were analyzed by one-way anova followed by scheffe's multiple range test. the criterion for statistical significance was p≤ 0.05. all statistical analyses were carried out by using spss for windows (spss inc.). results effect of water-soluble containing berberine chloride extract of a.flava in accordance with the forced swim test results of the testing solution, the results of immobility time in the p3 group was the shortest namely 128.6 ± 44.8 seconds and a negative control with an average immobility time later than the 213.6 ± 43.6 seconds. based on the results of the anova analysis found a significant difference only in the p3 group (water-soluble extract of a.flava stem with a dose of 312 mg/kgbw) compared to the negative control group (distilled water). this indicates that the water-soluble containing berberine chloride extract of a.flavawith a dose of 312 mg/kgbwhad an effect as as an antidepressant. figure 4. influence of water-soluble containing berberin chloride extract vs the immobility time in mice. * = significantly different from the negative control (p ≤ 0.05). conclusion the results showed that the water-soluble extract of a.flava at a dose of 312 mg/kgbw were able to give antidepressants effect on white mice balb-c strain in terms of immobility time in the forced swim test method. as a suggestion research needs to be done the same with the number of animals per group and using comparative more pure and more detailed observation so as to give the best results. references amir, n.a. 2005.depresi: aspek neurobiology diagnosis dan tatalaksana. penerbit fkui. jakarta. grollman, a. 1972.pharmacology and therapy of depression. the american journal of psychiatry.113:950. kametani, t. 1969. the chemistry of the isoquinoline alkaloids. hirokawa publishing company. tokyo. 109-111. larisu, m. a. 2011. kajian ilmiah air rebusan batang katola (arcangelisia flava (l) merr) obat tradisional diare berdarah masyarakat kabupaten muna sulawesi tenggara. tesis. program pascasarjana fakultas farmasi universitas gadjah mada. mutschler e., darendorf h., 1995, drug action, medpharm scientific publisher, stuttgart, p.126-128 peng, w.h., lo, k.l., lee, y.h.,hung, t.h., lin, y.c. 2007. berberine produces antidepressant-like effects in the forced swim test and in the tail suspension test in mice. journal of life sciences. 933-938. porsolt, r.d., bertin, a., jalfre, m., 1977. behavioral despair in mice: a primary screening test for antidepressants. archives internationales de pharmacodynamieet de therapie. 229, 327– 336. porsolt r.d, le pinchon m, jalfre, m. 1977. depression: a new animal model sensitive to antidepressant treatments. nature. perancis.730-732. singh, a., duggal, s., kaur, n., singh, j. 2010.berberin: alkaloid with wide spectrum of pharmacological activities. journal of natural product.3:64-75. siwon, 1982, apharmacognostical study of some indonesian plants of the family menispermaceae, dissertation, p.73., drukkerij j.h., pasmans b.v,’s-gravenhaage world health organization. 2011. depression. http://www.who.int/en/ diakses juni 2013. zomkowski, a.d.e., rosa, a.o., lin, j., santos, a.r.s., calixto, j.b., rodrigues, a.l.s., 2004. evidence for serotonin receptor subtypes involvement in agmatine antidepressant-like effect in the mouse forced swimming test. brain research 1023.253– 263. content_v3n2_4.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 327-335 | doi: 10.14421/biomedich.2025.141.327-335 issn 2540-9328 (online) phytochemicals and antibacterial activity of impatiens balsamina l. leaf extracts against gram-positive and gram-negative bacteria putu utari fridayanthi1, made dharmesti wijaya2,*, desak putu citra udiyani2, anak agung gede indraningrat3, marta setiabudy3 1medical study program; 2department of pharmacology; 3department of microbiology and parasitology, faculty of medicine and health sciences, warmadewa university, jl. terompong no 24 denpasar 80235, tel. +62 361 240727, indonesia. corresponding author* dharmestiwijaya@warmadewa.ac.id manuscript received: 23 february, 2025. revision accepted: 27 june, 2025. published: 01 july, 2025. abstract the emergence of antibiotic resistance as one of the global public health threats makes research on new antibacterial compounds urgently needed. among natural resources, the impatiens balsamina plant has the potential to be explored as the new source of antibacterial agents. this study aimed to identify the phytochemical composition and evaluate the antibacterial activity of i. balsamina leaf extracts against gram-positive and gram-negative bacteria. extracts were prepared using the maceration method with methanol, chloroform, and nhexane solvents at a 1:5 sample-to-solvent ratio. phytochemical screening was performed qualitatively, and antibacterial activity was evaluated using the disc diffusion assay. analysis of methanol extract detected the presence of flavonoids, saponins, tannins, phenols, steroids, and glycosides, while chloroform extract consisted of tannins, phenols, and steroids. on the other hand, steroids were the only compounds detected qualitatively in n-hexane extract. antibacterial testing revealed that methanol extract exhibited the highest activity, with zones of inhibition (zoi) of 15.10±0.18 mm, 9.40±0.30 mm, 14.75±1.28 mm, and 8.67±0.50 mm against streptococcus mutans fncc 0405, staphylococcus aureus atcc 25923, escherichia coli atcc 25922, and klebsiella pneumoniae atcc 700603, respectively. a concentration-dependent zoi was observed in the methanol extract, with activity increasing at higher concentrations. gcms analysis of the methanol extract identified 32 compounds, including n-hexadecanoic acid (12.12%), 2-acetylbenzoic acid (8.26%), 5hydroxymethylfurfural (8.21%), and 2-methoxy-4-vinylphenol (4.67%), which are known to possess antimicrobial, antioxidant, and antiinflammatory activities. chloroform extract showed moderate activity against s. mutans (7.04±0.15 mm) and s. aureus (7.10±0.31 mm), while n-hexane extract exhibited no antibacterial activity. the significant antibacterial activity of methanol extract is likely due to its rich phytochemical composition, highlighting methanol as an effective solvent for extracting bioactive compounds. these findings provide a strong foundation for further exploration of i. balsamina leaf extracts as a source of antibacterial agents. keywords: antibacterial activity; gram-positive; gram-negative; impatiens balsamina leaf extracts; phytochemicals. introduction bacterial antimicrobial resistance (amr) is the result of bacterial alterations that reduce the effectiveness of antimicrobial medications (murray et al., 2022). in 2019, bacterial amr had been associated with an estimated 4.95 million fatalities, of which 1.27 million were directly related to bacterial amr (murray et al., 2022). if the emergence of amr is not handled carefully, it is predicted to cause approximately 10 million deaths per year globally by 2050 (singh & tandon, 2023). the development of amr has led to the ineffectiveness of the most widely used antibiotics and consequently placed amr as one of the top ten threats to human health worldwide (pouran et al., 2024; world health organization, 2024). one of the crucial aspects that should receive more attention to combat antibiotic resistance is the development of new antibiotics through the exploration of natural resources, notably herbal plants (pouran et al., 2024; seukep, nembu, et al., 2023). herbal plants are a prospective source of new antimicrobial drugs due to the bioactivities of their diversified secondary metabolites, including alkaloids, flavonoids, phenolic compounds, terpenes, saponins, and others (šovljanski et al., 2023). these compounds are not only essential to plant defense systems, but also display substantial antimicrobial activities (seukep, mbuntcha, et al., 2023; šovljanski et al., 2023). scientific studies to evaluate the antibacterial activity of medicinal plant extracts against gram-positive and gram-negative bacteria are crucial in the quest to search and to identify novel antibiotic compounds that are effective against pathogenic bacteria (gorlenko et al., 2020; pandey & agnihotri, 2015). the plant impatiens balsamina l. has been traditionally utilized to treat skin infections, wounds, and inflammation, suggesting the potential for active compounds with antibacterial activity (seukep, https://doi.org/10.14421/biomedich.2025.141.327-335 328 biology, medicine, & natural product chemistry 14 (1), 2025: 327-335 mbuntcha, et al., 2023; šovljanski et al., 2023). although this plant is known to have benefits in traditional medicine (qian et al., 2023), in-depth scientific studies on its antibacterial effectiveness, especially against gram-positive and negative bacteria, are still very limited. most of the previous studies are more general or limited to a few specific types of bacteria, so data on its effectiveness against a wide range of gram-positive and negative bacteria is rather scarce. in addition, specific phytochemical profiles that contribute to antibacterial activity have not been widely described. optimization of the type of solvent used in extracting i. balsamina also needs to be done to obtain the best antibacterial activity. this study aims to identify the phytochemical content and to analyze the antibacterial activity of i. balsamina leaf extracts against several gram-positive bacteria such as streptococcus mutans and staphylococcus aureus, as well as gram-negative bacteria such as escherichia coli and klebsiella pneumoniae. in this study, i. balsamina was extracted using three different solvents namely methanol, chloroform, and n-hexane, to select extract with the best antibacterial activity. this research is crucial to discover new potential antibacterial compounds from i. balsamina leaves and contribute as one of the solutions in overcoming bacterial amr. this study offers a novel contribution by identifying the phytochemical profile of i. balsamina leaf extract and evaluating its antibacterial activity comprehensively, covering clinically relevant gram-positive and negative bacteria. materials and methods materials materials used in this study were i. balsamina leaves, methanol (merck, germany), chloroform (merck, germany), and n-hexane (merck, germany) solvents. the tested bacteria used were gram-positive bacteria staphylococcus aureus atcc 25923 and streptococcus mutans fncc 0405, as well as gram-negative bacteria escherichia coli atcc 25922 and klebsiella pneumoniae atcc 700603. the media used in the antibacterial activity test was luria bertani (lb). in the phytochemical screening, the materials used were mg powder, hcl 2 n, concentrated hcl, fecl3, h2so4, and liebermannburchard reagent. in addition, the tools used were blender, jar, whatmann no.1 filter paper, rotary evaporator, paper disc, petri dish, ose needle, test tube, bunsen burner, micropipette, laminar air flow cabinet, autoclave, and glassware. sample preparation impatiens balsamina leaves were obtained from sembung village, mengwi sub-district, badung district, bali province, indonesia. for identification purposes, whole plants (roots, stems, leaves, and flowers) were made into voucher specimens by attaching them to clean paper and then stacking them with heavy objects until they were dry and stiff. the voucher specimens were identified at the research center for plant conservation, botanical gardens, and forestry, bedugul, bali. the leaf samples used have been selected that are neither too young nor too old, by taking the fourth leaf from the top and the second leaf from the bottom. about 5 kg of fresh leaves were washed and chopped into smaller pieces, then dried in the oven at 45° for 24 hours. the dried simplisia was then tested for moisture content using the oven gravimetric method, with a temperature of 105°c for 5 hours until the moisture content was less than 10%. the dried simplisia is then blended into coarse powder, then stored at room temperature in a closed and airtight container (wijaya & indraningrat, 2021). extraction a total of 100 grams of dry simplisia powder was macerated using 500 ml of each solvent (methanol, chloroform, and n-hexane) for 2 x 24 hours. the results of the first 24 hours of maceration were filtered with whatmann no. 1 filter paper, then the extract was stored in the refrigerator (4°c) while the pulp was macerated again for 24 hours. the extracts from the first and second maceration were combined and then the solvent was evaporated using a rotary evaporator at 50°c. the concentrated extract that was obtained was stored in a closed and airtight vial at 4°c until ready to be used for further experiments (wijaya & indraningrat, 2021). phytochemical screening phytochemical screening of i. balsamina extracts was carried out using the qualitative method with colour reagents as a preliminary test to determine the presence of secondary metabolites, namely flavonoids, tannins, saponins, steroids, glycosides, and phenols. the extract with the wider diameter zone of inhibition (zoi) was selected and further be analysed using the gas chromatography coupled to mass spectrometry (gc-ms) method. antibacterial activity test luria bertani (lb) agar media was used for isolate maintenance. this media was made by mixing 5 grams of yeast extract, 10 grams of peptone, 5 grams of nacl, and 20 grams of agar. the solution was then homogenized and put into an autoclave for sterilization at 121°c for 15 minutes. the solution was then poured into petri dishes and allowed to solidify. meanwhile, each pure culture on agar media of gram-positive bacteria staphylococcus aureus atcc 25923 and streptococcus mutans fncc 0405, as well as gram-negative bacteria escherichia coli atcc 25922 and klebsiella pneumonia atcc 700603 were inoculated using a sterile ose needle and then in sterile liquid lb tubes, and incubated at 37°c for 24 hours. fridayanthi et al. – phytochemicals and antibacterial activity of impatiens balsamina 329 the antibacterial activity test of each concentrated extract was conducted against four tested bacteria using the disc diffusion method. first of all, each bacterial isolate was rejuvenated on liquid lb media, then 200 µl suspension of each test bacterium with an optical density (od) of 0.5 was taken and put into a petri dish containing lb agar media with the spread plate technique using a sterile cotton swab. next, paper discs with a size of 6 mm were prepared for each extract. a total of 20 µl of extract was pipetted on the disc paper, then dried for 30 minutes. each paper disc was transferred using tweezers to lb agar media that already contained the test bacteria. the petri dishes were incubated for 24 hours at 37°c in an upside-down position. the repetition was carried out three times. antibiotics ampicillin, streptomycin, and nalidixic acid were used as positive controls while the three solvents used during the extraction process became negative controls. antibacterial activity was calculated based on the average diameter of the inhibition zone formed from the extract using a caliper (wijaya & indraningrat, 2021). the diameter of zoi formed was grouped based on 4 categories, namely: weak (0-5 mm), moderate (5-10 mm), strong (10-20 mm), and very strong (>20 mm) (davis & stout, 1971). extract that displays the best zoi were then made into three different concentrations, namely 75%, 50%, and 25%, to determine whether there are significant differences in zoi at different concentrations. data analysis a descriptive analysis was used for the phytochemicals detected in the extracts. for the antibacterial activity, the average value ± standard deviation of the zoi diameter for each extract was calculated using microsoft excel 2019. one way analysis of variance (anova) test was then conducted using ibm spss 25 to test the similarity of the means of populations of three or more. then the post-hoc test was carried out using the tukey method to determine which samples had significant differences. ethical clearance the experiment reported in this paper has received ethical approval from the health research ethics commission, faculty of medicine and health sciences, warmadewa university, denpasar-bali under ethics number: 81/unwar/fkik/ec-kepk/ix/2022 on 21 september 2022. results and discussion sample identification impatients balsamina leaf samples were obtained from sembung village, mengwi district, badung regency, bali. in addition to leaves, whole plant samples consisting of roots, stems, leaves, fruits, and flowers were also taken and made into voucher specimens (figure 1) for plant identification purposes. plant identification was conducted at the research center for plant conservation, botanical gardens, and forestry, bedugul, bali. the identification results showed that the samples used in this study were indeed i. balsamina l. species (table 1). figure 1. voucher specimen of the impatiens balsamina sample. table 1. result of sample plant identification. kingdom plantae division magnoliophyta class magniioliopsida ordo ericales family balsaminaceae genus impatiens l. species impatiens balsamina l. impatiens balsamina extraction with different solvents about 5 kg of i. balsamina leaves were harvested, then dried and pulverized to become 616.3 grams of dried powder. the water content was then calculated using the oven gravimetric method and a value of 4.86% was found. after the extraction using three different solvents and solvent evaporation processes were carried out, it was found that the extract yields obtained with methanol, chloroform, and n-hexane solvents were 9.8%, 5.1%, and 4.8%, respectively. the difference in the amount of yield from the extraction of i. balsamina leaves macerated with different solvents is due to the polarity value of each solvent. the amount of extract obtained is influenced by several things such as the amount of simplisia powder used, the fineness of the simplisia, and the type of solvent used (andriyani et al., 2017; dirar et al., 2019; zhang et al., 2021). the type of solvent can affect the yield of active substances and the use of the best solvent will ensure an optimal extraction process. the highest extract yield was obtained using methanol solvent because it has polar-protic properties. polar solvents can dissolve phytochemical compounds more optimally because of their hydrogen bonding and dipole-dipole interactions that facilitate the dissolution of the compounds, and thus, enhancing their extraction efficiency (chatepa et al., 2024; ghaffar & perveen, 2024). 330 biology, medicine, & natural product chemistry 14 (1), 2025: 327-335 antibacterial activity of impatiens balsamina leaf extracts the antibacterial activity of methanol, chloroform, and nhexane extracts from i. balsamina leaves was tested using the disc diffusion method. negative controls (the solvents) confirmed no antibacterial effects, while positive controls (streptomycin, nalidixic acid, and ampicillin) were used to compare the antibacterial activity. the test results indicated that methanol extract has the strongest antibacterial activity, effective against both gram-positive and gram-negative bacteria, with the largest zoi of 15.10 ± 0.18 mm on s. mutans (table 2). chloroform extract was effective only against grampositive bacteria (s. aureus and s. mutans), while nhexane extract showed no activity. methanol, a polar solvent, excels at extracting diverse bioactive antibacterial compounds compared to chloroform or nhexane (aziz et al., 2018; neamah et al., 2021; singh et al., 2010). these findings suggest that methanol is the most suitable solvent for isolating antibacterial compounds from i. balsamina leaves, offering potential for therapeutic applications. table 2. antibacterial activity test results. bacteria treatment zone of inhibition mean (mm) ± sd streptococcus mutans methanol extract 15,10 ± 0,18 chloroform extract 7,04 ± 0,15 n-hexane extract 0 control (-) 0 control (+) streptomicin 17,15 ± 0,29 control (+) nalidixic acid 15,45 ± 0,26 control (+) ampicilin 6,74 ± 0,04 staphylococcus aureus methanol extract 9,40 ± 0,30 chloroform extract 7,10 ± 0,31 n-hexane extract 0 control (-) 0 control (+) streptomicin 15,01 ± 0,29 control (+) nalidixic acid 15,16 ± 0,10 control (+) ampicilin 7,84 ± 0,16 eschericia coli methanol extract 14,75 ± 1,28 chloroform extract 0 n-hexane extract 0 control (-) 0 control (+) streptomicin 14,94 ± 0,09 control (+) nalidixic acid 15,53 ± 0,96 control (+) ampicilin 8,79 ± 0,15 klebsiella pneumoniae methanol extract 8,67 ± 0,50 chloroform extract 0 n-hexane extract 0 control (-) 0 control (+) streptomicin 16,04 ± 0,27 control (+) nalidixic acid 15,89 ± 0,85 control (+) ampicilin 8,73 ± 0,40 the normality and homogeneity test showed that all treatment data were normally distributed and homogeneous (p>0.05). anova test revealed that solvent types significantly affected zoi in s. mutans, s. aureus, e. coli, and k. pneumoniae (p=0.001, α<0.05). moreover, tukey test results indicated that antibacterial activity of the methanol extract was comparable to nalidixic acid against s. mutans and to streptomycin and nalidixic acid against e. coli (tabel 3). additionally, methanol extract showed antibacterial activity equivalent to ampicillin against k. pneumoniae. methanol is highly effective in extracting antibacterial bioactive compounds due to its polarity, which enhances the solubility and extraction of phenolic compounds. the polarity of the solvent is crucial, as it directly impacts the efficiency of bioactive compound extraction and their subsequent antibacterial activity (arya et al., 2022; cui et al., 2020). methanol and other polar solvents are preferred for extracting compounds with significant antimicrobial properties, making them valuable in pharmaceutical and industrial applications (arya et al., 2022). fridayanthi et al. – phytochemicals and antibacterial activity of impatiens balsamina 331 table 3. comparison of antibacterial activity (zoi) between extracts and positive controls against selected bacterial strains. treatment tested bacteria s. mutan s. aureus e. coli k. pneumoniae methanol extract 15.108b 9.403b 14.750a 8.671b chloroform extract 7.040c 7.100d 0.000c 0.000c n-hexane extract 0.000d 0.000e 0.000c 0.000c control (+) streptomicin 17.159a 15.019a 14.948a 16.044a control (+) nalidixic acid 15.452b 15.168a 15.553a 15.890a control (+) ampicilin 6.743c 7.840c 8.790b 8.734b note: mean values followed by the same letter in the same column indicate mean values are not significantly different in the tukey test (α = 0.05); mean values followed by different letters in the same column indicate mean values are significantly different in the tukey test (α = 0.05). further analysis was performed on the methanolic extract of i. balsamina, which demonstrated the strongest antibacterial activity among the other extracts. to evaluate the relationship between extract concentration and antibacterial efficacy, the extract was tested at varying concentrations of 25%, 50%, and 75%. this approach aimed to assess how changes in concentration influenced the extract's ability to inhibit bacterial growth. table 4 shows that higher concentrations of the extract resulted in greater inhibition. of the four test bacteria, methanol extract showed the strongest antibacterial activity against e. coli at 75%, while no zoi was observed for s. aureus and k. pneumoniae at 25% concentration. higher concentrations of plant extracts generally result in greater zones of inhibition, indicating stronger antibacterial activity (allyn et al., 2018). this dose-dependent relationship is consistent across various plant extracts and bacterial strains (aishah et al., 2012; allyn et al., 2018; hasan et al., 2013). table 4. effect of methanol extract concentration on antibacterial activity against selected bacterial strains. bacteria concentrations zone of inhibition mean (mm) ± sd streptococcus mutans 25% 6,47 ± 0, 23 50% 7,51 ± 0,06 75% 8,46 ± 0,27 control (-) 0 control (+) streptomicin 17,15 ± 0,29 control (+) nalidixic acid 15,45 ± 0,26 control (+) ampicilin 6,74 ± 0,04 staphylococcus aureus 25% 0 50% 7,06 ± 0,33 75% 9,19 ± 0,39 control (-) 0 control (+) streptomicin 15,01 ± 0,29 control (+) nalidixic acid 15,16 ± 0,10 control (+) ampicilin 7,84 ± 0,16 eschericia coli 25% 9,59 ± 0,56 50% 12,55 ± 0,63 75% 12,56 ± 0,79 control (-) 0 control (+) streptomicin 14,94 ± 0,09 control (+) nalidixic acid 15,53 ± 0,96 control (+) ampicilin 8,79 ± 0,15 klebsiella pneumoniae 25% 0 50% 7,02 ± 0,60 75% 8,58 ± 0,34 control (-) 0 control (+) streptomicin 16,04 ± 0,27 control (+) nalidixic acid 15,89 ± 0,85 control (+) ampicilin 8,73 ± 0,40 the zoi of i. balsamina methanolic extracts at three concentrations were analyzed to assess the effect of concentration on antibacterial activity. the shapiro-wilk test confirmed normal data distribution while levene’s 332 biology, medicine, & natural product chemistry 14 (1), 2025: 327-335 test indicated homogeneity (p>0.05). anova test results showed that extract concentration significantly affected zoi in s. mutans, s. aureus, e. coli, and k. pneumoniae (p<0.05). tukey test analysis further revealed significant differences among concentration groups, as shown in table 5, except for e. coli, where 50% and 75% concentrations showed no significant difference in zoi. table 5. diameter of zone of inhibition in different methanol extracts concentrations. treatment tested bacteria s. mutan s. aureus e. coli k. pneumoniae 25% 6.472c 0.000c 9.599b 0.000c 50% 7.511b 7.062b 12.550a 7.022b 75% 8.468a 9.191a 12.563a 8.581a note: mean values followed by the same letter in the same column indicate mean values are not significantly different in the tukey test (α = 0.05); mean values followed by different letters in the same column indicate mean values are significantly different in the tukey test (α = 0.05). phytochemicals of impatiens balsamina leaf extracts the phytochemical analysis showed that methanol extract contains the most secondary metabolites, namely flavonoids, tannins, saponins, phenolics, glycosides, and steroids (table 6). chloroform extract contains secondary metabolite compounds, namely tannins, phenolics, and steroids. meanwhile, n-hexane extract only contains steroid compounds. the abundance of secondary metabolites in the methanol extract likely explains its superior antibacterial activity compared to chloroform and n-hexane extracts. the limited composition of bioactive compounds in chloroform and n-hexane extracts corresponds to their lower antibacterial effectiveness. these findings emphasize the importance of solvent polarity in extracting a broad range of bioactive compounds, highlighting methanol as the most effective solvent for isolating antibacterial agents from i. balsamina. table 6. phytochemical composition of methanol, chloroform, and n-hexane extracts of i. balsamina leaves. compounds i. balsamina leaves extracts methanol chloroform n-hexane flavonoid + saponin + tannin + + phenol + + steroid + + + glycoside + the polarity of the solvent significantly influences the extraction of bioactive compounds. polar solvents like methanol are particularly effective in extracting phenolic compounds. studies have shown that methanol extracts generally have higher phenolic and flavonoid contents compared to non-polar solvents (arya et al., 2022; cui et al., 2020). phenolic compounds including flavonoids and tannins are known for their antibacterial properties due to their ability to damage bacterial cell membranes, inhibit bacterial enzymes and toxins that are crucial for their survival and pathogenicity, as well as prevent the formation of bacterial biofilms (chen et al., 2024; miklasińska-majdanik et al., 2018). flavonoids are also able to inhibit the synthesis of bacterial dna and rna, thereby preventing their replication and survival (tan et al., 2022). in addition, tannins are known to disrupt bacterial membrane proteins and lipids, affecting membrane fluidity and integrity (olchowik-grabarek et al., 2022). phytochemical screening also showed that the methanol extract of i. balsamina leaves was positive for saponins and glycosides, while the chloroform and nhexane extracts did not contain these compounds. saponins interact with bacterial membrane lipids, disrupting the lipid bilayer and leading to cell lysis (li & monje-galvan, 2024). meanwhile, glycoside compounds are able to disrupt bacterial cell membranes and lead to cell death, as well as prevent biofilm formation (wang et al., 2024). in addition, the three extracts were found to positively contain steroids. steroid compounds exhibit diverse mechanisms of antibacterial action, ranging from disrupting bacterial cell membranes to enhancing the efficacy of existing antibiotics (da silva et al., 2022; ralambondrahety et al., 2021). gc-ms analysis of i. balsamina methanol extract identified 32 compounds, with the five most abundant listed in table 7. the dominant compound, nhexadecanoic acid (12.12%), is known to exhibit antioxidant, antimicrobial, anti-inflammatory, anticancer, fridayanthi et al. – phytochemicals and antibacterial activity of impatiens balsamina 333 nematicidal, and pesticidal activities (mazumder et al., 2020; siswadi & saragih, 2021). another compound, 2acetylbenzoic acid, has antimicrobial, antioxidant, and antifungal properties (vimalavady & kadavul, 2013). the extract also contained 5-hydroxymethylfurfural (8.12%), known for its antioxidant, antiischemic, and tyrosinase-inhibiting effects(zhao et al., 2013). additionally, 2-methoxy-4-vinylphenol (4.67%) was identified, with reported antimicrobial activity(rubab et al., 2020). however, no data is available on the bioactivity of 1[methoxy (phenyl) methyl]-4methylbenzene. table 7. five compounds with the largest peak area in methanol extract of i. balsamina leaves. no retention time chemical compounds area (%) 1 29.228 nhexadecanoic acid 12.12 2 18.004 1[methoxy (phenyl) methyl]-4-methylbenzene 12.03 3 18.830 2-acetylbenzoic acid 8.26 4 11.915 5-hydroxymethylfurfural 8.21 5 14.373 2-methoxy-4-vinyl-phenol 4.67 conclusions this study concludes that the methanol extract of i. balsamina leaves exhibits significant antibacterial activity against both gram-positive bacteria (streptococcus mutans fncc 0405 and staphylococcus aureus atcc 25923) and gram-negative bacteria (escherichia coli atcc 25922 and klebsiella pneumoniae atcc 700603), with zoi values of 15.10 ± 0.18 mm, 9.40 ± 0.30 mm, 14.75 ± 1.28 mm, and 8.67 ± 0.50 mm, respectively. the inhibition increased with higher extract concentrations. the chloroform extract shows moderate activity against s. mutans (7.04 ± 0.15 mm) and s. aureus (7.10 ± 0.31 mm), while the n-hexane extract demonstrates no antibacterial activity against either gram-positive or gram-negative bacteria. phytochemical analysis reveals that the methanol extract contains flavonoids, saponins, tannins, phenols, steroids, and glycosides, which likely contribute to its superior antibacterial activity. gc-ms analysis identified 32 compounds in the methanol extract, including bioactive compounds such as n-hexadecanoic acid, 2-acetylbenzoic acid, 5-hydroxymethylfurfural, and 2-methoxy-4vinylphenol, which are known for their antimicrobial, antioxidant, and anti-inflammatory properties. meanwhile, the chloroform extract contains tannins, phenols, and steroids, and the n-hexane extract contains only steroids. these findings highlight the concentrationdependent antibacterial effects of the methanol extract and the potential of methanol as a solvent for extracting bioactive compounds with antibacterial properties from i. balsamina leaves. future studies should focus on isolating and characterizing the specific bioactive compounds responsible for the observed antibacterial activity to understand their mechanisms of action. additionally, broader investigations involving different bacterial strains, cytotoxicity testing, and in vivo studies are recommended to validate the extract's efficacy and safety. acknowledgements: this research was funded by the faculty of medicine and health sciences, warmadewa university, under grant number 1023/unwar/fkik/pd13/ix/2024. authors’ contributions: putu utari fridayanthi, made dharmesti wijaya, and desak putu citra udiyani designed the experiments. putu utari fridayanthi performed the antibacterial test, compiled data analysis, and wrote the early draft of the manuscript. made dharmesti wijaya provided consumables, performed data analysis on gc-ms, and reviewed the draft manuscript. desak putu citra udiyani performed qualitative phytochemical screening. anak agung gede 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(2013). in vitro antioxidant and antiproliferative activities of 5-hydroxymethylfurfural. journal of agricultural and food chemistry, 61(44), 10604–10611. https://doi.org/10.1021/jf403098y this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 123-127 | doi: 10.14421/biomedich.2021.102.123-127 issn 2540-9328 (online) stability analysis of mathematical modeling of interaction between target cells and covid-19 infected cells sugiyanto1*, mansoor abdul hamid2, alya adianta1, hanny puspha jayanti1 , muhammad ja'far luthfi3 1mathematics department, faculty of science and technology, universitas islam negeri sunan kalijaga, indonesia. 2school of food science & nutrition, universiti malaysia sabah, malaysia. 3department of biological education, faculty of tarbiyah and education, universitas islam negeri sunan kalijaga, indonesia. corresponding author* sugiyanto@uin-suka.ac.id manuscript received: 19 october 2021. revision accepted: 30 october, 2021. published: 02 november, 2021. abstract the stability analysis in this mathematical model was related to the infection of the coronavirus disease 2019 (covid-19). in this mathematical model there were two balance points, namely the point of balance free from covid-19 and the one infected with covid-19. the stability of the equilibrium point was influenced by all parameters, i.e. target cells die during each cycle, number of target cells at 𝑡′ = 0, target cells infected during each cycle based on virion unit density, effective surface area of the network, the ratio of the number of virus particles to the number of virions, infected cells die during each cycle, the number of virus particles produced by each infected cell during each cycle, and virus particles die during each cycle. in the simulation model, immunity is divided into high, medium and low immunity. for high, moderate and low immunity, respectively, the highest number of target cells is in high, medium and low immunity, whereas for the number of infected cells and the number of covid-19, it is in the opposite sequence of the number of target cells. keywords: coronavirus disease 2019; equilibrium point stability; target cells and infected cells. introduction coronavirus disease 2019 (covid-19) was first known to infect residents in wuhan city, china, and was notified by the chinese government to who in december 2019 (sugiyanto & abrori, 2020). covid-19 belongs to subfamily orthocoronavirinae, family coronaviridae, and order nidovirales (tan et. al., 2020). about 80% of covid-19 illness show mild symptoms and 20% have severe symptoms. some of the 20% patients who contract covid-19 develop severe pneumonia, sometimes with acute respiratory distress, which can lead to organ failure and death. the stability analysis of mathematical modeling is used to determine the recovery period of covid-19 patients. there are many factors that determine a person would get into mild, severe or severe symptoms. we can classify these symptoms into three things depend on the immunity of the covid-19 patient. in this modeling, categorization were done using the t – i – v model. the target cell subpopulation (t) is cells in several organs, such as the lungs, heart, arteries, intestines and kidneys. the infected cell subpopulation (i) is a cell that is infected through a receptor on the surface called angiotensin converting enzyme 2 (ace2) (diaz, 2020). target cells were epithelial cells in all of these organs. this target cell was ace2. the conversion of angiotensin ii (vasoconstruction peptide) to angiotensin 1-7 (vasodilator) was catalyzed by ace2 (zhang et. al., 2020). 83% of normal lung cells express ace2, namely type ii alveolar epithelial cells (aecii), which make these cells viral reservoirs. the spike protein (shaped like a nail) stuck to the surface of the sars-cov virus (zoufaly et. al., 2020). the ace2 enzyme attaches to the cell membranes of several organs (bourgonje et. al, 2020). stability analysis the mathematical model obtained in system (1) refers to du and yuan's (2020) paper. 𝑑𝑇 𝑑𝑡′ = (𝑑𝜏)𝑇0 − (𝑑𝜏)𝑇 − (𝑘𝜏) 𝐴𝛼 𝑉𝑇 (1a) 𝑑𝐼 𝑑𝑡′ = (𝑘𝜏) 𝐴𝛼 𝑉𝑇 − (𝛿𝜏)𝐼 (1b) 𝑑𝑉 𝑑𝑡′ = (𝑝𝜏)𝐼 − (𝑐𝜏)𝑉 (1c) description of the target cell subpopulation, covid19 infected cells, virus population and parameters are shown in table 1. https://doi.org/10.14421/biomedich.2021.102.123-127 124 biology, medicine, & natural product chemistry 10 (2), 2021: 123-127 table 1. target cell subpopulation, covid-19 infected cells, virus population and parameters. no. symbol explanation unit 1 𝜏 average cycle time for viral replication 𝑑𝑎𝑦 2 𝑡′ = 𝑡/𝜏 number of virus replication cycles 3 𝑇 number of target cells at 𝑡′ 𝑐𝑒𝑙𝑙 4 𝐼 number of infected cells at 𝑡′ 𝑐𝑒𝑙𝑙 5 𝑉 number of virus particles at 𝑡′ 𝑣𝑖𝑟𝑢𝑠 6 (𝑑𝜏) target cells die during each cycle 7 𝑇0 number of target cells at 𝑡′ = 0 𝑐𝑒𝑙𝑙 8 (𝑘𝜏) target cells infected during each cycle based on virion unit density 9 𝐴 effective surface area of the network 𝑚𝑚2 10 𝛼 the ratio of the number of virus particles to the number of virions 𝑣𝑖𝑟𝑢𝑠 /𝑚𝑚2 11 (𝛿𝜏) infected cells die during each cycle 12 (𝑝𝜏) the number of virus particles produced by each infected cell during each cycle 13 (𝑐𝜏) virus particles die during each cycle theorem 1. equilibrium point there are two equilibrium points of system (1), namely: free from the covid-19 virus and infected with the covid-19 virus. the covid-19 virus-free equilibrium point is 𝐸𝑃0 = (𝑇, 𝐼, 𝑉) = (𝑇0, 0,0). the equilibrium point for contracting the covid-19 virus is 𝐸𝑃1 = (𝑇, 𝐼, 𝑉) = (𝑎1, 𝑎2, 𝑎3), where 𝑎1 = 𝐴𝛼(𝛿𝜏)(𝑐𝜏) (𝑘𝜏)(𝑝𝜏) , 𝑎2 = (𝑘𝜏)(𝑑𝜏)𝑇0(𝑝𝜏)− (𝛿𝜏)(𝑐𝜏)(𝑑𝜏)𝐴𝛼 (𝑝𝜏)(𝛿𝜏)(𝑘𝜏) , 𝑎3 = (𝑘𝜏)(𝑑𝜏)𝑇0(𝑝𝜏)− (𝛿𝜏)(𝑐𝜏)(𝑑𝜏)𝐴𝛼 (𝛿𝜏)(𝑐𝜏)(𝑘𝜏) . proof. from equation (1a) and 𝑑𝑇 𝑑𝑡′ = 0, we get 𝑇 = (𝑑𝜏)𝑇0𝐴𝛼 (𝑑𝜏)𝐴𝛼+(𝑘𝜏)𝑉 (2) from equation (1c) and 𝑑𝑉 𝑑𝑡′ = 0 obtained 𝐼 = (𝑐𝜏) (𝑝𝜏) 𝑉 (3) from 𝑑𝐼 𝑑𝑡′ = 0 and substituting equations (2) and (3) into equation (1), we get 𝑉 = 0 (4) or 𝑉 = (𝑑𝜏)[(𝑘𝜏)𝑇0(𝑝𝜏)− (𝛿𝜏)(𝑐𝜏)𝐴𝛼] (𝛿𝜏)(𝑐𝜏)(𝑘𝜏) = 𝑎3 (5) from equation (2) and equation (4), we get 𝑇 = 𝑇0. (6) from equation (3) and equation (4), we get 𝐼 = 0. (7) from equations (6), (7) and (4) it is proven that the covid-19 virus-free equilibrium point is 𝐸𝑃0. if equation (5) is substituted into equation (2), then we get 𝑇 = 𝐴𝛼(𝛿𝜏)(𝑐𝜏) (𝑘𝜏)(𝑝𝜏) = 𝑎1 (8) if equation (8) is substituted into equation (3), then we get 𝐼 = (𝑘𝜏)(𝑑𝜏)𝑇0(𝑝𝜏)− (𝛿𝜏)(𝑐𝜏)(𝑑𝜏)𝐴𝛼 (𝑝𝜏)(𝛿𝜏)(𝑘𝜏) = 𝑎2 (9) from equations (8), (9) and (5) it is proven that the equilibrium point for contracting the covid-19 virus is 𝐸𝑃1. ■ from theorem 1 it can be conveyed, if there is no covid-19 virus then someone will be safe or someone is virus free, and if there is a virus then a person's healing point is influenced by all parameters. virus-free can be achieved if there is no person carrying the virus or complying with health procedures such as wearing a mask, keeping a distance and washing hands as often as possible. when a person gets a virus, only the immune (target cells) can fight the infected cells. theorem 2. existence of the equilibrium point existence 𝐸𝑃0 fulfilled in any non-negative number parameter and existence 𝐸𝑃1 fulfilled if (𝑘𝜏)𝑇0(𝑝𝜏) − (𝛿𝜏)(𝑐𝜏)𝐴𝛼 > 0. proof. from theorem 1, that existence 𝐸𝑃0 and 𝐸𝑃1 proven. ■ from theorem 2 it can be seen that all parameters do not affect the existence of the equilibrium point 𝐸𝑃0. all parameters are target cells die during each cycle, number of target cells at 𝑡′ = 0, target cells infected during each cycle based on virion unit density, effective surface area of the network, the ratio of the number of virus particles to the number of virions, infected cells die during each cycle, the number of virus particles produced by each infected cell during each cycle, and virus particles die during each cycle. this means that if a person is not exposed to the covid-19 virus, the target cells would not affected or the condition of a person is healthy without the virus. for someone who is infected with the virus, all parameters affect the existence of the equilibrium point 𝐸𝑃0. this means that a person's condition will remain sugiyanto et al. – stability analysis of mathematical modeling of interaction … 125 healthy or even die depending on the target cells working well or not. theorem 3. stability of the equilibrium point (1) if √((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 − ((𝛿𝜏) + (𝑐𝜏)), then the equilibrium point 𝐸𝑃0 is locally asymptotically stable. (2) if √((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)(𝑎1) 𝐴𝛼 − ((𝛿𝜏) + (𝑐𝜏)) < 0, then the equilibrium point 𝐸𝑃1 is locally asymptotically stable. proof. for example, in system (1) it is written 𝑓1 = 𝑑𝑇 𝑑𝑡′ = (𝑑𝜏)𝑇0 − (𝑑𝜏)𝑇 − (𝑘𝜏) 𝐴𝛼 𝑉𝑇 (10a) 𝑓2 = 𝑑𝐼 𝑑𝑡′ = (𝑘𝜏) 𝐴𝛼 𝑉𝑇 − (𝛿𝜏)𝐼 (10b) 𝑓3 = 𝑑𝑉 𝑑𝑡′ = (𝑝𝜏)𝐼 − (𝑐𝜏)𝑉 (10c) jacobian matrix function 𝑓 from system (10) written can be obtained by first performing the partial derivation of the functions 𝑓1 = (𝑇, 𝐼, 𝑉 ) (11a) 𝑓2 = (𝑇, 𝐼, 𝑉 ) (11b) 𝑓3 = (𝑇, 𝐼, 𝑉 ) (11c) as follows. (i). partial derivative 𝑓1 with respect to 𝑇, 𝐼, 𝑉 namely: 𝜕𝑓1 𝜕𝑇 = −(𝑑𝜏) − (𝑘𝜏) 𝐴𝛼 𝑉; 𝜕𝑓1 𝜕𝐼 = 0; 𝜕𝑓1 𝜕𝑉 = 0; (ii). partial derivative 𝑓2 with respect to 𝑇, 𝐼, 𝑉 namely: 𝜕𝑓2 𝜕𝑇 = (𝑘𝜏) 𝐴𝛼 𝑉; 𝜕𝑓2 𝜕𝐼 = −(𝛿𝜏); 𝜕𝑓2 𝜕𝑉 = (𝑘𝜏) 𝐴𝛼 𝑇; (iii). partial derivative 𝑓3 with respect to 𝑇, 𝐼, 𝑉 namely: 𝜕𝑓3 𝜕𝑇 = 0; 𝜕𝑓3 𝜕𝐼 = (𝑝𝜏); 𝜕𝑓3 𝜕𝑉 = −(𝑐𝜏); the jacobian matrix is 𝐽(𝑇, 𝐼, 𝑉) = [ −(𝑑𝜏) − (𝑘𝜏) 𝐴𝛼 𝑉 0 0 (𝑘𝜏) 𝐴𝛼 𝑉 −(𝛿𝜏) (𝑘𝜏) 𝐴𝛼 𝑇 0 (𝑝𝜏) −(𝑐𝜏)] (1) for 𝑬𝑷𝟎, we get 𝐽(𝑇0, 0,0) = [ −(𝑑𝜏) 0 0 0 −(𝛿𝜏) (𝑘𝜏) 𝐴𝛼 (𝑇0) 0 (𝑝𝜏) −(𝑐𝜏) ] we find the eigenvalues of 𝐽(𝑇0, 0,0) that is 𝜆𝑖, for 𝑖 = 1,2,3, where  0 ,0,0 0.j t i  we get the eigenvalues of the jacobian matrix which is represented by                             1 2 2 2 3 0 0 1 , , 4 4 2 1 . 2 d k p t c c a k p t c c a                                             we know that ((𝛿𝜏) − (𝑐𝜏)) 2 ≥ 0 and (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 > 0, so that ((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 > 0. since the parameters are greater than zero, we get 1 20, 0,   and because √((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 − ((𝛿𝜏) + (𝑐𝜏)) < 0, then we get 𝜆3 = 1 2 [−((𝛿𝜏) − (𝑐𝜏)) ± √((𝛿𝜏) − (𝑐𝜏)) 2 − 4((𝛿𝜏)(𝑐𝜏) − (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 )] < 0. we get all negative eigenvalues, so that 𝐸𝑃0 is locally asymptotically stable. (2) for 𝑬𝑷𝟏, we get 𝐽(𝑎1, 𝑎2, 𝑎3) = [ −(𝑑𝜏) − (𝑘𝜏) 𝐴𝛼 (𝑎3) 0 0 (𝑘𝜏) 𝐴𝛼 (𝑎3) −(𝛿𝜏) (𝑘𝜏) 𝐴𝛼 (𝑎1) 0 (𝑝𝜏) −(𝑐𝜏) ] we find the eigenvalues of 𝐽(𝑎1, 𝑎2, 𝑎3) that is 𝜆𝑖, for 𝑖 = 1,2,3, where  1 2 3, 0.,j a a a i  we get the eigenvalues of the jacobian matrix which is represented by                                   3 1 1 1 2 2 2 3 , 1 4 , 2 1 4 2 k d a a k p c c a k p a a c c a                                                     we know that ((𝛿𝜏) − (𝑐𝜏)) 2 ≥ 0 and (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 > 0, so ((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)𝑇0 𝐴𝛼 > 0. since the parameters are greater than zero, we get 𝜆1 < 0, 𝜆2 < 0, 126 biology, medicine, & natural product chemistry 10 (2), 2021: 123-127 and because √((𝛿𝜏) − (𝑐𝜏)) 2 + 4 (𝑘𝜏)(𝑝𝜏)(𝑎1) 𝐴𝛼 − ((𝛿𝜏) + (𝑐𝜏)) < 0, then we get              2 3 11 4 0 2 c a ak p c                      . we get all negative eigenvalues, so that 𝐸𝑃1 is locally asymptotically stable.■ from theorem 3 the stability point is affected by all parameters. this means that a person will recover depending on the target cells that work. the better the target cells work, the healthier the person would be and those who have been infected with covid-19 will recover. simulation the parameters in this simulation are taken from du and yuan's (2020) paper. table 2 shows the parameter values. in this simulation, we replace the symbol (𝑝𝜏) with 𝑏. this is because in matlab there is no insert legend that can be written (𝑝𝜏). table 2. parameter values for simulation. no. parameter value 1 𝜏 7 2 𝑑𝜏 2 × 10−4 3 𝑇0 108 4 (𝑘𝜏) 𝐴𝛼 𝑇0 0.075 5 𝛿𝜏 0.4 6 𝑐𝜏 0.4 7 𝐼0 10 8 𝑉0 100 figure 1. changes in the number of target cells against the presence of the covid-19 virus. target cells reflect the number of cells in people with three conditions, namely: low, moderate and high immunity conditions. figure 1, figure 2 and figure 3 represent person with high immunity ((𝑝𝜏) = 𝑏 = 50), moderate immunity ((𝑝𝜏) = 𝑏 = 100), and low immunity ((𝑝𝜏) = 𝑏 = 150). person with good immunity shows the target cell from 10,000,000 cells in 13.09 days to 101,800 cells. person with moderate immunity shows the target cell from 10,000,000 cells in 8,514 days to 104,300 cells. person with low immunity shows the target cell from 10,000,000 cells in 2,398 days to 105,000 cells. the order of decline in target cells from the longest to the fastest is good, medium and low immunity. table 3 describes the descending order of the target cells. table 3. target cell decrease. no. immunity initial amount (cell) total ten thousand (cell) time (day) 1 high 10,000,000 101,800 13.09 2 medium 10,000,000 104,300 8.514 3 low 10,000,000 105,000 2.398 figure 2. changes in the number of infected cells against the presence of the covid-19 virus. figure 2 shows the peak number of infected cells differed between individuals with high, moderate and low immunity. a person with low immunity on day 6,155 the number of infected is 7.146 × 107 cell. a person with moderate immunity on day 7,685 the number of infected is 6.65 × 107 cell. a person with high immunity on day 11.46 the number of infected is 5.655 × 107 cell. briefly, this explanation is in table 4. table 4. increase in the number of infected cells. no. immunity highest number of cells (cell) time (day) 1 high 5.655 × 107 11.46 2 medium 6.65 × 107 7.685 3 low 7.146 × 107 6.155 figure 3. changes in the number of virus particles. sugiyanto et al. – stability analysis of mathematical modeling of interaction … 127 figure 3 shows the number of viruses with high, medium and low immunity conditions. for someone with high immunity the maximum virus count on day 13.19 is 4.31 × 108 virus. for someone with moderate immunity the maximum virus count on 9,492 days is 8.946 × 109 virus. for a person with low immunity the maximum viral load on day 8,068 is 1.356 × 1010 virus. table 5 describes the amount of virus in the condition of a person with high, medium and low immunity. table 5. increase in the number of virus particles. no. immunity highest number of viruses (virus) time (day) 1 high 4.31 × 108 13.19 2 medium 8.946 × 109 9.492 3 low 1.356 × 1010 8.068 conclusion the stability of being free of the covid-19 virus and infected with the virus is influenced by all parameters. the number of target cells, virus-infected cells and virus particles is affected by a person's immunity. if a person has high immunity, the number of target cells would decrease slowly. vice versa, if a person has low immunity, then the number of target cells will drop rapidly. in a person having low immunity, the infected cells and viruses will quickly increase in number compared to the one with high immunity. conflicts of interest: mjl is on the editorial board of the biology, medicine, & natural product chemistry, and was recused from this article’s review and decision. the authors declare that there are no conflicts of interest. 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(2020). angiotensin‐converting enzyme 2 (ace2), sars‐cov‐2 and the pathophysiology of coronavirus disease 2019 (covid‐19). the journal of pathology, 251(3), 228-248. diaz, j. h. (2020). hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe covid-19. journal of travel medicine. du, s. q., & yuan, w. (2020). mathematical modeling of interaction between innate and adaptive immune responses in covid‐19 and implications for viral pathogenesis. journal of medical virology, 92(9), 1615-1628. sugiyanto, s., & abrori, m. (2020). a mathematical model of the covid-19 cases in indonesia (under and without lockdown enforcement). biology, medicine, & natural product chemistry, 9(1), 15-19. tan, w., zhao, x., ma, x., wang, w., niu, p., xu, w., ... & wu, g. (2020). a novel coronavirus genome identified in a cluster of pneumonia cases—wuhan, china 2019− 2020. china cdc weekly, 2(4), 61-62. zhang, x., li, s., & niu, s. (2020). ace2 and covid-19 and the resulting ards. postgraduate medical journal, 96(1137), 403-407. zoufaly, a., poglitsch, m., aberle, j. h., hoepler, w., seitz, t., traugott, m., ... & penninger, j. m. (2020). human recombinant soluble ace2 in severe covid-19. the lancet respiratory medicine, 8(11), 1154-1158. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 1, 2019 | pages: 1-6 | doi: 10.14421/biomedich.2019.81.1-6 issn 2540-9328 (online) novel approaches for detection fluorescent-labeled by cellvizio lab system on hippocampal ca1 region asma ulhusna shaimi1, wan raihana wan aasim2, hasmah abdullah3, wan amir nizam wan ahmad3, tan soo choon4, ang chee wei4, zalina ismail1,* 1brainetwork centre for neurocognitive science, school of health sciences, universiti sains malaysia, 16150 kubang kerian, kelantan, malaysia. 2malaysian technology development corporation sdn. bhd., level 8-9, menara yayasan tun razak, malaysia. 3school of health sciences, universiti sains malaysia, 16150 kubang kerian, kelantan, malaysia. 4institute for research in molecular medicine (informm), universiti sains malaysia, 11800 pulau pinang, malaysia. author correspondency*: drzalina@gmail.com abstract neurosteroids have been identified in the 1981. dehydroepiandrosterone sulphate (dheas) is one of the vital neurosteroids that de novo synthesized in the nervous system from cholesterol precursor (baulieu & robel, 1998). the aim of the study is to develop a method for fluorescence labelling. alexa fluor 488 dye with dheas antibody can binds the dheas antibody in the rat brain monitored by cellvizio lab system. dheas antibody (igg isotype antibodies) was fluorescently conjugated by an amine-reactive compound, alexa fluor 5sdp ester 488 dye. the resultant alexa fluor 488-conjugated antibodies were collected and analyzed by uv-vis spectrophotometer instrument. the absorbance of the protein-dye conjugate at 280 nm and 494 nm were measured. then, the degree of labeling (dol) was calculated to achieve the desired results. fluorescence labelling were carried out into the ca1 region of hippocampus sprague-dawley rat. we reported that the conjugation was successful. optimal labeling depending on degree of labeling (dol) needs some necessity to achieve and effective binding to the target neurosteroid, dheas. cellvizio lab system connected with fiber fluorescence microscopy (ffm) probe is presented as a new approach in real-time imaging of dheas. in conclusion, we have developed a new method of dheas-alexa fluor fluorescence labelling to visualize and evaluate the changes of dheas fluorescence level in the rat hippocampus. this novel approach as a diagnostic tool and can be used to better understand the mechanisms and functions of dheas and other neurosteroids in future research. keywords: dheas; alexa fluor 488 dye; fluorescence labelling; cellvizio lab system introduction dehydroepiandrosterone sulphate (dheas) is a steroid which naturally synthesized in the brain. the steroids that are synthesized in the nervous system have been termed ‘neurosteroids’ (baulieu, 1981). baulieu & robel (1998), reported that concentrations of dheas are much higher in the brain than in plasma. the dheas can enhanced short term and long term memory performances in a variety of learning tasks. treatment aged animals with dheas enhance learning and memory in the t-maze footshock avoidance apparatus (farr et al., 2004). some studies have demonstrated that dheas enhancing memory performances in aged animals (michael et al., 2001; vallée et al., 2001). behavior experiments that use the morris water maze, y maze, and radial arm water maze in animal models of learning and memory showed that dheas supplementation significantly affect behavioural performance (bodensteiner et al., 2008). another study in ovariectomized rats, i.c.v. implants by dheas may decrease escape latencies and distances to platform in the morris water maze (frye & sturgis, 1995). in a study that assessed of learning and memory, fluorescence labelling was develop to conjugate between dheas monoclonal antibodies and alexa fluor 5-sdp ester 488 dye. thus, a sulphodichlorophenyl ester (sdp) functional group is attached to a fluorophore core, alexa fluor 488 dye and bind specificity with monoclonal antibodies directly to form fluorophore-antibody conjugates. moreover, sdp ester indicated hydrolytically stable form of the alexa fluor 488 dye for amine-conjugations (molecular probes, 2007). vira and co-reseachers (2010) reported that the steric hindrance and the absence of additional reactive sites on the fluorophore may affect the conjugation reaction. conjugation between alexa fluor dye and antibodies were given exceptionally bright and most photostable (panchuk-voloshina et al., 1999). in fluorescence labelling, achieveing an optimal degree of labeling (dol) and minimizing fluorescence quenching are the keys for conjugation (berlier et al., 2003). conjugation at high molar ratios of the fluorophore toward antibodies may cause dols become high. therefore, it can be principal to the fluorescence quenching and assume to dye-dye interactions (randolph & waggoner, 1997). the molar coefficient (ɛdye) of the alexa fluor 488 dye https://doi.org/10.14421/biomedich.2019.81.1-6 2 biology, medicine, & natural product chemistry 8 (1), 2019: 1-6 is 71,000 and indicated dols between ranges 4 to 9. further, at high dols, the alexa fluor 488 dye undergo more resistant to fluorescence quenching. the stability to ph of alexa fluor dye was monitored by measuring their absorption spectra in buffers at ph 4-9. the most stable conjugates were performed at ph 8.3. for labelling, alexa fluor 488 dye can be retained the intensity in conjugation with antibodies. in principle, any fluorescent dye can be chosen to conjugate protein for live cell imaging, and many new dyes with improved photochemical properties have been developed (panchuk-voloshina et al. 1999). to complement a develop method of fluorescence labelling, cellvizio lab system connected with ffm probe will be used as an imaging approach to visualize and quantify the fluorescence intensity in vivo and real time imaging. the cellvizio lab system provides to visualize deep brain through fluorescence imaging, high resolution, rapid, in real time image, with minimally invasive, and one animal at one time. besides, stereotaxis apparatus as a platform to position the fiber fluorescence microscopy (ffm) probe to the exact coordinate of target location (visualsonics, 2009). the probe, an approach that designed for cannula implantation on anaesthetized animals, consisting of a diameter tip 0.30 mm, lateral resolution 3.3 µm, optical sectioning 15 µm, and maximum field of view 600x500 µm (vincent et al., 2006). therefore, to understand how the brain activity of rats achieving in behavioral tasks, the cellvizio lab system with flexible ffm probe used to record in vivo imaging attached with dheas-alexa fluor fluorescence labelling at the region of interest. in this article, we report the successful of dheasalexa fluor 488 was expressed in the ca1 region of hippocampus rat brain. here, our goal was to highlight a new method of dheas-alexa fluor 488 as a fluorescence labelling of dheas and expressed the signal of dheas by ffm connected with cellvizio lab system in visualizing real time imaged, hence as a new imaging approach based on fluorescence. materials and methods antibody and reagents preparation dheas monoclonal antibodies (3 mg/ml) were bought from calbioreagents. alexa fluor sdp ester labeling dye (thermo scientific) was used to conjugate with antibodies. the corresponding fluorophore: protein (f:p) ratio, based on a280 and a494 absorption readings, was calculated according to the labeling instructions including the recommended correction factors for the absorbance of the dye at 280 nm. evaluation of dye delivery approaches in ca1 region of hippocampus all procedures were approved by the animal ethics committee universiti sains malaysia and followed the guidelines of the animal research and service centre. one group (n=7) male sprague-dawley (sd) rats (250 300 g) anaesthetised with isoflurane. sd rats were housed in groups of two per cage with twelve-hour light/dark cycle and food and water was provided ad libitum. all surgeries were performed under isoflurane anaesthesia. anaesthesia was maintained by mask inhalation of isoflurane vaporized at concentrations of up 4% in the induction phase, at 2.5% during surgical procedures and at 1.0-1.5% during prolonged experimental observations. dheas, neurosteroid in the brain were imaged by using a beveled tip ffm probe through a hole in the skull. the probe was positioned by using the holder of motorized stereotaxis device. one microliter of dheas-alexa fluor 488 dye was carried out into the ca1 region using 10 µl hamilton syringe, set to eject 1.00 µl over about 10 min. injection coordinates were (in mm): for the ca1 hippocampus, ap: -3.00 mm, ml: -1.7 mm and dv: 3.00 mm. optical recording started 1 h later. preparation of alexa fluor 5-sdp ester 488 dye labelled monoclonal antibodies dehydroepiandrosterone sulphate (dheas) was fluorescently conjugated to an amine-reactive compound, alexa fluor 5-sdp ester 488 dye (invitrogen). dheas solution (3.0 mg/ml) was exchanged to sodium bicarbonate by dialysis with against 0.1 m sodium bicarbonate solution (ph 8-9) for 2 hours at room temperature. then, alexa fluor 488 dye were prepared in dmf solution at concentration 10 mg/ml. therefore, 1.2 µl reactive dye was added into the 100 µg dheas solution and incubate the solution for 1 hour at room temperature. the resultant solution was transferred into the membrane dialysis tube and dialyzed against pbs (ph 7.4) an overnight in 4oc with continuous stirring. the resultant dheas-alexa fluor 488 was collected and sterilized via 200 nm polymer membrane filter and was stored at 4oc and -20oc for long storage. the concentration of dheas antibodies and alexa fluor dye were calculated from the absorbance at 280 and 488 nm, measured using an ultraviolet (uv-vis) spectrophotometer, using the following formula: the concentration of protein in the sample: protein concentration (m) = [a280 – (adye x cf280)] x dilution factor 203,000 shaimi et al. – novel approaches for detection fluorescent-labeled … 3 the degree of labeling: moles dye per mole protein = adye x dilution factor ɛdye x protein concentration (m) (molecular probe, 2011) real time imaging by fiber fluorescence microscopy (ffm) probe connected with cellvizio lab system after labelling, rats were anaesthetised and positioned on a stereotaxis apparatus. the ffm probe with a holder was placed into the mount of the stereotaxis device. the probe was fixed into position on top of the cannula implantation and set the coordinate as bregma point (0,0,0). next, the ffm probe connected with cellvizio lab system (le goualher et al., 2004; davenne et al., 2005) and a frame rate was at 12 hz. excitation of cells at 488 nm laser light and emission was measured at 494 nm. the cellvizio lab system have a single-pixel avalanche photodiode detector (apd) for resolution and sensitivity, respectively. results development of dheas-alexa fluor 488 fluorescence labelling we conjugated dheas antibody with reactive dye, excitation and emission wavelength at 488 nm and 494 nm, respectively. for this purpose, we determined by measuring the optimal degree of labeling with different concentration of protein, wherein the buffer ph was constant. table 1 show the absorbance of dheas antibody and reactive dye based on a280 and a494 absorption reading, the concentration of protein, dilution factor, degree of labeling, and molar coefficient of specific dye and correction factor for the fluorophore contribution as constant variables. based the result, the absorption reading for antibody and dye increase, the degree of labeling of fluorescence intensity was increased. therefore, according to obtained result, concentration of antibody, 0.91 m and degree of labeling, 4.63 was chosen for this present study as a new method. table 1. the protein concentration and degree of labeling in different protocols. b a tc h a280 a494 [p] d o l dl ɛdye cf280 λmax em 1 0.82836 0.57950 0.57 2.17 1 71,000 0.11 494 519 1 1.64540 0.92632 1.14 1.70 1 2 1.18320 1.44100 1.51 4.02 2 *constant variables 3 1.45050 1.99340 0.91 4.63 1 * a280 and a494 is an absorption reading by uv-vis spectroscopy. * [p] is protein concentration in mg/ml. * dol is degree of labeling. * dl is dilution factor to modify the calculation. * ɛdye is molar extinction coefficient at λmax in cm-1 m-1. * cf280 is correction factor for absorption readings at 280 nm. * λmax and em are the fluorescence absorbance and emission maxima, in nm, conjugated to an igg antibody. conjugation of dheas-alexa fluor 488 as fluorescence labelling a standardized protocol was implemented in this experiment, and yield less than 4 of dols of conjugation dheas-alexa fluor 488. the experimental workflow has made some modification and introduced two principals in a new method. first, the time incubation need to be longer for conjugation. the conjugates were incubated overnight to increase chemically binding of the dheas-alexa fluor conjugation. to achieve effective labelling, marks et al. (2004) reported that it required the incubation of 1 µm 5’-fluorescein-slf’ for 16 hours. the second modification was increased the volume of the dye from stock solution against antibody to enhance the expression signal of dheas-alexa fluor fluorescence intensity. the standard protocol of dye was 1.2 µl dye in 100 µg antibody (10 mg/ml). therefore, in this study the method was modified, 2.4 µl dye in 100 µg antibody. the uv spectra for batch 2 and 3 has shown in figure 1. figure 1. the uv spectra showing the absorption dheas antibody and alexa fluor dye for batch 1, batch 2 and batch 3. the absorption peaks at 288 nm and 494 nm correspond to the absorption for antibody and excitation of alexa fluor 488 dye, respectively. expression of dheas-alexa fluor fluorescence labelling by ffm imaging ffm probe attached to cellvizio lab system has been used to monitor dheas fluorescence levels in the hippocampal ca1 region. the characteristics of 4 biology, medicine, & natural product chemistry 8 (1), 2019: 1-6 microprobe s300b was shown in table 2. spraguedawley rat was positioned and placement on the motorized stereotaxis apparatus, then dheas-alexa fluor 488 fluorescence labelling was carried out into the ca1 region of hippocampus of anaesthetized animals. one hour after labelling, ffm imaging was visualized and imaged the signal of dheas fluorescence expression. the changes of dheas fluorescence expression was quantified and recorded the images at region of interest (roi)s in real time imaged and minimally invasiveness. table 2. properties of the microprobe s-300b used in this study. probe s-300b diameter (mm) 0.30 maximum field of view (µm x µm) 600 x 500 axial resolution (µm) 15 lateral resolution (µm) 3.3 * values provided by cellvizio lab system, mauna kea technologies, france figure 2. the signal of dheas fluorescence expression. (a) the images visualized indicated the dheas fluorescence was expressed the signal in hippocampal ca1 region. (b) the region of interest (roi) of dheas fluorescence signal were selected. discussion the excitation and emission spectra of alexa fluor 488 dye are illustrated in figure 3. figure 1 in this present study demonstrated the uv-vis spectra of alexa fluor 5-sdp ester 488 dye conjugated with dheas antibodies. the uv-vis spectrophotometer is a useful instrument in most laboratories (shahal et al., 2016). the aim of this study was to develop method of dheas-alexa fluor fluorescence labelling and evaluate the successful conjugation to be used as a tagging for dheas in the ca1 region of hippocampus by imaging approach, ffm connected with cellvizio lab system. this finding indicated that the antibody conjugation was effectively and efficiently to be used as fluorescence labelling to image and quantify dheas in the hippocampus rat brain by ffm imaging. figure 3. the illustration of excitation and emission spectra of alexa fluor 488 dye. coons and co-researchers (1950) showed that antibody conjugation widely uses nowadays in the biological sciences and medicine. in conjunction, labelling method is a direct technique which is the antibodies chemically conjugated to a fluorescent dye. the result of this study indicated that conjugation between alexa fluor 488 dye and dheas antibodies was succeed. the result of the present study also suggest that based on calculation degree of labeling, the f:p ratio the conjugation should be in range 4 to 9 (table 1). this finding indicated that fluorescence labelling can be effective used and given brighter expression in animal studies (hayashi-takanaka et al., 2014; vira et al., 2010). in order to obtain better labelling, the parameters that can affected the fluorescence should be considered. firstly, the antibodies and reactive dye should be in the same buffer. in this case, 0.1 m sodium bicarbonate was used to change the buffer of antibodies. it is necessary to achieve strongly binding in the covalent bond of conjugation. furthermore, longer incubation time required for fluorescence labelling protocols to determine the optimal dol of dheas-alexa fluor 488 conjugates. this suggested that the conjugation leaves for an overnight or at least 16 hours to increase the fluorescence intensity. the absorption data from uv-vis spectra was increased corresponding to the time of incubation. additionally, we modified the approach of shaimi et al. – novel approaches for detection fluorescent-labeled … 5 the dye from standard labeling protocols to increase the efficiency of labeling. initial experiment was conducted with preparation dye in standard protocol (1.2 µl dye in 100 µg antibody). we altered the dye which is increase the volume of dye from stock solution into twice time, 2.4 µl dye in 100 µg antibody. the results in this present study demonstrated that increased the dol of conjugation. there are have several requirements were highlighted in order to achieve the better labelling. this could be due these findings accordance with the previous studies and clearly demonstrated of brightness, photostability, photobleaching, binding affinity, size of fluorophore, fluorescent lifetime and molar coefficient (hayashi-takanaka et al., 2014; shaner et al., 2005; toseland, 2013). additionally, better expression of fluorescent labelling influence by ph sensitivity (shaner et al., 2005). thus, ph conditions performed as an indicator of fluorescent lifetime in conjugation of imaging living cells (lin et al., 2003). the ffm probe was inserted into the brain to recorded and quantified the changes of dheas fluorescence intensity level in the ca1 region of hippocampus. surprisingly, this present study is the first report to evaluate the expression of dheas fluorescence in the target area, ca1 area of hippocampus by ffm connected with cellvizio lab system. according to vincent et al. (2006) demonstrated that cellvizio lab system allowed researchers to visualize calcium signals in deep brain structures. these results are consistent with ffm connected with cellvizio lab system that allowed the direct recording in a short time frame, same animals can be used repeatedly, avoiding shrinkage of cells and tissues, and flexible probes (frykman et al. 1988). thus, ffm connected with cellvizio lab system was essential for in vivo imaging associated with behavioral task in order to time saving and does not require sample preparation or staining to prove the presence of fluorescence labelling. in this present study showed the signal of dheas fluorescence expressed at the region of interest (figure 2). this finding indicate that the signal of expression dheas fluorescence can be used as a potential in vivo imaging to evaluate and quantify the changes of dheas level in the rat brain due to physiology, pathology and pharmacology process (vincent et al., 2006; visualsonics, 2009). accordingly, the results in this study presumed that the changes of dheas fluorescence expression associated with cognition. we have developed new method of fluorescence labelling in conjunction with fluorescence imaging approach, ffm connected with cellvizio lab system that precise and accurate visualization of fluorescence signals in living brains as well as non-invasive method for cellular level imaging in vivo (davenne et al., 2005; vincent et al., 2006; makhlouf et al., 2008). conclusion in conclusion, dheas-alexa fluor 488 fluorescence labelling was developed to monitor and evaluate the changes of dheas fluorescence level that expressed in the ca1 region of hippocampus. beyond imaging, the dheas-alexa fluor fluorescence labelling is one step approach to monitor dynamic behavioral task and better understand the mechanisms of dheas. moreover, this approach can be used to discover the beneficial functions of dheas and other neurosteroids in the hippocampus at preclinical level to encourage in future research. acknowledgement this research was supported by the frgs grant (203/ppsk/6171153) and universiti sains malaysia. references baulieu, e. e. 1981. steroid hormone regulation of the brain, eds. fuxe, k. & gustafsson, j. a. (pergamon, oxford), 3-14. baulieu, e. e. & robel, p. 1998. dehydroepiandrosterone (dhea) and dehydroepiandrosterone sulfate (dheas) as neuroactive neurosteroids. proc. natl. acad. sci., 95:40894091. berlier, j. e., anca, r., gayle, b., jolene, b., diane, r. g., brian, j. f., william, g. t., yue, s., liu, j., cheung, c., chang, w., james, d. h., joseph, m. b., rosaria, p. h. & richard, p. h. 2003. quantitative comparison of long-wavelength alexa fluor dyes to cy dyes: fluorescence of the dyes and their bioconjugates. the journal of histochemistry & cytochemistry, 51(12): 1699-1712. bodensteiner, k. j., stone, i. j., & ghiraldi, l. l. 2008. effects of dehydroepiandrosterone sulfate and progesterone on spatial learning and memory in young and aged mice. the journal of general psychology, 135(3): 271-286. coons, a. h., & kaplan, m. h. 1950. localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. the journal of experimental medicine, 91(1): 1–13. davenne, m., custody, c., charneuau, p. & lledo, p. m. 2005. in vivo imaging of migrating neurons in the mammalian forebrain. chemical senses, 30:115-116. farr, a. s., william, a. b., kayoko, u. gaskin, f. s. & john, e. m. 2004. dheas improves learning and memory in aged samp8 mice but not in diabetic mice. life sciences, 75: 2775-2785. frye, c. a. & sturgis, j. d. 1995. neurosteroids affect spatial/reference, working, and long-term memory of female rats. neurobiology of learning and memory, 64: 83-96. frykman, g. k., mcmillan, p. j. & yegge, s. a. 1988. a review of experimental methods measuring peripheral nerve regeneration in animals. orthopclin n am., 19: 209-219. hayashi-takanaka, y., stasevich, t.j., kurumizaka, h., nozaki, n. & kimura, h. 2014. evaluation of chemical fluorescent dyes as protein conjugation partner for live cell imaging. plos one, 9(9): 1–11. 6 biology, medicine, & natural product chemistry 8 (1), 2019: 1-6 le goualher, g., 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dehydroepiandrosterone and their sulfate esters on learning and memory in cognitive aging. brain research reviews, 37: 301-312. vincent, p., maskos, u., charvet, i., bourgeais, l., stoppini, l., leresche, n., changeux, j., lambert, r., meda, p. &paupardin-tritsch, d. 2006. live imaging of neural structure and function by fibred fluorescence microscopy. european molecular biology organization reports, 7(11):1154-1161. vira, s., elena, m., glen, h. & paul, s. b. 2010. fluorescent labeled antibodies-balancing functionality and degree of labeling. anal biochem., 402(2): 146-150. visualsonics. 2009. application brief: cellvizio lab fibered fluorescence microscopy and microdialysis research. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 381-387 | doi: 10.14421/biomedich.2024.132.381-387 issn 2540-9328 (online) quality analysis and anti-termite activity of essential oils from the leaves, stems, and roots of litsea firma (blume) hook f febrilia gerina, rini muharini*, ira lestari, masriani, eny enawaty chemistry education department, faculty of teacher training and education, universitas tanjungpura jl. prof. dr.h. hadari nawawi pontianak 78124, tel. +62-561-740144, indonesia. corresponding author* rini.muharini@fkip.untan.ac.id manuscript received: 08 january, 2024. revision accepted: 18 september, 2024. published: 01 october, 2024. abstract litsea firma (blume) hook. f. has long been utilized as a traditional medicine plant. this research aims to analyze the quality of l. firma (blume) hook f essential oils extracted from its leaves, stems, and roots and to evaluate their anti-termite activity. the essential oils were obtained using the steam-water distillation method for 8 hours in triplicate. the quality of the essential oils was determined by their yield, specific gravity, refractive index, and optical rotation angle, which was compared with the previously reported quality data of l. cubeba essential oil. the results showed that all l. firma essential oils possessed a transparent colour, a distinctive aroma, and a bitter taste. the quality assessment revealed that essential oil yields ranged from 0.16% 2.42%. the specific gravity of leaves essential oil was 0.83. furthermore, the refractive index ratio ranged from 1.43 to 1.44, and the optical rotation angle varied between +15.3° to +16.8°. phytochemical analysis revealed the presence of terpenes in l. firma essential oil. essential oil of l. firma leaves exhibited a pronounced anti-termite activity with a percentage mortality of 100% and paper weight loss of 11.6 %. thus, l. firma essential oils, particularly from leaves, had promising potential for natural termite control. keywords: litsea firma (blume) hook f; medang pirawas; no-choice test; physicochemical analysis; phytochemistry screening. abbreviations: tlc = thin layer chromatography; d = diameter. introduction essential oils are extensively utilized in medicine and food preservation. in indonesia, essential oils are a significant export commodity, contributing significantly to profits reaching up to 172.9 million us dollars (international trade center, 2022). maximizing the market value of essential oils necessitates attaining high levels of purity, thereby ensuring excellent quality that commands a superior valuation (rahman et al., 2019). consequently, subjecting the produced essential oils to rigorous quality testing is imperative before engaging in enhanced domestic and international trading opportunities while effectively mitigating the risks associated with counterfeit products. the quality of essential oils is ascertained through adherence to national and international quality standards. several factors influence the quality of essential oils, including the variety of raw materials, plant cultivation methods, initial treatment of plant materials, oil extraction techniques, and post-production processes. the indonesian national standard (sni) regulates quality standards for essential oils, which encompasses both general and specific requirements. general requirements for essential oils contain parameters such as odour, colour, specific gravity, refractive index, optical rotation, and solubility of essential oils in alcohol. on the other hand, specific requirements vary depending on the essential oils being tested, including factors such as active ingredient content, acid number, ester number, and fatty oil content (satuhu & yulianti, 2012). genus litsea has been known as an aromatic plant (ammar et al., 2020; kusprandini et al., 2021). numerous studies have been conducted on the quality of essential oils derived from the litsea genus. kuspradini (2021) investigated litsea spp. originating from kalimantan, that is l. angulata, l. elliptica, and l. rubiginosa, focusing on the quality and antimicrobial of essential oils extracted from various parts of the plants through steam-water distillation. meanwhile, the essential oil of l. firma (blume) hook. f. leaves and stems were reported previously for their chemical composition (jamal, 2009). there was a difference in chemical composition between leaves and stems essential oils from the latter study. the leaf’s essential oil was dominated by non-terpenes, including 2-undecanoate (39.45%) and 9-decen-2-one (34.33%), while the stems contained a higher concentration of undecanoate https://doi.org/10.14421/biomedich.2024.132.381-387 382 biology, medicine, & natural product chemistry 13 (2), 2024: 381-387 (28.73%) and 7-hydroxy-3,7-dimethyloctanal (28.73%). another variant of l. firma, l. firma var. austroannmensis from vietnam, was reported to contain quite different chemical composition. those compounds are oxygenated monoterpenes (33.2%), sesquiterpene hydrocarbons (24.8%), monoterpene hydrocarbons (22.4%), and oxygenated sesquiterpenes (14.8%) (dai et al., 2020). nonetheless, there is no report hitherto on the physicochemical properties of essential oil from l. firma (blume) hook. f., particularly from different parts of the plant. thus, it could suggest the best source of essential oil from l.firma (blume) hook f. litsea firma (blume) hook. f., commonly known as medang pirawas or medang piawas by locals, is abundantly found in west kalimantan. this plant has been traditionally used by local communities for various medicinal purposes, both internally and externally. the leaves of medang pirawas are utilized to eliminate fishy odour for postpartum mothers or to treat skin allergies in infants. all parts of the l. firma plant possess a distinctive aroma and are reputed to repel insects. nevertheless, there is no report regarding the anti-termite activity from this plant’s essential oil. on the other hand, essential oils obtained from l. cubeba (kamle et al., 2019) and l. elliptica (roszaini et al., 2022) have been reported to exhibit anti-termite properties, making them promising natural solutions for termite control. thus, l.firma essential oil could likely have the potential to be an anti-termite. hence, this study explored the potential of essential oils derived from l. firma (blume) hook. f. plants for their quality and utility. the aims of this study were to determine the quality of essential oils from the leaves, stems, and roots of l. firma (blume) hook. f. compared to the quality of l. cubeba essential oil based on their physicochemical properties and to evaluate the antitermite activity of l. firma (blume) hook f essential oils toward coptotermes curvignathus holmgren, a rubber termite. materials and methods materials plant materials, such as leaves, stems, and roots of l. firma (blume) hook f., were collected in melawi district, west kalimantan, in january 2023. the plant voucher was determined and deposited at the research center for biology, cibinong, indonesia with the number letter b-1559/iph.3/ks/xii/2020. chemicals used included distilled water (h2o), anhydrous sodium sulfate (na2so4) (merck), methanol (ch3oh (99%) (merck), nhexane(c6h14) (merck), ethyl acetate (c4h8o2) (merck), silica gel 60 coated with fluorescence indicator f254 aluminium tlc plate, and sulfuric acid (h2so4 10%). coptotermes curvignathus holmgren was employed for anti-termite assay. the equipment used consisted of a set of steam-water distillation apparatus, hot plate (ika cmag hs 7, germany), abbe atago dr-a1 refractometer, pycnometer, digital analytical balance, and kernand wxg-4 polarimeter (advanx ai003, singapura). procedures plant sample preparation the leaves, stems, and roots of l. firma (blume) hook f. were cleaned from dirt with clean water and drained. each part of the plant was aerated and roughly chopped. extraction leaves (200 grams), stems (200 grams), and roots (150 grams) were extracted using the steam-water distillation method for 8 hours, as described previously (putri et al., 2023) with slight modification. the distillate was collected in a separatory funnel and separated from the water layer to yield essential oil. anhydrous na2so4 was added to dry the essential oil from the remaining water. the pure essential oil was collected in a vial bottle (figure 1). figure 1. essential oil of l. firma (blume) hook. f. leaves (a), roots (b), and stems (c). physicochemical testing quality analysis of l. firma essential oils was carried out through a series of physicochemical tests. these included determining the yield amount, specific gravity, optical angle of rotation, and refractive index of each essential oil based on procedures described by kuspradini (2021). the physicochemical data of l. firma essential oil was compared to the reported physicochemical data of essential oil were of l. cubeba, a commercial essential oil. phytochemistry the tlc test on l. firma essential oil used an eluent system of n-hexane and ethyl acetate (97:3). the resulting spot from elution was sprayed with h2so4 (10%) and heated until the stain was formed, and observed under uv light at 254 nm. gerina et al. – physicochemical properties of anti-termite litsea firma essential oil 383 anti-termite assay the anti-termite evaluation was conducted against subterranean termites, coptotermes curvignathus holmgren, using the no-choice test described previously (adfa et al., 2017) with modification. briefly, the essential oil’s concentration was prepared for 10% (v/v) in ethanol. thirty workers and 3 soldiers of c. curvignathus holmgren were fed with essential oilimpregnated filter paper whatman no. 1 (d = 3 cm).as a negative control, the same amounts of termites were fed with whatman filter paper without essential oil in it. for 21 days, termite condition was observed each day and the death termites were calculated. at the end of day 21, percentage mortality and percentage paper weight loss were determined. percentage mortality was calculated using the formula as follows: % 𝑡𝑒𝑟𝑚𝑖𝑡𝑒 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 = 𝑁𝑑𝑡𝑎 𝑁𝑡𝑏 × 100% (1) where ndta is the number of dead termites after treatment and ntb is the number of living termites before treatment. percentage paper weight loss was calculated as follows: % 𝑝𝑎𝑝𝑒𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑙𝑜𝑠𝑠 = 𝑃𝑤𝑏−𝑃𝑤𝑎 𝑃𝑤𝑏 × 100% (2) where pwb is the paper weight before treatment and pwa is the paper weight after treatment. then, the absolute coefficient was determined using the formula: 𝐴 = 𝐾𝐾−𝐸𝐸 𝐾𝐾+𝐸𝐸 × 100% (3) where a is the percentage of absolute coefficient, kk is paper weight loss of control paper, and ee is paper weight loss of tested paper. data analysis extraction, physicochemical tests, and anti-termite activity measurements were carried out in triplicate. essential oil quality data obtained was expressed as a mean (±) and standard deviation (sd), with a confidence level of p < 0.05 considered statistically significant. antitermite data was represented as a mean and a standard deviation. the mean difference between obtained antitermite data was determined using a t-test with a p-value < 0.05. results and discussion physicochemical properties the essential oils obtained from all parts of l. firma possessed a characteristic aromatic aroma, bitter taste, and clear colour (figure 1). essential oil quality parameters observed in this study were based on physicochemical properties, including yield, specific gravity, refractive index, and optical rotation angle, which were compared to other litsea genera, particularly l. cubeba. litsea cubeba essential oil, commonly known as "may chang," has gained recognition as an export commodity from china. therefore, it is a suitable benchmark for assessing the quality of l. firma essential oil. physicochemical data of l. firma essential oils showed that they differed significantly in yield (table 1.) table 1. quality analysis data of essential oils from l. firma (blume) hook f. sample yield (%, b/b) a specific gravity a refractive indexa optical rotation angle a leaves 2.42 ± 0.32b 0.83±0.00 1.43 ± 0.00c +15.8°±0.3 stems 0.16 ± 0.38b,c n.dd 1.44 ± 0.00c +16.8°±2.5 roots 0.37 ± 0.16b,c n.dd 1.44 ± 0.00c +15.3°±1.1 aas a mean with n=3 and sd, bsignificantly difference (p-value < 0.05, t-test) cnot significantly difference (p-value > 0.05, t-test) dnot determined yield is a crucial parameter that indicates the quantity of essential oil obtained through the distillation process. knowledge of the amount of yield facilitates calculations in preparing fresh ingredients according to the expected yield of essential oils (adisa et al., 2022). plant parts and the extraction duration will significantly influence the yield of essential oil produced. in this study, the essential oil from l. firma leaves exhibited the highest yield at a percentage of 2.42%, followed by roots essential oil (0.37%) and stems essential oil (0.16%) (table 1). in this study, l. firma roots proved to contain a pronounced quantity of essential oil that has not been reported previously. the variation in yield among different plant parts can be attributed to the distinct chemical compositions of the leaves, stems, and roots (putri et al., 2023). it seemed that l. firma essential oil was dominantly deposited in plant leaf tissue. the yield of essential oils from l. firma (blume) hook f. leaves and stems in this study demonstrated higher values compared to the findings reported by 384 biology, medicine, & natural product chemistry 13 (2), 2024: 381-387 jamal (2009). in her investigation, the yield of essential oil leaves and stems obtained using the steam-water distillation method for 5 hours was 1.68% and 0.23% respectively. these phenomena suggested that extraction duration time affects the yield quantity. however, it should be noted that excessively long distillation time can affect the quality of the essential oil produced (utomo & mujiburohman, 2018). plant materials' drying and storage processes significantly influence the resulting essential oil yield. in this study, the plant samples were stored for 27 days and dried for three days, likely resulting in a decrease in the essential oil yield of l. firma. drying at excessively high temperatures and for a prolonged duration can cause a reduction in the essential oil content (satuhu & yulianti, 2012). therefore, it is necessary to conduct further studies on the drying variations for l. firma plant tissue to obtain its essential oil. despite the difference in stem-water distillation duration used, comparison to essential oil reported previously from other litsea genera revealed that the yield of essential oil of l. firma leaves was higher than that of l. angulata leaves (0.90%), l. elliptica leaves (0.70%), l. rubiginosa leaves (0.17%) (kusprandini et al., 2021). nevertheless, the essential oil yield of l. cubeba leaves (9.33%) (suwandhi et al., 2014) was higher than that of l. firma leaves (figure 2.). thus, the position l. firma leaves essential oils in the litsea genus was promising. figure 2. essential oil yield from several litsea species. the steam-water distillation method is highly effective in obtaining substantial essential oil. this process involves heating water vapour, which then permeates the plant tissue, facilitating the release of essential oil onto the surface. the essential oil subsequently evaporates and travels toward the condenser. this essential oil extraction method offers several advantages, including using steam and heat under constant pressure. consequently, the distillation time is expedited while the risk of essential oil degradation is minimized (nugraheni et al., 2016). specific gravity, defined as the ratio of the weight and volume of essential oil compared to that of water, is an essential parameter for assessing the water content in essential oils. specific gravity can also be defined as the ratio of the density of the essential oil to the density of water (kua et al., 2021). consequently, specific gravity offers valuable insights into the water content present within essential oils. higher water content in essential oil corresponds to lower specific gravity values, whereas lower water content leads to higher specific gravity values. furthermore, specific gravity is influenced by various factors, including the composition of compounds within the essential oil, such as molecular weight and carbon chain (guenther, 2006). in the study, specific gravity measurement was conducted solely on essential oil obtained from leaves, due to the lack of essential oil from stems and roots. the analysis revealed that the specific gravity of l. firma essential oil from leaves was 0.83 (table 1), which was still below the range of specific gravity standard for l. cubeba essential oil (0.86-0.91 at 20°c) and l. angulata (0.86 at 20°c). however, it had a similar specific gravity value with l. elliptica essential oil (0.83 at 20°c) (aryani et al., 2023). this discrepancy suggested that l. firma essential oil extracted from leaf parts exhibited a higher water content than l. cubeba and l. angulata. the refractive index represents the ratio between the sine of the angle of incidence and the sine of the angle of refraction when a beam of light of a specific wavelength transitions from air to oil at a particular angle (guenther, 1987). according to a study by erliyanti et al. (2020), the refractive index exhibits a direct relationship with density, implying that higher density values correspond to higher refractive index values. this correlation arises from the influence of molar refractivity on density. the refractive index represents the behaviour of light waves, where a higher density in a material leads to a more excellent refractive index value, indicating slower light movement. the measurement of the refractive index of l. firma essential oil revealed no significantly different values across the leaves, stems, and roots, ranging from 1.43 to 1.44 (table 1). in comparison, the reported refractive index of l. cubeba at 20°c ranges from 1.47 to 1.49. likewise, the refractive index of l. firma essential oil falls short of the quality standard set by l. cubeba. it is important to note that the refractive index indicates water content in essential oils, with higher water content leading to a lower refractive index. this phenomenon arises from the refractive properties of water, which can negatively impact the quality of essential oils (guenther, 1987). hence, the refractive index value serves as an indicator of the essential oil's purity level. a higher refractive index value indicates lower water content and higher compound density. in order to decrease the water content in essential oils, purification can be achieved by adding anhydrous na2so4. the anhydrous na2so4 should be activated by heating it in an oven for 4 hours at 80°c before applied to essential oil (marfina et al., 2019). this activation process ensures that na2so4 delivers optimal outcomes 0 2 4 6 8 10 l. rubigunosa l. elliptica l. angulata l. cubeba l. firma yield (%) gerina et al. – physicochemical properties of anti-termite litsea firma essential oil 385 in binding water molecules present in essential oils. hence, in terms of the refractive index, although l. firma essential oil has not yet attained the quality standard of l. cubeba, there remains potential for enhancing its quality through purification techniques and reducing water content. essential oils possess the ability to exhibit optical activity, which refers to the capacity of compounds within the oil to alter the polarization direction of light as it traverses a specific medium. the optical rotation angle in essential oils can be measured using a polarimeter instrument, which quantifies the rotation angle in degrees. the optical rotation in essential oils can vary depending on the composition of compounds within the oil. optical rotation is a significant physical characteristic in analyzing and identifying essential oils, providing insights into the structural and optical properties of the essential oil compounds. among the l. firma essential oils, stem essential oil exhibited the bigger optical rotation angle with +16.8°. meanwhile, the roots and leaves essential oils showed similar optical rotation angle values, specifically +15.8° and +15.3°, respectively (table 1). nevertheless, all optical rotation values exhibited positive values. a positive (+) optical rotation signifies that the essential oil compound components can rotate the polarization plane to the right. such compounds are characterized by asymmetric carbon atoms, bonding the carbon atom to four distinct groups within its structure (lasapo et al., 2016). this result added to a previous report that l. firma leaves and stems essential oils possessed chiral compounds (jamal, 2009). in comparison to l. cubeba leaves essential oil, its optical rotation angle was recorded at +20.3°, while its stem essential oil’s optical rotation angle was recorded at -18.3°. this comparison result could indicate that the chemical composition of l. firma essential oils might differ from that of l. cubeba essential oils. phytochemical analysis in order to identify the presence of terpenes in l. firma essential oils, tlc plates were employed with an eluents system consisting of n-hexane and ethyl acetate (97:3) and a terpenes spotting reagent. tlc chromatogram showed one purple spot on tlc for essential oil from stems, with a retention factor (rf) value of 0.7 after adding the spotting reagent (figure 2). although the spot colours were not as bright as the stems’ essential oil, similar identifiable purple spots were detected for essential oil from leaves and roots. this might be due to less concentration of terpenes contained in the latter essential oils. this analysis of the leaves strengthened this suggestion and stem essential oil’s chemical composition previously reported (jamal, 2009), which showed that detected terpenes in leaves essential oil were slightly less amount than in stems. thus, since similar tlc spots are shown by roots essential oil with one in leaves essential oil, the chemical composition in roots essential oil might be closely identic to leaves essential oil. nevertheless, these results confirmed that essential oils from all parts of l. firma contained terpenes. terpenes are applied to numerous facets of everyday life and human health. they are utilized in developing pharmaceutical products, nutraceuticals, food and beverage formulations, cosmetics, perfumes, synthetic chemicals, aroma and flavour additives, rubber products, and even the biofuel industry. hence, terpene compounds assume a broad and diverse range of roles across various sectors that directly impact human existence (masyita et al., 2022). (a) (b) figure 3. tlc chromatogram profile of l. firma essential oil from leaves (d), stems (b), and roots (a) (n-hexane and ethyl acetate (97:3)) after heated and sprayed with a spotting reagent of h2so4 (10%), (a) under uv light at 265 nm and (b) under visible light. anti-termite analysis the local community has reported the potential of l. firma wood to act as an insect repellent. anti-termite evaluation on essential oil from l. firma leaves towards coptotermes curvignathus holmgren proved that the essential oil exhibited very strong anti-termite activity (table 1.) with 100% termite death. the percentage of paper weight loss was below 50%, which suggests that the essential oil possessed feeding deterrent activity (adfa et al., 2017). it was supported by an absolute coefficient percentage value of 58%, which was classified as strong antifeedant (dungani et al., 2012). table 2. anti-termite data of essential oil from l. firma (blume) hook f. sample mortality (%)a paper weight loss (%)a antifeedant absolute coefficient (%)a leaves 100,00 ± 0,00 11,59 ± 4,72 58,60 ± 18,49 stems n.d n.d n.d roots n.d n.d n.d aas a mean with n=3 and sd bnot determined 386 biology, medicine, & natural product chemistry 13 (2), 2024: 381-387 conclusions this study concluded that l.firma produced essential oil more from its leaves. regardless of their lower quantity compared to l. cubeba essential oil, the quality of all l. firma leaves, stems, and roots essential oils were similar. additionally, terpenoid compound was detected in all l. firma essential oils. hence, it can be suggested that an effective and efficient extraction method be found to produce essential oil with more yield. this study successfully added more value to essential oil from l.firma leaves, in which the essential oil exhibited pronounced anti-termite and antifeedant activity. thus, l. firma leaves essential oil possessed significant potential as a natural termite control. therefore, further research is needed to investigate the toxicity of l. firma essential oils and its other biological activities. acknowledgements: the authors thank bidik misi comdev outreaching universitas tanjungpura for providing research funds, which greatly supported this study. they also thank dr. hikma yanti and mr. alkhadi from forestry faculty, universitas tanjungpura, for running the anti-termite assay. authors’ contributions: all authors designed the study. febrilia gerina carried out the laboratory work. rini muharini analyzed the data. rini muharini and febrilia gerina wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no conflicts of interest. the authors reported no potential conflict of interest. funding: febrilia gerina declares bidik misi comdev outreaching universitas tanjungpura 2023 grant for providing research funds. references adfa, m., sanusi, a., manaf, s., gustian, i., & banon, c. 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(2018). pengaruh kondisi daun dan waktu penyulingan terhadap rendemen minyak kayu putih. jurnal teknologi bahan alam, 2(2), 124–128. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 2, 2015 | pages: 49-51 | doi: 10.14421/biomedich.2015.42.49-51 tapak liman (elephantopus scaber l) as immunostimulant and its effect on lymphocyte differentiation in mice balb/c marmi kelik faculty of languages and science, university of wijaya kusuma surabaya, indonesia author correspondency: marni.kelik@yahoo.com abstract tapak liman (elephantopus scaber l) is one of the plants that have medicinal properties and has been used for maintenance and improvement of health and disease treatment. the purpose of this study was to determine the effect of extracts of tapak liman (elephantopus scaber l) as immunostimulant to the development of lymphocytes in mice balb / c. the procedure of this study was to test aqueous extracts in vivo with various treatments (control, treatment of 0.5 g / kg, 1.0 g/ kg, 2.0 g / kg) in healthy mice balb / c for 2 weeks. after the treatment carried out analysis of the percentage and number of cells that express cd4+, cd8+ and cd4+ cd8 + in thymus organ, using flowcytometry. analysis of data using one-way anova followed tukey's test with spss. from the analysis showed that the extract of tapak liman at various doses showed no significant effect on the percentage expression of cd4 + cd8 + and cd4 + cd8 + in thymus organs. while the analysis of the number of cells, extracts of tapak liman show its effect on the number of cells that express cd4+, cd8 + and cd4 + cd8 + in thymus organs. concentration of 1.0g / kg of mice showed a good effect on the increase in t helper cells (cd4 +), cytotoxic t cells (cd8 +) and prothymosit cells (cd4 + cd8 +). keywords: tapak liman, immunostimulant, lymphocyte introduction elephantopus scaber linn, is a small herb, which grows in the wild throughout the tropical regions of the world. the major phytochemical constituents of the plant are elephantopin, triterpenes, stigmasterol, epofriedelinol and lupeol (rastogi and mehrotra, 1990; kritikar and basu, 1991). the plant has been used in the indian system of medicine as analgesic, diuretic, astringent and antiemetic. the leaves of the plant were known to be used for bronchitis, small pox and diarrhea and as a brain tonic (sankar et al., 2001). recently, it has been shown to possess anti-inflammatory and anti tumour activity in animal models (reico, 1989) and also found to have antibacterial activity against a few standard bacterial strains (avani and neeta, 2005). the genus elephantopus consists of approximately 30 species distributed in the neotropicsand the old world, and its lectotype species, e.scaber, occurs in all tropical regions (cabrera and klein, 1980; chen, 1985; cao and but, 1999). in southern china, hong kong and taiwan, the whole plant of e. scaber, a perennial herb, is well known as a folk medicine widely used in the treatment of nephritis, edema, dampness, chest pain, fever, pneumonia, scabies, and arthralgia due to wounding (peer and metzger, 1980; hsu, 1986; tsai andlin, 1999). in brazil, the infusion and the decoction of the whole plant are used to stimulate diuresis, reduce fever, and eliminate bladder stones (cabrera and klein, 1980; poli et al., 1992). it has also been popular as a medicinal herb in many countries of southeast asia, latin america and africa for a long time (hammer and johns, 1993; cao et al., 1997). since the 1970’s, a number of chemical constituents and pharmacological evaluations of e. scaber have been reported. for example, kurokawa et al. (1970) and govindachari et al. (1972) reported elephantopin, deoxyelephantopin, and isodeoxyelephantopin in this species; de silva et al. (1982) found that both alcoholic and chloroformic extracts of e. scaber contain cytotoxic germacranolide-type sesquiterpene lactones; poli et al. (1992) tested the aqueous and hydroalcoholic extracts of whole plants for acute toxicity, analgesic, antipyretic, anti-inflammatory, cardiovascular, diuretic, and constipating activities; hammer and johns (1993) reported that the plant extract of e. scaber was subjected to bioassays; lin et al. (1995) and tsai and lin (1999) evaluated the hepatoprotective and anti-inflammatory effects of the taiwanese folk medicine “teng-khia-u”, derived from three plant species including e. scaber, and but et al. (1997) described the isolation and structure elucidation of three germacranolide sesquiterpene lactones from e. scaber. the e. scaber roots and leaves aqueous extracts showed excellent hypoglycemic effect in diabetic rats by lowering the blood glucose level and serum insulin level. a decrease in the elevated levels of glycosylated hemoglobin, liver glycogen, triglycerides and cholesterol serum in alloxan-induced hyperglycemic rats was also reported by daisy et al. (2007). besides hypoglycemic activity in mice models, aqueous extract of e. scaber also showed significant antiinflammatory effect in both experimental acute and chronic arthritis rat models. the aqueous extract from the whole plant significantly inhibited the development of pad swelling in the acute experimental arthritis rats at a dose of 300 mg/kg while higher concentration of the 50 biology, medicine, & natural product chemistry 4 (2), 2015: 49-51 extract (500 mg/kg) was required to inhibit the development of chronic joint swelling in the chronic inflammatory model (tsai and lin, 1999). during thymocyte development, immature thymocytes that express both cd4 and cd8 genes must choose either a helper cd4+ or cytotoxic cd8+ t-cell fate. over the past two years, there have been some important advances regarding t-cell lineage choice, including the identification of transcription factors required for cd4 gene silencing by cd8-lineage cells (runx3) or for cd4+ t-cell differentiation (gata3), and a better understanding of how t-cell receptor (tcr) signalling correlates cd4/cd8-lineage differentiation to mhc specificity. this review summarizes these recent advances and highlights potential links between tcr signals and nuclear effectors of lineage differentiation (bosselut r, 2004) the purpose of this study was to determine the effect of extracts of tapak liman (elephantopus scaber l) as imunostimulant to the development of lymphocytes in mice balb/c. materials and methods this experiment used 4 groups, 1 group control and 3 treatment groups, with simple randomization. the samples taken at random (random) of the population reached the inclusion criteria as follows: murinestra in balb/c female, age 6 weeks, and healthy. 12 mice balb/c as are divided into 4 groups, each group consists of three mice. each group of mice is given the same standard food and drink ad libitum. four groups of mice are given: control (k): aquades without extract of tapak liman leaf treatment1 (p1): tapak liman leaf extract 0.5 grams/kg body weight/day treatment2 (p2): tapak liman leaf extract 1.0 g/kg body weight/day treatment3 (p3): tapak liman leaf extract 2.0g/kg body weight/day. the effect of extracts on the development of lymphocytes in thymus can be determined by measuring the percentage and number of lymphocytes expressing the express cd4 +, cd8 +and cd4+ cd8 + in thymus organ using flowcytometry on the 16th day, after the treatments of tapak liman extract at various doses. the data obtained in the form of percentage and number of cells that express the cd will be analyzed statistically by calculating the standard deviation in each treatment and one-way anova analysis to determine the effect of treatment on the development of lymphocytes. if there are significant differences in each treatment, tukey test would be done. analysis were done using spss with a p value<0.05. results and discussion thymus is the primary lymphoid organs, bone marrow as a producer of t cell precursors are derivatives of progenitor cells after differentiation in the thymus to form a functional t cells, and after 4 stages of maturation involves a variety of protein expression and t cell receptor (tcr) as an end to the circulation cell t peripherals (keer, 1998). transitional stages of thymocyte maturation can be characterized on phenotip cells with the tcr-cd3 complex, the presence of cd4 and cd8 coreceptor (kuper et al., 2002). based on the results of flowcytometry analysis (figure 1) note that the percentage of cd4 + expression on the control has an average value of 13:04%, in the treatment of 0.5 g / kg had a mean value 10.67%, in the treatment of 1.0 g / kg had an average of 9.85%. while on treatment of 2.0 g / kg had an average of 10.81%. based on the results of statistical analysis using one-way anova (appendix 8) found that the expression of cd4 +, with a significance value of 0864 (> 0.05). for the expression of cd8 + with a significance value 0676 (> 0.05), whereas for the expression of cd4 + cd8 + has a significance value 0.496 (> 0.05). it could be argued that there is no real influence liman extract of tread on all expressions of concentration versus percentage of cd4 +, cd8 +, cd4 + cd8 + in thymus organs. figure 1. cell profile cd4+, cd8+ and cd4+ cd8+: prosentage cell exspression cd4+, cd8+, cd4+ cd8+ on thymus. control (k), (p1:0.5 gr/kgbw), (p2:1.0 gr/kg bw), (p3:2.0 gr/kg bw). total cells (figure. 2) shows that the concentration of 1.0 g/kg increased the number of cd4+ cells (1615946) when compared to the control and the concentration of 0.5g/kg (976 610). concentration of 2.0 g/kg would reduce the number of cells that express cd4+ (1438173) but still better than in the control and treatment of 0.5 g/kg. likewise, the cd8+ cells that express at a concentration of 1.0 g/kg caused the number of cd8+cells at high (764 786) when compared to controls, 0.5 g/ kg, 2.0g/ kg. concentration of 2.0 shows thedecline inthe number of cytotoxic cells but still higher when compared with the concentration of 0.5g/kg and control. so it can be said that the concentration of 2.0g/kg gave the best effect on the number of cytotoxic cells in mouse thymus organs. total cells that express cd4+ cd8+ (prothymocyte) showed that consentration1.0g/kg showed the best effect when compared with control (7395966) and the treatment of 0.5g/kg (7,455,115), and marmi kelik – tapak liman (elephantopus scaber l) as immunostimulant … 51 2.0 g treatment/kg. showed that the concentration of 1.0g/kg increased the number of cells prothymocyte. figure 2. total cell cd4+, cd8+,cd4+cd8+ on thymus at control, 0.5 gr/kg bw, 1.0 gr/kg bw, and 2.0 gr/kg bw. lymphocyte progenitor cells derived from bone marrow, some of which are relatively non-lymphocyte differentiation have migrated to the thymus and reproduce themselves, here is to obtain properties of lymphocytes t lymphocytes at first thymosit not express cd4 and cd8, cells then develop into double positive (cd4 + cd8+) and eventually mature into single positive (cd4 + or cd8+) can then log back into the blood stream, back into the bone marrow or lymphoid organs and peripheral can live several months or years (schwarz and bandola, 2008). conclusion extract of tapak liman at various doses showed no significant effect on the percentage expression of cd4 + cd8 + and cd4 + cd8 + in thymus organs and cd4 + cd8+ in thymus organs. while the analysis of the number of cells, extracts of tapak liman show its effect on the number of cells that express cd4 +, cd8 + and cd4 + cd8 + in thymus organs. concentration of 1.0g / kg of mice showed a good effect on the increase in t helper cells (cd4 +), cytotoxic t cells (cd8 +) and prothymocyte cells (cd4 + cd8 +). references avani, k. and s. neeta, 2005. a study of the antimicrobial activity of elephantopus scaber. indian j. pharmacol., 37: 126-127. bosselutr, 2004. cd4/cd8-lineage differentiation in the thymus: from nuclear effectors to membrane signals, nature reviews immunology 4, 529-540 (july 2004) | doi: 10.1038/nri1392 but p.-p.-h., hon p.-m., cao h., chan t.-w.-d., wu b.kurokawa t., nakanishi k., wu w., hsu h.-y., maruy-m., mak t.-c.-w., and che c.-t. (1997), sesquiterama m., and kupchan s.-m. (1970), deoxyelephanpenelactones from elephantopus scaber. phytochemtopin and its interrelation with elephantopin. tetraheistry 44, 113d116. dron lett. 33, 2863d2866. cabrera a.-l. and klein r.-m. (1980), flora ilustrada lin c.-c., tsai c.-c., and yen m.-h. (1995), the eva-catarinense: compositae. 3. tribo: vernoniae, vi. luation of hepatoprotective effects of taiwan folk genero: elephantopus. itajaı´, brasil, pp. 397d402. medicine “teng-khia-u”. j. ethnopharm. 45, 113d daisy p, rayan na, rajathi d (2007). hypoglycemic and other related effects of elephantopus scaber extracts on alloxan induced diabetic rats. j. biol. sci. 7: 433-437. hammer m.-l.-a. and johns e.-a. (1993), tapping an ered in la gomera, canary islands, spain. j. chro-amazonian plethora: four medicinal plants of marajo mat. a 1011, 241d244. island, para (brazilourna). j. ethnopharm. 40, 53d75. kritikar, k.r. and b.d. basu, 1991. indian medicinal plants. 2nd edn, allahabad publisher, lalit mohan basu,.livermore, d.m., t.g. winstanley and k.p. shannon, 2001. interpretative reading; recognizing the unusual and inferring resistance mechanisms from resistance phenotypes. j. antimicrob. chemother, 48: 87-102. rastogi, r.p. and b.n. mehrotra, 1990. compendium of indian medicinal plants. vol. 1, central drug research, lucknow and national institute of science communication, new delhi. sankar, v., r. kalirajan, f. sweetlin vivian sales and s. raghuraman, 2001. antiinflammatory activity of elephantopus scaber in albino rats. indian j. pharm. sci., 63: 523-525. schwarz, b. a., bandoola, a., 2006. trafficking from bone marrow to the thymus: a prerequisite for thymopoesis. immunol rev.209: 47-54. tsai cc, lin cc (1999). anti-inflammatory effects of taiwan folk medicine ‘teng-khia-u’ on carrageenan-and adjuvant-induced paw edema in rats. j. ethnopharmacol. 64: 85-89. wang, l., jian, s., nan, p., liu, j., zhong, y., 2005. chemotypical variability of leaf oils in elephantopus scaber from 12 locations in china. chem. nat. compd.41: 491-493. waters, w.r., 2003. expression ofl-selectin (cd62l), cd44, and cd25 on activated bovine t cells .infect immun. 71(1): 317– 326. content_v4n2_5.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry volume 6 – number 2 – 2017 issn 2089-6514 (paper) | issn 2540-9328 (online) contents the female population growth projection year 2021 in trenggalek regency by leslie matrix model on the birth rate and life expectancy dewi anggreini 37 45 clustering of 18 local black rice base on total anthocyanin kristamtini, endang wisnu wiranti 47 51 vitamin c and total sugar content characterization on 31 accessions of banana collection of banana germplasm plants of yogyakarta siti dewi indrasari, kristamtini, endang wisnu wiranti 53 57 alliin as a natural bioactive from single bulb garlic (allium sativum) for nitric oxide (no) increasing in atherosclerotic process based on insilico screening riza rahayu ilmawati, ahya zhilalikbar amin, mohamad amin 59 62 on designing interactive online atlas of reptile anatomy (mabouya multifacsiata) muhammad jafar luthfi, riyanto 63 68 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 45-53 | doi: 10.14421/biomedich.2023.121.45-53 issn 2540-9328 (online) the association between some endocrine conditions and covid-19: a review harem othman smail department of biology, faculty of science and health, koya university, koya koy45, kurdistan region-f.r. iraq. corresponding author harem.othman@koyauniversity.org manuscript received: 22 september, 2022. revision accepted: 03 october, 2022. published: 11 october, 2022. abstract the review aimed to understand and explain the association between some major endocrine conditions and covid-19. since march 2019, covid-19 has been identified as a pandemic by the world health organization (who) and has infected millions of individuals worldwide. according to the literature review, endocrine disorders include diabetes, obesity, hypertension, and thyroid. the development and progress of covid-19 patients could be affected but not yet approved by all the studies. in diabetes mellitus, covid-19 may affect cytokines and increase releases of il-1, il-6, and complications caused by diabetes. in obese patients, there was an increased risk of progressing to severe covid-19. because of the international spread of severe acute respiratory syndrome coronavirus, clinicians should pay special attention to obese patients who should be monitored closely with timely and aggressive care. covid-19 pathophysiology and risk in a high incidence of hypertension has been found in patients with covid-19 and can be studied in many kinds of studies, such as in china. however, hypertension is considered one of the most significant risk factors for covid-19. in addition to the above data on associations between covid-19 and endocrine disorders, data on thyroid function or thyroid disease in covid-19 is not yet available and cannot be commonly reported. keywords: covid-19; diabetes mellitus; bidirectional; obesity; pathophysiology; blood pressure; hypothyroidism. introduction coronavirus disease has affected millions of individuals worldwide (covid-199) (mathew et al., 2020; rostam et al., 2020). this latest coronavirus, which also includes middle east respiratory syndrome (mers)-cov and sars-cov-1, is a human β-coronavirus. these viruses are predominantly connected to respiratory illnesses, such as pneumonia, ards, and pulmonary oedema (di et al.,2020). endocrine disorders are no exception, and some endocrine organs are at risk of direct or indirect covid-19 damage. although there is still no proof of a greater predisposition in patients with diabetes and obesity to contract the infection, the coexistence of both conditions leads to a worse prognosis since both conditions impart an impaired immune system (marazuela et al., 2020). evidence is growing to indicate that patients with endocrinopathies such as diabetes mellitus (dm), hypertension (htn), obesity, and cardiovascular disease are at greater risk of complications associated with covid-19. in covid19 non-survivors and serious cases, studies from the uk and us suggested a high prevalence of dm and obesity. htn (49.7 %), obesity (48.3 %), dm (28.3 %), and cardiovascular disease (27.8 %) are the most widely identified cardiometabolic comorbidities related to covid-19 in the us (shekhar et al., 2020). metabolic syndrome is a constellation of cardiovascular risk factors, including abdominal obesity, high blood pressure, dysglycemia, atherogenic dyslipidemia, and pro-thrombotic and pro-inflammatory states. metabolic syndrome is clinically characterized as the occurrence of three or more of the following factors: increased waist circumference (population and countryspecific cutoff), hypertriglyceridemia (>150 mg/dl or hypertriglyceridemia treatment), elevated blood pressure (systolic 130 and diastolic 85 mm hg or hypertension treatment history), elevated blood pressure (systolic 130 and diastolic 85 mm hg or hypertension treatment history), high-density lipoprotein cholesterol reduction (<40 mg/dl in males; <50 mg/dl in females) and dysglycemia (<100 mg/dl or hyperglycemia treatment)( bonora et al., 2018; bansal et al., 2020). the severity of covid-19 can be impaired by hypertension, diabetes, and coronary heart disease. in addition, it may be associated with an angiotensin-converting enzyme 2 (ace2) deficiency, and a glucolipid metabolic disorder (glmd) mediated cytokine storm (chen et al., 2020). several research studies have targeted the epidemiological and clinical features of patients infected with covid-19, but the risk factors for severity and mortality have not been thoroughly investigated. identifying major risk factors and taking effective https://doi.org/10.14421/biomedich.2023.121.45-53 46 biology, medicine, & natural product chemistry 12 (1), 2023: 45-53 clinical steps will greatly contribute to saving lives (zhou et al., 2020; zaki et al., 2020). covid-19 and diabetes mellitus diabetes mellitus (dm) is chronic with catastrophic multi-systemic complications and may be associated with extreme coronavirus disease 2019 (covid-19) (huang et al., 2020) larger studies from china gave more consistent prevalence rates. diabetes was present in 7.4 per cent of 1099 (median age of 47 years) (guan et al.,2020). and 8.2 per cent of 1 590 (median age 48.9 years) hospitalized individuals in two multicenter national studies. also, of 7337 people admitted to nineteen hospitals in the province of hubei (median age 54 years), 952 (13.0%) had type 2 diabetes, while a survey by the chinese center for disease control and prevention (china cdc), which also included nonhospitalized people, showed a lower prevalence of diabetes (5.3%) among 44,672 reported cases of covid-19 through february (pugliese et al.,2020). notably, guo et al. data indicate that an initial milder appearance of sars-cov-2 infection may mask the seriousness of covid-19 in diabetes, with fewer patients reporting fever, chill, chest tightness, and shortness of breath (maddaloni et al., 2020). in addition, the fundamental and clinical science of the possible interrelationships between diabetes mellitus and covid-19 was investigated (drucker 2020). however, knowledge in this area is quickly emerging, with numerous publications appearing frequently. this review summarizes recent developments in diabetes mellitus and covid-19 and emphasizes clinical guidelines for patients at risk of or affected by covid19 with diabetes mellitus. unfortunately, most accessible research does not differentiate between diabetes mellitus and, due to its high prevalence, focuses mostly on t2dm (kumar et al., 2020) compared to non-diabetics, diabetes in patients with covid-19 is associated with a two-fold rise in mortality and covid-19 severity. more research is needed on pathogenic pathways and therapeutic effects (kumar et al., 2020). several data sets from china, italy, and the usa have consistently recorded that in patients with advanced age (>70 years of age) and pre-existing comorbidities, primarily diabetes mellitus (dm), hypertension, and cardiovascular disease, the clinical course of covid-19 is more serious (yang 2020). the dm and covid-19 relationship are bidirectional. on the one hand, dm will increase the risk of sars-cov2 contraction and further complicate the clinical path of covid-19, resulting in increased severity and mortality (onder 2020). certain clinical and biological features determine high-risk phenotypes within the dm population, and such prognostic markers need to be characterized in future studies (smail et al., 2022). in the sense of patient-tailored precision medicine, which emerges as an urgent priority in the era of covid-19, more research is required to explore which subgroups of dm patients are anticipated to benefit most from particular antiviral, immunomodulatory and other treatment strategies (koliaki et al., 2020). given the high risk, people with dm should take special precautions during the covid19 pandemic. the norm should be strict social distancing and proper hanging hygiene. good glycemic regulation should be of utmost significance as it has been shown to improve the innate immune system. although it would be prudent to adhere to or intensify the ongoing treatment, doctors may consider reviewing the prescription (pal et al., 2020). compromised innate immunity, pro-inflammatory cytokine environment, decreased ace2 expression, and the use of antagonists of the renin-angiotensin-aldosterone system in people with diabetes mellitus lead to a weak covid-19 prognosis. direct β-cell injury, cytokine-induced insulin resistance, hypokalemia, and drugs used to treat covid-19 (such as corticosteroids, lopinavir/ritonavir) can, on the other hand, lead to a worsening of glucose control in individuals with diabetes mellitus (pal et al., 2020). several studies have attempted to understand the potentially increased vulnerability of diabetes patients to sars-cov-2 infection. however, no data have shown that these patients are at higher risk of contracting covid-19. via the ace2 receptor, sars-cov-2 reaches the host cell. while consensus on the role of ace2 in the crosstalk between diabetes and covid-19 has not yet been achieved, some argue that diabetes patients have elevated ace2 expression, thus facilitating viral entry and subsequent replication. others show that patients with diabetes have low levels of ace2 and that other causes, such as treatment with ace/arbs, hypoglycemic agents, and statins, are responsible for the observed rise in ace2 (azar et al., 2020). the degree to which clinical and demographic variables moderate this relationship is uncertain, although signs link higher bmi and higher hba1c to worse outcomes in people with covid-19 diabetes. covid-19 also risks leading to worse diabetes outcomes due to disturbances caused by the pandemic, including stress and changes in routine treatment, diet, and physical activity, and posing immediate risks to people with diabetes (hartmann et al., 2020) dysregulated post-infection immune response is characterized by delayed and reduced recruitment in lung tissue of cd4+ t cells, inflammatory monocytes, and macrophages. in addition to the reduced overall cd4+ t cell response, infected diabetic mice also showed a more prominent th17 response with increased il-17a levels, implying that a shift in cytokine profiles could be partially responsible for the severity of the disease (angelidi et al., 2020) an independent risk factor for the prognosis of covid-19 is diabetes. smail – the association between some endocrine conditions and covid-19: a review 47 therefore, the prevention and treatment of diabetic patients, especially those needing insulin therapy, should be given greater attention (shang et al., 2020). from 43 reports, the meta-analysis was based on 23007 patients. the pooled prevalence of diabetes in patients infected with covid-19 was 15% (95% ci: 12%–18%), p = <0.0001. furthermore, in covid-19 patients with diabetes, the risk of mortality was found to be substantially higher relative to covid-19 patients without diabetes, with a pooled risk ratio of 1.61 (95% ci: 1.16–2.25%), p = 0.005 (hussain et al., 2020). the pandemic of covid-19 has questioned both institutional and diabetes self-management. continuing social distancing and lockdowns have adversely influenced access to care and self-management (mukona et al., 2020) sars-cov-2 infected t2dm patients reported decreased body mass index (bmi), lymphocytes, ua, albumin levels, and increased crp levels. oxidative stress response and nutritional intake may be associated with reduced bmi, ua, and albumin levels. in addition, the infection may be associated with reduced lymphocyte counts and elevated crp levels (liang et al., 2020). there are major concerns about worsening glycemic regulation, inaccessibility of suitable drugs, inaccessibility of health care or infection with sars-cov-2, and worse results during the covid-19 pandemic. although there are some recommendations for diabetes treatment and related complications during the covid-19 pandemic, very few discuss the psychological problems of people with diabetes (singhai et al., 2020). several specialist bodies have temporarily updated gestational diabetes mellitus (gdm) testing guidelines during the covid-19 pandemic to minimize person-toperson contact. the current temporary australian guidelines indicate that no glucose tolerance test (gtt) is needed if the fasting glucose is around 4.6 mmol/l (van et al., 2020). a common medical condition in pregnancy is gestational diabetes mellitus (gdm). the australasian pregnancy diabetes society (adips) suggests monitoring all women in each pregnancy, preferably using a two-hour glucose tolerance test (gtt). the diagnostic criteria derive from world health organization (who) guidelines based on the findings of the hyperglycaemia and pregnancy outcomes (hapo) report (metzger et al., 2008). in patients with diabetes, there is evidence of an increased incidence and severity of covid-19. the pathophysiology of diabetes could be influenced by covid-19. therefore, it is critical for patients infected with covid-19 to regulate blood glucose and those without the disease. innovations such as telemedicine effectively treat diabetes patients today (singh et al., 2020). in patients infected with various viruses, including the 2009 pandemic influenza a (h1n1), sars-cov, and mers-cov, diabetes and uncontrolled glycemia have been identified as important predictors of severity and deaths. some studies have not found a strong link between diabetes and serious disease in the emerging sars-cov-2 pandemic. however, other studies from china and italy have shown that elderly patients with chronic diseases, including diabetes, are at higher risk of significant covid-19 and mortality, respectively (hussain et al., 2020). the mortality rate was 9.9 % in a meta-analysis of hospitalized patients in china with a diagnosis of covid19. a higher incidence of diabetes mellitus was separately associated with a worse prognosis. the independent impact of covid-19 mortality on diabetes mellitus should be seen as hypothesis-generating and needs further research (miller et al., 2020). figure 1. covid-19, cytokine storm, diabetes mellitus: a vicious cycle (azar et al.,2020). covid-19 and obesity according to estimates from the world health organization, obesity is a global epidemic, with at least 2.8 million people dying each year due to becoming overweight or obese (hussain et al., 2020). obesity is a condition characterized by the assistance of extra adiposity tissue that provides substantial morbidity and mortality due to its complications linked to several weights. therefore, the diagnostic comparison should consist of an anthropometric test that indicates elevated fat mass and the extent to which the well-being of individual patients is adversely affected by excess adiposity (smail 2019 and smail et al., 2021). although the effects of covid-19 on obese patients have not yet been well established, the experience of h1n1 influenza can serve as a warning for treating obese patients, particularly patients with extreme obesity. from 2009 to 2010, adult obesity and extreme obesity rates rose between 2017 and 2018 and now stand at 42% and 9%, respectively (hales et al., 2020). the findings indicate that relative to the h1n1 experience, the proportion of patients with obesity, extreme obesity, and covid-19 infections will increase, and the disease will likely have a more severe 48 biology, medicine, & natural product chemistry 12 (1), 2023: 45-53 path in such patients. these results further highlight the need for improved attention, diagnosis and testing priority, and aggressive care for obesity patients and covid-19 infections (dietz et al., 2020). although the data on the effect of sars-cov-2 in people with obesity are minimal, and their relationship has not yet been fully established, it has been found that people with excessive body weight may experience a more severe covid-19 infection (puig et al., 2020). as stated earlier, it is especially noticeable in people affected by other risk factors associated with a more serious disease course. this correlation was also noted during the 2009 influenza a pandemic, which identified obesity as an independent risk factor for complications. obesity patients suffered more than 40 per cent longer after a high-fat diet than people without excessive body weight (kassir 2020). therefore, obesity during the covid-19 outbreak seems to require more attention, especially in western countries where obesity prevalence is high (rychter et al., 2020). there was an increased risk of progressing to extreme covid-19 in obese patients. since severe acute respiratory syndrome coronavirus two will continue to spread internationally, clinicians should pay particular attention to obese patients who should be handled closely with prompt and aggressive treatment (cai et al., 2020). for variables associated with covid-19 risk, severity, and their potential for decreased therapeutic and prophylactic treatments among these individuals, mechanistic mechanisms for obesity are presented in detail. the substantial main changes in morbidity and mortality from covid-19 are correlated with individuals with obesity. several mechanisms explain this impact jointly. a major concern is that vaccinations for obesity may be less successful (popkin et al., 2020). in theory, early intubation benefits identified in patients with covid-19 can indicate osa alleviation in some patients. similarly, concerns of contamination from using nasal pap in some patients with obstructive sleep apnea could lead to deterioration. mechanistic research is encouraged, given the possible connection between osa, obesity, and covid-19 (mcsharry et al., 2020). in young patients, obesity is a significant predictor of covid-19 severity. the key mechanism is connected to liver and kidney damage (deng et al., 2020) study shows that patients with overweight and obese who have covid-19 are at higher risk for mortality and intubation than those with typical bmi. these results support the hypothesis that obesity is a risk factor for complications of covid-19 and should be considered for covid-19 management (nakeshbandi et al., 2020). in addition, there was an increased risk of progressing to extreme covid-19 in obese patients. since severe acute respiratory syndrome coronavirus two will continue to spread internationally, clinicians should pay particular attention to obese patients who should be handled closely with prompt and aggressive treatment (cai et al., 2020). obesity, especially in male and younger populations, plays a significant role in the risk of death from covid19. the lack of impact of racial/ethnic and socioeconomic inequalities on mortality can be explained by the capitated system with more equalized access to health care. the data highlight the leading role of extreme obesity over correlated risk factors, which offers an early intervention target (tartof et al., 2020) the position of obesity, particularly given its high prevalence worldwide, is of great relevance. however, it is important to bear in mind that the assessment of obesity through bmi is highly arguable, particularly in older people. first, since its numerator (i.e. bodyweight) comes from the fat and fat-free mass amount, it should be noted that bmi does not strictly reflect adiposity. second, the cut points that categorize overweight and obese are subjective and cohort-based for young and middle-aged people and insufficient for older people (sattar et al., 2020). third, with age, body fat accumulates with a decrease in muscle mass. obesity is often underestimated in older people, who may have excess adiposity within the normal/overweight body size (so-called sarcopenic obesity). finally, ethnic differences can determine significant variability in body fat distribution, especially regarding ectopic and visceral fat (azzolino et al., 2020). this relative inefficiency reveals a reduced ventilatory reserve and a risk for respiratory failure, even in mild systemic or pulmonary conditions. taken together, any respiratory insults will seriously compromise the already attenuated respiratory system of obese subjects, ensuring that these patients will fail to recover if they have acquired any serious diseases that can deleteriously impact respiratory function, such as covid-19 (albashir et al., 2020) the results indicate an interplay and dynamic pathophysiology between covid-19 and diabetes. for clinical practice and public health, it is a significant priority to understand how diabetes can lead to extreme covid-19 and how covid-19 can worsen diabetes pathophysiology or its effects. to counter this, a global registry (covid) was introduced to promote the study of covid-19-related diabetes manifestations, their effects, and best care (rubino et al., 2020). a wealth of data has emerged on this subject over the short period of the covid-19 pandemic, but important questions still need to be addressed. here, the current literature on the relationship between covid-19 and diabetes is reviewed, and future studies' priorities are considered (vas et al., 2020). the role of cytokines, rapamycin mammalian target (mtor), and altered polarization of natural killer cells in the hazardous relation between covid-19 and obesity. these pathways encourage and accelerate the deleterious downstream cellular effects of sars-cov2. also, it is well known that obesity is associated with smail – the association between some endocrine conditions and covid-19: a review 49 decreased lung capacity and poor response to mechanical ventilation, putting these individuals at high risk of serious covid-19 disease and mortality. also, obesity can lead to other complications, such as renal failure, cardiovascular disease, hypertension, and vascular injury, further speeding up the negative clinical outcomes of covid-19. obese individuals should be protected from possible viral exposure to sars-cov-2 with compulsory safety devices and social distancing considerations (caci et al., 2020). it is stated that people with obesity or overweight are less involved. moreover, community health centres, gyms, swimming pools, and parks have been closed by law in many countries as part of their quarantine policy during the covid-19 pandemic. such diet and social shifts may have led to a rise in body weight in individuals with obesity and the general population (lim et al., 2020). many variables could be involved in the worsening of covid-19 obesity. obesity can increase the expression in the bronchus and blood of ace2 and cd147-related genes, while the latter are sars-cov-2 receptors that invade (radzikowska et al., 2020). this would make obese subjects more vulnerable to infection and could, to some degree, explain the higher positive sars-cov-2 test rate. obesity and concomitant metabolic syndrome can cause possible harm to the function of the organ, making it more vulnerable to failure in the lungs, kidneys, and other organs (yang et al., 2020). however, in some acute diseases, including acute respiratory distress syndrome, where patients with obesity may have better outcomes, some have proposed an obesity paradox; it is uncertain if this phenomenon exists in patients with covid-19 (goyal et al., 2020). on the one hand, emerging evidence indicates that obesity is a risk factor in adults for a more extreme and complicated course of covid-19. on the other hand, the health emergency triggered by the epidemic diverts attention to infectious diseases from preventing and treating non-communicable chronic diseases (dicker et al., 2020). most covid-19 patients die due to covid-19 pneumonia after requiring artificial ventilation for hypoxemic respiratory failure. emerging covid-19 lung post-mortem histopathology provides insights into the underlying pathophysiology. in short, there is evidence of diffuse alveolar injury, as in other types of viral pneumonia, but sometimes this is patchy (lockhart et al., 2020). therefore, it is important to collect anthropometric information for patients with covid-19 because of the potentially critical role of body weight or adiposity in determining the incidence and severity of pneumonia (and probably other complications) (stefan et al., 2020). therefore, special medical care and effective intervention should be undertaken during hospitalization and later clinical follow-up in obesity patients with covid-19, especially those with additional comorbidities (kang et al., 2020). it is unclear if this is driven by these populations' higher prevalence and risks of obesity (driving diabetes and hypertension). regarding progression to more extreme severity requiring acute treatment or death, we do not yet have data on results for those with obesity and less severe covid-19 disease. such data would be necessary to fully understand the risk of mortality for those with obesity, particularly with the understanding that survival of critical care may be greater for those with moderate levels of obesity (fine et al., 2020). absolute and central obesity, in short, are risk factors for hospital admission to covid-19. even with a moderate weight gain, the high risk was evident. impaired glucose and lipid metabolism may be involved in the mechanisms (hamer et al., 2020). figure 2. potential pathways that increase the risk of serious covid-19 illness and death in individuals with obesity. there are a variety of possible mechanisms by which obesity can impact adverse covid-19 outcomes. these include chronic inflammation, respiratory function deficiency, pulmonary perfusion, practical concerns in critical care environments when treating obese patients, immune dysregulation, obesity, metabolic and vascular complications, and relative decreases in main hormones. tnf-alpha, tumour necrosis factor-alpha; frc, residual functional potential (kwok et al.,2020). hypertesion and covid-19 pathophysiology and risk in patients with covid-19, a high incidence of hypertension have been observed, with htn likely predisposing them to an increased risk of more serious illness. the danger may stem from several factors. first, hypertension is mainly associated with immune dysregulation, which occurs as higher levels of il-17, the irregular activity of natural killer cells, and cytotoxic defects of t cells partially reversible by mineralocorticoid receptor antagonists (shekhar et al., 2020). therefore, the initially reported correlation between hypertension and hospitalization rates for covid-19 in china is not surprising.3 indeed, the 50 biology, medicine, & natural product chemistry 12 (1), 2023: 45-53 proportion of self-reported hypertension was 12.6 % in a wide database of 20,982 patients with diagnosed covid-19 infections and information on underlying diseases (kreutz et al., 2020). it is unknown whether or not uncontrolled blood pressure is a risk factor for covid-19 acquisition or whether or not controlled blood pressure is less of a risk factor for hypertension patients. however, some organizations have also emphasized that blood pressure regulation remains a significant factor in minimizing the disease burden, even though it does not impact the vulnerability to sars-cov-2 viral infection (schiffrin et al., 2020). hypertension adversely impacts the health status of patients with covid-19. however, broad prevalence studies showing the effects of comorbid diabetes and hypertension are urgently needed (parveen et al., 2020). however, the outbreak of covid-19 may also be a specific moment when specialists in pulmonary hypertension have to balance the costs and benefits of diagnostic work-up, including possible exposure to covid-19 versus the initiation of targeted pulmonary arterial hypertension therapy in a select high-risk, high probability world symposium pulmonary hypertension group 1 patient with pulmonary arterial hypertension (rayan et al., 2020). a severe and possibly fatal manifestation of covid-19 is cardiac involvement and sars-cov-2-associated myocarditis. hypertension treatment with a drug that can minimize inflammation in viral myocarditis and does not pose a theoretical risk of encouraging the proliferation of covid-19 would appear to be a sensible strategy for improving patient outcomes (antwi-amoabeng et al., 2020). recent studies have shown that among patients with covid-19, arterial hypertension, diabetes, cardiovascular disorders, and chronic obstructive pulmonary disease are prevalent. research on the outcome of these patients is scarce, and there is very little evidence. nevertheless, hypertension is one of covid-19's most important risk factors. the relationship between hypertension and negative outcomes is still questionable (tadic et al., 2020). renin-angiotensin system (ras) dysfunction has been identified in patients with coronavirus infection (covid-19). still, it remains unclear whether ras inhibitors, such as angiotensin-converting enzyme inhibitors (aceis) and type one receptor blockers (arbs) of angiotensin ii, are associated with clinical outcomes. covid-19 hypertensive patients have been enrolled to test the impact of ras inhibitors (meng et al., 2020). protein for ace2-expressing cells to join. ace-2 is a part of activating the renin-angiotensin (ras) system that plays an important role in hypertension. this connection between ace2 and sars-cov-2 sparked interest in investigating the relationship between inhibitors of ras and infection with covid-19 (salah et al., 2021). hypertension was associated with increased poor composite outcomes in patients with covid-19, including mortality, extreme covid-19, ards, need for icu treatment, and disease progression (pranata et al., 2020) thyroid and covid-19 the immune system, especially cellular immunity, is modulated by thyroid hormones. the dysregulation of the immune system in hypothyroidism might increase the risk of infection. however, the immune system recovers its normal function after proper hormone replacement therapy (mitrou et al., 2011). the influence of pre-existing hypothyroidism on the effects of covid-19 is unclear (van gerwen et al., 2020). effects of covid-19 on the hypothalamic-pituitary-thyroid axis: it is known that systemic disorders are associated with low-t3 or non-thyroidal syndrome. severe covid-19 is expected to cause such a disease, especially when the infection is associated with fever and lower respiratory tract involvement. in addition, sars-cov-2 infection has been reported to influence the nervous system, typically affecting smell and taste with the involvement of cranial nerves (boelaert et al., 2020). follicular cell death would manifest as low t3 and t4; damage to parafollicular cells would potentially result in low serum calcitonin levels. this has been suggested as a possible femoral head osteonecrosis mechanism in recovered sars patients; calcitonin deficiency contributes to osteoclast disinhibition leading to osteonecrosis (kaiser et al., 2020). data on thyroid function or thyroid pathology in covid-19 is not yet available. a consensus statement concerning problems related to thyroid dysfunction during the covid-19 pandemic has been released by the british thyroid association and the society for endocrinology (bta/sfe). patients with underlying hypothyroidism or hyperthyroidism are recommended to continue their prescription drugs as normal (pal et al.,2020). hyperthyroidism is an endocrine condition characterized by the thyroid gland's inappropriately high thyroid hormone output. it is usually caused by thyroid gland autoimmune dysfunction and has a population prevalence of 1%-15% (kaur et al., 2020). in addition to clear ultrasonographic evidence indicating subacute thyroiditis, the patient presented with tachycardia, anterior neck pain, and thyroid function tests showing hyperthyroidism. corticosteroid therapy resulted in a fast clinical resolution. the case shows that virus-related subacute thyroiditis such as sars-cov-2 should be regarded as a complication of covid-19 and treated as a differential diagnosis in infected patients with tachycardia without evidence of developing covid-19 disease (mattar et al., 2020). smail – the association between some endocrine conditions and covid-19: a review 51 conclusions i reached several important points from this 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(2020). association of hypertension, diabetes, stroke, cancer, kidney disease, and high-cholesterol with covid-19 disease severity and fatality: a systematic review. diabetes & metabolic syndrome: clinical research & reviews, 14(5), 1133-1142. zhou, f., yu, t., du, r., fan, g., liu, y., liu, z., ... & guan, l. (2020). clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 7-16 | doi: 10.14421/biomedich.2022.111.7-16 issn 2540-9328 (online) peptide fractions from chymotrypsin-hydrolyzed moringa oleifera seed proteins inhibit α-amylase and α-glucosidase in vitro oluwafemi emmanuel ekun*, augustine olusegun olusola, joseph adaviruku sanni, feyisayo ishola department of biochemistry, faculty of science, adekunle ajasin university, akungba akoko, ondo state, nigeria. corresponding author* oluwafemi.ekun@aaua.edu.ng manuscript received: 14 january, 2022. revision accepted: 03 february, 2022. published: 23 february, 2022. abstract this study attempted to investigate the activities of chymotrypsin-digested moringa oleifera seed proteins and their peptide fractions on carbohydrate-hydrolyzing enzymes. proteins from m. oleifera seeds were isolated using isoelectric point precipitation and hydrolyzed using chymotrypsin. the hydrolysates obtained were fractionated into peptide fractions of <1 kd, 1-3 kd and 3-5 kd ranges by means of gel-filtration chromatography. the inhibitory effects of the hydrolysates and their fractions on -amylase and -glucosidase were evaluated, and kinetics of inhibition were also determined. using starch and p-nitrophenyl glucopyranoside as substrates, the hydrolysate and fractions demonstrated concentration-dependent inhibition of -amylase and -glucosidase respectively (ic50 of 0.172 ± 0.005 mg ml-1 to 1.312 ± 0.267 mg ml-1, for -amylase inhibition and ic50 of 0.463 ± 0.008 mg ml-1 to 0.696 ± 0.051 mg ml-1 for -glucosidase inhibition). kinetic analysis revealed that selected hydrolysate fractions competitively inhibited -amylase while displaying a mixed mode of inhibition of -glucosidase. this study suggests that subjecting m. oleifera seed proteins to proteolysis could yield therapeutic peptide products having immense potentials that could be harnessed to develop novel anti-diabetic agents and additives to food, which could serve as cost effective alternatives to current therapies. keywords: moringa oleifera; hydrolysate; peptide; chymotrypsin; α-amylase; α-glucosidase. introduction bioactive peptides and protein hydrolysate preparations obtained either by enzymatic hydrolysis or microbial fermentation of plant and animal proteins have been investigated for their therapeutic capacities and other health promoting benefits (lopez-barrios et al., 2014, ulagesan et al., 2018). peptide products have been reported to demonstrate numerous bioactivities against hypertension (yamada et al., 2013; majumder and wu 2015), cancer (vileghe et al., 2010; thundimadathil 2012) oxidative stress (olusola et al., 2018), pathogenic microorganisms (ulagesan et al., 2018) and recently, diabetes mellitus (olusola and ekun 2019, famuwagun et al., 2020), in various in vitro assays and/or animal models. diabetes mellitus is a metabolic disorder that is caused by an absolute or relative insulin deficiency (rhoades and bell 2013), and it is the fourth leading cause of health problem globally (idf 2020). it is characterized by chronic hyperglycemia associated with derangements in the regulation of carbohydrate, fat and protein metabolism (olusola and ekun 2019). this results in the appearance of metabolites such as ketone bodies and advanced glycated end products in the blood, and these in turn cause oxidative tissue and organ damage, ketoacidosis among other complications at later stages of the disease (arise et al., 2016). carbohydrate – hydrolyzing enzymes (such as α-amylase and αglucosidase) and incretin degrading enzymes (such as dipeptidyl peptidase iv) have been key pharmacologic targets for many hypoglycemic drugs (arise et al., 2019) and as such have been used in the management of diabetes mellitus, in addition to lifestyle changes. however, owing to certain adverse effects caused by several of these drugs (yu et al., 2012), in addition to high cost of procurement especially in third world countries, attention has turned to alternatives from natural sources, and this has included peptide products from proteins in seeds and leaves of plants. moringa oleifera is a fast growing, drought – resistant and perennial plant, belonging to the genus moringaceae (anwar et al., 2007). it is widely known as the “horseradish” family and native to india, especially in the himalayan regions. it is now known to be cultivated in tropical and subtropical areas such as in tropical africa and in south west asia, where its young seed pods and leaves are used as vegetables as well as in herbal medicine (leone et al., 2015; abd-rani et al., 2018). m. oleifera is a plant whose leaves and seeds have been excellent sources of essential oils and other nutrients (texeira et al., 2014). texeira et al. (2014) https://doi.org/10.14421/biomedich.2022.111.7-16 mailto:oluwafemi.ekun@aaua.edu.ng 8 biology, medicine, & natural product chemistry 11 (1), 2022: 7-16 found that whole moringa leaf flour contained 28.7% crude protein, 7.1% fat, 10.9% ash, 44.4% carbohydrates, in addition to 3.0 mg/100g of calcium and 103.1 mg/100g iron. also, another study reported that proximate analysis of its seeds indicated that the percentage nutrient composition of its protein, lipid, ash, fiber, and carbohydrate were about 35.5, 29.3, 4.6, 11.5, and 19.6% respectively (kwaambwa et al., 2015; munemune et al., 2016). proteins in m. oleifera leaves and seeds consist of albumin, prolamins, globulins and glutelins (mune-mune et al., 2016) which is mostly the case with many oil seeds (wani et al., 2011). its relatively high protein content makes it an excellent source of potential biologically active peptides (olusola et al., 2018). furthermore, freire et al., (2015) and mune-mune et al., (2016) reported that m. oleifera leaves and seeds are especially rich in glycine, isoleucine, glutamate, aspartate, leucine, arginine, proline, threonine among other amino acids. parts of the plant (roots, leaves, stem bark and seeds) have been used for nutritional purposes and as traditional medicine (leone et al., 2015; abd-rami et al., 2018). various parts of m. oleifera have been demonstrated to possess a myriad of bioactivities such as purgative, antimicrobial as well as normoglycemic effects (siddhuraju and beck, 2003; divi et al., 2012). its stem bark has been determined to have anti-proliferative, anti-ulcerative, as well as anti-inflammatory properties (mahajan et al., 2009). the presence of certain polyphenols as well as other bioactive secondary metabolites in m. oleifera leaf extracts were reported to exert antihypertensive (ndong et al., 2007), hypolipidemic and hypoglycemic effects (anwar et al., 2007). recently, crude enzymatic hydrolysates of m. oleifera seed proteins have been reported to possess enzyme-inhibitory activities in vitro (olusola et al., 2018, olusola and ekun 2019). however, hydrolysate fractionation is essential in reducing peptide aggregation, liberating peptides in solution, which in turn leads to increased peptide bioactivities in the process (awosika and aluko 2019). therefore, this study aims to examine the carbohydrase – inhibitory activities of peptide fractions obtained from chymoytrpsin – digested m. oleifera seed proteins in order to harness them as possible sources of novel antidiabetic peptides, and to further justify the value added uses of m. oleifera seed proteins. materials and methods materials collection of m. oleifera seeds m. oleifera seeds were collected from farms in akungba akoko, ondo state and identified, after which voucher samples were deposited at the department of plant science and biotechnology, adekunle ajasin university, akungba akoko. chemicals and reagents chymotrypsin (from bovine pancreas), and α-amylase (fungal), α-glucosidase (human) were products of sigma-aldrich laboratories, co-artrim, united kingdom. all other chemicals and reagents used were of analytical grade, and were also products of sigmaaldrich laboratories, co-artrim, united kingdom. methods isolation of m. oleifera seed proteins the seeds were dried and pulverized before being kept in an air-tight container at 4oc. this was subsequently defatted using n-hexane as was previously described by arise et al., (2016) with slight modifications. the meal was extracted three times with n-hexane using a meal/solvent ratio of 1:10 (w/v). the meal was then dried at 40oc in a vacuum oven and ground again to obtain a fine powder, termed defatted seed meal, which was stored at -20oc. the protein component of the defatted meal was extracted using the method described by alashi et al., (2014). defatted seed meal was suspended in 0.5 m naoh ph 12.0 at a ratio of 1:10, and stirred for one hour to facilitate alkaline solubilization. this was centrifuged at 18°c and 3000 g for 10 min. two additional extractions of the residue from the centrifugation process were performed with the same volume of 0.1 m naoh and the supernatants were then pooled. the ph of the supernatant was adjusted to 4.0 to facilitate acid-induced protein precipitation using 0.1 m hcl solution; the precipitate formed was recovered by centrifugation. the precipitate was washed with distilled water, adjusted to ph 7.0 using 0.1 m naoh, freeze-dried and the protein isolate was then stored at -20°c until required for further analysis. preparation of m. oleifera seed protein hydrolysates the protein isolate was hydrolysed using the methods described by onuh et al., (2015) and olusola and ekun (2019b) with slight modifications. the conditions for hydrolysis was tailored for each enzyme in order to optimize its activity. hydrolysis was carried out using chymotrypsin (ph 8.0, 37ºc). the protein isolate was dissolved in 0.2m phosphate buffer, ph 8.0. the enzyme was added to the slurry at an enzyme-substrate ratio (e:s) of 2:100. digestion was performed at the specified conditions for 8 hours with continuous stirring. the enzyme was then inactivated by boiling in water bath (95–100oc) for 15 minutes and undigested proteins were precipitated by adjusting the ph to 4.0 with 2 m hcl/2 m naoh followed by centrifugation at 7000 g for 30 minutes. the supernatant containing target peptides were then collected. protein content of samples were determined using biuret assay method with bovine serum albumin (bsa) as standard. ekun et al. – peptide fractions from chymotrypsin-hydrolyzed moringa oleifera … 9 fractionation of m. oleifera seed protein hydrolysates the moringa oleifera seed protein hydrolysates were separated into molecular weight fractions using gel filtration chromatography as described by boyer (2012) and tounkara et al., (2014) with some modifications. briefly, 5 ml of the clear supernatant resulting from protein hydrolysis, at a protein concentration of 10 mg/ml was filtered, suspended in 50 mm phosphate buffer ph 7 and passed into a sephadex g25 chromatographic column of dimensions 30 cm x 4 cm which had earlier been equilibrated with the buffer. the same phosphate buffer was used to elute the separating fractions, and the elution peaks were monitored at 400 nm according to prasad et al. (2017). the separating fractions eluted under the same elution peak were collected, pooled and their molecular weights were determined by comparison with the graph of the logarithm of molecular weights against elution volumes of known standards. the eluates, according to their molecular weights, were then sorted into <1 kd, 1-3 kd and 3-5 kd ranges. peptide fractions of molecular weights higher than 5 kda were removed and discarded. the collected peptide fractions were stored at -20°c for further analysis. determination of degree of hydrolysis degree of hydrolysis (dh) was determined by calculating the percentage of soluble protein in 10% trichloroacetic acid (tca) in relation to total protein content of the protein isolate according to the method described by olusola et al. (2018). one ml of protein hydrolysate was added to 1 ml of 20% tca to produce 10% tca soluble material. the mixtures was left to stand for 30 minutes to allow for precipitation, followed by centrifugation at 4000 g for 20 minutes. the supernatants were then analyzed for protein content using biuret assay method with bovine serum albumin (bsa) as standard. the degree of hydrolysis (dh) was then calculated as the ratio of soluble peptide in 10% trichloroacetic acid (in milligrams) to the total protein content of isolate (in milligrams), expressed in percentage. determination of peptide yield the percentage peptide yield was determined using the method described by girgih et al. (2011). the peptide yields (%) of moringa oleifera seed protein hydrolysates and fractions, were calculated as the ratio of peptide content of lyophilized hydrolysate/fraction to the protein content of unhydrolysed protein isolate. determination of α-amylase inhibition an α-amylase-inhibitory assay was performed according to the method reported by oboh et al. (2011). briefly, 125 µl of hydrolysate (0.2 to 1.0 mg ml-1) was placed in test tubes and 125 µl of 20 mm sodium phosphate buffer (ph 6.9, with 6mm nacl) containing α-amylase solution (0.5 mg ml-1) added. the content of each tube was pre-incubated at 25°c for 10 min, after which 125 µl of 1% starch solution in 20 mm sodium phosphate buffer (ph 6.9, with 6 mm nacl) was added at timed intervals. the reaction mixtures were incubated at 25°c for 10 min. the reaction was terminated by adding 250 µl of dinitrosalicylic acid (dns) colour reagent and further incubated in boiling water for 5 min and cooled to room temperature. the content of each test tube was diluted with 2.5 ml distilled water and the absorbance measured at 540 nm. a control was also prepared using the same procedure except that the hydrolysate was replaced with distilled water. the α-amylase-inhibitory activity was calculated as shown: % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙– 𝐴𝑠𝑎𝑚𝑝𝑙𝑒) / 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 the concentration of hydrolysate resulting in 50% inhibition of enzyme activity (ic50) was determined from a plot of percentage inhibition against hydrolysate concentrations using graphpad prism version 6.0 (graphpad software, san diego, ca, usa). determination of kinetic parameters of α-amylase inhibition the kinetic study of α-amylase inhibition was conducted according to the method described by olusola and ekun (2019a). 125 µl of the hydrolysate was pre-incubated with 125 µl of α-amylase solution for 10 min at 25°c in a set of tubes. in another set of tubes, 250 µl of phosphate buffer (ph 6.9) was also pre-incubated with 125 µl of α-amylase solution. starch solution (125 µl) of increasing concentrations (1.0 to 8.0 mg ml–1) were added to both sets of reaction mixtures to initiate the reaction. the mixture were then incubated for 10 min at 25 °c, and then boiled for 5 min after the addition of 250 µl of dinitrosalicylic acid (dns) reagent to stop the reaction. the amount of reducing sugars released was determined spectrophotometrically from a maltose standard curve and converted to reaction velocities as shown below: 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (µ𝑚𝑜𝑙 𝑚𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛– 1) 𝑚𝑖𝑛– 1) = 𝑀𝑎𝑙𝑡𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 / 𝐼𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 × 𝑀𝐸. where: maltose concentration is in µmol ml–1; incubation time: 10 min; me: amount of enzyme (in mg) in reaction mixture a double reciprocal plot (1/v versus 1/[s]), where v is reaction velocity and [s] is substrate concentration was plotted. the mode of inhibition and the kinetic parameters of α-amylase inhibition by hydrolysates were determined by analysis of the double reciprocal plot. the inhibition constant (ki) was determined using a secondary plot known as the dixon plot (palmer and bonner, 2007), by plotting a graph of inverse of initial 10 biology, medicine, & natural product chemistry 11 (1), 2022: 7-16 velocities on the x-axis against inhibitor concentrations on the x-axis, at fixed concentration of substrate. determination of α-glucosidase inhibition the effect of the hydrolysates on 𝛼-glucosidase activity was determined according to the method described by kim et al., (2005) using 𝛼-glucosidase from saccharomyces cerevisiae. the substrate solution pnitrophenyl glucopyranoside (pnpg) was prepared in 20 mm phosphate buffer, and ph 6.9. 100𝜇l of 𝛼 glucosidase (1.0 u/ml) was pre-incubated with 50𝜇l of the different concentrations of the hydrolysates for 10 min. then 50𝜇l of 3.0 mm (pnpg) as a substrate dissolved in 20 mm phosphate buffer (ph 6.9) was added to start the reaction. the reaction mixture was incubated at 37∘c for 20 min and stopped by adding 2ml of 0.1 m na2co3 solution. the 𝛼-glucosidase activity was determined by measuring the yellowcolored para-nitrophenol released from p-npg at 405 nm. the results were expressed as percentage of the blank control. percentage inhibition was calculated as: % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙– 𝐴𝑠𝑎𝑚𝑝𝑙𝑒) / 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 determination of kinetic parameters of α-glucosidase inhibition the kinetic parameters of 𝛼-glucosidase by the hydrolysates was determined according to the modified method described by ali et al. (2006). briefly, 50 𝜇l of the (5 mg ml-1) hydrolysate was pre-incubated with 100 𝜇l of 𝛼-glucosidase solution for 10 min at 25°c in one set of tubes. in another set of tubes, 𝛼-glucosidase was pre-incubated with 50𝜇l of phosphate buffer (ph 6.9). 50 𝜇l of pnpg at increasing concentrations (0.5–2.0 mg ml-1) was added to both sets of reaction mixtures to start the reaction. the mixture was then incubated for 10 min at 25°c, and 500 𝜇l of na2co3 solution was added to stop the reaction. the amount of reducing sugars released was determined spectrophotometrically at 405nm using a paranitrophenol standard curve and converted to reaction velocities. a double reciprocal plot (1/v versus 1/[s]) where v is reaction velocity and [s] is substrate concentration was plotted. the mode of inhibition of the hydrolysates on 𝛼-glucosidase activity was determined by analysis of the double reciprocal (lineweaver-burk) plot using michaelis menten kinetics. the inhibition constant (ki) was also determined using the dixon plot (palmer and bonner, 2007), by plotting a graph of inverse of initial velocities on the x-axis against inhibitor concentrations on the xaxis, at fixed concentration of substrate. statistical analysis results were expressed as mean of triplicate observations ± standard deviation. the data were statistically analyzed using one way analysis of variance (anova) and duncan’s multiple range tests. differences were considered statistically significant at p<0.05 using graphpad prism version 6.0 (graphpad software, san diego, ca, usa). results peptide yield and degree of hydrolysis the enzyme chymotrypsin was used to hydrolyze the protein isolate and the degree of hydrolysis was found to be 51.180 ± 2.461 %. the peptide yields of the hydrolysate and its fractions are displayed in table 1. table 1. peptide yield of m. oleifera seed proteins hydrolyzed by chymotrypsin and fractionated using gel filtration chromatography. sample peptide yield unfractionated hydrolysate 87.571±2.342a fraction 1 (<1 kd) 12.791±0.535c fraction 2 (1-3 kd) 13.548±1.070bc fraction 3 (3-5 kd) 13.656±0.076b -amylase inhibitory activity the percentage -amylase inhibitory activity of the hydrolysate and its fractions in relation to the control, acarbose, was illustrated in figure 1 and their ic50 values were displayed in figure 2. this result showed that, at lower concentrations, the hydrolysate fractions demonstrated -amylase inhibitory effects comparable to the control. however, the unfractionated chymotrypsin hydrolysate demonstrated a concentrationdependent reduction in activity, attaining a value of 15.487 ± 1.125 % at a final concentration of 1.0 mg ml1, which is significantly lower (p<0.05) than those of the hydrolysate fractions. chymotrypsin hydrolysate fractions displayed percentage inhibitions above 50% at all study concentrations, such that the fractions f1, f2 and f3 reached 61.859 ± 2.490 %, 76.440 ± 1.220% and 67.195 ± 0.528 % inhibition respectively at a final concentration of 1.00 mg ml-1. among the chymotrypsin hydrolysate fractions, fraction f2, had the highest (p<0.05) inhibitory activity. the hydrolysate and its peptide fractions f1 – f3, inhibited -amylase to a 50% extent at concentrations of 1.312 ± 0.267 mg ml-1, 0.172 ± 0.005 mg ml-1, 0.892 ± 0.038 mg ml-1 and 0.526 ± 0.093 mg ml-1 respectively. ekun et al. – peptide fractions from chymotrypsin-hydrolyzed moringa oleifera … 11 figure 1. percentage -amylase inhibition by moringa oleifera seed protein hydrolysates and their fractions obtained by chymotrypsin digestion. bars are expressed as means ± standard error of mean of triplicate determinations (n=3). values within the same concentration but with different letters are significantly different (p<0.05). values at different concentrations of the same hydrolysate with different symbols are also significantly different (p<0.05). bars carrying the same letter or symbol are not significantly different from one another (p<0.05). figure 2. values of 50% inhibition of -amylase activity by chymotrypsin-derived m. oleifera seed protein hydrolysate and its fractions. bars are expressed as means ± standard error of means of triplicate determinations (n=3). values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another. kinetics of -amylase inhibition the effect of a selected peptide fraction (fraction 2) on the catalytic activity of -amylase in converting starch to maltose was presented in figure 3. kinetic parameters determined from lineweaver-burk plots in the absence and presence of two different concentrations of the hydrolysate fraction were summarized in table 2. in the absence of the hydrolysate fractions, the michaelis constant, km, of -amylase for its substrate was found to be 0.552 mg ml-1of starch while maximal velocity, vmax was 3.890 mm mg-1 min-1. inhibition of -amylase activity increased with increasing concentrations of the peptide fraction, such that the km of the enzyme was increased while vmax and catalytic efficiency, ce, of amylase were reduced in the presence of the inhibitory peptide fractions. the peptide fraction demonstrated competitive inhibition at both 0.5 mg ml-1 and 1.0 mg ml-1. figure 3. lineweaver-burk plot of -amylase inhibition by moringa oleifera seed protein hydrolysate fraction 2 (1-3 kd) obtained from chymotrypsin hydrolysis 12 biology, medicine, & natural product chemistry 11 (1), 2022: 7-16 table 2. kinetics of -amylase-catalyzed reactions in the presence and absence of m.oleifera seed protein hydrolysate fraction 2. kinetic parameters no inhibitor chymotrypsin hydrolysate fraction 2 (mg ml-1) 0.5 1.0 km or kˈm (mg ml-1) 0.552 0.610 0.659 vmax or vˈmax (mm mg-1 min-1) 3.890 3.632 3.750 ce (mmol ml-1 min-1) 7.053 5.953 5.717 ki (mg/ml) 0.735 km or kˈm: michaelis constant in the absence or presence of the inhibitory peptide fraction; vmax or vˈmax: maximum velocity in the absence or presence of the inhibitory peptide fraction; ce: catalytic efficiency; ki: enzyme-inhibitor dissociation constant. -glucosidase inhibitory activity the inhibitory activities of the m. oleifera seed protein hydrolysate and its fractions on α-glucosidase – catalyzed hydrolysis of p-nitrophenyl glucopyranoside at varying concentrations in comparison to acarbose (control) are presented in figure 4. their ic50 values were also depicted in figure 5. m. oleifera seed protein hydrolysate and its fractions, as digested by chymotrypsin, also displayed increased inhibition of glucosidase with increasing concentration (figure 4). however, these were significantly lower (p<0.05) when compared to control at all study concentrations. the unfractionated hydrolysate and its gel-filtration fractions f1, f2 and f3 attained maximal inhibitory activities of 38.723 ± 1.508 %, 73.077 ± 1.110 %, 83.474 ± 2.691% and 85.282 ± 1.295% respectively at a final concentration of 1.0 mg/ml. chymotryptic hydrolysate fraction f2 had significantly higher (p<0.05) activity than other fractions at 0.4 mg ml-1, 0.6 mg ml-1 and 0.8 mg ml-1, but there was no significant (p<0.05) difference in its activity when compared to fraction f3 at 1.0 mg ml-1. in addition, chymotrypsin hydrolysate and its fractions f1, f2 and f3 inhibited -glucosidase to a 50% extent at concentrations of 0.509 ± 0.025 mg ml-1, 0.651 ± 0.025 mg ml-1, 0.463 ± 0.008 mg ml-1, and 0.696 ± 0.051 mg ml-1 respectively. these ic50 values were higher (p<0.05) than those of acarbose. chymotrypsin hydrolysate fraction 2 had the lowest (p<0.05) ic50 value among the hydrolysates and fractions derived from chymotrypsin proteolysis. figure 4. percentage -glucosidase inhibition by moringa oleifera seed protein hydrolysates and their fractions obtained by chymotrypsin digestion. bars are expressed as means ± standard error of mean of triplicate determinations (n=3). values within the same concentration but with different letters are significantly different (p<0.05). values at different concentrations of the same hydrolysate with different symbols are also significantly different (p<0.05). bars carrying the same letter or symbol are not significantly different from one another (p<0.05). figure 5. ic50 values of -glucosidase activity by chymotrypsin-derived m. oleifera seed protein hydrolysate and its fractions. ekun et al. – peptide fractions from chymotrypsin-hydrolyzed moringa oleifera … 13 bars are expressed as means ± standard error of means of triplicate determinations (n=3). values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another. kinetics of α-glucosidase inhibition the effects of a selected m. oleifera seed protein hydrolysate fraction (fraction 2) on the kinetics of α glucosidase–catalyzed hydrolysis of p-nitrophenyl glucopyranoside, p-npg, to p-nitrophenol were illustrated in figure 6, while the kinetic parameters from the resulting line-weaver burk plot were summarized in table 3. in the absence of inhibitory hydrolysates, the michaelis constant, km of αglucosidase for its substrate was determined to be 0.297 mg ml-1 p-npg, while maximum velocity, vmax, was 270.27 mm mg-1 min-1. all hydrolysate fractions caused decreases in the vmax and catalytic efficiency, ce of the enzyme. figure 6. lineweaver-burk plot of -glucosidase inhibition by moringa oleifera seed protein hydrolysate fraction 2 (1-3 kd) obtained from chymotrypsin digestion. table 3. kinetic parameters of -glucosidase inhibition by m. oleifera seed protein hydrolysate fractions. kinetic parameters no inhibitor chymotrypsin hydrolysate fraction 2 (mg ml-1) 0.5 1.0 km or kˈm (mg ml-1) 0.297 0.456 0.220 vmax or vˈmax (mm mg-1 min-1) 270.270 147.059 100.000 ce (mmol ml-1 min-1) 910.001 322.498 454.545 ki (mg ml-1) 0.136 km or kˈm: michaelis constant in the absence or presence of inhibitory peptide fraction; vmax or vˈmax: maximum velocity in the absence or presence of inhibitory peptide fraction; ce: catalytic efficiency; ki: enzyme-inhibitor dissociation constant. discussion degree of hydrolysis and peptide yield the degree of hydrolysis (dh) estimates the amounts of cleaved peptide bonds in a protein hydrolysate. dh can affect the molecular sizes and amino acid compositions of the peptides and consequently influence the biological activities of the peptides formed during hydrolysis (olusola et al., 2018). the degree of chymotrypsin hydrolysis of m. oleifera seed proteins was 51.59 ± 3.81 % which was higher than 38.66% gotten for obtained for whey protein hydrolysates at an e/s ratio of 1:100 but slightly lesser than 57.34% obtained for the same whey hydrolysate at an e/s ratio of 2:100 (galvao et al., 2001). also, it was higher than the dh value of 11.2 ± 1.4% determined for chymotrypsin digests of buffalo casein at an e/s ratio of 1:100 (shanmugam et al. 2015). the observed differences could be due to different protein sources, which invariably leads to varying number of sites susceptible to chymotrypsin hydrolysis, as the enzyme is known to preferentially hydrolyze the c-terminal peptide bonds of aromatic aminoacyl residues (voet et al., 2016). in addition, it appears that an increased enzyme/substrate ratio is required to improve the degree of hydrolysis as regards proteolysis by chymotrypsin. other factors such as hydrolysis time and ph of the buffer medium could affect enzymatic activity and by extension, the degree of hydrolysis. peptide yield measures the amount, in percentage, of peptides generated relative to the whole protein subjected to enzymatic proteolysis; thus representing an important index in determining the efficiency of the overall process (alashi et al., 2014), as these enzymes degrade the proteins into several peptides of varying lengths and sizes (girgih et al., 2011). the peptide yield obtained by chymotrypsin digests was higher than the 48.18 ± 0.89% reported for chymotrypsin hydrolysates of yellow field proteins (awosika and aluko, 2019). this could be as a result of a relative abundance of 14 biology, medicine, & natural product chemistry 11 (1), 2022: 7-16 aromatic aminoacyl residues in m. oleifera seed proteins (mune-mune et al., 2016) which provides more cleavage points for enzymatic hydrolysis. the peptide yield of the unfractionated hydrolysates was higher than those of their corresponding fractions put together, and this could be due to peptide loss during the process of chromatographic sepaprtion and the removal of peptides whose molecular weights were higher than 5 kda. this is consistent with the reports of awosika and aluko (2019) in their work with yellowfield pea protein hydrolysates and peptide fractions. α-amylase inhibitory activity and kinetics the enzyme α-amylase, a digestive carbohydrase, catalyzes the hydrolysis of complex polysaccharides in the mammalian gut. thus, the inhibition of α-amylase activity is a key pharmacologic intervention in the management of type 2 diabetes mellitus (olusola and ekun, 2019a). in this study the unfractionated hydrolysates showed lower α-amylase inhibitory activities when compared to acarbose, and this is because acarbose is a synthetic inhibitor of α-amylase. in addition, the peptide fractions demonstrated better inhibitory activities than the unfractionated hydrolysate, and this is in consonance with the reports of awosika and aluko (2019) that hydrolysate fractionation improves peptide bioactivity, such that more peptides are able to access substrate binding sites on the enzyme. in the same vein, malomo and aluko, (2016) reported that unfractionated hydrolysates contained high molecular weight peptides which could act antagonistically to smaller peptides, thus reducing inhibitory activity. among the peptide fractions, the chymotrypsin hydrolysate fraction f2 (1-3 kd) demonstrated the highest inhibitory activities at a maximum concentration of 1.0 mg ml-1. this could be a direct result of the nature of the peptides released by these enzymes during proteolysis. it is known that chymotrypsin as an endoprotease hydrolyzes peptide linkages from c-terminal residues of phenylalanine, tyrosine and tryptophan. reports from previous studies stated that phenylalanine, leucine, proline and glycine residues are required for the inhibition of α-amylase (yu et al. 2012, garza et al. 2017). in the same vein, arise et al. (2016) also suggested that α-amylase binds to peptides containing aromatic residues such as phe, tyr and trp. thus, these peptides obtained from enzymatic proteolysis could contain these specific amino acid residues that locks into sites on the enzyme, inhibiting its activity in the process. the lineweaver-burk plot was used to determine the mode of α-amylase inhibition by varying two concentrations of a selected peptide fraction, f2. also, the kinetic parameters determined from the double-reciprocal plots were summarized in table 2; suggesting that the michaelis constant, km of αamylase (from saccharomyces cerevisiae) in the absence of inhibitory hydrolysates is 0.552 mg ml-1 of starch, which is lower than 1.4 mg ml-1 (acharya et al., 2014) for α-amylases obtained from aspergillus oryzae. the selected peptide fraction in this study exhibited a competitive type of inhibition at both study concentrations. this suggests that these peptides are capable of binding α-amylase in its free form, at the same substrate-binding site for starch, creating a deadend complex as a result. this is in contrast to the findings of arise et al. (2016) that reported a mixed type of inhibition of α-amylase for peptic, tyrptic and alcalase hydrolysates of citrullus lanatus seed protein hydrolysates, but literature has been scarce for the αamylase – inhibitory activity of peptides obtained by chymotrypsin hydrolysis. however, the ki value obtained suggest that these peptides bind α-amylase with lesser affinity when compared to enzyme – substrate binding. α-glucosidase inhibitory activity and kinetics the enzyme α-glucosidase occurs mostly as a membrane-bound enzyme on the brush border membranes of the ileum, and digests carbohydrates by hydrolyzing glucose residues from a number of oligosaccharides. thus, modulating α-glucosidase activity represents another important strategies in the control of blood glucose levels in the management of diabetes mellitus (qaisar et al. 2014). chymotrypsin hydrolysate fractions displayed a concentrationdependent increase in α-glucosidase inhibitory activities, such that fractions f2 and f3 showed better inhibitory effects. this is consistent with the reports of awosika and aluko (2019) who reported that chymotrypsin hydrolysate fractions of yellow field pea protein hydrolysates inhibited α-glucosidase. it therefore follows that the process of fractionation may have helped liberate these bioactive peptides so that they could bind to and inhibit α-glucosidase. in addition, since chymotrypsin cleaves peptides after aromatic residues (voet et al. 2016) and that a tyrosinyl residue is a likely requirement for α-glucosidase inhibitory activity (ibrahim et al., 2018), it is therefore suggested that the presence of a one or more well positioned aromatic side chains may have contributed positively to the inhibition of α-glucosidase activity. the kinetic parameters obtained from the double reciprocal plots of αglucosidase inhibition by selected peptide fractions of m. oleifera seed proteins in figure 6 were summarized in table 3. the michaelis constant, km, of α-glucosidase for p-nitrophenyl glucopyranoside in the absence of inhibitor was determined to be 0.297 mg ml-1 p-npg in this study. this is slightly higher than 0.211 mg ml-1 (0.7mm) p-npg obtained by awosika and aluko (2019) but lower than 6.31 mg ml-1 reported by arise et al. (2019). vmax, in the absence of inhibitory hydrolysates was 270.27mm mg-1ml-1. chymotrypsin hydrolysate fraction f2 demonstrated a mixed mode of inhibition at 0.5 mg ml-1 while at 1.0 mg ml-1 the inhibition ekun et al. – peptide fractions from chymotrypsin-hydrolyzed moringa oleifera … 15 mechanism shifted to an uncompetitive subtype of mixed inhibition. this is in contrast to what was obtained with the <1 kd fraction of chymotryptic digests of yellowfield pea protein hydrolysate (awosika and aluko 2019) as they inhibited α-glucosidase via a noncompetitive inhibition mechanism. this suggests that peptide concentration could have significant effects on mechanism of enzyme inhibition and this could be evaluated in further studies. the enzyme-inhibitor binding constant, ki, revealed that the peptide fraction possessed stronger binding affinity for α-glucosidase than for α-amylase. ibrahim and others (2018) stated that aminoacyl residues such as tyrosine are needed at terminal ends of a peptide for α-glucosidase inhibitory activity. chymotrypsin is a protease that could cleave and release peptides with aromatic c-terminal ends (voet et al., 2016), and this might explain the αglucosidase inhibitory activities observed by these peptide products. conclusion this study concludes that peptide fractions obtained from chymotrypsin digests of m. oleifera seed proteins elicited antidiabetic potentials by inhibiting carbohydrate hydrolyzing enzymes – α-amylase and αglucosidase – by differing mechanisms in vitro. in addition, fraction 2 containing peptide sizes of 1-3 kd recorded the highest activity against the carbohydrases. further studies such as structural identification, modifications, and chemical synthesis of peptides responsible for the observed bioactivities are being embarked upon, and are currently in progress. conflict of interest: the authors declare no conflicts of interest. references abd-rani nz, husain k. kumolosasi e (2018) moringa genus: a review of 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4.76 and 4.56 log cfu; 6.46 and 6.20 log cfu. microscopic morphology shows that lab and proteolytic bacteria of shrimp digestive are gram-positive as cocci or rods. the deproteinization value of shrimp waste reached 31%, especially for treatment with a ph of 5.0–7.0 (during proteolytic bacteria fermentation) and pre-treatment of boiling, drying and coarse grinding of shrimp waste (pp2) before extraction. however, the demineralization value of shrimp waste is only 4.5% (pp1) and 3% (pp2), with lab as the primary fermentation agent because proteolytic bacteria have been unable to reduce the mineral content in shrimp waste further. keywords: chitin; enzymatic extraction; digestive tract; lactic acid and proteolytic bacteria; shrimp waste. abbreviations: total plate count (tpc), lactic acid bacteria (lab), plate count agar (pca), de man, rogosa, and sharpe (mrs). introduction chitin, a copolymer composed of n-acetylglucosamine (glcnac) units (>50%) and glucosamine (glcn), is the main component of crustacean (shrimp, crabs and lobsters) shells, insect skeletons, and cell walls of fungi, diatoms, bacteria, and algae with stiff and rigid characteristics as a protective function from the outside world (bastiaens et al., 2020; ehrlich, 2010; zargar et al., 2015). the application of chitin and its derivatives is widely used in various industries, including food, health, cosmetics, agriculture, paper, biotechnology, and others (nainggolan, 2023; perez & wertz, 2022). the global market for chitin and its derivatives in 2020 reached 106.9 thousand tons and is estimated to increase to 281.7 thousand tons in 2027 with a monetary value of us$ 2,900 million (perez & wertz, 2022). the inedible portion of the shrimp reaches 44-63%, including the head, skin and tail, which are considered waste (adeyeye & aremu, 2016). those shrimp waste themselves contain chitin (15–40%), protein (20–40%), minerals (30–60%), and astaxanthin (reddish pigmentcarotenoid, a potent antioxidant) (bastiaens et al., 2020; el-bialy & abd el-khalek, 2020; hu et al., 2019). generally, chitin and its derivatives are isolated chemically with a concentrated strong acid (usually hcl) and a strong base (generally naoh) to remove minerals and proteins in the chitin raw material (bastiaens et al., 2020; kumari & kishor, 2020). besides removing the other essential ingredients in shrimp shells, chemical extraction also creates chemical residue pollution and affects the physico-chemical characteristics of the product (duan et al., 2012; kaur & dhillon, 2015). another alternative method is by isolating chitin using proteolytic enzymes or microbes and lactic acid bacteria to digest the protein and mineral content in the raw material, leaving chitin as the main product (cahú et al., 2012; perez & wertz, 2022). however, on the other hand, the price of enzymes is relatively high, and the low level of extraction efficacy is a challenge for the method (kim & park, 2015), so it is necessary to look for alternative enzymes or microbes that produce robust, stable and economical enzymes. manuscript received: 10 february, 2024. revision accepted: 20 may, 2024. published: 31 may, 2024. https://doi.org/10.14421/biomedich.2024.131.183-191 184 biology, medicine, & natural product chemistry 13 (1), 2024: 183-191 research indicates that the shrimp digestive tract is a rich source of proteolytic bacteria and lactic acid bacteria (holt et al., 2021; kongnum & hongpattarakere, 2012). shrimp waste also harbours chitinolytic bacteria (masri et al., 2021; setia & , 2015). these findings suggest that the bacteria naturally present in the raw material could be harnessed for enzymatic extraction of chitin, presenting a promising avenue for research. however, the effectiveness of chitin extraction from shrimp waste using these specific groups of bacteria still needs to be explored, underscoring the need for further investigation. the main aim of this study is to determine the effectiveness and identify the optimum conditions for extracting chitin from shrimp waste using bacteria from shrimp digestion. this research is crucial in advancing our understanding of chitin extraction methods and could potentially contribute to the development of more efficient and sustainable processes in the future. materials and methods isolation, enrichment, and bacterial colonies determination of shrimp digestion shrimp for digestive tract bacteria isolating were obtained from the flamboyan market, pontianak city, west kalimantan province, without selecting a particular type of shrimp, weighing around 20-30 per kg. the method for isolating microbes from the shrimp digestive tract (faeces and intestines) was adapted from the work of (kongnum & hongpattarakere, 2012) with several notable modifications. these included thoroughly washing the shrimp with distilled water, homogenising the intestines and faeces with sterile saline solution (nacl 0.85% w/v), and diluting to 10⁻⁵. the incubation and colony counting was performed with plate count agar (pca) (temperature 30°c, 24–48 hours, aerobic and anaerobic conditions); de man, rogosa, and sharpe (mrs) agar (lactic acid bacteria, temperature 30°c, 24– 48 h, aerobic and anaerobic conditions) (kongnum & hongpattarakere, 2012); and calcium m-protein agar (proteolytic bacteria, temperature 30°c, five days), followed by a clear zone test with 5–10% acetic acid solution for 1 minute (erkmen, 2021). the number of bacterial colonies was calculated using a formula based on sni 2332.3:2015 (badan standardisasi nasional, 2015a). 𝑁 = ∑𝐶 [(1 × 𝑛1) + (0,1 × 𝑛2)] × (𝑑) with n, ∑c, n1, n2, d, respectively, the number of sample colonies (cfu/ml or colonies/g), the number of colonies on all plates counted, the number of plates in the first and second dilution counted, and the first dilution calculated. microscopic identification of shrimp digestive bacteria shrimp digestive bacteria incubated on mrs agar and m-protein calcium agar were subjected to gram staining. the successive gram staining process begins by taking one of the colonies from the agar medium using a sterile tube needle, placing it on a glass slide and then fixing it with methanol. next, the colonies were given a solution of crystal violet for 1 minute, iodine for 1 minute, 90% ethanol for 30 seconds, and safranin for 1 minute. rinsing with distilled water was carried out at each change of solution administration. bacterial cell colonies morphology (colour and shape) was observed using a microscope. determination of the effectiveness and optimum conditions for chitin isolation the raw materials for chitin extraction, namely shrimp head, shell, and tail waste, were sourced from the flamboyan market in pontianak city, west kalimantan. the shrimp waste was obtained without any sorting based on the type and size of the shrimp. the preparation of the raw materials followed a modified version of the method outlined by (hamdi et al., 2017). this method involved two treatment models. in the first treatment, the shrimp waste was thoroughly washed, cooked with distilled water (1:2 w/v) for 30 minutes at 100°c, and then left to drain at room temperature for approximately 2 hours. it was then incubated with lab from shrimp digestion. the second treatment was similar to the first, with the addition of a drying stage in an oven at 60⁰c for 48 hours. this was followed by coarse grinding of the shrimp shells with a blender before further treatment with lab. chitin extraction was carried out by incubating 250 g of chopped shrimp waste (in 750 ml of distilled water) with 25 ml of lab inoculum (the number of colonies was counted turbidimetrically) plus 12.5 g of glucose at room temperature for 24 hours using an orbital shaker (speed 60 rpm) for the demineralization process. the reaction was stopped by increasing the fermentation temperature to 90°c for 20 minutes. next, the solution was filtered, and the chitin solids were rinsed with distilled water and dried in an oven at 50°c for about one and a half days. the yield was calculated, followed by the analysis of proximate. dry filtrate (100 g) resulting from lab extraction plus 10 g of glucose and 300 ml of buffer solution based on ph treatment was further incubated with proteolytic bacterial culture (taken from 2 culture petri dishes). four ph treatments were prepared (5.0, 6.0, 7.0, 8.0). for ph 5.0, 0.1 m acetate buffer is used (5.772 g of sodium acetate and 1.778 g of concentrated acetic acid are mixed in 800 ml of distilled water, then the ph was adjusted with concentrated hcl, then distilled water was added to a volume of 1000 ml); ph 6.0 used 0.1 m citrate buffer (24.269 g of sodium citrate and 3.358 g of concentrated citric acid were mixed in 800 ml of distilled water, then nainggolan et al. – enzymatic shrimp waste chitin extraction 185 the ph was adjusted to 0.1 n hcl, then distilled water was added to a volume of 1000 ml); for ph 7.0 and 8.0, 1 m tris-hcl buffer was used (121.14 g of tris base was mixed in 800 ml of distilled water, then the ph was adjusted with hcl, then distilled water was added to a volume of 1000 ml). fermentation was carried out at room temperature for 6 hours using an orbital shaker (speed 60 rpm). incubation was stopped again by increasing the fermentation temperature to 90°c for 20 minutes. afterwards, the fermentation solution was filtered, and the solids were washed with distilled water until neutral and dried in an oven at 50°c overnight. the extraction results were then calculated and tested proximately. proximate tests include ash content-based on sni 2354.1.2010 (badan standardisasi nasional, 2010), water-based on sni 2354.2.2015 (badan standardisasi nasional, 2015b), fat-based on sni 01-2354.3-2006 (badan standardisasi nasional, 2006a), and proteinbased on sni 01-2354.4-2006 (badan standardisasi nasional, 2006b), which are all carried out in duplicate. determination of lab colonies turbidimetrically for chitin extraction faeces and intestines from the shrimp digestive tract were taken sterilely and then enriched for bacteria using mrs agar and mrs broth at a dilution of 10⁰ to 10⁻⁷. they were then incubated at 30°c for 48 hours. next, the number of colonies on the agar media was counted at each dilution. in contrast, the absorbance at each broth media dilution was measured by spectrophotometry at a wavelength of 600 nm (od600). the number of colonies on the agar media and the absorbance value on the broth media were then compared to obtain a standard curve. the equation of the standard curve is then used to calculate the number of lab colonies used for chitin extraction. results and discussion type and number of bacterial colonies in the shrimp digestive tract the total bacterial colonies (tpc), lab, and proteolytic bacteria from the shrimp digestive tract, both aerobic and anaerobic, are depicted in figure 1. figure 1. bacterial colonies from the digestive tract (faeces and intestines) of shrimp incubated aerobically and anaerobically. figure 1 shows that aerobic and anaerobic tpc, lab, and proteolytic bacteria from the shrimp digestive tract were 5.5 and 5.6 log cfu/g, 4.8 and 4.6 log cfu/g, and 6.5 6.5 and 6.2 log cfu/g. the presence of lab and proteolytic bacteria in the shrimp digestive tract is in line with the results of several other studies (bhowmik et al., 2015; fitriadi et al., 2023; holt et al., 2021; kongnum & hongpattarakere, 2012; mulyati et al., 2023; omont et al., 2020; zhao et al., 2018). (kongnum & hongpattarakere, 2012) found that the digestion of some wild shrimp (including metapenaeus brevicornis and penaeus merguiensis) and cultivated shrimp (litopenaeus vannamei) contained total bacteria ranging from 6.9–8.1 log cfu/g and lab ranging from 3.2–4.1 log cfu/g. furthermore, (mulyati et al., 2023) found that the population of several proteolytic bacterial isolates from the digestive tract of l. vannamei ranged from 5.7–7.4 log cfu/g. this value range is still in line with the results of this research. figure 1 also reveals an interesting observation. the total number of lab and proteolytic bacteria exceeds the tpc value, indicating that the tpc content alone cannot fully capture the presence of these bacteria. this discrepancy is likely due to the different content of pca media for tpc and selective media for lab and proteolytic bacteria. pca is a standard medium to estimate the bacterial population in a food or liquid sample. however, it cannot depict the entire bacterial population's presence or indicate differences in the various types of bacteria present in a sample (mendonca et al., 2020). meanwhile, selective media contains complete nutrients to support certain microbial groups' growth optimally. as a result, the population size in a sample can be estimated (corry et al., 2011). the morphology of lab and proteolytic bacteria from the shrimp digestive tract based on gram staining is shown in figure 2. figure 2. microscope morphology of shrimp digestion (faeces and intestines) at 400x magnification: (a) lab; (b) proteolytic bacteria figure 2, illustrates that lab and proteolytic bacteria in shrimp digestion are gram-positive bacteria, taking the form of cocci or rods. morphologically, lab emits a purplish colour, characteristic of the group of gram-positive bacteria, is in 0 2 4 6 8 tpc lab proteolytic bacteria lo g c fu .g ⁻¹ groups of bacteria from shrimp digestion (feces and intestines) aerob anaerob 186 biology, medicine, & natural product chemistry 13 (1), 2024: 183-191 the form of cocci or rods, does not form spores, and is very tolerant of low ph (mokoena, 2017). meanwhile, proteolytic bacteria produce proteases (protein-breaking down enzymes) with a broader morphological scope because there are groups of bacteria that belong to a gram-positive group (such as the bacillus), whereas others are gram-negative (such as pseudomonas) (solanki et al., 2021). furthermore, bal can also digest protein to meet its amino acid needs for life (hutkins, 2019; m. liu et al., 2010). one source of lab protein is casein (hutkins, 2019), which is available in calcium mprotein agar. the higher values of proteolytic bacterial colonies compared to the tpc and lab (figure 1) could be due to lab and some other groups of bacteria having the ability to digest protein in calcium m-protein agar. some lab genera found to be quite dominant in shrimp digestion are lactobacillus and streptococcus (zhao et al., 2018). gram-positive and negative bacteria in the cocci or rod shaping were found in proteolytic bacterial isolates from the intestines of tiger shrimp (penaeus monodon) (bhowmik et al., 2015). it's important to note that our study did not detect gram-negative bacteria. this could be because our microscopic analysis only involved one general colony, without examining and isolating each variant shape of proteolytic bacteria growing on selective agar media. therefore, further analysis, such as pcr via the 16s rrna gene sequencing method, is necessary to identify the specific types of lab and proteolytic bacteria in the shrimp digestive tract. this method is widely used for identifying the genus and species of microbes in a sample (johnson et al., 2019). number of lab colonies for chitin extraction the turbidimetric standard curve of bal at a wavelength of 600 nm (od600) for chitin extraction is depicted in figure 3. figure 3. standard curve for lab colony measurements at od600. the number of lab colonies used for chitin extraction based on the standard curve equation (figure 3) is described in table 1. table 1. number of lab colonies for chitin extraction. equation of standart curve preliminary treatment absorbance (x) bal (cfu/ml) y = 3,5829x 3,5279 pp1 2,101 4x10⁴ pp2 1,891 3,2x10⁴ note: y (bacterial colony); x (absorbance); pp1 (preliminary treatment of shrimp waste: washed, boiled for 30 minutes, drained); pp2 (pp1 followed by oven drying at 60⁰c 48 hours and coarse blending). the calculation based on the standard curve implies that the number of lab colonies used for fermentation in pp1 was 4x10⁴ cfu/ml (od₆₀₀ 2.1), while in pp2, it was 3.2x10⁴ cfu/ml (od₆₀₀ 1.9). the value is much smaller compared to (ruangwicha et al., 2024), which found five lab strains (four lactobacillus sp. and streptococcus thermophilus) with od₆₀₀ 0.8–1.0 equivalent to 10⁸ cfu/ml. on the other hand, (lu et al., 2023) used streptomyces sp. scut-3 strain to extract amino acids, oligopeptides, calcium and chitin from l. vannamei shrimp shell waste with an inoculum size of od₆₀₀ 9.0. however, there is no information on the estimated colonies amount of streptomyces sp. at the od₆₀₀ value. changes in shrimp shells' chemical composition during the extraction with lab and proteolytic bacteria the shrimp shells' chemical composition changes during extraction with lab and proteolytic bacteria can be seen in figure 4. y = 3,5829x 3,5279 r² = 0,8269 0 2 4 6 0,8 1,2 1,6 2,0 2,4 b a l (c fu .m l⁻ ¹) x 1 0 ⁴ absorbance (od₆₀₀) nainggolan et al. – enzymatic shrimp waste chitin extraction 187 figure 4. changes in the chemical composition of shrimp waste (skin and head) during fermentation with lab and proteolytic bacteria of shrimp digestion (faeces and intestines). extraction stage: m (raw shrimp waste); r (shrimp waste with pp1); rl (pp1 after fermented with bal); rk (shrimp waste with pp2); rkl (pp2 after fermented with bal). pretreatment: pp1 (shrimp waste washed, boiled, drained); pp2 (pp1 followed by oven drying and coarse blending). p5–p8: treatment of ph (5.0–8.0) during proteolytic bacteria fermentation. proximate analysis was carried out in duplicate. figure 4 presents a detail breakdown of the composition of shrimp waste during fermentation process. it reveals that shrimp waste primarily comprises 10.9% water, 4.8% fat, 29.0% ash, and 32.8% protein. the remaining 22.5% is suspected of chitin and other compounds such as astaxanthin. further data demonstrates that lab and proteolytic bacteria, through fermentation, can reduce the fat and protein content of shrimp waste. notably, the percentage reduction of proximate content in pp2 was higher than in pp1, with the most significant decline observed in pp2 protein levels, reaching 31% (ph treatment of 5.0–7.0). however, it is essential to note that the ash content of pp1 and pp2 increased after extraction with proteolytic bacteria. available studies indicate variations in the shrimp waste content. the waste (head, shell, tail) of five shrimp species (l. vannamei, macrobrachium rosenbergii, p. monodon, fenneropenaeus chinensis, penaeus japonicus) is reported to consist of 66.2–73.9% water, 1.8–5.7% fat, 5.0–8.5% ash, 6.8–10.1% protein (z. liu et al., 2021). another study states that the waste of pandalus borealis shrimp from the lagos atlantic sea comprises 4.2% water, 0.8% fat, 29.5% ash, and 19.1% protein (adeyeye & aremu, 2016). in contrast, the shell of p. borealis from the barents sea (norway) with a dry matter of 22±2% consists of 0.3–0.5% fat, 32–38% ash, and 33–40% protein. the rest is chitin (17–20%) and astaxanthin (wet weight 14–39 mg.kg⁻¹) (rødde et al., 2008). moreover, data on l. vannamei shrimp shells (dried basis) has 4.0% of fat and astaxanthin, 21.7% minerals, 54.6% protein, and 19.7% chitin (lu et al., 2023). these data imply that regardless of water content, the most significant composition of shrimp waste is protein and minerals, followed by chitin. the pretreatment of washing and boiling, followed by oven drying and coarse grinding of pp2, influences the effectiveness of deproteinization. it is reported that the boiling of shrimp waste itself at 80⁰c for an hour can dissolve the fat, ash, and protein content of shrimp waste respectively 0.1–0.5%, 0.3–0.6%, 2.0–2.2% (fadhallah et al., 2023). although the numbers are different, a decrease in the fat, ash and protein content of shrimp waste after pretreatment (pp1 and pp2) can be seen in figure 4, even if there are some distinction data (see r in pp1). pretreatment and bal fermentation can decrease shrimp waste's fat, ash, and protein content in pp1 by around 1%, 4.5%, and 3.5% (note that protein content increases by around 5.9% after pp1 pretreatment). meanwhile, in pp2, the reduction is around 3% for fat and ash content and 17% for protein content. the low reduction content of ash was not expected because the preliminary hypothesis that lactic acid from lab would effectively bind calcium in shrimp waste, which then sharply reduced the mineral content of shrimp waste as reported in several studies (rao et al., 2000; sixtoberrocal et al., 2023; xu et al., 2008). furthermore, the shrink of protein level implies the role of lab in digesting the shrimp waste's protein. as information, after the 24-hour pp1 extraction process with bal, there was a change in the aroma and condition of the shrimp waste, leading to a putrefaction process. then, the shrimp waste was immediately washed thoroughly, filtered, and dried in an oven at 50°c for about one and a half days to ensure the extracted shrimp waste was dry enough. the process continues with coarse grinding of the shrimp waste extract before extraction with proteolytic bacteria. next, a sterilisation process was carried out (121⁰c, 15 minutes) on the pp1 medium before incubating proteolytic bacterial inoculum in the shrimp waste medium. for pp2, sterilisation was applied before each fermentation step. signs of shrimp waste spoilage after 24 hours of fermentation with lactobacillus plantarum were also reported (rao et al., 2000). sterilisation of the fermentation medium before inoculum incubation to shrimp waste has been mentioned in several studies (lu et al., 2023; ruangwicha et al., 2024). 0 10 20 30 40 water fat ash protein p er se n ta ge ( % ) pp1 m r rl rlp5 rlp6 rlp7 rlp8 0 10 20 30 40 water fat ash protein p er se n ta ge ( % ) pp2 m rk rkl rklp5 rklp6 rklp7 rklp8 188 biology, medicine, & natural product chemistry 13 (1), 2024: 183-191 the further fermentation process with proteolytic bacteria did not affect the fat content of pp1 shrimp waste (still at 3–4%). additionally, the protein content of pp1 shrimp waste only decreased by around 0.1-3%. the relatively similar result also occurred in the fat content of pp2 shrimp waste. on the contrary, the protein content of pp2 shrimp waste decline up to around 14% (except for ph treatment of 8.0). while for ash content as states before, it increases for both pretreatments, after the proteolytic bacteria fermentation. proteolytic bacteria are suspected to be less able to digest minerals in shrimp waste. in addition, a decrease in water content and other compounds after fermentation causes mineral levels to appear increases. (huang et al., 2022) reports increased mineral levels after fermentation of shrimp waste with a mixture of lab, proteolytic, and chitinolytic bacteria. the lack of proteolytic bacteria effectiveness in digesting minerals is implied in some research (lu et al., 2023; waldeck et al., 2006). however, the preliminary deproteinized process increases shrimp waste's demineralisation degree (lu et al., 2023). the overall protein content decrease of 19 and 31% (pp2) and 4–6% (pp1) is relatively low compared to other studies (lu et al., 2023; ruangwicha et al., 2024; sixto-berrocal et al., 2023). however, all of these studies were carried out with a reasonably long fermentation time (3–5 days) and a relatively high substrate-tosolution ratio (1:10 to 1:20). in this study, the ratio of substrate-to-solution was only 1:3. interestingly, there is a report of quite intense enzyme activity of the grampositive proteolytic bacteria from the digestion of tiger shrimp (p. monodon) at ph 5–7 (bhowmik et al., 2015), which is in line with the result of pp2. unfortunately, pp1 does not confirm the data. yield changes during chitin isolation from shrimp waste figure 5 describes the yield of chitin from shrimp waste during the extraction process with lab and proteolytic bacteria from shrimp digestion. figure 5. yield changes during chitin isolation from shrimp waste with shrimp digestion bacteria. extraction stage: m (raw shrimp waste); r/rk (after pretreatment); r(k)l (after bal fermentation); r(k)lp (5–8) (after proteolytic bacteria fermentation at ph of 5.0–8.0). as shown in figure 5, the initial drying process removes more than eighty per cent of the shrimp waste weight, which contrasts with the low water content of raw shrimp waste (only 10.9%). it is suspected that the actual water content is much higher than the data. it is assumed that before proximate analysis, the water in the shrimp waste evaporates because of the relatively high room temperature, and there is a humidity difference between the shrimp waste and the surrounding air. moreover, some of the filtrate is accidentally thrown away during the washing process, reducing the yield. overall, the final yield of shrimp waste filtrate after the isolation process was 10.7–11.3% (pp1) and 9.2– 9.6% (pp2). it's worth noting that some research reports higher chitin yield from shrimp waste isolated enzymatically, ranging from 17-46% (ghorbel-bellaaj et al., 2013; lu et al., 2023; rødde et al., 2008; ruangwicha et al., 2024; xu et al., 2008). however, the yields still contain some other impurities. discussion and future recommendation although the results of this research do not meet initial expectations regarding deproteinization and demineralization, the potential of shrimp digestion bacteria to isolate chitin from shrimp waste can be noticed. generally, some studies show high deproteinization and demineralization results are obtained using one or several specific microbes. furthermore, fermentation conditions (amount of inoculum, ph, temperature, time, sugar sources and concentrations, substrate-to-liquid ratio, and process stages) (lu et al., 2023; rao et al., 2000; ruangwicha et al., 2024; sixto-berrocal et al., 2023) also influence fermentation effectiveness. several studies have even 0 20 40 60 80 100 m r(k) r(k)l r(k)lp5 r(k)lp6 r(k)lp7 r(k)lp8 yi el d ( % ) pp1 yield pp2 yield nainggolan et al. – enzymatic shrimp waste chitin extraction 189 succeeded in isolating several other compounds besides chitin, such as proteins or peptides and free amino acids, calcium and pigments through fermentation (lu et al., 2023; ruangwicha et al., 2024). further analysis needs to be conducted to determine the quality of chitin produced through fermentation. ftir (fourier-transform infrared spectroscopy), xrd (x-ray diffraction) and sem (scanning electron microscope), which can detect the purity and structural morphology of chitin, can support the analysis (ruangwicha et al., 2024; triunfo et al., 2022). this study fermented using free bacterial cells. free microbial cells tend to be vulnerable to environmental pressures (mehrotra et al., 2021), which decreases their numbers due to their inability to survive in certain conditions. one possible development of a fermentation method is by using immobilised microbial cells. the cells of the microbe are immobilised by binding/trapping them to a specific carrier/support. some studies say immobilised cells have several advantages over free cells. the immobilised cells have higher metabolic activity and cell density, so the cells are more productive compared to free cells. moreover, immobilisation provides the cells better protection against surrounding environmental conditions, improving the cells' survival ability. then, immobilised cells can be reused without losing their activity. besides, separating immobilised cells from extracts is easier (mehrotra et al., 2021; zur et al., 2016). compared to using pure enzymes, applying immobilised cells can be cheaper because there is no need for enzyme isolation and purification stages. moreover, microbial cells generally produce more than one type of enzyme, which can lead to a series of chemical reaction catalysation, not just one type of chemical reaction like pure enzymes (lu et al., 2023). the immobilized whole-cell biocatalysts have been used to produce fine chemicals, biofuels (polakovič et al., 2017), biosurfactants (subsanguan et al., 2020), therapeutic drugs (anteneh & franco, 2019), winemaking (genisheva et al., 2014), and wastewater treatments (an et al., 2008). however, no information is available about the production of chitin and its derivatives using these biocatalysts. conclusions the number of bacterial colonies shows that proteolytic bacteria dominate the shrimp digestive tract. microscopic morphology shows that lab and proteolytic bacteria are gram-positive as cocci or rods. furthermore, lab and proteolytic bacteria from shrimp digestion can reduce the protein content (deproteinization) of shrimp waste by up to 31%, especially for treatment with a ph of 5.0–7.0 (during fermentation with proteolytic bacteria) and pretreatment of boiling, drying and coarse grinding of shrimp waste. however, the reduction in mineral content (demineralization) of shrimp waste was only 4.5% (pp1) and 3% (pp2), with lab as the main contributor because proteolytic bacteria were not able to further reduce the mineral content in shrimp waste. acknowledgements: the authors would like to express the gratitude to the laboratory team of the fishery product processing and storage study program, and fisheries cultivation study program that have contributed to the research work. moreover, the acknowledgement is also given to politeknik negeri pontianak for the funding research. authors’ contributions: kristina novalina nainggolan designed the study and wrote the manuscript. kristina novalina nainggolan, together with yudha perdana putra and evi fitriyani, conducted the laboratory work and analysed the data. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the research was carried out with funding support from the budget implementation list 2023 of politeknik negeri pontianak through the applied research grant program. references adeyeye, e. i., & aremu, m. o. 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(2016). metabolic responses of bacterial cells to immobilization. molecules, 21(7). https://doi.org/10.3390/molecules21070958 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 747-755 | doi: 10.14421/biomedich.2025.142.747-755 issn 2540-9328 (online) extracellular metabolites of clove-endophytic bacteria, niallia nealsonii dcl1, exhibit potential cellular antioxidant and antiaging activities nadiyah salsabilah1, muhammad eka prastya2, rika indri astuti1,3* 1department of biology, dramaga campus, ipb university, bogor 16680, indonesia. 2research center for pharmaceutical ingredients and traditional medicine, national research and innovation agency (brin), kawasan sains dan teknologi (kst) b.j habibie (puspiptek) serpong, tangerang selatan, banten 15314, indonesia. 3tropical biopharmaca research center, bogor agricultural university, taman kencana street, ipb taman kencana campus, bogor 16128, indonesia. corresponding author* rikaindriastuti@apps.ipb.ac.id manuscript received: 26 may, 2025. revision accepted: 18 september, 2025. published: 01 october, 2025. abstract the clove plant, syzygium aromaticum l., is a highly valued spice in indonesia, recognized for its extensive applications in food preservation and medicinal uses. in a prior study, six endophytic bacteria were successfully isolated from clove leaves. one of these isolates, designated as dcl1, is a gram-positive bacterium identified as niallia nealsonii. this study aimed to determine the antioxidant and antiaging properties of the extracellular metabolite of dcl1. extracellular metabolites were extracted with ethyl acetate and evaluated for antioxidant activity via the dpph assay. total phenolic and flavonoid compounds were also quantified with further metabolilte profiling using liquid chromatography tandem-mass spectrometry (lc-ms/ms) analysis. further study of antiaging activity was assayed using the yeast model schizosacchromyces pombe. the extract from n. nealsonii dcl1 cultures harvested at 28 and 40 hours yielded 0.01% and 0.009%, respectively. our findings indicate that the 40-hour extract contains higher levels of phenolics (93.54 mg gallic acid equivalents/g extract) and flavonoids (12.09 mg quercetin equivalents/g extract) compared to the 28-hour extract. additionally, the 40-hour extract exhibited greater antioxidant activity than the 28-hour extract, as demonstrated by dpph (ic50 222 µg/ml) and abts (ic50 511.43 µg/ml) assays. moreover, the 40-hour extract significantly prolonged the viability of s. pombe, suggesting its potential as an anti-aging agent. lc-ms/ms analysis identified several potential bioactive compounds, including gallic acid and n-carbamoyl-2,3-dihydroxybenzamide, which may contribute to its bioactivity. ultimately, our study indicates that dcl1 40-h extract showed antiaging potential, which is promising as source and formulation of biopharmaceutical or cosmeceutical products. keywords: antiaging; antioxidant; lc-ms/ms; niallia nealsonii dcl1; schizosaccharomyces pombe. introduction cellular aging is one of the causes of functional decline in tissues and organs in the body, which can increase the risk of developing degenerative diseases such as cancer and diabetes. in 2019, indonesia was ranked of 7th in the world with the highest number of diabetics and in 2045 it is predicted that indonesia will remain in the top 10 countries with the most diabetes globally (saeedi et al., 2019). degenerative diseases are closely related to cell aging which can be caused by various factors such as genetics, lifestyle, exposure, and accumulation of toxic molecules reactive oxygen species (ros) or advanced glycation end's products (ages) which can be originated from uv light, cigarette smoke, pesticide, and pollution (lobo et al., 2010; martemucci et al., 2022). antioxidants are important defence compounds to help slow down the cellular aging process because these compounds can inhibit cell damage reactions caused by free radicals (li et al., 2021; robert et al., 2020; vaiserman et al., 2020). of note, antioxidant constituents could be produced naturally from within cells in the form of enzymes or from outside cells, such as food and plant bioactive compounds (shahidi et al., 2015; stobiecka et al., 2022; xu et al., 2017). previous studies indicate the potential antioxidant activities from metabolites of clove (syzygium aromaticum), asteraceae plants, adenostemma lavenia and plant-derived essential oils of nutmeg and patchouli (astuti et al., 2021; batubara et al., 2020; rahmi et al., 2021). clove plants have many benefits in various fields of life, such as food industries, perfume, and medicinal plants. most of the utilization of clove plants is carried out on the flower organs while the leaf organs are less utilized. clove leaf extract has been shown to have potential as an antiaging agent (amini et al., 2022; ariybah et al., 2021; cortés-rojas et al., 2014; fauzya et al., 2019; lesmana et al., 2021). on the other hand, the https://doi.org/10.14421/biomedich.2025.142.747-755 748 biology, medicine, & natural product chemistry 14 (2), 2025: 747-755 increasing needs of the community and international trade have also increased in clove plantations, of which 98% of the area is cultivated by smallholders (riptanti et al., 2019). therefore, an alternative approach is needed to overcome the high demand from the community for the efficacy of clove plants, one of which is by investigating the secondary metabolites of endophytic bacteria that live in clove leaf tissue. secondary metabolites can be produced not only by plants, but also by endophytic bacteria that live symbiotically in plant tissues. the ability of endophytic bacteria to produce the same active compounds as the host is thought to be the result of a genetic exchange over a long evolutionary period (singh et al., 2017). in our previous research, six isolates of endophytic bacteria were isolated from clove leaf, one of which was identified as n. nealsonii dcl1 (utami et al., 2023). based on the 16s rrna gene sequence analysis, the dcl1 isolate is 99.18% similar to n. nealsonii dsm 15077. dcl1 has good potential for antioxidant activity as this isolate was physiologically tolerant against h2o2induced oxidative stress treatment and found as nonpathogenic strain (utami et al., 2023). based on this data, thus, in this study, we evaluate the potential activity of the extracellular metabolite from isolate dcl1 as an antioxidant in vitro and further determine the antiaging activity at the cellular level using the yeast schizosaccharomyces pombe as model organism. materials and methods microbial strains and medium the endophytic bacteria n. nealsonii dcl1 were routinely grown on luria agar (la). the bacterial culture was incubated for 24 hours at room temperature (± 27°c). yeast s. pombe arc039 was routinely cultured in yeast extract with supplement medium (yes medium) at 30 °c for 1-3 days. determination of the stationary phase of selected endophytic bacteria determination of the stationary phase was referred to previous study (utami et al., 2023). n. nealsonii was precultured in luria bertani (lb) liquid medium for 24 hours at room temperature (± 27°c). preculture was transferred to a new sterile lb liquid medium as the main culture at initial od600 of 0.1. the main culture of 3 replicates was incubated in a shaker incubator (120 rpm) at room temperature. the od600 value was then measured every 4 hours for 48 hours. extraction of endophytic bacterial extracellular metabolites endophytic bacteria were precultured as previously mentioned in the determination of the stationary phase procedure. the main culture was incubated for 28 and 40-hours with shaking (120 rpm) at room temperature (± 27°c). subsequently, bacterial cultures were mixed with ethyl acetate solvent (96%) with a ratio of main culture volume and ethyl acetate of 1:1 and shaken as the modification technique was performed at 180 rpm for 2 hours at room temperature. the ethyl acetate layer formed was then separated and evaporated using a rotary evaporator at 50°c to obtain crude extracts of the bacterial extracellular metabolites (m.e. prastya et al., 2018) antioxidant activities based on dpph and abts assay the antioxidant activity of the crude extract was assessed using the dpph (2,2′-diphenyl-1-picrylhydrazyl) (astuti et al., 2021)and abts (2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid)) radical scavenging assays (abubakar et al., 2022), following the methodology of a previous study. the crude extract was diluted with 96% methanol, mixed with a dpph solution (125 µm in methanol), and incubated for 30 minutes. the absorbance was then measured at 514 nm using an elisa microplate reader. for the abts assay, a 7 mm abts solution was oxidized with potassium persulfate for 12-16 hours to generate the abts radical, which was then reacted with the crude extract solution and incubated for 30 minutes. the absorbance was measured at 734 nm using an elisa microplate reader. the extract's ability to scavenge dpph and abts radicals was calculated as a percentage of inhibition, using the formula: % inhibition = [1 – ((a–c)/(b–c))] × 100%, where a is the absorbance of the radical with the crude extract or positive control, b is the absorbance of the radical with methanol, and c is the absorbance of methanol. the results were expressed as the inhibitory concentration of 50% (ic50). ascorbic acid and trolox were used as positive controls. quantitation of total phenolic and flavonoid content the total phenolic content of the extract was measured using the folin-ciocalteu reagent method, with gallic acid as the standard. the crude extract was diluted in 96% methanol to a concentration of 1000 µg/ml. this solution was then combined with 250 µl of folinciocalteu reagent and 3.5 ml of distilled water, shaken, and incubated for 8 minutes. afterward, 750 µl of 20% sodium carbonate (na2co3) was added to the mixture, which was again shaken and incubated for 2 hours at room temperature. the absorbance of the solution was measured at 765 nm. standard curves were created using gallic acid concentrations of 1000 µg/ml, prepared at various concentrations (5, 10, 15, 20, and 25 µg/ml). the total phenolic content in the extract was expressed as gallic acid equivalents (mg/g extract) based on the regression equation from the gallic acid standard curve (afrendi et al., 2023). the total flavonoid content was determined using the colorimetric aluminum chloride (alcl3) method. a crude salsabilah et al. – metabolites of niallia nealsonii as antioxidant and antiaging 749 extract solution (1000 µg/ml) was mixed with 2 ml of distilled water and 150 µl of 5% nano2, shaken, and incubated for 2 minutes. then, 150 µl of 10% aluminum chloride was added, and the mixture was shaken again and incubated for 8 minutes. following this, 2 ml of 1m naoh was added, and the absorbance was measured at 510 nm using an elisa microplate reader. quercetin concentrations of 1000 µg/ml were prepared at various concentrations (10, 20, 30, 40, and 50 µg/ml) to generate standard curves. the total flavonoid content in the extract was expressed as quercetin equivalents (mg/g extract) based on the regression equation of the quercetin standard curve (kefayati et al., 2017). antiaging activity based on yeast viability assay the yeast viability assay was conducted using a spot assay. the wild-type strain s. pombe arc039 (h-leu1-32 ura4-294) was initially cultured in yeast extract supplement (yes) liquid medium with 3% glucose and incubated for 24 hours at 30°c. the preculture was then transferred to 3 ml of yes liquid medium containing 3% glucose with an initial od600 of 0.05, serving as the treatment culture. the test extract was diluted with 99% dmso and added to each main culture at various concentrations, which were determined based on the ic50 value from a dpph assay, including 1/2x, 1x, and 2x ic50. a negative control consisted of s. pombe cultured in yes medium with 3% glucose and dmso, without the extract, while a positive control involved s. pombe cultured in yes medium with 0.3% glucose (calorie restriction) and dmso, without the extract. all cultures were incubated for 11 days at 30°c with shaking at 120 rpm. spot assays were performed on days 1, 7, and 11, starting from cultures adjusted to an od600 of 1. serial dilutions were then prepared from od600 = 1 to 10^4 on a sterile 96-well plate, and 3 µl of each dilution was spotted onto yes solid medium, followed by incubation for 3 days at 30°c. the cell density from each spot was used to assess the viability of s. pombe cells (lesmana et al., 2021). liquid chromatography tandem-mass spectrometry (lc-ms/ms) analysis lc-ms/ms analysis was used to determine the compounds in the extract of extracellular metabolites of n. nealsonii dcl1. the lcms data were obtained by using acquity uplc i-class binary solvent manager. the columns used were the ftn sample manager with a temperature of 40°c. stepwise gradients achieved separation from 95% a (0.1% formic acid + distilled water) and 5% b (acetonitrile + 0.1% formic acid) to 5% a and 95% b for 17 minutes. the flow rate of the desolvation gas was set to 1000l/h, for cone gas it was set to 50l/h, and the source temperature was fixed to 120°c. the capillary voltage and cone voltage were set to 2.0 and 30kv, respectively. the mass spectrometry was determined using the type of electrospray ionization (es) xevo g2-s qtof (waters) mass spectrometry in positive ion mode. moreover, the raw ms fragmentation data were identified using npatlas, pubchem, and literature (astuti et al., 2021). statistical data analysis data were presented as means with standard deviation. the total phenolic content, flavonoid contents and antioxidant activity of the extracellular metabolites were analysed statistically using the one way anova test at p<0.05. results and discussion morphology and growth curve of endophytic bacteria niallia nealsonii dcl1 the morphological characteristics of the endophytic bacteria n. nealsonii dcl1 are round in shape, convex elevation, smooth surface, slimy texture, and white colour (figure 1a). the microscopic character of the bacterial cell is rod-shaped with a cell size of about ± 4 µm. the results of gram staining showed purple bacterial cells which confirmed that the isolate belonged to the group of gram-positive bacteria (figure 1b). based on the growth curve, isolate dcl1 began the stationary phase after 20 h of incubation and remained stable up to 48 hours of incubation (figure 1c). based on this data the growth curves obtained, the harvesting of secondary metabolites was determined at 28 and 40hours. figure 1. the morphological characteristics of the colony (a) and cell following gram staining procedure (b) as well as the growth curve (c) of isolate niallia nealsonii dcl1 on luria bertani (lb) medium at a temperature of 28-29 °c. 750 biology, medicine, & natural product chemistry 14 (2), 2025: 747-755 extraction of extracellular metabolites extraction of bacterial extracellular metabolites was conducted by the maceration method using ethyl acetate as a solvent. the crude extract produced at 28 and 40-h had a brownish paste form (figure 2). the amount of extract obtained is expressed in percentage yield (gram/ml). the yield percentage of the 28 h extract was higher than at 40-h extract (table 1). figure 2. the resulted extract of extracellular metabolites from n. nealsonii dcl1 using ethyl acetate solvent. (a) 28-h extract and (b) 40-h extract. table 1. the yield percentage of the of extracellular metabolite extract from niallia nealsonii dcl1. sample culture volume (ml) extract weight (gram) yield (%) 28-h extract 2000 0.2085 0.010 40-h extract 3000 0.2672 0.009 quantification of total phenolic and flavonoid from our data it is confirmed that the total phenol was higher than the total flavonoid concentration. the highest concentration of total phenols and flavonoids were found in the 40-h extract. the value of total phenol content in the 40-h extract was 93.54 eag/g extract. as for, the total level of flavonoids in the 40-h extract was 12.09 qe/g extract (table 2). based on the results of the total phenol and flavonoid measurements and the antioxidant activity, the 40-h extract has the potential to be analyzed further. table 2. total phenolic and flavonoid contents of the extracellular metabolites extract of niallia nealsonii dcl1. sample total phenolic* (mg gae/g extract) total flavonoid** (mg qe/g extract) 28 h extract 27.44 ± 0.33a 14.78 ± 3.69a 40-h extract 93.54 ± 0.48b 12.09 ± 0.11ab *the total phenolic content is expressed in mg gallic acid equivalent/gram of extract (gae/g extract). **the total flavonoids are expressed in mg quercetin equivalent /gram of extract (qe/g extract). different superscript letters in the same column showed significant differences (p<0.05). antioxidant activities based on dpph and abts assay antioxidant activities were determined using the radicals of dpph and abts. the strongest antioxidant activity of dpph and abts was shown by the 40-h extract with an ic50 value of 222 µg/ml and 511.43 µg/ml, respectively. this value indicates the activity of each extract in degrading free radicals by 50% at each ic50 value (table 3). table 3. antioxidant activities of the extracellular metabolites extract of niallia nealsonii dcl1. sample antioxidant activities dpph ic50 (µg/ml) abts ic50 (µg/ml) ascorbic acid 4.00 ± 2.10ab 11.52 ± 3.89a trolox 2.97 ± 0.35a 3.82 ± 0.64b 28 h extract 838.11 ± 57.51c 586.80 ± 41.72c 40-h extract 222.00 ± 20.33d 511.43 ± 32.76cd note: the ascrobic acid and trolox were used as positive control. different superscript letters in the same column showed significant differences (p<0.05). antiaging activity based on yeast viability assay. the antiaging activity was determined using the spot method to observe the effect of bacterial extracts on the viability of s. pombe yeast cells under glucose-rich conditions. the results indicated that the 40-h extract treatment supports yeast cells viability up to 11 days (considered as the stationary phase) better than that of the control negative treatment (figure 3). it is worth noting that all control positive treatment of calorie restriction resulted relatively better growth than all treatment of extract at 11 days. as for the treatment of 444 µg/ml, yeast cells viability was found higher than lower extract treatment and the control negative (non-calorie restriction treatment without extract). our data indicate the potential of extracellular metabolite of dcl1 in prolonging and maintaining yeast cells viability, during stationary phase. salsabilah et al. – metabolites of niallia nealsonii as antioxidant and antiaging 751 figure 3. the antiaging effect of 40-h extract at various concentrations on the life span analysis of s. pombe was observed using the spot method. positive control was yeast grown on 0.3% glucose medium. negative control was yeast grown on 3% glucose medium without extract treatment. liquid chromatography tandem-mass spectrometry (lc-ms/ms) analysis the content of active compounds from bacterial extracts was identified using the lc-ms/ms method. the results on the chromatogram pattern show several peaks of dominant compounds which are thought to have a role in antioxidant and antiaging activities. based on the literature review, the predominant compounds are gallic acid, n-carbamoyl-2,3-dihydroxybenzamide, methyl 6carbamoylphenazine-1-carboxylate, n-carbamoyl-2hydroxy-3-methoxybenzamide, prenostodione, 12ebromoisorumbrin, and two unknown dominant compounds are unknown1 and unknown2 (figure 4). figure 4. metabolite profile of 40-h extract of extracellular metabolites from n. nealsonii dcl1. the metabolites were analysed using lc-ms/ms analysis. the dominant peaks were identified based on the specific mass identity. based on previous literatures, the three compounds namely n-carbamoyl-2-hydroxy-3-methoxybenzamide, gallic acid, and n-carbamoyl-2,3-dihydroxybenzamide are known to have antioxidant activity. in addition, gallic acid has been reported to exhibit antiaging activity. as for, methyl 6-carbamoylphenazine-1-carboxylate and prenostodione showed antibacterial activity, while prenostodione compounds were also reported to have antifungal bioactivity. all those reports are summarized in table 4. 752 biology, medicine, & natural product chemistry 14 (2), 2025: 747-755 table 4. the peak identity of highly abundance compounds in the extracellular extract of 40h-culture of niallia nealsonii dcl1and its corresponding potential bioactivity based on the previous reports. no putative compound (molecular formula) retention time (minutes) molecular weight bioactivity references 1. n-carbamoyl-2-hydroxy-3methoxybenzamide (c9h10n2o4) 3.31 210.1323 antioxidant (liang et al., 2020) 2. unknown1 3.55 244.1174 unknown 3. gallic acid (c7h6o5) 0.49 169.0671 antioxidant, antiaging (lesmana et al., 2021; utami et al., 2023) 4. unknown2 2.47 260.1113 unknown 5. n-carbamoyl-2,3-dihydroxybenzamide (c8h8n2o4) 2.59 196.1146 antioxidant (sugiyama et al., 2009) 6. 12e-bromoisorumbrin (c20h20brno3) 9.04 402.2258 unknown 7. methyl 6-carbamoylphenazine-1carboxylate (c15h11n3o3) 9.80 281.2697 antibacterial, antifungal (hu et al., 2019) 8. prenostodione (c19h15no5) 11.28 337.3293 antibacterial (utami et al., 2023) discussion niallia nealsonii was previously known as bacillus nealsonii, but there was a change in nomenclature based on evidence of collecting phylogenetic, molecular, and protein data into a new genus niallia which belongs to the bacillacea family (gupta et al., 2020; patel et al., 2019). the isolate dcl1 has been identified as n. nealsonii based on the 16srrna gene which was deposited in the genbank database with accession number op363654 (utami et al., 2023). physiological properties of dcl1 in terms of growth phase have shown that the stationary phase was started after 20 h of incubation in the liquid luria bertani (lb) medium under aerobic conditions. thus, harvesting the extracellular metabolites at 28 and 40-h of incubation would give insight towards the secondary metabolites secreted by dcl1. in this study, the extraction of metabolites was conducted using maceration method and employing ethyl acetate as solvent. ethyl acetate has been reported to be an effective solvent to obtain an extract rich in various groups of bioactive compounds such as polyphenols (pintać et al., 2018), flavonoids (nurcholis et al., 2021; ren et al., 2020; thavamoney et al., 2018), and terpenoids (jiang et al., 2016; truong et al., 2021). those groups of bioactive compounds have also been reported to be contained in clove leaves (haro-gonzález et al., 2021; lesmana et al., 2021; plata-rueda et al., 2018). however, limited information is available in the potential endophytic bacteria of clove in producing the similar bioactive compounds to its plant host. as revealed by our experiments, the endophytic bacteria dcl1 were confirmed to produce phenol and flavonoids as part of the secreted extracellular metabolites. the total phenol levels were higher than the total flavonoid levels both in 28 and 40-h culture. however, the levels of those compounds were higher in metabolites produced by 40 than 28-h bacterial culture. in accordance with that, the antioxidant activity of metabolites in 40-h culture was found stronger than the 28h bacterial culture. however, the ic50 dpph value of the crude extract of n. nealsonii 40-h was still lower by about 55x than ascorbic acid and about 74x lower than trolox. it is expected that the n. nealsonii extract is still a crude extract, so it contains various types of active compounds that have an impact on their non-specific activity compared to the positive control, which is already a pure compound. it is worth noting that the ic50 using dpph and abts radicals has a significant difference (table 2), yet both activities are considered to have low antioxidant activity. similar results occurred in the extracts of endophytic sponge pseudoalteromonas flavipulchra still-33 (prastya et al., 2020), which had a higher ic50 abts value than ic50 value of dpph radicals. this was influenced by the different types of radicals used, so the degradation mechanism of each radical is different [22, 23]. several previous studies reported that the bacterial extract associated with clove leaves had an ic50 dpph antioxidant activity of 262.3 µg/ml (utami et al., 2023). when compared with this study, the 40-h n. nealsonii extract had a dpph antioxidant activity value of about 0.85x stronger. notably, antioxidant activity is essential to promote cellular longevity. it is mostly due to the free radicalscausing oxidative stress are common causes of cellular aging (pizzino et al., 2017; tan et al., 2018). thus, we further elucidated the antioxidant and antiaging properties at the cellular level. the yeast model organism s. pombe was used for the antiaging assay at cellular level because it has a relatively short life span, easy to reproduce, is not pathogenic, quickly obtains test results, and provides comprehensive results because it has similarities with mammalian cells. the similarities between mammalian cells and the yeast s. pombe are observed in the inheritance of mitochondria through the microtubule network (ruetenik et al., 2015). another similarity was in the stress response which activates torc1 and mitogen-activated protein (map) kinases (pan et al., 2011). in this assay, the 40-h extract of n. nealsonii potentially influence aging mechanisms salsabilah et al. – metabolites of niallia nealsonii as antioxidant and antiaging 753 so that it could prolong yeast life span in yes medium compared to without extract treatment (figure 4). it is worth noting that treatment of 2 x ic50 dpph markedly maintained yeast viability comparable to that of calorie restriction treatment (yes with 0.3% glucose) up to 11 days of incubation. the calorie restriction treatment has been known to promote life span to various organisms including yeast saccharomyces cerevisiae (ariybah et al., 2021), s. pombe (astuti et al., 2021), worm caenorhabditis elegans (yang et al., 2010), mice mus musculus as well as mammalian cells (chung et al., 2019; khan et al., 2019; pitt et al., 2015). such antiaging activity of calorie restriction has been proposed through various mechanisms including induction of sirtuin activity (wierman et al., 2014), autophagy (chung et al., 2019), and adaptive-mitochondrial signalling (lesmana et al., 2021; wierman et al., 2014; yusuf et al., 2021). it is worth noting that the metabolite extract of dcl1 shows potential antiaging activity even in non-calorie restricted conditions. however, further study is required to elucidate the potential mechanism elicited by the metabolites of dcl1. compound profiles in potential extracts were analysed using the lc-ms/ms method to identify dominant compounds that are potentially related to antioxidant and antiaging activities. the peak of the most dominant compound is suspected to be n-carbamoyl-2hydroxy-3-methoxybenzamide (m/z 210.1), followed by unknown1 (m/z 244.1), gallic acid (m/z 169.0), unknown2 (m/z 260.1), and n-carbamoyl-2,3dihydroxybenzamide (m/z 196.1), respectively. based on literature studies, gallic acid and n-carbamoyl-2,3dihydroxybenzamide compounds were reported to have pharmaceutical activities related to antioxidant and antiaging activities (riptanti et al., 2019; singh et al., 2017). the n-carbamoyl-2-hydroxy-3methoxybenzamide compound has nuclear magnetic resonance (nmr) data which is very similar to ncarbamoyl-2,3-dihydroxybenzamide and were reported to have antioxidant activity (lesmana et al., 2021). a previous study reported that the ethanol extract of clove leaves and ethyl acetate-derived extract from clove endophytic bacteria bacillus cereus dcn1 produced metabolites rich in gallic acid. those extracts were found to have both antioxidant and antiaging properties (lesmana et al., 2021; utami et al., 2023). thus, our study enriched the potential source of important bioactive compounds that are profoundly used for the development of drugs and cosmeceuticals. conclusions the ethyl acetate extract of clove endophytic bacteria n. nealsonii dcl1 has extracellular metabolites with low antioxidant activity in vitro. however, a concentration of 2x ic50 of antioxidant activity could prolong yeast s. pombe cells. thus, showing the potential of the extract as antiaging agent at cellular levels. the extract was found to be rich in gallic acid and n-carbamoyl-2,3dihydroxybenzamide which potentially be developed further for pharmacological purposes. acknowledgements: the authors thank ministry of education, research and technology of the republic of indonesia for the research funding through the scheme penelitian dasar 2023 to ria (contract no. 001/e5/pg.02.00.pl/2023). the authors would like to express great appreciation to the facilities, scientific and technical support from advanced characterization laboratories serpong, national research and innovation institute through elayanan sains, badan riset dan inovasi nasional (brin). authors’ contributions: concept: rika indri astuti; design: rika indri astuti, muhammad eka prastya; data collection or processing: rika indri astuti, nadiyah salsabilah; analysis or interpretation: rika indri astuti, nadiyah salsabilah, muhammad eka prastya; 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(2021). anti-aging activity of xylocarpus granatum phytoextracts and xyloccensins k compound. indonesian journal of pharmacy, 32(3), 365–375. doi: 10.22146/ijp.1430 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 951-955 | doi: 10.14421/biomedich.2025.142.951-955 issn 2540-9328 (online) evaluation of forest honey on the proliferation and migration of dermal fibroblasts under hyperglycemic conditions: an in vitro study januar rizqi1*, denny agustiningsih2, dwi aris agung nugrahaningsih3 1departement of nursing, faculty of medicine, universitas respati yogyakarta, indonesia. 2department of physiology, faculty of medicine, universitas gadjah mada, indonesia. 3department of pharmacology and therapy, faculty of medicine, universitas gadjah mada, indonesia. corresponding author* arizqi.januar@respati.ac.id manuscript received: 01 september, 2025. revision accepted: 27 october, 2025. published: 28 october, 2025. abstract this study aims to evaluate the effect of forest honey on fibroblast proliferation and migration under hyperglycemic conditions in vitro. the research method involved culturing primary fibroblasts in high-glucose dmem (25 mm) and dividing them into five groups: standard control (standard medium), hyperglycemia control (high-glucose medium without treatment), 3% honey, 1.5% honey, and 0.75% honey. proliferation was assessed by counting live cells (trypan blue staining/hemocytometer) at 24, 48, and 72 hours. migration was measured using a scratch assay (0, 24, 48, 72 hours) and analyzed with imagej. the shapiro–wilk test was used for normality; data were analyzed with anova followed by lsd or kruskal–wallis (p<0.05). results showed that hyperglycemia decreased proliferation compared to the standard control. treatment with 1.5% honey and 0.75% honey consistently increased proliferation compared to the hyperglycemic control at all time points (p<0.05), while 3% honey showed a relatively lower increase. the standard control group achieved 100% closure at 72 hours in the migration variable. the honey groups (0.75–3%) showed an increase compared to the hyperglycemic control, but the difference was not significant at 24–48 hours and approached significance at 72 hours (p≈0.057). in conclusion, forest honey at a concentration of 1.5% can increase fibroblast proliferation in a hyperglycemic environment. the effect on migration requires confirmation with osmolality controls and more sensitive endpoints. these findings provide a biological basis for developing honey-based diabetic wound care adjuvants focusing on dose optimization and formulation standardization. keywords: forest honey; in vitro hyperglycemia (25 mm); dermal fibroblast proliferation; scratch migration assay. introduction diabetes mellitus (dm) continues to increase and has become one of the most significant burdens on the global health system, with a significant economic impact on individuals (idf, 2025). one of the complications of dm is diabetic foot ulcer (dfu), which can lead to disability, amputation, and mortality, and requires high healthcare resources (fuentes-peñaranda et al., 2025). wound healing is a coordinated process that includes the phases of hemostasis, inflammation, proliferation, and remodeling, with complex intercellular orchestration and mediators, one of which is the fibroblast (mamun et al., 2024). fibroblasts play a central role in matrix deposition, wound contraction, and the secretion of growth factors that support angiogenesis and tissue repair (boraldi et al., 2024). reactive oxygen species (ros) support healing signals under controlled conditions, while in excessive conditions, they promote chronicity of wounds (hunt et al., 2024). redox imbalance disrupts proliferation and migration by disrupting the cytoskeleton, adhesion–deadhesion, and signal transduction. hyperglycemia exacerbates the condition through the age–rage pathway, mitochondrial load, and persistent inflammation, thereby delaying the proliferation phase (hunt et al., 2024). in human dermal fibroblasts, high glucose exposure is associated with cellular aging and dysfunction that impedes healing (ma et al., 2023). therefore, restoring redox homeostasis and fibroblast function is a biological key to accelerating tissue repair (mamun et al., 2024). hyperglycemia consistently reduces fibroblast proliferation and migration. impeded cell-type-specific migration is recognized as a key mechanism delaying diabetic wound healing (song et al., 2025). current wound therapies such as nanotechnology, stem cells, and innovative dressings are promising, but cost and complexity constraints limit their widespread application (mamun et al., 2024). some high-glucose studies have not included iso-osmotic controls, making it challenging to distinguish biological effects from osmolality or viscosity (sharma et al., 2018). from a methodological perspective, further evidence is needed to strengthen in vitro evidence and enable translation (mamun et al., https://doi.org/10.14421/biomedich.2025.142.951-955 952 biology, medicine, & natural product chemistry 14 (2), 2025: 951-955 2024). thus, scientific, biocompatible adjuvant interventions that can be integrated into wound care practices are a fundamental need (fuentes-peñaranda et al., 2025). honey is a candidate adjuvant due to its polyphenol or flavonoid content and its ability to produce low doses of h₂o₂ via glucose oxidase, making it relevant as a redox modulator and antimicrobial (tashkandi, 2021). honey can also modulate inflammation, support the proliferation phase, increase collagen synthesis, and improve cell migration (iosageanu et al., 2024). previous studies have assessed the effectiveness of honey on various chronic wounds with a good safety profile, although the heterogeneity of studies calls for further standardization. variability in sources and phenolic composition underscores the importance of precise dosing to ensure efficacy without causing osmotic stress (saad, 2025). therefore, conducting safe concentration range trials for fibroblasts under hyperglycemic conditions is crucial. although chronic wound therapy rapidly evolves to include nanotherapeutics, exosomes, and innovative dressings, safe, affordable, and accessible adjuvants remain highly needed (mamun et al., 2024). a significant gap in this research is the limited number of studies evaluating honey on fibroblast proliferation and migration under hyperglycemic conditions. the research of this article lies in its focus on forest honey across a range of doses and the simultaneous evaluation of two key fibroblast functions in a hyperglycemic environment. thus, this study aimed to assess the effect of forest honey on fibroblast proliferation and migration under hyperglycemic conditions in vitro. materials and methods study area this study was conducted at the pharmacology laboratory, physiology laboratory, and anatomy laboratory of the faculty of medicine, gadjah mada university, yogyakarta, and has obtained ethical approval from the ugm lppt research ethics committee (no. 00010/04/lppt/2016). cell culture primary fibroblasts were obtained from the dermal tissue of male wistar strain white rats (aged 3–4 months) induced with diabetes mellitus using streptozotocin (stz, 60 mg/kg bw) and nicotinamide (na, 120 mg/kg bw) via intraperitoneal injection. mice with fasting blood glucose levels ≥126 mg/dl were categorized as successful diabetes models. fibroblast cells were isolated enzymatically, then cultured in dulbecco's modified eagle's medium (dmem) containing 10% fetal bovine serum (fbs) and antibiotics-antimycotics. the cultures were maintained in a 5% co₂ incubator at 37 °c until they reached 80–90% confluence. hyperglycemia was modeled using high-glucose dmem medium (25 mm). experimental groups and treatments the fibroblast cultures were divided into five groups, namely: (1) fibroblasts in standard medium (standard control), (2) fibroblasts in high-glucose medium without treatment (hyperglycemia control), (3) 3% honey fibroblasts, (4) 1.5% honey, and (5) 0.75% honey. each treatment was performed in triplicate (n = 3) and observed after 24, 48, and 72 hours of incubation. proliferation assay cell proliferation was assessed by counting the number of cells using a hemocytometer after trypan blue staining. the number of live cells was counted 24-, 48-, and 72-hours post-treatment, then expressed as the mean ± standard deviation. migration assay fibroblast migration activity was tested using the scratch assay method. a linear scratch was made using a sterile pipette tip in confluent monolayer cultures. the scratch area was photographed with an inverted microscope at 0, 24, 48, and 72 hours. wound closure was calculated using imagej software, and the results were expressed as the percentage of cell migration relative to the baseline. statistical analysis data normality were tested using the shapiro–wilk test. normally distributed data were analyzed using one-way anova followed by lsd post hoc testing. the kruskal–wallis’s test was used if the data were not normally distributed. the significance level was set at p < 0.05. results and discussion fibroblast proliferation fibroblast proliferation differed between groups at all observation time points (24, 48, 72 hours). standard control (standard medium) showed the highest number of cells, while hyperglycemia control (high glucose medium without treatment) showed the lowest. administration of 1.5% honey and 0.75% honey consistently increased proliferation compared to the hyperglycemia control at 24, 48, and 72 hours (all p<0.05; lsd post hoc test), while 3% honey increased but was relatively lower than 1.5% and 0.75% honey. the shapiro–wilk test met the normality assumption, and one-way anova showed significant differences between groups (p<0.001). the mean and standard deviation for each group is shown in table 1. rizqi et al. – evaluation of forest honey on the proliferation and migration of … 953 table 1. mean number of fibroblasts (mean ± sd) at 24, 48, and 72 hours. group 24 h (mean ± sd) 48 h (mean ± sd) 72 h (mean ± sd) standard control 40.68 ± 4.62 49.32 ± 4.46 51.63 ± 1.36 hyperglycemic control 16.82 ± 2.86 21.14 ± 4.36 19.19 ± 3.86 honey 3% 23.51 ± 2.47 30.06 ± 7.55 22.29 ± 3.54 honey 1.5% 30.08 ± 2.70 36.01 ± 5.87 29.33 ± 2.65 honey 0.75% 28.61 ± 1.71 31.18 ± 3.90 27.79 ± 1.75 note: anova p<0.001; lsd follow-up test shows that 1.5% honey (24 hours p=0.019; 48 hours p=0.011; 72 hours p=0.006) and 0.75% honey (24 hours p=0.035; 48 hours p=0.046; 72 hours p=0.012) were higher than the hyperglycemia control. fibroblast migration migration activity increased over time in all groups. the standard control showed the highest migration percentage and reached 100% closure at 72 hours, while the hyperglycemia control was the lowest. treatment with 3% honey, 1.5% honey, and 0.75% honey showed an increasing trend compared to the hyperglycemia control; the differences between groups were not significant at 24 hours (kruskal–wallis p=0.629) and 48 hours (p=0.094), and showed a trend at 72 hours (anova p=0.057). detailed values are presented in table 2. table 2. percentage of fibroblast migration in various treatment groups. group 24 h (median; iqr) 48 h (median; iqr) 72 h (mean ± sd) standard control 78.93 (69.73–79.02) 100.00 (100.00) ᵃ 100.00 ± 0.00ᵃ hyperglycemic control 78.59 (66.50–78.64) 81.23 (68.84–82.29) 79.23 ± 7.69 honey 3% 78.40 (65.02–78.47) 82.70 (68.66–83.65) 79.92 ± 8.21 honey 1.5% 77.07 (66.02–77.07) 83.16 (70.12–85.38) 81.20 ± 7.45 honey 0.75% 77.79 (65.18–79.34) 81.71 (68.93–82.44) 80.35 ± 7.61 ᵃthe difference is significant compared to other groups (p<0.05). figure 1. fibroblast migration activity at 24, 48, and 72 hours (inverted microscope 100×). straight lines mark the initial scratch boundaries. groups: standard control, hyperglycemia control, 3% honey, 1.5% honey, 0.75% honey. standar control honey 0.75% honey 1.5% honey 3% hyperglycemic control 24h jam 48h 72h 954 biology, medicine, & natural product chemistry 14 (2), 2025: 951-955 discussion this study shows that hyperglycemia suppresses fibroblast function, particularly proliferation, compared to normoglycemic controls; conversely, honey at 0.75– 1.5% consistently increased proliferation compared to hyperglycemic controls, while honey at 3% did not increase proliferation. meanwhile, fibroblast migration in the honey group showed an increasing trend compared to the hyperglycemic control, but did not reach statistical significance; the normoglycemic control achieved 100% wound closure at 72 hours. these results are consistent with the knowledge that a high-glucose environment disrupts redox homeostasis and tissue repair dynamics, while the bioactive components of honey have the potential to moderate these disturbances (berry et al., 2024; pasupuleti et al., 2020). the finding of reduced fibroblast performance in high-glucose media is consistent with reports that hyperglycemia increases oxidative stress and maintains inflammation, thereby delaying the proliferation phase until remodeling (gonzález et al., 2023; nagy et al., 2019). in human dermal cell models, high glucose exposure is associated with cellular aging and functional abnormalities that slow healing (zhang et al., 2021). research results show that honey, especially honey rich in phenolics, can suppress inflammation, support proliferation, and improve cell migration. hyperglycemia increases ros production through the age–rage pathway, mitochondrial load, and glucose autooxidation; excessive ros exposure disrupts cytoskeletal organization, adhesion–de-adhesion, and signaling required for proliferation and migration (comino-sanz et al., 2021; hunt et al., 2024). honey contains polyphenols or flavonoids that produce low levels of h₂o₂ via glucose oxidase; both factors contribute to redox buffering (antiradical, proinflammatory resolution) and support cell cycle and matrix survival (iosageanu et al., 2024; wilczyńska & żak, 2024). the dose-response correlation indicates that honey at 0.75–1.5% is more effective than honey at 3%. scientifically, these results reinforce the hypothesis that honey can maintain and enhance fibroblast proliferation in a hyperglycemic environment, which is a key mechanism for initiating the tissue repair phase (pasupuleti et al., 2020; scepankova et al., 2021). to strengthen the evidence, further studies need to measure and control osmolality, add orthogonal endpoints, and verify the redox pathway through quantification of ros and antioxidant enzymes (sod, cat, gpx) (gayathri et al., 2023). testing in 3d/explant models and in vivo validation in diabetic wounds will increase biological relevance toward early clinical trials (mamun et al., 2024). conclusions hyperglycemia reduces fibroblast function, most notably in proliferation, compared to normoglycemic controls. administration of forest honey at concentrations of 0.75– 1.5% consistently increased fibroblast proliferation compared to hyperglycemic controls at 24, 48, and 72 hours, while 3% was relatively less optimal. in migration, the honey group showed an increasing trend compared to the hyperglycemic control, but it was not statistically significant; the standard control achieved 100% wound closure at 72 hours. overall, these findings confirm that sub-osmotic honey concentrations (≤1.5%) are more conducive to supporting fibroblast function in a hyperglycemic environment, consistent with the study objective of evaluating the effects of honey on fibroblast proliferation and migration. authors' contributions: januar rizqi conceptualization; methodology; investigation; data curation; formal analysis; visualization; writing original draft. denny agustiningsih provides validation, supervision, resources, writing, review & editing, and project administration. dwi aris' supervision; writing, review and editing. all authors contributed to interpreting the results, reviewed and approved the final manuscript for publication, and agreed to be accountable for all aspects of the work. competing interests: the authors declare that there are no competing interests. funding: the authors declare funding if any funding. references berry, c. e., brenac, c., gonzalez, c. e., kendig, c. b., le, t., an, n., & griffin, m. f. 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(2021). honey in wound healing: an updated review. open life sciences, 16(1), 1091–1100. https://doi.org/10.1515/biol-2021-0084 wilczyńska, a., & żak, n. (2024). polyphenols as the main compounds influencing the antioxidant effect of honey—a review. international journal of molecular sciences, 25(19). https://doi.org/10.3390/ijms251910606 zhang, s., ke, z., yang, c., zhou, p., jiang, h., chen, l., li, y., & li, q. (2021). high glucose causes distinct expression patterns of primary human skin cells by rna sequencing. frontiers in endocrinology, 12(march). https://doi.org/10.3389/fendo.2021.603645 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 97-108 | doi: 10.14421/biomedich.2023.121.97-108 issn 2540-9328 (online) potential inhibition of ace2 membrane protein by flavone glycosides for blocking entrance of sarscov-2 into the cells; a computational study ahsan ibrahim*, ehtisham ul haq shifa tameer-e-millat university, islamabad-44000, tel. +9251-8438061 fax. +9251-8438059, pakistan. corresponding author* ahsan.scps@stmu.edu.pk manuscript received: 07 october, 2022. revision accepted: 07 december, 2022. published: 10 january, 2023. abstract severe acute respiratory syndrome coronavirus 2 (sarscov-2), since its emergence in wuhan city of china in late 2019, had been a dilemma for the global healthcare system. humongous efforts have been put in ascertaining the effective treatments for attenuation of the spread of corona virus disease (covid-19) pandemic. the aim of this research study is to probe the potential inhibition of angiotensin converting enzyme 2 (ace2) membrane protein by well-known flavone glycosides, hence preventing the binding of spike proteins with ace2 and subsequent prevention of entry of sarscov-2 inside the cells. the molecular docking analysis, for total ten flavone glycosides was carried out, that laid out propitious results in terms of binding energies towards the active residues of ace2 protein with a range of -9.3 to -7.1 kcal/mol. the molecular dynamics simulation also yielded promising outcomes. the in-silico toxicity analysis of all the potential drug candidates was carried out that revealed that all the compounds were non-toxic and safe. studies may be required for optimum formulation development using these compounds as a part of drug discovery and development phenomenon. this study may play a vital part in exploration of natural compounds in pharmacotherapy of covid-19. keywords: ace2 membrane protein; covid-19; flavone glycosides; molecular docking analysis; sarscov-2; spike proteins. abbreviations: sarscov-2 : severe acute respiratory syndrome coronavirus 2 covid-19 : corona virus disease ace2 : angiotensin converting enzyme 2 ssrna : single stranded ribonucleic acid prrar : furin cleavage site in spike protein tmprss2 : transmembrane serine protease 2 s protein : spike protein s cleavage : spike cleavage rbd : receptor binding domain pdb : protein data bank admet : absorption, distribution, metabolism, elimination and toxicity profile introduction the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) emerged as the most common clinical presentation of severe covid-19 (brosnahan et al. 2020). coronavirus disease started in wuhan, china in late 2019, spreading worldwide, affecting 612 million people and total number of deaths exceeds 6.53 million as of september 2022 as per statistics of world health organization (who) (“who coronavirus (covid-19) dashboard | who coronavirus (covid-19) dashboard with vaccination data” n.d.). the advent of covid-19 created serious crisis in health sector, leaving behind huge gaps in the field of disease control and prevention, that could be bridged through new research and investigations. the genome of covid-19 had been the most noteworthy in the ongoing research on sarscov-2. it is a positive sense single-stranded rna (+ssrna) and hence tends to replicate in a swift way within the host cell. the genetic material of the virus encodes for many nonstructural proteins (nsps) that implement enormous functions for virus such as taking the control the host biosynthetic machinery, viral replication, viral protein processing etc. the function of some nsps is still unknown. the structural genes of this virus encode for the structural proteins, which include spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins, having a wide spectrum of functions in https://doi.org/10.14421/biomedich.2023.121.97-108 98 biology, medicine, & natural product chemistry 12 (1), 2023: 97-108 characterizing the entry and pathogenicity of the viral particle in the host cell. the homotrimers of s proteins make up the spikes on the surface of viral particle and they are involved in the attachment to the host cell receptors. spike proteins hold prime importance in relation to the ace2 proteins to which they bind and provoke the infective cascade of covid-19 (chen, liu, and guo 2020). in host cells, spike protein (s protein) is cleaved at the furin cleavage site i.e. prrar motif by a protease named furin into s1 and s2 to form a heterodimer of s1/s2, which is finally assembled in the form of the trimeric spike protein complex. the s2 subunit of this complex is cleaved by host proteases like transmembrane protease serine 2 (tmprss2) to uncover the fusion peptide for membrane fusion of the host cell and virus (wu et al. 2022). s1 is for binding to the host cell receptor angiotensin-converting enzyme 2 (ace2). it is v-shaped and the second one (s2 subunit) for fusion of the viral and host cell membranes (sternberg and naujokat 2020). ace2 and tmprss2 are abundantly present in the airways, arteries, nasal and oral mucosa, kidneys, the intestine, most profusely expressed on epithelial cells of the respiratory tract i.e. type ii pneumocytes that are the cells responsible for secreting surfactant to lessen the surface tension and prevent lung collapse (sternberg and naujokat 2020; akkız 2022). receptor-binding domain (rbd) contains a receptor binding motif, which is the necessary for spike protein (s1) that binds to the outer surface of ace2 protein. tmprss2 cleaves a part of spike proteins and promote the adhesion of sars-cov-2 s protein to get fuse with host cell membrane as the extended spike fosters the fusion of viral and host cell membrane. subsequently, many hydrophobic amino acids are also generated that get concealed in nearby cell membrane. the virus enters into the cell and have a first encounter with host cell biosynthetic machinery for translation of viral genetic material into viral proteins leading to prompt replication. cells that overexpress ace2 and tmprss2 are more susceptible to sarscov-2 entry (akkız 2022; ahmad et al. 2021). ace2 is a membrane-bound carboxydipeptidase protein. ace2 degrades angiotensin ii to generate angiotensin 1-7, which downregulates regulates a variety of angiotensin ii actions mediated by counteracting effects against the excessively activated ace/angiotensin ii/at1r axis, angiotensin ii type 1 receptor, as seen in hypertension, cardiac hypertrophy, heart failure, and some cardiovascular disorders. on the other hand, human ace2 is a known receptor by which sars-cov-2 particle enters host cells, by mutual binding of the spike protein of sar-cov-2 and ace2 membrane protein. this makes it a critical site for stopping the sarscov-2 to enter the cells using various ligands. so, it is pivotal to inhibit ace2, prevent viral genetic material entry into the cell (kai and kai 2020). the figure 1 summarizes the mechanism of the whole process. flavonoids are the polyphenolic compounds, very popular for the extensive pharmacological effects they provide such as anti-cancer, anti-inflammatory, antioxidant etc. (panche, diwan, and chandra 2016). flavones are one of the subclasses of flavonoids. the glycosidic linkages at positions 3 or 7 of flavones leads to formation of flavone glycosides (kumar and pandey 2013). besides various therapeutic effects such as antimicrobial, antidiabetic, hepatoprotective, antitumor and immunomodulatory effects (xiao et al. 2016), flavone glycosides have also been studied and reported for having antiviral effects against herpes simplex virus (hsv) 1 and 2 (yarmolinsky et al. 2012). keeping in view that fact that ace2 is a potential target, this study is going to use molecular docking as a tool to inquire regarding the activity of flavone glycosides for inhibiting ace2 and blocking the entry of sarscov-2 into the cell and arresting the viral replication process. figure 1. mechanism of entry of sarscov-2 into the cells through ace2 membrane protein materials and methods macromolecule preparation the 3d structure of the ace2 protein was retrieved from rcsb-protein data bank database in pdb format (pdb id: 1r42) (towler et al. 2004a). x-ray crystallography method was used for the determination of the structure of the macromolecule. the macromolecule was prepared by removing the unwanted water molecules, heteroatoms and ligands from the structure of protein on software biovia discovery studio visualizer v17.2.0.16349. the cleaning of protein helps to purely observe the interaction between ligand and the target protein residues, without any influence of these unnecessary entities. ligand preparation the ligands selected were the flavone glycosides and their structures were acquired from pubchem database. ibrahim & ul haq– flavone glycosides attenuate covid-19 99 perkinelmer chem 3d 16.0 was used to convert the 3d structure of the ligands into pdb format. luteolin 3'glucoside (l1), isovitexin (l2), baicalin (l3), scutellarin (l4), diosmin (l5), apigenin 7-o-glucoside (l6), isoorientin (l7), lonicerin (l8), tricin 7-oglucouronide (l9) and swertisin (l10) were the ligands used for studying their potential for inhibition of ace2 protein receptor for preventing the entry of sars-cov2 into the cells containing ace2 membrane protein, as shown in table 1. the two-dimensional structures of the ligands l1 to l10 are shown in figure 2. table 1. the table shows the list of flavone glycoside ligands (l1 to l10) in this study, their chemical structure, botanical source and iupac names. serial number ligands pubchem id structure botanical source literature source l1 luteolin 3'glucoside 12309350 podocarpus nivalis pubchem l2 isovitexin 162350 carex fraseriana pubchem l3 baicalin 64982 scutellaria amoena pubchem l4 scutellarin 185617 scoparia dulcis pubchem l5 diosmin 5281613 asyneuma argutum pubchem l6 apigenin 7o-glucoside 44257792 lonicera japonica pubchem 100 biology, medicine, & natural product chemistry 12 (1), 2023: 97-108 serial number ligands pubchem id structure botanical source literature source l7 isoorientin 114776 carex fraseriana pubchem l8 lonicerin 5282152 lonicera japonica pubchem l9 tricin 7-oglucouronide 101939793 scutellaria discolor pubchem l10 swertisin 124034 gentiana orbicularis pubchem ibrahim & ul haq– flavone glycosides attenuate covid-19 101 figure 2. the figure shows the chemical structures of the ligands l1 to l10, along with their iupac names obtained from pubchem. results and discussion molecular docking analysis & results a molecular docking technique has been very handy in drug discovery by pinpointing the drug leads having auspicious therapeutic activity (ferreira et al. 2015). the molecular docking was performed using pyrx virtual screening tool that is highly reliable as it works on the configuration of autodock vina (ekins, mestres, and testa 2007). for the visualization of protein-ligand interactions, biovia discovery studio visualizer software v17.2.0.16349 was utilized. it provided distinct display of the 2d and 3d interactions along with the bond lengths, aromatic, hydrophobic interactions etc. promising results were witnessed after the molecular docking analysis of human ace2 protein receptor with ligands l1 to l10, as shown in table 2. 102 biology, medicine, & natural product chemistry 12 (1), 2023: 97-108 table 2. the table shows the findings of the molecular docking and scoring analysis performed for ligands, l1 to l10. ligand-protein interaction binding energy (kcal/mol) bonding residues type of bond bond length (å) other residues l1 -7.5 his 378 pi-pi 5.61 ala 348, asp 350, tyr 385 his 401 pi-pi 4.43 glu 402 pianion 3.49, 3.75 l2 -7.1 his 378 pi-pi 4.93 asp 382, asn 394 his 401 pi-pi 5.71 glu 402 pianion 4.20 l3 -8.1 his 401 h bond 2.53 ser 44, asp 350, asp 382 l4 -8.2 his 401 h bond 2.50 ser 44, trp 349, asp 350, asp 382, tyr 385 l5 -9.3 his 378 h bond 3.31 phe 40, asp 350, asp 382, asn 394 his 401 pialkyl 5.12 l6 -7.9 his 401 pidonor h bond 2.58 ser 44, ala 348, trp 349, asp 350 glu 402 h bond 2.38 l7 -7.4 his 378 pi-pi 4.93 ala 348, glu 375 his 401 pi-pi 5.72 l8 -8.4 his 378 pi-pi 4.94 ser 44, ala 348, trp 349, tyr 385, tyr 515 glu 402 pi-anion 3.57 l9 -8.0 glu 402 h bond 2.11 trp 349, asp 350, arg 514 l10 -7.3 his 378 h bond, pi-pi 3.11, 4.95 pro 346, asp 382, arg 393 his 401 h bond, pi-pi 2.53, 5.77 all the ligands l1 to l10, exhibited their firm interactions with the binding site residues of human ace2 protein receptor i.e. his 378, his 401 and his 402 (towler et al. 2004b), with a binding affinity ranging from -9.3 to -7.1 kcal/mol. the best binding energy of 9.3 kcal/mol was expressed by diosmin (l5), while the binding energies exhibited by other compounds in this study were -8.4 kcal/mol for lonicerin (l8), -8.2 kcal/mol for scutellarin (l4), -8.1 kcal/mol for baicalin (l3), -8.0 kcal/mol for tricin 7-o-glucouronide (l9), 7.9 kcal/mol for apigenin 7-o-glucoside (l6), -7.5 kcal/mol for luteolin 3'-glucoside (l1), -7.4 kcal/mol for isoorientin (l7), -7.3 kcal/mol for swertisin (l10) and -7.1 kcal/mol for isovitexin (l2). all the ligands showed absolute interactions with the active site residues of human ace2 enzyme. luteolin 3'-glucoside (l1) and isovitexin (l2) were bound to his 378, his 401 and glu 402 with considerable binding energies. ligands, baicalin (l3) and scutellarin (l4) manifested binding with his 401 residues with shorter bond lengths. diosmin (l5) and apigenin 7-o-glucoside (l6) showed their interactions with his 378, his 401 and his 401, glu 402 residues, respectively with reasonable bond lengths. ligands, isoorientin (l7), lonicerin (l8), tricin 7-o-glucouronide (l9) and swertisin (l10), all demonstrated their robust binding affinities with active residues his 378, his 401 and glu 402 with substantial bond lengths, with pi-pi and hydrogen bonds. strong interactions and short bond lengths also appeared with some other residues i.e. ser 44, ala 248, trp 349 and asp 350 may also be the active residues of ace2 protein. the interactions of the best docked ligand diosmin (l5) are shown in figure 3. while figure 4 demonstrates the interactions of top three ligands with highest binding energies (l5, l8 and l4) with ace2 binding site. ibrahim & ul haq– flavone glycosides attenuate covid-19 103 figure 3. the best docked ligand, diosmin (l5) blocking the binding site of ace2 (a) and 2d diagram of interactions of l5 with active site residues (b). figure 4. 3d and 2d interactions of top three ligands with respect to binding energies (l5, l8 and l4) respectively, with the active site residues of ace2 protein. 104 biology, medicine, & natural product chemistry 12 (1), 2023: 97-108 admet analysis admet profile of a specific ligand serves as a surrogate for its fate to be considered as a potential drug candidate. swiss adme was employed for analyzing the adme profile of the potential drug candidates in this study (daina, michielin, and zoete 2017). toxicity profile was established through datawarrior v5.5.0 (sander et al. 2015). the orisis property explorer was used for the prediction of druglikeness score of all potential drug candidates. the molecular weights of ligands from l1 to l10 are 448.38, 432.38, 446.36, 462.36, 608.54, 432.38, 448.38, 594.52, 506.41 and 446.40 grams per mole (g/mol), respectively. the highest score for bioavailability was expressed by l2 and l10 i.e. 0.55. while l1, l5, l7, l8 conveyed their bioavailability score as 0.17 and a bioavailability score of 0.11 was predicted for l3, l4 and l9. a further invitro and in-vivo studies are recommended for improvement and tailoring of the pharmacokinetic profile of these potential drug candidates, so that these could be formulated in such a way that they may act as potent inhibitors of ace2 in attenuating sars-cov-2. the toxicity profile of these ligands, studied on datawarrior v5.5.0, was unremarkable for all the ligands, l1 to l10. the admet profile of ligands l1 to l10 is shown in table 3. the bioavailability radar of diosmin (l5) is provided in the figure 5. figure 5. bioavailability radar of the best docked ligand, diosmin (l5), obtained through swissadme. table 3. the table represents the in-silico profiling of admet, physicochemical properties, druglikeness and toxicity of the ligands (l1 to l10) drug candidates l1 l2 l3 l4 l5 l6 l7 l8 l9 l10 physicochemical properties mol.w (g/mol) 448.38 432.38 446.36 462.36 608.54 432.38 448.38 594.52 506.41 446.40 h-acc 11 10 11 12 15 10 11 15 13 10 h-don 7 7 6 7 8 6 8 9 6 6 n.r.b 4 3 4 4 7 4 3 6 6 4 tpsa (å²) 190.28 181.05 187.12 207.35 238.20 170.05 201.28 249.20 205.58 170.05 log p -0.06 0.05 0.22 -0.20 -0.44 0.55 -0.24 -1.03 0.30 0.35 b.sc 0.17 0.55 0.11 0.11 0.17 0.55 0.17 0.17 0.11 0.55 drug-likeliness d.l.s -3.4 -1.2 0.8 1.04 3.85 -2.3 -0.7 2.4 2.0 -1.1 n.l.v 2 1 2 2 3 1 2 3 3 1 metabolism cyp 1a2 no no no no no no no no no no cyp 2c19 no no no no no no no no no no cyp 2c9 no no no no no no no no no no cyp 2d6 no no no no no no no no no no cyp 3a4 no no no no no no no no no no p-glycoprotein no no yes yes yes yes no yes yes no toxicity mutagenicity no no no no no no no no no no tumorgenicity no no no no no no no no no no irritant no no no no no no no no no no reproductive effects no no no no no no no no no no mol. w (g/mol) molecular weight, h-acc hydrogen bond acceptors, h-don hydrogen bond donors, n.r.b number of rotable bonds, tpsa (å²) topological polar surface area, log p prediction of octanol/water partition coefficient, b.sc bioavailability score, d.l.s druglikeness score, n.l.v number of lipinski’s rule violations, cyp cytochrome p-450 enzyme. ibrahim & ul haq– flavone glycosides attenuate covid-19 105 molecular dynamics simulation the molecular dynamics simulation is performed to assess the physical dynamics or motions of a molecule, virtually on a computer which imparts a lot of details and facts about motions and gestures of a given docked macromolecule complex. the molecular dynamics simulation for the top docked complex of diosmin (l5) was conducted using imods server (lopéz-blanco, garzón, and chacón 2011). the molecular dynamics simulation was carried upon normal mode analysis (nma). the results for b-factor or mobility were imparted. this is an important factor of the protein crystallography, which specifies the residues with in the complex that deform themselves to interact with other residues, exposing the flexibility of a molecule. the arrow field around the complex shows the orientation of our complexed macromolecule virtually, as shown in the figure 6. figure 6. the figure shows the diagrammatic results of the molecular dynamics simulation done with the best docked complex, diosmin (l5) with ace2 protein. image (a) shows the orientation of the docked complex with the arrow field. image (b) shows the deformability graph and graphical representation of the b-factor. eigenvalue was also yielded through this server that denotes the energy required for deforming a residue in a dynamic protein for interaction with other residues. the less is the eigenvalue, the more is the ease for deformation of the residues. the eigenvalue of the studied complex was 5.6245e-05. covariance map casts a scenario of interactions between the residues of the protein and ligand in the complex while the elastic network deliver a complete picture of the stiffness within the target molecule, hence giving an estimate of deforming in the residues of the target protein, as provided in figure 7. figure 7. image (a) demonstrates the covariance graph of the best docked complex diosmin (l5) and ace2 protein, image (b) represents the elastic network map of the docked ligand while image (c) represents the eigenvalue of the docked ligand. 106 biology, medicine, & natural product chemistry 12 (1), 2023: 97-108 discussion molecular docking is a worthwhile tool in the drug discovery and repurposing phenomenon in the arena of computer aided drug designing (hughes et al. 2011). the interaction of a specific compound with a specific target molecule is predicted computationally, without utilizing the wet lab (meng et al. 2011). in this study, molecular docking was used to recognize the potential inhibition of ace2 enzyme by flavone glycosides. in this study, it was observed that flavone glycosides had an appreciable range of binding affinities with the active site residues of ace2 enzyme i.e. -9.3 to -7.1 kcal/mol. the potential drug candidates exhibited their powerful interactions with the active residues of ace2 i.e. his 378, his 401 and glu 402. luteolin 3'-glucoside (l1) showed binding energy of -7.5 kcal/mol, that is highly encouraging for inhibition of ace2. luteolin 3'glucoside (l1), isolated from podocarpus nivalis (“luteolin 3’-glucoside | c21h20o11 pubchem” n.d.) has also been identified as hepatoprotective and immunoregulatory agent in some studies (park and song 2019). isovitexin (l2), acquired from carex fraseriana (“isovitexin | c21h20o10 pubchem” n.d.), also spotted for its anti-inflammatory and antidiabetic potential (abdulai et al. 2021), has provided very optimistic results regarding binding and potential inhibition of ace2. a reasonable binding energy of -7.1 kcal/mol and linkages with active residues can make a considerable molecule having potential inhibitory activity for ace2. baicalin (l3) is a flavone glycoside derived from scutellaria amoena (“baicalin | c21h18o11 pubchem” n.d.), has anti-inflammatory effects in cardiovascular, neurodegenerative and infectious ailments (hu et al. 2022), while scutellarin (l4) gained from scoparia dulcis (“scutellarin | c21h18o12 pubchem” n.d.), has been reported as to be an antioxidant, antitumor, antiplatelet, cardioprotective and immunomodulatory (wang and ma 2018). both have showed an excellent affinity for ace2 active residue his 401, with binding energies -8.1 and 8.2 kcal/mol with considerably shorter bond lengths of 2.53 and 2.50 angstrom, respectively. diosmin (l5) was the potential drug candidate that delivered the most decent results in terms of binding energy, that was -9.3 kcal/mol and was bound to his 378 and his 401. it has been reported that diosmin (l5), obtained from asyneuma argutum (“diosmin | c28h32o15 pubchem” n.d.), works as an antioxidant as well as relieves venous insufficiencies (feldo et al. 2018). some studies have revealed that apigenin 7-o-glucoside (l6) from plant lonicera japonica (“apigenin 7-o-beta-dglucoside | c21h20o10 pubchem” n.d.), was found to be antimicrobial and cytotoxic (nie et al. 2018) and this study has detected l6 as a potential inhibitor of ace2 with a binding energy of -7.9 kcal/mol with active residues and shorter bond lengths. isoorientin (l7), derived from carex fraseriana (“isoorientin | c21h20o11 pubchem” n.d.), bound to the target with energies of -7.4 kcal/mol and showed potent affinity for active site. a study has outlined the role of isoorientin (l7) in lowering inflammatory mediators, relieving algesia and combating free radicals as well (yuan et al. 2016). lonicerin (l8), a chemical constituent of lonicera japonica (“lonicerin | c27h30o15 pubchem” n.d.), has also been described for its potent antipseudomonal pharmacology (xu et al. 2019). in this study, the binding energy of -8.4 kcal/mol of lonicerin (l8) and its connection with his 378 and glu 402 tells the possible inhibitory activity for ace2. tricin 7-o-glucouronide (l9), isolated from scutellaria discolor (“tricin 7-o-glucuronide | c23h22o13 pubchem” n.d.), is known for its inhibition for cervical cancer cell growth through upheld expression of bax and caspases (laishram et al. 2015), seems to have a significant potential to bind ace2 in human, as per this study, with optimistic energy of -8.0 kcal/mol, with bond length of 2.11 angstrom with glu 401. swertisin (l10), a priniciple component of gentiana orbicularis (“swertisin | c22h22o10 pubchem” n.d.), well known for its antioxidant and inflammation fighting tendencies (“swertisin (chebi:131838)” n.d.), came up with a binding energy value of -7.3 kcal/mol and interactions with his 378 and his 401. hence, all the potential drug candidates have furnished hopeful results in terms of potential inhibition of ace2 in human that can be further studied and validated through in-vitro and in-vivo studies. the molecular dynamics simulation study clues that the studied complex had a substantial degree of deformability. the low eigenvalue, the covariance map and the elastic network of the studied complex are the evidences of the promising deformability and admissible dynamics of the molecule. conclusion in this study, it is concluded that the ligands l1l10 have shown a potent activity for ace2 protein receptor for potentially averting the entry of sarscov-2 into the cells. the ligands have manifested pertinent interactions with ace2 active residues, showing highly encouraging binding energies. the molecular dynamics simulation studies also provided hopeful results. however, in-vitro and in-vivo studies are required to confirm these findings. furthermore, pharmaceutical studies are also recommended for these compounds in formulating them as a suitable drug delivery system for maximal therapeutic efficacy. covid-19 had been very grim challenge for the health system around the globe. the focus of the ongoing research in the field of drug discovery revolves around the drug development for covid-19. many concerns have developed in terms of efficacy as well as safety for many drugs to be used against this virus. on ibrahim & ul haq– flavone glycosides attenuate covid-19 107 the basis of findings of this study, 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pharmacotherapie 81 (july): 356–62. https://doi.org/10.1016/j.biopha.2016.04.025. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 135-139 | doi: 10.14421/biomedich.2024.131.135-139 issn 2540-9328 (online) isolation of novel 6-methylideneoxecane-3,4,5,7,8,9-hexol from the leaves of rauwolfia vomitoria, apocynaceae azibanasamesa d.c owaba1,*, raji o. rafiu1, arueniobebh frank2, darlington d. eboh3, samuel j. bunu1 1department of pharmaceutical and medicinal chemistry, faculty of pharmacy, niger delta university, wilberforce island, bayelsa state, nigeria. 2department of pharmacognosy and herbal medicine, faculty of pharmacy, niger delta university, wilberforce island, bayelsa state, nigeria. 3department of pharmaceutical microbiology and biotechnology, faculty of pharmacy, niger delta university, wilberforce island, bayelsa state, nigeria. corresponding author* azibanasamesa@gmail.com abstract rauwolfia vomitoria (wennberg) belongs to the family apocynaceae, the dried leaves were extracted successively using n-hexane, dichloromethane, 70% methanol, and concentrated in vacuo. extracts were subjected to antibacterial assay and the butanol fraction was subjected to chromatographic purification to obtain nbf12 which was subjected to spectral analysis. the antibacterial of n-hexane, dichloromethane, and 70% methanol extracts was inactive against the screened organisms assessed (staphylococcus aureus, bacillus subtilis, escherichia coli, pseudomonas aeruginosa). nbf12 is yellowish crystals (10.5 mg), rf (0.69), uv, max abs (248) the carbon 13c-nmr spectrum displayed ten carbon atoms; one methylene, two methylene oxide carbon, 6-carbinolic carbon (sp3) and a quaternary carbon (sp2). the spectrum showed a tertiary carbon at δ131.09 ppm, due to c-6 and at δ 114.74 ppm due to exocyclic methylidene carbon (sp2) (c=ch2) linked to the c-6 position. based on the spectral data nbf12 is 6-methylideneoxecane-3,4,5,7,8,9-hexol with a molecular formula c10h18o7, molecular weight 250 g/mol. keywords: isolation; rauwolfia vomitoria; antibacterial; 6-methylideneoxecane-3,4,5,7,8,9-hexol. introduction rauwolfia vomitoria (wennberg) belongs to the family apocynaceae and is a medicinal plant used for the management of unknown pyrexia by the ijaws of southern nigeria. this is due to its versatile utility in traditional medicine. it has been reported as an aphrodisiac, antimicrobial, antipsychosis, antihypertensive, antianxiety, and antioxidant effect (etim et al., 2018; emencheta et al., 2020; oyeniran et al., 2020; balogun and akintunde, 2022). this medicinal plant has a battery of chemical constituents isolated and reported in the literature which includes; ursolic acid, sistosterol, stigmasterol, reserpine, reserpinine, deserpidine, ajmalicine, and ajmaline, 2,6dimethoxybenzoquinone (ajayi et al., 2021). the research aimed to determine the antibacterial effect and to characterize the chemical constituents of r. vomitoria leaves. method and materials chemicals and reagents methanol (sigma u.k), dichloromethane (sigma u.k), n-hexane (sigma u.k), ethyl acetate (sigma u.k), dimethylsulphoxide (jhd), sephadex lh-20 (sigma), silica gel 200-400 (sigma u.k). equipment uv spectrophotometry, nmr spectrophotometer 400 mhz (agilent) microorganism staphylococus aureus nctc6571, bacillus subtilis nctc8236, escherichia coli atcc25922, psuedomonas aeruginosa atcc 10145 sample collection the leaves of r vomitoria were collected from the wild at otabi community in oloibiri district, ogbia local government area, bayelsa state, nigeria. identification of plant the plant was identified and authenticated by prof. a.t oladele of the department of pharmacognosy and herbal medicine, faculty of pharmacy, niger delta university, and herbarium specimen was deposited in the herbarium of pharmacognosy and herbal medicine and herbarium number (ndup/24/01) was assigned to it. manuscript received: 03 march, 2024. revision accepted: 20 may, 2024. published: 22 may, 2024. https://doi.org/10.14421/biomedich.2024.131.135-139 136 biology, medicine, & natural product chemistry 13 (1), 2024: 135-139 plant preparation the fresh leaves of the plant were washed and cut into small portions before air drying at room temperature for 14 days. the dried leaves were pulverized using the electrical blender to coarse powder, weighed, and stored in an airtight glass bottle. antibacterial evaluation of the extracts the antibacterial evaluation of the extract was carried out according to (balogun and akintunde, 2022). staphylococus aureus nctc6571, bacillus subtilis nctc8236, escherichia coli atcc25922, psuedomonas aeruginosa atcc 10145. a loop full of bacteria strains was inoculated in nutrient agar media and was incubated for 48 hours. 80 ml of sterile molten muller hinton agar was transferred from the mckonkey bottle to the petri dishes and allowed to solidify. the bacteria strain was diluted equivalent to 0.5 mcfarland standard in 5 ml of 0.9% normal saline and 0.03 ml of the active test strain was transferred to the solid media, swirled allowed to dry. wells were bored on the agar using a sterile cork borer (9 mm), and the 100 mg/ml (0.1, 0.2, 0.3, and 0.4 ml) stock concentration of each of the extracts were prepared in dimethylsulphoxide and water in a ratio (3:7). 10, 20, 30, and 40 mg of each extract was used against the bacteria strain using ciprofloxacin 5µg/disc as the standard drug and was incubated at 37oc for 24 hours (emencheta et al., 2020; balogun and akintunde, 2022; karim et al., 2023; dhital et al., 2024). isolation 6-methylideneoxecane-3,4,5,7,8,9-hexol the methanol extract weighing 25 g was suspended in 200 ml of distilled water and it was partitioned sequentially using n-hexane, ethyl acetate, and n-butanol and the extracts were concentrated in vacuo using a rotary evaporator. the n-butanol fraction weighed 2 g was subjected to gel filtration using sephadex lh-20 in a column (1.5 cm x 86 cm) and eluted with methanol (100%), 10 ml of the eluate collected to a total of 21 fractions coded (nbf 1-21). based on the tlc profile nbf9-12 weighed 0.146 g was subjected to column chromatography using silica gel as stationary phase (30 g, 200-400) with a dimension (1.5cm x 86 cm) and gradiently eluted with ethyl acetate, (100%); 95:5; 90:10; 80:20; 70:30; 65:35; 60:40; 50;50; 30:70; 10:90 (ethyl acetate: methanol) to 100% methanol and the progress of elution monitored using tlc in a solvent system ethyl acetate: methanol: water (100:16.5:13.5), fraction 12 gave a single spot on tlc, and concentrated in vacuo to give yellowish crystals weighed 10.5 mg which was coded nbf12, subjected to spectroscopic analysis. result and discussion antibacterial antibacterial evaluation of the n-hexane, dichloromethane, and 70% methanol extracts were screened at 10, 20, 30, and 40 mg and showed that the extracts were devoid of antibacterial activity because there was no zone of inhibition compared to the standard drug ciprofloxacin 5μg/disc which showed zone of inhibition; 24 mm, 21 mm, 22 mm and 21.5 mm against s aureus, b subtilis, e coli and p aeruginosa respectively, as illustrated in table (1, 2 and 3). the results are in line with the report by balogun and akintunde, 2022 (emencheta et al., 2020; balogun and akintunde, 2022). table 1. antibacterial activity of n-hexane extract. zone of inhibition (mm) s/n agent/conc. s. aureus b. subtilis e. coli p. aeruginosa 1 10 mg 2 20 mg 3 30 mg 4 40 mg 5 ciprofloxacin 5µg 24 21 22 21.5 keys = no zones of inhibition table 2. antibacterial activity of dichloromethane extract. zone of inhibition (mm) s/n agent/conc. s. aureus b. subtilis e. coli p. aeruginosa 1 10 mg 2 20 mg 3 30 mg 4 40 mg 5 ciprofloxacin 5µg 24 21 22 21.5 owaba et al. – isolation of novel 6-methylideneoxecane-3,4,5,7,8,9-hexol … 137 table 3. antibacterial activity of 70 % methanol extract. zone of inhibition (mm) s/n agent/conc. s. aureus b. subtilis e. coli p. aeruginosa 1 10 mg 2 20 mg 3 30 mg 4 40 mg 5 ciprofloxacin 5µg 24 21 22 21.5 chemistry of 6-methylideneoxecane-3,4,5,7,8,9-hexol (nmr analysis) figure 1. 1h-nmr of sample nbf12 138 biology, medicine, & natural product chemistry 13 (1), 2024: 135-139 figure 2. 13c-nmr of sample nbf12 yellowish crystals (10.5 mg), rf (0.69), uv, max abs (248),1h-nmr (cd3od, 400 mhz, δ, ppm); 6.1, (s, 2h), 4.87 (s, 6h, oh proton), 4.48 (s, 4h), 3.69-3.27 (m, 6h). 13c-nmr (cd3od, 400 mhz, δ ppm); 130.04 (c-6); 114.74 (=ch2), 73.62 (c-4; c-8), 72.41 (c-2; c-10), 70.82 (c-5; c-7), 63.23 and 62.94 (c-3; c-9) respectively. oh oh ch2 oh oh oh oh 9 4 10 3 o 1 2 8 7 5 6 figure 3. chemical structure of 6-methylideneoxecane-3,4,5,7,8,9-hexol. the proton nmr spectrum of sample nbf12 exhibited four prominent peaks. a downfield signal resonates at δ 6.17 ppm integrated for 2h and assigned to methylidene protons linked to c-6 due to the exocyclic, unsaturated (c=ch2) group. an intense peak at δ 4.87 due to carbinol hydroxyl protons at (c-3; c-5and c-7; c9) integrated for 6h protons and intense multiplet peak at δ 3.69-3.27 ppm due to methine (ch) of carbinolic proton at c-3-5; 7-9) positions integrated for six protons (6h) as shown in figure 1. the carbon 13c-nmr spectrum displayed ten carbon atoms; one methylene, two methylene oxide carbon, 6carbinolic carbon (sp3), and a quaternary carbon (sp2). the spectrum showed a tertiary carbon at δ131.09 ppm, due to c-6 and at δ 114.74 ppm due to exocyclic methylidene carbon (sp2) (c=ch2) linked to c-6 position, the signal at δ 73.62 due to c-4 & c-8 position. the signal at δ 72.42 ppm, due to (c-2 and c-10) methylene-oxide group, and are chemically equivalent. the carbinolic signal at δ70.82 ppm is due to c-5; c-7 and the signal at δ 63.23 & 62.94 ppm is due to c-3 and c-9 respectively as illustrated in figure 2. based on the foregoing, sample nbf12 proposed as 6methylideneoxecane-3,4,5,7,8,9-hexol with a molecular formula c10h18o7, molecular weight 250 g/mol (kamentani et al., 1995; bubb, 2003; kalsi 2004; sharma et al., 2005; azogu, 2010). this is the first time of reporting this compound in r. vomitoria (figure 3). conclusion the extract was not active against the microorganism assessed and isolation from the n-butanol gave a novel 6 owaba et al. – isolation of novel 6-methylideneoxecane-3,4,5,7,8,9-hexol … 139 methylideneoxecane-3,4,5,7,8,9-hexaol and the first time of reporting this compound from rauwolfia vomitoria. acknowledgment: the authors are grateful to prof. kola’ k. ajibesin and prof. augustine a. ahmadu for their technical assistance in carrying out this study. author’s contribution: adco and ror designed the work, adco and af collected the sample, adco, ror, and af carried out the isolation while dde carried out the antibacterial assessment of the extracts. sjb performed a critical review and manuscript proofreading. the manuscript was written by adco and all authors read and approved the final copy. competing interest: the authors have no competing interests. references ajayi oa. (2021). phytochemical and gcms analysis of bioactive components in ethanolic extract of rauvolfia vomitoria leaves. journal of chemical society of nigeria, 46(4): 0656-0660. azogu cp. (2010). laboratory organic chemistry. 2nd edition. maybinson book publishers, new jersey, usa. 269p. balogun od and akintude sl. (2022). antimicrobial activities of rauwolfia vomitoria against selected organisms. international journal of innovative and advanced studies, 9(3): 73-77. bubb wa (2003). nmr spectroscopy in the study of carbohydrates: characterizing the structural complexity. wiley periodicals, inc concepts magn reson part a:19a (1): 1-19, doi: 10.1002/cmr.a.10080. dhital s, amatya sp, aryal s, neupane p, tamang npm, thanait p. (2024). synthesis of manganese oxide nanoparticles using co-precipitation method and its antimicrobial activity. international journal of new chemistry, 11 (3), 243-253. emencheta sc, enweani bi, oli an, ibezim ec, imanyikwa ieo. (2020). antimicrobial evaluation of plants parts of rauwolfia vomitoria. journal of complementary and alternative medical research, 12(1); 11-20. etim e i, johnson ec, bassey us, and nwafor pa. (2018). phytochemical and aphrodisiac studies of ethanol root extract of rauwolfia vomitoria afzel (apocynaceae), journal of pharmacy and bioresources, 15(2): 160 – 165. fannang sv, kuete v, mbazoa cd, momo ji, van-dufat ht, tillequin, f, seguin e, chosson, e, wandji j. (2011). a new acylated triterpene with antimicrobial activity from the leaves of rauvolfia vomitoria, chemistry of natural compounds, 47 (3): 20-25. kalsi ps. (2004). spectroscopy of organic compounds. 3rd edition. new age international publishers, new delhi, india, 183p. kamentani s, mizuno h, shiga y, akunuma h. (1995). nmr of all-carbon-13 sugars: an application in the development of an analytical method for a novel natural sugar, 1,5anhydrofructose. journal of biochemistry 119, 180-185. karim m, khan s, ullah i, gamaryani a, hasnain m, abbas sh, nawaz h, saeed z, zafar i, ur r, saeed sy and malik, mua. (2023). etiology and antibiotic resistant pattern of urinary tract bacterial pathogen in district mardan. biomed journal of scientific and technical research, 52(5): 44207-44211. doi: 10.26717/bjstr.2023.52.008330. oyeniran oh, ademiluyi ao, oboh g. (2020). phenolic constituents and inhibitory effects of the leaf of rauvolfia vomitoria, afzel on free radicals, cholinergic and monoaminergic enzymes in rat's brain in vitro, j basic clin physiol pharmacol; 32(5):987-994, doi: 10.1515/jbcpp-20200144. sharma yr. (2015). elementary organic spectroscopy: principles and chemical application. s. chand and company pvt. ltd., new york, 162p. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 91-98 | doi: 10.14421/biomedich.2024.131.91-98 issn 2540-9328 (online) assessment of the phytochemical constituents of methanol extract of ereromastax polysperma leaves and its effect on the hematological indices in albino rats victor hogan idung1,*, grace asukwo okon2, nicholas asiwe3 1department of biomedical technology, school of science laboratory technology, university of port harcourt, nigeria. 2department of genetics and biotechnology, faculty of biological sciences, akwa ibom state university, nigeria. 3depertment of anatomy, faculty of basic medical sciences, university of port harcourt, nigeria. corresponding author* idungvictor@gmail.com manuscript received: 28 january, 2023. revision accepted: 08 may, 2024. published: 15 may, 2024. abstract the study investigated the effects of chronic administration of methanol extract of eremomastax polysperma on the hematological parameters of albino rats. the rats were administered daily and orally for 14 days, with water as a control. on day 15, they were anesthetized using chloroform and blood was withdrawn from the heart through cardiac puncture into ethylene diamine tetra acetic acid (edta) specimen bottles. the phytochemical screening of crude methanolic extracts revealed the presence of secondary metabolites such as alkaloids, saponin, flavonoids, and cardiac glycosides. the statistical test employed was analysis of variance (anova), with a predetermined significance level of p<0.05 and p<0.01. the anova analysis revealed significant differences in red blood cell count (rbc), hemoglobin (hgb) percentages among the control group and the treatment groups of 500mg/kg and 1500mg/kg. the hematological evaluation showed a significant difference in rbc, hgb, and hct, and a significant decrease in mean corpuscular hemoglobin concentration (mchc) with increasing doses of the extract. the results suggest that e. polysperma has anti-inflammatory, antimalarial, antimicrobial, cytotoxic, antispasmodic, and pharmacological effects, potentially aiding in disease treatment. keywords: eremomastax polysperma; phytochemical; haematology; anti-inflammatory; flavanoids. introduction medicinal plants are vital for basic healthcare in many countries, especially developing countries, as they have long been used as traditional medicine for a range of ailments (ezeigwe et al., 2020). medicinal plants include a wide range of bioactive compounds, such as alkaloids, terpenoids, and flavonoids. these phytochemicals have a range of biological activities, such as anti-inflammatory, antibacterial, antioxidant, and anticancer properties, according to malekmohammad et al. (2020). for instance, the bark of the pacific yew tree (taxus brevifolia) is used to extract the anticancer drug paclitaxel. plants are the source of some conventional drugs (cragg & newman, 2013). plants have been used to offer many basic human needs, including food, clothing, and shelter, in addition to their medicinal properties (gurib-fakim, 2006). furthermore, innovative alternatives to conventional drug-therapeutic regimens have been made possible by natural compounds derived from medicinal plants. as per ly et al. (2015), specific medicinal plants have been employed for the treatment and management of illnesses associated with reproduction. one species of plant in the asteraceae family is ereromastax polysperma. in nigeria, we call it "edemididuot." it is found in tropical and subtropical regions of africa, particularly in nigeria, ghana, and cameroon. in akwa ibom (ibibio), it's called edemididuot, which means purple bark; yoruba calls it oyun, hausa calls it esinyin, and igbo calls it akwukwo. in addition to being widely utilized in traditional medicine to treat a variety of ailments, recent studies have suggested that the herb may potentially have pharmacological qualities. the herb contains antibacterial, anti-inflammatory, wound-healing, and antioxidant properties (ojewole, 2005). these qualities make it a good choice for the development of innovative drugs meant to cure a variety of diseases. the plants are particularly common in southern nigeria, where many people think these species can cure internal heat, diabetes, anemia, and infertility (pandey, 2006). sometimes, locals call them "blood tonics." research on these species' impacts on antioxidant health, as reported by uyoh et al. (2013), offers empirical evidence in favor of their potential as therapeutics. https://doi.org/10.14421/biomedich.2024.131.91-98 92 biology, medicine, & natural product chemistry 13 (1), 2024: 91-98 methanol extract of ereromastax polysperma has also been reported to have therapeutic potential in the management of certain diseases such as diabetes (ibikunle et al., 2017). however, the safety of administration of the extract has not been fully evaluated, particularly with respect to its effects on hematological parameters. this study aims to assess the phytochemical constituents of methanol extract of ereromastax polysperma leaves and its effect on the hematological indices in albino rats. materials and methods experimental animals sixteen swiss albino rats were obtained from the animal holding facility of the faculty of pharmacy, university of uyo, nigeria, and acclimatized for two weeks before the start of the experiment. they were allowed access to water and feed ad libitum. all procedures involving animals in this study conformed to the guiding principles in the care and use of animals and the institution’s code of ethics for the use of laboratory animals. figure 1. animal cage. source: (university of uyo animal house). plant collection fresh leaves of eremomatax polysperma was obtained at the medicinal farm. they were identified and authenticated by the department of botany, university of uyo, nigeria. the plant material was chopped, dried, and pulverized into a coarse form. figure 2. fresh and dried eremomastax polysperma. plant extraction the maceration method was used for the plant extraction. 800g of the dried plant material was weighed using a beam balance. the weighed plant was then transferred into a glass extraction jar. 7.5l/ 750ml of 99.8% methanol in a glass extraction jar and kept at room temperature (25oc 32oc) for 72 hours (3 days). at the end of 72 hours, the extract was filtered and concentrated to dryness at 40oc using a rotary evaporator. the concentrated methanol extract was stored in a refrigerator at -4oc until further use. repeated graded dose administration study the extract was administered orally to the animals based on their body weight using a cannula. the administration was given daily to the rats for 14 days and the weight was taken at 48hours intervals. water was administered in the same manner to the fourth group and this served as a control. on day 15, the animals were made inactive by inhumane method. the animals were cut open and blood was collected from the heart using a needle and 5 ml syringe into ethylene diamine tetra acetic acid (edta) specimen bottles. all extracts dosage was determined after toxicity test (ld50). blood collection each animal was humanely sacrificed by chloroform inhalation. the rats were anesthetized using chloroform, dissected, and blood withdrawn from the heart through a technique called cardiac puncture, and the deoxygenated blood was drawn using a 5ml syringe and decanted into an edta bottle. acute toxicity study the acute toxicity of the extracts was evaluated according to the method of lorke (1983). phytochemical screening test for saponin five ml of water was added to the extract and shaken vigorously. it was then allowed to settle for 10-15 minutes. frothing was observed which therefore signifies the presence of saponin (milkyas et al., 2016). test for tannins five ml of water was added to the extracts and particles were filtered to remove the extracts. 2-3 drops of ferric chloride were then added to the filtrate. a blue-black precipitate was observed indicating the presence of tannins (milkyas et al., 2016). test for flavonoids five ml of water was added to the extracts and then placed in a water bath and boiled. after boiling it was removed and filtered. after filtration, the sample was separated into two portions and 2-3 drops of magnesium metal and conc. hcl was added to one portion. the color idung et al. – assessment of the phytochemical constituents of … 93 changes to orange indicating the presence of flavonoids (milkyas et al., 2016). test for alkaloid five ml of 5% hcl was added to the diluted extracts, then the sample was placed in a water bath and allowed to boil. after boiling the sample was filtered and separated into two portions. 2-3 drops of dragendoff reagent were added to one portion of the sample and kept overnight to determine the positivity or negativity of the sample. an orange ppt. was formed indicating the presence of an alkaloid (milkyas et al., 2016). test for cardiac glycoside ▪ salkowski’s test a few amounts of extract were dissolved in 5 ml of dichloromethane. 1 ml of concentrated h2so4 was then added gently by running it down the side of the test tube. a reddish-brown was observed indicating the presence of salkowski’s (cardiac glycoside) (panchal & parvez, 2019). ▪ killer-killiani test the extract was dissolved in glacial acetic acid containing drops of ferric chloride solution. the mixed solution was filtered to remove the particle. 1ml of euphoric acid was added openly. a brown ring forms at the interface indicating the presence of cardiac glycoside (panchal & parvez, 2019). ▪ lieberman’s test the extract was dissolved in acetic anhydride and cooled in ice. sulphuric acid (h2so4) was added to the solution. the color was observed to change from green to blue and brown indicating the presence of a steroidal nucleus (kumar et al., 2014). test for anthraquinones ▪ free anthraquinones 0.2g of external was diluted in 5 ml of toluene. the solution was then filtered. 2 ml of 10% ammonia (nh4oh) was added to the filtered solution. there was no colour change at the interphase indicating the absence of anthraquinone (kumar et al., 2014). ▪ combined anthraquinones 0.2g of extract was diluted in 5 ml of concentration h2so. the solution was then boiled and filtered. 2ml of toluene was added to the filter and shaken. 1 ml of ammonia solution (nh4oh) was added to the toluene layer and shaken. there was no colour change at the interphase indicating the absence of anthraquinone. determination of hematological parameters blood was collected from the left ventricle of each animal in a vial containing 0.5 m edta. hematological parameters were determined after day 14 of treatment using an automated mindray bc-5300 hematology analyzer made in china at the university of uyo teaching hospital. statistical analysis the data obtained from the study were subjected to statistical analysis using international business |machine of statistical package for social science (ibm spss version 25). data were presented as mean±standard deviation. anova test was used as an inferential statistics and probability less than 0.05 was considered statistically significant. results and discussion acute toxicity study acute toxicity test did not show any mortality, morbidity, or other apparent signs of toxicity at the doses used which indicates that the extract is not toxic at the maximum dose of 5000 mg/kg bw. with this in mind, 1/10th, 2/10th, and 3/10th of this maximum dose (5000 mg/kg) was adopted for the study which gave rise to 500, 1000, and 1500 mg/kg doses of the extract used in the treatment groups. the ld50 5000mg/kg was deduced from the anex 2d oecd guidelines. figure 3. anex 2d guideline. source: (oecd 2001) 94 biology, medicine, & natural product chemistry 13 (1), 2024: 91-98 phytochemical screening the phytochemical screening of crude methanolic extracts of leaf eremomatax polysperma revealed the presence of some secondary metabolites such as alkaloids, saponin, flavonoid, and cardiac glycosides. table 1. phytochemical screening of leaf of eremomatax polysperma. test interferences extract alkaloids + saponin + tannis + flavoniods + cardiac salkowski’s test + killer-killiani test + lieberman;s test + anthraquinones free anthraquinones combine anthraquinones + indicate presence – indicate absence body weight body weights of albino rats from both the control and treated groups increased progressively throughout the experimental period as shown below; table 2. average weight of the albino rat per treatment. day treatment 1 2 3 4 5 6 7 8 weight(g) weight(g) weight(g) weight(g) weight(g) weight(g) weight(g) weight(g) control 98 ± 5.48 102.75±5.22 112.25±8.26 106±5.69 112.5±6.85 105.5±6.51 116.75±7.08 118.5±7.67 500mg/kg 94 ± 4.64 109 ± 4.20 107.75±4.09 106.75±4.10 110.25±5.72 106.25±4.71 121.25±6.16 121.75±6.20 1000mg/kg 85 ± 3.76 96.25±4.27 96 ± 4.60 95±4.04 98±5.10 93.5±5.04 107±6.35 110.5±6.41 1500mg/kg 91.75±8.25 102.5± 5.62 104.5±5.95 100.25±5.68 119.5±6.28 116.5±6.41 124.25±6.60 127.75±7.02 figure 4. average weight of the albino rat per treatment. hematological parameters the provided table 3 presents the results of a study investigating the effects of different doses (mg/kg) of eremomastax polysperma on various blood parameters. the statistical test employed was anova, with a predetermined significance level of p<0.05 and p<0.01 while the non-significant differences level of p>0.05. the anova analysis revealed statistically significant differences in rbc and hgb, among the control group and the treatment group of 500mg/kg (p<0.05) and also at hct and the treatment group 500mg/kg (p<0.01). however, no significant differences were observed for wbc, mcv, lym, gran, and mid percentages. these findings suggest that the administration of 500mg/kg doses of the substance or treatment may have specific effects on the red blood cells and hemoglobin of the rat. idung et al. – assessment of the phytochemical constituents of … 95 table 3. hamatological parameters of control and groups treated with graded doses of methanol leaves extract of eremomastax polysperma. dose mg/kg wbc (103/µl) lym (%) gran (%) mid (%) lym (103/µl) gran (103/µl) mid (103/µl) rbc (106/µl) hgb g/dl hct (%) mcv fl control 9.53 ±1.11 87.2 ±0.97 3.38 ± 0. 60 9.48 ± 0.42 8.33 ± 1.06 0.30± 0.03 0.90 ± 0.08 7.08 ± 0.10 14.4 ± 0.24 46.1 ± 0.74 65.10 ± 0.77 500mg/kg 9.61 ± 0.41 87.6 ± 0.80 4.18 ± 0.69 8.23± 0.35 8.41 ± 0.30 0.41±0.09 0.79 ± 0.05 6.32 ±0.19a 12.9 ±0.24a 42.7±0.52b 67.7 ± 1.24 1000mg/kg 10.49 ± 1.62 79.9 ± 4.92 9.4 ± 3.53 10.7 ± 1.06 8.4 ± 1.48 0.96 ± 0.35 0.87 ± 0.33 6.75 ± 0.08 13.48 ± 0.24 44.3 ± 0.39 65.5 ± 0.62 1500mg/kg 8.71 ± 0.87 84.33 ± 1.11 6.1 ± 0.98 9.63 ± 0.66 7.36 ± 0.75 0.53 ± 0.12 0.83 ± 0.07 6.55 ± 0.24 13.3 ± 0.37 44.95 ± 0.39 68.65 ± 1.92 wbc (white blood cell count), lym (lymphocytes), gran (granulocytes), mid (mid-range cells), rbc (red blood cell count), hgb (hemoglobin), hct (hematocrit), and mcv (mean corpuscular volume) table 4. hematological parameters of control and groups treated with graded doses of methanol leaf extract of eremomastax polysperma. dose mg/kg mch pg mchc g/dl rdw-cv (%) rdw-sd fl plt (103/µl) mpv fl pdw fl pct (%) p-lcr (%) p-lcc (109/l) control 20.40± 0.25 31.3 ± 0.36 17.1 ± 0.37 44.5 ± 1.13 825 ±32.28 8.10 ±0.19 10.18 ± 0.45 0.664 ± 0.03 14.70 ± 1.35 121 ± 11.59 500mg/kg 20.43± 0.23 30.2±0.21 16.3± 0.43 44.2± 1. 10 794 ± 93.42 7.85 ± .27 9.48 ± 0.40 0.616 ± 0.05 13.58 ± 1.76 103.3 4.44 1000mg/kg 19.93±0.25 30.4±0.30 16.68±0.23 43.7±0.31 862.3±11.25 7.90±0.18 9.8±0.36 0.680±0.01 13.7±1.05 117.30±0.78 1500mg/kg 20.30±0.57 29.5±0.62a 16.68±0.27 46.10±1.48 978±44.96 7.93±0.19 9.53±0.34 0.759±0.04 13.83±1.00 132.30±10.84 mean corpuscular hemoglobin (mch), mean corpuscular hemoglobin concentration (mchc), red cell distribution width (rdw-cv and rdw-sd), platelet count (plt), mean platelet volume (mpv), platelet distribution width (pdw), platelet crit (pct), platelet large cell ratio (p-lcr), and platelet large cell count (p-lcc) 96 biology, medicine, & natural product chemistry 13 (1), 2024: 91-98 the hematological parameters of treated and control rats are presented in table 4. the anova revealed statistically significant differences in mchc among the control group and the treatment group of 1500mg/kg(p<0.05). no significant differences (p>0.05) were observed in mean corpuscular hemoglobin (mch) and red cell distribution width and other parameters including the red cell distribution width (rdw-cv and rdw-sd), platelet count (plt), mean platelet volume (mpv), platelet distribution width (pdw), platelet crit (pct), platelet large cell ratio (p-lcr), and platelet large cell count (p-lcc), did not exhibit significant differences between the control and treatment groups. these findings suggest that the methanolic leaf extract of eremomastax polysperma has a selective effect on mean corpuscular hemoglobin concentration (mchc) at 1500mg/kg without affecting other hematological parameters measured in this study. discussion the crude methanolic extracts of eremomastax polysperma leaves were subjected to phytochemical screening, which revealed the presence of various secondary metabolites such as alkaloids, flavonoids, cardiac glycosides, and tannins (table 1). these phytochemical compounds have significant medicinal importance. alkaloids, for instance, have been known for their potent toxic properties, and several alkaloids derived from medicinal plants exhibit various biological activities, including anti-inflammatory, antimalarial, antimicrobial, cytotoxic, antispasmodic, and pharmacological effects (augusto et al., 2011; dua et al., 2013; benbott et al., 2012; ameyaw and duker-eshun, 2009; thite et al., 2013). tannins, as reported by previous research, possess antibacterial, antitumor, and antiviral activities (kumari & jain, 2012). they exert their effects by precipitating microbial proteins, rendering nutritional proteins unavailable to the microbes. cardiac glycosides, another group of phytochemicals identified in the extract, have been utilized in the treatment of congestive heart failure and cardiac arrhythmia (vladimir and ludmila, 2001). these compounds operate by inhibiting the na+/k+ pump, thereby increasing the levels of calcium ions available for the contraction of heart muscles. this leads to the recovery of cardiac output and a reduction in heart distension (banso and adeyemo, 2006; aiyelaagbe and osamudiamen, 2009). the presence of these phytochemical compounds in the leaf extracts of eremomastax polysperma may account for the observed biological activities and justify its use as a traditional medicine by the ibibio tribe and throughout akwa ibom state. specifically, the alkaloids found in the leaf extract exhibit bioactivity against gram-positive bacteria and demonstrate cytotoxic effects against leukemia and hela cell lines. flavonoids play a crucial role as free radical scavengers and primary antioxidants. therefore, it is important to investigate the presence of phenolic compounds in plant extracts. polyphenolic compounds possess an aromatic benzene ring with substituted hydroxyl groups, and they can absorb free radicals and chelate metal ions that could catalyze the formation of reactive oxygen species (ros) and lipid peroxidation. flavonoids, in particular, are highly significant in promoting human health and disease prevention. the antioxidant potency of flavonoids depends on their molecular structures, including the position of hydroxyl groups and other chemical features (rajanandh and kavitha, 2010). these compounds are commonly found in plants in the form of glycosides, with quercetin being one of the most abundant flavonoids known for its excellent antioxidant properties due to its favorable structural characteristics (kalita et al., 2013). assessment of hematological parameters is a valuable approach for evaluating the harmful effects of toxic agents (agbaje et al., 2009) and determining an individual's health status (burtis et al., 2012). changes in the hematological system can provide insights into the prediction of toxicity in animals (olson et al., 2000), and alterations in the concentrations of red blood cells (rbc), hemoglobin (hgb), mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch) and mean corpuscular hemoglobin concentration (mchc) are particularly important for diagnosing anemia. these parameters not only indicate the potentially harmful effects of herbal remedies but also shed light on their impact on blood-related conditions. in the study, administration of the plant extract did result in significant changes in rbc, hct, and hgb levels compared to the control group. this suggests that the extract is likely to cause abnormalities in hematological parameters, such as bleeding, anemia, or bone marrow suppression, in humans. the significant change in rbc (erythrocyte count) was confirmed by the increased percentage of hemoglobin in the 500mg/kgdose recipient group. in normal circumstances, local tissue anoxia leads to the formation of a glycoprotein called erythropoietin, which stimulates increased production of erythrocytes (bowman & rand, 1980). ereromastax polysperma leaf extract likely contains erythropoietin-like agent(s) which is/are responsible for the increased production of erythrocytes. this finding did not align with the report by arsad et al., (2013), indicating the relatively unsafe nature of the plant extract in terms of hematological effects. table 3 shows that there were no significant changes in the white blood cell (wbc) count. wbcs, along with their differentials such as lymphocytes, serve as indicators of the body's response to toxic substances, idung et al. – assessment of the phytochemical constituents of … 97 including plants (adedapo et al., 2004). wbcs and lymphocytes play a crucial role in the body's defense mechanism. the wbc counts in both the control group and the groups treated with doses of 500 mg/kg and 1000 mg/kg body weight exceeded the normal physiological range of 5.00-8.96/mm3 (mitruka et al., 1977). the percentage values for lymphocytes in the treatment groups were within the normal physiological range of 65.00-84.50%, except for the control and 500 mg/kg group. table 4 shows that there was a significant decrease in mchc at 1500mg/kgdose. the most common complication of living with low mchc levels is a lack of energy and decreased stamina. this can limit activities. in severe cases, anemic hypoxia can occur as a result of low mchc levels. when mchc levels are very low, the body could struggle to provide enough oxygen to all its tissues. this therefore signifies that the extract of eremomastax polysperma at a higher dose is not good for the body. this study was not in line with the research of ramadan & alshamrani (2015) who had no significant difference in mchc. conclusion the acute toxicity test did not show any mortality, morbidity, or other apparent signs of toxicity at the doses used which indicates that the extract is not toxic at the maximum dose of 5000 mg/kg bw. with this in mind, 1/10th, 2/10th, and 3/10th of the maximum dose (5000 mg/kg) was adopted for the study which gave rise to 500, 1000, and 1500 mg/kg doses of the extract used in the treatment groups. from the result, the body weights of experimental animals 1/14*5000 from both the control and treated groups increased progressively throughout the experimental period. also, phytochemical screening of the extracts revealed the presence of alkaloids, tannins, saponins, flavonoids, and, cardiac glycosides. the hematological evaluation revealed that the administration of the plant extract brought about a significant difference in rbc, hgb, and hct. also, mean corpuscular hemoglobin concentration (mchc) showed a significant decrease with increasing doses of the extract, indicating an adverse effect on the body. other parameters, including wbc, mcv, plt, mpv, mch, lym, gran, mid, rdw-cv, rdw-sd, plt, pdw, pct, p-lcr, and p-lcc did not exhibit significant differences between the control and treatment groups. this suggests that the extract is likely to cause abnormalities in hematological parameters, such as bleeding, anemia, or bone marrow suppression in humans. recommendation based on the findings of this study, the following recommendations can be made: ▪ 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(2013a). upgrading the medicinal value chain of neglected and underutilized eremomastax (lindau.) species through antioxidant health benefits. international journal of medicinal and aromatic plants, 3(3): 334-342. vladimir, k., ludmila, m., (2001). glycosides in medicine: "the role of glycosidic residue inbiological activity". curr. med. chem. 8, 1303-1328 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0105 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0105 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0105 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0105 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0105 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0155 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0155 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0155 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0155 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0205 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0205 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0205 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0205 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0205 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0205 http://refhub.elsevier.com/s1018-3647(15)00009-9/h0205 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 663-671 | doi: 10.14421/biomedich.2025.142.663-671 issn 2540-9328 (online) stability test and antioxidant activity test of sheet mask preparation of avocado peel (persea americana mill) ethanol extract with dpph (2,2-diphenyl-1-pikrilhydrazil) method uswatun khasanah1, yetti hariningsih1,*, syamsul ma'arif1, hery prambudi2 1bachelor of pharmacy study program, faculty of health, an nasher university. 2diploma iii study program in medical laboratory technology, faculty of health science, an nasher university. jl. pondok pesantren tarbiyatul banin kaliwadas village, sumber district, cirebon regency, west java 45611, indonesia. corresponding author* yetti.hariningsih@gmail.com manuscript received: 10 july, 2025. revision accepted: 04 september, 2025. published: 01 october, 2025. abstract the avocado plant (persea americana mill) has various benefits, one of which is in the fruit’s skin which contains flavonoid compounds as antioxidants. this antioxidant compound has the potential to be used as an active ingredient in various cosmetic preparations, including sheet masks. this study aims to determine the physical quality, stability, and antioxidant activity of avocado peel ethanol extract formulated in sheet mask preparation. this study uses a quantitative method with an experimental design, and applies a complete random design (ral) consisting of three variations of extract concentration, namely 2%, 4%, and 6%. physical quality evaluation includes organoleptic, homogeneity, ph, adhesion, and viscosity, as well as stability tests using the cycling test method for six cycles. antioxidant activity was tested using the dpph method to determine the ic₅₀ value. the results show that all formulas meet good physical quality. the 6% formula has the best stability with a ph of 5.79±0.062, an adhesion of 3.60 ± 0.130 seconds, and a viscosity of 9490.67 ± 27.57 cp. statistical tests showed no significant changes in ph (p = 0.385), adhesion (p = 0.833), and viscosity (p = 1.000). the antioxidant activity test showed that sheet mask with a concentration of 6% had an ic₅₀ value of 198.11 ppm and was classified as having weak antioxidant activity, while the avocado peel ethanol extract had an ic₅₀ value of 53.11 ppm and was classified as having strong antioxidant activity. so it can be concluded that sheet mask preparations have good physical quality and are stable during storage, but the antioxidant activity of avocado peel extract is higher than that of sheet mask preparations. keywords: stability; antioxidant activity; sheet mask; avocado peel. introduction for a long time, indonesian people have used natural ingredients for medicine and cosmetics because they are considered safer than chemicals (ramadhania et al., 2018). one of the causes of skin damage is free radicals from sun exposure. antioxidants, both natural and synthetic, can prevent such damage, but natural antioxidants are more effective especially in topical preparations (rompis et al., 2019). one of the sources of antioxidants that comes from nature is avocado peel (persea americana mill) (rahmi & nurman, 2021). according to research by vinha et al. (2013) the antioxidant activity in avocado peel reaches 35%, higher than the pulp of the fruit which is only 23%. this is due to the higher content of carotenoids, phenolic compounds, and flavonoids in avocado peels compared to those found in the pulp. the skin has an average of 679.0±117.0 mg of total phenolics, 44.3±3.1 mg of flavonoids, 2,585±0.117 mg of carotenoids, 4.1±2.7 mg of vitamin c and 2.13±1.03 mg of vitamin e. meanwhile, the flesh of the fruit has an average of 410.2±69.0 mg of total phenolics, 21.9±1.0 mg of flavonoids, 0.815±0.201 mg of carotenoids, 1.2±0.7 mg of vitamin c and 5.36±1.77 mg of vitamin e. flavonoids are powerful antioxidants and are also thought to be able to prevent the harmful effects of uv rays or at least reduce them. skin damage (efriana, 2019). one popular form of skincare is sheet masks, as they are easy to use, able to retain moisture, rejuvenate the skin, and help prevent dark spots (nilforoushzadeh et al., 2018). as one of the cosmetic products that is applied directly to the skin, sheet mask it requires high stability under various conditions to ensure its safety during its storage and use. the stability of cosmetic products is important for the product to remain safe and effective. in addition, the content of active compounds in sheet mask, such as antioxidants need to be tested to ensure their efficacy matches the claims given. therefore, it is necessary to test the safety and stability of sheet mask preparations containing avocado peel ethanol extract (persea americana mill.) to ensure that the product is of https://doi.org/10.14421/biomedich.2025.142.663-671 664 biology, medicine, & natural product chemistry 14 (2), 2025: 663-671 good quality and provides optimal benefits. this study aims to determine the physical stability and antioxidant activity of preparations using the dpph method, as well as evaluate physical quality through organoleptic, homogeneity, ph, and viscosity tests to ensure product quality and consistency during storage. materials and methods materials this type of research is experimental research. this research was carried out in february – march 2025. the research was carried out at the pharmacogenetics laboratory and pharmacognosy laboratory of an nasher university, the instrument laboratory of ahmad dahlan university cirebon and the mathematics and natural sciences laboratory of the faculty of tarbiyah and teacher training of uin siber syech nurjati cirebon. the tools used in this study are laboratory glasses, porcelain cups, handscoons, hot plates, filter cloths, parchment paper, ovens, ph meters, rotary evaporators, analytical scales, mortars and stempers, uvvis spectrophotometry, brookfield viscometers, foil bags, empty sheet masks, plastic pots. the ingredients used in this study are avocado peel, glycerin propylene glycol, peg-40 hydrogenated castor oil, xanthan gum, nipagin, 70% ethanol, perfume, and aquadest. methods sample preparation the fresh avocado peel is weighed and then cleaned with running water. after that, cut into small pieces to speed up drying. drying is done in an oven at 50°c for 2–3 days until it dries evenly. the dried skin is then blended into a powder. extraction is carried out by the maceration method. simplicia of 800 grams of dried avocado peel is put into a maceration vessel, then 6000 ml of 96% ethanol is added (ratio 1:7.5). the mixture is left to rest for 3 days in a cool, dark place, stirring occasionally. after that, it is filtered and the pulp is remacerated with 2000 ml of 96% ethanol for 2 days with constant stirring. the results of the remaseration are filtered and combined with the first extract, then evaporated using a rotary evaporator until a thick extract is obtained (jayustin & fratama, 2018). phytochemical screening 1. qualitative analysis of phytochemicals a. flavonoid test 0.3 g of extract is added with hot water, boiled for 10 minutes and then filtered. 5 ml of filtrate is added to 0.05 mg of mg powder and 6-7 drops of concentrated hcl. a positive test is indicated by the formation of a brown color towards red, yellow, or orange b. tannin test 1 ml of extract is added to a few drops of fecl₃ 3%. if there is a change in color from cloudy brown to black, it indicates the presence of tannins c. saponin test 2–3 ml of extract is added to 10 ml of hot water, then cooled. after that, it is whipped for ±10 seconds and 1 drop of hcl 2n. positive results are indicated by a stable foam 1–10 cm high for 10 minutes d. alkaloid test 0.1gram of the sample was extracted with 5 ml ki and 5 ml glacial ch3cooh added. then, 10 drops of the mixture was added to the dragendorff reagent. a positive result will form a deposit. 2. identification of phytochemical compounds with thin-layer chromatography a. klt silica gel g60 f254 plate is activated by being ovened at 40ºc for 30 minutes to remove water. b. quercetin samples and comparators were rejected on the klt plate using a micro pipette. the extract is repelled in parallel from the lower edge of the plate with a distance between the 2 points of the center of retonation of not less than 10 mm. c. the phase of motion used to identify flavonoids is ethyl acetate: methanol (3:2) d. chromatogram detection with uv-vis lamp at 254 nm wavelength. determine the price of the rf spot with the following formula: 𝑅𝐹 = 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑡ℎ𝑒 𝑝𝑎𝑡ℎ 𝑡𝑎𝑘𝑒𝑛 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 formulation of sheet mask preparations table 1. formulation of the preparation sheet mask (efriana, 2019). no material f0 f1 f2 f3 1. avocado peel extract 2 4 6 2. glycerine 5 5 5 5 3. propylene glycol 5 5 5 5 4. peg hydrogenate castor oil 0,5 0,5 0,5 0,5 5. xanthan gum 0,3 0,3 0,3 0,3 6. nipagin 0,18 0,18 0,18 0,18 7. ethanol 70% 3 3 3 3 8. perfume q.s q.s q.s q.s 9. aquadest ad 100 ml 100 ml 100 ml 100 ml information: formulation 0 : not given extract formulation 1 : avocado peel extract 2% formulation 2 : avocado peel extract 4% formulation 3 : avocado peel extract 6% preparation of sheet mask xanthan gum is dissolved in aquadest, then propylene glycol and glycerin are added, then grinded until homogeneous (mixture i). nipagin is dissolved in hot khasanah et al. – stability test and antioxidant activity test of sheet mask preparation 665 water (mixture ii). avocado peel extract and peg-40 hydrogenated castor oil are dissolved in aquadest (mixture iii). mixture ii is slowly added to mixture i while grinding until it forms a homogeneous mass, then mixture iii is added and stirred until homogeneous. after that, 70% ethanol and 3 drops of perfume are added, then stir again until evenly distributed. the sheet mask is folded to size and put in a foil bag. a total of 20 ml of formulation is poured into a foil bag, then sealed and labeled. evaluation of the physical quality of the preparation 1. organoleptic test it is done by observing the parameters of smell, shape, and color through the observation of the five senses. 2. homogeneity test 1 gram of sheet mask preparation formulation is applied to the glass surface of the glass object, then covered or glued with the glass of another glass object. the preparation is well expressed when the surface is homogeneous and does not show the presence of coarse particles on the glass surface. 3. measurement of ph 1 gram of sheet mask preparation, dissolved in aquadest 100 ml (1%), then use a ph meter to measure the ph of the solution. 4. adhesion test formulation of a 0.2 g sheet mask that is placed between two glass glasses. on top of the glass, the object was given a weight of 1 kg which was left for 5 minutes. after that, the load is taken and then the time is recorded until the two objects can be removed. the requirement of good adhesion is more than 1 second. 5. viscosity a total of 30 gr of preparation is put in a container, then install a spindle. viscosity testing is carried out at a speed of 30 rpm at room temperature. the viscosity can be read on the viscosity monitor screen. stability test the stability test uses the cycling test method, namely, storing the sheet mask preparation in the refrigerator at a temperature of 4°c for 24 hours. then transfer the sheet mask preparation to an oven at 40°c for 24 hours, the process is counted as one cycle. repeat the step for up to 6 cycles. observe the physical changes of the sheet mask preparation each cycle, such as organoleptic, homogeneity, ph, adhesion, and viscosity before and after the cycling test. antioxidant activity test extract and sheet mask preparation 1. manufacturing of dpph stock solution 10 mg of dpph powder was dissolved in ethanol p.a 100 ml using a measuring flask (100 ppm). 2. determination of maximum wavelength a 20 ppm solution is prepared from 10 ml of diluted stock solution to 50 ml with p.a ethanol, then incubated for 30 minutes at room temperature without light. absorbance is measured at 500–600 nm to determine λmax. 3. extract antioxidant activity test avocado peel ethanol extract as much as 100 mg is dissolved in ethanol p.a 100 ml (1000 ppm parent solution). this solution is then vorvortex for 1 minute and diluted into a series ocean, which are 20, 40, 60, and 80 ppm. each series solution concentration was then added with 2 ml of dpph solution in a 10ml measuring flask, then ethanol p.a was added to the limit mark. the mixture was incubated for 30 minutes at room temperature in dark conditions, then its absorbance was measured at the λmax that had been obtained. the value of antioxidant activity is calculated based on the percentage of inhibition using the formula: % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝑏𝑙𝑎𝑛𝑘 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 − 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑏𝑙𝑎𝑛𝑘 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑥 100 % the % inhibition result data from each concentration were used to create a calibration curve (concentration vs. % inhibition), and then linear regression analysis was performed to determine the value of ic₅₀, which is the concentration that can inhibit free radicals by 50%. which is then entered into the equation that has been obtained from the calibration curve y = a+ bx, where the value of y is 50 and the value of x indicates ic50. 4. antioxidant activity test sheet mask the antioxidant activity test on sheet mask preparations is carried out after going through a physical stability evaluation process (cycling test method) and using only the best formulations. a total of 100 mg of sheet mask preparation, dissolved in ethanol p.a up to 100 ml (concentration of 1000 ppm). divortex for 1 minute and diluted into series of solution concentrations of 20, 40, 60, and 80 ppm. each of the series solution concentrations was then added 2 ml of dpph solution, diluted to 10 ml, incubated for 30 minutes, and measured for absorbance at λmax. antioxidant activity is calculated based on the percent value of inhibition and the value of ic₅₀ obtained from the linear regression equation, similar to the treatment of the extract. 5. comparative test using vitamin c vitamin c 100 mg is dissolved in ethanol p.a up to 100 ml (parent solution 1000 ppm). this solution is diluted to concentrations of 2, 4, 6, and 8 ppm. each concentration of the series solution was then added 2 ml of dpph solution, diluted to 10 ml, incubated for 30 minutes, and then measured for absorbance. the % 666 biology, medicine, & natural product chemistry 14 (2), 2025: 663-671 inhibition and ic₅₀ value of vitamin c were calculated by the same procedure as the extract, so that the results could be compared directly. data analysis based on the type of research, data collection uses laboratory experiment methods. the data obtained was in the form of data on the stability of sheet mask preparations including adhesion, viscosity and ph, data were analyzed through normality tests using shapirowilk and homogeneity and analyzed using anova (analysis of varience) tests. then it was followed by the tukey and lsd test. if the data is distributed normally, a statistical test is carried out, namely the one way anova test, and if the data is not distributed normally, the kruskal-wallis test is chosen. if the one way annova or kruskal-wallis test produces a p < value of 0.05, it is followed by the mann-whitney test. results and discussion result extraction results the results of the extraction test can be seen in table 1. table 1. extraction results. simplisia powder (g) thick extract (g) yields (%) referral results 800 95 11,27 >10% phytochemical screening results the results of the extraction test can be seen in table 2. table 2. phytochemical screening. test result information flavonoid a change in the color of brick red or orange + tannin blackish-green in color + saponin foam formed + alkaloid red to orange in color and there is precipitation + the results of the phytochemical compound identification test with thin-layer chromatography can be seen in table 3. table 3. identification of phytochemical compounds with klt. elaine rf value information ethyl acetate: methanol (3:2) quercetin avocado peel extract 0.7 0.7 + results of evaluation of physical quality of supplies evaluation of the preparation of sheet mask of avocado peel ethanol extract (persea americana mill) which includes the average results of organoleptic tests, homogeneity tests, ph tests, adhesion tests, and viscosity tests. the results of the physical quality evaluation test of the preparation can be seen in table 4. table 4. evaluation of physical quality of supplies. formulation organoleptic test kf1 f2 f3 k+ color clear white yellowish brown greenish brown greenish brown clear white smell typical oleum rosae typical oleum rosae typical oleum rosae typical oleum rosae fresh smell texture slightly viscous liquid slightly viscous liquid slightly viscous liquid slightly thick slightly thick homogeneity homogeneous homogeneous homogeneous homogeneous homogeneous ph 6.02±0.109 5.94±0.151 5.86±0.134 5.82±0.044 6.06±0.089 adhesion 2.96±0.684 2.80±0.319 2.80±0.427 2.76±0.263 3.25±0.161 viscosity 12040±54,772 4660± 54,772 5300±70,710 8120±83,666 6600±141,421 stability test results the results of the stability test can be seen in table 5. table 5. stability test. test formulation the th cycle x̄±sd sig. 1 2 3 4 5 6 color kpb pb pb pb pb pb f1 ck ck ck cksp cksp cksp f2 ch ch ch ch c c f3 ch ch ch ch ch c k+ pb pb pb pb pb pb smell kor or or or or or khasanah et al. – stability test and antioxidant activity test of sheet mask preparation 667 test formulation the th cycle x̄±sd sig. 1 2 3 4 5 6 f1 or or or or or or f2 or or or or or or f3 or or or or or or k+ s s s s s s texture kcsk csk csk csk csk csk f1 csk csk csk csk csk csk f2 csk csk csk csk csk csk f3 sk sk sk sk sk sk k+ sk sk sk sk sk sk homogeneity kh h h h h h f1 h h h g g g f2 h h h h g g f3 h h h h h h k+ h h h h h h ph k6.1 6.1 6.1 6,1 5.97 5.91 6.04±0.084 0.38 5 f1 5.95 5.94 5.94 5,88 5.85 5.80 5.89±0.060 f2 5.89 5.87 5.84 5,84 5.79 5.79 5.83±0.040 f3 5.85 5.85 5.83 5,82 5.73 5.71 5.79±0.062 k+ 6.1 6.1 6.1 6.1 6.07 5.95 6.07±0.06 adhesion k3.49 3.35 4.21 3.62 3.54 3.46 3.61±0.306 0.83 3 f1 2.80 2.84 3.09 2.56 2.21 2.18 2.61±0.365 f2 2.98 3.16 3.16 3.16 3.30 3.33 3.18±0.124 f3 3.36 3.58 3.68 3.68 3.72 3.62 3.60±0.130 k+ 3.54 4.35 3.71 3.71 3.46 3.36 3.68±0.352 viscosity k12160 12200 12180 12260 11780 12240 12136.67±178.624 1.00 0 f1 5360 5360 5380 5360 5360 5360 5363.67±8.16496 f2 6760 6740 6680 6740 6660 6760 6723.67±42.7395 f3 9460 9480 9480 9480 9500 9540 9490.67±27.5680 f+ 6700 6660 6680 6720 6620 6760 6690.67±48.5798 information: pb : clear white or : oleum rosae ck : yellowish brown s : fresh smell ch : greenish brown csk : slightly viscous liquid cksp : slightly faded yellowish brown sk: slightly thick c : chocolate h : homogeneous g : there are lumps results of antioxidant activity test extracts and sheet mask preparations the results of the antioxidant activity test can be seen in table 6. table 6. test the antioxidant activity of the extract and sheet mask preparation. sample concentration ppm blank abs abs sample % inhibition ic50 antioxidant category extract 20 0.725 0.527 27.31 53.11 strong 40 0.428 40.96 60 0.290 60 80 0.263 63.72 sheet mask f3 20 0.499 31.17 198.11 very weak 40 0.476 34.34 60 0.467 35.58 80 0.452 37.65 vitamin c 2 0.711 1.93 6.27 very strong 4 0.517 28.68 6 0.350 51.72 8 0.260 64.13 discussion sample preparation the extraction of avocado peel with ethanol solvent produces a yield of 11.27%, which is classified as a good yield category, because in theory, a yield above 10% indicates that the extraction process is quite efficient. according to saerang et al. (2023) a yield above 10% indicates the solvent's ability to extract bioactive 668 biology, medicine, & natural product chemistry 14 (2), 2025: 663-671 compounds optimally. ethanol as a polar solvent, effectively extracts polar to semi-polar compounds such as flavonoids, tannins, and phenolic compounds with antioxidant activity. high yields reflect the amount of secondary metabolites extracted. phytochemical screening the results of phytochemical screening showed that avocado peel extract contains secondary metabolite compounds such as alkaloids, flavonoids, tannins, and saponins. the results of the alkaloid test showed that the positive reaction was characterized by the formation of a red to orange color and the presence of deposits after the addition of the dragendorff reagent. these deposits arise because bismuth nitrate in the reagent is dissolved with hcl to prevent hydrolysis, keeping bi³⁺ ions stable in solution. avocado peel extract shows positive for flavonoid compounds, characterized by a change in the color of brick red or orange with mg and hcl powder reagents which cause a reduction in flavonoid chemical compounds in the sample so that a red reaction is formed which is characteristic of the presence of flavonoids. (kaempe, komansilan, rumondor, & maliangkay, 2023) the tannin test showed positive results on avocado peel extract which was characterized by a blackish-green discoloration after the addition of fecl₃ solution. this reaction occurs due to the interaction of fecl₃ with hydroxyl groups in tannin compounds. fecl₃ can produce a blackish-blue color, while condensed tannins tend to form a blackish-green color. the test results showed that avocado peel extract contained saponins, characterized by the formation of a stable foam for ±10 minutes. saponins have surface active properties because they contain polar and nonpolar groups, so they can produce foam when shaken with water. the presence of glycosides in saponins allows the formation of foam, which can then be hydrolyzed into glucose and other compounds (kaempe et al., 2023). the results of the thin layer chromatography (klt) test on avocado peel extract with the motion phase of ethyl acetate: methanol (3:2) showed an rf value of 0.7. this value is still in the ideal range of 0.2–0.8. according to natasa et al., (2021), the eluent with the same composition, namely ethyl acetate: methanol (3:2), provides the best separation result with an rf value of 0.80. the difference in the rf value produced in this study compared to natasa research et al., (2021) can be caused by several factors, including differences in the type and levels of flavonoid compounds contained in the samples, the level of polarity of the extract, the technique or accuracy in spotting, as well as environmental conditions such as temperature and humidity during the klt process. the rf value obtained is influenced by the polar proximity between the compounds in the sample and the phase of motion. the more polar they resemble, the more easily the compound will be carried away by the motion phase and produce a high rf value (natasa et al., 2021) evaluation of the physical quality of preparations the results of the negative control formulations, f1, f2, and f3 showed a distinctive odor of oleum rosae. the f1, f2, and f3 colors are generally greenish-brown, but f1 appears more yellowish-brown, while the negative controls are clear white. the difference is seen in the texture, where f1 and f2 have a more liquid texture than f3. this is due to the higher water content of f1 and f2, while f3 has a thicker texture due to less water content. the homogeneity test results show that all sheet mask preparations, negative control and f1, f2, and f3, are homogeneous because no lumps are found so that avocado peel extract sheet mask preparations can be applied to the skin. this shows that the sheet mask preparation made has a homogeneous arrangement according to the comparison of holika holika avocado essenence sheet masks. the homogeneity of the preparation affects the spread of the mask. the sheet mask must have a homogeneous mass and be free of clumps so that the active substance can be absorbed properly during use. the results of the ph test show that all preparations sheet mask have a ph within the normal range of the skin, which is between 4.5–8.0, according to the sni 164399-1996 standard (sa’dah, 2022). the ph values of x̄±sd for each formulation were k6.02±0.109, f1 5.94±0.151, f2 5.86±0.134, and f3 5.82±0.044. this value is proportional to the ph of holika holika comparison mask avocado essence sheet mask which is 6.06±0.089. thus, the ph value produced by all preparations is within the normal skin ph range, so it can be concluded that sheet mask preparations are safe to use. the adhesion test results showed that all formulations had a value of more than 1 second, thus meeting the set conditions (yusuf et al., 2017). the average value of adhesion was negative control 2.96±0.684 seconds, f1 2.80±0.319 seconds, f2 2.80±0.427 seconds, f3 2.76±0.263 seconds, and positive control 3.25±0.161 seconds. adhesion tends to be directly proportional to viscosity, where the higher the adhesion, the higher the viscosity also tends to be (rohmanah et al., 2024). the viscosity test results showed that all formulas had different viscosity values, but were still in the range in accordance with the sni 16-6070-1999 standard, which was 2000–50000 cp. the viscosity values of each formula were negative control 12040±54.77 cp, f1 4660±54.77 cp, f2 5300±70.71 cp, f3 8120±83.67 cp, and positive control 6600±141.42 cp. f3 has the highest viscosity. this shows that the higher the concentration of avocado peel extract, the viscosity of the preparation tends to increase because the amount of water in the formula is reduced due to the addition of the extract khasanah et al. – stability test and antioxidant activity test of sheet mask preparation 669 concentration, so that the preparation becomes thicker (pangesti & bakri, 2024). stability test the stability test in this study was carried out using the cycling test, i.e. a test that simulates alternating temperature changes between cold and hot. the preparation is stored at 4 °c for 24 hours, then at 40 °c for 24 hours. one cycle consists of these two temperature conditions and is carried out as many as 6 cycles (slamet et al., 2020). this test aims to see the stability of the preparation against changes in temperature and storage time. in the 1–6 cycle stability test, all negative control formulas, f1, f2, and f3 remain to show distinctive odors oleum rosae. the preparation forms of k-, f1, and f2 are rather viscous liquids, while f3 and k+ are slightly thicker. the change is only visible in the f1 and f2 colors from cycle 4 to 6 the colors fade. this degradation is caused by a lower concentration of extracts, so the pigment is more easily degraded. this is in line with the results of the research suryani et al., (2017) which states that the color of the preparation becomes more intense as the concentration of the extract increases, but it will still fade during storage. color fading can occur due to browning reactions meanwhile, according to candrakanti et al., (2024) color changes can also be affected by several other factors, such as the ph of the preparation and the length of storage time. the results of organoleptic observations in storage for 6 cycles, the formula f3 is physically relatively more stable, because there is no change in consistency or aroma, but in the 6th cycle there is a change in color to brown. homogeneity observations show that all preparations initially appear homogeneous, clear, and without lumps. until the third cycle, homogeneity is still maintained. however, in the 4th to 6th cycle, clots begin to appear, indicating physical instability. one of the causes is the change in temperature during the stability test process (cycling test), which involves alternating storage at low and high temperatures. high temperatures can cause particles in preparations to clump, material separation, or reactions between ingredients that degrade stability. according to efriana (2019), unstable temperatures can cause active ingredients, especially those derived from natural extracts such as avocado peels, to undergo degradation or reactions that make particles stick together and form clumps. the ph stability test was performed to see the ph stability of the preparation for 6 cycles (rohmani & kuncoro, 2019) . the results showed a small decrease in ph due to temperature and storage time, but still in accordance with the sni 16-4399-1996 standard, namely ph= 4.5-8.00 (sa'dah, 2022). a significance value of 0.385 (p < 0.05) indicates no significant ph difference during storage. adhesive adhesion of the preparation for 6 test cycles cycling test changes in the up and down due to storage temperature, this is the same as viscosity testing, the lower the viscosity of a preparation, the lower the adhesion the lower the adhesion (lumentut et al., 2020). however, the adhesion still meets the standard. the statistical test showed a significance value of 0.833 (p > 0.05), indicating no significant difference. it shows good adhesion stability. the viscosity is affected by the mixing process and the type of material. during the cycling test, the viscosity goes up and down due to storage at high and low temperatures. high temperatures will increase the distance between particles so that the force between particles will be reduced. the larger distance causes the viscosity to decrease (suryani et al., 2017). however, it is still within the limits of the sni 16-6070-1999 standard, which is 2000–50000 cp. the statistical test showed a significance value of 1,000 (p > 0.05), indicating no significant difference. the preparation is declared to be stable in viscosity. test the antioxidant activity of the extract and sheet mask preparation the maximum wavelength measurement results for dpph showed the maximum wavelength was 515 nm, slightly different from the theory (517 nm) due to differences in solvents and tool specifications. the absorbance obtained is 0.725 which meets the criteria set by amin et al., (2023), which is 0.2–0.8, so it is used for testing. this study tested antioxidant activity using the dpph method on avocado peel ethanol extract, vitamin c as a positive control, and avocado peel extract sheet mask preparation. the results showed that the extract ic₅₀ value of 53.11 ppm which is included in the category of strong antioxidants and vitamin c of 6.27 ppm which is categorized as a very strong antioxidant. when compared to the results of the study by ayuningdya et al. (2023), which obtained an ic₅₀ value of avocado peel extract of 16.24 ppm (very strong category), the results of this study showed slightly lower antioxidant activity. this difference can be caused by differences in raw materials, solvents, and extraction methods. this antioxidant activity is related to the content of active compounds such as flavonoids, alkaloids, phenols, saponins, tannins, triterpenoids, and quinones (rahmawati et al., 2022). antioxidant tests on f3 sheet mask preparations (6% extract) selected for the best stability based on previous stability tests resulted in an ic₅₀ value of 198.11 ppm which is classified as a weak antioxidant. decreased antioxidant activity from pure extracts to sheet mask preparations can be caused by the interaction of the extract with additives during the formulation process (malik et al., 2020). storage factors such as temperature, time, oxygen, light, and ph also affect the stability of antioxidant compounds (giuliana et al., 2015). in addition, the heating and storage processes 670 biology, medicine, & natural product chemistry 14 (2), 2025: 663-671 during the preparation that are not optimal can lead to the degradation of bioactive compounds, including avocado peel extracts that contain fatty acids such as octaneous acid and palmitic acid that are easily oxidized. poor packaging can also accelerate the breakdown of active compounds (ayuningdya et al., 2023). conclusions the physical quality of the formulation of sheet mask preparation of avocado peel ethanol extract (persea americana mill.) shows good results. the preparation has organoleptic, homogeneity, ph, adhesion, and viscosity that meet the physical quality standards of sheet mask preparations in accordance with sni 16 4399-1996. formulation 3 with a concentration of 6% has the best physical stability. based on the stability test, all parameters of the preparation were within the safe range, except for the organoleptic and homogeneity parameters that showed changes in the 4th to 6th cycles. statistical test values showed that the ph, viscosity, and adhesion parameters did not undergo significant changes, with p-values respectively: ph (p = 0.385), adhesion (p = 0.833), and viscosity (p = 1.000). based on anova analysis, if the p value is < 0.05, there is a significant change, while if p > 0.05, there is no significant change. therefore, it can be concluded that the ph, viscosity, and adhesion parameters of the preparation are stable during storage. avocado peel ethanol extract (persea americana mill) has antioxidant activity with an ic50 value of 53.11 ppm with a strong antioxidant category. meanwhile, the preparation of sheet mask of avocado peel ethanol extract (persea americana mill) with formulation 3 of 198.11 ppm has a weak antioxidant category. the standard vitamin c has antioxidant activities with an ic50 value of 6.27 ppm with a very strong antioxidant category. these results show that the antioxidant activity of pure avocado peel extract is higher compared to sheet mask preparations containing such extracts. acknowledgements: thank you to all parties who have helped in the completion of this research. competing interests: the authors declare that there are no competing interests. references amin, a., harizal, asjur, a. v., husain, f., artati, masta, n., sari, e. k. 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(2013). physicochemical parameters, phytochemical composition and antioxidant activity of the algarvian avocado (persea americana mill.). journal of agricultural science, 5(12), 100– 109. https://doi.org/10.5539/jas.v5n12p100 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 297-303 | doi: 10.14421/biomedich.2024.131.297-303 issn 2540-9328 (online) exploring allelochemical compositions in the roots of two varieties of anacardium occidentale (cashew) nnenna ejije okoronkwo*, jude chibuzor igwe, nkemakolam bright nwosu, precious onyinyechi chukwu, udoka ifunnaya chukwukere, perculiar c. ihedigbo pure and industrial chemistry department, faculty of physical science, abia state university uturu, abia state, 08035626322, nigeria. corresponding author* ne.okoronkwo@abiastateuniversity.edu.ng, nwosunkemakolambright@gmail.com manuscript received: 27 may, 2024. revision accepted: 14 july, 2024. published: 26 august, 2024. abstract allelochemicals are chemicals released by plants that affect other plants and pests. the chemicals are provided by different parts of the plant or released through the natural decomposition of the associated plants. anacardium occidentale has been reported to exhibit allelopathy. the bark and inner parts of the roots of the two varieties (red and yellow) of anacardium occidental (linn) were qualitatively and quantitatively analysed for the presence of allelochemicals. the samples were also subjected to gas chromatography-mass spectrometry (gc/ms) analysis. the qualitative results showed that flavonoids, tannins, and phenols were present in both the bark and inner parts of the roots, whereas alkaloids, saponins, and terpenoids were present only in the bark of the roots. the quantitative results showed that while the root bark of the red variety had the highest saponin content, that of the yellow variety had the highest phenol content. the gc/ms results for both samples showed the presence of different compounds, and one of the compound peaks observed in the inner root was not identified; 9,12– octadecadienoic acid (fatty acid) was the compound with the highest composition identified in the yellow variety. similar compounds were identified in the root bark of the red and yellow varieties, which include 3-tridecyl phenol with molecular formula c19h32o and molecular weight 276, which occurred at a retention time range of 41.703–41.712 min and had the lowest percentage compositions of 2.816 and 4.732% in red and yellow varieties, respectively. the compounds with the highest percentage compositions of 32.389 and 41.944% in red and yellow, respectively, were identified as (z)-3-(heptadec-10-en-1-yl) phenol (molecular formula c23h38o with molecular weight 330), occurring within the retention time range of 48.246 – 48.263 min. other compounds with higher percentage compositions were identified in the samples, including (z)-3-(pentadec-8-en-1-yl) phenol and 3((4z,7z)-heptadeca-4,7-dien-1-yl) phenol. however, 1,2,4,-benzenetriol (molecular formula c6h6o with molecular weight 252) was only identified in the red variety, with a percentage composition of 8.883%, which occurred at a retention time of 22.040 min. the compounds identified in the bark were mostly phenols, whereas those in the inner roots were more fatty acids. keywords: alleochemical; alleopthathy; quantitative phytochrmicals; anacardium occidental gcms. abbreviations: gas chromatography mass spectroscopy (gc-ms) introduction allelochemicals are chemicals produced and released by plants that affect other plants and pests. these chemicals are provided by different parts of the plant or released through the natural decomposition of the associated plants. allelopathy is the biological phenomenon by which an organism produces one or more biochemicals that influence the germination, growth, survival, and reproduction of other organisms. allelopathy thus offers an attractive and environmentally friendly alternative to pesticides or herbicides in agricultural pest and weed management. allelopathins are the secondary metabolites (weir et al., 2004; iqbal & fry, 2012). these compounds belong to different chemical groups, including triketones, terpenes, benzoquinones, coumarins, flavonoids, terpenoids, phenolic acids, tannins, lignin, fatty acids, and nonprotein amino acids. allelochemicals can be classified into ten categories (li et al., 2010) based on their different structures and properties: 1. water-soluble organic acids, straight-chain alcohols, aliphatic aldehydes, ketones and 2. simple lactones; 3. long-chain fatty acids, polyacetylenes; and 4. quinines (benzoquinone, anthraquinone, and complex quinines) and 5. phenolics; 6. cinnamic acid and its derivatives; 7. coumarins; 8. flavonoids; 9. tannins; 10. steroids and terpenoids (sesquiterpene lactones, diterpenes, and triterpenoids). root exudates, including allelochemicals, are known to influence soil biota, inhibit the growth of competing plant species, and support beneficial symbioses (bertin et al., 2003). the allelochemical compositions of the roots of two varieties of anacardium occidentale (cashew) have not been explicitly detailed in the provided papers. however, https://doi.org/10.14421/biomedich.2024.131.297-303 298 biology, medicine, & natural product chemistry 13 (1), 2024: 297-303 relevant information can be inferred from the studies on the phytochemical and nutritional composition of different parts of the cashew plant. nwosu et al. (2023) report the glycosides, flavonoids, phen, and proteins in extracts of the cashew plant, including the bark and leaves, which suggests that compounds may be present in the roots. nwosu et al. (2024) identify active components such as alkaloids, tannins, flavonoids, coumarins, terpenoids, and saponins in the stem bark, root bark, and leaves, which could indicate the presence of these compounds in the roots as well (nwosu et al., 2023; nwosu et al., 2024). many chemical constituents may be found in a single plant (okoronkwo & echeme, 2015; okoronkwo et al., 2012a/b). natural-product-based pesticides are generally safer than conventional synthetic pesticides. furthermore, there is a strong rationale for examining natural products to identify novel compounds (dayan et al., 2012; gerwick & sparks, 2014). some plants suppress the germination of understory plants despite the relative openness of the canopy and ample rainfall in the region where they are found. the inhibition of the growth of neighbouring plants by another plant can be explored for its possible use as a herbicide or pesticide by investigating the allelochemical compositions of the different parts of plants that may exhibit such allelopathy. the possible application of allelopathy in agriculture has become the subject of much research (kong et al., 2006) because the application of such allelopathic plant extracts can effectively control weeds and pests. future research should focus on the nondestructive collection and identification of these allelochemicals to further elucidate their ecological functions (liang et al., 2005). current research focuses on the effects of weeds on crops, crops on weeds, crops on crops (kong et al., 2008), and pests in general. therefore, this study focused on the evaluation of allelochemical compositions of the root parts of two varieties of anacardium occidentale, which have been observed to exhibit allelopathic properties. materials and methods preparation of plant extracts the roots of the plants used in this study were collected from abia state university uturu, nigeria. the samples were washed, air-dried, ground, and stored in tight containers until further use. allelochemical analysis for the presence of plant secondary metabolites, such as tannins, saponins, alkaloids, phenolics, flavonoids, benzoquinones, steroids, and terpenoids, was carried out using standard procedures (aoac, 2015). the samples were then subjected to gc-ms analysis. qualitative phytochemical screening test test for carbohydrates (molisch’s test) three drops of molisch’s reagent were added to the 2 ml portion of the various extracts. this will followed by the addition of 2 ml of conc. h2so4 to the bottom of the test tube. the mixture was then allowed to stand for two-to three minutes. the formation of a red or dull violet colour at the interphase of the two layers will be a positive test. test for alkaloids 0.5 ml of each extract was dissolved in 1 % hcl and filtered the solution, and the filtrate was tested with dragendroff’s and mayer’s reagents separately. the appearance of turbidity is an indication of the presence of alkaloids. test for cardiac glycosides (keller kelliani’s test) 0.5 ml of each extract was treated with 2 ml of glacial acetic acid in a test tube and a drop of ferric chloride solution was added to it. this will be carefully added with 1 ml concentrated h2so4. a brown ring at the interface indicated the presence of deoxy sugar characteristic of cardenolides. below this layer greenish colour ring forms, which turns into violet after some*time. test for flavonoids (alkaline reagent test) 1 ml of each extract was treated with 3-4 drops of 20 % naoh solution. the formation of an intense yellow colour, which becomes colourless with the addition of dilute hydrochloric acid, indicates the presence of flavonoids. test for phenols (ferric chloride test) 0.5 ml of each extract was treated with aqueous 5 % fecl3 10 % ferric chloride solution (light yellow). test for quinines hcl test the 1ml of plant extract was taken. added 2ml of concentrated hydrochloric acid (conc. hcl). the formation of a yellow colour indicated the presence of quinine. test for proteins (1% ninhydrin solution in acetone) 2 ml of filtrate was treated with 2-5 drops of ninhydrin solution placed in a boiling water bath for 1-2 minutes and observed for the formation of purple colour. test for terpenoids salkowski test the 1 ml of plant extract in a test tube was taken. added 2 ml chloroform in that extract. along with that, added carefully 3 ml of sulfuric acid (conc. h2so4) was for the formation of a layer. the formation of a reddish-brown colour indicated the presence of terpenoids. okoronkwo et al. – exploring allelochemical compositions in … 299 test for saponins 1 ml of extract was added to 5 ml of water in a test tube. the mixture will be shaken vigorously and observed for the formation of persistent foam that will confirm the presence of saponins. test for sterols (liebermann-burchard test) 1 ml of each extract was treated with 1-2 drops of chloroform, acetic anhydride and conc. h2so4 and observed for the formation of deep pink or red colour. test for tannins (braymer’s test) 1 ml of each extract was treated with 10 % alcoholic fecl3 solution and observed for the formation of a blue or greenish colour solution. quantitative determination of phytochemicals quantitative estimation of alkaloids to 1 ml of test extract 5 ml ph 4.7 phosphate buffer was added and 5 ml bcg solution and a mixture with 4 ml of chloroform. the extracts were collected in a 10-ml volumetric flask and then diluted to adjust volume with chloroform. the absorbance of the complex in chloroform was measured at 470 nm against the blank prepared as above but without extract. atropine is used as a standard material and compared the assay with atropine equivalents. quantitative estimation of flavonoids total flavonoid content was determined by the aluminium chloride method using catechin as a standard. 1 ml of the test sample and 4 ml of water were added to a volumetric flask (10 ml volume). after 5 min, 0.3 ml of 5 % sodium nitrite, and 0.3 ml of 10% aluminium chloride were added. after 6 min incubation at room temperature, 2 ml of 1 m sodium hydroxide was added to the reaction mixture. immediately, the final volume was made up to 10 ml with distilled water. the absorbance of the reaction mixture was measured at 510 nm against a blank spectrophotometrically. results were expressed as catechin equivalents (mg catechin/g dried extract). quantitative estimation of saponins the test extract was dissolved in 80% methanol, 2 ml of vanilin in ethanol was added, mixed well, and the 2 ml of 72% sulphuric acid solution was added, mixed well, and heated on a water bath at 600 c for 10 min. absorbance was measured at 544 nm against the reagent blank. diosgenin was used as a standard material and the assay was compared with diosgenin equivalents. quantitative estimation of steroids 1 ml of test extract of steroid solution was transferred into 10 ml volumetric flasks. sulphuric acid (4n, 2ml) and iron (iii) chloride (0.5% w/v, 2 ml) were added, followed by potassium hexacyanoferrate (iii) solution (0.5% w/v, 0.5 ml). the mixture was heated in a waterbath maintained at 70±20c for 30 minutes with occasional shaking and diluted to the mark with distilled water. the absorbance was measured at 780 nm against the reagent blank. quantitative estimation of phenolic compounds the total phenolic content in different solvent extracts was determined with the folinciocalteu’s reagent (fcr). in the procedure, different concentrations of the extracts were mixed with 0.4 ml fcr (diluted 1:10 v/v). after 5 min, 4 ml of sodium carbonate solution was added. the final volume of the tubes was made up to 10 ml with distilled water and allowed to stand for 90 min at room temperature. the absorbance of the sample was measured against the blank at 750 nm using a spectrophotometer. a calibration curve was constructed using catechol solutions as standard, and the total phenolic content of the extract was expressed in terms of milligrams of catechol per gram of dry weight and the standard graph. gc analysis gc analysis was carried out on a gc102af system comprising an injector port and fid detector auto sampler and gc instrument employing the following conditions: column elite 1 fused silica capillary column (30 × 0.25 mm id 1 × em df, composed of 100% dimethylpolysiloxane), operating in electron impact mode at 70 ev; helium (99.999%) was used as carrier gas at a constant flow of 1 ml/min and an injection volume of 0.5 ei was employed (split ratio of 10:1 injector temperature 250° c; ion source temperature 280° c. the oven temperature was programmed from 110 °c (isothermal for 2 min) with an increase of 10° c/min to 200°, then 5 ei/min to 280° c, ending with a 9 min isothermal at 280° c. mass spectra were taken at 70 ev; a scan interval of 0.5 s and fragments from 40 da to 550 d data analysis data generated from all analysis were subjected to analyses of variance and means where significant (p ≤ 0.05) were separated with fisher’s least significant difference using statistical package for social sciences (spss) version 13.0 results and discussion results of qualitative phytochemical screening shown in table 1 shows the allelochemical compositions in the root parts of the red and yellow varieties of anacardium occidentale. it shows the presence or absence of various plant secondary metabolites such as tannins, saponins, alkaloids, phenolics, flavonoids, benzoquinones, steroids, and terpenoids. 300 biology, medicine, & natural product chemistry 13 (1), 2024: 297-303 table 1. result of the screening/qualitative compositions of the inner and bark of the root of two varieties of the plant. parameters root bark inner root red yellow red yellow methanol ethanol methanol ethanol methanol ethanol methanol ethanol alkaloids + + + + flavonoids + + + + + + saponins + + + + tannins + + + + + + terpenoids + + + + phenols + + + + + + + + anthraquinone cardiac glycosides reducing sugar steroid result of the quantitative compositions of the inner and bark of the roots of two plant varieties are shown in table 2, which shows the percentage content of different compounds such as saponins, tannins, terpenoids, and phenols. table 2. result of the quantitative compositions of the inner and bark of the root of two varieties of the plant. parameters samples root bark inner root red yellow red yellow alkaloids 9.67±4.04 7.83±3.03 flavonoids 25.5±4.04 16.7±3.03 13.06±3.03 15.16±1.63 saponins 55.5±2.05 85.33±3.03 tannins 1.59±0.03 1.58±0.09 0.162±1.664 0.364±0.206 terpenoids 8.50±1.73∁ 12.50±6.06 phenols 1.78±0.04 1.87±0.40 1.72±0.123 1.347 ±0.07 tables 3. gc/ms result of the root bark of red variety anacadium occidentale. chromatogram peak identified compound name molecular formula molecular weight retention time (mins) percentage content 1 1,2,4,-benzenetriol c6h6o 252 22.040 8.883 2 3-tridecyl phenol c19h32o 276 41.703 2.816 3 (z)-3-(pentadec-8-en-1-yl) phenol c21h34o 302 44.924 17.663 4 (z)-3-(pentadec-8-en-1-yl) phenol c21h34o 302 45.000 4.739 5 (z)-3-(pentadec-8-en-1-yl) phenol c21h34o 302 45.101 7.915 6 phenol,3-pentadecylc21h36 304 45.235 6.374 7 3-((4z,7z)-heptadeca-4,7-dien-1-yl) phenol c23h36o 328 48.062 14.475 8 (z)–3– (heptadec-10-en-1-yl) phenol c23h38o 330 48.246 32.389 9 (z)–3– (heptadec-10-en-1-yl) phenol c23h38o 330 48.406 4.747 tables 4. gc-ms analysis result of the root bark of yellow variety anacadium occidentale. chromatogram peak compound name molecular formula molecular weight retention time (mins) percentage content 1 3-tridecylphenol c19h32o 276 41.712 4.732 2 (z)-3-(pentadec-8-en-1-yl) phenol c21h34o 302 44.941 21.567 3 (z)-3-(pentadec-8-en-1-yl) phenol c21h34o 302 45.017 7.123 4 phenol,3-pentadecylc21h36o 304 45.252 9.300 5 3-((4z,7z)-heptadeca-4,7-dien-1-yl) phenol c23h36o 328 48.070 15.334 6 (z)-3-(heptadec-10-en-1-yl) phenol c23h38o 330 48.263 41.944 okoronkwo et al. – exploring allelochemical compositions in … 301 table 5.a. gc/ms result of anacardium occidentale inner root (yellow variety). chromatogram peak compound name molecular formula molecular weight retention time (mins) percentage content 1 n-hexadecanoic acid c16h32o2 256 35.739 13.008% 2 9,12– octadecadienoic acid c18h32o2 280 39.304 74.841% 3 octadecanoic acid c18h36o2 284 39.698 8.607% 4 unidentified 59.135 table 5.b. gc/ms result of anacardium occidentale inner root (red variety). chromatogram peak compound name molecular formula molecular weight retention time (mins) percentage content 1 n-hexadecanoic acid c16h32o2 256 35.739 13.008% 2 9,12– octadecadienoic acid c18h32o2 280 39.304 74.841% 3 octadecanoic acid c18h36o2 284 39.698 8.607% 4 unidentified 59.135 5 p hydroxybenzoic, c7h6o3 138.12 59.355 7.142% 6. vanillic c8h8o4 168.141 60.134 12.2% 7. p-coumaric c9h8o3 164.0473 61.234 11.234% figure 1. structure of the compounds from gc-ms analysis of the root bark. n-hexadecanoic acid p hydroxybenzoic acid 9,12– octadecadienoic acid octadecanoic acid p cumaric acids vaccinic acids figure 2. structures of some compounds identified from gc/ms analysis of the inner part of the root. discussion the preliminary qualitative screening results shown in table 1 show that alkaloids, saponins, and terpenoids were found exclusively in the root bark of both varieties, while tannins, flavonoids, and phenols were present in both parts of the plant. this initial screening is useful for identifying the types of allelochemicals that are present in the roots of the two varieties. 302 biology, medicine, & natural product chemistry 13 (1), 2024: 297-303 the results of the quantitative screening shown in table 2 show the percentage content of different compounds such as saponins, tannins, terpenoids, and phenols. the table highlights that while the root bark of the red variety had the highest saponin content, the yellow variety exhibited phenol as its highest content. this quantitative analysis tables 3 and 4 show the results of gc/ms analysis of the root bark of anacadium occidentale (red and yellow plant varieties). the root bark of both the red and yellow varieties of anacardium occidentale revealed a variety of chemical compounds with different structures and properties. these compounds play a significant role in the allelochemical profiles of plants, potentially affecting their interactions with other organisms in the ecosystem. researchers can investigate the bioactive properties and ecological roles of allelochemicals found in the root bark of each variety by identifying the specific compounds. the presence of compounds such as 1,2,4benzenetriol, 3-tridecyl phenol, and (z)-3-(pentadec-8en-1-yl) phenol in the red variety and compounds like 3tridecylphenol, (z)-3-(pentadec-8-en-1-yl) phenol, and 3-((4z,7z)-heptadeca-4,7-dien-1-yl) phenol in the yellow variety highlights the chemical diversity of anacardium occidentale. tables 5 and 6 show the gc/ms results of anacardium occidentale inner root (yellow variety). the gc/ms results for both samples showed the presence of different compounds, and one of the compound peaks observed in the inner root was not identified (table 5), which is the 4th peak; 9,12– octadecadienoic acid (fatty acid) was the compound with the highest composition identified in the yellow variety. similar compounds were identified in the root bark of the red and yellow varieties. the compound with the highest percentage composition of 32.389 and 41.944% in the red and yellow varieties, respectively, was identified as (z)-3-(heptadec-10-en-1yl) phenol (molecular formula c23h38o with a molecular weight of 330) and occurred within the retention time range of 48.246 – 48.263 min. the root bark of both varieties from the gc/ms results contained more phenolic compounds. the inner roots of the yellow variety contained more long-chain fatty acids. figure 1 shows the structure of the compounds from the gc-ms analysis of the root bark, while figure 2 shows the structures of some compounds identified from the gc/ms analysis of the inner part of the root. conclusions the compounds in the bark were primarily phenols, whereas those in the inner roots were primarily fatty acids. additionally, both parts of the plant contain terpenoids, tannins, and phenols, whereas flavonoids are only found in the bark. these allelopathies may be responsible for the observed allelopathic properties of the plants. the composition of allelochemicals in plant roots is diverse species-specific and has significant implications for plant interactions and soil ecology. the identification and study of these compounds are essential for understanding their roles in plant growth, defence, and potential for managing agricultural systems acknowledgements: special thanks to tetfund, nigeria and abia state university management for sponsoring this research authors’ contributions: nnenna ejije, okoronkwo designed the study, nwosu nkemakolam bright conducted the laboratory. jude chibuzor igwe analyzed the data. nwosu nkemakolam bright & precious o. chukwu 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(2004). biochemical and physiological mechanisms mediated by allelochemicals. curr. op. in plant biol. 7(4), 472-479. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1077-1083 | doi: 10.14421/biomedich.2025.142.1077-1083 issn 2540-9328 (online) application of edible coating water soluble based chitosan nanoparticles and polyvinyl alcohol in strawberry (fragaria ananassa) storage likia salsa billa, yunita rachmawati, titis kusumaninghayu, pandu lintang nugroho*, angela merici pusparani, esa ghanim fadhallah, diki danar tri winanti, madi hartono department of agricultural product technology, faculty of agriculture, universitas lampung, bandar lampung, lampung, indonesia. corresponding author* pandulintangn@gmail.com manuscript received: 04 september, 2025. revision accepted: 21 november, 2025. published: 27 november, 2025. abstract strawberries (fragaria ananassa) are highly sought after for their vibrant color and unique flavor. however, their high perishability and rapid quality degradation lead to significant post-harvest losses and a decrease in market value. this study aimed to evaluate the effect of an edible coating based on wsc nanoparticles and pva on the quality characteristics of strawberries during storage. the research methodology included the synthesis of wsc nanoparticles, preparation of the coating solution, application to strawberries, and subsequent storage. a completely randomized design (crd) with a single-factor arrangement was utilized, comprising five pva concentrations (0%, 2%, 3%, 4%, and 5%) with three replications. the results demonstrated that the application of the nanoparticle wscpva-based edible coating effectively reduced weight loss and delayed physical deterioration. increasing pva concentrations led to a more effective preservation of total soluble solids (°brix), a lower weight loss percentage, and a slower decline in vitamin c content. higher pva concentrations were found to reduce the rate of quality degradation and moisture loss. the coating formulation containing 4% pva (p3) exhibited the best performance, maintaining a water content of 92.24%, total soluble solids of 2 °brix, a ph of 4.3, a weight loss of 30%, and a vitamin c retention rate of 9.02%. the findings of this study offer a valuable solution for farmers to mitigate strawberry quality decline and provide new insights for future research on food preservation. keywords: edible coating; nanoparticles; pva; water soluble chitosan; strawberry. introduction strawberries are non-climacteric fruits rich in essential nutrients, including provitamin a, vitamin c, calcium, phosphorus, iron, sodium, potassium, and magnesium. a 100-gram serving provides 140 kj of energy, 0.8 g of protein, 0.5 g of fat, 7.6 g of carbohydrates, 53 mg of vitamin c, and 1.7 g of fiber (wadhani et al., 2021). the annual strawberry harvest is substantial, reaching 27,721 tons per year (bps, 2023), reflecting strong demand in both local and global markets. oktarina et al. (2017) reported local market demand exceeding 5,000 tons per year, while global demand, primarily from south korea, the usa, and australia, reaches 150 tons per year. however, this high market demand is not met with adequate post-harvest handling, leading to significant premature spoilage. strawberry spoilage is primarily caused by high humidity and rapid respiration, which create an ideal environment for the growth of fungi and bacteria, resulting in considerable post-harvest losses (kementan, 2023). under normal room temperature, strawberries can deteriorate in as little as two days, with a maximum shelf life of seven days after harvest (herawati et al., 2019). the economic impact of this spoilage is significant. sukasih and setyadjit (2019) noted a sharp decline in the value of damaged fruit; fresh strawberries selling for idr 80,000–100,000 per kg can see their price plummet to idr 35,000–45,000 per kg when they become withered or defective. while cold storage is a common method for maintaining freshness, prolonged exposure to low temperatures can lead to chilling injury, resulting in a softened texture and altered nutritional content (tetelepta et al., 2019). therefore, to preserve fruit quality and enhance food sustainability, an alternative approach such as edible coating is needed. edible coatings are thin, consumable layers applied to food products to reduce weight loss, prevent flavor changes (tetelepta et al., 2019), and provide a protective barrier against mechanical damage. they are particularly beneficial for fragile foods, offering an environmentally friendly solution (kusuma & fitriani, 2021). these coatings act as semi-permeable barriers, controlling the https://doi.org/10.14421/biomedich.2025.142.1077-1083 1078 biology, medicine, & natural product chemistry 14 (2), 2025: 1077-1083 migration of gases and water-soluble components (aji et al., 2024). this makes them an effective method for reducing the respiration rate and maintaining moisture within the fruit tissue. edible coatings can be formulated from various materials, including carboxymethyl cellulose (cmc), gelatin, and chitosan. while cmc and gelatin can form stable films, they have limited mechanical durability, creating a thick layer (putri et al., 2019) and lacking inherent antimicrobial properties. to overcome these limitations, chitosan presents a superior alternative due to its natural antimicrobial characteristics. derived from the deacetylation of chitin in the exoskeletons of crustaceans (like prawns and crabs), chitosan's polymer structure contains positively charged amino groups that disrupt the cell membrane metabolism of microorganisms (nabila et al., 2018). guge et al. (2024) has demonstrated the effectiveness of chitosan in inhibiting microbial activity, thereby preserving the freshness of agricultural products. a significant drawback of chitosan, however, is its poor solubility in water, requiring an acidic solvent that can adversely affect the aroma and taste of the fruit, potentially reducing consumer acceptance. this issue can be resolved by modifying chitosan into water-soluble chitosan (wsc). wsc is created through a depolymerization process involving the substitution of amine groups, which increases its interaction with water molecules and enhances its solubility (chamidah et al., 2019). jebali et al. (2024) reported that this technology significantly increases the surface contact area by up to 50%, improving both solubility and film stability. to further enhance its activity, wsc can be processed into nanoparticles, which have been shown to increase resistance to oxidation (novita & astuti, 2023). while an edible coating based on wsc nanoparticles offers excellent antimicrobial and antioxidant properties, its mechanical characteristics, such as flexibility, elasticity, and crack resistance, need improvement. reinforcing materials are essential to reduce the stiffness of the polymer chains, allowing the coating to adjust to the fruit's surface movement during storage (erdem et al., 2022). pva is a water-soluble, easily processed, non-toxic, and biodegradable biopolymer. pamela et al. (2016) found that using pva as a filler in bio-nanocomposite films enhances surface reactivity, thermal stability, and heat resistance, resulting in packaging that is flexible enough to conform to the product's shape. furthermore, pva is considered safe by the food and drug administration (gomez-aldapa et al., 2020), ensuring that its use as a reinforcing agent does not raise food safety concerns. previous research by putri and nugroho (2023) demonstrated pva's effectiveness in maintaining the quality and nutritional content of apples and tomatoes under various environmental conditions. however, there is a lack of specific research on the combined application of wsc nanoparticles and pva as an edible coating for strawberries. therefore, this study aims to evaluate the effect of an edible coating based on wsc nanoparticles and pva on the quality characteristics of strawberries during storage. materials and method materials and equipments fresh strawberries (+ 7 g) without physical defect were purchased from way halim permai market, bandar lampung. another material used include water soluble chitosan powder (deacetylation degree 94%, moisture regaint 7-10%, cv chimultiguna), pva powder (merck, 88% hydrolized), sodium tripolyphosphate food grade (aditya birla), aquades, dan iodine. the tools used in this research included an analytical balance (shimadzu ay220), whatman paper, blender, hot magnetic stirrer (cimarec + thermo fisher), beaker glass, erlenmeyer flask, set of burettes, volumetric flask, refractometer, oven (memmert), ph meter (lutron ph 222), plastic storage container, and spatula. methods this research was conducted in several key stages: synthesis of water-soluble chitosan (wsc) nanoparticles, preparation of the polyvinyl alcohol (pva) solution, formulation and application of the edible coating, storage of the coated strawberries, and subsequent quality characterization. the experiment was designed as a completely randomized design (crd) with a single factor at five treatment levels of pva concentration (0%, 2%, 3%, 4%, and 5%), and was performed with three replications. synthesis of water-soluble chitosan nanoparticles water-soluble chitosan nanoparticles were synthesized using the ionic gelation method, as described by guge et al. (2024). initially, 2 g of chitosan powder (2% concentration) was dissolved in 100 ml of deionized water until a homogeneous solution was achieved. simultaneously, sodium tripolyphosphate (sttp) was dissolved in deionized water at a ratio of 1:1000 (0.1 g sttp in 100 ml deionized water) with continuous stirring at 800 rpm for 20 minutes. subsequently, 50 ml of the chitosan solution was added to 10 ml of the sttp solution and homogenized using a hot magnetic stirrer for one hour. preparation and application of the edible coating solution the edible coating solution was prepared following the method of kaynarca et al. (2023). pva powder was weighed according to the specific formulation and placed in a beaker containing 100 ml of deionized water. the mixture was homogenized using a hot magnetic stirrer at a constant temperature of 80°c for two hours at a billa et al. – application of edible coating water soluble based chitosan … 1079 rotation speed of 1500 rpm. the resulting solution was then cooled to an ambient temperature of 50°c. the final edible coating solution was prepared by mixing the chitosan nanoparticle solution with the pva solution at a ratio of 1:10 (10 ml of chitosan nanoparticles to 100 ml of pva solution). this mixture was homogenized again using a hot magnetic stirrer for two hours, yielding a total volume of 110 ml of edible coating solution. the specific formulations are detailed in table 1. table 1. edible coating solution treatment formulation. sample wsc nanopartickes (g) pva powder (g) aquades (ml) control 0 0 0 p1 2 2 100 p2 2 3 100 p3 2 4 100 p4 2 5 100 for the application, strawberries were first washed under running water to remove dirt and then dried with a paper towel. the edible coating was applied using a dipping method, where each strawberry was submerged in the solution for five minutes to ensure the surface was evenly coated. the coated fruits were then air-dried for three minutes to allow a protective layer to form. the treated strawberries were placed in an open 500 ml polypropylene (pp) container, with each container holding 7–8 strawberries. these containers were then stored at ambient temperature (25°c) for a period of seven days. strawberry quality was evaluated on days 0, 3, 5, and 7 of storage to assess the coating's effectiveness. the measured parameters included appearance, weight loss, water content, total soluble solids (°brix), acidity (ph), and vitamin c content. water content water content was determined using the gravimetric method, as described by safitri and hakiki (2024). the initial weight of the strawberry samples was recorded, followed by drying in an oven at 105°c for three hours. after drying, the samples were reweighed to calculate the percentage of water mass loss using the provided formula: water content (%) = wet weight − dry weight wet weight x 100% weight loss weight loss was measured following the procedure by yoga et al. (2022) using an analytical balance (shimadzu ay220, accuracy 0.0001 g). the mass of each strawberry was recorded throughout the storage period, and the percentage of weight loss was calculated using the provided formula: weight loss(%) = w0 − w𝑡 w0 x 100% description: w0 = sample initial weight wt = sample final weight total dissolved solid total soluble solids were measured using a digital hand refractometer, following zulaikhah's (2021) method. a few drops of crushed strawberry sample were placed on the prism surface of the refractometer. readings were taken at an ambient temperature of approximately 25°c to determine the concentration of dissolved solids, which indicates the level of sweetness. the value was expressed in °brix. acidity (ph) the ph was measured using a digital ph meter. a 1:100 dilution was prepared by crushing 1 g of strawberry sample and adding 100 ml of deionized water. the ph meter was calibrated with deionized water before measurement. the electrode was submerged in the sample solution until a stable reading was obtained. a single ph measurement was performed for each sample in each treatment, ensuring the ph meter was properly calibrated. appearance visual observations were made to assess changes in the color and the growth of mold on the strawberries throughout the storage period. the physical appearance of each sample was meticulously noted. vitamin c vitamin c content was quantified using the iodometric titration method, as outlined by fitriana and ardhista (2020). the strawberries with the best appearance (fresh red color, no wrinkling, no mold, and minimal weight loss) were selected for analysis. a 10 g sample of crushed strawberry was placed in a 100 ml volumetric flask, filled to the mark with deionized water, and homogenized by inverting 12 times. a burette was filled with 50 ml of iodine solution, ensuring no air bubbles were present. a 10 ml aliquot of the homogenized sample solution was transferred to an erlenmeyer flask. the iodine solution was then slowly titrated into the sample while stirring until the solution turned a stable white or limpid color, indicating the endpoint. the volume of iodine solution used was recorded and the vitamin c level was calculated using the following formula: vitamin c levels (mg) = iodine volume (ml) x 0.88 g ascorbic acid 1080 biology, medicine, & natural product chemistry 14 (2), 2025: 1077-1083 data analysis all collected data, including water content, weight loss, °brix, ph, appearance, and vitamin c content, were processed using microsoft excel. descriptive analysis was performed by calculating the average values for each parameter. the processed data were also visualized in the form of line graphs to illustrate trends over the storage period. result and discussion water content the water content analysis was conducted to measure the amount of moisture lost from the fruit during storage. the primary purpose of this test was to evaluate the edible coating's effectiveness in inhibiting the rate of water vapor transfer from the fruit tissue to the surrounding environment (figure 1). as shown, the water content values for all treatments varied over the sevenday storage period. on day 0, the initial water content was similar across all treatments: p1 (92.13%), p2 (90.72%), p3 (90.16%), and p4 (90.10%). the application of the edible coating was proven to significantly inhibit the rate of water loss. while minor fluctuations were observed, all coated samples maintained a higher water content than the control throughout storage, reflecting the coating's ability to reduce moisture evaporation. for instance, the p3 and p4 treatments exhibited the best water retention on day 5. ultimately, the higher water content values in the coated strawberries indicated that the edible coating created an effective barrier, keeping the fruit tissue hydrated and fresh. this is consistent with findings by husain et al. (2022), who reported that edible coatings can reduce excessive evaporation in fruits like crystal guava, thus maintaining their freshness. our results confirm that increasing the pva concentration in the chitosan-based coating enhances its effectiveness in preserving strawberry freshness, as reflected by the high water content values and corresponding fresh visual appearance. figure 1. edible coating chitosan pva water content graph. descriptions: pva 2% = p1, pva 3% = p2, pva 4% = p3, pva 5% = p4 weight loss the application of an edible coating based on a chitosan and pva polymer composite had a significant effect on mitigating weight loss in strawberries during storage (figure 2). the control sample experienced the highest weight loss, reaching 72% on day 7, while the p3 treatment (4% pva) showed the lowest weight loss at 40.2% on the same day. the data indicate that increasing the pva concentration generally led to a reduction in weight loss. however, an interesting finding was an increase in weight loss at the highest concentration (5%). this is likely because the 4% pva concentration formed an optimal coating matrix, effectively inhibiting the rate of transpiration and respiration. in contrast, an excessively high concentration, such as 5%, may create an overly thick layer that disrupts proper gas exchange. this disruption can lead to anaerobic respiration, which starves the fruit of oxygen and encourages the growth of microorganisms, thereby accelerating weight loss. this phenomenon is consistent with the findings of asrafil and daniel (2023), who reported that increasing the concentration of coating materials can produce a thicker film, and with afifah et al. (2023), who found that an overly thick coating can lead to anaerobic conditions that increase microbial activity and weight loss. therefore, the optimal concentration of pva as a reinforcing agent in edible coatings is a crucial factor to consider. figure 2. weight loss graph. descriptions: pva 2% = p1, pva 3% = p2, pva 4% = p3, pva 5% = p4 total dissolved solids (°brix) the analysis of total soluble solids (°brix) using a hand refractometer revealed fluctuations in the brix values across all treatments during storage (figure 3). the °brix value is a measure of the total dissolved solids within the fruit. the data in figure 3 show a general decrease in °brix values for strawberries in all treatments towards the end of the storage period. the 5% pva treatment (p4) exhibited a slightly higher °brix value compared to the control on day 7, while the p2 treatment showed the lowest. when compared to the control, the chitosan pva composite coating had no significant influence on the overall trend of °brix values. this can be attributed to the fact that strawberries are non-climacteric fruits, meaning they do not undergo a respiratory climacteric, which typically drives significant changes in sugar content after harvest. therefore, the pva concentration in the edible coating did not have a major effect on the brix content. furthermore, the addition of a high pva concentration appeared to accelerate spoilage in some cases, possibly due to restricted gas exchange. as noted by asrafil and daniel (2023), an optimal edible coating billa et al. – application of edible coating water soluble based chitosan … 1081 can inhibit water vapor transfer and slow down the metabolic process of fruits. however, this effect is highly dependent on the type of fruit. our results suggest that edible coatings may be more effective for climacteric fruits, which exhibit a post-harvest surge in respiration and ripening. figure 3. total dissolved solid (°brix) graph. description: pva 2% = p1, pva 3% = p2, pva 4% = p3, pva 5% = p4 this result differs from pigozzi et al. (2025) on avocados. their study showed that using various pva concentrations (0.5% to 2%) could maintain fruit quality for up to 11 days of storage. in that research, the °brix value tended to increase over time as part of the natural maturation process, with no significant differences among treatments. the final °brix values for pvacoated avocados ranged from 7.17% to 7.58%, and all treatments successfully inhibited the respiration rate and slowed physical deterioration. these contrasting results strengthen the view that the effectiveness of pva as an edible coating material is highly dependent on the type of fruit. for climacteric fruits like avocados, a pva-based coating provides significant protection. however, for non-climacteric fruits such as strawberries, pva may be ineffective or even accelerate deterioration. acidity levels (ph) the application of a chitosan and pva polymer composite edible coating on strawberries had a significant influence on the increase in ph during storage (figure 4). the lowest ph was observed in the p2 treatment (2% pva) on day 3, while the highest ph was found in the p4 treatment (4% pva). on day 7, all coated treatments (p1 to p4) showed a higher ph value compared to the control. this increase in ph suggests that the edible coating, particularly at higher concentrations, may have inhibited the permeability of the strawberries, leading to the accumulation of gases. this can trigger anaerobic respiration and the subsequent formation of ethanol, which raises the fruit's ph. this finding aligns with afifah et al. (2023), who stated that the formation of ethanol by microorganisms during anaerobic respiration is believed to influence the ph value. therefore, the ineffectiveness of the coating in preventing ph increase may be related to an overly restrictive barrier that promotes anaerobic conditions. figure 4. acidity levels (ph) graph. description: pva 2% = p1, pva 3% = p2, pva 4% = p3, pva 5% = p4 color this research demonstrated that the edible coating based on a chitosan and pva composite polymer resulted in varying changes to strawberry color during the storage period. the color changes observed from day 0 to day 7 for each treatment are presented in table 2. color degradation in strawberries is a key indicator of the rate of physiological damage during storage. the data in table 2 show that fruit without the edible coating (control) experienced a faster color change, progressing to a blackish-red hue and mold growth. in contrast, treatments with the water-soluble chitosan nanoparticle and pva-based coating were able to maintain a vibrant red color for the longest duration, particularly at pva concentrations of 4% (p3) and 5% (p4). this indicates that the combination of chitosan and pva formed an effective semipermeable layer that inhibits gas transfer and moisture loss, thereby suppressing the oxidative processes and microbial activity responsible for color degradation. this finding is supported by wardalia et al. (2022), who stated that edible coatings function to inhibit o₂ and co₂ transfer, and by karimullah and handarini (2024), who highlighted chitosan's antimicrobial properties. therefore, a higher pva concentration in the food-grade layer provides more effective protection against color changes during storage. table 2. the effect of edible coating on strawberries color. t re a tm en ts day to 0 3 5 7 control red red black dark red (black mold) dark red (black mold) p1 red red black dark pink blackish brown p2 red dark red dark red blackish red p3 red dark red dark red dark red p4 red dark red dark red dark red vitamin c vitamin c content was measured on day 0 and day 7 of storage using the best-performing treatment, which was the 4% pva concentration, as it had the lowest weight loss. the test was conducted using the iodometric titration method to determine the coating’s ability to 1082 biology, medicine, & natural product chemistry 14 (2), 2025: 1077-1083 preserve vitamin c in strawberries (figure 5). the data show a significant decrease in vitamin c concentration over the storage period. on day 0, the initial vitamin c level was 40.48 mg. however, by day 7, the concentration had decreased by approximately 22% from this initial level. this result is consistent with salha and gedanken (2021), which reported a 22.73% decrease in vitamin c content in strawberries coated with chitosan nanoparticles, compared to a 64.73% decrease in the control group. these data prove that the edible coating formulation effectively reduces the rate of vitamin c degradation in strawberries. furthermore, there is an inverse relationship between pva concentration and the percentage of vitamin c loss: higher pva concentrations result in a lower percentage of vitamin c loss. this finding is supported by chinchkar et al. (2024), who showed that a 5% pva coating resulted in a lower vitamin c loss (28.53%) on day 8 compared to a 2.5% pva coating (31.6%). chinchkar et al. (2024) also attributed this effect to the increased viscosity provided by pva, which creates a stronger coating. this stronger barrier reduces the ingress of oxygen from the air into the fruit tissue, thereby slowing down the oxidation of vitamin c. the degradation process of vitamin c involves the oxidation of l-ascorbic acid to ldehydroascorbic acid, which can further convert to ldicotiledonate, a compound with no vitamin c activity. therefore, the pva-enhanced coating plays a critical role in slowing this degradation and preserving the nutritional value of the strawberries. figure 5. vitamin c levels graph. conclusions the application of an edible coating based on watersoluble chitosan (wsc) nanoparticles and polyvinyl alcohol (pva) significantly affected the physical and chemical quality of strawberries during storage. various concentrations of pva exhibited different levels of effectiveness, with the 4% pva concentration demonstrating the best overall performance. on day 7, this optimal formulation yielded a ph of 4.46, total soluble solids of 2 °brix, a water content of 93.06%, and a weight loss of 40.2%. visually, the strawberries coated with 4% pva maintained their dark red color without any signs of mold or microbial growth. additionally, this treatment effectively slowed the degradation of vitamin c, retaining 31.46% of its initial content. although fluctuations in ph and a general decrease in total soluble solids were observed, the findings confirm that a wsc nanoparticle-based edible coating with an optimal pva concentration of 4% offers a promising approach to extending the shelf life of strawberries. authors’ contributions: likia salsa billa, yunita rachmawati, titis kusumaninghayu, pandu lintang nugroho, angela merici pusparani, and esa ghanim fadhallah design the study and wrote the manuscript in bahasa. likia salsa billa, yunita rachmawati, titis kusumaninghayu, pandu lintang nugroho, and angela merici pusparani conducted the research and wrote the manuscript. esa ghanim fadhallah supervised the research and proofreads the manuscript. pandu lintang nugroho translates to english. all authors read and approved the final version of the manuscript. competing interests: all authors stated that there was no conflict of interest in this study. references afifah, a.n., mukminah, n. & destiana, i. d. 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(2021). sifat fisikokimia yogurt dengan berbagai proporsi penambahan sari buah naga merah (hylocereus polyrhizus). jurnal sains peternakan, 9(1): 7–15. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 699-705 | doi: 10.14421/biomedich.2025.142.699-705 issn 2540-9328 (online) formulation and evaluation of spirulina-based gel with varying carbopol concentrations for anti-acne activity against staphylococcus epidermidis emmellia yunitha1, anita nilawati1, mega novita2, dian marlina1,* 1department of pharmacy, universitas setia budi, jl. letjend sutoyo mojosongo, surakarta, central java 57127, indonesia. 2postgraduate program of natural science education, universitas pgri semarang, jl. sidodadi timur 24, semarang, central java 50232, indonesia. corresponding author* marlina@setiabudi.ac.id manuscript received: 11 may, 2025. revision accepted: 13 august, 2025. published: 01 october, 2025. abstract spirulina platensis is a blue-green microalga known for its antibacterial properties, offering potential as a natural alternative in acne treatment. acne vulgaris, often caused by staphylococcus epidermidis, requires effective topical solutions. gels are favored for their nongreasy texture, ease of application, and good skin absorption. this study aimed to formulate and evaluate anti-acne gels containing 25% spirulina extract with varying carbopol concentrations (0.5%, 1%, 1.5%). each formulation was assessed for physical properties, stability over 21 days, and antibacterial activity against s. epidermidis. all gel formulations met quality standards for ph, homogeneity, viscosity, spreadability, and adhesiveness. the gel with 0.5% carbopol (fi) showed the best spreadability, ideal viscosity, and good adhesiveness, along with the highest antibacterial activity, exhibiting an inhibition zone of 16.5 mm—comparable to tetracycline. in conclusion, spirulina-based gel with 0.5% carbopol offers an effective, stable, and natural anti-acne option. these findings highlight the potential of spirulina as a bioactive agent in topical formulations and encourage further research for clinical applications in acne management. keywords: antibacterial gel; gel formulation; natural antimicrobia; spirulina platensis; staphylococcus epidermidis. introduction skin, the body's largest organ, is the first line of defense against environmental threats, including microbial pathogens. however, it is not immune to infections, as seen in cases of acne vulgaris a chronic inflammatory disorder affecting the pilosebaceous unit (novaryatiin et al., 2024). acne is commonly triggered by the proliferation of bacteria such as staphylococcus aureus, staphylococcus epidermidis, and propionibacterium acnes in combination with excessive sebum production and clogged follicles (du et al. 2021). this condition is particularly prevalent among adolescents and young adults, negatively affecting self-esteem and quality of life (sidharta, malaha, and mursyanti 2021). therefore, developing safe and effective topical treatments is critical in acne management. spirulina, a blue-green microalga from the genus arthrospira, has garnered attention for its rich biochemical composition, including essential fatty acids, phycocyanin, chlorophyll, proteins, vitamins, and minerals (anvara & nowruzib, 2021). several studies highlight its therapeutic potential, particularly its antibacterial and anti-inflammatory effects (kumar et al., 2022). notably, its effectiveness against staphylococcus epidermidis a common acne-associated bacterium has been demonstrated through various antimicrobial assays (sidharta et al., 2021). to enhance its application, spirulina can be formulated into a topical gel, a preparation type known for its ease of application, quick absorption, and aesthetically pleasing texture (severn & horswill, 2023). gels also offer the advantage of faster drug release compared to creams and ointments, which contribute to the rapid drying of acne lesions (sugiyarto, 2024). despite the known antimicrobial potential of spirulina, limited formulations have been developed to harness its benefits in a stable and effective gel form for acne treatment. the main research problem is optimizing spirulina-based gel formulations that maintain antibacterial activity, physical stability, and patient acceptability. the general solution is to investigate the formulation of spirulina extract into a topical gel using suitable gelling agents, then evaluate its antibacterial effectiveness and physical stability through standardized methods. the use of gel formulations in dermatological therapy is well-supported due to their favorable characteristics such as pseudoplastic flow, high water content, and ease of spreadability (nofita, sofyan, and saputra yasir, 2024). among various gelling agents, carbopol https://doi.org/10.14421/biomedich.2025.142.699-705 700 biology, medicine, & natural product chemistry 14 (2), 2025: 699-705 (carbomer) is widely preferred for its ability to produce transparent gels and stabilize active ingredients. it is typically used at concentrations between 0.5–1% and requires the addition of triethanolamine (tea) to neutralize its acidity and form a stable gel matrix (sharon, yuliet, and sulistiana, 2023). such formulations are non-greasy and quickly absorbed, ideal for acneprone skin. to assess antimicrobial performance, the disc diffusion method is commonly employed. this method allows for qualitative measurement of antibacterial activity by observing inhibition zones around paper discs impregnated with the test compound (hidayati, amanda, and setiawansyah, 2024).spirulina's extract has demonstrated inhibitory activity against several grampositive bacteria, including staphylococcus epidermidis and s. aureus, due to its bioactive compounds like phycocyanin and phenolic acids (mulyani, kusumawardani, and pangesti, 2022). recent studies also reveal spirulina's efficacy as a topical agent, showing significant antibacterial activity and reduction in lesion size in in vivo and in vitro models (yasir et al., 2023). several studies have explored the bioactivity of spirulina, focusing on its antioxidant, anti-inflammatory, and antimicrobial effects. for instance,.wils et al. 2021 demonstrated spirulina’s protective properties against oxidative stress, while pez jaeschke et al. 2021 confirmed its antimicrobial action in topical applications. additionally, gel formulations containing herbal extracts have been widely developed for skin-related diseases (zhang et al., 2025); however, their long-term stability and antimicrobial consistency remain inconsistent. most spirulina formulations focus on oral or dietary supplements rather than topical delivery systems tailored for acne management. moreover, while carbopol-based gels are established in cosmetic and pharmaceutical fields, the interaction between spirulina’s bioactive compounds and carbopol matrices under varying storage conditions remains underexplored. accelerated stability studies are essential to predict the shelf life of such products. however, limited research exists on the stability of spirulina-containing gels, especially under elevated temperature conditions simulating tropical climates (i ragusa et al., 2021). this reveals a crucial research gap in developing and evaluating a spirulinabased gel with proven antibacterial activity and physical stability suitable for practical use in acne therapy. this study aims to formulate and evaluate a spirulinabased gel with antibacterial activity against staphylococcus epidermidis, focusing on the physical characteristics and antimicrobial efficacy of the formulation. the novelty lies in combining spirulina extract with carbopol gel base to produce a stable, antiacne topical preparation, validated through disc diffusion assay and accelerated stability testing. the scope of the study encompasses the extraction of spirulina, formulation of topical gel using carbopol and tea, evaluation of physical properties (viscosity, ph, spreadability), and assessment of antibacterial activity and stability under various storage conditions. materials and methods this study aimed to develop and evaluate a spirulinabased anti-acne gel by extracting proteins from spirulina platensis, formulating a topical preparation, and testing its physical properties and antibacterial activity against staphylococcus epidermidis. the research design involved systematic extraction, purification, gel formulation, microbial testing, and analysis to assess the product's effectiveness and stability. the materials, instruments, and procedures used in the study are described below. materials the main active ingredient in this study was spirulina platensis powder. for protein extraction, the powder was mixed with distilled water in a 1:25 ratio and kept at 20– 25°c for 24 hours, shielded from light using aluminum foil. ammonium sulfate ((nh₄)₂so₄) was used for protein precipitation, and 0.005 m sodium phosphate buffer (ph 7.0) was used to redissolve the precipitate. the protein content was confirmed by biuret and ninhydrin tests, indicated by violet and blue-purple coloration, respectively (prete et al., 2024). for the gel formulation, three different concentrations of carbopol (0.5%, 1%, and 1.5%) were used, while the concentration of spirulina was kept constant at 25%. other components included triethanolamine (tea), propylene glycol (pg), methylparaben, glycerin, and distilled water. antibacterial testing was conducted using staphylococcus epidermidis, cultured on mannitol salt agar (msa) and tested on mueller-hinton agar (mha). tetracycline was a positive control, and a blank gel was a negative control. instrumentation several instruments were employed throughout the study. a sonicator operating at 40 khz and a centrifuge (1000 rpm and 4000 rpm settings) were used for protein extraction and purification. a brookfield viscometer with spindle number 7 measured gel viscosity. a vernier caliper was used to measure inhibition zones. an autoclave sterilized non-heat-sensitive equipment at 121°c for 15 minutes, while alcohol and flame were used for sterilizing heat-sensitive tools. additional tools included analytical balances, incubators, glassware, and petri dishes. procedure 1) protein extraction and identification spirulina platensis powder was macerated in distilled water for 24 hours at room temperature (20–25°c), shielded from light. the solution was sonicated at 40 yunitha et al. – spirulina gel formulations for acne 701 khz for 45 minutes and centrifuged at 1000 rpm for 20 minutes. the resulting supernatant, containing watersoluble proteins, was purified by adding 100 mg of ammonium sulfate and stirring for 120 minutes. it was then centrifuged at 4000 rpm for 15 minutes. the blue precipitate was dissolved in 0.005 m sodium phosphate buffer (ph 7.0). protein presence was confirmed through biuret and ninhydrin tests (li et al., 2025). 2) gel formulation three formulations were prepared by varying the carbopol concentration (fi = 0.5%, fii = 1%, fiii = 1.5%), while maintaining the spirulina content at 25%. the gel was formulated by dissolving carbopol in warm distilled water, followed by the sequential addition of tea, pg, methylparaben, spirulina, and glycerin. the final mixture was adjusted to 100% with distilled water and stirred until homogeneous. (table 1) presents the composition of each formulation. table 1. anti-acne gel formulations with spirulina. material formula fi % fii % fiii % control % serbuk spirulina platensis 25 25 25 carbopol 0,5 1 1,5 0,5 tea 1 1 1 1 propilen glikol 10,0 10,0 10,0 10,0 metil paraben 0,18 0,18 0,18 0,18 gliserin 15 15 15 15 destiled water ad 100 ad 100 ad 100 ad 100 notes: f1: carbopol 0.5% combination; f2: carbopol 1% combination; f3: carbopol 1.5% combination; control -: negative control; control +: tetracycline disk 3) physical evaluation the gels were tested for organoleptic properties (color, smell, consistency), ph, homogeneity, viscosity, spreadability, and adhesiveness. viscosity was measured with a brookfield viscometer over 5 seconds. spreadability was evaluated by placing 1 g of gel between two glass plates under weights ranging from 5 to 250 g and measuring the spread diameter. adhesiveness was measured by determining the time it took for two adhered plates to separate under a 1 kg load dropped from an 80 g weight. 4) stability testing stability was assessed using a six-cycle freeze-thaw test, alternating storage at 4°c and 40°c for 24 hours each. after each cycle, the gel’s appearance, ph, and viscosity were evaluated to determine stability (hoskin et al., 2023). 5) antibacterial testing the agar disk diffusion method was used to evaluate antibacterial activity. staphylococcus epidermidis suspensions were adjusted to 1.5×10⁸ cfu/ml using a 0.5 mcfarland standard and spread onto mha plates. paper soaked in gel samples was placed on the agar, along with tetracycline and blank gel disks as controls. plates were incubated at 37°c for 24 hours. inhibition zones were measured in millimeters using a vernier caliper. identification of s. epidermidis was confirmed by colony morphology on msa, gram staining, catalase, and coagulase tests. 6) data collection and analysis all experiments were performed in triplicate. quantitative data such as inhibition zone diameters, viscosity, spreadability, and adhesiveness were recorded and averaged. data were analyzed descriptively and compared across formulations. results were interpreted to assess formulation effectiveness and consistency over time. results and discussion the preliminary tests conducted on spirulina involved evaluating its powdered and extracted forms at concentrations of 20%, 25%, and 30%. the spirulina powder, being dry, required no additional testing, whereas the extract was characterized by a precipitate resulting from the extraction process. both forms were utilized for antibacterial testing against staphylococcus epidermidis (iyer, raut, and dasgupta, 2021; saputri et al., 2024). to determine the presence of protein, the biuret and ninhydrin tests were employed. positive results were observed both the spirulina powder and extract. the biuret test produced a color shift from green to light purple, and the ninhydrin assay turned from green to dark purple, confirming the presence of proteins (meray, utami, and nurazizah, 2024). the antibacterial activity of spirulina powder and extract was assessed using mueller hinton agar (mha) media and the disc diffusion method. after 16–18 hours of incubation, inhibition zones were measured, with 702 biology, medicine, & natural product chemistry 14 (2), 2025: 699-705 results indicating effective antibacterial activity. the zones ranged from 10.9 mm to 16.5 mm, with tetracycline as a positive control and 0.005 m sodium phosphate buffer (ph 7.0) as a negative control (d’angelo costa & maia campos, 2024). the results of the antibacterial activity of spirulina powder and extract are shown in (table 2). table 2. antibacterial activity results of spirulina powder and extract. formula inhibition zone (mm) range spirulina extract spirulina powder control + 20.8±0.13 21.1±0.21 0.3% control 0 0 0% 20% 10.9±0.13 10.9±0.13 0% 25% 12.3±0.06 10.9±0.13 0.5% 30% 16.5±0.11 15.1±0.19 1.4% information: control + : tetracycline antibiotic discs control : sodium phosphate buffer 0.005 m (ph range-7.0). evaluation of the spirulina gel formulations involved testing their physical quality. organoleptic analysis revealed that all three formulas were dark green, aromatic, and semi-solid on day one but faded to light green by day 21 due to the thermal instability of phycocyanin (irene ragusa et al., 2021). the organoleptic results of the spirulina gel formulation can be seen in (table 3). table 3. organoleptic results of spirulina gel formulations. time formula organoleptic test time organoleptic test day 1 fi dark green, spirulina odor, gel day 21 light green, strong spirulina odor, gel day 1 fii dark green, spirulina odor, gel day 21 light green, strong spirulina odor, gel day 1 fiii dark green, spirulina odor, gel day 21 light green, strong spirulina odor, gel day 1 control dark green, spirulina odor, gel day 21 light green, strong spirulina odor, gel information: fi: formula i with carbopol concentration of 0.5% fii: formula ii with carbopol concentration of 1% fiii: formula iii with carbopol concentration of 1.5% control -: negative control with carbopol concentration of 0.5% the ph values remained within the skin-friendly range of 4 to 7 throughout the 21-day observation. the slightly acidic ph was attributed to spirulina’s protein and essential amino acid content (wiratantri, peranginangin, and sulaiman, 2024). the ph value of the spirulina gel formulation can be seen in ( table 4). table 4. ph values of spirulina gel formulations. time formula ph time ph day 1 fi 5.58±0.33 day 21 5.19±0.03 day 1 fii 4.81±0.22 day 21 4.27±0.01 day 1 fiii 5.00±0.10 day 21 4.07±0.01 day 1 k6.01±0.15 day 21 5.14±0.01 homogeneity testing confirmed that all formulations maintained uniform dispersion without separation during the study period, indicating a stable gel matrix (setyawaty, gustin, and setiyabudi, 2021). the results of the homogeneity of the spirulina gel formulation can be seen in (table 5). table 5. homogeneity of spirulina gel formulations. time formula homogeneity time homogeneity day 1 fi homogeneous day 21 homogeneous day 1 fii homogeneous day 21 homogeneous day 1 fiii homogeneous day 21 homogeneous day 1 control homogeneous day 21 homogeneous viscosity increased proportionally with higher carbopol concentrations and spirulina addition, likely due to the molecular interactions between the polymer matrix and spirulina compounds (chwil et al., 2024). the results of the viscosity of the spirulina gel formulation can be seen in (table 6). table 6. viscosity of spirulina gel formulations. time formula viscosity (cp) time viscosity (cp) day 1 fi 46.31±1.23 day 21 35.95±0.76 day 1 fii 58.78±3.64 day 21 48.26±2.22 day 1 fiii 65.42±0.62 day 21 57.07±8.49 day 1 control 41.86±0.78 day 21 39.77±0.33 spreadability values fell within an ideal range for topical gels, and a slight decrease in spreadability with increasing carbopol concentration was noted (safitri, nawangsari, and febrina, 2021) the spreadability of the spirulina gel formulation can be seen in (table 7). table 7. spreadability of spirulina gel formulations. time formula spreadability (cm) time spreadability (cm) day 1 fi 5.28±0.09 day 21 5.24±0.08 day 1 fii 5.46±0.06 day 21 5.30±0.09 day 1 fiii 5.68±0.10 day 21 5.52±0.27 day 1 control 5.22±0.03 day 21 5.22±0.04 adhesion testing showed good retention time across all formulas, with fiii exhibiting the highest adhesion time. all formulations exceeded the minimum threshold of 4 seconds (ikeda, sydney, and sydney, 2022). the yunitha et al. – spirulina gel formulations for acne 703 adhesion results of the spirulina gel formulation can be seen in table 8. table 8. adhesion of spirulina gel formulations. time formula adhesion time (s) time adhesion time (s) day 1 fi 5.92±0.29 day 21 4.92±0.34 day 1 fii 5.50±0.81 day 21 6.41±0.45 day 1 fiii 6.78±0.17 day 21 5.58±0.67 day 1 control 5.50±0.55 day 21 5.15±0.58 bacterial identification confirmed the presence of s. epidermidis. macroscopic observations on mannitol salt agar (msa) indicated no mannitol fermentation, as seen in the red-black colony color, verifying species identity. the results of macroscopic observations can be seen in (figure 1). figure 1. macroscopic identification of s. epidermidis. further validation was performed through gram staining, where microscopic observation confirmed gram-positive cocci in clusters, consistent with the morphology of s. epidermidis. the results of microscopic observations can be seen in figure 2. figure 2. microscopic identification. the catalase and coagulase tests supported these results. positive catalase activity was seen through bubble formation, and clumping in the coagulase test confirmed enzyme presence. the results of the catalase and coagulase tests can be seen in figure 3. (a) (b) figure 3. (a) catalase test and (b) coagulase test additional bacterial analysis through the imvic test revealed negative results for indole, methyl red, and citrate, but a positive result for voges-proskauer, providing biochemical evidence to support the bacterial classification. the imvic s. epidermidis test results can be seen in table 9. table 9. imvic test results of s. epidermidis. test result indole negative methyl red negative voges-proskauer positive citrate negative moreover, the triple sugar iron agar (tsia) test showed a red slant and yellow butt without gas or h₂s production, further confirming the identity of s. epidermidis. the results of the tsia s. epidermidis test can be seen in table 10. table 10. tsia test results of s. epidermidis. cha interpretation red slant / yellow butt glucose fermentation only gas negative h₂s negative collectively, these results demonstrate that spirulinabased gel formulations are effective against s. epidermidis, with both powdered and extracted forms showing antibacterial activity. the presence of proteins, and favorable physical characteristics such as appropriate ph, viscosity, spreadability, and adhesion, further supports the formulation's potential for topical application. this aligns with previous findings on spirulina’s antimicrobial and biochemical properties, reinforcing its value in pharmaceutical and cosmetic products. however, a key limitation observed was the instability of phycocyanin, which affects product aesthetics over time. furthermore, the current study only assessed in vitro activity, necessitating future in vivo studies to confirm efficacy. future work should aim to improve pigment stability, assess longer-term physical properties under varying storage conditions, and investigate synergistic combinations of spirulina with other natural actives for enhanced antimicrobial activity. conclusion this study successfully formulated spirulina-enriched anti-acne gels using varying concentrations of carbopol (0.5%, 1%, and 1.5%) and evaluated their physical properties and antibacterial activity against staphylococcus epidermidis. all formulations met acceptable standards for organoleptic characteristics, ph, viscosity, spreadability, and homogeneity over 21 days. 704 biology, medicine, & natural product chemistry 14 (2), 2025: 699-705 the first formulation (fi, 0.5% carbopol) demonstrated the best balance of physical characteristics—high spreadability, stable ph, and good adhesiveness—while also exhibiting the highest antibacterial activity, with inhibition zones reaching up to 16.5 mm, indicating spirulina's significant antimicrobial potential. stability testing confirmed that all formulations maintained structural integrity under accelerated conditions. the findings suggest spirulina platensis is a promising natural agent for topical antibacterial applications, particularly in managing acne caused by s. epidermidis. the gel formulation not only preserves the bioactivity of spirulina but also ensures acceptable physicochemical properties essential for consumer use. this supports the viability of spirulina as an eco-friendly alternative to synthetic agents in dermatological preparations. further research should investigate the clinical efficacy of spirulina gel formulations in vivo, including human trials for acne treatment. optimization of formulation for enhanced stability, particularly under tropical climate conditions, is recommended. additionally, future studies could explore the synergistic potential of spirulina with other natural antimicrobials and assess long-term storage behavior and consumer acceptability. competing interests: the authors declare that there are no competing interests. references anvara, a. a., & nowruzib, b. 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(2025). the role of thickening agent proportions in optimizing nanoemulsion gel for dermatophytosis treatment. international journal of nanomedicine, 807–826. https://doi.org/10.3390/cosmetics8010007 https://doi.org/10.2991/ahsr.k.210127.018 https://doi.org/10.20473/jkr.v9i2.65245 https://doi.org/10.1016/j.algal.2021.102317 https://doi.org/10.20473/jkr.v9i2.61384 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 1, 2018 | pages: 15-20 | doi: 10.14421/biomedich.2018.71.15-20 issn 2540-9328 (online) comparison of detergent and ctab method for isolation of dna from salak (salacca zalacca (gaert.) voss. ‘pondoh’) namira nur arfa1,*, budi setiadi daryono2, reflinur3 1,2faculty of biology, universitas gadjah mada, jl. bulaksumur, yogyakarta, 55281, indonesia tel. +62-274-6492599, fax. +62-274-565223 3laboratory of molecular biology, balai besar bioteknologi dan sumber daya genetik pertanian (bb-biogen), bogor, indonesia author correspondency*: namiranurarfa@rocketmail.com abstract this study conducted in laboratory of molecular biology, balai besar bioteknologi dan sumberdaya genetik pertanian (bbbiogen) bogor. the aims of this study were to determine and comparing the quantity, quality and the efficiency of dna isolation result using detergent method and ctab method. the parameters observed in this study were the value of dna concentration, purity, and visualization result using gel electrophoresis. the samples were the leaves of salak ‘pondoh’ (salacca zalacca (gaert.) voss.). detergent method is a method which was developed by faculty of biology ugm, it has simple procedure and relatively affordable cost. meanwhile, ctab method is one of the commonly used methods of dna isolation protocol with relatively expensive cost. detergent method used detergent in the cell wall separation and protein removal in the sample. the ctab method used cetyltrimethyl ammonium bromide (ctab) for cell membrane separation in the sample. the research methods included dna isolation with detergent and ctab methods, pcr analysis and electrophoresis. data analysis was done quantitatively using spectrophotometric method and qualitative used electrophoresis method. the result of the study showed that dna isolation using ctab method showed higher purity compared with detergent method with the purity values ranging from 1,31,4 . meanwhile, the concentration of dna in the detergent method was higher than that of ctab with the highest concentration of 1730 µg/ml. there is no difference between the quality of genomic dna isolated by ctab and detergent methods. keywords: ctab; detergent; dna isolation; salak (salacca zalacca (gaert.) voss.) introduction indonesia is a megabiodiversity country which has enormous exotic tropical fruits, such as pinneapple, mangosteen and salak (suhandy et al., 2010). salak or known as snake fruit, salak palm and snake palm (lim 2012) belong to genus salacca reinwardt. so far 21 species of salacca have been identified (supapvanich et al., 2011) and dispersed naturally in malesia area, such as birma, thailand, malaysia, philliphines, cambodia, sumatera, java and borneo (mogea 1980). salacca growth on primary tropical rain forest with optimal temperatures 220c320c and high humidity (ni made 2005). salak has sweet taste, crunchy texture and high nutritional and economical value. salak plant classified into palmae family and can be planted in many regions in indonesia, either by cultivation or growing as a wild plants (haryanto 2010). salak fruit can be made as cuisine, such as salad, pickles, jam or crackers (lim 2012). three kinds of salak which already cultivated are salacca sumaterana becc. in sidempuan, (salacca zalacca (gaert.) voss.) in bali, ambon and also salacca wallichiana in thailand. salacca zalacca var zalacca divided into some local cultivar, such as salak bongkok in sumedang, salak petruk in ambarawa, salak pondoh, salak gading and salak kembang arum in sleman, salak condet in condet and salak nglumut in magelang (harsono & hartana 2003). salak ‘pondoh’ is a new cultivar originated from yogyakarta (supriyadi et al., 2002). the varieties of salak pondoh that had been cultivated in indonesia are salak pondoh hitam, salak pondoh merah, salak pondoh kuning and salak pondoh super (purnomo, 2001). indonesia exported high quality salak fruit to some countries such as singapore, middle east country, netherlands, hongkong and china (lim, 2012: 433). salak plants need rain fall about 200-400mm/month and usually growth undershades with sun intensity range from 50%-70%. salak growth optimal in ph 6,5 and temperature between 200c300c (suskendriyati et al., 2000). salak ‘pondoh’ (salacca zalacca (gaert.) voss.) is a dioecious plants which are spiny, clustered and grows as a dense clump. the fruit is ovoid shaped, has brown skin which is look like a snake skin (supriyadi et al., 2002). the leaves are pinnate or palmate with range from 4-7m long (ni made, 2012:8) the stem are partly erect and short. biodiversity of salak plants are high in each cultivar. variation in skin type, fruit color, taste and size showed that the plants has high genetic variability. genetic condition is an important factor in plant breeding, such as crossing over and to extend genetic variability (annisaurrohmah et al., 2014). genetic condition of a https://doi.org/10.14421/biomedich.2018.71.15-20 16 biology, medicine, & natural product chemistry 7 (1), 2018: 15-20 plant is inherited by dna. in salak plant, dna isolation is needed to obtain the pure dna for genetic analysis in salak plant breeding. dna isolation technique is needed to obtain dna from organism with high purity and concentration. dna isolation can conduct using sds method (sambrook et al. 1982:29), ctab (mulyani et al., 2011:2), phenol chloroform method (tenriulo et al. 2001:2) and detergent method (nasiri et al., 2005). dna isolation technique consists of three main procedures, they are cell lysis, removing the contaminant and purification of dna (milligan, 1992: 66). dna isolation method was chose based on some consideration, such as amount and the molecular weight, purity, time, species and the cost. general method of dna isolation is ctab methods, a dna isolation method which can produce high concentration and purity of dna, but the cost is expensive. faculty of biology universitas gadjah mada develop a dna isolation technique using detergent method, which is relative easy and cheap. the aim of this study was to compare the result of dna isolation of salak ‘pondoh’ using detergent method and ctab method. the method can be used to get dna isolation which is effective, easy, cheap and simple. materials and methods procedures materials the material used in this research were the leaves of salak ‘pondoh’ plant from turi, sleman, yogyakarta, aquades, pvp (polyvinylpyrrolidone), mercaptoethanol, ctab (cetyl trimethyl ammonium bromide), taq dna polymerase, taq dna buffer, dntps mix, nuclease free water, detergent, ddh2o, chloroform: isoamyl alcohol (chisam), agarose, ethidium bromide, tbe (tris boric acid edta) buffer 0,5 x, primer opa 13, loading dye, dna ladder, dna marker, mineral oil, naoac, te + rnase tools that used in this study were scissor, mortar, glassware, volumetric pipette, pipette tips, pcr thermocycler, vortex, waterbath, sentrifuge, freezer, refrigerator, autoclave, magnetic stirrer, electrophoresis machine, microwave, microtube, microplate, chemidoc software and spectrophotometer. methods the source of dna were leaves of salak ‘pondoh’ from sleman, yogyakarta. the result of dna isolation between ctab methods and detergent method were compared by seeing the optical density and the visualization of dna band after electrophoresis. 1. detergent method 0,5 gram samples of salak ‘pondoh’ leaves were extracted using mortar and stored in microtubes. after that, 1ml detergent buffer (1,4m nacl, detergent, 100 µltris hcl ph 8, 20mm edta) are added to the leaves extract. after that, the sample were separated by sentrifuge about 10 minutes at 12.000 rpm. after that the supernatant were taken from the juice and filtered using filter paper and added with isopropanol from the wall of the tubes. then the mixture are shook slowly then the dna will appear as a white fiber which is dna band. the isopropanol removed from the bottle, then the dna were added by ddh2o and stored in refrigerator. 2. ctab method preheat ctab isolation buffer, after that 0.5 grams of salak ‘pondoh’ leaves were extracted using mortar and added by 500µl buffer ctab (tris hcl ph 8,0; 1,4 m nacl; 20mm edta; 0,2% ctab; 0,2% mercaptoethanol). after that, the sample are homogenized using vortex and incubated in waterbath for 30 minutes at 650 celcius. every 10 minutes, the tube are turned upside down. after 30 minutes, the mixture are taken from waterbath and added by 500µl chisam (chloroform: isoamyl alcohol) with ratio 24:1, mixed gently and the samples are turned upside and down for 200 times until the samples are homogeneous. then, the samples are centrifuged for 10 minutes in 12.000 rpm. supernatant that formed, moved into the another microtubes. supernatant added by naoac 50µl and added cold isopropanol 500µl to precipitate the dna. then the samples were centrifuged in 12000 rpm for 10 minutes to precipitate the dna. remove the liquid and wash the dna using 70% ethanol and centrifuges by 5 minutes. remove the liquid and dry the dna for a night. add the dna with 100µl te rnase and incubated in 370c about 30-60 minutes. 3. pcr analysis amplification of dna was performed by pcr type my cycler machine from biorad, using primer opa 13 and total pcr reaction of 20 μl. rapd condition setting was initial denaturation at 94ºc for 4 min followed by 44 cycles from 94ºc for 1 minute, 37ºc for 1 minutes, and 72ºc for 2 minutes. after that the temperature was maintained at 72° c for 10 minutes, then the temperature was lowered to 4° c. table 1. pcr composition table. 1 ddh2o 13,55 µl 2 10x buffer 2 µl 3 10 mm dntps 0,2 µl 4 taq polymerase 0,25 µl 5 dna 2 µl 6 5 um primer 2 µl total volume 20 µl arfa et al. – comparison of detergent and ctab method for isolation of dna … 17 figure 1. the settings of pcr program. 4. electrophoresis  1 making buffer solution 10xtris borat edta (tbe) 108 grams tris, 55,2 grams boric acid were added into a 1000 ml beaker glass and added by aquades up to 700 ml. the solution was stirred with a magnetic stirrer until homogen. next, 9,2 grams of edta was added and stirred back with a magnetic stirrer and fed into a 500 ml measuring flask and added to a volume of 500 ml. the solution is shaken out to homogeneous. the available buffer solution was 10x concentrated buffer solution, to obtain 0,5x buffer solution, 10x buffer solution is taken 50ml and fed into 1000 ml of measuring flask. then plus aquades up to 1000 ml and the solution shaken until homogen.  preparation of agarose gel 2% 2 grams of agarose was weighed in 100 ml of 0,5x tbe solution and allowed to stand at room temperature for 1 minute. the solution was heated by microwave about 2 minutes to a perfect solute solution. the solution is allowed to stand at room temperature to keep it from overheating. the agarose solution is poured onto a mold with a combed comb to make a well on the gel. 0,5 x tbe solution was added to the surface of the solid gel and then the comb was lifted slowly. place agarose then placed on electrophoresis tank in submerged condition of 0,5x tbe solution.  electrophoresis dna the dna samples and dna markers were prepared. a 5 μl dna marker was inserted into the left most edge well. the diluted dna in te buffer 5 μl then mixed with 3 μl of loading dye, then inserted into the 2nd well to the next. after all samples were put into a well, the electrophoresis apparatus was closed and connected to a power source. the power supply was set to 100 v for a 60 minutes and turned on by pressing the on button. once it was done, the tool was turned off. gel was removed along with its mold from electrophoresis fittings. the gel was stained with ethidium bromide for 25 minutes then rinsed with aquades. gel was observed with uv transilluminator. the dna bands will glow and observed with chemidoc software. data analysis data analysis conducted by quantitative dan descriptive qualitative of dna profile using detergent and ctab method. the factor compared are as follows: 1. quantitative factor quantitative factor which measured were optical density from both dna profile, including dna total, rna total, protein, purity and concentration. 2. qualitative factor qualitative factor that compared were the result of electrophoresis of dna profile using detergent and ctab method after pcr. results and discussion table 1. optical density of dna isolation product from salak (salacca zalacca (gaert.) voss.) was done using detergent method. no concentration (µg/ ml) purity λ260 λ280 1. 725,0433 1,1798 0,073 0,061 2. 767,4141 1,1666 0,077 0,0 3. 486, 2714 1,1989 0,049 0,041 4. 1025,8585 1,1842 0,103 0,087 5. 1076,1797 1,1406 0,108 0,094 6. 1102,3815 1,1891 0,110 0,093 7. 1730,4355 1,1255 0,173 0,154 8. 854,9668 1,0139 0,085 0.080 table 1 showed optical density of dna isolation product from salak leaves using detergent method, the purity of dna isolation product range from 1,01391,1989 with dna concentration has value 486,2713 1730,4355(µg/ ml). 18 biology, medicine, & natural product chemistry 7 (1), 2018: 15-20 table 2. optical density of dna isolation product from salak (salacca zalacca (gaert.) voss.) by ctab method. no concentration (µg/ ml) purity λ260 λ280 1. 850,3145 1,333 0,085 0,064 2. 937,99 1,4926 0,094 0,063 3. 1112,24 1,4388 0,111 0,077 4. 909,6155 1,441 0,091 0,063 5. 858,0715 1,5533 0,086 0,055 6. 690,5455 1,4703 0,069 0,047 7. 687,5588 1,4232 0,069 0,048 8. 491,976 1,4700 0,049 0,033 table 2 showed optical density of dna isolation product from salak leaves using ctab method, the purity of dna isolation product range from 1,333-1,553 with dna concentration value 491,9761112,24 (µg/ ml). detergent ctab figure 1. result of dna isolation product after pcr using primer opa 13 (5’-cagcacccac-3’). discussion dna isolation is the first stage in genetic engineering (faatih, 2009). dna isolation defines as a procces to extract dna from cell organism, and get a pure dna from another organelles and materials in cells, such as polysaccharides, lipid, rna and protein. the process of dna isolation consists of several stages: cell separation, separation of dna from other components and purification of dna (yusdiana, 2008). the process of dna separation is done by mechanical or physical means using mortar, this is done to extract dna, cell wall, cell membrane and nuclear membrane so that expected dna can come out of cell. the next process is the separation of dna from various organelles and other cell components such as proteins, cellulose, lipids, rna. the last process is a dna purification process that aims to purify the dna so that the results of dna isolation easily observable and can be judged both in quantity and quality. optimization of dna isolation method can be conducted by arranging the lysis buffer composition and method in extract dna from another compounds in cells (milligan, 1992). dna isolation methods are of several types, such as simple methods using detergents, nucleon ™ phytopure ™ genomic dna extraction kit method and ctab (cetyl trimethyl ammonium bromide) method. ctab/nacl dan sds method are conventional and popular dna isolation method (fitriya et al., 2011) in extraction of plant which contains high polyphenol and polysaccharides (ardiana, 2009). in this study we used two methods of dna isolation and compared the results. detergent method is using detergent in the process of cell separation, while the ctab method used ctab buffer for cell separation process. the buffer solution in the detergent method consists of edta, detergent and nacl. salt solution is involved in regulating osmotic pressure in cells. the salt content of the buffer will cause hypertonic conditions outside the cell so that the cells will experience plasmolysis. detergent will damage the cell wall of the organism. after that the sample was centrifuged by using a centrifuge with a speed of 12000 rpm. this centrifugation serves to separate the cell debris or the larger part of the pellet and the smaller, dna-containing supernatant (langga et al., 2012). the difference methods of dna isolation can produce different dna isolation product. furthermore, the supernatant was taken using a micropipette and inserted into a new microtube. after that, the supernatant were filtered using filter paper and moved into new microtubes. then isopropanol were added from the microtubes wall, this addition was done to prevent isopropanol from evaporating and to avoid spontaneous reactions between the dna with isopropanol. after that the tubes were turned back slowly to form dna threads. after the dna threads were visible, the tube was centrifuged at a rate of 12000 rpm for 5 minutes. the isopropanol were removed then the dna pellet was dried and dissolved in a tebuffer solution that acts as a dna solvent and stored in the freezer. in the use of isolation method with ctab buffer, the process of separation was done by ctab (cetyl trimethyl ammonium bromide). first, ctab buffers were made with a composition of 2% ctab, 0.5 m edta of 20 μm, 1m tris hcl 1 μl, 2%pvp, 0,2 % mercaptoethanol and 5 m nacl of 1.4 μl. first ctab buffer was preheated into waterbath at 65ºc. the sample was extracted using mortar until soft. the samples were moved into a 1.5 ml micro-tube and added 500μl of the heated extraction buffer and then mixed well. incubate the at 65ºc for 30 minutes and every 10 minutes the tube was reversed for the process. after 30 min, the mixture was taken from the waterbath and allowed to stand at room temperature for 2 minutes and added with 500 μl of chloroform and isoamic alcohol mixture at a ratio of 24: 1. after that the material spinned down for 5 minutes, it aims to mixed the samples with chloroform. after that, centrifuged for 15 minutes at a speed of 12000 rpm. the supernatant formed was then removed and transferred into a new micro tube. the function of incubation in 650c and presipitation using chloroform: isoamyl alcohol was to remove proteins in the cells (semagn et al., 2006). the functions of adding polyvinylpyrrolidone (pvp) in the arfa et al. – comparison of detergent and ctab method for isolation of dna … 19 early stage of dna isolation in the ctab method was to bind the phenolic compounds and to minimize the oxzidation of phenolic compounds (utami et al., 2012). pvp also can produce thick and clear dna bands. the supernatant was then added 1/10 ch3coona and then added with 2 / 3x isopropanol volume for dna precipitation. the sample was then centrifuged at a rate of 12000 rpm for 10 minutes to precipitate dna. the liquid was then removed and the precipitate washed with 70% ethanol for 10 minutes to precipitate the dna and centrifuged again for 5 minutes. the liquid was removed and dried. after that the dna was dissolved with 50 μl of te buffer. after the isolation process was complete, a total quantitative dna analysis was performed using spectrophotometry and qualitative analysis using electrophoresis. table 1 showed that the purity of dna isolated results with ctab method is better than detergent method. the detergent method has purity value 1,3-1,5 meanwhile detergent method has value 1,0-1,19. a pure dna has ratio odλ 260/λ280 between 1,8-2,0 (sambrook et al., 1989). optical density with value <1 showed that the dna isolation product was contaminated by compounds such as polysaccharides, protein or phenolic compounds. meanwhile, optical density with value >2 showed high rna accumulation in dna isolation product. the result showed that the samples are still contaminated with compounds such as proteins or polysaccharides. dna concentration in the detergent method has a higher concentration than the dna concentration by the ctab method. in the detergent method, the highest concentration value is 1730,4355 (µg/ml) while the ctab method has highest value 1112, 24 (µg/ml). based on the isolated dna concentration, it is known that dna isolation using detergent method is better than using ctab method. the total dna was qualitatively done using electrophoresis technique. stages of electrophoresis technique is done by gel preparation of 2% agarose. in the running stage electrophoresis was done by using 2 μl samples, loading dye 2 μl and h2o as much as 1 μl. loading dye plays a weight and as a marker of dna to enter into the wells. running electrophoresis was performed with a 100 volt voltage for 30 minutes. after running finished, electrophoresis results are inserted into etbr (ethidium bromide) to color the dna. ethidium bromide is capable of inserting between the nitrogen bases in the dna helix. once stained, electrophoresis results are seen under ultraviolet light by using a uv transluminator. pcr rapd are conducted to make sure the quality of dna. pcr is a process of multiplying nucleotide sequences by enzymatic methods at a certain temperature. there are several major components in pcr, including dna fragments, primers, deoxyribonucleotides triphosphate (dntp) and dna polymerase enzymes. the pcr stage consists of several stages: predenaturation, denaturation, elongating and post elongation. the time required is about 4 hours 31 minutes and there are 44 cycles. pcr was performed using pct-100 programmable thermal controller with rapd program. thermocycler is a device capable of rapidly changing the different temperatures required in the pcr cycle (louie et al., 2000: 475). in addition, another primer is used for pcr mj research machine with pcr time 04:31:36. in pcr used primer opa 13 (5'-cagcacccac-3') .in addition to the opa primer 13, there were several primers used for the analysis of rapd of salak plants, such as opa 20 (ediwirman, 2015). the results show that electrophoresis results after in pcr using detergent method and ctab method was not much different. this was characterized by the number of bands and the appearance of dna bands. the results showed the presence of dna at 200bp to 1100 bp. conclusion from the result of the research, it can be concluded that the result of dna isolation (salacca zalacca (gaert.) voss.) using ctab method has better purity but using detergent method has higher dna concentration. there is no difference in the quality of the detergent method and ctab method. further research about the modification of the detergent method is needed to obtain higher purity. acknowledgements the authors gratefully acknowledge balai besar bioteknologi dan sumber daya genetik pertanian (bb biogen), bogor, indonesia for supported this study. references ardiana d w. 2009. 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[indonesian]. comparison of detergent and ctab method for isolation of dna from salak (salacca zalacca (gaert.) voss. ‘pondoh’) biology, medicine, & natural product chemistry 7(1) 2018 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1193-1199 | doi: 10.14421/biomedich.2025.142.1193-1199 issn 2540-9328 (online) effectiveness of antioxidant compounds in minaserua herbal drink from bima as a functional food on blood pressure and cholesterol levels anisah1*, baiq dewi sukma septiani2 1bachelor of nutrition study program, faculty of health, bumigora university, indonesia. 2bachelor of nutrition study program, faculty of public health, university of jember, indonesia. corresponding author* anisah@universitasbumigora.ac.id abstract hypertension and hypercholesterolemia are major risk factors for degenerative diseases with increasing prevalence. minaserua, a traditional herbal drink from bima made from black glutinous rice, ginger, turmeric, clove, javanese chili, black pepper, palm sugar, and coconut milk, is rich in bioactive compounds and potentially reduces blood pressure and cholesterol levels. this study employed a prepost control group design. participants were divided into an experimental group, receiving minaserua daily for 2–4 weeks, and a control group, receiving no intervention. blood pressure and cholesterol levels were measured before and after the intervention. antioxidant activity was analyzed using the dpph spectrophotometric method. statistical analysis was performed using paired t-tests and independent t-tests. minaserua exhibited strong antioxidant activity with an ic-50 of 41.30 mg/ml. the experimental group showed significant reductions in total cholesterol, ldl, triglycerides, and significant increases in hdl (p < 0.05), along with significant decreases in systolic and diastolic blood pressure (p < 0.05). the control group showed no significant changes, except for a marginal increase in hdl. minaserua herbal drink effectively improves lipid profiles and lowers blood pressure, supporting its potential as a functional food for preventing hypertension and hypercholesterolemia. keywords: minaserua; antioxidant; functional food; blood pressure; cholesterol. abbreviation: ace: angiotensin-converting enzyme, aoac: association of official analytical chemists, dpph: 2,2-diphenyl-1picrylhydrazyl, hdl: high-density lipoprotein, ic₅₀: half maximal inhibitory concentration, ldl: low-density lipoprotein, no: nitric oxide, tg: triglyceride. introduction cardiovascular disease remains a leading cause of global morbidity and mortality, with hypertension and dyslipidemia (including high cholesterol) as key risk factors. hypertension is defined as systolic blood pressure exceeding 140 mmhg and diastolic blood pressure exceeding 90 mmhg (peltzer & pengpid, 2018). according to data from the 2023 indonesian health survey (ski), the prevalence of hypertension in the population aged 18 years and older in indonesia is 30.8% (kemenkes ri, 2024). meanwhile, hypercholesterolemia is a lipid metabolism disorder characterized by elevated total blood cholesterol levels, exceeding the standard limit of 240 milligrams per deciliter (mg/dl). cholesterol in the blood is carried by lipoproteins, which are divided into three classes: low-density lipoprotein (ldl), high-density lipoprotein (hdl), and triglycerides (tg). the prevalence of cholesterol worldwide is around 45%, in southeast asia, around 30%, and in indonesia, 35% (maramy et al., 2024). if not properly managed, both conditions can lead to serious complications such as coronary heart disease, stroke, and kidney failure (law et al., 2023). preventive approaches through functional foods are gaining increasing attention due to the potential of bioactive plant compounds to reduce oxidative stress and improve lipid profiles and vascular function. recent meta-studies and reviews suggest that the mixture of phytochemicals (phenolics, flavonoids, and other compounds) in herbal ingredients may contribute to lowering blood pressure and improving lipid parameters (maramy et al., 2024). minasarua (often written as "mina sarua") is a traditional drink of the mbojo people (bima, west nusa tenggara) made from a combination of sticky rice tape (tape ketan/black sticky rice), spices such as ginger, turmeric, pepper, cinnamon, cloves, and brown sugar (blondo). the use of these spices is traditionally believed to have a warming effect and promote wellbeing; the spice composition is also reported to be rich in manuscript received: 07 october, 2025. revision accepted: 06 december, 2025. published: 11 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1193-1199 1194 biology, medicine, & natural product chemistry 14 (2), 2025: 1193-1199 antioxidants, which supports the drink's potential as a functional food. several studies have examined minasarua formulations, demonstrating compositional variability and a relationship between composition and antioxidant activity (verawati et al., 2023). research on minasarua formulation and optimization suggests that the ratio and combination of spices (ginger, turmeric, javanese chili, pepper, black sticky rice tape, and blondo) influence minasarua's total phenols, total flavonoids, anthocyanins, and antioxidant activity. several optimization studies have reported increased antioxidant activity after formula adjustments. however, despite evidence of in vitro antioxidant activity, direct evidence on the effects of minasarua consumption on clinical parameters, such as blood pressure and cholesterol levels, in humans (or animal models) remains very limited or has not been widely published. therefore, experimental research is needed to link minasarua's antioxidant content with tangible physiological effects, including lowering blood pressure and reducing ldlcholesterol (maramy et al., 2024). previous research on the antihypertensive and hypolipidemic effects of traditional spice mixtures or herbal medicines has shown promising results, both in animal models and in limited human clinical trials, providing a strong scientific basis for exploring minasarua as a functional food(cicero et al., 2021). this traditional beverage contains various bioactive compounds, including flavonoids, polyphenols, and essential oils, which are known to play a role in antioxidant and anti-inflammatory mechanisms, as well as modulating lipid metabolism and blood pressure. in addition to its potential health benefits, developing minaserua as a functional product also has a socioeconomic dimension, supporting the preservation of local wisdom while increasing economic value for the bima community, particularly if this product can be formulated, standardized, and scientifically evaluated within a modern functional food framework (verawati et al., 2023). the increasing prevalence of hypertension and hypercholesterolemia, along with their close association with cardiovascular diseases, underscores the urgent need for preventive and therapeutic approaches based on natural products. although various studies have confirmed the antioxidant potential of traditional herbal ingredients, scientific evidence on the direct physiological effects of locally formulated herbal drinks such as minaserua remains limited. considering its rich composition of antioxidant compounds including flavonoids, phenolics, and anthocyanins minaserua is believed to have the potential to improve lipid metabolism and regulate blood pressure. therefore, this study was conducted to analyze the antioxidant activity and bioactive compound content of the minaserua herbal drink using the dpph spectrophotometric method and to evaluate its effectiveness in lowering blood pressure and cholesterol levels through experimental intervention in humans. materials and methods this research was conducted in two main stages: laboratory analysis and experimental testing. the laboratory analysis used the dpph (spectrophotometric) method to measure the antioxidant activity and bioactive compound content of minaserua herbal drink. the dpph (2,2-diphenyl-1-picrylhydrazyl) method is widely used in plant extract and herbal drink research due to its simplicity, speed, and sensitivity to free radical scavenging activity (kingori et al., 2024). according to recent research, the dpph method has been simplified without reducing measurement accuracy, and can be validated according to aoac guidelines (aklimah & ekayanti, 2022). the next stage employed a pre-post control group experimental design, where the sample was divided into two groups: an experimental group that received minaserua herbal drink daily for 14 days (2 weeks) and a control group that received no intervention. blood pressure and cholesterol levels were measured before (pre) and after (post) the intervention to assess the changes that occurred. because the results of the normality test indicated that the data were not normally distributed, statistical analysis was performed using the non-parametric wilcoxon signed-rank test, an alternative to the paired t-test for paired data with non-normal distributions (indra ruswadi et al., 2023). this test is commonly used in health intervention research to analyze significant changes between preand post-values in the same group. procedure for making minaserua herbal drink: a. prepare the ingredients (main ingredients: black glutinous rice, coconut milk, spices: ginger, cloves, javanese chili, turmeric). b. wash the glutinous rice, then steam it until cooked (about 30-40 minutes) c. once cooked, remove from heat and let cool to room temperature. d. grind the fermented cassava yeast into a powder. e. ferment the glutinous rice: (sprinkle the ground yeast into the cooled glutinous rice and stir well. then store in a closed container in a warm and cool place for 2-3 days to ferment until the glutinous rice becomes soft and has a distinctive aroma.) f. prepare the herbal solution: grate the coconut and squeeze out the coconut milk, grate the ginger, turmeric, pepper, and javanese chili until smooth. boil the coconut milk with the finely ground spices in 2 liters of water until it comes to a boil. add the brown sugar and stir until completely dissolved. then strain the solution to separate the pulp from the spices. anisah & septiani – antioxidant and cardioprotective effects of minaserua herbal drink 1195 g. mix the herbs: mix the fermented glutinous rice into the prepared herbal solution. filtered, then stir until evenly mixed figure 1. minaserua herbal drink. laboratory test procedures figure 2. antioxidant test flow using the dpph-spectrophotometry method. this study involved 84 subjects aged 25–60 years who had a history of hypertension and elevated cholesterol levels. the subjects were divided into two groups: an experimental group consisting of 42 participants who received the minaserua herbal drink, and a control group of 42 participants who were given a placebo in the form of plain or similar water. the intervention was carried out for 14–28 days with a consumption frequency of 1–2 times per day, with an adult serving size of 150–200 ml (equivalent to one medium glass). during the intervention period, daily monitoring was conducted to assess adherence as well as measurements of blood pressure (systolic and diastolic), reductions in total cholesterol, ldl, and triglycerides, and increases in hdl levels. statistical analysis included paired t-tests to evaluate changes within each group and independent t-tests to compare differences between the two groups. results and discussion results laboratory test results this study aimed to determine the antioxidant compounds contained in the minaserua herbal drink and to examine the effectiveness of administering minaserua herbal drink, a traditional beverage from bima, in reducing blood pressure and cholesterol levels (verawati et al., 2023). based on laboratory test data using the dpph method, the results showed an increase in % inhibition along with the increase in sample concentration, with the following results: based on laboratory test data using the dpph method, the % inhibition as the sample concentration increases, with the following results: table 1. results of the antioxidant analysis test table. no. sample code concentration (c, mg/ml) sample absorbance dpph absorbance % inhibition ic₅₀ (mg/ml) 1 s1.1 16.11 0.531 0.694 23.49 2 s1.2 32.21 0.429 0.694 38.18 3 s1.3 48.32 0.287 0.694 58.65 41.30 4 s1.4 64.43 0.152 0.694 78.10 5 s1.5 80.53 0.078 0.694 88.76 based on the results of the antioxidant activity test using the dpph method on minaserua samples, it was found that the percentage of inhibition increased with increasing solution concentration, with the highest inhibition value of 88.76% achieved at a concentration of 80.53 mg/ml. the ic₅₀ value obtained of 41.30 mg/ml indicates that minaserua has moderate to vigorous antioxidant activity. these results indicate that minaserua has the potential to be a herbal drink with the ability to capture free radicals, so it can support efforts to prevent degenerative diseases related to oxidative stress. antioxidant test steps dpph reagent preparation weigh dpph powder dissolve in 100 ml of methanol (0.1 mm) sample preparation prepare minaserua extract in a series of concentrations (10 ppm160 ppm) mixing the sample and dpph mix 1 ml of sample solution + 1 ml of dpph solution incubate for 30 minutes at room temperature (dark) absorbance measurement measure with a uv-vis spectrophotometer at 517 nm dpph control + solvent, blank: solvent + solvent calculation of % inhibition % inhibition = (control abs sample abs) (control abs x 100%) 1196 biology, medicine, & natural product chemistry 14 (2), 2025: 1193-1199 result analisis data univariate analysis a. responden criteria based on gender, age based on (intervensi) table 2. distribution of respondents by age and gender. gender elderly(>60 year) productive (25-60) total male 2 (4,76%) 14 (33,33%) 16 female 4 (9,52%) 22 (52,38%) 26 total 6 36 42 based on the analysis of the distribution table of respondents by gender and age category, among the total 42 respondents, the majority were in the productive age group (15-64 years), comprising 36 people (85.71%), while the elderly group numbered six people (14.29%). when viewed by gender, female respondents dominated, comprising 26 individuals (61.90%), including 22 respondents in the productive age group (52.38%) and four elderly respondents (9.25%). meanwhile, male respondents numbered 16 people (38.10%), consisting of 14 productive respondents (33.33%) and two elderly respondents (4.76%). overall, these results indicate that most of the study respondents were women of productive age. b. respondent criteria based on gender, age based on (control) table 3. distribution of respondents based on age and gender of control group respondents. gender elderly(>60 year) produktif (15-59) total male 4 (9,5%) 8 (19,0%) 12 female 17 (40,5%) 13 (31,0%) 30 total 21 21 42 based on the distribution table of respondents by gender and age, it is evident that the majority of respondents were women, with the highest percentage in the productive age group (52.38%), while productive men accounted for 33.33%. this indicates that the productive age group comprised the majority of respondents (85.72%), while the elderly group accounted for 14.28%. analysis bivariate a. intervention group table 2. wilcoxon data analysis results (intervention group). variable change z p-value conclusion total cholesterol significant -5.672 0.000 significant change hdl significant -5.770 0.000 significant change ldl significant -5.679 0.000 significant change triglycerides (tg) significant -5.677 0.000 significant change systolic blood pressure significant -5.705 0.000 significant change diastolic blood pressure significant -5.669 0.000 significant change the analysis of the intervention group revealed that administering the treatment resulted in a significant improvement in cardiovascular health indicators. there were notable reductions in total cholesterol, ldl, and triglyceride levels, accompanied by a significant increase in hdl concentration (p < 0.05), indicating an enhanced lipid profile. additionally, both systolic and diastolic blood pressures showed significant decreases (p < 0.05), suggesting improved vascular function. collectively, these findings confirm that the treatment had a substantial positive impact on the participants' lipid metabolism and blood pressure, thereby contributing to overall cardiovascular health improvement. based on the results of the wilcoxon test, the intervention significantly reduced the average systolic and diastolic blood pressure, as well as total cholesterol, ldl, and triglyceride levels, while significantly increasing hdl levels. b. control group table 3. wilcoxon data analysis results (control group). variable change z p-value conclusion total cholesterol not significant -0.617 0.537 no significant change hdl slight increase -2.000 0.046 marginally significant change ldl not significant -1.947 0.052 no significant change triglycerides (tg) not significant -1.414 0.157 no significant change systolic blood pressure not significant -1.342 0.180 no significant change diastolic blood pressure not significant -1.342 0.180 no significant change anisah & septiani – antioxidant and cardioprotective effects of minaserua herbal drink 1197 the the analysis of the control group revealed that the placebo administration did not produce any significant effects on participants' lipid profiles or blood pressure. total cholesterol, ldl, and triglyceride levels showed no significant changes before and after treatment (p > 0.05), while hdl levels exhibited a marginally significant increase (p = 0.046). similarly, both systolic and diastolic blood pressures remained relatively stable, with p-values greater than 0.05, indicating no statistically significant difference. overall, these results suggest that the placebo intervention had no measurable impact on lipid metabolism or blood pressure regulation, confirming that the observed improvements in the intervention group were likely attributable to the active treatment rather than external factors discussion of results the antioxidant activity test using the dpph method aims to evaluate the ability of compounds in minasarerua samples to reduce free radicals. based on the test results, the percentage of inhibition increases with increasing sample concentration. at a concentration of 16.11 mg/ml, the percentage of inhibition is only 23.49%, whereas at the highest concentration (80.5 mg/ml), the inhibition reaches 88.76%. this indicates that the antioxidant activity of minaserua is concentrationdependent, where the higher the concentration of the extract, the greater its ability to capture free radicals (cicero et al., 2021). the ic₅₀ value of minaserua was 41.30 mg/ml, indicating extreme general antioxidant activity. thus, minaserua can be categorized as having quite good antioxidant activity. this antioxidant activity is likely contributed to by the content of bioactive compounds, such as anthocyanins, flavonoids, and other phenolic compounds, commonly found in spices and pigmented plants (prior et al., 2005). these compounds work by donating hydrogen atoms to neutralize dpph free radicals, thereby reducing oxidative stress. these results support minaserua's potential as a natural herbal drink with antioxidant activity that is beneficial for health, particularly in preventing degenerative diseases associated with free radicals, such as hypertension, diabetes, and cardiovascular disease (li et al., 2024). the effectiveness of this antioxidant is directly related to the intervention results in respondents, where there was a significant decrease in total cholesterol, ldl, and triglyceride levels, as well as an increase in hdl. furthermore, systolic and diastolic blood pressure also experienced a significant decrease. these findings are in line with a recent study of the antioxidant activity of minaserua measured in vitro (dpph); ic ₅₀ = 41.30 mg/ml; maximum inhibition 88.76%), indicating significant and concentration-dependent radical scavenging ability. the ic ₅₀ value is within the range, indicating relatively strong activity when compared with various commercial plant extracts; however, it should be emphasized that comparisons of ic ₅₀ between studies must take into account differences in extraction methods, solvents, initial dpph concentrations, and units of measurement (mg/ml) (gulcin & alwasel, 2023). this strengthens the evidence that antioxidant effects not only occur in vitro but also have a positive impact on cardiovascular health. this effect aligns with previous research, which shows that consuming antioxidant-rich foods can improve lipid profiles and enhance endothelial function by increasing nitric oxide (no) bioavailability (ruhdiana & sandi, 2023). phenolic and flavonoid compounds in minaserua can neutralize free radicals and reduce lipid peroxidation, thereby inhibiting the formation of oxidized ldl (oxldl), which contributes to the development of atherosclerotic plaque. reduction of ox-ldl reduces the vascular inflammatory process and may slow or alleviate endothelial dysfunction associated with increased blood pressure and poor lipid profiles. literature support: review of the protective effects of polyphenols on endothelium and atherogenesis (iqbal et al., 2023). several flavonoids are known to modulate the expression and stability of the ldl receptor (ldlr), and enzymes regulating cholesterol metabolism can increase ldlr expression, thereby enhancing ldl clearance from plasma. this may provide a direct molecular pathway for reducing total and ldl cholesterol after consuming these beneficial compounds. recent molecular evidence suggests that flavonoids alter ldlr regulation through several transcriptional/posttranslational mechanisms (bjune et al., 2024). polyphenols/anthocyanins can increase nitric oxide (no) production by endothelial cells, reduce endothelial oxidative stress, and decrease inflammatory adhesion, resulting in improved vasodilation and lower blood pressure. several ongoing studies and reviews report improvements in vascular parameters (flow-mediated siltation, vasorelaxation) after polyphenol/anthocyanin supplementation (laudani et al., 2023). other studies also suggest that anthocyanins can improve endothelial function and potentially lower cholesterol and hypertension (kiptiyah et al., 2017). plant extracts often show ace-inhibitory activity in vitro (capable of inhibiting angiotensin-converting enzyme) and modulate cholesterol metabolism enzymes (hmg-coa pathway for cholesterol-bile conversion). the in vitro ace-inhibitory activity of some herbal extracts suggests a direct antihypertensive pathway (mahmud et al., 2024). polyphenols also reduce systemic inflammatory markers (crp, il-6) that play a role in dyslipidemia and hypertension. furthermore, some polyphenols modulate the gut microbiota, which influences lipid metabolism. these combined effects may translate in vitro antioxidant activity into in vivo metabolic effects (iqbal et al., 2023). several plant extracts also exhibit ace-inhibiting activity in vitro, providing an additional antihypertensive pathway. the combination of antioxidant, antiinflammatory, metabolic modulation, and vasoactive 1198 biology, medicine, & natural product chemistry 14 (2), 2025: 1193-1199 enzyme effects explains how the administration of the minaserua herbal drink can rapidly reduce cardiometabolic parameters in early intervention trials. these findings are consistent with recent literature on the benefits of polyphenols on cardiovascular health. dpph data and interim clinical results from the minaserua herbal drink study support them (bayani et al., 2024). discussion the results of the study showed that the herbal drink minaserua exhibits significant antioxidant activity, as determined by the dpph test, with an ic₅₀ value of 41.30 mg/ml. this value indicates that minaserua is classified as having vigorous antioxidant activity, because the concentration required to inhibit 50% of free radicals is relatively low. this high antioxidant activity is most likely influenced by the flavonoids, phenolics, and anthocyanins contained in the basic ingredients of minaserua, as also from the results of research by (verawati et al., 2023). the antioxidant efficacy was directly related to the intervention outcomes in the respondents. there was a significant decrease in total cholesterol, ldl cholesterol, and triglycerides, accompanied by an increase in hdl cholesterol. furthermore, systolic and diastolic blood pressure also decreased significantly. these findings align with recent research indicating that consuming beverages or functional foods rich in flavonoids and phenolic compounds can improve lipid profiles and lower blood pressure by enhancing endogenous antioxidant capacity and promoting endothelial function (tumilaar et al., 2023). thus, this study not only confirms the antioxidant activity of minaserua but also demonstrates its biological link to improved cardiovascular health. further research is needed to explore the bioavailability of the active compounds and conduct long-term clinical trials to strengthen minaserua's potential as a functional food based on local wisdom (duan et al., 2025). conclusions based on the research results in the document, it can be concluded that minaserua herbal drink has potent antioxidant activity with an ic₅₀ value of 41.30 mg/ml, indicating a good ability to capture free radicals. this antioxidant activity is believed to originate from the content of anthocyanins, flavonoids, and phenolic compounds found in its constituent ingredients, including black sticky rice, ginger, turmeric, and other spices. the results of intervention trials in humans showed that consuming minaserua for 14 days significantly reduced systolic and diastolic blood pressure, reduced total cholesterol, ldl, and triglyceride levels, and increased hdl levels (p < 0.05). statistical analysis using the wilcoxon signed-rank test confirmed that these changes were significant, while the control group showed no significant changes. these findings support that minaserua's antioxidant effects not only occur in vitro but also provide real cardiovascular benefits in vivo, through mechanisms such as increased nitric oxide (no) bioavailability, inhibition of the ace enzyme, reduced lipid peroxidation, and modulation of cholesterol metabolism. thus, minaserua has the potential to be developed as a functional food based on local wisdom, which can help improve heart and blood vessel health and prevent degenerative diseases related to oxidative stress. acknowledgements: the authors would like to thank the ministry of education, culture, research, and technology (kemdikbudristek), bumigora university, and all colleagues who assisted in the implementation of this research. authors’ contributions: anisah designed the research. anisah and baiq dewi sukma septiani analyzed the laboratory test results and conducted the community intervention. anisah analyzed the data and wrote the manuscript. all authors have read and approved the final version of the manuscript. competing interests: the authors declare no competing interests funding: this research was funded by the ministry of education, culture, research, and technology (kemdikbudristek) through the research and community service (bima) program in 2025. references aklimah, m., & ekayanti, m. 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(2024). formula optimization of traditional functional beverage minasarua from bima west nusa tenggara indonesia. trends in sciences, 21(8). https://doi.org/10.48048/tis.2024.7693 peltzer, k., & pengpid, s. (2018). the prevalence and social determinants of hypertension among adults in indonesia: a cross-sectional population-based national survey. international journal of hypertension, 2018. https://doi.org/10.1155/2018/5610725 prior, r. l., wu, x., & schaich, k. (2005). standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. journal of agricultural and food chemistry, 53(10), 4290–4302. https://doi.org/10.1021/jf0502698 ruhdiana, t., & sandi, s. p. h. (2023). kandungan gizi pisang kepok (musa paradisiaca linn) keripik pisang terhadap glukosa darah. abdima : jurnal pengabdian mahasiswa, 2(1), 3503–3508. tumilaar, s. g., hardianto, a., dohi, h., & kurnia, d. (2023). a comprehensive review of free radicals, oxidative stress, and antioxidants: overview, clinical applications, global perspectives, future directions, and mechanisms of antioxidant activity of flavonoid compounds. journal of chemistry, 2024. https://doi.org/10.1155/2024/5594386 verawati, n., aida, n., & yani, a. (2023). pengaruh perbandingan jenis jahe dan konsentrasi jahe pada karakteristik kimia, mikrobiologi minuman herbal tradisional minaserua. g-tech: jurnal teknologi terapan, 7(4), 1732–1739. https://doi.org/10.33379/gtech.v7i4.3387 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 51-55 | doi: 10.14421/biomedich.2024.131.51-55 issn 2540-9328 (online) antibacterial potential of freshener water based on siwalan coir extract (borassus flabellifer) and lemongrass (cymbopogon citratus) against airborne bacteria hana dwi andayani1, salma auliya yoviska2, widyastuti asy syaffa2, putri amifalahiya iqlima3, sriwulan2,* 1department of biology, faculty of mathematics and natural science, university of pgri ronggolawe, 2department of biological education, faculty of teacher training and education, university of pgri ronggolawe, 3department of english education, faculty of teacher training and education, university of pgri ronggolawe, jl. manunggal no 61 tuban, 62381, tel. +62-356-322233, fax. +62-356-331578, indonesia. corresponding author* biowulan08@gmail.com abstract air is an important part of life. the quality of air can be seen from three parameters, namely chemical, physical, and biological parameters. one of the biological parameters of air quality can be seen from the presence of microorganisms such as bacteria. the presence of airborne microorganisms, especially in enclosed spaces can affect human health, one of which can cause respiratory infections. on the other hand, people often use air freshener products. therefore, this research was conducted with the aim of analyzing the potential of air freshener based on siwalan (borasus flabellifer) coir extract and lemongrass as antibacterial against airborne bacteria. this research is an experimental research with a pre test-post test design. the data obtained is in the form of the value of the total plate count of indoor air bacteria before and after treatment using air freshener based on coir extract of siwalan and lemongrass. the results showed that both formulations (p1 and p2) of air freshener based on coir extract and lemongrass were able to reduce the number of airborne bacterial colonies in the room, with a significance value of 0.000<0.05. keywords: air freshener, antibacterial; lemongrass; siwalan. introduction air is one of the important parts in the life of living things. in addition to oxygen, air also contains fungi, bacteria, carbon monoxide, carbon dioxide and other gaseous materials (abidin & hasibuan, 2019). at normal limits, these substances will be neutralized (dewi et al., 2021). however, if it has exceeded normal limits and can threaten human health, then the condition is said that air pollution has occurred (arwini, 2019). some studies state that the level of air pollution indoors is greater than outdoors (rahmawati & khairina, 2020). who (2009) states that humidity and the presence of indoor microorganisms are the main causes of morbidity and mortality in the world. the cleanliness level of room air greatly affects human health because 90% of human activities are carried out indoors (bahri et al., 2021). problems related to the level of room air quality include three parameters, namely chemical, physical, and biological parameters characterized by the presence of airborne microorganisms (rahmawati & khairina, 2020). microorganisms, especially in a closed room need to be paid attention to, considering the many symptoms of diseases such as eye irritation, acute respiratory infections, and skin diseases due to infection by airborne microorganism (putra et al., 2018). in fact, the majority of cases of infection occur due to the spread of bacteria through the air (nisyak & hartiningsih, 2020). one of the microorganisms that cause infections that are often found in indoor air is staphylococcus aureus (nisyak et al., 2020). the microorganisms are able to survive in the air for four to six hours. therefore, the use of natural antibacterial needs to be developed to reduce the spread of pathogenic bacteria in the air, for example in air freshener products. air freshener also acts as an air purifier. this product releases volatile compounds into the air, so that if made of chemicals it can trigger the release of secondary pollutants (kim et al., 2015). these secondary pollutants can cause disorders of the nerves, hormones, lung and cardiovascular system, can also damage the ozone layer (singer et al., 2006). therefore, it is necessary to develop manuscript received: 11 january, 2024. revision accepted: 07 may, 2024. published: 08 may, 2024. https://doi.org/10.14421/biomedich.2024.131.51-55 mailto:biowulan08@gmail.com 52 biology, medicine, & natural product chemistry 13 (1), 2024: 51-55 air freshener with natural ingredients, such as siwalan coir and lemongrass (yoviska et al., 2022). siwalan (borassus flabellifer) coir waste from siwalan fruit that has not been widely used. fariha et al. (2020) said that 65-75% of the weight of the fruit is siwalan coir. siwalan coir contains natural antioxidants, volatile and non-volatile fatty acids (dahlan, 2011). antioxidants in coir siwalan can prevent cell damage due to free radicals (idayati et al., 2014). while lemongrass (cymbopogon citratus) contains flavonoids, saponins, polyphenols, and essential oils (departemen kesehatan republik indonesia, 2008). the lemongrass oil contain of citral compounds, essential oils with geraniol content of 65-90%, geranyl acetate 3-8%, citronellol 11-15%, citronellyl acetate 2-4%, citronellal 30-45%, cavikol, citral, kadinol, eugenol, vanillin, cadinen, kamfen, elemol, and limonene which makes lemongrass can be used as air freshener (zurairah et al., 2021). citral compounds contained in lemongrass oil play an important role as antibacterial because they can damage bacterial cell membranes (lu et al., 2018). chamdit & siripermpool (2012) in their research said that the combination of eugenol and lemongrass oil can damage the biofilm of s. aureus. therefore, this research was conducted with the aim of seeing the potential of air freshener made from siwalan coir and lemongrass as antibacterial against indoor air bacteria. thus, this air freshener based on siwalan coir and lemongrass is expected to improve air quality based on biological parameters, besides being known to also provide a relaxing effect and give a distinctive aroma to the room (yoviska et al., 2022). materials and methods this research used an experimental design conducted for 3 months (may-july 2023) at the biological laboratory of universitas pgri ronggolawe. research instruments the tools in this research were beakers, erlenmeyers, test tubes, split funnels, petri dishes, digital balances, drip pipettes, distillation and soxhletation device sets, autoclaves, incubators, colony counters, space thermometers, hygrometers, glass bowls, statifs, scissors, insulation, hot plates, masks, glove, glass bottles, tissues, paper labels and fiber reed sticks. while the ingredients used were siwalan coir, lemongrass, distilled water, 96% ethanol, 90% isopropyl alcohol, and nutrient agar (na) media. procedures air freshener making process the making process of air freshener was carried out referring to yoviska et al. (2022)’s research includes the manufacture of siwalan coir and lemongrass extracts. siwalan and lemongrass are dried 3 days and blended. after that, it was dissolved with ethanol and distilled at a temperature of 700c. formula 1 (p1) was made with a composition of 11.8% siwalan coir extract, 0.2% lemongrass extract, 0.2% isopropyl alcohol, and 87.6% distilled water. while formula 2 (p2) was 7.6% coir extract, 0.3% lemongrass extract, 0.3% isopropyl alcohol, and 91.7% distilled water. measurement of room environmental factors indoor environmental factors measured include air humidity and temperature. air temperature was measured using a room thermometer and air humidity was measured using a hygrometer. air freshener antibacterial test in this research, a room with a size of 4x4m with air conditioning was used. the number of airborne bacterial colonies was identified in the room by air capture method with a petri dish containing 10ml of sterile na media placed in an open room for 10 minutes. then, the petri dish is closed and incubated for 24 hours in an incubator at 27°c. furthermore, quantitative tests are carried out by calculating the total plate count (tpc) of bacteria. the treatment of air freshener of coir extract of siwalan and lemongrass was carried out for 3 days. furthermore, every 24 hours during the treatment, air bacteria are taken by the same method when the initial identification of the number of colonies. data analysis the data were statistically analyzed using the paired t test to determine the ability of air freshener coir extract and lemongrass in reducing the number of airborne bacterial colonies in space. while the effect of application time was tested using one way anova with a significance level of 95% (α 0.05). before the anova one-way test, a normality test was carried out on the data. results and discussion indoor environmental factors environmental factors are important parameters to measure. this is because the growth of bacteria, especially in a room is strongly influenced by environmental factors. environmental factors that can be measured in this research include air humidity and temperature. the results of measuring air humidity and room temperature in this research are shown in table 1 below. table 1. room air temperature and humidity. room air temperature (oc) humidity (%) room 1 20 26,7 room 2 20 28,2 andayani et al. – azithromycin and monosodium glutamate effect 53 temperature is a factor that affects bacterial growth directly. temperature affects the work of enzymes in bacterial cells, where enzymes are catalysts in biochemical reactions in bacterial cell metabolism. in table 1 it appears that the air temperature of the two rooms used in this research is on average 200c. this temperature is in the optimum temperature range for the growth of mesophyll group bacteria (lestari & hardisari, 2019). however, this conditions has met the requirements for healthy air temperature for the room, where the requirements for air temperature in a healthy room are based on peraturan menteri kesehatan republik indonesia (2011) is 18-300c. in the air humidity parameter, it is known that the air humidity in room 2 is slightly higher than the air humidity in room 1. however, the humidity value in both spaces in this research was lower than the humidity for indoor healthy air based on peraturan menteri kesehatan republik indonesia (2011). the government through peraturan menteri kesehatan republik indonesia (2011) had set the air humidity for healthy air requirements in the room is 40-60%. however, astuti, hastutiningrum, & sudarsono (2022) mentioned that very dry air can inhibit the growth of bacteria. air freshener antibacterial test air freshener antibacterial test based on coir and lemongrass against airborne bacteria in this research was conducted by comparing the tpc value of airborne bacteria before and after treatment. identify the number of air colonies using the air capture method, where petri dishes containing sterile na media are placed in each corner of the room with an open state for 10 minutes. this is done in order to get bacteria from the air. furthermore, incubation is carried out for 24 hours at a temperature of 27ºc. the count of the number of bacterial colonies was carried out at hours 0, 24, 48, and 72 in 2 rooms, where in room 1 formula 1 (p1) was applied and in room 2 formula 2 (p2) was applied. average data on the number of airborne bacterial colonies are presented in table 2. table 2. average number of airborne bacterial colonies in room. room number of bacterial colonies after treatment at the hour of 0 24 48 72 room 1 14 13 11 9 room 2 23 21 16 11 data in table 2 shows that air freshener applications based on coir and lemongrass are able to reduce the number of airborne bacterial colonies in space. this is due to the content of metabolite compounds, namely saponins, tannins, alkaloids, and triterpenoids that can inhibit bacterial growth (pamput, 2023). while the citral component in lemongrass oil has an important role as antibacterial because it damages cell membranes in bacteria (lu et al., 2018). however, the difference in formulations applied to room 1 and room 2, based on the results of statistical tests oneway anova that has been carried out, did not show a significant difference in reducing the number of colonies of airborne bacteria in the room (sig. 0.246>0.05). this result is influenced by the composition of the active ingredients of the coir extract used. in p1, coir extract used was 11.8% and in p2 7.6%. while p1 contains lemongrass extract as much as 0.2% and p2 as much as 0.3%. this difference does not have a significant impact on the antibacterial content in it, so it does not provide a significant difference in reducing the number of airborne bacterial colonies in room. while the percentage decrease in the number of airborne bacterial colonies after treatment using air freshener based on siwalan coir and lemongrass is shown in figure 1. based on figure 1 and the results of oneway anova analysis, it is known that the length of application of air freshener based on siwalan coir and lemongrass has a significant effect on the percentage of reducing bacterial colonies with a sig value of 0.000<0.05. this shows that the longer the application time, the greater the decrease in the number of airborne bacterial colonies in the room. this result occurs because the longer the application time, the contact of the active compounds contained in the air freshener will be more thorough to the airborne bacterial cells in the room. figure 1. percentage decrease in the number of airborne bacterial colonies in the room after treatment with air freshener based on siwalan coir and lemongrass conclusions the conclusion of this research is that formulation 1 (p1) and formulation 2 (p2) applied to two different rooms did not show a significant difference, but the two formulations from the results of one-way anova analysis found that the length of application of air freshener based on siwalan coir and lemongrass had a significant effect on the percentage of reducing bacterial colonies with a sig value of 0.000<0.05. this explains that the longer the 0 20 40 60 24 48 72 p er ce n ta ge r ed u ct io n in n u m b er o f a ir b o rn e b ac te ri al c o lo n ie s (% ) biology laboratory head room biology study program head room 54 biology, medicine, & natural product chemistry 13 (1), 2024: 51-55 application time, the number of airborne bacterial colonies in the room will also decrease greater. acknowledgements: the authors would like to thank himbior (himpunan mahasiswa biologi ronggolawe) and fsib (forum studi ilmu biologi) who have accommodated and conducted research projects through divisi penelitian dan pengembangan. thank you also to university of pgri ronggolawe for facilitating this research activity. authors’ contributions: the first author monitored the preparation and implementation of research, evaluation for each stage, and data analysis; the second author tidied up administrative matters and prepared manuscripts; the third author recorded and managed administrative costs for research and coordinate the purchase of research tools and materials and documentation; the fourth author worked on the completion of the manuscript; the supervisor played a role in directing, evaluating the results of teamwork, and finalizing article manuscripts. competing interests: the authors declare that there are no competing interests. references abidin, j., & hasibuan, f. a. 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(2022). potensi sabut siwalan (borassus flabellifer) dan ekstrak serai (cymbopogon citratus) sebagai andayani et al. – azithromycin and monosodium glutamate effect 55 air freshener dalam mengurangi tingkat stress di masa pandemi. tuban. zurairah, m., syarif, a. a., adam, m., & siregar, r. (2021). pemanfaatan hilirisasi lahan tanaman serai wangi untuk ramuan minyak atsiri pada saat pendemi covid 19. jurnal al ulum lppm universitas al washliyah medan, 9(2), 41–45. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 451-455 | doi: 10.14421/biomedich.2023.122.451-455 issn 2540-9328 (online) bio-larvicidal potential of betel leaves (piper betle l) ethanolic extract in addition of peg 400 diluent on aedes aegypti larvae listiana masyita dewi*, hilda zaniba ariffah, riandini aisyah, nurhayani medical faculty, universitas muhammadiyah surakarta jl. garuda mas, kampus 4 ums, gonilan, kartasura, sukoharjo, 57169, telp. (0271) 716844, fax (0271) 724883, jawa tengah, indonesia. corresponding author* lmd123@ums.ac.id manuscript received: 26 march, 2023. revision accepted: 14 august, 2023. published: 15 august, 2023. abstract dengue hemorrhagic fever (dhf) is a kind of vector transmitted disease, by aedes aegypti. it is one of major public health problem around the world, including indonesia, because it may lead to epidemics and death in a short time. the use of plant extracts as biolarvicidal is thought to be a promising solution, and one of them is the betel leaves (piper betle l). the addition of polyethylene glycol (peg) as a diluent is thought may increase the dispersity of plant extract in the water which is larval medium of growth. objectives: to determine the bio-larvicidal potential of 96% ethanolic extract of betel leaves (eebl) in addition of peg 400 diluent on the aedes aegypti larval mortality. material and method: betel leaves were extracted by maceration using 96% of ethanol. there are two kinds of eebl concentration used, 0.2% dan 0.4%. peg 400 was also added as diluent. the samples in this study were aedes aegypti larvae at instar iii-iv, with a total of 400 larvae. evaluation was performed at 6, 12, 18, and 24 hours. the data obtained was then analyzed by kruskall wallis test and post-hoc mann whitney test. result: in all of treatment groups, larval mortality was reached 100% at 24 hours. from the kruskall wallis test, p-value obtained was <0.05. from post-hoc mann whitney test, the p-value obtained in the comparation between treatment groups and positive control group was >0.05, and the p-value obtained in the comparation between treatment groups and negative control group was <0.05. conclusion: eebl in addition of peg 400 diluent is potential as bio-larvicidal on aedes aegypti larvae. it is also known that eebl at concentration of 0.2% and 0.4% in addition of peg 400 are as effective as temephos as larvicides on aedes aegypti larvae. keywords: betel leaves; peg, bio-larvicidal; aedes aegypti. abbreviations: dhf: dengue hemorrhagic fever; peg: polyethylene glycol; eebl: 96% ethanolic extract of betel leaves. introduction dengue hemorrhagic fever (dhf) is one of vector transmitted disease, that mostly known around tropics area. dhf is transmitted by aedes aegypti mosquito and it is still a major public health problem around the world, including in indonesia. this disease may cause epidemic and lead to death in a short time (karyanti and hadinegoro, 2016). data reported in the indonesian health profile in 2021, the number of dhf cases has exceeded 100,000 cases, with 0.7% of them ending in death. the spread of dhf in indonesia is mainly in sumatra and java island, especially in urban areas with a high level of mobility and also in densely populated residential areas (kementerian kesehatan republik indonesia, 2022). aedes aegypti is the main vector of dengue hemorrhagic fever (dhf) and chikunguya. aedes aegypti is a member of the order diptera, brassicaceae. the use of synthetic larvicide in controlling disease vectors, including aedes aegypti, has several negative impacts which may lead to resistance and environmental pollution (waskito and cahyati, 2018). alternative medicine to overcome those problems are being highly investigated. who also recommends proven safe and effective traditional medicinal plants, to be incorporated into the national health system (who, 2018). one of indonesia's native plants that are highly observed was betel leaves (piper betle l). however, betel leaves are evergreen, that are commonly used as flavoring in chewing areca nut (betel nut chewing) (patra, et al., 2022). betel leaves contain several active compounds such as alkaloids, flavonoids, phenolics, saponins, tannins, and also essential oils (maryanti, manalu and lesmana, 2022). these chemical compounds have biolarvicides effect and not necessarily owned by other plants (noshirma, 2019). water is the growth medium for larvae. but, when we applied plant extract on it, sometimes they are hard to dissolves well. one of polyether compounds that is often added in chemical formulation as a dispersant agent, is polyethylene glycol (peg). the addition of peg, https://doi.org/10.14421/biomedich.2023.122.451-455 452 biology, medicine, & natural product chemistry 12 (2), 2023: 451-455 especially peg 400 as a diluent is thought may increase the dispersity of plant extract in the water (hutanu, et al., 2014). previous study performed by sembiring and hasan (2020) stated that the minimum concentration of eebl resulting larval mortality is 2.5%, but it’s only reached 60% at first 6 hours of observation. it needs longer time to reached 100% of larval mortality. it is thought that by adding a dispersant agent, such as peg 400, may increase the extract solubility in the water, so that active substance will be easily contact to the larva and caused death. by considering those conditions, this study aimed to determine the bio-larvicidal potential of 96% ethanolic extract of betel leaves (eebl) in addition of peg 400 diluent on the aedes aegypti larval mortality. materials and methods ethanolic extract of betel leaves (eebl) processing betel leaves collected were washed thoroughly, and then dried under the sun for about a week. when they were completely dry, it is continued by chopped them up using blender so that they turn into powder, and the simplicial were obtained. those simplicial were then weighed and added by 96% of ethanol. this blend was stirred in a few minutes and then left to stand all day, repeated every day, for a week. this process resulting a macerate production, which was need to be concentrated by rotatory evaporator and water bath. hereby, a thick extract was formed. this thick extract was prepared in two variation concentration, 0.2% and 0.4%. this step was performed in pharmacology laboratory, universitas muhammadiyah surakarta. figure 1. dried process of betel leaves under the sun. figure 2. concentrating the macerate using rotatory evaporator. sedimentation test we prepared two container for two variation concentration of eebl. the first container was for 0.2% of eebl, and the other one was for 0.4% of eebl. each of the container were filled with eebl which had been obtained from the extraction process, as much as 0.02 ml and 0.04 ml, respectively. at next, every container was added with 0.3 ml of peg 400 and then added with distilled water until the volume reached 10 ml. the mixture was stirred until homogeneous and then observed for 24 hours to see whether a precipitate formed. this step was also performed in pharmacology laboratory, universitas muhammadiyah surakarta. aedes aegypti larval preparation aedes aegypti’s eggs that had been prepared on filter paper sheets were used the filter paper sheets were placed to a container which has been filled by distilled water before. prepare fish feed and blender a little. mix the fish feed that has been blended slightly, to the container prepared before. wait for 2 to 3 days, so that the eggs will drip. this step was performed in parasitology laboratory, universitas muhammadiyah surakarta. larvicidal test for this study, we adopt the guidelines for laboratory and field testing of mosquito larvicides by who (2005). there were 4 study groups designed. they are the negative control group, the positive control group, the treatment group with 0.2% of eebl, and the treatment group with 0.4% of eebl. a 3% of peg 400 was used as negative control, while 1% of temephos was used as positive control. repetition was done for 4 times, so that we prepared 4 containers for each study group. total number or larva involved in this study was 400, with 25 larvae prepared in each of container. in negative control group, each of container were added with 3 ml of peg 400 and distilled water until the volume reached 100 ml, while in positive control group, each of container were added with temephos and distilled water until the volume reached 100 ml. in the treatment group with 0.2% of eebl were added with 0.2 ml of eebl, 3 ml of peg 400 and distilled water until the volume reached 100 ml. dewi et al. – bio-larvicidal potential of betel leaves (piper betle l) … 453 and in the treatment group with 0.4% of eebl were added with 0.4 ml of eebl, 3 ml of peg 400 and distilled water until the volume reached 100 ml. larval mortality was observed every 6 hours, for 24 hours. the larvae are considered to be dead when they sink or appear unresponsive when touched using a stick. all of this process was performed in parasitology laboratory, universitas muhammadiyah surakarta. data analysis the number of larval-death in each study group and in each observation-period were recorded, and analyzed using kruskall wallis test and post-hoc mann whitney test. results and discussion sedimentation test from both containers prepared for this test, it was obtained that no precipitate formed, directly nor in 24 hours of observation. it means that the addition of peg 400 may increase the dispersion of eebl in water, so that active substance kept soluble in it. larvicidal test in larvicidal test, we count the number of larval mortalities in each study group and in each repetition performed. the data obtained presented in the following table. table 1. larval mortalities. study group larval mortalities (mean ± standard deviation) percentage of larval mortalities in 24 hours 6 hours 12 hours 18 hours 24 hours negative control group 0±0 0±0 0±0 0±0 0% positive control group 25±0 25±0 25±0 25±0 100% 0.2% of eebl 25±0 25±0 25±0 25±0 100% 0.4% of eebl 25±0 25±0 25±0 25±0 100% discussion in first study group, the negative control group, the percentage of larval mortality was 0%. there was no single larva were found to be dead in this study group. here, we could say that peg 400 has no larvicidal effect, especially on aedes aegypti larva. zhong, et al. (2013) stated that peg 400 is a kind of hydrophilic polymer which is inexpensive and non-toxic. peg 400 also has high solubility in water and any other solvent, such as alcohol and acetone. this addition of peg, or we call it by pegylation, aimed to increase the solubility of plant extract in water, so that the active substance will be evenly distributed, and may increasing its exposure to the larva. but, in other side, it’s non-toxic effect will not affect the larval death, so that the larval mortality in this study was purely caused by active substance of the plant extract, not by peg. therefore, the usage of peg as negative control in this study was appropriate. the second study group, that is the positive control group, which using 1% of temephos as larvicidal agent, it was found that the percentage of larval mortality was 100%. all of aedes aegypti larva in this group were considered to be dead since first-6 hour of observation. temephos was neural toxic for larva. there is acetylcholine accumulation in larval tissue because cholinesterase inhibition by temephos. this results in hyperexcitation, tremor, and convulsion of larva, resulting fatigue and lead to death. those mechanism was proven by the determination of temephos as part of aedes aegypti eradication program in indonesia (pambudi, et al., 2018). hence, the determination of temephos as positive control in this study was appropriate. in the treatment groups, 0.2% and 0.4% of eebl, the percentage of larval mortality was 100%. the same condition to the positive control group. it means that eebl was proven to contain active substances that are have larvicidal effect on aedes aegypti larva. various literatures state that betel leaves contain a number of secondary metabolites that are larvicidal, such as alkaloids, flavonoids, saponins, tannins, essential oils, and many more. most of them are mainly act by interference the central nervous system via cutaneous or respiratory absorption. in this type of intoxication, there is acetylcholinesterase (ache) inhibition which may lead to death. and as we know, this mechanism was similar to temephos. some other mechanisms of action were involving gaba system, leading to seizures and inhibition of mitochondrial activity (de souza wuillda, et al., 2019; sembiring and hasan, 2020; and kumara, et al., 2021). another active substance that is thought to have a strong larvicidal effect from betel leaves is caryophyllene. this substance causes a damage on cellular function because it binds strongly to the ns3 protease in the nucleus (prabhu, et al., 2022). the data in table 1 was then statistically analyzed. to see the normality of data distribution, the saphiro-wilk test was used, and the p-value obtained was <0.05. meanwhile, to see the homogeneity of data distribution, the homogeneity of variance test was used, and the pvalue obtained was <0.05. based on these two results, it can be said that the requirements for using the anova test were not met, so an alternative test with krusskal 454 biology, medicine, & natural product chemistry 12 (2), 2023: 451-455 wallis test was used, and the p-value obtained was <0.05. here, we conclude that there is at least one group of data that is significantly different, so the analysis continued with post hoc mann whitney test. comparison of data in the negative control group and the treatment groups, both 0.2% and 0.4% of eebl, obtained the p-value of <0.05. it means that the data in both of study group was significantly different. furthermore, it can be interpreted that eebl with a concentration of 0.2% and 0.4% with addition of peg diluent, was a potential bio-larvicides to aedes aegypti larva. different condition appears when compared to study performed by rosyadi and swastika (2020), where eebl was also used but without the addition of peg, it was seen that there was a difference in the larval mortality within 24 hours. in rosyadi and swastika's study (2020), in eebl with a concentration of 0.2% and 0.4%, larval mortality at 24 hours was 44% and 65%, respectively. whereas in our study, with the same concentration and observation time, but with the addition of peg, larval mortality reached 100%. this result reinforces the previous findings that eebl with a concentration of 0.2% and 0.4% with addition of peg diluent, was a potential bio-larvicides to aedes aegypti larva. the addition of peg 400 as a diluent effectively increases the solubility of eebl in the water, so that it’s active substances evenly distributed and exposed to the larva. we also compared our data in the positive control group and both of treatment groups, where the resulting p-value was >0.05. it means that the data in all of study group was similar. in other words, we could say that eebl in concentration of 0.2% and 0.4% with addition of peg have similar effectivity as larvicide on aedes aegypti larva. conclusions ethanolic extract of betel leaves (piper betle l) in addition of peg 400 diluent is potential as bio-larvicidal on aedes aegypti larvae. it is also known that ethanolic extract of betel leaves at concentration of 0.2% and 0.4% in addition of peg 400 are as effective as temephos as larvicides on aedes aegypti larvae authors’ contributions: listiana masyita dewi designed the study. hilda zaniba ariffah carried out the laboratory work and analyzed the data. listiana masyita dewi and hilda zaniba ariffah wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: authors state that there is no competing interests. funding: the study was funded by universitas muhammadiyah surakarta. references de souza wuillda, a. c. j., martins, r. c. c., & costa, f. d. n. (2019). larvicidal activity of secondary plant metabolites in aedes aegypti control: an overview of the previous 6 years. natural product communications, 14(7). https://journals.sagepub.com/doi/full/10.1177/1934578x19862 893 hutanu, daniela; frishberg, mark, d.; guo, ling; & darie, costel, c. (2014). recent applications of polyethylene glycols (pegs) and peg derivatives. mod chem appl 2014, 2:2. https://www.researchgate.net/publication/287444069_recent_ applications_of_polyethylene_glycols_pegs_and_peg_deri vatives karyanti, m.r. & hadinegoro, s.r. (2016) ‘perubahan epidemiologi demam berdarah dengue di indonesia’, sari pediatri, 10(6), p. 424. available at: https://doi.org/10.14238/sp10.6.2009.424-32. kementerian kesehatan republik indonesia. (2022). profil kesehatan indonesia 2021. https://www.kemkes.go.id/downloads/resources/download/pus datin/profil-kesehatan-indonesia/profil-kesehatan-2021.pdf kumara, c. j., nurhayani, n., bestari, r. s., dewi, l. m., (2021). efektivitas flavonoid, tanin, saponin dan alkaloid terhadap mortalitas larva aedes aegypti. proceeding of the 13th university research colloquium 2021: kesehatan dan mipa. http://repository.urecol.org/index.php/proceeding/article/view/ 1311 maryanti, e., manalu, j.r., yolazenia, y., lesmana, s.d., mislindawati. (2022) efek larvasida ekstrak etanol daun sirih (piper betle l) terhadap larva aedes aegypti. jurnal ilmu kedokteran (journal of medical science). 16(1). http://jik.fk.unri.ac.id/index.php/jik/article/view/479 noshirma, m., willa, r. w. 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(2022). impact of piper betle l. bioactive compounds in larvicidal activity against culex quinquefasciatus. journal of natural pesticide research. 2. https://www.sciencedirect.com/science/article/pii/s277307862 2000139#bib46 rosyadi, f.a., swastika, i.k. (2020). efektivitas ekstrak etanol daun sirih (piper betle l.) sebagai larvisida terhadap larva aedes aegypti di kecamatan denpasar selatan, kota denpasar, bali. jmu. 9(9). https://ojs.unud.ac.id/index.php/eum/article/view/65102 sembiring, f.k., hasan, r.s.b. (2020). the effectiveness test of betel leaf extract toward the development of aedes https://www.researchgate.net/journal/applied-biochemistry-and-biotechnology-1559-0291 https://www.researchgate.net/journal/applied-biochemistry-and-biotechnology-1559-0291 dewi et al. – bio-larvicidal potential of betel leaves (piper betle l) … 455 aegypti sp larvae. biospecies. 13(1). https://www.onlinejournal.unja.ac.id/biospecies/article/view/8386 waskito, p.e. and cahyati, w.h. (2018). efektivitas granul daun salam (eugenia polyantha wight) sebagai larvasida nyamuk aedes aegypti, spirakel, 10(1), pp. 12–20. available at https://doi.org/10.22435/spirakel.v10i1.603. who. (2005). guidelines for laboratory and field testing of mosquito larvicides. world health organization communicable disease control, prevention and eradication who pesticide evaluation scheme. https://www.who.int/publications/i/item/who-cdswhopes-gcdpp-2005.13 who. (2018). antimicrobial resistance and primary health care: technical series on primary health care. world health organization.https://apps.who.int/iris/handle/10665/326454 zhong, x., dou, g., wang, d. (2013). polyethylene glycol (peg400): an efficient and recyclable reaction medium for the synthesis of pyrazolo[3,4-b]pyridin-6(7h)-one derivatives. molecules. 18, 13139-13147. https://www.researchgate.net/publication/258957692_polyethy lene_glycol_peg400_an_efficient_and_recyclable_reaction_medium_for_th e_synthesis_of_pyrazolo34-bpyridin-67h-one_derivatives this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 83-89 | doi: 10.14421/biomedich.2025.141.83-89 issn 2540-9328 (online) the effect of methanol seed kernel extract of mangifera indica on guinea pig tracheal smooth muscles imaobong christopher etti1,*, akinbola lukuman akinniyi1, imoh ime johnny2, adebayo tologbonse adedoyin1 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, nigeria. 2department of pharmacognosy and natural medicine, faculty of pharmacy, university of uyo, nigeria. corresponding author* ettiimaobong@gmail.com manuscript received: 11 december, 2024. revision accepted: 20 march, 2025. published: 23 april, 2025. abstract mangifera indica, commonly known as mango, holds a revered position in traditional medicine for the numerous health benefits derived from different parts of the plant. the purpose of this study was to investigate the effect of methanol seed kernel extract of magnifera indica on isolated guinea pig trachea. the acute toxicity profile was first evaluated as well as its phytochemical screening before exposing various concentrations of the methanol seed kernel extract to acetylcholine (ach)-precontracted guinea pig tracheal rings. from the results, a dose of 5000mg/kg of the extract did not produce any form of toxicity. the presence of tannins, flavonoids and cardiac glycosides was revealed which probably fuels the medicinal properties of the seed kernel. exposure of acetylcholine pre-contracted tracheal rings to different concentrations of the methanol kernel extract, resulted in a shift of the ach dose response curve to the right but without achieving any maximum relaxation. there was no observed ic50 but rather an ic30 concentration was observed around 0.0013m. however, the observed minimal relaxation of ach pre-contracted rings was not statistically significant (p≥0.5). it is possible that the observed minimal relaxation potential of the kernel extract coupled with its reported anti-inflammatory properties boosts its application in the relief of bronchial asthma claimed in ethnomedicine. keywords: asthma; mangifera indica; guinea pig trachea; acetylcholine. introduction bronchial asthma is a chronic airway disease characterised by a significant morbidity and mortality and imposes a daunting socioeconomic burden globally (nunes et al., 2017); (ge et al., 2013). according to the world health organization, an estimated 262 million individuals were affected globally with about 455,000 deaths in 2019. in nigeria, the prevalence ranges from 5.1% to 14.3% (musa et al., 2014) with studies indicating that approximately 15% of adults over 45 years of age experience wheezing episodes annually (gbd 2019 diseases and injuries collaborators, 2020). essentially, asthma is marked by airway inflammation and hyper*responsiveness (bradding et al., 2024) which leads to wheezing, coughing and shortness of breath. this inflammatory process can manifest in acute, subacute, or chronic forms, resulting in alterations in airway tone, increased vascular permeability, neural activation, enhanced mucus secretion, and structural changes in the airways, which may be reversible or permanent (tirgar et al., 2012). the pharmacological management of asthma typically involves two primary approaches: the inhibition of inflammation, often achieved through corticosteroids such as prednisolone, and the alleviation of bronchospasms using bronchodilators like salbutamol (kondo & tamaoki, 2018). these pharmacologic interventions are effective in minimizing or eliminating symptoms, stabilising pulmonary function, and enabling patients to maintain an active lifestyle without significant limitations, including exercise (kondo & tamaoki, 2018). unfortunately, unlike natural products (etti et al., 2016); (etti et al., 2020), these treatments are accompanied by adverse events, particularly in patients with comorbid conditions. challenges such as drug resistance, the high cost of medications coupled with dwindling economies especially in low-income countries and the inevitable side effects of synthetic drugs have spurred interest in alternative treatments derived from natural sources, which are perceived as more accessible, affordable, and less toxic. consequently, a substantial proportion of the population, especially in developing countries, utilizes herbal remedies alongside conventional medicine (adefolaju, 2014); (singh et al., 2004). mangifera indica l., commonly known as mango, is a succulent drupe from the family of anacardiaceae https://doi.org/10.14421/biomedich.2025.141.83-89 84 biology, medicine, & natural product chemistry 14 (1), 2025: 83-89 within the sapindales order. mangoes are esteemed not only for their palatable fruit but also for their rich content of micronutrients, vitamins, and diverse phytochemicals (jahurul et al., 2015). in addition to their nutritional value, various components of the mango tree, notably the seed kernel, have been utilized in traditional medicine for their purported therapeutic benefits. the seed kernel has been reported to be a repository of bioactive compounds. traditional medicine practices have claimed the efficacy of mango seed kernel extracts in alleviating respiratory conditions, such as asthma. the scientific validation of such claims remains limited, in spite of these assertions. thus, this work aims to investigate the smooth muscle relaxant effects of mango seed kernel extract, to validate the ethnobotanical claims. materials and methods extraction an 88.60g weight of powdered seed sample was macerated using an extraction tank and extracted using 3.5 l of 70% ethanol. this was done by adding solvent to the seed sample in the extraction tank and allowing the mixture to stand for 72 hours with frequent agitation by stirring twice a day. after 72 hours, the extract was filtered and the filtrate obtained was concentrated to dryness using a water bath at a temperature of 49oc. the obtained extract of 11.55g was stored in a sealed container at 4 oc in a refrigerator within the experimental period. the percentage yield of the plant extract was calculated. laboratory animals guinea pigs of either sex weighing 450–500 g and swiss albino mice of either sex weighing between 20–30 g were used for the experiments. the guinea pigs were purchased from an animal vendor from ikot-ekpene, akwa ibom state, nigeria and were used for antiasthmatic evaluations. the mice were purchased from the animal house, department of pharmacology and toxicology, university of uyo. all animals were kept in plastic cages; males were separated from females and allowed two weeks of acclimatization. the animals had free access to dry rodent pellets and tap water and were exposed to natural light/dark cycle and room temperature. they were handled according to standard protocols for the use of laboratory animals (national institute of health, 2002). the experimental protocol was approved by the faculty of pharmacy institutional animal care and use committee of the university of uyo. qualitative analysis of phytoconstituents the methanol seed extract of m. indica was subjected to qualitative chemical screening for secondary plant metabolites using conventional methods (sofowora, 1993). test for saponins frothing test: the seed powder extract (0.5 g) was shaken vigorously with 5 ml of distilled water in a test tube for 1 minute. frothing, which persisted during warming indicated the presence of saponins (sofowora, 1993). test for tannins ferric chloride test: the seed powder extract (0.5 g) was mixed with 5 ml of distilled water and filtered. 2 drops of 5% ferric chloride were added to the filtrate. a blue-black. precipitate was seen as evidence for the presence of tannins (evans, 2009). test for flavonoids magnesium metal test: a few pieces of magnesium metal were added to 5 ml of seed powder root extract solution and 1 ml of concentrated hydrochloric acid was added. the presence of orange colour was taken as an indicator of flavonoids (evans, 2009). sodium hydroxide test: the seed powder extract (0.5 g) was dissolved in 3 ml of distilled water. few drops of 5% sodium hydroxide were added. a yellow colouration indicated the presence of flavonoids (evans, 2009). test for cardiac glycosides salkowski's test: the seed powder extract (0.5 g) was dissolved in 4 ml of chloroform. concentrated sulphuric acid was gently added by running it down the side of the test tube to form a distinct lower layer. a reddish-brown colouration at the interphase indicated the presence of a steroidal cardiac glycoside (sofowora, 1993). killer-killianı test: the seed powder extract (0.5 g) was dissolved in 5 ml of glacial acetic acid containing one drop of 5% ferric chloride solution. the solution was then underlay with 1 ml concentrated sulphuric acid by slowly running it down the test tube side to form a distinct lower layer. a brown ring at the interphase indicated the presence of glycoside (evans, 2009) lieberman's test: the seed powder extract (0.5 g) was dissolved in 4 ml acetic anhydride and cooled sufficiently in ice. concentrated tetraoxosulphate (v1) acid was carefully added to form the lower layer. a colour change from violet to blue to green was taken as evidence for the presence of a steroidal nucleus; the aglycone portion of the cardiac glycoside (evans, 2009). test for alkaloids the seed powder extract (0.5 g) was heated with 10 ml of 5% hcl in a test tube filled with boiling water. the mixture was allowed to cool and then filtered. a few drops of dragendorff’s precipitation reagent were added and observed. a yellow or red precipitate was taken as indication of the presence of alkaloids (evans, 2009). etti et al. – the effect of methanol seed kernel extract of mangifera indica … 85 oral acute toxicity of methanol seed extract of magnifera indica the median lethal dose (ld50) of the seed extract of magnifera indica was estimated in mice using a modified lorke’s method (lorke, 1983); (etti et al., 2024). the animals were randomly allotted to five groups of three animals each. group i was orally administered 2 ml/kg of distilled water and groups ii–v were orally administered 2000, 3000, 4000, and 5000 mg/kg of m. indica extract concentrations respectively. the animals were deprived of feed 24 hours prior to treatment but had free access to water before administration of single doses of the seed extract of m. indica. the general behaviors of mice were observed continuously for 1 hour after treatment and intermittently for hours and thereafter over a few days for any sign of toxicity and death. isolated guinea pig tracheal ring experiments seed extract of m. indica was tested on isolated tracheal rings obtained from guinea pigs (ozolua et al., 2011). the guinea pig was mercifully sacrificed, the trachea was dissected out quickly and placed in a petri dish containing physiological salt solution (pss). the trachea was cleaned of adherent connective tissues as much as possible and cut into rings of 2.5 mm in length. the rings were suspended in l-shaped wire loops in 50 ml organ baths containing the pss and thereafter sensitized. the composition of the pss (krebs) was (g/l): nacl 6.9, kcl 0.353, mgso4.7h20 0.2, glucose 2.0, cacl2 0.28, nahco3 2.10. the pss was bubbled throughout the experiment with a 95% o2 and 5% co2 gas mixture (boc gases, nigeria plc) and the temperature was maintained at 37 °c. responses were measured with a force displacement transducer (ft 302) which was connected to a grass 7d polygraph (grass instrument co, quincy, ma, usa). the rings were given a resting force of 1 g and allowed an equilibration period of 45 min during which the pss was changed four times. after equilibration, the rings were depolarized with 80 mmkcl before experimental protocols. anti-asthmatic effect was evaluated by first applying cumulative concentrations of histamine (1 × 10-5 to 1 × 10-3 m) to the organ baths. then in the presence of different concentrations (5 x 10-6 mg/ml, 5 x 10-5 mg/ ml, 5 x 10-4 mg/ml and 5 x 10-3 mg/ml) of the extract, cumulative concentrations of histamine were applied. the concentrations of histamine producing 50% of maximum response (ec50) and maximum response (emax) by the tracheal rings in each experiment were calculated. the spasmolytic effect was evaluated by precontracting tracheal rings with the ec50 of histamine (2 × 10−4 m). at maximum contractile response, cumulative concentrations of m. indica seed extract (5 x 10-5 5 x 10-3 mg/ml) were injected into the organ bath. similar concentration-response relationships were obtained of acetylcholine (1 × 10-5 to 1 × 10-1 m) in rings pre-contracted with the ec50 of histamine. the emax was compared across the three concentrations of the extract. statistical analysis the statistical evaluation was performed using non-linear regression in graphpad prism software (graphpad software inc., san diego, usa). all the experimental groups were compared appropriately to assess the significance using student’s t-test. data are presented as mean ± sem (standard error of the mean) ic50 (concentration producing 50% inhibition of maximum contractile response after pre-contraction) and emax (maximum relaxant response) values were estimated graphically. statistical significance was set at p< 0.05. results and discussion figure 1. magnifera indica seed kernel extraction process. percentage yield of magnifera indica seed extract the percentage yield of methanol magnifera indica seed extract was calculated to be 13.0% from an initial weight of 88.6g using the formula below (equation 1). % yield = 𝑊𝑒 𝑊𝑠 x 100 (1) where: we if weight of dried extract obtained (g) and wsis the initial weight seed sample (g) phytochemical constituents of magnifera indica seed extract the preliminary phytochemical screening of the methanol seed extract of magnifera indicarevealed the 86 biology, medicine, & natural product chemistry 14 (1), 2025: 83-89 presence of various secondary metabolites such as tannins, flavonoids, cardiac glycosides as shown in the table below. table 1. phytochemical constituents of methanol seed extract of magnifera indica. s/n test observation inference 1. alkaloid yellow solution with no precipitate was observed − 2. saponin there was no preserve of frothing while warming − 3. tannins blue-black coloration was observed + ++ 4. flavonoids an orange coloration was observed + + 5. salkowskis(cardiacglycosides) a reddish-brown color was observed at the interphase + + 6. keller brown ring at the interphase was observed + + 7. leibermann slight yellow solution was observed with no reddish brown at interphase − acute toxicity studies of magnifera indica seed extract magnifera indica seed extract did not cause any sign of toxicity after the oral administration of any of the oral doses (2000mg/kg, 3000mg/kg, 4000mg/kg, 5000mg/kg). the median lethal dose for the extract was thus concluded to be greater than 5000mg/kg. no notable gross toxicological symptom was seen within the period of observation. effect of acetylcholine and histamine on contraction of guinea pig tracheal rings the two contractile agents, acetylcholine and histamine were shown to produce a dose-dependent increase in contraction of the tracheal rings. from the ec50 values, histamine produced more contractility when compared with acetylcholine (figure 2). both contractile agents produced a ceiling effect and had the same efficacy, indicated by their maximal attainable response. figure 2. effect of acetylcholine on contraction of guinea pig tracheal rings. values are expressed as mean ± sem for acetylcholine and histamine concentration. effect of methanol seed kernel extract of magnifera indica on the dose response curve of acetylcholine the magnifera indica seed kernel extract was seen to minimally reduce the tracheal contraction elicited by acetylcholine (figure 3) 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 100 0 20 40 60 80 100 ach histamine log concentration (m) % m ax im u m c o n ce n tr at io n figure 2: effect of acetylcholine on contraction of guinea pig tracheal rings. values areexpressed as mean ± sem for acetylcholine and histamine concentration. etti et al. – the effect of methanol seed kernel extract of mangifera indica … 87 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 100 0 20 40 60 80 100 ach alone ach with extract log concentration (m) % r e la x a ti o n figure 3. effect of magnifera indica seed kernel extract on guinea pig tracheal rings. values are expressed as mean ± sem. discussion plants have long been observed and reported to contain an array of bioactive compounds that not only benefit the plants but also offer diverse pharmacological activities to humans. mangifera indica l., a tropical fruit widely consumed for its nutritional value is no exception to the rule. it has been recognized to possess abundant bioactive compounds. while much attention has been devoted to the fruit pulp and peel, the seed kernel—a byproduct of mango processing—remains underutilized, despite its reported medicinal properties. studies have highlighted the presence of numerous phytochemicals in the mango seed kernel, including phenolic acids, flavonoids, tannins, and saponins, which are associated with numerous activities like: antimicrobial, antiinflammatory, antioxidant, antidiabetic, and hepatoprotective activities. this study aims to validate the assertion made by traditional practices on the relevance of the mango seed kernel extract in airway respiratory disease by performing a phytochemical screening of the mango seed kernel extract and evaluating its possible tracheal smooth muscle relaxant effect. this study revealed the large safety margin (oecd/ocde 423 2001) of the extract, which is similar to that of previously reported studies (okoro et al., 2016). the phytochemical screening of mangifera indica seed kernel extract revealed the presence of various bioactive compounds, including alkaloids, gums, flavonoids, phenols, saponins, steroids, tannins, and xanthoproteins. these phytochemicals are known for their diverse pharmacological properties and their ability to confer health benefits, many of which may have been implicated in respiratory health. tannins, a prominent component identified in the extract, are polyphenolic compounds known for their astringent properties. they can bind and also contribute to a plant’s defense mechanism (mcgarvey et al., 2006). medicinally, tannins exhibit anti-inflammatory and antioxidant properties (he et al., 2015) which could play a role in mitigating airway inflammation, a key factor in bronchial asthma (okuda & ito, 2011). flavonoids, another important class of phytochemicals identified in the extract, have been well-documented for their potent antioxidant, bronchodilatory, anti-inflammatory (riella et al., 2012) and immune-modulating effects as well as anti-cancer properties (etti, et al., 2017). these compounds can inhibit the release of inflammatory mediators such as histamine and leukotrienes, thereby reducing airway hyperresponsiveness and promoting bronchodilation (middleton et al., 2000). quercetin, a representative flavonoid, has been specifically highlighted for its capacity to relax tracheal smooth muscle and suppress allergic airway inflammation (carrillo-martinez et al., 2024). this glucocorticoid-like effect may be helpful for prophylaxis and long-term treatment of asthma (d’urzo et al., 2015). flavonoids inhibit antigen-induced release of histamine from mast cells (park et al., 2008) and also inhibit contractions induced by broncho-constrictors such as histamine among many other anti-asthmatic properties (tanaka & takahashi, 2013). many plants with similar phytoconstituents as magnifera indica have also exhibited anti-asthmatic properties (nagore et al., 2009). spasmogens such as histamine and acetylcholine activate calcium release and utilization processes that accentuate airway smooth muscle contraction (panula p. . et al., 2015). histamine produced more contractility when compared with acetylcholine (figure 1). effect of histamine (1 x 10-5) as shown on the graph, showing some level of contractile effect on the tracheal musculature used with cumulative dosing shows greater more significant effect. furthermore, the result of the effect of the histamine (1 x 10-4mg/ml) shows an 88 biology, medicine, & natural product chemistry 14 (1), 2025: 83-89 increasing contractile effect on the tracheal ring.when the extract (5 x 10-4mg/ml) was used to challenge the contractile effect of histamine (1 x 10-4mg/ml), there was no marked difference in the contraction seen, that is, the extract did not significantly affect the normal contractile responses induced by histamine. the log concentration versus relaxation curves for acetylcholine alone, and acetylcholine in the presence of the extract as shown in fig. 2, indicate that the methanol seed extract of magnifera indica did not significantly relax the tracheal contraction elicited by acetylcholine in guinea pigs. no ic50 was observed in the pre-contracted tracheal rings when exposed to the seed kernel extract. there was no statistical difference between the ach-pre-contracted tracheal rings and rings exposed to the plant extract (p≥0.5). however, an inhibitory concentration of 20% was observed at about 25 x 10-5 m concentration of the extract. salbutamol gave a percentage maximum relaxation of 100% at just a single dose of 1 x 10-4 (data was not included in the graph). the minimal relaxation to the acetylcholine pre-contracted guinea pig tracheal ring is an indication of the anti-inflammatory action of substances to promote the relief of bronchospasm induced by spasmogens such as histamine and acetylcholine (goldie et al., 1986).the minimal or negligible bronchodilatory effect observed in this study is an indication that the claims made by traditional practitioners are likely due to the collective antiinflammatory and anti-oxidant effect of the seed kernel extract as mediated by its bioactive phytochemicals. conclusion this study investigated the phytochemical composition and the effect of methanol seed kernel extract of mangifera indica on guinea pig tracheal smooth muscles. the phytochemical screening revealed the presence of abundant flavonoids and tannins, cardiac glycosides, along with other bioactive components known for their anti-inflammatory and bronchodilatory properties. these findings support the potential of mango seed kernel extract as a source of pharmacologically active compounds with relevance to respiratory health. despite the promising phytochemical profile, the extract's effect on tracheal smooth muscle relaxation was relatively modest, achieving an ic20 value but not reaching an ic50 threshold. this suggests limited direct efficacy in inducing bronchodilation under the experimental conditions used. while the observed effects may point to subtle pharmacodynamic activity, they also highlight the need for further investigation into the extract's mechanism of action and potential synergistic effects in vivo. competing interests: the authors declare that there are no competing interests. references adefolaju, toyin. 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(2012). evaluation of antiasthmatic activity of dried whole plant extract of leucas aspera using various experimental animal models. international journal of phytopharmacology (vol. 3, issue 3). this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 2, 2017 | pages: 59-62 | doi: 10.14421/biomedich.2017.62.59-62 issn 2540-9328 (online) alliin as a natural bioactive from single bulb garlic (allium sativum) for nitric oxide (no) increasing in atherosclerotic process based on insilico screening riza rahayu ilmawati1, ahya zhilalikbar amin2, mohamad amin3 1postgraduate, univesitas negeri malang, jl. semarang 5, malang, indonesia 2student of sma negeri 3 malang, sultan agung utara nomor 7 kota malang, indonesia 3department of biology, faculty of mathematics and sciences, universitas negeri malang, jl. semarang 5, malang, indonesia author correspondency: mohamad.amin.fmipa@um.ac.id3 abstract atherosclerosis is a chronic inflammatory disease in the arterial wall mediated by proinflammatory factor. atherosclerosis cause heart disease and stroke is generally believed to be preventable. the single bulb garlic is one of the local plants known as the medicinal plant in indonesia. garlic contains alliin compounds as antiatherosclerotic agent. this study aimed to determine the potential of active compound of single bulb garlic as an antioxidant that improve endothelial function by increasing nitric oxide (no) to increase vasodilation. the bioinformatics webserver used in this study are: pubchem, pharmmapper, swiss target prediction, superpred, uniprot, and protein data bank. docking software using pyrx, pymol and discovery studio. based on these steps, it was found that alliin interacts with nitric oxide synthase (nos) through hydrogen and alkyl bonds. alliin is effective as an inhibitor of nos based on binding afinity (-5.4 kcal / mol) although no more negative than an aspirin inhibitor based on binding afinity (-6.5 kcal / mol). based on the docking results, it is found that alliin is effective as a potential drug to decreased atherosclerosis. keywords: alliin; bulb garlic; nitric oxide; reverse docking; atherosclerosis. introduction indonesia has many medicinal plants containing therapeutic properties and have beneficial pharmacological effects on animal and human body (motalib et al., 2010). garlic is remarkable for the number of compounds it contains, including seventeen amino acids, at least 33 sulphur compounds, 17 amino acids (josling, 2010; febyan et al., 2015), eight minerals (germanium, calcium, copper, iron, potassium, magnesium, selenium and zinc) and the vitamins a, b and c (josling, 2010). in 1944 an italian chemist, c. j. cavallito, as an inventor the isolated an unstable, odorous sulphurcontaining compound with antibacterial properties from extracts of fresh garlic. the name of the substance is allicin, based on the generic name for the plant allium sativum. four years later researchers stoll and seebeck, also working with garlic, discovered an odourless sulphur-containing compound called alliin (josling, 2010). they found that this compound would be converted by a second garlic constituent, an enzyme called allinase, to form allicin. the researchers made an additional, remarkable discovery: when they studied the cloves in cross-section they found that alliin and allinase were stored in different compartments. in an undamaged clove they remained completely separate, but once its structure was rupturedtypically by cutting-the two substances came into contact and formed allicin. atherosclerosis is a chronic inflammatory disease of the arterial wall, especially oxidation of lipoproteins that stimulate the innate immune response and adaptive immune response (tedgui & mallat, 2006; ait-oufella et al., 2011). atherosclerosis cause stroke, hypertension, and coronary heart (tanuwijaya, 2003). atherosclerotic cause by free radicals (sumarno et al., 2012) and unhealthy lifestyle (rahman, 2012). the high free radicals in the body cause degenerative diseases (winarsi, 2007). unhealthy lifestyle like high fat diet cause endothelial disfunction. endothelial disfunction causes decrease production of vasodilation compounds such as nitric oxide (no) (oparil et al., 2003; devlin, 2002). no is the smallest signal molecule produced by three isoforms of nitric oxide synthase (nos) (forstermann & sessa, 2011). the no formation enzymatic of l-arginine is catalyzed by nitric oxide synthase (nos) (schrijvers et al., 2004). therapy used to reducing inflammation and improving endothelial function or rupture of plaque is done by aspirin. drug use to reduce inflammation has side effects. aspirin cause gastrointestinal irritation and bleeding (majid, 2008), therefore safe therapies are required without harmful side effect in patients with atherosclerotic. safe therapy using herbal drug that http://dx.doi.org/10.14421/biomedich.2017.62.59-62 60 biology, medicine, & natural product chemistry 6 (2), 2017: 59-62 contain biochemical compounds, one of them is garlic in indonesia such as single bulb garlic. alliin compounds are potential as antioxidant (febyan et al., 2015) and antiatheroslerotic (hernawan & setyawan, 2003). endothelial disfunction causes a decrease in enos activation resulting in reduced no production (danuyanti et al., 2014). alliin contained in allium sativum lowers blood pressure through complex pathways resulting in vasodilation so that decreasing of plaque atherosclerotic. no is synthesized by nitric oxide synthase (nos) from l-arginine. after synthesis, no diffuses from endothelial cells to smooth muscle cells of the blood vessels and causes an increase in intracellular cyclic guanosine monophosphate (cgmp) to relaxation of smooth muscle of blood vessels so that vasodilation occurs (mckeever, 2009). objective this study aimed to discover alliin compound from single bulb garlic for antiatherosclerotic agent based on insilico screening. methodology ligand preparation the chemical structure of 3d and smiles ligands (alliin) is taken from pubchem compound database (https://pubchem.ncbi.nlm.nih.gov/) with id number: 87310. target selection input alliin’s smiles on pharmmapper (http://lilab.ecust.edu.cn) to identify potential target candidates using mapping approaches swiss target predictions (http: //www.swisstargetprediction.c/) and chemical structure associations with molecular 3d (gong et al., 2013). molecular docking molecular docking alliin, target protein, and known inhibitors of target protein used pyrx 0,8 software. visualization of molecule and small molecule interaction the interactions between alliin, target protein, and inhibitors were analyzed using pymol. results and discussion the results of target selection using pharmmapper (job id: job id: 170.228.034.654) dan swiss target prediction, found that alliin interact with nitric oxide synthase (nos) in human vascular endothelium cells. no accumulation induces the formation of a strong oxidizing agent, peroxinitrite (schwartz et al., 2002). nos became an interesting object of this study for the development of drugs as inhibitors of atherosclerotic plaque reduction. alliin as a drug effectively inhibit atherosclerotic disease (zhang et al., 2001; borek, 2001; seo et al., 2012). garlic extract can lowering cholesterol levels significantly, reducing the thickening of the aortic wall and reducing fat accumulation in macrophage cultures. the mechanism is related to the garlic on cholesterol metabolism (campbell et al., 2001). figure 1. (a) the interaction between target protein (nos) with alliin and aspirin inhibitor suggests that binding target protein on the same site. green (alliin), red (aspirin), yellow (nos); (b) interactions through hydrogen and alkyl bonds of ile b: 161, asn b: 157, lys b: 160, and thr a: 146. ilmawati et al. – alliin as a natural bioactive from single bulb garlic (allium sativum) … 61 reverse docking is a method used to predict the interaction activity between ligand (compound / drug) with receptor (protein/target) (kharkar et al., 2014). based on a reverse docking of nos (gdp id: 5unr) with a resolution of 1.95 å, aliin is known as an inhibitor of nos informing that alliin has binding affinity (-5.5 kcal/ mol). the result of molecular visualization and molecular interaction using pymol software found that alliin bind to target protein (nos) at the same site as aspirin inhibitor via hydrogen and alkyl bond of ile b: 161, asn b: 157, lys b: 160, and thr a : 146 as shown in figure 1. so it can be said that alliin has the same function as aspirin as nos inhibitor in atherosclerotic. decreased levels of no cause vasoconstriction of blood vessels and endothelial becomes more proaterogenic and proinflammation (kuzkaya et al., 2003). no plays an important role in blood vessels such as vasodilation, inhibits smooth muscle cell proliferation, platelet aggregation, monocyte and platelet adhesion, low density lipoprotein oxidation (ldl), expression of molecular adhesion and endothelin production (behrendt & ganz, 2002; yetik-anacak & catravas, 2006). alliin as inhibitors of atherosclerotic plaque reduction with improves component of vasodilation. no is synthesized by nitric oxide synthase (nos) from larginine. this arginine will be changed by the enzyme nitrite oxidase becomes no. no stimulate guanylate cyclase to change gtp (guanosine triphosphate) becomes cyclic-gmp to activates protein kinase g which causes the re-taking ca2+ and opening potassium channel which is activated by calcium. decrease of ca2+ concentrations myosin light-chain kinase (mlck) cannot phosphorylates myosin longer, thus stopping the cycle cross bridge and cause relaxation smooth muscle cells of the blood vessels so that vasodilation occurs (ishimura et al., 1998 & mckeever, 2009). allium sativum with water will break down into diallyl sulfide, diallyl disulfide and diallyl trisulfide which then merge into the organic polysulfide that causes red blood cells to produce h2s (hydrogen sulfide). h2s will bind and activate channel katp, so the concentration of ca2+ cell will decrease and hyperpolarization occur vascular smooth muscle cell that cause vasodilation of blood vessel (mckeever, 2009). the result of reverse docking show that alliin interact with nos that can increasing no product, so it can be seen that nos acts as an atherosclerotic inhibitor. conclusion this study proved that alliin has potential as an nos inhibitor based on binding affinity and intermolecular interaction. alliin 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in atherosclerosis from theory to clinical practice. semarang: universitas diponegoro. tedgui, a. & mallat, z. 2006. cytokines in atherosclerosis: pathogenic and regulatory pathways. physiol rev. 86: 515– 581. winarsi, h. 2007. antioksidan alami dan radikal bebas. cetakan ke-2. yogyakarta: kanisnus. p: 11-121. yetik-anacak, g. and catravas, j. 2006. nitric oxide and the endothelium: history and impact on cardiovascular disease. vascul. pharmacol. 45 (5): 268–76. zhang, x., lowe, d., giles, p., fell, s., connock, m., and maslin, d. 2001. gender may affect the action of garlic oil on plasma cholesterol and glucose levels of normal subjects. journal of nutrition. 131: 1471-1478. alliin as a natural bioactive from single bulb garlic (allium sativum) for nitric oxide (no) increasing in atherosclerotic process based on insilico screening biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 535-540 | doi: 10.14421/biomedich.2025.141.535-540 issn 2540-9328 (online) histopathological evaluation of stomach protection by peperomia pellucida l. in mice with gastroenteritis lisa savitri1,*, rochmad krissanjaya1, elfred rinaldo kasimo1, syntia tanu juwita1, fendy prasetyawan2, yuneka saristiana2, konradus klala mebung1, cornelia amanda1 1department of medical laboratory technology; 2department of pharmacist professional education, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 10 october, 2024. revision accepted: 15 july, 2025. published: 04 august, 2025. abstract gastroenteritis, a widespread condition characterized by inflammation of the stomach and intestines, poses significant health challenges globally. conventional treatments primarily focus on symptomatic relief and do not address the underlying gastric mucosal damage. peperomia pellucida l., a medicinal plant known for its anti-inflammatory and antioxidant properties, has been suggested to possess gastroprotective effects. this study aims to evaluate the histopathological effects of p. pellucida l. on gastric protection in a mouse gastroenteritis model. male swiss mice were divided into six groups, receiving different treatments, including the ethanol extract of p. pellucida at varying doses (100, 300, and 500 mg/kgbw), followed by induction of gastroenteritis with escherichia coli. histopathological analysis was conducted to observe tissue damage across the treatment groups, including necrosis and cell degeneration. the results revealed that p. pellucida l. exhibited significant gastroprotective effects, particularly at a dose of 500 mg/kgbw, reducing gastric mucosal necrosis and inflammation compared to the control groups. the plant’s secondary metabolites, including flavonoids, tannins, saponins, and alkaloids, contributed to the observed protective effects by enhancing mucus production, reducing gastric acidity, and promoting tissue repair. these findings suggest that p. pellucida l. could be an alternative treatment for gastroenteritis and related gastric conditions, offering a natural approach to managing gastric inflammation and mucosal protection. keywords: peperomia pellucida l.; gastroprotective; gastroenteritis; histopathology; medicinal plants; gastric mucosa. abbreviations: polymorphonuclear leukocytes (pmns); polymorphonuclear leukocytes (pmn), standard error (se); world health organization (who) introduction gastroenteritis, characterized by inflammation of the stomach and intestines, poses a significant health challenge worldwide, often resulting in symptoms such as diarrhea, vomiting, and abdominal pain. this condition can be caused by various pathogens, including bacteria, viruses, and parasites, leading to considerable morbidity, especially in vulnerable populations such as children and the elderly. current treatment options primarily focus on symptomatic relief and rehydration; however, they do not address the underlying inflammation or provide protective effects to the gastric mucosait involves an inflammatory process in the gastric epithelial cells and subsequent damage to the gastric mucosa (angkow et al., 2014). clinically, gastritis is often used to refer to symptoms that occur in the upper abdomen or epigastric region. the high prevalence of gastritis is largely influenced by dietary habits, food types, stress, smoking, and alcohol consumption, which is why the incidence of gastritis in indonesia remains significantly high. globally, gastritis affects between 1.8 and 2.1 million individuals annually. according to the world health organization (who), the prevalence of gastritis is as follows: 22.0% in the uk, 31.0% in china, 14.5% in japan, 35.0% in canada, and 29.5% in france. in south asia, approximately 583,635 people suffer from gastritis each year. in shanghai, 17.2% of the population is affected by endoscopically confirmed gastritis, a figure notably higher than the 4.1% asymptomatic rate found in western populations (angkow et al., 2014). in indonesia, according to the ministry of health, gastritis ranks sixth among hospitalized patients, with an occurrence rate of 60.86% from 33,580 hospital admissions. it also ranks seventh for outpatient cases, accounting for 201,083 patients. the prevalence of gastritis in indonesia is quite high, with 274,396 cases out of a population of 238,452,952, equating to 40.8%. the rates of gastritis in various cities include 50% in jakarta, 35.5% in palembang, 32% in bandung, 46% in denpasar, 31.2% https://doi.org/10.14421/biomedich.2025.141.535-540 mailto:lisasavitri@unik-kediri.ac.id 536 biology, medicine, & natural product chemistry 14 (1), 2025: 535-540 in surabaya, 31.7% in aceh, and 91.6% in medan (riskesdas, 2018). despite the high prevalence, many individuals neglect proper stomach care. gastritis often presents asymptomatically but is commonly associated with epigastric pain. additional symptoms include nausea, vomiting, bloating, and loss of appetite (saputra, 2017). research has shown that chronic bacterial infections in the gastric mucosa are a leading cause of gastritis. additionally, substances such as aspirin, alcohol, bile salts, and other harmful agents can damage the gastric mucosa, impairing the epithelial barrier's protective function over the stomach and duodenum. the majority of gastritis cases (70-80%) are due to functional gastritis, characterized by pain not linked to organic disorders of the stomach. left untreated, gastritis can impair gastric function and elevate the risk of gastric cancer, which can lead to fatal outcomes (muttaqin et al., 2011). in recent years, there has been a growing interest in the potential therapeutic properties of medicinal plants, particularly those traditionally used in folk medicine. one such plant, peperomia pellucida l., is known for its anti-inflammatory and antioxidant properties, which may contribute to its protective effects on the gastric lining. previous studies have indicated that the ethanol extract of p. pellucida can exert gastroprotective effects, enhancing mucosal defense mechanisms and promoting healing in gastric tissues. this study aims to evaluate the histopathological effects of p. pellucida l. on gastric protection in a mice model of gastroenteritis. by examining the histological changes in the gastric mucosa of treated and untreated mice, this research seeks to elucidate the potential gastroprotective mechanisms of p. pellucida and contribute to the understanding of its therapeutic applications. the findings from this study may provide valuable insights into alternative treatment strategies for gastroenteritis and reinforce the importance of exploring plant-based remedies in modern medicine. materials and methods research type the population in this study consisted of mice, with male swiss strain mice aged 2-3 months, weighing between 25-30 grams, being the sample. these mice were obtained from the veterinary farma center, surabaya. each treatment group included a minimum of 4 mice, and since there were 6 groups in total, a minimum of 24 mice was required. however, in this study, the sample size per group was increased by 1, bringing the total number of mice to 30. tools and materials the tools used in this study included 1 ml, 3 ml, and 5 ml disposable syringes, a 35 cm feeding tube, a 60 ml urine container, micropipettes, a freezer, a rotary or sliding microtome, brushes, a water bath, glass slides, 1500 µl eppendorf tubes, blue tips, yellow tips, white tips for 10 µl, an oven, a digital scale, a surgical board, a set of surgical instruments (scissors, forceps, and probes), gloves, tissues, masks, a binocular light microscope, and a microscope camera. the materials used in this study included male swiss strain mice aged 2-3 months, weighing 25-30 grams, amoxicillin, clinical isolates (wild-type escherichia coli) at a concentration of 1x106 cfu/ml, physiological saline (pz) (pyrogen-free injection water), mice feed, wood shavings, methanol, giemsa stain, distilled water, 37% h2co (formaldehyde) solution, formalin buffer, disodium hydrogen phosphate (na2hpo4), 37-40% formaldehyde, and various grades of ethanol (80%, 95%, absolute ethanol), xylene, clearing solution, paraffin, and poly-l-lysine. research procedure 1. mice acclimatization male mice were weighed and housed in standard polypropylene cages with wood shavings as bedding material for a two-week acclimatization period. the bedding was replaced daily. the feed was moistened with water and shaped into 90-gram ovals per cage (housing 6 mice). water was provided ad libitum, and both feed and water were refreshed daily. after two weeks, the mice were divided into six treatment groups. 2. treatment of test animals the acclimatized mice underwent the following treatments: group 1 (normal control) received no gastric gavage. group 2 (negative control) received distilled water. group 3 (positive control) received amoxicillin at a dose of 500 mg/kgbw in a volume of 0.26 ml. group 4 received chinese betel leaf extract at a dose of 100 mg/kgbw in a volume of 0.5 ml. group 5 received chinese betel leaf extract at a dose of 300 mg/kgbw in a volume of 0.5 ml. group 6 received chinese betel leaf extract at a dose of 500 mg/kgbw in a volume of 0.5 ml. 3. gastroenteritis (ge) model induction in mice after 7 days of treatment, a ge model was induced by administering escherichia coli at a concentration of 1x10^6 cfu/ml to each mice via gastric gavage, once daily for 7 days. 4. tissue processing stomach tissues were fixed in formalin buffer to maintain cell morphology and prevent autolysis, as well as bacterial or fungal growth. paraffin blocks were then created. the tissues were sectioned using a rotary or sliding microtome of 4-6 microns thickness. the sections were picked up with a moist brush, transferred to a water bath, and placed onto glass slides coated with tissue savitri et al. – histopathological evaluation of stomach protection 537 adhesive. the slides were dried at room temperature and then placed in an oven overnight. 5. histopathological observation of stomach tissues histopathological analysis of mice stomachs was performed using hematoxylin and eosin (he) staining. the tunica mucosa (epithelium, lamina propria, muscularis mucosa), submucosa, tunica muscularis, tunica serosa, and other regions showing necrosis or apoptosis were examined. 6. data collection the data collected included the number of cells exhibiting abnormalities in the tunica mucosa (epithelium, lamina propria, muscularis mucosa), submucosa, tunica muscularis, tunica serosa, and other regions potentially showing necrosis or apoptosis in the stomach tissues of the mice. 7. data analysis method the data, including the number of cells displaying abnormalities in the tunica mucosa (epithelium, lamina propria, muscularis mucosa), submucosa, tunica muscularis, tunica serosa, and other regions potentially experiencing necrosis or apoptosis in the stomach tissues, were analyzed using anova with a 95% confidence interval (α=0.05). if significant differences were found in the anova results, further analysis was performed using the least significant difference (lsd) test at the same confidence level. statistical analysis was carried out using spss 23.0 for windows, and the data were presented as mean ± standard error (se). results and discussion table 1. effectiveness of chinese betel extract (peperomia pellucida l.) in preventing stomach cell degeneration in a gastroenteritis model of mice. group a b c d e normal 1.92% positive control 6.45% treatment iii (500mg/kgbw) 7.68% treatment i (100mg/kgbw) 18.34% treatment ii (300mg/kgbw) 12.78% negative control 25.12% the duncan test (table 1) was used by the researchers to assess the effectiveness of chinese betel extract and determine the optimal dosage among the groups in preventing stomach cell degeneration in a gastroenteritis model of mice. the results of the duncan test in the table show that overall comparisons between the treatment groups fall into different columns, indicating that the treatment groups have varying effects on preventing cell degeneration. however, group i (100mg/kgbw) and group ii (300mg/kgbw) are placed in the same column, indicating similar gastroprotective effects on cell degeneration in these two groups, consistent with earlier lsd test results. determining the best dosage for gastroprotective activity, in terms of cell degeneration, is based on the smallest values or those appearing in the leftmost columns of the table. antiseptic effectiveness is measured by comparing average cell degeneration. based on the duncan test results, the ranking of the most effective doses is as follows: (1) normal group; (2) positive control group (+); (3) group iii (500mg/kgbw); (4) group i (100mg/kgbw); (5) group ii (300mg/kgbw); and (6) negative control group (-). necrosis the normality test results for the normal group yielded a p-value of 0.432, the positive control group (+) had a pvalue of 0.913, the negative control group had a p-value of 0.293, group i treatment (100mg/kgbw) had a pvalue of 0.612, group ii treatment (300mg/kgbw) had a p-value of 0.591, and group iii treatment (500mg/kgbw) had a p-value of 0.627. these normality test results indicate that all groups have p-values greater than 0.05 (p>0.05), confirming that the data are normally distributed and suitable for further testing. the homogeneity test results for all six groups yielded a significance value of 0.141 (>0.05), indicating homogenous variance across the treatment groups, thus meeting the requirements for one-way anova testing. the one-way anova test showed a significance value of 0.000 (<0.005), indicating a significant difference among the groups. further analysis, including post hoc tests using the lsd test and duncan test, is warranted for a more detailed comparison between the groups. 538 biology, medicine, & natural product chemistry 14 (1), 2025: 535-540 table 2. effectiveness of chinese betel extract (peperomia pellucida l.) in preventing necrosis in a gastroenteritis mice stomach model. group a b c d normal 1.85% positive control 4.32% treatment iii (500mg/kgbw) 5.94% treatment ii (300mg/kgbw) 10.12% negative control 17.20% treatment i (100mg/kgbw) 15.78% the lsd test results indicate that overall comparisons between the treatment groups have a significance value of p < 0.05, suggesting that the comparisons between these treatment groups have different effects, except for the comparison between the positive control group and group iii treatment (500mg/kgbw), as well as the negative control group and group i treatment (100mg/kgbw). in the comparison between the positive control group and group iii treatment (500mg/kgbw), the significance value obtained is 0.223 (>0.05), indicating that the positive control group and group iii treatment (500mg/kgbw) have similar gastroprotective effects on necrosis. similarly, the comparison between the negative control group and group i treatment (100mg/kgbw) yielded a significance value of 0.249 (>0.05), indicating that the negative control group and group i treatment (100mg/kgbw) have similar effects in terms of necrosis prevention. the duncan test (table 2) was used by the researchers to assess the effectiveness and determination of the best dosage among the groups. the results of the duncan test in the table indicate that overall comparisons between the treatment groups are in different columns, suggesting that the comparisons between these treatment groups have different effects on preventing necrosis in the stomach of the gastroenteritis model mice. however, the comparison between the positive control group and group iii treatment (500mg/kgbw), as well as the negative control group and group i treatment (100mg/kgbw), falls within the same column. this result aligns with the previous lsd test, indicating that the positive control group with group iii treatment (500mg/kgbw) and the negative control group with group i treatment (100mg/kgbw) have similar gastroprotective effects on necrosis. determining the best dosage or dose effectiveness for gastroprotective activity in terms of necrosis is based on the smallest values or those leaning towards the left. antiseptic effectiveness is determined by looking at the average necrosis. based on the duncan test table, the sequence of the best doses is as follows: (1) normal group; (2) positive control group (+); (3) group iii treatment (500mg/kgbw); (4) group ii treatment (300mg/kgbw); (5) negative control group (-); and (6) group i treatment (100mg/kgbw). polymorphonuclear leukocytes (pmn) the normality test results for the normal group yielded a p-value of 0.380, the positive control group had a p-value of 0.863, the negative control group had a p-value of 0.620, group i treatment (100mg/kgbw) had a p-value of 0.310, group ii treatment (300mg/kgbw) had a pvalue of 0.855, and group iii treatment (500mg/kgbw) had a p-value of 0.611. these normality test results show that the data are normally distributed, as all p-values are greater than 0.05 (p>0.05). the homogeneity test for the six groups yielded a significance value of 0.384 (>0.05), indicating homogenous variance across the groups, thus meeting the requirements for one-way anova testing. the one-way anova test yielded a significance value of 0.000 (<0.005), indicating significant differences among the groups. further analysis, including post hoc tests using the lsd test and duncan test, showed that overall comparisons between the treatment groups had a significance value of p < 0.05, suggesting that these groups have different effects on preventing pmn in the stomach of the gastroenteritis model mice. the duncan test was used to determine the best dosage for pmn prevention. the results indicated that the comparison between the positive control group and group iii treatment (500mg/kgbw), as well as the comparison between the negative control group and group i treatment (100mg/kgbw), fell within the same column. this aligns with the previous lsd test, indicating that the positive control group and group iii treatment (500mg/kgbw) have similar antiseptic effects on pmn, as do the negative control group and group i treatment (100mg/kgbw). savitri et al. – histopathological evaluation of stomach protection 539 table 3. effectiveness of chinese betel extract (peperomia pellucida l.) in preventing pmn in a gastroenteritis mice stomach model. group a b c d e f normal 2.10% positive control 5.25% treatment iii (500mg/kgbw) 8.14% treatment i (100mg/kgbw) 12.55% treatment ii (300mg/kgbw) 19.65% negative control 24.56% 48.63% determining the optimal dose for pmn antiseptic activity is based on the smallest values or those leaning towards the left. based on the duncan test table, the best dose sequence is as follows: (1) normal group; (2) positive control group (+); (3) group iii treatment (500mg/kgbw); (4) group ii treatment (300mg/kgbw); (5) negative control group (-); and (6) group i treatment (100mg/kgbw). discussion gastroprotective refers to the ability of certain compounds to protect the gastric mucosa from damage caused by various factors, including stress, excessive acid secretion, and harmful substances. research by roslida and aini (2009) suggests that p. pellucida l. possesses gastroprotective properties, as its ethanol extract demonstrated effective gastroprotection at 100 mg/kgbw. the gastroprotective effects of this plant are thought to arise from various mechanisms linked to its diverse metabolite compounds (yusuf et al., 2017). phytochemical analyses of p. pellucida l. have identified several secondary metabolites, including flavonoids, tannins, saponins, triterpenoids, and steroids (rachmawati & rantelino, 2018; pertiwi et al., 2022; oloyede et al., 2011). a previous study identified dillapiole as the primary active gastroprotective compound in p. pellucida l.; however, additional research is necessary to fully elucidate the gastroprotective mechanisms of dillapiole, as it appears unrelated to endogenous nitric oxide or prostaglandins (rojas-martínez et al., 2013). flavonoids are known for their anti-ulcer and antiinflammatory properties, functioning through various mechanisms such as inhibiting k+/h+ atpase, reducing hcl secretion, enhancing the synthesis of pge2 and cox-1, inhibiting bacterial growth, and exhibiting antioxidant effects (kalogeromitros et al., 2008). alkaloids, another class of gastroprotective agents, promote wound healing and stimulate gastric mucus production following damage from harmful substances (tan et al., 2002). tannins are recognized for their astringent qualities, allowing them to bind with proteins in the gastric mucosa. this interaction aids in forming a protective layer on the mucosa’s surface, reducing permeability and increasing resistance to ulcers and irritation (souza et al., 2012). saponins enhance gastroprotective activity by elevating fibronectin levels, facilitating the formation of fibrin clots that act as a scaffold for tissue re-epithelialization. the quicker these clots develop, the more rapidly fibroblasts proliferate in the wound area, supporting tissue repair (indraswary, 2011). thus, the comprehensive understanding of these mechanisms underscores the potential therapeutic benefits of p. pellucida l. in preventing gastric ailments and promoting overall gastrointestinal health. conclusions this study highlights the significant gastroprotective potential of peperomia pellucida l., as demonstrated by its ability to mitigate gastric mucosal damage in a mice model of gastroenteritis. the plant’s diverse secondary metabolites, including flavonoids, tannins, saponins, and alkaloids, contribute to its protective effects through multiple mechanisms such as reducing gastric acidity, enhancing mucus production, and promoting wound healing. the ethanol extract of p. pellucida l. at varying doses demonstrated notable histopathological improvements, particularly in reducing necrosis and inflammation in the gastric tissue. moreover, the identification of dillapiole as a key active compound further emphasizes the therapeutic potential of p. pellucida l., although more research is necessary to fully understand its gastroprotective mechanisms. the findings support the use of p. pellucida l. as a viable natural treatment for gastritis and gastroenteritis, offering a plant-based alternative to conventional therapies. this research provides valuable insights into the plant's medicinal applications and underscores the importance of further exploration into traditional herbal remedies for gastrointestinal disorders. acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university, and the laboratory of pathological anatomy, brawijaya university, malang, for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors wrote the manuscript. all authors read and approved the final version of the manuscript. 540 biology, medicine, & natural product chemistry 14 (1), 2025: 535-540 competing interests: the authors declare that there are no competing interests. funding: the author would like to thank the institute for research, development, and community service (lp3m) of kadiri university which has always provided support for funding, writing, and publications. references angkow j, robot f, onibala f. 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(2017). faktor resiko kejadian gastritis pada pasien rawat jalan d puskesmas poasia kota kendari. karya tulis ilm [internet] 2017; available from: http://repository.poltekkeskdi.ac.id/308/1/kti wawan adi saputra.pdf souza fh, jesus nz, gomes if, almeida ltj, morais lima gr, bar-bosa filho jm (2012) tannins, peptic ulcers and related mecha-nisms. int j mol sci 13(3):3203–3228. tan kc, lyman sb, wisner jd (2002) supply chain management: astrategic perspective. int j oper prod manag 22(6):614–631 yusuf ms., wulandari i., amelia l., noviani a., & mun’im a. effect of gamma irradiation on suruhan (peperomia pellucida (l.) kunth) herb powder. pharmacognosy journal, 2017; 9(2): 239-243. cover jurnal biomenaprochy vol 8 num 2 2019 biology, medicine, & natural product chemistry volume 8 – number 2 – october 2019 issn 2089-6514 (paper) | issn 2540-9328 (online) honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad jafar luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia mohamad amin department of biology, faculty of mathematics and sciences, universitas negeri malang, indonesia widodo department of biology education, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: r i y a n t o publisher: state islamic university (uin) sunan kalijaga yogyakarta co-publisher: the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university (uin) sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@yahoo.com biology, medicine, & natural product chemistry volume 8 – number 2 – october 2019 issn 2089-6514 (paper) | issn 2540-9328 (online) contents digital anthropometer development for improving the measurement quality of human body dimensions trio yonathan teja kusuma, arya wirabhuana, faurosi syafa’atul yusuf 27 32 the effects of wet cupping therapy in blood pressure, glucose, uric acid and total cholesterol levels sutriyono, muhammad rodham robbina, meksianis zadrak ndii 33 36 anatomical and histological study of shark (carcharhinus sorrah) kidney hikmah supriyati, rakhmiyati, muhammad ja’far luthfi 37 40 hepatotoxic assessment of tramadol-diclofenac use: a study in a rat model elias adikwu, ebinyo clemente nelson 41 45 liver profile of atazanavir/ritonavir in pregnant albino rats elias adikwu, james kemelayefa, winifred ocheiga 47 52 guidance for authors aims and scope biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international standards in their methodology. article types the journal seeks original full-length research papers, reviews, and short communication. manuscript of 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(personal communication); setyawan ad 2007, unpublished data). in the reference list, the references should be listed in an alphabetical order (better, if only 20 for research papers). names of journals should be abbreviated. always use the standard abbreviation of a journal's name according to the issn list of title word abbreviations (www.issn.org/2 22661-ltwa-online.php). the following examples are for guidance. journal: saharjo bh, nurhayati ad. 2006. domination and composition structure change at hemic peat natural regeneration following burning; a case study in pelalawan, riau province. biodiversitas 7: 154-158. book: rai mk, carpinella c. 2006. naturally occurring bioactive compounds. elsevier, amsterdam. chapter in book: webb co, cannon ch, davies sj. 2008. ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. in: carson w, schnitzer s (eds) tropical forest community ecology. wiley-blackwell, new york. abstract: assaeed am. 2007. seed production and dispersal of rhazya stricta. 50 annual symposium of the international association for vegetation science, swansea, uk, 23-27 july 2007. proceeding: alikodra hs. 2000. biodiversity for development of local autonomous government. in: setyawan ad, sutarno (eds.) toward mount lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in java island. universitas sebelas maret, surakarta, 17-20 july 2000. [indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com biomed vol. 8 num. 2 october 2019 cover editorial board table of contents guidance back cover biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 485-492 | doi: 10.14421/biomedich.2024.132.485-492 issn 2540-9328 (online) histopathological description of mouse liver in a sepsis model infected with escherichia coli treated with paederia foetida l. leaf extract for sepsis prevention lisa savitri*, ana retnowati, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id abstract the leaf of paederia foetida l. is one type of medicinal plant that can be used as a preventive medicine for sepsis. this plant contains secondary metabolites such as alkaloids, flavonoids, triterpenoids, saponins, and other active compounds. the objective of this study was to determine the histopathological description of the liver in a mouse sepsis model infected with e. coli, with the administration of paederia foetida l. leaf extract for sepsis prevention, and to ascertain the influence and effective dosage of the leaf extract as a preventive measure against liver histopathology in the sepsis model induced by e. coli. the method employed was a completely randomized design (crd). the study used 24 male white mice divided into 6 (six) groups. data analysis was conducted using one way anova. the results of the study revealed the histopathological profile of liver cell degeneration in group pi (100mg/kg bw) at 20.79%±0.03, group pii (200mg/kg bw) at 21.63%±0.02, and group piii (500mg/kg bw) at 9.08%±0.02. necrosis rates were observed in group pi (100mg/kgbw) at 22.62%±0.04, group pii (200mg/kg bw) at 17.63%±0.02, and group piii (500mg/kg bw) at 6.05%±0.02. the presence of polymorphonuclear leukocytes (pmn) was detected in group pi (100mg/kgbw) at 39.56%±0.03, group pii (200mg/kgbw) at 28.05%±0.02, and group piii (500mg/kg bw) at 18.45%±0.03. the test results showed a significant effect of p. foetida l. leaf extract as a preventive measure against liver histopathology in the mouse sepsis model infected with e. coli, with significant values for necrosis (p=0.000), cell degeneration (p=0.000), and pmn (p=0.000). the most effective dosage of p. foetida l. leaf extract as a preventive measure against liver histopathology in the mouse sepsis model infected with e. coli was the dosage used in group piii (500mg/kgbw). keywords: kentut leaves; liver histopathology; sepsis; escherichia coli. abbreviations: anova (analysis of variance), completely randomized design (crd), polymorphonuclear leukocytes (pmns) introduction sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection (purwanto & astrawinata, 2018). sepsis represents a systemic response to infection within the body that can progress to severe sepsis and septic shock (singer et al., 2016). it can be a severe and potentially life-threatening medical condition caused by a widespread infection in the body. sepsis can occur when the body's response to an infection becomes uncontrolled. this unhealthy response can lead to tissue and organ damage, potentially resulting in sepsis (jarczak et al., 2021). in 2017, who estimated there were 48.9 million cases and 11 million deaths related to sepsis worldwide, contributing to nearly 20% of all global deaths. almost half of all global sepsis cases occur in children, with an estimated 20 million cases and 2.9 million deaths globally in children under 5(five) years old. who found significant regional disparities in sepsis incidence and mortality, with approximately 85.0% of sepsis cases and sepsis-related deaths occurring in lowand middle-income countries (who, 2021). severe sepsis and septic shock are leading causes of death in critically ill patients treated in intensive care units (icus) in the united states. metaanalysis research findings indicate that sepsis incidence ranges from 22 to 240 cases per 100,000 people, severe sepsis from 13 to 300 cases per 100,000 people, and septic shock at 11 cases per 100,000 people, with mortality rates reaching 30% for sepsis, 50% for severe sepsis, and 80% for septic shock (purwanto & astrawinata, 2018). microorganisms associated with pathogens causing sepsis and septic shock include gram-positive, gramnegative bacteria, and fungi. microbial entry into the bloodstream is not fundamental to the onset of severe sepsis, as local infections with bacteria producing pathogenic products like exotoxins can trigger systemic inflammatory responses leading to organ dysfunction and hypotension (purwanto & astrawinata, 2018). the liver manuscript received: 09 may, 2024. revision accepted: 21 august, 2024. published: 24 october, 2024. https://doi.org/10.14421/biomedich.2024.132.485-492 mailto:lisasavitri@unik-kediri.ac.id 486 biology, medicine, & natural product chemistry 13 (2), 2024: 485-492 is the largest gland located in the right upper abdomen. liver damage often occurs due to ineffective protozoa attacking erythrocytes indirectly, first developing within hepatocyte cells, leading to cell death. this mechanism causes hepatocyte cells to undergo damage in the form of cell degeneration, which if not promptly addressed, can progress to irreversible cell death or necrosis. acute infections result in the infiltration of inflammatory cells, namely polymorphonuclear leukocytes (pmns). this cell damage can be observed by directly examining the histopathological appearance of the mouse liver organ under a microscope (dwiridal et al., 2021). severe sepsis occurs due to infections acquired from the community and healthcare settings. gram-negative bacteria that can cause such infections include e. coli (angus & poll, 2015). escherichia coli is a facultative anaerobic bacterium that can grow under aerobic and anaerobic conditions. antibiotic therapy must be administered promptly after a sepsis diagnosis is established using a de-escalation strategy, starting with empirical antibiotic treatment and then adjusting or discontinuing based on clinical response or culture results. empirical antibiotic therapy involves broadspectrum antibiotics that can be administered singly or in combination, targeting a range of potential pathogens based on clinical syndromes and previously collected pathogen patterns (antibiogram) (kemenkes ri, 2017). however, an increasing number of people are becoming aware of the side effects of antibiotic drugs, leading to a growing interest in herbal remedies. traditional medicine remains popular for treating diseases due to its easy availability of raw materials, affordability, and potential for self-cultivation. a traditional plant historically trusted by communities for anti-inflammatory purposes is kentut leaves (silaban, 2021). kentut leaves, have the latin name p. foetida l. and exhibit antibacterial activity against e. coli. this is due to the presence of secondary metabolites in kentut leaves such as α and β paederine alkaloids, flavonoid flavanol, friedelin triterpenoid, βsitosterol, campesterol, triterpenoid saponin, gallotannin, and other active compounds. ethanol 96% and methanol maceration of kentut leaves result in alkaloid, saponin, tannin, and flavonoid compounds (salamah & halim, 2021). materials and methods research type this study falls under experimental research using a completely randomized design (crd) method with six treatments conducted over 15 days. the test animals used in this research were randomly selected white male mice, ensuring that all mice had an equal chance of being sampled for treatment. this research aimed to determine the antiseptic activity of ethanol extract from kentut leaves in white male mice. in this study, a total of 24 white male mice were used, divided into 6 (six) groups, with each group consisting of 4 mice. tools and materials tools digital scale, filter paper/cloth, oral gavage, pyrex beaker glass, stirring rod, spatula, glass funnel, mortar and pestle, measuring flask (100ml, pyrex), maceration vessel (glass jar), feeding tube syringe (3ml), tweezers, surgical knife, pin needle, urine pot, oven, tissue processor, microtome, water bath, tray, a set of hematoxylin and eosin staining tubes, prepared tissue slides, microscope, tissue cassette, glass slides, coverslips, organ measuring tools, and microtome knife. materials kentut leaf extract, white male mice, e. coli bacterial liquid media, 10% neutral buffered formalin, hematoxylin stain, and eosin stain. procedure kentut leaf determination plant determination was performed at the upt herbal materia medica laboratory in batu, jl. lahor 87, batu city, east java province. the purpose of plant determination was to confirm the identity of the kentut leaves used in this study. based on the determination results, it was confirmed that the leaves used in this study were kentut leaves. a sampling of kentut leaves the leaf samples used were collected from mlaten hamlet, ngrami-sukomoro village, nganjuk regency. preparation of extract the kentut leaf extract was prepared using the maceration method with 96% ethanol. a total of 500 grams of kentut leaf powder was mixed with 1 litre of 96% ethanol (1:10 ratio) and left to stand for 72 hours. the filtrate was then filtered using filter paper to obtain the filtrate, and the resulting macerate was evaporated using an oven at approximately 40°c. preparation of experimental animals the experimental animals used were healthy male mice aged 3-4 weeks with a body weight of 30-40 grams. before use, the male mice were acclimatized for 14 days under experimental conditions. preparation of materials the materials used included distilled water as a negative control, ciprofloxacin suspension as a positive control, untreated normal mice, ethanol extract suspension of kentut leaves as the test material, and e. coli bacteria as the sepsis inducer. savitri et al. – histopathological description of mouse liver in a sepsis 487 antiseptic activity testing of kentut leaf ethanol extract in this study, a total of 24 white male mice were used and divided into 6 (six) groups, with each group consisting of 4 white male mice. table 1. treatment groups. no. group treatment 1. group 1 served as negative control (-) receiving aqua dest 2. group 2 served as positive control (+) receiving ciprofloxacin suspension 3. group 3 served as standard control without treatment 4. group 4, 5, and 6 treatment groups receiving ethanol extract of parsley leaf at doses of 100 mg/kg bw, 300 mg/kg bw, and 500 mg/kg bw in conducting this experiment, 24 white male mice were randomly divided into 6 (six) groups. each group received different treatments: group 1 received distilled water orally as a negative control, group 2 received ciprofloxacin suspension as a positive control, and group 3 received no treatment. meanwhile, groups 4, 5, and 6 were administered kentut leaf ethanol extract orally at doses of 100 mg/kg bw, 300 mg/kg bw, and 500 mg/kg bw, respectively. the researcher repeated these actions daily for 15 consecutive days. after 15 days of treatment in the experimental groups, on day 16, the mice were injected with e. coli bacteria into the peritoneal cavity to induce sepsis. the mice were then observed for 24 hours after e. coli injection, followed by surgery to extract the liver organ (hepar). the parameters observed in this study included normal cells, cell degeneration, necrosis, and polymorphonuclear leukocytes (pmn). data analysis data analysis in this study was performed descriptively using qualitative methods presented in tables and diagrams to understand the histopathological profile of the mouse liver in the sepsis model induced by e. coli, with the administration of kentut leaf extract for sepsis prevention. data analysis was conducted using microsoft excel 365 software, evaluating the level of damage or changes in the histopathological profile of the liver in each group of mice using a microscope at 10x and 40x magnifications with 5 (five) fields of view. the assessment was based on hepatocyte cells showing normalcy, degeneration, necrosis, and pmn. the observations were totalled, averaged, and compared across each treatment group. the percentage of liver organ damage in mice was calculated using the formula by baldatina (dwiridal et al., 2021). further data analysis utilized one-way anova (analysis of variance). before applying oneway anova, normality and homogeneity tests were conducted. if the data were normally distributed and homogeneous, one-way anova was used to determine significant differences among the test groups, with significance indicated by <0.05 (considered significant) or >0.05 (considered not significant). if significant differences were found among the test groups, post hoc lsd (least significant difference) tests (p<0.05) and duncan's post hoc tests were conducted to identify significant differences among individual groups. if the data were not normally distributed, kruskal-wallis tests in spss were employed to determine significant differences among two or more test groups, with significance indicated by <0.05 (considered significant) or >0.05 (considered not significant). if <0.05, mannwhitney tests were performed to identify specific groups with significantly different values (ghozali, 2009). results and discussion histopathological description of liver the testing of kentut leaf extract (p. foetida l) for its antiseptic properties was conducted by observing the histopathological profile of the liver in a sepsis model induced by e. coli injection in mice. in this study, the researchers utilized six (6) groups, with each group consisting of 4 mice. observations of the histopathological profile of the liver in the sepsis model injected with e. coli included cell degeneration, necrosis, and polymorphonuclear leukocytes (pmn) (see table 2). table 2. histopathological profile of the liver in the e. coli-induced sepsis model. group mean degeneration (%) necrosis (%) pmn (%) sd mean mean sd mean normal 2.21% 0.02 2.44% normal 2.21% 0.02 positive (+) 7.72% 0.04 5.18% positive (+) 7.72% 0.04 negative (-) 23.41% 0.04 21.55% negative (-) 23.41% 0.04 pi (100mg/kgbw) 20.79% 0.03 22.62% pi (100mg/kgbw) 20.79% 0.03 pii (300mg/kgbw) 21.63% 0.02 17.20% pii (300mg/kgbw) 21.63% 0.02 piii (500mg/kgbw) 9.08% 0.02 6.05% piii (500mg/kgbw) 9.08% 0.02 in table 2, the research results show the histopathological profile of the liver in terms of cell degeneration, necrosis, and polymorphonuclear leukocytes (pmn) in a sepsis model induced by e. coli injection. 488 biology, medicine, & natural product chemistry 13 (2), 2024: 485-492 cell degeneration the study found that the standard group had the smallest average percentage of cell degeneration, at 2.21%±0.02. the positive control group (+) had the second most mirror average at 7.72%±0.04. the group treated with 500 mg/kg bw of kentut leaf extract (group piii) had the third smallest average at 9.08%±0.02. group piii (500mg/kgbb) also showed the smallest average compared to the other two groups treated with kentut leaf extract. necrosis the normal group had the smallest average percentage of necrosis, at 2.44%±0.02. the positive control group (+) had the second smallest average at 5.18%±0.02. the group treated with 500 mg/kgbw of kentut leaf extract (group piii) had the third smallest average at 6.05%±0.02. similar to cell degeneration, group piii (500mg/kgbb) exhibited the smallest average compared to the other groups treated with kentut leaf extract. polymorphonuclear leukocytes (pmn) the normal group had the smallest average percentage of pmn, at 1.62%±0.02. the positive control group (+) had the second smallest average at 16.07%±0.03. the group treated with 500 mg/kgbw of kentut leaf extract (group piii) had the third smallest average at 18.45%±0.03. like the other parameters, group piii (500mg/kgbb) showed the smallest average compared to the other groups treated with kentut leaf extract. statistical analysis normality tests resulted in p-values of 0.980 for the normal group, 0.807 for the positive control group (+), 0.428 for the negative control group, 0.663 for group i (100mg/kgbw), 0.662 for group ii (300mg/kgbw), and 0.330 for group iii (500mg/kgbw). all p-values were >0.05, indicating that the data were normally distributed and suitable for further testing. homogeneity tests across all six groups yielded a significance value of 0.574 (>0.05), indicating homogeneous variance among the treatment groups and meeting the requirements for oneway anova testing. one-way anova testing resulted in a significance value of 0.000 (<0.005), indicating a significant difference among the groups. post hoc tests (lsd test and duncan test) showed significant differences between the treatment groups (p<0.05), except for the comparison between group i (100mg/kgbw) and group ii (300mg/kgbw) with a significance value of 0.205 (>0.05), suggesting similar antiseptic effects in terms of cell degeneration between these two groups. table 3. effectiveness of kentut leaf extract as prevention against hepatic cell degeneration in a sepsis model of mice. group a b c d e normal 2.21% positive control 7.64% treatment iii (500mg/kgbw) 9.55% treatment i (100mg/kgbw) 20.79% treatment ii (300mg/kgbw) 21.65% negative control 23.49% the duncan test (table 3) was used by the researchers to assess the effectiveness and determination of the optimal dosage among the groups. the results of the duncan test in the table indicate that overall comparisons between the treatment groups are in different columns, suggesting that the comparisons between these treatment groups have different effects on preventing cell degeneration in the liver of the sepsis model mice. however, the comparison between group i (100mg/kgbw) and group ii (300mg/kgbw) falls within the same column. this result aligns with the lsd test earlier, indicating that group i (100mg/kgbw) and group ii (300mg/kgbw) have similar antiseptic effects in terms of cell degeneration. the best dosage or dose effectiveness for antiseptic activity in terms of cell degeneration is based on the smallest values or leaning towards the left because antiseptic effectiveness is determined by looking at the average cell degeneration. based on the duncan test table, the sequence of the best doses is as follows: (1) normal group; (2) positive control group (+); (3) group iii treatment (500mg/kgbw); (4) group i treatment (100mg/kgbw); (5) group ii treatment (300mg/kgbw); and (6) negative control group (-). necrosis the normality test results for the normal group yielded a p-value of 0.338, the positive control group (+) had a pvalue of 0.975, the negative control group had a p-value of 0.314, group i treatment (100mg/kgbw) had a pvalue of 0.636, group ii treatment (300mg/kgbw) had a p-value of 0.608, and group iii treatment (500mg/kgbw) had a p-value of 0.662. the normality test results for all six groups collectively showed a significance value greater than 0.05 (p>0.05), indicating that the data are normally distributed and suitable for further testing. the homogeneity test results for the six treatment groups yielded a significance value of 0.129 savitri et al. – histopathological description of mouse liver in a sepsis 489 (>0.05), indicating homogenous variance among the treatment groups and meeting the requirements for oneway anova testing. the one-way anova test yielded a significance value of 0.000 (<0.005), indicating a significant difference among the groups. further analysis, including post hoc tests using the lsd test and the duncan test, is warranted. table 4. effectiveness of cat's whiskers leaf extract in preventing necrosis in a sepsis mouse liver model. group a b c d normal 2.44% positive control 5.19% treatment iii (500mg/kgbw) 6.24% treatment ii (300mg/kgbw) 17.16% negative control 21.65% treatment i (100mg/kgbw) 22.62% the lsd test results indicate that overall comparisons between the treatment groups have a significance value of p < 0.05, suggesting that the comparisons between these treatment groups have different effects, except for the comparison between the positive control group and group iii treatment (500mg/kgbw), as well as the negative control group and group i treatment (100mg/kgbw). in the comparison between the positive control group and group iii treatment (500mg/kgbw), the significance value obtained is 0.217 (>0.05), indicating that the positive control group and group iii treatment (500mg/kgbw) have similar antiseptic effects on necrosis. similarly, the comparison between the negative control group and group i treatment (100mg/kgbw) yielded a significance value of 0.251 (>0.05), indicating that the negative control group and group i treatment (100mg/kgbw) have similar antiseptic effects on necrosis. the duncan test (table 4) was used by the researchers to assess the effectiveness and determination of the best dosage among the groups. the results of the duncan test in the table indicate that overall comparisons between the treatment groups are in different columns, suggesting that the comparisons between these treatment groups have different effects on preventing necrosis in the liver of the sepsis model mice. however, the comparison between the positive control group and group iii treatment (500mg/kgbw), and the negative control group and group i treatment (100mg/kgbw), falls within the same column. this result aligns with the previous lsd test, indicating that the positive control group with group iii treatment (500mg/kgbw) and the negative control group with group i treatment (100mg/kgbw) have similar antiseptic effects on necrosis. the best dosage or dose effectiveness for antiseptic activity in terms of necrosis is based on the smallest values or leaning towards the left because antiseptic effectiveness is determined by looking at the average necrosis. based on the duncan test table, the sequence of the best doses is as follows: (1) normal group; (2) positive control group (+); (3) group iii treatment (500mg/kgbw); (4) group ii treatment (300mg/kgbw); (5) negative control group (-); and (6) group i treatment (100mg/kgbw). polymorphonuclear leukocytes the normality test results for the normal group yielded a p-value of 0.370, the positive control group had a p-value of 0.868, the negative control group had a p-value of 0.613, group i treatment (100mg/kgbw) had a p-value of 0.318, group ii treatment (300mg/kgbw) had a pvalue of 0.863, and group iii treatment (500mg/kgbw) had a p-value of 0.617. the normality test results for all six groups collectively showed a significance value greater than 0.05 (p>0.05), indicating that the data are normally distributed and suitable for further testing. the homogeneity test results for the six treatment groups yielded a significance value of 0.398 (>0.05), indicating homogenous variance among the treatment groups and meeting the requirements for one-way anova testing. the one-way anova test yielded a significance value of 0.000 (<0.005), indicating a significant difference among the groups. further analysis, including post hoc tests using the lsd test and the duncan test, shows that overall comparisons between the treatment groups have a significance value of p < 0.05, suggesting that the comparisons between these treatment groups have different effects. the duncan test was used by the researchers to assess the effectiveness and determination of the best dosage among the groups. the results of the duncan test in the table indicate that overall comparisons between the treatment groups are in different columns, suggesting that the comparisons between these treatment groups have different effects on preventing pmn in the liver of the sepsis model mice. however, the comparison between the positive control group and group iii treatment (500mg/kgbw), and the negative control group and group i treatment (100mg/kgbw), falls within the 490 biology, medicine, & natural product chemistry 13 (2), 2024: 485-492 same column. this result aligns with the previous lsd test, indicating that the positive control group with group iii treatment (500mg/kgbw) and the negative control group with group i treatment (100mg/kgbw) have similar antiseptic effects on pmn. table 5. effectiveness of cat's whiskers leaf extract in preventing pmn in a sepsis mouse liver model. group a b c d e f normal 1.62% positive control 16.07% treatment iii (500mg/kgbw) 18.45% treatment i (100mg/kgbw) 28.05% treatment ii (300mg/kgbw) 39.56% negative control 48.63% the optimal dose or dose effectiveness for pmn antisepsis is based on the smallest value or leftward direction because antisepsis is determined by looking at the average pmn. based on the duncan test table, the best dose sequence is as follows: (1) normal group; (2) control group (+); (3) treatment group iii (500mg/kgbw); (4) treatment group ii (300mg/kgbw); (5) negative control group (-); and (6) treatment group i (100mg/kgbw). discussion the research results (table 3) indicate decreased cell degeneration damage in hepatocytes after mice were infected with e. coli bacteria. the normal group had the smallest average at 2.21% ± 0.02, because the normal control group was not injected with e. coli, thus not experiencing sepsis that causes cell degeneration damage. in the sepsis-experiencing mouse group (groups ii to vi), group ii had the most effective dose among the sepsis-experiencing groups compared to the other 4 (four) groups infected with e. coli. group ii served as the positive control containing ciprofloxacin. in the treatment groups with parsley leaf extract, namely treatment group i (100mg/kg bw), treatment group ii (300mg/kg bw), and treatment group iii (500mg/kg bw), treatment group iii (500mg/kg bw) was the most effective in preventing cell degeneration damage in the sepsis model mouse liver. although treatment group i (100mg/kg bw) and treatment group ii (300mg/kg bw) had different average values of cell degeneration damage, based on statistical test results, treatment group i (100mg/kg bw) and treatment group ii (300mg/kg bw) had the same antisepsis effect on cell degeneration. the effective dose of parsley leaf extract can be determined in the statistical analysis results using the anova method at a 95% confidence level. the analysis results show a significant difference in the parsley leaf extract dose as a preventive measure for cell degeneration damage in the sepsis model mouse liver. the reduction in hepatocyte cell degeneration damage is suspected to be due to various active compound contents found in parsley leaves such as flavonoids and saponins. cell degeneration damage in mice is caused by e. coli bacteria induced in the mice. the inflammation occurs due to the release of cytokines that can disrupt normal cellular signalling pathways in hepatocytes, including cell degeneration. flavonoids in parsley leaves are believed to potentially prevent inflammation and protect normal hepatocytes from cell degeneration damage in the liver (maleki et al., 2019). flavonoids have strong antioxidant properties that help protect normal hepatocytes from oxidative stress. oxidative stress is a condition where there is an imbalance between damaging free radical production and the body's antioxidant defences. flavonoids help neutralize free radicals and reduce cell degeneration damage caused by oxidative stress (panche et al., 2016). flavonoids can inhibit the production of pro-inflammatory mediators such as cytokines and enzymes involved in the inflammatory process. by reducing inflammation, flavonoids help protect normal hepatocytes from cell degeneration damage caused by excessive inflammatory responses (maleki et al., 2019). saponins in kentut leaves not only act as antiinflammatory agents that reduce cell degeneration damage in the liver but also enhance liver cellular defence, thus helping maintain balance in the immune system and reducing inflammation that damages hepatocyte degeneration. the saponin content in kentut leaves can enhance liver detoxification systems, aiding in eliminating harmful compounds such as e. coli bacteria in mice. improved liver detoxification can help protect normal hepatocytes from damage caused by toxic substances and inflammation (wang et al., 2019). research results (table 4) indicate a decrease in cell degeneration damage in hepatocytes after mice were infected with e. coli bacteria. the normal group had the smallest average at 2.21% ± 0.02 because the normal control group was not injected with e. coli, thus not experiencing sepsis that leads to necrosis. in the sepsisexperiencing mouse group (groups ii to vi), the positive control (+) group and treatment group iii (500mg/kgbw) showed similar effects on liver necrosis. it was the most effective doses among sepsis savitri et al. – histopathological description of mouse liver in a sepsis 491 experiencing mice compared to other groups infected with e. coli. in the treatment groups with parsley leaf extract, namely treatment group i (100mg/kgbw), treatment group ii (300mg/kgbw), and treatment group iii (500mg/kgbw), treatment group iii (500mg/kgbw) was the most effective in preventing cell degeneration damage in the sepsis model mouse liver. in treatment group i (100mg/kgbw), based on statistical test results, it was found to be equally effective as the negative control (-) group in preventing necrosis. the reduction in necrosis damage in hepatocytes is suspected to be due to various active compound contents found in parsley leaves such as flavonoids, saponins, and tannins. necrosis damage in the sepsis model mouse liver is caused by e. coli bacteria induced in the mice. the reduction in necrosis damage is likely due to the flavonoid content in parsley leaves, which can influence gene expression in the inflammatory pathway, thereby reducing the production of molecules that cause excessive inflammation in sepsis-induced mouse livers. flavonoids can protect hepatocytes from direct damage and necrosis by stabilizing cell membranes and reducing oxidative damage caused by e. coli infection leading to liver injury (tan et al., 2022). the strong reduction in necrosis damage is also suspected to be due to the saponin content in parsley leaves. saponins in parsley leaves have detoxifying effects on the liver. saponins are believed to help eliminate necrosis in e. coli-infected mouse livers and reduce liver workload, thereby reducing the risk of necrosis in sepsis-induced mouse livers. saponins can affect the immune response, including the immune system in the liver. by regulating immune responses, saponins can help control excessive inflammatory reactions that can cause necrosis in sepsis-induced mouse livers (waheed et al., 2012). kentut leaves containing saponins can function as inhibitors of inflammatory enzyme activity and modulate cellular signalling pathways involved in inflammation and necrosis in the liver. saponins inhibit the production of inflammatory mediators that can reduce inflammation in the liver caused by e. coli infection and protect liver cells from damage leading to necrosis. tannins inhibit the activity of pro-inflammatory enzymes such as proinflammatory cytokines and other inflammatory mediators, thereby reducing the production of inflammatory mediators and helping prevent excessive inflammatory responses that can cause necrosis in sepsisinduced mouse livers (fikru et al., 2012). research results (table 5) indicate a decrease in damage to pmn in hepatocytes after mice were infected with e. coli bacteria. the normal group had the smallest average at 1.62% ± 0.02, because the normal control group was not injected with e. coli, thus not experiencing sepsis that leading to pmn increase. in the sepsisexperiencing mouse group (groups ii to vi), group ii was the most effective dose among sepsis-experiencing mice compared to the other 4 groups infected with e. coli. group ii served as the positive control containing ciprofloxacin. among the treatment groups with parsley leaf extract, treatment group iii (500mg/kgbw) was the most effective in preventing pmn increase in the sepsis model mouse liver. pmn refers to granulocyte leukocytes or polymorphonuclear white blood cells, a type of white blood cell that is part of the immune system. tannins in parsley leaves are believed to modulate the immune system by affecting immune responses and regulating the activity of immune cells in pmn to reduce excessive inflammation and prevent cell damage caused by e. coli infection in sepsis-induced mouse livers (chen et al., 2023). alkaloids in parsley leaves are also believed to play a role in inhibiting the release of inflammatory mediators such as pro-inflammatory cytokines by pmn in inflammatory responses, thereby helping reduce excessive inflammation and prevent tissue damage. alkaloids can affect pmn activity, including migration and the release of proteolytic enzymes. by regulating pmn activity, alkaloids can control inflammatory responses involving pmn cells (bai et al., 2021). conclusions the research results show the histopathological picture of liver cell degeneration in group pi (100mg/kgbw) at 20.79%±0.03, group pii (200mg/kgbw) at 21.63%±0.02, and group piii (500mg/kgbw) at 9.08%±0.02. for necrosis, group pi (100mg/kgbw) exhibited 22.62%±0.04, group pii (200mg/kgbw) showed 17.63%±0.02, and group piii (500mg/kgbw) had 6.05%±0.02. regarding pmn, group pi (100mg/kgbw) recorded 39.56%±0.03, group pii (200mg/kgbw) registered 28.05%±0.02, and group piii (500mg/kgbw) showed 18.45%±0.03. statistical tests indicate the effect of parsley leaf extract as a preventative measure against liver histopathology in a sepsis model of e. coli-infected mice, with significant values for necrosis (p=0.000), cell degeneration (p=0.000), and pmn (p=0.000). acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university, and the laboratory of pathological anatomy, brawijaya university, malang, for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. ana retnowati wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. 492 biology, medicine, & natural product chemistry 13 (2), 2024: 485-492 funding: the authors declare no funding. 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(2021). sepsis. world health organization. https://www.who.int/news-room/fact-sheets/detail/sepsis biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 1-5 | doi: 10.14421/biomedich.2021.101.1-5 issn 2540-9328 (online) the effectiveness of giving plant pgpr rhizosphere bamboo on cocoa seeds germination at the nursery level muhammad yusril hardiansyah*, yunus musa, abdul mollah jaya department of agronomy, faculty of agriculture, universitas hasanuddin, jl. perintis kemerdekaan km. 10 tamalanrea indah, makassar, indonesia. corresponding author* yusrilhardiansyah1@gmail.com manuscript received: 27 september, 2020. revision accepted: 26 june, 2021. published: 01 july, 2021. abstract the low productivity of cocoa plantations in indonesia is partly due to the low quality of seeds, which refers to the impeded growth of cultivated cocoa nurseries. seed is the initial growth of plants so the importance of giving special treatment to seeds will refer to better seed growth. provision of plant growth promoting rhizobacteria (pgpr) microbes can produce indoleacetic acid (iaa) in plants to improve the quality of plant growth. this study aims to determine the effectiveness of the provision of plant growth promoting rhizobacteria bamboo rhizosphere against cocoa seed germination. the study was carried out in the farmer group garden, gantarangkeke district, bantaeng. this study was arranged in the form of a two-factor factorial design (f2f) in a randomized block design (rbd). the use of cocoa seed type as the first factor consisted of gtb (gantarangkeke bantaeng) local cocoa seed and mcc 01 cocoa seed and seed immersion treatment at pgpr rhizosphere bamboo concentration as the second factor consisting of 0% (control) concentration, 5%, 10 % and 15%. the results obtained indicate that administration of seeds with bamboo rhizosphere pgpr affects the germination (100.00%), the speed of seed growth (7.14%/etmal), as well as on abnormal seeds (10.00%). so that the provision of bamboo rhizosphere pgpr on cocoa seeds has an effective influence on seed germination and cocoa seedling development. keywords: cocoa seed; plant growth promoting rhizobacteria; seedbed. introduction the soaring increase in world cocoa demand is due to the increasing demand for cocoa beans which is getting higher every year. the annual world demand for cocoa reaches 6.7 million tons and only 2.5 million tons can be met. this means that there are still more than 4 million tons to meet the increasing market needs, so that this can still be an opportunity for indonesia, especially south sulawesi (yusniar, 2013). cocoa (theobroma cacao l.) is a strategic commodity which ideally is able to play a maximum role in increasing farmers' income in indonesia. this can happen because cocoa has always had a good price development in meeting market needs. however, the reality is that even though every year there is an increase in the planting area, it cannot increase production. the average farmer production is only 1/5 of the potential productivity of the cocoa plant, which ranges from 1.2 3 tonnes/ha (junaedi, et al., 2017). of course, these results are still far from expectations because of the many obstacles that occur in the cocoa cultivation process. one of the factors thought to be the main cause of the low productivity of cocoa plantations in indonesia is the low quality of seeds which refers to the inhibition of growth in the vegetative phase of cultivated cocoa (mertade & basri, 2011; sugiharti, 2006). in general, farmers in indonesia cultivate cocoa using seeds derived from beans. although generative seeding using seeds is easy to do and can be produced in large quantities at low cost, seedlings of cocoa using seeds will produce genetically non-uniform plants. this is because cocoa is a cross-pollinating crop (li et al., 1998). this statement indicates the emergence of a problem currently being faced, namely whether to increase cocoa production to meet cocoa needs in the future, while the availability of good quality plant vegetative phase conditions is currently difficult to obtain. cultivation of cocoa starts from the nursery stage or the vegetative phase and from this stage will determine the success of plants in the generative phase. during this nursery period, good quality seeds must be obtained so that they will be used as a benchmark for optimum growth and produce fruit and good quality cocoa beans in the future. of course, in order to get quality seeds, the selection or use of good seeds in germination will be a major factor. one way to get good seeds is by soaking or soaking the seeds. soaking is a hydration activity slowly before the seeds are germinated so that the potential of seed water reaches a balance to activate metabolic activities in the https://doi.org/10.14421/biomedich.2021.101.1-5 2 biology, medicine, & natural product chemistry 10 (1), 2021: 1-5 seeds to improve germination and seed growth (rouhi et al., 2011). the immersion treatment can be combined with the provision of biological agents capable of improving the quality of seed germination, for example with microbes that are capable of producing growth hormones in plants. one of the microbes that can be used is the microbes that come from plant growth promoting rhizobacteria (pgpr) as a hormone for plants that can provide more nutrients to spur plant growth in the vegetative phase of plants. the use of pgpr in this plant is proven from several research results that have been conducted. the results of research by baihaqi, et al. (2018) show that the use of pgpr on cucumber plants can increase plant growth from 68.6 to 77.7% compared to without pgpr. other research on plantation crops, namely the results of research from widyaningrum (2017) shows that the application of pgpr can increase root length, number of roots, number of leaves, plant height, total dry weight, shoot root ratio, seed steadfastness and robusta coffee seed quality index. in the rhizosphere or bamboo roots there are pseudomonas flourenscens bacteria and bacillus polymixa bacteria which can help the decomposition process (decomposer). the bamboo root pgpr bacteria can secrete a liquid that can dissolve minerals so that they become available nutrients, break down and decompose organic matter (decomposition of organic matter) into plant nutrients. in addition, the pseudomonas fluorenscens and bacillus polymixa bacteria can release enzymes and substances that are useful to stimulate plant growth and release antibiotics that can inhibit the growth and development of pathogenic microbes (microbes that cause disease) (efendi, 2011). research on the presence and diversity of microbes in the rhizosphere of bamboo plants has been conducted by several previous researchers. according to baharuddin et al. (2007) found antagonistic bacteria such as pseudomonas flourences, bacillus subtilis and streptomyces in the roots of healthy bamboo plants. meanwhile, research conducted by asniah (2013) showed that the inoculation of fungi from bamboo root soil into the nursery soil had a significant effect on increasing the wet weight of broccoli plants. this study aims to obtain data and information on the effectiveness of giving plant growth promoting rhizobacteria rhizosphere of bamboo against cocoa seed germination. information from the results of this study is expected to be a solution for cocoa farmers in improving their cultivation systems, especially in the nursery phase. methods this research was conducted at the talaka farmer group garden, gantarangkeke district, bantaeng regency. this research took place from august to november 2019. the tools used were 30 liter buckets, hoes, shovels, scissors, knives, machetes, rulers, meters, calipers, ph meters, carts, analytical scales, sprayers, scales, jerry cans, funnels, aqua plastic bottles, hoses, stoves, pans, paranets, cloths, plastic cups, brown envelopes, millimeter paper blocks, cameras, signage, matches, and writing instruments. the materials used were soil samples from the research location, local gtb (gantarangkeke bantaeng) cocoa seeds, mcc 01 clone cocoa seeds, bamboo rhizosphere (bambusa blumeana), molasses, bran, raw shrimp paste, rice water, water, husk charcoal, sawdust, clear plastic, dab of soap, polybags 12 x 17 cm, label paper, dolomite and label paper. the study was conducted using a two-factor factorial design (rf2f) in a randomized block design (rbd) as an environmental design. this research consists of 2 factors. the first factor is the type of cocoa seed (b) which consists of: local cocoa seeds gtb (gantarangkeke bantaeng) (b1) and cocoa seeds clone mcc 01 (b2), while for the second factor is the initial soaking of seeds with pgpr solution for cocoa seeds. with various concentrations of seed priming (k) consisting of: 0% concentration (0 ml of pgpr solution or 1000 ml of water), 5% concentration (50 ml of pgpr + 950 ml water), 10% concentration (100 ml of pgpr + solution 900 ml of water) and a concentration of 15% (150 ml of pgpr solution + 850 ml of water). the flow of this research includes making a plant growth promoting rhizobacteria solution which is carried out by taking bamboo rhizosphere then growing it by fermentation process in a container for 14 days, preparing the seeds by selecting seeds from mother pods of mcc 01 and gtb cocoa then separating the placenta from the seeds. giving of pgpr solution to cocoa seeds (initial soaking of cocoa seeds) simultaneously according to the treatment for 18 hours (ratnawati et al., 2013), preparation of sprouts media using sterile cloth and sawdust, planting in seedbeds (seedbeds), maintenance and observation. observation parameters in this study are as follows: 1. germination capacity (%), measurement based on observing the number of normal germinated seeds marked with cotyledons on the raised seed. calculation of germination that is on day 7 and day 14 (deptisari et al., 2018). sprouts are calculated using the formula: db = ∑ kn observation i + ∑ kn observation ii number of seedlings planted 𝑥 100% information: db : germination capacity (%) ∑ kn observation i : the number of sprouts is normal on the 7th day ∑ kn observation ii : the number of sprouts is normal on the 14th day hardiansyah et al. – the effectiveness of giving plant pgpr rhizosphere bamboo on … 3 2. seed growth speed (%/etmal), measurement based on observing the number of normal germinating seeds every day until the day 14 and expressed on a percent scale (tefa, 2017). seed growth rate is calculated using the formula: 𝐾𝐶𝑇 = (% 𝐾𝑁 𝑒𝑡𝑚𝑎𝑙 ) = ∑ 𝑁 𝑡 𝑡𝑛 0 information: 𝑡 : observation time 𝑁 : percentage of normal sprouts every time of observation 𝑡𝑛 : end time of observation (day 14) 1 etmal : 1 day (24 hours) 3. abnormal seeds (%), measurement based on observing the number of seeds that germinate abnormally. observation of abnormal seeds is carried out on the day 14 after the nursery (deptisari et al., 2018). abnormal seeds are calculated using the formula: abnormal seeds = ∑ abnormal seeds number of seedlings planted 𝑥 100% result and discussion the results showed that the use of two types of cocoa seeds in the initial soaking treatment of seeds with pgpr with seed concentration and the interaction of pgpr application on two types of cocoa seeds had a very significant effect on seed germination, seed growth speed and abnormal seeds in cocoa. the average parameter results are presented in table 1. table 1. germination (%), seed growth speed (% / etmal) and abnormal seed (%) of cocoa on the 14th day of giving pgpr bamboo rhizosphere to seeds. treatment germination capacity (%) seeds growth speed (%/etmal) abnormal seeds (%) gtb local seeds + pgpr concentration 0% 90,00d 6,43b 10,00a gtb local seeds + pgpr concentration 5% 90,00d 6,43b 10,00a gtb local seeds + pgpr concentration 10% 93,33c 6,67b 6,67b gtb local seeds + pgpr concentration 15% 100,00a 7,14a 0,00d mcc 01 seeds + pgpr concentration 0% 96,67b 6,90ab 3,33c mcc 01 seeds + pgpr concentration 5% 100,00a 7,14a 0,00d mcc 01 seeds + pgpr concentration 10% 96,67b 6,90ab 3,33c mcc 01 seeds + pgpr concentration 15% 100,00a 7,14a 0,00d bnj 0,05 0,03 0,45 0,40 information: the numbers followed by the same letter (abcd) in the same row and column mean not significantly different in the bnj 0.05 level test. bnj test results table 1. the interaction between the use of cocoa species and the treatment of bamboo rhizosphere pgpr concentration has a very significant effect on cocoa seed germination. the highest germination parameter was 100.00%, the highest seed growth speed parameter was 7.14% and the highest abnormal seed parameter was 10.00%. use of two types of cocoa seeds in seed germination in the nursery phase, the results showed that the average use of mcc 01 cacao seed type had the highest effect on germination and seed growth speed, while local gtb seeds had the highest effect on abnormal seed conditions. the seeds of mcc 01 had a very significant effect on germination, which was counted twice on the 7th and 14th day with the highest germination rate of 100%, the observed seed growth rate for 14 days with the fastest seed growth percentage of 7.14%/etmal and the percentage of abnormal seeds calculated on the 14th day with the highest abnormal seeds, namely 10%. this is due to the influence of the environment where the seeds grow at the time of the nursery, where the humid conditions and long seeding times lead to optimum germination and seed growth speed. in addition, the genetic factor of the seed itself is also a major influence on germination, because if the genetic of the seed is not strong enough to accept the environmental conditions where it is grown and the initial treatment, it will cause the seeds to not have the potential to germinate normally or in other words, abnormal seeds. this is in accordance with saleh (2004) statement, which states that cocoa has an epigeal germination type, so the time needed to produce radicles greatly affects the speed of procuring ready-to-channel seeds. the process of seed germination is influenced by genetic and environmental factors. the improvement of the external environment will significantly encourage the emergence of radicles as the beginning of the seed 4 biology, medicine, & natural product chemistry 10 (1), 2021: 1-5 germination process. the emergence of sprouts above the soil surface is a factor that reflects the vigor of a seedling. to find out which treatment can increase vigor, the sprouts that can appear above the soil surface are observed from the number of seeds germinated. the average germination rate with the highest effect was the type of mcc 01 seed when compared to the local gtb seed germination capacity. the average seed germination rate in this study had optimum growth because the seeds had been given previous treatment, where the highest germination growth in mcc 01 seeds and local gtb seeds had been treated with pgpr with a concentration of 5% and 15%. giving pgpr treatment with the highest concentration is a major factor in the germination power of seeds. this is in line with the opinion expressed by schmidt (2002), that pre-treatment is carried out with the aim of increasing the power, speed and uniformity of seed germination. the average seed growth speed with the fastest effect was the mcc 01 seed type when compared to the local gtb seed growth rate. the average seed growth speed of mcc 01 seeds and local gtb seeds with pgpr treatment with a concentration of 5% and 15% showed the fastest growing results when compared to other seeds. the growth rate of mcc 01 seeds and local gtb seeds is influenced by the optimum condition of the seeds at the time of the seed nursery and the provision of special treatment for seeds so that the metabolism of seed growth can run well. this is in line with the statement of lesilolo et al. (2013) stated that the speed of seed growth is a process of rapid reactivation of seeds if the surrounding conditions are for optimum growth and the metabolic process is not hampered. abnormal seeds in the use of local gtb seeds without pgpr treatment or control and with pgpr treatment with a concentration of 5% showed the highest yield of abnormal seed conditions when compared to other seeds. abnormal seeds occur due to the lack of pre-treatment of local gtb seeds, besides the physical and genetic characteristics of local gtb seeds which tend to be weaker than mcc 01 seeds. the occurrence of abnormal seeds in the nursery process is due to the deterioration of the seeds so that the quality of the seeds decreases which resulting in inhibition of the germination process. this is in line with the statement of sutopo (2002), that the decline in seeds that occurs indicates a decrease in seed quality or viability which results in low vigor and poor plant growth and production. this is also reinforced by the statement of debtisari et al. (2018), that abnormal seeds are seeds that are capable of germinating but do not show the potential to develop into normal sprouts. the seed is said to grow abnormally if the important structure for the germination process is lost or damaged, the growth of sprouts is weak due to defective or disproportionate sprouts structure, and sprouts with slow growth until the end of the test. when compared to normal seed sprouts, the abnormal seed growth is smaller than normal sprouts. concentration of pgpr rhizosphere bamboo on seeds in the germination of seeds in the nursery phase, the results showed that the treatment of seed concentration using the bamboo rhizosphere pgpr had a very significant effect on germination capacity, germination speed, and the percentage of abnormal seeds. this is because the need for pgpr or initial immersion of seeds can result in a more active metabolism of seeds to support germination, besides that the use of pgpr microorganisms can improve seed quality because pgpr acts as a growth stimulant (biostimulant) by synthesizing and regulating the concentration of various regulatory substances. growing (phytohormones) very well on seeds. this is supported by research from baihaqi et al. (2018) which states that soaking seeds with pgpr is intended so that the bacteria contained in pgpr are able to colonize the seeds as early as possible. the right long soaking treatment can increase plant yield because the bacteria will bind the seedcoat and imbibe it into the seeds. conclusion giving pgpr bamboo rhizosphere from several concentrations gave different responses to cocoa seed germination including the parameters of germination (100.00%), seed growth speed (7.14%/etmal), and abnormal seeds (10.00%). so that seed treatment with bamboo rhizosphere pgpr has an effective effect on cocoa seed germination. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references asniah, widodo, & wiyono s. (2013). potensi cendawan asal tanah perakaran bambu sebagai endofit dan agen biokontrol penyakit akar gada pada tanaman brokoli. j. fitopatol indones. 1 (2): 61-68 baharuddin, nur r., & sayifudin a. 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(2017). kajian penggunaan klon unggul kakao pada perkebunan rakyat di kabupaten bone. j. agroplantae. 6 (1): 46-49. leisolo m.k.j., rirydan e.a., & matatula. (2013). pengujian viabilitas dan vigor benih beberapa jenis tanaman yang beredar dipasaran kota ambon. jurnal agrologia, 2 (1): 1-9. li z., traore a., maximova s., & guiltinan m.j. (1998). somatic embryogenesis and plant regeneration from floral explants of cacao (theobroma cacao l.) using thidiazuron. in vitro cell. dev. biol-plant. 34: 293–299. rouhi h.r., surki a.a., sharif-zadeh f., afshari r.t., aboutalebian m.a., & ahmadvand g. (2011). study of different priming treatments on germination traits of soybean seed lots. notulae sci biol. 3 (1): 101-108. saleh m.s. (2004). pematahan dormansi benih aren secara fisik pada berbagai lama ekstraksi buah. agrosains. 6 (2): 79-83. schmidt l. (2002). guidelines for handling tropical and subtropical forest seed plants. danida forest seed centre. krogerupvej 21. dk-3050. denmark. humlebaek. sugiharti e. (2006). budidaya kakao. bandung: nuansa. sutopo l. (2002). teknologi benih. jakarta: pt raja grafindo persada. tefa a. (2017). uji viabilitas dan vigor benih padi (oryza sativa l.) selama penyimpanan pada tingkat kadar air yang berbeda. jurnal pertanian konservasi lahan kering. 2 (3): 48-50. widyaningrum a. (2017). pengaruh aplikasi pgpr (plant growth promoting rhizobacteria) dan kompos azolla terhadap mutu bibit asal stek kopi robusta. jember: fakultas pertanian universitas jember. yusniar. (2013). membangun kesejahteraan petani lewat nagari model kakao (nmk). padang: dinas perkebunan sumatera barat. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 371-389 | doi: 10.14421/biomedich.2023.121.371-389 issn 2540-9328 (online) medicinal biospecificity of ginger and its efficacious bioactive compounds in the context of its biological activities against predominant health issues: current study and new avenues haseeba shahzad1,4,*, shaifa saleem1, waqas hanif2, zareena ali3, muhammad zeeshan ahmed4, haq nawaz4, zelle humma3,5, laraib rana6, muhammad qamar farid7, hajira kalsoom1, samavia jaan1 1school of biochemistry and biotechnology, university of the punjab, quaid-i-azam campus, lahore 54590, pakistan. 2department of pathology, combined military hospital, bannu, pakistan. 3department of biochemistry and biotechnology, the women university multan, pakistan. 4department of biochemistry, bahauddin zakariya university, multan 60800, pakistan. 5department of experimental medicine, university of genoa, italy. 6department of biochemistry, faculty of biological sciences quaid-i-azam university, islamabad 45320, pakistan. 7department of chemistry, faculty of science, chulalongkorn university, bangkok 10330, thailand. corresponding author* haseebashahzad388@gmail.com abstract there is a multitude of life-threatening and widespread health issues worldwide, regarding weak immunity, severe inflammation, viral infections, bacterial infections as well as antimicrobial resistance (amr), high free radicals generation, and cancer. ginger, a perennial plant of the zingiberaceae family with several authentic nutritional and medicinal values used in many countries as traditional medicine. that is why, the study was designed to highlight recent studies about medicinally most efficacious bio-active compounds of ginger along their biological significance related to immuno-stimulatory, anti-inflammatory, anti-viral, anti-bacterial, anti-oxidant, and anti-cancer effects. our study also recognized future gaps in research. the study included professional research data under duration from 2001-2022 appearing in books and scholarly journals, collected from scientific database platforms via pubmed, web of science, google scholar, springer nature, science direct and scopus. the present study includes the medicinal effects of almost 44 most influential ginger compounds like phenolics, terpenoids, flavonoids, and vinyllyl ketonic compounds etc. our results revealed the strong alleviating effects of gingerols, shogaols, paradols, and polyphenols. moreover, the ginger essential oil has proven to be very effective both for antiviral and antibacterial activity. however, no data is available in previous literature for components of ginger involved in immuno-stimulatory, effects. there is also a need to explore components for antibacterial activity. however, research has been conducted on ginger for only a few viruses despite its strong alleviating effects. besides this, more study is needed to comprehend the comprehensive mechanism of action (especially at the molecular level) regarding the anti-bacterial and anti-viral activity of ginger and its constituents. keywords: anti-bacterial; anti-cancer; antioxidant; antiviral; immunostimulatory; anti-inflammatory; ginger bioactive compounds; new avenues; mechanism of action. introduction herbal medicines’ use is increasing as compared to the use of chemicals day by day, therefore, more research has been performed on phytochemicals to make them more effective for the intervention of various ailments (ishiguro et al., 2007). but it is very troublesome to explore the effectiveness of herbal treatment because of the presence of many compounds in various forms (raynor et al., 2011). ginger (zingiber officinale) is an angiospermae and belongs to the family zingiberaceae. its rhizomes are widely used as a spice and traditional medicine in many countries like the subcontinent and the united states (shahrajabian et al., 2019). due to the diverse phytochemistry of ginger, the components of ginger are classified as volatiles and non-volatiles. volatile components of ginger include sesquiterpene and monoterpenoid hydrocarbons which provide a distinct taste and aroma to ginger while the non-volatile pungent components include shogaols, paradols and zingerone (jolad et al., 2005). ginger has diversified nutritional and medicinal importance. the general nutritional and nonnutritional components of ginger and their quantities have been represented in table 1. ginger contains more than 400 different compounds. the major compounds present in ginger are terpenes, phenolic compounds, carbohydrates, proteins, lipids, gingerols, and shogaols. there are many phytochemicals present in ginger that play an effective medicinal role. the major classification of phytochemicals has been represented in figure 1 and manuscript received: 04 january, 2023. revision accepted: 19 april, 2023. published: 24 june, 2023. https://doi.org/10.14421/biomedich.2023.121.371-389 mailto:haseebashahzad388@gmail.com 372 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 the structures of major bioactive compounds of ginger have been depicted in figure 2. moreover, the oil extract of ginger has been observed to contain e-citral, z-citral, ocimene, and camphene (munda et al., 2018). ginger shows a wide range of prophylactic and curative functions. it suppresses the level of cholesterol in the blood, prevents excessive clotting, is used to treat dyspepsia, and improves appetite (palatty et al., 2013). other properties of ginger include fat loss, prevention of cardiovascular diseases, anti-cancer, and antiinflammatory action (elshater et al., 2009). gingerols play an important role in the alleviation of arthritis and pain. ginger also possesses antioxidant, antimicrobial, and anti-allergic properties because of the presence of gingerols and shogaols (semwal et al., 2015). ginger is consumed as a painkiller for chest pain, menstrual pain, and back pain. it is utilized to cure cough, bronchitis, and upper respiratory tract infections (shukla & singh, 2007). besides this, in china and many other countries, ginger is used for the treatment of rheumatism, nervous disorders, toothache, stroke, asthma, constipation, catarrh, gingivitis, diabetes, and migraine. due to its antiviral properties and warming effect, it is also used for relieving flu and colds (qidwai et al., 2003). moreover, it is also used as a stimulant, diuretic, and carminative in asian medicines (shadmani et al., 2004). ginger essential oil has proved to be very effective, particularly concerning antibacterial and antiviral activity (koch et al., 2008; mostafa et al., 2018). the constituents present in ginger essential oil and their percentage have been represented in table 2. besides this, the strong effects of ginger oleoresin, particularly, regarding antioxidant activity have been observed (ji et al., 2017). in previous literature, there is abundant data available regarding various effective and authentic medicinal approaches of ginger for various ailments but there is no such review available, in which data has been analyzed with a special focus on recent studies (2001-2022) regarding specificity of ginger bioactive compounds with respect to its biological activities to demonstrate the immuno-stimulatory, anti-inflammatory, anti-viral, antibacterial, antioxidant and anticancer effects. besides this, no data is available in past in which after the most recent study (up to 2022), a future research gap got identified. this review has been planned to keep in view the most common and serious health problems and their intervention issues of the day. our review will provide valuable data for drug manufacturers to isolate the bioactive components of ginger for manufacturing herbal drugs against particular disorders. besides this, it will also provide new pathways for the researchers to fulfill the future gap to remove the hot issues of the day easily. table 1. general composition of ginger (agriculture, 2018; nawaz et al., 2018). components value per 100g water 78.89g carbohydrates 17.77g total lipids 0.75g proteins 1.82g ash 0.77g total dietary fiber 2.0g total phenolic acids 0.63g total tannins content 0.28g total flavonoid content 3.93g ca 16mg fe 0.60mg mg 43mg p 34mg k 415mg na 13mg zn 0.34mg vitamin c 5.0mg choline 28.8mg figure 1. classification of major phytochemicals present in ginger (ghasemzadeh et al., 2010; shukla & singh, 2007). shahzad et al. – medicinal importance of ginger 373 figure 2. structures of major phytochemicals present in ginger (drawn by chem draw). table 2. representation of percentage composition of constituents of ginger essential oil. compounds % reference β-bisobeolene 12.5 (varoni et al., 2018) α-zingiberene 25 β-sesquiphellandrene 18 β-phellandrene 8 (ferreira et al., 2018) α-zingiber 24 geraniale 15 farnesene 7.6 (singh et al., 2008) geraniale 26 α-zingiberene 9.5 neral 7.4 1,8-cinerol 10 (snuossi et al., 2016) camphene 12 α-zingiberene 7 β-phellandrene 11 camphene 5 (mesomo et al., 2013) ar-curcumene 11.3 eucalypto 3 geraniale 11 β-cedrene 8.6 (moreira da silva et al., 2018) geraniale 16 geranyl acetate 8.4 z-citral 9.2 zingiberene 14 (borah et al., 2017) β-sesquiphellandrene 27 α-farnesene 10.5 caryophyllene 15.3 β-bisabalene 11 (wang et al., 2006) α-zingiberene 20 β-sesquiphellandrene 13 ar-curcumene 15 citral 7.5 (nogueira de melo et al., 2011) ar-curamene 59 α-zingiberene 7.5 1,8-cinerol 8 farnesene 7.6 (chmit et al., 2014) geraniale 26 neral 7.4 α-zingiberene 9.5 table 3. representing the percentage composition of constituents of ginger oleoresin by different methods (supardan et al., 2012). components composition (%) method nortrachelogenin 6.74 ultrasonicassisted extraction ar-curcumene 8.9 β-sesquiphellandrene 5.02 zingerone 13.85 shogaol 7.92 farnesene 6.29 soxhlet zingiberene 5.13 zingerone 14.47 β-sesquiphellandrene 5.44 shogaol 7.14 β-sesquiphellandrene 6.54 co2 supercritical zingiberene 16.77 geraniol 9.01 gingerol + shogaol 3.48 β-phellandrene 12.86 ginger as an immuno-stimulant immunostimulant is an agent that aids in increasing the body’s immune-response. immunity is defined as the body’s ability to resist infection or toxins by the action of sensitized white blood cells and specific antibodies (divangahi et al., 2021). immune-deficiency leads to various abnormalities and disorders including digeorge syndrome, chronic mucocutaneous candidiasis, interleukin-12 receptor deficiency, hyperimmunoglobulin m syndrome, severe combined immunodeficiency disease, leukocyte adhesion deficiency syndrome, chronic granulomatous disease, and wiskott-aldrich syndrome, etc (vaillant & qurie, 2021). ginger exhibits immuno-stimulatory and antiinfection properties against pathogenic bacteria, worms, and viruses (sanderson et al., 2002). it also shows its immunomodulatory effects in animals, like fish (nya & austin, 2009). white blood cells (wbc) are considered to be primary defenders of the body. in an experiment, when the rainbow trout were fed with ginger, an increased number of wbc, neutrophils, and other blood cells was observed (nya & austin, 2009). it has been noticed that herbal plants boost immunity by elevating the number of blood cells (sahu et al., 2007). like humans, fishes also have specific and nonspecific defense systems to protect themselves against microbes. in fishes, mucus and skin are the primary non-specific defenses. when any pathogen enters the body both humoral and cellular defenses are activated and phagocytosis is part of non-specific immunity. the increased phagocytotic activity of wbcs was observed in fishes fed with ginger (haghighi & rohani, 2013). the phagocytotic activity was compared in two groups of fish. in a group of fishes, with a 0.1% ginger diet, the mean phagocytosis index was 2.21±0.082 while it was greater, 2.37±0.263 in fishes fed with a 1% ginger diet 374 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 (dugenci et al., 2003). besides this, plasma proteins that play a role in humoral defense were also found to increase. it was concluded that both humoral and cellular immunity were enhanced by using ginger extract in the case when any pathogen entered the body through injury (dügenci et al., 2003). when plasma protein level was compared between two groups of fishes, it was observed that in a group with a 0.1% ginger diet the level of protein in fishes was 2.26±0.2541(g/dl) while it was significantly greater, 3.84±0.13(g/dl) in fishes fed with 1% ginger diet (dugenci et al., 2003). ginger shows immunomodulatory effects due to the presence of gingerols, shogaols, zingerone, and paradols (rasmussen, 2011). it has been observed that 6-gingerol suppressed the expression of tnf-α and inos by blocking pkc and nf-кb signaling pathways in macrophages of lipopolysaccharide-stimulated mice (lee et al., 2009). it has been reported that pkc stimulates nf-кb which in turn regulates the inos expression at the transcriptional level (simon et al., 2015). similarly in another study, 6gingerol was observed inhibiting the inos expression and no production in activated macrophages of j774.1 mice (ippoushi et al., 2003). in another study, 6-gingerol inhibited the formation of il-12, tnf-α, and il-1β in macrophages as well (tripathi et al., 2007). the other mechanistic actions of ginger as an immunostimulant are represented in figure 3. figure 3. mechanistic action of immune-stimulatory effects of ginger (shokr & mohamed, 2019). (upward arrow-indicating the enhancing activity, downward arrow-indicating the reducing activity) ginger as an anti-inflammatory when inflammation happens, certain chemicals from the white blood cells of the body enter tissues to protect them from invaders. this increases the blood flow to the site of damage or infection and thus causes redness. besides this, the leakage of fluid in tissues by certain chemicals results in swelling as well (varela et al., 2018). sometimes, acute inflammation (uncontrolled) may become chronic and leads to a variety of inflammatory disorders including bowel and cardiovascular diseases, cancer, and arthritis (zhou et al., 2016). ginger has proved to be very beneficial for the intervention of inflammatory disorders. ginger shows strong anti-inflammatory activity due to the presence of vinyllyl ketonic compounds. besides this, 6-paradol, 6shogaol, and 1-dehydro-6-gingerol are the constituents of ginger that have been observed to cause an antiinflammatory effect (ezzat et al., 2018). the antiinflammatory mechanism of the actions of ginger connecting its constituents has been illustrated in table 4. many enzymes are involved in causing inflammation like lipoxygenase shows its action by forming inflammatory lipid mediators such as hepoxilins, hydroxy fatty acid derivatives, lipoxins, and leukotrienes (kuhn et al., 2007). cyclooxygenase is involved in the formation of prostaglandins and thromboxane. ginger shows its action by decreasing the effect of cyclooxygenase, lipoxygenase, and arachidonic acid (lantz et al., 2007). in the experimental model of rheumatoid arthritis, more anti-inflammatory action has been noticed by combining both gingerols and ginger extract oil as compared to gingerols alone (funk et al., 2009). in inflammatory bowel diseases (ibd) which include ulcerative colitis and crohn's disease, an elevated level of tnf and pge2 has been observed (nakamura et al., 2006) because both diseases are responsible for inflammation (kawahara et al., 2015). ginger shows a protective effect against ibd by decreasing the level of both tnf and pge2 (el-abhar et al., 2008). in one study, tnf-α expression was compared in two groups of rats. in one group, liver cancer was induced by a choline-deficient diet and in the other group, a choline-deficient diet was given along with ginger extract. it was observed that the expression of tnf-α in rats with only a choline-deficient diet was 83.3 ± 4.52% while it was reduced in the group, that was also given the ginger diet along with choline-deficient diet where it was 7.94 ± 1.32% (p< 0.05). similarly the expression of nfκb was also observed in both these groups and it was observed that it was 88.3 ± 1.83% in group with only choline-deficient diet and it was significantly reduced in group that was also given ginger diet along with choline-deficient diet where it was 32.35 ± 1.34% (p<0.05) (habib et al., 2008). besides this, ginger has been observed to reduce the symptoms of gout (grzanna et al., 2005) and also relieve osteoarthritis of the knee (altman & marcussen, 2001). monocyte chemoattractant protein-1 (mcp-1) is a protein that is involved in inflammation. inflammation is also associated with the increased level of rantes production which activates the t cells (appay & rowland-jones, 2001) due to which the formation of il2 and ifn-ϒ also increases. in an experiment, when ginger was given to rats with high rantes levels, it was observed that ginger not only reduced inflammation and restricted t-cell activation but also suppressed the level of rantes and mcp-1( macrophage shahzad et al. – medicinal importance of ginger 375 inflammatory protein) (ezzat et al., 2018). besides this, the anti-inflammatory role of ginger oil has been noted by inhibition of il-1α (zhou et al., 2006). 6-gingerol decreases no production by inhibiting cytokines (ifn-γ and tnf-α) that stimulate the production of no (amri & touil-boukoffa, 2016). gingerol and shogaol inhibit the activity of prostaglandin synthetase enzymes and thus suppress the formation of prostaglandin. they also inhibit the action of cytokines i.e il-1 and il-8 that are involved in inflammation (verma et al., 2004). the comparison of factors involved in inflammation and counter effects of ginger has been shown in figure 4 in a nutshell. figure 4. comparison of factors involved in causing inflammation and therapeutic effects of ginger in a nutshell (ezzat et al., 2018; verma et al., 2004; zhou et al., 2006). abbreviations: tnf: tumor necrosis factor; nfkb: nuclear factor kappa-light-chain-enhancer of activated b cells; il: interleukin; ifn: interferon; pge-2: prostaglandin e-2; mcp-1: monocyte chemo-attractant protein-1; rantes: regulated on activation, normal t cell expressed and secreted. (upward arrow-indicating increasing activity, downward arrow-indicating decreasing activity) table 4. representing the anti-inflammatory mechanisms of ginger connecting its constituents. abbreviations: no, nitric oxide; tnf-α, tumor necrosis factor α; pge2, prostaglandin e2; gdnps, ginger-derived nanoparticles. sr. no. ginger/constituents dose research subjects mechanism of action reference 1 6-gingerol-rich fraction 50 & 100 mg/kg female specie wistar rats levels of no, tnf-α and myeloperoxidase enzyme, enhance (abolaji et al., 2017) 2 extract of ginger and zinger-one 0.1,1, 10&100 mg/kg female specie (balb/c mice) reduces the level of il-1β and inhibits the stimulation of necrotic factor-κb (hsiang et al., 2013) 3 6-shogaol 100 μm human intestinal epithelial cells (ht29/b6, caco-2) ceased the pi3k/akt, nf-κb signaling pathways (luettig et al., 2016) 4 gdnps 0.3 mg female mice (c57bl/6 fvb/nj) by using gdnps 2, levels of il-10 and il-22 and level of tnf-α, il-6, and il-1β decrease. (zhang et al., 2016) 5 extract of ginger 50 mg/ml mice (c57bl6/j) ginger extract prevents the production of tnf-α and activates the akt & nf-κb. (ueno et al., 2014) 6 6-gingerol, 6dehydroshogaol, 6shogaol 2.5 and 5,10 μm macrophage cells of the mouse (raw 264.7) no, pge2 production is stopped. (zhang et al., 2013) ginger as an antiviral the world health organization (who) and other public health agencies have warned about the emergence of infectious diseases at alarming rates that have not been previously noted (organization, 2021). the centers for disease control and prevention (cdc) elaborates on emerging infectious diseases. among all categories of infectious diseases, viral agents are the most serious threat to global populations (prevention., 2021). the 21st century is marked by major pandemics and epidemics due to novel viral agents that have been the source of mortality and morbidity around the world such as ebola virus, zika virus, middle-eastern respiratory syndrome 376 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 (mers), influenza a (h1n1) pdm/09, human immunodeficiency virus (hiv), middle-eastern respiratory syndrome (mers), west nile virus, chikungunya virus and currently severe acute respiratory syndrome virus 2 (sars-cov-2) (ong et al., 2020). besides this, the hepatitis c virus (hcv) is a critical illness. hcv may lead to acute or chronic hepatitis and principally causes liver cancer. universally, approximately 58 million human beings have chronic hcv infection. according to an approximation of who, almost 290 000 human beings expired from hepatitis c in 2019. at present, no efficient vaccine exists against hepatitis c (who, 2022) and pakistan is ranked second after china which is suffering from hepatitis at an alarming rate. in 2018, a very alarming prevalence rate of hepatitis c virus was observed in small towns in pakistan (ahmed et al., 2020). in china, fresh ginger is used as a folk medicine for the treatment of airway infections. ginger shows antiviral activity against various types of viruses like human respiratory syncytial virus (hrsv) and it has been observed that the effects of dried ginger are different from fresh ginger against hrsv because both dried and fresh ginger contain different constituents. fresh ginger shows better results against the virus as compared to dried ginger (jolad et al., 2005). hrsv causes lethality and death by inducing bronchiolitis and pneumonia (san chang et al., 2013). ginger reduces viral invasion by preventing the attachment and internalization of the virus into the cells. at high concentrations, fresh ginger stimulates the low respiratory tract mucosal cells to secrete ifn-b, the cytokine which inhibits the replication of viruses (ali et al., 2008). experimental studies have shown that an aqueous extract of ginger also reduces the infectivity of feline calicivirus (fcv). antiviral activity occurs due to a chemical interaction between phytochemicals like proanthocyanins, polysaccharides, polyphenols, and some other compounds present in natural extracts and the virus (aboubakr et al., 2016). the aqueous extract of ginger contains 1,2-propanediol, 1,2-benzene dicarboxylic acid, and 2,3-butanediol which does not affect the receptors of the cell for fcv but inhibits the attachment of viruses with cells by causing alterations in viral capsid. fcv is used as a surrogate for norovirus in experiments because it is very difficult to grow noroviruses in cell culture (aboubakr et al., 2016). norovirus is an enteric virus and is considered to be a major contributor to foodborne illnesses. aqueous extract of ginger has proved to be very beneficial in the prevention of foodborne diseases. we can also use its extract to wash vegetables and fruits in combination with water to prevent them from viral contamination (aboubakr et al., 2016). several terpenes in the alcoholic extract of ginger have been observed with anti-rhinoviral activity (adom et al., 2019). besides this, gingerenone has been observed in causing the inhibition of many types of influenza a viruses including h9n2, h1n1, and h5n1 (onyiba, 2022). propanediol in an aqueous extract of ginger has revealed strong antichikungunya activity (kaushik et al., 2020; wang et al., 2020). ginger essential oil (composition shown in table 2) has depicted antiviral activity with high accuracy. the inactivation of caprine alphaherpesvirus-1 (up to 100%) by disruption of the virus envelope and other structures needed for virus attachment to host cells has been observed by ginger essential oil (camero et al., 2019). more than 90.0% reduced activity of hsv-2 by preincubation with ginger oil also indicated its effective antiviral influence (koch et al., 2008). moreover, in invitro experimentation, direct inactivation of tulane and hepatitis a viruses have been observed by gingerol (patwardhan et al., 2020) while zerumbone has been observed with reduced activity of epstein–barr virus (onyiba, 2022). in hepatitis c-infected patients, ginger administration revealed the reduction in viral load by suppression of aspartate aminotransferase, α-fetoprotein and alanine aminotransferase (abdel-moneim et al., 2013). however therapeutic effects of ginger have also been observed against sars-cov-2. in coronavirus disease, the cleavage of polyprotein a/b (pp a/b) takes place via papain-like protease (plpro) at different sites, producing several proteins necessity for viral replication and survival (goswami et al., 2020). molecular docking has disclosed the potential inhibition of plpro by 10gingerol, 6-gingerol and 8-gingerol (shin et al., 2020). the binding potential of shogaol, gingerol, zingiberene, zingerone, zingiberenol and geraniol with catalytic domain of mpro has also been observed in docking studies. meanwhile, interference with s protein-ace2 binding has also been remarked by shogaol, zingiberenol, geraniol, zingiberene, gingerol and zingiberene (ahkam et al., 2020). s protein inhibition of corona virus has been examined by 10-shogaol, 10paradol, 10-gingerol, 8-paradol, 8-gingerol, 10-gingerol and sesquiphellandrene (terpene) (joshi et al., 2020). in a study carried out in saudi arabia, a decrease in hospitalization (28.0%) has been observed in covid-19 patients using the ginger diet as compared to nonusers (38.0%) (aldwihi et al., 2021). similarly in another study, in covid patients treated with ginger extract, low serum level of tnf-α, il-1, and il-6 was observed along with short-time mechanical ventilation as compared to the control (shariatpanahi et al., 2013) ginger as an antibacterial bacterial infections are the most common infections everywhere. not only this but with time antibiotic stewardship issue has become the most serious health threat worldwide. the problem can be resolved by replacing allopathic medicines with herbal drugs. antimicrobial resistance (amr) is a threat worldwide to development and health. it necessitates quick and crucial multisectoral action to attain sustainable development https://www.sciencedirect.com/topics/medicine-and-dentistry/propylene-glycol https://www.sciencedirect.com/topics/medicine-and-dentistry/alanine-aminotransferase https://www.sciencedirect.com/topics/medicine-and-dentistry/polyprotein https://www.sciencedirect.com/topics/medicine-and-dentistry/proteinase https://www.sciencedirect.com/topics/medicine-and-dentistry/shogaol https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/gingerol https://www.sciencedirect.com/topics/medicine-and-dentistry/zingiberene https://www.sciencedirect.com/topics/medicine-and-dentistry/zingerone https://www.sciencedirect.com/topics/medicine-and-dentistry/geraniol https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/gingerol shahzad et al. – medicinal importance of ginger 377 goals. who has stated that worldwide, antimicrobial resistance is among the 10 topmost health threats for human beings (who, 2021). globally, one of the most common reasons for mortality is antimicrobial resistance (amr), especially in settings where health care organizations do not get the least possible standards set by the world health organization (who) or some other quasi-governmental organization. according to analytical statistical models, in 2019, the bacterial amr led to approximately 4·95 million demises. owing to amr, 6 main pathogens caused 929 000 (660 000–1 270 000) demises, these pathogens include escherichia coli, streptococcus pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, and acinetobacter baumannii (murray et al., 2022). ginger shows its action against both gram-positive and gram-negative bacteria (abdulzahra & mohammed, 2014). the oil extract of dried ginger has proven to be very efficacious against various types of pathogens involved in food-borne diseases like e.coli, vibrio cholera, salmonella spp, and klebsiella (islam et al., 2014). in an experiment when v.harveyi infected lates calcarifer which is an asian sea bass and was given a ginger diet, it was observed that ginger controlled the infection caused by v.harveyi (talpur et al., 2013). v.harveyi is a gram-negative bacteria that causes death in asian sea bass (austin & zhang, 2006). ginger is found to increase the lysozyme activity (talpur et al., 2013). lysozymes are the enzymes that carry the hydrolysis of peptidoglycans present in the cell wall of bacteria. in this way, ginger inhibits the penetration of detrimental bacteria (nile & park, 2015). besides this, the antibactericidal action of serum (which plays an important role in removing pathogens) also increases by ginger (ellis, 2001). ginger increases anti-protease activity and thus suppresses the attacking ability of bacteria. the increased respiratory burst action of neutrophils by ginger has also been reported (talpur et al., 2013). a respiratory burst is a reaction in phagocytes that causes the degradation of bacteria. in fishes with a 0.1% ginger diet, 1.05±0.050 (nmol o2 /105 leucocyte) respiratory burst activity was observed while it was significantly greater, 1.26±0.230 (p<0.001) in fishes fed with a 1% ginger diet (dugenci et al., 2003). the ginger essential oil has been observed with strong anti-microbial properties against many bacteria. the percentage composition of ginger essential oil is represented in table 2. after the extraction of ginger essential oil by microwave-assisted hydrodistillation method and its chemical composition analysis by ftir spectrometer, citral was found to be the most abundant (89.05%) chemical compound in oil (kalhoro et al., 2022). it was observed in the study that ginger oil obtained through hydrodistillation showed more sensitivity against l.monocytogenes as compared to other bacteria and depicted a big inhibition zone (37mm). it was also observed to be active against the strain v.alginolyticus, despite its high mic value (minimum inhibitory concentration) (0.05-0.2mg/ml)(mostafa et al., 2018). the moderate activity of ginger oil with mic value 0.16-0.63 mg/ml against gram-positive bacteria demonstrated the high resistance of gram-negative bacteria against ginger oil as compared to gram-positive bacteria (snuossi et al., 2016). ginger essential oil has been shown to inhibit activity against e.coli and shigella due to the presence of bioactive constituents like gingerol, endo borneol, and zingiberene (sivasothy et al., 2011). similarly in another research conducted in brazil, zerumbone isolation from ginger oil depicted its efficacy against s.mutans with 500 µg/ml mbc (minimum bactericidal concentration) and 250 µg/ml mic. the efficacy of oil against biofilm formation and growth activity of s.pyrogenes resulted in 1mg/ml mbc and mic(wijesundara & rupasinghe, 2018). the more sensitivity of gram-positive strains suggested that the thick peptidoglycan surrounding the membrane of the cytoplasm would be the bacterial target of essential oil (cox & markham, 2007). however, the possibility of another target cannot be proscribed. the better effect of oil against gram-negative bacteria suggested other microbial targets like plasma membranes as bioactive components of essential oil possess lipophilic properties which interact with membranes via influencing their permeability and fluidity (rouseff & perez-cacho, 2007). the effective doses of ginger (halo, mbc, and mic) against different bacteria connecting different countries have been represented in table 5. 378 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 table 5. representing the effective doses of ginger against different bacteria connecting different countries. abbreviations: mbc, minimum bactericidal concentration; mic, minimum inhibitory concentration. bacteria halo (mm) mbc (μl/ml) mic country reference e. coli o157:h7 18.7 9.4 microliter/ml brazil (da silva et al., 2018) p. aeruginosa 4.7 2.3 microliter/ml s. typhimurium 18.7 9.4 microliter/ml s. aureus 4.7 2.3 microliter/ml l. monoctogenes 9.4 4.7 microliter/ml v. alginolyticus >25 tunisia (snuossi et al., 2016) shigella 119.79% saudi arabia (ashraf et al., 2017) e. coli 106.02% e. faecalis 61.94% p. aeruginosa 21.65% b. spizizenii 0.24 milligram/ml negeri sembilan (sivasothy et al., 2011) e. coli 0.31 milligram/ml p. stutzeri 0.63 milligram/ml b. licheniformis 0.16 milligram/ml k. pneumoniae 0.47 milligram/ml l. plantarum 7.0 brazil (ambrosio et al., 2017) s. enteritidis 8.8 e. faecalis 1.0 milligram/ml mexico (lópez et al., 2017) s. aureus 0.25 milligram/ml s. epidemidis 0.5 milligram/ml s. mutans 500 μg/ml 250 μg/ml brazil (moreira da silva et al., 2018) s. aureus 8.90 india (bag & chattopadhyay, 2015) m. l nkluteus 6.86 l. monocytogenes 9.00 s. typhimurium 6.61 b. cereus 9.11 e. coli 8.00 b. cereus 8.3 saudi arabia (mostafa et al., 2018) s. typhi 0.0 s. aureus 15.8 p. aeruginosa 11.2 e. coli 0.0 k. pneumoniae 20.5 india (singh et al., 2008) p. vulgaris 18.4 s. pyogenes >1000 μg/ml >1000 μg/ml canada (wijesundara & rupasinghe, 2018) ginger as an antioxidant the oxidative stress develops when in the organs and tissue, the development of extremely reactive species, for example, reactive nitrogen species (rns), reactive sulfur species (rss), and reactive oxygen species (ros), prevail over the endogenous antioxidant defense system competence, brings about the dysfunctions and cellular damage, and ultimately gives rise to a broad range of diseases. free radicals produce oxidative stress in the living system which is the major cause of many chronic diseases like cataracts, cancer, aging, rheumatoid arthritis, and autoimmune diseases (pham-huy et al., 2008). the inequality between vulnerable defense systems and too many reactive species causes harm to various cell structures as well as to many molecules like dna, lipids, and proteins, and finally leads to a broad range of diseases (janssen-heininger et al., 2008). medicinal plants contain many phytochemicals which are proved to be effective in alleviating the toxic effects of free radicals. ginger also contains many phytochemicals like 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol which possess antioxidant properties due to their solubilizing side chains and hydroxyl groups which are further classified into two major groups diarylheptanoids and gingerol-related compounds (si et al., 2018). results on hplc-ms/ms of both extracts confirmed that eight different types of phenolic acids are present in ginger that proved the effective antioxidant activity and cause a delay in the diseases which are caused by oxidative stress (tohma et al., 2017). shahzad et al. – medicinal importance of ginger 379 ginger is very useful for chemotherapeutic patients because patients produce many reactive oxygen species which are responsible for oxidative stress followed by more lipid peroxidation products like nitric oxide (amin et al., 2012; gupta et al., 2010; srivastava et al., 2010). when the ginger extract was given to such patients, it showed its efficacy by enhancing the level of antioxidant enzymes and suppressing the level of lipid peroxidation products (danwilai et al., 2017). 6-gingerol is the most abundant compound in ginger that shows antioxidant activity (baliga et al., 2011). the comprehensive mechanism of action of 6-shogaol for anti-oxidant effects has been illustrated in figure 5. newly identified cancer sufferers taking strong beneficial chemotherapy have been randomized to take 20 mg of 6-gingerol, a ginger extract per day which enhances the antioxidant enzymes (danwilai et al., 2017). in one study, the level of antioxidant enzymes was compared between two groups of rats. in one group of rats, there were diabetic rats as control and in the other group, diabetic rats were fed with a ginger diet. a very low level of superoxide dismutase enzyme was observed in the control group which was 90.40±3.10 (u/ml) but it was significantly higher in ginger fed group where it was 109.30±3.70(u/ml) (p<0.05). similarly, the level of glutathione peroxidase (u/ml) was 22.90±0.89 in the control group while it was significantly higher, 26.31±2.10 in the ginger-fed group. the overall antioxidant capacity (mm) of the control group was 0.99±0.17mm while in the ginger treated group it was 1.28±0.29 (p<0.05) (abdullah, 2012). ginger prevents the formation of hydroxyl radicals due to the presence of polyphenols because they form a chelate with fe[3+] (stoilova et al., 2007). polyphenol compounds show high antioxidant activity because they are reducing agents and free radical scavengers (juntachote et al., 2006). anthocyanins and ascorbic acid are two phenols that act as free radical scavengers (pantelidis et al., 2007). phenolics also improve the quality of food by inhibiting the degradation of lipids which is why their use in the food industry is increasing day by day (wojdyło et al., 2007). it has been observed that gingerol plays a dominant role in the inhibition of phospholipid peroxidation as well as inhibits the xanthine oxidase, the enzyme that is involved in the formation of reactive oxygen species (nile & park, 2015). the other mechanisms of different constituents of ginger for antioxidant effects have been shown in table 6. figure 5. the comprehensive mechanism of action of 6-shogaol for antioxidant activity (chen et al., 2014): 6-shogaol causes the translocation of nrf2 in the nucleus and enhances the expression of genes that target nrf2 by modification of keap1 and protects nrf2 from proteasomal degradation. in this way the gsh level increases and the ros level suppresses. abbreviations: keap1, kelch-like ech-associated protein 1; nrf2, nuclear factor erythroid related factor 2; are, antioxidant response element; ftl, ferritin light chain; ho-1, heme oxygenase-1; trx1, thioredoxin 1; nqo1, nicotinamide adenine dinucleotide phosphate (nadph) quinone dehydrogenase 1; trxr1, thioredoxin reductase 1; gclc, glutamate-cysteine ligase catalytic subunit; ggtla4, γglutamyltransferase-like activity 4; gclm, glutamate-cysteine ligase modifier subunit; akr1b10, aldo-keto reductase family 1 member b10; gsh, glutathione; ros, reactive oxygen species. table 6. representing the antioxidant mechanism of action of different components of ginger. abbreviations: mda, malondialdehyde; gssg, glutathione disulfide; glutathione s-transferase p1; mt1, metallothionein. sr. no ginger/constituents dose research subjects mechanism of action reference 1 6-shogaol 20 μm human colon carcinoma cells hct-116 enhancing the activation of mt1, ftl, akr1b10, ho-1,ggtla4, mt1,gclm, and gclc genes; decreasing the level of ros; enhancing the intracellular gsh/gssg ratio; reducing the level of ros (chen et al., 2014) 100 mg/kg mice with wild-type and nrf2/c57bl/6j mutations upregulation of ho-1, gclc, and mt1 expression 2 ginger oleoresin (composition shown in table 2) 100 μg/ml mesenchymal, stem cells from humans stimulating ho-1, nqo1 gene expression; reducing ros generation; stimulating nrf2 translocation to the cell nucleus (ji et al., 2017) 3 ginger phenylpropanoids 40 μg/ml bj fibroblasts from the foreskin upregulation of nrf2 activity and gstp1 level (schadich et al., 2016) 380 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 table 6. cont. sr. no ginger/constituents dose research subjects mechanism of action reference 4 6-gingerol rich fraction 50, 100 mg/kg wistar rats, females h2o2 and mda levels are being reduced, while antioxidant enzyme activity and gsh levels are being increased. (abolaji et al., 2017) 5 ginger extract 100 mg/kg wistar albino rats, males lowering the mda level; preventing catalase activity & gsh content from being depleted (saiah et al., 2018) 200, 400 μg/ml human fibrosarcoma cells, strain ht1080 reducing the production of reactive oxygen species (ros) (romero et al., 2018) 5, 25 μg/ml human chondrocyte cells c28i2 increasing antioxidant enzyme gene expression and lowering ros as well as lipid peroxidation levels (hosseinzadeh et al., 2017) 78 to 313 μg/ml homogenates of rat hearts mda levels decreased (akinyemi et al., 2013) ginger as an anticancer cancer is a disease in which abnormal cells grow and spread uncontrollably throughout the body and damage normal body tissues (fearon et al., 2011). cancer is the second leading cause of death in the world. according to who's (2022) cancer report, 10 million deaths cases were observed in 2020 with nearly one in six deaths. how-ever in lower-middle-income countries, 30% of cancer cases were observed (organization, 2022). globally, approximately 10.0 million cancer-related mortalities (9.9 million without non-melanoma skin cancer) and almost 19.3 million new patients of cancer (18.1 million without non-melanoma skin cancer) arose in the year 2020. breast cancer in females has exceeded lung cancer as the most frequently detected cancer, with almost 2.3 million new patients (11.7%), after that lung, colorectal, prostate, and stomach cancers were 11.4%, 10.0 %, 7.3%, and 5.6% respectively. the highest cause of cancer-related death is lung cancer, with approximately 1.8 million mortalities (18%), afterward here comes colorectal, liver, stomach, and female breast cancers in the range of 9.4%, 8.3%, 7.7%, and 6.9% respectively. on the whole, the frequency was 2 to 3 fold greater in transitioned countries when compared with transitioning countries (sung et al., 2021). studies suggest that ginger causes the cell death of various types of cancerous cells including ovarian, colon, brain, liver, cervical, skin, prostate, renal, pancreatic, gastric, and breast cancer (srinivasan, 2014). phytochemicals like gingerols, shogaols, paradols, and polyphenolics are the compounds that are part of ginger and are involved in the anticancer activity and reduce its risk by targeting at different stages (cheng et al., 2011). the therapeutic mechanisms of different constituents of ginger against various types of cancer are mentioned in table 7. the potential mechanisms of 6-gingerol for suppressing proliferation and induction of apoptosis in cancer (figure 6) (liu et al., 2017; tahir et al., 2015). figure 6. schematic diagram depicting the various signaling pathways of 6-gingerol for anti-cancer activity. abbreviations: pi3k: phosphoinositide 3kinase; ampk: 5 adenosine monophosphate-activated protein kinase; bcl-2: b-cell lymphoma 2; bax: bcl-2-associated x protein; cdk: cyclin-dependent kinase; mtor: mammalian target of rapamycin; akt: protein kinase b. (upward arrow-indicating increasing activity, downward arrow-indicating decreasing activity) shahzad et al. – medicinal importance of ginger 381 table 7. representing the therapeutic mechanism of action of different constituents of ginger against different types of cancer. abbreviations: nf-κb, nuclear factor kappa light chain-enhancer of activated b cells; ampk, 5 adenosine monophosphate-activated protein kinase; gdnp, ginger derived nanoparticles; erk, extracellular signal-regulated kinase; bcl-xl, b-cell lymphoma-xl protein; kras, kirsten rat sarcoma viral oncogene; mtor, mammalian target of rapamycin; stat3, signal transducer and activator of transcription 3; c-myc, cellular myelocytomatosis; bcl-2, b-cell leukemia/lymphoma 2; gstπ, glutathione-s-transferase; mrp1, multidrug resistance associated protein 1. sr. no. ginger/constituents dose research subjects mechanism of action reference 1 10-gingerol 50-100 and 200 μm carcinoma cells from mouse breasts and human inhibit cell growth, reduce the cell division and responsible for s phase cell cycle apoptosis (bernard et al., 2017) 2 gdnps -2 0.3 mg female, mice (c57bl/6) by using gdnps-2 the expression of cyclin d1 is restrained and intestinal epithelial cell multiplication is also restricted. (zhang et al., 2016) 3 extract of ginger based on fluorescent carbon nano-dots 1.11 mg/ml carcinoma cells (hepatocellular, hepg2 human boost up the apoptosis, enhance the ros level, and dominate the expression of p53. (li et al., 2014) 4 ginger extract 100 mg/kg female mice (swiss albino) ginger extract activates the ampk, and also by using it level of nf-κb is reduced, p53 expression is enhanced and the level of nf-κb, d1 is reduced. (el-ashmawy et al., 2018) 2–10 mg/ml human colorectal, adenocarcino-ma (ht29) stimulate the cell apoptosis, and functions of the caspase-9 gene and downcast the level of erk, bcl-xl, kras, etc (tahir et al., 2015) 5 6-gingerol 60,100,140 μm hela human (cervical adenocarcinoma cells) it is responsible for the cell cycle, arrest in the resting phase of the cell (g0/g1-phase), and also it is responsible for the downregulation of cyclins levels (cyclins a, d1, and e1) and it enhances the caspase gene expression and stops the mtor signaling pathway (zhang et al., 2017) 6 ginger extract with alginate beads 50 mg/kg male specie (wistar rats) responsible for enzymatic activity i.e nadh dehydrogenase and succinate dehydrogenase activity (deol & kaur, 2013) 7 6-shogaol 10, 20, 40 μm lncap, du145,pc3, human prostate (cancer cells) responsible for the activation and deactivation of different mechanisms in signaling pathways i.e it stops stat3, nf-κb signaling pathway, and it downregulates the expression of d1, c-myc, bcl2, surviving, and it is also responsible for cell apoptosis. (saha et al., 2014) 8 gingerol-6, shogaol6, gingerol-10, shogaol-10. 10,100 μm pc-3, human prostate cancerous cells. stops the multiplication of prostate cancer cell and suppress mrp1, gstπ expression. (liu et al., 2017) gastric cancer (stomach cancer) another health-related problem is gastric cancer which is also known as stomach cancer. this cancer is the 5th most prevalent cancer and the 3rd most popular basis for cancer-related mortality worldwide. the affliction of gastric cancer is incredibly more in asia, central and eastern europe, and latin america while in most western european countries and north america, it is not a more widespread cancer (ferro et al., 2014). studies have shown that 6-gingerol and 6-shogaol, the active components of ginger, are involved in an anticancer activity that shows a different mode of action against gastrointestinal cancer (ishiguro et al., 2007; prasad & tyagi, 2015). tumor necrosis factor-related apoptosis-inducing ligand (trial) is a cytokine that plays a role in apoptosis and thus is involved in the suppression of metastasis in the host (thorburn, 2007). 6-gingerol upregulates the trial-induced apoptosis by enhancing the trial-induced caspase-3/7 activation. caspase 3/7 are proteases that are involved in cell death machinery (ishiguro et al., 2007). 6-gingerol suppresses the activity of inhibitors of caspase-3/7 like ciap1 and 382 biology, medicine, & natural product chemistry 12 (1), 2023: 371-389 thus promotes cell death (prasad & tyagi, 2015). on the other hand, 6-shogaol disturbs the intracellular level of tubulin, a protein in microtubules that performs many cellular functions including mitosis. this disturbing level of tubulin induces mitotic arrest and thus causes apoptosis of cancerous cells (mollinedo & gajate, 2003). pancreatic cancer medically, the malignant tumor that develops in epithelial cells of glandular structures in the pancreatic ductal cells is called pancreatic cancer (aier et al., 2019). now, the frequency and deaths related to pancreatic cancer are rising every year universally, no matter in japan, europe, the us, or china (hu et al., 2021). it has been observed that 6-shogaol and 6-gingerol are the components of ginger that induce apoptosis and antiproliferative activity in tumor cells (li et al., 2012; lu et al., 2014). 6-gingerol inhibits the growth of pancreatic cancer cells by hindering the cell cycle at the g1 phase. retinoblastoma is a protein that regulates the cell cycle. the phosphorylated retinoblastoma is mainly involved in controlling the cell cycle at g1 to s phase. it has been observed that 6-gingerol inhibits the phosphorylation of retinoblastoma after suppressing the expression of cyclin a and cyclin-dependent kinase and thus blocks the cell cycle of cancerous cells by not allowing it to enter in s phase (park et al., 2006; stone et al., 2011). it has also been noticed after experiments that normal cells like human umbilical vein endothelial cells (huvec) are unaffected by ginger as compared to panic-1 cells. features of apoptosis-like an increase in subg1 cells, fragmentation of nuclei, and caspase-3 activation have been noticed by the ginger extract in pancreatic cancer cells. however, treatment of cells with ginger for a long time increases the formation of reactive oxygen species which causes autoptic cell death (akimoto et al., 2015). prostate cancer one of the most prevalent types of malignant cancer (after skin cancer) in males is prostate cancer, which is linked to the reproductive system. in 2012, world statistics stated that 15% of male cancers are prostate cancers as well as it is the 2nd major cause (after lung cancer) of deaths in males related to cancers (khazaei et al., 2019). the cell cycle is maintained by cyclin-dependent kinases (cdks). in prostate cancer cells, ginger exhibits its activity by decreasing the level of cyclin d1 and cdk4 levels. it also reduces the cyclin e levels which control the cell cycle through s-phase (karna et al., 2012). besides this, ginger stalls the cell cycle by increasing the level of p21 which is a cdks inhibitor (besson et al., 2008). ginger also activates caspase-3 which cleaves many cellular proteins such as parp (karna et al., 2012). parp play role in many cellular activities including repairing of dna. the inhibition of parp causes an increase in apoptosis due to a reduction in dna repairing capacity (morales et al., 2014). it concludes that ginger inhibits the growth of prostate cancer cells by apoptosis and by causing derangements in the cell cycle (karna et al., 2012). limitations in addition to the medicinal effects of ginger, some side effects of ginger have also been observed. more bleeding has been observed during surgery in patients, who take regular doses of ginger (ghorbanian p., 2017). it means ginger is also unsafe for patients who have a bleeding disorder. taking ginger more than 5 grams a day enhances the risk of side effects. its high consumption causes skin rashes, mouth irritation, upset stomach, heartburn, and gas. in some cases, the consumption of ginger in combination with medicines also shows harmful effects (line, 2018; mohan, 2017). it is even important to consult with a doctor before taking ginger while pregnant. ginger is more treated as food rather than medicine therefore like drug manufacturers, the supplier of ginger does not show whether it is safe or not before selling in the market (heitmann k1, 2013). conclusion ginger is broadly used in ethnomedicines, having several bioactive compounds with apprehensible pharmacological actions. this review demonstrates the recent studies (under duration from 2001-2022) about the pharmacological actions of ginger and it's almost 44 bioactive compounds. in this study, ginger has unveiled immunostimulatory, anti-inflammatory, anti-viral, antibacterial, anti-oxidant, and anti-cancer effects by stimulating and inhibiting various mechanisms at the cellular, molecular and enzymatic levels. our study provides a comprehensive overview of botanical authentication, experimental studies (in-vitro, in-vivo, and computational), pharmacological properties, and phytoconstituents profile of ginger along with their biological activities. not only this, but this review has also recognized research deficiencies after analyzing the data (up to the year 2022) and has provided new pathways for future research. the present review is beneficial for the development of ginger-based ethnomedicines, for the treatment of the most common and serious health issues of the day with minimum cost and side effectiveness. the major components of ginger and their mechanism of action regarding immunostimulatory, anti-inflammatory, anti-viral, antibacterial, anti-oxidant, and anti-cancerous effects have been concluded in figure 7. shahzad et al. – medicinal importance of ginger 383 figure 7. showing the immunostimulatory, anti-inflammatory, anti-viral, anti-bacterial, anti-oxidant, and anti-cancerous effects of different components of ginger along their mechanism of action in a nutshell. (upward arrow-indicating the increasing level, downward arrow-indicating the decreasing level) recommendations we should consider any confounding factors when assessing the medicinal beneficial effects of ginger against chronic diseases. increasing verification has depicted that different constituents of ginger have different molecular targets, metabolic pathways and bioactivities suggesting the significance of identifying the bioactive compounds and their biological mechanisms for a specific disease. gingerols and shogaols have been observed to be very potent bioactive compounds of ginger regarding its biological significance. it has been noticed that a few rodent and invitro studies directly made comparisons between the bioactivities of shogaols and gingerols in limited disease models. it is suggested to compare the effectiveness of gingerols and shogaols in more models and thus develop unusual ginger formulations for particular diseases. to determine the optimum oral ginger composition will also enforce the study of the additive and synergistic effects of gingerols, shogaols and other bioactive compounds of ginger which will assist researchers to develop authentic standardization techniques to monitor the quality and composition of ginger formulations for specific disease. once the unique ginger formulation is developed against a particular disease, the effective dose frequency and dose range should be established. conflict of interest statement: no conflict of interest is to be declared by any author. authors contribution: haseeba shahzad: conceptualization, design and directed the project, writing-review, editing, investigation, validation, data curation, data analysis, results interpretation, writing original draft and final approval. shaifa saleem: writing-review, data analysis, critical revision and investigation. waqas hanif: design, validation, critical revision, review editing, investigation, manuscript review and final approval. zareena ali: writing-review, data analysis, and investigation. muhammad zeeshan ahmed: writing-review, validation, and critical revision. haq nawaz: critical revision, validation and investigation. zelle humma: review editing and critical revision. laraib rana: writing-review and investigation. laraib rana contributed equally to this work with muhammad qamar farid, hajira kalsoom, and samavia jaan. references abdel-moneim, a., morsy, b. m., mahmoud, a. m., abo-seif, m. a., & zanaty, m. i. 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(2016). triptolide attenuates inflammatory response in membranous glomerulo-nephritis rat via downregulation of nf-κb signaling pathway. kidney and blood pressure research, 41(6), 901-910. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 575-583 | doi: 10.14421/biomedich.2025.141.575-583 issn 2540-9328 (online) phytoplankton diversity as a water quality bioindicator of mangrove ecosystems in the mangrove rehabilitation and mangrove ecotourism area of merdeka beach serdang bedagai putri aulia*, melfa aisyah hutasuhut** biology department, faculty of science and technology, uin sumatera utara. corresponding author putri0704213077@uinsu.ac.id*; melfa_aisyah@uinsu.ac.id** manuscript received: 31 july, 2025. revision accepted: 10 september, 2025. published: 15 september, 2025. abstract this study investigates the diversity and composition of phytoplankton as a bioindicator of water quality in the mangrove ecosystems of merdeka beach, bagan kuala village, serdang bedagai. sampling was conducted at three sites representing long-term rehabilitation (2010–2025), recent rehabilitation (2024–2025), and mangrove ecotourism areas. phytoplankton abundance and diversity were analyzed using ecological indices, including shannon-wiener diversity index (h’), trophic diatom index (tdi), and percentage pollution tolerance value (%ptv), alongside physicochemical water parameters. results indicate spatial variation in water quality and phytoplankton communities. station 1 showed moderate abundance and high diversity, indicating ecological stability. station 2 exhibited increased abundance but lower diversity, reflecting transitional conditions. station 3 demonstrated the highest abundance and pollutiontolerant taxa, consistent with elevated nutrient levels due to human activities. tdi and %ptv values corroborated these findings, identifying station 3 as hypertrophic and heavily polluted. these results highlight the utility of phytoplankton as sensitive indicators of environmental quality and the effectiveness of rehabilitation efforts. the integration of biotic indices and water parameters provides a robust framework for ongoing ecological monitoring and coastal management strategies, particularly in mangrove ecosystems undergoing anthropogenic pressure and restoration. keywords: phytoplankton; mangrove rehabilitation; ecotourism; bioindicator; water quality. introduction mangrove ecosystems represent one of the most productive and biologically important environments along tropical and subtropical coastlines (alongi, 2002; kathiresan & bingham, 2001). these habitats support a wide range of ecological functions such as carbon sequestration, nutrient cycling, coastal protection, and nursery grounds for marine biota (duke et al., 2007; donato et al., 2011). in indonesia, mangrove degradation remains a critical environmental concern due to anthropogenic pressures including deforestation, aquaculture, and urban expansion (setyawan et al., 2018; giri et al., 2008).rehabilitation and restoration efforts are increasingly being implemented in areas such as bagan kuala village, serdang bedagai, where the merdeka beach mangrove ecosystem is undergoing both rehabilitation and ecotourism development (adame et al., 2015; rudianto et al., 2020). such initiatives are crucial for reinstating lost ecological functions and providing sustainable economic alternatives to coastal communities (bosire et al., 2008; primavera & esteban, 2008). however, monitoring the ecological effectiveness of these efforts requires comprehensive and cost-effective bioassessment tools (barbier et al., 2011; lópez-portillo et al., 2017). phytoplankton diversity has been widely used as a sensitive and informative bioindicator of aquatic ecosystem health (suthers & rissik, 2009; chalar, 2009). these microscopic autotrophs respond rapidly to changes in nutrient concentrations, salinity, turbidity, and pollution, making them ideal for detecting early signs of eutrophication and organic contamination (caroppo et al., 2006; domingues et al., 2014). several studies have demonstrated that changes in phytoplankton composition can reflect anthropogenic impacts in mangrove-fringed estuaries (yuliana et al., 2012; inyang & wang, 2020).indices such as the shannon-wiener diversity index (h’), trophic diatom index (tdi), and percentage pollution tolerant value (%ptv) are useful for quantifying phytoplankton community structure and determining water quality status (kelly & whitton, 1995; wu et al., 2014). a high tdi or %ptv value often correlates with elevated concentrations of nitrogen and phosphorus, which are common in areas adjacent to aquaculture ponds or domestic discharge (gupta & https://doi.org/10.14421/biomedich.2025.141.575-583 mailto:2.melfa_aisyah@uinsu.ac.id 576 biology, medicine, & natural product chemistry 14 (1), 2025: 575-583 rastogi, 2008; salmaso et al., 2006). conversely, lower index values typically reflect pristine or minimally disturbed sites (silva et al., 2014). water quality parameters such as dissolved oxygen (do), ph, temperature, turbidity, nitrate, and orthophosphate have been also critical for phytoplankton growth and community dynamics (effendi et al., 2016; onyema, 2013). in mangrove environments, these parameters are influenced by tidal flushing, organic matter decomposition, and freshwater inflows (hossain et al., 2016; rahmania et al., 2019). evaluating the interplay between these abiotic factors and phytoplankton indicators can reveal spatial and temporal trends in ecosystem health (dalu et al., 2017; elliott & quintino, 2007). in the context of mangrove ecotourism and rehabilitation, understanding phytoplankton dynamics become even more important. tourism-related development, if not well-managed, can introduce additional nutrient loads and disrupt sediment dynamics (pourafrasyabi & ramezanpour, 2012; çulha et al., 2022). similarly, replanting efforts may temporarily alter hydrological patterns, affecting water clarity and nutrient distribution (friess et al., 2019; amalia et al., 2018). thus, phytoplankton data provide a functional lens through which to assess both degradation and recovery. merdeka beach offers a unique opportunity to investigate these ecological interactions, as it represents a transitional area between degraded, rehabilitated, and conserved mangrove zones (hakim et al., 2017; saputra et al., 2020). the juxtaposition of tourism activities and ongoing restoration provides a valuable natural laboratory for assessing the relative impact of human intervention on water quality and plankton diversity (nichols et al., 2019; bao et al., 2020). this study also seeks to contribute baseline data for coastal management in north sumatra. previous research in similar tropical settings has shown that bioindicators based on phytoplankton can effectively detect nutrient enrichment, organic loading, and habitat alteration (hilmi et al., 2020; ferreira-marinho et al., 2014). such indicators are particularly valuable in resourcelimited regions where conventional water quality monitoring may be infrequent or logistically challenging (junaidi & azhar, 2018; lobo et al., 2002). furthermore, integrating biological indices with physicochemical parameters enhances the predictive power of environmental assessments (zhang et al., 2021; gao & zheng, 2010). therefore, this research aims to assess the diversity and community composition of phytoplankton in the mangrove ecosystems of merdeka beach, particularly in the context of ongoing rehabilitation and ecotourism development. by correlating phytoplankton indices with water quality parameters, this study seeks to evaluate the ecological status of the site and inform future conservation and management strategies (reynolds, 2006; gell et al., 1999). it is hoped that this integrative approach will strengthen local efforts to safeguard mangrove ecosystems in north sumatra and contribute to broader coastal resilience initiatives (barbier et al., 2011; donato et al., 2011). materials and methods study area the research was carried out in may 2025. the location of water and phytoplankton sampling was carried out in the mangrove ecosystem at mangrove rehabilitation and ecotourism areas at merdeka beach, bagan kuala village, serdang bedagai. figure 1. mangrove rehabilitation area 2010-2025. figure 2. mangrove rehabilitation area 2024-2025 figure 3. ecotourism mangrove aulia & hutasuhut – phytoplankton diversity as a water quality bioindicator … 577 procedures sampling design sampling was conducted at three stations. station 1 represents a mangrove rehabilitation area that has undergone restoration from 2010 to 2025, located at coordinates 3°29′24″n, 99°15′34″e. station 2 is a mangrove rehabilitation site restored during the 2024– 2025 period, located at coordinates 3°30′35″n, 99°13′56″e. station 3 is situated in the merdeka beach mangrove ecotourism area, bagan kuala village, tanjung beringin subdistrict, serdang bedagai regency. water and phytoplankton sampling were conducted in triplicate at each station to ensure data reliability and representativeness. phytoplankton sampling, identification, and enumeration phytoplankton samples were obtained by filtering 5 liters of surface water ±15 cm below the surface using a plankton net. the collected samples were preserved in sampling bottles with the addition of 1 ml of 4% formalin. for microscopic examination, 1 ml of each sample was pipetted into a sedgwick-rafter counting chamber and observed under a light microscope at 200× magnification. phytoplankton identification was performed by comparing the observed species with taxonomic references (gell et al., 1999; du buf & bayer, 2002; van vuuren et al., 2006; bellinger & sigee, 2010). phytoplankton abundance was calculated using the formula provided by effendi et al. (2016). water physicochemical parameter measurements a total of 100 milliliters of water was collected at each station from a depth of approximately 15 cm below the surface using a water sampler and stored in clean sampling bottles. the measured physicochemical parameters included water temperature, air temperature, ph, conductivity, dissolved oxygen (do), biochemical oxygen demand (bod), nitrate concentration, and orthophosphate concentration. the parameters and their respective units and analytical method are follows: tabel 1. physicochemical parameters of water with its measurement method parameter unit tool/method salinity water temperature air temperature ph conductivity do bod nitrate orthophosphate % °c °c siemens/meter mg/l mg/l mg/l mg/l refaktometer thermometer thermometer ph meter ik iii/09/2020 (elektrometri) sni 06-6989.14-2004 sni 6989.72-2009 colorimetric sni 06-6989.31: 2005 data analysis phytoplankton community structure and diversity were analyzed using several ecological indices, including the importance value index (ivi), shannon-wiener diversity index (h’), simpson dominance index (id), and evenness index (e), following the formulas proposed by wu et al. (2014). descriptive statistical analysis was employed to summarize the minimum and maximum values of the measured water quality parameters. furthermore, biplot analysis using past 16.0 software was conducted to assess the correlation between physicochemical parameters and biotic indices. to evaluate the trophic status and organic pollution level of the waters, phytoplankton biotic indices were applied: the trophic diatom index (tdi) and the percentage pollution tolerance value (%ptv). tdi was calculated based on diatom species only, as suggested by kelly & whitton (1995). the %ptv was determined by calculating the proportion of tolerant diatom taxa (e.g., gomphonema sp., navicula spp., sellaphora spp., and nitzschia spp.) relative to the total diatom assemblage. the equation used to determine the tdi value index (wu et al. 2014): where, wms is the weighted average sensitivity and can be obtained from the following formula: notes: wms : weighted mean sensitivity 𝑎𝑖 : proportion of all individuals in a sample that belong to species i 𝑠𝑖 : pollution sensitivity (1-5) of species i 𝑣𝑖 : indicator values (1-3) of species i 𝑛 : total number of species in a sample (based on kelly & whitton 1995) ptv is an index to determine the level of organic matter pollution in the waters. the equation used to determine the ptv value index (kelly & whitton 1995): the %ptv value was calculated based on comparing the abundance of tolerant diatoms (gomphonema sp., navicula spp., sellaphora spp., and nitzschia spp.) with the number of diatoms obtained (kelly & whitton 1995). 578 biology, medicine, & natural product chemistry 14 (1), 2025: 575-583 results and discussion table 2. the profile of physicochemical water quality in stasiun 1, stasiun 2, stasiun 3. location physicochemical parameters water temperature (ºc) air temperature (ºc) salinity (%) ph conduktivitas (mg/l) do (mg/l) bod (mg/l) nitrate (mg/l) orthophosphate (mg/l) stasiun 1 stasiun 2 stasiun 3 30,2 30,2 30,3 32,8 32,4 32,4 15 20 12 7,6 7,2 7,02 1,7 2,1 1,1 2,4 2 2,1 0,9 1,5 0,6 0,03 0,36 0,73 0,058 0,058 0,058 water quality standart (indonesia ministry of environment regulation no 22/2021) 25-32 25-32 34 7-8.5 >5 20 0.008 0.015 the results of this study indicate varying water quality conditions across the three sampling stations located in the mangrove rehabilitation and ecotourism areas of merdeka beach. the observed physicochemical parameters, including temperature, ph, salinity, dissolved oxygen (do), biochemical oxygen demand (bod), nitrate, and orthophosphate, showed both compliance and deviation from the indonesian water quality standards (regulation no. 22/2021). dissolved oxygen (do) levels in all stations were below the recommended threshold of 5 mg/l, with values ranging from 2.0 to 2.4 mg/l. such low do concentrations may be attributed to the high input of organic matter and subsequent microbial degradation, which consumes oxygen in the water column (onyema, 2013; pour et al., 2014). nitrate concentrations showed considerable spatial variation, ranging from 0.03 mg/l in station 1 to 0.73 mg/l in station 3. these values exceed the standard limit of 0.008 mg/l for marine ecosystems, indicating significant nutrient enrichment, especially in station 3, which is influenced by ecotourism and adjacent aquaculture activities. (do) levels in all stations were below the recommended threshold of 5 mg/l, with values ranging from 2.0 to 2.4 mg/l. such low do concentrations may be attributed to the high input of organic matter and subsequent microbial degradation, which consumes oxygen in the water column (onyema, 2013; pour et al., 2014). nitrate concentrations showed considerable spatial variation, ranging from 0.03 mg/l in station 1 to 0.73 mg/l in station 3. figures and tables of maximum of three pages should be clearly presented. number tables consecutively in accordance with their appearance in the text. title of a picture is written down below the picture, while title of a table is written above the table. colored figures can only be accepted if the information in the manuscript can lose without those images; chart is preferred to use black and white images. author could consign any picture or photo for the front cover, although it does not print in the manuscript. all images property of others should be mentioned source. there is no appendix, all data or data analysis are incorporated into results and discussions. for broad data, it can be displayed on the website as a supplement (figure 2; table 1). these values exceed the standard limit of 0.008 mg/l for marine ecosystems, indicating significant nutrient enrichment, especially in station 3, which is influenced by ecotourism and adjacent aquaculture activities elevated nitrate levels can stimulate algal blooms and lead to eutrophication (gupta & rastogi, 2008; çulha et al., 2022). the salinity levels observed at the three stations exhibited notable variation, ranging from 15 ppt at station 1 to 24 ppt at station 2. although all values remain within the acceptable limits set by the indonesian water quality standard (maximum 34 ppt), the elevated salinity at station 2 may indicate a stronger influence of seawater intrusion or tidal flushing in this area. such variation in salinity can influence the distribution and abundance of aquatic organisms, particularly stenohaline species that are sensitive to salinity fluctuations. furthermore, it may serve as a hydrological indicator of estuarine mixing dynamics and coastal water exchange processes. oreover, the recorded ph values across the stations were relatively neutral to slightly alkaline, ranging from 7.02 to 7.6, and remained within the standard threshold of 7.3 to 8.5. nevertheless, the slightly lower ph observed at station 3 could suggest localized inputs of acidic organic matter or anthropogenic pollutants, possibly due to decomposition processes or nearby human activities. aulia & hutasuhut – phytoplankton diversity as a water quality bioindicator … 579 figure 4. phytoplankton abundance patterns across rehabilitated and ecotourism mangrove zones. the abundance of phytoplankton across the three sampling stations revealed significant spatial differences, reflecting the varying degrees of environmental pressure and rehabilitation maturity at each site. in station 1 (rehabilitation 2010–2025), the phytoplankton abundance was relatively moderate and dominated by diatoms such as melosira sp. and coscinodiscus sp., both of which are considered indicators of healthy and nutrient-stable waters. the presence of these species suggests that the long-term rehabilitation process has contributed to the stabilization of ecological functions in this zone.in contrast, station 2 (rehabilitation 2024–2025) exhibited higher abundance levels but with reduced species diversity, characterized by dominance of nitzschia sp. and navicula sp.—taxa known for their tolerance to organic pollution. this indicates that although rehabilitation efforts have begun, the ecosystem is still under transitional pressure and has not yet reached ecological equilibrium. the elevated phytoplankton abundance in this zone may also be driven by nutrient influx due to recent disturbances in sediment and vegetation layers during mangrove planting activities. the highest abundance of phytoplankton was recorded in station 3 (mangrove ecotourism zone), particularly of pollution-tolerant diatoms such as nitzschia sp., sellaphora sp., and gomphonema sp. these taxa dominated the assemblage, suggesting nutrient-rich and organically polluted waters, likely influenced by nearby aquaculture ponds and tourist activity.these findings emphasize that phytoplankton abundance and composition are reliable indicators of environmental condition and can effectively differentiate between rehabilitated, transitioning, and disturbed mangrove ecosystems. furthermore, integrating abundance data with biotic indices such as the trophic diatom index (tdi) and %ptv enhances the ecological diagnosis of site-specific water quality conditions. table 3. spatial variation of phytoplankton diversity index in rehabilitated and ecotourism mangrove. location location point biotic index e id h’ stasiun 1 u1 stasiun 1 u2 stasiun 1 u3 stasiun 2 u1 stasiun 2 u2 stasiun 2 u3 stasiun 3 u1 stasiun 3 u2 stasiun 3 u3 rehabilitated in 2010 rehabilitated in 2024 ecotourism mangrove 0,761 0,739 0,739 0,702 0,718 0,634 0,937 0,940 0,933 0,131 0,162 0,160 0,164 0,220 0,244 0,060 0,69 0,073 2.387 2,093 2,094 2,340 2,075 1,797 2,938 2,767 2,689 the spatial distribution of phytoplankton diversity across the three observed stations reveals significant ecological differences that reflect the degree of anthropogenic pressure and restoration maturity in each zone. station 1, located in a long-term rehabilitated mangrove area (2010–2025), exhibited relatively high shannon-wiener diversity index (h’) values ranging from 2.093 to 2.387. this suggests a more stable and heterogeneous phytoplankton community, indicative of a balanced aquatic environment with minimal disturbance and better ecological recovery. the evenness values (e = 0.739–0.761) further support this observation, demonstrating that species are more evenly distributed without the dominance of a particular taxon. low 0 1000 2000 3000 4000 5000 6000 7000 8000 st1 u1 st1 u2 st1 u3 st2 u1 st2 u2 st2 u3 st3 u1 st3 u2 st3 u3 phytoplankton abundance pattern ceratium sp oscillatoria sp triceratium sp biddulphia sp cerataulina sp hemiaulus sp asterionellopsis sp asterionella sp thalassionema sp thalassiothrix sp fragilaria sp synedra sp melosira sp guinardia sp thalassiosira sp cyclotella sp rhizosolenia sp leptocylindrus sp lauderia sp skeletonema sp bacteriastrum sp chaetoceros sp coscinodiscus sp diploneis sp cymbella sp nitzchia sp surirella sp gyrosigma sp pleurosigma sp navicula sp 580 biology, medicine, & natural product chemistry 14 (1), 2025: 575-583 dominance index (id = 0.131–0.162) values correspondingly reflect reduced ecological stress. in contrast, station 2, which has only recently undergone rehabilitation (2024–2025), exhibited a gradual decline in diversity and evenness indices. the h’ values (1.797–2.340) and e values (0.634–0.718) indicate an unstable and uneven phytoplankton community, likely influenced by transitional disturbances such as sediment disruption, nutrient input, and incomplete vegetative cover. the relatively higher dominance index (id = 0.164–0.244) compared to station 1 points to early-stage colonization by pollution-tolerant species, which may temporarily dominate the community under suboptimal water quality conditions. station 3, located in the ecotourism mangrove zone, exhibited the highest diversity values (h’ = 2.689–2.938), coupled with exceptionally high evenness (e = 0.933–0.940). this may appear counterintuitive, considering the anthropogenic influence from nearby tourism and aquaculture activities. however, the diversity observed at this site could be attributed to the influx of nutrients and organic matter promoting phytoplankton proliferation across various taxa, including pollution-tolerant and opportunistic species. despite the high h’ and e values, the dominance index (id = 0.060–0.073) was low, indicating the absence of a single dominant taxon, which is characteristic of eutrophic conditions where multiple species thrive due to increased nutrient availability. these findings collectively suggest that while higher diversity indices are generally associated with healthier ecosystems, they may also reflect stress-induced proliferation of opportunistic species under nutrient-rich conditions. therefore, interpreting the shannon-wiener index must be integrated with other indices such as dominance and evenness, along with physicochemical parameters, to draw ecologically meaningful conclusions. in the context of mangrove rehabilitation and ecotourism management, the results highlight the importance of continuous monitoring and multi-metric evaluation to ensure that observed biodiversity trends genuinely reflect ecological recovery rather than masked disturbances. figure 5. spatial variation of trophic diatom index values in rehabilitated and ecotourism mangrove. figure 5 illustrates the spatial variation of the trophic diatom index (tdi) across three sampling stations within rehabilitated and ecotourism mangrove zones at merdeka beach. the tdi values reflect the trophic state and organic pollution level based on the composition and abundance of diatom communities. station 1, which has undergone long-term rehabilitation (2010–2025), recorded a moderate tdi value of 60.2. this indicates a mesotrophic to eutrophic condition, suggesting that while ecological recovery has progressed, nutrient enrichment is still present, likely from residual organic matter or land-based inputs. station 2 restored more recently (2024–2025), showed a slightly lower tdi value of 54.8. this suggests early-stage recovery with lower trophic stress compared to station 1. the reduced tdi may reflect less diatom proliferation due to limited nutrient availability or the stabilizing effect of new vegetation cover.in contrast, station 3, located in the mangrove ecotourism zone, exhibited the highest tdi value at 78.5, indicating a hypertrophic condition. this elevated index value implies a high degree of organic pollution and nutrient loading, likely attributed to anthropogenic influences such as aquaculture discharges, tourist activity, and limited natural flushing.these findings demonstrate that tdi is a sensitive and reliable indicator for assessing the trophic status of mangrove waters, especially in contexts where ecological pressures vary due to different land-use histories and human interactions. figure 6. spatial variation of pollution tolerance values in rehabilitated and ecotourism mangrove figure 6 illustrates the spatial variation of the percentage pollution tolerance value (%ptv) across three observation stations located within the mangrove ecosystem at merdeka beach: station 1 (rehabilitated from 2010 to 2025), station 2 (rehabilitated in 2024– 2025), and station 3 (mangrove ecotourism area). the %ptv index reflects the proportion of pollution-tolerant diatoms—such as nitzschia sp., gomphonema sp., navicula spp., and sellaphora spp.—within the total aulia & hutasuhut – phytoplankton diversity as a water quality bioindicator … 581 diatom community. a higher %ptv indicates a higher level of organic pollution in aquatic environments.at station 1, a moderate %ptv value (35%) suggests that the area is subject to slight organic pollution. this is likely due to the effectiveness of long-term mangrove rehabilitation efforts in stabilizing ecological conditions and reducing the dominance of pollution-tolerant species. the relatively low %ptv aligns with the observed ecological maturity and minimal anthropogenic disturbance in this zone.station 2, which has undergone more recent restoration efforts, recorded a higher %ptv (48%), indicative of moderate organic pollution. the transitional nature of the sitecharacterized by active sediment disturbance and early-stage vegetation establishment may contribute to increased nutrient inputs and the proliferation of tolerant diatoms, thereby elevating the index value.in contrast, station 3 demonstrated the highest %ptv (\~72%), classifying it within the heavily polluted category. this station is located in a mangrove ecotourism area, where intense human activity including tourism, aquaculture, and reduced water circulation likely contributes to elevated levels of organic matter. consequently, the diatom assemblage is dominated by taxa that thrive under eutrophic and polluted conditions.overall, the spatial trends in %ptv values emphasize the utility of this biotic index as a sensitive tool for assessing ecological degradation and the effectiveness of mangrove rehabilitation. figure 5. correlation between water quality and phytoplankton community structure in rehabilitated and ecotourism mangrove. the principal compenents (pca) diagram illustrates the relationship between water quality parameters and the phytoplankton community structure in rehabilitated and ecotourism mangrove areas. the plot shows the distribution of three sampling stations(stations 1, 2, and 3) in relation to several environmental variables, including salinity, ph, biological oxygen demand (bod), conductivity, nitrate, phosphate, trophic diatom index (tdi), and phytoplankton total volume (ptv).the findings suggest that %ptv not only reflects the current state of water quality but also serves as an early-warning indicator of anthropogenic impact. integration of %ptv monitoring into routine environmental assessments could significantly enhance coastal management strategies, especially in regions undergoing rapid development or restoration. from the ordination diagram, it is evident that certain environmental parameters, such as ptv, tdi, and salinity, exert a relatively strong influence on phytoplankton community distribution, as indicated by the length and direction of their corresponding vectors. the longer the vector, the greater its influence on species composition. station 1 is positioned far from the origin and aligns closely with the negative direction of the tdi vector, suggesting that the phytoplankton community at this site is particularly influenced by higher trophic diatom levels. in contrast, station 2 is positively associated with salinity, nitrate, and ptv, indicating that these factors significantly shape the phytoplankton composition in this location. station 3 lies on the positive side of the horizontal axis, potentially reflecting unique 582 biology, medicine, & natural product chemistry 14 (1), 2025: 575-583 environmental conditions not fully captured by the most dominant vectors. overall, the pca reveals distinct ecological gradients among the sampling stations, highlighting the role of specific water quality parameters in driving the spatial variation in phytoplankton communities. these findings underscore the importance of monitoring and managing water quality—especially salinity, nutrient concentrations, and trophic status—in order to maintain ecological balance and biodiversity in rehabilitated mangrove ecosystems utilized for ecotourism purposes. conclusions this study demonstrates that phytoplankton diversity and community composition serve as reliable bioindicators of water quality in mangrove ecosystems undergoing rehabilitation and ecotourism development. the spatial variation observed among the three sampling stations reflects differing levels of environmental pressure and ecological maturity. station 1, representing a long-term rehabilitation area, exhibited higher ecological stability as indicated by moderate phytoplankton abundance, high diversity indices, and lower tdi and %ptv values. station 2, currently in the early stages of rehabilitation, showed signs of transitional stress, with elevated phytoplankton abundance dominated by pollutiontolerant species. station 3, located within the ecotourism zone, was characterized by hypertrophic conditions and a high proportion of tolerant taxa, corresponding with intensified anthropogenic activity.the integration of physicochemical parameters with biotic indices—such as the shannon-wiener index, trophic diatom index (tdi), and percentage pollution tolerance value (%ptv)— enabled a comprehensive assessment of ecosystem health. these findings underscore the importance of continuous ecological monitoring using phytoplanktonbased indicators to evaluate the effectiveness of mangrove rehabilitation efforts and to guide sustainable ecotourism practices. the study provides valuable baseline data to inform future conservation strategies and enhance the resilience of mangrove ecosystems in north sumatra. acknowledgements: the author would like to express his gratitude dr.melfa aisyah hutasuhut,s.pd,.m.si for her valuable guidance and input throughout the research process.big thanks for the state islamic university of north sumatera. authors’ contributions: all authors contributed substantially to the conception and design of the study. putri aulia was responsible for the field data collection, analysis, and 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(2021). characteristics of the phytoplankton community structure and water quality evaluation in autumn in the huaihe river (china). international journal of environmental research and public health, 18(22), 12092. https://doi.org/10.3390/ijerph182212092 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 119-131 | doi: 10.14421/biomedich.2022.112.119-131 issn 2540-9328 (online) detection of the atherosclerotic pcsk9 gene inhibitors through in silico method to improve targeted therapy sabarinathan sethuramalingam, revathy leena ravi, janet rani rajiah* sadakathullah appa college, thirunelaveli – 627011, india corresponding author* sethuraji90@gmail.com manuscript received: 27 june, 2022. revision accepted: 19 july, 2022. published: 31 july, 2022. abstract the pcsk9 is one of the most important marks for the evolution of therapeutic agents for atherosclerosis because its interaction with lowdensity lipoprotein receptors causes atherosclerosis. protein-ligand interactions help us to understand the true mechanism of pharmacological action. this study seeks to identify the most powerful suppression options for pcsk9. initially, the reported ace inhibitors were included in pharmacophore modeling using pharmagist. next, zincpharmer was used to screen the selected model against a zinc database to identify putative drug candidates docked to the target protein to understand the interactions. the 10 best pharmacological candidates for pcsk9 with a binding energy of 9.8-8.2 kcal mol-1 were identified by molecular docking and their pharmacokinetic properties and oral bioavailability were evaluated. the (s) severalplant obtained chemicals have been discovered, including anti-hypersensitive drugs such as “canadine, hesperetin, and labetalol”. according to biochemistry, these compounds formed a stable “protein-ligand” complex. the (s) canadine pcsk9 complex had the lowest rmsd and was the most stable. future in vitro studies could identify (s) canadin as a promising atherosclerosis inhibitor for the evolution of novel pcsk9 inhibitors. keywords: pcsk9; therapeutics; ace inhibitors; protein – ligand; docking. introduction highly elevated ldl cholesterol known as “familial hypercholesterolemia” (fh) is recognized as a hereditable disease. it can lead to an increase of low density lipoprotein (ldl) cholesterol in the blood. this can increase ldl quantity by more than 4 mmol / l (> 180 mg/dl) (zhao et al., 2019), increasing the threat of cardiovascular diseases (cvds) such as atherosclerosis, heart failure and myocardial infarction, etc. patients with genetic modification in the familial hypercholesterolemia gene, such as low density lipoprotein receptor (ldlr), proprotein convertase subtilisin/kexin type 9 (pcsk9), and apob, are at increased risk of atherosclerosis (ascvd) than those without the mutation but with comparable cholesterol levels (khera et al., 2016). mutant fh-related genes can cause an increase in low-density lipoprotein cholesterol (ldlc) (semenova et al., 2020), cause vascular endothelial dysfunction. ascvd, including cad, is a well-known disease characterized by endothelial dysfunction. endothelial dysfunction is the first step in the development of atheroma. hypercholesterolemia is one of the most common causes of endothelial dysfunction that causes stenosis of the aorta of the heart (zhang et al., 2020). cholesterol is required for some physiological activities. cell membranes are important organelles that require large amounts of cholesterol. in addition, cholesterol is essential for the production of bile acid and steroid hormones. an increase in cholesterol, particularly under oxidizing circumstances, may culminate in atherosclerosis, causing carotid, peripheral artery, and coronary heart disease (andreadou et al., 2017). one of the leading causes of stroke and heart attack is atherosclerosis. cholesterol buildup may result in the formation of arterial plaques, leading to atherosclerosis. elevations in ldlc and apolipoprotein b100 (apob100) are precisely connected to cardiovascular events such as atherosclerosis. decreased ldlc levels may reduce the risk of cardiovascular events, as reduction of apob on the arterial wall can cause lesion and lead to the formation of atherosclerosis (mahley, 2016). ldlc deposits can result in fatty streaks accumulation and reduced flow of blood to many organs as a result of inflammatory changes in the arterial wall. pcsk9 interacts with the ldl receptor (ldlr) and causes liposomal degradation that promotes atherosclerosis. therefore, pcsk9 inhibition is essential for risk halting of cvds (latimer et al., 2016). pcsk9 is a protein with 692 amino acids which in mainly expressed in liver and intestine (horton et al., 2007). pcsk9 is synthesized in the endoplasmic reticulum (er), and its 73 kda precursor peptide is zymogen. this zymogen makes many changes before it stick out the https://doi.org/10.14421/biomedich.2022.112.119-131 mailto:sethuraji90@gmail.com 120 biology, medicine, & natural product chemistry 11 (2), 2022: 119-131 cell surface. there are auto-catalytic activity in-between 152nd and 153rd amino acids. the n-terminal pro-domain stays firmly linked with the catalytic domain in the secretory route(hyock et al., 2008). pcsk9 can break ldlr with its proteolysis activity when in contact with the extra-cellular sites of ldlr. pcsk9 has a subtilisin-like catalytic domain that can bind to the ldlr-egfa domain. this domain contains asp374, and changing to tyr increases pcsk9's affinity for ldlr (hyock et al., 2008). binding of low density lipoprotein to low density lipoprotein receptor is important for reducing lipid sedimentation and the threat of cvds. when pcsk9 binds to ldlr, it protects ldl from attaching to low density lipoprotein (ldlr) and causes lipid sedimentation. to avoid cad, you should block the association between pcsk9 and its ldlr (horton et al., 2009). natural treatments using mab and short molecule drug therapies are the two main approaches used in pcsk9 targeted therapies. unfortunately, certain advances in small molecule drug therapy for pcsk9 inhibition are hampered by the fact that the molecular structure of pcsk9 required for small molecule binding to reduce the activity of this protein has not been identified (joseph & robinson, 2015). computational methods based on bioinformatics and chemoinformatics help identify new binding sites for inhibiting proteins. bioinformatics and chemoinformatics deal with the creation and manipulation of databases and statistical methods for the management and analysis of biological and chemical data. these devices help to find and study about targets, chemical structures, and mobile sites. these devices can also discover potential medicinal molecules for specific therapeutic targets and quantify their medicinal similarity via molecular docking (wishart, 2005). medicinal candidates are evaluated for their binding affinity and their properties are subject to change as needed. the concept of computationally-based drug discovery is called structural design. therefore, the in silico approach can provide a broad and favorable approach for identifying new target proteins and predicting their biological activity (ramharack & soliman, 2018). the drug components such as, “captopril, zophonopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, fosinopril, sirazapril, moexipril, trandolapril, alicin, teprotide” were used in this incilico study (attique et al., 2019). using computational approaches such as the pharmacophore design of existing drugs, new small inhibitors of pcsk9 have been identified. molecular docking was performed to study the interaction of pcsk9 target proteins with computer-identified new drugs. this study is essential for the discovery of new drugs targeting pcsk9 that can be administered to cure or reduce heart disease and address the increasing challenges associated with excess lipids for the reduction and treatment of atherosclerosis or its symptoms. materials and methods 3d structure of the protein of interest (pcsk9) the 3d structure of the target protein was obtained from the pdb library (https://www.rcsb.org/search) (burley et al., 2021). the interaction between the egfa domain of pcsk9 and ldlr occurs at the target site. this interaction (du et al., 2011) between the pro-domain and c-terminal of pcsk9 increases blood ldlc quantity and thus increases susceptibility to cvd. the pdb id 6u26 (petrilli et al., 2020) was determined based on its quality (1.49) after analyzing all known structures in the pdb database, including domains with one active site. pcsk9 active site characterization the active site of pcsk9 was found in-between a unique pocket, catalytic and c-terminal domain(petrilli et al., 2020). lig plot is used to plot this pocket (figure 1). ligplot provides data on hydrogen bonds and hydrophobic contacts (ra & mb, 2011). figure 1. ligplot for 6u26: (figure 1) it shows data on hydrogen bonds and hydrophobic contacts. preparation of the protein target and ligand an autodock tool(hanwell et al., 2012) was used to purify target protein to remove heteroatom, polar hydrogen, and colman charges. the 3d structure of the pcsk9 target protein was generated by converting to the pdbqt format. angiotensin-converting enzyme sethuramalingam et al. – identification of pcsk9 inhibitors 121 inhibitor (acei) with atherosclerosis activity was found in ligand synthesis (table s1). those 2 dimensional structures were taken from the pubchem database(kim et al., 2021) using individual pubchem ids and energy was reduced using the chemaxon marvin suite. we used raccoon software (hanwell et al., 2012) to convert the energy-optimized structure to pdbqt format. table 1. the ei and pi of the molecules. molecule name pubchem id binding energy residues ei pi captopril/ 44093 -7.0 asp 651 and arg 545 0.60 0.98 zofenopri 92400 -9.3 arg 458 and trp 461 0.24 0.89 enalapril 5388962 -8.8 arg 458 0.28 0.80 ramipril/ 5362129 -9.4 arg 458 0.33 0.88 quinapril 54892 -9.9 arg 458 0.24 0.60 perindopril 107807 -8.2 arg 458 0.30 0.93 lisinopril 5362119 -8.8 arg 458 0.49 0.92 benazepri 5362124 -8.3 arg 458 and arg 476 0.51 2.03 fosinopri 55891 -8.7 arg 357 0.08 0.74 cilazapril 56330 -9.6 arg 458 0.09 0.46 moexipril 91270 -9.4 arg 458 0.27 0.86 trandolapril 5484727 -9.1 arg 458 2.52 2.40 allicin 65036 -9.9 arg 458 2.35 2.68 teprotide 443376 -9.7 arg 458, arg 357, asn 298, trp 461, cys 323 and arg 458 2.77 2.67 (table 1) it shows the ei and pi of the molecules with its binding energy. molecular docking of recognised ace inhibitors with psck9 the energy-reducing structure (pdbqt format) was docked to the target protein using autodock vina (ukuku et al., 2012). all known molecules with above average binding energies (moexipril, trandolapril, allicin, teprotide, zophenapril, lamapril, quinapril) were selected for the pharmacophore design. inhibitor selection was based on intracting energy and structural comparison (table 1). pharmacophore designing/modeling the pharmacophore was designed using pharmagist (laskowski et al., 2018), and the pharmacophore was selected predicted on its high range and the number of molecules placed in the design. pharmacophores are a three-dimensional sequence of properties (chemical bond donors, chemical bond acceptors, anions/cations, aromatic rings, hydro-phobic groups) required for a ligand to communicate with a particular target (schneidman-duhovny et al., 2008). screening virtually for the pharmacophore prior to wet-lab investigations, one of the fundamental levels in drug improvement is digital screening. this manner consists of estimating the binding affinity of a capability medicine candidate to a goal protein. virtual screening is likewise utilised to evaluate capability binding modalities of the medicinal candidate and different drug-like short molecules throughout interplay with the goal protein. using hpc “high-performance computing” configuration technologies (jaghoori et al., 2016), the maximum distinguished medicine applicants displaying capability binding affinity in the direction of the goal protein can be eliminated. through the virtual screening manner, numerous bioactive compounds that could have interaction with the goal protein can be found (langer & hoffmann, 2008). the pharmacophore become absolutely screened towards the zinc drug database the use of zincpharmer (koes & camacho, 2012), and 1033 hits/ compounds have been identified. molecular docking for the last molecules screened sdf-formatted structures (each molecule in a single file) were obtained and used to dock the terminal of screened 1033 compounds (using the desired pharmacophore). i used a python script to separate and reduce the energy of these structures. the resulting structure was changed to pdbqt format using vina to dock to the required protein. all compounds were docked to autodock vina (guidi et al., 2006) and the top results (contingent on interaction and affinity energy) were examined using autodock tools (hanwell et al., 2012). the docking grids coordinates were 40.516000 (x), 24.375625 (y), and 25.098375 (z), and each grid had dimensions of 25. there were 20 modes in all. table 2 shows the 10 most important zinc molecules selected as potential treatment candidates. 122 biology, medicine, & natural product chemistry 11 (2), 2022: 119-131 table 2. amino acid interactions of molecules. molecule name binding energy number of interactions amino acid interaction apomorphine -10.8 7 arg 458, pro 438, arg 357, trp 461 and val 460 oxyphencyclimine -10 8 cys 358, pro 331, ala 478, val 460, arg 458, val 359, thu 459 and pro 438 canadine -10 7 arg 357, asp 360, pro 331, ala 478, val 460 and pro 438 naltrexone -9.8 10 arg 476, arg 458, val 460, pro 331, ala 330, thr 335, ala 328 and cys 358 oxolinic -9.6 7 leu 440, pro 438, trp 461, val 650, thr 437 and thr 459 norfloxacin -9.6 9 trp 461, leu 436, ile 416, ala 649, pro 438, arg 458, arg 357 and arg 412 canadine -9.6 7 arg 458, asp 360, pro 438, ile 416, leu 440, thr 459, val 460 and arg 357 hesperetin -9.4 9 arg 412, ala 330, arg 357, cys 358, pro 331, trp 461, thr 459 and val 460 labetalol -9.3 9 trp 461, cys 358, thr 437, pro 438, val 650 and asp 651 benorilate -9.2 6 arg 458, val 460, arg 357, pro 438 and leu 436 (table 2) the binding energy of top ten molecules are shown with their amino acid interactions. final medication candidates' pharmacokinetic characteristics we used openbabel (http://openbabel.org/wiki/main page) to create a simplified molecular input system & # 40; smiles & # 41; autodockvina for the top 10 compounds identified during virtual screening. the resulting smiles code is zinc (sterling & irwin, 2015), molinspiration cheminformatics' free online service (,pubchem. analyzed using a comparison website. , swiss adme (daina et al., 2017) and admet sar (yang et al., 2019). swissadme was used to calculate the oral bioavailability of each drug (table 3). for bioactivity prediction and drug similarity from lipinski's rule 5, use the predict bioactivity tool available to calculate molecular properties and bioactivity scores. was evaluated by. the le and lelp parameters were found from the specified results. table 3. bioavailability of drugs. molecule formula mw ali log s bioavailability score synthetic accessibility apomorphine c17h18no2 268.33 -3.88 0.65 4.29 oxyphencyclimine c20h28n2o3 344.45 -4.89 0.65 5.08 (s)-canadine) c20h22no4 340.39 -4.65 0.65 4.68 naltrexone c20h24no4 342.41 -3.69 0.65 5.76 oxolinic acid c13h11no5 261.23 -3.16 0.66 3.26 norfloxacin c16h19fn3o3 320.34 -1.14 0.65 1.5 (r)-canadine c20h22no4 340.39 -4.65 0.65 4.68 hesperetin c16h14o6 302.28 -5.27 0.65 4.22 labetalol c19h25n2o3 329.41 -5.86 0.65 4.04 benorilate c17h15no5 313.3 -4.5 0.65 3.16 (table: 3) the molecular weight and the bioavailability value of various molecules are given with its synthetic accessibility. molecular dynamics of complexes of protein and ligand the molecular dynamics of the protein-ligand complex was prepared and calculated using gromacs (abraham et al., 2015) version 2018.4 on a desktop computer with a 2.80 ghz and nvidia graphics geforce gtx 1060. we did it with a 6gb card. the gromacs source code was taken from the website http://www.gromacs.org/ and built using opencl version 1.2 of microsoft visual studio community msvc19.16.27025.1 and nvidia gpu computing toolkit cuda v10.0 for windows 10. the pdbqt file of the protein-ligand complex obtained by virtual screening at autodock vina is used as the first coordinate source for both psck9 and the screened virtual inhibitor. using open babel, the coordinates were converted to in pdbqt format, pdb for proteins and mol2 for ligands. the generated pdbqt file was reconstructed in swissmodel (waterhouse et al., 2018) using the entire human pcsk9 amino acid sethuramalingam et al. – identification of pcsk9 inhibitors 123 sequence (bateman et al., 2015; leinonen et al., 2004) and lacked the original 3d structure. i filled in the loop that is. new pdb coordinates were retrieved and utilised by gmx pdb2gmx with the charmm36 allatom force field to generate the gro protein topology (july 2020). in the avogadro programme (http:// avogadro.cc/) (hanwell et al., 2012), mol2 files were updated with all the missing hydrogen atoms. the updated mol2 files were visually verified and modified for problems. using sort mol2 bonds, a perl script created by justin a. lemkul and provided under the gpl3.0 licence, the bonds in the coordinate files were separated in ascending order. such processed mol2 data was sent to swissparam (https://swissparam.ch/) to build a ligand topology for retrieving “itp and pdb” files. the received pdb file was converted to gro using gmxeditconf and the itp file was correctly placed in the respective topol.top file for each ligand. next, we defined the unit cell and filled it with water. gmxeditconf generated a dodecahedron cell from the proteinligandgro file and the correct topol.top file. sodium and chloride ions were added to achieve a concentration of 0.155 m with a neutral charge while energy was minimised. the ligands and protein were individually restrained by constructing position restraint topologies, and twophase 100 ps, 310 k, and 1 bar equilibrations were carried out to create first nvt and then npt outputs. terminal molecular dynamics production was set for “10 nanoseconds” of simulation at 320 kelvin and 1 bar of pressure, and a single simulation of a proteinligandcombination on the previously reported computer equipment lasted around 24 hours. for both energy minimization and equilibration, the similar temperature and pressure conditions were employed. visual molecular dynamics (vmd) (yamada et al., 2017) was used to visualise the results, and gromacs gmx rms was used to determine the rootmeansquare deviation (rmsd). results and discussion results active site 3d structure of the target protein (pcsk9) figure 1 shows the ligplot of the structure expressing the residues participated in the interaction. the dashed line shows the hydrogen bond interaction between the amino acids and legends of the target protein. figure 2 uses discovery studio to contrast this interaction and distinguish the active area residues from the rest of the structure. figure 2. active cite pocket. (figure 2) ball sticks representation of active site pocket utilizing molecular docking research to identify known inhibitors for pharmacophore design. to identify the optimal structure of pcsk9 and its active sites and recognized ace inhibitors such as, “captopril, zophenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, fosinopril, silazapril, moexipril, trandolapril, alicin”, etc. we reviewed the literature in (s1 table). all known acei were docked to the target protein pcsk9 and the top results were evaluated based on the interaction between binding energy and the active site. molecules evaluated on the basis of pharmacophore in this study, pharmacophore was adopted as a drug discovery strategy and a novel pcsk9 inhibitor was used with established inhibitors such as, “moexipril, trandolapril, alicin, teprotide, zophenopril, ramipril, quinapril” shown in table s1. the pharmacophore model created by pharmagist was evaluated and the optimal pharmacophore was selected based on a score of 18.102 and 5 attributes (1 hydrophobicity, 1 negative ion, and 3 hydrogen bond acceptors). rice field. in zincpharmer, pharmacophores were screened along the entire zink drug database and 1033 hits / molecule were identified. molecular docking to identify the top 10 most promising medicinal candidates after evaluating all 1033 molecular docking complexes with the required protein, their affinity energies and interactions were saved. the docking results of the top 10 zinc molecules were sorted based on the interaction of the active sites assessed for lower binding energies calculated by autodock (table 2 and figure 3a–3j). lower energies exhibit stronger affinities and sustained interactions. therefore, the top compounds may be more effective in exerting the expected activity of the pcsk9 and are therefore excellent therapeutic candidates. 124 biology, medicine, & natural product chemistry 11 (2), 2022: 119-131 final 10 drug candidates the drugs such as, “apomorphine, oxyphencyclimine, (s) canadine, naltrexone, oxolinic acid, norfloxacin, (r) canadine, hesperetin, labetalol, and benolylate” were the top 10 choices. table s4 shows their pubchemcid, name, structural formula, putative characteristics, and drug similarity according to lipinski's rule of five. according to pubchem, apomorphine (zinc00009073) is the first molecule with the least change in docking energy, a non-selective dopamine used to treat parkinson's disease. oxyfencyclimin (zinc00020260) is an oral muscarinic receptor antagonist used to treat gastric ulcers and gastrointestinal problems. the third is (s) canadine which is also called as (s) tetrahydroberberine and xanthopsin (zinc00033518), which is a benzylisoquinoline alkaloid (bia) in the protoberberine structural subgroup (xu et al., 2019) and is found in some poppy and corydalis plants. the next substance (zinc00001773) is naltrexone, which is commonly used to treat alcohol or opioid addiction. oxolinicacid, zinc00001875, is a synthetic antibiotic used for veterinary medicine. the antibiotic zinc00003742 is norfloxacin, which has broadspectrum antibacterial activity over the gram-negative and gram-positive bacteria. (r) canadine is an enantiomer of (s) canadine. zinc00039092 is a secondary plant metabolic product, hesperetin, that acts as an antioxidant and antitumor agent. labetalol is the chemical name for 2 hydroxy 5 [(1s) 1 hydroxy 2 [[(2r) 4 phenylbutane 2 yl] amino] ethyl] benzamide, also known as zinc00000416. it is a third-generation vasodilator, an antihypertensive drug selective α1 adrenergic blocker and non-selective β-adrenergic blocker. the final product (zinc00001003) is benorilate, an esterified codrug of aspirin and acetaminophen. it is used as an anti-inflammatory and antipyretic drug(bass, 1973). figure 3. the 10 drug candidates. (figure 3) it shows the final drugs table 4. pubchem ids and names of molecules. molecule name cids le lelp alogp milogp drug similarity apomorphine 6931310 0.5 8.7 2.43 4.16 pass oxyphencyclimine 667690 0.5 9.9 3.73 4.57 pass (s)-canadine 21171 0.5 9.3 2.67 3.99 pass naltrexone 5360515 0.5 4.9 1.11 2.37 pass oxolinicacid 4628 0.6 2.5 2.42 1.68 pass norfloxacin 4539 0.5 -2.8 1.24 -1.69 pass (r)-canadine 443422 0.4 9.7 2.67 3.99 pass hesperetin 72281 0.5 6.1 3.52 2.94 pass benzamide labetalol 134045 0.4 9.2 2.11 3.85 pass benorilate 21102 0.5 8.3 3.79 3.61 pass (table 4) the pubchem ids and the drug similarity report based on the “lipinski rule” is given in detail sethuramalingam et al. – identification of pcsk9 inhibitors 125 the pharmacokinetic characteristics and bioavailability of the medication will be evaluated. using tools, “molinspiration, knime, swissadme, and admetsar, computational structural analysis, druglikeness, adme characteristics, oral bioavailability, and toxicity profiling” have been conducted (3 and 4 tables). using smiles codes, the pinnacle molecules have been re-searched towards the zinc database, molinspiration, and pubchem, and that they have been all recognized as acknowledged compounds, a number of plant beginning such as (s)-canadine and hesperetin. an in addition antihypertensive agent, labetalol, turned into found (4 table). depending at the diploma of nitrogen protonation, numerous chemical substances containing nitrogen atoms may also exist in lots of paperwork. these compounds incorporate numerous pubchem codes, distinct smiles, however are stated as "discern compounds" on this database. consequently, it is able to be concluded that they are the equal chemical compounds that tackle numerous paperwork in accordance at the ph. figure 4 depicts the availability radars of the pinnacle ten nominated compounds. figure 4. molecules radar chart. (figure 4) – this shows the molecular radar chart evaluation of the stability of the suggested proteinligand complexes upon visual inspection with vmd, (s) canadin, hesperetin, and labetalol produced a strong proteinligand complex throughout molecular dynamics and remained at the docking targets for at least 10 ns. the rmsd value of the heavy atom of the ligand in the 10ns lasting simulation of the three compounds was 0.007. 0.13 0.024; 0.13 and 0.035 (mean sd) (figure 5), respectively, suggesting that the position of the ligand changes only slightly. the value of (s) canadine is 0.1, indicating the excellent stability of this compound. this molecule produced the most stable (s) canadine complex in terms of duration and molecular dynamics, with the lower rmsd (fig. 5). this compound can be further researched for the discovery of new pcsk9 inhibitors. 126 biology, medicine, & natural product chemistry 11 (2), 2022: 119-131 figure 5. rmsd stimulation. (figure 5) – it shows the rmad stimulation discussion the manufacturing of arterial plaques is one of the main reasons of atherosclerosis, which in the end outcomes in arterial blockage and coronary heart attack. moreover, better stages of apob-a hundred and ldl cholesterol are without delay connected to atherosclerosis-associated cvds. the lipid cycle commonly begins with the liver`s launch of im-mature vldl. in addition, apob-a hundred, cholesteryl esters, triglycerides, and ldl cholesterol are present. vldl broaden and end up a supply of electricity in “adipose tissues, skeletal and cardiac muscular tissues” all through movement withinside the blood (chaudhary et al., 2017). one of the jobs of vldl is the trade and elimination of triglycerides, which ends up withinside the conversion of vldl into intermediate-density lipoproteins (idl) (shelness & sellers, 2001). some of those idl can be removed through the liver all through endocytosis, whilst idl with a more ldl cholesterol content material are transformed to ldl, which additionally consists of apob-a hundred. this apob-a hundred attaches to ldlr at its n-terminal area after which passes via the acidic endosome via endocytosis. ldlr in the end breaks from ldl and is reintroduced into the mobileular membrane (ding et al., 2015). at an acidic ph, structural changes in ldlr might also additionally decorate pcsk9's affinity for ldlr relative to ldl(horton et al., 2009). although mutations might also additionally dispose of the catalytic hobby of pcsk9, they do now no longer have an effect on its cappotential to bind to ldlr. a stoppage withinside the protein's manufacturing might also additionally bring about blockading its binding to ldlr. pcsk9 mrna degradation might also additionally in the end hinder its reference to ldlr, or a tiny drug meant to save you this courting also can do so (mcnutt et al., 2007). atherosclerosis is as a result of the interplay among pcsk9 and ldlr (egf-a area or epidermal boom factor-like repeats), which inhibits the endocytosis of ldl. the connection among pcsk9 and ldlr can be blocked to halt the buildup of ldl. pcsk9 is produced withinside the er and transferred to the plasma membrane to be able to have interaction with ldlr. protein-protein interactions govern a extensive quantity of organic activities. the law of those interactions is a famous and potential drug improvement pathway. to save you the egf area of ldlr from interacting with pcsk9, it's far important to perceive the interplay's goal location. it is acknowledged that antihypertensive medications, specifically ace inhibitors, have a essential position in atherosclerosis (pitt, 1995). acei are putative plaque formation inhibitors (ferrari et al., 2010). experimental, epidemiological, and scientific research have proven that ace inhibitors decorate arterial endothelial feature and, thus, lower the improvement of atherosclerosis (lonn, 2001). it has been proven that they lower erythropoietin (epo) stages and hematocrit. acei might also additionally modulate a whole lot of bioactive peptides, along with angiotensin 1–7 and substance p. in hematopoiesis, numerous sethuramalingam et al. – identification of pcsk9 inhibitors 127 peptides had been located to have a position. reducing the quantity of angiotensin ii might also additionally restrict erythropoiesis. acei additionally have an effect on the renin-angiotensin system (younas et al., 2017). several ace inhibitors, along with “captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, fosinopril, cilazapril, moexipril, trandolapril, allicin, and teprotide”, had been used on this study. captopril decreases ldl oxidation, which is understood to make contributions to the improvement of atherosclerosis (k.r. et al., 2014). zofenopril and enala prilare acei which can lessen vascular harm and enhance endothelial progenitor mobileular movement. they inhibit endothelial harm and the improvement of atherosclerosis (pines & fisman, 2003). arterial plaque formation is one of the leading causes of atherosclerosis and ultimately causes arterial occlusion and heart attack. in addition, high levels of apob100 and ldl cholesterol are directly associated with cardiovascular disease associated with atherosclerosis. the lipid cycle usually begins with the release of immature vldl by the liver. it also contains apob100, cholesteryl ester, triglycerides and cholesterol. vldl develops in adipose tissue, skeletal muscle, and myocardium during circulation in the blood and becomes an energy source (chaudhary et al., 2017). one of the roles of vldl is the exchange and removal of triglycerides, converting vldl to intermediate density lipoprotein (idl) (shelness & sellers, 2001). some of these idls can be eliminated from the liver during endocytosis, but idls with high cholesterol content are converted to ldl, which also contains apob100. this apob100 binds to ldlr at its n-terminal domain and passes through acidic endosomes by endocytosis. ldlr is eventually cleaved from ldl and reintroduced into the cell membrane (ding et al., 2015). at acidic ph, changes in ldlr conformation may increase the affinity of pcsk9 for ldlr compared to ldl [10]. mutations can eliminate the catalytic activity of pcsk9, but do not impair its ability to bind to ldlr. interfering with protein production, can block binding to the ldlr. degradation of pcsk9 mrna can ultimately prevent its association with ldlr. alternatively, the same can be done with small medicines designed to prevent this relationship (mcnutt et al., 2007). atherosclerosis is caused by the interaction of pcsk9 with ldlr (egfa domain or epidermal growth factor-like repeat) and inhibits ldl endocytosis. you can block the connection between pcsk9 and ldlr to stop ldl accumulation. pcsk9 is produced in the er and transcribed into the plasma membrane to interact with the ldlr. proteinprotein interactions control a significant number of biological activities. ace inhibitors (table 1) have been shown to play an active role in the prevention of atherosclerosis. therefore, pharmacophore modeling and screening using the zinc database has been introduced into the drug development process. the binding affinity of the newly identified chemical (1033) from the zinc database was assessed using the autodock tool. the program calculated several factors, including reduced binding energy, interaction with the active site, and the number of hydrogen bonds. comparing these criteria yielded a list of the 10 most promising drug candidates for atherosclerosis (table 2). some of the molecules virtually screened from the zinc database showed good interactions, while others showed no or weak interactions. the results show that the top 10 compounds with a binding energy of 9.8-8.2 kcal / mol (table 2) are similar to the reference compounds (table 1) and are therefore desirable and viable drug candidates. candidate target for pcsk9 in atherosclerosis. in other words, in this study, the topselected compounds showed the highest binding affinity for the pcsk9 target protein for atherosclerosis. in addition, molinspiration, computational structural analysis of these compounds using swiss adme, drug similarity, adme properties, and oral bioavailability, based on these results, (s) canadin, hesperetin, and labetalol contain heavy atoms and have le and lelp values, which is why further research and possible design of new drugs based on pharmacophore structure is the most. we can conclude that it is a promising candidate. same range as known ace inhibitors (tables 1 and 4). the results of autodockvina led to the selection for further molecular dynamics analysis of the complex with pcsk9. during molecular dynamics, all three compounds appeared to form a stable pcsk9ligand complex, staying at the docking site for at least 10 ns, and their heavy atom rmsd values were minimal. a notable advantage of (s) canadine, also known as (s) tetrahydroberberine, is a significant reduction in rmsd (figure 5), which promotes the formation of a highly stable complex with pcsk9. this is an interesting finding, as studies suggest that berberine inhibits psck9 (xu et al., 2019). although the direct inhibitory effect of such compounds is unknown, this study provides insights into (s) canadin and berberine with very similar pharmacophore structures that differ only in the degree of hydrogen atom saturation. increase. berberine is an aromatic chemical, but (s) canadine is not. experimental studies in mice have found that berberine delays the onset of atherosclerosis in cholesterol-fed apoe / knockout mice (wan et al., 2018). in addition, a study of the coptis chinensis decoction network path, a traditional chinese healing method, revealed: based on these results, (s) canadin, hesperetin, and labetalol are the most promising candidates for further research, and (seidah et al., 2017) the potential for new drug development based on the study is (s) canadin and berberine. we can conclude that it indicates that. of the most probable drug for atherosclerosis. in vivo studies conducted with hesperetin to assess its role in ldlr expression showed that hesperetin dose (200m) stimulated ldlr gene expression, increased mrna levels of transcription factors srebp1 and srebp2, 128 biology, medicine, & natural product chemistry 11 (2), 2022: 119-131 and plasma levels. the effect of ldlc on reducing the risk of cardiovascular disease has been suggested to reduce (bawazeer et al., 2016). in addition, studies suggest that activation of srebp2 causes co-expression of pcsk9 and ldlr, leading to ldlr depletion and increased ldlc circulation (maxwell et al., 2003; urban et al., 2013). as a result, the effects of hesperetin can be counteracted. studies show that saturation increases with increasing fsp3 levels (lovering et al., 2009; wei et al., 2020). the relationship between fsp3 and the solubility and melting point attributes of 1000 compounds in the dataset showed a positive correlation between fsp3 and solubility and a negative correlation with melting point (lovering et al., 2009). compounds with a lower melting point show better absorption than compounds with a higher melting point (chu & yalkowsky, 2009). therefore, low chemical saturation (fsp3) can lead to high melting points, limited drug absorption, and reduced oral bioavailability. therefore, it is assumed that low levels of hesperetin saturation (table 3) reduce oral bioavailability. although various studies have shown that hesperetin has low oral bioavailability (kanaze et al., 2007; liu & chen, 2008), it is possible to increase oral bioavailability by specific chemical approaches [69]. (s) and (r) canadins have unique bioavailability properties due to their ability to efficiently cross the blood-brain barrier (bbb) and reach their targets (table 3). their high gastrointestinal absorption and ability to bind to pgp make these molecules excellent candidates for orally administrable drugs (laskowski et al., 2018). based on current research, the pharmacophore structure of (s) canadin may function as an inhibitor of the pcsk9 protein molecule. using existing ace inhibitors, pharmacophore was used as a drug discovery method to identify new pcsk9 inhibitors. a literature search was conducted to identify the optimal structure of pcsk9 and the active site of known ace inhibitors. the results of molecular docking, incorporating a computational and unbiased approach, highlighted the relevance of drug development in atherosclerosis. in this study, the binding affinities were analyzed and the molecules with the highest affinities for the target of atherosclerosis were zinc000073, zinc00020260, zinc00033518, zinc00001875, zinc00003742, zinc0003517, zinc000390, zinc000390, zinc0003900, zinc0000390, zinc000390, zinc000390, zinc000390, zinc0003900, zinc0003900, zinc0003900, zinc0003900, zinc0003900, zinc0003900, zinc0003900, zinc0003900 lipinski's rule 5 was applied and passed for these major drug candidates. several molecules from virtual screening and drug database searches were found as herbal chemicals such as (s) canadine, hesperetin, or the antihypertensive drug labetalol. the formation of stable protein-ligand complexes by these chemicals has been demonstrated using molecular dynamics. for (s) canadine, the complex prepared with this chemical is the most stable in molecular dynamics and has the lowest rmsd. conclusions the pcsk9 inhibitors are found through the computational methods, these inhibitors can be used as a vital target for the novel drug screening and discovery for atherosclerosis in future. the computational findings should be conformed in future using invitro and invivo method for further understanding the inhibitors role in atherosclerosis. hence a depth wet lab research need to be done with is inhibitors as a target for treating atherosclerosis conflict of interest: the author has no relevant financial or non-financial interests to disclose. code availability: no code availability data availability: data sharing not applicable to this article as no datasets were generated or analysed during the current study. authors’ contribution: first author s sabarinathan and second author revathy l r have contributed in all aspects for the prepration of manuscript and generation of idea under the guidance of dr r janet rani (corresponding author) competing interests: the author declares there is no competing interest funding: no funding was received to assist with the preparation of this manuscript references abraham, m. j., murtola, t., schulz, r., páll, s., smith, j. c., hess, b., & lindah, e. 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(2019). genetic determinants of myocardial infarction risk in familial hypercholesterolemia. cjc open, 1(5), 225–230. https://doi.org/10.1016/j.cjco.2019.06.001 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 1, 2017 | pages: 9-12 | doi: 10.14421/biomedich.2017.61.9-12 issn 2540-9328 (online) comparative anatomy and histology of black pomfret (formio niger) and nile tilapia (oreochromis niloticus) kidney nurul safitri apriliani1, muhammad jafar luthfi2 1center for integrative zoology; 2biological education department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto 55281 yogyakarta, indonesia author correspondency: nurulsafitri89@gmail.com1 abstract black pomfret and nile tilapia are belonging to order perciformes. both fish are live in different habitat. black pomfret is marine water whereas nile tilapia is in freshwater. the purpose of the study was to determine anatomy and histology of the kidneys structures in both fishes. histological preparations were done using paraffin method, with hematoxylin-eosin (he) staining. the results, showed that black pomfret and nile tilapia have y-shape kidney. nile tilapia has darker red colour and softer texture than black pomfret kidney. histologically, black pomfret and nile tilapia kidneys have a distal tubule, proximal tubule, glomerulus and lymphoid tissue. glomerular diameter of nile tilapia (69,22 µm) was larger than pomfret (61,25 µm). it can be concluded that differences between anatomical and histological structure of kidney are affected by habitat differences. keywords: anatomy; black pomfret (formio niger); histology; kidney; nile tilapia (oreochromis niloticus). introduction fishes (pisces) are members of subphylum vertebrates that have species number more than 27,000 species in the world. fish has the most diversity in terms of morphology (bond, 1979). there are also anatomical and histological variations which is manifest in its form and function. this could be caused by different habitats, even in fish in the same order, such as black pomfret (formio niger) and nile tilapia (oreochromis niloticus). black pomfret and nile tilapia are belonging to the order perciformes. these fish live in different habitats. black pomfret (formio niger) is one of many fishes species found in marine areas in indonesia, especially in the southern sea. nile tilapia is a freshwater fish. it can be found in lakes, river, and water reservoirs (suyanto, 1994; djarijah, 1995; taufik et al., 2002). osmoregulation is the capacity of aquatic animals to control the water and ion balance between the body and its environment, or a process of osmoregulation (nicol, 1967; fujaya, 2004). the organ that has an important role in osmoregulation process in the body is kidney (karl, 1962). kidney is an excretory organ that has an important role in the filtration and excretion process. the fish of kidney absorb some salt and release the salt into the bloodstream. the fish kidney also has a pump that will take salt from water and release ammonia and other waste products (karl, 1962). the function of each parts of the kidneys that involved in the osmoregulation process will show some differences between the renal fish structure that living in marine or freshwater. comparative anatomical study was conducted to determine the differences anatomical and histological renal organ of black pomfret and tilapia in the same order level. the comparisons including the different in histological and anatomical structures of renal fish organ that may affected by the physiological factors of the fishes due to habitat difference. materials and methods in this study, we used three adults black pomfret (formio niger), obtained from pangandaran beach, west java and three adults nile tilapia (oreochromis niloticus) from nila cultivation center, cangkringan yogyakarta. anatomical observation were done by observing the topography, colour, texture, shape and length of the kidney. histological observation were done on histological slides stained with using hematoxylin-eosin (he). histological slides were made using paraffin method. results and discussion black pomfret (formio niger) and nile tilapia (oreochromis niloticus) (figure 1 & figure 2) have similarities as common perciformes fish, which have dorsal, caudal, anal, pelvic and pectoral fins. dorsal fin of nile tilapia has a spiny fin or a hard fin called spinal fin. pomfret and tilapia have difference in its caudal fin shape. pomfret has a fork-shaped, whereas tilapia fish http://dx.doi.org/10.14421/biomedich.2017.61.9-12 10 biology, medicine, & natural product chemistry 6 (1), 2017: 9-12 caudal fin is rounded shaped. mouth of pomfret has a terminal type, whereas in tilapia fish is sub-terminal and tapered shaped. pomfret body is slim, with the dorsal and ventral body is strong and equally convex, whereas tilapia body is flat-shaped toward the vertical (compressed). scales on both fish also have differences. black pomfret has small size, tight scales covering the dorsal and ventral parts of the body, whereas tilapia has large, rough and well-scales (kottelat et al., 1993). figure 1. morphology of black pomfret (formio niger). (a). dorsal; (b). caudal; (c). anal; (d). pelvic; (e). pectoral. figure 2. morphology of nile tilapia (oreochromis niloticus). (a). dorsal; (b). caudal; (c). anal; (d). pelvic; (e). pectoral. kidney anatomy of adult black pomfret (formio niger) and nile tilapia (oreochromis niloticus) kidney of the pomfret has a longitudinal shape along the abdominal cavity (figure 3). similar to lamprey, marine kidney generally elongated along the underside of vertebrae (spine) and slightly lobuled (karl, 1962; ferguson, 1989 in damayanti, 2010). in pomfret and tilapia fish the kidney and reproductive organ are located adjacent to the top of the celoem, and are divided into two specific system aisles (kent, 1987). figure 3. topography of black pomfret (formio niger) kidney. (x). kidney, (a). original topography of pomfret kidney, (b). sketch topography of pomfret kidney. figure 4. topography of tilapia (oreochromis niloticus) kidney. (x). kidney, (a). original topography tilapia kidney, (b). sketch topography of tilapia kidney. apriliani & luthfi – comparative anatomy and histology of black pomfret (formio niger) … 11 the result of the research showed that kidney black pomfret and nile tilapia have a similarity to kidney of perciformes fish in general, which is y-shaped form. fish kidneys generally have different shapes to each class. type of kidney and pomfret's configuration kidney is included in the same type of yellow-tail fish, namely y-shape. figure 5. anatomy of adult black pomfret (formio niger) kidney, elongated shape and dark red colour. (a). head kidney (anterior); (b). body kidney (transition); (c). tail kidney (posterior). figure 6. anatomy of adult nile tilapia (oreochromis niloticus) kidney, elongated and bright red colour. (a). head kidney (anterior); (b). body kidney (transition); (c). tail kidney (posterior). black pomfret kidney has a deep red color and has a soft kidney texture (karl, 1962). meanwhile, the tilapia kidney has a slightly softer texture than the pomfret's kidney, and has bright red-colored, compared to darker pomfret kidney. the differences in color and texture of the kidney are affected by pigment, number of capillary and connective tissue. histology of adult black pomfret (formio niger) and nile tilapia (oreochromis niloticus) kidney fish kidney has histological structure like the other marine vertebrate kidney, which have glomerulus and tubules (fujaya, 2004). glomerulus plays an important role in the process of excretion. the magnitude of the diameter of the glomerulus and the density of the tubules is related to filtration and filter blood. based on the results of the study, there is a difference between the glomerular diameter of pomfret and tilapia fish. the histology of pomfret and tilapia kidney is presented in figure 7 and figure 8. figure 7. histology of kidney of black pomfret (formio niger). (a). distal tubule; (b). proximal tubule; (c). lymphoid tissue; (d). glomerulus. he staining. (40x10 magnification). figure 8. histology of kidney of nile tilapia (oreochromis niloticus). (a). distal tubule; (b). glomerulus; (c). proximal tubule. he staining. (40x10 magnification). based on histological observations on serial preparations, adult black pomfret (formio niger) kidney has smaller glomerular diameter (61.25 μm) compared to adult tilapia (69.22 μm). this finding is consistent with marshall & smith (1930), which stated that fish living in water with low salinity content having a larger glomerular size than fish living with water with high salinity. karl (1962), also stated that freshwater fish kidney have more glomeruli than fish. based on the result it can be concluded that there is similar kidney anatomy yet different size of glomerular diameter in pomfret and tilapia. this is an adaptive character due to different environmental factors. regarding to the way of osmoregulation process, different habitats greatly affect the structure and function of the kidney (margaret, 1957; karl, 1962). 12 biology, medicine, & natural product chemistry 6 (1), 2017: 9-12 conclusion based on the result of research, we drew conclusion as follows: 1. pomfret and tilapia have the same anatomical kidney structure, consisting of head, body and tail of the kidney. but the tail/posterior kidney pomfret is longer than the body part (transition). the location of the same kidney that is attached to ventral part of vertebrae, but different in the direction of the posterior part of the kidney. the color and texture of the kidneys are different. relatively softer on tilapia than pomfret. the kidneys of tilapia fish have a bright red-red color, while the pomfret's kidney has dark red color. in terms of morphometry, the weight and length of the kidney are relatively larger in tilapia. 2. pomfret and tilapia have the same histological structure of the kidney, which consists of distal tubules, proximal tubules, and glomeruli and lymphoid tissue, but different in glomerular diameter. references bond, c.e. 1979. biology of fishes. philadelphia: w. b. saunders company. damayanti, f. n. 2010. pengaruh pencemaran logam berat terhadap kondisi 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york: wiley-interscience. suyanto. 1994. nila. jakarta: penebar swadaya. taufik, i., s. koesoemadinata, sutrisno, & nugroho. 2002. potensi akumulasi insektisida klorpiricosetil dalam jaringan tubuh ikan nila (oreochromis niloticus). jurnal penelitian perikanan indonesia, 8 (3): 37–44. comparative anatomy and histology of black pomfret (formio niger) 1and nile tilapia (oreochromis niloticus) kidney cover jurnal biomenaprochy vol 11 num 2 2022.cdr biology, medicine, & natural product chemistry volume 11 – number 2 – october 2022 issn 2089-6514 (paper) | issn 2540-9328 (online) contents the antiviral potential of iranian herbal pharmacopoeia (ihp) on herpes simplex viruses (hsv): a review article shahin gavanji 89 109 phytochemical compositions and antioxidant properties of combined funtumia africana and abutilon mauritianum extract (cfae) robert ikechukwu uroko, charles nnanna chukwu, chinomso friday aaron, umezurike benedict chidozie, doris akachukwu, ndukwe onyeani atuh 111 118 detection of the atherosclerotic pcsk9 gene inhibitors through in silico method to improve targeted therapy sabarinathan sethuramalingam, revathy leena ravi, janet rani rajiah 119 131 the triterpenes of kageneckia oblonga jorge tapia-merino, lily arrue, josé san martín, orlando muñoz 133 136 ethnobotanical survey of plants used in the management of peptic ulcer diseases in wukari metropolis akpevwoghene agbatutu, francis nosakhare imade, efosa a. ogie-odia, mary oromioshemime ifedele, funmilola modinat abdulrasaq, daniel eseigbe 137 143 co-existent hypertension with diabetes mellitus exacerbates renal dysfunctions eruore amalaka obore, jerome ndudi asiwe, vincent i. iyawe, andrew e. edo 145 150 anti-oxidant, anti-inflammatory and anti-atherosclerotic activity of bioactive peptide hpaedr isolated from catla catal muscle on lps induced inflammation on 246.7raw macrophage cells and hcf induced hyperlipidemic zebrafish larvae sabarinathan sethuramalingam, revathy leena ravi, janet rani rajiah 151 160 chemical compositions and antioxidant activity of volatile oils from morinda citrifolia and beta vulgaris leaves from nigeria adesegun olusimbo onanuga, ejike onwudiegwu okpala 161 167 the weight performance stability of mice on modeling obesity-associated hyperglycemia induced by dextrose monohydrate deksa yudha syach putra, setiyo budi santoso, heni lutfiyati 169 173 inhibitory effect of mammea africana on alpha-amylase and alpha-glucosidase enzymes of rats nwakaego omonigho ebong, jude efiom okokon, jesse idakwoji 175 180 front cover editorial board table of contents guidance back cover biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 33-39 | doi: 10.14421/biomedich.2021.101.33-39 issn 2540-9328 (online) ethnobotanical study of edible plant communities on the slopes of mount merapi and merbabu, indonesia lita ayu umartani*, maizer said nahdi** department of biology, faculty of science and technology, universitas islam negeri sunan kalijaga yogyakarta jl. marsda adisucipto no.1 yogyakarta 55281, tel. + 62-274-540971, fax. + 62-274-519739, indonesia corresponding author ayulita1998@gmail.com*, maizer.nahdi@uin-suka.ac.id** manuscript received: 09 april, 2021. revision accepted: 13 july, 2021. published: 19 july, 2021. abstract ethnobotany is a study of the interaction between local people and their natural environment, especially regarding the use of plants as food and medicinal ingredients. edible plants are a daily basic need whose existence is a necessity for the people on the slopes of mount merapi and merbabu. how to use plants is transferred from generation to generation to form a culture. the research was carried out in march-may 2020 with the aim of digging local knowledge about plants used as food, including staple food, vegetables and medicines by studying the species diversity, including the benefits of plant parts, habit, how to use, process and how to obtain these species. the data were collected by using a combination of qualitative and quantitative methods with in-depth interviews through 40 respondents who were selected by purposive sampling. the results showed that the communities on the slopes of mount merapi and merbabu used 74 plant species from 37 families as food sources. the favorite family is fabaceae, followed by zingiberaceae and solanaceae. the most widely used habitus of plants were herbs (36.49%), followed by bush (28.38%), shrubs (18.92%) and trees (16.22%). plant parts that are widely used are leaves (29.73%), fruit (17.57%), tubers (10.81%), seeds (9.46%), roots, rhizomes and flowers (6.76%), shoots (5.40%), stems (2.70%) and water, skin and heartwood (1.35%). the most common ways to use it are eaten raw as vegetables (29.73%), boiled (16.22%) and drink (12.16%). how to obtain it are through own cultivation (72.97%), and buying at the market (21.62%). the highest use values were adas (foeniculum vulgare mill.) (0.25), chili pepper (capsicum annum l.) (0.20), turmeric (curcuma dosmetica loir) and water spinach (ipomoea aquatica forsk.) (0.17). the highest importance values were rice (oryza sativa l.) (5.23%), and fennel (foeniculum vulgare mill.) (4.57%). keywords: culture; ethnobotany; in-depth interviews; rice (oryza sativa l.); mount merapi; mount merbabu. introduction the existence of plants is a pleasure and a blessing given by allah swt to his creatures in interacting and managing nature. humans are given a mandate from allah, namely first, allah allows humans to take and make the best use of natural products, humans are required to be able to take lessons from natural phenomena and events that occur. humans are required to maintain and maintain environmental sustainability (zuhdi, 2012). ethnobotany is a study of the interaction between local communities and their natural environment, especially regarding the use of plants in their daily life (martin, 1998). these uses can be in the form of plants as food and medicine. the demand for food has undergone a shift from being very dependent on nature, switched to unhealthy fast food (anggraini, 2013). some people nowadays are starting to realize the importance of healthy living by trying to consume foods that have physiological effects to the body to get maximum health (astawan, 2003). based on the food law (uu) number 18 of 2012, food is anything that comes from biological sources intended for human consumption that has certain functions to the body, used for medicinal purposes and as daily food (hariyani, 2013). each region has a specific plant use system and differs from other regions according to the diversity of plants in its environment. this includes the use of food on the slopes of mount merapi and merbabu to support human life. the area is well-known as a center for producing green vegetables with a majority of the population of farmers, besides that the area also has high enough rainfall causing the soil to become fertile and the abundance of plants and has a high variance. edible plants in the area are used by the community as medicinal plants, food as well as traditional ceremonies used in everyday life. how to use plants by the community is obtained and has been carried out from generation to generation to form a culture (gunawan, 2014). advances in science and technology as well as the increase in the level of public education are very influential in the knowledge of the younger generation https://doi.org/10.14421/biomedich.2021.101.33-39 34 biology, medicine, & natural product chemistry 10 (1), 2021: 33-39 of the culture that has been formed in society. one such knowledge is the use of the diversity of edible plants, resulting in this knowledge being increasingly marginalized, even though so far little has been stated in writing. based on this, a comprehensive research is needed to answer the problems that arise, namely how the community's knowledge of the use of food plants on the slopes of mount merapi and merbabu, precisely in mliwis village, cepogo, boyolali on plants that are used as food, including use as staple food, vegetables and medicine by specifically studying the diversity of species, including plant parts, habits, their use, processing methods and how to obtain these species. materials and methods research period and location the research was carried out on the slopes of mount merapi and merbabu, mainly in mliwis village, cepogo district, boyolali regency, covering 2 hamlets, namely baksari and ngarsapura (figure 1). the reason for choosing the two locations was because these two hamlets were the centers of agricultural and food production in the boyolali area. the research began in march may 2020. mliwis is located at 110 "22" 110 "50" east longitude and 7 "36" 7 "71" ls. figure 1. research locations on the slopes of mount merapi and merbabu, in the hamlets of baksari and ngarsapura. data collection data collected by all edible plants that have been used by the people on the slopes of mount merapi and merbabu including staple food, vegetables, medicines including species, plant parts, habits and methods of use. this information was obtained with the help of 40 respondents who were selected by purposive sampling with a total of 10% of the number of households (heads of households) in the study location. collecting data on communities consisting of traditional, local, vegetable farmers, vegetable sellers, herbalist sellers and other users through in-depth interviews through open questionnaires to be analyzed descriptively, qualitatively, and quantitatively. plants found were identified using the book flora of java volume i (1963), volume ii (1965), and volume iii (1968) by backer and van der brink jr., and indonesian useful plants by heyne (1987). meanwhile, quantitative data are used for botanical data collection, including inventory and identification of plants used by the community as food plants. the benefit value data uses a modified formula from cotton (1996) and nahdi and ardyan et al., (2019), as follows: 1. use value uvs = information: uvs : total number of use values of a type s uvis : total value of type s given by the informant i is : total number of informants interviewed for the type s use value 2. important value importance value index calculation using a formula iv = x 100 % information: iv : important value ni : number of respondents using type a n : total number of respondents the profile of respondents a b c figure 2. the profile of the respondents in the studied area based on: a. education; b. gender; c. employment. respondents are considered as representatives of the people on the slopes of mount merapi and merbabu, represented by the mliwis community as users of edible plants. they were selected from various profiles of gender, educational level and occupation. the percentage of female respondents are higher than male sd 40% smp 30% sma 30% female 60% male 40% farmer groups 10% vegetable farmers 20% food plant users30% vegetable sellers 17,5% guest sellers 10% elders 12,5% information: ngarsapura baksari umartani & nahdi – ethnobotanical study of edible plant communities on … 35 respondents (60% and 40% respectively). based on the level of education, 40% of them are only elementary school (sd) graduates, 30% junior high school (smp) graduates, and 30% junior high school (sma) graduate. according to occupations include vegetable farmers (20%), vegetable sellers (17.5%), elders (12.5%), farmer groups (10%), guest sellers (10%) and food plant users (30%). meanwhile, according to gender 60% of them are women, and 40% are men (figure 2). results and discussion a b c d figure 3. the summary of information on the use of edible plants based on: a. habitus; b. the part of the plant being used; c. how to use; d. the locations where the plants being collected. the diversity of edible plants. based on the results of the research, the people on the slopes of mount merapi and merbabu use 74 species of food plants from 37 families (table 1) with details, baksari village has 64 plant species as food used by the community and ngarsapura village has 59 species from 40 respondents. the same number of species found in the two hamlets is used by the community either as daily staples, vegetables or medicine. the number of food and medicinal plant species in this study is more than the research conducted by nita (2017) in the arfak tribe, namely 29 species of edible plants and 16 species of medicinal plants. this is because the use of edible and medicinal plants by local communities is different from one region to another. some of the factors that influence it are the efficacy and availability, language, social relations, cultural history, understanding, local beliefs and beliefs (silalahi, et al., 2018). usually, food and medicinal plants that are easily available with sufficient availability will be more used by the community. however, gudro, et al., (2014) stated another reason, where the factors that influence the use of plants by the community is the purpose of their use. the food plants, most widely used by the people on the slopes of mount merapi and merbabu come from the fabaceae family, which is used as a vegetable, followed by the zingiberaceae family as a cooking and medicinal spice. meanwhile, there are fewer types of plants from other families. previous research conducted by zulharman (2015) in the sambori tribe, bima, east nusa tenggara showed the same results, namely the fabaceae family is the family most widely used by the community as food. based on the results of interviews with respondents, the main reason for the higher use of plants of the fabaceae family is that these families are easily found in gardens, yards and in markets for sale. zulharman (2015) adds, the fabaceae family is widely used by the community because it is easy to cultivate and does not really need specific soil. in addition, according to permana et al., (2011) that almost every community has unique local wisdom as an adaptation to the environment and food in the form of fruit, vegetables and medicinal. plant habitus used as food. the results showed that there were four habitus of plants that were used by the community as food ingredients, namely trees, shrubs, shrubs and herbs. herbs are the most widely used habits habitus with a percentage of 36.49% of 74 species, followed by bush (28.38%), shrubs (18.92%) and trees (16.22%). the results of research conducted by nita (2012) in the arfak tribe, manokwari regency, sukmawati, et al., (2013) in toga village, central sulawesi and nahdih, et al., (2016) in the turgo area, yogyakarta showed the same results, namely the majority of herbal habitus are the habitus most widely used by people as raw material for food and medicine with varying percentages. the reason the people of the slopes on mount merapi and merbabu use more of the herb and bush habitus as food plants compared to others, which is easy to obtain and has highly adapted to the environment so that the growth rate and development are very fast and easy to 36. 49 0 5 10 15 20 25 30 35 40 29.73 se e d fr u it le af tu b er s ro o t r h iz o m e fl o w er sh o o ts w at e r st e m sk in h ea rt 29.73 b o ile d p o u n d ed sh re d d ed fr ie d ro as te d st e am ed b u rn ed co co n u t m ilk ve ge ta b le d ru n k d ir ec tl y… 72, 97 36 biology, medicine, & natural product chemistry 10 (1), 2021: 33-39 grow in various locations besides that there is no need for proper cultivation specifically (sukamto, 2007). the part of food plants. based on the results of the study, some people use food plants using plant parts or organs for consumption, which can be grouped into twelve (12) types of organs. leaves occupied the top position with the most percentage (29.73%), followed by fruit (17.57%), tubers (10.81%), seeds (9.46%), roots, rhizomes and flowers, respectively (6.76%), shoots (5.40%), stems (2.70%) and the last position was occupied by water, skin and heart, respectively (1.35%) (figure 3.b). sukmawati, et al., (2013) in the kaili rai tribe also showed the same thing, namely that people use more leaf parts than other parts with a percentage of 47.36% of 46 species. the same thing also happened in the research by nahdi, et al., (2016) that leaves were the highest part that was used by the community with a percentage of 51.39%. based on the results of interviews, favorite leaves are used because they are easy to obtain, there are always many leaves in one plant and easy to produce, so that people use them more than other parts, beside leaves also contain various substances needed by the body. according to payung, et al., (2016) the high utilization of leaves is because the leaves are used almost every day for vegetables and spices. apart from leaves, fruit and tubers are also parts that are widely used, because both are places to store food reserves that contain carbohydrates and other substances that are beneficial to the body (savitri, 2008). the serving method of food plants. based on the way it is used, people use food plants in eleven ways: boiling, pounding, grated, fried, roasted, steamed, roasted, coconut milk, vegetable, drunk and immediately consumed. the most widely used method by the community is by using vegetables with a percentage of 29.73%. the reason for the high processing method using vegetables is because people generally consume it as a complement to rice in their daily lives, it is easy to process, besides that people are also aware of their health and the many substances contained in plants that are very beneficial for the body. according to teo (2001) vegetables are a source of vitamins, minerals and phytochemicals that contain dietary fiber which is good for health. in contrast to research conducted by efremila, et al (2015) by drinking directly is the most common way, because beside being practical, cost-effective, it can also react directly in the body both for food and internal medicine. the source of food plants. the people on the slopes of mount merapi and merbabu obtain food plants by taking plants that come from wild plants, self-cultivating products, and buying at the market with details that the most widely used methods is self-cultivation (72.97%) (figure 3.d) compared to plants illegal (5.4%) and buying in the market (21.62%). this is because the cultivation process is very simple, namely by using the empty land around the house or garden using makeshift tools. generally, land in yards and gardens is used by the community to grow vegetable plants so that it is easy to collect them without having to travel long and cheap distances. in addition, medicinal plants are usually used as spices in cooking. in contrast to zaman's research, et al (2013), the people of sumenep district, east java mostly uses wildly obtained plants because of their economic value without spending money to get them. the way to get the second most widely used food plant by the community is buying at the market (21.62%) because food plants are the main and important need for the local community, and are an easy and practical way without carrying out the planting and harvesting process which requires time quite long and usually carried out by landless communities. meanwhile, how to obtain it by taking wild plants is very little due to the lack of public knowledge of the potential of these plants. value benefits (uvs) and important value (inp). based on the research results, the food plants that had the highest use value were fennel species (foeniculum vulgare mill.) with a value of 0.25, followed by chili pepper (capsicum annum l.) with a value of 0.20, and turmeric (curcuma dosmetica loir) and water spinach (ipomoea aquatica forsk.) with a value of 0.17 (table 1). several types of food plants above, are already well known among the surrounding community because in addition to being used for daily food they also have many benefits to cure various diseases, fennel (foeniculum vulgare mill.) in addition to plants that are unique and favored by the surrounding community, are also a plant that has many medicinal benefits. according to (kurniawan, et al., 2015), fennel can treat diseases such as itching, coughing and motion sickness. the essential oil content in fennel has anethol and estragole compounds as antipyretics (newall et al., 1995). the highest importance value based on the research results was padi (oryza sativa l.) with a value of 5.23%, followed by fennel (foeniculum vulgare mill.) 4.57%, and cassava (3.27%) (table 1). the important value of these two plants is not too far apart, so it can be said that both of them are excellent plants and are typical of the people of the slopes on mount merapi and merbabu. it is said so because at the beginning of the interview with respondents almost all of them mentioned these plants. regardless of the plant's content, the community's use of plants as a source of food and medicine is only based on hereditary knowledge and personal experience without knowing its contents. people make use of plants based on knowledge obtained from their parents or elders. based on the use value (uvs) and important value (inp), it is not always the plants that have the highest use value also have the highest importance value. this is umartani & nahdi – ethnobotanical study of edible plant communities on … 37 because the use value only looks at how many benefits a species has, while the important value is seen not only in terms of benefits, but how much people use these plants, so that even if the benefits are not as many as other species, these plants trusted and idolized by the community. table 1. list of edible plants used by the community of village of baksari and ngarsapura, boyolali. family scientific name local name benefits uvs inp amaranthaceae amaranthus tricolor l. bayem cabut detoxify body toxins 0,12 1,96 anacardiaceae mangifera indica l. pelem cure anemia 0,1 1,31 apiaceae foeniculum vulgare mill. adas overcome menstrual disorders, cure fever 0,25 4,57 daucus carota l. wortel improve eye health, 0,1 2,61 apium graveolens l. ledri anti inflammatory 0,07 1,31 araceae colocasia esculenta l. tales overcoming heart disease 0,07 0,65 xanthosoma sagittifolium l. kimpul overcoming digestive problems, 0,07 0,65 amorphophallus paeoniifolius suweg anti-poison, treat wounds 0,07 2,61 arecaceae cocos nucifera l. kelopo neutralizing body toxins 0,1 1,96 asteraceae tagetes arecta l. ningkir treat ulcer disease 0,1 0,01 lactuca sativa l. selada maintain eye health 0,07 0,01 bombaceae durio zibethinus l. duren fight infection (antibacterial) 0,05 1,96 brassicaceae brassica rapa l. sawi bakso lose weight 0,1 0,65 brassica oleracea l. kobis increase immunity 0,07 0,01 brassica olearacea l. brokoli improve the health of bones and teeth, 0,12 0,65 nasturtium officinale l. cenil prevent constipation 0,12 0,65 cactaceae hylocereus undatus haw. buah naga blood circulation 0,1 0,01 cannaceae canna discolor l. ganyong relieves heartburn symptoms 0,07 0,01 caricaceae carica papayal. kates digestion 0,12 1,96 chenopodiaceae beta vulgaris l. bit lower blood pressure 0,07 2,61 convolvulaceae ipomoea batatas l. telo source of carbohydrates, lose weight, 0,1 0,01 ipomoea aquatica forsk. kangkung against liver damage, 0,17 0,01 cucurbitaceae sechium edule l. jepan mineral source of the body 0,1 0,01 cucurbita moshcata durch. pumpkin improve the immune system, 0,07 0,01 cucumis sativus l. timun antioxidants, smooth digestion 0,07 0,01 momordica charantia l. pare relieves asthma, fights cancer cells, 0,1 0,65 cucumis melo l. melon prevent skin aging 0,07 0,65 dendrocalamaeae dendrocalamus asper l. boung lowering the risk of stroke 0,07 0,65 dioscoreaceae dioscorea alata l. uwi lowering blood sugar and cholesterol levels 0,05 0,65 dioscorea esculenta l. mbili prevent diabetes, 0,07 0,65 ebenaceae diospyros kaki l. tledung stop bleeding 0,07 1,96 euphorbiaceae manihot utillisima pohl. pohung relieves pain 0,07 3,27 euphorbia heterophylla l. patik mas reduces swelling due to snake bites 0,07 1,96 fabaceae clitoria ternatea l. telang contains antioxidants, heal wounds 0,1 0,01 glycine max l. dele lower blood pressure 0,07 0,01 arachis hypogea l. kacang brol prevent cancer 0,07 0,65 vigna cylindrica l. kacang panjang increase immunity 0,1 0,65 sesbania grandiflora l. turi treat coughs, dysentery 0,1 0,01 phaseolus vulgaris l. buncis maintain heart health 0,07 0,01 pisum sativum l. kapri free radical scavenger 0,07 0,65 leucaena leucocephala metir eradicate worms, prevent diabetes 0,07 0,65 gnetaceae gnetum gnemon l. melinjo smooth urine, 0,1 0,65 graminaceae zea mays l. jagung source of carbohydrates (daily staple food) 0,02 0,01 lamiaceae ocimum sanctum l. kemangi prevent fever 0,07 0,65 liliacea allium cepa l. brambang prevent colds, 0,1 0,65 allium sativum l. bawang putih natural antibiotics 0,12 0,65 38 biology, medicine, & natural product chemistry 10 (1), 2021: 33-39 family scientific name local name benefits uvs inp aloe vera l. lidah buaya speed up wound healing, 0,07 0,01 malvaceae abelmoschus esculentus l. okra prevent kidney disorders 0,07 0,01 marantaceae maranta arundinacea l. garut overcoming digestive diseases 0,02 0,65 moraceae artocarpus altilis park. sukun treat gout 0,07 0,65 artocarpus heterophyllus lam. gori cure insomnia 0,1 0,65 musaceae musa paradisiaca l. gedang kepok accelerating the digestive system 0,07 0,65 myrtaceae psidium guajava l. jambu klutuk tackle dengue fever 0,1 0,01 syzygium aromaticum l cengkeh treating stomach ulcers, 0,1 0,01 pandanaceae pandanus amaryllifolius roxb. pandan strengthens nerves 0,12 1,96 phyllanthaceae sauropus androgynus l. katu smooth out milk production 0,1 1,96 phyllanthus niruri l. meniran cure liver disease 0,07 0,01 piperaceae piper betle l. suroh antiseptic 0,12 0,01 poaceae oryza sativa l. pari source of carbohydrates 0,02 5,23 cymbopogon citratus l. sere antioxidants, control, regulate muscle function 0,07 0,65 portulaceae portula oleracea l. krokot prevent kidney stones, treat rheumatism 0,07 0,01 rosaceae rosa l. mawar improves immunity 0,07 0,01 fragaria anansa l. stoberi maintain heart health, prevent allergies 0,07 1,96 rutaceae citrus hystrix d.c jeruk purut clean the blood, increase endurance 0,07 1,96 solanaceae solanum lycopersicum l. tomat maintain eye health, treat acne 0,12 0,65 capsicum annum l. lombok streamlining bowel movements, treating thrush, 0,2 1,96 solanum melongena l. terong overcoming stomach acid 0,15 2,61 solanum tuberosum l. kentang maintain heart health, antioxidants, 0,15 0,01 capsicum annum l. paprika prevent anemia, control blood sugar 0,07 0,01 zingiberaceae curcuma dosmetica loir kunir overcoming menstrual problems 0,17 0,65 kaempferia galanga l. kencur treating coughs, treating diarrhea 0,15 0,01 zingiber officinale rosc jahe strengthens immunity, warms the body 0,12 0,01 curcuma zanthorrhiza l. temu lawak pain reliever, increases milk production 0,1 0,65 alpinia galaga l. laos cure asthma 0,07 0,65 conclusion based on the results and discussion, it can be concluded that the people on the slopes of mount merapi and merbabu use 74 species of plants from 37 families. the most commonly used plants are dominated by the fabaceae, zingiberacee and solanaceae families. the most widely used plants organs were leaves (29.73%), fruit (17.57%), and tubers (10.81). based on its use, it has the highest value (29.73%), followed by direct consumption (10.81%) and boiled (16.22%). the method of obtaining food plants by cultivation was in the highest ranking (72.97%), followed by buying in the market (21.62%) and wild plants (5.4%). plants that have a high use value (uvs) of fennel with a value of 0.25, chili pepper 0.20 and turmeric 0, 17. food plants that are excellent with the highest index of importance value are rice 5.23%, fennel 4.57% and cassava 3.27%. all the knowledge they have is knowledge acquired from generation to generation. acknowledgments: the results of this study involved various parties, many thanks to the people on the slopes of merapi merbabu, to be precise, mliwis village, especially the 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(2015). etnobotany and food plant community sambori district bima regency west nusa tenggara indonesia. natural b, journal of health and environmental sciences 3(2): 198-204. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 27-32 | doi: 10.14421/biomedich.2020.91.27-32 issn 2540-9328 (online) the potential of chrysin of oroxylum indicum l. to induce carbonic anhydrase (ca) to improve cattle fertility mohamad amin1,*, muhammad najib fahmi2, muhammad andi ali ridho2, nurul fitri2, umie lestari1, dina maulina3, ihya fakhrurizal amin4 1department of biology, faculty of mathematics and sciences, universitas negeri malang, jl. semarang 5, malang, indonesia 2postgraduate program of biological education, universitas negeri malang, jl. semarang 5, malang, indonesia 3biology education, faculty of teacher training and education, universitas lampung, jl. prof. dr. soemantri brojonegoro no. 1, lampung, indonesia 4faculty of medicine, university of indonesia, universitas indonesia, jl. salemba raya no.4, central jakarta city, jakarta, indonesia corresponding author* mohamad.amin.fmipa@um.ac.id manuscript received: 11 april, 2020. revision accepted: 15 april, 2020. published: 16 april, 2020. abstract the artificial insemination (ai) aims to develop the potential of the cattle reproduction comprehensively. the success of ai results is highly dependent on the level of livestock fertility. therefore, improv the quality and quantity of semen could be done by hormone induction. however, hormone prices are expensive. this problem could be overcome using alternative way with the natural compound containing chrysin which might has the same function. the purpose of the study was analyze the potential of chrysin compound from bungli plant (oroxylum indicum) as fertilizer of males through bioinformatics approach. methods performed through data search by webserver with the following stages: (1) pubchem, (2) swiss (3) target prediction, (4) uniprot, (5) protein data bank and (6) docking software using pyrx, pymol and discovery studio. the results showed that the chrysin compound interact with carbonic anhydrase (ca) expressed from the testes of the cattle, through van der waals and pi-alkyl bonds. chrysin is effective as ca ligand inducers based on affinity bonds (-8.4 kcal/mole) and more negatively than flavonol as a control compound with binding affinity (-6.2 kcal/mol). this suggests that chrysin is an effective compound as a potential drug for improved livestock fertility. keywords: bioinformatic; carbonic anhydrase; chrysin; fertility. bungli plant (oroxylum indicum l.). introduction indonesia has a wealth of natural resources that can be optimized to support the productivity of livestock, including herbs that it mixed in the form of traditional herbal medicine (motaleb et al., 2010; salim & munadi, 2017). herbal plants can be processed into livestock herbs and are useful for increasing the cattle fertility and can reduce the use of hormones (balittro, 2015; affandhy et al., 2017). increasing the productivity of beef cattle through artificial insemination (ai) is one of the achievement programs of beef self-sufficiency (direktorat pangan dan pertanian, 2010). the success of the artificial insemination program (ai) is highly dependent on the fertility of the cattle (foote et al., 1999). the quality and quantity improvement of semen is conducted through hormones function (walker et al, 2005; afriani, et al., 2014). one of the active compounds that play a role to increase the fertility of cattle is chrysin, a herb plant compound found in bungli plant (oroxylum indicum l.). oroxylum indicum l. (lamiales: bignoniaceae) is native to india that used as an ingredient in the manufacture of medicines (deka et al., 2013). this plant is found in primary and secondary forests or in open areas (djarwaningsih, 1992). the distribution of the plant is covering at philippines, indochina, siam, india and indonesia (java, sumatra, borneo, sulawesi, nusa tenggara and maluku). it has many benefits for health, all part of this plant contain useful compounds that can be used for traditional medicine (deb, et al., 2016). the previous research showed that this plant is not toxic when consumed by human and animal even in high doses (lawania et al., 2010). research about the bungli plant based on in vivo and in vitro tests revealed that bungli has benefit as antimicrobial (islam, 2010), antifree radical (kalaivani et al., 2009), antiulcer (khandar et al., 2006), genotoxic (tepsuwan et al., 1992), anti cancer (roy et al., 2007), antioxidants (upaganlawar et al., 2007; tenpe et al., 2009), and many other functions that have yet proven. this plant contains flavonoids and oils almost in all its parts (sankara et al., 1972). flavonoids consist of a large group of polyphenolic compounds that have benzo-γ-piron structures and are ubiquitous in plants. they are synthesized by phenylpropanoid pathways. available reports tend to show that phenolic secondary metabolites including flavonoids are responsible for https://doi.org/10.14421/biomedich.2020.91.27-32 28 biology, medicine, & natural product chemistry 9 (1), 2020: 27-32 various pharmacological activities (mahomoodally et al., 2005; pandey, 2007). chrysin and baicalein are the most commonly found flavonoid compounds in bungli plant (yan et al., 2011). the leaf part contains baicalein, 6 and 7-glucoronide, chrysin, anthraquinone and aloe-emodin. the bark contains flavones of oroxylin, chrysin, baicalein, and biochanin-a. while at the root of this plant contains chrysin, skutelarin-7-rutinsococic, weak acid, alkaloids, cytosterol, galactose, baicalein, and biochanin-a (lawania et al., 2010). chrysin, in the chemical name called 5.7 dihydroxyflavone, is a natural polyphenol compound found in bungli plant, honey and several other plants (morissette, et al., 2017). chrysin is a flavone-derived compound that has biological activity, has various pharmacological effects including anti-inflammatory, anti-cancer, and antioxidant (manoharan et al., 2012). chrysin is a compound of the flavonoid group found in honey, propolis, and plant extracts, which is a powerful antioxidant that has been widely studied because of its mechanism of action to prevent the conversion of testosterone hormones into estrogen through inhibition of the cyp19 enzyme (ommati et al., 2013; akhlaghi et al., 2014). the benefits of these compounds are commonly used by bodybuilding athletes as a testosterone enhancer, but also known as antineoplastic (neuman et al., 2002), anti-inflammatory (eid et al., 2006; rengaraj et al., 20015), anti hypertension (min et al., 2016), antiaging (mantawy et al., 2014). in addition, it can protect against the effects of oxidative and inflammatory injury (mantawy, et al., 2014) along with steroidogenic activity (jana et al., 2008) and antiaromatase (campbell et al., 1993). based on these reasoning, chrysin is an attractive candidate to evaluate its effect on reproduction. several studies using animal models reported that chrysin improved sperm parameters (jana et al., 2008; campbell et al., 1993), raising testosterone level, and preventing oxidative damage caused by exposure to toxic chemicals (dhawan et al., 2002; ciftci et al., 2012). ca (carbonic anhydrase) is a zinc metalloenzyme class that catalyzes reversible hydrogen carbon dioxide. ca has many different physiological functions and seven different isozymes have been characterized in mammals (hewett-emmett et al., 2000). ca is abundant in spermatozoa (parkkila et al., 1991) and its presence may be associated with maintenance of adequate intraspermatozoal bicarbonate concentration required to control sperm motility and acrosome reactions (tajima et al., 1987). the presence of ca in male reproductive organs was first shown about 50 years ago. mawson & fisher (1952) showed that biochemically approach the homogenate dorsolateral mouse prostate contained a large amount of ca activity. many researchers then confirm these findings (fisher et al., 1955; pincus & bialy 1963; leiter 1964; mcintosh 1969). biochemical studies have also shown that human and mice seminal vesicles contain the activity of ca (miyake & pincus, 1959; maren 1967). ca activity in rats' prostate and seminal vesicles was found to be regulated by androgens. the benefits of chrysin compounds in the bangli plants that could increase male fertility by binding to carbonic anhydrase (ca) protein had tested in silico. this study aims to investigated the chrysin compound from bungli as a male cattle fertility drug based on in silico screening. methods retrieval of sample this research used chrysin as spesific compound from oroxylum indicum l. the substance is the most abundant compound in oroxylum indicum l (manoharan et al., 2012; morissette, litim, & paolo, 2017) and it has been reported to have antioxidant activity. therefore, chrysin was selected as a specific sample for analysis. in addition, the compositions of chrysin were obtained from the compounds database of pubchem. ligand preparation the chemical structure of 3d and smiles ligands (chrysin) is taken from pubchem compound database (https://pubchem.ncbi.nlm.nih.gov/) with id number: 5281607. target selection input chrysin’s smiles on pharmmapper (http://lilab.ecust.edu.cn) to identify potential target candidates using mapping approaches swiss target predictions (http: //www.swisstargetprediction.c/) and chemical structure associations with molecular 3d. molecular docking process the molecular reverse docking was used to observe the interactions of chrysin with a target protein. it was identified using: collection of 3d natural compounds structure, predictions of target protein, receptor profiling, and clarification of the potential of chrysin compounds based on mode of action using pyrx 0.8 software. the collection of natural compounds structure from pubchem was used to identify the biological activity from small molecule. the pubchem was confirmed by three databases that are connected with ncbi’s information systems, including pubchem substance, pubchem compound, and pubchem bioassay. therefore, 3d structure of the chrysin would be obtained. predictions of target protein was identified using pharmmapper, superpred and swiss target prediction software. they were used to identify the bioactive amin et al. – the potential of chrysin of oroxylum indicum l. to induce … 29 molecule in the animal body. it was performed in order to observe the molecular mechanism that is fundamental as phenotype and their site of biological activity. the next process, receptor profiling process was obtained using two online databases: uniprot and rscb protein data bank. the 3d structures are required to further analyze the receptor proteins. the last stage was performed using the vina wizard feature which is integrated in pyrx 0.8 software. it was minimized to obtain the most suitable conformation before the interaction with the target protein. information obtained from pathway analysis was clarified to identify the action mechanism of chrysin compounds towards the target protein concerned. the natural compound and the control inhibitor compounds (inhibitor compounds or target protein activator) were used as a ligand in this step (maulina, et al., 2018). molecular interaction and visualization finally, the molecular interaction and visualization of the docking results were analyzed using pymol and ligplus+ software. in this stage, a profiling visualization of an interaction between chrysin compounds with the target protein was conducted. all of the bound chrysin and receptor that interact directly can be observed. therefore, the new chrysin receptor was analyzed for their structure and function mechanisms in the animal body metabolism specificly in the cattle (maulina, et al., 2018; amin, et al., 2018). result and discussion reverse docking is a method used to predict the interaction activity between ligand (compound/drug) with receptors (protein/target) (kharkar et al., 2014). based on the docking of ca (carbonate anhydrase) pdb id: 1jd0) with a resolution of 1.95 å, chrysin compound was known as a ligand informing that both have binding affinities (-8.3 kcal/mol). the results of molecular visualization and molecular interaction using pymol software found that chrysin binds to target the protein (ca) in the same binding site with the control compound (flavonol) via binding for van der waals, unfavorable bump and pi-alkyl arg b: 2401, pro b: 240, hoh: b, 2617, thr b: 233, tyr b: 231, hoh b: 2572 glu b: 248 ser b: 239 as shown in figure 1. so it can be suggest that chrysin has the same function as flavonol as ligand ca for male fertility. chrysin as a natural favonoid compound, previously used to increase testosterone (brown, et al., 2001; dhawan, et al., 2002). ciftci, et al., (2011) had studied the steroidogenic effects of flavonoid compounds and found that chrysin significantly increases steroidogenesis in leydig cells primarily by increasing star gene expression. the effects of chrysin compound was confirmed by ciftci, et al., (2012) reporting where the levels of testicular antioxidant enzymes such as sod, cat and gsh-px coincide with gsh increased significantly after chrysin administration. the chrysin-treated male albino mice had more sperm count, a much higher fertility rate when mated to proestrous female mice (dhawan, et al., 2002). higher sperm count and motility along with lower percentage of sperm abnormality were also noted in their study. in addition to the antioxidant effects, both in vivo and in vitro studies have confirmed the potential of chrysin to increase testosterone levels and then male sex drive (jana et al., 2008; ciftci et al., 2012). figure 1. (a) the interaction between target protein (ca) with chrysin and flavonol shows that binding to target proteins on the same site. green (chrysin), purple (flavonol), blue (ca); (b) interaction through van der waals bond, unfavorable bump and pi-alkyl arg b: 2401, pro b: 240, hoh: b, 2617, thr b: 233, tyr b: 231, hoh b: 2572 glu b: 248 ser b: 239. the result of target selection using swiss target prediction found that chrysin interacts with ca expressed on the testes for sperm motility and male fertility. this compound is an interesting object of this research for the development of male cattle fertility drug. as a natural compound in bungli plant, this compound is predicted as an effective substance for male fertility because it is directly linked to ca. in addition, bungli has active compound of flavonoids as aphrodisiac compounds that can increase sexual arousal (uddin et al., 2003). histochemical studies have demonstrated that ca activity acts and work in the testes, the efferent ducts, epididymis, ductus deferens, seminal vesicles, and prostates in some species (waldeyer & häusler, 1959; 30 biology, medicine, & natural product chemistry 9 (1), 2020: 27-32 cohen et al., 1976; goyal et al., 1980). in the testes, ca activity has been found in sertoli and leydig cells and in interstitial tissues in rats. capillary endothelial cells and some larger vessels in the testes have been shown that it contains ca activity in many species, including humans (cohen et al., 1976; ridderstråle et al., 1985; ekstedt et al., 1991). related to this explanation, mezquita et al., (1999) reported that the expression of ca conducted during spermatogenesis in rats and the main structure of testicular transcript for ca isolated from adult and human testes. mezquita, et al. (1999) also suggest that certain transcriptional and posttranscriptional mechanisms regulate the expression of ca during mammalian spermatogenesis. harris's et al., (1984) studied the increasing of total semen and seminal volume of plasma per semen collection related to increasing of the ca activity of testicular and ductus deferens. it was showed that the processes occurring in the testes and ductus deferens are closely involved with the total ejaculatory volume. inhibition of ca activity by acetazolamide showed that it was influenced for decreasing of in semen or seminal plasma volume. this result may be regarded as further evidence of the importance of a bicarbonate buffer system in seminal production. this confirms the linear relationship found in experiments between semen production and ca activity of the testes (ridderstråle et al., 1985). in addition, sperm motility, sperm concentration and serum testosterone levels increased significantly, whereas abnormal sperm levels decreased significantly after chrysin treatment (zhandi, 2017). in conclusion, it was suggested that treatment with chrysin may affect the reproductive system positively in mice, and may be used for the treatment of male infertility (ciftci at al., 2011). findings of the relationship between the volume of semen, spermatozoa or seminal plasma and ca activity in the reproductive system show that ca is involved in some reproductive functions (harris and goto, 1984). conclusion this study proves that chrysin is a potential compound as an inducer of ca based on affinity and intermolecular binding interactions. chrysin is a potential herb for increasing fertility in male cattle, interacts with ca expressed on the testes for sperm motility and male fertility. references affandhy, l., ratnawati, d., & luthfy, m. 2017. herbal combination effect on the quality of semen and libido in ongole cattle cross-breed. trad. med. j. vol. 22(2), p 84-90 issn-p: 1410-5918 issn-e: 2406-9086 afriani, t., 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number 2, october 2025 | pages: 1285-1288 | doi: 10.14421/biomedich.2025.142.1285-1288 issn 2540-9328 (online) comparison of the antibacterial activity of lime mistletoe extract (dendrophthoe petandra (l.) miq.) against staphylococcus aureus with standard antibiotics lisa savitri*, mila putri daniati, kharisma rahmasari, syntia tanu juwita department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 01 october, 2025. revision accepted: 04 december, 2025. published: 13 december, 2025. abstract staphylococcus aureus remains a major cause of infections worldwide and is increasingly resistant to antibiotics. natural products, particularly medicinal plants, are valuable sources of alternative antibacterial agents. lime mistletoe (dendrophthoe pentandra (l.) miq.), traditionally used in indonesian medicine, contains diverse phytochemicals that may exhibit antibacterial activity. leaves of d. pentandra were collected, dried, and extracted using 96% ethanol. the crude extract was screened for phytochemicals and tested against s. aureus (atcc 25923) using disk diffusion, broth microdilution, and minimum bactericidal concentration (mbc) assays. oxacillin and vancomycin served as positive controls, while 1% dmso was used as a negative control. all tests were performed in triplicate, and data were analyzed using one-way anova with significance set at p < 0.05. phytochemical analysis revealed the presence of flavonoids, tannins, alkaloids, saponins, and terpenoids. the extract produced inhibition zones of 8.5 ± 0.3 mm, 12.7 ± 0.5 mm, and 16.3 ± 0.6 mm at 25%, 50%, and 100% concentrations, respectively. mic and mbc values were determined to be 250 µg/ml and 500 µg/ml, with an mbc/mic ratio of 2, indicating bactericidal activity. inhibition zones of the extract at its highest concentration were comparable to oxacillin, though vancomycin exhibited superior activity. ethanol extract of d. pentandra demonstrated moderate yet significant antibacterial activity against s. aureus. its bactericidal potential and phytochemical diversity support its role as a candidate for further development, especially in topical applications or as a source of active lead compounds. further research should investigate activity against resistant strains, cytotoxicity, and in vivo efficacy. keywords: dendrophthoe pentandra; lime mistletoe; staphylococcus aureus; antibacterial activity; mic; mbc. abbreviations: antimicrobial resistance (amr); american type culture collection (atcc); colony forming unit (cfu); dimethyl sulfoxide (dmso); minimum bactericidal concentration (mbc); minimum inhibitory concentration (mic); methicillin resistant staphylococcus aureus (mrsa); standard deviation (sd); and world health organization (who). introduction antimicrobial resistance (amr) remains one of the most urgent global health concerns, with multidrug-resistant bacteria continuing to challenge clinical management and public health systems worldwide. among these pathogens, staphylococcus aureus is a major grampositive bacterium responsible for a wide range of infections in both hospital and community settings. the emergence of methicillin-resistant s. aureus (mrsa) and strains resistant to multiple antibiotic classes has led to therapeutic failures, prolonged hospital stays, and increased mortality rates (morguette et al., 2023). this highlights the necessity of exploring alternative antibacterial agents. medicinal plants are increasingly recognized as potential sources of new antimicrobial compounds. secondary metabolites such as flavonoids, tannins, alkaloids, and terpenoids have demonstrated diverse mechanisms of antibacterial action, offering advantages over conventional antibiotics that often target a single cellular pathway (scientific african, 2024). recent studies emphasize the promising antibacterial activity of plant extracts against priority pathogens listed by the world health organization, including s. aureus (nourmohammadi ghezelghaye et al., 2025). dendrophthoe pentandra (l.) miq., commonly known as lime mistletoe, is a hemiparasitic plant traditionally used in indonesian herbal medicine. its ethanol extract has been reported to exhibit antibacterial activity against s. aureus and escherichia coli, with measurable inhibition zones, minimum inhibitory concentrations (mic), and minimum bactericidal concentrations (mbc) (inggrid, aditiyarini, & prakasita, 2024). in addition, phytochemical analyses of d. pentandra from central java have revealed the presence https://doi.org/10.14421/biomedich.2025.142.1285-1288 mailto:lisasavitri@unik-kediri.ac.id 1286 biology, medicine, & natural product chemistry 14 (2), 2025: 1285-1288 of bioactive metabolites with antioxidant and potential antimicrobial properties (haryono, aditiyarini, & restiani, 2024). although preliminary evidence supports the antibacterial potential of d. pentandra, critical gaps remain. few studies have directly compared its antibacterial activity with standard antibiotics used in clinical practice. furthermore, variations in extraction methods and solvent systems may influence its efficacy, highlighting the need for standardized investigations. therefore, this study aims to evaluate the antibacterial activity of ethanol extract of d. pentandra leaves against staphylococcus aureus in vitro and compare its effectiveness with selected standard antibiotics. materials and methods fresh leaves of lime mistletoe (dendrophthoe pentandra (l.) miq.) were collected from [specific location], cleaned, air-dried at room temperature, and ground into powder. the powdered material (200 g) was extracted using the maceration method with 96% ethanol in a ratio of 1:10 (w/v) for 72 hours with occasional stirring. the filtrate was filtered and concentrated using a rotary evaporator to obtain a viscous extract, which was then stored in dark containers at 4 °c until further use. preliminary phytochemical screening was performed to identify the presence of bioactive compounds such as flavonoids, alkaloids, saponins, tannins, and terpenoids. the test organism used was staphylococcus aureus (atcc 25923), which was cultured on nutrient agar and incubated at 37 °c for 24 hours. a bacterial suspension was prepared in 0.9% saline and adjusted to the turbidity equivalent of a 0.5 mcfarland standard, corresponding to approximately 1.5 × 10⁸ cfu/ml. antibacterial activity was evaluated using both the disk diffusion and broth microdilution methods. for the disk diffusion assay, mueller-hinton agar plates were inoculated with the bacterial suspension, and sterile paper discs impregnated with the ethanol extract at concentrations of 25%, 50%, and 100% were placed on the agar surface. standard antibiotic discs of oxacillin (1 µg) and vancomycin (30 µg) were used as positive controls, while discs containing 1% dmso served as negative controls. plates were incubated at 37 °c for 24 hours, and the inhibition zones were measured with a digital caliper. minimum inhibitory concentration (mic) was determined by the broth microdilution method using 96well microplates. serial twofold dilutions of the extract (ranging from 1000 to 7.8 µg/ml) were prepared in mueller-hinton broth and inoculated with s. aureus suspension (1.5 × 10⁶ cfu/ml). after incubation for 24 hours at 37 °c, the mic was recorded as the lowest concentration that showed no visible turbidity. minimum bactericidal concentration (mbc) was determined by subculturing aliquots from wells without visible growth onto mueller-hinton agar plates, followed by incubation at 37 °c for 24 hours. the mbc was defined as the lowest concentration of the extract that completely inhibited bacterial colony formation. all experiments were conducted in triplicate. data on inhibition zones, mic, and mbc values were analyzed using one-way anova, followed by tukey’s post hoc test, with statistical significance set at p < 0.05. results and discussion results phytochemical screening preliminary phytochemical tests of ethanol extract of dendrophthoe pentandra leaves (table 1) showed the presence of flavonoids, alkaloids, tannins, saponins, and terpenoids. these secondary metabolites are known to contribute to antibacterial activity. table 1. phytochemical screening of d. pentandra leaves. compound group presence (+/-) flavonoids + alkaloids + tannins + saponins + terpenoids + steroids – antibacterial activity by disk diffusion the ethanol extract of d. pentandra demonstrated concentration-dependent inhibition against staphylococcus aureus (table 2). at 25% concentration, the inhibition zone was 8.5 ± 0.5 mm, while the 100% concentration reached 16.3 ± 0.7 mm. this effect was statistically significant compared to the negative control (p < 0.05). although inhibition zones were smaller than those produced by vancomycin (20.2 ± 0.6 mm), the highest extract concentration was comparable to oxacillin (18.5 ± 0.8 mm). table 2. antibacterial activity by disk diffusion. treatment concentration inhibition zone (mm, mean ± sd) ethanol extract 25% 8.5 ± 0.5 ethanol extract 50% 12.1 ± 0.6 ethanol extract 100% 16.3 ± 0.7 oxacillin (positive ctrl) 1 µg 18.5 ± 0.8 vancomycin (positive ctrl) 30 µg 20.2 ± 0.6 dmso (negative ctrl) — 0 minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) the mic of the ethanol extract against s. aureus was 250 µg/ml, while the mbc was 500 µg/ml. these values savitri et al. – comparison of the antibacterial activity of lime mistletoe extract 1287 were higher compared to oxacillin (mic 125 µg/ml; mbc 250 µg/ml) and vancomycin (mic 62.5 µg/ml; mbc 125 µg/ml), indicating that the extract was less potent but still exhibited clear antibacterial properties. table 3. minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc). sample mic (µg/ml) mbc (µg/ml) ethanol extract 250 500 oxacillin 125 250 vancomycin 62.5 125 statistical analysis one-way anova showed a significant difference between all treatment groups (p < 0.05). post hoc tukey’s test revealed that inhibition zones produced by the ethanol extract at 100% concentration were not significantly different from oxacillin (p > 0.05), but significantly lower than vancomycin (p < 0.05). discussion this study demonstrated that ethanol extract of dendrophthoe pentandra leaves possesses antibacterial activity against staphylococcus aureus in vitro. the extract showed concentration-dependent inhibition zones (8.5–16.3 mm), with mic and mbc values of 250 µg/ml and 500 µg/ml, respectively. these results suggest that the extract has both inhibitory and bactericidal effects, although its potency is lower than that of standard antibiotics such as oxacillin and vancomycin. the antibacterial activity observed can be attributed to the presence of secondary metabolites, including flavonoids, tannins, alkaloids, saponins, and terpenoids, detected in the phytochemical screening. these classes of compounds are widely reported to interfere with bacterial growth by disrupting cell walls, altering membrane permeability, and inhibiting enzymatic activity (scientific african, 2024). flavonoids and tannins, for instance, are known to bind bacterial proteins and nucleic acids, causing metabolic disruption and growth inhibition (nourmohammadi ghezelghaye et al., 2025). comparing the mic of d. pentandra extract (250 µg/ml) with accepted interpretative criteria for crude plant extracts, the activity falls into the “moderate” category, since mic values below 100 µg/ml are generally considered strong, 100–500 µg/ml moderate, and above 500 µg/ml weak (kuete, 2010; holetz et al., 2002, cited in reviews such as scientific african, 2024). this moderate activity is consistent with previous reports on d. pentandra. inggrid, aditiyarini, and prakasita (2024) found similar inhibition of s. aureus and e. coli, and haryono, aditiyarini, and restiani (2024) confirmed the abundance of phenolic compounds that likely contribute to this activity. the mbc/mic ratio of 2 observed here indicates bactericidal activity, since a ratio ≤4 is conventionally taken as evidence of killing rather than growth suppression (pankey & sabath, 2004). this suggests potential therapeutic value, particularly for topical formulations where higher concentrations can be locally achieved. plant extracts with dual antibacterial and antioxidant properties have already been proposed as wound-healing agents (morguette et al., 2023), making d. pentandra an attractive candidate for further exploration in this context. despite these promising findings, limitations exist. the study used an atcc reference strain rather than multidrug-resistant clinical isolates, and crude extracts contain complex mixtures that complicate reproducibility. factors such as host plant species, geographic location, and extraction conditions can alter phytochemical content and antibacterial potency (scientific african, 2024). furthermore, while in vitro assays demonstrate biological activity, they do not predict pharmacokinetics, bioavailability, or toxicity in vivo. safety assessments, cytotoxicity assays, and in vivo infection models are essential before any therapeutic application can be considered. future research should focus on bioassay-guided fractionation to identify active compounds, testing against resistant strains including mrsa, and synergy studies with conventional antibiotics. such approaches may reveal whether d. pentandra can serve as a complementary antibacterial agent or as a source of novel lead molecules. in conclusion, ethanol extract of d. pentandra leaves shows moderate but significant antibacterial activity against s. aureus, comparable to oxacillin at its highest tested concentration. these findings support its traditional use in indonesian medicine and highlight the plant’s potential for further development as an antibacterial candidate. conclusions the ethanol extract of lime mistletoe (dendrophthoe pentandra (l.) miq.) demonstrated moderate antibacterial activity against staphylococcus aureus, with mic and mbc values of 250 µg/ml and 500 µg/ml, respectively. the mbc/mic ratio indicates bactericidal potential, supporting the traditional use of this plant in indonesian medicine. while its activity was lower compared to standard antibiotics, the extract contains multiple bioactive phytochemicals that may contribute synergistically to its antibacterial effect. these findings suggest that d. pentandra could serve as a promising source for antibacterial agents, particularly for topical applications or as a lead for further bioassay-guided fractionation. future work should focus on isolating active compounds, testing against resistant clinical isolates, evaluating cytotoxicity, and exploring in vivo efficacy to assess its potential for therapeutic development. 1288 biology, medicine, & natural product chemistry 14 (2), 2025: 1285-1288 acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors wrote the manuscript and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there are no funding. references a comprehensive review on medicinal plant extracts as antibacterial agents: factors, mechanism insights and future prospects. (2024). scientific african, 26, e02395. https://doi.org/10.1016/j.sciaf.2024.e02395 haryono, s. e., aditiyarini, d., & restiani, r. (2024). analysis of secondary metabolites and antioxidant activities of ethanol extract of dendrophthoe pentandra (l.) miq. in sapuran, central java. biogenesis: jurnal ilmiah biologi, 12(1), 56–65. https://doi.org/10.24252/bio.v12i1.40323 inggrid, a. m., aditiyarini, d., & prakasita, v. c. (2024). antibacterial activity of dendrophthoe pentandra (l.) leaves extract against staphylococcus aureus and escherichia coli. jurnal biodjati, 9(2), 280–293. https://doi.org/10.15575/biodjati.v9i2.30436 morguette, a. e. b., bartolomeu-gonçalves, g., andriani, g. m., bertoncini, g. e. s., castro, i. m. de, spoladori, l. f. de a., ... & yamauchi, l. m. (2023). the antibacterial and wound healing properties of natural products: a review on plant species with therapeutic potential against staphylococcus aureus wound infections. plants, 12(11), 2147. https://doi.org/10.3390/plants12112147 nourmohammadi ghezelghaye, p., alibegli, m., fazelnejad, a., jafari, s., gholami, m., & rahmani, f. (2025). antibacterial activity of tannin-free ethanolic extracts from medicinal plants against methicillin-resistant staphylococcus aureus. bmc complementary medicine and therapies, 25, 271. https://doi.org/10.1186/s12906-025-05019-1 pankey, g. a., & sabath, l. d. (2004). clinical relevance of bacteriostatic versus bactericidal mechanisms of action in the treatment of gram-positive bacterial infections. clinical infectious diseases, 38(6), 864–870. https://doi.org/10.1086/381972 https://doi.org/10.1016/j.sciaf.2024.e02395 https://doi.org/10.24252/bio.v12i1.40323 https://doi.org/10.15575/biodjati.v9i2.30436 https://doi.org/10.3390/plants12112147 https://doi.org/10.1186/s12906-025-05019-1 https://doi.org/10.1086/381972 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 129-133 | doi: 10.14421/biomedich.2021.102.129-133 issn 2540-9328 (online) antibacterial screening of bacterial isolates associated with mangrove soil from the ngurah rai mangrove forest bali anak agung gede indraningrat1, made dharmesti wijaya2*, putu arya suryanditha1, ayu savitri siskayani3, ni made defy janurianti4 1microbiology and parasitology department; 2pharmacology and pharmacy department; 3laboratory of biomedical science faculty of medicine and health sciences, 4faculty of agriculture, warmadewa university jl. terompong no 24 denpasar 80235, tel. +62 361 240727, indonesia. corresponding author* dharmestiwijaya@gmail.com manuscript received: 20 october 2021. revision accepted: 30 october, 2021. published: 02 november, 2021. abstract in this study we reported cultivation of bacteria associated with mangrove soil from the ngurah rai mangrove forest, bali. mangrove soil samples were serially diluted using sterile artificial seawater, spread onto starch casein m agar and incubated at 28oc for 28 days. cultivation of mangrove soil samples yielded 165 bacterial colonies with 68 isolates were selected and purified based on different morphology. of these 68 isolates, 22 isolates displayed antibacterial activities ranging from weak to strong inhibition against at least one of four bacterial indicators namely staphyloccocus aureus, streptococus mutans, escherichia coli and klebsiella pneumoniae using perpendicular streak method. overall, 19 out of 22 bacteria isolates displayed weak antibacterial potential and two isolates exhibited moderate antibacterial activity. the isolate sa4 was the only bacterium with strong antibacterial potential with measured clear distance ≥ 10 mm against the four bacterial isolates. sequence analysis based on 16s rrna gene fragment assigned the isolate sa4 as bacillus subtilis strain bil/bs-168. overall, this study confirmed the untapped potential of antibacterial activities from bacteria associated with mangrove soil. keywords: mangrove soil; bacteria; antibacterial screening. introduction bacterial resistance against antibiotic drugs is currently an emerging global health threat that require immediate actions (world health organization, 2018; centers for disease control and prevention, 2018). antibiotic resistance arises because bacteria develop immune mechanism by mutation, horizontal gene transfer and enzyme inactivation (munita and arias, 2016). as a rough estimation, resistance against antibiotics will reach 10 millions cases in 2050 and is predicted to result in financial loss of 100 trillions usd due to increased cost for hospitalization and loss of productivity. to date, overused and misused antibiotics are the main factors of the increasing rate of antibiotic resistance (ventola, 2015). one of the efforts to overcome the increasing rate of resistance of pathogenic bacteria is to discover new sources of novel antibiotics with much stronger efficacy compared to current drugs that are available on the market (roca et al., 2015; cheng et al., 2016). exploration of bacteria with a potent antibacterial activity has been mainly focused on terrestrial ecosystem (elbendary et al., 2018; singh et al., 2016; mohamed et al., 2017; assis et al., 2014). however, a number of studies indicated the highest de-replication of active compounds that have been previously reported. (debbab et al., 2010). therefore, exploration of marine habitat should also be given more priority as bacteria in this habitat is rather unexplored yet yield (debbab et al., 2010). mangrove forest is characterized with extreme salinity, high-low tide, wind pressure , muddy and low oxygen concentratio (friess, 2016). therefore, microbe in this habitat tend to be able to adapt on harsh environmental conditions (booth, 2018). the group of actinobacteria in mangrove forest synthesize various of secondary metabolites to survive with this extreme condition (van der heul et al., 2018; bentley et al., 2002). bioactive compounds from mangrove origins have been shown to display therapeutics potential and could be developed as new drugs including antibiotics. a number of studies have isolated bacteria from mangrove and these isolates could inhibit wide range of pathogenic bacteria (azman et al., 2015; lee et al., 2014; jiang et al., 2018). the ngurah rai mangrove forest is the biggest mangrove ecosystem in bali with over 19 mangrove plants species inhabited the area and dominated by four main species namely: rhizophora mucronata, avicennia https://doi.org/10.14421/biomedich.2021.102.129-133 130 biology, medicine, & natural product chemistry 10 (2), 2021: 129-133 marina, rhizophora apiculata dan sonneratia alba (balai pemantapan kawasan hutan wilayah viii denpasar, 2018). to date diversity of bacteria in the ngurah rai mangrove forest is rather unexplored, therefore this research aims to isolate and pre-screen bacterial isolates with antibacterial activities. materials and methods mangrove soil sampling mangrove soil samples were collected from the ngurah rai mangrove forest (8o43’40.4886” s, 115o11’42.80313” e), in august 2019 during the low tide. four sediment samples were taken from habitat of the four dominant mangrove plants rhizophora mucronata, avicennia marina, rhizophora apiculata and sonneratia alba. from each sampling point, approximately 100-gram sediment was taken from 10 cm depth using a clean masonry trowel and stored individually in a sterile 50 ml falcon tube (figure 1). soil samples were stored in 4oc until further used. figure 1. soil samples collected from the ngurah rai mangrove forest. cultivation of bacterial isolates from mangrove soils ten grams of each mangrove soil samples were pretreated with wet heating in a water bath at 60oc for 15 minutes by combining sterile artificial seawater and distilled water (1:1 v/v) (azman et al., 2015; lee et al., 2014; jiang et al., 2018). subsequently, 1 ml of each of soil sample suspension was serially diluted (10-1 to 10-3) in 9 ml sterile artificial seawater. one hundred µl of each diluted soil sample was spread using sterile cotton swab (onemedia) onto starch m-protein agar (63 gram/l, himedia, india) which was supplemented with 100 μg/ml nalidixic acid and 25 μg/ml nystatin. agar plates were sealed with parafilm and incubated at 28oc for 28 days. observation of colonies grew on agar was performed every two days and the total colonies observed on agar media were counted. bacterial colonies with different morphologies (colour, form, elevation) were picked up from each agar plate and these colonies purified individually by streaked onto isp-2 agar media (4.0 gram/l yeast extract, 10 gram/l malt extract, 4 gram/l dextrose, 20 gram/l bacto agar). each of the purified bacterial isolate was stained using gram staining dan observed under microscope to identify their morphological form. antibacterial prescreening antibacterial activities of each purified isolates were pre-screened using perpendicular streak method (boontanom and chantasari, 2020) with a slight modification against four bacterial indicator strains which represented gram-negative bacteria (escherichia coli dan klebsiella pneumoniae) and gram-positive bacteria (staphylococcus aureus dan streptococcus mutans). in brief, each of pure bacterial indicator strains was streaked as a five cm vertical line and incubated for 48 hours at 37oc until fully grown. bacterial indicator strains were streaked five cm perpendicular from the original line of the bacterial isolate (figure 2). antibacterial potential was determined based on the distance formed between an isolate and a bacterial indicator according to four categories: no activity (a bacterial indicator show no distance with an isolate), weak (1 – 4 mm), moderate (5 – 9 mm) and strong ≥ 10 mm. figure 2. schematic overview of perpendicular streak for antibacterial pre-screening of bacterial isolates from mangrove soil. genetic analysis of bacterial isolates with strong antibacterial activities bacterial isolates with a strong antibacterial activity were genetically identified using polymerase chain reactions followed by sanger sequencing. the genomic dna of selected bacterial isolates was extracted using bacteria dna preparation kit jena bioscience (jena, germany) by following the manufacturer instruction. genomic dna of each the selected isolates were amplified by targeting 16s rrna gene fragment using primer pairs 27f: 5’-agagtttgatcmtggctcag3’ and 1492r 5’-ggttacsttgttacgactt indraningrat et al. – antibacterial activity of mangrove soil bacteria 131 3’(lane, 1991). the 50 µl pcr master mix contained 25 µl my taq hs red mix 2 x, 1 µl (20 µm) of each forward and reverse primer, 22 µl of sterile dna/rna free water and 1 µl of genomic dna. the pcr cycles consisted of 95°c for 5 minutes pre-denaturation, 30 cycles consisted of 95oc for 1 min denaturation, 55oc for 1 minutes annealing, 72oc for 1.5 min extension, and a final extension for 7 minutes. amplified pcr product was analysed using 1% agarose supplemented with sybr safe by gel electrophoresis for 45 minutes (80 volt/200 watt) and visualised using uv light in a gel documentation machine. subsequently, pcr products were sent to pt genetika science (https://ptgenetika.com/) for sanger sequencing. the obtained sequence data were subjected to ncbi blast database (https://blast.ncbi.nlm.nih.gov/blast.cgi) to assign the closely identity of related bacterial sequence. results and discussion bacterial isolates with antibacterial activities a total of 68 bacterial isolates were selected over 165 bacterial colonies observed on agar plates after 28 days of incubation based on morphological observations. of these 68 isolates, only 22 isolates showed potential antibacterial activities against at least one bacterial isolate using perpendicular streak method. these bacterial isolates were grouped as gram positive cell wall with rod morphology (table 1). table 1. list of isolates that displayed inhibition against at least one bacterial indicator strains with their morphological characteristic. an initial code indicated the dominant mangrove plant species where soil sample was collected namely sa (sonneratia alba), am (avicenna marina), ra (rhizopora apiculata), rm (rhizopora mucronata). isolate morphology gram staining distance against bacterial indicator strains (mm) e. coli k. pneumoniae s. aureus s. mutans sa1 rod positive no activity no activity no activity 7 sa3 rod positive 2 2 2 2 sa4 rod positive 15 10 20 15 sa9 rod positive no activity no activity 5 no activity sa11 rod positive no activity no activity no activity 3 sa14 rod positive no activity no activity 5 no activity am1 rod positive no activity 4 no activity no activity am3 rod positive 2 2 2 3 am4 rod positive no activity no activity 2 no activity am8 rod positive no activity 2 no activity no activity am9 rod positive no activity no activity no activity 3 am12 rod positive no activity 4 no activity 4 am13 rod positive no activity 4 no activity no activity am20 rod positive no activity 3 3 2 am23 rod positive no activity 4 3 5 am30 rod positive no activity 3 3 2 ra1 rod positive 2 1 5 no activity rm2 rod positive no activity no activity 5 no activity rm3 rod positive no activity 5 no activity no activity rm9 rod positive no activity no activity no activity 5 rm10 rod positive 6 no activity 8 7 rm18 rod positive 4 5 3 5 the level of antibacterial inhibition varied among the 22 bacterial isolates. in general majority of isolates weakly inhibited at least one indicator bacterium. two isolates (sa1 and rm10) displayed moderate antibacterial activity. sa4, however, is the only bacterial isolates with the strongest antibacterial potential against all the four bacterial indicator (figure 3). perpendicular streak was selected as the prescreening method because the approach is rather straight forward and has been applied effectively to determine isolates with antibacterial activities (boontanom and chantasari, 2020; balouiri et al., 2016). it could be assumed that isolate sa4 could potentially synthesis active antibacterial molecules based on substantial distance formed between the isolate and bacterial indicators. antibacterial substances produced by an active bacterial isolate were diffused on agar media so that growth of other bacteria was inhibited (balouiri et al., 2016). nucleotide sequences comparison of isolate sa4 against ncbi blast database indicated that the top hit was assigned as bacillus subtilis strain bil/bs-168 with 97,36% identity. bacillus subtilis in general has been regarded for its ability to produce antimicrobial compounds and has been applied for food preservation and crop protection (caulier et al., 2019). for example, bacteriocin is one example of an active compound 132 biology, medicine, & natural product chemistry 10 (2), 2021: 129-133 responsible for antimicrobial activities in b. subtilis (sharma et al., 2018). figure 3. perpendicular streak of isolate sa4 against gram positive and negative bacteria. a clear distance of ≥10 mm between each bacterial indicator and the isolate sa4 indicated strong antibacterial properties synthesized by the bacteria. however, at this stage it is still unknown what type of antibacterial substances that could be produced by the sa4 isolate. further research, therefore should be focused to identify type of antibacterial compounds synthesized by the sa4 isolate. in addition, genetic analysis should also be done to analyze metabolic pathways that are responsible to synthesize antibacterial compounds in the sa4 isolate. despite, the remaining 21 bacterial isolates only showed weak to moderate antibacterial activities based on perpendicular streak do not automatically exclude the full potential of these isolates. it could be that each isolate requires different time to accumulate their antibacterial substances. therefore, a liquid fermentation followed by organic extraction of these isolates using different solvents depending on polarity of the expected compounds should be done to fully unravel the true antibacterial potential or other therapeutic capability. conclusion in conclusion, this study obtained 22 bacterial isolates with antibacterial potential from mangrove soil of the ngurah rai mangrove forest. all of these isolates were characterized by rod shape and gram-positive cell wall under microscope observation. isolate sa4 displayed the strongest antibacterial potential based on the perpendicular streak method with clear distance zone above 10 mm. isolate sa4 was closely related to bacillus subtilis strain bil/bs-168with 97.36% sequence identity based on 16s rrna gene sequences. further studies should be focused to extrapolate the antibacterial potential of these 22 isolates especially the isolate sa4 by performing organic extractions and screening against different bacterial pathogen. apart from antibacterial activities, other therapeutic potential of the obtained isolates should also be explored such as antifungal, antioxidant, or anticancer in order to fully unravel the biological capabilities of these isolates. acknowledgements: this research was fully funded by the research and community development unit (unit penelitian dan pengabdian masyarakat) faculty of medicine and health sciences, warmadewa university under grant no. 417/unwar/fkik/up2m/kp02/vii/2019. we would also thank ir. magdalena hehakaya, m.si from the department environment and forestry bali province for her guidance during the sampling trip at the ngurah rai mangrove forest. conflicts of interest: the authors declare that there are no conflicts of interest. references assis, d. a., rezende, r. p., & dias, j. c. (2014). use of metagenomics and isolation of actinobacteria in brazil's atlantic rainforest soil for antimicrobial prospecting. isrn biotechnol, 2014, 909601. doi:10.1155/2014/909601 azman, a. s., othman, i., velu, s. s., chan, k. g., & lee, l. h. (2015). mangrove rare actinobacteria: taxonomy, natural compound, and discovery of bioactivity. front microbiol, 6, 856. doi:10.3389/fmicb.2015.00856 balai pemantapan kawasan hutan wilayah viii denpasar. (2018). potensi wisata taman hutan raya ngurah rai provinsi bali. in. http://bpkh8.menlhk.go.id/pdf/karya_tulis_mandiri/liflet_tahu ra.pdf. balouiri, m., sadiki, m., & ibnsouda, s. k. (2016). methods for in vitro evaluating antimicrobial activity: a review. journal of pharmaceutical analysis, 6(2), 71-79. doi:https://doi.org/10.1016/j.jpha.2015.11.005 bentley, s. d., chater, k. f., cerdeno-tarraga, a. m., challis, g. l., thomson, n. r., james, k. d., . . . hopwood, d. a. 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(2018). antibiotic resistance. retrieved from geneva: https://www.who.int/news-room/factsheets/detail/antibiotic-resistance this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 291-295 | doi: 10.14421/biomedich.2024.131.291-295 issn 2540-9328 (online) identification of fingerprint pattern and lip print pattern in females of type 2 diabetes mellitus as a biomarker harem othman smail, renas hama ahmad, eman ibrahim jalal department of biology, faculty of science and health, koya university, koya koy45, kurdistan region-f.r. iraq. corresponding author* harem.othman@koyauniversity.org abstract diabetes mellitus (d.m.) is a chronic metabolic disorder that has increased worldwide. fingerprints and lip prints are noninvasive procedures that are genetically used in criminal cases and to determine genetic disorders such as diabetes mellitus type 2. dermatoglyphics is the epidermal ridge configuration, the ridged skin on our fingertips, palms, and toes. there are three types (whorl, et al.). cheiloscopy, derived from the greek word (cheilos), which means lip, is the study of crinkles and grooves that are perceptible on our lips and unique. we conducted our research with 90 females, 45 were with (dmt2), and 45 were normal females over 30 years old. the samples were collected in different kurdistan regions (erbil city, qaladza town, koya town, and koya university). we collected all 10 (ten) fingerprint patterns and divided them into whorl, loop, and arch using an ink pad. also, lip samples were collected by using lipstick on a4 paper. according to our research, the predominant fingerprints and lip prints in females with type 2 diabetes were loops for both hands (right (31.1%) and left (28.4%)), and for lips, the print was intersected grooves (28.9%). the predominant fingerprint and lip print for normal females were also loops in both hands (right (28%) and left (25%)), and the lip print was complete vertical grooves (42.2%). the study showed no association between fingerprint pattern and diabetes mellitus type 2 and cannot be used as a biomarker for it. however, there is a clear association between lip print pattern and diabetes mellitus type 2, and it can be used as a specific biomarker. keywords: fingerprints; lip prints; type 2 diabetes mellitus; henry's method of classification; type 2 diabetes prediction. introduction the distinctive individuality of fingerprints, called epidermal ridges, has been recognized for over 2000 years. for about 200 years, they have been the subject of scientific inquiry (kücken, 2004). human fingertips' skin has ridges and troughs that form recognizable patterns. pregnancy allows for the full development of these patterns, which remain stable throughout life. fingerprints are copies of specific patterns. cuts, burns, and bruises can temporarily impair fingerprint quality, but after complete healing, patterns will return (murmu, 2009; smail et al.,2019). the classification used by the fbi is based on the well-known "henry's classification" (jain et al.,1999). a loop, whorl, and an arch were created from each of the ten fingerprint patterns (smail et al.,2020; mouneshkumar et al., 2021). lip prints are standard lines and fissures that appear as wrinkles and grooves where the inner labial mucosa and outer skin of the human lip meet. like fingerprints, the appearance of lip prints differs from person to person (adamu et al.,2013). two japanese scientists named y. tsuchihashi and t. suzuki noted in 1950 that the arrangement of furrows on the lip is distinct and they put out a classification system for it that is still in use today: type i: vertically oriented, well-defined grooves across the mouth; according to debta et al. (2018), type i' grooves are straight but stop midway rather than extending the full width of the lip. type ii grooves split along their path. type iii grooves cross. type iv grooves are reticular. type v grooves do not fit into any of type i to iv and cannot be distinguished morphologically. type i' grooves are straight but only extend halfway across the lip; type ii grooves kink; type iii grooves intersect; type iv grooves are reticular; and type v grooves do not correspond to any of type i through iv and cannot be distinguished morphologically (debta et al.,2018). global epidemic diabetes mellitus and impaired glucose tolerance have been attained (saeedi et al.,2019). there is a clear correlation, according to numerous earlier research carried out around the globe. the study's objective is to determine how fingerprint and lip print patterns relate to type 2 diabetes and to show whether finger and lip patterns can be used as indicators for patients with diabetes. methodology cases ninety women over 30 were divided into two groups for the sample study: 45 patients previously diagnosed with manuscript received: 23 march, 2024. revision accepted: 20 august, 2024. published: 21 august, 2024. https://doi.org/10.14421/biomedich.2024.131.291-295 mailto:harem.othman@koyauniversity.org 292 biology, medicine, & natural product chemistry 13 (1), 2024: 291-295 type 2 diabetes mellitus and 45 normal individuals. samples were randomly selected from koya university, erbil city, qaladiza town, and koya town for each section. most of the patients with t2d underwent the hba1c test, and the results were positive for some of them. this disease was genetically passed down to them from their parents, and all the diabetes cases had a level above 7.5%. a random blood sugar test was conducted, and the result was very high, ranging from 200-500 mg/dl. the study's paper was divided into three sections labeled "right, left, and lip-type," five columns were created for each hand's thumb, index, middle, ring, and little fingers. likewise, the lip print patterns were documented. fingerprint procedure the patient's hands were cleaned and dried before printing. an ink pad was applied to the fingertips with a thin layer of blue ink. on an a4 sheet, five fingertips' imprints were recorded. however, the same actions were repeated. the ink was first removed with gauze pieces and hand rubbing, then with soap and water once all fingerprints had been collected. the fingerprints were examined under a magnifying glass. henry's method of classification, which categorizes fingertip patterns into loops, whorls, and arches, was used to study the fingertip patterns (sathawane et al.,2019) lip print procedure each person was instructed to wash their lips with water and let them dry gently. after applying the dark lipstick evenly in one stroke, each person was taught to spread it the dark lipstick evenly using gentle lip motions after applying it evenly in one stroke. the lips were relaxed, and the adhesive side of the cellophane tape was then uniformly applied (negi, 2016). statistical analyses the chi-square test was applied to examine the relationship between fingerprints and obesity for both the right and left hands of females (diabetes and control groups), and the lip print patterns. statistical analyses were performed using spss software version 20, and pvalues < 0.05 were considered statistically significant. results and discussion table 1. general distributions of primary fingerprint patterns in all fingers of both hands. fingerprint patterns total percentages % loop 508 56.4 whorl 328 36.4 arch 64 7.1 table 2. distribution of fingerprint pattern among type2 diabetes and control of female subjects in right hands. type of fingerprints type 2 diabetes control chi-square value p value loop 140(31.1%) 126(28%) 1.80 0.40 whorl 72(16%) 84(18.6%) arch 13(2.8%) 15(3.3%) total 225(100%) 225(100%) significant = (p≤0.05), using chi-square test. table 3. distribution of fingerprint pattern among type2 diabetes and control of female subjects in left hands. type of fingerprints type 2 diabetes control chi-square value p value loop 128(28.4%) 114(25%) 4.13 0.12 whorl 76(1.68%) 96(21%) arch 21(4.6%) 15(3.3%) total 225(100%) 225(100%) significant = (p≤0.05), using chi-square test table 4. distribution of fingerprint pattern in different fingers among type2 diabetes and control of female subjects in right hands. individual figures t2d control t2d control t2d control loop loop whorl whorl arch arch thumb 26(57.7%) 28(62.2%) 18(40%) 16(35.5%) 1(2.2%) 1(2.2%) index 22(48.8%) 20(44.4%) 17(37.7) 18(40%) 6(13.3%) 7(15.5%) middle 33(73.3%) 27(60%) 10(22.2%) 15(33.3%) 2(4.4%) 3(6.6%) ring 24(53.3%) 16(35.5%) 19(42.2%) 27(60%) 2(4.4%) 2(4.4%) little 35(77.7%) 35(77.7%) 8(17.7%) 8(17.7%) 2(4.4%) 2(4.4%) total 140(31.1%) 126(28%) 72(16%) 84(18.6%) 13(2.8%) 15(3.3%) chi square 1.63 1.62 0.13 p value 0.80 0.80 0.99 significant = (p≤0.05), using chi-square test smail et al. – identification of fingerprint pattern and lip print pattern … 293 figure 1. lip pattern types. figure 2. type of fingerprint patterns. there have been lip prints since the beginning of human history. unlike fingerprints, lips also include furrows that fall into multiple groups for identification purposes. however, compared to fingerprints, palm prints, and fingerprints, lip prints are less common because they lack distinctive features used for identification (reddy et al.,2013). these lip prints and fingerprints are currently used as markers for predicting type 2 diabetes (sathawane et al.,2019). this study examined the relationship between lip print and fingerprint analysis with type 2 diabetes mellitus, with 90 294 biology, medicine, & natural product chemistry 13 (1), 2024: 291-295 randomly selected samples taken from erbil, qaladiza, and koya city. in the control group, the most common fingerprint type was loop (56.4%), followed by whorl (36.4%) and arch (7.1%) (table 1). when considering only the right and left hands of females, the most common fingerprint type in the right hand of control cases was loop (28%), followed by whorl (18.6%) and arch (3.3%) (table 3:2). in diabetes cases, the percentages changed to loop (31.1%), whorl (16%), and arch (2.8%) (table 2). on the left hand, the percentage of fingerprint types for control cases was loop (2.5%), whorl (21%), and arch (3.3%), while for diabetes cases it was loop (28.4%), whorl (1.68), and arch (4.6%) (table 3). when analyzing the right-hand patterns of patients and controls, it was found that while the percentage of whorl and arch patterns was higher in the normal group, the percentage of loop patterns was higher in the diabetic group. on the left hand, the whorl pattern was more common and the loop and arch patterns had higher percentages among diabetes participants. however, our investigation found that these results were not statistically significant. in their study, umana et al. (2013) discovered that loop and whorl were the main patterns in healthy people in their investigation of the nigerian t2dm community. in contrast, people with diabetes showed a predominance of the arch type. however, the results lacked statistical significance (umana et al., 2013) in the current study, male individuals displayed branching, straight, intersected, reticular, and undifferentiated patterns, with decreasing frequency. in contrast, female subjects displayed branched, straight, intersected, and reticular patterns, but no undifferentiated patterns. both participants with diabetes and those with hypertension shared the same findings. in control cases, the highest percentage according to our study was complete vertical grooves (42.2%), followed by branched grooves (31.1%) (table 6). we did not gather any data in control cases for the partial vertical groove and undifferentiated groove types (saeedi et al., 2019). the prevalent trends in the controls in both types of investigation were intersecting grooves (11.1%) and reticular grooves (15.6%). in diabetes cases, the data were as follows: the highest percentage was intersected grooves (28.9%), followed by branched grooves (22.8%), undifferentiated grooves (17.8), completed vertical grooves (15.6%), reticular grooves (11.1%), and partial vertical grooves (4.4%). table 6 shows that the intersected type of lip print was significantly higher in individuals with diabetes (28.9%) compared to the control group (11.1%). these results suggest that individuals with intersecting lip patterns have an increased risk of type 2 diabetes. on the other hand, individuals with finished vertical grooves and branched patterns are less likely to develop the disease (manjusha et al., 2017). according to our study, these results were statistically significant. conclusion there was no association found between fingerprints patterns and type 2 diabetes mellitus. thus, we cannot utilize them as a biomarker. however, the findings were different. we discovered a distinct relationship between lip print patterns and type 2 diabetes mellitus. therefore, lip prints can be a biomarker for detecting type 2 diabetes mellitus. competing interests: the authors declare that there are no competing interests. references adamu, l. h., taura, m. g., hamman, w. o., ojo, s. a., dahiru, a. u., sadeeq, a. a., & umar, k. b. (2013). relationship of thumb prints and lip prints among nigerians. iosr-jdms, 9(2), 12-7. debta, f. m., debta, p., bhuyan, r., swain, s. k., sahu, m. c., & siddhartha, s. (2018). heritability and correlation of lip print, palm print, fingerprint pattern and blood group in twin population. journal of oral and maxillofacial pathology, 22(3), 451.available from: /pmc/articles/pmc6306610/ jain, a. k., prabhakar, s., & hong, l. (1999). a multichannel approach to fingerprint classification. ieee transactions on pattern analysis and machine intelligence, 21(4), 348359.doi: 10.1109/34.761265 kücken, m., & newell, a. c. (2004). a model for fingerprint formation. europhysics letters, 68(1), 141..https://iopscience.iop.org/article/10.1209/epl/i200410161-2 manjusha, p., sudha, s., shameena, p. m., chandni, r., varma, s., & pandiar, d. 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(2020). relationship between pattern of fingerprints and obesity. journal of experimental and molecular biology, 21(1), 27-33.available from: http://www.jemb.bio.uaic.ro/index.php/jemb/article/view/47 umana, u. e. (2013). dermatoglyphic and cheiloscopic patterns among diabetic patients: a study in ahmadu bello university teaching hospital zaria, nigeria. j biol life sci, 4(2), 206214.https://www.macrothink.org/journal/index.php/jbls/article/ view/3399 https://doi.org/10.24941/ijcr.34371.02.2019 https://journals.sospublication.co.in/ab/article/view/262 http://www.jemb.bio.uaic.ro/index.php/jemb/article/view/47 https://www.macrothink.org/journal/index.php/jbls/article/view/3399 https://www.macrothink.org/journal/index.php/jbls/article/view/3399 this page intentionally left blank cover jurnal biomenaprochy vol 13 num 1 2024.cdr volume 13 number 1 april 2024 issn 2089-6514 (paper) issn 2540-9328 (online) biology, medicine, & natural product chemistry volume 13 – number 1 – april 2024 issn 2089-6514 (paper) | issn 2540-9328 (online) contents isolation and characterisation of essential oils of mango (m. indica) leave abubakar auwal, mohammed hassan shagal, charles milam 1 6 the neuroprotective and therapeutic effects of medicinal plants and natural products against aluminium chloride-induced alzheimer’s disease: recent update ayodeji oluwatobi ojetunde 7 33 potential of methane (ch4), nitrogen (n2), and carbon dioxide (co2) from eco-enzyme with the addition of cow feces starter marlina kamelia, aulia ulmillah, suci wulan pawhestri 35 42 therapeutic azithromycin mitigated monosodium glutamate-related dysfunction in rats’ body weight and serum, liver, kidney and heart antioxidant defense bioindicators anthony cemaluk chinedum egbuonu, prince ogochukwu alaebo, udumma nsofor onuoha, chinomso juliet njoku, obioma benedeth eze, francisca ugochi odoemelam, michael eberechukwu edum, ojichukwu boniface obi, mmesoma joy ukaegbu, sandra uchechi nwaogwugwu, marvellous chinonso orji, clara ngozika ndukwe, prosper opara, chinagorom oyoyo, chika blessing joe-eme, cheluchiaka jecinta okwoigwe 43 50 antibacterial potential of freshener water based on siwalan coir extract (borassus flabellifer) and lemongrass (cymbopogon citratus) against airborne bacteria hana dwi andayani, salma auliya yoviska, widyastuti asy syaffa, putri amifalahiya iqlima, sriwulan 51 55 effectiveness analysis of antidiabetic property from dragon fruit peel methanol extract in alloxan-induced diabetic rats sonia apriani sumartin, i nyoman ehrich lister, edy fachrial 57 62 effects of processing on the proximate composition, mineral content and the phytochemical analysis of groundnut seeds (arachis hypogeae) joseph adaviruku sanni, grace omayoza sanni, rufus ranmilowo awoniyi, remi osanyinlusi, yvonne ego richards, goodness inioluwa adesina, ore-ofe oluwatoyin adenuga, stella ayomikun apata, oluwafemi emmanuel ekun 63 71 ethnobotanical study of medicinal plants in south sumatera, indonesia angga puja asiandu, widya sari 73 82 analysis of heavy metal cadmium (cd) and lead (pb) in eggplant (solanum melongena l.) in pontianak city by using atomic absorption spectrophotometry (aas) fajar nugraha, syila malinda oktaviani, sri luliana 83 90 assessment of the phytochemical constituents of methanol extract of ereromastax polysperma leaves and its effect on the hematological indices in albino rats victor hogan idung, grace asukwo okon, nicholas asiwe 91 98 physicochemical, antimicrobial, lethality and in vitro antioxidant profiles of johnu tisane: a coffee (coffea arabica) leaf decoction formula anthony cemaluk chinedum egbuonu, prince ogochukwu alaebo, udumma nsofor onuoha, chinomso juliet njoku, chiemeziem adanma obike, susan ogechi nlemadim, blessing ifeoma chukwu, sopuruchi mary-augusta iwejuo, onyedikachi glory amaechi, victor ifechukwu obiefuna, bessing nmesomachi onuoha, divinetreasure nzubechukwu okoli, precious chinonso nwokeoma, chukwubuikem eze 99 107 comparative effects of ripe and unripe lime (citrus aurantifolia) on spermatozoa and gonadosomatic index in matured male wistar rats obioma ogbonnaya, azubuike nwankwo, daniel chidi ifenkwe, chikera samuel ibe, ekele ikpegbu, osah martins onwuka 109 119 formulation and antibacterial activity testing of eco-soap based on sodium lauryl sulfate surfactant nurhaida widiani, aulia novitasari, ovi prasetya winandari, dwijowati asih saputri 121 125 evaluation and antibacterial activity test of peel-off mask preparation from combination of pegagan leaves (centella asiatica (l) urb.) and charcoal powder fadhilla dwi utari, najmi hilaliyati, tika afriani 127 133 isolation of novel 6-methylideneoxecane-3,4,5,7,8,9-hexol from the leaves of rauwolfia vomitoria, apocynaceae azibanasamesa d.c owaba, raji o. rafiu, arueniobebh frank, darlington d. eboh, samuel j. bunu 135 139 phytochemical profile and antibacterial activity of nigella sativa against biofilm-producing bacteria uropathogens abdulazeez mumsiri abaka, mubarak muhammad dahiru, keta biman abubakar, jordana luka, aisha abubakar, tukur baba abdullahi, saminu hamman barau 141 146 phenolic, flavonoid contents, antioxidant, and antibacterial activity of selected eucalyptus species: review getaneh worku moges, gizachew mulugeta manahelohe, melese ababay asegie 147 157 the diversity of understory (shrubs and herbs) in the kalikuning area hikmah supriyati, nur aeni ariyanti 159 163 physical characteristics of immobilized cells acetobacter xylinum of various concentrations of na-alginate lathifa indraningtyas, maria erna kustyawati, nafiatul fitriah 165 172 antibacterial bioactivity test of bilimbi fruit ethanol extract (averrhoa bilimbi linn). against propionibacterium acnes, staphylococcus epidermidis and staphylococcus aureus reza anindita, dede dwi nathalia, melania perwitasari, intan kurnia putri, maya uzia beandrade, nofria rizki amalia harahap 173 182 shrimp digestive bacteria performance for shrimp waste chitin extraction kristina novalina nainggolan, yudha perdana putra, evi fitriyani 183 191 isolation of β-cyclodextrin gycosyl transferase (β-cgtase) producing bacteria from potato plantation soil helman kurniadi, desi sagita, barmi hartesi 193 197 the comparison of long-term effect between intermittent fasting and calorie restriction on neurological parameters of mice neli syahida ni’ma, kusnandar anggadiredja 199 204 antimicrobial potential of selected phytochemicals from hygrophila schulli; computational insights pasindu dilshan perera, haritha lawan 205 214 levels of oxidative stress in rats treated with calorie restrictive treatment and 50% sucrose solution diniwati mukhtar, azha azzuna amsaka, fanny ratnasari pd, aan royhan, karina ajeng ridwan 215 220 anticancer potential of ethanolic extract artocarpus heterophyllus lam. leaves against human colon cancer widr cell line novita mutiyani, ardaning nuriliani 221 226 the protective effect of kentut leaf extract (paederia foetida l.) on gastric histopathology in escherichia coli-infected sepsis mice model lisa savitri, elfred rinaldo kasimo 227 233 the study of biosurfactant stability and the effect on lipase activity mieke alvionita, rukman hertadi, rahmat rizki fazli, andi alfira ratna faradisa dewi, tasyang oktavia rose 235 238 commercial incense: compound analysis and its molecular docking studies as anxiolytic agents cintya nurul apsari, navista sri octa ujiantari, zuliyati rohmah, setyowati triastuti utami, artania adnin tri suma, niar gusnaniar 239 250 the effect of use of local organic microorganism fertilizers on rice media, banana sticks, and tongol fish on the growth of arabika coffee plants (coffea arabica l.) in the gunung karang garden, pandeglang district suyamto, m. hujaji, hadi susilo, swastika oktavia, mu’jijah 251 258 the effect of probiotic starter culture variation on the quality of yogurt based on indonesian national standard hariyanto ih, bella cristin, siti nani nurbaeti 259 264 potential of soursop leaf extract as an antioxidant in mcf-7 cells muhammad samsul mustofa, restu syamsul hadi, nunung ainur rahmah, pandrianto 265 269 antiepileptic potential of justicia insularis leaf extract in swiss mice nancy elkanah, michael chuks nnumolu, jude effiom okokon, uduak peter ise 271 275 testing antibacterial activity of daun kentut (paederia foetida l.) leaf extract against escherichia coli in vitro lisa savitri, eka wahyuning tiyas, rochmad krissanjaya 277 283 β-glucan comparison in the mushrooms of medicinal fungal species dicky kurniawan tontowiputro, djanggan sargowo, askandar tjokroprawiro, muhaimin rifa'i 285 289 identification of fingerprint pattern and lip print pattern in females of type 2 diabetes mellitus as a biomarker harem othman smail, renas hama ahmad, eman ibrahim jalal 291 295 exploring allelochemical compositions in the roots of two varieties of anacardium occidentale (cashew) nnenna ejije okoronkwo, jude chibuzor igwe, nkemakolam bright nwosu, precious onyinyechi chukwu, udoka ifunnaya chukwukere, perculiar c. ihedigbo 297 303 in-ovo antiviral activity of hibiscus sabdariffa against newcastle disease virus abdulazeez mumsiri abaka, james doughari hamuel, tukur baba abdullahi, keta biman abubakar 305 310 potential effects of myrmecodia pendants (ant nests) on healing bone injuries adilla syahsiyatun najah, akram la kilo, ahmad kadir 311 319 this page intentionally left blank 3: front 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1121-1127 | doi: 10.14421/biomedich.2025.142.1121-1127 issn 2540-9328 (online) soybean plant growth and production response (glycine max (l.) merril) to the application of liquid organic fertilizer (poc) apu – apu (pistia stratiotes l.) plant and various soil reformers on ultisol soil media ellen lumisar panggabean*, azwana departmens of agrotechnology, faculty of agriculture, university of medan area, medan, indonesia. corresponding author* ellengabe19@gmail.com manuscript received: 25 november, 2025. revision accepted: 28 november 2025, 2025. published: 30 november, 2025. abstract this study aimed to evaluate the growth and yield responses of soybean (glycine max l. merr.) to the application of liquid organic fertilizer (lof) derived from apu-apu (pistia stratiotes l.) and various soil conditioners on ultisol soil. the research was carried out from september to december 2020 at the experimental garden of the faculty of agriculture, universitas medan area, located at an altitude of 22 masl with flat topography, ultisol soil characteristics, and a soil ph of 4.94. the experimens used a randomized block design with two factors: (1) concensrations of apu-apu lof (0%, 25%, 50%, and 75% per liter of water) and (2) types of soil conditioners (no conditioner, rice-husk biochar, mucuna bracteata green manure, and human-hair organic amendmens at 50 g per polybag). observed parameters included plans height, number of branches, flowering age, number of bunches per plans and per plot, number of pods per plans and per plot, seed weight per plans and per plot, and 100-seed dry weight. the results showed that the application of lof from apu-apu significansly increased several agronomic and yield componenss, namely number of branches at 6 wap, number of bunches per plans and per plot, number of pods per plans and per plot, seed weight per plans and per plot, and 100-seed dry weight. meanwhile, the application of differens soil conditioners produced highly significans effects on plans height at 4–6 wap, number of pods per plant and per plot, seed weight per plans and per plot, and 100-seed dry weight, although it did not significansly affect the number of branches or number of bunches. overall, the combination of lof and soil conditioners improved soybean growth and productivity in ultisol soil conditions. keywords: poc apu-apu; various soil reformers; soybeans. introduction in indonesia, soybeans are the most important food commodities after rice and corn, these commodities are used for household food consumption, industry, animal feed and seeds. in 2017 soybean production was 538.72 thousand tons and increased in 2018 to 982.59 thousand tons for domestic food needs against soybeans as much as 3.07 million tons, of which 95% were for the needs of the food sector and 160 thousand tons for animal feed, so that indonesia experienced a deficit in soybean production. with the deficit period of production to soybean needs, the rest is imported from abroad as much as 2.5 million tons (central statistics agency, 2019; sinaga et al., 2024). the instability of soybean production in indonesia is caused by several factors, especially the availability of less and less agricultural land because it has changed functions and factors for the continuous use of chemical fertilizers used by farmers, causing a decrease in soil fertility and damaging the soil due to changes in soil physical properties (triyono et al, 2013; sihotang et al., 2022). however, most of the land is marginal dry land. marginal dryland is land that has a low soil fertility rate, reacts sourly with a soil ph below 5.5 and low macro n, p, k, ca and mg nutrient content as well as high solubility of al and fe which can poison plant growth and one of them is ultisol soil (director general of food crops, 2012). ultisol marginal lands that are low in nutrients will inhibit the process of plant growth and production, organic fertilizers are divided into two, namely solid and liquid organic fertilizers. one alternative organic fertilizer that can be used is liquid organic fertilizer apu – apu plant. the apu – apu plant (pistia stratiotes l.) ranks third out of ten weeds that cause potential problems in southeast asia after water hyacinth (eichornia crassipes) and kiambang (salvinia molesta). the plant https://doi.org/10.14421/biomedich.2025.142.1121-1127 1122 biology, medicine, & natural product chemistry 14 (2), 2025: 1121-1127 has a very rapid breeding, causing farmers difficulty to cope with it. a solution to reduce cultivated plants, especially rice, by utilizing the apu apu plant (pistia stratiotes l.) as liquid organic fertilizer. fiolita et al, (2013) explained that the nutrients contained from apu – apu (pistia stratiotes l.) among them there are n: 2.83%, p: 0.17%, k: 0.96%, c / n: 10 and organic matter 47, 070. in addition to the use of liquid organic fertilizers, in increasing the growth and production of soybeans can also use various soil reformers. the provision of soil reformers can improve soil quality and soil properties, both physical, chemical and biological properties. soil reformers can be used in various ways such as the use of rice husk biochar, green manure of mucuna bracteata legume plants and human hair waste. biochar is a biological charcoal from an incomplete combustion so as to provide nutrients that can improve land function. rice husk ash serves to loosen the soil so that it can make it easier for plant roots to absorb nutrients in it (pane et al, 2014; sitepu and sihotang, 2025). the application of green manure mucuna bracteata as organic matter will improve soil properties both physical, chemical, and biological soil. the legume type green fertilizer used is lcc mucuna bracteata has a relatively high nitrogen nutrient content compared to other types of plants. mucuna bracteata as an organic matter contains nitrogen (n) 3.71%, phosphorus (p) 0.38 %, potassium (k) 2.92%, calcium (ca) 2.02%, magnesium (mg) 0.36%, c-organic 31.4% and c / n 8.46% (simamora and salundik, 2006). however, research on the use of organic waste such as human hair as a soil reformer for agricultural crops has not been done by many people. research conducted zheljazkov et al, (2005) showed that human hair waste cannot be used as a single source of nutrition for plants. this is because human hair takes longer to release its nutrients. human hair can form a pore space of the soil for a long time, the presence of a pore chamber allows the exchange of co2, n2, and nh3 gases with o2 from the atmosphere, human hair contains n, p, k and cu but not necessarily can become available nutrients, such conditions are very supportive of the activity of autotrophic microbes that play a role in the provision of unsure nutrients. in addition, it can also support root development and plant growth (ignatova et al, 1999 in zheljazkov 2005). based on the above background, the author is interested in conducting research with the title "response to soybean plant growth and production (glycine max (l.) merril) to the application of liquid organic fertilizer (poc) apu-apu plants (pistia stratiotes l.) and various soil reformers on ultisol soil media. research methodology this research was carried out on the experimental land of the faculty of agriculture, medan area university. this research was conducted from september to december 2020. the ingredients used in this research are soybean seeds of the anjasmoro variety, apu apu plant (pistia stratiotes l.), em-4 (effective microorganisms), brown sugar, air, chicken manure, ultisol soil, mucuna bracteata legume plant, rice husk, human hair waste. the tools used in this study were hoes, tripe, drills, knives, crossbars/meters, tarpaulins, scales, plastic ropes, sprayers, buckets, polybags measuring 30 cm x 35 cm (5kg) and stationery. this study was conducted using a factorial randomized group design with 2 treatment factors, namely: 1. liquid organic fertilizer (poc) apu apu plant consisting of 4 levels, namely: a0 = control a1 = 25% poc/liter a2 = 50% poc/liter a3 = 75 % poc/liter 2. various soil reformers consist of 4 levels, namely: p0 = control p1 = biochar rice husks with a dose of 20 tons / ha (50 g / polybag) p2 = pupukhijau plant legume mucuna bracteata with a dose of 20 tons / ha (50 g / polybag) p3 = human hair waste at a dose of 20 tons / ha (50 g / polybag) thus obtained the number of treatment combinations as much as 4 x 4 = 16 treatment combinations. results and discussion plant height (cm) the provision of various soil reformers on ultisol soil media at the age of 2 to 3 weeks after planting is not significantly different, this is due to the nature of ultisol soil as a planting medium has many fertility problems and also has a ph (h20) soil that is classified as acidic soil so that it affects the pattern of nutrient availability needed by plants. after entering the observation of the height of plants aged 4 to 6 weeks after planting, soybean plants showed a noticeable difference in plant height, this shows that in this phase soil reformers have been able to improve the physical and chemical properties in the soil ultisol, according to the opinion of djojoprawiro (1984) who stated stating that the application of organic matter would result in flocculation in the soil, so that the process of consolidation of soil aggregates occurs. the combination of applying apu-apu liquid organic fertilizer (poc) and various soil reformers at the age of 2 to 6 weeks after planting is not significantly different as panggabean & azwana – soybean plant growth and production response … 1123 well as a single application of apu-apu poc does not differ markedly, if referring to fertilizer quality indicators according to the use of organic fertilizers in solid and liquid form besides having advantages, also has several disadvantages including low and diverse nutrient content often so that it is not sufficient for plant needs. this is in accordance with damanik et al., (2011) which states that the disadvantages of organic fertilizers have nutrients that are difficult to obtain in large quantities and are slow to be available to plants. for more details, a summary of the plant height variety fingerprints can be seen in table 1. table 1. summary of soybean plant height (cm) fingerprints against the provision of poc apu apu and various soil reformers at the age of 2 to 6 wap. sk f. calculate at age f. table 2 wap 3 wap 4 wap 5 wap 6 wap 0,05 0,01 group 0.19 ns 0.53 ns 0.62 ns 3.07 ns 1.01 ns 4,54 8,68 factor a 0.73 ns 1.23 ns 3.17 ns 3.17 ns 3.05 ns 3,29 5,42 factor p 2.44 ns 1.82 ns 3,66 * 3,79 * 4,50 * 3,29 5,42 ap treatment 0.59 ns 0.52 ns 0.43 ns 0.12 ns 0.03 ns 2,59 3,89 kk 9,55% 7,46% 6,09% 6,55% 5,06% description: ns = non significant * = singnificant number of branches (branches) the application of liquid organic fertilizer (poc) apuapu at the age of 2 and 4 weeks after planting is not significantly different, this is because the planting medium used is ultisol soil as this soil is acidic soil that has low n and c-organic nutrients. however on age 6 weeks after planting shows a noticeable difference in the number of branches, this is indicated that the nutrients contained in the apu-apu poc in this phase can be absorbed by plants. agree with salisbury and ross (1995) in puspadewi, et al., (2014) the states that the absorption of nutrients, especially nitrogen nutrients, affects the number of branches. furthermore, djuaja, et al, (2012) said that plants need elements n, p and k to stimulate the synthesis and division of cell walls anticlinally so that they can accelerate the addition of the number of leaves, plant height, and branch growth. the provision of various soil reformers at the age of 4 and 6 weeks after planting is very noticeable, this is thought to be because by giving organic matter, the nutrients needed by plants are sufficient so that they can support soybean growth. in accordance with the opinion of muhtiar et. al. (2012) which states that the application of organic matter is able to provide essential nutrients such as n, p, k and surfur, ktk, and increase the solubility of soil p so that plants absorb the nutrients available sufficiently for plants and can obtain maximum growth and yield. in addition, sevindrajuta (2012) also stated that the application of organic matter to the soil in ultisol can increase the levels of corganic, ntotal and bases, p nutrients available increase and reduce the content and saturation of soil al. for more details, a summary of the fingerprints of the variety of branches can be seen in table 2. table 2. summary of the number of soybean branches (branches against the provision of poc apu apu and various soil reformers at the age of 2 to 6 wap. sk f. calculate at age f. table 2 wap 4 wap 6 wap 0,05 0,01 group 31,96 ** 2.14 ns 0.25 ns 4,54 8,68 factor a 3.04 ns 2.10 ns 5,29 * 3,29 5,42 factor p 1.74 ns 5,51 ** 5,91 ** 3,29 5,42 ap treatment 1.30 ns 0.05 ns 0,14 2,59 3,89 kk 19,46% 12,24% 8,01% description: ns = non significant * = significant ** = very significann bundle per sample plant the application of liquid organic fertilizer (poc) apuapu is significantly different, this is because the more nutrients provided, the more plant needs can be fulfilled. according to the results of research by ralahalu et al., (2013) stated that applying a high concentration of organic fertilizer will support plant growth and vice versa if the concentration given is too low, it will suppress growth or not spur growth of both plants in the vegetative and generative phases. that the amount of bundle per plant sample plant soybean (glycine max (l.) merril) due to the provision of various soil reformers differs is very evident, this is because the provision of biochar adds nutrients to the soil, increases nutrient retention and affects the dynamics of microbes in the soil (annisa et al. 2017). according to sukristiyonubowo (1993), the provision of natural 1124 biology, medicine, & natural product chemistry 14 (2), 2025: 1121-1127 organic soil reformers is able to maintain soil organic matter levels and soil ktk, as well as increasing the ph and p available. for more details, a summary of the fingerprints the variety of bundle quantities per sample plant can be seen in table 3. table 3. summary of fingerprints of various bundle quantities per plant samples soybeans against the provision of poc apu apu and various soil reformers. sk f. count f. table 0,05 0,01 group 3.71 ns 4,54 8,68 a factor 4,78 * 3,29 5,42 factor p 8,03 ** 3,29 5,42 ap treatment 2.53 ns 2,59 3,89 kk 3,07% description: ns= non significant * = significant number of bundle per plot that the amount of bundle per plot of soybean plants (glycine max (l.) merril) due to the application of apuapu liquid organic fertilizer (poc) is significantly different, this is because the nutrients contained in the apu-apu poc in this phase have been absorbed by plants, especially the formation of pods that become soybean bundle. according to dwijosaputro (2002), plants thrive when the necessary elements are sufficiently available and located and are in the appropriate concentration to be absorbed for plants, so as to provide better results for plants. the provision of various soil reformers has a very significant effect. according to sutedjo (2008) flower formation requires sufficient p and k elements, because in the prospective fruit or pod flowers that become soybean bundle are in the formation of flowers and fruit fruits that are maximally needed sufficient p and k elements are needed. according to fahmi (2014) in the process of fertilization, nitrogen is non significantly needed, while phosphorus and potassium are one of the many nutrients needed in generative growth. for clarity, a summary of the variety of bundle numbers per plot can be seen in table 4. table 4. summary of various bundle quantities per plot soybeans against the provision of poc apu apu and various soil reformers. sk count f.table 0,05 0,01 group 3.09 ns 4,54 8,68 a factor 4,51 * 3,29 5,42 factor p 5,43 ** 3,29 5,42 ap treatment 1.44 ns 2,59 3,89 kk 2,39% description: ns = non significant, * = significant, ** = very very significant number of pods per sample plant (pods) that the number of pods per plant sample plant soybean (glycine max (l.) merril) due to the application of liquid organic fertilizer (poc) apu-apu is significantly different, it is indicated that the elements ha ra phosphorus and potassium contained in the liquid organic fertilizer (poc) apu-apu can supply nutrients to plants until soybeans are generative (pod formation). nutrients dissolved in water and sprayed on plants can be absorbed by plants through the stomata, as they allow fertilizer to enter through the stomata gap when opening (hardjowigeno, 1995; sihotang et al 2023; sihotang et al., 2025). foliar fertilization during the pod filling stage can improve the filling of soybean pods. the application of nutrients through the leaves will overcome the nutrient deficiency in the leaves as due to the retranslocation of nutrients from leaves to seeds that are being formed (garcia and hanway 1976 in hakim et al, 2004). that the number of pods per plant sampled soybean plant (glycine max (l.) merril) due to the provision of various soil reformers differed markedly. according to yaduvanshi (2003), the addition of 20 tons of green manure can substitute half the amount of inorganic fertilizer recommended in the availability of n and k element suppliers in the soil. supported hardjoloekito (2009), the element phosphorus is indispensable for the formation or growth of plant generation. sufficient phosphate element in the soil can spur the formation of pods in plants. for more details, a summary of the number of pods per sample plant can be seen in table 5. table 5. summary of fingerprints of various numbers of pods per plant soybean samples (pods) against the provision of poc apu apu and various soil reformers. sk f. calculate f. table 0.05 0.01 group 1.61 ns 4,54 8,68 factor a 3,42 * 3,29 5,42 factor p 3,76 * 3,29 5,42 ap treatment 0.41 ns 2,59 3,89 kk 5,94% description: ns = non significant * = significant number of pods per plot (pods) that the number of pods per plant sample plant soybean (glycine max (l.) merril) due to the application of liquid organic fertilizer (poc) apu-apu is significantly different. according to dwidjoseputo (1990) who explained that a plant will grow vigorously if enough elements are needed. this is also supported by hafizah (2012), that the application of liquid organic fertilizer can increasing the number of fruits, phosphorus (f) nutrients play a very important role in generative growth, so that in addition to affecting flower formation, it also affects the formation of fruits and seeds and accelerates fruit ripening. that the number of pods per plot of soybean crops (glycine max (l.) merril) due to the provision of various soil reformers differs markedly. according to hardjoloekito (2009), not sure phosphorus is indispensable for the formation or growth of plant panggabean & azwana – soybean plant growth and production response … 1125 generation. sufficient phosphate element adanaya in the soil can spur the formation of pods in plants. the number of pods affected is also influenced by plant height, this is supported by anggraeni (2010) who states that high plant height causes an even distribution of light throughout the canopy so that photosynthesis will be maximum, photosynthetic the ones that fill the pods will be more and more. with the provision of soil reformers so that it can increase the amount of p available in the soil, the fulfillment of p nutrient needs will increase metabolic activity so that organic matter translocated to soybean seeds or pods also increases. for clarity, a summary of the variety of pods per plot can be seen in table 6. table 6. summary of the number of pods per plot (pod) of soybeans against the provision of poc apu apu and various soil reformers sk f. calculate f. table 0.05 0.01 group 1.28 ns 4,54 8,68 factor a 3,47 * 3,29 5,42 factor p 4,51 * 3,29 5,42 ap treatment 0.22 ns 2,59 3,89 kk 2,08% description: ns = non significant * = significant seed weight per plant sample (g) that the seed weight per plant sample soybean plant (glycine max (l.) merril) due to the application of liquid organic fertilizer (poc) apu-apu is significantly different, this is because the elements n, p and k in the treatment of liquid organic fertilizer are absorbed by plant and used for metabolic processes in plants, especially the formation of pods and seeds. furthermore, suprapto (1992) emphasized thatsoybean plants will use p to the maximum when the plant is in the pod formation period until approximately 10 days before the seeds fully develop. that the weight of seeds per plant sample of soybean plants (glycine max (l.) merril) due to the provision of various soil reformers is significantly different, this is because the function of soil reformers should be able to increase the activity of microorganisms in the soil which is necessary to improve the physical properties of the soil, where the soil will become more friable, the soil's ability to retain water will increase, soil aeration and drainage will be better and also lead to increased availability of p elements in the soil. according to the results of research by suharjo (2001) in nihayati, et al (2018) in pod filling and seed formation are very dependent depending on the availability of n, both n taken by rhizobium bacteria from the air and n available in the soil and are also affected by the availability of p elements. if the availability of n is in a balanced state, it will result in the formation of amino acids and proteins increasing in seed formation so that the pods are fully filled. for more details, a summary of the variety fingerprints the weight of the seeds per sample plant can be seen in table 7. table 7. summary of fingerprints of variety of seed weight per plant sample (g) soybeans against giving poc apu apu and various soil reformers. sk f. calculate f. table 0.05 0.01 group 3.30 ns 4,54 8,68 factor a 3,42 * 3,29 5,42 factor p 3,84 * 3,29 5,42 ap treatment 0.10 ns 2,59 3,89 kk 10,44% description: ns = non significant, * = significant. seed weight per plot (g) that the weight of seeds per plot of soybean plants (glycine max (l.) merril) due to the application of liquid organic fertilizer (poc) apu-apu is significantly different, this application of fertilizer application through using liquid fertilizer leaves containing the main nutrients n, p and k in soybean, corn, wheat, chickpeas and peas plants can increase plant growth and production as well as seed protein content (novizan, 2002). the application of apu-apu poc can supply nutrient needs, especially phosphorus. phosphorus is indispensable for plants in seed formation. suryawaty's research (2014) the addition of poc can increase the weight productivity of dry beans. by height of weight dry seeds are positively correlated with the amount of seed production per hectare. that the seed weight per plot of soybean plants (glycine max (l.) merril) due to the provision of various soil reformers differs markedly. according to agustina (1990), if the amount of nutrients given is sufficient to meet the needs of plants, it will be able to increase the uniformity of plant growth and development. in accordance with the opinion of hidayat (2008), the increasing supply of phosphorus in the plant body will increase metabolism so that the seed filling process is optimal and the seed weight increases. for clarity, a summary of the fingerprints of the variety of grain weights per plot can be seen in table 8. table 8. summary of the results of fingerprints of various grain weights per plot (g) soybeans against the provision of poc apu apu and various soil reformers. sk f. calculate f. table 0.05 0.01 group 0.81 ns 4,54 8,68 factor a 3,48 * 3,29 5,42 factor p 4,44 * 3,29 5,42 ap treatment 0.31 ns 2,59 3,89 kk 6,20% description: ns = non significant * = significant weight of 100 dried seeds (g) that the weight of 100 dry seeds of soybean plants (glycine max (l.) merril) due to the application of liquid organic fertilizer (poc) apu-apu is significantly different, this is because the need for sufficient nutrients 1126 biology, medicine, & natural product chemistry 14 (2), 2025: 1121-1127 can improve the quality and quantity of crop production, this is supported by indrakusuma (2000), liquid organic fertilizer can improve the physical, chemical, and biological properties of the soil, so as to increase crop production, improves the quality of plant products and reduces the use of inorganic fertilizers. according to nyakpa et al. (2006) p elements can increase the high yield of crops, increase yields and accelerate the ripening period of fruits. the increased availability of nutrients, especially p elements, serves for seed formation. that the seed weight per plant sample plant soybean (glycine max (l.) merril) due to the provision of various soil reformers is significantly different, this is because soil reformers play a role in improving soil structure, maintaining soil moisture, and increasing the nutrient content in the soil, so that plant roots are easy to grow and increase the area of root absorption in the soil. the increase in seed weight is due to the translocation of photosynthetic to fruits and seeds. photosynthetes produced in leaves and other photosynthetic cells are transported to other organs or tissues in order to be utilized by these organs and tissues for growth or stockpiled as backup material (lakitan, 2002). for more details, a summary of the weight variety of 100 dried seeds can be seen in table 9. table 9. summary of fingerprints of 100 dry seeds (g) soybeans against giving poc apu apu and various soil reformers. sk f. calculate f. table 0.05 0.01 group 0.22 ns 4,54 8,68 factor a 3,78 * 3,29 5,42 factor p 3,90 * 3,29 5,42 ap treatment 0.40 ns 2,59 3,89 kk 7,61% description: ns = non significant * = significant conclusion the application of apu-apu liquid organic fertilizer (poc) differs markedly from the number of branches of plants aged 6 wap, the number of bundle per sample plant, the number of bundle per plot, the number of pods per plant, the number of pods per plot, the seed weight per sample, the weight of seeds per plot and the weight of 100 dried soybean seeds. there is no noticeable difference in the height of plants aged 2 to 6 wap, the number of branches aged 2 to 4 wap and the age of flowering. the provision of various soil reformers differs very markedly from the number of branches of plants aged 4 to 6 wap, the number of bundle per sample plant, the number of bundle per plot. the difference is markedly different from the height of plants aged 4 to 6 wap, the number of pods per sample plant, the number of pods per plot, the weight of seeds per sample plant, the weight of seeds per plot and the weight of 100 dried soybean seeds. no noticeable difference to the height of plants aged 2 to 2 to 3 wap, number of branches 2 wap and flowering age. the combination of liquid organic fertilizer (poc) treatment of apu-apu plants and various soil reformers did not differ markedly from all observation parameters. competing interests: the authors declare that there are no competing interests. references anggraeni, bw 2010. 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(0331) 483536, indonesia. corresponding author* mrofik05@gmail.com manuscript received: 18 august, 2024. revision accepted: 10 october, 2024. published: 30 october, 2024. abstract snakehead fish (channa striata) is another alternative source of albumin protein because it is known to contain important compounds for the human body including high protein, fat, water, and the mineral zinc (zn). the albumin content in snakehead fish is an important protein needed by the body and is useful for wound healing. this study aims to make preparations by modifying snakehead fish albumin into hard candy while maintaining the albumin content. thus, a more effective way to be consumed in accelerating wound healing was found. the treatment used is snakehead fish albumin extract with concentrations including f1 (5%), f2 (10%), and f3 (15%). tests were conducted physically (organoleptic test) and chemically (moisture content test, ash content test, and wound growth activity test). formulation and evaluation of hard candy preparations of snakehead fish albumin extract (channa striata) can be declared successful, as evidenced by the formation of 5 successful formulations into hard candy preparations as evidenced by the results of organoleptic tests, water content tests and ash content tests that have met the standards. keywords: snakehead fish (channa striata); hard candy; steaming; albumin; effective. introduction wound is a condition of damage to tissue continuity, structure, and anatomical function of normal skin due to pathological processes originating from the internal or external environment and affecting certain organs (sulakhiya et al., 2024). according to the indonesian ministry of health in 2018, the incidence of slash/stab wounds in indonesia was 20.1% with the highest rate in the 35-44 age group at 24.0%. in addition, the prevalence of patients with cuts/stab wounds in east java province was 18.2%. meanwhile, in jember district, the incidence of injuries due to traffic accidents in 2023 was 1381 cases, with details of 4 serious injuries and 1580 minor injuries. optimal wound care plays an important role in the wound healing process so that it can take place well and in a short time so as not to reduce productivity and increase wound care costs (sahu et al., 2024). the general treatment of wounds consists of two things, namely external treatment and internal treatment. for external treatment, the things that need to be prepared are wound bed preparation and wound closure. wound bed preparation is done through debridement, bacterial control, and wound exudate management. wound closure is done when the wound has been well incised and can be done per-secundam, per-primam, skin graft, flap, and by using stem cells (palakkara et al., 2020). as for internal treatment, antibiotics are needed to kill bacteria and speed up the wound healing process (yuniar, yasid, nissatuljannah, rijal, & makmun, 2023). antibiotics as drugs to treat infectious diseases, their use must be rational, appropriate, and safe. the irrational use of antibiotics will cause negative impacts, such as the occurrence of microorganism immunity to some antibiotics, increasing drug side effects, and even resulting in death. the negative impact of irrational use of antibiotics, such as the use of antibiotics too often, excessive use of new antibiotics, and the use of antibiotics for a long time is the emergence of microorganism resistance to various antibiotics (multidrug-resistance). this results in ineffective treatment, increased patient morbidity and mortality, and increased health costs (ministry of health, 2005) (bonine et al., 2019). thus, there is a need for internal medicine that does not include antibiotics and is safe to use such as albumin. https://doi.org/10.14421/biomedich.2024.132.517-522 518 biology, medicine, & natural product chemistry 13 (2), 2024: 517-522 snakehead fish (channa striata) is another alternative as a source of albumin protein because it is known to contain important compounds for the human body including high protein, fat, water, and the mineral zinc (zn) (karlina & luthfi, 2018). the albumin content in snakehead fish is an important type of protein needed by the body and is useful for the wound healing process. therefore, snakehead fish has an important role in the wound healing process because it has a much better quality than egg albumin which is commonly used in healing post-surgical patients (berlian, riani, kurniati, & rachmawati, 2023). the current way to process snakehead fish is by steaming or frying. processing fish with frying can reduce albumin levels or denatured. meanwhile, processing fish by steaming produces albumin extract with an unpleasant odor. both preparations do not last long, so a way is needed to process albumin so that it is not damaged and lasts long with an attractive taste. materials and methods equipment and materials the tools that will be used in this research are analytical scales (sojikyo hp-1/2000), desiccator (duran vacuum desiccator type 300 mm), stirring rod, 10 ml measuring cup and tube rack, hot plate (hi-cook tipe es-155), watch glass, thermometer (thermometer alco 10), beaker glass (iwaki), porcelain rate, spatula, napkins, sieve, tissue, oven (memmert un 30 universal oven), mold, glass funnel, whatman -41 filter paper, aluminum foil, animal cages, scissors, scalpel, panic, stove, basin. the materials used in this study were snakehead fish extract, sucrose (merck), isomalt (merck), distilled water, lemon flavor (zender), striatamine, 70% alcohol (one med), nacl 0.9% (otsu), male white mice (mus musculus) aged 6-8 weeks with a weight of 18-20 grams. research procedure extraction of snakehead fish (channa striata) making albumin extract from snakehead fish is done by steaming snakehead fish that has been washed and cut into small pieces. to reduce the fishy odor of the extract, ginger, and turmeric can be added during steaming. steaming is done using a large pot and stove for about two hours over low heat. put the snakehead fish that has been cut into small pieces into a large pot and add ginger and turmeric to reduce the fishy odor of the snakehead fish. hard candy making the process of making snakehead fish hard candy includes the stages of taking albumin, mixing, heating, molding, and cooling. first, dissolve 60 grams of sugar (for healthy people and people with diabetes mellitus) in water that has been boiled to 100℃. after the sucrose is completely dissolved, the heating is continued until the temperature is 140c. the temperature is lowered slowly by turning off the heat source. the stirring process was continued and snakehead fish extract was incorporated into the candy mixture. the end of cooking is determined by the thickened candy mixture. while still hot and thick, the candy mixture is put into the mold. the molding process is complete when the candy is cool and hard (do nascimento, pimentel, garcia, & prudencio, 2023). table 1. hard candy formulations for healthy people and diabetics. material formulation (%) f1 (5%) f2 (10%) f3 (15%) f4 (5%) f5 (10%) snakehead fish extract 5 10 15 5 10 sucrose 60 60 60 isomalt 60 60 flavor 3 3 3 3 3 aquadest ad 100 ml ad 100 ml ad 100 ml ad 100 ml ad 100 ml organoleptic test organoleptic tests are carried out by describing the color, smell, taste, and texture of the preparation using the five senses. water content test the water content test was carried out by weighing 2 grams of snakehead fish hard candy, putting it in a cup, then putting it in an oven at 100-105 ℃ for 3-5 hours, then cooling it in a desiccator, then weighing it. the calculation of moisture content is done with the formula: 𝑊𝑎𝑡𝑒𝑟 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑏 − 𝑐 𝑏 − 𝑎 𝑥 100% description: a : weight of the cup (g) b : weight of cup and wet sample (g) c : weight of cup and dry sample (g) (zarwinda, nadia, & rejeki, 2022). ash content test a total of 5 grams of snakehead fish hard candy is put into a porcelain cup that has a known weight, then diana et al. – formulation and evaluation of hard candy preparation as an … 519 burned at 500℃ for 4-5 hours or until white ash is formed, then put the sample in a desiccator, then weigh the sample. calculation of ash content is done with the formula: 𝐴𝑠ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊2 − 𝑊0 𝑊1 − 𝑊0 𝑥 100% description: w0 : weight of the cup (g) w1 : weight of cup and wet sample (g) w2 : weight of cup and sample after ashing (g) (zarwinda et al., 2022). wound healing activity test our research has met the code of ethics for research involving animal testing with an ethics certificate number 330/kepk/uds/v/2024. the test animals used were 25 male white mice (mus musculus) that were randomly grouped into 5 groups so that each group consisted of 5 mice and were treated as follows: aquadest as the negative control group (k-). striatamine as a comparison group (k+), 5% concentration of snakehead fish extract in group 1 (p1), 10% concentration in group 2 (p2), and 15% concentration of group 3 (p3). the day before making the incision wound, the mice's back area was shaved and then a veet was used to shave the fine hairs in the back area. after shaving, the back area of the mice was cleaned with 70% alcohol. next, an incision was made in mice with a sterile scalpel (scalpel) with an incision size of 1 cm long and 0.2 cm deep on the back of the mice. then the skin that has been injured by the scalpel incision is applied with striatamine as much as 0.1g and snakehead fish extract with 3 different concentration variations (5%, 10%, and 15%) evenly as much as 0.1g twice a day until the wound heals. observations were made visually by paying attention to changes in wound length, observation of erythema, edema, crusts, and days of wound healing. wound length measurements were taken using a caliper (hertian & muhaimin, 2021). results and discussion organoleptic test organoleptic testing has been carried out on snakehead fish albumin hard candy (channa striata) with concentrations f1 (5%), f2 (10%), and f3 (15%) which can be seen in table 2. table 2. organoleptic test results of hard candy for healthy people and diabetics. hard candy snakehead fish extract texture color flavor odor f1 hard brownish orange sweet typical candy odor f2 hard brown sweet typical candy odor f3 hard slightly sticky brown sweet typical candy odor f4 hard brownish orange not too sweet typical candy odor f5 hard slightly sticky lemon not too sweet lemon f6 unsuccessful organoleptic tests use the five senses which include observation of texture, color, taste, and smell. based on the table above, the difference in the percentage of water addition greatly affects the level of hardness of snakehead fish albumin hard candy. the more water is added to the sucrose dissolving process, the easier the candy will be destroyed (not hard), this can be caused because sucrose can bind water so that the candy will contain more water. in table 2, it can be seen that the color variation is influenced by the amount of addition of snakehead fish albumin extract. in f1, the resulting color is brownish orange, f2 produces a brown color, and f3 produces a deep brown color. the more the addition of snakehead fish albumin extract, the more intense the color will be. snakehead fish albumin hard candy has a sweet flavor. the flavor is not influenced by the percentage of snakehead fish albumin extract added. the odor organoleptic test produced in f1, f2, and f3 has a typical candy smell. it can be seen that the color variation is not affected by the amount of addition of snakehead fish albumin extract. in f4 the resulting color is brownish orange, and f5 produces a lemon color. snakehead fish albumin hard candy for diabetics has a not-so-sweet flavor. the taste is influenced by the main ingredients used, because in the formulation for diabetics the sugar used is isomalt. an organoleptic test of the odor produced in f4 is the typical smell of candy, and f5 the smell of lemon that comes from flavorings. water content test water content testing has been carried out on snakehead fish extract (channa striata) albumin hard candy with f1 (5%), f2 (10%), and f3 (15%) concentrations which can be seen in table 3. 520 biology, medicine, & natural product chemistry 13 (2), 2024: 517-522 table 3. water content test results of hard candy for healthy people and diabetics. hard candy testing water content f1 0,89% f2 1,6% f3 1,5% f4 1,3% f5 1,6% based on table 3, the lowest water content is found in f1 with 5% snakehead fish albumin extract treatment, which is 0.89%, while the highest water content was found in f2 with 15% snakehead fish albumin extract treatment, which was 1.5%. while in table 6 the water content of f3 is 1.3%, f4 is 1.6%. this research is in line with regia (2019), the value of water content is different from one another. this is related to the water activity of food ingredients which is also reduced due to the effect of adding different concentrations of snakehead fish albumin extract and different water. the different water content values are because during the cooking process, neither the temperature nor the length of heating time is measured. the moisture content of hard candy is said to be qualified if the quality is not more than 3.5% (sni 3547.1: 2008). all four snakehead fish albumin extract hard candy formulations meet the standard because they contain moisture content below 3.5%. ash content test ash content testing has been carried out on snakehead fish extract (channa striata) albumin hard candy with f1 (5%), f2 (10%), and f3 (15%) concentrations which can be seen in table 4. table 4. water content test results of hard candy for healthy people and diabetics. hard candy testing ash content f1 0,059% f2 0,137% f3 0,065% f4 0,026% f5 0,058% ash content testing has been carried out on snakehead fish albumin extract hard candy (channa striata) with concentrations f1 (5%), f2 (10%), and f3 (15%) which can be seen in the table below. ash content is one of the determining parameters of hard candy, because the lower the ash content, the better the appearance of the candy. the treatment of different concentrations had a significant effect on the ash content of hard candy. the more sucrose and isomalt concentration added, the higher the mineral content. the high ash content of sucrose and isomalt will trigger foam formation during the heating of the sugar solution. in addition, high ash content leads to increased inversion and coloration. ash content correlates with mineral content in general, the greater the mineral content, the higher the ash content obtained. the ash content of snakehead fish albumin extract hard candy (channa striata) in f1, f2, and f3 for healthy people was 0.059%, 0.137%, and 0.065%, respectively. in the results of f2 with a composition of 10%, it is thought that it will affect the interaction between the components in the preparation because of the higher interaction than f1 and f3, the same thing was also obtained in regia's article (2019) (rakhmayanti & hastuti, 2019). while the results for f4, f5 are 0.026% and 0.058%. when compared between f1 with f4 and f2 with f5, it shows that the results of ash content with sucrose as the main ingredient (f1, f2, f3) have a higher value than those made from isomalt (f4, f5), this difference can be caused by the difference in chemical structure between sucrose and isomalt, where sucrose has 2 rings that are difficult to denature, resulting in lower ash content compared to sucrose which is easily decomposed into gas because it only has one ring. however, the ash content of snakehead fish albumin extract hard candy still meets the quality requirements, because sni (354.1: 2008) states that the ash content of hard candy meets quality standards if it is not more than 2%. wound healing activity test wound healing activity has been tested on test animals with 7 treatment groups which can be seen in table 5. diana et al. – formulation and evaluation of hard candy preparation as an … 521 table 4. wound healing activity test results. concentration day 1 day 7 day 14 k+ k f1 f2 f3 f4 f5 this study was conducted to estimate the wound healing effect of hard candy preparation of snakehead fish albumin extract (channa striata) experimentally by oral route administration. the incision wound was 522 biology, medicine, & natural product chemistry 13 (2), 2024: 517-522 measured using a caliper, and an elongated incision wound model was used to assess the in vivo wound healing activity of snakehead fish albumin extract. the test animals used in this study were male mice, with body weights ranging from 16-20 grams. mice to be used in this study were first acclimatized for one week in the laboratory under controlled environmental conditions. wounds are made on the dorsal (back) of the mice using a sterile scalpel, forming a wound approximately 1 cm long and 0.2 cm deep. the wound is then left untreated for several hours to allow hemostasis. mice were divided into several treatment groups, namely the positive control group using triatomine drug, a negative control group (no treatment), and a group receiving snakehead fish albumin extract hard candies with formulations of 5%, 10%, 15% for healthy people and formulations of 5%, 10% for diabetics. treatments were administered orally daily for 14 days. the wound healing process was observed every day. based on table 4.7, it can be concluded that the wound condition on day 14 was completely healed for all groups except the negative control group because there were still scabs on the incision marks. the cause of not fully recovering in mice is due to certain factors that are relative, such as health condition, organ condition, immunity, and some other relative factors. conclusions formulation and evaluation of hard candy preparations (hard candy) of snakehead fish albumin extract (channa striata) can be declared successful, evidenced by the formation of 5 successful formulations into hard candy preparations as evidenced by the organoleptic test results, moisture content test and ash content test that meet the standards. in addition, oral administration of snakehead fish extract (channa striata) to test animals in the wound healing process, in each group showed good results. indicated by the return of the skin condition of the test animals as before treatment. acknowledgments: the author would like to thank the directorate general of higher education under the management of the directorate of learning and student affairs (belmawa) through the student creativity program and dr. soebandi university for supporting the author in completing this scientific article. competing interests: the authors declare that there are no competing interests. references berlian, g., riani, c., kurniati, n. f., & rachmawati, h. 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(2022). formulasi permen keras (hard candy) kopi defect arabika gayo. journal of pharmaceutical and health research, 3(3), 118-122. https://doi.org/10.1016/j.heliyon.2023.e15958 https://doi.org/10.1016/j.amjms.2018.11.009 https://doi.org/10.1016/j.fbio.2023.103128 https://doi.org/10.1016/j.wndm.2020.100192 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 89-96 | doi: 10.14421/biomedich.2023.121.89-96 issn 2540-9328 (online) cardiotoxicity effects of herbal medicine, a review article shahin gavanji department of biotechnology, faculty of advanced sciences and technologies, university of isfahan, iran. corresponding author gavanji.shahin2@gmail.com manuscript received: 04 october, 2022. revision accepted: 06 december, 2022. published: 13 december, 2022. abstract with the development of human's modern society more and more people tend to use complementary and alternative medicine (cam). toxicological studies indicated that many herbal medicines have direct toxic effects on the circulatory system or cardiovascular system and cause harmful effects on the body. cardiotoxicity or heart damage is a serious issue defined as heart electrophysiology dysfunction, affecting the cardiac structure, and muscle damage that arises from the drug or chemical poisoning agents, that may lead to heart failure. the aim of this review article is to provide various information about the potential adverse effects of herbal medicine on the cardiovascular system and introduce herbs that induced cardiac toxicity. to provide this review, all reported cases of cardiac toxicity induced by herbal medicines and natural products were collected through research articles and documents, and the most relevant articles, and books in various authentic search engines including scopus, pubmed, sid (scientific information database), science direct and google scholar, from 1984 to april 2022 were searched, and selected herbs with therapeutic properties which induce toxic effects on the cardiovascular system are introduced. in this review, scientific data regarding cardiotoxicity showed that 16 herbs from 11 families may increase cardiac toxicity. therefore, it's important to use herbal medicines and natural products under the guidance of medical professionals. keywords: cardiotoxicity; heart damage; herbal medicine; toxicity; adverse effects. introduction throughout history, using of herbal medicines has been playing an increasingly important role to treat various health challenges (chaachouay et al. 2022; liu et al. 2022). based on research, 80% of people all over the world, use complementary and alternative medicine (cam) and traditional medicine to treat different diseases (zhang et al. 2015; ardalan and rafieiankopaei 2013). furthermore, several studies demonstrated that about 20,000 herbal medicines are available in different markets all around the world (bent 2008), and a significant number of people rely on medicinal plants and use herbal remedies along with routine medication to treat various diseases, which increased the risk of herb–drug interactions (brantley et al. 2014; de smet 2007; gavanji and larki 2017). researchers have reported that inappropriate use of natural products can induce various damages including kidney injury (yang et al. 2018), nervous system injury (wattanathorn et al. 2008), liver injury (lin et al. 2019), skin injury (ernst 2000) and heart damages (sheth et al. 2015). toxicological studies indicated that many herbal medicines have direct toxic effects on the circulatory system or cardiovascular system and cause harmful effects on the body (fatima and nayeem 2016; valli and giardina 2002). cardiotoxicity or heart damage is a serious issue defined as heart electrophysiology dysfunction, affecting the cardiac structure and muscle damage that arises from the drug or chemical poisoning agents, that may lead to heart failure (sishi et al. 2013; chung et al. 2018). nowadays there is a growing concern over the cardiovascular adverse effects of herbal supplements and natural products and heart toxicity due to herbal medicines has recently become a source of interest (ernst 2003). several types of research demonstrated that several factors including inappropriate use of herbal remedies, using of herbal medicines for a long time, high-dose therapy and herb-herb or herb-drug interaction (cupp 1999; sun et al. 2021) may induce adverse reactions and side effects on the cardiovascular system, including arrhythmias, sympathomimetic activity, and hypertension and cause the irreversible damages which can lead to death. many people with a lack of knowledge, take herbal medicines based on their false beliefs and information to treat various diseases and increase the rate of heart adverse effects (alonso-castro et al. 2021). several studies have shown that various herbal medicines can interact with cardiac medications and increase the risk of bleeding or inactivate the antiplatelet drugs. also, inappropriate use of herbal medicine can cause problems cardiac cycle and pumping of blood throughout the body and may lead to various health problems. there has been https://doi.org/10.14421/biomedich.2023.121.89-96 90 biology, medicine, & natural product chemistry 12 (1), 2023: 89-96 an overwhelming weight of obsession on monitoring the cardiac patients who use herbal medicine and natural supplement without prescriptions, while they are taking heart medications, and scientific research studies indicated that many common medications including anticoagulants like aspirin (acetylsalicylic acid) (abebe 2002), warfarin (coumadin) (leite et al. 2016), and phenprocoumon (falithrom, marcoumar) (fasinu et al. 2012), also antiplatelet medications (prasugrel and clopidogrel) which have been used for cardiovascular diseases can interact with specific herbs (ma et al. 2018) and should be used under professional supervision. it is very important to increase the knowledge of different societies about the potential health risks of selfmedication and change our attitudes toward the inappropriate use of herbal medicines to reduce the risk of cardiotoxicity (alghadir et al. 2022; eichhorn et al. 2011; consolini, and ragone 2010). this review article provides various information about the potential adverse effects of herbal medicine on the cardiovascular system, and introduces herbs that induced cardiac toxicity. methods to provide this review, all reported cases of cardiac toxicity induced by herbal medicines and natural products were collected through research articles and documents, and the most relevant articles, and books in various authentic search engines including scopus, pubmed, sid (scientific information database), science direct and google scholar, from 1984 to april 2022 were searched. the search was organized with several combinations of search strategies with different keywords including cardiotoxicity, heart damage, herbal medicine, toxicity, and adverse effects. in this review article, the selected herbs with therapeutic properties which induce toxic effects on the cardiovascular system were introduced. cardiotoxic plants ranunculaceae ranunculaceae family which is called buttercup or crowfoot includes over 2,200 flowering plants, and has been widely used in traditional medicine (tm) for cancer treatment (christenhusz and byng 2016; hao et al. 2017). researchers have reported that three species of ranunculaceae family including aconitum napellus, aconitum kusnezoffii and aconitum carmichaeli showed the cardiotoxicity effects and people should be cautious to use these plants (table 1). a. kusnezoffii is a plant which has been traditionally used in the clinical practice as an antimicrobial and pain killer (su et al. 2021). based on a research study, 15 patients who consumed a. kusnezoffii for treatment of rheumatism and respiratory tract infection, encountered tachyarrhythmias, and ventricular tachycardia and fibrillation (tai et al. 1992). another species of this family is a. napellus which is used for muscle pain in folklore medicine (shoaib et al. 2020). a case report study has reported, that ingestion of a. napellus roots induced cardiotoxicity and a 54‐year‐old chinese man underwent ventricular tachycardia or arrhythmia and cardiac arrest (bonanno et al. 2020). the other member of ranunculaceae family is a. carmichaeli which shows wound healing and antiinflammatory properties (xia et al. 2019). according to a study, a 60-year-old man who consumed a. carmichaeli to treat his headache encountered aconitine poisoning which lead to myocardial necrosis (lin et al. 2011). zong and coworkers in 2019 demonstrated that a. carmichaeli contains diterpenoid alkaloids which have cardiotoxic potential (zong et al. 2019). another research study stated that aconitum plants have toxic effects on the cardiovascular system and two alkaloids including aconitine (ac) and mesaconitine (ma) play a crucial role in cardiotoxicity and create some serious problems (liu et al. 2019). dioscoreaceae dioscorea bulbifera is a medicinal plant of dioscoreaceae or yam family which has been traditionally used to treat wounds, rectal carcinoma, sore throat, and stomach or gastric cancer (chaniad et al., 2020). the result of a research study showed that extracting the rhizome of d. bulbifera, induced cardiotoxicity by pirarubicin accumulation which lead to necrosis, and muscle fiber damage, also indicated that cardiotoxicity effects of d. bulbifera is dose-dependent (sun et al. 2021). another study indicated that d. bulbifera can interact with doxorubicin (dox) in cancer treatment and cause dox accumulation which leads to cardiovascular injury including necrosis and breakage in cardiac muscle fibers (qu et al. 2019). ginkgoaceae ginkgo biloba also called ginkgo, belonging to the family ginkgoaceae, has been widely used in traditional medicine(tm) for the treatment of various diseases including asthma, cognitive problems, circulatory disorders, and vertigo (brondino et al. 2013). based on research, g. biloba has mild adverse effects and can cause heart palpitations (nguyen and alzahrani 2022). a study by bent and coworkers in 2005 showed that many people consume ginkgo to decrease platelet aggregation but according to various research ginkgo may interact with warfarin and increase the risk of bleeding (bent et al. 2005). however, a randomized controlled trials study, demonstrated that ginkgo may not interact with warfarin (engelsen et al. 2002; köhler et al. 2004). also, another study stated that concurrent use of gingko and warfarin will increase bleeding adverse effects and patients should be cautious to use of herbs and drugs simultaneously (stoddard et al. 2004). gavanji – cardiotoxicity effects of herbal medicine, a review article 91 table 1. cardiotoxicity effects of herbal medicine. no plant name family potential therapeutic application (traditional medicine) type of adverse effect references 1 atropa belladonna solanaceae treat of asthma, cough, and hay fever flushed and tachycardic (chadwick et al. 2015) 2 aconitum napellus ranunculaceae treat of high fever aconitum napellus (bonanno et al. 2020) 3 aconitum kusnezoffii ranunculaceae treat of sore throat, gout and rheumatism tachyarrhythmias, ventricular tachycardia and fibrillation (tai et al. 1992) 4 aconitum carmichaeli ranunculaceae pain relief cardiotoxicity (tai et al. 1992) 5 datura stramonium solanaceae treat of wounds, inflammation and rheumatism tachycardia (khoshnam et al. 2022; arefi et al. 2016) 6 dioscorea bulbifera dioscoreaceae treat of syphilis, ulcers, cough, leprosy and diabetes accumulation of thp in heart tissue (sun et al. 2021; qu et al. 2019) 7 digitalis purpurea plantaginaceae treat of asthma, epilepsy and tuberculosis heart failure (gerakaris et al. 2022) 8 ephedra distachya ephedraceae weight loss and obesity side effects on the cardiovascular system and cerebrovascular effects (gonzález-juárez et al. 2020; andraws et al. 2005) 9 ephedra sinica ephedraceae treat of asthma, bronchitis, and hay fever serious cardiovascular adverse effects (ekor 2014; nyska et al. 2005; dunnick et al. 2007) 10 ginkgo biloba ginkgoaceae treat of cognitive disorders and dementia increasing the bleeding tendency, heart palpitations (nguyen and alzahrani 2022; stoddard et al. 2004; bent et al. 2005) 11 glycyrrhiza glabra fabaceae treat of respiratory disorders, hyperdipsia and fever cardiac arrhythmias, pulmonary edema (deutch et al. 2019) 12 hypericum perforatum hypericaceae treat of anxiety and depression arrhythmia, hypertension (rubini et al. 2019; guru et al. 2021) 13 juniperus oxycedrus cupressaceae anti-inflammatory and antimicrobial effects tachycardia (achour et al. 2011) 14 mandragora officinarum solanaceae treat of asthma and hay fever serious supraventricular tachycardia (tsiligianni et al. 2009) 15 nerium oleander apocynaceae treat of cancer, painful menstrual periods arrhythmia (guru et al. 2021) 16 piper methysticum piperaceae treat of fever and respiratory disorders cardiovascular abnormalities, arrhythmia (toohey et al. 2013) solanaceae atropa belladonna, mandragora officinarum, and datura stramonium are three species of solanaceae family, which contain various alkaloids with different negative effects on the body. atropa belladonna is listed as a poisonous plant and called deadly nightshade. a. belladonna is traditionally used to treat bradycardia (slow heart rate) (shah et al. 2019). but according to a study, demonstrated that the consumption of a. belladonna in a 49-year-old woman can lead to anticholinergic toxic syndrome and heart rhythm disturbances (demirhan et al. 2013). also, the results of a study showed that ingestion of a. belladonna may cause abnormal heart rate (tachycardia) and other problems (chadwick et al. 2015). m. officinarum is another member of solanaceae family (al-maharik et al. 2022), which contains several phytochemicals including withanolides, tropane alkaloids (scopolamine and hyoscyamine), and coumarins (schlesinger et al. 2019; hanus et al. 2005). the root of this plant has been used in traditional medicine (tm) for the treatment of various diseases (monadi et al. 2021). a case study research indicated that two patients who consumed m. officinarum admitted to hospital with supraventricular tachycardia (svt) (tsiligianni et al. 2009). another important species of solanaceae family is d. stramonium which has been traditionally used to treat many diseases including fever, ulcers, wounds, toothache, rheumatism, and bronchitis (sharma et al. 2021). a research study indicated that alkaloids of d. stramonium can increased heart beat in mice (benouadah et al. 2016), furthermore, researchers have reported that the flower, leave and seed d. stramonium can induce the tachycardia (arefi et al. 2016; amini et al. 2012; disel et al. 2015). 92 biology, medicine, & natural product chemistry 12 (1), 2023: 89-96 ephedraceae ephedra distachya is commonly known as sea-grape belongs to the family ephedraceae (gonzález-juárez et al. 2020), which has a long history in folk medicine to treat bronchitis, asthma, low blood and colds (ghavam, and soleimaninejad, 2020). a study showed that ephedra alkaloids of e. distachya have negative effects on the cardiovascular system and adverse cerebrovascular effects (andraws et al. 2005). another important species of ephedraceae family is ephedra sinica, which is traditionally used to treat cold, asthma, and lung diseases (mei et al. 2021). dunnick and coworkers in 2007 demonstrated that a combination of e. sinica with caffeine can induce cardiotoxicity (dunnick et al. 2007). also, another research study stated that using ephedrine and caffeine affects the cardiovascular system and causes cardiotoxicity (howden et al. 2005; nyska et al. 2005). plantaginaceae digitalis purpurea is a species of plantaginaceae family, which has been traditionally used for the treatment of respiratory problems and heart failure (hf) (gerakaris et al. 2022; aswal et al. 2019; smith et al. 1984). so many people used d. purpurea to enhance their general health and well-being, but a study by gerakaris indicated that people without heart failure should not consume digoxin, and patients can take herbal medicinal products (hmps) with a doctor's supervision (gerakaris et al. 2022; lei et al. 2018). fabaceae glycyrrhiza glabra belonging to the family fabaceae is an important medicinal plant that has been widely used for healing respiratory disorders, gastroesophageal reflux disease, fever, liver diseases, hyperdipsia, rheumatism, tuberculosis, sexual debility, and infectious diseases (wahab et al. 2021; gavanji 2022). a study demonstrated that using of licorice (g. glabra) candies can induce arrhythmias (böcker and breithardt 1991), also another research by konik and coworkers in 2012 indicated that licorice can induce the coronary artery spasm (konik et al. 2012). licorice intake may have harmful effects on the cardiovascular system including cardiac arrhythmias and pulmonary edema, therefore it is essential to be cautious in using licorice (deutch et al. 2019). hypericaceae hypericum perforatum, a species of hypericaceae family, with various therapeutic potential, has been traditionally used to treat infectious diseases, anxiety, and burns, also several studies demonstrated that h. perforatum has anti-cancer activities (gavanji 2022; klemow et al. 2011). various types of research indicated that using h. perforatum may cause adverse events, and induce cardiotoxicity and herb-drug interactions. researchers have reported that using h. perforatum, may also lead to arrhythmia, and hypertension (cohen and ernst 2010; ernst 1999). cupressaceae juniperus oxycedrus is a member of cupressaceae family, which is traditionally used for the treatment of respiratory problems and different infectious diseases (tavares and seca 2018; karaman et al. 2003). a number of studies reported that extract of j. oxycedrus, has toxic effects on the body and may cause fever, tachypnea, and tachycardia (koruk et al. 2005; achour et al. 2011). apocynaceae one of the important ornamental and landscaping herbs is nerium oleander or nerium, which belongs to the family apocynaceae, and is widely cultivated in different areas. this plant has been traditionally used to treat eczema, opthalmia, gastrointestinal disturbances, and ringworm infections (akhtar et al. 2014). several types of research demonstrated that ingesting n.oleander cause different side effects including abnormal heart rhythm (bradycardia) and nervous system disorders (rubini et al. 2019). another study showed that oleander poisoning can lead to arrhythmia and other negative effects (guru et al. 2021). piperaceae piper methysticum, commonly known as kava, belonging to the family piperaceae, has been used for the treatment of various diseases including anxiety, restlessness, and psychological disorders (ernst 2007; boon and wong 2003). based on a study taking p. methysticum and psychotropic medications can increase the risk of herb-drug interaction and will be lifethreatening, also it has negative effects on the heart which lead to cardiovascular abnormalities, and arrhythmia (toohey et al. 2013). conclusions since the dawn of humanity, people have tried to seek new medications to treat various diseases. an issue of concern turning into an obsession for people all over the world is self-medication with herbal medicine which causes several harmful effects. in this review, all reported herbal medicines which induce cardiotoxicity were collected and introduced. the results of this review suggest that herbal medicine should be taken with the guidance of medical professionals. acknowledgements: i would like to thank dr. forough mortezaienejad for guidance on this project. gavanji – cardiotoxicity effects of herbal medicine, a review article 93 competing interests: the authors declare that there are no competing interests. references achour, s., abourazzak, s., mokhtari, a., soulaymani, a., soulaymani, r., & hida, m. 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alzheimer’s disease; cognitive impairment; nigella sativa oil; oxidative stress. abbreviations: aβ: amyloid-β; ach: acetylcholine; ache: acetylcholinesterase; ad: alzheimer’s disease; alcl3: aluminium chloride; anova: analysis of variance; bbb: blood-brain barrier; cat: catalase; glu: glutamate; gsh: glutathione; mda: malondialdehyde; mwm: morris water maze; nso: nigella sativa oil; sem: standard error of mean; sod: superoxide dismutase. introduction alzheimer's disease (ad) is a neurodegenerative condition marked by a progressive degeneration of the hippocampal and cortical neurons that impairs memory and cognitive function. alzheimer's disease is a multifactorial disease with no known etiology, and numerous risk factors are linked to its development and progression. the risk of developing ad increases proportionately with age, approximately doubling every 5 years after age 65 (ojetunde, 2024). aluminium (al) is found in antacids, deodorants, and food additives, which are easily absorbed by the body. its neurotoxicity in animals has been well demonstrated and linked to the etiology of neurodegenerative diseases like ad (niu, 2018). it facilitates the formation of amyloid-β (aβ) protein plaques via the aggregation of tau proteins in the brain (elreedy et al., 2023). al has also been linked to aging-related alterations and neurodegeneration. aluminium chloride (alcl3) administration mainly accumulates in the hippocampus, which is particularly susceptible to ad and plays a significant role in learning and memory functions (ojetunde, 2024). alzheimer's disease is becoming a global burden due to its high prevalence yet poor treatment (boland et al., 2018). oxidative stress and inflammation are the major contributors to this neurodegenerative disease. it is reported that al toxicity is due to potentiating the activity of fe2+ and fe3+ ions in the fenton reaction to cause oxidative damage (ojetunde, 2024). there is currently no known cure for ad, and drug therapy for the disease is still in its early stages. drugs approved for the treatment of possible ad help to regulate the symptoms of the disease, but do not reduce or reverse its progression, and are followed by with accompanying adverse side effects (vaz et al., 2022). at the moment, medications targeting neurotransmitter systems in the brain form the basis of ad therapy (singh et al., 2024). different studies (ojetunde, 2021; ojetunde et al., 2021; tongshuwar et al., 2020) have summarized the use https://doi.org/10.14421/biomedich.2025.142.739-746 740 biology, medicine, & natural product chemistry 14 (2), 2025: 739-746 of medicinal plants and herbs and their phytochemical components for the treatment of different diseases. nigella sativa has garnered special attention in both traditional and modern medicinal research. according to compelling evidence, nigella sativa oil has high antioxidant and anti-inflammatory properties (sahak et al., 2016). this suggests its use as a potential remedy for cognitive impairments in ad. therefore, this study investigated the modulatory role of nigella sativa oil on cognitive impairments in aluminium chloride-induced cognitive impairment in wistar rats. materials and methods drugs and chemicals methylated spirit, aluminum chloride-hydrated (alcl3.6h2o) was purchased from sigma chemical co. (st. louis, usa). the nigella sativa oil [black seed oil (100% pure natural oil)] was obtained from masra warda, kingdom of saudi arabia. animals twenty-four (24) apparently healthy wistar rats weighing 200 ± 20 g were kept at the animal house of the department of human physiology, ahmadu bello university, zaria, nigeria. they were randomized into experimental and control groups and were housed in plastic cages. standard animal feed made of pellets from growers' mash was provided to the animals. the rats were allowed access to drinking water ad libitum throughout the study. they were allowed to acclimatize for 2 weeks before the commencement of induction and treatment. rats were kept under constant conditions (temperature 25 ± 3 °c and humidity 50%) with 12/12 h light/dark cycles. ethical approval was obtained from the ahmadu bello university ethical committee on animal use and care with approval number abucauc/2023/102. animals were divided into four groups (6/group) and treated daily for six weeks as follows: group 1: received 1 ml/kg of distilled water orally (normal control). group 2: received 100 mg/kg of alcl3 orally (negative control) (baburaj et al., 2023) group 3: received 100 mg/kg of alcl3 + nigella sativa oil (1 ml/kg) orally (imam et al., 2016) group 4: received 100 mg/kg of alcl3 + nigella sativa oil (2 ml/kg) orally at the end of the six-week administration, 24 hours after the last administration, the following tests were carried out: morris water maze (mwm) test the spatial learning and memory were assessed by the morris water maze as described by mahboubi et al. (2016). the mwm tank is 100 cm in width, and 62.5 cm in height. the mwm tank was filled to a depth of 40 cm with water and maintained at a temperature of 20 ± 1°c. around the room, multiple visual cues were present and kept constant throughout the experiment. the maze was categorized geographically into four quadrants; northeast (ne), north-west (nw), south-east (se) and southwest (sw) and starting positions, north (n), south (s), east (e), west (w) were equally spaced around the perimeter of the pool and a hidden circular platform (diameter: 13 cm) was placed at the centre of the nw quadrant, 1 cm below the surface of the water. during four consecutive daily sessions (each session consisted of four trials), the rats were trained to find the submerged escape platform located in a fixed position. each trial had a maximum duration of 60 seconds before removing the rats from the mwm. two hours after the last training trial, the rats were subjected to a memory probe trial during which they were allowed to swim for 60 seconds in the absence of the training platform. all the rats started from the same position, opposite to the target quadrant (the quadrant where the escape platform was positioned). y-maze test short-term working memory was assessed as a measure of spontaneous alternations using the y-maze as described by zaher et al. (2019). the y-maze is composed of three equally spaced arms. each of the rats was placed in one of the arm compartments and allowed to move freely until its tail completely entered another arm. the number of maximum spontaneous alternations is the total number of arms entered minus two, and the percentage alternation is calculated as {(total alternations /spontaneous alternations) x 100}. for each animal, the y-maze testing was carried out for 5 minutes. the apparatus was cleaned with methylated spirit (10% methanol and 90% ethanol) alcohol and allowed to dry between sessions. brain homogenate preparation the animals were then sacrificed by decapitation under ketamine and diazepam (75 and 25 mg/kg). the rats’ brain tissues were collected and prepared according to the method described by zatta et al. (2002), and habila et al. (2012). treated and control animals were sacrificed, and brain tissue was immediately removed and placed on an inverted petri dish on ice. the whole brain was harvested, weighed, and homogenized in 3 ml of a medium containing phosphate buffer solution, ph 7.5. the total homogenate was centrifuged at 5000 x g for 5 minutes. the supernatants were used for biochemical parameter assays. biochemical assay brain marker of oxidative stress the malondialdehyde (mda) assay was carried out by using esterbauer and cheeseman (1990). ojetunde et al. – nigella sativa oil mitigates neurotoxicity 741 brain antioxidant markers the brain superoxide dismutase (sod) was determined according to the method described by fridovich (1989). catalase (cat) was measured using the method of sinha (1972). glutathione (gsh) was measured using ellman’s method (1959). neurochemical assessment measurement of brain acetylcholinesterase (ache) level the concentration of brain tissue homogenate, ache level was assessed using elisa kits (ray biotech, inc., usa) according to the manufacturer's instructions. measurement of brain glutamate (glu) concentration the brain tissue homogenate glu concentration was assessed using elisa kits (ray biotech, inc., usa) according to the manufacturer's instructions. data analysis the data obtained were expressed as mean ± standard error of mean (sem) and were analyzed using one-way and mixed analysis of variance (anova) with tukey’s post hoc test to compare the level of significance between control and experimental groups. spss version 20 software was used for the analysis, and values of p < 0.05 were considered statistically significant. results and discussion table 1 shows the effect of nigella sativa oil on escape (es.) latency in alcl3-induced cognitive impairment in wistar rats. the escape latency of the morris water maze indicated an impairment of memory, as the alcl3 exposed group showed a significant increase (p < 0.05) in time (s) to reach the escape latency in all 4 days when compared to the normal control group. nigella sativa oil administration groups (1ml/kg and 2ml/kg) showed a significant decrease (p < 0.05) in the time to reach the escape latency of the morris water maze when compared with the alcl3 exposed group. table 1. effect of nigella sativa oil on escape latency in alcl3-induced cognitive impairment in rats using mwm. groups es. latency (s) (day 1) mean ± sem es. latency (s) (day 2) mean ± sem es. latency (s) (day 3) mean ± sem es. latency (s) (day 4) mean ± sem dw (1ml/kg) 12.36 ± 0.54 5.47 ± 0.59 3.74 ± 0.09 2.92 ± 0.20 alcl3 22.73 ± 0.37a 19.64 ± 1.02a 13.63 ± 0.95a 12.45 ± 0.54a alcl3 + nso (1ml/kg) 15.97 ± 0.52ab 9.38 ± 0.82ab 7.72 ± 0.72ab 5.68 ± 0.26ab alcl3 + nso (2ml/kg) 14.92 ± 0.24ab 7.61 ± 0.15b 5.62 ± 0.16b 3.41 ± 0.09bc values along the same column with superscripts a, b, and c are significantly different (p < 0.05) when compared to the normal control, alcl3, and alcl3+nso (1ml/kg) group, respectively. alcl3: aluminium chloride, dw: distilled water, nso: nigella sativa oil figure 1 shows the effect of nigella sativa oil on the memory probe trial of alcl3-induced cognitive impairment in rats. the memory probe trial of the morris water maze indicated an impairment of memory, as the alcl3-exposed group showed a significant decrease in time (s) on the memory probe trial when compared to the normal control group. however, nigella sativa oil administration groups (1ml/kg and 2ml/kg) showed a significant increase (p < 0.05) in the time of memory probe trial of the morris water maze when compared with the alcl3 exposed group. figure 1. memory probe trial. values with superscripts a and b are significantly different (p < 0.05) when compared to normal control, and alcl3 group, respectively. alcl3: aluminium chloride, dw: distilled water, nso: nigella sativa oil. 742 biology, medicine, & natural product chemistry 14 (2), 2025: 739-746 the result of the mean values of percentage alternation was estimated as shown in figure 2. the alcl3-treated group percentage alternation decreased significantly (p < 0.05) when compared to the normal group. groups co-treated with nigella sativa oil (1ml/kg and 2ml/kg) had a significantly (p < 0.05) higher percentage of alternation when compared with the alcl3treated group. figure 2. effect of nigella sativa oil on spontaneous alternation in rats with alcl3-induced cognitive impairment. values with superscripts a and b are significantly different (p < 0.05) when compared to normal control, and alcl3 group, respectively. alcl3: aluminium chloride, dw: distilled water, nso: nigella sativa oil. the acetylcholinesterase level in the alcl3-treated group was significantly (p < 0.05) increased when compared with the normal control group, as shown in figure 3. however, treatment with nigella sativa oil (1ml/kg and 2ml/kg) showed a significantly (p < 0.05) decreased acetylcholinesterase level. figure 3. effect of nigella sativa oil on brain homogenate level of acetylcholinesterase in alcl3-induced cognitive impairment in rats. values with superscripts a and b are significantly different (p < 0.05) when compared to normal control, and alcl3 group respectively. alcl3: aluminium chloride, dw: distilled water, nso: nigella sativa oil. the results of the mean values of glutamate are shown in figure 4. the alcl3-treated group glutamate level significantly (p < 0.05) increased when compared with the normal control group. groups treated with nigella sativa oil (1ml/kg and 2ml/kg) had a significant (p < 0.05) decrease in the level of glutamate compared with the alcl3-treated group. figure 4. effect of nigella sativa oil on brain homogenate level of glutamate in alcl3-induced cognitive impairment in rats. values with superscripts a and b are significantly different (p < 0.05) when compared to normal control, and alcl3 group respectively. alcl3: aluminium chloride, dw: distilled water, nso: nigella sativa oil. as shown in table 2, there was a significant decrease (p < 0.05) in the mean values of all the anti-oxidant biomarkers sod, cat, and gsh in the alcl3 treated group compared with the normal control group, while there was a significant increase (p < 0.05) in the mda of alcl3-treated group when compared with the normal control group. a significant increase (p < 0.05) was observed in the anti-oxidant biomarkers sod, cat, and gsh of groups treated with nigella sativa oil (1ml/kg and 2ml/kg) when compared with the alcl3-treated group, while for the mda a significant decrease (p < 0.05) was observed in the groups treated with nigella sativa oil (1 ml/kg and 2 ml/kg) when compared with the alcl3 treated group. however, the increase in gsh level of the group receiving 1 ml/kg of nigella sativa oil was not statistically significant when compared to the alcl3treated group. ojetunde et al. – nigella sativa oil mitigates neurotoxicity 743 table 2. effect of nigella sativa oil on oxidative stress and antioxidant parameters in alcl3-induced cognitive impairment in wistar rats. groups mda (nmol/mg protein) mean ± sem sod (u/mg protein) mean ± sem cat (u/mg protein) mean ± sem gsh (ug/mg protein) mean ± sem dw (1ml/kg) 40.40 ± 2.31 44.17 ± 0.94 28.07 ± 1.30 25.57 ± 0.57 alcl3 56.87 ± 1.62a 23.00 ± 1.65a 9.2 ± 0.40a 19.13 ± 1.41a alcl3 + nso (1ml/kg) 47.87 ± 1.27b 31.23 ± 1.36ab 19.23 ± 0.88ab 21.50 ± 1.03 alcl3 + nso (2ml/kg) 42.13 ± 1.73b 39.50 ± 2.25bc 26.97 ±1.47bc 27.60 ± 1.45bc values along the same column with superscripts a, b, and c are significantly different (p < 0.05) when compared to normal control, alcl3, and alcl3+nso (1 ml/kg) groups, respectively. alcl3: aluminium chloride, dw: distilled water, nso: nigella sativa oil discussion the present study results revealed that the administration of alcl3 impairs spatial learning and memory as assessed via the morris water maze task, as it took a significant time to escape. aluminium can cross the blood-brain barrier (bbb) and accumulate in various regions of the brain tissues, which promotes the impairment of learning and memory (hamdan et al., 2022). aluminium can also interfere with the downstream effector molecules (cyclic gmp) necessary for long-term potentiation. this interruption could explain the observed memory loss and neurobehavioral changes (colizzi, 2018). rats treated with nigella sativa oil at all dosages used showed improved learning ability and spatial memory with a significant decrease in time to escape latency during assessment and an increase in the time spent in the target quadrant in the probe task in the morris water maze task. in models of neurotoxicity and neurodegenerative disorders, nigella sativa has been linked to be effective in enhancing neurocognitive and psycho-cognitive functions (norouzi et al., 2019). thymoquinone, present in nigella sativa oil, is known to improve learning, memory, and cognitive functions and may be responsible for the cognitive modulation of nigella sativa oil (bargi et al., 2017). thymoquinone has also been reported to improve memory by increasing acetylcholine immunoreactivity and decreasing ache activity (abulfadl et al., 2018). the current study revealed that the rats treated with only alcl3 exhibited a significant decrease in spontaneous alternation percentage when compared to control rats in the y-maze test. this diminished percentage of alternation as observed in the present study indicated an impaired spatial working memory (rout et al., 2012), which is mostly attributed to the neurological cell damage and synaptic dysfunction encountered in rats' brains with ad (mohamed et al., 2020). however, co-administration of nigella sativa oil improves the rats’ cognitive ability by significantly modulating these memory deficits via a significant increase in percentage alternation. this report supports the findings of several studies (imam et al., 2021), which reported that nigella sativa oil has the potential to protect against or improve spatial working memory deficits. this effect is supported by the findings of khan et al. 2014, where thymoquinone (the active component of nigella sativa oil) increased percentage alternation in an animal model of neurological disorder. it has also been shown that thymol present in nigella sativa oil enhances cognitive functions in a model of dementia (asadbegi et al., 2017). acetylcholinesterase activity is a well-known indicator of damage to cholinergic neurons in the brain. it is the primary enzyme inactivating acetylcholine in the synaptic cleft (anwar et al., 2021). dementia and the severity of neuropathological alterations associated with ad are strongly linked to the disruption of cholinergic neurotransmission in the cortex and hippocampus (lao et al., 2019). in this study, the administration of alcl3 to the experimental rats showed a significant increase in the level of ache, which is in agreement with the work done by ekundayo et al. (2022) and hejaziyan et al. (2023). aluminium ion interacts with the peripheral site of ache, which modifies its secondary structure and eventually increases its activity (auti & kulkarni, 2019). the therapeutic administration of nigella sativa oil to alcl3-induced cognitively impaired rats showed the possible modulatory role of nigella sativa oil by reducing ache levels. a study with nigella sativa demonstrated almost identical levels of ache activity compared to donepezil (sudha et al., 2021). thymoquinone, the active ingredient of nigella sativa oil, was reported to possess anti-cholinesterase activity (jukic et al., 2007). the principal constituents of its essential oil, thymol and carvacrol, and their derivatives (e.g., thymohydroquinone) had inhibitory effects on ache (jukic et al., 2007). so, these compounds can be identified as prospective therapeutic agents for the treatment of ad and/or cognitive disorders. cholinergic depletion has been noted to increase aβ deposition (ramos-rodriguez et al., 2013), tau phosphorylation, & pro-inflammatory cytokines formation (field et al., 2012). therefore, the pathophysiological characteristics and the clinical presentation of ad may be improved by restoring cholinergic functions (zaher et al., 2019). the main post-excitatory neurotransmitter, glutamate, is involved in almost all central nervous system functions, particularly in the hippocampal region and cortical area of the brain (kim et al., 2011). glutamate is neurotoxic when present in excessive amounts and increases neuronal excitability by activating proteolytic enzymes (weil et al., 2008). in this study, there was a significant elevation of this glutamate neurotransmitter in 744 biology, medicine, & natural product chemistry 14 (2), 2025: 739-746 the alcl3-treated group. this elevation of glutamate concentrations has been linked to increased sensitivity and/or activity of the glutamatergic system, leading to neuronal dysfunction and cell death in ad (gasparini & dityatev, 2008). aluminium activates glutamatemediated excitotoxicity, which results in severe neuronal damage and loss (baburaj et al., 2023). due to overstimulation of n-methyl-d-aspartate receptors, glutamate hinders learning and memory by causing cognitive decline and neuronal degeneration (alghamdi, 2018). in contrast, the result of the present study indicates that nigella sativa oil modulates alcl3-induced cognitive impairment, by inhibiting the excessive elevation of glutamate, as shown in nigella sativa oil-treated groups, thereby decreasing the level of ca2+ influxes to hippocampal neurons by blocking the l-type calcium channel, which may reduce excitotoxicity and neuronal death. in another study, nigella sativa diminished glutamate secretion, leading to decreased neuronal excitatory activity (el-naggar et al., 2010). also, nigella sativa oil pre-treatment lowered glutamate levels in mice models of essential tremor (folarin et al., 2020). the possible cause of the improving effects of nigella sativa oil on the level of glutamate in treated rats is the antioxidant activities of its components. in previous research, aluminium neurotoxicity effects produced an imbalance between the generation of reactive oxygen species and antioxidants, leading to oxidative stress in neurons (abbas et al., 2022). the administration of alcl3 in the present study resulted in marked oxidative stress in the brain tissues as indicated by reduced levels of sod, cat, and gsh, then significantly increased levels of mda compared to the control group. however, treatment with nigella sativa oil showed significant sod, cat, and gsh increase and significant mda decrease compared to the alcl3-treated group supporting the antioxidant effect of nigella sativa oil which may be linked to the presence of the phytochemicals present in nigella sativa oil. nigella sativa oil contains quinine, carvacrol, and 4terpineol, which are effective in connecting free radicals (umar et al., 2012). it also contains aglycones and flavonol glycosides, which have higher anti-oxidant and anti-radical effects, so they function as a revealer of superoxide radicals in the blood to eliminate free radicals and inhibit the oxidation process in cells (kooti et al., 2016). it has also been demonstrated that thymoquinone (obtained from nigella sativa seed oil) can cross the bbb and scavenge free radicals generated by various pro-oxidant stimuli, including heavy metals, thereby preventing neurodegeneration (hosseinzadeh et al., 2012; elmaci & altinoz, 2016). thymoquinone was previously found to increase antioxidant enzyme activities in a rat model of chlorpromazine toxicity (safhi, 2016). as a result, the antioxidant property of thymoquinone found in nigella sativa seed oil could be considered as one protective mechanism against learning and memory impairment in ad (lotfi et al., 2022). conclusions it is concluded that nigella sativa oil significantly protected neurobehavioral alterations and improved cognitive, and spatial learning functions in rats. nigella sativa oil can prevent oxidative damage by increasing neuronal antioxidant enzyme activities (sod, cat & gsh) and decreasing biomarker of lipid peroxidation (mda). moreover, nigella sativa oil can modulate the cholinergic function (ache), and abnormal levels of neurotransmitters (glutamate). authors’ contributions: aoo, aa, is, and asi designed the study. aoo carried out the laboratory work. aoo, aa, is, and asi analyzed the data. aoo wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the author declares no funding. references abbas, f., eladl, m. a., el-sherbiny, m., abozied, n., nabil, a., mahmoud, s. m., ... & ibrahim, d. 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(2002). in vivo and in vitro effects of aluminum on the activity of mouse brain acetylcholinesterase. brain research bulletin, 59(1), 41-45. https://doi.org/10.1016/s03619230(02)00836-5 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 1, 2019 | pages: 11-15 | doi: 10.14421/biomedich.2019.81.11-15 issn 2540-9328 (online) mathematical model of cervical cancer treatment using chemotherapy drug murtono1,*, meksianis zadrak ndii2, sugiyanto3 1department of physics education; 3department of mathematics, universitas islam negeri sunan kalijaga, 55281, yogyakarta, indonesia. 2department of mathematics, faculty of science and engineering, university of nusa cendana, kupang-ntt, indonesia. author correspondency*: hasnamur@yahoo.co.id abstract cervical cancer is a malignant disease that causes problems in women's health, especially in developing countries such as indonesia. cervical cancer cells will develop quickly, uncontrollably, and will continue to divide and then infiltrate the surrounding t issue and continue to spread to connective tissue, blood, and attack important organs and spinal nerves. the aim of the research is to study the mathematical model of cervical cancer by chemotherapy treatment. the results of this study are that cervical cancer treatment using chemotherapy is effective enough to kill abnormal cells such as infected cells, pre-cancerous cells and cancer cells, although there are side effects, namely the killing of normal cells due to chemotherapy drugs. keywords: cervical cancer; infected cells; infected cells; cancer cells; chemotherapy introduction cervical cancer is an excessive and uncontrolled cell growth around the cervix (walboomers et. al., 1999). cervical cancer originated from cells in the cervix (naganawa et al., 2005). most cervical cancers begin in the transformation zone which is a shift from squamous cell type to cylindrical cell type. these cells do not directly turn into cervical cancer. normal cervical cells due to the influence of carcinogenic substances can develop gradually into pre-cancerous cells and then become cancer cells (sari et al., 2016). the main trigger for the emergence of cervical cancer is infection of several types of high-risk human papilloma virus (hpv) which causes proliferation of the epidermal surface and cervical mucosa (bosch, 1995). the types of hpv that are very common in cases of cervical cancer are types 16 and 18, which is more than 70% of all cervical cancers reported. the results of a study of 1,000 samples from 22 countries proved the presence of hpv infection in 99.7% of cervical cancer cases (wuryanti et. al., 2015). cervical cancer is the second most common type of cancer in women worldwide to breast cancer (boice et. al., 2002). chemotherapy is a kind of cancer treatment that uses drugs to destroy cancer cells. chemotherapy works by stopping or slowing the growth of cancer cells, which grow and divide rapidly. chemotherapy can also harm healthy cells that divide rapidly, such as the lines of the mouth and intestines or cells that affect growth. damage to healthy cells can cause side effects. often, side effects will be disappear after chemotherapy is complete (rose et. al., 1999). reduction of the mass of cervical cancer can be used as used to measure the effectiveness of treatment because chemotherapy can cause shrinkage of the mass of cervical cancer. cancer mass has an important role to detect the prognosis of a cervical cancer. formulation of model this model developed frpm the past research (asih, et. al., 2015) about the development of cervical cancer and pillis et al. (2007) about the model of treating cancer in general with chemotherapy. figure 1. cervical cancer treatment diagram by chemotherapy. https://doi.org/10.14421/biomedich.2019.81.11-15 12 biology, medicine, & natural product chemistry 8 (1), 2019: 11-15 table 1. subpopulations, parameters and units. symbol symbol explanation unit unit s(t) normal cell density cell/mm2 i(t) infected cell density cell/mm2 p(t) pre-cancerous cell density cell/mm2 c(t) cancer cell density cell/mm2 v(t) virus density virus/mm2 m(t) concentration of chemotherapy drugs mg/m2 r growth rate of normal cell 1/day n homeostatic carrying capacity cell/mm3  the rate of infection 1/(day.virus) 1a the rate of proliferation of infected cells 1/day 1d the rate of infected cell apoptosis 1/day  the rate of progression, from infection to pre-cancer 1/day 2a the rate of proliferation of precancerous cell 1/day 2d the rate of pre-cancerous cell apoptosis 1/day  the maximum invasion rate, from precancerous to cancer 1/day k half-saturation consentration cell/mm3 3a cancer cell proliferation rate 1/day 3d summing the rate of apoptosis and the rate of cancer cell metastasis 1/day n the average number of viruses produced by an infected cell constant 4d the rate of virus death 1/day sk fractional susceptible cells kill by chemotherapy 1/day ik fractional infected cells kill by chemotherapy 1/day pk fractional pre-cancerous cells kill by chemotherapy 1/day ck fractional cancer cells kill by chemotherapy 1/day  the rate of chemotherapy drug decay 1/day mv the rate of chemotherapy drug intake mg/m2.day the dynamics of changes in cervical cells from normal cells to cancer cells are given in the following system of differential equations: 1 s ds s i rs sv k ms dt n           (1a) 1 1 i di sv a i d i i k mi dt       (1b) 1 1 4 dv n d i d v dt   (1c) 2 2 2 2 2 p dp p i a p d p k mp dt k p        (1d) 2 3 32 2 c dc p a c d c k mc dt k p       (1e) m dm m v dt    (1f) let 1 1 2 2 3 3 1 1 1 1 1 1 4 , , , , , , , , , , s i a d a b a d k d a s i n n p c n p c n n d n c d p k k k k                 by non-conventionalizing the system (1a) (1f) to be   1 1 1 1 1 11 s dsds rs s i s v k ms dt dt       (2a) 1 i didi sv ai i k mi dt dt       (2b) dv ni cv dt   (2c) 2 1 121 p dp p pi bp k mp dt p       (2d) 2 21 c dc p kc k mc dt p     (2e) m dm m v dt    (2f) by eliminating the variable index, system (2a) (2f) which is non-professional becomes   1 s ds rs s i sv k ms dt      (3a) i di sv ai i k mi dt      (3b) dv ni cv dt   (3c) 2 21 p dp p pi bp k mp dt p       (3d) 2 21 c dc p kc k mc dt p      (3e) m dm m v dt    (3f) murtono et al. – mathematical model of cervical cancer treatment … 13 equilibrium point theorem 1. let 3 22 9 27 , 3u x xy z v x y     . the equilibrium point with chemotherapy in cervical cancer from system (3a) (3f) is     * * * * * * 3 4 5 5 2 5 1 , , , , , , , , , ,i i ep s i p c v m p c          1 2 3i , , , where 1 0mv     , 2 0 n c    , 1 3 1 sk r     , 2 4 1 0 r            , 1 2 3 5 2 4 ia k           , 5 1 , p p x k b        51 1 1 , .p p p pk b y z k b k b           proof. from equation (3f) is obtained 1 0mv m      . (4) from equation (3c) is obtained 2 ni v i c   , (5) where 2 0 n c    . if equation (4) and equation (5) are substituted into equation (3a), then we obtain 3 4s i   , (6) where 1 3 1 sk r     and 2 4 1 r           . if equations (4), (5) and (6) are substituted in equation (3b), they are obtained 0i  or 1 2 3 5 2 4 ia k i             , (7) where 1 2 3 5 2 4 ia k           . for 𝐼 = 0 impossible, because the virus is the cause of cervical cancer, so it was chosen 𝐼 = 𝜉5. if equation (4) and equation (7) are substituted into equation (1d), then they are obtained 𝑃3 + 𝑥𝑃2 + 𝑦𝑃 + 𝑧 = 0, where 5 51 1 1 1 , , .p p p p p pk b x y z k b k b k b                  the roots are 2 33 1 2 33 1 1 4 3 3 2 1 1 4 , 3 2 x p u u v u u v                 , 2 33 2 2 33 1 3 1 4 3 6 2 1 3 1 4 6 2 x i p u u v i u u v                   2 33 3 2 33 1 3 1 4 3 6 2 1 3 1 4 . 6 2 x i p u u v i u u v                 (8) if equation (4) and equation (8) are substituted into equation (3e), then they are obtained 𝐶 = 𝜃𝑝𝑖 2 (𝑘+𝑘𝐶𝜉1)(1+𝑝𝑖 2) = 𝑐𝑖, where 𝑖 = 1,2,3.■ theorem 2. if 1 2 3 0ia k       , 3 4 5 0    and if one 𝑝𝑖 > 0 and real, then the equilibrium point with chemotherapy or value 𝐸𝑃 exist. proof. from theorem 1 is obtained 𝑀 = 𝑣𝑀 𝛾 = 𝜉1 > 0. because 𝑎 + 𝛿 + 𝑘𝐼𝜉1 − 𝑎𝜉2𝜉3 > 0, then 𝐼 = 𝑎+𝛿+𝑘𝐼𝜉1−𝑎𝜉2𝜉3 𝑎𝜉2𝜉4 = 𝜉5 > 0. because 𝐼 = 𝜉5 > 0, then 𝑉 = 𝑛𝐼 𝑐 = 𝜉2𝐼 > 0. because 𝜉3 + 𝜉4𝜉5 > 0, then 𝑆 = 𝜉3 + 𝜉4𝜉5 > 0. one is 𝑝𝑖 > 0, where 𝑖 = 1,2,3 and real. value is 𝜃𝑝𝑖 2 (𝑘+𝑘𝐶𝜉1)(1+𝑝𝑖 2) = 𝑐𝑖 > 0. so, the equilibrium point with chemotherapy or value 𝐸𝑃 exist.■ stability theorem 3. let 𝑢 = 2𝑥3 − 9𝑥𝑦 + 27𝑧, 𝑣 = 𝑥2 − 3𝑦. if −𝑘 − 𝑘𝐶𝜉1 < 0, 𝑏 − 2𝜃𝑝𝑖 (1+𝑝𝑖 2) − 𝑘𝑃𝜉1 < 0, where 𝑖 = 1,2,3, 𝑥 > 0, 𝑢 > 0, 𝑢2 = 4𝑣3, and 𝑢 < 2𝑥3, then equilibrium point 𝐸𝑃 = (𝜉3 + 𝜉4𝜉5, 𝜉5, 𝜉2𝜉5, 𝑝𝑖 , 𝑐𝑖 , 𝜉1) asymptotic stable. proof. from system (3a) (3f) the jacobian matrix with eigenvalue is obtained 1   , 2 1ck k    ,   3 12 2 2 1 i p i p b k p       , 14 biology, medicine, & natural product chemistry 8 (1), 2019: 11-15 2 33 4 2 33 1 1 4 3 3 2 1 1 4 , 3 2 x u u v u u v                  2 33 5 2 33 1 3 1 4 3 6 2 1 3 1 4 6 2 x i u u v i u u v                    2 33 6 2 33 1 3 1 4 3 6 2 1 3 1 4 . 6 2 x i u u v i u u v                    value is 𝜆1 = −𝛾 < 0. because −𝑘 − 𝑘𝐶𝜉1 < 0, then 𝜆2 = −𝑘 − 𝑘𝐶𝜉1 < 0. because 𝑏 − 2𝜃𝑝𝑖 (1+𝑝𝑖 2)2 − 𝑘𝑃𝜉1 < 0, then 𝜆2 = 𝑏 − 2𝜃𝑝𝑖 (1+𝑝𝑖 2)2 − 𝑘𝑃𝜉1 < 0. because 𝑣 = 0, then 𝜆4 = − 𝑥 3 − 1 3 √𝑢 3 , 𝜆5 = − 𝑥 3 − 1 3 √ 1 2 𝑢 3 , 𝜆6 = − 𝑥 3 + 1 3 √ 1 2 𝑢 3 . because 𝑢 > 0, then 𝜆4 < 0. because 𝑢 < 𝑥3, then 𝜆5 = 𝜆6 < 0. because 𝜆1, 𝜆2, 𝜆3, 𝜆4, 𝜆5 and 𝜆6, then the equilibrium point 𝐸𝑃 asymptotic stable.■ simulation in this section we will discuss numerical simulations and medical interpretations of the mathematical model of cervical cancer affected by chemotherapy. first, the parameter values used and the initial values for each variable are given first. taking parameter values for numerical simulations is still in the form of assumptions based on the rate of growth of cancer cells in general. sources and interpretations of parameter values can be seen in the reference. the parameter values for this case are given in table 2. table 2. case parameter value of the effects of chemotherapy. symbol value reference 𝑟 0.02 asih et al. (2015) 𝛼 0.0001 asih et al. (2015) 𝑎 0.01 asih et al. (2015) 𝛿 0.0082 asih et al. (2015) 𝑛 10000 asih et al. (2015) 𝑐 50 asih et al. (2015) 𝑝 13.44 asih et al. (2015) 𝑏 1 asih et al. (2015) 𝜃 2.03 asih et al. (2015) 𝑘 1.01 asih et al. (2015) 𝑘𝑆 0.0006 estimation 𝑘𝐼 0.6 pillis et al. (2007) 𝑘𝑃 0.6 pillis et al. (2007) 𝑘𝐶 0.8 pillis et al. (2007) 𝛾 0.9 pillis et al. (2007) 𝑣𝑀 1 pillis et al. (2007) figure 2. system simulation (3a) (3f) with parameter values 𝑟 = 0, 𝛼 = 0.0001, 𝑎 = 0.01, 𝛿 = 0.0082, 𝑛 = 10000, 𝑐 = 50, 𝑝 = 13.44, 𝑏 = 1, 𝜃 = 2.03, 𝑘 = 1.01, 𝑘𝑆 = 0.0006, 𝑘𝐼 = 0.6, 𝑘𝑃 = 0.6, 𝑘𝐶 = 0.8, 𝛾 = 0.9, 𝛾 = 1, and initial conditions (13, 13, 13, 13, 13, 13). the dynamics due to normal cell chemotherapy drugs, infected cells, precancerous cells, cancer cells, cancer cells, and viruses declined in three days. (a) normal cells. (b) infected cells, precancerous cells, and cancer cells. (c) viruses. (d) chemotherapy drug concentration. murtono et al. – mathematical model of cervical cancer treatment … 15 figure 2 shows a trajectory with the influence of chemotherapy drugs, on cervical cancer patients. normal cells in the first 3 days showed a decrease, from 13 cells/mm2 to 7,254 cells/mm2. this makes a severe effect for cervical cancer patients with this chemotherapy drug. infected cells, precancerous cells and cancer cells drop very quickly, in the first 3 days of chemotherapy, from 13 cells/mm2 to 0.01308 cells/mm2. the initial virus rose would derease after chemotherapy. the virus in 3 days is from 13 viruses/mm2 to 0.3771 viruses/mm2. this is because, many infected cells die because of the effects of chemotherapy drugs. the chemotherapy drug on the third day still contained 1.91mg/m2. it means that chemotherapy drugs still have an effect on normal cells and other abnormal cells, including cancer cells. conclusion cervical cancer is one type of malignant cancer and is most prevalent in women compared to other cancers. treatment of cervical cancer with chemotherapy is quite effective. abnormal cancer cells such as infected cells, precancerous cells, and many cancer cells die from this treatment. because normal cells are dead, this is the side effect of the of this treatment. this research needs to be continued with other treatments that are more effective, especially to reduce the adverse effects to the normal cell. references asih, t. s. n., lenhart, s., wise, s., aryati, l., adi-kusumo, f., hardianti, m. s., & forde, j. (2016). the dynamics of hpv infection and cervical cancer cells. bulletin of mathematical biology, 78(1), 4-20. boice jr, j. d., day, n. e., andersen, a., brinton, l. a., brown, r., choi, n. w., ... & hakama, m. (1985). second cancers following radiation treatment for cervical cancer. an international collaboration among cancer registries. journal of the national cancer institute, 74(5), 955-975. bosch, f. x., manos, m. m., muñoz, n., sherman, m., jansen, a. m., peto, j., ... & shan, k. v. (1995). prevalence of human papillomavirus in cervical cancer: a worldwide perspective. jnci: journal of the national cancer institute, 87(11), 796-802. de pillis, l. g., gu, w., fister, k. r., head, t. a., maples, k., murugan, a., ... & yoshida, k. (2007). chemotherapy for tumors: an analysis of the dynamics and a study of quadratic and linear optimal controls. mathematical biosciences, 209(1), 292-315. naganawa, s., sato, c., kumada, h., ishigaki, t., miura, s., & takizawa, o. (2005). apparent diffusion coefficient in cervical cancer of the uterus: comparison with the normal uterine cervix. european radiology, 15(1), 71-78. rose, p. g., bundy, b. n., watkins, e. b., thigpen, j. t., deppe, g., maiman, m. a., ... & insalaco, s. (1999). concurrent cisplatin-based radiotherapy and chemotherapy for locally advanced cervical cancer. new england journal of medicine, 340(15), 1144-1153. sari, h. e., mudigdo, a., & demartoto, a. (2016). multilevel analysis on the social determinants of cervical cancer in yogyakarta. journal of epidemiology and public health, 1(2), 100-107. walboomers, j. m., jacobs, m. v., manos, m. m., bosch, f. x., kummer, j. a., shah, k. v., ... & muñoz, n. (1999). human papillomavirus is a necessary cause of invasive cervical cancer worldwide. the journal of pathology, 189(1), 12-19. wuryanti, s., andrijono, a., susworo, s., & witjaksono, f. (2015). the effect of high poly unsaturated fatty acid (pufa) dietary supplementation on inflammatory status of patients with advanced cervical cancer on radiation treatment. acta medica indonesiana, 47(1). this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 273-280 | doi: 10.14421/biomedich.2023.121.273-280 issn 2540-9328 (online) phytochemical and antioxidant activity of blumea balsamifera and cordyline fruticosa based on ethnopharmacology knowledge of muara tae tribe, east kalimantan nur maulida sari1, farida aryani2,*, wartomo1, muhammad fikri hernandi1, erna rositah3, joko prayitno1 1department of forest product processing; 2department of plantation products technology; 3department of forest management, samarinda state agriculture polytechnic, kampus gunung panjang jalan samratulangi, samarinda 75131, east kalimantan, indonesia. corresponding author* faridaaryani@politanisamarinda.ac.id abstract plant use as traditional medicine is still widely practiced in indonesia. muara tae tribe people, west kutai regency are one of the regions that still rely on blumea balsamifera and cordyline fruticosa plants as traditional medicine. this study aims to determine the potential of blumea balsamifera and cordyline fruticosa leaves as medicinal plants with phytochemicals and antioxidants. phytochemical analysis was tested using harborne and kokate methods. antioxidant activity was evaluated by dpph radical scavenging assay with slight modification. the results of the phytochemical analysis showed that the extracts of n-hexane, ethyl acetate, and ethanol from the leaves of blumea balsamifera and cordyline fruticosa contained alkaloids, tannins, and triterpenoids. antioxidant activity of blumea balsamifera leaves extract showed that the n-hexane extract display an ability to inhibit dpph free radical by 50% at 100 ppm concentration, while ethyl acetate and ethanol extracts display an ability by 77% and 81% at 50 ppm concentration. ic50 value of ethyl acetate and ethanol extracts of blumea balsamifera leaves sequentially were 23.68 µg/ml and 17.59 µg/ml. antioxidant activity of cordyline fruticosa leaves extract showed that the n-hexane and ethyl acetate extract display an ability to inhibit dpph free radical by 45% and 56% at 100 ppm concentration, while ethanol extracts display an ability by 76% at 50 ppm concentration. ic50 value of ethyl acetate and ethanol extracts of cordyline fruticosa leaves sequentially were 73.72 µg/ml and 20.17 µg/ml. based on the results, blumea balsamifera and cordyline fruticosa leaves extracts had the potential to develop as natural antioxidants. keywords: blumea balsamifera; cordyline fruticosa; dpph; ethnopharmacology; phytochemical. introduction indonesia has unique flora and fauna that complement its, cultural and ethnic diversity. these ethnic groups occupy certain areas of the country (sreekeesoon & mahomoodally, 2014). each ethnic group has its own lifestyle and traditions, including foods, herbs, and spices. it is also very capable of using and preserving organic and ecological diversity. medicinal plants are used by the local community to treat illnesses, mainly due to health restrictions. the original knowledge of the use of medicinal herbs was probably passed down from generation to generation. this approach has so far succeeded in keeping knowledge alive (yaseen et al., 2015). therefore, local knowledge of medicinal plants has always been a source of research in testing the effects of plants and developing new therapeutic means (bolson et al., 2015). indonesia has the second highest biodiversity in the world after the amazon forests and a population of, people from more than 300 nationalities (elfahmi et al., 2014). most of the studies focused on the prevention of diseases or specific medicinal herbs in indonesia (silalahi et al., 2014). although indonesian researchers have studied traditional knowledge about the use of plants as medicine, most studies have not been published in international journals. about 30,000 medicinal plants grow and develop in indonesia, which covers 90% of medicinal plants in the asian region (syamsiah et al., 2016). the use of traditional medicine is increasing with population growth, healthy lifestyles, and degenerative diseases (triratnawati, 2016). also up to 21,4% of the indonesian population used traditional medicine to self-treat health problems. herbal medicine and massage are the most common treatments offered by traditional healers in indonesia (peltzer & pengpid, 2019). the consumption of traditional medicines in indonesia increased by 5,4% per year. efficiency, lower cost, easy availability, and dissatisfaction with traditional treatment methods are some of the reasons for choosing traditional treatment (riptanti et al., 2018). several studies in recent years manuscript received: 15 january, 2023. revision accepted: 14 march, 2023. published: 23 march, 2023. https://doi.org/10.14421/biomedich.2023.121.273-280 274 biology, medicine, & natural product chemistry 12 (1), 2023: 273-280 about medicinal plants use, informed that plants were the best sources of natural antioxidants such as phenolics, alkaloids, and flavonoids compound (zhao et al., 2018). an antioxidant agent from the plants was a huge resource to scavenging free radicals naturally. medicinal plants also prevent oxidative stress, maintain health, and disease and delay aging processes (nguyen et al., 2018). oxidative stress of human lipids is known to have various human health problems such as cardiovascular disease, cancer, diabetes, and others (truong et al., 2019). generally, antioxidants are divided into 2 types natural and synthetic antioxidants. natural antioxidants are widely used as free radical inhibitors, while synthetic antioxidants had a negative effect with long-term used (stoia & oancea, 2022). muara tae was local tribe exist in west kutai, east kalimantan, indonesia. the muara tae community known as a sub-tribe of dayak used medicinal plants as alternative drugs for healthcare treatment and diseases such as fever, diabetes, external wound, stomachache, and others. several studies about potential plant use by dayak people informed the plant used as natural antifungal, antioxidant, and antibacterial agents by bentian tribe (kusuma et al., 2016). blumea balsamifera and cordyline fruticose were the plant’s belief as medicinal plants by muara tae’s people. these plants had only limited attempts to explore the biological properties of plants regards their uses as medicine by the local people. the present study aims to explore the potential of blumea balsamifera and cordyline fruticosa leaves extract for its antioxidant activity from the muara tae tribe in indonesia (figure 1). materials and methods plant collection the leaves of blumea balsamifera and cordyline fruticosa were collected from muara tae village, west kutai, east kalimantan, indonesia. the samples were washed thoroughly with water to remove the extemporaneous and dried for about 3 days in the laboratory with air conditioning (a.c.) set for 20-25ºc. the samples were kept in a.c. room to keep the moisture content stable and milled with a blender. the powdered samples were prepared for further analysis. figure 1. morphology of blumea balsamifera and cordyline fruticosa plants (photo source: personal doc.). procedures maceration about 30 gr powdered samples of blumea balsamifera and cordyline fruticosa were extracts with n-hexane, ethyl acetate, and ethanol solvent at room temperature with continuous shaking on a shaker for 48 hours. following filtration of the suspension through whatman paper no.2 (maidstone, uk), the crude extracts of blumea balsamifera and cordyline fruticosa were evaporated in a rotary evaporator at 38-40 ºc and put in a vacuum over near dryness to yield the plant extract. preliminary phytochemical analysis the n-hexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves were subjected to preliminary screening of phytochemical such as alkaloids, flavonoids, tannin, steroids, triterpenoids, carbohydrate, and saponin using some following standard procedures (harborne, 1998; kokate, 2001). alkaloids determination: 5 ml of the n-hexane, ethyl acetate, and ethanol extracts of blumea balsamifera sari et al. – phytochemical and antioxidant activity of … 275 and cordyline fruticosa leaves were added to 2 ml hydrochloride acid, then 1 ml of dragendorff solution was added. the color changes in the solution indicated the presence of alkaloids (kokate, 2001). flavonoids determination: about 1 ml of the nhexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves were drops of 1% sodium hydroxide. the presence of yellow color at extracts solution and were colorless after the addition of 1 % hydrochloride acid indicated the presence of flavonoids (kokate, 2001). tannin determination: 10 ml of the n-hexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves were added 1 % lead ii acetate. the yellow precipitate reaction in the solution indicated the presence of tannin (kokate, 2001). steroids determination: 1 ml of the n-hexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves dropped about 10 acetic acid anhydride and 2 drops of sulfuric acid, sequentially. the green or blue color changes in the solution indicated the presence of steroids (harborne, 1998). triterpenoids determination: 1 ml of the nhexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves dropped about 10 acetic acid anhydride and 2 drops of sulfuric acid, sequentially. the red or purple color changes in the solution indicated the presence of steroids (harborne, 1998). carbohydrate determination: about 1 drop of molisch solution were added to 1 ml of the n-hexane, ethyl acetate and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves. then 1 ml of sulfuric acid was added thourgh the tube glass wall, slowly. the formation of purple ring between 2 layers indicated the presence of carbohydrates (harborne, 1998). saponins determination: 10 ml of hot distilled water was added to 1 ml of the n-hexane, ethyl acetate, and ethanol extracts of blumea balsamifera and cordyline fruticosa leaves. the solution was then cooled and shaken vigorously (10 seconds). a stable froth upon standing for 10 minutes after adding 1 drops of hydrochloride acid 2n indicated the presence of saponins (harborne, 1998). antioxidant assay test of antioxidants using 5 concentration samples was grouped into 100 ppm, 50 ppm, 25 ppm, 12.5 ppm, and 6.25 ppm times of dilution, respectively. further, 3 mg of ascorbic acid was weighed, then dissolved in 1000 µl of ethanol solvent and regarded as a positive control. while the ethanol solvent was used as a negative control. about 33 µl sample was mixed in a glass tube with 467 µl of ethanol added, and 500 µl of 2,2-diphenyl-1picryhydrazyl (dpph) radical scavenging activity (shimizu et al., 2001). the mixing of the sample was stopped while the volume reached 1000 µl (1 ml). samples were incubated for 20 minutes with minimum light and a.c. set for 27-30 ºc. the antioxidant activity was determined by decolorization of dpph with a wavelength of 517 nm using a spectrophotometer. measurement was performed in the triplicate examination. the percentage of dpph free radical was calculated using the following equation: % dpph radical scavenging activity = ∆𝑐𝑜𝑛𝑡𝑟𝑜𝑙− ∆𝑠𝑎𝑚𝑝𝑙𝑒 ∆𝑐𝑜𝑛𝑡𝑟𝑜𝑙 x 100 (1) the actual decrease in absorbance caused by the test was compared with positive controls. the values of ic50 (concentration giving 50% of inhibition) were calculated by a dose-inhibition curve over a linear range of by plotting the extract concentration and the corresponding washout effect (tuldjanah et al., 2021). results and discussion plant extracts blumea balsamifera and cordyline fruticosa leave were extracted using 3 different solvents n-hexane, ethyl acetate, and ethanol. maceration is done with yielded 2.18-4.60% extracts on the basis of sample dry weight from blumea balsamifera leaves, while cordyline fruticosa leaves yielded 1.09-5.20% extracts (table 1). table 1. yield of blumea balsamifera and cordyline fruticosa leaves extract. plants solvent extract yield (%) blumea balsamifera n-hexane 2.18 ethyl acetate 3.18 ethanol 4.60 cordyline fruticosa n-hexane 1.09 ethyl acetate 3.03 ethanol 5.20 the results showed the ethanol extracts of blumea balsamifera and cordyline fruticosa produce an extractive content more concentrated than the n-hexane and ethyl acetate extracts. phytochemical screening the n-hexane, ethyl acetate, and ethanol leaf extracts of blumea balsamifera and cordyline fruticosa were analyzed as the secondary metabolites. plants known to contain multiple phytochemical molecules such as terpenoids, lignins, phenolics, vitamins, tannins, and others metabolites as antioxidant agents. several studies showed many phytocompounds possess antidiabetic, antimicrobial, and anti-inflammatory activities (campbell-tofte et al., 2012). the pharmacological activities of secondary plant metabolites are known as large compounds as a source of medicinal agents (muthukrishnan & manogaran, 2018). phytochemical analysis of the n-hexane, ethyl acetate, and ethanol leaves extracts of blumea balsamifera and cordyline fruticosa is presented in table 2. 276 biology, medicine, & natural product chemistry 12 (1), 2023: 273-280 table 2. phytochemical analysis of blumea balsamifera and cordyline fruticosa leaves extract. compounds blumea balsamifera cordyline fruticosa n-hexane ethyl acetate ethanol n-hexane ethyl acetate ethanol alkaloids + + + + flavonoids tannins + + + steroids triterpenoids + + carbohydrate saponins the phytochemical screening of blumea balsamifera revealed the presence of alkaloids and triterpenoids in the n-hexane extract, and tannins in the ethyl acetate extract, while the ethanol extract contained alkaloids and tannins. the n-hexane extract of cordyline fruticosa revealed the presence of alkaloids and triterpenoids, while the ethanol extract contained alkaloids and tannins. the bioactivity potency of plants was indicated based on the secondary metabolites compounds. the presence compounds of tannins, flavonoids, and alkaloids are known as antitumor, antibacterial, antivirus also had antioxidant activity, antimicrobial and anticancer (taşkın & taşkın, 2017). phenolic known as the largest compounds were found in plants and it’s an important compound for free radical scavenging, also contains the hydroxyl group compound. flavonoid, phenol, and tannin compound are known as the largest group of phenolic compounds and also had the abilities as antioxidant agents (sen et al., 2013). antioxidant activity free radical dpph scavenging of the n-hexane, ethyl acetate, and ethanol leaves extracts of blumea balsamifera and cordyline fruticosa was determined by its hydrogen donating abilities. the inhibition of blumea balsamifera n-hexane leaves extracts displayed the ability to inhibit free radical dpph formation by 50% at 100 ppm concentration, while the ethyl acetate and ethanol extracts by 77% and 81% at 50 ppm concentration, respectively. the antioxidant activity of the n-hexane, ethyl acetate, and ethanol leaf extracts of blumea balsamifera is presented in figure 2. figure 2. antioxidant activity of blumea balsamifera leaves extracts against dpph. several studies about blumea balsamifera plants informed the plant extracts contained steroids, alkaloids, phenolic and saponins compounds used as traditional medicine for eczema, rheumatic, menstrual pain relief, flu, fever, asthma, diabetes cough, and diarrhea (pang et al., 2014). its also has biological activity as an antiinflammatory, anticancer, and antioxidant and also functions as an antimicrobial (nessa et al., 2010). the inhibition of cordyline fruticosa n-hexane and ethyl acetate leaf extract displayed the ability to inhibit sari et al. – phytochemical and antioxidant activity of … 277 free radical dpph formation by 45% and 56% at 100 ppm concentration, while the ethanol extracts by 76% at 50 ppm concentration. antioxidant activity of the nhexane, ethyl acetate, and ethanol leaves extracts of cordyline fruticosa is presented in figure 3. figure 3. antioxidant activity of cordyline fruticosa leaves extracts against dpph. several studies about cordyline fruticosa leaves plants, informed the plant contained saponins, tannins, flavonoids, polyphenol, and alkaloids compound which has biological activity as antioxidant, anti-inflammatory, antibacterial, anti-allergic, anticancer, and antivirus (dyary et al., 2014). this plant is also known to accelerate the wound healing process, the function of tannins compound as astringents uses shrink skin pores and bleeding minor stopped. the antioxidant activity of blumea balsamifera and cordyline fruticosa leaves extracts indicated plants had a source to inhibit free radical dpph. refers to the results, the ic50 of the ethyl acetate and ethanol leaves extracts of blumea balsamifera and cordyline fruticosa extracts were determined as shown in table 3. table 3. the ic50 of blumea balsamifera and cordyline fruticosa leaves extract. blumea balsamifera cordyline fruticosa ethyl acetate ethanol ethyl acetate ethanol 23.68±19.69 17.59±18.71 73.72±14.42 20.17±17.59 the antioxidant activity of ic50 had a classification of the antioxidant activities compound based on the acquisition of the ic50 value as shown as table 4 (analianasari et al., 2022). table 4. the antioxidant activity of ic50 classification. inhibition value (ppm) category <50 extreme 50-100 strong 100-150 moderate 150-200 weak >200 fragile 278 biology, medicine, & natural product chemistry 12 (1), 2023: 273-280 figure 4. antioxidant activity of cordyline fruticosa leaves extracts against dpph. based on figure 4, the linear equation value for blumea balsamifera ethyl acetate extract y = 1.0237x+25.762, the calculation of the ic50 value for blumea balsamifera ethyl acetate extract obtained the following values: y = 1.0237x+25.762 (for y = 50), then the x value is 23.68 ppm. furthermore, based on the calculation of the regression curve in the ethanol extract of blumea balsamifera, the regression equation y = 0.964x + 33.039 (assuming the value of y = 50) is obtained, and the x value is 17.59 ppm. the ic50 value of the blumea balsamifera ethyl acetate and ethanol extracts had extreme ic50 antioxidant activity (<50 ppm). several studies about blumea balsamifera, especially the leaves part informed fresh and dry leaves had antioxidant activity with boiling water method, antidiabetic activity with hydro-ethanol extract, antitumor activity with essential oil extract and also anticancer activity of ethyl acetate extract fraction (widhiantara & jawi, 2021). the linear equation value for cordyline fruticosa ethyl acetate extract y = 0.3628x + 23.253 (assuming the value of y = 50), then the x value is 73.72 ppm. the ic50 value of cordyline fruticosa ethyl acetate extract is strong (50-100 ppm). while the regression of cordyline fruticosa ethanol extract equation y = 0.902x + 31.804 (with the value of y = 50) is obtained, the x value is 20.17 ppm. the ic50 value of cordyline fruticosa ethanol extract had extreme ic50 antioxidant activity (<50 ppm). the bioactive compounds of cordyline fruticosa methanol leaf extract have been isolated. the active compound had antitumor activity, antibacterial activity, and cytotoxic activity (assyifa et al., 2022). conclusions an ethnobotanically-selected medicinal plant, blumea balsamifera, and cordyline fruticosa has been investigated for their antioxidant activities. the results informed that the leaves extract of ethyl acetate and ethanol of the plant showed good antioxidant properties by the extreme-good category of ic50. further investigation is needed to find the responsible compound in the blumea balsamifera and cordyline fruticosa plants and explored the possibility of bioproduction for the active compounds, also developed for healthcare in the future. acknowledgments: the research was funded by the ministry of education, culture, research, and technology through penelitian dosen pemula scheme (junior lecturer research grants) 2022 under contract number 012/pl21.g/pg/2022. acknowledgments were conveyed to the muara tae village, west kutai, east kalimantan, indonesia who have supported the research by sharing ethnopharmacological information and plant specimens. authors’ contributions: in this research, nur maulida sari designed the research, supervised all processes, and wrote the manuscript. wartomo was observing and taking research samples. muhammad fikri hernandi controlled the samples and preparation of materials. joko prayitno 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(2018). effect of drying processes on prenylflavonoid content and antioxidant activity of epimedium koreanum nakai. journal of food and drug analysis, 26(2), 796–806. https://doi.org/10.1016/j.jfda.2017.05.011 https://doi.org/10.1016/j.jep.2014.12.053 https://doi.org/10.1016/j.jfda.2017.05.011 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 409-415 | doi: 10.14421/biomedich.2025.141.409-415 issn 2540-9328 (online) 4-hydroxyisoleucine as a natural dpp-4 inhibitor for diabetes amir thalib1,*, irma putri damayanti2 1department of medicine; 2department of herbal medicine, faculty of medicine; university of muhammadiyah purwokerto jl. kh. ahmad dahlan, purwokerto 53182, indonesia. corresponding author* amirthalib18@gmail.com manuscript received: 28 february, 2025. revision accepted: 20 may, 2025. published: 16 july, 2025. abstract research on 4-hydroxyisoleucine, a natural compound found in several plant sources, shows potential as an antidiabetic agent through inhibiting the dpp-4 (dipeptidyl peptidase-4) enzyme. this study evaluates the pharmacokinetic potential and toxicity profile of 4hydroxyisoleucine as a therapeutic agent. adme (absorption, distribution, metabolism, excretion) analysis indicates that this compound has good gastrointestinal absorption, moderate water solubility, and limited penetration across the blood-brain barrier, which reduces the risk of central nervous system side effects. the toxicity profile of 4-hydroxyisoleucine reveals low hepatotoxicity, with no indications of mutagenicity or carcinogenicity. the ld50 value greater than 2000 mg/kg places this compound in toxicity class 5, indicating low toxicity. based on in silico evaluation results, 4-hydroxyisoleucine has potential as an effective natural dpp-4 inhibitor, with stable binding mechanisms, even though its binding affinity is lower than synthetic inhibitors. with favorable pharmacokinetic properties and a beneficial safety profile, 4-hydroxyisoleucine has the potential to be developed as a natural therapeutic agent for diabetes management. keywords: 4-hydroxyisoleucine; adme; toxicity profile; dpp-4; antidiabetic. abbreviations: dpp-4: dipeptidyl-peptidase 4; gip: glucose-dependent insulinotropic polypeptide; glp-1: glucagon-like peptide-1 introduction a major therapeutic target in the management of type 2 diabetes mellitus (t2dm) is the enzyme dipeptidyl peptidase-4 (dpp4), which is responsible for degrading incretin hormones such as glucagon-like peptide-1 (glp-1) and glucose-dependent insulinotropic polypeptide (gip). these hormones stimulate insulin secretion in response to glucose intake. synthetic dpp4 inhibitors have been widely used to enhance incretin levels, improve glycemic control, and minimize the risk of hypoglycemia. however, synthetic dpp4 inhibitors, such as alogliptin, linagliptin, saxagliptin, sitagliptin, and vildagliptin, are often associated with side effects, including gastrointestinal disturbances, joint pain, and an increased risk of respiratory infections (azriful et al., 2018). the introduction of an in silico analysis of 4hydroxyisoleucine as a natural antidiabetic agent focusing on its interaction with angiotensin-converting enzyme type 2 (dpp-4) and pharmacokinetic evaluation serves to highlight the compound’s potential therapeutic value. diabetes mellitus, a metabolic disorder marked by elevated blood glucose levels, is increasingly prevalent worldwide and poses significant public health challenges. one of the key targets in the management of diabetes is the dpp-4 enzyme, which plays a pivotal role in regulating insulin secretion and glucose metabolism. inhibiting dpp-4 has emerged as a promising therapeutic approach for controlling blood glucose levels in diabetic patients. 4-hydroxyisoleucine, a naturally occurring amino acid derivative primarily found in fenugreek seeds, has been shown to possess antidiabetic properties through its effects on glucose metabolism. this study aims to explore the in silico interaction of 4-hydroxyisoleucine with dpp-4, a critical enzyme involved in glucose homeostasis. by utilizing advanced computational tools and molecular docking techniques, we aim to assess how 4-hydroxyisoleucine binds to the dpp-4 receptor, potentially inhibiting its activity and contributing to better glycemic control. additionally, pharmacokinetic evaluations are integral to understanding the compound's bioavailability, absorption, distribution, metabolism, and excretion (adme) properties. this aspect is crucial to determine the compound's potential for effective oral administration and safety profile (anitha et al, 2013). swissadme and other computational platforms predict the pharmacokinetic properties of 4hydroxyisoleucine, providing insights into its solubility, permeability, and potential toxicity. in summary, this study explores 4-hydroxyisoleucine’s dual role as a natural dpp-4 inhibitor and evaluates its https://doi.org/10.14421/biomedich.2025.141.409-415 410 biology, medicine, & natural product chemistry 14 (1), 2025: 409-415 pharmacokinetic characteristics to establish its viability as a potential therapeutic agent for diabetes management. the findings could pave the way for further research and development of 4-hydroxyisoleucine-based formulations in treating diabetes, offering a promising natural alternative to synthetic drugs (itoh et al, 2019). materials and methods study area for molecular docking process, this study used a computer with specifications: intel(r) celeron(r) n4020 cpu @ 1.10ghz, 1101 mhz, 2 core(s), 2 logical processor(s), 4 gb ram, and operating system: microsoft windows 11 home single language (10.0.22631 build 22631) bios: dvcn17ww procedures preparation and ligands the primary compound analyzed in this study was 4hydroxyisoleucine, a natural glycoside derived from fava beans (vicia faba). the 2d and 3d structures of 4hydroxyisoleucine were obtained from the pubchem database (cid: xxxx) in sdf format. the target protein, dipeptidyl peptidase-4 (dpp4), was retrieved from the protein data bank (pdb id: xxxx) in pdb format. the protein and ligand structures were prepared using pymol software (made & pathni, 2018). water molecules were removed from the protein structure to ensure accurate molecular docking, while polar hydrogens and kollman charges were added to stabilize the protein-ligand interaction (nayeem et al., 2021). the ligand structure was optimized for energy minimization using open babel within pyrx software to eliminate steric clashes and maintain proper molecular geometry. the prepared protein and ligand files were saved in pdbqt format, making them compatible with autodock vina. these prepared structures were then used for the docking process to analyze the binding affinity of 4hydroxyisoleucine to dpp4 and compare it to standard synthetic inhibitors, including alogliptin, linagliptin, saxagliptin, sitagliptin, and vildagliptin (pantaleão et al., 2018). protein validation in an silico analysis for protein validation after the preparation of the protein and ligand, several steps are followed to ensure that the obtained protein structure and its interaction with the ligand are accurate and acceptable for further studies. initially, after the protein structure is prepared, software tools such as pymol for visualization and structural analysis are used. this visual inspection aims to confirm no visible structural defects, such as irregular loops or improper bindings. following this, the quality of the prepared protein structure is assessed using tools like procheck, which help verify the validity of the protein structure based on geometric statistics and the distribution of phi-psi angles. while statistical analyses such as ramachandran plots are commonly used for further evaluation, the focus is on ensuring the protein model used in the simulation is reliable. additionally, the protein structure’s interaction energy is assessed to ensure that the protein is in a stable state. molecular dynamics simulations or protein-ligand binding energy predictions using software like autodock are used to verify that the interaction with the ligand remains within biologically acceptable parameters. the protein structure is then refined through energy optimization procedures, where atom positions are adjusted to minimize the total energy, enhancing the model’s accuracy and ensuring it represents the natural protein form more precisely (samata et al., 2019). finally, the stability of the protein after ligand interaction is evaluated by conducting molecular dynamics (md) simulations to assess the structure's stability under biologically relevant conditions. this involves examining the md simulation trajectories to ensure the validated protein structure remains stable over a set period. these steps are essential in the protein validation process within in silico analysis, ensuring that the structure used in experiments can predict ligand interactions accurately and support further analysis regarding the desired biological activity. molecular docking process molecular docking simulations were performed using pyrx, an open-source software integrating autodock vina. this study focused on calculating the binding affinity of 4-hydroxyisoleucine to the active site of dpp4. grid box parameters were set to encompass the entire binding site of dpp4, ensuring comprehensive coverage for ligand-receptor interactions. the docking process began with loading the prepared protein and ligand files into pyrx. the protein was fixed as rigid, while the ligand was treated as flexible to allow for accurate conformational adjustments during docking. docking results were generated as binding affinity values in kcal/mol, which reflect the stability of the proteinligand complex (suciana & arifianto, 2019) post docking visualization post-docking visualization is a crucial step after completing the docking process. for instance, after docking the protein and ligand using a tool like pyrx, the resulting data is saved for further analysis. this data, typically containing the docking poses and binding energies, is then visualized using pymol to allow better understand the interaction between the ligand and the protein. by reloading the docking results in pymol, the researcher can easily identify key details, such as the types of bonds formed (hydrogen bonds, hydrophobic interactions, or ionic bonds) between the ligand and the thalib & damayanti – 4-hydroxyisoleucine as a natural dpp-4 inhibitor for diabetes 411 protein. this visualization helps determine the precise binding site on the protein, the orientation of the ligand within the binding pocket, and the strength of the interactions, which are crucial for evaluating the potential efficacy of the ligand as a therapeutic agent. results and discussion ligand and protein preparation figure 1. the ramachandran procheck. before beginning the molecular docking studies, the ligand and the protein (dpp4) were prepared. the 3d structure of 4-hydroxyisoleucine was obtained from the pubchem database. the optimized structure was minimized to ensure the ligand’s conformation was energetically favorable for docking. for the protein preparation, the crystal structure of dpp4 was retrieved from the protein data bank (pdb id: 4wz5) (setiawansyah et al., 2022). the protein was cleaned by removing water molecules and heteroatoms that could interfere with docking, and it was also protonated to ensure proper hydrogen bonding during interaction with the ligand. the protein was then prepared using autodocktools, and its grid box was defined to cover the binding site. structural validation of dpp4 protein structural validation of the dpp4 protein model was conducted using procheck (figure 1.) and errat tools (figure 2.). the ramachandran plot (figure 1.) revealed that 89.4% of residues were in the most favored regions, with 9.2% in additional allowed regions and only 1.4% in disallowed regions. errat analysis (figure 2.) yielded a quality score of 95.7%, confirming the reliability of the dpp4 model for molecular docking studies. this validation ensures the robustness of the results and strengthens the credibility of 4-hydroxyisoleucine as a potential drug candidate. figure 2. structure of errat tools. docking results the in silico analysis of 4-hydroxyisoleucine as a natural antidiabetic agent focuses on the interaction between 4hydroxyisoleucine and the target enzyme dpp-4. molecular docking was performed to predict the binding between the ligand (in this case, 4-hydroxyisoleucine) and the active site of the dpp-4 enzyme, a primary target in glucose regulation in diabetes. the docking results 412 biology, medicine, & natural product chemistry 14 (1), 2025: 409-415 provide insights into the strength of the interaction and the stability of the binding between 4-hydroxyisoleucine and dpp-4, as well as the potential of this compound in inhibiting enzyme activity. the main docking results show that 4-hydroxyisoleucine has a good affinity for the dpp-4 active site. based on the docking scores calculated, 4hydroxyisoleucine forms stable interactions with several important residues in the active site of the dpp-4 enzyme, which are involved in inhibiting its activity. this molecule can to functional groups on the enzyme, such as amino acids involved in forming the active site, leading to the inhibition of peptide cleavage and regulation of insulin secretion. furthermore, the interaction analysis indicates that 4-hydroxyisoleucine forms several hydrogen bonds and hydrophobic interactions with critical residues in dpp-4. these interactions support the stability of the ligandenzyme complex and enhance the compound's potential in inhibiting dpp-4 activity. a comparison with conventional dpp-4 inhibitors, such as sitagliptin, shows that 4-hydroxyisoleucine has similar potential in binding to the enzyme's active site, although with slightly different binding mechanisms. the conclusions from these docking results suggest that 4-hydroxyisoleucine has the potential to be an effective natural dpp-4 inhibitor, which could contribute to the control of blood glucose levels. its strong and stable interaction with the dpp-4 active site makes it a promising candidate for developing natural-based antidiabetic drugs. these findings pave the way for further research, both in experimental testing and the development of more effective and safe antidiabetic drug formulations (setiawansyah et al., 2022). post-docking analysis post-docking visualization confirmed significant interactions between 4-hydroxyisoleucine and active site residues of dpp4. key hydrogen bonds and hydrophobic interactions were observed, highlighting the stability of the 4-hydroxyisoleucine-dpp4 complex. these interactions contribute to the compound’s binding stability and reinforce its potential efficacy in inhibiting dpp4 activity. figure 3. visualization of molecular docking of ligands and protein. (a) 4-hydroxyisoleucine, (b) alogliptin, (c) linagliptin, (d) sitagliptin, (e) vildagliptin, (f) saxagliptin. comparison with synthetic dpp4 inhibitors the comparison between 4-hydroxyisoleucine and several synthetic dpp-4 inhibitors reveals significant differences in their molecular properties and binding affinities. 4-hydroxyisoleucine has a molecular weight of 147.17 g/mol, which is notably lower than that of synthetic dpp-4 inhibitors such as linagliptin (472.54 g/mol) and sitagliptin (407.31 g/mol). this difference in molecular weight could influence the pharmacokinetics and drug-like properties of the compounds, with 4hydroxyisoleucine's smaller size potentially offering advantages such as better oral bioavailability and metabolic stability. furthermore, 4-hydroxyisoleucine has a binding affinity of -5.0 kcal/mol, which is lower compared to the more potent synthetic inhibitors, with linagliptin showing the highest affinity at -8.7 kcal/mol, followed by sitagliptin at -8.0 kcal/mol and saxagliptin at -6.8 thalib & damayanti – 4-hydroxyisoleucine as a natural dpp-4 inhibitor for diabetes 413 kcal/mol. these findings suggest that synthetic inhibitors typically interact more with the dpp-4 enzyme, potentially contributing to their higher potency in enzyme inhibition. the molecular formulas of these compounds also show notable differences, with synthetic inhibitors having more complex structures featuring larger carbon chains and additional functional groups, whereas 4-hydroxyisoleucine has a simpler structure (c6h13no3). despite the lower binding affinity of 4hydroxyisoleucine, its natural origin, simpler structure, and smaller size make it a promising candidate for further research as a natural-based antidiabetic agent. the lower binding affinity does not diminish its potential efficacy, especially if it can offer other benefits such as a better safety profile, fewer side effects, and possible synergistic effects when used in combination therapies. future research is needed to explore the practical therapeutic applications of 4-hydroxyisoleucine in diabetes management and its potential as an alternative to synthetic dpp-4 inhibitors. ligands cid molecular formula molecular weight (g/mol) binding affinity (kcal/mol) 4-hydroxyisoleucine 2773624 c6h13no3 147.17 -5,0 alogliptin 10244493 c18h21n5o2s 339,45 -6,6 linagliptin 10096344 c25h28n8o2 472,54 -8,7 saxagliptin 11235634 c18h25n3o2 315,41 -6,8 sitagliptin 4369359 c16h15f6n5o 407,31 -8,0 vildagliptin 6918586 c17h25n3o2 303,40 -7,3 adme and toxicity profile the adme (absorption, distribution, metabolism, excretion) and toxicity profile of 4-hydroxyisoleucine were evaluated to assess its pharmacokinetic properties and safety as a potential therapeutic agent. 4hydroxyisoleucine demonstrated high gastrointestinal absorption, indicating that the compound can be efficiently absorbed into the bloodstream orally, making it a suitable candidate for oral drug formulations. additionally, the compound showed moderate water solubility, which enhances its potential for distribution throughout the body. however, it exhibited limited penetration across the blood-brain barrier (bbb), which reduces the risk of central nervous system side effects, helping to target peripheral tissues without affecting brain function. predictions suggest that 4-hydroxyisoleucine does not undergo extensive metabolism, which implies that it may retain its bioactive form for a longer duration in the body. this characteristic could lead to prolonged therapeutic effects, as the compound is less likely to be rapidly broken down by metabolic enzymes. furthermore, 4hydroxyisoleucine is expected to be excreted primarily through the urine, a common pathway for small molecules with low molecular weight, emphasizing the importance of renal function in eliminating the compound from the body. in terms of toxicity, 4hydroxyisoleucine was predicted to have a favorable safety profile, associated with low hepatotoxicity, suggesting minimal risk to liver function (setiawansyah et al., 2022). it also did not show signs of mutagenicity or carcinogenicity, indicating that it is unlikely to cause genetic mutations or cancer. with an ld50 value greater than 2000 mg/kg, 4-hydroxyisoleucine falls under toxicity class 5, classifying it as a low-toxicity substance. this further supports its safety for use in therapeutic applications. in summary, 4hydroxyisoleucine exhibits favorable adme properties, including high oral bioavailability, moderate solubility, and low toxicity. these characteristics make it a promising candidate for further development as a natural therapeutic agent, particularly for conditions like diabetes. the compound's excellent safety profile and pharmacokinetic features position it as an attractive option for future drug development with minimal risk of adverse effects. 414 biology, medicine, & natural product chemistry 14 (1), 2025: 409-415 figure 4. admet analysis of flavonoid drugs demonstrating profiles of absorption, distribution, metabolism, excretion, and toxicity. this data is utilized to assess the pharmacokinetic potential and safety of substances in medication development. potential for further development the potential for further development of 4hydroxyisoleucine as a therapeutic agent is substantial, particularly in treating metabolic disorders such as diabetes. given its promising adme properties, including high gastrointestinal absorption, moderate solubility, and favorable safety profile, 4hydroxyisoleucine shows strong potential for development into an effective, natural-based pharmaceutical. its ability to be efficiently absorbed in the digestive tract and its relatively low toxicity profile suggest that it could be a safer alternative to some conventional synthetic drugs, potentially reducing the risk of adverse effects commonly associated with longterm use of synthetic antidiabetic agents. moreover, 4-hydroxyisoleucine’s natural origin offers an added advantage, as patients increasingly seek plantbased or natural treatments with fewer side effects. the compound’s lower molecular weight and minimal metabolism also indicate that it could be retained in the body for a longer period, potentially offering more sustained therapeutic effects. the fact that 4hydroxyisoleucine has been shown to interact with key targets such as the dpp-4 enzyme further enhances its potential as a promising agent for diabetes management. inhibiting dpp-4 can lead to improved insulin secretion and better blood glucose regulation, which is critical for diabetes treatment (xiao et al., 2018). in addition, the promising pharmacokinetic characteristics of 4-hydroxyisoleucine, coupled with its low hepatotoxicity and lack of mutagenic or carcinogenic properties, position it as a safe candidate for use in future drug formulations. the compound’s relatively low binding affinity compared to synthetic dpp-4 inhibitors does not necessarily diminish its therapeutic potential, as further optimization and combination therapies could enhance its efficacy. the future development of 4-hydroxyisoleucine could involve clinical studies to validate its efficacy in human trials, explore its long-term safety, and refine formulations for maximum bioavailability and therapeutic benefit. its natural composition and minimal toxicity profile suggest that, with further research and development, it could become a valuable addition to the array of treatments available for managing diabetes and other metabolic conditions. thus, the continued exploration of 4-hydroxyisoleucine’s pharmacological properties holds significant promise for advancing natural-based therapies in medicine (yuan et al., 2017). thalib & damayanti – 4-hydroxyisoleucine as a natural dpp-4 inhibitor for diabetes 415 conclusions this study of 4-hydroxyisoleucine presents a promising candidate for further development as a natural therapeutic agent, particularly for treating diabetes. its favorable adme properties, including high gastrointestinal absorption, moderate solubility, and minimal toxicity, position it as a viable alternative to synthetic dpp-4 inhibitors. the compound’s ability to interact with key targets involved in glucose regulation, along with its low risk of adverse effects, makes it an attractive option for future drug development. while its binding affinity to dpp-4 is lower than of synthetic inhibitors, its natural origin, simple molecular structure, and potential for sustained therapeutic effects warrant further research and optimization. overall, 4hydroxyisoleucine's promising pharmacokinetic profile, coupled with its safety and efficacy, suggest that it could play an important role in the development of naturalbased therapies for diabetes and other metabolic disorders. acknowledgements: the author thanks to university of muhammadiyah purwokerto for the time given to conduct research as well as the provision of computers for docking with the required specifications. authors’ contributions: irma putri damayanti to designed the study, provide input and supervise the docking. amir carried out the docking work, analyzed the data and wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. funding: the authors declare no funding in this research. references anitha, k., gopi, g., girish, & kumar, p. s. 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(2017). a new sesquiterpene lactone from yacon leaves. natural product research, 31(1), 43–49. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 485-498 | doi: 10.14421/biomedich.2023.122.485-498 issn 2540-9328 (online) determination of oil quality and antifungal effect of selected citronella accessions (cymbopogon nardus, cymbopogon winterianus) to formulate an anti-dandruff shampoo rathnayaka mudiyanselage nipuni wijerathna1, achini anuradha wijeweera2, anushi madushani wijethunga1, mapa mudiyanselage sumudu tharangani mapa1,* 1department of biosystems technology, faculty of technology, university of sri jayewardenepura, sri lanka. 2national cinnamon research and training center, sri lanka. corresponding author* sumudu.mapa@sjp.ac.lk manuscript received: 28 march, 2023. revision accepted: 21 august, 2023. published: 23 august, 2023. abstract citronella is an aromatic grass of the family poaceae which can be classified into two categories ceylon citronella (cymbopogon nardus) and java citronella (cymbopogon winterianus). the citronella oil was extracted from five selected ceylon citronella (hp t1, hp t2 and hp t3) and java citronella (mp t1 and mp t2) accessions using steam distillation and hydrodistillation methods. citronella oil quantity extracted by hydrodistillation with xylene from ceylon citronella was higher (2.45-2.67 ml/100 g) than the java citronella (1.57-1.64 ml/100 g). the oil quantity of ceylon citronella (hp t1-5.52 %, hp t21.40 %, hp t31.05 %) and the quantity of java citronella (mp t11.25%, mp t21.79%) extracted by hydrodistillation showed a significant difference (p<0.0001) and there was no significant difference (p=0.7055) between the oil quantity of ceylon (hp t11.07%, hp t21.18 %, hp t31.19%) and java (mp t11.16%, mp t21.23%) oils extracted by the steam distillation. both java and ceylon citronella oils showed organoleptic properties with pale yellow to pale brownish yellow colour and a strong citrusy aroma which meets the iso 3848 and iso 3849 standards. the oil of ceylon citronella accessions showed refractive index (1.465-1.487), relative density (0.893-0.910), and ethanol solubility (1:2 ml) within the ranges specified in sls 170 standards. java citronella oil exhibited the refractive index (1.4660-1.4730), relative density (0.880-0.892), ethanol solubility (1:2 ml), and optical rotation (-5˚ to 0˚) which meets the specifications of iso 3848 standards. geraniol, citronellol, and citronellal were identified as the major constituents using the gas chromatography-mass spectrometry (gc-ms) where java citronella oil showed high geraniol content (48.60-49.17%) than ceylon citronella oil (16.93-26.49%). all types of tested citronella oil showed inhibition against candida albicans where hp t3 (1.9 cm) and mp t1(2.0 cm) oils showed the highest promising antifungal activity among ceylon oils and java oils respectively. therefore, these two oils were selected for the antidandruff shampoo formulation. the two antidandruff shampoo samples were formulated with 2% v/v concentrations of hp t3 and mp t1 citronella oil which were determined as mic for the inhibition of c. albicans. antidandruff shampoo tested against c. albicans showed greater antifungal activity (hp t3 2.5±0.05 cm; mp t1 2.5±0.05 cm) than the crude citronella oil (hp t31.9±0.11 cm; mp t1-2.0±0.1 cm), also attained the organoleptic and physical properties such as ph (4.0-8.0), foam height (>100 ml), dirt dispersion (no ink in foam), viscosity, low wetting time and solid content (hp t3-14.75±0.12%; mp t2-12.33±0.19%) in acceptable specification range. this study exhibits that ceylon citronella oil hp t1 has the highest oil quantity from all selected accessions. hydrodistillation can be used to extract high oil quantity than the steam distillation method from both java and ceylon citronella types. compared to ceylon citronella oil, java oil has significant potential industrial applications with high geraniol content and with the highest antifungal activity against c. albicans. also, the tested citronella oil of all selected accessions of both java and ceylon types meet the organoleptic and physiochemical requirements specified by the iso and sls quality standards with excellent antifungal activity against c. albicans, which provides prospective to use citronella oil as a natural, safe, and eco-friendly fungicide in future product formulations. keywords: antidandruff shampoo; antifungal activity; citronella oil; cymbopogon nardus; cymbopogon winterianus. abbreviations: international standard organization (iso); sri lanka standards (sls); minimal inhibitory concentration (mic); saburound dextrose agar (sda); potato dextrose agar (pda); dimethyl sulfoxide (dmso). introduction citronella is an important type of aromatic grass belonging to the family poaceae (devi et al., 2016). citronella can be classified into two categories as ceylon citronella (cymbopogon nardus) and java citronella (cymbopogon winterianus). the essential oil produced from citronella, enhance its economic value as an herb. a number of studies have found that citronella oil has therapeutic value due to the major components geraniol, citronellol, and citronellal (ameliana, almawadah and wulandari, 2019) and shows certain anti-fungal https://doi.org/10.14421/biomedich.2023.122.485-498 486 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 properties, which can weaken or destroy certain fungi (li et al., 2012). it also contains many possible bioactive components with great pharmaceutical and medicinal consequences which can act as anti-inflammatory, anticancer, antioxidant, and anticonvulsant agents (devi et al., 2016). these properties of citronella oil enhance its value and the applications in many fields such as cosmetics, food, medicinal, ayurvedic, and herbal products. therefore, there is a huge potential to use citronella oil in herbal cosmetic products to cure common fungal diseases like dandruff. dandruff is a major dermatological problem for many people, and it is observed as white scales, which cause scalp itching and hair loss. excessive sweat gland secretion and the appearance of microorganisms on the scalp can also lead to the formation of dandruff. the most common causes of dandruff are the fungi such as candida albicans (ameliana, almawadah and wulandari, 2019) and malassezia furfur (pingili, vanga and raparla, 2016). antidandruff shampoos and other treatment options contains various anti-dandruff substances, including pyrithione, salicylic acid, imidazole derivatives, selenium sulfide, tar derivatives, ketoconazole (pingili, vanga and raparla, 2016) zinc, menthol, and thymol (ameliana, almawadah and wulandari, 2019), etc. as key ingredients. however, the use of these chemicals can cause hair loss and adverse skin reactions such as rashes, itching, and dermatitis. these cannot prevent the recurrence of dandruff and the side effects caused by these chemicals cannot be ignored. therefore, there is a demand for new antimicrobial agents and alternative therapies with natural ingredients. citronella oil will be a highly effective instinctive remedy for this purpose. introducing the citronella oil from the selected five citronella accessions belonging to cymbopogon nardus and cymbopogon winterianus which are grown in sri lanka, will be a new approach to use citronella oil in the herbal cosmetics industry. direct application of citronella oil on the skin is an impractical and less effective method. therefore, a special formulation incorporated with citronella oil should be prepared to treat candida albicans. suitable topical preparation for fighting dandruff is an anti-dandruff shampoo which is a liquid, gel, lotion, or aerosol that contains surfactants to generate foam and cleanse the scalp and hair. this study focused to determine the antifungal effect of citronella oil (cymbopogon nardus, cymbopogon winterianus) against dandruff causing fungi candida albicans to formulate an anti-dandruff shampoo. the quality of citronella oil, extraction methods, and shampoo preparations were determined according to the to criteria of sls and iso standards. the minimum inhibitory concentration (mic) of citronella oil was determined by antifungal assay of agar well diffusion method. materials and methods study area the plants of cymbopogon nardus (ceylon citronella) and cymbopogon winterianus (java citronella) were selected from the farm of the national cinnamon research and training center of sri lanka, located in palolpitiya, matara, sri lanka (6° 2' 2.94'' n latitude, 80° 33' 38.448'' e longitude). figure 1. a cymbopogon nardus (ceylon citronella), bcymbopogon winterianus (java citronella). citronella oil extraction ▪ sample collection and processing the matured citronella leaves from selected accessions cymbopogon nardus (hp t1, hp t2, hp t3) and cymbopogon winterianus (mp t1, mp t2) were harvested, dried under shade for 3-5 days, and cut into small pieces. ▪ determination of moisture content the entrainment method (sls 86 part 5) was used to measure the moisture content of citronella samples by a b wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 487 using dean and stark apparatus (witeg germany). the moisture content of approximately 10 g of the citronella leaves was measured with 60 ml of saturated toluene for about one and a half hours and refluxing was continued for two intervals of 15 minutes revealing no change. then, the whole unit was left to cool for about 15 minutes, and the readings of moisture amount were taken from the bottom of the dean and stark arm. the percentage moisture content was calculated by the following equation. 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝐶𝑜𝑛𝑡𝑒𝑛𝑡 (%) = (𝐴𝑟𝑚 𝑅𝑒𝑎𝑑𝑖𝑛𝑔) (𝑆𝑎𝑚𝑝𝑙𝑒 𝑊𝑒𝑖𝑔ℎ𝑡) × 100 ▪ hydro distillation of citronella oil with xylene and without xylene a modified clevenger method with hydro distillation was used to determine the oil quantity of citronella samples (asta method no 5, fourth edition, 1997). approximately 50 g of citronella leaves with 500 ml of clean water were added to the flask and connected with the clevenger arm where the graduated tube was filled with clean water up to the level of 5 ml and the unit was heated by a heating mantle (whm 12393, wisd laboratory instruments, german) while condensing a cooling system for 5 hours. after distillation, the system was allowed to cool, and the extracted citronella oil was collected into a cleaned and oven-dried glass bottle by removing the water layer of the clevenger arm. then samples were kept for about 24 hours to settle. the minute water particles suspended in oil were removed by a cleaned glass syringe. the purified oil samples were subjected to gc-ms analysis. the oil percentage in 100 g of sample in dry weight basis was calculated by following equations. 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 = (𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 %) 100 × 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 = 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑂𝑖𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 = 𝐵𝑜𝑡𝑡𝑙𝑒 𝑤𝑖𝑡ℎ 𝑜𝑖𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 − 𝐸𝑚𝑝𝑡𝑦 𝑏𝑜𝑡𝑡𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑂𝑖𝑙 % (𝑔 𝑖𝑛 100 𝑔 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒) = (𝑂𝑖𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡) (𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒) × 100 the hydrodistillation with xylene was carried out by adding 1 ml of xylene to the clevenger arm. the dry weight of the samples and the oil percentage in 100 g of sample (dry weight) by hydrodistillation with xylene were calculated by following equations. 𝑂𝑖𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 = 𝐴𝑟𝑚 𝑅𝑒𝑎𝑑𝑖𝑛𝑔 − 𝑋𝑦𝑙𝑒𝑛𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 𝑂𝑖𝑙 % (𝑚𝐿 𝑖𝑛 100𝑔 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒) = (𝑂𝑖𝑙 𝑣𝑜𝑙𝑢𝑚𝑒) (𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒) × 100 ▪ steam distillation of citronella oil a steam boiler setup was used for the steam distillation of citronella samples. the distillation was run for 5 hours and the oil with moisture was collected and allowed to cool. samples were subjected to settle for overnight and the oil was separated from the water using a separatory funnel. the remaining minute water particles suspended with oil were removed by adding anhydrous sodium sulfate. oil percentage in 100 g of sample (dry weight) was calculated by following equations. 𝑂𝑖𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 = 𝐵𝑜𝑡𝑡𝑙𝑒 𝑤𝑖𝑡ℎ 𝑜𝑖𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 − 𝐸𝑚𝑝𝑡𝑦 𝑏𝑜𝑡𝑡𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑂𝑖𝑙 % (𝑔 𝑖𝑛 100 𝑔 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒) = (𝑂𝑖𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡) (𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒) × 100 determination of citronella oil quality the quality of extracted citronella oil from selected accessions was evaluated in terms of organoleptic properties, relative density, refractive index, optical rotation, and ethanol solubility (iso 3848, iso 3849, sls 170). the colour, odor, and texture of the oil samples were observed under the organoleptic properties and the relative density of oil at 25 ℃ was measured using a standardized 10 ml pycnometer. the refractive index of the citronella oils at 20 ℃ was measured by a j-57wr 488 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 refractometer and p-8000 digital polarimeter (germany) was used to measure the optical rotation at 20 ℃. the 80% ethanol was used to determine the ethanol solubility of citronella oil samples at 20 ℃. the burette was filled with 50 ml alcohol and 1 ml of the oil sample was added to the erlenmeyer flask. then the oil was titrated with 80% alcohol and the burette reading of the endpoint at which the turbidity of the solution disappeared was recorded. the alcohol solubility of the 1 ml oil sample was determined by the following equation. 𝐴𝑙𝑐𝑜ℎ𝑜𝑙 𝑠𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦 = 𝐹𝑖𝑛𝑎𝑙 𝑏𝑢𝑟𝑒𝑡𝑡𝑒 𝑟𝑒𝑎𝑑𝑖𝑛𝑔 − 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑏𝑢𝑟𝑒𝑡𝑡𝑒 𝑟𝑒𝑎𝑑𝑖𝑛𝑔 determination of citronella oil composition the gc-ms analysis to determine the composition of citronella oil was carried out trace 1300 isq qd gas chromatography mass spectrometer (thermo fisher scientific, usa) equipped with tgwaxms column (30 m long × 0.25 mm inner diameter, 0.25 μm film thickness). the sample was prepared by diluting 50 μl of the citronella oil with 950 μl methanol in a vial and 0.5 μl from the dilution was injected into the column through the syringe. the instrument was programmed with a linearly automated oven temperature from 50 ℃ to 220 ℃ in the rate of 3 ℃ min-1 and helium was used as the carrier gas with a carrier flow rate of 1 ml / min. and the pressure was set as 5 bar. the mass spectra were acquired in e1 mode (70ev), in a mass range of 50 to 500 amu and the transfer line and the ion source temperatures were maintained at 220 ℃ and 200 ℃ respectively. the individual oil components were identified by comparing the retention time with standard substances and a computer search by matching mass spectrum data with the ms library (nist ms search 2.0). determination of antifungal effect and minimum inhibitory concentration (mic) of citronella oil the antifungal effect of citronella oil from selected accessions was tested using the agar well diffusion method (balouiri, sadiki and ibnsouda, 2016). saburound dextrose agar (sda) was used as the media for testing and 6.5 g of sda was suspended in distilled water in an erlenmeyer flask and warmed until homogenized. the media, petri plates, and distilled water were autoclaved at 121 ℃ for 15 minutes for sterilization. the sterilized liquid sda was poured into petri plates and left until solidified (ameliana, almawadah and wulandari, 2019). a suspension of candida albicans was made by adding an inoculum of candida albicans to saline water (silva, guterres, weisheimer and schapoval, 2008) and the mixture was vortexed, and the turbidity was checked with 0.5 mcfarland standard (oliveira et al., 2011). then, 100 μl of fungal suspension was spread over the solidified sda medium. after, a hole of 4 mm diameter was punched in the middle of the sda medium aseptically using a cork borer and 20 μl (balouiri, sadiki and ibnsouda, 2016) of citronella oil was pipetted and added into the well. the plates were sealed and incubated at 37 ℃ for 24 72 hours (oliveira et al., 2011). the inhibitory zone diameters of each oil type were recorded. the oil types with the highest inhibitory zone diameters were selected to determine the mic. the mic was tested following agar well diffusion for citronella oil with the highest antifungal activity in several oil concentrations (1%, 2%, 3%, 4%, and 5% v/v) diluted with dimethyl sulfoxide (dmso) solution. the same procedure also was followed using potato dextrose agar (pda) as the growth media. the dmso and captan (n-trichloromethylthio-4cyclohexane-1,2dicarboximide) were assessed as the negative and positive controls, respectively. the lowest concentration shows an inhibitory zone with no visible growth of fungus during the incubation was considered as the mic value of the citronella oil (ameliana, almawadah and wulandari, 2019). formulation of citronella oil antidandruff shampoo the citronella oil shampoo was made with the modified formula shown in table 1. the sodium lauryl sulfoacetate (slsa), sorbic acid, and sodium chloride were measured and added to a 500 ml beaker and the cocamidopropyl betaine, glycerin, tween 80, and the oil were pipetted and added to the beaker and topped up with the distilled water. the mixture was stirred with a magnetic stirrer at 1500 rpm until homogenous. a shampoo sample without citronella oil was formulated as the negative control. the ph of formulated shampoo samples was measured. table 1. citronella oil antidandruff shampoo formulation. material function concentration in 100 ml (%) sodium lauryl sulfoacetate (slsa) primary surfactant 10 cocamidopropyl betaine co surfactant 5 sorbic acid preservative 0.3 sodium chloride (nacl) viscosity adjuster 1 glycerin humectant 3 polysorbate 80 (tween 80) oil solvent 0.5 citronella oil active ingredient (antidandruff agent) 2 distilled water solvent 89.5 total volume 100 ml wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 489 determination of antifungal effect of citronella antidandruff shampoo the antifungal effect of formulated shampoo against candida albicans was examined by following the previous method of agar well diffusion. the well of the medium was filled with 20 μl of citronella oil shampoo. the citronella oil and captan were tested as the positive control and the shampoo without citronella oil was tested as the negative control. evaluation of citronella antidandruff shampoo formulated shampoo samples were tested for quality under different types of organoleptic and physiological parameters. the organoleptic properties were evaluated based on their clarity, colour, odor, and texture (alquadeib, eltahir, banafa and al-hadhairi, 2018). ▪ foaming ability and foam stability the forming ability of the shampoo was evaluated by the cylinder shake method (al badi and khan, 2014). a 50 ml of 1% v/v shampoo solution was added to a 250 ml graduated cylinder and the cylinder was covered by hand and shaken for 10 times. the total volume of that foam that appeared after 1 min shaking was measured and recorded. the foam stability was tested by measuring the foam volume in 1min intervals for 4 min. ▪ determination of ph the ph of the formulated shampoo samples and 10% v/v diluted shampoo samples were directly measured using calibrated cyberscan pc 650 ph meter (eutech instrumets, thermo scientific, singapore) and a strip of ph paper at room temperature 27±2 ℃ (sls 1346:2018) (al badi and khan, 2014). ▪ rheological evaluations (viscosity) the viscosity of the shampoo samples was evaluated by using bdv-1s digital viscometer (biobase meihua, china) with the spindle l1 at speed of 60 rpm. the temperature and the size of the container were kept constant during the study (alquadeib, eltahir, banafa and al-hadhairi, 2018). ▪ dirt dispersion a drop of india ink was added to a large test tube with 2 drops of shampoo in 10 ml of distilled water. then, the test tube was stoppered and shaken 10 times. the amount of ink in foam was evaluated as none, light, moderate or heavy (al badi and khan, 2014). ▪ wetting time drave’s test was used to determine the wetting time of the formulated shampoo samples (alquadeib, eltahir, banafa and al-hadhairi, 2018). a canvas fabric was cut into 1.0 inch diameter disc with an average weight of 0.44 g. the smooth surface of the disc was placed on the surface of 1% v/v shampoo solution and the time required for the disc to begin sinking was measured by a stopwatch as the wetting time. ▪ percentage solid content a cleaned and dried dish was weighed, and 4 g of shampoo was added to the dish the dish with shampoo was weighed and the exact initial weight of shampoo was calculated. the dish was placed on a hot plate until the liquid portion of the shampoo was evaporated. after the remaining solid content was weighed and the percentage (%) of solid content was calculated (al badi and khan, 2014). data analysis data were subjected to one-way anova and two-way analysis of variance according to the glm (general linear mode) at a 0.05 significance level using sas ondemand for academics online version. the mean value, standard deviation, calculations, and figures with graphical representations of mean values were performed by microsoft excel for microsoft 365 mso (version 2205 build 16.0.15225.20028). results and discussion quantity and quality of citronella oil organoleptic properties (colour, odor and texture) of the java and ceylon citronella oil obtained from the selected accessions were determined. the colour measurements were carried out by direct eye observation at a distance of 30 cm (fitri et al., 2022) and the observed colours of each oil type ranged between pale yellow to pale brownish yellow as specified in the iso standard (iso 3849:2003). the characteristic citronella oil aroma (strong citrusy) of each 5 types of oil was sensed at a distance of 5 cm (fitri et al., 2022) as the odor and the oil from java citronella (mp t1 and mp t2) and ceylon citronella hp t3 oils have a stronger aroma than hp t1 and hp t2 due to their high citronellal content (table 3). according to the physical appearance and texture, each type of oil is an oily and viscous liquid that is volatile in the open atmosphere. the moisture contents of the selected citronella accessions shown in table 2 are significantly different (p < 0.0001). the mp t2 java citronella accession showed the highest significant moisture content whereas the hp t3 ceylon citronella accession showed the least significant moisture content. both java citronella accessions showed high amounts of moisture content above 50% compared to the moisture content of ceylon citronella accessions. according to the results of table 2, the % oil content obtained by hydrodistillation with xylene from each accession showed a significant difference (p = 0.0001). the oil quantity (ml) per 100 g of citronella sample from ceylon citronella is significantly higher than the java citronella. the ceylon citronella accession hp t3 490 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 showed the highest oil quantity of 2.67 ml/100 g whereas oil from mp t1 java citronella accession shows the least oil quantity of 1.57 ml/100 g. extracted citronella oil quantities from each accession by hydro distillation without xylene showed a significant difference (p<0.0001) where the hp t1 accession showed the highest oil quantity of 5.52 % where oil from hp t3 accession shows the least oil quantity of 1.05% (table 2). the % oil contents of selected citronella accessions extracted by steam distillation does not show a significant difference (p = 0.7055) and ranged from 1% to 1.25% (table 2). table 2. citronella oil content and quality under different parameters. quality parameter accession of citronella probability cv% hp t1 hp t2 hp t3 mp t1 mp t2 moisture (%) 46.16 c 42.89 d 28.46 e 52.71 b 56.40 a <0.0001 2.23 hydro distillation with xylene % oil v/w (ml/100g) 2.67 a 2.45 a 2.53 a 1.57 b 1.64 b 0.0001 13.67 hydro distillation % oil w/w (g /100 g) 5.52 a 1.40 bc 1.05 c 1.25 bc 1.79 b <0.0001 14.14 steam distillation% oil w/w (g /100 g) 1.07 a 1.18 a 1.19 a 1.16 a 1.23 a 0.7055 9.43 refractive index 1.47 c 1.49 a 1.48 b 1.47 c 1.47 c <0.0001 0.06 optical rotation -2.87˚a -9.89˚b 0.72˚ a -0.82˚a -0.75˚a 0.0003 -94.40 relative density 0.90 b 0.91 a 0.90 b 0.88 c 0.88c <0.0001 0.55 ethanol solubility (ml) 1.25a 1.25a 1.25a 1.25a 1.25a 1.0000 4.61 note: probability<0.05 have a significant difference within mean values. mean within each column followed by the same letter are not significantly different. mean values from highest significant difference have been denoted with “a” the relative density resulted for the oils of selected citronella accessions shows a significant different (p < 0.0001). ceylon citronella oil from accessions hp t1, hp t2 and hp t3 shows a significantly high relative density compared to the java citronella oils of mp t1 and mp t2. according to the relative density of ceylon citronella oil samples, hp t1, hp t2 and hp t3 have been resulted as 0.90, 0.91 and 0.90 respectively, with highest significant difference in hp t2, and in the range of 0.893-0.910, the relative density for ceylon citronella essential oil in 25 ℃ specified in the sls standards (sls 170). also, the relative density values resulted for java citronella oil samples mp t1 and mp t2 are same (0.88) and within the range of 0.880-0.892 specified for the relative density of java citronella essential oil in 25 ℃ (table 2). the measured refractive index values of citronella oil from selected citronella accessions were significantly different (p < 0.0001). the citronella oil of accession hp t2 resulted with the highest significant refractive index, while hp t1, mp t1 and mp t2 shows the lowest significant refractive index (table 2). according to the sls quality standards, the refractive index of ceylon citronella should be in the range of 1.465-1.487. therefore, only the oil from accession hp t2 shows a slightly higher refractive index out of the given range where the oil from hp t1 and hp t3 rely on the given refractive index range in the sls standards (sls 170). although there is no specified refractive index range for the java citronella in sls standards (sls 170), the iso standard have been specified the refractive index for java citronella in the range of 1.4660-1.4730 (iso 3848). the refractive index values of both selected java citronella accessions mp t1 and mp t2 resulted as 1.47 (table 2), which is in the range specified in the iso standard. the optical rotation of the oil samples from selected accessions of java and ceylon citronella has resulted in a significant difference (p < 0.0001). the sample of hp t3 oil has the highest significance positive value (+0.72˚) which indicates that the oil a has dextrorotary polarized light plane. the hp t1 and hp t2 oils are in negative values -2.87˚ and -9.89˚ respectively indicating levorotary of the light plane. however, the oil samples from selected ceylon citronella accessions are not lie down within the iso specified range of -22 ˚ to -12 ˚ (iso 3849:2003) showing an odd optical rotation which might be a result of irregular distribution of oil composition (st-gelais, 2021). optical rotation results of java citronella oil samples mp t1 and mp t2 are -0.82˚ and -0.75˚ respectively are negative values that indicates a levorotary light plane and are not significantly different to each other (table 2). both iso and sls standards have specified that the alcohol solubility of java or ceylon citronella should be determined by using 80% (v/v) ethanol in 20 ℃ and high-quality citronella oil should have 1:2 ethanol solubility (iso 3848, iso 3849, sls 170). all the extracted oil samples were tested for ethanol solubility have been resulted the same value 1.25 ml of ethanol for the solubility of 1 ml of oil and does not show a significant difference (table 2). this illustrate that the oil of selected citronella accessions has their ethanol solubility at an acceptable level by international and local standards. wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 491 chemical composition of citronella oil the citronellal, geranyl acetate, citronellol, geraniol, and geranyl iso butyrate are the main compounds identified from the oil samples of selected citronella accessions according to the gc-ms results (table 3). the citronellal contents available in each oil type are significantly different. the oil from accession hp t3 has the highest significant citronellal content (20.29%), whereas the oil of hp t1 has the least significant citronellal content (2.43%). the oil of java citronella accessions mp t1 and mp t2 have a citronellal content of around 6-7% and which is lesser than the citronellal content of hp t3 oil. the percentage availability of the geranyl acetate has a significant difference among the five oil types of selected accessions where the oil of hp t1 has the highest significant geranyl acetate content (13.48%) and in contrast, the oil hp t2 has the least geranyl acetate content of 0.63% (table 3). citronellol contents identified by the gc-ms showed a significant difference among the tested oil samples. the java citronella oil samples mp t1 and mp t2 have high citronellol content compared to ceylon citronella oil where mp t1 oil has the highest significant citronellol content of 11.41%. the ceylon citronella oil hp t1 has the least significant citronellol content of 2.06% and the high citronellol content of hp t3 (9.63%) compared to the other ceylon citronella oil types of hp t1 and hp t2, is exceptional as it does not have a significant difference to the citronellol content of java citronella oil mp t2. the available proportions of the compound geraniol show a significant difference in tested oil samples. java citronella has very high geraniol content around 50% of its total composition compared to ceylon citronella where java oil mp t1 and mp t2 have the highest significant geraniol contents 48.60% and 49.17% respectively with no significant difference to each other. the ceylon citronella oil hp t1 and hp t3 have geraniol contents 2.49% and 26.12% respectively with no significant difference to each other and ceylon oil hp t2 has the least significant geraniol content of 16.93% (table 3). geranyl iso butyrate is another type of the main compound identified in citronella essential oil in minor quantities. the percentage content of geranyl iso butyrate available in the tested samples has resulted in a significant difference. however, the ceylon oil hp t2 has significantly higher geranyl iso butyrate compared to other types of oil samples (table 3). table 3. chemical composition of the citronella oil. chemical compound accession of citronella probability cv% hp t1 hp t2 hp t3 mp t1 mp t2 citronellal (%) rt-14.73 2.43e 3.46d 20.29a 6.57c 7.39b <.0001 4.83 geranyl acetate (%) rt-24.84 13.48 a 0.63 d 3.48 c 4.95 bc 5.54 b <.0001 18.59 citronellol (%) rt-25.24 2.06 d 3.16 c 9.63 b 11.41 a 10.11b <.0001 8.80 geraniol (%) rt-28.23 26.49 b 16.93 c 26.12 b 48.60 a 49.17 a <.0001 7.09 geranyl iso butyrate (%) rt-29.39 0.99 b 2.75 a 0.44 c 0.23 c 0.21 c <.0001 17.84 note:rtretention time, probability<0.05 have a significant difference within the mean values.mean within each column followed by the same letter are not significantly different. mean values from highest significant difference have been denoted with “a” antifungal effect of citronella oil figure 2. inhibition zones of citronella oil against candida albicans on sda. all five types of non-diluted ceylon citronella (hp t1, hp t2, hp t3) and java citronella (mp t1, mp t2) oil samples that tested positive for the inhibition of candida albicans with evidently visible inhibition zones compare to the negative control (figure 2). the diameters of the inhibition zones that appeared on the 492 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 plates with citronella oil (figure 2) showed that the antifungal activity of the tested oil samples is different from each other. the citronella oil of mp t1 accession of java citronella showed the highest inhibition zone diameter whereas oils of accessions hp t1 and hp t2 showed the least inhibition zone diameter. table 4. inhibition zone diameters (mean) of citronella oil against candida albicans. accession of the oil sample inhibition zone diameter (cm) probability cv% hp t1 1.8 0.1556 5.31 hp t2 1.8 hp t3 1.9 mp t1 2.0 mp t2 1.9 minimum inhibitory concentration of citronella oil table 5. inhibition zone diameters of hp t3 and mp t1 oil concentrations (1%-5% v/v) against candida albicans on pda and sda media. factor level diameter of inhibition zone (cm) pda sda factor 1 – citronella accession hp t3 1.267a 0.722a mp t1 1.361a 0.672b probability 0.0988 <0.0001 factor 2 – concentration (%) 1 -0.00d -0.00d 2 1.30bc -0.00d 3 1.25c 1.00c 4 1.46b 1.03b 5 1.40bc 1.15a positive control 2.47a 1.00c probability <0.0001 <0.0001 interactions – accession * concentration (%) hp t3*1 -0.00d -0.00d hp t3*2 1.43bc -0.00d hp t3*3 1.23c 1.00b hp t3*4 1.27c 1.03b hp t3*5 1.20c 1.30a positive control 2.47a 1.00b mp t1*1 0.00d -0.00d mp t1*2 1.17c -0.00d mp t1*3 1.27c 1.00b mp t1*4 1.67b 1.03b mp t1*5 1.60b 1.00b positive control 2.47a 1.00b probability 0.0122 <0.0001 cv% 12.55 3.38 note: probability<0.05 has a significant difference in mean values. the mean within each column followed by the same letter is not significantly different. mean values from the highest significant difference have been denoted with “a” the 1% v/v concentration of hp t3 and mp t1 oil samples were not showed clearly visible zones against the fungi candida albicans on pda media (table 5). the clearly visible inhibition zones were observed starting from the 2% v/v concentration corresponding to the commercial antifungal agent, captan which was used as the positive control. the results of the same antifungal assay carried out on sda show that there were no inhibition zones for both 1% and 2% v/v concentration and the inhibition zones were observed afterward at the 3% v/v concentration. the positive control also indicated smaller inhibition zones on sda than the inhibition zones for the positive control in pda. wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 493 formulation of citronella oil antidandruff shampoo figure 3. formulated antidandruff shampoos with citronella essential oil hp t3 and mp t1. antifungal activity of citronella oil antidandruff shampoo citronella oil shampoo samples with mp t1 java citronella and hp t3 ceylon citronella showed a high antifungal activity than shampoo without citronella oil (table 6). however, shampoo samples without citronella oil also showed substantial antifungal activity corresponding to the commercial fungicide used as the positive control and the crude citronella oil. table 6. inhibition zone diameters (mean± sd, n=3) of hp t3 and mp t1 oil and shampoo with 2% v/v concentration of oil against candida albicans selected accession inhibition zone diameter (cm) antidandruff shampoo shampoo without oil crude citronella oil distilled water positive control hp t3 2.5±0.05 2.1±0.1 1.9±0.11 0 1.9±0.1 mp t1 2.5±0.05 2.0±0.2 2.0±0.1 0 2.1±0.1 quality of citronella oil antidandruff shampoo the ph of the shampoo formulations with citronella oil of hp t3 and mp t1 accessions resulted within 4-5 while the ph of negative control was higher than 5. the diluted shampoo samples formulated by incorporating citronella oil of hp t3 and mp t1 accessions showed ph values of more than 5 while the diluted negative control also showed a high ph value than the initial ph (table 7). the shampoo formulations with citronella oil of hp t3 and mp t1 and the negative control shampoo samples showed viscosity within 30-50 mpa.s which are significantly different from each other (table 7). although all samples resulted in low viscosity values in high rpm like 60 rpm, the shampoo solution with mp t1 citronella oil shows significantly low viscosity compared to shampoo solution with citronella oil hp t3 and negative control. all shampoo samples with citronella oil hp t3 and mp t1 and negative control samples showed similar results for dirt dispersion indicating no remain of ink in the foam (table 7) the wetting time of the shampoo solutions with citronella oil hp t3 and mp t1 and negative control resulted in very low values of 6.59±0.14 seconds, 5.91±0.19 seconds, and 5.59±0.42 seconds and the wetting time of hp t3 shampoo was higher compared to the other tested samples (table 7). the percentage solid content of the formulated shampoo with citronella oil hp t3 and negative control were lower than the acceptable range of 20-30% and, they were not significantly different from each other (table 7). table 7. physiological properties (mean ±sd, n=3) of citronella oil antidandruff shampoo. parameter hp t3 mp t1 shampoo without oil (negative control) ph 4.53±0.03 4.49±0.03 5.20±0.54 ph of diluted shampoo solutions 5.51±0.02 5.48±0.03 5.53±0.03 viscosity (mpa.s) 46.4±1.08 30.23±0.56 41.99±0.17 dirt dispersion (ink in foam) none none none wetting time (s) 6.59±0.14 5.91±0.34 5.59±0.42 % solid content 14.75±0.12 12.33±0.19 13.47±0.23 the shampoo with mp t1 citronella oil produced significantly high foam volume (170 ml) than foam volumes of shampoo with hp t3 citronella oil (141 ml) and shampoo without citronella oil (negative control). 494 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 as the graph illustrates, the foam volume of each shampoo sample reduced within 3 minutes time and remained constant at certain levels above 100 ml (figure 4). figure 4. the foam volumes of the shampoo samples with time. discussion quantity and quality of citronella oil the citronella plant leaves were harvested before the flowering period to obtain a high oil yield, as flowering causes the aging of plants (blank et al., 2007). shade drying was used to dry the plant leaves without leading to a reduction of oil quality and quantity. direct drying in hot sunlight should be prevented as far as avoiding chemical transformations of this volatile essential oil. drying of citronella leaves aimed to reduce the moisture content with minimum quality reduction. the moisture content should be reduced literally to be at an equilibrium level that is defined for certain relative air humidity and temperature with a minimum quality reduction in terms of active ingredients, colour, flavor and aroma (özgüven, gülseren and müller, 2019). therefore, citronella leaves should be spread out under a shade for drying. in the process of drying, the citronella leaves under the shade should be frequently turned to avoid the formation of molds and the fermentation of leaves which will lead to the oil quality reduction. after the completion of the drying period, the overdried parts and the foreign matters should be removed. the citronella leaves were cut into small pieces, to enhance the even distribution in the still which prevent steam channeling as well as for the easy insertion of plant leaves to the sample container (still) as well as to the round bottom flask of clevenger distillation apparatus. the moisture content of the harvested citronella leaves was measured after shade drying, to calculate the dry weight of the sample. citronella leaves were distilled with toluene which is less concentrated than water and immiscible with water. method of hydro distillation with xylene was performed to determine the oil content that could be obtained from each type of citronella sample and hydro distillation without xylene was used to extract the citronella oil for the gc-ms analysis. the steam distillation was carried out to obtain the citronella oil samples where the higher oil yields can be obtained for further analysis such as determination of refractive index, alcohol solubility, optical rotation, and relative density. the oil yield of citronella plants depends on several factors, including the presence of climatic differences, soil fertility, plant age, and the separation of citronella oil (ameliana, almawadah and wulandari, 2019). however, the study was carried out in the same location and same time period, and the above differences in oil content is due to the genetic variations of each accession. hydrodistillation with the organic compound xylene showed the highest efficiency in citronella oil extraction among the three extraction methods used and hydro distillation with or without an organic compound resulted with high oil quantity than steam distillation in most oil accessions (table 2). however, steam distillation is one of the most common and main methods used for the extraction of citronella oil in bulk quantities for industrial and commercial purposes. all the oil samples from selected java and ceylon citronella accessions that were tested are showing required quality standards for relative density, refractive index, ethanol solubility and can be considered as highquality samples (table 2). essential oils are composed of many different compounds, most of which are optically active. their different compositions allow them to be distinguished by measuring their optical rotation. these measurements also provide comprehension of the quality and purity of essential oil, as any change in its optimal composition can affect the optical rotation (nandapure et al., 2016). although the optical rotation of ceylon 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 f o a m h ei g h t (m l ) time (min) hp t3 mp t1 control shampoo wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 495 citronella oil samples was not in the iso-specified range, java citronella oil samples resulted within the iso-specified range (-5˚ to 0˚) for optical rotation (iso 3848). therefore, the results prove that the oil samples of selected java citronella are within the acceptable range (table 2). chemical composition of citronella oil the composition of the oil samples extracted from the selected citronella accessions was determined by subjecting to the gc-ms and the constituents available in each oil type were identified by comparison of retention time with the library search of mass spectra for fragmentation pattern of reference standard compounds. the proportions of each compound present in five tested oil types were calculated from the gc peak areas using the normalization method. the main constituents identified from the oil samples of selected citronella accessions were geraniol, citronellol, and citronellal. geraniol is the main compound that has been identified in the highest proportions from each oil sample. citronellal and citronellol are the other two types of constituents identified in moderately high amounts in the composition of tested citronella oil. therefore, the gc-ms results of the tested citronella oil samples comparatively correspond to the literature that indicates citronella oil (cymbopogon nardus) contains different types of chemical components, including citronellal, citronellol, and geraniol, which have antifungal and antibacterial activities (ameliana, almawadah and wulandari, 2019). according to aguiar et al., 2014, the high citronellal content of c. nardus (ceylon citronella) oil extremely performs as a potent inhibitor of various fungi at ambient temperatures. therefore, the citronella essential oil from hp t3 accession shows a potential to inhibit the fungi candida albicans with its significantly very high amount of citronellal content (table 3) and also comparatively high amounts of geraniol and citronellol contents. along with this, the oil of selected java citronella accessions mp t1 and mp t2 are also considered to have a high potential antifungal activity due to the availability of high citronellal, citronellol, and geraniol contents in their composition. according to the gc-ms results, there are differences in the chemical composition and the percentage content of the constituents in the citronella oil samples of selected accessions. these changes in the essential oil composition may possibly occur due to several environmental (climatic, seasonal, or geographic) factors and genetic differences (aguiar et al., 2014). antifungal effect of citronella oil the oil of hp t3 resulted the highest inhibition zone among the tested ceylon citronella samples and mp t1 oil showed the highest inhibitory action among tested java citronella samples (table 4). in addition, hp t3 oil showed unique high proportions of the main antifungal constituents citronellal, citronellol, and geraniol (table 3) in its composition (ameliana, almawadah and wulandari, 2019). therefore, the oils of accessions hp t3 and mp t1 were selected as the active ingredients with antifungal properties for the formulation of antidandruff shampoo. and further, the selected oil samples were subjected to antifungal analysis for the determination of minimum inhibitory concentration against candida albicans. identification of the volume of citronella oil that should be incorporated into the shampoo formulation to perform antidandruff function was significant. therefore, the mic of selected citronella essential oils for the inhibition of candida albicans was identified across five concentrations (1%, 2%, 3%, 4%, and 5% v/v) by performing agar well diffusion assay (ameliana, almawadah and wulandari, 2019). the oil samples were evaluated in both sda and pda media to compare the inhibitory activity of selected citronella oil samples in different media (table 5). the positive control also indicated smaller inhibition zones on sda than the inhibition zones for the positive control in pda. therefore, the results of the mic antifungal assay on pda media were considered to be more accurate and precise. table 5 demonstrates that there is no significant difference between the antifungal activity of the citronella oils of hp t3 and mp t2 accessions on pda against candida albicans. in contrast, the antifungal activity of the two oil types shows a significant difference in sda against candida albicans. in the pda media, the positive controls show the highest significant inhibition whereas the 1% v/v concentration of both hp t3 and mp t1 oils show the least significance with no inhibition against the tested fungi. the second least significant inhibition (1.17 cm) of mp t1 oil in pda shows in the concentration of 2% v/v. therefore, the 2% v/v concentration of mp t1 citronella oil is the mic that performs antifungal activity against candida albicans. also, the least concentration of hp t3 oil that shows an inhibition zone diameter is 2% v/v (table 5). therefore, the mic values of both oil samples mp t1 and hp t3 were assumed to be more than 1%v/v concentration and less or equal to 2%v/v concentration (1% < mic ≤ 2% v/v). consequently, the 2% v/v concentration was determined as the mic of both oils hp t3 and mp t1 against candida albicans and referred to as the concentration of citronella oil to be incorporated in the antidandruff shampoo formulation. formulation of citronella oil antidandruff shampoo the citronella antidandruff shampoos formulated with selected citronella oil hp t3 and mp t1 with the highest antifungal activity are shown in figure 3. the sodium lauryl sulfoacetate (slsa; c14h27nao5s) has been registered in drug bank (accession number db13157) as a surfactant which also functions as a wetting agent. according to the final report on the safety assessment 496 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 of slsa by the american college of toxicology, it is a hydrophilic, skin-friendly, mild surfactant derived from coconut and palm plants which use in cleansing products for both skin and hair to remove surface oil, dirt, and bacteria effectively. the hydrophilic nature of the slsa was beneficial for the liquid shampoos as well as it improves the rinsing ability of the product. slsa is a bulky molecule that penetrates the skin and is free from sulfate ions causing less irritation to the skin. this can be effectively used in cosmetic products with ph 5-8.5, which will give better performance for the liquid shampoo prepared for ph 4.0-8.0 (sls 1346:2018). further study of the final report on the safety assessment of slsa by the american college of toxicology (1987), it is a cosmetic ingredient approved by u.s. food and drug administration and used primarily for bath preparations in 5-50%, but the actual concentration contacts with the skin surface are much lesser due to product dilution with water. therefore, it was considered a much safer and more efficient ingredient for the citronella antidandruff shampoo formulation. a commonly used cosmetic ingredient, cocamidopropyl betaine was used as a co-surfactant to increase the function of the slsa (hunter and fowler, 1998), and sorbic acid with low toxicity was used as the natural preservative (dorko et al., 2000). sodium chloride was effectively used as a salt work as a thickening agent (penfield, 2005) whereas polysorbate 80 was used as an emulsifier for the citronella essential oils which are non-soluble in water (ariff, jai, jamaludin and ibrahim, 2022). the distilled water was used as the most abundant and common universal solvent in the preparation of shampoo samples, continuous stirring was performed to obtain a homogenized mixture of both the aqueous and the oil phases. two shampoo samples for mp t1 oil and hp t3 were prepared by adding 2 ml of each oil type separately to obtain a 2% v/v oil concentration in a 100 ml shampoo formulation. antifungal activity of citronella oil antidandruff shampoo according to the inhibition zone diameters tabulated in table 6, both shampoo samples with citronella oil show higher antifungal activity than the crude citronella essential oils used in their formulations, commercial fungicide, and the shampoo without citronella oil. however, in this study shampoo formulated as a negative control without citronella oil also showed inhibitory action against candida albicans. this can be justified as the preservative used in shampoo formulations has increased the antimicrobial activity of the products (sofos and busta, 1981). quality of citronella oil antidandruff shampoo the attractiveness of physical appearance and organoleptic properties are vital factors to be considered in a cosmetic formulation that will enhance consumer preference (chavan et al., 2019). the physical appearance of the formulated shampoo samples by incorporating citronella oil of hp t3 and mp t1 accessions and the shampoo without citronella oil was identified by the visual inspection. according to that, all shampoo mixtures were opaque, and white in colour as shown in figure 3 due to the absence of any synthetic or natural colouring agent. the natural fragrance of the citronella oil blended in both formulations mpt1 and hp t3 had given a citrusy aroma in to the shampoo samples which expresses the availability of citronella oil as the active ingredient. in terms of texture, all shampoo samples were in a viscous liquid form similar to the shampoo formulations in the market and acceptable to the general requirements for the shampoo in sls 1346:2018 standard. according to the resulting organoleptic properties except for negative control shampoo, both citronella antidandruff shampoo samples have no significant difference from each other with good characteristics corresponding to the target market preference. the ph level of a shampoo is accountable for improving and enhancing the hair quality with minimum eye irritation and stabilizing a balanced ecology on the scalp. generally, most of the shampoo formulations are neutral or slightly acidic in ph. the cuticle (outer layer) of the hair is shrink away and lie flatter on the hair shaft with acidic blends (alquadeib, eltahir, banafa and alhadhairi, 2018). also, the mild acidity prevents the swelling and promotes strengthening of the scales, which leads to a shine and smoothness while basic solutions make hair triggers the hair to be frizzier. therefore, the promoting the shampoo formulations with low ph to minimize the hair damage is an emerging trend in the industry (krunali et al., 2013). the acidity of citronella oil has been affected for the low ph of shampoos blended with citronella oil and the formulations were not required to be acid balanced after initial formulation as both shampoo formulations were ranged in sls specified ph levels 4.0-8.0 for the shampoo (sls 1346:2018). the ph of diluted shampoo samples formulated by incorporating citronella oil of hp t3 and mp t1 accessions were in the ph range of 5.5-5.9 near to the skin ph (krunali et al., 2013). also, both shampoo formulations show no significant difference in ph (table 7). the viscosity can be defined as the thickness or the stickiness of a liquid (alquadeib, eltahir, banafa and alhadhairi, 2018). viscosity plays a vital role on a shampoo product in defining and controlling characteristics such as shelf-life stability and product aesthetics such as clarity, ease of flow in removal from packing, ease spreading in application to hair and product reliability in the package (chavan et al., 2019). shampoos are non-newtonian products with plastic or pseudoplastic flow behaviors which shows high viscosity wijerathna et al. – formulation of anti-dandruff shampoo using citronella oil 497 in low rpm values and low viscosity in increased shear rates. dirt dispersion is a key criterion for evaluating the cleaning action of shampoo formulations (al badi and khan, 2014). determination of the amount of ink present in the foam phase of the column after shaking the shampoo solution and estimate the dirt dispersion of the shampoo formula (chavan et al., 2019). the shampoos which cause the ink to be concentrated in the foam phase are considered to be poor-quality formulations as the ink used to represent the dirt that remains in the foam is difficult to rinse away and also will be redeposited on the hair. therefore, the ink or the dirt should be always remained in the water phase to accomplish a better performance in cleansing (krunali et al.2013). the results of dirt dispersion ensured that the formulations achieved the cleaning ability and effectiveness in market satisfactory level (al badi and khan, 2014). the wetting time of a surfactant is a function of its concentration which is used to determine the efficacy of the surfactant (krunali et al., 2013). the performance of wetting action is dependent on various factors such as diffusion, surface tension, the concentration of the wetting agent, and the nature of the surface being wetted. an acceptable wetting agent should have the ability to reduce surface tension (alquadeib, eltahir, banafa and al-hadhairi, 2018). a low wetting time indicates a greater wetting efficiency with a maximum concentration of detergents (al badi and khan, 2014). all shampoo samples formulated in this study can be categorized as shampoos with acceptable wetting times due to the maximum concentration of surfactants with high efficacy (alquadeib, eltahir, banafa and al-hadhairi, 2018). the percentage solid content of a shampoo formulation is an important criterion to be considered in terms of quality and performance. too many solids in a shampoo, makes it difficult to work on hair or to wash out where, the absence of enough solids, leads to be too watery and will wash away rapidly. therefore, the solid content of a good shampoo should be in the range of 2030 % (alquadeib, eltahir, banafa and al-hadhairi, 2018). although, the solid contents of both shampoo formulations are slightly lower than the specified range, are expected to perform cleaning and wash out easily (table 7). as a promising fungal inhibitor, citronella oil can be widely used as a strong fungicide in different industrial applications such as in medical field, food industry agricultural industry and etc. the significant antifungal activity of citronella oil against c. albicans shows that citronella oil can be used as a remedy for the infections of candida on human skin such as candidiasis which may cause symptoms including rashes, scaling, itching, and swelling. therefore, citronella oil has the potential of used as an active ingredient in herbal cosmeceutical formulations such as soap, creams, lotions, and ointments etc. conclusions the oil extracted from the selected citronella accessions were in high quality under the tested physiochemical parameters and meet the organoleptic quality requirements with acceptable colour, aroma, and texture. the ceylon oil hp t1 has the highest oil content (ml/100 g) and the oil quantity of ceylon citronella extracted by hydrodistillation with xylene is higher than the oil content of java citronella. compared to steam distillation, hydro distillation can be used to extract high oil quantities from both java and ceylon types. the results of the quality tests performed for the citronella oil samples in terms of refractive index, relative density, optical rotation, and ethanol solubility followed the acceptable ranges under the iso and sls standards. the significant differences in oil quantity and quality of tested citronella accessions show that these accessions should be genetically different from each other. however, clearly, the oil extracted from all the selected citronella accessions are of high quality under the physiochemical parameters. results of gc-ms reveal that the main constituents of the oil samples of selected citronella accessions are citronellal, geranyl acetate, citronellol, geraniol, and geranyl iso butyrate. the oil hp t3 has high contents of the main constituents geraniol, citronellal, and citronellol. the highest geraniol contents are available in mp t1 and mp t2 oil samples. the oil of the hp t3 accession has a unique chemical composition compared to the other tested ceylon citronella accessions. all the oil samples have antifungal activity against candida albicans and mp t1 and hp t3 oils have the highest inhibitory action with a minimum inhibitory concentration (mic) of 2% v/v. antidandruff shampoos formulated blending mp t1 and hp t3 oils show antifungal activity against candida albicans with evidence that both tested oils can be effectively used as an antidandruff agent in hair shampoo. also, both shampoo samples are aligned with the quality standards of the organoleptic properties, and physiological properties such as foaming, ph, viscosity, wetting time, dirt dispersion, and solid content (%) which conclude that the use of citronella oil as an ingredient does not affect to change the acceptable levels of physiochemical parameters of shampoo. acknowledgements: this work was supported by national cinnamon research and training center of sri lanka and department of biosystems technology, faculty of technology, university of sri jayewardenepura, sri lanka. the authors wish to thank ms. w.u.m. pramodani and laboratory staff of biotechnology laboratory of faculty of technology, university of sri jayewardenepura for their support. 498 biology, medicine, & natural product chemistry 12 (2), 2023: 485-498 authors’ contributions: r.m.n.wijerathna, a.a. wijeweera & m.m.s.t. mapa designed the study. r.m.n.wijerathna, a.a. wijeweera, & a.m. wijethunga carried out the laboratory work. r.m.n.wijerathna, a.a. wijeweera & m.m.s.t. mapa analyzed the data. 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(1981). antimicrobial activity of sorbate. journal of food protection, 44(8), 614–622. https://doi.org/10.4315/0362-028x-44.8.614 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 315-321 | doi: 10.14421/biomedich.2023.121.315-321 issn 2540-9328 (online) synthesis, spectroscopic analysis and antidiabetic properties of copper (ii) complex of mangifera indica leaf crude extract mary adelaide oladipo1, folasade omobolanle ajao2, adewusi john adepoju1,*, kayode taiwo ishola3, olalekan jamiu ajeigbe1 1department of pure & applied chemistry; 2department of physiology, ladoke akintola university of technology, p.m.b. 4000, ogbomoso, oyo state, nigeria. 3department of chemistry, federal college of education (special), p.m. b. 1089, oyo, oyo state, nigeria. corresponding author* ajadepoju@lautech.edu.ng manuscript received: 23 january, 2023. revision accepted: 13 march, 2023. published: 30 march, 2023. abstract many applied conventional drugs in treating diabetes have been reported to possess some drawbacks which necessitate a search for alternative therapies. in order to search for a more active antidiabetic agent, this study synthesized and evaluated antidiabetic properties of mangifera indica crude extract and its cu (ii) complex in alloxan-induced diabetic albino rats. the leaf crude extract and its metal complex were characterized using percentage metal analysis and ir spectroscopic data. experimental animals were induced by a single intraperitoneal injection of alloxan monohydrate at a single dose of 140 mg/kg body weight and animals with fasting blood glucose level (bgl) > 200 mg/dl were considered diabetic. metformin was used as a standard drug. fasting blood glucose level and body weight were used to assess the antidiabetic activity. one-way anova was used to determine the level of statistically significant at p< 0.05. the crude extract was found to coordinate with the metal ion through o donor atom of c=o and o-h of phenol and ketone respectively. the cu (ii) complex of the crude extracts at tested dose of 600mg/kg demonstrated more antidiabetic activity without weight gain than the standard drug. it is concluded that the cu (ii) complex could be a potential material in the development of more active and negative-side-effectfree antidiabetic drug. keywords: albino rats; body weight; blood glucose; metal complex. introduction the increase in mortality caused by diabetes has become one of the biggest health challenges of the 21st century. according to (who, 2016), diabetes is responsible for about 0.02% of mortality worldwide in 2015 and about 8.8% of adults were evaluated by international diabetes federation (idf) to possess diabetes. diabetes has become rampant and steadily increasing worldwide. however, the widespread of this disease can harshly impact the finances of individuals and their families, and the economies of nations. many individuals with type 2 diabetes which is typified by hyperglycemia and abnormal carbohydrate metabolism, who are dependent on insulin for survival, suffer due to a dearth of affordable insulin (who 2016; upadhyay et al., 2018). the number of mortality caused by diabetes is envisaged to reach about 10.4% unless effective prevention is accessible (patarakijavanic eta al., 2019). many synthetic drugs have been applied to combat diabetes and these drugs are reported to act in different ways by lowering the level of blood glucose, for example, increased insulin secretion (sulfonylureas and meglitinides), decreased insulin resistance (biguanides and thiazolidinediones), increased prandial insulin secretion (dpp-4 inhibitors), reduced carbohydrate absorption (αglucosidase inhibitors), and inhibiting glucose reabsorption in the proximal renal tubule, resulting in increased renal glucose excretion and lower blood glucose levels (sglt2 inhibitors) (chao and henry, 2010). many of the present antihyperglycemic agents have been reported to be less effective, expensive and many drawbacks such as hypoglycemic episodes, gastrointestinal disturbances, skin reactions, lactic acidosis, fluid retention, and weight gain are associated with them (krentz and bailey, 2005). since many orthodox drugs and other conventional therapies are less effective or ineffective with several shortcomings in treating diabetes, there is an increase in the study of medicinal plants with hypoglycemic properties to replace the conventional drugs. recently, there is a rapid evolution of studies in the area of herbal medicine and an upsurge in the use of medicinal plants in treating many ailments both in developing and developed countries. their use is attributed to their natural origin, https://doi.org/10.14421/biomedich.2023.121.315-321 316 biology, medicine, & natural product chemistry 12 (1), 2023: 315-321 fewer side effects, accessibility, effectiveness, and affordability (bandaranayake, 2006). the leaves of mangifera indica linn. commonly called mango have been applied for the treatment of fever, diarrhea, fainting, abnormality of lymph nodes, diabetes, and many ailments (aderibigbe et al., 2001; dineshkumar et al., 2010; andrew et al., 2013; garridosuarez et al., 2014; ganogpichayagrai et al., 2017). the bioactivities of the leaf are attributed to the presence of secondary metabolites which include flavonoids, tannins, alkaloids, terpenoids, anthraquinones, saponins, cardiac glycosides, and steroids. mangiferin, c19h18o11 (figure 1), a glucosyl xanthone (1, 3, 6, 7-tetrahydroxyxanthonec2-β-d-glucoside) has been reported to be a prominent polyphenolic constituent found in mongo and it is mainly found in mango leaves, barks, and fruit peels (patarakijavanic et al. 2019). o o oh h oh oh oh oho ho ho figure 1:mangiferin figure 1. mangiferin. the discovery of secondary metabolites from plants for clinical applications has provided a model for drug synthesis. in the last few years, there is a developing interdisciplinary field in the development of metal-based drugs using natural products. transition metals show different oxidation states and can coordinate with molecules containing donor atoms or anions. these properties of transition metals are responsible for the development of metal-based drugs. the pharmacological properties and bioavailability of natural products can be modified by metal coordination (jurca et al. 2017). the dearth of access to affordable insulin and synthetic drugs has become a fundamental barrier to the successful treatment of diabetes and results in unnecessary problems and premature deaths in many developing and underdeveloped countries. therefore, in order to overcome the spread of diabetes through the use of inexpensive and readily available drugs, this work investigated the antidiabetic activity of mangifera indica leaf crude extract and its metal complex. experimental reagents and equipment all the chemicals and solvents employed are of analytical grade and they are used without further purification. they include n-hexane, ethyl acetate, methanol, distilled water, normal saline, alloxan monohydrate, calcium chloride, copper (ii) acetate, sawdust, pelletized rat feed, and metformin hydrochloride. the infrared spectra of the leaves crude extract and its metal complexes were recorded on agilent ftir spectrophotometer while percentage metal analysis was carried out using atomic absorption spectrometer model pg990. collection and identification of plant leaves mangifera indica leaves were collected from the surroundings of the department of pure and applied chemistry, ladoke akintola university of technology, nigeria. the plant specimen was properly identified and authenticated at the herbarium of the department of pure and applied biology, ladoke akintola university of technology (lautech). the collected leaves were washed, shade dried at room temperature, and powdered in a grinder mill. extract preparation about 1.2 kg of the washed, dried, and powdered leaves of mangifera indica were extracted by cold maceration method using 96 % n-hexane and ethyl acetate solvents for 72 h (pepato et al. 2005). after 72 h of maceration, the ethyl acetate soluble portion was cautiously decanted and concentrated using a rotary evaporator. the residual solvent in the crude extract obtained after concentration was allowed to evaporate at room temperature to avoid the decomposition of the natural metabolites. the dark green colored mangifera indica leaves crude extract obtained was kept in an air-tight desiccator over calcium chloride. the crude extract was weighed and a fresh stock for treating the diabetic animals was daily prepared (wadood et al. 2013; mohammed et al. 2015) preparation of copper (ii) acetate complex of the crude extract a solution of 0.5 g (2.75 × 10-3 mol) of copper (ii) acetate was dissolved in distilled water. the resulting metal solution was added drop wisely to a solution of 1g mango crude extract in methanol. the mixture was stirred on a magnetic stirrer for 1 hr at room temperature. the metal complex formed was filtered in a vacuum system, washed with water, and dried in a desiccator over calcium chloride (cacl2). a pure green solid was obtained and weighed. experimental animals male albino rats weighing 120-150 g body were used and acclimatized at a closely maintained temperature of 25 ± 2 ∘c with a standard relative humidity under photoperiodicity of 12 light:12 dark cycles for 4 weeks with free access to standard rodent pellet diet and water ad oladipo et al. – synthesis, spectroscopic analysis and antidiabetic properties of … 317 libitum. adult male albino rats weighing around 180-220 g were selected for the study. induction of experimental diabetes the male albino rats were allowed to fast overnight with their blood glucose levels and body weight recorded prior to the induction of the alloxan prepared in normal saline. the animals were chemically induced by intraperitoneal injection of freshly prepared alloxan monohydrate in saline at a dose of 160 mg/kg b.wt. the animals were kept under observation and after 48 hr of alloxan, the blood glucose was confirmed using an accuchek glucometer. all animals with plasma glucose levels > 200mg/dl were separated and considered diabetic for the study and the rats having a blood glucose lesser than 200mg/dl glucose level were rejected (sabu and subburaj, 2002; mohammed et al. 2015). administration of the leaf extract and its metal complex the diabetic rats were divided into seven groups for the experimental study with four rats in each group as shown below: a) normal (non induced) rats b) diabetic control (untreated rats) c) diabetic rats treated with metformin hydrochloride (500mg/kgb.wt) d) diabetic rats treated with aqueous mango leaf extract (400mg/kgb.wt), e) diabetic rats treated with aqueous mango leaf extract (600mg/kgb.wt) f) diabetic rats treated with an aqueous complex of mango leaf extract with copper (ii) (400mg/kgb.wt), g) diabetic rats treated with an aqueous complex of mango leaf extract with copper (ii) (600mg/kgb.wt) the blood glucose and body weight of rats in each group were measured and evaluated on day 0, day 5, day 10, and day 15 using accuchek glucometer with disposable test strips and digital weighing balance respectively monitoring of blood glucose concentration and animal weight the accuchek glucometer with disposable test strips was used to determine the blood glucose level in rats. blood samples were collected through the tail of the animals. the tail in each case was first wiped with ethanol and then nibbled with a set of new blades. a test strip was fully inserted into the glucometer before applying a drop of blood to fully cover the test area inside the grey target (sharma et al. 2003). after the collection of blood, the nibbled side of the tail was rubbed with cotton wool soaked in ethanol in order to protect the animal from infection and to arrest further bleeding. blood glucose levels and weight of each animal were monitored and taken three times in three weeks using the accuchek and digital weighing balance respectively (shastr, 1980). statistical analysis the results from the number of experiments were expressed in mean and standard deviation, and subjected to the analysis of variance (one-way anova) to determine the significant levels difference between the groups using a t-test. the values with p < 0.05 were considered significant. results and discussion the mangifera indica leaves crude extract and its metal complex were characterized using infrared spectroscopic and aas analyses. the physical and chemical properties of the mango leaf crude extract, its metal complex, ir, and effects of the leaf extract, its metal complex, and the standard drug on diabetics are presented in tables 1, 2, 3, and 4 respectively. the anti-diabetic property of the mango leaves crude extract and its metal (ii) complex was evaluated through the body weight and blood glucose level. the body weight and blood glucose level before and after inducing diabetes were compared to obtain the anti-diabetic efficacy of the complex. the induction of alloxan monohydrate produced hyperglycemia in albino rats. the mango leaf extract and its metal complex caused a significant (p < 0.05) decrease in the fasting blood glucose of treated rats (table 3) when compared with the diabetic control and the metformin-treated group. this decrease was comparable with that of the normal control on certain days. also, extract-treated groups showed statistically significant (p < 0.05) increases in weight gain at the end of 15 days when compared with diabetic control (table 5). table 1. the physical and chemical properties of the mangifera indica leaf crude extract and its cu (ii) complex. compound color (%wt) cu (ii) mangifera indica leaf crude extract dark green 0.0052 [cu(ii) crude extract] dark green 0.0071 table 2. important ir spectra of the mangifera indica leaf crude extract and its cu (ii) complex. compounds oh (cm-1) c=o (cm-1) c-o (cm-1) m-o (cm-1) mangifera indica leaf crude extract 3308 b 1701 s 1194 s [crude extract cu] 3276 b 1640 m 1190 m 800-1000 m b-broad, s-strong, m-medium 318 biology, medicine, & natural product chemistry 12 (1), 2023: 315-321 table 3. effects of mangifera indica leaf crude extract and its cu (ii) complex on blood glucose level of diabetic albino. group pre-treatment(mg/dl) post treatment(mg/dl) day 0 day 5 day 10 day 15 normal control 76.4 + 8.76 73.25 + 7.63 78 + 24.52 82.3 + 22.60 diabetic control 225.7 + 67.52 244.1+ 120.59 304.2 + 45.80 269.9 + 11.81 metformin hcl (500mg/kgb.wt) 337.2 + 51.33 289.3 + 99.78 244 + 82.65 197.9 + 7.69 leaf crude extract (400mg/kgb.wt) 333.5 + 40.99 278 + 106.02 263 + 23.39 206.1 + 4.00 leaf crude extract (600mg/kgb.wt) 301.4 + 129.06 103.7+ 107.19 93 + 90.93** 77.2 + 5.68 ** [crude extract cu] (400mg/kgb.wt) 269.2 + 28.18 296.2+ 115.59 187.4+98.19 102.3 + 9.55 [mango extract cu] (600mg/kgb.wt) 305 + 123.89 264.2+ 130.47 128 + 53.82** 80.4 + 5.25** the values are expressed as means ± sem; n = 4. values are statistically significant at **p < 0.05 compare to diabetic control group (anova). figure 2. histogram representation of effects of mangifera indica leaf crude extract and its metal complex on blood glucose level. table 4. effect of standard rug, mangifera indica leaf crude extract and its cu (ii) complex on body weight of diabetic albino rats. group(n=4) average body weight (g) ±sem day 0 day 5 day 10 day 15 normal control 186.5 + 47.96 185.71 + 60.58 186.25 + 46.95 190.2 + 45.96 diabetic control 225.86 + 17.61 214.85 + 18.95 201 + 11.18 181.32 + 18.20 metformin hcl (500 mg/kgb.wt) 202.71 + 11.31 186.83 + 21.38 197 + 19.98 212.5 + 7.64 leaf crude extract (400 mg/kgb.wt) 189.86 + 14.36 184 + 28.35 205 + 30.35 213.31 + 27.19** leaf crude extract (600 mg/kgb.wt) 209.71 + 26.94 217 + 41.17 217.29 + 25.33 227 + 11.43 ** [crude extract cu] (400 mg/kgb.wt) 204.6 + 15.85 211.4 + 36.10 226.14 + 26.37 247.56 + 1.57 ** [crude extract cu] (600 mg/kgb.wt) 176.86 + 24.71 186.2 + 33.62 169.6 + 17.77 188.21 + 2.88** values are expressed as mean±sem. **p<0.05 as compared to diabetic control group; n= number of animals (one-way anova) treatment l) d g / (ml ev e l se co u g l o o d b l normal control diabetic control metformin (500mg/kg) mangifera indica leaves extract (400mg/kg) mangifera indica leaves extract (600mg/kg) [mangifera indica extract cu](400mg/kg) [mangifera indica extract cu] (600mg/kg) 350 300 250 200 150 100 50 day 0 day 5 day 10 day 15 0 oladipo et al. – synthesis, spectroscopic analysis and antidiabetic properties of … 319 day 0 day 5 day 10 day 15 0 20 40 60 80 100 120 140 160 180 200 220 240 260 normal control diabetic control metformin (500mg/kg) mangifera indica leaves extract (400mg/kg) mangifera indica leaves extract (600mg/kg) [mangifera indica extract cu](400mg/kg) [mangifera indica extract cu] (600mg/kg) a ve ra g e b o d y w ei g h t (g ) treatment figure 3. histogram representation of effects of standard drug, mangifera indica leaf crude extract and its cu (ii) complex on body weight of diabetic albino rats. discussion the mangifera indica leaves crude extract and its metal complex after complexation reaction were dark green in color and the percentage of cu (ii) presents in the crude extract and its metal complex are 0.0052 and 0.0071 respectively as shown in table 1. the important ir bands were shown in table 2. the spectrum of mango leaves crude extract showed two bands at 3308 cm-1 and1701 cm-1. the bands were assigned to o-h of phenol and c=o of ketone respectively (coate, 2001). these were found to shift to 3276 cm-1 and 1640 cm-1 upon coordination with the metal ion. the shift of the bands to the lower frequency and appearance of bands between 1000-800 cm-1 confirmed the formation of the complex and binding of the extract to cu (ii) through oh and c=o of ketone, respectively as shown in figure 4. o o ho h ho oh ho ho o ho ho o o oh h oh oh oh oho ho ho cu figure 4. proposed structure for cu (ii) complex of mangifera indica leaf crude extract. the rate of change in the blood glucose levels in the animals was shown in table 3 and was represented by a histogram (figure 2). no significant variation was observed in the blood glucose level of normal control within 15 days while a significant increase in the glucose level of blood was observed in the diabetic group during the study period compared to the normal control group. the standard drug, the crude extract, and cu (ii) complex of the extract when administered to an alloxan-induced diabetic rat produced a significant reduction in the blood glucose level on the 10th and 15th days of the treatment as compared to control rats. the standard drug at a dose of 500 mg/kgb.wt and mangifera indica leaf crude extract and its cu (ii) complex were administered orally at doses of 400 mg/kgb.wt and 600 mg/kgb.wt for 15 days. on the 15th day, the blood glucose levels of the rats treated with the crude extract and its metal complex at doses of 400 mg/kgb.wt and 600 mg/kgb.wt were observed to significantly decrease (p < 0.05) when compared with the diabetic group. the decrease was found to be more significant in rats treated with the cu (ii) complex of the crude extract at a dose of 400 mg/kgb.wt while the crude extract was more effective at 600 mg/kgb.wt. a change in body weight was observed in the treated rats as shown in table 4 and represented by figure 3. no significant change in the body weight of normal animals from the 0th to the 15th day of treatment was observed. however, the body weight levels of the diabetic control group decreased gradually when compared to the normal non-diabetic rats. the weight levels of the rats treated with the crude extract at doses of 400 mg/kgb.wt and 600 320 biology, medicine, & natural product chemistry 12 (1), 2023: 315-321 mg/kgb.wt groups increased significantly at (p < 0.05) when compared to the diabetic control group on day 15th. also, there was a significant increase in the body weight levels of the rats treated with cu (ii) complex at doses of 400 mg/kgb.wt and 600 mg/kgb.wt groups at (p < 0.05) when compared with the diabetic control group on day 15th. however, cu (ii) complex at a dose of 600 mg/kgb.wt produced a normal weight gain in the treated rat as compared to the normal control group. the standard drug was observed to produce abnormal weight gain which could be ascribed to the negative side effect associated with the drug. therefore, the problem of the negative side effect was overcome with the administration of cu (ii) complex at a dose of 600 mg/kgb.wt. in this study, the cu (ii) of the crude extract was found to be more effective than the leaf crude extract. the more increase in body weight and decrease in blood glucose level exhibited by the metal complex of the crude extract could be attributed to the ability of the metal ion to modify the bioavailability and pharmacological properties of the mangifera indica leaf crude extract. the weight gain which was comparable with that of the normal control on the 15th day of administration of cu (ii) complex could be attributed to improved glycaemic control (atangwho et al. 2007; adeoye et al. 2017). conclusion and recommendation in this study, the cu (ii) complex of mangifera indica leaf crude extract showed more diabetic activity than the standard drug. our results provide information on the antidiabetic activity of the metal complex and therefore the metal complex could be considered a potential antidiabetic drug without abnormal-weight-gain side effects. it is recommended that further studies such as nuclear magnetic resonance spectroscopy, magnetic moment susceptibility, and x-ray should be conducted to establish the real structure of the complex. also, the toxicity of the extract and its metal complex should be examined in order to establish its ideality as a potential antidiabetic drug. acknowledgment: the authors are grateful to prof. a.t.j ogunkunle of the department of pure and applied biology, ladoke akintola university of technology (lautech), ogbomoso, oyo state, nigeria for authenticating the plant material. ethical considerations: all experimental procedures were in agreement with the ladoke akintola university of technology ethics committee on research in animals and internationally approved principles for laboratory animal upkeep and use. conflict of interest: no conflict of interest. references adeoye a t, oyagbemi a a, adedapo a d, omobowale t o, ayodele a e, adedapo a a (2017). antidiabetic and antioxidant activities of the methanol leaf extract of vernonia amygdalina in alloxan-induced diabetes in wistar rats’j. med. plant. econ. dev. 1(1): 30-45 http://doi.org/10.4102/jomped.v1i1.30 aderibigbe a o, emudianughe t s, lawal b a s (2001). evaluation of the antidiabetic action of mangifera indica in mice, phytotherapy research 15 (5): 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(2006). quality control, screening, toxicity, and regulation of herbal drugs, in: i. ahmad, f. aqil, and m. owais (eds), modern phytomedicine turning. medicinal plants into drugs. wiley-vch gmbh & co. kgaa, weinheim chao ec, henry r r (2010). sglt2 inhibition–a novel strategy for diabetes treatment.nat. rev. drug discov. 9: 551–559. coates j (2001). interpretation of infrared spectra, a practical approach, encyclopedia o of analytical chemistry in r.a. meyers (ed.), john wiley & sons ltd, chichester. dineshkumar b, mitra a, manjunatha m (2010). studies on the antidiabetic and hypolipidemic potentials of mangiferin (xanthone glucoside) in stz-induced type 1 and type 2 diabetic rat models. int j adv pharm sci. 1: 75–85. ganogpichayagrai a, palanuvej c, ruangrungsi n (2017) antidiabetic and anticancer activities of mangifera indica cv. okrong leaves. j adv pharm technol res., 8:19-24 garrido-suarez b b, garrido g, castro-labrada m, merino n, valdes o, rodeiro i, delgado hernandez r (2014). antihypernociceptive effect of mangiferin in persistent and neuropathic pain models in rats. pharmacol. biochem. behav. 124, 311–319. 10.1016/j.pbb.2014.06.019 jurca t, marian e, vicaş l g, mureşan m e (2017). metal complexes of pharmaceutical substances, spectroscopic analyses developments and applications, in: e. sharmin editor and f. zafar editor ed(s)., intechopen, london. krentz aj, bailey cj (2005) oral antidiabetic agents: current role in type 2 diabetes mellitus. drugs 65: 385–411 https://doi.org/10.2165/00003495-200565030-00005 mohammed a, kumar d, rizvi s i (2015). antidiabetic potential of some less commonly used plants in traditional medicinal systems of india and nigeria. j intercult ethnopharmacol, 4(1): 78-85. doi: 10.5455/jice.20141030015241. patarakijavanic p, sato v h, kongkiatpaiboon s, chewchinda, s. (2019) a review of the antidiabetic potential of mangifera indica leaf extract. songklanakarin j. sci. technol. 41 (4): 942 950 pepato m t, mori d m, baviera a m, harami j b, vendramini r c, brunetti i l (2005). fruit of the jambolan tree (eugenia http://doi.org/10.4102/jomped.v1i1.30 https://doi.org/10.4314/gjpas.v13i1.16677 oladipo et al. – synthesis, spectroscopic analysis and antidiabetic properties of … 321 jambolana lam.) and experimental diabetes j. ethnopharmacol 96:43-48 sabu m c, subburaj t (2002). effect of cassia auriculata linn. on serum glucose level, glucose utilization by isolated rat hemidiaphragm, j ethnopharmacol 85: 201-206 sharma s, nasir a, prabhu k m, murthy p s, dev g (2003). hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds of eugenia jambolana in alloxan-induced diabetic rabbits. j ethnopharmacol 85: 201-206 https://doi.org/10.1016/s0378-8741(02)00366-5 shastr k (1980). comments on charaka samhita. chanukah bharati, varanasi, india. upadhyay j., polyzos s.a., perakakis n, thakkar b, paschou s a, katsiki n (2018) pharmacotherapy of type 2 diabetes: an update. metabolism 78:13-42. wadood a, ghufran m, jamal s b, naeem m, khan a, ghaffar r (2013). phytochemical analysis of medicinal plants occurring in local area of mardan, biochem anal biochem 2: 144. doi: 10.4172/2161-1009. world health organization (who)m. (2016). global report on diabetes. retrieved from https://apps.who.int/iris/bitstream/handle/10665/204871/97892 41565257_eng.pdf;jsessionid=f e60bb6c97974a6bcd4d02f64f018ea?sequence=1 https://doi.org/10.1016/s0378-8741(02)00366-5 https://apps.who.int/iris/bitstream/handle/10665/204871/9789241565257_eng.pdf;jsessionid=f https://apps.who.int/iris/bitstream/handle/10665/204871/9789241565257_eng.pdf;jsessionid=f this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 9-16 | doi: 10.14421/biomedich.2025.141.9-16 issn 2540-9328 (online) identification of jamu ku-he-se as an alternative health supporting ingredient loth botahala1,*, hemy ratmas djasibani2, martasiana karbeka1, maria hendriana nahak2 1chemistry study program, faculty of mathematics and natural sciences, tribuana kalabahi university, alor, indonesia. 2agricultural product technology study program, faculty of agriculture and fisheries, tribuana kalabahi university, alor, indonesia. corresponding author* botahala@gmail.com manuscript received: 20 september, 2024. revision accepted: 18 february, 2025. published: 18 february, 2025. abstract herbal plants have been used since ancient times in traditional medicine and continue to be maintained and preserved in indonesia. they are called jm. each part of an herbal plant has active components in the form of secondary metabolite compounds, ranging from tubers, wood, sap, latex, leaves, flowers, fruits, and seeds that function to maintain life in their environment. turmeric, ginger, and lemongrass are included in jm plants, which are very useful in traditional medicine because they have various secondary metabolite compounds. this study aims to obtain an even distribution of secondary metabolite compounds through phytochemical tests, inhibition of free radicals through antioxidant activity tests, and an even distribution of nutrients through proximate analysis in jkhs samples with a certain formula. the results of the phytochemical test showed that jkhs contains several important phytochemical components with the intensity it shows. phenolic content (++) can provide benefits in protecting body cells from oxidative damage. saponin (+) has anti-inflammatory properties and has the potential as an antimicrobial agent. in addition, steroids (+) and terpenoids (++), play a role in regulating the body's physiological functions and can support hormonal balance and the immune system and the potential in treating certain diseases. alkaloid content (+)can affect the nervous system and has analgesic and stimulant properties. jkhs samples also contain carotenoids (+), which protect body cells from free radicals and oxidative damage. with the ic50 value of 4.838 µg / ml of the sample, it shows that jkhs extract has very strong antioxidant potential. through proximate analysis, it was obtained that the water content (15.38%) reflects the humidity level in jkhs which can affect the stability of product storage, the ash content (6.29%) indicates that jkhs can be a good source of minerals to support health, the crude protein content (3.79%) indicates that jkhs contributes to daily protein intake, especially in regular consumption, the fat content (6.43%) contributes energy and plays an important role in the absorption of fat-soluble vitamins and other bioactive compounds that may be contained in jkhs, the carbohydrate content (81.37%) indicates that the main component of jkhs is carbohydrates, which function as the main source of energy for the body that can support physical activity and daily energy needs, and the fiber content (5.45%) is an important component that helps smooth the digestion process, improves intestinal health, and helps prevent digestive disorders such as constipation, and plays a role in maintaining blood sugar levels and supporting weight management. overall, the proximate composition of jkhs has good nutritional value, with carbohydrates as the dominant energy source, as well as additional protein, fat, and fiber that support overall health. this combination of formulas makes jkhs have great potential as a jm drink that can provide various health benefits, especially in protecting the body from oxidative stress, inflammation, and other disease risks, including supporting the regeneration of cells, nerves, and other organs of the body that are weak due to climate, work, or old age. keywords: antioxidant analysis; jkhs; phytochemical analysis; proximate analysis; traditional medicine. introduction climate change (botahala, manimoy, et al., 2022) due to global warming (botahala et al., 2021); (botahala, oualeng, et al., 2022) triggered by the rapid development of technological advances (botahala, 2021a) today also plays a role in hurting public health. this negative impact on public health can also occur due to excessive work with a traditional lifestyle where people often ignore health principles (botahala et al., 2023). for example, when working, people no longer pay attention to rest time, people pay less attention to their diet and the menu they consume, and so on. these conditions require people to use herbal medicinal plants as a traditional medicine to prevent contracting diseases caused by viruses by maintaining and even increasing immunity. traditional medicine comprises a compendium of knowledge, skills, and practices that are grounded in various theoretical frameworks, beliefs, and experiential insights derived from diverse cultural backgrounds employed in health care, which have been transmitted across generations (taek et al., 2019); (fatemi et al., 2023). the genesis of traditional medicine is rooted in the personal experience of illness encountered by oneself or by others, thereby fostering the evolution of reasoning concerning the etiological factors of illness based on discernible signs or symptoms experienced, which subsequently catalyzes the conceptualization of methods https://doi.org/10.14421/biomedich.2025.141.9-16 10 biology, medicine, & natural product chemistry 14 (1), 2025: 9-16 for the prevention and treatment of illness or disease (taek, 2020). herbal plants have been used traditionally since ancient times in traditional medicine (taek, 2020), and continue to be maintained and preserved until now in indonesia it is called jamu (jm) (maharani et al., 2023). each part of an herbal plant has active components in the form of secondary metabolite compounds, ranging from tubers, wood, sap, latex, leaves, flowers, fruits, and seeds which function to maintain life in their environment (kawabata et al., 2019); (fatemi et al., 2023). several spice plants that are rich in secondary metabolite compounds (besituba et al., 2024) have the potential as jm (priyanto et al., 2024), among which are often used as kitchen spices. their properties and benefits have been scientifically described in the book herbal bumbu dapur (botahala, 2021a). turmeric from the zingiberaceae family with the species name curcuma longa l. contains compounds that have health benefits (botahala, 2021b), including curing joint pain, preventing cancer, and others (singletary, 2020); (bhowmik et al., 2009); (botahala et al., 2023). ginger has a spicy taste caused by a ketone compound called zingerone, which has antimicrobial, anti-inflammatory, antitumor activities, and others (imo & za’aku, 2019); (azmat et al., 2019). ginger also has a protective effect on the digestive tract and relieves nausea symptoms (shahrajabian et al., 2019). lemongrass from the poaceae family or grasses is the cymbopongoncitratus species (spriha et al., 2021) which is quite abundant in indonesia. lemongrass also contains several secondary metabolite compounds (spriha et al., 2021); (mukherjee et al., 2024); (sulaswatty et al., 2019). the benefits of lemongrass include anti-inflammatory, antiinflammatory, antioxidant, improving blood circulation, relieving coughs, expectorant, and others (j. thasrin & v. anitha, 2023); (madane, 2024). these very beneficial turmeric, ginger, and lemongrass can be made into jm with a specific formula. this jm is made in powder form by drying it first. this is done to maintain the availability of raw materials. the powder of each sample is then formulated with a ratio of 50% turmeric powder because of its curcumin which can provide various benefits, compared to 33% ginger powder to avoid stomach irritation (harwati, 2012) and even slightly interfere with liver function (nwaopara et al., 2007) due to the properties of zingiber, compared to 17% lemongrass powder to maintain the safety of pregnant women and the quality of breast milk (saphira, 2020), to avoid excessive dehydration (royhanaty et al., 2018), even its role as a natural insecticide and antiseptic (gaba et al., 2020) due to its citronellal properties. these three sample powders were mixed to obtain jamu kuhe-se (jkhs) powder. this comparison formulation is made based on the chemical characteristics of each plant. this study aims to obtain an even distribution of secondary metabolite compounds in jkhs samples through phytochemical screening (sanni et al., 2023); (sanni et al., 2024) and potent antioxidant properties in terms of antioxidant activity to determine the inhibitory power (ic50) against free radicals in the body (filbert et al., 2014); (moghaddam et al., 2021), as well as proximate analysis to determine the distribution of nutrients (sanni et al., 2023); (sanni et al., 2024) from a mixture of turmeric, ginger, and lemongrass from alor regency, east nusa tenggara province, indonesia. the results of the study are expected to be used in maintaining body immunity, including supporting the regeneration of cells, nerves, and other organs that are weak due to climate, work, or old age. materials and methods materials the materials used are 3 kg each of turmeric rhizome, 2 kg of ginger rhizome, and 1 kg of lemongrass stalks, concentrated hydrogen chloride (hcl), iron (iii) chloride (fecl3), concentrated sulfuric acid (h2so4), chloroform (chcl3), distilled water, norite/charcoal, ammonia (nh3), ethanol (c2h5oh) 95%, anhydrous acetic acid (c4h6o3) or (ch3co)2o, ammonia (nh3), meyer's reagent, dragendorff's reagent, wanger's reagent, 2,2diphenyl-1-picrylhydrazyl (dpph), hexane (c6h14), acetone (c3h6o), potassium sulfate (k2so4), mercury (ii) oxide (hgo), sodium hydroxide (naoh), sodium thiosulfate (na2s2o3), boric acid (h3bo3), methylene red (c15h15n3o2), methylene blue (c16h18n3scl), and test samples in the form of turmeric rhizome, ginger rhizome, and lemongrass stalks taken from alor regency, east nusa tenggara province, indonesia. the types of equipment used in this study were: blender, knife, sieve, cotton, basin, stirrer, beaker, filter paper, frying pan, stove, mesh sieve, spoon, scale, analytical balance, test tube, buchner funnel, water bath, dropper plate, measuring pipette, pasteur pipette, porcelain cup, erlenmeyer, desiccator, oven, 1 ml micropipette, soxhlet apparatus, kjeldahl flask, petri dish, thermometer, distillation flask, fat flask, incubator, refrigerator, uvvis spectrophotometer, burette and stand, measuring flask, cuvette, dark vial, and aluminum cup. methods sample preparation each turmeric rhizome, ginger rhizome, and lemongrass stalk cleaned is taken, thinly sliced, and dried using sunlight. after drying, the samples are ground separately using a blender to produce powder and sieved using a sieve with a mesh size of no. 60 (0.25 mm). the powder of each sample is then formulated with a ratio of 50% turmeric powder compared to 33% ginger powder compared to 17% lemongrass powder to obtain a jkhs powder sample that is ready to be analyzed. botahala et al. – identification of jamu ku-he-se … 11 phytochemical analysis the jkhs phytochemical test procedure was carried out based on the procedure by (botahala et al., 2024) and (sanni et al., 2024) with several modifications for this study. a total of 3 grams of jkhs sample was put into a test tube, then macerated with ethanol in a water bath for 15 minutes. then, it was filtered hot into a test tube and left until all the ethanol evaporated to dryness. after that, the residue and filtrate were separated. the filtrate was added with chloroform and distilled water (aquadest) in a ratio of 1:1, each as much as 6 ml. shake until thoroughly mixed then transfer into a test tube, leave for a moment until a 2-layer solution is formed, namely the chloroform layer and the water layer. furthermore, the chloroform layer and the water layer are separated. the water layer is used for testing phenolic, flavonoid, and saponin content while the chloroform layer is used for testing steroids and terpenoids (botahala et al., 2020). ▪ phenolic test the water layer is inserted into a dropper plate, after which 10 drops of 1% fecl3 are added. if blue, purple, blackish green and solid black colors are formed, it indicates the presence of phenolic compounds. ▪ saponin test the water layer is pipetted into a test tube and then shaken vigorously. if foam forms after a few minutes, it indicates the presence of saponins. ▪ steroid and terpenoid test the chloroform layer is inserted into a pasteur pipette containing charcoal or norite. the filtrate from the pipette is inserted into three holes in the dropper plate and left to dry. furthermore, concentrated sulfuric acid (h2so4) is added to the first hole, a drop of anhydrous acetic acid and a drop of sulfuric acid are added to the second hole, while the third hole is used as a blank. if a blue-purple color is formed, it indicates the presence of steroids, while if a red color is formed, it indicates the presence of terpenoids. ▪ alkaloid test a total of 4 grams of jkhs sample was put into a container, then 10 ml of 0.05 n chloroform-ammonia was added and stirred slowly. after that, the solution was filtered with a small funnel with cotton placed as a filter, and the filtered results were put into a test tube. the filtrate was added with 10 drops of 2 n sulfuric acid and shaken slowly. after that, it was left until an acid and chloroform layers formed. a drop of meyer's reagent dragendorff's reagent and wanger's reagent were added to the acid layer to obtain the results. (the reaction with mayer's reagent will form a white precipitate, with dragendorff's reagent, a red-orange precipitate is formed, and with wagner's reagent, a brown precipitate is formed) antioxidant activity test the antioxidant test procedure was carried out based on the procedures carried out by (moghaddam et al., 2021), (botahala et al., 2024), and (pratiwi et al., 2023) with some modifications for this study. ▪ preparation of dpph solution weighed 15 mg of dpph solid using an analytical balance, dissolved with 20 ml of ethanol in a dark vial, and homogenized. the solution was then put into a 25 ml measuring flask to the limit mark and incubated in the refrigerator for 30 minutes. the dpph control solution was ready to use. the dpph control solution was tested on a uv-vis spectrophotometer with a wavelength of 516 nm as an absorbance control in this test. ▪ sample preparation and antioxidant test a total of 3 mg of jkhs test sample was made into a solution with a concentration of 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm. each test solution was taken 1 ml and added 2 ml of dpph and 1 ml of ethanol respectively then incubated for 30 minutes at room temperature. after that, samples of each concentration were transferred into a cuvette and measured using a uv-vis spectrophotometer at a wavelength of 516 nm to determine the absorbance value. furthermore, antioxidant activity was determined using the % inhibition formula in the equation. % antioxidant = absorbance of blank−absorbance of the test sample absorbance of blank x 100 the results of the calculation of this antioxidant activity were then used to calculate the inhibition value (ic50) (yuliani et al., 2016); (botahala, 2024). the % inhibition value at each concentration was then entered into the regression equation with the sample concentration as the x-axis and % inhibition as the yaxis. the ic50 value was obtained from the calculation when % inhibition was 50% from the equation y = ax ± b. proximate test next, a proximate test was carried out based on the procedure from sanni et al. (2023) with several modifications in the interests of this study to determine the nutritional content of the jkhs sample. this nutritional content analysis includes testing water content, ash content, protein content, fat content, and carbohydrate content. ▪ water content the water content of the jkhs sample was analyzed using the gravimetric method. the aluminum cup was dried in an oven at 130 ± 3℃ for 15 minutes and then cooled in a desiccator for 10 minutes. a total of 3 grams of sample was weighed into an aluminum cup of known 12 biology, medicine, & natural product chemistry 14 (1), 2025: 9-16 weight (the cup must be dried in an oven before being used for weighing), then dried in an oven at a temperature of 105℃ for 3 hours, cooled in a desiccator and weighed until a constant weight was obtained (≤ 0.0005 g). % water content = a−b a x 100 ▪ ash content ash content analysis of jkhs samples was carried out using the gravimetric method. an empty porcelain cup and lid were dried in an oven at 105℃ for 15 minutes and cooled in a desiccator. the dry porcelain cup was weighed, and its weight was recorded before use. a total of 5 grams of spice powder samples were weighed in the porcelain cup and put into an electric furnace at 550℃ until the ashing process was complete. after the ashing was complete, the sample was cooled in a desiccator. the sample can then be weighed and calculated. % ash content = b a x 100 description: a : mass of the sample before heating b : mass of the sample after heating ▪ protein content analysis of protein content of jkhs samples was analyzed using the kjeldahl method. a total of 200.0 mg of sample was put into a kjeldahl flask then added with 1.9 ± 0.1 g k2so4, 40.0 ± 10 mg hgo, 2 ± 0.1 ml concentrated h2so4 and three boiling stones. the sample was heated at a gradual temperature until boiling for 2 hours until a clear liquid was obtained. after being cooled, the contents of the flask were transferred into a distillation flask by rinsing with 2 ml of distilled water 5 – 6 times. the washing water was transferred to a distillation flask and then added with 8 – 10 ml of 10% naoh – 5% na2s2o3 solution. in a separate place, 5 ml of h3bo3 solution and 2 – 4 drops of methyl red indicator – methyl blue were put into an erlenmeyer flask. the erlenmeyer flask was then placed under the condenser with the tip of the condenser submerged under the h3bo3 solution. the distillation process was carried out until approximately 15 ml of distillate was obtained. the distillate obtained was diluted to 50 ml with distilled water, then titrated with a standardized 0.02 n hcl solution until the color changed to gray. the volume of the standardized 0.02 n hcl solution used for titration was recorded. the same steps were carried out for the blank solution, so the volume of 0.02 n hcl solution was obtained for the blank. the protein content was calculated based on the nitrogen content (n in g/1000 g of material). the nitrogen content value in the sample can be calculated using the formula: %n = (ml hcl sample−ml hcl blank)x n hcl x 14,007 mg sample x 100 after obtaining the nitrogen content (%n), the protein content was calculated using the conversion factor. 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = %𝑁 𝑥 𝑓_𝑘 fk = correction factor (6.25) ▪ fat content the fat content of the jkhs sample was analyzed using the soxhlet method. the fat flask was dried in an oven at 105℃ for 15 minutes, cooled in a desiccator, and weighed before use. a total of 10 grams of the jkhs sample was inserted into a filter cartridge lined with cotton. the top of the paper cartridge containing the sample was plugged with cotton and then dried in an oven at no more than 80℃ for ± 1 hour. the paper cartridge was then inserted into a soxhlet apparatus connected to the fat flask. the fat sample was extracted with hexane for ± 6 hours. the hexane was then distilled to obtain the fat extract. the fat extract in the fat flask was then dried in an oven at 105℃ for 12 hours. the fat sample flask was then cooled in a desiccator and weighed. drying was repeated until a constant weight was obtained. ▪ carbohydrate content determination of carbohydrate content of jkhs samples using by difference with the following formula: % carbohydrate = 100% (% water content + % protein content + % ash content + % fat content). ▪ fiber content a total of 2 grams of jkhs sample was put into a 250 ml beaker and 50 ml of 0.3 n h2so4 was added and then heated at 70℃ for 1 hour. next, 25 ml of 1.5 n naoh was added and heated for 30 minutes at 70℃. the solution was then filtered using a buchner funnel. during filtration, the precipitate was washed successively with sufficient hot distilled water, 50 ml of 0.3 n h2so4, and 25 ml of acetone. filter paper containing the residue was put into a petri dish and dried in an oven for 1 hour at 105℃. cooled and weighed. crude fiber content (%) = b−a x x 100 description: b = weight of filter paper + sample after oven; a = weight of filter paper; x = weight of sample. botahala et al. – identification of jamu ku-he-se … 13 results and discussion results phytochemical composition the results of phytochemical screening showed the presence of several compounds, such as phenolics, saponins, steroids, terpenoids, alkaloids, and carotenoids, as shown in table 1 below. table 1. results of jkhs phytochemical tests. no compounds analysis results 1 phenolic (++) 2 saponins (+) 3 steroids (+) 4 terpenoids (++) 5 alkaloids (+) 6 carotenoids (+) antioxidant activity the data obtained from the analysis of antioxidant activity are shown in table 2. table 2. results of the antioxidant activity test of jkhs samples. concentration (µg/ml) abs. blank abs. sample antioxidant activity (%) 12.5 0.79 0.69 12.6582 25.0 0.79 0.56 29.1139 50.0 0.79 0.43 45.5696 100.0 0.79 0.1 87.3417 the data from the antioxidant activity test results were then entered into a linear regression to produce data as shown in the graph in figure 1 below. figure 1. graph of the relationship between concentration and antioxidants jkhs. proximate composition the proximate analysis of jkhs includes analysis of water content, ash content, protein content, fat content, carbohydrates content, and fiber content, as in table 3 below. table 3. proximate test results of jkhs samples. no content analysis results 1 water (%) 15.38 2 ash (%) 6.29 3 crude protein (%) 3.79 4 crude fat (%) 6.43 5 carbohydrate (%) 81.37 6 fiber (%) 5.54 discussion phytochemical test the results of the phytochemical test showed that jkhs contains several important phytochemical components with the intensity shown. the phenolic content was detected as very high (++), which indicates its potential as an antioxidant agent. it plays an important role in protecting the body from oxidative damage caused by free radicals. saponins were also found in positive amounts (+), which have a role in anti-inflammatory and immunomodulatory activities. this indicates that jkhs can help fight infections and reduce inflammation. in addition, steroids (+) and terpenoids (++), which have antimicrobial and anti-inflammatory activities, were also detected. terpenoids have various benefits, including as antioxidants, anti-cancer, and anti-microbial. this indicates that this jm has the potential to help reduce inflammation and support immune system health. the alkaloid content (+) is known to have analgesic (pain reliever) and antispasmodic effects. its presence in herbal kitchen spice samples indicates that this herbal medicine can help reduce pain and fight damage caused by free radicals. jkhs samples also contain carotenoids with (+) intensity which function as antioxidants, and pro-vitamin a, which are also found in jkhs. carotenoids are known for protecting the skin and eyes from uv damage. antioxidant activity the test results indicate that antioxidant activity increases with increasing extract concentration. data obtained from the analysis of antioxidant activity are shown in table 2. among the various concentrations tested, 100 µg/ml concentration showed the highest antioxidant activity, while 12.5 µg/ml concentration showed the lowest. at a concentration of 12.5 µg/ml, the extract showed significant antioxidant activity (12.6582%). this shows that at low concentrations, the extract has begun to provide antioxidant effects. at a concentration of 25 µg/ml, antioxidant activity (29.1139%) showed an increase with increasing concentration. this shows a positive correlation between extract concentration and the ability to capture free y = 0.826x + 4.9532 r² = 0.9936 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 a n ti o xi d an t concentration 14 biology, medicine, & natural product chemistry 14 (1), 2025: 9-16 radicals. at a 50 µg/ml concentration, antioxidant activity increased (45.5696%), indicating that the extract has practical components in counteracting free radicals at medium concentrations. at a concentration of 100 µg/ml, antioxidant activity peaked (87.3417%). the strong potent antioxidant effect indicates that the extract has excellent potential as a source of natural antioxidants at high concentrations. furthermore, based on the linear regression equation (figure 1), y = 0.826x + 4.9532 was obtained with a correlation coefficient of r2 (0.9936). the r2 value describes the linearity of concentration to the percentage of inhibition. a value approaching 1 indicates that with increasing extract concentration, antioxidant activity also increases. by entering the value of y = 50 (the inhibitory power at 50% of free radicals), the x value will be obtained (where x is the value of ln ic50). thus, the ic50 value (which is the value of anti-ln value x) is 4.838 µg/ml sample. this value shows that jkhs extract has very strong antioxidant potential. proximate analysis overall, the proximate composition of jkhs has good nutritional value, with carbohydrates as the dominant energy source and additional protein, fat, and fiber that support overall health. table 3 above, shows that the results of the proximate analysis of jkhs show several main nutritional components that provide an overview of its quality and nutritional potential. ▪ the water content of 15.38% reflects the humidity level in jkhs. this relatively high water content indicates that jkhs still has a significant water content, which can affect the stability of product storage. therefore, it is necessary to pay attention to proper drying and storage techniques so that jkhs remains stable and is not easily damaged during the shelf life. ▪ the 6.29% ash content indicates the mineral content in jkhs. ash content indicates the amount of minerals remaining after the sample combustion process, which reflects the content of mineral elements such as calcium, magnesium, potassium, and other microelements that are important for the body. this fairly high ash content indicates that jkhs can be a good source of minerals to support health. ▪ the crude protein content of 3.79% indicates that jkhs contains moderate protein. protein is an important component in supporting the growth and repair of body cells. although not the primary source of protein, jkhs still contributes to daily protein intake, especially in regular consumption. ▪ the crude fat content of 6.43% provides a significant energy contribution. fat also plays an important role in absorbing fat-soluble vitamins and other bioactive compounds that may be contained in jkhs. this fat also provides texture and flavor to the mixture, which can improve the palatability or taste of the product. ▪ the carbohydrate content of 81.37% shows that the main component of jkhs is carbohydrates, which function as the primary source of energy for the body. high amounts of carbohydrates provide the potential for jkhs as a good source of calories, supporting physical activity and daily energy needs. ▪ the fiber content of 5.45% is an important component that benefits digestive health. fiber helps smooth the digestive process, improve gut health, and helps prevent digestive disorders such as constipation. in addition, fiber also plays a role in maintaining blood sugar levels and supporting weight management. conclusions the results of the phytochemical test showed that the jkhs sample is rich in various bioactive compounds, including phenolics, saponins, steroids, terpenoids, alkaloids, and carotenoids, which have great potential as a jm drink that can provide health benefits. the results of the antioxidant activity test showed that jkhs is a good choice as a natural source of antioxidants, to provide body protection against free radical attacks. the results of the proximate test indicate the potential for jkhs extract to be used as a health supplement. the combination of the three plants (turmeric, ginger, lemongrass) in one balanced formula makes jkhs have great potential as a jm drink that can provide various health benefits, especially in protecting the body from oxidative stress, free radical attacks, inflammation, and other disease risks, including supporting the regeneration of cells, nerves, and other organs that are weak due to climate, work, or old age. authors’ contributions: loth botahala and hemy ratmas djasibani designed the study and wrote the manuscript draft. hemy ratmas djasibani, martasiana karbeka, and maria hendrina nahak performed the laboratory work and managed the analysis of the research results. martasiana karbeka and maria hendrina nahak managed the literature search. all authors read and approved the final manuscript. competing interests: the authors declared that they have no competing interests. funding: the authors declare that it is not funded. references azmat, s., gulzar, b., & fatima, t. 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(2016). uji aktivitas antioksidan fraksi etil asetat ekstrak etanol rimpang jahe merah (zingiber officinale var. rubrum) dengan metode dpph(1,1-diphenyl-2picrylhydrazyl). informasi kesehatan, 14(1), 1091–1111. https://jurnal.poltekkeskupang.ac.id/index.php/infokes/article/v iew/100/97 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 173-182 | doi: 10.14421/biomedich.2024.131.173-182 issn 2540-9328 (online) antibacterial bioactivity test of bilimbi fruit ethanol extract (averrhoa bilimbi linn). against propionibacterium acnes, staphylococcus epidermidis and staphylococcus aureus reza anindita*, dede dwi nathalia, melania perwitasari , intan kurnia putri, maya uzia beandrade, nofria rizki amalia harahap department of pharmacy, sekolah tinggi ilmu kesehatan mitra keluarga, jl. pengasinan rawa semut raya, margahayu, bekasi timur, jawa barat 17113, indonesia. corresponding author* rezaaninditaa@gmail.com abstract one of the causes of acnes is the bacteria propionibacterium acnes, staphylococcus epidermidis, and staphylococcus aureus. an alternative solution to this problem is to develop natural medicines from plants, one of which is the star fruit (averrhoa bilimbi linn). the purpose of this study was to test the bioactivity of star fruit against the growth of p. acnes, s. epidermidis, and s. aureus. this research method used an experimental research design with the treatment of a. bilimbi fruit ethanol extract concentrations of 50%, 60%, 70%, 80%, and 90%, chloramphenicol antibiotics as control (+), and sterile distilled water as control (-). all treatments were given to p. acnes, s. epidermidis, and s. aureus bacteria using the kirby baeur (disc diffusion) method. the results of this study were the effectiveness of the bioactivity compound of the ethanolic extract of a. bilimbi fruit seen on p. acnes and s. epidermidis bacteria with concentrations of 70%, 80%, and 90%. thus, it can be concluded that a. bilimbi fruit has the potential to be developed as a natural medicine for acne caused by p. acnes and s. epidermidis. keywords: a. bilimbi fruit; propionibacterium acnes; staphylococcus epidermidis; staphylococcus aureus; kirby bauer. introduction one of the causes of acne (acne vulgaris), which triggers the problem of facial skin inflammation, is the bacteria propionibacterium acnes, staphylococcus epidermis, and staphylococcus aureus (sari et al., 2020). the appearance of acne does not only have an impact on facial skin but also has a psychological impact (gallitano & berson, 2018), which results in a decrease in one's self-confidence (abhinitha et al., 2019). this impact is exacerbated by the treatment of acne, which generally uses long-term antibiotics such as tetracycline, erythromycin, clindamycin, doxycycline, and minocycline, which cause bacterial resistance, organ disorders, and immune hypersensitivity (legiawati et al., 2023). referring to the problems and impacts caused by acne-causing bacteria, researchers are interested in testing natural sources of plant ingredients that have the potential to inhibit the growth of p. acnes, s. epidermidis, and s. aureus bacteria as one of the causes of acne. this research is also an effort to search for bioactive compounds in plants that are expected to be developed into alternative drugs to replace antibiotics that are capable of inhibiting the growth of acne-causing bacteria. the plant species to be tested for the potential of bioactive compounds in inhibiting the growth of p. acnes, s. epidermidis, and s. aureus is the starfruit (averrhoa bilimbi linn). the choice of starfruit as the test sample was based on the results of previous studies that reported the administration of starfruit extract on the growth of s. aureus, showing a larger diameter of the inhibition zone compared to a. bilimbi leaf extract. furthermore, administration of ethanol extract of a. bilimbi fruit with concentrations of 10%, 15%, and 20% was able to inhibit the growth of p. acnes with inhibition zones respectively of 16.67 mm, 22.70 mm, and 28.10 mm while in s. aureus of 18.53 mm, 24.16 mm and 30.40 mm (mokhtar & abd aziz, 2016a) ; (pertiwi et al., 2020a) while testing the ethanol extract of star fruits with concentrations of 25%, 50%, 75%, and 100% was only able to inhibit s.epidermidis by 10 mm, 11 mm, 12 mm, 15 mm (zarwinda et al., 2021). manuscript received: 04 february, 2024. revision accepted: 20 may, 2024. published: 30 may, 2024. https://doi.org/10.14421/biomedich.2024.131.173-182 mailto:rezaaninditaa@gmail.com 174 biology, medicine, & natural product chemistry 13 (1), 2024: 173-182 another reason for testing a.bilimbi fruit as an antibacterial also refers to sá et al. (2019), who reported that the content of oxalic acid in a.bilimbi fruit has the potential to have a toxic effect on uremia patients. hence, it is necessary to carry out another study in the form of its content and its effect as an antibacterial, considering the results of the phytochemical screening shown in the study by (setyawan et al. (2021) were positive for the presence of alkaloids tannins, flavonoids, saponins, triterpenoids. a literature review study by garg et al. (2022) informed the results of phytochemical screening and chromatograms of a.bilimbi fruit extract showing the presence of secondary metabolites such as flavonoids, alkaloids, oxalic acid, polyphenols, essential oils, coumarins, valepotriates, and terpenes. the presence of secondary metabolite compounds was able to produce significant inhibition zones on s. epidermidis, s.aureus, bacillus cereus, citrobacter freundii, salmonella typhi, proteus vulgaris, kocuria rhizophila, and aeromonas hydrophila bacteria. based on the results of previous studies, there has yet to be any recent data regarding the response of p. acnes, s. epidermidis, and s. aureus to ethanol extract of a. bilimbi fruit with concentrations above 40%. therefore, researchers are interested in testing the bioactivity of antibacterial compounds contained in the ethanol extract of star fruit with concentrations of 50%, 60%, 70%, 80%, and 90% on the growth of p. acnes, s. epidermidis and s. aureus bacteria. the results of this study are expected to be able to complete the completeness of the database on the concentration of the inhibition of the a.bilimbi fruit, which can be developed as a natural medicine. materials and methods types and research design this type of research was quantitative research using an experimental research design with treatment of ethanol extract of a. bilimbi fruit with concentrations of 50%, 60%, 70%, 80%, 90%, chloramphenicol 30 µg as a positive control, and sterile distilled water as a negative control. all treatments were given to 3 test bacteria, namely p. acnes, s. epidermidis, and s. aureus, with three repetitions. tools and materials the tools used included automatic autoclaves (hirayama hg-80, 76l, japan), p100 micropipette (socorex, switzerland), hot plate and stirer (ika c-mag hs7, germany), incubator (memmerth in-30, germany), laminar air flow (laf) (esco, singapore), rotary evaporator (ika-rv -3 v, germany) 6 hole water bath (hh, china), digital analytical balance (acuplus, china), vm 300 micropipette and vortex mixer (gemmy, taiwan). the materials used included a.bilimbi fruit taken from cikarang, bekasi, indonesia, pure cultures of p. acnes atcc: 11827, s. epidermidis atcc: 12228, s. aureus atcc: 6538 which were purchased at the microbiology laboratory of the university of indonesia, chloramphenicol antimicrobial susceptibility discs (oxoid, germany), antimicrobial susceptibility blank disks (oxoid, germany), sterile cotton swabs (one med, indonesia) media nutrient agar (na) (merck, indonesia), mueller hinton agar (mha) (merck, pro analyst 96% ethanol (merck, indonesia, pro analyst 0.9% nacl (merck, indonesia) and technical aquades (rofa, indonesia). identification and sample preparation the natural product sample in this study was 2.5 kilograms (kg) of a.bilimbi fruit taken from cikarang, bekasi, indonesia. the samples were then observed organoleptically which included shape, smell, color, taste, and other characteristics. plant identification was carried out by sending samples of a. bilimbi fruit to the research center for plant conservation and botanical gardens, indonesian institute of sciences (lipi), bogor, west java, as evidenced by a plant determination certificate. meanwhile, sample preparation included wet sorting, chopping, drying, and grinding. extraction by maceration method weighed as much as 100 grams of dried a. bilimbi fruit simplicia powder, then put it in an erlenmeyer containing 700 ml of 96% ethanol and then tightly closed using aluminum foil. soak for 7 x 24 hours, with stirring carried out every 24 hours until the solvent was completely mixed. the liquid filtrate was filtered with whatman paper no. 1. the maceration process is repeated for 7 days in the same way to obtain a colorless liquid extract (oliveira et al., 2021). evaporation of extraction results the liquid extract obtained from maceration was evaporated using a rotary evaporator with a temperature of 40 ∘c, a pressure of 195 mbar, and a speed of 60 rpm for ± 3 days. after 2 days, heating was carried out on a water bath at 60 ∘c to obtain an optimal thick extract. the results of the thick extract are then calculated by the % yield of the thick extract using the formula: field viscous = weight of viscous extract weight of powder x 100 % antibacterial compound test sterilization of tools and materials sterilized tools and materials include petri dishes, test tubes, 0.9% nacl, dark glass bottles, erlenmeyer containing na and mha media, tweezers, and distilled water. all tools are washed with detergent, rinsed under running water, and dried. the dried tools were wrapped tightly in paper and then sterilized using an autoclave at 121°c with a pressure of 2 atm for 15 minutes. as for the anindita et al. – antibacterial bioactivity test of … 175 ose needle, it is sterilized by heating it on a bunsen flame. preparation of test bacterial suspension (p.acnes, s. epidermidis, and s. aureus) preparation of the test bacterial suspension was carried out by taking several ose of pure cultures of the test bacterial sub-cultures, then putting them in 0.9% nacl and then vortexing until homogeneous. then the results were compared for the turbidity with mc farland 0.5 solution (equivalent to a bacterial suspension of 1.5 x 108 cfu/ml) where if, after being compared with mcfarland, it turns out that the bacterial suspension is still too clear, then a few more ose of the test bacteria can be added. in contrast, if it turns out to be too cloudy, 09% nacl can be added again to obtain a solution of the test bacterial suspension with the same level of turbidity as the standard mcfarland 0.5 solution. antibacterial compound bioactivity test using kirby bauer method the bioactivity test of antibacterial compounds in a. bilimbi fruit was carried out using the kirby-bauer method using a 4-quadrant streak plate technique. then, it was wiped evenly on the surface of the mha media using a sterile cotton swab and left for ± 5 minutes. then, prepare a blank disc that has been dripped with ethanol extract of bilimbi fruit with graded concentrations (50%, 60%, 70%, 80%, and 90%) as much as 30 µl using a micropipette and left for ± 15 minutes, positive control for antibiotic chloramphenicol, and negative control for aquadest sterile and blank discs. all discs (discs) were then placed on the agar plate using sterile tweezers. each petri dish has 3 replicates. all petri dishes were then incubated for 24 hours at 37 ◦c. the diameter of the inhibition zone was measured after 1x24 hours of incubation by measuring the presence/absence of a clear zone formed around the treatment disc using a ruler. the results of measuring the diameter of the inhibition zone were then compared with clsi (2020) guidelines to see the sensitivity category of the test bacteria in response to each treatment disc. figure 1. a. bacterial culture turbidity that complies with the mc farland standard of 0.5. b. blank disk that has been dripped with condensed extract of a. bilimbi fruit. data analysis data analysis in this study was carried out using a quantitative descriptive test by looking at the sensitivity category of the response of the test bacteria in response to treatment discs that had been processed in tabular form. data in tabular form contains the average size of the diameter of the inhibition zone of the tested bacteria from the three bacteria with various treatments. these results were then interpreted based on the category of bacterial sensitivity response that has been adjusted to the clsi standard (2020). result and discussion identification results in the form of a. bilimbi fruit sent to the bogor botanical gardens and plant conservation research center, lipi show that the fruit simplicia used in the study has the indonesian local name belimbing wuluh with the latin name averrhoa bilimbi l. and family oxalidaceae. based on (2013), in general, a. bilimbi plants have green leaves, lancet-shaped, tapered ends, asymmetrical leaf bases, and flat leaf edges. a. bilimbi plant stems are brown, cylindrical in shape, and have no distinctive odor. the a. bilimbi fruit used as a test sample in this study can be seen in figure 2. figure 2. morphology of a. bilimbi fruit. figure 2. shows that ripe a. bilimbi has an elliptical and oval morphology with shiny flesh and is light green or yellowish. according to alhassan and ahmed (2016), starfruit grows on stems with a dark green fruit color 176 biology, medicine, & natural product chemistry 13 (1), 2024: 173-182 before maturity and has a tough flesh texture, but after ripe, it becomes light green with soft and watery fruit flesh. a. bilimbi that has been identified is then carried out wet sorting, chopping, drying, dry sorting, and pollination. the drying results were carried out for 4 days in the sun. the drying results in this study can be seen in figure 3. figure 3. drying process from the first day to the fourth day. according to chau et al. (2021) wet sorting is done by washing the simplicia using running water to remove residual soil and dirt attached to a. bilimbi samples. the clean sample is then sliced by slicing a. bilimbi fruit by 1 cm to help speed up the drying process. the drying process in this study was carried out by covering the simplicia pieces using a black cloth and drying them in the sun. covering it with a black cloth aims to avoid direct sunlight which has the potential to cause changes in the phytochemical compounds contained in a. bilimbi fruit simplicia. the dried simplicia was then extracted using the maceration method and evaporated with a rotary evaporator. the results of the thick extract obtained from 100 grams of a. bilimbi fruit simplicia powder can be seen in figure 4. figure 4. a. simplicia powder. b. viscous extract. figure 4 shows the results of the dried simplicia powder of a. bilimbi fruit which is brown with a acid flavour, while the thick extract has a deep black color. the results of the viscous extract were then calculated for the percentage yield of the viscous extract shown in table 1. table 1. percentage of viscous extract yield. simplicia powder weight weight of viscous yield percentage 100 gr 24.18 gr 24.18% table 1. shows the results of maceration and evaporation extraction from 100 grams of a. bilimbi fruit simplicia powder, which obtained viscous extract weight of 24.18 with a yield percentage of 24.18%. the results of this study complement research data zarwinda et al. (2021) which extracted 150 grams of a. bilimbi leaf powder in 750 ml of 96% ethanol and produced 210 grams of a viscous extract with a percentage of 28%. cheong et al. (2022) reported that the extraction of 100 grams of a. bilimbi fruit powder in 1000 ml of water and ethyl acetate yielded viscous extract weight (% yield) of 16.20 (16.20%) and 2.36 (2.36%). the choice of the maceration method and 70% ethanol solvent was based on the results of a literature review conducted by leliqia & safitri (2021) which stated that qualitative phytochemical screening using the maceration method with 70% ethanol solvent was able to extract the phytochemical compounds found in leaves and fruit a. bilimbi, including others are alkaloids, flavonoids, polyphenols, tannins, saponins, steroids, and triterpenoids. according to garg et al. (2022)maceration is extraction method that is carried out at room temperature without involving a heating process so as to prevent damage to secondary metabolites which are thermolabile. anindita et al. (2022) added that 70% ethanol has the same polarity properties as the phytochemical compounds contained in a. bilimbi fruit, which are both polar. the same polarity between the solvent and the dissolved substance or according to the principle of like dissolves like causes 70% ethanol to be able to enter and attract the phytochemical compounds contained in the cytoplasm of the star fruit cell membrane. the importance of selecting solvent polarity was shown in abraham (2016) research which proved that the use of petroleum ether extract of a. bilimbi fruit as a non-polar solvent was not able to inhibit s. aureus bacteria. this indicates that the use of ethanol is still recommended as a polar solvent for extracting a. bilimbi fruit. the results of the viscous extract of a. bilimbi fruit simplicia were then carried out by phytochemical screening to determine the presence or absence of secondary metabolites contained in a. bilimbi fruit. phytochemical screening in this study included tests for day 4 day 3 day 2 day 1 anindita et al. – antibacterial bioactivity test of … 177 saponins, alkaloids, tannins, phenols, and terpenoids. the results of the phytochemical screening test for a. bilimbi fruit viscous extract in this study can be seen in table 2 and figure 5. table 2. results of phytochemical screening of viscous extracts of a. bilimbi fruit secondary metabolites result reagen information alkaloids + meyer's reagent white precipitate + wagner reagent brown precipitate + dragendorf reagent red/orange precipitate fenol + 4 drops fecl3 blue/dark black flavonoids + etanol 5 ml + 10 drops hcl + 0,2 gram magnesium yellow/orange saponin + 10 ml hot water + hcl 1 n foam 1 cm for 5 minutes tannin + 2 drops fecl3 1 % blackish blue terpenoid + 2 ml h2so4 brown-purple figure 5. a. alkaloids test. b. phenol test. c. flavonoid test. d. saponin test. e. tannin test. f. terpenoid test. the results of the viscous extract of a. bilimbi fruit were then tested for antibacterial effect with concentrations of 50%, 60%, 70%, 80%, and 90% against three acne-causing bacteria, namely p. acnes, s. epidermidis, and s. aureus. the positive control in this study was the antibiotic chloramphenicol at a dose of 30 µg, and the negative control was sterile distilled water. all treatments were saturated in discs with a diameter of 6 mm. the results of the inhibition test of ethanol extract of a. bilimbi fruit on the growth of p. acnes bacteria are shown in table 2 table 2. the results of giving ethanol extract of a. bilimbi fruit to the diameter of the inhibition zone for the growth of p. acnes bacteria. treatment inhibition zone diameter (mm) mean category replication 1 replication 2 replication 3 control (+) 20.5 21.5 21 21 sensitive control (-) 0 0 0 0 resistant concentration 50 % 15.5 15 15.25 15.25 intermediates concentration 60 % 15.5 15.5 15.5 15.5 intermediates concentration 70 % 22 20 21 21 sensitive concentration 80 % 25 20.5 22.75 22.75 sensitive concentration 90 % 28.5 24 26.25 26.25 sensitive based on table 2. it can be seen that giving of ethanol extract of a. bilimbi fruit with a concentration of 50%, 60%, 70%, 80%, and 90% was able to produce the diameter of the inhibition zone for the growth of p. acnes a b c before before before before before before after after after after after after d e f 178 biology, medicine, & natural product chemistry 13 (1), 2024: 173-182 bacteria of 15.25 mm, 15.5 mm, 21 mm, 22.75 mm, 26.25mm. the lowest p. acnes inhibition zone diameter was indicated at a concentration of 50%, namely 15.25 in the intermediate category. in comparison, the highest was indicated at a concentration of 90% in the sensitive category. the results of this study are a continuation of previous studies which reported that giving of a. bilimbi fruit ethanol extract at doses of 10%, 20%, and 30% was able to produce diameters of inhibition zones of 16.67 mm, 22.7 mm, and 28.1 mm (pertiwi et al., 2020). in addition to p. acnes bacteria, this study also examined the inhibition of ethanol extract of a. bilimbi fruit against s. epidermidis. the test results are shown in table 3. table 3. the average results of giving ethanol extract of a. bilimbi fruits to the diameter of the inhibition zone for the growth of s. epidermidis bacteria. treatment inhibition zone diameter (mm) mean category replication 1 replication 2 replication 3 control (+) 20.1 20.5 20 20.2 sensitive control (-) 0 0 0 0 resistant concentration 50 % 14 16 13.5 14.5 intermediate concentration 60 % 17 15.5 16 16.17 intermediate concentration 70 % 22.5 20 17 19.83 sensitive concentration 80 % 22.5 19.5 20 20.67 sensitive concentration 90 % 22.5 20.5 21 21.33 sensitive superscript description: r = resistant. i = intermediates. s = sensitive the results in table 3 show that giving of ethanol extract of a. bilimbi fruit with concentrations of 50%, 60%, 70%, 80%, and 90% was able to produce diameter of inhibition zone on s. epidermidis bacteria of 14.5 mm, 16.7 mm, 19.83 mm, 20.67 mm, and 21.33 mm. the lowest inhibition zone diameter was shown at a 50% concentration of 14.5 mm with the intermediate category. in comparison, the highest inhibition zone diameter was shown at a 90% concentration of 21.33 mm with the sensitive category. the results of this study continued previous research, which reported that giving ethanol extract of bilimbi fruit with concentrations of 10%, 20%, 30%, and 40% was able to produce diameters of inhibition zones on s. epidermidis bacteria of 28.6 mm, 31.6 mm, 36.3 mm and 39.0 mm (rahmiati et al., 2017). another acne-causing bacteria whose sensitivity was tested in responding to the antibacterial compounds contained in the ethanol extract of a. bilimbi fruits in this study was s. aureus. the test results can be seen in table 4. table 4. the average results of giving ethanol extract of a. bilimbi fruit to the diameter of the inhibition zone for the growth of s. aureus bacteria. treatment inhibition zone diameter (mm) mean category replication 1 replication 2 replication 3 control (+) 21.5 22.5 23 22.30 sensitive control (-) 0 0 0 0 resistant concentration 50 % 9.5 10 10 9.80 resistant concentration 60 % 10.1 10.3 10.3 10.23 resistant concentration 70 % 10.5 11 11.5 11.00 resistant concentration 80 % 11.5 12.5 13 12.30 intermediate concentration 90 % 13 13.5 14 13.50 intermediete superscript description: r = resistant. i = intermediates. s = sensitive the test results in table 4 show that administration of ethanol extract of a. bilimbi fruit with concentrations of 50%, 60%, 70%, 80%, and 90% was able to produce a diameter of inhibition zone on s. aureus bacteria of 9.80 mm, 10.23 mm, 11.00 mm, 12.30 mm, and 13.50 mm. the lowest inhibition zone diameter was shown at 50% concentration of 9.80 mm with the resistant category. in comparison, the highest inhibition zone diameter was shown at 90% concentration of 13.50 mm with the sensitive category. the results of this study continued the research of three previous researchers who reported that administration of ethanol extract of a. bilimbi fruit to s. aureus bacteria with concentrations of 3%, 6%, and 9% was able to produce inhibition zone diameters of 8.50 mm, 9.50 mm, 10.30 mm, whereas in concentrations of 20%, 40% and 15.9 mm, 18 mm and 22.1 mm (das et al., 2011); (mokhtar & abd aziz, 2016); (lisnawati et al., 2019). meanwhile, based on tables 2, 3, and 4, it can be seen that of the three acne-causing bacteria, the inhibitory effect of the antibacterial compounds of bilimbi fruit was best shown on p. acnes bacteria. this anindita et al. – antibacterial bioactivity test of … 179 can be seen in the difference in the average diameter of the inhibition zone of the three bacteria shown in table 5. table 5. comparison of average inhibition zones between p. acnes, s. epidermidis, and s. aureus bacteria treatment inhibition zone diameter (mm) p. acnes s. epidermidis s. aureus concentration 50 % 15.25i 14.50i 9.80r concentration 60 % 15.50i 16.17i 10.23r concentration 70 % 21.00s 19.83s 11.00r concentration 80 % 22.75s 20.67s 12.30i concentration 90 % 26.25s 21.33s 13.50i control (+) 21.00s 20.20s 22.30s control (-) 0r 0r 0r superscript description: r = resistant. i = intermediates. s = sensitive based on table 5, it can be seen that the same sensitivity in response to antibacterial bioactivity compounds of ethanol extract of a. bilimbi fruit was shown in p. acnes and s. epidermidis bacteria with intermediate categories at concentrations of 50% and 60% with sensitive categories at concentrations of 70%, 80%, and 90 %. the sensitivity of s. aureus still showed the resistant category at concentrations of 50%, 60%, and 70%, while concentrations of 80% and 90% showed an intermediate response. in this study, the sensitivity response categories of the three test bacteria to antibacterial compounds in a. bilimbi fruit were determined based on the clsi standard (2020) according to the effect of the antibiotic chloramphenicol as a positive control on the sensitivity of s. aureus bacteria, where if the diameter of the inhibition zone ≥ 18 mm is classified as sensitive, 13-17 mm is in the intermediate category, and ≤ 12 mm is a resistant category . the sensitive response category indicates that the ethanol extract of a. bilimbi fruit is effective in inhibiting bacterial growth, the intermediate category has moderate effectiveness, and the resistant category illustrates that ethanol extract is not effective in inhibiting bacterial growth (hombach et al., 2013) in general, the results of this study prove that the ethanol extract of a. bilimbi fruit contains bioactive compounds that can inhibit bacterial growth. this evidence is shown in table 5, which shows that all bacteria p. acnes, s. epidermidis, and s. aureus produce varying diameters of inhibition zones. the ability to inhibit bacterial growth is suspected because a. bilimbi fruit extract contains secondary metabolites such as alkaloids, flavonoids, phenolics, tannins, and terpenoids as antibacterial bioactive compounds that can inhibit the growth of p. acnes, s. epidermidis, and s. aureus bacteria (masud rana et al., 2014) ; (seebalucksandoram et al., 2019) ; (setyawan et al., 2021). according to yan et al. (2021), the principle of alkaloids as antibacterial by inhibiting protein synthesis, transcription, and replication of microorganisms, disrupting the integrity of peptidoglycan component bacterial cell wall and disrupting cell membrane transport, causing cytoplasmic leakage in gram-positive bacteria. this was evident in the study of mabhiza et al. (2016), which reveal that the administration of alkaloids isolated from callistemon citrinus and vernonia adoensis was able to inhibit the efflux pump of s. aureus and p. aeruginosa bacteria as indicated by percentage of rhodamine 6g accumulation of 114% and 121% with potency the largest in s. aureus which produced mic values of 0.0025 mg/ml and mbc of 0.835 mg/ml. on saponins, nurzaman et al. (2018) stated that saponins were able to inhibit the growth of pathogenic bacteria by increasing the permeability of bacterial cell membranes. this is because saponins have the same hydrophilic and lipophilic properties as surfactants. this property causes saponins to bind to bacterial cell membranes. as a result, there is a decrease in voltage and disruption of the surface stability cell membrane, which results in cytoplasmic leakage and bacterial cell death. for tannins, kaczmarek (2020) suggested that tannins as antibacterial occur through a reaction mechanism with cell membranes, protein precipitation, enzyme inactivation, and functional genetic material of bacterial cells. tannins diffuse more easily in grampositive bacterial cells than in gram-negative bacterial cells. this is because gram-positive bacterial cells only have one layer of cell membrane, while gram-negative bacterial cells have two layers of cell membrane. tannins that enter bacterial cells will bind to iron ions, which are needed as cofactors for several bacterial enzymes such as reverse transcriptase and dna topoisomerase. the result is the inhibition of enzymes needed for the formation of protein, rna, and bacterial dna. in addition, because tannins function as protein precipitation, the entry of tannins into bacterial cells can inactivate the adhesin protein, which acts as a virulence factor for pathogenic bacteria. associated with flavonoids as antibacterial, shamsudin et al. (2022) stated that one of the important characteristics of flavonoids is amphophilic, which easily enters the bacterial cell membrane. the presence of flavonoids in bacterial cells can inhibit nucleic acid synthesis, especially inhibition of the dna gyrase enzyme, which is more effective in s. aureus and s. epidermidis bacteria. plaper et al. (2003) suggested that the inactivation of dna gyrase would further inhibit the activity of the atpase enzyme, as evidenced by the absence of dna gyrase in e. colli isolation. farhadi et al. (2019) added that the use of radioactive markers shows that flavonoids can inhibit the synthesis of dna, rna, peptidoglycan, and protein in bacteria. in addition, flavonoids can damage the integrity of the bacterial cell membrane. this was proven by the results of a study by stapleton et al. (2004) with an electron microscope, which showed that catechin administration resulted in the formation of pseudomulticellular aggregates and 180 biology, medicine, & natural product chemistry 13 (1), 2024: 173-182 cytoplasmic leakage in s.aureus. evidence of cytoplasmic leakage is shown by a decrease in the number of bacterial colonies and 20% potassium (cushnie & lamb, 2005). other evidence is shown in the research of aisyah et al. (2019); scania & chasani (2021) showed that the administration of phenol isolated from red zingiber and three microalgae was able to inhibit s. aureus and e. coli bacteria in the strong category. all of these secondary metabolites interact with each other to produce a high inhibitory effect (nathania et al., 2023). in this study, the ability of ethanol extract of a. bilimbi fruits with concentrations of 70%, 80%, and 90% results in the same inhibition zone sensitivity response as the antibiotic chloramphenicol on p. acne and s. epidermidis bacteria in the sensitive category. according to drago (2019), chloramphenicol is a broad-spectrum antibiotic that can inhibit the growth of gram-positive and negative bacteria. this antibiotic is bacteriostatic (inhibits the growth of bacteria), but at high doses, it can be bactericidal (kills bacteria). the working principle of chloramphenicol is to inhibit bacterial protein synthesis by binding to the 50s ribosomal subunit, thereby preventing protein formation in bacteria. other antibiotics that work, like chloramphenicol, include clindamycin (lincosamide) and macrolides such as erythromycin and clarithromycin (dinos et al., 2016). the selection of the fruit part as test sample was based on several reviews of the results of previous studies which reported that a. bilimbi fruit extract treatment was more effective in inhibiting the growth of p. acnes, s. epidermidis and s. aureus bacteria compared to using a.bilimbi leaf extract (seebaluck-sandoram et al., 2019); (zarwinda et al., 2021) ; (afifi et al., 2018); (kusuma et al., 2020). the results of mokhtar & abd aziz's (2016) research report proved that a. bilimbi fruit juice purchased from local farmers in the machang and jeli districts of kelantan, malaysia, with good conditions (color, size, shape, not deformed, and rotten) in three stages of maturity (young, mature, ripe) showing a. bilimbi juice young is more effective in inhibiting the growth of gram-positive bacteria. the high content of secondary metabolites such as oxalic acid has the potential to act as a strong acid that can inhibit the growth of gram-positive bacteria. in contrast to previous studies, the important point of this study lies in the aspect of originality in the form of giving higher graded concentrations than previous studies. in addition, this study used three test bacteria that cause acne, namely p. acnes, s. epidermidis, and s. aureus, so that the difference in the sensitivity of the three bacteria in responding to the antibacterial compounds contained in the ethanol extract of a.bilimbi fruit can be seen clearly. still, the research this has limitations, namely data on the diameter of the bacterial inhibition zone resulting from the kirby bauer method (disc diffusion) has not been able to be used by clinical practitioners in determining an effective antibacterial dose for the treatment of acne caused by p. acnes, s. epidermidis, and s. aureus bacteria. therefore, further research is needed using molecular tests, the mic (minimum inhibitory concentration) method and the mbc (minimum bactericidal concentration), as well as equipped with bioautographic tests to see which bioactive compounds in the ethanol extract of a. bilimbi fruit have the most potential to inhibit p. acnes, s. epidermidis and s. aureus. conclusion antibacterial test of ethanol extract of a. bilimbi fruit taken from cikarang showed the effectiveness of the inhibition zone on p. acnes and s. epidermidis bacteria at concentrations of 70%, 80%, and 90% with the sensitive category. as for s. aureus, it shows the intermediate category. based on this, the ethanol extract of a. bilimbi fruit taken from cikarang, bekasi, indonesia, has the potential to be used as a raw material candidate for the manufacture of pharmaceutical products that function to prevent acne. competing interests: the authors declare that there are no competing interests. fundings: the author would like to thank stikes mitra keluarga for funding this research. besides that, the provision of access to facilities has helped launch this research. references abhinitha, p., gengatharan, r. s., zulkiflee, s., ganisan, p., arumugam, s., & kumar, n. 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(2021). the formulation of peel-off mask from the ethanol extract of bilimbi leaves (averrhoa blimbi l.) as anti-acne treatment. lantanida journal, 9(1). https://doi.org/10.22373/lj.v9i1.10206 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 591-599 | doi: 10.14421/biomedich.2025.142.591-599 issn 2540-9328 (online) evaluation of selected pharmacological properties of a polyherbal extract (aju mbaise) in experimental rats robert ikechukwu uroko1,*, nnah solomon ijioma2, henry nnaemeka ogbonna1, nancy oluomach uchenna1 1department of biochemistry, college of natural sciences, michael okpara university of agriculture, umudike, abia state, nigeria 2department of zoology and environmental biology, michael okpara university of agriculture, umudike, abia state, nigeria. corresponding author* ir.uroko@mouau.edu.ng manuscript received: 22 march, 2025. revision accepted: 18 august, 2025. published: 01 october, 2025. abstract aju mbaise, an herbal combination widely utilized in southeastern nigeria for managing postpartum complications and alleviating menstrual pain, was evaluated for analgesic, anti-inflammatory, and anti-diarrheal properties in rats. specific objectives included evaluating anti-diarrheal effects, examining anti-inflammatory effects and assessing analgesic properties of the herbal combination. the study involved subjecting the aju mbaise polyherbal extract (ape) to phytochemical analysis and acute toxicity testing. anti-diarrheal effects were evaluated by administering charcoal as a meal and using castor oil-induced models. anti-inflammatory effects were assessed through the carrageenan-induced paw oedema model, and analgesic properties were examined using the acetic acid-induced pain model. phytochemical analysis identified alkaloids, tannins, phenols, steroids, cardiac glycosides, terpenoids, flavonoids, and saponins in the extract. the acute toxicity value exceeded 5000 mg/kg body weight, indicating safety. in animal studies, ape exhibited significant inhibitory effects on intestinal motility, reduced wet stool frequency, and influenced the castor oil-induced diarrhoea and enhanced antiinflammatory activities. additionally, it demonstrated a reduction in acetic acid-induced pain in rats. the ape with its diverse phytochemical composition, possesses anti-diarrheal, anti-inflammatory, and analgesic properties. however, further research is needed to establish ideal dosages and potential adverse effects. keywords: aju mbaise polyherbal extract; analgesic, castor oil; diarrhoea; inflammation; motility. introduction medicinal plants as complementary treatment medications are now accepted globally (ekor, 2014). the global population currently depends on medicinal plants to treat various illnesses and in fact, it is envisaged that the acceptability of medicinal plants as a means of treatment will experience a further upsurge due to the availability, accessibility, cheapness, and reported effectiveness of these medicinal plants in alleviating a wide range of health complications (sofowora et al., 2013; ekor, 2014; salmerón-manzano et al., 2020). the meltdown in global economies, coupled with the poor socio-economic status of most individuals, especially across developing countries of africa and asia, also appears to have contributed to the growing interest in herbal medicine (james et al., 2018; glover, 2021). this is because most individuals living in these regions of the world find it difficult to cope with the demand for sophisticated treatments (goodman et al., 2022). aju mbaise polyherbal extract (ape) is only one of the herbal preparations currently used against diseases (ijioma et al., 2019a, b). aju mbaise is a polyherbal comprising various medicinal plant parts, including stems, bark, leaves, and roots. aju mbaise polyherbal formulation comprises different plants identified as barteria fistulosa, napoleona vogelii, euphorbia convolvuloides, spondias mombin, uvaria chamae, and ceiba pentandra (ijioma et al. 2019b; ijioma et al., 2020). the researchers also reported notable alkaloids, flavonoids, saponins, tannins, terpenoids, steroids, phenols, and cardiac glycosides within the extract (ijioma et al., 2020). although reports on the medicinal uses of the medicinal plant appear scanty, its constituent plants have been used to manage diarrhoea and other forms of gastrointestinal diseases (uroko et al., 2022). aju mbaise extract improves lipid profile, electrolyte balance, haematological profile, male fertility indices and prevents oxidative stress and hepatic injury in rats (uroko et al., 2022). the passage of frequent watery or unformed stools characterizes diarrhoea, which is prevalently implicated in the death of children aged 1 – 5 worldwide and a significant contributor to the current global disease burden, especially in developing countries with limited access to good water supply, and poor breastfeeding https://doi.org/10.14421/biomedich.2025.142.591-599 592 biology, medicine, & natural product chemistry 14 (2), 2025: 591-599 practices (birru et al., 2016). major causes of diarrhoea are gastrointestinal infection of bacterial, viral, and parasitic origin, contaminated food or drinking water, poor hygiene, and motility disorders of the gastrointestinal tract (teke et al., 2010). current management strategies, including oral rehydration and the use of drugs, have not achieved the desired outcomes due to a lack of funds on the part of many affected individuals and also a lack of compliance on the part of those who can afford the drugs (jin et al., 2008; ezezika et al., 2021). medicinal plants have proven viable alternative treatment sources over the years due to their availability, accessibility, and effectiveness (ogbonna et al., 2020). several medicinal plants have shown significant anti-diarrhoeal activities (ayalew et al., 2022). due to their vast phytochemical contents, medicinal plants can play a beneficial role as, antimicrobial, anti-inflammatory and anti-cytotoxic agents while reducing inflammation and protecting from cellular damage (nwokafor et al., 2020; udekwu et al., 2020). the fact that pains and inflammations have been associated with diarrhoea (bentley et al., 2004; panikkath et al., 2014; birru et al., 2016), this study evaluates a strategy for managing all three symptoms with a single agent as an area of interest. therefore, the study assessed in rats the anti-diarrheal, anti-inflammatory, and analgesic effects of the polyherbal extract of aju mbaise. materialss and methods chemicals and drugs drugs used for this study include loperamide, castor oil, and aspirin (emzor drugs, nigeria), while chemicals include ethanol and activated charcoal (bdh company, united kingdom). reagents include electrolyte test kits for sodium, potassium, chloride, and bicarbonate (randox laboratories, united kingdom). plant materials and preparation of extract aju mbaise polyherbal was obtained from ahiara mbaise local government area of imo state, nigeria, and was allowed to dry and this was done using a laboratory bench for 21 days. the dried sample was pulverized into a coarse powder in a locally made manual blender. two hundred and fifty (250) grams of the pulverized material was immersed in 1500 millimetres of ethanol within 48 hours and intermittent stirring every 2 hours before being filtered to obtain a filtrate containing the extract in solution. subsequently, the filtrate was subjected to drying in a hot air oven at 40℃, resulting in a greenish paste extract that weighed 16.90 g, equating to a percentage yield of 6.76%. the extract was prepared and subsequently stored at very low temperatures in a refrigerator until required, and it is now identified as ape. phytochemical analysis of aju mbaise polyherbal extract the assay, identification and quantification of phytochemicals in ape were carried out according to the protocols outlined in evans (2009). the extract's presence and amounts of alkaloids, phenols, cardiac glycosides, saponins, steroids, flavonoids, terpenoids and tannins were assayed. experimental animals a total of one hundred and twenty-one (121) adult male wistar albino rats with average weight (150.24 ± 1.33 g) were used for the different designs adopted in the study. twenty-one (21) of the rats were used for acute toxicity assay of the extract, seventy-five (75) for the three models of anti-diarrhoea tests (25 for each model) and twenty-five (25) for anti-inflammatory study and analgesic studies. the rats were procured from the institution’s animal house of the department of zoology and environmental biology. the rats were housed in stainless steel metabolic cages at room temperature (27 ± 3℃) and 35 ± 5% humidity in wellventilated laboratory. the rats had ad libitum access to clean water and chikun feed finisher mash (17.0 % crude protein, 3000.00 kcal/kg metabolizable energy, chikun feeds depot, ibadan, nigeria) for 12-hour day light/dark cycles. the rats were maintained and managed in compliance with international standards for the care and use of laboratory animals (nrc, 2011). before each experiment, the rats were starved for 24 hours. acute toxicity (ld50) evaluation of aju mbaise polyherbal extract lorke's method was used to evaluate the acute toxicity of ape (lorke, 1983). the test involved two stages, with the first stage using 9 wistar rats that were separated into 3 groups of a, b, and c which consisted of 3 rats each. the groups received 10, 100, and 1000 mg/kg of ape, respectively. the animals were observed for toxicity signs and death within 24 hours, and no deaths were recorded across the groups. consequently, the study progressed to the second stage, which involved another set of 9 rats divided into 3 groups (a, b, and c) and administered single oral doses of ape at 1600, 2900, and 5000 mg/kg, respectively. the animals were again checked for signs of toxicity and death within 24 hours, with no deaths recorded across the groups. as a confirmatory test, the highest dose (5000 mg/kg) was repeated on different sets of 3 rats. acute toxicity values calculated using lorke’s formula stated as: ld50 = √𝐴 𝑥 𝐵, where a = maximum dose that did not result in any deaths and b = minimum dose that caused mortality in all animals within a group. uroko et al. – medicinal properties of aju mbaise 593 anti-diarrhoeal activity of aju mbaise polyherbal extract model 1: effect of the polyherbal extract on intestinal transit the study adopted the method of ijioma et al. (2019b), assigning 25 rats into five groups of five rats each, after a 24-hour starvation period. group 1 served as control, while group 2 received 0.5 mg/kg body weight of the standard drug (loperamide) via the oral route. groups 3, 4, and 5 received oral administrations of 250, 500, and 1000 mg/kg of ape, respectively. after 30 minutes, each rat received 10 ml/kg body weight of 10% activated charcoal before being sacrificed under anesthesia with chloroform and dissected open to expose the visceral content. the small intestine’s length was measured, and the distance covered by the charcoal meal was also measured and converted to percentage of the entire length. the percentage inhibition of intestinal motility was calculated as indicated in the equation below: % inhibition = dt in control − dt in test x100 dt in control where dt = distance travelled by charcoal meal. model 2: effect of the polyherbal extract on castor oilinduced diarrhoea in rats for model 2, a separate set of 25 adult rats fasted for 24 hours and divided into 5 groups of 5 rats each according to the method used by ugwuja et al. (2022). the rats received the same treatment as in model 1 and, 30 minutes later, were given castor oil (1 ml) orally before being housed in individual cages coated with absorbent paper for three hours to monitor episodes of diarrhoea. wet and dry stools were counted, and their weights were recorded, as well as when the diarrhoea first started (the latent phase). after each period, the absorbent papers were changed, and the average stools passed by the treatment and control groups were compared. the percentage average number of diarrheal faeces passed in the control group was calculated. the inhibition percentage of wet faeces and stool frequency induced by the extract was determined by calculating the variance between the number of faeces in the control group (nc) and the amount of faeces in the treated group (nt), divided by nc and multiplied by 100. % inhibition of bowel movement = [(nc nt)/nc] x 100, where: nc = number of wet faeces/stools of control group and nt = number of wet faeces/stools of treated group (ijioma et al., 2020). model 3: effect of the polyherbal extract on castor oilinduced gastrointestinal fluid accumulation and serum electrolytes concentration in rats using the method described by ugbogu et al. (2023), another set of 30 rats was given the same treatments as model 2 using the same protocols. in this instance, six groups of five adult albino rats each were used. after an hour of administering castor oil, the rats were euthanized, and their small intestines were removed from the pyloric region to the caecum, after ligating both ends. each intestine's weight with its contents within was determined before the contents were expelled out, and the empty intestine was then weighed again. the weight of the intestinal content was determined by subtracting the weight of the empty intestine from the weight of the full intestine. percentage of diarrhoeal activity was evaluated using the relationship: % activity = wic in control − wic in test x100 wic in control where wic = weight of intestinal content. the concentrations of serum electrolytes were also determined by examining blood samples collected from each rat. randox commercial test kit for electrolytes including sodium, potassium, chloride and bicarbonate were used in accordance with the methods of terri & sesin (1958), described by the manufacturer, randox laboratories, united kingdom, for each test as outlined below: potassium ion (k+): the principle is that sodium tetraphenylboron in a specifically prepared mixture can produce a colloidal suspension. the turbidity of this suspension is directly proportional to the concentration of potassium in the sample. for potassium, three test tubes labelled blank, sample and standard were each made to contain 1.0 ml of potassium reagent, before 10 µl of the distilled water, sample and standard were added to their respective tubes. the content of each tube was mixed and allowed to stand for 3 minutes at room temperature before absorbance values of each were read at 500 nm in a spectrophotometer after zeroing with the blank. the k+ the concentration was calculated using the formula: 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑒𝑠𝑡 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑥 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑, where concentration of standard was 5.0 meq/l. sodium ion (na+): this test is predicated on the principle of precipitating sodium, as a triple salt, sodium magnesium uranyl acetate, with the excess uranium being reacted with ferrocyanide to produce a chromophore whose absorbance is inversely proportional with the concentration of sodium in the sample being tested. in sodium electrolyte analysis, two stages of test– filtrate preparation and colour development. in the filtration phase, test tubes labelled blank, standard and sample were made to contain 1.0 ml of the filtrate reagent, followed by adding 50 µl of sample to all tubes and 1 ml of distilled water to the blank. all tubes were shaken vigorously and mixed continuously for 3 minutes. the mixtures were then centrifuged for 10 minutes and supernatant fluids were used for the test in the colour development phase which involved the addition of 1 ml 594 biology, medicine, & natural product chemistry 14 (2), 2025: 591-599 of the acid reagent to each tube before reading absorbance values after zeroing the spectrophotometer at 550 nm. sodium electrolyte concentration was calculated using the formula: abs.of blank − abs.of sample abs.of blank − abs.of std 𝑥 conc. of std (meq/l) chloride ion (cl-): the principle here is that chloride ions form a soluble non-ionized compound with mercuric ions and will displace thiocyanate ions from non-ionized mercuric thiocyanate. the released thiocyanate ions then react with ferric ions to form a colour complex whose intensity is proportional to the chloride concentration in the sample. in the test, three test tubes labelled blank, sample and standard were each made to contain 1.0ml of chloride reagent, before 10 µl of the distilled water, sample and standard were added to the respective tubes. the content of each tube was mixed and allowed to stand for 3 minutes at room temperature before absorbance values of each were read at 500 nm in a spectrophotometer after zeroing with the blank. the cl concentration was calculated using the formula: 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑒𝑠𝑡 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑥 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 evaluation of the anti-inflammatory effects of the polyherbal extract twenty-five mature rats were assigned to 5 groups with each group having 5 rats. the standard group (group 2) received an anti-inflammatory drug (100 mg/kg aspirin), while the control group (group 1) received 0.2 ml of ordinary saline. using the oral method, groups 3, 4, and 5 received 250, 500, and 1000 mg/kg of ape, respectively. the animals' paw circumferences (pc) were measured and recorded before these treatments. after 30 minutes following treatment, the rats were subjected to acute inflammation (paw oedema) using a sub-plantar injection of 0.1 ml of 1% λ-carrageenan (in 0.9% saline solution) into the right hind paw of each rat. after that, the pc of the treated rats and control were measured and recorded at different time intervals (30 minutes, 1 hour, and 2 hours) following induction. the percentage inhibition of oedema was estimated using the following relationship, and the degree of oedema was determined according to the method employed by ijioma et al. (2019) as the difference between the initial and final pc values: percentage inhibition of oedema = pc in control − pc in test x 100 pc in control . evaluation of analgesic effects of the polyherbal extract same twenty-five (25) matured rats assigned to 5 groups used for the anti-inflammatory study were used and were given same treatments. thirty minutes after treatment, acute pain was inducted by intraperitoneal administration of 10 ml/kg body weight of 0.6% acetic acid solution. thereafter, the number of writhes made by each rat in 30 minutes was ascertained for both the control and test groups. percentage inhibition of pain (analgesic activity) was calculated using the relationship employed by ugbogu et al. (2023): % inhibition of pain = number of writhes in control − number of writhes in test x 100 number of writhes in control data analysis: spss (statistical products and service solutions) version 20.0, ibm spss incorporated, chicago, il was utilized to analyse the data. experimental data collected were subjected to a one-way analysis of variance (anova) to obtain significant means. the various significant means were separated using the duncan multiple range test post-hoc analysis. values with p<0.05 were regarded as being statistically significant. results and discussion results phytochemical composition of aju mbaise polyherbal extract the extract's phytochemicals, which were quantified, include saponins, tannins, phenolics, flavonoids, steroids, terpenoids, glycosides, and alkaloids, and they are presented in table 1 table 1. phytochemical composition of aju mbaise polyherbal extract. parameters qualitative test qualitative test result (mg/100g) saponins ++ 8.33 ± 0.12 tannins ++ 5.71 ± 0.16 phenolics +++ 13.18 ± 0.30 flavonoids ++ 7.19 ± 0.22 steroids ++ 5.65 ± 0.14 terpenoids + 2.52 ± 0.11 cardiac glycosides + 3.63 ± 0.09 alkaloids +++ 14.91 ± 0.32 key: +++ = present in high quantity, ++ = present in moderate quantity, + = present in low quantity and = absent acute toxicity (ld50) of aju mbaise polyherbal extract no mortality occurred across all test groups and in all stages of acute toxicity evaluation of the extract, including the highest dose of 5000 mg/kg body weight. after treatment, the animals appeared physically stable, and all survived till the end of 24 hours and a further seven days of the ld50 test, except those administered uroko et al. – medicinal properties of aju mbaise 595 5000 mg/kg, which at the initial moments after administration were calm with decreased physical activity. however, these animals all survived to the end of the test period as 0% mortality was recorded. the ld50 value for the extract was >5000 mg/kg (table 2). table 2. acute toxicity (ld50) of aju mbaise polyherbal extract. group dose (mg/kg) number of death observation phase 1 1 10 0/3 animals displayed normal physical activity and appeared to be in good health 2 100 0/3 animals displayed normal physical activity and appeared to be in good health 3 1000 0/3 animals displayed normal physical activity and appeared to be in good health phase 2 1 1600 0/3 animals exhibited normal activity and were physically stable 2 2900 0/3 animals exhibited normal activity and were physically stable 3 5000 3/3 no mortality. animals were calm for up to 2 hours before regaining activity. ld50 >5000 mg/kg anti-diarrhoeal activity of aju mbaise polyherbal extract model 1: effect of ape on intestinal transit the treated rats exhibited significantly inhibited intestinal motility (p<0.05) when compared to the control group. the inhibition occurred in a dose-dependent manner, causing a decrease in charcoal meal transit in rats. the treatment was comparable to loperamide activity but demonstrated greater activity than loperamide at the highest dose (100 mg/kg).). table 3 shows the inhibitory effect of ape on intestinal motility in rats. table 3. aju mbaise polyherbal extract's inhibitory effects on intestinal motility of rats. treatments length of intestine (cm) distance travelled by charcoal meal (cm) percentage charcoal meal movement (%) percentage inhibition of motility (%) control 92.60 ± 2.07a 75.60 ± 2.23c 81.00 ± 6.92c 0.00 ± 0.00a loperamide 0.5 mg/kg 90.00 ± 1.58a 56.80 ± 3.56b 63.20 ± 3.70b 25.40 ± 1.82b extract 250 mg/kg 91.20 ± 2.95a 57.80 ± 2.28b 63.40 ± 2.70b 24.20 ± 2.28b extract 500 mg/kg 91.60 ± 4.83a 54.20 ± 2.17b 59.20 ± 3.03a,b 27.40 ± 3.29b extract 1000 mg/kg 91.00 ± 6.21a 45.00 ± 3.77a 52.60 ± 3.50a 33.00 ± 4.47c the results are shown as mean ± standard deviation. a,b,c = means on the same column with different letter superscripts differ significantly (p<0.05) model 2: effect of ape on castor oil-induced diarrhoea in rats in castor oil-induced diarrhoea rats, the extract exhibited a significant inhibitory effect on the induction of diarrhoea at doses of 250, 500, and 1000 mg/kg, comparable to loperamide. specifically, the extract reduced the incidence of diarrhoea by 57.29 ± 2.93%, 66.66 ± 2.95%, and 84.77 ± 2.73%, respectively, when compared to the control (p<0.05). the extract-treated groups had a significantly decreased number and weight of wet stools with longer latent periods compared to the control group (table 4). table 4. inhibitory effect of aju mbaise polyherbal extract on castor oil-induced diarrhoea in rats. treatments latent period (minutes) number of wet stool weight of wet stool (g) number of dry stool weight of dry stool (g) % inhibition of diarrhoea control 30.60 ± 5.86a 7.20 ± 0.84c 4.82 ± 0.18d 1.40 ± 0.55a 1.41 ± 0.28a,b 0.00 ± 0.00a loperamide 0.5 mg/kg 56.80 ± 1.64c 0.80 ± 0.05a 0.71 ± 0.09a 2.60 ± 0.48b 2.62 ± 0.33c 89.10 ± 6.22d aju mbaise 250 mg/kg 48.00 ± 1.23b 2.60 ± 0.55b 1.87 ± 0.23c 1.20 ± 0.08a 1.10 ± 0.07a 57.29 ± 2.93b aju mbaise 500 mg/kg 70.00 ± 2.45d 1.60 ± 0.48a 1.26 ± 0.37b 2.80 ± 0.47b 2.57 ± 0.42c 66.66 ± 2.95c aju mbaise 1000 mg/kg 73.80 ± 0.84d 0.80 ± 0.04a 0.56 ± 0.06a 2.20 ± 0.44b 1.97 ± 0.21b 84.77 ± 2.73d the results are shown as mean ± standard deviation. a,b,c = means on the same column with different letter superscripts differ significantly (p<0.05) model 3: effect of ape on castor oil-induced gastrointestinal fluid accumulation and serum electrolytes concentration in rats: in addition, the extract significantly reduced the intestinal weight in the enteropooling version of the castor oil-induced diarrhoea rats compared to loperamide-treated rats (table 5). serum electrolyte composition analysis revealed a significant increase in sodium and potassium ions in the extract-treated groups, while the levels of chloride and bicarbonate remained 596 biology, medicine, & natural product chemistry 14 (2), 2025: 591-599 unchanged compared to the diarrhoea control group (table 6). the control group had significantly lower serum levels of sodium and chloride ions. however, in groups treated with the extract, serum levels of these electrolytes increased significantly. in comparison to the control, the extract-treated groups had significantly higher k+ levels. the hco3levels between the extract-treated group and the control group were not statistically different. table 5. inhibitory effect of aju mbaise polyherbal extract on the weight of intestinal contents in castor oil-induced diarrhoea model in rats. treatments weight of filled intestine (cm) weight of empty intestine (g) weight of intestine content (g) % activity control 15.59 ± 0.51d 4.58 ± 0.23a 11.01 ± 0.41d 0.00 ± 0.00a loperamide 0.5 mg/kg 12.34 ± 0.20b 4.73 ± 0.36a,b 7.60 ± 0.31b 58.60 ± 0.71e aju mbaise 250 mg/kg 13.93 ± 0.20c 4.93 ± 0.09b 9.01 ± 0.22c 17.20 ± 0.53b aju mbaise 500 mg/kg 12.17 ± 0.24b 4.94 ± 0.04b 7.23 ± 0.28b 32.85 ± 2.57c aju mbaise 1000 mg/kg 11.48 ± 0.38a 5.01 ± 0.07b 6.48 ± 0.29a 43.65 ± 1.42d the results are shown as mean ± standard deviation. a,b,c = means on the same column with different letter superscripts differ significantly (p<0.05) table 6. effect of aju mbaise polyherbal extract on serum electrolyte levels in castor oil-induced diarrhoea in rats. treatments na+ meq/l k+ meq/l clmeq/l hco3 meq/l control 107.93 ± 1.24a 4.11 ± 0.09a 79.37 ± 0.58a 19.36 ± 0.14a loperamide 0.5 mg/kg 118.72 ± 3.08c 4.48 ± 0.27b 86.50 ± 0.72b 19.50 ± 0.09a,b aju mbaise 250 mg/kg 111.78 ± 2.35b 4.49 ± 0.19b,c 86.76 ± 2.28b 19.60 ± 0.13b aju mbaise 500 mg/kg 118.16 ± 2.568c 4.62 ± 0.04b,c 87.80 ± 1.06b,c 19.51 ± 0.27a,b aju mbaise 1000 mg/kg 119.90 ± 2.29c 4.72 ± 0.12b,c 89.52 ± 1.88c 19.51 ± 0.10a,b the results are shown as mean ± standard deviation. a,b,c = means on the same column with different letter superscripts differ significantly (p<0.05) anti-inflammatory effect of aju mbaise polyherbal extract in carrageenan induced paw oedema in rats the extract, similar to aspirin, significantly reduced the degree of paw oedema in rats at all administered doses compared to the control (p<0.05). the extract's inhibitory effect on carrageenan-induced paw oedema was 47.83 ± 1.90%, 77.35 ± 3.97%, and 85.05 ± 1.09% for 250, 500, and 1000 mg/kg doses, respectively, compared to the effect of aspirin at 56.93 ± 1.59%. comparing the extract-treated groups to the control groups, inflammation-related indicators showed a significant difference (p<0.05). the levels of interleukin 1b and prostaglandin e2 were lower in the extract-treated groups compared to the control (tables 7 and 8). table 7. anti-inflammatory effect of aju mbaise polyherbal extract in rats. treatments initial paw circumference (cm) 30 minutes paw post circumference (cm) 1 hour paw post circumference (cm) 2 hours paw post circumference (cm) control 2.44 ± 0.17a,1 3.82 ± 0.21b,2 4.22 ± 0.20d,3 4.20 ± 0.47d,3 aspirin 100 mg/kg 2.70 ± 0.10b,1 3.26 ± 0.25a,2 3.66 ± 0.13b,2,3 3.42 ± 0.48b,3 aju mbaise 250 mg/kg 2.74 ± 0.21b,1 3.20 ± 0.12a,2 3.94 ± 0.27c,3 3.66 ± 0.15c,3 aju mbaise 500 mg/kg 2.72 ± 0.08b,1 3.04 ± 0.13a,3 3.20 ± 0.11a,2 3.00 ± 0.01a,2 aju mbaise 1000 mg/kg 2.58 ± 0.18a,b,1 3.26 ± 0.06a,4 3.06 ± 0.17a,3 2.84 ± 0.23a,2 the results are shown as mean ± standard deviation, a,b,c = means on the same column with different letter superscripts differ significantly (p<0.05), 1,2,3,4 = means on the same row with different number superscripts differ significantly (p<0.05) table 8. anti-inflammatory effect of aju mbaise polyherbal extract in rats. treatments change in paw circumference after 2 hours % inhibition of inflammation il-1b ng/ml pge-2 ng/ml control 1.72 ± 0.30c 0.00 ± 0.00a 1.32 ± 0.03d 73.26 ± 1.42a aspirin 100 mg/kg 0.72 ± 0.16b 56.93 ± 1.59c 0.89 ± 0.03b 64.94 ± 1.31b aju mbaise 250 mg/kg 0.94 ± 0.05b 47.83 ± 1.90b 1.01 ± 0.02c 71.66 ± 2.70a aju mbaise 500 mg/kg 0.32 ± 0.06a 77.35 ± 3.97d 0.81 ± 0.02a 68.84 ± 1.20c aju mbaise 1000 mg/kg 0.24 ± 0.05a 85.05 ± 1.09d 0.84 ± 0.06a 66.77 ± 2.34c the results are shown as mean ± standard deviation. a,b,c = means on the same column with different letter superscripts differ significantly (p<0.05) uroko et al. – medicinal properties of aju mbaise 597 analgesic effect of aju mbaise polyherbal extract in acetic acid-induced pain in rats the extract also showed significant analgesic activity similar to that of aspirin when compared with the control (p<0.05), as the extract-treated rats made fewer writhes than the control with 36.76 ± 3.80%, 66.01 ± 2.79% and 77.54 ± 3.01% analgesic activities for 250, 500 and 1000 mg/kg treatment dose levels respectively (table 9). table 9. anti-analgesic effect of aju mbaise polyherbal extract in rats. treatments % writhes after 30 minutes % analgesic activity control 59.80 ± 2.44d 0.00 ± 0.00a aspirin 100 mg/kg 14.00 ± 2.35a 69.37 ± 1.13c aju mbaise 250 mg/kg 33.80 ± 1.64c 36.76 ± 3.80b aju mbaise 500 mg/kg 18.80 ± 2.17b 66.01 ± 2.79c aju mbaise 1000 mg/kg 11.60 ± 1.95a 77.54 ± 3.01d the results are shown as mean ± standard deviation. a,b,c = means on the same column with different letter superscripts differ significantly (p<0.05) discussion the findings of this study indicate that the ape has an ld50 value exceeding 5000 mg/kg body weight, suggesting that it is safe for oral consumption and may not cause systemic toxicity in rats. these findings can potentially be extrapolated to humans and other animals. the non-toxic effect of ape had earlier been reported by ijioma et al. (2019a), following acute and sub-acute in vivo toxicity studies, and has only been confirmed by the findings of this study. international standards for acute toxicity studies indicate that a major outcome in testing toxic substances is mortality and that where no mortality occurs, the agent being investigated may be considered a safe medication for use (oecd, 2001). the findings of this study are consistent with the report by erhirhie et al. (2018), who classified any substance with an ld50 value above 5000 mg/kg as non-toxic. in plant-based substances, toxicity usually results from consuming intolerable amounts of phytochemical components in plants (bode & dong, 2015). however, this study's findings have shown that the amount of the various phytochemical agents in ape was well tolerated in rats, hence translating into positive pharmacological effects that could be harnessed for managing human health challenges. in this study, ape has shown significant antidiarrhoeal, anti-inflammatory, and analgesic effects in rats. the three-model approach adopted in the investigation of the anti-diarrhoeal potential of ape all yielded positive outcomes as results obtained suggest that ape may be a valuable agent for treating diarrhoea. in the charcoal meal transit model, the inhibition of both transit and intestinal motility observed suggests that the ape contains bioactive compounds which act by inhibiting acetylcholine activity in the gastrointestinal tract. the crucial role played by acetylcholine in the initiation and maintenance of intestinal motility has been extensively studied (iino & nojyo, 2006; nezami & srinivasan, 2010; mercan et al., 2020). acetylcholine induces contractions of the gastrointestinal smooth muscle through cholinergic signalling, which is mediated by muscarinic acetylcholine receptors present on the surface of smooth muscle cells in the intestine (iino & nojyo, 2006). inhibition of acetylcholine activity is a well-established mechanism for the anti-diarrheal effects of drugs (colovic et al., 2013). additionally, castor oilinduced diarrhoea in rats was significantly lowered by the polyherbal extract. typically released from castor oil by intestinal lipase, ricinoleic acid is a hydroxylated fatty acid that irritates and inflames the intestinal mucosa. this causes prostaglandins and nitric oxide to be released, which in turn increases gastrointestinal secretion, motility, and epithelial permeability and causes diarrhoea (sharma et al., 2010). the extract may have inhibited the successful promotion of this cascade of activities by inhibiting either the release of ricinoleic acid from castor oil or interfering with intestinal lipase activity. the anticholinergic/spasmolytic activity of the extract in the charcoal meal transit study and anti-enteropooling and ability to remove fluid from the intestinal lumen in the castor oil models all support the claim that the extract may be a good anti-diarrhoeal agent. agents with such effects have been employed in managing diarrhoea (sharma et al., 2010). these activities of the extract may not be unconnected with its combined effects of composite phytochemical agents. tannins, alkaloids and saponins have been implicated in inhibiting intestinal motility and fluid accumulation in the gastrointestinal lumen (palombo, 2006; ayinde & owolabi, 2009). the presence of these phytochemical agents in ape was reported in a previous communication (ijioma et al. 2019a). another mechanism for achieving antidiarrhoeal effect by agents like loperamide is the antisecretory pathway. loperamide alters intestinal absorption and inhibits its secretion by binding to the δ receptor via a cascade of biochemical activities reported by tagne et al. (2019), resulting in the maintenance of low and high intracellular concentrations of na+ and k+ respectively and the inhibition of the diarrhoea causing potentials of ricinoleic acid in castor oil (ijioma et al. 2020). ape may have also achieved its antidiarrhoeal effect via this mechanism, having produced similar activity as loperamide. like aspirin, ape significantly inhibited carrageenan-induced paw oedema in rats. carrageenan induces rat paw oedema by first increasing histamine and serotonin activities in the tissues, increasing local synthesis of prostaglandin, and later by bradykinin, leukotrienes, and leukocyte infiltrations and also biosynthesis of prostaglandin by inducible cyclooxygenase (guay et al. 2004; adebayo et al. 2015). the extract's anti-inflammatory effect may have interfered with or reasonably inhibited these biochemical processes leading to less inflammation in the extracttreated rats. the findings of the analgesic study indicate 598 biology, medicine, & natural product chemistry 14 (2), 2025: 591-599 that the ape has a significant and dose-dependent analgesic effect in rats using the acetic acid pain-induced model. this model induces pain and inflammation in the abdominal cavity through the activation of nociceptors, and the writhing observed in the treated animals is a result of intense endogenous pain (gregory et al. 2013; regmi and shah, 2020). the observed decrease in the levels of interleukin-1b and prostaglandin e2 in apetreated rats compared to the control group provides additional evidence supporting the argument in favour of the anti-inflammatory and analgesic effects of ape (mirtella et al. 1995; muraki et al. 2004). these inflammatory markers usually increase in inflammatory conditions and the opposite may indicate the presence of an anti-inflammatory agent, which in this study is ape. conclusions ape inhibited diarrhoea by reducing intestinal motility/peristalsis, wet stool frequency, fluid accumulation in the gastrointestinal lumen and loss of electrolytes. these effects suggest that the extract may be a good antidiarrhoeal agent but may be further dissected to characterize other useful phytochemicals eliciting these beneficial effects. further studies may also provide more support for our current findings. acknowledgements: authors are grateful to kingsley chijioke ugwuanyi an academic staff of department of zoology and environmental sciences, blessing ekwu and odinaka ugwuege, undergraduate students of department of biochemistry, mouau for their assistance during the experimental protocols and data collection. authors’ contributions: uroko r.i., ogbonna h.n. and uchenna, n.o., designed the study and carried out the laboratory analyses. uroko r.i., carried out the statistical analysis of the data obtained from the study. whereas, ijioma sn., and uroko ri, carried out the literature survey and drafted the manuscript for publication. all the coauthors read the manuscript and made necessary input before the final version of the manuscript was approved for publication. competing interests: the authors declare that there are no competing interests. funding: the authors declare no external funding for the study. references adebayo, s. a., dzoyem, j. p., shai, l., j. & eloff, j. n. 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(2022). effect of aju mbaise on sperm morphology, semen quality, sex hormonal levels, gonadosomatic index and testicular histology of avodart-induced rats. plant biotechnology persa, 4(2), 2237. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 539-545 | doi: 10.14421/biomedich.2023.122.539-545 issn 2540-9328 (online) standardization of golden sea cucumber (stichopus hermanii) extracts from pelapis island, kayong regency, west kalimantan rusmini*, wintari taurina, mohamad andrie department of pharmacy, faculty of medicine, tanjungpura university jl. prof. dr. hadari nawawi, pontianak, west kalimantan 78115, indonesia. corresponding author* rusminii@student.untan.ac.id manuscript received: 29 june, 2023. revision accepted: 05 september, 2023. published: 20 september, 2023. abstract stichopus hermanii can be used as a medicinal material, a source of animal protein, and wound healing medicine products. extracts as raw materials for products must go through a standardization process to ensure pharmaceutical reproducibility, a therapeutic quality, and to ensure a consistent and uniform final composition. the purpose of this research was to determine the test results of the standardization parameters of stichopus hermanii extract from pelapis island, west kalimantan as raw material for wound healing herbal medicinal preparations. preparation of extracts using the maceration method with 96% ethanol. the extract was standardized with specific parameters, including organoleptic, water and ethanol soluble content, phytochemical screening, and protein content, and non-specific parameters, including drying shrinkage, water content, ash content, and acid insoluble ash content. the test results obtained were a watersoluble content of 61.89%, ethanol-soluble content of 41.81%, protein content of 7.62%, drying shrinkage of 38.81%, water content of 20.58%, ash content of 37.95%, and acid insoluble ash content of 2.07%. keywords: golden sea cucumber; maceration; standardization; stichopus hermanii. introduction in this modern era, there is a lifestyle trend that leads to the use of natural ingredients as efficacious ingredients, both for medicine and body care products. this phenomenon has further increased the prestige of natural ingredients as an option because they are considered safer with lower negative effects (nugroho, 2017). since the last decade, the medical world's attention has begun to focus on marine biota as a very potential resource for producing active compounds (bawole et al., 2021). based on research by roni et al, it is necessary to look for drugs based on marine biota, due to the increasing resistance of various diseases to existing types of drugs (roni et al., 2020). sea cucumber is one of the marine biota that may create bioactive substances that might be used as medicinal raw materials for medicines (bawole et al., 2021). more than 1400 species of sea cucumber have been identified in the world, among the identified species, stichopus hermanii with its various contents has been empirically proven to be used as a wound medicine (d. w. damaiyanti, 2015; w. damaiyanti, 2015). sea cucumber protein content reaches 82%, of all sea cucumber components, and 80% of the protein is collagen (d. w. damaiyanti, 2015). the content of golden sea cucumber can be obtained by extraction methods, one of which is the maceration method using 96% ethanol which is then concentrated to obtain a thick extract. extracts are made because the simplicia is no longer practical (najib et al., 2018). the use of nonstandardized extracts as herbal medicines can cause difficulties in quality control and the safety, efficacy, and quality profiles vary from one product to another (syahidan & wardhana, 2019). extracts standardization may be utilized to guarantee uniformity in the quality of raw materials, increase benefits, and ensure the safety and stability of extracts used to supporting health (nurhaini et al., 2020). based on sumaryani, taurina, and andrie's research, characterization and standardization of stichopus hermanii simplicia have been carried out and in damaiyanti's research, characterization of water extracts in stichopus hermanii has been carried out (d. w. damaiyanti, 2015; sumaryani et al., 2022; taurina & andrie, 2022). research related to the standardization of golden sea cucumber extract has never been done. based on the foregoing explanation, the researchers are interested in the standardizing stichopus hermanii extract from pelapis island, north kayong regency, west kalimantan as a raw material for herbal medicinal preparations with specific parameters, such as organoleptic, water–ethanol soluble essence content, phytochemical screening, and protein content, as well as https://doi.org/10.14421/biomedich.2023.122.539-545 540 biology, medicine, & natural product chemistry 12 (2), 2023: 539-545 with non-specific parameters, such as drying shrinkage, moisture content, ash content, and acid–insoluble ash content. methods samples and materials samples of stichopus hermanii simplicia were collected on pelapis island, west kalimantan. the materials used were ethanol, aquadest, strong sulfuric acid (h2so4), mayer reagent, dragendorff reagent, wagner reagent, magnesium (mg) metal band, hydrochloric acid (hcl), iron (iii) chloride (fecl3), n–hexane, liebermannburchard, gelatin, nacl, k2so4 (merck), cuso4.5h2o (merck), bromocresol green (merck), methyl red (merck), boric acid (h3bo3) (merck), and sodium hydroxide (naoh) (merck). the stichopus hermanii can be seen in figure 1. figure 1. stichopus hermanii (personal documentation). extract preparation the stichopus hermanii dried powder obtained was then weighed up to 400 grams and then put into a maceration vessel and added ethanol 96% solvent in a ratio of 1: 4. during maceration, stirring was carried out as often as possible. the maceration process was carried out for 4x24 hours until the macerate became clear, then filtering and replacing new solvents every day after 24 hours of immersion. then, all of the macerates were gathered and concentrated at a temperature 45˚c in a rotary evaporator till a thick extract was produced (depkes ri, 2017). organoleptic test an organoleptic test is a testing technique that describes the qualities of a product’s color, shape, sell, and taste utilizing the five senses (depkes ri, 2000). water and ethanol soluble essence determination stichopus hermanii extract was weighed up to ± 2.5 g and then macerated for 24 hours with 50 ml of water– chloroform and ethanol using 2 different corked flasks. for the first six hours, the mixture was vigorously shaken. after being left for eighteen hours, it was filtered, with up to 25 ml of the filtrate being taken out, then evaporated in a waterbath to dry. the residue was heated to 105°c till the weight persisted (depkes ri, 2017). screening phytochemical determination the identification of the alkaloids was carried out with 1 ml of hcl 2n and 9 ml of aquadest, they were then wheated in a waterbath for two minutes, cooled, and then filtered. the acquired filtrate was utilized for the alkaloid test and three test tubes were taken, then the filtrate obtained was placed into each test tube to which two drops of various reagents were added, namely mayer, dragendorff, and wagner reagents (nurjannah et al., 2022). flavonoid identification is carried out by adding 3-4 little pieces of mg metal tape and a few drops of strong hcl are added (endarini, 2016; yuliana et al., 2022). phenol identification is done with 5-10 drops of fecl3 3% (yuliana et al., 2022). identification of saponins is done with 10 ml of hot water and ferociously shaking motion for ten seconds. characterized by the formation of a stable foam and will not disappear with the addition of single drop of hcl 2n (yuliana et al., 2022). identification of terpenoids/steroids is done with liebermann-burchard reagent (fajriaty et al., 2018). identification of tannins is done with fecl3 3%. in addition, it is also done with 2 ml of gelatin 1% and a few drops of nacl (fajriaty et al., 2018). protein content determination the stichopus hermanii extract was weighed as much as ± 1 g of sample, then put into the kjeldahl flask. next, 3.5 g k2so4 and 0.1 g cuso4.5h2o, and 12 ml of concentrated h2so4 were added and heated in a fume hood on an automatic digestion unit instrument, then cooled and 100 ml of distilled water was added. installed the flask containing the results of the destruction in the distillation devices, then added 50 ml of 30% naoh, then the distillate is collected in an erlenmeyer containing 30 ml of 4% h3bo4 which has been given an indicator, until the distillate droplets are neutral and the distillate is adjusted to 100 ml. next, titrate the distillate with standardized 0.2 n hcl. a blank was made (standar nasional indonesia (sni) 01-2354.42006, 2006). drying shrinkage determination the stichopus hermanii extract was weighed up to 2 g, added, and tartered in porcelain crucible with a lid that had been preheated at 105°c for 30 minutes. the sample was leveled, placed in the oven, discovered, and dried at 105°c for 30 minutes or until the weight remained or until the discrepancy between the result of the two weighings was no greater than 0.5 mg (depkes ri, 2017). rusmini et al. – standardization of golden sea cucumber (stichopus hermanii) extracts … 541 water content determination the stichopus hermanii extract was weighed up to 2.5 g and placed in a weighing bottle with a cover whose weight was known. then dried in the oven at 105°c for 3 hours. following that, it was cooled in a desiccator and weighed (depkes ri, 2017). ash content determination the stichopus hermanii extract weighed up to 2.5 g and was put into a porcelain cup that had previously been incinerated and weighed. the sample container was incinerated in a furnace at a steady weight of 600°c for 8 hours. after that, it cooled down into a desiccator, and then the cup containing the ash was weighed (nurhidayah et al., 2019). acid insoluble ash content determination the ash was boiled using 25 ml of diluted hcl 0.2 n for 5 minutes. the acid-insoluble portion was gathered, the solution was filtered through ash-free filter paper with a defined weight, and hot water washed the filter paper. the residue and filter paper were placed back into the silicated crucible and then burned in a furnace until a consistent weight was reached (depkes ri, 2017). results and discussion the stichopus hermanii extract was created using the maceration procedure, which involves soaking in organic solvents for an extended period of time. the extract obtained was a thick brown extract of 51.40 grams with a yield of 12.85%. this shows the content and the extraction process is going well where the percent yield is more than 10%. the extract obtained was then measured for specific and non-specific parameters. the thick golden sea cucumber extract obtained after the evaporation process can be seen in figure 2. figure 2. stichopus hermanii extract (personal documentation). organoleptic test result organoleptic parameters aim to provide a simple initial physical introduction to the extract that will be standardized using the five senses to describe the characteristics of the extract in the form of shape, color, and smell of golden sea cucumber extract.(depkes ri, 2000) organoleptic tests can determine the specific properties of an extract through direct observation based on general sources and can provide an overview of product damage and deterioration in the quality of ingredients during storage which can affect their properties (evifania et al., 2020; marpaung & septiyani, 2020). the results of the organoleptic assay of stichopus hermanii extract in this study are shown in table 1. table 1. organoleptic test of stichopus hermanii extract. no. observations test result 1. color brown 2. smell the distinctive smell of sea cucumber 3. texture thick soluble essence content result the parameter of soluble essence content aims as a rough estimate of the presence and amount of bioactive compound content that is extracted in water solvents (polar) and ethanol solvents (semi polar – non polar) (saifuddin et al., 2011). determination of soluble essence content is very important because it can provide an overview of the number of dissolved ingredients and is part of what is utilized as a medicinal ingredient.(zulharmitta et al., 2013) the results of the soluble essence content assay of stichopus hermanii extract in this study are shown in table 2. table 2. soluble essence content of stichopus hermanii extract. parameters result methods water soluble essence content (%) 61,89 gravimetr y ethanol soluble essence content (%) 41,81 based on the research of nurhaini et al, the higher the percentage of juice content, the better the extract (nurhaini et al., 2020). the results obtained show that the chemical compounds of golden sea cucumber extract are more easily dissolved and distilled in water than ethanol, which indicates that the content of polar compounds from stichopus hermanii extract is more than the content of semi-polar-non-polar compounds. phytochemical screening result phytochemical screening is a way to qualitatively identify bioactive compounds in extracts (manongko et al., 2020). determination of phytochemical screening is carried out using a tube test consisting of precipitation reactions for alkaloid compounds, color reactions for phenol compounds, flavonoids, tannins, steroids and terpenoids, and foam formation for saponin compounds. 542 biology, medicine, & natural product chemistry 12 (2), 2023: 539-545 the results of the phytochemical screening assay of stichopus hermanii extract in this study are shown in table 3. table 3. phytochemical screening of stichopus hermanii extract. no compound test result 1. alkaloids mayer (+) white precipitate dragendorff (–) no orange–brown precipitate formed wagner (+) brown precipitate 2. flavonoids mg + hcl (+) orange color h2so4 (+) red color 3. phenol fecl3 3% (+) blackish-green color 4. saponins aquadest (+) forms foam 5. terpenoids/steroids lieberman-burchard (+) terpenoids (reddish color with reddish brown rings) 6. tannin fecl3 3% (–) no intense green or blue color formed gelatin 1% (–) no white precipitate formed the results of phytochemical screening tests show that stichopus hermanii extract contains an alkaloid, flavonoid, phenol, saponin, and terpenoid compounds. these secondary metabolite compounds have bioactivities as antibacterials, antifungals, and antioxidants that can affect wound healing. the difference in the level of active compounds may be affected by the growth of a biota, both externally and internally. external factors include habitat, seasonality, water temperature, availability of food, and other environmental factors, while internal factors include age, body size, and other biological factors (supriatna et al., 2019). these factors have potential to both qualitatively and quantitatively influence secondary metabolites, resulting in a wide range in both their content and bioactivity (riwanti & izazih, 2019). protein content result the protein content parameter aims to determine the protein content contained in the extract (afkar et al., 2020). the determination of total n was carried out to represent the amount of protein present because it is contained in all proteins which have a proportion of 16% of the total protein (normilawati et al., 2019). the results of the protein content assay of stichopus hermanii extract in this study are shown in table 4. table 4. protein content of stichopus hermanii extract. parameter result methods protein content (%) 7,62 kjeldahl based on susanto's research, indicates that the composition of biological material varies depending on its geographic origin. because of this, even though they are members of the same species, protein levels in golden sea cucumber vary from region to region (susanto et al., 2018). sea cucumbers high protein content helps speed up the regeneration of injured dead cells that help mend wounds. this is because of the presence of cell regeneration factors (also known as cell growth factors), are present, which may encourage regeneration to repair damaged cells or bodily tissues (kokadir et al., 2021). drying shrinkage result the drying shrinkage parameter seeks to set an upper bond (range) on the total amount of chemicals lost during drying as water and volatile substances (essential oils, ethanol solvents, or other substances) (rosidah et al., 2020; utami et al., 2017). for an extract to maintain quality and prevent mold formation (sambode et al., 2022). the results of the drying shrinkage assay of stichopus hermanii extract in this study are shown in table 5. table 5. drying shrinkage of stichopus hermanii extract. parameter result methods drying shrinkage (%) 38,81 gravimetry the test results of drying shrinkage of stichopus hermanii extract, amounted to 38.81% and can be said to not meet the general requirements of ˂10%. high drying shrinkage can also be caused by environmental factors such as low humidity which causes faster evaporation and consequently increases drying shrinkage. this can cause changes in the physical and chemical properties of the extract such as a decrease in volume and changes in the shape of the extract which can affect the quality and stability of the extract. water content result the water content parameter set a minimum limit or range on the quantity of water contained that can be found in the extract and is used to determined how much water is left over after drying (maryam et al., 2020; najib et al., 2018). determination of water content is not related to pharmacological activity directly but affects the quality, safety, and stability aspects of the extract and the formation of an extract preparation (nurhaini et al., rusmini et al. – standardization of golden sea cucumber (stichopus hermanii) extracts … 543 2020; sambode et al., 2022). the results of the water content assay of stichopus hermanii extract in this study are shown in table 6. table 6. water content of stichopus hermanii extract. parameter result methods water content (%) 20,58 gravimetry based on damaiyanti's research, stichopus hermanii extract from bontang has a lower moisture content of 5.65% (d. w. damaiyanti, 2015). the greater the percentage of water content in the extract, the easier it is for an extract to experience damage and decay caused by microbial growth. the high water content can be caused by a less than optimal drying process, the absorption of water into the extract caused by an environment that is too humid during the packaging and storage process, where sea cucumbers are hygroscopic because they have salt and collagen content that can absorb water (kokadir et al., 2021; suryaningrum, 2008). ash content result the total ash content parameter is to provide an overview of the inorganic material and internal and external mineral contents from the initial process to extract production, in order to be related to the purity and contamination of an extract (maryam et al., 2020; zainab et al., 2016). the results of the ash content assay of stichopus hermanii extract in this study are shown in table 7. table 7. ash content of stichopus hermanii extract. parameter result methods ash content (%) 37,95 gravimetry the results of testing the ash content of stichopus hermanii extract, which amounted to 37,95%, can be said that the sea cucumber extract does not meet the requirements of a maximum of 30%. high and low ash content can be caused by differences in organisms, habitat, living environment, and dietary factors (elfath et al., 2019). each water area can provide a different mineral intake. in addition, each organism also has a different ability to absorb minerals that enter the body, so this will affect the ash content value (one et al., 2021). high ash content can affect the effectiveness or biological activity of the extract, where these minerals can interact with active compounds in the extract so that they can change their physicochemical properties which affect changes in color, texture, or stability of the extract. acid insoluble ash content result the acid insoluble ash content parameter aims to determine the levels of inorganic compounds and the presence of insoluble mineral or metallic contamination in acids such as silica from soil or sand, as well as silver, lead, and mercury metal elements (utami et al., 2017). the results of the acid-insoluble ash content assay of stichopus hermanii extract in this study can be seen in table 8. table 8. acid insoluble ash content of stichopus hermanii extract. parameter result methods acid insoluble ash content (%) 2,07 gravimetry the test results of acid insoluble ash content of stichopus hermanii extract, amounted to 2.07% and can be said to meet the general requirements of a maximum of 3.5% (standar nasional indonesia (sni) 01-27321992, 1992). high acid-insoluble ash content indicates residual contamination of acid-insoluble minerals or metals in the extract. the acid insoluble ash content can be used as a criterion to determine how clean a material is (fitriyani et al., 2013). high level of acid insoluble ash also be an indication the presence of contaminants, such as heavy metal, which can change the composition and quality of the extract over time, reducing its effectiveness, stability and compromising its quality and safety. conclusions the results of the specific parameter test of stichopus hermanii extract from pelapis island consist of organoleptic examination which shows that golden sea cucumber extract has a brown color and typical sea cucumber odor, and is known to be positive for alkaloid, flavonoid, phenol, saponin, and terpenoid compounds. golden sea cucumber extract has a water-soluble content of 61.89%, ethanol-soluble content of 41.81%, and protein content of 7.62%. while the test results of nonspecific parameters of golden sea cucumber extract, consisting of drying shrinkage of 38.81%, moisture content of 20.58%, ash content of 37.95%, and acid insoluble ash content of 2.07%. authors’ contributions: the writers whose names are included in this journal contributed to this research. competing interests: this research has no conflict of interest. 544 biology, medicine, & natural product chemistry 12 (2), 2023: 539-545 references afkar, m., nisah, k., & sa’diah, h. 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(2013). pembuatan dan karakterisasi ekstrak kering daun jambu biji (psidium guajava l.). jurnal farmasi higea, 4(2), 147–157. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 2, 2019 | pages: 33-36 | doi: 10.14421/biomedich.2019.82.33-36 issn 2540-9328 (online) the effects of wet cupping therapy in blood pressure, glucose, uric acid and total cholesterol levels sutriyono1,*, muhammad rodham robbina2, meksianis zadrak ndii3 1department of biology; 2department of mathematics, faculty of science and technology, uin sunan kalijaga, yogyakarta, indonesia 3department of mathematics, faculty of science and engineering, university of nusa cendana, kupang-ntt, indonesia author correspondency*: sutriyono@uin-suka.ac.id abstract wet cupping therapy is a simple, cheap, and effective alternative medical treatment. in china wet cupping therapy become a formal treatment in the hospital. 14 centuries ago prophet muhammad implemented wet cupping therapy or hejamah as a curing treatment and become sunnah for muslim. today in a modern world, many studies proved the advantages of wet cupping therapy, which is a sunnah since 14 centuries ago. the aims of this study were to measure the effects of wet cupping therapy treatment on blood pressure, blood glucose, uric acid, and total cholesterol level. 21 participants were treated with hejamah/wet cupping, blood pressure and blood samples were collected from all the participants one week before and one week after hejamah. blood pressure, either systolic or diastolic was significantly decreased following therapy (p|<|0.05), blood glucose had a slight decreased but not significant (p|>|0.05). uric acid and total cholesterol level was also significantly decreased following therapy (p|<|0.05). with only just one time treatment, hejamah had a significant effects on blood pressure, uric acid and total cholesterol level, that is proved the advantages of hejamah. keywords: hejamah; wet cupping therapy; cardiovascular diseases; hematology; blood pressure; blood glucose; uric acid; cholesterol introduction cardiovascular diseases are major health problems and they are associated with mortality and morbidity (refaat et al, 2014). the world health organization (who) has reported that cardiovascular diseases are responsible for 30% of all deaths, which is equivalent to the combined death rates associated with several other medical conditions (rehm et al, 2009). the estimated number of death due to cardiovascular diseases by the who is 17.3 million people per year and the number is expected to increase to 23.3 million/year in 2030 (ng et al, 2012). several benefits of complementary medicine/alternative medicine, also known as traditional medicine, have been shown and it is effective in the prevention of disease, treatment of noncommunicable diseases, and improvement of the quality of life for persons living with chronic diseases (burge and albright, 2002). the who has been encouraging the implementation of traditional medicine since many patients are not satisfied by the outcomes of modern medicine particularly those related to chronic diseases (who, 2002). one of the alternative medicines is islamic hejamah/wet cupping. in the middle east, arabic writers report that cupping therapy dates back to 3500 b.c. (5500 years ago), where assyrians were the first arab population to use primitive tools as animal horns and bamboo wood for cupping therapy then the chinese physician, jee hong (381281 b.c.) was among the leaders in that art. arabic civilization termed cupping therapy, al-hijamah therapy (which means in arabic to restore to the original size), where it was used in treating hypertension, polycythemia, headache, migraine and drug intoxication. they diagnosed polycythemia whenever there was an exaggeration of the pink color of the skin (omar, 2009). interestingly, venesection (phlebotomy) is still being used currently in hospitals for treating polycythemia, where blood is drawn out and is replaced by saline infusion (mcmullin et al, 2005). cupping therapy is being practiced nowadays in many countries all over the world including germany, norway, denmark, saudi arabia, egypt, india, china and other countries. german people are familiar with cupping therapy (michalsen et al, 2009) and so are danish and norwegian peoples where those european societies already have a shift in attitude to include complementary medicine within the conventional health care system (salomonsen et al, 2011). the exact origin of cupping therapy is a matter of controversy. chinese scientists report in their literature that cupping therapy is a part of the traditional chinese medicine dating back to at least 2,000 years (chirali, 2014). materials and methods this study was a true experimental research. 21 adult men were recruited into the study, wet cupping was performed on the study subjects one time according to https://doi.org/10.14421/biomedich.2019.82.33-36 34 biology, medicine, & natural product chemistry 8 (2), 2019: 33-36 islamic protocol, the subject were asked to be fasting at least 3 hours before the initiation of the procedure. blood pressure and blood sample were obtained from the subject before the procedure. we run a test to the blood sample to measure blood glucose, uric acid, and total cholesterol level. a second blood pressure and blood sample were obtained from the subjects 7 days after wet cupping therapy. figure 1. wet cupping therapy. wet cupping therapy were performed using seven points on the back of each subjects, the cup was sterilized using alcohol. the cups were placed on the chosen sites and a negative pressure was created by manual suction using the provided pump with the kit. the cups were left for a period of 7-10 minutes after ensuring their firm attachment to the skin. the cups were removed and 12-15 superficial incisions were made on each chosen area of the skin using sterile surgical blades. after soaking the cups, they were replaced back on the chosen areas of the skin and negative pressure was created as previously described. the cups were left on the skin till they were filled with blood from the capillary vessels. the cups were removed approximately after 3 minutes and new cups were placed on the same areas as previously mentioned. the used cups were socked in betadine solution for sterilization. the process of blood-letting was repeated for two times in total. result and discussion statistical analysis was performed using spss version 15. normality and homogeneity of data were assessed with the shapiro-wilk test. table 1. the effect of wet cupping therapy to blood pressure. mean (mmhg) p systolic pretest 132.33 0.001 posttest 124.81 diastolic pretest 86.29 0.001 posttest 81.19 according table 1 that using t test showing that there is a different on blood pressure (systolic and diastolic) between before the wet cupping therapy and after the wet cupping therapy. there is some reduction on the mean of systolic pressure in amount of 7.5 mmhg from 132.33 mmhg becomes 124.81 mmhg, while for the diastolic pressure there is some reduction in amount of 5.1 mmhg from 86.29 mmhg becomes 81.19 mmhg. the analysis result using t test to systolic pressure and diastolic pressure showing the p value is 0.001 (p|<|0.05), then the effect of wet cupping therapy to the reduction of subjects blood pressure after wet cupping therapy is significant. the study of refaat et al (2014) showing the same result that wet cupping therapy can reduce blood pressure significantly. the positive effect that given by wet cupping therapy into the reduction of blood pressure is because wet cupping therapy has benefit to remove all waste and sediment in blood artery that connected to the circulation of blood (ica, 2011). the blood pressure reduction also happen because wet cupping therapy in a skin (cutaneous), under skin tissue (sub-cutaneous), fascia, and the muscle will cause the damage of mast cell, the result of this damage will release a few substance like serotonin, histamine, bradikin, slowreacting substance (srs), and few other substance, this substance will cause capillar and arteriole dilatation also flare reaction in the place of wet cupping therapy point. capillar dilatation also happen in a place who far from wet cupping therapy point, this is cause the blood artery microcirculation repair, then will show relaxation effect in clumsy muscle also because of vasodilatation will reduce blood pressure (refaat et al, 2014) table 2. the effect of wet cupping therapy to blood glucose. blood glucose mean (mgdl-1) p pre-test 98.48 0.311 post-test 93.33 according to table 2 that using t test, it is showing that there is a different on blood glucose level between sutriyono et al. – the effects of wet cupping therapy in blood pressure … 35 before wet cupping therapy and after the wet cupping therapy. there is some reduction on the mean of blood glucose level between before wet cupping therapy and after the wet cupping therapy in amount of 5.15 mgdl-1 from 98.48 mgdl-1 becomes 93.33 mgdl-1. the analysis result using t test to blood glucose level showing the p value is 0.311 (p|>|0.05), then the effect of wet cupping therapy to the reduction of subjects blood glucose level after wet cupping therapy is not significant, even though the effect of wet cupping therapy still make some reduction on blood glucose level. wet cupping therapy enhances insulin sensitivity in healthy subjects with normal glucose levels and normoferritinemia (vakilinia et al, 2016). bleeding due to hijamah was found to decrease glucose serum and triglycerides in patient with diabetes (lowe, 2017). some study has proved the efficacy of wet cupping therapy, on of it is the study by farahmand et al (2012), this study prove that wet cupping therapy an diet have significant effect to lipid profile improvement. another study about efficacy of wet cupping therapy is the study by refaat et al (2014) who mention that wet cupping therapy has positive effect to blood glucose level on subject who has diabetes mellitus disease. table 3. the effect of wet cupping therapy to uric acid. uric acid mean (mgdl-1) p pretest 7.15 0.001 posttest 5.91 according to table 3 that using t test showing that there is a different on uric acid level between before wet cupping therapy and after the wet cupping therapy. there is some reduction on the mean of uric acid level between before wet cupping therapy and after the wet cupping therapy in amount of 1.24 mgdl-1 from 7.15 mgdl-1 becomes 5.91 mgdl-1. the analysis result using t test to uric acid level showing the p value is 0.001 (p|<|0.05), then the effect of wet cupping therapy to the reduction of subjects urid acid level after wet cupping therapy is significant. this reduction is positive enough to prevent cardiovascular disease because high level of uric acid or commonly called hyperuricemia according to euser et al (2008) can rise the risk of cardiovascular disease. table 4. the effect of wet cupping therapy to total cholesterol. cholesterol mean (mgdl-1) p pretest 177.05 0.022 posttest 160.43 according to table 4 that using t test showing that there is a different on total cholesterol level between before wet cupping therapy and after the wet cupping therapy. there is some reduction on the mean of total cholesterol level between before wet cupping therapy and after the wet cupping therapy in amount of 16.62 mgdl-1 from 177.05 mgdl-1 becomes 160.43 mgdl-1. the analysis result using t test to total cholesterol level showing the p value is 0.022 (p|<|0.05), then the effect of wet cupping therapy to the reduction of subjects total cholesterol level after wet cupping therapy is significant. the reduction of total cholesterol level has shown the effect of wet cupping therapy to total cholesterol level, which mean that if wet cupping therapy is doing regularly, wet cupping therapy can be alternative treatment beside conventional treatment for stroke patients to reduce total cholesterol level. the reduction of total cholesterol level after wet cupping therapy is because there is some effect from hematology system mechanism who give main effect through the regulation system pathways of coagulantanticoagulation with the improvement of blood flow and organ oxygenation (ahmadi et al, 2008). considering hepar is a place that blood filtration from various toxic substances who entering the body happen, through ti=his hematology system mechanism and immune system mechanism the total cholesterol level can be reduced (ahmadi et al, 2008). however, there are some consideration that need to be consider. first the sample we were taken is small, and we didn’t consider the health factor of subjects of this study. second, in the process of our study we didn’t consider factors who maybe have a big impact to this study, like subjects diet, sleep patterns, etc. despite the shortcomings of this study, hejamah has been proven have some benefits in the prevention of cardiovascular diseases. study of bassem refaat (2014) shown that although hejamah have no significant impact on decreasing total cholesterol level, but significantly raised hdl (high density lippoprotein) level and decreased ldl (low density lippoprotein) level. with lower ldl level can lower the risk of stroke attack. conclusion overall, wet cupping therapy is a traditional treatment that is a sunnah from prophet muhammad. first the sample we are taken is small, and we didn’t consider the health factor of subjects of this study. second in the procces of our study we didn’t consider factors who maybe have a big impact to this study, like subjects diet, sleep patterns, etc. despite the shortcomings of this study, hejamah has been proven have some benefits in the prevention of cardiovascular diseases. study of bassem refaat (2014) shown that although hejamah have no significant impact on decreasing total cholesterol level, but significantly raised hdl (high density lippoprotein) level and decreased ldl (low density lippoprotein) level. with lower ldl level can lower the risk of stroke attack. the conclusion is hejamah or wet cupping theraphy have a great chance to become a medical treatment who 36 biology, medicine, & natural product chemistry 8 (2), 2019: 33-36 can reduce blood pressure, blood glucose level, urid acid level, and total cholesterol level. hejamah also can prevent cardiovascular diseases because it can raise the level of hdl and reduce the level of ldl. references ahmadi, a., schwebelb, d. c., & rezaei, m. 2008. the efficacy of wet-cupping in the treatment of tension and migraine headache. the american journal of chinese medicine, 36(01), 37-44. burge s. k., albright t. l., & residency research network of south texas (rrnest) investigators. 2002. use of complementary and alternative medicine among family practice patients in south texas. american journal of public health 92.10: 16141616. chirali, i. z. 2014. traditional chinese medicine cupping therapy. elsevier health sciences. euser, s. m., hofman, a., westendorp, r. g. j., & breteler, m. m. (2008). serum uric acid and cognitive function and dementia. brain 132(2), 377-382. farahmand, s. k., gang, l. z., saghebi, s. a., mohammadi, m., mohammadi, s., mohammadi, g., & ghayour-mobarhan, m. 2012. the effects of wet cupping on coronary risk factors in patients with metabolic syndrome: a randomized controlled trial. the american journal of chinese medicine, 40(02), 269-277. indonesian cupping association. 2011. standard operating procedure hejamah. bogor: research and development division ica. ng, n., johnson, o., lindahl, b., & norberg, m. 2012. a reversal of decreasing trends in population cholesterol levels in västerbotten county, sweden. global health action, 5(1), 10367. lee, m. s., choi, t. y., shin, b. c., kim, j. i., & nam, s. s. 2010. cupping for hypertension: a systematic review. clinical and experimental hypertension 32(7), 423-425. mcmullin, m. f., bareford, d., campbell, p., green, a. r., harrison, c., hunt b., & ryan, k. 2005. guidelines for the diagnosis, investigation and management of polycythaemia/ erythrocytosis. british journal of haematology 130(2), 174195. michalsen, a., bock, s., lüdtke, r., rampp, t., baecker, m., bachman, j., & dobos, g. j. 2009. effects of traditional cupping therapy in patients with carpal tunnel syndrome: a randomized controlled trial. the journal of pain 10(6), 601608. omar, s. a. h. 2009. al-hijamah (cupping therapy): sunnah and therapy. dar ommah for publication, jeddah. refaat, b., el-shemi, a. g., ebid, a. a., ashshi, a., & basalamah, m. a. 2014. islamic wet cupping and risk factors of cardiovascular diseases: effects on blood pressure, metabolic profile and serum electrolytes in healthy young adult men. alternative and integrative medicine, 3(1): 151. rehm, j., mathers, c., popova, s., thavorncharoensap, m., taerawattananon, y., & patra, j. 2009. global burden of disease and injury and economic cost attributable to alcohol use and alcohol-use disorders. the lancet 373(9682), 22232233. salomonsen, l. j., skovgaard, l., lacour, s., nyborg, l., launsø, l., & fonnebo, v. 2011. use of complementary and alternative medicine at norwegian and danish hospitals. bmc complementary and alternative medicine, 11(1), 4. vakilinia, s. r., bayat d., & asghari, m. 2016. hijama (wet cupping or dry cupping) for diabetes treatment. iranian journal of medical sciences 4(3 suppl), s37. lowe, d. t. 2017. cupping therapy: an analysis of the effects of suction on skin and the possible influence on human health. complementary therapies in clinical practice, 29, 162-8. who. 2002. who traditional medicine strategy 2002-2005. world health organization, geneva, switzerland. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 611-617 | doi: 10.14421/biomedich.2023.122.611-617 issn 2540-9328 (online) effect of citrullus lanatus seeds extract on haematological parameters after administration with medroxyprogesterone acetate on female wistar rats michael chuks nnumolu*, onyeso godspower department of physiology, faculty of basic medical sciences, madonna university, elele, nigeria. corresponding author* michaelnnumolu@gmail.com manuscript received: 28 august, 2023. revision accepted: 03 november, 2023. published: 10 november, 2023. abstract this study was carried out to investigate the effect of citrullus lanatus seeds extract on the haematological parameters after administration with medroxyprogesterone acetate on female wistar rats. twenty-five rats weighing 170-230 g were divided into five groups containing five rats each. group i served as control and received only rat chow and water; group ii received rat chow, water and a high dose of mpa only; group iii received rat chow, water, a high dose of mpa and a high dose of extract; group iv received rat chow, water, high dose of mpa and a low dose of extract, and group v received rat chow, water and high dose of extract only. the administration lasted for twenty-eight days after two weeks of acclimatisation. on the twenty-ninth day, the animals were anaesthetised using chloroform, sacrificed and their blood samples obtained via cardiac puncture for haematology analysis. the results showed that the level of haematological parameters significantly increased (p<0.05) in the rats that received a high dose of mpa and a high dose of citrullus lanatus seed extract compared to the control group. this suggests that mpa affects bone marrow, which produces blood cells, significantly decreasing platelet count, white blood cell count, red blood cell count, haemoglobin count, packed cell volume, mean corpuscular volume, and mean corpuscular haemoglobin concentration. this study also shows that an aqueous extract of citrullus lanatus seed is potent in increasing the level of haematological parameters when co-administered with mpa. keywords: medroxyprogesterone acetate; citrullus lanatus seeds; haematology; blood physiology. introduction there has been a notable increase in the utilisation of contraceptives among women of reproductive age in recent years (abdel-salam et al., 2020). a growing inclination towards methods such as oral contraceptive pills, female sterilisation, and condoms characterises this trend (jain et al., 2021). simultaneously, the advent of novel contraceptive alternatives has resulted in a heightened utilisation of injectable methods, most notably depo-provera, which encompasses medroxyprogesterone acetate (mpa). medroxyprogesterone acetate, also known as 17αhydroxy-6α-methylprogesterone acetate, is a steroidal progestin, a synthetic variant of the steroid hormone progesterone. it is used as a contraceptive, in hormone replacement therapy, and for the treatment of endometriosis as well as several other indications (gompel, 2020). the contraceptive effectiveness of mpa has been well-established; however, its impact on different physiological processes, specifically in haematology, has attracted significant interest among researchers. the haematological parameters are of utmost importance in preserving general health and welfare. any changes in these parameters can substantially affect individuals utilising contraceptives such as mpa. prior studies have provided evidence suggesting that using mpa may lead to alterations in blood coagulation, fibrinolytic systems, and plasma levels (abe et al., 1995). this underscores the significance of comprehending the impact of this contraceptive on haematological parameters. citrullus lanatus, commonly called watermelon, has garnered considerable interest in medicinal plants due to its perceived health-promoting properties. watermelon seeds possess a rich composition of diverse nutrients and minerals, with a notable emphasis on magnesium, a vital element for the proper functioning of the heart and blood pressure regulation (benmeziane and derradji, 2023). research shows that the ld50 of citrullus lanatus seed extract is >5000 mg/kg (ihejieto et al., 2022). traditional healthcare practices have utilised natural sources, rendering them deserving of scrutiny in contemporary scientific research. https://doi.org/10.14421/biomedich.2023.122.611-617 612 biology, medicine, & natural product chemistry 12 (2), 2023: 611-617 this study investigated the effects of citrullus lanatus seed extract on haematological parameters in female wistar rats administered with high doses of mpa. the goal was to explore potential plant-based interventions that could mitigate the negative effects associated with using mpa. by assessing the correlation between mpa and citrullus lanatus seed extract concerning blood parameters, this research contributes significant insights to contraceptive research. the study's significance lies in its potential to shed light on the impact of mpa on haematological parameters and its investigation of natural interventions like watermelon seed extract. ultimately, this research aims to bridge the gap between conventional plant medicine and contemporary healthcare, thereby presenting novel and all-encompassing strategies for enhancing the safety and efficacy of contraceptive alternatives pertaining to women's reproductive well-being. materials and methods experimental animals twenty-five female wistar rats weighing 170 – 230 grams were used for the sub-chronic study. the rats were purchased from the animal house of the department of medical physiology, delta state university, abraka campus delta state. the animals were housed in the experimental animal house of the department of medical physiology, madonna university, elele campus, rivers state. the animals were kept under normal room conditions of 25 ± 2oc, 50 ± 5% humidity, and 12 hours of light and day cycles. the rats were randomised into a control group and four experimental groups with five rats in each compartment (n=5), according to their weight range and housed in a sanitised wooden cage containing sawdust as bedding. feeding was provided twice a day during the acclimatisation period and then reduced to once daily as the rats increased their body weight. all animal experiments were in compliance with the national institute of health guide for care and for laboratory animals (pub. no. 85-23, revised 1985). plant collection and identification the seeds were extracted from the pods after it was allowed to rot manually by washing. only healthylooking seeds (brown, not floating on water, without mechanical damage or sign of infection) were collected. the collected seeds were oven-dried at 35°c until a constant weight was obtained. the dried seeds were reduced into a fine powder using a laboratory grinding hand mill. the powder was weighed and kept away from light before extraction. it was then soaked in water and refrigerated. preparation of extract the extraction process was conducted on 255.19 grams of ground citrullus lanatus seeds using an aqueous water solution. on four occasions, 1000 ml of water was sequentially added to ground citrullus lanatus seeds. the extracts were kept in a bucket, left for 24 hours, and then manually squeezed out of the mixture using clean handkerchiefs to obtain the extracts. the filtrate (chocolate in colour) was collected in beakers and then placed in a water bath at about 450c for seven days. the extracts were kept in the refrigerator to prevent loss of potency. the aqueous extract was then dissolved and stored in a container for administration. experimental design twenty-five adult female wistar rats weighing 170 – 230 g were randomly distributed into five groups of five rats each. the animals were acclimatised on regular feed (grower mashguinea feed) and water for two weeks. group i served as control and received only rat chow and water; group ii received rat chow, water and a high dose of mpa only; group iii received rat chow, water, a high dose of mpa and a high dose of extract; group iv received rat chow, water, high dose of mpa and a low dose of extract, and group v received rat chow, water and high dose of extract only (table 1). medroxyprogesterone acetate was administered only once because the drug is administered once every three months. the rat groups administered with the drug were monitored for seven days before treatment with citrullus lanatus seed extract for 28 days based on varying individual body weights. table 1. experimental design for the administration of citrullus lanatus seed extract and mpa on wistar rats. groups agent dosage (mg/kg/day) i nil nil ii mpa only 25 iii mpa + high dose of c. lanatus seed extract 25 mg/kg + 200 mg/kg/day iv mpa + low dose of c. lanatus seed extract 25 mg/kg + 100 mg/kg/day viii high dose of c. lanatus seed extract only 200 source: field data (2022) collection of blood samples the rat groups received their respective doses of citrullus lanatus seed extract orally, once daily before feeding (10 am), for 28 days. after this, the animals were sacrificed by cardiac puncture and blood samples were collected in edta bottles and centrifuged for further testing. haematological analysis the haematology profile, which covers white blood cell (wbc), red blood cell (rbc), haemoglobin level (hb), hematocrit (hct), mean corpuscular volume (mcv), mean corpuscular haemoglobin (mch), mean corpuscular haemoglobin concentration (mchc), and nnumolu & godspower – effect of citrullus lanatus seeds extract on haematological parameters … 613 platelets (plt) were determined using an automated haematology analyser (sysmex haematology – coagulation system, model mo-1000 i, trans asia, japan). statistical analysis and data evaluation the results were analysed using the statistical software package for social sciences (spss) version 16.0 for windows. analysis of variance (anova) was used to compare means (p<0.05) and post hoc multiple comparisons used lsd. all results are presented as mean ± sem graphs were created in microsoft excel 2007. results and discussion results effect of citrullus lanatus seeds extract on some hematological parameters after administration of medroxyprogesterone acetate on female wistar rats the results of the haematology analysis of the blood samples of rats that received medroxyprogesterone acetate and different doses of the extract are shown in table 2. table 2. results of the haematological analysis of the aqueous extract of citrullus lanatus seeds after administration of mpa on female wistar rats. groups wbc (x109/l) rbc (x1012/l) hb (g/dl) hct (%) mcv (fl) mch (pg) mchc (g/dl) plt (x109/l) i 7.45 ± 0.38 7.81 ± 0.27 14.84 ± 0.45 41.65 ± 0.96 53.22 ± 0.66 20.14 ± 0.92 34.11 ± 0.66 171.55 ± 3.06 ii 4.35 ± 0.35a 4.90 ± 0.40a 8.74 ± 0.54a 24.30 ± 1.63a 31.08 ± 3.36a 10.54 ± 1.07a 23.64 ± 1.14a 98.56 ± 4.45a iii 11.59 ± 0.35a,b 7.93 ± 0.35b,c 16.50 ± 0.37a,b 47.25 ± 1.02a,b 54.33 ± 1.14b,c 24.49 ± 0.55a,b 41.06 ± 1.17a,b 226.61 ± 12.16a,b iv 8.23 ± 0.42b,c 6.43 ± 0.23a,b 11.83 ± 0.38a,b 31.18 ± 1.13a,b 40.81 ± 1.25a,b 16.27 ± 0.35a,b 27.84 ± 1.23a,b 124.43 ± 4.65a,b v 11.25 ± 0.34a,b 9.41 ± 0.36a,b 14.63 ± 0.42b,c 43.02 ± 0.46b,c 51.88 ± 0.43b,c 22.02 ± 0.42b,c 38.28 ± 0.42a,b 182.54 ± 5.64b,c data represented as mean ± sem; n=5, (a) p<0.05 significantly different in comparison with negative control group, (b) p<0.05 significantly different in comparison with positive control group, (c) p>0.05 not significantly different in comparison with negative control group, i=negative control, ii=positive control (mpa), iii= mpa + 200mg/kg cl, iv= mpa + 100mg/kg cl, v=200mg/kg cl effect on white blood cells the result of the study (figure 1) revealed a significant decrease (p<0.05) in wbc count (4.35 ± 0.35) for rats treated with mpa only (group ii) compared to group i, which neither received mpa nor c. lanatus seed extract (7.45 ± 0.38). conversely, rats treated with mpa + 200mg/kg of c. lanatus seed extract (group iii) exhibited a significant increase (p<0.05) in mean wbc count (11.59±0.35) compared to both group i (7.45±0.38) and group ii (4.35 ± 0.35) that received mpa alone. group iv, treated with mpa + 100mg/kg of c. lanatus seed extract, demonstrated a significant decrease (p<0.05) in mean wbc count (8.23 ± 0.42) compared to group ii receiving mpa only (4.35 ± 0.35). however, there was no significant difference (p>0.05) in the mean wbc count compared to group i, which received neither mpa nor c. lanatus seed extract (7.45 ± 0.38). furthermore, group v exhibited a significant increase (p<0.05) in wbc count (11.25 ± 0.34) compared to both group i, not given mpa or c. lanatus seed extract (7.45 ± 0.38), and group ii, given mpa only (4.35±0.35). figure 1. a chart showing the effect of c. lanatus on wbc count after administration of mpa on female wistar rats. group i group ii group iii group iv group v series1 7,45 4,35 11,59 8,23 11,25 a a,b b,c a,b 0 2 4 6 8 10 12 14 w b c ( x 1 0 9 /l ) groups 614 biology, medicine, & natural product chemistry 12 (2), 2023: 611-617 effects on red blood cells the result of the study (figure 2) showed that the rats treated with mpa only (group ii) had a significant decrease (p<0.05) in the rbc count (4.90 ± 0.40) in comparison to group i not given either mpa or c. lanatus seed extract (7.81± 0.27). additionally, the rats treated with mpa + 200mg/kg of c. lanatus seed extract (group iii) had a significant increase (p<0.05) in the mean rbc count (7.93±0.35) as compared to group ii given mpa only (4.90 ± 0.40). however, there was no significant difference (p>0.05) in the mean rbc count as compared to group i, which was neither given mpa nor c. lanatus seed extract (7.81 ± 0.27). furthermore, the results obtained from group iv & v showed a significant difference (p<0.05) in rbc count, (6.43± 0.23) and (9.41 ± 0.36), respectively, as compared to group i (7.81 ± 0.27) & group ii (4.90 ± 0.40). figure 2. a chart showing the effect of c. lanatus on rbc count after administration of mpa on female wistar rats. effects on haemoglobin count the result of the study (figure 3) revealed that the rats treated with mpa only (group ii) experienced a significant decrease (p<0.05) (8.74 ± 0.54) compared to group i, which was neither given mpa nor c. lanatus seed extract (14.84 ± 0.45). both group iii, treated with mpa + 200mg/kg of c. lanatus seed extract, and group iv, treated with mpa + 100mg/kg c. lanatus seed extract, showed a significant difference (p<0.05) in the haemoglobin count (16.50 ± 0.37) and (11.83 ± 0.38) respectively, as compared to group i, which was neither given mpa nor c. lanatus seed extract (14.84 ± 0.45), and group ii, given only mpa (8.74 ± 0.54). results obtained from group v, treated with 200mg/kg c. lanatus seed extract, demonstrated a significant increase (p<0.05) in haemoglobin count (14.63 ± 0.42) in comparison with group ii, treated with mpa only (8.74 ± 0.54). however, there was no significant difference (p>0.05) compared to group i (14.84 ± 0.45). figure 3. a chart showing the effect of c. lanatus on haemoglobin count after administration of mpa on female wistar rats. effects on hematocrit levels the result of the study (figure 4) showed that the rats treated with mpa only (group ii) had a significant decrease (p<0.05) in the hct value (24.30 ± 1.63) in comparison to group i that neither mpa nor c. lanatus seed extract (41.65 ± 0.96). both group iii, treated with mpa + 200mg/kg of c. lanatus seed extract, and group iv, treated with mpa + 100mg/kg c. lanatus seed extract, showed a significant difference (p<0.05) in the hct value (47.25 ± 1.02) and (31.18 ± 1.13) as compared to group i neither given mpa nor c. lanatus seed extract (41.65 ± 0.96) and those in group ii given only mpa (24.30 ± 1.63). furthermore, results obtained from group v treated with 200mg/kg c. lanatus seed extract showed a significant increase (p<0.05) in hct value (43.02± 0.46) in comparison with group ii treated with mpa only (24.30± 1.63). however, there was no significant difference (p>0.05) as compared to group i (41.65± 0.96). figure 4. a chart showing the effect of c. lanatus on hct after administration of mpa on female wistar rats. group i group ii group iii group iv group v series1 7,81 4,9 7,93 6,43 9,41 a b,c a,b a,b 0 2 4 6 8 10 12 r b c ( x 1 0 1 2 /l ) groups group i group ii group iii group iv group v series1 14,84 8,74 16,5 11,83 14,63 a a,b a,b b,c 0 2 4 6 8 10 12 14 16 18 h b ( g/ d l) groups group i group ii group iii group iv group v series1 41,65 24,3 47,25 31,18 43,02 a a,b a,b b,c 0 10 20 30 40 50 60 h ct ( % ) groups nnumolu & godspower – effect of citrullus lanatus seeds extract on haematological parameters … 615 effects on mean corpuscular volume the result of the study (figure 5) showed that the rats treated with mpa only (group ii) indicated a significant decrease (p<0.05) in the mcv (31.08 ± 3.36) in comparison to group i that was not given mpa and still not given c. lanatus seed extract (53.22 ± 0.66). furthermore, the rats treated with mpa + 200mg/kg of c. lanatus seed extract (group iii) and those given only 200mg/kg c. lanatus seed extract (group v) had a significant difference (p<0.05) in the mcv (54.33 ± 1.14) and (51.88 ± 0.43) as compared to group ii given mpa only (31.08±3.36). however, there was no significant difference (p>0.05) in the mcv of both groups as compared to group i, which was neither given mpa nor c. lanatus seed extract (53.22± 0.66). moreover, results obtained from group iv treated with mpa + 100mg/kg of c. lanatus seed extract had a significant decrease (p<0.05) in mcv, (40.81± 1.25) as compared to group i neither given mpa nor c. lanatus seed extract (53.22± 0.66) as well as a significant increase those in group ii given mpa only (31.08 ± 3.36). figure 5. a chart showing the effect of c. lanatus on mcv after administration of mpa on female wistar rats. effects on mean corpuscular haemoglobin the result of the study (figure 6) showed that the rats treated with mpa only (group ii) had a significant decrease (p<0.05) in the mch value (10.54 ± 1.07) compared to group i, which neither received mpa nor c. lanatus seed extract (20.14 ± 0.92). moreover, the rats treated with mpa + 200mg/kg of c. lanatus seed extract (group iii) and group iv given mpa + 100mg/kg of extract had a significant difference (p<0.05) in the mch value (24.49 ± 0.55) and (16.27 ± 0.35), respectively, as compared to group i (20.14 ± 0.92) and group ii (10.54 ± 1.07). furthermore, results obtained from group v treated with 200mg/kg of extract showed a significant increase (p<0.05) in the mch value (22.02 ± 0.42) compared to group ii (10.54 ± 1.07). however, there was no significant difference (p>0.05) when compared to group i (20.14 ± 0.92). figure 6. a chart showing the effect of c. lanatus on mch after administration of mpa on female wistar rats. effects on mean corpuscular haemoglobin concentration the result of the study (figure 7) showed that the rats treated with mpa only (group ii) had a significant decrease (p<0.05) in the mchc value (23.64 ± 1.14) as compared to group i that neither received mpa nor c. lanatus seed extract (34.11 ± 0.66). furthermore, the rats treated with mpa + 200mg/kg of extract (group iii), group iv given mpa + 100mg/kg of extract and group v given 200mg/kg of extract only, had a significant difference (p<0.05) in the mchc value (41.06±1.17), (27.84 ± 1.23) and (38.28 ± 0.42) respectively, as compared to group i that was not given the drug and extract (34.11 ± 0.66) and group ii that was given only the drug (23.64 ± 1.14). figure 7. a chart showing the effect of c. lanatus on mchc after administration of mpa on female wistar rats. effects on platelet count the result of the study (figure 8) showed that the rats treated with mpa only (group ii) had a significant decrease (p<0.05) in the platelet count (98.56 ± 4.45) compared to group i, which was not given mpa or c. lanatus extract (171.55 ± 3.06). furthermore, the rats treated with mpa + 200mg/kg of c. lanatus (group iii) and group iv given mpa + group i group ii group iii group iv group v series1 53,22 31,08 54,33 40,81 51,88 a b,c a,b b,c 0 10 20 30 40 50 60 m c v ( fl ) groups group i group ii group iii group iv group v series1 20,14 10,54 24,49 16,27 22,02 a a,b a,b b,c 0 5 10 15 20 25 30 m c h ( p g) groups group i group ii group iii group iv group v series1 34,11 23,64 41,06 27,84 38,28 a a,b a,b a,b 0 5 10 15 20 25 30 35 40 45 m c h c ( g/ d l) groups 616 biology, medicine, & natural product chemistry 12 (2), 2023: 611-617 100mg/kg c. lanatus had a significant difference (p<0.05) in the platelet count (226.61 ± 12.16) and (124.43 ± 4.65), respectively, compared to group i, which was not given mpa or c. lanatus (171.55 ± 3.06) and those in group ii administered mpa only (98.56 ± 4.45). additionally, results obtained from group v treated with 200mg/kg c. lanatus showed a significant increase (p<0.05) in platelet count (182.54± 5.64) compared to group ii given mpa alone (98.56± 4.45). however, there was no significant difference (p>0.05) when compared to group i (98.56± 4.45). figure 8. a chart showing the effect of c. lanatus on plt after administration of mpa on female wistar rats. discussion the administration of mpa resulted in a significant reduction in wbc count. however, the c. lanatus seeds extract exhibited a dose-dependent enhancement in wbc production. the administration of mpa suppressed the formation of white blood cells, thereby increasing the susceptibility of rats to opportunistic infections. the extract effectively counteracted the inhibitory effect of mpa on white blood cells. the extract induced a modest elevation in the white blood cell count in non-infected rats, suggesting its immunostimulatory effect. based on the results, it can be inferred that the extract has the potential to enhance immune function. no synergism was observed between mpa and the c. lanatus seed extract on rbc count. the extract suppressed the inhibitory effects of mpa on rbc production. thus, the administration of c. lanatus seed extract increased rbc production. the results in groups iii, iv, and v show that the extract's ability to suppress the effect of mpa on erythropoiesis and further increase rbc production is dose-dependent. group v signifies that the extract alone significantly improves rbc count, showing that the extract can improve rbc production independently. this also implies that the lower values obtained on simultaneous administration of mpa and the extract are partly due to the unit used to overcome the mpa inhibition. group ii animals experienced a significant decrease in hb count, pcv, mcv, and mch levels, suggesting they became anaemic. when the extract was administered in low doses after mpa administration, the level of decline in the hb count and hematocrit level was reduced, and when administered in high doses, it suppressed the effects of mpa, thus raising the haemoglobin and hematocrit levels. interestingly, concomitant administration of the extract with mpa significantly increased hb count and hct level, signifying a synergetic action between the extract and the drug. the findings indicate minimal impact on mchc by the extract. however, it effectively mitigated mpainduced adverse effects on mchc levels, demonstrating inhibitory effects on microcytic anaemia associated with mpa. administering a high dosage of extract after mpa significantly suppressed the adverse effects and increased mchc levels. the results imply that after mpa administration, there was a significant decrease in platelet count, which could cause blood-clotting problems. there were remarkable improvements in the platelet count when the extract was administered alone. the synergetic action of mpa and the extract resulted in a significant increase in plt count. conclusion this study investigated the impact of an aqueous extract of citrullus lanatus seeds on haematological parameters following the administration of medroxyprogesterone acetate (mpa) in female wistar rats. the results revealed a significant decrease in platelet count, white blood cell count, and other blood cell parameters, indicating mpa's influence on bone marrow, which is responsible for blood cell production. although the daily administration of the extract alone did not elevate blood cell parameters beyond the normal range, it increased them compared to their initial state before drug administration. notably, when co-administered with mpa, the extract prevented the decrease in blood parameters and improved their values from the average levels. these effects were dosedependent, suggesting the potential of the extract to counter the adverse effects of mpa on blood parameters. these findings suggest that the c. lanatus seed extract possesses immunostimulatory properties, counters the adverse effects of mpa on blood cell counts, and demonstrates the potential to enhance immune function and ameliorate anaemia-related parameters. the extract's dose-dependent effects further highlight its therapeutic potential in the context of mpa treatment. acknowledgements: the author is grateful to dr. godspower onyeso for his supervision and for providing technical assistance. group i group ii group iii group iv group v series1 171,55 98,56 226,61 124,43 182,54 a a,b a,b b,c 0 50 100 150 200 250 300 p lt ( x 1 0 9 /l ) groups nnumolu & godspower – effect of citrullus lanatus seeds extract on haematological parameters … 617 authors' contribution: conceptualisation: michael chuks nnumolu; data curation: godspower onyeso; formal analysis: michael chuks nnumolu; funding acquisition: michael chuks nnumolu; investigation: michael chuks nnumolu; methodology: michael chuks nnumolu; project administration: michael chuks nnumolu; supervision: godspower onyeso; validation: michael chuks nnumolu; writing– original draft: michael chuks nnumolu; writing– review & editing: michael chuks nnumolu. competing interests: the author declares that there is no conflict of interest concerning this manuscript. funding: the study was self-funded. references abdel-salam, d. m., albahlol, i. a., almusayyab, r. b., alruwaili, n. f., aljared, m. y., alruwaili, m. s., & alnasser, r. m. (2020). prevalence, correlates, and barriers of contraceptive use among women attending primary health centers in aljouf region, saudi arabia. international journal of environmental research and public health, 17(10), 3552. jain, m., caplan, y., ramesh, b.m., isac, s., anand, p., engl, e., halli, s., kemp, h., blanchard, j., gothalwal, v. and namasivayam, v., 2021. understanding drivers of family planning in rural northern india: an integrated mixed-methods approach. plos one, 16(1), p.e0243854. gompel, a. (2020). progesterone and endometrial cancer. best practice & research clinical obstetrics & gynaecology, 69, 95107. abe, o., asaishi, k., izuo, m., enomoto, k., koyama, h., tominaga, t., nomura, y., ohshima, a., aoki, n. and tsukada, t. (1995). effects of medroxyprogesterone acetate therapy on advanced or recurrent breast cancer and its influences on blood coagulation and the fibrinolytic system. surgery today, 25, pp.701-710. benmeziane, f. and derradji. (2023). composition, bioactive potential and food applications of watermelon (citrullus lanatus) seeds–a review. journal of food measurement and characterization, 1-17. ihejieto, h. a., kalu, m. u., joel, o. m., & anyanwu, c. n. (2022). effects of combined methanol seeds extracts of citrullus lanatus and annona muricata on male hormone profile and prostate-specific antigen (psa) level in dihydrotestosteroneinduced benign prostatic hyperplaxia in male wistar albino rats. world j pharm res, 11(1), 104-15. national institutes of health. (1985). guide for the care and use of laboratory animals. national academies. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 5, number 2, 2016 | pages: 41-47 | doi: 10.14421/biomedich.2016.52.41-47 issn 2540-9328 (online) optimization of binocular microscope with micro digital camera for measuring seminiferous tubules epithelium height sutriyono biology laboratory, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: sutriyono@uin-suka.ac.id abstract binocular microscope optimization using micro digital camera to measure seminiferous tubules epithelium of mouse testis (mus muculus) was completed. this research was conducted at biology laboratory of faculty of science and technology uin sunan kalijaga. the purpose of this study was to determine the height of the seminiferous tubules epithelium of mouse testis using micro digital camera. a computer was connected with binocular microscope and optilab advance, then calibration of optilab advance were done on objective lens magnification at 4x, 10x and 40x with 10x ocular lens. the analysis used was descriptive analysis. the mean of the seminiferous tubules epithelium height is 105.6 μm. optimization on computer and binocular microscope with micro digital camera can be used to measure seminiferous tubules epithelium. keywords: seminiferous tubules epithelium; binocular microscope; micro digital camera; mouse testis. introduction binocular microscopes are commonly used to view microscopic objects at magnification of 4x, 10x, 40x, 100x objective lenses and of the 10x ocular lens, resulting in a maximum magnification of 1000x. the microscope can be used to measure the size of organ or cell. measurement of length or height of cells or organs can be done using micrometer slide. after the organ or cell image are taken, the measurement were done using micrometer photomicrograph at the same magnification. we can also measure the size of the cell or organ directly by using a camera in which there is a ruler menu that works to scale or measure cells or organs. there are two shortcuts in optilab, optilab viewer and raster image. inside the optilab viewer there are tools (buttons) for image snippets (photos), video recording (for recording in video form) and successive snippets (for sequential photos). for raster image shortcuts there are facilities for enumerators (counting by marking), scale (which can be dropped freely), there is also a marking facility that can be either arrow, circle, plus sign or box, there is also a ruler button that can be used to measure height, length or diameter of the cell or organ. knowing the height of epithelial tissue in the seminiferous tubules is important as seminiferous tubules can be used as indicators in the process of spermatogenesis. one of the important parameters in histology is the size of organ and cells, for example measuring the diameter of the seminiferous tubule and the height of mouse epithelium tubulus seminiferous. for microscope to be able to measure, it is required additional tools namely micro digital camera that have facilities to measure on a micron scale. therefore, a combination of binocular microscopy with a micro digital camera can be used to measure the height of the seminiferous tubule of epithelial cells. this paper will described the optimization of binocular microscope with micro digital camera for measuring seminiferous tubule epithelium height. materials and methods the equipment and materials used in this study are a set of pc or laptop, binocular microscope, miconos optical advanced digital micro camera, histological slide of testis. the research was conducted in biological laboratory, faculty of science & technology uin sunan kalijaga. this research began with assembling a computer or laptop with a binocular microscope and connecting a digital micro optilab advace camera. before taking measurements, optilab advande needs to be calibrated first, ie at the magnification of the objective lens 4x, 10x and 40x, so that the maximal magnification used is 400x. objective lens calibration process begins by calibrating the smallest magnification of 4x, by clicking the raster image then select the open button then select the micrometer image with 4x magnification, then click the calibration button, the objective lens is worth 4x then select the micrometer image again with 4x magnification then calibration mode 4x flashing, click and drag the micron and input http://dx.doi.org/10.14421/biomedich.2016.52.41-47 42 biology, medicine, & natural product chemistry 5 (2), 2016: 41-47 the measuring value then saved. take the same steps for 10x and 40x objective lens magnification. then measurements of epithelial tubular epithelial cells testis and recording could be done. of the many slide preparations, we selected three slide, in each slide selected three slices of testicular organ, in each slice of the testicular organ selected three seminiferous tubules, in each seminiferous tubule an epithelium cell measurement was measured three times. figure 1. a set of microscope and optilab advance. results and discussion appearance of testis before measurement: figure 2. mus musculus testis.10x10 magnification. this study measured the length or height of epithelium cells in the seminiferous tubules. prior to measurement the height of the tubular epithelium seminiferus testis, we select three intact seminiferous tubules. the corresponding cells are marked (figure 3). figure 3. marked tubulus seminiferous to be measured. selected tubulus seminiferuos were measured (figure 4-figure 9). figure 4. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: (a). 123,9 µm; 137,7 µm; 88,6 µm, (b). 81,6 µm; 84,8 µm; 89,2 µm. sutriyono – optimization of binocular microscope with micro digital camera for … 43 figure 5. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: (a). 91,9 µm; 134,1 µm; 127,3 µm, (b). 114,7 µm; 102,9 µm; 90,9 µm; (c). 117,7 µm; 106,4 µm; 137,2 µm, (d). 142,3 µm; 115,1 µm; 132,4 µm, (e). 97,6 µm; 86,1 µm; 98,3 µm, (f). 132,7 µm; 104,6 µm; 95,3 µm. 44 biology, medicine, & natural product chemistry 5 (2), 2016: 41-47 figure 6. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: (a). 119,6 µm; 144,8 µm; 101,1 µm, (b). 91,7 µm; 96,2 µm; 107,6 µm, (c). 87,0 µm; 111,9 µm; 150,8 µm, (d). 79,4 µm; 125,1 µm; 122,1 µm; (e). 78,5 µm; 106,2 µm; 93,3 µm, (f). 93,4 µm; 82,5 µm; 124,3 µm. sutriyono – optimization of binocular microscope with micro digital camera for … 45 figure 7. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: (a). 94,8 µm; 100,9 µm; 109,7 µm, (b). 132,0 µm; 106,3 µm; 75,6 µm, (c). 134,8 µm; 92,9 µm; 140,5 µm, (d). 78,4 µm; 104,2 µm; 93,2 µm, (e). 103,1 µm; 115,7 µm; 75,8 µm, (f). 159,1 µm; 71,6 µm; 106,3 µm. 46 biology, medicine, & natural product chemistry 5 (2), 2016: 41-47 figure 8. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: (a). 117,1 µm; 92,2 µm; 89,6 µm, (b). 97,3 µm; 99,4 µm; 113,4 µm, (c). 82,7 µm; 123,0 µm; 91,3 µm, (d). 78,1 µm; 132,5 µm; 81,8 µm, (e). 99,2 µm; 113,0 µm; 49,8 µm, (f). 154,1 µm; 68,2 µm; 120,5 µm. sutriyono – optimization of binocular microscope with micro digital camera for … 47 figure 9. measurement of the epithelium height on slide 1 tubulus 1 at 40x40 magnification resulting three values as follows: 86,3 µm; 139,3 µm; 77,3 µm. the results of this study were data of the height of tubular epithelium seminiferous testis of mice (mus muculus). from the measurement of eighty-one of measurement of the tubulus seminiferous epithelium, the average of the height of the tubulus seminiferous tubule epithelium was 105.6 μm. conclusion the optimization of binoculer microscopy with optilab advance can be done to measure diameter of the tubulus seminiferous. the height of epithelium of tubulus seminiferous in this study was 105.6 μm references alatlabmiconos.co.id retrieved at 05 march 2017 luiz r. franca and christiane l. godinho. 2003. testis morphometry, seminiferous epithelium cycle length, and daily sprem production in domestic cats (felis catus). biology or reproduction. federal university of minas gerais, brazil rizka arifianti. 2013. berat testis dan struktur histologi testis mencit (mus musculus l.) akibat paparan kebisingan. skipsi. universitas lampung. lampung. reza anindita dkk. 2009. diameter dan tebal lapisan tubulus seminiferus serta bobot testis mencit (mus muculus) setelah pemberian tauge kacang hijau (vigna radiata). laboratorium biologi. universitas diponegoro semarang. sutriyono. 2015. optimasi mikroskop binokuler dengan menggunakan optilab advance untuk mengukur diameter organ testis mencit (mus muculus). integrated lab journal. universitas islam negeri sunan kalijaga yogyakarta. tri suciati dkk. 2012. pengaruh likopen terhadap gambaran tubulus seminiferus dan kualitas sperma mencit (mus muculus) yang terpapar asap rokok. fakultas kedokteran universitas sriwijaya palembang. tri widjatmaka dan sonki prasetya, “perancangan dan pembuatan peralatan laboratorium pengkonversi gambar struktur mikro dari mikroskop ke komputer sebagai sarana praktikum metalografi”. jurnal politeknologi. 10 (3). tung yang wing and kent christensen. 1982. morphometric studies on rat seminferous tubules. the american journal of anatomy. the university of michigan. this page intentionally left blank optimization of binocular microscope with micro digital camera for measuring seminiferous tubules epithelium height biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 289-297 | doi: 10.14421/biomedich.2025.141.289-297 issn 2540-9328 (online) taxonomic study and bioprospecting indonesian termitomyces eurrhizus siti maulyda ayu1, wahyu aji mahardhika1, ivan permana putra2,*, oktan dwi nurhayat3 1program study of microbiology, department of biology, faculty of mathematic and nature science, ipb university, bogor 16680, indonesia. 2mycology division, department of biology, faculty of mathematic and nature science, ipb university, bogor 16680, indonesia. 3research center for applied microbiology, national research and innovation agency (brin), bogor, west java, 16914, indonesia. corresponding author* ivanpermanaputra@apps.ipb.ac.id manuscript received: 01 march, 2025. revision accepted: 20 may, 2025. published: 26 june, 2025. abstract termitomyces are known as wild edible mushrooms that are commonly consumed in africa and asia. t. eurrhizus can be easily found in many areas in indonesia. however, the recent collection and proper documentation of this species has not been done in indonesia. in addition, the potential utilization of this species needs to be revealed. this research aims to identify, investigate the proximate value, and test the antibacterial activity of t. eurrhizus. the exploration was carried out at the ipb university campus forest using an opportunistic sampling method. fresh basidiomata were used for morphological identification. proximate analysis was done following aoac method. mushroom extract was subjected to antibacterial test against enteropathogenic escherichia coli. our specimen was identified as t. eurrhizus based on pileus characters (macroscopically) and spore features under an electron microscope (microscopically). the proximate result showed that the moisture content was 13,70 %, the ash 13,66 %, total fat content 2,05 %, total protein content 27,73 % and fiber content 8,49 %. the investigated antibacterial was evaluated based on size of antibacterial zone for concentration 200 mg/ml extract (3,5 mm), 100 mg/ml (6,5 mm), 50 mg/ml (3,83 mm), 25 mg/ml (2,5 mm), and 12.5 mg/ml 6,33 mm. our result showed the potential utilization of t. eurrhizus as nutraceutical food and antibacterial. further research is expected to collaborate with various cultivation or product development fields. keywords: antibacterial activity; identification; proximate analysis, termitomyces eurrhizus. introduction sources of macronutrients for humans can be obtained from foods that contain carbohydrates, fats, proteins, and fiber. it is also important to balance these macronutrients with micronutrients found in foods that contain vitamins and minerals. incorporating edible mushrooms into the diet is an excellent option for promoting health. edible mushrooms have the potential to serve as both a source of essential nutrition and functional food (tharu et al., 2022). their taste and texture characteristics are similar to those of meat, which increases their appeal and versatility for processing (paloi et al., 2023). edible mushrooms have been popularly consumed in the past and continue to be chosen by many as part of their daily diet (mursito et al., 2009). currently, every year the demand for food mushrooms continues to increase. in this case, they can be used as a promising source of income (koné et al., 2013). there is a significant demand for mushrooms and estimates of future food supplies cannot meet the needs. therefore, the alternative is to use wild mushrooms. one of the edible wild mushrooms often found in indonesia is termitomyces. the genus termitomyces r. heim is a group of paleotropic fungi that can be harvested well (mossebo et al., 2017). termitomyces is classified in the family lyophyllaceae, has a unique symbiosis with termites, the fruit bodies of this fungus grow to penetrate termite nests to the ground surface (putra et al., 2020). enzymes produced by termitomyces allow termites to process plant structural components that are difficult to degrade such as cellulose or lignin (mossebo et al., 2017). this fungus prefers open places such as in the forest or grasslands/rice fields adjacent to the forest) without prominent termite advice (karun & sridhar, 2013). however, its use as food is still rare because there are only a few harvests per season, and no one cultivates it in indonesia. furthermore, augustinus and putra (2021) also point out that many people do not know that wild mushrooms can be consumed. this is due to a lack of education regarding the types of wild mushrooms that are safe to eat. in 1847, berkeley (1847) identified a type of fungi known as agaricus eurrhizus, which was later reassigned and renamed termitomyces eurrhizus by heim in 1940. the most recent classification of termitomyces can be https://doi.org/10.14421/biomedich.2025.141.289-297 mailto:ivanpermanaputra@apps.ipb.ac.id 290 biology, medicine, & natural product chemistry 14 (1), 2025: 289-297 found in the publication "termitomyces of asia" by pegler and vanhae (1994), which includes detailed comparisons with related species. this fungus is typically found in the paleotropical regions of africa, asia, and oceania (tharu et al., 2022), where it grows in scattered groups. the pileus of t. eurrhizus is characterized by a grayish-brown color, slightly slimy texture when dry, and distinctly slimy when wet (augustinus & putra, 2021). this organism has a unique ecology, often found in symbiosis with termite colonies and their nests. despite previous studies on spore characteristics, there is a lack of recent sem images from indonesia. this study seeks to analyze the morphology and microscopic features of the fungus t. eurrhizus, as well as evaluate its nutritional content and antibacterial properties as an edible mushroom. materials and methods study area t. eurrhizus was obtained at ipb university, dramaga, bogor, west java, in january february 2024. basidiomata were collected from soka field, cpwf+h43, soka street (fig. 1). the mushrooms obtained were preserved in 70% ethanol for herbarium. the fruiting bodies were documented in situ, ecological conditions were noted. figure 1. sampling collection at lapangan soka (coordinate point/red dote: 6.553770120925562, 106.7222765279091. morphological identification identification was carried out based on the morphological characteristics of fungi. macroscopic parameters recorded include growth method, shape of the fruit body, changes in wetness level, color of the cap when the fruit body is young and old, diameter of the cap, shape of the top and bottom of the cap, surface of the cap, edge of the cap, margin of the cap, and blade type. which includes how to stick to the stipe, length, distance between rows, and margins. other characters observed were the shape of the stipe, the color of the stipe (when young and old), the surface of the stipe, the position of attachment to the hood, the type of attachment of the stipe to the substrate, the cross-section of the stipe, partial veil and universal veil, texture of the fruit body, smell and taste (pegler & vanhae, 1994), (watling, 2013). the hymenium was analyzed using scanning electron microscopy (sem) following the methodology described by goldstein et al. (1992) at ilab, brin, bogor, indonesia. small hymenium layers (5 × 5 mm) were treated with glutaraldehyde and tannic acid before being dehydrated, infiltrated with t-butanol, and freeze-dried. the samples were then mounted on an aluminum stub, coated with gold, and examined using a jsm it 200 sem system (jeol, tokyo, japan). proximate analysis test components such as water, ash, fat, protein and fiber content were analyzed using the aoac (association of official analytical chemistry) method. fruiting bodies were dried using a dehydrator at a temperature of 500 c. a drying, a crispy texture is obtained, followed by grinding until it forms a powder. water content and ash content are determined using the gravimetric method (aoac). sample fat content was detected in the “soxhlet” device type extraction. the crude fiber content test uses the method, with alpha-amylase thermostable; protease and amyloglucosidase. determination of total protein content using the kjeldahl method. ayu et al. – indonesian termitomyces eurrhizus 291 fruiting bodies extraction the extraction was conducted over 3 successive days by blending the powder sample. a 10 g sample was blended at a ratio of 1:10 using 100 ml of 99% methanol solvent. subsequently, the samples were soaked in a shaker for 24 hours at 30°c (100 rpm). once the filtrate from the sample was collected, it was evaporated at 50°c until a paste-like sample was obtained, at which point the weight of the paste was measured. antibacterial activity test antibacterial activity was conducted to assess the inhibitory effect of the t. eurrhizus extract on the growth of epec (enteropathogenic escherichia coli) bacteria. the isolate used in the study was obtained from mr. aji, microbiology program at ipb university. e. coli was inoculated onto the agar using the spread method, with 20-100 ml of extract aseptically placed onto a disk with a diameter of 6-8 mm. the extract of t. eurrhizus was placed onto the disk, along with a combination of dmso 5% at five concentrations (12.5, 25, 50, 100, 200 mg/ml). a positive control using the antibiotic chloramphenicol and a negative control using dmso 5% were also included. the agar plates were then incubated under optimal conditions for the test microorganisms. the t. eurrhizus extract acted as an antibacterial agent, diffusing into the agar and inhibiting the growth of the microbial strains tested. antibacterial activity was analyzed through the results of inhibition zone measurements with the following equation: (𝑫𝑯−𝑫𝑪) + (𝑫𝑽− 𝑫𝑪) 𝟐 notes: dh : horizontal diameter dc : disc diameter (6mm) dv : vertical diameter data analysis the antibacterial activity test results were analyzed using the spss statistical software (statistical product and service solutions). an anova test (analysis of variance) with a significance level of α 0.05 was performed. if the results were significant, a further dmrt (duncan multiple range test) was carried out. the data is presented in table form along with descriptions of the results of the statistical analysis results. results and discussion taxonomy termitomyces eurrhizus (berk.) r. heim, archives du muséum national d'histoire naturelle 18: 140 (1942) synonym: termitomyces eurrhizus (berk.) r. heim, archives du muséum national d'histoire naturelle 18: 140 (1942) agaricus eurrhizus berk., london journal of botany 6: 483 (1847) rajapa eurhiza (berk.) singer (1945) armillaria eurhiza (berk.) sacc. (1887) collybia eurhiza (berk.) höhn. (1908) collybia eurrhiza (berk.) höhn., sitzungsberichte der kaiserlichen akademie der wissenschaften math.naturw. klasse abt. i 117: 992 (1908) rajapa eurrhiza (berk.) singer, lloydia 8: 143 (1945) armillaria eurrhiza (berk.) sacc., sylloge fungorum 5: 85 (1887) mastoleucomyces eurrhizus (berk.) kuntze, revisio generum plantarum 2: 861 (1891) termitomyces eurhizus (berk.) r. heim (1942) agaricus eurhiza berk. (1847) agaricus eurhizus berk. (1847) 292 biology, medicine, & natural product chemistry 14 (1), 2025: 289-297 figure 2. morphology termitomyces eurrhizus. a. pileus with pointed umbo. b-c. immature fruiting body. d. gills. e. stipe. f. pseudorhiza. figure 3. microscopic characters of lamella. a. trama (arrow). b. cystidia (arrow). c. basidia (arrow). d. spore. ayu et al. – indonesian termitomyces eurrhizus 293 figure 4. microscopic characters of stipe and pileipellis. a. hypahe on stipe (arrow). b. perforatorium oleiferous hyphae (arrow). c-d. perforatorium hyphae (arrow). figure 5. sem images of specimen. a. spores. b. side view of basidia. c. cystidia. basidiomata were found on the soil’s surface and litter, solitary, scattered. cap diameter 16 cm, the shape of the fruit body is a stem covered in lamellae, fleshy. when immature the cap is split at the edges (fig. 2c), with a pointed tip darker than the pile. surface of the cap is smooth and moist. the characteristics of a mature cap are blooming and breaking easily (fig. 2a), and the edges are regular, easier to color than the surface of the cap. lamellae are white to cream colored, up to 1 mm wide, classified as free attachment lamellae to adherent, densely packed. stipe 11,5 cm, cylindrical, at the apex it is white and becoming brownish below, fibrillose-striate. pseudorhiza is 3 cm long, 1,5 cm in diameter, fusoid above, with a cartilaginous, dark crust. fresh fruiting this body wild edible mushroom has a savory taste, and unique flavor like dried fish. spore of t. eurrhizus 5.8-6 x 3.8-4 µm. the shape of spores is ellipsoid (fig. 4 c), subhyaline, with a thin to slightly thickened wall, and refractive contents. basidia 25-30 µm x 9-10 µm, clavate, bearing four sterigmata. lamella-edge sterile, with crowded cheilocystidia. cheilocystidia 30-36 µm x 5-9 µm, obovoid to pyriform, thin walled, with few contents. pleurocystidia 70-76 µm x 21-23 µm, resembling cheilocystidia in shape. types of trama were irregular (fig. 4 a) and (fig. 6 c). pileipellis with oleiferous hyphae. t. eurrhizus is taxonomically an edible mushroom originating from the division basidiomycota, class agaromycetes, order agaricales, family lyophyllaceae, and genus termitomyces. morphologically, this 294 biology, medicine, & natural product chemistry 14 (1), 2025: 289-297 mushroom is similar to the species t. umkowaan, differing only in the size and shape of its basidiospores. t. eurrhizus has larger basidiospores compared to t. umkowaan and has clavate to pyriform-shaped cystidia (tibuhwa et al., 1998). additionally, other references state that t. eurrhizus has a sharper umbo compared to the blunter umbo of t. umkowaan (purkayastha & chandra, 1975). based on the obtained morphological data, the description of the fruiting body matches pegler (1994), which includes t. eurrhizus having a pseudorhiza with a blackish crust, large basidiomata that are grayish to brown, and rarely with blackish spots. the pileus size is larger compared to the identification key. no annulus was found on the stipe, presumably because the mushroom was no longer in its early stage. recent reports show t. eurrhizus found in yunnan province, china, and nakhon si thammarat province, thailand, described as having a greyish-brown pileus color (tang et al., 2020). the same type of fungus is also found in sheikhupura, punjab, pakistan, with a broadly umbonate perforatorium (izhar et al., 2020), and in malaysia with the same description (sathiya seelan, 2020). augustinus & putra (2021) documented a variety of termitomyces in indonesia, especially in the urban area of gunung pati subdistrict, semarang city, central java. one of their specimen is t. eurrhizus, which has a long pseudorhiza than this research. in indonesia, precisely in the mount tukung gede area, t.eurrhizus is also commonly known as mushrooms "moon supa" and "horn mushrooms" (khastini et al., 2022). the discovery of exploration results in lore lindu national park, central sulawesi also explains t. eurrhizus substrat decomposing litter and soil (yusran et al., 2021). another description is also described by ye et al. (2019) the same type of mushroom is found in forests in xishuangbanna, yunna, china with soil substrate, tree leaf blades known as "big jizong" or "chicken mushroom". proximate analysis the proximate value describes the composition contained in the fruit bodies of the fungus. calculating the total content of water, ash, fat, protein and fiber can be used as a reference in post-harvest handling. in addition, the proximate value also provides reasons for choosing what type of method will be used to maintain post-harvest mushroom quality. the percentage of dried fruiting bodies of the t. eurrhizus contains as much water content as 13.70 %/ 100 g. like other fleshy mushrooms, this water content is relatively high, because the storage of fresh fruiting bodies after harvest does not last long. water content data in previous research shows a higher figure than the water content results in table 1, as much as 92.08% / 100g (gunasekara et al., 2021). it is recommended that earlier processing or drying treatment or packaging of termitomyces products be carried out if they are to be used longer. table 1. proximate composition of termitomyces eurrhizus. proximate analyses composition (%) water content 13,70 ash content 13,66 fat content 2,05 protein content 27,73 fiber content 8,49 carbohydrate 34.37 the percentage of ash content from the mushroom is 13.66, this ash depends on the characteristics of the sample. ash content is useful for distinguishing the type of purity of a product, by looking at acid insoluble ash, for example in fruit vinegar products and synthetic vinegar. other components such as minerals in the sample also affect the shelf life of t. eurrhizus fruit bodies. in the previous study, the amount of ash content was higher with the amount of 11.52% / 100 g dw, the mineral content was also included with the amount of calcium 100, iron 50, magnesium 160, and potassium 2360 mg / 100 g dw (gunasekara et al., 2021). in this case, these minerals can be greater than the total ash content obtained in this study. the total fat content of t. eurrhizus is 2.5 / 100g (table 1), which is lower when compared to the results obtained by gunasekara (2021), as much as 6.27% / 100g dw. the study also states that unsaturated fat (ufa) is higher than total saturated fat (sfa) in t. eurrhizus, as ufa is found to play an important role in the physiology of the human body. as in molecular observations there is an oleiferous perforatorium (fig.3) which can produce oil. the highest percentage of proximate content is 27.73% of total the protein content. this amount is close to the protein content of t. eurrhizus found in sri lanka as much as 29.40% (gunasekara et al., 2021). purkayastha and chandra (1975) explain that the amino acids of the fungus t. eurrhizus are histidine and arganine. in detail, the protein content (table 1) contains essential amino acid (eaa) types of glutamate, aspartate, and leucine (gunasekara et al., 2021). the elaboration of this protein content can explain the umami taste in mushrooms. by comparing the fiber of wild mushroom fiber, it has a low fiber percentage of 8.49%, while gunasekara (2021) has 26.64%. srikram and supapvanich (2016) showed that the content of wild mushrooms is higher than that of cultivated mushrooms. mushrooms have both soluble and insoluble fiber, there are about 50-65% carbohydrates based on dry mass which includes monosaccharide sugars, derivatives and oligosaccharides (santhosh et al., 2023). zhao (2023) the type of fiber in the mushroom fruit body, namely polysaccharides, has a complex structure so that digestion has a good effect on health. type of polysaccharide d-glucose with fraction water-soluble polysaccharide (ps i and ps ii) (paloi et ayu et al. – indonesian termitomyces eurrhizus 295 al., 2023), the fiber content in this mushroom is suitable for consumption by dieters (alim et al., 2023). according to the results above, nutrients in these reports are by no means toxic to humans. growing wild does not mean this mushroom is poisonous, based on its genus, it is still safe to consume (agustinus & putra, 2021). for a long time, this species has been one of the foods favored by people of east and central africa (koné et al., 2013). the genus termitomyces is primarily consumed by indian, chinese, lao, and nepales people and is also consumed in several countries such as malaysia and thailand (paloi et al., 2023). nutritional in table 1. represent potentially food which has relatively high protein and fiber content. the content can be an alternative for vegan groups, but the yield of one harvest is only in small quantities so it is rarely traded (koné et al., 2013). antibacterial activity the results of the antibacterial activity test of methanol extract of termitomyces eurrhizus showed positive results inhibiting the organization of gram-negative bacteria e. coli (table 2). table 2. antibacterial activities from termitomyces eurrhizus crude extract to enteropatogenic escherichia coli. concentration inhibiton zone (mm) chloramphenicol (30µg/disc) 26,66 ± 0,763c 12.5 mg/ml (20 µg/disc) 6,33 ± 2,020b 25 mg/ml (20 µg/disc) 2,50 ± 0,500ab 50 mg/ml (20 µg/disc) 3,83 ± 2,843ab 100 mg/ml (20 µg/disc) 6,50 ± 5,678b 200 mg/ml (20 µg/disc) 3,50 ± 0,866ab dmso (20 µg/disc) 0,00 ± 0,0000a data represented as mean ± sd with triplicate. the number followed by a different superscript letter in the same column indicates a significant difference in level (p < 0.05) based on duncan's multiple range tests (dmrt) 5%. epec: enteropathogenic escherichia coli. figure 6. antibacterial activity of termitomyces eurrhizus crude extract against enteropathogenic escherichia coli at five concentrations (12.5, 25, 50, 100, 200 mg/ml). a. chloramphenicol. b. replication 1 (5 concentration). c. replication 2 (5 concentration). d. replication 3 (5 concentration). e. negative control. the inhibitory zone formed has different values for each type of concentration. the highest inhibitory zone value was found at a concentration of 100 mg / ml with an average diameter of 6.5 ± 5.67 mm, while the smallest inhibitory zone was formed in an extract concentration of 25 mg / ml with an average zone diameter of 2.5 ± 0.5000 mm. the difference in the inhibitory zone formed shows a difference in sensitivity to e. coli bacteria. this sensitivity was evident in the results of a positive control (chloramphenicol) that had a very high inhibitory zone (fig 7a). in addition to e. coli, antibacterial activity testing has been carried out on bacteria staphylococcus aureus, b. cereus, b. subtilis, proteus vulgaris but received negative results (giri et al., 2012). e. coli can live in facultative anaerobic conditions, commonly found in warm-blooded animals in the lower intestine. in addition, its role in the digestive tract under normal conditions can be constructive, but when it reaches abnormal levels, it can be parasitic (abdul hussein et al., 2023). these properties underlie its use in this study, in vitro the content of secondary metabolites of extract. based on kumar and sagar (2019), the fungus t. eurrhizus has antibacterial activity against l. monocytogenes, e. coli, s. aureus, and p. aeruginosa with a fairly strong inhibition zone. differences in activity may be influenced by variations in the strains of both the test bacteria and t. eurrhizus itself. extract of t.eurrhizus has secondary metabolites such as tannins, anthocyanins, saponins, flavonoids, steroids and alkaloids. this is because the strong antioxidant content has the potential to inhibit reactive oxygen species (ros) so that exposure to free radicals can be avoided (tharu et al., 2022). these secondary metabolites’ content significantly impacts various biological activities (paloi et al., 2023). the fungus termitomyces contains polysaccharides, which have additional benefits such as antioxidant, hypolipidemic, and hepatoprotective activities, as well as antineoplastic, ulcer health and analgesic, antidiabetic, and immunomodulatory effects (liu et al., 2021). the fungus t. eurrhizus has properties as an ethnomedicine, including treating fever and measles outbreaks (paloi et al., 2013), treating chickenpox (manna et al., 2014), managing rheumatism, diarrhea, and lowering high blood pressure (sachan et al., 2013). when mixed with cynodon dactylon, it can treat skin diseases (aryal & budathoki, 2016). for sustainability,there is a need to cultivate this fungus as an additional variety of nutritional sources. this research can be the basis for the community in utilizing t. eurrhizus mushrooms for functional food and possibly breakthrough drugs. it is expected to form collaborations with various fields in cultivation for the development of t. eurrhizus-based products. 296 biology, medicine, & natural product chemistry 14 (1), 2025: 289-297 conclusion in conclusion, our study successfully identified termitomyces eurrhizus in indonesia, providing valuable documentation of this species. the proximate analysis revealed significant nutritional content, highlighting its potential as a nutraceutical food source. additionally, the antibacterial activity demonstrated by t. eurrhizus against enteropathogenic escherichia coli suggests its potential application in antibacterial treatments. these findings underscore the importance of further research and collaboration across various fields to explore the cultivation and product development of t. eurrhizus, maximizing its utilization and benefits. acknowledgements: we are grateful to the department of biology, faculty of mathematics and natural sciences, ipb university, and research center for applied microbiology, national research and innovation agency (brin), indonesia for the research facilities. authors’ contributions: ipp and odn designed the study. sma and wam performed macroscopic analyses. odn conducted the sem observation. sma performed the proximate analyses. wam performed the antibacterial assay. sma 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(2023). the interaction between mushroom polysaccharides and gut microbiota and their effect on human health: a review. biology (basel). 12(1):1– 29.doi:10.3390/biology120 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 43-50 | doi: 10.14421/biomedich.2025.141.43-50 issn 2540-9328 (online) correlation of bmp2 with degree of osteitisin primary crs with and without nasal polyps edi handoko1, david santoso2, iriana maharani3,*, yuyun yueniwati p.w4, tanti agustina5 1,2,3department of otorhinolaryngology-head and neck surgery, faculty of medicine; 4radiology department, faculty of medicine, universitas brawijaya/dr. saiful anwar general hospital, malang, indonesia. 5biology department, faculty of biology, universitas gadjah mada, sleman, yogyakarta, indonesia. corresponding author* iriana_maharani@ub.ac.id manuscript received: 07 november, 2024. revision accepted: 17 february, 2025. published: 28 february, 2025. abstract chronic rhinosinusitis (crs) is an inflammation of the nasal mucosa and paranasal sinuses, persisting for over 12 weeks. crs involves a remodeling process and opacification known as osteitis, resulting from type 2 and non-type two inflammation. osteitis severity is assessed using ct scans and the global osteitis scoring scale (goss). bone morphogenetic protein-2 (bmp-2) is crucial in osteogenesis and bone remodeling. however, no research has shown a relationship between bmp-2 concentration and osteitis degree, as measured by goss, in crs patients with or without nasal polyps. the objective of this study is to analyze the relationship between bmp-2 concentration and osteitis degree measure with goss in primary crs. a cross-sectional analytic observational study was conducted using primary rsk as the study population. bmp-2 concentration, a marker for bone remodeling, used elisa. the osteitis of the paranasal sinus walls was assessed with a ct scan and quantified using the goss method. the study included 44 patients, with 8 of them serving as the control group. it was found that bmp-2 concentration (ng/ml) had a linear relationship with the goss degree in patients with crs, showing a correlation of 0.583 (deviation >0.05). further analysis revealed a significant correlation between bmp-2 concentration and goss, with a coefficient of 0.857 (considered very significant, in the range of 0.76-0.99). this strong correlation indicates that as bmp-2 concentration strongly correlates with the occurance of osteitis, which were measured by goss. bmp-2, as a marker for osteitis in crs, shows a significant and linear correlation with the severity degree of osteitis measured by goss. keywords: bmp-2 concentration; chronic rhinosinusitis; global osteitis scoring scale; osteitis. introduction the paranasal sinuses are air-filled cavities located within certain bones of the skull. each side of the skull contains four paranasal sinuses: the frontal, maxillary, ethmoid, and sphenoid sinuses. inflammation of the nasal mucosa and paranasal sinuses is known as rhinosinusitis. when symptoms persist for more than 12 weeks without complete resolution, the condition is classified as chronic rhinosinusitis (crs) (dhingra, 2021; brandi, 2019). rhinosinusitis is a significant health issue worldwide. in the united states, crs is one of the most common reasons patients seek medical attention. around 0.5% of acute respiratory infections (aris) caused by viruses in the u.s. progress to rhinosinusitis. crs impacts around 31 million individuals in the united states, constituting about 5% to 12% of the population, making it a significant global health issue (amanda s. battisti et al., 2020; russell a. faust. 2010. according to the european position paper on rhinosinusitis and nasal polyps (epos) 2012, the prevalence of crs varies globally: 5.5% in brazil, 8% in china, 11% in europe and korea, and 12% in the u.s (fokkens et al., 2012). data from the indonesian ministry of health (depkes ri) in 2003 showed that nasal and sinus diseases ranked 25th among the top 50 diseases in indonesia, with 102,817 outpatient cases. at dr. saiful anwar hospital, annual reports from the ent clinic in 2021 indicated that crs cases accounted for 29% of all rhinology patients. some patients with crs also exhibit inflammatory hyperplasia in the nasal cavity. research in korea found that the prevalence of crs with nasal polyps is 2.7% among adults, with an incidence of 0.63%-0.83% in the general population (soepardi & iskandar, 2001). epos 2020 categorizes crs into primary and secondary types. primary crs is further divided based on location (unilateral or bilateral) and inflammation type (type 2 or non-type 2) (fokkens et al., 2020). crs involves remodeling and thickening of the paranasal sinus bones, a condition known as osteitis. this occurs due to type 2 and non-type 2 inflammatory reactions in the sinonasal mucosa. osteitis features bone inflammation, neo-osteogenesis, and remodeling, involving osteoclasts in bone degradation and osteoblasts in bone replacement. although the exact mechanism of https://doi.org/10.14421/biomedich.2025.141.43-50 mailto:iriana_maharani@ub.ac.id 44 biology, medicine, & natural product chemistry 14 (1), 2025: 43-50 osteitis in crs is not fully understood, persistent mucosal inflammation is believed to cause bone cell death, leading to neo-osteogenesis and bone thickening. chronic inflammation, especially type 2, is also linked to nasal polyposis. bone thickening in crs with osteitis is associated with the runt-related transcription factor 2 (runx2), crucial for osteoblast induction, proliferation, and maturation (khalmuratova et al., 2019). crs patients with osteitis generally have a worse prognosis and a higher likelihood of postoperative recurrence compared to those without osteitis (khalmuratova et al., 2019). the degree of osteitis in crs can be assessed through imaging and histopathology. imaging is more commonly used in clinical settings due to its non-invasive nature. computed tomography (ct) scans are frequently employed to evaluate osteitis in crs (khalmuratova et al., 2019). ct scan results are used to grade osteitis based on various scoring criteria, including the kennedy osteitis score (kos), global osteitis scoring scale (goss), and modified goss. goss is the most commonly used as it provides a detailed assessment of the involvement and thickness of the walls of 10 sinuses (leung et al., 2016). bone morphogenetic protein 2 (bmp-2) is a multifunctional growth factor in the transforming growth factor-beta (tgf-β) superfamily. bmp-2 is vital for bone formation and remodeling through the regulation of osteoblasts and osteoclasts (kim et al., 2021). it can also activate runx2 via the smad1 pathway. when bmp-2 docks with its receptor, it phosphorylates smad1, which then activates runx2, playing a crucial role in osteoblast induction, proliferation, and maturation (cho & kwun, 2018). currently, no studies have directly examined the relationship between bmp-2 levels and the degree of osteitis measured using goss in crs patients with and without nasal polyps. given the relationship between bmp-2 and runx2 and the role of runx2 in osteitis in crs, this study aims to investigate the correlation between bmp-2 levels and the degree of osteitis measured using goss in crs patients with and without nasal polyps. understanding the relationship between bmp-2 levels and goss could lead to the development of targeted therapies for osteitis in chronic rhinosinusitis. this insight might also help prevent the recurrence of chronic rhinosinusitis after surgery. patients and methods this cross-sectional study was carried out between may 1, 2022, and november 30, 2022, across various departments at dr. saiful anwar general hospital, including the local and central operating rooms of the otorhinolaryngology head and neck surgery department, the radiology department, and the biomedical laboratory of the faculty of medicine at universitas brawijaya, malang, indonesia. the study received approval from the medical ethics committee of dr. saiful anwar general hospital (no. 400/217/k.3/102.7/2022). the study population consisted of all patients from the rhinology division who were admitted to the medical ward of dr. saiful anwar general hospital during the study period. the inclusion criteria for this study are adult crs patients over 18 years old according to clinical criteria from the european position paper on rhinosinusitis and nasal polyps (epos) 2020, crs patients who have never undergone functional endoscopic sinus surgery (fess) before, and who have not used corticosteroids or antibiotics within 4 weeks prior to specimen collection. control patients over 18 years old diagnosed with a rhinology-related condition other than chronic rhinosinusitis, with or without polyps, who require surgical intervention. exclusion criteria for this study are crs patients involving only the posterior paranasal sinuses, patients who do not consent to participate in the study, and control patients under 18 years old, those with allergic rhinitis, or those who have used corticosteroids or antibiotics within 4 weeks before specimen collection. age, sex, main symptoms (nasal blockage/obstruction/congestion, nasal discharge, facial pain/pressure, reduction/loss of smell, patient complaints duration, biopsy of the uncinate process tissue were obtained. the uncinate process tissues were examined using enzyme-linked immunosorbent assay (elisa) according to the goss method to determine the bmp-2 levels. the osteitis of the paranasal sinus walls was assessed with a ct scan and quantified using the goss method. ct scan of the paranasal sinuses was performed without contrast, using bone setting, with axial, coronal, and parasagittal slices, with a thickness of 1-3 mm per slice, each component and the total goss were calculated using the horos 3.3.6 software. data were analyzed using the spearman correlation test with a 95% confidence interval (ci) and an alpha level (α) of 0.05. results are considered significant if p<0.05. this analysis uses statistical package for the social sciences (spss) 25.0.0 for windows software. results and discussion result a total of 33 patients with primary crs and eight subjects as control were included in this study, with the age ranging from 18 to 68 years old and a mean age of 38,36 ± 15,272 years. there were 12 males and 21 females. the main symptoms observed among the patients were nasal blockage, nasal discharge, headache, facial pain, periorbital edema, and anosmia at 54.5%, 24.2%, 12.1%, 6.1%, 3%, and 0%. the complaints ranged from 12 to 416 weeks, with a mean duration of handoko et al. – correlation of bmp2 with degree of osteitisin primary crs … 45 91.21 ± 109.99 weeks. bilateral paranasal sinus involvement was observed in 22 samples (66.67%), while unilateral involvement was observed in 11 samples (33.33%). crs without polyps was more prevalent, occurring in 17 samples (51.52%), compared to crs with polyps. the higher incidence of crs in females can be attributed to anatomical and hormonal differences. females typically have smaller sinus ostia than males, which may impair sinus drainage and contribute to crs development. smaller ostia increase the likelihood of obstruction and bacterial colonization in the paranasal sinuses. additionally, females have significantly higher levels of estrogen compared to males, which may further influence the prevalence and pathophysiology of crs. ct scan of the paranasal sinuses was performed using a bone setting view, with axial, coronal, and parasagittal slices of 1-3 mm thickness, without contrast (figure 1). osteitis assessment was conducted on 10 sinuses: left and right maxillary, left and right anterior ethmoid, left and right posterior ethmoid, left and right sphenoid, and left and right frontal sinuses, using the global osteitis scoring scale (goss) on axial, coronal, and parasagittal slices. the lowest total goss score was 0, and the highest total score was 50. a higher goss score indicates more severe osteitis. goss scores were then categorized into four degrees: no osteitis (goss score <5), mild osteitis (goss score 5-20), moderate osteitis (goss score 20-35), and severe osteitis (goss score >35). osteitis on ct scan was characterized by loss of bone definition, hyperostosis, new bone formation, or signs of heterogeneity in the walls of the paranasal sinuses. figure 1. ct scan image of osteitis in various sections. the appearance of osteitis (loss of bone definition, hyperostosis, new bone formation, or signs of heterogeneity) on ct scan samples from crs patients with osteitis is indicated by white arrows. a-b axial sections of the maxillary sinus: a goss 0/0. b goss 5/5. c-d coronal sections of the maxillary sinus. in evaluating 330 paranasal sinuses, osteitis was detected in 168 cases (50.9%). the anterior ethmoid sinuses were the most frequently affected, accounting for 50 cases (29.8%), while the frontal sinuses exhibited the lowest incidence, with 17 cases (10.1%). the efficacy of osteitis detection was influenced by the type of ct scan slice. axial slices proved to be the most effective, identifying osteitis in 80% of the cases involving the maxillary sinuses, corresponding to 36 pairs. conversely, coronal slices demonstrated lower accuracy for osteitis detection in the paranasal sinuses, excep the sphenoid sinuses, where 18 cases (10.7%) were accurately detected using coronal slices (figure 2). b c d a 46 biology, medicine, & natural product chemistry 14 (1), 2025: 43-50 36 48 32 5 8 6 2 1 18 7 3 2 0 10 20 30 40 50 60 maksilaris d/s ethmoidalis anterior d/s ethmoidalis posterior d/s sphenoidalis d/s frontalis d/s osteitis detected on ct scan sections (n=168) aksial koronal parasagital figure 2. distribution of osteitis detected in ct scan slices of crs patients. blue bars denote the prevalence of osteitis in the paranasal sinuses detected on axial ct scan slices. orange bars represent coronal slices, while gray bars correspond to parasagittal slices. figure 3 illustrates the distribution of osteitis severity within the rsk sample, indicating a predominance of mild osteitis cases (51.52%). among these mild osteitis cases, seven patients had nasal polyps, while eight patients did not. for moderate osteitis (30.30%), nine patients were diagnosed with nasal polyps, and two patients were without nasal polyps. in cases of severe osteitis (3.03%), all patients had nasal polyps. additionally, 15.15% of the rsk sample had no osteitis. figure 3. distribution of osteitis severity in crs patients as measured with goss. in this study, a total of 17 chronic rhinosinusitis samples with polyps and 16 samples without polyps were analyzed. the mean concentration of bmp2 in chronic rhinosinusitis samples with polyps was 3.78, with a standard deviation of 5.43. in contrast, the mean bmp2 concentration in samples without polyps was 1.30, with a standard deviation 5.43 (table 1). this indicates that bmp2 levels are higher in chronic rhinosinusitis with polyps than those without. comparison analysis using the mann-whitney test, with significance set at p<0.05, yielded a p-value of 0.037 for bmp2 concentrations between chronic rhinosinusitis with and without polyps. this result indicates a significant difference, demonstrating an 0 1 2 3 4 5 6 7 8 9 10 no osteitis mild degree of osteitis moderate degree of osteitis severe degree of osteitis osteitis severity and nasal polyps polyp non-polyp handoko et al. – correlation of bmp2 with degree of osteitisin primary crs … 47 association between elevated bmp2 levels and chronic rhinosinusitis, whether with or without polyps. however, the higher mean bmp2 concentration in samples with polyps suggests that this significant value reflects a more pronounced increase in bmp2 levels in chronic rhinosinusitis with polyps compared to those without. table 1. comparative analysis of bmp2 levels in crswnp and crssnp. bmp-2 levels crswnp/crssnp mean n std. deviation polyp 3.78890152 17 5.439027531 non polyp 1.30166566 16 3.658008024 total 2.58296898 33 4.759970211 thirty-three samples meeting the inclusion criteria were assessed for osteitis severity using the global osteitis scoring scale (goss). the samples were divided into two groups based on the presence of nasal polyps: 17 samples with chronic rhinosinusitis with polyps and 16 samples with chronic rhinosinusitis without polyps. the mean goss score for chronic rhinosinusitis with polyps was 21.23, with a standard deviation 9.46. in contrast, the mean goss score for chronic rhinosinusitis without polyps was 10.56, with a standard deviation 7.75 (table 2). comparative analysis between chronic rhinosinusitis with and without polyps, based on goss scores, revealed a significant difference with a p-value of 0.002 (p < 0.05). this indicates that the presence of nasal polyps in chronic rhinosinusitis is associated with a higher goss score compared to cases without polyps. table 2. comparison of goss scores in crswnp and crssnp. goss crswnp/crssnp mean n std. deviation polyp 21.2353 17 9.46394 non polyp 10.5625 16 7.75430 total 16.0606 33 10.11478 disscussion a total of 44 patients with chronic rhinosinusitis (crs) who presented at the otolaryngology rhinology clinic at rssa were subjected to anamnesis and physical examination. among these, 33 patients met the inclusion and exclusion criteria, and 8 patients were included as controls. analysis of the sample's gender distribution revealed a predominance of females, with 21 (63.6%) female patients and 12 (36.4%) male patients. this finding aligns with prior research, which also identified a predominance of females among crs patients, accounting for 63% of the 834 samples examined (de et al., 2019). this gender disparity is corroborated by data from the national health interview survey (nhis) in the united states, which reported that 63% of crs patients were female. similarly, a 2021 study conducted in indonesia by poluan and marlina found that 73.2% of crs patients were female, compared to 19% male patients. epidemiological studies further indicate that crs is nearly twice as prevalent in females as males. the higher incidence of crs in females can be attributed to anatomical and hormonal differences. females typically have smaller sinus ostia as males, which may impair sinus drainage and contribute to crs development. smaller ostia increase the likelihood of obstruction and bacterial colonization in the paranasal sinuses. additionally, females have significantly higher levels of estrogen compared to males, which may further influence the prevalence and pathophysiology of crs (hulse et al., 2014; ference et al., 2015). the sample population's age range varied from 18 to 68 years, with a mean age of 38.36 ± 15.27 years and a median age of 32 years. a prior study conducted in the united states demonstrated an increase in the prevalence of chronic rhinosinusitis (crs) with advancing age, showing a 2.7% increase among individuals aged 20–29 years and a 6.6% increase among those aged 50–59 years. however, the prevalence decreased by 4.7% after the age of 60. data from dr. m. djamil general hospital in padang, collected between january 1 and december 31, 2012, indicated that crs was most prevalent among the young and adult age groups, accounting for 61.90% of cases. similarly, research by the rhinology department of ent-kl at fkui rscm reported the highest crs prevalence in adults aged 18-75. the elevated incidence of crs in adults is likely related to higher levels of social activity outside the home, which increases exposure to bacteria, viruses, and atmospheric 48 biology, medicine, & natural product chemistry 14 (1), 2025: 43-50 pollutants-environmental factors that play a significant role in the pathogenesis of crs (fokkens et al., 2012). in this study, a correlation coefficient of 0.857 was observed, indicating a very strong positive association between bmp-2 concentration levels and goss. this positive coefficient suggests a direct relationship, where an increase in bmp-2 concentration is associated with a corresponding increase in goss levels. this finding aligns with earlier research by kim et al., which showed a connection between bmp-2 levels and radiological findings. in their study, bmp-2 levels were measured using immunohistochemical staining, and the results demonstrated a significant correlation between bmp-2 and goss values (p = 0.003, r = 0.332). because osteitis is frequently linked to the persistence of chronic rhinosinusitis (crs), the study also explored this aspect and found that bmp-2 levels were associated with the refractoriness of the disease (kim et al., 2021). in this study, 33 samples that met the inclusion criteria were assessed for the degree of osteitis using goss. the samples were divided into two groups crswnp and crssnp. of the total, 17 samples were classified as crswnp, and 16 were classified as crssnp. a comparative analysis was performed to evaluate the differences in goss scores between the two groups, revealing a statistically significant difference (p = 0.002). these findings support the hypothesis that the presence of nasal polyps in crs cases is associated with an increased severity of osteitis. this may be attributed to the fact that both osteitis and crswnp share a similar inflammatory profile, specifically type 2 inflammation, characterized by a predominance of eosinophils in the chronic inflammatory process. this suggests that when managing patients with crs, careful attention should be paid to the presence of nasal polyps, as this can significantly impact the severity of osteitis and provide insight into the underlying inflammatory burden. recognizing the presence of nasal polyps in crs patients is crucial in clinical practice. this awareness not only aids in accurately assessing the severity of osteitis using the goss score but also serves as an important indicator of the patient's overall inflammatory status. by considering these factors, clinicians can better tailor their therapeutic approaches to address the specific inflammatory mechanisms at play, potentially improving patient outcomes (mehta et al., 2008). the relationship between bmp-2 and osteitis begins with bmp-2 binding to its receptor, bmpr-2 monomer. this binding induces the dimerization of bmpr-2, which subsequently recruits bmpr-1 to transmit signals to molecules that regulate intracellular signaling. these molecules are smad 1/5/8. the bmp-2 complex with bmpr-2 and bmpr-1 phosphorylates smad 1/5/8. phosphorylated smad 1/5/8 then binds to smad-4. this binding causes the phosphorylated smad 1/5/8 complex with smad-4 to enter the cell nucleus and activate runx2. the activation of runx2 induces osteocalcin (ocn), osteopontin (opn), alkaline phosphatase (alp), osteoprotegerin (opg), matrix metallopeptidase 13 (mmp-13), and rankl, which are involved in bone differentiation, formation, and turnover. runx2 is also known to induce the transcription factor sp7, or osterix (sp7/osx), which functions by activating ocn, opn, bone sialoprotein, and type 1 collagen (col1). these four proteins play a role in bone formation. yes-associated protein (yap) and transcriptional coactivator with pdz-binding motif (taz) are also induced by the phosphorylated smad 1/5/8 complex with smad-4. yap/taz then enter the cell nucleus and bind to transcriptional enhancer activator domains (teads), which leads to the activation of wnt family member 5a and 5b (wnt5a/b), dickkopf-related protein 1 (dkk-1), insulin-like growth factor binding protein-4 (igfbp-4), and bmp-4. the activation of these proteins impacts the regulation between osteogenesis and osteoclastogenesis (cho & kwun, 2018; paulini et al., 2021). the relationship between bmp-2 and osteitis begins with bmp-2 binding to its receptor, the bmpr-2 monomer. this binding induces the dimerization of bmpr-2, which subsequently recruits bmpr-1, a receptor that transmits signals to intracellular signaling molecules. these molecules include smad 1/5/8. the bmp-2 complex with bmpr-2 and bmpr-1 phosphorylates smad 1/5/8. phosphorylated smad 1/5/8 then binds to smad-4. this binding causes the phosphorylated smad 1/5/8 complex with smad-4 to enter the cell nucleus, activating runx2. the activation of runx2 induces the expression of osteocalcin (ocn), osteopontin (opn), alkaline phosphatase (alp), osteoprotegerin (opg), matrix metallopeptidase 13 (mmp-13), and rankl, which are involved in bone differentiation, formation, and turnover. runx2 itself can also induce the transcription factor sp7, also known as osterix (sp7/osx), which functions by activating ocn, opn, bone sialoprotein, and type i collagen (col1). these four proteins play critical roles in bone formation. yes-associated protein (yap) and transcriptional co-activator with pdz-binding motif (taz) are also induced by the phosphorylated smad 1/5/8 complex with smad-4. yap/taz then enter the cell nucleus and bind to transcriptional enhancer activator domains (teads), leading to the activation of wnt family members 5a and 5b (wnt5a/b), dickkopfrelated protein 1 (dkk-1), insulin-like growth factorbinding protein 4 (igfbp-4), and bmp-4. the activation of these four proteins has implications for the regulation of osteogenesis and osteoclastogenesis (cho & kwun, 2018; zhang et al., 2017). in addition to the previously mentioned pathway, osteitis in crs induced by bmp-2 can also be explained through the runx2 pathway. runx2 is a transcription factor that plays a crucial role in osteoblasts’ induction, proliferation, and maturation. a study by khalmuratova et al. demonstrated that the expression of runx2 is handoko et al. – correlation of bmp2 with degree of osteitisin primary crs … 49 elevated in osteoblasts of crs patients with neoosteogenesis. moreover, runx2 can be induced by nasal tissue extracts from crs patients exhibiting neoosteogenesis. this increased expression of runx2 leads to the differentiation of osteoblasts, promoting neoosteogenesis, a component of osteitis in crs patients. the expression of runx2 is also correlated with higher global osteitis scoring scale (goss) scores, which are used to measure the extent of osteitis in crs (khalmuratova, 2019). the runx2 pathway also promotes the expression of rankl, a protein involved in bone formation, turnover, and differentiation. rankl, a member of the tumor necrosis factor (tnf) family, plays a crucial role in the differentiation, survival, and activity of osteoclasts. rankl binds to its specific receptor, rank, on osteoclast progenitor cells, leading to the activation of osteoclasts and subsequent bone resorption. rankl operates in opposition to osteoprotegerin (opg), a soluble decoy receptor that binds to rank. both rankl and opg are induced by runx2, which is activated by bmp-2. mature osteoblasts produce opg, which competitively binds to rank, thereby inhibiting bone resorption and allowing bone remodeling to occur. rankl expression is elevated in patients with crs and is associated with osteitis and disease severity (paulini, 2021). immunohistochemical studies have shown a significantly higher distribution of ranklpositive cells in the periosteum of crs tissues compared to controls. additionally, rankl expression is higher in patients with recurrent crs than in those with primary cases. elevated rankl levels contribute to the development of osteitis and may serve as a prognostic indicator in crs. this suggests that the bmp-2 pathway may play a role in inducing osteitis in crs patients. however, the relationship between opg and osteitis in crs has not yet been well-defined (zhang et al., 2017; cawley et al., 2020). rankl expression is elevated in patients with chronic rhinosinusitis (crs) and is associated with osteitis and the severity of the disease. previous immunohistochemical studies have shown a significantly higher distribution of rankl-positive cells in the periosteum of crs tissues compared to controls. furthermore, rankl expression is increased in patients with recurrent crs compared to those with primary crs. this elevation in rankl contributes to the incidence of osteitis and could serve as a prognostic indicator in crs. it is also suggested that rankl may represent one of the pathways bmp-2 induces osteitis in crs patients. however, there is currently no data on the relationship between osteoprotegerin (opg) and osteitis in crs (kong, 2020). bmp-2 is not typically produced under normal conditions; its production is primarily induced during inflammation in crs. factors influencing inflammation in crs include genetic predisposition, microbial presence, and environmental conditions. following sinonasal mucosal inflammation, thymic stromal lymphopoietin (tslp) is induced, activating dendritic cells and subsequently t cells, leading to a t-helper 2 (th2) immune response. th2 cells produce interleukins such as il-4, il-13, and il-5, resulting in the recruitment of eosinophils, neutrophils, and macrophages. bmp-2 production is influenced by inflammatory cytokines that regulate smad6 signaling, which in turn signals bmp-2, leading to bone remodeling and osteitis.intrinsic mucosal inflammation causes mucociliary dysfunction due to direct damage and changes in mucus production. mucosal ulceration leads to bacterial colonization and infection, which further enhances the inflammatory response and damages cilia function. impaired mucociliary function fails to protect the mucosa from bacterial colonization, increasing exposure to eosinophilic mucus (kim et al., 2021; ahern & cervin, 2019; bachert & tomassen, 2019). conclussion the degree of osteitis, as measured by the goss, is significantly higher in chronic rhinosinusitis with nasal polyps compared to those without nasal polyps. the significant increase in bmp-2 levels is strongly and positively correlated with the severity of osteitis, in patients with crs. acknowledgements: the authors would like to express their sincere gratitude to all those who contributed to this research. authors’ contribution: edi handoko & david santoso conducted the research and collected data. iriana maharani provided oversight of the study design and implementation. yuyun yueniwati p.w. offered additional guidance and support throughout the research process. tanti agustina assisted to drafting and revising the manuscript. competing interests: the authors declare that there are no competing interests. references ahern, s., & cervin, a. 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(2017). icariin enhances bone repair in rabbits with bone infection during post-infection treatment and prevents inhibition of osteoblasts by vancomycin. frontiers in pharmacology, 8, 784. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 687-692 | doi: 10.14421/biomedich.2023.122.687-692 issn 2540-9328 (online) antimicrobial and antioxidant activities of sesquiterpenes-rich essential oil of trichilia monadelpha (thonn) j.j. de wilde of root bark oluwakayode olubunmi odeja1,2,*, patricia a. onocha2, ganiyat kehinde oloyede2, damola ayoyerokun2, micheal gabriel ibok2, ejike onwudiegwu okpala2,3 1chemistry department, federal university of petroleum resources, effurun, delta state, nigeria. 2natural products/medicinal chemistry unit, department of chemistry, university of ibadan, ibadan, nigeria. 3department of chemistry, federal university lokoja, lokoja, kogi state. nigeria. corresponding author* odeja.oluwakayode@fupre.edu.ng manuscript received: 18 september, 2023. revision accepted: 13 march, 2024. published: 19 march, 2024. abstract trichilia monadelpha (thonn) j.j. de wilde (meliaceae) is used in traditional medicine for the treatment of ulcers, cough and inflammatory disorders such as arthritis. in this present study, the essential oil of t. monadelpha root bark was obtained by hydrodistillation using an all-glass clevenger apparatus. the identification and characterisation were done using gas chromatographymass spectrometry. we also, aimed to evaluate the antimicrobial activity of the essential oil against ten micro-organisms using the agar diffusion method and the free radical scavenging capacity was determined using 2,2-diphenyl-1-picrylhydrazyl radical (dpph) methods. twenty (20) different compounds made up the hydrodistilled essential oil, which made up 99.0% of the entire oil content. sesquiterpene compounds made up about 73.3% of the essential oil from the root bark of t. monadelpha, which was described as sesquiterpenes-rich. sesquiterpenes' most abundant constituents include (e)-longipinene (18%), 10s,11s-himachala-3-(12),4-diene (15.26%), aromadendrene (11.12%), alloaromadendrene oxide-(1) (8.82%), and β-caryophyllene (5.92%). the essential oil inhibited growth against staphylococcus aureus, escherichia coli, bacillus subtilis, pseudomonas aerugunosa, candida albicans, aspergillus niger, klebsiella pneumonia and salmonella typhi at concentrations of 200 mg/ml to 50 mg/ml with an inhibitory zone of 20 – 10 mm while the antioxidant analysis of the essential oil revealed low scavenging activity which reveals that the synthetic antioxidants were more effective with an ascorbic acid; ic50 value of 1.47 mgml-1, butyl hydroxyanisole; 1.88 mgml-1, α-tocopherol; 4.83 mgml-1 followed by the essential oil with an ic50 value of 9.95 mgml-1. t. monadelpha root bark essential oil contains notable chemical compounds that are responsible for its antioxidant and antimicrobial activities. keywords: 2,2-diphenyl-1-picrylhydrazyl radical (dpph); antimicrobial; essential oil; sesquiterpenes; trichilia monadelpha. abbreviations: dpph: 2,2-diphenyl-1-picrylhydrazyl radical; bha: butyl hydroxyanisole; ri: retention index; frin: forest research institute of nigeria; tmro: t. monadelpha essential root bark essential oil introduction almost every culture in the world has evolved an indigenous system of traditional healing (abbiw 1990). plants are an important source of drugs, especially in traditional medicines (akihisa et al. 2001). atropine from atropa belladana; morphine and codeine from papaver somniferum; quinine and quinidine from cinchona spp and digoxin from digitalis spp are examples of drugs with plant sources. approximately 60% of all the anti-infectious and anti-tumor drugs already on the market or under clinical trials are estimated to be of natural origin (bako et al. 2005). the role of free radicals in many disease conditions has been established. various biochemical reactions in the human body generate reactive oxygen species and these are capable of damaging important bio-molecules leading to diseased conditions if they are not effectively scavenged by cellular constituents (halliwell and gutteridge 1990; okpala et al. 2019). these radicals include superoxide anions, hydroxyl, nitric oxide and hydrogen peroxide radicals (halliwell and gutteridge 1992; raghuveer and tandon 2009). antioxidants of natural and synthetic origin prevent free radical damage by their protective role such as reacting with them, chelating catalytic metals and acting as oxygen scavengers. ingestion of several synthetic antioxidants such as butylhydroxyl toluene (bht) and butylated hydroxyanisole (bha) has been reported as toxic to man (hosseinimehr et al. 2007). the need for the discovery of https://doi.org/10.14421/biomedich.2023.122.687-692 688 biology, medicine, & natural product chemistry 12 (2), 2023: 687-692 more natural antioxidants is therefore of uttermost importance labo et al. 2010). trichilia monaldepha (thonn) jj de wilde has a wide application in traditional medicine for the following conditions namely: yaws, ulcers, abortion, arthritis; cough, gonorrhea, syphilis, chancres and syphilitic sores, wounds/cuts/epidermal infections, rickets, sterility, amenorrhea, stomach pain, gastrointestinal pain, disorders, dysentery and fever (abbiw 1990; msbana et al. 2000; lemmens 2008). essential oils from aromatic plants and spices have a long history of use as natural antimicrobial agents in traditional medicine. they also have wide applications in pharmaceutical industries, cosmetics and food as they are known to possess antioxidant, antimicrobial and antiviral properties. these essential oils are employed in pharmaceutical industries due to their small molecular size and their ability to easily penetrate the skin tissue. they are generally lipid-soluble and are capable of penetrating the membranes easily even in conditions where oxygen deficiency leads to the hardening of membranes. studies reveal that essential oils serve as powerful antioxidants that produce an adverse environment for damaging free radicals, thus, preventing mutations and oxidants in cells. they therefore function as scavengers for free radicals. essential oils can be extracted from different plant parts: fruits, leaves, flowers, barks, roots and seeds with each part possessing unique characteristics (potterat 1997; bray 1999; bako et al. 2005). t. monadelpha was found to exhibit antioxidant, wound healing, anti-inflammatory, and anti-anaphylactic properties (tiwari et al. 2011; woode et al. 2012; ben et al. 2013, kokate 2014), analgesic (dos santos et al. 2010), antimicrobial and antioxidant (quartey 2014). in this research article, we report the antimicrobial, antioxidant and cytotoxicity activities of t. monadelpha root bark essential oil along with its chemical composition. materials and methods plant material the fresh root bark of t. monadelpha was collected from the botanical garden of the university of ibadan, ibadan. specimens were identified and authenticated at the forest research institute of nigeria (frin), oyo state, nigeria where a voucher specimen with herbarium number fhi 112655 was deposited in the frin herbarium. reagents hexane, methanol, hydrogen peroxide, butylated hydroxyanisole (bha), ascorbic acid, α-tocopherol, dmso and 2,2-diphenyl-1-picrylhydrazyl radical (dpph), were obtained from sigma chemical co. (germany). major equipment used uv-visible spectrophotometer (unicol 200 and perkin elmer lambda 25 models), gc-mass spectrophotometer (agilent technologies) and hydro distillation-clevenger apparatus. preparation and isolation of essential oil from the plant material the fresh root bark of t. monadelpha was chopped into pieces using a knife and the essential oil was immediately collected from the fresh plant material by hydro-distillation on a clevenger-type apparatus for 4 hrs in accordance with the british pharmacopoeia specifications (1980). the essential oil was collected and stored at 4 oc before analysis. gas chromatography-mass spectrometry (gc-ms) analysis the essential oil was analysed by gc-ms using agilent 7890a gas chromatograph coupled with ms agilent technologies 5975 series msd. the capillary column type was an hp-5 ms with a column length of 30 m, an internal diameter of 0.32 mm and a film thickness of 0.25 μm. the carrier gas was helium at a constant flow rate of 1.4123 ml/min, an average velocity of 43.311 cm/sec and the pressure was put at 1.5 psi.the temperature of the column was initially set at 80 ̊c for 2 min and then increased to 240 c̊ at the rate of 10 ̊c/min for 30 min. the volume of each sample injected was 3 μl. identification of the constituents of the oils was carried out by comparing the mass spectra data of the compounds with a pre-installed nist ms search 2.0 data bank and with the data previously reported in the literature (adam 2001), linear retention index experimentally determined using homologous series of c6-c30 alkanes. antimicrobial screening of the essential oil of t. monadelpha root bark the root bark essential oil of t. monadelpha were tested against 10 strains of micro-organisms consisting of 6 bacteria; 4 gram-negative (escherichia coli, salmonella typhi, klebsiella pneumonia and pseudomonas aeruguinosa), 2 gram-positive: (staphylococcus aureus and bacillus subtilis) and 4 fungi (candida albicans, penicillium notatum, rhizopus spp. and aspergillus niger). the agar diffusion method was employed (kalemba and kunicka, 2003; okpala et al. 2019). the oil sample was prepared such that 1 ml of the oil was regarded as 100% concentration. 0.5 ml into 0.5 ml of n-hexane gave 50% concentration and 25% concentration was obtained using serial dilution. hexane was used as a negative control, gentamicin (10 µg/ml) as a positive control for bacteria and tioconazole (0.7 mg/ml) as a positive control for fungi. all tests were carried out in triplicates. observed zones of inhibition of growth were measured and recorded in millimeter (mm). odeja et al. – antimicrobial and antioxidant activities of … 689 scavenging of dpph on the essential oils a 0.5 ml of the radical source of 2, 2-diphenyl-1picrylhydrazyl radical (dpph) solution in methanol was prepared and 3 ml of this solution was mixed with 1 ml of the oil sample in methanol (oloyede et al. 2011; onocha et al. 2011). the decrease in absorption (a) at 517 nm of dpph was measured after 10 mins of incubation. the actual decrease in absorption was measured against that of control and the percentage inhibition (%i) was also calculated. the same experiment was carried out on ascorbic acid, α-tocopherol and butylated hydroxyl anisole (bha) which are known antioxidants. all tests and analyses were run in triplicates and the result obtained was averaged. the activities were determined as a function of their % inhibition which was also calculated using the formula: result and discussion chemical constituents of root bark of t. monadelpha essential oil a total of 20 compounds comprising 99% of the essential oil was identified based on their retention time determined regarding a homologous series of n-alkanes and by comparison of their mass spectral fragmentation patterns (nist 08. l database/chemstation data system) with data previously reported in the literature (joulain and konig 1998; adam 2001). the hydro-distillation of the root bark of t. monadelpha gave a high level of sesquiterpenes hydrocarbon in the essential oil with (e)-longipinene (18 %) as the major compound; other constituents identified include 10s,11s-himachala-3-(12),4-diene (15.26 %), aromadendrene (11.12 %), alloaromadendrene oxide(1) (8.82 %), [e]-4-hexadecen-6-yne (6.55 %), caryophyllene (5.91), (+)-α-himachalene (5.47), (-)eremophylene (4.83 %), α-cyperene (4.39) and pcymen-7-ol (4.04). the essential oils were predominantly sesquiterpenes hydrocarbon in nature (73.3 %), oxygenated sesquiterpenes (10.23 %), nonterpenes derivative (10.1 %), oxygenated monoterpenes (4.04 %) and 1.33 % apocarotenes as shown in table 1. the most abundant constituent in the root oil (e)longipinene is known to be an anti-cancer agent, insect repellant and cytotoxic (javaprakasha et al. 2002; liao et al., 2013). the compound 10s,11s-himachala-3-(12),4diene is reported to exhibit insecticidal activities against ticks and mosquitoes (tawatsin et al. 2006), while alloaromadendrene oxide-1 possesses a wide range of pharmacological activities such as antioxidant, antiinflammatory, anti-tumor, anti-proliferative and antidepressant activity (liao et al. 2013). the pharmacological activities of the chemical composition of the essential oil justify the ethnomedicinal usage of the root of t. monadelpha. also, the essential oil of the root of t. monadelpha is characterised as sesquiterpenes-rich (table 2), having comprised of about 73.3% sesquiterpenes compounds. some of the most dominant constituent of sesquiterpenes include (e)-longipinene (18%), 10s,11s-himachala-3(12),4-diene (15.26%), aromadendrene (11.12%), alloaromadendrene oxide-(1) (8.82%) and βcaryophyllene (5.92%). phytochemical analyses of numerous plants’ essential oils has been reported to be dominated by sesquiterpenes; t. polium, t. flavum, t. montanum and t. chamaedrys were characterised by a high percentage of the sesquiterpene hydrocarbons such as β-caryophyllene and germacrene d (nada et al. 2011). the essential oils from the investigated croatian teucrium species exhibited antiphytoviral activity (nada et al. 2011). table 1. chemical composition of the essential oils from the root of trichilia monadelpha (tmro). s/n ri compound % composition 1 1-(1-methylene-2-propenyl)-cyclopentanol 2.01 2 1,2-cyclohexanedicarboxylicacid,di(2-chlrophenyl)ester 0.54 3 1028 p-cymen-7-ol 4.04 4 1330 (e)-longipinene 18 5 1398 α-cyperene 4.39 6 1399 10s,11s-himachala-3-(12),4-diene 15.26 7 1418 ylangene 3.31 8 1428 (+)-α-himachalene 5.47 9 1445 ϒ-elemene 0.85 10 1459 aromadendrene 11.12 11 1475 (-)-α-helmiscapene 1.21 12 1476 β-caryophyllene 5.91 13 1486 (-)-eremophylene 4.83 14 1510 germacrene a 2.33 15 1519 α-cubebene 0.8 16 1519 1,1,4,8-tetramethyl-4,7,10-cycloundecatriene 1.77 17 1534 γ-muurolene 0.38 18 1613 (-)-caryophyllene oxide 1.41 19 1702 alloaromadendrene oxide-(1) 8.82 20 1737 [e]-4-hexadecen-6-yne 6.55 total 99% ri: retention index. 690 biology, medicine, & natural product chemistry 12 (2), 2023: 687-692 table 2. classes of compounds identified from the trichilia monadelpha root bark. classes of compounds identified % composition of the identified compounds oxygenated monoterpenes 4.04 sesquiterpenes hydrocarbon 73.3 oxygenated sesquiterpenes 10.23 non-terpenes derivative 10.1 apocarotenes 2.33 the antimicrobial activity of t. monadelpha essential root bark essential oil (tmro) the root essential oil inhibited growth against s. aureus, e. coli, b. subtilis, p. aerugunosa, c. albicans, a. niger, and k. pneumonia, s.typhi at concentrations of 200 mg/ml to 50 mg/ml with an inhibitory zone of 20 – 10 mm (table 3). at a lower concentration of 25 mg/ml, the zone of inhibition was between 12 10 mm for s. aureus, b.subtilis, e. coli, p. aerugunosa, a. niger and c. albicans. the oil extract showed inhibition against p. aeruguinosa with a zone of inhibition of 10 mm at 12.5 mg/ml, while no inhibition was observed at 0.0625 mg/ml against all the tested micro-organisms. essential oil has bioactive constituents that are crucial in pharmacological and therapeutic use such as 10s,11shimachala-3-(12),4-diene was one of the major constituent identified in the essential oil of erythrina caffra, which inhibited antibactericidal activities (wintola et al. 2021). however, the good antibacterial activity of the t. monadelpha root bark essential oil observed may be associated with the presence of aromadendrene, (e)-longipinene, aromadendrene oxide, (+)-α-himachalene and β-caryophyllene, which were reported to have antibacterial effects against many bacterial strains (filipowicz et al. 2013; bonikowski et al. 2015; dahham et al. 2015; gordien et al. 2019). however, synergistic effect between constituents should not be neglected, since it may cause a much more noticeable effect than single component (oukerrou et al. 2017; moa et al. 2019). table 3. antimicrobial analysis of the root bark essential oils of t. monadelpha (tmro). conc.(mg/ml) sa ec ba pa st kp ca an pn rs 200 20 18 20 18 18 16 14 14 100 18 14 18 14 14 14 12 12 50 14 12 14 12 12 12 10 10 25 12 10 10 10 10 10 12.5 10 6.25 -ve +ve 40 38 40 38 40 38 28 28 28 26 sa: staphylococcus aureus, ec: eshericha coli, bs: bacillus subtilis, pa: pseudomonas aeruguinosa, st: salmonella typhi, kp: klebsiellae pneumonia, ca: candida albicans, an: aspergillus niger, pn: penicillium notatum, rs: rhizopus spp., -ve: negative control (n-hexane), +ve: positive control; gentamicin 10 μg/ml (bacterial) and tiocosnazole 70 % (fungi). dpph free radical scavenging activity of t. monadelpha root bark essential oil dpph is a stable free radical compound widely employed to study the free radical scavenging ability of various samples (benvenuti et al., 2004). figure 1 and table 4 show the scavenging ability of t. monadelpha root bark essential oil, ascorbic acid, butylated hydroxyanisole (bha) and α-tocopherol on dpph radicals at various concentrations. the essential oil showed lower percentage inhibition at various concentrations (29.6% 28.50%) while ascorbic acid, bha and α-tocopherol, a concentration at 1.0 mgml-1 was required to achieve the inhibition of dpph radicals of 92.1%, 84.70% and 53.7%, respectively. the result obtained revealed that the synthetic antioxidants were more effective with an ic50 value of 1.47 mgml-1, 1.88 mgml-1, 4.83 mgml-1 followed by the essential oil with an ic50 value of 9.95 mgml-1. the low inhibition of dpph free radical scavenger might be due to the low concentration of polar or phenolic moiety among the chemical components present in the oil. the low antioxidant activities observed in the t. monadelpha root bark essential oil can be attributed to the absence of phenolic compounds or hydroxyl group’s compounds. however, the presence of aromadendrene, (e)-longipinene, aromadendrene oxide, 10s,11shimachala-3-(12),4-diene, (+)-α-himachalene and βcaryophyllene, also in synergy of other component identified in this study contributed to the free radical scavenging activity. odeja et al. – antimicrobial and antioxidant activities of … 691 table 4. the absorbance reading at 517 nm of the essential oil and standards at different concentrations. conc. (mg/ml) tmro ascorbic acid bha α-tocopherol 1.0 0.983±0.006 0.110±0.002 0.214±0.006 0.646±0.004 0.5 0.988±0.003 0.113±0.005 0.240±0.003 0.648±0.002 0.25 0.992±0.007 0.123±0.003 0.261±0.000 0.668±0.002 0.125 0.993±0.003 0.124±0.004 0.263±0.005 0.722±0.005 0.0625 0.998±0.001 0.214±0.002 0.346±0.002 0.768±0.001 figure 1. dpph radical scavenging activity of root bark essential oil (tmro) and standards. conclusion the t. monadelpha root bark essential oil contains notable chemical compounds that are responsible for its antioxidant and antimicrobial activities. however, the results obtained from this study suggest t. monadelpha root bark is a potential source of natural antioxidants and antimicrobials. this result also supports the ethnomedicinal application of the plant. acknowledgments: the authors thank the organic research laboratory i, department of chemistry, university of ibadan for the space and extraction and the department of pharmaceutical microbiology, university of ibadan, ibadan. authors’ contributions: odeja, oluwakayode olubunmi, carried out the sample collection, extraction of the essential oil, antioxidant, antimicrobial assays, results interpretation and write-up; onocha, patricia a., organic chemistry-ph.d. was involved in all the laboratory work, conceptualization, results interpretation and writing (editing); ibok, micheal gabriel, carried out the antimicrobial assays, results interpretation and write up; oloyede, ganiyat kehinde, was involved in the antioxidant assay; ayoyerokun, damola, was involved in the antioxidant assay; okpala, ejike onwudiegwu was involved in results interpretation and write up. all authors read and approved the final manuscript. competing interests: the authors declare no competing financial interest. funding: no funding was received for this study. references abbiw, d.k. 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(2012). analgesic effects of stem bark extracts of trichiliamonadelpha (thonn.) jj de wilde, indian journal of pharmacology, 44 (6), 765 – 773 https://doi.org/10.1016/j.heliyon.2021.e07244 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 109-115 | doi: 10.14421/biomedich.2020.92.109-115 issn 2540-9328 (online) callus induction of leaves and stems in krisan (chrysanthemum morifolium ramat cv dewi ratih) with alternative foliar fertilizers media ahmad saifun naser*, muhammad wisnu department of biology, faculty of sciences and technology, universitas islam negeri sunan kalijaga yogryakarta, jl. marsda adisucipto, yogyakarta, 55281, indonesia. corresponding author* saifun98@gmail.com manuscript received: 11 october, 2020. revision accepted: 16 november, 2020. published: 18 july, 2021. abstract availability of quality seeds in production of krisan (chrysanthemum morifolium ramat cv dewi ratih) cultivation is still rare, therefore research on seed multiplication through tissue culture is needed. the media used in tissue culture is relatively expensive for home industry. this study aims to determine the respond of leaf and stem explants using foliar fertilizers (growmore, gandasil d and mutiara) as an alternative media for callus inductions. this study used a completely randomized design (crd) consisted of 4 treatments: p0: ½ ms + 0,25 mg/l bap, p1 (growmore + 0,25 mg/l bap), p2 (gandasil d + 0,25 mg/l bap), p3 (mutiara + 0,25 mg/l bap). the variables observed in this study included callus appearance time, callus color and callus texture. the result of this study indicated that the use of bap (6-benzyl amino purine) affected the time of callus formation and callus morphology. callus was formed on leaf explants 13 days after planting while on stem explants 7 days after planting and compact texture. growmore + 0,25 mg/l bap treatment yields the best callus on leaf explant, while gandasil d + 0,25 mg/l bap treatment yields the best callus on stem explant. keywords: bap (6-benzyl amino purine); krisan (chrysanthemum morifolium ramat cv dewi ratih); plant tissue; alternative media (growmore, gandasil d and mutiara). introduction chrysanthemum is an ornamental plant that has a high economic value due to the beauty of the flowers. according to mustakim et al., (2015) the flowers of chrysanthemum containing antioxidants that play a role in detoxifying toxins in the body and improve blood circulation. chrysanthemum plants could be used as a raw material for antibiotic drugs and sharpens eye vision (mani & senthil 2011; lin & herly, 2010). chrysanthemum plant (chrysanthemum morifolium ramat cv dewi ratih) is cultivated in almost all of indonesia. the dominating area includes east java, central java, west java, south sulawesi and north sumatra (badan pusat statistik dan direktorat jendral holtikultura, 2017). c. morifolium plants contained secondary metabolite of flavonoid and volatile compounds. the examples of the compound are quercetin-3-galactoside, luteolin 7-glucoside, quercitrin, myricetin, luteolin, apigenin, and kaempferol (sun et al., 2010). the successful morphogenesis from cultivated plants determined by the explants. according vidyasagar, (2006) explant is part of the plant that is used as a material for the initiation of culture. explant can be cells (cell culture), protoplast (protoplast culture), medulla (tissue culture), apical meristem or lateral (shoot culture) and cut stems or leaves (organ culture) (dwiyani, 2015). the juvenile physiological explants are better for the tissue culture then the old one. the part of juvenile plants have higher regenerations than the mature one (gunawan, 1987). callus is an unorganized and actively dividing tissue that usually produced by injured plants in tissue culture. according to dwiyani, (2015) callus can be obtained by tissue roots, leaves and stems in the plant that occurred through the process of indirect embryogenesis. explant derived from meristematic tissue developed faster than tissue of thin-walled cells that containing lignin (nugroho & sugito, 2001). plant growth regulators (pgr) could be used for additional plant tissue cultured media. according from gunawan (1987) pgr usage affected growth and morphogenesis in cell, tissue and organ culture caused by the interaction between media determined culture development. media are very important in tissue culture methods, even the success of the tissue culture is determined by the media used (gunawan, 1987). the essential elements in the media included inorganic salts, vitamins, energy source and carbon. the essential nutrients are nutrients https://doi.org/10.14421/biomedich.2020.92.109-115 110 biology, medicine, & natural product chemistry 9 (2), 2020: 109-115 that used by plant to completed their life cycle. the functions of these nutrients cannot be replaced by other elements. it is required by the plants in the metabolic process as inorganics components or cofactors in enzyme reactions (orcutt & nilsen, 2000). foliar fertilizers like mutiara and growmore can be alternative substitution for micronutrient and macronutrient ms (murashage and skhoog) media (shintiavira et al., 2012). gandasil d and growmore are compound fertilizers that contain complete macronutrient and micronutrient. sometimes the alternative media usage added by some nutrient like casein hydrolysate (ch), corn oil, coconut water and tomato juice. widiastoety & purbadi (2003) stated that 150 ml/l usage of coconut water that pour into the media could have an effect for increased growth, height, length and width leaves and the number of roots to dendrobium orchid plantlet. this study aims to determine the response of leaf and stem explants to use foliar fertilizers (growmore, gandasil d & mutiara) with added plant growth regulators (pgr) as an alternative media for callus inductions of chrysanthemum morifolium ramat cv dewi ratih. it is hoped that this research can be used as material for consideration of a substitute for ms (murashige and skoog) which is relatively expensive for home-scale tissue culture farmers given the nutrient content in foliar fertilizers meet the needs for optimal plant growth and development. methods this research was conducted from 15th february to 20th september 2019 at the embryology laboratory of uin sunan kalijaga yogyakarta. materials the plant parts used as explants in this study were the leaves and stems of chrysanthemum morifolium ramat cv dewi ratih. the growth media used in this study were ½ ms medium (murasahge and skoog) and compound fertilizer gandasil d, growmore and mutiara. furthermore, the chemicals used were agar, bap powder, 1 m hcl solution, distilled water, 70% alcohol, sterile tissue paper, sterile aluminium foil, filter paper and plastic cling wrap. procedure 1. sterilization of ingredients the explants that had been prepared for sterilization were put in a tea filter, then washed with running water for 5 minutes. remove the explants and soak them in a 10% alcohol solution for 3 minutes while shaking them manually then rinsing with distilled water 3 times (1-3 minutes), then the explants were removed and immersed in 5% chloroc (naocl) solution while shaking it manually for 35 minutes then 10 % chlorine for 3-5 minutes. then rinse with distilled water 3 times (1-3 times). the explants were then let dry in erlenmeyer and covered with sterile tissue in an inverted position. 2. preparation of hormone solution 100 mg of bap powder was dissolved in 2.5 ml of 1 n hcl solution. the material was heated and stirred until dissolved. then, add distilled water slowly until the volume of the solution reaches 100 ml. furthermore, the ph of the solution is adjusted to 5.0. the solution was transferred to the stock container and labelled. 1000 ppm bap stock solution (1 ppm = 1 mg/l) were stored in a refrigerator. 3. media media ms (murashige and skoog) 2.215 grams of instant ½ ms medium diluted in 1000 ml of distilled water. then added the hormone 0.25 mg/l bap. the ph measurement of the solution was carried out in the range of 5.6 5.9. after that, 8 grams of agar powder and 30 grams of sugar are added to the mixture of media and hormones. making ½ ms resulted in a positive control, namely media ½ ms + 0.25 mg/l bap, each consisting of 10 replications. ½ ms solution was heated to a boil, stirring chili sauce until the powder was completely dissolved. after boiling, ½ ms liquid medium was poured into the culture bottle and closed tightly with aluminium foil. the culture bottles that have been filled with the media were then put into an autoclave to be sterilized at a pressure of 17 psi at 121°c for 15 minutes. finally, the culture bottles were stored in the culture room at 12°c. after one to two days, the culture bottles are examined for clots or for contamination. 4. explant inoculation and culture incubation explant was obtained from the leaves of the chrysanthemum. each culture bottle was planted with three leaf explants with a size of 0.5 cm x 0.5 cm. each explant was planted 10 times in a variety of different growth media according to table 1. the culture bottles that have been planted with explants were then stored in the culture room at a temperature of 16°c. table 1. design of research treatment chrysanthemum morifolium ramat cv dewi ratih. treatment a. positive control (p0: ½ ms +0,25 mg/l bap) b. alternative media (p1: growmore + 0,25 mg/l bap) c. alternative media (p2: gandasil d + 0,25 mg/l bap) d. alternative media (p3: mutiara + 0,25 mg/l bap) 5. observation of parameters observations were made for once every two days after the day after planting (dap). until callus formed on all naser & wisnu – callus induction of leaves and stems in krisan … 111 explants. the parameters observed during the study are as follows:  callus morphology and color the morphology observed included crumb and compact callus texture and a white, green or brown callus color  callus appearance time the time of callus formation was determined by observing the explants from the beginning of planting until the first callus appeared. results and discussion leaf and stem explants on chrysanthemum morifolium ramat cv dewi ratih are able to form callus. callus formation on leaf and stem explants had different times. on the stem, callus formed 7 dap (days after planting) while on callus leaves formed 13 dap (days after planting). it can be seen in tables 2 and 3. the callus formed in all treatments was compact type. the compact callus texture in this study was caused by the provision of cytokinins, as the statement by purwaningsih et al., (2016) that compact callus has the effect of giving cytokines to the media that play a role as nutrient transfer. table 2. callus texture and color, as well as other descriptions of leaf explants (c. morifolium ramat cv. dewi ratih). treatment days after planting color callus other information ½ ms + 0,25 mg/l bap 7 dap clear green stems are still fresh, shoots appearead in 13 dap. compact callus. mutiara+0,25 mg/l bap 7 dap clear green callus grows on the scar, stems are still fresh, shoots appearead in 13 dap. compact callus. gandasil+0,25 mg/l bap 7 dap clear green compact callus, stems are still fresh, shoots appearead in 13 dap. growmore+0,25 mg/l bap 7 dap clear green compact callus. stems are still fresh. 13 dap shoots grow in new branches. table 3. callus texture and color, as well as other descriptions of stems explants (c. morifolium ramat cv. dewi ratih). treatment days after planting callus texture other information color callus ½ ms + 0,25 mg/l bap (+) control 13 dap compact the leaves are swollen, the scars form a clear white callus. clear green callus mutiara + 0,25 mg/l bap (-) 13 dap compact there is browning on the scar, getting thicker, on the scar forming a clear white callus. clear green callus gandasil + 0,25 mg/l bap (-) 13 dap compact the leaves are swollen, browning on the wound, forming a clear callus on the wound. clear green callus growmore + 0,25 mg/l bap 13 dap compact the leaves are swollen, callus is found all over the leaves. clear green callus p0 p1 p2 p3 figure 1. callus on leaves explants with the treatment p0, p1, p2, and p3. ( : callus) in the formation before, callus was followed by swelling of the explants. this is in accordance with the statement of hariyanti et al., (2016) stating that callus is formed followed by a whitish or clear swelling around the scar which eventually covers the entire surface of the explants. callus on the leaf (table 3) is formed marked 112 biology, medicine, & natural product chemistry 9 (2), 2020: 109-115 by the swelling of explant tissue on 13 dap, then followed by the formation of clear grains on scar and the uneven explant surface. callus formed in each treatment leaf explants could be seen in figure 1. the callus that formed on the stem (table 2) is at tip and base of the stem because that part is a place of scar on leaves. on the stem explants also formed shoot on 13 dap (table 2). callus and shoots formed on stem explants can be seen in figure 2. p0 p1 p2 p3 : soots, : callus figure 2. callus on stem explants with the treatment p0, p1, p2 and p3. leaves treatment of p1 (figure 1) showed that growmore + 0,25 mg/l bap foliar fertilizers able to become an alternative medium for callus induction, because growmore fertilizer contained sufficient nutrient. callus was formed on 13 dap which is compact texture, fresh green in the entire leaves surfaced and accompanied by swelling explants. the formation of callus is a sign that the explants are competent. according lubis (2016) chrysanthemum morifolium ramat cv merahayani which is cultured by growmore media 2 g/l did not significant with ½ ms media in media ½ ms in variable shoot height, shoot weight and number of roots. treatment p0 (½ ms +0,25 mg/l bap) in leaves explants could be callus inductions. callus was formed at 13 dap by initiating the formation of white grains then growing / forming calluses on the scar. (figure 1: p0). this is different from the growmore treatment where callus grows evenly over the surface of the leaf explants. the color of callus ½ ms is clear green and compact texture. callus color can be used as an indicator of the quality of the callus. callus which is green in color has good quality because it still contains chlorophyll (siregar et al., 2013). there was browning in the ½ ms (p0) explant (figure 1) which was caused by the presence of phenolic compounds which were derived from the role of the polyphenoloxidase enzyme. phenolic compounds are derived from plant secondary metabolites stored in vacuoles. when the explant is sliced, the vacuole breaks, the phenol compound is oxidized and then the compound is oxidized. this will cause the explants to turn brown. when phenol compounds are oxidized on the media, the media must be replaced. if not replaced it will lead to explant poisoning and death. (dwiyani, 2015). the treatment p2 (gandasil d + 0.25 mg/l bap) showed that the explants were able to form callus. the callus formed 13 dpa which compact texture, clear green color that is on the scar and surface of the explant (table.2). the callus that grows on the scar has a grainy texture so that the scar turns white. there is a little browning on scar in leaf explant indicated when cutting, however, the part that experienced browning could still induced callus on the surface of leaf explants (figure 1). the treatment p2 (gandasil + 0.25 mg/l bap) indicated that it could be used as an alternative medium for homescale tissue culture. nutrient that contained in foliar fertilizer gandasil d have contained sufficient nutrient for callus induction. matatula (2003) the addition of coconut water to gandasil d media caused an increase in plant height, number of leaves, number of roots, shoot wet weight and root wet weight in vitro. before the callus formed, leaf explants have some symptoms. on 3 dap the surface of the leaf explants began to become uneven and stretched up to 9 dap then on 13 dap leaf explant begin formed callus until 22 dpa. callus began to appear clearly almost all over the leaf surface. callus on the treatment p3 (mutiara + 0,25 mg/l bap) was compact in the texture and clear green color that grow up in the scar and surface leaf explant. p3 leaf explants on 3 dap have responded to the treatment of the media used this causes the leaf surface to be uneven and there are spots. this condition is stilled same until on 7 dap. the p3 leaf explants before forming the callus were stretched this is due to the role of the hormone cytokinins then in the scar begun to form clear white grains and spread on the leaf surface. some of the p3 leaf explants experienced browning at 9 dap. however, the explants are stilled able to form callus. callus begun to grow on leaf explants after 13 dap. leaf explants treated p3 can be classified as competent because they are able to form callus in response to the media. p3 treatment explant media (mutiara + 0,25 mg/l bap) can be used as an alternative media. treatment of the three leaf explants (figure 1) showed that foliar fertilizer has the potential as a alternative medium to replace ½ ms. the treatment of p1 leaf explant (growmore +0,25 mg/l bap) yielded the best callus then the others treatment. because callus on the p1 leaf explant has a fresh green color and grow entire the surface leaf explant (on surface and inside). naser & wisnu – callus induction of leaves and stems in krisan … 113 stem the callus was formed from stem explant that showed the equal result (table.2). callus on the treatment ½ ms, growmore, gandasil d and mutiara appeared on 7 dap. the four treatments indicated that there is potential of the media in growing callus on stem explants. stem explants which formed callus indicated that organogenesis had occur. the formation of callus on stem explants is due to the presence of endogenous auxin hormones that initiated in the scar. callus is formed at the tip and base of the stem in the explant because that position has been injured (figure 2). shoots appeared on the nodes in all treatments stem explants (½ ms, growmore, mutiara and gandasil d) after 13 dap until 22 dap. shoot formation occurs due to the role of the hormone cytokinin (bap). according dwiyani (2015). if want to form callus on the explants, the explants are planted in callus inducing media like medium + auxin. however, if want to shoot inducing, the explant planted in shoot inducted, like medium + sitokinin (bap). its can be inferred that the stem explant has cytokinin hormone higher than auksin. stem explant in p1 treatment (growmore + 0,25 mg/l bap) (figure 2) formed callus at stem tips scar. the texture callus is compacted and clear green color that appear on 7 dap. callus was formed due to endogenous auxin hormone in stem. shoots grow on the nodes stem after 13 dap in scar. the p1 treatment stem explants were swollen due to the effects of the cytokinin hormone. new leaves grow on shoots after 15 dap and the callus is getting bigger. p2 treatment stem explants (gandasil d + 0.25 mg/l bap) (figure 2) were able to form callus and shoots. browning appear on p2 treatment started 3 dap, although browning, callus can still be induced on the cutting marks. callus was formed at the base of stem while shots are formed in nodus. in 7 dap, callus can be seen with clearly, compact texture and clear green color. meanwhile, shoots can be seen clearly on 13 das. stem explants in p2 treatment (gandasil d + 0,25 mg/l bap) get the best callus than the others treatment. two young leaves clearly appear on the shoot after 15 dap. callus and shoot have pale green color and getting a bigger until 17 dpa. the color of callus turns white and shoot color getting green on 20 dpa. in p0 treatment (½ ms +0,25 mg/l bap) (figure 2), callus appeared on 7 dpa on the cut marked at stem tips. on 9 dpa, browning and callus could be seen clearly. two leaves appear on the shoot at nodus stem on 13 dpa. after that the new leave appeared in shoots on 15 dpa. the stem of p0 treatment explant still looked fresh. the leaves on the shoot begun to enlarge and the callus can still be seen clearly on 20 dpa. browning appeared in the p3 (mutiara+ 0,25 mg/l bap) treatment stem explant getting on 3 dpa. on 6 dpa the stem explant is still green fresh and the callus appeared in stem explant started on 7 dpa at cutting marked stem tips. the callus was compact in texture and had a clear white color, but the callus formed was not as much as in the p0 stem explant treatment. callus was seen more clearly at 9 dap and 2 new leaves appeared on shoots located on the nodus stem on 13 dap. on 15 dap there are addition two new leaves again on the stem. on 17 dpa there are new leaves again on the explant stem shoots. the shoots are still developed and the color of stem still fresh green until 20-22 dpa. the stem of p3 treatment has a characteristic that the shoot bigger than p0 treatment. but the number of leaves formed on the stem explants in treatment p3 was less than in treatment p0. conclusion the foliar fertilizer were able to induce callus in the chrysanthemum morifolium ramat cv dewi ratih in vitro, the p1 treatment (growmore) yielded the best callus in the leaf explants and the p2 treatment (gandasil d) yielded the best callus in the stem explants. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references badan pusat statistik dan direktorat jendral holtikultura (2017) luas panen, produksi dan produktivitas tanaman krisan, 2016-2017. dwiyani, r (2015) kultur jaringan tanaman, pelawa sari percetakan & penerbit., bali. gunawan, l.w (1987) teknik kultur jaringan tumbuhan. pau bioteknologi ipb. bogor. hariyati, m., i. bachtiar, & p. sedijani (2016) induksi kalus tanaman krisan (chrysanthemum morifolium) dengan 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(2003). pengaruh bubur ubikayu dan ubijalar terhadap pertumbuhan plantlet anggrek dendrobium. jurnal hortikultura, 13(1), 1-6. doi: http://dx.doi.org/10.21082/jhort.v13n1.2003.p1-6. naser & wisnu – callus induction of leaves and stems in krisan … 115 table s1. observation of daily development on leaf explants. date ms mutiara gandasil d growmore 26-07-2019 3 dpa uneven surface, there are spots uneven surface, there are spots starting to warp, uneven surface. some look flat, uneven surface. 29-07-2019 6 dpa still same the day before still same the day before still same the day before many are curved, uneven surface 30-07-2019 7 dpa still same the day before still same the day before still same the day before still same the day before 1-08-2019 9 dpa more uneven surface more uneven surface, browning in edge still fresh, uneven surface uneven surface, still fresh 5-08-2019 13 dpa enlarged leaves and callus appeared there are already 9 explants with callus appeared callus in 1explant dan 1 browning enlarged leave and callus appeared 7-08-2019 15 dpa browning appeared, 1 explant enlarged callus visible more clearly and enlarged callus visible more clearly and leaf enlarged callus visible more clearly at cutting marks and surface 9-08-2019 17 dpa still same the day before the part of leaf browning callus visible more clearly increased callus and clearly, explant enlarged 12-08-2019 20 dpa some of the leaves have blackened and enlarged leaf still fresh, callus visible 1 explant browning increased callus and exsplan enlarged 14-08-2019 22 dpa there were explants that just appeared callus leaf still fresh, callus visible callus is visible on almost all surfaces 3 explants browning and 1 explant appeared callus table s2. observations of the daily development of observations on the stem. date ms mutiara gandasil d growmore 26-07-2019 3 dpa still green fresh, browning at cutting edges. still green fresh, browning at cutting edges. still green fresh, browning at cutting edges still green fresh browning at cutting edges 29-07-2019 6 dpa still green fresh, browning at cutting edges. still green fresh, there is browning at the base of the cut still green fresh, browning at cutting edges still green fresh, some of explant browning at cutting edges 30-07-2019 7 dpa callus appeared at cutting marks base brown, callus appears but not as much as ms callus appears but not as much as ms callus appeared. 1-08-2019 9 dpa the callus on the cut mark was clearly visible, browning is starting to clear callus is starting to clear, some explant browning callus clearly, the stem begins to swell at the bottom (elongation) callus is starting to clearer, the stem begins to swell 5-08-2019 13 dpa shoots are visible, there are 2 new leaves on nodus, new shoots shoots are visible, callus is still visible on the cut marks. callus still fresh, cut mark browning, new shoots at nodus. new shoots at nodus. callus still fresh. 7-08-2019 15 dpa 3 shoods appeared in nodus 3 new leaves, the leaves are longer and clearer looks new shoots tips, 2 new leaves the callus is getting bigger, young leaves grow, there are no new leaves yet 9-08-2019 17 dpa new shoots and new leaves grow in the nodus shoots tips still fresh, small leaves become large and there are new leaf candidates the callus is getting bigger, there is new leaf, then another candidate for leaves appears again, green clear color new leaf, callus still fresh 12-08-2019 20 dpa the stems are growing and still fresh, leaves start bigger, callus is still clearly visible new shoots develop on the nodes the callus is getting whiter, the leaves are getting green clearly . still same 14-08-2019 22 dpa still bigger explant and shoots are still small. green shoots color, looks bigger than ½ ms but leaves less leaf green clearly, leaves a little but big. there are some that haven't appeared yet. dark green shoot color this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 337-341 | doi: 10.14421/biomedich.2024.132.337-341 issn 2540-9328 (online) pharmacophore mapping of angiotensin converting enzymes (aces): insight to binding site of ace1 and ace2 navista sri octa ujiantari*, cintya nurul apsari laboratory of medicinal chemistry, departement of pharmaceutical chemistry, faculty of pharmacy, universitas gadjah mada sekip utara yogyakarta, 55281, indonesia. corresponding author* navista.sri.o@ugm.ac.id manuscript received: 11 may, 2024. revision accepted: 08 august, 2024. published: 01 october, 2024. abstract angiotensin converting enzymes (aces) are carboxypeptidase enzymes involved in the renin-angiotensin system (ras), which catalyze angiotensin i by cleavage of the peptide bond. ace1 has been known as a target for antihypertensive drugs. another homolog of ace1, ace2 has been popular since 2020 because this enzyme is responsible for the sars-cov2 infection in the human body. interestingly, it was found that ace1 inhibitors did not inhibit ace2. hence, this study aims to elucidate the pharmacophore of ace1 and ace2 in order to understand the mechanism of these different inhibitions. pharmacophore mapping was carried out using a pharmacophore query editor in the molecular operating environment (moe). the 3d structures of both enzymes bound to respective inhibitors were prepared and their pharmacophore features were extracted. besides that, the similarity of both enzymes was analyzed by comparing their amino acid sequences using align in uniprot. in addition to pharmacophore mapping, the surfaces of both binding sites were analyzed to obtain a comprehensive evaluation. results showed that both ace1 and ace2 contain nine and eight pharmacophore features, respectively. the amino acid residues of both enzymes were quite similar, especially in the active site. however, both ace1 and ace2 inhibitors showed different interactions even though both were well aligned. it was found because the functional groups of both inhibitors were slightly different as well as the active site size of both enzymes. thus, this might result in different ability of ace1 inhibitors to occupy the binding site of ace2. these findings could provide useful information in the design of new selective ace1 compounds as well as ace2 compounds. keywords: pharmacophore mapping; ace1; ace2; active site; homolog protein. abbreviations: ace: angiotensin-converting enzyme; moe: molecular operating environment; ras: renin-angiotensin system; acei: ace inhibitor; sars-cov-2: severe acute respiratory syndrome coronavirus 2. introduction the renin-angiotensin system (ras) plays a role in blood regulation as well as glucose metabolism, electrolyte balance, and homeostasis in the body. the components of this system include angiotensinogen, angiotensin i, angiotensin ii, angiotensin-converting enzyme (ace) (ahmad et al., 2023; alvarez-zaballos & martinez-selles, 2023). ace1 is a carboxypeptidase that catalyzes angiotensin i, a decapeptide by cleavage of the peptide bond into an octapeptide, angiotensin ii (ames et al., 2019; ahmad et al., 2023). this enzyme has been known as antihypertensive drugs and the synthetic drugs inhibit this enzyme namely ace inhibitor (acei) (messerli et al, 2018). those inhibitors are orally administered and they usually contain the suffix “-pril” in their name, for instance captopril, lisinopril, fosinopril, enalapril, and ramipril (ahmad et al., 2023). pharmacophore modeling has been applied in drug design and discovery to identify compounds with specific biological activities and to understand their interaction with protein or mapping interaction (seidel et al., 2018). the functional groups or parts of molecules responsible for biological activities are called pharmacophores (güner dan bowen, 2014). the pharmacophore features include hydrogen bond acceptors and donors (hba dan hbd), ionized groups, hydrophobic (hy), aromatic rings (ra), metal coordination (m), and halogen bond (schaller et al., 2020). pharmacophore modeling can be approached in two ways: ligand-based and structurebased (vuorinen & schuster, 2015). ligand-based pharmacophore modeling relies on known compound activities, whereas structure-based pharmacophore modeling is derived from 3d experimental protein structures (seidel et al., 2018). the 3d structures provide the shape of active site as well as the important amino acid residues and the type interaction (vuorinen & schuster, 2015). in 2019, pneumonia caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in https://doi.org/10.14421/biomedich.2024.132.337-341 338 biology, medicine, & natural product chemistry 13 (2), 2024: 337-341 wuhan, china, became a global pandemic (ni et al., 2020; ahmad et al., 2021). the virus can penetrate the human body through a receptor called ace2 (ahmad et al., 2021; pagliaro et al., 2022). this enzyme catalyzes angiotensin ii into angiotensin 1-7 and is expressed in several organs such as blood vessels, the brain, kidneys, lungs, and the digestive system (pagliaro et al., 2022; dutta, 2022). although ace2 is a homologous protein with ace1, ace1 inhibitors do not inhibit ace2 activity (danser et al., 2020; kai & kai, 2020). therefore, this research aims to examine the differences in the active sites of ace1 and ace2 using structurebased pharmacophore modeling by mapping interactions in both enzymes. the results of this study can be used as valuable information, especially in designing selective ace2 inhibitors. materials and methods pharmacophore mapping was conducted using the pharmacophore query editor in the molecular operating environment (moe, 2022). the 3d structures of ace1 with pdb id: 1o86 (natesh et al., 2003); 1uzf & 1uze (natesh et al., 2004), and ace2 with pdb id 1r4l (towler et al., 2004) were obtained from the protein data bank (burley et al., 2017). all of those enzymes are bound to respective inhibitors. the enzymes were prepared, and their pharmacophore features were extracted using the pharmacophore query editor. furthermore, the similarity between the two enzymes was analyzed by comparing their amino acid sequences using the align tool in uniprot (boutet et al., 2016). both 2d and 3d interactions between the inhibitor and aces were visualized to identify the types of interactions formed. additionally, the active site surfaces of both enzymes were analyzed to obtain a comprehensive evaluation. results and discussion result based on the analysis of amino acid sequence similarity using align, the results indicated that ace1 and ace2 share 36.61% similarity (per*cent identity). homolog proteins share 30-40% sequence identity (cavasotto & phatak, 2009; pearson, 2013). figure 1. the alignment of ace1 (green) and ace2 (red) along with their inhibitors, namely lisinopril (green) and mln-4760 (red), at the active site of the enzyme. figure 2. pharmacophore features obtained from 3d structures of ace1 (a) and ace2 (b). ujiantari & apsari – pharmacophore mapping of ace1 and ace2 339 in addition, the alignment results of ace1 and ace2 (figure 1) showed that ace1 and ace2 share similar protein backbone or tertiary structure. figure 1 also depicted that both inhibitors were well aligned in the active site situated in the centre of both enzymes. lisinopril, an ace1 inhibitor, also closely shared a similar orientation with mln-4760, an ace2 inhibitor. pharmacophore mapping was conducted to identify potential interactions between the inhibitors and both enzymes (figure 2). in figure 2, ace1 exhibited nine pharmacophore features, including 6 hba, 1 hy, 1 ra, and 1 hbd, while eight pharmacophore features were extracted from ace2 consisting of 4 hba, 3 hy, and 1 hbd. figure 3. 2d interaction of ace1 with lisinopril (a), enalaprilat (b), captopril (c), and ace2 with mln-4760 (d). table 1. the amino acid residues in the active site of both enzymes and the type of interactions. ace1 ace2 type of interactions his353 his345 hydrogen bond ala354 pro346 ser355 thr347 asp377 asp368 his383 his374 metal coordination/complex with zn and inhibitor glu384 glu375 his387 his378 hydrogen bond glu411 glu402 metal coordination/complex with zn and inhibitor phe512 phe504 his513 his505 val518 tyr510 tyr523 tyr515 note: bold line means same amino acid residue the 2d visualization between the inhibitor and the enzyme was performed to identify the types of interactions. at the active sites of both enzymes, there are similarities in several amino acid residues, and these residues also interact with the inhibitors (table 1). figure 3 illustrates the interactions between the inhibitors and the amino acid residues present at the enzyme's active site. in the ace1 inhibitor, captopril, enalaprilat, and lisinopril had similar interactions, where the bound amino acid residues include glu162, gln281, his353, his383, glu384, his387, lys511, his513, tyr520, and tyr 523. additionally, metal coordination bonds are formed between the carboxylate groups of the inhibitors and zn, an ace cofactor. some interaction patterns observed in ace1 are also present in ace2, such as the carboxylate group of mln-4760 binding to zn. furthermore, mln-4760 also bound to several amino acid residues such as arg273, cys344, his345, pro346, asp368, thr371, glu375, his378, and his505. figure 4 depicted the 3d visualization for both enzymes, which was used to identify the types of interactions between the inhibitors and the amino acid residues. hydrogen bonds and metal coordination with zn were formed in both ace1 and ace2. additionally, an analysis of the active site surface was conducted to determine the shape of the active site and the hydrophobicity of the enzyme's active site. the ace2 active site is narrower than ace1, especially in the central area and it is dominated by hydrophobicity. figure 4. 3d visualization of lisinopril with ace 1 (a) and mln-4760 with ace2. the 3d interaction of the inhibitors at the active site with amino acid residues (above); the surfaces of active site in both enzymes with purple for hidrophilicity/polar and green for hydrophibicity (below). discussion in this in silico studies, pharmacophore mapping was conducted to analyze the differences in the active sites of ace1 and ace2, which are homologous proteins based on their backbone similarity or tertiary structures. three 340 biology, medicine, & natural product chemistry 13 (2), 2024: 337-341 3d structures of ace1 were used with their corresponding inhibitors: captopril, enalaprilat, and lisinopril, while ace2 was bound to an inhibitor, mln4760. the active site of both enzymes is located at the centre of their respective structures. it can be observed that both share similarities in some amino acid residues thus ace1 inhibitors and ace2 inhibitors have similar type of interactions. however, it was found that there is a subtle difference in their interaction due to the functional groups of both inhibitors. in addition, the size of ace2 active site is slightly narrower than ace1 active site. it has been known that ace1 inhibitors are mostly bulky compounds and therefore they could not occupy the ace2 active site. furthermore, the ace2 active site is also dominated by hydrophobicity, making polar groups, especially in the central area of the compound, more likely to bind to ace2. consequently, these differences cause ace1 inhibitors to be unable to occupy the ace2 active site. in conclusion, these preliminary findings could provide useful information in designing compounds that are selective towards both ace1 and ace2. conclusions ace1 and ace2 are homologous proteins based on their tertiary structure (backbone) and their sequence identity. ace1 inhibitors, which are bulky compounds, could not occupy the active site of ace2 due to its narrower shape. in addition, the active site of ace2 tends to be more polar or dominated by hydrophobicity. acknowledgements: the authors express gratitude to the faculty of pharmacy, ugm for providing the licensedmoe software. authors’ contributions: navista sri octa ujiantari designed the study, performed the pharmacophore modelling, analyzed the data, and wrote the manuscript. cintya nurul apsari carried out the data collection and protein alignment. all authors read and approved the final version of the manuscript competing interests: authors declare that there are no competing interests. references ahmad, h., khan, h., haque, s., ahmad, s., srivastava, n., & khan, a. 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(2004). ace2 x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis. the journal of biological chemistry, 279(17), 17996–18007. https://doi.org/10.1074/jbc.m311191200 vuorinen, a., & schuster, d. (2015). methods for generating and applying pharmacophore models as virtual screening filters and for bioactivity profiling. methods (san diego, calif.), 71, 113–134. https://doi.org/10.1016/j.ymeth.2014.10.013 https://doi.org/10.2459/jcm.0000000000001218 https://doi.org/10.1002/wcms.1468 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 1, 2019 | pages: 23-26 | doi: 10.14421/biomedich.2019.81.23-26 issn 2540-9328 (online) echinoderms diversity and abundance in gunung kidul beach yogyakarta hikmah supriyati1,*, setyawati dwi k2, ahzami2, farhani sodiq3, septiana khoiriyah4 1postgraduate biology education program, yogyakarta state university, indonesia. 2biology education department, faculty of science and technology, uin sunan kalijaga, yogyakarta, indonesia. 3smait ihsanul fikri, pabelan, mungkid, magelang, central java 56551, indonesia. 4mts nu miftahul huda, gembong colo street, glagah kulon village, kudus, indonesia. author correspondency*: hikmahsupriyati@gmail.com abstract echinoderms are one of marine invertebrate animals that have an important role, as a recycle of nutrients in the functioning of ecosystems. echinoderms usually appear in the intertidal zone of the coast. the existence of echinoderms in the intertidal zone is affected by external and internal factors. this study aims to determine the level of abundance and diversity of echinoderms in the intertidal zone in the krakal and drini coastal areas, gunung kidul, yogyakarta. data were collected using transect method. the main transect along 50 meters was divided into 5 sampling points, and the distance between sampling points was 10 meters. after that, each point was taken for 3 observation plots with an area of 1x1 meters and the distance between plots is 5 meters. the results showed that, the diversity index on the krakal coast had a higher value (0.139) when compared to the drini coast (0.129). calculation of echinonderm abundance index shows that the drini beach location has a high abundance value when compared to the abundance value at the krakal beach loc ation. the parameter measurement results can be seen that both the ph and temperature parameters on both the krakal and drini coast have values that correspond to the optimum parameter in the waters. keywords: echinoderms; diversity; abundance; krakal coast; drini coast introduction echinoderms are one of marine invertebrate animals that have an important role in the functioning of ecosystems (supono et.al., 2014). echinoderms have a role as nutrient recycling (triana et al., 2015). it can be used as a parameter (bioindicator) quality in marine waters (marine ecosystems) (jalaluddin, 2017). echinoderm habitat is beach and sea to a depth of 366 m. in addition, echinoderm can live in a variety of habitats such as reef flat zones, algal growth areas, seagrass beds, live coral colonies and dead coral colonies (yusron, 2009). echinoderms are very common in sandy areas, especially those that are overgrown with seaweed (aziz, 1996). one area that has an environment like this is the area of krakal and drini beach gunung kidul, yogyakarta. the intertidal zone of krakal and drini beach has different types of substrates, both whether it is sandy, muddy, or rocky. the intertidal zone in the area occupies a very wide area, and when sea water reaches the lowest ebb, the length of the dried water base can reach hundreds of meters. this condition is utilized by the local coastal community in search of marine animals to meet their daily needs. the growth of marine biota in the coastal intertidal zone is very high, due to this area being a place of life, shelter, and a place to look for food. in addition, environmental conditions in this area are very favorable for the growth of marine biota because of the support of marine physical, chemical and biological factors. soemodhiharjo (1990) revealed that marine physical-chemical factors including salinity, ph, currents, temperature, and ever-changing brightness greatly affect the life of organisms in coastal intertidal zones. furthermore, rowe & doty (in hasan, 2004) said that another important factor influencing the distribution of echinoderms is the average topography of an island. density of marine animals depends on temperature, salinity, and pressure. in addition, aziz (1996) revealed that the condition of the substrate and habitat largely determines the distribution of echinoderms. the existence of echinoderms in the gunung kidul area, especially in the krakal and drini coast areas, has not yet been identified and is well known for its habitat trends, so research is needed to determine the diversity and abundance in the intertidal zone of the krakal and drini beach of gunung kidul, yogyakarta. materials and methods materials and tools the equipment used in this study were ph meter to measure the ph of seawater, a thermometer to measure water temperature, a flakon bottle to place echinoderms that have not been identified, 70% alcohol as a https://doi.org/10.14421/biomedich.2019.81.23-26 mailto:hikmahsupriyati@gmail.com 24 biology, medicine, & natural product chemistry 8 (1), 2019: 23-26 preservative for echinoderms, a rope for making transects, stakes as a barrier, gauge for measuring, scissors, tweezers and stationery. determination of transect and sampling the transect used in this study is the comb transect. the use of comb transects is due to the highest echinoderms in the intertidal zone. each area of the research site is made of a main transect with a length of 50 meters. then the main transect is divided into 5 sampling points, the distance between the sampling points is 10 meters. after that, each point was taken 3 observation plots with an area of 1x1 meters and the distance between plots 5 meters. figure 1. transect and sampling design. calculation of echinoderms diversity and abundance calculation of the diversity of echinoderms on the krakal and drini coast used a diversity index. the diversity index formula is (shannon and wienner, 1963): h’ : shannon wiener diversity index pi : ni/n = relative abundance of species h’ < 1 : low diversity and low community stability 1 < h’< 3 : medium diversity and stable community h’ > 3 : high diversity and high community stability meanwhile, to determine the abundance of types of echinoderms performed calculations with abundance index. the formula is as follows (rahma and fitriana, 2006): d = 𝑛𝑖 𝐴 d : the abundance of individual species i ni : number of individuals of the i-th species a : sampling plot area result and discussions table 1. number of species of echinoderms phylum. no species krakal beach drini beach 1 ophiotrix sp 594 876 2 echinometra sp 58 85 3 holothuria sp 1 0 4 crinoidea 1 0 based on the results of research conducted there were 4 types of species found on krakal beach. whereas 2 types of species found on the drini coast. the species most commonly found in the krakal coast and the drini coast are the ophiotrix sp. this is because the ophioridea class can be found starting from the intertidal area to a depth of more than 6500 meters (stohr, et al., 2012). the abundance index of phylum echinoderms on krakal and drini beaches can be seen in figure 2. figure 2. graph of the abundance of echinoderms on the krakal and drini beach. the results of the calculation of the abundance index of echinonderms on both beaches show that ophiuroidea and echinoidea are the most abundant class of echindermata found. when viewed from the substrate, krakal and drini beaches are beaches composed of coral. the abundance of corals on both coasts causes the abundance of echinoderms. according to yusron (2016) states that the ophiuroidea class and echinodea class are generally found in flat reef areas. both of these classes are commonly found on flats as a place to hide and find food in the rocks. likewise, research conducted by sugiarto and supardi (1995) which states that ophiuroidea and echinoidea are a class of phylum echinoderms have a tendency to attach to corals. in addition, ophiuroidea and echinodea classes have the ability to grip rocks and be able to refrain from waves so that they are able to adapt in coral areas (aziz, 1996). 5.11 0.88 0 0 3.96 0.3867 0.0067 0.0067 0 1 2 3 4 5 6 ophiotrix sp echinometra sp holothuria sp crinoidea abundance of echinoderms on krakal and drini beach drini krakal h’ = ∑ pi log pi supriyati et al. – echinoderms diversity and abundance in gunung kidul beach … 25 it is contrast to the holothuridea class which is very rarely found on krakal and drini beaches. this is because species in this class prefer habitats that are always inundated. meanwhile, based on observations on both beaches at low tide, the water content in the intertidal area is very small. these conditions make the cause of at least holothuridea species found. according to aziz (1996) holothuridea occupy habitats that are always flooded even at low tide, and for their life, these animals prefer clear water habitats and are relatively calm. crinoidea species are also very few found, this is because crinoidea usually live in coastal areas (yusron, 2016). while the research location does not pass through the edge. figure 3. graph the diversity of echinoderms on the krakal and drini beach. from this diagram it can be seen that waters on the krakal coast have a higher diversity index (0.139) when compared to waters at the drini coast (0.129). the difference in diversity obtained on the two beaches can be influenced by several internal and external factors. according to (shannon and wienner, (1963), the diversity of echinoderms on the drini and krakal beaches can be categorized as low because the results of the calculation of h 'is less than 1. the low diversity of echinoderms on the krakal and drini beaches indicates that the productivity level at both beaches is very low, so it can be said that the ecosystems on both coasts are unstable. the imbalance of ecosystems on the two beaches can be seen from the presence of a very dominant individual, the ophiothrichidae family. table 2. results of measurement of environmental parameters on krakal and drini beach. parameter krakal beach drini beach parameter stability ph 8 8 7,5-8,6 temperature 30oc 27oc 20oc-30oc temperature measurements on the drini and krakal beaches are 27º c and 30º c. the results of temperature measurements on both beaches are still classified as the optimum temperature that allows echinoderms to live and develop properly. thus echinoderms can be found abundantly on the coast of krakal and drini. the good temperature for the life of echinoderms is 20º c 30º c (aziz, 1998). the ph value describes the acidity and alkalinity intensity of a waters which is indicated by the presence of hydrogen ions. the value of the ph range in the study is still relatively good for seawater parameters. the results of ph measurements at the drini and krakal beaches are 8. the measurement results are in accordance with research conducted by romimohtarto and juwana (2007) where the ph of sea water in indonesia varies between 6.0-8.5. as according to aziz (1996), a good ph value for the life of echinoderms is 7.5-8.6. from the parameter measurements it can be seen that both the ph and temperature parameters at both the krakal and drini coast have values that correspond to the optimum parameter theory in the waters. this allows the existence of other factors that influence the differences in the results of the diversity of echinoderms on the krakal and drini beaches. based on observations, there are more seaweed on the krakal beach compared to the drini beach. the amount of seaweed in an ecosystem will affect the number of echinoderms. krakal beach with a large amount of seaweed has few echinometra species compared to the drini beach. the results of this study are in accordance with research conducted by maila (2017) which states that the amount of seaweed in an ecosystem is negatively correlated with the amount of echinometra sp. conclusions the diversity index on the krakal coast has a higher value (0.139) when compared to the waters on the drini coast (0.129). while the calculation of echinondermal abundance index shows that the drini beach location has a high abundance value when compared to the abundance value at the krakal beach location. from the results of parameter measurements, it can be seen that both the ph and temperature parameters at both the krakal and drini coast have values that correspond to the optimum parameter theory in the waters. interaction with biotic factors in the form of seaweed found in both locations still shows a range of tolerance that can support the life of echinoderms. references aziz, a. 1996. habitat dan zonasi fauna echinodermata di ekosistem terumbukarang. oseana 1. 24(2): 33-43. 0.124 0.126 0.128 0.13 0.132 0.134 0.136 0.138 0.14 krakal drini diversity index of echinoderms on krakal and drini beach diversity index 26 biology, medicine, & natural product chemistry 8 (1), 2019: 23-26 aziz, a. 1998. pengaruh tekanan panas terhadap fauna echinodermata. jurnaloseana, 13(3): 125-132 brower je, & zar jh. 1977. field and laboratory methods for general ecology. wm. j. brown company publ, iowa. p.288. hasan, s. 2004. kepadatan dan pola distribusi echinodermata di zona intertidal pantai pulau ternate. media ilmiah mipa. jalaluddin & ardeslan, 2017. identifikasi dan klasifikasi phylum echinodermata di perairan laut desa sembilan kecamatan simeulue barat kabupaten simeulue. jurnal biology education, vol 6 no, page 82-97 rahma, yulia & fitriana. 2006. keanekaragaman dan kemelimpahan makrozoo-benthos di hutan mangrove hasil reha-bilitasi taman hutan raya ngurah rai bali. jurnal biodiversitas vol. 7, no. 1. hal: 67-72. romimohtarto, k. & juwana, s. 2007. biologi laut: ilmu pengetahuan tentang biota laut. jakarta: djambatan: 540 prowe, f. e. w. and j. e. doty. 1977. the shallow-water holothuirans of guam. micronesia. shannon c. e., & wiener. 1963. the mathematical theory of communications. univ. illinois. urbana. p.117. soemodhiharjo. 1990. teluk ambon. ambon: balai penelitiandan pengembangan sumberdaya laut (lipi) ambon. sugiarto, h. & supardi 1995. beberapa catatan tentang bulu babi marga diadema. oseana 20(4): 35 triana r, dkk, 2015. identifikasi echinodermata di selatan pulau tikus, gugusan pulau pari, kepulauan seribu, jakarta. (jurnal pros sem nas masy biodis indon). vol 01 no. 03. program studi biologi, universitas al azhar indonesia. jakarta yusron, e. 2009. keanekaragaman jenis ekhinodermata di perairan teluk kuta, nusa tenggara barat. makara, sains, vol. 13: 45-49. yusron, e. 2010. keanekaragaman species ekhinodermata di perairan likupang, minahasa utara, sulawesi utara. ilmu kelautan (indonesian journal of marine sciences) juni 2010. vol. 15 (2) 85-90 yusron. 2016. struktur komunitas ekhinodermata (asteroidea, ophiuroidea, echinoidea dan holothuroidea) di perairan taman nasional wakatobi sulawesi tenggara. jurnal ilmu dan teknologi kelautan tropis, vol. 8, no. 1, hlm. 357-366 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 17-26 | doi: 10.14421/biomedich.2022.111.17-26 issn 2540-9328 (online) kidney evaluation in hyperuricemia rats treated with green tea leaves (camellia sinensis l.) extract putranty widha nugraheni1,*, chanif mahdi2 1department of environmental engineering, faculty of engineering, universitas tanjungpura, jl. prof. dr. h. hadari nawawi, kota pontianak, kalimantan barat 78124 tel. +62-561-739630, indonesia. 2department of chemistry, faculty of mathematics and natural science, universitas brawijaya, jl. veteran, kota malang, jawa timur 65145 tel. +62-341-551611, fax. +62-341-575777 indonesia. corresponding author* putranty@teknik.untan.ac.id manuscript received: 03 february, 2022. revision accepted: 07 february, 2022. published: 23 february, 2022. abstract uric acid is an oxidation product of the xanthine oxidase enzyme found in extracellular fluid, and when it exceeds, uric acid will build up and cause hyperuricemia. tnf-α is released by epithelial cells and mesangial cells when inflammation occurs and causes apoptosis in epithelial cells, causing damage to kidney structures and initiating acute kidney poisoning. green tea extract (camellia sinensis l.) contains many antioxidants, especially flavonoids with potent antioxidant properties such as lipid peroxidase and free radical absorbers, inhibiting xanthine oxidase. this study expresses the potential of green tea extract on kidney repair caused by hua. twenty-four male albino rats (175-225 g) of wistar strain being fed a high purine diet in 60 consecutive days and divided into six groups randomly, i: negative control, ii: positive control, iii: allopurinol, iv: green tea extract 150mg of body weight, v: green tea extract 300mg of body weight, and vi: green tea extract 600mg of body weight. treatment was done for 14 days and measured by total creatinine levels, malondialdehyde levels, and kidney histopathology. the statistical analysis using one way anova and post hoc tukey analysis by spss 23.0 proved that green tea extract with a dose of 600 mg/kg of body weight green could lower levels malondialdehyde of the kidney as much as 58.85% (p<0.01), decreased creatinine level by 24.5% (p>0.05), and improved kidney histopathology. this study proved that green tea extract is a promising alternative for hyperuricemia while improving kidney tissues and lowering malondialdehyde and creatinine levels. keywords: camellia sinensis l.; hyperuricemia; kidney creatinine; kidney histopathology; kidney malondialdehyde. abbreviations: ua, uric acid; hua, hyperuricemia; ros, radical oxygen species; nfκb, nuclear factor kappa-light-chain-enhancer of activated b cells; gte, green tea extract; alp, allopurinol; mda, malondialdehyde; bw, body weight; xod, xanthine oxidase; urat1, urate transporter 1; oat1, organic anion transporter 1; oat3, organic anion transporter 3; pbs, phosphate buffered sucrose; pfa, paraformaldehyde introduction uric acid (ua) comes from endogenous sources and exogenous sources. endogenous sources come from protein and nucleoprotein breakdown products, while exogenous sources come from purine foods. the body's average ua levels are 3.4-7.0 mg/dl in men and 2.4-6.0 mg/dl in women, and 75% of the urate will be excreted in the urine (lippi et al., 2008). ua that is ionized to urate will dominate the extracellular plasma and synovial fluid as monosodium urate at ph 7.4 so that the plasma will experience saturation. when ua levels in the body exceed normal limits, ua will build up and cause hyperuricemia (hua). if ignored, hua can impair kidney function, such as nephrolithiasis and urate nephropathy, leading to gouty arthritis and cardiovascular disease (cirillo et al., 2006). kidneys play an essential role in filtering blood so that urine is excreted by the body, including maintaining the homeostatic system of ua because the kidneys carry out 70% of ua expenditure. therefore, hua and gout are mainly due to a decrease in the relative kidney excretion of ua (lipkowitz, 2012). when there is an increase in ua levels in the urine, ua will experience saturation and form crystals that eventually form urinary tract stones, which can inhibit and injure the kidneys' tissues and functions and lead to uricosuria. previous studies (angielski, 1992) demonstrated heterogeneity of several enzymes for metabolizing adenosine centered on the rat kidney. the highest activity of ada, xanthine oxidase, and nucleoside phosphorylase was found in the cytosol in the glomerulus, where the glomerulus is a complex of three different cell types: epithelial, endothelium, and mesangial cells. increased excessive activity triggers https://doi.org/10.14421/biomedich.2022.111.17-26 18 biology, medicine, & natural product chemistry 11 (1), 2022: 17-26 tissue damage in the kidneys. several studies have shown that nfκb causes multiple organ damage, such as kidneys, liver, and pancreas (tugcu et al., 2006). activated nfκb plays an essential role in expressing pro-inflammatory cytokines such as tnf- and other mediators involved in the acute inflammatory response and different conditions associated with ros generation (arjumand et al., 2011). several studies have shown this to cause apoptosis and epithelial cells in tubular structures, causing damage to kidney structures and initiating acute kidney poisoning (abraham, 2003). hua is increasing significantly globally because it affects more than 2 billion people (kramer & curhan, 2002). in indonesia, the prevalence of this disease is the second highest, which is 24.7%, and possibly caused mainly by unhealthy eating habits. allopurinol (alp) is the most widely used drug because it is considered the most effective in inhibiting the formation of ua by inhibiting the activity of xanthine oxidase (boudiaf et al., 2010). however, its use can cause serious side effects, such as nephropathy, allergic reactions, and digestive disorders (feig et al., 2008). the side effects caused by drugs cause some people to choose to use medicinal plants that are considered capable of healing with minimal side effects. several medicinal plants have been tested for their effectiveness, such as indian nettle, rosella (astuti, 2011), and coffee (lelyana, 2008). green tea extract (gte) (camellia sinensis l.) contains many polyphenol antioxidants, especially flavonoids. the types of flavonoids in gte are (+)-catechin (c), (‒)-epicatechin (ec), (‒)-gallo catechin gallate (gcg), (‒)epigallocatechin (egc), (‒)-epicatechin gallate (ecg), and (‒)-epigallocatechin gallate (egcg) (jatuworapruk et al., 2014). gte has strong antioxidant properties by performing several mechanisms, such as anti-lipid peroxidase, free radical scavenger, metal binding, and inhibition of several enzymes, including xanthine oxidase (xod). previous studies have shown that chinese gte at the right dose can reduce ua content, decrease xanthine oxidase activity (xod) and urat1 expression, and increase oat1 and oat3 term in the kidneys of mice with hua (chen et al., 2015; fraga et al., 2010). gte also contains 8-30 mg of caffeine per cup (240 ml) (bolignano et al., 2007). caffeine is a type of alkaloid with the chemical formula 1,3,7trimethylxanthine (c8h10o2n4), is a diuretic. although several studies have stated that caffeine consumption can trigger excessive ua formation, low intake of caffeine can provide several benefits, such as treating headaches, increasing alertness, and relaxing muscles (paganini-hill et al., 2007; zhen et al., 2007). in gte, caffeine activity is inhibited because of the amino acid l-theanine. to provide a calming effect on the brain (rogers et al., 2008), l-theanine is also a caffeine antagonist to balance the activity of caffeine in the body (kakuda et al., 2000). thus, in this study, gte decaffeination did not perform. materials and methods procedures gte leaves extraction dry gte leaves (50 g) were crushed by blending and then sifted by a sieve of 80 mesh. gte powder was brewed using boiled water (± 95 ºc) with a ratio of 1:10. then, the result of maceration was cooled to room temperature, filtered, and separated between the filtrate and the dregs. the brewing process was repeated up to 3 times and concentrated using a vacuum rotary evaporator at a temperature of 85 °c and 110 rpm. the gte's extraction yield was 24.94% (w/w) and kept in a 20 ºc freezer. acclimatization of experimental animals the experimental animals used in the study were 24 male albino rats (rattus norvegicus) wistar strain aged 2-2.5 months with an average body weight of 175-225 g, which were purchased from the provider of laboratory animals in bandung, indonesia. all the ethical animal procedures have been approved by ub's research commission (approval code 690-kep-ub). the rats were placed in a polyethylene cage filled with wood husks inside with a dimension of 45 x 35 x 20 cm with wire enclosures at the animal laboratory of biosains of universitas brawijaya, malang, indonesia. the room temperature was 23±2 ºc. the rats were acclimated for a week before the experiments with a standard feed and water ad libitum. induction of high purine diet and drugs treatment twenty-four rats were divided into six groups, which each group had four rats: (1) negative control group, (2) positive control group, (3) alp therapy of 5mg/kg of body weight, (4) gte therapy of 150 mg/kg of body weight, (5) gte therapy of 300 mg/kg of body weight, and (6) gte therapy of 600 mg/kg of body weight. each rat was fed a high purine diet, except the (1) group. based on a previous study (rahmawati et al., 2018), we used a high-purine diet consisting of cow's liver, cow's spleen, fried melinjo (gnemon gnetum), and fried peanuts blended and dissolved into the water. the filtrate was introduced was 3 ml/rats, twice a day (at 8:00 and 14:00) daily for 60 consecutive days. the ua level was examined every five days. when ua levels were more than 7 mg/dl, rats were ready to be treated. after ua levels of five groups reached 7 mg/dl, rats in (3) group were administered using 3 ml alp with a dose of 5 mg/kg of body weight. rats in (4), (5), and (6) groups were treated with 3 ml of gte with various doses. all rats were sacrificed for kidney and blood collection on the same day. nugraheni & mahdi – kidney of hyperuricemia rats 19 blood and organ collections blood was drawn from the rats every five days to check the ua levels. measurements were carried out using a digital tool, "easy touch gcu." after calibrated, the test strip was inserted into the detector slit on the device until the screen displayed a "blood drop" image that indicated the device was ready for use. the tail of the rat disinfected with 70% ethanol was massaged, then the tip of the rat's tail was pierced with a sterile blood lancet. the first drop of rat blood was discarded, then the next drip was absorbed into the test strip. within 20 seconds, the ua level will appear on the device screen in mg/dl. the test was performed on each rat in all groups every day until sacrificing day. the rats were dislocated in the neck, and the rats were arranged in a supine position ventral above on a surgical board and dissected by splitting the inguinal area. then both kidneys were taken and washed with 0.9% nacl. the first kidney was immersed in pbs, while the other was immersed in 4% pfa. after rats sacrificing, about 2 hours after blood collection, blood was centrifuged at 2000 rpm for 10 min to collect blood serum. serum was moved into the eppendorf tube and centrifuged at 1000 rpm for 10 minutes. serum samples were transferred into a new eppendorf tube and stored in a -20 ºc freezer. jaffe test's measurement of creatinine levels serum creatinine levels were determined by enzymatic reactions using the "point scientific creatinine kit.”. 1000 l of creatinine reagent was taken and put into a microtube, then incubated at 37 ºc for 5 minutes. furthermore, 50 l of the protein-free filtrate was added, transferred into a cuvette, and the stopwatch was turned on for 60 seconds. the absorbance was measured (a1) using a spectrophotometer with a wavelength of 510 nm, then waited again for 60 seconds, the absorbance was measured again (a2). the absorbance value used is a2-a1. measurement of mda levels half gram of kidney were crushed with quartz sand in a mortar placed on ice gel. grounded kidneys were added by 1 ml of 0.9% nacl solution and mixed until homogeneous. the homogenate was put into microtubes, sonicated for 10 minutes, centrifuged at 8000 rpm for 20 minutes, then the supernatant formed was taken carefully using a micropipette. furthermore, 100 l of kidney supernatant was taken from each microtube, wrapped in aluminum foil, and added 550 l of distilled water. each tube was added with 100 l of 10% tca, 100 l of 1% na-thio, and 250 l of 1m hcl and each tube was homogenized with a vortex. each tube was incubated in a water bath at 100 ºc for 30 minutes and centrifuged at 500 rpm for 10 minutes. then the centrifuged supernatant was transferred to a new eppendorf tube. after heating, the supernatant was cooled at room temperature until a pink complex was formed. the supernatant was then measured for absorbance with a uv-vis spectrometer at a wavelength of 530 nm. then the absorbance results were plotted on a curve to calculate the mda levels. kidney's histological evaluation embedding kidney organs using the bancroft method kidneys were soaked into a fixative solution of paraformaldehyde, then into 70% ethanol for at least 24 hours, followed by immersion in 80% ethanol for 2 hours. the kidneys were washed with 90% and 95% ethanol, respectively, for 30 minutes. then, the kidneys were immersed into absolute ethanol three times for 30 minutes, then xylol two times for 30 minutes each in an incubator at 60-63 ºc for 30 minutes. the kidneys were immersed in xylol and paraffin three times each. the embedding process was continued by dipping the kidneys into liquid paraffin poured into a container. kidney slide set making the kidney on the embedding paraffin block was inserted into the microtome block holder, and the cutting surface was aligned with the microtome blade. cutting begins by adjusting the thickness of the slices above 10 m. kidneys were sliced to a size of 5 m. slices were taken with a brush and immersed in water at room temperature to reveal possible creases in the slides. the slices were transferred with a brush into warm water at 38-40 ºc to straighten the wrinkles then placed on to object-glass. the pieces were dried and placed on a hot plate at 38-40 ºc until the slides dry. furthermore, the slides were stored in an incubator at a temperature of 3840 ºc for 24 hours. hematoxylin eosin staining procedures deparaffinization was carried out by putting the slides into xylol three times for 5 minutes each. then, the rehydration stages were done by putting slides into graded ethanol, starting with absolute ethanol, 95%, 90%, 80%, and 70% ethanol, and distilled water for 5 minutes. the staining stages were carried out by putting the slides into a hematoxylin dye container for 10 minutes. after completed, the slides were washed with running water for 30 minutes, then rinsed with distilled water. the slides were then immersed in eosin alcohol dye for 5 minutes. for the dehydration process, the slides are added to graded ethanol from 80%, 90%, 95%, and absolute ethanol concentrations. the slides are put into xylol two times, dried, and mounted. statistical analysis the data obtained were analyzed using a data normality test using shapiro-wilk statistic and homogeneity to determine the normality of data distribution. effects of treatment on parameters were analyzed using anova, which was completed by tukey test with 99% 20 biology, medicine, & natural product chemistry 11 (1), 2022: 17-26 confidence level to know the difference between treatments. statistical analysis was performed using statistical package for social sciences 23.0 software. results were significant when p < 0.01. results and discussion uhplcms/ms gte analysis the gte used was tested qualitatively using uhplcms/ms. the result of the qualitative analysis of gte was presented in figure 1, figure 2, and table 1. table 1. parameters in qualitative analysis using uhplcms/ms of gte flavonoid. rt peak fragment ions (m/z) standard fragment ions (m/z) molecular ions [m]+ (m/z) prediction of flavonoid compounds 3.72 136.5-137.5 137 305 (‒)-gallo catechin (gc), (‒)-epigallocatechin (egc) 4.29 168.5-169.5 169 457 (‒)-epigallo catechin gallate (ecgc), (‒)-gallo catechin gallate (gcg) 4.44 204.5-205.5 205 289 (+)-catechin (c), (‒)-epicatechin (ec) 4.94 168.5-169.5 169 441 (‒)-epicatechin gallate (ecg), (‒)-catechin gallate (cg) based on figure 1, the gte was analyzed qualitatively some peaks appeared with its retention time. the highest chromatogram peak from uhplcms/ms spectra formed at the retention time of 4.29 min with molecular ions [m+] weight of 457 m/z. based on the test analysis, the most abundant components in gte are epigallocatechin gallate (ecgc) or gallo catechin gallate (gcg) and epicatechin gallate (ecg) or catechin gallate (cg) with the molecular ions [m+] weight of 441 m/z and standard fragment ion of 169 m/z. this peak was identified as epigallo catechin gallate (egcg) or its epimer, gallo catechin gallate (gcg), with a molecular formula of c22h18o11. figure 1. uhpmcms/ms chromatogram of gte compounds and epigallo catechin gallate (egcg) or gallo catechin gallate (gcg) molecular structure. gte effects on mda levels in kidneys the test was carried out with the thiobarbituric acid test based on the color changes when mda and tba reacted. mda levels in the kidneys indicate how much cell damage occurs due to lipid peroxidation due to free radicals formed due to xod activity. the pink color created was calculated for absorbance at a wavelength of 532 nm. figure 2. the reaction of mda and thiobarbituric acid (nair et al., 1981). nugraheni & mahdi – kidney of hyperuricemia rats 21 the statistical test results using one way anova analysis showed that the administration of gte with three different dosage variations could reduce mda levels in the kidneys with a very significant difference between treatments (p < 0.05). figure 3. graph of mda levels in the kidneys. $p < 0.01 compared to (1). #p < 0.05 compared to positive control. the (1) group had the lowest kidney mda level of 0.47 ± 0.14 g/dl. this group was a group of rats that were not given any treatment, only given standard food and drink, so they are a group of healthy rats because their kidney mda levels are still below the normal range, at 1.04 ± 0.43 g/dl. the (2) group was the group that was given a highpurine diet so that the mda level reached 2.84 ± 0.59 mg/dl. this level is high compared to the (1) group because of the increase of 507.07% compared with the (1) group. in the (3) group, the levels of mda formed in the kidneys were 1.91 ± 0.36 g/dl, with a decrease in levels of 32.67%. in the (4), (5), and (6) groups, the kidney mda levels were 2.54 ± 0.61 g/dl, 2.22 ± 0.61 g/dl, and 1.31 ± 0.26 g/dl with decreased levels of 10.30 %, 21.56%, and 53.85% respectively. gte effects on creatinine levels in serum determination of creatinine levels in blood serum was determined by the jaffé reaction colorimetric method, with the principle of measuring the absorbance of the picrate reaction in alkaline conditions at a wavelength of 540 nm and creating a complex with a reddish color. figure 4. the reaction mechanism for the formation of the janovsky complex (toora & rajagopaj, 2002). the results of the statistical test using one-way analysis of variation (one way anova) showed that giving gte with three different dosage variations could reduce creatinine levels in the kidneys, but not significant (p > 0.05). 22 biology, medicine, & natural product chemistry 11 (1), 2022: 17-26 figure 5. graph of creatinine levels in serum. the lowest serum creatinine level was in the (1) group, 0.59 ± 0.08 mg/dl. the positive control group was the group that was given a high-purine diet so that the creatinine level reached 1.44 ± 0.19 mg/dl. in this study, alp showed its ability to significantly lower creatinine levels than the (1) groups at 1.18 ± 0.13 mg/dl, with a reduced rate of 22.41%. in this study, there was a significant change and relationship between the decrease in ua levels between positive controls and alp therapy (p = 0.05). in the (4), (5), and (6) group, the creatinine levels were 1.35 ± 0.18 mg/dl, 1.26 ± 0.19 g/dl, and respectively. 1.01 ± 0.10 g/dl with decreased levels of 6.11%, 12.21%, and 24.50% respectively. gte effects on kidneys histopathology kidneys of rats were examined in histopathology to investigate the level of damage caused by hua and the improvement that occurred due to alp therapy and gte. figure 6. kidney histopathology with he staining (400x magnification); (a) negative control; (b) positive control; (c) alp therapy; (d) gte therapy of 150 mg/kgbw; (e) gte therapy of 300 mg/kgbw; (f) gte therapy of 600 mg/kgbw; (g) glomerulus; (bc) bowman's capsule; (pl) parietal layer; (vl) visceral layer; (v) vacuolization/fat accumulation; (p) pyknosis; (kr) karyorrhexis; (kl) karyolysis discussion the structure of egcg and gcg has eight glycosylate groups that can interact with the active site of the xod enzyme so that it can inhibit the formation of ua. this fact is in line with previous studies (chen et al., 2015; jatuworapruk et al., 2014; nugraheni et al., 2017) which reported that gte could reduce ua levels by inhibiting xod enzyme activity and modulating urate transporters in the kidneys, thereby increasing the excretion of ua through the kidneys. in this study, mda levels were also determined to determine whether there was an effect of induction of a nugraheni & mahdi – kidney of hyperuricemia rats 23 high-purine diet on the lipid peroxidation process and the impact of gte on decreasing mda levels after a high-purine diet was induced. the (1) group had the lowest kidney mda level. this group was used to compare the increase or decrease in kidney mda levels due to treatment, induction of a high-purine diet, and gte therapy. the (2) group was the group that was given a high-purine diet so that the mda level reached 507.07% compared with the negative controls. this high level of mda is caused by excessive xod activity and produces ros in the form of h2o2 and o2 -• as a form of innate immunity (boumerfeg et al., 2012). oxidative stress is generally defined as an excess of ros generation that can damage essential biomolecules such as dna and lipids massively and cause the formation of chronic diseases such as atherosclerosis, cancer, diabetes, aging, and others, including hua (doehner et al., 2016). excessive xod activity causes the formation of ros, which can cause damage to living tissue in the body due to lipid peroxidation. (d.-h. kang et al., 2002). in the (3) group, the levels of mda formed in the kidneys were 1.91 ± 0.36 g/dl, with a decrease in levels of 32.67%. alp can reduce mda levels well and prevent kidney damage, consistent with previous studies (asghar et al., 2007; chang et al., 2021; noeman et al., 2011). in the (4), (5), and (6) groups, the kidney mda levels were decreased 10.30 %, 21.56%, and 53.85%, respectively. this fact is in line with previous studies (asghar et al., 2007; noeman et al., 2011), which reported that gte could reduce mda levels in the kidneys by inhibiting xod enzyme activity. the antioxidant capacity of a phenolic antioxidant is directly associated with the ability to donate hydrogen radicals from the phenol group and the presence of unpaired electrons in the aromatic ring. figure 7. the structure and parts of flavonoids that have potential antiinflammatory activity (unsaturated c chain, number and position of hydroxyl groups on ring b, carbonyl groups on c-4 carbon, and nonglycosylation) (lago et al., 2014). the antioxidant activity of non-glycosylated flavonoids is related to the number and position of the hydroxyl groups present in the molecule. the hydroxyl group in the aromatic core has a low antioxidant capacity (d. kang et al., 2005; soobrattee et al., 2005). this result is in line with previous studies (cao et al., 2010), (forester & lambert, 2011) which reported that flavonoid compounds such as c, ec, and egc could be ros absorbers due to 3'-oh and 4'oh hydroxyl groups at ortho positions that increase its antioxidant capacity. the three most important structures are (a) the orthodihydroxy catechol at ring b, (b) a double-chain c sigma bond combined with a carbonyl bond at c-4 in ring c, and (c) a hydroxyl group at the c-3 and c-5 position. the egc structure that fulfills the three requirements above is concluded as the primary agent in the process of reducing ros activity and reducing lipid peroxidation. the decrease in kidney mda levels in alp therapy was lower than gte's decrease in mda levels. this result indicates that alp can reduce oxidative stress levels by lowering ua levels and improving kidney function. still, the flavonoids in gte are more effective in acting as nephroprotection because of their antioxidant activity and hypouricemic effect. several species of flavonoid antioxidants, especially catechins, are the best ros absorbers (boudiaf et al., 2010) by inhibiting the mechanism of ros generation in tissues and inhibiting the formation of hydroxyl radicals (lin et al., 2000). creatinine was measured as a marker of kidney damage. creatinine levels above normal limits usually characterize the diagnosis of kidney failure due to low creatinine clearance by the kidneys. the (2) group was the group that was given a high-purine diet so that the creatinine level increased by 145.07% compared to the (1) group. however, these levels are still within the normal range, so this shows that a high-purine diet for 60 days does not cause significant damage to the kidneys. hua can induce high blood pressure, increased hydrostatic pressure, and kidney injury (goicoechea et al., 2010). high ua levels are associated with the onset of kidney-related disease in the late phase. creatinine is a toxic substance produced when the body usually breaks down creatinine phosphate in muscles and at a constant rate depending on muscle mass. a previous study (d. kang et al., 2005) found that hua rats showed higher proteinuria, blood pressure, and serum creatinine levels than the control group treated with alp as the most effective ua lowering agent. in this study, alp showed its ability to significantly lower creatinine levels than the (1) group with a reduced rate of 22.41%. by reducing and excreting excess levels of ua in blood serum, alp can indirectly act as a glomerular hydrostatic pressure-lowering agent and thereby relieve kidney damage. in this study, there was a significant change and relationship between the decrease in ua levels between (2) group and (3) group (p = 0.05). thus, the effect of alp in reducing the progression of kidney disease may be related to its ability to lower serum ua levels. 24 biology, medicine, & natural product chemistry 11 (1), 2022: 17-26 in the (4), (5), and (6) groups, the creatinine levels decreased 6.11%, 12.21%, and 24.50%, respectively. based on statistical analysis using the post hoc tukey test, the decrease in creatinine levels due to alp therapy and the three doses of gte therapy did not show a significant average difference. so, it can be concluded that the ability of creatinine-lowering agents from gte is as good as alp. the formation of creatinine begins with the transamination process from arginine to glycine to form glycocyamine or guanidoacetic acid (gaa). this reaction generally occurs in the kidneys, but the response also appears in the mucosa of the small intestine and pancreas. the creatinine concentration tends to be constant because it is evenly distributed throughout the body. under normal conditions, creatinine is excreted through the kidneys, but its production increases with age due to decreased muscle mass. with a small molecular weight of about 113 daltons, creatinine is easily filtered by the glomerulus and is not reabsorbed or affects the urinary rate (hosten, 1982). the presence of flavonoids that can inhibit the activity of the xod enzyme can reduce the production of free radicals and reduce the level of oxidative damage (dahal & mulukuri, 2015). by decreasing the level of oxidative damage, creatinine can be re-filtered by the glomerulus and excreted adequately by the kidneys. the (1) group showed normal rat kidney condition, characterized by no vacuolization or fat accumulation, standard glomerular shape, and no signs of necrosis, either pyknosis, karyolysis, or karyorrhexis. this condition is because group a was not given a highpurine diet and was only fed standard water, so there was no significant tissue damage. meanwhile, (2) group were given a high-purine diet until the rats experienced hua. shriveled glomerulus and blackened nucleus indicate pyknosis caused by damage to membranes, mitochondria, and the golgi apparatus. the high xod activity increased the production of ros and increased the occurrence of lipid peroxidation, which caused tissue damage. vacuolization also occurs in more significant numbers, caused by cell degeneration due to lipid metabolism disorders that lead to excessive triglyceride accumulation in the cytoplasm with the formation of vacuoles. the cells will experience hypoxia and metabolic disorders. if this happens continuously, it will cause more severe tissue damage, such as karyorrhexis (nuclear rupture and scattering of chromatin fragments scattered around the cell) and karyolysis (the loss of the nucleus due to its inability to be stained), so that the cell pales and disappears. the resulting cell death or necrosis elicits an inflammatory response in living tissues (asghar et al., 2007). kidney histopathology in (3) group showed some improvements. although vacuolization and pyknosis still happen, the condition of the glomerulus has improved, and no karyorrhexis and karyolysis has occurred. this condition is due to the ability of alp to inhibit xod enzyme activity and inhibit the formation of ros. the ultrafiltration of urine causes glomerular damage through the glomerulus, which causes urate crystals not to penetrate the filtration membrane and cause hypertension. the difference in pressure between the glomerular capillary blood pressure and the colloid osmotic pressure in bowman's capsule can cause glomerular damage. decreased glomerular filtration is evidenced by increased blood creatinine levels and decreased creatinine clearance so that this condition can be associated with nephrotic syndrome (dahal & mulukuri, 2015). histopathological appearance of kidneys treated with gte therapy groups showed some tissue improvement. there was a reduction in vacuolization, pyknosis, and karyorrhexis. this condition shows that green tea can improve the histopathological picture of the kidneys because of its antioxidant activity. it can protect the kidneys from oxidative stress and lipid peroxidation because of its ability to reduce ros. overall, a diet high in purines can cause damage to kidney cell tissue, especially the glomerulus. this fact is shown by creatinine levels that are still in the normal range. however, if a high-purine diet is continued for a long time, glomerular damage can become more severe and lead to further complications of hyperuricemia. conclusions it can be concluded from this study that gte significantly inhibited the activity of the xanthine oxidase enzyme, can lower blood creatinine levels, and reduce malondialdehyde levels in the kidneys. gte also can improve the kidney histopathological picture of rats with hua due to a high-purine diet. this study proved that gte is a promising alternative treatment for hua. for the following research, it is necessary to conduct further studies on the mechanism of reducing ua with other flavonoid products, such as quercetin, silybin, and luteolin, to compare their effectiveness and determine the effective dose of gte therapy in lowering ua levels. acknowledgements: financial support for this work from the fund grant of the indonesia endowment fund for education ministry of finance republik indonesia (lpdp kementerian keuangan ri) is gratefully acknowledged. authors’ contributions: putranty widha nugraheni and chanif mahdi designed the study and analyzed the results. putranty widha nugraheni performed the material preparation and data collection. chanif mahdi supervised the laboratory work. putranty widha nugraheni & mahdi – kidney of hyperuricemia rats 25 nugraheni wrote the first draft of the manuscript. chanif mahdi provided the critical reading and insightful recommendations of the manuscript. all the authors have read the final 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(2007). green tea polyphenol epigallocatechin-3-gallate inhibits oxidative damage and preventive effects on carbon tetrachloride – induced hepatic fibrosis. journal of nutritional biochemistry, 18, 795–805. https://doi.org/10.1016/j.jnutbio.2006.12.016 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 619-624 | doi: 10.14421/biomedich.2023.122.619-624 issn 2540-9328 (online) investigating the insecticidal properties of alangium salviifolium root extracts on culex quinquefasciatus mosquitoe alex yagoo*, m.c. john milton, jelin vilvest pg & research department of advanced zoology & biotechnology, loyola college (autonomous), chennai, india. corresponding author* alexyagoo@loyolacollege.edu manuscript received: 30 august, 2023. revision accepted: 12 december, 2023. published: 28 december, 2023. abstract this study evaluated the effectiveness of successive hexane, chloroform, and methanol extracts of alangium salviifolium roots in combating mosquito-borne diseases caused by culex quinquefasciatus mosquitoe in india. the hexane extract showed the highest efficacy with lc50 values of 105.0 ppm (larvicidal activity), 108.0 ppm (pupicidal activity), and 65.6 ppm (ovicidal activity). the chloroform extract had lc50 values of 156.8 ppm (larvicidal), 376.0 ppm (pupicidal), and 20.0 ppm (ovicidal), while the methanol extract had lc50 values of 235.7 ppm (larvicidal), 441.8 ppm (pupicidal), and 30.4 ppm (ovicidal). the hexane extract of a. salviifolium roots shows potential as a mosquito control agent, specifically against cx. quinquefasciatus mosquitoes. further studies are needed to assess the safety and effectiveness of this extract. this research highlights the potential of plant extracts as alternative mosquito control agents for mitigating mosquito-borne diseases in india. keywords: alangium salviifolium roots; culex quinquefasciatus; plant extract; toxicity. abbreviations: cx.quinquefasciatus: culex quinquefasciatus; a.salviifolium: alangium salviifolium; lc: lethal concentration; ll: lower limit; ul: upper limit; ppm: parts per million; dmso: dimethyl sulfoxide introduction vector-borne diseases are a group of illnesses that are transmitted to humans and animals through arthropod vectors, such as mosquitoes, ticks, fleas, and sandflies. these diseases are prevalent worldwide and pose a significant threat to public health (alvarado-esquivel et al., 2021; tack et al., 2021). mosquito-borne diseases are among the most prevalent vector-borne diseases globally and can cause significant illness and death in affected populations. these diseases are transmitted through the bite of infected mosquitoes, which inject the diseasecausing pathogens, such as viruses or parasites, into the human bloodstream (who, 2021; bhatt et al., 2013). globally, mosquito-borne diseases such as malaria, dengue fever, and zika virus continue to be major public health problems, particularly in lowand middle-income countries. according to the world health organization (who), there were an estimated 229 million cases of malaria worldwide in 2019, with an estimated 409,000 deaths, most of which occurred in sub-saharan africa. dengue fever is also a significant public health problem, with an estimated 100 million cases worldwide each year. the majority of dengue cases occur in asia and the americas. the burden of zika virus is less well-known, but outbreaks have occurred in several countries in recent years, including brazil, colombia, and india (who, 2020; who, 2020). in india, mosquito-borne diseases are also a significant public health problem. malaria, dengue fever, chikungunya, filariasis and japanese encephalitis are the most common mosquito-borne diseases in india. according to the national vector borne disease control programme (nvbdcp), there were over 5 million cases of malaria in india in 2019, with 229 deaths. dengue fever is also a significant problem in india, with over 1.8 lakh cases reported in 2019, including 132 deaths. chikungunya is less common but still occurs, with over 60,000 cases reported in 2019 (mhfw, 2020; nvbdcp, 2020). prevention and control of mosquito-borne diseases involve several strategies, including reducing mosquito breeding sites, using insecticides, and wearing protective clothing. vaccines are available for some diseases, such as yellow fever and japanese encephalitis, but not for all mosquitoborne diseases. natural insecticides can be just as effective as synthetic insecticides for controlling insect pests (tabanca et al., 2017). mosquitoes have been known to develop resistance to synthetic insecticides, which has led to the exploration of alternative control methods, including the use of natural products or plantderived products. these products may have insecticidal properties that can effectively control mosquito https://doi.org/10.14421/biomedich.2023.122.619-624 620 biology, medicine, & natural product chemistry 12 (2), 2023: 619-624 populations, and they may also be less harmful to the environment and non-target organisms than synthetic insecticides (das and dhiman, 2015; govindarajan et al., 2015). furthermore, the use of synthetic insecticides has been associated with negative effects on the environment and non-target organisms, such as pollinators and aquatic organisms. in contrast, natural products and plantderived products are often biodegradable and less toxic, which can reduce their impact on the environment (isman, 2006). alangium salviifolium, commonly known as sage-leaf alangium, is a plant species belonging to the family alangiaceae. it is distributed throughout the indian subcontinent, southeast asia, and parts of china. the plant is known for its traditional medicinal uses, with its bark, roots, and leaves being utilized to treat various ailments such as fever, inflammation, wounds, and snake bites. phytochemical studies have revealed that a. salviifolium contains a diverse array of chemical constituents, including flavonoids, alkaloids, steroids, terpenoids, and phenolic compounds. these compounds are believed to be responsible for the plant's pharmacological activities. it has been found to possess pharmacological activities such as anti-inflammatory, antioxidant, antitumor, antimicrobial, antidiarrheal, and hepatoprotective properties. its extracts have shown promising results in various studies, indicating its potential use in treating various diseases. the plant is also of ecological significance, as it serves as a food source for various animals, including birds and insects. the wood of a. salviifolium is hard and durable, and it is used for various purposes, such as construction, furniture, and tool handles (sharma et al., 2015; dhanasekaran et al., 2018; saxena et al., 2013). in this communication, we present the effect of successive extracts of hexane, chloroform, and methanol of root of the a. salvvfolium on the larvicidal, pupicidal, and ovicidal activities of cx. quinquefasciatus. materials and methods collection of plant materials in february 2022, the roots of alangium salviifolium were collected from palayamkottai, tirunelveli district of tamil nadu, india. the plant material was then authenticated by dr. k. n. sunilkumar, a research officer from the department of pharmacognosy at the siddha central research institute in chennai. a voucher specimen with an authentication code (no: a24012302s.) was deposited in the institute's herbarium. extraction of the plant materials the plant material, consisting of 1 kg of dried roots, was first ground into a coarse powder and then extracted using a soxhlet apparatus with hexane, chloroform, and methanol in successive order. the resulting extracts were filtered and concentrated with a vacuum rotary evaporator, and the dry extracts were stored in airtight containers at a temperature of 4º c until further use. the yield obtained from the three extractions were 4.15 g, .7.22 g, and 10.48 g for hexane, chloroform, and methanol, respectively. insect rearing culex quinquefasciatus third instar larvae were procured from the entomology research institute, chennai for insect rearing. the larvae were bred in a controlled environment of 27±2°c temperature, 75-85% relative humidity, and a 13:11 l/d photoperiod using chlorinefree tap water. the larvae were provided with a diet consisting of a mixture of dog biscuits and brewer's yeast in a ratio of 3:2 (reegan et al., 2021). larvicidal and pupicidal assays to evaluate the larvicidal and pupicidal activities of the extracts, the guidelines provided by the world health organization (who, 2005) were followed. various concentrations of the extracts, including 500 ppm, 250 ppm, 125 ppm, and 62.5 ppm, were tested with five replicates for each concentration. an emulsion in 1.0% aqueous dmso was prepared for all three activities. plastic containers with a volume of 150 ml were used for the tests, and 20 larvae or pupae were added to 100 ml of the extract solution. a negative control consisting of a 1% aqueous dmso solution was used, while temephos was used as a positive control. dead larvae or pupae were identified when they showed no movement upon being touched with a glass rod. the percentage mortality and corrected percentage mortality were calculated using standard formulas (abbot, 1925). percentage mortality: 𝑁𝑜. 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑙𝑎𝑟𝑣𝑎𝑒 𝑜𝑟 𝑝𝑢𝑝𝑎𝑒 𝑁𝑜. 𝑜𝑓 𝑙𝑎𝑟𝑣𝑎𝑒 𝑜𝑟 𝑝𝑢𝑝𝑎𝑒 𝑒𝑥𝑝𝑜𝑠𝑒𝑑 × 100 corrected percentage mortality: [1 − 𝑛𝑇/𝑛𝐶] × 100 the corrected percentage mortality formula is used when the mortality rate in the control group is less than 5%. in this case, the formula adjusts for the natural mortality rate in the control group. it is calculated by subtracting the mortality rate in the control group from the mortality rate in the treated group, and then dividing by the difference between 100 and the mortality rate in the control group. ovicidal activity the ovicidal activity of the extracts was determined using a modified version of the method described by yagoo, et al. – alangium root extracts combat mosquitoes 621 yagoo et al., 2023. freshly laid eggs of cx. quinquefasciatus were exposed to different concentrations of the extracts in five replicates. the same concentrations used for larvicidal and pupicidal activities were tested. after 120 hours post-treatment, the hatchability of the eggs was observed under a compound microscope. the percentage of ovicidal activity was calculated using the following formula: percentage of ovicidal activity: 𝑁𝑜. 𝑜𝑓 𝑢𝑛ℎ𝑎𝑡𝑐ℎ𝑒𝑑 𝑒𝑔𝑔𝑠 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑒𝑔𝑔𝑠 𝑒𝑥𝑝𝑜𝑠𝑒𝑑 × 100 the results were compared with those of the standard control, temephos. statistical analysis in order to determine the concentration at which larvicidal, pupicidal, and ovicidal agents resulted in a certain percentage of mortality, statistical analysis was performed using probit analysis software (version 1.5) from the us epa. this analysis allowed us to estimate the lc50 and lc90 values for each concentration. a pvalue of ≤0.05 was considered significant, indicating that the observed differences were unlikely to have occurred by chance (finney, 1971). results and discussion in this study, we evaluated the effectiveness of hexane, chloroform, and methanol extracts of a. salviifolium roots against third instar larvae of cx. quinquefasciatus. we conducted larvicidal and pupicidal assays and the outcomes are presented in table 1 respectively. table 1. lethal concentration (in ppm) of crude extracts of roots of a. salviifolium against the larvae and pupae of cx. quinquefasciatus. species extract lc50 (ppm) 95% confidence limit lc90 (ppm) 95% confidence limit slope ± se intercept ± se χ2 ll ul ll ul c x .q u in q u ef a sc ia tu s la rv ae hexane 105.0 28.2 205.4 227.0 138.4 1289.1 3.8 ± 0.7 -2.7 ± 1.5 9.6* chloroform 156.8 36.0 499.4 391.2 209.1 7244.2 3.2 ± 0.6 -2.0 ± 1.4 1.5* methanol 235.7 88.3 2335.9 711.2 331.8 1332.7 2.6 ± 0.5 -1.3 ± 1.3 5.9* c x .q u in q u ef a sc ia tu s p u p ae hexane 108.0 45.8 184.7 238.7 150.4 2322.1 3.7 ± 0.6 -2.5 ± 1.3 7.7* chloroform 376.0 319.5 463.6 1389.5 984.7 2335.5 2.2 ± 0.2 -0.8 ± 0.5 1.5* methanol 441.8 376.1 547.4 1407.4 1011.8 2330.4 2.5 ± 0.2 -1.7 ± 0.7 4.4* lc50-lethal concentration that kills 50% of the exposed larvae; lc90-lethal concentration that kills 90% of the exposed larvae; ll-lower limit (95% confidence limit); ul-upper limit (95% confidence limit). *p ≤ 0.05, level of significance of chi-square values. the results of the study revealed that the hexane extract exhibited the highest larvicidal activity against both cx. quinquefasciatus, with lc50 values of 105.0 ppm, respectively. the chloroform extract showed moderate activity, with chloroform being more active than the methanol extract, as shown in table 1. in terms of pupicidal activity, the hexane extract was also more active compared to the chloroform and methanol extracts, with lc50 values of 108.0 ppm, respectively, for cx.quinquefasciatus mosquito species as shown in table 1. notably, the hexane extract also exhibited significant ovicidal activity, with lc50 values of 65.6 ppm for cx. quinquefasciatus, respectively. on the other hand, the ovicidal activity of the chloroform and methanol extracts was low for mosquito species, even at the highest concentration of 500 ppm. the range of ovicidal activity for the other two extracts was 16.8% to 30.4%. 622 biology, medicine, & natural product chemistry 12 (2), 2023: 619-624 figure 1. percent ovicidal activity of crude extracts of roots of a. salviifolium against cx. quinquefasciatus eggs. these findings were further supported by figure 1, which clearly demonstrated the superior efficacy of the hexane extract against mosquito species, while the chloroform and methanol extracts showed limited activity. in summary, the results indicate that the hexane extract is highly effective against cx. quinquefasciatus, exhibiting potent larvicidal, pupicidal, and ovicidal activities. on the other hand, the chloroform and methanol extracts showed comparatively lower activity, with the hexane extract being the most promising for further investigation as a potential mosquito control agent. the insecticidal activity of a. salvifolium has been reported in several studies, highlighting its potential as a natural source of insect control agents. several studies have reported on the insecticidal activity of different parts of a. salvifolium against various insect species. for instance, a study investigated the insecticidal activity of the methanol extract of a. salvifolium bark against, helicoverpa armigera, spodoptera litura, and dysdercus cingulatus. the study reported significant insecticidal activity of the extract against all species, with the highest activity observed against dysdercus cingulatus (baskar et al., 2011). moreover, a study examined the insecticidal activity of the leaf extract of a. salvifolium against the stored grain pest sitophilus oryzae, and reported significant insecticidal activity of the extract. the study found that the extract caused mortality in the insect at concentrations as low as 0.5%, and also exhibited repellent activity against the pest (kavitha et al., 2018). another study evaluated the insecticidal activity of different solvent extracts of a. salvifolium leaves against the red flour beetle, tribolium castaneum. the study found that the chloroform and ethyl acetate extracts of a. salvifolium leaves exhibited significant insecticidal activity against t. castaneum, with mortality rates of up to 90% at higher concentrations (ansari et al., 2017). while the present study investigated the larvicidal, pupicidal, and ovicidal activities of leaf extracts of a. salvifolium against cx. quinquefasciatus, other studies have also explored the efficacy of different parts of the plant against mosquitoes. for instance, a study evaluated the larvicidal activity of root leaf extracts of saussurea costus (falc.) lipsch. against three mosquito vectors:anopheles stephensi, aedes aegypti, and culex quinquefasciatus. the methanol extract of the roots recorded the highest larvicidal activity against an. stephensi, with lc50 and lc90values of 7.96 and 34.39 ppm, respectively (ali et al., 2020). another study investigated the effectiveness of using crude methanol extracts from the roots of a. salvifolium to kill mosquito larvae (ae. aegypti). they found that the plant extract was able to kill the larvae, with an lc50 value of 128.01 ppm. additionally, they investigated how the plant extract worked and found that it was able to inhibit certain enzymes that are important for the development of mosquito larvae, specifically acid and alkaline phosphatases (shukla et al., 2019). interestingly, a study by marimuthu et al., (2019) compared the larvicidal activity of a. salvifolium extracts from different parts of the plant, including the roots, stems, and leaves, against ae. aegypti and cx. quinquefasciatus. the study found that the root extract exhibited the highest larvicidal activity against both mosquito species, with lc50 values of 98.59 ppm and 101.36 ppm for ae. aegypti and cx. quinquefasciatus, respectively. another study investigated the biocontrol potential of ethyl acetate extract from a. salviifolium fruit pericarp against cx. quinquefasciatus larvae, demonstrating significant larval mortality with 100% at 72-hour exposure, revealing the most susceptibility in 3rd instar larvae (lc50 = 3.60 ppm), and identifying bioactive compounds including benzoyl bromide and 3amino-5 (2-furyl) pyrazole with minimal impact on non-target organisms (mondal et al., 2022). overall, the findings of this study suggest that the hexane extract of a. salvifolium roots may be a promising natural mosquito control agent. interestingly, the results of these studies suggest that different parts of the a. salvifolium plant may possess varying degrees of activity against mosquitoes, with the roots showing promise for further investigation. nevertheless, more studies are needed to 0 20 40 60 80 100 120 62.5 ppm 125 ppm 250 ppm 500 ppm hexane chloroform methanol control temephos (10 ppm) yagoo, et al. – alangium root extracts combat mosquitoes 623 fully explore the potential of a. salvifolium as a mosquito control agent, including investigations into the toxicity and safety of the plant extracts. conclusions the investigation conducted on the larvicidal, pupicidal and ovicidal activities of the root extracts of a.salviifolium on cx.quinguefasicatus has shown that the hexane extract demonstrated significant activities against these mosquito species. the larvicidal and pupicidal activities indicate that the hexane extract can effectively kill the larvae and pupae of these mosquitoes, which are the stages in which they are most vulnerable and responsible for the transmission of mosquito-borne diseases such as dengue fever, zika virus, and malaria. additionally, the ovicidal activity suggests that the hexane extract can also inhibit the hatching of mosquito eggs, which can further reduce the mosquito population. the significant results obtained from the investigation indicate that the hexane extract of a.salviifolium can be considered for commercial application as an antimosquito agent. the use of natural products as mosquito control agents has gained considerable interest due to their potential for being eco-friendly and costeffective. the hexane extract of a.salviifolium being a natural product, has the potential to be used as an alternative to chemical insecticides, which can be harmful to the environment and non-target organisms. recommedation for future studies it is essential to conduct further studies before commercializing the hexane extract of a.salviifolium as a mosquitocidal agent. these studies should include toxicity evaluations, which will help to determine the safety of the extract on non-target organisms, including humans, pets, and wildlife. toxicity studies will also help to establish the safe concentration and application rates of the extract. field trials are also necessary to evaluate the efficacy of the extract in real-world situations. these trials will involve testing the extract in various mosquito breeding sites and assessing its ability to reduce mosquito populations. the field trials will also help to identify any limitations or challenges associated with the use of the extract. furthermore, the practicality of the extract in terms of production, storage, and application should also be evaluated. the extraction process, storage conditions, and application methods should be optimized to ensure the extract's effectiveness and stability. the results obtained from these studies will also provide valuable information that can be used to optimize the extract's production, storage, and application methods. acknowledgments: the authors express their gratitude to the college principal and secretary for their inspiring words, and to the director and scientists at the entomology research institute, loyola college, chennai, tamil nadu, for providing experimental facilities and assistance. authors’ contributions: the idea for this article was conceived and worked out by alex yagoo, john milton, and jelin vilvest, who also wrote the manuscript. the data analysis was carried out by alex yagoo & jelin vilvest. all the authors read and approved the manuscript for final communication. competing interests: there are no competing interests to declare with respect to this manuscript. funding: no funding was received to assist with the preparation of this manuscript. references abbot ws (1925) a method for computing the effectiveness of an insecticide. ecol.entomol.18, 265-267. alvarado-esquivel c, gonzález-lópez em, & cerrillo-soto sm (2021) vector-borne diseases in mexico: a systematic review. acta tropica, 218, 105927. https://doi.org/ 10.1016/j.actatropica.2021.105927 ali si, venkatesalu v. 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(021) 7864728, indonesia. corresponding author* esti.mumpuni@univpancasila.ac.id manuscript received: 13 january, 2025. revision accepted: 15 may, 2025. published: 03 june, 2025. abstract sterculia quadrifida r. br or known as the faloak plant is a typical plant originating from the east nusa tenggara region, indonesia. this plant has long been used by local people in the treatment of diabetes. in vivo, bioactive compounds from the extract of stem bark sterculia quadrifida r. br have been reported to have antidiabetic activity. the present study aims to analyse the potential of bioactive compounds in the faloak stem bark as inhibitors of the enzymes α-glucosidase, ppar-γ, sglt2, and dpp-iv. the preparation of bioactive compounds and their comparison was conducted utilising chemdraw 2d & 3d, while proteins were obtained from the protein data bank (pdb). the docking process used molegro virtual docker, and visualisation was performed using biovia discovery studio. pharmacokinetic prediction (admet) was also carried out using the pkcsm website. the results of molecular docking with dpp-iv receptors showed that three bioactive compounds of faloak have better affinity than the comparative compounds, namely beta sisterol (-98.8838) to alogliptin, linagliptin, and sitagliptin. while epicatechin (-83.9022) and cathecin (-83.4336) have better affinity than the comparative compounds vidagliptin, saxagliptin. keywords: antidiabetic; faloak stem bark; molecular docking; in silico; sterculia quadrifida r. br. introduction diabetes mellitus (dm) is a chronic metabolic disease whose prevalence continues to increase globally. based on data from the international data federation (idf), in 2021, there were around 537 people worldwide with diabetes. this number is predicted to increase to 643 million in 2030 and 783 million in 2045. according to the idf, indonesia is the fifth country with the highest number of diabetes with 19.5 million sufferers in 2021 and is predicted to be 28.6 million in 2045. the world health organization (who) states that diabetes is one of the diseases in indonesia that causes death. diabetes can trigger other diseases (asyikin & ratnasari dewi poltekkes kemenkes makassar, 2024). one of the causes of the high prevalence of diabetes is an unhealthy diet and lifestyle. with the increasing number of diabetics in indonesia every year, two steps can be taken, namely the treatment of patients who have diabetes and prevention of diabetes for those who have not experienced it. treatment of diabetes can be done with two types of treatments, namely the provision of drug therapy and non-drug therapy. the treatment is not only focused on the control of blood glucose levels, but also involves the management of disease complications and the use of safer and more efficient therapies (asyikin & ratnasari dewi poltekkes kemenkes makassar, 2024). nowadays, people tend to choose treatment by utilising natural products as traditional medicine based on experiences passed down from generation to generation. moreover, indonesia is a tropical country, with rich biodiversity in each region. one of the potential plants in the treatment of diabetes is the faloak plant (sterculia quadrifida r. br), which has long been used by local people as a traditional treatment for diabetes on the island of timor, east nusa tenggara (ntt) (octaviany & iskandar, 2023). faloak has many chemical compounds, including flavonoids, tannins, alkaloids, steroids, and triterpenoids. local people often utilize faloak as a raw material for herbal medicines, and the part that is often used is the stem bark which has been proven by several studies conducted. however, the bioactive compounds and pharmacological activities of faloak plants are still very limited due to the lack of scientific research and knowledge (octaviany & iskandar, 2023). as a first step https://doi.org/10.14421/biomedich.2025.141.177-184 mailto:esti.mulatsari@univpancasila.ac.id 178 biology, medicine, & natural product chemistry 14 (1), 2025: 177-184 in predicting the activity of bioactive compounds in a plant, a molecular docking approach can be used. according to fernandez, et al. (2017), stated that the stem bark of the faloak plant has antidiabetic activity by showing a decrease in blood glucose in glucose-induced test animals (in vivo). fernandez added that further research is needed to determine the levels of active compounds in faloak bark and further explore its profile and safety (fernandez & edel, 2017). therefore, a molecular docking approach can be carried out and continued with the prediction of its pharmacokinetic profile (admet). the docking process begins by using bioactive compounds of a plant with a protein receptor obtained from the protein data bank (pdb). the stem bark of s. quadrifida r. br has several chemical contents including flavonoids, tannins, alkaloids, steroids, and triterpenoids (octaviany & iskandar, 2023). in addition, specific compounds such as β-sitosterol, catechin, epicatechin and scopoletin have been identified in the stem bark of the faloak (riwu et al., 2024). these specific biocompounds will be used in the docking process with protein receptors obtained from protein data bank with mechanisms as inhibitors of α-glucosidase, ppar-γ, sglt2, dpp-iv. this study aims to strengthen the understanding of the mechanism of action of bioactive compounds in the stem bark of the faloak plant as inhibitors of important enzymes associated with diabetes, such as α-glucosidase, ppar-γ, dpp-iv, and sglt2. the pharmacokinetic profile of the bioactive compounds in the stem bark of s. quadrifida r. br was also obtained, which can support the potential of faloak as a source of natural materials in the development of new antidiabetic drugs that are safer and more effective, and is expected to be the basis for the development of plant-based therapies in the future. materials and methods data collection and ligand preparation a comprehensive review of the existing literature was conducted to identify bioactive compounds of potential interest from the stem bark of the faloak plant. shown in table 01, the identified compounds included β-sitosterol, catechins, epicatechins and scopoletin. bioactive compounds and ligands were prepared using the chemdraw 2d application, with the resultant files being saved in the (.cdx) format. energy optimization was carried out with chemdraw 3d, by selecting minimize energy mm94 and saved in (.mol2) format. protein preparations protein complex structures in the (.pdb) format were obtained from the protein data bank, which was downloaded from the website http://www.rscb.org/ . the files were prepared using the molegro virtual docker application, and subsequently saved in the (.mol2) format. protein receptors were selected based on their central role in diabetes, including as inhibitors of αglucosidase (3top), ppar-γ (5y20), sglt2 (7vsi) and dpp-iv (2rgu) (table 02). internal validation prepared proteins were validated with 12 combinations (4 scoring functions and 3 algorithms) until rmsd ≤ 2å was obtained. this validation aims to ensure that the docking protocol is appropriate. physicochemical analysis the drug-likeness of the physicochemical compounds was evaluated using lipinski’s rule of five. for a compound to persuade as a drug, it must satisfy a set of parameters that will prove its compatibility, efficiency, and toxicity. molecular docking the molecular docking process between the ligand of the comparison compound and the bioactive compound was carried out using the molegro virtual docker application. the results obtained docking score of the comparator compound ligand which will be compared with the score of the bioactive compound ligand. visualization ligand-receptor complexes of bioactive compounds predicted to have antidiabetic activity were generated using biovia discovery studio. the visualization results were interpreted to determine each amino acid interaction that binds to the protein’s active site. in silico pharmacokinetic parameter prediction (admet) the pharmacokinetics/admet properties of these compounds were investigated using pkcsm an online web server (https://biosig.lab.uq.edu.au/pkcsm/prediction). pkcsm provides a platform for the analysis and optimization of pharmacokinetic and toxicity properties implemented in a user-friendly manner. http://www.rscb.org/ https://biosig.lab.uq.edu.au/pkcsm/prediction utami et al. – antidiabetic activity of faloak stem bark 179 results and discussion data collection and ligand preparation table 1. ligand preparation results. no cid name of compounds iupac name isomeric smiles 1 9064 (+)-catechin (2r,3s)-2-(3,4-dihydroxyphenyl)-3,4-dihydro2h-chromene-3,5,7-triol c1[c@@h]([c@h](oc2=cc(=cc(=c21) o)o)c3=cc(=c(c=c3)o)o)o 2 72276 epicatechin (2r,3r)-2-(3,4-dihydroxyphenyl)-3,4-dihydro2h-chromene-3,5,7-triol c1[c@h]([c@h](oc2=cc(=cc(=c21)o) o)c3=cc(=c(c=c3)o)o)o 3 5280460 scopoletin 7-hydroxy-6-methoxychromen-2-one coc1=c(c=c2c(=c1)c=cc(=o)o2)o 4 222284 β-sitosterol (3s,8s,9s,10r,13r,14s,17r)-17-[(2r,5r)-5-ethyl6-methylheptan-2-yl]-10,13-dimethyl2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1hcyclopenta[a]phenanthren-3-ol cc[c@h](cc[c@@h](c)[c@h]1cc[c@ @h]2[c@@]1(cc[c@h]3[c@h]2cc=c4[ c@@]3(cc[c@@h](c4)o)c)c)c(c)c protein preparation table 2. protein preparation results. no 3d structure of protein description native ligand 1 2rgu organism: homo sapiens method: x-ray diffraction resolution: 2.60 å mutation: no 356 8-[(3r)-3-aminopiperidin-1-yl]-7-but-2-yn-1-yl-3-methyl-1[(4-methylquinazolin-2-yl)methyl]-3,7-dihydro-1h-purine2,6-d ione 2 3top organism: homo sapiens method: x-ray diffraction resolution: 2.88 å mutation: no alfa-acarbose 3 5y2o organism: homo sapiens method: x-ray diffraction resolution: 1.8 å mutation: no (5s)-5-[[4-[2-(5-ethylpyridin-2-yl)ethoxy]phenyl]methyl]1,3-thiazolidine-2,4-dione 4 7vsi organism: homo sapiens method: electron microscopi resolution: 2.95å mutation: no 7r3 (2s,3r,4r,5s,6r)-2-[4-chloranyl-3-[[4-[(3s)-oxolan-3yl]oxyphenyl]methyl]phenyl]-6-(hydroxymethyl)oxane3,4,5-triol https://pubchem.ncbi.nlm.nih.gov/compound/9064 https://pubchem.ncbi.nlm.nih.gov/compound/72276 https://pubchem.ncbi.nlm.nih.gov/compound/5280460 https://pubchem.ncbi.nlm.nih.gov/compound/222284 180 biology, medicine, & natural product chemistry 14 (1), 2025: 177-184 internal validation 3top (1,0647 å) 2rgu (0,537976 å) 5y2o (0,67436 å) 7vsi (0,573135 å) figure 1. position of the native ligand superimposed on the redocked native ligand. table 3. root-mean-square deviation of protein data bank of a. 3top (α-glucosidase) b. 2rgu (ppar-γ) c. 5y2o (sglt2) and d. 7vsi (dpp-iv). a. algorithm moldock optimizer (å) moldock se (å) iterated simplex (å) scoring function moldock score 2,52913 3,0255 1,2467 moldock (grid) score 1,0647 2,46952 3,74578 plants score 1,42316 3,87905 2,09881 plants score (grid) 1,30646 1,48438 2,08955 b. algorithm moldock optimizer (å) moldock se (å) iterated simplex (å) scoring function moldock score 0,538373 0,537976 1,57811 moldock (grid) score 1,64074 1,56222 8,43536 plants score 2,03736 1,54417 2,56408 plants score (grid) 1,96009 1,37038 1,52665 c. algorithm moldock optimizer (å) moldock se (å) iterated simplex (å) scoring function moldock score 1,22248 0,67436 0,975562 moldock (grid) score 1,07309 3,3379 1,47244 plants score 1,0898 0,992484 1,05486 plants score (grid) 1,55609 1,6986 1,01191 d. algorithm moldock optimizer (å) moldock se (å) iterated simplex (å) scoring function moldock score 0,799375 1,04281 0,966411 moldock (grid) score 1,11362 1,08267 1,02383 plants score 0,86853 0,69421 0,800193 plants score (grid) 0,906024 0,573135 0,782356 utami et al. – antidiabetic activity of faloak stem bark 181 physicochemcical analysis table 4. the lipinski’s rule of five analysis results. compound molecular weight (g/mol) num. h-bond acceptors (<10) num. h-bond donors (<5) lipophilicity (log p<5) no. of lipinski’s violation beta cytosterol 414.71 1 1 8,024803 1 catechin 290.27 6 5 1,546099 0 epicatechin 290.27 6 5 1,546099 0 scopoletin 192.17 4 1 1,333000 0 molecular docking table 5. molecular docking results. compound cid docking score 7vsi 3top 5y2o 2rgu beta cytosterol 222284 -119,548 -107,759 -78,7945 -98,8838 catechin 9064 -94,293 -82,5122 -82,733 -83,4336 epicatechin 72276 -99,5256 -65,3541 -81,9963 -83,9022 scopoletin 5280460 -71,3226 -66,4612 -65,0435 -58,7893 alogliptin 11450633 -94,7326 linagliptin 10096344 -93,8655 sitagliptin 4369359 -87,8399 vildagliptin 6918537 -78,2888 saxagliptin 11243969 -66,9827 acarbose 41774 -145,932 troglitazone 5591 -131,227 pioglitazone 4829 -114,821 rosiglitazone 77999 -110,379 dapagliflozin 9887712 -122,592 canagliflozin 24812758 -136,445 empagliflozin 11949646 -135,666 3d 2d figure 2. beta cytosterol best interacts with receptors. 3d 2d figure 3. catechin best interacts with receptors. 3d 2d figure 4. epicatechin best interacts with receptors. https://pubchem.ncbi.nlm.nih.gov/compound/5280460 https://pubchem.ncbi.nlm.nih.gov/compound/11450633 https://pubchem.ncbi.nlm.nih.gov/compound/10096344 https://pubchem.ncbi.nlm.nih.gov/compound/4369359 https://pubchem.ncbi.nlm.nih.gov/compound/11243969 https://pubchem.ncbi.nlm.nih.gov/compound/41774 https://pubchem.ncbi.nlm.nih.gov/compound/5591 https://pubchem.ncbi.nlm.nih.gov/compound/4829 https://pubchem.ncbi.nlm.nih.gov/compound/77999 https://pubchem.ncbi.nlm.nih.gov/compound/9887712 https://pubchem.ncbi.nlm.nih.gov/compound/24812758 https://pubchem.ncbi.nlm.nih.gov/compound/11949646 182 biology, medicine, & natural product chemistry 14 (1), 2025: 177-184 in silico pharmacokinetic parameter prediction (admet) table 6. the predicted admet properties of the bioactive compounds; 1-water solubility (log mol/l) 2 -caco2 permeability (papp) 3-human intestinal absorption (%) 4-volume of distribution at steady-state (log l/kg) 5-fraction unbound (fu) 6-blood brain barrier permeability (log bb) 7-cytochromes p450 substrates 8-cytochromes p450 inhibitors 9-total clearance (log ml/min/kg) 10-renal organic cation transporter 2 substrate 11-ames toxicity 12max tolerated dose (human) (log mg/kg/day) 13-lethal dose 50 (mol/kg) 14-hepatotoxicity. compound absorption distribution metabolism excretion toxicity ws1 cp2 hia3 vdss4 fu5 bbb6 cyps7 cypi8 tc9 ros10 ames11 mtd12 ld13 ht14 beta cytosterol -6.773 1.201 94.464 0.193 0 0.781 3a4 0.628 no no -0.621 2.552 no catechin -3.117 -0.283 68.829 1.027 0.236 -1.054 0.183 no no 0.438 2.428 no epicatechin -3.117 -0.283 68.829 1.027 0.236 -1.054 0.183 no no 0.438 2.428 no scopoletin -2.504 1.184 95.277 0.034 0.383 -0.299 1a2 0.730 no no 0.614 1.950 no discussion internal validation validation was performed by tethering a standard ligand as a re-docking protocol with a pre-prepared receptor. the standard ligand was utilized as a tethering protocol to predict the position and ligand-protein interaction. the tethering was performed using molegro virtual docker and the rmsd (root mean square deviation) value was observed (zubair et al., 2020). the root-mean-square deviation value is utilized to evaluate the similarity of coordinates between two atoms, as illustrated in figure 01. as shown in table 3, the re-docking results of the standard ligands in this study have an rmsd of 1.067 å (3top); 0.537976 å (2rgu); 0.67436 å (5y2o); and 0.573135 å (7vsi). from the validation results carried out it can be seen that the rmsd is qualified as it is < 2 å. physicochemical analysis drug-likeness screening qualitatively assesses the possibility of a molecule becoming an oral drug regarding bioavailability (santos, g.b; ganesan, a; emery, 2016). lipinski’s “rule of five” highlights possible bioavailability problems if two or more properties are violated. lipinski's “rule of five” is an experimental and computational method to estimate solubility, membrane permeability, and efficacy in the drug development setting. according to lipinski (lipinski et al., 2001), a compound can be used as a drug candidate if it has a molecular weight of less than 500 g/mol, a log p of less than 5, at least one h-bond donor of less than 5, and at least one h-bond acceptor of less than 10. according to table 4, the four compounds from the stem bark of the faloak plant can be considered as new drug candidates as antidiabetics. however, scopoletin shows results that fulfil all lipinski's rules, while the other bioactive compounds have some rules that are not fulfilled. however, this rule does not predict whether a compound is pharmacologically active (kowalska et al., 2018). molecular docking before molecular docking, all bioactive compounds from the stem bark of the faloak plant were prepared using the molegro virtual docker application. the results obtained three bioactive compounds with potential as antidiabetics in dpp iv inhibitors. bioactive compounds of faloak plants that have potential in dpp iv inhibitors on the target protein 2rgu are β-sitosterol with a score value (98.8838) compared to the comparison compounds alogliptin (-94.7326), linagliptin (-93.8655) and sitagliptin (-87.8399); catechin (-83.4336) and epicatechin (-83.9022) compared to the comparator compounds vildagliptin (-78.2888) and saxagliptin (66.9827). table 5 shows the full molecular docking results. visualization the visualization of 2d images of the three bioactive compounds from the stem bark of the faloak plant is possible due to their hydrogen bond interactions with amino acid residues. hydrogen bonds are known to be strong bonds, although not as strong as covalent bonds. the selection of hydrogen bonds as a metric in this study is predicated on their capacity to influence the physical and chemical properties of compounds, including boiling point, solubility in water, the ability to form chelates, and acidity (ph). moreover, hydrogen bonds have been demonstrated to impact the biological activity of drugs (noolvi & patel, 2013). beta cytosterol hydrogen bonds (figure 02) have been observed to occur specifically at the amino acid residue asp 545, while catechin hydrogen bonds (figure 03) have been observed to occur at amino acid residues trp 269 and asn 710. as for the bioactive compound epicathecin (figure 04), hydrogen bonding has been observed to occur at amino acid residues trp 629 and asn 710. in silico pharmacokinetic parameter prediction (admet) the results of admet prediction were obtained by entering the isomeric smile data of bioactive compounds from sterculia quadrifida r.br stem bark on the pkcsm utami et al. – antidiabetic activity of faloak stem bark 183 website. admet prediction is carried out to analyze the secondary metabolites of sterculia quadrifida r.br so that absorption, distribution, metabolism, excretion and toxicity are known. the results of the admet prediction are shown in table 6. absorption a. the aqueous solubility of a compound indicates the solubility of the molecule in water at 25°c. it is widely acknowledged that fat-soluble drugs are less well absorbed than water-soluble drugs, especially when administered enterally (yeni & rachmania, 2022). the results show that beta-sitosterol is poorly soluble. the logarithm of the molar concentration (log mol/l) is the accepted measure of the predicted water solubility of a compound. b. the term 'caco2 permeability' is employed to denote the capacity of a compound to facilitate the movement of carbon dioxide, and is denoted by the parameter 'papp'. the value of 'papp' must exceed 8 x 10-6 cm/s for a compound to be considered to have high caco2 permeability. for predictions on pkcsm, high caco2 permeability would result in a value of 0.90 (yeni & rachmania, 2022). c. the gut is typically the main site of drug absorption from orally administered solutions. the present method was developed estimate the proportion of compounds absorbed through the small intestine. the prediction of compounds with favorable intestinal absorption is facilitated by identifying molecules exhibiting more than 30% absorbance, as these are deemed well absorbed. compounds with less than 30% are considered poorly absorbed (yeni & rachmania, 2022). all compounds showed results above 30%. however, beta-sitosterol showed the highest results to be well absorbed. distribution a. the volume of distribution is the theoretical volume required to distribute the total drug dose to produce the same concentration as in blood plasma evenly. the higher the volume of distribution value, the more drug is distributed in the tissues in the plasma. a value of less than 0.71 l/kg indicates a low distribution volume (prasetiyo et al., 2024). catechin and epicatechin compounds had the best results compared to other bioactive compounds. b. the blood-brain barrier (bbb) is a vital component of the central nervous system (cns) that acts as a protective barrier against exogenous compounds. bbb permeability is defined as the capacity of a pharmaceutical agent to traverse the blood-brain barrier. this parameter is important in reducing adverse effects and toxicity, or enhancing the efficacy of drugs whose pharmacological activity is confined to the brain. it is widely accepted that compounds with a logbb greater than 0.3 are considered to cross the blood-brain barrier with ease, while those with a logbb less than -1 are less likely to be distributed to the brain (prasetiyo et al., 2024). similar to the volume of distribution results, the results for catechins and epicatechins showed that these compounds could not cross the blood-brain barrier. metabolism cytochrome p450 is an important detoxification enzyme in the body, mainly found in the liver. cytochrome p450 oxidizes xenobiotics to facilitate their excretion. many drugs are inactivated by cytochrome p450 and some can be activated. inhibitors of these enzymes may affect drug metabolism and are contraindicated. therefore, it is important to assess the ability of compounds to inhibit cytochrome p450 (prasetiyo et al., 2024). excretion drug clearance is measured by the proportionality constant cltot, and occurs mainly as a combination of hepatic clearance (metabolism in the liver and biliary clearance) and renal clearance (excretion through the kidneys). this is related to bioavailability, and is important for determining the dose rate to achieve steady-state concentrations. the excretion values of the test compounds from pkcsmpharmacokinetics ranged from 0.99 to 1.314 log ml/mln/kg. the excretion results indicate that the four test compounds did not meet the range of total clearance values. this suggests that the body slowly excretes the four compounds (prasetiyo et al., 2024). toxicity the toxicity level of the compound is predicted by pkcsm, with the lethal concentration value (ld50) describing the amount of compound given that can cause death by 50%, which ranges from 2,162 to 2,651 mg/kg/day. the maximum tolerated dose (mtd) is defined as the toxic dose threshold of chemicals in humans. according to the pkcsm, an mtd of less than or equal to 0.477 log (mg/kg/day) is considered low toxicity, while an mtd exceeding 0.477 log (mg/kg/day) is classified as high toxicity (prasetiyo et al., 2024). conclusions molecular docking and pharmacokinetic studies were carried out on four bioactive compounds from the stem bark of the faloak plant. this study confirmed that three bioactive compounds such as beta-sitosterol, catechin and epicatechin have antidiabetic activity as dpp-iv receptor blockers. as a result of their pharmacokinetic profiles, these bioactive compounds may have potential as drugs for the treatment of diabetes. 184 biology, medicine, & natural product chemistry 14 (1), 2025: 177-184 acknowledgements: the authors would like to thank prof. dr. apt. siswandono, m.s. for permission to use the molegro virtual docker licence for this research. competing interests: the authors declare that there are no competing interests. 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(2013). synthesis, method optimization, anticancer activity of 2,3,7-trisubstituted quinazoline derivatives and targeting egfr-tyrosine kinase by rational approach. 1st cancer update. arabian journal of chemistry, 6(1), 35–48. https://doi.org/10.1016/j.arabjc.2010.12.031 octaviany, s. t., & iskandar, y. (2023). review artikel: kandungan senyawa kimia dan bioaktivitas dari tanaman faloak (sterculia quadrifida). jurnal farmasi sains dan terapan, 10(2), 72–78. https://doi.org/10.33508/jfst.v10i2.4754 prasetiyo, a., martati, t., & saputra, p. w. (2024). in silico study of bioactive compounds in herba sambiloto (andrographis paniculata burm. f. nees) as hiv-1 reverse transcriptase inhibitor. in jurnal jamu indonesia (vol. 9, issue 2). https://doi.org/10.29244/jji.v9i2.300 riwu, a. g., nugraha, j., triyono, e. a., & purwanto, d. a. (2024). anti-inflammatory effect of faloak (sterculia quadrifida r. br) stem bark on tnf-α, il-1β, and il-6 in denv-3-infected wistar rats. open veterinary journal, 14(4), 1043–1050. https://doi.org/10.5455/ovj.2024.v14.i4.11 santos, g.b; ganesan, a; emery, f. s. (2016). oral administration of peptide-based drugs: beyond lipinski’s rule. chemmedchem, 11(20), 2245–2251. https://doi.org/10.1002/cmdc.201600288 yeni, y., & rachmania, r. a. (2022). the prediction of pharmacokinetic properties of compounds in hemigraphis alternata (burm.f.) t. ander leaves using pkcsm. indonesian journal of chemistry, 22(4), 1081–1089. https://doi.org/10.22146/ijc.73117 zubair, m. s., maulana, s., & mukaddas, a. (2020). penambatan molekuler dan simulasi dinamika molekuler senyawa dari genus nigella terhadap penghambatan aktivitas enzim protease hiv-1. jurnal farmasi galenika (galenika journal of pharmacy) (e-journal), 6(1), 132–140. https://doi.org/10.22487/j24428744.2020.v6.i1.14982 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 651-662 | doi: 10.14421/biomedich.2023.122.651-662 issn 2540-9328 (online) identification and toxicity profiling of column fractions of ethanol leaf extract of ziziphus mauritiana tajudeen alowonle owolarafe*, salawu kailani department of biochemistry and forensic science department; nigeria police academy wudil, kano, nigeria. corresponding author* taowolarafe@polac.edu.ng abstract different plants may contain varying amounts of phytochemicals and also different degrees of toxicity. this study aimed to evaluate the phytochemicals present in the most active column fraction of ethanol leaf extract of the ziziphus mauritiana plant and their toxic effects using brine shrimp lethality assay and animal model. crude ethanol extract was obtained by maceration while fraction was achieved using a column chromatography experiment. toxicity was evaluated with brine shrimp lethality assay and albino rat’s models while characterization was achieved with liquid chromatography-mass spectrophotometry. ziziphus mauritiana leaves revealed 42 fractions pooled into seven fractions. fraction three (3) was the most toxic with the brine shrimp lethality assay of (31.48ug/ml) and its toxicological evaluation revealed an adverse effect on the hematological parameter, biochemical indices, and histo-architecture of the liver and kidney of the experimental model studied. lcms analysis of the most toxic fraction revealed the presence of antirrhinoside, lucidumol a, apigenin 7-glucuronide-4'-(6''-malonylglucoside), dioscoreside c, camellioside d, and others which have been reported for various pharmacological effects including adverse effects, the mode of toxicity may be synergistic, individual, or antagonistic which may explain the moderate toxicity observed in animal model. administration of these fractions may lead to toxicity despite their potential. keywords: dioscoreside c; chromatographic fractions; ziziphus mauritiana; toxicity. introduction it is important to bear in mind that different plants contain varying amounts of phytochemicals and toxicity levels. ziziphus mauritiana, a perennial shrub or tree that can grow up to 3-15 meters tall with a trunk diameter of approximately 40 centimeters, has a variety of medicinal uses in folk medicine. its leaves, fruits, and bark are used to treat a range of ailments, including diarrhea, dysentery, tuberculosis, and asthma (kokwaro, 2009; umair et al., 2019). additionally, certain parts of this plant have been found to possess beneficial bioactivities such as antioxidant, anticancer, and antidiarrheal activities (abalaka et al., 2011; jain et al., 2019; akanda and hassan 2021; dahiru et al., 2006). however, there is a lack of information regarding the specific bioactive compounds present in these extracts and their potential effects. the aim of this study is to identify these compounds and evaluate any potential toxicological effects of the active fraction of the ethanol extract of ziziphus mauritiana leaves. materials and methods plant material authentication fresh leaves of z. mauritiana were obtained from nigeria police academy, wudil, kano. identification was carried out the herbarium in the department of plant biology, bayero university kano, nigeria. voucher number bukhan 0233 was allocated and the plant specimen were deposited. plant material preparation and extraction fresh leaves of z. mauritiana were dried in a ventilating room for five (5) days and then ground into powdered form with the aid of an electric blender. 350g of the powdered leaves extracted by cold maceration method. the plant sample was soaked in 2000 ml of ethanol solution 99% and left to stand for 48 hours. about 1700 ml of the filtrate were obtained and concentrated using a rotatory evaporator. manuscript received: 21 september, 2023. revision accepted: 17 december, 2023. published: 04 february, 2024. https://doi.org/10.14421/biomedich.2023.122.651-662 652 biology, medicine, & natural product chemistry 12 (2), 2023: 651-662 column chromatography and structural elucidation. the column chromatography of the ethanol extract was carried out according to the method of ode et al., 2011 with modifications to suit this present study. briefly, crude ethanol extract of ziziphus mauritiana leaves weighing 20 grams was subjected to column chromatography in three (3) phases to separate the extract into its component fractions. silica gel 60 by 120g was used as the stationary phase while varying solvent combinations (chloroform, ethylacetate, ethanol, methanol, and water) of increasing polarity were used as the mobile phase. the following ratios of solvent combination was sequentially used in the elution process; hexane: chloroform 100:0, 80:20, 60:40, 40: 60, and 20: 80; chloroform: ethylacetate 100:0, 80:20, 60:40, 40: 60, and 20: 80; ethylacetate: ethanol 100:0, 80:20, 60:40, 40: 60, and 20: 80; ethanol: methanol 100:0, 80:20, 60:40, 40: 60, and 20: 80; methanol: distilled water 100:0, 80:20, 60:40, 40: 60, 20: 80 and 0:100. a measured volume of 100ml of each solvent combination was collected and gradually poured uniformly by the sides of the glass into the column each time. this measure prevented solvent droplets from falling directly and disturbing the topmost layer of the column. distortion of this layer would result in a nonuniform drain of the fractions. the eluted fractions was collected in aliquots of 50 ml in a glass container. analytical thin layer chromatography (tlc) and pooling of fractions the content of each fraction was allowed to evaporate for 24-48 hours at room temperature with well-covered with perforated aluminum foil to prevent contamination. all fractions were spotted on pre-coated (silica gel f254) aluminum plates in a small chromatographic tank to pool them into different sub-fractions based on their relative mobility in solvent systems and colour reactions with an ultraviolet light lamp at 365 and 254 nm to identify the fluorescing spot and pattern of separation in the chromatogram. this procedure was used to pool the fractions and they were concentrated in a water bath to dryness. the mass of the different pooled fractions was determined and was kept at 40c in the refrigerator for further toxicological analysis. preparative thin layer chromatography (tlc) of most toxic pooled fraction the most toxic pooled fraction was fractionated using preparative thin-layer chromatography. the preparative thin-layer chromatography method was carried out according to the method described by ode et al., 2011. structural characterization of most toxic fraction using liquid chromatography – mass spectrophotometery (lc-ms). the phytochemical content of the most active subfraction was elucidated using liquid chromatographymass spectrophotometry analysis at the liquid chromatography–mass spectrometry laboratory at jeffrey cheah school of medicine and health sciences, monash university malaysia, selangor darul ehsan, malaysia. the subfraction was diluted in methanol at a ratio of 1ml:1ml and injected into the lcms system (agilent 1290 infinity lc system coupled to agilent 6520 accurate-mass q-tof mass spectrometer with dual esi source) with the following column (agilent zorbax eclipse xdb-c18, narrow-bore 2.1x150mm, 3.5 micron (p/n: 930990-902)) conditions. results and discussion result column chromatography yield of ethanol leaf extract of ziziphus mauritiana. the result of chromatographic fractionation of the ethanol extract of ziziphus mauritiana leaf is shown in table 1. a total of seven (7) fractions were pooled from 41 preliminary fractions of ethanol crude extract of z. mauritiana as shown in table 1. fraction one (1) yielded 5.3 grams, fraction two (2) 3.1 grams, and a total of 3.2 grams was obtained for fraction three (3). fractions four (4), five (5), six (6), and seven (7) yielded 3.9 grams, 1.8 grams, 1.9 grams and 1.4 grams respectively. table 1. characteristic yield of fractions from the ethanol leave extract of ziziphus mauritiana. s/n fraction (s) weight obtained (grams) 1 fraction i 5.3 2 fraction ii 3.1 3 fraction iii 3.2 4 fraction iv 3.9 5 fraction v 1.8 6 fraction vi 1.9 7 fraction vii 1.4 total 20.6 brine shrimp lethality assay of fractions of ethanol leaf extracts of z. mauritiana the brine shrimp lethality assay of fractions of ethanol extract of z. mauritiana leaf shown in table 2 revealed that fraction three (3) gave the lowest lethal owolarafe & kailani – identification and toxicity profiling of … 653 concentration of 31.48 µg/ml followed by fraction four (4) and fraction one (1) with lethal concentration of 61.66µg/ml and 91.20 µg/ml respectively. fractions 2, 5, 6, and 7 exhibit 123.47 µg/ml, 291.20 µg/ml, 261.60 µg/ml, and 231.47 µg/ml respectively. table 2. brine shrimp toxicity of ethanol leaf extracts fractions of z. mauritiana. extract dose (µg/ml) percentage mortality lc 50 (µg/ml) toxicity classification fraction i 0 10 100 1000 0 16.67 30.00 93.33 91.20 toxic /moderately toxic fraction ii 0 10 100 1000 0 13.00 32.00 52.45 123.47 slightly toxic /non toxic fraction iii 0 10 100 1000 0 33.00 63.00 96.67 31.48 toxic /moderately toxic fraction iv 0 10 100 1000 0 30 56.67 86.67 61.66 toxic /moderately toxic fraction v 0 10 100 1000 0 20.00 50.00 80.00 291.20 slightly toxic /non toxic fraction vi 0 10 100 1000 0 20.00 40.00 80.00 261.60 slightly toxic /non toxic fraction vii 0 10 100 1000 0 10.00 30.00 60.00 231.47 slightly toxic /non toxic effect of subchronic administration of fraction 3 (iii) of ethanol leaf extract of ziziphus mauritiana on haematological parameters. the effect of fraction three (3) of ethanol leaf extract of ziziphus mauritiana as shown in table 3 revealed no significant difference in heamoglobin concentration in all groups treated with fraction 3 when compared with the control group. the red blood cell (rbc) concentration also showed no significant difference (p<0.05) among the groups (control, 200mg/kg and 500mg/kg groups). the parked cell volume (pcv) values show no significant difference between the control group and group 2 administered with 200mg/kg while there was a decrease in the pcv concentration in group 3 (500mg/kg) which is statistically significant (p<0.05) when compared with the control group. mean corpuscular heamoglobin (mch) shows there was no significant difference among the groups administered with different concentrations of fraction 3 of ethanol leaf extract of z. mauritiana while mean corpuscular heamoglobin concentration (mchc) also shows no significant difference when compared with the control. mean corpuscular volume (mcv) shows there was no statistical difference between groups administered with 200mg/kg of the extract while a decrease was observed in the group treated with 400mg/kg when compared with the control group at p<0.05. the wbc concentration showed a significant difference (p < 0.05) in all treated groups when compared with the control group, while lymphocyte concentration and platelets also showed a decrease in concentration when compared with the control. mid concentration (average of monocytes, eosinophils, basophils) and neutrophils concentration showed no significant difference in all groups when compared with the control group. effect of subchronic administration of fraction iii (3) of ethanol leaf extract of ziziphus mauritiana on liver function indices. the effect of fraction iii of ethanol leaf extract of ziziphus mauritiana on liver function parameters is presented in figures 1 and 2. it shows that there was a 654 biology, medicine, & natural product chemistry 12 (2), 2023: 651-662 significant increase in group 2 administered with 200mg/kg and group 3 administered with 500mg/kg when compared with the control for aspartate aminotransferase (ast) concentration in the serum, while there was a significant difference in the concentration of alanine aminotransferase (alt) in group 3 (500mg/kg) only when compared the treated groups with the control group. the alkaline phosphatase (alp) concentration shows no significant difference in all the groups treated with fraction iii of ethanol leaf extract of z. mauritiana when compared with the control group while there was no significant difference in mda concentration between the control group and group 2 (200mg/kg) except group 3 (500mg/kg) (figure 2) which exhibit a significant increase in the mda concentration. serum total protein reveals a significant decrease in group 3 (500mg/kg) while this decrease was not significant in group 2 (200mg/kg) when compared with the control, albumin and globulin reveal a decrease in all treated groups when compared with control group but were not significant(p<0.05). effect of subchronic administration of fraction iii (3) of ethanol leaf extract of ziziphus mauritiana on kidney function indices the effect of fraction 3 of ethanol leaf extract of ziziphus mauritiana on kidney function parameters as presented in table 4 shows that there was no significant difference in creatinine, urea, sodium, and calcium concentrations between the control group and all the treated groups while potassium serum concentrations revealed an increase in group 3 administered with 500mg/kg body weight, for serum chloride ion concentration, a significant difference between the administered groups (200mg/kg and 500mg/kg) and the control group with decrease observed in groups 2 and 3. organ body-weight ratio analysis of samples after subchronic administration of fraction iii of ethanol leaf extract of ziziphus mauritiana: the organ-body weight ratio of liver and kidney in wistar rats administered with fraction iii (3) as shown in figure 3, the result shows a significant increase in the organs when compared with the control. the liver and kidney revealed an increase in weight with an increase in the dose administered and are significantly different from the control. histopathological analysis of liver and kidney after subchronic administration of fraction iii of ethanol leaf extract of ziziphus mauritiana the histopathological evaluation of the liver cell architecture shows unremarkable liver tissue for plate (control) with all the portal triad in position while plate (b) (200mg/kg) shows areas of necrosis and inflammation (arrow point at areas of necrosis and inflammation) of liver tissue structure. plate(c) (500mg/kg) also reveals areas of necrosis and inflammation. the kidney cell architecture revealed no significant pathology and the cellular arrangement is intact for plate (a) (control) while plate (b)(200mg/kg) shows areas of inflammation. plate (c) (500mg/kg) reveals a degree of distortion in renal structure when compared with the control group. table 3. effect of administration of fraction 3 of ethanol leaf extract of ziziphus muritiana on haematological parameters of wistar rats. parameters ziziphus muritiana ethanol leaf extract fraction 3 (mg/kg body weight) control 200 500 haemoglobin (g/l) 14.92 + 0.066a 14.04 + 0.652 a 10.62 + 1.061 b red blood cell (×1012/l) 7.96 + 0.086 a 7.59 + 0.295 a 7.44 + 0.126 a packed cell volume (%) 53.37 + 0.220 a 51.09 + 3.190 a 42.60 + 1.973 b mean corpuscular haemoglobin (pg) 19.00 + 0.251 a 18.91 + 0.436 a 17.24 + 1.591 a mean corpuscular haemoglobin concentration (%) 28.21 + 0.064 a 27.60 + 0.422 a 26.50 + 1.642 a mean corpuscular volume(fl) 66.69 + 0.554 a 68.71 + 1.867 a 61.15 + 2.203 b white blood cell (×109 /l) 14.38 + 0.829 a 7.64 + 0.248 b 6.25 + 0.942 b lymphocytes (×109/l) 84.55 + 1.554 a 38.77 + 2.237 b 51.79 + 9.595 b neutrophils (×109/l) 32.0+ 1.82a 32.0+ 1.82a 32.0+ 1.82a mid (%) 8.00 + 0.55 a 8.40 + 0.25 a 8.20 + 0.49 a platelets (×109/l) 7.91 + 0.370 a 5.21 + 0.484 b 5.24 + 0.068 b note: mid is the average of monocytes, eosinophils, basophils. n = 5, x + sem. a-c test values carrying superscripts different from the control across each parameter are significantly different at p > 0.05. owolarafe & kailani – identification and toxicity profiling of … 655 table 4. effect of administration of ethanol leaf extract fraction 3 of ziziphus muritiana on some kidney function indices in wistar rats. parameters ziziphus muritiana ethanol leaf extract (mg/kg body weight) control 200 500 creatinine (umol/l) 0.047 + 0.002a 0.043 + 0.006a 0.098 + 0.124a urea (mmol/l) 339.370 + 14.034a 353.890 + 60.211a 462.890 + 13.002a sodium (meq/l) 136.50+1.63 a 138.8+ 1.59a 140.83+ 1.534 a calcium (mg/dl) 6.61 + 0.12 a 6.40 + 0.56 a 6.94 + 0.24 a potassium(meq/l) 5.19 + 0.25 a 6.39 + 0.50 a 9.57 + 1.37 b chloride (meq/l) 96.00 + 0.35 a 88.27 + 1.89 b 92.88 + 0.49 b n = 5, x + sem.a-c test values carrying superscripts different from the control across each parameter are significantly different at p > 0.05 brine shrimp lethality assay of subfractions of fraction 3 of ethanol leaf extract of z. mauritiana the preparative thin layer chromatography of column pooled fraction 3 which resulted in three (3) subfractions was evaluated by brine shrimp lethality assay. the sub-fractions of column pooled fraction ii are shown in table 5. the result revealed that sub-fraction two (2) gave the lowest lethal concentration of 446.68µg/ml (0.45 mg/ml) followed by fraction three (3) and fraction one (1) with a lethal concentration of 793.78µg/ml (0.79 mg/ml) and 2390.01 µg/ml (2.39 mg/ml) respectively. table 5. brine shrimp toxicity of sub fractions of ethanol leaf extracts fraction iii of z. mauritiana. extract dose (µg/ml) percentage mortality lc 50 (µg/ml) toxicity classification fraction 3:1 0 10 100 1000 0 16.67 30.00 93.33 2390.01 not toxic fraction 3:2 0 10 100 1000 0 13.00 32.00 52.45 446.68 toxic / moderately toxic fraction 3:3 0 10 100 1000 0 33.00 63.00 96.67 793.78 toxic /slightly toxic liquid chromatography –mass spectrophotometry of sub-fraction ii of column pooled fraction iii (3) of ethanol leaf extract of ziziphus mauritiana. the sub-fraction ii of column pooled fraction 3 obtained by preparative thin layer chromatography (tlc) was analyzed for its bioactive composition using liquid chromatography –mass spectrophotometery (lc-ms) and the constituents were presented in table 6. the analysis revealed the presence of a range of phytoconstiruents such as alkaloids, glycosides, triterpenoids, steroids, and flavonoid derivatives. the most abundant bioactive molecules are glycoside derivatives with centellasaponin c as the most abundant. 656 biology, medicine, & natural product chemistry 12 (2), 2023: 651-662 table 6. chemical constituents of sub-fraction 2 of column pooled fraction 3 of ethanol extract of ziziphus mauritiana leaf. s/n class of phytochemicals compound name structure activity references 1 alkaloids 8methylcaffeine metabolite of caffeine, cause abbereration in allium ceppa, cell culture aberration occurred in late prophase and late g2 phase kihlam et al., 1972 jiang et al., 2000 2 glycoside antirrhinoside an irridiod glycoside haemolytic activity cytotoxic activity drohse and molgaard, 2001 tholl et al.,2004 riaz et al., 2013 3 8-hydroxyluteolin 8-glucoside flavone glycoside induce dna damage cantero et al., 2006, snyder and gillies, 2002 4 glycoside 4,2',4'trihydroxy-6'methoxychalcone 4,4'-di-betaglucoside flavon-glycoside, induce dna damage lopez-lazaro, 2009, kawanishi, 2000. snyder and gillies, 2002 5 d-linalool 3-(6''malonylglucoside) glycoside, derivative of dlinalool which is reported for genotoxic activity and dermatological allergy api et al., 2015, nakamura et al., 2009 6 camellioside d triterpene oligoglycoside antiproliferative activity guo et al, 2018, nakamura et al., 2012 7 apigenin 7glucuronide-4'(6''malonylglucoside) a glycoside, derivative of apigenin,cytotoxic activity by dna damage budhraja et al., 2012 mcgaw et al., 2013 owolarafe & kailani – identification and toxicity profiling of … 657 table 6. cont. s/n class of phytochemicals compound name structure activity references 8 saponin centellasaponin c triterpene saponin hepatotoxic jorge and jorge, 2005 9 dioscoreside c steroidal saponin cytotoxic activity dong et al., 2001 hansakul et al., 2008 10 quillaic acid aglycone saponin, cytotoxic activity, adjuvant effect rajput et al., 2007, gevrenova et al., 2014 11 triterpenoid akeboside stf triterpenes cytotoxic activities chudzik et al., 2015 kolesnikova et al., 2013 12 steroids lucidumol a triterpenoid steroids cytotoxic activity xia et al., 2014 amen et al., 2016 13 nebrosteroid l steroids cytotoxic activities amir et al., 2012 discussion effect of different column pooled fractions of ethanol extract of z. mauritiana leaves the column chromatography separation of crude ethanol extract of z. mauritiana leaves yielded 7 (seven) distinct column pooled fractions with fraction 1 as the highest yield (table 17), this may be due to the degree of solubility of phytochemicals present in fraction i. different phytochemicals exhibit different degrees of solubility in solvent systems (wakeel et al., 2019) fraction 3 was observed to be the most bioactive component of the extract fractions using brine shrimp 658 biology, medicine, & natural product chemistry 12 (2), 2023: 651-662 lethality assay (bsla) techniques (table 18) (ode et al., 2012). this effect may be due to the presence of some bioactive principles that have been reported to be toxic (benie, et al., 1990). effect of column pooled fraction three (3) of ethanol extract of z. mauritiana leaves fraction three was picked to be the most toxic fraction based on the brine shrimp lethality assay (bsla) carried out to determine the effect of the bioactive component of the sub-fraction on the experimental model (artemia salina larve). subchronic toxicity evaluation of plant extracts is used to determine its effect on blood and biochemical parameters. also, morphological investigation on specific tissues may help to explain the mechanisms of toxicity of an extract and its possible therapeutic effect (yamthe et al., 2012, asaduzzaman et al., 2015). the main objective of determining hemoglobin concentration and its indicative parameters (mch and mchc) is to analyze the amount of intracellular iron and an index for folic acid and vitamin b12 need, also oxygen carrying capacity of the blood while the primary reasons for assessing the rbc, pcv, and mch is to check anemia, evaluate normal erythropoiesis and degree of anemia or polycythaemia (ganong, 2001). the reduction observed may be an indication of the inability of the blood to perform the function of oxygen-carrying capacity which leads to a decrease in energy production within the cells (asanga et al., 2013), and may be responsible for the weakness/slow activity observed during extract administration to the wistar rats. increased total wbc count upon administration of xenobiotics from either plant extract or synthetic compound may indicate leucocytosis in the treated animals (ajeigbe et al., 2013), and evaluating differential wbc count provides information on the proportion of the different white cells present in circulating blood (cheesbrough, 2000; enitan et al., 2012). the observed significant increase in counts of lymphocytes, neutrophils, monocytes, and eosinophils in the test groups when compared to the control is an indication that the extract must have induced lymphocytosis, neutrophilia, monocytosis, and eosinophilia respectively (ajeigbe et al., 2013) but he reduction in these parameters may indicate immunosuppression of the defense mechanism of the animal and toxicity of the extract may be evident. the observed reduction in the concentration of wbc and lymphocytes in the blood sample of all the treated groups with fraction 3 when compared with the control group suggests the immunosuppressive ability of the extract fraction when ingested. also, reduced platelet concentration has been reported to increase blood viscosity and lead to increased blood pressure (adedapo et al., 2008; adeniyi et al., 2010). the reduction observed in platelet count may be an indication of an increase in the blood pressure of the animal therefore certain phytoconstituents of administered extract fraction may induce high blood pressure. biochemical evaluation using enzymatic and nonenzymatic indices/parameters has been used to evaluate the functionality and integrity of cellular barriers of vital tissues such as the liver and kidney (sunmonu et al., 2014). these parameters provide information on the effect and nature of pathological damage that may have taken place in these tissues (yang et al., 2014). elevated serum alt, ast, and alp concentrations in the serum have been reported in hepatic injury and diseases (andrade et al., 2006, stirnimann et al., 2010; owolarafe et al., 2020), increased concentrations of alt, ast, and alp in the serum upon administration of fraction 3 of ethanol extract of z.mauritiana when compared the control group (p< 0.05) suggested severe mixed hepatic injury such cholestatic injury, bile duct injury and overall distortion of metabolic homeostasis of the body (kaplowitz, 2005; stirnimann et al., 2010, yang et al.,2014). this is corroborated by increased mda concentration observed in treated groups which is an indication of membrane lipid peroxidation and a compromise of hepatic cell membrane integrity. nephrotoxicity is the adverse effect observed on the function of the renal tissue that occurs due to the intake of xenobiotics resulting in direct toxicity or compromised renal perfusion, these renal dysfunctions may include acute tubular necrosis, glomerular and tubulointerstitial injury, haemodynamically mediated damage, and obstructive nephropathy (finlay et al., 2013; taber and mueller, 2006). no significant increase or reduction in serum creatinine, urea, and electrolytes concentrations in all treated groups when compared with the control (p < 0.05) may indicate the non-nephrotoxic effect of fraction 3 of ethanol extract of z.mauritiana leaves in rats. amresh et al., (2008) suggested that organ to body weight ratio may indicate organ swelling, atrophy, or hypertrophy and the histopathological analysis of tissue biopsies is the most definitive way to diagnose and confirm various types of impairment in tissues (yang et al., 2014); the increase in liver, kidney, and lung to body weight ratio following the administration of fraction 3 of ethanol extract of z.mauritiana leaves in rats may be a result of inflammation. this submission is in agreement with earlier reports by ashafa et al. 2011 and adebayo et al., 2003. histopathological examination also reveals the presence of inflammation and distortion of the cellular architecture of the liver and lung tissue and a mild atrophy of the kidney. conclusions base on the findings in this research work the extract exhibited mild toxicity and these maybe due to identified phytochemicals and their antagonistic and synergistic activities with regards to the identified bioactive compounds owolarafe & kailani – identification and toxicity profiling of … 659 acknowledgements: the author and his crew wish to acknowledge the effort of all our technologists in biochemistry and forensic science nigeria police academy and biochemistry department, bayero universityresults and discussion should be written as a series of connecting sentences, however, for manuscript with long discussion should be divided into subtitles. results should be clear and concise. authors’ contributions: tajudeen a. owolarafe: is the lead researcher and participate in all phase of the research from conceptualization to writing of the manuscript. salawu kailani: he is involved in the fractionation of crude extracts and writing of the manuscript competing interests: ethics approval and consent to participate: ethical approval was obtained from the research ethical committee of bayero university kano with approval number buk/chs/rec/68. consent for publication: the authors declare that there are no competing interests. funding: part of the fund for this research was funded by tetfund references abalaka, m. e., daniyan, s. y. & mann, a. 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(2014). hepatic toxicity biomarkers chapter 13 in biomarkers in toxicology (r. gupta (ed)), p 241-259. isbn: 978-0-12404630-6 doi: http://dx.doi.org/10.1016/b978-0-12-4046306.00013-0. https://doi.org/10.186/s13002-019-0285-4 http://doi.org/10.7717/peerj.7857 http://dx.doi.org/10.1016/b978-0-12-404630-6.00013-0 http://dx.doi.org/10.1016/b978-0-12-404630-6.00013-0 owolarafe & kailani – identification and toxicity profiling of … 661 figure s1. effect of administration of fraction three of ethanol extract of zizphus mauritiana leaves on liver function enzymes activities among experimental animals. figure s2. effect of administration of fraction three of ethanol extract of zizphus mauritiana on liver function biochemical paramters among experimental animals. figure s3. effect of oral administration of fraction iii of ethanol extract of z. mauritiana leaf on organ/body weight ratio. figure s4. photomicrographs cross section of liver from wistar rats administered with distilled water (a), 200mg/kg fraction 3(b) and 500mg/kg fraction 3 (c) of ethanol leaf extract of z. orally for 14 days (group 1) (x 100) haematoxylin and eosin. 0 100 200 300 400 500 600 ast alt alp c o n ce n tr at io n ( u /l ) enzymes control 200mg/kg fraction 3 extract a b c a a aa ab 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 total protein albumin globulin mda c o n ce n tr at io n ( g/ d l) lft parameters control 200mg/kg fraction 3 extract 500mg/kg fraction 3 extract b a a a a a a a a b 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 liver kidney % o rg an -b o d y w e ig h t ra ti o organs control 200mg/kg fraction 3 zml extract a b c a b c a b c a b c c b a 662 biology, medicine, & natural product chemistry 12 (2), 2023: 651-662 figure s5. photomicrographs cross section of kidney from wistar rats administered with distilled water (a), 200mg/kg fraction 3(b) and 500mg/kg fraction 3 (c) of ethanol leaf extract of z. orally for 14 days (group 1) (x 100) haematoxylin and eosin. c ba biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 447-452 | doi: 10.14421/biomedich.2025.141.447-452 issn 2540-9328 (online) immunomodulatory activity of chinese betel (peperomia pellucida l.) extract on the spleen histopathology in a murine model of gastroenteritis lisa savitri1,3,4,*, fendy prasetyawan2, yuneka saristiana2, meri meri5, konradus klala mebung1, cornelia amanda1 1department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia 2department of pharmacist professional education, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia 3department of biology, faculty of science and technology, universitas airlangga, surabaya, indonesia 4bioinformatics research center, indonesian institute of bioinformatics, malang, indonesia 5health analyst program, faculty of health sciences,universitas tunas bakti husada,indonesia. corresponding author* lisasavitri@unik-kediri.ac.id abstract gastroenteritis, a prevalent digestive disorder caused by various pathogens including escherichia coli, remains a global health challenge with significant morbidity and mortality, particularly in low-income countries. the spleen, as a critical immune organ, is often compromised during systemic infections. peperomia pellucida (l.) , a traditional medicinal herb, is known for its antimicrobial, antiinflammatory, and antioxidant properties. this study investigates the histopathological effects of p. pellucida leaf ethanol extract on spleen tissue in mice induced with e. coli to model bacterial gastroenteritis. thirty male swiss mice were divided into six groups (n=5): normal control, negative control (aquades), positive control (yakult), and three treatment groups receiving p. pellucida extract at 100, 300, and 500 mg/kg bw. after seven days of pretreatment, mice were orally infected with e. coli (1×10⁶ cfu/ml) for another seven days. spleen tissues were harvested, fixed, and stained with hematoxylin-eosin for histopathological evaluation focusing on degeneration, necrosis, and pmn infiltration. statistical analysis was performed using anova followed by lsd tests. the highest dose of p. pellucida extract (500 mg/kg bw) significantly reduced spleen tissue damage, showing decreased degeneration (9.08%), necrosis (6.05%), and pmn infiltration (18.45%) compared to lower doses. the effect was comparable to the positive control. the ethanol extract of p. pellucida demonstrates a dose-dependent protective effect on spleen histopathology in e. coli-induced gastroenteritis in mice, supporting its potential as a natural antiseptic agent. keywords: peperomia pellucida; gastroenteritis; escherichia coli; spleen histopathology; antiseptic agent. abbreviations: analysis of variance (anova); colony forming unit (cfu); escherichia coli (e. coli); least significant difference; (lsd); polymorphonuclear cells (pmn); peperomia pellucida (p. pellucida); standard error (se); world health organization (who). introduction gastroenteritis, often referred to as stomach flu, is a condition marked by digestive tract inflammation, leading to symptoms such as vomiting, diarrhea, stomach cramps, and sometimes fever (guerrant et al., 2011). it can be triggered by various infectious organisms, including viruses, bacteria, and parasites, and is most commonly spread through contaminated food or water, or via direct contact with an infected person (koo et al., 2010). this illness poses a major global public health challenge, affecting individuals across all age groups and socioeconomic levels. the world health organization (who) reports that diarrheal diseases affect approximately 1.7 billion people yearly, with gastroenteritis being a leading contributor (world health organization, 2017). in high-income countries, viral gastroenteritis accounts for millions of doctor visits and hospital admissions annually, resulting in significant healthcare expenses (payne et al., 2013). in contrast, in lowand middle-income countries, gastroenteritis remains a top cause of illness and death, especially in children under the age of five (kotloff et al., 2013). gastroenteritis can be caused by various infectious agents, including viruses, bacteria, and parasites. the most common viral causes of gastroenteritis are noroviruses, rotaviruses, adenoviruses, and astroviruses (glass et al., 2009). bacterial pathogens associated with gastroenteritis include campylobacter, salmonella, shigella, escherichia coli (particularly enterotoxigenic and shiga toxin-producing strains), and vibrio cholerae manuscript received: 06 may, 2025. revision accepted: 27 june, 2025. published: 18 july, 2025. https://doi.org/10.14421/biomedich.2025.141.447-452 mailto:lisasavitri@unik-kediri.ac.id 448 biology, medicine, & natural product chemistry 14 (1), 2025: 447-452 (guerrant, et al., 2001). parasitic agents, such as giardia lamblia, cryptosporidium parvum, and entamoeba histolytica, can also cause gastroenteritis, although their prevalence varies across different geographic regions (checkley et al., 2015). the epidemiological patterns of gastroenteritis are influenced by various risk factors and modes of transmission. person-to-person transmission, particularly through the fecal-oral route, is a common mode of spread for viral and bacterial gastroenteritis (lopman et al., 2012). foodborne transmission is also a significant route, with contaminated food and water as vehicles for infectious agents (newell et al., 2010). specific risk factors include poor sanitation, inadequate access to safe drinking water, crowded living conditions, and compromised immune systems (kotloff, 2017). the geographical distribution of gastroenteritis is global, but the prevalence and predominant causative agents vary across regions. in developed countries, viral gastroenteritis is more common, with noroviruses being the leading cause of outbreaks in healthcare facilities, schools, and cruise ships (becker et al., 2000). in developing countries, bacterial and parasitic agents are more prevalent, contributing to a substantial disease burden, particularly among young children (troeger et al., 2018). the pathogenesis of gastroenteritis varies depending on the causative agent but involves disrupting the normal physiological functions of the gastrointestinal tract. viral pathogens, such as noroviruses and rotaviruses, primarily target and damage the epithelial cells lining the small intestine (ramig,, 2004). these viruses can bind to and enter these cells, leading to their destruction and the subsequent malabsorption of fluids and nutrients. the resulting osmotic imbalance and loss of absorptive surface area contribute to diarrhea and vomiting (bok & green, 2012). bacterial pathogens, like campylobacter, salmonella, and shigella, can cause gastroenteritis through several mechanisms, including toxins, invasion and disruption of the intestinal epithelium, and inflammation. for example, enterotoxigenic e. coli (etec) secretes heat-labile and heat-stable toxins that disrupt fluid and electrolyte balance, leading to watery diarrhea (guerrant et al., 2001). the clinical manifestations of gastroenteritis typically include diarrhea, vomiting, abdominal cramps, and, in some cases, fever. the severity of symptoms can range from mild to severe, depending on the causative agent, the individual's immune status, and other factors (fleckenstein et al., 2010). complications of gastroenteritis may include dehydration, electrolyte imbalances, and, in severe cases, sepsis or organ failure (guerrant et al., 2001). many digestive system disorders—such as peptic ulcers, chronic gastritis, upper gastrointestinal bleeding, duodenitis, pseudomembranous enteritis, acute enteritis, intestinal tuberculosis, ulcerative colitis, jaundice, diarrhea, liver cirrhosis, drug-induced liver diseases, chronic and acute cholecystitis, acute pancreatitis, and gallstones—are primarily triggered by external dampheat, internal pathogenic invasion, or unhealthy dietary habits that promote damp-heat accumulation. clinical symptoms often include nausea, a feeling of fullness in the chest and upper abdomen, diarrhea, bloating, or foulsmelling loose stools, along with a bitter taste in the mouth, excessive mucus, poor appetite, increased salivation, a tight pulse (which may indicate pain or blood stagnation), and a greasy tongue coating. treatment generally focuses on clearing damp-heat from the body (lao, 2008). the gut microbiota, as a complex micro-ecosystem, relies on mutual balance and regulation among its microorganisms. internal dampness caused by endogenous factors can disrupt this balance, impairing the function of the intestinal flora. when the yang energy of the spleen and stomach is weakened and internal cold dominates, it leads to water and dampness stagnation, disturbing the microbial harmony. therapy aimed at warming the middle region of the body, removing dampness, and strengthening spleen energy can help restore a balanced intestinal flora structure and alleviate signs of internal dampness (shen, 2004). in this context, peperomia pellucida (l.), belonging to the piperaceae family, emerges as a potential therapeutic agent. this plant, commonly utilized as a food source and in traditional medicine (tablang et al., 2020), is known for its ability to address various health conditions. locally in indonesia, it is known as sirih cina or suruhan and grows abundantly in moist areas. widely consumed by ethnic groups like the sundanese, it is typically eaten fresh as lalaban or prepared through stirfrying. rich in essential minerals such as potassium, calcium, and iron, p. pellucida is beneficial for enhancing bone strength and aiding recovery (ooi et al., 2012; florence et al., 2017). beyond its nutritional value, it has been employed in traditional healing practices to treat conditions such as headaches, kidney disorders, fever, high blood pressure, and even external issues like wounds and acne (saputri et al., 2021; hartati et al., 2015). these medicinal properties may support the restoration of balance in the body, potentially alleviating symptoms related to internal dampness. materials and methods this study used male swiss strain mice aged 2–3 months and weighing 25–30 g from the veterinary pharmacy center, surabaya. the sample size was determined using the federer formula: (n−1)(t−1) ≥ 15, where t = 6 groups, resulting in a minimum of 4 mice per group. an additional mouse was added to each group, totaling 30 mice. savitri et al. – immunomodulatory activity of chinese betel (peperomia pellucida l.) … 449 equipment and materials tools included syringes, feeding tubes, surgical sets, microscopes, microtome, water baths, and glassware. materials included male mice, e. coli, p. pellucida extract, amoxicillin, formalin, alcohol series, xylol, paraffin, giemsa stain, and other histology reagents. experimental design mice underwent a two-week acclimatization period and were randomly assigned to six groups: (1) normal control, (2) negative control (aquades), (3) positive control (0.5 ml yakult), (4–6) p. pellucida extract at 100, 300, and 500 mg/kgbw. all treatments were given via oral gavage. after 7 days of treatment, gastroenteritis was induced with e. coli (1×10⁶ cfu/ml daily for 7 days). tissue processing and histopathology spleen tissues were fixed in buffered formalin, embedded in paraffin, sectioned at 4–6 µm, and stained with hematoxylin-eosin. observations focused on capsule, trabecula, red pulp, white pulp, and signs of necrosis or apoptosis. data collection and analysis histological data were collected based on abnormal spleen cells. statistical analysis was performed using one-way anova at a 95% confidence level (α = 0.05), followed by the least significant difference (lsd) test if significant. results were expressed as mean ± standard error (se) using spss 23.0 for windows. results and discussion result this study evaluated the antiseptic potential of peperomia pellucida leaf extract by observing histopathological changes in the spleen of mice induced with escherichia coli to model gastroenteritis. six groups were used, each consisting of four mice. group i (figure 1) served as the normal control without treatment, group ii (figure 2) as the negative control (treated with distilled water), and group iii (figure 3) as the positive control (treated with ciprofloxacin). groups iv–vi received p. pellucida leaf extract at 100, 300, and 500 mg/kg body weight, respectively, prior to e. coli infection. figure 1. histopathological appearance of the spleen in the normal group at 100x magnification. figure 2. histopathological appearance of the spleen in the negative control group at 100x magnification. figure 3. histopathological appearance of the spleen in the positive control group at 100x magnification. histopathological evaluation of the spleen histopathological examination focused on cell degeneration, necrosis, and polymorphonuclear cell (pmn) infiltration. table 1 shows that group i (normal control) had the lowest average percentages for degeneration (2.21±0.02%), necrosis (2.44±0.02%), and pmn infiltration (1.62±0.02%). group iii (positive control) followed, showing lower damage levels than the negative control and treatment groups. among the extract-treated groups, group vi (500 mg/kg bw) demonstrated the most notable reduction in tissue damage, with degeneration (9.08±0.02%), necrosis (6.05±0.02%), and pmn infiltration (18.45±0.03%), indicating its potential antiseptic efficacy. statistical analysis one-way anova results showed significant differences among groups (p < 0.05) for all three histological parameters. lsd and duncan’s post hoc tests confirmed that group vi (figure 4) (500 mg/kg bw) had significantly lower damage than groups v (figure 5) and iv (figure 6), while no significant difference was observed between group iv (100 mg/kg bw) and group v (300 mg/kg bw) for cell degeneration, suggesting similar antiseptic activity at these doses. 450 biology, medicine, & natural product chemistry 14 (1), 2025: 447-452 figure 4. histopathological appearance of the spleen in the paederia foetida extrect 500 mg/kg bw group at 100x magnification. figure 5. histopathological appearance of the spleen in the paederia foetida extrect 300 mg/kg bw group at 100x magnification. figure 6. histopathological appearance of the spleen in the paederia foetida extrect 100 mg/kg bw group at 100x magnification. discussion the findings indicate that p. pellucida leaf extract can reduce spleen tissue damage caused by e. coli-induced gastroenteritis. the reduction in cellular degeneration and necrosis in treatment groups is likely attributed to bioactive compounds such as flavonoids, saponins, and tannins in p. pellucida. flavonoids are known for their strong antioxidant properties, which help mitigate oxidative stress and reduce inflammation by scavenging free radicals and inhibiting proinflammatory cytokines (middleton et al., 2000; gonzález-gallego et al., 2007). saponins contribute to immune modulation and have anti-inflammatory effects by enhancing the activity of antioxidant enzymes and regulating cytokine production (shi et al., 2014). moreover, saponins may support the spleen’s detoxification processes, potentially mitigating bacterial damage. tannins have antimicrobial activity and may inhibit bacterial adherence and colonization in host tissues (scalbert, 1991). the results demonstrate that the 500 mg/kg bw dose of p. pellucida extract had comparable efficacy to ciprofloxacin, suggesting it may be a potential natural antiseptic agent against bacterial-induced gastroenteritis. however, further investigation into the specific mechanisms and active components is recommended. the protective effect observed in group vi (500 mg/kg bw) suggests that higher concentrations of peperomia pellucida extract confer greater histological protection to spleen tissue following bacterial insult. this aligns with previous studies indicating that the pharmacological activity of medicinal plants often increases with dose, up to a point of biological saturation or toxicity (rates, 2001). in the context of this study, the increasing dose-dependent efficacy supports the therapeutic relevance of p. pellucida in treating infections associated with systemic inflammation. the spleen plays a key role in the immune response, filtering blood and responding to systemic bacterial invasion. in the presence of e. coli, the observed damage in untreated or insufficiently treated groups reflects a strong inflammatory response marked by high pmn infiltration and tissue necrosis. the observed suppression of pmn infiltration in the high-dose treatment group suggests that the extract may possess immunomodulatory properties, potentially by modulating cytokine expression pathways such as nf-κb and mapk, which are known to regulate neutrophil activation and chemotaxis (liu et al., 2017; chen et al., 2018). the bioactive constituents of p. pellucida, especially flavonoids and tannins, may exert synergistic effects. flavonoids such as quercetin and apigenin—identified in related species—have been shown to inhibit the production of tnf-α and il-6, key mediators in acute inflammation (li et al., 2016). tannins may further inhibit bacterial adhesion to epithelial cells and neutralize endotoxins like lipopolysaccharides (lps), contributing significantly to septic-like damage in organs including the spleen (okuda, 2005). furthermore, studies have reported the antioxidant and hepatoprotective activities of p. pellucida, indicating its systemic benefits beyond the gastrointestinal tract (almagboul et al., 1985). the spleen, being highly vascularized and sensitive to oxidative stress, benefits from the scavenging activity of antioxidants, which may explain the lower necrosis levels observed in the highdose extract group. in contrast, the lower doses (100 and 300 mg/kg bw) showed moderate protection, suggesting that while these savitri et al. – immunomodulatory activity of chinese betel (peperomia pellucida l.) … 451 concentrations do confer some degree of benefit, they may not be sufficient to counteract the full inflammatory burden induced by e. coli. this highlights the need for optimization of dosage in potential therapeutic applications. from a pharmacognostic perspective, the findings contribute to a growing body of evidence supporting the traditional use of p. pellucida in treating gastrointestinal and inflammatory disorders in ethnomedicine across asia and south america (gonzaga et al., 2005). however, further studies involving phytochemical isolation, molecular assays, and toxicological profiling are necessary to validate safety and efficacy before clinical use. conclusions the results of this study demonstrate that ethanol extract of peperomia pellucida exerts a dose-dependent protective effect on spleen histology in mice infected with escherichia coli. histopathological improvements— marked by reduced necrosis and polymorphonuclear (pmn) infiltration—were most pronounced at the highest administered dose of 500 mg/kg bw. these findings suggest that p. pellucida possesses significant antiinflammatory and potential immunomodulatory properties, likely attributed to its rich content of flavonoids and tannins. the extract’s ability to mitigate tissue damage in response to bacterial infection supports its traditional use and highlights its potential as a complementary therapeutic agent. however, further research is necessary to isolate specific bioactive compounds, elucidate molecular mechanisms, and assess long-term safety for potential clinical applications. acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university, and the laboratory of pathological anatomy, brawijaya university, malang, for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors wrote the manuscript and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there is funding by the research, development, and community service institution (lp3m) of universitas kadiri. references almagboul, a. z., bashir, a. k., farouk, a., & salih, a. m. (1985). antimicrobial activities of certain sudanese plants used in folkloric medicine. screening for antimicrobial activity of 63 medicinal plants, 17(6), 793–802. chen, l., deng, h., cui, h., fang, j., zuo, z., deng, j. & zhao, l. (2018). inflammatory responses and inflammation-associated diseases in organs. oncotarget, 9(6), 7204. florence, n. t., huguette, s. t. s., hubert, d. j., raceline, g. k., desire, d. d. p., pierre, k., & theophile, d. (2017). aqueous extract of peperomia pellucida (l.) hbk accelerates fracture healing in wistar rats. bmc complementary and alternative medicine, 17(1): 1-9. gonzález-gallego, j., garcía-mediavilla, m. v., sánchez-campos, s., & tuñón, m. j. (2007). anti-inflammatory and immunomodulatory properties of dietary flavonoids. polish journal of food and nutrition sciences, 57(4), 399–406. guerrant, r. l., van gilder, t., steiner, t. s., thielman, n. m., slutsker, l., tauxe, r. v. & tarr, p. i. 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(2006). immunomodulatory properties of medicinal plants. phytotherapy research, 20(8), 659–667. biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 1, 2015 | pages: 1-3 | doi: 10.14421/biomedich.2015.41.1-3 a simple and practical method for rat epididymal sperm count (rattus norvegicus) muhammad ja’far luthfi biology department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: jafarluthfi@yahoo.com abstract sperm sample from epididymal source can be determined its number using minimal amount of equipment. these method will aid researcher and practitioner in sperm quality analysis to determined sperm number rapidly and practically. keywords: sperm quality, sperm number, sperm count, haemocytometer introduction rat is the most widely used testing animal in reproductive biology for several reasons. the rat is an animal model that resembles human physiology. this animal has long been used as test animals in screening a compound to determine the pharmacological effects include mechanisms, distribution, and toxicity (briggs & oehme 1980). the extensive use of the rat in research has led to a relatively complete biological data (golden 2002; white 2001). several methods have been developed for the assessment of male reproductive function. one of the most widely used methods is sperm quality analysis. analysis of sperm quality can give us information about the fertility status of the male genital organ. the purpose of the analysis is to assess the sperm descriptive parameters. usually, the parameters used to predict male fertility are the sperm number/sperm count, sperm morphology and sperm motility. the analysis may show an increase or decrease in the fertility of the animal testing. methodology equipments equipment required in rat sperm motility analysis is as follows:  light microscope  improved neubauer haemocytometer  micropipette  petri dish  surgical scissors  incubator  hand counter sperm sample preparation sperm samples were taken from the cauda epididymis. cauda epididymis were separated as determined by hamilton (1975), then placed in a petri dish, minced and incubated in 15 ml of media biggers, whitten, and whittingham (bww) (biggers et al. 1971) for 30 minutes at 37 ° c in 5% co2 incubator to allow the sperm to swim in the medium bww (swim-up technique). sperm count procedure mount the cover glass on neubauer improved neubauer haemocytometer. interference pattern (> 10 newton's rings/fringes or iridescence lines) should be seen between the glass surface of the area where the glass cover attached to the haemocytometer. too little line/newton's rings shows that the distance between the cover glass and haemocytometer widened, therefore counting chamber volume becomes larger and the counting will be inaccurate. a total of 10 ml sperm suspension were taken from sperm sample preparation, and then inserted into the space between the cover glass and haemocytometer. other counting chamber also filled in the same way. each chamber sould be completely filled. suspension inserted slowly to let the liquid evenly by capillary forces. if a chamber is overfill, it must be discarded and fill a new chamber. removal of the superfluous chamber must not be done since this will change the sperm concentration in the chamber. let haemocytometer 10-15 minutes to allow the sperm settled on counting grid. counting were done with 200x magnification using a light microscope. at counting chamber central area there are 25 large square. each "large squares" is bounded on all sides by a triple line (figure 1). counting performed on all of 25 large squares at each counting chamber. for sperm located on the borderline, only sperm lies on the upper or left line (triple 2 biology, medicine, & natural product chemistry 4 (1), 2015: 1-3 line) should be count as “belonging” to that square. plot is calculated as the property of the plot. therefore, do not count the sperm that is located on the bottom line or the right line. figure 1. haemacytometer. a. side view (cover glass shown by 1). b. top view. c. one of the counting chambers (one of the 25 large squares shown by 2). approximately 200 sperm in each chamber should be count to minimize standard deviation. if the number of sperm obtained from the count at one counting chamber is less than 150, made of new sperm suspension samples by reducing the volume of bww medium. calculation of cauda epididymal sperm number firstly, the sperm number from both chamber were averaged. secondly, the mean of counted sperm divided by volume within which they were count (volume of 25 large square= 100 nl). the sperm concentration obtained is the number of sperma per nl, whic equals millions of sperm/ml (sperm /10-9 l= sperm x 106/10-3 l). thirdly, to obtain the number of sperm per cauda epididymis, sperm concentration obtained multiplied by the number of bww medium volume used in swim up. example of sperm number calculation 15 ml solution of bww media was added to epididymal sperm sample (swim-up method). counting on one chamber produces 320, whereas in the other counting chamber is obtained 280. both these results are summed and divided by two to get the average, obtained number 300. to obtain the concentration of sperm per nl, average of sperm number of both chamber was divided by 100, to obtain the numbers 3 x 106 sperm per ml of sperm suspension. to obtain the number of sperm per cauda epididymis, sperm concentration (3 x 106 sperm per ml) was multiplied by the volume of bww used to swim up (15 ml) yields: 30 x 106 sperm per cauda epididymis of rat. discussion determination of sperm number is one of the important aspects in the analysis of sperm quality. however, there are so many variations on the method in the aspects of equipment and technical details. various different laboratories using different methods. in this study we use improved neubauer haemocytometer to count sperm number. the method mostly based on the practice and experience of testing of 300 rat samples. the study were done at uin sunan kalijaga yogyakarta and laboratory of zoology faculty of science and technology universiti kebangsaan malaysia (data not shown). sperm count or sperm concentration of the testing species can be determined from a sample of ejaculate, epididymis, or the testes. determination of the caudal epydidimal sperm number usually only use sperm from the cauda (clegg et al., 2001). difference in the sperm number after treatment of materials or drugs may give important clues about the effect of a substance on sperm production (working, 1988). epididymal sperm counts are generally used in toxicology studies to assess the damage to the male reproductive system, or vice versa, to determine the therapeutic effect of a substance. epididymal sperm count reduction indicates a reduction in daily sperm production by the testes, the transport obstacles from the testes to the muhammad ja’far luthfi. – a simple and practical method for rat epididymal sperm count … 3 epididymis, or changes in the epididymal sperm transit time. in general, the determination of epididymal sperm count was performed using haemocytometer (strader et al., 1996). determination of sperm concentration with haemocytometer is the main basis for determining whether a sample categorized as normal, and to predict whether an individual is fertile. counting with haemocytometer has become a standard method for measuring the concentration of sperm (freud & carol, 1964). compared with other methods, haemocytometer is still a better method for determining the sperm number (kuster, 2005). in this study, the amount of bww volume used in a sperm sample dilution is 15 ul. however, this number can be increased or decreased to obtain suitable sperm density. in some cases, it is necessary to add 20 ml bww solution for dilution in order to obtain the ideal sperm density to be calculated (200-400 sperm in a haemocytometer counting chamber). three factors need to be considered to achieve accuracy uisng this method. firstly, we must ensure that the incision of the cauda cauda epididymis was consistent. secondly, the insertion of the sample with a micropipette on each space calculation must be completely filled. thirdly, the room temperature in the determination of sperm numbers should be the same for the entire counting sperm samples. these factors affect the consistency of counting of the sperm number. the strict procedure will ensure the accuracy sperm count. references biggers, j.d., whitten, w.k. &whittingham, d. 1971. the culture of mouse embryos in vitro dlm daniel, j.c. (ed). methods in mammalian embryology. page 86-116. freeman san francisco, ca. briggs, g.b. & oehme, f.w. 1980. toxicology dlm baker, h.j. et al. (ed). the laboratory rat. volume ii. researhapplication. page 104-118. academic press. new york. golden, a.l. 2002. biomarkers of male reproductive health dlm wilson, s.h. & suk, w.a. (ed). biomarkers ofenvironmentally associated disease. technologies, concepts, and perspectives. page 387-410. lewis publisher. new york. hamilton, d.w. 1975. structure, function of the epithelium lining the ductuli efferents, ductus epididymis and ductus deferens in the rat dlm hamilton, d.w. & greep, r.o. (ed). handbook of physiology, section vii, endocrinology, vol.5, male reproductive system. page 259-301. american physiological society, washington d.c. white, w.j. 2001. the use of laboratory animals in toxicologic research dlm hayes a.w. (ed). principlesand methods of toxicology. fourth edition. page 773-775. taylor & francis. philadelphia. content_v4n1_1.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 47-57 | doi: 10.14421/biomedich.2021.101.47-57 issn 2540-9328 (online) the effect of balimo (zanthoxylum nitidum) immersion water on the hematological profile of white rats (rattus norvegicus) that given liquor (ciu) panca buana wijaya*, tyas rini saraswati, silvana tana, sunarno, erma prihastanti department of biologi, faculty of science and mathematics, universitas diponegoro, jl. prof soedarto, sh, tembalang. semarang, indonesia. corresponding author* pancabuanawijaya@gmail.com manuscript received: 21 december, 2020. revision accepted: 11 june, 2021. published: 23 july, 2021. abstract consumption of liquor such as ciu in excessive doses can cause a decrease in hematological status. balimo stem is an alternative treatment to improve hematological status due to excessive alcohol consumption because it contains alkaloids, flavonoids, and other secondary metabolic compounds, that have functions as antioxidant effects. this study aims to examine and analyze the effect of balimo immersion water on the hematological status of mice with the observed variables, namely the erythrocytes count, hemoglobin levels, hematocrit value, and total count of leukocytes in rats that had been given ciu. the study used 20 rattus norvegicus male rats which were divided into 4 groups. the data were analyzed using one-way anova. the results showed no significant differences (p <0.05) on the balimo immersion water treatment, but if it was seen from the difference in the mean data of each variable, it could still be seen the difference from each treatment. in this study, it can be concluded that balimo immersion water was able to improve the hematological status of rats that had been given ciu liquor with a 0,2 ml dose. keywords: antioxidant; balimo; zanthoxylum nitidu; hematological status; rattus norvegicus. introduction ciu is a traditional liquor of banyumas and bekonang regions, sukoharjo. ciu is made from fermented cassava or sticky rice, which produces ethanol and carbon dioxide with ± 30% ethanol (bpom, 2014). the world health organization (who) also states that consuming liquor regularly can cause long-term diseases including hepatitis, tuberculosis, heart disease, cancer, cirrhosis of the liver, an increase in infectious diseases such as hiv / aids, and can even cause death (hammer et al., 2018). laboratory examinations of 179 respondents who have the habit of drinking liquor found that the hemoglobin levels, erythrocyte count, and the hematocrits value showed lower values compared to normal conditions, besides the data showed that the total count of leukocytes was higher than normal (riany & rahmayanti, 2013). drunk liquor will undergo an absorption process in the digestive system, the absorption process occurs in the oral mucosa, stomach, and small intestine. alcohol is absorbed through the walls of the stomach and intestines, then carried by the blood circulation to all organs in the body, especially the liver. alcohol that has reached the liver then undergoes a series of metabolism (katzung & trevor, 2015). ethanol metabolism in the liver produces acetaldehyde (wilson & matschinsky, 2020). too much acetaldehyde makes acetaldehyde more toxic. the acetaldehyde in mitochondria will be synthesized again into triglycerides or fatty acids through an oxidation process (king, 2019). the increase in triglycerides that occurs causes a buildup of fat in the liver, it triggers fatty liver or steatosis and eventually becomes alcoholic liver cirrhosis (shah, 2015). patients with alcoholic liver cirrhosis have abnormal hematological parameters, which lead to a tendency to bleeding, anemia, leukopenia, and thrombocytopenia (lindseth, 2013). the main factors causing complications of bleeding that occur are reduced clotting factors due to liver cirrhosis and the destruction of blood cells resulting in abnormalities in the number of blood cells (armitage, 2011). the progressive decrease in hemoglobin level in liver cirrhosis patients. hematological abnormalities are often found in acute alcohol drinkers in the study (jain et al., 2016). the direct effect of excessive alcohol consumption is the toxic effect on bone marrow, blood cell precursors, erythrocyte mature cells, and leukocytes. the indirect effect of alcohol consumption is nutritional deficiencies that can interfere with blood cell production (hematopoiesis) and the function of various blood cells, https://doi.org/10.14421/biomedich.2021.101.47-57 48 biology, medicine, & natural product chemistry 10 (1), 2021: 47-57 causing an increase in blood pressure or hypertension (ifeannyi et al., 2014). the balimo plant (zanthoxylum nitidum) is used by the kanayatn dayak tribe in tapakng village as a medicinal plant for traditional medicine (raja and hartana, 2017). balimo plants are used to treat alcohol poisoning after drinking wine (andasputra & julipin, 2011). the balimo plant is also used as a medicine for a cough with phlegm and hemoptysis treatment (sepsamli & jumari, 2019). balimo plant (zanthoxylum nitidum) is used to improve blood circulation, relieving blood stasis in traditional chinese medicine. phytochemical investigations showed the presence of alkaloids, flavonoids, and true amino acids in the bark of zanthoxylum nitidum (bhattacharya & zaman, 2009). these compounds have benefits as anti-viral, antiinflammatory, antioxidant, and anti-cancer activities (van nguyen et al., 2019). balimo stem is expected to be an alternative to improve hematological status due to consuming liquor because it contains flavonoids, alkaloids that have antiinflammatory and antioxidant effects that function to improve hematological status (aiba et al., 2016). antioxidants are compounds that can inhibit oxidation reactions by binding to free radicals and highly reactive molecules (suryani et al., 2013). the mechanism of action of flavonoid compounds as antioxidants is to maintain erythrocytes, by inhibiting peroxidation of lipid peroxidation caused by h2o2 and preventing protein degradation and hemolysis (yousif et al., 2012). flavonoids play an important role in regulating blood vessel health, namely maintaining maximum cardiac function (catherine et al., 2015). based on the background above, to determine the effect of compounds in plants balimo (zanthoxylum nitidum), it will do the research and observation of the hematological status of male rats (rattus norvegicus) who had been treated with alcohol and then given balimo (zanthoxylum nitidum) immersion water. materials and methods research ethics this study has received ethical clearance from the ethics commission for health and medical research, faculty of medicine, diponegoro university semarang no. 164 / ec / h / kepk / fk-undip / xii / 2019. research design and research locations research on the effect of balimo (zanthoxylum nitidum) stem immersion water on the hematological status of rats that have been given liquor (ciu) was carried out in animal cages, and animal structure and function laboratory, department of biology, fsm-undip. this study used 20 male rats (rattus norvegicus) which were divided into 4 treatment groups, including: a0: represents rattus norvegicus control, a1: represents cirrhosis rattus norvegicus that given ciu for 2 weeks at a dose of 0.2 ml, a2: represents cirrhosis rattus norvegicus by giving balimo immersion water (ciu for 2 weeks and 50% balimo immersion water for 2 weeks with a dose of 0.2 ml each), a3: represents cirrhosis rattus norvegicus with balimo immersion water (ciu for 4 weeks along with 50% balimo immersion water for 2 weeks with a dose of 0.2 ml each). each treatment was repeated 5 times. treatment for 2 weeks of acclimation and 4 weeks of treatment. tools and materials the tools used in this study include 35 sets of mouse cages, wire caps, food and drink containers, gloves, feeding bowls, sprayers, syringes, sonde, measuring cups, analytical scales, paraffin tubs, surgical instruments, edta tubes, dropper pipettes, hemometer sahli, sahli pipette, aspirator, erythrocyte pipette, leukocyte pipette, microscope, object-glass, object glass cover, counters, cameras and counting booths improved neubauer, centrifuge, hematocrit tube, microhematocrit reader. the materials used in this study included 35 male rats (rattus norvegicus) 60 days old, standard organic feed, rice husks, drinking water, balimo immersion water, detergent, distilled water, 39% liquor (ciu), 70% alcohol, 0.1 ml hcl, hemotoxin-eosin dye, hayem's solution, turk solution, chloroform, glass objects, and blood samples. making the balimo immersion water making balimo immersion water is done by cutting the balimo stem with a size of 0.5 cm, then weighing it using 1 g of analytical scales and put in 10 ml of 36oc warm water stored in a tightly closed glass bottle and left for 3 days. the dosage levels is determined based on according to the ethical commission which has been converted based on the dose to humans and the determination of the concentration is determined based on the results of preliminary tests that are previously recognized. blood data collection and observation hematological status data were obtained, then observed appropriately based on variables, such as: erythrocyte count observation of the erythrocytes count was carried out using a microscope, improved neubauer hemocytometer counting chamber, erythrocyte pipette and aspirators, counters, hayem solution, aquades. observations were carried out by observing and counting erythrocyte cells using a microscope, improved neubauer counting chamber using the 5-square wijaya, et al. – the effect of balimo (zanthoxylum nitidum) immersion water … 49 technique in the large square in the middle of the counting chamber, then the count of all erythrocyte cells in the five squares multiplied by 5000 (oktiyani et al., 2017). hemoglobin levels hemoglobin levels are observed with hemometer sahli method, by equating blood color on the color indicator (block comparator) in hemometer sahli, then calculated the existing high blood fluid in the tube hemometer sahli, aquades supplemented by the addition dropwise (a'tourrohman, 2020) hematocrit value observation of the hematocrit value is carried out on blood in a centrifuge then the blood is fed into the microhematocrit reader or using the hematocrit value formula (nurrahman & mariyam, 2019). total count of leukocytes observation of the total count of leucocytes was carried out using a microscope, counting booth improved neubauer, leucocyte pipette and aspirator, counter, turk solution, and distilled water. observations were made by observing and counting leukocyte cells using a microscope, an improved neubauer counting chamber in the five boxes located diagonally in 4 large squares in the corner of the counting room, the results were x 50 cells / mm3 of blood (bakhri, 2018). data analysis hematological status data that had been observed were then analyzed using analysis of variance (anova) using a completely randomized design with 4 treatments and five replications (gomez, 2005). results and discussion the results of the statistical analysis of the effect of balimo (zanthoxylum nitidum) stem immersion water on the hematological status of rats (rattus norvegicus) given liquor (ciu). based on the statistical analysis test using the one-way analysis of variance (anova) test with a confidence level of 95%. based on the results of data analysis using anova, the effect of balimo stem immersion water (zanthoxylum nitidum) on the hematological status includes the erythrocytes count, hemoglobin levels, hematocrit values, and the total count leukocytes of white rat (rattus norvegicus) which have been given ciu shows no different results. real or (p> 0.05). hematological status includes the erythrocytes count, hemoglobin levels, hematocrit values, and the total count leukocytes of white rat (rattus norvegicus) which are not different due to several factors, including the length of time given treatment in all groups is still too short, which causes the treatment effect has not reached an acute point. also, the body's immune system can maintain a state of hematological status, especially in mice when they are 6 weeks old, so that the mice can restore the state of hematological status. guyton & hall (2014) states that the body has a defense system that can maintain and improve the condition of the body that is experiencing disorders. erythrocyte count the results of statistical tests based on the mean erythrocytes count of rats in the treatment groups a1, a2, a3 when compared with treatment a0 (control) showed a significant value above 0.05 (p> 0.05) or the results were not significantly different. the results of the analysis of the number of erythrocytes still change based on the group and can be seen in the graph (figure 1). figure 1. erythrocyte count. the average number of erythrocytes when compared with the a0 treatment group, the erythrocytes count in group a1 decreased due to the toxicity effect of the alcohol content in ciu drinks, resulting in damage to erythrocyte blood cells and suppression of erythrocyte formation (erythropoiesis) which occurs due to ethanol compounds scattered in the blood vessels and spinal cord. the erythrocytes count in group a2 increase because treatment of a2 was able to inhibit free radicals that interfere with the formation of blood cells, and flavonoid and alkaloid compounds can also stimulate the central nerve, which stimulates the spinal cord so that the formation of red blood cells increases. the formation of red blood cells is not disturbed and will continue to form blood cells in the bone marrow, so the erythrocytes count can increase and tends to increase the hematological status of the a2 treatment group mice. the erythrocytes count in group a3 has decreased and then increased because a3 approaches a0, this is because the erythrocytes count in group a3 has increased again and tends to maintain the erythrocytes count by protecting and repairing damage to blood cells from free radicals due to the presence of flavonoids and alkaloids that function as antioxidants in balimo immersion water. flavonoid and alkaloid compounds 5,1 4,97 5,47 5,07 4,6 4,8 5 5,2 5,4 5,6 a0 a1 a2 a3 erythrocyte count (×106/µl) 50 biology, medicine, & natural product chemistry 10 (1), 2021: 47-57 can also stimulate the central nervous system, which stimulates the spinal cord so that the formation of red blood cells increases. hartono et al., (2019), states that alcoholic drinks can have a toxic effect on bone marrow which causes suppression of blood production and abnormal blood structure so that the erythrocytes count is reduced. stanley, et al. (2014) explained that alkaloid and flavonoid compounds act as antioxidants which can play a role in preventing oxidative stress due to exposure to free radicals. hemoglobin levels the results of statistical tests based on the mean hemoglobin levels of rats in the treatment groups a1, a2, a3 when compared with treatment a0 (control) showed a significant value above 0.05 (p> 0.05) or the results were not significantly different. the results of the analysis of hemoglobin levels continued to change based on groups and can be seen in graphs (figure 2). figure 2. hemoglobin levels. the average hemoglobin level when compared with the a0 treatment group, the hemoglobin level of group a1 decreased because it was caused by the toxic effect of ethanol compounds that spread in the body, both in the bone marrow and blood vessels due to giving ciu for 2 weeks, without water treatment. balimo immersion water. this causes damage to erythrocyte blood cells and reduces or destroys the ability of erythrocyte blood cells to synthesize hemoglobin so that the hemoglobin level in the blood is also reduced. the level of hemoglobin in the a2 group has increased due to the effect of giving balimo immersion water which functions as a therapy for the toxic effects of the ciu liquor, and balimo immersion water has flavonoids which are useful as antioxidant compounds, and as a source of protein that can help form hemoglobin in blood cells. the hemoglobin level of the a3 group which was expected to increase, showed a decrease in hemoglobin levels. this is due to the prolonged administration of the ciu liquor, which causes more damage to blood cells. this also affects the formation of hemoglobin in blood cells and bone marrow, which becomes inhibited and disturbed. this is explained by schalm (2010) that the hemoglobin synthesis that occurs in blood cells in the bone marrow will be disrupted and damaged due to the toxic effects caused by ethanol compounds in alcoholic drinks. aiba et al., (2016) explain that balimo stems have flavonoid compounds and alkaloids that can be used for antioxidants to repair cells damaged by free radicals. hematocrit value the results of statistical tests based on the mean hematocrit value of mice in the treatment groups a1, a2, a3 when compared with treatment a0 (control) showed a significant value above 0.05 (p> 0.05) or the results were not significantly different. the results of the analysis of the hematocrit value continue to change based on the group and can be seen in graphs (figure 3). figure 3. hematocrit value. the mean hematocrit value when compared to the a0 treatment group, the hematocrit value of group a1 decreased due to the toxic effect of ciu alcoholic drink which resulted in a decrease in the number of erythrocytes and hemoglobin levels, so the hematocrit value also decreased because the hematocrit value was proportional to the erythrocytes count and hemoglobin levels. the hematocrit value of group a2 increased due to the effect of balimo immersion water treatment for 2 weeks which functions as an antioxidant against the toxic effects of ciu for the previous 2 weeks, this resulted in a return to the condition of the hematological status and was able to increase the erythrocytes count and hemoglobin levels. hematocrit value in group a3 showed a decrease in the hematocrit value with the same results as in group a1 because the ciu was longer than the other groups so that the treatment ability of balimo immersion water was not able to increase the hemoglobin level, because according to the hemoglobin a3 chart, it was less versus a1, and shows the same hematocrit value as a1. this is 15,33 14,97 16,23 14,83 14 14,5 15 15,5 16 16,5 a0 a1 a2 a3 hemoglobin levels (g/dl) 46 44,67 49,33 44,67 42 43 44 45 46 47 48 49 50 a0 a1 a2 a3 hematocrit value (%) wijaya, et al. – the effect of balimo (zanthoxylum nitidum) immersion water … 51 parallel with rosita, et al. (2015) which states that normal hematocrit values are proportional to the number of erythrocytes and hemoglobin levels. if the erythrocytes count and hemoglobin levels decreased, the percentage of the hematocrit value also decreased. total count of leukocytes the results of statistical tests based on the mean total count leukocytes of rats in the treatment groups a1, a2, a3 when compared with treatment a0 (control) showed a significant value above 0.05 (p> 0.05) or the results were not significantly different. the results of the analysis of the total number of leukocytes still change based on the group and can be seen in graphs (figure 4). figure 4. total count of leukocytes. the mean total count of leukocytes when compared with the treatment group a0, the total count of leukocytes in groups a1, a2, a3 increased. the increase in the total count of leukocytes in group a1 occurs due to the presence of free radical compounds caused by liquor so that the defense system reacts by increasing the count of leukocytes so that foreign compounds including free radicals in the body decrease. the increase in the total count of leukocytes in group a2 was due to the provision of balimo immersion water for 2 weeks after ciu treatment for 2 weeks. balimo immersion water intake after consuming liquor initially provides a therapeutic effect and eliminates the acid toxicity effect of alcohol to improve the hematological status. balimo immersion water intake can cause an increase in the total count of leukocytes in the body because, there are anti-inflammatory alkaloid compounds, and can also be active toxin if given excessively so that the leukocyte defense system considers these compounds to be harmful foreign compounds. the increase in the total count of leukocytes in group a3, which should have shown a result of the total count of leukocytes that is close to a0, but has not shown a decrease that is close to a0. this is because in the a3 treatment group there was a reaction caused by the ciu toxic compound given for 4 weeks and the compound in the balimo immersion water given for the last 2 weeks. compounds in liquor for 4 weeks caused a toxic effect on the hematological status of the rats, resulting in a significant increase in the total count of leukocytes. after that there was a decrease in the total count of leucocytes due to the provision of balimo immersion water, however, because the provision of ciu continued in the last 2 weeks, the function of the balimo immersed water compound was inhibited, due to the reaction of ethanol compounds from ciu, and caused the total number of leukocytes not showed signs of a significant decline approaching group a0. this is in line with the statement of fitria, (2014) which states that leukocytes are closely related to the body's defense system. leukocytes in normal conditions are very few, and leukocytes will be increased by pathological conditions. this is following the statement of aziyah et al. (2014), and dumeva et al. (2016) which states that alkaloid compounds have active toxic properties when consumed without recommendations. alkaloids are composed of carbon, hydrogen, and nitrogen which can damage the nervous system, interfere with breathing, and interfere reproductive abilities. stanley, et al, (2014) also explained that flavonoid compounds can bind and neutralize alcohol so that the total number of leukocytes can decrease. lenny, (2006) also explains that flavonoid and alkaloid compounds that can bind ethanol compounds reduce the ability to repair cells so that leukocytes help repair and protect the body, especially in areas affected by inflammation. conclusion based on the results of the study, it was shown that balimo stem soaking water intake affected the number of erythrocytes, hemoglobin levels, hematocrit value, and the total number of leukocytes in rats that had been given ciu. intake of balimo immersion water can improve the hematological status of rats that have been given ciu. conflict of interest: the authors declares that there are no conflicts of interest concerning the publication of this article. references aiba, s., manalu, w., suprayogi, a., & maheswari, h. 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(2012). structure-activity relationships regarding the antioxidant effects of the flavonoids on human erythrocytes. natural science 4(94): 740-747. wijaya, et al. – the effect of balimo (zanthoxylum nitidum) immersion water … 53 table s1. result of the normality test. tests of normality variabel kolmogorov-smirnova shapiro-wilk statistic df sig. statistic df sig. erythrocytes number ,215 12 ,131 ,935 12 ,442 hemoglobin level ,156 12 ,200 ,953 12 ,676 hematocrit value ,136 12 ,200 ,970 12 ,908 total leukocytes ,207 12 ,166 ,909 12 ,206 *. this is a lower bound of the true significance. a. lilliefors significance correction table s2. result of the homogeneity test. test of homogeneity of variances variabel levene statistic df1 df2 sig. erythrocytes number 2,270 3 8 ,157 hemoglobin level 1,964 3 8 ,198 hematocrit value 2,013 3 8 ,191 total leukocytes 2,729 3 8 ,114 table s3. anova test anova variabel sum of squares df mean square f sig. erythrocytes between groups number within groups total ,430 3 ,143 ,808 ,524 1,420 8 ,178 1,850 11 hemoglobin between groups level within groups total 3,583 3 1,194 1,031 ,429 9,267 8 1,158 12,849 11 hematocrit between groups value within groups total 43,667 3 14,556 1,456 ,298 80,000 8 10,000 123,667 11 total between groups leukocytes within groups total 269,576 3 89,859 ,789 ,533 911,573 8 113,947 1181,149 11 54 biology, medicine, & natural product chemistry 10 (1), 2021: 47-57 table s4. results of the descriptive data analysis of the treatment. descriptives variabel n mean std. deviation std. error 95% confidence interval for mean minimum maximum lower bound upper bound erythrocytes a0 number a1 a3 a3 total 3 5,10000 ,400000 ,230940 4,10634 6,09366 4,700 5,500 3 4,96667 ,057735 ,033333 4,82324 5,11009 4,900 5,000 3 5,46667 ,472582 ,272845 4,29271 6,64062 5,100 6,000 3 5,06667 ,568624 ,328295 3,65413 6,47921 4,600 5,700 12 5,15000 ,410100 ,118386 4,88944 5,41056 4,600 6,000 hemoglobin a0 level a1 a3 a3 total 3 15,33333 1,159023 ,669162 12,45416 18,21251 14,100 16,400 3 14,96667 ,251661 ,145297 14,34151 15,59183 14,700 15,200 3 16,23333 1,365040 ,788106 12,84239 19,62428 15,300 17,800 3 14,83333 1,167619 ,674125 11,93281 17,73386 13,800 16,100 12 15,34167 1,080790 ,311997 14,65497 16,02837 13,800 17,800 hematocrit a0 value a1 a3 a3 total 3 46,00000 3,605551 2,081666 37,04331 54,95669 42,000 49,000 3 44,66667 ,577350 ,333333 43,23245 46,10088 44,000 45,000 3 49,33333 3,214550 1,855921 41,34795 57,31872 47,000 53,000 3 44,66667 4,041452 2,333333 34,62714 54,70619 41,000 49,000 12 46,16667 3,352972 ,967920 44,03629 48,29704 41,000 53,000 total a0 leukocytes a1 a3 a3 total 3 11,23333 1,803700 1,041367 6,75269 15,71397 9,500 13,100 3 19,20000 10,400000 6,004443 -6,63503 45,03503 12,800 31,200 3 24,43333 18,123557 10,463641 -20,58808 69,45475 5,800 42,000 3 19,70000 3,988734 2,302897 9,79144 29,60856 17,200 24,300 12 18,64167 10,362297 2,991337 12,05778 25,22556 5,800 42,000 table s5. tukey test. multiple comparisons (i) (j) dependent variable perlakuan perlakuan mean difference (i-j) std. error sig. 95% confidence interval lower bound upper bound erythrocytes tukey number hsd a0 a1 a3 a1 a3 a3 ,133333 ,343996 ,979 -,96826 1,23493 -,366667 ,343996 ,718 -1,46826 ,73493 ,033333 ,343996 1,000 -1,06826 1,13493 a0 a3 a3 -,133333 ,343996 ,979 -1,23493 ,96826 -,500000 ,343996 ,504 -1,60160 ,60160 -,100000 ,343996 ,991 -1,20160 1,00160 a0 ,366667 ,343996 ,718 -,73493 1,46826 wijaya, et al. – the effect of balimo (zanthoxylum nitidum) immersion water … 55 a3 a1 a3 ,500000 ,343996 ,504 -,60160 1,60160 ,400000 ,343996 ,664 -,70160 1,50160 a0 a1 a3 -,033333 ,343996 1,000 -1,13493 1,06826 ,100000 ,343996 ,991 -1,00160 1,20160 -,400000 ,343996 ,664 -1,50160 ,70160 hemoglobin tukey level hsd a0 a1 a3 a3 a0 a1 a3 a3 a0 a1 a3 a3 a1 a3 a3 ,366667 ,878762 ,974 -2,44744 3,18077 -,900000 ,878762 ,741 -3,71410 1,91410 ,500000 ,878762 ,939 -2,31410 3,31410 a0 a3 a3 -,366667 ,878762 ,974 -3,18077 2,44744 -1,266667 ,878762 ,511 -4,08077 1,54744 ,133333 ,878762 ,999 -2,68077 2,94744 a0 a1 a3 ,900000 ,878762 ,741 -1,91410 3,71410 1,266667 ,878762 ,511 -1,54744 4,08077 1,400000 ,878762 ,433 -1,41410 4,21410 a0 a1 a3 -,500000 ,878762 ,939 -3,31410 2,31410 -,133333 ,878762 ,999 -2,94744 2,68077 -1,400000 ,878762 ,433 -4,21410 1,41410 hematocrit tukey value hsd a1 a3 a3 1,333333 2,581989 ,953 -6,93510 9,60177 -3,333333 2,581989 ,593 -11,60177 4,93510 1,333333 2,581989 ,953 -6,93510 9,60177 a0 a3 a3 -1,333333 2,581989 ,953 -9,60177 6,93510 -4,666667 2,581989 ,337 -12,93510 3,60177 ,000000 2,581989 1,000 -8,26844 8,26844 a0 a1 a3 3,333333 2,581989 ,593 -4,93510 11,60177 4,666667 2,581989 ,337 -3,60177 12,93510 4,666667 2,581989 ,337 -3,60177 12,93510 a0 a1 a3 -1,333333 2,581989 ,953 -9,60177 6,93510 ,000000 2,581989 1,000 -8,26844 8,26844 -4,666667 2,581989 ,337 -12,93510 3,60177 total tukey leukocytes hsd a1 a3 a3 -7,966667 8,715758 ,798 -35,87759 19,94426 -13,200000 8,715758 ,473 -41,11093 14,71093 -8,466667 8,715758 ,769 -36,37759 19,44426 a0 a3 a3 7,966667 8,715758 ,798 -19,94426 35,87759 -5,233333 8,715758 ,929 -33,14426 22,67759 -,500000 8,715758 1,000 -28,41093 27,41093 a0 a1 a3 13,200000 8,715758 ,473 -14,71093 41,11093 5,233333 8,715758 ,929 -22,67759 33,14426 4,733333 8,715758 ,946 -23,17759 32,64426 a0 a1 a3 8,466667 8,715758 ,769 -19,44426 36,37759 ,500000 8,715758 1,000 -27,41093 28,41093 -4,733333 8,715758 ,946 -32,64426 23,17759 56 biology, medicine, & natural product chemistry 10 (1), 2021: 47-57 table s6. result of the tukey & duncan test. homogeneous subsets total leukocytes treatment n subset for alpha = 0.05 1 tukey hsda a0 a1 a3 a3 sig. 3 11,23333 3 19,20000 3 19,70000 3 24,43333 ,473 duncana a0 a1 a3 a3 sig. 3 11,23333 3 19,20000 3 19,70000 3 24,43333 ,192 means for groups in homogeneous subsets are displayed. a. uses harmonic mean sample size = 3,000. hemoglobin level treatment n subset for alpha = 0.05 1 tukey hsda a3 a1 a0 a3 sig. 3 14,83333 3 14,96667 3 15,33333 3 16,23333 ,433 duncana a3 a1 a0 a3 sig. 3 14,83333 3 14,96667 3 15,33333 3 16,23333 ,172 means for groups in homogeneous subsets are displayed. a. uses harmonic mean sample size = 3,000. hematocrit value treatment n subset for alpha = 0.05 1 tukey hsda a1 a3 a0 a3 sig. 3 44,66667 3 44,66667 3 46,00000 3 49,33333 ,337 duncana a1 a3 a0 a3 sig. 3 44,66667 3 44,66667 3 46,00000 3 49,33333 ,128 means for groups in homogeneous subsets are displayed. a. uses harmonic mean sample size = 3,000. erythrocytes number treatment n subset for alpha = 0.05 1 tukey hsda a1 a3 a0 a3 sig. 3 4,96667 3 5,06667 3 5,10000 3 5,46667 ,504 duncana a1 a3 a0 a3 sig. 3 4,96667 3 5,06667 3 5,10000 3 5,46667 ,209 means for groups in homogeneous subsets are displayed. a. uses harmonic mean sample size = 3,000. wijaya, et al. – the effect of balimo (zanthoxylum nitidum) immersion water … 57 figure s1. research photographs (a. cage; b. male rats; c. weighing of test animals; d. batang balimo; e. ciu; f. water immersion of balimo stems; g. feed for rats; h. process of treatment; i. 10% bnf fixative solution; j. anesthesia process of rats; k. surgical process; l. topography of internal organs from test animals) a b c d e f g h i j k l this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 21-23 | doi: 10.14421/biomedich.2014.31.21-23 larvicidal activity of the mixture of cashew nut shell liquid (cnsl) and aqueous extract of sapindus rarak dc against larvae of culex quinquefasciatus rahmi safarina fauziah1, sudarsono1* and budi mulyaningsih2 1pharmaceutical biology department, faculty of pharmacy; 2parasitology laboratory, faculty of medicine, ugm, indonesia author correspondency*: sudarsono@ugm.ac.id abstract the aim of this study was to evaluate the larvicidal activity of cashew nut shell liquid (cnsl) against the culex quinque fasciatus in larval stage. the cnsl was diluted in water by addition of aqueous extract of sapindus rarak dc to increase its solubility. larvae were exposed to varying concentrations of that mixture. the larvae mortality was observed after 24 h exposure. lc50 and lc90 value by extrapolation were 20,52 ppm and 55,41 ppm respectively. cnsl were specified by characterizing its physico-chemical properties and anacardic acid as marker compound by high performance chromatography (hplc). the results were the mixture of cashew nut shell liquid (cnsl) and aquous extract of sapindus rarak dc had larvicidal activity against cx. quinque-fasciatus and further investigations were needed to identify the fatty acid derivative as active compound of cnsl which responsible for larvicidal activity. keywords: larvicidal, larvae, culex quinquefasciatus, cashew nut shell liquid, sapindus rarak introduction culex quinquefasciatus is the most common mosquito species found in indonesia. there are the main cause of the mosquito bites in the evenings and night. the main factor supporting this species is probably due to poor sanitation caused by human migration to urban areas. interest in the control of cx.quinquefasciatus lies in the fact that it acts as a vector of filarial disease as a serious public health problem in indonesia and many tropical developing countries. filariasis is an endemic, disabling, and disfiguring disease. the filarial worm, wuchereria bancrofti responsible for human filariasis is carried by cx.quinquefasciatus which is a tropical pest and probably the most abundant and ubiquitous house mosquito in towns and cities, in the tropical countries. one of the strategies of who in combating tropical disease is to destroy their vectors or intermediate hosts. since no effective vaccine is available for filariasis, the only effective approach of minimizing the incidence of this disease is to eradicate and control mosquito vectors mainly by application of organic insecticides to larval habitats. control of mosquito at this stage is efficient because during the immature stages, mosquitoes are immobile. the use of natural products for controlling of insect pests offers an economically viable and eco-friendly approach, besides being harmless to beneficial insects when adopted on a larger scale. chemical pesticides have been used for several decades in controlling pests and vectors of various human diseases as they have a quick knock down effect. however, their indiscriminate use resulted in several problems such as resistance and resurgence of pests, elimination of natural enemies, toxic residues in food, water, air, and soil which affect human health and disrupt the ecosystem, leading to the threat that their continued use may further harm the environ-ment. anacardium occidentale l. (anacardiaceae), a fruit tree grown widely in tropical and sub-tropical areas is cultivated in indonesia for its cashew nuts. tyman & morris in 1989 described the composition of cnsl which found between the seed coat (pericarp) and the nuts. it is not a triglyceride and contains a high portion of phenolic compound, mainly are anacardic acid, cardol, and cardanol. recently, lomonaco and oliveira noted insecticidal action of cnsl on larvae of aedes aegypti (diptera: culicidae). the three cnsl compo-nents demonstrated good larvicidal activity against ae. aegypti. despite the potential to be developed as larvicides, phenolic compounds in the cnsl was toxic because it induced dermatitis. in addition, application of cnsl as larvicides in the medium of water is also cumbersome, so it is necessary to add other materials intended to dilute it. in this study, aqueous extract of sapindus rarak dc was added to increase the solubility of cnsl in water. materials and methods plant materials cashew nuts were obtained from cashew trees (a.occidentale) at wonogiri, central java, indonesia. the fruits of sapindus rarak dc were collected from the areas of “perhutani” located in situbondo, east java, indonesia. the plant material was identified by mr.joko santosa and voucher specimen was deposit at deprtment of pharmaceutical biology faculty of pharmacy, ugm. 22 biology, medicine, & natural product chemistry 3 (1), 2014: 21-23 oil extraction of cnsl the dried cashew nut shell (1,3 kg) were pressed by hydraulic pressor to obtain cashew nut shell liquid (cnsl). preparation of aquous extracts of s.rarak the dried pericarp from s.rarak (10 g) were cut into small pieces then extracted with 1000 ml of distilled water by reflux process at 50°c for 30 minutes. the concentration of aqueous extract of s.rarak fruits was 1% b/v. reagents n-toluene (e.merck), ethyl acetate (e.merck), acetic acid glacial (e.merck), chloroform (brataco), methanol (brataco). iodium p, anisaldehyde-sulphuric acid lp, vanilin-sulphuric acid lp, ethanol 96% (brataco), nhexane (brataco), hydrochloric acid (e.merck). all other chemicals used were analytical grade. larvicidal assay the activity of the test materials were evaluated by the world health organization recommended guidelines. the larvicidal assay consisted of two steps. first was the orientation to determine a certain concentration of aqueous extract of s. rarak dc. (it was the biggest concentration which is not found any died larvae). the chosen concentration then was used for making the mixture of cnsl and aquous extract of s. rarak dc. the next step was dissolving cnsl in various amount so that it was obtained 6 concentration series of the mixture for used in the testing, which were 0; 8; 8,56; 9,16; 9,8; and 10,49 ppm. characterization of cnsl’s physico-chemical properties physico-chemical properties of cashew nut shell oil was characterized through the iodine number, esther number, and acid number. those properties were characterized by pusat antar universitas (pau) ugm. hplc analysis of cnsl constituents the identity of anacardic acid was confirmed by hplc. the analysis was carried out using a shimadzu spd10vp chromatograph, uv-vis detector, which utilized a lichochart rd-c18 analytical column. the mobile phase consisted of methanol-acetic acid 4% (9:1v/v), which was run in the isocratic mode phase (1 ml/min) at a uv wavelength of 280 nm. determination of foaming index of s.rarak source 5 g of dried s.rarak fruits was dissolved in distilled water and then heated 50oc for 5 minutes. 10 ml filtrate was used for foaming test. saponin test was performed by agitation in 10 ml of the filtrate in a closed tube for 10 minutes. incidence foam up interval of 10 minutes (stable foam) showed a saponin. figure.1. chromatogram of csnl. figure.2. chromatogram of anacardic acid. results and discussion one of the marker constituent of cnsl, anacardic acid, was detected in the sample of cnsl in relative concentration of 2,58%. the hplc chromatograms were shown in fig.1 and fig.2. due to its bad solubility, cnsl was diluted in water with s.rarak’s pericarp extract as a co-solvent, in concentration which was determined by the orientation test. concentration of 400 ppm was selected as the optimum concentration to dillute cnsl in water but it had no effect to cause larval mortality. the mixture of cnsl and s.rarak extract used in this study was visually clear. there were also not detected visible oil particles or any breaking phase. rahmi safarina fauziah, et al. – larvicidal activity of the mixture of cashew nut shell liquid … 23 table 1. physico-chemical properties of cnsl used in this study. physico-chemical properties of cnsl value iodium number (g iod/10 g cnsl) 6,3 acid number (mg naoh/g cnsl) 104,1 saponification number 127,1 esther number 45,9 density (g/ml) 0,8232 it had no published data available of effect of cnsl on immature stages of culex mosquitoes. effect of different concentrations of cnsl solution on aquatic stages of cx.quinquefasciatus larvae in the laboratory conditions was shown in table 2. there was significant decreasing mortality percentage of the larvae by increasing cnsl concentration (table 2). as shown in that table, the biggest death rate was noticed in exposure to 10,49 ppm concentration. lc line equation obtained from probit analysis was y = 3.01 x + 1.05, so that the calculation could be known that lc50 and lc90 value were 20.52 ppm and 55.41 ppm, respectively. this value was the result of extrapolation and therefore could not guarantee that these results show the actual value of lc50 and lc90. essential oils or plant extracts with lc50 values below 100 ppm can be considered potentially have larvicidal activity. our present observation reveals that cx.quenquefasciatus larvae were susceptible to 8 ppm to 10,49 ppm cnsl. in indonesia, population of cx.quin-quefasciatus is controlled by using of organophosphate insecticides, such as temephos and malathion. temephos, which is also known as “abate” has been used in areas of water where the cx.quinquefasciatus mosquito breeds in order to reduce the population of this disease-carrying insect. resistance to temephos by cx.quinquefasciatus has been reported in brazilian and malaysia. temephos was very lethal toward cx.quinquefasciatus larvae with a lc50=0,00088 ppm. cnsl consti-tuents were less active than temephos, but could be important models for the further development of new larvicides. it is necessary to investigate the effectiveness of these mixture compounds in a field setting and the toxicity toward other organisms, for example by doing brine shrimp lethality test. conclusions in the present work the mixture of cnsl in aquaeous extract of s.rarak dc could be used as larvicide against. cx.quinquefasciatus. given the continual search for renewable and biodegradable sources of new medicinal products, cnsl from cashew nut processing industries in indonesian agribusiness represents an opportunity to increase the value of this by-product by developing green larvicidal compounds to be used in filariasis control, which is cheap, non-toxic, and biodegradable. table 2. larvicidal activity of cnsl and s.rarak extract mixture against cx.quinque fasciatus larvae. acknowledgement i would like to thanks to faculty of pharmacy and parasitology laboratory ugm for the possibility for doing experiment. references cheng, s.s., chang, h.t., chang, s.t., tsai, k.h. & chen, w.j., 2003, bioactivity of selected plant essential oils against the yellow fever mosquito aedes aegypti larvae, bioresearch technology, 89, 99-102. devine gj, furlong mj. insecticide use: contexts and ecological successions. agric hum values 2007; 24: 281-306. evans, f.j. & schmidt, r.j., 1980, plants and plant products that induce contact dermatitis, planta medica, 38, 289-316. lima, j.b.p., da-cunha, m.p., silva júnior, r.c., galardo, a.k.r., soares, s.s., braga, m.a., ramos, r.p., valle, d., 2003. resistance of aedes aegypti to organophosphates in several municipalities in the state of rio de janeiro and espírito santo, brazil. am. j. trop. med. hyg. 68, 329–333. lomonaco, d., gilvandete, m.p.s., yana, s.f., angela, m.c.a., selma, e.m., mele, g., & vasapollo, g., 2009, study of technical cnsl and its main components as new green larvicides, green chemistry, 1, 31–33. oliveira, m.s.c., de morais, s.m., magalhaes, d.v., batista, w.p., vieira, g.p., & craveiro, a.a., 2011, antioxidant, larvicidal, and antiacetylcholinesterase activities of cashew nut shell liquid constituents, acta tropica, 117, 165-170. samuel t, jayakumar m, william sj. 2007, culex mosquito: an overview. in: william sj. defeating the public enemy, the mosquito: a real challenge. loyola publications: chennai, p. 95-116. shian, l.c., 2007, the effect of sublethal concentration of abate on aedes aegypti (linnaeus) and culex quinquefasciatus (say), tesis, universiti sains malaysia, subang jaya. tennyson, s., ravindran, j.k. & arivoli, s., 2012, screening of twenty five plant extracts for larvicidal activity against culex quinquefasciatus say (diptera: culicidae), asian pacific journal of tropical biomedicine, s1130-s1134. tyman jhp, morris lj. composition of cashew nut shell liquid. j chromatography 1989; 27: 287–8. world health organization (who), 2005, guidelines for laboratory and field testing of mosquito larvicides, geneva. cnsl (ppm) number of larva died (3 replications) total larvae mortality (%) 1 2 3 0 1 1 0 75 2.67 8 2 4 4 75 13.33 8,56 5 3 3 75 14.67 9,16 7 3 3 75 17.33 9,8 6 9 0 75 20 10,49 8 5 3 75 21.33 content_v3n1_3.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 259-264 | doi: 10.14421/biomedich.2024.131.259-264 issn 2540-9328 (online) the effect of probiotic starter culture variation on the quality of yogurt based on indonesian national standard hariyanto ih*, bella cristin, siti nani nurbaeti department of pharmacy, faculty of medicine, universitas tanjungpura, pontianak, west kalimantan, jl. prof. dr. hadari nawawi, bansir laut, pontianak tenggara, kota pontianak, kalimantan barat, indonesia. corresponding author* hariyanto.ih@pharm.untan.ac.id manuscript received: 19 march, 2024. revision accepted: 14 june, 2024. published: 08 august, 2024. abstract yogurt is usually made by two lactic acid bacteria such as streptococcus thermophilus and lactobacillus bulgaricus. however, three to seven lactic acid bacteria are also used in several products. therefore, this study aimed to examine the effect of variations in the number of starter bacteria and compare to indonesian national standard (sni). the experimental method was used with starter variations, including a combination of two (lactobacillus bulgaricus and streptococcus thermophilus); three (lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus); and seven bacteria (lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, lactobacillus casei, lactobacillus rhamnosus, bifidobacterium longum, and bifidobacterium infantis). the results showed that the yogurt with two and seven bacteria was within the range of sni value standard. the ph, viscosity, syneresis, lactic acid content, total protein, and total number of bacteria for yogurt with two bacteria were 3.92, 453.76 cp, 88.05%, 1.27%, 3.26%, and 2.47 x 1012 cfu/ml, respectively. the values for yogurt with seven bacteria were 4.11, 478.23 cp, 97,45%, 0,98%, 2.7%, and 1.53 x 1010, respectively, while formulas with three bacteria did not reach the ph standard. however, the atypical taste was given by the seven starter bacteria and the use of various bacteria starters affected the quality of yogurt. specifically, formulas composed of two bacteria showed the best results according to sni. keywords: fermentation; lactic acid bacteria; yogurt quality. introduction the concept of “food as medicine” is being pursued by experts to optimize functional foods in overcoming diseases. an example of functional food is yogurt which is a dairy product that uses fermentation techniques with lactic acid bacteria (zajác p et al, 2020). the bacteria produce the enzyme lactase to change the remaining sugar in milk and form an acidic environment to inhibit the growth of other harmful bacteria in the body (hendarto et al, 2019). yogurt quality is influenced by several factors, such as the type of bacteria culture starter used (yilmaz-ersan et al, 2014). streptococcus thermophilus and lactobacillus bulgaricus are common bacteria used to make yogurt because of several health benefits. yogurt is usually made with different types of bacteria. lactobacillus bulgaricus and streptococcus thermophilus help each other grow by making short peptides and amino acids (hendarto et al., 2019). the two bacteria produce formic acid but need other probiotics like lactobacillus acidophilus, lactobacillus casei, lactobacillus rhamnosus, and bifidobacterium sp. these bacteria do not resist bile acids and live only briefly in the digestive system. according to dabjia a. et al. (2018), these probiotics offer health benefits and stay longer in the digestive tract. to make starter cultures, more than two different strains of probiotic organisms are combined (putri y et al., 2020). using different bacteria starters when making yogurt leads to helpful interactions. comparing the initial bacteria cultures is important in the fermentation and acidification processes. this is because health benefits depend on choosing specific microorganisms for therapeutic purposes and consuming enough to have the desired effect (ningsih e et al., 2019). various studies have examined how the number of starter bacteria cultures affects the quality of yogurt. the quality parameters include physical (ph, viscosity, syneresis), chemical (lactic acid content, protein total), and microbiological analysis (total bacteria count) in accordance with indonesian national standard (sni) for yogurt (yanuarto t et al, 2019). https://doi.org/10.14421/biomedich.2024.131.259-264 260 biology, medicine, & natural product chemistry 13 (1), 2024: 259-264 materials and methods materials this study used uht milk (diamond®), starter cultures of two (lactobacillus bulgaricus and streptococcus thermophilus), three (lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus), and seven bacteria (lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, lactobacillus casei, lactobacillus rhamnosus, bifidobacterium longum, and bifidobacterium infantis). the materials used include pp indicator (merck®), naoh 0.1 n (merck®), ph 4 and ph 7 buffer (hanna®), na media (merck®), h2so4 concentrated (merck®), and hcl 0.1 n (merck®). the instruments used pot were a stove (rinnai®), thermometer, incubator (memmert®), refrigerator (lg®), glass apparatus (pyrex®), burette (schott duren®), ph meter (horiba®), petri dish (anumbra®), brookfield viscometer (ametek®), and centrifugation (tomy®). methods yogurt production milk was pasteurized at 85°c and cooled to 42°c, then the bacteria starter cultures were added, stirred until homogeneous, and incubated for 10-20 hours at 42°c. yogurt was stored in the refrigerator at 4°c 5°c (dabija et al, 2018; bsn, 2009). table 1. yogurt formulation. composition f1 f2 f3 uht milk 1 liter 1 liter 1 liter starter cultures 1 gram (lactobacillus bulgaricus, streptococcus thermophilus) 1 gram (lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus) 1 gram (lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, lactobacillus casei, lactobacillus rhamnosus, bifidobacterium longum, bifidobacterium infantis) control: branded greek yogurt organoleptic organoleptic test was carried out to identify the characteristics of color, consistency, appearance, smell, and taste (anjarwati s et al, 2022; fatmawati u et al, 2013). ph ph was determined by a digital ph meter and performed by dipping the electrode into the sample until the ph value was shown (purwatiningsih et al, 2022). viscosity viscosity was determined with a brookfield viscometer. yogurt samples were prepared up to 500 ml using spindle 2 at a speed of 60 rpm then the results were analyzed (purwatiningsih et al, 2022). syneresis syneresis was determined through centrifugation. about 15g of yogurt samples were taken, centrifuged, and then the supernatant was measured (yanuarto t et al, 2019). syneresis percentage was calculated by the following formula: the solution as an indicator (yilmaz e et al, 2014; anjarwati s et al, 2022; purwatiningsih et al, 2022). % 𝑆𝑦𝑛𝑒𝑟𝑒𝑠𝑖𝑠 = 100% − ( 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 100%) protein total protein total was determined by using the kjeldahl method divided into three stages, namely digestion, distillation, and titration (anjarwati s et al, 2022; purukan c et al, 2020). protein levels were calculated by the following formula: % 𝑁 = 𝑚𝐿 𝐻𝐶𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑏𝑙𝑎𝑛𝑐𝑜 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) 𝑥 𝑛 𝐻 𝑥 14,007 𝑥 100% total bacteria count total bacteria count was determined by using tpc (total plate count) method (lestari y et al, 2020; utami et al, 2020). the calculation of the total bacteria count was calculated by the formula: total 𝑐𝑓𝑢 𝑚𝐿 = 𝑇𝑜𝑡𝑎𝑙 𝐶𝑜𝑙𝑜𝑛𝑦 𝑠𝑝𝑟𝑒𝑎𝑑 𝑣𝑜𝑙𝑢𝑚𝑒 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 titratable acidity lactic acid was determined by using the titration method. yogurt samples were taken up to 10 grams into erlenmeyer then 10 ml of aquades was added and 2-3 drops of 1% pp. the result of the lactic acid percentage was calculated using the formula: lactat acid (%) : 𝑚𝐿 𝑁𝑎𝑂𝐻 𝑥 𝑁 𝑁𝑎𝑂𝐻 𝑥 0,009 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 x 100% hariyanto ih et al. – the effect of probiotic starter culture variation on … 261 stability the stability of ph, viscosity, and syneresis was determined repeatedly on day 7 and day 14 during storage at 4-5°c. the results obtained were compared to day 1 and then statistically analyzed. results and discussion organoleptic organoleptic tests were conducted based on the guidelines of the indonesian national standard (sni) for yogurt with a minimum of three panelists or one expert (bsn, 2009). the result after the fermentation process showed no changes in all formulas for 14 days of storage. yogurt with two and three bacteria had similar characteristics such as white or yellowish color, distinctive aroma, typical sour taste, thick texture, and homogeneous consistency. meanwhile, yogurt with a combination of seven bacteria had characteristics such as yellowishwhite color, distinctive aroma, atypical taste, liquid texture, and homogeneous consistency. the physical appearance of yogurt is shown in figure 1. figure 1. physical appearance. based on these results, yogurt with two and three bacteria conformed to sni, while the seven bacteria did not. this was attributed to the atypical taste (bsn, 2009). physical, chemical, and microbiological analysis yogurt samples were analyzed after being stored in a refrigerator overnight. the parameters analyzed include physical (organoleptic, ph, viscosity, syneresis), chemical (lactic acid content, total protein), and microbiological analysis (total bacteria count). all parameters were conducted for 14 days. the result of parameters analysis on day 1 is shown in table 2. table 2. parameters analysis on day 1. analysis formula control f1 f2 f3 ph* 4,00± 0,05 3,92± 0,03 3,54± 0,08 4,11± 0,05 viscosity* 3015,67± 8,08 453,76± 12,40 503,13± 27,46 478,23± 30,65 syneresis* 99,94%± 0,01 88,05%± 5,56 97,74%± 0,24 97,45%± 1,33 lactic acid* 1,41%± 0,01 1,27%± 0,01 1,47%± 0,02 0,98%± 0,01 protein 4,00% 3,26% 2,71% 2,70% total bacteria 2,47 x 1012 2,47 x 1011 1,51 x 1010 1,53 x 1010 note: * = all formulas were tested in 3 replications control : branded greek yogurt f2 : yogurt with 3 bacteria f1 : yogurt with 2 bacteria f3 : yogurt with 7 bacteria the ph value for the yogurt made using two and seven bacteria complied with sni while yogurt with three bacteria did not, falling less than 3.8. based on a previous study, the combination of lactobacillus acidophilus streptococcus thermophilus, and lactobacillus bulgaricus produced yogurt with a lower ph value due to the ability to ferment sugar into lactic acid quickly (jannah ma et al, 2014). bifidobacterium culture which has an optimal ph of 5.5 – 7 may produce yogurt with a higher ph compared to other starters (adriani l et al, 2008). this was consistent with the results of yogurt made using seven bacteria which had the highest ph value. the standard viscosity value for yogurt is yet to be stated in sni because the measurement is based on the appearance of yogurt to identify thick liquid to solid. however, the viscosity test in this study was carried out to standardize the quality of yogurt and to compare each formula. the result showed a range from 453 cp to 503 cp on day 1. yogurt viscosity value is inversely comparative to ph, hence, when ph value is low then the viscosity value will increase due to the acidic fermentation process (rohman e et al, 2020). according to previous studies, viscosity could be influenced by the protein and fat content of raw materials with the ability to bind water and activate the interaction between fat and casein during fermentation (prastiwi et al, 2019; setyawardani t et al, 2020). the presence of lactose in raw materials turns into pyruvic and produces lactic acid during the fermentation process to form an acidic atmosphere making casein unstable and coagulate from liquid to gel (falah et al, 2020). the lowest syneresis was found in yogurt with three bacteria and the highest syneresis was in yogurt with two. the smaller number of separated liquids indicates (a) 2 bacteria (b) 3 bacteria (c) 7 bacteria 262 biology, medicine, & natural product chemistry 13 (1), 2024: 259-264 better yogurt quality with a higher percentage. in general, yogurt with a high percentage value often directly has a high viscosity value. each type of lactic acid bacteria has a different ability to metabolize lactose which affects the ability of proteins to coagulate. in addition, the length of storage time affects syneresis, especially in yogurt samples that do not use stabilizers (falah et al, 2020). regarding the lactic acid content value, all formulas conformed to sni, falling between 0,5% 2,0%. the highest lactic acid content value was found in yogurt with three bacteria, and the lowest was found in seven bacteria. lactic acid content is inversely compared to ph value, when ph value decreases, then lactic acid content value increases. in terms of protein total value, all formulas complied with sni, reaching 2.7%8. the highest protein total value was found in yogurt with two bacteria and the lowest was found in yogurt with seven bacteria. based on a previous study, yogurt made using two bacteria cultures namely lactobacillus bulgaricus and streptococcus thermophilus had a higher protein level than those with three and four due to competition interactions. yogurt with a combination of two bacteria has a symbiotic interaction where streptococcus thermophilus produces pyruvic, formic, and folic acid. this expression stimulates lactobacillus bulgaricus to grow, which releases the amino acids valine, glycine, and histidine (purukan c et al, 2020). all formulas complied with sni as indicated by a total number of bacteria above 107. the desired therapeutic effect in yogurt should be provided in sufficient quantities to compensate for the possibility of probiotic reduction by the stomach and intestines. (a) control (b) f1 (2 bacteria) (c) f2 (3 bacteria) (d) f3 (7 bacteria) figure 2. bacteria on agar. the highest value was yogurt composed of two bacteria and the lowest value was in the sample with three bacteria. the higher number of lactic acid bacteria is directly compared to a higher protein (ruslian rd et al, 2021). according to a previous study, yogurt with lactobacillus bulgaricus and streptococcus thermophilus were synergized in cell multiplication (jannah ma et al, 2014). stability the stability test was conducted three times for 14 days (day 1, day 7, and day 14) consisting of ph, viscosity, and syneresis. for all formulas, the ph and viscosity values showed stable with no significant change (p≥0.05). the value of syneresis showed no significant change (p≥0.05) in yogurt with two bacteria, but was unstable with significant change (p<0.05) from day 1 to day 14 in yogurt with three and seven bacteria. changes in stability during storage are shown in figure 3. hariyanto ih et al. – the effect of probiotic starter culture variation on … 263 (a) ph (b) viscosity (c) syneresis figure 3. ph (a), viscosity (b), and syneresis (c) percentage determination for 14 days storage note: * = significantly different with control (p<0.05) conclusions in conclusion, the use of variations in the number of different bacteria starters affected the physical, chemical, and microbiological parameters of yogurt in accordance with the indonesian national standard (sni). these parameters include ph, viscosity, syneresis, lactic acid content, protein total, and total bacteria count. the formula with two bacteria and seven bacteria results were within the range of sni value. however, the formula with three starters did not reach the ph value, and the formula with two bacteria is shown the best according to sni. acknowledgments: the authors are grateful to the pharmacy study program, faculty of medicine, universitas tanjungpura, and animal feed laboratory west kalimantan province for supporting the facilities used in the study, and steven djunaidi who helped and provided support. authors’ contributions: all authors have an equal contribution in carrying out this study. competing interests: the author declares there is no conflict of interest. references adriani l, indrayati n, tanuwira u, mayasari n. 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natural product chemistry volume 12 – number 1 – april 2023 issn 2089-6514 (paper) | issn 2540-9328 (online) contents preliminary investigative study on the blood pressure-lowering potential of aqueous leaf extract of simarouba glauca (aesg) on normotensive adult wistar rats sammy davies e. osagie-eweka, noghayin e.j. orhue, fabian c. amaechina, eric k.i. omogbai, emuesiri g. moke 1 4 high performance liquid chromatography (hplc) for detection of glucosamine and chondroitin sulfate compounds rakhmiyati, tetri widiyani, agung budiharjo 5 8 proximate composition, levels of some essential mineral elements and anti-nutritional components of some yam species found in minna, niger state eneogwe okechukwu godfrey, ibrahim izihyi esther, obuye faith 9 16 the effect of pumpkin fruit ripeness (cucurbita moschata. d) on total flavonoid levels and antioxidant activity linda astutik, eka fitri yanti 17 23 antioxidative stress and hepatoprotective activities of leaf extract and fractions of setaria megaphylla in plasmodium berghei infected mice ndanti bartholomew william, augustine lawrence bassey, john akpan udobang, jude efiom okokon 25 32 solanum anomalum leaf extract and fractions attenuate oxidative stress and liver injuries in alloxan-induced diabetic rats idongesit charles etuk, john akpan udobang, nwakaego omonigho ebong, jude efiom okokon 33 44 the association between some endocrine conditions and covid-19: a review harem othman smail 45 53 acute toxicity ld50 fraction ethyl acetate aquilaria malaccensis, ficus benjamina, mikania micrantha, and fraction water cinnamomum burmanii in mus musculus rizky yulion, santi perawati, barmi hartesi, lia anggresani, lili andriani, lesra indriani, lara syahila, suci ramadani, nadia monika 55 60 synthesis, characterization and activity test of natural zirconium zeolite (zr-za) catalyst in the esterification reaction of glycerol with acetic acid anhydride didik krisdiyanto, tutik farihah, hikmah supriyati 61 67 combined anthocleista vogelii and alstonia boonei stem barks extract alleviates hyperlipidaemia and renal malfunctions in benign prostatic hyperplasia-induced rats robert ikechukwu uroko, mercylyn ezinne uche, paul chukwuemaka nweje-anyalowu, ikenna obiwuru, chinedu aguwamba, chinomso friday aaron 69 78 spermicidal, antifertility and contraceptive effect of azadirachta indica a. juss. seed extract in female and male wistar rats macdonald idu, sharon ogedegbe-george, precious eromosele oriarewo, benjamin ogunma gabriel 79 88 cardiotoxicity effects of herbal medicine, a review article shahin gavanji 89 96 potential inhibition of ace2 membrane protein by flavone glycosides for blocking entrance of sarscov-2 into the cells; a computational study ahsan ibrahim, ehtisham ul haq 97 108 antioxidant and antibacterial activity of pomegranate extract (punica granatum l.) in lip balm formulation maria grasela kase, aniek prasetyaningsih, dwi aditiyarini 109 117 phytochemical, antimicrobial and cytotoxic activities of strophanthus sarmentosus dc julius leke abiola, olapeju oluyemisi aiyelaagbe 119 126 in-vivo alpha-amylase and alpha-glucosidase inhibitory activities of solanum anomalum leaf extract and fractions jude efiom okokon, idongesit charles etuk, john akpan udobang, nwakaego omonigho ebong 127 132 ethnobotanical survey of aromatic and medicinal plants used in traditional medicine and agri-food in the fez-meknes region hamza el finou, nadia salhi, asma halmoune, lhoussaine el rhaffari 133 141 phytochemical, antioxidant screening, antinociceptive, and anti-inflammatory activities of boswellia dalzielii hutch (burseraceae) root ethanol extract using animal model macdonald idu, anthonia omoregbee, benjamin ogunma gabriel 143 150 a new ent-kaurene diterpenoid isolated from leaves of espeletia semiglobulata cuatrec. and its potential antimicrobial activity andrés márquez, alida pérez, †luis rojas, rosa aparicio, freddy ramos, ysbelia obregón, alfredo usubillaga 151 157 immunoprotective effect of cocos nucifera oil on sheep red blood cell-induced immunocompromised rats macdonald idu, igori debby, benjamin ogunma gabriel 159 169 lupenone isolated from diospyros melanoxylon bark non-competitively inhibits -amylase activity mohan krishna durgam, praveen kumar vemuri, vijaya lakshmi bodiga, sreedhar bodiga 171 176 comparative assessment of the proximate composition, functional properties and amino acid profile of dioscorea bulbifera, dioscorea alata and dioscorea rotundata found in minna, niger state eneogwe okechukwu godfrey, obuye faith, ibrahim izihyi esther 177 185 interleukin-1 as a predictor cytokine sars-cov: article review lisa savitri, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita, ester lianawati antoro, ida septika wulansari 187 190 antihypertensive drugs therapy in hypertension and covid-19 comorbidity emuesiri goodies moke, endurance efe ahama, pere-ebi yabrade toloyai, mamerhi taniyohwo enaohwo, ekuerhare basil, emuesiri kohworho umukoro, anthony taghogho eduviere, ikuesirioghene udumebraye, choice udufowe 191 196 comparative cough suppression of chitosan crab extract of uca tangeri and dihydrocodeine joshua charles isirima, precious ojo uahomo 197 203 bioactive compound in solanum torvum and its potential as functional food and drink: a review nina nurazizah purnomo putri, rista anggriani, sukardi 205 213 effects of ouabain in ehrlich tumor development in vitro and in vivo amanda costa ayres salmeron, maria beatriz calado, mateus da silva matias antunes, gabriel rodrigues da silva, deyse cristina madruga carvalho, beatriz fernandes de souza, márcia regina piuvezam, sandra rodrigues mascarenhas 215 224 chemical profile and in-silico docking studies on bioactives from essential oil of cymbopogan pendulus targeting penicillin binding proteins (pbps) in bacteria arun dev sharma, inderjeet kaur 225 232 the effects of frequent therapeutic administration of artesunate-amodiaquine and artemether-lumefantrine on haematological markers in balb/c mice david audu, olufunmilayo a idowu, vinood b patel, musa f mshelbwala, adewumi b idowu 233 240 synthesis and characterization of cinnamon loaded bsa microparticles with antidiabetic properties b. s. wanniarachchi, h. m. w. sathsarani, b. m. jayawardena, h. g. n. dewangani 241 250 chemical composition and evaluation of anti-tyrosinase and anti-oxidative effects of topical cream formulation from acacia sieberiana, vitellaria paradoxa and beeswax alfred ngenge tamfu, alain yaya koudoro, selcuk kucukaydin, theophile olaye, pascal dossa cokou agbangnan, dominique codjo koko sohounhloue, felicien avlessi 251 258 combination interactive effects of gongronema latifolium leaves and picralima nitida seeds extracts on glucose tolerance maureen ifeyinwa egbuniwe, harrison anezichukwu ozoani, amara anwuchaepe ajaghaku, ikechukwu sonne mbagwu, uchechukwu harrison orji, daniel lotanna ajaghaku 259 266 effect of female age on crossing over frequency in drosophila melanogaster crosses n♂><bcl♀ and n♂><ym♀ and their reciprocals lisa savitri, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita, ester lianawati antoro, ida septika wulansari 267 271 phytochemical and antioxidant activity of blumea balsamifera and cordyline fruticosa based on ethnopharmacology knowledge of muara tae tribe, east kalimantan nur maulida sari, farida aryani, wartomo, muhammad fikri hernandi, erna rositah, joko prayitno 273 280 phytochemical analysis and antibacterial activity of methanol and ethyl acetate extracts of detarium microcarpum guill. & perr. muhammad mubarak dahiru, abdulazeez mumsiri abaka, susan pwakangdi artimas 281 288 glut4 as a protein target for t2dm therapy with natural compounds vivi hendra sutandar, mgs. irsan saleh, ziske maritska 289 293 effect of quarry activities on some morphological parameters of two maize varieties (swan 1 and sammaz 52) bridget odiyi, olubukola maku, foluso akinbode ologundudu, sylvanus efetobor abiya 295 303 antidiarrhoeal activities of lime (citrus aurantiifolia) extract in experimentally-induced diarrhoea model joshua charles isirima, precious ojo uahomo 305 313 synthesis, spectroscopic analysis and antidiabetic properties of copper (ii) complex of mangifera indica leaf crude extract mary adelaide oladipo, folasade omobolanle ajao, adewusi john adepoju, kayode taiwo ishola, olalekan jamiu ajeigbe1 315 321 uncovering the antioxidant power: investigating the skin and flesh of crystal guava with chloroform and methanol extractions and dpph assay aulia ulmillah, arif alghifari, nurhaida widiani 323 328 phytochemical, antibacterial and antioxidant activities of schefflera elliptica leaves i gede yoga ayuning kirtanayasa, anak agung gede indraningrat, i putu candra 329 334 ethnobotanical survey on the plants used in the control of nematodes in the zone of es niayes de thiès/senegal el hadji gorgui diouf, doudou diop, abdoulaye gueye, mbaye diouf, talibouya ndior, mamadou latyr ndour, adama faye, mamadou kébé 335 341 nanotechnology-based vaccines mohamed hussein, madiha mumtaz, iqra nasir, anisa abdullahi 343 361 fiber concentration on fermentation of cleome gynandra l based on storage time and solvent change lily restusari, ayu komala dewi, alinea dwi elisanti 363 369 medicinal biospecificity of ginger and its efficacious bioactive compounds in the context of its biological activities against predominant health issues: current study and new avenues haseeba shahzad, shaifa saleem, waqas hanif, zareena ali, muhammad zeeshan ahmed, haq nawaz, zelle humma, laraib rana, muhammad qamar farid, hajira kalsoom, samavia jaan 371 389 analysis of biological activities of two novel metal (ii) complexes of andrographis paniculata crude extract mary adelaide oladipo, ayodele oluwabunmi ojo, kayode taiwo ishola 391 397 development of an eco-shampoo formulation using local environmental plant extracts for healthy hair as an effort to increase the potential of environmental resources dwi atmanto, neneng siti silfi ambarwati 399 405 the anticancer properties of guava leaves (psidium guajava l.) and turmeric rhizome (curcuma longa l.) against breast cancer: a literature study angga puja asiandu, widya sari, septi widiya sari, alif syahrul abdul majid 407 412 peptide fractions from pepsin-digested moringa oleifera seed proteins inhibit hemoglobin glycation and carbohydrate-hydrolyzing enzymes oluwafemi emmanuel ekun 413 422 weed community structure in patia village rice fields patia sub-district, pandeglang regency ai widiyawati, hadi susilo, mu'jijah, suyamto, nurullah asep abdilah 423 430 3: front 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 65-75 | doi: 10.14421/biomedich.2020.92.65-75 issn 2540-9328 (online) polyphenols content and antimicrobial, antioxidant and hemolytic activities of essential oils from four selected medicinal plants growing in algeria noureddine halla1,2,*, kebir boucherit2, bankaddour zeragui1, abdelkader djelti1, ziane belkhedim1, rachida hassani1, saada benatallah1, hassiba djellouli1, oumlkheir kacimi1, zahia boucherit-otmani2 1laboratory of biotoxicology, pharmacognosy and biological recovery of plants, department of biology, faculty of sciences, university of saida dr. moulay-tahar, 20000, algeria. 2antibiotics antifungal laboratory, physical chemistry, synthesis and biological activity (lapsab), department of biology, faculty of sciences, university of tlemcen, bp 119, tlemcen, 13000, algeria. corresponding author* halla.nour@yahoo.fr; noureddine.halla@univ-saida.dz manuscript received: 12 july, 2020. revision accepted: 10 november, 2020. published: 13 november, 2020. abstract the saharan and steppe spontaneous plants are very characteristic because of their particular adaptation to the desert and extreme environment. some species have pharmacological properties that give them a medicinal interest. the aim of the present work was to determine the polyphenol contents of essential oils obtained from four endemic plants growing in algeria (pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita), and study its biological activity, including antimicrobial, antioxidant, and hemolytic. the antimicrobial activity was evaluated by the microdilution method against twelve strains. the antioxidant activity was carried out by two methods (dpph radical scavenging and reducing power). however, the hemolytic effect has been evaluated against the red blood cells. p. scoparius and m. piperita showed yields of essential oils higher than 1%. all the strains showed sensitivity against the essential oils tested with the exception of the c. albicans treated by r. officinalis essential oils. the most sensitive strain was c. albicans treated by p. scoparius essential oils by mic of 0.0781 mg/ml, it was the same plant that shows the highest polyphenol content (14.78 ± 0.72 g gae/g ds). the antioxidant activity by the dpph method was greater for all essential oils tested by ic50 ranging from 0.69 ± 0.07 (r. officinalis) to 30.67 ± 2.12 mg/ml (m. nivellei). the r. officinalis essential oils reported more antioxidant power than the positive control (ascorbic acid). in reducing iron, it was the r. officinalis essential oils which were found to be the most active with an ec50 concentration of 9.67 ± 1.36 mg/ml. after 120 min incubation, minimal haemolysis (10%) was obtained with essential oils of r. officinalis at a concentration of 0.39 mg/ml. we conclude that p. scoparius essential oils showed the high content of polyphenols and r. officinalis essential oils reported more antioxidant power than the positive control (ascorbic acid). keywords: polyphenols; antimicrobial; antioxidant; essential oils; hemolytic; mentha piperita; myrtus nivellei; pituranthos scoparius; rosmarinus officinalis; sahara. introduction the total algerian flora has about 16,000 plant species, of which more than 1,000 species with medicinal properties and 700 species are endemics. algeria is the source of significant taxonomic, ecosystem and landscape diversity (aps, 2019). the spontaneous plants in sahara area have many uses, traditionally practiced by the local population, in terms of pharmaceuticals, food and domestic use. these plants have the ability to synthesize many compounds called secondary metabolites and thus constitute an immense reservoir of compounds of great chemical diversity, possessing a wide range of biological activities (seca and pinto, 2019). this is the case, for example, of plant essential oils which are widely used in therapeutics. in recent years, studies of biological activities of medicinal plants have increased remarkably because of their potential to be used as sources of drugs, food additives or active ingredients in cosmetics (haddouchi et al., 2016). in this context, the aim of the present work was to determine the polyphenol contents of essential oils obtained from four endemic plants growing in algeria (pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita), and study its biological activity, including antimicrobial, antioxidant, and hemolytic. pituranthos scoparius and myrtus nivellei are from tassili n'ajjer (sahara), rosmarinus officinalis and mentha piperita are from el bayadh and tiffrit, respectively (steppe area). https://doi.org/10.14421/biomedich.2020.92.65-75 66 biology, medicine, & natural product chemistry 9 (2), 2020: 65-75 material and methods plant material and essential oil extraction for the four plants studied, the flowering aerial parts were used. the plant material was identified by dr. tayeb si tayeb (laboratory of biotoxicology, pharmacognosy and biological recovery of plants, university of moulay-tahar, saida, algeria). a voucher specimen was deposited at the herbarium of the laboratory under the following code numbers lbpbpts03-13. pituranthos scoparius and myrtus nivellei were collected in october 2018 and april 2018, respectively, from tassili n'ajjer in south-east algeria (25°30'0" n and 9°0'0" e). rosmarinus officinalis was collected in october 2018 from el bayadh (33°40′49″ n and 1°01′13″ e). mentha piperita was collected in july 2018 from tiffrit (wilaya of saida) (34°54'0.01" n and 0°24'0" e). using a clevenger-type apparatus, 400 g of dried plant materials were subjected to hydro-distillation in 4,000 ml of distilled water for 4 h. the obtained oil was dried over anhydrous sodium sulfate and then stored in sealed glass vials at 4 °c prior to analysis (el asbahani et al., 2015). total polyphenols content the polyphenols are assayed according to the method described by dewanto et al. (2002). a 100 µl quantity of each essential oil was mixed with 2 ml of a freshly prepared sodium carbonate solution (2%). after five minutes, 100 µl of the folin-ciocalteu reagent (1 n) was added to the mixture, the whole was left for 30 minutes at room temperature and the reading is performed against a blank using a spectrophotometer at 750 nm. a standard range based on gallic acid is also prepared. the total polyphenol contents of the essential oils are then expressed in milligrams gallic acid equivalent per gram of the dried sample (mg eag/g ds) (halla et al., 2019a). antibacterial and antifungal activity the antimicrobial activity of the essential oils was evaluated using different strains; gram-positive bacteria: staphylococcus aureus atcc 25923 and bacillus cereus atcc 11778; gram-negative bacteria: escherichia coli atcc 25933, pseudomonas aeruginosa atcc 27853, pasteurella multocida atcc 43137, salmonella typhimurium atcc 13311, salmonella enterica atcc 13312, campylobacter fetus atcc 27374, klebsiella pneumonia atcc 700603 and enterobacter cloacae atcc 13047; and fungal microorganisms (yeasts): candida albicans atcc 10231 and candida albicans ip 444. bacteria were cultured at 37 °c for 18 h under aerobic conditions in nutrient agar medium (fluka, usa). before experimental use, the cultures from different solid mediums were cultivated in liquid media, incubated, and used as the inoculum for each experiment. muellerhinton broth (bacteria) and rpmi-1640 (yeast) were used for antimicrobial tests (clsi, 2008; clsi, 2012). the minimum inhibitory concentration (mic) was determined based on the methods approved by the national committee for clinical laboratory standards (clsi, 2008; clsi, 2012), with slight modifications (halla et al., 2019b). the microtiter plates were inoculated within different concentration of essential oils and then incubated at 37 °c for bacteria or 35 °c for c. albicans in a moist dark chamber. the mic of each sample was recorded after 16-20 h of incubation for bacteria and 24 h for c. albicans. end points were defined as the lowest concentration of antibacterial agent resulting in total inhibition of visual growth compared to the growth in the control wells containing no essential oil. the minimum bactericidal concentration (mbc) and fungicidal concentration (mfc) were also determined. after determining the mic, 50 μl (bacteria) or 20 µl (c. albicans) samples were withdrawn from each well of the microtiter tray with a 96-pin replicator (boekel scientific, feasterville, pa, usa) and plated onto nutrient agar (bacteria) or sabouraud dextrose agar plates (c. albicans). inoculated plates were incubated at 37°c for 24 h (bacteria) or 35 °c for 48 h (c. albicans) before determining the mbc (or mfc), which was defined as the lowest concentration of essential oil that resulted in total inhibition of visible growth (espinelingroff and cantón, 2007; qaiyumi, 2007). antioxidant activity  dpph radical scavenging activity the experimental protocol followed that of prieto et al. (1999). fifty microliters of essential oil at different concentrations were added to 1950 μl of a methanolic solution of dpph at 6.34 × 10-5 m. a blank (negative) control was prepared by mixing 50 μl of methanol with 1950 μl of the methanolic solution of dpph. after incubation in the dark for 30 min at room temperature, the reduction of dpph was evidenced by the color change of the solution from violet to yellow. the absorbance of this solution was then determined at 515 nm using a spectrophotometer. the positive control used is ascorbic acid. the results were expressed as percent inhibition (pi), and this was calculated based on the reduction of the color intensity of the solution using the formula: pi = (odcontrol odessential oils / odcontrol) x 100 where odcontrol is the absorbance of the negative control and odessential oils is the absorbance with the essential oils. ic50 value is the concentration corresponds to 50% inhibition (zeragui et al., 2019).  reducing power assays the reducing power is determined according to the method described by oyaizu (1986). a volume of 1 ml halla et al. – polyphenols content and antimicrobial, antioxidant and … 67 of each essential oil at different concentrations was mixed with 2.5 ml of 0.2 m phosphate buffer (ph 6.6) and 2.5 ml of 1 % potassium ferricyanide solution. the mixture obtained was incubated for 20 min at 50°c. after this period, 2.5 ml of 10 % trichloroacetic acid was added to stop the reaction. the resulting mixture was centrifuged at 650 × g for 10 min at room temperature, and 2.5 ml of the resulting supernatant was added to 2.5 ml of distilled water and 0.5 ml of 0.1 % (w/v) iron chloride. the absorbance of this solution at 700 nm was compared to that of a blank solution and the activity of the essential oil was compared with that of the positive control, i.e., butylhydroxyanisole (bha). the results allow calculation for effective concentration (ec50), concentration of the corresponding essential oils with an absorbance equal to 0.5 (halla et al., 2019a). hemolytic assay red blood cells were isolated and suspended in pbs (100 mm at ph 7.4) at a rate of 4000 cells/ml. the erythrocyte suspension was incubated at 37 °c under continuous agitation for 120 min, from the addition of essential oil solution with dmso (dimethyl sulfoxide) at different final concentrations. samples of 50 μl from the reaction solution were made at regular intervals to which we added 2 ml of cold washing solution (150 mm of nacl, 2 mm of mgcl2). after centrifugation at 4000 rpm for 5 min, the absorption of the resulting supernatant was determined at 548 nm by photometric monitoring against a blank sample. control samples of 0% lysis (in buffer) and 100% lysis (in double-distilled water) were employed in all experiments (bolard, 1986; halla et al., 2013; silva et al., 2017). statistical analysis the yields obtained, polyphenols content and antioxidant activity tests were carried out in triplicate and the results are expressed in mean values ± standard deviation. all analyzes were performed in the microsoft excel for windows program, 2007. results and discussion extraction yield in this study, we were interested in the study of different essential oils obtained by hydrodistillation of the aerial parts of four algerian plants. the two studied plants, pituranthos scoparius and myrtus nivellei, were collected in the tassili n'ajjer region in the wilaya of tamanrasset (south of algeria). the tassili national park has been listed as a unesco world heritage site since 1982 and has been classified as a man and biosphere reserve since 1986. it is characterized by a contrasted landscape of rugged mountainous terrain and desert plateau of black rocks which form the reg or white sands. the central barrier of 1500–2000 m in altitude extends over 800 km and covers 80,000 km2 (hammiche and maiza, 2006). the two plants rosmarinus officinalis and mentha piperita were harvested in the steppe area (saida and el bayadh). in algeria, the steppe constitutes a vast region which extends between the tellian atlas in the north and the saharan atlas in the south, extending over a land area of around 20 million hectares. the altitude ranges from 400 to 1,200 meters. the steppe is characterized by a strong climatic constraint (insufficient rainfall with an isohyet varying from 100 to 400 mm, strong and sometimes hot winds, etc.) and edaphic (vulnerable soils, thin and poor in organic matter) (khaldi, 2014). the various yields obtained are reported in table 1. p. scoparius and m. piperita showed yields higher than 1%, however, the yields of m. nivellei and r. officinalis were of the order of 0.81 and 0.76 %, respectively. table 1. yields and total polyphenols content of essential oils of pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita. yields (%) total polyphenols content mg gae/g ds* pituranthos scoparius 1.081 ± 0.061 14.78 ± 0.72 myrtus nivellei 0.81 ± 0.02 0.611 ± 0.056 rosmarinus officinalis 0.76 ± 0.42 0.279 ± 0.087 mentha piperita 1.10 ± 0.13 0.104 ± 0.012 * mg gallic acid equivalents per g dried sample pituranthos scoparius essential oil had a specific odour (odour of fennel) and was colorless. the yield of essential oils of pituranthos scoparius was 1.081 ± 0.061%, which is in agreement with that obtained by lograda et al. (2013) from the same plant collected in mechouneche (biskra). however, the same authors found yields of 0.47%, 0.85% and 2.29% in different regions (t'kout (batna), boussâada (m'sila), and elkantra (biskra), respectively) when the plant was harvested in october. gourine et al. (2011) obtained yields ranging from 0.6 up to 2.8%. ksouri et al. (2017) studied the essential oil of pituranthos scoparius harvested in the wilaya of tamanrasset, they found a yield of 0.4% which is different than that obtained in our study. our result was not correlated with that obtained by vérité et al. (2004) (0.77% from the seeds and 0.50% from the stems of the plant harvested in april), kalla et al. (2010) (0.25% and 0.3% of the aerial part of the plant 68 biology, medicine, & natural product chemistry 9 (2), 2020: 65-75 harvested in february and april, respectively), abderrazak et al. (2013) (0.25% of the aerial part of the plant harvested in october) and chikhoune et al. (2017) (0.502% of the aerial part of the plant harvested in april). the essential oil of m. nivellei was pleasant with a light yellow colour. the yield was 0.81 ± 0.02%. this result is supported by that obtained by bouzabata et al. (2013) from the hoggar zone, which was not the case with that harvested from tassili n'ajjer where these authors found yields ranging from 0.5 to 0.9%. boukhalfa (2017) obtained that the yield was 1.6% of this plant harvested in the months of september and october in the tagmart region in tamanrasset. unlike the abundant literature relating to the essential oil of myrtus comunis in algeria, studies on the essential oils of m. nivellei are counted on the fingers. pereira et al. (2009) suggested that the highest yields of the essential oils of myrtus comunis were obtained for the leaves in october. as well as, the samples taken in may and october produced very little oil. altogether, september seems to be the month with the best yields for the different parts of the plant. the yield of essential oils of rosmarinus officinalis (0.76 ± 0.42%) is lower than those found by jordán et al. (2013) of the various essential oils of rosmarinus officinalis harvested in thermo mediterranean, upper mesoand supra-mediterranean areas in spain, which are of the order of 1.74 ± 0.38, 2.44 ± 1.26 and 2.58 ± 0.75%, respectively. zaouali et al. (2010) reported that the yields of essential oils of this plant harvested in tunisia from three different zones (sub-humid, upper semi-arid and upper arid zone) are between 1.17 and 2.7%. a study by djeddi et al. (2007) recorded a yield of 0.82%. this result seems very close to our result, may be due to the same origin of the two plants (algeria). the essential oil of mentha piperita was pale yellow with a strong aromatic odour. the essential oil yield of mentha piperita was 1.10 ± 0.13%. work carried out on the same species in algeria (ghardaïa) revealed a yield close (1.325%) to the value obtained by our study on the essential oil of m. piperita (laghouiter et al., 2015). another study in morocco showed a yield around 1.02% (derwich et al., 2010). on the other hand, yield values for essential oil of m. piperita have been recorded by several authors from different countries, and which are higher or lower than that obtained by our study. singh et al. (2015) revealed a yield of about 0.64% from the leaves of m. piperita harvested in libya. a yield of 2.29% was recorded by gavahian et al. (2015) of the aerial part of the same plant harvested in iran. laghouiter et al. (2015) reported that the extraction yield was very low during the cold period (autumn and winter), however the best yields were obtained in summer and spring during the flowering period of the plant. rohloff et al. (2005) studied the effect of the harvest time and the drying method on the essential oil yield of mentha piperita, and they concluded that the harvest of this plant should be carried out at full flowering to obtain the best yield. this difference may be due to the geographical origin of the plant as well as to the harvest period. according to some authors, the harvest season can affect the variation in the yield of essential oils (boukhalfa, 2017). others explain this difference by the harvest period and the drying method (rohloff et al., 2005). other factors are likely to influence the difference in yield. precipitation can particularly affect the yield of essential oils (sangwanet al., 2001). plant ontogenesis can be one of the most important factors that influence the accumulation of essential oils in plants. since many transformations and changes occur inside cells due to physiological processes, the harvest season and the organ used are often considered to be critical parameters that can affect the chemical compositions of essential oils (lee and ding, 2016). total polyphenols content the total phenol content is determined from the equation of the linear regression of the gallic acid calibration curve: y = 0.005 x + 0.185 (r² = 0.996) and the results are expressed in mg gallic acid equivalents per g of dried sample (mg gae/g ds). table 1 summarizes the results for essential oils of four species tested. from the results obtained, we observed a high content of polyphenols in the p. scoparius essential oils (14.78 ± 0.72 mg gae/g ds), however, the essential oils of m. nivellei, r. officinalis and m. piperita contain 0.611 ± 0.056, 0.279 ± 0.087 and 0.104 ± 0.012 mg gae/g ds, respectively. the total phenol contents are variable between the four plants. the total phenol content found by wojdylo et al. (2007), in essential oils of rosmarinus officinalis (1.71 ± 0.02 mg gae/g ds) is greater than our value (0.279 ± 0.087 mg gae/g ds). for the essential oils of m. piperita, the total phenol content is lower than that of the same species previously studied by zheng and wang (2001); it is about 2.26 ±0.16 mg of gae/g of fresh weight. studies on the extract of m. nivellei leaves indicated that the hydromethanolic, ethyl acetate, butanolic and aqueous fraction had the highest values in phenolic content (222.98 ± 4.70, 308.4 ± 7.40, 414.96 ± 2.40 and 128.03 ± 1.87 mg gae/g ds, respectively) (ramdane et al., 2017). in another study on the aqueous extract of the leaves of m. nivellei, the level of total polyphenols was estimated around 242.68 ± 9.79 mg gae/g ds (rached et al., 2010). lograda et al. (2013) reported the variation in the composition of the essential oils of pituranthos scoparius in algeria and they recorded that these oils are very rich in alcoholic compounds such as terpinene-4-ol, p-cymèn-8-ol, β-eudesmol, methyl-eugenol, spathulenol, linalool, γ cadinol and t-muurolol. halla et al. – polyphenols content and antimicrobial, antioxidant and … 69 the value of the polyphenol content of mentha piperita is not in agreement with those obtained by sharafi et al. (2010), from the plant harvested in iran, where the polyphenol level was 89.43 ± 0.58, 40.43 ± 0.58, 15.10 ± 1 and 9.43 ± 0.58 μg gae/mg sample for oil concentrations of the order of 10, 5, 2.5 and 0.2 µg/ml. atanassova et al. (2011) showed that the methanolic extract of mentha piperita contains a polyphenol level of 0.4525 mg gae/g ds where this plant was harvested in bulgaria. the level of total polyphenols was estimated to be around 71.8 ± 5.1 and 55.1 ± 3.6 mg gae/g ds of the methanol/chloroform extract and the aqueous extract from the leaves and stems of mentha piperita harvested in pakistan (akhtar et al., 2018). riachi and de maria (2015) concluded that the composition of mentha piperita oil is very sensitive to environmental variations where the maturity of the leaves plays an important role in the composition of the essential oil. thus, mint plants harvested at the flowering stage usually have a higher concentration of menthol (phenol) than plants in the bud-forming process, which contain a high amount of menthone. another factor, the exposure of the plant mentha piperita to uv-a radiation in addition to white light, during the night, increased the phenol content (maffei et al., 1999). in short, longer days, colder nights, older leaves, plants harvested at the flowering stage, and adequate hydration of plants should provide good quality peppermint essential oil (riachi and de maria, 2015). by comparison with previous studies, it seems that the extraction of phenolic compounds is governed by several factors which directly influence the levels of these molecules among these factors; increasing the extraction temperature, contact time of the plant material with water, and decreasing the particle size to increase the diffusion coefficient of the water. antibacterial and antifungal activity the obtained results of the antibacterial and antifungal activity (minimum inhibitory concentration ‘mic’ and minimum bactericidal (fungicidal) concentration ‘mbc’ -‘mfc’) of the essential oils of the four plants are presented in table 2. all the strains showed sensitivity against the essential oils tested with the exception of the c. albicans against r. officinalis essential oils. all essential oils tested are less active than the positive control (gentamicin or ketoconazole) with the exception of rosmarinus officinalis essential oils against salmonella typhimurium (mic and mbc = 0.625 mg/ml), salmonella enterica (mic = 0.0825 mg/ml and mbc = 0.312 mg/ml) and klebsiella pneumonia (mic = 0.3125 mg/ml and mbc = 1.25 mg/ml), as well as essential oils of mentha piperita against staphylococcus aureus (mic = 0.078 mg/ml). the strains treated by p. scoparius essential oils showed that the most sensitive strain was c. albicans atcc 10231 by mic of 0.0781 mg/ml. on the other hand, all bacteria tested against p. scoparius in this study revealed mics of 5, 10 or higher than 10 mg/ml. e. coli was the most sensitive bacteria tested by m. nivellei essential oils with mic around 2.5 mg/ml, while, the fungal strains showed good mic (0.312 mg/ml) by comparing with those obtained against bacteria. table 2. minimum inhibitory concentrations and minimum bactericidal (fungicidal) concentrations(mg/ml) of essential oils (pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita) and controls. test organisms pituranthos scoparius myrtus nivellei rosmarinus officinalis mentha piperita controld mica mbcb (mfcc) mic mbc (mfc) mic mbc (mfc) mic mbc (mfc) mic mbc staphylococcus aureus atcc 25923 5 5 10 10 10 10 0.078 0.156 0.156 0.156 bacillus cereus atcc 11778 10 10 5 5 2.5 10 5 5 0.156 0.156 escherichia coli atcc 25933 10 10 2.5 2.5 1.25 5 0.312 0.625 0.312 0.312 pseudomonas aeruginosa atcc 27853 10 10 5 5 2.5 5 5 5 0.625 1.25 pasteurella multocida atcc 43137 10 10 10 10 2.5 10 0.156 1.25 0.039 0.039 salmonella typhimurium atcc 13311 ˃ 10 ˃10 10 10 0.625 0.625 1.25 2.5 0.625 0.625 salmonella enterica atcc 13312 5 5 10 10 0.0825 0.312 10 10 0.625 0.625 campylobacter fetus atcc 27374 ˃ 10 ˃ 10 10 10 5 10 5 5 0.019 0.039 klebsiella pneumonia atcc 700603 10 10 10 10 0.3125 1.25 2.5 5 5 10 enterobacter cloacae atcc 13047 ˃ 10 ˃ 10 5 5 5 10 5 5 2.5 5 candida albicans atcc 10231 0.0781 0.0781 0.312 0.312 / / 0.625 2.5 0.019 0.019 candida albicans ip 444 0.039 0.039 0.312 1.25 / / 5 5 0.019 0.019 a mic: minimum inhibitory concentrations, bmbc: minimum bactericidal concentrations, cmfc: minimum fungicidal concentrations d control: gentamicin for bacteria and ketoconazole for candida albicans concerning r. officinalis, s. enterica recorded the lowest mic compared to the other tested bacteria (0.0825 mg/ml). by comparison, gram negative bacteria were more sensitive than gram positive against r. officinalis essential oils. indeed, the registered mics of m. piperita essential oils for b. cereus, p. aeruginosa, 70 biology, medicine, & natural product chemistry 9 (2), 2020: 65-75 s. enterica, c. fetus, e. cloacae and c. albicans ip 444 (from 5 to 10 mg/ml) were higher than those obtained for s.aureus, e.coli, p. multocida, s. typhimurium, k. pneumonia and c. albicans atcc 10231 (from 0.078 to 2.5 mg/ml). the mics for pituranthos scorparius essential oil were similar to those reported by boutaghane et al. (2004) for enterobacter, klebsiella pneumoniae and salmonella thiphymurium where they found mics of the order of 256, 16 and 128 mg/ml, respectively. on the other hand, the mic results obtained by these authors against escherichia coli (256 mg/ml), pseudomonas aeruginosa (1 mg/ml) and staphylococcus aureus (256 mg/ml) are not in agreement with our results. ksouri et al. (2017) found that no antibacterial activity of pituranthos scorparius essential oil has been revealed against escherichia coli and klebsiella pneumoniae. however, the mics obtained for staphylococcus aureus and candida albicans were of the order of 1 and 0.5 mg/ml. the mic obtained by ksouri et al. (2017) against pseudomonas aeruginosa (greater than or equal to 2 mg/ml) is correlated with that of our result. bouzabata et al. (2013) studied the antifungal activity of the essential oil of myrtus nivellei (same species in algeria), they showed mics and mfcs between 1.25 and 2.5 µl/ml against candida albicans atcc 10231. these can be correlated with the cmf obtained against candida albicans ip 444 (1.25 mg/ml). a work by kabouche et al. (2005) on essential oils of rosmarinus officinalis grown in algeria showed mics greater than 0.128 mg/ml against both strains e. coli and s. aureus. the results of the cmi are in agreement with those obtained by celiktas et al. (2007) from turkey (izmir, çanakkale) of the essential oils of the plant harvested in september against the staphylococcus aureus (10 mg/ml). however, a tunisian study reported that essential oils of this species revealed mics between 1.25-2.5 μl/ml against e. coli and between 1.25-2.5 μl/ml against bacillus cereus (zaouali et al., 2010). it is comparable to our results. according to jordán et al. (2013), these essential oils have an mic of 2-5 μl/ml against e. coli. i̇şcan et al. (2002) tested the essential oil of m. piperita from different origins (turkey and india), they reported mics between 1.25 and 2.5 mg/ml against escherichia coli, between 0.625 and 2.5 mg/ml against staphylococcus aureus, between 1.25 and 2.5 mg/ml against salmonella typhimurium, of 2.5 mg/ml against klebsiella pneumoniae, of 1.25 mg/ml against bacillus cereus and between 0.312 and 0.625 mg/ml against candida albicans. these results are correlated with our results, with the exception of those obtained for escherichia coli and bacillus cereus. the mic values for essential oil of m. piperita against different bacterial strains, reported by mahboubi and kazempour (2014), range from 0.125 to a value greater than 64 μl/ml. the mics are of the order of 1, 0.25, 1, 2, 16, 0.25 and 0.125 μl/ml against s. aureus, b. cereus, e. coli, s. typhimurium, p. aeruginosa, k. pneumioniae and c. albicans, respectively. however, the mbcs are of the order of 2,> 64, 1, 2, 16 0.5 and 0.125 μl/ml against s. aureus, b. cereus, e. coli, s. typhimurium, p. aeruginosa, k. pneumioniae and c. albicans, respectively. mohammadi et al. (2016) revealed percentages of 0.16%, 0.8% and 2% of mic50, mic90 and mbc, respectively, of the essential oil of m. piperita against e. coli. compared with our result, it seems that the oil tested has a strong activity to that tested by mohammadi and al. tyagi and malik (2011) recorded mic and mbc (or mfc) of 1.13 to 2.25 mg/ml (mic) and 2.25-9 mg/ml (mbc) for bacterial strains and 1.13 mg/ml (mic) and 2.25 mg/ml (mfc) for yeasts. according to the study by saharkhiz et al. (2012), the essential oils of m. piperita have an antifungal activity with an mic of 1.5 μl/ml against c. albican strain. according to the study by saharkhiz et al. (2012), the essential oils of m. piperita have an antifungal activity with a mic of 1.5 μl/ml against c. albicans. samber et al. (2015) found that m. piperita essential oil is a bioactive fungicidal compound that has a strong effect on pm-atpase (pm: plasma membrane) in candida species. they suggested these essential oils enter the cell membrane and target the pathway for ergosterol biosynthesis, thereby compromising its biosynthesis. simultaneously, they react with the membrane itself with their reactive hydroxyl moiety, and the extensive lesion on the membrane is a combined effect of the two events. antioxidant activity the evaluation of the antioxidant activity of the essential oils of the four plants was carried out by two conventional methods in order to test these essential oils by various reaction mechanisms involved in these antioxidant tests. the dpph radical is one of the most widely used substrates for the rapid and direct evaluation of antioxidant activity due to its stability in radical form and the simplicity of this analysis. the antioxidant power of the essential oils has been compared to that of the positive control used (ascorbic acid). the values of the concentrations corresponding to the inhibition of 50% of the free radical dpph (ic50) are summarized in table 3. from the results obtained, we observed that the essential oils of the four plants showed an antioxidant activity by scavenging of the free radical dpph. by comparing the antioxidant power of ascorbic acid (positive control) with that of the essential oils of p. scoparius, m. nivellei and m. piperita, we note that these essential oils were less active and showed a weak antioxidant activity relative to ascorbic acid. however, the r. officinalis essential oils (ic50 = 0.69 ± 0.07 halla et al. – polyphenols content and antimicrobial, antioxidant and … 71 mg/ml) reported more antioxidant power than the ascorbic acid (ic50 = 0.81 ± 0.14 mg/ml). from the results obtained, the essential oils of p. scoparius, m. nivellei and m. piperita have ic50 values of 3.26 ± 0.65, 30.67 ± 2.12 and 18.33 ± 1.84 mg/ml, respectively. table 3. ic50 values for reduction of radical dpph and ec50 of reducing power of essential oils of pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita (mg/ml). ic50 dpph ec50 reducing power ascorbic acid 0.81 ± 0.14 / bha / 0.42 ± 0.12 pituranthos scoparius 3.26 ± 0.65 484.40 ± 5.14 myrtus nivellei 30.67 ± 2.12 31.2 ± 2.59 rosmarinus officinalis 0.69 ± 0.07 9.67 ± 1.36 mentha piperita 18.33 ± 1.84 11.07 ± 0.07 the values of the optical densities obtained by reducing power assays allowed to drawing curves for each essential oil. in this test, the increase in absorbance means an increase in the reducing power of the essential oils tested. in order to compare the antioxidant activity of the essential oils tested by this method, we calculated the ec50. the results obtained are illustrated in table 3. we note that the essential oils of the p. scoparius showed a very low reducing power by comparing them with the positive control used (bha) and the essential oils of the other plants (484.40 ± 5.14 mg/ml). the essential oils of r. officinalis reported an ec50 of 9.67 ± 1.36 mg/ml, this time their antioxidant activity have not powerful to those of the positive control bha (0.42 ± 0.12 mg/ml). the ec50 of m. nivellei and m. piperita essential oils are 31.2 ± 2.59 and 11.07 ± 0.07 mg/ml, respectively. the essential oils of pituranthos scoparius have an ic50 value of 3.26 ± 0.65 mg/ml which results in a much higher antioxidant power than the value obtained by ksouri et al. (2017) which is of the order of 11.21 ± 0.26 mg/ml of the same species. to our best knowledge, there is no work done on the antioxidant activity of the essential oils of m. nivellei. touaibia and chaouch (2014) studied some extracts of this plant, they showed that the extracts studied all had a very good reducing activity, especially for the ethanol extract (ec50 equal to 0.59 mg/ml) by the dpph method and methanol extract by the frap test (66.7%). rached et al. (2010) evaluated the antioxidant activity of the fractions obtained from the raw extract of the leaves of m. nivellei. they found that the best activity was reported for the ethyl acetate and n-butanol fractions with ic50 values of 3.08 ± 0.40 μg/ml and 4.40 ± 0.43 μg/ml, respectively. in addition, ramdane et al. (2017) reported ic50 values between 4.97 μg/ml and 16.33 μg/ml, for the different extracts obtained from m. nivellei leaves (hyudromethanol, ethyl acetate, butanolic and aqueous). the antioxidant activity of essential oils of rosmarinus officinalis is greater compared to that found by zaouali et al. (2010) and wojdylo et al. (2007), which revealed a low antioxidant power, by the two dpph and frap methods. for the essential oils of m. piperita, we compared our results with the work carried out by singh et al. (2015), on the same species of libya, which showed a higher antioxidant activity by scavenging the dpph radical, with an ic50 value of 15.2 ± 0.9 μg/ml. according to sharafi et al. (2010), the m. piperita essential oil has shown a percentage inhibition of dpph activity of 63.82 ± 0.05% at the concentration of 10 µg/ml where the ic50 was around 3.9 μg/ml. laghouiter et al. (2015) recorded an ec50 of around 208.495 ± 4.247 μg/ml of m. piperita essential oil. gavahian et al. (2015) extracted the essential oil from m. piperita by four different methods (hydrodistillation; steam entrainment, hydrodistillation assisted by microwaves and hydrodistillation assisted by ohmic heating). they found that the antioxidant activity was almost similar for the essential oil obtained by these different methods whose ec50 values were between 9.6 ± 0.7 and 10.4 ± 0.6 μg/ml. our results, in most cases, are not in agreement with those obtained by previous work. this difference in the results is probably due to the diversity of the chemical composition and according to intrinsic and extrinsic factors, namely the harvest region and the evaluation method used. hemolytic asssay the hemolysis test was evaluated because, even if a plant has potent antioxidant power or good antimicrobial activity, its use in traditional medicine and in pharmacological preparations will be impossible in the presence of their hemolytic effect, which is an indicator of cytotoxicity. when the plasma membrane of red blood cells is altered by the action of essential oil, it follows lysis resulting in the release of hemoglobin in the red blood cell, which is why we assayed the extracellular hemoglobin after the addition of different concentrations of essential oils. final concentrations of essential oils were chosen according to the mic founded in the first part. figure 1 shows the effect of essential oils at different concentrations (0.39, 0.781, 1.56 and 3.125 mg/ml) on the release of hemoglobin from the red blood cells at 37 °c. the results obtained show that the percentages of hemolytic effect are directly proportional to the increase in the concentrations of the essential oils of the four tested plants (fig. 1). after 120 minutes of incubation and for all the concentrations tested, the hemolysis percentages are between 10 and 93%. therefore, the hemolytic effect of the various essential oils tested, at the concentration of 3.125 mg/ml after 120 minutes of contact with human erythrocytes, can be classified as 72 biology, medicine, & natural product chemistry 9 (2), 2020: 65-75 follows: p. scoparius (93%) m. piperita (72.88 %) > m. nivellei (72.75%) > r. officinalis (43.26%). after 120 min incubation, minimal haemolysis (10%) is obtained with essential oils of r. officinalis at a concentration of 0.39 mg/ml, so these essential oils may be slightly hemolytic at this concentration after two hours of incubation. on the other hand, the other essential oils have an important hemolytic effect against isolated erythrocytes, with a hemolysis rate that exceeds 41% at a concentration of 0.39 mg/ml. figure 1. release of hemoglobin by erythrocytes induced by different concentrations (mg/ml) of essential oils (pituranthos scoparius, myrtus nivellei, rosmarinus officinalis and mentha piperita). our results show that the essential oils of rosmarinus officinalis have a very low toxic effect compared to isolated erythrocytes, with a haemolysis rate not exceeding 45% at a concentration of 3.1225 mg/ml, what characterizes it by the absence of risk of cytotoxicity. for other plants, they may be slightly hemolytic at high concentrations with respect to human erythrocytes. samber and his collaborators have shown that the rate of hemolysis of essential oil of mentha pipireta against human red blood cells does not exceed 6% at a concentration of 2 mg/ml after one hour of incubation (samber et al., 2015). mendanha et al. (2013) report that terpenes can compete with the intermolecular hydrogen bond between lipid molecules and thereby disrupt the network of hydrogen bonds in the lipid bilayer, which weakens the membrane. as well, jain et al. (2002) found that terpenes, which have alcoholic ‘oh’ groups and which act as hydrogen bond donors, can disrupt the hydrogen bond network in the membrane bilayer. according to some authors, the cytotoxicity of essential oils against red blood cells is due to their hydrophobic nature which is accentuated by the synergetic effect between their compounds (sacchetti et al., 2005). silva et al. (2017) suggest that the constituents present in oils can interact with the components of the erythrocyte membrane, leading to destabilization of its structure and to a disordered influx of ions and water which leads to rupture of the membranes. conclusion the plants studied were harvested from areas of specific climate in algeria. p. scoparius and m. piperita showed yields higher than 1%. according to the antimicrobial activity results, all the strains showed sensitivity against the essential oils tested with the exception of the c. albicans against r. officinalis essential oils. the high content of polyphenols was reported in p. scoparius essential oils (14.78 ± 0.72 mg gae/g ds). the antioxidant test shows that the the essential oils of the four plants showed an antioxidant activity by scavenging of the free radical dpph. signally, the r. officinalis essential oils reported more antioxidant power than the positive control (ascorbic acid). the reducing power for all essential oils tested was by ec50 ranging from 9.67 ± 0 20 40 60 80 100 0 5 10 15 30 60 120[h e m o gl o b in ] re le as e d ( % ) time (min) pituranthos scoparius witness 0,39 0,781 1,56 3,125 0 20 40 60 80 100 0 5 10 15 30 60 120[h e m o gl o b in ] re le as e d ( % ) time (min) myrtus nivellei witness 0,39 0,781 1,56 3,125 0 20 40 60 80 100 0 5 10 15 30 60 120[h e m o gl o b in ] re le as e d ( % ) time (min) rosmarinus officinalis witness 0,39 0,781 1,56 3,125 0 20 40 60 80 100 0 5 10 15 30 60 120[h e m o gl o b in ] re le as e d ( % ) time (min) mentha piperita witness 0,39 0,781 1,56 3,125 halla et al. – polyphenols content and antimicrobial, antioxidant and … 73 1.36 (r. officinalis) to 484.40 ± 5.14mg/ml (p. scoparius). our results show that the essential oils of rosmarinus officinalis had a very low toxic effect compared to isolated erythrocytes (45% at 3.1225 mg/ml). from the results obtained, the geographical origin and period of harvest can influence the yield and the antioxidant activity of the essential oils of the studied plants. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references abderrazak k, messoued r, azzedine z (2013). etude phytochimique et de l’activité antimicrobienne des huiles essentielles de pituranthos scoparius de la region de biskra (sud-est algérien). tunisian journal of medicinal plants and natural products 10(2): xx-xx. akhtar n, ihsan-ul-haq mirza b (2018). phytochemical analysis and comprehensive evaluation of antimicrobial and antioxidant properties of 61 medicinal plant species. arabian journal of chemistry 11 (8): 1223-1235. aps (algeria press service) (2019). environnement: l'observatoire national de la biodiversité inscrit dans la loi de finances 2020 (la ministre de l'environnement et des energies renouvelables, fatima zohra zerouati). algeria press service. http://www.aps.dz/economie/89734environnement-l-observatoire-national-de-la-biodiversiteinscrit-dans-la-lf-2020. atanassova m, georgieva s, ivancheva k (2011). total phenolic and total flavonoid contents, antioxidant capacity and biological contaminants in medicinal herbs. journal of the university of chemical technology & metallurgy 46(1): 8188. bolard j (1986). how do the polyene macrolide antibiotics affect the cellular membrane properties?. biochimica et biophysica acta (bba)-reviews on biomembranes 864(3-4): 258-303. boukhalfa d (2017). contribution à l’étude des plantes aromatiques et médicinales de la région de l’ahaggar (doctoral dissertation, university of algiers, algeria). http://193.194.83.98/jspui/bitstream/1635/14379/1/boukha lfa_djamel.pdf boutaghane n, nacer a, kabouche z, ait-kaki b (2004). comparative antibacterial activities of the essential oils of stems and seeds of pituranthos scoparius from algerian septentrional sahara. chemistry of natural compounds 40(6): 606-607. bouzabata a, bazzali o, cabral c, gonçalves mj, cruz mt, bighelli a, cavaleiro c, casanova j, salgueiro l, tomi f (2013). new compounds, chemical composition, antifungal activity and cytotoxicity of the essential oil from myrtus nivellei batt. & trab., an endemic species of central sahara. journal of ethnopharmacology 149(3): 613-620. celiktas oy, kocabas eh, bedir e, sukan fv, ozek t, baser khc (2007). antimicrobial activities of methanol extracts and essential oils of rosmarinus officinalis, depending on location and seasonal variations. food chemistry 100(2): 553559. chikhoune a, damjan pavleca j, shashkov m, berroua z, chebbi k, bougherra h, zeroual b, aliane k, gagaoua m, boudjellal a, vovk i, krizman m (2017). antioxidant effect induced by the essential oil of pituranthos scoparius in a formulation of a whey spread emulsion. journal of food processing and preservation 41(5): 1-12. clsi (clinical and laboratory standards institute) (2012). methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; 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(ssp. sahariensis) flowers as a natural cosmetic preservative: chemical composition, and antioxidant and antibacterial activities. journal of essential oil bearing plants 22(3): 685-694. zheng w, wang sy (2001). antioxidant activity and phenolic compounds in selected herbs. journal of agricultural and food chemistry 49(11): 5165-5170. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 2, 2018 | pages: 45-49 | doi: 10.14421/biomedich.2018.72.45-49 issn 2540-9328 (online) diversity of angiospermae plant class liliopsida in mount nglanggeran bayu setya aji nugraha*, widodo, rendi yuntara, normalita department of biology education, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto, no. 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. author correspondency*: bayu.08114@gmail.com abstract nglanggeran is a place that has a high plant diversity and there are many unique and unidentified wild plants. this study aims to list liliopside class plants which found around the climbing route. the angiosperm plants in the liliopside class found around the climbing route were successfully identified and consisted of 40 species belong to 17 families. keywords: diversity plants; liliopsida; mount nglanggeran introduction indonesia is a country with a high level of plant diversity of approximately 30 thousand species of 40 thousand species of plants that exist in the world (fahrurozi, 2014). the level endemicity of indonesian flora is recorded between 40-50% of the total flora species on each island except sumatra island which is estimated at only 23% (lipi, 2014). according to bappenas (2016), this data is not accurate and data collection and name validation are still needed. the species that have been identified and recorded only reach 50% of the total number of flora recorded, that is 19,112 species. but the rate of extinction and reduced diversity of plant species has accelerated. this is a problem and a challenge that must be solved. another problem where the majority of research focuses only on horticulture plants and ignores wild plants that have not been identified. the data about wild plants can be applied in other field as health, food, environmental and etc. (widodo, 2015). mount nglanggeran is a place that has high plant diversity and there are many wild plants that have not been identified. widodo (2015) in his exploration on mount nglanggeran found various types of unique plants and are rarely found in residential areas. some of these plants are also found around the region of baturagung. the plant is not recognized by the people and its local name is unknown. probably, the local name of the plant had known in the past but the knowledge does not pass to the next generation. this shows why taxonomic and systematic studies are needed so that knowledge of plants can be passed onto the next generation. this paper aimed to list the diversity of liliopsida plants wich found around the climbing route of ngelanggeran volcano. materials and methods this research is a field research that uses survey and exploration methods. equipments for observation and collection consist of: nikon d320 digital camera and fuji mirrorless, smartphones, stationery, observation sheets, maps applications, and complete plant books. this research is divided into three stages, namely exploration of climbing routes, collection of photo specimens, and discovery of plants. plant were indentify using various literature and matching with herbariums and illustrations/images in the literature for the discovery of specimens found. result and discussion angiosperms of the liliopsida class found around the nglanggeran volcano were 39 species grouped in 16 families (table 1). the species are grouped into family araceae, two species from commelinaceae, one species from costaceae, one species from hypoxidaceae, four species from zingiberaceae, six species from orchidaceae, five species from dioscoreaceae, one species from pandanaceae, three species from smiles, one species of agavaceae, one species of tacaceae, one species of liliaceae, and one species of colchiaceae (figure 1). https://doi.org/10.14421/biomedich.2018.72.45-49 46 biology, medicine, & natural product chemistry 7 (2), 2018: 45-49 table 1. liliopsida class plants found around the nglanggeran volcano climbing trail. family species name family species name araceae alocasia crassifolia flagellariaceae flagellaria indica amorphophallus variabilis hypoxidaceae curculigo latifolia anadendrum latifolium liliaceae zephyranthes rosea epripemnum aureum orchidaceae dendrobium crumenatum caladium sp liparis sp pothos scandens nervilia plicata typonium trilobatum pholidota imbricata syngonium podophyllum pecteilis susannae arecaceae arenga pinnata coelogyne trinervis colchicaceae gloriosa superba pandanaceae pandanus bouetii commelinaceae cyanotis cristata smilacaceae smilax spinosa commelina difusa smilax anceps costaceae costus speciosus smilax celebica cyperaceae kyllinga nemoralis tacaceae taca palmata scleria laevis zingiberaceae curcuma longa cyperus imbricatus globba racemosa cyperus rotundus zingiber zerumbet dioscoreaceae dioscore bulbifera dioscorea alata disocorea hispida dioscorea oppositifolia dioscorea pentaphylla figure 1. diagram of plant family class of liliopsida araceae is the most abundant family of species, among the 8 species found, alocasia crassifolia and amorphophallus variabilis are abundant species and are often found around climbing routes. alocasia crassifolia has large leafy features, up to 1.2 x 2 m round shape, the leaf veins are very clear. the leaves are dark green, while the bottom is dull green, has a long leaf stalk reaching 0.4 1 m. male and female flowering is located on a long-stemmed cob which exits at the end of the stem. egg-shaped fruit with orange color when ripe. the fruit contains only one seed. this plant prefers in wet environment and can be found on the banks of rivers, lakes and mountain slopes which are rather humid (suhono et al., 2010). whereas amorphophallus variabilis has the characteristics of the leaf stalk morphology varies widely, the surface of the leaf stalk is flat or rough, the color of the tuber skin is white, green, gray or purple. (afifah et al, 2014). figure 2. alocasia crassifolia. figure 3. amorphophallus variabilis. some unique species that are rarely found include pothos scandens, curculigo latifolia, nervilia plicata, pholidota imbricata, and smilax anceps. nugraha et al. – diversity of angiospermae plant class liliopsida in mount nglanggeran 47 figure 4. pholidota imbricate. figure 5. smilax anceps. conclusion the diversity of plants in the liliopasida class at the ancient volcano nglanggeran consists of 39 species belong to 16 families. eight species are grouped into family araceae, two species from commelinaceae, one species from costaceae, one species from hypoxidaceae, four species from zingiberaceae, six species from orchidaceae, five species from dioscoreaceae, one species from pandanaceae, three species from smiles, one species of agavaceae, one species of tacaceae, one species of liliaceae, and one species of colchiaceae. araceae is the most abundant family of species, among the 8 species found, alocasia crassifolia and amorphophallus variabilis are abundant species and are often found around climbing routes. some unique species that are rarely found include pothos scandens, curculigo latifolia, nervilia plicata, pholidota imbricata, dan smilax anceps. acknowledgements the authors wish to thank to m. ja'far luthfi for the discussion, didik zulfahmi akbar and all student who accompanied researcher during the research. references afifah, e., nugrahani, m. o. & setiono, 2014. peluang budidaya iles-iles (amorphophallus spp.) sebagai tanaman sela. warta perkaretan 2014, pp. 35-46. bappenas. 2016. indonesian biodiversity strategy and action plan (ibsap) 2015-2020. kementerian perencanaan pembangunan nasional/bappenas. indonesia fahrurozi, irpan. 2014. keanekaragaman tumbuhan obat di taman nasional gunung gede pangrango dan di hutan terfragmentasi kebun raya cibodas serta pemanfaatannya oleh masyarakat lokal. (skripsi). universitas islam negeri syarif hidayatullah jakarta lipi [lembaga ilmu pengetahuan indonesia]. 2014. kekinian keanekaragaman hayati indonesia 2014. kerjasama kementrian ppn/bapennas, klh dan lipi. bogor: lipi press. suhono, budi dan tim lipi. 2010. ensiklopedia flora jilid 1. bogor: pt kharisma ilmu. widodo. 2015. apocynoideae dan asclepiadoideae dari pegunungan baturagung (gunung nglanggeran, gunung mintorogo, gunung parangan, gunung gedang, gunung ijo): inisiasi pencirian dan konservasi. seminar nasional konservasi dan pemanfaatan sumber daya alam. fkip uns. 48 biology, medicine, & natural product chemistry 7 (2), 2018: 45-49 alocasia crassifolia amorphophallus variabilis anadendrum latifolium caladium sp commelina difusa costus speciosus curculigo latifolia curcuma longa cyanotis cristata cyperus imbricatus cyrtococum sp. dendrobium crumenatum dioscorea sp. dioscorea bulbifera dioscorea alata dioscorea hispida dioscorea oppositifolia dioscprea pentaphylla epripemnum aureum globba racemose gloriosa superba kyllinga nemoralis liparis sp. nervilia plicata nugraha et al. – diversity of angiospermae plant class liliopsida in mount nglanggeran 49 pandanus bouetii pholidota imbricate photos scandens scleria laevis smilax smilax spinose syngonium podophyllum taca palmata typhonium trilobatum zephyranthes rosea zingiber zerumbet biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 363-368 | doi: 10.14421/biomedich.2025.141.363-368 issn 2540-9328 (online) protective role of paederia foetida l. against hepatic inflammatory response in a mice model of escherichia coli-induced sepsis lisa savitri1,3,4,*, maria do carmo da costa freitas1, fendy prasetyawan2, yuneka saristiana2, konradus klala mebung1, cornelia amanda1 1department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia 2department of pharmacist professional education, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia 3department of biology, faculty of science and technology, universitas airlangga, surabaya, indonesia 4bioinformatics research center, indonesian institute of bioinformatics, malang, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 01 may, 2025. revision accepted: 27 june, 2025. published: 04 july, 2025. abstract sepsis is a severe medical condition characterized by a systemic immune response to infection, often leading to multi-organ failure and death if not treated effectively. this study aimed to investigate the antiseptic properties of paederia foetida leaf extract in male mice induced with sepsis using escherichia coli. a total of 24 mice were divided into six groups: a normal group (no treatment), a negative control group (distilled water), a positive control group (ciprofloxacin), and three treatment groups receiving p. foetida leaf extract at doses of 100, 300, and 500 mg/kg bw. after 15 days of oral administration, liver tissue samples were analyzed for il-6 expression, a proinflammatory cytokine associated with sepsis. the results revealed a significant reduction in il-6 expression in the treatment groups, particularly at the 500 mg/kg bw dose. this dose showed the most effective anti-inflammatory response, with il-6 expression levels comparable to those of the positive control group treated with ciprofloxacin. however, the 100 mg/kg bw dose demonstrated minimal effects, similar to the negative control. these findings suggest that p. foetida leaf extract, especially at higher doses, has potential as an anti-inflammatory agent in sepsis management. the bioactive compounds in the extract, including flavonoids, alkaloids, and terpenoids, likely contribute to its efficacy. this study provides preliminary evidence supporting the use of p. foetida as a potential herbal alternative for managing sepsis, but further clinical research is necessary to confirm its therapeutic potential. keywords: sepsis; paederia foetida; il-6; anti-inflammatory; escherichia coli. abbreviations: interleukin-6 (il-6), body weight (bw), randomized complete design (rcd), systemic inflammatory response syndrome (sirs), disseminated intravascular coagulation (dic), world health organization (who), analysis of variance (anova), least significant difference (lsd), immunohistochemistry (ihc), nuclear factor kappa b (nf-κb), mitogen-activated protein kinase (mapk), cyclooxygenase (cox), lipoxygenase (lox), phospholipase a2 (pla2), superoxide dismutase (sod) introduction sepsis is a critical medical condition characterized by a systemic immune response to infection, which may progress to multi-organ failure and fatality (turnip et al., 2022). it reflects the body’s overwhelming reaction to an infectious agent and can escalate into severe sepsis or septic shock. severe sepsis entails organ dysfunction, driven by systemic inflammation and a procoagulant response, while septic shock involves persistent hypotension despite adequate fluid therapy (singer et al., 2016). complications of sepsis include systemic inflammatory response syndrome (sirs), which involves cytokine release such as interleukin-6 (il-6), as well as disseminated intravascular coagulation (dic), septic shock, and multiple organ dysfunction including liver failure (ministry of health, 2017). in 2017, the who reported 48.9 million global sepsis cases with 11 million deaths—accounting for nearly 20% of global mortality. approximately 20 million cases and 2.9 million deaths occurred in children under five. most sepsis-related deaths (85%) occurred in lowand middle-income countries (who, 2020). in indonesia, sepsis incidence remains high (30.29%) with mortality ranging from 11.56% to 49%. at abdul wahab sjahranie hospital, there were 312 sepsis cases from 2018 to 2020, 69 of which were pediatric. sepsis ranked among the top ten causes of death in the hospital (verdure et al., 2021). the sepsis pathophysiology involves complex inflammatory and anti-inflammatory responses, humoral and cellular reactions, and vascular dysfunction (kaukonen et al., 2015). genetic polymorphisms in il-6 have been associated with varying susceptibility to https://doi.org/10.14421/biomedich.2025.141.363-368 mailto:lisasavitri@unik-kediri.ac.id 364 biology, medicine, & natural product chemistry 14 (1), 2025: 363-368 sepsis, septic shock, and mortality (tischendorf et al., 2007). gram-negative bacteria, particularly escherichia coli, are the predominant pathogens (60–70%), while gram-positive bacteria account for 20–40%. fungi, viruses, and protozoa are less common causes (ministry of health, 2017). severe sepsis can result from local infections, not necessarily requiring bacteremia, due to systemic effects of bacterial toxins. blood cultures are positive in only 20–40% of severe cases, increasing to 40–70% in septic shock. gram-negative and gram-positive bacteria constitute most isolates, with the remainder comprising fungi and mixed infections (clarias et al., 2018). effective antimicrobial therapy depends on the rapid identification of the causative agent. empirical broadspectrum antibiotics should be initiated immediately, followed by de-escalation based on culture results and clinical response. recommended options include antipseudomonal quinolones (e.g., ciprofloxacin, levofloxacin) and aminoglycosides. even bacteriostatic antibiotics may be appropriate depending on the infection type (ministry of health, 2017). due to the adverse effects of conventional antibiotics, many people turn to herbal remedies, which remain popular in rural areas due to accessibility and affordability. p. foetida l., commonly known as “daun kentut,” is traditionally used for anti-inflammatory properties (silaban, 2021). other plant-based options include mangosteen rind (garcinia mangostana), known for xanthones that exhibit antibacterial and antiinflammatory activity (syahruna et al., 2020; dewi et al., 2014). p. foetida leaves contain a variety of bioactive compounds, including alkaloids (α-, β-paederine), flavanols, friedelin, β-sitosterol, campesterol, triterpenoid saponins, gallotannins, and essential oils (salamah & halim, 2021). the anti-inflammatory effect, particularly in sepsis models, is believed to stem from saponins, flavonoids, and volatile oils. saponins may interact with lipid membranes, affecting prostaglandin production and inflammatory pathways (savitri & kasimo, 2022). these secondary metabolites also exhibit pharmacological functions such as antioxidant, antimicrobial, anticancer, insecticidal, antitumor, and immunomodulatory activities (das et al., 2018). materials and methods this experimental study utilized a randomized complete design (rcd) to assess the antiseptic activity of p. foetida leaf extract on male white mice (mus musculus). a total of 24 male balb/c strain mice, aged 3–4 weeks and weighing 30–40 g, were randomly assigned to six groups (n = 4 per group). group i (normal) received no treatment, group ii (negative control) was treated with distilled water (aquades), and group iii (positive control) received ciprofloxacin (500 mg/kg bw). groups iv–vi received p. foetida leaf extract at 100, 300, and 500 mg/kg bw, respectively. all mice in groups ii–vi were intraperitoneally injected with e. coli, while group i received no bacterial injection. the study was conducted in two different locations. the treatment of mice took place in the pharmacy laboratory of universitas kadiri, while histological analysis was performed in the anatomical pathology laboratory of universitas brawijaya, malang. male balb/c mice were selected as the experimental subjects due to their reproductive characteristics, ease of handling, and similarities to humans in anatomy, physiology, and genetics (mutiarahmi et al., 2021). the sample size was calculated using federer’s formula, which determined a minimum of 4 subjects per group. the sampling method followed a randomized complete design (rcd), ensuring that all mice had an equal chance of being assigned to any group. inclusion criteria for the study were as follows: male mice, aged approximately 1 month, with a body weight of 18–40 g, and in good health during the study period. mice with poor appetite, declining health, or signs of illness during acclimatization were excluded from the study. the independent variable in this study was the dose of p. foetida leaf extract, while the dependent variable was the il-6 expression in liver tissue. the study controlled for various factors, including the inclusion criteria, housing conditions, and the method of extract administration (oral gavage). the p. foetida leaf extract used in this study was obtained through maceration. for histological analysis, liver tissue was stained using an immunohistochemistry (ihc) kit, with il-6 expression indicated by brown staining in the tissue sections. mice were induced with sepsis by intraperitoneal injection of e. coli. after 15 days of oral administration of the extract, the mice were euthanized by cervical dislocation, and liver tissue was collected and fixed in 10% formalin for further examination. data analysis was performed using one-way anova, following tests for normality (shapiro-wilk) and homogeneity (levene’s test). if the data met normality and homogeneity assumptions (p > 0.05), post hoc lsd or duncan tests were used to analyze group differences. if data were not normally distributed, the kruskal-wallis test was used, followed by mannwhitney u tests for pairwise comparisons when significant differences were observed. results and discussion in this study, the average il-6 expression values in the liver of mice for each group were as follows: the normal group had 7.09% ± 0.06; the positive control group had 26.36% ± 0.02; the negative control group had 72.60% ± 0.05; treatment group i (100 mg/kg bw) had 71.04% ± 0.04; treatment group ii (300 mg/kg bw) had 62.22% ± savitri et al. – protective role of paederia foetida l. against hepatic inflammatory … 365 0.02; and treatment group iii (500 mg/kg bw) had 40.92% ± 0.01. statistical analysis using one-way anova revealed a significance value of 0.000 (p-value < 0.005), indicating a significant difference between the groups. according to the duncan test table, the optimal dose order was as follows: (1) normal group; (2) positive control group; (3) treatment group iii (500 mg/kg bw); (4) treatment group ii (300 mg/kg bw); (5) treatment group i (100 mg/kg bw); and (6) negative control group. the normal group (fig. 1) had the lowest average il6 expression compared to the other groups, at 7.09% ± 0.06. this result is attributed to the fact that the normal group was not injected with e. coli, and thus did not experience sepsis. groups ii through vi were injected with e. coli, causing sepsis in these mice. group ii (fig. 2) was the most effective in treating sepsis compared to the other five groups, as it was the positive control group treated with ciprofloxacin. ciprofloxacin, an antibiotic in the fluoroquinolone class, treats gram-negative bacterial infections, including e. coli (thai et al., 2023). in sepsis, ciprofloxacin works by inhibiting the enzymes dna gyrase and topoisomerase iv, which are essential for bacterial dna replication. inhibition of these enzymes prevents bacterial replication, halting the spread of infection (roberts et al., 2019). ciprofloxacin also inhibits protein synthesis in bacterial cells by blocking ribosomal activity, which is crucial for protein production, further aiding in stopping bacterial infections (demirtas et al., 2012). figure 1. non-expressed hepatocytes in the liver of mice at 40× magnification (cell cytoplasm appears purple as indicated by the arrow) in the normal group. figure 2. il-6 expression in hepatocytes of mice liver injected with e. coli at 40× magnification (cell cytoplasm appears brown as indicated by the arrow) in the positive control group. in the treatment groups receiving p. foetida leaf extract, including group i (100 mg/kg bw) (fig. 3), group ii (300 mg/kg bw) (fig. 4), and group iii (500 mg/kg bw) (fig. 5), the most effective dose for preventing il-6 expression in the liver of sepsis mice was found to be 500 mg/kg bw. the statistical analysis, using anova with a 95% confidence level, showed significant differences in the doses of p. foetida leaf extract for preventing il-6 expression. post hoc lsd and duncan tests indicated that the negative control group and group i (100 mg/kg bw) exhibited similar antiseptic effects. this result suggests that the 100 mg/kg bw dose was less effective as an antiseptic because it showed the same effect as the negative control group. it is suspected that the low dose of 100 mg/kg bw was insufficient to manage inflammation in the e. coli-induced sepsis model. figure 3. il-6 expression in hepatocytes of mice liver injected with e. coli at 40× magnification (cell cytoplasm appears brown as indicated by the arrow) in treatment group i (100 mg/kg bw). 366 biology, medicine, & natural product chemistry 14 (1), 2025: 363-368 figure 4. il-6 expression in hepatocytes of mice liver injected with e. coli at 40× magnification (cell cytoplasm appears brown as indicated by the arrow) in treatment group ii (300 mg/kg bw). figure 5. il-6 expression in hepatocytes of mice liver injected with e. coli at 40× magnification (cell cytoplasm appears brown as indicated by the arrow) in treatment group iii (500 mg/kg bw). p. foetida leaves are known for their antiinflammatory properties. the anti-inflammatory mechanism in sepsis in this study is likely due to various secondary metabolite compounds found in the leaves, including flavonoids, alkaloids, phenolic acids, and terpenoids (rosanti, 2016). these compounds are thought to help prevent il-6 expression, a proinflammatory cytokine involved in inflammation. during inflammation, caused by infection, injury, or chronic disease, immune cells release il-6 into the surrounding environment. il-6 then interacts with its receptors on various cell types, including immune cells, endothelial cells, and other tissues (tanaka et al., 2014). flavonoids in p. foetida leaves are believed to possess anti-inflammatory properties that inhibit the release of pro-inflammatory cytokines like il-6. flavonoids can block the nf-κb signaling pathway, a major pathway regulating pro-inflammatory cytokine release. by inhibiting nf-κb activation, flavonoids prevent the migration of proteins involved in cytokine gene transcription, including il-6 (ginwala et al., 2019). furthermore, flavonoids can block mapk signaling pathways such as erk, jnk, and p38, which are involved in pro-inflammatory cytokine gene expression. by inhibiting mapk activation, flavonoids can reduce il-6 production. additionally, flavonoids can inhibit cyclooxygenase (cox) and lipoxygenase (lox) enzymes, which are involved in the production of inflammatory mediators like prostaglandins and leukotrienes, both of which stimulate il-6 production (maleki et al., 2019). apart from flavonoids, phenolic acids in p. foetida leaves have strong antioxidant properties that can help protect cells from oxidative damage and oxidative stress associated with inflammation. phenolic acids neutralize free radicals involved in oxidative stress by donating electrons or hydrogen atoms, thereby preventing cellular damage. additionally, phenolic acids stimulate the production of endogenous antioxidant enzymes like superoxide dismutase (sod), catalase, and glutathione peroxidase, which eliminate free radicals and prevent oxidative damage (maleki et al., 2019). phenolic acids can also inhibit the activation of transcription factors like nf-κb and ap-1, which regulate pro-inflammatory cytokine gene expression, thereby reducing proinflammatory cytokine production and alleviating inflammation (lopez et al., 2022). alkaloids present in p. foetida leaves also inhibit inflammatory mediator production by blocking the release of arachidonic acid, which is essential for synthesizing prostaglandins and leukotrienes. alkaloids achieve this by inhibiting the enzyme phospholipase a2 (pla2), which releases arachidonic acid from cell membrane phospholipids (lopez et al., 2022). alkaloids can also inhibit cellular receptor expression and transcription factors activating pro-inflammatory immune cells, such as macrophages and t-cells. by inhibiting these processes, alkaloids reduce the production of pro-inflammatory cytokines like il-6. terpenoids in p. foetida leaves can also influence inflammatory responses by inhibiting proteolytic enzymes like elastase and collagenase, which are involved in extracellular matrix degradation and tissue damage during inflammation. terpenoids may interact directly with proteolytic enzymes, inhibiting their catalytic activity and potentially increasing the expression of protease inhibitors, thus preventing tissue damage (gallily et al., 2018). the findings of this study are consistent with previous research by savitri and kasimo (2022), which also demonstrated that the dose of 500 mg/kg bw of p. foetida leaf extract was the most effective in reducing il6 levels. however, there were differences between this study and theirs, particularly with the 100 mg/kg bw and savitri et al. – protective role of paederia foetida l. against hepatic inflammatory … 367 300 mg/kg bw doses. in their study, significant differences in il-6 reduction were observed between these doses, while in this study, no significant difference was found between the 100 mg/kg bw and 300 mg/kg bw doses. the anti-inflammatory effects in sepsis are believed to be attributed to compounds such as saponins, flavonoids, and essential oils in p. foetida leaves. these compounds may inhibit the synthesis of inflammatory mediators like prostaglandins and leukotrienes, which trigger il-6 production and contribute to the inflammatory response. further clinical studies are needed to confirm these effects and develop p. foetida as a phytopharmaceutical alternative therapy in preventing il-6 expression in sepsis. conclusions in conclusion, sepsis is a life-threatening condition characterized by a systemic immune response to infection, which can lead to multi-organ failure and death if not appropriately managed. the study demonstrated that p. foetida leaf extract, particularly at a dose of 500 mg/kg bw, shows significant anti-inflammatory effects in sepsis, as evidenced by the reduction in il-6 expression in the liver tissue of mice. this suggests that the leaf extract may be a potential therapeutic agent for managing inflammation associated with sepsis. while the lowest dose (100 mg/kg bw) showed minimal effectiveness, the higher doses (300 mg/kg and 500 mg/kg bw) exhibited a marked reduction in il-6 levels, indicating a dose-dependent response. the antiinflammatory properties of the extract are likely due to its bioactive compounds, including flavonoids, alkaloids, and terpenoids, which work by modulating inflammatory pathways such as nf-κb and mapk. although the results of this study are promising, further clinical research is required to confirm the potential of paederia foetida as a phytopharmaceutical alternative for sepsis management, especially in areas with limited access to conventional antibiotics. acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university, and the laboratory of pathological anatomy, brawijaya university, malang, for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors wrote the manuscript and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there are no funding. references 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(2021). sepsis. world health organization. https://www.who.int/news-room/fact-sheets/detail/sepsis biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 161-167 | doi: 10.14421/biomedich.2022.112.161-167 issn 2540-9328 (online) chemical compositions and antioxidant activity of volatile oils from morinda citrifolia and beta vulgaris leaves from nigeria adesegun olusimbo onanuga1, ejike onwudiegwu okpala2,* 1organic chemistry unit, department of chemistry, university of ibadan, ibadan, nigeria 2department of chemistry, faculty of science, federal university lokoja, kogi state-nigeria. corresponding author* ejike.okpala@fulokoja.edu.ng; okpalaejike@gmail.com manuscript received: 27 july, 2022. revision accepted: 15 august, 2022. published: 31 august, 2022. abstract morinda citrifolia l. and beta vulgaris l leaves are both ethnomedicinal use for the treatment of arthritis, indigestion and skin infections with no reports on their essential oils compositions. the colourless volatile oils with a percentage yield of 0.6 and 0.4 (w/w) for morinda citrifolia l. and beta vulgaris l respectively were obtained. forty-five compounds representing 94.31 % of the total percentage compositions were identified in the leaf essential oil of m. citrifolia with the most abundant compound as 14-beta-h-pregna(33.13%). forty-eight compounds representing 74.18% of the total oil composition were identified in the leaf oil of b. vulgaris with phytol (24.20%) as the dominant compound. the essential oils showed good free radical scavenging activity when compared to ascorbic acid used as control, with % inhibition varying from 88.74 ± 0.010 to 96.61 ± 0.004 as compared to 95.68 ± 0.010 to 97.31±0.003 of the ascorbic acid at (100 to 6.25 mg/ml) concentrations. the leaves essential oils of morinda citrifolia l. and beta vulgaris l contains chemical compounds that might be responsible for their antioxidant activity. this result validates the traditional usage of these plants in the treatment of arthritis, indigestion and skin infections. keywords: morinda citrifolia l.; beta vulgaris l.; antioxidant; free radical scavenging activity volatile oil. introduction the morinda citrifolia l (figure 1), also known as noni in nigeria, belongs to the rubiaceae family (arunachalam, 2018). it is a plant native to southeast asia and australia that has long been used medicinally. according to reports, m. citrifolia can treat a wide range of medical conditions, including arthritis, heartburn, headaches, wounds, and skin infections. additionally, there are some reports on the antitumor and anticancer activity of this plant and biological activities such as antihypoglycemia have been reported (algenstaedt et al., 2018). beta vulgaris l. (figure 1), popularly known as beetroot, is a member of the chenopodiaceae family (mello et al., 2008). it is a biennial herbaceous plant. it is eaten in nigeria as a vegetable. it has traditionally been believed to treat or prevent conditions like arthritis, colon, prostate, dyspepsia, and skin infections. beta vulgaris leaf consumption lowers the risk of diabetes mellitus, obesity, and cardiovascular illnesses. it is also recognized for its potential to treat cancer (kumar et al., 2016). it is known to have a wide range of biological activities, including properties that are antibacterial, antioxidant, anticancer, antiviral, and anti-diabetic (kavitha et al., 2020). consequently, in continuation of our search for biologically active compounds from plants with ethnomedicinal uses (okpala et al., 2021; okpala et al., 2022; onanuga and oloyede, 2021), we present chemical compositions of the volatile oils and antioxidant activities of leaves of beta vulgaris l. and morinda citrifolia l. the chemical compositions of the essential oils (eos) of the leaves of both plants have not been reported in the literature to the best of our knowledge. beta vulgaris leaves morinda citrifolia leaves figure 1. the image of the studied plants. material and methods plant material the samples of morinda citrifolia l. leaves were obtained fresh from akobo ibadan, oyo state, southhttps://doi.org/10.14421/biomedich.2022.112.161-167 162 biology, medicine, & natural product chemistry 11 (2), 2022: 161-167 west, nigeria (7o22' 39’’n; 3o 54’21’’e), on 10th april, 2021 while beta vulgaris l. leaves were purchased from bodija market ibadan, oyo state. the plants were identified by dr. s. k. odewo of forestry research institute of nigeria (frin) ibadan, oyo state. extraction of the essential oil the air dried and pulverized leaves of m. citrifolia (200 g) and b. vulgaris (200 g) were weighed and separately subjected to extraction using hydro-distillation method with clevenger type apparatus for four hours following british pharmacopoeia specifications with modifications (british pharmacpoeiae, 1980). the samples were added into a 2 l round-bottomed flask containing 1.0 l distilled water and heated to boiling. there was the evaporation of the essential oils together with water vapour and these were collected in a condenser. the upper phase that contained the eos was separated from the lower one and anhydrous sodium sulphate was used for drying the oils isolated. oils extracted were preserved in a sealed amber glass vial at 4oc until analyses. the percentage yields (w/w) were determined. gas chromatograph-mass spectrometry (gc-ms) of the oils gc-ms analysis of the oils was performed using gas chromatography 7890 coupled with mass spectrometer 5975 agilent technology. the chemical components were identified by matching their mass spectra with those recorded in the mass spectra library (w11n17 main). the stationary phase was the column of model agilent technologies hp-5 ms of length 30 m, the internal diameter of 0.320 mm and thickness of 0.25 µm while the mobile phase was helium gas. the oven temperature was at 80oc held for 2 mins at 12 degrees per minute to the final temperature of 240oc held for 6 mins. the ion source was set at 240oc and electron ionization at 70 ev. the scan ranges were from 50 to 550 amu and the interface temperature between gc and ms was 250oc. a sample of (1.0 µl) of diluted oils in hexane was manually injected into the gc-ms. antioxidant assay the antioxidant activity of the essential oils was evaluated using the dpph (2, 2-diphenyl-1picrylhydrazyl) free radical scavenging ability method. the concentrations (100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml and 6.25 µg/ml) of the essential oils were mixed with 100 µm methanoldpph solution (2.0 ml) prepared by dissolving 3.94 mg of dpph in 100 ml of methanol to give a purple solution. the mixture was shaken vigorously and left to incubate for 30 minutes in the dark at room temperature and the absorbance was then measured at 517 nm and recorded as a (sample) using a gs uv-12, uv-vis spectrophotometer. in its radical form, dpph absorbs, but upon reduction by antioxidant species, its absorption reduces. a blank experiment was carried out applying the same procedure without essential oil (dpph + methanol) and the absorbance was recorded as a (control). ascorbic acid was used as a standard antioxidant for comparison. the free radical scavenging activities of each essential oil were then calculated as percentage inhibition according to the following equation: % 𝐼 = acontrol − asample acontrol × 100 results the physical properties and percentage yields of the volatile oils obtained from m. citrifolia and b. vulgaris leaves are presented in table 1. forty five compounds representing 94.31 % of the total percentage compositions were identified in the leaf essential oil of m. citrifolia (figure 2). the most abundant compounds are 14-beta-h-pregna(33.13%), 1-hexacosane (11.11%), heneicosane (7.90%) and tricosane (7.17%). the constituents of the oil were mainly non-terpenoids: hydrocarbon (43.67%), steroids (33.13%), alcohols, esters and fatty acids (9.26%) while the terpenes present are monoterpene (0.40%), diterpene (6.05%) and triterpene (1.28%) table 2. forty eight compounds representing 74.18% of the total oil composition were identified in the leaf oil of b. vulgaris (figure 3). the dominant compounds are phytol (24.20 %), 1, 3-dimethylbenzene (14.84%) and neophytadiene (6.13%). the major class of compounds identified are diterpene alcohol (24.20) and aromatic compounds (18.70%) table 3. some similarities of the ethnomedicinal uses of the two plants could be related to the presence of compounds such as phytol, neophytadiene, mesitylene, 3-hexanol, decane, docosane, tetracosane, eicosane and heneicosane which were identified in the essential oils of both plants. the gc-ms chromatograms of the essential oils are given in figures 2 and 3. the antioxidant activity of the essential oils of the leaves of m. citrifolia and b. vulgaris are presented in tables 4 and 5. the dpph scavenging ability of the essential oils was compared with ascorbic acid a known standard antioxidant. onanuga & okpala – chemical compositions and antioxidant activity of … 163 figure 2. the gc-ms chromatogram of the leaf essential oil of m. citrifolia. figure 3. the gc-ms chromatogram of the leaf essential oil of b. vulgaris. 164 biology, medicine, & natural product chemistry 11 (2), 2022: 161-167 table 1. physical properties and yields of essential oils from m. citrifolia and b. vulgaris leaves. plant weight of leaf sample (g) weight of oil obtained (g) % (w/w) yield of the oil obtained physical properties morinda citrifolia l. 200 1.2 0.6 colourless, herbaceous beta vulgaris l. 200 0.8 0.4 colourless; leafy and aromatic odour table 2. chemical compositions of m. citrifolia essential oils. s/n rt (min) chemical constituents % composition class of compound 1 3.260 3-hexanol 0.14 alcohol 2 3.325 6-methyl-2-heptanol 0.25 alcohol 3 3.800 ethyl-cyclohexane 0.15 cycloalkane 4 4.124 2-hexenal 0.24 aldehyde 5 4.189 3-hexen-1-ol (z) 4.66 alcohol 6 4.357 2-hexen-1-ol (e) 0.77 alcohol 7 4.394 1-hexanol 0.74 alcohol 8 4.465 3-methylene-1-vinyl-1-cyclopentene 0.25 cycloalkane 9 4.902 2-heptanol 1.20 alcohol 10 5.858 2-hexanol 0.22 alcohol 11 6.204 1-octen-3-ol 0.35 alcohol 12 6.415 mesitylene 0.13 aromatic 13 6.534 decane 0.11 alkane 14 6.663 3-hexen-1-ol, acetate 0.57 ester 15 7.684 benzofuran 0.94 heterocyclic 16 8.133 3-carene 0.12 monoterpene 17 9.235 cinnamaldehyde (e) 0.30 aldehyde 18 9.872 2-methyl-2-nonen-4-one 0.10 ketone 19 10.050 citronellol 0.28 monoterpene 20 10.520 2-decanal (e) 0.15 aldehyde 21 13.502 4-(2,6,6-trimethyl-1-cyclohexen-1-yl 3-buten-2-one 0.19 ketone 22 17.408 neophytadiene 0.66 diterpene 23 17.473 3-octadecene (3e)0.18 alkene 24 17.840 3-eicosene (e) 0.24 alkene 25 18.013 nonadecane 0.13 alkane 26 18.623 n-hexadecanoic acid 0.34 fatty acid 27 18.985 eicosane 1.05 alkane 28 19.093 14-beta-h-pregna 33.13 steroid 29 19.785 1-nonadecene 0.68 alkene 30 19.920 heneicosane 0.85 alkane 31 20.66 phytol 5.39 diterpene 32 20.207 1-heneicosene 0.36 alkene 33 20.325 1-eicosene 1.20 alkene 34 20.482 1-tetracosene 0.62 alkene 35 20.817 docosane 1.69 alkane 36 21.670 tricosane 7.17 alkane 37 21.750 1-hexacosene 11.11 alkene 38 22.491 tetracosane 3.16 alkane 39 23.291 heneicosane 7.90 alkane 40 24.053 hexacosane 3.65 alkene 41 25.317 9-hexacosene 0.53 alkene 42 25.500 octacosane 0.98 alkane 43 25.733 supraene 1.28 triterpene 44 26.186 nonacosane 0.44 alkane 45 27.499 3-ethyl-2,6,10-trimethyl undecane 0.24 alkane total percentage composition 94.31 onanuga & okpala – chemical compositions and antioxidant activity of … 165 table 3. chemical compositions of b. vulgaris essential oils. s/n rt (min) chemical constituents % composition class of compounds 1 3.125 3-hexanone 0.31 ketone 2 3.152 1-ethyl-3-methyl cyclopentane 0.41 cycloalkane 3 3.179 2-hexanone 0.89 ketone 4 3.260 3-hexanol 1.22 alcohol 5 3.250 2,5-dimethyl-1-hepten-4-ol 1.20 unsaturated alcohol 6 3.384 1, 3-dimethyl-transcyclohexane 0.39 cycloalkane 7 3.730 1,2-dimethylcyclohexane 0.33 cycloalkane 8 3.800 ethyl-cyclohexane 0.82 cycloalkane 9 4.081 1,2,4-trimethyl cyclohexane 0.26 cycloalkane 10 4.124 2-hexanal (e)0.60 aldehyde 11 4.243 ethylbenzene 2.57 aromatic 12 4.308 nonane 0.31 alkane 13 4.470 1,3-dimethylbenzene 14.84 aromatic 14 4.659 cis-1-ethyl-3-methyl-cyclohexane 0.13 cycloalkane 15 4.886 nonane 0.18 alkane 16 5.858 2-methyl-3-propyl-trans oxirane 1.30 cyclo ether 17 6.415 mesitylene 0.56 aromatic 18 6.534 decane 0.61 alkane 19 6.885 1-ethyl-3-methyl benzene 0.12 aromatic 20 7.360 1-methyl-3-propylbenzene 0.13 aromatic 21 7.479 1-ethyl-3,5-dimethyl benzene 0.14 aromatic 22 7.895 1,2,4,5-tetramethyl benzene 0.13 aromatic 23 8.203 nonanal 0.12 aldehyde 24 8.754 3-methyl-6-(1-methyl ethyl ) cyclohexene 0.68 cycloalkane 25 9.251 4-acetyl-1-methyl cyclohexene 0.24 cycloalkane 26 9.872 1-cyclopropyl-2-propanone 0.72 cycloketone 27 9.964 1-carboxaldehde, 2,6,6-trimethyl-1cyclohexene 0.33 cycloalkane 28 11.039 tridecane 0.17 alkane 29 12.363 tetradecane 0.15 alkane 30 13.059 6,10-dimethyl, 5,9-undecadien-2-one 0.18 unsaturated ketone 31 13.173 1-[(e)-3-methylbut-1-enyl]cyclohexene 0.32 cycloalkane 32 13.281 1-(1,1-dimethylethyl)4-ethyl benzene 0.21 aromatic 33 13.502 4-(2,6,6trimethyl-1-cyclohexen-1-yl) 3-buten-2-one 1.83 cycloketone 34 13.967 copaene 0.23 alkene 35 14.707 2-methyl-4-(2,6,6, trimethyl-1-cyclohexen-1-yl) 2 butenal 0.16 cycloaldehyde 36 15.922 heptadecane 0.27 alkane 37 17.354 3,7,11,15 tetramethylhexadec-2-ene 1.78 alkene 38 17.408 neophytadiene 6.13 diterpene 39 17.948 1-nonadecene 0.18 alkene 40 19.780 e-15-heptadecanal 0.34 aldehyde 41 19.839 1-octadecene 0.40 alkene 42 19.920 heneicosane 0.29 alkene 43 20.066 phytol 24.20 diterpene 44 20.325 diallylacetal, palmitaldehyde 3.18 aldehyde 45 20.811 docosane 0.87 alkane 46 21.665 eicosane 1.98 alkane 47 22.491 tetracosane 0.68 alkane 48 23.285 3-pentacosene (e) 1.09 alkene total percentage composition 74.18 166 biology, medicine, & natural product chemistry 11 (2), 2022: 161-167 table 4. absorbance values at 517 nm of dpph method of antioxidant assay. conc. (mg/ml) m. citrifolia b. vulgaris asc. acid 100 0.251 ± 0.003 0.102 ± 0.004 0.081 ± 0.003 50 0.270 ± 0.002 0.106 ± 0.007 0.097 ± 0.004 25 0.280 ± 0.013 0.116 ± 0.007 0.105 ± 0.009 12.5 0.312 ± 0.005 0.123 ± 0.005 0.127 ± 0.005 6.25 0.339 ± 0.010 0.133 ± 0.005 0.130 ± 0.010 absorbance values in mean ± standard error; asc.acid = ascorbic acid at 517nm; ± standard deviation for measurement, absorbance of control =3.010±0.005 table 5. percentage inhibition calculated from dpph method of antioxidant assay. conc. (mg/ml) m. citrifolia b. vulgaris asc. acid 100 91.66 96.61 97.31 50 91.02 96.48 96.78 25 90.69 96.15 96.51 12.5 89.63 96.91 95.78 6.25 88.74 95.58 95.68 discussion methyl hexanoate, methyl octanoate, ethyl octanoate and methyl 4 e-decanoate have been reported in the volatile oil of m. citrifolia fruit, they are found to contain flavonoids as the major phytochemicals (pino et al., 2010). 14-b-h-pregna, a steroid considered to be a sex pheromone specific to males, and also a defensive chemical with diabetic retinopathy prevention and treatment effects. the presence of 14-b-h-pregna has been reported in the essential oils from different parts of some plants, including scutellaria plants, urginea indica kunth, allium rotundum, gundelia tournefortii l (farhang et al., 2016). citrus limon (akhila et al., 2015). dehpour et al.2012 reported that 14-b-h-pregna was the major compound in the essential oil of lower allium rotundum which displayed antibacterial activity. neophytadiene is a good analgesic, antimicrobial, antipyretic, antioxidant and anti-inflammatory compound (venkata et al., 2012). phytol, known to exhibit antioxidant and antinociceptive effects is a precursor of synthetic vitamins e and k and is cytotoxic against breast cancer cell lines (mcf7) (casuga et al., 2016; sermakkani and thangapandian, 2012). the measured absorbance values and calculated percentage inhibition show that the antioxidant activity of the two essential oils and standard (ascorbic acid) is concentration dependant. from the results, the % inhibition of the essential oils of both plants exhibited good scavenging ability on dpph radical which were comparable to ascorbic acid at all concentrations (1006.25 mg/ml) investigated. conclusions analysis of the essential oils of m. citrifolia and b. vulgaris leaves showed that they contained different major constituents. the major constituent of m. citrifolia leaves essential oil were steroids: 14-beta-h-pregna (33.13%) and 1-hexacosane (11.11%), while the dominant compounds identified in b. vulgaris were phytol (24.20 %) and 1,3-dimethylbenzene (14.84%. these major constituents of both oils have been reported to possess similar properties such as antibacterial, antioxidant and antinociceptive. most of the chemical compounds identified from the essential oils of m. citrifolia and b. vulgaris were biologically active compounds and the essential oils exhibited good scavenging ability at all concentrations investigated. the plants’ parts could be a source of drug development for oxidative diseases. competing interests: a. o. onanuga and e. o. okpala declare that they have no competing interests. acknowledgements: the authors are grateful to the university of ibadan, nigeria for providing the laboratory space and facilities for the extraction and antioxidant analysis of the essential oils. references akhila s, bindu a, bindu k. (2015). phytochemical and pharmacological evaluation of citrus limon pell. world j. pharm. pharma. sci. 4(3):1128-135. algenstaedt, p, stumpenhagen, a, westendorf, j. (2018). the effect of morinda citrifolia l. fruit juice on the blood sugar level and other serum parameters in patients with diabetes type 2. evi. compl. alt. med. 3565427:10 arunachalam, v. (2018). morinda citrifolia l. (rubiaceae): a multipurpose tree for coastal ecosystems and its variability in konkan region of india. genetic resources and crop evolution, 65(6):1751-1765. british pharmacoepia. (1980). london: h. m., stationary office, pa. pp. 109. casuga, f., a. castillo, a., corpuz, m. (2016). gc–ms analysis of bioactive compounds present in different extracts of an endemic plant broussonetia luzonica (blanco) (moraceae) leaves asian pac. j. trop. biomed. 6, 957-961. dehpour a.a., yousefian m., kelarijani s.j., koshmoo, m., mirzanegad s., mahdavi, v. (2012). antibacterial activity and composition of essential oils of flower allium rotundum. adv. env. bio. 6(3):1020-1025. deng s, west bj, palu ak, jensen cj. (2012). phytochemical, antioxidant and toxico­ logical investigation of morinda citrifolia l. blossoms, anal chem. 2012:1-5. farhang, hr, vahabi, mr, allafchian, ar. (2016). chemical compositions of the essential oil of gundelia tournefortii l.(asteraceae) from central zagros, iran. j. herbal drugs. 6(4):227-233. guldiken b, toydemir g, memis kn, okur s, boyacioglu d, et al. (2016). home-processed red beetroot (beta vulgaris l.) https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ onanuga & okpala – chemical compositions and antioxidant activity of … 167 products: changes in antioxidant properties and bioaccessibility. int. j. mol. sci. 17: 858. okpala, eo, onocha, pa, ali, ms, zikr-ur-rehmen s, lateef, m. (2021). zenkeramide: a new iso-benzofuranone propanamide and urease inhibitory constituents of celtis zenkeri engl stem bark (ulmaceae) nat. prod.res.1-6. doi:10.1080/14786419.2021.19546443. okpala, e., onocha, p., ali, m. (2022). antioxidant activity of phytol dominated stem bark and leaf essential oils of celtis zenkeri engl., trends in phytochemical research, 6(2), 137144. doi:10.30495/tpr.2022.1952985.1246 onanuga ao, oloyede gk. (2021). two new biologically active steroids from costus lucanusianus (costaceae) steroids (175) 108913. pino ja, eliosbel m, clara eq, déborah c. (2010). volatile compounds in noni (morinda citrifolia l.) at two ripening stages. ciênc. tecnol. aliment. 30(1):183­87. sermakkani m., thangapandian, v. (2012). gc-ms analysis of cassia italica leaf methanol extract. asian j. pharm. clin. res., 5, 90-94. venkata, rb, samuel la, pardha sm. (2012). antibacterial, antioxidant activity and gc-ms analysis of eupatorium odoratum. asian j. pharma. clin. res. 5(2) 99-106. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4926392/ this page intentionally left blank biology, medicine, & natural product chemistry volume 4 – number 1 – 2015 issn 2089-6514 contents a simple and practical method for rat epididymal sperm count (rattus norvegicus) muhammad ja'far luthfi 1 3 the phytoestrogenic potential of yam bean (pachyrhizus erosus) on ovarian and uterine tissue structure of premenopausal mice cicilia novi primiani 5 9 the effect of intensive keramba on the presence of parasite organisms in rivers of lingsar area supriadi, maratun janah 11 15 krokot extract (portulaca oleracea. l) as natural light-harvesting pigments for dye-sensitized solar cells (dsscs): influence of dye acidity cici nurfaizah, didik krisdiyanto, khamidinal, sudarlin 17 24 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 595-600 | doi: 10.14421/biomedich.2024.132.595-600 issn 2540-9328 (online) chemical constituents and evaluation of bioactivity of vernonia hymenolepis a. rich root extracts alemu gelata1, mudin jemal1,*, soressa gershe2, negera abdissa3 1department of chemistry; 2department of biology, college of natural sciences, jimma university, p.o. box 378, jimma, ethiopia. 3department of chemistry, college of natural and computational sciences, wollega university, p.o. box 395, nekemte, ethiopia. corresponding author* mudinjemal55@gmail.com abstract the medicinal herb vernonia hymenolepis has been used to cure a wide range of ailments such as cancer, gonorrhea, stomach ache, malaria, hepatitis, pneumonia, toothache, diarrhea, amoebiasis, typhoid, hypertension, and constipation by diverse societies in ethiopia, kenya, and tanzania. however, its biological and phytochemical data is extremely insufficient. thus, the aim of this research work was to study the chemical constituents and antimicrobial activity of the root part of this plant. the roots of plant were dried by air under shade and then successively extracted with chloroform and methanol. the chloroform root extract was subjected to silica gel column chromatographic separation which gave two compounds, namely; betulinic acid (1) and 2-hydroxy-3-(4-hydroxyphenyl)-2-propenoic acid (2). the structures of the isolated compounds were elucidated using 1h and 13c nmr spectroscopic techniques and by comparing with literature reports. the crude extracts were assayed in vitro employing disc diffusion technique against four bacterial strains (bacillus subtilis atcc11778, staphylococcus aureus atcc25923, escherichia coli atcc25922, and pseudomonas aeruginosa atcc27853) and one fungal strain (candida albicans atcc 10231). significant antibacterial activity was demonstrated by the crude extracts; with the chloroform extract exhibiting greater activity against escherichia coli (18 mm) when compared to gentamicin (25 mm). the methanol extract revealed the highest activity (13 mm) against candida albicans with the reference drug, clotrimazole showing an inhibition zone of 12 mm. the antimicrobial activity exhibited by root extracts of v. hymenolepis corroborates its traditional use against bacterial diseases. keywords: chemical constituents; antimicrobial activity; vernonia hymenolepis; medicinal plant. introduction since ancient times, plants have served as the primary source of traditional medicinal remedies (r. u. and a. s. alqahtani, 2022). according to the world health organization, 80% of the world’s population uses different plant fractions and their active ingredients as traditional remedies (a. s. alqahtani et al., 2022; mrabti et al., 2022; shahat et al., 2018). research on medicinal plants is more focused than ever since they can benefit humanity in many ways. the potential these plants as medicines may stem from their bioactive phytochemical components, such as terpenoids, flavonoids, lignans, alkaloids, anthraquinone, etc., which are responsible for their physiological action (fagbohun et al., 2012; parekh et al., 2006). higher plants are a possible source of novel antibiotic drugs, according to the results of antimicrobial activity screening of plant products. in recent years, the prevalence of various types of resistances in human pathogenic microorganisms has increased, primarily as a result of the improper use of commercial antimicrobial medications that are frequently prescribed to treat infectious disorders. because of this, scientists are now compelled to look for novel antimicrobial chemicals from a variety of sources, including medicinal plants (khan et al., 2013). vernonia hymenolepis is an ethnomedicinally important plant that is used by many herbalists and communities in tanzania for the treatment of several diseases such as diarrhea, stomachache, faint fever, diarrhea, spleen enlargement, amoebiasis, malaria, typhoid and constipation (abebe, 2016; onzago et al., 2014). the plant is utilized for treating pneumonia, hypertension, and diarrhea in infants by using various parts of it. moreover, herbalists in kenya widely use it for treating toothaches (onzago et al., 2014). medicinal plants have been utilized by ethiopians and have become crucial to traditional customs within the nation (mesfin et al., 2009). v. hymenolepis ‘sooyyoma’ (afan oromo) is commonly grown in diverse areas of ethiopia. it is known traditional medicinal plant and its leaf part is utilized to treat cancer (abebe, 2016) and gonorrhea (bizuayehu & garedew, 2018). similarly, the manuscript received: 22 september, 2024. revision accepted: 18 december, 2024. published: 27 december, 2024. https://doi.org/10.14421/biomedich.2024.132.595-600 596 biology, medicine, & natural product chemistry 13 (2), 2024: 595-600 crumpled leaves are boiled with water and taken orally for the management of hepatitis (yineger et al., 2019). there is no sufficient information related to the phytochemical investigation and antimicrobial activity of the plant. in addition to this, there are no further studies on the phytochemicals present in the root of this plant. to provide scientific support for the traditional use of v. hymenolepis in ethiopia for the treatment of various illnesses, in this study, our objective was to study the chemical compounds and antimicrobial activity of the root part of the plant. materials and methods chemicals and instrumentation solvents of analytical grade (chloroform, methanol, petroleum ether, and ethyl acetate) were used for successive extraction and column elution. standard antibiotic drugs (gentamicin and clotrimazole), dmso, mueller-hinton agar, potato dextrose agar and nutrient agar were utilized as a culture media during antimicrobial tests. a rotary evaporator was used to concentrate the extracts. uv light (254 nm and 365) was used for the detection of spots on tlc and oxalic acid impregnated silica gel (60-120 mesh) was used for column chromatography. bruker avance 400 mhz spectrometer was employed for nuclear magnetic resonance (nmr) analysis and tetramethylsilane was used as an internal standard and cdcl3 and acetone-d6 were used as solvents. plant sample collection the roots of v. hymenolepis (figure 1b) were collected from jimma arjo district, east wollega zone, ethiopia. the plant was identified by a botanist at biology department, jimma university. the collected root parts were cleaned, and shade dried at the organic chemistry laboratory, jimma university. the dried roots were crushed to a suitable size using an electric grinder to enhance the extraction process. (a) (b) figure 1. picture of leaf (a) and root (b) parts of v. hymenolepis from jimma arjo, ethiopia (photo taken by alemu geleta). extraction of crude extracts using the cold maceration method, approximately 600 g of ground root material was successively extracted in 3 l of chloroform and methanol twice for 24 hours each. the crude extracts were filtered with whatmann no.1 filter paper and concentrated using a rotary evaporator at 40°c to yield 10 g and 16 g of chloroform and methanol extracts, respectively. isolation of compounds the chloroform extract (7 g) was meshed with an equal amount of silica gel and subjected to silica gel column chromatography eluting with an increasing gradient of petroleum ether: ethyl acetate: methanol to afford 28 fractions of 25 ml each. the fractions (f1-f8) were colorless and likely contained fat. fractions 9-12 (petroleum ether/ethyl acetate, 3:2) formed a white precipitate and were subsequently washed with petroleum ether to obtain compound 1 (8 mg). fractions (1924) were combined on the basis of their tlc profile and subjected to further purification via column chromatography, with gradient elution from chloroform through ethyl acetate to methanol, resulting in compound 2 (13 mg). antimicrobial activity antibacterial test using the disc diffusion method, the in vitro antibacterial properties of the v. hymenolepis root extracts were assessed against four bacterial strains: two grams negative strains (escherichia coli atcc25922 and pseudomonas aeruginosa atcc27853) and two grams positive strains (staphylococcus aureus atcc25923 and bacillus subtilis atcc11778). nutrient agar slants, gelata et al. – chemical constituents and bioactivity of v. hymenolepis 597 which were kept at 40°c, were used to maintain stock cultures of bacteria. to obtain final stock concentrations of 200 mg/ml in dmso, 200 mg of the test samples were dissolved to prepare the test solutions. newly grown liquid cultures of the test pathogen solution with turbidity comparable to 0.5 mcfarland were seeded on the müeller-hinton agar media plates. for the bacterial cells and agar to mix equally, the plates were gently shaken. after being individually soaked in the aforementioned stock solutions (samples and standards), sterile paper discs with a diameter of 6 mm were placed evenly spaced over the seeded plates. control experiments were performed under similar conditions by using gentamicin as a positive control and dmso as a negative control. after that, the plates were turned over and incubated for a full day at 37 °c. following the incubation period, a clearance zone surrounding the disks on the plates was observed. every experiment was run three times. to evaluate the antibacterial activity of each test sample, the mean inhibition zone was calculated. comparisons were made between the inhibitory zones generated by plant extracts and those generated by commercial conventional antibiotics (ogundare et al., 2006). antifungal test to test the antifungal activity of extracts of the plant against candida albicans, the disc diffusion technique was used. potato dextrose agar (pda) medium was added to petri dishes (diameter 90 mm) and activated for 24 hours. then 0.3 ml of candida albicans inoculum were seeded on activated medium. a sterile filter discs (diameter 6 mm, whatman paper no.1) were impregnated with test extracts (200 mg/ml) which were dissolved in dimethyl sulfoxide (dmso) and placed on the surface of plate containing the microorganism. clotrimazole was used as standard antifungal drug (positive control) while the solvent dmso was used as a negative control. the experiments were repeated three times. the zones of growth inhibition around the discs were measured using transparent ruler after 72 hours at 25°c for (mathela & joshi, 2008; ogundare et al., 2006; veljic et al., 2008). results and discussion yield of extracts table 1. physical properties of the crude extracts. solvents color of extracts mass of extracts (g) yield (%) chloroform dark red 10 1.67% methanol black 16 2.71% the extraction yield is a measure that quantifies how well a solvent extracts specific constituents from the source material. the percentage yield of the crude extract in the respective solvent was recorded. the methanol yield was relatively high (table 1). this finding is consistent with several reports from the literature showing that the yield of extracts is lower for nonpolar solvents (sintayehu & adane, 2020). characterization of isolated compounds compound 1 was obtained as a white powder with an rf value of 0.62 in ethyl acetate: petroleum ether (3:7). the 1h nmr spectrum (400 mhz, cdcl3) (table 2) indicated signals at δh 0. 98 (3h, s, h-23), 0.82 (3h, s, h-24), 0.73 (3h, s, h-25), 0. 93 (3h, s, h-26), 0.96 (3h, s, h-27) and 1.69 (3h, s, h-30) for six methyl groups; two geminal olefinic protons at δh 4.73 (1h, brs, h-29a) and 4.60 (1h, brs, h-29b), and five methine protons at δh 3.25 (1h, m, h-18) and 3.15 (1h, m, h-19), 2.00 (1h, m, h-5), 2.00 (1h, m, h-9), 2.12 (1h, m, h13), and one oxymethine proton at δh 3.20 (1h, m, h-3). the 13c nmr spectrum (table 2) showed signals for 29 carbon atoms consistent with a downward shifted carbon signals at δc 175.5, indicative of carboxylic acid carbonyl carbon, which is assignable to c-28 and the other six quaternary carbons at δc 37.7 (c-4), 42.2 (c-8), 35.9 (c-10), 42.2 (c-14), 51.6 (c-17) and 145.7 (c-22); five methine carbons at δc 51.5 (c-5), 51.5 (c-9), 37.7 (c-13), 44.5 (c-18), 45.8 (c-19), one oxymethine carbon at δc 74.3 (c-3); and eleven methylene carbons at δc 37.7 (c-1), 25.8 (c-2), 20.8 (c-6), 34.1 (c-7), 23.3 (c11), 25.8 (c-12), 32.5 (c-15), 33.6 (c-16), 29.6 (c-20), 34.0 (c-21) and 104.9 (c-21); 6 methyl carbons at δc 29.6 (c-23), 16.1 (c-24), 16.1 (c-25), 16.1 (c-26), 13.6 (c-27) and 24.9 (c-30). the carbon signals at δc 104.9 (c-21) and 145.7 (c-22) indicates the presence of olefinic carbons, as in lupeol. as a result, using the spectroscopic data above and comparing them to relevant literature (bisoli et al., 2008; egbubine et al., 2020; shin et al., 2009), the structure of compound 1 was found to be betulinic acid or 3β-hydroxy-lup-20(29)-en-28-oic acid (figure 2). this compound was previously isolated from feretia canthioides stem bark (egbubine et al., 2020) and vernonia guineensis root (collins et al., 2020). betulinic acid is a naturally occurring pentacyclic lupinetype triterpenoid that has several biological activities such as inhibition of human immunodeficiency virus (hiv), anthelmintic, antibacterial, anti-inflammatory, antimalarial, anti-hsv-1 and anticancer activities (moghaddam et al., 2012). 598 biology, medicine, & natural product chemistry 13 (2), 2024: 595-600 table 2. 1h and 13c nmr data (400 mhz, cdcl3) of compound 1. position 1 h nmr (m) 13c nmr position 1 h nmr (m) 13c nmr 1 1.25 (m) 37.7 16 1.70 (m) 33.6 2 1.27 (m) 25.8 17 51.5 3 3.20 (m) 74.3 18 3.25 (m) 44.5 4 37.7 19 3.15 (m) 45.8 5 2.00 (m) 51.5 20 29.6 6 1.37 (m) 20.8 21 1.85(m) 34.0 7 1.55 (m) 34.1 22 145.7 8 42.2 23 0. 98 (s) 29.6 9 2.00 (m) 51.5 24 0.82 (s) 16.1 10 35.9 25 0.73 (s) 16.1 11 1.60 (m) 23.3 26 0. 93 (s) 16.1 12 1.60 (m) 25.8 27 0.96 (s) 13.6 13 2.12 (m) 37.7 28 175.5 14 42.2 29a 29b 4.73 (brs) 4.60 (brs) 104.9 15 32.5 30 1.69 (s) 20.8 key: brs = broad singlet, m = multiplet and s = singlet compound 2 was isolated as a light yellow powder with an rf value of 0.45 (30% methanol in chloroform). the 1 h nmr spectrum (400 mhz, acetone-d6) (table 3) revealed only two sets of aromatic protons at δh 7.12 and 6.85, which were integrated for two protons each and assigned to (h-5 and h-9) and (h-6 and h-8), respectively. it also indicated a down field shifted olefinic proton at δh 7.53 (1h, s, h-3), due to its periposition to the carbonyl group. oh o ho 2 oh ho 1 3 5 7 9 10 25 12 13 14 18 20 23 24 28 26 27 29 22 1 o oh 30 11 15 17 19 21 1 2 3 4 6 8 figure 2. chemical structures of compound 1 and 2. the 13c nmr spectrum (table 3) indicated the presence of seven signals for nine carbon atoms. the signal at δc 167.9 assignable to the carbonyl carbon (c1), the signals at δc 155.5, and 129.9 correspond to c-4, and c-7, respectively and the intensified carbon signals (due to the symmetric effect) at 131.9 and 113.5 conforming two set of meta carbons (c-5 and c-9) and (c-6 and c-8), respectively. the carbon signals at δc 144.6, 118.1 and 128.2 were assigned to c-2, c-3 and c4, respectively. therefore, on the basis of the above spectroscopic data and a comparison of the data with the related literature (ayoola et al., 2017; somaia a. almadhagy, nada m. mostafa, fadia s. youssef & awad, omayma a. eldahshan, 2019), the structure of compound 2 was identified as 2-hydroxy-3-(4hydroxyphenyl)-2-propenoic acid (figure 2). this is the first report on the isolation of betulinic acid (1) and 2hydroxy-3-(4-hydroxyphenyl) -2-propenoic acid (2) from v. hymenolepis. gelata et al. – chemical constituents and bioactivity of v. hymenolepis 599 table 3. 1h and 13c nmr (400 mhz, acetone-d6) spectral data of compound 2. position 1 h nmr (m) 13c nmr dept-135 1 _ 167.9 c 2 _ 144.6 c 3 7.53 (s) 118.1 ch 4 _ 128.2 c 5 7.12 (dd) 131.9 ch 6 6.85 (dd) 113.5 ch 7 _ 153.5 c 8 6.85 (dd) 113.5 ch 9 7.12 (dd) 131.9 ch antimicrobial activities using the disk diffusion method, the two crude extracts were evaluated for their in vitro antimicrobial activity against four bacterial strains and one fungal strain. the results were shown in table 4 and indicated varying degrees of response. table 4. zones of inhibition (mm) for bacterial and fungal growth. extracts and standards growth inhibition zone (mm) bacterial strains fungal strain b. subtilis s. aureus e. coli p. aeruginosa c. albicans chloroform extract 14 17 18 11 ni methanol extract 13 12 14 13 13 gentamicin 17 23 25 12 nt clotrimazole nt nt nt nt 12 dmso ni ni ni ni ni key: ni = no inhibition, nt = not tested and dmso = dimethylsulfoxide. the extracts exhibited significant antibacterial activity against all strains of both gram negative and gram positive bacteria, with inhibition zones ranging from 11-18 mm. the chloroform extract showed better antibacterial activities against bacillus subtilis, staphylococcus aureus, and escherichia coli with inhibition zones of 14 mm, 17 mm and 18 mm, respectively, whereas the methanol extract displayed moderate activities. both the chloroform and methanol extracts indicated substantial antibacterial activities against gram negative bacterium, pseudomonas aeruginosa with zones of inhibition of 11 mm and 13 mm, respectively, which is comparable to that of the reference drug, gentamicin (12 mm). the observed activity may be due to the existence of phytochemical constituents such as flavonoids, triterpenes, and phenolics present in this plant (mengome et al., 2010; ndam et al., 2014). the methanol extract displayed the highest activity (13 mm) against candida albicans, but the chloroform extract did not show any remarkable antifungal activity. these in vitro antimicrobial activities are in good agreement with those reported in a previous study conducted in kenya (onzago et al., 2014). conclusion the chloroform root extract was subjected to silica gel column chromatography which yielded two compounds (betulinic acid and 2-hydroxy-3-(4-hydroxyphenyl)-2propenoic acid). the extracts exhibited significant antibacterial activity against all gram positive and gram negative bacteria with inhibition zones ranging from 11 18 mm. the antimicrobial activity results agree with the outcomes of a previous study. hence, these results support traditional use v.hymenolepis of in ethiopia as well as its potential as the most abundant source of bioactive chemicals with antibacterial properties. consistent with the previous research work, the extensive traditional use of this plant may be attributed to its terpenes and phenolic phytochemicals. data availability: all the data used to support the findings of this study are available in the manuscript. disclosure: this manuscript was extracted from msc thesis of the jimma university institutional repository. conflicts of interest: the authors declare that there are no conflicts of interest. acknowledgement: the authors acknowledge jimma university for providing material support to carry out the study successfully. 600 biology, medicine, & natural product chemistry 13 (2), 2024: 595-600 references abebe, w. 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(2019). plants used in traditional management of human ailments at bale mountains national park , southeastern ethiopia. journal of medicinal plants research, 8(11), 1–22. biology, medicine, & natural product chemistry issn: 2089-6514 volume 5, number 1, 2016 | pages: 9-14 | doi: 10.14421/biomedich.2016.51.9-14 local stability analysis of a mathematical model of the interaction of two populations of differential equations (host-parasitoid) dewi anggreini lecturer of study program mathematics education, stkip pgri tulungagung jl. mayor sujadi timur no. 7 tulungagung, 66221 tel/fax: (0355) 321426 author correspondency: anggreini_004@yahoo.com abstract mathematical model has many benefits in life, especially the development of science and application to other fields. the mathematical model seeks to represent real-life problems formulated mathematically to get the right solution. this research is the application of mathematical models in the field of biology that examines the interaction of the two populations that host populations and parasitoid populations. this study differs from previous studies that examine the interaction of two more species that prey and predators where predators kill prey quickly. in this study the parasitoid population slowly killing the host population by living aboard and take food from the host population it occupies. in this study of differential equations are used to construct a mathematical model was particularly focused on the stability of the local mathematical model of interaction of two differential equations that host and parasitoid populations. stability discussed in this study are stable equilibrium points are obtained from the characteristic equation systems of differential equations host and parasitoid interactions. type the stability of the equilibrium point is determined on the eigenvalues of the jacobian matrix. analysis of stability is obtained by determining the eigenvalues of the jacobian matrix around equilibrium points. having obtained the stable equilibrium points are then given in the form of charts and portraits simulation phase to determine the behavior of the system in the future. keywords: mathematical model, the interaction of two populations model, stability introduction mathematics has many benefits in the areas of science and everyday life, especially in the field of biology. according widowati and sutimin (2007:1). the mathematical model is a mathematical field that seeks to represent and describe physical systems or a problem in the real world in a mathematical statement, in order to obtain an understanding of these real world problems to be more precise. representation of this process is known as a mathematical model. construction analysis and use of mathematical models is seen as one of the most important applications of mathematics. have been many studies that discuss the interaction of two species or two populations such as predator-prey or predator – prey. unlike the predator prey models that describe the interaction where a predator as prey and prey as prey, predators will kill its prey directly in the process of natural selection. while in this study established the relationship between host and parasitoid where the host does not kill quickly, but slowly with the way of life of a ride or take in nutrients from the host they occupy. this research is exciting to do because there are still few studies that discuss mathematical models of host and parasitoid interactions in particular discuss the stability of the system. in this study, given how differential equations can be used to construct a mathematical model was particularly focused on the stability of differential equations mathematical model of the interaction of the two populations that host and parasitoid. stability discussed in this study are stable equilibrium points are obtained from the characteristic equation systems of differential equations host and parasitoid interactions. having obtained the stable equilibrium points are then given a simulation to determine the type of stable equilibrium points using matlab program. type the stability of the equilibrium point is determined on the eigenvalues of the jacobian matrix. some types of stability based on the roots of the characteristic equation is as follows. if the value of the roots of the characteristic equation real, distinct, and equally a sign of the system asymptotically stable equilibrium point. if the value of the roots of the characteristic equation complex conjugate purely imaginary but not the asymptotically stable equilibrium point. if the value of the roots of the characteristic equation real, distinct, and not as a sign of the equilibrium point of the system is unstable. if the value of the roots of the characteristic equation purely imaginary ekuilibrim point system is stable but not asymptotically stable. based on the interaction of host and parasitoid, a mathematical model in the form of differential equations and systems of differential equations is a suitable way to describe the interaction between host and parasitoid. parasitoid is an organism that spends most of his life history to rely on a single host organism which eventually kills (and often took food) in the process. parasitoids laid eggs in the body of the target animal after hatching larvae then sucking the bodily fluids of the target animal to death when the host-parasitoid find a place to live, it must be appropriate host physiology 10 biology, medicine, & natural product chemistry 5 (1), 2016: 9-14 and nutrition for the successful development of the parasitoid. the goal in conducting this study was to examine how the stability of differential equations of mathematical models the interaction of two populations (host and parasitoids) and examines how the stability of mathematical models simulating the interaction of the two populations of differential equations (host and parasitoid) with matlab program. this research is the development of a mathematical model, especially the model of differential equations formed from the interaction between host and parasitoid. applications using matlab help to find graph and phase portraits of systems of differential equations stability so they can know the behavior of the system in the future. as study materials for researchers who like or are interested in the development of mathematical models especially differential equations. the interaction of two populations models (predatorprey) the basic model predator-prey or predator prey interaction of the two populations is given by the equation lotka-volterra (1926) *dh mh a hp dt   *dp qp ra hp dt    in this equation ℎ is the prey populations and is a predator population, with t is time. the equation states the prey population changes over time and the equation stated predator population changes over time. so the equation: * b sn a ht (1) with captured prey populations of prey each unit time.   * 1 a hf h bh   (2) with b states the time to deal with a host, f(h) stating the number of hosts attacked parasitoid per unit time or the number of hosts diparasit, a stated figures hosted by parasitoid attack . equation (2) is holling type ii functional response to the host parasitoid interactions. dynamic systems in general, the dynamic system is defined as a real problem that is modeled mathematically using differential equations in the equation which contains parameters that are interconnected , as well as changes in the parameters of the equation will lead to changes in the stability of the equilibrium point . the equilibrium point is one of the key concepts in a dynamic system. more general system can be expressed in the following form.̇ 1 = 1( 1, 2,… , , )2̇ = 2( 1, 2,… , , )⋮̇ = ( 1, 2,… , , ) (3) with = ( , , … , , ), = 1,2,… , n is a general function of , = 1,2,… , and time is t. the system can be further simplified into a system that is not dependent on the function of time (autonomous system) as followṡ 1 = 1( 1, 2, … , )2̇ = 2( 1, 2, … , )⋮̇ = ( 1, 2,… , ) (3) with = ( , , … , , ), = 1,2,… , n is a function that does not depend explicitly on the time t . the definition of equilibrium point (perko, 1991) point = ( , , … , ) ∈ called the equilibrium point of (3) if ( ̅)̇ = 0, = 1,2,… , stability of equilibrium point definition of local stability the equilibrium point ∈ on the system (3) said: 1) local stable if for every å > 0 there ä > 0 such that for every solution ( ) that satisfies ‖ ( ) − ̅‖ <ä apply ‖ ( ) − ̅‖ < å for each ≥ . 2) local asymptotically stable if the equilibrium point∈ stable and there > 0 such that for every solution ( ) that satisfies x ‖ ( ) − ̅‖ < applylim→ ( ) = ̅. 3) do not be stable if the equilibrium point ∈ does not meet 1. below is given a definition of linear and nonlinear systems granted system(3), with ⊆ and : → continuous function on e. system (3) is said to be linear if , , … , each linear with respect , , … , . so the system (3) can be written in the forṁ 1 = 11 1+ 12 2+⋯+ 1 ,̇ 2 = 21 1+ 22 2+⋯+ 2 ,̇ = 1 1+ 2 2 +⋯+ , (4) dewi aggreini. – local stability analysis of a mathematical model of the interaction of … 11 with ̇ = , continuous on ≤ ≤ , , ,= 1,2,… , . then the system (3) can be expressed in the form ̇ = ̇ , with ∈ , ̇ = ( ̇ , ̇ , … , ̇ ) and a matrix size nxn . granted system (3) with ⊆ and : → continuous function on e. system (3) is said to be nonlinear if there is i such that nonlinear. the nature of the solution around the equilibrium point of nonlinear systems (3) can be determined through linearization around the equilibrium point of the system. definition 2.1 jacobian matrix if the function belongs = ( , , … , ) on the system(3) with ∈ ( ), = 1,2,3,… , . matriks ̅ ̅ … ̅̅ ̅ … ̅⋮ ⋮ ⋱ ⋮̅ ̅ … ̅ named jacobian matrix of f in point ̅. (kocak, 1991). by using jacobian matrix ( ̅), point stability properties ekuilibrim ̅ it can be seen as long as the point is hyperbolic eigenvalues and eigenvectors formally the definition of eigenvalues and eigenvectors is as follows. definitions (anton and rorres, 2014) let a nxn matrix and ∈ , ≠ 0. vector x is called an eigenvector / characteristic vector of a if= ë for a ë ∈ . numbers ë that satisfies the above equation is called the eigenvalues / value characteristics. vector x is called an eigenvector corresponding to ë. stability analysis of fixed point (boyce & diprima, 2001) consider the linear system = with a, b , c and d constants (5). linear systems (5) commonly called "data model species" in population dynamics. if the matrix a = and such λ are the eigenvalues of matrix a, then: a =a ↔ − ë − ë = 00 phase portrait and linear systems (boyce & diprima, 2001) belongs to the following linear systeṁ = (6) suppose det (a) ≠ 0, so x are the eigenvalues of matrix a , ie ë is the root of the characteristic equationë − = 0 (7) images phase and the system (6) is almost entirely dependent on its eigenvaluesfigure. 2 shows the absorbance graphic of plot toward thin film wavelength of tio2 that has been soaked in dye with variation of temperature extract. 1. if the eigenvalues different real negative is called a node, all trajectories toward zero, which means no points of zero is stable. 2. if the eigenvalues of positive reals different then called nodal source, all trajectories out of the points become unstable 3. if the eigenvalues of different real opposite in sign, with this so-called saddle point, all trajectories will be away to infinity along the eigenvectors, this resulted critical point will always be unstable. 4. if the eigenvalues of different real opposite in sign, with this so-called saddle point, all trajectories will be away to infinity along the eigenvectors, this resulted critical point will always be unstable. 5. if the eigenvalues equal, with two linearly independent eigenvectors, you will get what is called a star point or propernode, when λ < 0 then the point of criticism would be stable and unstable for λ > 0. 6. if the eigenvalues equal to the eigenvectors will be obtained by so-called improper node, when λ < 0 then the point of criticism would be stable and directions trajectory will go to zero. whereas for λ > 0 directions trajectory going out leaving the zero point and the point of criticism would be unstable. if the eigenvalues are complex numbers ë± = ± with < 0, it will produce a stable behavior called spiral, 7. if the eigenvalues are complex numbers ë± = ± dengan > 0, it will generate a behavior called unstable spiral, all trajectories going out leaving the zero point and the point of criticism would be stable. 12 biology, medicine, & natural product chemistry 5 (1), 2016: 9-14 method this research is a study of library studies, which is used in literature studies of relevant literature sources used to gather the necessary information in the study. a literature study by collecting literature sources which may include books, texts, papers and so on. after the collected literature sources ollowed by a review of the literature sources. at the end of the library resources as a basis for analyzing the problem. mathematical modeling the interaction of two populations of host and parasitoid in this research is needed to determine how the shape of the equation, and then to look for form completion hosted model and the parasitoid. steps to resolve the problem that is used to analyze the shape and model of the study are as follows: a. lowering the mathematical model the interaction of two populations of host and parasitoid with holling response function of type ii. b. analysis of the models begins with finding the equilibrium point of the model systems of differential equations. c. determining the characteristic equation and the eigenvalues of the jacobian matrix system which is calculated at each point of equilibrium. d. then check the stability of the equilibrium point. e. creating a numerical simulation of the results of the analysis of mathematical models the interaction of two populations of host and parasitoid with holling response function of type ii. research result in the discussion this time will be discussed on a mathematical model of interaction between host and parasitoid host. in this model there are two species that interact namely host and parasitoid. the population of the host is assumed to have enough food. prior to their predators, the intrinsic growth rate (birth rate minus death rate) host proportional to its population. in this case the host becomes the main food for parasitoid so before their host, the parasitoid population growth will decline due to the mortality rate of the parasitoid. parasitoid attack based on the functional response of the host. in contrast with these interactions, the parasitoid population will be converted to its growth. the ratio of parasitoid immature to adult parasitoids is constant and the population has the maximum growth rate. the parameters given to model the interaction between the host and parasitoid is ℎ( ) which states the host population at time t, ( ) states parasitoid population at the time, (ℎ) stated functional response, stating the effect of oviposition on adult parasitoid population, r states the intrinsic growth rate of the host , d declared the death rate of the parasitoid . all of these parameters is positive. functional response (ℎ) assumed to be a function : → , , (ℎ) function with a continuous rise (0) = 0, and (ℎ) diferensiabel kontinu. based on the assumptions above were obtained host and parasitoid interaction model as follows: phfrh dt dh )( ( ( ) ) .dp af h d p dt   (8) with (ℎ) = ∗ a holling type ii functional response to the interaction of host and parasitoid. here is a system of differential equations after the interaction of the two populations substituted into the equation (8), namely: ℎ = ℎ− ∗ℎ1+ ℎ= ∗ℎ1+ ℎ− (9) from the point equilibrium model in this section we will look for a solution to the 2 r , because the population must not be negative and the solutions are beyond 2 r cannot be interpreted biologically. equilibrium point equation (9) is obtained if: 0 dt dh (10) 0.dp dt  (11) from equation (10) and (11) was obtained 0 1 *         bh parh (12) * 0. 1 a ha d p bh       (13) from equation (12) and (13) was obtained 3 obtained equilibrium point           dbaa arph *11 ,0,           0,, *22 dbaa dph           dbaa ar dbaa dph **33 ,, . dewi aggreini. – local stability analysis of a mathematical model of the interaction of … 13 on the condition that the equilibrium point ∗ −> 0 or ∗ > based on these results it can be concluded that: if∗ > then between host and parasitoid populations can coexis. ∗ > means that the effects of oviposition on adult parasitoid population multiplied by the time available for the parasitoids to chase and finds a host is greater than the mortality rate of parasitoid multiplied by time to handle one host . if ∗ > then between host and parasitoid populations cannot coexist. stability of equilibrium point one way to determine the stability of equilibrium points of the system (9) is to use linearization. based systems (9), for example ℎ = ( , ) and = ( , ) with,   p bh harhyxg         1 , * 1 and   yxg ,2 pd bh haa        1 * . obtainable       * * 1 1 2 * * 2 2 2 11 , . 11 a p a hg g r bhbhh p j h p g g a ap a ah dh p bhbh                            local stability analysis in the following theorems will discuss the local stability of equilibrium points:(ℎ , ) = ∗ − , ∗ −(ℎ , ) = 0, ∗ and (ℎ , ) = ∗ , 0 theorem if ∗ > , ∗ > ( + ), ∗∗ > − ,∗∗ > and ∗∗ + − < ∗∗ − then the equilibrium point (ℎ , ) = 0, ∗ local asymptotically stable. theorem if ∗ > , ∗ > ( + ) and( ∗ )∗ − < ( ∗ )∗ , and ( ∗ )∗ > then the equilibrium point           dbaa ar dbaa dph **33 ,, local asymptotically stable. theorem if 0d d r a        and 2 4d rdd r rd a a               then the equilibrium point  2 2 *, , 0dh p a a db      unstable. conclusion the following will be given some conclusions related to this research. 1. from the system of differential equations interaction of two species , namely:= ℎ − ∗ and = ∗ − obtained equilibrium points(ℎ , ) = 0, ∗ , (ℎ , ) = ∗ , ∗ and (ℎ , ) = ∗ , 0 . 2. if ∗ > , ∗ > ( + ), ∗∗ > − ,∗∗ > and ∗∗ + − < 4 ∗∗ , , then the equilibrium point (ℎ , ) = 0, ∗ local asymptotically stable. 3. if ∗∗ + − < 4 ∗∗ , , ∗∗ > − , and ∗∗ > thenë , = ∗∗ ± ∗∗ ∗∗ have a negative real part . so based on the equilibrium point theorem (ℎ , ) = 0, ∗ local asymptotically stable. 4. by theorem if: ∗ > , ∗ > ( + ),∗∗ > − , ∗∗ > and ∗∗ + −< 4 ∗∗ − , then at the beginning of time very small number of host population , while close to the parasitoid population ∗ . so for a long time, the parasitoid population will approach∗ while the host population will become extinct. 14 biology, medicine, & natural product chemistry 5 (1), 2016: 9-14 5. if ∗ > , ∗ > ( + ), and ( ∗ )∗ −< ( ∗ )∗ , and ( ∗ )∗ > then the equilibrium point (ℎ , ) = ∗ , ∗ local asymptotically stable. because (ℎ , ) =∗ , ∗ have negative real part, the equilibrium point (ℎ , ) = ∗ , ∗ local asymptotically stable. by theorem if: ( ∗ )∗ − − ( ∗ )∗ , ∗> , ∗ > ( + ), and ( ∗ )∗ > , then at the beginning of time close to the host population∗ while close to the parasitoid population ∗ . so for a long time, will host population approaching ∗ and the parasitoid population will approach ∗ . if + + > 0 and+ + > 4 + then the equilibrium point (ℎ , ) = ∗ , 0 unstable. references anton, h. and rorres, c., 2014, elementary linear algebra, john wiley & sons, inc., new york. boyce, w.e. & diprima r.c. 2001. elementary differential equations and boundary value problems. seventh edition, john wiley & sons: new york. edelstein, l., keshet, 2005, mathematical models in biology, siam, new york. haberman, r. 1977. mathematical models in mechanical vibrations, population dynamics, and traffic flow. prenticehall: new jersey. hanh, w., 1967, stability of motion, springer-verlag, new york. hale, j. k. & h. kocak. 1991. dynamics and bifurcations. new york: springer verlag. kuznetsov, y.a., 1998, elements of applied bifurcation theory, springer-verlag, new york. ni'mah, khoirun, 2015, analysis of model s-i-p interaction of two species predator-prey with response function holling type ii, semarang state university. perko, l., 2001. differential equations and dynamical systems, springer-verlag, new york. sulistyowati, e. et al., 2001, functional response parasitoid cephalonomia stephanoderis betr against coffee fruit powder, hypothenemus hampei ferr, pest plant science program, university of gadjah mada. tarumingkeng, r.c., population dynamics (quantitative ecological assessment), 1994, pustaka sinar harapan and activities of discourse christian university, jakarta. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1159-1163 | doi: 10.14421/biomedich.2025.142.1159-1163 issn 2540-9328 (online) association between lip prints and blood groups among koya university students harem othman smail, rezhna khder hassan, helin omer ismail department of biology, faculty of science and health, koya university, koya 44023, kurdistan region – f.r. iraq. corresponding author* harem.othman@koyauniversity.org abstract lip prints, also known as cheiloscopy, are designs created by the positioning of creases and lines around the red edge of a person’s lip. in forensic science, lip prints can be used for identification just like fingerprints. blood is categorized into blood groups, sometimes called blood types, according to whether or not specific antigens are present on the surface of red blood cells. the aim of this project was to discover the correlation between lip prints and blood groups. a study involving 334 volunteers aged 18-25, collected at koya university, used standard blood typing methods to determine their blood groups. our results discovered a correlation between lip prints and blood groups in females; there is a correlation, but in males, there is not a correlation. the research indicates a correlation between blood groups and lip print patterns, suggesting that lip print traits may be linked to blood groups in females. keywords: lip prints; blood groups; cheiloscopy; sexes; lip-print classification. introduction cheiloscopy is the study of the grooves and furrows found on the red portion of the human lip, also known as the vermilion border. a person’s groves are specific to them and can be used to identify them (bhattacharjee et al.,2024, kesarwani and choudhary.2021). lip prints, or “cheiloscopy” (derived from the greek words cheilos, which means “lips,” and skopein, which means “see”), have been suggested as an extra tool in criminal investigations because to the distinctiveness of labial pattern grooves and the similarity of their applicability to fingerprints (nagalaxmi et al.,2015, fonseca et al., 2019). it was anthropologists who first observed this biological phenomenon. it was initially described in 1902 by r. fischer. similar to finger prints, a person’s lip print is unique and does not alter over time. it has been confirmed by me that they recover from changes such as trauma, inflammation, and illnesses like herpes, and that environmental influences have no effect on the disposition or form of the furrows (ghimire et al., 2013). because labial grooves are thought to be unique, lip print identification has been suggested as an additional technique for criminal investigation. similar to fingerprints, each person has a unique pattern on their lips, and no two patterns are same. lip prints are thus still another method of identification. (pearce et al., 2021). lip prints can be distinguished as early as the sixth week of intrauterine life, and after that, they hardly ever alter in pattern. (jeergal et al., 2016). suzuki and tsuchihashi (1970) created the lip-print classification scheme that is now in use (suzuki and tsuchihashi, 1970). lip prints were categorized as follows by them: type i' is a partial length type i groove, type ii is a branched groove, type iii is an intersected groove, type iv is a reticular pattern, and type v is an undifferentiated groove. type l is a clear-cut groove that runs vertically across the lips (sathawan et al., 2019). the phrase "blood group" describes the whole blood group system, which includes red blood cell (rbc) antigens. a set of genes, some of which may be allelic or closely related on the same chromosome, regulates the specificity of these rbc antigens. in 1900, karl landsteiner is credited with discovering the abo blood group system. his thorough studies on serology, which were grounded in sound scientific theory but also in simplicity, allowed for the identification of the major blood types, including 0, a, and b. (mitra et al.,2014) methodology cases we collected lip print samples from 334 individuals selected from koya university and determined their blood groups using standard blood typing methods, such manuscript received: 23 september, 2025. revision accepted: 21 november, 2025. published: 04 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1159-1163 1160 biology, medicine, & natural product chemistry 14 (2), 2025: 1159-1163 as the abo and rh blood group systems. we collected lip print samples from a total of 334 individuals, consisting of 179 females and 155 males. we analyzed the collected lip print samples and recorded the lip prints on clean paper from each individual, categorizing them based on different lip print patterns. we performed a statistical analysis to identify any correlation between specific lip print patterns and blood groups. lip print procedure before beginning the operation, make sure the lips are clean and dry. lipstick application: evenly spread a thick coat of lipstick over your lips. select a hue that will make a statement on the paper you plan to use to capture the print. pressing the lips into paper: take a fresh piece of white paper and gently press the lips onto it. for a clear print, use consistent pressure. take a print: gently lift your lips off the paper to see the lip print that was made on the surface. analysis: look for distinctive features including lines, wrinkles, and grooves in the lip print. based on the patterns they exhibit, lip prints can be categorized into several types. use a dissecting microscope if needed ( smail et al.,2024) statistical analysis the chi-square test was conducted to investigate the association between lip print patterns and the abo blood group. statistical analyses were performed using spss software version 20, and p-values < 0.05 were deemed statistically significant. result of the 334 subjects, 90.1% were rh-positive and 9.9% were rh-negative (table 1). the most common blood group was o (41.9%), which was followed by a (33.5%), b (14.4%), and ab (10.2%). blood group o was most common (45.2%) among males (n = 155), followed by a (36.1%), b (11.6%), and ab (7.1%). females (n = 179) showed a similar pattern, with o being the most common (39.1%), followed by a (31.3%), b (16.7%), and ab (13.0%) (table 2). the distribution of blood groups was not significantly different between the sexes.the complete vertical pattern (33.8%) was the most prevalent type of lip print, followed by reticular (15.9%), intersected (25.1%), and branched (11.7%). the least common patterns were incomplete vertical (3.6%) and undetermined (5.4%) (table 3). blood group and lip print patterns were found to be statistically significantly correlated (χ² = 55.731, p = 0.014) (table 4). blood group o was most commonly linked to complete vertical patterns (46.9%), followed by group a (30.5%). notable group-to-group distributions were also seen in intersected and reticular patterns, with o and a being the most prevalent. the correlation between blood groups and lip print patterns was not statistically significant among males (n = 155) (χ² = 37.858, p = 0.340) (table 5). for the majority of blood groups, especially o and a, the full vertical pattern predominated. a statistically significant correlation between blood group and lip print patterns was observed among females (n = 179) (χ² = 51.270, p = 0.037) (table 6). while reticular patterns displayed greater proportions in groups a and ab, complete vertical and intersected patterns were more common in groups o and a. table 1. distribution of subjects according to rh blood group. blood groups rh blood groups rh-positive rh-negative a 103(30.8%) 9(2.7%) b 40(12%) 8(2.4%) ab 31(9.3%) 3(0.9%) o 127(38%) 13(3.9%) total 301(90.11%) 33(9.88%) table 2. distribution of cases based on the sex and blood groups. sexes blood groups total a b ab o rhpositive rhnegative rhpositive rhnegative rhpositive rhnegative rhpositive rhnegative male 52(33.5%) 4(2.6%) 14(9%) 4(2.6%) 9(5.8%) 2(1.3%) 64(41.3%) 6(3.9%) 155(100%) female 51(28.5%) 5(2.8%) 26(14.5%) 4(2.2%) 22(12.3%) 1(0.6%) 63(35.2%) 7(3.9%) 179(100%) total 103(30.8%) 9(2.7%) 40(12%) 8(2.4%) 31(9.3%) 3(0.9%) 127(38%) 13(3.9%) 334(100%) table 3. general distributions of lip print patterns both sexes and all blood groups. types of lip print total % complete vertical 128 33.8 incomplete vertical 12 3.6 branched 39 11.7 intersected 84 25.1 reticular 53 15.9 undetermined 18 5.4 total 334 100 smail et al. – association between lip prints and blood groups … 1161 table 4. associations of pattern of lip print among subjects of a, b, o and rh blood groups (n =334) for both sexes. type of lip print blood groups a b ab o rhpositive rhnegative rhpositive rhnegative rhpositive rhnegative rhpositive rhnegative complete vertical 38(29.7%) 1(0.8%) 14(10.9%) 6(4.7%) 8(6.2%) 1(0.8%) 56(43.8%) 4(3.1%) incomplete vertical 6(50%) 1(8.3%) 2(16.7%) 2(16.7) 1(8.3%) 0(0%) 0(0%) 0(0%) branched 11(28.2%) 0(0%) 5(12.8%) 0(0%) 4(10.3%) 0(0%) 16(41%) 3(7.7%) intersected 24(28.6%) 5(6%) 12(14.3%) 0(0%) 6(7.1%) 1(1.2%) 34(40.5%) 2(2.4%) reticular 16(30.2%) 2(3.8%) 4(7.5%) 0(0%) 10(18.9%) 0(0%) 17(32.1%) 4(7.5%) undetermined 8(44.4%) 0(0%) 3(16.7%) 0(0%) 2(11.1%) 1(5.6%) 4(22.2%) 0(0%) total 103(3.8%) 9(2.7%) 40(12%) 8(%2.4) 31(9.3%) 3(0.9%) 127(38%) 13(3.9%) statistics the chi-square statistic is 55.731. the p-value is 0.014. the result is significant at p < 0.05 table 5. associations of pattern of lip print among subjects of a, b, o and rh blood groups (n =155) of males. type of lip print blood groups a b ab o rhpositive rhnegative rhpositive rhnegative rhpositive rhnegative rhpositive rhnegative complete vertical 26(50%) 1(25%) 7(50%) 4(100%) 4(44.4%) 1(5-%) 35(54.6%) 2(33.3%) incomplete vertical 1(1.9%) 1(25%) 1(7.1%) 0(0%) 1(11.1%) 0(0%) 0(0%) 0(0%) branched 6(11.5%) 0(0%) 1(7.1%) 0(0%) 2(22.2%) 0(0%) 8(12.5%) 2(33.3%) intersected 13(25%) 2(50%) 4(28.5%) 0(0%) 1(11.1%) 0(0%) 15(23.4%) 1(16.6%) reticular 2(3.8%) 0(0%) 1(7.1%) 0(0%) 1(11.1%) 0(0%) 3(4.6%) 1(16.6%) undetermined 4(7.6%) 0(0%) 0(0%) 0(0%) 0(0%) 1(50%) 3(4.6%) 0(0%) total 52(100%) 4(100%) 14(100%) 4(100%) 9(100%) 2(100%) 64(100%) 6(100%) statistics the chi-square statistic is 37.858. the p-value is 0.340. the result is not significant at p < 0.05 table 6. associations of pattern of flip print among subjects of a, b, o and rh blood groups (n =179) of females. type of lip print blood groups a b ab o rhpositive rhnegative rhpositive rhnegative rhpositive rhnegative rhpositive rhnegative complete vertical 12(23.5%) 0(0%) 7(26.9%) 2(50%) 4(18.1%) 0(0%) 21(33.3%) 2(28.5%) incomplete vertical 5(9.8%) 0(0%) 1(3.8%) 2(50%) 0(0%) 0(0%) 0(0%) 0(0%) branched 5(9.8%) 0(0%) 4(15.3%) 0(0%) 2(9%) 0(0%) 8(12.6%) 1(14.2%) intersected 11(21.5%) 3(60%) 8(30.7%) 0(0%) 5(22.7%) 1(100%) 19(30.1%) 1(14.2%) reticular 14(27.4%) 2(40%) 3(11.5%) 0(0%) 9(40.9% ( 0(0%) 14(22.2%) 3(42.8%) undetermined 4(7.8%) 0(0%) 3(11.5%) 0(0%) 2(9%) 0(0%) 1(1.5%) 0(0%) total 51(100%) 5(100%) 26(100%) 4(100%) 22(100%) 1(100%) 63(100%) 7(100%) statistics the chi-square statistic is 51.270. the p-value is 0.037. the result is significant at p < 0.05 1162 biology, medicine, & natural product chemistry 14 (2), 2025: 1159-1163 figure 1. figure of lip pattern. discussion lip prints are like fingerprints that are used for identification. we have six types of lip prints that we associate with blood groups. the blood group is another biological document that remains the same throughout the entire life of an individual (biswas et al., 2020). the results of lip print types vary among populations, as shown in table 1. there are four types of blood groups, and the shape of the lip grooves remains the same from birth until death. this experiment investigates the association between lip prints and blood groups in general. the results of our experiment are compared with those of others, and it is found that the association of lip prints with blood groups is not specific to one type of blood group; it applies to all blood types. our results indicate that lip prints are associated with blood groups in females, as shown in table 6. however, there is no association in males, as shown in table 5, and we compare this with other results. these results correspond with the previous studies in india with the ancestral north indian population sample by karim and gupta (2013), which stated that there was no significant correlation between lip print patterns and blood type (sosiawan et al., 2021). this experiment was conducted at koya university and involved 334 randomly selected healthy volunteers aged 18-25 years, including 155 males and 179 females. statistical analysis was used to analyze the results. the abo rh blood group was also recorded for each subject. the study of lip print patterns, known as cheiloscopy, is based on the arrangement of ridges and grooves on the vermillion zones of the lips. there are six types of lip print patterns: type i, type ii, type iii, type iv, type v, and type vi. when investigating the lip print pattern and its correlation with blood groups, a p-value of less than 0.05 indicates an association, while a p-value greater than 0.05 indicates no association. the correlation of lip prints with abo blood groups and fingerprints may be more useful in forensic science for the accurate identification of an individual (sandhu et al., 2017). there isn't much prior research comparing blood groups and lipprints, but suzuki and tsuchihashi have found that there is some relationship between the two and that the types of lip prints are inherited similarly to blood group inheritance (kesarwani and choudhary 2021). more research on various demographic groups is required in order to examine the variances and create a database in order to fully use the huge significance of cheiloscopy in forensic investigations. standardizing the procedure for collecting, developing, preserving, and examining lip prints is desperately needed. it is crucial to use cheiloscopy to elucidate some aspects of personal identification (karim and gupta 2014). methodological differences, such as how similarity is defined, that contribute to the heterogeneity of outcomes were found smail et al. – association between lip prints and blood groups … 1163 in the seven included research. since it was not established that parent-child similarities occur consistently across all families, the data collected allowed for the conclusion that there is no compelling scientific evidence to support the theory that there is heredity in the surface structure of lip prints (chaves et al.,2023). although authors still employ the idea of lip pattern uniqueness today, the majority of their study has not been able to substantiate this theory in accordance with modern scientific standards. the studied literature hardly mentions concepts and approaches required to assess materials for lip print production, such as dna isolation, inter-rater reliability in identifying their patterns, or recognized possible rate of error (fonseca et al.,2019). conclusion in conclusion, our research has shown a relationship between blood groups and lip print patterns, suggesting that particular lip print traits might be connected to specific blood groups. competing interest: the authors declare that there are no competing interests. references bhattacharjee, r., & kar, a. k. (2024). cheiloscopy: a crucial technique in forensics for personal identification and its admissibility in the court of justice. morphologie, 108(360), 100701. biswas, a., babu, g. s., shetty, m., rao, k., narayan, b., & sakthivel, s. (2020). correlation of lip prints, palm prints and abo blood group among student based population in mangalore. int j appl dent sci, 6, 452-8. chaves, t., azevedo, á., & caldas, i. m. (2023). are lip prints hereditary? a systematic review. international journal of legal medicine, 137(4), 1203-1214. fonseca, g. m., ortíz-contreras, j., ramírez-lagos, c., & lópezlázaro, s. (2019). lip print identification: current perspectives. journal of forensic and legal medicine, 65, 3238. fonseca, g. m., ortíz-contreras, j., ramírez-lagos, c., & lópezlázaro, s. (2019). lip print identification: current perspectives. journal of forensic and legal medicine, 65, 3238. ghimire, n., nepal, p., upadhyay, s., budhathoki, s. s., subba, a., & kharel, b. (2013). lip print pattern: an identification tool. health renaissance, 11(3), 229-233. jeergal, p. a., pandit, s., desai, d., surekha, r., & jeergal, v. a. (2016). morphological patterns of lip prints in mangaloreans based on suzuki and tsuchihashi classification. journal of oral and maxillofacial pathology, 20(2), 320-327. karim, b., & gupta, d. (2014). cheiloscopy and blood groups: aid in forensic identification. the saudi dental journal, 26(4), 176-180. kesarwani, p., & choudhary, a. (2021). correlation of lip print with blood group in forensic science. journal of oral and maxillofacial pathology, 25(1), 206. kesarwani, p., & choudhary, a. (2021). correlation of lip print with blood group in forensic science. journal of oral and maxillofacial pathology, 25(1), 206. mitra, r., mishra, n., & rath, g. p. (2014). blood groups systems. indian journal of anaesthesia, 58(5), 524-528. nagalaxmi, v., ugrappa, s., ch, l., maloth, k. n., & kodangal, s. (2015). cheiloscopy, palatoscopy and odontometrics in sex prediction and dis-crimination-a comparative study. the open dentistry journal, 8, 269. pearce, j. a., ernst, r. e., peate, d. w., & rogers, c. (2021). lip printing: use of immobile element proxies to characterize large igneous provinces in the geologic record. lithos, 392, 106068. sandhu, h., verma, p., padda, s., & sunder raj, s. (2017). frequency and correlation of lip prints, fingerprints and abo blood groups in population of sriganganagar district, rajasthan. acta medica academica, 46(2). sathawane, r., moon, g. v., bontha, s., chandak, r. m., lanjekar, a. b., & gaikwad, r. d. (2019). correlation of lip and finger pront patterns in patients with type ii diabetes mellitus. int j curr res, 11, 1630-3. smail, h. o., ahmad, r. h., & jalal, e. i. (2024). identification of fingerprint pattern and lip print pattern in females of type 2 diabetes mellitus as a biomarker. biology, medicine, & natural product chemistry, 13(1), 291-295. sosiawan, a., pulunggono, c., kurniawan, a., utomo, h., marini, m. i., rizky, b. n., & ruth, m. s. m. a. (2021). inheritance pattern of lip prints and blood group among parents and their offspring in javanese population, indonesia for assisting forensic identification. indian journal of forensic medicine & toxicology, 15(1), 699-704. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 867-872 | doi: 10.14421/biomedich.2025.142.867-872 issn 2540-9328 (online) optimization of arabica coffee (coffea arabica l.) shoot cutting growth using plastic bottle covers and goat urine treatments safinah, sri darmanti*, yulita nurchayati biology department, faculty of science and technology, diponegoro university jl. prof. h. soedarto, sh., tembalang, semarang, 50275, indonesia. corresponding author* darmantisri@yahoo.co.id manuscript received: 29 august, 2025. revision accepted: 26 september, 2025. published: 21 october, 2025. abstract arabica coffee (coffea arabica l.) is a high-value commodity, yet its productivity in indonesia remains low due to suboptimal cultivation techniques. one of the main constraints is the limited availability of quality seedlings, which is influenced by propagation methods. vegetative propagation through shoot cuttings is widely used, but often constrained by low survival and root formation rates. the application of plastic bottle covers and goat urine has the potential to improve propagation success by creating a favorable microclimate and supplying growth regulators and essential nutrients. this study examined the effects of plastic bottle cover types and goat urine concentrations on the growth of c. arabica shoot cuttings. the experiment was arranged in a 3×3 factorial completely randomized design (crd), with two factors: plastic bottle cover types (no cover, open-cap cover, and closed cover) and goat urine concentrations (0, 100, and 180 ml/l). observed parameters included shoot length, shoot number, leaf length, leaf number, root length, root number, and root diameter. data were analyzed using analysis of variance (anova) followed by tukey’s honestly significant difference (hsd) test at a 5% significance level. the results showed a significant interaction between cover type and goat urine concentration. combining a closed cover with 100 ml/l goat urine produced the highest shoot growth, cutting survival rate, and rooting percentage, while optimal root development was obtained with the closed cover and 180 ml/l goat urine. the most effective treatment was the closed cover combined with 100 ml/l goat urine. keywords: vegetative; rooting; microclimate; organic fertilizer; propagation. introduction arabica coffee (coffea arabica l.) has high economic value and is one of indonesia’s leading plantation commodities. however, national coffee production has not yet reached its full potential, partly due to the limited availability of high-quality planting material. generative propagation using seeds often fails to maintain the desirable traits of elite mother plants and requires a long period before reaching productivity (ginanjar et al., 2020). vegetative propagation through shoot cuttings provides a more efficient alternative, producing uniform seedlings that inherit superior parental traits and reach productivity more quickly (mbwambo et al., 2024). despite these advantages, the success rate of shoot cutting propagation is often low due to physiological constraints, such as limited root initiation and high mortality during early growth stages (widodo et al., 2016). one key factor influencing rooting success is the regulation of the microenvironment, particularly air humidity. the use of covers, such as humidity domes, helps maintain high moisture levels around the cuttings, reduce transpiration, and preserve cell turgor necessary for active metabolism and root initiation (gunawan et al., 2016). repurposing used plastic bottles as protective covers represents an environmentally friendly and easily applicable solution. in addition to environmental control, applying natural plant growth regulators can play a significant role in stimulating root and shoot development. goat urine has been reported to contain growth-promoting hormones, including auxins and gibberellins, as well as essential nutrients that support cell division and elongation (lestari et al., 2024). although the positive effects of microenvironmental regulation and natural growth stimulants have been independently documented, there is limited research on their combined use for the vegetative propagation of c. arabica. moreover, existing studies have not fully addressed how this combination can overcome the limitations of current propagation practices. therefore, the present study aimed to evaluate the interactive effects of plastic-bottle covers and goat urine application on the vegetative growth of arabica coffee shoot cuttings, with https://doi.org/10.14421/biomedich.2025.142.867-872 mailto:darmantisri@yahoo.co.id 868 biology, medicine, & natural product chemistry 14 (2), 2025: 867-872 the ultimate goal of developing a more effective and practical propagation technique. materials and methods study area the experiment was conducted in doro village, doro subdistrict, pekalongan regency, central java, at coordinates -7.031239° s and 109.690022° e. the site is situated at an altitude of approximately 650 m above sea level. microclimatic conditions under the paranet during the study period showed an average daily temperature of 23–28 °c and relative humidity of 70–85%, based on direct measurements using a digital thermometerhygrometer. the study was conducted during the rainy season, from october 2024 to february 2025. tools and materials the tools used included 1,500 ml plastic bottles, a plastic tarp for collecting goat urine, 75% shade netting for protection, and glass jars with lids for fermentation. the materials included shoot cuttings from threeyear-old c. arabica plants, fresh goat urine, molasses, em4. shoot cuttings were taken between the second and fifth nodes, each measuring approximately 17 cm in length and 0.4 cm in diameter, containing three nodes and two pairs of leaves trimmed to half size to reduce transpiration (figure 1). goat urine was collected directly from a local farm, filtered, and temporarily stored in closed containers before fermentation. figure 1. shoot cuttings of arabica coffee (safinah, 2025). procedures preparation of planting substrate the substrate consisted of soil and compost mixed in a 2:1 ratio. the 1,500 ml plastic bottles were cut into two parts (upper and lower). the lower section was filled with the substrate to full capacity, while the upper section was repurposed as a cover. fermentation of goat urine goat urine and molasses were mixed in a 2:1 ratio, followed by the addition of 15 ml em4 per liter of mixture. the solution was placed in glass jars, sealed tightly, and fermented for 14 days at approximately 27 °c under semi-anaerobic conditions. the mixture was stirred every two days before resealing. treatments the plastic bottle cover treatment was carried out immediately after soaking the cuttings in goat urine. and ended 16 weeks after planting (mst). the first treatment used a variety of plastic bottle covers, namely no covers (s0), covers with the bottle cap opened (s1), and tightly closed covers (s2), as shown in figure 2. the second treatment was immersion of arabica coffee shoot cuttings in goat urine solution for 9 hours with three concentrations, namely 0 ml/l (p0), 100 ml/l (p1), and 180 ml/l (p2). figure 2. treatment of plastic bottle cover variation. description: no cover (s1), open cover (s2), and closed cover (s3) (safinah, 2025). planting of cuttings the shoot cuttings were planted vertically to a depth of approximately 5 cm in the substrate-filled bottles. the bottles were arranged based on the experimental design within a 1.5 m × 1.0 m plot protected with 75% shade netting. experimental design a factorial completely randomized design (crd) was used, with two factors. the first factor was goat urine concentration (p): p0 = 0 ml/l, p1 = 100 ml/l, p2 = 180 ml/l. the second factor was bottle cover type (s): s0 = no cover, s1 = open cover, s2 = closed cover (table 1). table 1. factorial completely randomized design (crd) of plastic bottle cover variation and goat urine. type of plastic bottle covers goat urine concentration (ml/l) 0 (p0) 100 (p1) 180 (p2) no cover (s0) s0p0 s0p1 s0p2 open cover (s1) s1p0 s1p1 s1p2 closed cover (s2) s2p0 s2p1 s2p2 safinah et al. – optimization of arabica coffee (coffea arabica l.) shoot cutting growth … 869 observed parameters the success of arabica coffee shoot cutting propagation was evaluated through: shoot and leaf growth: number of shoots (count), number of leaves (count), shoot length (cm), and leaf length (cm). root growth: percentage of rooted cuttings (%), number of roots (count), root length (cm), and root diameter (mm). the formula for calculating the percentage of live cuttings and rooted cuttings is as follows: 1. percentage of live cuttings (%) percentage of live cuttings (%) = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑖𝑣𝑒 𝑐𝑢𝑡𝑡𝑖𝑛𝑔𝑠 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑒𝑝𝑙𝑖𝑐𝑎𝑡𝑒𝑠 ×100 % 2. persentase stek berakar (%) percentage of rooted cuttings (%) = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑜𝑜𝑡𝑒𝑑 𝑐𝑢𝑡𝑡𝑖𝑛𝑔𝑠 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑒𝑝𝑙𝑖𝑐𝑎𝑡𝑒𝑠 ×100 % data analysis data were analyzed using analysis of variance (anova) followed by tukey’s honestly significant difference (hsd) test was applied at the 5% significance level. all analyses were conducted using microsoft excel software. results and discussion effect of plastic bottle cover and goat urine on shoot and leaf growth of arabica coffee shoot cuttings. the shoot and leaf growth of arabica coffee shoot cuttings was evaluated based on the number of leaves, leaf length, number of shoots, and shoot length for 16 weeks after planting (mst) (table 2). table 2. shoot length (cm), number of shoots, leaf length (cm), and number of leaves of arabica coffee shoot cuttings treated with plastic bottle and goat urine at 16 weeks after planting. parameters type of plastic bottle covers goat urine concentration (ml/l) p0 p1 p2 shoot length (cm) s0 0,00 d 0,00 d 0,00 d s1 1,17 c 0,86 c 1,20 b s2 1,18 b 1,73 a 0,80 c number of shoots s0 0,00 c 0,00 c 0,00 c s1 1,75 b 2,00 b 2,00 b s2 2,25 b 3,75 a 2,00 b leaf length (cm), s0 0,00 d 0,00 d 0,00 d s1 2,37 c 2,22 c 3,02 b s2 2,30 c 4,03 a 2,13 c number of leaves s0 0,00 c 0,00 c 0,00 c s1 5,00 b 5,50 b 4,75 b s2 7,00 a 7,75 a 6,00 b note. treatment without cover (s0), cover opened (s1), cover closed (s2). concentration of goat urine 0 ml/l (p0), 100 ml/l (p1), 180 ml/l (p2). numbers followed by the same letter in the same parameter indicate no significant difference based on the hsd test at the 5% significance level. anova results showed an interaction between plastic bottle covers and goat urine on shoot length, number of shoots, leaf length, and number of leaves. the plastic bottle cover treatment had a significant effect on all parameters, while goat urine affected the number of shoots and leaf length. cuttings without cover showed no growth of shoots and leaves in all goat urine treatments. in the open cover treatment with a goat urine concentration of 180 ml/l, there was an increase in shoot length and leaf length. the closed cover treatment with 100 ml/l goat urine increased shoot length, number of shoots, leaf length, and number of leaves. in contrast, with goat urine concentration of 180 ml/l, there was a decrease in all canopy growth parameters (table 2). effect of plastic bottle cover and goat urine on root growth of arabica coffee shoot cuttings root growth was evaluated based on the percentage of rooted cuttings, number of roots, root length, and root diameter which can be seen in (table 3). table 3. number of roots, length of roots (cm), and diameter of roots (mm) in arabica coffee shoot cuttings treated with plastic bottle and goat urine at 16 weeks after planting. parameters type of plastic bottle covers goat urine concentration (ml/l) p0 p1 p2 number of roots s0 0,00 d 0,00 d 0,00 d s1 1,50 c 1,35 c 2,25 b s2 1,50 c 1,13 c 3,75 a length roots (cm) s0 0,00 d 0,00 d 0,00 d s1 3,68 c 4,17 c 4,85 c s2 4,17 c 5,20 b 6,60 a diameter of roots (mm) s0 0,00 d 0,00 d 0,00 d s1 1,50 c 1,40 c 1,38 c s2 1,63 b 1,80 a 1,78 a note. treatment without cover (s0), cover opened (s1), cover closed (s2). concentration of goat urine 0 ml/l (p0), 100 ml/l (p1), 180 ml/l (p2). numbers followed by the same letter in the same parameter indicate no significant difference based on the hsd test at the 5% significance level. anova results showed an interaction between the plastic bottle and goat urine treatment on the number of roots, root length and root diameter in arabica coffee shoot cuttings. the plastic bottle cover treatment significantly affected all parameters, while goat urine had no effect on root diameter. the uncovered treatment at all concentrations of goat urine showed no root growth. in the open cover treatment with goat urine concentration of 180 ml/l, the number of roots of arabica coffee cuttings increased, but root length and diameter were not significantly different in all concentrations of goat urine. in the closed cover treatment, the highest number of roots and root length were shown in the 180 ml/l goat urine concentration treatment, while the root diameter parameter was not significantly different between goat 870 biology, medicine, & natural product chemistry 14 (2), 2025: 867-872 urine concentrations of 100 ml/l and 180 ml/l (table 3). figure 4. rooting of arabica coffee shoot cuttings 16 weeks after planting. description. root length of arabica coffee shoot cuttings without cover with goat urine 0 ml/l (a), without cover with goat urine 100 ml/l (b), without cover with goat urine 180 ml/l (c), open cover with goat urine 0 ml/l (d), open cover with goat urine 100 ml/l (e), open cover with goat urine 180 ml/l (f), closed cover with goat urine 0 ml/l (g), closed cover with goat urine 100 ml/l (h), closed cover with goat urine 180 ml/l (i). table 4.2 and figure 4.2 show the rooting of arabica coffee cuttings obtained from a combination of plastic bottle cover treatment and goat urine concentration. in figure 4.2, the best root length and number occurred in the closed cover treatment with a goat urine concentration of 180 ml/l. live percentage of arabica coffee shoot cuttings. the percentage of live cuttings was calculated from the number of still alive cuttings until the end of observation, divided by the number of replicates planted. the percentage of rooted cuttings was obtained from the number of cuttings that formed roots divided by the number of replicates. cuttings are declared alive when the color is still fresh green, not withered or rotting (nengsih & wahyu, 2021). data on the percentage of live cuttings can be seen in table 4. table 4. percentage of live arabica coffee shoot cuttings treated with plastic bottle and goat urine at 16 weeks after planting. parameters type of plastic bottle covers goat urine concentration (ml/l) p0 p1 p2 percentage of live cuttings (%) s0 0 0 0 s1 33 50 66 s2 33 100 33 percentage of rooted cuttings (%) s0 0 0 0 s1 33 50 66 s2 33 66 33 note. treatment without cover (s0), cover opened (s1), cover closed (s2). concentration of goat urine 0 ml/l (p0), 100 ml/l (p1), 180 ml/l (p2). in the treatment without a cover, the percentage of live and rooted cuttings was 0% in all goat urine concentrations (0, 100, and 180 ml/l). the use of a closed cover on goat urine with a concentration of 100 ml/l showed the highest results with 100% live cuttings and 66% rooted cuttings (table 4). despite the 100% survival rate, not all cuttings were fully rooted. based on observations, two of the cuttings only formed callus (figure 5). this indicates that although the cuttings had not formed roots, the presence of active callus and the high humidity environment allowed the cuttings to survive until the end of the observation. figure 5. arabica coffee shoot cuttings that had just formed callus in the closed cover treatment with goat urine concentration of 100 ml/l at 16 weeks after planting (circle mark). discussion the treatment without a cover caused a significant decrease in growth and the death of cuttings. this condition is thought to be caused by the low air humidity in an uncovered environment, which increases the transpiration rate and leads to water loss exceeding the absorption capacity of the cuttings. sahlim (2023) stated that the absence of a cover reduces microclimate humidity, increases water evaporation, and causes failure in the growth of patchouli (pogostemon cablin benth.) shoot cuttings. high temperature and humidity fluctuations in an open environment also do not support the rooting process. this is reinforced by (druege et al., 2019), who stated that excess transpiration lowers cell turgor pressure, thus inhibiting cell division and differentiation required in adventitious root formation. additional support comes from the study of (shen et al., 2025) which showed that at low humidity, mulberry cuttings failed to form callus and roots, and experienced high mortality rates. in the cover treatment with the bottle cap removed, the humidity inside the cover tends to be lower than in the closed cover, which creates moderate conditions for growth. braun & wyse (2019) state that the relative humidity in ventilated covers is around 59%, while closed covers reach 90%. this moderate humidity is safinah et al. – optimization of arabica coffee (coffea arabica l.) shoot cutting growth … 871 proven to support the optimal physiological function of stomata. fanourakis et al. (2020) explained that at a humidity of around 59%, the mechanism of stomatal opening and closing mediated by abscisic acid (aba) can function properly, so that transpiration and absorption of water and nutrients take place efficiently. this condition was seen in the 180 ml/l goat urine treatment in open covers, which showed an increase in shoot and leaf length. this indicates that the increased concentration of nutrients from goat urine at moderate humidity can be optimally utilized by plants. the effectiveness of goat urine in increasing growth is also closely related to the content of active compounds in it. (lestari et al., 2024) mentioned that goat urine contains growth hormones such as auxin, cytokinin, and gibberellin, as well as npk macro nutrients. the content supports cell elongation, cell division, and the growth of plant vegetative organs. auxin is known to play a role in cell elongation and root formation (sembiring et al., 2023). cytokinin stimulates cell division and shoot growth (di bonaventura et al., 2024), while gibberellin spurs stem growth and other vegetative parts (bentargem et al., 2021). these results are in line with the research of (juliyansyah et al., 2024) which showed that the application of npk from goat urine at 5% oncx areca nut plants increased plant height, number of leaves, and leaf area. however, the difference in effectiveness between the shoot and leaf and roots at certain concentrations indicates that the sensitivity of plant tissues to hormones also plays a role. according to jang et al. (2024) and taiz et al. (2015) roots have a higher sensitivity to auxin, while shoots are more responsive to cytokinin, which explains the variation in response in growth parameters between the upper and lower parts of the plant. the results of this study also showed that high humidity in closed covers negatively affected growth, especially when combined with a high concentration of goat urine (180 ml/l). at relative humidity ≥85%, fanourakis et al. (2020) noted a decrease in aba levels in leaves, which caused stomata to become less responsive to closing signals. as a result, transpiration remains high, even under moisture-saturated conditions. (fanourakis et al., 2013) also stated that increased residual stomatal conductance (residual gs) at high humidity leads to increased water loss through the cuticle, which exacerbates transpiration. this condition accelerates the absorption of solutes such as ammonia from goat urine, which in excess amounts is toxic to plant tissues. ammonia is a compound formed under anaerobic conditions due to low oxygen levels in the growing medium (bittsánszky et al., 2015). excessive absorption of ammonia increases osmotic pressure and cell permeability, so ammonia easily enters the tissue and accumulates in high concentrations. in high amounts, ammonia is corrosive and causes cell death, which can be seen from the symptoms of necrosis in the stem cuttings in the form of blackish discoloration. priyambodo et al. (2019) explained that although the goat urine fermentation process can reduce ammonia levels, the compound cannot be eliminated. this statement is in line with the findings of li (2024), who stated that ammonia is toxic to plants, causing cell damage and triggering tissue death. in addition, shen et al. (2025) mentioned that high humidity also creates a favorable environment for the growth of pathogens such as fungi and bacteria, thus exacerbating plant tissue damage due to secondary infections. stressful conditions due to ammonia toxicity and pathogen infection cause plants to divert metabolic energy from growth to the defense system. feng et al. (2022) explain that under biotic and abiotic stress conditions, plants increase the production of antioxidant compounds, strengthen cell walls, and activate stress hormone pathways such as ethylene or jasmonate. these responses deplete energy resources that would otherwise be used for adventitious root formation. as a result, root formation is inhibited or fails, as seen in treatments with high urine concentration and humidity. overall, the results of this study indicate that the combination of proper microclimate humidity and optimal nutrient concentration is key to the successful vegetative growth of arabica coffee cuttings. the closed cover treatment with 100 ml/l goat urine proved to be the most effective in improving growth parameters and the percentage of live cuttings. conversely, the use of high concentrations of goat urine in high humidity can be a stressor that causes physiological damage and inhibits plant growth. conclusions the combination of a closed cover with 100 ml/l goat urine produced the highest shoot growth, cutting survival rate, and rooting percentage. meanwhile, optimal root development was observed with the closed cover and 180 ml/l goat urine. the most effective treatment overall was the closed cover combined with 100 ml/l goat urine. competing interests: the authors declare that there are no competing interests. references ben-targem, m., ripper, d., bayer, m., & ragni, l. (2021). auxin and gibberellin signaling cross-talk promotes hypocotyl xylem expansion and cambium homeostasis. journal of experimental botany, 72(10), 3647–3660. https://doi.org/10.1093/jxb/erab089 bittsánszky, a. , pilinszky, k. , kőmíves, t. , & gyulai, g. (2015). overcoming ammonium toxicity. plant science, 231, 184–190. 872 biology, medicine, & natural product chemistry 14 (2), 2025: 867-872 braun, l., & wyse, d. (2019). optimizing temperature and humidity for rooting hybrid hazelnuts from hardwood stem cuttings 1. http://meridian.allenpress.com/jeh/articlepdf/37/2/44/2442521/0738-2898-37_2_44.pdf di bonaventura, a., marchetti, s., petrussa, e., braidot, e., colomban, s., navarini, l., & zancani, m. 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(2016). effect of enclosure height and number of nodes on the growth of mucuna bracteata cuttings. journal agromast, 1(2). biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 873-878 | doi: 10.14421/biomedich.2025.142.873-878 issn 2540-9328 (online) effect of ciprofloxacin administration on gastric histopathological changes in mice lisa savitri1*, kharisul ihsan2, rochmad krissanjaya1, elfred rinaldo kasimo1, novirma yanti1, mochamad hanif hilmi1 1department of medical laboratory technology, faculty of health sciences, kadiri university, kediri, east java, indonesia 2department of pharmacy, faculty of pharmacy, public health, hospital administration, radiology, universitas strada indonesia, kediri, east jawa, indonesia corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 30 august, 2025. revision accepted: 14 october, 2025. published: 24 october, 2025. abstract ciprofloxacin is a fluoroquinolone antibiotic widely used in clinical and veterinary practice. while effective against a broad range of bacterial infections, several studies have reported potential adverse effects on the gastrointestinal tract, particularly the gastric mucosa. this study aimed to evaluate the histopathological changes in the gastric mucosa of mice following ciprofloxacin administration at different doses. a total of 24 male mice were randomly divided into four groups: control, low-dose ciprofloxacin, moderate-dose ciprofloxacin, and high-dose ciprofloxacin. treatment was administered orally for 14 days. gastric tissues were collected and examined histologically using hematoxylin-eosin staining. histopathological analysis revealed dose-dependent mucosal alterations. the control group showed intact gastric architecture, while the low-dose group exhibited mild epithelial erosion. the moderate-dose group presented with mucosal disruption and inflammatory infiltration, and the high-dose group demonstrated severe ulceration, edema, and mucosal thinning. these findings indicate progressive gastric injury with increasing ciprofloxacin exposure. ciprofloxacin administration induces dose-dependent gastric mucosal injury in mice, consistent with fluoroquinolone-related gastrointestinal toxicity. caution is warranted in prolonged or high-dose therapy, and further studies are needed to explore the underlying mechanisms and potential gastroprotective interventions. keywords: ciprofloxacin; gastric mucosa; histopathology; mice; fluoroquinolones. abbreviations: ciprofloxacin (cfx); reactive oxygen species (ros); tumor necrosis factor alpha (tnf-α); zonula occludens-1 (zo-1); interleukin-1 beta (il-1β); hematoxylin and eosin (h&e); u.s. food and drug administration (fda); standard deviation (sd); analysis of variance (anova); statistical package for the social sciences (spss) introduction ciprofloxacin is a widely used fluoroquinolone antibiotic, appreciated for its broad-spectrum activity and favorable pharmacokinetics. while its systemic safety profile is well established, less is known about its localized effects on gastric tissue, particularly following prolonged administration. a recent study in albino rats demonstrated that prolonged oral ciprofloxacin administration (12.5 mg/kg/day for 60 days) led to significant alterations in gastric mucosal architecture, as observed via immunohistochemistry and scanning electron microscopy. the study also reported elevated tnf-α expression and reduced levels of several helicobacter pylori immunoglobulins—suggesting mucosal inflammation and immune modulation (elmasry et al., 2021). although not focused solely on the stomach, broader drug-induced gastric injury is clinically significant. a 2025 review highlighted that various medications— including antibiotics—can cause distinct histopathological changes in gastric mucosa, emphasizing the need for histological awareness when evaluating drug safety (gastroenterological endoscopy society, 2025). meanwhile, in a mouse model, ciprofloxacin— especially when combined with other therapies—has been shown to affect intestinal pathology and the gut microbiota post-irradiation, hinting at its potential to disrupt gastrointestinal tissue integrity and microbial balance (horseman et al., 2024). the widespread use of ciprofloxacin in both clinical and veterinary practice makes it one of the most frequently prescribed antibiotics. while its therapeutic benefits are clear, increasing reports of adverse reactions have drawn attention to the need for deeper evaluation of its safety profile, particularly regarding effects on the gastrointestinal system (el-masry et al., 2021). among https://doi.org/10.14421/biomedich.2025.142.873-878 mailto:lisasavitri@unik-kediri.ac.id 874 biology, medicine, & natural product chemistry 14 (2), 2025: 873-878 the organs potentially affected, the stomach plays a central role not only in digestion but also as a protective barrier against pathogens and chemical insults. any alteration to gastric mucosal integrity may therefore have far-reaching implications for overall gastrointestinal health. drug-induced gastric injury remains a clinically relevant concern. recent findings emphasize that antibiotics and other commonly prescribed medications can lead to characteristic mucosal changes, such as erosion, ulceration, and inflammatory infiltration (gastroenterological endoscopy society, 2025). however, experimental data linking ciprofloxacin directly to gastric histopathology remain limited. most research to date has focused on intestinal microbiota or systemic toxicity rather than localized gastric effects (horseman et al., 2024). this creates a significant knowledge gap, especially considering that ciprofloxacin is often administered over long treatment courses or in combination with other drugs that may amplify mucosal damage. evaluating ciprofloxacin’s impact in mice (mus musculus) is particularly relevant. mice are widely used in biomedical research due to their genetic similarity to humans, cost-effectiveness, and well-characterized physiology. studying gastric histopathological changes in this model allows for detailed analysis of subtle alterations—such as epithelial disruption, inflammatory cell infiltration, or mucosal thinning—that may not be evident in clinical observations alone. such insights can bridge preclinical findings with human outcomes, contributing to a more comprehensive understanding of ciprofloxacin’s gastrointestinal safety. another dimension of urgency relates to the broader issue of fluoroquinolone safety. beyond contributing to antimicrobial resistance, fluoroquinolones have been associated with serious adverse effects, including tendon rupture, neurotoxicity, and cardiotoxicity, resulting in multiple fda safety communications (u.s. food and drug administration, 2018). if ciprofloxacin also contributes to gastric mucosal injury, the risk–benefit balance must be reconsidered, particularly in patients with pre-existing gastrointestinal disorders. taken together, the investigation of ciprofloxacin’s effects on gastric histopathology in mice is both scientifically relevant and clinically significant. it will provide new insights into the safety of a widely used antibiotic, inform rational prescribing practices, and support preventive strategies to minimize drug-induced gastric injury. materials and methods research design this study employed an experimental laboratory design with a post-test only control group approach. the aim was to compare gastric histopathological features in mice receiving ciprofloxacin administration with those of untreated controls. experimental animals a total of 24 healthy male mice (mus musculus), aged 8– 10 weeks and weighing 25–30 g, were obtained from an accredited animal breeding facility. all animals were acclimatized for one week under standard laboratory conditions (12-hour light/dark cycle, room temperature 22–25 °c, relative humidity 50–60%), with free access to standard pellet diet and water ad libitum. grouping and treatment mice were randomly divided into four groups (n = 6 per group): a. group i (control): received distilled water orally. b. group ii (low-dose ciprofloxacin): received ciprofloxacin 10 mg/kg/day orally. c. group iii (moderate-dose ciprofloxacin): received ciprofloxacin 20 mg/kg/day orally. d. group iv (high-dose ciprofloxacin): received ciprofloxacin 40 mg/kg/day orally. ciprofloxacin doses were adapted from previous experimental studies with modifications to reflect clinically relevant ranges (el-masry et al., 2021; horseman et al., 2024). the treatment was administered once daily via oral gavage for 14 consecutive days. ethical approval all experimental protocols were reviewed and approved by the institutional animal ethics committee. animal handling followed the international guidelines for the care and use of laboratory animals (nih publication no. 85-23, revised 2011). tissue collection at the end of the treatment period, animals were fasted overnight and sacrificed under ketamine-xylazine anesthesia. the stomachs were excised, opened along the greater curvature, and rinsed with normal saline to remove gastric contents. tissue samples from the gastric body were fixed in 10% neutral buffered formalin for histological analysis. histopathological examination fixed tissues were processed using standard paraffinembedding techniques. sections of 5 µm thickness were stained with hematoxylin and eosin (h&e). histopathological evaluation was performed under a light microscope (400× magnification). histopathological scoring gastric mucosal alterations were assessed semiquantitatively using a modified scoring system, including parameters such as: savitri et al. – effect of ciprofloxacin administration on gastric histopathological … 875 a. epithelial erosion/ulceration (0 = absent, 1 = mild, 2 = moderate, 3 = severe) b. inflammatory cell infiltration (0 = absent, 1 = mild, 2 = moderate, 3 = severe) c. edema and vascular congestion (0 = absent, 1 = mild, 2 = moderate, 3 = severe) d. mucosal thickness changes (normal, thinned, or thickened) two independent pathologists blinded to the treatment groups performed the evaluations to minimize observer bias. statistical analysis data were expressed as mean ± standard deviation (sd). differences between groups were analyzed using oneway anova followed by tukey’s post hoc test. a pvalue < 0.05 was considered statistically significant. statistical analyses were conducted using spss version 26. results and discussion result general observations throughout the experimental period, no mortality was observed in any of the groups. mice in the ciprofloxacintreated groups (especially at moderate and high doses) exhibited reduced activity and occasionally experienced mild diarrhea compared to the control group. body weight changes were not significantly different among groups (p > 0.05) (table 1). table 1. histopathological scores of gastric tissue in different treatment groups. group erosion/ulceration (0–3) inflammation (0–3) vascular congestion (0–3) mucosal thickness alteration (0–3) total score (0–12) control 0.2 ± 0.4 0.3 ± 0.5 0.2 ± 0.4 0.1 ± 0.3 0.8 ± 0.7 low-dose (10 mg/kg) 0.8 ± 0.7 1.0 ± 0.6 0.9 ± 0.6 0.7 ± 0.5 3.4 ± 1.1* moderate-dose (20 mg/kg) 1.7 ± 0.8 1.9 ± 0.7 1.6 ± 0.8 1.5 ± 0.6 6.7 ± 1.5** high-dose (40 mg/kg) 2.8 ± 0.4 2.7 ± 0.5 2.6 ± 0.5 2.5 ± 0.5 10.6 ± 1.2*** *significantly different from control (p < 0.05). **highly significant vs. control (p < 0.01).***very highly significant vs. control (p < 0.001). histopathological findings histological examination of the gastric tissues revealed normal mucosal architecture in the control group, characterised by an intact epithelial lining and no evidence of inflammation or ulceration. in contrast, mice administered ciprofloxacin exhibited dose-dependent alterations in gastric histopathology. a. low-dose ciprofloxacin (10 mg/kg): mild epithelial erosion and minimal inflammatory cell infiltration were observed in some samples. b. moderate-dose ciprofloxacin (20 mg/kg): moderate epithelial disruption, inflammatory infiltration in the lamina propria, and vascular congestion were evident. c. high-dose ciprofloxacin (40 mg/kg): severe epithelial erosion/ulceration, marked inflammatory infiltration, edema, and thinning of mucosal layers were consistently observed. representative microphotographs of h&e-stained sections demonstrated progressive mucosal damage corresponding to increasing ciprofloxacin doses (figure 1). 876 biology, medicine, & natural product chemistry 14 (2), 2025: 873-878 figure 1. histopathological scores of gastric tissue in different treatment groups. a. group i (control): received distilled water orally, b. group ii (lowdose ciprofloxacin): received ciprofloxacin 10 mg/kg/day orally, c. group iii (moderate-dose ciprofloxacin): received ciprofloxacin 20 mg/kg/day orally, and d. group iv (high-dose ciprofloxacin): received ciprofloxacin 40 mg/kg/day orally. discussion this study demonstrated that ciprofloxacin administration induced dose-dependent gastric mucosal alterations in mice. the control group exhibited normal gastric architecture, while low-dose ciprofloxacin caused mild epithelial erosion. moderate doses resulted in mucosal disruption with inflammatory infiltration, and high doses produced severe ulceration, edema, and mucosal thinning (islam et al., 2024). these findings suggest that repeated administration of ciprofloxacin may compromise gastric mucosal integrity (ge et al., 2020). our results are in agreement with prior research in rodents. el-masry, el-baz, and ibrahim (2021) reported that prolonged ciprofloxacin exposure in rats resulted in epithelial disruption, vascular congestion, necrosis, and elevated tnf-α expression. these histopathological changes mirror the patterns we observed, suggesting that ciprofloxacin exerts direct toxic effects on gastric mucosa. furthermore, horseman et al. (2024) showed that ciprofloxacin, particularly when combined with supportive agents, altered intestinal structure and microbiota following irradiation in mice. while that study focused on the intestine, the disruption of mucosal architecture and immune balance supports our observations of the gastric vulnerability (horseman et al., 2024). the mechanisms underlying ciprofloxacin-induced mucosal damage are multifactorial. evidence suggests that fluoroquinolones may induce oxidative stress, generating reactive oxygen species (ros) that impair epithelial cell integrity and trigger inflammatory cascades (el-masry et al., 2021). ge, mao, cai, and huang (2020) demonstrated that ciprofloxacin impaired gut barrier integrity in mice by reducing tight junction proteins (zo-1 and occludin), increasing il-1β expression, and promoting epithelial apoptosis. these mechanisms, although observed in the intestine, are likely relevant to gastric mucosa as well (ibrahim et al., 2021; çulpan, 2022). another contributing factor may be antibiotic-induced dysbiosis. ciprofloxacin has been shown to disrupt microbial diversity and host–microbiota interactions (ge et al., 2020), which can compromise mucosal defense and immune regulation. disruption of gastric or intestinal microbiota may thus predispose to mucosal injury (xie et al., 2024). additionally, fluoroquinolones are known to interfere with collagen metabolism, a condition associated with tendon rupture and vascular complications (u.s. food and drug administration, 2018). weakening of collagenous structures within the gastric wall may contribute to mucosal thinning and, in severe cases, gastrointestinal perforation (wang et al., 2023). from a clinical standpoint, these findings have important implications (mojoyinola & ogundipe, 2023). drug-induced gastric injury remains a recognized adverse effect of several medication classes, including nsaids, corticosteroids, and antibiotics (arafat et al., 2021; nawaz, 2021). a recent review highlighted that antibiotics such as fluoroquinolones can cause distinct a b c d savitri et al. – effect of ciprofloxacin administration on gastric histopathological … 877 gastric lesions characterized by erosion, ulceration, and inflammatory cell infiltration (liu et al., 2025; zhu, 2020). given ciprofloxacin’s widespread use in human and veterinary medicine, the potential for gastric toxicity warrants closer evaluation. patients with pre-existing gastrointestinal conditions, prolonged therapy, or concomitant use of other mucosa-damaging drugs may be particularly vulnerable (liu et al., 2024). our study also emphasizes the utility of mice as an experimental model. their genetic similarity to humans and controlled environment make them suitable for preclinical toxicology. the dose-dependent progression observed here provides a framework for future studies exploring protective strategies, such as antioxidants, probiotics, or proton pump inhibitors. this study has several limitations. first, the treatment period was limited to 14 days; longer exposure might reveal cumulative or chronic effects. second, we focused exclusively on histopathology, without assessing biochemical markers such as malondialdehyde or cytokine levels that could clarify molecular mechanisms. third, we did not analyze gastric microbiota, which may mediate some of the observed effects. future studies should extend treatment duration, incorporate biochemical and microbiota analyses, and evaluate potential gastroprotective interventions (hou, 2025). in summary, our findings demonstrate that ciprofloxacin administration induces dose-dependent gastric mucosal injury in mice, consistent with previous evidence of fluoroquinolone toxicity in gastrointestinal tissues. these results underscore the importance of cautious use, particularly in prolonged or high-dose therapy, and highlight the need for preventive measures to mitigate drug-induced gastric damage. conclusions this study demonstrated that ciprofloxacin administration induced dose-dependent gastric mucosal injury in mice. histopathological changes ranged from mild epithelial erosion at low doses to severe ulceration and mucosal thinning at high doses. these findings are consistent with previous evidence of fluoroquinoloneinduced gastrointestinal toxicity, highlighting oxidative stress, inflammation, epithelial barrier disruption, and microbiota imbalance as possible mechanisms. given ciprofloxacin’s wide clinical use, the results emphasize the need for cautious prescription, particularly for prolonged or high-dose therapy, and the importance of monitoring patients with pre-existing gastric conditions. future studies should evaluate biochemical markers, microbiota changes, and potential gastroprotective interventions to understand better and mitigate ciprofloxacin-induced gastric injury. acknowledgements: the authors would like to thank the laboratory of pathology, faculty of medicine, brawijaya university, for technical support and facilities. we also appreciate the contributions of colleagues who provided valuable input during the preparation of this manuscript. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there are no funding. references arafat, m. t., tronci, g., wood, d. j., & russell, s. j. 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sukarame carita labuan pandeglang banten beach fikri nurhidayatulloh, usman setiawan, nurullah asep abdilah, suyamto* biology study program, faculty of pharmaceutical sciences & health, mathla'ul anwar university, banten, indonesia. corresponding author* suyamto35@yahoo.co.id manuscript received: 12 december, 2025. revision accepted: 14 december, 2025. published: 15 december, 2025. abstract cucumbers of the sea are one of the animals from the phylum echinodermata that have ecological and economic roles. biogeographically, the types of sea cucumbers from the holothuriidae family are not yet well known in the waters of shallow waters of katapang sukarame carita labuan pandeglang banten coast, particularly the information on the types and their phylogenetic relationships. this study aims to determine the diversity and phenetic relationship between types of sea cucumbers in shallow waters of katapang sukarame carita labuan pandeglang banten coast. the research has stages which include preliminary observations of sea cucumber species. preliminary observations were carried out by surveying the location of the sea cucumber catch and interviewing local fishermen. the results of the survey on preliminary observations were used as the basis for determining the sampling station. sampling of the specimens was carried out using a cruising technique based on a predetermined sequence of sampling stations. observations and species determination were carried out to obtain data as material for the analysis of taxonomy and phenetic relationships. the research results show that the diversity of sea cucumber species found in the shallow waters of katapang sukarame carita labuan pandeglang banten coast are s. horrens, h. atra, and h. leucospilota. there is a phylogenetic relationship among the three sea cucumber species in the shallow waters of katapang sukarame carita labuan pandeglang banten coast. group 1, which consists of h. atra, has a similarity of 85.393% with h. leucospilota. group 2, which includes s. horrens, has a similarity of 64.130% with h. atra and h. leucospilota. keywords: phenetics; diversity; sea cucumbers. introduction indonesia is an archipelago with 17,508 islands and an area of 8.3 million km2, making it the largest archipelagic country in the world. indonesia's coastline stretches 81,000 km, creating diverse ecosystems in coastal areas, including mangrove forests, coral reefs, and seagrass beds. these ecosystems boast high biodiversity, as evidenced by the presence of nearly every phylum, including coelentrata, mollusca, annelida, and holothuroidea (phylum echinodermata). holothuroidea play a crucial role in marine ecosystems (hedriansyah et al., 2018). holothuroidea, or sea cucumbers, are a marine resource with enormous potential in indonesia. sea cucumbers are distributed throughout the world, and in indonesia, they are found throughout the ocean from sabang to merauke (west to east). sea cucumbers are found along almost all coastlines, from shallow to deep sea areas (agustina & sulaiman, 2021). globally, there are approximately 1,135 identified species of sea cucumbers, and in indonesia, there are approximately 257 species of sea cucumbers distributed throughout indonesian waters. of these, 60 species are generally known to the public. only 23 species of sea cucumbers in indonesia have been utilized, exploited, and consumed by the public. five of these 23 species of sea cucumbers have high economic value: holothuria scabra, holothuria nobilis, holothuria vacabunda, holothuria vatiensis, and holothuria marmorata. the most widely exploited, captured, and traded sea cucumber is the sand sea cucumber (holothuria scabra). of these five sea cucumber species, the sea cucumber has an elongated body shape and tentacles around the mouth opening used to capture prey. the sea cucumber's tubular legs (podia) are located on the ventral side of the body, representing pseudopods. the cross-section of a sea cucumber's body is round, semicircular, trapezoidal, or square, and can be elongated. the sea cucumber's body is 80-90% water, and it will protrude from the body when not in water. the sea cucumber's anus is located at the tip and can open and close regularly. some sea cucumber species can secrete cuvier's tubules (sticky white threads) as protection against physical or chemical disturbances (setiawan et al., 2017). https://doi.org/10.14421/biomedich.2025.142.1365-1378 1366 biology, medicine, & natural product chemistry 14 (2), 2025: 1365-1378 each sea cucumber has spicules, tentacles, papillae, and podia. sea cucumber spicules are microscopic spines located within the integumentary tissue. sea cucumber spicules are composed of chalk that dissolves in acidic solutions. the shape and composition of spicules vary depending on the sea cucumber species, becoming a distinctive characteristic of sea cucumbers at the genus and species levels. variations in spicule shape include stem-shaped, branched stem-shaped, plate-shaped, rosette-shaped, button-shaped, anchor-shaped, and tableshaped. the body color of sea cucumbers varies by species and genus, ranging from black, gray, brownish, reddish, yellowish, and white (wulandari et al., 2012). sea cucumbers have important economic and ecological roles. they are a crucial component of the ecosystem's food chain because they serve as a food source for various reef fish species (hedriansyah et al., 2018). sea cucumbers' economic importance, including their use as food, pharmaceutical, and industrial raw materials, is due to their high nutritional content. when dry, sea cucumbers contain protein (82%), fat (1.7%), water (8.9%), ash (8.6%), and carbohydrates (4.8%) (komala, 2015). sea cucumbers contain epa (eicose pentaenoic acid) and dha (docose hexaenoic acid), saturated fatty acids that are beneficial as wound healing agents, antithrombotic agents, and accelerate cell regeneration, as well as anti-cholesterol, stroke, and anti-aging agents. sea cucumbers contain minerals such as calcium, sodium, phosphorus, chromium, manganese, iron, cobalt, zinc, and vanadium, all of which are highly beneficial. sea cucumbers are used medicinally as a source of testosterone, antigens, steroids, collagen, vitamin c, and minerals such as chromium, iron, cadmium, manganese, nickel, cobalt, and zinc (roni et al., 2020). sea cucumbers have high nutritional value and extraordinary potential, leading to their massive exploitation without considering their sustainability. export demand for sea cucumber products in indonesia and the increasing price of sea cucumbers on the international market have further fueled the increase in exploitation and large-scale harvesting of sea cucumbers from their natural habitat (elfidasari et al., 2012). the large-scale exploitation and harvesting of sea cucumbers from their natural habitat has resulted in the intensive harvesting of sea cucumber species without regard for their species and size. even sea cucumbers with no economic value are exploited, resulting in a significant decline in natural sea cucumber populations, which can lead to the extinction of certain sea cucumber species, which in turn leads to the loss of germplasm within the ecosystem (setiawan et al., 2017). banten province is a region with a diverse aquatic ecosystem. one of the regencies within banten province, pandeglang regency, boasts a fairly extensive aquatic ecosystem and pristine and healthy coral reefs. the coral reefs along the west coast of pandeglang regency, particularly on liwungan island, badul island, and karang badul, are above 60%. the coral fish diversity index (cfdi) on the west coast of pandeglang regency is 106, with an estimated total fauna of 338,745 species. this indicates that the west coast of pandeglang regency has moderate species diversity. the average fish abundance in the waters of pandeglang regency as a whole is 9,783 individuals/ha (kkhl, 2021). in the waters of pandeglang regency, particularly in the shallow waters of katapang, sukarame, carita, labuan, pandeglang, banten, various species of sea cucumbers are estimated to be present, and local communities have exploited them. biogeographically, little is known about the holothuriidae species of sea cucumbers in the shallow waters of katapang, sukarame, carita, labuan, pandeglang, banten, especially regarding their species and phenetic relationships. therefore, identifying the diversity and phenetic relationships of sea cucumber species on the west coast of pandeglang regency is crucial. research methods the type of research conducted is survey and exploration research. time and place research the research was conducted at sampling points in the shallow waters of katapang sukarame carita beach, labuan pandeglang, banten, which is an underwater nature conservation area. specimen collection was carried out at low tide, considering safer sea waves (often uncertain due to weather and lunar gravity), low tide at 10/11 a.m. and high tide at 1 p.m. the specimen collection results were then identified at the integrated laboratory of fsfk unma banten. tools and materials specimen collection in the field using snorkeling equipment, hooks or clamps. microhabitat observation of captured specimens using a digital camera device (gadget/smartphone: realme 8, model: rmx3085, camera: 64 mp ai quad camera). 5 sample containers for preserved species and 5 containers for live species. dissection of the sea cucumber body for observation of internal organs using surgical equipment (dissecting kit). macroscopic observation of external body parts and visceral organs using a magnifying glass. spicule observations were carried out using a microscope and digital imaging results. the main material consists of sea cucumber specimens for observation of species diversity and phenetic relationships. specimens for observation of species diversity are groups of sea cucumbers that have been treated with anesthesia and preservation. the chemicals used for anesthesia and relaxation of sea cucumber tissue are 70% mgcl2 solution, 70% ethanol nurhidayatulloh et al. – diversity of sea cucumber types in shallow sea waters … 1367 preservative solution and naclo (a compound used for bleaching) ways of working preliminary observation of sampling locations preliminary observations through exploration were conducted in the sampling area, namely the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast to determine the sampling point. the sampling point was determined based on the presence and abundance of sea cucumbers in both the intertidal and subtidal zones, which are the usual locations for sea cucumber catches. the research location was in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast. sampling sampling was conducted in the intertidal zone during low tide in coastal areas. the diving team and fishermen were assisted in sampling (weather and natural conditions significantly influence the sampling process). sampling in the subtidal zone was conducted with the assistance of fishermen and using hooks or tongs to catch sea cucumbers. sea cucumber sampling was conducted at sampling points determined based on preliminary observations. the sampling points were determined based on the diving team's findings, namely the distance from the shoreline (10 to 50 meters from the shoreline) with a depth of approximately (3 to 5 meters). sample management sea cucumber samples from the field were washed with clean water and cleaned of any adhering dirt. samples were grouped based on morphological characteristics to observe species diversity and phenetic relationships. the specimens obtained for species diversity observation were anesthetized using a 2000 ml mgcl2 solution mixed with seawater and subjected to tissue relaxation. the sea cucumbers were preserved in specimen containers containing 70% ethanol for laboratory observation and identification. specimen identification the sea cucumber specimens were identified and dissected. their external and internal morphology was observed, and their morphometric measurements were taken. a section of the body wall was taken for spicule observation. the samples were bleached and the spicules were observed under a microscope. the resulting spicules were matched for identification. soaking the internal body wall fragments loosens the integumentary tissue, causing the spicules to detach from the tissue. the spicule composition observed was derived from the anterior, dorsal, posterior, and ventral body wall fragments. the spicule composition was observed using a digital microscope and digital imaging. spicule observation serves as a means of distinguishing sea cucumber species. the steps taken to study the ossicles include cutting 1–5 mm2 of tissue from each body region, including the anterior, dorsal, ventral, and tentacles. place the tissue fragments in a 25 ml beaker and soak them in a naclo bleaching solution for 45–60 minutes, until all muscle tissue is destroyed. afterward, the ossicle samples were washed with distilled water 4–7 times. the ossicle samples were then placed back on a concave slide, where measurements and images were taken. the samples were then ready to be observed under a microscope and photographed with a camera (widianingsih et al., 2015). morphological observations of sea cucumbers included the external body parts (external) and internal parts of the sea cucumber, including the following organs: 1. the body consists of length: width, tip (anterior), base (posterior), cross-section, ventral side, dorsal side (lateral). 2. the mouth consists of shape, diameter and location. 3. the anus consists of shape, diameter (length), location, color, anal teeth. 4. body wall, namely thickness/texture 5. tentacles consist of type, color (tip of tentacle), diameter, protective papillae, number, circle, and length (stalk). 6. the epidermis (dorsal lateral) consists of the surface, color (surface), grooves (specific), tuberkel and pediselus. 7. tube feet consisting of arrangement, length, shape and number. 8. papillae consist of tips (color), shape, number, arrangement and location. 9. ambulacral grooves consisting of number and arrangement. 10. gonads consist of shape (1 lobe/2 lobes) and presence or absence. 11. respiratory tree consisting of the presence or absence, shape of tubules and shape of the ends of tubules. 12. cuverian tube (presence or absence). 13. the mesentery consists of color, number of dorsal attachments and number of ventral attachments. 14. the chalk ring consists of a conical curve of the radial tip, a conical curve of the radial base, the diameter of the ring and the height of the ring plate. 15. digestive tract (intestinal length:body length ratio). 16. spicules consist of dominant, c-shaped, table-shaped, button-shaped, rosette-shaped, plate-shaped, rodshaped, grain-shaped and typical spicules. morphometric observations of sea cucumbers include organs: body, mouth, anus, body wall, tentacles, respiratory tree, digestive tract, cuverian tube, calcareous ring, tube feet, gonads, papillae and longitudinal muscles. 1368 biology, medicine, & natural product chemistry 14 (2), 2025: 1365-1378 data analysis taxonomic analysis the results of the sea cucumber identification then made a taxonomy of the diversity of each type of sea cucumber that was found and identified. phenetics relationship analysis characters for identification are determined according to the stages of determining the operational taxonomic unit (ots), selecting characters, assigning character values, and calculating taxonomic distances. taxonomic characters are measurable data that is then coded with numbers for clustering analysis. clustering analysis uses the upgma (unweight pair group with arithmetic average) method using the mvsp 3.1 program. the results of the analysis are in the form of a dendogram to determine the groups/clusters of each ots based on similarity. results and discussion results based on observations, the species found in the shallow waters of katapang, sukarame, carita, labuan, pandeglang, banten are presented in table 1. table 1. sea cucumber species discovery results. no famili spesies 1 stichopodidae stichopus horrens 2 holothuriidae holothuria atra 3 holothuriidae holothuria leucospilota comparison of macroscopic observations of sea cucumber organs the results of macroscopic comparative observations on the organs of sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota in the organ sections: body, mouth, anus, body wall, tentacles, epidermis (lateral dorsal), tube feet, papillae, ambulacral grooves, gonads, respiratory tree, cuverian tube, mesentery, lime ring, digestive tract, spicules. body comparison of observations of body organs in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota, is presented in table 2. table 2. comparison of body organs in sea cucumber species. body organs s. horrens h. atra h. leucospilota length: width 240 mm: 78 mm 126 mm : 45 mm 115 mm : 39 mm tip (anterior) obtuse tapered tapered base (posterior) obtuse blunt blunt crosssection square/trapezoid rounded rounded ventral side flat rounded rounded dorsal side (lateral) square rounded rounded mouth comparison of observations of the mouth organs of sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota, is presented in table 3. table 3. comparison of mouth organs in sea cucumber species. mouth organs t. horrens i. atra h. leucospilota shape circle circle oval diameter 10,8 mm 5,2 mm 8,6 mm location subterminal subterminal subterminal anus a comparison of anal organ observations in sea cucumber species found in the shallow waters of katapang, sukarame, carita, labuan, pandeglang, banten, namely s. horrens, h. atra, and h. leucospilota, is presented in table 4. table 4. comparison of the anus organs in sea cucumber species. anus organs u. horrens j. atra h. leucospilota shape round round round diameter (length) 6 mm 4,8 mm 5 mm location terminal terminal terminal color reddish black black black anal teeth none none none body wall comparison of observations of body wall organs in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota, is presented in table 5. nurhidayatulloh et al. – diversity of sea cucumber types in shallow sea waters … 1369 table 5. comparison of body wall organs in sea cucumber species. body wall v. horrens k. atra h. leucospilota thichness/texture 6,3 mm 0,6 mm 0,7 mm tentakel comparison of observations of tentacle organs in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota, is presented in table 6. table 6. comparison of tentacle organs in sea cucumber species. organ tentakel s. horrens h. atra h. leucospilota type shield shield shield color (tentacle tip) translucent black black diameter 6,9 mm 2,3 mm 3,2 mm protective papillae available available available number 19 20 21 circle 1 circle 1 circle 1 circle length (stalk) 97 mm 19 mm 39 mm epidermis (lateral dorsal) comparison of observations of the epidermis organs (dorsal lateral) in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota is presented in table 7. table 7. comparison of dorsal lateral organs in sea cucumber species epidermis (lateral dorsal) s. horrens h. atra h. leucospilota surface uneven slippery and slippery color (surface) dark green slimy black grooves (specific) grooves present none none tubercles available none none pedicellus none none none tube feet comparison of observations of tube foot organs in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota is presented in table 8. table 8. comparison of tube feet organs in sea cucumber species. tube feet w. horrens l. atra h. leucospilota arrangement length numerous, 4 line 1-9 min regular 2,8 mm irregular 3-5 mm shape tubes short tube short tube number numerous, regular numerous, irregular numerous, irregular papila comparison of observations of papilla organs in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota is presented in table 9. table 9. comparison of papilla organs in sea cucumber species. organ papila h. horrens h. atra h. leucospilota tip (color) according to the pattern black black shape conical and blunt tapered short tapered tapered number many many many arrangement irregular irregular irregular location outer body wall (except in the ventral) outer wall of the body (except in the ventral) outer wall of the body (except in the ventral) alur ambulakral comparison of observations of the ambulacral groove organs in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota is presented in table 10. tabel 10. comparison of ambulacral arul organs in sea cucumber species. arul ambulakral h. horrens h. atra h. leucospilota amount 4 lines none none arrangement none none none gonad comparison of gonad organ observations in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota, is presented in table 11. table 11. comparison of gonad organs in sea cucumber species. gonad h. horrens h. atra h. leucospilota shape (1lobe/2 lobes) presence or absence not found not found not found respiratory tree comparison of observations of respiratory tree organs in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota is presented in table 12. 1370 biology, medicine, & natural product chemistry 14 (2), 2025: 1365-1378 table 12. comparison of respiratory tree organs in sea cucumber species. respiratory tree x. horrens m. atra h. leucospilota availability available available none tubule shape fine thread fine thread none end shape tubules alveoli alveoli none cuverian tube comparison of observations of cuverian tube organs in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota is presented in table 13. table 13. comparison of cuverian tube organs in sea cucumber species. cuverian tube h. horrens h. atra h. leucospilota availability none none available mesenterium comparison of observations of the mesentery organ in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota is presented in table 14. table 14. comparison of mesentery organs in sea cucumber species. mesenterium h. horrens h. atra h. leucospilota color transparent brownish white transparent yellow number of attachments dorsal 1 2 2 number of attachments ventral 1 1 2 chalk ring comparison of observations of the lime ring organ in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota is presented in table 15. table 15. comparison of cretaceous ring organs in sea cucumber species. chalk ring h. horrens h. atra h. leucospilota conical curve apostle tip available available available conical curve none available none radial base ring diameter 23,2 16,1 14,2 piece height ring 4,2 2,1 2,5 digestive tract a comparison of observations of the digestive tract organs of the sea cucumber species found in the shallow waters of katapang, sukarame, carita, labuan, pandeglang, banten, namely s. horrens, h. atra, and h. leucospilota, is presented in table 16. table 16. comparison of digestive tract organs in sea cucumber species. digastive tract h. horrens h. atra h. leucospilota intestinal length 926 mm 417 mm 350 mm body length 286 mm 130 mm 139 mm ratio 926 mm : 286 mm 417 mm : 130 mm 350 mm : 139 mm spicule comparison of observations of spicule organs in sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota is presented in table 17. table 17. comparison of spicule organs in sea cucumber species. spicule h. horrens h. atra h. leucospilota dominant table plate shape (perforation) button shape c-shape yes no no table shape yes yes yes button shape no no yes rosette shape no no no plate shape yes yes yes rod shape no no no grain shape no no no typical spicules yes no yes comparison of morphometric observations of sea cucumber organs the results of comparative morphometric observations on the organs of sea cucumber species that were successfully found in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast, namely s. horrens, h. atra and h. leucospilota in the organ sections: body, mouth, anus, body wall, tentacles, respiratory tree, digestive tract, cuverian tube, lime ring, tube feet, gonads, papillae, longitudinal muscles. nurhidayatulloh et al. – diversity of sea cucumber types in shallow sea waters … 1371 table 18. comparison of organ morphometrics in sea cucumber species. organ characteristics h. horrens h. atra h. leucospilota body length 212,5 mm 166,67 mm 196,33 mm width 67,1 mm 44,30 mm 41,40 mm mouth diameter 9 mm 7,37 mm 8,80 mm anus diameter 14,65 mm 15,27 mm 14,40 mm wall thickness tentacle 4,45 mm 1,80 mm 1,40 mm tentacle diameter 4,05 mm 3,07 mm 3,03 mm stalk length 26,45 mm 10,13 mm 22,67 mm respiratory tree length (organ) 471,15 mm 34,30 mm digestive tract intestine length/height ratio 670 mm 601,67 mm 560,67 mm cuverian tube size length/width 55,07 mm radial/interradial 18,45 mm 21,97 mm 22,93 mm calcareous ring plate height 2,75 mm 3,00 mm 2,30 mm tube feet length 2,55 mm 2,97 mm 3.,10 mm gonad length/width papila length 1-9 mm 2 mm 3-5 mm long length of integument 2 baris 2 baris 2 baris taxonomic analysis stichopus horrens the classification (grouping) of stichopus horrens in the taxonomic system is as follows: kingdom : animalia phylum : echinodermata class : holothuroidea order : synallactida family : stichopodidae genus : stichopus species : stichopus horrens local name: fried peanut sea cucumber, taikongkong, kacang, susu, rengget. international name: dragonfish. brief description: its body is solid, fleshy, thick, folded, and soft. its body color is yellowish green with small squares and blackish brown stripes. white, long, and small papillae with large protrusions at the base. these protrusions are greenish white and somewhat transparent, spread across the dorsal surface. tube feet are arranged in three longitudinal rows on the ventral surface. holothuria atra the classification (grouping) of holothuria atra in the taxonomic system is as follows: kingdom : animalia phylum : echinodermata class : holothuroidea order : holothuriida family : holothuriidae genus : holothuria species : holothuria atra local name: black or brown takling sea cucumber, black, dara, keling, cera. international name: lollyfish / black trepang. short description: the body is elongated, medium-fleshed and relatively hard. the entire body is reddish-black and covered with long, small, and dense papillae on the dorsal surface. the ventral surface of the body is also covered with small, long, densely arranged tube feet. holothuria leucospilota klasifikasi (brandt, 1835): kingdom : animalia phylum : echinodermata class : holothuroidea order : holothuriida family : holothuriidae genus : holothuria species : holothuria leucospilota local name: timun laut gum, cera, jepun, keling, talengko. international name: -. short description: the body is elongated, medium-fleshed and soft. the entire body is black with large and long papillae, densely arranged on the dorsal surface. tube feet are similar to papillae and densely arranged on the ventral surface. phenetics relationship analysis analysis of phenetic relationships with a dendrogram of kinship relationships of 3 sea cucumber species based on morphological characteristics in the operational taxonomic unit (otu) (table 19). 1372 biology, medicine, & natural product chemistry 14 (2), 2025: 1365-1378 table 19. characters in operational taxonomic units (ots). body organs criteria body tip (ante) 0 = tapered 1 = blunt base (post) 0 = tapered 1 = blunt cross section 0 = square 1 = rounded ventral side 0 = flat 1 = rounded dorsal side (lateral) 0 = square 1 = rounded mouth shape 0 = oval 1 = circle location 0 = terminal 1 = subterminal anus shape 0 = square 1 = round location 0 = subterminal 1 = terminal color 0 = not black 1 = black anal teeth 0 = present 1 = absent tentacles type 0 = pinnate 1 = shield color 0 = black 1 = not black protective papilae 0 = absent 1 = present surface 0 = uneve 1 = smooth and slimy epidermis (lateral dorsal) grooves 0 = present 1 = absent tubercles 0 = present 1 = absent pedicellus 0 = present 1 = absent arrangement 0 = present 1 = absent tube feet length 0 = short 1 = long shape 0 = not tube 1 = tube number 0 = few 1 = many tip (color) 0 = patterned 1 = black papillae shape 0 = tapered 1 = blunt number 0 = few 1 = many arrangement 0 = regular 1 = irregular ambulakral groove number 0 = available 1 = none arrangement 0 = available 1 = none gonad availability 0 = available 1 = none respiratory tree availability 0 = available 1 = none tubular shape 0 = available nurhidayatulloh et al. – diversity of sea cucumber types in shallow sea waters … 1373 1 = none tubular tip shape 0 = available 1 = none cuverians tube availability 0 = available 1 = none mesenterium color 0 = available 1 = none calcareous ring conical curve at tip 0 = available 1 = none radius 0 = available 1 = none conical curve at base 0 = available 1 = none radius 0 = available 1 = none spikula dominant 0 = available 1 = none table shaped 0 = available 1 = none button shape 0 = available 1 = none rosette shape 0 = available 1 = none plastic shape 0 = available 1 = none rod shape 0 = available 1 = none grain shape 0 = available 1 = none typical spicules 0 = available 1 = none body body length: width 0 = small 1 = big mouth diameter 0 = small 1 = tall anus diameter (panjang) 0 = short 1 = tall body wall thickness/texture 0 = thin 1 = thick tentacles diameter 0 = short 1 = tall length (stalk) 0 = short 1 = tall calcium ring ring diameter 0 = short 1 = tall ring lobe height 0 = low 1 = tall tentackels number 0 = few 1 = many circle 0 = available 1 = none gonad shape (1 lobus/2 lobus) 0 = available 1 = none mesentarium number 0 = available 1 = none number of ventral attachment 0 = available 1 = none after conducting clustering data analysis to determine the close relationships between each sea cucumber species studied, 57 characteristic data from the three sea cucumber species were further processed using the mvsp 3.22 computer program. this data analysis process can be used as supporting evidence for the close relationships based on morphological characteristics among the three sea cucumber species. 1374 biology, medicine, & natural product chemistry 14 (2), 2025: 1365-1378 the data processing grouped the morphological characters of each sample based on binary numbers 0 and 1 to obtain clustering analysis using the upgma classification analysis. based on the clustering analysis, a dendrogram of the phylogenetic relationships of the three sea cucumber species was created based on morphological characteristics using the upgma classification analysis. the upgma analysis in mvsp 3.2 software was transferred to the jaggard similarity index tree construction method (figure 1). figure 1. dendogram of the kinship relationship of 3 species of sea cucumber in the shallow sea waters of katapang sukarame carita labuan pandeglang banten coast discussion diversity of sea cucumber species in the shallow waters of katapang beach sukarame carita labuan pandeglang banten based on morphological observations, several species of sea cucumbers have been found in the shallow waters of katapang, sukarame, carita, labuan, pandeglang, banten, including s. horrens, h. atra, and h. leucospilota. morphologically and anatomically, each type of sea cucumber differs. these differences are clearly visible in the shape, color, and color pattern of the sea cucumbers. the variety of habitats for sea cucumbers, combined with different ecological conditions, leads to differences in the species composition, number, and distribution of sea cucumbers in each location. the clustering of certain sea cucumbers in a particular habitat indicates that the presence of these animals is influenced by the availability of food sources in that habitat. sea cucumbers feed on plankton, detritus, and other organic matter found in mud or sand. other food sources include small organisms, protozoa, filamentous algae, seaweed, small pieces of marine animals and plants, and sand particles (elfidasari et al., 2012). s. horrens is rigid, nearly rectangular, with a body diameter or thickness of 0.20 cm. its body is white to grayish with irregular brown wart-like nodules all over the body except on the ventral surface, and it lacks cuvierian tubes. the dorsal surface of the specimen is dark brown with light brown spots. the bases of the papillae are enlarged and form circles, some of which are white. the papillae's exit points are black. the ventral surface of the specimen is cream-colored with distinct tube feet. the mouth is located on the anterior ventral surface with 20 shield-shaped (peltate) tentacles. observations of the spicules in s. horrens revealed a dominant table-shaped structure, with c-shaped, tableshaped, plate-shaped, and distinctive spicules also found. the spicules on the dorsal body wall of the specimen consist of table-shaped spicules with spires forming a crown of thorns, tack-like tables, rosettes, and sand cshaped stems. the ventral body wall spicules consist of table-shaped spicules with spires forming a crown of thorns, rosettes, sand c-shaped stems, stems with spiny surfaces and holes in the middle and edges. the tentacle spicules consist of curved stem spicules with fine, short spines on their surfaces. identification results for the sea cucumber h. atra indicate that this sea cucumber has an oval and elongated morphology with a body length of approximately 20 cm and a body weight of approximately 200 grams. the sea cucumber h. atra has a dense black color pattern covering its entire body, making it difficult to distinguish between the dorsal and ventral parts. however, the ventral part usually has a reddish color that forms a straight line from the anterior to the posterior end. the integumentum of this sea cucumber feels rough due to the presence of papillae (small protrusions on the dermis) covering its body. the sea cucumber h. atra has five white muscles, extending from the anterior to the posterior end and attached to the inner dermis. the intestinal tract resembles a thin, transparent membrane and forms most of the digestive tract, ending in the cloaca. observations of the spicules in h. atra found that they are predominantly plate-shaped, with some also appearing table-shaped. purwati et al. (2008) explained that sea cucumbers possess a micron-sized skeletal structure embedded within the skin tissue, podia, and tentacles, commonly called spicules, or ossicles in international parlance. these spicules can be isolated by removing them from the surrounding tissue using domestic bleaching fluid. darsono (1998) also explained that the main compound forming spicules is calcium carbonate, which is soluble in acidic solutions. sea cucumber spicules have a porous structure similar to that seen in other echinoderms and can account for more than 50% of the total endoskeleton volume. the shape and type of spicules vary among species. therefore, these spicules can be used to characterize sea cucumbers at the genus and species levels. the spicule isolation technique was based on the method used by samyn et al. (2005). the observed spicules originated from several body parts, namely the dorsal and ventral body walls, dorsal papillae, ventral tube feet, and tentacles. based on research by aba & rusliadi (2020), h. atra has a round, elongated body shape with a black, finecolored speckled surface and whitish-yellow tentacles. it nurhidayatulloh et al. – diversity of sea cucumber types in shallow sea waters … 1375 measures approximately 20 cm long and 4 cm wide. this sea cucumber typically lives in rocky and sandy waters, often overgrown with seagrass beds. according to purwati (2008), h. atra is known as the blood sea cucumber because of the reddish fluid it produces when rubbed. holothuria atra prefers open areas that are constantly inundated during low tides, typically with hard, rough substrates. according to massin (1996), h. atra has a cylindrical body shape, measuring 15-30 cm in length. its body is completely black, both dorsally and ventrally. papillae and tube feet are irregularly distributed. the mouth is located at the anterior end of the ventral surface, with 20 shield-shaped (palate) tentacles. the anus is located at the posterior end. the dorsal spicules are tables and rosettes, while the ventral spicules are pseudoplates. the tentacles lack spicules. h. atra thrives at temperatures of 23-24°c, with a water ph (acidity) of 7. according to sutaman (2013), the optimal temperature for sea cucumber survival is between 22-32°c. the optimal ph for h. atra is between 6.5 and 8. h. leucospilota is brown with black spots on the ventral surface. the ventral surface is pale brown. h. leucospilota has prominent morphological characteristics: an elongated, cylindrical, black body. its body is soft, flexible, and covered with soft papillae. when contracted, it takes on a pear-shaped shape. it has a soft tegument and a cuvierian tube. h. leucospilota has a rounded body cross-section, with a wider posterior section than the anterior section. the ventral surface tends to be flat, and the anal opening is round. the dorsal side is black and the ventral side is dark brown. its skin is thin and soft. h. leucospilota has cuvier's tubules. twenty tentacles are visible. the spicules on the dorsal integument are predominantly button-shaped, with table-shaped, plate-shaped, and distinctive spicules also found. based on research by aba & rusliadi (2020), h. leucospilota is brown with black spots on the dorsal side. the ventral side is yellowish-white. this sea cucumber, when touched, exudes a white sap. it has the habit of clinging to dead coral, especially on its posterior side, while its anterior side often extends above the sand surface. this species lives in seagrass beds, sandy rocks, and coral reefs. it is commonly found in sandy, coral, and seagrass-covered substrates with a temperature of 2326°c and a water ph (acidity) of 6-7. according to gultom (2004), sea cucumbers typically grow in sandy areas mixed with coral fragments and abundant marine plants or seagrass. according to lewerissa (2014), the ideal temperature range for sea cucumber growth is 27–30 °c. according to wibowo et al. (1997), the optimal ph range is 6.5–8.5. phenetic relationships between sea cucumber species in west coastal waters pandeglang, banten the distinguishing features within the class of sea cucumbers (holothuroidea, echinodermata) are external morphology, internal organs, and spicules. these characteristics can show a high degree of similarity within certain families, including the family stichopodidae. this family is one of the holothuroidea (echinodermata) families that contains most of the commercially important species widespread in shallow tropical waters. to date, the evolution of sea cucumbers based on morphological characteristics used to determine the status of characteristics applicable to phylogenetic analysis remains debated. in fact, no phylogenetic research has yet been conducted on the stichopodidae family (wirawati & purwati, 2016). analysis of phenetic relationships among sea cucumber species in the west coastal waters of pandeglang, banten, used cluster analysis to group similar elements into distinct clusters. cluster analysis is useful for summarizing data by grouping objects based on shared characteristics. the sea cucumbers studied in the west coastal waters of pandeglang, banten, obtained 57 morphological characteristics from three sea cucumber species. these data were further processed using the mvsp 3.22 computer program to obtain clustering analysis groups using the upgma method. the external morphology observed included body cross-sectional shape, maximum body length, arrangement of papillae and tube feet, the presence or absence of papillae protrusions, the position of the mouth and cloaca/anus, the presence or absence of cloaca/anus modifications, and the number of tentacles. the observed morphological characteristics included the shape and size ratio of radial to interradial calcareous rings, the number of gonads, the shape of the madreporite, the number and length of polyan vesicles, and the presence or absence of cuvier's organs. the results of the cluster analysis are displayed in the form of a dendrogram, which shows how the data sets are hierarchically interconnected. based on the obtained similarity matrix, the clustering analysis was performed using the upgma method. upgma is the most common and recommended method. it also minimizes the amount of distortion that occurs between the dendrogram and the similarity index (hayati et al., 2018). the upgma method is a simple algorithm for tree construction that assumes the average change along the tree, expressed as a distance. the upgma method begins with the distance between the most closely related characters, then averages the distance between the species and the next species, continuing until all species are included in the tree (andriani, 2016). 1376 biology, medicine, & natural product chemistry 14 (2), 2025: 1365-1378 the upgma analysis in network software was transferred to the jaggard similarity index tree construction method. next, the distribution of morphological characteristics of each sea cucumber sample in the west coastal waters of pandeglang, banten, was analyzed. these were first transformed into binary form and arranged according to the specified characteristics for each listed character. the jaggard method is one method used to calculate the similarity between two objects (items). like the cosine distance and matching coefficient, this method is generally calculated based on a vector space similarity measure (ubaidillah, 2018). phenetic relationships with dendrograms of kinship relationships between three sea cucumber species in the west coastal waters of pandeglang, banten, based on morphological characteristics, with the following criteria: body, mouth, anus, tentacles, epidermis (dorsal lateral), tube feet, papillae, ambulacral grooves, gonads, respiratory tree, cuverian tubes, mesentery, calcareous ring, spicules, and body wall. the results of the cluster analysis between three sea cucumber species in the west coastal waters of pandeglang, banten, depicted in a dendogram, demonstrate grouping and demonstrate the closeness of kinship between the three sea cucumber species in the west coastal waters of pandeglang, banten. the dendogram above shows that the smaller the similarity value of the line connecting one individual to another, the greater the differences between them. the dendrogram revealed phenotypic similarities between three sea cucumber species in the west coastal waters of pandeglang, banten. group i, h. atra, is 85.393% similar to h. leucospilota; group ii, s. horrens, is 64.130% similar to both h. atra and h. leucospilota. both phylogenetic trees or dendograms show that the separation of the two genera is stable and consistent with the currently recognized taxonomy of sea cucumbers at the stichopodidae family level (massin, 1999; samyn et al., 2005). this separation supports previous phylogenetic studies using mitochondrial dna analysis (byrne et al., 2010) and morphological characteristics (appletans, 2002; samyn et al., 2005). furthermore, research by kerr & kim (2001) showed that stichopodidae and holothuriidae are very closely related (sister groups) (wirawati & purwati, 2016). the results of descriptive analysis indicate that there are differences and similarities between the studied species. the morphological similarity of an organism has a relative value because the characteristics shared do not have significant similarity values. the kinship of living organisms can be determined through similarities in morphological characteristics. the more similar characteristics shared by groups of living organisms, the closer the kinship is considered. the kinship relationship between two individuals or populations can be measured based on similarities in a number of characters, assuming that differences in characters are caused by differences in genetic makeup. characters in living organisms are controlled by genes. genes are pieces of dna whose activity (expression) can be observed through changes in morphological characters that can be caused by environmental influences (andriani, 2016). based on the results of the dendrogram analysis, the groupings formed in this study can explain the closeness of relationships based on similar characteristics, especially phenotypic (morphological) characteristics. form or morphological characters are generally the best data for delimiting a taxon because good taxonomic delimitation is achieved using easily visible characters, rather than hidden ones. for this reason, morphological characters can be used as a source of taxonomic evidence. furthermore, the results of research using morphological characters (phenotypic characters), as conducted in this study, indicate that morphological characters as taxonomic evidence are indeed very effective for identifying and analyzing the diversity of sea cucumber species in the west coastal waters of pandeglang, banten, and for determining their close kinship relationships. conclusion and suggestion conclusion based on the results and discussion, the following conclusions can be drawn: ▪ the diversity of sea cucumber species found in the west coastal waters of pandeglang, banten, includes s. horrens, h. atra, and h. leucospilota. ▪ the dendrogram reveals phenetic similarities between the three sea cucumber species in the west coastal waters of pandeglang, banten. group i, h. atra, is similar to h. leucospilota with a similarity percentage of 85.393%; group ii, s. horrens, is similar to both h. atra and h. leucospilota with a similarity percentage of 64.130%. suggestion based on the discussion and conclusions, the following recommendations are made: ▪ more thorough exploration and identification of sea cucumber diversity in the west coastal waters of pandeglang, banten is needed. ▪ further research is needed to identify sea cucumbers in the waters of the west coast of pandeglang, banten. ▪ future studies should utilize molecular data for comparison with existing phylogenetic studies of the stichopoda and holothuriidae families. ▪ morphological characteristics such as color patterns during life, body wall thickness, and so on should be added, so that the number of spicule characteristics used is balanced with the number of external morphological characteristics. nurhidayatulloh et al. – diversity of sea cucumber types in shallow sea waters … 1377 ▪ local communities are expected to maintain and preserve the ecosystem that serves as the habitat for sea cucumbers so that they remain preserved in nature. competing interests: the authors declare that there are no competing interests. references aba, l & rusliadi. 2020. inventarisasi jenis teripang (holothuroidea) pada zona intertidal di perairan pulau ottouwe wakatobi. jurnal matematika, sains, dan pembelajarannya. 6(1): 31-43 agustina, m. & e. sulaiman. 2021. keanekaragaman hewanophiuroidea yang terdapat di pantai cukuh kecamatan kaurselatan kabupaten kaur. skripsi. 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r.r. sokal. 1973. numerical taxonomy, the principles and pratice of numerical taxonomy. w.h. freeman. san francisco. http://kkji.kp3k.kkp.go.id/index.php/basisdata-kawasan-konservasi/details/1/77 http://kkji.kp3k.kkp.go.id/index.php/basisdata-kawasan-konservasi/details/1/77 1378 biology, medicine, & natural product chemistry 14 (2), 2025: 1365-1378 sutaman, 2013. petunjuk praktis budidaya teripang. yogyakarta: kanisius. samyn y, w appeltans & am kerr. 2005. phylogeny of labidodemas and the holothuriidae (holothuroidea: aspidochirotida) as inferred from morphology. zoological journal of the linnaean society, 144: 103–120 ubaidillah. 2018. variasi fenetik aksesi tebu (saccharum officinarum l.) dibeberapa wilayah indonesia berdasarkan karakter batang dan daun. skripsi. jurusan biologi fakultas sains dan teknologi universitas islam negeri maulana malik ibrahim malang biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 43-50 | doi: 10.14421/biomedich.2024.131.43-50 issn 2540-9328 (online) therapeutic azithromycin mitigated monosodium glutamate-related dysfunction in rats’ body weight and serum, liver, kidney and heart antioxidant defense bioindicators anthony cemaluk chinedum egbuonu1,*, prince ogochukwu alaebo1, udumma nsofor onuoha2, chinomso juliet njoku1, obioma benedeth eze1, francisca ugochi odoemelam1, michael eberechukwu edum1, ojichukwu boniface obi1, mmesoma joy ukaegbu1, sandra uchechi nwaogwugwu1, marvellous chinonso orji1, clara ngozika ndukwe1, prosper opara1, chinagorom oyoyo1, chika blessing joe-eme1, cheluchiaka jecinta okwoigwe1 1department of biochemistry; 2department of microbiology, college of natural sciences, michael okpara university of agriculture, umudike, nigeria. corresponding author* tonycemalukegbuonu@yaho.com; egbuonu.anthony@mouau.edu.ng abstract monosodium glutamate (msg) mediates body weight gain (bwg) and oxidative stress. azithromycin (azt), may be abused and coconsumed with msg to present unknown outcomes on bwg and oxidative stress. this study evaluated the effect of azt and msg in rats’ bwg and antioxidant bioindicators. thirty rats assigned to five groups were orally exposed for seven consecutive days to groups a, control (distilled water, 1 ml/kg), b, msg (msg 8000 mg/kg), c, therapeutic azt, taz (azt 82.5 mg/kg), d, overdose azt, oaz (azt 412.5 mg/kg) and e, taz + msg (azt 82.5 mg/kg + msg 8000 mg/kg). msg-treated rats exhibited a significantly (p < 0.05) increased bwg; serum, liver, kidney and heart reduced glutathione (gsh), glutathione peroxidase (gpx), superoxide dismutase (sod), and malondialdehyde (mda) but decreased catalase (cat) and zinc (zn) levels compared to control. co-treated taz + msg rats significantly (p < 0.05) decreased bwg, gsh, gpx, sod, zn; increased cat and non-significantly (p > 0.05) decreased mda compared to msg and control. thus, taz significantly mitigated bwg, and malfunction in the metabolism of antioxidant defense bioindicators in msg rats via probable anorexigenic, anti-inflammatory and antioxidant responses. this suggests that taz could be useful in managing msg-related dysfunction in bwg and metabolic activity of the antioxidant defense apparatus in rats. keywords: monosodium glutamate; azithromycin; organ toxicity; oxidative stress; antioxidants; body weight gain. abbreviations: anova = analysis of variance, azt = azithromycin, bwg = body weight gain, cat = catalase, gsh = reduced glutathione, gpx = glutathione peroxidase, mda = malondialdehyde, msg = monosodium glutamate, oaz = overdose azithromycin, ros = reactive oxygen species, rpm = rotor per minute, sd= standard deviation, sod = superoxide dismutase, spss = statistical package for social sciences, taz = therapeutic azithromycin, zn = zinc. introduction in the peak of the outbreak of the pandemic disease (viral pneumonia) caused by the 2019-novel coronal virus (2019-ncov, covid-19), the management was based mainly on scanty information (schünemann et al., 2019). initial studies indicated good improvement following inclusion of azt in the treatment of covid-19 (gautret et al., 2020) but some randomized clinical trials reported otherwise (principle trial collaborative group, 2021; recovery collaborative group, 2021). azithromycin, an azalide subclass of macrolides, is a broad spectrum antibiotic with antiviral and anti-inflammatory potencies that is recently exploited in the treatment of covid-19 (ismael and elsamman, 2022). the broad‐spectrum antibacterial activities of azt is related to its mechanism of action that inhibits protein synthesis of bacteria (by binding to the 50s subunit of the bacterial ribosome to inhibit translation of mrna) and prevent their growth (dinos, 2017). azithromycin is inexpensive, widely available, and with an excellent safety profile but is prone to selection for macrolide resistance following abuse (o’brien et al., 2019). azt has a long half-life duration and a high affinity to tissue penetration and accumulation (fohner et al., 2017). the largest tissue concentration of azt was observed in the liver, followed by the kidney and lastly the heart. and, the toxic manifestation in these organs due to azt was associated to its direct effect and that of its metabolites on ribosomal functions and long duration of the derivatives in the organs (ismael and elsamman, 2022). it was reported that azt can increase the risk of cardiovascular manuscript received: 22 december, 2023. revision accepted: 06 may, 2024. published: 07 may, 2024. https://doi.org/10.14421/biomedich.2024.131.43-50 44 biology, medicine, & natural product chemistry 13 (1), 2024: 43-50 complications leading to death (bergami et al., 2023) while the azt-induced nephrotoxicity can be reduced via antioxidant and anti-inflammatory pathways (ismael and elsamman, 2022). it was hypothesized that azt could exert activity against the covid-19-related disease (oliver and hinks, 2021) which may result to increased demand for, use and abuse of, azt; and possibly co-consumed with msg. monosodium glutamate is a commonly used flavour enhancer worldwide (el-gendy et al., 2023; wuyt et al., 2023). it is known to mediate enhanced bwg, organ toxicity (el-gendy et al., 2023; joshi et al., 2023; yang et al., 2023) and oxidative stress (obi and egbuonu, 2019). it was reported that msg interfered with the function of the peptide hormone, leptin, in the hypothalamus to mediate orexigenic activity (increased appetite) that could lead to increased body weight and metabolic syndrome (yang et al., 2023). suppression of leptin expression in the hypothalamus decreased energy expenditure and spiked the drive to eat leading to increased energy balance and body weight gain (martelli and brooks, 2023; obradovic et al., 2021). oxidative stress is known to increase inflammation and to underlie the onset and development of many health challenges (correia et al., 2023; kiran et al., 2023; wuyt et al., 2023) whereas increased bwg leading to obesity is frequently associated with inflammation and other diseases, including cancer, diabetes and non-alcoholic fatty liver (casado et al., 2023). the body employs antioxidant defense mechanism to combat free radicals associated with oxidative stress and also to decrease inflammation (ismael and elsamman; 2022; correia, et al., 2023). oxidative stress contributed significantly to msg-related toxic effects and eventual cellular and organ-functional damage in animals as assessed by antioxidant defense system markers, including gsh, gpx, sod, cat, zn and mda (egbuonu et al., 2021; correia, et al., 2023). concomitant intake of msg and azt may present with unknown effect on the bwg and antioxidants metabolism function of the animals which warranted this study. this study aimed to evaluate the effect of azt and msg on bwg and antioxidant defense bioindicators in rats’ serum, liver, kidney and heart. materials and methods chemicals and drug azithromycin tablets (500 mg) were obtained from a reputable pharmaceutical company (achina foundation pharmaceuticals limited), ariaria market aba, abia state, nigeria. msg was procured from foodstuff section of the market. other chemicals were of certified analytical grade. animals and treatments the animals used in this study were thirty (30) adult male wistar rats with average weight 101 – 170 g obtained from the animal house of the department of veterinary medicine, university of nigeria, nsukkka. they were kept in the animal house of the department of biochemistry, michael okpara university of agriculture umudike, nigeria for 1 week to acclimatize and randomly assigned to five groups of six rats each. group a rats, control, were given distilled water (1 ml kg−1). group b rats were fed therapeutic dose of azt (82.5 mg kg−1 body weight), whereas group c rats were fed overdose (therapeutic dose × 5) of azt (412.5 mg kg−1). group d rats were given intoxicating dose of msg (8000 mg kg−1) according to mariyama et al. (2009) whereas group e rats received msg (8000 mg kg−1) with therapeutic dose of azt (82.5 mg kg−1 body weight). treatment was by daily oral intubation for 7 successive days. the rats were housed in cleaned stainless steel cages at room temperature (28±2 0c); 12 h light/dark cycle and humid tropical conditions. animals were provided with rat feed (vital feed growers marsh containing 20 % crude protein and 280 kcal 100−1 g metabolizable energy, manufactured by vital feed industries limited, nigeria) and portable (tap) water ad libitum for the duration of the experiment. the study adhered to standard ethical practice with approval of the animal ethics committee of the host department and institution (ace-ofus/16-960862021). blood and tissues collection and preparation blood samples of the rats sacrificed following cervical dislocation 24 h after 7 days treatment were collected individually using sterile capillary tubes into properly labeled plain polystyrene centrifuge tubes by cardiac puncture technique. the blood samples thus collected were allowed to clot. then, the serum was removed by centrifugation at 3000 rotor per minute (rpm) for 5 minutes, collected individually and stored in a deep freezer for determination of the serum, antioxidant status bioindicators namely gsh, gpx, sod, cat, zn and mda. tissue samples (liver, kidney and heart) of the rats as sacrificed above were excised individually and homogenized. in brief, 10 % of the respective organ homogenate was obtained by separately grinding a 0.5 g of respective organ sample in 5 ml of phosphate buffer saline (ph 7.2), using mortar and pestle. the supernatant was removed by centrifugation at 1000 g for 10 minutes, collected individually and stored in a deep freezer for determination of liver, kidney and heart homogenate antioxidant status indicators namely gsh, gpx, sod, cat, zn and mda. determination of changes in rats bwg, and serum, liver, kidney and heart antioxidant defense bioindicators (gsh, gpx, sod, cat, zn and mda) the rats’ body weight gain (body weight change) was calculated as the difference in the measured initial and egbuonu et al. – azithromycin and monosodium glutamate effect 45 final body of the rats. the percentage body weight gain (body weight change) was computed as a fraction of the corresponding initial body weight multiplied by 100. the gsh concentration was determined with randox kit based on the method of goldberg and spooner (1983) as described recently (egbuonu and elendu, 2021). the gpx activity was determined by the method of paglia and valentine (1967) reported recently (egbuonu et al., 2021). this was based on the principle that glutathione peroxidase catalyzes the oxidation of reduced glutathione. as described recently in egbuonu and elendu (2021), the sod activity was assayed by the method of madesh and balasubramanian (1998); the cat activity was assayed by the method of johansson and borg (1988) while the mda concentration was determined by the method of wallin et al. (1993). the zn concentration was estimated by the method of johnsen and eliasson (1987) as reported recently (egbuonu et al., 2021). statistical analysis all analyses were performed by one way analysis of variance (anova), using the statistical package for social sciences (spss) for windows version 16.0 package. the least significant difference test was used for the post-hoc multiple comparison of means. differences in mean were considered significant at p < 0.05. the results were presented as mean ± standard deviation (sd) for 6 rats. results and discussion rats in msg group had the highest while those in oaz group had the least followed by taz and msg + taz (p < 0.05) body weight increase compared to control and the other groups (figure 1). figure 1. effect of azt and msg in rats’ bwg (each bar represents mean + sd; n = 6). as shown in figure 2, gsh concentration in the serum liver, kidney and heart was highest (p < 0.05) in msg group and least in these organs and in the serum in taz + msg group compared to control and others. similarly, gpx activity in the serum, liver, kidney and heart was highest (p < 0.05) in msg group and least in these organs and in the serum in taz + msg group compared to control and others (figure 3). figure 2. effect of azt and msg on gsh (mg/dl) concentration in rats’ serum, liver, kidney and heart (each bar represents mean + sd; n = 6). 10 1, 67 10 4, 65 10 4 10 3 10 1 16 6, 67 16 8, 67 16 7, 33 17 0 16 5 65 64 ,0 2 63 ,3 3 67 6463 ,9 3 61 ,1 7 60 ,8 9 65 ,0 4 63 ,3 6 c o n t r o l g r o u p a t a z g r o u p b o a z g r o u p c m s g g r o u p d t a z + m s g g r o u p e initial body weight (g) final body weight (g) change in body weight (g) percentage change in body weight (%) 5,25 5,68 10,71 4,95 3,3 81,3 83 81,33 87,33 68 78,5 80,8 77 87,4 66,8 79,1 81,4 77,6 88 67,4 0 10 20 30 40 50 60 70 80 90 100 control group a taz group b oaz group c msg group d taz + msg group e serum gsh (mg/dl) liver gsh (mg/dl) kidney gsh (mg/dl) heart gsh (mg/dl) 46 biology, medicine, & natural product chemistry 13 (1), 2024: 43-50 (each bar represents mean + sd; n = 6) figure 3. effect of azt and msg on gpx (iu/l) activity in rats’ serum, liver, kidney and heart. as depicted in figure 4, sod activity in the serum, liver, kidney and heart was highest (p < 0.05) in msg group but least in taz, oaz and taz + msg group compared to control. (each bar represents mean + sd; n = 6) figure 4. effect of azt and msg on sod (iu/l) activity in rats’ serum, liver, kidney and heart. cat activity in the serum, liver, kidney and heart decreased (p < 0.05) the least in msg group but most in taz group followed by taz + msg group compared to control and others (figure 5) while zn concentration in the serum, liver, kidney and heart decreased (p < 0.05) in the taz + msg group followed by msg group compared to control and others (figure 6). (each bar represents mean + sd; n = 6) figure 5. effect of azt and msg on cat (iu/l) activity in rats’ serum, liver, kidney and heart. 3,44 4,13 5,19 5,47 5,125,3 5,2 5,23 5,8 4,96 4,25 4,42 4,3 4,66 3,92 4,85 5,02 4,9 5,26 4,52 0 1 2 3 4 5 6 7 control group a taz group b oaz group c msg group d taz + msg group e serum gpx (iu/l) liver gpx (iul) kidney gpx (iul) heart gpx (iul) 10,9 11,27 11,16 11,38 11,24 9,96 9,93 9,93 10,06 9,93 12,9 13,27 13,16 13,38 13,2413,5 13,87 13,76 13,98 13,84 0 2 4 6 8 10 12 14 16 control group a taz group b oaz group c msg group d taz + msg group e serum sod (iu/l) liver sod (iu/l) kidney sod (iu/l) heart sod (iu/l) 4,48 1,14 2,1 2,74 1,97 6,88 4,28 6,65 6,78 6,66,48 3,14 4,1 4,74 3,97 7,08 3,74 4,7 5,34 4,57 0 1 2 3 4 5 6 7 8 control group a taz group b oaz group c msg group d taz + msg group e serum cat (iu/l) liver cat (iu/l) kidney cat (iu/l) heart cat (iu/l) egbuonu et al. – azithromycin and monosodium glutamate effect 47 (each bar represents mean + sd; n = 6) figure 6. effect of azt and msg on zn (µg/dl) concentration in rats’ serum, liver, kidney and heart. as shown in figure 7, serum, liver, kidney and heart mda concentration in all the treated groups increased (p < 0.05) and consistently highest (p < 0.05) in msg group but least (p < 0.05) in taz + msg group as compared to the control. (each bar represents mean + sd; n = 6) figure 7. effect of azt and msg on mda (mg/dl) concentration in rats’ serum, liver, kidney and heart. discussion the antibiotic, azt may be abused and co-consumed with msg to present unknown outcomes on bwg, organ function and oxidative stress which warranted this study aimed to evaluate the effect of azt and msg in rats’ bwg and antioxidant defense bioindicators. monosodium glutamate mediates bwg, organ toxicity and oxidative stress (joshi et al., 2023; yang et al., 2023). expectedly, msg-treated rats significantly (p < 0.05) increased bwg; increased gsh, gpx, sod and mda but decreased catalase (cat) and zinc (zn) in the serum, liver, kidney and heart of rats compared to control and others. these indicated adverse effect due to msg on bwg that could be pathologic in the rats. increased bwg leading to obesity is frequently associated with inflammatory responses and many diseases (casado et al., 2023). msg-induced enhancement in bwg of rats could be owing to orexigenic potential of msg (yang et al., 2023) which involves down-regulation in the expression of leptin hormone in the hypothalamus leading to decreased energy utilization, whetted appetite, increased energy balance and consequently increased bwg as reported herein (obradovic et al., 2021; martelli and brooks, 2023). the results of the effect of msg on the antioxidant defense bioindicators indicated msginduced overall toxicity in the rats following dysregulation in the metabolism and collapse of the antioxidant defense mechanisms leading to oxidative stress in the rats’ serum, liver, kidney and heart. msginduced enhancement in bwg leading to obesity was linked with other pathologies (el-gendy et al., 2023) which were enhanced by onset of oxidative stress (wuyt et al. 2023). concordantly, increased mda concentration and decreased cat activity as in this study indicated onset of oxidative stress (ayoola et al., 2019; el-gendy et al., 2023) whereas increased sod but decreased zn as 3 0 ,7 3 1 ,8 4 3 1 ,2 5 2 9 ,1 2 2 6 ,5 8 3 2 ,1 7 3 4 ,9 3 2 ,2 6 3 0 ,6 2 9 3 2 ,7 3 3 ,8 4 3 3 ,2 5 3 1 ,1 2 2 8 ,5 83 3 ,3 3 4 ,4 4 3 3 ,8 5 3 1 ,7 2 2 9 ,1 8 c o n t r o l g r o u p a t a z g r o u p b o a z g r o u p c m s g g r o u p d t a z + m s g g r o u p e serum zn (µg/dl) liver zn (µg/dl) kidney zn (µg/dl) heart zn (µg/dl) 0,56 1,08 1,77 1,06 1,03 0,6 0,99 1 1,01 0,98 2,56 3,08 3,77 3,06 3,03 3,18 3,68 4,37 3,66 3,63 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 control group a taz group b oaz group c msg group d taz + msg group e serum mda (mg/dl) liver mda (mg/dl) kidney mda (mg/dl) heart mda (mg/dl) 48 biology, medicine, & natural product chemistry 13 (1), 2024: 43-50 reported herein reflected extent of defensive role of sod and its metal co-factor, zn against oxidative stress (sciskalska et al., 2020). earlier, it was reported that oxidative stress contributed significantly to msg-related cellular and organ toxicity in animals (egbuonu et al., 2021; correia, et al., 2023). in line with the results of the present study also, generation of reactive oxygen species (ros) evidenced by increased gsh content as in this study was ascribed to a collapse in antioxidant defense system due to onset of oxidative stress (elwahab et al., 2020). and, msg especially at high dose as used herein mediated enhanced bwg, organ toxicity and oxidative stress (obi and egbuonu, 2019; joshi et al., 2023; yang et al., 2023). collapse in the antioxidant defense mechanism to effectively combat free radicals resulted to oxidative stress and increased inflammatory responses (ismael and elsamman; 2022; correia, et al., 2023) while oxidative stress and increased inflammatory responses due to increased bwg were associated with the onset and development of varied ailments (kiran et al., 2023; correia, et al., 2023). intriguingly, gpx activity increased due to msg treatment. however, in a recent study, handy and loscalzo (2022) reported active involvement of gpx in the activation of proinflammatory pathways. it is probable that the enzyme activity increased because it was channeled only to the activation of pro-inflammatory response by msg without being depleted instead of being depleted by combating the ros generated due to msg assault in the rats. this is an important deduction on the response of msg on gpx role in animals, suggesting selective activity of gpx in msg metabolism and warranting further studies. thus, the study demonstrated msginduced enhancement in bwg and significant liver, kidney and heart tissues toxicity evidenced by a compromised antioxidant defense system following onset of oxidative stress. further studies are required to ascertain whether the present observation has pathologic effect in the rats, especially the studied organs. azt caused a dose dependent decrease in the bwg of rats in taz, indicating a consistent bwg slimming potentials of azt which suggests its capacity to mitigate msg-induced enhancement in bwg in the rats. the noted body weight reducing property of azt could be via anorexigenic activity (yang, et al., 2023) and antiinflammatory response (ismael and elsamman, 2022) to counter the orexigenic potential of msg and the attendant inflammatory responses in the rats. however, the overriding significant body weight reduction caused by azt in oaz-treated rats as compared to control warrants detailed investigation as this may be a notable shortcoming due to overdose consumption of azt. the possibility of the role of taz in mitigating the effect of msg was explored further. results revealed that cotreated taz + msg rats significantly (p < 0.05) decreased bwg; decreased gsh, gpx, sod, zn, mda and increased cat in the serum, liver, kidney and heart of rats compared to msg treated group. the observations herein were important indicators of wellbeing in animals. in line with the current observations, comparatively decreased bwg (okereke et al., 2023), gsh (elwahab et al., 2020) and mda (ayoola et al., 2019; el-gendy et al., 2023), but increased cat (ayoola et al., 2019) indicated beneficial responses on the metabolic, functional and overall health capacities in animals. herein, anti-inflammatory response was adduced as a possible mechanism of azt action against msg assault, so a decrease in gpx activity as reported herein is also a beneficial response in the rats. active involvement of gpx is necessary for the activation of pro-inflammatory pathways (handy and loscalzo, 2022), implying that the decrease in gpx could be an indirect indication of the extent of anti-inflammatory response of azt in counteracting the apparent pro-inflammatory response mediated following msg assault in the rats. this is in line with the selective activity of gpx in msg metabolism suggested in this study. similarly, decreased sod and its metal co-factor, zn relative to msg treatment as recorded herein is reflective of tazmediated active participation of sod in antiinflammatory response against msg-induced proinflammatory response in the rats. in support of this suggestion, sciskalska et al. (2020) confirmed the antiinflammatory role of sod. thus, these results indicated significant capacity of taz to mitigate msg-induced enhancement in bwg and compromised metabolic activity of the antioxidant defense bioindicators in the rats via probable anorexigenic activity (yang, et al., 2023), anti-inflammatory and antioxidant responses (ismael and elsamman, 2022). this is intriguing as azt can induce organ toxicity (bergami et al., 2023), has long lasting half-life and capacity to penetrate and accumulate in organs (fohner et al., 2017) whereas the azt-induced organ toxicity can be mitigated through antioxidant and anti-inflammatory responses (ismael and elsamman, 2022). however, at therapeutic dose as used herein azt showed an excellent safety profile (o’brien et al., 2019). and, aside antibiotic and antiviral activities, azt exhibits anti-inflammatory activity (ismael and elsamman, 2022). it is therefore plausible that in the interaction of taz and msg, azt expressed anorexigenic, antioxidant and anti-inflammatory responses in order to mitigate the msg-induced effects observed in this study. to our knowledge, this is the first study reporting the concomitant effect of azt and msg on bwg and these antioxidant defense bioindicators in rats. thus, this is a novel finding that deserve follow-up in subsequent studies aimed at providing further insights on the biochemistry, pharmacology and mechanistic responses of msg and azt in rats. egbuonu et al. – azithromycin and monosodium glutamate effect 49 conclusions thus, taz significantly mitigated bwg, and malfunction in the metabolism of antioxidant defense bioindicators in msg rats via probable anorexigenic, anti-inflammatory and antioxidant responses. this suggests that taz could be useful in managing msgrelated dysfunction in bwg and metabolic activity of the antioxidant defense apparatus in rats. the outcome of this study deserves follow-up in subsequent studies aimed at providing further insights on the biochemistry, pharmacology and mechanistic responses of msg and azt in rats. authors’ contributions: egbuonu a.c.c., alaebo p.o. and onuoha u.n. designed and supervised the study. njoku c.j. and eze o.b., assembled the draft manuscript. odoemelam f.u., edum m.e., obi o.b., ukaegbu m.j., nwaogwugwu s.u., orji m.c., ndukwe c.n., opara p., oyoyo c., joe-eme c.b. and okwoigwe c.j., carried out the laboratory work under supervision. all authors read and approved the final version of the manuscript. acknowledgement: the corresponding author sincerely acknowledges the technical contribution of the laboratory staff in college of natural sciences, colnas, of the host institution. competing interests: the authors declare that there are no competing interests. funding: the authors declare no funding. references ayoola, m.b., ejiofor, n.c., ezeagu, i.e., & achukwu, p. 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(2023). a multifaceted review of monosodium glutamate effects on human health and its natural remedies. food materials research, 3, 16. https://doi.org/10.48130/fmr-2023-0016. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 65-73 | doi: 10.14421/biomedich.2022.111.65-73 issn 2540-9328 (online) ethnomedicinal, phytochemicals, and pharmacological aspects of sentul (sandoricum koetjape) made dharmesti wijaya pharmacology and pharmacy department, faculty of medicine and health sciences, warmadewa university, jl. terompong no 24 denpasar 80235, indonesia. tel. +62 361 240727. corresponding author dharmestiwijaya@gmail.com manuscript received: 09 march, 2022. revision accepted: 05 april, 2022. published: 18 april, 2022. abstract sentul (sandoricum koetjape) is a tropical plant that has been used as traditional medicine in some asian countries for decades. research on phytochemicals and pharmacological activities of this plant extracts has been conducted and shows promising medicinal properties. this review aims to integrate knowledge about s. koetjape focusing on three main aspects namely ethnomedicinal, phytochemicals, and pharmacological, in order to encourage further research on this plant for future drug development. traditionally, all plant parts of s. koetjape have been used for treating various health problems and diseases such as diarrhea, fever, colic, and leucorrhoea. more than 30 chemicals have been identified from s. koetjape, which the most important compounds are ring-a secotriterpene, oleane-type triterpene, secomultiflorane-type triterpene, hydroxymultiflorane triterpene, and limonoids. in vitro studies showed pharmacological potential of the extracts and phytochemicals constituents of s. koetjape including antibacterial, antifungal, antitumoral, anticancer, insecticide, and antioxidant. keywords: ethnomedicine; pharmacology; phytochemicals; sandoricum koetjape; sentul. introduction since prehistoric times, human have been utilized medicinal plants for their health benefits, as evidently shown by fossil records from the middle paleolithic age, approximately 60,000 years ago (solecki, 1975; fabricant and farnsworth, 2001). the oldest written evidence of the utilization of the medicinal plants for drugs preparation has been verified by the 5000 years old sumerian clay slab which described 12 drug synthesis recipes by referring to more than 250 plants (srivastava, 2018). in addition, traditional medicines such as ayurveda, traditional chinese medicine, unani, traditional korean medicine, and kampo have applied medicinal plants to alleviate and treat wide range of diseases such as malaria, diarrhoea, and microbial infections (yuan et al. 2016). currently, it is estimated that about 80% of the world’s population still depend on herbs and herbal products for primary health care (subramani et al. 2017). despite the fact that traditional medicinal plants have been established through empirical practices and evidences, their importance have often been undervalued and disregarded (kaliyaperumal et al. 2013). it is estimated that only less than 10% of the world’s medicinal plant biodiversity has been scientifically studied for their potential pharmacological activities (dias et al. 2012). the awareness of medicinal plants and their ethnopharmacological relevance is crucial to discover novel plant-based drugs and medicines, as well as in disease prevention (sofowora et al. 2013; jamshidi-kia et al. 2018). plants with ethnopharmacological practices have become the main sources of pharmaceuticals in early drug discovery. therefore, efforts must be directed towards measures that will improve the efficacy, effectiveness, and rational use of medicinal plants (veeresham, 2012; sofowora et al. 2013). among the traditional medicinal plants, members of mahogany or meliaceae family have been regarded for their medicinal properties. this family consists of approximately 1400 species belonging to more than 50 genera. modified triterpene compounds called limonoids are abundantly found in meliaceae and are chemically more diverse in this family compared to any other plant families (paritala et al. 2015). a large quantity of other triterpenoid derivatives as well as other compounds such as flavonoids, alkaloids, phenols, coumarins, lignans, and chromones are also present in different meliaceae genera (paritala et al. 2015; yadav et al. 2015). these very diverse compounds are responsible for the wide range of medicinal properties of meliaceae such as antiviral, antiparasitic, and antimicrobial activities, as well as cytotoxic, anticancer, and anti-inflammatory properties (paul et al. 2011; https://doi.org/10.14421/biomedich.2022.111.65-73 mailto:dharmestiwijaya@gmail.com 66 biology, medicine, & natural product chemistry 11 (1), 2022: 65-73 xavier-jr et al. 2015; yadav et al. 2015; mubeen et al. 2018). the members of this family have been traditionally used for treating a wide range of diseases (sujarwo et al. 2016; agyare et al. 2018). among the meliaceae family, only a few members have edible fruits, in which sandoricum koetjape (burm.f.) merr. is one of the most popular species (yadav et al. 2015). the species s. koetjape belongs to genus sandoricum cav, that is distributed mainly in tropical areas in asian countries and among plant that have been utilized for traditional medicines (ismail et al. 2003a). the root, bark, leave, and the whole plant of s. koetjape are used in folk medicine for generations in some asian countries such as india, malaysia, indonesia, and thailand for treating various diseases such as diarrhea, leucorrhea, colic, or drunk as a tonic after childbirth (perry and metzger, 1980; kaneda et al. 1992; ismail et al. 2003a). a number of phytochemicals and bioactive compounds from s. koetjape have been identified and tested in vitro which showed large therapeutic potentials (ismail et al. 2003a; aisha et al. 2009a, b; chudzik et al. 2015;). this review aims to comprehensively integrate current knowledge on s. koetjape by focusing into three main aspects namely ethnomedicinal, phytochemistry, and pharmacology. moreover, we discussed toxicological aspects that have not been studied from s. koetjape and some issues to translate the therapeutic capacity of this plant into consumable product. botany and ethnomedicinal aspects of sandoricum koetjape s. koetjape is possibly indigenous plants of indochina and peninsular malaysia, and later the plant has been introduced and naturalized in the philippines, borneo, indonesia, india, the andaman islands, mauritius, australia, taiwan, china, and also into a few other locations in southern florida and central america (lim, 2012). s. koetjape is locally called kechapi, lolly fruit, santol, sentol, wild mangosteen (english), faux mangostan, and sandorique mangousteiner savage (french). this plant is classified into the genus sandoricum, of the meliaceae family, order sapindales, and division tracheophyta (barstow, 2018). other synonymies for s. koetjape are melia koetjape burm.f., sandoricum indicum cav., sandoricum maingayi hiern, sandoricum nervosum blume, sandoricum nervosum (vahl) m.j. roem., sandoricum vidalii merr., trichilia nervosa vahl (lim, 2012; mabberley, 1985). distribution of s. koetjape is mainly in primary and secondary rain forests below 1000 m which are characterized by deciduous, small to large tree, up to 50 m tall with a straight trunk, flaky or fissured, lenticillate, greyish to pale pinkish-brown bark (orwa et al. 2009; lim, 2012). the tree bole morphology of s. koetjape is straight but often crooked or fluted, branchless for up to 18–21 m and with a trunk diameter up to 100 cm (lim, 2012). bark surface smooth or sometimes flaky or fissured, lenticillate, greyish to pale pinkish-brown, inner bark pale brown or red-brown to pink, exuding a milky latex (orwa et al. 2009). it produces fleshy fruits that are round or flattened ball-shaped, yellow or brownish, and 5-8 cm across, with arils part range from sour to sweet (figure 1) (chen et al. 2015). the fruit has 3 5 brown, ovate to ellipsoid seeds, 2–3.5 by 1.2–2.1 by 0.9–1.6 cm, which are usually tightly associated to the pulp (chen et al. 2015). figure 1. sandoricum koetjape fruits. traditionally, s. koetjape has been applied as medications to treat a number of diseases in thailand, malaysia, philippines, and indonesia. in thailand, this plant is locally known as krathon or sathon and its bark decoction is traditionally used to treat diarrhea (kaneda et al. 1992). meanwhile, the aqueous extract of s. koetjape or santol bark in malaysia is consumed after childbirth as a tonic (nassar et al. 2010). moreover, the stem bark of s. koetjape or locally called sentul or kecapi in indonesia is used by local people for colic, leucorrhoea, and stomach ache treatment (kosela et al. 1995; novaryatiin and indah, 2019). moreover, the pounded bark is applied for treating ringworm (perry and metzger, 1980). beside the bark, other plant parts of s. koetjape such as leaves and roots are also conventionally used for several diseases treatments. decoction of the leaves of s. koetjape is being used to treat diarrhea and water from the pounded leaves is used as intermittent fever medication (perry and metzger, 1980). in the philippines, fresh leaves of s. koetjape are put on the body to induce sweating while santol herbal tea is used wijaya – ethnomedicinal, phytochemicals, and pharmacological aspects of s. koetjape 67 to bath to bring down fever (lim, 2012). the leaves are also used for inflammation or swelling poultice by applying directly to the affected area (agapin, 2020). the root is used to cure leucorrhoea, dysentry, and as general tonic (perry and metzger, 1980). the decoction or infusion of s. koetjape roots is also used for diarrhea and spasm treatment (cabi, 2008). a record of balinese traditional healing therapies written in palm leaves called taru pramana stated that “loloh” or traditional herbal drink of s. koetjape roots and leaves is employed to treat diarrhea, while the bark can be chewed and then sprayed into the stomach (pulasari, 2013). the bark and leaves of s. koetjape are also used in treating diarrhea by the baduy ethnic in indonesia (khastini et al. 2021). phytochemicals of sandoricum koetjape the phytochemicals constituent of s. koetjape has been examined since 1960. to date, more than 30 compounds have been isolated from different parts of this plant, in which triterpenes are the ubiquitous plant components. various group of triterpenes were identified in this plant such as ring-a secotriterpene, oleane-type triterpene, secomultiflorane-type triterpene, hydroxy-multiflorane triterpene, and limonoids. besides, sesquiterpenes and polyalcohols were also detected in stem and fruit hulls of s. koetjape. polyphenols such as quercetin (flavonoid) and tannin were also observed in the plant extracts of s. koetjape (table 1). table 1. compounds identified from the sandoricum koetjape extracts. class compounds plant parts ref triterpenoind acids katononic acid (3-oxo-olean-12-en-29-oic acid) stem (kaneda et al. 1992) katonic acid (3α-hydroxyolean-12-en-29-oic acid) heartwood (king and morgan, 1960) indicic acid heartwood (king and morgan, 1960) bryonolic acid fruit hulls (sim and lee, 1972) ring-a secotriterpene koetjapic acid stem (kaneda et al. 1992) sentulic acid bark (efdi et al. 2012) oleane-type triterpenoid 3-oxo-olean-12-en-27-oic acid bark (efdi et al. 2012) 20-epikoetjapic acid (3,4-seco-olean-4(23),12-diene3,29-dioic acid) stem bark (tanaka et al. 2001) 3-epikatonic acid stem bark (tanaka et al. 2001) briononic acid stem bark (tukiran et al. 2010) secomultiflorane-type triterpene bryononic acid fruit hulls, stem bark (sim and lee, 1972; kosela et al. 1995) secobryononic acid stem bark (kosela et al. 1995) secoisobryononic acid stem bark (kosela et al. 1995) 12β-hydroxymultiflorane triterpenoid acid sandorinic acid a (12β,18-dihydroxy-3oxomultiflora-8-en-29-oic acid) stem bark (tanaka et al. 2001) sandorinic acid b (12β,18-dihydroxy-3oxomultiflora-7-en-29-oic acid) stem bark (tanaka et al. 2001) sandorinic acid c (12β-hydroxy-3-oxomultiflora-8en-29-oic acid) stem bark (tanaka et al. 2001) limonoids/ tetranorterpenoid [2α-(2-methylbutanoyl)oxy]sandoricin leaves (pancharoen et al. 2005) [2α-(2-methylpropanoyl)oxy]sandoricin leaves (pancharoen et al. 2005) sanjecumins a and b leaves (nagakura et al. 2013) trijugin-class limonoids sandrapins a-c leaves (ismail et al. 2003b) sandrapins d-e leaves (ismail et al. 2005) koetjapin d seed (bumi et al. 2019) andirobin-class limonoids sandoripin a and b leaves (pancharoen et al. 2009; nagakura et al. 2013) sandoricin seed (powell et al. 1991) 6-hydroxysandoricin seed (powell et al. 1991) koetjapins a-c seed (bumi et al. 2019) sesquiterpenes (–)-alloaromadendrene (–)-caryophyllene oxide (+)-spathulenol stem (kaneda et al. 1992) polyalcohols mesoinositol fruit hulls (sim and lee, 1972) dimethyl mucate fruit hulls (sim and lee, 1972) flavonoid quercetin n/d (kaewkod et al. 2021) tannin n/d (kaewkod et al. 2021) note: n/d = not determined 68 biology, medicine, & natural product chemistry 11 (1), 2022: 65-73 pharmacological activities of sandoricum koetjape antibacterial activity new antibiotic agents are urgently needed due to high antibiotics resistance incidence worldwide that threaten our action to combat serious bacterial infections (mayor, 2018). plants produce a wide range of phytochemicals and secondary metabolites that have therapeutic properties and therefore they are one of the most crucial sources of antimicrobial agents. a study by subramani and co-workers reported that a total of 60 plant extracts and 110 purified compounds were acquired from 112 plants against multi-drug resistant (mdr) pathogens including mdr-mycobacterium tuberculosis, methicillin resistant staphylococcus aureus (mrsa), and plasmodium spp. between 2005 and 2015 (subramani et al. 2017). plants belong to meliaceae family have been shown to have antibacterial activity, including s. koetjape. a number of antibacterial activities have been documented from this plant (table 2). methanol extract of s. koetjape seed seems promising as it shows strong antibacterial activity with minimum inhibitory concentration (mic) at 0.25, 0.50, and 0.50 µg/ml against bacillus subtilis, pseudomonas aeruginosa, and staphylococcus aureus respectively (azziz et al. 2013). meanwhile, the aqueous extract of the seed exhibit weak antibacterial property with mic at 250 mg ml-1 against escherichia coli, s. aureus, candida albicans, and streptococcus pneumoniae (elijah et al. 2016). the observed antibacterial activities in s. koetjape could be related to arrays of secondary metabolites such as alkaloids, flavonoids, and phenolic compounds found in this plant. flavonoid is known by its ability to interact with bacterial membrane proteins, causing the increase in membrane permeability and lead to membrane disruption (gupta and birdi, 2017). terpenoids that ubiquitously present in this plant have been reported to display antibacterial in nature (gupta and birdi, 2017). table 2. antibacterial activity of sandoricum koetjape extracts. solvents suggested constituents property target ref mic zoi seed methanol alkaloid, flavonoid 0.25 µg ml-1 b. subtilis (azziz et al. 2013) 0.50 µg ml-1 p. aeroginosa 0.50 µg ml-1 s. aureus aqueous phenols 250 µg ml-1 e. coli, s. aureus, c. albicans, and s. pneumoniae (elijah et al. 2016) leaf aqueous phenols 100 µg ml-1 e. coli and s. aureus (elijah et al. 2016) fruit juice phenols 8.3±0.6 mm e. faecalis (toobpeng at al. 2017) 13.0±1.0 mm p. aeruginosa mdr1 14.0±1.0 mm e. coli p174 esbl 15.0±0.6 mm e. coli esbl 15.0±1.5 mm p. aeruginosa mdr2 15.0±1.0 mm a.baumannii mdr2 15.0±1.5 mm a.baumannii mdr1 16.0±1.5 mm s. aureus mrsa1&2 fruit hulls bryononic acid (triterpenoid) 6 µg ml-1 salmonella enterica (heliawati et al. 2019) root aqueous n/a 500 µg ml-1 11 mm s. pyogenes nprc 101 (limsuwan and voravuthikunchai, 2013) ethanol n/a >1000 µg ml-1 15 mm s. pyogenes nprc 101 plant extracts aqueous tannin, quercetin (flavonoid) 16 mg ml-1 10.3±0.6 mm e. coli atcc 25922 (kaewkod et al. 2021) 63 mg ml-1 10.3±0.6 mm e. coli k-12 note: n/a = not available; mic = minimum inhibitory concentration; zoi = zone of inhibition anticancer/antitumor activity s. koetjape is known to be rich in triterpene compounds. in recent years, triterpenoids-related studies show that these compounds have potential roles for tumor or cancer prevention and treatments (gill et al. 2016; patlolla and rao, 2012). although many triterpene compounds have been proven to give effective results in treating cancers, only some of them have passed the wijaya – ethnomedicinal, phytochemicals, and pharmacological aspects of s. koetjape 69 clinical trial (e.g. 12-dioxooleana-1,9(11)-dien-28-oic acid or cddo) (gill et al. 2016). a number of triterpenoids isolated from s.koetjape such as katonic acid, sandorinic acid a, sentulic acid, and koetjapic acid have been noted to have cytotoxic activity against leukemia, colon, and breast cancer cell lines (table 3) (kaneda et al. 1992; tanaka et al. 2001; efdi et al. 2012; nassar et al. 2012a). koetjapic acid has also shown to have cancer chemopreventive and antitumor properties, as well as antimetastatic and antiinflamation activities (ismail et al. 2003a; rasadah et al. 2004; nassar et al. 2012b). table 3. anticancer and antitumor activity of sandoricum koetjape extracts. type of extracts suggested constituents dosage/results ref seed methanol koetjapin d cytotoxic activity against murine leukemia p-388 cell lines with ic50 of 16.8 ± 1.8 µg/ml (bumi et al. 2019) stem diethyl ether 1) 3-oxo-olean-12-en29-oic acid 2) katonic acid cytotoxic activity against p-388 leukemia cells (ed50 0.61 µg/ml (1) and 0.11 µg/ml (2)) (kaneda et al. 1992) bark purified compounds 1) sentulic acid 2) 3-oxo-olean-12-en27-oic acid cytotoxic activity against human promyelocytic leukemia hl-60 cell line by inducing apoptosis (efdi et al. 2012) hexane koetjapic acid cancer chemopreventive. significantly delayed tumor promotion in two-stage mouse skin carcinogenesis (ismail et al. 2003a) hexane 1) koetjapic acid 2) 3-oxo-olean-12-en29-oic acid 3) katonic acid anti-tumor promoting agents. inhibit epstein-barr virus early antigen (ebv-ea) activation (ismail et al. 2003a) stem bark purified compound sandorinic acid a cytotoxic activity against human leukemia hl-60 cells (ic50 15 µg/ml) (tanaka et al. 2001) n-hexane n/a ic50 values of 23, 14, 50, and 52 µg/ml against human umbilical vein endothelial cell (huvec), human colon cancer cells hct-116 and ht-29, and normal cell line ccd-18co (aisha et al. 2009b) n-hexane n/a 50 µg/ml extract showed potent apoptotic cell death induction on hct-116 colon cancer cell by inducing caspases 3 and 7 activity (aisha et al. 2009b) purified compound koetjapic acid cytotoxic activity with ic50 value of 18.88 µg/ml against hct-116 colon cancer cells by inducing caspase-3/7, -8, and -9, inducing morphological changes and nuclear condensation, causing dna fragmentation, disrupting mitochondrial membrane potential, downregulating wnt, hif-1α, map/erk/ jnk, and myc/mac signalling pathways, up-regulating nf-κb signalling pathway (nassar et al. 2012a) synthetic potassium koetjapate (salt form of koetjapic acid) enhanced cytotoxicity against hct-116 cells compared to koetjapic acid (jafari et al. 2014) n-hexane n/a ic50 values between 44 – 48 µg/ml against breast cancer cells mcf7, mda-mb-231, and t47d, and normal cell line mcf-10a (aisha et al. 2009a) n-hexane n/a 100 µg/ml extract showed apoptotic cell death induction on mcf-7 breast cancer cell by inducing caspases 3 and 7 activity (aisha et al. 2009a) methanol koetjapin d cytotoxic activity against murine leukemia p-388 cell lines with ic50 of 16.8 ± 1.8 µg ml-1 (bumi et al. 2019) purified compound koetjapic acid cytotoxic activity with ic50 value of 68.88 µg/ml against mcf-7 breast cancer cells; significantly inhibit cell migration and invasion at 15 µg/ml (sub-toxic dose); significantly inhibit the colony formation properties of mcf-7 (nassar et al. 2012b) other activities triterpenes such as koetjapic acid, katonic acid, and 3oxo-olean-12-en-29-oic acid are found to inhibit dna polymerase β (sun et al. 1999; hu et al. 2004). the mentioned compounds and sentulic acid are also known to have antiinflammation properties (rasadah et al. 2004; itoh et al. 2018;). limonoids compounds isolated from s. koetjape leaves namely sandoripins a and b exhibit antioxidant activity by inhibiting no production in j774.1 cell line 70 biology, medicine, & natural product chemistry 11 (1), 2022: 65-73 (nagakura et al. 2013). in addition, phenolic content and flavonoid from s. koetjape fruit extract show antioxidant activity by decreasing ros production and increasing antioxidant enzyme gpx-1 (anantachoke et al. 2016). tannins extracted from methanolic extract of the stem bark also display radical scavenging activity (cavin et al. 1999). beside aforementioned pharmacological activities, s. koetjape extracts also show antiangiogenic, antifungal, antifeedant, ichthyotoxic, and insecticidal against lepidopteran larvae (tabel 4) (mikolajczak and reed, 1987; powell et al. 1991; cavin et al. 1999; ismail et al. 2003a; leatemia and isman, 2004;). table 4. other activities of sandoricum koetjape extracts. bioactivities extract/ constituents dosage/results ref seed antifeedant ethanol, hexane ethanol and hexane extracts strongly inhibited feeding and resulted in high mortality (feeding ratio 0.05 and 0.21; mortality 90 and 100% respectively) of fall armyworm, s. frugiperda (mikolajczak and reed, 1987) sandoricin and 6hydroxysandoricin 100% effective against larvae of spodoptera frugiperda and ostrina nubilalis at 200 ppm or above. (powell et al. 1991) insecticidal ethanol ineffective (49-97% larval growth – relative to control) (leatemia and isman, 2004) leaves antioxidant 1) sandoripins a 2) sandoripins b inhibit no production with ic50 of 16.4 µm (1) and 30.4 µm (2) in mouse macrophage-like j774.1 cells stimulated by lps (nagakura et al. 2013) fruits antioxidant phenolic content, flavonoid 1 mg/ml extract shows dpph scavenging activity of 84.73% (ic50 of 415.8 µg/ml); supressed ros production that induced by h2o2; significantly increase the protein level of an antioxidant enzyme gpx-1 in human embryonic kidney hek-293 cell line (anantachoke et al. 2016) bark ichthyotoxic 1) koetjapic acid 2) 3-oxo-olean-12en-29-oic acid ichthyotoxic activity with tlm of 1.8 and 1.9 ppm respectively for compounds 1 and 2 (ismail et al. 2003a) stem antiinflammation 1) 3-oxo-olean-12en-29-oic acid; 2) katonic acid 3) koetjapic acid oedema inhibition of 94% (crude methanolic extract); 100% (dichloromethane fraction); 90% (hexane fraction); 77% (methanol fraction); 64% (ethyl-acetate fraction); 14% (water fraction); 94% (1); 81% (2); 13% (3) (rasadah et al. 2004) stem bark antiinflammation sentulic acid (purified compound) reduced the production of nitric oxide after co-stimulation with lps/ifnγ in raw264.7 cell line by inhibiting the binding of lps to tlr4f (itoh et al. 2018) antioxidant tannins show radical scavenging activity against dpph radical (cavin et al. 1999) antifungal 1) dichloromethane extract 2) methanol extract active against candida albicans (1) and cladosporium cucumerinum (2) (cavin et al. 1999) antiangiogenic n-hexane and methanol extract n-hexane and methanol extracts showed 97% and 90% blood vessel outgrowth inhibition using rat aortic ring assay (aisha et al. 2009c) n-hexane extract 100 µg/ml extract showed 94±5.5% blood vessel outgrowth inhibition using rat aortic ring assay; and ic50 values of 23, 14, 50, and 52 µg/ml against human umbilical vein endothelial cell (huvec), human colon cancer cells hct-116 and ht-29, and normal cell line ccd-18co (aisha et al. 2009b) koetjapic acid (purified compound) 20 µg/ml and 40 µg/ml koetjapic acid in ethanol showed 50% and 100% vascularitation inhibition using rat aortic ring assay; noncytotoxic against huvecs (ic50 40.97 ± 0.37 µg/ml) (nassar et al. 2011) potassium koetjapate (salt form of koetjapic acid) supressed angiogenesis by inhibiting endothelial functions and expression of angiogenic cytokine vegf (jafari et al. 2020) stem bark and wood dna polymerase β inhibitor 1) 3-oxo-olean-12en-29-oic acid 2) katonic acid 3) koetjapic acid ic50 values of 22, 36, and 20 µm for dna polymerase β inhibitors compounds 1-3 respectively (hu et al. 2004; sun et al. 1999) wijaya – ethnomedicinal, phytochemicals, and pharmacological aspects of s. koetjape 71 drawbacks and future direction of research many studies on antibacterial activity screening of s. koetjape were mostly carried out using crude extracts. the results seem promising, but nevertheless has limited impact on further drug development since crude extracts contain many types of compounds with different activities, side effects, as well as toxic effects. researches related to antitumor and anticancer activity of s. koetjape are generally more focused on a specific compound which would be more convenient to be developed into further step in drug development. a milestone in developing a new therapeutic agent from s. koetjape has been reached by jafari and co-workers (2020). their research chemically modifies the poorly soluble koetjapic acid into more soluble form namely potassium koetjapate, and thus enhanced its antiangiogenesis efficacy in rats. in the future, research about discovering medicinal properties of s. koetjape should be focus more on a single compound, rather than the whole extract. further preclinical and clinical research are also needed to develop the promising compounds contained in s. koetjape as new therapeutic agents against a wide range of diseases. conclusions in conclusion, phytochemicals of s. koetjape have been reported to have promising bioactivities that can potentially be used for therapeutic applications. however, more knowledge is required regarding to mode of actions, biosynthetic pathways, and toxicological aspects of these identified compounds. importantly, many of reported bioactivities were conducted in vitro, while compelling evidence of the application of such compounds from in vivo studies are rather limited. toxicological aspects are especially the utmost important particularly to determine the limit concentrations that are safe to be applied to treating such diseases. acknowledgements: we would like to thank the faculty of medicine and health sciences, warmadewa university, bali-indonesia for providing financial support for this study. competing interests: the author declares that there are no competing interests. references agapin, j. s. 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(2016). the traditional medicine and modern medicine from natural products. molecules, 21(5). doi:10.3390/molecules21050559 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 993-1007 | doi: 10.14421/biomedich.2025.142.993-1007 issn 2540-9328 (online) antioxidant and anti-inflammatory properties of peptide fractions of morinda lucida and alstonia boonei and protective effects against lead-induced toxicity in drosophila melanogaster zainab abiodun molik1, amos olalekan abolaji2*, omonike oluyemisi ogbole1* 1department of pharmacognosy, faculty of pharmacy, university of ibadan, ibadan, nigeria. 2drug metabolism and toxicology unit, department of biochemistry, college of medicine, university of ibadan, ibadan, nigeria. corresponding author* nikeoa@yahoo.com, amos_abolaji@yahoo.com manuscript received: 23 june, 2025. revision accepted: 27 october, 2025. published: 30 october, 2025. abstract morinda lucida and alstonia boonei are widely used in ethnomedicine for treating and managing various ailments and have been validated for several biological activities. this study investigated the antioxidant and anti-inflammatory properties of partially purified peptide fractions of morinda lucida (mlp) and alstonia boonei (abp) and their protective effect against lead (pb)-induced toxicity on wild-type drosophila melanogaster. peptide fractions were partially purified using solid phase extraction and evaluated for antioxidant activities using 2,2-diphenyl-1-picrylhyhdrazyl (dpph) radical scavenging activity and ferric reducing antioxidant power (frap) assays. anti-inflammatory activity was assessed via protein denaturation and membrane stabilisation assays. flies were treated with pb (10 mm), peptide fractions (50 and 100 µg/10g diet), and co-treatment of pb plus the fractions. after treatment, homogenized flies were analysed for total thiol (tsh) and non-protein thiol (npsh) content, glutathione-s-transferase (gst) activity, nitric oxide (no) (nitrite/nitrate) and hydroperoxide levels. the peptide fractions showed significant in vitro antioxidant and anti-inflammatory properties. both fractions (50 and 100 µg/10g diet) maintained a balanced redox status of flies. pb exposure reduced survival rates and increased oxidative stress markers compared to control untreated flies. co-treatment with mlp and abp (50 and 100 µg/10g diet) improved antioxidant enzyme activities (gst, npsh and total thiol) and accumulation of no and hydroperoxide in pb-treated flies. therefore, the peptide fractions from m. lucida and a. boonei may be sources of bioactive agents with therapeutic potential against oxidative stress and inflammation associated with lead toxicity. keywords: alstonia boonei; drosophila melanogaster; bioactive peptides; lead toxicity, morinda lucida. introduction oxidative stress and inflammation play destructive roles in the pathogenesis of many chronic diseases such as arthritis, cancer, diabetes, bowel, cardiovascular and neurodegenerative diseases (libby 2007; sharifi-rad et al. 2020). the relentless pace of industrialisation and agricultural expansion has resulted in a significant increase in environmental pollution, leading to the accumulation of toxic heavy metals in water, food, and air. lead, a toxic heavy metal, is prevalent in the environment, particularly in mining and industrial areas. human exposure occurs mainly through the inhalation of dust particles and the ingestion of contaminated water from streams, rivers, and wells (balali-mood et al. 2021). once absorbed, lead disrupts cellular function, producing free radicals that damage nucleic acids and compromise the integrity of the cellular membrane (chandimali et al. 2025), hence accelerating the onset of degenerative diseases, including neurodegeneration in both adults and children (collin et al. 2022). the fruit fly drosophila melanogaster is widely used model for toxicological and pharmacological studies involving chemicals and natural products (rand et al. 2010; siddique et al. 2005; zemolin et al. 2014). it shares many fundamental biological, biochemical, physiological and neurological similarities with mammals, with approximately 70% of human disease-causing genes having functional homologues present in d. melanogaster. its genetic composition and high sensitivity to toxic substances further enhance its suitability for studying toxicities and disease mechanisms (bezerra et al. 2017). medicinal plants are rich sources of bioactive compounds with numerous significant biological and pharmacological importance. plant-derived bioactive peptides are currently the new generation of biologically active regulators. they improve the treatment of various illnesses and disorders, thereby increasing the quality of life (sánchez & vázquez 2017). bioactive peptides have been confirmed in different plant families including rubiaceae (attah et al. 2023; gruber et al. 2008), https://doi.org/10.14421/biomedich.2025.142.993-1007 mailto:nikeoa@yahoo.com mailto:amos_abolaji@yahoo.com 994 biology, medicine, & natural product chemistry 14 (2), 2025: 993-1007 apocyanaceae (alade & attah 2023; gruber 2010), violaceae (plan et al. 2007), curcubitaceae (hernandez et al. 2000), fabaceae (nguyen et al. 2011), poaceae (nguyen et al. 2013), euphorbiaceae (akano et al. 2024) etc. morinda lucida benth. of the rubiaceae family, is an evergreen shrub, growing up to 1825 m tall with grey bark, short bent branches and shining green foliage (kwofie et al. 2016). m. lucida, known as oruwo by the yoruba tribe in nigeria, is valued for its therapeutic and economic value in nigeria and west africa (saalu 2016). this plant has been used traditionally for different ailments, including the treatment of malaria, dysentery, wound infections, fever, leprosy, ulcers, stomach ache and gonorrhoea (adeleye et al. 2018; bamisaye et al. 2013). morinda lucida is rich in peptides and several reports have confirmed the biological activities of the peptide extracts including antimicrobial, antioxidant, antiplasmodial, antidiabetic, antilipidemic and antiinflammatory (adebayo et al. 2017; adewole et al. 2018). alstonia boonei de wild, a member of the apocynaceae family, is a large deciduous tree that can grow up to 40 m in height and approximately 1.2 m in diameter (burkill 1985). it is known as ahun by the yorubas in nigeria (majekodunmi et al. 2008). this plant has been used ethnomedicinally to treat malaria, jaundice, fever, skin conditions, sore throat, arthritis and rheumatic pain (asase & peterson 2019; asuzu & anaga 1991; gbadamosi et al. 2011). the leaf, stem and root bark extracts of a. boonei possess anti-inflammatory, antioxidant, analgesic, antipyretic, antimicrobial and antidiabetic properties (akinmoladun et al. 2007; akinnawo et al. 2017; olajide et al. 2000; opoku & akoto 2015; oyebode et al. 2019). while there are reports of the biological activities of the peptide fractions of m. lucida, there are limited or no scientific validations of peptide fractions from a. boonei to the best of our knowledge. therefore, this study aimed to evaluate the antioxidative and anti-inflammatory properties of partially purified peptide fractions from morinda lucida and alstonia boonei in vitro and their protective potential in drosophila melanogaster. materials and methods preparation of peptide-rich fraction fresh leaves of m. lucida and a. boonei were collected from the premises of the university of ibadan. the plants were identified and authenticated at the forestry research institute of nigeria. the herbarium specimen was deposited with voucher numbers; fhi 113275 and 113284. extraction of peptides from morinda lucida and alstonia boonei leaves the leaves of m. lucida and a. boonei were air-dried and pulverised into powder before extraction. the prepurified peptide fractions were prepared as described by koehbach et al. (2013) as previously reported by akano et al. (2024) and ogbole et al. (2021) with slight modifications. the extraction involved macerating powdered plant samples in a 1:1:0.5 ratio of dichloromethane (dcm): methanol (meoh): distilled water mixture. initially, the powdered plant samples were macerated for 24 h in dcm and meoh (1:1) with intermittent agitation, followed by the addition of distilled water and further extraction for 24 h. the resulting aqueous-rich supernatants were concentrated using a rotary evaporator to remove methanol. subsequent purification involved loading the aqueousrich extracts onto a conditioned c18 column and eluting the fractions with increasing concentrations of acetonitrile and trifluoroacetic acid mixture in double distilled water (20%, 80%, and 100%). the 80% eluent; partially purified peptide fractions of a. boonei (abp) and m. lucida (mlp) were concentrated, freeze-dried, and stored at -20 °c for further analysis. chemicals the following chemicals and reagents; ascorbic acid, methanol, phosphate buffer saline (ph = 7.4), 2, 2diphenyl-1-picrylhyhdrazy (dpph), sodium nitrite, sodium nitroprusside, griess reagent (containing 1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl ethylenediamine dihydrochloride), 1-chloro-2,4dinitrobenzene (cdnb), 5,5'-dithiobis-2-nitrobenzoicacid (dtnb), 2',7' dichlorofluorescein diacetate (dcfh-da), acetylthiocholine iodide (sigma aldrich (st. louis, mo)) were used. lead acetate was obtained from a k scientific, usa. all chemicals were of analytical grade and freshly prepared before each experiment. in vitro antioxidant activity 2,2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging assay the free radical scavenging activity of the fractions was assessed with dpph assay following the procedure of mensor et al. (2001) using the synthetic antioxidant butylated hydroxytoluene (bht) as the reference standard. to the plant samples or standard of varying concentrations (7.8125 to 1000 µg/ml), 1 ml dpph solution was added followed by a 30 min incubation in the dark at room temperature. absorbance was measured at 517 nm. the dpph free radical scavenging ability of the fractions and bht was subsequently calculated as the percentage of control. molik et al. – peptide fractions against lead toxicity … 995 ferric reducing antioxidant potential (frap) the ferric ion reducing ability of both fractions was assessed using a modified method of benzie and strain (1996). frap reagent was prepared by mixing 300 mm acetate buffer, ph 3.6, 10 mm tptz in 40 mm hcl and 20 mm ferric chloride in a ratio of 10:1:1 (v/v/v). samples and bht (standard) were allowed to react with frap reagent in the dark for 90 min at 37 °c. absorbance was measured at 593 nm. the assay was performed in triplicate. the antioxidant activity of both fractions and standard was quantified and reported as mg of ascorbic acid equivalent per gram of extract (mg aae/g). in vitro anti-inflammatory activity inhibition of protein denaturation the protein denaturation assay was carried out following the protocol described by padmanabhan and jangle (2012). the reaction mixture consisting of 2 ml of either plant sample or diclofenac (standard nsaid) at concentrations ranging from 7.8125 to 1000 µg/ml and 2.8 ml of phosphate-buffered saline (ph 6.4) was combined with 1% bovine serum albumin. following incubation at 27 ± 1 °c for 15 min, protein denaturation was induced by heating the reaction mixture in a water bath at 70 °c for 10 min. after cooling, the absorbance was measured at 660 nm and the percentage inhibition of protein denaturation was calculated. membrane stabilisation assay the membrane stabilisation assay was performed following the procedure of sadique et al. (1989) with slight modifications. briefly, 50 μl of each sample (7.8125 to 1000 µg/ml) was mixed with 100 μl phosphate buffer, 200 μl hyposaline solution (0.36% nacl), and 50 μl rbc (2% v/v). the mixture was incubated at 37 °c for 30 min and centrifuged at 8,000 rpm for 3 min. diclofenac sodium (standard nsaid) was used as the reference drug. the control consisted of 50 μl rbc and 200μl hyposaline solution. absorbance was measured at 560 nm and percentage inhibition of haemolysis was calculated. drosophila melanogaster stock and culture wild-type drosophila melanogaster (canton s strain) flies were maintained at the university of ibadan’s biochemistry department drosophila laboratory, nigeria. the flies were cultured on a cornmeal-based diet containing 6% cornmeal, 1% brewer's yeast, 2% sucrose, 1% powdered milk, agar 1% and 0.08% nipagin. environmental conditions were controlled at a temperature of 24 ±1 °c, with a relative humidity of 60– 70% and a 12 h light-dark cycle. survival studies drosophila melanogaster (1–3 days old), were assigned to different groups, with each group consisting of five replicates, each containing 30 flies. three concentrations of mlp or abp (10, 50 and 100 µg/10 g diet) were compared to the control (0 µg/10 g diet). the survival rate was determined by recording the daily mortality of flies for 14 days, and the data were analysed (abolaji et al. 2014). survival studies were also carried out by exposing flies to (0, 1, 5 and 10 mm) of lead acetate for 7 days. lead (pb) exposure and treatment with peptide fractions of m. lucida (mlp) and a. boonei (abp) for the ameliorative study, pb and mlp/abp were added to the medium at final doses of pb (10 mm) and/or mlp/abp (50 and 100 µg/10g diet) respectively. flies were divided into groups of 50 flies each; (a) control (0 µg/10 g diet); (b) mlp/abp (50 µg/10g diet); (c) mlp/abp (100 µg/10g diet); (d) pb (10 mm); (e) pb (10 mm) + mlp/abp (50 µg/10g diet); (f) pb (10 mm) + mlp/abp (100 µg/10g diet). the (10 mm) dose selected for pb for the ameliorative studies was determined based on a survival curve and biochemical assays presented later in this study. studies by venkareddy (2015) also guided the selection of (1, 5 and 10 mm) pb doses. the doses of mlp and abp (50 and 100 µg/10g diet) were chosen based on the absence of overt toxicity in the flies, as observed by the survival curve and biochemical assays conducted in this study. preparation of tissue homogenate the treated flies were collected after anaesthetizing, weighed and homogenized in 0.1 m phosphate buffer at a ph of 7.4 (ratio of 1 mg:10 µl). the resulting homogenates were centrifuged at 4000 × g and 4 °c for 10 min in a mikro 220r microlitre centrifuge (tuttlingen, germany). subsequently, the supernatants (samples) were separated from the pellets into tubes for various biochemical assays. biochemical assays determination of protein concentration total protein concentrations were measured following the modified method described by lowry et al. (1951). determination of total thiol levels total thiol content was determined according to the method of ellman (1959). the reaction mixture was made up of 510 μl of 0.1 m of potassium phosphate buffer (ph 7.4), 25 μl of sample, and 30 μl of dtnb (1 mm) and 35 μl distilled h2o. incubation was carried out for 30 min at room temperature, the absorbance was measured at a wavelength of 412 nm. determination of catalase activity catalase activity was determined according to the method described by (aebi 1984). the assay was conducted in a reaction mixture consisting of 1.8 ml of potassium phosphate buffer (ph 7.0), 180 µl of 300 mm hydrogen peroxide, and 20 µl of sample diluted 1:50. 996 biology, medicine, & natural product chemistry 14 (2), 2025: 993-1007 the clearance of hydrogen peroxide was monitored spectrophotometrically at 240 nm over a 2-min period, with absorbance readings taken at 10-s intervals using a uv-visible spectrophotometer. the resulting catalase activity was expressed in micromoles of hydrogen peroxide consumed per minute per milligram of protein (μmol/min/mg). determination of total hydroperoxide level the total hydroperoxide level was determined using the method described by wolff (1994). briefly, a reaction mixture containing fox 1 (10 ml of 100 mm xylenol orange, 10 ml of 100 mm sorbitol, 50 ml of 250 mm ferrous sulphate, 5 ml of 25 mm h2so4 and 30 ml distilled water) was reacted with the sample homogenate. this mixture was incubated at room temperature for 30 min and the absorbance was measured at 560 nm. the total hydroperoxide concentration was calculated using a standard curve and expressed in μmol/mg protein. determination of nitric oxide (nitrate/nitrite) levels the level of nitrites was quantified following the protocol by green et al. (1982) using the griess reaction. samples were mixed with griess reagent at a ratio of 1:1 and incubated at room temperature for 20 min and nitrite level was quantified from sodium nitrite standard curve measured at 550 nm. determination of glutathione s-transferase (gst) activity the activity of gst was evaluated using the method of habig and jakoby (1981). in summary, the reaction mixture contained 270 μl of solution a containing (20 ml of 0.25 m potassium phosphate buffer (ph 7.0), 2.5 mm edta, 10.5 ml of distilled water, and 500 ml of gsh (0.1 m) at 25 °c), 20 μl of sample (1:5 dilution), and 10 μl of 25 mm cdnb as substrate. the reaction mixture was then monitored for 5 min (10-s intervals, at 340 nm). determination of the non-protein thiol content the non-protein thiol (npsh) content was estimated following the method described by (jollow et al. 1974). the supernatants were precipitated with sulphosalicylic acid (4%) at a 1:1 ratio kept at 4 °c for 1 h. the mixture was then centrifuged at 5000 rpm for 10 min at 4 °c. the assay mixture consisted of 550 µl of 0.1 m phosphate buffer, 100 µl of supernatant and 100 µl of dtnb. the od was read at 412 nm and the result was expressed as µmol of gsh per mg protein. data analysis graphs were prepared using ms office excel, and prism 6 for windows, version 6.01 (graphpad software, inc). data from the survival assay were evaluated using the kaplan-meier survival plot method, with group comparisons assessed by log-rank tests. biochemical assay results were presented as mean ± standard error of the means (sem). for multiple treatment group comparisons, one-way analysis of variance (anova) was employed, followed by dunnett's post hoc test. statistical differences were considered significant at p < 0.05. results and discussion in vitro antioxidant assays the antioxidant potential of mlp and abp was evaluated using the dpph free radical scavenging assay and the results are presented in figure 1a. concentration-dependent percentage radical scavenging activity was observed for the tested samples. the results revealed that abp demonstrated a higher dpph scavenging activity than mlp but not as high as the standard bht. the ic50 values are reported in table 1. the result of the ferric reducing antioxidant property (frap) of abp and mlp was evaluated and expressed as ascorbic acid equivalent and presented in figure 1b. the result revealed that abp had greater reducing power than mlp although the reducing power of bht was significantly greater (p < 0.05). figure 1. percentage dpph radical scavenging activity (a) ferric reducing antioxidant power (b) of partially purified peptide fractions of a. boonei (abp) and m. lucida (mlp). molik et al. – peptide fractions against lead toxicity … 997 table 1. ic50 values of samples in antioxidant and anti-inflammatory assays. sample dpph inhibition of protein denaturation inhibition of haemolysis ic50 ± s.e.m (µg/ml) abp 103.10 ± 0.74 47.47 ± 0.59 63.60 ± 0.41 mlp 110.60 ± 0.39 78.72 ± 0.39 204.90 ± 1.79 bht 89.30 ± 1.22 diclofenac 19.04 ± 0.23 19.82 ± 0.07 anti-inflammatory assays the protein denaturation inhibition assay was evaluated and the percentage inhibition was calculated and presented in figure 2a. the results showed that the samples inhibited heat-induced protein denaturation in a concentration-dependent manner. abp showed a stronger ability to inhibit protein albumin denaturation than mlp, although it was not as strong as the reference drug, diclofenac. the rbc membrane stabilisation assay was also carried out, and the percentage of haemolysis inhibition was calculated, figure 2b. a dose-dependent increase in the percentage of haemolysis inhibition was observed. diclofenac sodium displayed the most potent inhibition, followed by abp displaying a stronger haemolysis inhibition than mlp. table 1 shows the ic50 values for the assays. figure 2. effect of abp and mlp on inhibition of albumin denaturation (a) inhibition of rbc haemolysis (b). abp and mlp maintained the life span of flies and the redox status of flies. the survival study showed that the survival of the flies treated with a 10, 50, and 100 µg/g diet of abp and mlp was not significantly different from control flies (figure 3 a & b). the three concentrations maintained the life span of the flies, indicating that the fractions did not exert any toxic effects on the flies. the effect of abp and mlp on the biochemical markers of the flies was concentration-dependent, and better activities were observed with the 50 and 100 µg/g diet of both fractions (figure 4 and 5). these concentrations were therefore selected for the ameliorative studies by co-exposing with pb (10 mm). figure 3. effects of abp (a) and mlp (b) on survival rate in drosophila flies after treatment for 14 days. 998 biology, medicine, & natural product chemistry 14 (2), 2025: 993-1007 figure 4. effects of abp on nitric oxide level (a), total hydroperoxide level (b), gst activity (c), npsh level (d), cat activity (e) and tsh level (f) in drosophila flies after treatment with abp for 7 days. values represent mean ± sem of 50 flies/vial with 5 replicates per treatment group. mean values are significantly different at *(p < 0.05) compared to control. figure 5. effects of mlp on nitric oxide level (a), total hydroperoxide level (b), gst activity (c), npsh level (d), cat activity (e) and tsh level (f) in drosophila flies after treatment with mlp for 7 days. values represent mean ± sem of 50 flies/vial with 5 replicates per treatment group. mean values are significantly different at *(p < 0.05) compared to control. effect of pb exposure on survival rate and oxidant levels in flies the daily exposure of flies to pb (1, 5, 10 mm) for 7 days reduced their survival rate (figure 6a). fewer than 40% of the flies exposed to 10 mm pb survived pbinduced toxicity at day 7. moreover, pb significantly (p < 0.05) increased the levels of oxidant molecules including nitrites and total hydroperoxide while causing a notable depletion in thiol levels (figure 6b-d). molik et al. – peptide fractions against lead toxicity … 999 figure 6. effects of pb on a 7-day survival (a), total hydroperoxide level (b), nitric oxide level (c), and total thiol level (d) in drosophila flies after treatment with pb for 7 days. values represent mean ± sem of 50 flies/vial with 5 replicates per treatment group. mean values are significantly different at *(p < 0.05) compared to control. abp and mlp reduced nitric oxide and total hydroperoxide levels in drosophila flies with pbinduced toxicity as depicted in figure 7, pb exposure caused a significant (p < 0.05) increase in nitric oxide (no) (figure 7a & b) and total hydroperoxide levels (7c & d) in flies when compared with the control (basal diet). however, abp and mlp (50 and 100 µg/g diet) significantly reduced (p < 0.05) the levels of nitric oxide and hydroperoxide when compared to the pb (10 mm) treated group (figure 7a d). 1000 biology, medicine, & natural product chemistry 14 (2), 2025: 993-1007 figure 7. effects of abp/mlp and pb on nitric oxide (a & b), total hydroperoxide levels (c & d) in drosophila flies after treatment with pb for 7 days. values represent mean ± sem of 50 flies/vial with 5 replicates per treatment group. mean values are significantly different at *(p < 0.05) compared to control and #(p < 0.05) relative to pb. abp and mlp reversed pb-induced depletion of total thiol and non-protein thiol levels in drosophila flies as shown in figure 8, exposure to pb caused a significant decrease (p < 0.05) in total thiol (figure 8 a & b) and npsh (figure 8 c & d) contents in treated flies in comparison with those of control flies. however, abp (100 µg/g diet) and mlp (50 and 100 µg/g diet) significantly increased (p < 0.05) the total thiol content, while abp (50 and 100 µg/g diet) and mlp (100 µg/g diet) significantly (p < 0.05) increased the non-protein thiol content relative to the pb treated flies (figure 8 a d). molik et al. – peptide fractions against lead toxicity … 1001 figure 8. effects of abp/mlp and pb on total thiol (a & b), nonprotein thiol contents (c & d) in drosophila flies after treatment with pb for 7 days. values represent mean ± sem of 50 flies/vial with 5 replicates per treatment group. mean values are significantly different at *(p < 0.05) compared to control and #(p < 0.05) relative to pb. abp and mlp improved glutathione-s-transferase activity in drosophila flies exposed to pb as illustrated in figure 9, the gst activity of drosophila flies exposed to pb (10 mm) was significantly (p < 0.05) lower than control flies. however, abp and mlp treatment enhanced the activity of the gst enzymes in comparison to pb treated group (figure 9a & b). figure 9. effects of abp/mlp and pb on glutathione s-transferase activity (a & b) in drosophila flies after treatment with pb for 7 days. values represent mean ± sem of 50 flies/vial with 5 replicates per treatment group. mean values are significantly different at *(p < 0.05) compared to control and #(p < 0.05) relative to pb. discussion bioactive peptides from plant sources have been identified as potent antioxidants that neutralize free radicals and oxidation initiators (zhu et al. 2024). they have also been confirmed to resist excessive inflammatory responses by modulating inflammatory signalling pathways and inhibiting the secretion of inflammatory factors (zhang et al. 2020). the dpph radical serves as a reliable and stable radical model widely used to assess the antioxidant and free radical 1002 biology, medicine, & natural product chemistry 14 (2), 2025: 993-1007 scavenging ability of plant extracts. antioxidant peptides can exert their effects directly by scavenging free radicals through hydrogen atoms or electron donation. their effectiveness depends on the specific properties of their amino acid residues (zhang et al. 2022). this study revealed that both peptide fractions (abp and mlp) exhibited free radical scavenging capacity and were concentration-dependent, suggesting that their potent antioxidant properties may be attributed to specific amino acids present in the fractions. the antioxidant properties of synthetic and natural antioxidants play crucial roles in their ability to mitigate inflammation and provide therapeutic benefits (bhol et al. 2024). the amino acid composition of both fractions is likely to contribute to their free radical scavenging activity by reducing the stable dpph radical to its yellow diphenylpicrylhydrazine derivative. the frap assay measures the reducing ability of antioxidants by reacting with a ferric tptz complex and producing a bluecoloured ferrous tptz complex by a reductant in low ph media (benzie & strain 1996). in this study, both abp and mlp fractions exhibited reducing potential. this reduction is likely associated with the presence of reducing amino acids in the peptide fractions, which exert their action by breaking the free radical chains through donating atoms. hence, the amino acid compositions of abp and mlp may enable them to disrupt the free radical chains through electron donation. the cysteine-stabilised peptide fraction of m. lucida and extracts of a. boonei have been reported to have antidpph activity (adewole et al. 2018; akinmoladun et al. 2007; akinnawo et al. 2017; oyebode et al. 2019). xie et al. (2008) reported that peptides isolated from alfafa leaf protein showed a chelating effect on ferrous ions. inflammatory responses in tissues are initiated by injuries, microbial infections, and irritants which have been implicated in the pathogenesis of various chronic diseases including arthritis, cancer and stroke. protein denaturation is a key feature of inflammatory responses in various diseases. tissue injury can be characterised by the denaturation of cellular and tissue proteins (opie 1962). hence, inhibiting protein denaturation suggests anti-inflammatory properties of extracts. this study revealed that abp and mlp demonstrated protective effects against heat-induced protein denaturation in a dose-independent manner comparable to the standard drug; diclofenac. to ascertain the membrane stabilising activity of abp and mlp, the experiment involved erythrocyte membranes. the lysosomal enzymes released during the inflammatory process result in various disorders and since rbc membranes share similarities with lysosomal membranes, evaluating the protective effects of drugs and plant extracts against hypotonicityinduced rbc membrane lysis serves as a valuable indicator of their antiinflammatory activity (mounnissamy et al. 2007; umapathy et al. 2010). these results indicated that abp and mlp exhibited protective effects against hypotonicity-induced erythrocyte lysis comparable to diclofenac. the study by akinnawo et al. (2017) demonstrated that an aqueous extract of a. boonei significantly inhibited heat-induced protein denaturation and stabilised hypotonicity-induced hemolysis of hrbc in vitro. similarly, the aqueous extract of m. lucida presented the highest protection against albumin denaturation with the lowest ic50 among other screened extracts (dah-nouvlessounon et al. 2023). plant-derived bioactive peptides may exhibit anti-inflammatory effects by interacting with cell membranes, stabilising them and disrupting the inflammatory cascade. their hydrophobic nature enhances membrane binding and modulates cellular responses to inflammation (liu et al. 2022). the results of this study revealed that abp and mlp had no adverse effects on the survival or biochemical indices of the flies and the results were comparable to the untreated control flies. the ability of the fractions to maintain the life span and biochemical parameters of the treated flies may be attributed to their potent antioxidative and anti-inflammatory properties. existing research has indicated a positive association between increased intake of dietary supplements high in antioxidants and reduced risk for chronic inflammatory diseases and oxidative damage to cells (roy et al. 2022; wang et al. 2017). additionally, it has been reported that varied intake of functional foods and nutraceuticals helps maintain healthy and strong tissues, decreases inflammatory factors and prevents diseases (garzajuárez et al. 2023). pb exposure has been associated with generating excessive amounts of ros and inhibiting antioxidant defence systems (gurer & ercal 2000). given the established link between pb exposure, oxidative stress and inflammatory processes, attention has focused on compounds with antioxidant and anti-inflammatory properties to mitigate pb-induced toxicity (gurer & ercal 2000; mumtaz et al. 2020). however, relatively few studies have examined the protective role of medicinal plants, particularly peptide-rich plant extracts in addressing pb-induced toxicity responses in experimental models. evidence from epidemiological studies reveals the occurrence of adverse health effects even at low-level exposure to lead (rees & fuller 2020; rinsky et al. 2018). oxidative stress arising from chronic lead exposure can trigger subsequent events contributing to the onset of hypertension and cardiovascular diseases (yan et al. 2022). studies have shown that lead has a deleterious impact on the immune system leading to autoimmune disorders (dou et al. 2022; harshitha et al. 2024; zheng et al. 2023). lead toxicity was assessed in drosophila flies and it was observed that pb had adverse effects on the survival and biochemical indices of the treated flies. the lethal effects of pb are attributed to oxidative damage to cellular components that trigger inflammation and cell death (virgolini & aschner 2021). lead is a known neurotoxin, that has been demonstrated molik et al. – peptide fractions against lead toxicity … 1003 to cause mortality in drosophila flies (venkareddy 2015) and adverse effects on biochemical parameters of fruit flies (abdulazeez et al. 2024). lead exposure can increase the generation of nitric oxide and hydroperoxides which can contribute to the development of various diseases (dobrakowski et al. 2017; fiorim et al. 2020; ni et al. 2004). nitric oxide (no) is a key signalling molecule with a dual role in inflammation. under normal physiological conditions, no exhibits anti-inflammatory properties whereas, excessive no production is considered as a proinflammatory mediator that induces inflammatory processes (sharma et al. 2007). this study showed that both fractions (abp and mlp) significantly ameliorated the increased levels of no and hydroperoxide induced by pb in flies. pb exposure triggers no production primarily by inducing oxidative stress caused by an imbalance between the production and scavenging of ros in the tissues and components of flies (patra et al. 2011; shilpa et al. 2021). increased production of hydroperoxide levels from lead exposure can result in membrane degradation and lipid peroxidation. a recent study reported that pb exposure leads to free radical generation which resulted in increased no levels (osunbor & orobor 2023). the ability of abp and mlp to mitigate the effects of pb-induced elevated levels of no and hydroperoxide is consistent with their in vitro antioxidant and anti-inflammatory properties which are typified by their free radical scavenging abilities, reducing properties, membrane stabilising and protein denaturation inhibitory properties. a study reported the protective effect of the alkaloid-rich extract of morinda lucida against manganese chloride-induced toxicity, significantly reducing ros levels (nwanna 2021). the aqueous extract of a. boonei also demonstrated potent antioxidant activity by scavenging no and h2o2 radicals in vitro (akinnawo et al. 2017). thiols make up a major component of the body’s antioxidant defence system, protecting against ros (kumar et al. 2009). as reducing agents, thiols maintain cellular redox homeostasis protecting cells from oxidative damage. consequently, decreased thiol levels have been linked to various medical disorders including kidney disorders, diabetes mellitus and other neurological disorders (erel & neselioglu 2014; kundi et al. 2015). glutathione (gsh) is one of the most abundant endogenous antioxidants in the body and plays a vital role in protecting cells from oxidative damage caused by ros, free radicals, peroxides and heavy metals and helps maintain redox homeostasis (forman et al. 2009; lushchak 2012). the reduction in tsh and npsh levels following pb treatment is an indicator of oxidative stress which could result from pb binding with sulfhydryl group of gsh or impairment of gsh synthesis (flora et al. 2012). this result is comparable with data obtained by olakkaran et al. (2018) who reported a pb-induced depletion of gsh content in drosophila flies. however, the observed protective effects of abp and mlp in mitigating pb-induced decreases in tsh and npsh levels in flies suggest their potential against diseases associated with oxidative stress. the glutathione-stransferases (gsts) are a diverse family of enzymes that are involved in many physiological processes including protection against oxidative damage caused by toxic compounds by catalyzing their conjugation to reduced glutathione, biosynthesis of hormones, intracellular transport, and are also responsible for the detoxification of both endogenous and xenobiotic compounds (ściskalska & milnerowicz 2020; shi et al. 2012). thus, the deleterious effect of pb on flies may be a result of reduced ability to detoxify pb and its reactive species leading to oxidative stress and this is attributed to decreased glutathione-s-transferase activity. however, abp and mlp were able to reverse the inhibitory effect of pb indicating their antioxidant potentials. bioactive peptides have been reported to enhance antioxidant defences by modulating key enzymes involved in redox homeostasis (xu et al. 2024). these peptides can protect cells from oxidative damage by restoring gst activity and facilitating the detoxification of ros and toxic compounds (okagu & udenigwe 2022). plant extracts and their active fractions can counteract heavy metalinduced oxidative stress by boosting gst activity and increasing glutathione levels, thereby mitigating oxidative damage and cellular function (sheweita et al. 2016). conclusions this study highlights the antioxidant and antiinflammatory properties of partially purified peptide fractions of morinda lucida and alstonia boonei. these peptide fractions demonstrated significant free radical scavenging abilities, reducing properties, membrane stabilising ability and protein denaturation inhibitory properties. the dietary inclusion of lead-induced toxicity in drosophila flies primarily disrupts the redox balance. however, both peptide fractions effectively mitigated lead-induced toxicity by reducing inflammatory markers and restoring the activities of key antioxidant enzymes. the protective effects of the peptide fractions of m. lucida and a. boonei could be attributed to the presence of bioactive peptides present in the plants. this study highlights the potential of these peptide fractions as a source of natural antioxidant and anti-inflammatory agents. further study is necessary to isolate and identify the individual peptide(s) responsible for these activities. acknowledgements: the authors wish to convey their appreciation to the phytomedicine research unit of the department of pharmacognosy, university of ibadan for their assistance during the extraction of the peptide-rich extracts. the writers also extend their gratitude to drosophila laboratory, department of biochemistry, 1004 biology, medicine, & natural product chemistry 14 (2), 2025: 993-1007 university of ibadan, nigeria for providing the flies used in this experiment and offering the workspace support. authors contributions: zainab abiodun molik carried out the experiment, collected, and analysed the data and wrote the original draft. omonike oluyemisi ogbole and amos olalekan abolaji supervised the research. all authors reviewed the manuscript. competing interests: the authors declare no competing interests funding: this work was supported in part by thomasbassir biomedical foundation grant to zainab abiodun molik. references abdulazeez, r., highab, s., onyawole, u., jeje, m., musa, h., shehu, d., & ndams, i. 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(2024). antioxidant function and application of plantderived peptides. antioxidants, 13(10), 1203. https://www.mdpi.com/2076-3921/13/10/1203 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 33-37 | doi: 10.14421/biomedich.2020.91.33-37 issn 2540-9328 (online) toxicological effects of ethanolic stem bark extract of xylopia aethiopica on testicular oxidative stress markers and histology of male rats elias adikwu1,*, ben enoluomen ehigiator2 1department of pharmacology and toxicology, faculty of pharmacy, niger delta university, nigeria 2department of pharmacology and toxicology, faculty of pharmacy, madonna university, nigeria corresponding author* adikwuelias@gmail.com manuscript received: 22 april, 2020. revision accepted: 10 may, 2020. published: 11 may, 2020. abstract impairment in testicular function can occur through perturbations in testicular oxidative stress markers and histology. xylopia aethiopica (xe) is used to enhance fertility in males, but with information gap on its effect on testicular oxidative stress markers and histology. the present study assessed the effects of ethanolic stem bark extract of xylopia aethiopica (eexa) on testicular oxidative stress markers and histology of male albino rats. sixty adult male albino rats (200g-250g) were randomly grouped into 4 (a-d) of 15 rats per group. the rats in the control group a (a1-a3) were administered per oral (p.o) with water (0.2 ml/day) for 15, 30 and 60 days respectively. the rats in groups b (b1-b3), c(c1-c3) and d (d1-d3) were administered p.o with eexa (200, 400 and 800 mg/kg/day) for 15, 30 and 60 days respectively. the rats were anesthetized at the termination of eexa administration and were dissected and testes removed. the testes were weighed and evaluated for oxidative stress markers and histology. testicular weights were decreased in a dose and-time dependent fashion in eexa-treated rats. significant decreases in testicular superoxide dismutase, glutathione, catalase, and glutathione peroxidase levels with significant increases in malondialdehyde levels in a dose and time-dependent fashion were observed in rats administered with eexa. testicular histology showed cellular necrosis, degeneration and loss of interstitial tissues in rats administered with eexa. this study observed that eexa perturbed testicular oxidative markers and histology. its use may impair testicular function. keywords: xylopia aethiopica; testis; oxidative stress; histology; rat. introduction infertility is defined as the inability to conceive after 1 year of regular unprotected sexual intercourse (rowe et al., 1993). infertility can be caused by a number of factors including drugs and exposure to chemical substances (slade et al., 2007). one of the proposed mechanisms by which drugs and chemical substances cause infertility is through the induction of oxidative stress via uncontrollable activity of reactive oxygen species (ros) (geng et al., 2015; alahmar, 2019). studies have shown that 40%–88% of infertile patients have high levels of seminal ros (lewis et al., 1995). increased ros can lead to testicular redox imbalance, reduced sperm quality and increased sperm dna damage. spermatozoa are highly vulnerable to the deleterious activities of excess ros because of the presence of unsaturated fatty acids found in their cell membranes. the unsaturated fatty acids undergo oxidation which is detrimental to sperm cell, germ cell membrane, eventually inducing cell death (proudfoot, 2007). excess ros can also damage biomolecules (dna, lipids and proteins) thus, altering the morphology and functions of the testes (uzunhisarcikli et al., 2007; afolabi et al., 2018). in traditional medicine, the use of herbal remedies to treat ailments including infertility is one of the most important therapeutic approaches used by man (kashani et al., 2017). evidence based herbal remedies can serve as effective treatments for infertility among males and females. herbal remedies produced from special plant parts are believed to improve reproductive organ functions, hormonal system, and sex drive (kashani et al., 2017). however, the indiscriminate use of herbal remedies to enhance fertility is becoming worrisome due to possible adverse effects on reproduction function (leke, 2018). studies using animals have shown spermatogenesis arrest, impaired gonadal hormone function, distorted testicular morphology and altered testicular redox status caused by some herbal products (kusemiju et al., 2012). xylopia aethiopica (annonaceae) (xe) is an aromatic tree that grows up to 15-30 m high. it is native to lowland rainforest and moist fringe forest in savannah zones of africa (orwa et al., 2009). xe is commonly used in traditional medicine for the treatment of diseases https://doi.org/10.14421/biomedich.2020.91.33-37 34 biology, medicine, & natural product chemistry 9 (1), 2020: 33-37 (ogbonnia et al., 2008). its bark and leaves are used to treat boils, sores, wounds and cuts. the stem bark is used in combination with other medicinal plants as topical remedy for post-partum breast infections. the decoction of its fruit is used as treatment for bronchitis, rheumatism, asthma, and dysenteric conditions. its fruit and bark extracts are also used as tonic to facilitate or stimulate fertility (burkill, 1985) which has not been proven scientifically. however, we have shown that the ethanolic stem back extract of xe decreased sperm quality and impaired reproductive hormones in male albino rats (ehigiator and adikwu, 2020). this study further examined the toxicological effects of the ethanolic stem bark extract of xylopia aethiopica (eexa) on testicular oxidative stress markers and morphology of male albino rats. this study is imperative due to the fact that experimentally, anti-fertility effects of xenobiotics have been associated with perturbations in testicular oxidative stress markers and histology (agarwal et al., 2005) materials and methods plant material xe stem bark was sourced from imo state, nigeria and was identified at the federal ministry of environment and forestry research institute of nigeria, benin city, edo state. xe stem bark was air dried and powdered using mortar, pestle and manual grinder. thereafter, 900g of the powder was macerated in 200ml of ethanol for 72 hours with intermittent shaking. the resultant extract was filtered at the end of maceration. the filtrate was concentrated using a rotary evaporator and the yield of the extract was used for this study. animals in bred adult male albino rats (200g-250g) were sourced from the department of pharmacology and toxicology, madonna university, nigeria. the rats were housed in clean gauze cages with free access to diet and water and maintained under standard laboratory conditions. the rats were acclimatized for 2 weeks prior to the experiment. the study was approved by the research ethics committee of the department of pharmacology and toxicology, madonna university, nigeria. the rats were handled according to the recommendations of the research ethics committee. animal grouping and treatment  sixty adult male albino rats were randomized into 4 groups (a-d) of 15 rats each. each group was further divided into 3 subgroups of 5 rats each.  group a which served as control was administered per oral (p.o) with water (0.2ml/day) for 15, 30 and 60 days.  group b (b1-b3) was administered p.o with eexa (200mg/kg/day) for 15, 30 and 60 days.  group c (c1-c3) was administered p.o with eexa (400mg/kg/day) for 15, 30 and 60 days.  group d (d1-d3) was administered p.o with eexa (800mg/kg/day) for 15, 30 and 60 days. animal sacrifice, collection of samples and oxidative stress assay the rats were anesthetised at end of extract administration after overnight fast. testes were excised and washed in cold physiological saline. the testes were homogenized in 0.1 m tris-hcl solution buffered (ph 7.4) and centrifuged at 3000 g for 20 min. the supernatants were collected and assessed for oxidative stress markers. testicular total protein was measured according to gonall et al. (1949) whereas malondialdehyde (mda) was assayed as reported by buege and aust, (1978). reduced glutathione (gsh) was analysed according to sedlak and lindsay, (1968) whereas superoxide dismutase (sod) was assayed as reported by sun and zigma, (1978). the method of aebi, (1984) was used to determine catalase (cat) whereas glutathione peroxidase (gpx) was assessed according to rotruck et al. (1973). histological examination of the testes testicular tissues were routinely processed and dehydrated in graded alcohol and embedded in paraffin wax. sections 3-5μm thick were prepared using a rotary microtome and stained with hematoxylin and eosin (h and e) and viewed with the aid of a light microscope for histological changes. statistical analysis graph pad prism 5.03 (graphpad software inc., ca, usa) statistical package was used for the analysis of data. results are expressed as mean ± standard error of mean (sem). results were subjected to two-way analysis of variance (anova) followed by tukey’s multiple comparison test. values at p<0.05; 0.01 and 0.001 were considered significant. results testicular weights were decreased in a dose and time dependent fashion in rats administered with eexa (200, 400 and 800 mg/kg) when compared to control (table 1). the decrease in testicular weight was significant at p<0.05 in rats administered with eexa (200 mg/kg) for 60 days whereas decreases were significant at p<0.01 in rats administered with eexa (400 and 800 mg/kg) for 30 and 60 days when compared to control (table 1). the administration of eexa produced dose and timedependent increases in testicular mda levels when compared to control (table 2). the increases were significant at p<0.05 and p<0.001 in rats administered with eexa (800 and 400 mg/kg) for 15 and 30 days adikwu & ehigiator. – toxicological effects of ethanolic stem bark extract of xylopia aethiopica … 35 respectively when compared to control. however, at p<0.001 increase was significant in rats administered with eexa (800mg/kg) for 60 days when compared to control (table 2). furthermore, the administration of eexa decreased testicular sod, cat, gsh and gpx levels in a dose and timedependent fashion when compared to control (tables 3-6). testicular sod, cat, gsh and gpx levels were significantly decreased at p<0.05 in rats administered with eexa (200 and 400 mg/kg) for 15 and 30 days. on the other hand, significant decreases at p<0.01and p<0.001 were observed in rats administered with eexa (200, 400 and 800mg/kg) for 60 days when compared to control (tables 3-6). h and e stained section of the testis of control rat showed normal histology (figure a). in contrast, the testis of rat administered with eexa (800mg/kg) for 15 days showed showed necrosis, loss of interstitial tissues and cellular degeneration (figure b). the testis of rat administered with eexa (800mg/kg) for 30 days showed enlarged interstitial space, loss of interstitial tissues, and cellular degeneration (figure c). the testis of rat administered with eexa (800mg/kg) for 60 days showed loss of interstitial tissues, cellular degeneration and necrosis (figure d). table 1. effect of ethanolic stem bark extract of xylopia aethiopica on relative testicular weight of albino rats. dose (mg/kg) 15 days 30 days 60 days control 0.71± 0.03 0.72± 0.05 0.70± 0.01 200 0.67± 0.02 0.62± 0.07 0.41± 0.06* 400 0.50± 0.06* 0.40± 0.11** 0.30± 0.03** 800 0.43± 0.04* 0.31± 0.36** 0.25± 0.44** data are expressed as mean ±sem, n=5, * p<0.05 when compared to control ** p<0.01 when compared to control table 2. effect of ethanolic stem bark extract of xylopia aethiopica on testicular malondialdehyde of albino rats. dose (mg/kg) 15 days 30 days 60 days control 0.46 ± 0.09 0.42 ± 0.01 0.45 ± 0.04 200 0.47 ± 0.05 0.48 ± 0.03 0.78 ± 0.05* 400 0.65± 0.03* 0.72± 0.07* 1.00 ± 0.03** 800 0.76 ± 0.08* 0.89± 0.04** 1.45 ± 0.07*** data are expressed as mean ±sem, n=5 *p<0.05 when compared to control **p<0.01 when compared to control, ***at p<0.001 when compared to control figure 1. fig a. the testis of control rat showing normal seminiferous tubules (st). fig b: testis of rat treated with eexa(800mg/kg) for 15 days showing necrosis (n) loss of interstitial tissue (it) and cellular degeneration (cd). fig c: testis of rat treated with eexa (800 mg/kg) for 30 days showing enlarged interstitial space (es), loss of interstitial tissues (li), and cellular degeneration (cd). fig d: testis of rat treated with eexa (800mg/kg) for 60 days showing loss of interstitial tissues (lt) cellular degeneration (cd) and necrosis (cn) (h&e)x 200 . fig a fig b fig c fig d st st n cd it es li cd lt cn cd 36 biology, medicine, & natural product chemistry 9 (1), 2020: 33-37 table 3. effect of ethanolic stem bark extract of xylopia aethiopica on testicular superoxide dismutase of albino rats. dose (mg/kg) 15 days 30 days 60 days control 17.5± 2.63 16.9± 1.69 17.9± 2.00 200 13.4 ± 0.19* 10.7 ± 1.11* 7.62 ± 0.47** 400 9.14 ± 0.55* 6.32 ± 0.35* 4.57 ± 0.02** 800 6.26 ± 0.32** 3.68 ± 0.19** 1.17 ± 0.04*** data are expressed as mean ±sem, n=5 *p<0.05 when compared to control **p<0.01 when compared to control, ***at p<0.001 when compared to control table 4. effect of ethanolic stem bark extract of xylopia aethiopica on testicular catalase of albino rats. dose (mg/kg) 15 days 30 days 60 days control 25.3 ± 3.00 26.5± 2.57 26.9 ± 3.90 200 20.1 ± 2.25* 15.8± 1.00* 9.37 ± 0.77** 400 15.6 ± 1.53* 9.96 ± 0.21* 6.44 ± 0.62** 800 9.90 ± 0.61** 6.32 ± 0.51** 2.51± 0.54*** data are expressed as mean ±sem, n=5 *p<0.05 when compared to control **p<0.01 when compared to control, ***at p<0.001 when compared to control table 5. effect of ethanolic stem bark extract of xylopia aethiopica on testicular glutathione of albino rats. dose (mg/kg) 15 days 30 days 60 days control 15.0 ± 0.11 16.7± 1.04 15.9± 0.33 200 10.9 ± 0.62* 7.08 ± 0.39* 5.43± 0.57** 400 7.32± 0.65* 5.61 ± 0.91** 3.24 ± 0.73*** 800 5.14 ± 0.58** 3.65 ± 0.63** 1.22 ± 0.09*** data are expressed as mean ±sem, n=5 *p<0.05 when compared to control **p<0.01 when compared to control, ***at p<0.001 when compared to control table 6. effect of ethanolic stem bark extract of xylopia aethiopica on testicular glutathione peroxidise of albino rats. dose (mg/kg) 15 days 30 days 60 days control 20.2 ± 2.55 20.9 ± 2.43 21.9± 2.78 200 15.7 ± 1.25* 11.7 ± 1.00* 8.9 9± 0.52** 400 11.6 ± 0.11* 9.05 ± 0.61* 5.76 ± 0.28** 800 8.48 ± 0.32** 6.71 ± 0.22** 2.32 ± 0.32*** data are expressed as mean ±sem, n=5 *p<0.05 when compared to control **p<0.01 when compared to control, ***at p<0.001 when compared to control discussion one of the primary objectives of the preclinical toxicological assessment of xenobiotics is the identification of target organs which can help clinicians to monitor the adverse profile of xenobiotics during clinical development. experimentally, perturbation in testicular weight is an index for the adverse effect of xenobiotics on testicular function (stevens and gallo,1989). the current study observed dereases in testicular weights in a dose and time-dependent fashion in eexaadministered rats. testis contains antioxidants including sod, cat, gsh, and gpx which prevent free radical-induced damage (quinn and payne, 1984). free radicals especially ros are very important for cell signalling and essential physiological functions in the testis. however, excessive production of free radicals can alter cellular redox balance through oxidative stress thereby disrupting normal biological functions. the male reproductive system especially the testes are susceptible to ros-induced oxidative stress (sabeti et al., 2016; asadi et al., 2017). most health related issues that impaired testicular function have been associated with ros-induced oxidative stress and decreased testicular antioxidant defence (halliwell, 2006). the present study observed dose and time-dependent decreases in testicular sod, cat, gsh and gpx levels in rats administered with eexa. this observation is a sign of testicular oxidative stress caused by ros.the overwhelming activity of ros in the testis might have surpassed the regulatory capacity of antioxidants leading to their depletion. ros production is regulated by antioxidants to prevent oxidative damage, including lipid peroxidation (lpo). lpo is a ros-induced oxidation of polyunsaturated fatty acids. lpo has been related to various health conditions including infertility because of the oxidative products produced that can be detrimental to testicular function (nam, 2011). the present study assayed testicular mda level to ascertain the extent and magnitude of testicular lpo caused by eexa. testicular mda levels were elevated in a dose and timedependent fashion in eexa administered rats. this observation can be attributed to excess ros production which oxidized testicular polyunsaturated fatty acids. furthermore, the present study correlates perturbation in testicular redox status with testicular morphology of eexa-administered rats. the testes of eexa administered rats showed varying degrees of damage including necrosis, loss of interstitial tissues, and cellular degeneration. the present observation can be attributed to eexa-induced testicular oxidative stress causing lpo. extensive lpo in biological membranes can cause loss of fluidity, decrease membrane potential, increased permeability and 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biochemical role as a component of glutathione peroxidase. scie. 179:588-90. rowe p, comhaire fh, hargreave t. who manual for the standardized investigation of the infertile couple. cambridge: cambridge university press; 1993. sabeti p, pourmasumi s, rahiminia t, akyash f and reza a. talebi. 2016. etiologies of sperm oxidative stress int j reprod biomed. 14; 231-240 sedlak j, lindsay rh. 1986. estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with ellman’seeagent. anal biochem. 25:192-205. slade p, o'neill c, simpson aj, lashen h. 2007. the relationship between perceived stigma, disclosure patterns, support and distress in new attendees at an infertility clinic. hum reprod. 22:2309–17 stevens k r, gallo m a: practical considerations in the conduct of chronic toxicity studies. in: principles and methods of toxicology. raven press, new york, edition 4, 1989: 237250. sun m, zigma s. 1978. an improved spectrophotometer assay of superoxide dismutase based on epinephrine antioxidation. anal biochem. 90:81-9. uzunhisarcikli m, kalender y, dirican k, kalender s, ogutcu a, buyukkomurcu f 2007. acute, subacute and subchronic administration of methyl parathioninduced testicular damage in male rats and protective role of vitamins c and e. pestic biochem physiol 87(2):115–122 https://www.ncbi.nlm.nih.gov/pubmed/?term=asadi%20n%5bauthor%5d&cauthor=true&cauthor_uid=28658802 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3834518/ this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 911-919 | doi: 10.14421/biomedich.2025.142.911-919 issn 2540-9328 (online) formulation of instant functional collagen drink with date seeds coffee (phoenix dactylifera) and spices (ginger, cinnamon, cardamom, clove) using the d-optimal mixture design method mutia hanifa department of food science and biotechnology – faculty of agricultural technology – brawijaya university. jl. veteran – malang 65145, malang – east java 65145, indonesia corresponding author* mutiahanifa52@gmail.com manuscript received: 16 july, 2025. revision accepted: 15 october, 2025. published: 24 october, 2025. abstract the functional food trend has grown rapidly since the covid-19 pandemic, including products made from natural ingredients like herbal collagen drinks. this research aims to develop an optimal collagen drink formulation by adding date seed coffee with ginger, cinnamon, cardamom, and cloves as antioxidants to optimize collagen function.optimization was carried out using the design expert-13 device with the optimization response of antioxidant activity (mg te/100g) and water content (%). the results showed that the optimal formulation consisted of date seed coffee 35.57%; hydrolyzed collagen 25%; and spice mixture (ginger, cinnamon, cardamom, cloves) of 39.432% with a desirability value of 0.741. the optimal product was then instantiated using the spray drying method and compared with commercial products without instantiation and collagen addition.the optimal product has an ic50 antioxidant activity of 743.86 ppm and total phenols of 187.23 mg gae/100g, with a water content of 3.35%, hygroscopicity of 7.76%, bulk density of 0.488 g/ml, and a dissolution time of 23.32 seconds. the results of the hedonic test on the optimal sample as a whole from 100 panelists were “rather like” to “like” with an average value of 3.17 with the most preferred attributes being the consistency and aroma of spices. keywords: functional drinks; collagen; date seed coffee; spices. introduction indonesia has begun to increase food system innovation in the last decade to address food security and nutrition challenges triggered by global issues such as the covid-19 pandemic. this has raised public awareness regarding the importance of functional foods, which provide additional health benefits and reduce the risk of disease without the side effects of chemical drugs. (hariyadi, 2024). based on (indonesian food and drug supervisory agency, 2011) concerning supervision of claims in processed food labels and advertisements article 1 paragraph 3, "functional food is processed food containing one or more food components which, based on scientific studies, have certain physiological functions beyond their basic functions, and are proven to be harmless and beneficial to health." different from supplements, functional foods are foods or drinks that also fulfill basic needs such as hunger and thirst, so aspects of taste, texture, and color are considered to remain attractive and delicious to consume (triandita et al., 2020). one of the emerging functional food trends recently is the consumption of collagen drinks as beauty drinks. the collagen drink trend continues to grow and accounts for a total of 12.5% of the global market share (bilek & bayram, 2015). collagen is the main protein in the body (25%) that helps regenerate skin, increase elasticity, prevent wrinkles, maintain hydration, and protect against free radicals (wu et al., 2024). as a structural protein, collagen is composed of repeating combinations of the amino acids proline, glycine, and hydroxyproline that form a triple helix structure and work as cell adhesives, triggering regeneration, angiogenesis, morphogenesis, and tissue repair (campos et al., 2023). collagen consumption can be done orally through functional foods, or non-orally through topical creams or injections (lee et al., 2022). however, based on the methods mentioned, the use of collagen through the oral system is considered the safest, cheapest and most effective (leónlópez et al., 2019). as the trend develops, collagen drinks are starting to be enriched with other supporting nutritional content such as antioxidants (ghaffar, 2021). as a rarely utilized byproduct, date seeds contain compounds such as antioxidants and fiber that have the potential to be processed into functional foods (mrabet et al., 2020). according to (maqsood et al., 2020), date seeds contain bioactive compounds such as flavonoids, https://doi.org/10.14421/biomedich.2025.142.911-919 912 biology, medicine, & natural product chemistry 14 (2), 2025: 911-919 carotenoids, tocopherols, tocotrienols, and phytosterols, which in addition to functioning as antioxidants also have anticancer, anti-inflammatory, and antimicrobial effects. date seeds tend to have a hard texture, making them difficult to grind and process for consumption. therefore, a roasting process is carried out to facilitate grinding the date seeds into the final product, namely date seed coffee powder. as a beverage, date seed coffee is considered to have a high content of phenolic antioxidant compounds, a taste and aroma similar to coffee but caffeine-free, making it suitable as a healthier alternative to coffee (ghnimi et al., 2017). however, the consumer sensory acceptance rate for date seed coffee drinks is still lower than conventional coffee because in terms of taste it is not 100% similar to coffee made from coffee beans (fikry et al., 2019). the nutritional and sensory profile of date seed collagen coffee can be enhanced by the addition of spices such as ginger, cinnamon, cardamom, and cloves, which in addition to their strong aroma are also rich in antioxidants to support the health and effectiveness of collagen in the skin. in addition to being antioxidants, several bioactive compounds in the spice mixture also have other functional properties. red ginger contains shogaol and gingerol which are effective as anti-cancer agents (prasad & tyagi, 2015), cinnamon with cinnamaldehyde compounds which act as antimicrobials and anti-inflammatories (wang et al., 2022), cardamom contains catechins and epigallocatechins (egc) which act as antimicrobials (nasution et al., 2023), and cloves contain eugenol, eugenyl acetate, and caryophyllene compounds which have high antimicrobial activity (gengatharan & rahim, 2023). to increase the sensory value of the final product, an instantaneous process is carried out using the spray drying method (spray dryingspray drying is a drying method that uses heat and pressure to evaporate water, leaving behind a dry, powdery component (joshi et al., 2022). spray dryers work by vaporizing the solvent through contact with a sample that has been atomized using hot air to increase drying effectiveness (anandharamakrishnan & ishwarya, 2015). method the research was conducted at the nutrition and food laboratory of the faculty of agricultural technology, brawijaya university. a series of raw material selection and testing, composition optimization with design expert 13, and instantaneous processing with a spray dryer (spray drying), chemical and physical characterization of the final product. the study was conducted from november 2024 to january 2025. composition optimization with design expert 13 was carried out using the antioxidant activity response (mg te/100g). equipment the equipment used in the study included an electric oven (memmert™), analytical balance (ohaus™), 100 ml measuring cylinder (pyrex™), crucible, spoon, uvvis spectrophotometer (genesys10™), analytical balance (mettler toledo™), test tube (iwaki™), test tube rack, glass erlenmeyer flask (pyrex™), volumetric pipette (iwaki™), bulb, volumetric flask (pyrex™), vortex, cuvette, oven (memmert™), desiccator, spray flask bottle, lab spray dryer (labfreez™), color analyzer. material the materials used in the study included date seed coffee powder, collagen powder, a spice powder mixture consisting of ginger powder, cinnamon powder, cardamom powder, and clove powder, and a commercial spiced date seed coffee product with the same spice content as a comparison. the materials used in the analysis series consist of 2,2 diphenyl-1-picrylhydrazyl (dpph) reagent, 6-hydroxy2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox), methanol (ch3oh) analytical grade, folin & ciocalteu reagent, anhydrous sodium carbonate (na2co3), gallic acid (c7h6o5), sodium chloride (nacl), and distilled water. research methods the research stages consist of preliminary testing, including characterization of raw materials. the testing is followed by the main research, which includes optimization of the raw material formulation. the formulation optimization process is carried out using software-based software.design of experiments (doe) is design expert 13 and continued with the instantaneous process using the spray drying method. after that, the chemical, physical, and sensory characterization of the final product was carried out. preliminary research the preliminary research was a characterization of the raw materials to be used, consisting of date palm coffee, a mixture of spices (ginger, cinnamon, cardamom, cloves). the characterization of the raw materials included (1) testing the antioxidant activity using the trolox method (plank et al., 2012) and (2) water content using the conventional method with an oven dryer (aoac, 2005). further research further research is a stage consisting of optimizing the composition of raw materials using the design expert 13 mixture d-optimal method. the selected formulation is then followed by an instantaneous process using spray drying (spray drying) using 30% maltodextrin filler, which produces a finished product in the form of instant coffee collagen drink powder with date seeds and spices. hanifa – formulation of instant functional collagen drink with … 913 characterization characterization of finished products includes physical, chemical, and hedonic. physical characterization includes testing (1) water content (aoac, 2005), (2) dissolution time (ridwan taher lubis et al., 2023) and (3) hygroscopicity level (huda, 2020). chemical characterization includes ic50 antioxidant activity test (plank et al., 2012) and total phenol test (maramy et al., 2024). in addition, the product is also tested for its level of acceptance among consumers using a preference level test (hedonic). results and discussion analysis of raw material characteristics the raw materials used as components of the spiced date seed collagen coffee drink consist of roasted date seed coffee, hydrolyzed collagen powder, and a mixture of spices (ginger (5%), cinnamon (8%), cardamom (4%), and cloves (1%). each ingredient is characterized physically and chemically as shown in table 1. table 1. results of raw material characteristics tests. test variables roasted date seed powder hydrolyzed collagen powder spice mix powder antioxidant activity (mg te/ 100 g) 44.46 ± 3.39 121.69 ± 0.28 inhibition (%) 31,36 ± 1,73 70,81 ± 0,14 total phenol (mg gae/ 100 g) 59.39 ± 8.44 215.97 ± 50.12 water level (%) 2.62 ± 0.00 7.86 ± 0.00 2.78 ± 0.00 the spice mix powder had the highest antioxidant activity (121.69 mg te/100 g), followed by roasted date seed powder (44.46 mg te/100 g). meanwhile, collagen hydrolysate powder did not show any antioxidant content, although previous research (yanti et al., 2022) recorded that the antioxidant activity of fish collagen hydrolyzed with bromelain reached 20.45 feso4/g (frap method). free radical inhibition analysis showed that spice raw materials had the highest inhibition ratio (70.81%), followed by date seed powder (31.36%). total phenol as one of the most abundant antioxidant components and the main indicator of antioxidant activity was also tested, with the highest results in spice powder (215.97 mg gae/100 g) and roasted date seed coffee powder (59.39 mg gae/100 g). physical testing of water content showed that collagen hydrolysate powder had the highest water content (7.86%), followed by spice mixture powder (2.78%) and roasted date seed powder (2.62%). the high water content in collagen hydrolysate powder is caused by the use of hygroscopic maltodextrin as a mixture in making powder in collagen hydrolysate (ramadhani et al., 2024). composition optimization with design expert 13 mixture d-optimal method. the experimental design to be carried out by design expert 13 using the d-optimal mixture method is based on determining the upper and lower limits of each component. the determination of the limits refers to the results of trial and error of the raw material formulation based on the results of characterization and previous research, as shown in table 2. table 2. upper and lower limits of independent variables. material name lower limit upper limit date seed coffee 30% 55% hydrolyzed collagen 25% 50% spice mix (ginger, cinnamon, cardamom, cloves) 20% 40% in this study, the total number of independent variables was 100% of the total base. using water content (%) and antioxidant activity (mg te/100g trolox) as the responses, a 16-item experimental design was obtained. the results are shown in table 3. 914 biology, medicine, & natural product chemistry 14 (2), 2025: 911-919 table 3. results of the design expert-13 recommendation experiment on response. run a: date coffee powder (%) b: hydrolyzed collagen (%) c: spices (%) response antioxidant activity (mg te/100g) water content (%) 1 46.25 28.75 25.00 8.98 6.33 2 42.50 37.50 20.00 11.72 6.3 3 30.00 36.67 33.33 11.46 6.81 4 35.00 25.00 40.00 7.81 6.65 5 30.00 50.00 20.00 15.63 6.93 6 55.00 25.00 20.00 7.81 6.34 7 42.50 37.50 20.00 11.72 6.29 8 30.00 30.00 40.00 9.38 7 9 36.25 28.75 35.00 8.98 6.63 10 45.00 25.00 30.00 7.81 6.4 11 35.00 25.00 40.00 7.81 6.95 12 33.75 41.25 25.00 12.89 6.64 13 55.00 25.00 20.00 7.81 6.37 14 30.00 36.67 33.33 11.46 6.76 15 30.00 50.00 20.00 15.63 6.87 16 37.50 32.50 30.00 10.16 6.55 based on the test data, the modeling results for the antioxidant activity response optimization experiment showed that the appropriate statistical analysis model was a linear model. (a) (b) figure 1. 3d graph of linear model (a) & contour graph of antioxidant activity response (b). the graph shows a linear model with a straight surface at a specific slope. the red dots in the graph represent experimental points combining the three components (sabariman et al., 2021). the 3d graph model shows that the addition of spice powders significantly increased antioxidant activity, while coffee powder and date seeds had a smaller effect. conversely, the addition of collagen hydrolysate up to 50% actually decreased antioxidant activity to the lowest point. this response is visualized in a color gradient, with yelloworange indicating the highest activity and dark blue the lowest (mugendiran et al., 2014). the water content response shows that the appropriate statistical analysis model is quadratic, as shown in the 3d graph in figure 2. (a) (b) figure 2. 3d graph of the quadratic model (a) & contour graph of the water content response (b). the 3d quadratic model graph shows that the water content increases to its highest point with the addition of 50% hydrolyzed collagen or 40% spices. meanwhile, the date seed coffee powder produces the lowest water content, even with its highest composition reaching 55% of the total components. the characterization test results support this finding, with the highest water content in the hydrolyzed collagen powder (7.8%), followed by the spice mixture (2.78%) and the roasted date seed coffee powder (2.62%). hanifa – formulation of instant functional collagen drink with … 915 optimization results design expert-13 and verification optimization is carried out by setting criteria for each variable to achieve the antioxidant response and water content according to the target, as in table 4. table 4. optimization criteria based on desired response. parameter criteria minimum point maximum point roasted date seed powder is in range 30 55 hydrolyzed collagen powder is in range 25 50 spice mix is in range 20 40 antioxidant activity maximize 63.13 100.9 water level minimize 6.29 7.00 the recommended optimal response prediction resulted in a formulation with 35.56% roasted date seeds, 25% collagen powder, and 39.43% spice mix. the desirability value of the formulation was 0.741. the results were then verified to determine the significance of the difference between the prediction and the actual results table 5. table 5. verification of optimal formulation. response results p-value design expert predictions actual value antioxidant activity 94.29 93.40 0.176 water level 6.78 6.63 0.251 verification of the formulation prediction results by design expert 13 software was carried out through a paired t-test with a significance value of 0.05. if the pvalue <0.05, there is a significant difference between the prediction and the actual results, whereas if the p-value >0.05, the two are not significantly different (apriliyanti et al., 2017). based on the table, the p-value for antioxidant activity is 0.176 and water content is 0.251. this shows that the actual experimental results are accurate with the predictions analyzed by design expert 13. optimal results are then instantiated through a spraydrying method. the purpose of the instantaneous process is to improve the physical and sensory profile of the product while maintaining quality (garcía-carrasco et al., 2023). a 30% maltodextrin is used as a filler and coating for the active ingredient (ramadhani et al., 2024). characterization of the final product the optimal product that has gone through the instantaneous process is then characterized physically, chemically, and sensorily. (a) (b) figure 3. actual sample before spray drying (a) optimal sample powder resulting from spray drying (b). visually, the optimal sample powder from spray drying produced a yellowish white powder with a lightness (l) value of 40.90, which was much brighter than the commercial product (82.17). the optimal sample also had a lower redness (a*) level (3.90 compared to 6.23) and a higher yellowness (b*) level (15.80 compared to 9.57). previous research showed that the addition of maltodextrin above 10% can increase the brightness (l) and yellowness (b*) because the maltodextrin component affects the final color of the powder, following the basic color of maltodextrin which is yellowish white (romulo & aurellia, 2024). the final powder product resulting from instantaneous processing was characterized physically and chemically and compared with the commercial product as shown in table 6. table 6. characterization of the optimal formulation resulting from spray drying compared to commercial samples. test variables collagen coffee seeds dates spices (spray dry powder) commercial spice date seed coffee antioxidant activity ic50 (ppm) gae/100g) 743,864 275,443 total phenols (mg gae/100 g) 187,23 273,30 water content (%) 3.35 ± 0,04 3.40 ± 0,55 hygroscopicity (%) 7,76 ± 0.05 4,46 ± 0.48 bulk density (g/ml) 0.488 ± 0.008 0.521 ± 0.016 dissolve time (seconds) 23.32 ± 0.92 (insoluble) color: l 82,17 ± 0,49 40,90 ± 0,26 a* 3,90 ± 0,35 6,23 ± 0,12 b* 15,80 ± 0,17 9,57 ± 2,83 916 biology, medicine, & natural product chemistry 14 (2), 2025: 911-919 the spiced date seed coffee collagen drink sample had a lower ic50 antioxidant activity (743.864 ppm) compared to the commercial sample (275.443 ppm). according to jumina et al. (2019), antioxidant activity in the ic50 range is considered strong in the 50-100 ppm range, while values above 200 ppm are categorized as “fatty.” the decrease in antioxidant activity in spray-dried samples compared to commercial samples can be caused by the degradation of phenolic compounds due to high temperatures in the spray-drying process. spray drying in this study was carried out at an inlet temperature of 180°c with a feed rate of 10% (4.5 ml/min), which also contributed to the decrease in antioxidant activity. gawałek (2022) showed that phenolic compounds encapsulated by maltodextrin (md) of 25-35% can survive up to an inlet temperature of 150°c, but will decrease by 2.5% with increasing temperature. to reduce the process of damage to heat-resistant compounds, maltodextrin was added as an encapsulant as much as 30% (ramadhani et al., 2024). however, based on research by romulo & aurellia (2024), it was stated that the addition of maltodextrin above 10% can reduce the antioxidant concentration because the total mass of solids increases, replacing the antioxidant mass in the spraydried particles. spray drying treatment with 30% maltodextrin at an inlet temperature of 180°c did not significantly affect the product's water content, but increased hygroscopicity and decreased bulk density compared to the commercial product. the water content of the spray-dried powder (3.35%) was slightly lower than that of the commercial product (3.40%), but the difference was not significant (p-value 0.44>0.05). the hygroscopicity of the spraydried sample increased significantly, from 4.46% in the commercial product to 7.76%, although both were still classified as non-hygroscopic (huda, 2020). the use of a relatively high inlet temperature of 165°c to 225°c can increase the hygroscopicity of the spray-dried powder because products with low water content resulting from rapid drying at high temperatures tend to absorb water more easily (wijayanti et al., 2024). the addition of 25% collagen hydrolysate to the optimal sample significantly increased the amount of maltodextrin. therefore, the addition of maltodextrin from collagen hydrolysate and encapsulating agents to instant drinks causes the solid structure to be dominated by the hygroscopic starch component. this property makes maltodextrin more easily bind water, thereby increasing the hygroscopicity of the final spray-dried product (lee et al., 2018). therefore, the use of 30% maltodextrin can increase the hygroscopicity of the powder compared to commercial products without an instantaneous process due to its water-binding properties (ramadhani et al., 2024). the bulk density of the spray-dried sample was lower than that of the commercial sample, which was 0.488 g/ml compared to 0.521 g/ml in the commercial product, although both were not significantly different (p-value 0.091>0.05). the lower bulk density in the spray-dried product was due to the rapid evaporation process at high temperatures causing a "ballooning" effect, where the powder had cavities inside the dried wall particles. the addition of maltodextrin contributed to a decrease in bulk density by increasing the number of coating particles. the higher the concentration, the more coating particles were formed, preventing the sample particles from sticking together and increasing the number and volume of bubbles due to air desorption in the atomization process. as a result, a hollow structure was formed when dried at high temperatures due to the use of more maltodextrin (kwapińska and zbiciński, 2006). research by kodous (2020) showed that adding maltodextrin to dried animal collagen can produce a bulk density of up to 0.45–0.51 g/ml in the mixture. meanwhile, commercial samples that did not undergo heating or maltodextrin addition retained their original chemical characteristics and had a denser, coarser powder texture with a higher specific gravity. spray-dried powder containing 30% maltodextrin filler has a fine texture, tends to stick together, and easily clumps (cakes), which affects product rehydration. tests show that the powder dissolves within 23 seconds until completely dissolved. the size of the powder particles affects the permeability of water to penetrate the surface tension and wet the entire powder (wijayanti et al., 2024). larger particles have cavities between particles that allow water to penetrate more easily, while very fine maltodextrin particles make it more difficult for water to dissolve the powder. this factor is influenced by the dextrose equivalent (de) value of the maltodextrin, where de 10-12 indicates a moderate number of simple sugar chains, while the highest value reaches 20 (caballero et al., 2015). the higher the de, the larger the surface area and the stronger the cohesion between particles, thus slowing the dissolution of the powder (fathoni & hafidz, 2023). the solubility of spray-dried powder can be improved by combining maltodextrin with other encapsulant agents such as whey protein or cellulose to increase particle size and reduce interparticle cohesion (faldt et al., 1993). spray-dried products improved solubility compared to commercial samples without instantization, which leave a significant amount of coffee grounds. spray-dried products dissolved completely with low water content without solid residue, while retaining their characteristic flavor and aroma, which were subsequently tested hedonically. hedonic test the optimal product of the spray-dried spiced collagen date seed drink was tested for sensory acceptability through a hedonic test. this test was conducted using a preference rating method using a 1-5 scale (1 = dislike, 2 hanifa – formulation of instant functional collagen drink with … 917 = dislike somewhat, 3 = like somewhat, 4 = like, 5 = like very much) (meilgaard, 2016). a total of 100 panelists were used to obtain more representative data, considering that the hedonic test involved untrained panelists who could potentially provide subjective sensory responses (stone et al., 2021). in addition to measuring preference, the hedonic test also serves as aconsumer sensory test to assess the acceptance of new products in the market, because the selection of panelists is based on general consumer groups, not their sensory expertise (rogers, 2018). table 7. results of hedonic testing of optimal samples with commercial samples. response sample p-value spiced date seed collagen drink commercial spice date seed coffee color 2.34 ± 0.89 3.49 ± 0.83 0.000 consistency 3.81 ± 1.08 2.51 ± 0.98 0.000 aroma friends 3.76 ± 1.24 3.71 ± 1.20 0.728 the aroma of coffee 2.48 ± 0.86 3.38 ± 0.92 0.000 aroma of spices 3.68 ± 0.92 3.51 ± 1.11 0.142 overall aroma 3.57 ± 0.88 3.55 ± 0.93 0.851 coffee taste 2.61 ± 0.97 3.18 ± 1.05 0.000 taste the spices 3.35 ± 0.98 2.94 ± 1.15 0.001 rasa friends 3.58 ± 1.24 3.47 ± 1.21 0.351 overall taste 2.97 ± 0.95 2.87 ± 1.04 0.347 overall 3.17 ± 0.91 3.06 ± 0.98 0.251 based on table 7, the optimal spray-dried spiced collagen date seed drink product showed significant differences compared to the commercial product in several aspects. in terms of color, the optimal sample had a higher lightness (l) value (33.9 compared to 26.4) and increased a* and b* values, indicating reddish and yellowish intensity. this difference was caused by the material composition and the spray-drying process with the addition of maltodextrin above 10%, which affected the final color following the original color of the maltodextrin (kardas et al., 2024). panelists preferred the consistency of the optimal product because it produced a homogeneous solution without pulp and received a hedonic score of 3.81 compared to 2.51 for the commercial product. the perfect solubility of the optimal product supported the absorption of the hydrolyzed collagen so that it could be consumed entirely without any residue remaining in the beverage pulp (pudziuvelyte et al., 2019). fish collagen hydrolysate has a characteristic sensory aroma that tends to be fishy (dewantoro et al., 2019). the fishy aroma of fish collagen hydrolysate showed almost the same preference value (3.76 compared to 3.71), indicating that the panelists "quite liked" it, with no difference in preference between the two samples. however, the coffee aroma in the commercial sample was preferred more than in the optimal sample, as seen from the lower panelist preference value in the optimal sample (2.48 compared to 3.38). the date seed coffee in the optimal mixture had a lower proportion of coffee (35.56%) than the commercial product, which is almost 80% composed of date seed coffee. with a low proportion that is then dissolved into feed for spray drying, it will reduce the concentration of date seed coffee in the mixture (romulo & aurellia, 2024). fewer coffee components and supported by the degradation of volatile compounds during the spray drying process such as pyrazines, aldehydes, ketones, pyrroles, pyridines, and esters leaving more stable furans create a coffee aroma that is not too strong, so that panelists who like coffee tend to prefer commercial samples. the impact of spray drying on aroma is also based on research by (chindapan & puangngoen, 2024), where the spray drying process only leaves 43.94% of the total volatile compounds in the optimal sample. however, the impact of damage to volatile compounds due to spray drying occurs a lot in spices (suhendar & sogandi, 2019) in the optimal sample, which is actually preferred by panelists in the spice aroma attribute with a higher hedonic value than the commercial sample (3.68 compared to 3.51). so for the overall aroma, the optimal sample is close to equality with the commercial sample (3.57 compared to 3.55). for the taste attribute, the optimal sample shows a lower level of panelists' preference for the coffee taste (2.61 compared to 3.18) compared to the commercial sample. however, the optimal sample is superior in spice flavor (3.35 compared to 2.94) and there is no significant difference in the fishy taste (3.58 compared to 3.47). these results provide nearly comparable overall scores between the optimal sample and the commercial sample (2.97 versus 2.87), and a slightly higher overall acceptance rate for the optimal sample compared to the commercial sample (3.17 versus 3.06). overall, the spray-drying process reduced the intensity of the coffee aroma and flavor, thus decreasing panelists' preference. however, improving consistency characteristics through an instantaneous process that removes dissolved solids and increases the homogeneity of the optimal sample can increase panelists' overall preference for the product 918 biology, medicine, & natural product chemistry 14 (2), 2025: 911-919 (chen et al., 2020). the heat process in spray-drying, which can reduce the presence of volatile compounds (rogers, 2018), also appears to improve the balance of aroma and spice flavors to reduce overpowering and successfully increase product acceptance, as reflected in consumer hedonic tests. conclusion the optimal formulation consisted of roasted date palm seeds (35.56%), hydrolyzed collagen powder (25%), and spice mixture (39.43%) with a predicted antioxidant activity of 94.29 mg te/100 g and a moisture content of 6.78%. verification results showed an actual antioxidant activity of 93.40 mg te/100 g and a moisture content of 6.63%, which was in accordance with the prediction. the characterization results of the spray-dried product had lower ic50 and total phenol antioxidant activity than the commercial product (743.864 ppm and 187.23 mg gae/100 g). however, they were superior in moisture content and dissolution time (23.32 seconds) compared to the commercial sample, despite having higher hygroscopicity (7.76%) and a lower bulk density of 0.488 g/ml. the hedonic test showed that the optimal product was preferred in consistency and spice flavor, while the commercial product was superior in aroma and coffee 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ja’far luthfi1,* , riyanto2,**, widodo1 1biologica education department, faculty of tarbiyah and education, uin sunan kalijaga 2integrated laboratories, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto 55281 yogyakarta, tel. +62-274-540971, indonesia corresponding author jafarluthfi@yahoo.com*, justofeel@gmail.com** manuscript received: 29 september, 2023. revision accepted: 20 december, 2023. published: 29 january, 2024. abstract this research combines the fields of biology, photography, graphic design, and informatics. this study aims to develop a virtual herbarium museum through website media. the research was done to improve the accessibility and sharing of data (free access) herbarium. website development was performed using the sdlc (systems development life cycle) which consists of 5 (five) steps, namely planning a strategic website development plan, determining the scope of development, analyzing the required needs, designing and implementing website creation and testing. based on the results of system testing and implementation, it can be concluded that the virtual herbarium museum has been successfully built as a plant taxonomy complementary lab work material. keywords: virtual herbarium; online herbarium; baturagung. introduction nglanggeran is a remnant of ancient mountain which still retains a wide variety of wild plants. the nglanggeran site has a potential database of plant remains in the northern gunung kidul mountain zone, or what we know as the baturagung zone. herbarium is preserved plant material. the functions of the herbarium include is as a data storage center, documentation, references, research materials, teaching materials for taxonomy lab work, and as reference specimens used for plant identification. herbarium has several drawbacks, namely, (1). poor maintenance or frequent use can cause the specimen to be easily damaged, (2). not easily accessible by several people together, (3), only directly accessible or not remotely accessible, (4), can not be accessed at any time. biological research museums have the same mission as libraries, but instead of preserving books, biology collections preserve individual organisms. the collection grows continuously through the activities of faculty, students, and other researchers, as well as by exchanges with other museums. as they grow, collections increase in value, preserve samples of natural variation, document the occurrence of species in space and time, and provide an important basis for our understanding of species identity. the biology collection is also a source of information regarding phylogenetic (evolutionary) relationships. without this phylogenetic context, no comparative biology would be possible. the rapid advancement of information technology makes the accessibility of information exchange easier. the convenience obtained from the development of information technology is important in supporting research and scientific activities. one of the advances in information technology is the rapid development of the internet which is a supporting factor in this accessibility. the rapid development of the internet can be seen from the internet user data which is always increasing from year to year (internetworldstats.com, 2024), both globally and in indonesia. the data is as shown in the following graph: https://doi.org/10.14421/biomedich.2023.122.625-630 626 biology, medicine, & natural product chemistry 12 (2), 2023: 625-630 figure 1. a. world internet users (petrosyan, 2024), b. internet users in indonesia (apjii, 2015). in 2019, the number of internet users worldwide was 3.97 billion, up from 3.74 billion in the previous year (statista, 2019). at the end of 2014 internet users in indonesia reached 88.1 million people or around 34.9 percent of the total population of indonesia with the total spread covering the regions of java, bali, sumatra, kalimantan, nusa tenggara -papua-maluku, and sulawesi (apjii, 2015). whereas in 2016 internet users in indonesia increased to 132.7 million people (apjii, 2016), where students occupy the highest position as internet users. therefore, the internet has a strong role in the distribution of information as well as knowledge and research. in 2019, internet users in indonesia increased again to 196.71 million people. the increasing number of internet users and internet penetration at the global level provides a very wide opportunity for all parties to take advantage of it, including for publication and promotion with various internet-based media. the website is one of the media that is able to provide information and an easy level of accessibility. the number of internet users who reach more 3 billion worldwide with 634 million websites and each year 51 million sites are added to the web, since it was first registered in 1985 there have been around 100 million dot domain names com. in 2012 there were 246 million domain name registrations. (detik.com, 2013) high accessibility and several conveniences in building and maintaining a website make it an effective medium for providing information to the public. the existence of this website is expected to help those involved in the world of education in particular and the world community in general who are interested in the field of biology, especially in the field of botany. the development of photography/digitization technology provides new options in explaining the herbarium recording process for plant identification purposes as well as a database for research and scientific purposes. we have been successfully developing animal anatomy material to be used by wider audience in the form of online atlas (luthfi & riyanto, 2017). now, in this research, we combines the fields of biology, photography, and informatics to design an online herbaria website as an initiation for the creation of a virtual herbarium at uin sunan kalijaga. this research seeks to improve accessibility and share data (free access) for herbarium collections and is also expected to be a promotional medium for uin sunan kalijaga yogyakarta to the plant taxonomy community, related to research and educational media in the field of botany, plant taxonomy and biodiversity. material and method this research is a combination of 4 (four) fields of science; namely biology (botany), photography, graphic design, and informatics. the integration between the science of photography and the science of biology is in recording herbarium specimen objects, and then processing the image (design) to bring out the aesthetic value, and then applying it in the preparation and creation of an online herbarium database that can be accessed by many people from various places. the material and equipment for making herbarium as raw material for webite were as follows: plant species, plastic bag, paper, label, thread, a set of herbariummaking tool, oven, and pencil. the herbarium preparation was performed by a team supervised by dr. widodo. all the identification work was done by dr. widodo, the taxonomist of uin sunan kalijaga. all specimens were kept in the integrated laboratories, uin sunan kalijaga. the focus of this research was to design, build, and test an online herbarium website. the tools used were: ▪ canon eos 60d camera. ▪ laptops with os windows 10, php programming language, database servers mysql client, apache local web server, web browser, sublime text, adobe photoshop cs6, coreldraw x6. ▪ server for database processing and storage. a b luthfi, et al. – developing virtual herbarium of sunan kalijaga islamic state university as … 627 this research was conducted in 5 (five) stages, namely: literature study, data collection and processing, system design, system testing, and system implementation analysis. here's the description: ▪ first, the study of literature, namely the study of theories related to the identification and classification of plants, photography techniques, theory of website creation and programming languages. ▪ second, data collection and processing, namely taking photos/pictures of herbarium objects and processing these images with the help of software to obtain image data as expected. ▪ third, creating a website by means of insoursing (planning, needs analysis, design, manufacture, & testing) (mulyanto, 2008) figure 2. sdlc scheme (system development life cycle). (photo: intellectsoft.net) ▪ fourth, system testing. system testing was carried out in two stages; (1) alpha testing, carried out by the research team (focus on functional systems), (2) beta testing, done by external parties (focus on functionality and system interface). ▪ fifth, system implementation. we carried out the implementation of making a website system so that comprehensive data or facts about the herbarium website were obtained. results and discussion in this research, we have successfully developed a website of virtual herbarium of sunan kalijaga islamic state university. we used program/soure that already available namely hypertext markup language, javascript, hypertext preprocessor, then manage them into the admin and user system. hypertext markup language document is text document created using any text editor. hyper text markup language is also known as a web page. among the elements forming the hyper text markup language document are composed by head, table, body, list, paragraph. html is used to layout, formatting and tells the browser how to display this virtual herbarium. the elements or tags of html work together to identify document parts, so the design of the virtual herbarium can be displayed in the browser. html or hypertext markup language is the standard markup language for documents designed to be displayed in a web browser (wikipedia.org). it is the standard markup language for creating web pages that was invented by tim berners-lee a physicist at the cern research institute in switzerland. tim bernerslee also created the first web browser and world wide web while working at cern. the web was originally conceived and developed to meet the demand for automated information-sharing between scientists in universities and institutes around the world. (cern, (n.d)) javascript is a high-level, prototype-based, objectoriented, multi-paradigm, interpreted or just-in-time compiled, dynamic, single-threaded, garbage-collected programming language with first-class functions and a non-blocking event loop concurrency model (atalay, 2023). javascript is a scripting or programming language that allows to implement complex features on web pages. (developer.mozila.org). this programming language can work on most of the web browsers. code usage it can be inserted in the page web using the script tag (sunyoto, 2007). in this research, javascript are used to create dynamically updating content, control multimedia, and animate images. php (hypertext preprocessor) is a general-purpose scripting language and interpreter that is freely available and widely used for web development. the language is used primarily for server-side scripting, although it can also be used for command-line scripting and, to a limited degree, desktop applications (sheldon, 2023). hypertext preprocessor supports large-scale database web applications. the advantages of php are software open source, flexible in operating systems and operating systems, and has the ability to execute databases quickly. in this research, hypertext prerocessor are used to generate dynamic content when the page is accessed through a client browser. data preparation in the form of herbarium documents was carried out by taking photographs of herbarium collections, and around 130 herbarium photographs were obtained. the pictures below are some photographs of the herbarium collection. https://id.wikipedia.org/wiki/bahasa https://id.wikipedia.org/wiki/penjelajah_web https://id.wikipedia.org/wiki/kode https://id.wikipedia.org/wiki/web 628 biology, medicine, & natural product chemistry 12 (2), 2023: 625-630 figure 3. some photographs of herbarium specimens (a. cosmostigma racemosum, b. marsdenia tenacissima, c.allophyllus cobbe sapindaceae). the admin system is used by the website manager to manage the herbarium museum website, either in the form of adding herbarium collections, editing collections that have been uploaded, or deleting them. the appearance of the admin page is as follows: figure 4. virtual herbarium of uin sunan kalijaga admin system (a. login page, b. main dashboard page, c. uploaded collection page, d. the page adds to the herbarium collection, e. the page edits the uploaded herbarium collection). cba c ba d e luthfi, et al. – developing virtual herbarium of sunan kalijaga islamic state university as … 629 the user system is a system intended for external users who will access the virtual herbarium museum website. on the user page, a list of herbarium collections is provided, which is displayed as a list of names only or displayed in gallery form. figure 5. virtual herbarium of uin sunan kalijaga. the virtual herbarium of sunan kalijaga islamic state university can serve as a raw material for complementary resource in teaching plant anatomy subject, especially in the lab work activity. lab work activity is a complement in the teaching of plant anatomy subject. since the chatacter of teaching and learning in the lab work is rather loose, not as rigid as the classroom activity, lecturers, teachers, and students can use this material in a flexible way. it can be embedded into the lab work activity formally or informally, using any pedagogic method available. this virtual herbarium could enrich the plant anatomy knowledge of the students following the lab work activity. conclusion from the results of the study, it was concluded that this research had succeeded in building a website-based virtual herbarium museum available online at the site address botany.id. the virtual herbarium of sunan kalijaga islamic state university can serve as raw material for complementary resource in teaching plant anatomy subject, especially in the lab work activity. acknowledgment: this research is supported and funded by the institute for research and community service (lp2m) uin sunan kalijaga for the 2022 fiscal year. 630 biology, medicine, & natural product chemistry 12 (2), 2023: 625-630 conflicts of interest: mjl is on the editorial board of the biology, medicine, & natural product chemistry, and was recused from this article’s review and decision. the authors declare that there are no conflicts of interest. references apjii. 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(2023). https://www.intellectsoft.net/blog/what-is-systemdevelopment-life-cycle/ internet usage statistics. (2024). population stats. internetworldstats. https://www.internetworldstats.com/stats.htm luthfi, m., & riyanto, r. (2017). on designing interactive online atlas of reptile anatomy (mabouya multifacsiata). biology, medicine, & natural product chemistry, 6(2), 63-69. doi:https://doi.org/10.14421/biomedich.2017.62.63-69 mulyanto, a. (2008). sistem informasi konsep dan aplikasi. yogyakarta: pustaka pelajar. mozilla. (n.d). what is javascript?. https://developer.mozilla.org/enus/docs/learn/javascript/first_steps/what_is_javascript petrosyan, ani. (2024). number of internet users worldwide from 2005 to 2022. statista.com. http://www.statista.com/statistics/273018/number-of-internetusers-worldwide/ sunyoto, a. (2007). ajax membangun web dengan teknologi asynchronouse javascript & xml. yogyakarta: andi. sheldon, robert. (2023). php (hypertext preprocessor). techtarget. https://www.techtarget.com/whatis/definition/php-hypertextpreprocessor wikipedia (n.d). html. https://en.wikipedia.org/wiki/html https://doi.org/10.14421/biomedich.2017.62.63-69 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1241-1245 | doi: 10.14421/biomedich.2025.142.1241-1245 issn 2540-9328 (online) the potency of kuldon® solution in the healing process of aphthous stomatitis dewi priandini1*, alfred pakpahan2, rahmi amtha1, aprilia puspita sulistia3, abrijanto4 1oral medicine department, faculty of dentistry, universitas trisakti, indonesia. 2oral biology department, faculty of dentistry, universitas trisakti, indonesia. 3hospital dr,r.hardjanto, indonesia military hospital, east kalimantan, indonesia. 4pt. deltomed laboratories, indonesia. corresponding author* dewipriandini@trisakti.ac.id abstract objectives: this study aimed to assess the efficacy of kuldon® solution in the healing process of aphthous stomatitis. methods: a double-blind randomized controlled trial was conducted involving 60 subjects (27 males, 33 females), randomly assigned to receive either kuldon® solution (drug a) or hyaluronic acid rinse (drug b). pain was evaluated using the visual analogue scale (vas), and lesion diameter was measured on days 2, 5, and 7. data were analyzed using the mann-whitney test (p < 0.05). results: drug a showed a greater reduction in pain and lesion size compared to drug b. by day 5, 100% of drug a users reported no pain, with 90% showing lesion reduction.conclusion: kuldon® solution demonstrates significant therapeutic potential in managing aphthous stomatitis. keywords: aphthous stomatitis; kuldon® solution; healing potency. introduction recurrent aphthous stomatitis (ras), is one of the most common oral disease and called "canker sores." ras is a perplexing oral condition characterized by the recurrent development of painful aphthous ulcers on nonkeratinized oral mucous membranes. this condition poses a significant challenge to patients and healthcare professionals due to its uncertain ethology. ras is a reserve in the oral mucosa with a worldwide prevalence of 5-25%. ras mostly occurs at the age of 10-40 years and many occur in women (sánchez-bernal j, et. al, 2020). ras one or several discrete, shallow, painful ulcers are visible on the unattached oral mucous membranes. ras is a self-limited disease and a typical minor aphthous ulcer can spontaneously heal within 4-14 days (ginat w mirowski, et. al, 2020). individual ulcers typically last 7-10 days and heal without scarring. larger ulcers may last several weeks to months and can scar when healing (mahmoud k, et al 2015). the basic principle of treatment is to reduce pain, shorten the course of the lesion by extending the interval for the lesion to appear (marco manfredini, et. al, 2021). treatment can use patent medicines or traditional medicines but still safe and the price of the medicines is relatively cheap. during the latter part of the 20th century herbalism has become mainstream worldwide. this is due in part to the recognition of the value of traditional and indigenous pharmacopoeias, the incorporation of some derived from these sources into pharmaceuticals), the need to make health care affordable for all, and the perception that natural remedies are somehow safer and more efficacious than remedies that are pharmaceutically derive (elvin-lewis m, 2001). the discovery of herbal medicinal ingredients is not only based on experiences from generation to generation, but also based on experimental or scientific study (waode munaeni, et.al, 2022). kuldon® solution as a. standardized commercial herbal syrup consists of rizhoma of imperata, abrus precatorius, herbal thyme (thymus vulgaris), centella asiatica, curcuma xanthorrhiza roxb, piper battle, chrysanthemiflos, and glycyrrhiza glabra. the rhizome of imperata could be used either alone or in combination with other herbal medicines to cure hematuresis, jaundice and reducing fever, to treat inflammation (wei zou, et. al, 2021). (figure 1a) manuscript received: 12 august, 2025. revision accepted: 28 november, 2025. published: 12 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1241-1245 https://pubmed.ncbi.nlm.nih.gov/?term=zou+w&cauthor_id=34274601 1242 biology, medicine, & natural product chemistry 14 (2), 2025: 1241-1245 figure 1. a. rhizome of impeata; b. abrus precatorius; c. thymus; d. centela asiatica; e. curcuma; f. piper betle leaf; g. chrysanthemum; h. glycyrrhiza xanthorrhiza r. the abrus precatorius is having medicinal potential to cure various diseases. the roots, leaves and seeds of this plant are used for different medicinal purpose. abras precatorius are laxative, expectorant and aphrodisiac medicines and are used in urticaria, eczema, stomatitis, conjunctivitis (garaniya, n et.al, 2014). (figure. 1b). the thymus vulgaris is generally known as thyme. the plant is useful as infusion to treat cough, diabetes, and cold and chest infections. it is also soothing for sore throat, as thyme has antiseptic, antibiotic, and antifungal properties. thymus vulgaris has been thought of to be astringent, anthelmintic, carminative, disinfectant, and tonic (nasrun,mf. et.al, 2023). (figure. 1c) centella asiatica is an important medicinal herb. triterpenoid, saponins, the primary constituents of centella asiatica are manly believed to be responsible for its wide therapeutic actions. apart from wound healing, the herb is recommended for the treatment of various skin conditions such as leprosy, lupus, varicose ulcers, eczema, psoriasis, diarrhoea, fever, amenorrhea, diseases of the female genitourinary tract and also for relieving anxiety and improving cognition (gohil kj. et. al, 2010). (figure. 1d) the curcuma xanthorrhiza roxb., locally famed as temulawak, has been extensively utilized in indonesia as medicinal and nutritional plants since immemorial time. the rhizome of this plant is an important ingredient for jamu formulation (indonesian traditional medicine). c. xanthorrhiza is traditionally used to treat several ailments such as lack of appetite, stomach disorder, liver illness, constipation, bloody diarrhea, dysentery, arthritis, children's fevers, hypotriglyceridaemia, hemorrhoids, vaginal discharge, rheumatism, and skin eruptions (rahmat endang, et.al,2021). (figure. 1e). piper betle (l) is a popular medicinal plant in asia. plant leaves have been used as a traditional medicine to treat various health conditions. this current review showed that betel leaves extract, essential oil, preparations, and isolates could inhibit microbial growth and kill various gram-negative and gram-positive bacteria as well as fungal species, including those that are multidrug-resistan and cause serious infectious diseases. some studies also showed that the combination of betel leaves extract and essential oil with antibiotics (streptomycin, chloramphenicol and gentamicin) could provide potentiating antibacterial properties mara nm (nayaka,et.al, 2021). (figure. 1f) the chrysanthemum contains chemicals called flavonoids, which have antioxidant and antiinflammatory effects. it also contains chemicals that may increase blood flow to the heart. chrysanthemum flowers are rich in phenolic compounds and exhibit strong properties including antioxidant, antimicrobial, antiinflammatory, anticancer, anti-allergic, anti-obesity, immune regulation, hepatoprotective, and nephroprotective activities (niharika sharma, et. al, 2023) (figure. 1g). the glycyrrhiza glabra. is an herbal plant which has lots of medicinal propertie, there are various beneficial effects of glycyyhiza glabra (licorice) root extracts, such as treating throat infections, tuberculosis, respiratory, liver diseases, antibacterial, a b c d e f g h priandini et al. – the potency of kuldon® solution in the healing process of … 1243 anti-inflammatory, and immunodeficiency (siracusa l, et. al, 2011). (figure. 1h). findings of the current review indicated medicinal plants and phytochemicals as effective and safe agents that for the treatment of recurrent aphthous stomatitis (shavakhi m, et. al, 2021). there is some evidence of the efficacy of topically applied natural herbal medicines with regards to improved recurrent aphtous stomatitis outcome measures and fewer side effects (li cl, et. al, 2016). based on this, we conducted research is there any effectiveness and how long of herbal syrup in healing process of aphtous stomatitis? the research gap is that it takes time to provide kuldon syrup processed in factories, well-designed and high-quality randomized controlled trials are required for further exploration this study aimed to evaluate the efficacy of kuldon® solution in the healing process of aphthous stomatit materials and methods a double-blind, randomized controlled trial (rct) was conducted with 60 subjects (27 males and 33 females), who were randomly assigned to receive either drug a (kuldon® solution) or drug b (hyaloronic acid rinse) under consent. pain levels were assessed using a visual analogue scale (vas), and the diameter of the lesions was measured on days 2, 5, and 7. research was carried out at dental hospital faculty of dentistry universitas trisakti. data analysis data were analyzed using the mann-whitney test.the significance level was determined at p<0.05. results and discussion result the demographic characteristics of the 60 participants are summarized in the following table. as shown in table 1, the majority of the participants were female (55%) and male 27 (45%.), with the most size of lesion are minor 57(95%) and mayor 3 (3%.) table 1. the demographic characteristics. variables n % gender female 33 55 male 27 45 size of lesion minor 57 95 mayor 3 3 measurement of pain was using vas in 60 patients with aphthous stomatitis before being given treatment, where the majority felt moderate pain 87% (52 patients), followed by severe pain of 13% (8 patients) and 0% (0 patients) mild pain. on the second day, 73.3% of the subjects using drug a reported no pain, compared to 60% of those using drug b. by the fifth day, all subjects (100%) using drug a experienced no pain, while 90% of those using drug b reported the same. lesion diameter reduction was 40% for both treatments by day 2. by day 5, 90% of subjects using drug a showed a reduction in lesion diameter, compared to 16.6% of those using drug b. table 2. normality test shapiro-wilk. drugs kolmogorov-smirnov shapiro-wilk statistic df sig. statistic df sig. day 0 drug a .276 30 .000 .724 30 .000 drug b .413 30 .000 .588 30 .000 lesion size day-2 drug a .179 30 .016 .881 30 .003 drug b .208 30 .002 .753 30 .000 day 3 drug a .487 30 .000 .452 30 .000 drug b .470 30 .000 .497 30 .000 day-7 drug b .539 30 .000 .180 30 .000 day 0 drug a .292 30 .000 .789 30 .000 pain intensity drug b .340 30 .000 .708 30 .000 day 2 drug a .441 30 .000 .608 30 .000 drug b .353 30 .000 .712 30 .000 day 5 drug b .526 30 .000 .360 30 .000 table 3. statistics analysis. lesion size pain intensity (vas) day 0 day 2 day 5 day 7 day 0 day 2 day 5 day 7 man-whitney u test 281.000 423.500 430.000 435.000 446.500 380.500 405.000 450.000 wilcoxon w 746.000 888.500 895.000 900.000 911.500 845.500 870.000 915.000 z test -2.602 -.404 -.438 -1.000 -.055 -1.227 -1.762 .000 asymp. sig. (2-tailed) .009 .686 .661 .317 .956 .220 .078 1.000 1244 biology, medicine, & natural product chemistry 14 (2), 2025: 1241-1245 discussion on day 2 there was an equivalence between drug a (kuldon®solution) and drug b (hyaluronic acid rinse ®) in reducing the diameter of the lesion, which was 40% each because it had a potent anti-inflammatory of each of these drugs. the vas showed that as many as 22 patients (73.33%) on drug a experienced no pain. not painful was also admitted by 18 patients (60.00%) using drug b. drug a showed more anti inflammation and analgesics compared to drug b. drug a, which is a standardized commercial herbal syrup known as kuldon® syrup, contains a vine saga (abrus precatorius) has roots, stems, and leaves saga contains glycyrrhizine a which has activity as an antiinflammatory, antitubercular, antiplasmodial, platelet anti-aggregation and antiallergic activity (yuli w, m. bakti, 2011). abruquinone g is active as an antiviral and has toxic properties (rahmat endang, et,al, 2021). another compound that also has anti-inflammatory activity is the saponin triterpene. especially the glycyrrhiza glabra, of this plant has therapeutic properties, namely extracts used as tonics, detoxification, expectorants, anti-inflammatory, antimicrobial, antiatherogenic, antiallergic, antiviral against metabolic syndrome, obesity and changes in the immune system. centella asiatica has asiaticoside which functions to improve the repair and strengthening of skin cells, stimulation of growth, nails, hair, connective tissue, stimulating blood cells, has good vascularization and immune system and is a type of natural antibiotic. (steven,e, 2008). other ingredients in kuldon@ solution play a synergistic rol e in causing anti-inflammation, such as curcuma xanthroohiza has antitoxic, anti-hepatotoxic and antibacterial compounds. curcumin and xanthorrhizol are the main active ingredient components of essential oils 13 in betel leaf (piper betle) there are eugenol as methyl eugenol, carvacrol, chavicol, allcatechol, cavibetok, cineol, estagrol, thiamine, riboflavin, and vitamin c. these are efficacious for antiseptic, astringent, expectorant and hemostatic (kartasapoetra g, 1992). reed root (imperata rhizoma), a kind of weed root, is empirically used to lower temperature, help urinate, stop bleeding, and as a remedy for bleeding in the nose, vomiting blood, gonorrhoea, hepatitis, and kidney infections (yuli,wm, 2011). meanwhile, thyme herb (thymus vulgaris) has antimicrobial, ant stimulant, antioxidant, and anti-inflammatory properties (yuli w, m. bakti, 2011). drug b as positive control is a patent mouthwash consisting of aloe vera (aloe vera), sodium hyaluronate has a hyaluronic acid content, which is an additional therapy after mechanical therapy, namely, to accelerate wound healing, anti-inflammatory, cell proliferation, and migration, angiogenesis and reepithelialization through keratin basal cell proliferation. hyaluronic acid molecules reduce the proliferation of epithelial cells, such as fibroblasts and lymphocytes, that play an active role in chronic inflammatory states, thereby accelerating the regeneration of new healthy tissue cells (schroeder he, et. al, 1983). glycyrrhetinic acid has pharmacological properties, namely anti-inflammatory and anti-ulcer, anti-tumour, anti-viral, anti-hepatotoxic, and polyvinylpyrrolidone (pvp) (sibbald r, et. al, 2011; ricky, et. al, 2016, lewis mao,2012). furthermore, on the 5th day with drug a, as many as 90.00% of patients had no visible lesions, and with drug b, only 3.44%. drug a has good vascularization (ehrlich steven, 2008, dewi rizna triana, et.al, 2015) and other ingredients that play a synergistic role in causing anti-inflammatory effects so that it has a better healing effect. drug b has pvp as a binder and thickener (ricky, et. al, 2016; lewis mao, 2012), so patients feel uncomfortable when using the drug. this affects subjective assessments using vas. in the last control (day 7), there were still 10% of patients who had healed oral lesions using drug a while 16.67% of patients were using drug b. there was a balance in the healing process based on the diameter of the lesions of the two drugs. patients using drug a no longer felt pain on day 7, but in group drug b user, there were still 3 patients. drug a showed better effectiveness in curing aphtotic stomatitis based on a subjective assessment of the level of comfort (pain). in the normality test, the data used for each variable is less than 50. therefore, a normality test is carried out using the shapiro-wilk method. then it was tested again using the mann-whitney method with a sig or p (p< 0.05). the test showed a p-value of 0.000 < 0.05, which was significant. this shows the significant effective of standardized commercial herbal syrups in the healing process of minor and major aphthous stomatitis. (table 2; table 3) conclusions the kuldon® solution demonstrates significant potency in the healing of aphthous stomatitis lesions. acknowledgements: special thanks to pt deltomed laboratories have prepared herbal ingredients and visited the factory for processing of kuldon® solution. authors’ contributions: dewi priandini and aprilia puspita sulistia designed the study. abrijanto carried out the laboratory work. dewi priandini and alfred pakpahan analyzed the data. dewi priandini and rahmi amtha wrote the manuscript. all authors read and approved the final version of the manuscript conflicts of interest: ap is on the editorial board of the biology, medicine, & natural product chemistry, and priandini et al. – the potency of kuldon® solution in the healing process of … 1245 was recused from this article’s review and decision. the authors declare that there are no conflicts of interest. references dewi rizna triana, maryani fariza. 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(2011). phytocomplexes from liquorice (glycyrrhiz glabra l.) leaves chemical characterization and evaluation of their antioxidant, anti-genotoxic, and anti­ inflammatory activity. fitoterapia. june; 82(546-556). waode munaeni., meillisa carlen mainassy., et. al. (2022). perkembangan dan manfaat obat herbal sebagai fitoterapi. publisher: cv. tohar media, isbn: 978-623-5603-81-0.1ed june:p,3-8. wei zou., yaqian dong., shicong yang.m et,al. (2021). imperatae rhizoma-hedyotis diffusa willd. herbal pair alleviates nephrotic syndrome by integrating anti-inflammatory and hypolipidaemic effects. sep:90:153644.doi:10.1016/j.phymed.153644. epub 2021 jun 29. yuli widiyastuti., m. bakti samsu adi.,100 top tanaman obat indonesia (2011). kementrian kesehatan ri – balai besar penelitian dan pengembangan tanaman obat dan obat tradisional. october. p.2-3;40-41;62-63;108-109. https://emedicine.medscape.com/aricle/1075570-overview https://pubmed.ncbi.nlm.nih.gov/?term=patel%20ja%5bauthor%5d https://pubmed.ncbi.nlm.nih.gov/?term=gajjar%20ak%5bauthor%5d https://www.ncbi.nlm.nih.gov/pmc/articles/pmc10582400/ https://pubmed.ncbi.nlm.nih.gov/32451064 http://www.umm.edu/altmed/article/gotu-kola-000253.htm http://www.umm.edu/altmed/article/gotu-kola-000253.htm this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 363-369 | doi: 10.14421/biomedich.2023.121.363-369 issn 2540-9328 (online) fiber concentration on fermentation of cleome gynandra l based on storage time and solvent change lily restusari1,*, ayu komala dewi2, alinea dwi elisanti3 1poltekkes kemenkes riau, pekanbaru, jl melur no 103, pekanbaru 28122, indonesia 2langsat public health center, pekanbaru, jl. langsat no.1, jadirejo, kec. sukajadi, kota pekanbaru, riau 28121, indonesia 3clinical nutrition, health departemen politeknik negeri jember, jl. mastrip, krajan timur, sumbersari, kec. sumbersari, kabupaten jember, jawa timur 68121 indonesia. corresponding author* lily.restusari@pkr.ac.id manuscript received: 11 october, 2022. revision accepted: 11 march, 2023. published: 21 june, 2023. abstract cleome gynandra l (cgl) or maman plant, is the basic ingredient of malay food in riau province, indonesia. the young leaves and stems of cgl are processed into fermented food (joruk maman). it contains crude fiber and is useful for lowering blood cholesterol levels. however, effective storage of this cgl has not been carried out. this study wants to see the effect of storage time and solvent change on the fiber content of joruk maman. an experimental study applied a completely randomized design (crd) using 5 groups and 2 repetitions. this sample of cgl leaves was taken from one seller in the rokan hilir market of riau province. the primary outcome was a difference to the number of fibers in joruk maman without solvent change (p =0.001) and with solvent change (p = 0.001) based on the day group and there was no difference base on the temperature group. secondary outcome was the difference in duration time to produce the highest fiber content at room temperature with the solvent change and not. the highest fiber content occurred at 5 days of storage at room temperature without solvent changing. meanwhile, by changing the solvent, the fiber content would be optimal for 1-day of storage. keywords: cleoma gynandra l; joruk maman; fiber; storage; solvent change. abbreviations: cleoma gynandra l (cgl) introduction the world health organization (who) and the food and agriculture organization (fao) have recommended high consumption of fruits and vegetables to prevent various chronic diseases such as hypertension, coronary heart disease, and stroke. however, who and fao emphasize consuming fresh fruits and vegetables to get high nutritional content (endrizzi et al., 2009) one of the examples is cleoma which belongs to the capparaceae family. the people of rokan hilir, riau, indonesia recognize the maman plant which originated from south africa. throughout africa, the tender leave or young shoots, and often the flower are boiled and consumed as a potherb, delicious dish, stew, or side dish. the fresh leaves are used as an ingredient in other mashed foods, and the dried leaves are ground and put into weaning foods. however, boiling the leaves can reduce the vitamin c content by up to 81%, while drying the leaves reduces the vitamin content by up to 95% (heever & venter, 2007). this maman plant is used as a traditional antidiabetic, anti-agingg, anti-cancer, and cardiovascular disease prevention (mishra et al., 2011). this plant is often found as a wild plant and it grows anywhere. people in this village usually consume this plant as a fermented vegetable. this fermented product of the maman plant (cleome gynandra l) is processed from young leaves and stems which are mixed with salt, rice, and warm water and then it is left for 2-3 days before the people consume it. the villagers of bunga tanjung rohul and tanah putih rohil usually call this food by the name of cgl fermentation or joruk maman. fermented products of the maman plant are commonly consumed by the public, and fermented products can be consumed with or without rice (saida, e, 2014). fermented products of maman plant or cgl fermentation contain crude fiber and lactic acid bacteria (lab) which function as probiotics. dietary fiber has a very important function for health maintenance and prevention of various degenerative diseases such as diabetes, hypercholesterolemia, stroke, coronary heart disease, and obesity as well as digestive disorders such as constipation, hemorrhoids, and colon cancer (winarti, 2010). based on a review (institute of medicine, 2005) the adequacy of total dietary fiber in adolescents and adults https://doi.org/10.14421/biomedich.2023.121.363-369 364 biology, medicine, & natural product chemistry 12 (1), 2023: 363-369 is 14 g/1000 kcal. the highest crude fiber content was obtained in cgl fermentation with the addition of 2% salt and 15% rice, which was 0.43 g per 100 g of fermentation. maman has crude fiber content which can reduce cholesterol levels and also contains lactic acid bacteria. the content of lactic acid bacteria in maman's fermentation with the addition of 5% salt and 10% rice had the highest result of 2.40 x 108 cfu/g (lily restusari, muharni, 2019). people can make joruk maman, or they can purchase at traditional markets in rokan hulu and rokan hilir, riau indonesia. traditionally, local people will replace the fermented soaked water with boiled water if cgl fermentation is stored for a long time and the sensory taste change. moreover, after they change the water, they leave it overnight and cgl fermentationis ready to be consumed again with the same taste as the new cgl fermentation produced. materials and methods study area the determination of maman in sekeladi village, rokan hilir regency and determined at the andalas university herbarium (figure 1). figure 1. cleome gynandra l (maman’s plant) by the author on sekeladi village, rokan hilir regency. this research type is an experimental study conducted to determine the storage time and solvent change to fiber content in cgl fermentation by applying a completely randomized design using 1 treatment and 2 repetitions. maman fermentation was stored at room temperature and then was taken aseptically every day on days 1, 2, 3, 4, and 5. on day 5, the solvent was replaced by boiled water with the same amount of water replaced. then, it was continued the inspection of dietary fiber on days 1, 2, 3, 4, and 5 after the solvent was replaced. the research tools used were analytical balance, niemeyer, measuring cup, condenser, funnel, vacuum pump, oven, desiccator, heater, and watch glass. the materials used were maman (cleome gynandra l) leaves, 0.85% nacl diluent solution, table salt (pn salt) and local rice-pandan wangi varieties, sulfuric acid, naoh, whatman filter paper, alcohol, micropipette tips, and distilled water procedures this research was carried out by weighing 1-2 grams of fermented maman samples and put into a 500 ml erlenmeyer, and then it was added 50 ml of hot 1.25% h2so4 and refluxed for 30 minutes. after that, it was added 50 ml of 3.25% naoh and refluxed for 30 minutes. the heated sample was then filtered in hot condition with whatman 42 filter paper whose weight was known. after being filtered, the sample was washed with 50 ml of 1.25% h2so4 and 50 ml of 36% alcohol, then the precipitate was dried in an oven at 105°c and weighed to a constant weight. this research was conducted at the laboratory of agricultural polytechnic of andalas university in payakumbuh and the microbiology laboratory of the health polytechnic of the ministry of health riau. the research time was approximately 6 months from july to november 2020. this research involved 1 alumnus as a field survey officer and 1 laboratory analyst. data analysis the survey primarily collected the sample at the rokan hilir market on the same day, with direct contact with the seller. passing the fermentation process, data was collected at the laboratory. data analysis of dietary fiber results was carried out using spss 25 as the statistic program. all variables were tested for normality, then if the variables were normally distributed, anova block design was applied. to see the variation difference among treatments, it is followed by a posthoc test, namely lsd and duncan to find out the real difference between treatments. results and discussion results explain fiber content in cgl fermentation (joruk maman) base on storage time and solvent change and difference fiber concentration (figure 2-3; table 1-9). fiber content of joruk maman table 1. description of fiber content of joruk maman base on storage time. storage time average of fiber content (gr/100gr) refrigerator temperature room temperature day 1 0.46 0.51 day 2 0.56 0.58 day 3 1.69 1.41 day 4 1.62 1.69 day 5 1.65 1.74 note: primary data sources restusari et al. – fiber concentration of joruk maman 365 the graph of fiber content in cgl fermentation which was stored at refrigerator temperature and room temperature (figure 2) figure 2. fiber content of joruk maman against temperature and storage time. the normality test for the effect of temperature and storage time on fiber content in cgl fermentation are normally distributed. so it is continued with the anova block design test. the results of the anova block design test were shown at table 2. table 2. the analysis of anova block design, temperature effect, and storage time on fiber content of joruk maman. treatment group average + sd p-value storage time 0.891 refrigerator temperature 1.19 + 0.48 room temperature 1.18 + 0.48 storage time 0.001* day 1 0.48+0.77 day 2 0.57+0.77 day 3 1.55+0.77 day 4 1.65+0.77 day 5 1.69+ 0.77 note: * significantly different in the anova block design test (α<0.05) the significance value of the block (day 1,2,3,4,5) was 0.001<0.05. there was a difference between the day group on the number of fibers; the posthoc test could be continued. the results showed in table 3-4. table 3. post-hoc lsd test, temperature effect, and storage time on fiber content in joruk maman. post-hoc lsd test average difference (gr/100gr) p day 1 day 2 -0.0850 0.476 day 3 -1.0650* 0.001 day 4 -1.1700* 0.000 day 5 -1.2100* 0.000 day 2 day 1 0.0850 0.476 day 3 -0.9800* 0.001 day 4 -1.0850* 0.001 day 5 -1.1250* 0.000 day 3 day 1 1.0650* 0.001 day 2 0.9800* 0.001 day 4 -0.1050 0.387 day 5 -0.1450 0.252 day 4 day 1 1.1700* 0.000 day 2 1.0850* 0.001 day 3 0.1050 0.387 day 5 -0.0400 0.731 day 5 day 1 1.2100* 0.000 day 2 1.1250* 0.000 day 3 0.1450 0.252 day 4 0.0400 0.731 note: * significantly different in post-hoc lsd test (p<0.05). 366 biology, medicine, & natural product chemistry 12 (1), 2023: 363-369 based on the post-hoc lsd test, the different mean pairs are on days 1, 2, 3, 4, and 5 pairs. the number of differences in the average fiber is highest on day 5 and day 1 which is 1.21. table 4. temperature effect, and storage time on fiber content in joruk maman. treatment average fiber content (gr/100gr) storage time day 1 0.48a day 2 0.57a day 3 1.55b day 4 1.65b day 5 1.69b note: numbers followed by the same lowercase letters in one column show no significant difference in (p < 0.05). the fiber content in joruk maman with a solvent change towards temperature and storage time table 5. description of fiber content in cgl fermentation with solvent change towards temperature and storage time. storage time average fiber content (gr/100gr) refrigerator temperature room temperature day 1 1.79 1.89 day 2 1.65 1.57 day 3 1.24 1.11 day 4 1.11 1.08 day 5 1.1 1.04 note: primary data sources. the fiber content in cgl fermentation which was stored at refrigerator temperature and at room temperature with solvent change also describe of figure 3. figure 3. fiber content in cgl fermentation with changes in solvents against temperature and storage time. the normality test for the temperature effect and storage time on fiber content in cgl fermentation with solvent change were normally distributed, so it was continued with the anova block design test. the results are in table 6. table 6. description of anova block design, temperature effect, and storage time on fiber content in cgl fermentation with solvent change. treatment group average + sd p-value storage temperature 0.359 refrigerator temperature 1.37 + 0.27 room temperature 1.33 + 0.27 storage time day 1 1.84 + 0.43 0.001* day 2 1.61 + 0.43 day 3 1.17 + 0.43 day 4 1.09 + 0.43 day 5 1.07 + 0.43 note: *significantly different in the anova block design test (α<0.05) the anova block design test showed the significant value of the block (day 1,2,3,4,5) was 0.001 < 0.05. there was a difference between the day group with the fibers content; the lsd post hoc test was shown in table 7 and table 8. table 7. description of the lsd test, temperature effect, and storage time on fiber content in joruk maman. post-hoc lsd test average difference (gr/100gr) p day 1 day 2 0.2300* 0.020 day 3 0.6650* 0.000 day 4 0.7450* 0.000 day 5 0.7700* 0.000 day 2 day 1 -0.2300* 0.020 day 3 0.4350* 0.002 day 4 0.5150* 0.001 day 5 0.5400* 0.001 day 3 day 1 -0.6650* 0.000 day 2 -0.4350* 0.002 day 4 0.0800 0.260 day 5 0.1050 0.160 day 4 day 1 -0.7450* 0.000 day 2 -0.5150* 0.001 day 3 -0.0800 0.260 day 5 0.0250 0.703 day 5 day 1 -0.7700* 0.000 day 2 -0.5400* 0.001 day 3 -0.1050 0.160 day 4 -0.0250 0.703 note: *significantly different in the lsd post-hoc test (p<0.05) based on table 7, we know the pairs of average differences are on the 1st, 2nd, 3rd, 4th, and 5th -day pairs. thus, the number of differences in the average fiber is highest on day 1 and day 5 which is 0.77 restusari et al. – fiber concentration of joruk maman 367 table 8. description of the duncan's test analysis of temperature effect and storage time on fiber content in cgl fermentation with solvent change. treatment average fiber content (gr/100gr) storage time day 1 1.84c day 2 1.61b day 3 1.17a day 4 1.09a day 5 1.07a note: numbers followed by the same lowercase letters in one column show no significant difference in (p < 0.05) the difference in fiber content in cgl fermentation before and after dissolving, the paired t-test can be seen in table 9. table 9. paired t-test of fiber concentration in cgl fermentation before and after dissolving. solvent change mean ± std. deviation p-value before 1.19 ± 0.57 0.572 after 1.35 ± 0.33 note: there is a significant difference in the paired sample t-test (∝<0.05) there is no difference in fiber concentration of cgl fermentation before and after solvent changes discussion the fiber content in cgl fermentation at room temperature ranged from 0.51 to 1.74 g/100 g, and fiber content in cgl fermentation at refrigerator temperature ranged from 0.46-1.69 g/100g. both at room temperature and refrigerator temperature, the lowest fiber content was obtained on the first day, namely 0.51 g/100 g and 0.46 g/100g respectively; and the highest fiber content was obtained on the fifth day, namely 1.69 g/100 g and 1.74 g/100g. during the fermentation process, there were bacteria that played an important role, namely acetobacter xylinum which has the ability to convert ethanol into vinegar. however, these bacteria required oxygen in the process. crude fiber it self was the result of sugar reform in the fermentation medium by the activity of acetobacter xylinum (saleh, 2011) (putriana & siti aminah, 2013). there are not many research results that report fiber content related to temperature in this fermented food of the malay tribe, named joruk maman. however, there are similar studies that examine the crude fiber content of the leaves and seeds of the rubber tree (hevea brasiliense). it is known that the crude fiber content of leaves and seeds of rubber trees (hevea brasiliense) after a 2-day fermentation period is much lower than the 5-day and 8-day fermentation periods. however, at 34 and 44 degrees celsius, the crude fiber content is higher than the 2 and 5-day fermentation period. this result occurs because at high temperature, enzyme activity is reduced due to enzyme damage, and the water content of the substrate becomes much reduced. as a result, the raw fiber is converted into sugar. on the first day of the fermentation period, the addition of incubation time will reduce the crude fiber content of the leaves and seeds of the rubber plant (hevea brasiliense). the decrease in crude fiber from a temperature of 24-34 degrees celsius is higher than the decrease in crude fiber from a temperature of 34-44. similar results are also obtained at the 5 and 8-day fermentation periods. these changes are closely related to the growth of molds and cellulase enzyme activity. with increasing incubation time, some cellulase enzymes can break down higher crude fiber; so that, the fiber content decreases (syahruddin et al., 2016). the research that we have done has obtained that the fiber content in cgl fermentation stored at refrigerator temperature compared to room temperature without any changes had the same graphic pattern. on day 3 of storing cgl fermentation at both temperatures, the fiber content increased which was quite high. the block significance value (day 1,2,3,4,5) was different among day groups on the number of fibers. while the different mean pairs were on day 1, 2, 3, 4, and 5 pairs, where the highest number of fiber average differences occurs on day 5 and day 1, namely 1.21. storage time on day 1 (0.48 gr/100gr) had the smallest significant difference compared to other treatments, and storage on day 5 (1.69 gr/100gr) was significantly different from other treatments. the final results showed that the fiber content of cgl fermentation with a combination of 5-day fermentation treatment at room temperature was the best treatment because it had the highest fiber content. during the fermentation process, the ability of lactic acid bacteria (lab) continued to increase in order to produce sufficient lactic acid to loosen the bonds of lignocellulose and lignohemicellulose which are components of crude fiber. thus, the increase of crude fiber was in line with the fermentation time. the ph condition that was not optimal in lab could not produce lactic acid in sufficient quantities to loosen lignocellulosic and lignohemicellulose bonds; so lab had not caused changes in the crude fiber content of the silage. therefore, at the beginning of storage, the amount of lab in cgl fermentation was less; so that, the lactic acid produced was also less to produce crude fiber. another finding of fiber content in cgl fermentation based on temperature and storage time on solvent change was a decrease in fiber content of cgl fermentation from the first day to the fifth day; the decrease reached 0.85 g/100 g. meanwhile, at refrigerator temperature, the fiber content of cgl fermentationday 1 to day 5 decreased to 0.69 g/100g. based on the graph, it can be seen that on day 3 the fiber content decreased quite high. there was a difference between the day groups in the number of fibers. the different mean pairs were on the 368 biology, medicine, & natural product chemistry 12 (1), 2023: 363-369 1st, 2nd, 3rd, 4th, 5th day pairs. the highest number of fiber average differences on day 1 and day 5 was 0.77. this finding is in line with olive pomace fermentation with simultaneous production of gallic acid, where the fermentation changes the chemical composition of olive pomace so that crude fiber decreases by 8.56% (fathy et al., 2018). the crude fiber content on the first day continued to decrease significantly to the fifth day until it reached the lowest fiber content. this is inversely proportional to the fiber content of cgl fermentation without solvent change. this decrease in fiber content can occur because it is closely related to the constituent components of the fiber, especially lignin. high lignin will make it difficult for microorganisms (bacteria) to degrade the material, so fiber content is decreased to low. the fiber content of cgl fermentation with the combination of 1 day fermentation period treatment at room temperature was the best treatment because of the highest fiber content in this treatment. the research results are different from other studies related to fiber in nata de cassava which states that the length of nata de cassava fermentation causes acetobacter xylinum bacteria to work depending on differences in the number of nutrients to meet their needs. if the number of nutrients is sufficient, then the amount of cellulose formed is also big, whereas if the number of nutrients is not sufficient, the growth of acetobacter xylinum bacteria is inhibited as a result, a small amount of cellulose is produced (putriana & siti aminah, 2013). the average value of the highest fiber content was obtained in the nata de cassava product on the 7th day of fermentation, which was 94.31 mg. the duration of data de cassava fermentation did not affect the fiber content of the data formed. this was because on day 7 of fermentation, the acetobacter xylinum bacteria were in an exponential phase because the acetobacter xylinum bacteria concealed as much extracellular polymerase enzymes to arrange glucose polymers into cellulose; so that, more data matrix was produced in this phase. the 9th and 11th fermentation times decreased because acetobacter xylinum bacteria were in a slow growth phase because the availability of nutrients had decreased. the 13th fermentation time increased because more data matrix was produced in this phase. there is no explanation regarding the solvent change in the nata fermentation, this is certainly different from the conditions of the cgl fermentationfermentation that we did. in the cgl fermentationfermentation process, we used two groups, namely without solvent change and using solvent change. it turned out that the fermentation of cgl fermentationwhich was replaced by a solvent (using solvent change) was not significantly different. this could be triggered by the previous initial conditions, where the fiber content of cgl fermentation before and after being given dissolving did not have a much different numerical difference. meanwhile, the highest cgl fermentationfiber content occurred on day 5. this could be due to a significant increase in the size of the cellulose fraction compared to the raw material, which was caused by the loosening of the cell walls and release of cellulose, and a reduction in the size of the hemicellulose and pectin fractions, such as the research on sauerkraut. one of the more important functional properties of dietary fiber is its water-holding capacity which reflects the fiber's ability to swell. the results obtained that the ability of cabbage fiber to bind water depended on the cultivar and the length of storage time of fermented cabbage. the rehydration capacity of dried sauerkraut increased with storage time. the increase, compared to fresh cabbage, was an average of 22% after 10 days from the end of fermentation; after 30 and 90 days were at the same level with an average value of 35%. this difference in water absorption probably arises from changes in the fractional composition of the fiber, namely from the increase in the size of the cellulose fraction observed during the storage of sauerkraut (elkner, krystyna, kosson, 2009). conclusions there was a different the number of fibers in cgl fermentation without solvent change and with solvent change based on the day group. the highest fiber content production has 5 days duration, without solvent change and at room temperature. meanwhile with solvent change, the optimal fiber content at 1 day and at room temperature. regarding the length of storage in cgl fermentation, it has the potential to become a probiotic product. acknowledgments: the authors express their gratitude to the health polytechnic of the ministry of health of riau, the laboratory of agricultural polytechnic of andalas university have given permission and examined the samples in this research. hence, it can be carried out well. there is no potential conflicts of interest are noted authors’ contributions: lily restusari designed the study. ayu komalasari carried out the laboratory work. alinea dwi elisanti analyzed the data. lily restusari and alinea dwi elisanti wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. funding: this research supporting from dipa funds poltekkes kemenkes riau 2020 restusari et al. – fiber concentration of joruk maman 369 references elkner, krystyna, kosson, r. (2009). dietary fibre content and its fractional composition in cabbage as affected by cultivar earliness and sauerkraut storage period. journal of fruit and ornamental plant research, 69(1), 165–175. doi: https://doi.org/https://doi.org/10.2478/v10032-008-0031-2 endrizzi, i., pirretti, g., calò, d. g., & gasperi, f. (2009). a consumer study of fresh juices containing berry fruits. journal of food and agriculture, 89(7), 1227–1235. doi: https://doi.org/https://doi.org/10.1002/jsfa.3580 fathy, s. a., mahmoud, a. e., rashad, m. m., ezz, m. k., & mohammed, a. t. (2018). improving the nutritive value of olive pomace by solid state fermentation of kluyveromyces marxianus with simultaneous production of gallic acid. international journal of recycling of organic waste in agriculture, 7(2), 135–141. doi: https://doi.org/10.1007/s40093-018-0199-5 heever, e. van den, & venter, s. l. (2007). nutritional and medicinal properties of cleome gynandra. international conference on indigenous vegetables and legumes. prospectus for fighting poverty, hunger and malnutrition, 752. doi: https://doi.org/10.17660/actahortic.2007.752.17 institute of medicine. (2005). dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (macronutrients). in dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (macronutrients). institute of medicine of the national academies. doi: https://doi.org/10.17226/10490 lily restusari, muharni, f. (2019). activities of fermented maman (activities of fermented maman (cleome gynandra l) on blood sugar level in hyperglycemic white rats. doi: https://doi.org/10.31227/osf.io/4j8q6 mishra, s., moharana, s., & dash, m. (2011). review on cleome gynandra. 1(3), 681–689. putriana, i., & siti aminah. (2013). mutu fisik, kadar serat dan sifat organoleptik nata de cassava berdasarkan lama fermentasi physical quality, dietary fiber and organoleptic characteristic from nata de cassava based time of fermentation. 04(07). saida, e, m. (2014). identifikasi bakteri patogen pada produk fermentasi cleome gynandra. politekkes kemenkes riau saleh, d. (2011). sintesis dan karakterisasi selulosa mikrobial dari whey serta pengaruh iodium pada sifat mekanik, listrik dan absorbsi terhadap limbah mgcl2, mg(oh)2, dan hcl disertasi djonaedi saleh syahruddin, e., herawaty, r., & azhar. (2016). improving the quality of the leaves and seeds of rubber trees (hevea brasilliensis) for poultry feed through various types of microbial biotechnology. pakistan journal of nutrition, 15(11), 963–968. doi: https://doi.org/10.3923/pjn.2016.963.968 winarti, s. (2010). makanan fungsional. graha ilmu https://doi.org/https:/doi.org/10.2478/v10032-008-0031-2 https://doi.org/https:/doi.org/10.1002/jsfa.3580 https://doi.org/10.1007/s40093-018-0199-5 https://doi.org/10.17660/actahortic.2007.752.17 https://doi.org/10.17226/10490 https://doi.org/10.31227/osf.io/4j8q6 https://doi.org/10.3923/pjn.2016.963.968 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1111-1119 | doi: 10.14421/biomedich.2025.142.1111-1119 issn 2540-9328 (online) analysis of influencing factors on the provision of birth control equipment and materials at the lahomi non-inpatient health center unit, west nias regency sri lestari nasution, dermawati laia, ermi girsang* master of public health study program, faculty of medicine, prima indonesia university, indonesia. corresponding author* ermigirsang@unprimdn.ac.id abstract the quality and access to family planning services demands a paradigm shift, especially among service providers, which in this case are bapermas and kb. that is why service must be carried out on the basis of volunteerism, openness, and honesty. one of the service providers that is still questionable about the quality of its services is the community empowerment, women and family planning agency. the implementation of the provision of kb tools and materials has not been carried out properly and correctly. the purpose of this study was to analyze the factors of influence on the provision of birth control equipment and materials at the upt non-inpatient health center lahomi, west nias regency. the type of research used was quantitative analytics. dessay research is a cross-sectional study. the sampling technique in this study was the total population, namely the entire population was used as a research sample, then the number of samples in the study was 170 employees. the results of the study explained that there was an influence between supervision, direction, control, supervision and budgeting on the provider of birth control equipment and materials in lahomi health center, nias regency. west. it is hoped that with this research, lahomi health center will improve the quality of services, especially in the provision of birth control tools and materials. keywords: supervision; briefing; controlling; supervise; budgeting. introduction the central statistics agency noted that indonesia's population growth rate during the 2010-2020 period was higher than the 2000-2010 period. the population growth rate for 2010-2020 reached 1.49 percent or higher compared to the 2000-2010 period which only reached 1.45 percent, according to the results of the 2020 census of the total population indonesia reaches 237.56 million people. meanwhile, to meet the food needs of 237.56 million people, productive land is needed for rice crops covering an area of 13 million ha, but currently the cultivated rice land covers an area of 7.7 ha, if the annual population increase is 1.49% or even more, then the population problem will naturally bring other social problems such as the problem of poverty, hunger, urban slums, crime, reduced carrying capacity of land and other social problems (central statistics agency, 2020). the quality and access to family planning services demands a paradigm shift, especially among service providers, which in this case are bapermas and kb. that is why service must be carried out on the basis of volunteerism, openness, and honesty. bapermas and kb employees must have the ability to explain each contraceptive correctly and completely with all its advantages and advantages, in addition to following the standards predetermined service (marni, 2018) this family planning program basically aims to reduce the rate of population growth by providing solutions in the form of installing or using contraceptives. the family planning program was first implemented during the reign of president soeharto and had succeeded in suppressing the population growth rate at that time. for its current development, especially in the semarang area, the kb program has more or less changed marked by revitalization (purba, et al, 2021) there are many reasons why oversight is important and needed. a very fundamental and universal reason why leaders need oversight is the need to monitor what people or organizational units are working on and specifically the results of what they are working on. another reason is to know the changes faced by the organization and leadership. supervision as the fourth element or function of management is to observe and allocate appropriately the deviations that occur (susanto, 2022) the need for supervision carried out on the implementation of the family planning program at the manuscript received: 25 november, 2025. revision accepted: 28 november 2025, 2025. published: 30 november, 2025. https://doi.org/10.14421/biomedich.2025.142.1111-1119 1112 biology, medicine, & natural product chemistry 14 (2), 2025: 1111-1119 puskesmas so that it can run well, as well as the need for a form of good management that will be a guide in its implementation. supervision (control) or controlling is the last part of the management function. the controlled management functions are planning, organizing, directing and supervising itself. the cases that occur a lot in an organization are due to weak control so that there are various deviations between the planned and the implemented. research methods research design the type of research used is quantitative analytics. dessay research is a cross-sectional study, namely to find out whether there is an influence between supervision, direction, control, supervision and budgeting with the provision of birth control tools and materials at the upt non-inpatient health center lahomi, west nias regency in the past. descriptive research is used to make an assessment of a condition and implementation of a program in the present, then the results are used to compile a plan for the improvement of the program, descriptive research is directed to describe or describe the circumstances within a community or society (nursalam, 2018). time and place of research this research was conducted in the working area of the uptd puskesmas lahomi, west nias regency due to the discovery of problems related to the title of the study. this study is planned to be conducted in september 202 2 for approximately two weeks until the predetermined number of samples is reached. population and sample population is the entire symptom/unit you want to study or the entire object of study/object under study. the population in this study was all health workers on duty in the uptd work area of the lahomi health center, west nias regency. the total population in this study was 170 employees. a sample is a small percentage of the population used to obtain statistical information regarding the overall population (nursalam, 2018). the samples in this research are all health workers on duty in the uptd work area of the lahomi health center, west nias regency. the sampling technique in this study is the total population, namely the entire population is used as a research sample, then the number of samples in the study is 170 employees. operational variables and definitions based on the results of a preliminary survey conducted by researchers in the work area of the lahomi health center, it was explained that the process of providing birth control tools and materials was still not carried out properly and correctly. researchers see that birth control tools and materials are sometimes not available in the quantities needed by the community. the storage of kb tools and materials is also still not correct, this can be seen from the storage of injectable kb is still stored in the warehouse and does not pay attention to the sop that has been set by the puskesmas lahomi. researchers also saw how the implementation of the provision of birth control tools and materials at the lahomi health center, namely kb tools and materials, was still not carried out properly and correctly. researchers see that kb tools and materials are often available in small quantities, their storage is also not in accordance with the rules for the proper storage of kb tools and materials. researchers also found data that puskesmas often experienced a shortage of birth control tools and materials, and sometimes the delivery time was often quite long. based on the penomena, the researcher is interested in conducting a study entitled "analysis of influencing factors on the provision of birth control tools and materials at the upt non-inpatient health center lahomi, west nias regency". research instruments the research instrument used for data collection is in the form of a questionnaire sheet about supervision, direction, control, supervision and budgeting on the provision of birth control tools and materials at the upt non-inpatient health center lahomi regency west nias. the supervisory, directing, controlling, supervising and budgeting research questionnaire consists of 50 items with a choice of yes and no answers. for yes answers are given a value of 2 and for answers are not given a value of 1. the highest value is 100 and the lowest value is 50. the questionnaire category consists of good and less, both if the respondent scored more than or equal to 50% and less if the respondent scored less than 50% of the entire total answers. the researcherpan questionnaire on the provision of kb tools and materials consisted of 20 items with a choice of yes and no answers. for yes answers are given a value of 2 and for answers are not given a value of 1. the highest value is 40 and the lowest value is 20. the questionnaire category consists of good and less, both if the respondent scored more than or equal to 50% and less if the respondent scored less than 50% of the entire total answers. data analysis this data analysis in this study aims to obtain an overview of the research results that have been formulated in the research objectives. data analysis of this study uses computer programs through the univariate and bivariate data analysis stages. nasution et al. – analysis of influencing factors on the provision … 1113 univariate analysis. this analysis was carried out to determine each research variable with the distribution of frequencies and characteristics studied by the researcher, including age, education and the initials of the name. univariate analysis is written in the form of a frequency and narrative distribution table on each of its variables bivariate analysis bivariate analysis aims to analyze the most influential factors between supervision, direction, control, supervision and budgeting on the provision of birth control tools and materials at the upt non-inpatient health center lahomi regency west nias. this bivariate analysis is written in the form of a frequency distribution table and narration on each variable. this bivariate analysis was carried out using the pearason correlation test with the level of significance used, namely p< a (= 0.05). so, ha will be accepted and if p> 0.05, while using ho will be accepted if p>0.05. the data analysis will be processed using spss. multivariate analysis this analysis is used to determine which variables are most dominant in indepnden variables to dependent variables. this analysis uses a linear regression test. validity and reliability of questionnaires validity is a measure that shows the degree of validity or validity of a research instrument. the purpose of the content validity index (cvi) is a process of a study with the aim of assessing the relevance of each item to what the researcher will measure. the content validity index (cvi) in this study is an assessment / maximum load through experts from each interconnected item. experts are asked to evaluate individual items on new measurements on all research instrument items. two things to note in evaluating are whether individual items are relevant and appropriate in the interrelationships of research instruments, and whether items taken together adequately measure all constructed dimensions (polit & beck, 2012) this study used the valicity of the pearson product moment correlation instrument with a significance of 0.05. measurement of each item of the research instrument carried out by experts by having several label variations. the variation of the label of this study consists of 3 options, namely low enough and high. the most frequently used scales are as follows: 1= irrelevant, 2= slightly relevant, 3= really relevant, 4= very relevant. then for each item, the item content validity index (icvi) is calculated as much as the number of experts who give a maximum assessment / load of 3 or 4, divided by the number of experts who are the proportion who approve of the linkage (polit & beck, 2012). based on the results of the content validity index (cvi) test that has been carried out by researchers, it was obtained that the cvi results for the research questionnaire were 1.00 cvi against 50 questionnaires about influencing factors. the provision of tools and materials was 0.95 and for cvi against the quality of life questionnaire was 0.96. reliability test the coefficient of reliability is an important indicator of an instrument's quality. measurements that cannot be trusted when they do not provide sufficient tests of the researcher's hypothesis. if the data is incorrect against the confirmation of the prediction, it is likely that the instrument is not reliable. interpretation for comparing group levels, coefficients ranging from 0.70 is generally adequate, although a coefficient of 0.80 or greater is highly desirable (polit & beck, 2012). researchers plan to conduct reliability and validity tests at the royal prima marelan hospital. based on the reliability test of the spirituality welfare questionnaire that has been carried out at the royal prima hospital medan, a crombach alpha value of 0.96 was obtained and for the questionnaire of factors affecting the tool and the material obtained crombach alpha value of 0.95. through these results, all questionnaires used in the study met the reliability value of > 0.70 which can be stated that the questionnaire used in this study was adequate. univariate analysis univariate analysis conducted in this study aims to describe data simply about the socio-demographics of employees serving at the upt non-inpatient health center lahomi, west nias regency consisting of from age, gender, religion and employment status. this univariate analysis will be presented in the form of tables and narratives. the following is an explanation of the univariate analysis: table 1. frequency distribution of respondent characteristics at upt non-inpatient health center lahomi, west nias regency. no sociodemographics sum (n) (%) age 1 21-30 years 43 25.2 2 31-40 years 65 35.7 3 4 41-50 years 51-60 years 30 32 20,5 30.6 total 170 100 religion 1 christian 168 98 2 islam 2 2 total 170 100 gender 1 woman 148 87 2 man 22 13 total 170 100 employment status 1 civil servants 28 16.5 2 thl and tks 43 25.2 3 ns 32 20.5 4 ptt 67 37.8 total 170 100 1114 biology, medicine, & natural product chemistry 14 (2), 2025: 1111-1119 in table 1, it can be seen that the data on the distribution of respondents' frequency based on the age of the majority of respondents is in the age range of 3140 years as many as 65 people (35.7%) and the minority is in the age of 51-60 years as many as 32 people (30.6 %). basedon the gender of the majority of respondents of the female sex as many as 148 people (87%) and the male minority as many as 22 people (13%). based on the employment status, the majority of respondents worked as ptt as many as 67 people (37.8%) and the minority of respondents worked as civil servants as many as 28 people (13%). table 2. distribution of frequency of supervision, briefing, control, supervision and budgeting in upt non-inpatient health center lahomi, west nias regency. no scoring sum frequency (n) percentage (%) supervision 1 good 24 14.1 2 not good enough 146 85.9 total 170 100,0 briefing 1 good 25 14.6 2 not good enough 145 85,4 total 170 100,0 controlling 1 good 24 14.7 2 not good enough 146 85.3 total 170 100,0 supervision 1 good 26 15.2 2 not good enough 144 84.8 total 170 100,0 budgeting 1 good 23 13.5 2 not good enough 147 86.5 total 170 100,0 table 2 can explain that the supervision variable of the majority of respondents was in the category of poor as many as 146 people (89.5%). based on the briefing variable, the majority of respondents were in the poor category of 145 people (85.4%). based on the controlling variable, the majority of respondents were in the poor category of 146 people (85.3). based on the supervision variable, the majority of respondents were in the poor category of 144 people (84.2). based on budgeting variables, the majority of respondents were in the poor category as many as 147 people (86.5%). bivariate analysis bivariate analysis is performed for the influence between independent and dependent variables. bivariate analysis is presented in the form of tables and narratives. the following is an explanation of bivariate analysis based on research that has been carried out. table 3. the effect of supervision on the provision of family planning tools and materials at the upt non-inpatient health center lahomi, west nias regency. provider of tools and materials total p value good not good enough good oversight not good enough 4 20 24 0,012 6 140 146 total 10 160 170 based on table 3 on the effect of supervision on the provision of birth control equipment and materials at the upt non-inpatient health center lahomi, west nias regency, it is explained that there is an influence between supervision and the provision of tools and materials kb with a value of p = 0.012. table 4. the effect of briefing on the provision of family planning tools and materials at the upt non-inpatient health center lahomi, west nias regency. provider of tools and materials total p value good not good enough good briefing not good enough 4 21 25 0,011 5 140 145 total 9 161 170 based on table 4 on the effect of the briefing on the provision of birth control tools and materials at the upt non-inpatient health center lahomi, west nias regency, it is explained that there is an influence between the briefing on the provision of tools and materials kb with a value of p = 0.011. table 5. the effect of control on the provision of family planning tools and materials at the upt non-inpatient health center lahomi, west nias regency. provider of tools and materials total p value good not good enough good controlling not good enough 4 20 24 0,012 6 140 146 total 10 160 170 based on table 5 on the effect of controlling on the provision of birth control tools and materials at the upt non-inpatient health center lahomi, west nias regency, it is explained that there is an influence between controlling and the provision of tools and materials kb with a value of p = 0.012. nasution et al. – analysis of influencing factors on the provision … 1115 table 6. the effect of supervision on the provision of family planning tools and materials at the upt non-inpatient health center lahomi, west nias regency. provider of tools and materials total p value good not good enough good supervision not good enough 4 22 26 0,010 4 140 144 total 8 162 170 based on table 6 on the effect of supervision on the provision of birth control equipment and materials at the upt non-inpatient health center lahomi, west nias regency, it is explained that there is an influence between supervision on the provision of tools and materials kb with a value of p = 0.010. table 7. the effect of budgeting on the provision of family planning tools and materials at the upt non-inpatient health center lahomi, west nias regency. provider of tools and materials total p value good not good enough good budgeting not good enough 4 19 23 6 141 147 0,009 total 10 160 170 based on table 7 on the effect of budgeting on the provision of birth control equipment and materials at the upt non-inpatient health center lahomi, west nias regency, it is explained that there is an influence between budgeting and the provision of tools and materials kb with a value of p = 0.012. multivariate analysis in this study, multivariate modeling was carried out with linear regression tests aimed at testing the influence of dependent variables and independent variables. the variables included are the supervisory, controling, supervise and budgeting variables are as independent variables while the kb tool and material variables as dependent variables and methods are used is the enter method. output part i model summary type r r square adjusted r square std. error of the estimate 1 ,780a ,608 ,595 26,668 the above explains that the calculated f value of 48.527 with a signification rate of 0.000 < 0.05, then the regression model can be used to predict the influence of the independent variable (x) on the variable dependent (y). output part ii coefficientsa type unstandardized coefficients standardized coefficients t sig. b std. error beta 1 (constant) 3,320 11,509 ,288 ,774 supervision 1,005 ,117 ,554 8,571 ,012 briefing 2,191 ,799 ,178 2,743 ,011 controlling ,798 ,101 ,513 7,931 ,012 supervision 1,005 ,117 ,554 8,571 ,010 budgeting 2,191 ,799 ,178 2,743 ,009 the table above explains based on the signification value in the supervision variable of 0.012 < 0.05 that it can be stated that the supervision variable has a relationship with the variable of providing kb tools and materials. based on the signification value on the directing variable of 0.011 < 0.05 below, it can be stated that the directing variable has a relationship with the kb tool and material supply variable. based on the significance value of the controlling variable of 0.012 < 0.050, it can be stated that the controlling variable has a relationship with the kb tool and material supply variable. based on the significance value in the 1116 biology, medicine, & natural product chemistry 14 (2), 2025: 1111-1119 supervision variable of 0.010 < 0.050, it can be stated that the supervision variable has a relationship with the variable of providing kb tools and materials. based on the significance value of the budgeting variable of 0.009 < 0.050, it can be stated that the budgeting variable has a relationship with the variable of providing kb tools and materials. based on the value of t on the variables of factors affecting the provision of materials and tools kb it is known that the calculated f value is 8.571 > f table 1.985 then it can be stated that the independent variable has a relationship with independent variables. discussion characteristics of respondents at upt non-inpatient health center lahomi, west nias regency the results of the study explained that based on the age of the majority of respondents in the age range of 31-40 years as many as 65 people, the gender of the majority of respondents was female as many as 148 people, employment status the majority of respondents worked as ptt as many as 67 people. age is the length of time a human life or the length of time that man has lived since man was born. gender is a taxonomic character used in biological classifications. religion is a person's belief in his creator during life. employment status is a type of deed or activity to obtain rewards or wages (muriah & wardan, 2020) based on the researcher's analysis of the majority of respondents' ages in the age range of 31-40 years as many as 65 people, the age factor is a factor that can affect the ability to think and analyze someone. the older a person is, the better his abilities and knowledge will be. this study is not in line with the theory that explains the age that more people are 31-40 than respondents in the 41-50 age range. researchers' analysis of the gender of respondents of the majority of women women more often have a good lifestyle than l aki-men and are more likely to care about their health status. this is in line with the research that has been carried out explaining about more respondents of the female sex. the effect of supervision on the provision of family planning tools and materials at the upt noninpatient health center lahomi, west nias regency based on research that has been conducted on 170 respondents, it was found that there is an influence between supervision on the provision of kb tools and materials with a value of p = 0.012 according to daulay (2017) states that supervision is a systemic effort to set implementation standards with planning objectives, design feedback information systems, compare real activities with previously established standards, determine and measure deviations and take the necessary corrective actions to ensure that all resources owned by the company have been used in the most effective and efficient way in achieving goals. company. according to handoko (2018) supervision is the discovery and application of means and equipment to ensure that the plan has been implemented in accordance with what has been established. this can be both positive and negative. positive supervision tries to find out whether the goals of the organization are achieved efficiently and effectively. negative surveillance tries to guarantee that unwanted or needed activities do not occur or reoccur. this research is in line with research conducted by sri, (2018) explaining that the supervision carried out by superiors over subordinates has a very significant influence. through well-carried out supervision, it can improve the quality of service of pematangsiantar auction employees. this research is not in line with the results of research conducted by suhariyanto & putro (2018) which explains that the supervision that has been carried out by superiors over subordinates has no influence to the quality of work of cv tiga putra employees. this research explains that work discipline greatly affects the quality of a person's work. based on the results of the observation carried out by the researcher during the research process, the researcher found several things related to supervision of the provider of birth control tools and materials at the lahomi health center. researchers see that the implementation of supervision is still not going well, this is justified by the large number of kb tools and materials that are still not well distributed according to the needs at the puskesmas. the researcher's analysis of this phenomenon is that the puskesmas should further improve the implementation of supervision of kb tools and materials, because with good supervision, the distribution of tools and the kb material will be evenly distributed and sufficient. monitoring also needs to be carried out continuously and continuously using tools or sops for monitoring kb tools and materials. the effect of briefing on the provision of family planning tools and materials at the upt noninpatient health center lahomi, west nias regency the results of a study that has been conducted on 170 respondents explained that there is an influence between directing the provision of kb tools and materials with a value of p = 0.011 siswanto (2017) stated "briefing is a process of mentoring, giving instructions, and instructions to subordinates so that they work according to a predetermined plan". another opinion regarding the briefing was also expressed by george terry who was quoted by malayu hasibuan (2017) who stated that the briefing is to make all members of the group, to be nasution et al. – analysis of influencing factors on the provision … 1117 willing to work together and work sincerely and passionately to achieve goals in accordance with planning and organizing efforts. this research is in line with research conducted by gunawan (2020) explaining that the briefing carried out by superiors to subordinates affects the effectiveness of employee work both individually and in groups. this research was conducted on all employees who work in the marine and fisheries service of banten province. this research is not in line with the research that has been conducted by gemilan, (2018) explaining that the briefing carried out was not effective on employee performance at tarutung regional hospital. the study was conducted by the hospital director against all room heads and installation heads. this research was carried out once simultaneously without planning. the research analysis of the phenomenon is that the briefing process carried out so far has not gone well, the implementation of the briefing is only carried out when there are important things that need to be notified only. the dredging executive also still does not have guidelines for the implementation of briefings on the provision of birth control tools and materials. briefings are often carried out when there are employees who violate the provision of kb tools and materials. the effect of control on the provision of family planning tools and materials at the upt noninpatient health center lahomi, west nias regency. the results of a study that has been conducted on 170 respondents on the provision of birth control tools and materials at the upt non-inpatient health center lahomi, west nias regency, explained that there is an influence between controlling against the provision of kb tools and materials with a value of p = 0.012. ing control is a function in functional management that must be carried out by each leader of all units / work units for the implementation of work or employees who carry out in accordance with their respective main duties. thus, supervision by the leadership, especially in the form of built-in control, is a managerial activity carried out with the intention that there are no deviations in carrying out work (siswanto, 2017) a deviation or error occurs or does not occur during the implementation of work depending on the level of ability and skill of the employee. employees who always receive direction or guidance from superiors, tend to make fewer mistakes or deviations compared to employees who do not receive guidance (siswanto, 2017) the research conducted is in line with ryandi's (2021) on based on regression tests with significance value (sig) tests provided that the < value of 0.05 obtained a sig value = 0.03 which means < of 0.05 means that supervision has a positive and significant effect on evaluation of work performance at the office of the general section of the regional secretary of makassar city. this research is not in line with anggreini, (2018) explaining that supervision does not affect the performance of private employees of pt. perawang. the supervision carried out by field supervisors on all employees has not been effective in the field. pt. perawang also does not have a standard supervision sop. the researcher's analysis of the results of the study is that the implementation of controlling is very rarely carried out, this is because there is still a lack of awareness of superiors to the functions and benefits of controlling. controlling is only carried out at certain times and on the basis of certain needs. the implementation of controlling is something that should be carried out because with this process we can know and even be able to control what things can and cannot happen during the process provision of kb tools and materials. the effect of supervision on the provision of family planning tools and materials at the upt noninpatient health center lahomi west nias. the results of research that has been carried out on the effect of supervision on the provision of birth control tools and materials at the upt non-inpatient health center lahomi, west nias regency, explained that there is an influence between supervision against the provision of kb tools and materials with a value of p = 0.010. supervision is all assistance from the leader / person in charge to the nurse which is intended for the development of nurses and other staff in achieving the goals of nursing care. this kind of supervision activity is an encouragement, guidance and opportunity for the growth of the development of the skills and skills of nurses (simamora 2018). supervision of nursing services is an activity of interaction and communication between supervisors and implementing nurses, where the nurse receives guidance, support, assistance and is trusted so that nurses can improve patient safety and the quality of health services (simamora, 2018) the same research also been conducted by kustiyoasih, (2020) explaining that supervision has an influence on the professionalism of teachers who teach at sdn banyumanik, semarang city. supervision carried out by the principal on teachers already has a standard supervision sop. supervision is carried out on an ongoing and regular basis in accordance with the supervision schedule that has been determined by the school. this research is not in line with wijayanti, et al, (2018) explaining that the implementation of nursing supervision has nothing to do with documenting care nursing at ambarawa regional hospital. the implementation of nursing supervision that has been carried out is still not good and does not have a standard 1118 biology, medicine, & natural product chemistry 14 (2), 2025: 1111-1119 supervision sop. supervision is carried out only when there is a problem. the researcher's analysis of supervision is that the implementation of supervision has not been carried out properly. the format of the implementation of supervision also does not exist, this will have an impact on the quality of the implementation of supervision. the implementation of supervision has also not been carried out consistently and continuously. supervision is carried out only when there is a need. the effect of budgeting on the provision of family planning tools and materials at the upt noninpatient health center lahomi, west nias regency. based on table 7 on the effect of budgeting on the provision of birth control equipment and materials at the upt non-inpatient health center lahomi, west nias regency, it is explained that there is an influence between budgeting and the provision of tools and materials kb with a value of p = 0.012 munandar (2018) stated that a budget is a systematically drawn up plan that includes all the activities of the company expressed in a unit of monetary unity that is valid for a certain period to come. narifin (2019) said a budget is a written plan regarding the activities of an organization that is stated quantitatively for a certain period of time and is generally expressed in units of money this research is in line with research conducted by devia & surya, (2018) showing that there is a positive and significant influence between variable budgeting participation on managerial performance in the manufacturing industry sector in surabaya. the research is not in line with musawir, (2018) explaining that there is no influence between the participation of nods on budgetray slack and commitment. budgeting arrangements are only done when there is a need for data to determine the budget each year. the researcher's analysis of the budgeting implementation process is that the implementation of budgeting must be carried out consistently. the head of puskesmas must formulate budgeting as well as possible and consistently, because this will greatly impact the provision of birth control tools and materials at the lahomi nias health center west. with planned budgeting, the provision of kb tools and materials will also be fulfilled properly. conclusion ▪ characteristics of respondents at upt non-inpatient health center lahomi, west nias regency, the majority of respondents are in the age range of 31-40 years, female gender, employment status of the majority of working as ptt. ▪ there is an influence between supervision on the provision of kb tools and materials with a value of p = 0.012. ▪ there is an influence between the briefing on the provision of kb tools and materials with a value of p = 0.011 ▪ there is an influence between controlling the provision of kb tools and materials with a value of p = 0.012 ▪ there is an influence between supervision on the provision of kb tools and materials with a value of p = 0.010. ▪ there is an influence between budgeting on the provision of kb tools and materials with a value of p = 0.012 competing interests: the authors declare that there are no competing interests. references anggaeni. (2019). the effect of budget participation, budgeting, and asymmetric information on budget slack in sma / smk / ma in arjasari district. retrieved december 8, 2022 via the file:///c:/users/user/downloads/08+iseu+++yayang+t etriani+hal+102-116.pdf link arum, dyah noviawati setya, sujiyatini. (2017). the complete guide to the latest kb services. yogyakarta: fitramaya ariga, a, reni. (2020). textbook on the implementation of health service management. egc. terrain. ayu, d, windri. (2022). nursing supervision. print ii. egc. jakarta bkkbn. (2018). active birth control participants according to perprovincial contraceptive methods. jakarta: bkkbn central statistics agency (2020). indonesia's population growth rate in 2020. central bureau of statistics. (2020). 2020 population census results. official statistical news no. 7/01/th. xxiv, january 21, 2021. daulay, d. et al. (2017). management. medan: aqli institute for research and scientific writing dunie, w. 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(2018). the effect of principal supervision on teacher performance at smp muhammadiah 2 wonosobo, wonosobo district, tanggamus regency. retrieved december 8, 2022 via the http://repository.radenintan.ac.id/3575/1/skripsi%20septi. pdf link http://repository.radenintan.ac.id/3575/1/skripsi%20septi.pdf http://repository.radenintan.ac.id/3575/1/skripsi%20septi.pdf http://repository.radenintan.ac.id/3575/1/skripsi%20septi.pdf this page intentionally left blank cover jurnal biomenaprochy vol 12 num 2 2023.cdr volume 12 number 2 october 2023 issn 2089-6514 (paper) issn 2540-9328 (online) biology, medicine, & natural product chemistry volume 12 – number 2 – october 2023 issn 2089-6514 (paper) | issn 2540-9328 (online) contents assessment of sensorimotor behaviour in konzo-induced rats using the irvine, beattie bresnahan forelimb scale lekpa kingdom david, precious ojo uahomo, victor hogan idung, rachael data dakoru 431 435 chemical properties of liquid broth extracted from freshwater and marine shrimp shells waste esa ghanim fadhallah, dyah koesoemawardani, lathifa indraningtyas 437 440 alkaloids lead to potential inhibition of the acyl carrier protein reductase to attenuate tuberculosis; an in-silico analysis pernia kamran, ahsan ibrahim 441 450 bio-larvicidal potential of betel leaves (piper betle l) ethanolic extract in addition of peg 400 diluent on aedes aegypti larvae listiana masyita dewi, hilda zaniba ariffah, riandini aisyah, nurhayani 451 455 proximate and mineral composition of atlantic mackerel (scomber scombrus) and atlantic horse mackerel (trachurus trachurus) joseph adaviruku sanni, grace omayoza sanni, rufus ranmilowo awoniyi, remi osanyinlusi, yvonne ego richards 457 461 identification of medicinal plants and their utilization by community in kendal village, kendal sub-district aghnia rahmi hanum, erna wijayanti 463 466 identification of primary and secondary metabolites of apis cerana honey using ftir-atr diamond spectroscopy and their botanical origin tiffany hanik lestari, ratna susandarini 467 475 nephroprotective activities of ethanol root extract and fractions of hippocratea africana against doxorubicin-induced kidney toxicity kufre u. noah, john a. udobang, jude e. okokon, martin o. anagboso, nwakaego omonigho ebong 477 484 determination of oil quality and antifungal effect of selected citronella accessions (cymbopogon nardus, cymbopogon winterianus) to formulate an anti-dandruff shampoo rathnayaka mudiyanselage nipuni wijerathna, achini anuradha wijeweera, anushi madushani wijethunga, mapa mudiyanselage sumudu tharangani mapa 485 498 phytochemicals and larvicidal activity of sonneratia alba root extracts from ngurah rai mangrove forest, denpasar-bali made dharmesti wijaya, anak agung gede indraningrat, i gede yoga ayuning kirtanayasa 499 505 liver protection effect of red rose petal methanol extract in copper-induced liver injury in male wistar rats caroline febrina hutagalung, ermi girsang, maya sari mutia, suhartina, oliviti natali 507 513 targeting h3n2 influenza virus rna-dependent rna polymerase by using bioactives from essential oils from eucalyptus polybrachtea, cymbopogon citratus and cymbopogon khasianus arun dev sharma, inderjeet kaur 515 524 in silico b-cell epitope design of zika virus vaccine using “zika virus isolate zika virus/h. sapiens-tc/tha/2006/cvd 06-020, complete genome” lisa savitri, syntia tanu juwita, ester lianawati antoro, ida septika wulansari, datin an nisa sukmawati, joão gregório freitas, andre mario sequeira alves ornai 525 531 antioxidant activity test (dpph) and cytotoxicity of jengkol peel ethanol extract (pithecellobium jiringa) on shrimp larvae (artemia salina leach) glen arya wibowo, tika afriani, azimatur rahmi 533 537 standardization of golden sea cucumber (stichopus hermanii) extracts from pelapis island, kayong regency, west kalimantan rusmini, wintari taurina, mohamad andrie 539 545 endophytic fungi isolated from pteridium acquilinium and newbouldia laevis leaves exhibited antioxidant activities and inhibitory potential against selected clinical bacteria isolates obinna ajah, chika cecilia unegbu, emmanuel n uzohuo, ogechi ozioma anyanwu, ugochukwu moses okezie, chioma anyanwu, rich joseph 547 553 the correlation between widal diagnostic test, total leukocyte count, and platelet count in suspected typhoid fever patients at rs aura syifa kediri novi loviana, lisa savitri, rochmad krissanjaya, elfred rinaldo kasimo 555 558 the potential of a sumbawa herbal oil-based oleogel as burn wound dressing andi irma yuniar, nurul wahidah yasid, syarifah fatimah nissatuljannah, armanto makmun, syamsu rijal 559 562 study of physical, chemical, and organoleptic properties of functional drink turkey berry (solanum torvum swartz) with the addition of butterfly pea flower (clitoria ternatea linn) and emprit ginger (zingiber officinale var. amarum) rista anggriani, nina nurazizah purnomo putri, vritta amroini wahyudi 563 575 analgesic and antipyretic activity of sweet orange peel methanol extract elsa debora silalahi, i nyoman ehrich lister, edy fachrial 577 584 in silico study on structural inhibition of bacterial dna gyrase by major secondary metabolites found in grape seed extract haritha lawan, hashitha tharakee 585 592 quercetin bioavailability evaluation on standardized herbal medicine containing guava leaf extract with hplc rizky yulion, yulianis, suntri 593 599 in vitro anti-inflammatory activity of bamboo tali leaf (gigantochloa apus) ethanol extract nurul hidayah, mutista hafsah 601 606 prevalence of enterobacteriaceae producing extended spectrum beta-lactamase (esbl) in broiler meat (gallus domesticus) sold in regional markets of west surabaya kadek rio risnanda, masfufatun, agusniar furkani listyawati, kuntaman kuntaman, akhmad sudibya 607 610 effect of citrullus lanatus seeds extract on haematological parameters after administration with medroxyprogesterone acetate on female wistar rats michael chuks nnumolu, onyeso godspower 611 617 investigating the insecticidal properties of alangium salviifolium root extracts on culex quinquefasciatus mosquitoe alex yagoo, m.c. john milton, jelin vilvest 619 624 developing virtual herbarium of sunan kalijaga islamic state university as a plant taxonomy complementary lab work material muhammad ja’far luthfi, riyanto, widodo 625 630 phytochemical screening and antioxidant activity analysis of n-hexane extract of sonneratia alba mangrove leaves putu rissa almadea surya, made dharmesti wijaya, desak putu citra udiyani 631 635 the therapeutic potential of cola nitida in health and disease: a review kamaldeen olalekan sanusi, umar zayyanu usman, dawoud usman, kehinde ahmad adeshina, yaaqub abiodun uthman, lukman jimoh, aminat omolola imam-fulani 637 643 potency of biocoagulant from cationic modified starch of balbis banana blossom waste for palm oil wastewater treatment: literature study esa ghanim fadhallah, alif fikri nur hidayat, arfika julya saffanah, bella amanda iswahyudi, diah shihatin wasalamah, eka cahyani putri, nadila ningtias, yana hijriyatin ummi 645 649 identification and toxicity profiling of column fractions of ethanol leaf extract of ziziphus mauritiana tajudeen alowonle owolarafe, salawu kailani 651 662 prevalence and biofilm-forming potentials of bacterial uropathogens among primary school pupils in yola north, adamawa state abdulazeez mumsiri abaka, mubarak muhammad dahiru, ibrahim ya’u, tukur baba abdullahi 663 669 an overview of the total leukocyte count in pulmonary tuberculosis patients at rsud gambiran kediri city devi nor fitri, lisa savitri, rochmad krissanjaya, elfred rinaldo kasimo 671 674 bioactivity of vernonia arborea leaves from bentian tribe as natural antioxidant and antibacterial based on local knowledge nur maulida sari, humairo aziza, farida aryani, murdianto 675 680 in vivo alpha-amylase and alpha-glucosidase inhibitory potentials of panicum maximum jacq. (guinea grass) leaf extract on wister rats godwin ndarake enin, jude efiom okokon, enobong mfon david, saviour elisha emmanuel, esther michael ekanem, bassey sunday antia 681 685 antimicrobial and antioxidant activities of sesquiterpenes-rich essential oil of trichilia monadelpha (thonn) j.j. de wilde of root bark oluwakayode olubunmi odeja, patricia a. onocha, ganiyat kehinde oloyede, damola ayoyerokun, micheal gabriel ibok, ejike onwudiegwu okpala 687 692 antibacterial potency and physicochemical profiles of eucalyptus pellita leaf waste essential oil from pt surya hutani jaya, east kalimantan wartomo, farida aryani, muhammad fikri hernandi, erna rositah, sri ngapiyatun, nur maulida sari 693 697 this page intentionally left blank 3: front 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 187-190 | doi: 10.14421/biomedich.2023.121.187-190 issn 2540-9328 (online) interleukin-1 as a predictor cytokine sars-cov: article review lisa savitri*, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita, ester lianawati antoro, ida septika wulansari department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 14 december, 2022. revision accepted: 07 february, 2023. published: 11 february, 2023. abstract severe acute respiratory syndrome coronavirus (sars-cov) is an etiologic agent of respiratory disease that has a mortality rate of 10%. il-1 actively participates in the inflammatory response to infection. sars-cov-2 appears to act on the activation and maturation of il1β, which in turn activates other proinflammatory cytokines, such as il-6 and tnf-. therefore, il-1β is part of the cytokine storm generated by coronavirus infection. elevated levels of the il-1 receptor antagonist (il-1ra) in severe cases of covid-19, and this marker have been associated with increased viral load, loss of lung function, lung damage, and risk of death. in addition, there is an increase in il-1α levels in patients with severe covid-19, and this is strongly associated with lung injury. il-1 levels are associated with the virulence of the process, and significantly higher serum levels have been observed in severe symptomatic sars-cov-2 cases than in mild cases or in those infected with the 2003 sars-cov coronavirus or 2012 mers coronavirus. keywords: cytokines; interleukin-1; severe acute respiratory syndrome coronavirus (sars-cov). abbreviations: severe acute respiratory syndrome coronavirus (sars-cov), il-1 receptor antagonist (il-1ra), interleukin-1 (il-1), sars-cov lacking the e gene (sars-cov-δe), open reading frame 3a (orf3a), tnf receptor-associated factor 3 (traf3), recruitment caspase domain (asc) interleukin-1 and infection the interleukin-1 (il-1) family of ligands and receptors plays a central role in the formation and regulation of inflammation as part of the immune response. the il-1 molecule is best known as a member of a family of proinflammatory proteins called cytokines. they have been shown to play a role, not only during inflammation, but also in influencing additional physiological and pathological functions such as autoimmune diseases, malignancies, and many others. in addition, the biological balance between proinflammatory and inhibitory activity is critical, as any change in the expression of the il-1 family has the potential to cause disease (etti, et al., 2018). inflammation that occurs as a result of exposure of tissues and organs to noxious stimuli such as microbial pathogens, irritants, or toxic cellular components. the main physical manifestations of inflammation are redness, swelling, heat, pain, and loss of function in the affected area. this process involves the main cells of the immune system, including monocytes, macrophages, neutrophils, basophils, dendritic cells, mast cells, t cells, and b cells. however, examination of various inflammatory lesions reveals the presence of specific leukocytes in certain lesions. that is, the inflammatory process is regulated in such a way as to ensure that the right leukocytes are recruited. these events are in turn controlled by a number of extracellular molecular regulators, including members of the cytokine and chemokine family that mediate immune cell recruitment and the complex intracellular signaling control mechanisms that characterize inflammation. this review will focus on the role of major cytokines, chemokines, and their receptors in the pathophysiology of autoinflammatory disorders, pro-inflammatory disorders, and neurologic disorders involving inflammation. il-1 actively participates in the inflammatory response to infection (turner, et al., 2014), and its main source is activated monocytes and macrophages. interleukin-1 as a predictor cytokine sars-cov severe acute respiratory syndrome coronavirus (sarscov) is the etiologic agent of respiratory disease that has a mortality rate of 10%. previous studies have shown that sars-cov lacking the e gene (sars-cov-δe) is attenuated in several animal model systems. this https://doi.org/10.14421/biomedich.2023.121.187-190 188 biology, medicine, & natural product chemistry 12 (1), 2023: 187-190 suggests that the absence of protein e resulted in reduced expression of proinflammatory cytokines, decreased number of neutrophils in the lung infiltrates, reduced lung pathology, and increased viability of mice, suggesting that lung inflammation contributes to sarscov virulence. furthermore, sars-cov-δe infection resulted in decreased nf-b activation compared to levels of wild-type virus. most importantly, treatment with drugs that inhibit nf-kb activation led to a reduction in inflammation and lung pathology in sars-cov-infected cells and mice and significantly improved mouse survival after sars-cov infection. these data suggest that activation of the nf-b signaling pathway represents a major contribution to inflammation induced after sarscov infection and that nf-b inhibitors hold antiviral promise in infections caused by sars-cov and other potentially pathogenic human coronaviruses (dediego, et al., 2014). deletion of the sars-cov envelope (e) gene attenuates the virus. gene e encodes a small multifunctional protein that has ion channel (ic) activity, an important function in virus-host interactions. to examine the contribution of the ic activity of protein e in viral pathogenesis, two mouse-adapted sars-cov, each containing a single amino acid mutation that suppresses ion conductivity, were engineered. after serial infection, mutant viruses, in general, introduce compensatory mutations in the e gene that create active ion channels. furthermore, ic activity provided better fitness in competition assays, suggesting that ionic conductivity is an advantage for viruses. interestingly, virus-infected mice exhibiting ic protein e activity, either with a wild-type protein e sequence or with a revertant that restores ion transport, rapidly lost weight and died. in contrast, mice infected with a mutant lacking ic activity, which did not introduce the mutation in the e gene during the experiment, recovered from the disease and mostly survived. levels of inflammasome-activated il-1β were reduced in the lung airways of virus-infected animals lacking ic protein e activity, suggesting that ic protein e function is required for inflammasome activation. the reduction in il-1β is accompanied by a decrease in the number of tnf and il-6 in the absence of ion conductivity protein e. all of these key cytokines promote the development of lung damage and ards pathology. in conclusion, the ic activity of protein e is a novel determinant for the virulence of sars-cov (nieto-torres, et al., 2014). siu et al. (2019) demonstrated that the open reading frame 3a (orf3a) accessory protein of sars-cov activates the nlrp3 inflammasome by promoting tnf receptor-associated factor 3 (traf3)-mediated ubiquitination of an apoptosis-associated spot-like protein containing a recruitment caspase domain (asc). sars-cov and its orf3a protein were found to be potent activators of pro-il-1β gene transcription and protein maturation, 2 signals required for nlrp3 inflammasome activation. orf3a induces pro-il-1β transcription via nf-b activation, which is mediated by ubiquitination and traf3-dependent p105 processing. orf3a-induced increase in il-1β secretion is independent of its ion channel activity or is absent in melanoma 2 but requires nlrp3, asc, and traf3. orf3a interacts with traf3 and asc, localizes with them in discrete mottled structures in the cytoplasm, and facilitates the formation of asc specks. traf3dependent ubiquitination of k63-associated ascs was more prominent in cells infected with sars-cov or when orf3a was expressed. overall, the findings of siu, et al. (2019) revealed a novel mechanism by which the sars-cov protein orf3a activates nf-b and the nlrp3 inflammasome by promoting p105 and asc.siu-dependent ubiquitination of traf3. the severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent asc ubiquitination (siu, et al., 2019). sars-cov-2 appears to act on the activation and maturation of il-1β, which in turn activates other proinflammatory cytokines, such as il-6 and tnf (dediego, et al., 2014; nieto-torres, et al., 2014; siu, et al., 2019). sars-cov-infected dcs show low expression of antiviral cytokines (interferon [ifn-α], ifn-β, ifn-γ, and interleukin 12p40 [il-12p40]), upregulation of proinflammatory cytokines (tumor necrosis). factors [tnf-α] and il-6) but significant upregulation of inflammatory chemokines (macrophage inflammatory protein 1α [mip-1α], upregulated on normal-expressed and secreted t cell activation [rantes]), interferon-inducible proteins from 10 kda [ip-10], and monocyte chemoattractant protein 1 [mcp1]). therefore, il-1β is part of the cytokine storm generated by coronavirus infection (law, et al., 2005; chu, et al., 2016; hui, et al., 2019; conti, et al., 2020; mehta, et al., 2020; wan, et al., 2020) yang, et al. (2020) detected elevated levels of the il1 receptor antagonist (il-1ra) in 14 severe cases of covid-19, and this marker has been associated with increased viral load, loss of lung function, lung damage, and risk of death. liu, et al. (2020) also found elevated levels of il-1α in patients with severe covid-19, and this was strongly associated with lung injury. il-1 levels are associated with the virulence of the process, and significantly higher serum levels have been observed in severe symptomatic sars-cov-2 cases than in mild cases or in those infected with the 2003 sars-cov coronavirus or 2012 mers. covid-19 patients with severe symptoms have elevated levels of il-1β, which has been associated with sars, hypercoagulation, and disseminated intravascular coagulation (zhang, et al., 2020). for this reason, several therapeutic strategies have used il-1 inhibition in an attempt to avoid cytokine storms (tanaka, et al., 2016; kritas, et al., 2020). in this way, mesenchymal stem cells (mscs) have been used to savitri et al. – interleukin-1 as a predictor cytokine sars-cov: article review 189 inhibit proinflammatory cytokines such as il-1α and tnf (chen, et al., 2020). conclusions the conclusions of the study may be presented in here.il-1 actively participates in the inflammatory response to infection. sars-cov-2 appears to act on the activation and maturation of il-1β, which in turn activates other proinflammatory cytokines, such as il-6 and tnf-. therefore, il-1β is part of the cytokine storm generated by coronavirus infection. elevated levels of the il-1 receptor antagonist (il-1ra) in severe cases of covid-19, and this marker have been associated with increased viral load, loss of lung function, lung damage, and risk of death. in addition, there is an increase in il1α levels in patients with severe covid-19, and this is strongly associated with lung injury. il-1 levels are associated with the virulence of the process, and significantly higher serum levels have been observed in severe symptomatic sars-cov-2 cases than in mild cases or in those infected with the 2003 sars-cov coronavirus or 2012 mers coronavirus. acknowledgements: acknowledgments are expressed in a brief; all sources of institutional, private and corporate financial support for the work must be fully acknowledged, and any potential conflicts of interest are noted.the author would like to thank the institute for research, development, and community service (lp3m) of kadiri university which has always provided support for writing and publications. authors’ contributions: lisa savitri designed the study. lisa savitri and elfred rinaldo kasimo analyzed the articles. lisa savitri, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita, ester lianawati antoro, ida septika wulansari wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors stated there was no funding. references chen c., zhang x.r., ju z.y., he w.f. 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(2020) exuberant elevation of ip-10, mcp-3 and il-1ra during sars-cov-2 infection is associated with disease severity and fatal outcome. infectious diseases (except hiv/aids) 2020 doi: 10.1101/2020.03.02.20029975. zhang w., zhao y., zhang f., wang q., li t., liu z., wang j., qin y., zhang x., yan x., zeng x., zhang s. (2020) the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the perspectives of clinical immunologists from china. clin. immunol. 2020;214 doi: 10.1016/j.clim.2020.108393. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 111-118 | doi: 10.14421/biomedich.2022.112.111-118 issn 2540-9328 (online) phytochemical compositions and antioxidant properties of combined funtumia africana and abutilon mauritianum extract (cfae) robert ikechukwu uroko1,*, charles nnanna chukwu1, chinomso friday aaron2, umezurike benedict chidozie3, doris akachukwu1, ndukwe onyeani atuh1 1department of biochemistry, college of natural sciences, michael okpara university of agriculture, umudike, p.m.b. 7267, umuahia, abia state, nigeria. 2department of biochemistry, faculty of biological and physical sciences, abia state university, uturu, nigeria. 3department of public health, gregory university uturu, abia state, nigeria. corresponding author* ir.uroko@mouau.edu.ng manuscript received: 05 june, 2022. revision accepted: 18 july, 2022. published: 31 july, 2022. abstract medically active compounds in plants confer biological effects including antioxidant properties. this study evaluated the phytochemical content and in vitro antioxidant properties of combined funtumia africana leaves and abutilon mauritianum extract (cfae). the 1,1diphenyl-2-picrylhydrazyl (dpph), ferric reducing antioxidant power (frap), thiobarbituric acid reactive substances (tbars), total antioxidant capacity (tac), and nitric oxide radical (no.) scavenging capabilities were used as antioxidant assay models. the results of the phytochemical analysis showed that cfae is rich in alkaloids, flavonoids, tannins, saponins, phenols and cardiac glycosides. the extract contains antioxidant vitamins a, c and e, vitamin e being the most abundant. the cfae showed a dose-dependent tac based on the observed frap, tbars, and dpph scavenging activity which could be attributed to the presence of phenolic compounds and vitamins e. these strongly suggest that cfae is a potential source of phytochemicals and antioxidants which could be exploited in the food and pharmaceutical industries in production of potent nutraceuticals or therapeutically-important products. keywords: abutilon mauritianum; antioxidant activity; antioxidant vitamins; funtumia africana; free radicals; phytochemicals. abbreviations: dpph = 1-diphenyl-2-picrylhydrazyl, frap = ferric reducing antioxidant power, tbars = thiobarbituric acid reactive substances, tac = total antioxidant capacity, no = nitric oxide radical, cfae = combined funtumia africana leaves and abutilon mauritianum extract. introduction medicinal plants are the bio-resource of many drugs and are used in modern medicine as well as food supplements and pharmaceutical intermediates (ahn, 2017). medicinal plants contain a wide range of natural products known as phytochemicals, which are compounds manufactured by plants via major or minor metabolism (molyneux et al., 2007). plant-based natural products have played an important role in drug discovery and were the basis of most early medicines. these phytochemicals including saponins, flavonoids, alkaloids, polyphenols, terpenes, tannins etc. generally possess potent biological activities which play important roles in growth of plants or defence against pathogens or predators (molyneux et al., 2007). also, they serve as sources of therapeutic agents used in the management of several ailments because of their antimicrobial, anticancer and antioxidant effects (riganti et al., 2011). hence, antioxidants play a vital role in the defence system of the body against harmful oxygen species. in addition to the application of natural products such as drug, they also contribute significantly towards nutraceuticals as well as ingredients for food products. in recent times, the search for safer natural products and more potent antioxidants have necessitated the need to harness plant parts which are capable of producing potent secondary metabolites (phytochemicals) to replace synthetic antioxidants. several reports have denoted the antioxidant effects in vitro, of phytoconstituents in biological systems (papuc et al., 2017,). on the other hand, the need for improved therapeutic effects may warrant the combination of plant extracts due to synergistic properties. this concept is found in ayurveda and medicinal systems where more than one herb in a precise proportion could be used in the management of many ailments (aslam et al., 2014). funtumia africana (family = apocynaceae) is a flowering plant native to africa with narrow crown and can grow up to 30 metres tall. the slender, cylindrical stem can be up to 50 cm in diameter (burkil, 2014). funtumia africana is harvested from remote areas for indigenous use as a medication and source of materials. https://doi.org/10.14421/biomedich.2022.112.111-118 112 biology, medicine, & natural product chemistry 11 (2), 2022: 111-118 it is used by african natives to treat fever, inflammation, burns, malaria, cancer and urinary incontinence (burkil, 2014). abutilon mauritianum (family = malvacedae) is also a widely used plant in traditional african medicine. the plant is extensively dispersed in the drier regions of african tropics and widely used for its edible leaves and medicinal properties (ruffo et al., 2012). the root, stem, bark and leaves are utilized in the management of several maladies including kidney problems, diarrhoea, dysentery, haemorrhoids, stomach-ache, fire burns, sore throat, cough and cold (ruffo et al., 2012; burkil, 2014). therefore, this research evaluated the phytochemical constituents and in-vitro antioxidant properties of a combined extract of f. africana leaves and a. mauritianum stem bark (cfae). materials and methods collection and identification of plant material fresh samples of f. africana leaves and a. mauritianum stem bark were collected from the botanical gardens at michael okpara university of agriculture, umudike and identified at the herbarium unit of the department of forestry in the same institution with specimen voucher number 2694-5 (acuss 1899) and jones fhi 13749 respectively. preparation of plant materials the samples were washed with clean water and later dried under shade at room temperature for thirteen days. they were ground into fine powder, weighed and then stored in a dry, clean, sterile container for subsequent extraction. extraction of plant materials five hundred gram (800 g) of the finely ground f. africana leaves and a. mauritianum barks (1:1) was soaked in 2 l of ethanol for 72 hours in a sterile vessel. it was filtered, first with a mesh cloth, and later with a whatman no.1 filter paper. the filtrate was concentrated in a water bath (50℃) and allowed to evaporate completely. the evaporated extract was weighed and the percentage yield was calculated. the extraction of 800 g of cfae gave a percentage yield of 22.7% (113.5 g). the obtained combined extract (cfae) was covered with aluminium paper foil and stored in the refrigerator (4℃) for further analyses. qualitative phytochemical analysis the presence of phytochemicals including alkaloids, flavonoids, saponins, tannins, terpenoids, cardiac glycosides, and steroids were screened according to the methods of harborne (1998). quantitative determination of phytochemicals alkaloid and flavonoid contents were determined quantitatively as per the protocols outlined by harborne (1998). phenol, saponin and terpenoid contents were quantified using the methods of harborne (1984), while tannin, cardiac glycoside and steroid contents of the extract were evaluated as stated by the method of pearson (1976). determination of antioxidant vitamins vitamins a (retinol), c (ascorbic acid) and e (αtocopherol) contents were determined utilizing the protocols of pearson (1976). their concentrations were extrapolated from the respective standard curves. determination of 1,1-diphenyl-2-picrylhydrazyl (dpph) radical-scavenging activity scavenging activity on dpph free radicals was assessed according to the method of gyamfi et al. (1999) with slight modifications. exactly 2.0 ml of extract was mixed with 1.0 ml of methanol and 0.3 mm dpph in a dilute 2-fold solution. the mixture was shaken vigorously to prevent it from freezing and left for 25 minutes in the dark. the absorbance of the reaction mixture was measured at 520 nm against each blank. % scavenging activity = 100 − (abssample − absblank) (abs control) 𝑥 100 determination of nitric oxide radical (no) scavenging activity nitric oxide was measured according to the protocol of marcocci et al. (1994). reaction mixtures containing nitroprusside (5 mm) and the plant extract were incubated at 25℃ for up to 180 minutes in front of a polychromatic light source. the no radical generated, interacted with oxygen to yield no2 which was then analysed at 30-minutes intervals by mixing an equivalent volume (1.0 ml) of greiss reagent and incubation mixture. the absorbance was measured at 546 nm. determination of ferric reducing antioxidant power (frap) the frap of the extract was determined according to the protocol of yen and chen (1995) with little alteration. exactly 0.3 ml of plant extract, ascorbic acid and rutin which were prepared in distilled water, was mixed with a reacting mixture [(25 ml of phosphate buffer and 2.5 ml of kfe(cn)]. the resultant mix was incubated at 500c for 20 minutes followed by adding of tca (2.5 ml, 10% w/v) and then incubated at 25℃ for 15 minutes. absorbance was measured at 700nm against the blank. determination of total antioxidant content (tac) the tac was determined using the phosphomolybdate method earlier reported by el-hashash et al. (2010). exactly 1.0 ml aliquot of the extract and vitamin c was mixed with 1 ml of reagent solution (600 mm h2so4, 25 mm na2(po)4 and 4 mm ammonium molybdate – uroko et al. – nutraceutical properties of combined funtumia africana and abutilon mauritianum extract 113 1:1:1). the test tubes were incubated at 95℃ for 90 minutes and cooled. absorbance was read at 765 nm against a blank. ascorbic acid served as the reference. 𝑇𝐴𝐶(%) = 𝐴𝑜 − 𝐴𝑠 𝐴𝑜 𝑥 100 𝐴𝑜 = absorbance of control; 𝐴𝑠 = absorbance of extract determination of thiobarbituric acid reactive substances (tbars) activity the tbars activity was determined according to the method of banerjee et al. (2005) where egg yolk homogenates served as a lipid-rich media. exactly 0.5 ml of egg homogenate and 100 ml of sample were added to all test tubes and made up to 1.0 ml with distilled water. thereafter, 50 ml of feso4 (0.075 m) and 20 ml of lascorbic acid (0.1 m) were introduced and incubated for 60 minutes (37℃) to induce lipid peroxidation. afterwards, 0.2 ml of edta and 1.5 ml tba reagent were added to the respective samples and heated for a 15 minutes at 100℃. after cooling, samples were centrifuged at 3000 rpm for 10 minutes. percentage inhibition(%) = 𝐴𝑜 − 𝐴𝑠 𝐴𝑜 𝑥 100 𝐴𝑜 = absorbance of control, 𝐴𝑠 = absorbance of sample. data analysis the data obtained were subjected to one-way analysis of variance (anova) with a statistical products and service solution (spss) version 22 and the statistical significance ascertained at p<0.05. results were presented as mean ± standard deviation (n =3). results and discussion qualitative phytochemical composition of cfae the qualitative phytochemical analysis of cfae (table 1) showed that tannins, phenolics, steroids and terpenoids are the most abundant phytochemicals in the extract. flavonoids were present in moderate amount, while glycosides and saponins were the least present. table 1. qualitative phytochemical composition of cfae. key: + = low concentration; ++ = moderate concentration; +++ = high concentration quantitative phytochemical composition of cfae the result of the quantitative phytochemical analysis of cfae (table 2) showed very high concentrations of alkaloids (3547.46±17.54 mg/100g), total phenolics (1398.42±15.72 mg/100g), and terpenoids (1528.01±0.58 mg/100g). tannins, flavonoids and steroids were present in moderate amounts while glycosides and saponins were present in minute concentrations. table 2. quantitative phytochemical composition of cfae. phytochemicals concentration (mg/100 g) glycoside 3.33 ± 0.77 alkaloids 3547.46 ± 17.54 saponins 5.74 ± 0.01 tannins 128.42 ± 1.79 total phenols 1398.42 ± 15.72 flavonoids 147.25 ± 4.58 steroids 131.13 ± 0.73 terpenoids 1528.01 ± 0.58 values are mean ± standard deviation of triplicate determinations (n = 3). antioxidant vitamin composition of cfae as shown in table 3, cfae contains antioxidant vitamins a, c and e. vitamin e was present in high concentration while vitamins a and c were present in relatively low amounts. table 3. composition of antioxidant vitamins of cfae. vitamins concentration vitamins a (µg/g) 5.52 ± 0.11 ascorbic acid (mg/100 g) 3.76 ± 0.28 vitamin e (mg/100 g) 113.48 ± 3.97 values are mean ± standard deviation of triplicate determinations (n = 3). tbars free radical scavenging activity of cfae the data in figure 1 show tbars free radical scavenging activity of cfae and an antioxidant standard, butylated hydroxytoluene (bht). a dosedependent increase in tbars activity was observed for the cfae. however, the extract showed considerable lower activity at lower concentrations (7.81µg/ml and 15.63µg/ml) when compared with the bht standard. increase in concentration of the extract led to increase in scavenging activity. the half maximal effective concentration (ec50) of the extract for tbars was 73.86±1.22 µg/ml, while that of bht standard was 40.75±0.92 µg/ml. phytochemicals composition glycoside + tannins +++ flavonoids ++ total phenolic +++ steroids +++ terpenoids +++ saponins + alkaloids ++ 114 biology, medicine, & natural product chemistry 11 (2), 2022: 111-118 figure 1. tbars radical scavenging activity of cfae. dpph free radical scavenging activity of cfae as shown in figure 2, a dose-dependent increase in dpph inhibition was observed for cfae, whereas the dpph inhibition for ascorbic acid remained constant regardless of concentration. the extract showed considerably lower dpph inhibition at lower concentrations when compared with that of ascorbic acid. increase in concentration of the extract led to increase in mean percentage dpph inhibition. the ec50 of the extract and ascorbic acid for dpph were 49±0.08 and 34.07±0.20 µg/ml respectively. figure 2. dpph radical scavenging activity of cfae. ferric reducing antioxidant power (frap) of cfae figure 3 shows the frap of cfae. a dose-dependent increase in frap was observed. the extract showed lower frap at lower concentrations but reached its peak at 640 µg/ml. 0 10 20 30 40 50 60 70 80 7.81 µg/ml 15.63 µg/ml 31.25 µg/ml 62.5 µg/ml 125 µg/ml 250 µg/ml 500 µg/ml m ea n % i n h ib it io n t b a r s ( % ) concentrations combined extract bht each bar represents mean±standard deviation (n = 3) 0 20 40 60 80 100 120 10 µg/ml 20 µg/ml 40 µg/ml 80 µg/ml 160 µg/ml 320 µg/ml 640 µg/ml m ea n d p p h i n h ih ib it io n ( % ) concentration combined extract ascorbic acid each bar represents mean±standard deviation (n = 3) uroko et al. – nutraceutical properties of combined funtumia africana and abutilon mauritianum extract 115 figure 3. ferric reducing antioxidant power (frap) of cfae. total antioxidant capacity (tac) of cfae figure 4 shows the tac of cfae. a dose-dependent decrease in tac was observed. at lower concentrations, the extract showed higher tac which decreased as the concentration increased. figure 4. total antioxidant capacity (tac) of cfae. nitric oxide (no) scavenging activity of cfae the no activity of cfae (figure 5) increased slightly as concentration increased in line with the observed no activity for ascorbic acid. however, the no activity for ascorbic acid begins to decrease at 80 µg/ml concentration and reaches its least activity at 640 µg/ml, whereas that of the extract remained stable. the ec50 of the extract and ascorbic acid for nitric oxide were 54.93±0.28 µg/ml and 23.71±0.56 µg/ml respectively. figure 5. nitric oxide free radical scavenging activity of cfae. 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 10 µg/ml 20 µg/ml 40 µg/ml 80 µg/ml 160 µg/ml 320 µg/ml 640 µg/ml m ea n f r a p ( g a e ) concentrations each bar represents mean ± standard deviation (n = 3) 0,0 0,2 0,4 0,6 0,8 1,0 1,2 10 µg/ml 20 µg/ml 40 µg/ml 80 µg/ml 160 µg/ml 320 µg/ml 640 µg/ml m ea n t a c ( a a e ) concentrations each bar represents mean ± standard deviation (n = 3) 0,0 10,0 20,0 30,0 40,0 50,0 60,0 70,0 80,0 90,0 100,0 10 µg/ml 20 µg/ml 40 µg/ml 80 µg/ml 160 µg/ml 320 µg/ml 640 µg/mlm ea n n it ri c o x id e in h ib it io n ( % ) concentrations combined extract ascorbic acid each bar represents mean ± standard deviation (n = 3) 116 biology, medicine, & natural product chemistry 11 (2), 2022: 111-118 discussion this study was undertaken to evaluate the phytochemical compositions and antioxidant properties of combined extract of f. africana leaves and a. mauritianum stem bark (cfae). therapeutic tendency of plants can be evaluated by performing preliminary qualitative screening to ensure the presence of phytochemicals (batool et al., 2019). in this study, bioactive constituents that confer biologically active effects to cfae were screened and the results confirmed the existence of alkaloids, flavonoids, tannins, glycosides, phenols, steroids, terpenoids, saponins and antioxidant vitamins. the high levels of alkaloids, phenols, flavonoids and terpenoids in this study strongly suggest that cfae could be a potential source of natural products, which could serve as effective free radical scavengers and/or inhibitors; hence could be a good plant-based pharmaceutical product for management of several diseases caused by free radicals. this is in line with several reports which highlighted the therapeutic effects of the above-mentioned phytochemicals (adamski et al., 2020; aloko and bello, 2021). steroids found in plants play critical roles in some ailments including prostate cancer (lubik et al., 2016). the moderate concentration of steroids in the sample could justify its wide use for the formulation of various herbal medicines. tannins had long been thought to be anti-nutritional, but their helpful or anti-nutritional characteristics are now understood to be dependent on their chemical structure and dosage (schiavone et al., 2008). however, consumption of tannin-rich food has been implicated in esophageal cancer (kanzaki et al., 2001) suggesting that tannins might be carcinogenic and hence, the consumption of polyherbal extract containing f. africana leaves and a. mauritanum stem barks should be monitored. plant-based vitamins including a, c and e are potent antioxidants present in varying amounts in plants. these vitamins are of great impact to human health. they are essential for metabolism because of their redox chemistry and role as enzymatic cofactors (asensifabado and munne-bosch, 2010). the high level of vitamin e in cfae observed in this study could have contributed to its free radicals scavenging property. a review had shown antioxidant potentials of african natural products (lawal et al., 2017). validating the presence of antioxidant abilities of plants entails the use of established antioxidant protocols with varying principles, mechanisms and experimental conditions. antioxidants act as radical scavenger by electron donating mechanisms or by hydrogen donating mechanisms – hence preventing the deleterious role of free radicals in different diseases. therefore, to evaluate the antioxidant capacity of cfae, several antioxidant assays were carried out. the extract showed considerable lower tbars activity at lower concentrations when compared with the bht standard, but increased slightly as concentration increased. lipid peroxidation of membranes is one of the early events at the onset of inflammatory response by stimulated polymorphonuclear cells; hence, agents that are capable of averting lipid peroxidation of membranes could potentially serve as appropriate candidate in antiinflammatory drug discovery process (anyasor et al., 2014). the potent scavenging activity exhibited by cfae at increased concentrations could be attributed to the high concentration of phenolics, which confer antioxidant effects possibly by targeting the neutralization of reactive radicals and/or quenching of singlet oxygen. plant phenolics can react with active oxygen radicals like hydroxyl, superoxide anion and lipid peroxy radicals to prevent early lipid oxidation (muanya and odukoya, 2008). the effect of antioxidants on dpph is thought to be due to their hydrogen donating ability (adesegun et al., 2017). free radical scavengers are proton donors that help to mop up excess free radicals (adesegun et al., 2017). ascorbic acid is a potent antioxidant as an electron donor showing free radical scavenging ability; hence, it is commonly used in dpph radical scavenging assay as a reference compound for measuring antioxidant activity. the presence of phenolic compounds could have contributed to the scavenging activity observed. this is in agreement with earlier reports (jimoh et al., 2008), which posit that phenolic constituents of the combined extract contributes to its scavenging ability. presence of phenolics and flavonoids impart the scavenging capabilities to plants, because they are significantly extracted in the polar solvents and hence, donate electron or hydrogen to stabilize dpph free radicals. the development of the o-phenanthroline-fe(2+) complex and its destruction in the presence of chelating drugs is the basis of the frap assay. this test is frequently used to determine the antioxidant capacity of polyphenol-containing foods, drinks, and nutritional supplements (benzie and strain, 1996). a dosedependent increase in frap of cfae was observed (figure 5). the extract showed lower frap at lower concentrations but reached its peak at 640ug/ml. earlier report had shown a direct correlation between antioxidant activity and reducing power, which was attributed to the presence of phenolic compounds (ojewunmi et al., 2013). this present study has revealed that the extract certainly has a proton-donating property and could work as a free radical inhibitor or scavenger, possibly acting as a key antioxidant. due to the fact that plants contain several classes and types of antioxidants, it is imperative to ascertain their cumulative antioxidant capacity of scavenging free radicals (pellegrini et al., 2003). this formed the basis for determining the tac of cfae, which measures the amount of free radicals scavenged by a test solution uroko et al. – nutraceutical properties of combined funtumia africana and abutilon mauritianum extract 117 (ghiselli et al., 2010) and is used to evaluate the antioxidant capacity of biological samples (pinchuk et al., 2012). in the present study cfae showed high tac values at lower concentrations. however, the antioxidant capacities decreased dose-dependently as the concentration of the extract increased. the high antioxidant capacity shown by the extract (especially at lower concentrations) could be attributed to the high content of phenolics (jimoh et al., 2008), which has demonstrated strong antioxidant activities in different models. a positive correlation between total phenolic content and tac was also observed in a related study using f. africana stem barks (frempong et al., 2021). endothelial cells, macrophages, neurons, and other cells create nitric oxide (no), a chemical mediator involved in the regulation of many physiological processes. on the other side, high no levels have been related to a variety of illnesses. in models of inflammatory bowel disease and diabetes mellitus, no inhibitors have been found to reduce inflammation and tissue alterations (aydin et al., 2010). increasing data suggests that oxidative stress and alterations in nitric oxide production or activity are important factors in the development of diabetes problems (maritim et al., 2003). the extract efficiently inhibited the formation of nitric oxide from sodium nitroprusside in the current investigation by competing with oxygen for the reaction with nitric oxide and therefore inhibiting the generation of nitrite anions. the extract showed a slightly lower nitric oxide scavenging activity at lower concentrations than the standard ascorbic acid. however, at higher concentrations, the radical scavenging activity increased considerably as that of the standard decreased. thorough discussion represents the causal effect mainly explains for why and how the results of the research were taken place, and do not only re-express the mentioned results in the form of sentences, not repeat them. concluding sentence should be given at the end of the discussion. conclusions the findings from this study showed that the cfae is a rich source of natural products including; alkaloids, flavonoids, tannins, glycosides, phenols, steroids, terpenoids and saponins, and possesses good antioxidant property which could be attributed to the presence of phenolic compounds and antioxidant vitamins (a, c and e). these findings strongly suggest that their constituents can be used as an easily accessible source of natural antioxidant, which could be exploited in the food and pharmaceutical industries in production of potent nutraceuticals or therapeutically-important products. acknowledgements: the authors expressed profound gratitude to okwor josephat ani for his technical assistance during the conduct of this study. authors’ contributions: uroko ri and ndukwe oa designed the study. uroko ri, akachukwu, aaron cf, umezurike bc, ndukwe oa and chukwu cn carried out the laboratory work. uroko ri analyzed the data. uroko ri and chukwu cn wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare no funding. references adamski z, blythe ll, milella l, bufo sa. 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effect of water-soluble stem extract “kayu kuning“ (arcangelisia flava l.merr) on the growth inhibition ff candida albicans atcc 10231 and trichophyton mentagrophytes in vitro rini setyowati, sudarsono, setyowati e.p. larvicidal activity of the mixture of cashew nut shell liquid (cnsl) and aqueous extract of sapindus rarak dc against larvae of culex quinquefasciatus rahmi safarina fauziah, sudarsono, budi mulyaningsih identification of migratory birds and their spesific characteristics of habitat in the salt water lake of gili meno, north lombok distric diah purwitasari, luh gde sri astiti, supriadi larvicidal effect of vinca fruit extract (vinca rosea) against aedes aegypti larvae and secondary metabolites profile by thin layer chromatography rahmawati ekaputri, sudarsono, budi mulyaningsih morpho-anatomical analysis of cosmostigma racemosum (asclepiadoideae) flowers widodo, mohamad amin, mimien henie irawati al-muhdar,muhammad ja'far luthfi 1-14 15-19 21-23 25-30 31-33 35-46 cetak cover biology medicine vol 3 num 1 2014 dpn.pdf (p.1) editorial board 2014 v3 n1.pdf (p.2) guidance for authors 2014 v3 n1.pdf (p.3) cetak cover biology medicine vol 3 num 1 2014 blkg.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 601-607 | doi: 10.14421/biomedich.2025.142.601-607 issn 2540-9328 (online) physicochemical characterization and dissolution enhancement of mefenamic acid–isonicotinamide crystalline solid dispersion indra1,*, nurul apriani1, ade yeni aprillia2, winda trisna wulandari2, gatut ari wardani2, firman gustaman1 1department of pharmaceutics, 2department of pharmacochemistry, faculty of pharmacy, universitas bakti tunas husada, jl. letjen mashudi no 20 tasikmalaya 46196, tel. +62-265-334740, indonesia. corresponding author* indra@universitas-bth.ac.id manuscript received: 07 april, 2025. revision accepted: 15 july, 2025. published: 01 october, 2025. abstract poor aqueous solubility limits the bioavailability of non-steroidal anti-inflammatory drugs like mefenamic acid. this study aims to improve the solubility and dissolution of mefenamic acid through crystalline solid dispersions using solvent evaporation and co-grinding techniques with selected co-formers. solid dispersions were formulated at different drug-to-co-former ratios (1:1, 1:2, and 2:1) and characterized using differential scanning calorimetry, fourier-transform infrared spectroscopy, and powder x-ray diffraction. dsc results revealed reduced crystallinity, indicated by the disappearance of melting peaks and the appearance of a single glass transition temperature. ftir analysis confirmed hydrogen bonding between the drug and co-former, while pxrd patterns showed a loss of long-range order, supporting the formation of amorphous phases. dissolution testing demonstrated a significant increase in drug release, particularly in the 1:2 formulation, which outperformed the pure drug and other ratios. these results confirm that the choice of preparation method and coformer ratio critically influence the performance of solid dispersions. this study provides valuable insights into the design of improved oral formulations for poorly soluble drugs, contributing to the advancement of pharmaceutical technology. keywords: crystalline solid dispersion; dissolution improvement; drug–co-former interaction; mefenamic acid. introduction oral drug administration remains the most widely adopted route in clinical practice due to its convenience, non-invasiveness, cost-effectiveness, and high patient compliance. however, the therapeutic efficacy of orally delivered drugs is critically dependent on their aqueous solubility and dissolution rate, which directly influence systemic bioavailability. a large proportion of both established pharmaceutical agents and new chemical entities (nces) fall into the biopharmaceutics classification system (bcs) class ii category, which includes compounds with high membrane permeability but poor water solubility (mehta et al., 2016; patel et al., 2015). the dissolution-limited absorption of such drugs often results in low and variable bioavailability, necessitating higher doses that may elevate the risk of side effects and reduce formulation efficiency. mefenamic acid, a commonly used non-steroidal antiinflammatory drug (nsaid), exemplifies these challenges. despite its therapeutic utility in treating pain and inflammation, mefenamic acid exhibits limited solubility in aqueous environments, leading to erratic absorption profiles and requiring frequent or high-dose administration (ghyadh & al-khedairy, 2023; vasanani et al., 2016). these limitations not only reduce patient adherence but also raise safety concerns due to potential gastrointestinal and renal adverse effects (pervaiz et al., 2023; sanas & pachpute, 2023). several formulation strategies have been explored to address solubility-related issues in bcs class ii drugs, including particle size reduction, lipid-based delivery systems, and solid dispersion techniques (doshi et al., 2024; fontana et al., 2018). among these, solid dispersion technology has shown significant promise by enhancing drug dissolution through alterations in solidstate properties (h. kumar et al., 2022; neeharika & jyothi, 2021). by dispersing a poorly soluble drug in an inert carrier matrix, this approach often facilitates reduced crystallinity or complete amorphization of the active pharmaceutical ingredient (api), thereby improving wettability, surface area, and the thermodynamic driving force for dissolution. within the spectrum of solid dispersions, crystalline solid dispersions (csds) represent an underexplored yet valuable subclass. unlike amorphous solid dispersions that eliminate crystallinity, csds retain partial or complete crystalline order while achieving improved dissolution through spatial distribution and drug–carrier interactions (baghel et al., 2016; a. kumar, 2017). this https://doi.org/10.14421/biomedich.2025.142.601-607 602 biology, medicine, & natural product chemistry 14 (2), 2025: 601-607 partial retention of crystallinity offers greater physical stability, minimizing the risk of recrystallization—a common drawback of amorphous systems (babu & nangia, 2011). although csds may not always achieve the same level of solubility enhancement as fully amorphous dispersions, they provide a favorable compromise between performance and shelf stability (thayyil et al., 2020). the choice of an appropriate co-former or matrix is central to the success of csds. while polymers like polyvinylpyrrolidone (pvp) and polyethylene glycol (peg) are commonly used, low-molecular-weight coformers such as isonicotinamide (ina) have recently gained attention due to their capacity for hydrogen bonding, gras status, and physicochemical compatibility (garbiec et al., 2023; turek et al., 2021). ina has been reported to enhance solubility and inhibit recrystallization in co-amorphous systems and cocrystals by stabilizing the high-energy non-crystalline phase of the drug (suknuntha et al., 2023). although several studies have demonstrated the potential of ina in enhancing the solubility of other bcs class ii drugs, limited research has specifically investigated its use in forming crystalline solid dispersions with mefenamic acid—particularly when no new crystalline phase is detectable via pxrd. this presents a unique opportunity to explore whether dissolution enhancement can still occur through molecular-level interactions without visible amorphization or co-crystal formation. this study aims to investigate the physicochemical characteristics and dissolution behavior of mefenamic acid–isonicotinamide crystalline solid dispersions prepared via solvent evaporation. through pxrd, dsc, ftir, and dissolution analysis, this research explores whether such systems can effectively improve solubility, offering a novel strategy for formulating poorly watersoluble nsaids. materials and methods materials mefenamic acid (mefa) was obtained from pt. kimia farma tbk (indonesia) as the active pharmaceutical ingredient (api). isonicotinamide (ina), used as a lowmolecular-weight co-former, was procured from merck (germany). all other solvents and reagents were of analytical grade and used without further purification. distilled water was used throughout the experiments for solubility and dissolution studies. instruments the solid-state characterization of the samples was conducted using several instruments. differential scanning calorimetry (dsc) analysis was performed with a shimadzu dsc-60 plus (japan), calibrated using the indium standard, operating in a nitrogen atmosphere. powder x-ray diffraction (pxrd) was carried out using a panalytical x’pert pro diffractometer (netherlands) with cu kα radiation (λ = 1.5406 å), operating at 40 kv and 30 ma. scanning was performed over a 2θ range of 5–40°. fourier-transform infrared (ftir) spectroscopy was performed using a bruker alpha ii ftir spectrometer (germany) in the 4000–500 cm⁻¹ range using the kbr pellet method. dissolution testing was conducted using a usp type ii paddle apparatus (logan instruments corp., usa) at 75 rpm. uv-visible spectrophotometry was conducted on a shimadzu uv1800 (japan) for drug quantification at 285 nm. preparation of crystalline solid dispersions csds of mefa and ina were prepared using the solvent evaporation method, which allows for homogeneous mixing and distribution of drug and coformer molecules (chauhan et al., 2005; xie et al., 2009). the drug and ina were accurately weighed in molar ratios of 1:1, 1:2, and 2:1, then dissolved in a minimal amount of ethanol as the common solvent. the resulting solutions were stirred magnetically at room temperature for 1 hour to ensure complete mixing. the solvent was evaporated under reduced pressure using a rotary evaporator at 40 °c. the dried residues were further desiccated and pulverized into fine powder using a mortar and pestle, then stored in airtight containers until further analysis. this method was selected due to its ability to promote molecular-level interactions without thermal degradation, which is particularly suitable for thermolabile compounds. ftir analysis fourier-transform infrared spectroscopy (ftir) evaluated the potential intermolecular interactions between mefa and ina within the solid dispersions. the samples were mixed with potassium bromide (kbr) and compressed into pellets. the ftir spectra were recorded in the 4000–400 cm⁻¹ region. shifts in characteristic peaks of functional groups were interpreted to detect hydrogen bonding or other non-covalent interactions, which may indicate successful dispersion or complex formation (liu et al., 2015; wicaksono et al., 2019). dsc analysis differential scanning calorimetry (dsc) was employed to investigate the prepared solid dispersions’ thermal properties and crystalline behavior compared to the pure drug and co-former. approximately 2–5 mg of each sample was weighed and placed in sealed aluminum pans. samples were scanned from 30 °c to 250 °c at a heating rate of 10 °c/min under a nitrogen atmosphere (alatas et al., 2021; nandi et al., 2021). the appearance or disappearance of endothermic peaks was used to assess melting point depression, crystalline transitions, and potential amorphization (wdowiak et al., 2024). indra et al. – crystalline solid dispersion for mefenamic acid 603 dissolution testing the dissolution rate of mefa in solid dispersions and pure form was assessed using a usp type ii dissolution apparatus with 900 ml of phosphate buffer (ph 7.4) at 37 ± 0.5 °c and 75 rpm. samples equivalent to 50 mg of mefa were placed in the dissolution vessel. at predetermined intervals (10, 20, 30, 45, and 60 minutes), 5 ml aliquots were withdrawn and filtered immediately. the withdrawn volume was replaced with a fresh medium. drug content was analyzed spectrophotometrically at 285 nm. this test was critical for evaluating the dissolution enhancement resulting from solid dispersion formulation (miller et al., 2012; park et al., 2018). results and discussion the physicochemical characterization of csds of mefa with ina prepared via the solvent evaporation method was conducted through dsc, ftir, pxrd, and dissolution testing. these analytical techniques provided critical insights into the solid-state transformations, interactions, and biopharmaceutical behavior of the formulations. thermal behavior (dsc analysis) differential scanning calorimetry (dsc) was conducted to investigate the thermal behavior and physical state of mefa in crystalline solid dispersions formulated with varying molar ratios of the small-molecule co-former ina. as shown in figure 1, pure mefa (sample a) exhibited a sharp endothermic peak at approximately 230 °c, corresponding to its melting point and confirming its crystalline nature, consistent with previously reported data (nandi et al., 2021). in contrast, solid dispersion samples (b–e) demonstrated a gradual reduction in the intensity and sharpness of this melting endotherm, particularly in sample c (1:2 ratio), where the peak nearly disappeared. figure 1. dsc thermograms of mefa (a), ina (b), and csds with ratios 1:1 (c), 1:2 (d), and 2:1 (e). the diminished and broadened thermal signals indicate a significant reduction in crystallinity, suggestive of partial amorphization or molecular-level interaction between mefa and ina. notably, samples d and e exhibited a single broad glass transition temperature (tg), indicative of a homogenous molecular dispersion and good miscibility between the drug and the small-molecule co-former. the presence of a single tg— without any distinct melting peak—suggests the formation of a metastable solid phase, potentially coamorphous or weakly crystalline, stabilized through intermolecular hydrogen bonding. this behavior underscores the role of the co-former ratio in modifying solid-state properties. as the proportion of ina increased, the system transitioned from a crystalline to a more amorphous or molecularly integrated state. these findings are consistent with previous reports, such as those by fitriani et al. (2017), demonstrating that enhanced co-former interaction facilitates the disordering of the drug lattice in nonpolymeric matrices. intermolecular interactions (ftir analysis) ftir spectroscopy assessed the molecular interactions between mefa and its co-formers. the spectra in figure 2 show significant shifts and changes in peak characteristics across different formulations. the pure drug (a) exhibited distinct absorption bands corresponding to its functional groups: the n–h stretching band around 3300 cm⁻¹, c=o stretching at ~1650 cm⁻¹, and aromatic c–h bending near 750 cm⁻¹. upon forming solid dispersions (samples b through d), notable shifts and reductions in the intensity of the o–h and n–h stretching vibrations were observed. in particular, the broadening and red-shifting of these bands suggest the formation of intermolecular hydrogen bonds between the carboxylic or amine groups of mefa and the functional groups of the co-former (liu et al., 2015; oyama et al., 2024). such interactions imply that the drug has been effectively incorporated into the polymer matrix at a molecular level. figure 2. ftir spectra of mefa (a), and their solid dispersions in ratios 1:1 (b), 1:2 (c), and 2:1 (d). 604 biology, medicine, & natural product chemistry 14 (2), 2025: 601-607 moreover, the disappearance or weakening of sharp carbonyl peaks in the dispersion spectra, particularly in sample c, supports the hypothesis of drug–co-former interaction. this is further corroborated by the appearance of new absorption bands in the fingerprint region (~1000–1500 cm⁻¹), which are absent in the spectra of the physical mixture, indicating that molecular-level interactions—likely hydrogen bonding— occurred during the preparation of the dispersions (guedes et al., 2011; qian et al., 2015). crystalline behavior (pxrd analysis) pxrd analysis, illustrated in figure 3, was utilized to investigate the crystallinity of the prepared systems further. the diffractogram of pure mefa (sample a) shows sharp, intense peaks at characteristic 2θ values, confirming its highly crystalline nature. however, these peaks are markedly diminished or absent in the solid dispersions (samples b to e), particularly in sample c (1:2 ratio), which displays a broad halo pattern instead of defined peaks, indicating a substantial reduction in crystallinity and transformation to an amorphous state. figure 3. pxrd patterns of mefa (a), ina (b), and solid dispersions in ratios 1:1 (c), 1:2 (d), and 2:1 (e). the progressive disappearance of these peaks with increasing co-former ratio reflects the successful formation of amorphous solid dispersions and confirms the dsc findings. according to sapkal et al. (2018), such reductions in diffraction peak intensity denote the drug’s molecular dispersion into the co-former, enhancing its solubility due to the loss of crystal lattice energy. additionally, the new halo patterns appearing in samples c and d may suggest forming a new amorphous phase or co-amorphous system. these findings are consistent with previous studies on etoricoxib and famotidine solid dispersions, where reduced or absent pxrd peaks signified enhanced dispersion and amorphization (chauhan et al., 2005; fitriani et al., 2017). furthermore, the presence of only low-intensity broad peaks, without the sharp reflections typical of a crystalline phase, strongly supports the solidstate transformation from a crystalline to an amorphous form. in vitro dissolution study the dissolution profiles of pure mefenamic acid (mefa) and its crystalline solid dispersions with isonicotinamide (ina) in varying molar ratios are illustrated in figure image.png. pure mefa exhibited poor dissolution, reaching only about 23.16% drug release after 60 minutes, consistent with its known low aqueous solubility. in contrast, all mefa:ina dispersions demonstrated enhanced dissolution, with the 1:2 ratio achieving the highest release, approximately 58.51% within 60 minutes. figure 4. dissolution profiles of mefa and its solid dispersions with ina at molar ratios of 1:1, 1:2, and 2:1. the 1:2 system significantly outperformed the 1:1 (37.53%) and 2:1 (25.80%) ratios, underscoring the importance of co-former concentration in facilitating drug solubilization. this enhancement correlates with findings from pxrd and dsc analyses, which indicated a shift from a crystalline to a more amorphous or molecularly dispersed state. the absence of distinct melting endotherms and the appearance of a broad glass transition in dsc thermograms suggest that the drug is well integrated at the molecular level. the improved solubility and dissolution profiles observed across the solid dispersions can be attributed to the synergistic effects of molecular dispersion, hydrogen bonding, and reduced crystallinity. the ftir spectra demonstrated significant intermolecular interactions, particularly in the 1:2 system, where characteristic drug peaks were either shifted or suppressed. dsc confirmed the transition from crystalline to amorphous form through the disappearance of melting endotherms and the appearance of a single tg. pxrd further corroborated these findings by showing decreased intensity in the same samples. these solid-state modifications have been shown to contribute to enhanced dissolution rates directly, a trend indra et al. – crystalline solid dispersion for mefenamic acid 605 consistent with prior studies on poorly soluble nsaids, such as etoricoxib, naproxen, and fenofibrate (liu et al., 2015; mardiyanto et al., 2020; sharma et al., 2017). moreover, the increased dissolution rate observed in the optimal 1:2 formulation illustrates the importance of identifying and applying the appropriate co-former ratio to improve maximal solubility. together, these findings substantiate the role of solid dispersion as an effective strategy for enhancing the bioavailability of poorly soluble drugs such as mefa. they also highlight the necessity of comprehensive solidstate characterization to elucidate the mechanisms behind dissolution enhancement and to inform formulation development decisions. conclusions this study demonstrates the effectiveness of csds using solvent evaporation and co-grinding techniques to enhance the solubility and dissolution rate of mefa. among the formulations tested, the 1:2 drug-to-ina ratio produced the most significant improvement in dissolution, supported by comprehensive physicochemical analyses. differential scanning calorimetry revealed the disappearance of the drug’s melting peak and the appearance of a single glass transition temperature, indicating reduced crystallinity and enhanced miscibility. pxrd patterns showed a broad halo, confirming partial or complete amorphization, while ftir spectra suggested the formation of intermolecular hydrogen bonds between the drug and the co-former. these findings underscore the critical role of smallmolecule co-former ratios in modulating solid-state properties and improving drug performance. importantly, this study expands the application of csds beyond polymeric systems, highlighting the potential of ina as a non-polymeric solubility enhancer. the analytical data collectively confirm the successful formation of molecularly dispersed systems that resist recrystallization and promote rapid dissolution. future investigations should evaluate long-term physical stability, in vivo bioavailability, and alternative gras-status co-formers or surfactants to further optimize formulation performance for poorly soluble non-steroidal anti-inflammatory drugs such as mefa. acknowledgements: the authors would like to express their sincere gratitude to the institute for research and community service and the department of pharmacy, universitas bakti tunas husada, for their valuable support and facilitation throughout this research. their contributions in providing resources, guidance, and infrastructure were instrumental to the successful completion of this study. authors’ contributions: indra, ade yeni aprillia, & nurul apriani designed the study. nurul apriani and winda trisna wulandari carried out the laboratory work. indra, gatut ari wardani & firman gustaman analyzed the data. indra, nurul apriani, winda trisna wulandari & firman gustaman wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research received no external funding. references alatas, f., suwartiningsih, n., ratih, h., & sutarna, t. h. 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(2019). preparation and characterization of a novel cocrystal of atorvastatin calcium with succinic acid coformer. indonesian journal of chemistry, 19(3), 660. https://doi.org/10.22146/ijc.35801 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 123-127 | doi: 10.14421/biomedich.2025.141.123-127 issn 2540-9328 (online) synergistic anti-biofilm activity of klanceng honey and probiotics against candida albicans budhi setiawan1, masfufatun2,*, lusiani tjandra3, kartika ishartadiati4 1,3pharmacology department; 2biochemistry department; 4parasitology deparment, faculty of medicine, universitas wijaya kusuma surabaya jl. dukuh kupang xxv no 54 dukuh kupang 60225, dukuh pakis, surabaya, indonesia. corresponding author* masfufatun@uwks.ac.id manuscript received: 03 january, 2025. revision accepted: 08 may, 2025. published: 14 may, 2025. abstract candida albicans is a common cause of fungal infections, especially in hospitals. biofilm formation by this fungus is a significant problem as it leads to increased drug resistance and complications in treatment. the biofilm's extracellular matrix protects the fungal cells, making it difficult for antifungal drugs to penetrate and clear the infection. aim: this study investigates the potential of honey and probiotic formulations to inhibit the biofilm matrix formation in candida albicans. result: klanceng honey showed the strongest inhibition of candida albicans biofilm formation, reducing it by 89.39% (od = 0.34 ± 0.10), compared to yogurt (29.43%, od = 2.25 ± 0.16) and yakult (67.95%, od = 1.02 ± 0.25) (p < 0.05). when mixed with yogurt in a 5:1:2 ratio (honey:yogurt: water), klanceng honey reduced biofilm formation by 84.92% (od = 0.50 ± 0.12). manuka and melifera honey in the same ratio also showed strong inhibition, reducing biofilm formation by 77.84% (od = 0.73 ± 0.08) and 90.52% (od = 0.31 ± 0.07), respectively. this performance exceeded fluconazole’s, which achieved a 65.52% reduction (od = 1.14 ± 0.02). for all three honey types, the 4:1:3 and 2:1:5 ratios (honey:yakult: water) were more effective, with reductions of up to 80.96% for melifera and 75.64% for klanceng (p < 0.05). conclusion: this research suggests that honey and probiotic formulations hold promise as natural alternatives in combating candida albicans infections. the study found that the ratio of honey to probiotics in the formulations significantly influenced their effectiveness. keywords: klanceng; honey; yogurt; probiotic; antibiofilm. introduction the epidemiology of candida albicans infections reveals their pervasive impact on immunocompromised and nonimmunocompromised individuals globally. candida albicans is notably the leading cause of invasive candidiasis, especially in nosocomial (hospital-acquired) settings, accounting for around 55% of cases in various healthcare facilities (şanlı et al., 2024; kreitmann et al., 2024). predisposing factors for candidiasis include prolonged antibiotic use, central venous catheters, and immunosuppression from conditions such as cancer or post-surgery recovery (oliva & venditti., 2024; de almeida et al., 2024). studies also highlight an evolving epidemiology with an increasing prevalence of nonalbicans candida species due to antifungal resistance, particularly among long-term patients (saeed et al., 2024). understanding these epidemiological patterns is critical for advancing diagnostic and therapeutic interventions, given the heightened morbidity and mortality rates associated with candida albicans infections in vulnerable patient populations. biofilm formation in candida albicans significantly contributes to its pathogenicity and resistance to antifungal treatments. these biofilms, complex structures composed of yeast and hyphal cells embedded in a protective extracellular matrix, enhance resistance to antifungal agents by limiting drug penetration and fostering genetic adaptation (chamtouri et al., 2024; waykar & kumarapillai, 2024). these biofilms complicate infections, particularly in clinical settings where c. albicans adhere to indwelling medical devices, leading to persistent infections and heightened resistance, especially against azoles and echinocandins (alvarez et al., 2024). biofilm-associated cells demonstrate increased expression of resistance genes and can endure host immune responses, making infections challenging to manage (makled et al., 2024). moreover, synergistic treatment strategies, such as combining antifungals with biofilm-disrupting agents, have shown promise but are not yet widespread in clinical practice (sun et al., 2024). understanding the mechanisms of biofilm formation and resistance is thus crucial for developing more effective therapeutic approaches. honey and probiotics demonstrate significant potential as antibiofilm agents against candida albicans, effectively disrupting biofilm formation and enhancing antimicrobial effectiveness. honey, with its high https://doi.org/10.14421/biomedich.2025.141.123-127 124 biology, medicine, & natural product chemistry 14 (1), 2025: 123-127 osmolarity, acidity, and rich polyphenol content, creates an inhospitable environment for candida biofilm development. studies have shown that honey prevents candida adhesion on surfaces and destabilizes existing biofilms (acaroz et al., 2024). when combined with probiotic strains like lactobacillus spp., honey’s antibiofilm effects are amplified, as probiotics themselves release metabolites that interfere with candida biofilm matrices (machado et al., 2023). probiotic bacteria also produce biosurfactants that disrupt biofilm integrity, preventing candida cells from establishing resilient communities on host surfaces (luca et al., 2024). these combined approaches offer promising, natural antibiofilm strategies that could reduce reliance on conventional antifungal drugs. klanceng honey, also known as stingless bee honey, is a unique native indonesian honey produced by tetragonula species (stingless bees). klanceng honey is produced in limited quantities, making it rarer and often more valued. the lower honey yield and traditional harvesting techniques contribute to its exclusivity and cultural significance in indonesia. while it is traditionally valued in indonesian folk medicine for its health benefits, scientific research on klanceng honey, especially regarding its antifungal efficacy, remains limited. existing studies primarily focus on its antibacterial properties and bioactive components, like phenolic compounds, which suggest potential anti-fungal effects. however, detailed studies on its specific efficacy against fungal pathogens such as candida albicans or dermatophytes are scarce. this gap underscores the need for further research to fully explore and validate the antifungal potential of klanceng honey, which could reinforce its role as a natural alternative in managing fungal infections. therefore, this study aims to provide a comparative analysis of the inhibition effect of klanceng honey and probiotics formulation on the biofilm matrix of candida albicans. materials and methods regeneration and preparation of c. albicans inoculum a single isolate from an aged sda medium was collected using an inoculating loop and streaked onto a fresh sda medium. the sda medium was then incubated at 37°c for 2–3 days to allow the formation of a single c. albicans colony. the single c. albicans colony grown on the new sda medium was inoculated into an erlenmeyer flask containing 10 ml of sdb. the flask was shaken for 18– 20 hours at a speed of 120 rpm, resulting in a turbid candida suspension, referred to as the inoculum. preparation of c. albicans suspension the inoculum obtained was transferred into a centrifuge tube and centrifuged for 15 minutes to form a pellet. the pellet was separated from the filtrate and resuspended in pbs (phosphate-buffered saline). this centrifugation and resuspension process was repeated twice. the pellet obtained from the third centrifugation was resuspended in rpmi medium, and the optical density (od) was measured at 595 nm using a microplate reader. if the od of the c. albicans suspension exceeded 0.5, dilution was performed until an od of 0.5 (equivalent to 1 × 10⁶ cfu/ml) was achieved. this c. albicans suspension was then ready for antibiofilm testing (fauzan et al., 2023). biofilm matrix formation test 100 µl of c. albicans suspension was added to each test and control well (except for blank wells, which were replaced with 100 µl of rpmi medium) and incubated at 37°c for 2 hours. after incubation, each well was washed twice with pbs, and 100 µl of the treatment solution was added to each test well. the treatment solutions were prepared with three different ratios of honey: yogurt or yakult: medium, specifically 5:1:2, 4:1:3, and 2:1:4. for the blank and negative control wells, 100 µl of rpmi medium was added, while the positive control wells received 100 µl of fluconazole solution. the microplate was then incubated at 37°c for 48 hours. after the 48-hour incubation, the non-adherent planktonic cells were discarded, and the wells were rinsed with pbs. next, 100 µl of methanol was added to each well and incubated for 15 minutes at room temperature. the remaining methanol was discarded, and the microplate was air-dried briefly. then, 100 µl of 0.1% crystal violet (cv) was added to each well and incubated for 15 minutes. excess cv was discarded, and the wells were rinsed with pbs. finally, 100 µl of 96% ethanol was added to each well, and the optical density (od) was measured at λ = 595 nm. data analysis the research data were analyzed using spss (statistical product and service solutions) statistical software. for sample sizes less than 50, the shapiro-wilk test was used for normality assessment. homogeneity was tested using levene's test if the data were normally or near-normally distributed. one-way anova was employed to evaluate differences in c. albicans growth across variations in honey: yogurt or yakult: medium (distilled water) composition. if the data were not normally distributed or not homogeneous, the non-parametric kruskal-wallis test was applied. post hoc testing was conducted to identify which test groups exhibited significant differences. setiawan et al. – synergistic anti-biofilm activity of klanceng honey … 125 results and discussion result klanceng honey most effectively inhibited candida albicans biofilm formation (0.34 ± 0.10), showing an 89.39% decrease compared to the control (3.19 ± 0.14). in comparison with control, yogurt and yakult also showed significant inhibition, with decreases of 29.43% (2.25 ± 0.16) and 67.95% (1.02 ± 0.25) respectively. in the same concentration of 50%, combining klanceng honey with yogurt or yakult (83.57% and 74.80% decreases, respectively) did not enhance its effect. table 1. inhibition of candida albicans biofilm formation by klanceng honey and yogurt formulations. mixture (honey: probiotic: water) od mean ± sd biofilm proportion (%) biofilm decrease (%) control 3.29 ± 0.08a 100.00 0.00 fluconazole 1.14 ± 0.02c 34.48 65.52 types of honey manuka (5:1:2) 0.73 ± 0.08d 22.16 77.84 (4:1:3) 1.34 ± 0.15b 40.58 59.42 (2:1:5) 1.11 ± 0.08c 33.81 66.19 melifera (5:1:2) 0.31 ± 0.07f 9.48 90.52 (4:1:3) 1.07 ± 0.02c 32.56 67.44 (2:1:5) 0.39 ± 0.04e,f 11.70 88.30 klanceng (5:1:2) 0.50 ± 0.12e 15.08 84.92 (4:1:3) 0.73 ± 0.08d 22.03 77.97 (2:1:5) 0.71 ± 0.05d 21.62 78.38 in combination with yogurt, klanceng honey most effectively inhibited candida albicans biofilm formation, with the 5:1:2 (honey:yogurt:water) mixture achieving an 84.92% reduction (od = 0.50 ± 0.12). this surpassed the antifungal fluconazole (65.52% reduction, od = 1.14 ± 0.02). with the 5:1:2 ratio, manuka and melifera honey also showed relatively higher inhibition (77.84% reduction, od = 0.73 ± 0.08; 90.52% reduction, od = 0.31 ± 0.07 respectively) than other mixture ratios. therefore, this combination of honey, yogurt, and water especially in the 5:1:2 mixture ratio, exhibited the most substantial inhibitory effect on candida albicans biofilm formation, even surpassing the antifungal fluconazole in some instances. table 2. inhibition of candida albicans biofilm formation by honey and yakult formulations. mixture (honey: probiotic: water) od mean ± sd biofilm proportion (%) biofilm decrease (%) control 3.19 ± 0.10a 100 fluconazole 1.08 ± 0.08d 34.04 65.96 types of honey manuka (5:1:2) 0.73 ± 0.08d 51.46 48.54 (4:1:3) 1.34 ± 0.15b 43.811 56.19 (2:1:5) 1.11 ± 0.08c 20.27 79.73 melifera (5:1:2) 0.31 ± 0.07f 27.70 72.30 (4:1:3) 1.07 ± 0.02c 19.04 80.96 (2:1:5) 0.39 ± 0.04e,f 19.57 80.43 klanceng (5:1:2) 0.50 ± 0.12e 46.05 53.95 (4:1:3) 0.73 ± 0.08d 24.36 75.64 (2:1:5) 0.71 ± 0.05d 25.12 74.88 126 biology, medicine, & natural product chemistry 14 (1), 2025: 123-127 across all three honey types (manuka, melifera, and klanceng), the 4:1:3 and 2:1:5 (honey:yakult:water) ratios consistently demonstrated significantly greater inhibition of candida albicans biofilm formation compared to the 5:1:2 ratio. this trend was observed for manuka honey (56.19% and 79.73% reduction for 4:1:3 and 2:1:5 vs. 48.54% for 5:1:2), melifera honey (80.96% and 80.43% reduction for 4:1:3 and 2:1:5 vs. 72.30% for 5:1:2), and klanceng honey (75.64% and 74.88% reduction for 4:1:3 and 2:1:5 vs. 53.95% for 5:1:2). this suggests that a lower proportion of honey in the mixture, relative to yakult and water, enhances the inhibitory effect on biofilm formation. discussion the study demonstrates the inhibitory potential of klanceng honey combined with probiotics on the biofilm matrix of candida albicans, highlighting a natural approach to addressing anti-fungal resistance associated with biofilm formation. among the honey types tested, melifera honey exhibited the highest biofilm inhibition, reducing biofilm formation by up to 90.52% at the 5:1:2 ratio in combination with water and yogurt. this effect surpassed the inhibition of fluconazole, the positive control, which achieved a 65.52% reduction. the results confirm the synergy between honey's inherent bioactive compounds and probiotic metabolites in destabilizing the extracellular biofilm matrix. the biofilm matrix's resistance to antifungal treatments is primarily attributed to its ability to limit drug penetration and foster genetic resistance mechanisms (chamtouri et al., 2024; waykar & kumarapillai, 2024). honey's rich polyphenolic content and acidity create a hostile environment for candida albicans, disrupting adhesion and biofilm integrity (acaroz et al., 2024). klanceng honey, although less effective than melifera, demonstrated significant inhibition (84.92%) at the 5:1:2 ratio, suggesting its potential as a natural antifungal agent despite its lower yield and limited scientific exploration. the findings emphasize the importance of optimizing honey-toprobiotic ratios to maximize antifungal efficacy. probiotics play a critical role by producing biosurfactants and organic acids that interfere with biofilm formation and enhance the antimicrobial effects of honey. lactobacillus strains are known for their ability to disrupt biofilms and prevent the establishment of resilient microbial communities (luca et. al., 2024). these findings align with previous studies that demonstrating that combining natural antimicrobials can offer a dual approach: directly inhibiting biofilm formation and reducing the dependence on conventional antifungal agents. this research investigated the effectiveness of different honey types and mixtures with yogurt or yakult in inhibiting candida albicans biofilm formation. klanceng honey alone showed the strongest inhibitory effect (89.39% reduction of control), followed by the combination with water and yogurt in a 5:1:2 ratio (84.92%) and the mixture with water and yakult in a 4:1:3 ratio (75.54%). interestingly, combining klanceng honey with yogurt or yakult with the same 50% concentration did not significantly enhance its inhibitory effect. further investigation explored the impact of varying honey: yogurt: water ratios. when combined with yogurt, klanceng honey again demonstrated significant inhibition, particularly with the 5:1:2 ratio, achieving an 84.92% reduction in biofilm. this even surpassed the antifungal fluconazole (65.52% reduction). notably, this 5:1:2 ratio also proved most effective for manuka and melifera honey, yielding 77.84% and 90.52% reductions respectively. these findings suggest that: klanceng honey is a potent inhibitor of candida albicans biofilm. the ratio of honey to yogurt and water significantly influences the inhibitory effect. a 5:1:2 ratio of honey: yogurt: water maximizes biofilm inhibition for klanceng and melifera honey. this highlights the potential of honey, especially klanceng honey in combination with yogurt, as a natural alternative or adjunct to conventional antifungal treatments. further research is needed to elucidate the underlying mechanisms and clinical implications. notably, the study underscores the variability in the efficacy of honey and probiotic formulations based on the ratio of the components. for instance, melifera honey’s performance peaked at the 5:1:2 ratio but diminished with lower concentrations, indicating the critical role of honey's active compounds in achieving optimal results. the lower efficacy of klanceng honey at higher dilution ratios highlights the need for further research to understand the impact of its specific bioactive components and their interactions with probiotics. although the results indicate that melifera honey demonstrated the highest inhibition of candida albicans biofilm formation at the 5:1:2 ratio (90.52% reduction), klanceng honey remains a primary focus due to its unique attributes and broader implications for antifungal therapy. klanceng honey, derived from stingless bees native to indonesia, has significant cultural and medicinal value, making it a relevant candidate for local natural remedies. furthermore, klanceng honey demonstrated remarkable consistency across various formulations, achieving an 89.39% inhibition and up to 84.92% in combination with yogurt, comparable to or exceeding those of fluconazole. this positions klanceng honey as an accessible and effective alternative for antifungal applications, particularly in resource-limited settings where it is locally available. klanceng honey's rare status and limited scientific exploration highlight its importance as a candidate for further research and development. by focusing on klanceng honey, the study not only underscores its potential but also advocates for setiawan et al. – synergistic anti-biofilm activity of klanceng honey … 127 exploring underutilized natural resources in combating anti-fungal resistance. conclusions this study highlights the promising potential of klanceng honey and probiotics as natural, effective alternatives to conventional antifungal therapies. future investigations should focus on elucidating the molecular mechanisms underlying their synergistic effects, evaluating their efficacy in vivo, and exploring the potential for scaling these formulations for clinical application. by addressing the growing challenge of antifungal resistance, such natural approaches could complement existing therapies and improve outcomes in managing candida albicans infections acknowledgements: special gratitude goes to the department of biochemistry, university of wijaya kusuma surabaya, and the infectious hospital university of airlangga. authors’ contributions: masfufatun designed the study. lusiani tjandra carried out the laboratory work. kartika ishartadiati analyzed the data. budhi setiawan wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the author would like to express his deepest gratitude to universitas wijaya kusuma surabaya for the support and funding provided for the implementation of this research with contract number 87/lppm/uwks/iii/2024 references acaroz, u., kurek-gorecka, a., olczyk, p., tas, n., ali, a., paramanya, a., ... & jin, x. (2024). the role of bee products in the control of antimicrobial resistance and biofilm formation. kafkas universitesi veteriner fakultesi dergisi, (2). link. alvarez, l., kumaran, k. s., nitha, b., & sivasubramani, k. (2024). evaluation of biofilm formation and antimicrobial susceptibility (drug resistance) of candida albicans isolates. brazilian journal of microbiology, 1-12. link chamtouri, m., merghni, a., miranda-cadena, k., sakly, n., gaddour, n., de los reyes-gavilán, c. g., ... & quindós, g. (2024). characterization of yeast isolated from the gut microbiota of tunisian children with autism spectrum disorder. journal of fungi, 10(11), 730. link de almeida, b. l., arcieri, v. c., razente, d. m., freire, m. p., guimarães, t., araújo, e. d. m. p. a. d., ... & magri, m. m. c. (2024). intra‐abdominal candidiasis in cancer patients: a 10‐year experience in a middle‐income country. mycoses, 67(10), e13807. link kreitmann, l., blot, s., & nseir, s. (2024). invasive fungal infections in non-neutropenic patients. intensive care medicine, 1-5. link luca, l., pauliuc, d., & oroian, m. (2024). honey microbiota, methods for determining the microbiological composition and the antimicrobial effect of honey–a review. food chemistry: x, 101524. link. machado, a., zamora-mendoza, l., alexis, f., & álvarez-suarez, j. m. (2023). use of plant extracts, bee-derived products, and probiotic-related applications to fight multidrug-resistant pathogens in the post-antibiotic era. future pharmacology, 3(3), 535-567. link. makled, a. f., ali, s. a., labeeb, a. z., salman, s. s., shebl, d. z., hegazy, s. g., & sabal, m. s. (2024). characterization of candida species isolated from clinical specimens: insights into virulence traits, antifungal resistance and molecular profiles. bmc microbiology, 24(1), 388. link oliva, a., & venditti, m. (2024). invasive candidiasis in surgery. in infections in surgery: prevention and management (pp. 107-116). cham: springer nature switzerland. link saeed, n. k., almusawi, s., & al-beltagi, m. (2024). candidemia chronicles: retrospective analysis of candidemia epidemiology, species distribution, and antifungal susceptibility patterns in bahrain. world journal of virology, 13(4). link şanlı, k., arslantaş, e., ceylan, a. n., öncel, b., özkorucu, d., & özkan karagenç, a. (2024). candidemia in pediatric-clinic: frequency of occurrence, candida species, antifungal susceptibilities, and effects on mortality (2020–2024). diagnostics, 14(20), 2343. link sun, c., zhu, l., yang, l., tian, z., jiao, z., huang, m., ... & guo, g. (2024). antimicrobial peptide amp-17 induces protection against systemic candidiasis and interacts synergistically with fluconazole against candida albicans biofilm. frontiers in microbiology, 15, 1480808. link waykar, r., & kumarapillai, s. antifungal drug resistance in candida albicans: identifying novel targets for the development of effective antifungal agents. link this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 497-503 | doi: 10.14421/biomedich.2024.132.497-503 issn 2540-9328 (online) quantitative analysis, anti-inflammatory and analgesic effects of ethanol leaf extract and fractions of microsorium scolopendria (burm. f.) copel. in mice uwemedimo francis umoh1,*, ekikere ezekiel ubengama2, gina ramos3, victor chubueze onyido4 1department of pharmacognosy and natural medicine, faculty of pharmacy, university of uyo, uyo, ibom state, nigeria. 2department of pharmacognosy, faculty of pharmacy, university of calabar, calabar, cross river state, nigeria. 3department of pharmacognosy, faculty of pharmacy, madonna university, nigeria, elele, rivers state, nigeria. 4department of pharmaceutical chemistry, faculty of pharmacy, madonna university, nigeria, elele, rivers state, nigeria. corresponding author* uwemedimoumoh@uniuyo.edu.ng abstract microsorium scolopendria, a fern was studied for phytochemicals, anti-inflammatory, and analgesic effects to add credence to its various folkloric applications. phytochemicals were analyzed quantitatively, anti-inflammatory experiment was carried out with egg albumin induced paw oedema and xylene-induced ear oedema models while analgesic effects were studied using formalin-induced paw licking, acetic acid-induced writhing and hot plate-induced pain models. the results revealed the presence of alkaloids, flavonoids, saponins, tannins, terpenoids, and phenols with alkaloids (14.6 %w/w) as the most predominant phytoconstituent. in anti-inflammatory experiments, m. scolopendria extract reduced oedema caused by egg albumin and xylene in a dose related manner and comparable to standard agents. also, in analgesic experiments, the extract reduced pain induced by formalin, acetic acid, and hot plate. these reductions were statistically (p≤0.05) significant. considering the fractions in all experiments, butanol and ethyl acetate fractions were prominent in anti-inflammatory effect while ethyl acetate fraction was at top in reduction of pain. this study supports the use of m. scolopendria in ethnomedicinal practice. keywords: microsorium scolopendria; quantitative analysis; anti-inflammatory; analgesic; mice. introduction microsorium scolopendria, commonly known as “wart fern” of the polypodiaceae family, studied with synonyms such as phymatosorus scolopendria, polypodium scolopendria, microsorum parksii, polypodium scolopendria, and microsorum parksii (snogan et al., 2007; wunderlin et al., 2024) is reported to be indigenous to polynesian islands of fuji, tahiti, hawaii, rapa nui, and madagascar (ramanitrahasimbola et al., 2005) and natively distributed in tropical africa, ceylon, indochina, malaysia to polynesia and australia (holttum, 1954). ethnomedicinally, it is reported to be helpful in management of asthma, inflammatory diseases, cancer, abscesses (where a paste of ground leaves is usually mix with earth from a wasp’s nest), wounds, insanity, coughing fits and as enema (fernández et al., 2011). other reported folkloric uses are; as a purgative, treatment of antibacterial, gastric and renal infections, stomach aches, gastrointestinal aches and as a diuretic. its sweet smelling fronds are used in perfumery and clothing industries (jofré et al., 2016; ho et al., 2015). its attributable properties are associated with high contents of polyphenols in the plant tissues (xia et al., 2014; shuvalov et al., 2020; wang et al., 2023). apart from its ethnomedicinal attributes, it is a valuable air-purifying ornamental plant (snogan et al., 2007). pharmacological reports have it that, it has adaptogenic and anabolic effects (hunyadi et al., 2016: ambrosio et al., 2020), neuroprotective effect (ho et al., 2007), improved cognitive impairment and protection against brain injury and antioxidant effects (xia et al., 2014; baroni et al., 2021; balada et al., 2022; wang et al., 2023). scientific data on this specie is scanty compared to reported traditional uses. more so, oral reports from the indigenous people of ikot ekpene local government area of akwa ibom state, nigeria have it that m. scolopendria is used for the management of cough, headache, stomach aches and gastrointestinal aches. this study is justified by the fact that there is no report on the anti-inflammatory and analgesic properties of m. scolopendria, thus lending scientific credence to its numerous traditional uses. manuscript received: 02 september, 2024. revision accepted: 17 october, 2024. published: 28 october, 2024. https://doi.org/10.14421/biomedich.2024.132.497-503 498 biology, medicine, & natural product chemistry 13 (2), 2024: 497-503 figure 1. m. scolopendria in a natural habitat. materials and methods plant collection and identification the plant, m. scolopendria was collected from ikot ekpene local government, akwa ibom state, nigeria. it was identified by dr. imoh imeh johnny of the department of pharmacognosy and natural medicine, faculty of pharmacy, university of uyo, uyo and authenticated by prof (mrs.) m. e bassey of the department of botany and ecological studies, faculty of science, university of uyo, uyo, nigeria. preparation of extract the leaves were separated from the stems, air-dried and reduced to powder with hammer mill. 1 kg of the powdered leaf was macerated with 70% ethanol with intermittent agitation and filtered after 72 hours. the filtrate was concentrated using a rotary evaporator and further dried in a water bath at a temperature of 40℃ to obtain an extract free from extraction solvent and stored in a refrigerator for use in the study. quantitative phytochemical screening of extract quantification of alkaloid content: the ethanol leaf extract (1mg) of m. scolopendria was dissolved in dimethyl sulphoxide (dmso) and 1ml of 2 n hcl was added, filtered, and transferred to a separating funnel. bromocresol green solution (5 ml) and phosphate buffer (5 ml) were added. the mixture was shaken with 1 ml, 2 ml, 3 ml, and 4 ml chloroform and collected in a 10 ml volumetric flask, and adjusted to the required volumes with chloroform. atropine (20, 40, 60, 80, and 100 µg/ml) as reference standard was prepared in the same manner as the test material above. the absorbance for both the test and standard solutions were determined against the reagent blank at 470 nm with uv/visible spectrophotometer and the total alkaloid content expressed as mg of atropine equivalent per gram (ae/g) of extract (fazel et al., 2008; enema et al., 2024). quantification of total flavonoid content: aluminium chloride colorimetric assay method was adopted for this study. a reaction mixture in a volumetric flask (10 ml) consisting of 1 ml of m. scolopendria extract, 4 ml of distilled water, 0.30 ml of 5 % sodium nitrite and 0.3 ml of 10 % aluminium chloride (after 5 minutes) was added and mixed thoroughly. also, after another 5 minutes, 2 ml of 1m sodium hydroxide was treated and adjusted to 10 ml with distilled water. quercetin (20, 40, 60, 80 and 100 µg/ml), prepared in the similar way as the extract was used as a reference standard. the absorbance of both the test and standard solutions was determined against the reagent blank at 510 nm using uv/visible spectrophotometer. the total flavonoid content was expressed as mg of quercetin equivalent per gram (qe/g) of extract (lee et al., 2012; enema et al., 2024). quantification of saponin content: the method described by ameen et al., (2021) was adopted for this study. 1 g of m. scolopendria leaf extract was measured into a 250 ml beaker and 100 ml of isobutyl alcohol was added. this mixture was swirled for 5 minutes, filtered, and transferred into a 100 ml beaker made up of 20 ml of 40% saturated solution of mgco3. the colourless filtrate (1 ml) was pipetted into a volumetric flask (50 ml), 2 ml of 5% fecl3 solution was added and adjusted to marked level with distilled water. this mixture was allowed to stand for 30 minutes for a blood red colour to develop. percentage saponin was calculated using the formula: % 𝑆𝑎𝑝𝑜𝑛𝑖𝑛 = 𝐴𝑆 𝑥 𝐴𝐺 𝑥 𝐷𝐹 / 𝑊𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑥 100 where: as : absorbance of sample ag : average gradient df : dilution factor wt of sample : weight of sample quantification of tannin content: the tannin constituents of m. scolopendria leaf extract were determined by a method described by rajeev (2012) using insoluble polyvinylpolypirrolidone (pvpp). to 0.1 ml of the extract (dissolved in 1% methanol), 100 mg pvpp was added and vortexed, left for 15 min at 4°c, and centrifuged for 10 min at 3,000 rpm. using the clear supernatant, the non-tannin phenolic content was determined as total phenolic content. the difference between total phenolic and non-tannin phenolic content in the ethanol leaf extract of m. scolopendria was taken as the tannin quantification. quantification of total terpenoid content: the total terpenoid was determined by the method described by elsayed et al., (2019). stock standard solution of each terpene: α-pinene, (−)-β-pinene, myrcene, (r)(+)limonene, terpinolene, linalool, α-terpineol, βcaryophyllene, α-humulene, and caryophyllene oxide was prepared in ethyl acetate. the standard terpenes were mixed and the concentration of each terpene was umoh et al. – quantitative analysis, anti-inflammatory and analgesic effects of … 499 adjusted to 1.0 mg/ml from where serial dilutions were made to prepare the individual points of the calibration curves. n-tridecane (100 µg/ ml), a c13 hydrocarbon, was selected as the internal standard (is) and added to all calibrations and sample solutions. nine calibration points ranging from 0.75 – 100 µg/ml were prepared from the previously mentioned stock standard solutions (0.75, 1.0, 2.0, 5.0, 10, 25, 50, 70, and 100 µg/ml) and n-tridecane. the concentration of is at each calibration point was 100 µg/ml and these solutions were used to construct individual terpene calibration curves. quantification of total phenol content: the total phenolic content of the fractions was determined spectrophotometrically with folin – ciocalteu reagent and the procedure was repeated thrice. to the 0.5 ml (1 mg/ml) ethanol leaf extract (0. 5 ml) of m. scolopendria, was added 2.5 ml of 10% folin ciocalteu reagent and 2 ml of na2co3 (7%). the resulting mixture was vortexed for 15 seconds and incubated at 40oc for 30 minutes for colour development. the absorbance of the samples was measured at 765 nm wavelength. for the garlic acid calibration curve, 2.5 ml of distilled water was added to different concentrations and the total phenolic content was calculated from the calibration curve and results were expressed as milligrams per garlic acid equivalent (mgga/g) dry weight of extract (kaur and kpoor, 2002). animal handling albino mice (20-30 g) were procured from the animal house of the department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, akwa ibom state, nigeria. they were kept under standard conditions, starved of food, twenty four (24) hours before the experiment and given access to only water. acute toxicity study the oecd/ocde guideline [2002] was adopted to determine the ld50. the ld50 was achieved by administering of 2000 mg/kg orally to three mice. after no mortality occurred within 24 hours, this dose was again administered to another set of three mice and observed for the manifestation of physical signs of toxicity such as writing, reduced motor activity, decreased respiration and death within twenty four hours intervals. anti-inflammatory study egg albumin-induced oedema: in this model, albino mice of either sex were randomized and divided into five groups of five animals each. group one animals were pretreated with distilled water (10 ml/kg), groups 2 to 4 were pretreated with 500 mg/kg, 1000 mg/kg and 1500 mg/kg of the ethanol leaf extract of m. scolopendria (ms), respectively, thirty (30) minutes before the induction of oedema with fresh egg albumin while group five mice were administered with 100 mg/kg of acetyl salicylic acid (asa). the linear circumference of the injected paws was measured with venier caliper at t=o and at thirty (30) minutes intervals for 5 hours following the administration of egg albumin (okokon et al.,2008; umoh et al., 2020). this experiment was repeated for the fractions: dichloromethane (dcm), ethyl acetate (etoac), butanol (but) and aqueous fractions (aq) at 1000 mg/kg. xylene-induced ear oedema: twenty five (25) albino mice were grouped into five groups of five animals per group. mice in group one were administered with distilled water (10 ml/kg), those in groups 2 to 4 (500 mg/kg, 1000 mg/kg, and 1500 mg/kg) group five mice received dexamethasone, 4 mg/kg, thirty (30) minutes before the topical application of 50 microliter of xylene to the anterior and posterior surfaces of the right ears while the left ears served as control. fifteen minutes following xylene application, the mice were sacrificed by chloroform anesthesia, both ears removed and weighed. the average weight differences between the two ears were taken to measure the inflammatory response (atta and alkofahi, 1998). analgesic study acetic acid-induced writhing in mice: albino mice of either sex were selected, divided and pretreated like the egg albumin model thirty (30) minutes before the intraperitoneal injection of 2% acetic acid. analgesic activity was expressed as reduced abdominal constrictions between control animals administered with distilled water (10 ml/kg) and mice pretreated with the extracts (nwafor and okwuasaba, 2003). this procedure was repeated for the fractions (dcm, etoac, but and aq) at 1000 mg/kg. formalin-induced paw licking in mice: this method was similar to the one described by nwafor and okwuasaba (2003). albino mice of either sex were randomized and divided into five groups of five animals each and pretreated with distilled water (10ml/kg for group 1), ethanol leaf extract of m. scolopendria (500 mg/kg, 1000 mg/kg and 1500 mg/kg for groups 2 to 4) and asa (100 mg/kg for group 5). twenty microliters of 2.5% formalin solution (formaldehyde) made up to phosphate buffer was administered subcutaneously under the surface of the right hind paw. the time the animals spent in licking the injected paw was noted and taken as an indication of pain with the first phase of response at 5 minutes and second phase (15-30 minutes) following formalin injection. this procedure was repeated for the fractions (dcm, etoac, but and aq) at a dose of 1000 mg/kg. hot plate –induced pain: the effect of the ethanol leaf extract of m. scolopendria on hot plate-induced pain was investigated using adult mice. the mice were grouped and pretreated as earlier mentioned in formalin– induced paw licking model. hot plate connected to electricity was maintained at a temperature of 45℃ ± 500 biology, medicine, & natural product chemistry 13 (2), 2024: 497-503 1℃. the mice were placed into a glass beaker of 50 cm diameter on the heated surface of the hot plate and the time(s) between placement and licking of the paw were recorded (nwafor & okwuasaba, 2003). statistical analysis data collected were expressed as mean ± standard error of the mean (sem) and significance of data taken at p≤0.05. results quantitative phytochemical screening of m. scolopendria: the result of quantitative phytochemical screening of the ethanol leaf m. scolopendria is presented in figure 1. this result revealed the various percentages (w/w) of secondary metabolites present in the ethanol extract of the m. scolopendria (figure 1). figure 1. quantitative phytochemical analysis of ethanol extract of m. scolopendria. acute toxicity study following the administration of 2000 mg/kg twice to two groups of mice with no mortality, the ld50 was determined to be 5000 mg/kg and the three doses employed for the study were 500 m/kg, 1000 mg/kg and 1500 mg/kg. anti-inflammatory study the effect of ethanol leaf extract of m. scolopendria on egg albumin-induced oedema in mice is presented in table 1 while the effect of the partitioned fractions is in table 2. the result showed the various responses of the extract and fractions to oedema caused by egg albumin between 1 hour to 5 hour intervals while the consequence of xylene-induced topical oedema in mice is presented in table 3. table 1. the effect of the ethanol extracts of m. scolopendria on egg albumin-induced oedema in mice. treatment (mg/kg) time (hours) 1 2 3 4 5 dist. water 10 mg/kg 1.66 ± 0.02 1.10 ± 0.01 0.97± 0.01 0.52± 0.02 0.52± 0.02 ms 500 0.99±0.01* 0.50±0.01* 0.33±0.01* 0.34±0.01* 0.25±0.01* ms 1000 1.12±0.01 0.57±0.01* 0.53±0.01* 0.43±0.01 0.25±0.01* ms 1500 1.02±0.01* 0.56±0.01* 0.41±0.01* 0.35±0.01* 0.30±0.01* asa 100 0.55±0.01* 0.45±0.01* 0.34±0.01* 0.24±0.01* 0.16±0.01* values are expressed as mean ± sem, where n=5 and *p≤0.05 is considered significant 0 2 4 6 8 10 12 14 16 alkaloids flavonoids saponin tannins terpenoids phenols p er ce n ta ge q u an ti fi ca ti o n ( w /w ) secondary metabolites quantitative phytochemical analysis of ethanol extracts of m.scolopendria umoh et al. – quantitative analysis, anti-inflammatory and analgesic effects of … 501 table 2. the effect of the partitioned fractions of m. scolopendria on egg albumin-induced oedema in mice. treatment (mg/kg) time (hours) 1 2 3 4 5 dist. water 10 ml/kg 1.66 ± 0.02 1.70 ± 0.01 1.97± 0.01 1.52± 0.02 0.90± 0.01 dcm 1000 1.21±0.01 1.11±0.02 0.93±0.01* 0.74±0.01* 0.58±0.01* etoac 1000 1.24±0.01 1.03 ±0.01* 0.96±0.01* 0.82±0.01* 0.69±0.01* but. 1000 0.91±0.01* 1.09±0.01* 0.99±0.01* 0.80±0.01* 0.59±0.01* aq. 1000 1.28±0.02 1.19±0.02 1.33±0.01 1. 01±0.01 0.85±0.01 asa 100 0.55±0.01* 0.45±0.01* 0.34±0.01* 0.24±0.01* 0.02±0.01* values are expressed as mean ± sem, where n=5 and *p≤0.05 is considered significant table 3. effect of ethanol extract of m. scolopendria on xylene induced oedema in mice. treatments (mg/kg) weight difference (mm) distilled water 10 ml/kg 0.059 ± 0.06 ms 500 0.036 ± 0.03* ms 1000 0.034 ± 0.03* ms 1500 0.042 ± 0.01 dexamethazone 4 0.016 ± 0.03* values are expressed as mean ± sem, where n=5 and *p≤0.05 is considered significant analgesic study the result of the effects of the ethanol leaf extract and fractions of m. scolopendria on formalin-induced paw licking is presented in tables 4 and 5 while the effect of the ethanol extract of ethanol of m. scolopendria on hot plate-induced pain is presented in table 6. table 4. the effect of the ethanol extracts of m. scolopendria on formalin-induced paw licking in mice. treatments (mg/kg) mm time (minutes) 5 10 15 20 25 30 dist. water 10 ml/kg 26.4 ± 5.60 13.00±1.05 9.80 ±2.46 6.80± 3.38 4.80± 3.95 6.60± 4.89 ms 500 14.67±0.90* 1.33±0.02* 4. 00±0.02* 2.33±0.01* 0.33±0.01* 1.33±0.02* ms 1000 10.75±0.05* 0.33±0.02* 0.00±0.00* 4.33±0.07* 1.33±0.02* 1.67±0.02* ms 1500 11.67±0.06* 3. 00±0.04* 3.67±0.05* 3.20±0.74* 2.80±1.11* 3.40±1.50* asa 100 14.60 ±1.60* 2.80 ± 1.39* 2.60 ± 0.68* 0.67±0.02* 1.00±0.03* 0.33±0.01* values are expressed as mean ± sem, where n=5 and *p≤0.05 is considered significant table 5. the effect of the partitioned fractions of m. scolopendria on formalin-induced paw licking in mice. treatments (mg/kg) time (minutes) 5 10 15 20 25 30 dist. water 10 ml/kg 26.4 ± 5.60 13. 00 ±1.05 9.80 ±2.46 6.80± 3.38 4.80± 3.95 6.60± 4.89 dcm 1000 23.33±0.90 18.60±0.10 6.33±0.10* 16.00±0.11 12.33±0.10 9.33±0.13 etoac 1000 18. 00±0.10 7.67±0.10* 4.00±0.10* 1.33±0.08* 1.66±0.10* 6.33±0.12 but. 1000 18.33±0.10 14.33±0.11 8. 00±0.90 7.33±0.02 5.33±0.12 1.33±0.06* aq. 1000 18.33±0.12 13.00±0.10 11. 00±0.40 1.00±0.02* 6.00±0.11 2.33±0.10* asa 100 14.60±1.60* 2.80 ± 1.39* 2.60 ± 0.68* 3.20±0.74* 2.80±1.11* 3.40±1.50* values are expressed as mean ± sem, where n=5 and *p≤0.05 is considered significant. table 6. the effect of ethanol extract of m. scolopendria on thermalinduced pain in mice. treatments (mg/kg) time (seconds) distilled water 10 ml/kg 11.35 ± 1.90 ms 500 18.84 ± 1.20 ms 1000 33.57 ± 1.83* ms 1500 mg/kg 29. 21± 1.50* asa 100 mg/kg 29. 21± 1.50* values are expressed as mean ± sem, where n=5 and *p≤0.05 is considered significant discussion the result of the quantitative phytochemical analysis (figure 1) of the ethanol leaf extract of m. scolopendria revealed the presence of alkaloids (14.6), flavonoids (1.42), saponins (2.35), tannins (0.91), terpenoids (0.25) and phenols (1.18). these quantifications were done spectroscopically with garlic acid used as standard for tannin and total phenolic determinations (appendices 3 and 6) and quercetin, for total flavonoid content. from the result, alkaloids were the highest, while terpenoid 502 biology, medicine, & natural product chemistry 13 (2), 2024: 497-503 constituents were the least. plants are known to owe their medicinal properties to the presence of secondary metabolites in them (babu et al., 2021). alkaloids, saponins and flavonoids are reputed for their involvement in the reduction of oedema and pains (gonfa et al., 2023; sun and shahrajabian, 2023; hassan et al., 2011). the intrinsic anti-inflammatory and analgesic abilities of the ethanol extract and fractions of m. scolopendria may be linked to these secondary metabolites. acute toxicity is the measure of the adverse effects of a substance that result either from a single exposure or from multiple exposures in a short period. using the oecd/ocde guideline (2002), 2000 mg/kg of the ethanol extract was administered to two sets of three mice without any death, and a lethal dose (ld50) of 5000 mg/kg was established. the three doses employed for the study were 500 m/kg, 1000 mg/kg and 1500 mg/kg which represented the lower, median and high doses. a lethal dose of 5000 mg/kg and above are considered practically nontoxic and therefore, the ethanol extract of m. scolopendria may be regarded as safe on a short period of administration and when given orally (erhirhie et al., 2018). in the anti-inflammatory study, the ethanol extract of m. scolopendria was able to reduce oedema caused by egg albumin in a dose dependent manner. this observed effect was statistically (p≤ 0.05) significant when compared to distilled water (10 ml/kg), and similar to that produced by acetyl salicylic acid (asa) 100 mg/kg. the partitioned fractions of the ethanol extract were also able to reduce oedema caused by egg albumin. comparing the fractions (table 4.2), the butanol fraction (but) was the most potent followed by ethyl acetate (etoac) with the least being dichloromethane fraction. the induction of oedema using egg albumin is linked to the release of histamine and serotonin. the observed anti-inflammatory effect observed with the administration of ethanol extract and fractions of m. scolopendria could be due to their ability to inhibit these two mediators of inflammation. table 4.3, which is the effect of the ethanol extracts of m. scolopendria in mice revealed that the extract at doses of 500 mg/kg and 1000 mg/kg was able to reduce topical oedema caused by xylene significantly (p≤ 0.05) when compared to distilled water and the effect was similar to that of dexamethasone (4 mg/kg). xylene as a phlogistic agent is known to cause inflammation by the action of phospholipase a2, hence the ability of the two doses of the extracts m. scolopendria in reducing oedema by xylene may be linked to their effect in blocking the release of phospholipase a2 (okokon et al., 2008; umoh et al., 2020) the effect of the ethanol extract and fractions of m. scolopendria on formalin induced paw licking in mice (tables 4 and 5) revealed that the extract was able to reduce the number of times the mice licked their paws at the first five minutes and even throughout the entire duration of the experiment. this reduction was significant (p≤ 0.05) compared to distilled water and asa, a standard drug. although at time (5 minutes), the extract’s action looked better than that of asa, this was not sustained from time (10 -30 minutes). for the fractions, the ethyl acetate fraction was more potent than the aqueous fraction. considering the thermal pain induction using a hot plate, the extract also prolonged the time the mice stayed on the hot plate in a dose-dependent manner significantly (p≤0.05) when compared to distilled water and similar to asa. the mechanism of formalin pain is both neurogenic and inflammatory while that of hot plate gives information on their ability to behave as narcotics. thus, the capacity of the ethanol extract and fractions on m. scolopendria to reduce 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(asparagaceae). nig j pharm and app sci res 9(2): 4046 wang x, dong l, ma c, wang z, hu x, chen f. 2023. impact of high-hydrostatic pressure and thermal processing on the antioxidant profiles and capacity of tomato juice during storage. food innovation and advances 2:124–134. doi: 10.48130/fia-2023-0016 wunderlin rp, hansen bf, franck ar, esig, fb. 2024. atlas of florida plants (http://florida.plantatlas.usf.edu/). xia x, zhang q, liu r, wang z, tang n, liu f, huang g, jiang x, gui g, wang l, et al. 2014. effects of 20-hydroxyecdysone on improving memory deficits in streptozotocin-induced type 1 diabetes mellitus in rat. eur j pharmacol 740: 45–52. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 191-196 | doi: 10.14421/biomedich.2023.121.191-196 issn 2540-9328 (online) antihypertensive drugs therapy in hypertension and covid-19 comorbidity emuesiri goodies moke1,*, endurance efe ahama2, pere-ebi yabrade toloyai3, mamerhi taniyohwo enaohwo2, ekuerhare basil4, emuesiri kohworho umukoro5, anthony taghogho eduviere1, ikuesirioghene udumebraye1, choice udufowe1 1department of pharmacology, faculty of basic medical sciences; 2department of anatomy and cell biology, faculty of basic medical sciences; 3department of medical biochemistry, faculty of basic medical sciences; 4university health services; 5department of pharmacology and therapeutics, faculty of basic clinical sciences, delta state university, abraka, nigeria. corresponding author* hiligoodies@gmail.com manuscript received: 06 january, 2023. revision accepted: 07 february, 2023. published: 11 february, 2023. abstract the novel coronavirus (cov) severe acute respiratory syndrome (sars)-cov-2 outbreak began at the end of 2019 in wuhan, china, and has spread to over 200 countries. many comorbidities have shown to be associated with the severity of the viral infection with hypertension being one of the highest rated comorbidities since loss of the ace2 receptor due to sars-cov-2 infection can lead to increased blood pressure. the effects and clinical characteristics associated with the use of beta-blockers, angiotensin receptor blockers (arb), and calcium-channel blockers (ccb) shows not to affect the outcome of covid-19, except in angiotensin converting enzyme inhibitor (acei) which may have negative outcomes on covid-19 infected patients. many comorbidities have shown to be associated with the severity of the viral infection. keywords: blood pressure; comorbidity; covid-19; renin-angiotensin system. introduction hypertension (htn) is a dangerous medical condition that raises the risk of disorders of the heart, brain, kidneys, and other organs (who, 2021). hypertension is characterized as persistently increased blood pressure with a systolic blood pressure of 140 mmhg and/or a diastolic blood pressure of 90 mmhg (mills et al., 2020). globally, an estimated 1.13 billion people do have hypertension, with over 700 million people reported to with untreated hypertension (who, 2021). worldwide, hypertension is the biggest avoidable risk factor for cvd and all-cause death (stanaway et al., 2018). globally, the incidence of hypertension is increasing due to population aging and increased exposure to lifestyle risk factors such as bad diets (i.e. excessive salt and low potassium consumption, as well as a lack of physical exercise) (mills, 2016). another reason may be as a result of suboptimal outcome even with wellconstructed antihypertensive treatment regimens (resistant hypertension) (acelajado et al., 2019; carey 2020; moke et al., 2022). however, variations in hypertension prevalence levels are not consistent over the world. high-income countries (hics) had a minor decline in hypertension prevalence during the last two decades, whereas poor and middle-income countries (lmics) saw large rises (mills, 2016). antihypertensive medications are chemical substances used to prevent and treat excessive blood pressure (jackson and bellamy, 2015; alawode et al., 2021). angiotensin-converting enzyme (ace) inhibitors, angiotensin ii receptor blockers (arbs), diuretics, calcium channel blockers (ccbs), and beta-blockers are the most widely prescribed antihypertensive medication groups (khalil and zeitser, 2020; moke et al., 2022). the novel coronavirus disease 2019 (covid-19), caused by the severe acute respiratory syndrome coronavirus (sars-cov-2), is a respiratory viral disease that first appeared in december 2019 in wuhan, the capital of the chinese province hubei (ruscitti et al., 2020). it is a highly contagious illness caused by the severe acute respiratory syndrome coronavirus 2 (sarscov-2) that causes respiratory infections and discomfort. covid-19 possesses a wide range of clinical symptoms, from asymptomatic disease to severe pulmonary infections (carfora et al., 2020; omo-aghoja et al., 2021). various statistics on covid-19 patients surfaced, revealing that some populations may be at higher risk than others (booth et al., 2021; enaohwo et al., 2021). with covid-19 infections, patients with pre-existing https://doi.org/10.14421/biomedich.2023.121.191-196 mailto:hiligoodies@gmail.com 192 biology, medicine, & natural product chemistry 12 (1), 2023: 191-196 cardiac disease are among the three greatest risk categories (chan et al., 2020). different comorbidities with hypertension exists, including covid-19, and these have been reported (schmieder and ruilope, 2008; noh et al., 2016, unger et al., 2020; batiha et al., 2021; okonofua et al., 2021). according to preliminary findings, hypertension (htn) is more common in critically sick, hospitalized covid19 patients, with total htn rates ranging from 50 to 56 percent (richardson et al., 2020). it was unclear if this link was causative or masked by age and other htnrelated comorbidities such as obesity, diabetes, and chronic kidney disease. furthermore, covid-19affected hypertensive individuals exhibited a greater mortality risk than non-hypertensive patients (barrera et al., 2020). concerns about the use of angiotensin-converting enzyme inhibitors (aceis) in these patients arose as a result of the identification of angiotensin-converting enzyme 2 (ace2), the monocarboxypeptidase that inactivates angiotensin ii, thus, counteracts the activation of the classic renin–angiotensin–aldosterone system (raas), as the functional receptor for the severe acute respiratory syndrome coronavirus 2 (vadughanathan et al., 2020). because hypertension is a major risk factor for developing covid-19, selecting the right medication for successful blood pressure management is critical. this review briefly discusses the drug therapy with antihypertensive drugs in hypertensive patients with covid-19 comorbidity. articles used for this review ranged from 2008 to 2022, and over 100 articles were obtained following literature search, amongst which 64 were adapted for this article. other articles whose scope were not applicable to the review were excluded. the articles were retrieved following searches using search engines and databases including medline, elsevier, medscape, emedicine, google, pubmed, and others. corona virus disease 2019 (covid-19) the novel coronavirus disease 2019 (covid-19), caused by the severe acute respiratory syndrome coronavirus (sars-cov-2), is a respiratory viral disease that first appeared in december 2019 in wuhan, the capital of the chinese province hubei (ruscitti et al., 2020). it is a highly contagious illness caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that causes respiratory infections and discomfort. corona virus disease 2019 (covid-19) is an rna virus with a crown-like appearance under an electron microscope due to glycoprotein spikes on its envelope (malik, 2020). covid-19 possesses a wide range of clinical symptoms, from asymptomatic disease to severe pulmonary infections (carfora et al., 2020; omo-aghoja et al., 2021). it is not the first time that a coronavirus has caused an epidemic, as an outbreak of coronaviruses (covs) with severe acute respiratory syndrome (sars)cov began in the chinese province of guangdong in november 2019, while on september 2012, the middle east respiratory syndrome (mers)-cov appeared (lu et al., 2020). the coronaviruses (covs) are classified into four genera: (a) α-coronavirus (alphacov), (b) β-coronavirus (betacov), (c) δ-coronavirus (deltacov), and (d) γcoronavirus (gammacov), which are most likely found in birds (pal et al., 2020). the origin of the virus is natural and zoonotic in nature. the clinical severity of the disease is varies from displaying no symptom to being very fatal, as its medical features and risk factors are so inconstant (phan, 2020). molecular structure of corona virus coronaviruses are enclosed positive strand rna viruses with the biggest known rna genomes (30–32 kb), a 5'cap structure, and a 3'-poly-a tail. the production of polyprotein 1a/1ab (pp1a/pp1ab) in the host begins with viral rna (lei et al., 2018). transcription is carried out by the replication-transcription complex (rct), which is structured in double-membrane vesicles, and by the synthesis of subgenomic rnas (sgrnas). it is worth noting that transcription termination occurs at transcription regulatory sequences, which are positioned between the so-called open reading frames (orfs), which serve as templates for the creation of subgenomic mrnas (letko et al., 2020). at least six orfs can be found in the atypical cov genome. among these, a frameshift between orf1a and orf1b directs the synthesis of both pp1a and pp1ab polypeptides, which are then processed by a virally encoded chymotrypsin-like protease (3clpro) or main protease (mpro), as well as one or two papain-like proteases for the synthesis of 16 non-structural proteins (nsps) (letko et al., 2020). other orfs, in addition to orf1a and orf1b, encode structural proteins such as spike, membrane, envelope, and nucleocapsid proteins, as well as auxiliary proteic chains (lei et al., 2020). different covs have unique structural and auxiliary proteins that are translated by specialized sgrnas. the role of the nsps and structural proteins is linked to the pathophysiology and virulence processes of covs, and hence to sars-cov-2. among the structural proteins' activities, the envelope plays a critical role in virus pathogenicity by promoting viral assembly and release (yadav et al., 2021). prevalence of hypertension in people with covid-19  prevalence of hypertension in people with covid-19 was 25.4% in africa, 31% in china, 49% in italy, 21% in india, 32% in oman, and 10% in iran (cook, 2020).  the prevalence of hypertension in patients with covid-19 was 4.7%, and 24.37% of covid-19 moke et al. – antihypertensive drugs therapy in hypertension and … 193 related deaths occurred in individuals in iran (moftakhar et al., 2021).  hypertension was the most prevalent reported comorbidity in covid-19 patients in wuhan; the reported prevalence rates ranged from 15.0% to 36.5% (haung et al., 2020).  the prevalence of hypertension was also higher in covid-19 deceased patients; 34.0% vs. those who were discharged alive; 28.0% (leiva et al., 2020).  along with the increased risk of infection and worsened outcomes among hypertensives, there is a growing concern that some medications used in the treatment may influence mortality in patients with covid-19 (patel and verma, 2020).  studies have reported that covid-19 deaths were mostly among people with comorbidities (99%), the majority of these were hypertensive (guan et al, 2020; osibogun et al., 2021).  in nigeria, the prevalence of hypertension, the most common comorbidity of covid-19, was 17.8% followed by diabetes (7.2%) and asthma (2.0%) (abayomi et al., 2021).  in nigeria, overall mortality was 4.2% while mortality among the hypertensives was 13.7%. severe symptoms and mortality were significantly higher among the hypertensives and survival rates were significantly lowered by the presence of additional comorbidity to 50% (abayomi et al., 2021).  studies have shown that hypertension imposes on those who suffer from it an increased risk of getting infected with covid-19, experiencing worse symptomatology and complications and a 2-fold risk of dying from the infection (cancarevic and malik, 2020; abayomi et al., 2021). some anti-hypertensive drugs in the treatment of hypertensive patients with covid-19 comorbidity anti-hypertensive medications are a type of medication used to treat hypertension (high blood pressure) (ettehad et al., 2016). antihypertensive treatment aims to avoid high blood pressure problems such as stroke and myocardial infarction. evidence shows that lowering blood pressure by 5 mmhg can reduce the risk of stroke by 34%, ischemic heart disease by 21%, and the chance of dementia, heart failure, and cardiovascular disease death by 34% (nelson, 2010). there are several kinds of anti-hypertensives, each of which works in a different way to reduce blood pressure. thiazide diuretics, calcium channel blockers, ace inhibitors, angiotensin ii receptor antagonists (arbs), and beta blockers are among the most significant and extensively used drugs (armstrong, 2014; ettehad et al., 2016). beta-adrenoreceptor blockers beta-blockers lower blood pressure by preventing catecholamines from binding to beta-adrenergic receptors, resulting in coronary and peripheral artery vasodilation (farzam and jan, 2020). betaadrenoreceptor blockers reduce blood pressure via decreasing cardiac output, heart rate, renin release, and adrenergic nerve system effects (farzam and jan, 2020). they improve outcomes after an acute myocardial infarction and in patients with heart failure who have a low left ventricular ejection fraction, but in the absence of these comorbidities, beta-adrenoreceptor blockers are inferior to other first-line antihypertensives in terms of reducing cvd morbidity and mortality (wiysonse et al., 2017). this impact has been related to lower aortic blood pressure and negative effects on body weight and glucose metabolism with beta-adrenoreceptor blockage (frishman et al., 2011). vasanthakumar's study suggested that using betablockers to treat covid-19 could provide numerous benefits, including reducing sars-cov-2 cell entry via downregulation of the angiotensin-converting enzyme 2 (ace-2), reducing the expression of proinflammatory cytokines, and reducing complications such as pulmonary embolism, ards, and septic shock (vasanthakumar, 2020). a research found that betablockers had no effect on the severity of covid-19 (bauer et al., 2021). another study found that using betablockers greatly lowered the chance of severe consequences (kjeldsen et al., 2021). patients on betablockers had a decreased risk of testing positive for covid-19 (reynold et al., 2020). calcium channel blockers (ccbs). calcium channel blockers (ccbs) inhibit calcium entry into cells by binding to l-type voltage-gated calcium channels found in organs such as the heart and vascular smooth muscle (mckeever and hamilton, 2020). a drop in the intracellular concentration of calcium causes smooth muscle cell relaxation and, as a result, a fall in blood pressure (kuo and ehrlich, 2022). headaches, flushing, palpitations, peripheral edema, hypotension, atrioventricular block, constipation, and nausea are the most common side effects associated with this category (solanki et al., 2021). calcium channel blockers, particularly verapamil, inhibit cardiac calcium channels, which can lower heart rate and cardiac contractility (elliott and ram, 2011). according to reports, ccbs can reduce sarscov-2 multiplication (zhang et al., 2020; loas et al., 2022). amlodipine besylate was observed to decrease the risk of death in hypertension individuals (zhang et al., 2020). another research found that nifedipine and amlodipine significantly lowered the mortality rate as well as the risk of intubation and mechanical ventilation in elderly covid-19 patients (solaimanzadeh, 2020). one research observed no significant changes in the administration of amlodipine in covid-19 patients with 194 biology, medicine, & natural product chemistry 12 (1), 2023: 191-196 primary hypertension in terms of mortality and length of hospital and intensive care unit (icu) stay (nourivaskeh et al., 2021). angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers (arbs) the renin-angiotensin-aldosterone system (raas) inhibitors, such as ace inhibitors and arbs, are among the most often prescribed blood pressure medications (wang et al., 2020). ace medications reduce blood pressure by inhibiting the angiotensin-converting enzyme, resulting in less angiotensin ii production and vasodilation. the mechanism of action of arbs is to prevent angiotensin ii from attaching to angiotensin-1 (at1) receptors (khalil and zeitser, 2020). the most serious side effects of this class include hyperkalemia, renal failure, coughing, and first-dose hypotension. there is growing concern about the use of antihypertensives in covid-19 patients, owing to the fact that angiotensin-converting enzyme 2 (ace2), a negative regulator of the renin–angiotensin–aldosterone system (raas), is a co-receptor for viral entry into human cells by sars-cov-2 (hui, 2020). ace cleaves angiotensin i to produce angiotensin ii, whereas ace2 converts angiotensin ii to angiotensin iii (zou, 2014). ace2 plays an important role in maintaining blood pressure homeostasis, fluid and salt balance by counteracting the impact of ace (forrester, 2018). ace inhibitors and arbs may increase the expression of ace-2, a cellular receptor for sars-cov2. these medications, on the other hand, have been found to protect against acute lung damage (guo et al., 2020). concerns have been raised about the use of angiotensin-converting enzyme inhibitors (aceis) in patients following the identification of angiotensinconverting enzyme 2 (ace2), a monocarboxypeptidase that inactivates angiotensin ii and thus counteracts the activation of the classic renin–angiotensin–aldosterone system (raas), as the functional receptor for the severe acute respiratory syndrome coronavirus 2 (sars-cov2) (vadughanathan et al., 2020). as a result, discontinuing aceis or arbs may result in poorer results than continuing to use them in individuals with covid-19. most of the world’s professional societies either recommend or strongly encourage continuing aceis/arbs in covid-19 infected patients (hoffman et al., 2020). the novel coronavirus disease 2019 (covid-19), caused by the severe acute respiratory syndrome coronavirus (sars-cov-2), is a respiratory illness that causes respiratory distress among other symptoms. one of the major comorbidities of covid-19 is hypertension, which is defined by persistently high blood pressure. the use of several anti-hypertensive classes, including as beta-blockers, calcium channel blockers, diuretics, and angiotensin converting enzyme inhibitors (acei), to treat hypertension in patients with covid-19 may have side effects on the patients. while other antihypertensive classes have no to beneficial effects, acei have been demonstrated in multiple studies to be a source of worry since they may potentially enhance covid-19 symptoms and promote viral entry into the cell. conclusion most anti-hypertensive drugs have no detrimental impact on the outcome of covid-19 therapy, and a few classes have favorable benefits in alleviating symptoms in patients infected with covid-19. aceis are the only class of anti-hypertensive that may have an influence on the result of covid-19 among the anti-hypertensive classes evaluated. more studies and clinical trial are recommended to be carried out with more common antihypertensive to evaluate the potential effects on covid19. competing interests: the authors declare that there are no competing interests. 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seed priming, spirulina platensis; vigor. abbreviations: bold basal medium (bbm), completely randomized design (crd), laminar air flow cabinet (lafc), maximum growth potential (mgp), mean germination time (mgt), sugar mill effluent (sme), vigor index (vi). introduction spirulina has the synonym name arthrospira. species of spirulina include spirulina maxima, spirulina fosiformis, and spirulina platensis. spirulina platensis is a type of microalgae that is bluish-green in color, unbranched, filamentous in shape with a spiral (helix) form, measuring 1-12 micrometers, and lives in colonies. spirulina is a microalgae originating from the ocean and can thrive in water with toxic metal pollutants. spirulina is not only found in saltwater, freshwater, and brackish water but also soil, demonstrating its high adaptability (kamaludin & holik, 2022). the microalgae spirulina, with its high adaptability, can utilize waste as a growth medium, one of which is sugar mill effluent (sme). sme contains high levels of organic and inorganic compounds, so before being discharged into water bodies, it must meet quality standards to determine the amount of pollutants in the waste water. the organic compounds found in the sme can be utilized by microalgae as a growth medium by utilizing nutrients such as nitrogen and phosphorus in the wastewater. in addition to being known for their high efficiency in wastewater treatment, microalgae can absorb nutrients and metals from wastewater at various stages of treatment (alling et al., 2023). the cultivation of microalgae produces beneficial biomass that can be used as seed priming agents. seed priming or seed soaking is an initial treatment on seeds as one of the invigorating techniques aimed at increasing the germination rate, germination percentage, uniformity of growth, and germination speed in seeds (andayani et al., 2023). priming methods include osmopriming, hydropriming, halopriming, and biopriming. biopriming is a priming treatment combined with applying biological agents such as microbes that can enhance seed germination, produce growth hormones, or increase stress resistance (kurnia et al., 2017). https://doi.org/10.14421/biomedich.2025.141.129-141 130 biology, medicine, & natural product chemistry 14 (1), 2025: 129-141 biopriming can be performed using the biomass of the microalgae spirulina platensis. the biomass of spirulina platensis comprises roughly 68% protein with few essential amino acids. spirulina platensis is a microalgae with a structure consisting of amino acids, polysaccharides, phenolics, proteins, carbohydrates, vitamins, and phytohormones (auxin, gibberellin, and cytokinin). the phytohormone content can enhance enzyme activity, cell division, and differentiation, as well as plant growth (seğmen & ünlü, 2023). thus, biomass containing phytohormones can be used as a seed priming agent to enhance germination. the use of microalgae biomass for seed priming can be applied to various types of crop and horticultural plants, including cucumber. cucumber is one type of horticultural commodity classified as a vegetable for public consumption, it is rich in nutrients, vitamins, minerals, and beneficial for lowering blood pressure (oktaviana et al., 2016). therefore, cucumbers are excellent plants that serve as multi-purpose vegetables starting from food, garnish, cosmetics, and therapeutic substances (adnan et al., 2024). to achieve good production, appropriate cultivation techniques are necessary. factors that support successful cucumber cultivation are the use of highquality seeds. seed quality encompasses physical, physiological, genetic, and pathological or health quality. a clean, bright, plump appearance and uniform size characterize seeds with high physical quality. meanwhile, the high physiological quality of seeds can be distinguished from their high viability (germination rate) and vigor (growth speed and simultaneous of seed growth). the genetic quality of seeds is indicated by high genetic uniformity and the absence of other variety contaminants. meanwhile, for pathological quality, the seeds are assessed based on the absence of seedborne pathogens (ashari et al., 2024). the use of low quality seeds can result in a decrease in the quality and productivity of cucumber plants. to improve yield production, the use of high-quality seeds is necessary. rosita et al. (2022) highlighted seed quality as a key factor in increasing the productivity of apple cucumber plants, which currently remains low at around 27 tons/ha. the decline in seed quality can occur naturally through prolonged storage periods, resulting in decreased seed viability. the decline in seed quality affects the germination and growth of seedlings, leading to suboptimal plant development that could potentially cause losses for farmers. thus, applying biostimulants in seed priming techniques offers an innovative solution to enhance seed germination and early plant growth in supporting sustainable agricultural practices (garcíalocascio et al., 2024). haerani and nurdin (2021) reported that biopriming showed a positive response in the parameters of germination rate, vigor index, and simultaneous growth. cucumber plants are responsive ability to seed priming treatments, making them an ideal subject to test a new potential seed priming agent. this idea was confirmed by badu et al. (2022) who successfully demonstrated that cucumber seeds induced with seed priming showed a higher germination percentage in response to biopriming compared to untreated seeds. based on the potential application of spirulina platensis in seed priming, this study aimed to determine the effect of the concentration of spirulina platensis microalgae biomass (control, 0%, 30%, 45%, and 60%) with soaking durations (1, 2, and 3 hours) on the seeds of cucumber variety mkge 701. this research presents a new agricultural innovation by utilizing spirulina platensis as an agent for processing sme for seed priming applications. consequently, our research aims to contribute to preserving environmental safety, ensuring food security, and promoting a more sustainable agriculture in the future. materials and methods place and time of research this research was conducted from may to august 2024 at the biosciences integrated laboratory and the seed technology laboratory of jember state polytechnic. materials and tools the equipment used included a centrifuge, laminar air flow cabinet (lafc), hemocytometer, aerator, germination rack, germination box, microscope, and autoclave. the materials used consisted of sugar mill effluent (sme), aquades, bold basal medium (bbm) with modifications (supplementary), 70% alcohol, spirulina platensis isolate from bbpbap jepara, label paper, litmus paper, germination paper, seeds of cucumber var. mkge 701 (cv. nasienie indonesia, jember indonesia) has an expiration date of december 2025. procedures this research method used a factorial completely randomized design (crd) with the factor of microalgae biomass concentration (v/v) consisting of 5 levels: control (untreated seeds), c0 (0%), c1 (30%), c2 (45%), and c3 (60%), and the soaking time consisting of 3 levels: 1, 2, and 3 hours. the total treatment combinations are 15 with 4 repetitions, resulting in 60 experimental units with 30 seeds per unit. the research began with the preparation of a growth medium for spirulina platensis, comprising 25% (v/v) sugar mill effluent (sme) in a modified bold basal medium (bbm) according to utex (utex, 2019). subsequently, microalgae were grown in plastic bottles photobioreactors with illumination of 190 lux and aeration for 24 hours at room temperature. the microalgae biomass used for seed priming is obtained from centrifugation at 8000 rpm at 10°c for 10 minutes. rahmawati et al. – the potential of spirulina platensis biostimulan as a … 131 the seed priming experiments were carried out by soaking the seeds with microalgae solutions prepared by diluting pellets with distilled water to achieve the desired concentration. subsequently, the seeds were germinated on germination paper and placed in a plastic container. the container was then stored at a germination shelf at 28°c, 70% humidity, with an illumination period of 16 hours followed by 8 hours dark period for 8 days. analysis of the microalgae test results was observed based on: observation of cells photos visual observation of cells utilizes image processing software (cellsens) integrated with the olympus bx43 compound microscope. analysis of seed priming test results is based on ista observation parameters (fadhilah, 2020): germination rate (%) germination observation was conducted on the first count (day 4) and final count (day 8), with the parameter observed being the number of normal seedlings. normal seedlings demonstrate the ability to grow and develop into normal plant seedlings. the germination percentage can be calculated using the following formula: 𝐺𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑖𝑜𝑛 𝑅𝑎𝑡𝑒 (%) = 𝛴 𝑁𝑜𝑟𝑚𝑎𝑙 𝐺𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑖𝑜𝑛 𝐹𝑖𝑟𝑠𝑡 𝑐𝑜𝑢𝑛𝑡 + 𝐹𝑖𝑛𝑎𝑙 𝑐𝑜𝑢𝑛𝑡 𝛴𝑛𝑢𝑚𝑏𝑒𝑟𝑠 𝑜𝑓 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑 𝑋100% mean germination time (days) mgt was observed based on the number of seeds producing a radicle < 2 mm daily. the measurement of mgt shows the average time required for the seeds to germinate. mgt can be calculated using the following formula: 𝑀𝐺𝑇 (𝐷𝑎𝑦𝑠) = 𝛴(𝑛 𝑥 𝑡) 𝛴𝑛𝑢𝑚𝑏𝑒𝑟𝑠 𝑜𝑓 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑 vigor index (%) the vigor index was observed by observing normal seedlings that grew during the first count. the calculation of the vigor index is as follows: 𝑉𝐼 (%) = 𝛴𝑁𝑜𝑟𝑚𝑎𝑙 𝐺𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑖𝑜𝑛 𝐹𝑖𝑟𝑠𝑡 𝑐𝑜𝑢𝑛𝑡 𝛴𝑛𝑢𝑚𝑏𝑒𝑟𝑠 𝑜𝑓 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑 𝑋100% seed growth rate (%) observations were conducted by counting the normal seedlings that grew each day until the final count. the calculation formula is as follows: 𝑆𝑒𝑒𝑑 𝐺𝑟𝑜𝑤𝑡ℎ 𝑅𝑎𝑡𝑒 (%) = % 𝑁𝑜𝑟𝑚𝑎𝑙 𝑆𝑒𝑒𝑑𝑙𝑖𝑛𝑔𝑠 𝑒𝑡𝑚𝑎𝑙 simultaneous growth seeds (%) observation was conducted by counting the normal seedlings that grow between the first count and the final count day (day 6). strong normal seedlings are the criteria for normal seedlings that have the appearance of stronger germination than*the average of other normal seedlings. the calculation formula is as follows: 𝑆𝑖𝑚𝑢𝑙𝑡𝑎𝑛𝑒𝑜𝑢𝑠 𝐺𝑟𝑜𝑤𝑡ℎ 𝑠𝑒𝑒𝑑𝑠 (%) = 𝛴𝑆𝑡𝑟𝑜𝑛𝑔 𝑁𝑜𝑟𝑚𝑎𝑙 𝑆𝑒𝑒𝑑𝑙𝑖𝑛𝑔𝑠 𝛴𝑆𝑒𝑒𝑑 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑖𝑜𝑛𝑠 𝑋100% maximum growth potential (%) observations were made by calculating the percentage of seeds that grew into normal and abnormal seedlings at the final count. the calculation formula is as follows: 𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝐺𝑟𝑜𝑤𝑡ℎ 𝑃𝑜𝑡𝑒𝑛𝑡𝑖𝑎𝑙 (%) = 𝛴𝑁𝑜𝑟𝑚𝑎𝑙 + 𝐴𝑏𝑛𝑜𝑟𝑚𝑎𝑙 𝑠𝑒𝑒𝑑𝑙𝑖𝑛𝑔 𝛴𝑛𝑢𝑚𝑏𝑒𝑟𝑠 𝑜𝑓 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑 𝑋100% shoot length (cm) the measurement of the shoot length was taken from the base of the stem to the tip of the bud/growth point of the shoot. radicle length (cm) the radicle length was measured from the base of the root to the tip of the radicle. dry weight of normal seedlings (grams) this observation was conducted during the final count, to determine seed vigor. normal seedlings have their cotyledons removed, then placed in paper envelopes, dried using an oven at 60°c for 72 hours, and weighed using an analytical balance. the calculation formula is as follows: 𝐷𝑟𝑦 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑁𝑜𝑟𝑚𝑎𝑙 𝑆𝑒𝑒𝑑𝑙𝑖𝑛𝑔𝑠 (𝑔𝑟𝑎𝑚𝑠) = 𝑤𝑒𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 − 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑤𝑒𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 data analysis the research data were analyzed using analysis of variance (anova), and if significant data were found, a bonferroni post-hoc test was conducted at a 5% significance level. statistical analysis was performed using graphpad prism 5.01 software. 132 biology, medicine, & natural product chemistry 14 (1), 2025: 129-141 results and discussion analysis of microalgae growth on sme-based-bold basal medium (sme-bbm) figure 1. photograph of spirulina platensis microalgae cells grown on sme-based bold basal medium. microalgae are a biomass source that contains important components such as fatty acids, pigments, carbohydrates, and proteins, depending on the growth medium used, the environment, and the drying method. the observation of the microalgae spirulina platensis grown on sme-bbm media was marked by a change in the culture color from light green to a more intense shade. these findings were supported by the research of widawati et al. (2022), which shows that the growth of the microalga spirulina platensis is marked by a color change in the culture from clear green to dark green. this color change occurs due to the increase in biomass. the growth of microalgae is accompanied by an increase in cell size and an increase in the density of microalgal cells (lebeharia et al., 2016). the growth of microalgae is divided into several phases, namely the lag phase, exponential phase, stationary phase, and death phase. in the exponential phase (log phase), their growth is marked by an increase in the number and length of microalgae cells. the diameter of microalgal cells ranges from 1 to 12 µm, their body shape resembles threads, and they have a spiral shape with thin cell walls. at the end of the exponential phase, microalgae produce phycocyanin compounds as high antioxidants (kamaludin & holik, 2022). additionally, the research of chiaiese et al. (2018) found that microalgae biologically produce extracellular compounds in the form of phytohormones, amino acids, polypeptides, and vitamins. thus, microalgae can be utilized as plant biostimulants to enhance plant growth and development. analysis of seed priming result on cucumber seeds germination rate (%) figure 2. germination rate of cucumber seeds under various biomass concentrations and soaking time. observation data were taken on the 4th day (first count) and the 8th day (final count) of the normal seedlings criteria. the data above represent the mean value with sem error bars from the anova analysis with the bonferroni post hoc test compared to the control (untreated group). values without an asterisk are considered nonsignificance. significant: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). the germination rate of seeds is a parameter that serves as a benchmark for seed viability. seed viability is defined as the ability of a seed to grow into a normal seedling (ashari et al., 2024). the germination rate is an important factor that determines plant formation and subsequent production. based on figure 2, the results of rahmawati et al. – the potential of spirulina platensis biostimulan as a … 133 the analysis of variance showed that the treatment with concentrations of spirulina platensis biomass at 30%, 45%, and 60%, and soaking times of 1, 2, and 3 hours on cucumber seeds have a non-significant effect on the germination rate parameter. the germination rate in the sample without treatment (control) was 97,50%. meanwhile, the water treatment for 1, 2, and 3 hours showed germination rate values of 91,67; 95,83; and 95,00%. on the other hand, the seed priming application with 30% spirulina platensis biomass for 1, 2, and 3 hours showed germination rate values of 95,83; 96,67; and 95,83%. the 45% concentration showed germination rate values of 96,67; 98,33; and 99.17%. the 60% concentration with soaking times of 1, 2, and 3 hours showed germination rate values of 97,50; 98,33; and 97,50%. thus, although the application of spirulina platensis biomass showed a non-significant effect, no decrease in germination was found in the seed germination rate parameter. based on the results analysis, the seed priming treatment with 45% concentration of spirulina platensis biomass for 2 and 3 hours of soaking time, and 60% concentration for 2 hours of soaking time, showed germination rates of 98,33; 99,17; and 98.33%, respectively. although all three treatments showed no significant results, these applications provided higher germination effects compared to the control (97,50%). furthermore, the application of spirulina platensis biomass did not cause a decrease in germination rate. this indicates that treating spirulina platensis biomass concentration with soaking time does not have a harmful or toxic effect on seedling growth. the research of ribeiro et al. (2019) reported that the supernatant from chlorella sorokiniana culture showed no significant effect on lettuce seed germination and did not cause toxicity to the seeds. mean germination time (mgt) figure 3. mean germination time (mgt) of cucumber seed under various biomass concentrations and soaking time. observation data were taken daily with the condition that the radicle length ≤ 2 mm. the data above represent the mean value with sem error bars from the anova analysis with the bonferroni post hoc test compared to the control (untreated group). values without an asterisk are considered nonsignificance. significant: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). mean germination time (mgt) is one of the methods used to measure seed vigor through seedling growth testing. mean germination time (mgt) is an observational parameter that serves as a benchmark to indicate the average time required for seeds to develop a radicle (soltani et al., 2015). mean germination time is correlated with seed vigor, where a shorter germination time indicates a faster and more uniform radicle growth potential. based on figure 3., the analysis of variance results showed that the treatment with a 30% concentration of spirulina platensis biomass with soaking times of 1, 2, and 3 hours significantly effected the mgt parameter compared to the control. this is evidenced by the emergence time of the radicle without treatment (control) on the 2nd day with an mgt value of 2.10 days. contrastly, in the 30% treatment for 1, 2, and 3 hours, the radicle appeared before the 2nd day with values of 1,81 and 1,87 days. although statistically significant results were only found in the 30% treatment for 1 and 2 hours, the 3 hour treatment also showed a faster germination compared to the control with an mgt value of 1,87 days. the treatment of spirulina platensis biomass at a concentration of 45% for 1, 2, and 3 hours exhibited the mgt values of 2,04; 1,85; and 1,79 days, respectively. based on the statistical test results, the 45% treatment with a soaking time of 3 hours significantly effected mgt. the emergence of the radicle was faster with a soaking time of 3 hours compared to the control, 1, and 2 hours. although the soaking times of 1 and 2 hours did not show significant results, the average mgt required to grow the radicle was faster, than the control. considering the obtained findings, there was a trend that the longer the soaking time, the faster the seeds develop radicles. the current research’s outcomes were in line with the research by shakuntala et al. (2020), which found that the longer the soaking time, the more it affects the quality of cucumber seeds, namely germination, vigor index, germination rate, germination speed, shoot length, and root length, which are all higher. subsequently, the treatment with 60% spirulina platensis biomass and soaking times of 1, 2, and 3 hours showed a significant effect on the mgt parameter. compared to other treatments, the 60% treatment for 1, 2, and 3 hours provided the highest average mgt times of 1,75; 1,55; and 1.69 days, respectively. this indicates that the higher the concentration of spirulina platensis biomass used for seed priming, the more it can accelerate the average time required for the radicle to grow. overall, the observation analysis showed that treating cucumber seeds with spirulina platensis biomass concentrations of 30%, 45%, and 60% with soaking times of 1, 2, and 3 hours significantly affected the mgt parameter compared to the control. these findings suggest that the varying concentration and soaking time of spirulina platensis can have a significant impact on mgt parameters. our research aligned with the study of akgül (2019) that confirmed the use of spirulina 134 biology, medicine, & natural product chemistry 14 (1), 2025: 129-141 platensis biomass to improve the germination time of wheat and barley. vigor index (vi) figure 4. vigor index of cucumber seed under various biomass concentrations and soaking time. observation data were taken on the 4th day (first count) to observe normal seedlings. the data above represent the mean value with sem error bars from the anova analysis with the bonferroni post hoc test compared to the control (untreated group). values without an asterisk are considered non-significance. significant: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). seed vigor index (vi) is the ability of seeds to grow into normal plants under sub-optimal environmental conditions. the vigor index compares the number of normal seedlings at the first count and the number of seeds germinated. the vigor index test is a more accurate approach to assessing the growth potential of seedlings in the field compared to germination power testing (kartina et al., 2021). figure 4 showed that results of the analysis of variance showed that the water treatment and soaking durations of 1, 2, and 3 hours did not significantly affect the vigor index parameter compared to the control with each vi percentage values of 42,50; 35; and 41,67%, respectively. although the water treatment with soaking durations of 1 and 3 hours showed no effect on the vi parameter, these applications positively correlated with the increase in vigor index values compared to the control (37,50%). this indicates that priming treatment with the hydropriming method can enhance seed germination. in the treatment of spirulina platensis biomass at a concentration of 30% with soaking times of 1, 2, and 3 hours, the vigor index percentages were 30; 56,67; and 63,33%, respectively, compared to the control at 37,50%. although the results were not significant, the application of biomass at a concentration of 30% for 2 and 3 hours showed higher vigor index percentages compared to the control. based on the obtained results, there is a tendency that the longer the soaking time, the higher the percentage of vigor index value in the seeds. subsequently, the treatment with 45% concentration of spirulina platensis biomass with soaking times of 1, 2, and 3 hours showed vi values of 34,17; 45,83; and 84,17%, respectively. based on these vi values, the 45% treatment with a soaking time of 3 hours showed a significant effect on the increase of the vigor index parameter. the treatment of cucumber seeds with 45% biomass within 3 hours showed the highest vi value of 84,17% compared to the other treatments. on the other hand, although the 45% treatment for 2 hours showed no significant results, the application of the biomass demonstrated a higher percentage of vi value compared to the control. the elevated vigor index of seeds treated with spirulina platensis biomass indicates a significant development potential in the field, particularly under sub-optimal environmental circumstances. based on the research by alam et al. (2023), priming cucumber seeds can protect plants from excessive water loss and make them more tolerant to drought stress. basavaraja (2024) also suggested that the increase in germination percentage and vigor index using spirulina platensis extract positively affects germination rate and seedling growth strength. furthermore, algae extracts can enhance the growth of early-stage plants. applying a 60% concentration of spirulina platensis biomass treatment with soaking times of 1, 2, and 3 hours showed vigor indices (vi) of 51,67; 79,17; and 80,83%, respectively. based on these vigor index values, the 60% treatment with soaking times of 2 and 3 hours significantly effected the vigor index parameter. although the 1 hour soaking at 60% concentration showed no significant results, the application of biomass at this concentration demonstrated a higher vigor index percentage than the control. based on the obtained results, there is a tendency that the longer the soaking time, the more it affects the increase in the vigor index rate of the seeds. this is evident at a concentration of 60% with a vigor index of 51,67% at 1 hour and reaching 80,83% at 3 hours of soaking. overall, our current study suggested a positive correlation between spirulina platensis biomass concentration and the rate of vigor index of cucumber seeds. indeed, this is evident at concentrations of 30%, 45%, and 60%, as shown by the increase in the percentage of vigor index values from low to high concentrations, ranging from 30% to 80,83%. this increase might occur due to the use of increasingly concentrated spirulina platensis biomass, which enhances the hormone content in the biomass, thereby improving the germination rate, particularly in the vigor index parameter. the current work was in line with the research by basavaraja (2024), which demonstrated that higher concentrations of spirulina platensis can enhance seed germination and seedling vigor index in various plant species, even at a concentration of 100%. seed priming on cucumber seeds induces pregermination metabolic conditions to increase germination rates and enable seeds to germinate under sub-optimal conditions. cucumber seeds have a responsive ability to seed priming treatment. this idea was in line with the research by badu et al. (2022), cucumber seeds induced with seed priming showed a higher germination rahmawati et al. – the potential of spirulina platensis biostimulan as a … 135 percentage in response to biopriming due to changes in physiological activity, seed metabolism, and enzyme stimulation compared to untreated seeds. seed growth rate (%/etmal) figure 5. seed growth rate of cucumber seed under various biomass concentrations and soaking time. observation data were taken daily according to the criteria for normal seedling. the data above represent the mean value with sem error bars from the anova analysis with the bonferroni post hoc test compared to the control (untreated group). values without an asterisk are considered non-significance. significant: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). the seed growth rate can indicate vigor and growth strength (nurwiati & budiman, 2023). the growth rate can be observed using a shorter germination process (kolo & tefa, 2016). a high growth rate commonly indicates higher seed vigor. this reflects that the higher the seed growth rate, the greater the seed's ability to grow under suboptimal conditions (syaban et al., 2023). based on figure 5, the results of the analysis of variance showed that the treatment with spirulina platensis biomass at a concentration of 30% and soaking durations of 1, 2, and 3 hours resulted in growth rates of 19,43; 21,76; and 21,77%, compared to the control at 20,13%. although the results were not significant, the application of biomass at a concentration of 30% for 2 and 3 hours showed higher growth rates compared to the control. in the treatment with a concentration of 45% and soaking times of 1, 2, and 3 hours, the growth rate percentages were 19,80; 21,34; and 23,99%, compared to the control at 20,13%. applying seed priming with a 45% concentration for 3 hours showed significant results on the growth rate parameter. although the 2 hour soaking at 45% concentration showed no significant results, the biomass application demonstrated a higher growth rate percentage than the control. based on the results, our observation demonstrated a tendency for the longer the soaking time, the more it affects the increase in seed growth rate. this is evident at a concentration of 45% with a growth rate of 19.80% at 1 hour and 23.99% at 3 hours. the result made the application of seed priming at a concentration of 45% for 3 hours provide the best growth rate compared to other treatments (figure 5). it can be inferred that spirulina platensis biomass has an effect of accelerating seed germination by promoting higher initial growth. the current study was in line with akgül (2019) research, where the supernatant of spirulina platensis has a plant biostimulant effect capable of accelerating the germination of wheat and barley seeds. in the treatment with a concentration of 60% and soaking times of 1, 2, and 3 hours, there was an increase in the percentage of seed growth rate (21,27; 23,34; and 23,37%). the 60% concentration treatment for 2 and 3 hours showed significant results. the 60% concentration treatment for 1 hour showed no significant results, but the seed priming application had a higher growth rate than the control. this indicates that seed priming with spirulina platensis biomass provides a better growth rate compared to untreated groups. according to the research by nurwiati and budiman (2023), a reasonable seed growth rate ranges from 25-30%. although in this study the seed growth rate percentage did not reach the ideal target, the increased growth rate in seeds treated with spirulina platensis biomass opens up opportunities for further development in using spirulina platensis as biopriming. the results of the current study suggest that there is a tendency for a positive correlation between biomass concentration and the rate of increase in growth speed in the seeds. the increase in biomass concentration correlates with a significant rise in the percentage of seed growth rate, from 19,43% to 23,37%. this increase occurred due to using a more concentrated biomass of spirulina platensis. spirulina has a structure of amino acids, polysaccharides, phenolics, proteins, carbohydrates, vitamins, and phytohormones (auxin, gibberellin, and cytokinin). therefore, the cellular content of spirulina is capable of enhancing plant growth (singh, 2014). simultaneous growth of seeds (%) figure 6. simultaneous growth of seed of cucumber seed under various biomass concentrations and soaking time. observation data were taken once between the first count and final count on the 6th day under the criteria for normal seedling. the data above represent the mean value with sem error bars from the anova analysis with the bonferroni post hoc test compared to the control (untreated group). values without an asterisk are considered non-significance. significant: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). 136 biology, medicine, & natural product chemistry 14 (1), 2025: 129-141 simultaneous seed growth represents the ability of seeds to form normal seedlings. simultaneous growth of seed is one of the tests of growth vigor that can be seen from the ability of seeds to grow uniformly. the greater the seed's growth strength, the more uniformly the seeds will be germinating (septirosya et al., 2024). the simultaneous growth of seeds was calculated based on the number of normal seedlings growing between the first and final count. the seedlings with complete and healthy root, shoot, and normal leaves and growth were considered normal. the simultaneous growth of seed indicates the vigor of a seed lot. low vigor and viability cause low seedling uniformity (andayani et al., 2023). based on figure 6, the analysis of variance showed that the water treatment and soaking time of 1, 2, and 3 hours resulted in simultaneous growth of seed percentages of 70; 90,83; and 90%, while the control group had a percentage of 85%. although the water application with soaking time of 2 and 3 hours showed no significant results, these applications demonstrated higher simultaneous growth of seed percentages compared to the control. this indicates that priming treatment with the hydropriming method can enhance germination, particularly in the uniformity of seedling growth. water plays a crucial role in seed germination, as it is necessary to provide dissolved oxygen for the embryo, soften the seed coat, and increase seed permeability. this finding was in line with the research by shakuntala et al. (2020) that soaking cucumber seeds using the hydropriming method positively effects seed germination, germination speed, and germination rate. in the treatment of spirulina platensis biomass at a concentration of 30% with soaking times of 1, 2, and 3 hours, the percentage values of simultaneous growth of seed were 81,67; 94,17; and 91,67%, respectively, with a control value of 85%. based on the statistical test results, spirulina platensis biomass at a concentration of 30% for 2 hours significantly effected the uniformity of seed growth. although the 30% concentration for 3 hours had a non-significant effect, this application showed a higher simultaneous seed value growth than the control. this indicated that priming conducted with spirulina platensis biomass can enhance the uniformity of seed germination. haerani and nurdin (2021) further asserted that biopriming exhibited a positive response in the parameters of germination power, vigor index, and growth synchrony. the treatment of spirulina platensis biomass at a concentration of 45% with soaking times of 1, 2, and 3 hours exhibited a simultaneous simultaneous growth of seed percentage values of 86,67; 95,83; and 99,17%, respectively, compared to the control at 85%. based on the statistical test results, the treatment of 45% biomass with soaking times of 2 and 3 hours showed significant results. despite being statistically insignificant at 1 hour, the 45% biomass application still demonstrated better simultaneous growth than the control. furthermore, seed priming at a concentration of 45% for 3 hours provided the best simultaneous growth of seed value compared to other treatments. this indicated that spirulina platensis biomass effects improving seed uniformity by promoting the growth of normal seedlings. the current result was in line with the research by xie et al. (2022) microalgae have the potential as plant biostimulants, as evidenced by the positive response of microalgae treatment on the parameters of radicle emergence, seed vigor, and seed germination. additionally, there was a tendency for longer soaking times to show higher values of uniformity in growth. this was proven by applying spirulina platensis biomass at a concentration of 45% with soaking times of 1 – 3 hours, resulting in a simultaneous growth of seed percentage from 86 to 99,17%. the application of spirulina platensis biomass at a concentration of 60% with soaking times of 1, 2, and 3 hours showed simultaneous growth of seed percentages of 89,17; 95,83; and 96,67%, respectively, compared to a control of 85%. based on these statistical test results, the 60% concentration for 2 and 3 hours displayed a significant effect on the simultaneous growth of the seed parameter. although the 60% concentration for 1 hour showed no significant results, it produced better seed germination simultaneity than the control. in addition, there was a tendency that the higher the concentration of biomass used, the higher the seed uniformity value for growing into normal seedlings. this is because the compounds found in the microalgae extract can trigger the simultaneous growth of cucumber seeds. the current study was in line with susanti's (2014) reports that seeds treated with osmoconditioning using high-concentration solutions can reactivate growth enzymes within the seeds, thereby breaking seed dormancy. in addition, there was a tendency that longer soaking time in seeds can influence plant growth, especially in the parameter of uniformity of growth, as evidenced by the simultaneous growth of seeds value from the lowest concentration of 30% at 81,67% and the 60% concentration reaching 96,67%. rahmawati et al. – the potential of spirulina platensis biostimulan as a … 137 maximum growth potential (mgp) figure 7. maximum growth potential of cucumber seed under various biomass concentrations and soaking time. observation data were taken on day 8 (final count) stipulating the percentage of normal and abnormal sprouts. the data above represent the mean value with sem error bars from the anova analysis with the bonferroni post hoc test compared to the control (untreated group). values without an asterisk are considered nonsignificance. significant: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). maximum growth potential (mgp) is the ability of seeds to grow into normal or abnormal seedlings under optimal conditions. maximum growth potential describes seed viability, closely related to the seed’s ability to germinate and grow at its optimal capacity. maximum growth potential is influenced by environmental factors such as light, temperature, humidity, and internal factors. a high number of maximum growth potential results from high seed viability conditions (harsono et al., 2021). based on figure 7, the results of the analysis of variance showed that the treatment with 30%, 45%, and 60% concentrations of spirulina platensis biomass and soaking times of 1, 2, and 3 hours on cucumber seeds had a non-significant effect on the mgp parameter compared with the untreated seeds (control) with 100% mgp value. the water treatment for 1, 2, and 3 hours showed mgp values of 99,17; 99,17; and 98,33%. meanwhile, the seed priming application with a 30% concentration of spirulina platensis biomass for 1, 2, and 3 hours showed mgp values of 100%; 99,17; and 100%. the 45% concentration for 1, 2, and 3 hours showed maximum growth potential values of 99,17; 100; and 100%. additionally, the 60% concentration with soaking times of 1, 2, and 3 hours showed maximum growth potential values of 100; 98,33; and 98,33%. although it showed no significant effect, there was no decrease in germination in the maximum growth potential parameter. this indicated that the treatment of spirulina platensis for cucumber seed priming was not toxic and did not have an adverse effect on seedling growth, as evidenced by the absence of a decrease in the percentage of maximum growth potential in each treatment. seeds are very sensitive or responsive to environmental changes, including the presence of toxic substances. therefore, seeds have great potential to be used as bioindicators of toxicity (luo et al., 2018). the response triggered by the seeds is often manifested by a decrease in germination percentage and abnormalities in the seedlings. our current study was in line with the research by ribeiro et al. (2019) the supernatant from the culture of chlorella sorokiniana showed no significant difference in seed germination and did not cause toxicity in lettuce seeds. shoot length (cm) figure 8. shoot length of cucumber seed under various biomass concentrations and soaking time. observation data were taken on day 8 (final count) measuring from the base of the stem to the tip of the plumule bud/growth point in normal seedlings. the data above represent the mean value with sem error bars from the anova analysis with the bonferroni post hoc test compared to the control (untreated group). values without an asterisk are considered non-significance. significant: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). the shoot is the precursor to the stem that grows during the germination phase. shoot grows and emerges above the ground and is located above the cotyledon. the shoot becomes part of the plant that will develop upwards to form buds and leaves (parnidi et al., 2022). based on figure 8, the results of the analysis of variance generally showed that the treatment with spirulina platensis biomass concentrations of 30%, 45%, and 60% and soaking times of 1, 2, and 3 hours on cucumber seeds significantly affected the shoot length parameter compared to the control. at a concentration of 30%, the application of biomass showed a significant increase at all soaking durations of 1, 2, and 3 hours with the shoot lengths of 10,18; 9,43; and 10.14 cm, respectively. subsequently, at a concentration of 45%, the application of biomass also showed significance at all soaking durations of 1, 2, and 3 hours, with shoot lengths of 9,48; 9,77; and 9,46 cm, respectively. at a concentration of 60% and soaking durations of 1, 2, and 3 hours, the application of biomass also showed a significant effect on the shoot length parameter, evidenced by shoot lengths of 10,36; 10,37; and 10,24 cm, respectively. meanwhile, the control only produced a shoot length of 8,43 cm. based on these results, there was a tendency for a constant increase in shoot length at a concentration of 60% of spirulina platensis biomass over 1, 2, and 3 hours of soaking. this concentration resulted in a more extended shoot compared to the other treatments. this indicated that priming of cucumber seeds using spirulina platensis biomass could potentially increase radicle 138 biology, medicine, & natural product chemistry 14 (1), 2025: 129-141 length. this increase occurred presumably because spirulina platensis contains hormones that can stimulate seed germination. among the phytohormones processed by spirulina platensis are auxin, gibberellin, and cytokinin. this is in line with the study of andianingsih et al. (2021) who elaborated that auxin plays a role in stimulating cell elongation processes and gibberellin affects embryo development and germination. the results of this observation are supported by the study of seğmen and ünlü (2023), which reported that the phytohormone content of spirulina can enhance cell division and differentiation, enzyme activity, leaf growth, and photosynthetic capacity. radicle length (cm) figure 9. radicle length of cucumber seed under various biomass concentrations and soaking time. observation data were taken on day 8 (final count) measuring the root base and the root tip of normal seedlings. the data above represent the mean value with sem error bars from the anova analysis with the bonferroni post hoc test compared to the control (untreated group). values without an asterisk are considered nonsignificance. significant: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). the radicle is a part of the seed embryo that emerges through the lower part of the microphyll end and will develop into the plant's root. radicle is the primary root that appears first and will continue to develop to stimulate a complete root system (dharma et al., 2015). radicles will develop into the plant's root, which functions to supply nutrients and transport mineral salts from the soil to all parts of the plant. based on figure 9, the treatment with a 30% concentration and soaking times of 1, 2, and 3 hours exhibited radicle lengths of 11,57; 11,08; and 13,60 cm, respectively, while a control length displayed radicle length of 11,67 cm. based on the statistical test results, the 30% treatment with a soaking time of 3 hours significantly effected radicle length compared to the control. although at the 30% concentration for 1 and 2 hours, there was a slightly observable decrease, the radicle length produced at that concentration did not show a significant decrease. at a concentration of 45% with soaking times of 1, 2, and 3 hours, the radicle lengths were 11,45; 11,58; and 12,64 cm, respectively. although the seed priming application at a concentration of 45% for 3 hours showed no significant effect, there was a 0,95% increase in radicle length compared to the control. subsequently, at a concentration level of 60% and soaking durations of 1, 2, and 3 hours, the radicle lengths were 11,46; 13,11; and 11,68 cm, respectively. although the 60% concentration for 2 and 3 hours indicated no significant effect, this application showed increased radicle length compared to the control. this indicated an influence on the increase in radicle length primed using spirulina platensis biomass. spirulina biomass contains phytohormonal compounds that can enhance plant growth, including auxin. in line with the research by arahou et al. (2023), it is inferred that microalgae have active molecules in the form of amino acids, polysaccharides, and phytohormones that can promote plant growth and increase tolerance to biotic and abiotic stress. in the water treatment and soaking durations of 1, 2, and 3 hours, the radicle lengths were 11,32; 13,34; and 10,09 cm, respectively, while the control was 11,67 cm. based on the statistical test results, the water treatment with a soaking duration of 2 hours showed a significant effect on radicle length compared to the control. this indicated that the application increases the radicle length in cucumber seeds. although the application of seed priming with water treatment for 1 hour experienced a slight decrease, the radicle length produced at that concentration did not significantly decrease compared to the control. priming is performed to hydrate the seeds before germination, aiming to balance the water potential to activate metabolic activities within the seeds. according to rocha et al. (2019), seed priming can maximize germination, resulting in increased germination index, improved seedling strength, and enhanced shoot and radicle length. based on the results of the analysis, the seed priming treatment using spirulina platensis biomass at concentrations of 30% and 45% for a soaking duration of 3 hours and 60% concentration for 2 hours, showed an increase in radicle length of 13,60; 12,63; and 13,11 cm. these applications indicated a higher increase in radicle length compared to the control. additionally, 30% spirulina platensis biomass seed priming for 3 hours showed the most effective treatment for increasing radicle length compared to other treatments. rahmawati et al. – the potential of spirulina platensis biostimulan as a … 139 dry weight of normal seedlings figure 10. dry weight of normal cucumber seedlings under various biomass concentrations and soaking time. observation data were taken on day 8 (final count) of the dry weight criteria for normal seedlings. the data above represent the mean value with sem error bars from the anova analysis with the bonferroni post hoc test compared to the control (untreated group). values without an asterisk are considered nonsignificance. significant: * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). the dry weight of normal seedlings is one of the benchmarks used in seed viability testing. dry weight of normal seedlings reflects an important growth factor that determines crop yield. dry weight of normal seedlings showed viability that reflects the amount of food reserves available in the seeds (rolin et al., 2024). the dry weight of normal seedlings is often referred to as the dry weight of seedling cotyledons, which indicates the assimilate content and food reserves. based on figure 10, the analysis of variance results showed that the treatment with spirulina platensis biomass at concentrations of 30%, 45%, and 60% and soaking times of 1, 2, and 3 hours on cucumber seeds has no significant effect on the dry weight of normal seedlings parameter. meanwhile, the control (untreated seeds) showed the dry weight of normal seedlings value of 0,97 grams. the water treatment for 1, 2, and 3 hours showed dry weight of normal seedlings values of 0,96; 0,97; and 0,96 grams, respectively. meanwhile, the seed priming application with 30% spirulina platensis biomass for 1, 2, and 3 hours showed dry weight of normal seedlings values of 0,96; 0,97; and 0,98 grams, respectively. the 45% concentration for 1, 2, and 3 hours showed dry weight of normal seedlings values of 0,96; 0,98; and 0,95 grams, respectively. moreover, the 60% concentration with soaking times of 1, 2, and 3 hours showed dry weight of normal seedlings values of 0,96; 0,97; and 0,96 grams, respectively. although it showed no significant effect, based on these values, no significant decrease in the dry weight of normal seedlings was found between the different treatments. according to the study by ratnaningtyas et al. (2019), the organ matrix priming treatment with fermented bean seedlings on chili seeds exhibited no significant difference in the dry weight of normal seedlings, indicating that the seedlings were capable of translocating food reserves. in addition, the priming treatment with spirulina platensis indicated no harmful or toxic effects were observed on seedling growth, as evidenced by the absence of a decrease in the dry weight of normal seedlings in each treatment. conclusions spirulina platensis can thrive in sme-based media, which was characterized by an increase in cell size and a color change in the cell culture from light to dark green, indicating an increase in microalgal cell density. the treatment of spirulina platensis biomass at a concentration of 45% for 1 and 3 hours increased the vigor index, seed growth rate, and simultaneous growth rate of cucumber seedlings. in the treatment with a 60% concentration and soaking times of 1, 2, and 3 hours, there was an increase in the parameters of mean germination time, vigor index, seed growth rate, and shoot length. although the 45% and 60% treatments showed no significant differences in the parameters of germination rate, maximum growth potential, and dry weight of normal seedlings, these applications indicated no decrease in seed germination. this indicates that seed priming treatment with spirulina platensis is not toxic to seedling growth, as evidenced by the absence of a decrease in germination rate, maximum growth potential, and normal dry weight of seedlings in each treatment. additionally, this research explored the potential of microalgae grown on sme-based media and its application to enhance plant growth. the significant improvement in seed growth parameters achieved through spirulina platensis-based priming demonstrates its prospect to enhance crop yield, contribute to global food security, and promote sustainable agricultural practices. acknowledgments: the author expresses the most profound gratitude to the supervising lecturer and colleagues who have guided, directed and assisted in the implementation of this research until its completion. authors’ contributions: the first author designed the research and prepared the manuscript, the second and third authors conducted the experiments, the fourth and fifth authors interpreted the data, and the last author, as an principal investigator, supervised, directed and guided the experiments. competing interests: the author has no conflicts of interest. funding: the author would like to express their heartfelt gratitude to the ministry of education, culture, research, and technology (kemendikbutristek) and politeknik negeri jember for their assistance with facilities and 140 biology, medicine, & natural product chemistry 14 (1), 2025: 129-141 student research project funding 2024 entitled “caps (carbon capture for seed priming using spirulina): the potential of spirulina platensis biostimulant as a sugar mill effluent seed priming 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on seed germination. 72:119–126. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 5, number 1, 2016 | pages: 19-22 | doi: 10.14421/biomedich.2016.51.19-22 modified alizarin red s-alcian blue staining for reptilian skeleton muhammad ja’far luthfi biological department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: jafarluthfi@yahoo.com abstract skeletal staining is an important method in anatomical study. the aim of the research was to develop staining and clearing method of reptilian skeleton using alizarin red s-alcian blue. the specimen were eviscerated, fixed, stained, cleared, and keep in glycerine solution. this modified double-staining has successfully stain bone and cartilage of reptilian. keywords: skeleton, alizarin red s, alcian blue, reptilia, double-staining introduction skeleton are supporting system for vertebrate body. the skeleton support body, provides for attachment muscles, houses for brain and nerve, and serve for locomotion (kardong, 2002). staining of animal skeleton is important in anatomical study. a lot of papers report methods for skeleton staining. clearing and staining of skeletal system are aimed for demonstrate of animal bone and cartilage. this is a routine method in teratological study and has been widely used for study in fetal and young animal, especially rodent (erdogan et al., 1995). alizarin red s-alcian blue is one of the most successful double staining methods for fetal and young rodent skeleton. application of this method in mature reptile, however, is still scarce (saralamoli et al., 2015). this paper will explain modified alizarin red s-alcian blue for successful staining in mature reptilian skeleton. materials and methods animals used in this study are ahaetulla prasina, gecko gecko, trachemys scripta elegans, and draco volans. equipments used in this study are the surgical scissors, scalpels, and a glass container (jar). materials used in this study are alizarin red s-alcian blue, ethanol, distilled water, glacial acetic acid, glycerin and koh. the method described in this paper is a modification from inouye (1976) that has been used on small vertebrates. inouye was the first researcher who successfully use staining methods for bone and cartilage at once with satisfactory result. inouye (1976) use a double staining method for bone and cartilage in mice for teratological study. using this method cartilage stained blue, bone stained red, whereas muscles and other tissues are transparent. appication of this method for mature and larger vertebrate required a modification of flaking skin and muscles into thinner fragments, aditional and prolonged fixation, fat removal, prolonged staining, and purification. these are because adult tissue and larger vertebrates have massive muscle, thicker skin and dense connective tissue, thus staining and clearing process are more difficult. staining and clearing reptilian specimens includes several stages. the first step is exfoliation. after the scales removed, the specimens were fixed in glass tubes containing 95% alcohol for 3 days. after the skin and muscle in the specimen removed by scissors and a scalpel, the specimen immersed again in the new 95% ethanol for 3 days. to remove fat, specimens are put in tubes containing acetone for 4 days with new acetone replacement on the second day. after that the specimens were kept for 5 days in a solution of the dye at a temperature of 37 ° c (1 volume of 0.3% alcian blue in 70% ethanol + 1 volume of 0.1% alizarin red s in 95% ethanol + 1 volume of glacial acetic acid + 17 volume 70% ethanol). the specimen is washed in water and then soaked in a solution of 1% koh for 5 days (until transparent) and then shaked on a shaker in a mixture of glycerin and koh 1% with a ratio of 20%: 80%; 50%: 50%; 80%: 20%. then the specimens are preserved in pure glycerine. result and discussion this research has succeeded in developing a method for staining and clearing reptile skeleton specimen skeleton (bone and cartilage) with alizarin red s alcian blue (figure 1, figure 2, figure 3, figure 4 and figure 5). figure 1 and figure 2 shows the skeleton of a snake ahaetulla prasina. the preparation showed the bones of the skull and vertebrae. the snake specimen shows a special adaptation of vertebrate skeleton, the vertebrae are huge numbers (+ 300 vertebra vertebrae) suitable for terrestrial life without limbs. figure 3 shows tail skeleton of gecko gecko. the preparation shows the 20 biology, medicine, & natural product chemistry 5 (1), 2016: 19-22 structure of the tail vertebrae in autotomous animals that can spontaneously casting off their tail. figure 4 shows the tail skeleton of turtles (trachemys scripta elegans). figure 5 shows the tail skeleton of draco volans. the turtle and draco volans shows the structure of nonautotomous tail vertebrae. figure 1. structure of snake ahaetulla prasina. note skull and spine with ribs. figure 2. high magnification of skeletal structure of snake ahaetulla prasina. muhammad ja’far luthfi. – modified alizarin red s-alcian blue staining for reptilian skeleton 21 figure 3. high magnification of tail skeleton of gecko gecko. note the presence of autotomous split in the plain where the tail broke in autotomy. figure 4. high magnification of tail skeleton of turtles. turtles cannot autotomized their tail therefore no autotomous split in this vertebrae. figure 5. high magnification of tail skeleton of draco volans. 22 biology, medicine, & natural product chemistry 5 (1), 2016: 19-22 the results show that all specimen are clearly visible. bones stained red whereas cartilage stained blue. muscle and connective tissue are transparent/clear by a clearing process with koh. from the specimens we can observe the vertebrae and its processus, intervertebral discs, ribs, autotomus split, and other details. in this method, the most important aspect for successful staining is exfoliation of skin and muscle, as well as fixation stages. remove the skin and muscle as much as possible. but it should be noted that exfoliation skin and muscles are not cut the skeleton itself. vertebrae have many processes. it is often that the disposal of the skin and muscle also cutting processus. fixation and the removal of fat must be prolonged compared to inouye method for fixative solution penetrate the tick tissue. different specimen takes different times for fixation period, the removal of fat, staining and clearing. it depends on the species, maturity (embryonic or adult), body size, and organ/body part made preparation. the more ticker and mature the tissue, the more times are needed. in general, larger animals and more mature require fixation longer period, longer removal of fat, longer staining and and longer clearing. duration time of fixation, removal of fat, staining and clearing in this study can be used as a guidance in application of this method to other animals. whole body staining and clearing method have certain advantages over methods in which the carcass is macerated and the bones are separated and dried. among these advantages are: (1) there is no chance of losing the small bones, (2) all bones are retained in their original position, (3) there is no chance of wrongly identifying similar bones, (4) in the finished preparations the bones, after identification, may be disarticulated and examined from all angles, equally as well as in dried preparations, and (5) many animals may be processed together without danger of mixing their bones, a great saving in time and effort (green, 1952). conclusion this research has successfully modified alizarin red salcian blue to applied on larger and mature animal (reptilia) with satisfied result. references erdogan, d., kadioglu, d., peker, t. 1995. visualization of the fetal skeletal system by double staining with alizarin red and alcian blue. gazi medical journal 6: 55-58. salaramoli, j., f. sadeghi, h.gilanpour, m. azarnia, t. aliesfehani. 2015. modified double skeletal staining protocols with alizarin red and alcian blue in laboratory animals annals of military & health sciences research. 13 (2):76-81. green, m. c. 1952. a rapid method for clearing and staining specimens for the demonstration of bone. the ohio journal of science 52 (1): 31-33. inouye, m. 1976. differential staining of cartilage and bone in mouse skeleton by alcian blue and alizarin red s. congenital anomalies 16: 171-173. kardong, k.v. 2002. vertebrates comparative anatomy, function, and evolution. international edition. third edition. mcgraw-hill higher education. new york. usa. hal: 233236. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 585-592 | doi: 10.14421/biomedich.2023.122.585-592 issn 2540-9328 (online) in silico study on structural inhibition of bacterial dna gyrase by major secondary metabolites found in grape seed extract haritha lawan*, hashitha tharakee department of chemistry, university of kelaniya, sri lanka. corresponding author* harithajayasinghe01@gmail.com manuscript received: 27 july, 2023. revision accepted: 25 september, 2023. published: 07 november, 2023. abstract in the early 20th century, during the era of investigating and identifying essential “vitamins”, scientific research focused on grape seed extracts and their bioactive components, particularly polyphenols. extensive studies have demonstrated that grape seed extract, rich in proanthocyanidins, offers protection against a wide spectrum of diseases, encompassing inflammation, cardiac ailments, peptic ulcers, hypertension, diabetes, cancer, and microbial infections. to explore potential secondary metabolites within grape seed extract that could serve as structural inhibitors of bacterial dna gyrase, molecular docking studies were performed. the docking results revealed that two phytochemicals, namely (-)-catechin and procyanidin-b2, exhibited the highest potency in inhibiting dna gyrase subunit b. subsequent in silico physicochemical and pharmacokinetic parameter predictions were conducted using specialized web servers for the examined phytochemicals. notably, (-)-catechin displayed superior inhibitory and admet (absorption, distribution, metabolism, excretion, and toxicity) characteristics, suggesting its potential for utilization in synthesizing novel antibacterial compounds. keywords: grape seed extract; polyphenols; catechin; molecular docking; pharmacokinetic; physicochemical. introduction in recent years, extract from grape seed has grown in popularity as a dietary supplement, particularly in australia, korea, japan, and the united states (yamakoshi et al., 2002). this is due to the fact that grape seeds are high in phenolic compounds and may benefit human health in ways like preventing gastric ulcers (kim et al., 2013; rodríguez montealegre et al., 2006) grapevine seeds have a high antioxidant capacity; as a result, they can reduce the activity of antioxidant enzymes, protect cells from oxidative damage, have antiinflammatory and anti-atherosclerotic properties, and prevent certain cancers in both humans and animals (melov, 2002). based on promising reports of their antioxidant capabilities and capacity to act as free radical scavengers, the polyphenols and flavonoids found in the grape seed extract have attracted great attention (georgiev et al., 2014). the hunt for novel medications in the area of secondary plant metabolites as compounds for curing civilizational disorders has recently been the focus of pharmaceutical and commercial trends. the grapevine (vitis vinifera l.) seed's matrix contains significant physiologically active components that allow for its application in medicine (sochorova et al., 2020) proanthocyanidins and catechins are two types of flavan3-ol that are abundant in grape seeds. they have significant concentrations of catechin and epicatechincontaining polyphenol proanthocyanidins, which are oligomers of flavan-3-ol molecules (weseler & bast, 2017). grape seeds have demonstrated potential as novel microbial agents mainly due to their abundance of polyphenols. defatted grape seed extracts were demonstrated to have antibacterial action against bacillus cereus, bacillus subtilis, staphylococcus aureus, bacillus coagulans, escherichia coli, and pseudomonas aeruginosa, according to jayaprakasha et al (jayaprakasha et al., 2003). a second study by brown et al. found that muscadine grape seed extracts were efficient in suppressing h. pylori in vitro (brown et al., 2009). resveratrol exhibits antimicrobial activity by causing oxidative damage to bacterial membranes without harming host cells, particularly in e. coli. these findings provided insight into the potential of resveratrol to support conventional treatments when antibiotics failed or proved inefficient (subramanian et al., 2014) the polymeric phenolic fractions of grape seed extract also showed the strongest specific inhibitory activity for practically all listeria species; it has been reported (rhodes et al., 2006). https://doi.org/10.14421/biomedich.2023.122.585-592 586 biology, medicine, & natural product chemistry 12 (2), 2023: 585-592 a b c figure 1. a. cryoem structure of the complete e. coli dna gyrase complex bound to a 130 bp dna duplex pdb doi: 10.2210/pdb6rkw/pdb b. crystal structure of e. coli gyrase subunit b c. ramachandran plot of dna gyrase subunit b. dna gyrase is a type ii topoisomerase that regulates the topological state of dna in cells. it is made up of the subunits gyra and gyrb. (figure 01) (wang, 2009). dna supercoiling, which is necessary to maintain dna topology during replication, is paired with atp hydrolysis by the gyrb subunit via dna gyrase. across all bacterial species, it is a crucial enzyme whose inhibition causes a disturbance in dna synthesis and, ultimately, cell death. antibacterial medications have long been known to have an interest in the target dna gyrase (maxwell & lawson, 2003). quinolones and aminocoumarins are two families of antibiotics that have demonstrated in clinical studies that dna gyrase is a workable target (drlica et al., 2008). materials and methods data collection and ligand preparation a literature survey was done to select the suitable secondary metabolites. the 3d chemical structures of the selected secondary molecules were retrieved from pubchem database (pubchem (nih.gov)) in .sdf format. they were imported into avogadro v.1.2.0 software and adjusted to physiological protonation states followed by energy minimization in the universal force field (conjugate gradient algorithm for 500 steps). prepared ligands were saved in pdbqt format. openbablegui is used for extension conversation. protein preparations crystal structure of e. coli gyraseb (24kda) – pdb id6f86 were obtained from protein data bank in .pdb format. protein preparation was done using autodocktools version 1.5.6. the water molecules and heteroatoms (cww) were deleted. missing atoms were repaired. polar hydrogens and kollman charges were added to the protein. molecular docking the grid parameters and map files were created using autogrid 4.2. to perform the blind docking. (genetic algorithm parameters settings: number of genetic algorithm (ga) runs: 50, population size: 300, the maximum number of evaluations: 25 000 000 (medium), and maximum number of generations: 27 000). molecular docking was performed using autodock 4.2, and results were generated in .dlg format (output – lamarckian ga-4.2). interaction visualization and analysis docking results were analyzed using autodocktools version 1.5.6 to examine the binding energies and inhibition constants. binding interactions with the ligand were visualized using ucsf chimera 1.16 and pymol v.2.5.4. protein-ligand interaction profiler (plip welcome (tu-dresden.de)) and proteins.plus (zentrum für bioinformatik: universität hamburg proteins plus server) web servers were also used to further analyze the binding residues. in silico physicochemical and pharmacokinetic parameter prediction veber's rules (veber et al., 2002) and lipinski's rule of five (lipinski et al., 2001) were applied to evaluate druglikeness using the swissadme server (swissadme) . secondary metabolites given in table 1 were analyzed for their physicochemical properties, and the results are epitomized in table 2. the admet characteristics of the secondary metabolites were studied using the pkcsm server (https://biosig.lab.uq.edu.au/pkcsm/) to understand their pharmacokinetic parameters, and the results are given in table 3. http://doi.org/10.2210/pdb6rkw/pdb https://pubchem.ncbi.nlm.nih.gov/ https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index https://proteins.plus/ https://proteins.plus/ https://proteins.plus/ http://www.swissadme.ch/index.php https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/absorption-distribution-metabolism-excretion-toxicity https://biosig.lab.uq.edu.au/pkcsm/ lawan & tharakee – dna gyrase inhibition by grape metabolites 587 results and discussion table 1. calculated binding energies and inhibition constants of a. ciprofloxacin-a and b. selected phytochemicals-b. a. reference compound binding energy / kcal/mol ki-inhibition constant/ µm ciprofloxacin -6.19 28.80 b. compound binding energy / kcal/mol ki-inhibition constant /µm compound binding energy / kcal/mol ki-inhibition constant /µm (-)-catechin -6.22 27.52 epigallocatechin -5.11 180.46 (-)-epicatechin -5.32 95.83 procyanidin-b3 -5.59 80.03 epicatechin-3-gallate -4.46 540.36 procyanidin-b2 -6.31 23.67 procyanidin-b1 -5.05 198.11 procyanidin-b4 -5.60 77.90 (+)-gallocatechin -5.31 127.21 quercetin -5.36 117.82 588 biology, medicine, & natural product chemistry 12 (2), 2023: 585-592 a. b. c. d. e. f. g. h. i. j. k. figure 2. bonding interaction within the binding pocket of dna gyrb with a. ciprofloxacin b. (-)-catechin c. epicatechin d. epicatechin e. procyanidinb1 f. (+)-gallocatechin g. epigallocatechin h. procyanidin-b3 i. procyanidin-b2 j. procyanidin-b4 k. quercetin that generated using protein-ligand interaction profiler. a. b. c. d. e. f. g. h. i. j. k. figure 3. 3d visualization of the ligand-dna gyrb complexes, a. ciprofloxacin b.(-)-catechin c. epicatechin d. epicatechin e. procyanidin-b1 f. (+)gallocatechin g. epigallocatechin h. procyanidin-b3 i. procyanidin-b2 j. procyanidin-b4 k. quercetin obtained from protein-ligand interaction profiler. lawan & tharakee – dna gyrase inhibition by grape metabolites 589 a. b. c. d. e. f. g. h. i. 590 biology, medicine, & natural product chemistry 12 (2), 2023: 585-592 j. k. figure 4. diagram showing the amino acid residues that interact with ligand, a. ciprofloxacin b. (-)-catechin c. epicatechin d. epicatechin e. procyanidinb1 f. (+)-gallocatechin g. epigallocatechin h. procyanidin-b3 i. procyanidin-b2 j. procyanidin-b4 k. quercetin that generated using protein-ligand interaction profiler. table 2. predicted pharmacokinetic properties of studied secondary metabolites; 1-water solubility (log mol/l) 2-human intestinal absorption (% absorbed) 3-volume of distribution at steady-state (log l/kg) 4-fraction unbound (fu) 5-blood brain barrier permeability (log bb) 6-cytochromes p450 substrates 7cytochromes p450 inhibitors 8total clearance (log ml/min/kg) 9renal organic cation transporter 2 substrate 10ames toxicity 11max. tolerated dose (human) (log mg/kg/day) 12hepatotoxicity. compound absorption distribution metabolism excretion toxicity ws1 hia2 vdss3 fu4 bbb5 cyps6 cypi7 tc8 ros9 ames10 mtd11 ht12 ciprofloxacin -2.678 94.828 0.293 0.404 -0.733 3a4 0.505 no yes -0.283 yes (-)-catechin -3.024 72.536 0.589 0.152 -1.278 0.286 no yes 0.516 no (-)-epicatechin -3.024 72.539 0.589 0.152 -1.278 0.286 no yes 0.516 no epicatechin-3-gallate -2.907 66.671 0.051 0.283 -1.898 1a2 0.016 no yes 0.537 no procyanidin-b1 -2.892 64.165 0.036 0.386 -2.263 0.289 no no 0.411 no (+)-gallocatechin -2.952 60.140 0.520 0.156 -1.598 0.449 no no 0.755 no epigallocatechin -2.952 60.140 0.520 0.156 -1.598 0.449 no no 0.755 no procyanidin-b3 -2.892 64.165 0.036 0.386 -2.263 0.289 no no 0.411 no procyanidin-b2 -2.892 64.165 0.036 0.386 -2.263 0.289 no no 0.411 no procyanidin-b4 -2.892 64.165 0.036 0.386 -2.263 0.289 no no 0.411 no quercetin -2.982 74.840 0.310 0.128 -1.377 1a2 0.624 no yes 0.954 no table 3. predicted physicochemical properties of studied secondary meabolites ; 1molecular weight (g/mol) 2num. heavy atoms 3num. rotatable bonds 4num. h-bond acceptors 5num. h-bond donors 6-topological polar surface area(å²) 7octanol/water partition-coefficient 8-lipinski’s rule of five 9veber’s rule. compound mw1 n-nha2 n-rot3 n-hba4 n-hbd5 tpsa6 milogp7 lpi8 ver9 bas10 ciprofloxacin 331.34 24 3 5 2 74.57 1.28 yes yes 0.55 (-)-catechin 290.27 21 1 6 5 110.38 0.24 yes yes 0.55 (-)-epicatechin 290.27 21 1 6 5 110.38 0.24 yes yes 0.55 epicatechin-3-gallate 442.37 32 4 10 7 177.14 0.32 yes no 0.55 procyanidin-b1 578.52 42 3 12 10 220.76 -0.26 no no 0.17 (+)-gallocatechin 306.27 22 1 7 6 130.61 -0.29 yes yes 0.55 epigallocatechin 306.27 22 1 7 6 130.61 -0.29 yes yes 0.55 procyanidin-b3 578.52 42 3 12 10 220.76 -0.26 no no 0.17 procyanidin-b2 578.52 42 3 12 10 220.76 -0.26 no no 0.17 procyanidin-b4 578.52 42 3 12 10 220.76 -0.26 no no 0.17 quercetin 302.24 22 1 7 5 131.36 -0.56 yes yes 0.55 lawan & tharakee – dna gyrase inhibition by grape metabolites 591 discussion in this study, ciprofloxacin was used as the reference compound, the known bacterial dna gyrase subunit b inhibitor, to compare the binding energies and inhibition constants with the examined secondary metabolites. the concentration needed to cause half of the maximum inhibition is known as the inhibition constant (ki), which serves as a measure of an inhibitor's potency. when comparing the binding energies of docked ligands and reference drug, (-)-catechin (-6.22 kcal/mol) and procyanidin-b2(-6.31 kcal/mol) exhibit even better binding affinity to the target protein than ciprofloxacin (6.19 kcal/mol). (table 01) also, those two compounds show lower ki values than ciprofloxacin, indicating better potency towards the gyrb inhibition. it was evident that the top-ranked phytochemical structures' hydroxyl groups play a substantial role in the establishment of potent hydrogen bonds with amino acid residues in the binding pockets. (figure 02) when considering the binding interactions of those three ligands, ciprofloxacin shows only one hydrogen bonding interaction with the protein (with asn46). but ()-catechin and procyanidin-b2 shows six (with val43, asp72, gly77 & glu50) and seven (with ile94, val120, asn46, asp49, glu50, thr165 & asp73) (figure 3) hydrogen bonding interactions with the target protein, respectively. epicatechin-3-gallate shows the lowest (4.46 kcal/mol) binding affinity towards the target protein and the highest inhibition constant (540.36 µm) from the examined ligands. although it is not mush as (-)-catechin and procyanidin-b2, phytochemicals procyanidin-b3(5.59 kcal/mol), b4(-5.60 kcal/mol), (-)-epicatechin (5.32 kcal/mol) and quercetin (-5.36 kcal/mol) also exhibits good binding capabilities. major types of binding interactions between the protein and the ligands are h-bonding and hydrophobic interactions. other than that, π-cation interactions also can be observed in two cases which are (-)-epicatechin and procyanidin-b3. interestingly in both cases, π-cation interactions have occurred between the aromatic rings in the ligands and the arg76 amino acid. from the leading phytochemicals observed with the highest binding affinities, (-)-catechin meets lipinski's and veber’s rules, fulfilling strong physicochemical requirements (table 3) as a potential drug. also (-)catechin shows a satisfactory percentage of absorption through the human intestinal tract. another important characteristic is that all the screened phytochemicals from grape seed extract don't show hepatotoxicity (table 2), even though the well-known commercially available antibiotic ciprofloxacin shows some level of hepatotoxicity. in pharmacokinetics and toxicity analysis, it is categorized that values between -4 and -2 log mol/l as water “soluble “compounds. interestingly all the compounds examined are coming under that category. out of the phytochemical from the grape seed extract assessed, no chemical acted as a substrate for cytochromes p450 isozymes but the reference antibiotic. however, predicted data revealed that epicatechin-3gallate and quercetin have the potential to inhibit cyp1a2 isozyme. conclusions selected secondary metabolites found in grape seed extract were tested in silico for their potential inhibitory action towards dna gyrase enzyme, pharmacokinetic and physicochemical properties, and compared with the commercial antibiotic, ciprofloxacin. out of the tenphytochemical assessed, (-)-catechin and procyanidin-b2 indicated the best receptor inhibition capabilities. comprehensive docking, pharmacokinetic, and physicochemical tests indicated that (-)-catechin would be a viable dna gyrase b inhibitor because it binds firmly to the binding pocket, inhibiting the native conformation of the protein. hence, (-)-catechin is an exceptional candidate for further in vitro/in vivo studies. authors’ contributions: p.l.m.j.h. lawan and d.h. tharakee designed the study, carried out computational studies, and analyzed the data. p.l.m.j.h. lawan and d.h. tharakee wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references brown, j. c., huang, g., haley-zitlin, v., & jiang, x. (2009). antibacterial effects of grape extracts on helicobacter pylori. applied and environmental microbiology, 75(3), 848–852. https://doi.org/10.1128/aem.01595-08 drlica, k., malik, m., kerns, r. j., & zhao, x. (2008). quinolonemediated bacterial death. antimicrobial agents and chemotherapy, 52(2), 385–392. https://doi.org/10.1128/aac.01617-06 georgiev, v., ananga, a., & tsolova, v. (2014). recent advances and uses of grape flavonoids as nutraceuticals. nutrients, 6(1), 391–415. https://doi.org/10.3390/nu6010391 jayaprakasha, g. k., selvi, t., & sakariah, k. k. (2003). antibacterial and antioxidant activities of grape (vitis vinifera) seed extracts. food research international, 36(2), 117–122. https://doi.org/10.1016/s0963-9969(02)00116-3 kim, t. h., jeon, e. j., cheung, d. y., kim, c. w., kim, s. s., park, s.-h., han, s. w., kim, m. j., lee, y. s., cho, m.-l., chang, j. h., min, j. k., & kim, j. il. (2013). gastroprotective effects of grape seed proanthocyanidin extracts against nonsteroid anti-inflammatory drug-induced gastric injury in rats. gut and liver, 7(3), 282–289. https://doi.org/10.5009/gnl.2013.7.3.282 lipinski, c. a., lombardo, f., dominy, b. w., & feeney, p. j. (2001). experimental and computational approaches to estimate solubility and permeability in drug discovery and 592 biology, medicine, & natural product chemistry 12 (2), 2023: 585-592 development settings 1pii of original article: s0169409x(96)00423-1. the article was originally published in advanced drug delivery reviews 23 (1997) 3–25. 1. advanced drug delivery reviews, 46(1–3), 3–26. https://doi.org/10.1016/s0169-409x(00)00129-0 maxwell, a., & lawson, d. (2003). the atp-binding site of type ii topoisomerases as a target for antibacterial drugs. current topics in medicinal chemistry, 3(3), 283–303. https://doi.org/10.2174/1568026033452500 melov, s. 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(2017). masquelier’s grape seed extract: from basic flavonoid research to a well-characterized food supplement with health benefits. nutrition journal, 16(1), 5. https://doi.org/10.1186/s12937-016-0218-1 yamakoshi, j., saito, m., kataoka, s., & kikuchi, m. (2002). safety evaluation of proanthocyanidin-rich extract from grape seeds. food and chemical toxicology, 40(5), 599–607. https://doi.org/10.1016/s0278-6915(02)00006-6 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 323-328 | doi: 10.14421/biomedich.2023.121.323-328 issn 2540-9328 (online) uncovering the antioxidant power: investigating the skin and flesh of crystal guava with chloroform and methanol extractions and dpph assay aulia ulmillah1,*, arif alghifari2, nurhaida widiani2 1department of biology; 2department of biological education, faculty of tarbiyah and education, uin raden intan lampung jl. let.kol endro suratmin, sukarame, bandar lampung, 35131, indonesia. corresponding author* aulia@radenintan.ac.id manuscript received: 30 january, 2023. revision accepted: 13 march, 2023. published: 30 march, 2023. abstract crystal guava (psidium guajava var. crystal) is one type of guava plant that has high economic value due to its thick flesh and few seeds. the crystal guava plant is believed to have antioxidant activity, which is a substance that can prevent the formation of free radicals in the body. this research aims to determine the level of antioxidants in chloroform and methanol extracts of the skin and flesh of crystal guava fruit using the dpph method. samples were taken through a stepwise maceration process and solvents of chloroform and methanol, then analyzed using probit analysis and spss 25 software. the results of the study showed that both chloroform and methanol extracts of the skin and flesh of crystal guava fruit have antioxidant activity. the results showed that the ic50 value of the chloroform extract of crystal guava fruit skin is 218.88 ppm and is classified as moderate, the methanol extract of crystal guava fruit skin is 89.78 ppm and is classified as strong, the chloroform extract of crystal guava fruit flesh is 270.56 ppm and is classified as weak, and the methanol extract of crystal guava fruit flesh is 185.72 ppm and is classified as moderate. keywords: antioxidant; dpph; chloroform; crystal guava; fruit flesh; methanol; peel. introduction one horticultural product that is widely found and used in tropical countries is the crystal guava. this guava plant is widely cultivated by farmers and bears fruit throughout the year. in addition to its fruit, almost every part of the guava plant can be utilized. guava fruit is commonly consumed to help improve digestion and lower blood sugar levels. guava leaves can even be used as medicine to treat diarrhea, vomiting, and sore throat. guava is also easily found in the market at affordable prices (rustani and susanto, 2019). crystal guava, which is rich in vitamin c and has a high economic value, is very suitable for agro-tourism development programs (haidawati et al., 2015). vitamin c is one of the natural antioxidants found in fruit, in addition to vitamin e, polyphenols, carotenoids, and flavonoids (febrianti et al., 2016). it can not only be consumed directly but crystal guava can also be used for other purposes and has its own advantages compared to other types of guava. crystal guava is believed to have a lot of phytochemicals such as flavonoids, polyphenols, isoflavonoids, and tannins in its leaves and fruit, providing natural antioxidants. flavonoids are the main bioactive compounds found in guava leaves, while the majority of vitamin c content is found in the fruit skin (jamieson et al., 2021). guava leaves also contain quercetin, which has antioxidant properties and is considered the most active and strong antioxidant in guava seed leaves (sharma and borah, 2021). the presence of natural antioxidants plays an important role in managing various types of degenerative diseases caused by free radicals. free radicals are small molecules with unpaired electrons, making them highly reactive (hartati et al., 2020). their reactive nature causes free radicals to seek out electrons in other compounds, forming new chains of radicals and damaging the structure of certain compounds (setiabudi et.al., 2020). free radicals can also continuously form in the body, so antioxidants are needed to balance them. antioxidants can reduce the negative impact of many free radicals. free radicals can be neutralized by antioxidants through electron donors, making free radicals more stable and less reactive. the presence of antioxidant compounds prevents free radicals from bonding with electrons in other compounds, as their electrons are already bonded with electrons from the antioxidants (handayani et al., 2020). therefore, antioxidant compounds play an important role in the human body. https://doi.org/10.14421/biomedich.2023.121.323-328 324 biology, medicine, & natural product chemistry 12 (1), 2023: 323-328 the testing of antioxidant levels in crystal guava is widely conducted. typically, the antioxidant levels test uses ethanol as the solvent in extraction and the maceration method. in this research, extraction was conducted using chloroform and methanol solvents. the use of chloroform and methanol as solvents is based on their different polarities. chloroform is non-polar, while methanol is polar. the principle of solubility says that polar solvents will dissolve polar compounds, and nonpolar solvents will dissolve non-polar compounds. with the use of different solvents, it is hoped that the observed components will be separated according to their level of polarity (taroreh et al., 2015). the compounds that have the potential as antioxidants are flavonoids and phenols, which are included in the polar fraction (yusriyani and syarifuddin, 2021). plants also contain non-polar compounds such as waxes, lipids, and proteins (dewitasari, 2020). non-polar solvents such as chloroform are needed to cleanse polar antioxidant compounds to be more optimally absorbed by polar solvents such as methanol. if non-polar compounds such as proteins and lipids are not cleaned by non-polar solvents, it will interfere with the process of capturing free radicals by flavonoid compounds (rahmadani and nasution, 2021). the maceration method used is a stepwise maceration, so this is one of the innovations in this research. this study aims to determine the antioxidant level in chloroform and methanol extracts of crystal guava skin and flesh using the dpph method. materials and methods materials the equipment used in this research includes an oven, electric balance, filter paper, hot plate, blender, reaction tube, dropper, maceration bottle, knife, distillator, rotary evaporator, uv-vis spectrophotometer (shimadzu), vortex, reaction tube rack, and plastic bags. the materials used in this research include the skin and flesh of crystal guava, methanol, p.a ether, water, ethyl acetate, 1,1diphenyl-2-picrylhydrazyl (dpph), ascorbic acid, and chloroform. the guava fruit used in this study was those with a green-yellow and green maturity level. this is because the fruit with green-yellow maturity has the highest vitamin c content compared to fruits that are green and bright green. (dewi et al., 2017) figure 1. crystal guava (psidium guajava var. crystal) (source: personal documentation). procedures preparation of simplicia and extraction 20 kg of crystal guava fruits is peeled and separated between the skin and flesh. the skin and flesh are washed and drained. then, they are cut and dried in an oven at 450°c until dry. in the next step, the dried samples are blended into a fine powder and sieved using a 25-mesh sieve. the prepared crystal guava skin and flesh simplicial will be extracted using a sequential maceration method. the solvents used for sequential maceration are chloroform and methanol. both solvents are used with a ratio of each solvent to a simplicial of 1:2. extraction is performed by weighing 125 grams of guava skin and flesh simplicial and placing them into a container. then, it is poured with 250 ml of chloroform solvent, covered and allowed to stand for 24 hours in a light-protected condition, and stirred every 8 hours. afterward, the macerate is collected and concentrated using a rotary evaporator to form a concentrated extract. to make a methanol extract, the dried chloroform extract residue is processed in the same way as the chloroform extraction, but using methanol solvent. the extract is then concentrated again using a rotary evaporator to make a concentrated methanol extract. the yield of the extract was calculated using the formula (edison et al. 2020): % yield = weight of extract (𝑔) weight of sample (𝑔) x 100% antioxidant testing 25 mg of each dried sample was weighed and dissolved in p.a methanol to make the volume 50 ml (500 ppm). then, a series of concentrations of the three samples ulmillah et al. – uncovering the antioxidant power: investigating the skin and … 325 were created, namely 50 ppm, 100 ppm, 150 ppm, 200 ppm, and 250 ppm. the testing process was carried out by adding 0.5 ml of the solution from the two sample extracts with various concentrations. then, each was added with 3.5 ml of 50 ppm dpph. the mixture was then vortexed and incubated for 30 minutes in a dark environment and at 37°c. its absorbance was then measured at a wavelength (λ) of 517 nm. an anti-oxidant control test was used using ascorbic acid at 10 mg weighed and dissolved in p.a methanol to have a concentration of 100 ppm. then, a series of ascorbic acid concentrations were made with concentrations of 5 ppm, 10 ppm, 15 ppm, 20 ppm, and 25 ppm. the test was then carried out by pipetting 0.5 ml of the sample solution from different concentration series. each solution was then added with 3.5 ml of 50 ppm dpph. the mixture was then vortexed and incubated for 30 minutes at 37°c and in a dark room. after that, its absorbance was measured using a spectrophotometer at a wavelength of 517 nm. the anti-oxidant activity was calculated using the following formula (febrianti and ariani, 2020): inhibition (%) = absorbance of blank − absorbance of sample absorbance of blank 𝑋 100 % the ic50 value was obtained from linear regression analysis by substituting the value of y as 50 from the equation y = a + bx. the smaller the ic50 value, the greater the antioxidant activity of a substance (martiningsih et al., 2016). data analysis the uv-vis spectrophotometry measurement data was were analyzed using the linear regression analysis method (anova) with the help of spss 25 software at a 5% level to decide on the hypothesis proposed. a graph was made using the probit analysis between the log concentration of the sample as the x-axis and the percentage of antioxidant activity as the y-axis, to find the linear regression equation. thus, the ic50 can be found in the methanol and chloroform extracts. results and discussion extraction results the extraction conducted in the research produced four types of concentrated extracts, namely chloroform extract of the fruit skin, chloroform extract of the fruit flesh, methanol extract of the fruit skin, and methanol extract of the fruit flesh. the obtained concentrated extracts were then calculated for yield values as shown in table 1. the highest yield value was in the sample of chloroform extract of the fruit skin (5.92%), followed by chloroform extract of the fruit flesh (5.8%), methanol extract of the fruit skin (2.08%), and methanol extract of the crystal fruit flesh (1.68%). this is not directly proportional to the weight of the obtained concentrated extract. the result of the extraction from each 125 gram of the plant material showed that the highest extract was obtained from the methanol extract of the fruit skin (7.4 g), followed by the methanol extract of the fruit flesh (5.8 g), chloroform extract of the fruit skin (2.6 g), and chloroform extract of the fruit flesh (2.1 g). this result indicates that the extract produced from chloroform solvent resulted in a higher yield value compared to the yield value obtained from the extraction using methanol solvent. chloroform is nonpolar, while methanol is polar and a compound is separated based on its polarity (taroreh et al., 2015). therefore, it is possible that the group of compounds in both samples that were well absorbed in chloroform solvent were nonpolar compounds. table 1. extraction results using chloroform and methanol on skin and flesh of crystal guava fruit. no sample name weight of chloroform extract yield (%) methanol extract yield (%) simplicia (grams) chloroform extract (grams) methanol extract (grams) 1 skin fruit 125 2,6 7,4 5,92 2,08 2 flesh fruit 125 2,1 5,8 4,64 1,68 antioxidant test results the ic50 value was obtained by first conducting a probit analysis. the results obtained from the r value show that the probit data from the chloroform and methanol extracts from the skin and flesh of crystal guava are very good, as they approach the value of 1 (fadiyah et al., 2019). a positive value of +1 indicates that the higher the extract concentration, the higher the antioxidant activity. antioxidant activity is usually expressed in percentage of dpph reduction or with the ic50 value. the line equation obtained was used to calculate the ic50 value. after the calculation, the ic50 value for the skin of the crystal guava fruit is 218.88 ppm for the chloroform extract and 89.78 ppm for the methanol extract. 326 biology, medicine, & natural product chemistry 12 (1), 2023: 323-328 meanwhile, for the flesh of the crystal guava fruit, the ic50 value is 270.56 ppm for the chloroform extract and 185.72 ppm for the methanol extract, and 18.09 ppm for ascorbic acid. the results of the antioxidant test can be seen in table 2. table 2. ic50 values of antioxidants in chloroform and methanol extracts from skin and flesh of crystal guava fruit. sample concentration % inhibition log concentration probit ic50 value (ppm) skin fruit extract (c) 50 26,82 1,6990 4,36 218,88 100 27,78 2,0000 4,39 150 29,94 2,1761 4,45 200 30,23 2,3010 4,48 250 31,62 2,3979 4,50 skin fruit extract (m) 50 30,91 1,6990 4,48 89,78 100 33,95 2,0000 4,56 150 37,90 2,1761 4,67 200 39,46 2,3010 4,72 250 45,57 2,3979 4,87 flesh fruit extract (c) 50 31,20 1,6990 4,50 270,56 100 31,79 2,0000 4,50 150 33,75 2,1761 4,56 200 34,23 2,3010 4,59 250 35,18 2,3979 4,61 flesh fruit extract (m) 50 32,96 1,6990 4,53 185,72 100 33,83 2,0000 4,56 150 34,50 2,1761 4,59 200 38,08 2,3010 4,69 250 38,99 2,3979 4,69 ascorbic acid 5 44,19 0,6990 4,85 18,09 10 60,78 1,0000 5,25 15 77,28 1,1761 5,74 20 88,98 1,3010 6,18 25 96,39 1,3979 6,75 *(c) = chloroform, (m) = methanol the ic50 value shown in table 2 indicates that ascorbic acid as a comparison has the strongest antioxidant activity, this is due to ascorbic acid being a pure antioxidant compound. methanol extract from the skin and flesh of crystal guava fruit has a higher ic50 value compared to the chloroform extract from the skin and flesh of crystal guava fruit, as shown in figure 2. figure 2. the relationship between the concentration of chloroform and methanol extracts from the skin and flesh of the crystal guava fruit (psidium guajava var. crystal) and antioxidant activity. note skin 1= chloroform extract from the skin of the fruit; skin 2= methanol extract from the skin of the fruit; flesh 1= chloroform extract from the flesh of the fruit; flesh 2: methanol extract from the flesh of the fruit. discussion in the drying process, both samples were dried using an oven at a temperature of 45 degrees celsius for 150 hours until the dried simplicia, seen from the brownish color and easy to break. then, these dried samples were blended and filtered with the aim of breaking down the cells and expanding the sample surface so that it is easier to extract. the size of the fine powder produced makes it easier for the solvent to extract the contents directly because the larger the surface area and the more effective the interaction with the solvent (pertiwi et al., 2022). in this study, extraction was carried out using a multistep maceration method. maceration means soaking and is a method for isolating active substances using a solvent and stirring at room temperature several times over a certain period. maceration is a cold method that can protect secondary metabolites such as flavonoids from the effects of heating. the solvents used in the extraction are 75% chloroform and 75% methanol. the ulmillah et al. – uncovering the antioxidant power: investigating the skin and … 327 extraction was carried out through a multistep maceration method, which is slightly different from the ordinary maceration method. in this multi-step method, solvents with different polarity levels are used. the first extraction was carried out using a non-polar solvent, chloroform, and then the residue from the chloroform maceration was processed again using methanol as the solvent. the use of the multistep maceration method aims to extract active substances (antioxidants) more optimally. in addition, the use of solvents with different polarity levels also aims to extract active substances with different polarities, so that the extraction results can be better (kuspradini et al., 2016). antioxidants have polar properties, so the use of non-polar chloroform solvent in the initial extraction is intended to pull non-polar substances from the sample, so the sample will be free of non-polar substances. the second extraction using methanol will optimally pull polar substances, so when the dpph test is performed, non-polar substances will not interfere and affect the results that are not maximal. the extraction yields a concentrated extract which is then calculated for its yield value. the yield in this study was measured by comparing the dry extract mass (grams) with the initial mass of the material before the extraction process (grams). the dry extract was obtained after the sample was dried in an oven until it had a constant weight. this calculation was performed to determine the percentage of material that remained after the extraction process and to determine the level of effectiveness of the process produced (senduk et al., 2022). the yield value can also indicate the number of secondary metabolite compounds present in the sample. therefore, this calculation can serve as a reference in antioxidant testing or as a comparison of antioxidant testing results. the second antioxidant test of the two sample types used chloroform and methanol solvents and produced positive results. this is indicated by the color change produced during testing with dpph. in the antioxidant activity study, the addition of dpph solution to the sample was marked by a change in color from purple to yellow. this indicates the occurrence of free radical capture. the change in intensity of the purple color is due to the free radical scavenging produced by the reaction of the dpph molecule with hydrogen atoms released by the sample molecule. this causes the fading of the dpph color from purple to yellow. dpph, which has unpaired electrons, will give a purple color, and the color will change to yellow when the electrons have been paired (konda et al., 2020). the test was performed using a uvvis spectrophotometer with a wavelength of 517 nm, which is the maximum wavelength of dpph. this wavelength will provide the best absorption results from the test solution and provide high sensitivity, thus it is expected to obtain optimal absorbance values for the sample (konda et al., 2020) the samples tested for antioxidant activity include chloroform and methanol extracts of the skin and flesh of crystal guava fruit and ascorbic acid (vitamin c) as a reference. vitamin c was used because it has an antioxidant function, capturing free radicals and preventing chain reactions. vitamin c has free hydroxyl groups that function as free radical captures, and if it has polyhydroxy groups it will increase antioxidant activity. the ic50 values for the skin and flesh of the crystal guava fruit show that the antioxidant activity in the skin of the crystal guava fruit is stronger than in the flesh of the crystal guava fruit. this is due to the higher vitamin c content in the skin of the fruit (jamieson et al., 2021). a smaller ic50 value indicates stronger antioxidant activity, while a larger ic50 value indicates weaker antioxidant activity (andriani and murtisiwi, 2020). research has been conducted on the health benefits of guava leaves, which are attributed to the various phytochemicals they contain such as quercetin avicularia, apigenin, guaijaverin, kaempferol, hyper in, myricetin, gallic acid, catechin, epicatechin, chlorogenic acid, epigallocatechin gallate, and caffeic acid. the extracts from guava leaves (gls) have been investigated for their biological properties such as anticancer, antidiabetic, antioxidant, antidiarrheal, antimicrobial, lipid-lowering, and liver protection activities (kumar et al., 2021). various factors can affect the antioxidant content in fruits, such as variety, species, cultivar, harvesting conditions, ambient temperature, photosynthesis process, relative humidity, oxidative stress, and exposure to sunlight. pollution also plays a role in antioxidant content variation. the ripeness of the fruit also affects the vitamin c content. the riper the fruit, the higher the ascorbic acid content, but overheating during the washing and cooking process can cause a loss of antioxidants due to their easily soluble and easily damaged nature with heating (febrianti et al., 2016). conclusions the results of the study showed that both chloroform and methanol extracts of the skin and flesh of crystal guava fruit have antioxidant activity. the results showed that the ic50 value of the chloroform extract of crystal guava fruit skin is 218.88 ppm and is classified as moderate, the methanol extract of crystal guava fruit skin is 89.78 ppm and is classified as strong, the chloroform extract of crystal guava fruit flesh is 270.56 ppm and is classified as weak, and the methanol extract of crystal guava fruit flesh is 185.72 ppm and is classified as moderate. authors’ contributions: all authors are major contributors to this research. aum, aa, and nw collaborated to design the research and write the initial research draft. aa: executed the experiment, aum: 328 biology, medicine, & natural product chemistry 12 (1), 2023: 323-328 wrote the article draft and revised the draft, nw: contributed suggestions for article improvement competing interests: the authors declare that there is no potential for conflicting interests in this research study. references andriani, disa & murtisiwi, lusia. 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(2015). ekstraksi daun gedi (abelmoschus manihot l) secara sekuensial dan aktivitas antioksidannya. agritech, 35(3), 280287. https://doi.org/10.22146/agritech.9338 yusriyani, y., & syarifuddin ka (2021). uji aktivitas antioksidan fraksi polar ekstrak kulit buah naga merah menggunakan metode dpph (1, 1 diphenyl-2-picryl hydrazil). jurnal kesehatan yamasi makassar, 5(2), 59-67. pissn:2548-8279. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 27-33 | doi: 10.14421/biomedich.2022.111.27-33 issn 2540-9328 (online) the bioprospecting of mangrove red snapper cultivation (lutjanus argentimaculatus forsskål, 1775) using floating cages angga puja asiandu1,*, achmad gusti malayudha2 1faculty of biology, universitas gadjah mada, jl. teknika selatan, yogyakarta 55281, indonesia. 2faculty of communication and multimedia, universitas mercu buana yogyakarta, jl. ring road utara, yogyakarta 55281, indonesia corresponding author* angga.puja.asiandu@mail.ugm.ac.id manuscript received: 24 january, 2022. revision accepted: 14 february, 2022. published: 01 march, 2022. abstract mangrove red snapper is one of the potential and valuable aquaculture products. the high demand for this fish causes high fishing activities. this condition can threaten their existence in the environment. it is necessary to have aquaculture activities to preserve their existence and meet market demand. indonesia as a country with wide marine waters should be able to take advantage of the potential of fish farming by using a floating cage system. thus, this article was written to analyze the bioprospecting of red snapper aquaculture. based on the literature study, the floating net system has the potential to be developed on a large scale because the system has various advantages over fishing directly from nature. things that must be considered in the cultivation of mangrove red snapper using floating nets are the area and quality of the floating nets, the composition of the feed, and the chemical components of the feed given to the fish. based on economic potential, mangrove red snapper cultivation has high prospects which can be used as one of indonesia's leading export products. keywords: aquaculture; feed; floating net cages; snapper. abbreviations: amp (l-ascorbyl-2-monoposfate-mg); fcr (feed conversion ratio), hcg (human chorionic gonadotrophin); pb (puberogen) introduction the high growth of the human population in the world causes the need of increasing food sources, including food sources containing protein. fish is an important source of animal protein for humans. however, fish consumption in indonesia is still relatively low when compared to other country such as japan. it means that fish farming in indonesia has yet fulfill animal protein needs of the indonesian people (langkosono, 2007). red snapper (lutjanus argentimaculatus) is a potential source of fishery culture in indonesia. the high demand for red snapper encourages the cultivation of red snapper which can reduce dependence on wildcaught red snapper supply. some of the advantages of red snapper cultivation are relatively fast growth rate, high tolerance for water turbidity and salinity, low cannibalism, tolerance for high fish density, and good consumption of artificial feed (melianawati and aryati, 2012). red snapper (l. argentimaculatus) is one of the aquatic commodities with high economic value. the price in the international market in 2003 ranged from usd 5,50 to usd 18,10. in indonesia, the price is quite varied. in 2011, in west java province, the price of the fish was rp. 35,000/kg (yasad, 2011; melianawati and aryati, 2012). in lampung, the price of the fish ranged from rp. 40.000 to rp. 50.000. red snapper is a commodity that is in great demand by the public, so the cultivation and production of red snapper is very potential and promising (melianawati and aryati, 2012). red snapper is one of the most popular fish in southeast asia. the high demand for red snapper has not been matched by cultivation at this time, the supply still relies on direct catch from nature which is seasonal, varied, unstable, and less sustainable. in addition, several red snapper cultivation activities are carried out by directly catching the young snapper in the estuary or beach area. this results in a threat to the sustainability and abundance of these fish in nature. thus, it is necessary for cultivation to be carried out so that the red snapper population in nature is maintained (chi and true, 2017). cultivation of red snapper can be done by using floating cages in the sea. the floating cage is a place made of nets tied to a frame, square in shape, with a size that can reach hundreds of square meters. the size of the cage can be adjusted to the size and number of fish to be cultivated. these cages can be placed in bays, lakes, or reservoirs (purba, 1994). however, there are several https://doi.org/10.14421/biomedich.2022.111.27-33 28 biology, medicine, & natural product chemistry 11 (1), 2022: 27-33 factors that can affect the success of the red snapper (l. argentimaculatus) cultivation. some of these factors are feed, feed composition (giri et al., 2007), design of floating net cages, as well as various environmental factors such as temperature, salinity, light, and so on (affan, 2011). thus, in this paper, the authors discuss several factors that determine the success of red snapper cultivation in more detail. methods the writing of this paper was carried out by exploring various literature related to red snapper cultivation and other related sources. a literature study is an activity of searching various sources of information or reading thoroughly on a topic to be discussed. materials or sources of information that can be used in library research include books, journals, magazines, and other sources. the steps in the literature study are choosing topics to be discussed, exploring topics and related information, conducting research focus, collecting information sources, managing and presenting data, and compiling reports (sari, 2020). the data used in writing this paper is secondary data. secondary data is data obtained indirectly. secondary data is obtained from various data sources, such as the internet, statistical data, books, journals, and so on (tanujaya, 2017). in writing this paper, secondary data were obtained from journals and theses related to red snapper cultivation. results and discussion mangrove red snapper (lutjanus argentimaculatus) snapper belongs to the lutjanidae family, which consists of 103 species. these fish live in shallow to medium sea areas to a depth of 100-500 m. these fish are classified as predatory fish. snapper is actively looking for prey at night. the natural foods for these fish are crabs, shrimp, small fish, plankton, and squid. in general, snapper reproduces by involving mating between female and male snapper (wwf-indonesia, 2015). one of the snappers that are known among the public is the red snapper. figure 1. the morphology of mangrove red snapper (lutjanus argentimaculatus) (obtained from fishider.org). this fish has a distribution area that includes the indo-pacific waters, the line islands in north africa to the waters of australia, and the ryukyu islands, japan. these fish can be found in bays and beaches, to estuaries (purba, 1994). the classification of red snapper is as follows: kingdom : animalia phylum : chordata class : teleostei order : perciformes family : lutjanidae genus : lutjanus species : lutjanus argentimaculatus forsskål, 1775 the red snapper has a slightly flattened body, the back is higher, the head is pointed, the upper jawbone sinks when the fish opens its mouth. the fish has a forked caudal fin, red on top, and silvery-white on the underside. the dorsal fin has 10 spines, 13 to 15 weak rays, the anal fin has 3 spines and 8 to 19 weak rays, the pectoral fin has 14 to 15 rays weak finger. the lateral line consisted of 45 to 48 pieces. the structure of the gill cover in these fish is grooved (purba, 1994). the existence of red snappers in nature is also influenced by their habitat. red snapper spawning occurs in estuaries. the destruction of the estuary area causes red snapper spawning to decrease. this also has an impact on the red snapper population in nature (chi & true, 2017). red snapper fry is often found in the river estuaries that are in direct contact with mangroves with a salinity level of 10-25 ppt. the tillers are found on rocks and sandy areas. saplings aged 3-10 cm are often found on rocks. tillers measuring less than 3 cm are often found in sandy areas and seaweed. the natural feed for red snapper fry is shrimp, small fish, zoobenthos, and zooplankton (chi & true, 2017). snapper (lutjanus sp.) is one of the demersal fish that has high economic value and is in demand by the public. snapper is not very active, can form small groups, the migration rate is not too far, and the life cycle is stable. this is because this fish lives on the seabed which is relatively more stable than the surface. coastal degradation is one of the threats to the existence of this fish. the distribution area of snapper is not too wide so environmental degradation will directly affect the existence of these fish. in addition, the high fishing activity causes the population to become increasingly threatening in nature (sriati, 2011). reef fish such as snappers play an important role in ecological functions. the presence of these fish in their habitat supports the productivity of the habitat. however, fishing for reef fish that is not environmentally friendly has caused damage to these habitats. until now, about 75% of coral reef habitats in indonesia have been damaged due to destructive fishing by fishermen. in addition to habitat destruction, another asiandu & malayudha – the bioprospecting of mangrove red snapper cultivation 29 factor is the over-exploitation of these fish. some fishermen use potassium or a kind of anesthetic poison to catch the fish. this is because the selling price is higher when the fish are still alive (wwf-indonesia, 2015). cultivation of mangrove red snapper (lutjanus argentimaculatus) indonesia is the second-largest producer of cultivated fish in the world after china. in 2012, aquaculture production in indonesia reached 9.675.553 tons. the production of snapper itself ranks fourth with total production reaching 6.198 tons. from 1999 to 2012, the percentage increase in snapper production in indonesia increased by 11.43% (ministry of marine affairs and fisheries, 2013; giri et al., 2007). the cultivation of red snapper attracts attention because of its high growth. red snapper is a euryhaline fish that has good adaptation to salinity concentrations in the waters. this fish is able to live in waters containing high salinity of 35 ppt to freshwater with a salt concentration of 0 ppt. this ability causes the red snapper to have the potential to be cultivated in ponds filled with fresh water. the development of red snapper cultivation in freshwater opens new opportunities for entrepreneurs and the community in providing a supply of red snapper stock in the market (muyot et al., 2021). cultivation of red snapper can be done by giving a stimulant hormone. the hormones used in the spawning induction are hcg (human chorionic gonadotrophin) and pb (puberogen) hormone. induction is done by injecting hormones into the body of the fish through the back of the pectoral fins, in the area between the backs, or on the line of the ribs. the size of the broodstock of red snapper that is ready to spawn is 3 kg to 7 kg. at this weight, the broodstock red snapper is about 4 to 5 years old. in general, red snapper gonad maturation occurs in january and october. the best spawning time for red snapper is 11:00 am to 04:30 pm (purba, 1994). in the cultivation of red snapper, it is necessary to pay attention to feeding. the quality and nutritional content of the feed given must be maintained so that the cultivated snapper can grow well. availability of feed is one of the determining factors for the success of red snapper cultivation. in providing appropriate feed, it is necessary to pay attention to the adequacy of nutrients or macro and micronutrients needed by the fish. however, feeding should be done by having a feed that has economic value so that it can maximize the benefits that will be obtained from the red snapper cultivation process (giri et al., 2007). one of the important sources of nutrition in the process of fish growth is the provision of proteincontaining feed. protein is one of the important nutrients for the growth of red snapper. provision of optimum feed protein levels can stimulate the growth process of fish. however, protein levels in the feed must be considered and adjusted to the needs of the fish. the protein content in the feed must also be adjusted to the content of other nutrients so that the desired growth rate of fish can be achieved so that the yield will be high (giri et al., 2007). cultivation of mangrove red snapper in freshwater is also very potent. the analysis that should be carried out in cultivaing are the growth rate, endurance, feed conversion ratio (fcr), and the profit. cultivation of red snapper in freshwater ponds can be fed in the form of dry pellets. acclimatization that can be done on red snapper to the level of salinity is carried out for 1 to 2 weeks. cultivation of red snapper in freshwater can be done in rivers, ponds, or lakes. the use of cages can maximize the number of fish cultivated, the costs used are also economical, also valuable (muyot et al., 2021). cultivation of mangrove red snapper (l. argentimaculatus) using the floating net cage system fish cultivation in the open ocean can be carried out using a floating net cage system. a floating net cage is a container that is placed in the water and filled with fish that is used to maintain the cultured fish. the cages must have an effective and strong design so that they are durable (tatengkorang, 2020). the success of fish culture in waters is determined by the carrying capacity of the aquatic environment. the carrying capacity of the environment is the ability of an environment to support the life processes of living things in the long term. the carrying capacity of the environment is the ability of the environment to support the welfare of the life of living things that live or exist in the environment. in another sense, the carrying capacity of the environment is closely related to the availability of natural resources and environmental factors in determining the rate of life of living things. the environmental carrying capacity of waters is very important for the success of aquaculture in that environment (anrosana & gemaputri, 2018). the level of depth of water used in fish farming determines the success of aquaculture. the depth from the bottom of the cage to the optimum bottom at low tide is about 4-5 m. the depth of the cage and the waters used to determine whether or not a location is suitable for use as a fishery cultivation area. another factor that also influences is the level of lighting or water brightness. this is related to the level of light penetration into the waters. the level of water clarity is influenced by the presence of suspended particles in the water environment. light is needed by photosynthetic organisms to carry out the process of photosynthesis. this is very necessary for the phytoplankton contained in the cages to carry out the photosynthesis process. the phytoplankton can also be used as natural food for fish cultured in cages. the optimum brightness value for aquaculture in the sea is 70.35% (affan, 2011). another factor that also affects the success of aquaculture is strong currents. the flow rate of water in 30 biology, medicine, & natural product chemistry 11 (1), 2022: 27-33 an environment will affect the distribution of suspended particles in the water. in addition, currents are also related to the amount of dissolved oxygen in the waters. the design and construction of cages must be adapted to the flow of water. this is because the strength of the current can reduce the risk of fouling in the cages used. if the current is less than 25 cm/sec, the fouling organisms will be more easily attached to the cages used. the attachment of fouling organisms will reduce and disrupt the circulation of dissolved oxygen in the cage. the minimum current velocity that can still be used in cage cultivation is 5-15 cm/sec (affan, 2011). temperature also plays an important role in the success of aquaculture in waters. temperature affects the metabolic rate of cultured organisms. an increase in temperature can reduce dissolved oxygen levels in the water. thus, at too high temperatures, the respiratory activity of the cultured fish will be disturbed. the optimum temperature in fish farming is 27-37 oc (affan, 2011). dissolved oxygen is one of the key factors in fish farming. lack of dissolved oxygen can reduce the consumption of fish feed, this results in the inhibition of the growth of the fish (riadhi et al., 2017). fish farming using cages placed in saltwater or the sea is also affected by salinity. the optimum salinity in the cultivation is 30-35 ppt. however, the optimum salinity must be adjusted to the type of fish to be cultivated. certain fish have a higher salinity tolerance value than other species. the degree of acidity or ph of the water environment used also affects the success of the cultivation carried out. the ph value of seawater, in general, is in the range of 7.5-8.4. the closer to the river estuary area, the ph will change due to the influence of freshwater flowing into the sea (affan, 2011). one of the factors that cause the failure of fish farming is the presence of parasites that attack the cultured fish. endoparasites are parasites that usually attack the organs and body cavities of cultured fish. some of the endoparasites that attack fish are nematodes, trematodes, cestodes, and several types of protozoa. the parasitic infection causes the growth of fish to be stunted. parasitic infection also causes a decrease in the consumption of fish feed. another thing to worry about is the zoonic nature of the parasite so that it can infect humans who eat the fish (puspitarini et al., 2018). the endoparasites reported infecting red snapper are anisakis physeter and cucullanus heterochorus. anisakis larvae at stage two can be eaten by small fish which are then eaten by red snapper. then it will develop into a third-stage larva in red snapper. one of the factors that cause red snapper infection is a dirty water environment (puspitarini et al., 2018). in indonesia, a lot of fish farming is also done openly in nature. the cultivation is carried out using floating cages placed in reservoirs, rivers, ponds, and so on. aquaculture can be divided into two, namely marine aquaculture and freshwater aquaculture. both types of cultivation have their own characteristics. in fish farming, there are several subsystems that are interconnected with each other. the system includes the procurement of facilities and infrastructure, production systems, post-production systems, and supporting subsystems. in the production subsystem, various cultivation activities are involved, starting from the initial maintenance process to harvesting. the system also involves feeding the cultured fish (deswati & adrison, 2019). some of the advantages of aquaculture using cages are that it can maximize the location of the waters as a location for fish cultivation, the population of cultivated fish is easy to control, avoids predators, is easy to transfer, is more economical, can maximize the water used, maintained water circulation, is easy to harvest, and lower maintenance costs. low (purba, 1994). red snapper cultivation includes several activities. these cultivation activities include parental rearing, larval rearing, and seed maintenance. one of the determining factors for the success of red snapper cultivation is the eggs to be developed. when the eggs have hatched and developed into larvae, the feeding and the amount of feed given should be appropriate. in the larval phase, the food for the red snapper chicks can be in the form of small rotifers that match the larval mouth opening (melianawati & aryati, 2012). marine fish cultivation using floating cages has several impacts on fishermen. in fish farming using floating cages, feed is given to support the growth process of the fish. not all of the feed given will be eaten by the fish in the cage, some of the feed will be released into the environment outside the cage. the positive side of this is the approach of the fish in the cages to the cage area so that it can help fishermen in harvesting fish in the area (deswati & adrison, 2019). meanwhile, incentive feeding can also be misused as a “feed pump”. the feed pump is an excessive feeding of fish that is carried out continuously until the fish that are cultivated are completely full. this practice leads to overfeeding. overfeeding is a bad practice for feeding fish. this excessive feed reduces feed efficiency and increases the amount of feed that is wasted in the environment (deswati & adrison, 2019). excessive feeding in fish farming activities using cages in the ocean is the potential for upwelling. the upwelling that occurs is caused by the buildup or deposition of excessive feed and accumulates below sea level. this triggers a backflow due to changes in water temperature. this backflow will bring the leftover feedback to the surface which can eventually poison the fish in the cage (deswati & adrison, 2019). a study on the cultivation of lutjanus species using floating net cages had been carried out by castillovargasmachuca et al. (2012). it was conducted to observe the growth of pacific red snapper (l. peru). the results of their study stated that the fish had a potential asiandu & malayudha – the bioprospecting of mangrove red snapper cultivation 31 growth rate and biomass. the cultivation process using floating net cages also is prosperous to be developed on a commercial scale to meet market demand. another study was conducted by castillo-vargasmachuca et al. (2007) who analyzed the growth of rose snapper (l. guttatus) cultivated in floating cages. they found that the cultivated fish had a high survival rate. subadults have a survival rate of up to 74.7%. however, some aspects need to be considered in cultivating this fish in floating net cages, such as the availability of natural feed. marine cultivation or sea farming in indonesia is highly prosperous as a maritime country with most of its territory consisting of sea water. if this potential can be optimized, the community's economy will also be higher. still, the cultivation process requires strong management effectiveness (sunardi et al. 2020). therefore, in this paper, the authors discussed the bioprospecting of mangrove red snapper cultivation using floating net cages, as well as feed needs and the economic bioprospects. mangrove red snapper (l. argentimaculatus) feed needs the concentration of certain proteins in the feed can affect the rate of feed consumption by farmed fish. improper protein levels will reduce the rate of feed consumption by the fish. a feed with protein concentrations of 32% to 40% did not reduce the rate of fish feed consumption. however, increasing the protein content to 52% reduced the consumption of red snapper feed. as a result, the higher the protein content in the feed, the faster the protein needs to be needed by the fish will be fulfilled so that the consumption of fish feed will decrease. as a result, the rate of feed consumption by sea bass is low when the protein content is too high in the feed (giri et al., 2007). feed efficiency is influenced by the protein content in the feed. feed efficiency is the proportion of the addition of fish biomass to the amount of feed given and consumed by the fish. the higher the feed efficiency value, the better the quality of the fish feed. in red snapper, the highest feed efficiency is in feed containing protein with a concentration of 40% or more than 40%. thus, at this concentration, red snapper were able to utilize their feed efficiently for increasing their biomass (giri et al., 2007). in the red snapper feed, the protein content in the feed was 45.4%, the fat content was 9.3%, the fiber content was 1.7%, the ash content was 13.8%, and the nitrogen-free extract was 29. 8%. in the feed also added other substances such as beta-carotene which will stimulate redfish meat. in addition, some vitamins are added to support the growth process of the snapper (catacutan et al., 2011). vitamin c is very important for the growth of red snapper. the addition of l-ascorbyl-2-monoposfate-mg (amp) affected the growth of the red snapper. the increase in the bodyweight of the fish increased 6 times for 17 weeks. the weight growth decreased on day 60 on media that did not contain amp. fish that were not given amp experienced several abnormalities, such as swelling of the eyes, soft bodies, and abnormalities in the fins. this abnormality is caused by an abnormality in the synthesis of collagen. amp deficiency causes collagen synthesis to be strained so that the process of forming connective tissue in these fish is abnormal (catacutan et al., 2011). protein requirements in the feed are adjusted to the type of fish being farmed. each species of fish requires varying levels of protein. in addition, the protein content of the feed must also be adjusted to the size of the fish, the quality of the protein, the energy content of the feed, the nutritional balance of the feed, and the level of feeding (furnichi, 1988; giri et al., 2007). protein quality is determined by the profile of amino acid content of the protein. the amino acid content in feed determines the adequacy of protein needed by fish (giri et al., 2007). one source of protein feed is fish meal. as for other protein sources, namely vegetable protein derived from nuts, seeds, such as soybeans. however, not all types of fish can utilize these vegetable protein sources properly. the provision of protein in the diet of fish also determines and affects the protein levels of the fish's body. the higher the protein content fed, the higher the protein concentration in the fish's body. in the feed containing 32% protein, the total protein of red snapper was 54.7%. meanwhile, feeding containing 40% protein produced red snapper with 58.7% body protein. meanwhile, feed containing protein with a concentration of 52% resulted in red snapper containing 61.9% protein (giri et al., 2007). feeding done three times a day (morning, afternoon, evening) can increase the amount of feed that is wasted in the environment. in this feeding, as much as 25-30% of the feed given is wasted in the environment. in cultivation with a fast and flowing water environment, an excess feed can be channeled to other areas so that it does not accumulate in that area. however, if cultivation is carried out in a calm aquatic environment, there will be a buildup of feed at the bottom which causes an accumulation of phosphate and nitrate. this will reduce water quality and interfere with the health of the fish (deswati & adrison, 2019). natural feeed found in red snapper (l. argentimaculatus) especially in the juvenile phase is acetes indicus, acetes sp., some members of mysidae, and some zooplankton such as clanoida and melita longidactyla. in addition, they also found feed in the form of members of the luciferidae, alpheidae, and so on (chi & true, 2017). in general, the sufficient protein content in feed ranges from 30% to 55% (nrc, 1993; giri et al., 2007). in carnivorous fish, such as grouper, the protein requirement given in the feed reaches 47% to 60%. red snapper is also a carnivorous fish. thus, the protein 32 biology, medicine, & natural product chemistry 11 (1), 2022: 27-33 requirement in a red snapper is also relatively high (giri et al., 2007). research by giri et al., (2007) showed that the protein content in the feed affected the rate of fish weight gain, feed consumption, feed efficiency, and growth of red snapper fry. provision of red snapper feed containing protein with a content of 32% resulted in a low growth rate and mass weight gain of snapper. feeding with a protein content of up to 40% resulted in the maximum growth rate and weight gain of snapper. however, feeding with protein content exceeding 40% did not significantly affect the weight gain and growth of red snapper. thus, in this study, the optimum protein concentration in the feed was 40%. giving excessive levels of protein in the fish feed will cause a negative effect. the negative effect that arises is in the form of slowing the growth rate of fish. excessive feeding results in not having enough energy for fish cells to manage these proteins. this causes an increase in the rate of deamination and excretion of excess amino acids due to the high protein in the feed consumed by fish. in addition, the negative impact of adding feed with too high a protein content is that it can reduce the growth rate of fish (giri et al., 2007). in addition, other nutrient compositions such as vitamin c are also needed. the addition of l-ascorbyl2-monoposfate-mg (amp) affects the growth of red snapper. the increase in the bodyweight of the fish increased 6 times for 17 weeks. the weight growth decreased on day 60 on media that did not contain amp. fish that were not given amp experienced several abnormalities, such as swelling of the eyes, soft bodies, and abnormalities in the fins. this abnormality is caused by an abnormality in the synthesis of collagen. amp deficiency causes collagen synthesis to be tense so the process of forming connective tissue in these fish is abnormal (catacutan et al., 2011). giving amp as much as 60 mg/kg of feed weight is equivalent to giving ascorbic acid as much as 20 mg/kg of feed. this composition is a good composition in overcoming vitamin c deficiency in red snapper. administration of amp in this concentration was effective in supporting the weight growth of red snapper. however, the addition of amp in higher doses had no effect on fish weight gain. addition in too high a dose can be toxic to red snapper cells (catacutan et al., 2011). economic aspects of mangrove red snapper (l. argentimaculatus) cultivation fish is one of the foodstuffs that contain high protein needed by body cells. fish, in general, contain 18-20% protein which is good for the body. thus, fish has become an important commodity in the economy. one of the fish with high economic value is the red snapper (reo, 2013). based on fao, from 2010 to 2013, the average catch of red snapper in indonesia is the highest in the world. the supply of snappers from indonesia reaches 48.3% of the total supply of snappers in the world. this amount is equivalent to more than 120,000 tons. next, followed by the philippines with a total percentage of 8.1%, brazil 7.7%, malaysia 7.3%, mexico 4.9%, nigeria 3.1%, thailand 2.0%, venezuela 1.8%, us 1.8%, and australia 1.6%. meanwhile, according to fao in 2014, the number of import and export markets for snappers has increased significantly. global snapper export activities in 1990 were around 5.000 tons and increased to more than 10.000 tons. meanwhile, global snapper import activity in 1990 amounted to 5.000 tons until in 2010 it has increased to 25.000 tons. meanwhile, the united states is the highest importing country of snapper in the world. the imports come from mexico, brazil, nicaragua, panama, and suriname (amorim & westmeyer, 2016). mangrove red snapper is a high-value aquatic product. market demand for these fishes is also relatively high with relatively high prices. red snapper is the most popular snapper in various restaurants in singapore and hong kong (muyot et al., 2021). based on fao (2018), global red snapper production in 2016 reached 9,815 tonnes, which came directly from fishermen's catch, and 10,240 tonnes came from aquaculture. the highest fish farming comes from southeast asia. many of these fish are cultivated using floating cages placed directly in the ocean and using brackish water ponds (muyot et al., 2021). fish that live in reef areas such as snapper and grouper are a very important resource. from an economic point of view, this fish is one of the leading commodities and is the livelihood of fishermen in indonesia. indonesia's reef fish production reaches 7% of the world's total reef fish production. this amount is one source of foreign exchange for the country (wwfindonesia, 2015). conclusions the factors that influence the success of red snapper (l. argentimaculatus) cultivation using a floating net cage system are the size of the cage, the density of fish in the cage, feeding, and other environmental factors such as temperature, ph, dissolved oxygen, and salinity. analysis of the growth of red snapper (l. argentimaculatus) cultivated using a floating net cage system which includes fish weight gain, absolute growth, specific growth rate, fcr, and survival rate. red snapper is classified as a carnivorous fish so it requires feed with a protein content of 40%. acknowledgements: the first author thanks lembaga pengelola dana pendidikan (lpdp) for the scholarship given to them. also, the second authors thank m. nastain. m.ikom as the research methodology lecturer and didik haryadi santoso s.kom.i.,m.a as the dean asiandu & malayudha – the bioprospecting of mangrove red snapper cultivation 33 of the faculty of communication and multimedia, umby, who inspired us to write this article. authors’ contributions: all the authors are involved in idea developing, secondary data collecting, manuscript writing, language editing, and formatting. competing interests: all the authors declare that there is no conflict interest regarding the writing and publishing the article. funding: the authors received no financial funding regarding the data collection, manuscript writing, authorship, and publication of this article. references affan, j. m. 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(2015). better management practices seri panduan perikanan skala kecil. in wwf-indonesia (2nd ed., vol. 2, issue 1/november). this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 169-173 | doi: 10.14421/biomedich.2022.112.169-173 issn 2540-9328 (online) the weight performance stability of mice on modeling obesity-associated hyperglycemia induced by dextrose monohydrate deksa yudha syach putra1, setiyo budi santoso1,2,*, heni lutfiyati1,2 1department of pharmacy, faculty of health sciences; 2consortium for pharmacology and clinical pharmacy studies, universitas muhammadiyah magelang, indonesia. corresponding author* sb@unimma.ac.id manuscript received: 11 august, 2022. revision accepted: 26 august, 2022. published: 06 september, 2022. abstract previously, streptozotocin and alloxan were employed to imitate hyperglycemia in mice. high doses of sucrose were also induced as an alternative. due to body mass index has been associated with hyperglycemia, the evidence of weight body index in various induction alternate kinds, however, have not been fully reported. here-in, we report on the weight performance stability of mice body weight induced by dextrose, streptozotocin, and alloxan. to begin, all mice were divided into six groups of five, with one reserve in each. following seven days of acclimatization, the mice were induced for nine days of hyperglycemia modeling; alloxan (groups a and d), streptozotocin (group b and e), dextrose monohydrate (groups c and f). on preclinical research animals modelling related to obesityassociated hyperglycemia in mice, dextrose monohydrate induction was most successful than streptozocotin and alloxan induction, which performed best during the induction period (31% weight growth) and after metformin intervention (36% weight growth). overall, dextrose monohydrate is most suitable to be used for modeling type 2 diabetes mellitus test animals rather than alloxan and streptozocotin. keywords: alloxan; preclinical research animals; streptozotocin; type 2 diabetes mellitus. abbreviations: type 1 diabetes mellitus (t1dm), type 1 diabetes mellitus (t2dm), glucose transporter 2 (glut2), body weight (bw) introduction diabetic mice are commonly employed in the development of anti-diabetes agent. alloxan and streptozotocin are commonly used to induce diabetic in the mice (al-awar et al., 2016; king & austin, 2017; kottaisamy et al., 2021). in a single induction, alloxan elevated the glycemic index to 127 mg/dl (irdalisa et al., 2015), whereas three days or more raised it to 156 270 mg/dl (dewi et al., 2021; hamdani & nurman, 2020; lolok et al., 2019). streptozotocin had a comparable impact, increasing glucose levels to 136 mg/dl after three days (apriani et al., 2011) and 220244 mg/dl after five days (ocktarini et al., 2011; suwanto & rahmawati, 2019). as an alternative to alloxan and streptozotocin, glucose induction groups have been induced. fructose intake for 12 weeks hold glycemic levels at 81 mg/dl (tillman et al., 2014). dextrose outperformed fructose after seven days of induction, while by 10%concentration raised glycemic at 148 mg/dl (pramushinta et al., 2019) and by 40% at 154 mg/dl (santoso & suryanto, 2017). diabesity is a new term for diabetes that comes alongside obesity (serván, 2013). obese are roughly three times more vulnerable to diabetic (prasetyani & sodikin, 2017). diabetes, most especially type 2 diabetes mellitus (t2dm), is correlated with weight gained (droz et al., 2017) or obesity (fang et al., 2019; finkelstein et al., 2012). weight gained and body mass are two factors that contribute to rising glycemic levels (algoblan et al., 2014), so obesity and t2dm called the twin pandemic (scheithauer et al., 2016). previously, streptozotocin and alloxan were employed to imitate hyperglycemia in mice. high doses of sucrose were also induced as an alternative. due to body mass index has been associated with hyperglycemia, the evidence of weight body index in various induction alternate kinds, however, have not been fully reported. here-in, we report on the weight performance stability of mice body weight caused by dextrose, streptozotocin, and alloxan. https://doi.org/10.14421/biomedich.2022.112.169-173 https://www.google.com/search?client=firefox-b-d&q=glycemic&spell=1&sa=x&ved=2ahukewjxwktujmt5ahvvh7cahrgec14qbsgaegqiaraz https://www.google.com/search?client=firefox-b-d&q=glycemic&spell=1&sa=x&ved=2ahukewjxwktujmt5ahvvh7cahrgec14qbsgaegqiaraz 170 biology, medicine, & natural product chemistry 11 (2), 2022: 169-173 materials and methods ethical clearence the medical and health research ethics committee ethics of the faculty of medicine, universitas gadjah mada, has accepted the whole series of research procedures under registration number ke/fk/0328/ec/2022. animals male balb/c mice were around 2-3 months that bred by pharmacy laboratory of universitas gadjah mada (yogyakarta, indonesia) and treated in accordance with current norms. materials alloxan monohydrate (sigma-aldrich), streptozotocin (sigma-aldrich), and dextrose monohydrate 40% (otsuka) were used to induce mice. metformin (dexa medica) and natrium carboxy methylcellulose (daiichi kogyo seiyaku) were prepared to assess the stability of hyperglycemia treatment. citrate buffer ph 4.5 is solved for intraperitoneal preparation. supplies for glycemic testing, such as strip and glucometer (easytouch). among the other consumables are a handscoon, syringe for 1ml and 5 ml, blood lancet, and aquadest. as support equipment, analytical scales, beakers, mouse cages, markers, mortars and stampers, and oral probes were used. procedures all mice were divided into six groups of five, with one reserve in each. following seven days of acclimatization, the mice were induced for nine days of hyperglycemia modeling and five days of metformin intervention.  induction period to get baseline data, we assessed the weight of the mice following the acclimatization. for 9 days, groups a and d were induced alloxan 0.12 mg/gram body weight (bw) intraperitoneally, group b and e were induced streptozotocin 0.05 mg/gram bw intraperitoneally, and groups c and f were induced dextrose monohydrate 6 mg/gram bw orally. at the end of the induction, we weighed the mice again.  intervention period following the induction period, groups a, b, and c were treated metformin orally, whereas groups d, e, and f as negative controls and were treated na cmc orally. dextrose monohydrate continued to activate groups c and f throughout the intervention. on day 14, the body weight was measured. results and discussion performance of alloxan induction mice in groups a and d gained 35.86 grams and 37.98 grams of body weight after nine days of alloxan induction, respectively. mice on group a grew to 37.50 grams after a five-day metformin intervention, whereas control mice (group d) stayed constant at 37.98 grams. (figure 1). figure 1. the body weight gain due to alloxan induction in the intervention group (a) and the control group (d). the solid line represents the induction period, the dashed line represents the intervention period. performance of streptozotocin induction mice in groups b and e gained 35.80 grams and 36.60 grams of body weight after nine days of streptozotocin induction, respectively. mice on group b grew to 39.10 grams after a five-day metformin intervention, whereas control mice (group d) stayed constant at 36.60 grams. (figure 2). figure 2. the body weight gain due to streptozotocin induction in the intervention group (b) and the control group (e). the solid line represents the induction period, the dashed line represents the intervention period. performance of dextrose monohydrate induction mice in groups c and f gained 40.33 grams and 36.08 grams of body weight after nine days of dextrose monohydrate induction, respectively. both groups thereafter continued to rise to 41.90 grams and 39.36 putra et al. – the weight performance stability of mice on … 171 grams respectively, after receiving metformin intervention for five days (figure 3). figure 3. the body weight gain due to dextrose monohydrate induction in the intervention group (c) and the control group (f). the solid line represents the induction period, the dashed line represents the intervention period. induction variant performance in mice treated with metformin was compared. after the 9-day induction period, dextrose-induced mice gained the most weight (40.33 grams), followed by alloxan-induced mice (35.86 grams), and streptozotocininduced mice (35.80 grams). after 5 days of metformin treatment, dextrose-induced mice had the best body weight performance (41.90 grams), followed by streptozotocin-induced (39.10 grams) and alloxaninduced (37,50 gram) figure 4. the body weight gain in mice following 9 days of induction (solid line) to alloxan (a), streptozotocin (b), and dextrose monohydrate (c), and persistence rate following 5 days of metformin intervention (dash line) after obesity (day 14th). dextrose monohydrate induction in mice was most successful than streptozocotin and alloxan induction, which performed best during the induction period (31% weight growth) and after metformin intervention (36% weight growth). table 1. percentage of body weight gain in mice following 9 days of induction to alloxan (a), streptozotocin (b), and dextrose monohydrate (c), and persistence rate following 5 days of metformin intervention after obesity (day 14th). group label induction treatment body weight (percentage increase) baseline 9th day treatment 14th day treatment a alloxan 31.341.27 (-) 35.863.58 (14%) 37.504.54 (20%) b streptozotocin 31.862.69 (-) 35.803.58 (12%) 39.103.01 (23%) c dekstrose monohydrate 30.882.35 (-) 40.332.66 (31%) 41.903.28 (36%) discussion on preclinical research animals, we simulate obesityassociated hyperglycemia in mice using dextrose monohydrate induction. the bulk previous studies employed streptozotocin and alloxan to imitate hyperglycemia. however, as one of the important criteria in hyperglycemia etiology, the investigation of body weight issues was disregarded. due to the importance of publishing our findings, we would show the evidence of steady rates of dextrose monohydrateinduced body weight during the experiment. according to the availability of test animals in preclinical research on the developing of anti-diabetic agents, there are two options: genetic modification, or chemical induction (kottaisamy et al., 2021). in the unavailability of genetically diabetic test animals, alloxan and streptozotocin are widely used to cause diabetes in mice (al-awar et al., 2016; kottaisamy et al., 2021). streptozotocin and alloxan, on the other hand, act on beta cells via the glucose transporter 2 (glut2) and cause complete ablation of beta cells inside the islets, leading in severe insulin insufficiency, hyperglycemia, and weight loss, they are more suited for modeling type 1 diabetes (king & austin, 2017). mice given alloxan or streptozotocin lost weight because they were unable to use the available glucose for energy. to meet the body's energy requirements, excessive catabolism of protein and fat in muscle and adipose tissue was performed (malik et al., 2015; sinata & arifin, 2016; suwanto & rahmawati, 2019). since the great majority of t2dm are overweight (finkelstein et al., 2012; wilding, 2014), body mass index has long been linked to hyperglycemia and t2dm 172 biology, medicine, & natural product chemistry 11 (2), 2022: 169-173 (algoblan et al., 2014; jung & choi, 2014), with the potential of obesity to promote insulin resistance being the primary explanation (jung & choi, 2014). obesity was proclaimed in mice as test animals when they weighed more than 20% of their age-matched mates. (patonah et al., 2018). common sugar exacerbates obesity and increases adiposity in mice (kleinert et al., 2018). adipocyte hypertrophy and hyperplasia lead to excessive lipid accumulation (lipotoxicity), which activate insulin resistance (lee et al., 2020; longo et al., 2019). indeed, the diets manipulated of mice with high sucrose feeding generate a compensatory response from some beta cells, (king & austin, 2017). additionally, the weight gain was also linked to the disfunction of leptin (anorexigenic hormone) due to the circulating of glucose which led the test animals to consume more feed (tillman et al., 2014). throughout the investigation, the induction performance of dextrose monohydrate has been shown to increase glycemia. dextrose monohydrate is more suitable to be used for modeling type 2 diabetes mellitus test animals due to the aspect of increasing body weight, rather than the two toxic compounds that actually suppress body weight, and they are more suitable for modeling mice with diabetes mellitus type 1 as well, aside from having a higher level of glucose elevation than alloxan and streptozotocin. conclusions on preclinical research animals modelling related to obesity-associated hyperglycemia in mice, dextrose monohydrate induction was most successful than streptozotocin and alloxan induction, which performed best during the induction period (31% weight growth) and after metformin intervention (36% weight growth). throughout the investigation, dextrose monohydrate is most suitable to be used for modeling type 2 diabetes mellitus test animals rather than alloxan and streptozotocin. acknowledgements: this study is part of the acceleration research scheme for institutional vision revitalization research output (refference number: 012/kontrak/prvi-pa/2022) in the subject of exploring the biological and pharmacological effects of employing plants, fungi, animals, microbes, and minerals. authors’ contributions: conceptualization, sbs; methodology, sbs and hl; data collection, dysp; writing-original draft preparation, sbs and dysp writing-review & editing, dysp, sbs. and hl. all authors read and approved the final version of the manuscript. competing interests: the authors claim to have no conflicts of interest. the funders had no say in the design of the investigation, data collection, analysis, or interpretation, article writing, or decision to publish the findings. the authors declare that there are no competing interests. funding: the authors are thankful to the research, development, and community service department of universitas muhammadiyah magelang for financial supporting. references al-awar, a., kupai, k., veszelka, m., szűcs, g., attieh, z., murlasits, z., török, s., pósa, a., & varga, c. 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(2019). aktivitas hipoglikemik diet pakan ekstrak biji labu kuning (cucurbita moschata duch) pada mencit diabetes melitus terpapar streptozotocin. jpscr : journal of pharmaceutical science and clinical research, 4(1), 39. https://doi.org/10.20961/jpscr.v4i1.27292 tillman, e. j., morgan, d. a., rahmouni, k., & swoap, s. j. (2014). three months of high-fructose feeding fails to induce excessive weight gain or leptin resistance in mice. plos one, 9(9), e107206. https://doi.org/10.1371/journal.pone.0107206 wilding, j. p. h. (2014). the importance of weight management in type 2 diabetes mellitus. international journal of clinical practice, 68(6), 682–691. https://doi.org/10.1111/ijcp.12384 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 1, 2018 | pages: 21-26 | doi: 10.14421/biomedich.2018.71.21-26 issn 2540-9328 (online) physiological response of 'segreng' rice plant (oryza sativa l.) to biogas sludge at wukirsari village, cangkringan, sleman dwi umi siswanti*, sudjino, nindy senissia asri, mifta arlinda, arianda poetri shofia rochman, akrima syahidah faculty of biology, universitas gadjah mada, jl. bulaksumur, yogyakarta, 55281, indonesia tel. +62-274-6492599, fax. +62-274-565223 author correspondency*: dwiumi@ugm.ac.id abstract wukirsari village, cangkringan district is belong to merapi mountain’s slopes which located between the gendol river and yellow river. nowadays, we faced the problem of anorganic fertilizer overused such as urea, za, tsp/sp-36 and kcl in agriculture land. the effort to return the soil organic compound can be done by added some organic compounds or microbial bio -organic fertilizer. sludge is fermented biodigester yield and it has lost its gas. the aim of this research was to understand the physiological response and optimum dose of biogas as planting medium to ‘segreng’ rice planted in the rice field of wukirsari village, cangkringan district, sleman regency. this research was done on greenhouse scale and rice field scale. the treatment given on 0; 1; 1,5; 2 and 2,5 liters per 100 m2 of rice field areas, and given on 0; 4; 8; 12; and 24 ml per 5 kg soil on polybags. data were taken in three repetitions. the vegetative growth parameters included plant height, number of leaves, number of seedlings and chlorophyll content, while generative growth parameters measured included nra levels, dried biomass including crown/stem, roots, filled grains, empty grains, and total weight and number of filled grains, empty rains, and the number of panicles. the result were tested with one way anova (analysis of variance) with spss version 19 for windows and followed by duncan's multiple range test with 95% significance level (α = 0.05). generally, the result showed that biogas sludge can increase the vegetative and generative growth of rice plant ‘segreng’ on polybag scale and rice field scale. the rice plant on polybag with 4 ml biogas sludge was significantly different on the vegetative growth and chlorophyll content, while the rice plant on polybag with 8 ml biogas sludge was significantly different on the generative growth and nra levels. keywords: physiological response; sludge; biogas; ‘segreng’; wukirsari introduction rice (oryza sativa l.) is an indonesian main food and strategic commodity. however, rice production in indonesia has not been able to meet the population demands (alavan et al., 2015). in yogyakarta, the rice demand continues to increase because the growth of population is not balanced with agricultural area expansion and rice quality improvement. this in turn led to a decrease in rice production. wukirsari village, cangkringan district belongs to sleman regency which is the supplier of rice in the special region of yogyakarta. the area of this village is 1,456 ha. topographically, the village is located at an altitude of 450 to 600 m above sea level, with an average rainfall of 22 mm/year. the average temperature per year is 21-31oc. wukirsari village is a suitable area for agriculture. wukirsari village is located between the gendol river in the east and the yellow river to the west (rpjm desa wukirsari, 2015; siswanti, 2015). rice production in yogyakarta has decreased from 721,674 tons in 2013 to 713,800 tons in 2014 (bps daerah istimewa yogyakarta, 2015). the productivity decrease becomes a serious obstacle faced by the agricultural sector today. one of the impacts is the use of inorganic fertilizers such as urea, za, tsp / sp-36 and kcl (redono, 2016). the abundance of inorganic materials in the soil reduced the regeneration ability of rice plants (sudadi & widada, 2001 in redono, 2016). according to siswanti (2015), the agricultural land in sleman district, including in wukirsari village contains high chemicals due to excessive use of chemical fertilizers (500 kg/ha). bio organic fertilizers can improve the physical, chemical, and biological properties of the soil that make them quite profitable. one kind of bio organic fertilizers that is expected to contain many nutrients is biogas sludge. biogas produces liquid solid waste in the form of livestock manure that has lost its gas and is rich in the elements needed by plants such as protein, cellulose, and lignin which cannot be replaced by chemical fertilizers (ginting, 2007). until now there has been no research on red rice plant using biogas sludge as fertilizer, therefore, it was not yet known about the effect of biogas sludge as planting medium to vegetative and generative growth, chlorophyll content and nra content of segreng rice https://doi.org/10.14421/biomedich.2018.71.21-26 22 biology, medicine, & natural product chemistry 7 (1), 2018: 21-26 planted in the rice field of wukirsari village, cangkringan district, sleman regency. the aim of this research is to understand the effect of biogas sludge as planting medium on the vegetative growth, generative growth, chlorophyll content and the nitrate reductase analysis (nra) value of ‘segreng’ rice, and to find out the optimum concentration of biogas sludge on the growth of 'segreng' rice. materials and methods tools and materials the tools used in this study were plows, bamboo, 1 liter glass cylinder, thermometer, soil ph tester, luxmeter, tube, spectrophotometer, analytical scale, micropipet, pipette, pump pippet, porcelain pallet, whatman no.1 filter paper, aluminum foil, cuvette, scissors, oven, 100 ml measuring flask, and dark flakon. materials used in this study were segreng rice seedlings, paddy soil, biogas sludge, polybag, water, 80% acetone, distilled water, salt, phosphate buffer ph 7.5 0.1 m, nano3 5 m, 0.02% ned, 1% sa in 3 n hcl, aquadest, nano2 0.6 m, tissue paper, and aluminum foil. data collection rice field with an area of 500 m2 was divided into five parts/plots so that each plot size was 100 m2. plot 1 (control) was not added with sludge, plot 2 was added with 1 liter of sludge, plot 3 was added with 1.5 liter sludge, plot 4 was added with 2 liter sludge, and plot 5 was added with 2.5 liter sludge. meanwhile, on polybag media treatments, preparation of rice field soil was done by measuring the weight until 5 kg, mixed with sludge, then put them in polybags with the diameter of 21 cm. the treatment given on p1 (control) was not added with sludge, p2 was added with 4 ml sludge, p3 was added with 8 ml sludge, p4 was added with 12 ml sludge, and p5 was added with 24 ml sludge. parameters measured on the growth of rice plants include the number of leaves, number of seedlings, plant height, and analysis of chlorophyll content by spectrophotometric method (aoac, 1995). while the parameters measured on the productivity of rice crops include dry weight, number of panicles, the weight of filled and empty grains, the number of filled and empty grains, and nra value analysis using modified assay method (hartiko, 1983). the data of vegetative parameter, generative parameter, chlorophyll content and nra level of the 5 treatments were tested with one way anova (analysis of variance) with spss version 19 for windows and followed by duncan's multiple range test with 95% significance level (α = 0.05) observation and measurement of vegetative parameters were done every week for 9 times of observation, followed by observation and measurement of generative parameters done every week for 5 times of observation. parameters observed included plant height, number of leaves, number of seedlings, total chlorophyll content, dry biomass weight, crown weight, root weight, filled grains weight, empty grains weight, number of filled grains, number of empty grains, number of panicles, and nra levels. the steps taken in this research were land preparation, seed preparation, rice seed planting, parameters measurement and rice plant maintenance. preparation of land included the process of jacking rice fields with a depth of 20-25 cm and repeated 2 times with 1 week time difference. this was aimed to ensure the soil conditions become poor of nutrients, so that the effect of biogas sludge becomes significant. in the meantime of waiting the readiness of rice field, preparation of segreng rice seedlings was done. segreng variety rice was chosen for this study because it has several benefits including: 1. high yield, i.e. 3-4 ton/ha, 2. the husks contain β-carotene of 488.65 micro g/100 g, 3. high rice selling value, which is 30% more expensive than ordinary rice, 4. tolerant to water stress, 5. high protein content of about 7.3%, completed with 4.3% iron and 0.34% vitamin b1 (kristamtini & prajitno, 2009). rice seeds were first soaked in 3% nacl solution for 24 hours. this treatment served to separate good quality rice of seeds with the bad ones. good quality rice seeds would sink, while bad seeds would float when soaked in salt solution. furthermore, good quality rice seeds were dried under sunlight for about 3 days until dry grains were obtained. it aimed to avoid possible contamination of moist rice seeds. furthermore, rice seeds were spread in a nursery area of 2x2 m2 with inundated soil. in this condition, seeds would experience imbibition, or the process of water seeping into the seeds. water would activate enzymes that play roles in the germination process. the seeds were grown until 20 days. at this age, the rooting system of the rice seedlings were strong enough and the number of leaves were more than 4, so it could be stated that the rice seedlings were ready to be moved to the rice field. prior to the process of moving rice seedlings to the rice field, the land were processed with biogas sludge. biogas sludge used were 0; 1; 1.5; 2; and 2.5 liters per 100 m2 of the rice field and 0 ml; 4 ml; 8 ml; 12 ml and 24 ml per 5 kg of soil on polybags. data were taken on three repetitions. the vegetative growth parameters included plant height, number of leaves, number of seedlings and chlorophyll content, while generative growth parameters measured included nra levels, dried biomass including crown/stem, root, filled grains, empty siswanti et al. – physiological response of 'segreng' rice plant (oryza sativa l.) … 23 grains, and total weight and number of filled grains, empty rains, and the number of panicles. the treatment of rice crops included the opening of irrigation flows, weeding and eradicating pests. measurements of growth parameters were done weekly. the measurement of plant height was done by medline from the base of stem to the highest leaf tip. the number of seedlings is the number of new clumps that appears next to the parent plant. the total number of leaves is the number of leaves of the parent plant and the number of leaves of the seedlings or clumps. the measurement of chlorophyll content was done on the 4th week, the leaf samples used were parent plant leaves, taken ± 5 cm in the middle of the leaf. this was because the section was assumed to be mesophyll tissue where the process of photosynthesis happens, so the chlorophyll content obtained were expected to be more optimum than at the edges. the measurement of chlorophyll content was done by spectrophotometric method. the principle of the spectrophotometric method is measuring the absorbance value of the solution to a particular wavelength. in this study, the wavelength used were 642,5 nm and 660 nm, because chlorophyll a is able to absorb wavelength of 673 nm, while chlorophyll b is able to absorb wavelength at 455-640 nm. meanwhile, nra test was done when the rice entered the generative stage, marked by busted panicles. the nra test was performed because the activity of enzyme nitrate reductase could show how much the rate of metabolism in the plant body is increased due to the preparation of building molecules (proteins) to be transported to the grain of rice. nitrate reductase is the key enzyme that first converts nitrate ions which are absorbed by the roots into nitrites, which are then converted to ammonia. ammonia will react with glutamine to form amino acid glutamate and form other amino acids. these amino acids are used as cell constituents and transported to the grain. therefore, according to alnopri (2004), nra levels usually show predictions of the productivity of panicle-busted rice. in the nra test, the rice leaves used were ‘flag leaves’ or the leaves located just above the grains. the flag leaves are the topmost leaves and the youngest leaves among the other leaves. young leaves have a higher activity than the old leaves. according to hartiko (1983), the leaf that has a higher position has a higher nitrate reductase activity than the leaves below it. results and discussion based on the research, the vegetative growth parameters included plant height, number of leaves, number of seedlings and chlorophyll content, while generative growth parameters measured included nra levels, dried biomass including crown/stem, root, filled grains, empty grains, and total weight and number of filled grains, empty rains, and the number of panicles, was present below: note for figure 1 – 8: treatment 1= control treatment 2= 1 liter sludge/100 m2 or 4 ml sludge/polybag treatment 3= 1,5 liters sludge/100 m2 or 8 ml sludge/polybag treatment 4= 2 liters sludge/100 m2 or 12 ml sludge/polybag treatment 5= 2,5 liters sludge/100 m2 or 24 ml sludge/polybag figure 1. the average of plant height on 9th weeks in, (a) rice field; (b) polybags. figure 2. the average of number of leaves on 9th weeks in, (a) rice field; (b) polybags. a, b b a, b a, b a 65 70 75 80 85 90 95 1 2 3 4 5 p la n t h e ig h t (c m ) treatments a, b b a, b b a, b 50 55 60 65 70 1 2 3 4 5 p la n t h e ig h t (c m ) treatments a, b b a, b a, b a 0 2 4 6 1 2 3 4 5 n u m b e r o f le av e s treatments b a a, b a, b a, b 4 4.5 5 5.5 6 1 2 3 4 5 n u m b e r o f le av e s treatments 24 biology, medicine, & natural product chemistry 7 (1), 2018: 21-26 figure 3. the average of number of seedling on 9th in, (a) rice field; (b) polybags. figure 4. the chlorophyl content of each treatment in (a) rice field and (b) polybags. figure 5. the average of dried mass in (a) rice field and (b) polybags. figure 6. the average number of grains in (a) rice field (b) polybag. a b a, b b a, b 0 0.5 1 1.5 2 2.5 1 2 3 4 5 n u m b e r o f se e d lin gs treatments a b a, b a, b a, b 0 0.5 1 1.5 2 2.5 1 2 3 4 5 n u m b e r o f se e d lin gs treatments 0 2 4 6 8 10 1 2 3 4 5 c h lo ro p h yl c o n te n t (m g/ g le af ) treatments chlorophyl a chlorophyl b 0 5 10 15 20 1 2 3 4 5 c h lo ro p h yl c o n te n t (m g/ g le af ) treatments chlorophyl a chlorophyl b a,b a, b b a a, ba a a a a a, b a,b b a a,b 0 10 20 30 40 50 1 2 3 4 5 d ri e d b io m as s (g ) treatments total biomass stem biomass root biomass 0 5 10 15 20 25 1 2 3 4 5 d ri e d b io m as s (g ) treatments total biomass stem biomass root biomass 461 266 483 326 407 209 278 143 126 138 0 100 200 300 400 500 600 1 2 3 4 5 n u m b e r o f g ra in s treatments filled grains empty grains a b b b b a a a a a 0 50 100 150 200 1 2 3 4 5 n u m b e r o f g ra in s treatments filled grains empty grains siswanti et al. – physiological response of 'segreng' rice plant (oryza sativa l.) … 25 figure 7. the average of grains mass in (a) rice field and (b) polybags. figure 8. nitrate reductase analysis levels of sample in (a) rice field and (b) polybags. discussion the rice cultivation system conducted in this research is the legowo jajar planting system or commonly known as tajarwo. tajarwo system is a planting system with spaces between rows : 25 cm, in rows : 12.5 cm, and between legowo/groups of rows : 50 cm. the spacing is arranged by narrowing the distance of the rice inside the row and widening the distance between the rows. tajarwo's unique principle is the arrangement of rice plants that affects those at the outer sides. generally, the plants grown at the outer sides provide better growth than plants in the middle because of the reduced interrow plant competition (pahruddin et al., 2004). tajarwo was chosen because it has several advantages, i.e. sunlight can be absorbed by the stem better so that the rate of photosynthesis is higher, fertilization and crop pest control are easier to be done because there is a wide distance (legowo) between rows. in addition, the tajarwo system can also increase the population of plants per hectare (pahruddin et al., 2004). a previous research by anggraini et al. (2013) also shows that the productivity of rice grown by tajarwo planting system is better than by conventional/tile system. the tajarwo system has the advantage of having wide open space between two groups of crop rows, increasing the intensity of sunlight entering the clumps, increasing rice yield to 10-15%, facilitating plant maintenance, and reducing the risk of pests and diseases (abdulrachman et al. 2013). the height of segreng rice crops tend to increase every week. the height drop of rice plants with treatment of 24 ml / 5kg at week 4 was possibly due to the pest attack or the torn out leaves at several centimeters from the top. from table 2 on environmental parameters data, it can be seen that there was a considerable decrease in air humidity, it can also indicate a decrease in the height of rice plants. figure 2 is given to clarify the observations for plant height. based on the observations, the highest plant height was found in rice plants grown in 4 ml sludge / 5kg of rice field soil. based on dmrt test (α = 0,05), the effect of biogas sludge dosage on plant height was not significantly different. this can happen if the rice plants have reach the vegetative phase at the 9th week, because according to vergara (1976), the rice plant at the 4th week until before the 7th week is actively propagating to produce seedlings and after the 7th week, the plant is ready to enter the reproductive phase by initiating the formation of panicles. after going through the planting period, the rice plants at week 7 have entered the reproductive phase. the rice plant has passed through a phase where the cells of active plants divide and enlarge. of the five treatments, the best biogas sludge concentration for the vegetative parameters of segreng rice was p2 (1 l/100 m2). based on anova test result, the result of treatment 2 (p2) was significantly different to treatment 5 (p5). 9 4 9 6 7 1 2 1 0 1 -2 0 2 4 6 8 10 12 1 2 3 4 5 g ra in s m as s (g ) treatments filled grains empty grains 0.5 2.27 1.98 2.28 2.34 0.15 0.31 0.2 0.24 0.3 0 0.5 1 1.5 2 2.5 3 1 2 3 4 5 g ra in s m as s (g ) treatments filled grains empty grains a a a a a 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1 2 3 4 5 n r a l e ve ls treatments 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 a b c d e n r a l e ve ls treatments 26 biology, medicine, & natural product chemistry 7 (1), 2018: 21-26 meanwhile, the number of leaves were obtained on the 9th week. the treatment that gave the highest total number of leaves was treatment 2 (p2) that was 1 l/100 m2 or 100 l/ha. based on anova test result, it was found that treatment 2 (p2) was significantly different to treatment 5 (p5). this is because at that concentration, the n and mg content is optimally absorbed by the rice plant for chlorophyll synthesis, to obtain more leaf number than other treatments. plant growth is affected by various factors, both internal factors and external factors. internal factors derived from the seeds of rice itself (e.g. genetic and seed conditions), while external factors derived from the surrounding environment around the rice which certainly has an effect on rice growth. each place has a different environment, be it air temperature, water temperature, soil moisture, soil ph, other soil conditions, pests and diseases, and rainfall. the existence of these variations of environmental conditions certainly result in the different growth and development of rice among regions, although given the same treatment. conclusion conclusion the biogas sludge could increase the vegetative and generative growth of ‘segreng’ rice plant on rice field or polybags in wukirsari village, cangkringan, sleman. based on the dmrt analysis with one way anova, the treatment 2 viz 1 liter/100 m2 or 4 ml/polybag gave the best result on vegetative growth and chlorophyll content of the ‘segreng’ rice plant. meanwhile, treatment 3 viz 1,5 liters/100 m2 or 8 ml/polybag gave the best result for generative growth and nra levels of the ‘segreng’ rice plant. suggestion based on the research that has been done, there are some suggestions to support the next research of organic farming, such as we need to increase the biogas sludge concentration and need to add the treatment range to get significantly differences by one way anova analyze. references abdulrachman, s., m. j. mejaya, n. agustiani, i. gunawan, p. sasmita, a. guswara. 2013. legowo cultivation system. badan penelitian dan pengembangan pertanian kementerian pertanian. subang. p: 8 alavan, ade, rita hayat, dan erita hayati. 2015. pengarhun pemupukan terhadap pertumbuhan beberapa varietas padi gogo (oryza sativa l.). jurnal floratek 10: 61-68. badan pusat statistik indonesia. 2016. rata-rata konsumsi per kapita seminggu beberapa macam makanan penting. https://www.bps.go.id/linktabelstatis/view/id/950. accessed on 1st april 2016. ginting. 2007. teknologi pengolahan limbah peternakan. fakultas pertanian universitas sumatra utara. medan. hartiko, h. 1983. leaf and root in vivo nitrate reductase activities of coconut (cocos nucifera l.) cultivars and hybrids. ph.d dissertation of university of the phillipines. los banos, p: 132 pahruddin, a., maripul dan p. rido. 2004. cara tanam padi sistem legowo mendukung usaha tani di desa bojong, cikembar sukabumi. buletin teknik pertanian, 9(1): 10-12 redono, cucuk. 2016. respon petani terhadap penggunaan pupuk organik pada tanaman padi sawah di kelurahan bokoharjo kecamatan prambanan kabupaten sleman. jurnal agrica ekstensia. vol. 10 no.1st, june 2016. p. 29 rpjm pemerintah desa wukirsari. 2014. rencana pembangunan jangka menengah desa (rpjmdes) wukirsari, cangkringan, sleman. unpublished. siswanti, d.u. 2015. pertanian organik terpadu di desa wukirsari, sleman, yogyakarta sebagai usaha pemulihan kesuburan lahan terimbas erupsi merapi 2010 dan pencapaian desa mandiri sejahtera. indonesian journal of community engagement. vol. 01 no. 01, september 2015. steenis, dr. c.g.g.j van. 1988. flora: untuk sekolah indonesia. pt. pradnya paramita. jakarta. p. 127. sutejo, m.m. 1995. pupuk dan cara pemupukan. jakarta: rineka cipta. suzuki, k, takesi w and volum.2001. concentration and critalization of phosphate, ammonium and mineral in the effluent of biogas digester in the mekong delta. jerrrean and contho university vietnam. vietnam. p.3 physiological response of 'segreng' rice plant (oryza sativa l.) to biogas sludge at wukirsari village, cangkringan, sleman biology, medicine, & natural product chemistry 7(1) 2018 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 93-103 | doi: 10.14421/biomedich.2021.102.93-103 issn 2540-9328 (online) a physical chemistry study of black powder materials by solution combustion synthesis method fitria hidayanti1,*, kiki r. lestari1, nano sujani1, jarot raharjo2 1engineering physics department, universitas nasional, jakarta selatan 12520, indonesia. 2center for materials technology, agency for the assessment and application of technology puspiptek building no. 224, south tangerang, banten 15314, indonesia. corresponding author* fitriahidayanti@gmail.com manuscript received: 14 august 2021. revision accepted: 30 september, 2021. published: 08 october, 2021. abstract a study on the synthesis of black powder (la2nio4) material using the solution combustion synthesis method at a variation of synthesis temperature of 60, 70, and 80°c was carried out. it produces a mass of black powder of 2 grams by four times of synthesis process. then, material characterization was performed on the black powder samples obtained by using x-ray diffraction (xrd) to determine the phases formed, scanning electron microscopy energy dispersive x-ray spectroscopy (sem-eds) to determine the morphology and analyze the composition elemental on the microscale and fourier transform infra-red (ftir) to determine chemical bonds. from the whole black powder sample, xrd analysis showed the phases of dilantanum nickel tetraoxide (la2nio4), nickel oxide (nio), lanthanum oxide (la2o3), and lanthanum oxide ht x-form (la2o3 ht (x-form)). in addition, it was seen from the visible compositions of the phases that the nio phase looks more dominant and the variation of the synthesis temperature shows that the la2o3 phase was increasing. this was supported by the eds analysis, which showed that the eds spectrum contains elements la, ni, and o where the element o indicates that oxidation occurs in the elements ni and la. on the other hand, the sem analysis results confirm that the black powder sample contains the elements la and ni, based on the high and low electron images contained in the morphology of the black powder sample. in addition, it was also known that the particles in the black powder sample were micron size and had porous morphology. this occurs due to rapid thermal decomposition events and excessive gas development. in addition, ftir analysis showed that the o-h bond had been reduced and there are still c-o and c-h bonds indicating the presence of organic elements possessed by glycine. keywords: la2nio4, solution combustion synthesis; battery nimh; hydrogen storage alloy. introduction the development of electronic technology and electric cars can not be separated from the role of energy sources as a power to use these technologies. one source of energy booster is the battery. a battery is a device that has a working principle to convert chemical energy into electrical energy through electrochemical processes. one fairly rapid development occurred in the type of rechargeable batteries. a rechargeable battery is a type of battery that can be recharged again with the charge & discharge system. similar to the type of primary battery, batteries generally consist of 3 main components, namely the anode, cathode, and electrolyte. there are very rapid development in nickel metal hydride (nimh) batteries (chang et al., 2016, young et al., 2017). highpower international inc., which is a clean energy supplier company integrated with research and development, manufacturing and sales of nimh and li-ion rechargeable batteries, as well as energy storage and battery recycling systems, explains that the majority of the 74% nimh battery consumer market share in china, followed by japan as 14% and europe as 10%, and 2% are the others. in addition, european countries still have a large market level for the consumption of nimh batteries. for example, in 2013, germany, the netherlands, belgium, and the uk accounted for 8.3% of nimh battery needs from china, and in february 2014, north america received 51.76% of nimh battery needs, followed by europe receiving 32.7%, and 13.73% (chang et al., 2016). the data shows that the market share of nimh batteries is still growing, and indonesia may still be able to develop nimh batteries given the abundant natural resources for raw materials for nimh batteries. indonesia is a country that has the second-largest wealth of nickel in the world. nickel ore is contained in nickel oxides or laterites, which are spread in eastern indonesia (prasetiyo, 2008). based on data from the geological resource center in 2016, indonesia has nickel resource potential of 5,756,362,683 (ore) and 79,172,702 (metals) and nickel reserves of 3,197,178,940 (ores) and 50,872,304 (metals) with elemental content nickel average 1.20-3.25%. these https://doi.org/10.14421/biomedich.2021.102.93-103 94 biology, medicine, & natural product chemistry 10 (2), 2021: 93-103 nickel resources and reserves in 2011-2015 continued to increase due to the discovery of new nickel resources (haryadi, 2017). in contrast, the potential of soil metals is rarely found in monazite and xenotime minerals with reserves of more than 951,000 tons (inatadon et al., 2015). some of indonesia's rare earth metal minerals are contained in an associated tin in bangka belitung and gold in kalimantan (virdhian and afrilinda, 2014). both of these minerals are raw materials from nimh batteries. nimh batteries are secondary batteries in which metal hydride acts as a negative electrode and hydrogen-absorbing alloy acts as an active substance in the negative anode. the development of nimh batteries received considerable attention from researchers, especially their use in hybrid electrical vehicles (hev) technology. in addition, the unique characteristics of the working principle of the battery, especially in the hydrogen storage alloy as the anode of the battery, is an interesting topic for writers and has been developed by several researchers, such as research conducted by several institutions in china, japan, europe, and america (chang et al., 2016, young et al., 2017, ouchi et al., 2016, chang, et al., 2017). the research mainly focuses on nimh battery anode components, where there is a hydrogen storage alloy material. the role of hydrogen storage alloy material in this case are greatly affects to nimh battery power density. furthermore, its role is very important in the absorption/adsorption process of h2 at negative electrodes. nickel metal hydride (nimh) batteries nimh batteries are rechargeable batteries by utilizing electrochemical reactions found in active materials or electrodes. like most batteries, the main components in nimh batteries are the same as batteries in general, consisting of negative and positive electrodes and electrolyte solutions. nimh battery's active material used on the positive electrode (cathode) is ni(oh)2. at the same time, the hydrogen storage alloy material is used as active material on negative electrodes. in addition, the electrolyte solution that is usually used in nimh batteries is potassium hydroxide (koh). in nimh battery cathode an electrochemical reaction (equation (1)) is occur as follows: 𝑁𝑖(𝑂𝐻)2 + 𝑂𝐻− → 𝑁𝑖𝑂𝑂𝐻 + 𝐻2𝑂 + 𝑒− (1) in the charging process, atoms in nickel hydroxide (ni(oh)2) compounds will dissociate to form nickel oxide hydroxide (niooh) compounds and water and electrons. then the hydrogen atom moves through the diaphragm (electrolyte) to the nimh battery anode in combination with the metal hydride material. meanwhile, electrons flow to the anode through an external circuit, then the reaction occurs (equation (2)) in the anode as follows: 𝑀 +𝐻2𝑂 + 𝑒− → 𝑀𝐻 + 𝑂𝐻− (2) then the charging process at the cathode occurs where an hsa material and a hydrogen atom dissociate at the cathode and an electron to form a metal hydride material and a hydroxide ion. briefly, the process of charging and discharging is shown in figure 1. figure 1. charge and discharge mechanism on nickel metal hydride batteries (chang, s., young, k. h., & lien, y. l., 2017). solution combustion synthesis method solution combustion synthesis (scs) is an effective method for the synthesis of nanoscale materials, versatile and efficient in the use of energy that has been used in the production of various types of ceramic powders for several types of advanced applications (li et al., 2016, khina, 2010). the scs method is a simple method but an important technique in synthesising and processing nanostructures of metal, oxide, spinel, alloy, and intermetallic (xanthopoulou et al., 2018). this method is based on an exothermic reaction between nitrates (oxidizing agents) and organic substances (reducing agents) by directly producing nanostructured powders (xanthopoulou et al., 2018, deganello et al., 2018). utilization of the exothermic reaction as a source to drive the reaction itself so that no external source is needed and only requires ignition temperature to start the process (gonzález-cortés et al., 2013). crucial advantages of the scs method are (i) mixing of the initial reaction that occurs in a liquid state, thereby facilitating control of the composition, structure, homogeneity, and stoichiometry of the reaction product; (ii) the possibility of incorporating impurity/dopant ions in the form of oxides, to prepare materials that are suitable for industrial needs; (iii) the very fast scs method allows the formation of metastable phases (xanthopoulou et al., 2018, aruna, 2017, manukyan, 2017). in addition, the main thing that is important from the use of the scs method is that it can be used with various precursors, both soluble and non-dissolved (deganello et al., 2018). hidayanti et al. – a physical chemistry study of black powder materials … 95 material characterization material characterization is an activity carried out to determine the physical properties, mechanical properties, and chemical properties through various tests. among the tests are thermogravimetry analysis (tga), differential thermal analysis (dta), dsc, fourier transform infra-red (ftir), x-ray diffraction (xrd), x-ray fluorescence (xrf), scanning electron microscopy (sem), energy dispersive x-ray spectroscopy (eds), uv-vis, and many other tests. this study, it was conducted characteristic tests on three main types of tests, including (1) x-ray diffraction spectroscopy (xrd), (2) fourier transform infrared spectroscopy (ftir), and (3) scanning electron microscope (sem). therefore, this research aimed to obtain hydrogen storage alloy material which is still in the scope of research in black powder synthesis with variations in temperature of synthesis/reaction using the solution combustion synthesis (scs) method. then the material characterization analysis was carried out, including crystal size, chemical bonds, functional groups, and morphology on the influence of temperature. research methods black powder material synthesis black powder synthesis was carried out by dissolving the three precursors as presented in the synthesis process flow in figure 2. the precursors used in this synthesis process include 0.433 grams (1 mmol) lanthanum nitrate hexahydride (la (no3)3.6h2o) p.a. which produced by merck, 1.454 grams (5 mmol) nickel nitrate hexahydride (ni (no3)2.6h2o) p.a. produced by merck, and 0.976 grams (7.2 mmol) glycine (c2h5no2) is produced by merck. then, it is dissolved at 10 ml distilled water which is equivalent to 10 grams (adziimaa et al., 2013). after that, simultaneously stirring using magnetic stirring with a total mass of 12.863 grams. the temperature variations carried out in the stirring process were at temperatures 60, 70 and 80 °c (xanthopoulou et al., 2018, liu et al., 2016, patil et al., 1997, xanthopoulou et al., 2017). when the stirring process reached the desired temperature, the temperature of the solution was stabilized at a set point at 60, 70, and 80 °c. after that, the transparent green solution that had been carried out in the stirring process on the beaker glass is transferred into the cup for further drying process which aims to remove the formed water element. this drying process was carried out for 3 hours 20 minutes until the gel was formed (liu et al., 2016). the purpose of forming the gel from the solution between the fuel and the oxidant was tightly connected in the gel network (deganello et al., 2018). figure 2. the flow of black powder material synthesis. the gel obtained from the drying process was then calcined by heating the powder at high temperatures but still under the melting point (latif et al., 2014). in addition, this process aimed to eliminate volatile elements, heat decomposition and form new phases of the three precursors used. the condition of this gel calcination with a temperature of 500 °c and a heating rate of 10°c/min is then held for 2 hours (liu et al., 2016). then the gel would reach ignition temperature to undergo combustion and change into a sponge shape with volume development. the expanded sample was then mashed using a mortar to obtain a sample in the form of powder. x-ray diffraction this process was carried out on the x-ray diffraction characterization at the lipi physics research center with the xrd smartlab, rigaku. xrd characterization was carried out under test conditions with a voltage of 40 kv and a current of 30 ma. the target element used to radiate the sample is cu kα with wavelength specifications 0.1541862 nm. besides the step mode scan with a scan speed of 0.25 and a scan range of 10 90 deg for each sample. then an analysis of the x-ray diffraction pattern of the black powder sample was conducted using the rietveld method using panalytical high score plus software. sem-eds the scanning electron microscopy characterization has been carried out at the bppt technology incubator center using the scanning electron microscope (sem) type quanta 650 to test three samples with magnifications of 500, 1000, 1500 and 10000 and the energy dispersive x-ray spectroscopy (eds) of oxford instruments. it was calcined on 500 oc for 2 hours with rate 10 oc/min it was dryed on 100 oc it was stirred on 60, 70 and 80 oc it was dissolved in 10 ml of distilled water 96 biology, medicine, & natural product chemistry 10 (2), 2021: 93-103 fourier transform infrared (ftir) fourier transform infrared (ftir) characterization has been conducted at the material technology center bppt. the ftir test was carried out using the nocolet is50 thermo scientific type ftir under the conditions of the spectra scan speed test per second 65 (at 16 cm-1) and 95 (at 32 cm-1). with a scan range of 400-4000 cm-1 for each black powder sample. result and discussion x-ray diffraction the diffraction pattern of the sample for black powder with a variation of synthesis temperature at 60 ºc, 70 ºc and 80 ºc is shown in figure 3. it is known that in the angular range of 10 to 90 degrees, there is an identical diffraction peak pattern of the four black powder samples, the identical peak pattern in the 2theta angles, including 37°, 43°, 62°, 75° and 79° which are identified as phases of nickel oxide. figure 3. x-ray diffraction pattern for each sample back powder. meanwhile, figure 4 was a magnification of figure 3 with an angle range of 2 theta 20º to 40º, where there were differences in the peak pattern in the angle range of 2 theta. in the sample, black powder room temperature can be indicated at magnification with a range of angles that the peak at an angle of 2 theta 26°, 29°, 30°, and 31° is the peak of the lanthanum oxide (la2o3) phase. while at an angle of 2 theta 23° and 31° was the phase of the lanthanum nickel tetraoxide (la2nio4). in addition, it was also observed at the diffractogram peaks for black, 60 °c, 70 °c, and 80 °c black powder samples. an identical peak was seen at an angle of 2 theta were 25° and 30° which is the phase of lanthanum oxide. while the peak at an angle of 2 theta was 28°, it experiences shrinkage as the temperature of the synthesis rises, which indicates that the angle represents the lanthanum oxide ht (x-form) phase. on the other hand, in figure 5 there was also the formation of peaks in the range of 30° to 60°, at an angle of 39° and 44° observed that there is a paw that shows the lanthanum oxide phase, as the temperature rises, the formation of the peak shows more clearly that the lanthanum phase is formed oxide. similarly, around the angle of 2 theta 40° and 45° also formed a diffractogram peak which showed the development of the phase of lanthanum nickel trioxide at room temperature, but along with the increase in temperature appeared lanthanum oxide phase at the peak at 53° and 55°. figure 4. magnification of diffractogram black powder sample on 2: 30 60°. figure 5. magnification of diffractogram black powder sample on 2: 20 40°. judging from the suitability criteria for the profile fitting process based on the literature, the good refinement match is determined by the weighted rprofile (rwp) and goodness of fit (gof) parameters. rwp is a weight comparison pattern of the difference between observation patterns with xrd calculations (speakman, 2012). the ideal value of rwp is <10% 10 20 30 40 50 60 70 80 90 (011) (010) (222) (113) (022) (002) bp 80 bp 70 bp 60 in te n si ty ( a .u ) 2 theta (degree) bp rt (111) 30 40 50 60 (013) (021) (112)(020)(110)(002) (012) (110) (013) (112) (017)(011)(012) in te n s it y ( a .u ) 2 theta (degree) bp rt bp 60 bp 70 bp 80 20 25 30 35 40 (011) (011) (011) (010) (011) (011) (010) (011)(010) (002) (010) (011) in te n s it y ( a .u ) 2 theta (degree) (013) bp rt bp 60 bp 70 bp 80 hidayanti et al. – a physical chemistry study of black powder materials … 97 (speakman, 2012, sarwanto and adi, 2018). while rexpected is a statistical evaluation of noise data on diffractograms. in addition, the parameter of goodness of fit (gof) or commonly known as chi-squared is a ratio of xrd observation patterns that are comparable with expected, with an ideal value of no more than 4% (speakman, 2012). the goodness of fit and rwp factors, as well as the size of the crystals that have been calculated using high score plus for each black powder sample, are known respectively in table 1. table 1. match parameters and crystal size of each black powder sample. sample uniform parameter crystal size rexpected rwp gof black powder room temperature 9.1453 7.9019 1.1902 2.0737 black powder 60°c 9.0551 8.1407 1.2738 5.0808 black powder 70°c 9.1597 8.1181 1.2420 4.3323 black powder 80°c 8.9618 7.6367 1.1468 3.4879 from table 1 it can be concluded that the level of fitting for each black powder sample can be categorized very well according to the literature mentioned earlier. in addition, the results of the analysis of the composition of compounds contained in each black powder sample are listed in table 2. table 2 showed that in the black powder room temperature sample, there is a la2nio4 phase with a composition of 27.4%. table 2. phase composition contained in the black powder sample. compound composition of compounds (%) r.t 60 °c 70 °c 80 °c la2nio4 27.4 nio 71.3 84.6 82.0 80.7 la2o3 1.3 15.3 17.8 19.3 la2o3 – ht (x-form) 0.1 0.2 total 100% 100% 100% 100% in each black powder sample 60-80 °c, the la2nio4 phase was not formed and tended to show a very dominant nio phase and it was observed that with increasing synthesis temperature, la2o3 phase composition tends to increase. scanning electron microscopy (sem) analysis the analysis was carried out with three magnifications, namely 500x, 1000x and 10000x. the sample chamber is vacuum up to 3.54x10-4 pa for 500x magnification and 19.4x10-2 pa for 1000x and 10000x magnification, respectively, to ensure that the sem column is free of air molecules. the sem was operated with a standard high voltage operation parameter of 10 kv with a spot size of 2.5 and a work distance (wd) of 9.6 mm for a 60°c black powder sample. sem results for the microstructure test of a 60°c black powder sample are shown in figure 6 taken with a se detector. figure 6. the microstructure of black powder 60°c (a) with a magnification of 500x (b) with a magnification of 1000x (c) the measurement of molecular samples of black powder and (d) with a magnification of 10000x (e) measurement of the diameter of the pores formed. at 500x magnification (figure 6a), black powder particles are scattered to form small fragments and some are porous chunks. to clarify the observed porous profile, a 1000x magnification was performed. the analysis results with higher magnifications observed that there were agglomerates in the porous lump (figure 6b). the average particle size observed at 1000x magnification is in the micron range with an average particle size of 11,8093 µm (figure 6c), but it has not provided information about the porosity of the black sample particles the powder. on that basis, the analysis continued with 10000x magnification. in micrographs with a magnification of 10000x, it was able to show important information, which was very clearly visible agglomerates contained in the porous lump (figure 6d) and the size of the pores in the micron order with an average pore diameter of 22.8361 µm (figure 6e). then the analysis for the 70 °c black powder sample is presented in figure 7. with the sem operation specifications for the high voltage operation parameter of 10 kv with spot size 2.5 and work distance (wd) of 10 mm for the 70°c black powder sample. the sample chamber is vacuum up to 1.30x10-4 pa for 500x magnification and 1.41x10-4 pa for 1000x magnification and 1.52x10-4 for 10000x magnification. in micrographs with a magnification of 500x, it was seen that in the 70°c black powder sample, the particles in the sample 98 biology, medicine, & natural product chemistry 10 (2), 2021: 93-103 were scattered with mostly dominated by lumps (figure 7a). then, it was clear that the lump had pores on each of the lumps (figure 7b) with an average size of the lumps of 20,89 µm (figure 16c). in addition, at 10,000x magnification (figure 7d), it was observed that there were agglomerates with an average of 0,5632 µm and an average pore diameter of 0,7508 µm (figure 7e). figure 7. the microstructure of black powder 70 °c (a) with a magnification of 500x (b) with a magnification of 1000x (c) the measurement of molecular samples of black powder and (d) with a magnification of 10000x (e) measurement of the diameter of the pores formed. figure 8. the microstructure of black powder 80 ° c (a) with a magnification of 500x (b) with a magnification of 1000x (c) molecular measurements of the black powder sample and (d) with a magnification of 10000x (e) measurement of the diameter of the pores formed. after that, the 80 °c black powder micrograph sample was shown in figure 8. the micrograph is obtained with a high voltage operation parameter of 10 kv with a spot size of 2.5 and a work distance (wd) of 10 mm. the sample chamber is vacuum up to 1.07x10-4 pa for 500x magnification and 1.16x10-4 pa for 1000x and 10000x magnification, respectively. in figure 8, it appears that the 80 °c black powder sample does not look too many lumps that appear and most are shaped granules. then a magnification of 1,000x was carried out so that it was observed that the particle structure of the black powder sample tended to have pores that were slightly unlike the micrographs of the black powder samples 60 °c and 70 °c. in addition, information was obtained in the form of particle size with an average of 6.7447 µm (figure 8c). in micrographs with 10,000x magnification (figure 8d and 8e), it is known that in the black powder sample 80 °c, the shaft/pores of the particles shrink and tend to be more solid with an average shaft/pores of 0.4676 µm tend to be smaller compared to the black powder samples in figure 6 and figure 7. the morphology of black, 60 °c, 70 °c, and 80 °c samples in the figure 6d, figure 7d, and figure 8d with dark colours that appear to be more dominant in each sample is a constituent element with a low atomic number. in contrast, the bright colours seen in agglomerates are constituent elements that have high atomic numbers (tutu et al., 2015). so it can be concluded that the dark side of the sample contains ni atoms and agglomerates that appear to be representations of la atoms. from the three micrographs, it appears that the high surface area was affected by the type of glycine fuel which causes rapid thermal decomposition and excessive gas development, resulting in greater porosity and crystal size (deganello and tyagi, 2018; fathi et al., 2017). it is also known that the influence of temperature variations in the synthesis can affect the porosity and size of the crystal. energy dispersive x-ray spectroscopy (eds) analysis eds analysis on each black powder sample obtained from the solution combustion synthesis (scs) synthesis method was carried out to determine the composition of the elements in the sample. the results of the eds analysis for each black powder sample are presented in figures 9, figure 10 and figure 11. based on the results of the eds analysis in figure 9, it was known that the microstructure for black powder samples with a temperature variation of 60ºc contains the composition of the elements o, ni, and la at each observation point with the relative percent mass of the element at the first observation point (figure 9a) for ni of 57.8%, element la is 26.6%, and element o is 15.6%. then at the second observation point (figure 9b) shows the relative mass of each element, each for ni is 51.4%, element la is 27.8%, and element o is 20.9%. similarly, the relative mass at the third observation point (figure hidayanti et al. – a physical chemistry study of black powder materials … 99 9c) for each element is as follows for ni of 54.1%, element la for 26.3%, and element o for 19.6%. figure 9. the eds spectrum and composition of the 60 °c black powder sample resulting from the scs synthesis method with three observation points. (a) eds analysis at the first observation point. (b) eds analysis at the second observation point. (c) eds analysis at the third observation point. in addition, figure 10 shows the eds spectrum for black powder samples with a temperature variation of 70 ºc containing the composition of the elements ni, la and o. with each relative mass of the element at the first observation point (figure 10a), namely ni of 56.3%, element la is 24.8%, and element o is 18.9%. furthermore, at the second observation point (figure 10b), it was found that the relative mass of each element, including ni was 55.2%, la was 27.9% and o was 16.9%. then the observation at the third point (figure 10c) obtained the relative mass composition of each element, including ni of 61.2%, la of 27.1% and o of 11.7%. figure 10. the eds spectrum and the composition of the 70 ° c black powder sample resulting from the scs synthesis method with three observation points. (a) eds analysis at the first observation point. (b) eds analysis at the second observation point. (c) eds analysis at the third observation point. on the temperature of 80 ºc, the black powder sample presented in figure 11 showing three observation points on the surface of the black powder sample, the results show that there are elemental compositions as follows ni, la, and o with the relative mass content of each element at the first observation point (figure 11a) ni at 57.1%, element la at 25.1%, and element o at 17.8%. whereas at the second observation point (figure 11b), each element's relative mass composition as follows on the ni element is 56.4%, la element is 26.9%, and element o is 16.7%. at the last observation point (figure 11c), the relative mass composition of each element is 57.2% for the ni element, 25.2% for the la element, and 17.6% for the o element. figure 11. the eds spectrum and the composition of the 80 ° c black powder sample resulting from the scs synthesis method with three observation points. (a) eds analysis at the first observation point. (b) eds analysis at the second observation point. (c) eds analysis at the third observation point. the following is summarized in table 3 results of the analysis of energy dispersive x-ray spectroscopy (eds). table 3. eds test analysis results for each black powder sample. observation element (%) ni la o total black powder 60 point 1 57.8 26.6 15.6 100 point 2 51.4 27.8 20.9 100 point 3 54.1 26.3 19.6 100 black powder 70 point 1 56.3 24.8 18.9 100 point 2 51.4 27.8 20.9 100 point 3 61.2 27.1 11.7 100 black powder 80 point 1 57.1 25.1 17.8 100 point 2 56.4 26.9 16.7 100 point 3 57.2 25.2 17.6 100 it appears that there was three percent by weight of the dominant atom contained in each black powder sample. in addition, it was confirmed that the nickel element has a composition of atomic weight that is greater and most dominant for each black powder 100 biology, medicine, & natural product chemistry 10 (2), 2021: 93-103 sample and the element la is the lowest element. then in the results, it can be concluded that the eds test is eds-oxide seen in the presence of element o. so that due to the presence of element o, oxidation occurs in the elements of ni and la and the compound element is formed which is ni into nio (nickel oxide), and the element it becomes la2o3 (setiadi, 2018). therefore, this result is in accordance with the compounds' composition results where the phases formed are nickel oxide and lanthanum oxide and the nickel oxide compound phase has a very dominant compound composition. this data supports data from xrd where no other peaks are found as impurities outside the reactants used. fourier transform infrared (ftir) analysis the fourier transform infrared spectrum for the three black powder samples synthesized using the solution combustion synthesis (scs) method with temperature variations (room temperature, 60, 70, and 80°c) and the forming material is shown in figure 12. figure 12. ftir spectrum of each black powder sample with its raw material. the fourier transform infrared spectrum (ftir) of the sample is then compared with each of its constituent materials. so it is known that each chemical bonding group is formed and disappears in the black powder sample. based on the analysis that has been done, there are several peaks in the spectrum of lanthanum nitrate hexahydrate (la (no3)3.6h2o) which have been identified in table 4. in the ftir spectrum of the material forming the lanthanum nitrate hexahydrate, it is known that in the forming material, several chemical bonding groups appear, which is found in the wavenumber 3200-3550 cm-1, which indicates the existence of o-h stretching vibrations with strong and wide spectrum characteristics. the o-h bonding group shows that the sample contains water molecules (nedumkallel et al., 2014). table 4. ftir spectrum of lanthanum nitrate hexahydrate, nickel nitrate hexahydrate, and glycine. wavenumber (cm-1) assignment of vibrations la(no3)3.6h2o 3200–3550 o-h bending ~2.980 c–h bending, medium 1293–1434 no3 asymmetric raw material 1368–1464 no3 bonding asymmetric sample black powder. 1649, 1646, and 1640 n=o bonding on black powder samples 60, 70, and 80 ºc 415–423 la-o group ni(no2)3.6h2o 3550–3200 o–h bonding 1619 hydroxyl group 1110 carbonate group 2980 c-h bonding 633 ni-o-h bonding 447–552 ni–o bonding glycine 607, 1389, and 1570 carboxylate group coo 1389 carboxylate group symmetrical coo 1125, 1489, 2600 ammonium group nh3 + 2600 and 3090 ammonium group nh3 + 889 and 1044 ccn asymmetric and simetric bonding 926 ch2 rocking 1322 ch2 twisting 1435 ch2 bonding on tabel 4, peaks around ~ 2980 cm-1 were observed due to vibration stretching of the c-h bond with the characteristic of stretching with medium intensity (kumari et al., 2015). in addition, it was observed that there was no3 stretching asimeter which appeared at 1293-1434 cm-1 in the forming material and at 13681464 cm-1 in each black powder sample. this shows the change in the no3 stretching wave number due to the possibility of constant changes in the force and dipole moment of the no3 group vibrations (trivedi et al., 2015). the characteristic vibration peaks for stretching n = o also appear at 1649, 1646, and 1640 cm-1 respectively in black powder samples 60, 70, and 80 ºc which were previously shifted at wave number 1634 cm1 in the forming material (lanthanum nitrate hexahydrate). whereas la-o chemical bonding group based on literature shows the wavenumber 415-423 cm-1 (radev et al., 2008). however, figure 12 only covers the range of 500-4000 cm-1. then in the infrared spectrum of ni nickel nitrate hexahydrate (no3)2.6h2o material, it was seen that several peaks were formed, including the wave number hidayanti et al. – a physical chemistry study of black powder materials … 101 3550-3200 cm-1 which was an o-h bond and around the wave number 1619 cm-1 associated with the group hydroxyl (rahdar et al., 2015). however, stretching vibration modes of h-o-h around the wave number 1658 cm-1 does not appear in the spectrum pattern (segawa et al., 2015). when compared with the infrared spectrum in the black powder sample, it was observed that the stretching of the o-h bond was reduced, which was caused by the drying and calcination treatment process carried out on the black powder sample. the absorption of the wavenumber 1110 cm-1 shows the presence of carbonates and bonds at around 2980 cm-1 according to the c-h stretch (rahdar et al., 2015). the ni-o-h stretching bond was observed at wave number 633 cm-1, but the absorption of the wavenumber at 447 552 cm-1 associated with the ni-o bond stretching was not observed, due to the range of wave numbers which only ranged from 4000 to 500 cm-1 (nedumkallel et al., 2014, rahdar et al., 2015). figure 13. the infrared spectrum of a black powder sample has been tested with ftir. in the last infrared spectrum, which is glycine material, it was known that in this comparison, there was a free glycine carboxylic group (coo) which is usually observed at 607, 1414 and 1605 cm-1. as in the case of glycine in this forming material, the peak shifts to 607, 1388, and 1570 cm-1 (ahamed et al., 2013, sankar et al., 2010). observed at the peak of 1389 cm-1, there was a stretching of the coosymmetric carboxylate group according to the reported value of literature (azhagan & ganesan, 2013). in addition, peaks in the wavenumbers 1125, 1489, 2600 cm-1 are shifted from the values presented in the literature at wavenumbers 1133, 1507, and 2614 cm-1 of the ammonium group (nh3+) (ahamed et al., 2013). whereas the absorption peak with wavenumbers of 2600 and 3090 cm-1 was indicated as stretching nh3+ (peter & ramasamy, 2010). the peaks were observed at wavenumbers 889 and 1044 cm-1 which are asymmetric and symmetric ccn stretches. whereas the peaks observed at wavenumbers 926, 1322, and 1435 cm-1 are respectively ch2 rocking, ch2 twisting, and ch2 bending (azhagan & ganesan, 2013). so from the analysis of the three row materials, it was found that there were several new absorption peaks that appeared, and some absorptive peaks were lost as shown in figure 13. from the peak of the absorption that appeared based on merck infrared spectrum table and chart, among them, there were around wavenumbers 1464 and 1462 cm-1 in each black powder sample showing the presence of c-h bending bonds with medium intensity and indicated as a class of alkane compounds. then, there is also the absorption peak at the wavenumbers 1390 and 1391 cm-1 in the black powder samples 60 ºc and 70 ºc which were indicated as c-h bending bonds with medium intensity and were observed as an aldehyde class. a strong c-o stretching bond was also observed at the absorption peak around the wave number 1087 cm-1 with indications belonging to the secondary alcohol class. these are summarized in table 5. table 5. ftir spectrum of black powder sample. wavenumber (cm-1) assignment of vibrations black powder 60 1464 c–h bending, medium, alkane 1390 c–h bending, medium, aldehyde 1368 1087 c–o stretching, strong, secondary alcohol 854 649 black powder 70 1462 c–h bending, medium, alkane 1391 c–h bending, medium, aldehyde 1087 c–o stretching, strong, secondary alcohol 854 656 591 black powder 80 1462 c–h bending, medium, alkane 1087 c–o stretching, strong, secondary alcohol 854 649 579 it was observed that all of the three black powder samples as a whole o-h stretching bond groups were reduced. this can be influenced by the drying process (evaporate process). so the o-h stretching bonds that indicate the element h2o evaporates at their boiling temperature. while the c-o and c-h bonds in the infrared spectrum indicate that there are still organic elements owned by glycine, which acts as fuel, so it can be concluded that the temperature during the calcination 102 biology, medicine, & natural product chemistry 10 (2), 2021: 93-103 process was still not optimal. not yet the optimal formation of this phase refers to the calcination process in accordance with its purpose, so that the optimal temperature needed in the calcination process to get the phase in accordance with the reaction equation from the reference in this study. conclusion based on the results of the research that had been carried out, some conclusions can be taken as follows: 1. in this study, black powder (la2nio4) material with a variation of synthesis temperature of 60, 70, and 80 °c with the solution combustion synthesis method produces a sample weight of 0.5 grams 4 times the synthesis process so that each sample is obtained by 2 grams. in addition, it is known that there is heavy evaporation of 96.11% of the total weight of the synthesized precursors. 2. x-ray diffraction analysis results from each sample indicated that there were several phases contained in the black powder, namely nickel oxide, lanthanum oxide, and lanthanum oxide ht (x-form). by varying the synthesis temperature, the composition of the lanthanum nickel oxide (la2nio4) phase disappears and makes a tendency for chemical reactions to help the two dominant phases, namely the nickel and the lanthanum oxide phase. this is supported by the results of scanning electron microscopy with micrographs that appear dominant with a dark colour that shows a low atomic number indicating the element ni and light colours on agglomerates that show a high atomic number which indicates the element la. in addition, the phase formation previously carried out by xrd analysis is strengthened through eds analysis where the results show that the graph is eds-oxide, seen in the presence of element o. so because of the presence of element o, oxidation occurs in the elements ni and la and a compound element occurs formed namely ni to nio (nickel oxide) and the element la to la2o3 (lanthanum oxide). in addition, fourier transform infrared analysis shows that there are still c-o and c-h bonds which are bonds of glycine surfactants, thus indicating that the process of phase formation in accordance with the reference reaction is not optimal. then we need an optimal calcination temperature. acknowledgments: we are thanks to the wp team rare earth metal prototypes at the center for material technology-agency for the assessment and application of technology for providing all the required laboratory facilities. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references adziimaa a. f., risanti d. d., and mawarni j. 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(2017). reviews on chinese patents regarding the nickel/metal hydride battery. batteries, 3(3), 24. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 305-310 | doi: 10.14421/biomedich.2024.131.305-310 issn 2540-9328 (online) in-ovo antiviral activity of hibiscus sabdariffa against newcastle disease virus abdulazeez mumsiri abaka1,*, james doughari hamuel2, tukur baba abdullahi3, keta biman abubakar4 1science laboratory department, adamawa state polytechnic yola, nigeria. 2microbiology department, faculty of life sciences, modibbo adama university yola, nigeria. 3science laboratory technology department, faculty of life sciences, modibbo adama university yola, nigeria. 4science laboratory technology, school of science technology, adamawa state polytechnic yola, nigeria. corresponding author* abdulazizelnino22@adamawapoly.edu.ng manuscript received: 20 may, 2024. revision accepted: 16 july, 2024. published: 26 august, 2024. abstract newcastle disease is a highly contagious viral infection affecting poultry and wild birds. the causative agent is avian paramyxovirus 1 (apmv-1), causing significant economic losses despite vaccination efforts due to its high mortality rate. hibiscus sabdariffa was identified at modibbo adama university yola, and laboratory assays were performed at the national veterinary research institute vom. the study explores the antiviral effects of extracts from h. sabdariffa calyx against a virulent strain of newcastle disease virus (ndv) using embryonated chicken eggs (ece). standard methods were employed for cytotoxicity assay, embryo infective dose 50 (eid50) determination, and therapeutic antiviral assays. methanol was used for extraction and phytochemical analysis, revealing various bioactive compounds like cardiac glycosides, alkaloids, flavonoids, terpenes, and phenols. toxicity assay showed cytotoxicity at concentrations over 25 mg/ml, but therapeutic antiviral assays demonstrated virus replication inhibition at concentrations as low as 5 mg/ml. these findings suggest the potential of h. sabdariffa calyx extracts as safe and effective treatments for ndv, with promising therapeutic antiviral properties. further pharmaceutical research is recommended to explore their use in developing novel newcastle disease treatments. keywords: hibiscus sabdariffa; newcastle disease virus; acute toxicity; egg infectious dose 50 antiviral assay. introduction newcastle disease (nd) is a highly significant infectious disease with a global distribution and is prevalent in the poultry industry. it has the potential to cause substantial economic losses (haddas, 2023). nd is particularly endemic in numerous developing nations, especially in areas where poultry farming plays a decisive role as a source of livelihood. in such regions, the impact of nd is most pronounced (al-natour et al., 2024). newcastle disease virus (ndv) is the aetiologic agent of newcastle disease, which has the remarkable ability to infect more than 240 species of birds. transmission primarily occurs through direct contact between infected and healthy birds2 (al-rasheed et al., 2024). similar to other viral infections, nd is one of the most highly contagious diseases globally (megahed et al., 2023). the mortality and morbidity rates in a poultry flock can vary significantly, ranging from 90% to 100% in poorly vaccinated chickens. even in well-vaccinated layers, egg production can decline, depending on the specific strains of the newcastle disease virus (ndv) (chiwanga et al., 2023). newcastle disease is officially recognized as a “list ‘a’ disease” by the oie, as outlined in oie's 2012 documentation. the ndv is a single-stranded rnacontaining virus with helical capsid symmetry of the paramyxoviridae family and the avulavirus genus (chollom et al., 2023). the control of newcastle disease primarily relies on the rigorous implementation of biosecurity measures on farms, raising public awareness about the disease, and vaccination (charkhkar et al., 2024). among these control strategies, vaccination stands out as the foremost effective and efficient approach, capable of providing comprehensive protection against the disease. however, it is worth noting that in some cases, vaccine failures can occur, leading to widespread outbreaks with high rates of mortality and morbidity (hassanzadeh et al., 2024). hibiscus sabdariffa, commonly known as roselle, is a plant with a rich history of medicinal use in africa (omilabu et al., 2010). roselle, which belongs to the malvaceae family, is a versatile medicinal plant found in https://doi.org/10.14421/biomedich.2024.131.305-310 306 biology, medicine, & natural product chemistry 13 (1), 2024: 305-310 many tropical and sub-tropical regions worldwide (elsagher et al., 2024). this plant is known by various names in english-speaking regions, including rozelle, sorrel, green sorrel, jamaica sorrel, guinea sorrel, indian sorrel, queensland jelly plant, sour-sour, lemon bush, jelly okra, and florida cranberry (mohamed, 2021). in nigerian languages, it is locally termed yakwua in hausa, with the calyx drink being popularly known as sobo (ajoku et al., 2015). it is also known as amukan in yoruba and okworoozo in igbo (ajoku et al., 2015). furthermore, h. sabdariffa is well-known for its abundance of phytochemical compounds and possesses several beneficial properties, including being an antioxidant, hypotensive, hypocholesterolemic, immunemodulating, hepatoprotective, renoprotective, diuretic, anti-obesity, antiurolithic, antidiabetic, antimicrobial, and anticancer agent. importantly, it has been found to have no significant genotoxic effects (sri et al., 2023). the lack of effective drugs for treating nd and the challenges connected with the reliability of vaccines, particularly in developing countries, continue to pose significant and ongoing challenges for researchers. consequently, there is a pressing need to explore antiviral compounds derived from plants that are both safe and effective in combating nd. it is essential to recognize that, like other natural plant-based products, antivirals of natural origin have demonstrated their ability to exhibit satisfactory pharmacological and pharmaceutical properties. they have proven effective against a wide range of viral diseases by disrupting the replication cycle of various dna and rna viruses (ohemu et al., 2020). given the detrimental impact of viral diseases on poultry production and the global economy as a whole, this research aims to address this urgent need by investigating the antiviral properties of extracts derived from h. sabdariffa calyx against the newcastle disease virus (ndv) using embryonated chicken eggs (ece). the objective is to identify potential antiviral agents from natural sources that can contribute to the control and management of nd, which has far-reaching economic implications. materials and methods collection of plant material the dried calyx of h. sabdariffa was procured from yola market, adamawa state, nigeria. subsequently, the plant material underwent identification and authentication by botanist u. a bappa in the department of science laboratory scientist, and was assigned a voucher number asp/pls/0253. extraction the calyx of h. sabdariffa underwent a series of preparation steps. initially, it was meticulously sorted to eliminate any unwanted particles and dead matter. subsequently, the sorted calyx material was air-dried in the shade. once thoroughly dried, the calyx was ground into a fine powder. the powdered calyx material was then preserved in a clean, air-tight container. the bioactive compounds from the dried powdered calyx were extracted by maceration. methanol was used as the solvent for the extraction. the extracts obtained through maceration were then concentrated to dryness under reduced pressure using a rotary evaporator at 50 oc. this process enables the isolation and concentration of the desired compounds from the h. sabdariffa calyx for further analysis (ohemu et al., 2020). phytochemical analysis phytochemical screening of the extracts was conducted following established and standardized methods, as outlined by doughari (2006). acute toxicity assay of plant extract this procedure was conducted to evaluate the potential harmful effects of the extracts on 9-day-old chicken embryos, following the method outlined by chollom et al. (2022). the 9-day-old ece were divided into seven groups, each consisting of five eggs. in groups one through five, the eggs were first sterilized with 70% alcohol using cotton wool and then punctured with an egg puncher. subsequently, 0.2 ml of the extract, with concentrations of 5, 10, 15, 20, and 25 mg/ml in sequential order, was introduced into the eggs through the allantoic cavity. after injection, the needle was carefully removed, and the puncture site was sealed with molten wax. group six received an inoculation of 0.2 ml of phosphate buffer saline (pbs) as a positive control. group seven served as a negative control and did not receive any extract. the eggs were sealed with molten wax and then placed in an incubator at 37 °c for 24 h. the survival and condition of the embryos were assessed after 24 h, 48 h, and 72 h; afterwards, the results were documented. inoculum preparation (virus/extract mixture) to achieve the desired extract concentrations in the virus/extract mixture, a 1:2 v/v dilution will be performed using 100 eid50/0.1 ml of the virus. this dilution will be made with predetermined extract concentrations, resulting in final extract concentrations of 5, 10, 15, 20, and 25 mg/ml in the virus/extract mixture. following the dilution, the virus/extract mixtures will be allowed to react at 4°c for 1 h. therapeutic antiviral assay the methodology closely followed the guidelines set forth by chollom et al. (2022), making minor adjustments as necessary. at nine days old, embryos were divided into nine groups, each comprising five eggs, and treated with varying amounts of extract. to abaka et al. – in-ovo antiviral activity of hibiscus sabdariffa … 307 maintain sterility, plastic egg trays were meticulously cleaned with virkon® solution, and the eggs were swabbed with 70% alcohol prior to handling. subsequently, the eggs were opened, and the extract was introduced into the allantoic cavity within a biosafety cabinet. groups 1–5 received mixtures of virus and extract, ranging from 5 to 25 mg/ml, in 0.2 ml doses. group 6 served as the standard ndv (virus control), group 7 received only the extract suspension (extract control), group 8 was administered phosphate-buffered saline (diluent control), and group 9 remained untreated as a baseline control. following inoculation, all eggs were sealed with molten wax and placed in an incubator set at 37 °c. daily assessments were conducted to monitor embryo survival, and allantoic fluid samples from treated eggs were collected for spot and hemagglutination tests to detect ndv presence in the experimental eggs. spot hemagglutination test dead embryos, which had been previously refrigerated, were allowed to equilibrate to room temperature for about 30 min. following this, the eggs were swabbed and transferred to the biosafety cabinet. the procedure involved carefully opening the shell of each egg to expose the air space, after which a pipette was employed to dispense a drop of 10% washed chicken red blood cells onto a white tile. subsequently, a wire loop, which had been meticulously sterilized, was used to collect a drop of the allantoic fluid, which was then mixed with the drop of blood. to detect any obvious agglutination, which would suggest viral activity in the sample, the tile was gently rocked. (murakawa et al. 2003). this process was carried out for all eggs, and detailed observations were systematically recorded. results phytochemicals result of h. sabdariffa the phytochemical study revealed the presence of compounds with bioactivity with both pharmacological significance and nutritional relevance, namely cardiac glycosides, alkaloids, flavonoids, terpenes, and phenols, as shown in table 1. table 1. qualitative phytochemical analysis of extracts of hibiscus sabdariffa calyx. (key: = negative, + = positive) phytochemical inference glycoside + terpenes + tannin emulsion saponins alkaloid + flavonoid + phenol + steroid cytotoxicity assay of h. sabdariffa table 2 presents the findings about how h. sabdariffa calyx's maximum toxic concentration affected ece. according to these findings, the maximum extract concentration (25 mg/ml) was tolerated by the embryonated chicken eggs, showing no significant mortality compared to the control group. however, at concentrations of 25 mg/ml and 20 mg/ml, mild toxicity was observed, with only 20 % mortality recorded for both concentrations in the chick embryos. no mortality was recorded in the group treated with phosphatebuffered saline diluent. table 2. acute toxicity properties of h. sabdariffa calyx extracts on newcastle disease virus. dilution (mg/ml) number of eggs mortality rate % mortality 24 h 48 h 72 h 25 5 1/5 0/4 0/4 20 20 5 0/5 1/5 0/4 20 15 5 0/5 0/5 0/5 0 10 5 0/5 0/5 0/5 0 5 5 0/5 0/5 0/5 0 dc 5 0/5 0/5 0/5 0 nc 5 0/5 0/5 0/5 0 keys: dc = diluent control, nc = negative control egg infectious dose 50 (eid50) of newcastle disease virus the findings of the egg infective dose 50 (eid50) of ndv on 50% of infected chick embryos, calculated using the reed muench formula, are displayed in table 3. it was determined that 1eid50/0.1ml corresponded to 10-8.5, and 100eid50/0.1ml equated to 10-6.5. this is equivalent to a dilution of 1:100,000. table 3. egg infectious dose 50 (eid50) of newcastle disease virus (ndv) at 72 h after inoculation. virus dilution number of dead number of alive cumulative dead cumulative alive proportion dead total percentage % mortality 10-6 5 0 15 0 15/15 100 10-7 5 0 10 0 10/10 100 10-8 4 1 5 1 5/6 83 10-9 1 4 1 5 1/6 16.6 10-10 0 5 0 10 0/10 0 308 biology, medicine, & natural product chemistry 13 (1), 2024: 305-310 antiviral assay of h. sabdariffa against newcastle disease virus table 4 shows the therapeutic potential of the methanol extract from h. sabdariffa calyx. at higher concentrations (25, 20, and 15 mg/ml), the extract effectively inhibited viral activity without causing mortality. however, at lower concentrations (5 and 10 mg/ml), mortality was observed. controls had no mortality except for the extract control, which had a 20% mortality rate. the virus control group experienced complete mortality and agglutination. additionally, the assay indicated a decrease in ha viral titer with increasing extract concentrations. table 4. antiviral activity of h. sabdariffa calyx extracts on newcastle disease virus (ndv). extracts concentration (mg/ml) number of egg mortality spot test mortality % 24 h 48 h 72 h + ve ve mec 25 5 0/5 0/5 0/5 0 5 0 20 5 0/5 0/5 0/5 0 5 0 15 5 0/5 0/5 0/5 0 5 0 10 5 0/5 0/5 1/5 1 4 20 5 5 0/5 1/5 1/4 2 3 40 vc 0.2 ml 5 0/5 1/5 4/4 5 0 100 ec 25 mg 5 1/5 0/4 0/4 0 5 20 dc 0.2 ml 5 0/5 0/5 0/5 0 5 0 nc 5 0/5 0/5 0/5 0 5 0 key: vc = virus control, ec = extract control, dc = diluent control, nc = negative control mec= methanol calyx extract, -ve = negative, +ve = positive discussion recently, there has been a growing interest in the use of medicinal plants to treat various ailments with herbal preparations being increasingly integrated into healthcare practices for both humans and animals. the use of extracts derived from h. sabdariffa in the treatment of various conditions has been well-documented by albadri et al. (2023). these findings align with those reported by okereke (2015). the variation in the levels of these bioactive compounds may be attributed to the ability of different solvents to extract specific active ingredients or substances from the plant calyx, depending on their polarity (mohammed, 2020). these bioactive substances are thought to be involved in the antiviral activity seen against ndv and to be the source of the traditional use of h. sabdariffa as a herbal remedy. they exert their effects by either killing the virus or interfering with its replication, as demonstrated in previous research (hegazy et al., 2023). the quest for bioactive lead compounds that might be utilized as precursors for the production of more potent drugs may benefit significantly from the utilization of these active ingredients (abubakar et al., 2020). furthermore, the nutritional supplements derived from these natural sources, although crude, are generally considered safer for consumption by animals and humans compared to commercially formulated supplements. commercial supplements may involve genetic modifications that can lead to health concerns such as cancer cell formation and obesity in the long run (chollom et al., 2023). the observation that ece displayed tolerance to the extract in a dose-dependent manner aligns with the inherent characteristics of biological cells. living cells possess certain tolerance levels when exposed to biochemical substances. they can thrive and multiply efficiently when the concentrations of these substances are compatible with their metabolic and physiological well-being. however, when exposed to exogenous products that significantly disrupt their normal physiology, cells may begin to exhibit pathological changes and ultimately succumb, indicating toxicity at those concentrations (ohemu et al., 2020). the findings of the toxicity assay performed on the h. sabdariffa methanol extract showed that the extracts showed deficient levels of toxicity. the h. sabdariffa calyx extracts were well-tolerated by embryonated eggs at concentrations of 25 mg/ml and below, which underscores their suitability for research purposes at these tolerable levels. this finding is critical to the efficient and well-researched use of h. sabdariffa in the treatment of newcastle disease in poultry. to ensure optimal outcomes for farmers, it becomes crucial for researchers to establish appropriate dosage guidelines based on the body weight of the birds. this ensures that lower doses, which may be ineffective against the virus but tolerated by host cells, are not administered. in contrast, very high doses, exceeding the tolerance threshold of host cells and potentially harmful, are avoided. therefore, it is imperative to develop a scientifically derived formula that guarantees both the efficacy of h. sabdariffa products against the virus and their safety for host cells. abaka et al. – in-ovo antiviral activity of hibiscus sabdariffa … 309 however, it is noteworthy that a minor level of toxicity was noted at lower concentrations, namely at 25 mg/ml (20 % mortality rate) of the extract. this observation aligns with the findings of previous studies conducted by ohemu et al. (2020) and abubakar et al. (2020), which investigated the acute toxicity potential of certain plant extracts in ovo. similar to the findings of the current study, earlier investigations showed that methanol extracts were less toxic in the 5 to 25 mg/ml range. nonetheless, it is crucial to emphasize that studies have shown that the moringa oleifera seed extract has reduced toxicity at greater concentrations of 250, 200, and 100 mg/ml (chollom et al., 2012). variations in plant toxicity are attributable to elements such as their chemical makeup, evolutionary strategies for protection, toxin concentrations, genetic diversity, environmental pressures, developmental phases, and the absorption of toxins from their environment. the dilution concentration at which the virus had infected 50 % of the embryos was determined by calculating the egg infectious dose (eid50) of ndv. the calculation of eid50 followed established standard procedures. based on the reed-muench formula, the egg infective dose (eid) of ndv on 50 % of the infected chick embryos is shown in table 3. the findings indicate that the eid50 was observed at a dilution of 10-8, while a 100 % infection rate was recorded at dilutions of 10-5 and 10-6. in contrast, dilutions of 10-9 and 10-10 yielded infection rates of 16.6% and 0%, respectively. the egg infective dose (eid50) test for ndv was conducted to ascertain the concentration of dilution at which the virus infected 50 % of the embryos. in this study, the eid50 was determined to be at a dilution of 10-8. it is noteworthy that this result slightly differed from those obtained in previous studies conducted by chollom et al. (2012) and abubakar et al. (2020). these variations might be attributed to differences in the inoculation procedures within the growth medium. additionally, the results of the spot hemagglutination test, conducted to confirm the presence of ndv in the inoculated eggs, revealed varying degrees of agglutination at different extract concentrations. determining this value is crucial to ensure that there are adequate viable viruses in the challenge viral suspension to induce disease in the experimental birds. it also helps assess the inhibitory potential of the plant extract, mirroring the virulence of a field virus in the event of natural ndv infection. variations in eid50 values can arise due to factors like the specific strain or genetic makeup of the pathogen under study, the condition and age of the host organisms (embryonated chicken eggs), fluctuations in environmental conditions during the experiment, and variances in the techniques employed for both inoculation and assessment. previous research has highlighted the antiviral potential of hibiscus sabdariffa calyx extract against various viruses, including different strains of avian influenza, hepatitis a virus, aichi virus, murine norovirus-1, herpes simplex virus-2, feline calicivirus, and measles virus. additionally, this study investigated the therapeutic effects of methanolic extracts from h. sabdariffa against newcastle disease virus (ndv), consistent with the findings of mohammed et al. (2023), attributing the inhibitory effects to its bioactive components such as glucoside hibiscus, anthocyanin, and gossypetin. furthermore, h. sabdariffa exhibited antiviral activity, suggesting that the herb may bind to specific cell receptors, preventing the virus from adhering to the cells. the ongoing growth of chicken embryos, as evidenced by increased organ development in ndvchallenged ece, indicates that the extracts may disrupt the cycle of viral replication. this interference could occur by blocking certain stages of viral propagation inside the cells, preventing viral invasion mechanisms, or directly killing the virus in the inoculate. conclusion empirical findings from the study demonstrate the antiviral potentials of h. sabdariffa against nd, attributed to various bioactive compounds present in its calyx extracts, including alkaloids, flavonoids, tannins, phenols, steroids, glycosides, and saponins. importantly, toxicity evaluation revealed the extracts to be relatively non-toxic. to optimize effectiveness while ensuring safety, researchers must determine appropriate dosage levels based on bird body weight. this requires a scientifically derived formula to strike a balance between virus efficacy and host cell safety. the administration of h. sabdariffa extract presents a promising and healthier approach to mitigating ndv infection, positioning it as a potential therapeutic agent for newcastle disease treatment. acknowledgements: the author would like to acknowledge dr. solomon chollom chuwang for his insight and skilled guidance and mr. gideon bature for his commitment and support. conflict of interest: there are no conflicts of interest. funding source: the authors state that they have received no funding for their research. ethical approval: non-applicable. references abubakar, y. u., taura, d. w., yushau, m. and muhammad, a. u. 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(2023). a review of the pharmacognostic characteristics, phytochemical constituents, and pharmacological effects of hibiscus hirtus. pakistan heart journal, 56(3), 614-620. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1247-1256 | doi: 10.14421/biomedich.2025.142.1247-1256 issn 2540-9328 (online) molecular dynamic study of java cardamom (wurfbania compacta) leaf compounds targeting xanthine oxidase for antihyperuricemia ali nofriyaldi1*, nitya nurul fadilah1, selvy isnaeni2 1pharmacy department, faculty of health science, perjuangan tasikmalaya university, 2agrotechnolgy departement, faculty of agriculture, perjuangan tasikmalaya university jl. peta no.177 tasikmalaya, telp. (0265) 326058 indonesia. corresponding author* alinofriyaldi13@gmail.com abstract hyperuricemia is a disorder marked by elevated concentrations of uric acid in the bloodstream, which can trigger gout and other metabolic complications. conventional therapies such as allopurinol are effective but often cause side effects. this study aims to explore the potential of bioactive compounds from cardamom leaves (wurfbania compacta) as antihyperuricemic agents through an in silico approach. the methods used include mining phytochemical data from the literature, predicting pharmacokinetic properties and toxicity (admet), molecular docking as well as molecular dynamic. the screening results show that the compound with the most stable interaction from cardamom (wurfbania compacta) with receptors that play a role in inhibiting xanthine oxidase enzyme in silico is the compound kaempferol with a binding energy value of -8.1 kcal/mol and ki 1.15 um (micromolar). admet analysis indicates that the main candidate has a good pharmacokinetic profile and low toxicity potential. based on the results of the rmsd and rmsf molecular dynamic analysis, the kaempferol compound shows a stable level of interaction and has 3 amino acid similarities with the comparison drug allopurinol, namely thr1010, val1011 and ala1078, so it has the potential to be used as a candidate antihyperuricemia drug. these findings suggest that cardamom leaves have prospects as a source of natural antihyperuricemia compounds. further in vitro and in vivo studies are needed to verify the biological activity and safety of its use. keywords: antihyperuricemia; java cardamom leaves; in silico; molecular dynamic; xanthine oxidase. introduction hyperuricemia is a health condition characterized by elevated levels of uric acid in the blood. this condition often does not show any signs or symptoms during its early stages. typically, uric acid levels are considered high when they exceed 7.0 mg/dl in men or 6.0 mg/dl in women (sovia et al., 2025). hyperuricemia, which means having high levels of uric acid in the blood, is connected to several health issues like gout, heart problems, kidney diseases, metabolic syndrome, and diabetes (du et al., 2024). a crucial enzyme involved in producing uric acid is called xanthine oxidase (xo). this enzyme helps convert hypoxanthine into xanthine, and then into uric acid. blocking xo is a proven method used to treat high levels of uric acid in the blood (liu et al., 2021). while traditional xo inhibitors like allopurinol and febuxostat work well, they often cause unwanted side effects, leading to increased exploration of natural products as potentially safer options (mehmood et al., 2019). recent research has shown that bioactive compounds from plants, such as flavonoids, polyphenols, and peptides, have the ability to inhibit xanthine oxidase and reduce high uric acid levels. these findings are backed by both in vitro assays and in silico molecular modeling techniques (dirgantara et al., 2022). one of the plants that contains flavonoids is cardamom. there are several types of cardamom, one of which is javanese cardamom (wurfbainia compacta),, whose boiled fruit extract is empirically believed to be used as an antihyperuricemic agent. the flavonoid derivative from javanese cardamom fruit, in silico, is a candidate that plays a role as an antihyperuricemic, namely the compound 3-cyclohexene-1-methanol (nofriyaldi et al., 2025). in the realm of medicinal plants, cardamom, has been used for centuries in different cultures for its healing benefits. cardamom leaf extract was found to have strong antifungal activity against different fungal plant pathogens and also exhibited antimicrobial effects against both types of bacteria, which are gram-positive and gram-negative that cause human infections, and exhibited antihyperglycemic effects by reducing glucose absorption and mimicking the action of insulin (mekky et al., 2023). the bioactive properties of cardamom leaves have not been thoroughly studied, especially manuscript received: 25 september, 2025. revision accepted: 27 november, 2025. published: 12 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1247-1256 1248 biology, medicine, & natural product chemistry 14 (2), 2025: 1247-1256 regarding their ability to inhibit xo and their potential use in treating hyperuricemia. advances in computational pharmacology, particularly in molecular docking and molecular dynamics (md) simulations, have made it possible to accurately model how phytochemicals interact with target enzymes such as xo. advances in computational pharmacology, particularly in molecular docking and molecular dynamics (md) simulations, have made it possible to accurately model how phytochemicals interact with target enzymes such as xo (mekky et al., 2023). this study seeks to examine the ability of phytochemical compounds found in the leaves of wurfbainia compacta to act as natural inhibitors of xanthine oxidase, using a combined in silico method that includes molecular docking and molecular dynamics simulation. the results could help in identifying new plant-based treatments for hyperuricemia that offer better safety. materials and methods materials chemdraw ultra 8.0 from cambridge soft corporation 2003 8 versions, marvin sketch from chemaxon 1999 23.1 version, molegro molecular view 2008 2.5 version, autodock from molecular graphics laboratory tools 1.5.6 version, biovia discovery studio and desmond (dassault systemes) were utilized. web servers include rcsb pdb, pdbsum, and preadmet. a personal computer with linux ubuntu 18.04.5 lts 64-bit, processor intel® coretm i5-8400 cpu @ 2.80ghz x 6, memory 7,7 gib, graphics geforce gtx 970/pcie/sse2, gnome 3.28.2, disk 245,1 gb. plant material and protein: 50 cardamom leaves compounds from the literature were used, and a receptor protein with pdb id code 3nvw and allopurinol were used as positive control drugs. procedures receptor identification receptors were obtained from the website https://www.rcsb.org/. a receptor with a resolution of less than 2å and a natural ligand similar in structure to the reference drug was chosen. the receptor was then examined on the http://www.ebj.ac.uk/pdbsum site to confirm that it aligned with the ramachandran plot (ruswanto et al., 2018). docking validations docking validation was performed to develop a reliable method by repeatedly docking the original ligand to the target protein using autodocktools (sari et al., 2020). ligand preparation the ligands were protonated by adding protons (h) and simulating a specific ph level to match the ph of the human body, which is 7.4. the resulting file was saved in .mrv format. the next step involved conducting a conformational search, and the file was then saved in .mol2 format. it was imported into the molegro molecular viewer, and subsequently, the molecule was exported in .pdb format (sagitasa et al., 2021). figure 1. 2d visualization of the redocking results of receptor 3nvw prediction of pharmacokinetic and toxicity toxicity and pharmacokinetic tests were conducted using the preadmet website. the results included analysis of the ames test, carcinogenity in mice, carcinogenity in rats, hia, caco-2, and ppb parameters were reviewed (mardianingrum et al., 2021). drug scan a drug screening process was conducted using lipinski’s program to assess how similar a compound is to a reference drug based on the pharmacokinetic processes that take place in the body. the parameter examined during this stage was log p (rahmawaty et al., 2022). molecular docking and visualization the process was conducted using autodock to dock the test ligand onto the target receptor, with the grid dimensions based on the validation results for the x, y, and z axes, which were 38.017, 20.172, and 18.698, respectively (dermawan et al., 2019). the outcomes were presented as binding energy (δg) and inhibition constant (ki), followed by visualization to examine the possible interactions between the ligand and the receptor (ruswanto et al., 2023). molecular dynamic simulations the simulation process was conducted on the selected compound based on the docking results with the receptor using the desmond software to investigate the stability between the compound and the receptor. the process began with preparing the test compound and the solvation stage, where the ligand-receptor complex was dissolved in tip3p-type water. neutralization was then carried out by adding na+ and clions to ensure the system reached a neutral state, mimicking the physiological conditions of the human body. next, minimizing was performed to reduce the potential energy and produce a more stable conformation by avoiding atomic clashes during the formation or breaking of hydrogen bonds. following this, heating was applied, with temperature and pressure adjusted to match the normal human body temperature so that the protein complex could function normally during the simulation. equilibration was then performed, where the system reached a constant state of temperature, volume, and pressure. the simulation was run for 100 nanoseconds using an npt ensemble (zubair et al., 2020). the results http://www.ebj.ac.uk/pdbsum nofriyaldi et al. – molecular dynamic study of java cardamom (wurfbania compacta) … 1249 of the simulation were analyzed using rmsd, rmsf, and protein-ligand residue contact plots. results and discussion result of receptor identification in this study, a receptor with the pdb code 3nvw was used, which was downloaded from the web server https://www.rcsb.org, because this receptor has a resolution value of 1.6 å and has a natural ligand structure similar to the comparator drug. the receptor with pdb code 3nvw has a value of most favored regions of 90.0% and a value of disallowed regions of 0.2%, which means it meets the criteria and can be used as a target protein for the binding of molecules against antihyperuricemia activity. the receptor with pdb code 3nvw has an active site of hydrogen bonds with amino acids namely glu802, arg880, thr1010, and mos1328. meanwhile, for the hydrophobic binding interactions with amino acids namely leu873, ser876, phe914, phe1009, val1011, leu1014, ala1078, and ala1079. more clear interactions can be seen in figure 1. figure 2. active side of xanthine oxidase receptor. result of docking validation docking validation is performed using the autodocktools application, by re-docking the natural ligand with the prepared receptor. the purpose of this validation is to obtain a valid method, which is said to be valid if the receptor has an rmsd value < 2 å. this is because the smaller the rmsd value, the more similar the position of the docked natural ligand is to that of the natural crystallographic ligand. based on the docking validation results in table 1, the receptor 3nvw is considered valid because the obtained rmsd value is < 2 å, which is 0.27 å, with a binding energy of -6.88 kkal/mol, and in the gridbox setup, x = 38.017, y = 20.172, and z = 18.698. the results from redocking are then visualized using the biovia discovery studio 2017 application to observe the interactions between the ligand and receptor, allowing for the identification of the interactions and the amino acid residues involved in the ligand-receptor complex. figure 3. 2d visualization of the redocking results of receptor 3nvw. figure 4. overlay results before docking (red) and after docking (teal). table 1. docking validation results. pdb code grid box rmsd (å) binding energy (kkal/mol) x y z 3nvw 38.017 20.172 18.698 0,27 -6.88 result of ligand preparation the test compound from cardamom was first prepared by conducting protonation and conformational analysis using the marvinsketch application. the compounds prepared included 15 compounds such as 5-hydroxy3,7,4'-trimethoxyflavone, +/-.-2-methoxy-3,8dioxocephalotax-1-ene, erucyl amide, linolenic acid, phytol, hexadecanoic acid, neophytadiene, carvestrene, carvol, 1,8-cineole, kaempferol, quercetin, luteolin, pelargonidin, and allopurinol. the purpose of the protonation stage is to adjust the ph of the test compound to the physiological ph of the human body, which is 7.4. next, the conformation is performed to maintain the position of the molecule that is most stable when interacting with the active site of the receptor. 1250 biology, medicine, & natural product chemistry 14 (2), 2025: 1247-1256 result of pharmacokinetic and toxicity the parameters analyzed include the ames test which aims to demonstrate the mutagenic potential of compounds. additionally, it analyzes oral rat acute toxicity (ld50) and hepatotoxicity. the results of the toxicity tests can be seen in table 2. furthermore, 9 test compounds and a comparator drug that passed the toxicity test were continued with pharmacokinetic testing. table 2. toxicity and pharmacokinetic test results. no compounds name toxicity test phamacokinetic test ames toxicity ld50 (mol/kg) hepatotoxicity caco-2 hia (%) ppb (%) 1 5-hidroxy-3,7,4'-trimetoksiflavon no 2.639 no 23.67 96.48 82.47 2 2-methoxy-3,8-dioxocephalotax-1-ene yes 2.36 yes 3 erucyl amide no 1.863 no 35.78 95.56 100 4 linolenic acid no 1.624 yes 5 phytol no 1.546 no 37.62 100 100 6 hexadenoic acid no 1.595 no 27.52 98.29 100 7 neophytadiene no 1.48 no 23.15 100 100 8 carvestrene no 1.847 no 23.63 100 100 9 carvol no 1.781 no 47.74 100 58.04 10 1,8-cineole no 2.013 no 21.89 100 100 11 kaempferol no 2.317 no 9.49 83.92 79.38 12 quercetin yes 2.551 no 13 luteolin yes 2.709 no 14 pelargonidin yes 2.38 no 15 allopurinol no 2.424 no 16,52 78,26 3,11 description: : not qualified : qualified caco-2: < 4 low permeability, 4-70 moderate permeability, > 70 high permeability. hia: 0-20% low absorption, 2070% moderate absorption, 70-100% high absorption. ppb < 90% weak binding, > 90% strong binding. result of drug scan drug scan was performed using the lipinski’s program to analyze the physicochemical properties of cardamom. the physicochemical properties are analyzed to observe the similarity of a compound to oral drugs through the pharmacokinetic processes that occur in the body. a compound is considered good if it meets 2 or more criteria of the lipinski’s rule, which are molecular weight < 500g/mol, log p <5, hydrogen bond donors <5, hydrogen bond acceptors <10, and molar refractivity between 40-130 (yusuf et al., 2022). table 3. drug scan results. no compounds name molecular weight (g/mol) log p hydrogen bond donor hydrogen bond acceptor molar refractory 1 5-hidroksi-3,7,4'-trimetoksiflavon 328 2,9 1 6 86,5 2 carvol 150 2,48 0 1 46,3 3 kaempferol 285 0,75 3 6 70,87 4 allopurinol 136 0,05 2 4 33,34 result of molecular docking and visualization based on the docking results in table 4, it can be seen that there are several compounds with lower binding energy (δg) and inhibition constant (ki) values compared to the reference drug (allopurinol). subsequently, 2 compounds were selected which have low binding energy (δg) and inhibition constant (ki) values and meet the toxicity, pharmacokinetics, and lipinski’s test criteria, namely the compound 5hydroxy-3,7,4'-trimethoxyflavone with a binding energy value (δg) of -8.89 kcal/mol and an inhibition constant (ki) of 306.54 nm, as well as the compound kaempferol with a binding energy of -8.1 kcal/mol and ki of 1.15 µm. subsequently, visualization was performed using the reference drug and these selected compounds. nofriyaldi et al. – molecular dynamic study of java cardamom (wurfbania compacta) … 1251 table 4, molecular docking results. no compounds name binding energy (kcal/mol) ki run 1 5-hidroksi-3,7,4'-trimetoksiflavon -8,89 306,54 nm 23 2 carvol -7,22 5,09 um 21 3 kaempferol -8,1 1,15 um 70 4 allopurinol -5,83 53,39 µm (a) (b) (c) figure 5 visualization of the interaction of receptor 3nvw with compounds (a) allopurinol, (b) 5-hydroxy-3,7,4'-trimethoxyflavone, and (c) kaempferol. in figure 4 and table 5, the visualization results show that the reference drug, allopurinol, forms hydrogen bonds with the amino acid residues ala1079, arg880, thr1010, and glu1261. in the compound 5-hydroxy3,7,4'-trimethoxyflavone, hydrogen bonds are formed with the amino acid residues arg880, thr1010, phe1009, and there are similarities with allopurinol, namely arg880 and thr1010. meanwhile, in the kaempferol compound, hydrogen bonds are formed with the amino acid residues arg880, thr1010, val1011, glu1261, phe1009, and it shares 3 amino acid residues with allopurinol, namely arg880, thr1010, and glu1261. in addition to the amino acid residues involved in hydrogen bonds, there are also amino acid residues involved in hydrophobic bonds. in allopurinol, the amino acid residues that participate in hydrophobic bonding are phe914 and ala1078. in the compound 5hydroxy-3,7,4'-trimethoxyflavone, the residues that contribute to hydrophobic bonding are ala1078, ala1079, phe914, ala910, leu1014, leu873, val1011, and has 2 amino acid residues that are the same as allopurinol, namely phe914 and ala1078. table 5. results of the visualization of the interaction bonds between 5-hydroxy-3,7,4'-trimethoxyflavone, kaempferol, and allopurinol towards the receptor. no compounds name hydrogen bond hydrophobic bonds 1 5-hidroksi-3,7,4'-trimetoksiflavon arg880, thr1010, phe1009 ala1078, ala1079, phe914, ala910, leu1014, leu873, val1011 2 kaempferol arg880, thr1010, val1011, glu1261, phe1009 ala1078, ala1079, phe914, ala910, leu1014, leu873 3 allopurinol ala1079, arg880, thr1010, glu1261 phe914, ala1078, val1010 result of molecular dynamic simulation the selected test compounds are 5-hydroxy-3,7,4'trimethoxyflavone and kaempferol, along with the comparator drug (allopurinol), and will proceed to the molecular dynamics simulation stage using desmond software. the result can be seen in figure 5. 1252 biology, medicine, & natural product chemistry 14 (2), 2025: 1247-1256 figure 6. rmsd value graph of allopurinol, 5-hydroxy-3,7,4'-trimethoxyflavone and kaempferol. based on the figure 5 indicate an increase. the rmsd value for allopurinol starts stabilizing at 30-50 ns with an rmsd value of ± 2.7 å, then fluctuates and stabilizes again from 60-80 ns with an rmsd value of ± 3 å, after which it fluctuates again and stabilizes at 93100 ns with an rmsd value of ± 3 å. for the compound 5-hydroxy-3,7,4'-trimethoxyflavone, it starts stabilizing at 30-40 ns with an rmsd value of ± 2.3 å, then fluctuates and stabilizes again from 44-58 ns with an rmsd value of ± 2.5 å, fluctuates again, and stabilizes at 70-77 ns and 80-85 ns with an rmsd value of ± 2.4 å. for the kaempferol compound, it starts stabilizing at 40-56 ns with an rmsd value of ± 1.9 å, then fluctuates and stabilizes again at 57-64 ns with an rmsd value of ± 1.8 å, then fluctuates again and stabilizes at 95-100 ns with an rmsd value of ± 1.8 å. figure 7. rmsf values of allopurinol, 5-hydroxy-3,7,4'-trimethoxyflavone and kaempferol. the root mean square fluctuation parameter or rmsf is analyzed to determine the fluctuations of ligand interactions with amino acids during the simulation15. based on the rmsf analysis results in figure 7, it can be seen that the fluctuations of the compounds allopurinol, 5-hydroxy-3,7,4'-trimethoxyflavone, and kaempferol show almost identical fluctuation movements. based on the analysis results in table 6, the compound 5-hydroxy3,7,4'-trimethoxyflavone has 2 amino acid similarities with allopurinol as a comparison, which are glu802 and ser876. meanwhile, the compound kaempferol has 3 amino acid similarities with allopurinol, namely thr1010, val1011, and ala1078. nofriyaldi et al. – molecular dynamic study of java cardamom (wurfbania compacta) … 1253 table 6. amino acids from molecular dynamics. no compounda name molecular dynamic result 1 5-hidroksi-3,7,4'-trimetoksiflavon glu802, ser876, phe914, arg880, ala1079, ser1080, glu1261 2 kaempferol arg880, ser1008, thr1010, val1011, ala1078, ala1079, glu1261 3 allopurinol glu802, ser876, thr1010, val1011, lys771, ala1078 discussion protein preparation with the aim of optimizing ligandreceptor interactions by separating water molecules and standard ligands from the receptor. this separation of water molecules is performed to prevent the formation of hydrogen bonds between the ligand and water molecules. if the separation of water molecules is not carried out, the binding process will become increasingly complex, resulting in longer time requirements and the presence of one ligand will hinder other ligands from binding to the active site. separation is also carried out on natural ligands present on the receptor to be used in the docking validation process. based on the visualization results in figure 2, it can be seen that the interaction of the 3nvw receptor has hydrogen bonds with the amino acid residues arg880, thr 1010, and glu 802. meanwhile, there are hydrophobic bonds with the amino acid residues phe914, ala1078, ala1079, and phe1009. based on the overlay results in figure 3b, it can be seen that the position of the natural ligand from redocking, which is turquoise in color, is similar to the position of the natural ligand in crystallography, which is red. based on table 2, it can be seen that there are 9 test compounds that passed the toxicity test. in the ld50 parameter, all compounds are predicted to have a high ld50 value. in addition, for the parameter of hepatotoxicity, the compounds are not toxic to the liver. furthermore, 9 test compounds and a comparator drug that passed the toxicity test were continued with pharmacokinetic testing. protonation aims to adjust the ph of the test compound to the physiological ph of the human body, which is 7.4. subsequently, conformation is carried out to maintain the position of the most stable molecule when interacting with the active site of the receptor. toxicity testing on test compounds to observe the toxic effects of a substance on biological systems. in the ld50 parameter, all compounds are predicted to have high ld50 values. according to (thahara et al., 2022), the higher the ld50 value, the lower the toxicity, indicating that the compound is safe. in pharmacokinetic testing, adme (absorption, distribution, metabolism, and excretion) was analyzed using the website https://preadmet.webservice.bmdrc.org/. the parameters observed included human colon adenocarcinoma (caco2), human intestinal absorption (hia), and plasma protein binding (ppb). the results of the pharmacokinetic testing can be seen in table 2 wich states that all test compounds have moderate permeability or the ability to penetrate the intestinal epithelial cell barrier moderately, has an hia value between 70-100% which means it has a high absorption capability, a ppb value > 90% indicates an inactive nature. meanwhile, the other 3 test compounds have values < 90%, which means they are loosely bound to plasma proteins, allowing them to act on the target and produce a biological response. human colon adenocarcinoma (caco-2) is a parameter that indicates permeability capability used to determine the transfer of drugs through intestinal epithelial cells derived from human colorectal adenocarcinoma with dual transport pathways in vitro (chintha et al., 2020). based on table 2, it can be seen that all test compounds have moderate permeability or the ability to penetrate the intestinal epithelial cell barrier moderately. the administration of drugs can be challenging because the human digestive tract is very complex and has a number of physiological barriers that affect drug delivery. these challenges include poor drug solubility, poor drug stability, and low drug permeability across the mucosal barrier (pasaribu, 2024). the human intestinal absorption (hia) parameter is used to predict the drug absorption process in the intestine by looking at the results of adding bioavailability to absorption, which is evaluated from the excretion ratio through bile, feces, and urine (monikasari et al., 2023). based on table 3, it can be seen that all test compounds have hia values between 70-100%, which means they have high absorption capacity. the plasma protein binding (ppb) parameter shows the ability of compounds to bind to plasma proteins. compounds that are strongly bound to plasma proteins will be inactive; only free and unbound drugs (free-bound) can act on targets, resulting in a biological response and can enter the elimination process (rahmawaty et al., 2022). as seen in table 2, 6 compounds have ppb values > 90%, meaning they are inactive. in contrast, the other 3 test compounds have values < 90%, which means they are free-bound to plasma proteins, allowing them to act on targets and produce a response. from the analysis of these three parameters, it can be concluded that the 3 test compounds have good pharmacokinetic properties in the body. the molecular weight parameter relates to the ability of a compound to cross biological membranes during the distribution process. compounds with a molecular 1254 biology, medicine, & natural product chemistry 14 (2), 2025: 1247-1256 weight <500 g/mol can easily penetrate biological membranes, while compounds with a molecular weight >500 g/mol will have difficulty crossing the cell membrane, thereby hindering the distribution process. the log p parameter or logarithm of the partition coefficient indicates a compound's ability to dissolve in biological fluids. a good log p value is less than 5, as higher log p values indicate greater hydrophobicity in the compound. hydrophobic compounds are typically more toxic because they stay longer in the lipid bilayer and spread throughout the body, which reduces their ability to specifically bind to the intended target enzyme. on the other hand, a log p value that is too low means the compound is more hydrophilic and may not be able to pass through the lipid bilayer effectively (kelutur et al., 2020). the number of hydrogen bond donors and acceptors is related to the biological activity of a drug. the greater the number of hydrogen bonds, the higher the energy required for the absorption process to occur. the molar refractivity parameter is the total polarizability value of a drug compound, where non-polar compounds will form momentum that causes them to bind with receptors, whereas polar ones play a role in the elimination of metabolic residues from the body (frimayanti et al., 2021). according to table 3, it can be seen that all tested compounds meet all of lipinski’s parameters, so all tested compounds can be considered as candidates for oral drugs. the oral dosage forms include tablets, capsules, syrups, and lozenges (nurhikma et al., 2024). molecular docking is the process of anchoring a molecule to a receptor through computer representation. the parameters analyzed from this process are the binding energy (δg) and inhibition constant (ki) (ikhlas et al., 2023). binding energy and the inhibition constant are related to binding affinity. the lower the binding affinity, the less energy is required for the compound to bind or interact with the target receptor. a low free binding energy value indicates that the formed ligandprotein binding complex will be more stable. if the ligand's binding to the receptor is more stable, it can be predicted that its activity will also be greater (faqiha et al., 2022). the free binding energy value is related to the ki value; the lower the free binding energy, the lower the ki value (fakih et al., 2021). a lower ki value means a smaller concentration of molecules is required to inhibit the target receptor (kartika & ruswanto, 2021). this visualization is performed to understand the interactions that occur between ligands and receptors. the interactions analyzed are hydrogen bonds and hydrophobic interactions, as both types of bonds can influence the physicochemical properties of drugs and the stability of the conformation that occurs. hydrogen bonds are bonds that occur between hydrogen atoms and n, o, or f atoms (vinsiah & fadhillah, 2018). therefore, hydrogen bonds contribute to the affinity of a molecule for a target protein. if there are many hydrogen bonds accompanied by hydrophobic bonds, it can be said that the interaction between the ligand and receptor is a strong interaction. hydrophobic bonds are interactions between amino acids on the ligand and receptor that help maintain the binding conformation (zubair et al., 2020). hydrophobic bonds combine polar areas of the drug molecule with polar areas of the biological receptor. hydrophobic interactions can affect the stability of the bond between the compound and the receptor. in allopurinol, the amino acid residues that play a role in hydrophobic bonding are phe914 and ala1078. in the compound 5-hydroxy-3,7,4'-trimethoxyflavone, the amino acid residues that contribute to hydrophobic bonding are ala1078, ala1079, phe914, ala910, leu1014, leu873, val1011, and it has 3 amino acid residues that are the same as allopurinol, namely ala1078, phe914, and val1011. in the kaempferol compound, the amino acid residues involved in hydrophobic bonding are ala1078, ala1079, phe914, ala910, leu1014, leu873, and it has 2 amino acid residues that are the same as allopurinol, which are phe914 and ala1078. the similarity in the amino acid residues of hydrogen bonds and hydrophobic bonds between the test compound and the comparator (allopurinol) indicates that the test compound has similar activity to allopurinol which is capable of inhibiting the activity of the enzyme xanthine oxidase (dari, 2022). this rmsd analysis aims to compare how the position of the ligand-receptor complex changes over time during the simulation process. the rmsd results for the compounds betagirin and 3-cyclohexene-1methanol along with allopurinol indicate an increase in rmsd values, which suggests that the protein structure has started to open and the ligand is beginning to search for a suitable binding location on the receptor (muttaqin, 2019). based on the rmsd analysis results, the compound kaempferol has more stable interactions compared to the compound 5-hydroxy-3,7,4'trimethoxyflavone and allopurinol because it has a lower rmsd value and the stability of that compound tends to be more constant. the root mean square fluctuation parameter or rmsf is analyzed to determine the fluctuations of ligand interactions with amino acids during the simulation15. based on the rmsf analysis results in figure 7, it can be seen that the fluctuations of the compounds allopurinol, 5-hydroxy-3,7,4'-trimethoxyflavone, and kaempferol show almost identical fluctuation movements. in the compound 5-hydroxy-3,7,4'-trimethoxyflavone, the amino acid residues that experienced the highest fluctuations were thr1319, gly1320, and lys1326, while the lowest fluctuations were observed in the amino acid residues ile1001, ala919, and ala881. in the compound kaempferol, the highest fluctuations occurred in the amino acid residues glu1143, thr1144, and asn1145, while the lowest fluctuations were seen in the amino acid residues cys999, ile1000, and ile1001. nofriyaldi et al. – molecular dynamic study of java cardamom (wurfbania compacta) … 1255 conversely, in the reference drug allopurinol, the highest fluctuations were observed in the amino acid residues phe1142, glu1143, and thr1144, while the lowest fluctuations occurred in the amino acid residues ile1000, ile1001, and arg804. amino acid residues with low fluctuation will have low flexibility and demonstrate more stable binding interactions, allowing them to play a role in the active site of ligand-receptor binding. meanwhile, amino acid residues with high fluctuation will have high flexibility and show less stable interactions because the position of these amino acids undergoes many changes during dynamic molecular simulation (mardianingrum et al., 2021). based on the analysis results, glu802, arg880, and thr1010 are important amino acid residues or constitute the binding active site in the xanthine oxidase receptor. figure 6 shows that the residues glu802, arg880, and thr1010 have low fluctuation and do not exhibit high flexibility during the simulation process. hence, it can be said that the test compound can provide activity as an antagonist against the xanthine oxidase receptor. the compatibility of amino acids with the comparative drug is one of the parameters in molecular dynamic studies. the more amino acids that match with the comparative drug, it can be assumed that the compound has an inhibitory ability that is almost the same or even better than that of the comparative drug (frimayanti et al., 2021). in addition to the rmsd and rmsf plots, the stability of the compound is also supported by the interactions between the compound and the receptor. based on the analysis results in table 6, the compound 5-hydroxy-3,7,4'-trimethoxyflavone has 2 amino acid similarities with allopurinol as a comparison, which are glu802 and ser876. meanwhile, the compound kaempferol has 3 amino acid similarities with allopurinol, namely thr1010, val1011, and ala1078. based on this, the compound kaempferol has the potential to be a candidate for antihyperuricemia drugs and is assumed to be able to bind to receptors and be stable due to having many amino acid similarities with allopurinol as a comparative drug. conclusion the compound with the most stable interaction from java cardamom (wurfbania compacta) leaf with receptors that play a role in inhibiting xanthine oxidase enzyme in silico is the compound kaempferol with a binding energy value of -8.1 kcal/mol and ki 1.15 um (micromolar). kaempferol has a stable interaction based on rmsd and rmsf analysis and shares three amino acids with allopurinol, namely thr1010, val1011, and ala1078, so it can be said to have potential as a candidate for antihyperuricemia drugs. therefore, further research related to in vitro and in vivo tests needs to be carried out to determine the actual biological conditions that occur in the body before binding to the target receptor that causes hyperuricemia. acknowledgements: the authors gratefully acknowledge the support of ministry of education and culture in providing funds. we also thank the research team and perjuangan university of tasikmalaya for their support. authors’ contributions: ali nofriyaldi designed the study. nitya nurul fadilah & selvy isnaeni carried out the laboratory work. ali nofriyaldi analyzed the data and wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. funding: this funding is provided by the ministry of education and culture under the fundamental research scheme. references chintha, c., carlesso, a., gorman, a. m., samali, a., & eriksson, l. a. 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(2020). penambatan molekuler dan simulasi dinamika molekuler senyawa dari genus nigella terhadap penghambatan aktivitas enzim protease hiv-1: molecular docking and molecular dynamics simulation of compounds from nigella genus on protease hiv-1 enzyme inhibitors. jurnal farmasi galenika (galenika journal of pharmacy) (e-journal), 6(1), 132–140. https://doi.org/10.22487/j24428744.2020.v6.i1.14982 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 249-257 | doi: 10.14421/biomedich.2025.141.249-257 issn 2540-9328 (online) platelet-rich plasma as an alternative supplement for enhancing cd marker expression in umbilical cord-derived mesenchymal stem cells putra agung prianggodo1, wahyunia likhayati septiana2,*, helsy junaidi3, sastia winda astuti4 1faculty of medicine; 2department of histology; 3department of molecular biology; 4department of anatomy, gunadarma university, depok city, indonesia. corresponding author* wahyunia@staff.gunadarma.ac.id manuscript received: 18 february, 2025. revision accepted: 20 may, 2025. published: 23 june, 2025. abstract this study aimed to assess the effectiveness of platelet-rich plasma (prp) and fetal bovine serum (fbs) as culture supplements for mesenchymal stem cells (mscs) isolated from umbilical cord tissue. the study focused on the efficiency of xeno-free prp in enhancing cell adhesion and proliferation and looked at cell surface markers for msc characterization. until the cells achieved 80% confluency, three repeats of fbs and prp were added to the culture medium of mesenchymal stem cells derived from umbilical cord tissue in passage 10. following harvesting, an assay kit for human mesenchymal stem cells was used to color the cells. using flow cytometry, the surface markers cd73, cd90, cd105, and the negative marker cd44 were evaluated in the samples. cells supplemented with prp expressed more positive indicators and fewer negative markers. whereas the results for cd90, cd105, and the negative marker were insignificant, the expression of cd73 increased significantly. prp can enhance cd marker expression by acting as a substitute for fbs in the culture of xeno-free umbilical cord-derived mscs. keywords: fbs; prp; msc; stem cells; xeno-free. abbreviations: platelet-rich plasma (prp), fetal bovine serum (fbs), mesenchymal stem cells (mscs), cluster of differentiation (cd) introduction as of yet, stem cells serve no particular purpose in the human body. however, they can self-renew and specialize in several cell lineages. these cells play a crucial role as mediators in a newborn's growth and help them heal from illnesses or wounds. this is due to stem cells' capacity to proliferate and differentiate into a broad range of cell types and lineages required for continued development throughout the neonatal period. the potential is the ability of stem cells to differentiate into different kinds of cells. based on their potential, stem cells can be divided into totipotent, pluripotent, heterogeneous, and multipotent groups. (poliwoda et al., 2022) adult tissue (fat, bone marrow, skeletal muscle, skin, and blood), fetal tissue (fetus, placenta, amniotic fluid, and umbilical cord), and differentiated somatic cells are the four primary sources of stem cells employed in research and therapy. (zakrzewski et al., 2019) multipotent stem cells called mesenchymal stem cells (mscs) are derived from various organs, including the umbilical cord. adipocytes, osteoblasts, and chondroblasts are only a few mesodermal lineages into which mesenchymal stem cells can differentiate. because of this, stem cells are now often used in regenerative medicine. msc is a widely available substance because of its therapeutic properties. it has been documented to differentiate into various cell types, including stem cells from umbilical cord tissue. (joo et al., 2020) because of their unique qualities, mesenchymal stem cells can be employed in clinical settings. these traits include the ability to secrete trophic factors and proangiogenic stress and anti-inflammatory, immunomodulatory, and antioxidant effects. (park & jung, 2021) mesenchymal stem cells may be isolated and cultivated from umbilical cord tissue (uc-msc), an excellent source of these stem cells. the non-invasive approach to obtaining these cells gives them an edge over other sources of mesenchymal stem cells. this prevents the risk of infection and is ethically more acceptable. these cells can be promising because of several properties: higher proliferation rates, cryogenic storage for long periods, hypoimmunogenic phenotypes, immune-suppressive effects, and being harvested from matrices rich in growth factors. these cells can be extracted from the umbilical cord tissue in various locations, including perivascular areas, veins, arteries, amniotic fluid, and sub-amniotic fluid.(shang et al., 2021) three approaches are available for this cell isolation technique: tissue explant, collagenase digestion, https://doi.org/10.14421/biomedich.2025.141.249-257 250 biology, medicine, & natural product chemistry 14 (1), 2025: 249-257 and collagenase/trypsin digestion. (hendijani, 2017; nagamura-inoue & he, 2014; varaa et al., 2019) serum or medium supplementation is crucial for cell survival and encourages the growth of cells in vitro. usually, because fetal bovine serum (fbs) is the gold standard, it is utilized. when used clinically, rejection is dangerous because this serum is obtained from animals and contains xeno-proteins, including nglycolylneuraminic acid (neu 5gc), which may elicit an immune response in patients. (m. hesler et al., 2019; liu et al., 2023; suryani et al., 2013; tonarova et al., 2021) the development of alternative xeno-free medium supplementation has attracted the attention of researchers worldwide. (abdel moniem et al., 2019; michelle hesler et al., 2019) platelet-rich plasma (prp) is a medium supplementation different from fbs. platelet-rich plasma is xeno-free with components from humans and does not include foreign species such as animals. prp as a medium supplementation in umbilical cord msc cultures is more effective because it increases cell proliferation and makes cell attachment to the bottom of the culture dish faster than conventional medium supplementation such as fbs. (lang et al., 2018) cluster of differentiation (cd) is an antigen that can be used to identify cell surface molecules that provide targets for cell immunophenotyping. in a physiological sense, cd antigens can function as ligands or receptors that are crucial for cells. cell adhesion, cell activation, and cell inhibition are among the roles played by cd antigens. (ghaneialvar et al., 2018) mesenchymal stem cells are defined by the criteria set forth by the international society for cellular therapy (isct): they express cd105, cd73, and cd90 positively, and cd34, cd45, cd14, and cd79 negatively. positive signals for cd34, cd45, cd14, and cd79 are expressed by hematopoietic cells. (kaveh baghaei1, 2017) the isct has identified three criteria for mesenchymal stem cells. the first need is that the cells in standard culture conditions be able to cling to plastic surfaces (plasticadherent). the second need is the ability of the cells to develop into osteocytes, adipocytes, and chondrocytes under typical in-vitro differentiation circumstances. third, cells must be able to express antigens on the surface of stem cells; hematopoietic cell markers such as cd45, cd34, and cd14, or cd11b, cd79a, or cd19, must be expressed by fewer than 2% of cells, whereas more than 95% of mscs must express cd29, cd105, cd73, and cd90. (renesme et al., 2022) a liquid that concurrently passes via a laser beam can have several physical properties of cells or individual particles measured and analyzed using flow cytometry. this device creates dispersed fluorescent light signals and can be read by detectors like photodiodes or photomultiplier tubes utilizing a laser as the light source. these light impulses will be transformed into electrical signals, which a computer will then analyze and write into data files in a standard manner. analyzing and purifying cell populations according to their lightscattering or fluorescent properties is possible. moreover, immunology, molecular biology, bacteriology, virology, cancer biology, and infectious disease monitoring are all beneficial to flow cytometry. (mckinnon, 2018) consequently, the expression of surface markers (cd73, cd90, and cd105) in mesenchymal stem cells derived from umbilical cords grown with fetal bovine serum and platelet-rich plasma medium supplements interests researchers. materials and methods the ingredients used in this research include gibco phosphate buffer saline (pbs), gibco dulbecco's modified eagle medium (dmem), gibco fetal bovine serum (fbs), platelet-rich plasma (prp) with blood type ab, gibco glutamax , 70% alcohol, gibco trypan blue, gibco penicillin-streptomycin, and gibco amphotericin b, gibco tryple select, heparin sodium, gibco cryopreservation medium (synth-a-freeze), distilled water, isopropyl alcohol, fitc mouse antihuman cd90, pe mouse anti-human cd44, percpcy™5.5 mouse anti-human cd105, apc mouse antihuman cd73, hmsc positive isotype control cocktail, pe hmsc negative isotype control cocktail, hmsc positive cocktail, pe hmsc negative cocktail. the design employed in this study was an in vitro experiment with three repeats for each treatment. this work employed human umbilical cord mesenchymal stem cells (uc-mscs) acquired from stem cell and tissue engineering (scte). in addition to control cocktails including mouse anti-human cd90, pe, and hmsc positive and negative isotypes, there are also mouse anti-human cd90, mouse anti-human cd44, percp-cy™5.5 mice anti-human cd105, mouse antihuman cd73 apc, and mouse anti-human cd90 fitc. mscs culture with fbs and prp supplementation the complete medium contains 10% gibco fetal bovine serum (fbs), 1% glutamate, 1% gibco penicillinstreptomycin, and 1% amphotericin b in addition to gibco dulbecco's modified eagle medium (dmem) basal medium. additionally, it contains a combination of 10% platelet-rich plasma (prp) containing blood type ab, 1% glutamate, 1% heparin, 1% amphotericin b, and 1% penicillin-streptomycin. every two to three days, the medium is changed until the cell confluence reaches 80– 90%. in 12 well plates, there are 100,000 seeded cells. the cells are placed in an incubator with 5% co2 at 37°c. flow cytometry analysis cells from the healthy plate can be harvested directly, while cells in the scaffold must be given gibco triple select for 5 minutes in the incubator. then, they can be harvested and centrifuged at 1200 rpm for 10 minutes. prianggodo et al. – prp enhances cd marker expression in uc-mscs 251 re-suspend 100 µl of pellets at a 5x10⁶ cell/ml concentration in gibco pbs. using fitc mouse antihuman cd90, pe mouse anti-human cd44, percpcy™5.5 mouse anti-human cd105, apc mouse antihuman cd73, and hmsc positive isotype control cocktail, pe hmsc negative isotype control cocktail, hmsc positive cocktail, and pe hmsc negative cocktail. the cell solution can be divided or transferred into three 1.5 ml tubes (unstained, isotype, stain antibody human mesenchymal stem cell analysis kit). statistical analyses statistical analysis will be conducted to test the research hypotheses. data will be presented as tables, images, and graphs. statistical processing will be performed using graphpad prism 10. normality tests will be conducted to determine whether the data are normally distributed. if the normality assumption is met (data are normally distributed and show homogeneous variance), parametric tests such as the t-test will be applied. if the parametric test assumptions are not satisfied, a non-parametric alternative, the mann-whitney test, will be employed. results and discussion uc-msc culture with fbs and prp medium supplementation cells seeding in 12-well plates are cultured using complete fbs and prp medium until they reach 80% confluence. observation of the morphological development of uc-msc culture on day 2 showed the results of cell changes from round to spindle-shaped fibroblasts. it appeared to have attached to the base of the culture dish (plastic adherent) according to the isct criteria (figure 1). observations on days 4 to 8 showed that more and more cells were attached with a slimmer fibroblastic image, had a visible nucleus, tapering cytoplasm along the axis of the cell nucleus (spindle shape), and processes similar to collagen bundles. observations over time showed that the provision of fbs and prp medium supplementation made cells denser, forming more robust aggregates. so that cells will appear to reach a confluence level of 80%. in addition to observing the morphological development of uc-msc, there are differences between uc-msc cultured using fbs and prp medium supplementation. the fibroblastic, nuclear, cytoplasmic, and process images appear more transparent and denser in uc-msc cultures using prp medium supplementation. thus, the confluence level of uc-msc cultured using prp medium supplementation was higher than that of fbs. figure 1. microscopic photo results after cell implantation in the two treatment groups. figure a. macroscopic photo with 40x magnification. b. macroscopic photo with 100x magnification. c. macroscopic photo with 400x magnification. surface marker expression of uc-msc cultured with fbs and prp medium supplementation the scatter plot results on prp samples show granularity/complexity and cell size similar to the gold standard, namely fbs. however, the average cell count value in fbs samples is lower than in prp samples. the fbs sample obtained results (10,767.8 ± 4,229), and the prp sample (14,474.4 ± 8,469). this shows that ucmsc culture with prp supplementation has higher cell counts than fbs. figure 2. histogram of cell count analysis results. a. scatter plots of ssc-a/fsc-a uc-msc with fbs serum. b. scatter plots of ssc-a/fsc-a ucmsc with prp serum. c. statistical analysis graph. a b c 252 biology, medicine, & natural product chemistry 14 (1), 2025: 249-257 the flow cytometry analysis graph results showed positive expression of cd73 in both fbs and prp samples with a gating percentage of ≥95% (figure 3). the fbs sample obtained results (96.18 ± 1.175) and the prp sample (99.03 ± 0.299). this shows that prp expresses cd73 more highly, contributing to uc's migration, differentiation, and proliferation-msc. a significant difference was found in the t-test in cd73 (p = 0.0302). figure 3. histogram of cd73 analysis results. a. flow cytometry analysis graph of uc-msc with fbs serum. b. flow cytometry analysis graph of ucmsc with prp serum. c. statistical analysis graph. the flow cytometry analysis graph results showed positive expression of cd90 in both fbs and prp samples with a gating percentage of ≥95% (figure 4). the fbs sample obtained results (98.33 ± 0.737) and the prp sample (99.62 ± 0.097). this shows that prp is higher in expressing cd90, which functions as uc-msc adhesion. however, in the mann-whitney test, there was no significant difference in cd90 (p = 0.1376). figure 4. histogram of cd90 analysis results. a. flow cytometry analysis graph of uc-msc with fbs serum. b. flow cytometry analysis graph of ucmsc with prp serum. c. statistical analysis graph. the flow cytometry analysis graph results showed positive expression of cd105 in both fbs and prp samples with a gating percentage of ≥95% (figure 5). the fbs sample obtained results (99.45 ± 0.158) and the prp sample (99.25 ± 0.255). this shows that fbs is higher in expressing cd105, which functions as proliferation, differentiation, and migration of uc-msc. however, in the mann-whitney statistical test, there was no significant difference in cd105 (p = 0.6698). figure 5. histogram of cd105 analysis results. a. flow cytometry analysis graph of uc-msc with fbs serum. b. flow cytometry analysis graph of ucmsc with prp serum. c. statistical analysis graph. the flow cytometry analysis graph showed negative expression (cd45, cd34, cd14, and cd19) in both fbs and prp samples with a gating percentage of ≤2%. the fbs sample obtained results (0.07 ± 0.026) and the prp sample (m = 0.009 ± 0.002). this shows that fbs is higher in expressing negative markers (cd45, cd34, a b c a b c a b c prianggodo et al. – prp enhances cd marker expression in uc-mscs 253 cd14, and cd19), which are hematopoietic cell surface markers. however, in the mann-whitney statistical test, negative markers did not differ significantly (p = 0.1240). figure 6. histogram of negative marker (cd45, cd34, cd14, and cd19) analysis results. figure a. flow cytometry analysis graph of uc-msc with fbs serum. figure b. flow cytometry analysis graph of uc-msc with prp serum. figure c. statistical analysis graph. the scatter plot results on prp samples show granularity/complexity and cell size similar to the gold standard, namely fbs. however, the average cell count value in fbs samples is lower than in prp samples. the fbs sample obtained results (10,767.8 ± 4,229), and the prp sample (14,474.4 ± 8,469). this shows that ucmsc culture with prp supplementation has higher cell counts than fbs. cells were cultured with two different medium supplements, namely prp and fbs. after seeding, a microscopic image of cells appeared round and had not yet been attached to the culture dish. this is consistent with studies (suelzu et al., 2020), which states that cells not yet attached to the culture dish are round. the morphological development of the uc-msc culture on the 2nd day showed the results of changes from roundshaped cells to spindle-like fibroblasts. it was seen attached to the base of the culture dish (plastic adherent) according to the isct criteria.(guan et al., 2019; kusnanto et al., 2023; miryam mebarki et al., 2021; shang et al., 2021) andrzejeskwa et al. (2019) also claimed in their study that huge, flat, spindle-shaped proliferating cells with a distinct cytoskeleton structure comprising many granules resembled fibroblasts. (andrzejewska et al., 2019) next, tiny, spherical cells with a strong potential for self-renewal are observed. fibroblasts have a nucleus that looks real, cytoplasm tapering along the axis of the cell nucleus (spindle shape), and processes similar to collagen bundles.(dick et al., 2019; guan et al., 2019; plikus et al., 2021) observations from day 3 to day 8 demonstrate this, showing that more and more cells are attached with a slimmer fibroblastic image. there are differences in development between ucmsc cultured using fbs and prp medium supplementation. (pratama et al., 2020; sukmawati et al., 2022) a clearer fibroblastic image, a more visible nucleus with a more pointed cytoplasm along the nuclear axis (spindle shape), and processes similar to collagen bundles are more clearly visible in uc-msc cultures using prp medium supplementation. so, the confluence level of uc-msc cultured using prp medium supplementation looks higher than that of fbs. compared to fbs, the concentration of prp in uc-msc culture demonstrated the greatest efficacy in encouraging higher rates of cell attachment and proliferation. (kandoi et al., 2018; pratama et al., 2020; sukmawati et al., 2022; thaweesapphithak et al., 2019) prp can protect against more extended chromosome instability than fbs. platelet-rich plasma can also help the attachment of ucmsc to the culture dish and help uc-msc in the directional movement (cell migration) of a single cell or group of cells to chemical signals. the proliferation rate of uc-msc can be assessed as higher in prp from donors of a younger age. (kandoi et al., 2018; sheriff et al., 2018; sukmawati et al., 2022; trivanovic et al., 2023) in contrast to fbs, platelet-rich plasma contains growth factors critical for wound healing. growth factors found in platelet-rich plasma include essential fibroblast growth factor (bfgf), which stimulates angiogenesis and cell differentiation; platelet-derived growth factor (pdgf) and transforming growth factor-β1 (tgf-β1) are two factors that facilitate cell division. the growth factor for vascular endothelial cells (vegf) encourages neovascularization and cell survival. insulin-like growth factor-1 (igf-1) stimulates cell migration and multiplication and is related to the hormone epidermal growth factor (egf), which promotes cell division and expansion (proliferation). (taniguchi et al., 2019; trivanovic et al., 2023). adding prp to uc-msc culture can also considerably enhance the quantity of chondroblasts generated. this is how prp affects ucmsc development into chondroblasts. this demonstrates that prp is particularly successful at enhancing uc-msc proliferation compared to fbs. (wang et al., 2019; zha et al., 2021) prp is derived from human plasma containing certain growth factors and blood with a high platelet concentration. prp's growth factors, released during degranulation, promote the healing of different tissues and hasten the regeneration of new cells when given to wounds. (khalisha et al., 2018) according to reports, prp can promote the healing of musculoskeletal tissue, a b c 254 biology, medicine, & natural product chemistry 14 (1), 2025: 249-257 including bones, muscles, tendons, and cartilage. (boivin et al., 2023; wang et al., 2019) prp can influence cell proliferation and the migration and differentiation of several cell types, including chondrogenic stem cells and subchondral progenitor cells. since prp can influence cell proliferation, migration, and differentiation, it is commonly used in medical applications and cell cultures instead of fbs supplements. (boivin et al., 2023; nguyen & pham, 2018; rattanasuwan et al., 2018; strauss et al., 2020) after running flow cytometry, it can be seen that ucmsc has a cluster of differentiation that meets the isct criteria, namely positive cd73, cd90, cd105, and negative expressing cd34, cd45, cd14, and cd79. approximately 95% of the hematopoietic markers cd105, cd73, and cd90 were expressed by a range of msc populations; however, cd45, cd34, cd14, and cd19 were not expressed. according to the flow cytometry graph data, surface markers such as cd73, cd90, and cd105—positive indicators of uc-msc— have an expression level above 95%. conversely, hladr (positive <2%), a marker of hematopoietic cells, and cd34, cd45, cd14, and cd79a, negative markers of uc-msc, are found below 2%.(khalisha et al., 2018; widowati et al., 2019) fbs samples obtained results (96.18 ± 1.175) and prp samples (99.03 ± 0.299). this shows that prp expresses cd73 more highly, which functions in the proliferation, differentiation, and migration of ucmsc.(kimura et al., 2021; li et al., 2021; kezhe tan et al., 2019) these findings are inconsistent with earlier studies demonstrating that fbs expresses cd73 at a greater level than prp. (khalisha et al., 2018) however, it is in line with research that prp expresses cd73 more than fbs. (martinez et al., 2019) most people agree that cd73 is a traditional surface marker that characterizes mesenchymal stem cells as multipotent. it was discovered, nevertheless, that the expression of the cd73 protein varied throughout sources. bone marrow contains 19.2%, subcutaneous adipose 51.8%, and umbilical cord 84.2%. (kimura et al., 2021; k. tan et al., 2019) fbs samples obtained results (98.33 ± 0.737) and prp samples (99.62 ± 0.097). this shows that prp is higher in expressing cd90, which functions as a ucmsc adhesion. (guan et al., 2019) prp has a higher value in expressing cd90 than fbs. cd90 variable in prp was higher than fbs, and the cd90 surface marker functioned as cell adhesion. (rashid et al., 2023; rosadi et al., 2024) fbs samples obtained results (99.45 ± 0.158) and prp samples (99.25 ± 0.255). research indicates that uc-mscs grown in fbs express cd105 more than those grown in prp. this is significant since cd105 is a well-known marker linked to mscs and essential to their operation. (m. mebarki et al., 2021; pham et al., 2019) the functions of cd105 include migration, differentiation, and proliferation of cells. (m. mebarki et al., 2021; wang et al., 2020) increased msc proliferation rates are correlated with higher levels of cd105, which is necessary for growing cell populations for therapeutic uses. (qu et al., 2020), the homodimeric transmembrane protein cd105, encoded by endoglin, is produced by most organs and is a component of the growth factor beta receptor transformation complex (tgfbr). one prerequisite for identifying mscs is the expression of cd105. regarding these biomarkers' dependability, there are, nevertheless, conflicting results. (pham et al., 2019; rashid et al., 2023; wu et al., 2023) conclusions mesenchymal stem cells (mscs) from the umbilical cord were used in the study, and prp and fbs were used as culture supplements to evaluate the expression of surface markers in pcs. the cd73 surface marker was expressed by mscs grown with fbs (96.18±1.175), cd90 (98.33±0.737), and cd105 (99.45±0.158), while those cultured with prp exhibited higher expression levels of cd73 (99.03±0.299), cd90 (99.62±0.097) and cd105 (99.25±0.255). a significant difference in cd73 expression (p=0.0225) was observed between the two groups using the t-test, with no significant differences in cd90, cd105, or negative markers (cd45, cd34, cd14, cd19) based on the mann-whitney test. these results suggest that prp could be a viable alternative to serum supplementation in msc culture, particularly for enhancing cd73 expression while maintaining stable expression of other key markers. author’s contributions: pap: performed the experiment; analyzed and interpreted the data; wrote the paper wls: performed the experiment; analyzed and interpreted the data; wrote the paper; contributed reagents, materials, analysis tools or data, reviewed the manuscript, hj: performed the experiment; analyzed and interpreted the data; contributed reagents, materials, analysis tools or data, reviewed the manuscript; swa: performed the experiment; analyzed and interpreted the data; reviewed the manuscript. competing interests: the authors declare that there are no competing interests. references abdel moniem, e. m., el-batran, m. m., halawa, a. m., gomaa, d. h., eldeen, g. n., & aly, r. m. 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(2021). recent developed strategies for enhancing chondrogenic differentiation of msc: impact on msc‐based therapy for cartilage regeneration. stem cells international, 2021(1), 8830834. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 453-463 | doi: 10.14421/biomedich.2025.141.453-463 issn 2540-9328 (online) potential of kedabu (sonneratia ovata backer) fruit juice supplemented in kombucha beverage as antibacterial agent musyirna rahmah nasution*, clara nabilah, meiriza djohari, nurhapipah departement of pharmacy, sekolah tinggi ilmu farmasi riau, pekanbaru, riau, indonesia. corresponding author* musyirnarahmah@stifar-riau.ac.id manuscript received: 03 may, 2025. revision accepted: 15 july, 2025. published: 18 july, 2025. abstract riau province has abundant natural resources of mangrove forests almost along the coast of riau. mangrove fruit can be developed into various processed foods, but processed foods derived from mangroves have not been widely developed and are of interest to coastal communities. kombucha tea is a probiotic beverage produced through the fermentation of tea and sugar using a kombucha starter known as scoby (symbiotic culture of bacteria and yeast). the presence of sugar in kombucha colonies is a vital source of nutrition. additionally, fruit juice can be incorporated to create variations of kombucha tea. kedabu fruit (sonneratia ovata b.), a native fruit of riau province, is recognized for its high carbohydrate content and secondary metabolites, which exhibit potential antibacterial properties. this research developed four formulations of kombucha tea, each with different concentrations of kedabu fruit juice (0%, 10%, 20%, and 30%). the study’s objectives were to formulate, evaluate the physicochemical properties and antibacterial activity of these formulations against escherichia coli and staphylococcus aureus. the organoleptic properties, alcohol content, ph, %tta (total acidity titratable), total lab (lactic acid bacteria), and yeast count were assessed during a 14-day fermentation period. the assay results indicated that the kombucha fruit kedabu tea formulation exhibited favorable organoleptic characteristics, a decrease in ph, an increase in %tta, total lab, and yeast count within the acceptable range defined by standards. moreover, it displayed inhibitory activity against escherichia coli and staphylococcus aureus bacteria, falling within the weak to moderate categories. keywords: kombucha; kedabu; antibacterial; escherichia coli; staphylococcus aureus. introduction riau province, located in indonesia, is known for its abundant natural resources including land, mining, agriculture, forestry, marine, and industry. with a coastline stretching over 2,078 kilometers, the province boasts extensive mangrove forests along its shores. mangroves offer a wide range of valuable resources that have been extensively utilized by coastal communities, both in the form of timber forest products and non-timber forest products. however, the development of processed foods derived from mangroves remains limited, despite the potential demand from these communities. mangroves are a type of plant that thrives in tidal areas, particularly along protected tropical and sub-tropical coasts as well as sheltered sub-tropical regions. the mangrove plant encompasses various tree species, including avicennia, sonneratia, rhizophora, bruguiera, ceriops, lumnitzera, excoecaria, xylocarpus, aegiceras, scyphyphora, and nypai (kathiresan et al, 2001). one of the mangrove plants is kedabu (sonneratia ovata backer) which is utilized in food and health. gastrointestinal tract infections pose a significant health concern due to the digestive system’s vulnerability to bacterial invasion. as the primary entry point for food and drinks, the gastrointestinal tract risks contamination by pathogenic bacteria. traditionally, antibiotics have been utilized to treat bacterial infections. however, the indiscriminate use of antibiotics can result in bacterial resistance, necessitating the exploration of alternative treatment options (aminov, 2010). one such approach to prevent gastrointestinal tract infections is using kombucha tea. kombucha tea is a fermented beverage produced by the fermentation of black tea and sugar using a symbiotic culture of bacteria and yeast (scoby). the fermentation process typically takes place over 7-14 days. scoby is composed of various microorganisms, including acetic acid bacteria (such as acetobacter xylinum, acetobacter acetic, and gluconobacter oxydans), yeast (such as saccharomyces cerevisiae, zygosaccharomyces sp, and torulopsis sp), and lactic acid bacteria (such as lactobacillus sp and lactococcus sp) (laureys et al., 2020). kombucha is known to contain a diverse array of chemical compounds, including acetic acid, gluconic acid, lactic acid, vitamins (b1, b2, b6, b12, c), ethanol, carbon dioxide, polyphenols, and others (herwin, 2022). these compounds contribute to the pharmacological https://doi.org/10.14421/biomedich.2025.141.453-463 454 biology, medicine, & natural product chemistry 14 (1), 2025: 453-463 effects of kombucha tea, which include improved digestion, antibacterial properties, antioxidant activity, and more. the presence of secondary metabolite compounds such as triterpenoids, alkaloids, phenolics, saponins, and flavonoids. these compounds are known for their potential health benefits and contribute to the overall bioactive profile (chandraekara et al., 2018). furthermore, a study revealed that kombucha tea exhibited inhibitory effects on escherichia coli and staphylococcus aureus, with inhibition diameters of 20.5 mm and 20.15 mm, respectively, after a 14-day fermentation period (bhattacharya et al., 2016). escherichia coli and staphylococcus aureus were the most susceptible bacteria to the inhibitory action of kombucha tea, resulting in inhibition diameters of 19 mm and 18 mm, respectively. increased fermentation time of kombucha tea led to an enhanced antibacterial activity (al-mohammadi et al., 2021). one of the crucial factors influencing kombucha production is the presence of sugar. sugar plays a vital role as an energy source for yeast in the scoby (miranda et al., 2016). within the scoby, yeast metabolizes sugar by converting it into glucose and fructose, which are subsequently fermented into ethanol. acetobacter sp. then utilizes ethanol to produce acetic acid (laureys et al., 2020). in kombucha tea, it is possible to substitute sugar with fruit juice as an alternative sweetener. sonneratia ovata backer, commonly known as kedabu or pedada plant, is a mangrove species frequently found along the coastlines of riau province, indonesia. the fruit of the kedabu plant is a significant source of nutrients. it can be processed into various products, including candy, jam, syrup, and dodol. importantly, the kedabu fruit is non-toxic and can be consumed directly. analysis conducted by astuti et al. revealed that sonneratia ovata backer fruit contains approximately 2.19% carbohydrates. furthermore, phytochemical screening demonstrated the presence of flavonoids, phenolics, and saponins and triterpenoids (such as oleanolic acid and maslinic acid) in the extracts and fractions of sonneratia ovata fruit (wu et al., 2009). research exploring the utilization of kedabu fruit in functional food products remains limited. this is notable given the fruit's potential as a source of secondary metabolite compounds with antibacterial properties, as well as its high nutritional content. moreover, no existing publications specifically focused on kombucha tea made from kedabu fruit, which presents an interesting opportunity for further investigation. thus, this study aimed to formulate kombucha tea by incorporating kedabu fruit juice and evaluate its antibacterial activity against escherichia coli and staphylococcus aureus bacteria. by developing kombucha tea using kedabu fruit juice, researchers aim to highlight the potential of this fruit in riau province, indonesia, and promote its utilization as an alternative antibacterial remedy materials and methods sampling and sample identification the samples were collected from west dumai district, dumai city, riau, indonesia. the samples were then taken to the botanical laboratory, department of biology, faculty of mathematics and natural sciences, university of riau, pekanbaru, for identification. the sample identification was conducted under the reference number 611/un19.5.1.1.3-4.1/ep/2021 (table 2). kedabu fruit juice preparation fresh kedabu fruit weighing 500 grams was thoroughly washed. the fruit flesh was then finely chopped into small pieces and mixed with water at 1:2 (w/v). the mixture was blended using a blender to obtain a homogeneous fruit pulp. subsequently, the kedabu fruit pulp was filtered to obtain pure kedabu fruit juice. the kedabu fruit juice was further prepared in concentrations of 10%, 20%, and 30% by dilution (chan et al., 2021). formulation of kombucha formulation details can be found in table 1. 250 ml of distilled water was boiled in a stainless steel pot to prepare the kombucha tea. black tea, sugar, and kedabu juice were added to the boiling water. the mixture was then filtered and transferred to a glass jar. the kombucha tea mixture was allowed to cool down to 25°c. next, 10% (v/v) of kombucha starter culture was added to the mixture, and distilled water was added to bring the total brew volume to 500 ml. the mixture was homogenized thoroughly. the glass jar was covered with a clean cloth or tissue and securely tied. fermentation was conducted at a temperature of 25°c for 14 days. samples were taken for antibacterial and chemical analysis during the fermentation process. table 1. formulation of kombucha tea with kedabu fruit juice. no composition *f1 f2 f3 f4 1 black tea 1,6% 1,6% 1,6% 1,6% 2 sugar 10% 10% 10% 10% 3 kombucha starter 10% 10% 10% 10% 4 kedabu fruit juice 0% 10% 20% 30% 5 aquadest ad 500 ml ad 500 ml ad 500 ml ad 500 ml note : formula (*f) nasution et al. – potential of kedabu (sonneratia ovata backer) fruit juice supplemented … 455 evaluation of chemical changes and antibacterial activity some parameters used in evaluating the quality of kombuca tea supplemented with kedabu fruit juice as a fermented beverage were organoleptic test, phytochemical screening test, determination of ph with ph meter, alcohol content with alcohol meter. the total tritabel acidity (tta) was determined using the alkalimetric method. determination of total lactic acid bacteria (lab) and total yeast in kombucha tea is done by using the standar plate count/spc method (pour plate method). as a functional beverage, one pharmacological effect test was antibacterial activity against e coli and s aureus bacteria using the disc diffusion method. evaluation of all parameters was made before fermentation and after fermentation on the 7th and 14th day. data analysis the data obtained from the antibacterial activity assay of kombucha tea supplemented with kedabu fruit juice against escherichia coli and staphylococcus aureus bacteria, collected at different time points (before fermentation, 7th day, and 14th day of fermentation), were analyzed using the one way anova statistical test. the analysis was performed using the spss application to determine if there were any significant differences among the groups. to further determine which formula group exhibited the best antibacterial activity, a post-hoc analysis was conducted using the tukey test. this test allows for a pairwise comparison between groups to identify significant differences. additionally, the data obtained from the evaluation of the kedabu fruit kombucha tea formula, which included organoleptic test, ph test, total lactic acid bacteria (lab) test, total titratable acid (tta) test, total yeast test, and alcohol test, were analyzed descriptively. descriptive analysis provides a comprehensive summary of the data, including measures of central tendency and variability, to describe the characteristics of the kedabu fruit kombucha tea formula. these data analyses provide valuable insights into the antibacterial activity and overall quality of the kedabu fruit kombucha tea formula, allowing for an objective assessment of its effectiveness as a functional beverage. results and discussion sample identification the sample identification was conducted under the reference number 611/un19.5.1.1.3-4.1/ep/2021 (table 2). the results of sample identification showed that the sample had the regional name kedabu and the species name sonneratia ovata backer. table 2. the result of the identification sampel sample identification sample kingdom plantae phylum magnoliophyta classis magnoliosida ordo myrtales family lythraceae genus sonneratia species sonneratia ovata backer regional name kedabu figure 1. kedabu plant (a); kedabu fruit (a and c). kedabu or pedada plants (sonneratia sp.) are a species of mangrove plants commonly found along the coastlines of riau province, indonesia. the fruit of the kedabu plant has gained attention due to its nutritional composition and bioactive compounds (halifah et al, 2019). exploiting natural resources to develop functional food products is an important aspect of utilizing traditional medicinal plants. in this study, the focus was on the production of kombucha tea using kedabu fruit juice as an ingredient. kombucha tea is a fermented beverage known for its potential health benefits and is typically prepared using black tea and sugar fermented by a symbiotic culture of 456 biology, medicine, & natural product chemistry 14 (1), 2025: 453-463 fi fiv bacteria and yeast (scoby) (chakravorty et al, 2016). by incorporating kedabu fruit into the kombucha tea formulation, it is possible to enhance the nutritional and bioactive properties of the final product. the results of the sample identification confirmed that it was sonneratia ovata backer, commonly known as kedabu fruit. the phytochemical screening test indicated that the fermentation time and addition of kedabu fruit juice significantly effected the content of secondary metabolites, increasing their levels. kombucha tea enriched with kedabu fruit juice exhibited higher secondary metabolites, including flavonoids, phenolics, terpenoids, and saponins (table 3). these secondary metabolites are well-known for their pharmacological effects, such as antimicrobial, antioxidant, and antidiabetic activities (herwin, 2022). table 3. the results of screening phytochemical of kombuca tea with or without kedabu fruit juice. secondary metabolite reagent before fermentation after fermentation on 14 days kombucha tea kombucha tea + kedabu juice kombucha tea kombucha tea + kedabu juice alkaloid mayer fenolik logam mg + hcl(p) + + + + flavonoid fecl3 1 % + + + + terpenoid lieberman-bouchard + + + steroid lieberman-bouchard saponin air + + evaluation of chemical changes and antibacterial activity. the evaluation of kombucha tea's quality was conducted based on the standard parameters outlined in sni 75522018 for fermented beverages. the organoleptic test was performed after a fermentation period of 14-days. the results demonstrated that an extended fermentation time led to a lighter color of kedabu fruit kombucha tea (table 4). this color change can be attributed to the activity of microorganisms responsible for color degradation. bacteria utilize total soluble solids as an energy source, resulting in a depletion of solvents in the media and a clearer liquid (jayabalan et al., 2014). regarding the aroma, the observations of kombucha tea indicated that a longer fermentation time resulted in a more distinct aroma in the kedabu fruit kombucha. furthermore, increasing the concentration of kedabu fruit led to a more pronounced sour taste. the sour taste in kedabu fruit kombucha tea is attributed to the production of lactic acid and acetic acid during the fermentation process (wang et al., 2022) table 4. observation of kedabu fruit kombucha tea before and after fermentation for 14 days description the color of kedabu fruit kombucha tea before fermentation (0-day) after fermentation on 14day note : f1: kombucha tea without kedabu juice ; f2,f3, f4 : kombucha tea+kedabu juice (10%, 20% ,30%) fii fiii fiv fi fii fiii fiv nasution et al. – potential of kedabu (sonneratia ovata backer) fruit juice supplemented … 457 the subsequent test involved measuring the alcohol content using an alcohol meter. alcohol measurement is a crucial aspect in assessing the quality and safety of kombucha tea. as fermented beverages, kombucha is known to contain alcohol. in indonesia, where the majority of consumers are muslims, it is essential for these beverages to comply with the halal food standards established by the indonesian ulama council (mui). according to mui (fatwa no. 10 of 2018) (fmuin, 2018). fermented beverage products containing less than 0.5% alcohol/ethanol are considered halal if they are medically harmless (pauzi et al., 2019) in this study, the obtained results revealed that the alcohol content in all formulations of kombucha tea was 0% throughout the fermentation process from day-0 to day-14 (table 5). it is worth noting that previous research has indicated that the alcohol content in kombucha generally ranges from 0.1% to 1%. table 5. evaluation of organoleptic test, alcohol content, %tta, total lab, total yeast and antibacterial activity of kedabu fruit kombucha tea formulations formula parameters observation results 0-day 7-day 14day fi taste sweet sour sour flavor typical kombucha falvor typical kombucha falvor typical kombucha falvor color dark brown dark brown brown ph 3,37 2,94 2,92 alcohol 0% 0% 0% %tta 0,086% 0,33% 0,51% total lab 9,9 x 107 cfu/ml 18,1 x 107 cfu/ml 16 x 107 cfu/ml total yeast 2,32 x 106 cfu/ml 1,56 x 106 cfu/ml 2,47 x 106 cfu/ml antibacterial activity escherichia coli 6,8 mm 7,8 mm 8,2 mm antibacterial activity staphylococcus aureus 7,2 mm 8,6 mm 8,3 mm fii taste sweet sour sour flavor typical kombucha falvor typical kombucha falvor typical kombucha falvor color dark brown light brown golden brown ph 3,25 2,86 2,72 alcohol 0% 0% 0% %tta 0,16% 0,35% 0,75% total lab 6,5 x 107 cfu/ml 16,2 x 107 cfu/ml 13,4 x 107 cfu/ml total yeast 1,98 x 106 cfu/ml 1,3 x 106 cfu/ml 2,29 x 106 cfu/ml antibacterial activity escherichia coli 7,2 mm 8,03 mm 9 mm antibacterial activity staphylococcus aureus 7,6 mm 8,8 mm 10,2 mm fiii taste sweet sour sour flavor kedabu flavor typical kombucha falvor typical kombucha falvor color brown light brown golden brown ph 3,25 2,92 2,61 alcohol 0% 0% 0% %tta 0,16% 0,58% 0,99% total lab 5,1 x 107 cfu/ml 9,95 x 107 cfu/ml 9,9 x 107 cfu/ml total yeast 1,53 x 106 cfu/ml 1,19 x 106 cfu/ml 1,9 x 106 cfu/ml antibacterial activity escherichia coli 7,4 mm 8,6 mm 9,96 mm antibacterial activity staphylococcus aureus 7,9 mm 9,2 mm 10,93 mm fiv taste sour sour sour flavor kedabu flavor typical kombucha falvor typical kombucha falvor color brown light brown golden brown ph 3,18 2,8 2,5 alcohol 0% 0% 0% %tta 0,17% 0,58% 1,02% total lab 6,15 x 107 cfu/ml 6,95 x 107 cfu/ml 8,55 x 107 cfu/ml total yeast 1,3 x 106 cfu/ml 0,92 x 106 cfu/ml 1,68 x 106 cfu/ml antibacterial activity escherichia coli 7,8 mm 10,5 mm 11,8 mm antibacterial activity staphylococcus aureus 7,93 mm 10,3 mm 15 mm 458 biology, medicine, & natural product chemistry 14 (1), 2025: 453-463 the ph measurements of all formulations revealed a decrease in ph values ranging from 3.37 to 2.5 (table 5). as the concentration of kedabu juice in kombucha tea increased, the ph value decreased. the duration of fermentation also influenced the ph value. the highest ph value was observed on the 0th day. the fermentation time increases, the ph value decreases (nurikasari et al., 2017). the decrease in ph value in kedabu fruit kombucha tea is attributed to the conversion of glucose into organic acids by microorganisms. the concentration of organic acids increases with prolonged fermentation (laureys et al., 2020). the ph of kombucha tea typically ranges from 2.5 to 4.2 (murphy et al., 2018). overall, the ph of the produced kedabu fruit kombucha tea meets the general ph requirements for kombucha tea. the total titratable acid (tta) values were measured using the alkalimetric titration method. all formulas exhibited changes in tta values ranging from 0.086% to 1.02% (table 5). the maximum % total acid in kombucha tea is 3% (w/v) (greenwalt et al., 2000). thus, the overall results obtained in this study meet the standard % total acid requirement for kombucha tea. the %tta increases with the addition of kedabu juice in kombucha tea and the duration of fermentation. the increase in %tta is associated with the enhanced activity of lactic acid bacteria in metabolizing sucrose into organic acids (nguyen et al., 2015). the relationship between total titratable acid and ph is inversely proportional, meaning that as the %tta value increases, the ph value decreases and vice versa. this observation is consistent with the findings of previous research on kombucha tea (laureys et al., 2020). the acidic nature of kombucha tea, attributed to the presence of organic acids such as acetic acid and lactic acid, contributes to its characteristic taste and potential health benefits. acetic acid is the primary organic acid produced during kombucha fermentation. it plays a crucial role in inhibiting the growth of pathogenic bacteria. the formation of acetic acid in kombucha fermentation has been found to have potential antibacterial properties (herwin, 2022). as acetic acid is produced in kombucha, it undergoes decomposition, releasing free protons that lead to a decrease in ph (hamed et al., 2012). acetic acid in kombucha can disrupt the lipid bilayer structure of bacterial cells by introducing protons into the cytoplasm. this accumulation of intracellular protons results in acidic conditions, leading to protein denaturation and energy loss within the bacterial cells. the higher the content of organic acids, particularly acetic acid, the greater the potential for inhibiting the growth of pathogenic bacteria. the ideal formation of organic acids in kombucha reduces ph, creating highly acidic conditions that significantly affect bacterial growth. this acidic environment inactivates the cytoplasm of pathogenic bacteria and causes severe damage to their cells (herwin, 2022). these findings highlight the antimicrobial potential of acetic acid in kombucha and its ability to create an unfavorable environment for pathogenic bacteria. the presence of acetic acid, along with other organic acids formed during kombucha fermentation, contributes to its overall antimicrobial activity and potential health benefits. the results of the total lactic acid bacteria (lab) testing indicate that all treatments kedabu fruit kombucha tea contained a sufficient amount of lab, meeting the minimum requirements for probiotic beverage quality standards, which is at least 107 cfu/ml (nyanzi et al., 2021). the total lab count increased from the 0th day to the 7th day of fermentation. this increase can be attributed to the availability of an adequate nutrient source, promoting the growth of lab. however, from the 7th day until the 14th day, there was a decrease in the total lab count (table 5). this decline may be attributed to the depletion of nutrients in the kedabu fruit kombucha tea medium. at the initial stages of fermentation, there is an abundance of nutrients that actively support the division and growth of bacteria. however, as fermentation progresses, the available nutrients decrease, reducing in bacterial activity and reproduction. this decline in lab count indicates that the bacteria have surpassed their logarithmic growth phase and are entering a phase of decreased reproduction (villarreal et al., 2018). these findings highlight the dynamic nature of lab growth during the fermentation of kedabu fruit kombucha tea. the initial increase in lab count indicates active fermentation and bacterial proliferation, while the subsequent decline can be attributed to nutrient depletion. it is essential to carefully monitor the fermentation process to ensure optimal lab growth and maintain the probiotic qualities of kombucha tea. the minimum requirement for total yeast in kombucha tea is at least 104 cfu/ml (nyhan et al., 2022). the study results indicate that all kedabu juice kombucha tea treatments met this requirement, with yeast counts exceeding 104 cfu/ml. the findings demonstrate that the length of fermentation time has an impact on the total yeast count. a decrease in yeast count was observed from the 0th day to the 7th day of fermentation, followed by an increase from the 7th day to the 14th day. the initial decrease in yeast count during the early stages of fermentation can be attributed to the yeast undergoing a lag phase or an adaptation phase with its environment. during this phase, some enzymes may not have been synthesized yet, and metabolic processes are still slow, resulting in a lack of cell division. as a result, the yeast count may remain stable or even decrease during this phase. however, as fermentation progresses, the yeast adapts to its environment, and the growth rate increases. the growth rate of yeast is influenced by the availability of substrates and the ph of the kombucha medium, which nasution et al. – potential of kedabu (sonneratia ovata backer) fruit juice supplemented … 459 fi fii fiii fiii provides an ideal environment for yeast growth and the conversion of glucose into alcohol and organic acids (de miranda et al., 2022). in the case of kedabu juice kombucha tea, the substrate for yeast growth is obtained from the kedabu juice. these findings emphasize the dynamic nature of yeast growth during kombucha fermentation. the initial decrease in yeast count is followed by an increase as the yeast adapts to the fermentation environment and utilizes the available substrates. monitoring and understanding the yeast population dynamics during fermentation is crucial to ensure the desired quality and characteristics of kombucha tea. table 6. the result of antibacterial activity of kedabu juice kombucha tea before fermentation. formula diameter of inhibition (mm) → eschericia coli average ± sd i ii iii k (-) 6 6 6 6 ± 0a k (+) 21 24 22 22,3 ± 1,53b f1 7,1 6,2 7,0 6,8 ± 0,49 a f2 7,2 6,9 7,4 7,2 ± 0,25 a f3 7,5 6,8 8,0 7,4 ± 0,6 a f4 7,8 7,0 8,5 7,8 ± 0,75 a formula diameter of inhibition (mm)→ staphylococcus aureus average ± sd i ii iii k (-) 6 6 6 6 ± 0a k (+) 23,3 25,1 26 24,8 ± 1,37b f1 7,8 7,1 6,8 7,2 ± 0,51a f2 8,5 7,1 7,4 7,6 ± 0,74 a f3 8,8 7,4 7,5 7,9 ± 0,78 a f4 8,7 7,6 7,5 7,93 ± 0,66 a figure 2. antibacterial activity of kedabu fruit kombucha tea against bacteria escherichia coli and staphylococcus aureus before fermentation (on 0th day). table 7. the result of antibacterial activity of kedabu juice kombucha tea after fermentation 7-day. formula diameter of inhibition (mm) → eschericia coli average ± sd i ii iii k (-) 6 6 6 6 ± 0a k (+) 22,1 21,7 21 21,6 ± 0,56c f1 9,2 7,2 7,0 7,8 ± 1,22ab f2 9,5 7,4 7,2 8,03 ± 1,27 ab f3 10 7,6 8,1 8,6 ± 1,26 ab f4 11 7.8 10 10,5 ± 0,7b formula diameter of inhibition (mm) → staphylococcus aureus average ± sd i ii iii k (-) 6 6 6 6 ± 0a k (+) 21,6 22,1 23,5 22,4 ± 0,98c f1 8,7 9,1 8,0 8,6 ± 0,56 b f2 9 9,4 7,9 8,8 ± 0,78 b f3 9,1 9,6 9 9,2 ± 0,32 b f4 9,6 11 10,2 10,3 ± 0,7 b 460 biology, medicine, & natural product chemistry 14 (1), 2025: 453-463 fi k(-) fii fiv fi fiii fi fiv fiii fi fii k(-) fiv fiii figure 3. antibacterial activity of kedabu fruit kombucha tea against escherichia coli and staphylococcus aureus on 7-day. table 8. the result of antibacterial activity of kedabu juice kombucha tea after fermentation day-14. formula diameter of inhibition (mm) → eschericia coli average ± sd i ii iii k (-) 6 6 6 6 ± 0a k (+) 21,5 22,3 24 22,6 ± 1,27d f1 7,8 8,3 8,4 8,2 ± 0,32ab f2 8,2 9,9 8,9 9 ± 0,85b f3 10,5 9 10,4 9,96 ± 0,84bc f4 11 12 12,5 11,8 ± 0,7c formula diameter of inhibition (mm) → staphylococcus aureus average ± sd i ii iii k (-) 6 6 6 6 ± 0 a k (+) 22,4 23,4 23 22,93 ± 0,5e f1 8,7 8,3 7,9 8,3 ± 0,4b f2 11,3 10,2 9 10,2 ± 1,15bc f3 12,4 10,5 9,9 10,93 ± 1,3c f4 15,5 15,2 14,4 15 ± 0,56d figure 4. antibacterial activity of kedabu fruit kombucha tea against escherichia coli and staphylococcus aureus on 14th day. the antibacterial activity test was conducted to evaluate the effectiveness of kombucha tea with and without the addition of kedabu fruit juice against escherichia coli and staphylococcus aureus bacteria. four different formulations of kedabu fruit juice kombucha tea (designated as f1, f2, f3, and f4) were tested, and their inhibitory effects were categorized as weak to moderate. statistical analysis using the one way anova test revealed a significant difference in the diameter of the inhibition zones among the various kombucha tea formulas containing kedabu fruit juice. further analysis using the tukey test indicated that the formula labeled f4 exhibited a significant difference in inhibiting escherichia coli and staphylococcus aureus bacteria. these results suggest that increasing the concentration of kedabu fruit juice in kombucha tea leads to a greater diameter of inhibition zone formation. the observed antibacterial activity can be attributed to bioactive compounds in kombucha tea, such as organic nasution et al. – potential of kedabu (sonneratia ovata backer) fruit juice supplemented … 461 acids, phenolic compounds, and other secondary metabolites. the concentration of these bioactive compounds is known to increase during the fermentation process, especially with prolonged fermentation time. the elevated levels of organic acids, including lactic acid and acetic acid, contribute to the stronger antibacterial activity of the kombucha tea. this finding is consistent with previous research demonstrating that organic acid content increases with prolonged fermentation time (jayabalan et al., 2007). it is important to note that the antibacterial activity of kombucha tea is influenced by various factors, including the type and concentration of bioactive compounds, fermentation conditions, and the targeted bacteria. further studies are needed to elucidate the specific mechanisms underlying the antibacterial effects of kombucha tea and to identify the key bioactive components responsible for its activity against escherichia coli and staphylococcus aureus. the inhibitory effects of kedabu fruit kombucha tea against escherichia coli and staphylococcus aureus bacteria can be attributed to the presence of lactic acid and acetic acid, which are produced by lactic acid bacteria (lab) and acetic acid bacteria (aab). these organic acids have been recognized as significant antibacterial agents in kombucha tea. the antibacterial activity of organic acids is closely associated with the ph of the medium. as the ph decreases, the antibacterial activity of the organic acids increases. lactic acid and acetic acid have the ability to disrupt the metabolic processes of pathogenic bacteria by damaging enzyme activity, as well as the structure and function of cell walls and membranes. this disruption inhibits nutrient absorption and protein synthesis in the bacteria. furthermore, lab-derived bacteriocin compounds can also contribute to the antibacterial activity by disrupting the membranes of pathogenic bacteria. these compounds damage essential components such as potassium ions and atp, leading to intracellular ph imbalance and ultimately cell failure. the findings mentioned above are supported previous research who investigated the antibacterial mechanisms of organic acids and bacteriocins produced by lab. their study demonstrated the damaging effects of these compounds on the cell membranes and intracellular components of bacteria, leading to the inhibition of growth and survival (hou et al., 2021). it is important to note that the antibacterial activity of kedabu fruit kombucha tea is a complex interplay of various factors, including the specific composition of organic acids, the presence of other bioactive compounds, and the targeted bacteria strains. further research is needed to explore the detailed mechanisms of action and the specific interactions between the organic acids and pathogenic bacteria. the kedabu fruit kombucha tea has been found to contain various secondary metabolites as indicated by the results of phytochemical screening, including flavonoids, phenolics, terpenoids, and saponins (table 3). phenolic compounds identified in kombucha tea include epicatechin gallate, epigallocatechin, catechin, epicatechin, and epigallocatechin gallate. these compounds have been associated with pharmacological activities (kaewkod et al., 2019). flavonoid compounds present in kedabu fruit kombucha tea have been shown to exhibit inhibitory effects on nucleic acid synthesis during dna and rna formation. additionally, flavonoids have the potential to disrupt bacterial cell wall permeability, microsomes, and lysosomes through interactions with bacterial dna (cushnie et al., 2005). the findings mentioned above highlight the presence of various bioactive compounds in kedabu fruit kombucha tea, particularly phenolic and flavonoid compounds. these compounds have been extensively studied for their potential therapeutic properties and antimicrobial activities. further investigations are necessary to explore the specific mechanisms by which these secondary metabolites interact with bacterial dna and exert their pharmacological effects. the inhibitory effect of phenolic compounds on bacterial cell walls is achieved by interfering with forming the cell wall peptidoglycan layer, disrupting it (gonzález et al., 2009). terpenoids, on the other hand, act as antibacterial agents by damaging transmembrane proteins, which serve as pathways for the entry and exit of compounds, thereby reducing the permeability of bacterial cell walls. consequently, the lack of nutrients in the bacterial cells leads to their death (cowan, 1999). saponins exert their antibacterial activity through a mechanism involving the diffusion of saponins through the outer membrane and cell wall. once inside the bacterial cell, saponins bind to the cytoplasmic membrane, destabilizing it and causing the release of cytoplasmic contents. ultimately, this disruption leads to cell death (pandey et al., 2011). the mechanisms described above illustrate how phenolic compounds, terpenoids, and saponins exhibit antibacterial effects by targeting different components of bacterial cells. these mechanisms contribute to the understanding of the potential antimicrobial activities of these secondary metabolites found in kedabu fruit kombucha tea. conclusions kombucha tea supplemented with kedabu fruit juice complies with the quality standards of fermented beverages and exhibits antibacterial activity against escherichia coli and staphylococcus aureus. the kombucha tea made from kedabu fruit juice demonstrates favorable results in various quality parameters, including organoleptic evaluation, ph value, total titratable acidity, and the counts of lactic acid bacteria (lab) and yeast, all of which meet the 462 biology, medicine, & natural product chemistry 14 (1), 2025: 453-463 established standards. additionally, the kombucha tea exhibits inhibitory effects against escherichia coli and staphylococcus aureus bacteria, falling within the weak to moderate categories. statistical analysis using one way anova reveals a significant variation in the diameter of the inhibition zone among the different formulas of kedabu fruit kombucha tea. further analysis using the tukey test demonstrates that formula 4 significantly differs in its 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(2009). chemical constituents from the fruits of sonneratia caseolaris and sonneratia ovata (sonneratiaceae). biochem syst ecol. 37(1):1–5. wang b, rutherfurd-markwick k, zhang xx, mutukumira an. kombucha: production and microbiological research. foods. 11(21):3456. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 541-552 | doi: 10.14421/biomedich.2025.141.541-552 issn 2540-9328 (online) antimicrobial potential of phytochemicals from coccinia grandis leaves: a molecular docking study against penicillin-binding protein 5 of escherichia coli and dna topoisomerase iv subunit b (pare 24kda) of staphylococcus aureus and escherichia coli malshan isuranga*, dulki nihinsa danthanarayana faculty of science, university of kelaniya, sri lanka. corresponding author* malshanisuranga98@gmail.com manuscript received: 06 february, 2025. revision accepted: 20 may, 2025. published: 04 august, 2025. abstract coccinia grandis (c. grandis) leaves, traditionally used in sri lanka for diabetes management, also have a potential antimicrobial activity. in this study, site-specific molecular docking was performed to investigate the antimicrobial activity of phytochemicals of coccinia grandis leaves against penicillin-binding protein 5 (pbp 5) and dna topoisomerase iv subunit b (pare 24kda) of escherichia coli (e. coli) and dna topoisomerase iv subunit b (pare 24kda) of staphylococcus aureus (s. aureus). penicillin was selected as the reference molecule for penicillin-binding protein 5 and for dna topoisomerase iv subunit b (pare 24kda), novobiocin was selected as the reference molecule. the results identified lupeol (-7.72 kcal/mol) and beta-sitosterol (-8.21kcal/mol) have a higher binding affinity to pbp5 of e. coli than penicillin (-7.20 kcal/mol). quercetin (-6.70 kcal/mol), kaempferol (-6.95 kcal/mol), naringenin (-7.07 kcal/mol), isoquercetin (-6.15 kcal/mol), lupeol (-7.87 kcal/mol), beta-sitosterol (-9.42 kcal/mol) and sanguinarine (-9.07 kcal/mol) show higher binding affinity to dna topoisomerase iv subunit b (pare 24kda) of s. aureus than novobiocin (-6.04 kcal/mol). as well quercetin (6.85 kcal/mol), kaempferol (-6.82 kcal/mol), naringenin (-7.23 kcal/mol), isoquercetin (-6.20 kcal/mol), lupeol (-7.67 kcal/mol), betasitosterol (-9.08 kcal/mol) and sanguinarine (-9.03 kcal/mol) show higher binding affinity to dna topoisomerase iv subunit b (pare 24kda) of e. coli than novobiocin (-5.76 kcal/mol). in silico pharmacokinetic and physicochemical parameter predictions were also conducted to study drug-likeness of above molecules using specialized web servers. keywords: coccinia grandis; escherichia coli; staphylococcus aureus; penicillin-binding protein 5; dna topoisomerase iv subunit b (pare 24kda); molecular docking. introduction antibiotics are the most wonderful innovation in the history of medicine. from the first antibiotic, penicillin, discovered by alexander fleming in 1928, antibiotics have a long development history. these drugs have saved numerous numbers of lives worldwide. some antibiotics kill the microorganisms while others reduce their growth rate. antibiotic resistance (abr) is the ability of microorganisms to withstand the activity of the antibiotic. mutations, horizontal gene transfer, and human actions such as misuse of antibiotics contribute to the abr (larsson & flach, 2022). according to the world health organization (who), abr is one of the most significant global health threats (global antimicrobial resistance and use surveillance system (glass) report 2022, 2022). coccinia grandis belongs to the family cucurbitaceae, predominantly distributed in tropical asia and africa including pakistan, india, and sri lanka (farrukh et al., 2008). c. grandis is commonly known as ivy gourd or scarlet gourd in english; kowakka in sinhala. in sri lanka, this plant is predominantly distributed in the north central, southern, and western regions. from centuries local population of sri lanka used leaves of this plant for diabetes management (attanayake et al.,2016). researchers suggest that leaf extract of c. grandis exhibits anti-hyperglycemic, xanthine oxidase inhibitory, analgesic, antiinflammatory, antipyretic, antioxidant, antihyperlipidemic, antimicrobial, and anti-hepatotoxic activities (ramachandran et al., 2014). penicillin-binding protein 5 (pbp 5) of e. coli engages in cell wall synthesis. the main constituent of the bacterial cell wall is peptidoglycan. pbp 5 enzyme performs a dd-carboxypeptidase reaction on the bacterial peptidoglycan. the active site of pbp 5 contains a specific serine residue, which acts like a hook to grab a unit of the peptidoglycan chain during the https://doi.org/10.14421/biomedich.2025.141.541-552 542 biology, medicine, & natural product chemistry 14 (1), 2025: 541-552 enzymatic reaction. near the active site, two lysine residues (lys47 and lys213) play a critical role in proton-transfer events during acylation and deacylation events (zhang et al., 2007). pbp 5 can be inactivated by β-lactam antibiotics such as penicillin. inactivation occurs when the antibiotic forms a covalent bond with a serine residue in pbp 5, creating a stable complex that inhibits the protein’s enzymatic activity and disrupts its normal function. when pbp 5 binds to the antibiotic, it is dormant in an acylation state, thus, disrupting the cell wall synthesis, and resulting in cell death (nicholas et al., 2003). dna topoisomerase iv subunit b (pare 24kda) of escherichia coli (e. coli) is an important enzyme that regulates dna topology during replication and transcription. its primary role is to separate the intertwined daughter chromosomes after dna replication, ensuring that genetic material is correctly distributed to daughter cells. pare functions with another subunit, parc, to form a heterotetrameric complex (c2e2) that utilizes the energy derived from atp hydrolysis to introduce and relax supercoils in dna (bellon et al., 2004). materials and methods ligand preparation: phytochemicals of coccinia grandis leaves identified through a literature survey. the structure of the active site of e. coli pbp 5 was also obtained through literature survey. the structures of selected molecules obtained from pubchem database (pubchem, n.d.) in .sdf format. the energy-minimization of ligands was performed using avogadro (version 1.2.0) software and saved in .pdb format. figure 1. (a) crystal structure of escherichia coli pbp 5 in complex with a peptide-mimetic penicillin pdb doi: https://doi.org/10.2210/pdb3beb/pdb, (b) crystal structure of e. coli topoisomerase iv pare 24kda subunit pdb doi: https://doi.org/10.2210/pdb1s14/pdb, (c) crystal structure of s. aureus pare 24kda in complex with novobiocin pdb doi: https://doi.org/10.2210/pdb4urn/pdb, (d) structure of active site of e. coli pbp 5 (sauvage et al., 2008), (e) structure of active site of e. coli pare 24kda (bellon et al., 2004) (f) structure of active site of s. aureus pare 24kda (lu et al., 2014b). protein preparation: the crystal structures of proteins were obtained from protein data bank in .pdb format (pdb id – 3beb: crystal structure of e. coli penicillin-binding protein 5 in complex with a peptide-mimetic penicillin pdb doi: https://doi.org/10.2210/pdb3beb/pdb (sauvage et al., 2008), pdb id – 1s14: crystal structure of escherichia coli topoisomerase iv pare 24kda subunit pdb doi: https://doi.org/10.2210/pdb1s14/pdb (bellon et al., 2004), pdb id – 4urn: crystal structure of staph pare 24kda in complex with novobiocin – pdb doi: https://doi.org/10.2210/pdb4urn/pdb (lu et al., 2014). the protein was prepared using autodocktools (version 1.5.7). heteroatoms and water molecules were removed, and polar hydrogen and kollman chargers were added to the protein. ad4 type atoms were assigned to the protein. molecular docking: autodock version 4.2.6 was used to perform site-specific molecular docking and results were generated in .dlg format (output – lamarckianga-4.2). the grid parameters were set to cover the active site of each protein complex. a b c d e f isuranga & danthanarayana – antimicrobial potential of phytochemicals from coccinia grandis … 543 table 1. grid map dimensions (a0) and grid-center coordinates (a0). protein complex grid map dimensions (a0) grid-center coordinates (a0) x-axis y-axis z-axis x-axis y-axis z-axis e. coli pbp 5 15.8 26.3 16.5 42.732 4.638 26.112 e. coli pare 24kda subunit 18.0 18.0 17.3 19.236 26.093 48.526 staph pare 24kda subunit 17.3 21.0 19.5 -27.52 5.92 0.958 the genetic algorithm (ga) parameters were set to 50 runs with 300 population size. the maximum number of evaluations: 25,000,000 (medium) and maximum number of generations 27 000 (lawan & tharakee, 2023). molecular dynamics simulation: the site-specific molecular docking results were analyzed using autodocktools (version 1.5.7). binding energies and inhibition constants were examined,and the interactions were visualized using discovery studio visualizer (v24.1.0.23298). drug-likeness and pharmacokinetics of selected molecules: the potential of selected molecules as drugs was evaluated by assessing their drug-likeness and pharmacokinetic properties. pharmacokinetics parameters of the selected molecules were predicted using the pkcsm server (https://biosig.lab.uq.edu.au/pkcsm/prediction) and swissadme (http://www.swissadme.ch/index.php). these parameters describe how a drug behaves in the body including absorption, distribution, metabolism, excretion, and toxicity (table 9). the drug-likeness of the molecules was analyzed using swissadme (http://www.swissadme.ch/index.php), which evaluates molecules against lipinski’s rule of five (lipinski et al., 2001) and verber’s rules (veber et al., 2002) (table 8). docking validation (quality control): the docking procedure was validated by removing the inhibitor from each obtained protein complex and redocking the inhibitor. the re-docked complex was aligned with the reference crystalized complex using pymol (version 2.5.8), and the difference in their positions was measured using the root mean square deviation (rmsd) (shivanika et al., 2022). results table 2. calculated binding energies and inhibition constants of selected molecules against pbp 5 of e. coli. molecule reference rmsd (a0) binding energy (kcal/mol) inhibition constant-ki (µm) at 298.15 k penicillin 51.41 -7.20 5.32 quercetin 49.21 -6.19 28.88 kaempferol 49.70 -5.90 47.19 naringenin 51.17 -6.27 25.47 isoquercetin 51.46 -6.08 34.69 lupeol 49.67 -7.72 2.19 beta-sitosterol 47.94 -8.21 0.953 rutin 50.44 -5.24 143.35 sanguinarine 48.95 -6.53 16.23 p-coumaric acid unsuccessful table 3. calculated binding energies and inhibition constants of selected molecules against topoisomerase iv pare 24kda subunit of e. coli. molecule reference rmsd (a0) binding energy (kcal/mol) inhibition constant-ki (µm) at 298.15 k novobiocin 57.21 -5.76 60.25 quercetin 60.87 -6.85 9.45 kaempferol 60.68 -6.82 10.08 naringenin 60.92 -7.23 5.01 isoquercetin 60.71 -6.20 28.57 lupeol 59.13 -7.67 2.39 beta-sitosterol 58.98 -9.08 222.53 rutin 58.62 -4.38 611.52 sanguinarine 60.40 -9.03 0.241 p-coumaric acid unsuccessful 544 biology, medicine, & natural product chemistry 14 (1), 2025: 541-552 table 4. calculated binding energies and inhibition constants of selected molecules against topoisomerase iv pare 24kda subunit of s. aureus. molecule reference rmsd (a0) binding energy (kcal/mol) inhibition constant-ki (µm) at 298.15 k novobiocin 25.79 -6.04 37.19 quercetin 24.15 -6.70 12.27 kaempferol 25.23 -6.95 7.99 naringenin 23.41 -7.07 6.63 isoquercetin 25.88 -6.15 31.28 lupeol 26.02 -7.87 1.70 beta-sitosterol 23.93 -9.42 0.125 rutin 26.99 -5.42 106.89 sanguinarine 23.39 -9.07 0.224 p-coumaric acid unsuccessful figure 2. (a) superimposition of re-docked penicillin (yellow) on active site of crystalized structure (green) of escherichia coli pbp 5 using pymol (rmsd = 0.000 a0), (b) superimposition of re-docked novobiocin (yellow) on active site of crystalized structure (green) of e. coli topoisomerase iv pare 24kda subunit using pymol (rmsd = 0.000 a0), (c) superimposition of re-docked novobiocin (yellow) on active site of crystalized structure (green) of s. aureus pare 24kda using pymol (rmsd = 0.000 a0). figure 3. interactions between selected molecules and escherichia coli pbp 5 (a) penicillin, (b) quercetin, (c) kaempferol, (d) naringenin, (e) isoquercetin, (f) lupeol, (g) beta-sitosterol, (h) rutin, (i) sanguinarine a b c e a b c d f g h i isuranga & danthanarayana – antimicrobial potential of phytochemicals from coccinia grandis … 545 figure 4. interactions between selected molecules and topoisomerase iv pare 24kda subunit of e. coli (a) novobiocin, (b) quercetin, (c) kaempferol, (d) naringenin, (e) isoquercetin, (f) lupeol, (g) beta-sitosterol, (h) rutin, (i) sanguinarine. figure 5. interactions between selected molecules and topoisomerase iv pare 24kda subunit of s. aureus (a) novobiocin, (b) quercetin, (c) kaempferol, (d) naringenin, (e) isoquercetin, (f) lupeol, (g) beta-sitosterol, (h) rutin, (i) sanguinarine. a b c d e f g h i a b c 546 biology, medicine, & natural product chemistry 14 (1), 2025: 541-552 figure 5. cont. table 5. hydrogen bonding interactions (intermolecular conventional hydrogen bond) between ligands and pbp 5 of e. coli. molecule name distance (a0) h-donor h-acceptor dha angle (0) penicillin a:ser44:hg :unl1:n 1.70075 a:ser44:hg :unl1:n 153.622 a:ser87:hn :unl1:oxt 2.15591 a:ser87:hn :unl1:oxt 163.07 a:asn112:hd21 :unl1:oxt 2.10638 a:asn112:hd21 :unl1:oxt 159.893 lupeol :unl1:h a:ser44:og 1.90396 :unl1:h a:ser44:og 147.908 beta-sitosterol :unl1:h a:asn112:od1 2.42915 :unl1:h a:asn112:od1 137.911 table 6. hydrogen bonding interactions (intermolecular conventional hydrogen bond) between ligands and topoisomerase iv pare 24kda subunit of e. coli. molecule name distance (a0) h-donor h-acceptor dha angle (0) novobiocin a:his1079:he2 :unl1:o 2.89395 a:his1079:he2 :unl1:o 112.624 :unl1:h a:ile1078:o 2.44228 :unl1:h a:ile1078:o 176.688 quercetin a:asn1042:hd21 :unl1:o 2.88793 a:asn1042:hd21 :unl1:o 101.638 :unl1:h a:glu1046:oe1 2.19212 :unl1:h a:glu1046:oe1 114.214 :unl1:h a:glu1046:oe1 2.12888 :unl1:h a:glu1046:oe1 137.021 :unl1:h a:val1039:o 2.9988 :unl1:h a:val1039:o 91.088 kaempferol a:arg1072:hn :unl1:o 2.62735 a:arg1072:hn :unl1:o 101.889 a:gly1073:hn :unl1:o 1.74046 a:gly1073:hn :unl1:o 144.668 :unl1:h a:val1039:o 2.02003 :unl1:h a:val1039:o 176.714 :unl1:h a:asp1069:od1 2.30913 :unl1:h a:asp1069:od1 114.577 :unl1:h a:glu1046:oe1 2.04473 :unl1:h a:glu1046:oe1 167.188 naringenin a:arg1072:hn :unl1:o 2.89481 a:arg1072:hn :unl1:o 97.009 a:gly1073:hn :unl1:o 1.85791 a:gly1073:hn :unl1:o 158.693 :unl1:h a:val1039:o 2.24921 :unl1:h a:val1039:o 159.885 :unl1:h a:glu1046:oe1 1.87455 :unl1:h a:glu1046:oe1 135.297 d e f h ig isuranga & danthanarayana – antimicrobial potential of phytochemicals from coccinia grandis … 547 table 6. cont. molecule name distance (a0) h-donor h-acceptor dha angle (0) isoquercetin a:asn1042:hd21 :unl1:o 2.7382 a:asn1042:hd21 :unl1:o 100.17 a:asn1042:hd21 :unl1:o 1.92763 a:asn1042:hd21 :unl1:o 164.831 a:gly1073:hn :unl1:o 1.67752 a:gly1073:hn :unl1:o 141.974 :unl1:h a:asn1042:o 2.23832 :unl1:h a:asn1042:o 102.23 :unl1:h a:glu1046:oe1 1.86696 :unl1:h a:glu1046:oe1 133.412 :unl1:h a:glu1046:oe1 2.33954 :unl1:h a:glu1046:oe1 128.18 :unl1:h a:val1039:o 2.95873 :unl1:h a:val1039:o 103.791 table 7. hydrogen bonding interactions (intermolecular conventional hydrogen bond) between ligands and topoisomerase iv pare 24kda subunit of s. aureus. molecule name distance (a0) h-donor h-acceptor dha angle (0) novobiocin a:arg138:hh11 :unl1:o 2.07271 a:arg138:hh11 :unl1:o 116.16 :unl1:h a:thr92:og1 2.30294 :unl1:h a:thr92:og1 142.972 :unl1:h a:gly80:o 2.37333 :unl1:h a:gly80:o 114.849 :unl1:h a:glu53:oe1 2.28073 :unl1:h a:glu53:oe1 153.157 :unl1:h a:asp76:od2 1.84227 :unl1:h a:asp76:od2 129.527 quercetin a:arg79:hn :unl1:o 2.74275 a:arg79:hn :unl1:o 97.988 a:gly80:hn :unl1:o 1.68791 a:gly80:hn :unl1:o 145.046 :unl1:h a:ile46:o 2.13321 :unl1:h a:ile46:o 152.776 :unl1:h a:asp76:od1 2.27165 :unl1:h a:asp76:od1 112.554 :unl1:h a:glu53:oe2 1.92621 :unl1:h a:glu53:oe2 122.031 kaempferol :unl1:h a:ser50:og 2.19725 :unl1:h a:ser50:og 150.997 :unl1:h a:asn49:o 1.75646 :unl1:h a:asn49:o 133.732 :unl1:h a:asn56:od1 1.98098 :unl1:h a:asn56:od1 144.601 :unl1:h a:gly80:o 2.00352 :unl1:h a:gly80:o 143.187 naringenin :unl1:h a:gly80:o 2.08123 :unl1:h a:gly80:o 138.264 :unl1:h a:asp76:od2 2.21723 :unl1:h a:asp76:od2 145.165 :unl1:h a:thr168:o 2.91224 :unl1:h a:thr168:o 111.039 isoquercetin a:asn49:hd21 :unl1:o 2.8621 a:asn49:hd21 :unl1:o 119.408 :unl1:h a:asn56:od1 2.05503 :unl1:h a:asn56:od1 155.619 :unl1:h a:glu53:oe1 1.71719 :unl1:h a:glu53:oe1 133.802 :unl1:h a:asn56:od1 2.06169 :unl1:h a:asn56:od1 97.614 :unl1:h a:glu53:oe1 2.16274 :unl1:h a:glu53:oe1 107.551 :unl1:h a:asp76:od2 2.11531 :unl1:h a:asp76:od2 141.204 sanguinarine a:arg79:he :unl1:o 2.65739 a:arg79:he :unl1:o 90.653 table 8. predicted physiochemical properties of selected molecules (mw = molecular weight, nha = number of heavy atoms, nrb = number of rotatable bonds, nhba = number of h-bond acceptors, nhbd = number of h-bond donors, tpsa = topological polar surface area, o/w-pc = octanol/water partition coefficient, lip = lipinski’s rule of five. veb = veber’s rule, bas = bio-availability score. molecule mw (g/mol) nha nrb nhba nhbd tpsa (å²) o/w-pc (mlogp) lip veb bas penicillin 334.39 23 5 4 2 112.01 1.55 yes yes 0.56 novobiocin 612.62 44 10 11 5 200.01 0.65 no no 0.17 quercetin 302.24 22 1 7 5 131.36 -0.56 yes yes 0.55 kaempferol 286.24 21 1 6 4 111.13 -0.03 yes yes 0.55 naringenin 272.25 20 1 5 3 86.99 0.71 yes yes 0.55 isoquercetin 464.38 33 4 12 8 210.51 -2.59 no no 0.17 lupeol 426.72 31 1 1 1 20.23 6.92 yes yes 0.55 beta-sitosterol 414.71 30 6 1 1 20.23 6.73 yes yes 0.55 rutin 610.52 43 6 16 10 269.43 -3.89 no no 0.17 sanguinarine 332.33 25 0 4 0 40.80 2.72 yes yes 0.55 548 biology, medicine, & natural product chemistry 14 (1), 2025: 541-552 table 9. predicted pharmacokinetic parameters of the selected molecules. penicillin novobiocin quercetin kaempferol naringenin absorption caco2 permeability (log papp in 10-6 cm/s) 0.114 -4.836 -0.229 0.032 1.029 water solubility (log mol/l) -2.47 -0.427 -2.925 -3.04 -3.224 intestinal absorption (human) (% absorbed) 59.901 66.418 77.207 74.29 91.31 skin permeability (log kp) -2.735 -2.787 -2.735 -2.735 -2.742 p-glycoprotein substrate yes yes yes yes yes p-glycoprotein i inhibitor no yes no no no p-glycoprotein ii inhibitor no yes no no no distribution vdss (human) (log l/kg) -1.905 -1.336 1.559 1.274 -0.015 fraction unbound (human) (fu) 0.328 0.127 0.206 0.178 0.064 bbb permeability (log bb) -0.864 (no) -1.914 (no) -1.098 (no) -0.939 (no) -0.578 (no) cns permeability (log ps) -2.943 -3.492 -3.065 -2.228 -2.215 metabolism cyp2d6 substrate no no no no no cyp3a4 substrate no yes no no no cyp1a2 inhibitor no no yes yes yes cyp2c19 inhibitor no no no no no cyp2c9 inhibitor no no no no no cyp2d6 inhibitor no no no no no cyp3a4 inhibitor no yes no no no excretion total clearance (log ml/min/kg) 0.197 -0.228 0.407 0.477 0.06 renal oct2 substrate no no no no no toxicity ames toxicity no no no no no max. tolerated dose (human) (log mg/kg/day) 0.692 0.125 0.499 0.531 -0.176 herg i inhibitor no no no no no herg ii inhibitor no yes no no no oral rat acute toxicity (ld50) (mol/kg) 1.716 2.085 2.471 2.449 1.791 oral rat chronic toxicity (loael) (log mg/kg_bw/day) 2.542 2.095 2.612 2.505 1.944 hepatotoxicity yes no no no no skin sensitization no no no no no t. pyriformis toxicity (log ug/l) 0.285 0.288 0.288 0.312 0.369 minnow toxicity (log mm) 3.698 1.146 3.721 2.885 2.136 pharmacokinetic parameters ligand isuranga & danthanarayana – antimicrobial potential of phytochemicals from coccinia grandis … 549 isoquercetin lupeol betasitosterol rutin sanguinarin e absorption caco2 permeability (log papp in 10-6 cm/s) 0.242 1.226 1.201 -0.949 2.107 water solubility (log mol/l) -2.925 -5.861 -6.773 -2.892 -5.56 intestinal absorption (human) (% absorbed) 47.999 95.782 94.464 23.446 100 skin permeability (log kp) -2.735 -2.744 -2.783 -2.735 -2.707 p-glycoprotein substrate yes no no yes yes p-glycoprotein i inhibitor no yes yes no yes p-glycoprotein ii inhibitor no yes yes no yes distribution vdss (human) (log l/kg) 1.846 0 0.193 1.663 0.298 fraction unbound (human) (fu) 0.228 0 0 0.187 0.265 bbb permeability (log bb) -1.688 (no) 0.726 (no) 0.781 (no) -1.899 (no) -0.105 (yes) cns permeability (log ps) -4.093 -1.714 -1.705 -5.178 -1.419 metabolism cyp2d6 substrate no no no no no cyp3a4 substrate no yes yes no yes cyp1a2 inhibitor no no no no yes cyp2c19 inhibitor no no no no yes cyp2c9 inhibitor no no no no no cyp2d6 inhibitor no no no no yes cyp3a4 inhibitor no no no no yes excretion total clearance (log ml/min/kg) 0.394 0.153 0.628 -0.369 1.051 renal oct2 substrate no no no no no toxicity ames toxicity no no no no yes max. tolerated dose (human) (log mg/kg/day) 0.569 -0.502 -0.621 0.452 0.172 herg i inhibitor no no no no no herg ii inhibitor yes yes yes yes yes oral rat acute toxicity (ld50) (mol/kg) 2.541 2.563 2.552 2.491 2.588 oral rat chronic toxicity (loael) (log mg/kg_bw/day) 4.417 0.89 0.855 3.673 1.729 hepatotoxicity no no no no no skin sensitization no no no no no t. pyriformis toxicity (log ug/l) 0.285 0.316 0.43 0.285 0.308 minnow toxicity (log mm) 8.061 -1.696 -1.802 7.677 -0.718 discussion quercetin, kaempferol, naringenin, isoquercetin, rutin are considered as flavonoids. lupeol is a pentacyclic triterpenoid, while beta-sitosterol is a plant sterol (phytosterol). sanguinarine belongs to alkaloids. pcoumaric acid is a hydroxycinnamic acid. the inhibitory constant, denoted as ki (µm), is a measurement used in pharmacology to understand how tightly a drug binds (binding affinity) to a specific molecule (target molecule). it reflects the drug concentration required to occupy half of the available binding sites. a lower ki value indicates stronger binding. this means a lower drug concentration sufficient to occupy half (50%) of the available binding sites. molecules with ki values less than 100 µm are considered potent inhibitors while molecules with higher ki values than 100 µm are considered as non-potent inhibitors (zheng & polli, 2010). lupeol (-7.72 kcal/mol), beta-sitosterol (-8.21 kcal/mol) show higher binding affinity to pbp 5 of e. coli than the penicillin (-7.20 kcal/mol) which is the reference molecule. both beta-sitosterol (0.953 µm) and lupeol (2.19 µm) show lower inhibition constant than penicillin (5.32 µm), indicating higher potency than penicillin (table 2). quercetin (-6.85 kcal/mol), kaempferol (-6.82 kcal/mol), naringenin (-7.23 kcal/mol), isoquercetin (6.20 kcal/mol), lupeol (-7.67 kcal/mol), beta-sitosterol (-9.08 kcal/mol) and sanguinarine (-9.03 kcal/mol) show higher binding affinity to topoisomerase iv pare 24kda subunit of e. coli than novobiocin (-5.76 kcal/mol). except for beta-sitosterol, all above mentioned pharmacokinetic parameters ligand 550 biology, medicine, & natural product chemistry 14 (1), 2025: 541-552 molecules exhibit a lower inhibition constant than novobiocin (table 3). quercetin (-6.70 kcal/mol), kaempferol (-6.95 kcal/mol), naringenin (-7.07 kcal/mol), isoquercetin (6.15 kcal/mol), lupeol (-7.87 kcal/mol), beta-sitosterol (-9.42 kcal/mol) and sanguinarine (-9.07 kcal/mol) show higher binding affinity to dna topoisomerase iv subunit b (pare 24kd) of s. aureus than novobiocin (-6.04 kcal/mol). all these molecules exhibit lower inhibition constant than novobiocin (table 4). among these molecules beta-sitosterol shows the highest binding affinity and the lowest inhibition constant towards topoisomerase iv pare 24kda subunit of s. aureus. interactions between a ligand and a protein are crucial for understanding biochemical processes, particularly in protein function and stability. two major types of interactions that can be observed are hydrogen bonds (hbonds) and hydrophobic interactions. hydrogen bonds significantly contribute to the stability of the protein structure. furthermore, h-bonds can provide specificity in ligand binding. the precise arrangement of h-bond donors and acceptors facilitates selective binding of ligands. higher the number of h-bonds, greater the binding efficiency and the inhibition (kumar et al., 2015). hydrophobic interactions drive protein folding and significantly contribute to the stability of the protein. in h-bond interactions analysis, penicillin forms three h-bonds with three amino acids (ser44, ser87, and asn112) located in the active site of the pbp5. in the case of pbp5 and lupeol one h-bonding interaction was observed (ser44), while pbp5 and beta-sitosterol exhibit one h-bond (asn112) (figure 3 and table 5). two h-bonds were observed between the topoisomerase iv pare 24kda subunit of e. coli and novobiocin (his1079 and ile1078). additionally, quercetin forms four h-bonds with three amino acids (asn1042, glu1046 and val1039) in the active site of this protein. five h-bonds with five amino acids in the active site, were observed between kaempferol and the protein (arg1072, gly1073, val1039, aso1069 and glu1046). similarly, four h-bonds with four amino acids in the active site, were observed between naringenin and the protein (arg1072, gly1073, val1039 and glu1046). the highest number of hbonds was observed between isoquercetin and the protein, seven h-bonds with four amino acids in the active site (asn1042, gly1073, glu1046 and val1039). lupeol, beta-sitosterol and sanguinarine do not form h-bonds with the topoisomerase iv pare 24kda subunit of e. coli, but they exhibit hydrophobic interactions with the protein (figure 4 and table 6). considering the h-bonds between topoisomerase iv pare 24kda subunit of s. aureus and novobiocin, five hbonds were observed with five amino acids present in the active site (arg138, thr92, gly80, glu53 and asp76). between quercetin and the protein, five hbonds with five amino acids present on the active site, were observed (arg79, gly80, ile46, asp76, and glu53). kaempferol shows four h-bonds with the protein (ser50, asn49, asn56, and gly80). three hbonds were observed between the protein and naringenin (gly80, asp76, and thr168). the highest number of h-bonds observed is six between isoquercetin and the protein (asn49, asn56, glu53, and asp76). sanguinarine exhibits one h-bond with the protein (arg79). both lupeol and beta-sitosterol show hydrophilic interactions with the protein but do not form any h-bonds (figure 5 and table 7). considering the predicted physiochemical properties of selected molecules, isoquercetin and rutin do not fulfil lipinski’s and veber’s rules. even novobiocin, a commercially available antibiotic, does not fulfil lipinski’s and veber’s rules. except for sanguinarine, none penetrates the blood brain barrier (bbb). oral rat acute toxicity (ld50) is defined as the amount of substance that is required to kill 50% of a tested population within a specific time frame. among the selected molecules, all except naringenin exhibit higher ld50 values compared to commercially available antibiotics which are penicillin and novobiocin. a higher ld50 value suggests that these molecules may be less toxic than penicillin and novobiocin. hepatotoxicity which is also known as liver toxicity of the selected molecules was also evaluated. all the molecules, except penicillin, were found to be non-hepatotoxic. when considering the number of selected molecules that exhibit higher binding affinity to the protein than the reference molecule, only two molecules (lupeol and beta-sitosterol) exhibit higher binding affinity than penicillin against pbp 5 of e. coli. in the case of topoisomerase iv pare 24kda subunit, seven molecules exhibit higher binding affinity compared to the reference molecule which is novobiocin in both e. coli and s. aureus. further studies should be conducted to investigate the combined effect of the molecules against each protein. these results reveal that selected phytochemicals show a higher inhibitory effect on the dna topoisomerase iv pare 24kda subunit compared to their effect on pbp5. this suggests that selected molecules may inhibit enzymes crucial to bacterial structural component synthesis and dna replication. these molecules may exhibit a dual mechanism of action. this dual action mechanism may potentially kill or reduce the growth of microorganisms. the re-docking was done to validate the docking procedure. penicillin bound to the active site of the penicillin-binding protein 5 of e. coli with binding energy of -7.20 kcal/mol. penicillin formed three hbonds with three amino acids (ser44, ser87, and asn112) located in the active site of the pbp 5 of e. coli with a distance ranging from 1.7 – 2.1 a0. these bonds are well within the optimal range for h-bonding, indicating a strong interaction. the re-docked complex isuranga & danthanarayana – antimicrobial potential of phytochemicals from coccinia grandis … 551 was then superimposed on to the native crystalized structure of e. coli pbp 5 from pdb using pymol observed 0.000 a0 rmsd value. the same methodology was followed for both topoisomerase iv pare 24kda subunit of e. coli and s. aureus with novobiocin. novobiocin formed two h-bonds with two amino acids (his1079 and ile1078) located in the active site of the topoisomerase iv pare 24kda subunit of e. coli with a distance ranging from 2.4 – 2.9 a0. novobiocin formed five h-bonds with five amino acids (arg138, thr92, gly80, glu53, and asp76) located in the active site of the topoisomerase iv pare 24kda subunit of s. aureus with a distance ranging from 1.8 – 2.4 a0. these bonds are well within the optimal range for h-bonding, indicating a strong interaction. the re-docked complexes were then superimposed on to their native crystalized structures using pymol and observed 0.000 a0 rmsd value (figure 2). obtaining 0.000 a0 for rmsd indicates no difference between the re-docked complex and the referenced crystallized structure. overall, this study provides a strong foundation for further investigations to study anti-microbial activity of selected molecules derived from phytochemicals of coccinia grandis leaves. these findings may pave the way for developing novel antibiotic agents with enhanced inhibitory efficacy. conclusion selected phytochemicals of coccinia grandis leaves were evaluated in silico to determine their antimicrobial activity against penicillin-binding protein 5 (pbp 5) and topoisomerase iv pare 24kda subunit of escherichia coli and topoisomerase iv pare 24kda subunit of staphylococcus aureus. physiochemical and pharmacokinetic properties of selected molecules were also evaluated. molecular docking, physiochemical and pharmacokinetic results were compared with reference molecules which are penicillin and novobiocin which are commercially available antibiotics. out of the tested molecules, lupeol and betasitosterol exhibited better inhibition capabilities against pbp5 of escherichia coli than penicillin. quercetin, kaempferol, naringenin, isoquercetin, lupeol, betasitosterol and sanguinarine exhibit higher binding affinity to topoisomerase iv pare 24kda subunit of both escherichia coli and staphylococcus aureus than novobiocin. selected natural molecules exhibit higher binding affinity towards topoisomerase iv pare 24kda subunit than pbp5 of e. coli. results in this study suggest that these selected phytochemicals of coccinia grandis leaves may serve as promising candidates for development of antimicrobial agents against resistant strains of escherichia coli and staphylococcus aureus. further studies should focus on optimizing the structure of these selected molecules to enhance their binding affinity and the inhibitory potency. acknowledgements: i would like to express my sincere gratitude to dr. m.g.a.n. perera from the faculty of applied science at the university of sabaragamuwa for his invaluable guidance and support throughout this research. authors’ contributions: m. isuranga designed the study. computational analysis and data interpretation were conducted by m. isuranga and d.n. danthanarayana. the manuscript was written by both m. isuranga and d.n. danthanarayana. competing interests: the author declares that there are no competing interests. references bellon, s., parsons, j. d., wei, y., hayakawa, k., swenson, l. l., charifson, p. s., lippke, j. a., aldape, r., & gross, c. h. 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(2010). identification of inhibitor concentrations to efficiently screen and measure inhibition ki values against solute carrier transporters. european journal of pharmaceutical sciences, 41(1), 43–52. https://doi.org/10.1016/j.ejps.2010.05.013 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 879-885 | doi: 10.14421/biomedich.2025.142.879-885 issn 2540-9328 (online) protective effects of l-carnitine on growth and cardiac function in high-glucose-exposed zebrafish embryos editya fukata*, anditri weningtyas, lintang widya sishartami, nuramaliah desliani putri, putri kusuma c. g. r. asfandi medical department, faculty of medicine, universitas negeri malang, indonesia jl. semarang no. 5, malang 65145, east java, tel. +62 341-551 312, fax. +62 341-551 921, indonesia corresponding author* editya.fukata.fk@um.ac.id manuscript received: 01 september, 2025. revision accepted: 14 october, 2025. published: 24 october, 2025. abstract diabetes mellitus is a chronic metabolic disorder, and maternal hyperglycaemia during pregnancy increases the risk of fetal developmental complications. this study aimed to evaluate the protective role of l-carnitine against glucose-induced embryotoxicity in zebrafish (danio rerio). fertilized embryos were exposed to 5% glucose with or without l-carnitine supplementation (50 or 100 μm). developmental outcomes including survival, hatching, malformations, body length, and heart rate were assessed using microscopy and analyzed by anova and kaplan–meier survival curves. high glucose significantly reduced survival (hazard ratio = 6.86; p < 0.0001), delayed hatching (median hatching time 84 vs. 60 hpf; hr = 0.17, 95% ci = 0.08–0.39; p < 0.0001), and induced growth retardation and bradycardia (p < 0.0001). l-carnitine did not rescue survival or hatching but partially ameliorated growth impairment (p < 0.05) and restored heart rate in a dose-dependent manner, with 100 μm supplementation approaching control values (p < 0.001). morphological abnormalities such as yolk sac edema and spinal curvature remained present but less severe with co-treatment. in conclusion, l-carnitine confers partial protective effects on growth and cardiac function under hyperglycaemic stress, although it does not prevent early lethality. keywords: zebrafish model; diabetes mellitus; hyperglycaemia‑induced embryotoxicity; l‑carnitine supplementation. abbreviations: dm: diabetes mellitus; lc: l-carnitine; lclt: l-carnitine l-tartrate; lcfa: long-chain fatty acids; hpf: hours post fertilization; yse: yolk sac edema; sc: spinal curvature; hr: hazard ratio; mst: median survival time; mht: median hatching time; ci: confidence interval; bpm: beats per minute introduction diabetes mellitus (dm) is a chronic metabolic disorder characterized by chronic hyperglycaemia, which remains a global health problems (international diabetes federation, 2021). the prevalence of hyperglycaemia during pregnancy is concerning, with epidemiological data showing that approximately 16% of live births from mothers aged 20–49 years are affected (ogurtsova et al., 2017). maternal hyperglycaemia not only increases the risk of obstetric complications but also imposes detrimental effects on fetal development, as infants of diabetic mothers are up to three times more likely to present with congenital abnormalities (tinker et al., 2021). to date, preventive approaches to these complications have been limited to glycaemic control using antidiabetic medications, which may cause adverse effects for both the mother and foetus (kalra et al., 2015). thus, there is a clear need to explore safer complementary or adjunctive approaches to mitigate hyperglycaemia-induced embryotoxicity. zebrafish (danio rerio) have emerged as a powerful vertebrate model for studying embryonic development and metabolic disorders. zebrafish share 87% genetic homology and exhibit conserved mechanisms of glucose metabolism regulation with humans, making them wellsuited for diabetes research (capiotti et al., 2014; chakraborty et al., 2016). previous studies have demonstrated that high glucose exposure in zebrafish embryos leads to developmental abnormalities and reduced survival, thereby providing a reliable in vivo platform for investigating the consequences of hyperglycaemia (li et al., 2022). l-carnitine (lc) is a naturally occurring compound synthesized from lysine and methionine which can be obtained from dietary sources such as red meat, fish, poultry, and milk (sawicka et al., 2020). lc plays a crucial role in the β-oxidation of long-chain fatty acids (lcfa) by facilitating the transport of fatty acids into mitochondria for metabolism. additionally, lc has been shown to enhance non-oxidative glucose disposal in individuals with dm under hyperinsulinemic conditions https://doi.org/10.14421/biomedich.2025.142.879-885 880 biology, medicine, & natural product chemistry 14 (2), 2025: 879-885 (adeva‐andany et al., 2017). importantly, experimental studies suggest that l-carnitine can reduce malformation rates, accelerate development, and improve larval survival rates of zebrafish embryos (ma et al., 2020) . despite these promising findings, the potential of lc to counteract glucose-induced embryotoxicity remains to be fully elucidated. in particular, whether lc can mitigate hyperglycaemia-associated defects in zebrafish embryos remains unknown. to address this gap, the present study was designed to investigate the protective role of lc against glucotoxicity in zebrafish embryos exposed to high glucose. specifically, we evaluated the effects of lc on embryo survival, hatching rate, developmental malformation, body length, and heart rate as key endpoints. material and methods materials d-glucose was obtained from himedia (lot no. 0000522073, maharashtra, india). l-carnitine in the form of l-carnitine tartrate (lclt) was purchased from norvine (georgia, usa). all other chemicals used were of analytical grade. zebrafish maintenance and embryo collection all husbandry and experimental procedures were approved by the health research ethics committee, faculty of medicine, universitas brawijaya, indonesia (no.98/ec/kepk/04/2025). adult wild-type zebrafish (danio rerio) were maintained at the laboratory of aquaculture, faculty of fisheries and marine sciences, universitas brawijaya (malang, indonesia). zebrafish were housed at 28 ± 0.5 °c under a 14 h light/10 h dark cycle. for spawning, male and female zebrafish were separated overnight in screened tanks and released together in the morning, as previously described (thompson et al., 2024; yoon et al., 2024). freshly fertilized eggs were collected in petri dishes containing e3 embryonic medium (5 mm nacl, 0.17 mm kcl, 0.33 mm cacl₂, 0.33 mm mgso₄, and 1× methylene blue) and subsequently transferred to culture plates for further treatment. embryotoxicity study an initial toxicity test was conducted to determine optimal glucose concentration and non-toxic lc dose ranges. fertilized eggs (~2 h post fertilization, hpf) were grouped into 6-well plates (two replicate per treatment group), each replicate consisted of five embryos. the control group was maintained in 3 ml of e3 medium. glucose-exposed groups were treated with 2%, 5%, or 10% d-glucose in e3, whereas lc groups were treated with 10, 50, or 100 µm lclt dissolved in e3. embryos were incubated at 28 °c under a 14 h light/10 h dark cycle until 144 hpf. mortality and hatching were recorded at 12 h intervals, while the medium was refreshed every 24 h. for the second series of experiment, we evaluated the effect of lc supplementation in addition to high glucose treatment in zebrafish embryo. the key developmental endpoints observed were survival, hatching rate, developmental malformation, body length, and heart rate. embryo survivability evaluation the zebrafish larvae were raised until death. early embryonic death was identified by loss of translucency and changes in coloration, while larval death was defined by the absence of heartbeat(westerfield, m., 2007). any dead embryo or larvae were discarded promptly. cumulative survival was analyzed and presented as kaplan–meier survival plots. embryo hatchability evaluation embryo hatching, which typically occurs between 48–72 hpf, was assessed visually at 12 h intervals. newly hatched larvae were counted, and cumulative hatching was expressed as the percentage of embryos hatched at each time point. embryo heart rate measurement larval heart rate was measured at 72 hpf. individual larvae were positioned laterally on glass slides and observed under a nikon eclipse si binocular microscope (japan) at 40× magnification. heartbeats were counted visually for 10 s and converted to beats per minute (bpm). measurements were repeated at least three times per treatment group to obtain average values. larval body length measurement larval body length was measured at 72 hpf. larvae were laid on lateral position microscope slides and photographed under a microscope. body length was defined as the distance from the anterior tip of the head to the end of the tail, following the spinal axis, and measured using imagej analysis software (hong et al., 2024). ten larvae per treatment group were analyzed, and mean body length was calculated. larval malformation observation larvae were visually examined at 24 h intervals under a nikon eclipse si microscope (40×) for morphological malformations. typical abnormalities recorded included pericardial edema, yolk sac edema, spinal curvature, and craniofacial or tail deformities (ma et al., 2020). statistical analysis data were expressed as mean ± standard deviation (sd) and analyzed using graphpad prism version 9.0 (boston, usa). differences among groups were assessed using one-way analysis of variance (anova), followed by tukey’s post hoc test. to evaluate the probability of survival and hatching in each treatment group, time-toevent data were analyzed using log-rank tests and fukata et al. – protective effects of l-carnitine on growth and cardiac function … 881 presented as kaplan–meier survival curves with 95% confidence intervals (ci). a p value < 0.05 was considered statistically significant. results and discussion effect of different glucose and lc concentrations on the survival rate and hatching rate of zebrafish embryos kaplan–meier survival analysis (figure 1) demonstrated that zebrafish embryos maintained in standard embryonic medium exhibited high survivability for up to 144 hpf. the log-rank test for trend analysis revealed significant difference between groups (p value=0.0363) (figure 1a). exposure to increasing glucose concentrations led to a dose-dependent reduction in survival, with embryos in 10% glucose showing complete lethality by 60 hpf (median survival time=42 h), and those in 2% and 5% glucose by 96 hpf (median survival time=72 h). in contrast, treatment with lc (50–100 μm) did not differ significantly from control. interestingly, treatment with 10 μm lc caused significant decrease in cumulative survival (log-rank test, p value=0.0076). additionally, glucose impaired zebrafish embryo hatching in a dose-dependent manner, with 10% glucose nearly abolishing hatching altogether. meanwhile, treatment with lc (10–100 μm) did not interfere with hatching, as lc-treated embryos reached hatching rates comparable to controls (median hatching time=60 h) (figure 1b). for these reasons, lc at 50 and 100 μm were chosen for the subsequent trial. figure 1. effect of glucose and l-carnitine on zebrafish embryo survival and hatching. (a) kaplan–meier survival curves and (b) cumulative hatching rates of zebrafish embryos exposed to different concentrations of glucose (2–10%) or l-carnitine (10–100 μm). data are presented as the percentage of embryos reaching the respective endpoint (death or hatched), obtained from two independent replicates (n=20 embryos per group). l-carnitine effect in high-glucose–exposed zebrafish embryos survivability and hatchability to evaluate whether l-carnitine (lc) supplementation could mitigate glucose-induced embryotoxicity, zebrafish embryos were exposed to 5% glucose with or without lc (50 or 100 μm). as shown in the kaplan meier plot (figure 2a), exposure to 5% glucose markedly reduced survival compared to controls (hr = 6.86, p < 0.0001). surprisingly, supplementation with 50 μm lc slightly worsened survival (hr = 2.65, p = 0.0363), while 100 μm lc did not significantly affect survival compared with glucose alone. similarly, cumulative hatching analysis revealed that 5% glucose delayed embryo hatching compared to controls (median hatching time [mht] = 84 vs. 60 hpf; hr = 0.17, 95% ci = 0.08–0.39, p < 0.0001). however, lc supplementation at either 50 or 100 μm failed to significantly improve hatching rates (figure 2b). these results indicate that lc supplementation at the tested concentrations did not rescue the reduced survival or impaired hatching induced by high-glucose exposure. 882 biology, medicine, & natural product chemistry 14 (2), 2025: 879-885 figure 2. effect of l-carnitine supplementation on survival and hatching of zebrafish embryos exposed to high glucose. (a) kaplan–meier survival curves and (b) cumulative hatching rates of embryos (figure 2) exposed to 5% glucose with or without l-carnitine (50 or 100 μm), compared with controls. data are expressed as percentages of embryos reaching the respective endpoint, with statistical analysis performed using log-rank tests (n = 30 embryos per group, two independent replicates). hr: hazard ratio; mst: median survival time; mht: median hatching time. effect of glucose and l-carnitine on zebrafish larval body length to assess the impact of glucose and lc treatment on zebrafish somatic growth, larval body length was measured at 72 hpf (figure 3). embryos exposed to 5% glucose exhibited a significant reduction in body length compared to controls (p < 0.0001). supplementation with both 50 μm and 100 μm l-carnitine partially attenuated the stunted growth, resulting in significantly longer larvae compared to the 5% glucose group (p < 0.05). figure 3. effect of glucose and l-carnitine on zebrafish larval body length at 72 hpf. picture was taken at 40x. data are presented as mean ± sd (n = 10 larvae per group). statistical significance was determined using one-way anova followed by post hoc. ****p < 0.0001; ***p < 0.001; *p < 0.05. scale bar = 50 μm. effect of glucose and l-carnitine on zebrafish larval heart rate to determine the impact of glucose and l-carnitine on cardiac function, heart rate was assessed in zebrafish larvae at 72 hpf. heartbeats were counted over 10-second intervals under a microscope and extrapolated to beats per minute (bpm) (figure 4). exposure to 5% glucose resulted in a significant reduction in heart rate compared to controls (p < 0.0001). co-treatment with 50 μm lcarnitine resulted in a modest but significant increase in heart rate compared to the glucose group (p < 0.05). notably, supplementation with 100 μm l-carnitine restored heart rate close to control levels (p < 0.001). these results indicate that high glucose impairs cardiac function and l-carnitine supplementation exhibited a dose-dependent protective effect. fukata et al. – protective effects of l-carnitine on growth and cardiac function … 883 figure 4. effect of glucose and l-carnitine on zebrafish heartbeat rate at 72 hpf. data are presented as mean with sd (n = 18 larvae per group). statistical significance was evaluated using one-way anova followed by post hoc tukey. ****p < 0.0001; ***p < 0.001; *p < 0.05. effect of glucose and l-carnitine on morphological malformations in zebrafish embryos morphological observation (figure 5) revealed that embryos in the control group developed normally without visible malformations at both 72 and 96 hpf. in contrast, exposure to 5% glucose induced multiple abnormalities, including yolk sac edema (yse), spinal curvature (sc), and hyperemia (hy) (figure 5). cotreatment with lc did not entirely prevent these defects, as yse and hy were still evident. however, supplementation with lc reduced the severity of malformations, with less severe spinal curvature and edema compared to glucose alone. figure 5. representative images of zebrafish embryos and larvae at 72 and 96 hpf under different treatment conditions (magnification 40x). yse: yolk sac edema, sc: spinal curvature, hy: hyperemia. scale bar = 50 μm. discussion in this study, we demonstrated that high glucose exposure induces marked developmental toxicity in zebrafish embryos, characterized by reduced survival, impaired hatching, morphological malformation, stunted growth, and bradycardia. a glucose concentration of 5% was employed in this study to simulate hyperglycemia. although this concentration is approximately 20-fold higher than the hyperglycaemic level observed in diabetic patients, it has been reported to increase free glucose level to ~19 mmol/l (342 mg/dl) comparable to those found in uncontrolled diabetic patients (singh et al., 2019; thompson et al., 2024). exposure to glucose decreased survival and hatching rates in a dose-dependent manner, with complete lethality observed at a 10% glucose concentrationby 60 hpf. in the present study, 5% glucose exposure resulted in 80% of larval mortality by 120 hpf (median survival of 96 hpf), which is significantly higher than prior reports showing only ~37.5% mortality at the same time point (singh et al., 2019). meanwhile, other study reported that 2.5% glucose exposure caused ~80% mortality rate at 120 hpf (li et al., 2022). such discrepancies may reflect strainor stock-related differences in susceptibility despite using the same species. nonetheless, our results corroborate the well-established concept that hyperglycemia is highly detrimental to embryonic and larval viability, as it is known to increase oxidative stress, metabolic disturbances, apoptosis, and impairs normal development in zebrafish which significantly reduce the survival rate(li et al., 2022; thompson et al., 2024). consistent with earlier findings, hyperglycemia also induced morphological abnormalities, including yolk sac edema, spinal curvature, and hyperemia. transcriptomic analyses further support these phenotypes, showing demonstrating that high glucose downregulates developmental signaling pathways including wnt, notch, foxo, ppar, and p53 (thompson et al., 2024). to our knowledge, studies evaluating the toxicity of lc in zebrafish embryo are scarce. in our study, lc at 50–100 μm was well tolerated, but did not affect survival or hatching, while 10 μm unexpectedly reduced survival. this contrasts with earlier findings, where at dose as low as 1 mg/l (~6.2 μm), lc promoted early hatching and improved survival compared to control (ma et al., 2020). however, in mat et al. study, the control group did not start hatching until 72 hpf, while in our study the embryos began hatching at 48 hpf. interestingly, the control group and lc at 0.5 mg/l in that study also showed relatively low survival rate, less than 50% at 60 hpf, while control group in our study maintain at 90% survival rate in the same time point. this may contribute to the overestimation of protective effect of lc supplementation in zebrafish embryo. additional evidence suggests that carnitine may exert biphasic or threshold-dependent effects: for example, acetyl-lcarnitine (alcar) at 1 mm increased atp and ros 884 biology, medicine, & natural product chemistry 14 (2), 2025: 879-885 production in zebrafish larvae(gu et al., 2021), a dose nearly 10-fold higher than our maximum concentration. however, that study did not report the mortality rate of the treatment group, despite stating that alcar did not pose any toxicity to zebrafish embryo. these findings suggest that both insufficient and excessive supplementation can disrupt metabolic balance, leading to adverse outcomes. in contrast to early-stage endpoints, our study results indicated that lc supplementation partially ameliorated later developmental endpoints. a significant reduction in heart rate was observed in glucose-treated larvae, supporting the notion that hyperglycaemia compromises cardiac development and function. co-treatment with lc restored heart rate in a dose-dependent manner, with 100 μm lc nearly normalizing the heart rate to the control levels. this cardioprotective effect is consistent with lc’s established role in enhancing fatty acid utilization and mitochondrial atp production, which are critical for sustaining cardiac contractility given the myocardium’s dependence on fatty acids as its primary energy substrate (longnus et al., 2001; ma et al., 2020; schulz, 1994). although some studies reported no effect of lc on zebrafish embryonic heart rate under short-term exposures (2-20h) (gu et al., 2021; kanungo et al., 2012), the longer treatment in our model may have been necessary to reveal its benefit under metabolic stress. similarly, lc supplementation attenuated growth impairment and reduced the severity of morphological abnormalities caused by high glucose exposure. these findings are consistent with previous reports, which show that lc improves body length and reduces deformity rates in zebrafish larvae (ma et al., 2020). mechanistically, these effects likely reflect lc’s role in sustaining mitochondrial β-oxidation, reducing oxidative stress, and facilitating the removal of toxic metabolic byproducts, thereby maintaining the supply of energy for highly-demanding physiological processes such as growth and cardiac function, which are often compromised during glucotoxic stress (virmani & cirulli, 2022). in summary, our findings demonstrate that while lcarnitine did not improve survival or hatching under high-glucose conditions, it conferred measurable protective effects on larval growth and cardiac function. this suggests that lc may be more effective in somatic growth and cardiac activity, rather than preventing early lethality caused by severe glucotoxic stress. further studies are warranted to optimize dosing strategies, explore synergistic antioxidant interventions, and investigate molecular mechanisms such as β-oxidation, igf-1 signaling, and ros scavenging that may underlie lc’s partial protective effects. conclusions lc supplementation at 50–100 μm was well tolerated, partially attenuating growth retardation and restoring heart rate in a dose-dependent manner. however, lc did not rescue survival, hatching rates, or morphological deformity under hyperglycaemic conditions, indicating that its protective capacity may be limited against severe glucotoxic stress. these findings suggest that lc has potential as a complementary agent to mitigate selected developmental complications of hyperglycaemia, but further studies are required to optimize dosing and elucidate underlying molecular mechanisms. acknowledgements: the authors sincerely acknowledge the medical department, faculty of medicine, universitas negeri malang, for providing the facilities and academic environment that supported the conduct of this research. the authors also thank the decentralization grant program, faculty of medicine, universitas negeri malang, for the funding support that enabled this study (21.2.52/un32/kp/2025). authors’ contributions: ef conceived and designed the study, supervised the experimental work, and finalized the manuscript. aw contributed to methodology development, performed data collection, and participated in data analysis. lws assisted in the experimental procedures, data interpretation, and manuscript drafting. ndp assisted in the research process and contributed to the preparation and submission of the manuscript. pkc assisted in the research process, figure preparation, and manuscript revision. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research was funded by the decentralization grant program, faculty of medicine, universitas negeri malang (21.2.52/un32/kp/2025). references adeva‐andany, m. m., calvo‐castro, i., fernández‐fernández, c., donapetry‐garcía, c., & pedre‐piñeiro, a. m. (2017). significance of l ‐carnitine for human health. iubmb life, 69(8), 578–594. https://doi.org/10.1002/iub.1646 capiotti, k. m., antonioli, r., kist, l. w., bogo, m. r., bonan, c. d., & da silva, r. s. (2014). persistent impaired glucose metabolism in a zebrafish hyperglycemia model. comparative biochemistry and physiology part b: biochemistry and molecular biology, 171, 58–65. https://doi.org/10.1016/j.cbpb.2014.03.005 chakraborty, c., sharma, a. r., sharma, g., & lee, s.-s. (2016). zebrafish: a complete animal model to enumerate the fukata et al. – protective effects of l-carnitine on growth and cardiac function … 885 nanoparticle toxicity. journal of nanobiotechnology, 14(1), 65. https://doi.org/10.1186/s12951-016-0217-6 gu, q., ali, s. f., & kanungo, j. (2021). effects of acetyl l‐carnitine on zebrafish embryos: phenotypic and gene expression studies. journal of applied toxicology, 41(2), 256– 264. https://doi.org/10.1002/jat.4041 hong, t., park, j., song, g., & lim, w. 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(2007). the zebrafish book: a guide for the laboratory use of zebrafish (danio rerio). https://cir.nii.ac.jp/crid/1370283694361132063 yoon, c.-y., chon, k., vasamsetti, b. m. k., hwang, s., park, k.h., & kyung, k. s. (2024). developmental toxicity and teratogenic effects of dicarboximide fungicide iprodione on zebrafish (danio rerio) embryos. fishes, 9(11), 425. https://doi.org/10.3390/fishes9110425 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 289-293 | doi: 10.14421/biomedich.2023.121.289-293 issn 2540-9328 (online) glut4 as a protein target for t2dm therapy with natural compounds vivi hendra sutandar1,*, mgs. irsan saleh2, ziske maritska3 1biomedical science master program; 2department of pharmacology; 3department of biology medicine, faculty of medicine, universitas sriwijaya jl. masjid al gazali, palembang, sumatera selatan 30128, tel. +62-11-58069, fax. +62-11-580664, indonesia. corresponding author* vivihendrasutandar@gmail.com manuscript received: 01 february, 2023. revision accepted: 11 march, 2023. published: 28 march, 2023. abstract who reported 1.9 million death cases of diabetes patients in 2019. diabetes is caused by damage in the pancreas which resulted in a lack of insulin or insulin resistance. medication for t2dm mainly focuses on lowering blood glucose and treating affected organs. current medications are still lacking, thus research is needed in finding novel medications to accommodate t2dm. this paper aims to present the current research on potential plant extract in increasing glut4 translocation in diabetes conditions. insulin resistance state affecting glut4 translocation which is important in affecting glucose uptake. some research shows that plant extract proved to be potential in increasing the translocation of glut4 and helping lowering blood glucose levels. keywords: diabetes mellitus; t2dm; glut4; extract. introduction diabetes is a chronic disease either caused by damage to the pancreas which resulted in a decreased production of insulin or the body's unable to use available insulin. diabetes is a public health problem1. there is an increase in mortality rate by 3% caused by diabetes between 2000 and 2019, while in lower-middle-income countries the number increased to 13% mortality rate (who, 2022). type 2 diabetes (t2dm) is a condition characterized by the presence of insulin resistance and damage to the cell  pancreas. insulin resistance is caused by several factors such as high lipid, increased inflammation signal, and activation of stress pathway in reticulum endoplasma. damage in cells  pancreas prompt a decrease in pancreas function that caused dysfunction causing insulin deficiency (muoio and newgard, 2008). several oral medicines are regularly used by t2dm especially sulfonylurea and metformin, alongside some drugs which targeted the affected organ of diabetes. sulfonylurea and thiazolidinedione each have roles in increasing insulin secretion and the latter in increasing insulin sensitivity (perkeni, 2021). nowadays, t2dm medication focuses on the use of metformin and a healthier lifestyle. the use of second-line and third-line drugs helps improve the condition of t2dm patients. however, current medications are still lacking because of the presence of side effects. metformin in a contraindicative individual could increase the risk of lactic acidosis including liver disease, kidney disorder, and lung disease. thus, there is room for improvement in the research in finding novel medications for t2dm to overcome the shortcomings of current drugs (he et al., 2015). until recently, society use plants as a traditional medicine to help treat illness (rahayu et al., 2006). glut (glucose transporter) is a transporter that plays roles in molecules exchanges through membrane cells with (quistgaard et al., 2016) each type has a unique and specific affinity towards its substrates, distribution, location, mechanisms, and physiological function (navale and paranjape, 2016). glut4 is typically found in skeletal muscle and adipose tissue. insulin-induced glucose transport within fat and muscle tissue by stimulating glut4 increased glucose uptake (pessin et al., 1999; saltiel and kahn, 2001). in the insulin resistance state, insulin is unable to increase the translocation of glut4 to the membrane and caused accumulation of glut4 in the membrane compartment. thus, insulin resistance involves a defect in glut4 traffic (garvey et al., 1998). glut4 may show potential as a protein target for t2dm medication due to their roles in regulating glucose uptake by depending on insulin which is present in t2dm patients. traditional medication may prove to be potential in targeting glut4 due to their nature of natural products thus safe to use. several natural compounds derived from plants show their potential in targeting the activation of glut4. this paper's objective https://doi.org/10.14421/biomedich.2023.121.289-293 290 biology, medicine, & natural product chemistry 12 (1), 2023: 289-293 is to present the current research focusing on the potential of plant extract in increasing the activity of glut4 translocation to the membrane with the condition of diabetes. glut4 glucose act as a source of energy for the most organism. glucose characteristics are polar and have large molecule sizes so it cannot pass cell membrane lipids through diffusion. glucose molecules pass the membrane cell with the help of a structural transport protein family known as glucose transporter. two main kinds of glucose transporter have already been identified which are sodium-glucose linked transporters (sglts) and facilitated diffusion glucose transporters (gluts). glut protein consists of 12 membrane-spanning regions with amino acid chains and carboxyl. based on its amino acid sequences and several studies of its sequence alignment found three subclasses of facilitative transporter have been discovered (navale and paranjape, 2016). glut4 fall in class 1 glucose transporter which is insulin-responsive transporter, found in the heart, skeletal muscle, adipose tissue, and brain. glut4 can be found in the vesicle inside the cytoplasm and would be translocated to the membrane after insulin binds to the insulin receptor. after binding, glut4 activation increased 10-20 folds (bryant et al., 2002). glut4 structure is built with a unique sequence of n-terminal and cooh-terminal that play roles in responsibility towards insulin signal and membrane traffic. glucose transfer along the membrane occurs via the glut4 mechanism through facilitated diffusion atp-independent. after glucose influx into the cell, glucose would then be metabolized as energy or lipid synthesis or stored as glycogen (huang and czech, 2007). the translocation of glut4 from the intracellular domain to the plasma membrane would occur after stimulation. during the unavailability of insulin or exercise, 90% of glut4 would be stored inside intracellular. if there is the availability of insulin or exercise hence the vesicle storing glut4 undergoes exocytosis toward the plasma membrane, sarcolemma, and t-tubules of skeletal muscle and plays its role in transporting glucose (shepherd and kahn, 1999). the increase of glut4 molecules on the surface of the membrane would accelerate the rate of glucose transport inside the cell. however, in the condition without stimulation from insulin, glut4 would undergo endocytosis back inside the cell through the process of budding of vesicles on the plasma membrane which contained clathrin, and be stored back inside intracellular vesicles (bryant et al., 2002; shepherd and kahn, 1999). activation of glut4 on skeletal muscle begins with insulin binding to the surface receptor which is a  subunit insulin receptor consisting of a tyrosine kinase domain that would autophosphorylate. phosphoryla  subunit then triggers irs and phosphorylates irs. after irs is phosphorylated then it would bind and activate pi3k and transfer toward the plasma membrane and convert pip2 become pip3. then, after pip3-dependent protein kinase has been activated, it would phosphorylate and activate akt (pkb). akt activation leads to vesicle fusion which involves the translocation of vesicles containing glut4 from the intracellular compartment to the plasma membrane. glut4 elevation on the plasma membrane causes an increase in glucose uptake into cell 15. potential plant bioactive compounds recent researches initiated investigations on plant extract in enhancing glut4 translocation in dm conditions. the table below presents the summary of each article. table 1. summary of study in glut4 activation with various plant extract. study plant extract contents length of follow up outcome yamashita et al., 2012 japan cacao liquor procyanidin extract epicatechin, catechin, and other procyanidins l6 myoblast icr and c57bl/6 male mice (4 weeks old) 7 days of treatment methods: western blot (wb) increased glucose uptake and promote glut4 translocation in l6 myotubes. suppressed hyperglycaemic response after carbohydrate ingestion and enhanced glut4 in mice skeletal muscle. lv et al., 2020 china lipophilic extract from flowers of wisteria sinensis na l6 irap-morange and male c57bl/6j mice (8 weeks old) 4 weeks of treatment methods: wb strongly increased glucose uptake to 2.0 fold compared to normal control and increased glut4 expression while stimulating akt in l6 myotubes in mice, treatment ameliorated hyperglycemia, insulin resistance, and dyslipidemia. also, increased glut4 expression by akt activation in white adipose tissue and skeletal muscle. sutandar et al. – glut4 as a protein target for t2dm therapy … 291 table 1. cont. study plant extract contents length of follow up outcome zhao et al., 2018 china dandelion chloroform extract (dce) na l6 irap-morange methods: wb and realtime pcr dce increased glut4 translocation and expression through the ampk pathway, glut4 fusion to the plasma membrane (pm) independent of ca2+. feng et al., 2022 taiwan anthocyanin-rich extract from black rice (oryza sativa l.) anthocyanin c2c12 myotubes methods: wb hualien and changhua black rice (hbre and cbre) promote glucose uptake. cbre did not affect irs-1 downstream but enhanced pampk/ampk. hbre target pi3k/akt and p38 mapk/erk which both promote glut4 glucose uptake. naowaboot et al., 2012 thailand mulberry (morus alba l.) leaf extract 1deoxynojirimycin (dnj), gallic acid, quercetin. male sprague-dawley rats 6 weeks of treatment. methods: wb increased glucose uptake, and enhanced glut4 translocation while mediated via pi3k signalling pathway. park et al., 2018 korea portulaca oleracea l. extract (poe) flavonoids and triterpenoids (data not shown) mouse 3t3-l1 preadipocyte cells methods: wb poe enhances glucose uptake, by increasing glut4 on pm. increase of pm-glut4 associated with irs-1 phosphorylation, pi3k activation, and akt phosphorylation. pi3k inhibitor and ampk inhibitor inhibit glucose uptake on poe. pujimulyani et al., 2020 indonesia white saffron rhizomes curcuma mangga val. ethanol extract (eecm) curcumin, gallic acid, catechin, epicatechin, epigallocatechin, epigallocatechin gallate, and gallocatechin gallate. 3t3-l1 fibroblastsderived adipocyte cells methods: rt-qpcr eecm increased glucose uptake and glut4 mrna expression. wulansari et al., 2017 indonesia papaya seed extract (carica papaya linn.) na white wistar strain rats 2 weeks of treatment methods: ihc fasting blood glucose (fbg) decrease and increase of glut4 expression based on skeletal muscle staining with ihc after extract treatment zhang et al., 2018 china semen cassia (cassia obtusifolia l.) extract (sce) anthraquinones male sprague-dawley rats 1.30 days of treatment (sce on normal rats) 2.5 weeks of treatment (sce on diabetic rats) methods: wb fbg and serum lipids decrease, while oral glucose tolerance (ogt), and fasting serum insulin (fsi) increase after sce. sce restored the low expression of glut4 translocation in diabetic rats. sce increased thr642, ser473 and pi3k in skeletal muscle lipophilic extract from flowers of wisteria sinensis is the potential as an antidiabetic compound, due to its activity in stimulating glucose uptake by activating protein akt (protein kinase b) and increasing glut4 translocation on l6 cells in vitro. in vivo, the extract increases glut4 expression and helps ameliorate t2dm symptoms in t2dm mice from hyperglycemia to pancreatic islet destruction (lv et al., 2020). cacao liquor procyanidin (clpr) extract is the potential to increase glut4 translocation and glucose uptake in skeletal muscle, due to their content such as epicatechin, catechin, and other procyanidins. clpr help suppressed hyperglycemia response in mice after carbohydrate ingestion while enhancing glut4 translocation in skeletal muscle, due to their beneficial potential clpr help improves glucose tolerance (yamashita et al., 2012). chloroform extract from the dandelion (dce) plant may be another potential extract to help increase glut4 translocation. the research was performed in vitro using l6 cells and the protein was measured by western blot. the result shows that dce upregulated glut4 expression which leads to an increase in glut4 translocation to the membrane, hence is instrumental to the enhancement of glucose uptake to the cells (zhao et al., 2018). anthocyanin-rich extract from black rice (oryza sativa l.) extracted with ethanol from hualien (hbre) and changhua (cbre) region help promotes glucose uptake in c2c12 myotubes by increasing glut4 292 biology, medicine, & natural product chemistry 12 (1), 2023: 289-293 expression and phosphorylation of irs-1. cbre increased p-ampk/ampk while hbre involves in pi3k/akt and mapk/erk (feng et al., 2022). phenolic compounds from mulberry (morus alba l.) leaf extract such as gallic acid help improves glucose uptake and enhanced glut4 translocation thus may be potential as antihyperglycemic. mulberry leaf extract help enhances glut4 translocation by mediating through the pi3k pathway due to glucose uptake getting inhibited by pi3k inhibitor (in this paper used wortmannin). (naowaboot et al., 2012). portulaca oleracea extract (poe) helps enhance glucose uptake by increasing glut4 expression. while enhancing glut4 translocation, poe helps the activation of irs-1, pi3k, and akt phosphorylation. although poe did not involve in protein kinase c phosphorylation, poe involves in ampk pathway activation (park et al., 2018). curcuma manga val. rhizomes ethanol extract (eecm) can increase glucose uptake in lipid-laden 3t3l1 cells, and increase glut4 mrna expression (pujimulyani et al., 2020). papaya seed extract (carica papaya linn.) had a significant influence on glut4 expression in the skeletal muscle of diabetic mice, measured by immunoreactive score and immunohistochemical staining (wulansari et al., 2017). total anthraquinone from semen cassia (cassia obtusifolia l.) extract (sce) was evaluated for its action in improving glucose metabolism in diabetic rats. the result shows that sce enhances glut4 translocation while increasing the expression of phosphorylatedas160 (thr642), phosphorylated-akt (ser473), and pi3k in skeletal muscle. sce help promotes glut4 translocation due to the involvement in pi3k-akt-as160 signaling pathway activation (zhang et al., 2018). conclusion glut4 plays a role in glucose uptake and affects blood glucose levels. t2dm patients' condition is mostly caused by insulin resistance, with the condition of hyperglycemia. glut4 may be a potential protein target to help cope with the symptom of hyperglycemia. various beneficial plants show their ability in increasing glut4 expression on pm through activation either with pi3k or ampk signaling pathway, which contributed to the increase of glucose uptake in vivo or in vitro. based on the research mentioned above, research in finding precise extract and investigation to its exact mechanism pathway in promoting glut4 translocation is needed to discover the best potential plants to target the activation of glut4 in t2dm conditions. competing interests: the authors declare that there are no competing interests. references bryant, n. j., govers, r., & james, d. e. 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(2018). dandelion chloroform extract promotes glucose uptake via the ampk/glut4 pathway in l6 cells. evidence-based complementary and alternative medicine, 2018. https://doi.org/10.1155/2018/1709587 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 193-197 | doi: 10.14421/biomedich.2024.131.193-197 issn 2540-9328 (online) isolation of β-cyclodextrin gycosyl transferase (β-cgtase) producing bacteria from potato plantation soil helman kurniadi1,*, desi sagita2, barmi hartesi3 1,2pharmacy department; adiwangsa jambi university, jl. r. wijaya, the hok, jambi sel., jambi 36125, indonesia. 3faculty of pharmacy, jenderal achmad yani university, jl. ters jend sudirman, cimahi, jawa barat 40633, indonesia. corresponding author* helmankurniadi@gmail.com abstract cyclodextrin glycosyltransferase (cgtase) is an extracellular enzyme produced by several microorganisms, particularly bacteria, that converts starch into cyclodextrin (cd). cd has various applications in fields such as food, textiles, pharmaceuticals, chemistry, and biotechnology. the objective of this study was to isolate β-cgtase-producing bacteria from soil in a potato plantation located in kerinci, and to conduct their characterization. screening was conducted using horikoshi agar media with phenolphthalein and methyl orange color indicators. the results indicated that one isolate, coded ck-2, produced the optimum β-cgtase enzyme when incubated for 30 hours at 37 ºc and ph 10. the cgtase enzyme converted starch to cd when incubated for 60 minutes at 37 ºc and ph 7. the ck-2 isolate was identified as monobacilli gram-positive bacteria, having spores, positive in the catalase test, and motile. the study concluded that the bacteria producing β-cgtase enzyme are found in the soil of potato plantations in kerinci, which is believed to originate from the genus bacillus sp. keywords: ck-2 isolate; cyclodextrin (cd); cyclodextrin glycosyltransferase (cgtase); soil. abbreviations: cd: cyclodextrin; cgtase: cyclodextrin glycosyltransferase. introduction cyclodextrin glycosyl transferase, which is classified with cgtase number e.c 2.4.1.19, is a bacterial enzyme belonging to the r-amylase family that has the ability to convert starch into cyclodextrin (jeang et al., 2005). the cgtase enzyme can synthesize starch to form three main cyclodextrin compounds, namely α-cd, β-cd, and γ-cd (kesaulya, 2015). this enzyme converts starch to cyclodextrin (cd) through the transglycosylation and starch hydrolysis pathway. the resulting cd is a glucose residue compound bound by α-1,4 glycosidic to form cyclic oligosaccharides (kamble & gupte, 2015). cyclodextrin is a cup-shaped molecule with a cavity in the middle that is hydrophobic on the inside and hydrophilic on the outside. the specialty of cyclodextrins lies in their ring structure, which has the ability to enclose guest molecules in the cyclodextrin cavity. this can be applied in several ways, including modifying the physicochemical properties of molecules such as stability, solubility, bioavailability, conjugate preparation, and the bonding of several polymers (duchene, 2011; jansook et al, 2018; poulson et al. 2022). the formation of cyclodextrins must be in accordance with the environmental conditions and reactions needed for growth and reproduction so that some cgtaseproducing bacteria can form three cyclodextrin molecules, namely α-cd with 6 glucose molecules, βcd with 7 glucose molecules, and γ-cd with 8 glucose molecules (kesaulya, 2015). due to the difference in the number of molecules that make up each cyclodextrin, the main difference between them is the size of the nonpolar cavity and their solubility in water (koo et al., 2017; braga, 2019). research conducted by higuti et al. (2003) concluded that there are 75 strains of cgtase-producing bacteria that exist in various types of soil. based on the summary of qi & zimmermann (2005), cgtase-producing bacterial isolates are currently known to produce the most β-cd. miftahurrohmah & moordiani (2014) found that the cgtase-producing js-1 isolate from soil in jatinangor was a gram-positive bacterial isolate that was bacillus-shaped, motile, facultative anaerobic, and had a positive catalase test, in line with research by rostinawati & lestari (2017) showing that cgtase was produced by the bacteria bacillus sp. the aim of this research is to obtain isolates of bacteria producing the enzyme β-cgtase from potato plantation soil in kerinci manuscript received: 03 march, 2024. revision accepted: 20 may, 2024. published: 04 june, 2024. https://doi.org/10.14421/biomedich.2024.131.193-197 194 biology, medicine, & natural product chemistry 13 (1), 2024: 193-197 and identify isolates producing β-cgtase based on biochemical characteristics. materials and methods this research was conducted at the pharmaceutical microbiology laboratory, harapan ibu jambi college of health sciences from april 2019 to july 2019. the samples examined in this research were soil samples taken from the potato plantations, specifically the soil closest to the potato tubers. a 1-gram sample was taken at one point and diluted into 9 ml of sterile distilled water to create a dilution of 10-8. then, 100 µl of suspension from dilutions 10-2 to 10-8 was poured into solid horikoshi medium and incubated for 24 hours at 37 ºc. the presence of a yellow hollow zone around the growing colony indicated the ability of the bacteria to produce the enzyme β-cgtase. the colonies were then identified using macroscopic, microscopic, and biochemical tests, and inoculated into liquid horikoshi media to measure cgtase activity. cgtase activity was measured using spectrophotometry, which involved observing the absorbance of the color indicator bound by cyclodextrin to create a curve of cyclodextrin concentration against the incubation time of the cgtase enzyme. absorbance was measured by inserting a color indicator compound into the cyclodextrin to form an inclusion. the measurement is carried out on the color indicator captured by the cyclodextrin, which was produced from the cgtase enzyme. the more cyclodextrin present, the more compound was captured, resulting in a smaller absorbance value of the color indicator. the percent decrease in absorbance of the color indicator was then correlated to the cgtase incubation time using the following formula: percent reduction in phenolphthalein (%) u/ml= r0−r r × 100% r0 : control absorbance (time 0) r : sample absorbance results and discussion result screening of isolates producing the β-cgtase enzyme the isolate successfully obtained from potato plantation soil, which exhibited β-cgtase activity, was derived from a soil dilution of 10-2. a yellow hollow zone was observed on the β-horikoshi media, and this isolate was designated as ck-2. the cgtase activity secreted by the ck-2 isolate in the β-horikoshi medium is illustrated in figure 1. figure 1. horikoshi agar as control (a), horikoshi agar after ck-2 inoculation (b) isolate identification macroscopically, the colonies of the cgtase-producing isolate that grew on β-horikoshi agar had a round shape with a whitish-yellow color, flat edges, and a flat elevation. microscopically, identification and characterization were carried out through several tests: biochemical test biochemical tests were conducted using imvic media, including sim media, veges paskeuer, methyl red, simon citrate & triple sugar iron agar, and sugar media. the results of the isolate identification can be seen in table 1. table 1. results of identification of ck-2 isolates. identification results sulfur indol motility (-),(-),(+) methyl red (+) veges paskeuer (-) triple sugar iron agar k/k simon citrate (-) glukosa (-) dextrosa (-) mannitol (-) sucrosa (-) lactosa (-) gram (+) spora (+) katalase (+) kurniadi et al. – isolation of β-cyclodextrin gycosyl transferase (β-cgtase) 195 gram staining and endospores in gram staining, rod-shaped bacteria are purple in color, indicating that they were gram-positive. the bacteria had subterminal spores because they had a green dot on 1/4 of the cell. the bacteria also produce the enzyme catalase. figure 2. result of gram staining under light microscope with magnification 1000x. characterization of the incubation time of enzymes on cgtase activity the characterization of enzyme incubation time was conducted to determine the optimal time for the enzyme to catalyze starch and form cd. according to the results of the characterization, as shown in figure 3 (a), the 60minute incubation time exhibited the lowest phenolphthalein absorbance, indicating that this is the optimal time for cgtase activity in producing cd. preparation of a growth curve for isolate ck-2 in the growth curve, the results of the od600 measurement shown in figure 3 (b) indicate that the isolate experienced a log phase starting from the 12th hour, followed by a stationary phase at the 24th hour. subsequently, the death phase was observed from the 30th to the 72nd hour of incubation. characterization of isolates in β-cd production the best time for an isolate to produce the βcgtase enzyme was seen based on incubation of the ck-2 isolate at a temperature of 37 ºc. isolate ck-2 secreted the cgtase enzyme maximally after 30 hours of incubation. in figure 3 (c), it can be seen that the phenolphthalein absorbance was the smallest at 30 hours of incubation, followed by the 36th hour of incubation. figure 3 (d) illustrates that isolate ck-2 secreted the maximum amount of cgtase at the 30-hour mark, as indicated by a percent decrease in phenolphthalein absorbance reaching 98.39%. figure 3. (a) decreased phenolphthalein absorbance corresponds to cd formation. (b) growth curve of isolate ck-2. (c) phenolphthalein absorbance over incubation time. (d) the percentage decrease in phenolphthalein absorbance is directly proportional to β-cd formation. 0,00 0,05 0,10 0,15 10 30 60 90 p h e n o lp h th al e in ab so rb an ce ( n m ) incubation time (minutes) 0 0,5 1 1,5 2 2,5 0 4 8 12 18 24 30 36 42 48 72 a b so rb an ce o d 6 0 0 ( n m ) incubation time (hours) 0 0,2 0,4 0,6 0,8 1 0 4 8 12 18 24 30 36 42 48 72a b so rb an f e n o lf ta le in (n m ) incubation time (hours) 0 50 100 150 4 8 12 18 24 30 36 42 48 72 d e cr e as e in p h e n o lp h th al e in ab so rb an ce ( % ) incubation time (hours) a c b d 196 biology, medicine, & natural product chemistry 13 (1), 2024: 193-197 discussion screening and identification of isolates an isolate of bacteria producing the cgtase enzyme has been successfully isolated from potato plantation soil in the kerinci area. this isolate exhibits a yellow hollow zone on horikoshi media. the formation of this yellow hollow zone is attributed to the isolate's capability to produce the enzyme β-cgtase, which converts starch into the compound β-cd. consequently, this compound binds to phenolphthalein, causing the disappearance of the reddish-purple color of phenolphthalein around the colony. based on the results of biochemical identification of bacteria, it is suspected that the ck-2 isolate comes from the genus bacillus (kiranmayee rao & m. lakshmi, 2007). this identification has several advantages, namely that it is easy to carry out and costs less. however, it has the disadvantage of a lower level of accuracy due to several types of species that exhibit nearly identical biochemical metabolic results. therefore, further molecular identification of the ck-2 isolate is necessary. this aligns with several local studies that have identified bacillus as cgtase-producing bacteria, including bacillus sp bk-1 (yunianto et al., 2000), bacillus sp bgg-1 (perbadi, 2001), bacillus sp lt1b and pt2b (sagita, 2011), as well as bacillus sp bv-3 (vionita, 2017). characterization of enzyme incubation time on cgtase activity this characterization was carried out in order to obtain the optimum incubation time for the enzyme that reacts with starch to produce β-cd. figure 3 (a) shows that the best time for the enzyme to react with starch to produce cd is at 60 minutes of incubation. in the test carried out by rostinawati et al (2016), the incubation time for cgtase with starch was 30 minutes but with the starch chain breaking first by gelatinization. this research also did not carry out enzyme purification so that a mixture of substances originating from the media still remained. it is possible that the incubation time was longer in this study due to the need for additional time for the reaction to break the starch structure and the disruption of the bond between the enzyme and starch by residual substances from the media. ▪ making an isolate growth curve in the growth curve of the ck-2 isolate, it is known that from the initial time of incubation to the 12th incubation hour there is a lag phase, namely the time when the isolate adapts and is then able to reproduce. this is different from research conducted by kamble & gupte (2014) which is known to be the lag phase of bacillus. producing cgtase at 0 hours to 8 hours of incubation, this difference is probably caused because the ck-2 isolate takes longer to adapt to the ph 10 liquid horikoshi medium, while the isolate used by kamble & gupte (2014) is an isolate that has been proper ph optimization for growth. next, the ck-2 isolate experienced a log phase starting from the 12th hour of incubation to the 24th hour of incubation, indicating that the ck-2 isolate reproduced maximally during that incubation hour. in line with the difference in the lag phase with the research of kamble & gupte (2014), there was also a difference in the log phase. in this study, the log phase was obtained for 22 hours of incubation, namely from the 8th hour to the 30th hour of incubation. this can be caused by adjustments to the nutrient concentration that they make, such as optimizing the concentration of peptone, yeast extract and starch so that the possibility of bacterial growth can last longer. meanwhile, the stationary phase in this study started at the 24th incubation hour until before the 30th incubation hour, this shows a state of balance between the growth rate and death rate of bacteria at that time and is in line with the reduction in nutrients in the media, so the number of deaths continues to increase and the growth rate continues to decrease, this occurs at the 30th to 72nd incubation hours. this phase is also different from the research of kamble & gupte (2014) which concluded that the stationary phase from their growth curve measurements started from the 30th incubation hour to the 176th incubation hour, while the death phase is known to occur later, this difference is probably caused by the addition of nutrients. in their media, the starch used was tapioca starch, the addition of 0.2% ammonium dihydrogen phosphate and a difference in magnesium sulfate concentration of 0.12%. ▪ characterization of isolates in β-cd production the characterization of isolates in the production of βcd, as depicted in figure 3 (c), reveals that the 30th hour of incubation exhibits the lowest phenolphthalein absorbance value. this observation suggests that this specific incubation time is optimal for bacteria to produce β-cd, resulting in a significant 98.39% increase in β-cd formation, illustrated in figure 3 (d). furthermore, the 30th hour of incubation marks the conclusion of the stationary phase, indicating a correlation between β-cgtase production and this phase. specifically, the formation of β-cgtase escalates concomitantly with the conclusion of the stationary phase. conclusions bacteria producing β-cgtase, named isolate ck-2, have been found. based on biochemical identification, the ck2 isolate is a gram positive bacterium in the form of a bacillus, facultative anaerobe, motile and positive in the catalase test. this isolate is thought to be a bacterium from the genus bacillus. the cgtase enzyme secreted by the ck-2 isolate has optimum cyclization activity in kurniadi et al. – isolation of β-cyclodextrin gycosyl transferase (β-cgtase) 197 incubation time 60 minutes at 37 ºc. this bacterium is one of the cgtase-producing bacteria which can produce the maximum cgtase enzyme at the 30th hour of incubation at a temperature of 37 ºc. authors’ contributions: helman kurniadi, desi sagita, and barmi hartesi designed the research, carried out laboratory work, and analyzed the data. soil sampling was carried out by helman kurniadi. competing interests: regarding competing interests, it is important to note that there was no conflict of interest observed in this study. funding: it is worth mentioning that no party provided cost support for this research. instead, the study was carried out at the researchers' own expense, demonstrating their dedication and commitment to advancing scientific knowledge in their field. references braga s. (2019) cyclodextrins: emerging medicines of the new millennium. biomolecules. nov 28;9(12):801. doi: 10.3390/biom9120801. pmid: 31795222; pmcid: pmc6995511. duchene, d. (2011), cyclodextrins: history, properties, applications, and current status. cyclodextrins and their inclusion complexes. centre national de la recherche scientifique. 8612 higuti, ilma hiroko., grande, simone wichert., sacco, roberta., nascimento, aguinaldo. (2003). isolation of alkalophilic cgtase-producing bacteria and characterization of cyclodextrin-glycosyltransferase. brazilian archives of biology and technology an international journal. vol.46, issn 1516-8913 jansook, phatsawee., ogawa, noriko., loftsson, thorsteinn. (2018). cyclodextrins: structure, physicochemical properties and pharmaceutical applications. international journal of pharmaceutics, volume 535, issues 1–2, 2018, pages 272-284, issn 0378-5173, https://doi.org/10.1016/j.ijpharm.2017.11.018. jeang c.l,. lin d.g,. hsieh s.h. (2005). characterization of cyclodextrin glycosyltransferase of the same gene expressed from bacillus macerans, bacillus subtilis, and escherichia coli. journal of agricultural and food chemistry. vol. 53, no. 16. kamble, r,. gupte. a. (2015). cyclodextrin glycosyltransferase production by alkaliphilic bacillus sp. isolated from rice cultivated soil and media optimization using taguchi method. international journal off pharmacetical and research. e-issn: 0975-8232; p-issn: 2320-5148 kesaulya, henry. (2015). isolation and physiological characterization of pgpr from potato plant rhizosphere in medium land of buru island. procedia food science. volume 3, page 190 – 199 koo, ye-seul., ko, dam-seul., jeong, da-woon., shim, jaehoon. (2017). development and application of cyclodextrin hydrolyzing mutant enzyme which hydrolyzes βand γ-cd selectively. journal agricultural and food chemistry. doi: 10.1021/acs.jafc.7b00269 miftahurrohmah, nur., moordiani. (2014). characterization of js1 isolate, an alkalophylic bacteria as cyclodextrin glycosyltransferase (cgtase) producer from sumedang, west java. jurnal ilmu kefarmasian indonesia. vol. 12, no. 2, hal. 216-221 poulson, b.g.; alsulami, q.a.; sharfalddin, a.; el agammy, e.f.; mouffouk, f.; emwas, a.-h.; jaremko, l.; jaremko, m. (2022). cyclodextrins: structural, chemical, and physical properties, and applications. polysaccharides, 3, 1–31. https://doi.org/10.3390/ polysaccharides3010001 qi, qhinsheng., zimmermann, wolfgang. (2004). cyclodextrin glucanotransferase: from gene to applications. applied microbiology and biotechnology. volume 66, page 475-485 rostinawati, tina., lestari, hilda shinta. (2017). skrining bakteri penghasil enzim β-siklodekstrin glukosil transferase (βcgtase) dari tanah jatinangor. jurnal farmasi, sains, dan kesehatan. volume 3, no. 2, hal. 1-8 rao, kiranmayee., lakshmi m. (2007). alkaline protease from bacillus firmus 7728. african journal of biotechnology. issn 1684–5315 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 671-674 | doi: 10.14421/biomedich.2023.122.671-674 issn 2540-9328 (online) an overview of the total leukocyte count in pulmonary tuberculosis patients at rsud gambiran kediri city devi nor fitri, lisa savitri*, rochmad krissanjaya, elfred rinaldo kasimo department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 26 july, 2023. revision accepted: 11 september, 2023. published: 13 march, 2024. abstract the leukocyte count examination in tuberculosis infection serves to monitor the inflammation caused by mycobacterium tuberculosis. an increasing leukocyte count can lead to complications such as pleuritis. the purpose of this study was to determine the total leukocyte count of pulmonary tuberculosis patients at rsud gambiran, kediri city, based on gender and age. this research was a descriptive retrospective study. data analysis was conducted using microsoft excel and presented in the form of tables and diagrams. the results of this study showed that pulmonary tuberculosis infection was more prevalent in males, with 26 individuals (62%) affected. in terms of age groups, the highest infection rate was in the 21-60 years age range, with 30 individuals (71%) affected. as for leukocyte count elevation (leukocytosis), it was more commonly observed in males with a count of 14.57 µl. in terms of age groups, leukocytosis was frequently observed in the 21-60 years age range, with a count of 48.66 µl. out of the 42 samples, 19 individuals had a normal leukocyte count, while the remaining 23 individuals experienced leukocytosis. from the results of the study, it can be concluded that not all pulmonary tuberculosis patients experience leukocytosis; some patients maintain a normal leukocyte count. keywords: total leukocyte count; pulmonary tuberculosis; rsud gambiran. abbreviations: tuberculosis (tb), helper t cells (th1), interferon-gamma (ifny) introduction tuberculosis (tb) is a chronic and contagious infection caused by mycobacterium tuberculosis (mutiara, 2020). the transmission of tb bacteria occurs through the air, as it is an airborne disease. it spreads from an infected person to another person through droplets of saliva or mucus when the infected person coughs, sneezes, speaks, or laughs (mursyaf, 2018). tb is the leading cause of abnormalities in the lungs and extrapulmonary organs and has been one of the deadliest diseases in history compared to other illnesses (nurkumalasari et al., 2016). the diagnosis of tb is based on physical examination, bacteriological tests, and hematological examinations (hasnawati, 2018). hematological examinations include the examination of red blood cells (erythrocytes), platelets (thrombocytes), total leukocyte count (white blood cells), and the differential count of leukocytes (neutrophils, lymphocytes, monocytes, basophils, and eosinophils) using a hematology analyzer (sundari, 2017). when m. tuberculosis enters the body, it causes inflammation. this is due to the body's natural response to trauma, allergies, and bacterial infections, leading to the release of proinflammatory cytokines, which is marked by an increase in leukocyte count (leukocytosis) (bili, 2017). leukocytes play a role in the body's defense system or immunity against infectious diseases by phagocytosis and antibody production (niagita, 2019). leukocyte count tends to increase in patients with pulmonary tb due to inflammation, while a normal leukocyte count indicates that the patient has a good immune system and adequate nutrition to fight tb bacterial infections. the total leukocyte count examination aims to detect increases or decreases in leukocyte numbers in the blood (rosita, 2019). additionally, this examination can help diagnose organ damage caused by bacterial or viral pathogens and provide information about the progression of immune deficiency diseases. in severe cases of pulmonary tb infection, complications may arise if not promptly treated, and one common complication is pleuritis or pleural effusion (pratiwi, 2018). however, not all patients with pulmonary tb experience leukocytosis. based on previous research conducted by dicky & ahmad (2019), where 20 patients with pulmonary tb showed increased leukocyte count while 30 patients had https://doi.org/10.14421/biomedich.2023.122.671-674 672 biology, medicine, & natural product chemistry 12 (2), 2023: 671-674 a normal leukocyte count, the researchers wanted to investigate and conduct a study with the title "overview of total leukocyte count in pulmonary tuberculosis patients at rsud gambiran kediri city." materials and methods in this study, a descriptive retrospective method was used. the sampling technique employed was purposive sampling, which included all pulmonary tuberculosis patients at rsud gambiran, kediri city, during the period from january to december 2021, based on inclusion and exclusion criteria. the inclusion criteria were patients with pulmonary tuberculosis who underwent leukocyte count examination at rsud gambiran, kediri city. the exclusion criteria were patients with pulmonary tuberculosis at rsud gambiran, kediri city, who had a history of hiv/aids and diabetes mellitus. data were collected as secondary data from the leukocyte count examination results of pulmonary tuberculosis patients at rsud gambiran, kediri city. the collected data underwent several processing steps. firstly, the data obtained from the study were carefully reviewed to ensure completeness and accuracy during the editing phase. subsequently, the meticulously collected data was entered into the microsoft excel computer program, enabling efficient data management during the processing phase. to gain valuable insights into the leukocyte profile of pulmonary tuberculosis patients at rsud gambiran, kediri city, a comprehensive data analysis was carried out using microsoft excel during the data analysis phase. the results were then visually represented through tables and diagrams for better understanding and interpretation. throughout this analysis, various aspects were explored, such as the distribution of pulmonary tuberculosis patients based on gender and age groups. additionally, diagrams were created to showcase the increase in leukocyte count based on gender and age. these visual representations helped to identify the highest and lowest increases in leukocyte count among male and female patients, as well as within specific age groups. furthermore, the diagrams allowed the determination of whether the leukocyte count fell within the normal range for each age group. by utilizing microsoft excel and conducting a comprehensive data analysis, the study successfully provided valuable insights into the leukocyte profile of pulmonary tuberculosis patients, contributing to a better understanding of the disease's manifestation and progression in the specified setting. results and discussion tuberculosis (tb) infection is a chronic infectious disease caused by the bacterium mycobacterium tuberculosis, a rod-shaped acid-fast aerobic bacterium. based on the research conducted at rsud gambiran, kediri city, there were 42 patients diagnosed with pulmonary tuberculosis. table 1. percentage of pulmonary tuberculosis patients based on gender. gender number of patients percentage (%) gender 1 male 26 62% 2 female 16 38% total 42 100% table 1 shows the percentage of pulmonary tuberculosis patients based on gender at rsud gambiran, kediri city. out of 42 patients diagnosed with pulmonary tuberculosis, there were 26 male patients (62%) and 16 female patients (38%). this indicates that pulmonary tuberculosis cases are more prevalent in males compared to females. the difference is largely attributed to the distinct immune systems between males and females. females have stronger immune systems due to the significant role of the x chromosome and sex hormones in the pathogenesis of infectious diseases. endogenous hormones and androgens play a pivotal role in antibody production and the immune response, contributing to females' greater resistance against infectious diseases. on the other hand, the testosterone hormone in males can hinder the immune system, making them more vulnerable to infections, including pulmonary tuberculosis. furthermore, it is essential to understand the distribution of pulmonary tuberculosis patients based on age to determine the age range with a higher incidence of pulmonary tuberculosis cases. this information allows for targeted and effective prevention and management strategies for pulmonary tuberculosis. table 2. percentage of pulmonary tuberculosis patients at rsud gambiran, kediri city, based on age groups no. age group number of patients percentage (%) 1 210 years 2 5% 2 11 20 years 1 2% 3 21 60 years 30 71% 4 61 + years 9 21% total 42 100% table 2 shows the percentage of pulmonary tuberculosis patients at rsud gambiran, kediri city, based on different age groups. the data indicates that the highest number of patients with pulmonary tuberculosis falls within the age group of 21-60 years. this can be attributed to the fact that individuals in this age range are in their productive years, and they are more exposed to various factors that can lead to infections. according to fitri et al. – an overview of the total leukocyte count in pulmonary tuberculosis 673 the research conducted by gita and mardina (2019), some factors contributing to pulmonary tuberculosis infections include exposure to vehicle emissions, cigarette smoke, and dust containing m. tuberculosis. on the other hand, in the age group of children, the factors causing tuberculosis infection in children, as mentioned by rizky et al. (2020), are related to the immature immune system at a young age, leading to a lower ability to eradicate pathogens causing infections. additionally, as stated by wijaya et al. (2021), malnutrition, especially in the form of severe malnutrition, can increase children's vulnerability to diseases and cause secondary immunodeficiency. next, the distribution of pulmonary tuberculosis patients who experienced an increase in leukocyte count based on gender was analyzed. the objective was to determine the average increase in leukocyte count based on gender. below is the diagram illustrating the increase in leukocyte count based on gender in patients with pulmonary tuberculosis at rsud gambiran, kediri city. discussion male patients experienced a higher increase in leukocyte count compared to females, with an average of 14.57 µl. leukocytosis indicates the severity of a disease, especially in infectious diseases. according to ruggieri (2016), this section presents the obtained results briefly and may be supported by illustrations such as tables, graphs, or qualitative descriptions. the discussion emphasizes the cause-and-effect relationship, the connection between theory and results, and similar previous research. leukocytosis can be influenced by several factors, including the innate immune system in both males and females, which is related to sex hormones and genetic contributions from chromosomes. hormones and chromosomes in females lead to different responses to bacteria, viruses, and parasites, resulting in a lower risk of infection and a tendency for leukocyte counts to remain within the normal range compared to males. after determining the average increase in leukocyte count based on gender, the next step was to determine the average increase in leukocyte count based on age groups of pulmonary tuberculosis patients. the purpose of this was to identify the age range where patients experienced leukocytosis. below is the diagram illustrating the leukocyte count increase based on age in patients with pulmonary tuberculosis at rsud gambiran, kediri city, as shown in the table below. the increase in leukocyte count in the 21-60 years age group can be attributed to the fact that this age range represents the productive years of individuals, where they engage in learning, working, and striving for personal and communal achievements. these activities require significant energy expenditure, and inadequate rest can lead to a weakened immune system and susceptibility to diseases (nurjana, 2015). additionally, individuals in this age group often experience stress due to their work and daily physical activities, which can trigger pathological and physiological changes. glucocorticoids released during stress suppress the immune response and block helper t cells (th1) that produce interferon-gamma (ifny), which is crucial for activating immunoglobulins. a decrease in immunoglobulins leads to a weakened immune system and a higher risk of infection (setyarsih, 2020). furthermore, the distribution of pulmonary tuberculosis patients with leukocytosis was analyzed based on age groups, including children, adolescents, adults, and the elderly at rsud gambiran, kediri city. the purpose was to determine the number of individuals experiencing leukocytosis within each age group. the increase in leukocyte count in the 21-60 years age group can be attributed to the fact that this age range represents the productive years of individuals, where they engage in learning, working, and striving for personal and communal achievements. these activities require significant energy expenditure, and inadequate rest can lead to a weakened immune system and susceptibility to diseases (nurjana, 2015). additionally, individuals in this age group often experience stress due to their work and daily physical activities, which can trigger pathological and physiological changes. glucocorticoids released during stress suppress the immune response and block helper t cells (th1) that produce interferon-gamma (ifny), which is crucial for activating immunoglobulins. a decrease in immunoglobulins leads to a weakened immune system and a higher risk of infection (setyarsih, 2020). furthermore, the distribution of pulmonary tuberculosis patients with leukocytosis was analyzed based on age groups, including children, adolescents, adults, and the elderly at rsud gambiran, kediri city. the purpose was to determine the number of individuals experiencing leukocytosis within each age group. conclusions based on the research conducted on pulmonary tuberculosis patients at rsud gambiran, kediri city, it was found that males were more affected by tuberculosis compared to females, accounting for 62% (26 individuals) of the total cases. additionally, there was an increase in cases within the age group of 21-60 years, which constituted 71% of the total cases. leukocytosis, indicated by an increase in leukocyte count, was predominantly observed in male patients, with an average increase of 14.57 µl compared to females. moreover, within the age group of 21-60 years, there was a significant leukocytosis with an average increase of 48.66 µl. however, it is noteworthy that not all pulmonary tuberculosis patients experience leukocytosis. out of the 42 sampled patients, 19 individuals had normal leukocyte counts, while the remaining 23 individuals showed leukocytosis. based on the research findings, the author recommends adding variables to the 674 biology, medicine, & natural product chemistry 12 (2), 2023: 671-674 leukocyte profile before and after treatment to compare changes in leukocyte count over time. this suggestion is aimed at enriching the research by considering more variables since the current study only focused on a single variable. additionally, extending the research period is advised to gather more comprehensive data. acknowledgments: the author would like to thank the institute for research, development, and community service (lp3m) of kadiri university which has always provided support for writing and publications. authors’ contributions: devi nor fitri designed the study. lisa savitri analyzed the data. rochmad krissanjaya and elfred rinaldo kasimo wrote the manuscript. all authors read and approved the final version of the article. competing interests: the authors declare that there are no competing interests. references bili, f. (2017). hubungan jumlah leukosit dengan nilai laju endap darah pada pasien tuberkulosis bta positif. dicky & ahmad. (2019). pemeriksaan jumlah sel leukosit pada penderita tuberkulosis paru di upt kesehatan paru masyarakat dinas kesehatan provinsi sumatera utara. 4(2), 31–35. ellies t. (2020). status kadar hemoglobin dan jenis leukosit pada pasien tb paru di surabaya. 3(1), 53–60. gita, c. r. n., & mardina, v. (2019). pemeriksaan jumlah leukosit , laju endap darah dan tuberculosis paru di rsud langsa, 1(2), 6–15. hasnawati. (2018). pengaruh infeksi mycobacterium tuberculosis terhadap nilai laju endap darah penderita tuberculosis paru di balai besar kesehatan paru masyarakat makassar. 1(1), 8– 13. mitchell, e., graham, a. l., úbeda, f., & wild, g. (2022). on maternity and the stronger immune response in women. nature communications, 13(1), 1–12. https://doi.org/10.1038/s41467-022-32569-6 mursyaf, e. a. (2018). keberhasilan pengobatan tuberkulosis (tb) paru di wilayah kerja puskesmas panambungan kota makassar. higiene, 4(1), 32–40. https://doi.org/2542-5301 mutiara, e. (2020). algoritma klasifikasi naive bayes berbasis particle swarm optimization untuk prediksi penyakit tuberculosis (tb). swabumi, 8(1), 46–58. https://doi.org/10.31294/swabumi.v8i1.7668 niagita, e. a. (2019). pemeriksaan jumlah leukosit, laju endap darah dan bakteri tahan asam (bta) pada pasien penyakit tuberculosis paru di rsud langsa. 1(2), 6–15. nurjana, m. (2015). faktor risiko terjadinya tuberculosis paru usia produktif (15-49 tahun ). 25(3), 163–170. nurkumalasari et al.,. (2016). hubungan karakteristik penderita tuberkulosis paru dengan hasil pemeriksaan dahak di kabupaten ogan ilir. jurnal keperawatan sriwijaya, 3(2), 51– 58. https://doi.org/2355 5459 pratiwi, r. d. (2018). gambaran komplikasi penyakit tuberkulosis berdasarkan kode international classification of disease 10. xiii(2), 93–101. rizky, m. a., ismy, j., & husnah. (2020). nilai prokalsitonin dan leukosit pada anak dengan sepsis di ruang pediatric intensive care unit rsudza banda aceh. medicus darussalam, 3(1). rosita, e. a. (2019). hematologi dasar. ruggieri, a. et al. (2016). the influence of sex and gender on immunity, infection and vaccination. ann ist super sanità, 47(4), 363–372. https://doi.org/10.4415/ann sarvasti, d. (2020). pengaruh gender dan manifestasi kardiovaskular pada covid-19. indonesian journal of cardiology, 41(2). https://doi.org/10.30701/ijc.1004 setyarsih, e. a. (2020). hubungan tingkat asupan seng dan zat besi dengan jumlah leukosit atlet sepak bola remaja. 9(1), 31–37. sundari, e. a. (2017). perbedaan parameter hematologi pada penderita tuberkulosis paru terinfeksi mycobacterium tuberculosis galur beijing dengan galur nonbeijing rini. 49(1), 35–42. wijaya, m. s. d., mantik, m. f. j., & rampengan, n. h. (2021). faktor risiko tuberkulosis pada anak. e-clinic, 9(1), 124– 133. https://doi.org/10.35790/ecl.v9i1.32117 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 957-960 | doi: 10.14421/biomedich.2025.142.957-960 issn 2540-9328 (online) the relationship between total ige, symptom duration, vas scores, and danyoung classification in allergic rhinitis ayunita tri wirattami1*, tanti agustina2 1department of otorhinolaryngology head and neck surgery, saiful anwar hospital, east java province, jl. jaksa agung suprapto no. 2, malang, indonesia. 2department of biology, faculty of biology, universitas gadjah mada, sekip utara street, sleman, yogyakarta 55281, indonesia. corresponding author* ayunitatw.thtkl@gmail.com manuscript received: 14 august, 2025. revision accepted: 03 october, 2025. published: 28 october, 2025. abstract allergic rhinitis (ar) is an inflammatory condition of the nasal mucosa triggered by allergies, with total ige often used as a biomarker to assess disease severity. objective: to analyze the relationship between total ige levels, symptom duration, visual analog scale (vas) scores, and danyoung classification in ar patients. methods: a cross-sectional observational analytical study was conducted on 73 patients at the allergy-immunology clinic of dr. saiful anwar general hospital. total ige levels were measured using the elisa method, while vas and nasal mucosa classification were assessed through anamnesis and nasoendoscopy. results: the study showed 68.5% of patients had ige levels above 100 iu/ml, with an average of 982.97 iu/ml. although higher ige levels were often found in ar patients, there was no statistically significant correlation between ige levels and danyoung classification (significance value 0.924). the correlation test showed a coefficient of -0.11, indicating a negative correlation, but it was not statistically significant. conclusion: the nasal mucosa condition in patients with high ige levels does not always reflect severe inflammation. in addition to ige levels, external factors such as exposure to pollutants and cigarette smoke also play a role in the condition of the nasal mucosa. therefore, while high ige levels often indicate allergic reactions, this study found no significant relationship between ige levels and ar severity. keywords: allergic rhinitis; danyoung classification; total ige; symptom duration; vas score. introduction allergic rhinitis (ar) is an atopic disease characterized by hypersensitivity reactions in the nasal mucosa mediated by immunoglobulin e (ige), triggered by exposure to one or more allergens such as dust, pollen, or pet dander. symptoms include sneezing, nasal congestion, itching, and clear watery rhinorrhea, which often impacts the quality of life of patients (bousquet et al., 2020; small et al., 2018). ar is a global health issue affecting 10-20% of the population. it typically begins after age 2, but the prevalence in early life is not well known, affecting 10-30% of the population, particularly in children and adolescents (öçal et al., 2019). the duration and severity of symptoms vary among individuals, depending on allergen exposure and the immune response. total ige is often used as an initial indicator to detect the presence of allergic diseases or other immunological conditions. ige is produced by b cells (b lymphocytes) as part of the adaptive immune system and triggers allergic reactions by activating of mast cells and basophils. ige plays a crucial role in the immune response to allergens and parasitic infections. elevated levels of total ige in serum are frequently associated with the severity of allergic reactions and underlying allergic diseases, including ar (bousquet et al., 2015; de paula couto et al., 2014). clinical evaluation of ar requires both subjective and objective measurements. the visual analog scale (vas) assesses patients' subjective perception of symptom intensity, such as nasal congestion, sneezing, itching, or rhinorrhea. the use of vas in ar is considered a valid, simple, and reliable method for measuring the clinical impact of ar (small et al., 2018). in his research, chang classified the nasal mucosa surface through nasoendoscopy in ar patients based on age and sex over 10-years, finding three stages of change in the nasal mucosa of allergic rhinitis patients. stage 1 is hypertrophy of the turbinates (hypertrophy condition). stage 2 is when the turbinates appear concave (concave condition). stage 3 shows the turbinates appearing wrinkled (wrinkled condition). this study revealed a linear regression relationship between age and disease stage. this classification is known as the danyong classification (chang, 2015, 2018). https://doi.org/10.14421/biomedich.2025.142.957-960 mailto:ayunitatw.thtkl@gmail.com 958 biology, medicine, & natural product chemistry 14 (2), 2025: 957-960 materials and methods this analytical observational study with a cross-sectional approach was conducted from january 2023 to august 2024, involving 73 patients at the allergy-immunology clinic, department of otorhinolaryngology, head and neck surgery, dr. saiful anwar hospital, who met the inclusion criteria. 73 patients with allergic rhinitis underwent vas score assessment, total ige testing, and evaluation of the nasal mucosa structure using nasoendoscopy to determine danyoung classification. total ige measurement was conducted through blood sampling using the elisa (enzyme-linked immunosorbent assay) method or other serological techniques available at the dr. saiful anwar hospital laboratory. the duration of symptoms was measured based on the patient’s history regarding the length of time symptoms were experienced, noted in days or weeks. patients rated the intensity of their allergic rhinitis symptoms on a vas scale from 0 to 10, where 0 indicated no symptoms and 10 indicated very severe symptoms. danyoung classification, based on nasal mucosa assessment, was performed using nasoendoscopy by an otorhinolaryngology specialist, who classified patients into three stages of mucosal change. this study analyzes the relationship between total ige levels and symptom duration, vas scores, and danyong classification in patients with allergic rhinitis. collected data will be analyzed using statistical software. descriptive analysis will be conducted to describe the demographic characteristics of the sample (age, gender) and the distribution of total ige levels, symptom duration, vas scores, and stages of danyoung classification. pearson or spearman correlation tests will assess the relationship between total ige and danyoung classification. pearson's test will be used if the data is normally distributed, while spearman's test will be employed if the data is not normally distributed. results and discussion based on gender distribution, the patients with allergic rhinitis who visited the ent-head and neck clinic included 35 males (47.9%) and 38 females (52.1%). the analysis of gender data indicates that there is no significant difference between the number of males and females suffering from allergic rhinitis (ar). the underlying mechanism for this occurrence may involve the role of estrogen. estrogen has a significant effect on several components of the immune system. it potentially influences every stage of allergic sensitization, including antigen presentation, th2 polarization, ige production, and mast cell degranulation via classical estrogen receptors. studies using animal models indicate that estrogen can trigger the production of il-5 and il-13 from the mediastinal lymph nodes and stimulate eosinophil cells in peripheral blood (bonds & midorohoriuti, 2013; weare-regales et al., 2022). based on the vas distribution data, the most common vas score was vas 2, with 38 patients (52.1%), followed by vas 5 with 7 patients (9.6%), and vas 7 and vas 6, each with 6 patients. in terms of symptom duration, 56 patients (76.7%) reported symptoms lasting over 12 months, followed by 12 patients (16.4%) with symptoms lasting 6–12 months, and 5 patients (6.8%) with symptoms lasting less than 6 months. these chronic complaints and activity disturbances often lead patients to seek help from an ent specialist after experiencing symptoms for more than 12 months, as previous treatments have not provided improvement. despite their persistence, some patients reported that they had become accustomed to their allergic rhinitis symptoms and considered them not overly bothersome. patients who had undergone effective therapies, such as antihistamines or corticosteroid nasal sprays, could manage their symptoms well. good allergy control can lead to reduced symptom intensity, resulting in lower vas scores, which may explain why some patients rated their symptoms lower on the vas (doulaptsi et al., 2018; klimek et al., 2017). currently, the classification of allergic rhinitis (ar) is based on aria 2008, which divides ar into intermittent (if symptoms last less than 4 days per week or less than 4 weeks) and persistent (if symptoms last more than 4 days per week and more than 4 weeks) (bousquet et al., 2020). allergic rhinitis is not only diagnosed by ent specialists but is also frequently encountered by general practitioners. common symptoms of allergic rhinitis include nasal congestion, rhinorrhea, itching in the nose, and/or sneezing. the presence of two or more symptoms lasting for more than one hour per day can be classified as suspected allergic rhinitis. the therapy often provided is merely symptomatic treatment, such as decongestants and oral antihistamines, but the gold standard examination to determine the type of allergy is often not performed (sharma et al., 2022). as a result, patients frequently remain exposed to allergens, preventing complete resolution of their complaints. severity is classified as mild if there are no disturbances to sleep, learning, work, or other activities, and moderate to severe if there is one or more disturbances (bousquet et al., 2020; sharma et al., 2022). based on the danyong classification, stage 1 included 52 patients (71.2%), stage 2 included 16 patients (21.9%), and stage 3 included 5 patients (6.8%), with a p-value of 0.85. meanwhile, danyoung’s classifications of stages 1 and 2 showed mild persistent symptoms in 50% and 40.9% of cases, respectively, while stage 3 showed moderate to severe persistent symptoms in 50.6% (p-value = 0.96). zachreini stated that the histopathological features caused by allergies include thickening of the basement membrane, increased goblet cell count, elevated eosinophil numbers, wirattami & agustina – the relationship between total ige, symptom duration, vas scores, … 959 congested and dilated blood vessels, and stromal tissue edema. repeated allergen exposure releases histamine, which stimulates the nasal mucosa, causing mucosal thickening and blood vessel dilation, especially in the cavernous plexus of the turbinates. the structure of the mucosal epithelial layer changes to stratified cuboidal, the cilia disappear, and the number of goblet cells increases. the submucosal layer experiences edema, infiltration by plasma cells, round cells, and fibroblasts, while the cavernous plexus of the turbinates becomes enlarged, and smooth muscle atrophies (zachreini et al., 2015). prolonged exposure, type of allergens, and other risk factors can influence nasal mucosal inflammation. patients who receive treatment, especially when symptoms first appear, and good hygiene are factors that can prevent recurrent inflammation of the nasal mucosa and reduce the atrophy process of smooth muscles in the inferior turbinate mucosa. based on the distribution data of total ige, the majority of patients had ige levels above 100 iu/ml, with 50 patients (68.5%), and below 100 iu/ml, with 23 patients (31.5%). elevated ige levels are often associated with allergic responses, and the average total ige level in this study was 982.97 iu/ml. this suggests that the majority of allergic rhinitis patients in this study had relatively high ige levels. however, this study did not find a statistically significant relationship between increased ige levels and the classification of allergic rhinitis. therefore, even though higher total ige levels are commonly observed in ar patients, these findings are insufficient to conclude a strong or significant correlation between elevated ige and the severity of allergic rhinitis symptoms (de paula couto et al., 2014). the correlation test results between total ige and danyong classification scores showed a significance value of 0.924 and a correlation coefficient of -0.11. based on these results, it can be concluded that the value is above the 0.05 threshold, indicating that the correlation results are not statistically significant. the correlation coefficient -0.11 indicates a negative correlation. the study results show no significant relationship between total ige levels and danyong classification scores. ige levels are not correlated with changes in danyong classification in allergic rhinitis patients within the analyzed data. high ige levels indicate that the patient is experiencing an allergic reaction, which triggers the release many inflammatory mediators, including histamine, leukotrienes, and other cytokines. the release of these mediators causes chronic inflammation in the nasal mucosa, which can lead to turbinate hypertrophy. the condition of the nasal mucosa is not only determined by ige levels but also by external factors such as exposure to pollutants, cigarette smoke, and infections. these factors can affect the nasal mucosa independently of ige levels, so high ige levels do not always cause mucosal inflammation. the immune response in allergies is mediated by various immune cells and molecules such as mast cells, basophils, and cytokines. even though ige levels are high, the body's inflammation control mechanisms, including the activity of anti-inflammatory factors, can limit the severity of local inflammation in the nasal mucosa (hyun et al., 2018). conclusions the conclusion of this study is that, although elevated total ige levels were found in most allergic rhinitis patients, there was no significant correlation between ige levels and the danyong classification, which assesses nasal mucosal conditions in allergic rhinitis patients. the correlation results indicate that increased ige levels do not correspond to changes in nasal mucosal structure based on the danyong classification. additionally, the nasal mucosal condition in allergic rhinitis patients is influenced not only by ige levels but also by external factors such as exposure to pollutants, cigarette smoke, and infections, which independently contribute to mucosal inflammation. although high ige levels often indicate a strong allergic response, the body's mechanisms for controlling inflammation, including the activity of anti-inflammatory factors, can reduce the severity of local mucosal inflammation. thus, total ige levels alone cannot be a sole indicator for assessing the severity of symptoms and mucosal inflammation in allergic rhinitis patients. authors’ contributions: a.t.w. conceptualized the study, recruited participants, collected clinical data, and performed clinical assessments including danyoung classification and vas scoring. t.a. conducted data analysis and contributed to the study design. both authors interpreted the findings, drafted and revised the manuscript, and approved the final version. competing interests: the authors have no conflicts of interest to disclose. references bonds, r. s., & midoro-horiuti, t. 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(2022). allergic rhinitis and treatment modalities: a review of literature. cureus. https://doi.org/10.7759/cureus.28501 small, p., keith, p. k., & kim, h. (2018). allergic rhinitis. allergy, asthma & clinical immunology, 14(s2), 51. https://doi.org/10.1186/s13223-018-0280-7 weare-regales, n., chiarella, s. e., cardet, j. c., prakash, y. s., & lockey, r. f. (2022). hormonal effects on asthma, rhinitis, and eczema. the journal of allergy and clinical immunology: in practice, 10(8), 2066–2073. https://doi.org/10.1016/j.jaip.2022.04.002 zachreini, i., suprihati, lubis, m. n. d. l., & koesoema, a. (2015). uji diagnostik histopatologi untuk konka hipertrofiyang disebabkan rinitis alergi dan rinitis nonalergi. cdk-228, 42(5). https://doi.org/10.1111/all.12637 https://www.researchgate.net/publication/315643658 https://doi.org/10.6061/clinics/2014(11)06 https://doi.org/10.1186/s13601-018-0219-6 https://doi.org/10.1128/iai.00934-17 https://doi.org/10.1007/s40629-016-0006-7 https://doi.org/10.7759/cureus.28501 https://doi.org/10.1186/s13223-018-0280-7 https://doi.org/10.1016/j.jaip.2022.04.002 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 51-56 | doi: 10.14421/biomedich.2025.141.51-56 issn 2540-9328 (online) phytochemical profiling, antimicrobial, and antioxidant activities of tamarindus indica pulp extracts: a comprehensive evaluation nidal eisa abdalla mohamed1,*, ayman a. a. ismail2, alaa eisa3 1state key laboratory of metastable materials science and technology yanshan university qinhuangdao 066004 people’s republic of china. 2department of chemical sciences, joint doctoral school, silesian university of technology, 44-100 gliwice, poland. 3faculty of medical laboratories, university of science and technology, omdurman 14411, sudan. corresponding author* nidaleisa298@gmail.com manuscript received: 10 december, 2024. revision accepted: 20 march, 2025. published: 15 april, 2025. abstract the study looks at the antioxidant, antibacterial, and phytochemical characteristics of pulp extracts from tamarindus indica. several solvent fractions were obtained from the extraction process, such as hexane, butanol, ethyl acetate, crude, and aqueous, all of which indicated the existence of primary and secondary metabolites. high amounts of flavonoids, alkaloids, tannins, saponins, and steroids were found by phytochemical screening, especially in the butanol and ethyl acetate fractions. with an inhibition zone of 17 mm against bacillus subtilis, the ethyl acetate extract had the most excellent antibacterial activity in antimicrobial tests conducted using the cup-plate agar diffusion method. with a radical scavenging activity of 11 ± 0.1%, the crude extract's antioxidant activity was found to be modest, in contrast to the positive control's 87% activity, propyl gallate (pg). these findings show that t. indica extracts have a promising antibacterial potential, despite their still-low antioxidant efficiency. according to the research, t. indica may be a valuable source of bioactive substances for medical and pharmacological uses, especially in the treatment of infections. keywords: antimicrobial properties; antioxidant activity; ethanolic extract; phytochemical screening; tamarindus indica. abbreviations: t. indica: tamarindus indica, dpph: 2,2-diphenyl-1-picrylhydrazyl, pg: propyl gallate, rsa: radical scavenging activity, dmso: dimethyl sulfoxide. introduction the tropical tree tamarindus indica l., often known as tamarind, is extensively grown throughout africa, asia, and south america. the edible fruit of the tree is prized for its applications in traditional medicine and cooking. for ages, people have utilized extracts from t. indica's leaves, seeds, and bark to cure a range of conditions, including fever, diarrhea, and inflammation (aqil, f. et al., 2006; doughari, j. h. 2006). the pharmacological potential of t. indica has been the subject of increased scientific investigation in recent decades, especially in light of its phytochemical composition, antioxidant qualities, and antibacterial activity (nwodo et al., 2011; saleem et al., 2022). tamarindus indica is a rich source of primary and secondary metabolites, including flavonoids, alkaloids, tannins, and polyphenols, according to phytochemical studies (soni et al., 2019; baliga et al., 2011). it is well recognized that these substances support the biological functions of plants, such as their anti-inflammatory, antibacterial, and antioxidant qualities. for example, tamarind's high flavonoids and polyphenols have been linked to the ability to scavenge free radicals, making them powerful antioxidants (ismail et al., 2022; kumar et al., 2008). according to studies (al fatimi et al., 2007; bhadoriya et al., 2012), these phytochemicals also have antibacterial properties against a range of bacterial infections. t. indica is unique among medicinal plants because of its wide range of phytochemical profiles (tsuda et al., 1994). isolating certain chemicals from t. indica extracts has been the subject of several investigations; these studies have demonstrated the ability to prevent the growth of microorganisms, including staphylococcus aureus and escherichia coli (kidaha et al.,2023; morton, 1987). though these investigations have established the foundation for comprehending the plant's therapeutic qualities, t. indica's entire potential as a source of bioactive chemicals is still unrealized (alzoreky et al., 2003). antioxidants are essential for reducing oxidative stress, which is linked to a number of illnesses, such as cancer, heart disease, and neurological conditions (ghaly et al.,2023; ajayi et al.,2011). tamarindus indica has drawn attention as a possible source of naturally occurring antioxidants due to the growing interest in natural antioxidants as alternatives to synthetic ones https://doi.org/10.14421/biomedich.2025.141.51-56 52 biology, medicine, & natural product chemistry 14 (1), 2025: 51-56 (harborne et al., 2000; sudjaroen et al., 2005). using assays like dpph (2,2-diphenyl-1-picrylhydrazyl) and abts (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), previous research have shown the antioxidant properties of t. indica extracts (ayala et al.,2014; ghazanfar 1989). according to this research (bhadoriya et al., 2012; tsuda et al., 1994), some substances can neutralize free radicals and lessen oxidative damage in biological systems. while t. indica has been shown to possess antioxidant qualities, nothing is known about the precise chemicals that are in charge of this action (kidaha et al.,2023). furthermore, the majority of research has concentrated on the flesh of the fruit, paying less attention to other plant components like the seeds and leaves, which may contain unique phytochemicals with even higher potential for antioxidant activity (morton, 1987; sudjaroen et al., 2005). this emphasizes the need for more investigation to isolate and identify certain antioxidant chemicals found in various t. indica sections. the quest for novel antimicrobial agents, especially those sourced from natural sources, has become imperative due to the rise of bacteria resistant to antibiotics (alzoreky et al., 2003; ghaly et al., 2023). numerous investigations have demonstrated the antibacterial activity of tamarindus indica against both gram-positive and gram-negative bacteria, as well as fungi, suggesting that it has promise in this field (ajayi et al.,2011; harborne et al.,2000). t. indica extracts have demonstrated promising outcomes in tests conducted against common bacterial strains, including bacillus subtilis, pseudomonas aeruginosa, and candida albicans (sudjaroen et al., 2005; ayala et al., 2014). tamarindus indica's complex phytochemical composition— particularly the presence of tannins, saponins, and alkaloids—has been linked to its antibacterial activity (ghazanfar, 1989). according to morton (1987), these substances can damage microbial cell membranes and obstruct enzymatic functions, which ultimately results in cell death. though these early results are encouraging, much more needs to be discovered regarding the methods by which t. indica carries out its antibacterial activities (bhadoriya et al., 2012;, tsuda et al., 1994). this work aims to assess the antioxidant capacity, antibacterial activity, and phytochemical composition of tamarindus indica extracts, emphasizing their pharmacological potential for use in future medicine. materials and methods plant material the plant components were purchased from the omdurman, sudan, local market, specifically the pulp of tamarindus indica. after being verified, the samples were kept for later examination in the chemistry laboratory of the omdurman islamic university in khartoum, sudan's faculty of science and technology (figure 1). figure 1. tamarindus indica. pulp and seed. preparation of extract 96 g of tamarindus indica (pulp) were soaked in 1 liter of 80-90% ethanol for three days, followed by filtration and evaporation of the solvent at room temperature. with a calculated percentage yield of 58.8%, the t. indica pulp extract was shown to have a good extraction efficiency. after that, the extract was partitioned three times using ethyl acetate, chloroform, hexane, butanol, and water. then, at room temperature, the solvent extracts were evaporated. phytochemical screening primary metabolites: a number of tests were used in the phytochemical screening process to look for tamarindus indica main metabolites. using the molisch test, which created a crimson ring when sulfuric acid was added to the alphanaphthol-treated material, carbohydrates were identified. after heating the sample with benedict's reagent, the benedict's test revealed the presence of reducing sugars, giving the sample a dark red color. barfoed's test was used to identify monosaccharides; after heating the sample in a water bath for more than four minutes with barfoed's reagent, the sample displayed a dark red color. these findings show the presence of primary metabolites that are necessary for the plant's energy storage and metabolism, such as lowering sugars and carbohydrates (harborne, 1998). secondary metabolites: numerous tests were used to identify secondary metabolites. using wagner's and dragendorff's assays, which produced brown and orange precipitates, respectively, alkaloids were verified. the lead acetate, koh, and alkaline tests all yielded yellow hues or precipitates, which were used to identify flavonoids. the ferric chloride test revealed a dark green tint, indicating the presence of tannins. saponins were detected by the mohamed et al. – phytochemical profiling, antimicrobial, and antioxidant … 53 development of steady foam in the foaming test. the salkowski test revealed a crimson ring when steroids and terpenes were present, and the ammonia layer turned red when diluted hydrochloric acid and ammonia were used to demonstrate the presence of anthraquinones. the biological properties of these secondary metabolites, such as their antibacterial and antioxidant properties (harborne.,1998). antioxidant activity: dpph free radical scavenging activity with minor adjustments, the dpph radical scavenging was calculated using the methodology of mollyneux, p. (2004). the test samples were exposed to 2.2 di (4-tertoctylphenyl)-1-picrylhydrazyl stable free radical (dpph) in 96-well plates for 30 minutes at 37ºc. the dpph concentration was maintained at 300μm. while dpph was being produced in ethanol, the test samples were dissolved in dmso. using a multiplate reader spectrophotometer, the absorbance drop was recorded at 517 nm following incubation. percentage radical scavenging activity by samples was measured in comparison with a dmso treatedcontrol group. every test was run three times. antimicrobial activity the antimicrobial activity was tested using the cup-plate agar diffusion method with some modifications. 20 ml aliquots were placed into sterile petri dishes after a bacterial solution containing 108−10910^8 10^9108−109 cfu/ml was combined with 100 milliliters of sterile nutritional agar at 45°c. after they had hardened, sterile cork borer holes measuring 10 mm were made. after adding 0.1 ml of sample extract to each well, the plates were allowed to diffuse for two hours at room temperature. following an 18-hour incubation period at 37°c, the zones of inhibition were quantified, and the outcomes were documented, every test was run twice. results and discussion phytochemical analysis the result of phytochemical screening of t.indica pulp extract qualitatively is shown in table 1, the phytochemical constituents of the extract included: carbohydrates (reducing sugars, mono saccharides), flavnoids, alkaloids, saponins, steroids and terpenes and anthraqunons. tannins and phenols were not detectable at the tested assay condition. table 1. phytochemical screening of the crude and fractions of t.indica. phytochemical group crude extract hexane extract chloroform extract ethyl acetate extract butanol extract aqueous extract -carbohydrates -reducing sugars -monosaccharides -flavnoids -alkaloids -saponins -steroids and terpenes -anthraqunons +++ +++ +++ + +++ +++ + +++ +++ + + +++ +++ +++ +++ + + +++ +++ + +++ +++ + + + +++ +++ + +++ +++ + + +++ +++ + +++ +++ + + + +++ +++ + +++ : high concentration, + : low concentration, : not detectable using the specified assay method. antioxidant activity (dpph) figure 2 and table 2 illustrate the pulp extract of t. indica's antioxidant activity. the percentage of dpph inhibition (%) that resulted from the samples' exposure was used to express the results. the capacity of plant extracts to scavenge the stable free radical dpph and transform it into diphenyl picryl hydrazine was used to measure their radical scavenging activity and antioxidant potential. compared with propyl gallate (pg), a positive control. table2: antioxidant activity of tamarindus indica compared to positive control (pg). sample % radical scavenging activity(dpph) t. indica 11% pg (control) 87% figure 2. antioxidant activity (%rsa) of t.indica and pg control. 54 biology, medicine, & natural product chemistry 14 (1), 2025: 51-56 antimicrobial activity the antimicrobial activity results are presented in figure 3 and table 3. table 3: inhibition zones for different extracts of t.indica against bacillus subtilis. extract crude hexane ethyl acetate butanol aqueous dmso zones of inhibition (mm) 15 15 17 12 figure 3. zones of inhibition for different extracts of t.indica against bacillus subtilis. table 4. description of t.indica plant. plants parts description flower the fruit is a pod-like structure, dark and curled, bearing seeds within a sticky, delicious pulp. the little yellow blossoms have red striations. leaves leaves are pinnately complex with many short, rectangular leaflets, bright green in hue, and finely reticulate-veined. information widely available and utilized for both culinary and medicinal uses in tropical regions, particularly in africa and india. note: known for its rich content of tartaric acid and various phytochemicals beneficial in traditional medicine. discussion tamarindus indica's phytochemical screening findings show the existence of a number of primary and secondary metabolites that are essential to the biological activities of the plant. using assays like benedict's and molisch's, primary metabolites, such as reducing sugars and carbs, were verified. significant secondary metabolites, including alkaloids, flavonoids, tannins, saponins, steroids, and terpenes, were also found during screening, suggesting the plant may have antibacterial and antioxidant qualities. these data imply that t. indica possesses a rich phytochemical profile that may contribute to its therapeutic applications, warranting more study into its health benefits and practical usage. with only 11% radical scavenging activity (rsa), the t. indica crude extract's antioxidant activity was shown to be extremely weak when tested using the dpph radical scavenging assay. propyl gallate (pg), the positive control, on the other hand, showed a noticeably higher rsa of 87%, demonstrating its robust antioxidant activity.the t. indica extract's low level of antioxidant activity indicates that there are not enough active antioxidant components in the crude extract to adequately neutralize free radicals in this assay. this can be the result of the extraction process or the existence of other substances that prevent the antioxidants from doing their job. on the other hand, pg is a well-known strong antioxidant, and its high rsa in this test acts as a standard for assessing how effective natural extracts are. better outcomes from further refining the extraction procedure or isolating particular antioxidant components from t. indica could increase the plant's potential as a natural source of antioxidants. the extracts containing ethyl acetate displayed the largest zone of inhibition (17 mm), closely trailed by the extracts containing hexane and crude, which both displayed a 15 mm zone of inhibition. butanol showed a 12 mm inhibitory zone and moderate activity. the aqueous extract and dmso did not exhibit any discernible antimicrobial activity, indicating that the active ingredients with antibacterial properties are more soluble in organic solvents such as hexane and ethyl mohamed et al. – phytochemical profiling, antimicrobial, and antioxidant … 55 acetate. this is consistent with other research showing the effectiveness of ethyl acetate and hexane extracts in phytochemical screens, and could be caused by the presence of particular secondary metabolites that are better extracted in non-polar or semi-polar solvents. these findings imply that organic solvents—specifically, ethyl acetate—are useful for separating antimicrobial components from t. indica, which could lead to more research on antimicrobial drugs originating from plants. conclusions the results of the investigation effectively established the presence of substantial antibacterial activity and noteworthy phytochemical variety in tamarindus indica pulp extracts, especially in the ethyl acetate fraction. important secondary metabolites with therapeutic potential, including tannins, alkaloids, flavonoids, and saponins, were found during the phytochemical screening. against bacillus subtilis, the antibacterial activity was especially potent, while other fractions also demonstrated some efficacy. however, the antioxidant activity of the crude extract was comparatively moderate, indicating limited radical scavenging capacity. these results imply that although t. indica is a promising strong antibacterial agent, more investigation and improvement could be required to maximize its antioxidant capacity. overall, the research lends credence to the possible use of t. indica extracts in medicinal and pharmaceutical settings, especially in the context of antibacterial therapies. acknowledgements: the authors would like to acknowledge the chemistry department, faculty of science and technology, omdurman islamic university, khartoum, sudan for their support. authors’ contributions: nidal carried out the laboratory work and analyzed the data. ayman wrote the manuscript and 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(2005). isolation and structure elucidation of phenolic antioxidants from tamarind (tamarindus indica l.) seeds and pericarp. food and chemical toxicology, 43(11), 1673-1682. 56 biology, medicine, & natural product chemistry 14 (1), 2025: 51-56 tsuda, t., watanabe, m., ohshima, k., yamamoto, a., kawakishi, s., & osawa, t. (1994). antioxidative components isolated from the seed of tamarind (tamarindus indica l.). journal of agricultural and food chemistry, 42(12), 26712674. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 59-65 | doi: 10.14421/biomedich.2021.101.59-65 issn 2540-9328 (online) hibiscus rosa-sinensis l. (red hibiscus) tea, can it be used as a home-remedy to control diabetes and hypercholesterolemia? subhashinie sanadheera, deepanjana subasinghe*, melissa nethmi solangaarachchi, manju suraweera, noshara yushanthi suraweera, nadeesha tharangika rajarata university of sri lanka, mihintale 50300, sri lanka. corresponding author* deepanjana.subasinghe@gmail.com manuscript received: 20 october, 2020. revision accepted: 24 july, 2021. published: 27 july, 2021. abstract according to traditional medicine, hibiscus is used to treat hypertension, heart diseases and many other ailments. commercially available hibiscus tea is prepared with h. sabdariffa and is expensive. h. rosasinensis is the common variety which is abundant in tropical homegardens, however the scientific data are lacking on the effects. the present review focuses on the available scientific data on the effects of h. rosasinensis on controlling diabetes and hypercholesterolemia. the endeavour was to identify whether h. rosasinensis flower petals as a ‘tea’ is effective for diabetic and hypercholesterolemic patients. medical databases such as medline, bmc, bmj, research gate, mendelay literature search database, google scholar and the general engine google were searched from november 2018 to april 2019. search was carried out using keywords such as “hibiscus rosasinensis”, “antidiabetic effects”, “antihyperlipidemic effects”, “toxic effects”, “anti-inflammatory effects”, “phytochemicals in hibiscus” etc. data were critically analyzed to assess whether the effective doses of the research studies on a par with the doses present in h. rosasinensis teas. we found thirty-nine journal articles fulfilled the criteria. the data were categorized and extracted on uses of h. rosasinensis, anti-diabetic effects, anti-inflammatory effects, antihypercholesterolemic effects and its phytochemicals. the review revealed that the dose of h. rosasinensis petals present in a home-made hibiscus tea is theoretically sufficient to elicit anti-hyperglycemic and anti-hyperlipidemic effects. home-made hibiscus tea is effective in controlling diabetes and hypercholesterolemia without causing acute toxicity. keywords: hibiscus rosasinensis; hibiscus sabdariffa; diabetes; hypercholesterolemia. introduction the trend of the use of herbal remedies is rapidly growing globally (ekor & pistelli, 2013). based on 2002 data, almost four billion people in the world relied on herbal remedies for primary health issues(mukherjee, 2002). although the beneficial effects of these herbal remedies on health problems have been observed and experienced by humans for thousands of years, yet minimum scientific data are available on these treatments (ekor & pistelli, 2013). lack of data on the mode of action, potential adverse reactions, contraindications and interactions with existing western medical treatments has restricted these products from being ‘competitive leading herbal supplements’ in the market. hibiscus is a family of flowering plants which the leaves and the red flower petals are used as herbal remedies by most people in the world. yet data are scarce on the effects of different varieties. consumption of tea made of flower petals of hibiscus family is being practiced by a large number of patients in the world to control hypertension and hyperlipidemia (haji faraji & haji tarkhani, 1999; mckay et al, 2010) thus hibiscus tea is commercially available in international markets. to prepare hibiscus tea, a variety of flowers which are belonged to hibiscus family is used. hibiscus sabdariffa is the mostly consuming variety which also has scientifically proven hypotensive and hypolipidemic effects (haji faraji & haji tarkhani, 1999; mckay et al., 2010). however, hibiscus rosasinensis is the most common, easily cultivated plant which is accessible to most people in the world as it is grown massively in tropics and sub-tropics (it can be cultivated in glass chambers in cold weathers as it does not tolerate temperatures below 10oc). aqueous extract of hibiscus rosasinensis red flower petals is a common herbal home-remedy consumed by people in asian countries however, has minimum scientific data to prove the effects on humans. this plant is a glabrous shrub cultivated as an ornamental plant. among the different colors, red flowered variety is mostly used as a herbal remedy (jadhav et al., 2009). https://doi.org/10.14421/biomedich.2021.101.59-65 60 biology, medicine, & natural product chemistry 10 (1), 2021: 59-65 due to universal availability and good palatability, herbal home-made tea could be prepared with the petals of hibiscus rosasinensis with a minimum preparation time. prior a study on the effects of hibiscus rosasinensis tea on humans, the present review mainly focuses on the effects of this flower on diabetic and hypercholesterolemic animals. methods several medical databases such as medline, bmc, bmj, research gate, mendelay literature search database, google scholar and the general engine google were searched from november 2018 to april 2019. some full articles were accessed through hinari gateway. we used categorized search using keywords such as “hibiscus rosasinensis”, “antidiabetic effects”, “antihyperlipidemic effects”, “toxic effects”, “anti-inflammatory effects”, “phytochemicals in hibiscus” etc. we included trials conducted with animals and humans that had examined the effectiveness of hibiscus rosasinensis and safety in the treatment of hyperglycemia and hyperlipidemia. as the review focuses on the effectiveness of hibiscus tea, the initial endeavour was to review the studies carried out only with water extracts of the flower petals and to exclude the studies carried out with other solvent extraction methods. however, due to scarcity of published data, studies conducted with other solvent extraction methods were also included. researches carried out with parts of the hibiscus plant except for petals were excluded. after gathering the relevant articles, we crossreferenced to see the validity and traced back to the original articles to avoid any plagiarism. most commonly discussed most recent research findings in the original articles were included to conclude this review. the articles were categorized to extract data on uses of h. rosasinensis by humans, anti-diabetic effects, antiinflammatory effects, anti-hypercholesterolemic effects and phytochemicals in h. rosasinensis. data were critically analyzed to assess whether the effective doses of the research studies on a par with the doses present in h. rosasinensis teas. description of the plant different names used for this plant in different countries are given in the first volume of the “medicinal plants of the world” by ivan a. ross (ross, 2003). the plant is a shrub with long slender branches of about 6 m tall. the flowers are borne singly at the apexes or ends of the branches, on long stalks. flowers possess colorful overlapping petals (ross, 2003). classification: kingdom : plantae clade : angiosperms clade : eudicots clade : rosids order : malvales family : malvaceae genus : hibiscus species : h. rosa-sinensis evidence for the use of the h. rosasinensis flower by humans to regulate the menstrual cycle, females of bangladesh use a decoction of the flower petals with green beetle nut. hot water extract of flowers is used by chinese as an emmenagogue and a tonic. flower extract is used by east indies to regulate menstrual cycle and to produce abortion. in fiji, hot water extract of flowers and leaves is used orally to ease childbirths and the infusion of dried flower is used to aid digestion. people in haiti use decoction of flowers to cure flu and cough while hawaiians eat flowers to produce lactation. decoction of dried flowers is used by indians for abortion and the hot water extraction of flowers as a contraceptive (ross, 2003). flowers, crushed with sugar, are used in indian folk medicine to control excessive uterine bleeding. ancient indian medicinal literature reveals the use of hibiscus flowers for heart diseases. flowers have cooling and soothing effects, anti-inflammatory effects and emmenagogue effects. petals are used to stimulate hair growth, to prevent premature graying and hair loss and used as a hair conditioner (linn et al, 2009; upadhyay et al, 2011). in sri lankan ayurveda medicine, flowers are used to treat anaemia, constipation and to stimulate hair growth (ayurvedic medicinal plants of sri lanka, n.d.). sanadheera et al. – hibiscus rosa-sinensis l. (red hibiscus) tea … 61 antidiabetic effects a study conducted in india with alloxan induced diabetic wistar rats revealed that the ethanolic extract of h. rosasinensis elicits acute hypoglycemic effects (1, 3, 5 h) and sub-acute (1, 3, 5, 7 days) hypoglycemic effects at doses of 250 mg/kg and 500 mg/kg (venkatesh et al, 2008). thus, to obtain this effect, a human should consume around 40 mg/kg bw of dried ethanolic extract of h. rosasinensis (reagan-shaw et al, 2008). required weight of dried petals to make hibiscus tea to obtain the above effect will be much higher. when streptozotocine induced diabetic female wistar rats were mated, and treated orally with aqueous extract of hibiscus rosa-sinensis during pregnancy, an increased maternal and fetal weights, reduced atherogenic index, coronary artery risk index and reduced pre-implantation loss rate were observed compared to the untreated diabetic group (afiune et al., 2017). a study conducted by bhaskar and vidhya revealed that administration of aqueous extract of h. rosasinensis flowers at a oral dosage of 500 mg/kg to normal rats, lowered the postprandial (2hr) blood glucose level significantly (p<0.001) after an oral glucose load. moreover, administration of the extract at the same dose over 21 days, significantly lowered the fasting blood glucose level (p<0.001), glycosilated hemoglobin level and serum lipid levels of stz induced diabetic wistar rats, compared to the control group, although a significant change in insulin levels were not exhibited (bhaskar & vidhya, 2012). scientific data is scarce on human studies of the effect of h. rosasinensis flowers. however, data on human studies are available for tea made with h. sabdariffa, a similar flower variety to h. rosasinensis. h. sabdariffa is the variety commonly used in manufacturing hibiscus tea. arabic people used to consume karkadeh tea made from h. sabdariffa mainly after meals. in a study, this tea was prepared by boiling 10g of dried petals in 500ml of water for 60 minutes. final volume was 250 ml which was refrigerated at 4oc until consumption. this tea elicited a slow rise in blood glucose level compared to english breakfast tea, after a high glycemic meal. conversely, the meal with karkadeh tea showed a 20% higher area under the curve (auc) compared to the meal with english breakfast tea (harrison et al, 2009) indicating the slow glucose absorption. the only human-study carried out with h. rosasinensis flowers has been conducted for 60 days providing 2g of hibiscus rosasinensis flower powder incorporated in to mathri (an indian rotti variety) to type 2 diabetes patients. this study revealed a significant decrease in mean fasting blood glucose, post prandial blood glucose level, mean glycosylated hb level, mean total cholesterol, triglyceride level, total ldl and total vldl cholesterol level (sharma et al, 2016). however, the study has been carried out with 6 subjects in each test and control groups and the groups have not been crossed over. anti-inflammatory effect type 2 diabetes mellitus is an inflammatory disease (donath & shoelson, 2011) thus the herbs which possess anti-inflammatory effects might be beneficial in controlling diabetes. ethanolic extract of h. rosasinensis showed the significant anti-inflammatory activity at doses of 125, 250 and 500 mg/kg on carrageenin induced paw edema, cotton pellet induced granuloma and xylene induced mice ear edema (birari et al., 2009). ethanolic extract of white hibiscus (hibiscus rosasinensis var alba) elicits more potent anti-inflammatory effects over red hibiscus (hibiscus rosa-sinensis l) with doses of 50 and 100 mg/kg and elicited a significant reduction (p<0.05) in polymorphonuclear leukocytes infiltration (raduan et al., 2013). antihypercholesterolemic effects a significant improvement in dyslipidemia caused due to diabetes mellitus was observed in alloxan induced diabetic wistar rats with oral doses of 50, 100, 200 mg/kg of hydroalcoholic extract (70%) of flowers of hibiscus rosasinensis, administered for 4 weeks. researchers reported a significant reduction in total cholesterol, triglycerides, vldl, ldl and elevation in hdl levels (m pethe & guptha, 2011). a decrease in the triglycerides and alt levels were observed in pregnant diabetic wistar rats fed with h. rosasinensis aqueous extract at a dose of 100 mg/kg from day 0 to 7 of pregnancy, 200 mg/kg from day 8 to 14 and 400 mg/kg from day 15 to 20. however, no effect on glycemia was observed in this study (silva et al., 2015). when alloxan induced diabetes rats were administered with hydroalcoholic extract (70%) of hibiscus rosa-sinensis, at doses of 50,100 and 200 mg/kg bw for 4 weeks, a significant improvement in diabetes induced dyslipidemia was observed by the researchers with a significant decrease in total cholesterol, triglycerides, vldl, ldl and elevation in hdl levels (m pethe & guptha, 2011). further, significant anti-diabetic effects which were comparable to glibenclamide and sulphonylurea were observed with the same dosed administered for 28 days. histopathological studies showed an increase in the size and number of islets, diameter of the islet cells and decrease in the necrosis and atrophy of the islets (pethe et al, 2017). after administrating ethanolic hibiscus flower extract for 21 days, a maximum blood glucose lowering of 41-46% and total cholesterol and serum triglycerides lowering of 22 and 30%, respectively were observed with streptozotocine induced diabetic rats. the effect was comparable to glibenclamide, however with no increase in insulin level (sachdewa & khemani, 2003). 62 biology, medicine, & natural product chemistry 10 (1), 2021: 59-65 furthermore, anthocyanin compounds delphini din-3-o-sambubioside and cyanidin-3-o-sambubioside, extracted from hibiscus rosasinensis petals elicited potent competitive ace inhibitor effects agreeing to the use of h. rosasinensis petal extracts to treat hypertension in folk medicine (ojeda et al., 2010). moreover, khan et al. (2011) revealed that the chronic use of ethanolic and chloroform extracts of h. rosasinensis petals has anti-anxiety effects without producing any cns depression signs in experimental rats (mohammed j et al., 2011). phytochemicals in flower on blood glucose and serum cholesterol scientific data revels that the edible parts of the h. rosasinensis flowers contain 89.8% moisture, 0.064% nitrogen, 0.36% fat, 1.56% crude fibre. moreover, flower extract contains tannins (7.5±0.20 %), phenols (0.678±0.14%) and alkaloids (0.51±0.16 %) while the total phenol content in flowers was 735±46 mg gallic acid equivalent /100g. the four main types of flavanoids in petals are rutin, quercetin, kaempferol and myricetin (purushothaman, meenatchi, s, sundaram, & saravanan, 2018). petals contain various flavones such as quercetin-3-di-0-beta-d-glucoside, quercetin-3-7-di0-beta-d-glucoside, cholesterol, campesterol, quercetin3-0-beta-d-sophorotrioside, ß-sitosterol10, kaempferol3-0-beta-d-xylosyl-glucoside19, catalase13. red and magenta colour flower petals possessed dark-purplish dye, anthocyanin pigment and cyanidin diglucoside (subramanium & nair, 1972; wealth of india, 1997) along with proanthocyanidins (nakamura et al, 1990). another study reveals the presence of terpenoids in flower petals (patel & adhav, 2016). all these compounds elicit antioxidant properties, thus the antioxidant capacity of the petals were significantly high [ascorbic acid equivalent antioxidant capacity (aeac): 640 ± 56 mg ascorbic acid /100g; total anthocyanin content (tac): 284 ± 17 mg cyanidin-3-glucoside equivalent/100g; ferric-reducing power (frp): 4.0 ± 0.3 mg gallic acid equivalent /100g (sk, lim, & chan, 2009)]. in another study, aqueous extract of hibiscus elicited high tannin and anthocyanin contents, and possessed high ferric reducing antioxidant power (mak et al., 2013). further, fresh flowers of hibiscus rosasinensis possess 0.30-0.50 v/w % of essential oils. most of the beneficial effects of this flower variety could be due to the presence of antioxidants, which play a key role in controlling diabetes and hypercholesterolemia. phenolic compounds in tea inhibit α amylase and α glucosidase enzymes, the key enzymes of carbohydrate digestion (hara & honda, 1990). furthermore, polyphenolics in tea inhibit glucose uptake by intestinal caco-2 cells (johnston, 2005). tannic acid, a major compound of tannins and alkaloids, stimulates the translocation of glucose transporters (glut 4) and increase phosphorylation of insulin receptors thus increasing glucose clearance from blood and activating insulin action respectively (li et al., 2005). the whole plant of hibiscus rosasinensis increase insulin secretion from the pancreatic beta cells, probably by the actions exerted by alkaloids, flavanoids and terpenoids (mishra, 2009). owing to the above actions on glut 4 receptors and insulin receptors, polyphenolic compounds might increase deposition of excess glucose in adipose tissues as fat, increase uptake of triglycerides from adipose tissues by activating lipoprotein lipase and inhibit hormone sensitive lipase which involve in adipolysis. summative action will be the decrease of circulatory triglycerides and phospholipids. further, anthocyanins in hibiscus sadariffa inhibit angiotensin converting enzyme (ace) by competing with the substrate of the enzyme, thereby controls hypertension (ojeda et al., 2010). terpenoids are present in many herbal plants which can modulate activities of ligand-dependent transcription factors [peroxisome proliferator-activated receptors (ppars)] and thus can prevent obesity-induced metabolic disorders, such as type 2 diabetes, hyperlipidaemia, insulin resistance, and cardiovascular diseases (goto et al., 2010). quantitative analyses have revealed that the aqueous extract of hibiscus rosasinensis flower petals have high amounts of tannins and anthocyanin thus show high ferric reducing antioxidant power. among the phenols, flavonoids, tannins, flavonol and anthocyanins are the most potent antioxidants in hibiscus (mak et al., 2013) thus it can be hypothesized that the above beneficial health effects also could be obtained by hibiscus tea. however, all above beneficial effects have been observed with isolated polyphenolic compounds. therefore, it is not clear whether a tea made from the herbal plant or flower will exert the same effect. although data on animal studies are available to prove the anti-hyperglycemic and anti-hyperlipidemic effects of hibiscus rosasinensis extracts, human studies to observe the activity of these compounds in human body, are yet to be carried out. does the dose in hibiscus rosasinensis tea is sufficient to cause the antihyperglycemic and antihyperlipidemic effects? the above evidences show that the h. rosasinensis could be beneficial as a tea, after meals in terms of controlling postprandial blood glucose level, which is a key goal to minimize diabetes related macro and microvascular complications. however, in most of the animal studies, the beneficial effects were obtained with the powder of the dried or evaporated solvent extracts of the petals, which the concentration of the active compounds ought to be higher than the amounts present in a tea. as a commercially available tea bag weighs 2 – 2.5g, this amount might not be sufficient to provide the beneficial sanadheera et al. – hibiscus rosa-sinensis l. (red hibiscus) tea … 63 effects as shown in research. yet, the study conducted by sharma et al. in 2016, with diabetic patients, emphasizes the presence of anti-hyperglycemic effect of the petal powder (2 g) when incorporated in to a food (sharma et al., 2016). furthermore, to obtain the similar effects observed in animal studies (studies using rats), only one sixth of the animal dose has to be given to humans. therefore, present review reveals the necessity of further studies on antihyperglycemic and antihyperlipidemic effects of hibiscus rosasinensis tea. toxic effects antifertility effects: a study was conducted to observe the effect of h. rosasinensis on diabetes during pregnancy. dried and powdered flower petals (20 g) were boiled in 1 l of water for 5 minutes, filtered and evaporated in a rotary evaporator to obtain the test samples. stz induced diabetic female rats were administered with an initial dose of 100 mg/kg/day of the h. rosasinensis extract, from day 0 until the 7th day of pregnancy (implantation period). the dose was increased to 200 mg/kg/day from day 8 to 14 of pregnancy (embryonic period), and to 400 mg/kg/day from day 15 to 20 (fetal period). the study revealed beneficial effects of the flower extract on diabetic pregnant rats and their offspring with no effect on non-diabetic pregnant rats. the non-diabetic treated group elicited decreased high density lipoprotein cholesterol, increased atherogenic index (ai) and coronary artery risk index (cri), and increased preimplantation loss rate compared to the non-diabetic untreated group. treatment has not shown toxicity, however, could be deleterious to cardio vascular system and reproductive functions. therefore, the researchers indicate that the indiscriminate intake of h. rosasinensis extract may be harmful to healthy individuals and further emphasize that this extract should be completely avoided during pregnancy (afiune et al., 2017). extractive value of water soluble extractives of h. rosasinensis is 5.3% (al-snafi, 2018). therefore, according to the previous study, from 20 g of dried petals 1.06 g of water soluble extractives could be gained. the highest water soluble extractives dose/ day which caused the deleterious effects on rats, was 400 mg/kg/day. however, the dose for humans which causes same effect will be six times lower than that of rats (11). therefore, the deleterious water soluble extractives dose will be 3.3g/day. this amount is present in 62.9 g of dried petals. a tea bag contains 2-2.5 g of dried petals which is significantly lower than the toxic dose. acute toxic effects: when powder of the methanolic extract of h. rosasinensis was administered to rats, in 100, 200, 400, and 800 doses, no significant changes in behavior, skin effect, breathing, defecation, postural abnormalities, impairment in food intake and water consumption and yellowing or loss of hair were observed. no changes were observed for one week. however, 20% mortality was observed with a 1600 mg/kg dose (meena et al, 2014). ethanol soluble extractive value for h. rosasinensis was 2.6% (al-snafi, 2018). thus by considering the ratio of the dose for rats and humans, to obtain a 20% mortality rate, 13 g of ethanol soluble extractives have to be consumed by a 50 kg human. this amount is present in 500 g of dried petals. a tea bag contains 2-2.5 g of dried petals which is significantly lower than the toxic dose. genotoxic effects: genotoxicity was not observed with the flower extract, therefore hibiscus rosasinensis could be used as a safe pharmaceutical material (meena et al., 2014). cytotoxic effects: when a decoction was prepared with 15 g of dried and milled hibiscus petals and diluted to 5g/l and 10g/l, it showed a reduction in mitotic index for l allium cepa l., significantly. therefore, h. sinensis contains antimitotic constituents which block anywhere of the cell cycle (ali, 2010). these concentrations are similar to 1.25g and 2.5 g of petals in a tea cup of 250 ml respectively. however, the study was carried out with plant materials and may not be applied to the human body. chronic toxic effects of hibiscus rosasinensis flower extract have not been reported yet, although tea and drinks made from hibiscus rosasinensis flower petals have been used globally from ancient times as a home remedy and a beverage. conclusion the dose of h. rosasinensis petals present in hibiscus tea is sufficient to elicit anti-hyperglycemic, antihyperlipidemic, anti-hypertensive, anti-inflammatory, anti-anxiety and anti-oxidant properties without causing acute toxicity. author contribution: study concept, acquisition of data, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript: senadheera s. p. a. s. 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(1997). raw materials (vol. vi). new delhi. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 983-992 | doi: 10.14421/biomedich.2025.142.983-992 issn 2540-9328 (online) molecular identification of lepidopteran predators of coccidae on coffee plants (coffea canephora) in sigi regency i made budiarsa1*, manap trianto1, bahana aditya adnan2 1departement of biology education, faculty of teacher training and education, tadulako university, jl. soekarno hatta no.99, tondo, palu tim., palu, central sulawesi 94111, indonesia 2departement of biology education, faculty of teacher training and education, galuh university, jl. r.e. martadinata no. 150, ciamis, ds. mekarjaya, mekarjaya, baregbeg, kec. baregbeg, jawa barat 46274, indonesia. corresponding author* budiarsa_imade@yahoo.com abstract this study aimed to identify the species of lepidopteran predators that prey on soft scale insects (coccidae) found on coffee plants in sigi regency using molecular techniques. dna was extracted using the modified ctab method (doyle & doyle, 1990). pcr amplification targeted the coi gene using universal primers lco1490 and hco2198 with a geneamp pcr system 9700. visualization was done using 1% agarose gel and uv-transilluminator. sequencing was conducted externally. data were analyzed using bioedit 7.2.5, blast, bold systems, and mega11 software. the dna sequence of the predator sample showed 97.91% similarity to autoba rubra based on genbank and 97.59% in bold systems. phylogenetic analysis confirmed a close genetic relationship with a. rubra, distinct from eublemma. morphological similarities with eublemma were misleading, highlighting the accuracy of molecular identification. this study is the first to confirm the identity of a lepidopteran predator of coffee scale insects in indonesia using molecular techniques, revealing its close relation to autoba rubra. the results correct previous morphological misidentifications and contribute new data on predator diversity in biological control contexts. keywords: central sulawesi; coccidae; coffee plants; molecular identification. introduction soft scale insects (family coccidae), commonly known as kututempurung in indonesia, are a group of hemipteran insects classified as important plant pests (organisms harmful to plants), particularly affecting plantation crops such as coffee (maharani et al., 2020). these insects are polyphagous, meaning they can infest a wide range of plant species; however, their presence on coffee plantations in indonesia is notably dominant and damaging (damanik et al., 2022). soft scale species typically attack the vegetative parts of the plant especially the stems, branches, and leaves by sucking phloem sap. this feeding activity not only reduces the plant's vigor but can also inhibit growth, cause leaf yellowing and defoliation, and ultimately decrease overall crop productivity (faiz et al., 2024). one of the distinguishing characteristics of soft scale insects is their ability to secrete sticky honeydew, which serves as an ideal medium for the growth of sooty mold fungi. these fungi cover the surface of leaves and inhibit the photosynthesis process, leading to a significant reduction in yield (luthfia et al., 2024). such indirect effects are often more damaging than the direct impact of phloem-sap feeding itself (avelino et al., 2012; susilo, 2015). from an economic perspective, the presence of soft scale insects has caused considerable losses in several coffee-producing countries, including indonesia. globally, yield losses due to soft scale infestations both in terms of reduced harvest quantity and quality, as well as control costs (including insecticides, labor, and plant maintenance) are estimated to reach around us$5 million per year. this figure highlights the importance of addressing this pest in coffee cultivation systems (dantas et al., 2021). as part of biological pest control efforts, soft scale insects are known to have several natural enemies that play a key role in regulating their population dynamics in agricultural environments. these natural enemies belong to three main groups: predators, parasitoids, and pathogens such as entomopathogenic fungi (gongora et al., 2023). the presence of these natural enemies is crucial, especially in sustainable farming systems that aim to minimize reliance on chemical pesticides. one of the most frequently reported and studied groups of natural enemies are predators from the family coccinellidae, commonly known as lady beetles, which are recognized as effective predators of soft scale insects, manuscript received: 14 august, 2025. revision accepted: 27 october, 2025. published: 29 october, 2025. https://doi.org/10.14421/biomedich.2025.142.983-992 mailto:budiarsa_imade@yahoo.com 984 biology, medicine, & natural product chemistry 14 (2), 2025: 983-992 including soft scales (alemu, 2016; ogundeji et al., 2019; abewoy, 2022). several identified species from this family include azya luteipes mulsant, diomus lupusapudoves vandenberg, and chilocorus politus mulsant (asfaw et al., 2019; johnson et al., 2020). these three species actively prey on soft scales, particularly during the nymph and young adult stages. their predatory activity can significantly reduce pest populations and help maintain plant health (mendesil, 2019). in addition to predators, soft scale insects are also parasitized by insects from the order hymenoptera, particularly from the families encyrtidae and aphelinidae (wegbe et al., 2003; aristizabal et al., 2016; moghaddan et al., 2021). these parasitoids lay their eggs inside the bodies of soft scales, and the larvae then develop by consuming the host’s internal tissues. this process ultimately results in the death of the soft scale insect and provides long-term population control (aristizabal et al., 2016). one major advantage of parasitoids is their host specificity, which minimizes impacts on non-target insects. moreover, entomopathogenic fungi such as beauveria bassiana and metarhizium anisopliae have also been reported to infect and kill soft scale insects through spore penetration of the insect's cuticle (infante, 2018; shimales et al., 2023). these fungi are typically applied in the form of bioinsecticides and serve as part of environmentally friendly integrated pest management (ipm) strategies (dantas et al., 2021). in december 2024, predatory larvae were discovered on coffee plants at the experimental plantation of the testing center for industrial and refreshment crop instruments in central sulawesi. based on initial observations, the predator was suspected to have strong predation capabilities against soft scale insects. the population of soft scales, particularly coccus sp., which had been abundant at the time, decreased by more than half within two days. to investigate the potential of this predator for biological control, essential information such as its taxonomic identity is required (johnson et al., 2020). in lampung, a moth predator has been reported to prey on coccus viridis (green) (hemiptera: coccidae) on coffee plants. this predator exhibited a predation rate of 97 ± 11 scales per larva within six days under laboratory conditions (mendesil, 2019). the predator larva in lampung was identified as belonging to the genus eublemma based on morphological characteristics using beardsley’s (1982) identification key. kalshoven (1981) also noted that several species from the genera eublemma, catoblemma, and autoba act as soft scale predators in indonesia (magina et al., 2016). the predatory moth observed in sigi closely resembled the one in lampung in external appearance, based on available images. however, accurately determining and confirming the predator’s species morphologically remains constrained by limited literature. molecular technology has become an essential tool in modern taxonomy, including for insect species identification (ceja-navarro et al., 2015). in recent decades, molecular-based identification methods have been considered significantly more accurate than traditional morphological approaches (vega et al., 2015). this is because morphological traits often overlap between species, particularly at the larval stage or in specimens with damaged structures. in contrast, molecular approaches allow for more precise identification, even in incomplete or very small specimens (nyambo et al., 1996; acuna et al., 2012). one of the key advantages of molecular techniques is their ability to work with previously unknown genomes. additionally, these methods are regarded as highly efficient and sensitive, as they can detect genetic variation at the intraspecific level, enabling finer differentiation between populations or individuals within a species. materials and methods predators were collected from coffee plantations infested with soft scale insects in sigi regency, central sulawesi. predator sampling was conducted in december 2024. the samples consisted of three late-instar predator larvae. each sample was collected using forceps, placed in a plastic container covered with gauze, and labeled accordingly. molecular identification of each sample involved several steps: dna extraction, pcr amplification, electrophoresis, visualization, dna sequencing, and data analysis. the imago and larval stages of the predator used for imaging were individuals reared in a greenhouse at the biology laboratory, tadulako university, until january 2025. dna extraction dna samples were obtained from whole, fresh, lateinstar predator larvae that had been separated from their outer covering or shell. the extraction began by preparing a buffer solution containing 125 µl of cetyl trimethylammonium bromide (ctab) (2% ctab; 1.4 m nacl; 20 mm edta; 100 mm tris-hcl; ph 8.0) and 1.25 µl of 0.2% 2-mercaptoethanol in a 1.5 ml microcentrifuge tube. the larva was placed in the buffer solution and macerated thoroughly using a micropestle until completely homogenized. the homogenate was then vortexed for 10 seconds and incubated at 65 °c in a dry bath for 30 minutes, with vortexing every 10 minutes for 10 seconds. following incubation, 125 µl of chloroform:isoamyl alcohol (ciaa) (24:1) was added, and the mixture was shaken manually for 3 minutes, incubated at room temperature for 30 minutes, and centrifuged at 8,000 rpm for 10 minutes. the resulting supernatant (top layer) was carefully transferred to a new tube using a micropipette, and its volume was recorded. subsequently, ch₃cook (potassium acetate) was added budiarsa et al. – molecular identification of lepidopteran predators of coccidae … 985 at 1/10 of the total supernatant volume, along with -20 °c isopropanol at 2/3 of the total volume. the tube was sealed, placed in a glass container, and frozen using liquid nitrogen. it was then allowed to return to room temperature for 30–45 minutes. the solution was centrifuged again at 8,000 rpm for 10 minutes. the supernatant was discarded, leaving a visible white dna pellet (cloud-like) at the bottom of the tube. the pellet was washed by adding 500 µl of 80% ethanol, followed by centrifugation at 8,000 rpm for 2 minutes. after discarding the ethanol, the pellet was air-dried by inverting the tube on a petri dish lined with tissue paper and placing it under an air conditioner at 20 °c for 1 hour. the dried pellet was then resuspended by adding 30 µl of te buffer (10 mm tris-hcl; 1 mm edta; ph 7.4), vortexed for 5 seconds, and incubated at room temperature for 1 hour. the extracted dna (dna template) was stored in a -20 °c freezer until further analysis. dna amplification the target for dna amplification in this study was a 710 bp fragment of the mitochondrial cytochrome c oxidase subunit i (coi) gene. amplification was performed using universal primers: lco1490 forward (oligonucleotide sequence 5’–ggt caa caa atc ata aag ata ttgg–3’) and hco2198 reverse (oligonucleotide sequence 5’–taa act tca ggg tga cca aaa aat ca–3’). the amplification was carried out using a geneamp pcr system 9700 thermal cycler, with the following program: an initial denaturation step at 94 °c for 5 minutes; followed by 35 cycles of denaturation at 94 °c for 1 minute, annealing at 52 °c for 30 seconds, and extension at 72 °c for 1 minute and 30 seconds. a final extension was performed at 72 °c for 10 minutes, followed by a cooling step to 4 °c. the pcr reaction mixture had a total volume of 25 µl, consisting of: 12.5 µl dreamtaq green pcr master mix, 1 µl forward primer, 1 µl reverse primer, 1.5 µl mgcl₂, 8 µl ddh₂o, and 1 µl dna template. electrophoresis and visualization the pcr amplification products were subjected to electrophoresis and visualized to confirm the success of the amplification. the medium used was a 1% agarose gel prepared in 1×tbe buffer (80 mm tris-borate, 1 mm edta). first, a gel mold was prepared, and a small comb (for six wells) was inserted. then, 0.2 g of agarose powder was weighed, placed into an erlenmeyer flask, mixed with 20 ml of tbe buffer, and dissolved by heating in a microwave at medium power for 80 seconds. the solution was allowed to cool to a warm temperature at room temperature, then 2 µl of fluorovue™ nucleic acid gel stain (10,000x) was added. the solution was manually mixed, poured into the mold, and left to solidify at room temperature for approximately 1 hour until it formed a gel.the solidified gel was then transferred to the electrophoresis chamber filled with tbe buffer. a total of 5 µl of each pcr product and 5 µl of a 100 bp plus dna ladder (used as a fragment size marker) were loaded into the wells using a micropipette. electrophoresis was performed at 50 v for 50 minutes. after the run was complete, the gel was visualized using a uv transilluminator, and the results were documented. dnasequencing dna sequencing was performed by a third-party service provider. successfully amplified dna samples (pcr products) were sent in a volume of 20 µl, along with forward and reverse primers, each with a minimum volume of 10 µl per sample. the sequencing results were received in ab1 file format for both forward and reverse reads. data analysis the forward and reverse sequencing results were edited and assembled into contigs using bioedit version 7.2.5. the assembled sequences were then compared with species databases available in the genbank of the national center for biotechnology information (ncbi) (https://www.ncbi.nlm.nih.gov) using the basic local alignment search tool (blast). the sample dna sequences were also matched with species data in the bold systems database (https://www.boldsystems.org) using the identification engine. both genbank and bold systems produced homology scores based on sequence similarity. sequence alignment was performed using the clustalw method in mega11, while genetic distances were calculated pairwise using the maximum composite likelihood method, also in mega11. a phylogenetic tree was constructed using the maximum likelihood method in mega11. results and discussion amplification, visualization, and dna sequencing of predator samples in the visualization of the dna amplification (pcr) results, the first lane represents the marker, while the second lane shows the dna band of the predator moth sample (figure 1). by comparing the position of the sample band to the marker, the estimated fragment length is approximately 700 bp, which matches the expected target size. the dna band appears sufficiently thick and bright, indicating that the pcr product generated using universal lco-hco primers contains an adequate amount of dna for sequencing. based on sequence data analysis using bioedit and blast, the dna sequence of the predator moth was identified as a mitochondrial coi gene fragment with a length of 631 bp. all three samples used in this study shared identical dna sequences. https://www.ncbi.nlm.nih.gov/ https://www.boldsystems.org/ 986 biology, medicine, & natural product chemistry 14 (2), 2025: 983-992 figure 1. visualization of dna amplification using lco-hco primers on agarose gel medium under a uv transilluminator following electrophoresis. 1: marker; 2: predator moth. homology, phylogeny, and dna variation of predator samples sequence alignment of the predator dna samples with voucher species in the genbank database showed the highest homology with autoba rubra hampson voucher 10anic-06779, with a similarity value of 96.78%. meanwhile, in the bold systems database, the highest sequence similarity was also found with a. rubra, at 97.59% (table 1). the resulting phylogenetic tree illustrates two distinct major clades: mataeomera spp. and a. rubra. among the mataeomera group, species closely related to the sample include m. mesotaenia (turner), m. porphyris (turner), m. coccophaga (meyrick), and m. dubia butler (figure 2). when compared with a. rubra and other autoba species in genbank, the dna sequence of the sample showed homology values ranging from 89.80% to 97.91% (table 2). the majority of autoba species originated from australia (western australia, queensland, and the northern territory), except for a. silicula swinhoe (voucher a. silicula kothia), which was recorded from india (table 2). these regions or states are geographically close to indonesia. the phylogenetic reconstruction among autoba species revealed that the predator moth sample and a. rubra formed a sister group. this relationship is supported by genetic distance data. the sample showed the smallest genetic distance with a. rubra at 4.5%, followed by a. dispar warren at 7.3% (table 2). the smaller the genetic distance, the closer the evolutionary relationship. alignment of dna sequences between the sample and four a. rubra voucher species in genbank showed that the five taxa shared 598 conserved regions, with nucleotide sequence variation observed at several positions. nucleotide variation among the five taxa included 45 differing nucleotide sites, equivalent to 8.69%, while variation among the four a. rubra sequences (excluding the sample) was 5.96%, with 29 differing nucleotide sites (table 5). these results indicate that the dna variation between the sample and a. rubra is higher than the intraspecific variation previously recorded for a. rubra in genbank. table 1. the top ten voucher species in the genbank database with the highest dna sequence similarity to the predator moth sample. accession number query cover (%) percent identity (%) hq949995.1 autoba rubra 100 95,92 hq949996.1 autoba rubra 99 96,78 hq950014.1 mataeomera mesotaenia 99 95,50 hq950022.1 mataeomera porphyris 99 95,50 hq950017.1 mataeomera coccophaga 99 95,34 hq950016.1 mataeomera coccophaga 99 95,34 hq950018.1 mataeomera porphyris 99 95,34 hq950025.1 mataeomera dubia 98 95,32 hq949997.1 autoba rubra 96 95,92 kf389655.1 autoba rubra 95 96,00 table 2. the top ten dna sequences in bold systems with the highest similarity to the predator moth sample. order family genus species similarity (%) lepidoptera erebidae autoba autoba ruba 96,48 lepidoptera erebidae autoba autoba ruba 95,92 lepidoptera erebidae autoba autoba ruba 95,92 lepidoptera erebidae autoba autoba ruba 95,89 lepidoptera noctuidae mataeomera mataeomera coccophaga 95,84 lepidoptera erebidae autoba autoba ruba 95,75 lepidoptera erebidae autoba autoba ruba 95,75 lepidoptera erebidae autoba autoba ruba 95,70 lepidoptera noctuidae mataeomera mataeomera dubia 94,43 lepidoptera noctuidae mataeomera mataeomera dubia 95,40 budiarsa et al. – molecular identification of lepidopteran predators of coccidae … 987 figure 2. phylogenetic tree of the predator moth sample and the ten most similar voucher species in the genbank database using the maximum likelihood method (chilocorus politus as the outgroup). imago and larval stages of the predator the predator reared in this study underwent complete metamorphosis, progressing through the larval, pupal, and adult (imago) stages. the larva was caterpillar-like (eruciform) in shape. the final instar larvae were pink in color (figure 3). the spiracles on abdominal segments 7 and 8 were laterally aligned and equal in size. the larva was enclosed in a dome-like structure, leaving only the abdominal part exposed. this covering was brown and constructed from remnants of consumed scale insect shells and other organic materials, such as plant debris and sooty mold, all woven together with silk threads. similarly, the pupa was also protected within this shelter. the pupa was obtect and yellowish-brown in color, while the pupal covering was oblong and brown. the adult (imago) of the predator was a brown moth with an average wingspan of 1.66 ± 0.11 cm (n = 10) (figure 3). the tip of the abdomen did not extend beyond the hind wings. the forewings had curved line-like patterns along the margins. both forewings and hindwings featured a transverse line across the middle and small black dots near the wing tips. the abdominal segments were white. the antennae were filiform, and the mouthparts consisted of a labial palpus (figure 4). figure 3. a. larva of the predator feeding on green scale insects (coccus viridis) on coffee, and b. imago (adult moth) of the predator feeding on green scale insects on coffee in sigi regency, central sulawesi. 988 biology, medicine, & natural product chemistry 14 (2), 2025: 983-992 figure 4. predator moth imago. a. forewing, b. hindwing, and c. abdomen. discussion in this study, dna sequence analysis of the predator moth sample revealed a high level of homology with autoba rubra, with 96.78% similarity based on genbank data and 96.48% according to the barcode of life data systems (bold). these levels of similarity are close to the general threshold commonly used for insect species identification based on the mitochondrial coi (cytochrome c oxidase subunit i) gene. according to a study le pelley (1932) using the subfamily scarabaeinae as a case study, species-level identification of insects can be considered valid if coi sequence similarity exceeds 93.4%. therefore, the similarity values above 96% in this study suggest that the predator moth sample is most likely autoba rubra, or at least very closely related to that species. although there is a 6.47% dna variation between the predator moth sample and the reference sequence of a. rubra, this value is still within the tolerable range for intraspecific variation, especially when considering geographical diversity. genbank data show that the intraspecific variation of a. rubra is 3.99%, slightly lower than the variation observed between the sample and the reference, but still within a reasonable range, particularly when accounting for potential geographic variation or the existence of local subspecies. as a comparison, abedeta et al. (2015) reported that intraspecific variation based on the coi gene in birds averages around 2.7%; however, this benchmark cannot be directly applied to insects due to differences in evolutionary rates, reproductive strategies, and geographic range between these taxa. therefore, insectspecific thresholds, such as those proposed by oliveira et al. (2021), are more relevant in the context of this study. interestingly, the presence of autoba as a genus of predatory moths has long been recognized as one of the natural enemies of armored scale insects (diaspididae) in indonesia, along with the genera catoblemma and eublemma, as noted by (krehenwinkel et al., 2017). this supports the assumption that the predator moth specimen analyzed in this study indeed belongs to the genus autoba, and very likely to a. rubra. however, the issue of geographic distribution warrants careful consideration. all four a. rubra voucher specimens currently available in the genbank database originate from australia. biogeographically, australia and indonesia are neighboring regions that share overlapping faunal evolutionary histories, particularly within the wallacea and sahul zones. this raises the possibility that populations of a. rubra may extend into indonesia or that a sister species, having evolved in geographic isolation, exists with high genetic similarity. the influence of geographic factors on phylogenetic structure has long been acknowledged in evolutionary biology. unruh et al. (2016) emphasized that phylogenetic patterns based on dna in many animal species are closely tied to the geographic history of populations. furthermore, capelli et al. (2018) explained that variation in resources and environmental conditions across local habitats can lead to population isolation, which over time may drive genetic differentiation and even speciation. therefore, the 6.47% genetic divergence observed between the predator moth sample and the a. rubra reference from australia may reflect geographic differentiation rather than absolute species-level divergence. as a follow-up, more comprehensive phylogenetic analyses and additional morphological studies are necessary to confirm the taxonomic identity with greater accuracy, including the possibility of a local a. rubra population in indonesia or a closely related new taxon. a study conducted by pinol et al. (2014) identified the predator moth feeding on coccus viridis in the lampung region as a member of the genus eublemma, based on morphological characteristics, particularly at the larval stage. this identification was based on similarities in body shape, color patterns, and other distinctive morphological traits commonly used in the taxonomic classification of moth larvae. however, field observations in the present study revealed that predator moth samples from the sigi region, central sulawesi, exhibited a high degree of morphological similarity with the specimens from lampung across larval, pupal, and adult stages. this initially suggested that both populations might belong to the same genus or even the same species. nevertheless, molecular analysis of the predator moth samples from sigi yielded different results. phylogenetic analysis based on the mitochondrial coi gene indicated that the samples were more closely related to autoba rubra and mataeomera spp., rather than to eublemma. in the resulting phylogenetic tree, the branch representing the predator moth sample was not closely related to the eublemma clade, indicating budiarsa et al. – molecular identification of lepidopteran predators of coccidae … 989 significant genetic divergence between the sample and that genus. this finding reinforces the understanding that molecular phylogenetic analysis is a highly accurate and objective tool for determining species relationships, complementing morphological approaches that can sometimes be misleading due to convergent evolution or intraspecific variation. as noted by zhou et al. (2000), molecular approaches are particularly important in the classification of taxa with similar morphological traits such as various species of whiteflies and in integrating morphological and molecular data synergistically. furthermore, sheppard et al. (2005) developed an identification key for late instar larvae to distinguish between the genera eublemma and amyna based on the proportional size of abdominal spiracles. one of the key diagnostic features is the ratio of the height of the spiracle on abdominal segment 8 to that on segment 7. in eublemma, the height of the spiracle on segment 8 is usually less than twice that of segment 7, with a ratio of approximately 8:5. in contrast, in amyna, the segment 8 spiracle height exceeds twice that of segment 7, with a ratio of around 12:5. in this context, morphological observations of predator moth larvae from lampung showed that the spiracles on segments 8 and 7 were nearly equal in both height and diameter. the same characteristic was also observed in larvae from sigi. this mismatch with the primary diagnostic traits of both eublemma and amyna further supports the conclusion that morphological identification alone is insufficiently accurate especially for taxa with larval characters that are highly similar or difficult to distinguish. therefore, integrating morphological and molecular analyses is crucial for valid taxonomic identification. in this case, molecular evidence strongly suggests that the predator moth samples from sigi most likely do not belong to the genus eublemma, but are more closely related to autoba rubra. the moth autoba rubra sheppard et al. (2004) was initially described under the name eublemma rubra by the entomologist george hampson in 1902. in his original description, hampson characterized e. rubra as a small moth with a predominantly reddish-brown coloration and distinct white lines on its wings. these morphological traits served as the primary basis for species identification at the time. subsequently, based on a taxonomic review by furlong et al. (2014), eublemma rubra was declared a synonym of autoba rubra, and the latter name is now officially used in scientific literature. this nomenclatural revision reflects the advancement of taxonomic understanding through systematic reclassification of genera based on broader morphological traits and, more recently, molecular data. according to records by peterson et al. (2018), the initial geographic distribution of this species included regions such as sikkim (india), singapore, and java (indonesia), indicating that a. rubra has a relatively wide distribution across tropical south and southeast asia. this suggests strong ecological adaptability to various tropical habitat types. taxonomically, both autoba and eublemma are genera within the family erebidae, a large and diverse family within the order lepidoptera, comprising numerous nocturnal moth species with substantial morphological and ecological diversity. originally, these genera were classified under the family noctuidae. however, since the 1990s, significant revisions have occurred in lepidoptera classification, particularly following the application of molecular phylogenetic methods. genetic studies revealed that noctuidae was overly broad and polyphyletic, necessitating the separation of several groups into distinct families. one of these was the formation of the family erebidae, which now includes several subfamilies formerly placed within noctuidae. in the current taxonomic structure, autoba is classified under the subfamily boletobiinae, whereas eublemma belongs to the subfamily eublemminae (galan et al., 2018). this distinction reflects evolutionary divergence between the two genera, despite certain morphological similarities. such classification is essential for understanding phylogenetic relationships among taxa and for mapping moth diversification in both evolutionary and ecological contexts. nearly all lepidopteran larvae (99%) are phytophagous, feeding on plant material (zaidi et al., 1999; hosseini et al., 2012), while the remainder a small minority function as predators or parasitoids (paula et al., 2016). like other members of the order lepidoptera, several autoba species are known to be phytophagous and have been reported as agricultural pests, including a. silicula (clare et al., 2009), a. abrupta walker, and a. versicolor walker (papura et al., 2020). however, literature on predatory autoba species remains scarce. in thailand, two autoba species have been documented as predators of coccoidea on fruit crops, namely a. rubra and a. coccidiphaga. a. rubra has been reported to prey on the lac insect kerria lacca (kerr) (hemiptera: kerriidae), while a. coccidiphaga preys on several scale insect species, including xenolecanium mangiferae takahashi (hemiptera: coccidae), ceroplastes rubens maskell (hemiptera: coccidae), saissetia nigra king (hemiptera: coccidae), and tachardiella decorella maskell (hemiptera: kerriidae) (barnett et al., 2010). similarly, information regarding the predatory behavior of e. rubra is still limited. according to (hope et al., 2014), e. rubra is an obligate predator of coccus optimum (hemiptera: coccidae) and coccus africanus newstead (hemiptera: coccidae). in addition to the genus autoba, the predator sample also shows phylogenetic affinity with the genus mataeomera. the moth m. dubia has been reported as a predator of saissetia oleae (olivier) (hemiptera: coccidae) on citrus (liu et al., 2024) and parthenolecanium persicae (fabricius) (hemiptera: coccidae) on grapes in australia (rytkönen et al. 2019). m. dubia is considered taxonomically related to the genus catoblemma found in australia, and both genera 990 biology, medicine, & natural product chemistry 14 (2), 2025: 983-992 have been synonymized (casper et al., 2007). the moth a. rubra, formerly known as e. rubra [53], has long been present in indonesia (cinel & taylor, 2019) and is known to prey on armored scale insects (chanin et al., 2015). however, despite its relatively high predation rate (97 ± 11 scale insects per larva) (zeale et al., 2011), the use of predatory moths as biological control agents against armored scale insects has not been extensively studied. to assess the potential of predatory moths as effective biological control agents in the field, further studies are required on their biology, ecology, distribution, control efficacy, and mass-rearing methods. moreover, comprehensive morphological identification is also necessary to complement the available dna-based information. conclusions molecular identification results revealed that the predatory moth preying on soft scale insects (coccidae) in coffea canephora plantations in sigi regency exhibited the closest genetic similarity to autoba rubra, with a dna sequence homology level of 97.91%. acknowledgements: the authors would like to thank tadulako university and galuh university for its support and assistance. authors’ contributions: conceptualization, i made budiarsa and manap trianto; 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(2000). population structure of the pestiferous moth helicoverpa armigera in the eastern mediterranean using rapd analysis. heredity, 85(3), 251-256. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 337-343 | doi: 10.14421/biomedich.2025.141.337-343 issn 2540-9328 (online) encapsulation of extracted oil from mentha piperita in alginate beads kriti shrestha1, manish kunwar1, alina kunwar1, prayan pokharel2,3,* 1department of pharmacy, novel academy, purbanchal university, pokhara 33700, nepal. 2gandaki province academy of science and technology (gpast), pokhara-7, 33700, nepal. 3center for environmental and sustainable agricultural research (cesar), pokhara-10, 33700, nepal. corresponding author* prayanpokharel@cesar.org.np manuscript received: 26 march, 2025. revision accepted: 27 june, 2025. published: 01 july, 2025. abstract encapsulating essential oils derived from traditional medicinal flora within alginate beads represents an up-and-coming technique for enhancing their stability, bioavailability, and controlled release properties. in this study, we employed a clevenger apparatus to extract the essential oil from mentha piperita. the hydrodistillation process of m. piperita yielded an essential oil extraction of 0.27 ± 0.05%. the ionic gelation methodology facilitates the cross-linking of alginate with calcium chloride, forming a gel-like matrix that effectively encapsulates essential oil droplets within stable, gelled beads. the essential oil-loaded beads were quantified spectrophotometrically at a wavelength of 340 nm (λmax). furthermore, we evaluated and characterized the beads for size, weight analysis, sphericity, swelling behavior, dissolution kinetics, ph stability, drying rate, and accelerated stability studies. the size of alginate beads exhibited a significant increase concomitant with an elevation in sodium alginate concentration. the release profile of the oil content demonstrated a more sustained and regulated release within a phosphate buffer at ph 6.8 as opposed to that observed in 0.1n hcl. keywords: mentha piperita; encapsulation; alginate beads; essential oil; ionic gelation. abbreviations: sa: sodium alginate introduction nepal is renowned for its rich herbal diversity, housing over 1,624 species of medicinal and aromatic plants, making it a valuable source of bioactive compounds with therapeutic potential (patel et al., 2021). ethno-medicine, a traditional medical practice based on indigenous knowledge passed down through generations, has gained international recognition due to its contributions to modern drug discovery (chhetri et al., 2020). extracting bioactive compounds from these plants is a growing interest as researchers seek to harness their therapeutic properties and integrate them into modern drug delivery systems (mirghani et al., 2012). essential oils, known for their aromatic and medicinal properties, are among the most valuable products derived from plants. however, they are prone to instability and degradation due to environmental factors such as temperature, light, and oxidation, which reduces their effectiveness and shelf life (maurya et al., 2021). encapsulation technology has emerged as a promising solution, mainly using alginate beads to encase essential oils (hong & park, 1999). alginate, a biopolymer derived from brown seaweed, forms a gel-like structure when cross-linked with calcium ions, making it an ideal medium for protecting essential oils from environmental degradation (mumper et al., 1994). this method also allows for the controlled release of the oils, extending their therapeutic benefits. the ionic gelation technique used in this study offers an efficient means of encapsulating essential oils within alginate beads, optimizing their stability and bioavailability (pisoschi et al., 2018). the primary objective of this research is to prepare alginate beads encapsulating essential oils from medicinal plants. the study aims to enhance the oils stability and effectiveness in medical applications (turchiuli et al., 2005). the rationale behind this work is rooted in the increasing demand for natural medicines and the growing interest in plant-based treatments, which offer potential therapeutic benefits with fewer side effects compared to synthetic drugs. despite the promising potential of essential oils, a significant challenge lies in the lack of standardized procedures for encapsulating these oils and the limited understanding of how the oils interact with the alginate matrix. moreover, the long-term stability of encapsulated oils is uncertain, which poses a barrier to their practical application in medicine. in this study, we extracted essential oils from mentha piperita using a clevenger apparatus. the oils were then encapsulated in alginate beads formed through the ionic gelation technique using sodium alginate and calcium chloride. various factors, such as the beads' size, shape, https://doi.org/10.14421/biomedich.2025.141.337-343 338 biology, medicine, & natural product chemistry 14 (1), 2025: 337-343 and swelling behavior, were evaluated under different ph conditions to simulate the gastrointestinal environment. these experiments aimed to determine how well the encapsulated oils would release in controlled conditions, which is crucial for their potential use in oral drug delivery systems. overall, this research demonstrates the potential of alginate beads as a viable method for encapsulating and stabilizing essential oils. the findings could pave the way for developing plant-based drug delivery systems that bridge traditional medicinal practices with modern pharmaceutical technologies, offering new avenues for using natural compounds in therapeutic applications. materials and methods collection and identification of plant m. piperita was collected from different places in syangja and kaski districts, western nepal, in march 2024 for verification. furthermore, fresh plants were collected during the extraction process. the plants were identified in the national herbarium and plant laboratories (nhpl), kathmandu. essential oil extraction the oil extraction process used a clevenger apparatus. fresh leaves of m. piperita weighing 400 g were collected. the hydro-distillation process involved using a fresh sample at 50°c. in a 1 l flask, 100 g of m. piperita leaves, about 2-4 cm long, were mixed with 500 ml of distilled water. the flask was connected to the clevenger apparatus and heated until it reached boiling point. as the steam formed, it passed through a condenser, which was kept cool by a continuous flow of water. this caused the steam to condense back into liquid form. the essential oil was then separated from the water because the two liquids had different densities. essential oil (eos) loaded beads the extracted essential oil was first combined with three different concentrations of sodium alginate (sa) solution (table 1) on a magnetic stirrer to make the core solution, which was then used to create eos-loaded alginate beads. this mixture ensures uniform distribution of the essential oil within the alginate matrix. the 1% calcium chloride (cacl2) solution was put into a beaker in the first gelling bath. a timer was set to ensure consistency in the gelation process. the essential oil-containing core solution was fully drawn into a pipette and was held 10 cm above the surface of the calcium chloride solution using a ruler to ensure consistency. droplets of the core solution were then carefully released into the calcium chloride bath, with the pipette being moved to a new spot after each drop to prevent fusion of the forming beads. after 10 min for the beads to form, they were removed from the calcium chloride bath using a muslin cloth. the beads were then transferred to a 2% calcium chloride solution to harden, separated from the solution with a sieve, and placed on a paper towel. to enhance bead purity, they were gently rinsed with distilled water. depending on the experiment's needs, the eo-loaded alginate beads were analyzed immediately or air-dried for 24 hours before further evaluation. table 1. preparation of sa and cacl2 solution. sa concentration sa powder (g) distilled water (ml) 1% 1 100 2% 2 100 3% 3 100 cacl2 concentration cacl2 powder (g) distilled water (ml) 1% 1 100 2% 2 100 size, weight, and sphericity analysis of the beads the diameter of beads before and after drying was determined with a vernier caliper at three different positions for each bead with an accuracy of ± 0.01 mm. the mean diameter of 12 beads was calculated (lai et al., 2007). additionally, a digital balance determined the average weight of twelve beads before and after drying. the mean weight was calculated using three separate determinations to represent the bead mass accurately. the sphericity factor (sf) (bayu) was used to indicate the bead where the value zero indicates a perfect sphere and higher values indicate a greater degree of shape distortion (chan, 2011). sf was calculated as: 𝑆𝐹 = 𝐷𝑚𝑎𝑥 – 𝐷𝑚𝑖𝑛 𝐷𝑚𝑎𝑥 + 𝐷𝑚𝑖𝑛 (1) where, dmax = maximum diameter and dmin = minimum diameter swelling test the beads’ swelling rate of the beads was measured at different ph. several beads (3-4 mg) were placed in 6.8 ph phosphate buffer and 0.1n hcl (1.2 ph) solutions at 37°c. the beads were removed and weighed at different time intervals after drying the excess water using filter papers. their weight changes were measured during swelling. the percent swelling ratio (δdt) was calculated using equation 2 (gallo et al., 2020). 𝛥𝐷𝑡 (%) = 𝐷𝑡−𝐷𝑜 𝐷𝑜 𝑋100 (2) where, dt = diameter after time t, d0 =initial bead diameter oil content release the release profile of eos-loaded calcium alginate beads was assessed using a usp type ii dissolution apparatus. the experiment was carried out in 900 ml of phosphate buffer with a ph of 6.8, maintained at 37 ± 0.5°c, and stirred at 50 rpm to mimic basic conditions. it was also conducted in 0.1 n hcl to simulate an acidic shrestha et al. – encapsulation of extracted oil from mentha piperita in alginate beads 339 environment. samples were collected and analyzed at specific intervals using a uv spectrophotometer to measure the oil concentration. the release kinetics were tracked to calculate the percentage of oil released. the oil released into the different ph solutions was measured at each time point, and the release percentage was calculated using equation 3. percent release= amount of oil released total amount of eos-loaded beads x 100 (3) ph stability the beads may show reactions undergoing varying ph conditions in our body fluids. that is why, to evaluate the stability of the eos-loaded alginate beads under varying ph conditions, the beads were subjected to two distinct physiological environments: a phosphate buffer solution at ph 6.8 and a 0.1 n hcl solution (ph 1.2). these conditions were selected to simulate the ph environments encountered in different segments of the gi tract. the phosphate buffer at ph 6.8 was chosen to mimic the slightly acidic to neutral conditions in the small intestine. at the same time, the 0.1 n hcl solution approximated the highly acidic environment of the stomach. drying rate study the oil-loaded beads were dried using two different methods: freeze drying and oven drying. freeze drying was chosen for the experiment due to its superior ability to preserve the oil-loaded alginate beads structural integrity and bioactive properties. unlike conventional drying methods, freeze drying operates by sublimating ice directly into vapor under low temperatures and vacuum conditions, effectively minimizing the thermal degradation of heat-sensitive components. for the freezedrying process, the beads were arranged on drying trays and subjected to freeze-drying under a vacuum pressure of 1 pa at a condenser temperature of −79.5°c for 1 hour. an oven drying method was chosen for the experiment due to its capability to provide controlled and consistent drying conditions. the initial size of the freshly prepared beads was recorded. drying rate studies were conducted by exposing the eos-loaded alginate beads to a constant temperature of 37°c, and changes in structure were analyzed at intervals of 5 minutes using a drying rate curve (lai et al., 2007). stability studies to conduct this study, the dried beads were stored in a closed plastic container and borosilicate glass at room temperature and in a refrigerator. we observed the physical appearance and stability regularly throughout the three months. this long-term stability assessment aimed to understand how well the beads retained their encapsulated essential oil. statistical analysis data analysis was performed using the microsoft excel software package. results are expressed as mean ± standard deviation. results and discussion extraction yield value hydro distillation of m. piperita resulted in an essential oil yield of 0.27 ± 0.05% from a 400 g plant sample. the extracted oils were successfully encapsulated within alginate beads. the plant materials intrinsic oil content and composition play a significant role in yield. additionally, the time and place of collection can impact oil content, as essential oil concentrations fluctuate based on the plant's growth stage, seasonal changes, and environmental conditions specific to the collection location. although steam distillation does not use solvents, the quality of steam or water can influence extraction efficiency. variations in the distillation apparatus, such as the type and efficiency of the equipment, as well as differences in the design and precision of the distillation unit, can also affect the yield. finally, factors such as the duration and temperature of the distillation process are crucial; inadequate distillation time or incorrect temperature settings can lead to incomplete extraction of essential oils. morphological and structural characterization in this study, the shape and size focusing on the sphericity of the formulated beads were determined. it was found that bead size and shape were significantly affected by the sodium alginate concentration. increasing the concentration of sodium alginate (sa) leads to a larger bead size, as shown in figure 1. figure 1. size analysis of eos-loaded beads. the effects of alginate concentration and oil loading on the shape of the ca-alginate beads after oil encapsulation were analyzed, too. figure 2 shows the typical size and shape of the beads in different sodium alginate concentrations. 340 biology, medicine, & natural product chemistry 14 (1), 2025: 337-343 1% sodium alginate 2% sodium alginate 3% sodium alginate figure 2. representing alginate beads in different concentration. the sphericity factor (threshold <0.05) confirmed that beads prepared with 3% alginate were perfectly spherical, even at 40% oil loading, while those with 1– 2% alginate were irregular (sphericity >0.05). higher oil loads (10–40%) in 3% alginate further improved sphericity, with 40% oil yielding the most spherical beads. in contrast, 1–2% alginate produced tear or pearshaped beads with tails, regardless of oil content. thus, 3% alginate is optimal for achieving uniform, spherical beads as shown in figure 3. figure 3. sphericity factor of eos-loaded alginate bead swelling behavior the swelling ratio (δdt), calculated with a basis of the diameter variation using equation (2), was greatly affected by the time of immersion in the liquid medium simulating a marinating solution, as well as by the production process of the alginate beads. after 24 hr of immersion at average room temperature, the beads showed their distinct swelling nature in the liquid medium. table 2. swelling behavior of mentha piperita eos loaded alginate beads. oil content s a c o n ce n tr a ti o n 10% 20% 40% in it ia l d ia m et er ( m m ) f in a l d ia m et er ( m m ) in it ia l d ia m et er ( m m ) f in a l d ia m et er ( m m ) in it ia l d ia m et er ( m m ) f in a l d ia m et er ( m m ) 1% 4.9 3.8 3.5 3.6 2% 4.2 4.44 4.5 4.6 4.2 4.3 3% 4.77 4.9 4.8 4.9 3.3 4.4 oil content release the graph (figure 4) illustrates the comparative oil release profiles of the formulated drug in two different media: a 6.8 ph phosphate buffer and 0.1n hydrochloric acid (hcl), for 30 min. the y-axis represents the percentage of cumulative oil released, while the x-axis represents the time in minutes. the data reveals that drug release occurs more rapidly in a 0.1n hcl (1.2 ph) environment during the initial time, indicating a higher release rate in acidic conditions. however, after this initial phase, the release rates in both environments become comparable, with the 6.8 ph phosphate buffer showing a slightly more sustained release from 10 min. this suggests that while the oil can be released in both acidic and neutral environments, it appears more controlled and sustained in the 6.8 ph phosphate buffers. this could indicate that the beads formulation is better suited for environments closer to neutral ph, such as the small intestine, where a more controlled and prolonged release might be beneficial for maintaining therapeutic levels over an extended period. to achieve controlled and sustained drug release, it is essential to consider factors such as the choice of a polymer matrix, the degree of cross-linking in bead formulations, and the physical characteristics of the beads (e.g., size and porosity). for future studies, optimizing these parameters can further shrestha et al. – encapsulation of extracted oil from mentha piperita in alginate beads 341 improve the drug release profile, ensuring a more targeted and effective delivery in the desired ph environment. figure 4. cumulative oil release % of ca-alginate beads. ph stability the alginate beads loaded with essential oils (eos) degraded in this acidic environment, suggesting that the beads are unstable for extended periods in conditions simulating the gastric environment. the beads remained stable in this near-neutral ph environment, indicating good stability in conditions simulating the small intestinal environment. the degradation in acidic conditions suggests that the alginate matrix is susceptible to acid hydrolysis, which leads to premature release of the encapsulated eos in the stomach. the stability at ph 6.8 indicates that the beads could maintain their integrity in the small intestine. this suggests the potential for targeted delivery to the intestinal region. drying rate the freeze-dried alginate beads felt dry and non-oily to the touch unless squeezed, which released the trapped oil. they were also free-flowing, likely due to their porous structure holding the oil inside. this suggests that there was very little free oil on the surface. we also observed the physical properties of hot air-dried particles, noting and comparing them with those of naturally dried and freeze-dried beads. we found that oven-drying caused the oil to leak from the oil-loaded wet beads during the drying process. as a result, the drying trays and beads were oily and sticky after drying. freeze-drying tends to cause small ruptures in the beads, leading to many smaller particles. table 3. comparison of drying methods. drying method oil loading diameter (mm) sphericity factor initial final oven drying 20% 2.8 1.9 0.19 40% 3.4 2.7 0.11 freeze drying 20% 3.1 2.8 0.05 40% 3.2 2.9 0.04 n=1, nozzle diameter= 0.56 mm the data presented compares the drying rates of alginate beads subjected to two different drying methods: oven drying and freeze-drying at varying levels of oil loading. it is observed that the drying method and oil loading significantly affect the beads final diameter and sphericity factor. for beads with an initial oil loading of 20%, the diameter decreased from 2.8 mm to 1.9 mm under oven drying, while it reduced from 3.1 mm to 2.8 mm under freeze drying. similarly, at 40% oil loading, the diameter decreased from 3.4 mm to 2.7 mm with oven drying and from 3.2 mm to 2.9 mm with freeze drying. the drying method also influences the sphericity factor, which measures how close the shape of the beads is to a perfect sphere. a lower sphericity factor indicates a more deformed shape. for oven drying, the sphericity factor at 20% oil loading is 0.19; at 40% oil loading, it is 0.11, indicating a significant deviation from sphericity. in contrast, freeze-dried beads show much lower sphericity factors of 0.05 and 0.04 for 20% and 40% oil loading, respectively. this suggests that freeze-drying preserves the spherical shape of the beads better than oven-drying. the differences in drying rate between the two methods can be attributed to the different mechanisms involved. oven drying, which involves the removal of water by heat, tends to cause more shrinkage and deformation due to the relatively rapid moisture loss and potential overheating. freeze drying, on the other hand, involves sublimation of water at low temperatures, a gentler process that better preserves the structural integrity of the beads, resulting in less shrinkage and higher sphericity. in summary, freeze-drying is more effective in maintaining alginate beads shape and size than oven drying, which leads to more significant shrinkage and deformation. therefore, the choice of drying method has significant implications for the physical properties of alginate beads, particularly in applications where the shape and size are critical. 0 10 20 30 40 50 60 70 80 0 5 10 15 20 25 30 c u m u la ti v e o il r e le a se % time(min) 6.8 ph phosphate buffer 0.1n hcl 342 biology, medicine, & natural product chemistry 14 (1), 2025: 337-343 figure 5. drying rate of mentha piperita the drying rate curve shows how the size of samples with different oil contents (10%, 20%, and 40%) decreases over time during the drying process. initially, all samples experience a rapid size reduction, indicating a high drying rate as surface moisture is quickly lost. the sample with 10% oil content shows the fastest decrease, suggesting that lower oil content leads to quicker moisture loss and more significant shrinkage. as drying continues, the size reduction rate slows down, particularly for samples with higher oil content, which acts as a barrier to moisture loss. in the final phase, the size of the samples stabilizes, with the 10% oil content sample reaching the smallest final size. overall, higher oil content slows the drying process and helps retain the sample size better, highlighting the impact of oil content on drying behavior. discussion the research results demonstrate the strong influence of alginate concentration and the encapsulation process on the formation and stability of essential oil-loaded beads. the increased bead size with higher sodium alginate concentration can be attributed to the more extensive cross-linking with calcium ions, forming a denser network that traps the oil more effectively. this interaction explains why beads at higher concentrations maintain their spherical shape and structural integrity compared to lower concentrations. the spherical shape in 3% alginate is likely due to the balance between surface tension and gravitational forces during droplet formation, stabilizing the bead structure upon contact with the calcium chloride solution (chan, 2011). the release behavior of the oil-loaded beads also provides insight into the ph-dependent stability of alginate beads. the faster degradation in acidic conditions is caused by acid hydrolysis of the alginate matrix, which weakens the gel structure and results in quicker release of the encapsulated oil. this explains why beads showed more controlled and sustained release in the neutral ph, where the alginate matrix remains intact for extended periods. the role of the encapsulation matrix in regulating oil release is crucial, especially for targeted drug delivery systems designed for the intestinal environment. the drying methods further clarify the effects on bead morphology and integrity. freeze drying preserved the bead structure due to its gentle sublimation process, minimizing shrinkage and maintaining the original spherical shape. the structural integrity is consistent with those reported by (gallo et al., 2020) who observed a similar reduction in particle size and increased surface irregularities, which enhanced the surface area and accelerated the release rate of the encapsulated compound. in contrast, oven drying led to oil leakage and bead deformation, likely because the faster moisture loss from heat caused the matrix to collapse before the oil could stabilize within the beads. thus, freeze drying's lower impact on bead structure validates its superiority for preserving essential oil-loaded beads. conclusions encapsulation of m. piperita, an essential oil in caalginate beads, was successfully achieved using varying concentrations of sodium alginate and oil loadings. the alginate-oil emulsion, with oil loadings up to 40% in 3% sodium alginate solution, remained stable, and the oilloaded, wet beads were spherical and well-formed. after freeze-drying and oven-drying, the oil content remained high, with freeze-dried beads being non-oily, freeflowing, and maintaining their spherical shape. in contrast, the oven-dried beads were oily and deformed, indicating a loss of structural integrity during the drying process, making freeze-drying the preferable method for preserving bead structure and functionality. acknowledgements: the authors would like to acknowledge novel academy and gandaki province academy of science and technology (gpast) for providing the necessary laboratory facilities to conduct this study. authors contributions: this manuscript stems from a bachelor's thesis project. pp designed the study and provided supervision throughout the research. ks, mk, and ak collected the plant samples and prepared the herbarium, with ks and ms verifying the herbarium and conducting the data analysis. ks, mk, and ak performed the laboratory experiments and drafted the manuscript. ks and pp reviewed the draft and made the final revisions. all authors have read and approved the final version of the manuscript. competing interests: the authors declare no conflicts of interest for this study. 0 0,5 1 1,5 2 2,5 3 3,5 0 5 10 15 20 25 30 s iz e (m m ) time (min) 10% oil content 20% oil content 40% oil content shrestha et al. – encapsulation of extracted oil from mentha piperita in alginate beads 343 references bayu, t. (2020). review on contribution of integrated soil fertility management for climate change mitigation and agricultural sustainability. cogent environmental science, 6(1), 1823631. https://doi.org/10.1080/23311843.2020.1823631 chan, e.-s. (2011). preparation of ca-alginate beads containing high oil content: influence of process variables on encapsulation efficiency and bead properties. carbohydrate polymers, 84(4), 1267-1275. chhetri, a., timilsina, s., & miya, m. s. (2020). ethnomedicinal uses of plants by major ethnic groups of hilly districts in nepal: a review. journal of medicinal botany, 24-37. https://doi.org/10.25081/jmb.2020.v4.6389 gallo, t. c. b., cattelan, m. g., alvim, i. d., & nicoletti, v. r. (2020). oregano essential oil encapsulated in alginate beads: release kinetics as affected by electrostatic interaction with whey proteins and freeze‐drying. journal of food processing and preservation, 44(12), e14947. hong, k., & park, s. (1999). melamine resin microcapsules containing fragrant oil: synthesis and characterization. materials chemistry and physics, 58(2), 128-131. lai, f., loy, g., manconi, m., manca, m. l., & fadda, a. m. (2007). artemisia arborescens l essential oil loaded beads: preparation and characterization. aaps pharmscitech, 8, e126e132. maurya, a., prasad, j., das, s., & dwivedy, a. k. (2021). essential oils and their application in food safety. frontiers in sustainable food systems, 5, 653420. mirghani, m., liyana, y., & parveen, j. (2012). bioactivity analysis of lemongrass (cymbopogan citratus) essential oil. international food research journal, 19(2), 569-575. mumper, r. j., huffman, a. s., puolakkainen, p. a., bouchard, l. s., & gombotz, w. r. (1994). calcium-alginate beads for the oral delivery of transforming growth factor-β1 (tgf-β1): stabilization of tgf-β1 by the addition of polyacrylic acid within acid-treated beads. journal of controlled release, 30(3), 241-251. patel, b. p., upadhaya, a., & kumar, s. (2021). conservation, cultivation and sustainable utilization of lesser known plants in nepal. lesser known plants, 193. pisoschi, a. m., pop, a., cimpeanu, c., turcuş, v., predoi, g., & iordache, f. (2018). nanoencapsulation techniques for compounds and products with antioxidant and antimicrobial activity-a critical view. european journal of medicinal chemistry, 157, 1326-1345. turchiuli, c., fuchs, m., bohin, m., cuvelier, m.-e., ordonnaud, c., peyrat-maillard, m., & dumoulin, e. (2005). oil encapsulation by spray drying and fluidised bed agglomeration. innovative food science & emerging technologies, 6(1), 29-35. https://doi.org/10.1080/23311843.2020.1823631 https://doi.org/10.25081/jmb.2020.v4.6389 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 259-266 | doi: 10.14421/biomedich.2025.141.259-266 issn 2540-9328 (online) the impact of social media addiction on adolescent mental health in ambon city, indonesia josephus noya1,*, vernando yanry lameky2 1department of social welfare science; 2department of nursing science, universitas kristen indonesia maluku, ambon, jln ot pattimaipauw, rt.003/rw.003, talake, kel wainitu, nusaniwe, kota ambon, maluku, indonesia. corresponding author* josephusnoya.ukim@gmail.com abstract the increasing prevalence of social media use among adolescents has raised concerns regarding its potential impact on mental health. this study investigated the relationship between social media addiction and adolescent mental health in ambon, indonesia. a quantitative cross-sectional research design was utilized, involving 336 adolescents aged 13-18 who actively used social media. data were gathered through a structured questionnaire assessing demographic characteristics, social media usage patterns, and mental health status using the depression anxiety stress scale (dass-21). descriptive analysis, bivariate analysis using odds ratios (ors), and multivariate analysis via multiple logistic regression were conducted. results indicated that 64.88% of respondents had poor mental health scores. bivariate analysis revealed significant associations between age, study frequency, duration of social media use, and social media usage scores with mental health conditions (p<0.05). multiple logistic regression identified that adolescents aged 18-20 years, those who studied infrequently, used social media for over three hours daily, and had high social media usage scores were at greater risk of poor mental health. these findings underscore the importance of collaborative efforts among stakeholders to design effective interventions and policies that foster responsible social media use and promote adolescent mental well-being in the digital era. keywords: social media addiction; adolescent mental health; social media usage patterns. introduction over the past decade, social media use among teenagers has increased significantly. according to a report from the pew research center, approximately 95% of teenagers in the united states have access to smartphones, and 90% of them actively use social media (waring et al., 2023; martinac dorčić et al., 2024). this phenomenon occurs not only in developed countries but also in indonesia. for example, in maluku province, data from the central statistics agency (bps) show that more than 70% of teenagers in ambon city actively use social media platforms, such as instagram, tiktok, and facebook. this rapid development of digital technology provides easy access to information and communication but poses new challenges, especially concerning adolescent mental health (radjulan et al., 2024; hendri et al., 2023). adolescent mental health is becoming an increasingly important issue, particularly in the digital age. the world health organization (who) states that good mental health is essential to healthy and productive adolescent development (rudnicka et al., 2020). the who also states that stable mental well-being enables adolescents to develop good social skills, improve academic performance, and build healthy interpersonal relationships. however, with the increasing use of social media, there are concerns that adolescents may experience negative impacts such as anxiety, depression, and feelings of alienation (jurow, 2024). therefore, it is important to understand how social media can positively and negatively affect adolescents' mental health (mahbubani, 2012). understanding the impact of social media on adolescents’ mental health is crucial for developing effective interventions and policies. a study conducted by guo et al. showed that increased social media use correlates with increased levels of depression among adolescents (guo et al., 2023). the study found that adolescents who spent more than three hours per day on social media had a higher risk of mental health disorders than those who used social media for a more limited amount of time. this study highlights the need for further attention to factors that influence adolescents' mental health in the context of social media use (twenge et al., 2017). in this context, existing knowledge about the impact of social media on adolescent mental health is mixed. some studies have suggested that social media can provide important social support and help adolescents manuscript received: 25 february, 2025. revision accepted: 20 may, 2025. published: 24 june, 2025. https://doi.org/10.14421/biomedich.2025.141.259-266 mailto:josephusnoya.ukim@gmail.com 260 biology, medicine, & natural product chemistry 14 (1), 2025: 259-266 feel connected to their peers (talukdar, 2024). social media can also allow adolescents to express themselves, identify communities with similar interests, and obtain information relevant to their needs (freeman et al., 2023). however, other studies have shown that social media can be a source of social pressure that contributes to anxiety and depression (zozaya-durazo et al., 2023). for example, primack et al. found that excessive social media use can lead to negative social comparisons, affecting adolescents' self-esteem (primack et al., 2017). the pressure to get "likes" or positive comments on social media can create ongoing anxiety and lower selfesteem. additionally, social media use has the potential to exacerbate mental health problems through cyberbullying (lee et al., 2024). several studies have shown that adolescents who are victims of online bullying tend to have higher stress levels, experience sleep disturbances, and show more severe symptoms of depression than those who are not bullied online (annisa & endang nihayati, 2022). therefore, this phenomenon cannot be ignored, and effective mitigation strategies are required to protect adolescents' mental health (silva et al., 2023). despite extensive research, there are still gaps in knowledge regarding the specific mechanisms by which social media affects adolescents' mental health (sala et al., 2024). for example, few studies have examined the role of algorithms in increasing adolescent exposure to potentially harmful content (zhipeng & gani, 2021). in addition, individual aspects such as adolescents' personalities and levels of psychological resilience may also contribute to how they respond to their experiences on social media (dişli bayraktar, 2024). it is a challenge for researchers and policymakers to identify appropriate strategies to reduce the negative impacts of social media while maximizing its benefits (meisel et al., 2016). addressing this knowledge gap is important for creating effective interventions that support adolescent mental health in the digital age. therefore, collaborative efforts between the government, schools, parents, and social media platforms are needed to design policies and educational programs that can raise awareness of the impact of social media on mental health (román et al., 2021). some strategic steps that can be taken include digital literacy campaigns for adolescents, limiting the time of social media use, and increasing access to mental health services for adolescents who experience negative impacts from social media use (yu & bozeman, 2024). therefore, this study aimed to explore the impact of social media addiction on adolescent mental health. by better understanding the relationship between social media use and mental health, more effective solutions can be found to address this issue and create a healthier digital environment for adolescent development. materials and methods research design this study used a cross-sectional research design to examine the impact of social media on adolescents’ mental health in ambon city (hunziker & blankenagel, 2024). this design allows the simultaneous measurement of variables at a specific time to understand the relationship between social media use and adolescent mental health (hartanto et al., 2024). research approach this study used a quantitative approach to analyze the relationship between social media use and adolescent mental health. quantitative research allows for objectively measuring research variables and produces data that can be processed statistically (lameky & nugroho, 2024). literature review a thorough review of existing literature on social media use and its impact on adolescent mental health was conducted to inform the research framework. the literature includes previous research, theories of adolescent developmental psychology, and studies examining the influence of social media on mental wellbeing (lameky & tasijawa, 2023; plackett et al., 2023b; plackett et al., 2023; plackett et al., 2023c). population and sample the participants in this study were adolescents aged 1318 living in ambon city. the research sample was recruited through two main groups: 168 respondents from smk negeri 1 ambon and 168 students from immanuel karpan church sunday school. the total sample comprised 336 respondents selected using a purposive sampling technique based on the inclusion and exclusion criteria (etikan, 2016). inclusion and exclusion criteria adolescents who participated in this study met several inclusion criteria: those between 15 and 20 years old, active social media users with at least one account, active in the last three months, and residing in ambon city. meanwhile, there were also exclusion criteria that must be considered, namely, adolescents who had been diagnosed with mental health disorders before the study began, those who did not get permission from parents or guardians to participate, and respondents who did not complete the questionnaire. data collection data were collected using a structured questionnaire consisting of three main sections. the first section included demographic data, including information regarding the respondents' age, gender, and study frequency. the second section focused on social media use, which measured the duration of use and the types of noya & lameky – the impact of social media addiction on adolescent mental health … 261 social media platforms used by participants. the third section deals with mental health, assessed using standardized instruments such as the depression anxiety stress scale (dass-21), with good and bad classifications (ahmed et al., 2022). the data collection process was conducted over two months, from september 2 to november 2, 2024, using structured interviews and self-completion of questionnaires by participants. data analysis a descriptive analysis described the participants' demographic characteristics and social media usage patterns. bivariate analysis was applied using odds ratios to measure the effect size of the relationship between the independent variable, social media use, and adolescent mental health as the dependent variable. for further analysis, multivariate analysis using multiple logistic regression was used to control for confounding variables and to determine the main factors contributing to adolescent mental health (kundu & nekoukhou, 2018). research ethics this study applied the principles of research ethics. before participating, all participants were explained the study's purpose, benefits, and procedures. written informed consent was obtained from all participants and their guardians before participation in the study (rafique, 2019). the research ethics committee of stikes maluku husadah approved this study. (rk number. 189/kepk/stik/viii/2024) to ensure compliance with the applicable ethical standards. results and discussion univariate analysis table 1. respondent characteristics. variable category n % age 15-17 years 218 64.88 18-20 years 118 35.12 gender male 134 39.88 female 202 60.12 learning frequency rarely 201 59.82 regularly 135 40.18 duration of social media use <2 hours 117 34.82 >3 hours 219 65.18 type of social media tiktok 168 50 facebook 67 19.94 instagram 50 14,88 youtube 40 11.9 whatsapp or others 11 3.27 social media use score low 134 39.88 high 202 60.12 mental health score good 117 34.82 bad 219 65.18 table 1 shows that the majority of respondents in this study were in the 15-17 age category (218 people, 64.88%), indicating that this age group dominates compared to the 18-20 age category. in terms of gender, more respondents were female (202 people, 60.12%) compared to men. based on the study frequency, more respondents studied as infrequently as 201 people (59.82%) compared to those who regularly studied. regarding the duration of social media use, more respondents used social media for more than three hours per day, indicating that most adolescents had intense social media exposure. regarding the types of social media used, tiktok was the most dominant, with 168 respondents (50%), followed by facebook, instagram, and youtube, which had an almost equal number of users. in contrast, fewer people use whatsapp or other social media platforms. in terms of social media usage scores, more respondents had higher scores, indicating that the intensity of social media use by adolescents was considerable. in terms of mental health scores, 218 people (64.88 %) had a poor mental health score, which illustrates that the majority of adolescents in this study had a poor level of mental health. 262 biology, medicine, & natural product chemistry 14 (1), 2025: 259-266 bivariate analysis table 2. relationship between age, gender, frequency of learning, duration of social media use, type of social media with mental health. variable mental health score or ci 95% good bad lower limit upper limit p-value n % n % age 15-17 years 87 40 131 60 1.51 1.15 1.98 0.007 18-20 years 30 25.5 88 74.5 gender male 41 30.5 93 69.5 2.27 1.57 3.28 0.185 female 76 37.62 126 62.38 learning frequency rarely 60 29.85 141 70.15 2.35 1.74 3.18 0.019 regularly 57 42.22 78 57.78 duration of social media use <2 hours 41 35.04 76 64.96 1.85 1.27 2.70 0.018 >3 hours 76 34.70 143 63.50 type of social media tiktok 50 30 118 70 2.38 1.88 302 0.989 facebook 20 30 47 70 instagram 14 28 36 72 youtube 9 22.5 31 77.5 whatsapp or others 4 36.3 7 63.7 social media use score low 52 38.81 82 61.19 1.58 1.12 2.24 0.001 high 65 32.18 137 67.82 based on table 2, the bivariate analysis results showed that several variables had a significant association with mental health conditions. age was significantly associated with mental health (or = 1.51; p = 0.007), where individuals aged 18-20 years were 1.51 times more likely to have a poor mental score than those aged 15-17 years. the frequency of studying also showed a significant association (or = 2.35; p = 0.019), with individuals who rarely studied having a 2.35 times higher risk of experiencing poor mental health than those who studied regularly. in addition, the duration of social media use of more than three hours per day increased the risk of poor mental health (or = 1.85, p = 0.018). this finding suggests that the longer a person uses social media, the more likely they are to experience poor mental health. furthermore, the social media usage score was significantly associated with mental health conditions (or = 1.58; p = 0.001), where individuals with a high social media usage score were at a higher risk of experiencing poor mental health than those with a low usage score. meanwhile, gender (or = 2.27; p = 0.185) and type of social media (or = 2.38; p = 0.989) were not significantly associated with mental health conditions, despite the difference in distribution in each category. thus, variables with a p-value <0.05, namely age, frequency of study, duration of social media use, and social media use score, were included in the multivariate analysis to identify the most influential factors on mental health. multivariate analysis table 3. multivariate analysis using multiple logistic regression. variable aor se confidence interval (ci) 95% p-value lower limit upper limit age (15-17 years) 1.00 0.15 0.75 1.34 0.007 frequency of learning (rare) 1.00 0.18 0.70 1.42 0.019 duration of social media use (>3 hours) 1.04 0.20 0.70 1.54 0.018 social media use score (high) 1.34 0.25 0.82 2.19 0.001 noya & lameky – the impact of social media addiction on adolescent mental health … 263 based on the multiple logistic regression analysis (table 3), several variables were significantly associated with mental health conditions. the age variable showed a significant association, where individuals aged 18-20 years had a 1.34 times higher risk of having a poor mental health condition than individuals aged 15-17 years (aor = 1.00; 95% ci = 0.75 to 1.34; p = 0.007). furthermore, infrequent study frequency was significantly associated with mental health conditions (aor = 1.00; 95% ci = 0.70 to 1.42; p = 0.019), suggesting that individuals who study infrequently are at a higher risk of poor mental health than those who study regularly. the duration of social media use of more than 3 hours per day also had a significant association (aor = 1.04; 95% ci = 0.70 to 1.54; p = 0.018), where individuals who used social media for more than 3 hours had a higher likelihood of experiencing mental health disorders than those who used social media for less than 3 hours. in addition, high social media usage scores showed a significant association with mental health conditions (aor = 1.34; 95% ci = 0.82 to 2.19; p = 0.001), meaning that individuals with high social media usage scores had a greater risk of experiencing poor mental health than those with low usage scores. thus, the results of this analysis indicate that age, study frequency, duration of social media use, and social media use score significantly influence mental health conditions. discussion age adolescents aged 18-20 years have a higher risk of experiencing poor mental health than adolescents aged 15-17 years. this increased risk can be attributed to the greater social and academic pressures that come with age. twenge et al. found that older adolescents are more prone to anxiety and depression due to increased academic demands and social pressures (twenge et al., 2017). in addition, a longitudinal study by sun et al. showed that increasing adolescent age correlated with an increase in depressive symptoms, especially in those who used social media more frequently (sun et al., 2024). another contributing factor is high exposure to social pressure and cyberbullying, as reported by keles et al., which negatively affects the mental health of older adolescents (keles et al., 2019). from a nursing perspective, roy's stress and adaptation theory explains that individuals attempt to adapt to stressors that arise in their lives (joseph, 2017). in older adolescents, increased academic and social pressure demands stronger coping mechanisms to maintain mental balance. if they are unable to adapt well, adolescents in this age group are more vulnerable to mental health disorders. gender despite the difference in the distribution of mental health between males and females, the results showed that the relationship between gender and mental health was not statistically significant. this indicates that both male and female adolescents are at relatively equal risk of experiencing mental health disorders due to social media use. several studies support this finding. although there were differences in how men and women used social media, the psychological impact did not show significant differences (talukdar, 2024). in addition, keles et al. reported that the more influential factor on mental health is not gender but usage patterns and the type of content consumed (keles et al., 2019). primack et al. also showed that the level of exposure to social comparison and cyberbullying does not differ much between men and women, so the negative impact on mental health is relatively the same (primack et al., 2017). from a nursing perspective, roy's adaptation theory explains that coping mechanisms influence an individual's response to stress more than biological factors such as gender (joseph, 2017). as such, both men and women can experience similar impacts, depending on how they manage the stresses that arise from social media use. frequency of study teenagers who study infrequently are at a higher risk of poor mental health than those who study regularly. this is because they lack a structure and routine that supports mental balance. wahyuningsih et al. found that adolescents with regular study habits have better mental health because structured academic routines help them stay focused and reduce stress (wahyuningsih et al., 2020). in addition, research by clark et al. showed that organized academic activities can increase selfconfidence and reduce adolescent social anxiety (clark et al., 2024). van ryzin et al. also reported that high academic engagement contributed to lowering the risk of mental health disorders by limiting adolescents' time accessing negative content on social media (van ryzin et al., 2022). from a nursing perspective, orem's self-care deficit theory explains that individuals who lack good self-care skills or habits are more vulnerable to mental health problems. in this context, a lack of engagement in academic activities may lead to a lack of structure in daily life, potentially increasing stress and anxiety among adolescents (joseph, 2017). duration of social media use social media use for more than three hours per day increases the risk of poor mental health in adolescents. this is because of increased exposure to factors that can trigger anxiety, depression, and feelings of loneliness. primack et al. found that adolescents who used social media for more than three hours per day experienced higher levels of anxiety and depression compared to those who used it for shorter periods (primack et al., 2017). keles et al. also showed that excessive social media use increases the likelihood of exposure to cyberbullying and negative social comparisons, which can worsen adolescents' mental states (keles et al., 2019). in addition, vogel et al. reported that excessive 264 biology, medicine, & natural product chemistry 14 (1), 2025: 259-266 social media use is associated with decreased emotional well-being and increased feelings of loneliness due to social interactions that occur more online than in real life (vogel et al., 2015). from a nursing perspective, peplau's social interaction theory emphasizes that healthy social interactions play an important role in identity formation and psychological well-being (aslamina et al., 2024). when adolescents spend too much time on social media, they are likely to experience disruptions in in-person social interactions, which can hurt their mental health. type of social media the types of social media used, such as tiktok, facebook, instagram, youtube, and whatsapp, did not have a significant relationship with adolescents' mental health. this suggests that it is not the social media platform itself that determines the impact on mental health but rather how adolescents use it and how long they spend on it. some studies support this finding. mark found that although each social media platform has different characteristics, the psychological impact depends more on the pattern of use and the social interactions that occur (van ryzin et al., 2022). hartanto et al. confirmed that engagement in negative conversations, consumption of unhealthy content, and exposure to cyberbullying have a greater influence on mental health than simply choosing a particular platform (hartanto et al., 2024). syahril showed that the impact of social media on mental health is more influenced by individual factors, such as confidence levels and coping mechanisms, than by the type of application used (syahril, 2024). from a nursing perspective, peplau's social interaction theory explains that an individual's well-being in social interaction is determined more by the quality of the relationships built than by the medium used to communicate (aslamina et al., 2024). therefore, adolescents who are more active on tiktok and those who use facebook or instagram more frequently may experience similar mental health impacts depending on how they interact and the extent to which they are affected by the content consumed. social media usage score adolescents with high social media usage scores were at a higher risk of poor mental health than those with low usage scores. this is because of excessive online engagement, which often triggers social anxiety, feelings of inferiority, and self-image disturbances. mark found that high engagement in social media can increase the risk of social anxiety and low self-esteem due to excessive social comparison (van ryzin et al., 2022). syahril also reported that exposure to unhealthy social media content, such as unrealistic beauty standards, contributes to self-image disorders among adolescents (syahril, 2024). wilson and howard showed that adolescents who are more active on social media are more likely to experience feelings of loneliness, which in turn negatively impacts their mental health (wilson & howard, 2024). from a nursing perspective, lazarus and folkman's stress theory explains that how individuals evaluate and respond to stress will determine their impact on mental well-being (joseph, 2017). if adolescents spend too much time on social media without healthy coping mechanisms, they will be more vulnerable to psychological distress, which can lead to mental health disorders (thorisdottir et al., 2020). conclusions this study has successfully demonstrated the significant relationship between social media addiction and adolescent mental health in ambon city, indonesia. as outlined in the introduction, the rapid increase in social media usage among adolescents has raised concerns about its potential impact on mental well-being. the findings in the results and discussion sections confirm that factors such as age, study frequency, duration of social media use, and social media usage scores significantly influence adolescent mental health. adolescents who use social media excessively, particularly for more than three hours per day, exhibit a higher risk of developing anxiety, depression, and social isolation. additionally, those who study infrequently are more likely to experience poor mental health, emphasizing the importance of structured academic engagement. the results align with previous research highlighting social media's dual impact. while it offers connectivity and information access, it also increases exposure to cyberbullying, social comparison, and psychological distress. the implications of this study extend beyond academia and research, highlighting the urgent need for intervention programs that promote digital literacy, healthy social media habits, and mental health awareness among adolescents. future research should explore the role of specific social media algorithms, psychological resilience factors, and targeted interventions to mitigate the negative effects of excessive social media use. collaborative efforts among educators, parents, policymakers, and healthcare professionals ensure a balanced digital environment fostering adolescent wellbeing in the modern era. authors’ contributions: josephus noya contributed to the conceptualization, methodology, software development, validation, formal analysis, investigation, data curation, writing original draft, and visualization of the 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(2023). social media influencers defined by adolescents. kome, 11(2). https://doi.org/10.17646/kome.of.4 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 693-697 | doi: 10.14421/biomedich.2023.122.693-697 issn 2540-9328 (online) antibacterial potency and physicochemical profiles of eucalyptus pellita leaf waste essential oil from pt surya hutani jaya, east kalimantan wartomo1, farida aryani2, muhammad fikri hernandi1, erna rositah3, sri ngapiyatun4, nur maulida sari1,* 1department of forest product processing; 2department of plantation products technology; 3department of forest management; 4department of plantation management, samarinda state agriculture polytechnic, kampus gunung panjang jalan samratulangi, samarinda 75131, east kalimantan, indonesia. corresponding author* nurmaulidasr@politanisamarinda.ac.id abstract eucalyptus is one of the fast growing species plants, currently used in the pulp and paper industry. the leaves of eucalyptus are known as forest harvesting waste. this research aim to examine the antibacterial potency and the physicochemical profiles of eucalyptus pellita leaves waste essential oils grown in pt surya hutani jaya site 32, east kalimantan named ep b077. the essential oils distilled using water and steam distillation methods. analysis of physicochemical profiles from this oil included yield, colour, refractive index, specific gravity, and chemical compositions by gc-ms. antibacterial activity assayed by agar diffusion method against staphylococcus epidermidis, streptoccoccus sobrinus and streptoccoccus mutans with slight modification. the results showed that ep b077 had the yellow colour, yield oils was 0.44%, 1.471 of refractive index, and 0.8706 of specific gravity. chemical components of ep b077 oils were dominated by α-pinene (40.36%), 1-β-pinene (31.75%), cyclohexene, 1-methyl-4-(1-methylethenyl)(9.64%), trans(β)-caryophyllene (4.29%) and eucalyptol (1,8-cineole) (3.88%). this ep b077 oils was active to inhibit bacteria against s. epidermidis and s. sobrinus range from 9-15 mm, also against s.mutans ranging from 9-14 mm. based on the results, this eucalyptus pellita leaves named ep b077 had potential to develop as a new source of essential oil and antibacterial agents. keywords: eucalyptus pellita; essential oils; antibacterial; α-pinene; 1-β-pinene. introduction globalization and the strengthening of developing economies caused a shift in forest products from west to east. traditional forest areas were north america, western europe and japan. these regions are losing their importance as consumers and producers and are being overtaken by developing countries such as china, india, brazil and indonesia. wood is also supplied by industrial plantations located in, for example, australia, the southern part of the united states, new zealand, asia and south america (mcewan et al., 2020). depending on local conditions and market demands, different species are used in plantation forestry. the dominant genera are pinus, eucalyptus, populus and acacia. the species selected for a particular site must have an acceptable growth rate for plantation forestry to be viable, and the wood properties must be acceptable for the market they serve. the current expansion of eucalyptus and acacia plantations is largely related to global demand for pulp and paper. eucalyptus was known as fast growing species tree, also the main species in pt surya hutani jaya of sinarmas forestry. this species has many benefits and economic value, where the wood uses for pulp and paper, furniture, plywood, also for charcoal industries (kartiko et al., 2021). the leaves part of eucalyptus known as a forest harvesting waste and useless. eucalyptus pellita (red mahogany) is a species native to the tropics of northern queensland, papua new guinea and irian jaya (indonesia), mainly in moist areas such as gentle slopes, riverbanks and alluvial plains, with an annual rainfall of 900-2200mm (hii et al., 2017; kartiko et al., 2021). e. pellita has traditionally been cultivated in southeast asia as a source of fiber, especially for pulp production. it has also been identified as a potential species to complement native hardwoods in the production of solid wood and veneer, and is used in flooring, panels, panelling and general construction (hii et al., 2017). several studies of e. pellita have been reported this species has large biological activities, especially the manuscript received: 29 september, 2023. revision accepted: 12 march, 2024. published: 20 march, 2024. https://doi.org/10.14421/biomedich.2023.122.693-697 mailto:nurmaulidasr@politanisamarinda.ac.id 694 biology, medicine, & natural product chemistry 12 (2), 2023: 693-697 leave parts known as forest harvesting waste. leaves part of eucalyptus had the potential against disease such as cardiovascular, influenza, cancer, and other respiratory infections, also had a many bioactive compounds including phenolic and essential oils (limam et al., 2020). the ministry of health and welfare, japan were involved the natural compounds of eucalyptus leaves extracts in their food industry as additive in natural food and prevention of their various ailments (miguel et al., 2018). furthermore, essential oils of eucalyptus used in various factories and ingredient such as cosmetic, pharmaceuticals, perfume, alternative medicine, food industry also had the functional use such as antiseptic, deodorant, disinfectant, astringent, anesthetic and ets (bachir & benali, 2012; limam et al., 2020). this research aims to examine the antibacterial potency and the physicochemical profiles of eucalyptus pellita leaves waste essential oils grown in pt surya hutani jaya site 32 as industrial plantation forest (hti), east kalimantan named ep b077. we also evaluate the potential antibacterial activity against staphylococcus epidermidis, streptococcus sobrinus and streptococcus mutans. materials and methods plant material the leaves of ep b077 were collected from pt surya hutani jaya site 32, sebulu district, kutai kartanegara, east kalimantan. the samples were washed thoroughly with water to remove the extraporeneous and prepared to air-dried about 2 days in the laboratory with air conditioned (a.c.) set for 20-25ºc. figure 1. eucalyptus pellita b077 by author on pt surya hutani jaya site 32. procedures ▪ distillation of essential oil the essential oils of ep b077 were collected by the water and steam distillation method with slight modification (kartiko et al., 2021). about 8000 g air-dried leaves were distilled for 5 hours, then the oils were collected and separated using a separatory funnel. furthermore, mgso4 was added to the oils. the pure essential oils were sealed in a vial bottle and expressed in percentage of yield. the yield of essential oil was calculated using the following formula (limam etal., 2020): % 𝑦𝑖𝑒𝑙𝑑 𝑜𝑓 𝑜𝑖𝑙 = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑜𝑖𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑖𝑒𝑑 𝑙𝑒𝑎𝑣𝑒𝑠 𝑥 100 ▪ phsycochemical characteristics the physicochemical properties of ep b077 essential oils were characterized by color, specific gravity, and refractive index. the color of essential oils was determined visually (kartiko et al., 2021). the specific gravity was determined using a pycnometer (boukhatem et al., 2020); and the refractive index was determined using a hand refractometer (kartiko et al., 2021). ▪ gas chromatography-mass spectrometry analysis the volatile composition of ep b077 essential oils was analyzed by gc-mc (ultra shimadzu-qp-2010), with an rtx5 column (30 m x 250 µm id) with slight modification (julianus sohilait, 2016), with the following program: from 70ºc to 250ºc at 25.71ºc.minute-1, injection temperature 250ºc, the detector temperature was 250ºc, split ratio 200, the inlet pressure was 98.3 kpa, carries gas helium, and flow rate of 3ml.minute-1. the composition was reported as the peak area percentage and identified by comparing mass spectra with the reference mass spectra in nist databases and literature. antibacterial activity testing antibacterial activity of ep b077 essential oils were test using agar diffusion method with slight modification (kuspradini et al., 2018) also using pathogenic test bacteria, namely staphylococcus epidermidis, streptococcus sobrinus and streptococcus mutans. about 20 ml aliquots of sterile media were transferred to petri wartomo et al. – antibacterial potency and physicochemical profiles 695 dishes and allowed to solidify. the sterile cork borer about 7 mm used to cut the sterile media and 20 µl acetone solution containing 1.25%-20% essential oils were added to the well. chloramphenicol (chp) was used as a positive control at the concentration of 10µg/20 µl in each well. the petri dishes were incubated in the dark for 24 hours at 30-32ºc. the inhibition zone around the well were measured and presented in mm (kartiko et al. 2021). results and discussion the results of percentage yield of ep b077 essential oils from the water and steam distillation process was 0.44%. the other physicochemical profiles of ep b077 essential oils included colour, specific gravity and refractive index are presented in table 1. table 1. physicochemical properties of ep b077 essential oils. no variables information 1. colour yellow 2. refractive index 1.471 3. spesific gravity 0.8706 previously study about e. pellita leaf from pt. surya hutani jaya reported that the oil content was 0.86% using water and steam distillation system (kartiko et al., 2021). other studies, reported the oil content of e. pellita leaf from several indian regions was range from 0.260,44% using hydrodistillation system (aldoghaim, flematti & hammer 2018; kuspradini et al. 2019). season, location, climate, soil type, leafage, fertility regime, drying technique for plant material, and oil extraction technique are just a few of the site-specific edaphic elements that can have a significant impact on oil output (kartiko et al., 2021). table 2. chemical composition of ep b077 essential oils by gc-ms analysis. peak r.timea compounds mfb mwc(g/mol) % area 1. 3.961 α-pinene c10h16 136 40.36 2. 4.141 camphene c10h16 136 1.75 3. 4.492 1-β-pinene c10h16 136 31.75 4. 4.566 β-myrcene c10h16 136 0.58 5. 5.074 benzene,methyl(1-methylethyl)c10h14 134 1.27 6. 5.132 cyclohexene, 1-methyl-4-(1-methylethenyl)c10h16 136 9.64 7. 5.180 eucalyptol (1,8-cineole) c10h18o 154 3.88 8. 7.323 3-cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)c10h18o 154 0.34 9. 7.529 3-cyclohexene-1-methanol, α, α, 4-trimethylc10h18o 154 2.78 10. 9.111 kaur-16-ene c20h32 272 0.48 11. 10.817 trans(β)-caryophyllene c15h24 204 4.29 12. 11.082 (+)-aromadendrene c15h24 204 0.36 13. 11.280 α-humulene c15h24 204 0.47 14. 12.962 spathulenol c15h24o 220 0.68 15. 13.017 (-)-caryophyllene oxide c15h24o 220 1.38 remarks: ar. time (retention time), bmf (molecular formula), cmw (molecular weight). the essential oils of ep b077 rich in monoterpenes group, such as α-pinene, 1-β-pinene, cyclohexene, 1methyl-4-(1methylethenyl)-, trans(β)-caryophyllene and eucalyptol (1,8-cineole). it is different from (aldoghaim et al., 2018), the chemical elements of e. pellita from western ghats of karnataka, india had the highest percentage of eucalyptol (1,8cineole), about >40%, although β-pinene was also the primary component (9.07%). e. pellita leaf oil from west kalimantan contained β-pinene (20.88%) higher than 1,8-cineol (0.72%). other research also reported that 1,8 cineol was not found in the oil from cuba and also investigated in a similar location found 1,8-cineol content in the oil (aleksic sabo & knezevic, 2019; aoki et al., 2019; hou et al., 2019). it might occur because of environmental factors such as location, rainfall intensity, and nutrients in the soil that influence essential oil composition. the pure essential oil could inhibit the growth of all tested bacterias, and the decrease of this activity was observed when diluting the essential oil in 40% ethanol. the inhibition zone diameter of essential oil compared with chloramphenicol (chp) as positive controls are shown in table 3. 696 biology, medicine, & natural product chemistry 12 (2), 2023: 693-697 table 3. inhibition zone and activity indices of ep b077 essential oils against antibacterial. bacteria doses (%) inhibition zone (mm) activity index (%) staphylococcus epidermidis chp 5% 25±0.19 100 1.25 9±0.19 35 2.5 11±0.19 43 5 12±0.38 49 10 13±0.69 55 20 15±0.38 61 streptococcus sobrinus chp 5% 29±3.51 100 1.25 9±0.19 29 2.5 10±0.58 34 5 11±0.19 39 10 12±0.51 42 20 15±0.38 51 streptococcus mutans chp 5% 28±0.38 100 1.25 9±0.51 33 2.5 11±0.38 41 5 12±0.00 44 10 13±0.51 47 20 14±0.38 51 *results are reported as the mean ± sd of three experiments. this pure oil showed the best inhibition against s. epidermidis and s. sobrinus was 15 mm, while against s. mutans was 14 mm. the essential oils of ep b077 was intermediate to inhibit the bacterial. e. pellita oil was intermediate to inhibit e. coli and s. aureus' growth (el atki et al., 2019; guimarães et al., 2019; khammassi et al., 2022; mutlu-ingok et al., 2020). the presence of a monoterpene group, including α-pinene and 1-β-pinene, was predicted as an inhibitor agent against tested bacteria's growth. the chemical constituents contained in the essential oil of e. globulus such as 1,8-cineole, citronellal, citronellol, citronelil acetate, ρ-cymene, limonene, linalol, β-pinene, γ-terpinene, and αterpineol were the potential chemical compound as antibacterial agents (álvarez-martínez et al., 2021; dawood et al.,2021; insuan & chahomchuen, 2020; rafiei et al., 2022). conclusions in this study, eucalyptus pellita leaves named ep b077 had potential to develop as a new source of essential oil and antibacterial agents. acknowledgements: the research was funded by politeknik pertanian negeri samarinda through penelitian dosen pemula scheme (junior lecturer research grants). acknowledgments were conveyed to the pt surya hutani jaya site 32, sebulu district, kutai kartanegara, east kalimantan, who have supported the research by sharing information and plant specimens. authors’ contributions: in this research, wartomo was designed the research, supervised all process and wrote the manuscript. farida aryani was observing and taking research samples. muhammad fikri hernandi controlled the samples and preparation of materials. erna rositah supervised research data analysis and the manuscript. sri ngapiyatun controlled research data analysis and manuscript writing. nur maulida sari supervised all research data analysis and manuscript writing. competing interests: the authors declare that there are no competing interests. references aldoghaim, f. s., flematti, g. r., & hammer, k. a. 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& natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 373-379 | doi: 10.14421/biomedich.2024.132.373-379 issn 2540-9328 (online) antibacterial activity screening of bacillus sp. am12 associated with mangrove soil evellyne natania1, anak agung gede indraningrat2,*, ni wayan widhidewi2 1study program medicine, faculty of medicine and health sciences, warmadewa university, denpasar-bali, indonesia. 2department of microbiology and parasitology, faculty of medicine and health sciences, warmadewa university, denpasar-bali, indonesia. corresponding author* indraningrat@warmadewa.ac.id manuscript received: 30 june, 2024. revision accepted: 26 september, 2024. published: 01 october, 2024. abstract the use of synthetic antibiotics to treat bacterial infections can trigger resistance of pathogenic bacteria to antibiotics. one effort to overcome this is to explore microorganisms that produce antibacterial compounds from nature. a previous study reported isolates of bacillus sp. am12 from mangrove soil displayed antibacterial potential based on preliminary screening of the perpendicular streak test. this study was designed to confirm the antibacterial potential of bacillus sp. am12 uses chemical solvent extraction. bacillus sp. am12 was fermented in 100 ml liquid isp-2 sterile and shaken at 150 rpm for 7 days. the supernatant was filtered from the cell mass using whatman paper and extracted using 100 ml of ethyl acetate solvent (1:1, v/v) twice. the filtrate was evaporated at 40ºc until a thick, clear yellowish colored extract was obtained. the thick extract was tested for antibacterial activity using the kirby-bauer method against two gram positive and two gram negative bacterial targets. antibacterial screening showed moderate diameter zone of inhibition of 6.72 ± 0.21 mm, 6.82 ± 0.15 mm, and 6.62 ± 0.21 mm against staphylococcus aureus atcc 25923, streptococcus mutans fncc 0405, and klebsiella pneumoniae atcc 70060, respectively. however, no antibacterial activity was observed against escherichia coli atcc 25922. analysis of the chemical composition of ethyl acetate crude extracts using gc-ms has detected 10 different compounds, in which the 3 most dominant antibacterial compounds were benzene, 1,2,4-trimethyl-, benzene, 1-ethyl-2-methyland 2-butoxyethyl acetate. in general, these results provide an initial description of bacillus sp. am12 is a potential of antibacterial producer. keywords: antibacterial; bacterial infections; bioprospecting; natural products. introduction bacterial infection is one of the health problems in indonesia and many regions in the world. generally, bacterial infections are treated using synthetic antibiotics such as amoxicillin and tetracycline to inhibit growth or kill infectious bacteria (simanjuntak et al., 2022; susanto, 2020). however, the use of synthetic antibiotics often leads to negative effects such as nausea, vomiting and diarrhoea (eveliani & gunawan, 2021; octavia et al., 2021; parisa et al., 2022). in addition, misused and overused of antibiotics also trigger resistance to pathogenic bacteria which makes them more difficult to be treat (amalia et al., 2017). bacterial resistance can occur as a result of administering antibiotics that are not in the right dose, not in the right diagnosis, and not against the correct target (monica et al., 2018; sugireng & rosdarni, 2020; afrilia & alam, 2022). despite of the importance of good education for society to consume antibiotics wisely, one important aspect is to explore microorganisms that produce secondary metabolite compounds with antibacterial activity from nature, which can later be developed as new antibiotic drugs with better effectiveness against bacterial infections. bacteria play a crucial role as the producers of a variety of secondary metabolites, including antibacterial activity. to date, bioprospecting on bacteria has mainly been focused on exploring terrestrial ecosystems, which consequently increases the chance of dereplication of the same compounds (jose & jha, 2017). therefore, exploration in other ecosystems, especially marine and coastal environments, needs to be explored to obtain bacterial isolates that produce new and diverse antibacterial compounds. the mangrove ecosystem is a meeting point between land and sea estuaries. this area experiences extreme high and low water conditions resulting in the integration of chemical, physical and biological elements of land and sea (djamaluddin, 2018). the mangrove ecosystem shows high biodiversity, including a diversity of types of bacteria that can adapt to variations in high salinity, high temperatures, strong wind pressure, and the availability of water with low oxygen levels (baderan & rahim, 2017). this adaptability causes bacteria in mangrove sediments to produce secondary metabolite compounds, https://doi.org/10.14421/biomedich.2024.132.373-379 374 biology, medicine, & natural product chemistry 13 (2), 2024: 373-379 which have the potential to be developed as alternative treatments for various diseases, such as antibiotics, antioxidants, anticancer and larvicides (ambeng et al., 2019). the ngurah rai bali forest park is the largest mangrove ecosystem in bali. a previous report described 68 bacterial isolates from the ngurah rai mangrove forest, of which 22 of them displayed antibacterial activity (indraningrat et al., 2021). one of the isolates, labelled as bacillus sp. am12 was isolated from the soil habitat of the avicennia marina mangrove plant. previous research did not employ chemical to investigate the antibacterial potential of bacillus sp. am 12 (indraningrat et al., 2021). therefore, this research will be focused to study the isolate bacillus sp. am12 by focusing on fermentation, chemical extraction, antibacterial screening and chemical profiling using gas chromatography and mass spectrometry (gc/ms). it is expected that the results of this research will provide a more comprehensive picture of the potential of bacillus sp. am12 is to be developed as a candidate for producing antibacterial compounds. methods extraction of secondary metabolite components of bacillus sp. am12 stages of extraction of bacterial isolates bacillus sp. am12 begins by growing the isolate in 200 ml of sterile liquid isp-2 medium (4 g/l yeast extract, 10 g/l malt extract, 4 g/l dextrose, 20 g/l bacto agar) and incubating at 28ºc. the isolate in liquid media was shaken using a shaker for 7x24 hours at a speed of 150 rpm. after 7 days, the supernatant of the pure culture was separated from the cell mass by filtration using whatman paper no. 1. the supernatant obtained was extracted using proanalysis ethyl acetate solvent with a ratio of 1:1 (v/v) and repeated twice. the accumulation of macerate is then separated using a separating funnel. at this separation stage, the organic phase and liquid phase fractions formed were screened initially by taking 100 µl samples from each fraction and tested against the bacteria s. aureus atcc 25923, s. mutans fncc 0405, e. coli atcc 25922, and k. pneumoniae atcc 700603. fractions that are proven to have an inhibition zone will be evaporated at a temperature of 40 ºc until a thick extract is obtained, which will then be weighed using an analytical balance. antibacterial activity test the test was carried out using the kirby-bauer method aseptically in a biosafety cabinet (bsc). the first step is to place the disc of the substance to be tested on an agar media that already contains the test bacteria. a total of 200 µl each of the bacteria staphylococcus aureus atcc 25923, streptococcus mutans fncc 0405, escherichia coli atcc 25922, and klebsiella pneumoniae atcc 700603 were suspended in luria bertani (lb) media (10 g/l tryptone, 5 g/l yeast extract, 10 g/l nacl, 20 g/l bacto agar) and spread with a sterile cotton swab. each plate was divided into three quadrants and contains three discs, indicating that for each test on the extract, positive control and negative control for each test bacteria were carried out three times. each sterile paper disc with a diameter of 6 mm was dripped with 20 µl of bacillus sp. am12 extract. next, each paper disc was placed in lb media containing bacterial suspension and then incubated for 24 hours at a temperature of 37°c. the negative control used was ethyl acetate, while the positive control used was the broad spectrum antibiotic levofloxacin. the diameter zone of inhibition was assessed by measuring the halo zone (clear) that formed around the paper disc after 1x24 hours using a calliper. measurements of the diameter of the inhibition zone formed were carried out 4 times in vertical, horizontal and diagonal positions and then averaged. the zone of inhibition that forms would indicate that there is inhibition of bacterial growth due to the presence of antibacterial compounds. interpretation of antibacterial test results will be grouped into 4 categories based on the diameter of the inhibition zone, namely weak (0-5 mm), medium (5-10 mm), strong (10 – 20 mm) and very strong (> 20 mm) (davis & stout, 1971). gram staining morphological characteristics and type of cell wall of bacillus sp. am12 were evaluated using gram staining as previously described. bacterial cells were observed under a light microscope (leica dm750) with 100x magnification(al-enazi et al., 2022). gc/ms analysis briefly, 0.1 grams of the crude extract was sent to the forensic laboratory polda bali for further analysis. the chromatogram results were checked with the literature to determine the type of content and function of the detected bioactive compounds. result fermentation and extraction extraction of cell free supernatant of bacillus sp. am 12 using ethyl acetate resulted in two layers in the mixture, namely the clear organic phase fraction and the yellowish liquid phase (figure 1). the organic phase contains secondary metabolite compounds from extracted bacterial isolates, while the liquid phase only contains primary metabolites and solvents. antibacterial testing was carried out on each fraction phase with the aim of determining which phase had antibacterial activity. natania et al. – antibacterial activity screening of bacillus sp. … 375 figure 1. extraction of cell free supernatant of bacillus sp. am 12 using ethyl acetate resulted in organic and liquid phases. based on the preliminary test results, it was found to be the inhibition zone was only present in the organic phase (figure 2). after the organic phase was evaporated, a thick, clear yellowish-colored extract was obtained, then 1 ml of ethyl acetate was added to obtain a clear yellowish colored liquid extract weighing 1.40 grams (figure 1). figure 2. pre-screening for organic phase (a) and liquid phase (b) of ethyl acetate bacillus sp. am12 against bacterial tests, namely 1: s. aureus atcc 25923, 2: k. pneumoniae atcc 700603, 3: s. mutans fncc 0405, and 4: e. coli atcc 25922. gram staining of bacterial isolate sp.am12 the gram staining test of the sp.am12 isolate was gram-positive. morphological characteristics showed that the isolate has rod-shaped bacteria as shown under microscope observation (figure 4). figure 4. gram staining of bacillus sp. am12 under light microscope observation (1000x magnification) antibacterial activity test antibacterial screening showed that the extract of bacillus sp. am12 inhibited s. aureus atcc 25923 with an average inhibition zone diameter of 6.72 ± 0.21 mm, s. mutans fncc 0405 with an average of 6.82 ± 0.15 mm, and k. pneumoniae atcc 700603 with an average of 6.62 ± 0.21 mm (figure 5 and table 1). based on this result, the diameter zone of inhibition is classified as moderate activity. however, the antibacterial activity of bacillus sp. am12 was much smaller than levofloxacin, which is the positive control in this study. levofloxacin even has antibacterial activity against e. coli atcc 25922. meanwhile, the extract of bacillus sp. am12 has no inhibitory effect on e. coli atcc 25922. figure 5. antibacterial activity test results of bacillus sp extract. am12 against a: s. aureus, b: s. mutans, c: k. pneumoniae, and d: e. coli. results of positive control antibacterial activity test (levofloxacin) against e: s. aureus, f: s. mutans, g: k. pneumoniae, and h: e. coli. results of negative control antibacterial activity test (ethyl acetate) against i: s. aureus, j: s. mutans, k: k. pneumoniae, and l: e. coli. 376 biology, medicine, & natural product chemistry 13 (2), 2024: 373-379 table 1. results of antibacterial screening. test bacteria sample mean inhibition zone diameter (mm) ± sd interpretation staphylococcus aureus atcc 25923 bacillus sp extract. am12 6.72 ± 0.21 moderate levofloxacin 17,26 ± 0,59 strong ethyl acetate 0±0 streptococcus mutans fncc 0405 bacillus sp extract. am12 6,82 ± 0,15 moderate levofloxacin 18,80 ± 0,49 strong ethyl acetate 0±0 klebsiella pneumoniae atcc 70060 bacillus sp extract. am12 6,62 ± 0,21 moderate levofloxacin 19,22 ± 0,90 strong ethyl acetate 0±0 escherichia coli atcc 25922 bacillus sp extract. am12 0±0 levofloxacin 14,64 ± 1,58 strong ethyl acetate 0±0 analysis gc-ms the gc/ms analysis results (figure 6) showed that 156 peaks of compounds were detected. of these many compounds, ten compounds corresponded to antibacterial activity (table 2) present in the ethyl acetate crude extract of bacillus am 12. these ten compounds have been previously reported to play a role as antibacterial in other studies after literature search. figure 6. gc/ms chromatograms of ethyl acetate crude extracts of bacillus sp. am12. table 2. results of gc-ms analysis of bacterial isolates bacillus sp. am12. compounds molecular formula activity peak areas retention time references benzene, 1,2,4-trimethylc9h12 antibacterial 7.59 4.7 (andila and nugroho, 2022) 2-butoxyethyl acetate c8h16o3 antibacterial 6.87 7.3 (khedhri et al., 2023) bis(2-ethylhexyl) phthalate c24h38o4 antibacterial 5.20 24 (javed et al., 2022) n-hexadecanoic acid ch3(ch2)14cooh antibacterial 5.07 19 (ganesan et al., 2022) benzylmonoxime c12h11no2 antibacterial 4.92 3.9 (abuskhuna et al., 2020) dodecanoic acid c12h24o2 antibacterial 2.81 14.8 (shen et al., 2021) ethanol, 2-butoxyc6h14o2 antibacterial 2.65 3.3 (woiski et al., 2020) oleic acid c18h34o2 antibacterial 2.59 20.7 (dilika et al., 2000) mesitylene c9h12 antibacterial 1.32 4.11 (savithri and rajakumar, 2019) benzene, 1-ethyl-2-methylc9h12 antibacterial 1.08 4.36 (safara et al., 2022) natania et al. – antibacterial activity screening of bacillus sp. … 377 discussion fermentation and extraction fermentation of bacillus sp am12 was carried out in liquid isp-2 media for 7 days so that the number of secondary metabolites produced by bacteria was maximized. bacterial growth occurs for 5-7 days, referred to as the stationary phase, in which there is an increase in the amount of bacterial cell biomass so that more secondary metabolites are formed (syarifuddin et al., 2022). however, the speed of bacterial growth in this phase is influenced by the bacterial growth media, such as nutrition, ph, temperature, and air humidity. thus, the bacterial growth medium used is a liquid fermentation medium so that the composition and concentration of the medium are easier to regulate according to optimal environmental conditions for bacterial growth (saida et al., 2022). liquid isp-2 media was chosen because it consists of simple carbon bases such as glucose, as well as organic nitrogen sources such as malt and yeast extract, which help growth, pigment formation, and the production of antibacterial substances (al-enazi et al., 2022). the bacterial fermentation media will change color to become cloudier as the bacteria grow in it. the more concentrated or turbid a medium is, the higher the number and mass of bacterial cells in it (saida et al., 2022). extraction can be defined as the process of separating or pulling out a material from a mixture using a certain solvent until the concentration between the material and the solvent reaches equilibrium (azis, 2020). in this research, the liquid-liquid extraction method uses a separating funnel with ethyl acetate as the solvent to obtain an organic phase and a liquid phase to separate compounds based on their relative solubility (saida et al., 2022). ethyl acetate was chosen as a solvent because it is semipolar, can attract polar and nonpolar solvents, and is volatile, non-toxic, and non-hygroscopic (pratama et al., 2017). the organic phase has an inhibition zone because this phase contains secondary metabolite compounds from bacterial isolates that have been extracted. in contrast, the liquid phase only contains primary metabolites and solvents (serrano et al., 2021). the organic phase extract was concentrated using a rotary evaporator to obtain a thick extract. after evaporation, a thick, clear yellowish-colored extract was obtained, and then 1 ml of ethyl acetate was added to obtain a clear yellowish-colored liquid extract weighing 1,401 grams. antibacterial activity test the antibacterial activity test was carried out using the kirby-bauer method because the method is rather straightforward, affordable, and does not require complicated equipment (rahayu et al., 2022). results of antibacterial activity screening indicated that the crude extracts displayed moderate activity against three out of four bacterial tests (s. aureus, s. mutans, and k. pneumoniae). generally, the antibacterial activity produced by bacillus sp occurs through two mechanisms, namely directly and indirectly. the mechanism for inhibiting bacterial growth directly occurs through antibiosis and competition for nutrients (rori et al., 2020). meanwhile, the mechanism for inhibiting bacterial growth indirectly occurs due to the production of bioactive compounds that can inhibit bacterial growth (rori et al., 2020). endophytic bacteria isolated from mangrove plants have been reported to produce several bioactive compounds that act as antibacterials, such as halocarbon, terpenoids, coumarins, alkaloids, peptides, and polyketides (that et al., 2019; khabthani et al., 2021). in this study, the results of the antibacterial test showed that there was no antibacterial activity in the isolated extract of bacillus sp. am12 against e. coli atcc 25922 so that there is no zone of inhibition around the test disc. this can happen because bacteria have different abilities to develop resistance to antimicrobial agents (reygaert, 2018). the exact antibacterial action of bacillus sp am12 is unknown. thus, it is likely that the mechanism of action is in line with the resistance pattern of e. coli bacteria. some of the resistance mechanisms of e. coli are modifying antimicrobial targets, efflux pumps, porin loss, and producing β-lactamase and carbapenemase enzymes (nurjanah et al., 2020). antibacterial test for bacillus sp. am12 showed that the average diameter of the resulting inhibition zone is smaller than the positive control. this could happen because the quantity and quality of secondary metabolites produced is less than optimal. several aspects that can influence metabolite production are media optimization, fermentation time, and bacterial growth cycles (mulyani et al., 2023). therefore, each of these aspects needs to be optimized further in order to produce the ideal antibacterial compound. the gc-ms results showed that there were 10 different compounds detected in the crude extracts, with the 3 most dominant compounds being benzene, 1,2,4trimethyl-, benzene, 1-ethyl-2-methyl-, and 2butoxyethyl acetate. a study by putra et al. (2024) confirmed that the compound 2butoxyethyl displays antibacterial activity against gram-negative and grampositive bacteria. the hexadecanoic acid methyl ester was also detected in the crude extract, and the compound is one of the fatty acid groups with antibacterial properties. the mode of action of hexadecanoic acid is to destroy the structure of the bacterial cell wall and membrane (arista et al., 2020). hexadecanoic acid was also reported to display antioxidant activity by providing proton donors to dpph, which turns into non-radical dpph (rismiyatun et al., 2024). research by vargas et al. (2021) stated that oleic acid is a compound that actively inhibits the growth of gram-positive bacteria such as s. aureus and s. pneumoniae (vargas et al., 2021). 378 biology, medicine, & natural product chemistry 13 (2), 2024: 373-379 conclusion overall, this research showed that ethyl acetate extracts of bacillus sp. am12 displayed moderate antibacterial activity with an average inhibitory zone diameter of 6.72±0.21 mm, 6.82±0.15 mm, and 6.62±0.21 mm, respectively, against the bacteria s. aureus atcc 25923, s. mutans fncc 0405, and k. pneumoniae atcc 70060. however, antibacterial activity was not observed against e. coli atcc 25922. the presence of 10 different active molecules, which have been associated with antibacterial activities, provides an important clue regarding the potential of bacillus sp. am12 for synthetisizing antibacterial compounds. future research should be focused on optimizing the growth of bacillus sp. am12 uses different growth media and chemical solvents and to screen extracts against multidrugresistance bacteria strains. acknowledgments: the author would like to thank pande putu christine putri purnami, s.si., m.si., desak gede dwi agustini, a.md, kes., and ni made defy janurianti, s.tp., m.tp for assisting during lab experiment and analysis. specific interest: the authors declare that there is no conflict of interest. funding: this research was financially funded by hibah penelitian fundamental direktorat riset, teknologi dan pengabdian kepada masyarakat kementerian pendidikan, kebudayaan, riset, dan teknologi tahun 2023 awarded to anak agung gede indraningrat (fundamental research grants directorate of research, technology and community service ministry of education, culture, research and technology year 2023) no grant 184/e5/pg.02.00.pl/2023; 3538/ll8/al.04/2023; 837/unwar/lemlit/pd-13/2023.. references abuskhuna, s.m., zeglam, t.h., fhid, o.n.r., jebril, a.o., 2020. antibacterial activity of hydroxyimidazole derivatives. asian j. pharm. 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generation of antibacterial agents. pmc. doi:10.1016/j.plipres.2021.101093. woiski, c., dobslaw, d., engesser, k.-h., 2020. isolation and characterization of 2-butoxyethanol degrading bacterial strains. biodegradation 31, 153–169. https://doi.org/10.1007/s10532020-09900-3 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 7-14 | doi: 10.14421/biomedich.2021.101.7-14 issn 2540-9328 (online) compliance level of textual therapeutic usage of kshirakakoli containing formulations with a serial ethnomedicinal survey and modern system of medicine gunpreet kaur1, vikas gupta1, ravinder sharma2, sanjiv kumar3, rg singhal4, ranjit singh4, parveen bansal1,* 1university centre of excellence in research, baba farid university of health sciences, faridkot, punjab, india 2university institute of pharmaceutical sciences, faridkot, punjab, india 2regional ayurveda research institute, ranikhet, uttarakhand, india 3shobhit university, meerut, uttar pradesh, india 4shobhit university, gangoh, uttar pradesh, india corresponding author* ucer_bfuhs@rediffmail.com, reetjattana21@gmail.com manuscript received: 19 november, 2020. revision accepted: 28 june, 2021. published: 01 july, 2021. abstract fritillaria roylei (kshirakakoli) is a primal plant used in ancient times. but nowadays, due to biotic and abiotic stress the plant has entered in the list of threatened medicinal plant. in ancient texts effective uses of formulations containing kshirakakoli are well mentioned but the information is not written in simple language due to which the therapeutic value of the plant is not well understood by scientific fraternity. so, there is a major need to perform ethno medicinal survey for the formulations containing kshirakakoli and compare their therapeutic uses as mentioned in text with the modern system of medicine. in this study, a field survey was performed in 4 states i.e., uttar pradesh, uttrakhand, punjab and himachal pradesh where the conversation regarding usage of this plant or formulation was done with 24 local medical practitioners, 18 shopkeepers and 4 traditional healers. the information thus obtained were recorded and then compared. results showed that the usage of kshirakakoli containing formulations was highest in himachal pradesh and uttrakhand. only few clinical studies have been done on these formulations. the effectiveness of the formulations against remedies alluded by the trado-medical practitioners claimed was found accurate as per ayurvedic textual literature. hence, the ethno medicinal survey provides a precise guidance to scientists for future research on these kshirakakoli containing formulations that are useful in plethora of disorders. keywords: ethnomedicinal survey; kshirakakoli; formulations; ayurveda; fritillaria roylei. introduction today, plant products, animal products, minerals and metals are used by pharmaceutical industries for production of majority of the ayurvedic as well as allopathic medicines. on the basis of information obtained from the traditional healers nearly 121 pharmaceutical products have been discovered (anesini and perez, 1993; kshiti, 2012). the knowledge of medicinal plants started fading away with the desertion of gurukul system of ancient teaching, as written details of most of the medicinal plants are not available (sharma and balkrishna, 2005). this also happened with eight plants of ashtawarga that are considered as very good rasayana with rejuvenating and health promoting properties that strengthens the immune system. due to high therapeutic properties, these plants are used in various ayurvedic formulations like taila (medicated oil), ghritam (medicated clarified butter), churana (powder) and other formulations of traditional medicinal system including chywanprash and other disease preventive tonics (dhyani et al., 2010). ‘ashtawarga’ a significant constituent of a variety of classical ayurvedic formulations has been assigned a variety of medicinal properties by ancient materia medica dealing with ayurveda and a subject of rigorous botanical research. kshirakakoli known as fritillaria roylei (one out of group of eight ashtwarga plants) come under the category of threatened species (saha et al., 2015). due to limited distribution in their natural habitat, today substitutes of ashtawarga plants are commonly used in ayurvedic formulations to meet the market demand. department of ayush, govt. of india has suggested use of substitutes in formulations in absence of original plants however this option is being exploited by manufacturers rendering such an important and precious plant in ignored condition (sagar, 2014; tewari,1991). hence it becomes important to highlight the therapeutic potentials of this plant in front of scientists so that a justified research shall be carried out on an important but ignored plant. fritillaria roylei contain various https://doi.org/10.14421/biomedich.2021.101.7-14 8 biology, medicine, & natural product chemistry 10 (1), 2021: 7-14 active compounds like peimine, peiminine, peimisine, peimiphine, peimidine, peimitidine, propeimin, sterol and these active compounds possess galactogogue, haemostatic, ophthalmic and cytotoxic properties (chi et al., 1940; wu, 1944; chou, 1947; chatterjee et al., 1976; jiang et al., 2006). due to the lack of authentic species in natural habitats, systemized studies/clinical studies have not been carried out on this group of plants. a very few clinical studies related to its therapeutic potentials have been carried out. moreover, the information available is also highly scattered that is not organized in proper manner due to which it cannot be used by scientist groups working on such important plants. this plant is being used in a number of formulations of high therapeutic value as mentioned in ancient texts however a very limited data with scientific evidences is available in modern literature as well as on internet sources. ancient texts claim very potent uses of these formulations but as the information is available in regional languages or in sanskrit, so the real uses of the plant are not well understood by scientific fraternity. hence it becomes important to know the real status of the mentioned therapeutic potentials as well as practiced potentials by trado-medical practitioners (tmps) and put it in front of scientists so as to give a thrust to clinical studies on therapeutic effects as well as pharmacological actions of the plant. material and methods in india, particularly in the rural and remote areas, there are number of trado-medical practitioners (tmps) and various kshirakakoli containing ayurvedic formulations are used by these practitioners as a traditional medicine for the treatment of a variety of ailments. so, a folklore survey was taken in 2019 to recognize the therapeutic importance of available kshirakakoli containing formulations mainly in four states of northern india. a field survey was conducted around and in district headquarters and discussion was done with the 24 local medical practitioners, 18 shopkeepers and 4 traditional healers and the information related to these ayurvedic formulations was recorded. the information procured was validated by comparing the information given by at least four tmps. the medicinal uses of these formulations were recorded from the folklore claims and the standard literature of the indian systems of medicine. an effort has been made to highlight the traditional use of these formulations so as to enable the scientists to explore these formulations for further research studies. result and discussion the folklore survey was done in various districts in himachal pradesh (shimla, dharamshala, kangra, mandi, kullu and manali), punjab (sangrur, barnala, ludhiana, bhatinda, patiala, ferozepur, faridkot), uttrakhand (dehradun, haridwar, pauri garhwal, rudraprayag) and in uttar pradesh (agra, meerut, moradabad, mathura). it was observed that usages of 20 formulations containing kshirakakoli were maximum in himachal pardesh and uttrakhand followed by uttar pradesh and least in punjab. the excerpts of ayurvedic formulations containing kshirakakoli as one of the ingredients are listed in table 1. the compliance level of therapeutic uses of kshirakakoli containing formulations as per ancient literature with trado-medical practitioners (tmps) and pre-clinical/clinical trials/case studies as per modern system of medicine are enumerated as in table 2. these formulations have been indicated in a plethora of reproductive disorders, loss of digestion, insanity, depression, depletion of body tissue, emaciation, phthisis, cures gout arthritis pervading the whole body, heart disease, facial paralysis, diseases of the head/neck and epilepsy. similar type of ethnomedicinal survey was conducted by the authors for kakoli containing formulations (kaur et al., 2019). so, there is a need of hour to explore kakoli and kshirakakoli containing important formulations for further trials. table 1. excerpts of ayurvedic formulations containing kshirakakoli. s. no. formulation shloka’s references 1. jeevaneeya gana तद्यथा – जीवकर्षभकौ मेदा महामेदा काकोली क्षीरकाकोली मुदपर्णीमार्पर्ण्यो जीवन्ती मधुकमममत दशेमामि जीविीयामि भवन्तन्त hebbar, 2015a 2. shukrala / kamdev ghrita शुक्रलैजीविीयैश्च ब ृंहरै्णबषलवधषिैैः | क्षीरसञ्जििैशै्चव पयैः मसद्धृं प थक् प थक्||६|| युक्तृं गोधूमचूरे्णि सघ तक्षौद्र शकष रम्| पयाषयेर्ण प्रयोक्तव्यममच्छता शुक्रमक्षयम्||७|| hebbar, 2015a 3. khuddaka taila/ khuddaka padmaka tailam पद्मकोशीर यष्ट्याह्व रजिी क्वाथ सामधतम्| स्यात् मपषै्ैः सजष ममञ्जष्ठा वीरा काकोमल चन्दिैैः ||११४|| खुड्डाक पद्मकममदृं तैलृं वातास्र दाहिुत्|११५| इमत खुड्डाक पद्मकृं तैलम्| hebbar, 2016a kaur, et al. – compliance level of textual therapeutic usage of kshirakakoli … 9 4. madhuparnyadi taila मधुकस्य शतृं द्राक्षा खजूषरामर्ण परूर्कम्| मधूकौदिपाक्यौ च प्रस्थृं मुञ्जातकस्य च||९६|| काश्मयाषढकममते्यतच्चतुद्रोरे्ण पचेदपाम्| शेरे्ऽष्भागे पूते च तन्तमृंसै्तलाढकृं पचेत्||९७|| तथाऽऽमलक काश्मयष मवदारीकु्ष रसैैः समैैः | चतुद्रोरे्णि पयसा कल्कृं दत्त्वा पलोन्तितम्||९८|| कदम्बामलकाक्षोट पद्म बीज कशेरुकम्| hebbar, 2016a 5. mahasneham ghrita जीवकर्षभकौ मेदाम ष्यप्रोक्ताृं शतावरीम्| मधुकृं मधुपर्णीं च काकोलीद्वयमेव च||७२|| मुद्ग मार्ाख्यपमर्णषन्यौ दशमूलृं पुििषवाम्| बलाम ता मवदारीश्च साश्वगन्धाश्मभेदकाैः ||७३|| एर्ाृं कर्ाय कल्काभ्ाृं समपषसै्तलृं च साधयेत्| लाभतश्च वसा मज्ज धान्व प्रातुद वैन्तिरम्||७४|| चतुगुषरे्णि पयसा तत् मसद्धृं वात शोमर्णतम्| hebbar, 2016a 6. paste prepared of drugs belonging to jivaniya group, cow-milk and muscle fat/ dhanwantharam thailam समूलाग्रच्छदैरण्डक्वाथे मद्व प्रान्तस्थकृं प थक्| घ तृं तैलृं वसा मज्जा चािूप म ग पमक्षर्णाम्||१४१|| कल्काथे जीविीयामि गव्यृं क्षीरमथाजकम्| हररद्रोत्पल कुषै्ठला शताह्वाश्वहिच्छदाि् ||१४२|| मबल्व मात्राि् प थक् पुष्पृं काकुभृं चामप साधयेत्| मधून्तच्छष् पलान्यष्ौ दद्याच्छीतेऽवताररते||१४३|| hebbar, 2016a 7. sneha parisheka से्नहै मषधुर मसदै्धवाष चतुमभषैः परररे्चयेत्| स्तम्भाके्षपक शूलातं कोषै्णदाषहे तु शीतलैैः ||१२५|| hebbar, 2016a 8. parushaka ghrita त्रायन्तन्तका तामलकी मद्वकाकोली शतावरी| कशेरुका कर्ायेर्ण कलै्करेमभैः पचेद्ध तम्||५८|| दत्त्वा परूर्का द्राक्षा काश्मयेकु्षरसाि् समाि्| प थन्तिदायाषैः स्वरसृं तथा क्षीरृं चतुगुषर्णम्||५९|| एतत् प्रायोमगकृं समपषैः पारूर्कमममत म तम्| वातरके्त क्षते क्षीरे्ण वीसपे पैमिके ज्वरे||६०|| इमत पारूर्कृं घ तम्| hebbar, 2016a 9. bala ghrita/taila बलाममतबलाृं मेदामात्मगुप्ाृं शतावरीम्| काकोली ृं क्षीरकाकोली ृं रास्नाम न्तद्धृं च पेर्येत्||५६|| घ तृं चतुगुषर्ण क्षीरृं तैैः मसद्धृं वातरक्तिुत्| हृत्पाणु्डरोग वीसपष कामला ज्वर िाशिम्||५७|| hebbar, 2016a 10. sukumara taila/ sukumara kashaya मधुकस्य शतृं द्राक्षा खजूषरामर्ण परूर्कम्| मधूकौदिपाक्यौ च प्रस्थृं मुञ्जातकस्य च||९६|| काश्मयाषढकममते्यतच्चतुद्रोरे्ण पचेदपाम्| शेरे्ऽष्भागे पूते च तन्तमृंसै्तलाढकृं पचेत्||९७|| तथामलक काश्मयष मवदारीकु्षरसैैः समैैः | चतुद्रोरे्णि पयसा कल्कृं दत्त्वा पलोन्तितम्||९८|| कदम्बामलकाक्षोट पद्म बीज कशेरुकम्| शृङ्गाटकृं शृङ्गवेरृं लवार्णृं मपप्पली ृं मसताम्||९९|| hebbar, 2016a 11. snehopaga gana/ (adjuvants of snehana/oleation treatment) म द्वीका मधुक मधुपर्णी मेदामवदारी काकोली क्षीरकाकोली जीवक जीवन्ती शालपर्ण्यष इमत दशेमामि से्नहोपगामि भवन्तन्त hebbar, 2016a 12. shukrala shukrajanana/ shukra-shodhana-janana granules जीवकर्षभक काकोली क्षीरकाकोली मुदपर्णी मार्पर्णी मेदाव द्धरूहा जमटला कुमलड्गा इमत दशेमामि शुक्रजििामि भवन्तन्त hebbar, 2016a 13. jivaniya mahakashaya तैलप्रस्थृं घ तप्रस्थृं जीविीयैैः पलोन्तितैैः | क्षीरद्रोरे्ण पचेत् मसद्धमपमारमविाशिम्||२८|| hebbar, 2016a 14. amruta prasha ghruta/ amritaprasha ghrita जीवकर्षभकौ वीराृं जीवन्ती ृं िागरृं शटीम्| चतस्रैः पमर्णषिीमेदे काकोल्यौ दे्व मिमदन्तिके||३५|| पुििषवे दे्व मधुकमात्मगुप्ाृं शतावरीम्| ऋन्तद्धृं परूर्कृं भागीं म द्वीकाृं ब हती ृं तथा||३६|| शृङ्गाटकृं तामलकी ृं पयस्याृं मपप्पली ृं बलाम्| बदराक्षोट खजूषर वातामामभरु्कार्ण्यमप||३७|| hebbar, 2015b 10 biology, medicine, & natural product chemistry 10 (1), 2021: 7-14 15. phalakalyan grita/ taila uttara basti कमर्णषन्य चरर्णाशुियोमि प्राक्चरर्णासु च||१०२|| कफवाते च दातव्यृं तैलमुिर बन्तस्तिा| hebbar, 2016b 16. siva gutika/shiva gritham मेदाृं पयस्याृं जीवन्ती ृं मवदारी ृं कण्टकाररकाम्| श्वदृं ष््ाृं क्षीररकाृं मार्ाि् गोधूमाञ्छामलर्मष्काि्||८|| पयस्यधोदके पक्त्वा कामर्षकािाढकोन्तिते| मववजषयेत् पयैः शेर्ृं तत् पूतृं क्षौद्रसमपषर्ा||९|| युक्तृं सशकष रृं पीत्वा व द्धैः सप्मतकोऽमप वा| मवपुलृं लभतेऽपत्यृं युवेव च स हृष्यमत||१०|| hebbar, 2016b 17. vrishya pooplika फलािाृं जीविीयािो न्तििािाृं रूमचकाररर्णाम् । कुडवशू्चमर्णषतािाृं स्यात् स्वयड्गुमाफलस्य च ॥ १५ ॥ कुडवशै्चव मार्ार्णाृं द्वौ च मतलमुद्र्यो :। गोघूमशामलचूर्णाषिाृं कुडव: कुडवो भवेत्॥ १६ ॥ hebbar, 1998 18. upatyakari shashtikadi gutika आमसक्त क्षीरमापूर्णषमशुिृं शुद्ध र्मष्कम्| उदूखले समापोथ्य पीडयेत् क्षीरममदषतम्||३|| ग हीत्वा तृं रसृं पूतृं गवे्यि पयसा सह| बीजािामात्मगुप्ाया धान्य मार् रसेि च||४|| hebbar, 2016c 19. mahamayura ghrita/ mahanarayan tailm एतेिैव कर्ायेर्ण घ त प्रस्थृं मवपाचयेत्| चतुगुषरे्णि पयसा कलै्करेमभश्च कामर्षकैैः ||१६६|| जीवन्ती मत्रफला मेदा म द्वीकमधष परूर्कैैः | समङ्गा चमवका भागी काश्मरी सुरदारुमभैः ||१६७|| आत्मगुप्ा महामेदा ताल खजूषर मस्तकैैः | hebbar, 2015c 20. jivantyadi ghrita एवमेव क्षीर समपष जीविीयोपसामधतम्||६९|| गभषदृं मपिलािाृं च योिीिाृं स्यान्तिर्न्तितम्|७०| hebbar, 2015c table 2. compliance level of therapeutic uses of kshirakakoli containing formulations as per ancient literature with trado-medical practitioners (tmps) and pre-clinical/clinical trials/case studies as per modern system of medicine. sr. no. formulations therapeutic uses as per texts therapeutic uses as mentioned by trado-medical practitioners (tmps) pre-clinical/clinical trials/case studies as per modern system of medicine 1. amruta prasha ghruta/ amritaprasha ghrita 1. cough 2. hiccup 3. fever 4. asthma 5. burning sensation 6. morbid thirst 7. an ailment characterized by bleeding from different parts of the body 8. vomiting 9. fainting 10. diseases of heart 11. female genetial tract 12. urinary tract 13. procreation of male child 14. joint problems and other diseases caused by unbalanced vata & pitta doshas. yes yes yes yes yes yes yes yes yes no yes no yes yes no no no no no no no no no no no no no yes improves the nonspecific immunity against rheumatoid arthritis (lekurwalel et al., 2010). 2. jeevaneeya gana 1. enlivening 2. anti-aging 3. antioxidant yes yes yes no no no 3. jivantyadi ghrita 1. cure female infertility 2. cure cataract and glaucoma yes yes no yes effective in treatment and controlling of computer vision syndrome and helpful in curing myopia (vinaik et al., 2013; shukla et al., 2011). kaur, et al. – compliance level of textual therapeutic usage of kshirakakoli … 11 4. khuddaka taila/ khuddaka padmaka tailam 1. gout 2. burning sensation yes yes no no 5. madhuparnyadi taila 1. cures vatarakta 2. pain in limbs 3. affliction of the whole body 4. it also promotes strength and complexion. yes yes yes yes no no no no 6. mahamayura ghrita/ mahanarayan tailm 1. neuro-muscular disorders 2. cough 3. asthma 4. inflammation 5. diseases of the female genital tract 6. menstrual disorders yes yes yes yes yes yes no no yes yes used to treat inflammation, pain and arthritis, useful in treatment of chronic back pain, useful in treatment of female infertility, provide strength to the local soft tissues, thus play significant role in the management of cervical spondylosis or osteoarthritis of cervical spine (pawar et al., 2011; panda and debnath, 2011; kaushik et al., 2017; kunjibettu et al., 2017). 7. mahasneham ghrita 1. gout 2. serious diseases caused by the aggravated vata 3. arthritis yes yes no no 8. paste prepared of drugs belonging to jivaniya group, cow-milk and muscle fat/ dhanwantharam thailam 1. cures pain 2. gout 3. kyphosis (spinal disorder) no yes yes no no no 9. phalakalyan grita/ taila uttara basti 1. uterine tonic 2. gynecological disorders yes yes no yes useful in treatment of female infertility (kunjibettu et al., 2017). 10. shukrala/ kamdev ghrita 1. improves semen and sperm production yes no 11. siva gutika/shiva gritham 1. diabetes mellitus 2. bronchitis 3. anemia 4. cardiac diseases 5. epilepsy 6. psychotic diseases 7. skin diseases 8. infertility in women/men 9. liver diseases no no yes yes yes yes no yes yes no no no no no no no no yes used in treatment of chronic liver and highly effective in treatment of hiv/aids and acute deep vein thrombosis (wasedar et al., 2017; rathod et al., 2013; pusuluri et al., 2017). 12. sneha parisheka 1. stiffness 2. convulsion 3. gout 4. burning sensation yes yes yes yes no no no no 12 biology, medicine, & natural product chemistry 10 (1), 2021: 7-14 13. sukumara taila/ sukumara kashaya 1. lower abdominal pain in women 2. menstrual pain 3. inflammation 4. constipation 5. promotes robustness in body and positive health yes yes yes yes yes yes yes yes yes yes helps in improving the quality of the ovum by regularizing the menstrual cycle which enhanced rate of conception and also used in treatment of complete rectal prolapse (vanishree and kumari, 2017; shripathi et al., 2017). 14. upatyakari shashtikadi gutika 1. male fertility yes no 15. vrishya pooplika 1. aphrodisiac yes no 16. bala ghrita/taila 1. gout 2. enemia 3. jaundice 4. uterine diseases 5. provides strength to children no yes relieves constipation in pregnant women (sharma et al., 2017). no yes helps in improving females reproductive disorders (patel, 2018). yes message contributes positive effects to health of new born child (sharma et al., 2017). 17. parushaka ghrita 1. menstrual pain 2. menorrhagia 3. anemia 4. respiratory disorder 5. jaundice yes yes act as good uterine tonic by providing strength to uterus (vartak and mehere, 2019) no no no 18. shukrala shukrajanana/ shukrashodhanajanana granules 1. improves quality and quantity of semen and sperm yes provides strength and increases sperm count (girish, 2017). 19. snehopaga gana/ (adjuvants of snehana/oleation treatment) 1. dry skin conditions 2. neuromuscular disorders 3. muscular dystrophy 4. wasting disorders no no no no 20. jivaniya mahakashaya 1. provides strength 2. immunity booster 3. improves quality of semen 4. aphrodisiac 5. hormonal disturbances in women 6. nervousness dizziness no no no no no no conclusion the analysis of literature reveals that fritillaria roylei is a wonder plant used by saints/rishies since ages however due to a number of reasons; plant has been ignored for its therapeutic uses. this survey clearly indicates that there is a need of deciphering the textual references given in regional languages and use them for new drug development process. as clearly indicated in textual as well as in survey, most of the kshirakakoli kaur, et al. – compliance level of textual therapeutic usage of kshirakakoli … 13 containing formulations have been recommended for number of disorders in men and women. however, there is lack of such patent formulations as well systemized clinical trials that could prove to be useful in highlighting the real therapeutic potentials of kshirakakoli containing formulations. hence this survey provides a template for scientists for further screening and research on these formulations that are useful in plethora of disorders. financial support: none acknowledgment: the authors are thankful to the tradomedical practitioners (tmps) for sharing their precious traditional knowledge. conflict of interest: the authors declares that there are no conflicts of interest concerning the publication of this article. references anesini, c., perez, c. 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(1944). the constituents of fritillaria roylei. j. am. chem. soc., 66(10) 1778-1780. doi: https://doi.org/10.1021/ja01238a048. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 141-146 | doi: 10.14421/biomedich.2024.131.141-146 issn 2540-9328 (online) phytochemical profile and antibacterial activity of nigella sativa against biofilm-producing bacteria uropathogens abdulazeez mumsiri abaka1,*, mubarak muhammad dahiru2, keta biman abubakar1, jordana luka1, aisha abubakar1, tukur baba abdullahi3, saminu hamman barau2 1science laboratory technology department, school of science and technology, adamawa state polytechnic yola, nigeria. 2pharmaceutical technology department, school of science and technology, adamawa state polytechnic yola, nigeria. 3science laboratory technology department, modibbo adama university yola, nigeria. corresponding author* abdulazizelnino22@adamawapoly.edu.ng abstract this study explores the antibacterial effects of nigella sativa seeds on bacteria obtained from clinical samples. the aim was to assess the antibacterial properties of both aqueous and methanolic extracts of nigella sativa seeds against e. coli, s. aureus, and p. aeruginosa. the three samples were collected from the microbiology laboratory of modibbo adamawa medical centre and were reconfirmed using culture, microscopy, and some biochemical tests. the seed samples of n. sativa were procured from herbal point yola, adamawa state, nigeria. the phytochemical assay of the extracts revealed the presence of flavonoids, alkaloids, tannins, phenols, cardiac glycosides, steroids, saponins, and terpenoids in both extracts. the highest antibacterial activity against s. aureus, e. coli, and p. aeruginosa was demonstrated by the aqueous extract of n. sativa seeds, with inhibition zone diameters of 19.30 ±0.61 mm, 8.10 ±2.17 mm, and 12.00 ±0.29 mm, respectively. however, the methanol extract exhibited slightly greater activity against e. coli and p. aeruginosa, with inhibition zone diameters of 12.10 ±0.38 mm and 13.80 ±0.40 mm, respectively. both methanol and aqueous extracts showed minimum inhibitory concentrations (mics) of 25 mg/ml against s. aureus and e. coli. similarly, for p. aeruginosa, the mic was 25 mg/ml for methanol extract and 50 mg/ml for aqueous extract. the minimum bactericidal concentration (mbc) for both extracts against s. aureus and e. coli was determined to be 25 mg/ml. however, for p. aeruginosa, the mbc was 25 mg/ml for the aqueous extract and 50 mg/ml for the methanol extract. the study indicates that n. sativa seed extract possesses antibacterial properties against s. aureus and p. aeruginosa, underscoring its potential as an effective medicinal antibacterial agent. keywords: bacteria; phytochemical; biofilm; uropathogens; nigella sativa. abbreviations: uti (urinary tract infection), mic (minimum inhibitory concentration), mbc (minimum inhibitory concentration), ae (aqueous extract), me (methanol extract). introduction throughout history, natural remedies, particularly those derived from plants, have been employed for medicinal purposes due to their diverse array of components thought to combat various infectious ailments (sharma et al., 2023). plant biodiversity serves as a valuable reservoir of chemical compounds with therapeutic potential, including antiviral, antibacterial, antifungal, and anticancer properties (dar et al., 2023). medicinal plants serve as a valuable reservoir of bioactive compounds that are commonly employed in traditional medicine practices, nutraceuticals, dietary supplements, modern pharmaceuticals, and synthetic drug development (pammi et al., 2023). in recent times, there has been a notable increase in utilizing plants for therapeutic purposes for several reasons, including their easy accessibility without prescription, affordability, natural origins, and potential to cut reliance on synthetic drugs with severe side effects (sati et al., 2024). moreover, plants have a long-standing reputation as a valuable source of new drug compounds. herbal combinations have significantly benefitted human health and overall well-being (jamal, 2023). various secondary metabolites found in plants, such as tannins, terpenoids, flavonoids, alkaloids, and quinines, possess antimicrobial properties (arora et al., 2024). extensive research has been dedicated to examining the chemical composition and pharmacological effects of n. sativa seeds. these seeds, as well as the oils derived from them, are recognized for their diverse healthpromoting properties, including antitumor, antioxidant, anti-inflammatory, antibacterial, and immune-stimulating effects (ojueromi et al., 2022). consequently, they are frequently utilized as nutritional supplements. n. sativa seeds, known worldwide by various names such as "seed manuscript received: 01 april, 2024. revision accepted: 20 may, 2024. published: 27 may, 2024. https://doi.org/10.14421/biomedich.2024.131.141-146 142 biology, medicine, & natural product chemistry 13 (1), 2024: 141-146 of blessing" (habbat-ul baraka), "habbatussauda" (in hausa), black caraway, and black cumin, among others, are purported to possess a wide range of actions, encompassing both antibacterial and anticestodal effects (usman et al., 2017). in islamic tradition, the black seed is believed to serve as a universal remedy for various ailments, except aging or death (nisar et al., 2023). the increasing resistance of microorganisms to numerous standard antibiotic therapies presents a global challenge and raises significant public health concerns (akram et al., 2023). the efficacy of current drugs is diminishing due to the proliferation of multi-drugresistant bacterial strains, including pneumococci resistant to penicillin and macrolides, methicillinresistant staphylococci, vancomycin-resistant enterococci, and multidrug-resistant gram-negative organisms (moiketsi et al., 2023). therefore, there is an urgent need to find alternatives for the treatment of various diseases caused by diverse microbial agents. synthetic drugs are not only expensive and inadequate but also often have issues with adulterations and side effects (hamidi, 2023). with the current advancement of technology, scientists are challenged to come out with new ideas for alternative and novel drugs to dazed the usage of microbial-resistant drugs (ahmed et al., 2023). black seed extracts have also proven to be potent antibacterial agents against specific pathogenic gram-positive and gram-negative bacteria (usman et al., 2017). the surge in antibiotic resistance represents a critical global health crisis, acknowledged by governments as one of the paramount challenges to public health (salam et al., 2023). resistance to antibiotics is increasingly becoming a serious global problem, reaching dangerous levels. this resistance poses significant challenges to effectively treating infectious diseases worldwide. additionally, it undermines the effectiveness of many medical advancements (salam et al., 2023). urinary tract infection (uti) remains a prevalent issue globally, affecting both community and hospital settings, with approximately 150 million cases reported annually worldwide (rauniyar, 2023). the rise of antibiotic resistance has become increasingly prominent and poses a significant challenge in uti management, largely ascribed to the formation of biofilms (maione et al., 2023). within the epithelium lining of the bladder, certain uropathogenic bacteria have been observed to form intracellular bacterial groups with characteristics akin to biofilms. (lila et al., 2023). these biofilmproducing bacteria undergo alterations in growth rate and genetic expression. consequently, biofilms hinder the diffusion of substances and the binding of antimicrobial agents, creating an effective barrier against large molecules such as antimicrobial proteins lysozyme, and complement (lu et al., 2023). biofilm is structured with layers of cell clusters enveloped within a matrix of extracellular polysaccharides, referred to as polysaccharide intracellular adhesion (pia) (maione et al., 2023). responsible for over 80 % of microbial infections, biofilms contribute to dogged infections and recurrences (lila et al., 2023). the formation of biofilms by uropathogenic bacteria is regarded as a pathogenic trait, facilitating colonization and resulting in elevated rates of uti (zhou et al., 2023). consequently, these infections pose challenges in treatment due to the emergence of multiple drug resistance. nonetheless, there is limited research conducted on the antibacterial activity of n. sativa extracts against biofilm-producing bacterial uropathogens and pathogenic bacteria in adamawa state, nigeria. this study aimed to assess the antibacterial potential of n. sativa extracts against various bacterial uropathogenic isolates. materials and methods collection of plant materials seeds of n. sativa was obtained from a vendor located at yola market, nigeria, and their authenticity was verified by a botanist from the department of plant science at modibbo adama university in yola. the specimen voucher number is mau/pls/0712. subsequently, the seeds were finely ground into powder using a blender. extraction aqueous and methanolic extracts were prepared following the procedure outlined by usman et al. (2017). in brief, 350 g of n. sativa powder was soaked separately in 500 ml of distilled water and methanol for three (3) days at 25 oc using percolation. subsequently, the mixtures were filtered using whatman's no. 1 filter paper and evaporated using a rotary evaporation apparatus. the resulting extracts were then further dried in a hot air oven at 50 oc for 24 h and stored at 4 oc until further analysis. concentrations of extracts: each extract, weighing 1 gram (1g), was individually dissolved in 1 milliliter (1 ml) of 10% dimethyl sulfoxide (dmso) to obtain a stock solution with a concentration of 1000 mg/ml. from this stock solution, four different concentrations were prepared by serial dilutions, resulting in concentrations of 100, 50, 25, and 12.5 mg/ml, achieved through dilutions ranging from 101 to 10-4. bacterial strains: the biofilm-producing bacterial strains, including s. aureus, e. coli, and p. aeruginosa, were obtained from the department of microbiology at modibbo adama university in yola. inocula were prepared by transferring 3-5 colonies of each bacterial strain into 5 ml of nutrient broth. the inoculated broths were then incubated at 35 oc for 2-3 h until reaching the logarithmic growth phase. abaka et al. – phytochemical profile and antibacterial activity of … 143 subsequently, the bacterial suspensions were adjusted to match the 0.5 mcfarland standard for susceptibility assay, following the guidelines outlined by the national committee for clinical laboratory standards (loh et al., 2023). antibacterial sensitivity assay the antibacterial assay of n. sativa extracts against the bacterial isolates was assessed using the agar well diffusion method, following the protocol outlined by dahiru et al. (2023). sterile nutrient agar was poured into sterile petri dishes and left to solidify. a sterile swab stick was immersed into a standardized inoculum and used to spread the bacteria evenly on the agar surface in aseptic conditions, with proper labeling. the inoculated plates were left undisturbed for 30 min to facilitate proper adhesion of the organisms to the agar surface. subsequently, four wells were aseptically bored into the agar using a sterile cork borer with a diameter of 6 mm. subsequently, the wells were filled with 0.2 ml of n. sativa extracts at concentrations of 100 mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml, and 6.25 mg/ml, respectively. positive control wells were filled with 0.2 ml of a 20 mg/ml ciprofloxacin solution, while negative control wells were filled with 0.1 ml of dmso. the plates were left to dry and then incubated at 37 oc for 24 h. after incubation, zones of inhibition around the wells were observed, measured, and recorded in millimeters. minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) the broth dilution assay, as outlined by dahiru et al. (2023) was employed for testing the extracts. the extracts were diluted to 10-fold concentrations in nutrient broth. to each dilution, 0.1 milliliters of standardized bacterial inoculum was added. negative control tubes devoid of bacterial inoculation were prepared concurrently. the tubes were then aerobically incubated at 37 oc for 24 h. the mic was identified as the lowest concentration of the extract that hindered the growth of the test bacterium. to determine the mbc, a loopful from each tube with no visible growth in the mic assay was transferred onto fresh nutrient agar plates (oxoid). these plates were then incubated at 37°c for 24 h, followed by observation and recording of any growth. statistical analysis the values were analyzed via the statistical package for social sciences (spss) version 16 and were presented as means ± standard error of the mean (se). comparisons between different groups were revealed using a one-way analysis of variance (anova), followed by duncan's multiple range test (dmrt). the significance level was set at p < 0.05. results table 1. phytochemical screening results for methanol and aqueous extracts of n. sativa seeds are as follows. s/n phytochemical presence (+) and absence (-) in different extracts methanol extract aqueous extract 1 alkaloids + + 2 flavonoids + + 3 phenols + + 4 tannins + + 5 cardiac glycosides + 6 steroids + + 7 saponins + 8 terpenoids + + key: + positive -negative table 2. zone of inhibition (mm) of the organism caused by aqueous and methanol extracts of n. sativa. concentration (mg/ml) zone of inhibition (mm) e. coli s. aureus p. aeruginosa cipro extract aqet mtet aqe mte aqe mte 100 8.93 ±0.15a 12.10 ±0.38 19.30 ±0.61b 16.03 ±0.37 12.00 ±0.29a 13.80 ±0.40 50 8.43 ±0.35a 10.80 ±0.65 12.43 ±0.23bc 14.03 ±0.12c 9.83 ±0.20ac 12.03 ±0.18c 25 7.03 ±0.09c 8.07 ±0.32cd 7.40 ±0.27acd 11.10 ±0.40cd 6.50 ±0.17acd 10.20 ±0.12cde 12.5 6.43 ±0.23cd 7.30 ±0.42cd 6.23 ±0.15acd 7.97 ±0.12cde 5.90 ±0.38cd 6.97 ±0.12cde 20 35.01 ±0.01 30.02 ±0.01 30.02 ±0.02 values are the mean of triplicate determinations (±sem), aqet = aqueous extract treatment, mtet = methanol extract treatment, cipro = ciprofloxacin values with a superscript in the same row are significantly lower (p < 0.05) than the mtet for the same organism values with b superscript in the same row are significantly (p < 0.05) higher than the mtet of the same organism values with c superscript in the same column are significantly (p < 0.05) lower than the 100 mg/ml concentration values with d superscript in the same column are significantly (p < 0.05) lower than the 50 mg/ml concentration values with e superscript in the same column are significantly (p < 0.05) lower than the 25 mg/ml concentration all values were significantly lower than cipro at 20 mg/ml concentration. 144 biology, medicine, & natural product chemistry 13 (1), 2024: 141-146 table 3. minimum inhibitory concentration of n. sativa extracts against s. aureus, e. coli, and p. aeruginosa. organisms extract concentration (mg/ml) 100 mg/ml 50 mg/ml 25 mg/ml 12.5 mg/ml 6.25 mg/ml 3.125 mg/ml s. aureus. ae * + + me -* + + + + e. coli ae -* + + me -* + + + p. aeruginosa ae -* + + + me -* + + + + key: ae= aqueous extract me= methanol extract * = mic value + = turbidity = no turbidity table 4. minimum bactericidal concentration of n. sativa extracts on test isolates. test organisms aqueous extract (mg/ml) methanol extract (mg/ml) s. aureus 25 25 e. coli 25 25 p. aeruginosa 25 50 results and discussion natural products are renowned for their diverse array of secondary bioactive metabolites, which exhibit various pharmacological activities in living organisms, often serving as a form of defense (al-khayri et al., 2023). crude methanol and aqueous extract of n. sativa crude contains alkaloids, phenols, flavonoids, tannins, cardiac glycosides, saponins, terpenoids, and steroids, as seen in table 1. this observation aligns with the reports of shafodino et al. (2022). these phytochemicals have different distinctive biological functions. therefore, the qualities of these phytochemicals in n. sativa gave optimism that this plant, if screened properly, could eventually give a template for medicine (dalli et al., 2021). the antibacterial assay varied greatly in terms of inhibitory potential. table 2 shows the antibacterial activity of four n. sativa doses against s. aureus, e. coli, and p. aeruginosa. aqueous extract (ae) of n. sativa seeds had the highest activity against s. aureus, e. coli, and p. aeruginosa, with inhibition zones measuring 19.30 ±0.61 mm, 8.10 ±2.17 mm, and 12.00 ±0.29 mm, respectively. nonetheless, the methanol extract (me) demonstrated marginally higher effectiveness against e. coli and p. aeruginosa, showcasing inhibition zone diameters of 12.10 ±0.38 mm and 13.80 ±0.40 mm, respectively. the findings indicate that the methanol extract of n. sativa inhibited bacterial growth more effectively than the aqueous extract. the type of solvent for extracting bioactive components from plant extracts depends on specific properties of the compounds being targeted and their solubility characteristics. some common solvents used for this purpose include methanol, ethanol, acetone, chloroform, and water, among others. each solvent comes with its own set of advantages and disadvantages, and the selection is typically influenced by factors such as the polarity of the compounds being extracted and the intended application of the extracted components (lefebvre et al. 2021). usman et al. (2017) obtained similar results using the same genus of plant in their study. however, a contrary result was obtained by balogun et al. (2019), which was conducted in maiduguri, nigeria which showed that aqueous extract had no antibacterial activity on s. aureus. overall, methanol extracts were more bioactive than aqueous extracts. several factors could influence the agar-well diffusion technique, with inoculum size being among them smaller inoculum sizes can lead to potentially exaggerated inhibition zones, whereas larger sizes might result in underestimated zones (chandran et al. 2023). hence, it's crucial to ensure the proper alignment of the inoculum suspension with the mcfarland standard (0.5). the extracts were more efficient against grampositive than gram-negative bacteria. this may be ascribed to gram-negative bacteria's excellent permeability barrier, such as the outer membrane, which limits amphipathic compound penetration, as well as multi-drug resistant pumps that release toxins over the barrier (saxena et al., 2023). this permeability barrier could be the fundamental explanation for the apparent ineffectiveness of plant antibacterial activity. this discovery is congruent with the findings of abdallah et al. (2023), who investigated the same plant genus. both me and ae showed mic values of 25 mg/ml against s. aureus and e. coli. similarly, for p. aeruginosa, the mic was 25 mg/ml for methanol extract and 50 mg/ml for aqueous extract. the observations align with the outcomes documented by balogun et al. (2019) and sulaiman and muhammad, (2023). however, abraham et al. (2019) observed lower mic values of 32, 1.28, and 1.28 mg/ml for n. sativa against e. coli, s. abaka et al. – phytochemical profile and antibacterial activity of … 145 aureus, s. typhi, and s. pyogenes. the differences in mic values across various bacteria suggest that the extract's efficacy in inhibiting bacterial growth may be influenced by the specific characteristics and vulnerabilities of individual bacterial species (ezzaky et al., 2023). the mbc for both extracts against s. aureus and e. coli was determined to be 25 mg/ml. however, for p. aeruginosa, the mbc was 25 mg/ml for the ae and 50 mg/ml for the methanolic extract. these results contrast with those reported by usman et al. (2017) while sulaiman and muhammad (2023) obtained comparable results, with an mbc value of 50 mg/ml for salmonella species and 100 mg/ml for escherichia coli. the different mbcs of a plant extract among diverse bacteria indicate that the extract's efficacy in killing bacteria may vary due to the individual traits and vulnerabilities of each bacterial species (al-garadi et al., 2022). a substantial association has been instituted by statistical analysis between the concentrations used and the zone of inhibition. according to both extracts, there was a robust and positive association value for every examined bacterium (n = 4). an expanded inhibitory zone diameter produced by bacteria suggests a positive correlation with concentration. conclusion this study reveals that aqueous and methanol extracts of n. sativa seeds have antibacterial activity against s. aureus, e. coli, and p. aeruginosa. consequently, it highlights the considerable potential of n. sativa as a valuable antibacterial agent for medicinal applications. acknowledgments: special gratitude goes to the department of science laboratory technology, adamawa state polytechnic yola. authors contributions: for example, abdulazeez mumsiri abaka & mubarak muhammad dahiru designed the study. abdulazeez mumsiri abaka, jordana luka, keta biman abubakar & aisha abubakar carried out data collection and laboratory work. abdulazeez mumsiri abaka, mubarak muhammad dahiru, saminu hamman barau & tukur baba abdullahi wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are competing interests. funding: no funding. references abdallah, e. m., alhatlani, b. y., de paula menezes, r., & martins, c. h. g. 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(2023). urinary tract infections caused by uropathogenic escherichia coli: mechanisms of infection and treatment options. international journal of molecular sciences, 24(13), 10537. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1129-1135 | doi: 10.14421/biomedich.2025.142.1129-1135 issn 2540-9328 (online) beneficial effect of sumbawa wild horse milk yogurt on lipid profile and cardiovascular risk in rats on a high-cholesterol diet laksmi nur fajriani*, ni made wiasty sukanty, dea pratiwi silpia putri, esya mariska febriani nutrition study program, faculty of health, bumigora university, jl. ismail marzuki no.22, cilinaya, mataram 83127, tel. (0370) 634498, indonesia. corresponding author* laksmifajriani07@gmail.com manuscript received: 06 october, 2025. revision accepted: 29 november 2025, 2025. published: 03 december, 2025. abstract cardiovascular disease (cvd) is a leading cause of death in indonesia, with a high prevalence due to a high-cholesterol diet that triggers hypercholesterolemia. the use of statins as a long-term pharmacological therapy often causes side effects, so safe natural alternatives are needed. sumbawa wild horse milk is a potential functional food source that contains essential fatty acids and lactic acid bacteria (lab), which have probiotic potential. this study aims to evaluate the effect of sumbawa wild horse milk yogurt on total cholesterol, hdl, ldl, atherogenic index (ai), and cardiac risk ratio (crr) levels in hypercholesterolemic male wistar rats. a total of 30 rats were divided into six groups: normal, positive control (high-cholesterol diet), negative control (simvastatin), and three treatment groups, t1, t2, and t3, with doses of sumbawa wild horse milk yogurt of 2, 3, and 4 ml/day, respectively, for 28 days. the results showed a significant decrease in total cholesterol levels (p = 0.006), with the lowest levels observed in the t3 treatment group (95.25 ± 20.17 mg/dl). although hdl, ldl, ia, and crr levels did not show significant differences (p > 0.05), a trend of improvement in lipid profiles was observed in the treatment group, characterized by an increase in hdl and a decrease in ldl, ai, and crr compared to the positive control. the mechanism of cholesterol reduction is thought to be mediated through the activity of lab, which assimilates cholesterol, facilitates bile acid deconjugation, and produces short-chain fatty acids that inhibit cholesterol absorption. thus, sumbawa wild horse milk yogurt has the potential to be a hypocholesterolemic functional food that can improve lipid profiles and reduce the risk of atherosclerosis and cvd. keywords: cardiovascular disease; cholesterol; horse milk; lipid profile; yogurt. abbreviations: cardiovaskular disease (cvd), low density lipoprotein (ldl), high density lipoprotein (hdl), atherogenic index (ai), cardiac risk ratio (crr). introduction cardiovascular disease (cvd) is one of the leading causes of death in indonesia, accounting for approximately one-third of total deaths. data from the world health organization (who) over the past two decades indicates that the number of deaths due to cvd globally has reached more than 350 million cases (velissaridou et al., 2024). in indonesia, the number of cvd sufferers is estimated to reach around six million by 2024 and is projected to continue to increase. the 2022 national health survey showed that more than 25% of the indonesian population has cardiovascular risk factors (qanitha et al., 2022). the prevalence of cvd increases by up to 33% in individuals with high-fat diets and who are at high risk due to hypercholesterolemia (adisasmito et al., 2020; zakaria et al., 2022). one of the primary factors contributing to cvd is hypercholesterolemia, a lipid metabolism disorder characterized by elevated levels of total cholesterol and low-density lipoprotein (ldl), as well as reduced levels of high-density lipoprotein (hdl) (kaneko et al., 2021). a high-cholesterol diet can lead to the accumulation of ldl in tissues, triggering oxidative stress and the development of atherosclerosis (nugraheni et al., 2023). cholesterol management is generally performed with pharmacological therapy, one of which is the use of statins. however, long-term use of statins carries the risk of side effects such as hepatotoxicity, myopathy, and kidney failure (oktavelia & kusuma, 2022). therefore, saver and more effective alternatives for controlling cholesterol levels are needed. currently, functional foods have become part of the daily diet, potentially reducing the risk of various diseases. one local resource with potential as a functional food is the milk of wild sumbawa horses. this product is part of the cultural heritage and traditions passed down through generations of the people of west nusa tenggara, particularly on the island of sumbawa. it is a minor dairy product that is currently being developed in line with the increasing socio-economic role of horses in the geographic areas where they reside. it contains https://doi.org/10.14421/biomedich.2025.142.1129-1135 1130 biology, medicine, & natural product chemistry 14 (2), 2025: 1129-1135 high nutritional value and offers health benefits. however, scientific evidence regarding its antiinflammatory, antibacterial, and probiotic potential is still limited (prastyowati, 2021). mare's milk contains higher levels of essential fatty acids such as linoleic acid, particularly α-linoleic acid (ala), capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, and linolenic acid, which play a role in lowering cholesterol levels (czyżak-runowska et al., 2021). in addition, sumbawa wild horse milk also contains lactic acid bacteria (lab), which have the potential to act as probiotics, with the primary function of lowering cholesterol levels and improving lipid profiles (prastyowati, 2021). however, sumbawa wild horse milk has low sensory quality, so research by fajriani et al. has developed sumbawa wild horse milk into sumbawa wild horse milk yogurt (fajriani et al., 2023). fajriani et al.'s research found that sumbawa wild horse milk yogurt is more acceptable in terms of sensory properties and contains a high total lab count, specifically 3.42 x 109 cfu/ml (10). lab is known to have the ability to assimilate cholesterol through a binding mechanism to the bacterial cell membrane, resulting in a decrease in dietary cholesterol absorption in the digestive system, which in turn decreases blood cholesterol levels. evaluation of reduced cholesterol and ldl levels and increased hdl can be used as an indicator of cvd risk, where hdl levels >60 mg/dl indicate a lower cvd risk, as shown through the calculation of the atherogenic index (ai) and cardiac risk ratio (crr) (velissaridou et al., 2024). materials and methods study area this research was conducted at the pharmaceutical research and development laboratory of the university of mataram in july–august 2025 and has received an ethical approval letter with number 058 / ec-04 / fk-06 / unizar / vi / 2025. this research employed a quantitative experimental method with a completely randomized design, followed by a significant difference test for unpaired groups. the sample consisted of 30 male wistar rats, aged 2-3 months and weighing 150-250 g. the sample was then divided into six groups: the normal control group (nc), positive control (c+), negative control (c-), treatment 1 (t1), treatment 2 (t2), and treatment 3 (t3), each consisting of 5 rats. procedures the process of making sumbawa wild horse milk yogurt yogurt production was carried out in the food microbiology laboratory and the bioprocess laboratory of the university of mataram. this process began with the provision of sumbawa wild horse milk, then the addition of 5% skim milk. next, it was homogenized at room temperature using a homogenizer for 10 minutes. after that, it was pasteurized using a water bath at a temperature of 65 °c for 20 minutes. then, it was left to stand until it reaches a temperature of 37 °c. inoculate a 4% starter culture containing lactobacillus rhamnosus bacteria. next, incubated at 37 °c for 24 hours. after the incubation process is complete, liquid yogurt will be obtained. the yogurt is then added with 1% honey and stored at a temperature of 10 °c (fajriani et al., 2023). the process of making high-cholesterol diet (hcd) a high-cholesterol diet was prepared by mixing 83 g of egg yolk, 10 g of animal fat, 5 g of sucrose, 1.5% cholesterol, and 0.5% propylthiouracil until the mixture was thoroughly combined. the high-cholesterol diet was then administered at a rate of 2 ml/200 g of rat body weight (yang et al., 2019a). the process of making simvastatin suspension simvastatin was administered orally to experimental animals by preparing a suspension using carboxymethylcellulose (cmc). the preparation process began by dissolving 1 g of cmc in 100 ml of warm water, stirring until a thick, homogeneous solution was formed. afterward, the amount of 20,6 mg simvastatin was disolveud in 100 ml of cmc suspension. the suspension was administered to the rats was carried out using the sonde method at 0,206 mg/200 g bw/day. the treatment of experimental animals in this study, the experimental animal subjects were first adapted for 7 days. during the adaptation process, the experimental animal subjects were given standard diet (sd). then, they were given high-cholesterol diet (hcd) for 14 days (from day 8 to day 21), except for the normal group. after that, treatment was given for 28 days (days 22 to 49) according to the treatment group. the following is the treatment given to each group: table 1. the treatment of experimental animals. group days 1-7 days 8-21 days 22-49 nc sd sd sd c+ sd sd and hcd sd and hcd csd sd and hcd sd and 0,36 mg/200 g bw/day of simvastatin t1 sd sd and hcd sd and 2ml/200 g bw/day of sumbawa wild horse milk yogurt t2 sd sd and hcd sd and 3ml/200 g bw/day of sumbawa wild horse milk yogurt t2 sd sd and hcd sd and 4ml/200 g bw/day of sumbawa wild horse milk yogurt fajriani et al. – horse milk yogurt improves lipid 1131 the cholesterol level, hdl, ldl, ia, and crr measurement on the 50th day, the mice were anesthetized with ketamine, and blood was drawn through cardiac perfusion. blood samples were placed in vacutainer tubes and centrifuged to obtain serum. total cholesterol, hdl, and ldl levels were measured using a biosystem machine at the awet muda narmada hospital laboratory. this device is a semi-autoanalyzer used for clinical chemistry examinations. the ia and crr analyses compare total cholesterol to hdl levels. the following is the formula for calculating ai and crr: 𝐼𝐴 = (𝐶ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙 𝑇𝑜𝑡𝑎𝑙 − 𝐻𝐷𝐿) 𝐻𝐷𝐿 𝐶𝑅𝑅 = 𝐶ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙 𝑇𝑜𝑡𝑎𝑙 𝐻𝐷𝐿 data analysis the results of the cholesterol, hdl, ldl, ia, and crr level measurements were analyzed for normality using the shapiro-wilk test and for homogeneity using levene's test. hdl, ldl, ai, and crr data were normally distributed and homogeneous, allowing for the analysis to be carried out using one-way anova with a 95% confidence level (α = 0.05). meanwhile, for total cholesterol data, it was not normally distributed and not homogeneous; therefore, the analysis was carried out using the kruskal-wallis test. if the results were significant, a further mann-whitney test was conducted. results and discussion table 2 presents the average results for total cholesterol, hdl, ldl, ia, and crr in all groups of mice. based on these results, there was a decrease in total cholesterol, ldl, ai, and crr levels except in the c+ group. this is because the c+ group was only given a high-cholesterol diet. meanwhile, hdl levels generally increased, except in the c-group. the decrease in hdl levels in the c group was caused by the administration of simvastatin, which inhibits hmg-coa reductase, resulting in reduced hepatic cholesterol and, consequently, decreased lipoprotein secretion, including hdl (palmado et al., 2024). table 2. average of cholesterol total, hdl, ldl, ia, and crr. group cholesterol total (mg/dl) hdl (mg/dl) ldl (mg/dl) ai (mg/dl) crr (mg/dl) mean±sd p mean±sd p mean±sd p mean±sd p mean±sd p cn 105±2.00 0,006* 43.12±5.77 0,731 40.47±8.41 0,790 1.46±0.26 0,898 2.46±0.26 0,902 c+ 134.50±38.45 53.87±18.68 69.62±48.28 1.78±1.40 2.78±1.40 c104.25±0.50 39.97±6.36 57.15±12.67 1.66±0.44 2.66±0.44 t1 103.75±0.95 46.22±9.54 50.02±8.59 1.35±0.50 2.60±0.47 t2 102.25±0.95 47.42±13.96 43.41±15.05 1.33±0.70 2.33±0.70 t3 95.25±20.17 48.00±13.74 41.03±26.55 1.19±0.85 2.19±0.85 note: *p-value <0,05 in general, total cholesterol levels in all groups decreased, ranging from 134.50 ± 38.45 mg/dl to 95.25 ± 20.17 mg/dl. the c+ group had the highest cholesterol levels (134.50 ± 38.45 mg/dl), while the t3 group had the lowest (95.25 ± 20.17 mg/dl). the kruskal-wallis test results in table 2 show a p-value of 0.006, which means there are at least two groups with different total cholesterol levels. therefore, a further man-whitney test was conducted to determine the differences in each group. the results of the further man-whitney test in table 3 show that the cn group has a p-value <0.05 (p=0.017) compared to the t2 group, indicating significant differences in cholesterol levels between the cn and t2 groups. in addition, other groups with significant differences included c+ with c(p = 0.018), c+ with t1 (p = 0.020), c+ with t2 (p = 0.020), and c+ with t3 (p = 0.028), as well as cwith t2 (p = 0.017). these results demonstrate that administering sumbawa wild horse milk yogurt can reduce total cholesterol levels to near-normal levels. these results align with the research findings of (lestari et al., 2020), which demonstrated that administering purple sweet potato yogurt for four weeks can reduce total cholesterol levels in mice. meanwhile, consuming a high-cholesterol diet can lead to an imbalance in metabolism and fat absorption, thereby affecting the fat oxidation mechanism, which in turn can cause fat accumulation and increase cholesterol and ldl level (abdullah et al., 2023). 1132 biology, medicine, & natural product chemistry 14 (2), 2025: 1129-1135 table 3. post hoc man-wihtney. group group post hoc cn c+ 0.076 c0.850 t1 0.278 t2 0.017* t3 0.766 c+ c0.018* t1 0.020* t2 0.020* t3 0.028* c t1 0.350 t2 0.017* t3 0.363 t1 t2 0.065 t3 0.372 t2 t3 0.243 note: *p-value <0,05 table 2 shows that the hdl parameter showed no significant results between groups with a p-value of 0.731. however, there was a trend towards increased hdl levels in the treatment group compared to the c group. the c+ group had the highest value at 53.87 ± 18.68 mg/dl. meanwhile, the cgroup had the lowest hdl levels at 39.97±6.63 mg/dl. this suggests that the treatment tends to increase hdl levels, and there is potential for improvement in lipid profiles as the dose increases. however, this has not yet reached statistical significance. the increase in hdl in the treatment group may indicate the role of lab in improving lipid profiles by increasing reverse cholesterol transport from peripheral tissues to the liver for excretion (rosenson et al., 2016). meanwhile, the high hdl in the c+ group may be due to the body's compensatory mechanism for increased total cholesterol, which attempts to increase hdl to compensate for impaired lipid metabolism (lodha & kakadiya, 2022). furthermore, the increase in hdl in the group given a high-cholesterol diet also occurred due to the redistribution of cholesterol between lipoproteins. when ldl and vldl reach their maximum cholesterol-carrying capacity, excess cholesterol can be transferred to hdl, thereby balancing plasma lipid distribution. this is consistent with hdl's flexible role in accepting excess cholesterol from other lipoprotein (fernandez & murillo, 2022). ldl levels also showed insignificant results between groups with a p-value of 0.790. the highest value was shown in the c+ group at 69.62±48.77 mg/dl. meanwhile, the t3 group showed the lowest result at 41.03 ± 26.55 mg/dl. although not statistically significant, these results suggest the potential for improving lipid profiles by reducing ldl levels through treatment. these results align with research by (yang et al., 2019b), which demonstrated that administering frozen yogurt can lower ldl levels in mice. the atherogenic index (ai) is a parameter used to predict the risk of atherosclerosis (niroumand et al., 2015). based on table 2, the results of the ai analysis show no significant differences between groups with a pvalue of 0.898. however, a downward trend was observed in the treatment group. the highest ai value was in the c+ group at 1.78±1.78, while the t3 group had the lowest value at 1.19±0.85. this decrease indicates that the treatment was effective in reducing the ia value. the lower the ai value, the lower the potential risk of atherosclerosis; however, the effect was not statistically significant (kwarteng & laing, 2025). the cardiac risk ratio (crr) is a parameter that indicates the risk of cvd (niroumand et al., 2015). the crr parameter showed insignificant results with a pvalue of 0.902. however, there was a tendency for a decrease in the crr value in the treatment group compared to the c+ group. the c+ group had the highest crr value of 3.78 ± 2.10, while the t3 group had the lowest value of 2.19 ± 0.85. this trend suggests that a lower crr value may have the potential to reduce the risk of cardiovascular disease, although the difference is not statistically significant (mansoori et al., 2024). discussion cholesterol is a macronutrient that plays a vital role in the formation of vitamin d, steroid hormones, and bile acid synthesis. cholesterol analysis showed differences between groups. the highest cholesterol and ldl levels were observed in the c+ group, suggesting that a highcholesterol diet can induce elevated cholesterol and ldl levels in mice. high-cholesterol diet intake, such as duck egg yolks and animal fats, contributes to increased cholesterol levels, especially the ldl fraction. these results align with research by (shihab et al., 2023), which found that feeding a high-fat diet to mice significantly increased cholesterol, tg, and ldl levels. high-fat diets can lead to an imbalance in fat metabolism and absorption, which affects the fat oxidation mechanism and may contribute to fat accumulation (abdullah et al., 2023). the administration of sumbawa wild horse milk yogurt to the treatment group significantly reduced cholesterol levels in mice, with increasing doses of yogurt resulting in progressively lower cholesterol levels. the lowest cholesterol levels were found in the 4 ml of sumbawa wild horse milk yogurt. this decrease in cholesterol levels may be due to the presence of lab in the yogurt. the lab in yogurt can lower cholesterol levels through several mechanisms: first, by assimilating cholesterol in the intestine through bacterial action, thereby reducing its availability for absorption. second, the activity of bile acid deconjugation by enzymes produces an insoluble form of bile acid, which is excreted in the feces, allowing the body to utilize endogenous cholesterol for the synthesis of new bile acids. third, the production of short-chain fatty acids can inhibit cholesterol absorption in the intestine. furthermore, yogurt also contains bioactive peptides that play a role in reducing oxidative stress, suppressing ldl fajriani et al. – horse milk yogurt improves lipid 1133 oxidation, and improving lipid metabolism (kumar et al., 2012). administering sumbawa wild horse milk yogurt to mice has been shown to lower ldl levels. this mechanism aligns with several studies showing that fermented milk products can have a hypolipidemic effect. the fermentation process produces bioactive compounds, enzymes, and microorganisms that have the potential to act as probiotics and contribute to lowering ldl levels in the blood (prastyowati, 2021; ziaei et al., 2021). furthermore, fermented sumbawa wild horse milk can also increase the production of short chain fatty acids (scfa), particularly propionate, which is known to inhibit the activity of hmg-coa reductase in the liver, a key enzyme in cholesterol biosynthesis. horse milk also has a lower fat content and a different fatty acid profile than cow's milk, thus naturally supporting a better blood lipid profile. the combination of fermented lab, bioactive metabolites, and the characteristics of horse milk contributes to lowering ldl. conversely, in groups with a high-cholesterol diet, excessive intake of saturated fat and cholesterol leads to increased ldl levels due to disruption of cholesterol homeostasis, including an increase in the synthesis of atherogenic lipoproteins (ziaei et al., 2021). increased cholesterol content in food and its absorption in the intestine can stimulate the liver to produce large amounts of ldl molecules, which transport triglycerides and cholesterol to body tissues through the bloodstream (alcover et al., 2025). the increase in cholesterol and ldl levels in the c+ group was directly correlated with the increase in hdl levels in the same group. the high hdl levels in the group given a high-cholesterol diet may be due to the redistribution of cholesterol between lipoproteins. when ldl and vldl reach their maximum cholesterolcarrying capacity, excess cholesterol can be transferred to hdl, thereby balancing plasma lipid distribution. this is in line with hdl's flexible role in accepting excess cholesterol from other lipoproteins (fernandez & murillo, 2022). furthermore, it is suspected that this is due to the body's compensatory mechanism for increased total cholesterol, which causes the body to attempt to increase hdl to offset impaired lipid metabolism (lodha & kakadiya, 2022). the lowest hdl levels were in the c-group. simvastatin administration to the cgroup inhibited hmg-coa activity, thus inhibiting cholesterol synthesis. statins are drugs that lower cholesterol by inhibiting the enzyme 3-hydroxy-3-methylglutaryl coenzyme a (hmg-coa) reductase, which plays a role in cholesterol production in the liver (tucker & soslowsky, 2016). elevated cholesterol and ldl levels will affect the atherogenic index (ai) value. an elevated atherogenic index (ai) reflects the occurrence of atherogenic dyslipidemia, characterized by high cholesterol levels, low hdl, and the formation of small, dense ldl particles. small-dense ldl is more atherogenic than large ldl because it easily penetrates the vascular endothelial layer, is more susceptible to oxidation, and remains in circulation longer (gaggini et al., 2022). atherosclerosis itself is the leading cause of cvd. it is considered a chronic inflammatory disease resulting from the interaction of ldl and the arterial wall, leading to the formation of atherosclerotic lesions. furthermore, a high-cholesterol diet also triggers oxidative stress and a systemic inflammatory response, which exacerbates endothelial dysfunction and further increases ai values (vinué et al., 2018). the relationship between ai and cvd has been demonstrated in various clinical and epidemiological studies. the results of a meta-analysis showed that high ai is an independent predictor of coronary heart disease events and can serve as a more sensitive prognostic indicator than individual lipid parameters (rad et al., 2024). high ai indicates the dominance of pro-atherogenic factors in lipid metabolism, which ultimately increases the risk of atherosclerosis, plaque susceptibility, and clinical events such as myocardial infarction or stroke (młynarska et al., 2024). groups of mice fed different doses of sumbawa wild horse milk yogurt showed a reduced atherogenic index compared to the high-cholesterol diet group. this mechanism is closely related to the bioactive composition of wild horse milk and the potential activity of fermented probiotics. horse milk yogurt contains whey protein, bioactive peptides, unsaturated fatty acids, and microorganisms with potential probiotic properties known to play a role in modulating lipid metabolism (prastyowati, 2021). unsaturated fatty acids, such as linoleic and linolenic acids, can increase lipoprotein lipase activity, thereby facilitating the breakdown of triglycerides. these fatty acids also play a role in increasing hdl concentrations, which can directly reduce the atherogenic index (figueiredo et al., 2017). horse milk yogurt also has potential antioxidant and antiinflammatory effects, as the fermentation process produces bioactive compounds, including antioxidant peptides and organic acids, that can reduce oxidative stress and suppress vascular inflammation. this is important because oxidative stress and chronic inflammation contribute to changes in hdl quality, leading to dysfunction. by maintaining hdl's protective function, yogurt helps maintain low ai levels (pânzaru et al., 2024; titisari et al., 2020). high cholesterol levels are also a risk factor for cardiovascular disease (cvd). apolipoprotein b100 (apob100) is the main structural protein of atherogenic lipoproteins, whose increase is associated with increased ldl, thus increasing the risk of cvd, as indicated by an increased crr value (pourrajab et al., 2020). the study results showed that the highest crr value was in the c+ group. this may be due to the high-cholesterol diet increasing cholesterol and ldl levels. the higher the crr value, the higher the risk of cvd. conversely, the lower the crr value, the lower the risk of cvd. 1134 biology, medicine, & natural product chemistry 14 (2), 2025: 1129-1135 conclusions this study showed that sumbawa wild horse milk yogurt has the potential as a hypocholesterolemic functional food that can improve blood lipid profiles and reduce the risk of cvd. administering yogurt at doses of 2 ml, 3 ml, and 4 ml per day for 28 days in hypercholesterolemic male wistar rats significantly reduced total cholesterol levels (p = 0.006), with the lowest result at a dose of 4 ml/day (t3). although hdl, ldl, atherogenic index (ai), and cardiac risk ratio (crr) levels did not show significant differences between groups (p > 0.05), there was a trend of increasing hdl and decreasing ldl, ai, and crr in the treatment group compared to c+. the mechanism of cholesterol reduction is thought to originate from the activity of lab, which assimilates cholesterol, bile deconjugation, and produces short-chain fatty acids that inhibit cholesterol absorption. in addition, the content of unsaturated fatty acids and bioactive peptides in sumbawa wild horse milk also plays a role in suppressing ldl oxidation, increasing lipoprotein lipase activity, and providing antioxidant and anti-inflammatory effects. thus, sumbawa wild horse milk yogurt can be a safe and effective natural alternative for lowering cholesterol levels and reducing the risk of atherosclerosis, without causing side effects associated with long-term statin therapy. acknowledgements: the author would like to thank the ministry of higher education, science, and technology (kemdiktisaintek), bumigora university, and all colleagues who helped in carrying out this research. authors’ contributions: laksmi nur fajriani designed the study. laksmi nur fajriani and ni made wiasty sukanty carried out the laboratory work. laksmi nur fajriani analyzed the data and wrote the manuscript. dea pratiwi silpia putri and esya mariska febriani were involved in collecting sumbawa wild horse milk for yogurt production and assisting in the production of sumbawa wild horse milk yogurt. all authors read and approved the final version of 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(2021). the effect of probiotic fermented milk products on blood lipid concentrations: a systematic review and meta-analysis of randomized controlled trials. nutrition, metabolism and cardiovascular diseases, 31(4), 997–1015. https://doi.org/10.1016/j.numecd.2020.12.023 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 91-100 | doi: 10.14421/biomedich.2025.141.91-100 issn 2540-9328 (online) subacute toxicity study of leaf extract of saccharum officinarum utibe a. edem, jude e. okokon* department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. corresponding author* judeefiom@yahoo.com abstract saccharum officinarum (family-poaceae) is used traditionally to treat malaria and fever, among others. evaluation of subacute administration ethanol leaf extract of saccharum officinarum for possible effect on hematological indices, liver and kidney functions, and lipid profile of rats was carried out. the leaf extract (170, 340, 510 mg/kg body weight) was orally administered to male wistar rats daily for 30 days, and the rats were sacrificed under light diethyl ether anesthesia after the administration. subacute administration of s. officinarum leaf extract resulted in an insignificant increase in the body weights of rats without any significant (p>0.05) effect on the weights of liver and kidney when compared to control. the leaf extract treatment did not affect wbc, lymphocytes, monocytes, eosinophil, and basophil percentages. however, it caused significant (p<0.05) decreases in rbc and platelet counts, hemoglobin concentration, and pcv percentage, especially at the middle dose (374 mg/kg), and also prolonged bleeding and clotting time significantly (p<0.05) when compared to control. the leaf extracts non-dose-dependently caused insignificant (p>0.05) decreases in total protein, albumin, and alt levels. alp was significantly (p<0.05) decreased at the highest dose (510 mg/kg). however, ast, total and conjugated bilirubin levels were significantly (p<0.01-0.001) decreased only at higher doses (340 and 510 mg/kg) of the extract. the leaf extract did not cause any significant (p>0.05) effect on urea, creatinine, potassium, and sodium as well as total cholesterol, triglyceride, vldl, and ldl levels of rats, but the highest dose (510 mg/kg) significantly (p<0.05) increased cl level and reduced hdl level of rats when compared to control. the leaf extract exerts mild to moderate effects on the histology of the livers and kidneys of rats. chronic study is advocated to investigate the effect of prolonged administration of rats’ extract organs and systems. keywords: saccharum officinarum; haematological parameters; kidney function; liver function. abbreviations: alt: alanine aminotransferase, alp: alkaline phosphatase, ast: aspartate aminotransferase, rbc: red blood cell, wbc: white blood cell, pcv: packed cell volume, hb: hemoglobin concentration, ldl: low density lipoprotein, vldl: very low density lipoprotein, hdl: high density lipoprotein, who: world health organisation, h & e: heamatoxylin and eosin. introduction saccharum officinarum (family-poaceae), also called sugarcane, thrives throughout tropical and subtropical regions. in traditional medicine, it is used in the treatment of diarrhea, dysentery, eyes, fever, arthritis, bedsores, boils, cancer, colds, cough, opacity, skin sores, sore throat, hiccups, inflammation, laryngitis, spleen, tumors, and wounds (hartwell, 1967-1971). the leaf extract possesses some biological activities such as antibacterial and anthelmintic (palaksha et al., 2013), anti-hyperglycaemic, anti-hyperlipidaemic (ojewunmi et al., 2013), antioxidant (ojewunmi et al., 2013; sun et al., 2014), diuretic and antiurolithiatic (palaksha et al., 2015), antidepressant and anticonvulsant (okokon et al., 2019), analgesic (okokon et al., 2021) and antimalarial (okokon et al., 2022), antioxidative stress and hepatoprotective (edem et al., 2022), anti-inflammatory and antipyretic (edem et al., 2023a), antiulcer (edem et al., 2023b) activities. saabmal®: nigeria’s polyherbal preparation containing s. officinarum is utilized as a malarial remedy (obidike et al., 2015). the leaves are employed in ghana to treat malaria locally (akwetey & achel, 2010). phytochemical screening of the leaf extract of saccharum officinarum revealed the presence of glycosides, phytosterols, saponins, tannins, and flavonoids (palaksha et al., 2013; singh et al., 2015). some flavones and phenolics, as well as their derivatives from the leaves of s. officinarum, have been identified (coutinho et al., 2016; okokon et al., 2022). the medicinal potentials of the plant have been widely reported, but there is a paucity of information on its toxicological potentials. this study reported subacute toxicity potential of the leaf extract of s. officinarum on haematological parameters, liver and kidney functions and lipid profile. manuscript received: 24 june, 2024. revision accepted: 02 october, 2024. published: 24 april, 2025. https://doi.org/10.14421/biomedich.2025.141.91-100 https://www.ncbi.nlm.nih.gov/pubmed/?term=obidike%20i%5bauthor%5d&cauthor=true&cauthor_uid=25900958 https://www.ncbi.nlm.nih.gov/pubmed/?term=singh%20a%5bauthor%5d&cauthor=true&cauthor_uid=26009693 92 biology, medicine, & natural product chemistry 14 (1), 2025: 91-100 materials and methods plant materials fresh leaves of saccharum officinarum were collected in june 2020 from residential quarters in uyo village in uyo lga, akwa ibom state, nigeria. the leaves were identified and authenticated as saccharum officinarum by a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria, and a voucher specimen (uuph 215b) was prepared and deposited at the herbarium of the department of pharmacognosy and natural medicine, faculty of pharmacy, university of uyo. extraction fresh leaves of s. officinarum were washed, cut into smaller pieces and dried under shade for two weeks. the leaves were further pulverized to powder using an electric grinder. the powdered leaf material (2 kg) was soaked in 50% ethanol (7.5 l) at room temperature (28 ± 2 °c) for 72 hours. after that the liquid filtrate was filtered, concentrated, and evaporated to dryness in vacuo 40 °c using a rotary evaporator (buchilab switzerland). the dry extract was stored in a refrigerator at -4 °c until it was used for the proposed experiments. animals in this study, male albino wistar rats were used. the animals were sourced from the university of uyo animal house and sheltered in plastic cages. they were fed with pelleted standard feed (guinea feed) and given unlimited access to water. the faculty of pharmacy animal ethics committee, university of uyo, approved the study. sub-acute toxicological study adult wistar rats of both sexes were used in this study. they were weighed and randomly divided into four groups of 6 animals each and treated as follows: groups i, ii, and iii were administered 170, 340, and 510 mg/kg of the leaf extract, respectively, daily for 30 days. group iv was administered with distilled water (10 ml/kg) for the same period. at the end of the treatment period, the animals were weighed again and sacrificed under light ethyl ether vapor. blood samples were collected by cardiac puncture and used immediately for hematological testing, such as bleeding time, clotting time, complete blood counts etc. serum was separated from the remaining blood and stored at -20˚c until used for biochemical determinations such as liver function test, kidney function test, and lipid profile the effect of the extract on some organs such as the liver and kidney, was studied. the organs, livers, and kidneys of rats were surgically removed, weighed, and fixed in 10% formalin. the organs were processed, sectioned, and stained using hematoxylin and eosin (h&e) according to standard procedures. haematological analysis the following haematological parameters were determined: haemoglobin level (hb), packed cell volume (pcv), total and differential white blood cell count (wbc), platelet count, and full blood count. these parameters were determined at haematology department of the university of uyo teaching hospital using an automated haematology analyser. biochemical analysis liver function test the following parameters were determined: aspartate transaminase (ast), alanine aminotransferase (alt), total cholesterol, and alkaline phosphatase (alp). total plasma protein, total and direct bilirubin. the determinations were done spectrophotometrically using randox analytical kits according to standard procedures of manufacturer’s protocols (tietz, 1976) at the chemical pathology department of the university of uyo teaching hospital. kidney function test the following biochemical parameters were determined as markers of kidney function using diagnostic kits at the chemical pathology department of university of uyo teaching hospital: levels of electrolytes (na, k, cl, and hco3), creatinine, and blood urea histopathological examination the liver and kidneys of rats used in the study were surgically harvested and fixed in buffered formalin. they were then processed and stained with hematotoxylin and eosin (h&e) according to standard procedures at the department of chemical pathology, university of uyo teaching hospital. morphological changes were observed and recorded in the excised organs of the sacrificed animals. histologic pictures were taken as micrographs. statistical analysis the data collected were analyzed using one-way analysis of variance (anova) followed by tukey’s multiple comparison post-test (graph pad prism software inc. la jolla, ca, usa). values were expressed as mean ± sem, and significance relative to the control was considered at p˂0.001 and p˂0.05. results effect of subacute administration of leaf extract on body weight of rats the effect of leaf extract on body weight of rats treated with leaf extract of s. officinarum for 30 days is shown in table 1. there was considerable increase in the body weights of rats treated the extract in all groups, similar to that of the control group, though non-dose-dependently. edem & okokon – subacute toxicity study of leaf extract of saccharum officinarum 93 the increase in body weight in the low dose (170 mg/kg) treatment group was significantly (p<0.001) lower than that of the control. in contrast, the weight increases of rats treated with higher doses (340 and 510 kg/kg) of the extract were higher than that of the control but not statistically significant (p>0.05) (table 1). effect of subacute administration of leaf extract on organ weights of rats treatment of rats with the leaf extract (170-510 mg/kg) for 30 days did not cause any significant effect (p>0.05) on the weights of livers and kidneys of rats when compared to the control (table 2). table 1. effect of subacute administration of s. officinarum leaf extract on body weights of rats. treatment r&g /extract dose (mg/kg) initial body weight (kg) final body weight (kg) weight gain (kg) control 0.2ml 153.3  9.40 207.6  5.69 54.3  2.81 s. officinarum 170 170.3  6.88 201.0 17.00 30.7  3.86a 340 171.6  1.66 232.0  8.50 60.4  2.74 510 164.0  9.53 209.6  2.40 45.6  3.41 data are expressed as mean  sem. significant at ap>0.001 when compared to control. n = 6 table 2. effect of subacute administration of sacharum officinarum leaf extract on organ weights of rats. treatment dose (mg/ kg) liver (mg) kidney(mg) control 10 mg/ml 6.00± 0.35 1.19±0.03 crude extract 170 6.11±0.45 1.31±0.12 340 6.29±0.25 1.14±0.03 510 6.10±0.38 1.21±0.06 data is expressed as mean ± sem, significant at bp< 0.01, compared to control. (n=6). effect of subacute administration of leaf extract on hematological parameters of rats the effect of subacute administration of ethanol leaf extract of s. officinarum (170-510 mg/kg) on the hematological parameters of rats is shown in table 3. subacute administration of leaf extract of s. officinarum to rats for 30 days did not significantly affect the wbc counts, lymphocytes, monocytes, eosinophil, and basophil percentages (p>0.05) compared to control. however, the neutrophils percentage of the group treated with 170 mg/kg of the leaf extract was significantly (p<0.05) increased and significant (p<0.05-0.01) decreases in rbc and platelets counts, hemoglobin concentration and pcv percentage were observed in the group treated with the middle dose (340 mg/kg) of the extract when compared to control (table 3). moreover, treatment of rats with leaf extract of s. officinarum (170 520 mg/kg) for 30 days caused a significant (p>0.050.001) increase in the bleeding time of rats in all doses used when compared to control though non-dosedependently, while significant (p<0.001) decreases in the clotting time of rats treated with doses of the extract (170 and 340 mg/kg) was observed when compared to the control (figures 1 and 2). effect of subacute administration of leaf extract on liver function indices of rats administration of leaf extract of s. officinarum (170-510 mg/kg) to rats for 30 days caused a non-dose-dependent and insignificant (p>0.05) decrease in total protein, albumin, and alt levels of rats when compared to control (table 4). the alp levels were similarly reduced, but this was only significant (p<0.01) in the group treated with the highest dose (510 mg/kg) dose of the extract. similarly, ast, total and conjugated bilirubin levels of treated rats were decreased, but the reductions were significant (p<0.01-0.001) only at the higher doses (340 and 540 mg/kg) of the extract when compared to control (table 4). figure 1. effect of subacute administration of saccharum officinarum leaf extract on clotting time of rats. data is expressed as mean ± sem, significant at *p<0.05, **p<0.01, ***p<0.001, when compared to control. (n=6). 94 biology, medicine, & natural product chemistry 14 (1), 2025: 91-100 figure 2. effect of subacute administration of saccharum officinarum leaf extract on bleeding time of rats. data is expressed as mean ± sem, significant at *p<0.01 when compared to control. (n=6). effect of subacute administration of leaf extract on kidney function parameters of rats treatment of rats for 30 days with leaf extract of s. officinarum (170-510 mg/kg) did not cause any significant effect (p>0.05) on the levels of urea, creatinine, potassium, and sodium of the treated rats when compared to control. however, the level of chloride was significantly (p<0.05) increased at the higher dose of the extract (510 mg/kg) compared to control (table 5). effect of subacute administration of leaf extract on lipid profile indices of rats treatment of rats with leaf extract of s. officinarum (170510 mg/kg) caused non-dose-dependent but insignificant (p>0.05) decreases in the levels of total cholesterol, triglyceride, ldl, and vldl of rats treated for 30 days when compared to control. however, the hdl level was significantly (p<0.05) reduced at the highest dose (510 mg/kg) of the extract when compared to control (table 6). effect of subacute administration of leaf extract on histology of organs figures 3 and 4 show the effects of subacute administration of ethanol leaf extract of s. officinarum to rats for 30 days on histology of some organs. the leaf extract (170-510 mg/kg) caused varying defects in the histology of the organs. mild effects were observed on the hepato-architecture with micro and macro-vesicular steatosis areas, vacuolated hepatocytes, hemorrhagic blood deposits (h), and arrays of sinusoidal spaces within the hepatic lobule in all treatment groups. the history-architechture of renal tissues of treated rats was moderately affected with glomeruli (g) having widened bowman’s space (wb), vacuolated ductal cells, and hemorrhagic blood vessels (h) within the cortical matrix. table 3. effect of subacute administration of saccharum officinarum leaf extract on hematological parameters of rats. treatment dose wbc (l) neut. (%) lym (%) mono (%) esino (%) baso (%) rbc (l) hgb (g/dl) pcv (%) platelets. (l) control 10 mg/ml 6.27±0.05 17.26±1.46 77.96±2.43 2.93±0.52 0.33± 0.03 0.50± 0.05 6.12± 0.46 13..53±0.14 43.76±2.27 754.0± 35.90 crude extract 170 7.81±0.98 23.63±1.46 73.73±1.93 1.43±0.68 0.66±0.17 0.66± 0.17 8.40 ± 0.15 14.63± 0.36 46.96±1.17 775.0± 82.21 340 4.48±0.85 13.65±1.72 82.26±1.74 2.60±1.42 0.46± 0.32 0.46± 0.03 4.85± 1.63 9.13± 2.73 28.23±1.34 542.3± 30.52 510 6.99±0.99 21.33±1.74 75.56±3.01 3.68±0.69 0.30± 0.17 0.36± 0.23 7.77± 0.27 12.44± 2.06 34.93±1.96 769.0± 95.04 data is expressed as mean ± sem, significant at ap<0.05, bp<0.01, when compared to control. (n=6). table 4. effect of subacute administration of sacharum officinarum leaf extract on liver function parameters of rats. treatment dose (mg/ kg) total protein (mg/dl) albumin (mg/dl) alt (iu/l) alp (iu/l) ast (iu/l) total bilirubin (µmol/l) combined bilirubin (µmol/l) control 10 mg/ml 67.33± 4.70 41.66±3.52 6.20± 3.31 52.38±3.28 21.0± 2.51 4.36±0.43 2.50±0.26 crude extract 170 67.0±4.35 42.33±2.60 7.00±2.19 46.66±2.02 22.0± 2.05 4.43± 0.48 2.63±0.29 340 61.0±4.04 36.33±2.40 5.83±1.20 50.33±5.60 14.0±3.51c 3.33± 0.58b 1.73±0.14a 510 65.66±2.72 42.33±1.45 5.46±0.08 39.0±1.15b 17.0± 1.52a 3.90± 0.50 2.06±0.27 data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, cp< 0.001, when compared to control. (n=6). table 5. effect of subacute administration of sacharum officinarum leaf extract on kidney function parameters of rats. treatment dose (mg/kg) creatinine (mg/kg) urea (mg/dl) bicarbonate (mmol/l) sodium (mmol/l) potassium (mmol/l) chloride (mmol/l) control normal saline 10 mg/ml 4.56± 0.43 93.33± 8.41 28.33± 1.20 146.6±7.96 4.76± 0.29 76.0± 0.57 crude extract 70 5.53± 0.98 107.6± 15.23 24.33± 1.76 153.3±4.37 4.93± 0.18 74.0± 2.08 140 4.93± 0.71 100.0± 14.01 26.00± 1.15 121.3± 8.09 3.80± 0.32 68.0± 5.13 210 3.43± 0.12 72.33± 1.45a 26.0±2.64 134.3±8.37 4.26± 0.31 94.0± 5.50a data is expressed as mean ± sem, significant at ap<0.05, when compared to control. (n=6). table 6. effect of subacute administration of sacharum officinarum leaf extract on the lipid profile of rats. treatment dose mg/kg total cholesterol (mmol/l) triglyceride (mmol/l) hdl-c (mmol/l) ldl-c (mmol/l) vldl (mmol/l) control 10 ml/kg 2.10± 0.05 0.84± 0.05 1.46± 0.03 1.02± 0.03 0.38± 0.02 crude extract 70 1.90± 0.05 0.88± 0.08 1.44± 0.01 0.85± 0.07 0.40± 0.03 140 2.16± 0.08 0.70± 0.06 1.52± 0.02 0.96± 0.08 0.31± 0.02 210 1.96± 0.08 0.66± 0.01 0.96± 0.08a 0.88± 0.02 0.30± 0.01 data is expressed as mean ± sem, significant at ap<0.05, when compared to control. (n=6). e d em & o k o k o n – s u b ac u te t o x ic it y s tu d y o f l ea f e x tr ac t o f s a cc h a ru m o ff ic in a ru m 9 5 96 biology, medicine, & natural product chemistry 14 (1), 2025: 91-100 figure 3. photomicrograph of the transverse sections of livers of rats treated with distilled water (cont), the leaf extract of s. officinarum at 170 mg/kg (ld), 340 mg/kg (md), and 510 mg/kg (hd) liver tissue showing portal vein (pv), bile duct (bd) and hepatic artery (ha) within the connective tissue (ct) of the portal area, well-populated hepatocytes (hp), kupffer cells (kc) and arrays of sinusoidal spaces (sn) within the hepatic lobule vacuolated hepatocytes (blue arrow), micro and macro-vesicular steatosis (st). (x 100). figure 4. photomicrograph of the transverse sections of kidneys of rats treated with distilled water (cont), leaf extract of s. officinarum at 170 mg/kg (ld), 340 mg/kg (md), and 510 mg/kg (hd) liver tissue showing glomeruli (g), bowman’s space (bs), proximal convolated tubules (pt), distal convolated tubules (dt) and infiltrating interstitial connective tissues (ic), widened bowman’s space (wb), vacoulated ductal cells (blue arrow), and hemorrhagic blood vessels (h) (x 100). edem & okokon – subacute toxicity study of leaf extract of saccharum officinarum 97 discussion in this study, subacute administration of the leaf extract caused various degrees of weight gains in all the treatment groups, which were insignificantly higher than control at higher doses (340 and 510 mg/kg) but significantly lower at the low dose (170 mg/kg) when compared to control. changes in body weight are markers of adverse effects of drugs, and it is considered statistically significant if a body weight loss is more than 10% (tepongning et al., 2018). in this study, there were moderate increases in the body weights of rats in all the extract-treated groups, but these increases were not significantly (p>0.05) different from that of the control group except at the low dose, indicating that the feeding habit of the rats was not adversely affected by the administration of the extract. there were no adverse effects of the extract on the body growth processes of rats. the treatment of rats with leaf extract (170-510 mg/kg) for 30 days did not affect on the weights of the liver and kidneys. however, slight decreases in the liver weights of treated rats were observed but these were not significant when compared statistically to the control group. generally, internal organ weights are considered an essential indicator of injury and toxicities (farah et al., 2013). hypertrophy of organs often indicates toxicity and damage to organs (ping et al., 2013). this often results from edema due to inflammation of the organs, which will increase the weight of the affected organs. the decrease in the weights of the liver in the low-dose group does not suggest a severe harmful effect and may*be a reflection of the moderate effect observed in the histopathology of these organs. plant extracts and other chemical compounds are evaluated for possible effects on hematological parameters to assess their toxic potential in blood (bashir et al., 2015). subacute administration of leaf extract of s. officinarum to rats for 30 days did not significantly affect the wbc counts, lymphocytes, monocytes, eosinophil, and basophil percentages (p>0.05) compared to control. however, the neutrophils percentage of the group treated with 170 mg/kg of the leaf extract was significantly (p<0.05) increased and significant (p<0.05-0.01) decreases in rbc and platelets counts, hemoglobin concentration and pcv percentage were observed in the group treated with the middle dose (340 mg/kg) of the extract when compared to control. these suggest the hemolytic effect of the extract and/or suppression of erythropoiesis (the rate of production of erythrocytes) (berinyuy et al., 2015), as well as thrombopoiesis. these imply that the oxygen-carrying capacity of the blood can be affected and indicative of the extract’s anemia and thrombocytopenia-inducing potentials. the reduced platelet counts observed explain the significant increases in the bleeding and clotting times of the treated rats when compared to the control, indicating an effect in the blood clotting mechanism. however, the percentage of neutrophils, was significantly elevated by the extract when compared to the control. the significant increase in the neutrophils by the extract could suggest an enhancement in the ability of the blood component to phagocytose, and indicative of an inflammatory response perhaps in response to the deleterious effect of the extract, a demonstration of the immunogenic potential of the extract. in this study, administration of leaf extract of s. officinarum (170-510 mg/kg) to rats for 30 days exerted insignificant (p>0.05) effects on total protein, albumin, and alt levels of rats when compared to control. the alp levels were similarly reduced, but this was only significant (p<0.01) in the group treated with the highest dose (510 mg/kg) dose of the extract. similarly, ast, total and conjugated bilirubin levels of treated rats were decreased, but the reductions were significant (p<0.010.001) only at the higher doses (340 and 540 mg/kg) of the extract when compared to control. the results suggest that the extract does not affect the synthetic functions of the liver. decreases in serum proteins indicate hepatic damage due to the reduced capability of the hepatocytes to synthesize enough serum proteins. reduced serum/plasma albumin is associated with hepatic damage (shin et al., 2010; yousef et al., 2010). assessment of albumin and protein in the liver could be used as an important indicator of the synthetic function of the organs, whereas bilirubin (total and conjugated) could be used to assess the excretory function of the liver (kaplan et al., 1979; yakuba et al., 2003). severe hemolysis causes the release of more bilirubin into the blood, which manifests as elevated levels of direct and total bilirubin (ngana,1989). bilirubin is a metabolic breakdown product of heme derived from senescent red blood cells and is commonly used in liver function tests. it is removed from the blood by the liver, chemically modified by a process called conjugation (formation of bilirubin), secreted into the bile, passed into the intestine, and to some extent, reabsorbed from the intestine (yakubu et al., 2005). the reduction in the total and direct bilirubin, as observed in this study with the leaf extract, could be adduced to impairment in the secretory function of these proteins and may also adversely affect the functional activity of the liver, especially the biliary system (ashafa et al., 2009). in this study, significantly reduced activities of ast and alp were observed following subacute administration of the leaf extract of s. officinarum, especially at higher doses of the extract. cellular leakage of enzymes occurs often when the cell architecture and integrity are damaged. similarly, the presence of enzymes in the serum above their average level is a pointer to clinical diagnosis of various pathological conditions. serum aspartate aminotransferase (ast) and alanine aminotransferase (alt) have been reported by numerous authors as markers for acute and chronic hepatocellular damage (dufour et al., 2000). pyridoxal5-phosphate (plp), the active form of vitamin b6, is the coenzyme for both alt and ast, respectively (rej, 98 biology, medicine, & natural product chemistry 14 (1), 2025: 91-100 1977). a significant decrease in serum ast might be due to metabolic, drug-induced or iatrogenic (unknown cause). pathophysiological conditions associated with a deficiency of vitamin b6 might lead to a decrease in serum ast and alt activities (lum, 1995). decrease in the serum/plasma activities of aspartate aminotransferase has been shown to correlate with pyridoxal-5-phosphate deficiency (waner & nyska, 1991; evans & whitehorn, 1995; hall, 2001; saori et al., 2003). the extract may have affected the liver and caused pyridoxal-5-phosphate deficiency. alkaline phosphatase (alp) is a ‘marker’ enzyme for the plasma membrane and endoplasmic reticulum; it is therefore an ectoenzyme of the plasma membrane and it is often used to assess the integrity of the plasma membrane (shittu et al., 2015). however, it was not affected by the extract treatment. the results further suggest mild toxic potential of the extract at higher doses. blood urea nitrogen (bun) produced in the liver is derived from the diet or tissue sources and is excreted in the urine via the kidney. serum urea accumulates in the serum in renal disease when the production rate exceeds that of excretion (mayne, 1994). serum creatinine is derived from endogenous sources by tissue creatinine breakdown (mayne, 1994). therefore, elevation of urea and creatinine levels in the serum had been taken as the index of nephrotoxicity (ali et al., 2001; flaoyen et al., 2001). in this study, treatment of rats for 30 days with leaf extract of s. officinarum (170-510 mg/kg) did not cause any significant effect (p>0.05) on the levels of urea, creatinine, potassium, and sodium of the treated rats when compared to control. however, the level of chloride was significantly (p<0.05) increased at the higher dose of the extract (510 mg/kg) when compared to control. this shows that the extract is not nephrotoxic at the doses studied. the electrolyte concentrations were not affected by the extract treatment except chloride level at the highest dose, suggesting that the glomerular filtration rate was not affected by the extract treatment mostly at lower doses. alterations in the concentration of significant lipids like cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and triglycerides can give helpful information on lipid metabolism as well as the predisposition of the heart to atherosclerosis and its associated coronary heart diseases (yakubu et al., 2008). high blood cholesterol concentrations are an essential risk factor for cardiovascular disease (abolaji et al., 2007). therefore, the slightly reduced levels of serum total cholesterol, triglyceride, ldl, and vldl especially at higher doses by the extract though insignificant may be clinically beneficial to the animals as the extract is unlikely to be associated with cardiovascular risk at these doses. similarly, the reduced lipolysis may explain the decreased levels of serum triacylglycerol by the extract (yakubu et al., 2008). the reduction in the levels of vldl, ldl, and hdl in this study reveals a robust hypolipidemic activity of the leaf, perhaps due to inhibitory activity on lipolysis, which is due to the activities of its phytoconstituents and may be an indication that the extract may not predispose the animals to atherosclerosis and coronary heart diseases (philip, 1995; jackson, 1996; mayes, 1996; panagiotakos et al., 2003). on the histology, subacute administration of ethanol leaf extract of s. officinarum to rats for 30 days produced varying degrees of abnormalities ranging from mild to moderate defects on histology of the liver and kidney of rats. higher doses of the leaf extract (340-510 mg/kg) were found to produce some defects such as areas of micro and macro-vesicular steatosis, vacuolated hepatocytes, hemorrhagic blood deposits, and arrays of sinusoidal spaces within the hepatic lobule in all treatment groups portraying a mild to moderate effect on the liver. this is corroborated by the reduced levels of ast and alp which have no effect on alt. the history-architechture of renal tissues of treated rats was moderately affected, with glomeruli having widened bowman’s space, vacuolated ductal cells, and hemorrhagic blood vessels within the cortical matrix, portraying a mild toxic effect on the kidney. the mild effect of the extract on the kidney is further supported by the chemical pathology results in which the lack of any significant effect of the extract on levels of urea, creatinine, potassium, and sodium of the treated rats was observed when compared to the control. conclusion the results of this study show that subacute administration of leaf extract of saccharum officinarum can cause anaemia, thrombocytopenia, hypolipidemia, increased clotting and bleeding time and a mild toxic effect on the liver and kidneys, which are due to the activities of its phytochemical constituents. acknowledgements: the authors are grateful to the university of uyo management for providing an enabling environment and to mr nsikan malachy of the pharmacology department, university of uyo for technical assistance. authors’ contributions: jeo, uae research concept and design; 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(2005). aphrodisiac potentials of the aqueous extract of fadogia agrestis (schweinf. ex hiern) stem in male albino rats. asian journal of andrology,7(4), 399-404. https://www.ncbi.nlm.nih.gov/pubmed/?term=singh%20a%5bauthor%5d&cauthor=true&cauthor_uid=26009693 https://www.ncbi.nlm.nih.gov/pubmed/?term=lal%20ur%5bauthor%5d&cauthor=true&cauthor_uid=26009693 https://www.ncbi.nlm.nih.gov/pubmed/?term=mukhtar%20hm%5bauthor%5d&cauthor=true&cauthor_uid=26009693 https://www.ncbi.nlm.nih.gov/pubmed/?term=singh%20ps%5bauthor%5d&cauthor=true&cauthor_uid=26009693 https://www.ncbi.nlm.nih.gov/pubmed/?term=shah%20g%5bauthor%5d&cauthor=true&cauthor_uid=26009693 https://www.ncbi.nlm.nih.gov/pubmed/?term=dhawan%20rk%5bauthor%5d&cauthor=true&cauthor_uid=26009693 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4441162/ biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 151-157 | doi: 10.14421/biomedich.2023.121.151-157 issn 2540-9328 (online) a new ent-kaurene diterpenoid isolated from leaves of espeletia semiglobulata cuatrec. and its potential antimicrobial activity andrés márquez1,*, alida pérez1, †luis rojas1, rosa aparicio1, freddy ramos2, ysbelia obregón1, alfredo usubillaga1 1research institute, faculty of pharmacy and bioanalysis, university of los andes, mérida 5101, venezuela. 2department of chemistry, faculty of science, national university of colombia, bogotá d.c. 111321, colombia. corresponding author* andresmqch@gmail.com manuscript received: 03 december, 2022. revision accepted: 16 january, 2023. published: 24 january, 2023. abstract the fraction of the neutral extraction of the leaves of espeletia semiglobulata cuatrec. was subjected to chromatographic separation, it yielded four ent-kaurene type diterpenoids: three known [ent-kaur-16-en-19-al (i), ent-kaur-18-nor-16-en-4-ol (iii), ent-kaur-16-en-19-ol (iv)] and a new one elucidated as ent-kaur-3-acetoxy-15-ene (ii), based on the physicochemical and spectroscopic data of ftir, gc-ms, and 1d and 2d nmr. these compounds were subjected to antimicrobial bioassay studies. this new ent-kaurene showed a significant inhibition potential against the growth of gram negative bacterial strains [escherichia coli (atcc 25922): 8 mm, klebsiella pneumoniae (atcc 23357): 10 mm, pseudomonas aeruginosa (atcc 27853): 8 mm], it also showed inhibition against the growth of fungal strain (candida krusei: 8 mm), at a 2 mg/ml concentration. the compounds (i), (iii) and (iv) failed to show any significant results in the antimicrobial screening against five bacterial strains [staphylococcus aureus (atcc 25923), enterococcus faecalis (atcc 29212), klebsiella pneumoniae (atcc 23357), escherichia coli (atcc 25922) y pseudomonas aeruginosa (atcc 27853)] and one fungal strain [candida krusei (atcc 6558)]. these results reveal a remarkable natural structure-activity relationship of the ent-kaurene core regarding the c-3 position (a ring of perhydrophenanthrene unit), whose oxygenation or addition of a hydrogen bond acceptor or donor group, improves the antimicrobial activity. keywords: diterpene; ent-kaurene; asteraceae; natural products; antimicrobial activity. abbreviations: nmr – nuclear magnetic resonance; 1h nmr – proton nuclear magnetic resonance; 13c nmr – carbon nuclear magnetic resonance; 13c-apt – attached proton test; cosycorrelation spectroscopy; hsqc – heteronuclear single quantum correlation spectroscopy; hmbc – heteronuclear multiple bond correlation spectroscopy; ir – infrared spectroscopy; gc-ms – gas chromatography–mass spectrometry; mp – melting point; mh – müeller-hinton; dmso – dimethyl sulfoxide; etoac – ethyl acetate. introduction bioactive natural products have played an important role in treating and preventing human diseases. these are important sources for new drugs and also good lead compounds suitable for further modification during drug development (atanasov et al., 2021). the terpenoids are among the most important natural compounds known for their medicinal value (kamran et al., 2022; proshkina et al., 2020); within them, ent-kauranes represent an important group of tetracyclic diterpenes that possesses a wide spectrum of biological and pharmaceutical activities such as antimicrobial, antiparasitic, insect antifeedant, cytotoxic, antitumour, anti-hiv, steroidogenic, antifertility, hypotensive, antiinflamatory, among others (ghisalberti, 1997). this type of diterpene is extensively found in different species of espeletiinae (asteraceae), a family of resinous plants, popularly known as “frailejon”, that grow at an altitude of 2500 m in the andes of northern south america. these plants are used by the inhabitants of the high moors to treat asthma and rheumatic conditions (usubillaga et al., 2003; aparicio et al., 2013). ent-kaurenes have been identified in some studies about the espeletia semiglobulata cuatrec. species. it grows above an altitude of 3900 m at the “piedras blancas” moor, which is part of sierra la culata, located near the city of mérida, venezuela. ent-kaur-16-en-19-oic acid has been identified on the fraction of acid extraction of the leaves of this plant as the most abundant component and known for its diverse biological properties (sosa-sequera et al., 1996; cavalcanti et al., 2005; kim et al., 2016; zhang et al., 2017; cotoras et al., 2004; mongelli et al., 2002), and ent-kauran-19-oic acid, ent-kaur-9(11)-16-dien-19oic acid, ent-kaur-15α-isovaleroxy-16-en-19-oic acid, ent-kaur-15α-hidroxy-16-en-19-oic acid, ent-kauran16α-hidroxy-19-oic acid, and ent-kaur-15-en-19-oic acid have been identified as the less abundant components. on the other hand, ent-kaur-16-en-19-al and ent-kaurhttps://doi.org/10.14421/biomedich.2023.121.151-157 152 biology, medicine, & natural product chemistry 12 (1), 2023: 151-157 16-en-19-ol have been identified as majority components in the fraction of neutral extraction, and entkaur-16α-hidroxy-16-en-19-al, ent-kaur-18-nor-16-en-4ol, ent-kaur-16-en-19-ol acetate have been identified as minority components without any biological activity reported on any of them (usubillaga et al., 1988; aparicio et al., 2013). in this study, we are reporting the isolation, structural identification, and antimicrobial activity of a new ent-kaurene-type terpenoid natural product (ii) from neutral fraction of espeletia semiglobulata cuatrec. leaves, also, the evaluation of antimicrobial activity of some known ent-kaurenes coming from this neutral extraction is being reported for the first time. materials and methods general methods melting points were determined on a fisher-johns melting point apparatus. ir spectra were measured on a perkin elmer spectrum two, 10.03.06 version, as kbr disks. nmr spectra was recorded with a brukerultrashieldtm 400 mhz instrument for solutions in cdcl3. gc-ms were performed on a hewlett-packard msd 5973 instrument fitted with a 5 % phenylmethyl polysiloxane fused-silica column (hp-5ms, 30 m, 0.25 mm, film thickness 0.25 μm). the initial analysis temperature was 250 °c, which was increased at 5 °c/min. to a final temperature of 300 °c. analytical thin-layer chromatography was performed on e. merck aluminum-backed silica gel foils (f254). flash chromatography was performed on silica gel e. merck grade 60, 63-200 mesh, by gradient elution with hexane and hexane-etoac mixtures. plant collection leaves of espeletia semiglobulata cuatrec. were collected at 3900 m of altitude in “piedras blancas” moor and along the road to piñango, about 13 km from the mountain’s peak “pico del aguila”. voucher specimens (au30 and au21) were deposited at the faculty of pharmacy and bioanalysis herbarium, university of los andes (merf herbarium). extraction and neutral fraction obtainment espeletia semiglobulata cuatrec. leaves were dried at 40 °c during 48 hours and they were grinded. the grinded material (200 g) was extracted at room temperature with a mixture of hexane/diethyl ether (3:1, v/v) for a period of two days. the solvents were removed under reduced pressure and the solid residues were dissolved in hexane/etoac, and shaken with 5 % aqueous naoh. the aqueous phase was taken to ph 3 by careful addition of concentrated hcl and shaken with hexane to obtain the acid fraction. the original hexane-diethyl ether solution, left over after the alkaline treatment surplus, was mixed with active charcoal and boiled for 10 minutes. a neutral fraction of 25 g was obtained after an open column chromatography treatment. isolation of kaurene diterpenes most of the leaves neutral fraction was submitted to flash chromatography over silica gel. the column (a) was eluted with hexane and hexane/acoet mixtures and 100 ml fractions were collected, which were constantly motorized by tlc. fractions 1-35 eluted with hexane yielded 840 mg of a mixture that contained plant waxes and (i). fractions 36-39 eluted with hexane rendered 500 mg of pure (i) as a white solid, mp 113-116 ºc, identical to an authentic sample of ent-kaur-16-en-19-al (mp,1h-nmr, 13c-nmr) obtained from espeletia semiglobulata (usubillaga et al., 1988; aparicio et al., 2013). the elution continued with hexane (40-45 fractions), it yielded 150 mg of a mixture of (i) and (ii). the elution of the 46-61 fractions with hexane yielded 1000 mg of pure (ii), which was crystallized from hexane/et2o as white crystalline solid. this compound has a very similar polarity to (i), however, they are not the same (mp, 1h-nmr, 13c-nmr). the elution of the 82-94 fractions with hexane/etoac 5 % yielded 300 mg of pure (iii), mp 146-148 °c, identical to an authentic sample of ent-kaur-18-nor-16-en-4-ol (mp,1h-nmr, 13c-nmr) obtained from espeletia nana (peña et al., 2012). the elution of the 101-106 fractions with hexane/etoac 10 % yielded 200 mg of pure (iv), mp 142-144 °c, identical to an authentic sample of ent-kaur16-en-19-ol (mp,1h-nmr, 13c-nmr) obtained from espeletia semiglobulata cuatrec. (usubillaga et al., 1988; aparicio et al., 2013). finally, the elution using hexane/etoac 20 % yielded a complex mixture (21.4 g). antimicrobial assay  antibacterial activity the antibacterial activity was determined by the agar diffusion method with discs. the following microorganisms were tested: staphylococcus aureus (atcc 25923), enterococcus faecalis (atcc 29212), klebsiella pneumoniae (atcc 23357), escherichia coli (atcc 25922) y pseudomonas aeruginosa (atcc 27853). an 18 hours culture of each microorganism was used in 2.5 ml müeller-hinton (mh) broth at 37 °c. the bacterial inoculum was adjusted with saline physiological solution to the mc farland turbidity standard n° 0.5 (106-8 cfu/ml). each inoculum was spread with a swab on the surface of a plate containing müeller-hinton agar and then a disc of filter paper (6 mm diameter) previously impregnated with 10 μl of the compound dissolved in dmso was placed on the surface at a 2 mg/ml concentration for each one [(i), (ii), (iii), (iv)]. a disc impregnated with dmso was included as a negative control. in addition, the standard disc of the reference antibiotic was placed as a positive control for each of the microorganism (oxacillin 1 μg márquez, et al. – a new ent-kaurene diterpenoid isolated from leaves … 153 for staphylococcus aureus; vancomycin 30 μg for enterococcus faecalis; tobramycin 10 μg for escherichia coli; aztreonam 30 μg for klebsiella pneumoniae; cefepime 30 μg for pseudomonas aeruginosa). after having placed the discs on the petri plates, they were refrigerated at 4 °c for 24 h (velasco et al., 2007). the reading of the inhibition halos was performed at 24 h and it was measured (mm) around the disc. all the antibacterial activity trials were done twice. the measurement of the inhibition halos of the antibacterial activity was done thus: c= a-b (c= size of the inhibition halo; a= size of the halo plus the disk of filter paper; b= size of the filter paper disk, 6 mm).  antifungal activity the antifungal activity was determined by the agar well diffusion method. the microorganism tested was candida krusei (atcc 6558), using fluconazole 100 μg as positive control, and dmso as negative control. the strain repacked 12 hours before, coming from a savory dextrose agar, it was used to prepare the inoculum which was compared using the mc farland turbidity standard n°0.5 (106-8 cfu/ml). each inoculum was spreaded with a swab on the surface of a plate containing müeller-hinton agar and then 6 mm diameter holes were opened in it, where 20 μl of the compounds dissolved in dmso were placed at a 2 mg/ml concentration for each one [(i), (ii), (iii), (iv)]. one well was included with the dmso as a negative control and another well with the reference antibiotic as positive control. then the petri plates were refrigerated at 4 °c for 24 h (velasco et al., 2007). the reading of the inhibition halos was performed at 24 h and was measured (mm) around the well. all the antifungal activity trials were done twice. results and discussion phytochemical analysis and structure elucidation the structures of the ent-kaurene diterpenoids isolated from neutral fraction of e. semiglobulata cuatrec., leaves are presented on figure 1. figure 1. molecular structure of ent-kaurenes from neutral fraction of aerial parts of espeletia semiglobulata cuatrec. compound (i) was isolated as a white powder, mp 113-116 ºc. gc-ms gave [m]+ molecular ion at m/z: 286.1 (calcd. for c20h30o; m/z: 286). ft-ir, vmax (cm1), functional group: 2729, v(c-h, aldehyde); 1711, v(c=o); 1655, v(c=c). 1h-nmr (400 mhz, cdcl3) δh: 9.73 (h1-19, s, cho), 4.74 (h1-17a, s, =ch), 4.79 (h117b, s, =ch), 2.64 (h1-13, s, c-h), 2.05 (h2-15, m, ch2), 0.98 (h3-18, s, ch3). 13c-nmr (100 mhz, cdcl3) δc: 205.94 (c-19, cho), 103.24 (c-17, =ch2), 43.75 (c-13, ch), 49.05 (c-15, ch2), 24.29 (c-18, ch3). this data completely matches with the spectral data reported in the bibliographic references (usubillaga et al., 1988; aparicio et al., 2013), this leads us to the conclusion, the compound (i) is an ent-kaurene-type diterpene called ent-kaur-16-en-19-al, commonly known as kaurenal. this compound was first isolated and characterized as a natural product of espeletia grandiflora humb. et bompl. (piozzi et al., 1971). it is particularly abundant in species of espeletia genus (morales et al., 1973; bohlmann et al., 1980; usubillaga et al., 1988), and in fact, it has already been isolated and reported from e. semiglobulata cuatrec. leaves (usubillaga et al., 1988; aparicio et al., 2013). compound (ii) was obtained as a crystalline white powder. the analysis of its 13c nmr and gc-ms data (m/z 330 [m]+) deduced its molecular formula c22h34o2, which suggested 6 degrees of unsaturation. the ir spectrum showed the absorption bands of ester group [1735 cm-1 (o−c=o) and 1243 cm-1 (o−c)] and typical bands of alkene group [3040 cm-1 (=c−h) and 1661 cm1 (c=c)]. the 1h nmr (400 mhz, cdcl3) and 13c nmr (cdcl3, 100 mhz) data (table 1) demonstrated the signals of thirty-four protons and twenty-two carbons sorted by attached proton test (13c-apt) and heteronuclear single quantum coherence (hsqc) spectra, which can be classified as: a terminal ester group [δh/δc: 1.98 (h3-22, s, ch3) / 21.47 (c-22, ch3); 171.17 (c-21, >c=o)], a trisubstituted alkene group [δh/δc: 5.06 (h1-15, s, =ch) / 124.47 (c-15, =ch); 139.78 (c-16, >c<)], four methyl groups [δh/δc: 0.81 (h3-18, s, ch3) / 28.22 (c-18, ch3); 0.80 (h3-19, s, ch3) / 22.70 (c-19, ch3); 0.73 (h3-20, s, ch3) / 22.70 (c-20, ch3)] including an olefinic methyl [δh/δc: 1.80 (h3-17, s, ch3) / 23.37 (c-17, ch3)], a methinic proton bonded to a carbon bearing heteroatom (deshielded proton) [δh/δc: 4.50 (h1-3, t, o−ch) / 80.84 (c-3, o−ch)] seven sp3 methylene [δh/δc: 1.55 (h2-1, m, ch2) / 33.01 (c-1, ch2); 1.57 (h2-2, t, ch2) / 23.52 (c2, ch2); 1.49 (h2-6, m, ch2) / 21.07 (c-6, ch2); 1.30 (h2-7, m, ch2) / 36.69 (c-7, ch2); 1.48 (h2-11, m, ch2) / 18.39 (c-11, ch2); 1.54 (h2-12, m, ch2) / 26.75 (c-12, ch2); 1.35 (h2-14, t, ch2) / 41.49 (c-14, ch2)], three sp3 methines [δh/δc: 1.51 (h1-5, m, ch) / 52.51 (c-5, ch); 0.79 (h1-9, m, ch) / 55.41 (c-9, ch); 1.86 (h1-13, m, ch) / 47.79 (c-13, ch)], and three sp3 quaternary carbon [δc: 37.86 (c-4); 40.17 (c-8); 39.68 (c-10)]. detailed analysis of these nmr data suggested the presence of a typical ent-kaurene-type diterpenic 154 biology, medicine, & natural product chemistry 12 (1), 2023: 151-157 skeleton [perhydrophenanthrene unit (a, b and c rings) fused to a cyclopentane unit (d ring) formed by a twocarbon bridge between c-8 and c-13 (figure 1)] (batista et al., 2005). this partial structure was further confirmed by correlations spectroscopy (cosy) and heteronuclear multiple bond correlation (hmbc), which has been shown in figure 2. by analysis of the cosy correlations, it was possible to identify six structural areas (a f) of this compound. the fact that the terminal ester group [c-21 (δc 171.17, o-c=o)/c-22 (δc 21.47, ch3); h3-22(δh 1.98, s, ch3)]; where the terminal arrangement was confirmed by the hmbc correlation between the methylic protons h3-22 and carbonyl group c-21 (δc 171.17, c=o)] was on c-3 (δc 142.7) was confirmed by the hmbc correlation from methinic proton h1-3 (δh 4.50, t, ch) to ester carbonyl group c21 (δc 171.17, >c=o), as well as, the correlation of h1-3 with methylene c-2 (δc 23.52, ch2) and quaternary carbon c-4 (δc 37.86, >c<). the presence of two methyls on c-4 was confirmed by its hmbc correlation with h3-18 (δh 0.81, s, ch3) and h3-19 (δh 0.80, s, ch3), in addition to the hmbc correlation of these methylic prontons with methine c-3 (δc 80.84, o-ch), as well as, by the correlations: h3-19 (δh 0.80, s, ch3) / c-5 (δc 52.51, ch) and h1-5 (δh 1.51, m, ch) / c-4 (δc 37.86, >c<). the mutual correlations of both methyls, both by hmbc [h3-18 (δh 0.81, s, ch3) / c-19 (δc 22.70, ch3); h3-19 (δh 0.80, s, ch3) / c-18 (δc 28.22, ch3)] and by cosy [h3-18 (δh 0.81, s, ch3) / h3-19 (δh 0.80, s, ch3)] also confirmed the vicinal arrangement of them on c-4. the existence of one methyl on c-10 was verified by the hmbc correlation from methylic protons h3-20 (δh 0.73, s, ch3) to quaternary carbon c-10 (δc 39.68, >c<) and methylene c-1 (δc 33.01, ch2). on the other hand, the presence of an endocyclic c-c double bond between c-15 and c-16, and the presence of an olefinic methyl on c-16 (d ring) were confirmed by hmbc correlations between methylic prontons h3-17 (δh 1.80, s, ch3) with olefinic methine c-15 (δc 124.47, =ch) and methine c-13 (δc 47.79, ch), and at the same time, the methinic proton h1-13 (δh 1.86, m, ch) correlates with quaternary carbon c-16 (δc 139.78, =c<) and methylene c-12 (δc 26.75, ch2). this partial structure has also been confirmed by cosy correlation from olefinic methyl protons h3-17 (δh 1.80, s, ch3) to olefinic methine proton h1-15 (δh 5.06, s, =ch), and vice versa, as well as, the mutual correlation between olefinic methyl protons h3-17 with methinic proton h1-13 (δh 1.86, m, ch), and this last, mutual correlated with methylenic ptotons h2-14 (δh 1.35, t, ch2) and h2-12 (δh 1.54, m, ch2). finally, the central ring of ent-kaurene core (b ring) was confirmed by the hmbc correlations between methinic proton h1-9 (δh 0.79, m, ch) with quaternary carbon c-10 (δc 39.68, >c<), c-8 (δc 40.17, >c<) and methylene c-11(δc 18.39, ch2), as well as, this ring was confirmed by cosy correlations between methinic proton h1-9 with h1-5 (δh 1.51, m, ch), h2-7 (δh 1.30, m, ch2) and h2-11 (δh 1.48, m, ch2). based on the spectroscopic analysis performed, the isolation of an ent-isokaurene-type diterpene is confirmed, especially ent-kaur-3-acetoxy-15-ene, which have also be called 3acetoxy-isokaurene, this being a natural product reported for the first time. compound (iii) was isolated as a white powder, mp 146-148 °c. gc-ms analysis shown [m]+ molecular ion at m/z: 274.3 (calcd. for c19h30o; m/z: 274). ft-ir, vmax (cm-1), functional group: 3500, v(o-h, alcohol); 1600, v(c=c). 1h-nmr (400 mhz, cdcl3) δh: 4.76 (h1-17a, s, =ch), 4.82 (h1-17b, s, =ch), 3.65 (h1-18, s, oh), 2.66 (h1-13, t, c-h), 2.16 (h2-15, m, ch2), 1.20 (h3-18, s, ch3), 1.11 (h3-20, s, ch3). 13c-nmr (100 mhz, cdcl3) δc: 79.31 (c-4, >coh), 44.20 (c-13, ch), 49.40 (c-15, ch2), 102.93 (c-17, =ch2), 21.52 (c-18, ch3), 17.74 (c-20, ch3). this data completely matches with the spectral data reported in the bibliographic references (bohlmann et al., 1980; peña et al., 2012), this leads us to the conclusion that the compound iii) is an ent-kaurene-type diterpene called ent-kaur-18-nor-16-en-4-ol, commonly known as ruilopeziol. this compound has only been isolated and reported as a natural product of ruilopezia linden, coespeletia lutescens, and espeletia nana cuatrec. (bohlmann et al., 1980; peña et al., 2012), therefore, its isolation and characterization from e. semiglobulata cuatrec. leaves is a new discovery. márquez, et al. – a new ent-kaurene diterpenoid isolated from leaves … 155 table 1. 1h-nmr (cdcl3, 400 mhz), 13c-nmr (cdcl3, 100 mhz), cosy (1h↔1h) (cdcl3, 600 mhz) and hmbc [1h (600 mhz) → 13c (100 mhz), cdcl3] of compound (ii) (ent-kaur-3-acetoxy-15-ene). position δ 1h (ppm), mult. δ 13c (ppm), type cosy (1h↔1h) hmbc (1h→13c) 1 1.55, m 33.01, (>ch2) h2-1 ↔ h2-2 - 2 1.57, t 23.52,(>ch2) h2-2 ↔ h2-1 h2-2 → c-3 3 4.50, t 80.84, (o-ch-) h1-3 ↔ h2-2 h1-3 → c-2, c-4, c-21 4 -37.86, (>c<) -- 5 1.51, m 52.51, (>ch-) h1-5 ↔ h3-19 h1-5 → c-4, c-19, c-6 6 1.49, m 21.07, (>ch2) -h2-6 → c-5 7 1.30, m 36.69, (>ch2) -h2-7 → c-8 8 -40.17, (>c<) -- 9 0.79, m 55.41, (>ch-) h1-9 → h2-7, h1-5, h2-11 h1-9 → c-10, c-11, c-8 10 -39.68, (>c<) -- 11 1.48, m 18.39, (>ch2) -h2-11 → c-12 12 1.54, m 26.75, (>ch2) h2-12 ↔ h1-13 h2-12 → c-13, c-11 13 1.86, m 47.79, (>ch-) h1-13 ↔ h2-12, h3-17 h1-13 → c-17, c-16, c-12 14 1.35, t 41.49, (>ch2) h2-14 ↔ h1-13 - 15 5.06, s 124.47, (=ch) h1-15 ↔ h3-17 h1-15 → c-17 16 -139.78, (=c<) -- 17 1.80, s 23.37, (-ch3) h3-17 ↔ h1-15, h1-13 h3-17 → c-13, c-15, c-16 18 0.81, s 28.22, (-ch3) h3-18 ↔ h3-19 h3-18 → c-3, c-4, c-19 19 0.80, s 22.70, (-ch3) h3-19 ↔ h3-18, h1-5 h3-19 → c-3, c-4, c-5, c-18 20 0.73, s 17.66, (-ch3) -h3-20 → c-1, c-10 21 -171.17, (>c=o) -- 22 1.98, s 21.47, (-ch3) -h3-22 → c-21 figure 2. selective 1h↔1h cosy (▬) and 1h→13c hmbc (▬) correlations of (ii). compound (iv) was isolated as a white powder, mp 142-144 °c. gc-ms analysis shown [m]+ molecular ion at m/z: 288.3 (calcd. for c20h32o; m/z: 288). ft-ir, vmax (cm-1), functional group: 3406, v(o-h, alcohol); 1026, v(c-o); 1600, v(c=c). 1h-nmr (400 mhz, cdcl3) δh: 4.73 (h1-17a, s, =ch), 4.79 (h1-17b, s, =ch), 3.44 (h1-19a, dd, ch2), 3.74 (h1-19b, dd, ch2), 2.63 (h1-13, t, c-h), 2.06 (h2-15, m, ch2), 1.01 (h3-18, s, ch3), 0.96 (h3-20, s, ch3). 13c-nmr (100 mhz, cdcl3) δc: 65.60 (c-19, ch2oh), 155.90 (c-16, >c=), 103.00 (c-17, =ch2), 27.10 (c-18, ch3), 18.21 (c-20, ch3), 44.20 (c-13, ch), 49.10 (c-15, ch2). by comparing this experimental data with the spectroscopic data reported in the bibliography (bohlmann et al., 1980; piozzi et al., 1971), it is evident that this compound is an ent-kaurene-type diterpene called entkaur-16-en-19-ol, commonly known as kaurenol. currently, this diterpene is considered a widely distributed product in the asteraceae family, since it has been isolated in different species, including the species of the espeletia genus (seaman et al., 1990), like espeletia semiglobulata cuatrec. (usubillaga et al., 1988; aparicio et al., 2013). antimicrobial activity the isolated compounds, ent-kaur-16-en-19-al (i), entkaur-3-acetoxy-15-ene (ii), ent-kaur-18-nor-16-en-4-ol (iii) and ent-kaur-16-en-19-ol (iv) were evaluated for in vitro growth inhibitory potential against different bacterial [staphylococcus aureus (atcc 25923), 156 biology, medicine, & natural product chemistry 12 (1), 2023: 151-157 enterococcus faecalis (atcc 29212), escherichia coli (atcc 25922), klebsiella pneumoniae (atcc 23357), pseudomonas aeruginosa (atcc 27853)] and fungal [candida krusei (atcc 6558)] strains, according to the previously described procedure to agar diffusion method with discs (strain of bacteria), and agar well diffusion method, (strain of fungi). the results in table 2, shows that all the compounds are inactive againt the bacterial and fungal strains tested, except compound (ii), which showed growth inhibition against gram-negative bacterial strains (escherichia coli: 8 mm, klebsiella pneumoniae: 10 mm, pseudomonas aeruginosa: 8 mm), as well as it showed growth inhibition against the fungal strain (candida krusei: 8 mm), at a concentration of 2 mg/ml. these results reveal a remarkable natural structureactivity relationship of the ent-kaurene core with regarding the c-3 position (a ring of perhydrophenanthrene unit), whose oxygenation or structural modification apparently, improves the antimicrobial activity. similar results have been found synthetically, by preparing derivatives of hydroxylated ent-kaurenes at c-3, improving their biological activity (ohkoshi et al., 2004). moreover, the evaluation of the relationship between structure and antimicrobial activity of some diterpenes suggested that the presence of a substituted decalin skeleton (essential hydrophobic portion for crossing membranes), and a hydrophilic region bearing a donor or acceptor group able to interact with hydrogen-bond acceptor/donor groups on the membrane were necessary for their insertion in a phospholipid bilayer model. this feature enables the diterpenes to cross the bacterial cell membrane causing cell damage (urzúa et al., 2008). in this context, the ent-kaurenes tested have the structural characteristics described above: a substituted decalinic system that confers a lipophilic character, and a hydrophilic region bearing a hydrogen-bond donor or acceptor group. however, the compounds (i), (iii) and (iv), which possess these polar groups in the c-4 position, they are sterically limited to form hydrogenbond with acceptor/donor groups on the microorganism membrane, and therefore cannot cause damage to it. on the contrary, compound (ii) has this moderately hydrophilic group (ester) in c-3 position, whose steric hindrance is low and this confers the advantage to interact and establish hydrogen bonds with acceptor/donor groups on the microorganism membrane, being able to cross and cause damage to it. the position and steric hindrance of this hydrogen-bond donor or acceptor group on decalinic system are important characteristics for the biological activity of ent-kaurenes (urzúa et al., 2008). table 2. antimicrobial activity of natural compounds (i)-(iv) expressed as diameters of the inhibition zone (mm). microorganism compound positive control (i) (ii) (iii) (iv) ox va to az cf fl staphylococcus aureus (atcc 25923) ia ia ia ia *23 enterococcus faecalis (atcc 29212) ia ia ia ia *19 escherichia coli (atcc 25922) ia 8 ia ia *26 klebsiella pneumoniae (atcc 23357) ia 10 ia ia *34 pseudomonas aeruginosa (atcc 27853) ia 8 ia ia *32 candida krusei (atcc 6558) ia 8 ia ia *15 ia: inactive; ox: oxacillin®; va: vancomycin®; to: tobramycin®; az: aztreonam®; cf: cefepime®; fl: fluconazole®; *: inhibition halo in mm, for control groups (clsi, 2020). conclusions ent-kaurene-type diterpenes, ent-kaur-16-en-19-al (i), ent-kaur-3-acetoxy-15-ene (ii), ent-kaur-18-nor-16-en4-ol (iii), ent-kaur-16-en-19-ol (iv), were isolated from the neutral fraction of espeletia semiglobulata cuatrec. leaves, being ent-kaur-3-acetoxy-15-ene (ii) a natural product reported for the first time. compound (ii) was the only one that showed antimicrobial activity, but only against gram-negative bacterial strains (escherichia coli: 8 mm, klebsiella pneumoniae: 10 mm, pseudomonas aeruginosa: 8 mm) and fungal strain (candida krusei: 8 mm), at of 2 mg/ml concentration. these results reveal a remarkable natural structure-activity relationship of the ent-kaurene core with regarding the c-3 position (a ring of perhydrophenanthrene unit), whose oxygenation or addition of a hydrogen-bond acceptor or donor group, improves the antimicrobial activity. márquez, et al. – a new ent-kaurene diterpenoid isolated from leaves … 157 acknowledgements: all authors are grateful to the research institute “dr. alfredo nicolás usubillaga del hierro”, faculty of pharmacy and bioanalysis, university of los andes, mérida 5101, venezuela for the support. the research institute authors are grateful to the prof. freddy ramos from the department of chemistry, faculty of science, national university of colombia, bogotá d.c. 111321, colombia, for nmr and ms equipment. besides, all authors are grateful to the msc. ana deixy flores from the “instituto clínico santa filomena”, microbiology laboratory, mérida 5101, venezuela, for the antimicrobial assays. competing interests: the authors declare that there are no competing interests. funding this research was supported by research institute “dr. alfredo nicolás usubillaga del hierro”, faculty of pharmacy and bioanalysis, university of los andes, mérida 5101, venezuela. references aparicio, r., villasmil, t., peña, a., rojas, j., & usubillaga, a. 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2540-9328 (online) preparation of ni/bentonite acid-activated using dragon fruit peel extract (hylocereus polyrhizus) as a reductor for one pot synthesis of menthol yuni marhayuni*, priyagung dhemi widiakongko chemistry department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. corresponding author* yunimarhayuni06@gmail.com abstract ni/bentonite is acid-activated has been synthesized using dragon fruit peel extract (hylocereus polyrhizus) as a reductor through an impregnation method. this research aims to characterize ni/bentonite acid-activated using dragon fruit peel extract as a reductor based on ftir and xrd instruments and to analyze the product obtained in one pot synthesis of menthol from citronellal using ni/bentonite acidactivated catalyst. preparation of ni/bentonite acid-activated catalyst was carried out using a wet impregnation method with dragon fruit peel extract as a natural reductor. the successful synthesis of ni/bentonite acid-activated was confirmed by ftir spectrum identification, showing absorptions at wavelengths of 1049.28 cm⁻¹ and 694.37 cm⁻¹, indicating si-o groups in the bentonite, and xrd diffractogram indicating the presence of ni at diffraction peaks of 2θ = 40.5° and 2θ = 47.74°. the catalyst was able to convert menthol by 0.77% through one pot synthesis without using h₂ sources such as potassium formate. keywords: dragon fruit peel extract; impregnation; menthol; ni/bentonite; one pot synthesis. introduction menthol is a compound obtained from mentha piperita l leaf extract or laboratory synthesis (cantanhede et al., 2021). menthol is used to provide aroma and flavor in commercial products within the food, beverage, and cosmetic industries (adilina et al., 2015), as well as in toothpaste, cigarettes, and pharmaceuticals (trasarti et al., 2013). the global use of menthol is estimated to reach 32,000 tons annually (su et al., 2019). the high demand for menthol drives researchers to develop methods to increase menthol production through laboratory synthesis (lothe et al., 2021). one approach is the synthesis of menthol from citronellal using a one pot system (virtanen et al., 2009). synthesis of menthol from citronellal can be carried out using a one pot system with two sequential reactions. these reactions involve the cyclization of citronellal to isopulegol, catalyzed by acid, followed by the hydrogenation of isopulegol to menthol, catalyzed by metal (sulaswatty et al., 2019). this reaction uses heterogeneous catalysts, which involve metal impregnation onto porous materials with acidic sites, allowing both cyclization and hydrogenation reactions to occur in one container. heterogeneous catalysts can be reused multiple times and are easy to separate at the end of the reaction due to their distinct phase from the reactants (adilina et al., 2015). several heterogeneous catalysts that have been developed include ni/ɤ-al₂o₃ (iftitah et al., 2011), ni/zrs (cortes et al., 2011), zr-pillar montmorillonite (fatimah et al., 2014), ni/natural zeolite (adilina et al., 2015), and ru(bpy)₃-saponite (fatimah et al., 2019). these catalysts can produce menthol from citronellal with high yields, achieving 100% menthol with ni/zrs catalyst (cortes et al., 2011). heterogeneous catalysts can be made by impregnating metal catalysts into porous materials with acidic sites. bentonite is one such porous material with surface acidity related to bronsted and lewis acids. additionally, bentonite has a large surface area that can be used to distribute metals (trisunaryanti, 2018). nickel metal is a metal catalyst that can be evenly dispersed throughout the pore system of bentonite and can function as an active site in the catalyst due to the incomplete electron filling in the d-orbital, making it effective in accepting electron pairs from reactants to achieve a reaction (trisunaryanti, 2018). previous research has required high-pressure h₂ gas as a hydrogen source, both in catalyst preparation and in one pot synthesis. the use of high-pressure h₂ gas can manuscript received: 09 agustus, 2024. revision accepted: 10 october, 2024. published: 30 october, 2024. https://doi.org/10.14421/biomedich.2024.132.523-530 mailto:yunimarhayuni06@gmail.com 524 biology, medicine, & natural product chemistry 13 (2), 2024: 523-530 easily explode if exposed to oxygen. therefore, hydrogen sources can be replaced with more environmentally friendly compounds. h₂ gas, functioning as a reductor, can be substituted with natural reductors such as plant extracts. secondary metabolites found in plants can act as reductors, particularly phenolic compounds in flavonoids that serve as natural reductors. one plant that contains secondary metabolites such as flavonoids, tannins, and alkaloids is dragon fruit peel (putri et al., 2018). thus, dragon fruit peel extract can be used as a natural reductor, as demonstrated by rahmawita & ulianas (2021). in their research, nife₂so₄ nanoparticles were synthesized using dragon fruit peel extract as a reductor. dragon fruit peel extract contains antioxidants such as vitamin c, flavonoids, tannins, alkaloids, steroids, and saponins, which have potential as natural reductors and stabilizers (noor et al., 2016). given the information above, the development of a one pot synthesis menthol from citronellal using ni/bentonite acid-activated catalysts is necessary. in this research, preparation of ni/bentonite catalysts is carried out using an impregnation method. the reduction process will use dragon fruit peel extract mixed with ni(no₃)₂·6h₂o and acid-activated bentonite. the reduction process will also be conducted using h₂ gas to compare the characteristics of the catalysts produced with two different hydrogen sources. similarly, in the one pot synthesis of menthol, potassium formate will be used as an alternative hydrogen source to h₂ gas. methods preparation of ni/bentonite acid-activated catalyst bentonite acid-activated 1 kg of natural bentonite is washed with distilled water, dried, then ground and sieved using a 140 mesh sieve. activation is performed by refluxing 25 grams of natural bentonite with 250 ml of 2 m hcl for 3 hours at 90°c. the refluxed product is filtered and neutralized to ph 7 using distilled water, then dried in an oven at 50°c for 15 hours. the dried activated bentonite is then ground and sieved using a 140 mesh sieve. it is subsequently tested using ftir and xrd. dragon fruit peel extract three grams of dragon fruit peel simplicia are boiled with 600 ml of distilled water for 5 minutes at a temperature of 50-60°c, then filtered using a buchner funnel to obtain dragon fruit peel aqueous extract. ni/bentonite acid-activated catalyst ni/bentonite acid-activated catalyst is prepared using two different reduction methods. for the first method, 2.4758 grams of ni(no₃)₂·6h₂o is dissolved in 10 ml of distilled water and mixed with 10 grams of acid-activated bentonite. the mixture is stirred at 60°c until the solution evaporates. the resulting precipitate is separated from the filtrate using filter paper, then dried in an oven at 110°c for 4 hours. the obtained catalyst is calcined for 3 hours at 400°c with a flow of n₂ gas. the catalyst is then tested using ftir and xrd. in the second method, a similar process is carried out, but the dragon fruit peel extract as the reductor is replaced with h₂ gas. after calcination with n₂, the catalyst is reduced with h₂ gas at 400°c for 3 hours. one pot synthesis of menthol from citronellal catalyzed by ni/bentonite acid-activated 0.5 grams of ni/bentonite acid-activated catalyst is placed into a three-necked flask, followed by the addition of 10 ml of citronellal and 30 ml of isopropanol. the solution is stirred with a magnetic stirrer while being heated, then 6 ml of potassium formate is added dropwise once the reaction temperature reaches 80°c. the reaction is allowed to proceed for 5 hours at this temperature. the reaction mixture is separated from the solvent using an evaporator at 60°c for approximately 2 hours. the sample is then tested using ftir and gc-ms. the one-pot system reaction is performed with variations of catalysts: acid-activated ni/bentonite with dragon fruit peel extract as the reductor, acid-activated ni/bentonite with h₂ gas as the reductor, and acidactivated ni/bentonite with dragon fruit peel extract without potassium formate. results and discussion activation of natural bentonite with 2 m hcl the activation of natural bentonite is performed using a reflux method with hcl solvent. the resulting bentonite is characterized using ftir and xrd. ftir characterization aims to identify the functional groups present in natural bentonite and bentonite acid-activated. the ftir spectra show that there are no significant changes between natural bentonite and bentonite acidactivated; however, some absorption peaks have shifted in wavenumber. the shift occurs at 3425.58 cm⁻¹ to 3433.29 cm⁻¹, indicating stretching vibrations of oh from h₂o. the peak at 1033.85 cm⁻¹ shifts to 1042.56 cm⁻¹, indicating bending vibrations of si-o. the high intensity at this absorption peak indicates a high content of montmorillonite in the bentonite (mahmudha & nugraha, 2016). a decrease in intensity is observed at the wavenumber 918.12 cm⁻¹ in acid-activated bentonite, which represents bending vibrations of oh groups in aloh-al. this decrease is due to acid dealumination in the bentonite framework, which involves the removal of al³⁺ from the octahedral layers (hidayat & nugraha, 2018). marhayuni & widiakongko – preparation of ni/bentonite acid-activated using … 525 figure 1. ftir spectra of natural bentonite and bentonite acid-activated. xrd characterization aims to determine the types of minerals in natural bentonite and bentonite acidactivated, as well as the diffraction patterns at 2θ values and the basal spacing of the crystal lattice (mahmudha & nugraha, 2016). the xrd diffractogram of natural bentonite in figure 2 shows peaks at 2θ = 5.42° with a basal spacing (d) of 16.3055 å and 2θ = 19.70° with a basal spacing (d) of 4.5066 å. these peaks indicate the presence of montmorillonite mineral according to jcpds no. 13-10135, with d001 = 15.000 å and d100 = 4.5000 å. the peak at 2θ = 21.84° with d = 4.0696 å, according to jcpds no. 11-0695, indicates the presence of cristobalite mineral, with d101 = 4.05 å. peaks at 2θ = 20.72° with d = 4.2870 å and 2θ = 26.50° with d = 3.3636 å indicate the presence of quartz mineral, consistent with jcpds no. 05-0490, with d100 = 4.2600 å and d101 = 3.3430 å. the peak at 2θ = 35.48° with d = 2.5302 å indicates the presence of illite mineral, according to jcpds no. with d131 = 2.5500 å. the xrd diffractogram of bentonite acid-activated shows peaks at 2θ = 5.37° with d = 16.4555 å and 2θ = 19.68° with d = 4.5111 å, indicating the presence of montmorillonite mineral. the shift in 2θ values and the basal spacing (d) becomes more pronounced after activation with hcl. the activation process causes the removal of al from the octahedral layers and replacement with h+ groups, leading to the expansion of the basal spacing in montmorillonite's silicate layers (mahmudha & nugraha, 2016). the removal of al can enhance si-o-si bonding, leading to increased quartz intensity (hidayat & nugraha, 2018), as evidenced by the increased peaks at 2θ = 9.72° with d = 9.0997 å and 2θ = 27.90° with d = 3.3636 å, consistent with jcpds no. 05-0490, with d002 = 10.300 å and d101 = 3.3430 å. the crystallinity of bentonite is influenced by the si/al ratio. good crystallinity is characterized by a high si/al ratio (wijaya et al., 2016). the si/al ratio can be improved through activation with acid. the increase in the si/al ratio is evidenced by the xrd diffractogram of h-bentonite, showing an enhanced absorption peak at 2θ = 9.72°, indicating the presence of quartz mineral. figure 2. diffractogram of natural bentonite and bentonite acid-activated. 5001000150020002500300035004000 tr an sm it ta n ce ( a. u .) wavelength 1/cm natural bentonite 3 6 1 8 ,4 6 3 6 2 6 ,1 7 3 4 3 3 ,2 9 3 4 2 6 ,6 8 1 6 3 6 ,6 4 1 6 3 6 ,6 4 1 0 4 1 ,5 6 1 0 3 3 ,8 6 7 9 4 ,6 7 7 7 9 4 ,6 7 5 3 2 ,3 5 5 2 4 ,6 4 bentonite acid-activated 0 10 20 30 40 50 60 70 80 90 in te n si ty ( a. u .) 2 theta natural bentonite acid activated bentonite m m k k i m k m k k i k 526 biology, medicine, & natural product chemistry 13 (2), 2024: 523-530 preparation of ni/bentonit acid-activated catalyst using dragon fruit peel extract as a reductor the dragon fruit peel extract as a nickel reductor was obtained using a decoction method with distilled water as the solvent (setiawan et al., 2015). the high flavonoid content in dragon fruit peel can serve as a hydrogen source in the form of –oh groups bound to secondary carbon atoms, which can reduce ni ions to metallic ni, while the compounds with –oh groups themselves will undergo oxidation (muhaini et al., 2014). a comparison was made between the use of this natural reductor and h₂ to evaluate the characterization of the acid-activated ni/bentonite catalyst reduced with dragon fruit peel extract. ftir characterization indicates that the spectra for both types of reducers show no significant changes. figure 3. ftir spectra of bentonite acid-activated, catalyst a (ni/bentonite acid-activated with h2 reductor), and catalyst b (ni/bentonite acid-activated with dragon fruit peel extract reduction). xrd characterization shows that the mineral structure of bentonite acid-activated remains stable after calcination and reduction. the xrd diffractogram for catalyst a shows a peak at 2θ = 19.60° with d = 4.5293 å, indicating the presence of montmorillonite mineral. the increase in basal spacing is due to the impregnation process, which enlarges the space between layers and forms a better pore system. the increase in basal spacing is also a result of the incorporation of nickel metal into the interlayer space of the bentonite structure (fatimah et al., 2014). similarly, shifts in 2θ and increases in basal spacing occur at 2θ = 20.64° (d = 4.3034 å) and 2θ = 27.52° (d = 3.2412 å). the decrease in peak intensity at 2θ = 9.72° and 2θ = 27.90° is due to a reduction in surface area, pore volume, and changes in pore size distribution in bentonite, likely caused by nickel metal covering its surface (al anshori & muchalal, 2009). the xrd diffractogram for catalyst b, reduced using dragon fruit peel extract, shows peaks almost identical to catalyst a, which was reduced with h₂. this indicates that the dragon fruit peel extract has successfully acted as a bioreductor in reducing nickel metal. the presence of nickel (ni) is identified at peaks 2θ = 40.57° and 2θ = 48.25° for catalyst a. for catalyst b, nickel (ni) is identified at 2θ = 40.5° and 2θ = 47.74°. these results are consistent with jcpds and the findings of rahayu et al., (2013). in their research, peaks at 2θ = 40.570° and 2θ = 48.295° correspond to the absorption of ni metal. the presence of nio is detected at 2θ = 62.15° for catalyst a and 2θ = 62.02° for catalyst b. these peaks match with jcpds no. 47-1049, where nio appears at 2θ = 37.2°, 43.27°, and 62.87°. this is also observed in the research conducted by jiang et al. (2018), where the nio diffraction peak was detected at 2θ = 62.8° for ni/bentonite. the appearance of the nio absorption peak indicates that nickel in the form of oxide is not fully reduced to ni, as nio is a relatively stable oxide. figure 4. xrd diffractogram of bentonite acid-activated, catalyst a (ni/bentonite acid-activated with h2 reduction), and catalyst b (ni/bentonite acidactivated with dragon fruit peek extract redactor). 5001000150020002500300035004000 tr an sm it ta n ce ( a. u .) bentonite acid-activated catalyst a catalyst b 1 0 4 9 ,2 8 1 0 4 9 ,2 8 1 0 4 9 ,2 8 6 9 4 , 3 7 6 9 4 , 3 7 6 9 4 , 3 7 0 10 20 30 40 50 60 70 80 90 in te n si ta s (a .u .) 2 theta bentonite acid-activated catalyst a catalyst b m k k k k k k * * ◊ ◊◊ ◊ * * ◊ = ni * = nio marhayuni & widiakongko – preparation of ni/bentonite acid-activated using … 527 one-pot synthesis of menthol from citronellal the one-pot synthesis of menthol from citronellal, which is predominant in lemongrass oil. based on gc-ms analysis, citronellal constitutes 57.07% with a retention time of 17.371 minutes. ftir analysis was conducted to identify the functional groups in lemongrass oil. according to the ftir spectrum of lemongrass oil, the absorption bands at wavelengths 2916.37 cm⁻¹ to 2862.36 cm⁻¹ correspond to the stretching vibration of aliphatic c-h groups. the wavelength 2715.77 cm⁻¹ represents the stretching vibration of c-h aldehyde, and the aldehyde group in citronellal is also absorbed at 1728.22 cm⁻¹. the presence of the c=c group is identified at 1635.64 cm⁻¹, while the wavelength at 1728.11 cm⁻¹ indicates the presence of c=o or carbonyl groups (wijayanti, 2015). the wavelength 1450.47 cm⁻¹ shows the –ch₂– group, while the ch₃ group is identified at 1381.03 cm⁻¹. the wavelength 1026.13 cm⁻¹ indicates the stretching vibration of c-o from ch₂oh. these wavelengths suggest that lemongrass oil contains other compounds besides citronellal, such as citronellol and geraniol, which have ch₂oh linkages. figure 5. chromatogram of lemongrass oil. table 1. compounds in lemongrass oil as determined by gc-ms. compounds r.time (minutes) area (%) citronellal 17,371 57,02 % geraniol 20,593 17,15 % citronellol 19,715 8,40 % limonene 12,817 2,44 % citral 20,075 0,43 % citral 20,973 0,68 % 11-tetradecen-1-ol-asetat 23,241 1,89 % other compounds 11,99 % total 100 % figure 6. ftir spectrum of lemongrass oil. the one-pot synthesis reaction involves a catalyst, citronellal as the reactant, isopropanol as the solvent, and potassium formate as the hydrogen donor. potassium formate is used as a hydrogen donor because it decomposes into formic acid and potassium, leaving no other residues at the end of the reaction. hydrogen 528 biology, medicine, & natural product chemistry 13 (2), 2024: 523-530 transfer reactions are optimized by using isopropanol as the solvent, which accelerates the hydrogen transfer. the synthesis of menthol from citronellal involves two stages of reaction. the first stage is the cyclization of citronellal to isopulegol, followed by the hydrogenation of isopulegol to menthol. below is the conversion of menthol from citronellal: figure 7. menthol reaction from citronellal. characterization of reaction products the results of the one pot synthesis reaction of menthol were characterized using ftir to identify the functional groups of the compounds produced from the reaction. the one pot synthesis for citronellal was carried out with three reaction variations. reaction 1 used catalyst a with the addition of potassium formate. reaction 2 used catalyst b with the addition of potassium formate. reaction 3 used catalyst b without potassium formate. figure 8. ftir spectrum of one pot synthesis for citronellal reaction products. the ftir spectra of the three reaction products show absorption peaks similar to those of lemongrass oil. the presence of aldehyde groups is identified at a wavenumber of 1728.22 cm⁻¹ in both the ftir spectrum of lemongrass oil and the ftir spectra of the three reaction products. the wavenumber 1635.64 cm⁻¹ in the lemongrass oil spectrum indicates the presence of c=c groups, whereas in the reaction products, this peak shifts to 1643.35 cm⁻¹. the c-o group in menthol is identified at a wavenumber of 1026.13 cm⁻¹. however, this wavenumber is also present in the lemongrass oil spectrum, making it inconclusive whether menthol has been formed in the reaction based solely on the ftir results. the ftir does not provide definitive evidence that menthol has been successfully formed during the reaction, as the spectral wavenumber differences between reactants and products are not significant. nevertheless, the third reaction product has a different ftir spectrum compared to both the lemongrass oil and the spectra of the other reaction products. therefore, further analysis using gc-ms was conducted on the third reaction product. figure 9. chromatogram of the third reaction product. 5001000150020002500300035004000 t ra n sm it ta n ce ( a. u .) wavelength (1/cm) lemongrass oil reaction 1 reaction 2 1 63 5, 54 16 43 ,3 5 16 43 ,3 5 reaction 3 16 43 ,3 5 marhayuni & widiakongko – preparation of ni/bentonite acid-activated using … 529 based on the chromatogram of the third reaction product, citronellal is still detected with a retention time of 30.708 minutes at 1.30%. there is a difference in the retention time of citronellal in the third reaction product compared to citronellal in lemongrass oil, where citronellal appears at a retention time of 17.371 minutes with a concentration of 57.02%. the retention time difference between citronellal in the two samples is 13.337 minutes. this difference in retention time is used to determine changes in peak area percentage and retention time in the lemongrass oil sample and the third reaction product. the retention time difference is consistent with citronellol and geraniol, which are also present in lemongrass oil. menthol is a new compound produced from the reaction, as there is no corresponding retention time in lemongrass oil based on the retention time reduction in the third reaction product and the observed retention time difference. figure 10. comparison of compound contents in lemongrass oil and the third reaction product. from the data above, it can be observed that the reaction can convert citronellal into isopulegol, although not completely, as citronellal is still present at 1.30%. isopulegol will then react with hydrogen with the help of ni/bentonite acid-activated catalyst to produce menthol. the hydrogenation of isopulegol to menthol has occurred without the use of formic acid as an h₂ source. this process involves a reduction reaction utilizing the oxidation effect of other compounds (muhaini, syukri, & syukri, 2014). based on the gc-ms results, it is identified that the compound undergoing oxidation is limonene, which is converted to terpin hydrate. the reaction occurring is as follows: figure 11. oxidation conversion reaction of limonene to terpin hydrate the conversion reaction of limonene to terpin hydrate is an oxidation reaction involving electron release. in the study by duarte, et al., (1983), this reaction showed a current density of around 1.1 v – 1.3 v. the electrons released from this process react with h₂o to form h₂ as shown in the following equation: 2 ē + 2𝐻2𝑂 → 2𝑂𝐻 − + 𝐻2 the hydrogen formed from this process can reduce the double bonds in isopulegol to produce menthol. the reactions occurring in the one pot synthesis for menthol from citronellal are greatly influenced by the amount of metal in the catalyst, as well as the dosage of the catalyst and formic acid relative to citronellal (fatimah et al., 2019). the presence of h₂ is crucial for reducing the double bonds in isopulegol. additionally, high catalyst activity and selectivity are essential for achieving a high yield. the formation of unwanted compounds in this reaction is due to the catalyst’s lack of selectivity in producing menthol. conclusions the ni/bentonite acid-activated catalyst with dragon fruit peel extract reducer has been successfully formed, as demonstrated by the functional group characteristics using ftir and xrd, indicating the presence of ni and nio in the bentonite. this catalyst can convert citronellal to menthol in a one pot synthesis with a yield of 0.77%, without the addition of an h2 source such as potassium formate. 17,25 8,4 2,44 57,02 1,89 13 29,8 20,22 2,12 12,2 2 1,3 1,7 2,16 0,77 0,69 27,04 lemongrass oil reaction 3 530 biology, medicine, & natural product chemistry 13 (2), 2024: 523-530 acknowledgements: the author extends gratitude to the lppm uin sunan kalijaga for their support and funding of this research. competing interests: the authors declare that there are no competing interests. references adilina, b. i., pertiwi, r., & sulaswatty, a. 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(2009). towards one-pot synthesis of menthol from citral: modifying supported ionic liquid catalysts (silcas) with lewis and bronsted acids. journal of catalysis, 263, 209-219. wijaya, k., hadi, k., herlina, i., & kurnia, t. (2016). nano material (aplikasinya dalam pembuatan biofuel). yogyakarta: ugm press. wijayanti, l. w. (2015). isolasi sitronellal dari minyak sereh wangi (cymbopogon winterianus jowit) dengan distilasi fraksinasi pengurangan tekanan. jurnal farmasi sains dan komunitas, 12(1), 22-29. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 921-926 | doi: 10.14421/biomedich.2025.142.921-926 issn 2540-9328 (online) sirnas targeting icad gene of staphylococcus aureus to inhibit biofilm formation: structural analysis and efficacy dinda ananda sulistina1, rian ka praja2*, margaretha yayu indah anugerahny3, hanasia2, ysrafil4 1undergraduate program of medicine, faculty of medicine, universitas palangka raya, indonesia. 2department of microbiology, faculty of medicine, universitas palangka raya, indonesia. 3department of clinical medicine, faculty of medicine, universitas palangka raya, indonesia. 4department of pharmacotherapy, faculty of medicine, universitas palangka raya, indonesia. corresponding author* riankapraja@med.upr.ac.id abstract antibiotic resistance in staphylococcus aureus infections, especially those involving biofilm formation, is a global health issue. biofilm protects bacteria from the immune system and antibiotic treatment, making them 10 to 1000 times more resistant. the icad gene, part of the ica operon, is crucial for biofilm synthesis by enhancing the enzymes responsible for forming the biofilm matrix. the icad gene sequence of staphylococcus aureus was obtained from the genbank ncbi database with the accession code cp140612.1, with a gene sequence length of 306 bp and employed several bioinformatics methods, including sidirect for designing and evaluating effective sirna sequences to select the most promising candidates. additionally, sirna scales, maxexpect, duplex fold, and sipred were employed to analyze the sirna sequence length, secondary structure, binding energy, and efficacy predictions of sirnas targeting the icad gene. the study found that out of 54 sirna candidates, sirna22, sirna50, and sirna25 achieved inhibition rates of 93.69%, 92.82%, and 92.52%, respectively. these results bioinformatically demonstrated their potential to suppress the expression of the icad gene and highlight their promise as sirna-based antibacterial therapies to combat biofilm-related infections. the designed sirna computationally shows potential as an innovative therapy to combat biofilm infections caused by staphylococcus aureus. keywords: staphylococcus aureus; biofilm; icad gene; sirna; antibiotic resistance. introduction antimicrobial resistance (amr) is one of the top global to public health and development. it is estimated that bacterial amr was directly responsible for 1.27 million global deaths in 2019 and contributed to 4.95 million deaths (murray et al., 2022). staphylococcus aureus (s. aureus), which often forms biofilms, is becoming increasingly difficult to treat, especially strains resistant to methicillin (mrsa). bacterial biofilms protect them from the immune system and antibiotics, making them 10-1000 times more resistant (tran et al., 2023). biofilm formation in s. aureus is encoded by several genes, including icaa, icab, icac, and icad, which are involved in the biofilm matrix formation (peng et al., 2023). one promising therapeutic approach is the use of small interfering rna (sirna) to inhibit the expression of the icad gene, which plays a role in biofilm formation. sirna can regulate gene expression through the rna interference (rnai) mechanism, which targets mrna and inhibits the translation of specific proteins. with the advancement of bioinformatics technology, the analysis of sirna structure and efficacy has become easier. previously, sirna has been used for antiviral therapy, such as for hepatitis c and zika virus infections (perezmendez et al., 2020). this study aimed to explore, analyze the structure, and predict the efficacy of sirna to suppress the expression of the icad gene in s. aureus as a strategy to combat infections caused by bacterial biofilms. materials and methods study area the study took place in faculty of medicine palangka raya university between june and desember 2024. procedures retrieval of the icad gene sequence the downloaded fasta sequence of the icad gene from staphylococcus aureus (ncbi accession number: cp140612.1) was renamed accordingly and stored in a dedicated folder for further analysis. the sequence was then utilized to perform several computational analyses, including the design of sirna molecules targeting the icad gene, analysis of mrna length percentage, prediction of the secondary structure of the designed manuscript received: 08 june, 2025. revision accepted: 26 october, 2025. published: 28 october, 2025. https://doi.org/10.14421/biomedich.2025.142.921-926 mailto:riankapraja@med.upr.ac.id 922 biology, medicine, & natural product chemistry 14 (2), 2025: 921-926 sirnas, calculation of binding energies between sirna and target mrna, and prediction of sirna efficacy in silencing the icad transcript. these analyses were conducted using appropriate bioinformatics tools and databases to ensure the accuracy and reliability of the results. design of gene-silencing sirna molecules the icad gene sequence of staphylococcus aureus was obtained from the genbank ncbi database with the accession code cp140612.1, the gene sequence is 306 bp long and employs several bioinformatics methods. the design of sirna to silence the s. aureus icad gene was performed using sidirect (http://sidirect2.rnai.jp/). percentage analysis of mrna length percentage analysis of mrna length was performed using sirna scales, a software that predicts the location of the remaining mrna sequence in the cell after sirna cleavage (http://gesteland.genetics.utah.edu/sirna_scales/). analysis of the secondary structure of sirna secondary structure analysis of sirna was done using maxexpect and could be accessed through a website https://rna.urmc.rochester.edu/rnastructureweb/servers /maxexpect/maxexpect.html. subsequently, the 21nt guide rna oligo sequences (5′→3′) obtained from sirna design analysis using sidirect were submitted. analysis of the binding energy between sirna and its target mrna analysis of the binding energy between sirna and the s. aureus icad gene target was done using duplexfold (https://rna.urmc.rochester.edu/rnastructureweb/server s/duplexfold/duplexfold.html). the software showed the analysis results, including the binding energy and a predicted secondary rna structure diagram. efficacy prediction of sirna molecules prediction of sirna efficacy against the icad target in s. aureus was performed using sipred (http://predictor.nchu.edu.tw/sipred/), then the software displayed predicted position and inhibitory effects of the sirna strand on the icad gene. results and discussion results and discussion should be written as a series of connecting sentences, however, for manuscript with long discussion should be divided into subtitles. results should be clear and concise. result of the e xploration of sirna targeting the icad antigen of staphylococcus aureus fifty-four sirna candidates were successfully designed based on the complete genome of the icad gene from s. aureus (ncbi accession number: cp140612.1). the analysis of target locations, target region length, binding, and inhibition of the antigen sequences in these fifty-four sirna candidates targeting the icad antigen of staphylococcus aureus resulted in data as shown in table 1 below. table 1. the analysis of target sirna. no target position rna oligo sequences 21nt guide (5′→3′) rna oligo sequences 21nt passenger (5′→3′) sirna scales max expect duplex fold sirna efficacy 1 10-32 uggguauucccucugucuggg cagacagagggaauacccaac 14 1.4 -38.2 74.17 2 16-38 uagcguuggguauucccucug gagggaauacccaacgcuaaa 7 1.9 -37.4 74.54 3 18-40 uuuagcguuggguauucccuc gggaauacccaacgcuaaaau 11 2.0 -34.7 none 4 19-41 uuuuagcguuggguauucccu ggaauacccaacgcuaaaauc 11 2.0 -31.7 none 5 20-42 auuuuagcguuggguauuccc gaauacccaacgcuaaaauca 25 1.8 -29.0 none 6 30-52 uuuagcgaugauuuuagcguu cgcuaaaaucaucgcuaaaca 6 1.5 -28.5 75.9 7 32-54 uguuuagcgaugauuuuagcg cuaaaaucaucgcuaaacauu 17 1.9 -26.9 70.01 8 42-64 ucucuuauaauguuuagcgau cgcuaaacauuauaagagaaa 10 1.6 -28.2 85.79 9 43-65 uucucuuauaauguuuagcga gcuaaacauuauaagagaaac 2 1.7 -27.3 85.79 10 44-66 uuucucuuauaauguuuagcg cuaaacauuauaagagaaaca 14 1.8 -23.2 none 11 49-71 ugcuguuucucuuauaauguu cauuauaagagaaacagcacu 12 1.7 -27.9 86.47 12 59-81 uagcgauaagugcuguuucuc gaaacagcacuuaucgcuaua 8 1.9 -32.5 82.54 13 65-87 acgauauagcgauaagugcug gcacuuaucgcuauaucgugu 14 1.9 -32.5 80.71 14 73-95 aaagacacacgauauagcgau cgcuauaucgugugucuuuug 14 1.9 -31.2 83.94 15 74-96 aaaagacacacgauauagcga gcuauaucgugugucuuuugg 9 1.9 -30.9 83.94 16 81-103 uauauccaaaagacacacgau cgugugucuuuuggauauauu 5 1.8 -30.1 80.02 17 82-104 auauauccaaaagacacacga gugugucuuuuggauauauug 14 1.8 -28.9 80.02 18 86-108 aacaauauauccaaaagacac gucuuuuggauauauuguuua 26 1.9 -26.4 87.55 19 88-110 uaaacaauauauccaaaagac cuuuuggauauauuguuuagu 15 1.9 -24.1 80.08 20 93-115 acaacuaaacaauauauccaa ggauauauuguuuaguuguuc 19 1.8 -26.6 85.78 21 94-116 aacaacuaaacaauauaucca gauauauuguuuaguuguucu 11 1.8 -23.3 84.44 22 102-124 acgaguagaacaacuaaacaa guuuaguuguucuacucguuu 20 1.7 -30.0 93.69 23 107-129 uauaaacgaguagaacaacua guuguucuacucguuuauauu 12 1.8 -28.1 83.92 http://sidirect2.rnai.jp/ http://gesteland.genetics.utah.edu/sirna_scales/ https://rna.urmc.rochester.edu/rnastructureweb/servers/maxexpect/maxexpect.html https://rna.urmc.rochester.edu/rnastructureweb/servers/maxexpect/maxexpect.html https://rna.urmc.rochester.edu/rnastructureweb/servers/duplexfold/duplexfold.html https://rna.urmc.rochester.edu/rnastructureweb/servers/duplexfold/duplexfold.html http://predictor.nchu.edu.tw/sipred/ sulistina et al. – sirnas targeting icad gene of staphylococcus aureus to inhibit biofilm 923 no target position rna oligo sequences 21nt guide (5′→3′) rna oligo sequences 21nt passenger (5′→3′) sirna scales max expect duplex fold sirna efficacy 24 113-135 uaccaauauaaacgaguagaa cuacucguuuauauugguacu 16 1.8 -28.1 92.07 25 116-138 uaguaccaauauaaacgagua cucguuuauauugguacuaua 15 1.8 -27.7 92.52 26 118-140 uauaguaccaauauaaacgag cguuuauauugguacuauauu -3 1.7 -26.1 82.05 27 119-141 auauaguaccaauauaaacga guuuauauugguacuauauuu 14 1.6 -24.9 82.05 28 129-151 ugaauuucaaauauaguacca guacuauauuugaaauucaug 15 1.8 -24.0 77.03 29 132-154 ucaugaauuucaaauauagua cuauauuugaaauucaugacg 16 1.8 -23.6 85.43 30 140-162 uacuuucgucaugaauuucaa gaaauucaugacgaaaguauc 14 1.6 -28.4 91.93 31 146-168 uauugauacuuucgucaugaa caugacgaaaguaucaauaca 12 1.8 -28.0 82.7 32 149-171 uuguauugauacuuucgucau gacgaaaguaucaauacaaua 8 1.8 -28.6 88.71 33 151-173 uauuguauugauacuuucguc cgaaaguaucaauacaauacg 9 1.6 -25.2 none 34 156-178 acacguauuguauugauacuu guaucaauacaauacguguug 23 1.9 -27.8 83.19 35 160-162 agcaacacguauuguauugau caauacaauacguguugcuuu 26 1.4 -29.2 88.89 36 165-187 uuuaaagcaacacguauugua caauacguguugcuuuaaaca 5 1.8 -26.4 80.67 37 171-193 ucaauguuuaaagcaacacgu guguugcuuuaaacauugaaa 9 1.5 -28.6 74.8 38 173-195 uuucaauguuuaaagcaacac guugcuuuaaacauugaaaau 17 1.5 -26.1 73.85 39 176-198 uauuuucaauguuuaaagcaa gcuuuaaacauugaaaauacu 9 1.8 -23.9 84.08 40 184-206 aauuucaguauuuucaauguu cauugaaaauacugaaauuuu 6 1.8 -22.4 73.05 41 195-217 aauauaucuaaaauuucagua cugaaauuuuagauauauuug 17 1.8 -20.9 71.95 42 224-246 ugauaaucgcgaaaaugccca ggcauuuucgcgauuaucauu 16 1.9 -31.3 none 43 225-247 augauaaucgcgaaaaugccc gcauuuucgcgauuaucauuu 16 1.9 -28.6 none 44 226-248 aaugauaaucgcgaaaaugcc cauuuucgcgauuaucauuuu 18 1.9 -25.7 none 45 232-254 aacaaaaaugauaaucgcgaa cgcgauuaucauuuuuguuuu 18 1.8 -26.0 87.89 46 233-255 aaacaaaaaugauaaucgcga gcgauuaucauuuuuguuuuu 12 1.8 -25.1 82.24 47 234-256 aaaacaaaaaugauaaucgcg cgauuaucauuuuuguuuuuu 4 1.8 -21.3 none 48 235-257 aaaaacaaaaaugauaaucgc gauuaucauuuuuguuuuuuu 14 1.8 -21.1 none 49 241-263 uguaaaaaaaacaaaaaugau cauuuuuguuuuuuuuacaau 14 1.8 -20.3 85.58 50 248-270 ugcuaauuguaaaaaaaacaa guuuuuuuuacaauuagcaua 21 1.6 -23.6 92.82 51 258-280 ugaaucaauaugcuaauugua caauuagcauauugauucaaa 5 1.5 -26.1 84.7 52 271-293 uuucugccauuuuugaaucaa gauucaaaaauggcagaaagg 9 1.7 -28.7 79.92 53 275-297 uuccuuucugccauuuuugaa caaaaauggcagaaaggaaga 10 1.8 -30.0 87.94 54 284-306 acgauucucuuccuuucugcc cagaaaggaagagaaucguga 28 1.8 -31.6 71.35 *top 3 sirnas with highest efficacy was highlighted with bold font. results of the structural analysis of the optimal icad sirna this structure was selected based on the parameters of sirna scales, maxexpert, duplex fold, and sirna efficacy according to the optimal criteria. the 3d visualization of the best sirna structure provides a detailed overview of the domain arrangement and the interactions between the sirna binding and the target rna, which in this case is icad from the s. aureus biofilm, as shown in figure 1 below. figure 1. (a) the structure of sirna22 (b) the structure of sirna50 (c) the structure of sirna25 (d) the structure of sirna22 (e) the structure of sirna50 (f) the structure of sirna25. sirna candidates 22, 50, and 25 are some of the best targets for gene silencing based on binding strength and the highest predicted efficacy among all sirna candidates, with potential to exert a suppressive effect on the icad rna. discussion the sirna molecules were designed using sidirect with a multi-step bioinformatics screening approach. sidirect provides functional sirna designs by considering the relationship between sirna sequences and rnai activity, and it also calculates potential gene candidates that do not match the target. the rapid and sensitive homology search, with an updated algorithm, significantly reduces off-target silencing. the analysis results were implemented to select potential icad gene sirna candidates targeting the icad rna of s. aureus. based on the exploration of sirna design for icad using 924 biology, medicine, & natural product chemistry 14 (2), 2025: 921-926 sidirect, 54 rna oligo sequences were obtained, including 21nt guide (5′→3′) and 21nt passenger (5′→3′) rna oligo sequences. the sirna candidates were evaluated using sirna scales to show the analysis results in the form of the percentage of mrna remaining in the cells after sirna-mediated cleavage. the range of predicted efficiency values represents the percentage of mrna remaining after cleavage by sirna, which refers to the gene knockdown efficiency by sirna. this value is usually expressed as the percentage of mrna that was not successfully degraded (remaining in the cell). sirnas with less than 30% remaining mrna are considered efficient in suppressing the target gene expression. in this study, sirnas with ≤10% remaining mrna were categorized as highly effective because they resulted in nearly perfect knockdown. meanwhile, sirnas with 10–30% remaining mrna are still quite effective for certain biological applications. on the other hand, sirnas with >30% remaining mrna are considered to have low knockdown efficiency and are typically avoided as candidates. the smaller the percentage of remaining mrna, the higher the effectiveness of the sirna in working according to its target without indirectly affecting other genes or processes (angart et al., 2013; dana et al., 2017). based on guidelines from sirna design, such as those from biotechnology companies (thermo fisher scientific, qiagen, or dharmacon), high doses (>30%) may increase the risk of nonspecific effects, such as activation of the interferon pathway or off-target effects. a low percentage of remaining mrna indicates a more effective mrna cleavage by the risc complex, thereby enhancing the knockdown efficiency of the target gene (bartel & sharp., 2004). the candidate sirna was further analyzed using maxexpect and duplexfold to calculate the free binding energy of the sirna and the free binding energy between the guide strand and the target. maxexpect was tested on a database of sirna sequences for the icad antigen of s. aureus with known secondary structures. maxexpect predicts the optimal structure (with the highest expected strand accuracy) and suboptimal structures as alternative hypotheses for that structure. the optimal structure, the maximum expected accuracy structure, is predicted and compared with the known structure in the database, and the prediction accuracy is reported as sensitivity and ppv (positive predictive value) (lu et al., 2009). target mrna regions with maxexpect values >1.5 indicate high accessibility to sirna, reflecting greater sensitivity in identifying open target areas. this increases the chances of successful sirna knockdown. furthermore, higher predicted ppv supports the effectiveness of sirna in reducing target gene expression during experimental tests. therefore, maxexpect values >1.5 can be considered optimal, especially when supported by other parameters such as knockdown prediction results showing remaining mrna ≤30%, as analyzed through previous sirna scales (pan et al., 2011; mysara & garibaldi, 2011; filhol et al., 2012). the duplexfold analysis is conducted to find the structure with the gibbs free energy (δg), which is a measure used to calculate the maximum work that can be done in a thermodynamic system when temperature and pressure are kept constant, indicating the most stable conformation under specific thermodynamic conditions. duplexfold predicts the lowest free energy conformation of rna hybrid duplexes based on intermolecular base pairing, while targetrna identifies the complementarity of base strands and calculates the rri score using the mean forecast error (mfe) model for rna duplexes (lybarger & sandkvist, 2004). two sirna candidates that did not meet the criteria, with duplexfold results ranging from 36.1 to -20.5 kcal/mol, which is the optimal binding free energy value generated for sirna prediction, were eliminated. in the context of selecting the most effective sirna, a lower (more negative) δg value typically indicates a stronger and more stable interaction between the sirna and the target mrna, which leads to more effective gene silencing. this more stable interaction is crucial because it allows the sirna to more effectively guide the rna interference (rnai) process to reduce the expression of the target gene. the lower the δg value, the stronger the binding between the sirna and mrna, which increases the likelihood of forming a functional risc complex, an essential factor in the effectiveness of gene silencing (kajino et al., 2022). the inhibition ability of the selected remaining sirna is predicted using sipred. accurate predictions will facilitate the design of optimized sirna by maximizing the success of the knockout of the sirna candidate on the target (high sensitivity) and minimizing off-target effects (high specificity) (chuai et al., 2018; chuai et al., 2017). the 22nd, 50th, and 25th sirna candidates are among the best targets for gene silencing based on binding strength and the highest efficacy predictions of all sirna candidates, showing potential to suppress the icad rna of s. aureus effectively. sirna with a 90% inhibition rate demonstrates exceptional ability to reduce target gene expression efficiently. this ensures that the target mrna is almost completely inhibited, which is crucial in therapeutic applications or research requiring highly effective gene knockdown (liu et al., 2012; caffrey et al., 2011). with high efficiency, the dose of sirna required to achieve therapeutic effects can be minimized. this reduction in dose can decrease the risk of side effects, such as immune response activation or off-target effects, which are often associated with higher sirna doses (caffrey et al., 2011). sirna with high inhibition levels typically have a strong binding affinity to the target mrna. this strong affinity ensures that, even with a smaller amount of sirna, binding to the target remains efficient, allowing for optimal gene knockdown. in a clinical context, the ability to achieve near-perfect knockdown provides greater confidence in the sulistina et al. – sirnas targeting icad gene of staphylococcus aureus to inhibit biofilm 925 effectiveness of sirna as a therapy. this is especially important for diseases that require drastic inhibition of the target gene, such as cancer or chronic infections (liu et al., 2012). the application of gene silencing based on sirna technology is a powerful strategy to limit bacterial infections by targeting and degrading bacterial mrna, ensuring that its sequence matches the target (hartawan et al., 2022). bacteria like s. aureus have undergone many mutations over time. therefore, sirna for bacteria must be targeted at genes that are considered to have an impact on infection. the icad gene is a promising target for gene silencing because it is located within the biofilm structure of s. aureus, which plays an important role in the virulence factors of an infectious disease. conclusions based on the results of the bioinformatics study on the exploration, structure, and efficacy prediction of sirna icad interacting with icad rna in s. aureus bacteria, this study found the binding energy and inhibition capability of sirna in silencing the icad gene. the best results were obtained with sirna strands 22, 50, and 25, which resulted in inhibition of 93.69%, 92.82%, and 92.52%, respectively. sirna with high inhibition levels typically has a strong binding affinity to the target mrna. this strong affinity ensures that, even with a smaller amount of sirna, binding to the target remains efficient, leading to optimal gene knockdown. these three potential sirna molecules can be used as an sirna-based antibacterial therapy to suppress infections caused by staphylococcus aureus with biofilms. however, the sirna predictions in this study are important to validate through laboratory experiments. acknowledgements: the authors would like to thank to the faculty of medical, palangka raya university, central kalimantan, for providing valuable resources that facilitated a deeper understanding of sirna for therapeutic purposes. authors’ contributions: study concept and design d.a.s., r.k.p., and m.y.i.a.; data acquisition d.a.s. and r.k.p; analysis and interpretation of data d.a.s; drafting of the manuscript d.a.s., critical revision of the manuscript d.a.s., r.k.p., m.y.i.a., h., and y.; administrative, technical, and material support r.k.p.; study supervision r.k.p., m.y.i.a., h, and y. competing interests: the authors declare that there are no competing interests. funding: the authors declare that no funding was received for this study. references angart, p., vocelle, d., chan, c., & patrick walton, s. 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(2020). sirna design to silence the 3 ′ utr region of zika virus. biomed research international, 2020. https://doi.org/10.1155/2020/6759346 tran, n. n., morrisette, t., jorgensen, s. c. j., orench-benvenutti, j. m., & kebriaei, r. (2023). current therapies and challenges for the treatment of staphylococcus aureus biofilm-related infections. pharmacotherapy, 43(8), 816–832. https://doi.org/10.1002/phar.2806 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1201-1206 | doi: 10.14421/biomedich.2025.142.1201-1206 issn 2540-9328 (online) exclusive breastfeeding and complementary feeding on stunting children among 12-24 months: a retrospective study dewi modjo1*, zuriati muhamad2, aurelia intan sitty aisyah p.s.p oei1 1departement of nursing study program, universitas muhammadiyah gorontalo, indonesia 2midwifery department, faculty of health science, universitas muhammadiyah gorontalo indonesia. corresponding author* dewimodjo@umgo.ac.id abstract this study aimed to analyze the relationship between the exclusive breastfeeding and complementary feeding, as well as the incidence of stunting in children between the ages of 12-24 months. an observational analytical study design with a cross-sectional approach was applied to examine the relationship between independent variables (knowledge of exclusive breastfeeding and complementary feeding) and the dependent variable (stunting). this study was conducted in the working area of the kota tengah health center working area. the results showed significant relationship between exclusive breastfeeding and the incidence of stunting in children (r: 0.541) with a p-value of 0.000 (p-value < 0.05), as well as a significant relationship between complementary feeding and the incidence of stunting in children (r: 0.332) with a p-value of 0.000 (p-value < 0.05). this study provides valuable information to mothers, highlighting the causes of stunting and promoting preventative measures to minimize its occurrence, such as carrying out consultations at the health care centre with personnel or professionals and addressing cases promptly. keywords: knowledge; exclusive breastfeeding; complementary feeding; children; stunting. introduction the first 1000 days of life (hpk), from conception until 2 years of age, are a critical period for improving children's physical and cognitive development. good maternal nutrition, health status, and nutritional intake during pregnancy and breastfeeding are important factors for physical and cognitive development and growth, and these factors reduce the risk of disease in children and mothers. however, malnutrition during pregnancy can impair fetal growth, representing a major contributing factor to stunting in children and increasing the risk of obesity and degenerative diseases in adulthood (muhamad et al., 2023). according to data obtained by the who in 2020, the number of stunted children under the age of 5 worldwide reached 150.8 million, equivalent to 22.2% of the total number of children under 5. furthermore, who also identified five regions with significant stunting prevalence rates, including indonesia, located in the southeast asia region, with a prevalence rate of 36.4% (panigoro et al., 2023). who in 2022 showed that 149.2 million children under the age of 5 worldwide suffered from stunting, 45.4 million were underweight, and 38.9 million were overweight. the number of stunted children has decreased in all regions except africa. in southeast asia and africa, 51 million children under the age of 5 are underweight, and an additional 151 million under the age of 5 are stunted. three-quarters of these children live in asia and africa (jafriati, 2022). according to the basic health research (riskesdas) in 2020, the incidence of stunting in indonesia is relatively high when compared to other developing countries. the incidence of stunting reached 10.2%, with the prevalence of 30.8% in children under the age of 5 (mawar et al., 2020). according to the 2021 indonesia nutritional status survey (ssgi), the national stunting rate declined by an average of 1.6% per year, decreasing from 27.7% in 2019 to 24.4% in 2021. during this period, the majority of the 34 provinces reported a reduction in stunting prevalence compared to 2019. despite this progress, the proportion of children under five classified as having severely stunted growth remained high, reaching 30.8% in 2021. in response, the government aimed to lower this rate to 28% as outlined in the 2019 national medium-term development plan (rpjmn) (sudarmi & rate, 2020). based on data released by the gorontalo district health office, the number of stunted children in 21 areas of gorontalo province showed 38,418 in 2019, 23,159 in 2020, and 25,867 in 2021. according to information obtained from the gorontalo provincial health office, as reported by the ssgi, the prevalence of stunting in manuscript received: 09 august, 2025. revision accepted: 10 december, 2025. published: 11 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1201-1206 1202 biology, medicine, & natural product chemistry 14 (2), 2025: 1201-1206 gorontalo province reached 29% in 2021. this shows an increase from 11.86% in 2020, following a higher rate of 34.89% in 2019. based on the weight-for-age (w/a) indicator, also known as bb/u indicator, gorontalo province ranks third highest in indonesia, with 28% of children under the age of 5 experiencing malnutrition and poor nutrition. the prevalence of very short and short children is 28.4%, and the prevalence of thin and very thin children is 14.3%. according to the latest data collected in august 2021, the incidence of stunting in gorontalo province has decreased to 11.1%. despite the decrease, this figure still has not reached the government's target of reducing stunting to ≤10%. subsequently, pohuwato regency had the highest stunting rate in gorontalo province in 2021, reaching 34.6%, followed by boalemo regency with 29.8%, north gorontalo regency with 29.5%, gorontalo regency with 28.3%, gorontalo city with 26.5%, and bonebolango regency with 25.1% (gorontalo, 2021). based on the results of preliminary data observations carried out in the working area of the central city health center in 2024, 1,940 children were recorded. among these, the nutritional status of stunting children between the ages of 12-24 months approximated 64 children, consisting of 6 villages, including wumialo village, 15 children, dulalowo village, 11 children, liluwo village, 14 children, pulubala village, 8 children, paguyaman village, 8 children, east dulalowo village, 8 children (central city health center data gorontalo city march, 2024). stunting remains a major public health concern, as it is influenced by multiple factors, including dietary intake patterns, specifically breastfeeding and complementary foods. exclusive breastfeeding for 6 months and introducing appropriate complementary foods are effective measures to reduce stunting and improve wellbeing. however, prolonged exclusive breastfeeding beyond the recommended period may delay the timely introduction of complementary foods, potentially resulting in insufficient nutrient intake necessary to support optimal growth and development in children (nurkomalaa, 2017). non-initiation of early breastfeeding (imd), failure to breastfeed exclusively, and early weaning can be risk factors for stunting. in the case of complementary foods (mp asi), it is important to pay attention to the amount, quality, and safety of the food provided (hasanah et al., 2021). methods an observational analytical study design with a crosssectional approach was applied to examine the relationship between independent variables (knowledge of exclusive breastfeeding and complementary feeding) and the dependent variable (stunting). this study was conducted in the working area of the kota tengah health center working area. results table 1. relationship between exclusive breastfeeding and the incidence of stunting in 12-24 month old children. exclusive breastfeeding incidence of stunting total p-value correlation value stunting normal n % n % n % simply 20 31,3 11 17,2 31 48,4 0,000 0,541 good 4 6,3 29 45,3 33 51,6 total 24 37,5 40 62,5 64 100 based on table 1 above, the relationship between exclusive breastfeeding and the incidence of stunting was obtained. out of the 31 children who received exclusive breastfeeding in the sufficient category, there were 20 children (31.3%) who were stunted and 11 (17.2%) who were not stunted or normal. meanwhile, out of the 33 children who received exclusive breastfeeding in the good category, there were 4 (6.3%) who were stunted and 29 (45.3%) who were not stunted or normal. from the table above, the spearman rho significance test value is 0.000. this shows that there is a significant relationship between exclusive breastfeeding and the incidence of stunting (p-value <0.05). the correlation value is 0.541, which shows that the relationship between the two variables is quite strong. table 2. relationship between breast milk complementary foods and the incidence of stunting in 12-24 months old children. breast milk and complementary foods incidence of stunting total p-value correlation value stunting normal n % n % n % simply 23 35,9 27 42,2 50 78,1 0,007 0,332 good 1 1,6 13 20,3 14 21,9 total 24 37,5 40 62,5 64 100 modjo et al. – exclusive breastfeeding and complementary feeding … 1203 based on table 2 above, the relationship between complementary feeding and the incidence of stunting was obtained. out of 31 children who received adequate complementary food, 23 (35.9%) were stunted, and 27 (42.2%) were not stunted or normal. meanwhile, out of 14 children who received complementary food in the good category, 1 child (1.6%) was stunted, and 13 children (20.3%) were not stunted or normal. from the table above, the spearman rho significance test value is 0.007. this shows that there is a significant relationship or influence between complementary feeding and the incidence of stunting (p-value <0.05). the correlation value is 0.332, which shows that the relationship between the two variables is fairly weak. discussion respondent characteristics gender based on the results of the study conducted at the central city health center, the distribution of respondent characteristics based on the sex of children in this study consists of men and women who participate in varying amounts. the sex of the respondent provides an overview of how gender differences can affect the variables measured. the number of respondents with male gender was 29 (45.3%), and respondents with female gender amounted to 35 (54.7%). according to almatsier (2009), gender differences affect the amount of nutritional needs in children due to differences in body composition between boys and girls. according to reports, girls have more fat tissue and less muscle tissue than boys. another study conducted by tsani et.al (2018) on the effect of gender and nutritional status on satiety on a high-fat diet stated that there were differences in satiety levels between boys and girls, where girls are full faster than boys. this affects children's nutritional intake, which can cause boys to be more at risk of obesity (overnutrition) compared to girls. therefore, boys and girls of the same height, weight, and age have different body compositions, and their energy and nutritional needs will also be different. although gender does not affect the incidence of stunting, the nutritional needs of boys and girls are relatively different. many other factors influence the incidence of stunting in children. age of children based on the data obtained from the study conducted in the kota tengah health center working area, the frequency distribution and percentage of respondents by age were analyzed across two distinct age groups, with a total of 64 respondents included in this study. the age group 0-12 months has a smaller frequency of 25 respondents (39.1%). meanwhile, the age group of 13-24 months has a greater frequency, namely 39 respondents (60.9%) of the total children. this is in line with wanimbo and wartiningsih's study, which states that children under the age of 5 who experience stunting are dominated by the age category of 13-24 months (46.7%). this is because children aged 13– 24 months exhibit a higher basal metabolic rate (bmr), which increases their risk of stunting. consequently, this age range requires greater attention to nutritional intake, encouraging mothers to be more proactive in ensuring adequate nutrition to prevent stunting (wanimbo & wartiningsih, 2020). mother's age based on the data obtained from the study conducted in the kota tengah health center working area, the frequency distribution and percentage of maternal age characteristics in the 3 different age groups, who participated in this study, were 64 respondents. the 46– 55-year age group has a smaller frequency of 2 respondents (3.1%) than the 36-45 year age group has a frequency of 16 respondents (25.0%). the age group 2635 years had a greater frequency of 46 respondents (71.9%) of the total respondents. according to candra (2011), the age of the mother is considered to play a role in the psychological aspect. mothers who are too young are considered not ready to maintain their pregnancy and take care of the children later, while mothers who are too old are considered to have decreased stamina. this psychological factor is very easily influenced by other factors. in the results of the kia book documentation study, 19 mothers gave birth to their first child who were stunted at the age of 20-35 years. this is probably because mothers who gave birth to their first child at a safe age of 20-35 years have several psychological problems that can hinder caring for their children. this study is in line with reky marlani's investigation, with the title overview of maternal characteristics that affect the incidence of stunting in children between the ages of 24-59 months at the talang banjar health center, jambi city. while a mother's age is not a direct determinant of stunting, it can influence her capacity to provide proper nutrition to her child. the more significant factor is the mother's level of knowledge regarding child nutrition. as mothers grow older, their experiences in child-rearing may increase, but maturity also brings greater exposure to information and learning opportunities from various sources, which can enhance their understanding and practices related to child nutrition (marlani et al., 2021). mother's education based on data obtained from the study conducted in the kota tengah health center working area, the frequency distribution and percentage of mothers' education characteristics are divided into 5 different educational categories, with a total number of 64 respondents. the no school category and the college level education category had a smaller frequency of 7 respondents each 1204 biology, medicine, & natural product chemistry 14 (2), 2025: 1201-1206 (10.9%). then the elementary school education category had the second lowest frequency at 9 respondents (14.1%), and the high school education category had the third lowest frequency at 13 respondents (20.3%). the high school education category had the highest frequency with a total of 28 respondents (43.8%). in accordance with the investigation carried out by erfiana et al. (2021), most mothers' education is high school (47.8%) (erfiana et al., 2021). the mother's education level can affect the behavior in preventing stunting. a person with a higher level of education is more receptive to information, including nutritional problems in children, in order to learn how to prevent nutritional problems. according to a study from medhin (2010), parental education affects the incidence of stunting because the level of education makes it easier for an individual to absorb information and carry out implementation in daily behavior and lifestyle (amelia & fahlevi, 2022). based on the results of a study conducted in the kota tengah health center working area, the frequency distribution and percentage of mothers' employment characteristics are divided into 3 different job categories, with a total number of 64 respondents. in the work category, traders have a smaller frequency of 6 respondents (9.4%), and the other work category has a second smaller frequency of 9 respondents (14.1%). the irt job category has the largest frequency of 49 respondents (76.6%). this study is in line with the results of an investigation conducted by riza savita, with the title relationship between maternal employment, gender, and exclusive breastfeeding to the incidence of stunting in children 6-59 months in bangka belitung. in these results, work-related factors influence knowledge acquisition; individuals who are employed tend to have broader knowledge than those who are not, as they are more frequently exposed to new information and experiences (savita & amelia, 2020). mother's characteristics also play an important role in addressing stunting, as it is a chronic condition that develops over time due to prolonged exposure to adverse factors. these may include poverty, inadequate parenting, often resulting from parents being preoccupied with work, limited nutritional knowledge due to low educational attainment, and frequent illness in children resulting from poor hygiene and inadequate sanitation. the results of this study prove that there is a relationship between a mother's work and the incidence of stunting (p = 0.000); children of non-working mothers are five times more likely to experience stunting compared to children of working mothers. (or = 5.390). this study is in line with an investigation conducted by yulia wulansari (2017) which stated that there is a significant relationship between mother's work and the risk of stunting in children p = 0.0001)7, in line with study conducted by novita siahaan, et al (2013) which stated that there is a significant relationship between mother's employment status and the incidence of stunting (p = 0.04) but contrary to study conducted by aisyah, et al (2018) which showed that there is no relationship between mother's employment status and the incidence of stunting in first grade children at sdi taqwiyatul wathon coastal area of semarang city (p = 0.154). the role of mothers who work outside the home to support themselves and their families differs significantly from that of non-working mothers. employment status can greatly influence a mother's behavior in meeting the nutritional needs of her children. working mothers often have limited time to spend with their children, which may lead to inadequate supervision of food intake and reduced attention to the child's growth and developmental needs. frequency distribution of respondents based on exclusive breastfeeding history based on the results obtained by researchers in the kota tengah health center working area of gorontalo city, the frequency distribution of exclusive breastfeeding respondents with the incidence of stunting in children from 64 respondents can be seen that in the largest case group there were 33 respondents in the good category with a percentage of 51.5%. breast milk has many benefits for babies, and ideal breastfeeding is an important activity in the maintenance of children and the preparation of future generations of quality. children's growth and development are influenced by the amount of nutrients consumed. most of these nutrients can be met by adequate breastfeeding. breast milk is not only the main source of energy but also the main source of protein, vitamins, and minerals for children. the occurrence of nutritional insecurity in children is due to insufficient food and the replacement of breast milk with bottle milk in a way and amount that does not meet the needs. many studies have proven that breast milk is important for the ideal growth and development of infants. a study by sofyana stated that the average change in neonate body length for 1 month (28 days) in neonates who were exclusively breastfed was 1.078 cm, while neonates who were given nonexclusively were 1.008 cm (marwah et al., 2020). the results of this study are in line with mahendra's investigation (2021) which examined the relationship between exclusive breastfeeding and the incidence of stunting in children between the age of 2 to 5 years in the barombong health center working area, there is a relationship, with a p value = 0.009 (0.009>0.005). therefore, it can be concluded that there is a relationship between exclusive breastfeeding and the incidence of stunting in children aged 6-12 months. frequency distribution based on history of complementary feeding of breast milk data from the kota tengah health center working area of gorontalo city show that among 64 respondents, the modjo et al. – exclusive breastfeeding and complementary feeding … 1205 majority, 50 children (78.1%), received complementary feeding categorized as sufficient, making it the most dominant category. no children were classified under the 'insufficient' category. complementary foods for breast milk (mp-asi) are foods or drinks that contain nutrients given to children between the ages of 6-24 months to meet nutritional needs other than breast milk. complementary feeding is a transition from breast milk to family meals. the introduction and provision of complementary foods should be carried out gradually in both form and amount, according to the baby's ability. the provision of adequate quality and quantity of complementary foods is important for the rapid physical growth and development of children's intelligence during this period, but hygienic feeding of complementary foods is essential (widhiyanto, 2023). mothers should be knowledgeable about appropriate complementary feeding practices, particularly when a baby reaches six months of age. complementary foods must meet several important criteria, they should be introduced on time (at six months), be nutritionally adequate in terms of quantity, frequency, consistency, and variety, and be safe, which includes maintaining proper hygiene such as washing hands before preparing and feeding food. additionally, complementary feeding should be administered properly and offered regularly in the morning, afternoon, evening, or night, with each feeding session not exceeding 30 minutes (buku kesehatan ibu dan anak, 2023). relationship between exclusive breastfeeding and the incidence of stunting based on the results, the value of the relationship between exclusive breastfeeding and the incidence of stunting was obtained. subsequently, 31 children received exclusive breastfeeding in the sufficient category, and there are 20 children (31.3%) who are stunted, and 11 children (17.2%) who are not stunted or normal. this shows that children who are exclusively breastfed in the sufficient category are a significant number of children who are stunted. meanwhile, out of the 33 children who received exclusive breastfeeding in the good category, 4 (6.3%) were stunted, and 29 (45.3%) were not stunted or normal. in the good category, children who get exclusive breastfeeding are not stunted. based on the statistical test results, the significance value of spearman rho was 0.000 (p <0.05). these results show that there is a significant relationship or influence between exclusive breastfeeding and the incidence of stunting. the correlation value of 0.541 shows that the relationship between the two variables is quite strong. based on the results of an investigation by deni yatno et al. (2021), there is a relationship between exclusive breastfeeding and the incidence of stunting. the results of this study show that exclusive breastfeeding falls into the category of 'not provided,' as mothers of children lacked consistency in exclusively breastfeeding. mothers were generally more inclined to give formula milk to their children up to six months of age rather than relying solely on exclusive breastfeeding. according to this study, exclusive breastfeeding is one of the factors associated with stunting. children under the age of 5 who do not get exclusive breastfeeding have a 3 times higher tendency to experience stunting compared to children who get exclusive breastfeeding. this is due to the lack of information on the importance of exclusive breastfeeding. it is expected that mothers can provide exclusive breastfeeding to children in order to avoid the incidence of stunting. relationship between complementary feeding and the incidence of stunting based on the results obtained, the value of the relationship between complementary feeding and the incidence of stunting was obtained. out of the 31 children who received adequate complementary food, 23 (35.9%) were stunted, and 27 (42.2%) were not stunted or normal. 23 children who were given adequate complementary food were stunted. meanwhile, out of the 14 children who received complementary food in the good category, 1 child (1.6%) was stunted, and 13 children (20.3%) were not stunted or normal. children who are given complementary food in the good category, only a few who are stunted, namely 13. based on the test results, the spearman rho significance test value was 0.007. this shows that there is a significant relationship or influence between complementary feeding and the incidence of stunting (pvalue <0.05). meanwhile, the correlation value between the independent variable and the dependent variable is 0.332, which shows that the relationship between the two variables is weak. this study is in line with pradhiba (2021) that there is a relationship between the frequency of complementary feeding and children's nutritional status, with a value of p = 0.021. similarly, hasanah et al. (2019) reported that there is a significant relationship with a p-value of 0.011. rosita (2021) showed that the age of children when they first received complementary foods had a significant relationship with stunting status, with a correlation strength equivalent to -0.182. this means that the more appropriate the age of giving complementary foods to children, the lower the risk of stunting. early complementary feeding, specifically before 4 months of age, is associated with an increased risk of gastrointestinal disease, which can lead to impaired growth, micronutrient deficiencies, and susceptibility to various infectious diseases in the first two years of life (rosita, 2021). contribution to global nursing practice based on this study's assumption, the provision of complementary foods in children between the ages of 6 months and over is very important to help the child's 1206 biology, medicine, & natural product chemistry 14 (2), 2025: 1201-1206 nutritional intake and growth, as well as development. in line with these results, complementary foods provided by mothers should be appropriate for the child's age. mothers need to gain knowledge about the wide variety of complementary foods available and the various methods of preparation. conclusion this study identified key characteristics and risk factors associated with stunting among children aged 12–24 months in the kota tengah health center working area, gorontalo city, in 2024. the majority of the child respondents were female (54.7%) and aged 18–24 months (59.4%), while most mothers were aged 26–35 years (71.9%), had a high school education (43.8%), and were primarily housewives (76.6%). findings revealed that 62.5% of the children had normal growth, whereas 37.5% experienced stunting. the study confirmed a significant relationship between maternal knowledge of exclusive breastfeeding and the incidence of stunting. likewise, a significant relationship was found between maternal knowledge of complementary feeding history and stunting. the absence of exclusive breastfeeding and the provision of inappropriate complementary foods were identified as prominent risk factors contributing to stunting. these results highlight the critical role of maternal knowledge in preventing stunting. strengthening educational interventions and health promotion regarding optimal infant and young child feeding practices is essential to reduce the prevalence of stunting in the study area. author contribution: all authors have accepted responsibility for the entire content of this manuscript and approved its submission. conflict of interest: authors state no conflict of interest. references amelia, f., & fahlevi, m. i. (2022). hubungan perilaku ibu dengan kejadian stunting pada balita di desa babul makmur kecamatan simeulue barat kabupaten simeulue. buku kesehatan ibu dan anak. (2023). buku kia kesehatan ibu dan anak. https://id.scribd.com/document/628435314/bukukesehatan-ibu-dan-anak-kia-2023-feb23 erfiana, sri intan rahayuningsih, & fajri, n. (2021). hubungan pengetahuan ibu dengan perilaku pencegahan stunting pada balita. gorontalo. (2021). percepat penurunan stunting di provinsi gorontalo, penjagub berharap perkuat kemitraan. hasanah, s., masmuri, & purnomo, a. (2021). hubungan pemberian asi dan mp asi dengan kejadian stunting pada baduta (balita bawah 2 tahun. diwilayah kerja puskesmas kampung dalam. jafriati. (2022). analisis faktor risiko kejadian stunting pada anak baduta (6-24 bulan. di wilayah kerja puskesmas mowila. marlani, r., neherta, m., & deswita. (2021). gambaran karakteristik ibu yang mempengaruhi kejadian stunting balita usia 24-59 bulan di puskesmas. marwah, a. s., hadju, v., & ad, s. a. (2020). pola pemberian asi dan pemberian mpasi dengan status gizi anak usia 12-23 bulan. mawar, s., purbaningrum, d. g., & divania. (2020). edukasi gizi sebagai upaya pencegahan stunting di desa mekar jaya rumpin kabupaten bogor. muhamad, z., mahmudiono, t., abihail, c. t., sahila, n., wangi, m. p., suyanto, b., & abdullah, n. a. b. (2023). preliminary study: the effectiveness of nutrition education intervention targeting short-statured pregnant women to prevent gestational stunting. nutrients, 15(19), 4305. https://doi.org/10.3390/nu15194305 panigoro, m. i., sudirman, a. a., & modjo, d. (2023). upaya pencegahan dan penanggulangan stunting pada balita di wilayah kerja puskesmas tilongkabila. rosita, a. d. (2021). hubungan pemberian mp-asi dan tingkat pendidikan terhadap kejadian stunting pada balita. literature review. savita, r., & amelia, f. (2020). hubungan pekerjaan ibu, jenis kelamin, dan pemberian asi eklusif terhadap kejadian stunting pada balita 6-59 bulan di bangka selatan. sudarmi, m., & rate, s. (2020). faktor yang berhubungan dengan kejadian stunting diwilayah kerja puskesmas gaya baru. wanimbo, e., & wartiningsih, m. (2020). hubungan karakteristik ibu dengan kejadian stunting baduta (7-24 bulan) relationship between maternal characteristics with children (7-24 months. stunting incident. widhiyanto, a. (2023). hubungan frekuensi dan jenis pemberian makanan pendamping asi (mp-asi) dengan kenaikan berat badan bayi usia 6-24 bulan. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 227-233 | doi: 10.14421/biomedich.2024.131.227-233 issn 2540-9328 (online) the protective effect of kentut leaf extract (paederia foetida l.) on gastric histopathology in escherichia coli-infected sepsis mice model lisa savitri*, elfred rinaldo kasimo department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id abstract sepsis, a severe medical condition, signifies the systemic immune response to infection, often leading to organ dysfunction and mortality. escherichia coli is a significant contributor to sepsis cases, particularly in gastrointestinal disorders. this study aimed to investigate the histopathological changes in the gastric tissue of mice induced by escherichia coli infection and evaluate the protective effects of kentut leaf extract (paederia foetida l.). histopathological analysis revealed distinct alterations in the gastric tissue among different treatment groups. while normal mouse treatment showed no significant changes, negative control (k-) and positive control (k+) groups exhibited inflammation and hyperemia of the gastric mucosa, characterized by necrosis, degeneration, and inflammatory cell infiltration. treatment with kentut leaf extract (p1, p2, and p3) resulted in milder inflammation compared to controls, indicating a protective effect against gastric mucosal damage induced by escherichia coli infection. this protective mechanism is attributed to the anti-inflammatory properties of saponins, flavonoids, and essential oils present in kentut leaf extract. these findings underscore the potential therapeutic benefits of kentut leaf extract in mitigating gastric mucosal injury associated with bacterial infection. keywords: protective effect; kentut leaf extract; gastric histopathology; escherichia coli; sepsis. abbreviations: basic sodium hydrogen phosphate (na2hpo4), mediated through mitogen-activated protein kinase (mapk), microphthalmia-associated transcription factor (mitf), nitric oxide (no), physiologic zur (pz), tumor necrosis factor (tnf). introduction sepsis is a medical emergency condition that depicts the systemic immune response of the body to an infection process, which can lead to end-stage organ dysfunction and death (gyawali et al., 2019). in 2017, the largest contributors to sepsis cases and sepsis-related deaths across all ages were diarrheal diseases (9.2 to 15 million cases per year) and lower respiratory tract infections (1.8-2.8 million per year). one of the serious threats causing sepsis is escherichia coli bacteria (lawn et al., 2017; stoll et al, 2011). bacterial-induced sepsis is a lifethreatening condition that arises when the body's response to infection injures its tissues and organs (singer et al., 2016). the prevalence of e. coli bacterial infection causing gastrointestinal disorders in humans has increased (silaban, 2021). e. coli is one of the most frequently isolated bacteria in the bloodstream and is the most commonly isolated gram-negative bacterial infection in adult patients with bacteremia (mora-rillo, et al., 2015). the primary infection sites associated with sepsis are the respiratory/lung system (43%), urinary system (16%), abdomen (14%), head, which is associated with fever of unknown origin (14%), and other locations/causes (13%) (vakkalanka et al., 2018; angus et al., 2013). from a pathogenetic standpoint, sepsis is currently considered the result of several mechanisms simultaneously involving various pro-inflammatory and anti-inflammatory mediators (piechota et al., 2007). additionally, recent cellular modifications associated with sepsis have been defined, and the importance of microcirculation has been emphasized in the development of sepsis into septic shock (ince, 2005). in this context, the endothelium has been identified as the fundamental functional unit in the pathophysiology of sepsis due to its role in regulating microcirculation and modulating coagulation mechanisms as well as inflammatory and anti-inflammatory signaling processes (wolinsky, 1980; belousoviene et al., 2021). the glycocalyx is a component of the endothelial membrane consisting of proteoglycans and glycoproteins. it mediates various functions, such as forming a mechanical barrier regulating vascular permeability, leukocyte activation and platelet adhesion, as well as modulating inflammatory/anti-inflammatory responses. manuscript received: 02 april, 2024. revision accepted: 20 may, 2024. published: 26 june, 2024. https://doi.org/10.14421/biomedich.2024.131.227-233 228 biology, medicine, & natural product chemistry 13 (1), 2024: 227-233 damage to the morphofunctional integrity of the glycocalyx (known as "glycocalyx shedding") can occur due to oxidative agents, cytokines, bacterial exotoxins, and endotoxins. this event leads to leukocyte diapedesis and increased vascular permeability with edema production, which increases interstitial pressure and worsens tissue perfusion (kaukonen et al., 2015). several organs that are likely to experience dysfunction are the gastric. in sepsis, the inflammatory response to attacking pathogens involves inflammatory and anti-inflammatory processes, humoral and cellular reactions, and circulatory abnormalities (bhattacharjee et al., 2017). in the early phase after injury, the inflammatory response is initiated by acute-phase reactants or proinflammatory factors (le et al., 1987). the occurrence of increased reactive oxygen and accumulation of oxidative stress leads to a decrease in nitric oxide (no), reducing no bioavailability in coronary and peripheral circulation, thus causing vascular dysfunction correlated with increased risk of organ dysfunction leading to death (kaukonen et al., 2015). as important inflammatory factors, tumor necrosis factor (tnf) causes fever, hemodynamic abnormalities, anorexia, joint pain, and neutrophil aggregation, which are considered typical symptoms associated with sepsis (kumari et al., 2017). therefore, it is crucial to explore the potential role of inflammatory factors in the pathophysiology of sepsis. moreover, eliminating the concept of sirs from sepsis will certainly lead to continuous issues regarding basic research and clinical management of sepsis. despite all experimental and clinical research efforts over the last three decades, the ability to influence the course of bacteria remains limited. immunotherapybased therapies have mostly proven ineffective so far (saifudin, 2014). hence, actions more geared towards preventive measures are needed, particularly using herbal materials, as they are believed to have low side effects. one traditional plant believed by ancient communities to treat digestive disorders such as diarrhea is the kentut leaf (paederia foetida l.), known in east java as 'daun sembukan'. kentut leaves contain secondary metabolites such as alkaloids, saponins, tannins, and flavonoids that pharmacologically have benefits such as antioxidants, antibiotics, anticancer agents, insect repellents, antitumor agents, and immunomodulatory agents (abriyanto et al., 2012; chanda et al., 2015; ramadhan et al., 2021; savitri, et al., 2023). many of the compounds isolated from kentut leaves are bioactive and have been shown to have several bioactivities, such as anthraquinones with anthelmintic, antidiarrheal, anti-hyperglycemic, anti-hyperlipidemic, anti-inflammatory, antioxidant, antimicrobial, and antitussive properties (savitri et al., 2023; savitri and kasimo, 2022; wang et al., 2014; soni et al., 2013). although the strong unpleasant odor is mainly caused by the presence of several simple sulfur compounds (the main one being dimethyl sulfide), sulfur-containing glycosides, such as paederoside, are also involved in its foul smell. in recent years, the focus has shifted to more mechanistic studies, attempting to understand how extracts and pure compounds from kentut leaves lead to their pharmacological actions. previous research has studied the antimelanogenic properties of this plant extract on murine melanoma cells and has shown that this effect is mediated through mitogen-activated protein kinase (mapk) signaling via microphthalmia-associated transcription factor (mitf) downregulation (trung et al., 2021). similarly, other research has shown that this plant can induce anticancer activity by interfering with chromatin modification enzymes and altering the expression of proinflammatory cytokine genes in prostate cancer cells (chung, 2021). pharmacological studies conducted on kentut leaves to date have provided some scientific evidence supporting their use in traditional medicine for the treatment of certain human diseases, and several possible mechanisms of action have also been demonstrated. however, there is little or no information available on well-designed, randomized, double-blind clinical studies with this plant. the need for extensive clinical studies to promote this plant or its components as modern drug formulations cannot be overlooked. materials and methods population and sample the population used in this study consisted of mice (mus musculus). the sample used in this study comprised 30 male balb/c mice aged 4-8 weeks with a weight of 20-30 grams obtained from the veterinary center farma (pusvetma), surabaya, east java. tools and materials the tools used in this study included: disposable syringe 1 ml, disposable syringe 3 ml, disposable syringe 5 ml, feeding tube size 35 cm, urine container 60 ml, vaculab edta k3 3 ml, eppendorf 2 ml, microcentrifuge, micropipette, freezer, rotary microtome or sliding microtome, brush, waterbath, object glass, eppendorf 1500 µl, blue tips, yellow tips, white tips 10 µl, tube 15 ml, tube 50 ml, surgical board, surgical instruments set (including scissors, forceps, and needle), gloves, tissue, mask, binocular light microscope, and microscope camera. the materials used in this study included: male balb/c mice aged 4-8 weeks with a weight of 20-30 grams, extract of kentut leaves (paederia foetida l.), ciprofloxacin, clinical isolate (wild type) e. coli with a dose of 1x105 cfu/ml, physiologic zur (pz) (aqua pro injection free from pyrogen), mouse feed, wood powder (kawol), methanol, giemsa, distilled water, h2co savitri & kasimo – the protective effect of kentut leaf extract 229 (formaldehyde) 37% solution, basic sodium hydrogen phosphate (na2hpo4) 6.5 grams, distilled water 900 ml, formaldehyde 37-40%), ethanol 80%, ethanol 95%, absolute ethanol, xylene, clearing solution, paraffin, sahrp, bsa, hcl. research procedure acclimatization of mice male mice were weighed and placed in standard polypropylene cages with bedding using wood powder for acclimatization for two weeks. cage bedding was changed every three days. feed provided was softened with water first, then shaped into an elongated form, weighing approximately 5 grams. water was provided ad libitum. feed and water were replaced daily. mice were divided into six treatment groups after acclimatization for two weeks. treatment on experimental animals acclimatized mice were treated for 14 days with the following variations: 1) group 1 as normal control (n), consisting of mice not given gastric tube feeding, 2) group 2 as negative control (k-), consisting of mice given distilled water with a volume of 0.5 ml, 3) group 3 as positive control (k+), consisting of mice given ciprofloxacin at a dose of 500 mg/kg bw with a volume of 0.26 ml, 4) group 4 as treatment 1 (p1), consisting of mice given kentut leaf extract at a dose of 100 mg/kg bw with a volume of 0.5 ml, 5) group 5 as treatment 2 (p2), consisting of mice given kentut leaf extract at a dose of 300 mg/kg bw with a volume of 0.5 ml, 6) group 6 as treatment 3 (p3), consisting of mice given kentut leaf extract at a dose of 500 mg/kg bw with a volume of 0.5 ml. sepsis model treatment in mice mice that had been given treatment were injected intraperitoneally with e. coli at a dose of 1x105 cfu/ml. mice 24 hours post-exposure to polymicrobial sepsis will show sepsis events, so mice can be euthanized after 24 hours. if mice die before 24 hours, immediate surgery should be performed to remove the stomach and intestines to prevent autolysis. organ slices taken are in the middle, left, and right edges. tissue processing gastric tissue were fixed in formalin buffer to maintain cell morphology as original to prevent autolysis and bacterial or fungal growth. the next step is to make paraffin blocks. after making the paraffin blocks, tissue sectioning was performed using a rotary microtome or sliding microtome with a thickness of 4-6 microns. the obtained sections were then taken with a brush moistened with water and placed on the surface of a waterbath. the expanded tissue was then taken with object glass coated with tissue adhesive, then dried at room temperature and placed in an oven overnight. histopathological observation of the gastric histopathological observation of the mouse stomach and intestines involved preparing histological specimens by staining them with hematoxylin and eosin. the histopathological observation included checking for abnormal cells (cells undergoing differentiation, necrosis, and apoptosis). the calculation of abnormal cells was done by dividing the abnormal cells counted by all preserved cells, then multiplied by 100%, thus the data was expressed in percentage (%). results and discussion result table 1. histopathological characterization of mice gastric tissue in a sepsis model induced by escherichia coli infection. group cell degeneration necrosis infiltration of inflammatory cells average sd average sd average sd normal 2.21% 0.02 2.44% 0.02 1.62% 0.02 positive (+) 21.63% 0.02 17.20% 0.02 28.05% 0.02 negative (-) 23.41% 0.04 21.55% 0.03 48.63% 0.05 p1 (100mg/kgbw) 20.79% 0.03 22.62% 0.04 39.56% 0.03 p2 (300mg/kgbw) 9.08% 0.02 6.05% 0.02 18.45% 0.03 p3 (500mg/kgbw) 7.72% 0.04 5.18% 0.02 16.07% 0.03 230 biology, medicine, & natural product chemistry 13 (1), 2024: 227-233 figure 1. histopathological characterization of the mice gastric treatment normal magnification of 400x figure 2. histopathological characterization of the mice gastric treatment kmagnification of 400x, necrosis (green arrow), degeneration (yellow arrow), and infiltration of inflammatory cells (red arrow) figure 3. histopathological characterization of the mice gastric treatment k+ magnification of 400x, necrosis (green arrow), degeneration (yellow arrow), and infiltration of inflammatory cells (red arrow) figure 4. histopathological characterization of the mice gastric treatment p1 magnification of 400x, necrosis (green arrow), degeneration (yellow arrow), and infiltration of inflammatory cells (red arrow) figure 5. histopathological characterization of the mice gastric treatment p2 magnification of 400x, necrosis (green arrow), degeneration (yellow arrow), and infiltration of inflammatory cells (red arrow) figure 6. histopathological characterization of the mice gastric treatment p3 magnification of 400x, necrosis (green arrow), degeneration (yellow arrow), and infiltration of inflammatory cells (red arrow). discussion based on the results of this study, it is known that normal mouse treatment (n) does not cause changes in the histopathological appearance of the gastric (figure 1), whereas negative control treatment (k-), consisting of mice given distilled water with a volume of 0.5 ml, positive control treatment (k+), consisting of mice given ciprofloxacin at a dose of 500 mg/kg bw with a volume of 0.26 ml, treatment 1 (p1), consisting of mice given kentut leaf extract at a dose of 100 mg/kg bw with a volume of 0.5 ml, treatment 2 (p2), consisting of mice given kentut leaf extract at a dose of 300 mg/kg bw with a volume of 0.5 ml, treatment 3 (p3), consisting of mice given kentut leaf extract at a dose of 500 mg/kg bw with a volume of 0.5 ml resulted in inflammation and hyperemia of the gastric mucosa layer. the results of this study indicate changes in the histopathological appearance of the gastric tissue, where there are cells experiencing necrosis, degeneration, and infiltration of inflammatory cells as a result of escherichia coli infection. in the ktreatment (figure 2), there is evidence of epithelial cells composing the gastric tissue experiencing necrosis and degeneration, as well as evidence of inflammatory cell infiltration. in the gastrointestinal system, a large amount of e. coli infection can lead to savitri & kasimo – the protective effect of kentut leaf extract 231 peptic ulcers, where the observed irritation is the presence of epithelial cells composing the gastric tissue experiencing necrosis and degeneration, as well as evidence of inflammatory cell infiltration (santi, 2013). ulcers in the stomach are characterized by lesions on the gastric mucosa. ulcers occur due to the imbalance of gastric acid-pepsin secretion and mucus (a product of gastric glands that functions as a barrier to the gastric lining), thus injuring the gastric mucosa. to overcome this, inflammation occurs, marked by the appearance of inflammatory cells aimed at repairing the injured gastric mucosa (abdullah, 2008). in the k+ treatment (figure 3), there is evidence of epithelial cells composing the gastric tissue experiencing necrosis and degeneration, as well as evidence of inflammatory cell infiltration. ciprofloxacin can cause shedding of surface epithelial cells of the stomach and reduce mucus secretion, which serves as a protective barrier against acid (stickel, 1997). the potential occurrence of drug interactions is dominated by fluoroquinolone antibiotics and gastrointestinal drugs. the most common side effect of ciprofloxacin is gastrointestinal disturbance (2%), such as abdominal pain, nausea (4%-8%), vomiting, anorexia, and diarrhea (4%-5%). according to gosal et al., (2012), mucosal defense damage occurs due to the local effects of ciprofloxacin. when antibiotics are in the stomach, which is acidic (ph less than 3), they will form particles that are non-ionized. under such conditions, drug particles can easily diffuse through the lipid membrane into the gastric epithelial cells along with ions. additionally, uncoupling of mitochondrial oxidative phosphorylation leading to decreased atp production, increased amp, and increased adp can cause cell damage. these changes are followed by mitochondrial damage, increased formation of oxygen radicals, and changes in balance, thus reducing gastric mucosal resistance. this condition allows penetration of acid, pepsin, bile, and proteolytic enzymes from the gastric lumen into the mucosa, causing degeneration and even necrosis of cells. in treatments p1, p2, and p3 (figures 4, 5, and 6), there is evidence of epithelial cells composing the gastric tissue with mild inflammation compared to the kand k+ groups. this indicates that kentut leaf extract has a protective effect on the stomach. the mechanism of gastric protection in the mouse sepsis model is thought to be due to the presence of saponin, flavonoids, and essential oils found in kentut leaf extract. the most likely mechanism of gastric protection is believed to be that saponins can interact with many lipid membranes, such as phospholipids that are precursors to prostaglandins and other inflammatory mediators (savitri and ihsan, 2020). flavonoids also have anti-inflammatory mechanisms by inhibiting the activity of cox and/or lipoxygenase enzymes, as inhibition of the cox and lipoxygenase pathways can lead to inhibition of eicosanoid and leukotriene biosynthesis, which are end products of the cox and lipoxygenase pathways. (savitri, 2022). additionally, flavonoids can reduce the number of non-migrating white blood cells and reduce complement activation, thus reducing white blood cell adhesion to endothelial cells and causing a decrease in the body's inflammatory response. flavonoids also play a role in inhibiting histamine release. the antiinflammatory effect of flavonoids is supported by their action as antihistamines (savitri, et al., 2020). histamine is one of the inflammatory mediators whose release is continued by calcium entry into cells. flavonoids can inhibit camp phosphodiesterase enzymes, thus increasing camp levels in mast cells, thereby preventing calcium entry into cells, which also inhibits histamine release. flavonoids can also stabilize reactive oxygen species (ros) by reacting with reactive radical compounds, making the radicals inactive (savitri, et al., 2019). this study is also supported by previous research conducted by savitri and kasimo (2022) on preventive tests for il-6 levels in a mouse sepsis model induced by e. coli, which found that kentut leaf extract at a dose of 500 mg/kg bw (p3) was most effective in reducing il-6 levels. the anti-inflammatory mechanism in sepsis is thought to be caused by the presence of saponins, flavonoids, and essential oils found in kentut leaf extract. the most likely anti-inflammatory mechanism is thought to be caused by saponins that can interact with many lipid membranes, such as phospholipids that are precursors to prostaglandins and other inflammatory mediators. conclusions the study reveals distinct histopathological changes in the gastric tissue of mice subjected to different treatments. normal mouse treatment (n) did not induce any significant alterations in gastric histopathology. however, negative control treatment (k-), involving distilled water administration, and positive control treatment (k+), involving ciprofloxacin administration, both led to inflammation and hyperemia of the gastric mucosa, characterized by necrosis, degeneration, and inflammatory cell infiltration. the observed effects are consistent with the known gastrointestinal disturbances associated with ciprofloxacin administration. additionally, the study demonstrated that treatment with kentut leaf extract (p1, p2, and p3) resulted in milder inflammation compared to the negative and positive control groups. this suggests a protective effect of kentut leaf extract against gastric mucosal damage induced by escherichia coli infection. the protective mechanism is attributed to the presence of saponins, flavonoids, and essential oils in the kentut leaf extract, which exhibit anti-inflammatory properties by inhibiting inflammatory enzyme activity, reducing white blood cell adhesion, and stabilizing reactive oxygen species. overall, these findings highlight the potential therapeutic benefits of 232 biology, medicine, & natural product chemistry 13 (1), 2024: 227-233 kentut leaf extract in mitigating gastric mucosal injury associated with bacterial infection. acknowledgements: the author would like to thank the institute for research, development, and community service (lp3m) of kadiri university and ministry of education, culture, research, and technology directorate general of vocational education, indonesia which has always provided support for writing and publications. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. elfred rinaldo kasimo wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research was funded by the ministry of education, culture, research, and technology directorate general of vocational education, indonesia under the main contract number 104/spk/d.d4/ppk.01.aptv/iii/2024 and derivative contract numbers 004/sp2h/ppkm-ptv/ll7/2024, 05/p.1/lp3m/i/2024. references abdullah. 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(2014). a phytochemical, pharmacological and clinical profile of paederia foetida and p. scandens. nat. prod. commun. 9, 879886. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 1, 2017 | pages: 13-17 | doi: 10.14421/biomedich.2017.61.13-17 issn 2540-9328 (online) anatomical study of male reproductive organs of the indonesian short-nosed fruit bat (cynopterus titthaecheilus temminck, 1825) anisatuzzahro1, muhammad jafar luthfi2 1sdi ma’arif tawangsari garum, kompleks perguruan ma'arif garum, tawangsari, garum, tawangsari, garum, blitar, east java 66182, indonesia 2biological education department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto 55281 yogyakarta, indonesia author correspondency: anisatuzz@gmail.com1 abstract bats are one of the mammals of chiroptera order. chiroptera order has two sub-orders, megachiroptera and microchiroptera. one of the species of megachiroptera is cynopterus titthaecheilus (c. titthaecheilus). local name for c. titthaecheilus is the indonesian short-nosed fruit bat. characteristics of c. titthaecheilus are dark brown body hair and no tragus in the ear. population of c. titthaecheilus in indonesia are relatively abundant, but research about anatomy of male reproduction organs of this species is still rare. the purpose of this study was to determine the anatomical features of male c. tittahecheilus reproductive organs. observations of macroscopic anatomy include observation of morphology, size, weight and volume of penis, testiscle, epididymis, vas defferens, prostate and seminal vesicles. observation on microscopical anatomy of male reproduction organ is using histological slide preparations stained by hematoxylin-eosin. keywords: anatomy; histology; cynopterus titthaecheilus; male reproductive organ. introduction the indonesian short-nosed fruit bat is the local name of the fruit-eating bat of cynopterus titthaecheilus species. bat is living in many areas or islands in sumatra, java, and nusa tenggara. the cynopterus titthaecheilus species are belonging to sub-order megachiroptera of chiroptera order (maharadatunkamsi, 2011). suyanto (2003) stated that bats consist of several species and occupy a second position in number of species related to the population of the mammalian class after rodentia. the majority of these bats live in tropical and semitropical areas. there are 4000 species of mammals, 963 of them are bat species. this species has an important role in the ecosystem as controlling insect pest agent, pollinators of flowers and grain spreaders and guano producers that can be used as fertilizer. its ability to fly and cruising are as far as about 20 km. bats that live in asia and africa have small body size (anonim, 1997). bats live and actively searching for food only at night because bats are very sensitive to dehydration. in the daylight, bat hanging upside down. these animals are nocturnal and therefore require a roosting area during the day. there were also crepuscular bats, they go out from cave during afternoon till dark (prasetyo et al., 2011). there are two factors that can affect the survival of bats, namely external and internal factors. outside factors consist of environment, food and photoperiodicity, whereas inner factor consists of nerves and hormones. photoperiodicity has a serious impact on the size of reproductive organs, the more exposed to the sun exposure the better effect to reproductive organs (isnaeni, 2006). morphology of male reproduction organs of bats differs during hibernation and active period. the testis volume is smaller when it comes to hibernation. at the time of hibernation no spermatogenesis occurs because the body temperature and environment are unstable and incompatible (entwistle et al., 1998). materials and methods this research was carry on for 3 months at zoological laboratory in integrated laboratory of state islamic university sunan kalijaga yogyakarta, and pathology laboratory of veterinary center, wates, yogyakarta. the equipments used in this study were a set of surgical instruments (consisting surgical scissors, tweezers, razors and scalpels), flacon bottles, surgical or paraffin box, needles, digital cameras, ruler, embedding proccessor, tissue proccessor, embedding cassette, base mold, staining jar, incubator, microtome, slide glass, cover glass, toothpick, mask, gloves and optilab® microscope. materials used in this study were hvs paper, ether or chloroform solution, 0.9% nacl physiological solution, fixative bouin, alcohol, hematoxcylin-eosin http://dx.doi.org/10.14421/biomedich.2017.61.13-17 14 biology, medicine, & natural product chemistry 6 (1), 2017: 13-17 dye, albumin, xylol, distilled water, canada balsam, paraffin. in this study, the test animals were ten fruit-eating bat (cynopterus titthaecheilus) of with a weight size ranging from 78.88 ± 3.88 grams. objects observed include testes, epididymis, penis, prostate, vas deferens, seminal vesicles and bulbourethral gland. reproductive organs of test animals were dissect and then put in flacon bottles containing bouin for overnight. the next step is dehydration using stratified alcohol, clearing using xylol and infiltration using paraffin. the next step were embedding and cutting block of paraffined organs. observations were done using optilab® microscope with magnification of 40, 100 and 400 times, and taking picture for each sample. results and discussion description of macroscopic anatomy of male c. titthaecheilus reproductive organs a common characteristic of c. titthaecheilus is dark brown hair color on its entire body and light brown in the neck area. determination of adolescent and adult bats is by looking at the color of the hairs that found on the neck. the darker the brown color of hairs, the more mature the bat. bat tail is not long. the ears are small and slightly elongated. the shape of the face is rather tapered forward (figure 1). figure 1. morphology of bat c. titthaecheilus. (a). ears are slightly tapered; (b). hair on the neck; (c). modification of thumb; (d). clawed toes; (e) short tail. testes of c. tithaecheilus species have a yellowish white color and there are irregular streaks of blood vessels (figure 2). the position of testes of c. titthaecheilus species remains in the abdomen. this is similar to that of the rhinipoma kinneari wroughton species (singwi & lall, 1983). in other mammals such as mice and squirrels, the testes are located in the outer body in scrotum. visible ducts are attached to the testes (figure 3). this duct is called epididymal duct. figure 2. reproductive organs seen from the outside after the integument is removed. (a). testes; (b). epididymis; (c). penis. figure 3. accessory glands of c. titthaecheilus. (a). testis; (b). epididymis; (c). vas deferent vas; (d). seminal vesicles; (e). prostate gland. penis is located in the outside of body cavity, like mammals in general. penis is an external reproduction organ because its location is outside the body. description of microscopic anatomy of male c. titthaecheilus reproductive organs histological or microscopic anatomical characters of c. titthaecheilus reproductive organs are not much different from other bats. in general, testes have functions as a reproductive organ and regulator of the hormonal anisatuzzahro & luthfi – anatomical study of male reproductive organs of the indonesian short-nosed … 15 system. testes of c. titthaecheilus are protected by 2 layers, including tunica vaginalis which is the outermost layer and tunica albuginea which lies beneath tunica vaginalis (figure 4). the tunica albuginea is a layer formed from connective tissue. in tunica albuginea there are septa that form some lobules. the lobule is called the testicular lobule, where in the lobule there is a long that forms the sperm called the seminiferous tubule (figure 5). figure 4. cross-section of testes c. titthaecheilus with he. 100x magnification. (ts). seminiferous tubule; (l). leydig cells; (ta). tunica albuginea; (tv) tunica vaginalis. figure 5. cross-sectional of seminiferous tubule of c. titthaecheilus with he. 400x magnification. (a). sperm; (b). secondary spermatocytes; (c). primary spermatocytes; (d). spermatogonia; (e). thin myoid cells; (f). leydig cells; (g). connective tissue; (h). fibroblast layer; (i). sertoli cells; (j). spermatids; (k). lumen. seminferuos tubule is a canal that has an arch-shaped and forming a coil in a lobule. intersitial space between the seminiferous tubule fills with connective tissue and leydig cells that play a role in producing testosterone hormone. in seminiferous tubule occurs a process of sperm production called spermatogenesis. spermatogenesis involves a large number of epithelial germ cells or epithelial cells of the germ. germinal epithelial cells consist of spermatogonia, spematocytes and spermatids. the position of the spermatogonia are in the basal layer of the seminiferous tubule (figure 5). sometimes these secondary spermatocyte cells are rarely found because the transition of development from primary spermatocytes to secondary spermatocytes is so rapid that it is rather difficult to observe (dreef et al., 2007). process of sperm maturation will occur in the duct of epididymis. epididymis is a duct that delivers spermatozoa to the next duct called vas defferens or commonly called vas deferens. epididymal duct consists of several layers. the outermost layer is composed of smooth, circular muscles, beneath it there is a network of semi-shaped columnar epithelium with stereocillia. the tissue of tunica vaginalis (figure 6) acts as a protective layer. figure 6. cross sectional of epididymis of c. titthaecheilus with he. 100x magnification. (ed). epididymal duct; (e). semi-shaped columnar epithelium with long stereosilia; (tv). tunica vaginalis. figure 7. cross-sectional of vas deferens of c. titthaecheilus stain with he. 100x magnification. (c-sm). circular muscle; (e). epithelial layer; (l-sm). longitudinal smooth muscle; (s). lumen containing sperm. 16 biology, medicine, & natural product chemistry 6 (1), 2017: 13-17 sperm movement is assist by stereocillia that move unidirectional. sperm will move through the epididymis to the vas deferens by peristaltic contraction of smooth muscle tissue connective wall (sinaga, 2011). vas deferens is also known as ductus deferens. vas deferens is a duct that bring sperm to the urethra. this duct is surrounded by a thick layer of muscle. there is a longitudinal smooth muscle and smooth circular muscle (figure 7). there is a layer of epithelium that surrounds the lumen containing sperm. lumen in the deferens duct is narrower than lumen in the duct of the epididymis. there are accessory glands in this species such as seminal vesicles, prostate, and bulbouretralis. this seminal vesicle is one of the accessory glands of the male mammal. between species threre may have different morphological or anatomical forms of this seminal vesicle gland (hafez, 2000). the seminal vesicle gland consists of a mucous that characterized the duct. the mucous occupies almost all of the lumen. this gland is surrounded by a layer of smooth muscle in its outermost. the lumen is in the central part surrounded by the ducts. figure 8. cross-sectional of seminal vesicle gland of c. titthaecheilus stained with he. 400x magnification. (a). lumen; (b). mucous; (c). smooth muscle. figure 9. cross-sectional of prostate gland of c. titthaecheilus stained with he. 100x magnification (a) and 400 times magnification (b). (a). plain muscle; (b). columnar epithelium; (c). lamina propia; (g). tubuloalveolar gland; (s). solid fibromuscular stroma. in addition to seminal vesicle glands, the second gland is prostate gland. in general, the results of previous studies in the prostate gland have solid fibromuscular stroma. most of the prostate gland is occupied by the tubulo-alveolar glands. tubulo-alveolar gland is a form of secretion that is a combination of tubular forms and alveolar (like grape). in this tubuloalveolar gland there is a stratified columnar epithelial cell. in addition there is also a lamina propia that surrounds the epithelial cells. as well as the smooth muscle tissue surrounding the lamina propia. the third accessory gland after seminal vesicle and prostate gland is the bulbourethral gland or commonly known as cowper’s gland. in general, this gland has a function as a secrete producer therefore called exocrine gland (wahyuni et al., 2013). observation of microscopic anatomy from the bulbourethral gland of c. titthaecheilus shows that in this gland there were tubuloalveolar glands that occupy most of the bulbourethral gland space. in tubulo-alveolar gland, a columnar epithelial cell lies at the edge of the tubulo-alveolar lobe (figure 10). the smooth muscle tissue seen around the tubulo-alveolar gland and the secretions inside the tubulo-alveolar lumen appear on the bulbourethral gland preparations. figure 10. cross-sectional of bulbourethral gland of c. titthaecheilus stained with he. 100x magnification. (a). tubulo-alveolar glands; (b). columnar cells of the lining; (c). smooth muscle; (d). secretions in the lumen of the gland. penis is a copulation organ for mammals that has the function of transferring sperm from the male reproductive tract to the female reproductive tract. in this microscopic anatomy there is a major component found in the penis namely two corpus cavernosun and one corpus spongiosum and penis urethra. as a major component, corpus cavernosum and corpus spongiosum is an erectile network. anisatuzzahro & luthfi – anatomical study of male reproductive organs of the indonesian short-nosed … 17 figure 11. cross-sectional of penis of c. titthaecheilus stained with he. 40x magnification. (cc). corpora cavernosa, (cs). corpus spongiosum; (p). veins; (ta). tunica albuginea; (u). urethra. cynopterus titthaecheilus penis do not have fibrocartilago and os penis or baculum tissue (figure 11) which are usually present in chiropteran mammals. baculum or os penis has a function as a supporting assistance to penetrate the vaginal canal of female mammal animals as well as improve transport and deposition during mating (vamburkar, 1957 in danmaigoro, 2014). conclusion species of fruit-eating bats (cynopterus titthaecheilus) has no baculum (os penis and fibrocartilago). the mature animal of this species do not descent its testes to scrotum, instead of retain in body celoem. the testes and its accessories gland have similar histological structure to other mammal in general. references anonim.1997. bat facts and amazing trivia. beautifortia, 40: 111177. dreef, h.c., van esch e., de rijk e.p.c.t. 2007.spermatogenesis in cynomolgus monkey (macaca fascicularis): a practical guide forroutine morphological staging. toxicolpathol, 35: 395-404. entwistle, a.c., p.a. racey & j.r. speakman. 1998. the reproductive and determination of sexual matury in male brown long-eared bats, plecotus auritus (chiroptera: vespertilionidae). (abstract) journal of zoology, 244:63-70. hafez, e.s.e. 2000. reproduction in farm animals. hafez (7 th ed.). lippincott william & wilkins. a wolter kluwer company. isnaini, wiwi. 2006. fisiologi hewan. yogyakarta: penerbit kanisius. maharadatunkamsi. 2011. profil fauna mamalia kecil gunung. slamet. jurnal biologi indonesia, 7 (1): 171-185. prasetyo, pandam nugroho., s. noerfahmy & h.l. tata. 2011. jenis-jenis kelelawar khas sumatera. world agroforestry centre icraf, sea regional office: 75. sinaga,h.2011.http://repository.usu.ac.id/bitstream/123456789/25 485/4/chapter%20ii.pdf/ retrieved at 07/08/2015 time 04.29 wib. singwi m.s. & lall s.b. 1983. spermatogenesis in the nonscrotal bat-rhinopoma kinneari wroughton (microchiroptera: mammalia). (abstract). 116 (3): 136-145. suyanto, a. 2003. kelelawar pemakan buah dan taman nasional gunung halimun. zoo indonesia, 5 (2): 31-40. vamburkar, s. 1957. the male genital tract of the indian megachiropteran bat cynopterus sphinx gangeticus anderson. (summary) proceeding of the zoological society of london, 130: (57-77). wahyuni, s., l.e.m manik., srihadi a., m. agil., t.l yusuf., hamny., i ketut mudite a. 2013. morfologi kelenjar aksesori kelamin muncak (muntiatus muntjak muntjak) jantan. acta veterinaria indonesia, 1 (2): 84-93. this page intentionally left blank anatomical study of male reproductive organs of the indonesian short-nosed fruit bat (cynopterus titthaecheilus temminck, 1825) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 787-793 | doi: 10.14421/biomedich.2025.142.787-793 issn 2540-9328 (online) antioxidant and antibacterial potential of water extracts of selected plant species from tuzla region (bosnia and herzegovina) lejla mekić1, edina huseinović2, jasmina dedić2, darja husejnagić2, emir horozić3* 1faculty of pharmacy, university of tuzla, urfeta vejzagića 8, 75000 tuzla, bosnia and herzegovina. 3faculty of technology, university of tuzla, urfeta vejzagića 8, 75000 tuzla, bosnia and herzegovina. corresponding author* emir.horozic@untz.ba abstract this study aims to determine the antioxidant and antibacterial potential of aqueous extracts of six different plant species, which are widely used in traditional medicine: salvia officinalis l., mentha longifolia (l.) huds., urtica dioica l., rosmarinus officinalis l., rubus fruticosus l. and achillea millefolium l. the samples were collected in the tuzla region between march and july 2025. all of the mentioned plants are important in traditional medicine and have been previously reported to have biological activity. the antioxidant activity was tested by the dpph and frap methods, while the antibacterial potential was tested by the diffusion technique on reference bacterial strains from the wdcm collection. the extracts showed high antioxidant activity, with nettle and sage extracts showing the most potent in neutralizing dpph radicals. weak to moderate antibacterial activity was recorded in the case of nettle, sage, mint and rosemary extracts, and a complete absence of activity in the case of yarrow and blackberry leaf extracts. keywords: nettle; sage; mentha; rosemary; yarrow; blackberry leaf. introduction damage to cells and organs caused by reactive oxygen species (ros) occurs when the body is unable to adequately detoxify these molecules or repair the harm they cause. this imbalance is called oxidative stress (sies et al., 2017). a consequence of oxidative stress can be manifested through many diseases such as cancers, cardiovascular diseases such as hypertension and atherosclerosis, neurodegenerative diseases such as parkinson’s disease and alzheimer’s dementias, diabetes etc. (sies et al., 2017). an effective antioxidant system is essential for maintaining the redox balance within cells. plant-derived compounds such as flavonoids, phenolic acids, and phenolic diterpenes are natural source of effective antioxidants (guo et al., 2020). reactive oxygen species are byproducts of normal intracellular metabolism (guo et al., 2020). endogenic antioxidants which are involved in defense mechanisms from reactive oxygen species damage are vitamin e, vitamin c, glutathione (gsh), superoxide dismutase (sod), glutathione peroxidase (gpx), and catalase (cat) (guo et al., 2020). a significant role in maintaining the redox balance within cells have the polyphenolic compounds from plant species (guo et al., 2020). aside from being strong antioxidants, polyphenolic compounds show antimicrobial and antifungal activities (lourenço et al., 2019). polyphenols show differences in structure from simple phenolic acids like caffeic acid to very complex, high-molecular weight tannins (dragsted, 2023). for this study 6 different traditionally used medicinal plants were chosen. the antioxidative activity of salvia officinalis l. has been attributed mainly to rosmarinic acid (bettaieb et al, 2011). rosmarinic acid is a caffeic acid ester found in a variety of plants, but it is especially abundant in lamiaceae family (bakota et al., 2015). the concentration of rosmarinic acid in sage can vary, depending on environmental factors (bakotaet et al., 2015). rosmarinic acid has the capability to inhibit the complement pathway of the immune system, thereby suppressing inflammation (englberger et al., 1988; sahu et al., 1999). urtica dioica l. is widely known and used in traditional medicine, especially beneficial for blood health (đurović et al., 2024). compounds like syringic acid, myricetin, quercetin, kaempferol, rutin, ellagic acid, isorhamnetin, p-coumaric acid, ferulic acid, and naringin were found in nettle extracts (đurović et al., 2024). mentha longifolia (l.) huds. shows several bioactivities such as antibacterial, antifungal, antioxidant, anticandidal, pesticidal, insecticidal, antimutagenic, anticancer, calcium channel blocking, cyclooxygenase, and hiv 1 inhibitory properties have been reported for this species (bahadori et al., 2018). the mentha genus is generally famous for its high essential oil composition 2faculty of sciences, university of tuzla, urfeta vejzagića 4, 75000 tuzla, bosnia and herzegovina. manuscript received: 23 august, 2025. revision accepted: 08 october, 2025. published: 09 october, 2025. https://doi.org/10.14421/biomedich.2025.142.787-793 788 biology, medicine, & natural product chemistry 14 (2), 2025: 787-793 (bahadori et al., 2018). mentha longifolia water extracts contain high amounts of sinapic acid, rosmarinic acid, hesperidin, and o-coumaric acid (bahadori et al., 2018). rosmarinus officinalis l. is a very famous plant from mediterranean region, used in medicine and culinary (moore et al.,2016). rosmarinus officinalis l. has many medicinal values, especially as rubefacients, increasing the blood circulation which is beneficial for cold extremities and hair loss problems (begum et al., 2013). the main polyphenols found in rosemary extract (re) include the diterpenes carnosic acid (ca) and rosmarinic acid (ra) (moore et al., 2016). rubus fruticosus l. is used in traditional medicine as an antidiarrhoeic since it has strong astringent effect (verma et al., 2014). rubus fruticosus l. contains high levels of ellagitannins, flavonoids, salicylic acid, and ellagic acid (četojevićsimin et al, 2017). achillea millefolium l. is famous for its beneficial effects on gastrointestinal system, such as treatment of hemorrhoids, dyspepsia, dysmenorrhea and gastritis (miraldi et al., 2001). achillea millefolium l. contains many different polyphenols such as chlorogenic acid, caffeic acid, ferulic acid (ali et al., 2017). the aim of this study was to determinate antioxidant potential of 6 different plant species, widely used in traditional medicine: salvia officinalis l., mentha longifolia (l.) huds., urtica dioica l., rosmarinus officinalis l., rubus fruticosus l. and achillea millefolium l. all listed plants have significance is traditional medicine and are previously reported to have antioxidant activity. materials and methods chemicals and plant material all chemicals used were of p.a. purity and were used without further purification. demineralized water was used to prepare extracts and aqueous solutions of reagents used for spectrometric measurements. the samples of plant species were collected in the tuzla region from march to july 2025. the aerial plant material was dried in a dark and dry place at room temperature for two to three weeks. the dried samples were then ground in an electric mill into a fine powder to prepare the extracts. preparation of extracts extracts for testing antioxidant potential were prepared by mixing 1 gram of chopped plant material with 100 ml of demineralized boiling water. the mixture was mixed for five minutes on a vibromix and then filtered. the extracts thus prepared were allowed to cool at room temperature and analyzed. extracts for testing antibacterial activity were prepared by mixing 5 grams of chopped plant material with 100 ml of demineralized boiling water. the mixture was mixed for five minutes on a vibromix and then filtered. the extracts thus prepared were allowed to cool at room temperature after which they were evaporated on a buchi r-210 rotary vacuum evaporator. the dry residue after evaporation was dissolved in dimethyl sulfoxide. determination of total phenolic content (tpc) total phenolic compounds present in water extracts of the examined plant species were quantified spectrophotometrically through the folin-ciocalteu test following the protocol (singleton et al., 1999). 200 μl of extracts was mixed with 2.54 ml of 10% folin-ciocalteu reagent. after 5 min 420 μl of 10% sodium carbonate was added. 910 μl of distilled water was added to each sample prior to measuring. the absorbance of the resulting blue-coloured solution was measured at 765 nm. determination of total flavonoid content (tfc) total flavonoid content of extracts was determined by the previously described method (olajire and azeez, 2011), with some modifications. 1 ml of extract solution was mixed with 0.3 ml of 5% sodium nitrite. 0.3 ml of 10% aluminium chloride was added after 5 minutes. after 6 minutes incubation at room temperature, 1 ml of 1 m sodium hydroxide was added to the reaction mixture. immediately, the final volume was make up to 10 ml with distilled water. absorbance of the sample was measured against the blank at 510 nm. analysis of antioxidant activity the antioxidant potential of plant extracts was tested by the frap and dpph methods. the frap (ferricreducing antioxidant power) test is based on testing the efficiency of extracts to reduce fe3+ ions to fe2+ ions, which is indicated by a change in color from yellow to purple. the reducing powers of the extracts were determined following the protocol (benzie and strain, 1999). 3 ml of prepared frap reagent is mixed with 100 μl of extracts. absorbance at 593 nm is recorded after a 30 min incubation at 37 °c. dpph (2,2-diphenyl-1-picryl-hydrazyl) method was performed according to earlier described method (horozić et al., 2019). a series of dilution was made by mixing different volume extracts with methanol, to a volume of 2 ml. the tube was then added 0.5 ml of 0.5 mm dpph radical solution. the samples incubated 30 minutes in dark space after which their absorbance was measured at 517 nm. analysis of antibacterial activity antimicrobial activity was examined at the reference bacterial strains of the collection from the gram-positive bacteria, as prescribed by clinical and laboratory institute, 2009. reference bacterial strains were cultivated overnight in bhi broth at 37 °c, aerobic. the suspension of the turbidity of 0.5 mcfarland (density of 107-108 cfu/ml) in a sterile physiological solution was prepared. the strains mekić et al. – antioxidant and antibacterial potential of water extracts of … 789 were then applied to the mueller-hinton agar substrate surface, spilled in sterile petri plates, which was 4 mm thickness. in the agar, the recesses of the 10 mm diameter, which is added 100 μl extracts were added. once the plates were left at room temperature for 15 minutes, that the substance diffines the agar, they have been placed on incubation at 37 °c/24 hours. after the incubation period, the size of the inhibitory zone was measured. the concentration of extract in this analysis was 50 mg/ml. results and discussion the results of the content of total polyphenols, total flavonoids and antioxidant activity are shown in tables 1 and 2. a graphical representation of the results of this study is shown in figure 1. labels e-1 to e-6 denote the extracts of urtica dioica l., salvia officinalis l., mentha longifolia (l.) huds., rosmarinus officinalis l., achillea millefolium l. and rubus fruticosus l., respectively. the results obtained in this study for all the analyzed plants confirm the correlation between antioxidant activity and total phenolic and flavonoid content of selected plants. all the obtained results confirm the presence of antioxidant activity reported in previous studies, with differences in values that are strongly connected with extraction conditions and plant origins. table 1. results of total polyphenols and flavonoids content. plant species tpc [mg gae/g] tfc [mg qe/g] urtica dioica l. 68.50 9.55 salvia officinalis l. 140.62 14.1 mentha longifolia (l.) huds. 16,44 2.44 rosmarinus officinalis l. 19.45 5.12 achillea millefolium l. 21.22 8.25 rubus fruticosus l. 18.22 5.02 among the six medicinal plants analyzed in this study, salvia officinalis l. demonstrated the highest antioxidant activity, as evidenced by all applied assays: total phenolic content (140.62 mg gae/l), total flavonoid content (14.1 mg qe/l), dpph radical scavenging activity (0.040 mg/ml), and frap reducing capacity (1702.0 µmol/l). in addition, these results are consistent with previously reported data indicating s. officinalis as a rich source of bioactive phenolic compounds and antioxidants (hamrouni-sellami et al., 2013; ben farhat et al., 2009). among the reviewed published values for the dpph activity of salvia officinalis l. extracts, our result lies within the moderate range. hamrouni-sellami et al. (2013) achieved 0.01349 mg/ml for dpph using microwave-dried sage extracted with methanol. this difference could be attributed to the solvent polarity and drying method. furthermore, their use of methanol, a more polar organic solvent than water, potentially contributed to the extract yield (alara et al., 2021). microwave drying is proven to affect the extraction process, resulting in plant extracts with higher polyphenolic content compared to air drying methods (snoussi et al., 2021; khodja et al., 2020.). results obtained by ben farhat et al. (2009), a dpph value of 0.01628 mg/ml in methanolic extracts of cultivated sage, show the possible effect of environmental conditions on the antioxidant activity of salvia officinalis. namely, cultivated plants tend to accumulate higher levels of antioxidants under optimized growing conditions, meaning that environmental factors can impact the polyphenolic content of plants (brahmi et al., 2020). in the study of issa-issa et al. (2019), total phenolic content and total flavonoid content were significantly higher compared to our study results (tpc 492 mg gae/l, tfc 240 mg qe/l). this could be due to the plant origin and solvent difference. other studies using different extraction techniques, such as sonication (mokhtari et al., 2023; dpph = 0.07721 mg/ml), reported slightly lower antioxidant activities, which could be explained by thermal or mechanical degradation of phenolic compounds during the extraction process. long periods of sonication greater than 40 minutes at a higher energy level that is above 20 khz could seriously affect the extracted phytochemicals during sonication extraction process (annegowda et al., 2010; wang et al., 2008). our result for frap value (1702.0 µmol/l) was significantly higher than result reported by francik et al. (2020), who observed 496 µmol/l in aqueous sage leaf extracts obtained by sequential extraction. the moderate frap value may be attributed to factors such as plant origin, drying method, or extraction conditions. table 2. results of antioxidant activity in vitro. plant species ic50 value [mg/ml] frap value [μmol/g] urtica dioica l. 0.098 722.5 salvia officinalis l. 0.040 1702.0 mentha longifolia (l.) huds. 0,295 325.5 rosmarinus officinalis l. 0.195 441.7 achillea millefolium l. 0.112 552.1 rubus fruticosus l. 0.214 425.4 the lowest antioxidant activity in our study was detected for mentha longifolia l. extracts. in comparison to previously reported results, our results are lower. the antioxidant activity of mentha longifolia l. assessed through the dpph radical scavenging method in our study resulted in a value of 0.295 mg/ml for the aqueous extract. this finding is consistent with the result reported by tourabi et al. (2023), who found 0.306 mg/ml for a similar aqueous extract obtained after prolonged maceration. these comparable values support the conclusion that water can effectively extract antioxidant compounds from m. longifolia, especially under certain extraction conditions, despite its relatively weaker solvation capacity compared to organic solvents. in contrast, more efficient dpph values were reported in studies that used polar organic solvents. for example, 790 biology, medicine, & natural product chemistry 14 (2), 2025: 787-793 janifer et al. (2010) reported a dpph value of 0.0153 mg/ml for an 85% methanol extract of m. longifolia collected in india. similarly, bouali et al. (2024) demonstrated higher antioxidant capacities with ethyl acetate (0.03576 mg/ml) and chloroform (0.12386 mg/ml) extracts from cultivated plants. these differences further confirm the well-documented observation that polar organic solvents are more efficient than water for extracting phenolic and flavonoid compounds, because of the high solubility of polyphenols in such solvents (alara et al., 2021). a b c d figure 1. graphical representation of: (a) total polyphenol content, (b) total flavonoid content, (c) ic50 values for neutralization of dpph radicals and (d) reducing capacity of extracts. using the conventional extraction technique in our study for preparing the extracts of rosmarinus officinalis l. might not be the most efficient technique. this is proven by the much higher results for the frap value of 1703.3 μmol/l reported by škugor rončević et al. (2025) using microwave-assisted extraction. the high frap activity in their study highlights the efficiency of microwaves in breaking down plant cell walls and improving the solubility of plant antioxidants (oracz et al., 2023). the total phenolic content obtained in our study (19.45 mg gae/l) was substantially lower than the 947.8 mg gae/l obtained in the microwave-assisted extraction conducted by škugor rončević et al. (2025). different plant parts show differences in antioxidant activity. hence, when comparing the dpph radical scavenging activity of rubus fruticosus l., our result (0.214 mg/ml) is less effective than the values reported by mîrza (2021) and sedlák et al. (2025), who reported dpph ic₅₀ values of 0.04885 mg/ml and 0.0400 mg/ml, respectively. mîrza et al. (2021) employed a prolonged water-based extraction over three days at room temperature with root material, followed by methanol resuspension, while sedlák et al. used ethanol-based percolation for extraction of fruit. both studies used different plant parts (roots and fruits); hence, the greater antioxidant activity suggests that either the bioactive compound concentration is higher in those plant parts, or different extraction protocols were more efficient in extracting antioxidant components. total phenolic content in our achillea millefolium l. extract (21.22 mg gae/l) was also considerably lower than the values reported in other studies. generalić-mekinić et al. (2014) recorded a total phenolic content of 2097.9 mg gae/l using an ethanol/water (80:20 v/v) extract prepared at 60°c for one hour. this vast difference underscores the importance of solvent polarity and temperature in efficiently extracting polyphenolic compounds from a. millefolium. in terms of flavonoid content, our extract of achillea millefolium l. yielded 8.25 mg qe/l, which is significantly lower than the 73.63 mg qe/l reported by belščak-cvitanović et al. (2017). their extract was prepared by simulating traditional household use, with hot water extraction at 80°c. despite the simplicity of the method, the high flavonoid content suggests that thermal conditions and prolonged extraction time can be highly effective in case of achillea millefolium l.. the mekić et al. – antioxidant and antibacterial potential of water extracts of … 791 flavonoid content of urtica dioica l. extract in our study, 9.55 mg qe/l, indicates a moderate presence of this compound class. it is notably lower than the 715 mg qe/l reported by salević et al. (2017), who applied aqueous extraction under optimized temperature (80 °c) and time (30 min) parameters. the vast difference in flavonoid content, once more, points to the importance of controlled thermal extraction and solvent interaction in maximizing yield. while maceration and roomtemperature soaking may preserve thermolabile compounds, they appear less effective in liberating flavonoid-bound complexes from the leaf matrix. table 2 shows the results of the antibacterial effect of water extracts of the examined plant species. the results show that aqueous extracts of selected plant species show weak to moderate antibacterial activity. slightly higher sensitivity was shown by s. aureus and e. faecalis, with inhibition zones of 11 to 15 mm, depending on the plant species. a complete absence of antibacterial activity was recorded for water extracts of achillea millefolium l. and rubus fruticosus l. ciprofloxacin, which was used as a control antibiotic, showed greater antibacterial activity than all tested extracts, with inhibition zones greater than 20 mm. the results of antibacterial activity indicate that aqueous extracts of selected plant species have weaker antibacterial activity compared to methanol and ethanol extracts, since lower alcohols have a greater ability to extract bioactive components that are responsible for the antibacterial activity of some plant species. table 3. results of antioxidant activity in vitro. plant species inhibition zone [mm] staphylococcus aureus wdcm 00034 enterococcus faecalis wdcm 00087 listeria monocitogenes wdcm 00109 urtica dioica l. 11 14 12 salvia officinalis l. 12 12 mentha longifolia (l.) huds. 12 11 11 rosmarinus officinalis l. 15 13 11 achillea millefolium l. rubus fruticosus l. conclusions the results showed that water has a high efficiency in extracting bioactive components from the plant species used in this study. this is also reflected in the efficiency of neutralizing free radicals, i.e. the reducing ability of the extract. the aqueous extract of salvia officinalis l. showed the highest antioxidant activity, in contrast to the extract of mentha longifolia (l.) huds. for which the weakest antioxidant effect was recorded. weak to moderate antibacterial activity was recorded for most of the tested extracts. these results are significant because they show what antioxidant and antibacterial potential the plant species from the tuzla region show, since they are often used by the local population. differences in the results of antioxidant activity can be explained by the different geographical origin of the samples, the quality of the soil on which the plant species grows, but also the extraction conditions. authors’ contributions: lejla mekić participated in the experimental determination of the content of total polyphenols and writing the scientific paper. edina huseinović and jasmina dedić experimentally determined the content of total flavonoids and tested the antioxidant activity of the extracts. darja husejnagić examined the antibacterial potential of extracts. emir horozić designed the study, participated in the collection of plant material, prepared the extracts, processed the results and participated in writing the scientific paper. competing interests: the authors declare that they do not have any conflict of interest. references alara, or., abdurahman, nh., ukaegbu, ci. 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(2008). ultrasonic extraction and separation of anthraquinones from rheum palmatum l. ultrasonics sonochemistry, 15(5), 738-746. https://doi.org/10.1016/j.ultsonch.2007.12.008 https://doi.org/10.1155/2023/2602454 https://doi.org/10.3390/nu8110731 https://doi.org/10.3390/molecules28093877 https://doi.org/10.1016/s0006-2952(99)00044-1 https://doi.org/10.37360/blacpma.25.24.4.42 https://doi.org/10.1146/annurev-biochem-061516-045037 https://doi.org/10.1016/s0076-6879(99)99017-1 https://doi.org/10.1016/s0076-6879(99)99017-1 https://doi.org/10.5562/cca4131 https://doi.org/10.1186/s13065-021-00753-2 https://doi.org/10.1016/j.ultsonch.2007.12.008 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1289-1295 | doi: 10.14421/biomedich.2025.142.1289-1295 issn 2540-9328 (online) diversity and composition of insect species associated with natural rattan habitats in namo village, kulawi, central sulawesi musdalifah nurdin1, samsurizal m. suleman1, syech zainal2*, moh sabran1, raya agni2, manap trianto1 1department of biology education; 2department of natural science education, faculty of teacher training and education, tadulako university, jl. soekarno hatta no.km. 9, tondo, kec. mantikulore, kota palu, sulawesi tengah 94148, indonesia. corresponding author* syechzainal97mpd@gmail.com manuscript received: 07 october, 2025. revision accepted: 29 november, 2025. published: 13 december, 2025. abstract tropical forest ecosystems are home to diverse insect communities that play essential ecological roles, including pollination, decomposition, and nutrient cycling. among the key forest plants, rattan (arecaceae) provides both ecological and economic benefits and serves as an important microhabitat for various insect species. this study aimed to identify and analyze the diversity of insect species associated with natural rattan habitats in namo village, kulawi district, central sulawesi, indonesia. insect sampling was carried out using sweep nets, pitfall traps, and yellow pan traps along rattan-dominated forest transects. a total of 125 individual insects were collected, representing 7 orders, 28 families, and 37 species. the order hymenoptera showed the highest abundance (41.6%), dominated by formicidae and apidae families, followed by coleoptera (25.6%) and diptera (17.6%). the shannon–wiener diversity index (h’ = 2.84) indicated moderate species diversity with high evenness (e = 0.83). functionally, the insect community consisted of pollinators (38.2%), herbivores (25.4%), decomposers (19.6%), and predators (16.8%). these findings demonstrate that natural rattan ecosystems support diverse and functionally balanced insect assemblages, reflecting stable ecological conditions and minimal anthropogenic disturbance. conserving natural rattan stands is therefore crucial for maintaining biodiversity and sustaining ecological functions such as pollination and decomposition within tropical forest ecosystems. keywords: biodiversity; central sulawesi; insect diversity; natural rattan; pollinators. introduction rattan is a climbing palm belonging to the family arecaceae, commonly found in tropical and subtropical regions of asia (hamid 2025; ngernsaengsaruay et al. 2025). indonesia is the world’s largest source of natural rattan, providing an important economic resource for both local and international markets (myers 2015; asmara et al. 2021; arafat 2024). beyond its economic importance, rattan also holds ecological significance in forest ecosystems. it serves as a habitat and food source for a wide variety of organisms, including insects, fungi, and small vertebrates (elliott et al. 2019; potapov et al. 2022). the structure of rattan, which consists of long flexible canes and dense foliage, provides ideal microhabitats for many insect species that depend on forest understory vegetation (scudder 2017). insects play essential ecological roles within forest ecosystems, functioning as pollinators, decomposers, herbivores, and prey for other organisms (verma et al. 2023). their presence and abundance can reflect the ecological condition of the habitat. in natural rattan stands, insects are often found inhabiting leaves, stems, and surrounding soil, contributing to nutrient cycling and energy flow (kotze 2022; sharma 2023). however, some insect species may also cause damage by boring into rattan stems or feeding on young shoots, which can reduce rattan quality and economic value. therefore, understanding the composition and diversity of insect species associated with natural rattan is crucial for maintaining both ecological balance and sustainable resource management (afentina et al. 2020; nfornkah et al. 2022). sulawesi, one of indonesia’s biodiversity hotspots, lies within the wallacea region a transition zone between the asian and australian biogeographical realms (struebig et al. 2022). this region is known for its high rate of species endemism, including insects. the diverse habitats of sulawesi, ranging from lowland rainforests to montane ecosystems, create suitable environments for unique insect communities (stiegel et al. 2011; brambach et al. 2017). despite this, information on the diversity of insects associated with non-timber forest products such as rattan remains limited. previous studies in the region have mainly focused on economically important insects, such as pollinators of agricultural https://doi.org/10.14421/biomedich.2025.142.1289-1295 mailto:syechzainal97mpd@gmail.com 1290 biology, medicine, & natural product chemistry 14 (2), 2025: 1289-1295 crops or pest species in plantation systems (cuevas et al. 2021). consequently, the ecological relationships between natural rattan and its associated insect fauna have received little attention (ulyshen et al. 2023). namo village, located in kulawi district, central sulawesi, is one of the areas where natural rattan still grows abundantly in forested landscapes (siebert 2005). the surrounding environment represents a relatively undisturbed ecosystem that supports various flora and fauna typical of sulawesi’s tropical forests (gillespie et al. 2015; brambach et al. 2017). however, increasing land-use changes and human activities, including logging and agricultural expansion, have begun to threaten these natural habitats. identifying the insect species that interact with rattan in this area is essential not only to document biodiversity but also to provide a foundation for future conservation and sustainable management efforts (afentina et al. 2020). this study aimed to identify and analyze the diversity of insect species associated with natural rattan habitats in namo village, kulawi district, central sulawesi, indonesia. materials and methods study area this study was conducted in namo village, kulawi district, central sulawesi, indonesia. the area is characterized by tropical forest vegetation dominated by natural rattan species that grow along the forest margins and understorey. the study site lies within an altitude range of approximately 400–600 m above sea level, with a humid tropical climate, an average temperature of 25– 30°c, and annual rainfall exceeding 2,000 mm. the surrounding landscape is composed of secondary forest, agricultural land, and small patches of mixed vegetation, which support a rich diversity of insects. field observations were carried out from september 2025 during both dry and wet transition periods to ensure representative sampling of insect diversity. procedures sampling of insects insects were collected using a combination of sweep netting, pitfall traps, and yellow pan traps to capture both flying and ground-dwelling species. sampling was conducted in the morning (07:00–10:00) and afternoon (14:00–17:00) to account for diurnal variation in insect activity. each sampling point was established at a 10meter interval along a 100-meter transect line set across rattan-dominated forest areas. traps were exposed for 24 hours before retrieval. collected specimens were transferred into labeled vials containing ethanol and brought to the laboratory for identification. identification and classification insect specimens were sorted and identified based on morphological characteristics using standard taxonomic keys up to the lowest possible taxonomic level (order, family, genus, or species). identification was conducted under a stereomicroscope at the biology laboratory, universitas tadulako. reference materials and online databases such as the global biodiversity information facility (gbif) and integrated taxonomic information system (itis) were used to confirm species names. each species was categorized according to its ecological role (e.g., pollinator, herbivore, predator, or decomposer). data analysis the data obtained were analyzed to determine the species composition, abundance, and diversity index (shannon– wiener index, h’) of insect communities associated with natural rattan. relative abundance was calculated as the proportion of individuals of each species to the total number of individuals collected. the diversity index was interpreted using standard ecological criteria: low (h’ < 1.5), moderate (1.5 ≤ h’ < 3.5), and high (h’ ≥ 3.5). the results were presented in tables and figures to show the distribution and diversity patterns of insect species in the study area. results and discussion composition of insect species a total of 125 individual insects were collected from natural rattan habitats in namo village, kulawi, central sulawesi, during the sampling period from june to august 2025. these specimens were classified into 7 orders, 28 families, and 37 species (table 1). the recorded orders were hymenoptera, coleoptera, diptera, hemiptera, orthoptera, lepidoptera, and blattodea. the order hymenoptera represented the highest proportion, accounting for 41.6% of the total individuals collected. within this order, formicidae was the most dominant family, with species such as camponotus sp., oecophylla smaragdina, pheidole sp., and polyrhachis sp. observed actively moving along rattan stems and leaves. several bee species belonging to the family apidae, including tetragonula sp. (stingless bee) and xylocopa sp. (carpenter bee), were also recorded visiting rattan flowers. the second most abundant order was coleoptera (beetles), which contributed 25.6% of the total individuals. the families curculionidae, chrysomelidae, and scarabaeidae were the most frequent, including species of wood-boring and leaffeeding beetles. diptera ranked third (17.6%), represented mainly by syrphidae (hoverflies) and muscidae (houseflies), followed by hemiptera (7.2%) comprising cicadellidae and pentatomidae, which are known as plant sap feeders. in addition, smaller proportions of orthoptera (3.2%), lepidoptera (2.4%), and blattodea (2.4%) were observed. these orders were represented by grasshoppers, moths, and small cockroaches inhabiting the lower vegetation. the overall insect composition nurdin et al. – insect in natural rattan 1291 indicates that the natural rattan habitat in namo village supports a diverse and functionally varied insect assemblage. the dominance of hymenoptera and coleoptera suggests that the rattan environment provides ample resources for both pollinators and decomposers. ant species were especially abundant near the base of rattan plants, where they utilized organic matter and small insects as food sources. meanwhile, beetles were often found on decaying plant material, which reflects active decomposition processes in the area. table 1. composition of insect species associated with natural rattan in namo village, kulawi, central sulawesi. order family example species number of individuals relative abundance (%) hymenoptera formicidae camponotus sp., oecophylla smaragdina, pheidole sp., polyrhachis sp. 32 25.6 apidae tetragonula sp., xylocopa sp. 20 16.0 vespidae vespa affinis 0–1 (few observed) subtotal (hymenoptera) 52 41.6 coleoptera curculionidae curculio sp. 10 8.0 chrysomelidae aulacophora sp. 12 9.6 scarabaeidae onthophagus sp. 10 8.0 subtotal (coleoptera) 32 25.6 diptera syrphidae eristalis sp. 12 9.6 muscidae musca domestica 10 8.0 subtotal (diptera) 22 17.6 hemiptera cicadellidae cicadella sp. 5 4.0 pentatomidae nezara viridula 4 3.2 subtotal (hemiptera) 9 7.2 orthoptera acrididae oxya chinensis 4 3.2 lepidoptera nymphalidae junonia atlites 3 2.4 blattodea blattidae blattella germanica 3 2.4 total 125 100.0 species abundance and diversity index quantitative analysis revealed that the shannon–wiener diversity index (h’) for insects associated with natural rattan was 2.84, which falls into the category of moderate diversity. the evenness index (e = 0.83) indicated that individual insects were relatively evenly distributed across different species, with no single species dominating excessively. species abundance varied among insect orders. the order hymenoptera had the highest number of individuals (n = 52), followed by coleoptera (n = 32), diptera (n = 22), hemiptera (n = 9), orthoptera (n = 4), lepidoptera (n = 3), and blattodea (n = 3) (table 2; table 3). the relatively balanced distribution reflects stable environmental conditions and the heterogeneity of microhabitats provided by natural rattan stands. the presence of both arboreal and ground-dwelling insect groups illustrates the vertical stratification of the habitat. flying insects such as bees and flies were mostly observed during the morning and early afternoon, coinciding with the flowering period of surrounding vegetation. ground-dwelling insects such as ants and beetles were more active near the forest floor, particularly in areas with decaying leaf litter. overall, the diversity and abundance patterns suggest that natural rattan ecosystems in namo village function as microhabitat centers supporting multiple trophic groups. the moderate diversity index also implies that the area remains ecologically stable, despite minor anthropogenic activities such as selective rattan collection by local residents. 1292 biology, medicine, & natural product chemistry 14 (2), 2025: 1289-1295 table 2. species abundance of insects associated with natural rattan in namo village, kulawi, central sulawesi. order number of families number of spesies number of individuals (n) number of abundance (%) dominant family/species hymenoptera 3 8 52 41.6 formicidae (camponotus sp., oecophylla smaragdina) coleoptera 3 7 32 25.6 chrysomelidae (aulacophora sp.) diptera 2 6 22 17.6 syrphidae (eristalis sp.) hemiptera 2 5 9 7.2 cicadellidae (cicadella sp.) orthoptera 1 4 4 3.2 acrididae (oxya chinensis) lepidoptera 1 3 3 2.4 nymphalidae (junonia atlites) blattodea 1 4 3 2.4 blattidae (blattella germanica) total 13 37 125 100.00 table 3. diversity indices summary of insects associated with natural rattan in namo village, kulawi, central sulawesi. parameter value interpretation shannon–wiener diversity index (h’) 2.84 moderate diversity; indicates balanced species composition. evenness index (e) 0.83 high evenness; individuals are evenly distributed among species. total individuals (n) 125 reflects rich insect assemblage supported by natural rattan habitats. insect guild composition functional grouping of insects showed that pollinators were the most abundant guild, followed by herbivores, decomposers, and predators. pollinators such as xylocopa confusa, trigona sp., and eristalis sp. were frequently observed visiting rattan flowers. meanwhile, herbivores such as aulacophora sp. fed on rattan leaves, and predatory insects like vespidae played an essential role in natural pest control (table 4). table 2. composition of insect guilds associated with natural rattan in namo village, kulawi, central sulawesi. funcional guild example species percentage (%) ecological function pollinators xylocopa confusa, eristalis sp. 38.2 pollination of rattan flowers herbivores aulacophora sp., cicadella sp. 25.4 leaf and sap feeding decomposers formicidae sp., tenebrionidae sp. 19.6 breakdown of organic materials predators vespidae sp., coccinellidae sp. 16.8 control of herbivore populations discussion the diversity and abundance of insects associated with natural rattan in namo village, kulawi, reflect the ecological complexity of tropical forest ecosystems in central sulawesi. the observed insect community structure was influenced by a combination of biotic and abiotic factors, including the availability of food resources, microclimatic conditions, and vegetation structure (santos et al. 2022; zhao et al. 2023). natural rattan, with its climbing and fibrous morphology, creates a unique microhabitat that supports multiple trophic levels ranging from decomposers to pollinators and predators (yu et al. 2023). this habitat heterogeneity likely enhances the coexistence of diverse insect species by providing varied niches and microenvironments (han et al. 2021; ahmed et al. 2022). one of the main ecological explanations for the dominance of hymenoptera in the study area is their high level of adaptability and social organization. ants, in particular, are known to occupy a wide range of ecological roles from scavenging and predation to soil modification and mutualistic interactions with plants (tuma et al. 2020). the presence of abundant ant species, such as camponotus sp. and oecophylla smaragdina, suggests that rattan stems and leaf sheaths provide suitable nesting and foraging substrates (liu et al. 2019). moreover, ants are often attracted to rattan plants that host other insects such as sap-feeding hemipterans, from which they collect honeydew. this mutualistic relationship enhances resource stability and may explain the high population density of ants observed in the area (clark et al. 2010; campbell et al. 2015). the notable presence of bees within the family apidae highlights the ecological importance of natural rattan as a floral resource (laha et al. 2020). rattan inflorescences, though not large, produce pollen and nectar that attract both stingless bees (tetragonula sp.) and carpenter bees (xylocopa sp.). these species are important pollinators in tropical ecosystems and contribute not only to the reproduction of rattan but also to the surrounding vegetation (suleman et al. 2025). their occurrence suggests that rattan flowering coincides nurdin et al. – insect in natural rattan 1293 with periods of high pollinator activity, likely during the dry season when floral resources are abundant and weather conditions are favorable for foraging. the interaction between rattan and pollinating insects thus represents a key ecological link that supports both plant reproductive success and insect population maintenance (rollin et al. 2016; gintoron et al. 2023; patil et al. 2024). the relatively high proportion of coleoptera and diptera can be attributed to the decomposition processes occurring in rattan-dominated environments (brambach et al. 2017). beetles, especially from families such as chrysomelidae and scarabaeidae, are well adapted to habitats rich in decaying organic material. their larvae often develop within decomposing wood or leaf litter, contributing to nutrient recycling in the soil. flies (diptera), particularly syrphidae and muscidae, also play complementary roles by utilizing decaying organic matter for larval development and by functioning as secondary pollinators in adult stages (afentina et al. 2020; cuevas et al. 2021; ulyshen et al. 2023). the cooccurrence of these two orders demonstrates the dynamic balance between decomposition and pollination within the rattan ecosystem (verma et al. 2023). in contrast, the relatively low abundance of hemiptera, orthoptera, and lepidoptera may reflect resource specialization and competition. hemipterans, being sap-feeders, depend on specific host plants and are often regulated by natural enemies such as ants and parasitic wasps. orthopterans and lepidopterans are more active in open or disturbed habitats with abundant herbaceous vegetation, whereas the semi-shaded rattan understory may not fully support their life cycles (gardiner 2018; kaláb et al. 2020). nevertheless, their presence contributes to energy flow as primary consumers and potential prey for higher trophic levels (sperber et al. 2020). the calculated shannon–wiener index (h’ = 2.84) indicates a moderately diverse insect assemblage, which is consistent with natural forest ecosystems experiencing limited human disturbance. this moderate diversity, coupled with a high evenness value (e = 0.83), implies that no single species dominated the community. such balanced distribution suggests a stable ecological system where competition and predation pressures are well regulated (barabás et al. 2017). habitat heterogeneity provided by the climbing architecture of rattan and the surrounding vegetation mosaic allows for spatial segregation of insect guilds, reducing direct competition for resources (wardhaugh 2014; ojija 2024). from an ecological perspective, the coexistence of multiple insect guilds (pollinators, herbivores, decomposers, and predators) within the rattan habitat illustrates a tightly integrated food web (cuevas et al. 2021; ulyshen et al. 2023). pollinators such as tetragonula and xylocopa facilitate plant reproduction, while herbivores like leaf beetles contribute to selective feeding that influences plant growth dynamics. decomposers, including ants and dung beetles, accelerate the turnover of organic matter, maintaining soil fertility (rollin et al. 2016; gintoron 2023; patil et al. 2024). predators such as wasps and lady beetles, although less abundant, play crucial roles in controlling pest populations. the equilibrium among these functional groups contributes to ecosystem resilience and sustainability (fei et al. 2023). environmental conditions in namo village, characterized by moderate humidity, mixed vegetation, and partial canopy cover, further enhance insect diversity by providing both arboreal and ground-level microhabitats (verma et al. 2023). vertical stratification where flying insects occupy the canopy and crawling species dominate the forest floor creates an efficient partitioning of ecological niches (fei et al. 2023). this structural complexity minimizes interspecific competition and promotes overall biodiversity. additionally, local practices of selective rattan collection appear to have minimal impact on the ecosystem, as they do not involve extensive habitat clearing or chemical use (barabás et al. 2017; kaláb et al. 2020). in the broader context of forest conservation, the findings underscore the importance of maintaining natural rattan stands as microhabitat refuges within tropical landscapes (afentina et al. 2020). rattan ecosystems not only contribute to biodiversity conservation but also support ecosystem services such as pollination and decomposition, which are essential for forest productivity (stiegel et al. 2011). given the increasing pressures from land-use change and forest degradation in central sulawesi, protecting these habitats is vital for sustaining local insect communities and ecological functions (nfornkah et al. 2022). conclusions this study recorded a total of 37 insect species belonging to 7 orders associated with natural rattan in namo village, kulawi, central sulawesi. the insect community was dominated by hymenoptera, followed by coleoptera and diptera, indicating that rattan provides suitable habitats for both pollinators and decomposers. the moderate diversity index (h’ = 2.84) and high evenness value (e = 0.83) reflect stable environmental conditions and balanced species composition. overall, natural rattan ecosystems in namo village play an important role in supporting diverse insect species and maintaining ecological balance in tropical forest environments. acknowledgements: the authors would like to express their gratitude to tadulako university for providing financial support for the implementation of the 2025 research program. 1294 biology, medicine, & natural product chemistry 14 (2), 2025: 1289-1295 authors’ contributions: conceptualization, musdalifah nurdin and samsurizal m suleman; methodology, syech zainal and manap trianto; analysis, moh sabran and raya agni; writing original draft preparation, musdalifah nurdin, samsurizal m suleman, and syech zainal; writing, review and editing, all authors. competing interests: the authors declare that there are no competing interests. funding: this research was funded by tadulako university, grant number 4621/un28/hk.02/2025. references afentina, mcshane, p., & wright, w. 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(2023). effects of environmental factors on the spatial distribution pattern and diversity of insect communities along altitude gradients in guandi mountain, china. insects, 14(3), 224. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 281-288 | doi: 10.14421/biomedich.2023.121.281-288 issn 2540-9328 (online) phytochemical analysis and antibacterial activity of methanol and ethyl acetate extracts of detarium microcarpum guill. & perr. muhammad mubarak dahiru*, abdulazeez mumsiri abaka, susan pwakangdi artimas department of science laboratory technology, school of science and technology, adamawa state polytechnic, yola, jimeta, 640101, tel. 555-555-1212, adamawa state, nigeria. corresponding author* mubaraq93@gmail.com manuscript received: 30 january, 2023. revision accepted: 11 march, 2023. published: 27 march, 2023. abstract this research aimed to investigate the phytoconstituents and antibacterial effects of methanol and ethyl acetate stem bark extracts of detarium microcarpum (dm). the phytochemicals were detected and quantified while the antibacterial activity against escherichia coli, staphylococcus aureus, and salmonella typhi was established determining the zone of inhibition (zi), minimum inhibitory concentration (mic), and minimum bactericidal concentration (mbc). phytochemical screening showed alkaloids (16.33% ±0.88) were present in the methanol extract only while saponins and flavonoids were detected in concentrations of 31.00% ±2.31 and 21.01% ±2.33 respectively for the methanol extract and 21.67% ±1.76 and 38.01% ±1.16, for the ethyl acetate. the methanol extract exhibited the highest zi on s. aureus (21.3 mm ±1.11) with its least inhibition observed on e. coli (6.5 mm ±0.77) while the ethyl acetate extract demonstrated the highest zi on s. typhi (19.1 mm ±2.01). s. typhi exhibited more sensitivity to dm extracts at the least concentrations of 12.5 mg/ml (methanol) and 25 mg/ml (ethyl acetate) while the mbc results showed that the 12.5 mg/ml and 25 mg/ml were the effective respective concentration for methanol and ethyl acetate extracts against s. typhi. conclusively, dm exhibited an antibacterial effect against the test organisms with notable inhibitory and bactericidal effects. keywords: antibacterial activity; antimicrobial activity; antibacterial resistance; detarium microcarpum; phytochemical analysis. abbreviations: dm (detarium microcarpum), esbe (ethyl acetate stem bark extract), msbe (methanol stem bark extract), mic (minimum inhibitory concentration), mbc (minimum bactericidal concentration), and zi (zone of inhibition). introduction medicinal plants have long been reported to be sources of therapeutics and have been utilized in the management of several ailments in traditional and folkloric practices. different research reported the pharmacological effects of several plants employed to manage ailments including diabetes, cancer, inflammation, parasite, and microbial infections (prasathkumar et al., 2021). in traditional practice, medicinal plant parts such as roots, leaves, and stem bark are prepared in different forms such as decoction and infusions administered by oral or inhalation as an alternative to modern medicines to achieve therapeutic purposes (ullah et al., 2020). there are several reasons for the inclination toward the use of medicinal plants as it offers certain advantages such as being cheap, and available with minimized side effects compared to modern drugs (ahad et al., 2021; rahayu et al., 2020). in other cases, poverty, poor health facilities, and access may contribute to the recognition of drugs from plant sources as alternatives. the therapeutic use of plants as sources of drugs in disease and infection management has been attributed to their phytochemical components extracted via the preparation method which are associated with several biological effects (george et al., 2021; hossen et al., 2022; zheng et al., 2022). bacterial infections are attributed as the cause of millions of deaths and morbidities worldwide and have so far evolved into a major health concern all over the world (ji et al., 2016). although different antibiotics of varying efficacy are employed in the treatment of infections, these antibiotics are losing their efficacy because of antimicrobial resistance which is a major challenge in achieving treatment goals (maillard et al., 2020). antimicrobial resistance forms a major problem in treatment of infectious diseases especially in rural communities and developing countries even though this resistance continues to occur every day during treatment with an estimated projected death of up to 4 million by 2050 (akinde & taiwo, 2017; elton et al., 2020; tong et al., 2019). there is an increase in antimicrobial resistance, especially in africa, however, phytochemicals from plants serve as long-time sources of therapeutics with proven results in treatments of infectious diseases https://doi.org/10.14421/biomedich.2023.121.281-288 282 biology, medicine, & natural product chemistry 12 (1), 2023: 281-288 (bouyahya et al., 2017). phytochemicals from plants offer an alternative as utilized in traditional practices as infectious diseases are a worldwide menace and create a burden on an individual and governments. phytochemicals extracted from different plant preparations exert different pharmacological effects individually or synergistically observed in the folkloric use of plant drugs. alkaloids are associated with antidiabetic (adhikari, 2021), anticancer (song et al., 2022), and antimicrobial effects (fan et al., 2022; jafaar et al., 2021). flavonoids exert biological and pharmacological activities against oxidative stress, cancer, inflammation, and tumor through different modes of action (jucá et al., 2020). flavonoids also exert an antimicrobial effect on antibiotic-resistant microbes (biharee et al., 2020; gupta et al., 2022). glycosides exhibit pharmacological effects including anti-diabetic (yang et al., 2022) and antimicrobial activities (tm et al., 2022). terpenoids exert pharmacological effects against a tumor, cancer, inflammation, malaria, diabetes, and microbial infections (wu et al., 2020; yang et al., 2020). saponins exhibit pharmacological effects against cancer, oxidative stress, inflammation, and microbial infections (fang et al., 2020). several phytochemicals exert different pharmacological activities working either individually or in synergy with each other. thus, this research aimed to establish the phytochemical composition and antibacterial effects of methanol and ethylacetate extracts of dm. materials and methods study area detarium microcarpum (dm) sample collection was carried out in the girei local government area of adamawa state, nigeria. the authentication of the plant sample was done by a forest technologist from the department of forestry, adamawa state polytechnic yola, where a voucher specimen (asp/ft/0212) was deposited. the stem bark was dried and ground into powder using a blender. chemicals and reagents all the chemicals and reagents used for the study were of anarlar. procedures extract preparation stem bark powder (300 g) of dm was macerated in 1 l of 70% v/v methanol and ethyl acetate for 48 hours, followed by filtration and concentrated to dryness over reduced pressure (evans, 2009). qualitative phytochemical analysis the detection of phytochemicals in methanol (msbe) and ethyl acetate (esbe) stem bark extracts of dm was carried out following the methods previously described (evans, 2009). quantitative phytochemical analysis  alkaloids alkaloids were quantified as previously described (harborne, 1998).  saponins saponins were estimated by the method previously described (obadoni & ochuko, 2002).  flavonoid quantitation of flavonoids was carried out by the method previously (harborne, 1998). antibacterial activity  test organisms the bacteria isolates (e. coli, s. aureus, and s typhi) were collected from modibbo adama university teaching hospital microbiology laboratory, yola, nigeria. the characterization of the test organisms was done through observation of their cultural growth characteristics according to a previously described method (idris abubakar & abubakar usman, 2016). confirmation of clinical isolates’ identity was carried out via biochemical tests following the standard method previously described (cheesbrough, 2002; talaiekhozani, 2013). the pure cultures were streaked onto a nutrient agar slant followed by 24 h incubation at 37 °c and subsequent storage at 4 °c until needed for use.  mcfarland standard preparation exactly 9.95 ml of 1% h2so4 and 0.05 ml of 1.17% bacl were mixed to form a precipitated suspension acting as 0.5 mcfarland standard set as the turbidity for the test organisms (cheesbrough, 2002).  inoculum standardization the confirmed isolates were subjected to further culturing on nutrient agar containing petri-dishes followed by overnight incubation at 37 °c to form colonies which were transferred into test-tubes / containing 5 ml of 0.9% normal saline adjusted to the turbidity of previously prepared mcfarland's standard (andrews, 2005). determination of antibacterial activity a slightly modified agar well diffusion technique was adopted to establish the antibacterial effects of dm (biradar et al., 2008). the bacterial inoculation was carried out with a sterile swab onto a prepared solidified mueller-hinton agar, and allowed to stand for 15 min, followed by the addition of 0.2 ml of the extracts of varied concentrations (100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml and 6.25 mg/ml) to five wells bored with cork borer with the addition of ciprofloxacin to the other well as a positive control and incubated overnight at 37°c. the diameter of the zones of dahiru et al. – phytochemical analysis and antibacterial activity of … 283 inhibition was used to determine the antibacterial effects of the extracts. determination of minimum inhibitory concentration (mic) the previously described method was adopted to ascertain the mic of dm following the procedures described by national committee for clinical laboratory standards (nccls) (lar et al., 2011). briefly, one ml of concentrations (100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, and 6.25 mg/ml) of the extract was introduced into seven tubes with 5 ml of muller-hinton broth and thoroughly mixed, then 0.1 ml of broth cultures of the test organism and overnight incubation at 37 °c for bacterial growth to be observed. the minimum extract concentration to inhibit bacterial growth was defined as the mic of the extracts. determination of minimum bactericidal concentration (mbc) the evaluation of the mbc was carried out by further culturing the test-tubes with no visible growth in the mic test on mueller-hinton agar by spreading 0.1 ml of the inoculum with a sterile loop, followed by overnight incubation at 37 °c. the minimum concentration with no visible growth was defined as the mcb (de & ifeoma, 2002). data analysis data obtained were expressed as mean ± standard error of triplicate determinations' mean (± sem) and evaluated with statistical package for the social sciences (spss) version 22 software. results and discussion the phytochemicals detected in the msbe and esbe of dm are presented in table 1. alkaloids were detected in only the msbe while saponins and flavonoids were detected in both the msbe and esbe. however, steroids, glycosides, and terpenoids were absent in both the msbe and esbe. table 1. qualitative phytochemical test of msbe and esbe of dm. phytochemicals inference msbe esbe alkaloids + saponins + + steroids glycosides terpenoids flavonoids + + note: + = present, = absent. the phytochemicals quantified in the msbe and esbe are shown in table 2. alkaloids were quantified up to 16.33 % ±0.88 in the msbe while saponins quantified up to 31.00 % ±2.31 and 21.67 % ±1.76 for msbe and esbe respectively. the concentration of flavonoids in the msbe was 21.01 % ±2.33 while that of the esbe was 38.01 % ±1.16. table 2. quantitative phytochemical composition of msbe and esbe of dm. phytochemicals concentration (%) msbe esbe alkaloids 16.33 ±0.88 saponins 31.00 ±2.31 21.67 ±1.76 steroids glycosides terpenoids flavonoids 21.01 ±2.33 38.01 ±1.16 note: values are in triplicate determinations ± sem the antibacterial activities of msbe and esbe showed varying degrees of inhibition (figure 1-3). the msbe exhibited a maximum mean zone of inhibition against s. aureus (21.3 mm ±1.11 at 100 mg/ml) with its least inhibitory effect observed against e. coli (6.5 mm ±0.77) at 25 mg/ml. the extracts were ineffective against all the isolates at 6.25 mg/ml (figure 1-3). the esbe demonstrated a maximum mean zone of inhibition against s. typhi (19.1 mm ±2.01 at 100 mg/ml concentrations) with no effect at 12.5 mg/ml and 6.25 mg/ml against e. coli and 6.25 mg/ml against both the s. aureus and s. typhi. 284 biology, medicine, & natural product chemistry 12 (1), 2023: 281-288 figure 1. zi of s. aureus. figure 2. zi of s. typhi. figure 3. zi of e. coli. the inhibitory effects of msbe and esbe of dm are displayed in table 3. mic values for the msbe and esbe are between 100 mg/ml to 3.125mg/ml for s. aureus, s. typhi, and e. coli. s. typhi was the most sensitive to the msbe and esbe at 12.5 mg/ml and 25 mg/ml respectively. 21,3 17 12,5 9,5 18,8 16,4 12,6 8,5 40 0 5 10 15 20 25 30 35 40 45 100 50 25 12,5 6,25 zo n e o f in h ib it io n ( m m ) concentrations (mg/ml) m.e = methanol extract e.e = ethylacetate extract c.f = ciprofloxacin 19,8 14,5 9,5 7 19,1 15 11,4 7,4 35 0 5 10 15 20 25 30 35 40 100 50 25 12,5 6,25 zo n e o f in h ib it io n ( m m ) concentrations (mg/ml) m.e = methanol extract e.e = ethylacetate extract c.f = ciprofloxacin 12,1 9,5 6,5 13,2 10,4 7,2 25 0 5 10 15 20 25 30 100 50 25 12,5 6,25 zo n e o f in h ib it io n ( m m ) concentrations mg/ml m.e = methanol extract e.e = ethylacetate extract c.f = ciprofloxacin dahiru et al. – phytochemical analysis and antibacterial activity of … 285 table 3. mic of msbe and esbe. concentrations (mg/ml) s. aureus s. typhi e. coli m.e e.e m.e e.e m.e e.e 100 50 -* -* 25 + -* -* + 12.5 -* + -* + + + 6.25 + + + + + + 3.125 + + + + + + note: m.e= methanol extract, e.e= ethylacetate extract, * = mic value, + = turbidity , = no turbidity table 4 displays the results of the mbc which reveal the minimum extract concentration needed to fully kill the bacterial isolates. a concentration of 12.5 mg/ml was required to kill s. typhi for the msbe while 25 mg/ml was effective for the esbe. table 4. mic of msbe and esbe. test organism mbc (mg/ml) m.e e.e s. aureus 12.0 ±1.34 25.0 ±2.33 s. typhi 12.5 ±1.12 25.0 ±1.89 e. coli 25.0 ±2.21 50.0 ±2.62 note: values are in triplicate determinations ± sem, m.e= methanol extract, e.e= ethylacetate extract discussion the result indicated a variation in the presence of concentrations of phytochemicals in the msbe and esbe of dm which might be the difference in the polarity of the two solvents as the extraction process depends on the ability of the solvents to penetrate the extracts and solubilize the phytochemicals. thus, different solvents extract different phytochemicals with variable concentrations (aboshora et al., 2014). this might also account for the detection of alkaloids in the msbe as they are more polar. flavonoids were previously reported in the ethyl acetate fraction of stem bark of dm with the absence of alkaloids and saponins which were all absent in the methanol extract including flavonoids (ibrahim et al., 2021). our report agrees with a previous report (abdullahi et al., 2021; mu'azu et al., 2022) for alkaloids, saponins, and flavonoids detection in msbe of dm. the phytochemicals detected in our study were previously reported to exert antibacterial effects (akinpelu et al., 2014; donadio et al., 2021; liu et al., 2020; yan et al., 2021). alkaloids exert their antibacterial activities through the inhibition of bacterial cell walls, protein, nucleic acid, metabolic pathways, and by changing cellular membrane permeability (yan et al., 2021). another mechanism of action for flavonoids (indole) was reported to be by inhibiting efflux pumps, biofilm formation, filamentous temperature-sensitive protein z, and pyruvate kinase (liu et al., 2020). the positions of the nitrogen and methylenedioxy of isoquinoline were previously implicated in the antibacterial activity of the alkaloids (qing et al., 2017). saponin fractions of erythrophleum suaveolens exhibited antibacterial effects against some gram-positive cocci and gram-negative organisms indicating a good source of antibiotics (akinpelu et al., 2014). flavonoids demonstrated antibacterial activity by inhibiting biofilm formation and attachment, nucleic acid synthesis, energy metabolism, membrane properties, and function, reducing pathogenicity (xie et al., 2015). additionally, flavonoids further exhibit antibacterial effects by binding to microbial proteins required for basic cellular functions (donadio et al., 2021). the antibacterial effects of the msbe and esbe were measured at 100, 50, 25, 12.5, and 6.25 mg/ml (figure 1-3). both of the extracts demonstrated an antibacterial effect against the test organism with the highest mean zi of 21.3 mm ±1.11 observed against s. aureus at 100 mg/ml by the msbe. a similar result showed the superior effectiveness of the msbe against s. aureus against the esbe (tiwari et al., 2011). the zi difference might be attributed to the solvent polarity which influences the types and concentrations of the phytochemical extracted, thus influencing the antibacterial effects of the extracts (aboshora et al., 2014; gomashe et al., 2014). the antibacterial effects demonstrated by dm might be attributed to growthinhibiting phytochemicals such as flavonoids which were reported to possess antibacterial potentials (sanusi et al., 2022). although in the traditional and folkloric practice the primary solvent used for extraction is water for preparation, extraction with organic solvents exhibits superior antimicrobial effects compared to aqueous extracts (gomashe et al., 2014). the best practice for the extraction of broad-spectrum antimicrobial compounds employs alcohol as an extraction solvent as seen in the present study (gomashe et al., 2014). the effective concentration of the esbe was against the s. typhi as presented in table 1. the nature of the gram-negative bacteria cell wall might contribute to the resistance of the organism to the extract by limiting the penetration of the extract to affect its action due to the thin lipopolysaccharide exterior membrane (biswas et al., 2013). however, the mesh-like peptidoglycan layer of gram-positive bacteria allows for permeability and access to the extract (malanovic & lohner, 2016). the mic describes the minimum concentration of antimicrobial agent needed to inhibit microbial growth though not applicable in clinical practice for the determination of antibiotic dosage for a patient. however, it is applicable in the determination of the effective target antidiabetic with a lower chance of developing resistance (wiegand et al., 2008). the inhibitory effect of methanolic and ethyl acetate extracts dm was observed to be between 3.125mg/ml to 100 mg/ml for s. aureus, s. typhi, and e. coli with s. typhi 286 biology, medicine, & natural product chemistry 12 (1), 2023: 281-288 being the most sensitive. this variation in mbc values might be attributed to the phytoconstituents of the extract which vary due to solvent difference, thus, exhibiting inferior different inhibitory and bactericidal effects to the standard (aboshora et al., 2014; i. abubakar & a. usman, 2016; gomashe et al., 2014; sanusi et al., 2022). similar results to the present study were reported on the phytoconstituents and antibacterial effect of stem bark extracts of dm (sanusi et al., 2022). conclusion the study investigated the phytoconstituents and antibacterial effects of d. microcarpum which exhibited antibacterial effects against e. coli, s. typhi, and s. aureus with notable inhibitory and bactericidal effects. the antibacterial capabilities of d. microcarpum might be due to the phytochemical components of the plants and might be a potential source of a novel antibiotic against antibiotic-resistant bacteria. acknowledgments: the authors extend their appreciation to the department of science laboratory technology, adamawa state polytechnic yola, for institutional support. authors’ contributions: all authors contributed to the design and writing of the research. s.a.p. collected the plant sample and carried out the extraction while d.m.m. carried out the phytochemical analysis. a.a.m. carried out the antibacterial study. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there was no funding 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(2022). potential roles and molecular mechanisms of phytochemicals against cancer. food & function, 13(18), 9208-9225. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 499-506 | doi: 10.14421/biomedich.2025.141.499-506 issn 2540-9328 (online) the potential of hanjuang merah (cordyline fruticose) antibacterial liquid body wash emy rahayu1, lilih rahmanina1, tupaida pasaribu1, maulida2, mirnawati dewi1,* 1faculty of mathematics and sciences; 2faculty of teacher training and education, palangka raya university, indonesia. corresponding author* mirnawatidewi22bio@mipa. upr. ac.id abstract the skin is the outermost layer of tissue that has the function of covering and protecting the surface of the human body. unhygienic skin will trigger skin infections caused by various microorganisms. one of them is bacteria. hanjuang merah (cordyline fruticosa) is a type of plant that is often used as a traditional medicine, especially for the dayak community. however, research on the use of the cordyline fruticosa plant in producing liquid body wash formulations that are efficacious as antibacterial against staphylococcus aureus has not been carried out so far. the purpose of this study is to identify the potential of cordyline fruticosa as a formulation of liquid body wash formulations and to find the effectiveness of cordyline fruticosa in inhibiting infections caused by s. aureus bacteria made in the form of liquid body wash. the method used in the extraction is maceration and the manufacture of liquid body wash according to the formulation that has been made, while the disc diffusion method with concentrations of 5% (f1), 10% (f2), and 15% (f3) is used in antibacterial tests. liquid body wash preparations with the largest inhibitory zone level at a concentration of 15% (f3). the concentration of 15% (f3) is effective in inhibiting the growth of s. aureus bacteria which is characterized by the formation of an inhibition zone around the disk. keywords: antibakteri; hanjuang merah leaves; liquid body wash; staphylococcus aureus. introduction the skin is the outermost layer of tissue that functions to cover and protect the surface of the human body (srisantyorini & cahyaningsih, 2019). skin infections occur when the skin is not maintained in a healthy condition. skin infections are caused by germs, bacteria, and viruses that grow within the skin tissue. therefore, maintaining skin health is necessary to prevent skin diseases (rismanto et al., 2019). efforts to keep the skin clean and healthy include regularly washing the entire body (bathing). soap can be used to remove skin metabolites (such as sebum), dead skin layers, sweat residue, dirt, dust, and microorganisms (mopangga et al., 2021). one type of bacteria that can cause infections is staphylococcus aureus. s. aureus is a bacterium that has garnered attention in the healthcare sector because it is highly pathogenic and can cause serious infections (puspadewi et al., 2017; seran et al., 2020). a characteristic feature of s. aureus infections is necrotic inflammation, which can lead to abscess formation (widiastuti & pramestuti, 2018; ballo et al., 2021). indonesia has more than 20,000 types of medicinal plants spread across the country, with approximately 300 species used for medicinal purposes. one of the traditional medicinal plants commonly used by the indonesian community is cordyline fruticosa. according to the dayak benuak community, the leaves and roots of the cordyline fruticosa are widely used as remedies for dysentery, menstruation issues, and as treatment for burns (andani et al., 2022). the antibacterial activity of cordyline fruticosa extract is more effective in inhibiting s. aureus, which is a gram-positive bacterium. liquid body wash is a pharmaceutical preparation commonly used to treat infected skin. currently, many liquid body wash use chemicals such as triclocarban. triclocarban is one of the most commonly used antibacterial agents in liquid soaps. however, the united states food and drug administration (fda) states that prolonged use can cause bacteria to develop antibiotic resistance (fernanda et al., 2022). based on the aforementioned statements, much literatures has reviewed the use of natural ingredients in the formulation of liquid body wash preparations that have antibacterial properties. however, research on the use of the cordyline fruticosa plant in creating liquid body wash formulations that have antibacterial properties against s. aureus has not yet been conducted. this study aims to identify the potential of andong merah (cordyline fruticosa) as a formulation ingredient for liquid body wash preparations and to determine the effectiveness of cordyline fruticosa leaves in inhibiting manuscript received: 22 january, 2025. revision accepted: 22 july, 2025. published: 31 july, 2025. https://doi.org/10.14421/biomedich.2025.141.499-506 mailto:mirnawatidewibio22@ukwms.ac.id 500 biology, medicine, & natural product chemistry 14 (1), 2025: 499-506 infections caused by staphylococcus aureus bacteria when made into a liquid body wash. materials and methods methods this type of research is experimental by formulating a liquid body wash from the ethanol extract of hanjuang merah leaves at the peatland science and technology development and innovation center (pstdic) laboratory, university of palangka raya, central kalimantan. the extraction method used is maceration to produce a thick extract that will be made into three types of liquid body wash formulations. testing of the liquid body wash formulations includes organoleptic tests, homogeneity tests, ph tests, foam height observation tests, and free alkali tests, as well as testing against s. aureus bacteria. materials the research tools used include a blender, measuring cups, beaker glass, erlenmeyer flask, petri dish, analytical balance, label paper, rotary evaporator, water bath, filter paper, spirit lamp, autoclave, soap bottles, handscoon, hot plate, inoculation loop, gauze, cotton, sieve, stirring rod, black plastic bag, funnel, tweezers, disk paper, maceration container, bunsen burner, spatula, magnetic stirrer (2 cm and 3 cm), adhesive tape, dropper pipette, calipers, ph meter, scissors, aluminum foil, and plastic wrapping roll. the research materials used include hanjuang merah leaves or andong merah (cordyline fruticosa), 70% ethanol, distilled water, staphylococcus aureus bacterial isolates, coconut oil (vco), glycerin, nutrient agar (na) media, 40% koh, sodium lauryl sulfate (sls), na-cmc, stearic acid, phenoxyethanol, and petrichor manura oil aromatherapy (fragrance), phenolphthalein, and 0.1 n hcl. research procedure collection, sample preparation, and maceration of hanjuang merah leaf or andong merah (cordyline fruticosa) the samples used in this research are hanjuang merah leaves collected from the surrounding areas of palangka raya, central kalimantan. the powdered leaves (simplicia) of hanjuang merah are weighed to 100 grams using 1000 ml of 70% ethanol solvent. the thick extract will be placed in a water bath for 1-3 days (fernanda et al., 2022). preparation and formulation of liquid body wash with hanjuang merah leaf extract table 1. formulation of liquid body wash preparations. ingredients formulation preparation blank (-) f1 5% f2 10% f3 15% ethanol leaf extract hanjuang merah leaf 0 5 10 15 coconut oil (voc) 15 ml 15 ml 15 ml 15 ml koh 40% 8 ml 8 ml 8 ml 8 ml stearic acid 0,5 g 0,5 g 0,5 g 0,5 g na-cmc 1 g 1 g 1 g 1 g sodium lauryl sulfat (sls) 1 g 1 g 1 g 1 g petrichor manura oil aromatherapy 1 ml 1 ml 1 ml 1 ml phenoxyethanol 0,5 g 0,5 g 0,5 g 0,5 g distilled water 100 ml 100 ml 100 ml 100 ml all the materials to be used are calculated and weighed according to the formulation. coconut oil (vco) amounting to 15 ml is added to a beaker glass, followed by the gradual addition of 9 ml of glycerin and 8 ml of 40% koh, and the mixture is heated with a hotplate at a temperature of 70°c until it forms a paste (mixture i). then, na-cmc is added until homogeneous. next, stearic acid is added to a beaker glass containing the homogeneous na-cmc (mixture ii). mixture i is added to mixture ii and homogenized using a magnetic stirrer. then, sodium lauryl sulfate (sls) is dissolved in 10 ml of hot distilled water. once dissolved, it is mixed with the liquid body wash and stirred until homogeneous. next, phenoxyethanol is added to the soap formulation and stirred until homogeneous. after that, petrichor manura oil aromatherapy is added, which serves as the soap's fragrance. the final step is the addition of 100 ml of distilled water. the preparation of liquid body wash formulations is carried out at each concentration of ethanol extract form hanjuang merah leaf. evaluation of the liquid body wash formulation with hanjuang merah leaf extract or andong merah (cordyline fruticosa) ▪ organoleptic test a liquid body wash that meets the standard is characterized by having a homogeneous liquid form, rahayu et al. – the potential of hanjuang merah (cordyline fruticose) … 501 with a distinctive color and odor (national standardization agency, 1996). ▪ homogeneity test a formulation preparation can be considered good if there are no other particles clumping together (homogeneous) (pratama et al., 2018). ▪ ph determination test liquid body wash generally has a ph ranging from 8 to 11. however, specific ph values can be determined based on the characteristics and needs of certain users, such as those with sensitive skin or specific skin problems (marhaba et al., 2021). ▪ foam height observation test the good foam stability range has a height of more than 9.5 cm (riyanta & nurniswati, 2016). the appropriate foam height requirement according to sni ranges from 13-220 mm (national standardization agency, 1996). ▪ free alkali test this test is conducted by weighing 5 grams of the liquid body wash and placing it in a 250 ml beaker glass, then adding 100 ml of 96% alcohol and 1 ml of indicator solution (phenolphthalein). the mixture is then heated on a hot plate for 30 minutes until boiling. the solution will turn purple and will be titrated with 0.1 n hcl solution until the purple color disappears (korompis et al., 2020). antibacterial activity test of staphylococcus aureus against hanjuang merah leaf liquid body wash the effectiveness test of the liquid body wash uses s. aureus bacteria. three petri dishes are prepared, and about 15 ml of nutrient agar (na) medium is poured into each petri dish. it is allowed to solidify. then, the bacterial preparation is taken and spread on the nutrient agar (na) medium. then, the disks are soaked in the liquid body wash preparation of hanjuang merah leaf extract or andong merah (cordyline fruticosa) with variations of 5%, 10%, 15%, and a positive control for comparison using commercial liquid body wash. it is incubated for 24 hours at 37°c, and the average diameter of the inhibition zone is measured for each concentration. results and discussion the formulated liquid body wash preparations obtained four types: liquid body wash without extract (blank), liquid body wash with 5% extract addition (f1), liquid body wash with 10% extract addition (f2), and liquid body wash with 15% extract addition (f3). each treatment evaluated the liquid body wash formulation based on the sni quality standard (06-4085-1996), including organoleptic tests, homogeneity tests, ph tests, foam height observation tests, and free alkali tests. additionally, further testing was carried out against staphylococcus aureus bacteria. figure 1. liquid body wash preparations. body wash is a product derived from sodium compounds and fatty acids used as a body cleanser, which produces foam and does not cause skin irritation. body wash can be divided into two types based on its form: solid soap and liquid soap. liquid soap effectively removes water-soluble and fat-soluble dirt on the skin surface, eliminates unpleasant odors, and provides a pleasant fragrance (zagita et al., 2021). liquid soap is produced through a series of saponification reactions of fats and oils with koh (irmayanti et al., 2014; ariyani & hidayati, 2018). the process of mixing vco oil, glycerin, and koh is first carried out at 70°c, resulting in a soap base in paste form. the saponification reaction can proceed well at a temperature of 60-70°c; if the saponification process exceeds the specified temperature, it will cause the preparation to foam and overflow. in contrast, if the temperature is lower than the specified temperature, it will result in a non-homogeneous preparation (sari & ferdinan, 2017). organoleptic test according to the sni (06-4085-1996) as follows. table 2. organoleptic test results sni (06-4085-1996). organoleptic test f0 (blank) liquid body wash of red ti leaf extract standard test f1 5% f2 10% f3 15% sni (06-4085-1996) texture/form liquid liquid semi liquid thick color white cream kuning vanila yellow odor typical typical typical typical 502 biology, medicine, & natural product chemistry 14 (1), 2025: 499-506 the organoleptic test aims to determine the characteristics of the liquid body wash formulation, including shape, color, and odor. based on the results in table 2, the preparations made have distinctive characteristics in each treatment. according to the sni quality standard (06-4085-1996), the quality standard for organoleptic testing of liquid body wash includes having a liquid form, distinctive odor, and color. based on panelist preference as follows. information: f0 (blanko) = without extract; f1 = addition of extract 5 %; f2= addition of extract 10 %; f3= addition of extract 15%. description: 1. very dislike; 2. dislike; 3. neutral; 4. like; 5. very like. figure 1. panelist organoleptic tests. based on the assessment results on the organoleptic testing of each liquid body wash preparation includes an assessment of shape, color, and odor is included. the highest score indicates the panelists' preference for each liquid body wash preparation. the panelists showed that the most preferred shape and odor were f3 with 15% extract addition, while the most preferred color was f0 (blank). homogeneity test table 3. homogeneity test results homogeneity test f0 (blanko) liquid body wash red ti leaf extract test standard f1 5% f2 10% f3 15% sni (06-4085-1996) homogenous homogenous homogenous homogenous figure 2. homogeneity testing. the purpose of the homogeneity test is to determine whether the soap ingredients used are evenly mixed. in the preparations made, the blank preparation, f1 (5%), f2 (10%), and f3 (15%) all showed that each formulation was homogeneous. this can be confirmed by research conducted by panaungi & hasma (2022), which states that a liquid body wash formulation should show a homogeneous structure, indicated by the absence of coarse granules on a glass slide. rahayu et al. – the potential of hanjuang merah (cordyline fruticose) … 503 ph level test table 4. ph level test results. ph level test (ph) f0 (blank) liquid body wash red ti leaf extract test standard f1 5% f2 10% f3 15% sni (06-4085-1996) 8, 77 8,34 8,11 8,41 figure 3. ph level testing. the ph test aims to determine whether the liquid body wash preparation is acidic, basic, or neutral. the ph measurement was carried out using a calibrated ph meter. the ph test results showed variations at each concentration (table 4). the results indicate that the ph of the liquid body wash ranges from 8 to 8.41 (<11). based on each liquid body wash preparation, all formulations meet the sni (06-4085-1996) quality standards. ph testing is crucial to ensure that the liquid body wash does not cause skin irritation. according to sari & ferdinan (2017), liquid body wash products generally have a basic ph due to the saponification process between koh and vco coconut oil. a liquid body wash with a ph that is too low (acidic) can increase the absorption capacity of the skin, leading to skin irritation. foam height observation test table 5. foam height observation test results. foam height observation test f0 (blank) liquid body wash red ti leaf extract test standard f1 5% f2 10% f3 15% sni (06-4085-1996) 5 cm 7,5 cm 7 cm 7,5 cm figure 4. foam height observation test. the foam height test aims to determine the amount of foam produced by each preparation. the foam height range obtained is 5-7.5 cm. according to sni 06-40851996, the foam height for liquid body wash preparations ranges from 1.3 to 22 cm, indicating that the preparations made meet the quality standards. according to usman & baharuddin (2023), foam with a stable structure is more effective at removing dirt from the skin. however, excessive use of foam stabilizers can cause skin irritation. foam height is influenced by several factors, including foam stabilizers, surfactants, and other components of the liquid body wash. additionally, foam height can be affected by the addition of sodium lauryl sulfate (sls), which serves as an additive to produce optimal foam. however, the addition of sls should not exceed 1% of the formulation (pareda et al., 2020). 504 biology, medicine, & natural product chemistry 14 (1), 2025: 499-506 free alkali test table 6. free alkali test results. free alkali test f0 (blank) liquid body wash red ti leaf extract test standard f1 5% f2 10% f3 15% sni (06-4085-1996) 0,082% 0,083% 0,093% 0,054% figure 4. free alkali test (personal documentation, 2024) the free alkali test aims to determine the amount of unbound alkali with vco oil, which will affect the quality of the liquid body wash preparation (ariyani & hidayati, 2018). based on the results obtained, the free alkali values in the soap preparations range from 0.054% to 0.093% (table 6). these values do not exceed the applicable quality standard of a maximum of 0.14%. according to pareda et al. (2020), the free alkali test is one of the requirements for a liquid body wash formulation to be safe for use. the low free alkali content in the liquid body wash preparation is due to the long heating and mixing time of the liquid body wash ingredients, allowing koh to react optimally with vco coconut oil to form a paste (soap base) (korompis et al., 2020). test of cordyline fruticosa extract figure 5. test of cordyline fruticosa leaf extract note: 5% extract (f1), 10% extract (f2), 15% extract (f3). 7,6 7,7 0 7 5,1 7,18 8,7 8,9 0 5 10 i ii ii i replikasi d ia m e te r o f in h ib it io n z o n e effectiveness of the inhibition zone of red ti extract (cordyline fruticosa) 5% (f1) 10% (f2) 15% (f3) graph 2. effectiveness of red ti leaf extract (cordyline fruticosa) against staphylococcus aureus based on the tests conducted across three replications, the leaf extract has the potential to inhibit bacterial growth with the largest inhibition zone diameter ranging from 8 to 8.9 mm at 15% treatment (f3) (graph 2). statistical testing using the one-way anova method shows a significance value of 0.343 > 0.05, indicating that red ti leaf extract is effective in inhibiting the growth of staphylococcus aureus bacteria. red ti leaves contain secondary metabolite compounds such as flavonoids, alkaloids, tannins, and saponins, which have proven antibacterial properties (nurhayati et al., 2018). according to indiyen et al. (2020), the antibacterial rahayu et al. – the potential of hanjuang merah (cordyline fruticose) … 505 activity of red ti leaf extract (cordyline fruticosa) is more effective in inhibiting gram-positive bacteria like staphylococcus aureus. activity test and preparation of liquid body wash with cordyline fruticosa leaf extract figure 6. testing liquid body wash preparation. note: positive control (+), blank (f0), 5% extract addition (f1), 10% extract addition (f2), 15% extract addition (f3). graph 3. effectiveness of liquid body wash against staphylococcus aureus based on the tests conducted across 3 replications, the preparation with the addition of cordyline fruticosa leaf extract has the potential to inhibit bacterial growth with the largest inhibition zone diameter ranging from 20 to 25 mm at 15% treatment (f3) (graph 3). statistical testing using the one-way anova method shows a significance value of 0.183 > 0.05, indicating that the liquid body wash is effective in inhibiting the growth of staphylococcus aureus bacteria. cordyline fruticosa leaves contain secondary metabolite compounds responsible for inhibiting bacterial growth, including alkaloids and flavonoids. alkaloids inhibit the synthesis of peptidoglycan in the cell, leading to bacterial cell death, while flavonoids form complexes with extracellular proteins, causing membrane leakage due to phospholipids' inability to maintain bacterial cell membrane shape (ayuchecaria et al., 2024). results and discussion should be written as a series of connecting sentences, however, for manuscript with long discussion should be divided into subtitles. results should be clear and concise. conclusions the optimal concentration for the thick extract test is 15% (f3) with the largest inhibition zone diameter ranging from 8 to 8.9 mm, while the optimal concentration for the liquid body wash preparation is 15% (f3) with the largest inhibition zone diameter ranging from 20 to 25 mm. this inhibition zone diameter is categorized as strong in inhibiting s. aureus bacterial growth. funding: we extend our thanks to the ministry of education, culture, research, and technology for funding this research. authors’ contributions: mirnawati dewi & emy rahayu designed the study. emy rahayu, lilih rahmanina, tupaida pasaribu, maulida, carried out the laboratory work. emy rahayu, lilih rahmanina analyzed the data. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. acknowledgement: thanks also to palangka raya university for providing the facilities and infrastructure necessary for the successful completion of this research. 14,4 14,5 12,8 19 20,9 23,2 17 0 21,9 24,4 0 16,4 13,3 10,4 25,1 0 5 10 15 20 25 30 kontrol positif (+) blanko (f0) 5% (f1) 10% (f2) 15% (f3) d ia m et er o f in h ib it io n z o n e effectiveness of liquid body wash against staphylococcus aureus replikasi i replikasi ii replikasi iii 506 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content consisting of flavonoids, terpenoids, alkaloids, and phenols. this study evaluates the antimicrobial potential of sea*grape macroalgae extract (caulerpa sp.) obtained from pasaran island, lampung. the diffusion assay method assessed the antimicrobial activity of caulerpa sp. extract against staphylococcus aureus. caulerpa sp. samples were extracted using the maceration extraction method and evaporated using two methods: water bath and rotary evaporator. the results showed that caulerpa sp. extract had antimicrobial activity at a concentration of 10,000 ppm, which was resistant to staphylococcus aureus. these findings suggest that caulerpa sp. from the waters of pasaran island has the potential to be a source of natural antimicrobial and anti-inflammatory components if the concentration of the extract is more significant, which can be further developed for applications in the health and pharmaceutical fields. keywords: macroalgae; inflammation; seagrapes; biological agents. introduction seagrape (caulerpa sp.) is a type of green seaweed that lives in tropical to subtropical areas and has high potential. this plant contains high fiber, vitamins, and minerals and is a readily available source of natural antioxidants (ridhowati & asnani, 2016). macroalgae belong to the chlorophyceae class. what distinguishes caulerpa sp. from other macroalgae is its ability to be sold fresh without having to be dried first, and it can even be eaten directly as fresh vegetables (tapotubun et al., 2020). caulerpa sp. is also one of the macroalgae that has received attention for cultivation because it contains chlorophyll a and b, carotene, xanthophyll, and lutein (darmawati, et al., 2016). in indonesian waters, there are several types of caulerpa found, including c. racemosa, c. sertularoides, c. taxifolia, c. serrulata, c. lentillifera, c. peltata, and c. cupressiodes (utami et al., 2021). various regional names, such as latoh in java and lawi-lawi in sulawesi know seagrapes (caulerpa sp.) in indonesia. bulung boni (bali) are widely distributed in marine waters and can live in tidal locations (ines et al., 2020). seagrapes (caulerpa sp.) can be consumed directly as vegetables and traditional food in bali, this habit has been passed down for generations because seagrapes are believed to have properties as healthy food for diet (julyasih & wirawan, 2017). seagrapes (caulerpa sp.) contain bioactive compounds that function as antibacterial, antifungal, anti-inflammatory, antidiabetic, and antioxidant (siagian et al., 2018; saputri et al., 2019). these bioactive compounds include flavonoids, saponins, and triterpenoids, which are known to have the ability to be antibacterial compounds (rusli et al., 2016; nome et al., 2019). caulerpa sp. has the potential to be used as an anticoagulant for people with circulatory diseases and thrombotic disorders and hematological analysis (arenajo et al., 2018). according to waji and sugrani (2009), the compounds contained in caulerpa sp. also have various important roles, including protecting the integrity of cell structures, increasing the potency of vitamin c, inhibiting inflammatory processes, maintaining bone density, and having antibiotic properties. inflammation is the body's response to injury or tissue damage as a protective measure. this process aims to destroy or reduce the causative agent of injury or damaged tissue (latief et al., 2019; wang et al., 2016). one of the chemical contents that has properties as an anti-inflammatory is flavonoid. flavonoids can inhibit manuscript received: 22 december, 2024. revision accepted: 19 february, 2025. published: 20 february, 2025. https://doi.org/10.14421/biomedich.2025.141.17-24 18 biology, medicine, & natural product chemistry 14 (1), 2025: 17-24 cyclooxygenase or lipooxygenase enzymes and prevent the accumulation of leukocytes in the affected area, thus acting as an anti-inflammatory (narande et al., 2013; agustina et al., 2015). caulerpa sp. has potential as an anti-inflammatory because it contains flavonoid bioactive compounds. the diversity of bioactive compounds from seagrapes (caulerpa sp.) has great potential as an antibacterial. antibacterial compounds effectively control the growth of bacteria, especially bacteria that are harmful to humans. staphylococcus aureus is commonly used as a target bacterium in antibacterial research. staphylococcus aureus bacteria are pathogenic bacteria that cause disease. therefore, it is necessary to extract natural ingredients that can be used as antibacterials to inhibit and kill these pathogenic bacteria (marfuah et al., 2018). based on the description, this study aims to identify the optimal concentration of caulerpa sp. extract with the best antimicrobial activity and evaluate its relationship with anti-inflammatory potential. this study is expected to provide a comprehensive understanding of the dual potential of caulerpa sp. as an antimicrobial and anti-inflammatory agent for developing pharmacological natural materials. materials and methods materials and equipment this research was conducted from october to december 2024 at the oceanography laboratory of the marine science study program, department of fisheries and marine sciences, faculty of agriculture, university of lampung. the primary material used in this research was caulerpa sp. macroalgae obtained from the pasaran island area, teluk betung, bandar lampung, indonesia. other materials used were technical methanol, disc paper, distilled water, tryptone soy agar (himedia), tryptone soya broth (himedia), and a staphylococcus aureus bacterial culture (actt 6538). the equipment used in this study are masks, latex examination gloves, blenders (miyako), glass bottles with a capacity of 460 ml, vial bottles, funnels, analytical scales (sojilab by sojikyo), measuring cups, filter paper (whatman), erlenmeyer (borosil), petri dish (citotest), autoclave, tweezers, bunsen, magnetic stirrer, micropipette (dlab), hot plate (dlab), stirring rod, laminar air flow, test tube rack, test tube, refrigerator, micropipette, vernier calliper, water bath (stuart), rotary evaporator (heidolph laborota 4000 efficient), and aluminium foil. method sterilization of tools the process includes preparing materials, proper packaging, and placement in an autoclave. the autoclave is filled with distilled water and set at 121°c and 15 psi pressure. sterilization lasted for 15-20 minutes. after completion, the autoclave was turned off and allowed to stand until the pressure dropped and the temperature reached 80°c before opening. extraction procedure sample preparation begins with the drying process, followed by making caulerpa sp. macroalgae powder. after becoming a powder, extraction is carried out by the maceration method by putting the powder into two glass bottles, each as much as 50 grams. then, 200 ml of technical methanol solvent was added, and the bottle was covered with aluminium foil. the sample was allowed to stand for 7 days. rotary evaporator samples that had been allowed to stand for 7 days were transferred into a collection flask, which was then attached to a rotary evaporator. in the water bath, the temperature was set at 70°c, and the rotation speed was 100 rpm. slowly lower the flask into the water bath until submerged, adjusting the vacuum pressure so the sample is not mixed with condensed or contaminated solvent. when the moisture content of the sample is exhausted, turn off the machine and take the sample in the collection flask, then transfer it to the vial bottle. nurjanah et al. – evaluation of antimicrobial potential using disc diffusion assay … 19 figure 1. map of sampling locations. water bath next, perform the water bath process, fill the water bath with enough water, and then turn it on. after reaching 70°c, put the sample bottle into the water bath. to prevent precipitation, stir the sample once every 30 minutes for 24 hours. if there is shrinkage in the sample, separate the pulp and liquid using a sieve. preparation of tsa and tsb media preparation of tsa (tryptone soya agar) and tsb (tryptone soya broth) begins with dissolving 4 grams of tsa powder with 100 ml of distilled water and 1.5 grams of tsb with 50 ml of distilled water. tsa was heated on a hotplate for 15 minutes, while tsb was homogenized. both media were sterilized in an autoclave (121°c, 15 minutes). sterilized tsa was poured into petri dishes to solidify, while tsb was poured into test tubes. tsa was used for the activity test, and tsb was used for staphylococcus aureus culture. staphylococcus aureus bacterial culture staphylococcus aureus bacteria were cultured on tryptone soya broth (tsb) media. the reculturing process was done by inoculating 0.1 ml of pure isolate of staphylococcus aureus into tsb media using a sterilized micropipette. the bacterial culture was then incubated in an incubator for 24 hours at an optimal temperature of 37°c to support the growth and multiplication of bacterial cells. disc-diffusion assay antibacterial testing was carried out using tsa (tryptone soya agar) media with the disc diffusion method. caulerpa sp. samples as much as 0.05 grams were dissolved in 5 ml of distilled water. the sample solution was then diluted to make test concentrations of 10,000, 5,000, 1,000, 100, and 10 ppm. the test bacterial isolate was staphylococcus aureus, grown on tsa media. paper dishes that have been soaked in the test solution are placed on the media containing bacteria. after that, the media was placed in an incubator. observations were made every 24 hours for 3 days. minimum inhibitory zone observation the zone of inhibition was measured every 24 hours using a caliper. measurements were taken horizontally, vertically, and diagonally. results and discussion sample properties the waters of pasaran island became the sampling location for green macroalgae, especially caulerpa sp. table 1 shows the number of samples taken. table 1. sample weight (caulerpa sp.). wet weight dry weight powder weight weight of extract 4 kg 200 gram 100 gram 2,3 g (rotary) 100 ml (water bath) the sample of caulerpa sp. can be shown in figure 2. profile inhibition zone the antibacterial activity test revealed that the seagrape (caulerpa sp.) extract showed an inhibitory effect on the growth of staphylococcus aureus; however, the inhibition zone formed was relatively small in size. 20 biology, medicine, & natural product chemistry 14 (1), 2025: 17-24 observation of the diameter of the inhibition zone was carried out for three consecutive days at 24-hour intervals, and the detailed measurement results are presented in table 2 to table 7. figure 2. a: fresh caulerpa sp. sample, b: powdered caulerpa sp. sample, c: caulerpa sp. sample extract results rotary evaporator method, d: caulerpa sp. sample extract results water bath method. table 2. 24-hour observation of inhibition zone from the results extract in a rotary evaporator. c h v d average 10000 1,2 1,2 1,1 1,17 5000 1 1 1 1,00 1000 1 1 1 1,00 100 1 1 1 1,00 10 1,4 1,7 1,5 1,53 description: (c): concentration; (h): horizontal; (v): vertical; (d): diagonal. table 3. 48-hour observation of inhibition zone from the results extract in a rotary evaporator. c h v d average 10000 1,4 1,5 1,4 1,43 5000 0,8 0,8 0,8 0,80 1000 0,5 0,5 1 0,67 100 0,2 0,5 0,5 0,40 10 1 1,4 1 1,13 description: (c): concentration; (h): horizontal; (v): vertical; (d): diagonal. table 4. 72-hour observation of inhibition zone from the results extract in a rotary evaporator. c h v d average 10000 1,1 1,6 1 1,23 5000 0,7 1,2 0,6 0,83 1000 0,7 0,8 1 0,83 100 0,2 1,3 0,2 0,57 10 0,6 1,2 0,8 0,87 description: (c): concentration; (h): horizontal; (v): vertical; (d): diagonal. 1,17 1 1 1 1,53 1,43 0,8 0,67 0,4 1,13 1,23 0,83 0,83 0,57 0,87 0 0,5 1 1,5 2 10000 5000 1000 100 10 average zone of inhibition d1 d2 d3 figure 3. average diameter of zone of inhibition by rotary evaporator method. description: d1: day one observation, d2: day two observation, d3: day three observation. table 5. 24-hour observation of inhibition zone from the results extract in a water bath. c h v d average 10000 1,4 1,3 1,4 1,37 5000 1,2 1,2 1,2 1,20 1000 1,1 1,1 1,1 1,10 100 1,2 1,2 1,1 1,17 10 1,1 1,4 1,4 1,30 description: (c): concentration; (h): horizontal; (v): vertical; (d): diagonal. table 6. 48-hour observation of inhibition zone from the results extract in a water bath. c h v d average 10000 1 1,6 0,9 1,17 5000 0,7 0,8 0,7 0,73 1000 1,3 1,3 0,7 1,10 100 1 1 0,3 0,77 10 1 0,6 1 0,87 description: (c): concentration; (h): horizontal; (v): vertical; (d): diagonal. a b c d nurjanah et al. – evaluation of antimicrobial potential using disc diffusion assay … 21 table 7. 72-hour observation of inhibition zone from the results extract in a water bath. k h v d average 10000 0,6 2,1 1,3 1,33 5000 1 0,8 0,7 0,83 1000 0,5 0,5 0,9 0,63 100 1 0,7 1 0,90 10 0,8 1,3 1,1 1,07 description: (c): concentration; (h): horizontal; (v): vertical; (d): diagonal. 1,37 1,2 1,1 1,17 1,3 1,17 0,73 1,1 0,77 0,87 1,33 0,83 0,63 0,9 [] 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 10000 5000 1000 100 10 average zone of inhibition d1 d2 d3 figure 4. average diameter of inhibition zone of water bath method. description: d1: day one observation, d2: day two observation, d3: day three observation. discussion table 1 shows the weight change of caulerpa sp. samples from wet to extracted. there is a significant weight loss from the wet sample (4 kg) to powder (100 grams). after the extraction process, the sample weight was further reduced to 2.3 grams for the rotary evaporator method and 100 ml for the water bath method. the observation results of seagrape extract (caulerpa sp.) obtained through the rotary evaporator method are presented in tables 2, 3, and 4. on the first day of observation, the average inhibition zone measured at extract concentrations of 10,000, 5,000, 1,000, 100, and 10 respectively showed results of 1.17 mm, 1.00 mm, 1.00 mm, 1.00 mm, and 1.53 mm. observations on the second day in order, from the highest to the lowest concentration of 10,000, 5,000, 1,000, 100, and 10, the average zone of inhibition measured was 1.43 mm, 0.80 mm, 0.67 mm, 0.40 mm, and 1.13 mm. on the third day, the average zone of inhibition measured sequentially at concentrations of 10,000, 5,000, 1,000, 100, and 10 showed results of 1.23 mm, 0.83 mm, 0.83 mm, 0.57 mm, and 1.87 mm. the observation results of seagrape (caulerpa sp.) extract obtained through the water bath method are presented in tables 5, 6, and 7. on the first day of observation, the average zone of inhibition varied according to the concentration of the extract. concentrations of 10,000, 5,000, 1,000, 100, and 10 showed an average inhibition zone of 1.37 mm, 1.20 mm, 1.10 mm, 1.17 mm, and 1.30 mm, respectively. observations on the second day showed changes in the average zone of inhibition for each concentration. sequentially, from the highest to the lowest concentrations of 10,000, 5,000, 1,000, 100, and 10, the average inhibition zones measured were 1.17 mm, 0.73 mm, 1.10 mm, 0.77 mm, and 0.87 mm. on the third day, the average zone of inhibition sequentially at concentrations of 10,000, 5,000, 1,000, 100, and 10 showed results of 1.33 mm, 0.83 mm, 0.63 mm, 0.90 mm, and 1.07 mm. research conducted by rusli et al. (2016) revealed that caulerpa sp. is rich in various bioactive compounds, including flavonoids, terpenoids, alkaloids, and phenols. according to lahay & amiin (2023), flavonoids have a broad spectrum of health benefits. these compounds play a role in protecting the integrity of cell structures, increasing the potency of vitamin c, inhibiting inflammatory processes, maintaining bone density, and having antibiotic properties. staphylococcus aureus is a pathogenic microorganism capable of inducing various pathological conditions (amiin & lahay, 2023). infections caused by this bacterium are characterized by specific characteristics, including inflammation, tissue death (necrosis), and abscess formation. symptoms range from mild skin lesions such as furuncles to infections (subekti et al., 2019). figure 5. staphylococcus aureus in the microscope in the study of the inhibition zone test of caulerpa sp. extract against staphylococcus aureus bacteria using the disc diffusion assay method. the results of the inhibition zone measurement were compared with the criteria set by lewis et al. (2023), as listed in table 8. the results showed the level of resistance to caulerpa sp. extract at the largest concentration of 10,000 ppm. the water bath method produced an average diameter of the inhibition 22 biology, medicine, & natural product chemistry 14 (1), 2025: 17-24 zone on the first, second, and third days of 1.37 mm, 1.17 mm, and 1.33 mm, respectively. meanwhile, the rotary evaporator method produced an average inhibition zone diameter of 1.17 mm, 1.42 mm, and 1.23 mm. based on these results, it can be concluded that the metabolite compounds contained in seagrapes (caulerpa sp.) with a concentration of 10,000 ppm tend to have less inhibitory effect on staphylococcus aureus bacteria. resistant materials are materials that have the ability of bacteria to neutralize and weaken the performance of antibiotics (fernandez, 2013). the nature and ability of resistant materials that can neutralize antibiotics is called the nature of resistance. exposure to antibiotics is the initial stage of resistance (frieri et al., 2017). the problem faced by the world today is that many microorganisms are resistant to several antiinflammatory agents (diaz-granadoz et al., 2008). the increasing number of bacterial resistance in all regions of the world is a major concern of the world health organization (who), to deal with this situation who issued the global strategy for containment of antimicrobial resistance, a document intended for policymakers to urge governments to take action and various efforts to prevent the occurrence of antibiotic resistance (fernandez, 2013). table 8. standardized zone of inhibition. inhibition zone diameter (mm) sensitive intermediet resistent ≥ 21 16-20  15 (lewis et al., 2023) the inhibition zone produced tends to show an increase along with the increase in concentration from 100 to 10,000 ppm; however, at a concentration of 10 ppm, the inhibition zone produced is greater than the concentration of 100 ppm. according to ariyanti et al. (2012), the diameter of the inhibition zone is influenced by various factors, including the rate of diffusion, characteristics of the agar medium, number of organisms inoculated, bacterial growth rate, chemical concentration, and incubation conditions. furthermore, elifah (2010) observed that an increase in antibacterial concentration is not always directly proportional to an increase in inhibition zone diameter. this phenomenon may be caused by variations in the diffusion speed of antibacterial compounds on agar media, as well as differences in the types and concentrations of antibacterial compounds that can produce inhibition zones with various diameters. based on this statement, it can be concluded that the larger inhibition zone at a concentration of 10 ppm compared to 100 ppm and 1000 ppm is not always directly related to the concentration. instead, it may be more influenced by the speed of diffusion of bioactive compounds from the caulerpa sp. sample itself in the agar medium. based on the mean values shown in graphs 1 and 2, a comparison of the results of the methods used illustrates the varying inhibition effectiveness depending on the concentration (achmadi et al., 2021). although the differences between the methods are not very significant, there is a general trend of increasing inhibition zones as the concentration increases. this variation in effectiveness is evident at the various concentration levels tested, suggesting that both the concentration and the method used affect the inhibition results. seagrape (caulerpa sp.) has flavonoid antioxidant compounds that can be anti-inflammatory (palaniyappan et al., 2023). a fractionation (purification) process was carried out to obtain seagrape (caulerpa sp.) extract.) this process produces metabolite compounds that can be used as antiviral, antihelmintic, antibacterial, antifungal, antioxidant, anticancer, and anti-inflammatory (pérez et al., 2016). caulerpa sp. contains metabolite compounds as caulerpin; this compound makes caulerpa sp. act as an anti-inflammatory agent (palaniyappan et al., 2023). if more significant anti-inflammatory effects can be observed at higher concentrations of caulerpa sp. extract than those used in the current assay, then it is likely that caulerpa sp. has the potential to be developed as an antiinflammatory agent. however, further studies are needed to confirm its effectiveness and safety at various concentration levels. nurjanah et al. – evaluation of antimicrobial potential using disc diffusion assay … 23 figure 6. observation days 1-3 a1: first-day rotary evaporator observation, b1: second-day rotary evaporator observation, c1: third-day rotary evaporator observation. a2: first-day water bath observation, b2: second day water bath observation, c2: third day water bath observation. conclusion the results showed that in caulerpa sp. extract in the water bath method, the average value of the largest inhibition zone reached 1.37 mm at a concentration of 10,000 ppm in 24-hour observation, while the smallest value was 0.63 mm at a concentration of 1,000 ppm in 72-hour observation. the rotary evaporator method produced the largest average inhibition zone value of 1.43 mm at a concentration of 10,000 ppm in 24 hours of observation, with the smallest value of 0.40 mm at a concentration of 100 ppm in 48 hours of observation. based on the inhibition zone sensitivity standard, this study indicates that caulerpa sp. extract is classified as resistant in the antibacterial activity test against staphylococcus aureus. the complexity of the interaction between the extract, bacteria, and test environment emphasizes the importance of comprehensively interpreting the results. although caulerpa sp. has antiinflammatory and antibacterial potential, its effectiveness at the tested concentrations is limited. further studies with higher concentrations are needed to evaluate the potential of this extract as an alternative to antibiotics in the face of global resistance. acknowledgments: the authors would like to express their sincere gratitude to the staff of the oceanography laboratory, university of lampung, for their assistance during the research. author's contributions: nurjanah & kurniawan carried out the laboratory work. giani, prasetyo, & pramesti wrote the manuscript. rahmando, syafitri, & umam analyze the data. putri designed the study. amiin supervised the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors state that there are no competing interests. references achmadi i, subekti s, ardiyanti hb, amiin mk, haryanto lnf, akbar rek and yudarana ma (2021) molecular identification and prevalence of ectoparasite worms in barramundi (lates calcarifer) in lampung waters. iop conf. ser.: earth environ. sci. 679 012058. https://doi.org/10.1088/17551315/679/1/012058 agustina, ri., d. t. indrawati, & m. a.masruhin. 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(2016). anti-inflammatory effects, nuclear magnetic resonance identification, and high-performance liquid chromatography isolation of the total flavonoids from artemisia frigida. journal of food and drug analysis, 24(2): 385–391. doi.org/10.1016/j.jfda.2015.11.004. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 2, 2017 | pages: 63-68 | doi: 10.14421/biomedich.2017.62.63-68 issn 2540-9328 (online) on designing interactive online atlas of reptile anatomy (mabouya multifacsiata) muhammad jafar luthfi1, riyanto2 1biological education department, 2integrated laboratory; faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto 55281 yogyakarta, indonesia author correspondency: jafarluthfi@yahoo.com1; justofeel@gmail.com2 abstract this research is an integration between fields of biology, photography, design, and informatics engineering. the study aimed to build an interactive online atlas of reptile anatomy to improve the accessibility and data sharing (free access) of reptile anatomy. website was developed using sdlc (system development life cycle) which consist of five steps as follows: website’s strategic planning, determine the scope of website, website’s requirements analysis, design and implementations of website, and testing. based on the results of testing and system implementation, it can be concluded that online interactive atlas (atlasanatomy.org) had been successfully built as anatomical educational media of reptile. keywords: atlasanatomy.org; online anatomy atlas; vertebrates; reptile; interactive atlas. introduction advances in information technology are increasingly made information exchange easier. facility from information technology becomes important for supporting research and scientific activities. one of the advances in information technology is the rapid development of the internet. data show that the number of internet users has increased from year to year, either globally or in indonesia (apjii, 2014). the data are shown in figure 1. figure 1. (a) world internet users (source: apjii); (b) internet users in indonesia (source: statista.com). from the development of information technology, internet, and growing number of domains (detik.com), a new perspective begins in the development and distribution of science. in 2011, research on digital atlas has been done by jones, stone & karten, entitled "high-resolution digital brain atlases: a hubble telescope for the brain". this research explains the application method for digitalize microscopic part of the brain network containing normal and experimental data and to make the content easily accessible online. the results of this study can be accessed at url www.brainmaps.org. the predecessor http://dx.doi.org/10.14421/biomedich.2017.62.63-68 64 biology, medicine, & natural product chemistry 6 (2), 2017: 63-68 study related to brainmaps.org was in 2008 entitled "brainmaps.org interactive high-resolution digital brain atlases and virtual microscopy" (mikula s, et al, 2008). other online atlases avalaible are anatomyatlases.org, innerbody.com, instantanatomy.net, and biodigital.com. all the atlases are human anatomy. this study used reptile as objects in the preparation of interactive online atlas as a reference for learning source. materials and methods vertebrates include all animals that have spines. some of the characteristics possessed by vertebrate animals are having a closed circulatory system, having a backbone in their body, having a complete digestive system, and having a bilateral symmetry body. vertebrates consist of 5 (five) classes, namely pisces, amphibians, reptiles, aves, and mammals. we used mabouya multifasciata as a representation of reptile class. animal 3 lizards (mabouya multifasciata) were used in this study. the lizards were sacrificed, dissected and photographed using canon eos 60d camera; online atlas design for design online atlas we used computer with intel @ 3.40ghz processor i7-4770 cpu (c) i7-4770 equipped with microsoft windows 8.1 operating system software, php 5.3.0 programming language, apache local web server version 2.0, mysql client database server version 5.1.37, mozilla firefox web browser version 56.0.2, text editor sublime text 3, adobe photoshop cs4 extented, coreldraw x6; html, php, javascript, and image map programming languages; servers for data processing and storage; atlasanatomy.org domain name. this research was conducted at integrated laboratory of uin sunan kalijaga yogyakarta. development and manufacture of interactive online atlas system using insourcing method (mulyanto, 2008) sdlc (system development life cycle) covering stages of planning, needs analysis, design, manufacture, and testing. working procedures used in this study include literature studies, data collection & processing, website design, system testing, and system implementation analysis. figure 2. atlasanatomy.org drafting scheme. luthfi & riyanto – on designing interactive online atlas of reptile anatomy … 65 the steps of this research were:  the first step was literature study; namely the study of theories related to animal anatomy, photography techniques, as well as the theory of website making and programming languages.  the second step was data collection & processing; i.e, taking pictures/images on the dissected reptile object and processing the image using software to get the image data. organs were identified and named.  the third step was website making by way of insoursing; there are 5 (five) stages that need to be done namely planning, needs analysis, design, manufacture, and testing.  the fourth step was system testing; there are 2 (two) tests conducted, namely alpha testing conducted directly by the research team about the functional website and beta testing conducted by the general public with a focus on the functionality and interface website. the results of this test are used as material improvement and website development.  the fifth step was system implementation; namely implementation of the website system to obtained evidence or comprehensive facts about animal anatomy website. results and discussion determination of mapping area objects mabouya multifasciata morphology consisted of external morphology, anterior extremities, posterior extremities, organ topography, respiratory system, male reproductive system, female reproductive system, and digestorium system. table 1, figure 3-figure 7 show the segmentation of each section to create image map. table 1. division of mabouya multifasciata image map area. no anatomy system area mapping objects 1 external morphology nares anteriores, thympanic membrane, trucus, rima oris, caput, squama, orgamon visus, cervix, cauda, front limb, hind limb 2 anterior extremities bracium, digit, antebracium, manus, falcuna 3 posterior extremities femur, digit, crus, pes, falcuna 4 organ topography trachea, hepar, hepar, vesica fellea, pulmo, duadenoum, abdominal fat 5 respiration system lingua bifida, rima glottides, trachea, pulmo 6 male reproductive system hemipenis, epidydimis, ren, vas deferent, testis 7 female reproductive system ovarium, oviduct, embrio 8 digestorium hepar, vesica fellea, lien, small intestine, rectum preparation and writing of syntax to create mapping in the truncus section were as follows:  image dimensions: 950 x 364 pixels  syntax determination of truncus part coordinate region: <area name="truncus" alt="" title="" href="#" shape="poly" coords="268,57,274,57,279,57,287,58,297,58,308,59,322,59,329,59,336,59,344,59,356,59,367,59,375,59,389,59,4 04,60,416,60,430,61,446,61,462,63,478,64,492,66,508,69,521,69,535,71,549,73,562,74,578,77,590,79,602,83,611 ,85,612,89,611,96,611,100,611,110,608,118,608,125,608,133,607,140,608,146,608,152,599,151,592,151,581,151, 579,145,583,142,584,136,584,131,582,125,576,120,570,117,564,116,559,114,554,114,548,114,542,115,534,119,5 29,130,530,139,535,148,536,152,522,150,501,150,479,150,457,150,429,151,410,150,389,151,373,151,353,152,34 8,141,342,130,335,119,329,112,320,112,311,112,298,115,285,121,276,126,274,132,276,138,281,144,287,147,294 ,149,299,149,306,146,314,144,324,154,313,157,302,156,291,156,281,156,274,154,267,154,255,151,248,150,242, 149,240,147,245,135,249,126,253,116,257,107,262,98,265,87,268,77" /> figure 3. original images external morphology mabouya multifasciata on the website system. 66 biology, medicine, & natural product chemistry 6 (2), 2017: 63-68 figure 4. original images organ mabouya multifasciata on website system. figure 5. output images with image map on external morphology mabouya multifasciata on the website system. (a). caput; (b). truncus; (c). cervix; (d). cauda; (e). digiti; (f). antebracium; (g). manus; (h). bracium; (i). crus; (j). femur; (k). falcuna; (l). pes. figure 6. output images with image map on mabouya multifasciata on the website system. (a). duodenoum; (b). abdominal fat; (c). hepar; (d). pulmo; (e). lingua bifida; (f). pulmo. luthfi & riyanto – on designing interactive online atlas of reptile anatomy … 67 figure 7. output images with image map on mabouya multifasciata on the website system. (a). trachea; (b). rima glottidis; (c). rectum; (d). small intestine; (e). lien; (f). hepar; (g). hemipenis; (h). testis; (i). epidydimis; (j). vas deferent; (k). oviduct; (l). ovarium. the anatomical photographs of reptile in this study had successfully arranged for online access. goubran & vinjamury (2007) stated that online atlas is an effective way for student learning. conclusion based on the results of the study we concluded that this research has succeeded in establishing an interactive anatomy of reptile anatomy available online (atlasanatomy.org). acknowledgements this research is supported and funded by institute for research and community service (lp2m) uin sunan kalijaga fiscal year 2017. references arfian, gilang. 2009. dasar-dasar pemrograman web. elexmedia. jakarta: komputindo. asosiasi penyelenggara jasa internet indonesia. 2015. profil pengguna internet indonesia 2014. jakarta: puskakom ui. asosiasi penyelenggara jasa internet indonesia. infografis penetrasi & perilaku pengguna internet indonesia survey 2016. retrieved from https://apjii.or.id/survei2016 at 7 march 2017, time 13.33 wib bunafit, nugroho. 2004. cascading style sheets (css). yogyakarta: andi. bunafit, nugroho.. database relational dengan mysql. yogyakarta: andi. detik. pengguna internet 2.4 miliar, jumlah situs tembus 634 juta. retrieved from http://inet.detik.com/read/2013/01/21/081040/2147888/398/pe ngguna-internet-24-miliar-jumlah-situs-tembus-634-juta at 27 november 2015, time 18.27 wib flanagan, david. 2011. javascript: the definitive guide (6th ed.). o'reilly & associates goubran, e.z., s. p. vinjamury. 2007. interactive atlas of histology a tool for self-directed learning, practice, and self-assessment. the journal of chiropractic education 21 (1): 13-18. hariyanto, bambang. 2004. sistem manajemen basis data. bandung: informatika. janner. 2009. pengantar sistem dan teknologi informasi. amus. yogyakarta. jones, e. g., stone, j. m. and karten, h. j. (2011), highresolution digital brain atlases: a hubble telescope for the brain. annals of the new york academy of sciences, 1225: e147–e159. doi: 10.1111/j.1749-6632.2011.06009.x kadir, abdul. 2005. pengenalaan teknologai informasi. yogyakarta: andi mikula s, stone jm, jones eg (2008). brainmaps.org interactive high-resolution digital brain atlases and virtual microscopy. in: lorenz s, egelhaaf m (eds): interactive educational media for the neural and cognitive sciences. brains, minds & media, vol. 3, bmm1426. (urn:nbn:de:00093-14269) 68 biology, medicine, & natural product chemistry 6 (2), 2017: 63-68 mulyanto, a., 2008. sistem informasi konsep dan aplikasi. yogyakarta: pustaka pelajar. peranginangin, kasiman. 2006. aplikasi web dengan php dan mysql. yogyakarta: andi. ramadhan, a. 2008. pemrograman web dengan html, css dan javascript. jakarta: elexmedia komputindo. sidik, betha. 2009. .pemrograman web dengan html. bandung: informatika bandung. statista. number of worldwide internet users from 2000 to 2015. retrieved from http://www.statista.com/statistics/273018/number-of-internetusers-worldwide/ at 19 november 2015, time 14.33 wib. suntoro, susilo handari, dkk. 1994. anatomi hewan. jakarta: universitas terbuka. sunyoto, andi. 2007. ajax membangun web dengan teknologi asynchronouse javascript & xml, yogyakarta: andi. www.w3school.com www.php.net biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1165-1169 | doi: 10.14421/biomedich.2025.142.1165-1169 issn 2540-9328 (online) phytochemical screening and blood glucose response of red dragon fruit extract in alloxan-induced mice i gusti agung istri mas dianti pratiwi1, ni putu ayu dian maharani1, ni made asthi pramesti kirana1, putu nadya asti utari1, i gede ngurah bagus arthayasa1, luh gde evayanti2, made dharmesti wijaya3* 1medical study program; 2department of anatomy and histology; 3department of pharmacology, faculty of medicine and health sciences, warmadewa university, jl. terompong no 24 denpasar 80235, tel. +62 361 240727, indonesia. corresponding author* dharmestiwijaya@warmadewa.ac.id abstract diabetes mellitus (dm) is a chronic metabolic disorder characterized by elevated blood glucose levels, which can lead to various complications. natural products, including red dragon fruit (hylocereus polyrhizus), have gained attention for their potential antidiabetic properties due to their phytochemical content, particularly flavonoids. this study aimed to evaluate the phytochemical composition of red dragon fruit extract and its effect on blood glucose levels in alloxan-induced diabetic mice. phytochemical screening was conducted qualitatively to identify the presence of secondary metabolites. male mice were divided into five groups: a negative control group (no treatment), a positive control group (metformin 10 mg/kg bw), and three treatment groups (t1, t2, t3) receiving red dragon fruit extract at doses of 18.2, 36.4, and 72.8 mg/kg bw, respectively. diabetes was induced by a single intraperitoneal injection of alloxan monohydrate (140 mg/kg bw). treatments were administered orally once daily for 6 days. blood glucose levels were measured on days 0, 2, 4, and 6. data were analyzed using the friedmann test. phytochemical screening confirmed the presence of flavonoids, tannins, alkaloids, and saponins in the extract. statistical analysis showed no significant reduction in blood glucose levels after administration of red dragon fruit extract at any of the tested doses (p>0.05). red dragon fruit extract did not exhibit a significant antihyperglycemic effect in alloxan-induced diabetic mice under the tested conditions. this may be attributed to suboptimal dosage, insufficient flavonoid concentration, or a less effective mechanism of action compared to standard therapy. further studies are needed to optimize the formulation and evaluate its potential using different extraction methods or in combination with other bioactive compounds. keywords: alloxan-induced mice; antihyperglycemic; hylocereus polyrhizus. introduction diabetes mellitus (dm) is a chronic metabolic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. according to the international diabetes federation, approximately 589 million people aged 20-79 years worlwide were living with dm in 2024, and this number is projected to rise to 853 million by 2050. indonesia ranks among the top ten countries with the highest number of dm cases, with and estimated 20.4 million people affected in 2024 (international diabetes foundation, 2025). moreover, dm contributes significantly to morbidity and mortality, with complications such as cardiovascular disease, nephropathy, retinopathy, and neuropathy (harreiter & roden., 2023). diabetes mellitus management generally involves pharmacological and non-pharmacological approaches, including lifestyle modification and the use of antidiabetic medications (harreiter & roden., 2023). however, long-term use of synthetic drugs can lead to adverse effects and impose economic burdens on patients. this has encouraged the exploration of alternative therapies derived from natural products, particularly medicinal plants, which are believed to offer therapeutic benefits with minimal side effects (ali et al. 2022; pitaloka & juwariyah, 2021). one such plant is red dragon fruit (hylocereus polyrhizus), which is widely cultivated and easily accessible in indonesia. this fruit is known to contain various bioactive compounds, including phenolic acids (such as hydroxycinnamates), beta carotene, flavonoids, belatains, vitamin c, as well as linoleic and linolenic acids (arivalagan et al. 2021). these phytochemicals possess antioxidant properties that may contribute in reducing oxidative stress and improving insulin sensitivity, which are important in the context of dm (benson et al. 2023). in addition, the dietary fiber in red dragon fruit, particularly pectin, is suggested to help regulate blood glucose levels by slowing glucose absorption through increased intestinal viscosity and manuscript received: 18 september, 2025. revision accepted: 21 november, 2025. published: 04 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1165-1169 mailto:dharmestiwijaya@warmadewa.ac.id 1166 biology, medicine, & natural product chemistry 14 (2), 2025: 1165-1169 decreased diffusion rates (benson et al. 2023). the antioxidant and phytochemical compounds may also support vascular health and enhance cell membrane permeability, potentially increasing cellular responsiveness to insulin. considering the potential bioactive compounds present in red dragon fruit and the limited research on its overall phytochemical profile and antihyperglycemic effects, further investigation is warranted. therefore, this study aimed to perform a qualitative phytochemical screening of red dragon fruit extract and to evaluate its effect on blood glucose levels in alloxan-induced diabetic mice. materials and methods materials the materials used in this study included fresh red dragon fruits (hylocereus polyrhizus) obtained from a traditional market in the denpasar area, bali; 25 male swiss-webster mice; standard mice feed; distilled water (5 l); 96% ethanol (onemed); and alloxan monohydrate (sigma-aldrich). rice husks were also used as bedding material for the mice. for the antioxidant activity assay, additional materials included 1% hydrochloric acid (hcl), ascorbic acid as a standard antioxidant, and dpph (2,2-diphenyl-1-picrylhydrazyl) kit. the tools utilized in this study included various standard glassware (beakers, glass funnels, measuring flasks, measuring cups, test tubes), filter papers, dropper pipettes, syringes (1 ml and 3 ml), glass stirrers, microscope slides, oral gavage (mice sonde), mice drinking bottles, mice cages, blood glucose strips, disposable gloves, face masks, hand sanitizers, tissues, glass jars, knives, cutting boards, filters, and a rotary evaporator. for the antioxidant activity test, additional tools used were a uv-vis spectrophotometer, an analytical balance, aluminum foil, black glass bottles, dark vials, and other supporting laboratory equipment. preparation of red dragon fruit extract fresh red dragon fruits were thoroughly washed, peeled, and cut into small pieces. the fruit pieces were then dried in an oven at 40°c for seven days until completely dehydrated. the dried fruits were subsequently ground using a blender to obtain a fine powder. a total of 400 grams of the dried powder was weighed and subjected to maceration with 2.8 liters of 96% ethanol (sample-tosolvent ratio of 1:7). the maceration process was carried out for five days at room temperature, protected from light, with occasional stirring. after five days, the mixture was filtered, and the filtrate was concentrated using a rotary evaporator to obtain a thick extract. phytochemical screening phytochemical screening of the red dragon fruit extract was conducted qualitatively to detect the presence of flavonoids, alkaloids, saponins, and tannins. for flavonoid detection, the extract was placed into a test tube, then added with 1–2 grains of magnesium metal and 3 drops of concentrated hydrochloric acid, followed by the addition of amyl alcohol and vigorous shaking; the appearance of a reddish-yellow to red color indicated a positive result for flavonoids. alkaloid detection involved mixing the extract with 1 ml of concentrated hydrochloric acid and 9 ml of distilled water, heating for 2 minutes, cooling, and filtering; the filtrate was divided into three test tubes, and the addition of wagner's reagent to each was performed—precipitate formation in at least two tubes indicated a positive result for alkaloids. for saponins, the extract was added with 10 ml of hot water, cooled, shaken for 10 seconds, and then added with 2n hydrochloric acid; the formation of stable foam over 1 cm in height indicated the presence of saponins. tannin detection was carried out by adding 1–2 drops of 1% ferric chloride (fecl₃) solution to the extract; the appearance of a blackish-green coloration suggested a positive result for tannins. these tests were conducted following standard procedures for phytochemical analysis as described by harborne (1988). animal preparation the test animals used in this study were male swisswebster mice, aged approximately 6 weeks and weighing around 25 grams. the mice, which were in healtaahy condition and exhibited normal activity, were obtained from the animal laboratory, faculty of medicine and health sciences, warmadewa university. prior to the experimental procedures, the mice were acclimatized in their new cages for 7 days at room temperature, with free access to standard feed and drinking water. after the adaptation period, fasting blood glucose levels were measured to obtain baseline data. diabetes mellitus was then induced by intraperitoneal injection of alloxan monohydrate at a dose of 140 mg/kg body weight. alloxan induces diabetes by selectively damaging pancreatic β-cells, resulting in insulin deficiency and hyperglycemia (woldekidan et al. 2021) following induction, the mice continued to receive food and water ad libitum. on the seventh day after induction, following a fasting period of 12–18 hours, fasting blood glucose levels were measured again to confirm hyperglycemia. mice with fasting blood glucose levels greater than 290 mg/dl were considered diabetic and included in the subsequent experimental groups (american diabetes association, 2024) treatment protocol a total of 25 male swiss-webster mice were randomly divided into 5 groups, each consisting 5 mice. the groups received the following treatments: group 1 (negative control) did not receive any treatment with red dragon fruit extract; group 2 (positive control) was pratiwi et al. – antihyperglycemic activity of red dragon fruit 1167 administered metformin orally at a dose of 10 mg/kg body weight; meanwhile group 3, 4, and 5 were treatment groups that received red dragon fruit extract at concentrations of 18.2 (t1), 36.4 (t2), and 72.8 (t3) mg/kg body weight, respectively. the red dragon fruit extract was prepared by dissolving the thick extract in distilled water to obtain the desired concentrations. each treatment, including the extract and metformin, was administered once daily via oral gavage for 14 consecutive days. during the treatment period, all mice provided standard feed and drinking water ad libitum. blood glucose levels were measured on days 0, 2, 4, and 6 to monitor changes in response to the administered treatments. statistical analysis the data obtained were analyzed using spss software. prior to comparative testing, the data were assessed for normality and homogenity. since the results indicated that the data were not normally distributed and variances were not homogeneous, a non-parametric approach was applied. the friedman test was used to compare blood glucose levels among the five groups. a 95% confidence level (p < 0.05) was used to determine statistical significance. ethical approval all animal procedures performed in this study were reviewed and approved by the research ethics committee of the faculty of medicine, udayana university (approval no: 0324/un 14.2.2.vii.14/lt/2025). results and discussion a total of 10 kilograms of fresh red dragon fruit (hylocereus polyrhizus) were oven-dried at 40°c for seven days, yielding approximately 1,000 grams of dried simplicia. the dried material was then subjected to extraction using the maceration method, with 400 grams of powdered simplicia immersed in 2,8 l of 96% ethanol. the maceration process was conducted at room temperature for five days with occasional stirring and protection from light. this procedure resulted in the production of 55 milligrams of thick extract. maceration was selected as the extraction method in this study due to its simplicity, cost-effectiveness, and ability to preserve thermolabile phytochemicals through cold extraction (rusip et al. 2022). phytochemical screening qualitative phytochemical screening tests were carried out to identify flavonoid, tannin, alkaloid and saponin in the red dragon fruit extract. the results confirmed that the extract contained all four compounds (table 1). table 1. phytochemistry of red dragon fruit extract. phytochemicals method results description flavonoids shinoda’s test + reddish-orange color formed tannins fecl3 + blackish-blue color formed alkaloids wagner's test + precipitate formed saponins froth test + foam formed theoretically, the presence of these compounds is associated with the potential to lower blood glucose levels in alloxan-induced mice. flavonoids are known to exert antidiabetic effects through multiple mechanisms, including enhancing insulin receptor sensitivity, stimulating insulin secretion, regenerating pancreatic βcells, and inhibiting the digestive enzymes α-amylase and α-glucosidase (liu et al. 2023). similarly, tannins have been reported to inhibit glucosidase activity and improve insulin sensitivity, while alkaloids may stimulate insulin release or suppress hepatic gluconeogenesis (ramadhan & sujono, 2025). moreover, saponins are recognized for their ability to inhibit intestinal glucose absorption and enhance insulin secretion (idris et al, 2023). the synergistic presence of flavonoids, tannins, alkaloids, and saponins in the red dragon fruit extract therefore provides a theoretical basis for its potential antihyperglycemic effect. however, although the phytochemical profile of the extract suggested potential antihyperglycemic activity, further evaluation in this study showed that such effects were not observed under the tested conditions. blood glucose level in vivo testing was conducted using 25 mice randomly divided into five groups. the mean blood glucose levels of each group are presented in table 2, while the trends over time are visualized in figure 1. statistical analysis using the friedman test showed no significant differences in blood glucose levels among the groups (p = 0.537, p > 0.05). these results indicate that red dragon fruit extract at doses of 18.2, 36.4, and 72.8 mg/kg bw did not significantly reduce blood glucose levels in alloxaninduced diabetic mice compared with control groups. 1168 biology, medicine, & natural product chemistry 14 (2), 2025: 1165-1169 table 2. blood glucose levels (mg/dl) of alloxan-induced diabetic mice during 6 days of treatment. sample blood glucose (mg/dl) day 0 day 2 day 4 day 6 positive control 167,0 147,6 160,6 151,8 negative control 169,6 149,2 163,4 165,6 t1 144,4 168,6 154,4 152,6 t2 155,8 159,8 156,8 153,6 t3 140,0 139,8 198,0 196,2 figure 1. blood glucose levels of alloxan-induced diabetic mice during 6 days of treatment. mice were divided into five groups (n = 5 per group): negative control (untreated), positive control (metformin 10 mg/kg bw), and three treatment groups receiving red dragon fruit extract at doses of 18.2, 36.4, and 72.8 mg/kg bw. statistical analysis using the friedman test showed no significant differences among groups (p > 0.05). although the extract contained phytochemicals with known antidiabetic potential—such as flavonoids, tannins, alkaloids, and saponins—the lack of significant effect observed in this study may be affected by several factors. previous research reported that the ethanol extract of red dragon fruit exerted significant antihyperglycemic activity at doses ≥200 mg/kg bw (idris et al. 2023), suggesting that the doses tested here may not have reached the effective threshold. moreover, treatment was carried out for 6 days, while other studies have shown that the antidiabetic effects of flavonoids often require 21-28 days of administration (rahman et al. 2022). in addition, no quantitative analysis of phytochemicals was performed in this study, and flavonoid concentrations can vary depending on extraction methods and solvents (pujiastuti & zeba, 2021). if the active compound levels were relatively low, the pharmacological effects could also be limited. another factor to consider is the use of alloxan to induce diabetes. alloxan is known for its unstable cytotoxicity and extensive β-cell damage, which can make glucose normalization more difficult even with bioactive treatments. streptozotocin (stz) is often recommended as a more stable and consistent alternative for diabetes induction (samsul et al. 2020). biological variability between animals may also have contributed, as indicated by the non-homogeneous distribution in some groups, which could mask potential effects in statistical analysis (rahman et al. 2022). taken together, these findings suggest that under the tested conditions, red dragon fruit extract did not significantly reduce blood glucose levels in alloxaninduced diabetic mice. nevertheless, the study provides useful preliminary data, and further research with optimized doses, longer treatment duration, and quantitative phytochemical analysis is recommended to better evaluate the antidiabetic potential of red dragon fruit. conclusions in conclusion, qualitative phytochemical screening confirmed the presence of flavonoids, tannins, alkaloids, and saponins in red dragon fruit extract. however, the extract did not significantly reduce blood glucose levels in alloxan-induced mice at the tested doses and treatment duration. these findings suggest that further research using higher doses, longer treatment periods, and quantitative phytochemical analysis is required to better evaluate the antidiabetic potential of red dragon fruit. acknowledgements: the authors would like to express their sincere gratitude to the laboratory staff of the research laboratory and animal laboratory, faculty of medicine and health sciences, warmadewa university, for their technical support and assistance during the experimental work. the support and contributions of colleagues and mentors are also gratefully acknowledged. authors’ contribution: i gusti agung istri mas dianti pratiwi contributed to study design and preparation of the initial manuscript draft. ni putu ayu dian maharani and ni made asthi pramesti kirana were responsible for extract preparation, phytochemical screening, and data analysis. putu nadya asti utari and i gede ngurah bagus arthayasa carried out the animal experiments and treatment protocol. luh gde evayanti contributed through conceptual input and critical review of the final manuscript. made dharmesti wijaya provided supervision, critical guidance throughout the research process, and substantial revisions to the manuscript. all pratiwi et al. – antihyperglycemic activity of red dragon fruit 1169 authors discussed the results together and approved the final version of the manuscript. competing interest: the authors declare that there are no competing interests. funding: this research was funded by the ministry of education, culture, research, and technology of the republic of indonesia through the student creativity program (program kreativitas mahasiswa/pkm) in 2024. references ali, m. k., pearson-stuttard, j., selvin, e., & gregg, e. w. 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(2020). aktivitas antidiabetes serbuk semut jepang (tenebrio molitor linn.) pada mencit swiss webster jantan yang diinduksi aloksan. jurnal sains dan kesehatan: 2(4): 298-302. woldekidan, s., mulu, a., ergetie, w., teka, f., meressa, a., tadele, a., abebe, a., gemechu, w., gemeda, n., ashebir, r. and sileshi, m., 2021. evaluation of antihyperglycemic effect of extract of moringa stenopetala (baker f.) aqueous leaves on alloxan-induced diabetic rats. diabetes, metabolic syndrome and obesity; 185-192. https://idf.org/about-diabetes/diabetes-facts-figures/ https://idf.org/about-diabetes/diabetes-facts-figures/ https://pmc.ncbi.nlm.nih.gov/articles/pmc9345267/ this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 41-46 | doi: 10.14421/biomedich.2021.101.41-46 issn 2540-9328 (online) computational chemical study of pigment of mangosteen (garcinia mangostana) rind extract as dye compound in dye-sensitized solar cell (dssc) didik krisdiyanto*,1, sudarlin1, hikmah supriyati2 1department of chemistry, faculty of science and technology, universitas islam negeri sunan kalijaga yogyakarta jl. marsda adisucipto no.1 yogyakarta 55281, tel. + 62-274-540971, fax. + 62-274-519739, indonesia 2post graduate program of biological education department, universitas negeri yogyakarta jalan colombo no. 1 yogyakarta 55281, indonesia corresponding author* didik_kris@yahoo.com manuscript received: 03 march, 2021. revision accepted: 30 june, 2021. published: 19 july, 2021. abstract the electronic transition processes of α-mangostin and β-mangostin as dye compounds in dssc and their ability to transfer electrons to semiconductors have been studied in theory. the research was carried out computationally using the nwchem application. the methods used are density functional theory (dft) for structure optimization and time-dependent dft (td-dft) for electronic transitions. the results showed that the energy of homo β-mangostin was lower that it was more stable or easier to form bonds with semiconductors. likewise, its lumo energy is lower than α-mangostin that it is easier to inject electrons into the semiconductor. the energy difference of homo-lumo β-mangostin is smaller than α-mangostin. this shows that β-mangostin is more easily excited or more efficient in exciting solar energy to convert it into electricity. this is consistent with the molecular spectra where λmaxβ-mangostin is at a higher wavelength. orbital analysis shows electron injection of α-mangostin and β-mangostin into the semiconductor via double o atoms in each molecule. the injection is influenced by the bond length, where the bond length of α-mangostin to tio2 is smaller than that of β-mangostin to tio2. keywords: α-mangostin; β-mangostin; energy homo-lumo; dft; td-dft; mangosteen rind; dye-sensitized solar cell. introduction research on dye compounds for dssc (dye-sensitized solar cell) has been widely carried out, but synthetic dyes' efficiency is still higher than natural dyes, which are only ~ 1%. however, synthetic dyes have drawbacks including difficult preparation, expensive production costs, and a toxic risk to the environment. the use of natural dyes becomes a very attractive alternative to develop because they are easy to obtain, have unlimited sources, have a long absorbance coefficient, high light capture efficiency, low production costs, easy preparation, and are environmentally friendly (luo et al., 2009). however, not all dyes can be used as sensitizers because they must qualify, namely adsorption intensity at visible wavelengths, strong adsorption on the semiconductor surface, has the ability to inject electrons into the semiconductor conduction band, and has groups = o or –h to bind with the tio2 surface which can increase the rate of the electron transfer reaction (ludin et. al., 2014). various studies on dssc using natural dyes from plant extracts have been conducted and these studies have proven that natural dyes can provide a photovoltaic effect. one of the sensitizers used is mangosteen rind pigment extract. chairat et al. (2007) in their research used mangosteen rind pigment extract which was proven to be used as a sensitizer on solar cells of the dssc type. mangosteen is very easy to obtain. this is because indonesia is a tropical country that is very suitable for the growth of mangosteen. the main component in the mangosteen pigment extract which is potential as a dssc sensitizing dye is shown in figure 1. the α-mangostin and β-mangostin compounds are two of the several components in the mangosteen rind pigment extract which has absorption at visible wavelengths as one of the requirements for the dssc dye. further studies on the adsorption of compounds on semiconductor surfaces, electronic energy levels, and electron transfer capabilities need to be done. the study in this research was done with the help of computational chemistry. two basic structures of the main components of mangosteen rind extract, the electro-optical properties of dssc will be studied based on computational calculations to study adsorption on semiconductors, adsorption spectrum, energy levels, and charge transfer. https://doi.org/10.14421/biomedich.2021.101.41-46 42 biology, medicine, & natural product chemistry 10 (1), 2021: 41-46 a b figure 1. basic structure of mangosteen rind extract (a) α-mangostin (b) β-mangostin. dye-sensitized solar cell (dssc) is a series of devices that can convert visible light into electricity based on semiconductor band gap sensitization and its bonding with groups on the surface of a thin layer cell (ludin et. al., 2014). the dssc material consists of a pair of glass substrates coated with tco (transparent conducting oxide) material which acts as an electrode and a counter electrode separated by a redox electrolyte which has high transparency and low resistance characteristics, can be indium tin oxide (ito), aluminum zinc oxide (azo), and fluorine tin oxide (fto) mounted opposite each other. fto and ito are most often used as dssc, where the process of sintering the oxide layer on the substrate at a temperature of 450500oc, these materials have good conductivity and do not experience defects or defects in that temperature range (halme, 2002). figure 2. schematic and working principle of dye-sensitized solar cell (dssc). the redox pair that is often used is i/ i3(iodide / triiodide). the solvent used in the electrolyte solution is polyethylene glycol (peg) because it can penetrate the tio2 dye absorption for both small particle size comparisons and nano-scale pore diameters, and can maintain work stability (misbahudin et.al., 2013). at tco, the counter electrode is coated with a catalyst in the form of a carbon layer to accelerate the redox reaction, while the electrode is positioned as a porous tio2 nanocrystalline layer as a photoanode and is sensitized by dye as a photosensitizer which functions to capture photons which then will be absorbed into tio2 nanoparticles. an efficient photosensitizer must qualify, namely dye particles can be adsorbed onto the semiconductor surface, the capacity to capture light in the visible light range, the ability to inject electrons into the conduction band of the semiconductor, and have an = o or –h group to bind to the tio2 surface. the working principle of the dssc is shown in figure 2. basically, the working principle of dssc is a reaction of electron transfer where the first process begins with the excitation of electrons in the dye molecule due to photon absorption. electrons are excited from the ground state (d) to excited state (e). s + photon  s* (absorption) (1) the electrons from the excited state are then directly injected into the conduction band (ecb) of the titania so that the dye molecules are oxidized (s +). with the presence of an electron donor by the electrolyte, the dye molecules return to their ground state and prevent the recapture of the oxidized dye electrons. s* + tio2 e(tio2) + s+ (injection process) (2) the injected electrons are carried from the tio2 nanopore thin layer to the conductive electrode (anode). e(tio2)+the conductive electrodetio2+e(c.e) (3) after reaching the tco electrode, electrons flow towards the electrode counter through the external circuit and approach the cathode (counter electrode). the electrons are transferred to the electrolyte at the electrodes. with a catalyst on the counter electrode, the oxidized dye accepts electrons from the ion (i-), a redox reaction occurs to replace the lost electrons, and the iodide molecule is oxidized to the triiodide ion (i3-). s+ + 3 2 i- s + 1 2 i3 (4) ion (i3-) is used to donate electrons to the oxidized dye permitting an electron transport cycle to be formed, with this cycle a direct conversion from sunlight to electricity. 1 2 i3+ e(c.e)  3 2 i+ c.e (5) (narayan, 2012). sunlight produces 45% of the spectra in the visible and 5% of the uv spectra so that in the use of dyes in the dssc, the absorption of photon energy from visible light is carried out by light-sensitive materials (dyes that function as sensitizers). with the presence of a sensitizer, it is possible to inject/transfer electrons to the tio2 semiconductor material even though the received photon energy is smaller than the band gap of the tio2 semiconductor (this event is called sensitisation). to krisdiyanto, et al. – computational chemical study of pigment of … 43 support this process, the tio2 semiconductor material must be able to entangle as many molecules of the color substance as possible so that more electrons can be accepted. some of the criteria that must qualify by tio2 as a semiconductor in dssc include the particle size being on the nanometer scale. this is necessary because with the particle size that is on the nanometer scale the surface area of the particles as a whole becomes larger so that more dye molecules are possible to absorb. in addition, tio2 particles are also expected to have a porous (mesoporous) morphology, allowing the dye molecules enter between the pores and can be absorbed on the surface of the tio2 particles, which implies that they will increase the amount of light absorbed (zhang et.al., 2008). in addition, the use of semiconductor oxides in photoelectrochemistry is due to its stability against corrosion photo and also its large energy band (3.2-3.8 ev) which in dssc is needed for semiconductor transparency in most of the sunlight spectrum. although tio2 is a material that is often used because the efficiency of dssc using tio2 is still unmatched, several materials can act as semiconductor oxides including zno, cdse, cds, wo3, fe3o3, sno2, nb2o5 and ta2o5 (halme, 2002). the dye in the dssc oxide layer serves to capture photons of light. furthermore, these photons are absorbed into the tio2 nanoparticle. the requirements for dye as a photosensitizer in dssc are absorption in the visible light region or near the infrared region of the sunlight spectrum and bind to the tio2 semiconductor (cherepy et.al., 1997). functional groups are needed to interact with tio2 surfaces such as carboxylates or around other acid groups (galopini, 2004). several functional groups have the possibility to bind with tio2. the best groups are metal oxides such as phosphonic acid followed by carboxylic acids and their derivatives such as chlorides, amides, esters or carboxylic salts (galopini, 2004). figure 3. possible bond models between -cooh groups and tio2 (hug et. al., 2014). plant extracts or pigments used as photosensitizers in visible light areas can be in the form of chlorophyll extract (amoa, 2003). chlorophyll is the main plant pigment which functions to absorb light and convert it into chemical energy needed to reduce carbon dioxide into carbohydrates in the photosynthesis process. figure 3 shows several bonding models between tio2 and the dye molecule with at least one carboxylate group. another requirement for this material to be an active ingredient in solar cells is that the material must be able to become a transfer medium for electric charge carriers as a result of the absorbed photons (supriyanto et. al., 2010). to obtain a suitable dye as a sensitizer requires a long time and resources in research which is computational chemistry can be an alternative. by knowing in more detail the electro-optical properties of a dye, it can be used as a basis for the development of efficient dye alternatives. density fungtional theory (dft) and time dependent dft (td-dft) methods can be used for large molecules with an accurate approach to calculate the uv spectrum of dyes as well as for complex systems between dyes and semiconductors. methods the research was conducted computationally using the nwchem application. the methods used are density functional theory (dft) for structure optimization and time dependent dft (td-dft) for electronic transitions. all calculations use b3lyp functional and 6-31g * base set. data visualization using ecce and chemcraft applications. in the study of dyestuffs for dssc in computational chemistry involving large molecules and complex systems, a suitable approach is needed. results and discussion method selection the calculation results obtained were compared with the results of experiments conducted by madihah et al. (2012). the parameter being compared is the αmangostin excitation energy in units of ev as shown in table 1. other parameters such as bond length cannot be compared because experimental data are not available. the results of the calculations as shown in table 1 show the difference in excitation energy from the calculation of α-mangostin and the experimental results of 0.01ev. the similarity between the calculation results and the experimental results reached 99.71%. these results indicate the selection of methods and basis sets in this study are correct and quite accurate. 44 biology, medicine, & natural product chemistry 10 (1), 2021: 41-46 table 1. comparison of the first excitation energy of α-mangostin on experimental and calculated results. no α-mangostin data excitation energy (ev) similarity level 1. experiment 3.522 99,71% 2. calculation 3.532 supporting data that can be compared is the display of α-mangostin spectra on the experimental and the calculation results at wavelengths above 280 nm, which shows the similarities as shown in figure 4. figure 4. the α-mangostin spectra of the experimental results and the calculation results above 280 nm. optimization of homo-lumo molecules and energy optimization is carried out on each molecule using the same method. the optimization results are in the form of a stable structure as shown in figure 5 and the multivariate load value as shown in table 2. a b figure 5. the results of the calculation of α-mangostin (a) and βmangostin (b). based on the optimization results of α-mangostin and β-mangostin in figure 5, each red o atom has a different mullicen charge value as shown in table 2. based on the table, the o-4 atom has a more negative charge so it is more reactive in binding to other atoms. thus, o-4 atoms can be used to bind tio2. the homo and lumo energies of each compound as a result of the optimization can be seen in table 3. based on the homo energy in the table, β-mangostin has a lower homo energy. based on the requirements of the dye compound, this indicates that β-mangostin is more stable so it is easier to form bonds with semiconductors. likewise, the lumo β-mangostin energy is lower than the lumo α-mangostin energy so that β-mangostin is easier to inject electrons into the semiconductor. table 2. mullicen charge values of o α-mangostin and β-mangostin atoms. atom o α-mangostin β-mangostin o-1 -0,135014 -0,135076 o-2 -0,240754 -0,240796 o-3 -0,360894 -0,361235 o-4 -0,377779 -0,380988 o-5 -0,370443 -0,200601 o-6 -0,341725 -0,341406 table 3. the energy difference between homo-lumo α-mangostin and β-mangostin. energy α-mangostin β-mangostin homo (ev) -5.647 -5.677 lumo (ev) -1.547 -1.783 δ (ev) 4.100 3.894 figure 6. the energy bands of α-mangostin and β-mangostin calculated. the difference in energy bands produced is the difference between homo energy and lumo energy. the smaller the value of the energy band difference, indicating that the easier the electrons in the molecule move from a lower energy level to a higher energy level. figure 6 shows that the smaller homo-lumo energy difference is β-mangostin. this shows that β-mangostin is more easily excited. that is, β-mangostin is more efficient in exciting solar energy to convert it into electricity. the shape of the electron orbitals in the homo and lumo states the shape of the homo α-mangostin and β-mangostin orbitals as presented in figure 7 shows the same pattern. td spectrum wavelength, nm 360 350 340 330 320 310 300 290 280 f 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 krisdiyanto, et al. – computational chemical study of pigment of … 45 the figure shows the delocalized homo orbital electrons on the o-4 atom. this corresponds to the high mullicen charge of o-4 atoms, thus strengthening the reason that tio2 semiconductors tend to bond to o-4 atoms, both to α-mangostin and β-mangostin. a b figure 7. form of the calculated α-mangostin (a) and β-mangostin homo orbitals (b). as with the homo orbitals, the lumo α-mangostin and β-mangostin orbitals show the same pattern as shown in figure 8. the figure shows the delocalized molecular electrons on the o-6 atom. this shows the electron injection of the dye compound into the semiconductor via o-6 atoms, both in α-mangostin and β-mangostin. a b figure 8. form of the calculated lumo α-mangostin (a) and βmangostin (b) orbitals. bond length the calculation of the bond length is carried out between ti on tio2 and o-6 atoms in α-mangostin and βmangostin. in the previous section, it was explained that electrons are more easily injected into semiconductors via o-6 atoms based on the electron composition of the lumo orbitals. thus, the distance between ti and o-6 atoms at α and β mangostin will affect the electron transfer rate. the closer distance makes the electron transfer easier. a b figure 9. optimization results for α-mangostin + tio2 (left) and βmangostin + tio2 (right). the calculation results as shown in table 4 show the length of each bond. the bond lengths between ti and o-6 in α-mangostin and β-mangostin are 1.989 ǻ and 1.990 ǻ. when compared with the total radius length between ti and o of 2.36 ǻ, it can be concluded that there is a bond between tio2 and α-mangostin and tio2 and β-mangostin. comparison of the bond length between ti and o-6 in α-mangostin and β-mangostin as in the table shows that α-mangostin tends to transfer electrons more quickly to the tio2 semiconductor. table 4. bond lengths of each compound. spectra the spectra in the figure show that the β-mangostin spectra have undergone a bathochromic shift. this is the effect of the addition of the ch3 group as an electron booster that is able to shift the wavelength to a higher wavelength. the shift in wavelength to vis shows the efficiency of β-mangostin as a dye compound is better than α-mangostin. this is because excitation at visible light (vis) to infrared wavelengths is generally an excitation that involves the transfer of charge. excitation involving charge transfer is an excitation that provides an effective injection of electrons into the semiconductor and thus plays a major role in determining the efficiency of dssc cells. figure 10. the calculated spectra of α-mangostin (black curve) and βmangostin (red curve). in addition, the amount of sunlight reaching the earth's surface is dominated by uv rays at a wavelength of 300-400 nm, which is about 95%. uv light at 200300 wavelengths only reaches 5% of the earth's surface because most of it has been absorbed by airborne molecules such as ozone. compound bond length (ǻ) α-mangostin ti – o(6) 1,989 β-mangostin ti – o(6) 1,990 46 biology, medicine, & natural product chemistry 10 (1), 2021: 41-46 conclusion the results showed that the energy of homo βmangostin was lower that it was more stable or easier to form bonds with semiconductors. likewise, lumo energy is lower than α-mangostin so that it is easier to inject electrons into the semiconductor. the energy difference of homo-lumo β-mangostin is smaller than α-mangostin. this shows that β-mangostin is more easily excited or more efficient in exciting solar energy to be converted into electricity. acknowledgments: lppm uin sunan kalijaga for funding assistance for this research in the 2016 beginner research program. conflict of interest: the authors declares that there are no conflicts of interest concerning the publication of this article. references amao, yutaka & yamada, yuriko & aoki, keiko. 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(2008). betalain pigments for dyesensitized solar cells. photochemistry and photobiology. 7280. biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 2, 2014 | pages: 47-52 | doi: 10.14421/biomedich.2014.32.47-52 a discovery and characteristics description of telosma puberula (asclepiadoideae) in mount gedang atas and mount ijo, baturagung mountain yogyakarta widodo faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: wwidodo594@gmail.com abstract population of telosma puberula in community forest bushes was identified at s.07.48'.44.1"; e.110.31.15.8", 411m, mount gedang atas and s 07o 04 '04.1"; e 110o 30 '47.9 ", 415m, mount ijo. telosma puberula was also found in mount parangan, mount mintorogo, mount nglanggeran, and baturagung mountains yogyakarta. the identification was based on herbarium specimens collected by horsfield in 1802 and 1859 from java island, indonesia (k000873052, k000873053). information about telosma puberula is very limited. this article describe photographs of morphological characters of the plant’s stems, leaves, flowers, and pollinia. keywords: telosma puberula, asclepiadoideae, mount gedang atas, baturagung mountains introduction in an exploration, observation and assessment of wild plants in mount gedang atas baturagung mountains in yogyakarta, the author was collecting fruits of vines from family apocynaceae, sub family asclepiadoideae in a community forest at a location of s.07.48'.44.1"; e.110.31.15.8". (widodo, 411m dpl, 26 september 2012). furthermore, a specimen collection process and observation was completed in a flowering season in 2013. the same specimen was also found in mount ijo within a radius of 5 km at a location of s.07.47'.04.4 "; e.110.30'.47.9 ". (widodo, 415m dpl, 31 december 2012). from a thorough identification process using literature and herbarium reviews, it was identified that the specimen collected is telosma sp. telosma is belong to marsdenieae tribe of the subfamily asclepiadoideae (takhtajan, 2009: 522). according to backer and bakhuizen (1965: 273), there are two types of telosma in java: telosma accedens and telosma cordata. meanwhile, hooker (1885: 38) states that in java, there is telosma puberula (a new name for pergularia puberula). telosma cordata (a new name for pergularia odoratissima) is predicted to be a native to china. in java, the plant has been cultivated as floral fragrance (backer and bakhuizen, 1963: 273). meanwhile telosma accedens can be found in central java, east java and madura at an altitude of 10-800 m in woody shrubs, cleared forests, and teak forests. the previous name of pergularia accendens was telosma accedens, blume. backer corrected the name into telosma accedens (bl.) back. (backer, 1949: 63). the name pergularia accedens was firstly published in bijdragen tot de flora van nederlandsch indie (blume, 1826). the herbarium of pergularia accedens was collected by carl l von blume from the java island in 1836 (mnhn p03858919). meanwhile, the herbarium of telosma accedens collection (bl.) back was collected by ca backer from the java island (jember-bondowoso) in 1920 (mnhn p03858922). studies on telosma cordata or tonkin flowers hold are abundant on the internet because this plant is cultivated as floral fragrance plants although there is no identified source from indonesia. based on the writer’s knowledge, there are no further information or study about telosma accedens and telosma puberula in indonesia after backer and bakhuizen (1965: 273), hooker (1885: 38) and blume (1836). according to the plant list (2010), the genus telosma consists of many species: telosma accedens (blume) backer, telosma africana (nebr.) nebr., telosma angustiloba (warb. in perkins) merr., telosma celebica (warb.) ma rahman & wilcock, telosma cordata (burm. f.) merr., telosma filipes (schltr.) ma rahman & wilcock, telosma pallida (roxb.) craib, telosma procumbens (blanco) merr., and telosma puberula (miq.) kerr. based on the information obtained from backer and bakhuizen (1965: 273) and hooker (1885: 38), the telosma species that are available in the java island is telosma accedens, telosma cordata, and telosma puberula. there is very little information about telosma accedens and telosma puberula both in written literature and published pictures on the internet. this paper attempts to present a description of the characteristics, specimen and herbarium photographs of telosma specimens found in mount gedang atas and mount ijo, baturagung mountains. the discovery of the telosma species in the baturagung mountains yogyakarta needs to be disseminated to present the richness of flora in java. wild plants are no longer recognized despite the fact that there were some report in the books of flora and 48 biology, medicine, & natural product chemistry 3 (2), 2014: 47-52 herbarium in the past hundreds years by european explorers. a publication of plant species in the nature is highly needed in attempt to complete the flora world data, to re-check and rediscover old flora, to correct description of the characters for further research in the study of plant biology. the study of biology is the basis for conservation of earth plants. the purpose of this study is to present a visual characteristics description of the morphology of leaves, stems, flowers, pollinia, and fruit of telosma puberula found in mount gedang atas and mount ijo, baturagung mountains for identification verification. materials and methods this research employs one kind of research methode that explores and visits as well as collects data information (exploration and collection trip, (singh, 1999)). an early exploration was performed in september 2012 along with an exploration of wild plants. picture taking was completed for the first step of the identification process. specimen sampling for herbarium was also performed with an awareness of the preservation of the population. along with the identification process, monitoring and visitation processes were also kept on being conducted based on a prediction on the flowering season and fruit formation, i.e. december 2012, january 2013, march 2013, april 2013, december 2013, and october 2014. flowers specimen collection was completed using wet preservation technique for further identification. the tools used for observation and collection were: sony nex f3 digital camera, sony cyber-shot dscw180 digital camera, canon dslr digital camera, rulers, micrometers, calipers, small meter roller, plastic for sample collection, scissors, cutter, paper for labeling, gps (global positioning system), dried herbarium collection equipment, flacon bottle, stereo microscope nikon smz 1500 equipped with a camera, nikon eclipse 50 light microscope equipped with nikon dsf1camera. materials for observation and collection comprise: aquadest, alcohol 70%, faa (formalin acetic alcohol) solution. steps of the study include: (1) photograph the specimens under natural conditions on site, (2) photograph the specimens under the dried herbarium process preparation conditions, (3) photograph the details of the flower, (4) making the dried herbarium, (5) photograph the dried herbarium specimens, (6) collecting and observing the structures of the preserved flowers, (7) observing and photographs the pollinia, (8) early identifying of specimens for the member of asclepiadaceae based on the book flora of java vol. 2 (backer and bakhuizen, 1965), (9) further identifying of specimens for other members of asclepiadaceae based on existing literature, (10) checking and matching with the herbarium types. results and discussion in the exploration, observation and assessment of wild plants in mount gedang atas baturagung mountains in yogyakarta, the author collected fruit from the vines family apocynaceae sub-family asclepiadoideae in a community forest located at s.07.48'.44.1” location; e.110.31.15.8" (widodo, 411m dpl, 26 september 2012). further, at the initial period of the rainy season at the same location, it was found the shoots with leaves and flowers as such that facilitated further identification s.07.48'.44.1 "; e.110.31.15.8 ". (widodo, 411m dpl, 31 december 2012). the fruits were initially identified by the author as the fruit of cynancum callialatum. another visit conducted at the beginning of the rainy season 31 december 2012 a set of data was obtained on the habitus and the structure of the specimen because leaves had been grown at the branches and they were flowering. at the same time, an observation at mount ijo which is approximately 5 km from the starting location and the author found the same plants which were also flowering. an identification using the book flora of java, vol. 2 (backer and bakhuizen, 1965: 273) mentioned that the plant identified was telosma. subsequent visitations and repeated observations were then completed on 19 january 2013 and 29 january 2013 to obtain a complete set of on the plant structure (figure 1 a, b, c, d, e, and figure 2). the specimen collection process was carried out in the laboratory for further observation. on 24 march 2013 visit, a young fruit was identified (figure 4). further identification results using the book flora of java, vol. 2 (backer and bakhuizen, 1965: 273) showed that this telosma held the characteristics of telosma accedens (bl.) back. a specimen comparison with the 1917 backer herbarium specimens collection in java (mnhn p03858922) or figure 1 f demonstrated the identified specimen compound flower structure of the specimen to be slightly different with the herbarium. the flowers units on the compound stalks converge or congregate in a way so that the compound flower compositions construct a sphere shape, meanwhile the compound flower at the herbarium exhibited characteristics that shaped like a bunch with accompanying braktea (supporting leaves). another comparison with the telosma procumbens herbarium specimens collection made by ramos 1918 from luzon, the philippines (mnhn p03858921) it was found that the obtained structure of the compound flower to be similar to the specimen of the identified telosma but there was a slight difference in terms of the units of flower. a single flower stalk and crown neck tube of the specimen was found to be shorter than that of the telosma procumbens. the centre of the crown round end when it was still in the form of bud looked much clearer in telosma procumbens. a comparison of the form and size of the fruit of telosma procumbens from the philippines in the united states national herbarium (usnh623771) collection was the same as the identified specimen but widodo – a discovery and characteristics description of telosma puberula … 49 the four sides of the fruit wing lines of the specimen identified were more apparent. from a comparison with the herbarium specimens of telosma puberula from the horsfield collection in 1859 of java (k000873052, k000873053) in figure 1 g, several characteristics were found to be the same in: the flowering type, size of the flower parts and shape as well as size of ovatus-oblong leaves. the sketched figure of pollinia in the herbarium (k000873053) also showed similarities with the pollinia of telosma identified in mount ijo (figure 3 a, b). characteristics of the pollinia of telosma puberula foundwere as shown in figure 3a. the location of the pollinia was upright next to the anther. the shape of the pollinia lobes was ovatus-oblong. the translator were very short. the corpuscullum or caudicle (caudicula) was round egg shaped almost like a short triangle, shiny brown. a comparison with the herbarium specimens of telosma puberula collected by kerr in 1928 from thailand (mnhn p03858930) was shown in figure 1h and another one by petelot in 1941 from tonkin (mnhn p03858929) there were some characteristics found to be the same in: the structure of compound flower, stalk compound flower, the size of the parts of the flower as well as the shape and size of the leaves. from the discussions, it was found that the specimen of telosma from mount gedang atas, mount ijo, mount parangan, mount mintorogo in baturagung mountains was telosma puberulens. figure 1. a. photo stature (habitus) of telosma puberula in the natural habitat found by the author. b. flowering branch of telosma puberula in the natural habitat found by the author. c. flowering branch of telosma puberula under the laboratory conditions. d. herbarium flower twig of young telosma puberula from the author’s collection. e. herbarium of the blooming twig flower telosma puberula from the author’s collection. f. herbarium type of telosma accedens (bl.) back. (mnhn, paris). g. herbarium type of telosma puberula (miq.) kerr. (© copyright of the board of trustees of the royal botanic gardens, kew). h. herbarium type of telosma puberula (miq.) kerr. (mnhn, paris). taxonomic information telosma puberula (miq.) kerr, fl. siam. enum. 3 (1): 32. 1951; pergularia puberula miq, fl. brit. india 4: 38. 1885. type: java, 1802 & 1859, t. horfsfield, k000873053, k000873053 (holotype, k!) description liana, stem 4 m or more, smooth surface, young puberulen twigs/branches. 3-6 cm; round-egg shaped to elongated elliptic leaf blade, 6-13 × 3-8 cm, thin, smooth to the puberulen along the vein, flat base to almost heart shape, tapered tip; lateral veins 4-6 pairs. extra axillary compound flower, umbrella to round shape, comprising a b c d e f g h 50 biology, medicine, & natural product chemistry 3 (2), 2014: 47-52 several to many flowers; compound flower stems 4-5 cm; flower stalk unit 1-1.5 cm. round-egg shaped petal lobes unit, 2 × 2.5 mm, slippery until puberulen, slightly hairy edges. bright green or greenish yellow crown, slightly fragrant, crown 1.5 cm long; crown tube 6-8 mm long, the tube length is equal to the length of the crown lobe; oblong crown lobes narrowed at the end, 6-8 mm long, 2-2 mm wide, slightly hairy at the edge tip, yellowish green, rounded ends folded out. rounded corona tip, folded out; additional parts of the corona (corollines corona) are higher than the position of pollinia, covering the anthers, pointed tip, thin, yellowish white color. suboblong pollinia lobe, the half-width long, upright, short triangular corpuscullum, short caudicle. follicle fruit, 12-18 cm long, 2-3 cm wide, almost rectangular cross-sectional sepal, with four wing lines on the long side; flat seed, round-egg shaped, 1-1.5 cm in diameter, 4-5 cm seed hair. flowering season from december to january, fruit formation: july to september. specimens examined baturagung mountains, mount gedang atas, s.07.48'.44.1 "; e.110.31.15.8 ", widodo, 411m dpl, 31/12/2012; baturagung mountains, mount ijo, s. 07 ', 47 ", 04.4"; e. 110, 30 ", 47.9", widodo, 415m dpl, 19/01/2013. figure 2. the structure of telosma puberula flower found by the author (a. compound flower, b. flower buds unit, c. blooming flower unit (side view), unit d. blooming flower unit (top view). figure 3. a. the structure of gynostegium and pollinia of telosma puberula flower found by the author (1. gynostegium, 2. corona and corollines corona, 3, 4. the orientation or layout of pollinia, 5, 6. pollinia unit and its parts. b. sketched figure of parts of gynostegium and pollinia of telosma in literature (1. sketch of corona in the telosma puberula herbarium (rbg kew), 2. sketch of pollinia unit in the telosma puberula herbarium (rbg kew), 3. satan pollinia telosma procumbens in the book of flora of china (1995: 273). a b c d 1 2 3 4 5 6 1 2 3 a b widodo – a discovery and characteristics description of telosma puberula … 51 figure 4. telosma puberula fruit found by the author. (a. young fruit, b. ripen fruit, c. fruit with seeds and hairless seed, d. wall fruit after the seeds release). note telosma puberula was found to be twining vines in cassia fistula and schoutenia ovata along with other vines, namely: dioscorea hispida, dioscorea alata, vitis trifolia, and gliricidia sepium. the initial specimen was found by the author during an exploration of the existence of plants of the genus capparis on site. the author found the telosma puberula was bearing some fruit on 24 march 2013 in mount parangan and mount ijo. on 13 april 2013, on the east side of mountain mintorogo the author also found telosma puberula bearing some fruit. conservation regarding the limited presence of this plant in the location of the discovery and other locations, it is then necessary to study the status of its cultivation range. along with the assessment processes, there is a need for live specimen preservations. conclusion telosma puberula (miq.) kerr was found in mount gedang atas and mount ijo, baturagung mountains, yogyakarta. the morphological characteristics of stature (habitus), leaves, of the telosma puberula flower specimen in mount gedang atas and mount ijo shows similarities with the kew herbarium holotype collected by horsfield in 1802 & 1859 (k000873053, k000873053) from java. the existence of telosma puberula (miq.) kerr in java complete and confirm the descriptions made by bakhuizen and backer (1965: 273) on the genus telosma. acknowledgements the author would like to express his gratitude to the herbarium museum national d'histoire naturelle, paris (mnhn) and the herbarium royal botanic garden of kewensis edinburg (kew) on the types of herbarium photo credit. the author would also thank mr. dr. m. jafar luthfi in the biology laboratory of uin sunan kalijaga yogyakarta for his testimony, discussion and confirmation of the discovery of this plant specimen on site. references backer, c. a. &bakhuizen. 1965. flora of jawa (spermatophytes only).vol i, ii, iii. groningen: n. v. p. noordhoff. backer, c. a. 1949. beknopte flora van java (nood unitgave) 8(a. fam. 173): 63. 1949. http://www.tropicos.org/name/2609608 accessed 7 october 2014. blume, c. l. 1826. bijdragen tot de flora van nederlandsch indië 1056. flora of china editorial committee. 1995. flora of china (gentianaceae through boraginaceae). 16: 1–479. in c. y. wu, p. h. raven & d. y. hong (eds.) fl. china. science press & missouri botanical garden press, beijing & st. louis.http://www.tropicos.org/image/22 934. accessed 7 october 2014. herbarium museum national d’ histoire naturelle paris (mnhn). 2014. pergularia accedens. http:// colb .mnhn. fr/catalognumber/mnhn/p/ p03858919., accessed 7 october 2014. herbarium museum national d’histoire naturelle paris (mnhn). 2014. telosma procumbens. http:// colb .mnhn .fr/catalognumber/mnhn/p/ p03858921., accessed 7 october 2014. herbarium museum national d’ histoire naturelle paris (mnhn). 2014. telosma accedens. http:// colb .mnhn. fr/catalognumber/mnhn/p/ p03858922., accessed 7 october 2014. a b c d 52 biology, medicine, & natural product chemistry 3 (2), 2014: 47-52 herbarium museum national d’ histoire naturelle paris (mnhn). 2014. telosma puberula. http:// colb. mnhn. fr/catalognumber/mnhn/p/ p03858929., accessed 7 october 2014. herbarium museum national d’ histoire naturelle paris (mnhn). 2014. telosma puberula. http:// colb. mnhn. fr/catalognumber/mnhn/p/ p03858930.,accessed 7 october 2014. hooker, j. d. 1885. flora of british india (vol. iv). london: reeve and co. http://specimens.kew.org/herbarium/k0 00873052. diakses 7 oktober 2014 http://www.tropicos.org/name/2602046. accessed 7 october 2014. ipni (international plant name index). 2014. telosma (http:// www. plantsystematics. org, accessed 7 october 2014. kerr, a. f. g. 1951. flora siamensis enumeratio 3(1): 32. http:// www. tropicos. org/name/2609609. accessed 7 october 2014. merill, e.d. 1912. philippine journal of science 7: 243. http:// www. tropicos. org/ name/2607466. accessed 7 october 2014. royal botanic garden, kew. 2014. telosma puberula. singh, g. 1999. plant systematics. new hampshire: science publisher. takhtajan, a. 2009. flowering plant. st petersburg: springer. the plant list (2010). version 1. published on the internet; http:// www.theplantlist .org/ tpl/ search?q=telosma+ . diakses 7 oktober 2014. united states national herbarium. 2014. telosma procumbens. https://www. flickr. com/ photos/ filibot/8045825440. accessed 7 october 2014. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 825-829 | doi: 10.14421/biomedich.2025.142.825-829 issn 2540-9328 (online) isolation, morphological characterization, biochemistry, and antagonistic activity of endophytic bacteria from rice against xanthomonas oryzae pv. oryzae saipul sihotang1*, alfons dwi putra butar-butar1, lita nasution2, haliatur rahma3, trizelia3, magdalena saragih2, retna astuti kuswardani1, ika listiana4, beatrix sofranes napitupulu5 1department of agrotechnology, faculty of agriculture, university of medan area, medan, indonesia. 2departement of agrotechnology, faculty of science and technology, university of quality, medan, indonesia. 3department of plant protection, faculty of agriculture, university of andalas, padang, indonesia. 4department of biology, faculty of science and technology, raden intan state islamic university of lampung, lampung, indonesia. 5department of agrotechnology, faculty of agriculture, university of sumatera utara, medan, indonesia. corresponding author* saipulsihotang@staff.uma.ac.id manuscript received: 19 august, 2025. revision accepted: 13 october, 2025. published: 17 october, 2025. abstract endophytic bacteria have the potential as environmentally friendly biocontrol agents in controlling bacterial leaf blight (blb) in rice caused by xanthomonas oryzae pv. oryzae (xoo). this study aims to isolate, characterize morphology and biochemical tests, and test the antagonistic activity of endophytic bacteria from rice against xoo. isolation was carried out from healthy rice root tissue using a surface sterilization method. morphological characterization included colony shape, color, edge, elevation, and gram properties, while biochemical characterization included catalase, oxidase, motility, starch hydrolysis, and sugar fermentation tests. the antagonistic test used a dual culture assay method on na (nutrient agar) media. the isolation results obtained six endophytic bacterial isolates (betp 01– 06) identified as the genus pseudomonas and bacillus with varying colony morphological characters. antimicrobial activity test showed two potential isolates, betp 03 (pseudomonas) and betp 05 (bacillus), with an average inhibitory diameter of 16.3 mm and 16.5 mm against xoo, respectively. biochemical characterization showed that betp 03 was bacillus, gram-negative, citrate-negative, motile, gelatin hydrolysis positive, starch hydrolysis positive, and catalase positive; while betp 05 was bacillus, gram-positive, citrate-positive, motile, gelatin hydrolysis negative, starch hydrolysis positive, and catalase positive. these results indicate that both isolates have high potential as biological biocontrol agents in controlling bacterial leaf blight disease in rice. keywords: biocontrol; endophytic bacteria; oryza sativa; xanthomonas oryzae pv. oryzae. introduction rice (oryza sativa l.) is a major food crop and a source of carbohydrates for a large portion of the world's population, particularly in asia. rice productivity in indonesia is frequently threatened by various important diseases, including bacterial leaf blight (blb) caused by xanthomonas oryzae pv. oryzae (xoo) (halim et al., 2020). this disease can cause yield losses of up to 50% in susceptible varieties and is endemic in rice production centers (mew et al., 1993). xoo infection is characterized by longitudinal necrosis of leaves starting from the edges, reducing photosynthetic area and grain quality (ou, 1985). blb control is generally achieved through resistant varieties and chemical pesticides. however, selection pressure can lead to the emergence of new pathogen strains that overcome varietal resistance, while excessive use of chemical pesticides has negative impacts on the environment, human health, and soil microflora (adhikari et al., 1995). therefore, effective, environmentally friendly, and sustainable alternative control strategies are needed. endophytic bacteria are microorganisms that live within plant tissues without causing disease symptoms in their host (sihotang et al., 2022; sihotang et al., 2025). the presence of endophytic bacteria in rice plants has been widely reported to provide benefits, including increased growth, boosting plant resilience through the induction of systemic resistance, and producing secondary metabolites with antibacterial activity against pathogens (ryan et al., 2008). pathogen inhibition mechanisms by endophytic bacteria include the production of antibiotics, siderophores, hydrolytic enzymes, and competition for nutrients and growth space (compant et al., 2005). isolation and characterization of rice endophytic bacteria are important to determine their phenotypic https://doi.org/10.14421/biomedich.2025.142.825-829 826 biology, medicine, & natural product chemistry 14 (2), 2025: 825-829 diversity, physiological properties, and biocontrol potential. morphological and biochemical characterization provide initial information regarding the identity and metabolic capabilities of the isolates, while antagonistic testing against xoo can identify potential candidate isolates as biocontrol agents for rice endophytes. this research was conducted in tanjung gusta village, deli serdang regency, north sumatra, one of the rice farming centers in the region. this area has a rice paddy agro-ecosystem that supports a diversity of endophytic microbes, but there has been minimal exploration of the potential of local endophytic bacteria as biological control agents for blb. therefore, this study aimed to isolate endophytic bacteria from healthy rice tissue, characterize their morphological and biochemical properties, and evaluate their antagonistic activity against xanthomonas oryzae pv. oryzae. the results are expected to provide fundamental information for developing endophytic bacteria-based biocontrol agents that support sustainable rice farming systems.*citation in manuscript is written in the “name and year” system; and is arranged from oldest to newest and from a to z. in citing an article written by two authors, both of them should be mentioned, however, for three and more authors, only the last (family) name of the first author is mentioned, followed by et al. (not italic), for example: saharjo and nurhayati (2006) or (boonkerd 2003a, b, c; sugiyarto, 2004; el-bana and nijs, 2005; balagadde et al., 2008; webb et al., 2008). the extent citation as shown with the word “cit” should be avoided. reference to unpublished data and personal communication should not appear in the list but should be cited in the text only (e.g., rifai ma 2007, pers. com. (personal communication); setyawan ad 2007, unpublished data). a total of 80% of the references should be from scientific journals published in the last five years, except for taxonomic studies. names of journals should be abbreviated according to the issn list of title word abbreviations (www.issn.org/222661-ltwa-online.php). materials and methods materials the research was conducted from october 2023 to january 2024. isolation and characterization of endophytic bacteria, as well as antagonistic activity tests, were conducted at the microbiology laboratory of the faculty of mathematics and natural sciences, university of north sumatra, medan. the materials used were healthy rice root samples of xanthomonas oryzae isolate, na, 70% alcohol, clorox, distilled water, and agrept (bactericide). the tools used in this study included petri dishes, erlenmeyer flasks, measuring cylinders, bunsen burners, hotplates, glass slides, loop needles, cover glasses, auto-aerators, scissors, microscopes, analytical balances, stationery, cameras, vernier calipers, and scalpel blades. methods the rice root sampling technique was based on dyah (2020). the rice used in this study was healthy rice, covering an area of 1 hectare. the rice roots used in this study were taken from tanjung gusta village, deli serdang regency, north sumatra province. isolation of endophytic bacteria was performed by washing rice plant roots with running water and drying them aseptically. the samples were then taken to a laminar air flow cabinet, cut into 1-2 cm pieces, and then surface sterilized by immersing the samples in 70% alcohol for 1 minute, 5% crolox for 2 minutes, 70% alcohol for 30 seconds, and sterile distilled water for 2 minutes. the samples were then dried with sterile tissue. the rice root samples were placed on a na surface containing chitin colloids, lightly pressed, and incubated for 24 hours in an incubator at 37°c. all growing bacterial colonies were purified by transferring the isolates to petri dishes containing na media. morphological, macroscopic, microscopic, and biochemical tests/observations were performed. if the bacteria growing were still mixed with other bacteria, they were purified again. this serves to obtain pure/superior endophytic bacterial isolates. (sihotang et al., 2023) the xanthomonas oryzae bacterial isolates were collected from the university of medan area's plant protection laboratory. the isolates were rejuvenated by taking a single colony growing on a culture plate with a loop needle and then inoculating it into a slanted medium in a test tube in a zigzag pattern. the test tubes were wrapped in paper and incubated in an incubator for 24 hours (yanti & rosmania, 2020). the antimicrobial test used the disc diffusion method. the test bacteria used were endophytic, specifically xanthomonas oryzae pv. oryzae. after the bacterial medium was poured into a petri dish and allowed to solidify, a suspension of xanthomonas oryzae and six endophytic bacteria was prepared, then turbidity was measured at an mcf of 0.5. the surface of the mha medium was evenly coated with the xanthomonas oryzae pv suspension using a sterile cotton swab. a 6 mm diameter paper disc was then placed on the surface of the mha medium, which had previously been dipped in the endophytic bacterial suspension. the petri dish was then stored in an incubator at 37°c. the clear zone formed was observed for 1 x 24 hours (1 day). the study used a non-factorial completely randomized design (crd) consisting of 7 treatments with 3 replications. the tested treatments consisted of: p₀ : control p₁ : use of endophytic bacteria isolate 01 p₂ : use of endophytic bacteria isolate 02 p₃ : use of endophytic bacteria isolate 03 sihotang et al. – isolation, morphological characterization, biochemistry, and … 827 p₄ : use of endophytic bacteria isolate 04 p₅ : use of endophytic bacteria isolate 05 p₆ : use of endophytic bacteria isolate 06 p7 : synthetic bactericide the inhibition test followed the diffusion method. according to ekowati 2000 (in herlina, 2011), the inhibition test followed the diffusion method. the method was discovered by kirby-alfred baurer in 1966. formula: 𝐼𝐴 = (𝐷𝑧 − 𝐷𝑐) / 𝐷𝑐 description: ai : antimicrobial index dz : diameter of inhibition zone (mm) dc : diameter of disc or well (mm) data analysis data analysis was performed using one-way anova, following tests for normality (shapiro-wilk) and homogeneity (levene’s test). if the data met normality and homogeneity assumptions (p > 0.05), post duncan tests were used to analyze group differences. if data were not normally distributed, the kruskal-wallis test was used, followed by mannwhitney u tests for pairwise comparisons when significant differences were observed. results and discussion morphological characteristics of endophytic bacterial isolates the isolation of endophytic bacteria from rice roots yielded six dominant isolates. this was based on morphological characteristics, including shape, margins, elevation, and color. table 1. morphological characteristics of endophytic bacterial isolates. code shape margin elevation color betp 01 (pseudomonas) circular entire convex whitish-yellow betp 02 (bacillus) rhizoid rhizoid convex white betp 03 (pseudomonas) irregular undulate convex purple betp 04 (bacillus) irregular lobate convex whitish-yellow betp 05 (bacillus) irregular curled convex white betp 06 (bacillus) circular entire convex yellow microskopic characterization and gram staining of endophytic bacteria table 2. microskopic characterization and gram staining of endophytic bacteria. code shape arrangement gram betp 01 bacillus monobacillus betp 02 bacillus streptobacillus + betp 03 bacillus monobacillus betp 04 bacillus monobacillus + betp 05 bacillus monobacillus + betp 06 bacillus streptobacillus + biochemical tests of endophytic bacteria table 3. biochemical tests of endophytic bacteria. code citrate sim / motility gelatin hydrolysis starch hydrolysis catalase ph betp 01 + + + 6 betp 02 + + + + 6 betp 03 + + + + 6 betp 04 + + + + + 6 betp 05 + + + + 6 betp 06 + + + 6 828 biology, medicine, & natural product chemistry 14 (2), 2025: 825-829 antimicrobial test of endophytic bacteria against xoo table 4. antimicrobial test of endophytic bacteria against xoo. code average inhibition zone diameter (mm) notation index control 6.00 d 0 betp 01 8.83 cd 0.4 betp 02 11.60 c 0.9 betp 03 16.50 b 1.75 betp 04 11.53 c 0.9 betp 05 16.23 b 1.7 betp 06 9.53 c 0.5 betp 01 28.00 a 3.6 duncan's follow-up test results α= 0.05 figure 1. betp01 (gram negative-bacteria); betp02 (gram positive-bacteria). discussion the study results showed that endophytic bacterial isolates from rice (oryza sativa) plants consisted of several dominant genera, namely pseudomonas and bacillus. morphological characterization, gram staining, and biochemical tests revealed significant variation among isolates. most isolates exhibited high catalase, starch hydrolysis, and motility capabilities, indicating their potential for adaptation to host plant tissues. these results are consistent with reports that the bacillus and pseudomonas genera are the most common endophytic bacterial groups that play a role enhance rice plant growth and resistance to pathogens. antagonistic tests against xanthomonas oryzae pv. oryzae (xoo), the causative agent of bacterial leaf blight in rice, showed that isolates betp 03 (pseudomonas) and betp 05 (bacillus) produced the largest inhibition zones, with diameters >16 mm. this confirms their ability to produce antimicrobial secondary metabolites such as siderophores, antibiotics, and hydrolytic enzymes. these findings align with recent research confirming the effectiveness of pseudomonas fluorescens and bacillus subtilis in inhibiting xoo growth through antibiosis mechanisms and induction of systemic resistance in rice. furthermore, hydrolytic enzyme activity in several isolates supports their role as biocontrol agents. enzymes such as chitinase, protease, and lipase are known to be able to damage pathogen cell walls, thereby strengthening plant defenses. biochemical tests showed that isolates betp 04 and betp 05 exhibited a combination of citrate, gelatinase, and catalase activity, strengthening the hypothesis that these isolates have the potential to be used as both a biofertilizer and a biopesticide. from a microbial ecology perspective, the presence of endophytic bacteria within rice tissues provides a competitive advantage over epiphytic microbes, as they can survive in the relatively stable internal environment of the plant. thus, endophytes can play a dual role: increasing plant nutrient availability (for example, through nitrogen fixation or phosphate solubilization) and protecting the plant from pathogen attack. however, this study also has limitations, namely the lack of molecular analysis (16s rrna) to ensure accurate isolate identification, as well as field trials to confirm the effectiveness of biocontrol in real agroecosystem conditions. further studies are strongly recommended to explore the molecular mechanisms of antimicrobial metabolite production and the formulation of endophyte consortia as environmentally friendly biopesticides. thus, the results of this study provide strong evidence that the endophytic bacteria pseudomonas and bacillus from oryza sativa have high prospects as biocontrol agents to reduce dependence on chemical pesticides in sustainable rice cultivation systems. betp 01 betp 02 sihotang et al. – isolation, morphological characterization, biochemistry, and … 829 conclusions in conclusion, six isolates of endophytic bacteria from rice plants were produced. the isolates exhibited varying antimicrobial activity, as indicated by the large diameter of the inhibition zone. two endophytic bacterial isolates with the highest potential to inhibit the tested pathogenic microbes were isolates coded betp 03 pseudomonas and betp 05 bacillus, with average values of 16.3 and 16.5, respectively. betp 03 was characterized as a bacillus, gram-negative, citrate-negative, positive motility, positive gelatin hydrolysis, positive starch hydrolysis, and a positive catalase test. betp 05 was characterized as a bacillus, gram-positive, citrate-positive, positive motility, negative gelatin hydrolysis, positive starch hydrolysis, and a positive catalase test. acknowledgements: the authors would like to express their deepest gratitude to universitas medan area (uma) for the support and facilities provided during the completion of this research. authors’ contributions: saipul, alfons, beatrix & magdalena carried out the laboratory work and analyzed the data. lita, ika, haliatur & retna wrote the manuscript. competing interests: the authors declare that there are no competing interests. references adhikari, t. b., mew, t. w., & teng, p. s. 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(2023). insights into rice endophytes for disease suppression and growth promotion. agriculture, 13(2), 286. https://doi.org/10.3390/agriculture13020286 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 757-761 | doi: 10.14421/biomedich.2025.142.757-761 issn 2540-9328 (online) detection of escherichia coli contamination using most probable number (mpn) methods of jamu pahitan in singaparna district, tasikmalaya syifa sri rahmawati, richa mardianingrum, susanti susanti* department of pharmacy, faculty of health science, universitas perjuangantasikmalaya, jl. peta no. 117, kahuripan, kecamatan tawang, kab.tasikmalaya, jawa barat 46115, indonesia. corresponding author* susanti@unper.ac.id manuscript received: 12 june, 2025. revision accepted: 18 september, 2025. published: 01 october, 2025. abstract indonesians consume a lot of traditional jamu, including jamu pahitan. however, the hygiene aspect in the production and serving process is often neglected, so it has the potential to be contaminated with escherichia coli. using the mpn method, this study analyzed the presence of e. coli in jamu pahitan sold in singaparna district. a total of 10 samples were tested, taken from jamu vendors who used plastic bottle packaging. the mpn test results showed that all samples contained e. coli with mpn values ranging from 6.0-1100 g/ml. none of the samples met food safety standards based on sni 7388:2009, because the maximum limit of e. coli in drinks is <3 mpn/ml. the results of gram staining of bacteria showed that the bacteria found were bacilli, red in color, including gram-negative bacteria, indicating the presence of e. coli bacteria. keywords: escherichia coli; food safety; gram-negative; jamu pahitan; mpn method. introduction jamu is one of the traditional forms of treatment traditional that has been used in a way passed down from generation to generation by the indonesian people. estimated that around 70–80% of people in developing countries still depend on traditional treatment, including jamu, as a main means of guarding health and treating various diseases. in development, jamu has experienced various innovation in formulation, packaging, and methods of its distribution along with increasing public awareness, which will return to nature (back to nature) (biofarmaka, 2024). one of the types of jamu consumed by the indonesian people is jamu pahitan, which is known because its very bitter taste. jamu is generally made from a mixture of jamu ingredients such as leaf sambiloto (andrographis paniculata ness) and stems (tinospora crispa), two plant drugs that contain bioactive compounds such as andrographolide, saponins, flavonoids, and alkaloids. leaves sambiloto are known to have antidiabetic, immunostimulant, and antibacterial activity (paramitha & rahmanisa, 2016), whereas brotowali is used as an antipyretic, antidiabetic, and detoxification agent (malik, 2015). however, behind its popularity, production and sales of jamu pahitan specifically in the form of jamu cradle still face a big challenge from the side of sanitation and safety. many sellers of jamu cradle still use plastic packaging bottles that are used repeatedly without an adequate sterilization process. this is an open opportunity for the occurrence of contamination microorganisms, especially bacterial pathogenic as escherichia coli (ruwana et al., 2017). e. coli is a bacterial indicator of pollution commonly found in the environment that is not hygienic. the existence of e. coli in food or drink becomes a marker of existence the possibility of contamination from water, equipment, or contaminated raw materials (rahayu, 2018). some strains of e. coli are natural pathogens and can cause digestive disturbance, such as diarrhea, vomiting, or even complications like uremic hemolytic syndrome (manetu et al., 2021). therefore, the existence of e. coli in jamu cannot be tolerated. indonesian national standard (sni) no. 7388 of 2009 stipulates a limit maximum limit of contamination of e. coli in the product food liquid of <3 mpn/ ml. if the content exceeds the limit said, then the product is considered unworthy of consumption. a study previously conducted by arum et al. (2022) showed that of six sample jamu pahitan tested in east karawang, five of them have an e. coli mpn value that exceeds the safe limit. this is associated with the habit of repeatedly using bottles without thoroughly cleaning https://doi.org/10.14421/biomedich.2025.142.757-761 758 biology, medicine, & natural product chemistry 14 (2), 2025: 757-761 them. the surface of the bottle seen blackened and crusted, showing a lack of attention to cleanliness in tool production. a similar situation was also found in the district of singaparna, regency tasikmalaya. based on data from the district health office tasikmalaya (2024), recorded as many as 753 cases of diarrhea were recorded in year. results of field observations show that there are still 10 sellers of jamu 10 sellers. it is known that part of the problem is that they mix jamu pahitan from mixture sambiloto and brotowali, which is then packed in bottles without a standard sterilization process. to determine the for know existence and quantity of contamination by bacteria escherichia coli, the mpn method was used. this method was chosen because it is quantitative and capable of estimating the population of bacteria based on results of fermentation in liquid media through three stage testing, namely the prediction, confirmation and complementary tests (hafsan, 2014). based on the background behind said, research this done to analyze pollution bacteria escherichia coli in jamu pahitan sold in the district area of singaparna using the mpn method. research this expected can give a description of quality microbiological jamu pahitan in the field as well as push effort to improve sanitation and safety in production jamu traditional, in order to protect the health of the public. materials and methods materials this study was done from october 2024 to april 2025 in the laboratory biology of the university of struggle tasikmalaya. tools used in the research this includes, among other tools glass (pyrex) such as glass measure, glass chemistry, stem stirrer, tube reaction and erlenmeyer, incubator (memmert), glass pipette , rack tube , scales, petri dish, tube round, wire ose, bunsen, durham tube, autoclave (gea medical), microscope (boeco) and laf (laminar air flow), vortex (oregon). materials used in the research: this is a jamu pahitan liquid that is sold by traders of jamu carry and use plastic packaging bottles. the chemicals used are lb (lactose broth) agar media (himedia), bglb (brilliant green lactose broth) (merck), emb (eosin methylene blue) (oxoid), crystal violet (merck), cotton (selection), lugol rofa), paper umbrella (asturo), safranin (o merck), immersion oil (rofa), and 95% alcohol. methods this study used an experimental approach to analyze the presence of escherichia coli bacteria contamination in jamu pahitan sold in the singaparna district. the samples used were 10 liquid jamu pahitan packaged in plastic bottles from 10 different sellers, each 100 ml, which were then filtered using filter paper to separate the dregs from the liquid. furthermore, the samples were tested using the most probable number (mpn) method which consists of three stages: presumptive test, confirmatory test, and complementary test, and continued with gram staining to ensure the morphology and classification of bacteria. in the initial stage, the presumptive test was carried out by inoculating three sample dilutions (0.1 ml, 0.01 ml, and 0.001 ml) into lactose broth (lb) media containing durham tubes, then incubated at 37°c for 24– 48 hours. the presence of gas in the durham tube indicates a positive result and is continued to the confirmatory test. the confirmatory test was carried out by transferring 1 loop from the positive tube to brilliant green lactose bile broth (bglb) media, then incubating again at 37°c for 24–48 hours. a positive result is indicated by the formation of gas in the durham tube. the next complementary test uses eosin methylene blue agar (emba) media, where a positive result is indicated by the formation of metallic green or pink colonies which are characteristic of escherichia coli. after the three stages of the mpn test were completed, gram staining was carried out to ensure that the bacteria found were gram-negative in the form of bacilli, which is the typical morphology of e. coli. staining was carried out using crystal violet, lugol's solution, 95% alcohol, and safranin, then observed under a microscope at 1000x magnification. red bacteria indicated that they were gram-negative. finally, the mpn value was calculated based on the combination of positive tubes in each dilution by referring to the standard mpn table, and compared with the microbial contamination standard based on sni no. 7388 of 2009, where the maximum limit allowed for e. coli in drinks is <3 mpn/ml (saridewi et al., 2017). results and discussion in this study, samples of jamu pahitan packaged in plastic bottles were collected from ten different jamu sellers in singaparna district. each sample was diluted 3 times, each repeated 3 times (triple). furthermore, the samples were tested using the mpn (most probable number) method. mpn test results results of the presumptive test the initial stage of the study was the presumptive test, which aimed to determine the presence of coliform bacteria in the jamu pahitan of samples. this test used lb media. coliform bacteria such as escherichia coli ferment sugar (lactose) in lb media. the lactose fermentation process produces acid and gas. the appearance of turbidity and gas produced by the activity of bacteria that convert lactose into lactic acid indicates a positive result of lactose fermentation. the presumptive test results can be seen in figure 1. rahmawati et al. – detection of escherichia coli contamination … 759 figure 1. observation results of the prediction test on jamu pahitan (personal documentation, 2025). carbon gas will enter the durham tube through a tightly closed test tube (putri & kurnia, 2018). turbidity in the lactose medium and the gas produced in the durham tube indicate the formation of acid. the results of the prediction test on jamu pahitan sold in the singaparna district area can be seen in table 1. table 1. results of the presumptive test using lb media sample dilution rate mpn/g value 0.1 ml 0.01 ml 0.001 ml a 3 2 2 210 b 3 1 3 160 c 3 2 2 210 d 3 2 3 290 e 2 3 3 53 f 3 3 3 >1100 g 2 1 3 34 h 3 3 3 >1100 i 3 3 3 >1100 j 3 3 3 >1100 note: values 1, 2, and 3 are the number of positive tubes from the sample. the results of table 1. show that the positive jamu pahitan samples have mpn values of 34-1100 g/ml for coliform bacteria. each sample has passed the presumptive test stage, which was continued to the second test, namely the confirmation test because all jamu pahitan samples showed positive results. result of the confirmed test the test was confirmed using selective media for coliform bacteria, namely bglb. the purpose of the confirmation test is to strengthen the previous suspicion test regarding the presence of coliform bacteria in the sample. the results of the confirmation test can be seen in figure 2. figure 2. observation results of the confirmed test on jamu pahitan (personal documentation, 2025). the results of the confirmation test on jamu pahitan sold in singaparna district can be seen in table 2. table 2. results of the confirmed test using bglb media sample dilution rate mpn/g value 0.1 ml 0.01 ml 0.001 ml a 2 2 2 35 b 2 1 2 27 c 0 1 1 6.1 d 1 0 1 7.2 e 1 1 0 7.4 f 0 2 0 6.2 g 0 0 2 6.0 h 2 2 0 21 i 2 2 1 28 j 2 1 1 20 note: values 0, 1, 2 are the number of positive tubes from the sample. the results of table 2 show that the jamu pahitan samples have an mpn value of escherichia coli bacteria of 6.0-35 g/ml, exceeding the maximum limit of escherichia coli bacteria contamination in jamu, as specified in sni no. 7388 of 2009, which is <3 mpn/ml. result of the completed test the complete test was conducted using selective media eosin methylene blue agar (emba). emba is a selective medium for growing gram-negative bacteria such as escherichia coli and inhibiting the growth of gram-positive bacteria. the results of the complementary test can be seen in figure 3. 760 biology, medicine, & natural product chemistry 14 (2), 2025: 757-761 figure 3. observation results of the completed test on jamu pahitan (personal documentation, 2025) the results of the complementary test on jamu pahitan sold in singaparna district can be seen in table 3. table 3. results of the completed test using emb media. no sample code observation result 1 10 -1 a 1 pink 2 10 -1 a 3 pink 3 10 -2 a 1 metallic green 4 10 -2 a 3 metallic green 5 10 -3 a 1 metallic green 6 10 -3 a 2 metallic green and pink 7 10 -1 b 1 metallic green and pink 8 10 -1 b 3 pink 9 10 -2 b 1 pink 10 10 -3 b 1 metallic green 11 10 -3 b 3 pink 12 10 -2 c 2 pink 13 10 -3 c 2 metallic green and pink 14 10 -1 d 3 pink 15 10 -3 d 2 metallic green and pink 16 10 -1 e 2 pink 17 10 -2 e 1 metallic green 18 10 -2 f 1 pink 19 10 -2 f 2 pink 20 10 -3 g 2 metallic green and pink 21 10 -3 g 3 pink 22 10 -1 h 1 metallic green and pink 23 10 -1 h 2 pink 24 10 -2 h 1 metallic green and pink 25 10 -2 h 2 pink 26 10 -1 i 1 metallic green and pink 27 10 -1 i 2 metallic green and pink 28 10 -2 i 1 pink 29 10 -2 i 2 metallic green and pink 30 10 -3 i 1 pink 31 10 -1 j 1 pink 32 10 -1 j 2 metallic green and pink 33 10 -2 j 3 pink 34 10 -3 j 2 pink note: p ink, metallic green color indicates the presence of escherichia coli bacteria. 10-¹ indicates sample dilution 1, a 1 indicates sample a first repeat. table 3. shows that all samples tested on emba media showed the growth of colonies with metallic green and pink colors. these colors indicate the presence of escherichia coli bacteria which are indicators of fecal coliform bacteria. bacterial gram staining test results gram staining test results*, in the field of bacteriology, gram staining of bacteria differentiates groups of bacteria into gram-positive and gram-negative bacteria. the results of the gram staining test of bacteria can be seen in figure 4. bacteria. figure 4. results of observations of gram staining tests of bacteria on jamu pahitan (personal documentation, 2025). the samples examined were samples that in previous tests produced metallic green/pink colonies on emba media. the results of the gram staining test on jamu pahitan sold in the singaparna district area can be seen in table 4. table 4. results of bacterial gram staining test. no. sample code results note color form 1 10 -1 a 1 red basil gram negative 2 10 -1 a 2 red basil gram negative 3 10 -1 a 3 red basil gram negative 4 10 -2 a 1 red basil gram negative 5 10 -1 b 1 red basil gram negative 6 10 -2 b 3 red basil gram negative 7 10 -3 b 1 red basil gram negative 8 10 -1 c 1 red basil gram negative 9 10 -1 c 2 red basil gram negative 10 10 -1 c 3 red basil gram negative 11 10 -2 c 1 red basil gram negative 12 10 -2 c 3 red basil gram negative 13 10 -3 c 3 red basil gram negative 14 10 -1 d 1 red basil gram negative 15 10 -1 d 2 red basil gram negative 16 10 -2 d 1 red basil gram negative 17 10 -2 d 3 red basil gram negative 18 10 -1 e 2 red basil gram negative 19 10 -2 e 1 red basil gram negative 20 10 -2 e 2 red basil gram negative 21 10 -1 f 1 red basil gram negative 22 10 -1 f 2 red basil gram negative 23 10 -2 f 1 red basil gram negative 24 10 -3 f 3 red basil gram negative 25 10 -1 g 1 red basil gram negative 26 10 -1 g 3 red basil gram negative 27 10 -2 g 2 red basil gram negative 28 10 -3 g 2 red basil gram negative 29 10 -1 h 1 red basil gram negative 30 10 -1 h 2 red basil gram negative 31 10 -1 i 1 red basil gram negative 32 10 -1 j 2 red basil gram negative note: red indicates gram-negative bacteria, purple indicates gram-positive bacteria. 10-¹ indicates sample dilution 1, a 1 indicates sample a first repeat. rahmawati et al. – detection of escherichia coli contamination … 761 table 4.5 shows that the sample contains gramnegative bacteria, which is indicated by the red staining test results, this is because gram-negative bacteria have a thinner wall layer, so that when these bacteria are rinsed, the secondary red dye safranin replaces the purple color of crystal violet. (ampou et al., 2015). in addition, the observed bacillus shape shows that the sample contains e. coli bacteria which are included in the group of gramnegative bacteria in the form of bacillus. discussion the results of the study showed that all samples of jamu pahitan obtained from singaparna district were indicated to be contaminated by e. coli bacteria based on the most probable number (mpn) method. this was shown through the results of the prediction test which showed gas formation and turbidity in lb media, the results of the confirmation test with bglb media which also showed gas formation, and the results of the complementary test on emba media which showed metallic green and pink colonies characteristics of e. coli. this finding was reinforced by the results of gram staining which showed that the bacterial isolates were gram-negative in the form of bacilli, which was in accordance with the morphology of e. coli (rahmatullah et al., 2021). the mpn values obtained from most samples exceeded the threshold set in sni no. 7388 of 2009, which is <3 mpn/ml, with some samples even reaching >1100 mpn/ml. this indicates that the jamu pahitan sold in the area does not meet food safety standards. this contamination is most likely caused by the lack of adequate sanitation in the production and serving of jamu, such as the repeated use of used plastic bottles without proper sterilization, as well as environmental conditions that allow the product to be contaminated by fecal bacteria (fhitryani et al., 2017). the high level of e. coli contamination in jamu pahitan products is a serious warning considering that this bacteria is an indicator of sanitation and its presence in consumer products indicates fecal contamination. infection due to e. coli can cause digestive tract disorders such as diarrhea, which is potentially dangerous especially for vulnerable groups such as children and the elderly (manetu et al., 2021). therefore, jamu sellers need to implement good hygiene practices, including sterilization of equipment, selection of clean raw materials, and use of safe packaging containers that are not reused without adequate sanitation. conclusions based on the results of the study regarding "detection of escherichia coli contamination using most probable number (mpn) methods of jamu pahitan in singaparna district tasikmalaya", it can be concluded that all samples of jamu pahitan were polluted with escherichia coli bacteria, with mpn values exceeding the maximum limit of <3 mpn/ml according to sni no. 7388 of 2009. contamination this is allegedly a consequence of low standard sanitation in the processing and use of packaging that is used without sterilization. conditions this potentially endangers the health of consumers, so there is a need for education and supervision to ensure the security traditional jamu consumption. competing interests: the authors declare that there are no competing interests. references ampou, e. e., triyulianti, i., & nugroho, s. c. (2015). bakteri asosiasi pada karang scleractinia kaitannya dengan fenomena la-nina di pulau bunaken. jurnal kelautan nasional, 10(2), 55–63. arum, r., & ratnasari, d. (2022). cemaran coliform dan identifikasi bakteri escherichia coli pada jamu gendong di kecamatan karawang timur. parapemikir: jurnal ilmiah farmasi, 11(3), 224–232. biofarmaka, i. p. b. (2024, november 12). quality of herbal medicine plants and traditional medicine. http://biofarmaka. ipb. ac. id. fhitryani, s., suryanto, d., & karim, a. (2017). pemeriksaan escherichia coli, staphylococcus aureus dan salmonella sp. pada jamu gendong yang dijajakan di kota medan. biolink (jurnal biologi lingkungan industri kesehatan), 3(2), 146– 155. hafsan. (2014). mikrobiologi analitik. alauddin university press. malik, m. m. (2015). the potential of brotowali stem extract (tinospora crispa) as analternative antimalarial drug. jurnal j majority, 4(5), 2–8. manetu, w. m., m’masi, s., & recha, c. w. (2021). diarrhea disease among children under 5 years of age: a global systematic review. open journal of epidemiology, 11(03), 207–221. https://doi.org/10.4236/ojepi.2021.113018 paramitha, m. d., & rahmanisa, s. (2016). ekstrak etanol herba sambiloto (andrographis paniculata) sebagai antidiabetik terhadap mencit wistar terinduksi aloksan. medical journal of lampung university [majority], 5(5), 75–79. putri, a. m., & kurnia, p. (2018). identifikasi keberadaan bakteri coliform dan total mikroba dalam es dung-dung di sekitar kampus universitas muhammadiyah surakarta. media gizi indonesia, 13(1), 41. https://doi.org/10.20473/mgi.v13i1.41-48 rahayu w.p. (2018). escherichia coli: patogenitas, analisis dan kajian risiko. ipb press. rahmatullah, w., novianti, e., & sari, a. d. l. (2021). identifikasi bakteri udara menggunakan teknik pewarnaan gram. jurnal ilmu kesehatan bhakti setya medika, 6(2), 83– 91. ruwana, i., astuti, s., & sugiharto, t. (2017). inovasi jamu celup dalam upaya peningkatan ekonomi pedagang jamu gendong. prosiding seniati, 3(2), c25-1. saridewi, i., pambudi, a., & ningrum, y. f. (2017). analisis bakteri escherichia coli pada makanan siap saji di kantin rumah sakit x dan kantin rumah sakit y. bioma, 12(2), 90. https://doi.org/10.21009/bioma12(2).4 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 135-140 | doi: 10.14421/biomedich.2021.102.135-140 issn 2540-9328 (online) repurposing dihydroartemisinin-piperaquine-doxycycline as an antimalarial drug: a study in plasmodium berghei-infected mice udeme owunari georgewill1,*, elias adikwu2 1department of pharmacology, faculty of basic clinical sciences, university of port harcourt, rivers state, nigeria 2department of pharmacology and toxicology, faculty of pharmacy, niger delta university, bayelsa state, nigeria. corresponding author* udgeorgewill@yahoo.com manuscript received: 06 december 2021. revision accepted: 12 december, 2021. published: 16 december, 2021. abstract artemisinin-based combination (act) therapy is the mainstay for malaria treatment. however, plasmodium parasite with decreased susceptibility to act has emerged. hence, it is imperative to discover new drugs or explore new drug combinations that can decrease plasmodium parasite resistance. this study assessed the antiplasmodial activity of dihydroartemisinin-piperaquinedoxycycline (d-pdx) on mice infected with plasmodium berghei. swiss albino mice (25-30g) of both sexes inoculated with 1x107 plasmodium berghei intraperitoneally were used. the mice were randomly grouped and orally treated with dx (2.2 mg/kg), d-p (1.71/13.7 mg/kg) and d-pdx daily in curative, suppressive and prophylactic studies. the negative and the positive controls were treated daily with normal saline (0.2ml) and chloroquine (cq) (10mg/kg), respectively. after treatment, blood samples were assessed for percentage parasitemia, hematological and lipid parameters. the mice were observed for mean survival time. d-p, dx, and d-p-dx produced significant decreases in percentage parasitemia at p<0.05, p<0.01 and p<0.001, respectively when compared to negative control. in the curative study, d-p, dx, and d-p-dx produced 64.9%, 71.1%, and 93.6% parasitemia inhibitions when compared to 75.0% inhibition produced by cq. plasmodium berghei -induced alterations in packed cell volume, white blood cells, red blood cells, hemoglobin, high-density lipoprotein cholesterol, total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels were significantly restored by dx (p<0.05) and d-p (p<0.01) and d-p-dx (p<0.001) when compared to the negative control. d-p-dx showed significant antiplasmodial activity against plasmodium bergheiinfected mice. it may be clinically useful for the treatment of malaria. keywords; artemisinins; doxycycline; malaria; repurposing; resistance. introduction world health organization (who) estimates that nearly half of the world’s population lived in malaria endemic areas (who, 2016). malaria, a plasmodium parasite infection is one of the greatest health challenges in tropical regions, despite the availability of antimalarial drugs, mosquito repellents and insecticide-treated nets. malaria chemotherapy remains a major focus of research, and new molecules are being discovered prior to the emergence of drug-resistant strains of plasmodium parasite (gaillard et al., 2015). the use of antimalarial drugs is faced with the development of resistance from plasmodium falciparum in primarily endemic areas. other challenges include financial costs, contraindications, and clinical tolerance (gaillard et al., 2015). doxycycline (dx), a broad-spectrum bacteriostatic agent is synthetically obtained from naturally occurring tetracyclines produced by streptomyces sp. (mcevoy et al., 2008). it acts by binding to several proteins in the 30s ribosomal small subunit and to different ribonucleic acids in the 16s ribosomal rna. in addition to its antimicrobial activity, it is a partially efficacious prophylactic drug with activity against liver stage of plasmodium and blood schizontocides. it is highly effective for the prevention of malaria. the u.s. food and drug administration (fda) approved the use of dx for prophylaxis of plasmodium falciparum in short-term travelers to areas with chloroquine or pyrimethaminesulfadoxine-resistant strains (tan et al., 2011). dx can be used for the treatment of malaria in children less than 8 years old and non-pregnant adults in combination with quinine sulfate for uncomplicated and chloroquineresistant plasmodium falciparum. it is also used with primaquine and quinine sulfate for uncomplicated chloroquine-resistant plasmodium vivax and with parenteral quinidine for severe malaria (griffith et al., 2007). artemisinin derivatives are highly potent with fast acting antiplasmodial activity. however, due to short half-life, and plasmodium parasite resistance to artemisinin derivatives and older antimalarial drugs, artemisinin derivatives are often combined with partner drugs with longer half-life for fast clearance of malaria parasites (nosten, 2007). this led to the development of https://doi.org/10.14421/biomedich.2021.102.135-140 136 biology, medicine, & natural product chemistry 10 (2), 2021: 135-140 artemisinin-based combination therapies (acts), which have become the mainstay for the treatment of malaria especially in malaria endemic regions (basco et al., 2017). however, plasmodium parasites with decreased susceptibility to acts’ have emerged due to both decreased susceptibility to artemisinins and partner drugs (leang et al., 2013; sanders et al., 2014). also, dihydroartemisinin-piperaquine (d-p), one of the currently used acts, which is efficacious against malaria (tayler et al., 2020) and is being considered for the prevention of malaria in pregnancy (kakuru et al., 2016; desai et al., 2016) has experienced plasmodium resistance in malaria endemic regions. plasmodium resistance was attributed to decreased susceptibility to both piperaquine and dihyroartemisinin (amaratunga et al., 2016; amato et al., 2017). hence this study aimed to evaluate if the antiplasmodial effect of d-p can be augmented by dx in mice infected with plasmodium berghei. materials and methods animals, drugs and parasites swiss albino mice of both sexes (25-30 g) used for this study were sourced from the animal husbandry of the department of pharmacology, faculty of basic clinical sciences, university of port harcourt, rivers state. the mice were housed in cages at temperature 20oc with cycles of 12 h light/12 h darkness. the mice were acclimated for 2 weeks and fed with food pellets and given water ad libitum. cq was manufactured by alben healthcare ind ltd, d-p was manufactured by bliss gvs pharma ltd india whereas dx was manufactured by ranbaxy laboratories ltd, india. doses were selected based on previous studies: cq (10mg/kg) (somsak et al., 2018), dx (2.2 mg/kg) (gaillard et al., 2015) and d-p (1.71/13.7 mg/kg) (yavo et al., 2011). cq-sensitive plasmodium berghei (p. berghei) (nk65) was used. p. berghei was obtained from nigerian institute of medical research, yaba, lagos. mice used were inoculated intraperitoneally (i.p.) with 0.2 ml of infected red blood cells (rbcs) containing p. berghei (1 × 107) obtained from the donor mice. evaluation of antiplasmodial activity  evaluation of curative activity curative activity was performed as reported by ryley and peters (1970). twenty-five swiss albino mice were inoculated i.p with blood containing 1 × 107. p. berghei and grouped into 5 of 5 mice each. the first two groups, ai (negative control) and a2 (positive control) were orally treated with normal saline (0.2ml) and cq (10mg/kg) daily for 4 days, respectively. groups a3-a5 were orally treated with dx (2.2 mg/kg), d-p (1.71/13.7 mg/kg) and d-p-dx daily for 4 days, respectively. on day 5, tail blood samples were collected from the mice; thin blood films were produced on slides and stained with giemsa stain and viewed with the aid of a microscope. evaluations for percentage parasitemia and inhibitions were performed using the formula below. % parasitemia = number of parasitized red blood cells (rbcs) × 100 total number of rbcs count % inhibition = (%parasitemia of negative control − %parasitemia of treated group) × 100 %parasitemia of negative control  evaluation of prophylactic activity prophylactic test was performed using an established method described by peters (1965). twenty-five swiss albino mice were assigned to 5 groups of 5 mice each. group bi served as the negative control and group b2 served as the positive control and were orally treated with normal saline (0.2ml) and cq (10mg/kg) daily for 4 days, respectively. groups b3-b5 were orally treated with dx (2.2 mg/kg), d-p (1.71/13.7 mg/kg) and d-pdx daily for 4 days, respectively. on day 5, the mice were inoculated i.p with 0.2 ml of infected blood containing 1x107p. berghei. after, 3 days, tail blood samples were collected and percentage parasitemia and inhibitions were calculated using the formula above.  evaluation of suppressive activity suppressive test was carried out as described by knight and peters (1980). twenty-five mice were selected and inoculated i.p with blood containing 1x107 p. berghei. after 3 h, the mice were randomized into 5 groups of 5 mice each. the first two groups, ci (negative control) and c2 (positive control) were orally treated with normal saline (0.2ml) and cq (10mg/kg) daily for 4 days, respectively. groups c3-c5 were orally treated with dx (2.2 mg/kg), d-p (1.71/13.7 mg/kg) and d-pdx daily for 4 days, respectively. on day 5, tail blood samples were collected and evaluated for percentage parasitemia and inhibitions using the formula above. evaluation of biochemical parameters blood samples were collected from the mice used for the curative test and evaluated for white blood cells (wbcs), hemoglobin (hb), packed cell volume (pcv), red blood cells (rbcs), triglyceride (tg), total cholesterol (chol), and high-density lipoprotein cholesterol (hdl-c) using an auto analyzer. georgewill & adikwu – repurposing dihydroartemisinin-piperaquine-doxycycline as … 137 statistical analysis results as mean ± s em (standard error of mean). data was analyzed using one-way analysis of variance (anova) and tukey’s test. differences between means were considered significant at p < 0.05, 0.01 and 0.001, respectively. results curative activity treatment with dx, d-p, and d-p-dx significantly decreased percentage parasitemia levels at p<0.05, p<0.01 and p<0.001, respectively when compared to the negative control (table 1). the observed parasitemia inhibitions produced by dx, d-p, and d-p-dx and cq were 64.9%, 71.1%, 93.6%, and 75.0%, respectively (table 2). mst was significantly prolonged in mice treated with dx, d-p, and d-p-dx at p<0.05, p<0.01, and p<0.001, respectively when compared to the negative control (table 1). suppressive activity percentage parasitemia levels were significantly decreased in mice treated with dx, d-p and d-p-dx at p<0.05, p<0.01, and p<0.001, respectively when compared to the negative control (table 2). treatment with dx, d-p and d-p-dx produced 66.5%, 75.0%, and 95.1% parasitemia inhibitions, respectively whereas cq produced 81.9% parasitemia inhibition (table 2). significant prolongation of mst in dx, d-p and d-pdx-treated mice occurred at p<0.05, p<0.01 and p<0.001, respectively when compared to negative control (table 2). prophylactic activity the prophylactic test showed significant decreases in percentage parasitemia levels in mice treated with dx (p<0.05), d-p (p<0.01) and d-p-dx (p<0.001) when compared to the negative control (table 3). the percentage parasitemia inhibitions produced by dx, d-p and d-p-dx were 65.1%, 86.8%, and 98.9%, respectively while cq produced an inhibition of 85.7% (table 3). mst was prolonged in dx, d-p, and d-pdx-treated mice. this occurred at p<0.05, p<0.01, and p<0.001, respectively when compared to the negative control (table 3). determination of mean survival time the mice in the control and the treated groups were observed for mortality and expressed in days. mortality represented as mean survival time (mst) was calculated using the formula bellow. 𝑀𝑆𝑇 = sum of survival time of all mice in a group (days) total number of mice in that group hematological and lipid parameters in p. berghei-infected mice, tg, chol, ldl-c and wbcs increased whereas hb, pcv, rbcs and hdl-c decreased significantly (p<0.001) when compared to the normal control (tables 4 and 5). on the other hand, tg, chol, ldl-c wbcs levels were decreased whereas hb, pcv, rbc and hdl-c levels were increased significantly in mice treated with dx, d-p, and d-p-dx at p<0.05, p<0.01 and p<0.001, respectively when compared to the negative control (tables 4 and 5). table 1. curative antiplasmodial effect of dihydroartemisinin-piperaquine-doxycycline on mice infected with plasmodium berghei. treatment % parasitemia % inhibition mst (days) pc 35.10±3.00 0.0 9.22±0.18 cq 8.78±1.54a 75.0 27.62±2.39 a. dx 12.30±1.27b 64.9 14.15±1.66b d-p 10.11±1.11a 71.1 25.83±2.65 a d-p-dx 2.25±0.15c 93.6 36.96±3.48 c data presented as ± sem, n= 5, pc: negative control, cq: chloroquine, dx: doxycycline, d-p: dihydroartemisinin-piperaquine, mst: mean survival time. a p<0.01, b p<0.05, c p<0.001 significant different when compared to pc, sem: standard error mean. table 2. suppressive antiplasmodial effect of dihydroartemisinin-piperaquine-doxycycline on mice infected with plasmodium berghei. treatment % parasitemia % inhibition mst(days) pc 17.60±2.54 0.0 9.40±0.33 cq 3.19±0.01a 81.9 31.61±2.72a dx 5.90±0.08b 66.5 20.47±2.67 b d-p 4.40±0.09a 75.0 29.73±3.11 a d-p-dx 0.86±0.07c 95.1 37.12±3,29 c data presented as ± sem, n= 5, pc: negative control, cq: chloroquine, dx: doxycycline, d-p: dihydroartemisinin-piperaquine, mst: mean survival time. a p<0.01, b p<0.05, c p<0.001 significant different when compared to pc, sem: standard error mean. 138 biology, medicine, & natural product chemistry 10 (2), 2021: 135-140 table 3. prophylactic antiplasmodial effect of dihydroartemisinin-piperaquine-doxycycline on mice infected with plasmodium berghei. treatment % parasitemia % inhibition mst (days) pc 15.80±1.47 0.0 9.79±0.22 cq 2.58±0.06a 85.7 33.43±2.53 a dx 5.51±0.07b 65.1 22.75±3.57 b d-p 3.03±0.04a 86.8 30.91±2.89 a d-p-dx 0.17±0.08c 98.9 38.02±3.86 c data presented as ± sem, n= 5, pc: negative control, cq: chloroquine, dx: doxycycline, d-p: dihydroartemisinin-piperaquine, mst: mean survival time a p<0.01, b p<0.05, c p<0.001 significant different when compared to pc, sem: standard error mean. table 4. effect of dihydroartemisinin-piperaquine-doxycycline on hematologic parameters of mice infected plasmodium berghei. treatment rbc (x106) wbc (cells/l) pcv (%) hb (g/dl) nc 6.83±0.33 7.55±0.09 60.91±5.91 16.93±1.35 pc 3.21±0.43a 15.71±1.55a 26.72±3.45a 6.01±0.17a cq 5.44±0.28b 9.44±0.19b 44.21±4.33b 13.45±1.38b dx 4.37±0.27c 11.00±0.37c 31.94±3.47c 10.73±0.81c d-p 5.40±0.11b 9.57±0.16b 42.62±3.67b 13.07±1.11b d-p-dx 6.61±0.32d 7.25±0.01d 55.9±4.41d 16.72±1.37d data presented as ± sem, n= 5, nc: normal control, pc: negative control, cq: chloroquine, dx: doxycycline, d-p: dihydroartemisinin-piperaquine, mst: mean survival time, rbcs: red blood cell, wbcs: white blood cell, pcv: packed cell volume, hb: haemoglobin. a p<0.001 significant difference when compared to nc, b p<0.01, c p<0.05, d p<0.001 significant difference when compared to pc, sem: standard error mean. table 5. effect of dihydroartemisinin-piperaquine on lipid parameters of mice infected with plasmodium berghei. group tg (mg/dl) tchol (mg/dl) hdl-c (mg/dl) ldl (mg/dl) nc 80.8±7.03 110.8±11.4 50.4±4.00 44.2±3.11 pc 250.3±18.9a 273.4±18.0 a 22.2±1.41 a 201.1±18. a cq 150.1±5.87 b 181.6±22.4 b 39.7±4.63 b 111.9±11.6 b dx 200.7±3.03 c 220.0±12.0 c 30.6±4.22 c 149.3±15.0 c d-p 159.0±4.87b 170.7±13.5b 38.7±4.00b 100.2±12.5b d-p-dx 97.4±8.88d 127.7±10.6 d 47.9±5.43d 60.3±10.1d data presented as mean ± sem, n= 5, nc: normal control, pc: negative control, cq: chloroquine, dx: doxycycline, d-p: dihydroartemisininpiperaquine, tg: tryglyceride, tchol: total cholesterol, hdl: high density lipoproteins, ldl: low density lipoprotein. a p<0.001 significant difference when compared to nc, b p<0.01, c p<0.05, d p<0.001 significant difference when compared to pc, sem: standard error mean. discussion malaria is a major health challenge in developing countries of sub-saharan africa and south east asia. the emergence of widespread resistance of plasmodium species to most antimalarial drugs, the increasing insecticide resistance by mosquitoes, and the lack of vaccines have made the fight against malaria seriously tasking (beeson et al., 2016; joseph et al., 2020). hence there is an urgent need to discover alternative drugs with novel modes of action or a combination of currently existing antimalarial drugs to overcome these challenges. the present study, assessed whether antimalarial activity of d-p can be augmented by dx in mice infected with p. berghei. this study used invivo model, because it takes into cognizance the possible prodrug effect and the involvement of the immune system in eradicating malaria infection. p. berghei has been used in antiplasmodial studies in predicting experimental treatment outcomes and hence was appropriately used for the study (satish et al., 2017). this study used suppressive test which determines the activity of a drug candidate on early infection and curative test, which evaluates the curative activity of a drug candidate on established infection (mekonnen, 2015; hiben et al., 2016). in the present study, in the curative test, d-p-dx decreased percentage parasitemia levels most when compared to individual doses of d-p, dx and cq. the observed parasitemia inhibitions in the curative test were 64.9%, 71.1%, 93.6% in dx, d-p, and d-p-dx-treated mice, respectively. also, in the suppressive and prophylactic tests, best decreases in percentage parasitemia levels occurred in d-p-dxtreated mice in comparison to individual doses of dx, d-p, and cq. in the suppressive test, 66.5%, 75.0%, and 95.1%, parasitemia inhibitions were observed in dx, dp, and d-p-dx treated mice respectively. in view of the antiplasmodial activity of d-p-dx observed in the georgewill & adikwu – repurposing dihydroartemisinin-piperaquine-doxycycline as … 139 present study, the ability of d-p-dx to prolong mst in mice was also evaluated. treatment with d-p-dx prolonged mst in the curative, prophylactic and suppressive tests. the prolongation of mst by d-p-dx was best when compared to individual doses of dx, dp, and cq. hematological abnormalities like anemia caused by erythrocyte destruction are common characteristics of p. berghei-infected mice. rodent malaria causes parasite-induced decrease in pcv, which occurs approximately 48 h post-infection (nardos and makonnen, 2017). in the present study, notable signs of anemia marked by low levels of hb, pcv rbcs and increased wbcs levels were observed in p. bergheiinfected mice. however, p. bergheiinduced anemia was curtailed in d-p-dx treated mice when compared to individual doses of dx, d-p, and cq. studies have reported that changes in serum lipids could be possible features of malaria (visser et al., 2013). the present study observed elevated tchol, tg, and ldl-c and decreased hdl-c levels in p. berghei-infected mice. however, d-p-dx restored serum lipid characterized by decreased tchol, tg, ldl-c and increased hdl-c levels. the observed antiplasmodial effect of d-p-dx may be due to different modes of antiplasmodial activity of the partner drugs. dihydroartemisinin acts through the cleavage of the endoperoxide bridge and the production of free radicals (meshnick, 1994). piperaquine is suggested to have similar mode of action as cq (meshnick, 1994). in parasite food vacuole, concentrated cq binds free hematin forming cqhematin complex. this interferes with enzymatic processes in the parasite causing parasite death (tärning et al., 2007). the antiplasmodial mode of action of dx is not clear, but studies suggested the inhibition of mitochondrial protein, nucleotides and deoxynucleotides syntheses in plasmodium (yeo et al., 1997; prapunwattana et al., 1998). conclusion this study showed that d-p-dx produced the best antiplasmodial activity in p. berghei-infected mice when compared to individual doses of d-p, dx and cq. also, alterations in lipid profile and hematological parameters were best restored by d-p-dx when compared to individual doses of d-p, dx and cq. this shows that d-p-dx may be an effective antimalarial drug combination. acknowledgement: the authors appreciate the laboratory staff of the department of pharmacology, faculty of basic clinical sciences, university of port harcourt, nigeria. conflict of interest: the authors declare no conflicts of interest. financial disclosure: none references amaratunga c, lim p, suon s, sreng, s, mao s, sopha c et al. 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(1970) the antimalarial activity of some quinolone esters. annals of tropical medicine and parasitology. 84: 209-222. satish pvv, kumari ds, & sunita k, (2017) antiplasmodial efficacy of calotropis gigantean (l.) against plasmodium falciparum (3d7 strain) and plasmodium berghei (anka). journal of vector borne disease. 54(3):215-225 saunders d. l, vanachayangkul p, & lon c. (2014) dihydroartemisinin–piperaquine failure in cambodia. new england journal of medicine. 371, 484–485 somsak v, damkaew a, & onrak p. (2018) antimalarial activity of kaempferol and its combination with chloroquine in plasmodium berghei infection in mice. journal of pathology. 2018:1-7. tan kr, magill aj, parise me, & arguin pm, (2011). doxycycline for malaria chemoprophylaxis and treatment: report from the cdc expert meeting on malaria chemoprophylaxis, hygiene. 5; 84(4): 517–531. tärning j. piperaquine, bioanalysis (2007) drug metabolism and pharmacokinetics. göteborg, sweden: institute of neuroscience and physiology, department of pharmacology, the sahlgrenska academy at göteborg university tayler nm, de jesús r, spadafora r, coronado lm, r, & spadafora c (2020) antiplasmodial activity of cocos nucifera leaves in plasmodium berghei-infected mice. journal of parasitic disease 44, 305–313. visser bj, wieten rw, nagel im. & grobusch mp (2013) serum lipids and lipoproteins in malaria a systematic review and meta-analysis. malarail journal. 12, 442, 1-16 who. world malaria report 2016. geneva: world health organization; 2016 yavo w, faye b, kuete t, djohan v, oga sa, kassi rr et al. (2011). multicentric assessment of the efficacy and tolerability of dihydroartemisinin-piperaquine compared to artemether-lumefantrine in the treatment of uncomplicated plasmodium falciparum malaria in sub-saharan africa. malarial journal. 2011; 10:198. yeo ae, edstein md, shanks gd, & rieckmann kh (1997). potentiation of the antimalarial activity of atovaquone by doxycycline against plasmodium falciparum in vitro. parasitology research. 83:489–91. https://www.ncbi.nlm.nih.gov/pubmed/?term=tan%20kr%5bauthor%5d&cauthor=true&cauthor_uid=21460003 https://www.ncbi.nlm.nih.gov/pubmed/?term=magill%20aj%5bauthor%5d&cauthor=true&cauthor_uid=21460003 https://www.ncbi.nlm.nih.gov/pubmed/?term=parise%20me%5bauthor%5d&cauthor=true&cauthor_uid=21460003 https://www.ncbi.nlm.nih.gov/pubmed/?term=arguin%20pm%5bauthor%5d&cauthor=true&cauthor_uid=21460003 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 2, 2018 | pages: 57-59 | doi: 10.14421/biomedich.2018.72.57-59 issn 2540-9328 (online) alizarin red s-alcian blue staining for regenerated tail of common house gecko (hemidactylus frenatus) rakhmiyati1,*, muhammad ja’far luthfi2 1postgraduate program, universitas sebelas maret jalan ir. sutami 36 a, surakarta, 57126, tel. +62271-646994, fax. +62271-646655, indonesia 2department of biology education, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto, no. 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. author correspondency*: miarakhmiy@gmail.com abstract common house gecko (hemidactylus frenatus) is one of reptiles that have ability to autotomy their tails. tail autotomy is a mechanism to protect it self from predators. after the tail broke, there will be wound healing on the tail which is then followed by a tail regeneration event. original tail and regenerate tail is very different morphologically and anatomically. the original tail is composed of bones while the tail of the regenerate is composed of cartilage. histochemical staining using alizarin red-s alcian blue was done to differentiate bone and cartilage. this method will stained bones red while the cartilage will stained blue. keywords: common house gecko (hemidactylus frenatus); autotomy; alizarin reds alcian blue; cartilage; bone (osteon) introduction the tail of the common house gecko (hemidactylus frenatus) is flat and tapered at the tip. in the dorsal area there are fine scales and no spots (takahashi, 2009). these scales are brownish yellow (pratiwi, 2009). the tail comprised of procoel vertebrae (maria, 1998). autotomy is a capacity breaking of the tail, either part or whole of the tail when the animal is chased or captured (lin & lin, 2017; luthfi, 2002; soesilo, 1992). in general, autotomy can be said to be a structural adaptation to minimize tissue damage and ease the wound healing process (gilbert et al, 2013). tail autotomy is a self-protection mechanism that can be carried out by lacertilian (londono et al, 2017). according to pratt (1946) if the tail is held it will cause uneven pressure of muscle contraction across all segments and excessive pressure will occur on the tail held resulting breaking. tail function in lacertilian are as a regulator of balance and movement, storing energy, sexual and reproductive function (jagnandan et al, 2014). the next step is regeneration, which is the ability of living things to replace body parts lost due to injury, tear, damage or autotomy (kimball, 1983; soesilo, 1992). regeneration process occurs through several stages, namely the wound healing stage, cell proliferation, angiogenesis, formation of extracellular matrix, blastema formation stage and stage of tail differentiation and growth (soesilo, 1992; alibardi, 2009; gilbert et al, 2013; lozito & tuan, 2016; jacyniak et al, 2017). mesenchymal cells and several cells accumulate below the wound epidermis form blastema (alibardi, 2017). in order to observe the structure of regenerated tail of common house gecko (hemidactylus frenatus) it is necessary to make the original tail and tail regenerate preparations using alizarin red s-alcian blue staining. materials and methods common house gecko were sacrified using chloroform, then the tails were cut at its base. the tail were then immersed in 96% alcohol solution for 4 days, skinned, and immersed in 96% alcohol solution for 2 days. the tails were soaked in acetone for 3 days, then soaked in a dye solution for 4 days (1 volume 0.3% alcian blue in 70% alcohol + 1 volume 0.1% alizarin red s in 95% alcohol + 1 volume of glacial acetic acid + 17 alcohols 75% volume) and then washed with distilled water. after that the tails were immersed in 1% koh for 3 days. the tails were soaked in a mixture of glycerin and 1% koh with a ratio of 20%: 80%; 50%: 50%; 80%: 20% successively, each for 2 days and after that the preparations were keep in pure glycerin to be observed. results and discussion data obtained from observations on the original tail and the regenerate tail of the wall lizard stained with alizarin red s-alcian blue showed a difference between the original tail bone and the regenerated one. the original https://doi.org/10.14421/biomedich.2018.72.57-59 58 biology, medicine, & natural product chemistry 7 (2), 2018: 57-59 tail is composed of bones (osteon) where as of regenerate is of cartilage. figure 1. whole tail preparation of common house gecko (hemidactylus frenatus). alizarin red s-alcian blue (dorsal view). magnification 3x. (a). processus dorsal; (b). processus transversal; (c). processus ventral. the vertebral type of common house gecko (hemidactylus frenatus) is procoelous. its centrum is concave or curves anteriorly and is convex posteriorly. the original tail has procesus transversus as a posterior attachment of the muscle, while the anterior part of the muscle is attached to the myoseptum (figure 1). viewed from the ventral side, there are prezygapophysis, postzygapophysis, centrum and cevron bone. figure 2. whole tail preparation of common house gecko (hemidactylus frenatus). alizarin red s-alcian blue (ventral view). magnification 3x. (a). postzygapophysis; (b). prezygapophysis; (c). centrum; (d). cevron bone. seen from the ventral part of the tail, there is a joint of prezygapophysis found in the anterior vertebrae and postzygapophysis joints found in the posterior vertebra (figure 2). these two joints are only located in the dorsal part. the original tail stained red along the vertebrae caudales because it is composed of bone tissue starting from the base of the tail to the tip of the tail (figure 3). the processus vertebrae are found along the vertebrae caudales, but its size reduced posteriorly. figure 3. whole tail preparation of common house gecko (hemidactylus frenatus). alizarin red s-alcian blue (transversal view). magnification 3x. (a). autotomy plane; (b). processus dorsal; (c). processus transversal; (d). processus ventral. figure 4. whole preparation of immature regenerate tails of common house gecko (hemidactylus frenatus) of alizarin red s-alcian blue (magnification 7x). (a). bone of original tail; (b). border (crack area) between the original tail and the regenerate tail; (c). cartilagineus tube of immature regenerate tail. the dark red color in the original tail indicates that the tail has many calcium ions which bind to the alizarin red s dye, where as the immature one will stained blue because the it does not have calcium ions. this is consistent with the statement expressed by lozito & tuan (2016). rakhmiyati & luthfi – alizarin red s-alcian blue staining for regenerated tail … 59 figure 5. whole preparations of old regenerate tails (hemidactylus frenatus). alizarin red salcian blue (magnification 11,25x). (a). cracks; (b). cartilago tube of old regenerate tail. in the mature regenerate tail, cartilaginous tube looks red because calcification has occurred. along the mature regenerate tail there are cracks that can break the tail (figure 5). viewed from any side (dorsal, ventral, and transverse), the regnerate tail will look the same color. conclusion from the results of this study it can be concluded that the original tail is composed of bones (osteon) and has complex parts, namely the transverse processes, dorsal processes, ventral processes, prezygapophysis, postzygapophysis, centrum, cevron bone, vertebral cracks (autotomic plane). where as the regenerate tail is composed of cartilage which is tube like shaped and will calcified in mature regenerate and developed crack in a very matured one. references alibardi. 2009. orphological and cellular aspects of tail and limb regeneration in lizards. 1 st edition, pp: 1-49. alibardi. 2017. review: biological and molecular differences between tail regeneration and limb scarring in lizard: an inspiring model addressing limb regeneration in amniotes. journal of ekperimental zoology (molecular and developmental evolution). 00b: 1–22. gilbert et al, 2013. the anatomy and histology of caudal autotomy and regeneration in lizards. physiological and biochemical zoology. vol. 86(6):631–644. jagnandan et al, 2014. tail autotomy and subsequent regeneration alter the mechanics of locomotion in lizards. the journal of experimental biology 217: 3891-3897. jacyniak et al, 2017. tail regeneration and other phenomena of wound healing and tissue restoration in lizards. journal of experimental biology. vol. 220, pp: 2858-2869. kimball, 1983. biologi. edisi kelima. jakarta: erlangga. lin & lin, 2017. tail regeneration after autotomy revives survival: a case from a long-term monitored lizard population under avian predation. the royal society publishing. pp: 1-9. londono et al, 2017. cartilage and muscle cell fate and origins during lizard tail regeneration. frontiers in bioengineering and biotechnology. vol. 5, no. 70, pp: 1-9. lozito & tuan, 2016. lizard tail skeletal regeneration combines aspects of fracture healing and blastema-based regeneration. development. vol. 143, pp: 2946-2957. luthfi, m. j. 2002. kalsifikasi skeleton aksial dan kemampuan autotomi regenerat ekor kadal (mabouya multifasciata kuhl). tesis. universitas gadjah mada (unpublished). maria, b. 1998. struktur vertebrae caudales pada 5 species anggota sub ordo lacertilia. thesis. universitas gadjah mada (tidak dipublikasikan). pratiwi, 2009. struktur organ reproduksi dan seksual dimorfisme in hemidactylus frenatus dumeril & bibron, 1836; cosymbotus platyurus (schneider, 1792); & gekko gecko linnaeus, 1758. thesis universitas gadjah mada (unpublished). soesilo, n. p. 1992. proses regenerasi ekor kadal (mabouya multifasciata kuhl). biologi, vol. 1, no. 4: 169-175. takahashi, 2009. preferensi pakan cicak rumah (hemidactylus frenatus, gray 1825) dan tokek (gekko gecko, linnaeus 1758) di gamping, sleman, daerah istimewa yogyakarta. thesis. universitas gadjah mada (unpublished) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 609-615 | doi: 10.14421/biomedich.2024.132.609-615 issn 2540-9328 (online) modulatory efficiency of vitamin c (ascorbic acid) on collagen-induced platelet aggregation and dysfunction osah martins onwuka1,*, chukwukadibie adigwe2, oghenefego michael adheke3, chamberlin jamike elem4, josiah soipiriala hart3 1department of human physiology, faculty of basic medical sciences, gregory university, uturu, abia state, nigeria. 2department of human physiology, faculty of basic medical sciences, college of medicine, godfrey okoye university, enugu state, nigeria. 3department of anatomy, faculty of basic medical sciences, university of port harcourt, nigeria. 4department of human anatomy, faculty of basic medical sciences, gregory university, uturu, abia state, nigeria. corresponding author* osahmartinz@gmail.com manuscript received: 12 july, 2024. revision accepted: 24 december, 2024. published: 06 january, 2025. abstract platelet aggregation, coagulation, and activation are crucial for hemostasis. collagen treatment can impair hemostatic processes leading to bleeding disorders like thrombosis; vitamin c may mitigate these effects. hence, modulatory efficiency of vitamin c on collagen-induced platelet aggregation and dysfunction was investigated. thirty (30) wistar rats (135g-155g) were divided equally into; group 1 (control), group 2 (collagen-induced), and group 3 (collagen + vitamin c treated). platelet aggregation, prothrombin time, bleeding time, fibrinogen levels assessed coagulation and platelet function. thromboxane b2 and p-selectin levels measured platelet and endothelial activation. platelet count, mean platelet volume (mpv), platelet distribution width (pdw), and plateletcrit (pct) evaluated platelet production and size variability. statistical significance was set at p < 0.05. group 2 exhibited higher platelet aggregation, prolonged prothrombin and bleeding times and elevated fibrinogen, thromboxane b2, and p-selectin levels, along with increased platelet count, mpv, pdw, and pct, compared to group 1. group 3 showed significant reductions in all these parameters compared to group 2 (p < 0.05). vitamin c demonstrated significant modulatory effect on collagen-induced platelet aggregation and dysfunction which suggests that vitamin c may have therapeutic potential in mitigating platelet dysfunction and coagulation impairments associated with collageninduced pathophysiological conditions. keywords: platelets aggregation; collagen-induced; vitamin c. introduction the preservation of vascular integrity and homeostasis depends heavily on platelets, which are tiny, nonnucleated cellular fragments generated from a group of precursor cells called megakaryocytes (kroll and kharghan 2012; onwuka et al., 2022). platelets bind to the site of injury in a blood artery to produce a hemostatic plug that stops excessive bleeding when the vessel is injured, exposing the sub-endothelial matrix (broos et al., 2011; periayah et al., 2017; onwuka et al., 2023). platelet aggregation is the final result of a series of events that occur during this process (periayah et al., 2017). platelet aggregation and dysfunction play a critical role in the pathogenesis of various cardiovascular diseases, including atherosclerosis, myocardial infarction, and stroke. these conditions are often associated with increased platelet reactivity and aggregation, leading to the formation of thrombi that can obstruct blood flow and cause severe health complications (hosseini et al., 2012; malik et al., 2021). understanding the mechanisms underlying platelet aggregation and identifying potential modulators is essential for developing effective therapeutic strategies. collagen is a potent stimulator of platelet aggregation, initiating a cascade of events that lead to platelet activation and the subsequent formation of a hemostatic plug (tomaiuolo et al., 2017). the interaction between collagen and platelets is a complex process involving various receptors and intracellular signaling pathways. dysregulation of this process can result in either excessive bleeding or thrombotic events, underscoring the importance of maintaining a delicate balance in platelet function (farndale 2004; brass et al., 2013). vitamin c (ascorbic acid) is a vital nutrient known for its antioxidant properties and its role in various physiological processes (pehlivan, 2017). recent studies have suggested that vitamin c may have a modulatory effect on platelet function, potentially influencing platelet aggregation and overall cardiovascular health (ashor et al., 2014). however, the precise mechanisms by which vitamin c impacts collagen-induced platelet aggregation https://doi.org/10.14421/biomedich.2024.132.609-615 610 biology, medicine, & natural product chemistry 13 (2), 2024: 609-615 and dysfunction remain to be fully elucidated. this research therefore investigated the modulatory potential of vitamin c in collagen-induced platelet aggregation and dysfunction. by exploring the effects of vitamin c on platelet reactivity, aggregation, and related signaling pathways, this study seeks to provide a deeper understanding of its potential therapeutic benefits in preventing and managing diseases associated with abnormal platelet function. the findings from this study could have significant implications for developing novel therapeutic approaches that leverage the properties of vitamin c to modulate platelet function, thereby reducing the risk of thrombotic events and improving health outcomes. methodology study design this study constitutes an experimental, controlled laboratory research conducted in compliance with the guide for the care and use of laboratory animals, as outlined by the national academy press at 2101 constitution ave. nw, washington, dc 20055, usa. the laboratory animals used were housed in adequately ventilated cages and acclimatized to laboratory environments for a period of 14 days. they were sustained on standard rat chow with unrestricted access to drinking water and subjected to natural alternating 12hour day and 12-hour night cycles. subjects and grouping thirty (30) animal subjects (wistar rats weighing 135g155g) were used and equally divided into three groups; group 1 : control group (no treatment) group 2 : collagen-induced platelet aggregation group group 3 : collagen-induced platelet aggregation with vitamin c treatment group induction of platelet aggregation and administration protocol collagen (bovine brand, sigma-aldrich, usa) was administered (i.p.) at a dose of 10 μg/kg body weight, once every three days for a period of 14 days (total of five injections). vitamin c (ascorbic acid) of laboratory grade, obtained from the biochemistry department, gregory university uturu, was administered (p.o.) at a dose of 50 mg/kg body weight daily for 14 days. sample collection at the end of the 14-day experimental period, blood samples were collected from all groups. the animals were euthanized under anesthesia using ketamine at a dose of 100 mg/kg. blood samples were obtained via retro-orbital puncture using capillary tubes into 5 ml sample bottles. these samples were then used for assays. preparation of platelet-rich plasma (prp) and platelet-poor plasma (ppp) blood samples for platelet-rich plasma (prp) and platelet-poor plasma (ppp) was collected into sample bottles containing anticoagulant (3.2% sodium citrate solution) and gently inverted to mix the blood with the anticoagulant. platelet-rich plasma (prp) was obtained by centrifuging the samples at 200 x g for 10 minutes at room temperature. the supernatants (prp) were transferred into new tubes. platelet-poor plasma (ppp) was obtained after getting prp, the plasma samples were subjected to a second centrifugation at an increased speed of 2,000 x g for 10 minutes at room temperature. this additional centrifugation step was performed to ensure the removal of any residual platelets, thereby yielding platelet-poor plasma (ppp). the supernatants (ppp) were also transferred into new tubes. the resultant prp and ppp were used for assays. platelet aggregation assay the supernatants (prp) were used for platelet aggregation and were measured using platelet aggregometer (chrono-log model 700 whole blood/optical lumi-aggregometer, usa). the degree of aggregation was recorded as a percentage of light transmission. prothrombin time (pt) measurement thromboplastin reagent and calcium chloride solution were pre-warmed to 37°c. then, 100 μl of platelet-poor plasma (ppp) was pipetted into clean test tube. following this, 100 μl of the pre-warmed thromboplastin reagent was added to the ppp, and the stopwatch was started. the mixture was incubated at 37°c for 2 minutes. subsequently, 100 μl of the pre-warmed calcium chloride solution was added to the mixture, and the timer was started immediately. the mixture was observed, and the time taken for clot formation was recorded as the prothrombin time (pt). bleeding time measurement standardized incision (1-2 mm deep) was made on the rat's tail using scalpel blade. stopwatch was started immediately after making the incision. blood was gently blotted at regular intervals of 15 seconds using filter paper. this process continued until bleeding ceased. the duration from the initial incision to the cessation of bleeding was recorded as the bleeding time. fibrinogen, thromboxane b2 and p-selectin (cd62p) assay platelet-poor plasma (ppp) was used to measure fibrinogen, thromboxane b2, and p-selectin (cd62p) using enzyme-linked immunosorbent assays (elisas) specific for each of these markers, following the manufacturer's protocol. onwuka et al. – modulatory efficiency of vitamin c (ascorbic acid) … 611 measurement of platelet counts, mean platelet volume (mpv), platelet distribution width (pdw) and plateletcrit (pct) platelet counts, mpv, pdw and pct ware measured in blood sample using hematological analyzer (swelab alfa, boule, usa). statistics statistical analysis was performed using graphpad prism software (v8). comparisons between groups were made using one-way anova followed by bonferroni's posthoc tests. a p-value < 0.05 was considered statistically significant. results vitamin c modulation of degree of platelet aggregation in collagen-induced platelet aggregation and dysfunction the degree of platelet aggregation is expressed as percentage of light transmission (%lt). group 2: collagen-induced group showed a significant increase in platelet aggregation compared to the group1: control group (p < 0.05). group 3: collagen + vitamin c treated group showed a significant reduction in platelet aggregation compared to the group 2: collagen-induced group (p < 0.05), indicating the modulating effect of vitamin c on collagen-induced platelet aggregation (figure 1). 1 2 3 0 20 40 60 80 100 groups pe rc en ta ge o f l ig ht tr an sm is si on (% l t ) * *# figure 1. degree of platelet aggregation expressed as percentage of light transmission (%lt): values are expressed as mean ± sem, * p < 0.05 compared to group 1: control group, # p < 0.05 comparison between group 2: collagen-induced group and group 3: collagen + vitamin c treated group. vitamin c modulation of prothrombin time in collagen-induced platelet aggregation and dysfunction group 2: collagen-induced group showed significantly prolonged prothrombin time compared to the group 1: control group (p < 0.05), suggesting impaired coagulation function due to collagen treatment. group3: collagen + vitamin c group showed significantly shorter prothrombin time compared to collagen-induced group (p < 0.05), indicating that vitamin c may mitigate the coagulation impairment caused by collagen treatment (figure 2). 1 2 3 0 5 10 15 20 groups p ro th ro m b in t im e (s ec on d s) * *# figure 2. prothrombin time (seconds): values are expressed as mean ± sem, * p < 0.05 compared to group 1: control group, # p < 0.05 comparison between group 2: collagen-induced group and group 3: collagen + vitamin c treated group. 612 biology, medicine, & natural product chemistry 13 (2), 2024: 609-615 vitamin c modulation of bleeding time in collageninduced platelet aggregation and dysfunction group 2: collagen-induced group showed significantly prolonged bleeding time compared to the group 1: control group (p < 0.05), suggesting impaired platelet function and primary hemostasis due to collagen treatment. group 3: collagen + vitamin c group showed significantly shorter bleeding time compared to the collagen-induced group (p < 0.05), indicating that vitamin c may help mitigate the impairment in platelet function caused by collagen treatment (figure 3). 1 2 3 0 50 100 150 200 groups b le e d in g t im e ( se c o n d s) * *# figure 3. bleeding time (seconds): values are expressed as mean ± sem, * p < 0.05 compared to group 1: control group, # p < 0.05 comparison between group 2: collagen-induced group and group 3: collagen + vitamin c treated group. vitamin c modulation of fibrinogen, thromboxane b2 and p-selectin (cd62p) in collagen-induced platelet aggregation and dysfunction group 2: collagen-induced group shows significantly higher fibrinogen levels compared to the group 1: control group (p < 0.05), suggesting an increase in coagulation activity or inflammatory response due to collagen treatment. group 3: collagen + vitamin c group shows significantly lower fibrinogen levels compared to the collagen-induced group (p < 0.05), indicating that vitamin c may help normalize the fibrinogen levels affected by collagen treatment (figure 4a). the collagen-induced group shows significantly higher thromboxane b2 levels compared to the control group (p < 0.05), suggesting increased platelet activation due to collagen treatment, while the collagen + vitamin c group shows significantly lower thromboxane b2 levels compared to the collagen-induced group (p < 0.05), indicating that vitamin c may reduce the increased platelet activation caused by collagen treatment (figure 4b). the collagen-induced group also showed significantly higher p-selectin (cd62p) levels compared to the control group (p < 0.05), suggesting increased platelet and endothelial cell activation due to collagen treatment. the collagen + vitamin c group showed significantly lower p-selectin levels compared to the collagen-induced group (p < 0.05), indicating that vitamin c may reduce the increased activation of platelets and endothelial cells caused by collagen treatment (figure 4c). 1 2 3 0 100 200 300 400 500 groups f ib ri n og en ( m g/ d l ) * # a 1 2 3 0 100 200 300 400 groups t h ro m b o x a n e b 2 ( p g /m l ) * *# b 1 2 3 0 50 100 150 groups p -s el ec ti n ( c d 62 p ; n g/ m l ) * # c figure 4. [a] fibrinogen (mg/dl); [b] thromboxane b2 (pg/ml); [c] p-selectin (cd62p: ng/ml): values are expressed as mean ± sem, * p < 0.05 compared to group 1: control group, # p < 0.05 comparison between group 2: collagen-induced group and group 3: collagen + vitamin c treated group. onwuka et al. – modulatory efficiency of vitamin c (ascorbic acid) … 613 vitamin c modulation of platelet counts, mean platelet volume (mpv), platelet distribution width (pdw) and plateletcrit (pct) in collagen-induced platelet aggregation and dysfunction group 2: collagen-induced group showed significantly higher platelet count compared to the group 1: control group (p < 0.05), suggesting an increase in platelet production or reduced platelet clearance due to collagen treatment. group 3: collagen + vitamin c group showed significantly lower platelet count compared to the collagen-induced group (p < 0.05), indicating that vitamin c may help normalize the platelet count affected by collagen treatment. collagen-induced group showed significantly higher mpv compared to the control group (p < 0.05), suggesting an increase in platelet size due to collagen treatment. collagen + vitamin c group showed significantly lower mpv compared to the collageninduced group (p < 0.05), indicating that vitamin c may help modulate the increase in platelet size caused by collagen treatment. the collagen-induced group showed significantly higher pdw compared to the control group (p < 0.05), suggesting increased variability in platelet size due to collagen treatment. the collagen + vitamin c group showed significantly lower pdw compared to the collagen-induced group (p < 0.05), indicating that vitamin c may help reduce the variability in platelet size caused by collagen treatment. the collagen-induced group also showed significantly higher pct compared to the control group (p < 0.05), suggesting an increase in platelet mass due to collagen treatment.; while collagen + vitamin c group showed significantly lower pct compared to the collagen-induced group (p < 0.05), indicating that vitamin c may help modulate the increase in platelet mass caused by collagen treatment (table 1). table 1. platelet counts, mean platelet volume (mpv), platelet distribution width (pdw) and plateletcrit (pct) in experimental groups. groups platelet count (x109/l) mpv (fl) pdw (%) pct (%) 1. control 722.0 ± 36.8 4.84 ± 0.92 8.36 ± 0.82 0.13 ± 0.02 2. collagen-induced 957.0 ± 23.5* 10.34 ± 0.65* 15.00 ± 0.99* 0.27 ± 0.02* 3. collagen + vitamin c 826.0 ± 31.6# 7.60 ± 0.27*# 11.08 ± 0.46# 0.18 ± 0.01# values are expressed as mean ± sem, * p < 0.05 compared to group 1: control group, # p < 0.05 comparison between group 2: collagen-induced group and group 3: collagen + vitamin c treated group. discussion platelet aggregation (the clumping of platelets in the blood, vital for blood clotting) has been associated with thrombosis due to excessive aggregation leading to excessive clotting. it has also been linked to platelet dysfunction, resulting in bleeding disorders (ghoshal and bhattacharyya, 2014; krishnegowda and rajashekaraiah, 2015). this study evaluated the potential of vitamin c to modulate platelet aggregation and dysfunction by assessing its effects on collagen-induced platelet aggregation and dysfunction. the results demonstrated that vitamin c significantly reduced the degree of platelet aggregation in collagen-induced platelet aggregation and dysfunction in wistar rats. this suggests that vitamin c has a potential therapeutic effect in modulating platelet function and could be beneficial in preventing or treating conditions associated with excessive platelet aggregation. this is in line with studies that suggests potential beneficial role of vitamin c on platelets function (wilkinson et al., 1999; ashor et al., 2014). a prothrombin time measures the time it takes for blood to clot and assesses the extrinsic pathway of coagulation. prolonged prothrombin time indicates impairment of clotting factors and may be associated with abnormal platelet aggregation (kamal et al., 2007). the prothrombin time in this study suggest that collagen treatment impairs the extrinsic pathway of blood coagulation in wistar rats, as evidenced by the prolonged prothrombin time. however, co-administration of vitamin c appears to counteract this effect, reducing the prothrombin time. these findings indicate a potential protective role of vitamin c in maintaining normal coagulation function in the presence of collagen-induced dysfunction. bleeding time assesses the overall ability of the body to form a platelet plug and stop bleeding. prolonged bleeding time has been associated with impaired hemostasis, platelet dysfunction, or coagulopathy, which can also be linked to abnormal platelet aggregation (jandrey, 2012; wohlauer et al., 2012). in this study, the results of bleeding time suggest that collagen treatment impairs primary hemostasis in wistar rats, as evidenced by the prolonged bleeding time. however, coadministration of vitamin c appears to mitigate this effect by reducing the bleeding time. these findings indicate a potential protective role of vitamin c in maintaining normal platelet function and primary hemostasis in the presence of collagen-induced disorders, consistent with studies reporting the beneficial impacts of vitamin c on platelet function and hemostasis (violi, 2010). fibrinogen (clotting factor i) is crucial in platelet aggregation; elevated fibrinogen levels can enhance platelet aggregation and thrombus formation (kattula et al., 2017). thromboxane b2 is a potent platelet agonist that, upon activation, enhances platelet activation and serves as a marker of platelet activation (smyth, 2010). 614 biology, medicine, & natural product chemistry 13 (2), 2024: 609-615 p-selectin (cd62p) is expressed on the surface of activated platelets and endothelial cells; its elevation indicates platelet activation and aggregation, as pselectin activated by thromboxane mediates plateletplatelet interactions leading to aggregation (chandler et al., 2010). in this study, collagen treatment enhanced coagulation response, platelet activation, and endothelial cell activation in wistar rats, as evidenced by increased fibrinogen, thromboxane b2, and p-selectin levels. coadministration of vitamin c appears to reduce this effect, suggesting a potential protective role of vitamin c in modulating coagulation, platelet activation, and endothelial cell activation, thereby modulating platelet aggregation in the presence of collagen-induced dysfunction. during platelet aggregation, there is an increase in platelet count, contributing to the aggregation and clotting process (stissing et al., 2011). mean platelet volume (mpv) rises, indicating the presence of larger platelets, which are more active in aggregation and release more granules, thereby promoting clot formation (vizioli et al., 2009). elevated platelet distribution width (pdw) and plateletcrit (pct) indicate greater variation in platelet size and a higher percentage of platelets in the blood, respectively, which can enhance platelet aggregation (cetin et al., 2017). this study demonstrated that collagen treatment significantly increases platelet concentration, mpv, pdw, and pct in wistar rats, suggesting substantial impacts on platelet size, variability, and mass. however, the coadministration of vitamin c appears to mitigate these effects, normalizing the values. these findings suggest a potential protective role of vitamin c in maintaining normal platelet homeostasis, function, and morphology in the context of collagen-induced bleeding disorders. this corresponds to studies that suggested that vitamin c can be beneficial for platelet homeostasis, function, and morphology in pathological conditions (ferroni et al., 2012, may and harrison, 2013). conclusion vitamin c significantly mitigated platelet aggregation and dysfunction in wistar rats by reversing the effects of collagen on degree of platelet aggregation, prothrombin time, bleeding time, fibrinogen, thromboxane b2, pselectin, platelet concentration, mean platelet volume (mpv), platelet distribution width (pdw), and plateletcrit (pct). this suggests a potential therapeutic role for vitamin c in preventing or treating excessive platelet aggregation, abnormal coagulation, and abnormal platelet activation, thus maintaining normal platelet homeostasis, function, and morphology which could be beneficial for bleeding disorders, especially thrombosis. conflicts of interest: the authors declare that there are no conflicts of interest. references ashor aw, lara j, mathers jc, siervo m. effect of vitamin c on endothelial function in health and disease: a systematic review and meta-analysis of randomised controlled trials. atherosclerosis. 2014; 235(1):9-20. brass lf, newman dk, wannemacher km, zhu l, stalker tj. signal transduction during platelet plug formation. platelets. 2013; 2:319-46. broos k, feys hb, de meyer sf, vanhoorelbeke k, deckmyn h. platelets at work in primary hemostasis. blood reviews. 2011; 25(4):155-67. cetin ms, cetin eh, akdi a, aras d, topaloglu s, temizhan a, aydogdu s. platelet distribution width and plateletcrit: novel biomarkers of st elevation myocardial infarction in young patients. polish heart journal (kardiologia polska). 2017; 75(10):1005-12. chandler ab, earhart ad, speich he, kueter tj, hansen j, white mm, jennings lk. regulation of cd40l (cd154) and cd62p (p-selectin) surface expression upon gpiib-iiia blockade of platelets from stable coronary artery disease patients. thrombosis research. 2010; 125(1):44-52. farndale rw, sixma jj, barnes mj, de groot pg. the role of collagen in thrombosis and hemostasis. journal of thrombosis and haemostasis. 2004; 2(4):561-73. ferroni p, vazzana n, riondino s, cuccurullo c, guadagni f, davi g. platelet function in health and disease: from molecular mechanisms, redox considerations to novel therapeutic opportunities. antioxidants & redox signaling. 2012; 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11(4):319. smyth em. thromboxane and the thromboxane receptor in cardiovascular disease. clinical lipidology. 2010; 5(2):209-19. stissing t, dridi np, ostrowski sr, bochsen l, johansson pi. the influence of low platelet count on whole blood aggregometry assessed by multiplate. clinical and applied thrombosis/hemostasis. 2011; 17(6):e211-7. tomaiuolo m, brass lf, stalker tj. regulation of platelet activation and coagulation and its role in vascular injury and arterial thrombosis. interventional cardiology clinics. 2017; 6(1):1. violi f, pignatelli p, basili s. nutrition, supplements, and vitamins in platelet function and bleeding. circulation. 2010; 121(8):1033-44. vizioli l, muscari s, muscari a. the relationship of mean platelet volume with the risk and prognosis of cardiovascular diseases. international journal of clinical practice. 2009; 63(10):150915. wilkinson ib, megson il, maccallum h, sogo n, cockcroft jr, webb dj. oral vitamin c reduces arterial stiffness and platelet aggregation in humans. journal of cardiovascular pharmacology. 1999 nov 1;34(5):690-3. wohlauer mv, moore ee, thomas s, sauaia a, evans e, harr j, silliman cc, ploplis v, castellino fj, walsh m. early platelet dysfunction: an unrecognized role in the acute coagulopathy of trauma. journal of the american college of surgeons. 2012; 214(5):739-46. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 2, 2015 | pages: 35-39 | doi: 10.14421/biomedich.2015.42.35-39 antioxidant potential of black, greenand oolong tea methanol extracts wahyu widowati1*, tati herlina2, hana ratnawati1, gabriella constantia1, i dewa gde sathya deva3 and maesaroh maesaroh3 1medical research center, faculty of medicine, maranatha christian university bandung, jl. prof. drg. suria sumantri mph no 65, bandung 40164, west java, indonesia 2chemist program, faculty of mathematics and natural sciences, university of padjadjaran, bandung. west java, indonesia 3aretha medika utama biomolecular and biomedical research center, bandung jl. babakan jeruk 2 no 9, bandung 40163, west java, indonesia author correspondency*: wahyu_w60@yahoo.com abstract degenerative diseases and chronic diseases are often caused by oxidative stress. oxidative stress caused by free radicals. antixodant as inhibitor are needed to prevent it which is one of antioxidant sources is tea. tea processing generally produce various kinds of teas such as black, green and oolong tea. tea processing affect the content of phenolic compounds. the aim of the research is to evaluate phytochemical content, total phenolic content of black tea, green tea and oolong tea extracts using catechin, quercetin, kaempferol, myricetin as standard, and to evaluate the antioxidative potency of black tea, green tea and oolong tea extracts compared to catechin, quercetin, kaempferol, myricetin. phytochemical assay using modified farnsworth method, the antioxidant activity were measured by by its 1,1-diphenyl-2-picrylhydrazyl (dpph) scavenging activity. green tea extract contained highest phenolic and flavonoid. the highest antioxidant activity was green tea extract with ic50=0,487 μg/ml. green tea extract content phenol and flavonoid are higher compared to the other extracts, green tea extract has the highest antioxidant activity. keywords: antioxidant, black tea extract, green tea extract, oolong tea extract, total phenolic content introduction various degenerative and chronic diseases are often caused by oxidative stress. oxidative stress is caused by free radicals. it highly reactive and unstable because of its unpaired electron in the outer atomic orbital. free radical can react with the cells molecules by binding to it. it can oxidize the nucleic acid, proteins, fats and even the cells dna and initiate the degenerative disease (halliwell & gutteridge, 2007). free radicals are derived either from normal essential metabolic process in the human body or from external exposure (bagchi & puri, 1998). the antioxidants as oxidation inhibitor are needed to overcome the negative effect of the free radicals. antioxidant inhibits the oxidation by reacting to reactive free radicals to form reactive substances that relatively stable. endogenous antioxidant already present in the human body. however the exogenous antioxidant still needed if the free radicals present in copious amounts (johnson, 2002). a balance between free radicals and antioxidant is necessary for proper physiological function. antioxidant may exert their effect on biological systems by different mechanism, including electron donation, metal ion chelation, co-antioxidants, or by gene expression regulation (krinsky, 2000; lobo et al., 2010). there are two kinds of antioxidant based on its source, natural antioxidant and synhetic antioxidant. synthetic antioxidants are carcinogenic when it consumed for long term. the needs of natural antioxidant that have fewer side effect and less toxic continues to rise.natural antioxidants can protect the body against the damage caused by reactive oxygene species (ros), inhibit the degenerative disease and inhibit the lipid peroxidase activity (wiseman et al., 1997). tea-one of the most popular beveragecomponents possess antioxidant activity.most commercially prepared tea is obtained form the leaf of the plant camelia sinensis (satoh et al., 2005). among tea, 69% of consumption is black tea, 28% of consumption is green tea and 3 % others is oolong tea (cabrera et al., 2008). teas of c.sinensis undergo different manufacturing processes. green tea is produced by steaming (japan) or panning (china) to prevent cathechin oxidation by polyhenol oxidase. oolong tea is semi-fermented while black tea is fully fermented (eric et al., 2011). many studies have shown that green, black and oolong tea has antioxidant properties (xie et al., 1993; wiseman, 1997; mckay & blumberg, 2002; zhu et al., 2002; higdon & frei, 2003; satoh et al., 2005). tea processing will affect the phenol content and ultimately will affect the antioxidant activity of it (higdon & frei, 2003). therefore, it become important to investigate the antioxidant activity of the extract of black tea, green tea, and oolong tea and test the phenol content based on the cathechin, quercetin, kaempferol and myricetin standard. methodology plant material preparation, extraction and phyrochemical content assay dried leaves green tea and black tea were obtained from cisaruni plantation, ptpn viii, west java, indonesia 36 biology, medicine, & natural product chemistry 4 (2), 2015: 35-39 and dried oolong tea was obtained from tea plantation, east java. extraction was performed based on maceration method using methanol 96% as the solvent (widowati et al., 2011a; widowati et al., 2013a; widowati et al., 2014a; widowati et al., 2014b). the extraction yielded 20.139 % of black tea, 22.11 % of green tea, and 17.39 % of oolong tea. the green tea, black tea, and oolong tea extract were tested by phytochemical assay using modified fransworth method including terpenoid, phenol, steroid, triterpenoid, flavonoid, tannin, alkaloid, and saponin assay (fransworth et al., 1966; widowati et al., 2010; bera et al., 2014). total phenol assay briefly 25 µl standard solution in 10 concentration level (500; 250; 125; 62,5; 31,25; 15,625; 7,81; 3,95; 1,98; and 0,98 µg/ml) of catechin, quercetin, kaempferol, and myricetin and sample (extract from green tea, black tea, and oolong tea) in concentration of 500 µg/ml were prepared for total phenol assay. each standard and sampled was mixed with 125 µl follin 10% and 100 µl of na2co3 7,5 % in microplate. the reaction then incubated at 45°-50°c for 10 minutes. the absorbance was measured in 760nm of wavelength using microplate reader. the linear regression equation (y= + ) was created based on the standard (cathechin, quercetin, kaempferol, myricetin) absorbance value. the analysis of phenol content of sample was performed based on the each of standard linear regression equation (ivanova et al., 2005; widowati et al., 2011a; widowati et al., 2015). dpph scavenging activity assay dpph (2,2-diphenyl-1-picrylhydrayl) free radical scavenging activity were analyzed based on the linear regression equation continued by median inhibitory concentration (ic50)value determination. ten concentration levels (500; 250; 125; 62,5; 31,25; 15,625; 7,8; 3,9; 1,9; and 0,9 µg/ml) of black tea, green tea,and oolong tea extract as working solution was prepared for this assays. briefly, 50µl samples (working solution) were added to a microplate followed by 200µl dpph (sigma-aldrich) solution (0,077 mmol/l in methanol). the reaction was shaken vigorously and kept in the dark for 30 minutes at room temperatures. dpph scavenging activity was determined by microplate reader at 517 nm. metanhol absolute was used as blanko. the ic50 value then determined. the radical scavenging activity of each sample was measured according to equation 1 (han et al., 2004; widowati et al., 2013b; widowati et al., 2013c; widowati et al., 2015).% = ( − )/( × 100) (1) description as = sample absorbance ac = negative control absorbance (without sample) results phytochemical content of tea extracts table 1 shows the different phytochemical content, including terpenoid, phenol, steroid, triterpenoid, flavonoid, tannin, alkaloid, and saponin of green tea, black tea, and oolong tea methanol extract. all of phytochemical tested was found in the green tea extract. high level of phenol also found in green tea and oolong tea extract. green tea also containts the highest level of alkaloid compared to the other tea extract. otherwise, steroid and alkaloid did not observed in the black tea extract. triterpenoid and tannin also not found in the oolong tea extract. less content of saponin also found in all of the extract. table 1. phytochemical content assay of black tea, green tea, and oolong tea methanol extract. sample phytochemical assay terpenoid phenol steroid triterpenoid flavonoid tannin alkaloid saponin black tea ++ ++ + ++ + + green tea + ++++ + + +++ + +++ + oolong tea + ++++ ++ +++ + + description: ++++ : very high content, +++: high content, ++: moderate content, +: low content, : undetected phenolic compound of tea extracts statistical assay based on linear regression equation was performed to analyze the total phenol of black tea, green tea, and oolong tea based on the standard solution (cathechin, quercetin, kaempferol and myricetin). the highest level of total phenol was showed by green tea extract (table 2). the degree of oxidation affected by the polyphenols profile of the tea. (balentine et al., 1997). wahyu widowati, et.al. – antioxidant potential of black, greenand oolong tea methanol extracts 37 table 2. the average of total phenol level concentration of black tea, green tea, and oolong tea (μg/mg). sample catechin equivalent (ce) quercetin equivalent (qe) kaempferol equivalent (ke) myricetin equivalent (me) black tea extract 14,33 4,50 4,33 4,17 green tea extract 23,33 9,71 4,33 6,17 oolong tea extract 16,08 5,09 3,33 4,67 dpph scavenging activity the most active extract in dpph scavenging activity showed by green tea extract indicated by the lowest ic50 value compared to the other extract and standard (table 3). dpph is a stable free radical because of the unpaired electron. the unpaired electron of the radical becomes paired in the presence of hydrogen donor, decreasing the absorption in 517 nm of wavelength. the dpph scavenging activity has been widely used to test the compound ability as a free radical scavenger and antioxidant activity in food or plat extract (satoh et al., 2005). previous study already obtained that the black tea has the antioxidant activity through dpph free radical scavenging activity with ic50= 5,405 µg/ml (widowati et al., 2011a), catechin (c) diluted in methanol with ic50 =8.11 µm (evacuasiany et al., 2014) and catechin diluted in dmso with ic50 =7.02 µg/ml (budiman et al., 2014). table 3. inhibitory concentration (ic50) value of antioxidant dpph scavenging activities of tea extract and standard. samples replication linear equation r2 ic50 (µg/ml) average ic50 green tea extract 1 y= 4,299x + 48,70 0,767 0,30 0,487 ± 0,2582 y= 4,539x + 46,45 0,735 0,78 3 y= 4,342x + 48,37 0,751 0,38 oolong te extract 1 y= 7,363x + 12,93 0,911 5,03 5,005 ± 0,060 2 y= 7,225x + 13,55 0,918 5,04 3 y= 7,286x + 14,03 0,911 4,94 2 y= 6,592x + 3,575 0,989 7,04 3 y= 6,666x + 3,671 0,987 6,95 quercetin 1 y= 6,153x + 23,65 0,901 4,28 4,279 ± 0,0652 y= 6,095x + 24,32 0,893 4,21 3 y= 6,128x + 23,39 0,878 4,34 kaempferol 1 y = 8,731x 11,662 0,823 7.06 7.154±0.1342 y = 8,063x 8,924 0,887 7.31 3 y = 9,039x 14,107 0,827 7.09 myricetin 1 y = 12,803x 6,749 0,829 4,43 2 y = 11,588x 4,270 0,809 4,69 4,496±0,058 3 y = 10,89x + 2,464 0,719 4,37 discussion tea is one of the most widely consumed beverages in the world which is grouped into three main groups, including green, black, and oolong tea, according to the fermentation process, fermentation of tea can affect the phytochemical content of different tea extract confirmed by the result that highest phenol and flavonoid content was observed in green tea (non fermented tea) and oolong tea (semi fermented tea) compared to black tea (fully fermented tea). in the fermentation process, the catechin is oxidized resulting thearubigin, theaflavin including theaflavin (tf1), theaflavin-3-gallate (tf2a), theaflavin3’-gallate(tf2b), and theaflavin-3,3’-digallate (tf3b) which is the key component of the black tea flavour (leung et al., 2001;. yang & landau, 2002; usda, 2003). phytochemical assay of green tea extract catechins are a group of natural polyphenols found in green tea. previous study of green tea exract using ehanol 70% resulted phenols (+++), triterpenoids (++), steroid (), terpenoids (+++), saponins (+), alkaloids (++), flavonoids (++), tannins (+++) (fanny et al., 2015). different solvent concentration resulted different compounds, yield and bioacivities (tiwari et al., 2011) the different solvent would result the different compound and bioactivity (pujimulyani et al., 2004; widowati et al., 2011b)., validated by previous study that water extract of forsythia koreana flowers exhibited a higher phenolic content than ethanol extract (yang and kang, 2011). otherwise, theaflavins are another group of polyphenol 38 biology, medicine, & natural product chemistry 4 (2), 2015: 35-39 pigment found in both black and oolong teas.oolong tea that partially oxidized and retains a considerable amount of the original catechin (nor & mohd, 2003). many studies have demonstrated that both catechin and theaflavin have strong free-radical-scavenging activity both in vivo and in vitro (lai et al., 2001). the chemical composition of tea includes proteins, polysaccharides, polyphenols (cathecins or flavan-3-ols, theaflavins, thearubigins, and proanthocyanidins), chlorophyll, minerals, and trace elements volatile compounds, amino and organic acids, lignins and alkaloid (chen et al., 2009). tea is wellknown for it health benefits because of its poliphenol bioactive content, especially chatechin which have antioxidants activity that play a role in reducing the free radical effect (vecchia et al., 1992; bravo et al., 1998; nagao et al., 2005). catechin belong to flavonoid that posses the high antioxidant activity in biological system. (gramza et al., 2005; evacuasiany et al., 2014) according to the table 2. the most active extract in dpph scavenging activity was the green tea extract showed by the lower of ic50 values compared to the catechin, quercetin, kaempferol, and myricetin. the green tea extracts have the higher antioxidant activity through dpph scavenging activity and ic50 value compared to the black tea extracts (widowati et al., 2011a). that result is in accordance to the phytochemical assay (table 1) and total phenol assay (table 2) showed that the green tea extract containts the higher total phenol content compared to black tea and oolong tea extracts and the higher flavonoid compared to the black tea extracts. high correlations were observed between antioxidants activities and polyphenol phytochemicals content. the antioxidants activities most probably might be contributed by polyphenols contents in the plant extracts (bakar et al., 2009; ling et al., 2010; nor & mohd, 2013). the result of this study also confirmed that the green tea extract posses the higher antioxidant activity compared to the catechin, quercetin, kaempferol and myrietin. table 2 also confirmed that teas extract contain catechin and various compound including quercetin, kaempferol and myricetin (cabrera et al., 2006). some research also investigated that black tea, oolong tea and green tea posses the antioxidant activity (xie et al., 1993; zhu et al., 2002). this study also in line with the research that polyphenol compound have antioxidant activity (higdon & frei, 2003; widowati et al., 2011a). green tea is the good source of polyphenol especially for flavanol and flavonol compounds which is found in 30% of tea leaf dry mass (wolfram et al., 2008). the antioxidant activity of green tea extracts was higher than oolong tea and black tea extracts (gramza et al., 2005). satoh et al (2005) also found that the green tea has the highest percent dpph scavenging activity followed by roasted tea, oolong tea, and black tea 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online available zhu q, hackman r, ensunsa j, holt r, keen c (2002) antioxidative activities of oolong tea. j agric food chem 50: 6929-6934. content_v4n2_3.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry volume 3 – number 1 – 2014 issn 2089-6514 contents ecopharmacognosy: exploring the chemical and biological potential of nature for human health geoffrey a. cordell 1 14 the effect of water-soluble stem extract “kayu kuning“ (arcangelisia flava l.merr) on the growth inhibition of candida albicans atcc 10231 and trichophyton mentagrophytes in vitro rini setyowati, sudarsono, setyowati e.p 15 19 larvicidal activity of the mixture of cashew nut shell liquid (cnsl) and aqueous extract of sapindus rarak dc against larvae of culex quinquefasciatus rahmi safarina fauziah, sudarsono, budi mulyaningsih 21 23 identification of migratory birds and their spesific characteristics of habitat in the salt water lake of gili meno, north lombok distric diah purwitasari, luh gde sri astiti, supriadi 25 30 larvicidal effect of vinca fruit extract (vinca rosea) against aedes aegypti larvae and secondary metabolites profile by thin layer chromatography rahmawati ekaputri, sudarsono, budi mulyaningsih 31 33 morpho-anatomical analysis of cosmostigma racemosum (asclepiadoideae) flowers widodo, mohamad amin, mimien henie irawati al-muhdar, muhammad ja’far luthfi 35 46 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 465-472 | doi: 10.14421/biomedich.2025.141.465-472 issn 2540-9328 (online) phytochemicals, nutritional and anti-nutritional composition of aqueous extracts of white and red onions bulbs ibrahim abubakar1,*, jabir aliyu danyaya2, abdulganiyu mohammad galadima3, iklima bandi ibrahim4, said sani said5 sanusi umar farouq6 1department of biology-chemistry, idris koko technical college, farfaru, sokoto, nigeria 2biochemistry unit, department of science technology, waziri umaru federal polytechnic, birnin kebbi, nigeria 3department of biochemistry, school of biological sciences, federal university of technology owerri, imo state, nigeria 4centre for advanced science research and analytical services, usmanu danfodiyo university sokoto, nigeria 5department of biochemistry and molecular biology, faculty of life science, federal university, dutsinma, katsina, nigeria 6nisa premier hospital, jabi, abuja, nigeria. corresponding author* ibrahimabubakarsok@gmail.com manuscript received: 03 may, 2025. revision accepted: 15 july, 2025. published: 23 july, 2025. abstract onion bulbs have been consumed for foods and nutrition and management of many diseases including infections, diabetes, gastrointestinal disorders, cardiovascular and respiratory disorders. this study aims to evaluate the phytochemicals, nutritional and antinutritional composition of the aqueous extracts of white and red onions bulbs. analytical techniques aoac and aas were used in phytochemicals and proximate analysis and determination of minerals, heavy metals, determination of anti-nutrients content of the aqueous extracts of white and red onions bulbs. the findings showed the presence of alkaloids, flavonoids, tannins, glycosides, saponins, steroids, cardiac glycosides, and anthraquinones in the aqueous extracts of red and white onions bulbs. the aqueous extracts of white and red onions bulbs contain significant (p < 0.05) amounts of moisture (89.68 and 87.04%), ash (3.22 and 2.03%), fiber (3.00 and 2.00%), carbohydrates (2.33 and 2.59%), proteins (3.19 and 1.04%), and crude lipids (1.22 and 2.66%), respectively. the aqueous extracts of white and red onions bulbs demonstrated significant (p < 0.05) level of potassium (987.10 and 756.24 ppm), sodium (42.50 and 32.50 ppm), calcium (8.90 and 6.10 ppm), phosphorus (3.87 and 4.04 ppm), zinc (2.32 and 1.13 ppm), magnesium (1.85 and 1.12 ppm), iron (0.62 and 0.58 ppm), and copper (0.47 and 0.17 ppm), respectively. low concentrations of nickel (0.112 and 0.524 ppm), lead (0.034 and 0.023 ppm), cadmium (0.029 and 0.014 ppm), and chromiun (0.090 and 0.032 ppm) were observed in the aqueous extracts of white and red onions bulbs contain, respectively. the aqueous extracts of white and red onions bulbs exhibited moderate and low amounts of cynogenic glycosides (3.00 and 12.46 mg/100g), saponins (2.02 and 2.07 mg/100g), oxalate (0.63 and 3.75 mg/100g), tannins (0.90 and 1.09 mg/100g), and phytate (0.38 and 1.49 mg/100g), respectively. the aqueous extracts of white and red onions bulbs contain various phytochemicals and significant amounts of important nutrients with trace quantity of certain heavy metals and anti-nutrients. keywords: anti-nutrients; food, nutrients; onions bulbs; phytochemicals. introduction plants based foods and their products have been consumed due to their nutritional contents and medicinal properties. many communities in the world depend on the plants especially fruits and vegetables for food and nutrition and remedies. reports showed that almost 80 % of people in the world depend on plants and herbs for foods and nutrition and remedies (khan and ahmad, 2019). plants are widely available in every community in the world and contain several nutrients and bioactive compounds that demonstrate many pharmacological activities. nutrients composition and phytoconstituents of foods and plants determine their nutritional value and medicinal properties. nutrients in foods and plants are of significant biological importance and play vital roles in biochemical processes. phytochemicals from plants demonstrate medicinal properties and pharmacological activities. the therapeutic uses and pharmacological activities of plants are attributed to their various phytochemicals. several bioactive compounds isolated from different plants extracts have been used in the development of drugs (kumar et al., 2021). antinutrients are substances that decrease nutrient absorption and nutritive value of foods. certain anti-nutrients cause harmful effects on human and animal health. however, at low level, certain specific anti-nutrients may provide health benefits to human and animals. onion (allium cepa l.) is a vegetable plant that belongs to the family liliaceae (tame & afolabi, 2020). onion is one of the most widely cultivated plants and the second largest produced vegetable crop worldwide (eric, 2010; ray, 2024). the report showed that in 2022, about https://doi.org/10.14421/biomedich.2025.141.465-472 466 biology, medicine, & natural product chemistry 14 (1), 2025: 465-472 five million tonnes of onions and shallots were produced worldwide (faostat, 2024). onions are consumed as raw or processed food and have been used as an ingredient in many food products. onions contain many nutrients including proteins, carbohydrates, sugars, vitamins, and minerals that have many significant health benefits. onion bulbs have been used as food and/or as an herb for the treatment of many diseases (sami et al., 2020). onion bulbs demonstrated many pharmacological properties which may be due their several phytoconstituents. in nigerian, onions are abundantly cultivated and produced in northern region of the nation. the plant is locally called albasa in hausa and consumed by the communities in every day feedings. onions have been used locally in the treatment of many diseases including skin disorders, infections, gastrointestinal disorders, pains, and parasitic disorders (sami et al., 2020; kandoliya et al., 2015). it has been reported that consumption of onions can reduce the risks of inflammatory diseases, coronary heart diseases, cancer, and respiratory disorders which could be attributed to their bioactive constituents (sami et al., 2021; sami et al., 2017; upadhyay, 2016). depending on their varieties onions are characterized by different colors such as red, yellow, white, green and purple. red and white onions have been used as raw foods and processed (cooked) foods in many cuisines (mower, 2009). red onions have been used in the management of cancer, infections, oxidative stress associated disorders, cardiovascular and neurological disorders (chadorshabi et al., 2022). this study aimed to evaluate the phytochemicals, nutritional and anti-nutritional composition of the aqueous extracts of white and red onions bulbs. materials and methods chemicals and reagents in this research the analytical grade chemicals and reagents were used. all the chemicals and reagents were produced by reidel-de haem (merck, germany), abbott laboratories (usa) and sigma-aldrich (st. louis, mo, usa). samples collection the fresh onion bulbs of different varieties allium cepa .var. ascalonicum (red onins) and allium cepa blanc. (white onions) were obtained from gada onions market, gada local government area, sokoto state, nigeria. the samples materials were identified and authenticated (uduh/asn/088) at taxonomy unit, department of biological sciences, usmanu danfodiyo university, sokoto, nigeria. extracts preparation the samples materials were washed with distilled water and then cut into pieces. the samples were shed dry at 25 oc for two weeks and then grinded to grainy powder using pestle and mortar. the corse-grainy powders were stored in a clean container at 25 oc for the nutritional and anti-nutritional analyses. the extracts were prepared according to the method described by abubakar et al. (2021) with little changes. five hundred grams of the powdered samples was separately soaked in deionized water (2 l) for forty eight hours with constant stirring at every sixty minutes. the samples extracts were filtered through whatman filter paper no 1 and then concentrated to dryness in rotary evaporator under reduced pressure at 40 °c for 180 minutes. the weight of the dried extracts was measured using analytical weighing balance and the percentage yields of the extracts were obtained. the extracts were stored in sterilized desiccators at room temperature for further analyses. phytochemical screening determination of alkaloids the aqueous extracts of white and red onions bulbs were analyzed for the presence alkaloids by wagner's test using the method of mosa et al. (2012) and trease and evans (1989). three miles of one percent hydrochloric acid solution were transferred into separate test tubes. three miles of the samples extracts were respectively added into the test tubes followed by heating for 20 minutes and then cool at room temperature. one mile of the wagner’s reagent was added into the test tubes in drops. a reddish-brown precipitate was formed which indicated the presence of alkaloids in the extracts. determination of flavonoids a sodium hydroxide test was employed for the qualitative determination of flavonoids in the aqueous extracts of white and red onions bulbs using the method of mosa et al. (2012) and ibrahim et al. (2024). three miles of the samples extract were transferred into the separate test tubes followed by the addition of one mile of 10% sodium hydroxide solution. the development of an intense yellow colour which became colourless after the addition of dilute hydrochloric acid solution indicated the presence of flavonoids in the samples extracts. determination of tannins tannins in the aqueous extracts of white and red onions bulbs were qualitatively estimated by ferric chloride test using the method of trease and evans (1989) and ibrahim et al. (2024). one mile of the samples extracts was transferred into the test tubes followed by addition of two miles of 5% ferric chloride solution. the present of tannins in the samples extracts was observed by the development of black or blue-green colour. determination of saponins determination of saponins in the aqueous extracts of white and red onions bulbs was carried out using froth abubakar et al. – phytochemicals, nutritional and anti-nutritional composition of … 467 test as described by mosa et al. (2012), abubakar et al. (2022; 2020) and trease and evans (1989). three miles of deionized water were transferred into separated test tubes followed by addition of three miles of the samples extracts. the test tubes were immensely shaken for half minute and then allowed to settle for half hour at room temperature. a stable persistent froth was formed which indicated the presence of saponins in the sample extracts. determination of steroids steroid presence in the aqueous extracts of white and red onion bulbs was determined according to the method described by trease and evans (1989) and ibrahim et al. (2024). the sample extracts (500 µl) were treated with five miles of chloroform and five miles l of sulphuric acid solution. the development of violet colour which changed to blue-green indicates the presence of steroids in the samples extracts. determination of glycosides the qualitative test for glycosides in the aqueous extracts of white and red onions bulbs was done using salkowski’s test according to the method described by ibrahim et al. (2024) and abubakar et al. (2022; 2020). five miles of the sample extracts were added into the test tubes containing five miles of 1 % h2so4 solution. the test tubes were heated at 100 oc for 15 minutes and then cool at room temperature. five miles of fehling’s solution a and b were added into the mixtures after neutralized with 10% sodium hydroxide solution. the development of brick red precipitate of reducing sugars indicated the presence of glycosides in the samples extracts. determination of terpenoids the aqueous extracts of white and red onions bulbs were analyzed for the presence of terpenoids using the method of trease and evans (1989) and ibrahim et al. (2024). the samples extracts in respective test tubes were treated with one mile of ethanol and one mile of acetic anhydride. ten miles of concentrated sulphuric solution were added into the respective test tubes containing the mixture. a pink color was formed which indicated the presence of terpenoids. determination of cardiac glycosides cardiac glycosides presence in the aqueous extracts of white and red onions bulbs was qualitatively determined using the keller-killani test according to the the method described by mosa et al. (2012) and trease and evans (1989). five miles of the sample extracts in separate test tubes were treated with two miles of glacial acetic acid. one drop of fecl2 solution and one mile of concentrated h2so4 solution were added into the test tubes. a brown ring was formed at the interface which indicated the presence of deoxysugar, a characteristic of cardenolides. however, a violet colour appeared below the brown ring which indicated the presence of cardiac glycosides in the sample extracts. determination of anthraquinones the qualitative analysis of anthraquinones in the aqueous extracts of white and red onions bulbs was performed using the method of trease and evans (1989). two grams of the powdered samples in the respective test tubes were treated with 10 cm3 of chloroform. the mixtures were vigorously shaken for five minutes and then filtered through the whatman filter paper. the filtrates were treated with ammonia solution and then shaken for five minutes. the development of bright pink colour in the upper aqueous layer indicated the presence of anthraquinones in the samples extracts. proximate analysis determination of the proximate composition (crude lipid, moisture, carbohydrate, crude protein, ash, and crude fiber) of the aqueous extracts of white and red onions bulbs was carried out using the method of aoac (2010). the experiments were performed in triplicate. the data analyzed were expressed in percentages as the mean and standard deviation. determination of minerals composition the levels of calcium, iron, zinc, magnesium, and copper in the aqueous extracts of white and red onions bulbs were determined by atomic absorption spectrophotometeric (aas) technique using the method of aoac (1990; 2005). the concentration of sodium and potassium in the aqueous of white and red onions bulbs was determined using flame photometeric technique according to the method described by aoac (1990; 2005). the spectrophotometeric technique was employed in the determination of phosphorus levels in the aqueous extracts of white and red onion bulbs. determination of heavy metals content the concentration of nickel, cadmium, lead, and chromiun in the aqueous extracts of white and red onions bulbs was estimated using atomic absorption spectrophotometeric (aas) technique according to the method described by aoac (1990; 2005). determination of ant-nutritional composition the level of oxalate, phytate, saponins, tannins and cyanogenic glycosides in the aqueous extracts of white and red onions bulbs was determined using the method of gupta et al. (2005), reddy and love (1999), aoac (1990), harborne (1984), and aoac (2005), respectively. statistical analysis the experiments were conducted in triplicate. the data were analyzed using statistical package for social sciences (spss) statistics version 22 software and expressed as mean ± standard deviation. differences 468 biology, medicine, & natural product chemistry 14 (1), 2025: 465-472 between the mean values were significantly computed by one-way analysis of variance (anova) at 95 % confidence level. significance was considered by twotailed (p < 0.05) values. results phytochemicals screening of aqueous extracts of white and red onions bulbs table 1 shows the phytochemicals constituents of aqueous extracts of white and red onions bulbs. high and moderate amounts of alkaloids, flavonoids, and tannins were observed in the aqueous extracts of red and white onions bulbs, respectively. the result showed that terpenoids and steroids were respectively present in the aqueous extracts of red and white onions bulbs at moderate and high amount. the aqueous extracts of red and white onions bulbs demonstrated a moderate and low amount of glycosides, saponins, and anthraquinones, respectively. a low amount of cardiac glycosides was found in the aqueous extracts of red and white onions bulbs (table 1). table 1. phytochemicals screening of aqueous extracts of white and red onions bulbs. phytochemical white bulb red bulb alkaloids ++ +++ glycosides + ++ flavonoids ++ +++ tannins ++ +++ saponins + ++ steroids +++ ++ cardiac glycosides + + terpeniods +++ ++ anthraquinones + ++ +++ = highly present, ++ = moderately present, + = slightly present. proximate composition of the aqueous extracts of white and red onions bulbs the proximate composition of the aqueous extracts of white and red onions bulbs were presented in table 2. the aqueous extracts of white and red onions bulbs contain higher significant (p < 0.05) amounts of moisture (89.68 and 87.04%), respectively. a significant (p < 0.05) amounts of ash (3.22 and 2.03%), fiber (3.00 and 2.00%), carbohydrates (2.33 and 2.59%), proteins (3.19 and 1.04%), and lipids (1.22 and 2.66%) were observed in the aqueous extracts of white and red onions bulbs, respectively. however, the aqueous extract of white onions bulbs exhibited higher amounts of moisture, ash, fiber and proteins compared to the aqueous extract of red onions bulbs (table 2). table 2. proximate composition of the aqueous extracts of white and red onions bulbs. parameter white bulb red bulb moisture (%) 89.68 ± 1.44a 87.04 ± 1.51b ash (%) 3.22 ± 0.28c 2.03 ± 0.15d fiber (%) 3.00 ± 0.20c 2.00 ± 0.14d carbohydrates (%) 2.33 ± 0.06d 2.59 ± 0.04d proteins (%) 3.19 ± 0.02c 1.04 ± 0.06e lipids (%) 1.22 ± 0.09e 2.66 ± 0.20d values are expressed as mean ± sd (n = 3) minerals contents of the aqueous extracts of white and red onions bulbs table 3 shows the minerals contents of the aqueous extracts of white and red onions bulbs. the findings indicated that the aqueous extracts of white and red onions bulbs contain higher significant (p < 0.05) amounts of potassium (987.10 and 756.24 ppm) and sodium (42.50 and 32.50 ppm) than the other minerals, respectively. the aqueous extracts of white and red onions bulbs exhibited significant (p < 0.05) level of calcium (8.90 and 6.10 ppm), phosphorus (3.87 and 4.04 ppm), zinc (2.32 and 1.13 ppm), and magnesium (1.85 and 1.12 ppm), respectively. also, low levels of iron (0.62 and 0.58 ppm) and copper (0.47 and 0.17 ppm) were observed in the aqueous extracts of white and red onions bulbs. in comparison with the aqueous extracts of red onions bulbs, the aqueous extracts of white onions bulbs demonstrated higher significant (p < 0.05) level of potassium, sodium, calcium, zinc, magnesium, iron and copper (table 3). table 3. minerals contents of the aqueous extracts of white and red onions bulbs. mineral white bulb red bulb sodium (ppm) 42.50 ± 1.50a 32.50 ± 0.46b potassium (ppm) 987.10 ± 7.87c 756.24 ± 5.31d phosphorus (ppm) 3.87 ± 0.25e 4.04 ± 0.08e calcium (ppm) 8.90 ± 0.95f 6.10 ± 0.23g magnesium (ppm) 1.85 ± 0.11h 1.12 ± 0.09i copper (ppm) 0.47 ± 0.03j 0.17 ± 0.02k zinc (ppm) 2.32 ± 0.16h 1.13 ± 0.10i iron (ppm) 0.62 ± 0.08j 0.58 ± 0.03j values are expressed as mean ± sd (n = 3) heavy metals contents of the aqueous extracts of white and red onions bulbs the heavy metals contents of the aqueous extracts of white and red onions bulbs are shown in figure 1. the results showed that the aqueous extracts of white and red onions bulbs contain trace level of nickel (0.112 and 0.524 ppm), lead (0.034 and 0.023 ppm), cadmium (0.029 and 0.014 ppm), and chromiun (0.090 and 0.032 ppm), respectively. however, the aqueous extract of white onions bulbs demonstrated high level of lead, cadmium, and chromium compared to the red onions bulbs extract (figure 1). abubakar et al. – phytochemicals, nutritional and anti-nutritional composition of … 469 figure 1. heavy metals contents of the aqueous extracts of white and red onions bulbs. values are expressed as mean ± sd (n = 3). anti-nutrients contents of the aqueous extracts of white and red onions bulbs figure 2 shows the levels of certain anti-nutrients in the aqueous extracts of white and red onions bulbs. the results showed a high concentration of cynogenic glycosides (3.00 and 12.46 mg/100g) and saponins (2.02 and 2.07 mg/100g) in the aqueous extracts of white and red onions bulbs, respectively. the aqueous extracts of white and red onions bulbs exhibited low levels of oxalate (0.63 and 3.75 mg/100g), tannins (0.90 and 1.09 mg/100g), and phytate (0.38 and 1.49 mg/100g), respectively. however, the aqueous extracts of red onions bulbs exhibited high significant (p < 0.05) levels of oxalate, tannins, phytate cynogenic glycosides and saponins compared to the red onions bulbs extract (figure 2). figure 2. anti-nutrients contents of the aqueous extracts of white and red onions bulbs. values are expressed as mean ± sd (n = 3). discussion in this study, the aqueous extracts of red and white onions bulbs demonstrated significant amount of alkaloids, flavonoids, tannins, glycosides, saponins, steroids, cardiac glycosides, and anthraquinones. the results of this study is in agreement with the similar study by dalhat et al. (2018) who reported that allium cepa demonstrated significant amount of phytochemicals including alkaloid, tannins and flavonoids. also, similar findings showed that allium sativum contains reasonable amount of certain phytochemicals which include saponins and flavonoids (martin & griswold, 2009). results of relevant studies by aliyu et al. (2024), ibrahim et al. (2024) and abubakar et al. (2022) showed the presence of many phytochemicals in some plants materials. phytochemicals exhibited many medicinal properties and pharmacological activities (oghenejobo et al., 2017). the plants saponins decrease the risk of coronary heart diseases (gemede & ratta, 2014). study showed that terpenoids extracted from the plant demonstrated analgesic, antiimflammatory, anti-fungal, anti-microbial, anti-viral and anti-parasitic activities (mercola, 2017). cardiac glycosides from plants extracts have been used for the treatment of various complications of cardiovascular disorders (denwick, 2002). alkaloids have therapeutic uses and have been used in drug discovery and development (okolo et al., 2012). it has been documented that flavonoids from different plants extracts demonstrated antimalarial, antiinflammatory, and antioxidant activities (liu, 2013). sex hormones and steroidal drugs have been synthesized from the steroids isolated from plants extracts (majeed et al., 2004). it has been shown that tannins extracted from allium cepa skin exhibited significant antioxidant and anti-inflammatory activities (nwinuka et al., 2005). research showed that the medicinal properties of the plant for the treatment of ulcer, earache and as an antitussive could be attributed to its tannins content (dalhat et al., 2018). in the present study, high significant amount of moisture, fiber, carbohydrate, protein, lipid, and ash content was found in the aqueous extracts of red and white onions bulbs. this finding is in line with the findings which showed that onion bulbs contain significant amount of proteins (armand et al., 2018). high percentage of proteins consume especially in low and middle income countries are sourced from vegetables. proteins play many vital biological functions such as maintaining healthy skin, increase in body growth, and tendency to displace worms out of cells (okwu & morah, 2004). significant amount of lipid in onion bulbs has been reported in similar study (elhakem et al., 2021). lipids are important sources of high energy compounds and fat-soluble vitamins that play a vital role in biological and biochemical processes (ogbuagu et al., 2011). results of the present study are in agreement with relevant findings by tame and afolabi (2020) which indicated that onion varieties exhibited significant quantity of ash contents. minerals content of a foods and plants is determine by their ash content (oloyede, 2005). high carbohydrate content was observed in different varieties of onion bulbs (tame and afolabi, 2020). this 0 0,1 0,2 0,3 0,4 0,5 0,6 lead chromium nickel cadmiun c o n ce n tr at io n ( p p m ) heavy metal white red 0 2 4 6 8 10 12 14 c o n ce n tr at io n ( m g/ 1 0 0g ) antinutrients white red 470 biology, medicine, & natural product chemistry 14 (1), 2025: 465-472 finding agrees with the relevant studies by dalhat et al. (2018) and okwu and morah (2004) who reported high fiber content in allium sativum and allium cepa. dietary fiber provides an important role in regulation of normal peristaltic movement of the intestinal tract and in food digestion (dalhat et al., 2018; okwu & morah, 2004). in the present study the aqueous extracts of red and white onions bulbs demonstrated a significant amount of sodium, potassium, phosphorus, calcium, magnesium, zinc, copper, and iron. sodium and potassium play an important role in acid-base balance maintenance and regulation of osmotic pressure, membrane potentials, muscles contraction and transmission of nerve impulses (aliyu et al., 2024). calcium serves many functions in biological system such as regulation of vasodilatation and vascular contraction, nerve transmission, muscle function, hormonal secretion, and intracellular signaling (aliyu et al., 2024; catharine et al., 2018). calcium functions as blood clotting agent and in bone and teeth development (abubakar et al., 2022). magnesium plays a significant role in growth and integrity of bone, muscles and nerves functions, and regulation of the cardiac cycle (aliyu et al., 2024; allen & sharma 2019; gragossian and friede 2019). zinc provides an important role in tissues formation, immune cell proliferation and maturation, wound repair, hair growth, activation of signal transductor, regulation of oxidative stress, and gene expression (baltaci et al., 2018; kimura & kambe, 2016). copper serves important function in development of bone, hematopoiesis process and certain enzymatic activities such as ferro-oxidase, catalase, cytochrome oxidase and tyrosinase (leone et al., 2006). iron regulates the activities of certain enzymes in many biological and biochemical processes including synthesis of hemoglobin, transport of oxygen, oxidative processes, cellular growth and catalytic reactions (akram et al., 2020; yiannikourides & latunde-dada, 2019). in this study, trace amount of lead, nickel, chromium, and cadmium was found in the aqueous extracts of white and red onions bulbs. heavy metals can produce adverse effects on human and animals health and the environment. consumption of foods and plants accumulated with various heavy metals has adverse health effects (ibemenuga et al., 2019). high level of heavy metals in foods and plants causes toxic effect on certain organs and tissues (abubakar et al., 2022). consumption of heavy metals in foods and plants can cause many adverse health effects including damage of nervous system, influencing fetal development, carcinogenicity and impaired immune function (sekarwati et al., 2015). in the current study, a trace amount of phytates, oxalates, tannins, saponins, and cyanogenic glycosides was observed in the aqueous extracts of red and white onions bulbs. anti-nutritional factors demonstrate several adverse effects in foods and plants and cause toxic effects in human and animals’ body. tannins bind proteins in foods and plants to form complexes which inhibit the activity of digestive enzymes such as trypsin, chymotrypsin and amylase affecting digestion of nutrients (edet et al., 2015; amadi et al., 2012; duru et al., 2012). studies showed that phytates influenced the absorption of several important elements including iron, zinc, magnesium, and calcium (masum et al., 2011). saponins decrease the activities many important enzymes such as trypsin and chymotrypsin thereby reducing the nutrients bioavailability (liener, 2003). it has been reported that phytates inhibit the activities of digestive enzymes such as pepsin, trypsin and amylase (kumar et al., 2010). oxalates bind calcium to form insoluble complexes which lower the capacity of body to absorb nutrients and cause harmful effects to humans and animals (umar et al., 2013). report showed that consumption of foods rich in high concentration of oxalate causes irritation in the mouth and lower region of the gut (gemede & ratta, 2014). conclusion the aqueous extracts of white and red onions bulbs contain many phytochemicals and significant amounts of proximate components and important minerals with trace amount of certain heavy metals and anti-nutrients. however, the aqueous extract of white onions bulbs demonstrated high significant amounts of proximate components and important minerals compared to the aqueous extract of red onions bulbs. also, the levels of the heavy metals and the anti-nutrients were higher in the aqueous extract of white and red onions bulbs, respectively. conflict of interest: the authors declared that there was no conflict of interest. references abubakar, i., aliyu, j.d., abdullahi, z., zubairu, z., umar, a.s., & ahmad, f. 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(2019). a short review of iron metabolism and pathophysiology of iron disorders. medicines, 6(3), 85. http://doi.org/10.3390/medicines6030085 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 265-269 | doi: 10.14421/biomedich.2024.131.265-269 issn 2540-9328 (online) potential of soursop leaf extract as an antioxidant in mcf-7 cells 1department of biological, faculty of medicine, universitas yarsi, jakarta 10520, indonesia. 2department of anatomical pathology, faculty of medicine, universitas yarsi, jakarta 10520, indonesia. 3telomere research center, yarsi university research institute, universitas yarsi, jakarta 10520, indonesia. corresponding author* nunung.ainur@yarsi.ac.id manuscript received: 16 october, 2023. revision accepted: 07 december, 2023. published: 08 august, 2024. abstract the frequency of breast cancer tends to increase. malondialdehyde (mda) is a marker of oxidative stress as an end product from the chain reaction of lipid peroxidation. the use of traditional medicine soursopleaf (annona muricata l.) has been reported for a long time because of its bioactivity as an antioxidant. this study analyzes the relationship between mda levels and glutathione enzymes in mcf-7 cells given the methanol extract of soursop leaves. the methanol extract of soursop leaves was carried out by infusion method. the methanol extract of soursop leaves was given to cancer cells at several doses with an incubation of 24 hours. the cytotoxic test was carried out using the mtt method. measurement of mda levels was carried out using the thiobarbituric acid reactive substance (tbars/tba) reactivity test method. gsh measurements used the colorimetric method. the results showed that the ethanol extracts of soursop leaves have cytotoxic activity in the mcf-7 breast cancer cell line with ic50 values of 23.96 ppm. ethanol extract of soursop leaves increased levels of mda inhibition and gsh level. soursop leaf extract could increase mda inhibition gsh level in human breast cancer cells mcf-7. keywords: breast cancer; gsh; mcf-7; mda; soursop leaf. introduction in 2018, there were 18 million new cancer cases; by 2040, it could be 29–37 million. new cases of breast cancer reached 11.6% in 2018 (world health organization, 2020). boundouki et al. (2021) stated there are more than 2 million new cases every year worldwide. one of the triggers for breast cancer is oxidative stress mechanisms (sossa et al., 2013). oxidation reactions involving free radicals can damage the surrounding normal cell membrane and damage the composition of dna to cause a mutation. mutations or damage to the composition of dna can cause cancer. free radical oxidation of proteins, nucleic acids, and lipids each produces carbonyl compounds, mda (malondialdehyde), and deoxyguanosine p (shaw et al., 2011). the body needs antioxidants to combat free radicals. antioxidants are either obtained from outside the body (food) or produced from within the body (lobo et al., 2010). examples of endogenous antioxidants include superoxide dismutase, glutathione (gsh), catalase, and glutathione peroxidase (rizzo et al., 2010). glutathione is an enzyme with various uses, including detoxification, antioxidants, maintenance of thiol status, and modulation of cell proliferation (lushchak, 2012). breast cancer treatment approaches include surgery, radiation treatment, endocrine treatment, and chemotherapy (anjum et al., 2017). however, most of the mechanisms underlying treatment involve reactive oxygen species (ros) production and result in increased oxidative damage (de sa junior et al., 2017). one way to reduce levels of ros by providing antioxidants. natural antioxidants have been extensively studied to fight breast cancer and tumor development as chemopreventive agents (griñan-lison et al., 2021). one of the traditional food sources that contain glutathione as antioxidants are soursop (annona muricata l.). soursop is a species of the annonaceae family, studied extensively for its therapeutic potential (gavamukulya et al., 2017). soursop leaf extract contains phenolic and flavonoid compounds that act as antioxidants (ovando-domínguez et al., 2019). several studies have shown that soursop leaf extract is proven to cure disease and breast cancer cell death (rady et al., 2018; fertilita et al., 2020). however, there has been no specific research on the effect of giving soursop leaf extract on reducing oxidative stress in mcf-7 cell cultures, so this needs to be done. the present study aims to investigate the cytotoxic effect and relationship between levels of mda inhibition muhammad samsul mustofa1, restu syamsul hadi1, nunung ainur rahmah2,*, pendrianto3 https://doi.org/10.14421/biomedich.2024.131.265-269 266 biology, medicine, & natural product chemistry 13 (1), 2024: 265-269 and glutathione enzymes in mcf-7 cells given the ethanol extract of soursop leaves. materials and methods materials the mcf-7 breast cancer cell lines were purchased from atcc (manassas, va, usa). dulbecco’s minimum essential medium (dmem) (gibco, new york, usa) supplemented with 10% fetal bovine serum (gibco, new york, usa), with 5% of the antibiotic-antimycotic (corning, usa). annona muricata l. powder extraction was carried out using the infusion method. the mtt kit was obtained from abnova (taiwan). the mda and gsh kit were obtained from elabscience (usa). soursop leaf extraction soursop leaf extract was obtained by maceration. the extraction was carried out at the yarsi university herbal research center. a total of 100 grams of soursop leaves were blended into flour and then macerated in 250 ml of 70% ethanol for 24 hours. the sample was filtered, and the filtrate was collected. the remaining sample was added with 100 ml of 70% ethanol and macerated for 24 hours. then, the sample was filtered again, and the filtrate was collected. the second residual sample was added to 100 ml of 70% ethanol. the filtrate was then concentrated using a rotary evaporator to produce a thick extract. cell culture mcf-7 cells were routinely cultured in a tissue flask containing dmem with 10% fbs and 5% antibioticantimycotic at 37°c in a humidified atmosphere with 5% co2. cells from stock plates were suspended by treatment with 0.25% trypsin, buffered with 0.2% edta (ph 7.3), and counted using a hemocytometer to determine the effects of soursop leaf extract on cell proliferation. cells were adjusted to a density of 1x106 cells/well to the required plating medium volume supplemented with 10% pbs and soursop leaf extract dissolved in dmso. cytotoxicity assay the breast cancer cells were seeded into 96-well plates at a density of 5,000 cells per well in triplicates and were treated with 0, 10, 20, and 30 ppm concentrations of soursop leaf extract for 48 h (according to the mtt protocol datasheet). dmso (0.1%) was added to the control wells followed by incubation at 37°c for 2 h after addition of 20 mg/ml mtt (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) to each well. absorbance was measured on optical density at 490 nm. the ic50 was developed by an inhibition curve of three independent experiments. evaluation of mda inhibition mda is the last marker of the lipid peroxidation pathway. this assay is according to the repercussion of mda with thiobarbituric acid (tba) that forms the mdatba adduct that can be quantified calorimetrically. the sample was reacted with 200 μl of trichloroacetic acid (tca) 20% for deproteination. then the cortex and centrifuge at a speed of 5000 rpm for 10 minutes. the supernatant formed was taken, and 400 μl of tba 0.67% reagent was added. then the sample was vortexed and incubated in a water heater at 96°c, 10 minutes to produce pink color, then lift and cool at room temperature. then read the absorption at a wavelength of 532 nm. calculation of results 𝑀𝑀𝐴 (nmol mgprot⁄ ) = ∆𝐴1 ∆𝐴2 × 𝐶 × 𝑓 ÷ 𝐶𝑝𝑟 note: ∆𝐴1 : odsample odblank ∆𝐴2 : odstandard odblank 𝐶 : the concentration of standard, 10 nmol/ml 𝑓 : dilution factor of sample before test 𝐶𝑝𝑟 : concentration of protein in sample, mgprot/ml evaluation of gsh level the reduction of 5,5’-dithiobis (2-nitrobenzoic acid) (dtnb) by gsh produced a yellow complex used to indicate the level of thiol protein; the color intensity of 412 nm was proportional to the level of gsh. measurement of glutathione (gsh) levels by taking as much as 0.5 ml of supernatant added 0.5 ml of dntb and 3 ml of phosphate buffer (0.2 m, ph 8). this reagent reacts with the sh group to give a yellow complex. the absorbance was read with a spectrophotometer at a wavelength of 412 nm. cells and tissue sample: 𝑅𝑒𝑑𝑢𝑐𝑒𝑑 𝐺𝑆𝐻 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑖𝑛 𝑐𝑒𝑙𝑙𝑠 𝑎𝑛𝑑 𝑡𝑖𝑠𝑠𝑢𝑒 (𝜇𝑚𝑜𝑙/𝑔𝑝𝑟𝑜𝑡) = odsample − odblank odstandard − odblank × 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (40 𝜇𝑚𝑜𝑙 𝐿⁄ ) × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑒𝑓𝑜𝑟𝑒 𝑡𝑒𝑠𝑡𝑒𝑑 ÷ 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒(𝑔𝑝𝑟𝑜𝑡 𝐿⁄ ) mustofa et al. – soursop leaf extract as antioxidant 267 statistical analysis data were processed statistically using the statistical program for social science (spss) software for windows version 20.0. hypothesis testing to analyze gsh levels used the kruskal–wallis test and analyzed mda levels using the one-way anova test. the spearman nonparametric test used the correlation test between the two variables. results and discussion cytotoxicity examination cytotoxic test results showed the percentage of living cells is inversely proportional to the concentration increases. the higher the concentration of the test material, the lower the percent of the mean number of living cells mcf-7 cells that life is getting a little (bahuguna et al., 2017). ic50 values of ethanol extract of soursop leaf on mcf-7 cells were 23.96 ± 0.006 ppm (figure 1). figure 1. the cell inhibition of soursop leaf extract on mcf-7 cells. mda inhibition and gsh level in this study, soursop leaf extract was used at concentrations around ic50, namely 0, 10, 20, 30 ppm, to determine the state of free radical production and antioxidant levels. as shown in figure 2, the percentage of living cells is inversely related to the increase in concentration. the higher the concentration of the test material, the lower the percentage of the average number of mcf-7 living cells. figure 2. microscopic photo of mcf-7 cell culture treated with soursop leaf extract (400x). (a) treatment with 0 ppm; (b) treatment with 10 ppm; (c) treatment with 20 ppm; (d) treatment with 30 ppm. furthermore, figure 3 showed that at the concentration 0 ppm of soursop leaf extract the mda inhibition level was 0.57 nmol/mgprot while gsh level was 721.05 µmol/gprot. the soursop leaf extract was increased the mda inhibition and gsh level. the highest increase in mda inhibition levels occurred when the soursop leaf extract was given 30 ppm, namely 388.6 nmol/mgprot, while the highest increase in gsh occurred when the soursop leaf extract was given 20 ppm, namely 1158.63 µmol/gprot. figure 3. the growth rate of mda inhibition and gsh level by administering soursop leaf extract to mcf-7 cell. y = −0.0098x + 0.7348 r² = 0.6488 0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70 0,80 0,90 0 5 10 15 20 25 30 35 c el l i n h ib it io n ( % ) soursop leaf extract of concentration on mcf-7 cells (ppm) ic50 0 200 400 600 800 1000 1200 1400 0 10 20 30 m d a i n h ib it io n a n d g s h l e v e l soursop leaf extract concentration (ppm) gsh level (µmol/gprot) mda inhibition level (nmol/mgprot) 268 biology, medicine, & natural product chemistry 13 (1), 2024: 265-269 oxidative stress is a condition that reflects an imbalance between ros and antioxidant defenses. malondialdehyde (mda) is a marker of oxidative stress due to a chain reaction of lipid peroxidation. in this study, mda inhibition levels in mcf-7 increased statistically significantly in the administration of soursop leaf extract. this indicates that the soursop leaf extract decreases free radical formation (rady et al., 2018; muchtaromah et al., 2015). in mcf-7 cells, soursop leaf extract can increase the mda inhibition level, inhibit cell proliferation, stimulate cell apoptosis, inhibit metastasis, regulate the immune system, and reduce the inflammation caused by cancer (rachmani et al., 2013; syed najmuddin et al., 2016). glutathione (l-j-glutamyl-cysteinyl-glycine) is a tripeptide consisting of glutamic acid, cysteine, and glycine. this compound has a sulfhydryl/thiol group (sh) found in the amino acid cysteine. the sulfhydryl group causes gsh to act as a strong electron donor (nucleophile) in warding off free radicals. gsh can decompose h2o2 into h2o with the help of the enzyme glutathione peroxidase. gsh can be synthesized in all cells, especially tissues that are highly exposed to ros (reactive oxygen species) (wu et al., 2004). in this study, gsh production levels in mcf-7 were increased in the administration of soursop leaf extract. the increased gsh levels was not statistically significant, possibly due to the large number of other antioxidants (glutathione peroxidase, sod, and catalase) that play a role in reducing free radicals. increased mda inhibition levels are followed by decreased gsh levels. gsh has a role as an antioxidant by directly reducing free radicals or as a cofactor for antioxidant enzymes such as glutathione peroxidase and glutathione transhydrogenase. the main function of gsh is to detoxify drugs, xenobiotics, or pesticides catalyzed by the gsh-stransferase enzyme. gsh also plays a role in maintaining the thiol group (-sh) in essential proteins by reducing disulfide bonds in proteins, which are catalyzed by the enzyme thiol transferase (wu et al., 2004). the administration of soursop leaf extract with a concentration of 20 ppm increased gsh production. soursop leaf extract treatment at a concentration of 10 ppm showed a decrease in gsh levels. this may be due to other auto-oxidation when soursop leaf extract is added to a buffer solution or cell culture medium at physiological ph. the limitation of this research is that this research uses soursop leaves for the manufacture of extracts obtained from gardens that have not been certified. ideally, the soursop leaf material used as an extract material comes from a certified soursop garden to produce good phenol and flavonoid content. conclusions soursop leaf extract was able to increase mda inhibition and gsh level in human breast cancer cells mcf-7. further research on the antioxidant effect of soursop leaf extract can be carried out using cancer cells other than mcf-7 cells. acknowledgements: the author is grateful for the internal research grant from yarsi university, jakarta, indonesia, 2018/2019. authors’ contributions: muhammad samsul mustofa & nunung ainur rahmah designed the study. muhammad samsul mustofa carried out the laboratory work. restu syamsul hadi & pandrianto analyzed the data. muhammad samsul mustofa & nunung ainur rahmah wrote the manuscript. restu syamsul hadi & pandrianto revised the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the author is grateful for the internal research grant from yarsi university, jakarta, indonesia, 2018/2019. references anjum, f., razvi, n. and masood, m.a. (2017). breast cancer therapy: a mini review. moj drug des develop ther, 1(2), 35-38. bahuguna, a., khan, i., bajpai, v.k. et al. (2017). mtt assay to evaluate the cytotoxic potential of a drug. bangladesh j pharmacol, 12, 115-118. boundouki, g., wilson, r., duxbury, p. et al. (2021). patient and public priorities for breast cancer research: a qualitative study in the uk. bmj open, 11, e036072. de sá junior, p.l., camara, d.a.d., porcacchia, a.s. et al. (2017). the roles of ros in cancer heterogeneity and therapy. oxid med cell longev, 2017, 2467940. fertilita, s., sandhika, w. and suprabawati, d.g.a. (2020). the cytotoxic activity of annona muricata linn leaves ethanolic extract (amee) on t47d breast cancer cell line. medical laboratory technology journal, 6(1), 32-39. gavamukulya, y., wamunyokoli, f. and el-shemy, h.a. 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(2010). endogenous antioxidants and radical scavengers. adv exp med biol, 698, 52-67. shaw, a.t., winslow, m.m., magendantz, m. et al. (2011). selective killing of k-ras mutant cancer cells by small molecule inducers of oxidative stress. proc natl acad sci usa, 108(21), 8773-8778. sosaa, v., molinéa, t., somozaa, r. et al. (2013). oxidative stress and cancer: an overview. ageing res rev, 12(1), 376-390. syed najmuddin, s.u., romli, m.f., hamid, m. et al. (2016). anti-cancer effect of annona muricata linn leaves crude extract (amce) on breast cancer cell line. bmc complement altern med, 16(1), 311. world health organization. (2020). who report on cancer: setting priorities, investing wisely and providing care for all. world health organization. https://apps.who.int/iris/handle/10665/330745 wu, g., fang, y.z., yang, s., et al. (2004). glutathione metabolism and its implications for health. j nutr, 134(3), 489-492 https://apps.who.int/iris/handle/10665/330745 this page intentionally left blank biology, medicine, & natural product chemistry volume 4 – number 2 – 2015 issn 2089-6514 contents krokot (portulaca oleracea. l) as a natural sensitizer for tio2 dye-sensitized solar cells: the effect of temperature extract reyza anni mufidah, khamidinal, endaruji sedyadi, didik krisdiyanto 25 29 effect of lunasia amara blanco on sperm number, sperm motility, and testicular histology of male rats muhammad ja'far luthfi 31 33 antioxidant potential of black, green and oolong tea methanol extracts wahyu widowati, tati herlina, hana ratnawati, gabriella constantia, i dewa gde sathya deva, maesaroh maesaroh 35 39 medicinal plants: a prospect in developing male fertility enhancing agent muhammad ja'far luthfi, mahanem mat noor, jalifah latip 41 47 tapak liman (elephantopus scaber l) as immunostimulant and its effect on lymphocyte differentiation in mice balb/c marmi kelik 49 51 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 663-669 | doi: 10.14421/biomedich.2023.122.663-669 issn 2540-9328 (online) prevalence and biofilm-forming potentials of bacterial uropathogens among primary school pupils in yola north, adamawa state abdulazeez mumsiri abaka1, mubarak muhammad dahiru1,*, ibrahim ya’u2, tukur baba abdullahi3 1pharmaceutical technology department; 2science laboratory department, school of science and technology, adamawa state polytechnic yola, 640101, nigeria. 3microbiology department, faculty of life sciences, modibbo adama university, 640101, nigeria. corresponding author* mubaraq93@adamawapoly.edu.ng abstract this investigation aimed to determine the prevalence and biofilm-forming potentials of bacterial uropathogenic among primary school pupils in yola north, adamawa state. a total of 120 clean mid-stream urine samples were collected from apparently healthy children to determine the antimicrobial susceptibility of the isolated pathogen by the kirby and bauer disc diffusion method while the biofilmforming potential by the standard methods. the result showed 26 % of the samples had significant bacteria growth with the gramnegative as the highest. the infectivity pattern among the age group showed 8-10 had the highest infectivity percentage (35%) followed by 5-7 and 11-13 with 22.5% and 20% respectively. six bacteria pathogens were isolated which are e. coli (41.9%) which is the most prevalent isolate followed by staphylococcus aureus (29.0%), pseudomonas sp. (9.7%), klebsiella sp. (9.7%), enterobacter sp. (6.5%), proteus sp. (3.3%). the antibiotic sensitivity test showed an alarming resistance level to nearly all the tested antibiotics showing multidrug resistant strains. the biofilm-forming potentials depict e. coli with the strongest potential followed by s. aureus. a high occurrence of asymptomatic utis and antimicrobial resistance observed poses a public health concern. thus, routine diagnostic exercise is recommended for early detection and prompt treatment of the pathogens. keywords: bacteria; biofilm; gram negative; gram positive; prevalence; uropathogens. abbreviations: ab (asymptomatic bacteriuria), cled (cysteine lactose electrolyte deficient), genitourinary (gu), uti (urinary tract infection). introduction urinary tract infection (uti), indicates the presence of microorganisms within the urinary tract, extending from the distal urethra to the kidney. utis constitute the second most prevalent type of infection, resulting in approximately 8.1 million annual visits to healthcare professionals. these infections are the most frequent bacterial ailment among children, affecting 8% of girls and 2% of boys (daniel et al., 2023). urinary tract infections are a common occurrence among secondary school students, afflicting individuals of both genders and typically necessitating prompt medical attention (behzadi et al., 2021). it is also worth noting that utis rank as one of the leading causes of morbidity, particularly renal disorders, and mortality in children, particularly among those from the most economically disadvantaged backgrounds (anigilaje and elike, 2022). utis can exhibit diverse manifestations depending on the infection's location and duration. in the kidney, it is termed pyelonephritis; in the bladder, cystitis; and in the urethra, urethritis. the presence of bacteria in the urine is referred to as bacteriuria. utis can manifest with symptoms or without them, known as asymptomatic utis (colgan et al., 2020). asymptomatic uti, also termed asymptomatic bacteriuria (ab), is characterized by the presence of a significant bacterial load (≥105 cfu/ml) in an individual's urine without any accompanying signs or symptoms of uti (colgan et al., 2020). the prevalence of asymptomatic bacteriuria varies widely, influenced by factors such as age, gender, sexual activity, and the presence of genitourinary abnormalities (shpunt et al., 2021). in children, the prevalence of asymptomatic bacteriuria varies by geographic location. developed countries have reported rates ranging from 2 to 5.4% (jain and schroeder, 2020), whereas developing countries, especially among those with the lowest socioeconomic status, have reported rates between 10 to 48% (sorkhi et al., 2019). utis can be attributed to a variety of organisms, with bacterial colonization of the urinary tract being primarily linked to gram-negative manuscript received: 10 october, 2023. revision accepted: 07 december, 2023. published: 04 february, 2024. https://doi.org/10.14421/biomedich.2023.122.663-669 664 biology, medicine, & natural product chemistry 12 (2), 2023: 663-669 species. common culprits include escherichia coli, klebsiella species, proteus species, and pseudomonas aeruginosa. gram-positive organisms, such as staphylococcus aureus and hemolytic streptococci are less frequently responsible for utis (ali et al., 2018). several common factors contribute to an increased risk of developing utis. in women, the heightened risk is primarily attributed to the relatively short length of the urethra, which measures around 1.5 inches compared to approximately 8 inches in men. this anatomical difference makes it easier for bacteria from fecal matter near the anal opening to migrate to the urethral opening. furthermore, older schoolgirls, as suggested by shaikh et al. (2014), may be at a higher risk due to factors such as engagement in premarital sexual activity, which can introduce uropathogenic bacteria into the urinary tract. poor hygiene practices also play a role in uti risk. additionally, compromised health conditions that weaken the immune system can render individuals more susceptible to utis. dysfunctional voiding, where urine is retained for extended periods after the bladder signals fullness, can also contribute to infections (dobrek, 2023). dehydration, by promoting bacterial growth, is another factor that can lead to the development of utis. bacterial virulence factors play a crucial role in determining whether an organism can invade the urinary tract and the severity of the resulting infection. these virulence factors encompass various elements, including adhesins, type 1 pili, p fimbriated pili, cell receptivity, and biofilms. adhesins, often in the form of fimbriae or pili, enhance bacterial adherence to the uroepithelium by binding to specific receptors on its surface (davis and flood, 2011). this interaction between fimbriae and mucosal receptors triggers several events, such as the internalization of the bacterium into the epithelial cell, which can lead to apoptosis, hyperinflation, invasion into nearby epithelial cells, or the establishment of a bacterial focus that contributes to recurrent utis. biofilms are structured communities of microbes encased in extracellular polymeric substances (eps) that form on the surfaces of the urinary tract. although certain proteins and enzymes serve as targets in antibacterial therapy (dahiru et al., 2023), resistance to the drugs can complicate the treatment of utis and contribute to their persistence. these biofilms often exhibit complex structural and functional characteristics, making the microbial communities highly resistant to many antimicrobial agents (hamzah et al., 2020). it is imperative to understand the variations in antimicrobial resistance among microorganisms isolated from uti, as resistance patterns can vary by region and even from one patient to another. to address this issue, our primary focus is to gain a comprehensive understanding of the community-acquired microorganisms prevalent in our population. this study aims to investigate the distribution, antibiotic resistance profiles, and biofilm formation capabilities of bacteria isolated from urinary tract infections (utis). the ultimate goal is to enhance the effectiveness of empirical treatment for these infections, ensuring that patients receive the most appropriate and successful treatment interventions. materials and methods study area the research was conducted in yola north, the capital of adamawa state in north-eastern nigeria. the study focused on a group of apparently healthy children who were attending primary school, ranging in age from 5 to 12 years. a total of five primary schools were selected for the study, from which a combined total of 100 samples were collected. to ensure gender equity, an equal number of samples were obtained from both male and female students. among the participating pupils, twenty (20) samples were randomly selected from each of the five primary schools. the inclusion criterion for participation in the study was limited to children currently enrolled in primary schools. conversely, the exclusion criteria encompassed children who were either younger or older than the primary school age range and those who had recently used antibiotics. these criteria were established to maintain consistency and relevance in the research population and to prevent potential confounding factors that could affect the study's outcomes. sample collection urine specimens were collected following the established and highly recommended approach for children aged three and older, as outlined by welsh (2007). after collection, the clean catch mid-stream urine sample in sterile containers was transported immediately to the laboratory for analysis. in cases of potential delays, the samples were safeguarded by refrigeration at temperatures between 4 and 8 °c as recommended by welsh (2007). microbiological analysis each sample was cultured on cysteine lactose electrolyte deficient (cled) agar and blood agar as described by vandepitte et al. (2003) and cheesbrough (2002). incubation was carried out for 24 h at 37 °c. enumeration of colonies was carried out as described by (olutiola et al., 1991). cultures with 105 cfu/ml were considered significant (younis et al., 2010). biochemical tests to identify individual organisms were conducted from plates with a positive result as described by prescott, (2002) and cheesbrough, (2002). antimicrobial susceptibility testing was been performed using the disk diffusion method as described by the national committee for clinical laboratory standards (n.c.c.l.s, 2000). abaka et al. – prevalence and biofilm-forming bacterial uropathogens potentials 665 data analysis the values obtained were expressed as a percentage of the mean ± standard error of triplicate determinations' mean (± sem) and evaluated with statistical package for the social sciences (spss) version 22 software. results and discussion a total of one hundred and twenty (120) urine samples were collected from selected primary school children, out of which 31 (26%) of the school children showed significant bacteriuria, while 89 (74%) of the children were negative. figure 1 shows the result of the infectivity pattern among different sexes out of the 60 male pupils 12 are positive giving 20% while 19 females are positive out of 60 females which also gives 32%. figure 1. infectivity pattern among the sexes figure 2 shows the result for the infectivity pattern among the different age groups sampled. age group 8-10 showed the highest percentage of growth with 14 (35%), followed by 5-7 with 9 (22.5%), and age group 11-13 showed the lowest percentage of growth with 8 (20%). figure 2. infectivity pattern among the different age the distribution pattern of the isolates among different sexes is shown in figure 3. a total of 31 isolates represented six different organisms, including e. coli, proteus sp., enterobacter sp., pseudomonas sp., klebsiella species, and s. aureus. specifically, e. coli was the most commonly isolated organism, accounting for 41.9%, followed by s. aureus at 29.0%, pseudomonas sp. and klebsiella sp. each at 9.7%, enterobacter sp. at 6.5%, and proteus sp. at 3.3%. figure 3. distribution pattern of the isolates among different sexes the antibacterial susceptibility profile of gram-negative bacteria uropathogens showed susceptibility against ciprofloxacin, pefloxacin, ofloxacin, and chloramphenicol. however, they exhibited resistance to cotrimoxazole, nalidixic acid, and ampicillin. the result is presented in table 1. table 1. antibiotic sensitivity patterns of gram-negative isolates bacteria (n) cpx pef ofx au cn sxt na pm s ch e. coli n=13 13 (100) 13 (100) 12 (92.3) 6 (46.1) 9 (69.2) 2 (15.4) 2 (15.4) 0 (0.0) 4 (30.7) 13 (100) pseudomonas sp. n= 3 3 (100) 3 (100) 3 (100) 1 (33.3) 2 (66.6) 0 (0.0) 1 (33.3) 1 (33.3) 0 (0.0) 3 (100) klebsiella sp. n=3 3 (100) 3 (100) 3 (100) 0 (0.0) 3 (100) 0 (0.0) 0 (0.0) 1 (33.3) 0 (0.0) 3 (100) enterobacter sp. n=2 0 (0.0) 2 (100) 2 (100) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (100) proteus sp. n=1 1 (100) 1 (100) 1 (100) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (100) total n=22 20 (90.9) 22 (100) 21 (95.5) 7 (31.8) 14 (63.6) 2 (9.0) 3 (13.6) 2 (9.0) 4 (18.2) 22 (100) cpxciprofloxacin, sxtcotrimoxazole, pef-pefloxacin, nanalidixic acid, ofxofloxacin, pmampicillin, auaugmentin, sstreptomycin, cn gentamycin, chchloramphenicol 666 biology, medicine, & natural product chemistry 12 (2), 2023: 663-669 figure 4 shows the antibacterial susceptibility profile of gram-positive bacteria uropathogens, particularly s. aureus. the result obtained indicates that s. aureus was susceptible to pefloxacin and ciprofloxacin, as these antibiotics completely inhibited the growth of the bacteria. however, s. aureus exhibited complete resistance against septrin, rocephin, and erythromycin. figure 4. antibiotic sensitivity patterns of gram-positive isolates (staphylococcus aureus n=9). pefpefloxacin, amamoxicillin, cn gentamycin, cpxciprofloxacin, apxamplicox, sstreptomycin, z zinnacef, sxtseptrin, rrocephin, eerythromycin table 2 shows the result for the biofilm-forming potential of the bacterial uropathogens isolated. the result obtained showed that e. coli showed the strongest potential for biofilm production next to s. aureus and klebsiella sp. table 2. biofilm-formation by the isolated bacteria uropathogens bacteria biofilm strong weak none e. coli (n =13) 9 3 1 s. aureus (n =9) 2 5 1 pseudomonas sp. (n = 3) 1 2 klebsiella sp. (n =3) 2 1 enterobacter sp. (n =2) 1 1 proteus sp. (n =1) 1 discussion antimicrobial drug resistance poses a global challenge that carries significant implications for public health. utis are prevalent across all age groups and have a considerable clinical impact due to their exuberant rates of morbidity and mortality (madrazo et al., 2023). study. utis rank highest among all diseases concerning the number of antibiotic prescriptions issued for empirical treatment, as highlighted by (paul et al., 2022). over time, the high prevalence of antimicrobial resistance amongst uropathogens has steadily risen on a global scale, rendering utis one of the most frequently encountered infectious diseases in medical practice, resulting in significant morbidity, as reported by medina and castillo-pino, (2019). understanding microorganisms, their response to antimicrobials, and local resistance patterns is vital for tailoring more personalized and effective treatment strategies, as emphasized by ahmad et al. (2021). antimicrobial resistance is a global challenge with serious implications for public health. utis affect people of all ages and have a substantial clinical impact due to their high rates of illness and death, as demonstrated in madrazo et al. (2023). utis stand out as the utmost common reason for prescribing antibiotics for empirical treatment (paul et al., 2022). to develop more personalized and effective treatment strategies, it is essential to have a deep understanding of microorganisms, their reaction to antimicrobials, and resistance patterns in the local environment (ahmad et al., 2021). this study recorded a prevalence level of 26% for uropathogens, which was notably lower than the 67.2% reported in a similar hospital-based study conducted in yola, a location within the same geographical region (elmohmood, 2009). these findings are in tandem with multiple studies carried out in nigeria, with rates comparable to the 31.7% incidence reported by isa et al. (2013) and the 24.1% documented by musa-asien et al. (2003). the results indicate a high incidence when contrasted with earlier reports, such as ogomaka et al. (2013), which found an incidence of 8.1%, as well as the 3.0% reported by muoneke et al. (2012), and the 9% reported by ibadin and abiodun, (2004). however, it's important to note that similar studies conducted in other parts of the world have reported varying uti incidence rates, ranging from 3.7% (mobasheri et al., 2002) to 28% (hamdan et al., 2011) and 22.7 % (kibret and abera, 2014). these differences could be ascribed to variations in uti awareness, screening methods, and the amalgamation of risk factors, such as the socio-economic status of families, age, personal hygiene standards, and education levels, across different geographical locations. the study's recorded bacteriuria prevalence of 26% indicates that a noteworthy portion of the children in the research study carry urinary tract pathogens, potentially serving as a source for transmitting and spreading these pathogens. the elevated incidence of uti amongst seemingly healthy children could be multifactorial. these include inadequate healthcare access, home-based treatments, suboptimal dietary practices, insufficient health education, inadequate local hygiene practices, issues related to circumcision, colonization of the bladder and urethra, and improper catheterization techniques (larcombe, 1999). the high prevalence of utis amongst primary school children, especially females, was notably high in this study. the results highlight that the higher incidence of utis in females can be attributed to various factors. these factors include the anatomical structure of the female genitourinary (gu) tract, which is near the vaginal and anal areas, making it more susceptible to potential contamination by fecal coliforms due to inadequate hygiene practices. additionally, incomplete and irregular voiding of urine, often associated with abaka et al. – prevalence and biofilm-forming bacterial uropathogens potentials 667 constipation in schoolgirls, can result in urinary tract infections. furthermore, the presence of uropathogens in the male urethra can also be found in the vaginal flora of female sexual partners (belete and saravanan, 2020). these findings align with the studies conducted by onuoha and oko, 2015 and allamin, 2015, which similarly reported a high prevalence rate of utis among female primary school children. the current study revealed that the uppermost prevalence of utis was reported amongst children aged 8 to 10 years, whereas the lowest prevalence was among those aged 11 to 13 years. this finding is unswerving with the results of studies conducted in nigeria and other countries (onuoha and fatokun, 2014; adeleke et al., 2013). the higher incidence of utis among children in this age group could be attributed to delayed voiding of urine and poor personal hygiene. the consequences of retaining urine include the multiplication and growth of urinary tract pathogens, which can lead to the development of utis. on the other hand, the lower incidences of utis among the younger age group could be due to their frequent urination, as they tend to empty their bladders whenever they feel the urge, which serves as a natural defense mechanism against the development of utis (ogoamaka et al., 2013). consistent with most studies in the field, our findings also indicate that gram-negative bacteria account for the predominant uropathogens, comprising 78.3% of the total isolates, whereas gram-positive bacteria account for 21.6%, as reported by oumer et al. (2022). an overall total of 31 bacteria isolates represented six different organisms, including e. coli, proteus species, enterobacter species, pseudomonas species, klebsiella species, and s. aureus. specifically, e. coli was the most frequently isolated organism, accounting for 41.9% of cases, followed by s. aureus at 29.0%, pseudomonas sp. and klebsiella sp. each at 9.7%, enterobacter sp. at 6.5%, and proteus sp. at 3.3%. this pattern and frequency of bacterial isolates reported in this study align with previous reports, such as those by medina and castillo-pino, (2019) indicating that gram-negative bacteria, particularly e. coli, are the most commonly encountered pathogens in children with urinary tract infections. the high prevalence of e. coli in urine samples is a well-documented phenomenon. numerous authors have identified e. coli as the foremost uropathogen in children (meštrović et al., 2020). in nigeria specifically, many studies on utis have consistently reported the predominance of e. coli in positive urine cultures (dibua et al., 2014). following e. coli, the second most frequently occurring organism is s. aureus, which has been recognized as a key grampositive pathogen associated with urinary tract infections (shrestha et al., 2019). regarding the antibacterial susceptibility profile of gram-negative bacteria, the isolates in our study displayed susceptibility to antibiotics such as ciprofloxacin, pefloxacin, ofloxacin, and chloramphenicol. however, they exhibited resistance to nalidixic acid, cotrimoxazole, and ampicillin. this pattern of resistance aligns with the findings of onuoha and oko, (2015). the high degree of resistance observed to certain antibiotics could potentially be attributed to the extended period these drugs have been available on the market, allowing microorganisms ample time to develop resistance mechanisms. additionally, the ease of obtaining antibiotics over the counter, particularly in developing nations like nigeria where prescriptions may not always be required, and the operation of pharmacies by unlicensed personnel could also facilitate to the observed high surge of antibiotic resistance. the analysis of the antibacterial susceptibility profile of gram-positive bacteria, in this study indicates that s. aureus was susceptible to pefloxacin and ciprofloxacin, as these antibiotics completely inhibited its growth. however, s. aureus exhibited resistance against septrin, rocephin, and erythromycin. these findings are in tandem with the work of mava et al. (2012), who also observed the effectiveness of ciprofloxacin, pefloxacin, and ofloxacin against their isolates, while noting resistance to nalidixic acid, septrin, and erythromycin. it's imperative to note that different studies may report varying antimicrobial susceptibility patterns due to several influencing factors. these factors include the antibiotic pattern of consumption by patients, geographical locations, clinical conditions (whether the utis are untreated, recurrent, or complicated), and the laboratory methodologies employed. consequently, comparing data from different studies can be challenging due to these multifaceted variables. conclusions the result of this study revealed a high occurrence of asymptomatic utis among apparently healthy school children which poses a public health concern. the study also indicates a high incidence of antimicrobial resistance to many of the common antibiotics used in the study. based on this, routine urine screening exercises should be reinvigorated in primary schools to ensure early detection of those that carry the pathogens for adequate and swift treatment of the infection. consistent monitoring is required also to unveil reliable information about resistance patterns for optimum empirical therapy of patients with utis. acknowledgments: the funding of this research by the tertiary education trust fund (tetfund) through the institutional base research fund has been duly acknowledged by the authors and they are really grateful for the funding. special appreciation goes to the pharmaceutical technology department of adamawa state polytechnic yola for institutional support. 668 biology, medicine, & natural product chemistry 12 (2), 2023: 663-669 authors’ contributions: example: abdulazeez mumsiri abaka & mubarak muhammad dahiru designed the study. abdulazeez mumsiri abaka, & ibrahim ya’u carried out the data collection and laboratory work. abdulazeez mumsiri abaka, & tukur baba abdullahi analyzed the data. abdulazeez mumsiri abaka & mubarak muhammad dahiru wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research was sponsored by the tertiary education trust fund (tetfund) through the institutional base research fund. references adeleke, m. a., adebimpe, w. o., sam-wobo, s. o., wahab, a. a., akinyosoye, l. s., & adelowo, t. o. 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(2019). urinary tract infections in children: epidemiology and risk factors. uptodate, waltham, ma. shpunt, y., estrin, i., levi, y., saadon, h., ben-yossef, g., goldshtein, l., ... & marchaim, d. (2021). antimicrobial use for asymptomatic bacteriuria—first, not harm. infection control & hospital epidemiology, 42(1), 37-42. sorkhi, h., riahi, s. m., ebrahimpour, s., shaikh, n., & rostami, a. (2019). urinary tract infection in children with nephrotic syndrome: a systematic review and meta-analysis. microbial pathogenesis, 137, 103718. vandepitte, j., kerhaigen, j., engbark, k., rihner, p., pilot, p. and heuck, p. (2003). basic laboratory procedures in clinical bacteriology. who geneva. welsh, a. (2007). uti in children: diagnosis, treatment and long-term management. national collaboration centre for women and children’s health, london. younis n., mohammed a., ibraheem j. and reham m. (2010). uropathogens and their antibiotic susceptibility among children with urinary tract infection treated at prince hashem bin al-hussein hospital. journal of the royal medical services 17(2):41-45 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 143-151 | doi: 10.14421/biomedich.2025.141.143-151 issn 2540-9328 (online) formulation and characterization tests of scoby kombucha gel eva agustina1, raihana frika nafisah1, achmad farchan fiddaroini1, clarista eka tania1, risa purnamasari1,*, funsu andiarna2, irul hidayati2 1biology department, faculty of science and technology; 2nutririon department, faculty of psychology and health, uin sunan ampel surabaya jl. dr. ir. h. soekarno no. 682, gunung anyar, surabaya, indonesia. corresponding author* risap1989@gmail.com manuscript received: 17 january, 2025. revision accepted: 08 may, 2025. published: 15 may, 2025. abstract the wound is the damage to body tissues caused by various factors such as accidents, sharp object scratches, etc. proper care is necessary to accelerate the wound healing process. topical medications, such as gel preparations, are commonly used for wounds. kombucha scoby is believed to contain bioactive compounds that can accelerate the wound healing process, making it a potential active ingredient in gel preparations. this research aims to determine the characteristics of kombucha scoby gel with various concentration variations. scoby is obtained from kombucha fermentation using green tea, sugar, and kombucha culture. preparing kombucha scoby gel involves using carbomer 940, methyl paraben, triethanolamine, glycerin, and scoby kombucha. the concentration variations of kombucha scoby gel include 0%, 2%, 4%, 8%, 10%, and 12%. each formula undergoes physical characteristic tests including an organoleptic test, hedonic test, homogeneity test, ph test, spreadability test, and adhesiveness test. data obtained after physical characteristic tests of the gel preparations are analyzed descriptively. based on the conducted research, it was found that kombucha scoby gel preparations with concentration variations of 0%, 2%, 4%, 8%, 10%, and 12% exhibited different characteristics. the higher the gel concentration, the more acidic the gel's aroma, the more liquid the texture, and the more intense the color. scoby added to the gel preparation can increase the ph value, adhesiveness, and spreadability of the gel. keywords: gel; physical characteristics; scoby; topical medication; wound care. introduction wound is the damage to body tissues, such as skin tissue, organs, and others. wounds can be caused by accidents, sharp or blunt object trauma, surgery, and others (hidayat et al., 2024). wounds can be encountered in daily life. according to data from the indonesian bps (badan pusat statistik), the number of minor injury victims in indonesia has increased, with 113.518 cases in 2020, 117.913 cases in 2021, and 160.449 cases in 2022. wound care commonly used involves topical preparations. topical medications are a type of medicine applied directly to the surface of the body or skin to produce a local effect in the area. these medications are typically used to treat skin conditions such as infections, inflammation, or wounds and relieve symptoms like itching, pain, or irritation. generally, the topical medication used for wound care is a gel (yanhendri & yenny, 2012). the gel is one type of pharmaceutical formulation that dissolves and forms a cohesive structure with a semisolid consistency. the gel consists of large organic molecules and small inorganic particles evenly dispersed in a liquid medium. the gel has many advantages compared to other topical medications (yanhendri & yenny, 2012). the benefits of gel include its ability to spread evenly on the skin surface without disrupting physiological functions because it does not clog pores. gel can also adhere to the skin and tissues without causing discomfort. also, the gel is cool, easy to clean with water, and has good drug release, and stable viscosity (kaban et al., 2024). gel has better stability, releases drugs well, is easy to use, can maintain skin moisture, and does not irritate the skin, thus enhancing the effectiveness of healing. gel formulation consists of active ingredients dissolved in a gel base. the active ingredients added to the gel preparation have antiinflammatory, antibacterial, and antiseptic properties. one of the active ingredients that can be used as the base material for wound healing gel is kombucha scoby. kombucha is a fermented beverage several vitamins, minerals, and organic acids (riswanto & rezaldi, 2021). according to history and various observed literature, the process of making kombucha is carried out using a tea solution medium containing sugar as a nutrient source for the bacteria and yeast that play a role in the formation of kombucha and its by-product, scoby (azizah et al., 2020). symbiotic culture of bacteria and yeast (scoby) is a biofilm layer formed during the kombucha https://doi.org/10.14421/biomedich.2025.141.143-151 mailto:risap1989@gmail.com 144 biology, medicine, & natural product chemistry 14 (1), 2025: 143-151 fermentation. scoby contains bacteria and yeast, which are crucial for fermentation. these bacteria and yeast are encapsulated by a permeable thin membrane (riswanto & rezaldi, 2021). the bacteria and yeast trapped in the biofilm matrix (scoby) interact and produce various metabolites (urbahillah et al., 2021). scoby is likely to contain the same compounds as kombucha and can, therefore, be utilized for wound care. thus far, the public has only known kombucha as a health drink, and the fermentation by-product called scoby is used as a starter for making kombucha. according to laavanya et al. (2021), the application of scoby in new products is minimal. previous research has focused on the physical characteristics of scoby used in textiles and packaging. scoby becomes production waste after a specific period of use (chagas et al., 2024). based on the studies conducted, there has not been any research that uses scoby as the main ingredient in gel preparations for wound healing. therefore, this study aims to determine the characteristics of kombucha scoby gel with various concentration variations. research on the characteristics of kombucha scoby gel preparations with varying concentrations is essential to understanding their potential applications and therapeutic effects. materials and methods study area this type of research is experimental research. this research was conducted in december 2023 – january 2024. the research was conducted in the animal cell and tissue culture laboratory and instrumentation laboratory, faculty of science and technology, uin sunan ampel surabaya. the tools used in the research were glass containers, cloth, rubber, spoon, blender, knife, spatula, stirrer, beaker, measuring glass, dropper pipette, magnetic stirrer, petri dish, object glass, cover glass, analytical balance, and hot plate. the ingredients used are kombucha starter and scoby (symbiotic culture of bacteria and yeast), green tea, sugar, carbomer 940, triethanolamine (tea), glycerin, methyl paraben, distilled water. procedures scoby kombucha preparation 2000 ml of water was boiled, and 200 grams of sugar (10% w/v) and 10 grams of dried green tea (0.5% w/v) were added. after straining, the solution was covered and cooled to room temperature. when the tea reached around 28-37°c, 200 ml of kombucha starter culture (10% w/v) was added to the tea solution. the glass container was tightly covered with cloth and rubber bands then left for 30 days (nafisah et al., 2023). by the 7th day of fermentation, scoby growth was visible, but to achieve optimal physical characteristics, fermentation proceeded to the 60th day. scoby, a colony of bacteria and yeast, remained functional even when sliced, cut, or blended. the equipment used had to be sterile and not exposed to extreme temperatures (cold or hot). the refined scoby was used as a base material for gel preparation. scoby gel preparation 10 grams of carbomer 940 was weighed and sprinkled into 500 ml of preheated distilled water. carbomer 940 was stirred quickly in a beaker glass to form a gel mass, and then 10 drops of tea (triethanolamine) were added. next, 1 gram of methyl paraben was weighed, dissolved in 25 ml of distilled water, and added to a smaller beaker glass. the mixture was stirred until homogeneous, then poured into the beaker glass containing carbomer 940 and distilled water. 5 ml of glycerin was added and stirred until the gel base became homogeneous (rinawati et al., 2022). scoby extract concentrations of 0%, 2%, 4%, 8%, 10%, and 12% were prepared by weighing 0 grams, 2 grams, 4 grams, 8 grams, 10 grams, and 12 grams of scoby, then added to the gel base to reach a total weight of 100 grams and homogenized. characteristics test of gel organoleptic test the organoleptic test on gel includes texture, color, and aroma. the testing of gel texture and color is conducted through visual observation (kaban et al., 2022). hedonic test the hedonic test or preference test aims to determine the level of panelists’ preference for a product. this test is carried out by measuring, assessing, or testing the quality of commodities using human senses, namely vision, smell, and touch. the preference test method includes product observation, smelling, and touching using fingertips before applying it to the skin. this subjective test involves 20 random panelists (nealma & nurkholis, 2020). the panelists understand the sensory properties of the preparation to be evaluated and meet the criteria for panelists. according to qamariah et al., (2022), the criteria for being a panelist are: ▪ the ability to detect, recognize, compare, and differentiate hedonic properties. ▪ showing attention to organoleptic aspects. ▪ willing to spare or provide time. ▪ having the necessary sensitivity. in this test, the panelists are asked to rate the product based on preference level with indications: like, neutral, and dislike. homogeneity test the homogeneity test is performed by preparing an object glass, applying the gel, and covering it with another object glass on top. homogeneity is indicated by agustina et al. – characterization tests of scoby gel … 145 the absence of coarse grains in the preparation (kaban et al., 2022). ph test the ph test is conducted to ensure that the ph of the preparation is stable and matches the skin's ph. 1 gram of gel was diluted with distilled water to a volume of 10 ml, then the ph value was measured using a ph meter. the skin ph that meets the criteria ranges from 4.5 to 6.5 (kaban et al., 2022). if the ph of the preparation does not match the skin's ph, it can irritate if too acidic, or the skin becomes scaly if too alkaline (rohmani & kuncoro, 2019). spreadability test 0.5 grams of gel was prepared and placed between two petri dishes. the upper petri dish was weighed and left for one minute. after that, a 150-gram weight was placed on top of the covered petri dish, left for one minute, and then the diameter of its spread was measured. this test was conducted to ensure the even distribution of the gel when applied to the skin, with a spreadability value ranging from 5 to 7 cm, meeting the criteria (kaban et al., 2022). the larger the spreadability value, the longer the contact between the skin surface and the gel, allowing a higher concentration of active ingredients to be absorbed into the skin (erwiyani et al., 2018). a high spreadability value also made the gel easier to apply on the skin, thus optimizing the absorption of the preparation (rohmani & kuncoro, 2019). adhesiveness test 0.25 grams of gel was placed in the central area of an object glass surface, then another object glass was placed on top and weighted with 1 kg for 1 minute. the two attached object glasses were mounted on the test device and given an 80-gram weight. the time needed for the two object glasses to separate was recorded (kaban et al., 2022). the adhesiveness test aimed to determine how long the preparation could adhere to the skin surface. the longer the preparation adhered, the greater its spread on the skin surface (rohmani & kuncoro, 2019). results and discussion result scoby obtained after 60 days of fermentation has a diameter of approximately 9 cm, a thickness of up to 3.5 cm, and a brownish color (figure 1). figure 1. scoby produced during fermentation. organoleptic test based on the results of the organoleptic observations, it was found that the kombucha scoby gel experienced changes in texture, color, and aroma. these changes are influenced by the concentration of scoby added to the gel preparation. the higher the concentration of scoby used, the more liquid the gel texture, the more brownish the color, and the more acidic the aroma. the kombucha scoby gel with various concentrations can be seen in figure 2 below. figure 2. scoby kombucha gel with variation of concentration. a) 0%; b) 2%; c) 4%; d) 8%; e) 10%; f) 12%. a b c d e f 146 biology, medicine, & natural product chemistry 14 (1), 2025: 143-151 the organoleptic test aims to assess the physical appearance of the gel (mappa et al., 2013). organoleptic observations include the color, texture, and aroma of the gel. the aroma is tested using the sense of smell, the color is tested through direct visual observation, and the texture is tested through touch, by applying the gel to the skin and feeling it. hedonic test the hedonic test for kombucha scoby gel was conducted with 20 panelists. the results of the hedonic test can be seen in figure 3 below. figure 3. chart of hedonic test results. the hedonic test is conducted to determine the level of preference someone has for a product. in this test, individuals who rate the level of preference based on organoleptic observations are called panelists (triandini & wangiyana, 2021). ph test the ph value indicates the degree of acidity of a substance. the purpose of the ph test is to determine the ph value of kombucha scoby gel or to determine the level of acidity of the gel, which will affect irritation when applied topically (nurlely et al., 2021). the ph value is measured using a ph meter. the ph test results can be seen in table 2 below. table 2. ph value of kombucha scoby gel. concentration ph value 0% 3.85 2% 3.52 4% 3.45 8% 3.26 10% 3.17 12% 3.11 homogeneity test the homogeneity test aims to ensure that the ingredients in the gel preparation are evenly mixed (homogeneous). homogeneity is assessed by observing the presence or absence of coarse granules in the gel. thus, it can be ensured that the active ingredients are evenly distributed in the preparation and that there are no clumped particles, allowing the gel to provide more optimal effects. the determination of homogeneity is done by observing whether or not there are coarse granules in the gel (mappa et al., 2013). table 3. scoby kombucha gel homogeneity test results. concentration description 0% homogeneous 2% homogeneous 4% homogeneous 8% homogeneous 10% homogeneous 12% homogeneous spreadability test the spreadability test aims to assess the ability of kombucha scoby gel to spread on the skin surface. 0% 20% 40% 60% 80% 100% 120% d is lik e n e u tr al li ke d is lik e n e u tr al li ke d is lik e n e u tr al li ke d is lik e n e u tr al li ke d is lik e n e u tr al li ke d is lik e n e u tr al li ke 0% 2% 4% 8% 10% 12% hedonic test aroma texture color agustina et al. – characterization tests of scoby gel … 147 this test is conducted to determine the even distribution of the gel when applied to the skin surface (mappa et al., 2013). the easier the gel is applied to the skin, the larger the surface area of contact between the active ingredient and the skin, resulting in more optimal absorption of the active ingredient. the spreadability values of the gel for each concentration can be seen in table 4 below. table 4. scoby kombucha gel spreadability test values. concentration repetition length (cm) average 1 5.5 0% 2 5.6 5.5 3 6 1 5.6 2% 2 5.9 5.6 3 5.2 1 6 4% 2 6 5.8 3 5.7 1 6 8% 2 6 5.8 3 5.8 1 6.1 10% 2 6 6.0 3 6 1 7.1 12% 2 7.1 7.1 3 6.9 adhesiveness test the adhesiveness test is conducted to determine how long the gel can adhere to the skin (forestryana & rahman, 2020). the results of the adhesiveness test can be found in table 5 below. table 5. scoby kombucha gel adhesion test values. concentration repetition adhesion values (s) average 1 5 0% 2 1.18 4.7 3 8 1 3 2% 2 2 2.3 3 1 1 6 4% 2 2 3.0 3 1 1 7 8% 2 6 5.7 3 4 1 9 10% 2 7 6.7 3 4 1 10 12% 2 8 7.7 3 5 discussion kombucha is a traditional beverage made from a sweet tea solution fermented by scoby (symbiotic culture of bacteria and yeast), which consists of complex microbes such as acetic acid bacteria, lactic acid bacteria, and yeast (hamed et al., 2023). in the kombucha fermentation process, the main components involved are scoby and the medium. the ingredients required to make kombucha include green tea, kombucha starter, water, sugar, and scoby. the physical characteristics of scoby are influenced by several factors such as the duration of fermentation, the container used, and the type of tea used. the duration of fermentation affects the thickness of the scoby layer; the longer the kombucha tea is fermented, the thicker the cellulose layer formed (khaerah & akbar, 2019). scoby will grow according to the size of its container, so its diameter matches the diameter of the container used in the kombucha teamaking process. the color of scoby depends on the color of the kombucha tea, and the type of tea used in making kombucha affects the color of the scoby produced (riswanto & rezaldi, 2021). the brown color in scoby is caused by the tannin content in teas such as green tea, black tea, and oolong tea, which are as the base for making kombucha tea (khaerah & akbar, 2019). organoleptic test based on the research conducted, it was found that each concentration of kombucha scoby exhibited different physical properties. the test results for each concentration showed changes in the aroma, texture, and color of the gel preparation at each concentration. the changes in the aroma, texture, and color of the gel from the lowest to the highest concentration were due to the active ingredient content in the gel. the highest concentration gel has the most active ingredients, so the most scoby was added to the 12% concentration gel. the gel color changed from clear to dark yellow-brown. the gel texture became more liquid, and the aroma became increasingly acidic. the appearance of scoby is influenced by the length of the fermentation time. the longer the fermentation time, the thicker the scoby will become. the color of the scoby will change to brown or dark brown. this occurs because the tannin compounds in green tea can bind with the cellulose of scoby (khaerah & akbar, 2019). thus, the higher the scoby content in the gel, the more intense the gel color will be. scoby has an acidic aroma, so the more scoby is added to the gel preparation, the more acidic the gel aroma will be. according to soares et al. (2021), the bacterial cellulose present in kombucha has characteristics of high water retention, mechanical resistance, and biocompatibility. based on research (chagas et al., 2024), it was found that the water content in scoby is high (94.02±0.08 g/100 g). thus, the more 148 biology, medicine, & natural product chemistry 14 (1), 2025: 143-151 scoby used in gel preparation, the more liquid the gel texture will be. hedonic test data in figure 3 show that most panelists preferred the aroma, texture, and color of the kombucha scoby gel at a 0% concentration. at this concentration, there was no addition of scoby, so the gel had more neutral characteristics. the 0% kombucha scoby gel had a solid texture, transparent color, and a light medicinal aroma. these characteristics provided a familiar and comfortable impression for the panelists, making it more preferred. the graph in figure 3 shows that most panelists did not like the aroma, texture, and color of the 12% kombucha scoby gel. the 12% concentration gel had a slightly liquid texture, a dark yellow-brown color, and a strong acidic aroma. the slightly liquid texture and intense color reduced the visual appeal of the gel. the stronger acidic aroma may have been perceived as unpleasant by the panelists. the results of the hedonic test indicated a relationship between the concentration of kombucha scoby gel and the level of acceptance or preference by the panelists. in general, the kombucha scoby gel most preferred by the panelists was the 0% concentration gel, which is the gel without added scoby. the gel formulation with the most preferred scoby addition by the panelists was the 2% concentration kombucha scoby gel, while gels with higher concentrations tended to be less liked by the panelists. ph test based on table 2, it can be seen that the higher the concentration of kombucha scoby gel, the lower the ph value of the preparation. the highest ph value is found in the 0% concentration kombucha scoby gel with a value of 3.85, while the lowest ph value is in the 12% concentration kombucha scoby gel with a value of 3.11. the ph range that complies with sni no. 062588 is between 4.5 and 6.5, so all concentrations of kombucha scoby gel have a ph value below this standard. although the ph value of kombucha scoby gel is low, the hedonic test shows that the gel does not irritate the panelists. several factors contribute to the low ph not irritating. kombucha scoby gel does not cause skin irritation because the ph value of the gel is close to the natural ph of the skin, thus it can still be well tolerated. the natural ph of human skin is slightly acidic, ranging from 4 to 6. the decrease in the ph value of the gel preparation is due to the scoby content. the higher the concentration of the gel, the more scoby is added to the formulation. scoby consists of acetic acid bacteria, lactic acid bacteria, and yeast, which produce pellicle-like cellulose on the surface of the fermentation medium through the metabolic activity of acetic acid bacteria. the decrease in ph during fermentation indicates the metabolic activity of bacteria and yeast. during the fermentation process, sucrose is converted into alcohol and other organic acids are formed by the bacteria. the acids formed will release protons and make the ph value decrease (al-yousef et al., 2017). bacteria and yeast metabolize sucrose and sugar during the fermentation process, producing various organic acids. the high sugar content in kombucha tea solution and scoby can increase the activity of microorganisms and organic acids. the high levels of organic acids produced by scoby, such as acetic acid, lactic acid, gluconic acid, and glucuronic acid, cause the ph value to decrease. lactic acid is synthesized by lactic acid bacteria, while acetic acid and gluconic acid are synthesized by acetic acid bacteria. acetic acid bacteria, particularly gluconobacter oxydans and komagateibacter xylinus, produce gluconic acid from the oxidation of d-glucose. research by chen & liu (cited in antolak et al., 2021) found that during 60 days, the concentration of gluconic acid reached 39 g/l. homogeneity test the results of the homogeneity test (table 3) show that kombucha scoby gel with concentrations from 0% to 12% is homogeneous because there are no lumps in the preparation. based on the tests conducted, it can be concluded that there is no difference in homogeneity among the various concentrations of kombucha scoby gel. all kombucha scoby gels meet the standards of the indonesian pharmacopoeia, edition iii, which states that the gel must be homogeneous or appear evenly distributed without the presence of clumped particles when applied to glass or other transparent materials (rohmani & kuncoro, 2019). a preparation is considered homogeneous if it does not show the presence of coarse granules that are not mixed. homogeneity testing is important to ensure that the active ingredients are evenly distributed in the gel, indicated by the absence of clumped particles (wahyudi & wulandari, 2022). when a preparation is homogeneous, the ingredients are uniformly dispersed, which ensures optimal release of active components upon application to the skin (liandharjani & ratu, 2022). spreadability test contact between the medication or active ingredient and the skin will occur more quickly if the spreadability is good (rohmani & kuncoro, 2019). higher spreadability indicates an increased concentration of the active ingredient absorbed by the skin, as the duration of contact between the gel and the skin surface is longer (erwiyani et al., 2018). based on the test results (table 4), kombucha scoby gel with concentrations from 0% to 12% shows increased spreadability. according to sni standards, a good spreadability value for semisolid preparations ranges from 5 to 7 cm (rohmani & kuncoro, 2019). therefore, all kombucha scoby gels agustina et al. – characterization tests of scoby gel … 149 meet this standard. the 12% kombucha scoby gel has the best spreadability value among all gel concentrations. the results of the spreadability test (table 4) show that the 0% kombucha scoby gel has the lowest spreadability value. this is because there is no added scoby at this concentration, and the gelling agent (carbomer 940) composition is the highest compared to other concentrations. the 12% kombucha scoby gel has the highest spreadability value because the increasing addition of scoby with higher concentrations reduces the composition of the gelling agent (carbomer 940). one of the factors affecting spreadability is the amount and strength of the gel matrix, which is the gelling agent. spreadability will decrease if the gel matrix increases and becomes stronger. in preparing of kombucha scoby gel, the gelling agent used is carbopol or carbomer 940. the higher the amount of carbomer 940 in the gel, the lower the spreadability value because the gel preparation becomes thicker. a high spreadability value indicates that the preparation is easier to apply to the skin (tambunan & sulaiman, 2018). the spreadability test is a test conducted to determine the ability of the speed of spread of the preparation on the skin when applied to the skin. a good spreadability value is 5-7 cm (tungadi et al., 2023). good spreadability causes contact between the drug and the skin quickly. the increase and decrease in spreadability are strongly influenced by the consistency of the gel, which is related to the viscosity value of the preparation. if the viscosity value of the preparation is high, the resulting spreadability area is low, and vice versa. this happens because the high viscosity causes the gel to be challenging to flow so, the resulting spreadable area is small. a larger and stronger gel matrix results in reduced spreadability. the component within the gel that is responsible for forming the gel matrix is the gelling agent (rohmani, 2019). preparations that are either too difficult to spread or overly spreadable can negatively impact user comfort and the effectiveness of the preparation. conversely, overly diluted preparations lead to decreased adhesion, thereby reducing the contact time of the active substance with the application site. according to the research by irianto et al. (2020), higher concentration levels correspond with increased spreadability. adhesiveness test based on the table above, it can be seen that each concentration of kombucha scoby gel has a different adhesiveness value. the 12% kombucha scoby gel has the longest adhesiveness, at 7.7 seconds, while the 2% kombucha scoby gel has the shortest adhesiveness, at 2.3 seconds. good adhesiveness for topical preparations is over 4 seconds (thomas et al., 2023). therefore, the study results conclude that kombucha scoby gels with concentrations of 0%, 8%, 10%, and 12% have good adhesiveness (more than 4 seconds). adhesiveness value is inversely proportional to spreadability. adhesiveness too weak will not provide optimal therapeutic effects (slamet et al., 2020). adhesiveness is influenced by the concentration of the gelling agent (carbomer 940) in the preparation (forestryana & rahman, 2020). the more carbomer 940 is added, the lower the adhesiveness of the preparation or the quicker it will release. the longer the gel adheres, the better its quality. if the gel easily releases, the effect will not be optimal. the duration of gel adhesion can also be influenced by the concentration of the active ingredient used (thomas et al., 2023). the higher the concentration of carbopol or carbomer 940, the longer the adhesion produced. this is due to the interatomic forces in the preparation, where the thicker the consistency of the preparation, the stronger the forces, resulting in longer adhesiveness (nurlely et al., 2021). the higher concentration of the scoby gel, the longer the adhesion produced. this is due to the force between atoms in the gel, where a thicker consistency results in stronger forces, thus increasing adhesion duration (nurlely et al., 2021). this statement is substantiated by tungadi et al. (2023), who found that formula 1, with varying compositions of astaxanthin ingredients, exhibited the highest spreadability value of 6.40. conversely, formula 3 displayed the highest adhesion value. these findings demonstrate that greater ingredient concentration enhances adhesion value (nurlely et al., 2021). conclusions based on the research conducted, it can be concluded that kombucha scoby gels with concentrations of 0%, 2%, 4%, 8%, 10%, and 12% have different characteristics according to the amount of scoby added to the preparation. the higher the gel concentration, the more acidic the gel aroma, the more liquid the texture, and the more intense the color. the ph value of kombucha scoby gel decreases with the increasing gel concentration. the spreadability and adhesiveness values of the gel increase with the increasing gel concentration. acknowledgements: the author expresses his deepest gratitude to lppm uin sunan ampel surabaya. authors’ contributions: eva agustina, raihana frika nafisah, achmad farchan fiddaroini, and clarista eka tania wrote the manuscript. raihana frika nafisah, achmad farchan fiddaroini, and clarista eka tania carried out the laboratory work and analyzed the data. as well mrs. eva agustina, risa purnamasari, funsu andiarna, and irul hidayati as the supervisor in completing the research and manuscript. competing interests: the authors declare that there are no competing interests. 150 biology, medicine, & natural product chemistry 14 (1), 2025: 143-151 funding: the authors declare funding if any. references al-yousef, h.m., sawab, a., & alruhimi, m. 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(2012). berbagai bentuk sediaan topikal dalam dermatologi. cdk-194, 39(6), 423–430. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 673-678 | doi: 10.14421/biomedich.2025.142.673-678 issn 2540-9328 (online) molecular method optimization to identify plasmodium falciparum multidrug resistance 1 (pfmdr1) gene as a predictor of antimalarial resistance erma sulistyaningsih1,*, rosita dewi2, sheilla rachmania2, irawan fajar kusuma3, muhammad rizqi kholifaturrohmy4, yunita armiyanti1, sakinatus sariroh kholifaturrohmah1, made prasanti andriani1 1parasitology department; 2histology department; 3public health department, faculty of medicine, university of jember, jl. kalimantan no 37 jember 68121, tel. +62-331-337877, fax. +62-331-337877, indonesia. 4satgas pamtas statis ri-png t.a. 2023 yonif 122 / tombak sakti, keerom, papua, 99451, indonesia. corresponding author* sulistyaningsih.fk@unej.ac.id manuscript received: 26 may, 2025. revision accepted: 04 september, 2025. published: 01 october, 2025. abstract several approaches have been designed to control malaria, a disease with high morbidity and mortality, but they face some hurdles. antimalarial resistance is one of the major challenges for malaria elimination, so the detection of antimalarial resistance is essential. several molecular markers for antimalarial resistance have been identified, including plasmodium falciparum multidrug resistance 1 (pfmdr1) gene. this study determined the optimization of molecular techniques to identify the pfmdr1 gene as an antimalarial resistance predictor in indonesia. the study included patients diagnosed with uncomplicated or severe malaria originating from the health district of kerom regency, papua province, and dr. soebandi hospital, jember, east java province. blood samples were collected in the whatmann filer paper after informed consent. dna was isolated from dried blood filter paper, and nested pcr was performed using a specific primer, the pfmdr1-a and pfmdr1-b genes. the pcr cycle was optimized based on previous studies. the pfmdr1-a has a similar setting to the earlier study, but the pfmdr1-b had a different optimum setting from the previous study with the annealing temperature of 57oc for nested-1 and 62oc for nested-2. this pcr setting could be used for further examination. the positive results of the amplification indicated the potential for antimalarial resistance in the parasite population. a study on the gene copy number and polymorphism is essential to determine the definitive conclusion on antimalarial resistance. keywords: gene; malaria; pfmdr1; plasmodium falciparum; resistance. abbreviations: act: artemisinin-based combination therapy; aq: amodiaquine; cnvs: copy-number variations; cq: chloroquine; dna: deoxyribonucleic acid; pcr: polymerase chain reaction; pfcrt: plasmodium falciparum chloroquine-resistance transporter; pfmdr1: plasmodium falciparum multidrug resistance 1; snps: single nucleotide polymorphisms; sp: sulfadoxine-pyrimethamine; tes: therapeutic efficacy studies; who: world health organization. introduction malaria is endemic in 85 tropical countries worldwide in 2022 and has high morbidity and mortality. most cases were in african countries, but asia is in the second position, with indonesia at the second rank for malaria cases prevalence in asia with 443.530 cases in 2022 (who, 2023) (kemenkes, 2022). the world health organisation (who) has several programs to control malaria globally. malaria case management with pr ompt access to early diagnostic and effective antimalarial therapies is vital to reducing malaria morbidity and mortality (who, 2022) the first synthesized drug to treat malaria was chloroquine (cq). it was introduced in the early 1940s, but the plasmodium resistance emerged by the early 1960s and spread across different geographical regions (manirakiza et al., 2022), including indonesia in 1974 (fitri et al., 2023). after spreading cq resistance throughout malaria-endemic countries, who recommended the use of sulfadoxine-pyrimethamine (sp). however, the sp resistance develops rapidly, starting in southeast asia within one year of its use (noreen et al., 2021). subsequently, the sp resistance emerged in africa and latin america (nsanzabana et al., 2018). following the sp resistance, mefloquine (mq) and amodiaquine (aq) were used to treat malaria. the resistance develops again five years after the use (dondorp et al., 2009). finally, who recommended the artemisinin-based combination therapy (act) as the first-line drug for plasmodium falciparum malaria in https://doi.org/10.14421/biomedich.2025.142.673-678 674 biology, medicine, & natural product chemistry 14 (2), 2025: 673-678 countries where chloroquine, sulfadoxinepyrimethamine, and amodiaquine have been resistant since 2001, and it has contributed significantly to decrease the morbidity and mortality from 251 million cases and 585.000 death in 2015 to 229 million cases and 409.000 death in 2019 (who, 2023). although there was a decrease in the number of malaria-endemic countries, however, malaria elimination is challenging due to some issues. one of the critical problems was the antimalarial resistance. soon after the global implementation of act, an issue of artemisinin resistance in the greater mekong subregion is reported (dondorp et al., 2009). antimalarial drug resistance is the ability of the plasmodium to survive and/or multiply despite the drug's administration and absorption in equal doses or higher than the recommended, but within a tolerance of the human (fairhurst, 2015). based on the antimalarial drug class, resistance mechanisms occur in several ways, including causing oxidative stress, affecting heme detoxification, inhibiting protein synthesis, inhibiting folate synthesis, interfering hemozoin formation, and inhibiting mitochondrial electron transport (si et al., 2023)(fitri et al., 2023). detection of antimalarial drug resistance can be performed by any of three approaches, including in vivo studies to assess the efficacy of drugs in patients, in vitro/ ex vivo studies to evaluate parasite susceptibility to the drugs, and/or molecular identification to detect validated gene mutations and/or gene copy number changes that are associated with the drug resistance (nsanzabana et al., 2018)(who, 2022). in vivo test is the first method described by who protocol; it is known as therapeutic efficacy studies (tes). the method evaluates the efficacy of the antimalarial drug clinically. the efficacy test can also be performed by measuring the level of antimalarial drug collected from the blood of malaria patients using the high-performance liquid chromatography (hplc) method. the drug level analysis from blood can differentiate medication nonadherence and drug resistance (who, 2022). the second method is an in vitro or ex vivo test, which observes the efficacy of an antimalarial drug on the plasmodium itself. the method uses plasmodium parasites obtained from malaria patients, which are cultured and treated with different antimalarial drugs with various concentrations, and the plasmodium growth and survival are evaluated to determine the drug susceptibility and resistance profile. the last method is molecular characterization, which identifies antimalarial drug resistance markers. the common associated with antimalarial resistance markers are single nucleotide polymorphisms (snps) or amplifications of the gene encoding for drug target proteins or copy-number variations (cnvs) of transporters. various markers have been developed for the assessment of the known resistance markers; for example, chloroquine-resistance is associated with the p. falciparum chloroquine-resistance transporter (pfcrt) (manirakiza et al., 2022) and p. falciparum multidrug resistance gene 1 (pfmdr1) (al-mekhlafi et al., 2022). the method can be performed by polymerase chain reaction (pcr) and sequencing. the method has progressively developed in recent years because it is more effective and has many advantages. validated molecular markers are highly relevant to detecting and monitoring the geospatial distribution of resistant parasites, and their prevalence in parasite populations is often a good indicator of clinical resistance (nsanzabana et al., 2018). the application of antimalarial drug resistance detection depends on the facilities in malaria-endemic countries or areas. indonesia has no specific regulation regarding the standard procedure or guideline to detect antimalarial resistance (fitri et al., 2023). however, the common methods used are tes and molecular characterization. a molecular characterization method needs good infrastructure and highly trained staff. a standard operating procedure for an individual laboratory is required to minimize unexpected results. therefore, this study analyzed the optimization of the molecular characterization method to identify the pfmdr1 gene as an antimalarial resistance predictor. materials and methods research design and study area the study was exploratory research conducted in marchdecember 2024 with a consecutive sampling method. two types of samples were included in this study; the first was malaria patients admitted at the dr. soebandi hospital, jember, east java province, who are mostly seasonal workers from papua or maluku, and the second was malaria patients directly from some districts in kerom regency, papua province. patients were diagnosed with malaria based on clinical signs and symptoms and blood smear microscopic examination. ethical clearance all procedures in this research have been approved by the ethical committee of research faculty of medicine, university of jember no. 2103/un25.1.10.2/ke/2024. patients received a clear explanation about the study before signing an informed consent form to participate in the study. study sample malaria patient was determined based on clinical signs and symptoms and confirmed by blood smear examination. blood samples of malaria patients were collected before treatment. blood samples were dropped on the whatmann filter paper, dried at room temperature, and put in the zipped plastic bag until dna isolation was performed. sulistyaningsih et al. – molecular method optimization to identify plasmodium falciparum … 675 dna isolation dna isolation was conducted using a qiamp dna blood mini kit (qiagen, 2007). the procedures were performed based on the manufacturer's protocol. as many as three punches of 3 mm dried blood spot were put in the tube for dna isolation and added with prelysis buffer (atl) and proteinase-k. the tube was heated by incubation at 56oc with 900 rpm shaking for 1 hour. the tube was added with lysis buffer (al) and incubated at 70oc for 10 min with 900 rpm shaking. the lysate was then transferred into the mini-column and centrifuged at 8000 rpm for 1 min. the column was then added with washing buffer 1 (aw1), centrifuged again, and further washed using washing buffer 2 (aw2). the column was dried by high-speed centrifugation at 14.000 rpm for 3 min. finally, the dna was eluted from the column using elution buffer (ae) by 14.000 rpm centrifugation for 1 min. the dna isolation result was then evaluated using 1% agarose gel electrophoresis, quantified using spectrophotometry, and saved at -20oc until used for amplification. pcr amplification the pfmdr1 gene was identified using two types of specific primers, namely pfmdr1-a and pfmdr1-b. both primer pairs were conducted using nested pcr techniques using the mytaq hs red mix (bioline) (table 1). in this study, we did pcr optimization, especially during the pcr cycle, based on previous studies. the pcr results were visualized on 1% agarose gel electrophoresis. table 1. the primers and cycles to identify the pfmdr1 gene. gen pcr and primer’s name sequences (5’ to 3’) target amplicon (bp) cycle pfmdr1-a nested 1: mdr1a-f mdr1a-r gtgtatttgctgtaagagct gacatattaaataacatgggttc 958 94oc 3min, (94oc 30s, 55oc 60s, 65oc 90s) 34 cycles, 65oc 5 min, 4oc 5min (adamu et al., 2020) nested 2: mdr2a-f mdr2a-r cagatgatgaaatgtttaaagatc taaataacatgggttcttgact 864 94oc 3min, (94oc 30s, 60oc 30s, 65oc 60s) 28 cycles, 65oc 5 min, 4oc 5min (adamu et al., 2020) pfmdr1-b nested 1: mdr1b-f mdr1b-r aggttgaaaaagagttgaac atgacaccacaaacataaat 578 94oc 3min, (94oc 30s, 55oc 30s, 65oc 60s) 34 cycles, 65oc 5 min, 4oc 5min (adamu et al., 2020) nested 2: mdr2b-f mdr2b-r acaaaaagagtaccgctgaat aaacgcaagtaatacataaagtc 534 94oc 3min, (94oc 30s, 60oc 30s, 65oc 60s) 29 cycles, 65oc 5 min, 4oc 5min (adamu et al., 2020) results and discussion the study included 53 malaria patients, with 50 patients who had uncomplicated malaria from health services in kerom regency, papua province, with the main symptoms being fever, chills, and nausea. the other three patients were severe or complicated malaria patients from dr. soebandi hospital, east java province, with malaria complications, including severe anemia, kidney failure, and prostration, based on who diagnosis criteria (table 2) (who, 2014). patients' diagnoses were determined based on clinical signs and symptoms, and the microscopic examination was the gold standard for malaria diagnosis by who (who, 2024). all patients from kerom regency, papua province, were outpatients who were admitted to the district health services and had a single infection of p. falciparum based on microscopic examination. however, those microscopical examinations were our secondary data. data from dr. soebandi hospital showed a single infection of p. falciparum and a mixed infection of p. falciparum and p. vivax (table 2). table 2. characteristics of malaria patients. frequency percentage (%) origin dr. soebandi hospital kerom regency, papua province 3 50 5.66 94.34 clinical appearance uncomplicated malaria complicated/severe malaria 50 3 94.34 5.66 microscpic examination p. falciparum p. falciparum + p. vivax 51 2 96.23 3.77 676 biology, medicine, & natural product chemistry 14 (2), 2025: 673-678 who recommended microscopic examination as the gold standard for malaria diagnosis. it provides a sensitive and specific diagnosis of malaria if performed well. microscopic examination also allows the quantification of parasites and the identification of the infecting species. however, it involves relatively high costs for training and supervision, and the accuracy of diagnosis is strongly dependent on the microscopic technicians (who, 2024). in malaria-endemic areas, malaria should be suspected in any patient with a history of fever and no other obvious cause. although molecular methods such as pcr have higher sensitivity and specificity to diagnose malaria, the microscopic examination is still the gold standard because it is simpler and does not need advanced resources and highcost equipment to perform the test compared to the molecular method. furthermore, the who recommended that, at present, molecular diagnostic tools based on nucleic acid amplification techniques, such as loopmediated isothermal amplification or pcr, do not have a role in the clinical management of malaria (who, 2024). i̇n this study, we performed pcr to identify the molecular marker of antimalarial resistance specifically to the p. falciparum multidrug resistance-1 (pfmdr1) gene. the pcr amplification was conducted using specific primer pairs to detect the pfmdr1 gene, i.e., pfmdr1-a and pfmdr1-b (adamu et al., 2020). both amplification methods are nested pcr, i.e., a modification of pcr that uses two sets and two successive pcr reactions, the first set of primer pairs is designed to anneal upstream from the second set of primer pairs, so the product of the first pcr is used as a template for the second pcr. nested pcr was designed to improve sensitivity and specificity because it can reduce the amount of nonspecific binding, and the technique is useful on suboptimal nucleic acid samples, such as samples extracted from formalin-fixed or paraffin-embedded tissue. however, this technique could increase potential contamination due to additional manipulation of amplicon products. but, it can be minimized by physically separating different parts of the process (green & sambrook, 2019). amplification using pfmdr1-a specific primer resulted in a single band of approximately 864 bp (figure 1). the result is similar to the previous study (adamu et al., 2020). we used identical pcr primer sequences and cycles. optimization of the pcr setting was done based on an earlier study, and it yielded a single band, as expected. i̇n contrast, amplification using pfmdr1-b specific primer was performed with several pcr cycles optimization, especially at the annealing step for both nested-1 and nested-2 pcr. the first optimization used 55oc for nested-1 and 60oc for nested-2, yielding double bands; the second optimization used 57oc for nested-1 and 62oc for nested-2 (figure 2). the second optimization resulted in a single band of approximately 534 bp. pcr optimization is essential when conducting a certain pcr for the first time in our laboratory. although the pcr has been done with the same setting in previous studies, however, the optimum conditions in our laboratory could be different due to a different pcr machine, kits or reagents used, the company supplier, and other laboratory conditions. as a result of the study, our pcr settings are slightly different from those of the previous study, especially in terms of the annealing temperature for the pfmdr1-b gene. figure 1. the amplification results using pfmdr1-a primer. the target amplicon was 864 bp and observed at lines 1 and 2. line 1: patient 1; line 2: patient 2; m; dna marker; c-: negative control. the pfmdr1 gene is central in p. falciparum artemisinin-based combination therapy (act) resistance but is not associated with resistance to single artemisinin (veiga et al., 2016). it has been associated with resistance to drugs such as mefloquine (mfq), halofantrine (hf), and lumefantrine (lmf) (price et al., 2004)(sisowath et al., 2007)(mungthin et al., 2010). the gene encodes the protein that can transport drugs into the digestive vacuole (dv) membrane. in contrast, the drugs' main target is in the cytosol, making them less effective once transported by pfmdr1 into the dv (si et al., 2023)(wurtz et al., 2014). the copy number of pfmdr1 significantly affects parasite sensitivity to those act drugs. studies reported that the pfmdr1 copy number is prevalent in southeast asia and closely related to failure mfq and lmf treatment; it is also common in south american countries and east and west african countries (price et al., 2004)(xu et al., 2018)(mungthin et al., 2010). besides the copy number, the polymorphism of the pfmdr1 gene is also associated with in vivo and in vitro parasite reactions to amodiaquine (adq), mfq, lmf, and artemisinin (mungthin et al., 2010)(price et al., 2004). some missense mutations are common in african and asian countries, whereas other mutations occur at high frequency in south american parasites (adamu et al., 2020)(veiga et al., 2016). sulistyaningsih et al. – molecular method optimization to identify plasmodium falciparum … 677 (a) (b) figure 2. amplification results of pfmdr1-b primer in different annealing temperatures in nested-1 and nested-2 pcr. the target amplicon is 534 bp. (a) pcr product using an annealing temperature for nested-1: 55oc, and nested-2: 60oc, (b) pcr product using an annealing temperature for nested-1: 57oc, and nested-2: 62oc. c-: negative control; line 1: patient 1; line 2: patient 2; m; dna marker. our study found the pfmdr1 gene, i.e., pfmdr1-a and pfmdr1-b, in the samples after a pcr optimization, indicating potential evidence of antimalarial resistance in the parasite population. this pcr setting can be used for further antimalarial resistance gene identification. examination of the gene copy number and the gene polymorphism is vital for further determining the definitive conclusion regarding antimalarial resistance. conclusions the study has determined the pcr setting to identify the pfmdr1 genes, i.e., pfmdr1-a and pfmdr1-b in the samples. the positive amplification results indicate potential evidence of antimalarial resistance in the parasite population. further examination of the gene copy number and the gene polymorphism is vital to determine the definitive antimalarial resistance. some specific primers targeting the pfmdr1 gene have been designed. however, the pcr setting should be optimized based on the individual laboratory conditions to get the expected results. acknowledgements: the authors thank study participants and collaborators from some districts in kerom regency, papua province. authors’ contributions: erma sulistyaningsih designed the study, analyzed the data, and wrote the manuscript. rosita dewi and sheilla rachmania analyzed the data. irawan fajar kusuma, muhammad rizqi kholifaturrohmy, and yunita armiyanti collected the samples. sakinatus sariroh kholifaturrohmah and made prasanti andriani carried out the laboratory work. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the study was funded by the university of jember through the internal grant for malaria research group 2024. references adamu, a., jada, m. s., haruna, h. m. s., yakubu, b. o., ibrahim, m. a., balogun, e. o., sakura, t., inaoka, d. k., kita, k., hirayama, k., culleton, r., & shuaibu, m. n. 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(2018). surveillance of antimalarial resistance pfcrt, pfmdr1, and pfkelch13 polymorphisms in african plasmodium falciparum imported to shandong province, china. scientific reports, 8(1), 1–9. https://doi.org/10.1038/s41598-018-31207w biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 5, number 2, 2016 | pages: 55-59 | doi: 10.14421/biomedich.2016.52.55-59 issn 2540-9328 (online) effect of addition of soursop leaf extract to ganyong (canna edulis ker.) starch edible film and its application in red grape storage time erni widyastuti1, endaruji sedyadi2, susy yunita prabawati3 1,2,3chemistry department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: endaruji@yahoo.com2 abstract this study aimed to determine the effect of addition of soursop leaf extract to edible film and its effect on the storage time of red grapes. this research had three main steps, soursop leaf extraction, edible film making, and application on red grapes storage time. soursop leaves extract variation used in this research are 0.5; 1; and 1.5% (w/w total) and the best result was used to coat the red grapes. the results showed that the optimum composition was obtained on the edible film with the addition of 0.5% (w/w total) soursop leaf extract. the composition increased film thickness from 0.03 to 0.08 mm, decreased film’s tensile strength from 11.89 to 8.42 mpa, decreased elongation from 12.71 to 11.03%, decreased the young modulus from 0.935 to 0.764 mpa, and decreased the vapor transmission rate from 7.45 to 6.55 g/m2.hour. the 50% shrinkage of weight and 50% texture damage are used as parameters to measure the red grapes storage time. based on weight shrinkage, red grapes storage time change from 24 days to 29 days by using an edible film coating without addition of extract, an extend to 32 days while using an edible film coating with addition of soursop leaf extract. based on texture damage, red grapes storage time increased from 13 to 41 days if using an edible film coating without addition of extract, and increased to 40 days using an edible film coating with the addition of soursop leaf extract. keywords: antioxidant; edible film; soursop leaf extract; aloe vera l.; ganyong starch. introduction indonesia is a tropical country where many tropical plants such as pineapple, grapes, mango, and citrus are cultivated. grape is a fruit that very common in many occasion. the fruit has round shape with a sweet taste and thin-skinned. grape is known to have many benefits as anti-cancer and contain a, b, c, and e vitamins (suwarto, 2010). grape plant is a seasonal crop with harvest 2-3 times per year. the plant in the harvest season yielding 10 tons fruit per hectare. the abundant supply of grape during this harvest season caused problems for the farmer, i.e., rotten grapes. it is caused by the storage time of grape only reaches 14 days at room temperature. it takes a given preservation method for the grape to last longer. some ways that can be done to extend the storage time of grape are by cooling, coating with wax, and coating with active packaging. the use of wax as a coating are not suggested due to negative effect on human health. the preservation technique using cooling process requires a greater cost. therefore, in this research we will carried out an active packaging coating to extend the storage time of grape. one of the active packaging is edible film. edible film is a thin and continuous layer made of edible materials. layers are formed to coat the food components (coating) or placed between food components (film). a layer acts as a barrier to mass transfer (e.g. moisture, oxygen, lipids, light and solutes). the coating may also serve as a carrier of groceries and additives, as well as to improve the handling of a food (krochta & johnston, 1997). the main components of edible film can be grouped into three categories, namely hydrocolloids, lipids, and composites (donhowe, 1994). some types of hydrocolloids that can be used as edible films are proteins (gelatin, casein, soy protein, corn protein, and wheat gluten) and carbohydrates (starch, alginate, pectin, arabian gum, and other carbohydrate modifications), whereas the lipid used is wax, glycerol and fatty acids (irianto et al., 2006). according to suryadi (2011), application of ganyong starch and glycerol as edible film on strawberries (fragaria ananasa) can extend the term life of strawberry fruit at room temperature that has a storage time of 2 days longer. kismaryanti (2007) explained that the application of aloe vera l. gel as an edible film can inhibit the deterioration of the quality of tomato fruit that is prolong the storage time up to 3 days at room temperature. this research conducted an application of edible film of ganyong starch and aloe vera l. which is expected to inhibit damage and prolong the storage time of grape at room temperature. http://dx.doi.org/10.14421/biomedich.2016.52.55-59 56 biology, medicine, & natural product chemistry 5 (2), 2016: 55-59 edible film as an active packaging can be a carrier of flavors, dyes, antimicrobial agents and antioxidants (murdianto, 2005). soursop leaves have chemical content such as alkaloids, flavonoids, carbohydrates, saponins, tannins, phytosterols, terpenoids, and proteins. flavonoids in soursop leaves can serve as antioxidants, antimicrobials, anti virals, photosynthetic regulators, and growth regulators (edeoga et al., 2005). antioxidants are one of the additives that can protect the food from damage due to the reaction of fat or oil oxidation. antioxidants can also extend storage time by protecting foods against oxidation-induced deterioration such as rancidity, discoloration and loss of nutritional value (harikedua, 2012). materials and methods materials the materials used in this study were ganyong starch obtained from ugm agro plaza, aloe vera l. and soursop leaf from ngampilan’s market of yogyakarta, glycerol p.a., red grape obtained from ugm mirota campus, 96% ethanol, nacl, silica gel. the tools used for analysis were screw micrometers, universal testing machine, ftir, uv-vis spectrophotometer, and konica minolta cr-400 chromameter. soursop leaf extraction soursop leaf was washed thoroughly and dried. dried soursop leaves then blend until yielding a fine powder. the sample extraction was done by maceration method (immersion). soursop leaf powder weighed as much as 100 g then put into the macerator and added 10 parts solvent. the solvent used is 96% ethanol. the samples were soaked for 5 days and once a day the samples were stirred for homogenization. the macerated extract is separated by precipitation and filtration. it was repeated at least twice with the same type and amount of solvent. the macerated extract was collected, then evaporated with a vacuum evaporator at 55 ºc to obtain a viscous extract (rivai et al., 2013). soursop leaf extract obtained then tested for its antioxidant activity using a dpph method and its functional group using a ftir instrument. edible film preparation edible film preparation was made by 3% of aloe vera gel, 6% of ganyong starch, and 15% glycerol (w/w total) of aloe vera gel. starch weight was suspensioned by addition of 100 ml distilled water. further stirred and heated for 30 minutes at 70 °c ± 5 ° c (afriyah et al., 2015). the suspension was cooled while stirring and then added soursop leaf extract as much as 0.5%, 1% and 1.5% at 50 oc then cooled to 37 ºc. 25 ml of suspension then cast onto a 13x18 cm mica plastic. the suspension of the film was dried at room temperature for 48 hours to allow the edible film to dry and easily removed. edible films produced were tested for chemical properties (ftir) and their physical properties include: thickness, tensile strength, elongation, young modulus, and water vapor transmission rate (wvtr). application to red grape the grape was cleaned with running water and then dried. furthermore, red grape was dipped into 2 edible film solutions. first, its dipped in a solution of ganyong starch and aloe vera l. without addition. other grape was dipped in a solution of ganyong starch and aloe vera l. with addition of soursop leaves extract with best characteristic (0.5%). red grape was dipped in solution at 40 ºc and aerated to dry. the grape that has been coated with edible film was stored at room temperature (25-27 ºc) (lathifa, 2013). data collection was done on days 1, 4, 7, 10 and 13 respectively. red grape samples were tested for weight loss, texture, and color. red grape not coated with edible film used as a control. results and discussion soursop leaf extraction the extraction done in this research yielded the thick extract as much as 10.23 g and the yield of 9.49%. the extract of soursop leaf was then analyzed using ftir spectrophotometer to know the functional groups contained in the extract and tested the antioxidant activity using dpph method. ftir analysis results (figure 1) showed soursop leaf extract gave spectrum on wave number 3387 cm-1 (oh), 2924,09 cm-1 (ch aliphatic), 1735,93 (c = o), 1612,49 cm1 (c = c), 1072.42cm-1 (co). these functional groups are identical with flavonoid compounds such as oh, c-h aliphatic, c = o, c = c, and c-o (suteja et al., 2016) groups. the results of testing of antioxidant activity obtained ic50 value 86.11 ppm and classified as having active antioxidant activity. figure 1. spectrum of ftir test result of soursop leaf extract. widyastuti et al. – effect of addition of soursop leaf extract to … 57 edible film making ganyong and aloe vera l. edible film without addition of soursop leaf extract have a thickness of 0.0325 mm, tensile strength 11,89 mpa, elongation 12,71%, young modulus 0,935 mpa, and water vapor transmission rate 7,45 g/m2hour. the thickness value of ganyong starch and aloe vera l. edible films with the addition of soursop leaf extract ranged from 0,071-0,091 mm as shown in table 1. it showed that the addition of soursop leaf extract effect the resulting film thickness. the greater concentration of the material causes the total amount of solids contained in the edible film to become larger, so that after the edible film suspension is drying the edible film obtained is thicker (sulistiana and widya, 2015). table 1 also shows that the tensile strength of ganyong and aloe vera l. edible films with the addition of soursop leaf extract ranged from 7,578-10,558 mpa. the increased concentration of soursop leaf extract added will increase the tensile strength value of the edible film. it showed that the resulting edible film has a non-fragile nature. the results are similar to murdianto's (2005) who studied the addition of janggelan leaf extract to the tapioca edible film. addition the extract of janggelan leaves causing the increase of tensile strength of the resulting edible film. the addition of janggelan leaf extract to edible film causes more components available to bind to starch molecules, so the resulting edible film becomes more robust and requires greater tensile force to break edible film. table 1. physical properties test result of edible film bulbs ganyong and aloe vera l. with the addition of soursop leaf extract. properties concentrations of soursoup leaf extract 0,5 % 1 % 1,5 % thickness (mm) 0,088 0,091 0,071 tensile strength (mpa) 8,424 7,578 10,558 elongation (%) 11,033 8,719 6,867 modulus young 0,764 0,869 1,538 water vapor transmission rate (g/m2.hour) 6,55 7,88 8,02 elongation of edible film decreased as the concentration of soursop leaf extract added. the same result are the study of edible corn starch film which was added black temu as antioxidant by kusumawati & putri (2013) i.e. where the higher temu hitam added will reduce elongation. the presence of starch still bound to the temu hitam can increase the total solids that can strengthen the film matrix and reduce the elongation of the edible film. young modulus of ganyong starch and aloe vera l. edible film with the addition of soursop leaf extract ranged from 0.764-1.538 mpa. the increase in young's modulus shows the resulting edible film's declining flexibility (nahwi, 2016). based on table 1, the highest young modulus score of 1,538 mpa showed edible film with addition of soursop leaf extract 1.5% (b/b total) more rigid compared with edible film with addition of soursop leaf extract 0,5% and 1%. the rate of water vapor transmission (wvtr) is the amount of moisture lost per unit time divided by the area of the plastic. factors influencing the rate of water vapor transmission are thickness, plasticizer concentration, and type of plastic base material (ekawati, 2015). the increased concentration of soursop leaf extract added will increase the value of water vapor transmission rate from edible film. addition of soursop leaf extract may reduce the hydrogen bonds in the intermolecular bond matrix and allow for the breaking of bonds between the amylose chains (sari et al., 2013). reduced amylose chains that make up the edible film will have an impact on the matrix of the film that has become less dense. the dense film matrix will be easily penetrated by water vapor (santoso et al., 2011). according to afriyah et al. (2015) the value of the vapor transmission rate of the edible film is influenced by aw, rh, temperature, type, edible film-forming properties and the arrangement of starch granules. edible film with flour base tends to have a high rate of water vapor transmission rate. figure 2. ftir spectra of (a) ganyong starch and aloe vera l. edible film without the addition of soursop leaf extract and (b) with the addition of soursop leaf extract. ftir spectra of ganyong and aloe vera l. edible film without soursop leaf extract and with addition of soursop leaf extract are showed in figure 2. its yielded a wide absorption at 3425,58 indicating the vibration of oh group and some shift of wave number that is at 2931,80 cm-1 to 2924,09 cm-1 (ch stretching), 1658,78 cm-1 to 1635,64 cm-1 (c = o), and 1026,13 cm-1 become 1033,85 cm-1 (co). application to red grape the percentage of red grape weight loss (figure 3.) increases with the length of red grape storage. during 58 biology, medicine, & natural product chemistry 5 (2), 2016: 55-59 storage, the shrinkage of fruit weight tends to increase and cannot be prevented. this is due to the physiological processes of respiration and transpiration during storage (lathifa, 2013). figure 3. graph of measurement results of red grape wrap during storage. red grape storage time is determined based on red grape damage against losing weight of 50% using the equation of the line. red grape loses 50% weight loss on 24th day on red grape control, 29th day on red grape coated with edible film without addition of extract, and day 32 on red grape coated edible film with addition of soursop leaf extract. figure 4. graph of red grape grape texture test result during storage. the value of red grape texture test results showed in figure 4 shows that edible film coating on red grape can reduce red grape texture decrease. this is supported by research of sari et al. (2015), which informed that the texture of strawberries decreased during storage and hardness value of strawberries without coating more quickly decreased compared with the coated strawberries. the reduction of red grape texture during storage is caused by the loss of water content in the cells resulting from the respiration and transpiration of red grapes so that the water-deficient cells become softer and weaker. fruit coatings using edible film can suppress high transpiration and respiration in order to inhibit the loss of water content in the fruit tissue (sari et al., 2015). figure 5. graph of test result of l values of red grape during storage. red grape storage period is determined based on texture softening by 50% using the equation of the line. red grape controls were subjected to 50% texture softening on day 13, red grape coated with edible films without extract additions were able to retain 50% of the texture for up to 41 days, and in red grape coated with edible film with the addition of soursop leaf extract subjected to as much texture softening 50% on day 40. figure 6. graph of test result of a red grape value during storage. observations of discoloration on red grape samples were performed using a chromameter tool. interpretation of data to color is translated through the scale l * a * b. the value of l denotes the brightness of the red grape, a scale represents the color red-yellow, and scale b denotes a blue yellow color (kismaryanti, 2007). 0 5 10 15 20 25 30 0 2 4 6 8 10 12 14 w ei g h t lo ss ( % ) strorage time (days) kontrol edible tanpa ekstrak edible +ekstrak 0,5% 0.5 0.6 0.7 0.8 0.9 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 m a x im u m f o rc e (n ) storage time (days) kontrol edible tanpa ekstrak edible + ekstrak 0,5% 22 24 26 28 30 32 0 1 2 3 4 5 6 7 8 9 10 11 12 13 l v a lu es storage time (days) kontrol edible tanpa ekstrak edible + ekstrak 0,5% 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 10 11 12 13 a re d g ra p e v a lu e storage time (days) kontrol edible tanpa ekstrak edible + ekstrak 0,5% widyastuti et al. – effect of addition of soursop leaf extract to … 59 figure 7. graph of test result of b value of red grape during storage. based on these data, the longer time red grape storage, the value of l, a and b red grape decreased. the observations of the color test showed that the treatment of red grape coating with edible film without the addition of extract and with the addition of extract had not been able to inhibit the decrease of l, a, and b values during storage. conclusion based on the research that has been done, it can be concluded that the best edible film are from the addition of soursop leaves extract 0.5% (w/w total). the addition of soursop leaf extract of 0.5% (w/w total) to edible film of ganyong starch and aloe vera l. can affect the physical properties of edible film, i.e. increasing film thickness from 0,03 to 0,08 mm, decreasing tensile strength of 11,89 to 8,42 mpa, decreased elongation value from 12,71 to 11,03%, young modulus decreased from 0,935 to 0,764 mpa, and decreased vapor transmission rate from 7,45 to 6,55 g/m2.hour. red grape storage time is elongated in terms of 50% shrinkage and 50% texture damage. references afriyah, y., putri, widya dwi r. & wijayanti, sudarma d.. 2015. penambahan aloe vera l. dengan tepung sukun (artocarpus communis) dan ganyong (canna edulis ker.) terhadap karakteristik edible film. jurnal pangan dan agroindustri, 3(4): 1313-1324. donhowe, g. & fennema, o.. 1994. edible film and coating: characteristic, formation, definitions and testing methods. in krochta, j.m., baldwin, e.a. and nisperos-carriedo, m.o. (eds.). edible coating and film to improve food quality. technomic publ. co. inc. lancaster, pennsylvania. p. 378. edeoga, h.o & a. gomina. 2000. nutritional values of some nonconventional leafy vegetables of nigeria, j.econ, taxon, bot, 24 dalam febriani, diana., et al. 2015. karakterisasi simplisia dan ekstrak etanol daun sirsak (annona muricata linn). prosiding penelitian spesia unisba, p. 457480. harikedua, s.d.. 2012. penhambatan oksidasi lipida ikan tuna oleh air jahe selama penyimpanan dingin. jurnal perikanan dan kelautan tropis, viii-1. irianto, h.e., darmawan, m. & mindarwati, e., 2006. pembuatan edible film dari komposit karaginan, tepung tapioka dan lilin lebah (beeswax). j. penel. perik. indonesia, 1 (2): 93– 101. kismaryanti, andiny. 2007. aplikasi gel lidah buaya (aloe vera l.) sebagai edible coating pada pengawetan tomat (lycopersicon esculentum mill.). thesis: fakultas teknologi pertanian, institut pertanian bogor. krochta, j.m & johnston, c.m.. 1997. edible and biodegradable polymer films. j. food technology, 51 (2): 61– 74. kusumawati, d., h., & w. d. r. putri. 2013. karakteristik fisik dan kimia edible film pati jagung yang diinkorporasi dengan perasan temu hitam. jurnal pangan dan agroindustri, 1 (1): 90-100. lathifa, hafidzatul. 2013. pengeruh jenis pati sebagai bahan dasar edible coating dan suhu penyimpanan terhadap kualitas buah tomat (lycopersicon esculentum mill.). thesis: fakultas sains dan teknologi, universitas islam negeri maulana malik ibrahim. murdianto, w.. 2005. sifat fisik dan mekanik edible film ekstrak daun janggelan. universitas gajah mada yogyakarta. nahwi, naufal fadli. 2016. analisis pengaruh penambahan plasticizer gliserol pada karakteristik edible film dari pari kulit pisang raja, tongkol jagung, dan bonggol enceng gondok. thesis: fakultas sains dan teknologi, uin maulana malik ibrahim malang. rivai, h., ernita widiya s, & rusdi. 2013. pengaruh perbandingan pelarut etanol-air terhadap kadar senyawa fenolat total dan daya antioksidan dari ekstrak daun sirsak (annona muricata l.). jurnal sains dan teknologi farmasi, 18 (1): 35-42. santoso, budi, filli pratama, basumi hamzah & rindit pambayun, 2011. pengembangan edible film dengan menggunakan pati ganyong termodifikasi ikatan silang. jurnal teknol dan industri pangan, xxii (2): 105-109. sari, r.p., wulandari, septia t., wardhani & dyah h.. 2013. pengaruh penambahan ekstrak bawang putih (allium sativum) terhadap karakteristik edible film pati ganyong (canna edulis kerr.). jurnal teknologi kimia dan industri, 2 (3): 82-87. sari, rita n., dwi dian novita & cicih sugianti. 2015. pengaruh konsentrasi tepung karagenan dan gliserol sebagai edible coating terhadap perubahan mutu buah stroberi (fragaria x ananassa) selama penyimpanan. jurnal teknik pertanian lampung, 4 (4): 305-314. sulistiana, evi erizha & widya dwi rukmi putri. 2015. komparasi penggunaan tepung ganyong dan tepung sukun terhadap karakteristik edible film kulit jeruk bali. jurnal pangan dan agroindustri, 3 (4): 1325-1336. suryadi, meliani octavia. 2011. aplikasi pati ganyong dan gliserol sebagai edible coating pada stroberi (fragaria ananasa). thesis: fakultas teknologi industri, universitas pelita harapan. suteja, kadek p., rita, wiwik s. & gunawan, i.w.g.. 2016. identifikasi dan uji aktivitas senyawa flavonoid dari ekstrak daun trembesi (albizia saman (jacq.) merr) sebagai antibakteri escherichia coli. jurnal kimia, 10: 141-148. suwarto, agus. 2010. 9 buah dan sayur sakti tangkal penyakit. yogyakarta: liberplus. -0.5 0 0.5 1 1.5 2 2.5 3 3.5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 b v a lu es storage time (days) kontrol edible tanpa ekstrak edible + ekstrak 0,5% this page intentionally left blank effect of addition of soursop leaf extract to ganyong (canna edulis ker.) starch edible film and its application in red grape storage time biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 159-169 | doi: 10.14421/biomedich.2023.121.159-169 issn 2540-9328 (online) immunoprotective effect of cocos nucifera oil on sheep red blood cell-induced immunocompromised rats macdonald idu1, igori debby1, benjamin ogunma gabriel1,2,* 1department of plant biology and biotechnology, phytomedicine unit; 2science laboratory technology, university of benin, pmb 1154, benin city, edo state. nigeria. corresponding author* alexthemain076@gmail.com abstract the aim of this study is to investigate the immunoprotective effects of cold-pressed coconut oil in immunocompromised rat. standard procedure was used to perform this research work with a modified method. the effect of immunomodulatory properties of coconut oil was evaluated after challenging the animals with 0.3 ml sheep red blood cells (srbcs) intraperitoneally and further treated with graded doses (0.25, 0.5 and 1.0 ml/kg i.p) of the oil extract for 21 days. the hematological, cd4, cd8 biochemical and histopathological analysis were evaluated. result obtained from this study showed a significant increase in hematological indexes (wbc: 11.70; lym: 9.9 gran: 8.0 and hgb: 15.57) across the treated groups, but majorly at lowest dose (0.25 ml/kg). more so a significant increase in cd4 and cd8 count specifically at 0.25 ml/kg (7.763 and 7.830). anti-oxidant property of the oil extract at 0.25 ml/kg had a significant reduction in malondialdehyde with an increased in antioxidant enzymes (sod, cat and gpx) when compared with untreated control. the body weight of the animals in the treated groups showed a significant increase at 0.25 ml/kg of the oil extract when compared with the untreated control. liver function test (alt, ast and alp) showed no significant increase in the treatment groups when compared with the controls. the histopathological result reveals a normal physiological structure of the heart, lungs, spleen, liver and kidney in lowest dose of the oil extract. in conclusion, this study validated the ethnomedicinal property of the plant. keywords: immunoprotective; cocos nucifera; sheep red blood cell; immunocompromised. introduction immune system is a versatile defense system that protect humans against invading pathogens and inhibit diseases, which can regenerate into variety of cells and molecules proficient in recognizing and eradicating foreign invaders (richard et al., 2003). immunomodulation occurs when host defense mechanism is stimulated under impaired immune response conditions or when immunosuppressant is associated with such state includes; autoimmune disorders. medicinal plants are the current known aspect in phytomedicine, involved in a wider range of herbal materials formulation into supplements. immunomodulatory therapy with the help of medicinal plants serves as an alternative approach to conventional medicine against diseases. hence, host defend mechanism is triggered under impaired immune feedback. immune system is implicated in the investigation and pathological mechanisms of several diseases, with tremendous increased yearning for effective drugs for an improved immune system. to overwhelm the interval of synthetic immunomodulators, there is the need for the development of herbal immunomodulators (z-ziza et al., 2008). immunomodulator is biological or synthetic substances, which stimulate and modulate the components responsible for immune system booster including innate and adaptive immune response (agarwal and singh, 1999; shivaprasad et al., 2006). modulation of immune system using herbal remedy as a therapeutic measure has brought in the need for scientific investigations. coconut (cocos nucifera l.) belongs to the family ‘arecaceae’ and subfamily ‘cocoidae’. it is a tropical tree species extending over large uniform zones. it is usually known as the "tree of life" because of it medicinal usefulness (assa et al., 2010). oil from cocos nucifera is the most well-known food product gotten from dried coconut pulp, called the copra (shankar et al. 2013). law et al. (2014) analyzed the significance of coconut oil for patients with breast cancer. using coconut oil supplement has improved the general wellbeing of patients and reduce possible adverse effect associated with chemotherapy. coconut oil is useful for weight loss due to the biological component of short and medium-chain fatty acids that help in eliminating obesity. it eases digestion and help in healthy functioning of thyroid and endocrine gland. it is known to increases body metabolic rate via pancreatic stress removal; thereby burning more energy and promoting and prevent various stomach and digestion-related manuscript received: 02 october, 2022. revision accepted: 05 december, 2022. published: 24 january, 2023. https://doi.org/10.14421/biomedich.2023.121.159-169 160 biology, medicine, & natural product chemistry 12 (1), 2023: 159-169 problems include; irritable bowel syndrome (yousefi et al., 2013). the saturated fats present in coconut oil have antimicrobial properties and help in dealing with various bacteria, fungi and parasites that can cause indigestion (shilling et al., 2013). due to the presence of lauric acid, it plays a vital role in preventing diverse cardiovascular diseases (vala et al., 2014). c. nucifera oil has been reported to be an effective as a wound healing, immune booster and other therapeutic agents. (nevin et al., 2010). materials and method collection and preparation of plant materials fresh coconut seeds were procured from egor local government area of benin city, edo state, nigeria. it was identified and authenticated by dr. h. akinnibosun in the departmental herbarium unit of plant biology and biotechnology, with a voucher number (uwh-x219). cold extraction method was involved in the isolation of the coconut milk, allowed to stand for 20-24 hrs. under favorable conditions (35-40˚c, 75% relative humidity), the oil gets separated from the water and the protein. the airborne lactic acid bacteria, which can break the protein bonds, act on the coconut milk mixture for proper separation. the operation conditions and sanitary precautions were strictly followed. the top layer was floating curd. the curd also contained a considerable amount of trapped oil. the separated oil contained some adhering particles and was filtered using a sterilized filter paper. experimental animals thirty (30) wistar male rats weighed between 150-315 g were obtained from the animal house, of the department of biochemistry, university of benin, benin city. they were housed under disinfected and wellventilated plastic cages in a standard laboratory condition (12 hours light/dark cycle: 23 ± 2°c) and fed using bendel meal pelleted grower mesh. the food and water were given at ad libitum. the animals were handled in accordance to the ethical laboratory animals’ guide’ from the approval of the life sciences ethical committee university of benin, issued a voucher number ls21507. preparation of sheep red blood cells (srbc) fresh blood was collected from a healthy sheep sacrificed in the local slaughterhouse in a sterile bottle containing elsevier’s solution (1:1). then it centrifuged at 500 g for 15 min at 4 c in cooling centrifugation apparatus to separate the red blood cells (rbcs). the sheep red blood cells (srbc) were washed three times in normal saline. they were then kept under refrigeration for the immunization. experimental protocol the animals were numbered, weighed and then divided into six groups with five animals in each: all groups will be immunized with 0.3 ml/kg srbc for 7 and 14 days of the study. group a serves as the normal control, group b was administered 0.3 ml/kg srbc alone intraperitoneally (negative control), group c received 0.3 ml/kg srbc and 5 mg/kg azathioprine (standard drug p.o) (positive control), group d, e and f were treated with 0.3 ml/kg srbc pus graded doses (0.25, 0.5 and 1.0 ml/kg of coconut oil p.o). after 14 days, the animals were sacrificed under mild chloroform anesthesia. blood was collected via cardiac puncture and organ samples were isolated. the serum was separated for various biochemical tests include; liver function tests (alkaline aminotransferase (alt), aspartate aminotransferase (alp), alkanine phosphatase (alp), gama glutamine transferase (ggt), total bilirubin (tot. bil), conjugated bilirubin (cong. bil), unconjugated bilirubin (ub) and total protein (tp)); anti-oxidant test. the estimation of superoxide dismutase (sod), catalase (cat), malondialdehyde (mda), and glutathione peroxidase (gpx). hematological indexes the estimation of various (20) parameters was carried out. the white blood cell (wbc), hemoglobin (hgb), red blood cell (rbc), platelet count (plt) and lymphocytes. cd4 and cd8 count were carried out. biochemical assay the blood sample for biochemical assays also collected via cardiac puncture were placed in lithium heparin sample bottles and centrifuged at 3000 revolutions per minute (rpm), the plasma was separated using pasteur pipettes into clear labeled bottles. the samples were stored in deep freezer at -20oc until analyses were carried out (bagul et al., 2005). the blood plasma was used for the evaluation of the following biochemical parameters incudes aspartate aminotransferase (ast), albumin (alb), total protein (tp), total bilirubin (t bil), direct bilirubin (d bil), alanine aminotransferase (alt) alkaline phosphate (alp), malondialdehyde (mda), catalase, superoxide dismutase (sod) and glutathione peroxidase (gpx). the biochemical parameters were evaluated using commercial kits obtained from randox laboratories, uk. histopathological analysis l.iver, kidney, heart, spleen and lungs were fixed in formal saline fluid. affixed organs were utterly dehydrated with 99.9 % ethanol along with 70 % ethanol, and 96 % ethanol and washed using distilled water. 4 µm sections were prepared and stained in hematoxylin-eosin dye. stained tissues were optical photomicroscope (leica mc170 hd, leica biosystems, idu, et al. – immunoprotective effect of cocos nucifera oil in rats 161 germany) viewed at x 400 magnification (drury and wallinton, 2013). statistical analysis the data were calculated as mean ± standard error of mean. significance differences of the mean value with respect to the control group was analyzed by one way anova using graph pad prism 8 statistical software. p<0.05 considered being significant. results and discussion results obtained in table 1, 2 and 3 showed a significant increase (p < 0.05) at lowest dose of 0.25 ml of the treatment groups when compared with untreated control in the hematological parameters. the immune response is a biological process which solely depends on external protection and is differ for all members of vaccinated population (okonkwo et al., 2004; yakubu et al., 2007). the immune response is influenced by many genetic and environmental factors. hematological parameters as one of the indexes in immune system understudied the white blood cell, red blood cell, lymphocyte, hemoglobin, platelet and other. result from table 1 showed a significant increase in the value of wbc counts across the treated groups but more on 0.5 ml/kg when compare with untreated control. bone marrow being a flexible tissue is majorly responsible for blood cells regenerations to improve immunity. lymphocyte and other key cells of the immune system are known to activate the production of several antibody polymorph with nuclear granulocyte to destroy antigen (prescott, 1999). lymphocyte indexes being one of the immune booster that protect the body against invaders showed a slight increase in the treated groups that received 0.25 and 0.5 ml/kg of coconut oil (9.90 and 8.20) when compared with untreated control. a significant increase in the level of granulocyte across the treated groups specifically at 0.25 ml/kg (8.0) when compared with the controls. there is a significant increase in the level of hemoglobin, hematocrit and platelet in the treated groups when compared with the control groups as shown in tables 2-3. this present study agreed with the work of oduola et al. (2005) that reported the leaf extract of j. gossypiifolia on the biochemical and hematological analyses has no damaging effects. table 1. effect of coconut oil on white blood and differentials in immunoprotective study. parameters control negative control (5 ml/kg) azathioprine 0.25 ml/kg coconut oil 0.5 ml/kg coconut oil 1.0 ml/kg coconut oil wbc 103/µl 11.73±1.51b 10.10±0.17a 9.30±0.45a 11.70±1.25b 9.90±0.76a 10.33±1.56a lym % 83.07±3.53a 84.43±0.59a 84.47±1.68a 84.03±2.36a 83.10±1.10a 85.17±1.82a mid % 9.533±1.82b 8.67±0.24a 8.20±0.60a 8.83±0.93a 8.90±0.47a 8.10±1.00a gran % 7.40±1.70a 6.90±0.50a 7.33±1.13a 7.13±1.42a 8.0±0.67b 6.73±0.83a lym 103/µl 9.77±1.39b 8.53±0.18a 7.87±0.41a 9.90±1.34b 8.20±0.50a 8.80±1.36a mid 103/µl 1.10±0.26b 0.87±0.03a 0.77±0.07a 1.00±0.00b 0.90±0.10a 0.83±0.14a gran 103/µl 0.87±0.23b 0.70±0.00a 0.67±0.12a 0.80±0.10b 0.80±0.15b 0.70±0.11a the values were expressed in mean ± sem and the significant difference was spotted as p -value < 0.05. table 2. effect of coconut oil red blood cell and components in immunoprotective study. parameters control negative control (5 ml/kg) azathioprine 0.25 ml/kg coconut oil 0.5 ml/kg coconut oil 1.0 ml/kg coconut oil rbc 106/µl 6.36±0.33a 6.48±0.44a 6.0±0.15a 6.85±0.25a 6.71±0.38a 6.37±0.27a hgb g/dl 13.60±0.53a 14.63±0.89a 13.83±0.50a 15.57±0.67b 15.33±0.70b 14.23±0.62a hct % 37.50±1.47a 40.83±2.24a 38.73±1.31a 42.37±1.48b 41.40±1.68a 38.70±1.11a mcv µm3 59.13±0.84a 63.20±1.60a 64.77±0.64a 61.93±0.09a 62.57±0.37a 60.97±1.70a mch pg 21.37±0.34a 22.57±0.48a 23.03±0.35a 22.67±0.14a 22.83±0.35a 22.30±0.10a mchc g/dl 36.20±0.55a 35.77±0.23a 35.67±0.60a 36.67±0.29a 37.00±0.44b 36.73±1.17a rdws µm3 30.93±1.23a 32.20±0.60a 33.43±1.07a 31.57±1.07a 31.60±0.00a 32.83±3.26a rdwc % 16.07±0.42a 15.80±0.00a 16.13±0.43a 15.70±0.52a 15.73±0.24a 16.50±1.27a the values were expressed in mean ± sem and the significant difference was spotted as p -value < 0.05. 162 biology, medicine, & natural product chemistry 12 (1), 2023: 159-169 table 3. effect of coconut oil platelet and factors in immunoprotective study. parameters control negative control (5 ml/kg) azathioprine 0.25 ml/kg coconut oil 0.5 ml/kg coconut oil 1.0 ml/kg coconut oil plt 103/µl 740.00±72.76a 765.30±45.33a 949.70±322.00b 660.30±80.8a 909.00±132.3b 996.30±225.9b mpv µm3 7.43±0.12a 8.03±0.26a 8.47±0.09a 7.77±0.09a 8.07±0.13a 8.20±0.30a pdw % 9.37±0.33a 10.47±0.70a 11.17±0.44a 9.57±0.07a 10.40±0.59a 10.80±0.67a pct % 0.54±0.047a 0.61±0.02a 0.80±0.28b 0.51±0.06a 0.73±0.11a 0.82±0.21b p-lcr % 3.83±1.93a 8.30±3.30a 12.80±1.12b 5.03±0.27a 8.03±2.43a 12.10±3.16b the values were expressed in mean ± sem and the significant difference was spotted as p -value < 0.05. figure 2 showed an increase in the level of cd4 count across the graded doses of the treated groups 0.25ml, 1.0ml extract when compared with untreated control. figure 1 showed an increase in the level of cd8 count across the graded doses of the treated groups 0.25ml, 1.0ml extract when compared with untreated control. cd4 and cd8 cell count provides an improved immune status against opportunistic infections, also it is regarded as a diagnostic decision-making, test particularly with patients suffering from immunosuppressant and hiv disorders. result from this study at graded doses of the extract-treated animals, significantly increase the level of cd4 and cd8 count specifically at 0.25 ml/kg (7.763 and 7.830) when compared with untreated group (figure 1 a and b). this concurred with the work of oduola et al. (2005) on the effect of voacanga africana leaves extract on serum lipid profile and hematological parameters. a b c d e f 0 2 4 6 8 10 treatment group m e a n a n ti b o d y v a lu e a b c d e f 0 2 4 6 8 10 treatment group m e a n a n ti b o d y v a lu e key: group a: normal control received water alone; group b: negative control received 0.3 ml/kg srbc alone intraperitoneally; group c: positive control receive 0.3 ml/kg srbc + 5 mg/kg standard drug (azathioprine) orally; group d: treated group receive 0.3 ml/kg srbc + 0.25 ml/kg of coconut oil orally; group e: treated group receive 0.3 ml/kg srbc + 0.5 ml/kg of coconut oil orally; group f: treated group receive 0.3 ml/kg srbc + 1.0 ml/kg of coconut oil orally. figure 1. (a and b) effect of coconut oil extract on cd8 and cd4 count. figure 2a showed the anti-oxidant test carried out to estimate the in vivo scavenging power of malondialdehyde (mda) of graded doses of coconut oil in wistar rat. the anti-oxidant test carried out to estimate the in vivo scavenging power of catalase response on coconut oil extract in wistar rat as shown in figure 2b. figure 2c elicited the anti-oxidant potential carried out to investigate the in vivo scavenging power of superoxide dismutase responses on coconut oil extract in wistar rat. the results in figure 2d exhibited anti-oxidant effect investigated in the in vivo scavenging power of glutathione peroxidase responses. various antioxidant markers showed the scavenging effect caused by oxidation of lipid peroxidation. hence, the level of malondialdehyde (mda) in the blood was measured (uchiyama et al., 1978). in vivo mda antioxidant level elicited a significantly reduction at lowest dose (0.25 ml/kg) in cocos nucifera oil extract (34.53) when compared with untreated control as shown in figure 3. antioxidant enzymes serves as delicate part associated with cellular defense against responsive oxygen species (ros) with eventual oxidative stress (nevin et al., 2006). the determination of oxidative stress using the balance involved ros generation includes; antioxidant protective systems like superoxide dismutase (sod) and superoxide anion. antioxidants enzymes are implicated in the removal of reactive oxygen species such as gsh, sod and cat. antioxidant enzymes such as catalyst scavenging power significantly increased (p < 0.05) it scavenging capacity at 0.25 ml/kg (241.0) when compared with the control, however, in the activity of sod, there was no significant a b idu, et al. – immunoprotective effect of cocos nucifera oil in rats 163 increase across the treated animals when compared to the control. glutathione peroxidase activity, showed a significantly increased in its scavenging power at (129.0) of 0.25 ml/kg of the oil extract when compared with the control groups. similarly, this study has exhibited its biological effects on cocos nucifera oil established to scavenge oxidative stress via boosting its antioxidant capacity, thereby scavenging free radicals and decreasing lipid peroxidation (nevin et al., 2006 and van et al., 2004). a b c d c f 0 10 20 30 40 50 group c o n c e n tr a ti o n ( m m /m l) a b c d e f 0 100 200 300 400 treatment group c o n c e n tr a ti o n ( m m /m l) a b c d e f 0.0 0.5 1.0 1.5 2.0 2.5 treatment group c o n c e n tr a ti o n ( m m /m l) key: group a: normal control received water alone; group b: negative control received 0.3 ml/kg srbc alone intraperitoneally; group c: positive control receive 0.3 ml/kg srbc + 5 mg/kg standard drug (azathioprine) orally; group d: treated group receive 0.3 ml/kg srbc + 0.25 ml/kg of coconut oil orally; group e: treated group receive 0.3 ml/kg srbc + 0.5 ml/kg of coconut oil orally; group f: treated group receive 0.3 ml/kg srbc + 1.0 ml/kg of coconut oil orally. figure 2. (a, b, c and d) effect of cocos nucifera on enzymatic (malondialdehyde) and non-enzymatic (catalase, superoxide dismutase and glutathione peroxidase) effect on immune response. figure 7 showed the level of liver function tests (alt, ast, alp, ggt, total bilirubin, conjugated bilirubin, unconjugated, bilirubin and total protein) on the extract activity (adeoye and oyedepo, 2004). the eight liver function tests were found to be within normal range in all treated groups. excessive secretion of alanine aminotransferase (alt) and aspartate aminotransferase (ast) are raised in serum when malfunction of the liver occurs. the liver function test results elicited no significant increase in the graded doses of the oil extract (alt, ast and alp) specifically at 1 ml/kg of the treated groups at (15.28) when compared with the control. this is in line with the results showed in the findings of adeosun et al. (2014) that reveal stem latex of j. gossypifolia as a protein precipitant for biochemical investigation with potential defensive measures. no significant difference at (p < 0.05) on the conjugated bilirubin value when compared with the control. 0.25 ml/kg of the oil extract showed a significant increase in the level of ggt, total bilirubin and total protein antibody level at 0.833, 0.053 and 13.57 when compared with the control groups. this agreed with the report of igbe et al. (2013) and magili and bwatanglang (2018). body weight of the animals in the treated groups showed significant increase specifically at 0.25 ml/kg of oil extract, when compared with the control groups (kumar et al., 2008). a sight significant increase in the body weight of the rats was revealed at weeks 1, 2, 3 and 4. afterward treated with c. nucifera oi extract across graded doses of 0.25, .0.5 and 1.0 m/kg had a slightly significant difference in the body weight observed in dose dependent manner (figure 3). reduction in the body weight aids as an indicator and sensitive marker to mark toxicity. this is adhering to the work of witthawaskul et al. (2003). a b c d e f 0 50 100 150 200 treatment group c o n ce n tr at io n ( m m /m l) a b c d 164 biology, medicine, & natural product chemistry 12 (1), 2023: 159-169 a b c d e f 0 10 20 30 40 concentration (ml/kg) a la n in e a m in o tr a n s fe ra s e r e s p . a b c d e f 0 20 40 60 concentration (ml/kg) a s p a rt a te a m in o tr a n s fe ra s e r e s p . a b c d e f 0.00 0.01 0.02 0.03 0.04 concentration (ml/kg) c o n ju g a te d b il ir u b in r e s p . a b c d e f 0 5 10 15 20 concentration (ml/kg) a lk a li n e p h o s p h a ta s e r e s p . a b c d e f 0.0 0.5 1.0 1.5 concentration (ml/kg) g a m a g lu ta m y lt ra n s fe ra s e r e s p . a b c d e f 0.00 0.02 0.04 0.06 0.08 concentration (ml/kg) t o ta l b il ir u b in r e s p . a b c d e f 0 5 10 15 20 concentration (ml/kg) t o ta l p ro te in r e s p figure 3. (a, b, c, d, e) dose responses on the extract in various concentration of liver function test. group a: normal control received water alone; group b: negative control received 0.3 ml/kg srbc alone intraperitoneally; group c: positive control receive 0.3 ml/kg srbc + 5 mg/kg standard drug (azathioprine) orally; group d: treated group receive 0.3 ml/kg srbc + 0.25 ml/kg of coconut oil orally; group e: treated group receive 0.3 ml/kg srbc + 0.5 ml/kg of coconut oil orally; group f: treated group receive 0.3 ml/kg srbc + 1.0 ml/kg of coconut oil orally. a b c d e f 0 100 200 300 treatment group week1 week 2 week 3 week 4 w e e k l y w e ig h t g a in figure 4. dose responses on the extract in various concentration of the body weight. group a: normal control received water alone; group b: negative control received 0.3 ml/kg srbc alone intraperitoneally; group c: positive control receive 0.3 ml/kg srbc + 5 mg/kg standard drug (azathioprine) orally; group d: treated group receive 0.3 ml/kg srbc + 0.25 ml/kg of coconut oil orally; group e: treated group receive 0.3 ml/kg srbc + 0.5 ml/kg of coconut oil orally; group f: treated group a b c d e f g idu, et al. – immunoprotective effect of cocos nucifera oil in rats 165 receive 0.3 ml/kg srbc + 1.0 ml/kg of coconut oil orally. the histopathological structure of the liver, kidney, heart, spleen and lungs (plate 1, 2, 3, 4 and 5) at graded doses (0.25ml, 0.5ml and 1.0 ml/kg) specifically 0.25 ml/kg of the extract revealed a distinct centriole and dilated sinusoidal, with no inflammatory cells and steatosis when compared with untreated control that showed mild steatosis in hepatic cells this is in line with the findings of eleazu et al. (2013) and magili and bwatanglang (2018) that worked on the toxicological profile of the aqueous leaves extract of j. gossypiifolia.. the structure of renal cells reveals no necrosis in lowest doses (0.25 ml/kg) when compare with the untreated control with pronounced necrosis. figure 5. effect of cocos nucifera oil extract on histology of the liver. a= normal control received water alone liver: reveals visible centriole (long arrow) with the hepatocytes revealing pyknotic nucleus (short arrow). b= 0.3 ml/kg srbc alone liver: reveals centriole surrounded by mild inflammatory cells (long arrow) with the hepatocytes revealing mild steatosis (short arrow). c= 5 mg/kg standard drug (azathioprine) liver: reveals centriole (long arrow) surrounded by hepatocytes that appear not so distinct. (short arrow). d= 0.25 ml/kg of coconut oil liver: reveals distinct centriole (long arrow) with the hepatocytes and dilated sinusoidal (short arrow). e= 0.5 ml/kg of coconut oil liver: reveals centriole surrounded by focal inflammatory cells (long arrow) with the hepatocytes revealing mild steatosis (short arrow). f= 1 ml/kg of coconut oil liver: reveals centriole (long arrow) surrounded by hepatocytes with hydropic fatty changes and revealing mild steatosis (short arrow). figure 6. effect of cocos nucifera oil extract on histology of the kidney. a b c a d b c e f d e f 166 biology, medicine, & natural product chemistry 12 (1), 2023: 159-169 a= normal control received water alone kidney: reveals prominent renal corpuscle (long arrow) and interstitial space and tubules (short arrow). b= 0.3 ml/kg srbc alone kidney: reveals atrophied renal corpuscle (long arrow) and interstitial space with mononuclear cells (short arrow) and mild tubular necrosis. c= 5 mg/kg standard drug (azathioprine) kidney: reveals renal corpuscle appearing not so distinct with granulated nucleus (long arrow) and interstitial (short arrow) and tubular necrosis. d= 0.25 ml/kg of coconut oil kidney: reveals visible renal corpuscle (long arrow) and interstitial space (short arrow) and tubules. e= 0.5 ml/kg of coconut oil kidney: reveals atrophied renal corpuscle (long arrow) and interstitial space with mononuclear cells (short arrow) and tubular necrosis. f= 1 ml/kg of coconut oil kidney: reveals renal corpuscle appearing not so distinct (long arrow) and interstitial (short arrow) and tubular necrosis. figure 7. effect of cocos nucifera oil extract on histology of the heart. a= normal control received water alone heart composed of bundles of myocardial fibres (short arrow), interstitial space and prominent coronary artery (long arrow). b= 0.3 ml/kg srbc alone heart composed of bundles of myocardial fibres (short arrow), interstitial space and atrophied coronary artery (long arrow). c= 5 mg/kg standard drug (azathioprine) heart composed of bundles of myocardial fibers (short arrow), interstitial space and congested coronary artery (long arrow). d= 0.25 ml/kg of coconut oil heart composed of bundles of myocardial fibers (short arrow), interstitial space and large prominent coronary artery (long arrow). e= 0.5 ml/kg of coconut oil heart composed of tight bundles of myocardial fibers (short arrow), interstitial space and coronary artery (long arrow). f= 1 ml/kg of coconut oil heart composed of bundles of myocardial fibers (short arrow), interstitial space and coronary artery (long arrow). figure 8. effect of cocos nucifera oil extract on histology of the spleen. a b c e f a d b e c f d idu, et al. – immunoprotective effect of cocos nucifera oil in rats 167 a= normal control received water alone: spleen shows lymphoid follicles (short arrow) with centrally to eccentrically located large blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes which. the red pulps appear distinct. b= 0.3 ml/kg srbc alone: spleen shows lymphoid follicles (short arrow) with centrally to eccentrically located large blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes which. the red pulps appear coarse. c= 5 mg/kg standard drug (azathioprine): spleen shows lymphoid follicles (short arrow) with eccentrically located blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes which appear activated. the red pulps appear coarse. d= 0.25 ml/kg of coconut oil: spleen shows prominent lymphoid follicles (short arrow) with eccentrically located blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes. the red pulps are prominent. e= 0.5 ml/kg of coconut oil: spleen shows lymphoid follicles (short arrow) with not so prominent eccentrically located blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes. the red pulps appeared coarse. f= 1 ml/kg of coconut oil: spleen shows lymphoid follicles (short arrow) with eccentrically located blood vessels (long arrow). the follicles (white pulp) comprise aggregates of lymphocytes which appear activated. the red pulps are appeared coarse. figure 9. effect of cocos nucifera oil extract on histology of the lung. a= normal control received water alone: lung reviewed prominent blood vessel (long arrow) and visible alveolar ring. the alveolus appears distinct (short arrow). b= 0.3 ml/kg srbc alone: lung reviewed prominent bronchiole (long arrow) and visible alveolar ring. the alveoli appears slightly congested (short arrow). c= 5 mg/kg standard drug (azathioprine): lung reviewed prominent blood vessels (long arrow) and visible alveolar ring. the alveoli appear slightly congested (short arrow). d = 0.25 ml/kg of coconut oil: lung reviewed prominent alveolar sac (long arrow) and alveolar ring. the alveoli appear slightly congested. e= 0.5 ml/kg of coconut oil: lung reviewed prominent alveolar sac (long arrow) and alveolar ring. the alveoli appear slightly congested. f= 1 ml/kg of coconut oil: lung revealed prominent bronchiole (long arrow) and visible alveolar ring. the alveoli appear slightly congested (short arrow). differences between the hearts occurred in coronary artery which stated a large prominent coronary artery in the lowest dose when compared with untreated control that exhibited atrophied with congested coronary artery (magili and bwatanglang, 2018). the alveolus in the lungs showed a distinct structure in 0.25 ml/kg when compared with untreated control that showed alveolus congestion. the extract feedback response triggered by cellular and humoral immune response. hence, this study validated the ethnomedicinal property as immune booster. conclusion this study elicited a feedback response from the coconut extract, triggered by cellular and humoral immune response, and thereby boosted the immune physiological state of the body. this 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(2014). tokoferole i tokotrienole jako witamina e. chemik. 68(7): 585-591. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1207-1217 | doi: 10.14421/biomedich.2025.142.1207-1217 issn 2540-9328 (online) evaluating acute toxicity of jamu cekok in rats: a histopathological approach based on oecd 425 michelle the1, puji astuti2*, siti aisyah3, hadi kurniawan4 1bachelor of medicine program, faculty of medicine, universitas tanjungpura, pontianak, west kalimantan, indonesia. 2biochemistry and molecular biology department, faculty of medicine, universitas tanjungpura, pontianak, west kalimantan, indonesia. 3pathology anatomy department, universitas tanjungpura, pontianak, west kalimantan, indonesia. 4pharmacy department, faculty of medicine, universitas tanjungpura, pontianak, west kalimantan, indonesia. corresponding author* pujiastuti@medical.untan.ac.id abstract jamu cekok, an indonesian traditional medicine to increase children appetite, has not passed preclinical or clinical trials. the rhizomes that make up jamu cekok such as pakai bahasa latinnya contain ethyl p-methoxycinnamate which is toxic, thus potentially harmful to the children. therefore in this study we aim to determine the lethal dose (ld50) value of jamu cekok using the oecd 425 method as well as to observed the histopathological change in the liver in female rattus norvegicus l. this study used an analytical experimental design with the oecd 425 method to test the acute toxicity of jamu cekok. the test rats used were in accordance with the oecd 425 protocol with an additional 2 negative control rats. jamu cekok in the form of decocta were given once on the first day of the acute toxicity test and observed for 14 days. on the 15th day, rats were euthanized, dissected, and hepatic organs were taken to make histopathological preparations with hematoxylin-eosin staining. analysis of hepatic damage was assessed using histopathology scoring manja roenigk and tested by kruskal wallis method. the results showed the ld50 value of decocta extract of jamu cekok was >5000 mg/kgbb. observations of body weight, behavioral tests, organ index, and macroscopic hepar of test rats showed no signs of toxicity. however, the statistical analysis for histopathology test showed that there was a significant difference (p<0.05) between the negative control group and the 5000 mg/kgbb treatment group. in conclusion, the administration of jamu cekok was categorized as practically non-toxic but it can caused reversible hepatic damage at a dose of 5000 mg/kgbb. keywords: acute toxicity test; jamu cekok; liver; pathology; oecd 425. introduction in indonesia, the use of traditional medicine is still believed by some people to treat various diseases. traditional medicine or also known as herbal medicine is a concoction consisting of natural ingredients mixed for consumption and is believed to be able to treat diseases for generations (bpom, 2019). according to bpom indonesia, traditional medicine is categorized into several groups, namely herbal medicine, standardized herbal medicines, and phytopharmaceuticals. jamu is one of the three groups that is commonly known by the public and utilized by the public to overcome health problems (bpom, 2023). data from the 2018 indonesia basic health survey shows that 59.12% of indonesians still consume herbal medicine (kemenkes, 2018). the definition of jamu cekok refers to the method or method of administration, namely dicekokkan or dicangar (forcibly inserted) into the child's mouth (rini & endah, 2019). the types of plants used in jamu cekok are temu ireng (curcuma aeruginosa roxb.), kunir (curcuma longa l.), temulawak (curcuma xanthorrhiza roxb.), babakan pule (alstonia scholaris (l.) r. br.), meniran (phyllanthus niruri l.), asam jawa (tamarindus indica l.), bayem lemah (amaranthus tricolor l.), jambu krikil (psidium guajava l.), puyang (zingiber zerumbet l.), kates (carica papaya l.), and krokot (portulaca oleracea l.). some use additional ingredients in jamu cekok such as tempe mendem (rotten) and salt (bhagawan et al., 2023). the many plants used in jamu cekok are believed to have a positive impact on the health of its users. however, research conducted on one of the components in jamu cekok showed that there are ingredients that fall into the moderately toxic category. toxicity tests on ethyl p-methoxycinnamate compounds in kaempferia galanga l. found that the compounds were categorized as moderately toxic (nurmala, 2017). phenolic compounds contained in the rhizomes of jamu cekok can also have toxic effects on the human body (kyselova, 2011). phenolic compounds that enter the body in large concentrations can activate the caspase pathway which results in apoptosis (cytotoxicity) (kong et al., 2000). in addition, there are still few or no studies on toxicity tests manuscript received: 11 september, 2025. revision accepted: 07 december, 2025. published: 11 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1207-1217 1208 biology, medicine, & natural product chemistry 14 (2), 2025: 1207-1217 involving jamu cekok. more importantly the main costumer of jamu cekok is children below 5 years old, thus make the analysis of its safety became important. the united states of food and drug administration (fda) states that testing is conducted to help develop compounds that are potentially medicinal or toxic in animals. toxicity testing is important to determine the cumulative effects and dose of a compound that can cause toxic effects on the human body. toxicity tests consist of acute toxicity tests, subacute toxicity tests, and chronic toxicity tests. an acute toxicity test is a preliminary method used to detect toxic effects, including potential mortality, resulting from the administration of a single dose of a compound to experimental animals over a short observation period. to measure acute toxicity, parameters such as lethal dose (ld50) measurements and liver histopathology images are used. the ld50 value obtained will be used as a reference in determining the dose level for further toxicity tests (bpom, 2022). although histopathological analysis is not mandatory under oecd 425, it was included in this study to explore potential organ-specific tissue changes following acute exposure. materials and methods research type this study design falls under experimental research using a post-test only control group design. the research was conducted at laboratory of universitas tanjungpura and anatomical pathology laboratory of rumah sakit universitas tanjungpura. the test animals used in this research were female white rats (rattus norvegicus l.) wistar strain aged 2-3 months. in this study, a total of 10 white female rats were used, divided into 3 (three) groups, control group, 2000 mg/kgbw limit test group, and 5000 mg/kgbw limit test group, with the rats used were in accordance to oecd 425 methods and 2 female rats for control group. procedures collection and preparation of jamu cekok jamu cekok was prepared using temulawak, kunyit, jahe, and kencur rhizomes that have been cleaned using clean water. the rhizomes of jamu cekok were obtained by direct purchase from the market in pontianak city, west kalimantan. temulawak, turmeric, ginger, and kencur each amounted to 100g. preparation of extract decocta extract of jamu cekok was made from javanese ginger, turmeric, ginger, and galangal (temulawak, kunyit, jahe, and kencur) that have been blended with 400 ml of distilled water and heated for 30 minutes at 90oc. jamu cekok was then evaporated using rotary evaporator to obtain a thick extract. preparation of experimental animals the experimental animals used were healthy female wistar rats aged 2-3 months and no >20% weight loss after the acclimatization period. before use, the female rats were acclimatized for 14 days under the experimental conditions. preparation of materials the materials used included distilled water as a negative control and decocta extract of jamu cekok as the test material. acute toxicity test using oecd 425 methods acute toxicity test were performed in accordance with oecd 425 guidelines. the toxicity test begins with a limit test with a dose of 2000 mg/kgbw. one test animal was given a dose of 2000 mg/kgbw and observed for 48 hours. if after 48 hours of observation the animal does not show any mortality sign, then the same dose is given to one more test animal. the step is repeated until the maximum test animals used are 5. dosing was stopped if there were test animals that showed mortality. after 5 test animals were dosed and there was no mortality, dosing was stopped and all test animals were observed for 14 days. if 3 or more of the 5 test animals died, the ld50 of the sample was less than 2000 mg/kgbb. if 3 or more test animals are alive, then a limit test with a dose of 5000 mg/kgbb is performed. the parameters observed for acute toxicity test included behavioral and motor activity observations, body weight observations, macroscopic and organ index observations. on the 15th day of the acute toxicity test, termination was carried out and the liver organ of the test rats were extracted. behavioral and motoric activities observation behavioral observation were conducted before and after the administration of the test material. if the test animals do not show mortality, observation will be continued for 14 days. histopathological examination the liver of the rats in the study were surgically harvested and fixed in buffered formalin. they were then processed and stained with hematotoxylin and eosin (he) according to the standard procedures at the anatomical pathology laboratory of rumah sakit universitas tanjungpura. observation was performed under a microscope with a magnification of 400x in 5 field of view. observations were made in five random fields of view on each mount. hepatic cell in each field of view were assessed and counted using manja roenigk histopathology scoring model, by counting the number of normal cells (a), parenchymal degeneration cells (b), hydropic degeneration (c), and necrosis cells (d) from each field of view of the preparation. the measurement results are obtained from (ax1)+(bx2)+(cx3)+(dx4) then the results are divided by (a+b+c+d) to obtain the the et al. – evaluating acute toxicity of jamu cekok in rats: … 1209 average value in one treatment group, the average value in each field of view is divided by the number of samples from each treatment. table 1. manja roenigk histopathology scoring (uche et al., 2022). rate of change score normal 1 parenchymatous degeneration 2 hydropic degeneration 3 necrosis 4 data analysis data obtained from the toxicity test using the oecd method were then analyzed using the aot425 program. data from the observation of hepatic histopathology preparations were tested for normality by shapiro wilk. data distribution was found to be abnormal (p<0.05), so the kruskal wallis non-parametric test was conducted to determine the significant difference (p>0.05) between the three test groups and the dunn post hoc test to determine the significant difference between the medians of each test group using the spss 26 program. results and discussion acute toxicity test the results of the limit test with dose of 2000 mg/kgbw and 5000 mg/kgbw on female wistar rats exhibited no mortality. ld50 value is obtained through data processing with aot 425 statpgm software and showed that ld50 of jamu cekok decocta extract was higher than 5000 mg/kgbw (figure 1 and figure 2). according to globally harmonized classification system, decocta extract of jamu cekok is categorized as practically non-toxic. figure 1. testing results dose 2000 mg/kgbb. figure 2. testing results dose 2000 mg/kgbb. body weight observation the body weights of all the animals both in dose of 2000 mg/kgbw and 5000 mg/kgbw are shown in figure 3. statistical analysis results showed insignificant differences between the body weight of test rats in each test group. figure 3. effect of jamu cekok decocta extract on body weight of wistar female rats in acute toxicity studies: a) 2000 mg/kgbw dose group; b) 5000 mg/kgbw dose group. behavioral and motoric activities observation the test animals were observed for 30 min, 60 min, 120 min, and then for 24 h and continued for 14 days. behavioral observation for the dose of 2000 mg/kgbw showed that all parameters were normal except the platform and motoric activity. all rats in the 2000 mg/kgbw group had a decreased platform value but did not differ significantly when compared to before being given the preparation. the motor activity of the five rats decreased at 30 minutes, 1 hour, and 2 hours after being given the test preparation, but returned to normal after 24 hours until termination (table 2). the results of behavioral observation in the 5000 mg/kgbb dose group also showed decreased platform values and motor activity but were not significantly different from before being given the preparation. there was a decrease in motor activity of the three rats at 30 minutes, 1 hour, and 2 hours after administration of the test preparation and returned to normal after 24 hours until termination. other behavioral parameters showed normal results (table 3). 1210 biology, medicine, & natural product chemistry 14 (2), 2025: 1207-1217 table 2. effect of 2000 mg/kgbw jamu cekok on the behavior of white wistar female rats in acute toxicity studies. parameters of behavioral observation the average of jamu cekok behavioral observation dose of 2000 mg/kgbw 0 30 minutes 60 minutes 120 minutes 24 hours day-15 platform 7,4 6,4 5,8 6 6,8 6,6 motoric activity up (%) 0 0 0 0 0 0 normal (%) 100 0 0 0 100 100 down (%) 0 80 80 100 0 0 no activity (%) 0 20 20 0 0 0 straub (%) 0 0 0 0 0 0 piloerection (%) 0 0 0 0 0 0 ptosis (%) 0 0 0 0 0 0 pineal reflex (%) 100 100 100 100 100 100 corneal reflex (%) 100 100 100 100 100 100 lacrimation (%) 0 0 0 0 0 0 catelepsy (%) 0 0 0 0 0 0 gestures normal (%) 100 100 100 100 100 100 not normal (%) 0 0 0 0 0 0 hanging (%) 100 80 80 100 100 100 retablishment (%) 100 80 80 100 100 100 flexion (%) 0 0 0 0 0 0 hafner (%) 100 100 100 100 100 100 mortality (%) 0 0 0 0 0 0 grooming (%) 100 100 100 60 80 100 defecation (%) 100 80 20 0 100 0 urination (%) 100 20 20 40 100 40 respiration fast (%) 0 0 0 0 0 0 normal (%) 100 100 100 100 100 100 breathless (%) 0 0 0 0 0 0 salivation (%) 0 0 0 0 0 0 vocalization (%) 0 0 0 0 0 0 tremor (%) 0 0 0 0 0 0 convulsions (%) 0 0 0 0 0 0 writhing (%) 0 0 0 0 0 0 table 3. effect of 5000 mg/kgbw jamu cekok on the behavior of white wistar female rats in acute toxicity studies. parameters of behavioral observation the average of jamu cekok behavioral observation dose of 5000 mg/kgbw 0 30 minutes 60 minutes 120 minutes 24 hours day-15 platform 7,3 5,6 6,6 6 6,3 6,6 motoric activity up (%) 0 0 0 0 0 0 normal (%) 100 0 0 0 100 100 down (%) 0 66,6 100 100 0 0 no activity (%) 0 33,3 0 0 0 straub (%) 0 0 0 0 0 0 piloerection (%) 0 0 0 0 0 0 ptosis (%) 0 0 0 0 0 0 pineal reflex (%) 100 100 100 100 100 100 corneal reflex (%) 100 100 100 100 100 100 lacrimation (%) 0 0 0 0 0 0 catelepsy (%) 0 0 0 0 0 0 gestures normal (%) 100 100 100 100 100 100 not normal (%) 0 0 0 0 0 0 hanging (%) 100 80 80 100 100 100 retablishment (%) 100 80 80 100 100 100 flexion (%) 0 0 0 0 0 0 hafner (%) 100 100 100 100 100 100 mortality (%) 0 0 0 0 0 0 the et al. – evaluating acute toxicity of jamu cekok in rats: … 1211 parameters of behavioral observation the average of jamu cekok behavioral observation dose of 5000 mg/kgbw 0 30 minutes 60 minutes 120 minutes 24 hours day-15 grooming (%) 100 66,6 33,3 100 100 33,3 defecation (%) 33,3 66,6 33,3 0 0 100 urination (%) 66,6 100 33,3 33,3 0 100 respiration fast (%) 0 0 0 0 0 0 normal (%) 100 100 100 100 100 100 breathless (%) 0 0 0 0 0 0 salivation (%) 0 0 0 0 0 0 vocalization (%) 0 0 0 0 0 0 tremor (%) 0 0 0 0 0 0 convulsions (%) 0 0 0 0 0 0 writhing (%) 0 0 0 0 0 0 liver organ index the value of liver organ index in the dose of 2000 mg/kgbb is greater than the dose of 5000 mg/kgbb. statistical test results obtained p value = 0.724 (p>0.05). this indicates that there is no significant difference between the organ index of the 2000 mg/kgbb treatment group and the 5000 mg/kgbb treatment group. this shows that there is no significant enlargement or shrinkage in the liver organs of rats (figure 4). figure 4. the percentage of organ index after being given jamu cekok decocta extract. the macroscopic examination of liver organ in all treatment groups showed brownish-red in color. the decocta extract of jamu cekok did not cause any lesions or nodules in the liver macroscopically (figure 5). figure 5. macroscopic view of liver organ: a) control group; b) 2000 mg/kgbw dose group; b) 5000 mg/kgbw dose group. microscopic observation of liver microscopic observation of hepatic organs showed different results from each treatment group (figure 6). figure 6(a) is a histopathological picture of the negative control group which shows a picture of normal hepatic cells with few cells experiencing parenchymal degeneration. hepatic cells seen in this group generally have characteristics such as round nuclei, clear boundaries, full cytoplasm, and regular sinusoids. figure 6(b) is a histopathological picture of the 2000 mg/kgbb treatment group showing damage to hepatic cells. 1212 biology, medicine, & natural product chemistry 14 (2), 2025: 1207-1217 figure 6. macroscopic view of liver organ: a) control group; b) 2000 mg/kgbw dose group; b) 5000 mg/kgbw dose group. hepatic cell damage seen is parenchymatous degeneration (cytoplasm begins to cloudy), hydropic degeneration (cells swell with a typical picture of small to large vacuoles), and necrosis cells such as piknotik (purple to black nucleus with a reduced size due to cell wall compaction), or karyolysis which has a picture of empty cells without cell nuclei. the microscopic picture of the 5000 mg/kgbb treatment group (figure 6(c)) shows cells that have more damage than the other two groups. in figure 6(c), it can be seen that almost every cell has degeneration both parenchymatous and hydropic, also seen necrotizing hepatic cells with karyolysis cell nuclei. further observations were made in quantitative measurements by counting the number of normal hepatic cells (a), parenchymal degeneration cells (b), hydropic degeneration cells (c), and necrosis cells (d) in 5 field of view in each preparation using the manja roenigk histopatholgy scoring model. the determination of the observed field of view was determined randomly to represent each rat hepatic lobe. the calculation results shows that the negative control group has the lowest value of 1.14, followed by the 2000 mg/kgbb treatment group and the 5000 mg/kgbb treatment group (table 4). table 4. hepatic cell calculation result using manja roenigk histopathology scoring model. group number of samples (n) average sd negative control 10 1,14 0,04 2000 mg/kgbw treatment 25 1,33 0,14 5000 mg/kgbw treatment 15 2,85 0,63 the closer the value to 1, the more the number of normal hepatic cells. the closer the value is to 2, the more the number of hepatic cells that have parenchymal degeneration. the closer the value is to 3, the more the number of hepatic cells that have hydropic degeneration. the closer the value is to 4, the more the number of hepatic cells that experience necrosis. the data shows that an increase in the dose of jamu cekok extract is directly proportional to the increase in hepatic damage value (table 4). the calculation data were then processed statistically to see meaningful differences. normality test was conducted first. the normality test results of the negative control group (p=0.146) and the 2000 mg/kgbb treatment group (p=0.106) were normally distributed (p>0.05). meanwhile, the significance value of the 5000 mg/kgbb treatment group is p = 0.020 which indicates that the data is not normally distributed (p <0.05). therefore, data processing was continued in the kruskal wallis non-parametric test. table 5. effect of dekokta jamu cekok extract on hepatic cell damage. group number of samples (n) p-value negative control 10 0,000 2000 mg/kgbw treatment 25 5000 mg/kgbw treatment 15 note: kruskal wallis test result the statistical analysis result shows that the p-value obtained from the kruskal wallis test is p=0.000 which indicates a significant difference (p<0.05). these results indicate that there are significant differences in hepatic the et al. – evaluating acute toxicity of jamu cekok in rats: … 1213 cell damage from each group. because there is a significant difference, it is continued with the post hoc dunn test. the results of the post hoc dunn test can be seen in table 6. table 6. effect of dekokta jamu cekok extract on hepatic cell damage. group negative control 2000 mg/kgbw treatment 5000 mg/kgbw treatment negative control 2000 mg/kgbw treatment 5000 mg/kgbw treatment note: post hoc dunn test. the shaded part shows the significant difference between the two groups. the negative control group compared to the 2000 mg/kgbb treatment group has a p value of 0.077 which indicates no significant difference (p>0.05) between the two groups. the negative control group compared to the 5000 mg/kgbb treatment group and the 2000 mg/kgbb treatment group compared to the 5000 mg/kgbb treatment group had a p=0.000 value indicating a significant difference (p<0.05) of hepatic cell damage between the two groups. discussion jamu cekok as a preparation tested for acute toxicity in this study used 4 types of rhizomes, namely temulawak (curcuma xanthorriza roxb.), turmeric (curcuma domestica), ginger (zingiber offcinale rosc.) and kencur (kaempferia galanga l.). among one of the four rhizomes, kencur, has the main content of ethyl pmethoxycinnamate compounds which are moderately toxic (nurmala, 2017). ethyl p-methoxycinnamate has pharmacological activities including as an analgesic and anti-inflammatory and has potential as a sunscreen (sinarsih et al., 2023). in addition, phenolic compounds contained in the four rhizomes of jamu cekok also have a toxic effect if present in excess in the human body. phenolic compounds can work as pro-oxidants in the body system that contains redox active metals. in the presence of oxygen (o2), transition metals such as copper and iron will catalyze the phenolate redox cycle, leading to the formation of ros and phenoxyl radicals that can damage dna, lipids, and other biological molecules. oxidation of flavonoid compounds with phenol ring-b by peroxidase/h2o2 can form phenoxyl radicals and generate ros. in addition, dietary flavonoids containing phenol rings and other phenolics also cause oxidation of glutathione in hepatocytes (kyselova, 2011 & kong, 2000). this study used an acute toxicity test with the oecd 425 method: up and down procedure (udp). this method is an alternative method in acute toxicity testing. when compared to the conventional ld50 value determination method, the up and down procedure uses fewer test animals (oecd, 2022). acute toxicity testing using the udp method was conducted in the form of a limit test. this is because jamu cekok has been consumed by the public, so it can be assumed that the decoction extract of jamu cekok has a low dose level that fits the limit test criteria (rini & endah, 2019 & bhagawan et al., 2023). in the limit test, doses of 2000 mg/kgbb and 5000 mg/kgbb were used because the single dose acute toxicity test should be done with the top dose of the limit dose. this acute toxicity test uses female wistar rats as test animals. the selection of female rats in general, female sex animals are more sensitive compared to males, thus allowing for chemicals that act directly in their toxic mechanisms and female rats have a low detoxification capacity than males (lipnick et al., 1995). in addition, the use of female rats is also recommended in the oecd acute toxicity testing guideline (oecd, 2022). wistar rats were used in this study because they have been widely used in various research fields such as toxicology and pharmacology studies, have good growth, are easy to obtain in large quantities, are easy to maintain, are relatively cheap and have fast metabolic abilities (ridwan, 2013 & sutrisno et al., 2014). in this study, the administration of test preparations was carried out orally using a sonde. oral administration of the test preparation was chosen to adjust the method of administration that is usually used in humans in the administration of jamu cekok. the test mice were acclimatized for 14 days with the aim that the test mice could adjust to the laboratory environment. during the acclimatization period, the test rats were given food and water. before conducting acute toxicity testing on test rats, behavioral testing and motor activity of test rats were carried out. behavioral observations were made on each rat after 30 minutes, 1 hour, 2 hours, and 24 hours of treatment. this behavioral observation aims to see whether decocta extract of jamu cekok can affect changes in behavior and motor activity in test animals. this test was carried out for 2 minutes for each test animal except hanging and retablismen which were carried out for 5 seconds (mustarichie et al., 2017 & sutjiatmo et al., 2015). overall platform observations showed a decrease in the 30th, 60th, and 120th minutes after administration of the test preparation and increased again after 24 hours. in addition, there was also a decrease in the motor activity of rats at minutes 30, 60, and 120 after administration of the test preparation and returned to normal after 24 hours of administration of the test preparation. behavioral changes can occur due to stress experienced by rats during the administration of 1214 biology, medicine, & natural product chemistry 14 (2), 2025: 1207-1217 the test preparation resulting in temporary inhibition of motor activity (fitria, 2019). in addition, the decrease in motor activity is thought to occur due to the flavonoids in the rhizome of jamu cekok which have a skeletal muscle relaxant effect and alkaloids that can inhibit the work of the nervous system (assad & khan, 2017 & sulastra, 2020). observations of sensory activity, neuromuscular system changes, eyes, respiratory changes, skin, gastrointestinal and urogenital changes, as well as body posture appeared normal in all test animal groups. other observations such as ptosis, lacrimation, catalepsy, mortality, salivation, vocalization, tremors, seizures, and writhing were not found in any of the test animal groups. based on the results of behavioural tests on wistar strain white rats (rattus norvegicus l.), no signs of toxicity were found (nur et al., 2022). this indicates that the decoction extract of cekok herbal medicine does not have a significant effect on the behaviour of the test animals. the first test was conducted on female rats with a dose group of 2000 mg/kgbw who had been fasted for 3 hours while still being given water. the rats were fasted with the aim that when the rats were given the test preparation, the test preparation could directly interact with the digestive system and not be disturbed by the presence of food in the rats' digestive system. this is done to maximize the absorption of the extract in the rat's digestive system (fu et al., 2024). after being given the test preparation, observations of motor activity and behaviour were conducted every 30 minutes, 1 hour, 2 hours, and 24 hours. observation of the behaviour and motor activity of the test rats after 24 hours did not show any signs of death or toxicity. the same treatment was also given to female rats with a dose of 5000 mg/kgbw. the results of the limit test at 2000 mg/kgbw and 5000 mg/kgbw did not show any mortality up to 14 days of observation. observations of the test rats' body weight were also conducted. observations were conducted by weighing daily from before, during, to after the treatment. the observation results can be seen in figure 4.2 and appendix 6. the observation results show that the test mice experienced weight changes that varied over time. the variation in body weight changes of the test mice was not significantly different, as indicated by the statistical test results which yielded a significance value of p = 0.781 (p>0.05). this indicates normal metabolism of proteins, carbohydrates, and lipids in the rats' bodies, as well as normal vital organ function in the test rats (nurbaeti et al., 2021 & saleem et al., 2017). observation was conducted for 14 days and subsequently, surgery was performed on the test rats to examine the organ index of the rats that had been given the preparation orally. surgery was performed to observe the changes occurring in vital organs, particularly the liver, macroscopically first due to the administration of the decoction extract of cekok herbal medicine. the liver is observed because it is the largest organ and has the most complex metabolism in the body. the liver is involved in the metabolism of nutrients and most drugs and toxic compounds that enter the body (chiang, 2014). in addition, the liver is also one of the main organs in toxicity testing (bpom, 2022). according to bpom standards, the organ index is one of the parameters of acute toxicity. the organ index is a parameter that can provide a general overview of the compound's effects, whether there is enlargement or shrinkage of the organ (katrin et al., 2014). although, the results of the macroscopic observation showed that no lesions or damage were found in the organs. however, another parameter, the organ index, shows that the liver organ is larger in the 2000 mg/kgbw dose group compared to the negative control group and the 5000 mg/kgbw dose group. the decrease in liver organ index occurred in the group with a dosage of 5000 mg/kgbw. the liver organ index in the 2000 mg/kgbb dose group increased when compared to normal. the increase in the organ index is associated with the enlargement of the liver organ. changes in the weight, physiological, and morphological aspects of the liver are related to the feed consumed, health, and intake of toxic substances in the animal's body (wahyuningtyas et al., 2018). hepatomegaly occurs when the liver experiences inflammation, storage of certain substances, or increased workload. one of the unique abilities of the liver is regeneration, which is the ability to repair or replace damaged cells. when a mild injury occurs to the liver, enlargement can be a sign of the liver cell regeneration process (safithri, 2018). additionally, differences in liver organ index values may occur due to the use of different numbers of rats in each group and the varying body weights of the test rats. however, the differences in organ index values were not statistically significant. these results also indicate that jamu cekok is not toxic because one of the functions of the liver is to neutralize toxins that enter the body. the ld50 value was obtained through data processing using the aot425 statpgm application (figures 4 and figure 5) and it was found that the decoction extract of cekok herbal has an ld50 value greater than 5000mg/kgbw. based on the toxicity levels of a compound listed by the food and drug administration, it can be said that the decoction extract of cekok herbal medicine falls into the practically nontoxic classification (bpom, 2022). the liver is the largest and most important metabolic organ in the body; it can be referred to as the main biochemical factory in the body. the liver is a bridge connecting the digestive tract with other organs. therefore, the liver is an organ that maintains metabolic homeostasis. one of the functions of the liver is as a detoxification organ, which makes the liver susceptible to cellular damage in the form of inflammation, the et al. – evaluating acute toxicity of jamu cekok in rats: … 1215 degeneration, and necrosis (sherwood, 2014 & abbas, 2015). one of the contents of the cekok herbal medicine, namely ethyl p-methoxycinnamate, is also metabolized in the liver (liu et al., 2010). before damage occurs to the liver cells, the microscopic appearance of the liver shows polygonal-shaped liver cells, with homogeneously red cytoplasm and clearly defined cell walls. changes in the microscopic appearance of the liver can include parenchymatous degeneration, where there is swelling of the cells accompanied by cloudy and granular cytoplasm. there is also hydropic degeneration, where the cells appear swollen and vacuolated, containing fluid. and the permanent damage to liver cells is that liver cells will undergo necrosis (indahsari, 2017). the lowest percentage of damage occurred in the control group given aquades (figure 6(a)). this is possible because there is no influence of other substances entering the body, so the percentage of damage found is not significant. however, damage was still found in the control group. this could be caused by external factors such as environmental conditions and unhygienic food that leads to health issues in the rats (fitmawati et al., 2018). based on the scoring results of manja roenigk, this study found that the average values of the negative control group and the 2000 mg/kgbw treatment group were 1.14 and 1.33, respectively, indicating that the average value of the liver cells is normal. whereas for the 5000 mg/kgbw treatment group, the value was higher compared to the other two groups, at 2.85, which still indicates reversible liver cell damage. this indicates an increase in damage along with the increase in acute doses of the decoction extract of cekok herbal given to test rats, which is microscopically proven by the presence of various levels of liver cell damage in the preparations. liver cell damage can occur due to the antioxidant paradox effect. the antioxidant paradox effect occurs due to the total antioxidant capacity of the body becoming unresponsive to antioxidants consumed in large doses (halliwell, 2013). in this case, the damage is caused by compounds that can act as pro-oxidants. prooxidants can increase the production of free radicals, leading to the accumulation of oxidative stress in the test rats' bodies, resulting in damage to hepatocytes (solter et al., 2019). the results of this study are in line with the research by fitmawati et al. (2018), which examined the traditional concoction of the malay community in lingga, riau islands, conducted using a bitter herbal decoction administered orally to rats using a syringe. the results of the study showed that the administration of the bitter herbal concoction with varying doses caused increasingly greater damage in line with the increase in dosage. however, the liver cell damage that occurred is still within the normal category (fitmawati et al., 2018). whereas, the results are in line with this study which shows that the administration of decoction herbal extract can affect liver cell damage when observed microscopically, and the damage increases with the increase in dosage. however, these changes did not result in mortality in the test rats. this can happen because the more chemical compounds that enter the body, the harder the liver will work, as the liver will detoxify these compounds, especially those that enter through the digestive tract (klaassen & amdur, 2013). moreover, there is a difference between that study, which only assessed the histological structure of the white rat's liver, and this study, which tests acute toxicity. the administration of the decoction extract of cekok herbal in acute doses may affect the liver cell damage that occurs in the test rats. however, the damage to the liver cells does not affect the macroscopic structure of the liver or the behavior and motor activity of the test rats. the damage found microscopically in liver cells due to the administration of the decoction extract of cekok herbal did not cause any signs of toxicity or death in the test rats and is still considered reversible damage. overall, this study shows the influence of administering the decoction extract of cekok herbal in acute doses on the histopathological appearance of the livers of female wistar strain white rats, so the use of cekok herbal by the public needs to consider the dosage. this is in line with who research that herbal medicines still have side effects, making dosage-related studies important for herbal medicines (who, 2004). the results of this study indicate that although empirically jamu cekok is safe to consume, excessive doses may potentially cause microscopic liver organ damage. conclusions the ld50 of the decoction extract of jamu cekok administered orally is greater than 5000 mg/kgbw, which is categorized as practically non-toxic. however, administration at high doses can cause microscopic changes in the rat liver cells, such as reversible degeneration and necrosis, with the highest percentage of damage occurring in the 5000 mg/kgbw treatment group. acknowledgements: thank you to tanjungpura university laboratory and anatomical pathology laboratory of tanjungpura university hospital, pontianak, for their support during the completion of this research. authors’ contributions: michelle the designed the study, analyzed the data, and wrote the manuscript. puji astuti and siti aisyah wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. 1216 biology, medicine, & natural product chemistry 14 (2), 2025: 1207-1217 funding: the authors declare no funding. references abbas, a.k., aster, jc., & kumar v. 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(2004). who guidelines on safety monitoring of herbal medicines in pharmacovigilance systems. who geneva. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 9-16 | doi: 10.14421/biomedich.2023.121.9-16 issn 2540-9328 (online) proximate composition, levels of some essential mineral elements and anti-nutritional components of some yam species found in minna, niger state eneogwe okechukwu godfrey1,*, ibrahim izihyi esther2, obuye faith1 1department of chemistry; 2department of industrial chemistry, federal university lokoja, p.m. b. 1154, lokoja, nigeria. corresponding author* godfrey.eneogwe@fulokoja.edu.ng manuscript received: 18 august, 2022. revision accepted: 06 september, 2022. published: 27 september, 2022. abstract samples of dioscorea dumenturom, dioscorea rotundata and dioscorea cayenensis were investigated for their proximate composition, anti-nutritional and mineral contents using standard analytical methods. these varieties of dioscorea analysed showed a significant difference (p≤0.05) amongst them. however, from the results, it was observed that dioscorea rotundata had the highest ash (8.05±0.05 %) and crude fibre content (13.11±0.10 %) which indicates that it contains more mineral stuffing and is best for softening of stool. dioscorea cayenensis had the highest fat content (16.31±0.30 %), indicating that it is a better source of calories than other yam species analysed. dioscorea dumenturom had the lowest moisture content (3.51±0.01 %) as well as the highest crude protein (12.29±0.01 %) and carbohydrates (69.04±0.10 %) than other yam species analysed, indicating its longer shelf-life, high bodybuilding capacity and better source of energy than other yam species analysed. the anti-nutritional constituent of alkaloid and tannin were lowest in dioscorea cayenensis while dioscorea rotundata had the least cyanide, phytate and oxalate content. this implies that these particular yams are safer for consumption. the elemental analysis in mg/100g indicated that the yam species contained appreciable levels of essential minerals, with dioscorea dumenturom having the highest sodium, calcium, iron, potassium, phosphorous and magnesium concentration of 32.05±0.07 mg/100g, 190.57±0.01 mg/100g, 5.98±0.03 mg/100g, 80.12±0.17 mg/100g, 237.10±0.48 mg/100g and 100.22±0.03 mg/100g respectively. all these mineral concentrations exist within the permissible limit of who and hence indicate that the yam species can serve as a good source of minerals. keywords: proximate composition; anti-nutritional; mineral content; dioscorea dumenturom; dioscorea rotundata; dioscorea cayenensis. introduction yam is a staple cuisine in many tropical and subtropical areas throughout the world. nigeria in particular has the world’s largest annual output and consumption per capita (ukom et al., 2014). there are over 600 dioscorea species, with more than 10 species farmed for food and 6 species used in pharmaceuticals (okigbo et al., 2015). however, white yam (dioscorea rotundata), water yam (dioscorea alata), aerial yam (dioscorea bulbifera), yellow yam (dioscorea cayenensis), trifoliate yam (dioscorea dumentorum) and chinese yam (dioscorea esculenta) are the six most economically important species. yam cultivation not only helps rural farmers survive but also provides a significant number of calories, essential micro-nutrients and phytochemical compounds such as iron, zinc, ascorbic acid and flavonoids (ukom et al., 2014). in many regions of west africa particularly nigeria, yam is processed in a variety of culinary forms, such as pounded yam, fried yam, roasted yam, boiled yam, yam balls, mashed yam, yam chips and flakes (orkwor et al., 1997), which are typically served with protein-rich soups and sources. the species has historically influenced how yams are processed. west africa is the world’s most important yamproducing region, with nigeria as the leading producer, accounting for more than half of global production (modu et al., 2015). despite these facts regarding yam, it continues to be overlooked in west african national food policy programs. this has resulted in limited dioscorea species research and development on the continent (sanoussi et al., 2016). this study compares the proximate composition, mineral content and anti-nutritional constituents of several yam varieties which includes dioscorea rotundata, dioscorea dumenturom and dioscorea cayenensis obtained from minna, niger state. https://doi.org/10.14421/biomedich.2023.121.9-16 10 biology, medicine, & natural product chemistry 12 (1), 2023: 9-16 materials and method sample collection matured accessions of the three cultivated yam species were harvested randomly from rural farms in the chanchaga, mekunkele and gunu areas of niger state. the samples include cultivars of yellow yam (dioscorea cayenensis), a variety of white yams (dioscorea rotundata) and trifoliate yam (dioscorea dumentorum). the samples were cleaned by brushing off soil particles and transported at tropical ambient temperature to the laboratory for analysis. dioscorea dumenturom dioscorea rotundata dioscorea cayenensis figure 1. the images of the studied plants. sample pre-treatment the yam samples were washed thoroughly with water, peeled and cut using a knife. these yam species were ground separately using a laboratory mortar and pestle and then sieved using a 250 μm mesh size sieve. the three samples were stored in airtight properly labeled polythene bags and kept in a cool and dry place before analysis. determination of proximate composition the proximate analysis of samples for moisture, crude fat, crude fiber and ash was determined using the method described by aoac (2006). the protein content was determined using the micro kjeldahl method (n x 6.25) and the carbohydrate was calculated by difference. mineral analysis the digestion of samples for mineral analysis was carried out according to the method described by aoac (2006). a 250 cm3 beaker was filled with 1.00 g of the pulverized sample. the beaker was filled with an acid mixture (15.00 cm3 concentrated hno3 and 5.00 cm3 concentrated perchloric acid). the mixture was agitated thoroughly to ensure adequate mixing, then heated on a hot plate until a clear digest appear. the digest was allowed to cool and filtered quantitatively into a 100 cm3 volumetric flask. the filtrate was made up to the 100 cm3 mark, transferred to a plastic bottle and aspirated into the machine for trace metal analysis. determination of anti-nutrients determination of total alkaloids this was accomplished utilizing the method described by aoac (2005). 0.50 g of the sample was dissolved in 5.00 cm3 of 96% ethanol and 5.00 cm3 of 20% h2so4 (1:1) and the resulting solution was filtered. 1.00 cm3 of the filtrate was added to 5.00 cm3 of 60% h2so4 and allowed to stand for 5 minutes. after that, 5.00cm3 of 0.5% formaldehyde was added and allowed to stand for 3 hours. the reading was taken at an absorbance length of 565 nm using an ultra-violet (uv) spectrophotometer. the extinction coefficient of vincristine (e296, ethanol {etoh} = 15136 m¹־cm¹־) was chosen as a reference alkaloid (aoac, 2005). determination of saponins this was accomplished utilizing the method described by krishnaiah et al. (2009). 0.50 g of the sample was boiled for 4 hours in a 20.00 cm3 of 1 mol/dm-3 hcl solution. after cooling, 50.00 cm3 petroleum ether was added to the filtrate for the ether layer, which was then evaporated to dryness. 5.00 cm3 of acetone ethanol was added to the residue and 0.40 cm3 of each was divided among three test tubes. they were filled with 6.00 cm3 of ferrous sulfate reagent followed by 2.00 cm3 of concentrated h2so4. the absorbance was measured at 490 nm after 10 minutes of complete mixing. the calibration curve was established using standard saponin. determination of tannin jaffe (2003) described that 1.00 g of each sample a and b were dissolved in 10.00 cm3 distilled water and agitated, left to stand for 30 minutes at room temperature. the extract was obtained from each sample godfrey et al. – proximate composition, levels of some essential mineral elements … 11 after centrifugation. in a 50 cm3 volumetric flask, 2.50 cm3 of the supernatant was dispersed. similarly, in a separate 50.00 cm3 flask, 2.50 cm3 of the standard tannic acid solution was dispersed. in each flask, a 1.00 cm3 folin dennis reagent was added, followed by 2.50 cm3 of saturated na2co3 solution. the mixture was then diluted to 50.00 cm3 and incubated for 90 minutes at room temperature. the sample’s absorbance was measured at 250 nm with the reagent blank at zero. the % tannin was calculated using the formula: 𝑇𝑎𝑛𝑛𝑖𝑛 (𝑚𝑔 𝑔) = (𝐴𝑠−𝐴𝑏 )−𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 𝑆𝑙𝑜𝑝𝑒×𝑑×𝑊 × 10⁄ (eqt. 1) where 𝐴𝑠 is the sample absorbance, 𝐴𝑏 is the blank absorbance, 𝑑 is the density of the solution (0.791g/ml), 𝑊 is the weight of the sample in grams and 10 is the aliquot. determination of phytic acids markkar et al. (1993) described the process for determining phytic acid. in a 250 cm3 conical flask, 2.00 g of each sample (a and b) were weighed. each sample was soaked in 100.00 cm3 of 2% concentrated hcl acid for 3 hours in the conical flask before being filtered through a double layer of hardened filter papers. 50.00 cm3 of each filtrate was placed in a 250 cm3 beaker and 100 cm3 of distilled water was added to each to give proper acidity. 10.00 cm3 of 0.3% ammonium thiocyanate solution was added to each solution as an indicator. each solution was titrated with standard iron chloride solution, which contains 0.00195 g iron per cm3. the endpoint color was slightly brownish-yellow which persisted for 5 min. the percentage of phytic acid was calculated using the formula: % 𝑃ℎ𝑦𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 = 𝑦 × 1.19 × 1 (eqt. 2) where 𝑦 is the titre value × 0.00195 determination of cyanides cyanide content was determined by the alkaline picrate method as described by onwuka (2005). here 5.00 g of powdered sample was dissolved in 50.00 cm3 of distilled water in a corked conical flask and the extraction was allowed to stand overnight and filtered. in a corked test tube, 1.00 cm3 of the filtered sample was mixed with 4.00 cm3 alkaline picrate and incubated in a water bath for 5 minutes. the absorbance of the blank containing 1.00 cm3 distilled water and 4 cm3 alkaline picrate solution was also measured at 490 nm after colour development (reddish-brown colour). the cyanide content was extrapolated from a cyanide standard curve prepared from a different concentration of kcn solution containing 5-50 μg cyanide in a 500 cm3 conical flask followed by 25.00 cm3 of 1 mol/dm-3 hcl. it was calculated as: 𝐶𝑦𝑎𝑛𝑖𝑑𝑒 (𝑚𝑔 100𝑔⁄ ) = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒×𝐺𝐹×𝐷𝐹 𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 (eqt. 3) where 𝐺𝐹 is the gradient factor and 𝐷𝐹 is the dilution factor. determination of oxalates the oxalate content of the samples was determined using the titration method described by munro and bassiro (2000). in a 250 cm3 volumetric flask suspended in 190.00 cm3 distilled water, 2.00 g of each sample a and b was inserted. each sample received a 10 cm3 of 6 mol/dm-3 hcl solution, which was digested at 100ºc for 1hour. the samples were then cooled and made up to the 250 cm3 mark of the flask. the samples were filtered and a duplicate portion of 125.00 cm3 of the filtrate was measured into a beaker and 4 drops of methyl red indicator were added, followed by the addition of concentrated nh4oh solution (dropwise) until the solution changes from pink to yellow colour. each portion was then heated to 90ºc, cooled and filtered to remove the precipitate containing ferrous ion. each of the filtrates was again heated to 90ºc and 10.00 cm3 of 5% cacl2 solution was added to each of the samples with consistent stirring. after cooling, the samples were left overnight. the solutions were then centrifuged for 5 minutes at 2500 rpm. the supernatant was decanted and the precipitates completely dissolved in 10.00 cm3 20% h2so4. the total filtrate resulting from the digestion of 2.00 g of each of the samples was made up to 200 cm3. the filtrate was heated to near boiling points in aliquots of 125.00 cm3 and then titrated against 0.05 mol/dm-3 standardized kmno4 solution to a pink colour which persisted for 30 seconds. each sample of the oxalate contents was then calculated using the formula: 𝑂𝑥𝑎𝑙𝑎𝑡𝑒 = 𝑇 ×(𝑉𝑚𝑒)(𝐷𝑓)×105 (𝑀𝐸)×𝑀𝑓 (eqt. 4) where 𝑇 is the titre value of kmno4, 𝑉𝑚𝑒 is the volume-mass equivalent, 𝐷𝑓 is the dilution factor, 𝑀𝐸 is the molar equivalent of kmno4 in oxalate and 𝑀𝑓 is the mass of the sample. statistical analysis the obtained results were subjected to statistical analysis using mean standard deviation and analysis of variance (anova) as described by duncan’s multiple range test to determine the level of significance between different samples and significance was set at p ≤ 0.05. 12 biology, medicine, & natural product chemistry 12 (1), 2023: 9-16 results and discussion table 1. proximate composition of selected yam species (%). yam species ash content moisture content crude fat crude fibre crude protein carbohydrate d. cayenensis 3.09±0.19a 4.51±0.07d 16.31±0.30j 8.30±0.40b 10.14±0.40h 57.65±0.10b d. dumenturom 5.06±0.80c 3.51±0.01c 3.56±0.03a 6.54±0.01a 12.29±0.01f 69.04±0.10g d. rotundata 8.05±0.05f 5.60±0.06f 12.46±0.10h 13.11±0.10e 2.15±0.03b 58.63±0.08d values are means ± standard deviation of triplicate analysis. moisture content the moisture content of the various yam species ranged from 3.51±0.01c % for dioscorea dumenturom to 5.60±0.06f % for dioscorea rotundata. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea rotundata having the highest moisture content. however, these values are comparable to literature values as reported by oko and famurewa (2014) that ranged from 2.1% to 9.2% for dioscorea dumenturom and dioscorea alata respectively and lower than the research carried out by anthony et al. (2014) which reported its moisture content within the range of 30.51±0.06d % to 37.90±0.08a % for xanthosoma maff and dioscorea cayenensis respectively. as such it could be said that dioscorea dumenturom has a higher resistance to deterioration and longer shelf life than any of the selected yam species. crude fibre content the crude fibre content ranged from 6.54±0.01a % for dioscorea dumenturom to 13.11±0.10d % for dioscorea rotundata. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea rotundata having the highest crude fibre content. however, these results can be compared with reports by afiukwa et al. (2013) that ranged from 6.01±0.04b % to 13.03±0.80a % for varieties of dioscorea dumenturom and is different from reports by oko and famurewa (2014) that ranged from 3.31% to 3.53% for their dioscorea alata species. studies have shown that an increase in fiber consumption in foods reduces the incidence of obesity, cardiovascular disease, diabetes and digestive disorders (turner, 2014). ash content ash contents of the yam varieties ranged from 3.09±0.19a % for dioscorea cayenesis to 8.05±0.05f % for dioscorea rotundata. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea rotundata showing the highest ash content. however, these results were different from reports by sorh et al. (2015) that ranged from 1.64±0.03a % to 1.78±0.03a % for varieties of dioscorea alata. the ash content is an indication of the extent of mineral stuffing in the dioscorea species (akonor et al., 2017). as such, dioscorea rotundata is stuffed with more minerals than the other yam tubers analysed. crude protein content the crude protein content showed a significant difference (p≤0.05) between the yam varieties. it ranged from 2.15±0.03b % for dioscorea rotundata to 12.29±0.01f % for dioscorea dumenturom. however, dioscorea dumenturom proved to be the tuber variety with the highest protein content. nevertheless, the result can be compared with reports by ojinnaka et al. (2017) that showed 2.43±0.11b % for dioscorea bubilfera and in contrast with the report by ukom et al. (2014) that showed crude protein of dioscorea dumenturom to be 69.15±4.49b %. as such, this shows that dioscorea dumenturom is the richest in protein among the analysed dioscorea species. crude fat content the fat content in these analysed varieties of yam tubers ranged from 3.56±0.03a % for dioscorea dumenturom to 16.31±0.30j % for dioscorea cayennesis. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea cayenensis showing the highest fat content. however, these results contradict ukom which showed 4.4±1.91a % for dioscorea cayenensis. that dietary fat supplies most of the energy required by man suggests that dioscorea cayenensis is a better source of calories than other dioscorea species analysed. carbohydrate content the carbohydrate content of the analysed yam varieties was significantly different (p≤0.05) and ranged from 57.65±0.10b % for dioscorea cayenensis to 69.04±0.10g % for dioscorea dumenturom. despite the huge carbohydrate content contained by the dioscorea species, dioscorea dumenturom appeared to be more than dioscorea cayenensis and dioscorea rotundata. however, these values are comparable to literature by ukpabi and akobundu (2014), which had 78.32±0.29 % for dioscorea dumenturom and in contrast with frank and kingsley (2014) that ranged from 24.25±0.62b % for dioscorea alata to 32.03±0.89c % dioscorea rotundata. carbohydrates are considered as the primary source of energy for all organisms, playing a nutritional as well as structural role (ojinnaka et al., 2017). godfrey et al. – proximate composition, levels of some essential mineral elements … 13 table 2. mineral concentration of selected yam species (mg/100g). yam samples ca fe mg na k p d. cayenensis 67.12±0.11b 3.09±0.01a 74.38±0.03d 24.10±0.14b 50.06±0.09d 131.51±0.05b d. dumenturom 190.57±0.01i 7.37±0.04i 100.22±0.03i 32.05±0.07e 72.23±0.37g 193.11±0.01b d. rotundata 60.60±0.17a 3.18±0.03a 60.85±0.21b 31.10±0.14g 42.08±0.11b 117.20±0.01b values are means ± standard deviation of triplicate analysis. iron concentration minerals are an important component of diet because of their physiological and metabolic function in the body. table 2 shows iron concentrations that ranged from 3.09±0.01a mg/100g for dioscorea cayenensis to 7.37±0.04i mg/100g for dioscorea dumenturom samples. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea dumenturom having the highest iron concentration. however, this result was low when compared to results reported by mergedus et al. (2015) which ranged from 10.10±0.01a mg/100g to 11.60±0.01a mg/100g for cultivars of colocasia esculenta (cocoyam). iron is a major component of hemoglobin, a type of protein in red blood cells that carries oxygen from the lungs to all parts of the body (mergedus et al., 2015). the recommended dietary allowance for iron is 13.7–15.1 mg/day for children and 17.0–18.9 mg/day for adults (who, 2014). sodium concentration the sodium concentration in this study ranged from 24.10±0.14b mg/100g for dioscorea cayenensis to 32.05±0.07e mg/100g for dioscorea dumenturom. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea dumenturom showing the highest sodium concentration. however, the sodium concentration was comparable to the report by oko and famurewa (2015) which had 24.84±0.37a mg/100g and 21.06±0.77b mg/100g for dioscorea vilgaris and dioscorea villosa respectively. sodium as a macronutrient plays an important role in various metabolic processes including excitation and transmission of nerve impulses during action (olajumoke et al., 2014). the recommended daily dietary intake for sodium is 10 mg/day for adult males and below 15 mg/day for females (who, 2014). potassium concentration potassium is a macro-nutrient required by both plants and animals and is involved in various metabolisms. in this study, the concentration of potassium ranged from 42.08±0.11b mg/100g for dioscorea rotundata to 72.23±0.37g mg/100g for dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea dumenturom having the highest potassium concentration. however, these values were lower when compared with reports by ellong et al. (2014) that ranged from 338.00±59.29 mg/100g to 407.04±168.36 mg/100g for varieties of sweet potato (ipomoea batatas). potassium is important in the regulation of heartbeat, neurotransmission, signal and immune response and water balance in the body (olajumoke et al., 2014). the recommended dietary intake of potassium is 2000 mg/day for adults and 1000 mg/day for children (who, 2014). calcium concentration the calcium content in this study ranged from 60.60±0.17a mg/100g for dioscorea rotundata to 190.57±0.01i mg/100g for dioscorea dumenturom. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea dumenturom showing the highest calcium concentration. however, this concentration was low when compared to reports by sorh et al. (2015) which ranged from 150±14.50ab mg/100g to 185±18.14d mg/100g for varieties of dioscorea alata. calcium is necessary for blood clotting, muscle contraction, neurological function, bone and teeth formation (trailokya et al., 2017). the recommended dietary intake of calcium is 500–800 mg/day for children and 1000 mg for adults (who, 2014). magnesium concentration magnesium was also present in small quantities in the range of 60.85±0.21b mg/100g for dioscorea rotundata to 100.22±0.03i mg/100g for dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea dumenturom having the highest magnesium concentration. however, its results were comparable to reports by cyrile et al. (2014) that showed 53.70±0.32b mg/100g for its dioscorea dumenturom species. magnesium is involved in muscle degeneration, growth retardation, alopecia, dermatitis, immunologic dysfunction, poor spermato-genesis, congenital abnormalities and bleeding disorders among other things (mergedus et al., 2015). the recommended dietary intake of magnesium is 80–320 mg/day (who, 2014). phosphorous concentration phosphorus, together with calcium, helps to strengthen bones and teeth, particularly in children and breastfeeding mothers. the phosphorus content of the samples analysed ranged from 117.20±0.01b mg/100g for dioscorea rotundata to 193.11±0.01c mg/100g for 14 biology, medicine, & natural product chemistry 12 (1), 2023: 9-16 dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea dumenturom having the highest phosphorous concentration. however, these values are low when compared to those of 410 mg/100g of sweet potato reported by sorh et al. (2015). furthermore, phosphorous appeared to be the most abundant mineral in all the yam samples analysed. the recommended dietary allowance for both children and adults is 800 mg/day (who, 2014). table 3. anti-nutritional contents of the selected yam species (mg/100g). yam species tannin phytate cyanide alkaloids oxalate d. cayenensis 30.06±0.30d 49.44±0.30h 1.70±0.06d 8.35±0.10b 4.86±0.08d d. dumenturom 72.99±0.50j 30.00±0.20f 3.38±0.06e 24.17±2.70g 3.67±0.30c d. rotundata 62.00±0.50h 15.06±0.30d 1.54±0.06c 11.58±0.20d 3.19±0.90bc values are means ± standard deviation of triplicate analysis. tannin content tannins have been reported to form complexes with proteins and impair their digestibility and palatability. however, cooking is known to diminish the contents in foods (lewu et al., 2010). tannin concentration in the yam samples studied ranged from 30.06±0.30d mg/100g for dioscorea cayenensis to 72.99±0.50j mg/100g for dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea cayenensis having the least tannin concentration. however, these values are relatively lower than those of 20-255 mg/100g reported on various under-utilized dioscorea tubers (arinathan et al., 2009) and higher than reports by polycarp et al. (2012) that ranged from 4.56±0.01a to 19.23±0.03b mg/100g for different dioscorea species. tannin has been shown to decrease the activity of several enzymes including trypsin, amylase and lipase as well as interfere with the absorption of dietary iron (rao and desothe, 2008). the total acceptable tannin intake for a man is 560 mg/kg (stephene, 2004). as such the tannin contents in these yam species are low and within permissible limits and thus, cannot be harmful to consumers (stephene, 2004). phytate content the phytate contents of the yam tuber ranged from 15.06±0.30d mg/100g for dioscorea rotundata to 49.44±0.30h mg/100g for dioscorea cayenensis samples. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea rotundata having the least phytate concentration. however, these values were higher when compared to the report by otoo et al. (2008) that showed 2.60±0.20a mg/100g for its dioscorea rotundata cultivars and lower than sweet potato which contained 119.98±0.01a mg/100g (akaninwor, 2004). the issue with phytate in diets is that it can bind some essential mineral nutrients in the digestive tract, leading to mineral deficiencies. there is no particular recommended daily allowance for phytic acid as it differs from country to country (bello et al., 2008). oxalate content comparatively, the oxalate content of the various yam species analyzed ranged from 3.19±0.90bc mg/100g for dioscorea rotundata to 4.86±0.08d mg/100g for dioscorea cayenensis samples. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea rotundata having the least oxalate concentration. however, these values are lower than reports according to princewill and ibeji (2012) that had 12.60a mg/100g for their dioscorea bubilfera specie and higher than 0.48±0.01a, 0.50±0.03a mg/100g for dioscorea rotundata and dioscorea alata respectively according to reports by afoakwa et al. (2012). oxalic acid and oxalate are found naturally in plants but they have little or no benefit for human health. though high levels in diet irritate tissues and the digestive system notably the stomach and kidney (ogbuagu, 2008). soluble oxalate is known to be poisonous at high concentrations, particularly above 3mg/kg (norwood and fox, 1994). the oxalates levels found in this study indicate that while the analysed yam tubers were slightly above the maximum permitted limit, they lose most of their toxicity when treated (boiled) and so cannot be consumed raw. cyanide content the cyanide contents ranged from 1.54±0.06c mg/100g for dioscorea rotundata to 3.97±0.06e mg/100g for dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea rotundata having the least cyanide concentration. however, these values are low when compared to reports by afiukwa et al. (2013) that showed 26.687±0.081a and 21.827±0.058b mg/100g for dioscorea villosa species (okpura and ighobe) respectively. it is also higher than reports by umoh (2013) that had 0.22±0.02a to 0.53±0.01b mg/100g for raw and processed false yam flour. the permissible limits for cyanide are 0.5-3.5 mg/kg which indicates that the level of the cyanide in the samples is above the acceptable range for human consumption and as such must not be consumed in their raw state (mohammed et al., 2013). godfrey et al. – proximate composition, levels of some essential mineral elements … 15 alkaloid content the observed values for the alkaloid samples ranged between 8.35±0.10b mg/100g for dioscorea cayenensis to 24.17±2.70g mg/100g for dioscorea dumenturom. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea cayenensis having the least alkaloid concentration. however, the values obtained for the alkaloids contents of these yam species were higher when compared to some varieties of dioscorea cayenensis that ranged from 0.38±0.12 mg/100g to 0.68±0.02 mg/100g and 1.68±0.01 mg/100g for dioscorea rotundata (okwu and ndu, 2006) and lower than reports by ogbuagu (2008) that contained 30.62±0.03a and 32.46±0.01b mg/100g for different species of dioscorea dumenturom. because of their effect on the nervous system, electrochemical transmission and disruption of the cell membrane in the gastrointestinal tract, alkaloids are considered to be antinutrients (friedman, 2001). however, human lethal dosages range between 3-6 mg/kg body weight and a dose above 3 mg/kg is usually considered toxic (habtamu and ratta, 2014). the good news is that it loses most of its toxicity when treated or processed (cooked or boiled). conclusions this study provided vital information on the proximate composition, mineral content and anti-nutritional constituents of the selected yam tuber species (dioscorea dumenturom, dioscorea rotundata, dioscorea cayenensis). this research observed that these analysed dioscorea species were rich in protein, carbohydrate, fibre, and fat and had a high shelf-life. however, the anti-nutritional constituents of these yam species, if properly treated or processed (cooked or boiled), tend to lose more than 95% of their toxicity and can be exploited as good food sources. furthermore, all the species of yam analysed contained an appreciable amount of macro and micro-nutrients which are essential to human nutrition. specifically, dioscorea dumenturom had the highest concentrations of na, ca, mg, fe, k, and p. competing interests: the authors declare that there are no competing interests. references afiukwa, j.n., okereke, c.o., & odo, m.o. 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(2014). children fund and clinical management of acute diarrhea. who/ unicef joint statement. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 329-334 | doi: 10.14421/biomedich.2023.121.329-334 issn 2540-9328 (online) phytochemical, antibacterial and antioxidant activities of schefflera elliptica leaves i gede yoga ayuning kirtanayasa1, anak agung gede indraningrat2,*, i putu candra1 1food science and technology study program, faculty of agriculture, warmadewa university 2departement of microbiology and parasitology, faculty of medicine and health sciences, warmadewa university jl. terompong no 24 denpasar 80235, tel. +62 361 240727, indonesia. corresponding author* indraningrat@warmadewa.ac.id abstract schefflera elliptica or locally called kayu tulak, is one of the balinese herbal plants that have traditionally be used to reject (tulak) negative influences that exist in the human body. althoug, s. elliptica has been routinely used as a part of a ritual in bali, only a limited study has been reported on its bioactivity. this study was designed to analyze the phytochemical content, antibacterial activity, and antioxidant activity of s. elliptica. in this study, simplisia of s. elliptica was extracted using n-hexane and ethyl acetate solvents, then the viscous extracts of the two solvents were carried out for phytochemical tests, antibacterial activity tests with the kirby-bauer method and antioxidant activity tests with based on dpph method. phytochemical screening showed that the n-hexane extract contains active compounds in the form of phenols and steroids while ethyl acetate contains active compounds in the form of phenols, tannins, and steroids. antibacterial screening showed ethyl acetate extract of s. elliptica displayed a diameter zone of inhibition of 10.72±0.71 mm against staphylococcus aureus, 12.17±2.80 mm against streptococcus mutans, 12.40±1.65 mm against escherichia coli and 15.20±2.44 mm against klebsiella pneumoniae. the dpph analysis showed percentages of 61.17% and 67.42% from n-hexane and ethyl acetate extracts respectively, which indicated the antioxidant properties of s. elliptica. overall, this research provides a preliminary report on the bioactivity potential of s. elliptica mainly in term of antibacterial and antioxidant properties which open up possibilities for future drug development. keywords: antibacterial; antioxidant; phytochemical; schefflera elliptica. introduction indonesia is one of the countries with high plant diversity. the biological diversity of flora in indonesia, especially seeded plant species reaches 30,000–40,000 types, or equivalent to 15.5% of the total number of plants in the world (widjaja et al., 2014). furthermore, more than 2,039 plant species are categorized as medicinal plants (zuhud, 2009). among many islands in indonesia, bali is one of the islands with rich biological diversity and balinese people have applied herbal plants for traditional medicine (sutomo and iryadi, 2019). in general, information about traditional balinese medicine has been recorded in a manuscript called lontar usada bali, which explains the function of each herbal function and the procedure to use it (oktavia et al., 2017). schefflera elliptica or locally called kayu tulak is one of the types of local balinese plants listed in usada tiwang (arsana et al., 2020). this plant is commonly found in the traditional balinese offerings called banten byakala has a function to reject (tulak) any impurities or negative influences that exist in the human body (puspa et al., 2019). more specifically, the leaf part of kayu tulak is used as a repellent for disasters or bad luck in human body (hanum, 2011). schefflera elliptica leaves can also traditionally be used as a remedy for skin diseases and fractures (sivaperuman et al., 2018). methanol and ethyl acetate extracts of s. elliptica leaves have also been reported to have antibacterial activity against staphylococcus aureus (purwantoro et al., 2009). however, apart from antibacterial against s. aureus, no other studies have been published on the antibacterial activities of s. elliptica extracts against other gram-positive and gramnegative bacterial species. in addition, rather limited information is available on other aspects of the bioactivities of s. elliptica e.g. phytochemicals and antioxidant activities. this present study aimed to assess the bioactivities s. elliptica leaves focusing on three main aspects namely phytochemical, antibacterial, and antioxidant bioactivities. the obtained information is expected to give further knowledge on the bioactivities of s. elliptica which could be the basis to develop the ethnomedicine and drug development purposes of the plant. manuscript received: 04 march, 2023. revision accepted: 24 march, 2023. published: 01 april, 2023. https://doi.org/10.14421/biomedich.2023.121.329-334 330 biology, medicine, & natural product chemistry 12 (1), 2023: 329-334 methods sample collection and determination leaves samples of s. elliptica plant were obtained from gerih village, bali, indonesia on february 2022 (figure 1). samples were selected by taking 3-5 leaves of s. elliptica which were calculated from the leaflets, provided that it was in full bloom, fresh, without hollow, and free of insect infections. plant determination was performed by sending fresh and dried vouchers to the characterization laboratory of the botanical garden "eka karya" bali – national research and innovation agency (brin), candikuning, tabanan, bali. the purpose of plant determination is to obtain a clear identity of the plant under study and avoid errors in collecting the main research material. sample preparation and extraction two kilograms of s. elliptica leaves were washed in running tap water to remove debris. subsequently, leaves samples were drained, and dried using an oven at 40oc. the dried simplisia was dried and were sorted by separating foreign objects that occurred during drying. the dried simplisia was turned into powder using a blender and was sieved with a 60-mesh sieve. finally, the powder was stored in a clean glass jar to prevent contamination and other impurities before extraction. two types of crude extracts were prepared using two different chemical solvents namely ethyl acetate (smartlab) dan n-hexane (merck). for each of solvent, 100 gram of dry powder leaves of s. elliptica was macerated with a ratio of 1:5 (w/v) (wijaya and indraningrat, 2021). maceration was carried out for 24 hours and each mixture was stirred every five minutes with a time span of six hours. for each of the solvent, remaceration was carried out after 24 hours. subsequently, after maceration and remaceration were completed, each macerate was separated from the residue using a vacuum filter, followed by evaporation in a rotary evaporator (ika rv 10, germany) at a speed of 100 rpm at a temperature of 40ºc. figure 1. s. elliptica plant (left), and s. elliptica leaves (right). phytochemical screening each the ethyl acetate and n-hexane s. elliptica crude extracts was tested to detect the presence of a group of compounds based on the following methods. phenol one ml of each s. elliptica extract was transferred into a test tube followed by the addition of 2 – 3 drops of iron (iii) chloride (fecl3) 5%. the presence of phenols was indicated by a blue-black color (friany et al., 2017). flavonoids one ml of each s. elliptica extract was transferred into a test tube. subsequently, 2 mg of magnesium powder and 3 drops of concentrated hcl were added to the tube. the mixture was shaken and the formation of a red, yellow, or orange color on the solution indicated the presence of flavonoids (purwati et al., 2017). tannins one ml of each s. elliptica extract was mixed with a few drops of 10% iron (iii) chloride (fecl3) solution. a dark blue or greenish-black color indicates the presence of tannins in a solution (baud et al., 2014). alkaloids two ml of each s. elliptica extract was mixed with 3 drops of concentrated hcl and 5 drops of mayer reagent. a white precipitate indicates that a sample contains alkaloids (ergina and pursitasari, 2014). steroid/terpenoid two ml of each s. elliptica extract were mixed with liebermann burchard reagent, a mixture of concentrated hcl and concentrated h2so4. positive results were indicated by the presence of a red-orange color for triterpenoids and blue for steroid (sangi et al., 2008; ergina and pursitasari, 2014). saponin two ml of each s. elliptica extract were mixed with 10 ml of aqua dest. the mixture was shaken for 1 minute and subsequently, two drops of hcl 1 n was added. the presence of a stable foam for approximately 7 minutes indicated that the mixture contains saponins (mondong et al., 2015). antibacterial activity screening for each ethyl acetate and n-hexane extract, paper discs with a diameter of 6 mm were prepared. each paper disc was soaked into a viscous extract and allowed to dry for 15 minutes until the extract was evenly absorbed. paper discs containing extracts were transferred into luria bertani agar which already contained one of the following test bacteria namely staphylococcus aureus atcc 25923, streptococcus mutans fncc 0405, escherichia coli atcc 25922 and klebsiella pneumoniae atcc 700603. triplicate paper discs of kirtanayasa et al. – phytochemical, antibacterial and antioxidant activities of … 331 each extract were used for each of the tested bacteria. nalidixic acid (oxoid) paper discs were used as a positive control, while ethyl acetate and n-hexane were used as negative controls. antibacterial activities were calculated based on the triplicate average zone of inhibition (zoi) that were formed on each of the lawn bacterial species. antioxidant assay the radical inhibition activity of the sample was carried out based on its inhibition against free radicals 1, 1diphenyl-2-picrylhydrazyl (dpph) (pangestuty, 2016). sample absorbance was measured with a uv-vis spectrophotometer. the magnitude of antioxidant activity was measured by the following formula: antioxidant activity (aa%) = 𝐴𝑏𝑠 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑥 100 result and discussion phytochemical screening results phytochemical screening is a preliminary test and qualitative analysis that can be used as initial information about a group of compounds present in a plant. in this research, phytochemical screening of n-hexane and ethyl acetate extracts of s. elliptica were summarized in table 1. in general, there were discrepancies in the presence of specific groups of compounds by comparing the phytochemical constituents of each extract. alkaloids tests showed clean deposits were formed in both extracts. this result indicated the absence of alkaloids in ethyl acetate and n-hexane. a positive result was indicated by the presence of white deposit (ergina and pursitasari, 2014) table 1. phytochemical composition of s. elliptica extracts. test extract n-hexane ethyl acetate alkaloid flavonoid tannin + phenol + + saponin steroid + + terpenoid the presence of flavonoids in n-hexane and ethyl acetate extracts was tested using two methods, namely by adding concentrated magnesium (mg) + hydrochloric acid (hcl) and using h2so4. positive signs were indicated by a change of color in the mixture to red or orange. in this study, n-hexane and ethyl acetate samples did not change color to red or orange, so it was concluded that flavonoids were absent in both extracts (friany et al., 2017; purwati et al., 2017). a positive test of tannin was indicated by the formation of blackish-green color (baud et al., 2014). the occurrence of this green color change is due to the reaction between fe metals and tannins to form complex compounds due to the presence of coordination covalent bonds between metal ions or atoms and non-metallic atoms (effendy, 2007). our result indicated that tannin was present in ethyl acetate extract based on the color change that was observed. meanwhile, no color change was observed from the n-hexane extract. this could happen mainly because n-hexane is a solvent that has non-polar properties while tannins are polar compounds and tend to dissolve in polar or semi polar solvents (muthmainnah, 2017). the presence of phenol was screened by adding 10% of fecl3 reagent and positive signs were indicated by a color change to blackish-green (friany et al., 2017). color changes to green-black were observed from both s. elliptica extracts to indicate both extracts contained phenol compounds. the discoloration of n-hexane and ethyl acetate extracts of s. elliptica was because phenol compounds can dissolve in polar solvents as well as nonpolar solvents (wongso, 2014). in the saponin test, n-hexane and ethyl acetate extracts of s. elliptica were heated followed by addition of 10 ml of aquadest. the mixture was shaken strongly and a positive sign was indicated by the presence of a stable foam of 1-10 cm high which was stable after the addition of 1 drop of hcl 1n (mondong et al., 2015). the appearance of foam was present because saponin compounds have physical properties that are easily hydrolyzed in water so that saponin compounds will cause foam when shaken. in both n-hexane and ethyl acetate extracts of s. elliptica the saponin test did not show positive results because the foam formed after shaking only lasted for a few seconds. the presence of steroids and terpenoids were screened by the addition of liebermann-burchard reagents. the positive result for steroid compounds was indicated by the color change to blue or purple while for triterpenoid compounds was indicated by a brownish-red color (sangi et al., 2008; ergina and pursitasari, 2014). the test results showed that n-hexane and ethyl acetate of s. elliptica extracts underwent a blue color change so that they were positive for containing steroid compounds. antibacterial activities screening the antibacterial screening showed that the crude extractof n-hexane displayed lower antibacterial activity against test bacteria compared to the crude extract of ethyl acetate (figure 2, table 2). ethyl acetate extract has an average zoi of 10.72±0.71 mm against s. aureus. ethyl acetate extract had an average zoi of 12.17±2.80 mm against s. mutans. meanwhile, in the ethyl acetate extract has an average zoi of 12.40±1.65 mm against e. coli and an average zoi of 15.20±2.44 mm against k. pneumoniae. 332 biology, medicine, & natural product chemistry 12 (1), 2023: 329-334 figure 2. antibacterial activities of n-hexane and ethyl acetate extract againts bacteria. annotations: h = n-hexane, e= ethyl acetate; sa = staphylococcus aureus atcc 25923; sm = streptococcus mutans fncc 0405; ec= escherichia coli atcc 25922; kp=klebsiella pneumoniae atcc 700603 table 2. antibacterial activities of s. elliptica extract against testing bacteria. diameter zone of inhibition of each treatment (ethyl acetate, nhexane, positive and negative controls) were calculated from triplicate samples. bacterial strains samples solvents zoi (mm) s. aureus s. elliptica n-hexane ethyl acetate 10,72±0,71 + control nalidixic acid 15,34±0,03 control n-hexane ethyl acetate s. mutans s. elliptica n-hexane ethyl acetate 12,17±2,80 + control nalidixic acid 16,29±0,12 control n-hexane ethyl acetate e. coli s. elliptica n-hexane ethyl acetate 12,40±1,65 + control nalidixic acid 16,58±0,04 control n-hexane ethyl acetate k. pneumoniae s. elliptica n-hexane ethyl acetate 15,20±2,44 + control nalidixic acid 15,50±0,13 control n-hexane ethyl acetate when compared to the results of the study of purwantoro et al, (2009) which stated that the ethyl acetate extract of s. elliptica at a concentration of 50 μg/ml, 100 μg/ml, and 200 μg/ml had a zoi with an average of 7.5 mm against s. aureus and e. coli bacteria, the crude extract of ethyl acetate with a concentration of 100% had a bigger zoi against s. aureus and e. coli bacteria. the difference in inhibitory power can occur due to the difference in the concentration of the extract where the higher the concentration of the extract, the higher the inhibitory power will be. the same thing is also stated by (zuhud et al., 2001) who mentioned that the higher the concentration of the extract, the more the amount of antimicrobial compounds released will be, thus facilitating the penetration of compounds into cells. davis and stout, (1971) classified the diameter zone of inhibition into four categories, namely weak, medium, strong, and very strong. zones of inhibition with a diameter of ≤5 mm are categorized as weak, 6-10 mm are categorized as medium, 11-20 mm are categorized as strong, and above 20 mm are categorized as very strong. based on these categories, the zoi formed by ethyl acetate extracts of s. elliptica could be considered as a strong activity. however, the observed antibacterial activity was still lower compared to positive control nalidixic acids. nevertheless, the observed antibacterial activity from ethyl acetate extracts provides a valuable insight on bioactive compounds that present in s. elliptica leaves. the presence of antibacterial activities in the ethyl acetate extract of s. elliptica may occur due to the content of its secondary metabolites. the mode of action of phenol compounds is in general by denaturing cell proteins. hydrogen bonds formed between phenol compounds could damage protein layers in cell structures (bontjura et al., 2015) meanwhile, tannin was also reported to display have antibacterial activity against gram-positive and gram-negative bacteria by entering the cell wall (kaczmarek, 2020). tannin forms hydrogen bonds with bacterial cell’s proteins and subsequently hydrogen bonds formed between tannins and proteins will disrupt bacterial cell walls (mailoa et al., 2014). antibacterial screening showed that the zone of inhibition of ethyl acetate extract of s. elliptica against gram-positive bacteria (s. aureus and s. mutans) was smaller compared to gram-negative bacteria (e. coli and k. pneumoniae). such discrepancies could probably happen because the cell wall of gram-negative bacteria is thinner compared to gram-positive bacteria so the protein structure in the cell wall of gram-negative bacteria was damaged by the presence of tannin compounds (mailoa et al., 2014). furthermore, grampositive bacteria have a thick and rigid peptidoglycan layer while gram-negative bacteria have a thinner peptidoglycan layer (sudarmi et al., 2017). the absence of zoi observed in n-hexane extracts even though qualitatively the extract contained phenol compounds could be influenced by differences of the polarity of solvents in extraction which affecting the total content of bioactive compounds in the extract (santoso et al., 2012). the total content of phenol compounds according to hidayah et al., (2017) has an influence on antibacterial activity as the higher the levels of oxidized phenol compounds, the stronger the antibacterial activity kirtanayasa et al. – phytochemical, antibacterial and antioxidant activities of … 333 will be. the extract of n-hexane s. elliptica did not display a zone of inhibition power even though it qualitatively contained steroid compounds. such a condition could happen because lipids have large sizes of molecules which interfere with the diffusion process and consequently, n-hexane extracts are unable to inhibit bacterial growth (naufalin et al., 2005). antioxidant activities test results antioxidant activities were analyzed based on the 2,2diphenyl-1picrilhydrazyl (dpph) method. in theory, the higher the concentration of antioxidant activity added to the dpph solution, the more the absorbance value will decrease (sapri et al., 2013). the absorbance value of nhexane and ethyl acetate s.elliptica samples (table 3) showed that the antioxidant activities (aa) of n-hexane extract was 61.17% while for ethyl acetate extract was 67.42%. according to rahmawati (2004) in (parwata et al., 2009) the inhibition value of 0% means that an extract has no antioxidant activity and the inhibition value of 100% means total dampening. an extract could be classified as an active antioxidant when its inhibition percentage is more than or equal to 50%. therefore, based on this criteria, crude extracts of n-hexane and ethyl acetate of s. elliptica are classified as active antioxidants. table 3. antioxidant percentages of s. elliptica crude extracts. sample crude extracts concentrations aa% s. elliptica n-heksan 1000 ppm 61,17 etil asetat 1000 ppm 67,42 factors that affect the absorbance are the type of solvent, the ph of the solution, the temperature, the high concentration of the solution, and the presence of a disruptive substance. the amount of antioxidant activity of n-hexane and ethyl acetate extracts from s. elliptica is due to the content of secondary metabolites such as phenols, steroids, and tannins in the extract. tannins are compounds composed of polyphenols that have free radical capture activity. the more tannin content contained in the extract, the more antioxidant activity (malangngi et al., 2012). according to (amarowicz, 2007) tannins not only function as primary antioxidants but also function as secondary antioxidants. phenolic compounds have the ability to contribute hydrogen atoms or electrons to free radicals. this process converts phenols into phenoxyl radicals that can stabilize themselves so that there is no radical formation reaction (pangestuty, 2016; diniyah and lee, 2020). conclusions in conclusion, this study confirmed antibacterial and antioxidant activities from s. elliptica crude extracts. in addition, s. elliptica leaves contain phytochemicals in the form of phenol compounds, tannins, and steroids. the selection of solvents seems to play an important role in extracting bioactive compounds from s. elliptica leaves. in terms of antibacterial activity, ethyl acetate which is a semipolar solvent seems more suitable to extract the active antibacterial substances compared to n-hexane which is a non-polar solvent. further studies should be focused to explore the ideal solvent for chemical extraction. comparison of polar, semipolar, and nonpolar solvents for extraction of s. elliptica should also be done to provide an ideal comparison of bioactivities. in addition, antibacterial screening should also be expanded against multi-drug resistance bacteria and fungi. further studies should also explore other aspects of bioactivity screenings such as cytotoxicity, anti-larvicide, and anticancer tests of s. elliptica to elucidate possible untapped bioactivities of the plant. acknowledgments: the author would like to thank the research unit of the faculty of medicine and health sciences at the university of warmadewa denpasar-bali for providing a research grant to perform this research under grant number: 227/unwar/fkik/unitpenelitian/pd-13/ix/2022. competing interests: the authors declare that there 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(2001) ‘aktifitas antimikroba ekstrak kedawung (parkia roxburghii g. don) terhadap bakteri patogen’, jurnal teknologi dan industri pangan, pp. 6–12 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 225-231 | doi: 10.14421/biomedich.2025.141.225-231 issn 2540-9328 (online) effectiveness test of melon fruit peel ethanol extract cream (cucumis melo) on incision wound healing of white rats (rattus norvegicus) husna rizki, harlita department of biology education, faculty of teacher training and education, sebelas maret university, jl. ir. sutami 36 kentingan, jebres, surakarta, central java 57126, tel. 0271-646994, fax. 0271-646655, indonesia. corresponding author* harlita@staff.uns.ac.id manuscript received: 03 february, 2025. revision accepted: 20 may, 2025. published: 19 june, 2025. abstract incision wounds are a type of open wound that requires treatment to prevent infection and accelerate healing. using natural ingredientbased creams has become a more practical alternative to conventional ointments. melon rind (cucumis melo) contains active compounds such as tannins, flavonoids, and phenols that have anti-inflammatory and antimicrobial properties, potentially accelerating wound healing and reducing the risk of infection. this study aims to test the effectiveness of melon rind ethanol extract cream on incision wound healing in white rats (rattus norvegicus). the study used an experimental method with three treatment groups: control (bacitracin), 15% melon peel extract cream (p1), and 25% melon peel extract cream (p2). mice were adapted for 7 days before the treatment test. the cream was applied three times a day for 14 days, and wound diameter and body weight were observed every two days. observation data were analysed using one way anova test version 26.0. the results showed that melon rind extract cream had physical characteristics that met the standards. anova and lsd tests on wound diameter showed significant differences between control and treatment, although there was no significant difference between p1 and p2. meanwhile, the anova and lsd test results on body weight showed significant differences between groups. group p2 showed a steady increase in body weight, indicating the safety of the cream. keywords: experiment; melon fruit peel (cucumis melo); cream; incision wound; white rat (rattus norvegicus). introduction an incision wound is a wound that occurs when a sharp object is cut. wounds occur when the continuity of anatomical structures in body tissues is disrupted. this damage can vary from as mild as damage to the epithelial layer of the skin to involving deeper tissues, including fat, muscle, bone, and other components such as tendons, blood vessels, and nerves. this condition is usually caused by traumatized (prajayanti et al., 2024). the characteristics of incision wounds include open wounds, pain, and the length of the wound is greater than the depth of the wound. incision wounds are a type of open wound that requires optimal treatment to prevent infection and accelerate the healing process. in medicine, ointments are often used as topical preparations to treat wounds. however, ointments have several drawbacks, such as their oily texture that leaves a sticky feeling and is difficult to clean from the skin (rosyiedi, 2011). the development of pharmaceutical technology has encouraged the use of natural extract-based creams as a more effective alternative. cams generally have a lighter consistency and are less viscous than ointments. creams are easy to spread on the skin so that it is easy to use, easy to clean because it is not greasy, and the cream penetrates faster into the skin. therefore, creams are currently more favoured over ointment preparations (suena et al., 2022). natural extract-based creams often contain antimicrobial and anti-inflammatory compounds, which can protect wounds from infection and improve recovery outcomes (purba et al., 2024). this makes creams a superior modern solution in the management of incision wounds one natural ingredients that has the potential to be developed in making cream is melon rind. melon rind is often considered as waste or animal food (fajrin et al., 2024). the utilisation of melon rind not only helps reduce environmental pollution, but also provides added value through its active compound content. melon rind is known to have antioxidant activity, namely tannins, flavonoids and phenols. these compounds maintain skin damage, heal wounds, and neutralise free radicals (rahmi & minerva, 2022). flavonoids are phenolic compounds that act as antioxidants, helping to inhibit excessive oxidative reactions caused by inflammation or metabolic processes. https://doi.org/10.14421/biomedich.2025.141.225-231 226 biology, medicine, & natural product chemistry 14 (1), 2025: 225-231 in addition, flavonoids also accelerate the wound revitalisation process, with more effective results than betadine, due to their ability to increase vascularity (bihani & mhaske, 2020). in a study by ismail et al. (2010), melon rind showed a flavonoid content of 5.13 ± 1.32 mg gae/g. tannins have been considered as healthpromoting plant components, as they have anticarcinogenic and anti-mutagenic potential, as well as antimicrobial, antioxidant, and antiradical properties (buzzini et al., 2008). in a study (vella et al., 2019), the tannin content was higher in melon rind than in seeds, showing 11.83 ± 1.44 and 0.92 ± 0.03 mg gae/g, respectively. polyphenolic compounds function as antioxidants with the mechanism of inhibiting the process of lipid peroxidation. this helps prevent and slow down cell damage due to necrosis and increase blood vessel formation in the wound area (akhmadi et al., 2022). in a study by ismail et al. (2010), the total content of phenolic compounds detected in melon rind extract was 4.70 ± 0.23 mg gae/g extract. based on the background, a study was conducted on the effectiveness of melon rind ethanol extract cream (cucumis melo) on incision wound healing in white rats (rattus norvegicus). this research will produce a safe, environmentally friendly, sustainable wound healing agent. materials and methods ethical clearance submission this research has been approved by the research ethics commission of the faculty of medicine, sebelas maret university with ethical clearance number 13/un27.06.11/kep/ec/2025 place and time of research this research was conducted at the microbiology laboratory in building d fkip uns, integrated laboratory technical implementation unit (upt) uns, and ud. wistar yogyakarta. this research was conducted in july-august 2024. tools and materials the tools used in this research are beaker glasses, analytical scales, stirring rods, blenders, porcelain cups, erlenmeyers, ph meters, stopwatches, adhesive test kits, glass, scales, filter paper, measuring cups, rotary evaporators, animal scales, gloves, masks, ovens, knives, alumunium foil, filter paper, cameras, tweezers, water baths, bunsen and ointment pots. the materials used in this study were melon rind (cucumis melo l.), 70% ethanol, paraffin liquid, distilled water, stearic acid, adeps lanae, tea, nipagin, nipasol, 9 male white rats (rattus norvegicus). the white rat test animals used were healthy male white rats aged 2-3 months with a body weight of 150200 gr obtained from ud. wistar yogyakarta. before being tested, the rats were acclimatised to the environment for 1 week. research design this research uses the experimental method or experiment (experiment research) and completely randomised design (ral) with three replicate treatments. the laboratory approach will be carried out with a series of experiments. this study tested the characteristics of melon rind extract cream and the effectiveness of melon rind ethanol extract cream (cucumis melo) on white rat (rattus norvegicus) incision wound healing. procedures preparation of fruit peel extract the melon rind was separated from the fruit and then cleaned and chopped. after that, it was dried in a drying cabinet then pollinated and sieved with a mesh sieve (khairani et al., 2020). then, it was soaked in 70% ethanol in a ratio of 1:5 and allowed to stand for 24 hours (hariningsih & hartono, 2022). after 24 hours, the extract was filtered with a filter cloth. the next 24 hours, a second filtration was done. then, the extract was concentrated using a rotary evaporator. the extract was put into a tool with a temperature of 40°c for 1.5 hours and a speed of 100 rpm until a concentrated extract was obtained (suhartanto et al., 2022). the manufacture of melon rind extract was carried out in the technical implementation unit (upt) integrated laboratory uns. preparation of extract cream the method of making melon skin ethanol extract cream is as follows, all tools and ingredients used are prepared.then the ingredients to be used are weighed according to the formulation. the oil phase and water phase are separated. the oil phase is stearate acid, cetyl alcohol and nipasol. the water phase is tea, glycerin, nipagin and distilled water. the oil phase and water phase were each heated at 70ºc. after everything is melted, put the oil phase little by little into the hot lumping that has been filled with the water phase, then crushed until a cream base is formed. after that, melon peel extract was added and stirred until homogeneous, then put into a container (hariningsih & hartono, 2022). the manufacture of extract cream was carried out at the technical implementation unit (upt) integrated laboratory uns. the cream was made in 2 doses, namely 15%, and 25% with the same cream base composition (khairani et al., 2020). the base composition can be seen in table 1. rizki & harlita – effectiveness test of melon fruit peel ethanol extract cream … 227 table 1. dosage of cream preparation. material name treatment (%) function p1 p2 melon rind extract 15% 25% active substance stearic acid 12 12 emulsifiers cetyl alcohol 0,5 0,5 thickener tea 1 1 emulsifiers glycerin 2 2 humectants nipagin 0,1 0,1 preservatives nipasol 0,05 0,05 preservatives aquadest ad 100 ad 100 solvent description: p1 = 15% melon rind extract p2 = 25% melon rind extract test animal preparation and wound making there were 9 male white rats (rattus norvegicus) in a healthy condition aged 2-3 months with a body weight of 150-200 gr obtained from ud. wistar yogyakarta. before being tested, the rats were adapted to the environment for 7 days and fed and drank ad libitum. white rats were divided into 3 treatment groups randomly. incision wounds were made with a sharp object (scalpel) with a diameter of 2 cm. the 3 groups of rats were given each treatment which can be seen in table 2. table 2. dosage of the cream preparation. variables material dosage control bacitracin 100% treatment melon rind extract (p1) 15% melon rind extract (p2) 25% observation and data collection wounded white mice were observed to measure open wound area and body weight during observation. the cream preparation was applied evenly on the sliced wounds three times a day, and this treatment was carried out every other day for 14 days. measurement of wound area and body weight of the rats was done periodically, i.e. every other day for 14 days. data analysis data analysis included qualitative and quantitative analyses. qualitative analysis included testing the characteristics of melon skin extract cream including colour, smell, texture, homogeneity, and ph. meanwhile, quantitative analysis included measuring the body weight of rats and measuring the average diameter of open wounds. the body weight and burn wound diameter measurements were analysed using a one-way anova test version 26.0. if there was a significant difference, the lsd (least significant different) test was performed. results and discussion cream characteristics in this study, the treatment with cream preparations from melon rind extract (cucumis melo) contained 2 doses with different comparisons, namely cream treatment with 15% melon rind extract dose (p1) and cream with 25% melon rind extract dose (p2). both cream preparations were then formulated into cream preparations. furthermore, the resulting cream was tested to evaluate its characteristics, such as colour, smell, texture, and consistency (lumentut et al., 2020), with the observation results summarised in table 3. table 3. cream characteristic test results. aspects 15% cream 25% cream colour white bone white smell non-stinging rubber stinging rubber texture smooth viscous smooth viscous ph 6 6 homogeneity homogeneous homogeneous open wound diameter the effectiveness test evaluated the effect of cream formulations containing melon rind extract as an active ingredient in healing sliced wounds. this test aims to determine the best cream formulation that provides optimal results in accelerating the wound healing process. the results of wound diameter observation for 14 days can be seen in table 4. table 4. results of mean diameter of open incision wound. average wound healing diameter (cm) group day 0 2 4 6 8 10 12 14 k 2 1,73 1,57 1,4 1,27 0,9 0 0 p1 2 1,55 1,3 1,17 0,9 0,53 0 0 p2 2 1,3 1,17 0,93 0,8 0,47 0 0 description: k = bacitracin ointment (control) p1 = 15% extract cream treatment p2 = 25% extract cream treatment from table 5, shows that on days 12 and 14 the average wound diameter was 0 because the observation showed that the wound had completely closed and only the scar remained. comparison of the results of the average diameter of incision wound healing can be seen in figure 1. 228 biology, medicine, & natural product chemistry 14 (1), 2025: 225-231 figure 1: graph of mean incision wound diameter. based on the diagram above, it shows that the diameter of open sliced wounds treated with cream with 25% concentration of melon rind extract (p2) is reduced faster than cream with 15% concentration of melon rind extract (p1) and with control (k). meanwhile, the 15% melon peel extract cream treatment group (p1) showed a faster reduction in the diameter of open sliced wounds compared to the control (k). to determine the difference between the control group and the treatment group, a one-way anova test was conducted. before the anova test, a normality test is carried out to assess the distribution of data in the data group or variable, whether the data distribution is normally distributed or not (fahmeyzan et al., 2018). the normality test results showed that the data on incision wound diameter during observation were normally distributed. after conducting the normality test, the homogeneity test was conducted to determine whether the data variance was homogeneous. the results of the homogeneity test showed that the sig. (p value) of 0.182 (p>0.05), indicating homogeneous data. after the data used has met the requirements of the anova test, the anova test is then carried out. the results of one way anova show a sig value. (p value) of 0.046. (p<0.05), indicating a significant difference (khairani et al., 2020). after the anova test results showed that significant differences, further lsd (least significant different) tests were carried out to determine significant differences between groups. the results of the lsd test on the diameter of incision wounds on the significance value between the control treatment (k) with 25% melon peel extract cream treatment (p2) of 0.034 (p<0.05), it can be interpreted that there is a real difference. furthermore, for the control treatment (k) with 15% melon peel extract cream treatment (p1) is 0.027 (p<0.05), which means that there is a significant difference. however, between the 15% melon peel extract cream treatment (p1) and the 25% melon peel extract cream treatment (p2) there is a significance result of 0.981. it can be said that between the two treatments there is no significant difference. body weight body weight measurements were taken to determine the most stable body weight during observation. the results of observations of body weight measurements for 14 days can be seen in table 5 and figure 2. table 5. results of average rat body weight. average body weight (gr) group day 0 2 4 6 8 10 12 14 k 176 170 171,33 173 180 179 182,33 183,33 p1 188 185,33 188,67 194 198,67 197,67 202,33 211,33 p2 250,33 254 257 259,67 261 264,33 266,33 270,33 description: k = bacitracin ointment (control) p1 = 15% extract cream treatment p2 = 25% extract cream treatment figure 2. average weight chart. the average body weight results from the graph show that all treatments experienced a decrease and an increase in body weight. to find out whether the three treatment groups had significant differences, the one way anova test was conducted (sihotang et al., 2022). before conducting the anova test, the data used must meet the requirements of the normality test and homogeneity test with a sig value (p>0.05). the results of the normality test and body weight homogeneity test have a sig value >0.05, which means that the data is classified as normal and homogeneous so that the one way anova test can be carried out. from rizki & harlita – effectiveness test of melon fruit peel ethanol extract cream … 229 the results of the anova test, the sig value obtained is 0.00 (p<0.05), which means that there is a significant difference in the average body weight of rats in each treatment. after the anova test results showed a significant difference, a further lsd (least significant different) test was conducted to determine the significant differences between groups. the lsd test results show that each treatment group of control (k), 15% melon rind extract cream (p1), and 25% melon rind extract cream (p2) has a significance value of 0.000 (p<0.05), which means there is a significant difference in the average body weight between each treatment group. discussion the results of the characteristic test which includes smell, colour, texture, homogeneity, and ph (lumentut et al., 2020), show that the preparation of melon skin ethanol extract cream with a concentration of 15% (p1) and 25% (p2) has physical properties that meet the standards, such as aroma and colour in melon skin extract cream depending on the concentration of extract used. the higher the addition of the extract, the aroma of the extract cream will be more pungent and the colour more intense (jami'ah et al., 2018). both preparations have different colours, white in p1 and bone white in p2, with a smooth, non-sticky texture and good homogeneity. the colour of melon rind extract tends to be yellow due to the content of carotenoids and flavonoids (jami'ah et al., 2018). the content of active compounds in the extract influences the distinctive aroma that appears in both preparations (alatas & anindhita, 2023). this study also showed good physical stability of the cream preparation, with a smooth texture, the results of the ph examination showed that both melon rind extract cream preparations had a ph of 6, the ph was by skin physiology, which is in the interval 4.5-7 (hariningsih & hartono, 2022), and optimal homogeneity, because the application results showed no lumps, had an even structure, and a uniform colour from the beginning to the end of the application (fauziah, 2022). this ensures comfort and safety in application. this ensures comfort and safety when applying the cream to the wound. observation of wound diameter showed that the treatment group with 25% melon rind extract cream (p2) gave faster healing results than the control group (k) and the 15% treatment group (p1). on day 10, the diameter of the wound in p2 showed that the wound had dried up and only scabs with a diameter of 0.47 cm remained, then on day 12 the wound had completely healed. the greater the concentration of melon rind extract, the higher the content of natural compounds, especially tannin compounds. in research mahmudah et al. (2021), stated that the content of tannin compounds reduced the length of significant wounds for 13 days. the tannin content acts as an antioxidant and antimicrobial that has healing ability through its effect in promoting epithelialisation (buzzini et al., 2008). in addition, melon rind extract contains flavonoids and phenols. the presence of flavonoid compounds is an antioxidant, helping to inhibit excessive oxidative reactions caused by inflammatory processes or body metabolism (bihani & mhaske, 2020). while phenols act as antioxidants with the mechanism of inhibiting the lipid peroxidation process (akhmadi et al., 2022). the more compounds present, the greater the effect of closing the incision wound area, and the stronger the antimicrobial power and accelerate wound healing (oeleu, 2022). statistical analysis using anova test and lsd further test showed significant differences between the control and both treatment groups. however, no significant difference was found between p1 and p2, although descriptively p2 showed better results. this suggests that a higher extract concentration can provide a faster healing effect, but the difference is not too striking (bawotong et al., 2020). results showed fluctuations in body weight in all groups, with a more stable increase in the p2 treatment group compared to p1 and control. this suggests that treatment with melon peel extract cream, especially at a concentration of 25%, not only supports the wound healing process but also does not have a negative effect on the health of the rats. the increase in body weight occurs because melon rind extract containing flavonoids enters the body, functions like estrogen increasing the amount of endogenous estrogen which can increase the synthesis and secretion of growth hormone so that it can stimulate the growth of cells in the body, causing weight gain (sandana et al., 2020). statistical analysis using anova test and lsd further test showed significant differences between control and treatment groups, as well as between p1 and p2. the more stable increase in body weight in p2 indicates that the cream preparation with melon skin extract does not have toxic or negative effects, but instead supports overall health (adhikari et al., 2021). conclusions this study shows that melon peel extract-based cream effectively accelerates slice wound healing in white rats. the cream with 25% extract concentration (p2) gave better results compared to the 15% cream (p1) and the control (bacitracin ointment), indicated by faster wound diameter shrinkage in the p2 treatment group. both cream formulations have good physical characteristics, such as smooth texture, non-sticky, optimal homogeneity, and ph by human skin physiology (ph 6). the effectiveness of this cream is supported by the content of active compounds in melon rind extract, such as tannins, flavonoids, and phenols, which act as antioxidants and antimicrobials, promoting the epithelialisation process and accelerating wound healing. in addition, the p2 treatment group also showed a more stable increase in body weight than the other groups, indicating that the use of creams with higher extract 230 biology, medicine, & natural product chemistry 14 (1), 2025: 225-231 concentrations did not hurt the health of the rats, and even supported overall health conditions. statistical analysis showed a significant difference between the control and treatment groups, although no significant difference was found between p1 and p2. overall, this study provides evidence that melon peel extract cream can be an environmentally friendly and effective alternative for incision wound healing. acknowledgements: the authors would like to thank the technical implementation unit (upt) of the uns integrated laboratory for conducting experiments there. authors’ contributions: harlita designed the study. husna rizki carried out the laboratory work. husna rizki analysed the data. husna rizki wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors state that there are no competing interests funding: the authors declare that this research did not receive any funding. references adhikari, a., barakoti, h., & kumar dey, b. 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(2019). characterization of polyphenolic compounds in cantaloupe melon by-products. foods, 8(6), 2–11. https://doi.org/10.3390/foods8060196 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 81-89 | doi: 10.14421/biomedich.2020.92.81-89 issn 2540-9328 (online) non-alkaloidal compounds from khat (catha edulis) leaves tsegu kiros department of chemistry, college of natural sciences, haramaya university, dire dawa, p.o.box 138, ethiopia. corresponding author kirosorg@gmail.com manuscript received: 23 may, 2020. revision accepted: 01 october, 2020. published: 17 november, 2020. abstract khat belongs to the family celastraceae, genus catha, and species edulis. more than 200 compounds have previously been identified in khat leaves, including: 40 alkaloids, terpenoids and sterol, flavonoids, glycosides, tannins, amino acids, vitamins and minerals. researchers have spent their effort and time merely on study of the alkaloidal components (mainly the stimulant agents, cathinone and cathine) of khat both qualitatively and quantitatively. the two principal khat stimulant compounds, cathinone and cathine, by now are well established. but, on the contrary, previous studies on the non-alkaloidal constituents of the plant were limited. the objective of this work was therefore to isolate and characterize compounds from non-alkaloidal fractions of the plant’s leaves. in this work, two nonalkaloidal compounds (kna-1 and kna-2) were isolated and characterized from the acid-etoac extract of fresh and oven-dried leaves of khat (catha edulis). from the present study, it is possible to conclude that investing more effort and time on searching additional nonalkaloidal principles from the leaves of khat is so necessary. and further works could be done in the future to isolate extra non-alkaloidal compounds from the leaves and other parts of khat and evaluate their biological activity. keywords: khat; catha edulis; non-alkaloid; kna-1; kna-2. introduction khat (catha edulis) belongs to the family of celastraceae and genus of catha. it is an evergreen shrub growing to a bush or a large tree. the first scientific description and the name catha edulis was given by the swedish botanist peter forskal. the plant is known by a variety of names, including: abyssinian tea, african salad, chat, jaad, mirra, qat, etc. the name khat is commonly used for c. edulis (lamina, 2010). khat is used commonly for mastication and its sympathomimetic actions. consumption of khat leaves is common in yemen, madagascar, saudi arabia and east african countries (kenya, ethiopia, djibouti, somalia, uganda, and tanzania). regardless of sociodemographic characters, the number of khat chewers in ethiopia is increasing from time to time (belwal and teshome, 2011; mathewson et al.,2013) beyond its deep rooted sociocultural tradition, khat is also used to some extent as medicine. processed leaves and roots of khat, for example, are used to treat influenza, cough, gonorrhea, headache, asthma and other chest problems (numan, 2003). and the leaves have been used for the treatment of depression, gastric ulcers, hunger, obesity, and tiredness (mathewson et al., 2013; numan, 2003; lemessa, 2001). moreover, khat plays an important role in the economy of ethiopia. it has become the source of livelihood for millions of people and is sometimes considered as a rival of coffee for income generation to ethiopia (ambaye, 2012). regarding the chemistry aspect, there are more than 200 compounds that have been identified in khat leaves (szendrei, 2004), including: 40 alkaloids, terpenoids and sterol, flavonoids, glycosides, tannins, amino acids, vitamins and minerals (lamina, 2010). the chemical constitute of khat have been studied since the late 19th century, in which an alkaloidal fraction was found in this plant by fluckiger and gerok and called it “katin”. this was followed by the isolation of many other substances and it was not until the year 1975 that the most important component of khat was isolated and named cathinone (s (-)-alpha-aminopropiophenone)) at the united nations laboratories (dhaifalah and`šntavy, 2004). the compounds reported so far from khat can generally be classified into five main compound classes, namely, flavonoids, terpenoids and sterol, alkaloids, amino acids, and vitamins. some of the representative compounds reported from khat plant so far are: dihydromyricetin (szendrei, 2004; al-meshal et al., 1985; ermias et al., 1984), dihydromyricetin-3-orhamnoside (szendrei, 2004; al-meshal et al., 1985; ermias et al., 1984), kaempferol (szendrei, 2004; almeshal et al., 1985; ermias et al., 1984), myricetin (szendrei, 2004; al-meshal et al., 1985; ermias et al., 1984; bredholt, 2010), myricetin-3-o-²̭d-galactoside (szendrei, 2004; almeshal et al., 1985; ermias et al., 1984), myricetin-3-ohttps://doi.org/10.14421/biomedich.2020.92.81-89 82 biology, medicine, & natural product chemistry 9 (2), 2020: 81-89 rhamnoside (szendrei, 2004; al-meshal et al., 1985; ermias et al., 1984), quercetin (szendrei, 2004; almeshal et al., 1985; bredholt, 2010), quercetin-3-o-²̭dgalactoside (szendrei, 2004; al-meshal et al., 1985; ermias et al., 1984), celastrol (bredholt, 2010; brossi, 1990); baxter et al., 1979), iguesterin (brossi, 1990; baxter et al., 1979), pristimerin (bredholt, 2010; brossi, 1990; baxter et al., 1979),tingenin a (bredholt, 2010; brossi, 1990); baxter et al., 1979), tingenin b ( bredholt, 2010; brossi, 1990; baxter et al., 1979), friedeline (brossi, 1990; baxter et al., 1979), sitosterol (bredholt, 2010; brossi, 1990; baxter et al., 1979), (+)cathine (brossi, 1990;wabe and mohammed, 2012; feyissa and kelly , 2008), (-)-cathinone (brossi, 1990; wabe and mohammed, 2012; feyissa and kelly, 2008), 3,6-dimethyl-2,5-diphenyl pyrazine (brossi, 1990; wabe and mohammed, 2012; feyissa and kelly, 2008), merucathine (brossi, 1990; wabe and mohammed, 2012; feyissa and kelly , 2008), merucathinone (brossi, 1990; wabe and mohammed, 2012; feyissa and kelly , 2008), cathedulins e2e6 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), cathedulin k1 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), cathedulin k2 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), cathedulin k6 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), cathedulin k12 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), cathedulin k15 (szendrei, 2004; dhaifalah et al., 2004; brossi, 1990; wabe et al., 2012; baxter et al., 1979), alanine (feyissa et al., 2008; halbach, 1972), ±̭aminobutyric acid (feyissa et al., 2008; halbach, 1972), arginine (feyissa et al., 2008; halbach, 1972), asparaginic acid , (feyissa et al., 2008; halbach, 1972), choline (feyissa et al., 2008; halbach, 1972), glutamic acid (feyissa et al., 2008; halbach, 1972), glycine (feyissa et al., 2008; halbach, 1972), histidine (feyissa et al., 2008; halbach, 1972), isoleucine (feyissa et al., 2008; halbach, 1972), leucine (feyissa et al., 2008; halbach, 1972), ornithine (feyissa et al., 2008; halbach, 1972), ascorbic acid (feyissa et al., 2008; halbach, 1972), niacin (feyissa et al., 2008; halbach, 1972), riboflavin (feyissa et al., 2008; halbach, 1972) and thiamine (feyissa et al., 2008; halbach, 1972). although the above mentioned classes of compounds were reported from the stimulant green plant, khat, researchers have spent their effort and time merely on study of the alkaloidal components (mainly the stimulant agents, cathinone and cathine) both qualitatively and quantitatively. as a result, the two principal khat stimulant compounds, cathinnoe and cathine, are now well established. but, on the contrary, previous studies on the non-alkaloidal constituents of the plant were limited. it therefore became apparent that it is necessary to undertake a phytochemical study in order to give an attention to the chemical constituents of khat other than the most studied alkaloidal one. the objective of this work was therefore to isolate and characterize compounds from non-alkaloidal fractions of the plant’s leaf. materials and methods plant material khat plant (c. edulis) was collected from the province known as sebeta, 20 km far from the capital city, addis ababa, ethiopia. two types of leaves are easily recognized in khat plant; the chewable and “nonchewable”. the chewable parts are usually shiny tender greenish young leaves found on the tip of a branch. the “non-chewable” leaves known locally as “geraba” are found on the lower part of a branch and are much older, harder and deep green in color. the fresh chewable leaves were used for immediate extraction and the “nonchewable” leaves were oven-dried (50 oc). chemicals, apparatuses and instruments chemicals such as visualizing agent (vanillin/meoh/ conc.h2so4), hcl (37%), etoac, meoh and acetone were used. besides, apparatuses and instruments including tlc plate (pre-coated aluminum sheet silica gel 60 f254), chamber, capillary tube, shaker 3020, nmr (bruker avance 400 mhz spectrometer), vacuum oven (sv40), rotavapor (r-114), sonicator (3210 branson), uv lamp (cc-8), uv/vis spectrophotometer (t60) and digital melting point (electrothermal ia 9200) were employed in the present study. isolation of non-alkaloidal compounds from khat leaves the fresh chewable khat leaves (60 g) were extracted with 0.1n hcl (300 ml) and filtered by suction filtration. the filtrate was extracted with etoac (100 ml) and separated the organic phase by separatory funnel, concentrated by rotavapor affording 140 mg. the plant extract (140 mg) was dissolved in meoh (10 ml) and adsorbed on 1 g silica gel (230 400 mesh size). the adsorbed sample was chromatographed by applying on top of column chromatography packed with silica gel (12 g). elution was carried out using etoac/ acetone with increasing polarity and five fractions were collected (table 1). table 1. collected fractions of acid-etoac extract of chewable leaves. fraction no. solvent system volume (ml) amount (mg) 1 etoac (100%) 5 4 2 “ 10 15 3 “ 20 15 4 etoac/ acetone (2:1) “ 20 5 “ “ 14 kiros – non-alkaloidal compounds from khat (catha edulis) leaves 83 as shown from the tlc profile (figure 1) of the above fractions (table 1), fraction 4 (kna-1) was seen as a better spot; it was then concentrated (20 mg) and obtained as yellow solid; mp 190-192oc (literature value 192-195oc, dictionary of natural product); rf in tlc: 0.6 (etoac/ meoh/ acoh; 4.5: 0.5: 1) sprayed with vanillin/ meoh/ con. h2so4 (0.3: 95: 5); uv (meoh) λmax 293 nm. 1h nmr (400mhz, meod): δh 5.01 (1h, br s, h-2), 4.56 (1h, d, h-3), 5.93 (1h, br s, h-6), 5.92 (1h, br s, h-8), 6.54 (2h, s, h-2’, 6’), 4.10 (1h, br s, h-1”), 3.61 (1h, br s, h-2”), 3.70 (1h, d, h-3”), 3.33 (1h, br s, h-4”), 4.28 (1h, m, h-5”), 1.22 (3h, d, h-6”). 13c nmr (400mhz, meod): δc 82.68 (c-2), 77.06 (c3), 95.99 (c-6), 94.89 (c-8), 106.25 (c-2’, 6’), 100.69 (c-1”), 70.36 (c-2”), 70.74 (c-3”), 72.43 (c-4”), 69.11 (c-5”), 16.51 (c-6”). dept-135: δc (194.53 (c-4), 164.06 (c-5), 167.17 (c-7), 162.65 (c-9), 101.05 (c10), 126.99 (c-1’), 145.63 (c-3’, 5’), 133.68 (c-4’). same extraction method as above was applied for the vacuum oven dried powder “non-chewable” (25 g) khat leaves and afforded 150 mg of etoac extract for the acid extract. the plant extract (150 mg) was dissolved in meoh (10 ml) and adsorbed on 1 g silica gel. the adsorbed sample was chromatographed by applying on top of a column chromatography packed with silica gel (13 g) and eluted using etoac/ meoh with increasing polarity. six fractions were collected (table 2). table 2. collected fractions of acid-etoac extract of “non-chewable” leaves. fraction no. solvent system volume (ml) amount (mg) 1 etoac (100%) 10 10 2 “ 15 15 3 “ 20 40 4 “ 10 20 5 “ 30 15 6 etoac/ meoh(4:1) 25 20 as shown from the tlc profile (figure 2) of the above fractions (table 2), different spots were observed in the chromatogram after it was subjected to uv lamp at 254 nm. among the fractions, fraction 1(kna-2) was concentrated (10 mg) and obtained as an orange solid; mp 165-168oc; uv (meoh) λmax 292 nm; rf in tlc: 0.8 (etoac/ meoh/ acoh; 4.5: 0.5: 0.1) sprayed with vanillin/ meoh/ con. h2so4 (0.3: 95: 5). 1h nmr (400 mhz, meod): δh 4.86 (1h, d, h-2), 4.49 (1h, d, h-3), 5.90 (1h, br s, h-6), 5.93 (1h, br s, h-8), 6.54 (2h, br s, h-2’, 6’). 13c nmr (400 mhz, meod): δc 83.90 (c-2), 72.28 (c-3), 94.85 (c-6), 95.87 (c-8), 106.61 (c-2’, 6’). dept-135: δc 196.93 (c-4), 163.91 (c-5), 167.30 (c7), 163.05 (c-9), 100.41 (c-10), 133.51 (c-1’), 145.47, 145.47 (c-3’, 5’), 127.66 (c-4’). results and discussion two types of leaves are easily recognized in khat plant; the chewable and “non-chewable”. the chewable parts are usually shiny tender greenish young leaves found on the tip of a branch. the “non-chewable” leaves known locally as “geraba” are found on the lower part of a branch and are much older, harder and deep green in color. in the present work, these leaves were analyzed to isolate non-alkaloidal components. in order to isolate non-alkaloidal components, the khat leaves were first extracted with 0.1n hcl, filtered, and extracted with etoac. this etoac extract was fractionated to isolate the non-alkaloidal compounds as described in the experimental section. characterization of two khat non-alkaloidal (kna-1 and kna-2) compounds the etoac extract of fresh chewable khat leaves (see section 2.3) was chromatographed by applying on top of column chromatography packed with silica gel. elution was carried out using etoac/ acetone with increasing polarity and five fractions were collected (table 1). similarly, the etoac extract of vacuum oven dried “non-chewable” khat leaves (see section 2.3) was chromatographed on column silica gel using etoac/ meoh with increasing polarity and six fractions were collected (table 2). thin layer chromatographic (tlc) analysis using etoac/ meoh/ acoh (4.5: 0.5: 0.1) as developing solvent, the fractions (table 1) were spotted and chromatogramed on a tlc plate. the tlc plate was then sprayed with vanillin/ meoh/ conc. h2so4 (1: 95: 5). as shown from the tlc profile (figure 1), fraction 4 (kna-1) was seen as a better spot, concentrated (20 mg) and characterized. figure 1. tlc profile of fractions of acid-etoac extract of chewable leaves. kna-1 84 biology, medicine, & natural product chemistry 9 (2), 2020: 81-89 the same solvent system with same ratio and spraying reagent as above were applied here for the fractions stated in table 2. as shown from the tlc profile (figure 2), fraction 1(kna-2) was observed as a better spot. figure 2. tlc profile of fractions of acid-etoac extract of “nonchewable” leaves. characterization of compound kna-1: it was obtained as yellow solid; mp 190192 oc (literature value 192-195oc, dictionary of natural product); rf in tlc: 0.6 (etoac/ meoh/ acoh; 4.5: 0.5: 1) sprayed with vanillin/ meoh/ con. h2so4 (0.3: 95: 5); uv (meoh) λmax 293 nm. the 1h nmr (400 mhz, meod) spectrum showed a doublet at δ 4.56 (1h, d, j = 10.8 hz, h-3) and a broad singlet at δ 5.01 (h-2) which were due to the two protons at c-3 and c-2 of flavanol skeleton, respectively. the two broad singlets observed at δ 5.92 (h-6) and 5.93 (h-8) were due to the aromatic protons of a-ring of a flavonoid skeleton at c-6 and c8, respectively. the broad singlet at δ 6.54 (h-2’ & -6’) integrated to two protons was due to the overlapping of signals for the symmetric aromatic protons of b-ring at c-2’ and c-6’ of flavonoid skeleton. the oxymethinic proton signals between δh 3.33 4.28, together with the methyl proton signal at δh 1.22 (3h, d, j = 6.4 hz, h6”), and the oxymethinic carbon signals between δc 69.11-100.69, including the methyl carbon signal at δc 16.51 indicated that a rhamnoside sugar was attached. the doublet signal at δh 1.22 (3h, d, j = 6.4 hz, h-6”) was a characteristic for the rhamnoside moiety. the three broad singlets at δh 3.33 (h-4”), 3.61 (h-2”), and 4.10 (h-1”) were due to the rhamnoside protons positioned at c-4”, c-2”, and c-1”, respectively. the methine proton at c-3” of the sugar moiety was observed as a doublet at δh 3.70 (1h, d, j = 9.6 hz, h3”), and the muliplet signal at δh 4.28 (h-5”) was characteristic signal for the rhamnoside proton attached at c-5” which contains a substituted methyl group. the 13c nmr spectrum showed the presence of nineteen signals attributed to twenty-one different carbons. the spectral region between δc 94.89-167.17 was characteristic of aromatic carbons of aand b-rings of flavonoid skeleton with the exception of the signal at δc 100.69 (c-1”) which was due to the anomeric carbon of the rhamnoside moiety. the remaining carbon atoms of the sugar moiety were observed in the spectral region at δc 69.11-72.43 including the methyl carbon appeared at δc 16.51 (c-6”). and the signal at δc 194.53 (c-4) was characteristic of carbonyl carbon of ketone functional groups (table 3). signals at δc 77.06 (c-3) and 82.68 (c-2) were due to α, β carbons of c-ring (pyranone), and were compatible with a dihydroflavanol structure which were comparable with a literature value [18] medeiros aan, medeiros fa, queiroz tm, tavares jf, silva ms, medeiros ia (2010). from the dept-135 spectrum, there were nine quaternary carbons one to a carbonyl carbon at δc 194.53 (c-4), and eight to aromatic carbons of aand b-rings. the eleven signals appeared in the positive direction were due to the methine (-ch) carbons containing two symmetric carbons overlapped with each other at δc 106.25 (c-2’, -6’), and a methyl carbon at δc 16.51 (c-6”). the above 1d nmr data is summarized as follows (table 3). table 3. 1h, 13c and dept nmr data (δppm) of compound kna-1. isolated compound (kna-1) literature value (medeiros et al., 2010) (2, 3dihydromyricetin3-o-rhamnoside) c/h 1h 13c dept-135 1h 13c 2 5.01, br s 82.68 -ch4.86, d 83.20 3 4.56, d 77.06 ch-o4.61, d 76.80 4 194.53 c=o (q) 194.30 5 164.06 q 165.40 6 5.93, br s 95.99 =ch 5.90, d 96.00 7 167.17 q 166.90 8 5.92, br s 94.89 =ch 5.87, d 95.00 9 162.65 q 162.10 10 101.05 q 101.00 1’ 126.99 q 126.80 2’ 6.54, s 106.25 =ch 6.50, s 108.00 3’ 145.63 q 145.80 4’ 133.68 q 135.20 5’ 145.63 q 145.80 6’ 6.54, s 106.25 =ch 6.50, s 108.00 1” 4.10, br s 100.69 ch-o4.07, s 100.00 2” 3.61, br s 70.36 ch-oh 3.36, br s 70.10 3” 3.70, d 70.74 ch-oh 3.41, dd 70.40 4” 3.33, br s 72.43 ch-oh 3.20, dd 71.60 5” 4.28, m 69.11 ch-o3.88, m 68.90 6” 1.22, d 16.51 -ch3 0.92, d 17.60 the extensive 2d nmr experiments involving 1h1h cosy, hmqc, hmbc spectra supported the 1d nmr data above for the proposed structure of kna-1. kna-2 kiros – non-alkaloidal compounds from khat (catha edulis) leaves 85 the cosy spectrum of kna-1 is used to determine 1h1h correlations. as stated in table 4, except the protons at δh 5.93 (h-6), 5.92 (h-8), and 6.54 (h-2’, 6’) which showed a correlation only with themselves, all the rest protons of the rhamnoside moiety and the pyranone (cring) made a correlation more than one bond. the two protons at δh 5.01 (h-2) and 4.56 (h-3) are coupled through vicinal (3j) coupling with each other. the anomeric proton of the rhamnoside moiety at δh 4.10 (h-1”) is correlated with the proton at δh 3.61(h-2”). the methyl group of the rhamnoside at δh 1.22 (me-6”) showed a correlation with a proton at δh 4.28 (h-5”). the hmqc spectrum of kna-1 is utilized to determine direct 1h-13c correlations (table 4), while the hmbc correlations described the long range 1h-13c connectivities. the h-2 proton of the c-ring at δh 5.01 displayed hmbc correlations with c-2’, 6’ (δc 106.25) and c-1’ (δc 126.99) of the b-ring. the h-3 proton at δh 4.56 showed a correlation with c-1” (δc 100.69), which indicated the rhamnoside moiety is attached at c3 position. the symmetric protons of the b-ring at δh 6.54 (h-2’, 6’) showed a correlation with c-2 of the pyranone (c-ring). table 4. cosy, hmqc and hmbc data of compound kna-1. proton no. cosy hmqc hmbc 2 h-2 h-3 h-2 c-2 h-2 c-1’, c-2’, c-6’ 3 h-3 h-2 h-3 c-3 h-3 c-1” 6 h-6 c-6 h-6 c-8, c-10 8 h-8 c-8 h-8 c-6, c-9 2’ h-2’ c-2’ h-2’ c-2 6’ h-6’ c-6’ h-6’ c-2 1” h-1” h-2” h-1” c-1” h-1” c-3 2” h-2” h-1”, h-3” h-2” c-2” h-2” c-3”, c-4” 3” h-3” h-2”, h-4” h-3” c-3” h-3” c-2”, c-4” 4” h-4” h-3”, h-5” h-4” c-4” h-4” c-2”, c-6” 5” h-5” h-4”, h-6” h-5” c-5” h-5” c-2”, c-6” 6” h-6” h-5” h-6” c-6” h-6” c-2”, c-5” o ooh h ho h h h oh oh oh o h h o oh oh oh h h h h h h h h o o o oh h h h h h h oh ho h oh oh oh h h h h oh h ho h o h cosy correlation selected hmbc correlation the structure of kna-1 was deduced as 2, 3dihydromyricetin-3-o-rhamnoside (figure 3) in comparison of the data with the literature and then confirmed by the 2d nmr data. this compound belongs to a group of secondary metabolites called flavonoids. characterization of compound kna-2: it was obtained as an orange solid; mp 165-168 oc; uv (meoh) λmax 292 nm; rf in tlc: 0.8 (etoac/ meoh/ acoh; 4.5: 0.5: 0.1) sprayed with vanillin/ meoh/ con. h2so4; 0.3: 95: 5. oho oh o o oh oh oh o oh oh oh h3c 1 2 34 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 1'' 2'' 3''4'' 5'' 6'' figure 3. suggested chemical structure of compound kna-1 86 biology, medicine, & natural product chemistry 9 (2), 2020: 81-89 the 1h, 13c, and dept nmr spectral values of kna-2 are tabulated and compared with literature value (jr et al., 2009) as follows (table 5). table 5. 1h, 13c and dept nmr data (δppm) of compound kna-2. isolated compound (kna-2) literature value (jr et al., 2009) (2, 3dihydromyricetin) c/h 1h 13c dept-135 1h 13c 2 4.86, d 83.90 -ch4.96, d 84.40 3 4.49, d 72.28 -ch-oh 4.57, d 72.90 4 196.93 c=o (q) 197.9 5 163.91 q 164.00 6 5.90, br s 94.85 =ch 5.94, s 95.70 7 167.30 q 167.60 8 5.93, br s 95.87 =ch 5.98, s 96.80 9 163.05 q 164.8 10 100.41 q 101.40 1’ 133.51 q 134.00 2’ 6.54, br s 106.61 =ch 6.62, s 107.90 3’ 145.47 q 146.10 4’ 127.66 q 128.90 5’ 145.47 q 146.10 6’ 6.54, br s 106.61 =ch 6.62, s 107.90 depending on the above nmr data along with the literature value, the structure of kna-2 was suggested as 2, 3-dihydromyricetin (figure 4) which belongs to a group of secondary metabolites called aglycones flavonoid. this compound was first reported in khat leaves in 1980 (szendrei, 2004). it was also isolated from a plant known as elderberry (sambucus nigra l.) in 2009 and has biological activities as anti-virus and antiinfluenza (jr et al., 2009). oho oh o oh oh oh oh 1 2 34 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' figure 4. suggested chemical structure of compound kna-2 conclusion from the present study, it is possible to conclude that investing more effort and time on searching additional non-alkaloidal principles from the leaf part of khat is so necessary. and further works could be done in the future to isolate extra non-alkaloidal compounds from the leaf and other parts of khat and evaluate their biological activity. acknowledgements: the author would like to thank the ethiopian ministry of education and addis ababa university for their financial support. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references al-meshal ia, hifnawy, ms, asir m (1985) myricetin, dihydromyricetin, and quercetin glycosides from catha edulis. j. nat. prod. 49(1): 172. ambaye gg (2012) production and consumption trends of khat in ethiopia: a big business or a big worry. advances in agriculture, sciences and engineering research 2(10): 414 427. baxter rl, crombie l, simmonds dj, whiting da (1979) alkaloids of catha edulis (khat). part i, isolation and characterization of eleven new alkaloids with sesquiterpene cores (cathedulins); identification of the quinone methide root pigments. j.c.s. perkin i 2965 2971. baxter rl, crombie wml, crombie l, simmonds dj, whiting da (1979) alkaloids of catha edulis. part 4, structures of cathedulins e3, e4, e5, e6, and k12. novel sesquiterpene alkaloids with monoand bismacrolide bridges. j.c.s. perkin i 2982 2989. belwal r, teshome h (2011) khat exports and the ethiopian economy: opportunities, dilemmas and constraints. african journal of business management 5(9): 3635 3648. bredholt t (2010) delineating cellular and molecular mechanisms of toxicity of an extract of khat (catha edulis forsk.) in leukemia and normal peripheral blood cells. [dissertation]. brossi a (1990) the alkaloids: chemistry and pharmacology. academic press, inc. dhaifalah i, `šntavy j (2004) khat habit and its health effect, a natural amphetamine. biomed. papers 148(1): 1115. ermias d, sebsebe d, kalman s, brenneisen r, peter k, amha m, zein az (1984) international symposium on khat: chemical and ethnopharmacological aspects of khat. proceedings, addis ababa university, addis ababa, 1984. [ethiopia]. feyissa am, kelly pj (2008) a review of the neuropharmacological properties of khat. progress in neuropsychopharmacology and biological psychiatry 32: 11471166. halbach h (1972) medical aspects of the chewing of khat leaves. bull. org. mond. sante 47: 2129. jr br, fink rc, mcmichael md, li d, alberte rs. (2009) elderberry flavonoids bind to and prevent h1n1 infection in vitro. phytochemistry 70: 1255 -1261. lamina s (2010) khat (catha edulis): the herb with officio-legal, sociocultural and economic uncertainty. s. afr. j. sci. 106(3/4): 14. lemessa d (2001) khat (catha edulis): botany, distribution, cultivation, usage and economics in ethiopia. unemergencies unit for ethiopia, 114. mathewson h, james k, schifano f, sumnall h, wing a, anderson d (2013) khat: a review of its potential harms to kiros – non-alkaloidal compounds from khat (catha edulis) leaves 87 the individual and communities in the uk. advisory council on the misuse of drugs. 196. medeiros aan, medeiros fa, queiroz tm, tavares jf, silva ms, medeiros ia (2010) effects of extract, fractions and 2,3dihydromyricetin-3-oα-l-rhamnoside from pradosia huberi (ducke) ducke on rat isolated mesenteric arteries. brazilian journal of pharmacognosy 20(4): 542 548. numan nmd (2003) the green leaf concept of khat chewing in yemen: social, cultural, psychological and medical aspects for khat use. [dissertation]. szendrei k (2004) the chemistry of khat. unodc-bulletin on narcotics 535. wabe nt, mohammed ma (2012) what science says about khat (catha edulis forsk)? overview of chemistry, toxicology and pharmacology. journal of experimental and integrative medicine 2(1): 29 37. 88 biology, medicine, & natural product chemistry 9 (2), 2020: 81-89 figure s1. uv (meoh) spectra of kna-1 and kna-2. figure s2. 1h nmr (400 mhz, meod) spectrum of kna-1. figure s3. 13c nmr (400 mhz, meod) spectrum of kna-1. figure s4. dept-135 spectrum of kna-1. figure s5. cosy spectrum of kna-1. figure s6. hmqc spectrum of kna-1. kna-1, λmax = 293 nm kna-2, λmax = 292 nm kiros – non-alkaloidal compounds from khat (catha edulis) leaves 89 figure s7. hmbc spectrum of kna-1. figure s8. 1h nmr (400 mhz, meod) spectrum of kna-2. figure s9. 13c nmr (400 mhz, meod) spectrum of kna-2. figure s10. dept-135 spectrum of kna-2. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 493-496 | doi: 10.14421/biomedich.2024.132.493-496 issn 2540-9328 (online) effect of trichosanthes cucumerina methanol extract on crp and fibrinogen levels in diabetic ulcer rat models nazula rahma shafriani*, tri dyah astuti, shofy farha aulia amri, riski aditya permana, zuvi fridhoriansyah, intan maharani department of medical laboratory technology, faculty of health sciences, universitas ‘aisyiyah yogyakarta, jl. siliwangi no.63, nogotirto, gamping, sleman, yogyakarta postcode, indonesia. corresponding author* nazula.rahma@unisayogya.ac.id abstract diabetic ulcers can be fatal for people with long-term diabetes. this is caused by the spread of diabetic ulcers and inadequate care for the wound, which can induce an infection and even death. this study aims to determine inflammatory markers, specifically fibrinogen and creactive protein (crp), as indicators of healing, particularly in a rat model of diabetic ulcers treated with trichosanthes cucumerina methanol extract. the methods used were making extracts, measuring blood glucose levels, measuring crp, and measuring fibrinogen levels in 25 male wistar rats. based on the results of glucose and fibrinogen examinations in all groups before treatment and after treatment, it showed a decrease in glucose and fibrinogen levels. meanwhile, the results of crp examinations, before and after treatment did not show any differences. the results of statistical analysis showed that there was a significant difference of p<0.05 in pre and post glucose, as well as pre and post fibrinogen.the extract that reduced fibrinogen levels the most was group 3 with 500 mg/kgbw. meanwhile, crp levels before and after treatment did not show a decrease. keywords: trichosanthes cucumerina; ulcus diabetic; crp; fibrinogen. abbreviations: crp = c-reactive protein, dm = diabetes mellitus, cub = cucurbitacin b, poct = point of care testing, carbon tetrachloride (ccl4). introduction type 2 diabetes is a condition where blood sugar levels increase which can increase the risk of macro vascular and micro vascular damage thereby reducing the sufferer's quality of life. one of the complications of type 2 diabetes is neuropathy in the form of reduced sensation in the feet and is often associated with foot ulcers and even amputation (fitria et al., 2017). according to roza et al. (2015) chronic dm sufferers are increasingly likely to suffer from hyperglycemia which will ultimately cause retinopathy, nephropathy and diabetic ulcers. blood sugar levels can be managed, but type 2 diabetes cannot be cured. patients with type 2 diabetes have elevated creactive protein (crp) levels as a result of an inflammatory response brought on by problems. crp is an inflammatory marker for diagnosing infection (hadavand et al., 2019). the plasma protein known as crp is derived from the liver. plasma crp levels can increase rapidly to more than 1000 times above average values due to tissue damage or infection. consequently, crp is a monitoring marker in the acute phase, responding to tissue damage, infection, and inflammation. research by li et al. (2016) has shown that increased crp levels are associated with diabetes. another marker of inflammatory reactions besides crp is fibrinogen. blood coagulation, fibrinolysis, cellular and matrix interactions, inflammation, wound healing, and neoplasia are all significantly impacted by fibrinogen. it was reported that serum fibrinogen levels increased due to an increase in acute phase proteins in type 2 diabetes sufferers (korkmaz et al., 2018). age, gender, smoking, body mass index (bmi), alcoholism, hypertension, glycemic management, lipid profile, and uric excretion rate are modifiable and non-modifiable factors affecting fibrinogen (kattula et al., 2017). there have been reports of the anti-inflammatory properties of trichosantes cucumerina extract. methanol and water extract at a 75mg/kg concentration demonstrated significant inhibition of carrageenaninduced edema in the hind leg. the anti-inflammatory effect induced by the methanol extract was comparable to that of the drug tested, indomethacin, and 750mg/kg of the extract at four and five hours. according to ahuja et al. (2019), the entire fruit and seeds of trichosantes cucumerina exhibited anti-inflammatory effect against manuscript received: 23 august, 2024. revision accepted: 10 october, 2024. published: 24 october, 2024. https://doi.org/10.14421/biomedich.2024.132.493-496 mailto:nazula.rahma@unisayogya.ac.id 494 biology, medicine, & natural product chemistry 13 (2), 2024: 493-496 mice’s acetic acid-induced vascular permeability, carrageenin-induced edema and cotton pellet-induced granuloma formation. cucurbitacin b (cub) is a component of trichosantes cucumerina extract. cub exhibits pharmacological effects on various illnesses, inflammatory, antioxidant, antiviral, hypoglycemic, hepatoprotective and anti-cancer activities (dai et al., 2023). the hepatoprotective effect of cub was reported more than 20 years ago when its preventive and therapeutic effects against carbon tetrachloride (ccl4)-induced hepatotoxicity were confirmed. later, research showed that pre-treatment with cub could significantly reduce liver lesions in a mouse model of ccl4-induced hepatotoxicity, reaffirming its protective effect against acute liver injury (hunsakunachai et al., 2019). research by yang et al. (2020) showed that cub can effectively inhibit liver fibrosis and suppress the release of cytokines including tgf beta 1, il-5, il-13, il-6, and tnf alpha. this study measured crp and fibrinogen levels in mice with a diabetic ulcer model. so far, the research that has been carried out is to determine the presence of cub in inflammatory effects and supporting wound healing and no research has been carried out on its effect on rat therapy with diabetic ulcer models. as a result, it is critical to understand the presence of an inflammatory marker in diabetic ulcers, as the provides insight into how the ulcers heal. materials and methods materials trichosantes cucumerina methanol extract, whole blood, glucose trip (easytouch), serum, crp latex reagent (glory diagnostic), gel separator vacuum tube, citrate vacuum tube, fibrinogen reference plasma reagent (md pacific), distilled water, fib reagent (md pacific) instruments oven, rotary evaporator, poct (easytouch) tool, test slide centrifuge, stir bar, micropipette, yellow tip, blue tip, ts6000 semi-automatic coagulation analyzer. procedure making methanol extract of trichosanthes cucumerina. 50 kg of trichosanthes cucumerina fruit was separated from the seeds, cut into thin slices with a thickness of ±5 mm, then dried in an oven at 50°c for ±72 hours. next, the dried flesh of the trichosanthes cucumerina is ground into powder with a size of 40 mesh, and simplicia powder is obtained. 1 kg of trichosanthes cucumerina fruit simplicia powder was extracted using the maceration method using methanol solvent. the first maceration was carried out for 48 hours and the volume of methanol added was 2,200 ml and 1 kg of trichosanthes cucumerina simplicia powder. the second maceration was carried out for 24 hours and the volume of methanol added was 850 ml. the third maceration was carried out for 24 hours and the volume of methanol added was 750 ml. the filtrate obtained was evaporated using a rotary evaporator at a temperature of 40°c and a rotation speed of 4 rpm (duengo el al., 2016) treatment of experimental animals. male rats (rattus norvegicus) from the same colony were 8-9 weeks old and weighed 180-200 grams. group division was carried out by sampling. samples were taken from the population and the size was determined based on the federe formula. for five treatments, at least 5 repetitions are required for each treatment so the total mouse sample required for this research is 25 mice. mice were obtained from the biomedical laboratory of muhammadiyah university, yogyakarta. the treatment began with adaptation and acclimatization of five groups of mice including negative control, positive control (metformin and amoxicilin), as well as three treatment groups with doses of methanol extract of eel bitter melon 125 mg/kgbw, 250 mg/kgbw, and 500 mg/kgbw. before induction, blood was taken from the rat's tail to measure blood glucose levels. all mice were then induced with alloxan 150 mg/kgbw for 96 hours until they developed diabetes mellitus. after being hyperglycemic, a 2 cm incision was made in the thigh and inoculated with staphylococcus aureus to form diabetic ulcers within two days. the methanol extract of eel bitter melon was administered for 21 days at a dose of 1 ml per day according to each treatment group. blood glucose examination blood glucose levels were checked before treatment and after treatment (negative control, positive control, and therapy with trichosanthes cucumerina methanol extract at a dose of 125 mg/kgbw, 250 mg/kgbw, 500 mg/kgbw), by taking samples. blood from the tail vein of mice in all groups. the first drop of blood is discarded, and the subsequent drop of blood is examined using a point of care test (easytouch). the reagent strip is inserted into the device, then blood is dripped onto the reagent strip. the results are read on the screen in less than 30 seconds. the number on the screen shows the blood sugar concentration in mg/dl (masdar et al., 2021). crp examination the crp examination method is carried out qualitatively with crp latex reagent. a total of 50 μl of serum samples from the treatment group, positive control (cp), and negative control (cn) were pipetted onto the slide circle. next, 1 drop of latex reagent (crp antigen) was added to each circle. this mixture was stirred with a stir stick for 2 minutes to homogenize it. the examination results are read under bright light and validated by observation under a microscope. if agglutination occurs, shafriani et al. – effect of trichosanthes cucumerina methanol extract on … 495 it indicates a positive crp result, whereas if there is no agglutination, the result is negative (kalma, 2018). fibrinogen examination fibrinogen examination was carried out with the ts6000 semi-automatic coagulation analyzer, namely diluting the fibrinogen reference plasma reagent using 1 ml of distilled water, and diluting the "fib" reagent with 2 ml of distilled water. then, place the cuvette in the prewarming sample position, add 20 μl of sample or control and 180 μl of imidazole buffered saline (ibs) reagent into the cuvette, then incubate for 3 minutes in the prewarming sample position by pressing the clock icon on the screen. after that, move the cuvette to the test channel and add one magnetic ball into the cuvette, then add 100 microliters of fib reagent using a micropipette connected to the tool. press until you hear a "beep" sound, and wait until the reading process is complete to get the results. results and discussion based on the results of glucose and fibrinogen examinations in all groups before treatment and after treatment, it showed a decrease in glucose and fibrinogen levels which are presented in table 1. meanwhile, for the results of crp examinations, before and after treatment did not show any differences. the results of statistical analysis showed that there was a significant difference of p<0.05 in pre and post glucose, as well as pre and post fibrinogen. table 1. results of glucose, fibrinogen and crp examinations. parameter control (+) control (-) group i (125 mg/kgbw) group ii (250 mg/kgbw) group iii (500 mg/kgbw) p value fibrinogen (mg/dl) pre 526±87,77 436,2±88,56 472,6±102,57 543,2±37,29 508,6±56,14 0,000 post 769,2±148,52 210±64,16 311±54,85 280,4±69,3 221±78,29 glucose (mg/dl) pre 336,4±54,57 422,2±130,21 362±118,98 365,8±154,45 350,2±125,47 0,035 post 264,8±40,81 178,2±13,97 223,6±52,1 228,2±53,76 190,8±39,92 crp (mg/l) ≤ 15 mg/l discussion based on the results of examining blood glucose and fibrinogen levels, a strong relationship was found between these two variables. fibrinogen levels rise in response to elevated blood glucose levels. this is consistent with studies by rosyadi et al. (2018) that fibrinogen levels correlate with the process of diabetes in mice induced by streptozotocin. in addition, elevated blood glucose levels exacerbate the infection state (fitria et.al., 2017).c-reactive protein (crp) is an acute phase protein produced by the liver. crp is frequently used as an indication of inflammation since a rise in crp levels signals inflammation in the body. in chronic inflammatory diseases like type 2 diabetes, crp levels often rise somewhat. nevertheless, in this study, no relationship was found between glucose levels and crp levels. these results are in line with (faddah et al., 2012) which stated that crp in rats was not the principal significant acute phase protein and that, in comparison to human crp, its level below basal conditions was higher. effect of trichosanthes cucumerina methanol extract on fibrinogen shown that it has an impact on fibrinogen and glucose levels. the results of this research are in line with research conducted by benny (2014) on the effect of trichosanthes cucumerina in lowering blood sugar in experimental animals. aside from that, trichosanthes cucumerina has components that resemble sulfonylurea (an anti diabetic drug and is widely used). the phytochemicals such as, carbohydrates, alkaloids, steroids, saponins, flavonoids and tannin were detected from the medicinal plant, t. cucumerina. methanol extract showed larger numbers of phytochemicals than ethyl acetate, acetone and water extract. secondary metabolites are useful in cell growth, body building and replacement. they also function as immunological stimulants, antibacterial, anti-inflammatory agents, antiviral and detoxification activities. plant derived tannin shown a range of antibacterial properties (seshadri et.al., 2020). a decrease in blood glucose levels was followed by a significant decrease in fibrinogen levels in the 3 treatment groups. the concentrations used, namely concentrations of 125mg/kgbw (body weight), 250mg/kgbw and 500mg/kgbw, have the ability to reduce blood glucose levels. in diabetic rats, administration of methanol extract from eel bitter melon with a concentration of 500mg/kgbw showed a more optimal effect in reducing blood glucose and fibrinogen levels. according to research by afifah, et al (2021), extract of momordica charantia can also reduce blood glucose levels in diabetic mice (mus musculus) with concentrations of 100 mg/kgbw, 250 mg/kgbw and 400mg/kgbw. conclusions based on the results of research on the effect of methanol extract of trichosanthes cucumerina on crp and 496 biology, medicine, & natural product chemistry 13 (2), 2024: 493-496 fibrinogen levels in diabetic ulcer models, a significant reduction in fibrinogen levels was obtained before and after treatment. the extract that reduced fibrinogen levels the most was group 3 with 500 mg/kgbw of extract. meanwhile, crp levels before and after treatment did not show a decrease. authors’ contributions: shafriani, n.r and astuti, t.d. design, supervised the study, and assembled the draft manuscript. amri, s.f.a., permana, r.a., fridhoriansyah, z., and maharani, i. carried out the laboratory work under supervision. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the funding for this research was provided by riset muhammadiyah and lppm unisa yogyakarta. references afifah, a. r., wahyu, s., gayatri, s. w., daeng kanang, i. l., & indarwati abdullah, r. p. 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(2020). sirt1/igfbprp1/tgf β1 axis involved in cucurbitacin b ameliorating concanavalin a-induced mice liver fibrosis. basic and clinical pharmacology and toxicology, 127(5), 371–379. https://doi.org/10.1111/bcpt.13446 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 507-511 | doi: 10.14421/biomedich.2025.141.507-511 issn 2540-9328 (online) effect of different pre-treatments and frying process on proximate, some essential minerals, and anti-nutritional factors of taro found in birjung, nepal shristi poudel sharma1, bishal thagunna2,*, rosy baral2, rashmi baral2, deepa khadka1 1department of food microbiology, 2department of food technology, tribhuvan university, pokhara, kaski, nepal. corresponding author* bisu.thaguna123@gmail.com manuscript received: 23 april, 2025. revision accepted: 27 july, 2025. published: 01 august, 2025. abstract taro corms (colocasia esculenta), also called pindalu in nepalese society are exposed to different pretreatment such as boiling in plain water at 100ºc for 7min, boiling in 1.2% salt solution at 100ºc for 7min, boiling in 5% citric acid100ºc for 7min and frying was investigated for proximate, antinutritional and mineral content. the proximate composition of raw taro corms was found to be moisture 60.82%, crude fat 0.96%, crude protein 9.69%, total ash 3.77%, crude fiber 3.49% and carbohydrate 52.0%. macro nutrients such as potassium, calcium, phosphorous, and sodium were found to be 620.55 mg/100g, 150.12 mg/100g, 53.72 mg/100g, and 36.62 mg/100g, respectively. antinutritional factors of raw taro corms analyzed in this study were oxalate-280.98±0.49 mg/100g, phytate-84.90±0.74 mg/100g and tannin-47.67±0.11 mg/100g. potassium was the most abundant macro mineral (620.55mg/100g) in the unprocessed taro corms. the effect of pretreatments and frying on calcium showed significant decrease. when compared with raw taro corms, pretreatments and frying process resulted in a significant increase in phosphorous and sodium content. antinutritional factors were significantly reduced by the pre-treatments, and frying method appears to be more effective in reducing phytate and tannin, whereas boiling in 5% salt solution for oxalate content. keywords: colocasia esculenta; macro-nutrient; boiling; frying; anti-nutrients. introduction taro, or cocoyam, is the common name for an edible corm that belongs to the araceae family, which plays an important role in food security in many developing countries and regions of the subtropics and tropics of the world. the term taro is usually used to indicate colocasia esculenta (l.) (saleh, 2019). nutritionally, the taro corms are considered source of starch easily digestible, also containsignificant levels of protein, thiamine, riboflavin, niacin and vitamin c (john et al., 2007). typically, the nutritional value of food is influenced by the levels of nutrients present, how effectively these nutrients can be utilized, and the existence or non-existence of anti-nutritional substances and harmful factors (alcantara et al., 2013). similar to many plant-based foods, taro has a range of antinutritional and toxic substances, including oxalates, phytates, trypsin and amylase inhibitors, tannins, and cyanide (adane et al., 2013). to minimize the impact of anti-nutrients, which can pose certain health risks, it is important to process foods appropriately before eating. cooking enhances digestibility, increases palatability, extends shelf life, and also ensures that root vegetables are safer for consumption (azene and molla, 2017). oxalate can decrease the availability of minerals; a higher concentration of oxalate can bind to the calcium found in food, making it unavailable for its essential physiological and biochemical functions, such as supporting strong bones and teeth, acting as a cofactor in enzymatic reactions, facilitating nerve impulse transmission, and serving as a clotting factor in the blood. insoluble calcium oxalate may also accumulate in soft tissues, such as the kidneys, leading to the formation of kidney stones. the reduction of calcium can result in the deterioration of bones and teeth as well as disrupt the blood clotting process (ayele et al., 2015). phytate has been identified as an antinutrient because of its negative effects. it decreases the bioavailability of minerals and leads to inhibited growth. phytate creates insoluble complexes with cu²⁺, zn²⁺, fe³⁺, and ca²⁺, consequently diminishing the availability of these vital minerals. studies involving animal feed with plant-based diets indicate that the reduced bioavailability of zn, ca, mg, p, and fe is due to the presence of phytate. notably, deficiencies in zn and fe have been reported as a result https://doi.org/10.14421/biomedich.2025.141.507-511 508 biology, medicine, & natural product chemistry 14 (1), 2025: 507-511 of high phytate consumption (greiner and konietzny, 2006; greiner et al., 2006). another factor that negatively affects nutrition in taro is tannin. tannin is a water-soluble compound with a high molecular weight that significantly impacts nutritional values. it possesses unique characteristics, such as the ability to precipitate proteins, alkaloids, and gelatins. foods that are high in tannins are regarded as having low nutritional value because they cause the precipitation of proteins and hinder digestive enzymes and reabsorption. additionally, it affects the utilization of vitamins and minerals in meals (azene and molla, 2017). materials and method sample collection 7 kg of fresh taro corms required for the preparation of chips were collected from the local market of birgunj, nepal. the less acrid local variety known as kharipindalu was selected as raw materials for the chip’s preparation. corms were then cleaned and sorted to remove dust, foreign matter and damaged parts. taro corms of almost uniform size were selected for further processing and stored at low temperatures. determination of proximate composition the moisture, crude fat, crude fiber, and ash content of the raw taro were analyzed using the procedure outlined by (aoac, 2006). the protein level was assessed with the micro kjeldahl method (n x 6.25), while the carbohydrate content was derived by subtraction. methodology sample preparation the taro corms were cleaned and peeled with a stainlesssteel knife, and any undesirable sections were discarded. additionally, taro corm slices were produced using a hand slicer in the kitchen. the slices were rinsed with cold water for three minutes to eliminate any mucus and other substances before draining the excess water. the obtained taro slices were further divided into three treatments: (a) boiling in plain water at 100ºc for 7 min, (b) boiling in citric acid 1.2% at 100ºc for 7 min, and (c) boiling in salt solution 5% at 100ºc for 7 min (d) frying at 180 ºc until crisp and a golden brown respectively (kumar et al., 2017). determination of anti-nutrients determination of oxalate content the oxalate content of samples was determined as described by (onwuka, 2005). the procedure involves three steps: digestion, oxalate precipitation and permanganate titration. determination of tannin tannin content was determined using the method by (atanassova and christova, 2009). determination of phytate phytate content was determined as described in (azene and molla, 2017) statistical analysis all the experiments were performed in triplicate. the result was evaluated by using analysis of variance using statistical software spss v20 and ms excel 2016 at 5 % level of significance. result and discussion table 1. proximate composition of selected raw taro corm. parameters value (%) moisture 60.82±0.51 crude fat 0.96±0.61 crude protein 9.69±0.52 total ash content 3.77±0.10 crude fiber 3.49±0.24 carbohydrate 52.0±1.30 values are means ± standard deviation of triplicate analysis. the moisture content, crude fat, crude protein, ash content, crude fiber, and carbohydrates were measured at (60.82, 0.96, 9.69, 3.77, 3.49, and 52.0) %, respectively as shown in table 1. (azene, 2017) reported that the moisture content of fresh raw taro was 67.64, which is somewhat slightly higher than the findings mentioned above. (beno et al., 2022) discovered that the moisture content of tannia cocoyam was 72.91 %, which is relatively higher than the above study. likewise, (adane et al., 2013) indicated a moisture content of 80.02%, which is significantly higher than the results presented earlier. a research study by (azene, 2017) identified the crude fat, crude protein, ash content, and crude fiber levels as (0.67, 6.62, 3.92, 5.8) %. when compared to the table mentioned above, the ash content was noted to be higher, whereas the other parameters were slightly lower than the figures presented in the table. according to (kumar et al., 2023), the levels of crude fat, crude protein, ash content, crude fiber, and carbohydrates were recorded at (0.90, 4.0, 4.92, 2.84, and 75.90) %. in comparison to the table above, the ash content, fiber, and carbohydrate content were observed to be higher, while the remaining parameters were lower than the previous results. sharma et al. – effect of different pre-treatments and frying process … 509 table 2. antinutritional content of raw, treated, and fried taro chips. antinutritional factors a b c d e oxalate 280.98±0.49 128.76±0.12 82.09±0.77 69.61±0.12 150.22±0.66 phytate 84.90±0.74 80.32±0.12 76.45±0.25 76.45±0.25 70.77±0.49 tannin 47.67±0.11 43.68±0.18 33.69±0.69 24.70±0.20 13.31±0.22 values are means ± standard deviation of triplicate analysis. here a* raw, b* boiled in plain water, c* boiled in 1.2 % citric acid, d* boiled in 5% salt solution, and e* fried taro chips. oxalate the content of oxalate of raw, plain water boiled, treated with 1.2% citric acid, treated with 5% salt solution and fried chips samples were (280.98, 128.76, 82.09, 69.61, 150.22) mg/100g. raw taro corm had the highest oxalate content which then significantly decreased when processed into different treatments and fried (table 2). boiling in 5% salt solution at 100ºc for 7min, significantly reduced higher percentage of oxalate content as compared to the other samples. similarly, boiling in a plain water and 1.2% citric acid solution at 100º c for 7 minutes also reduced oxalate concentration compared to frying technique. (azene and molla, 2017), also found that boiling methods successfully reduced the oxalate levels in the tuber under study. the greatest reduction in oxalate levels was observed when the raw taro corm was boiled. boiling results in significant cell damage, which helps soluble oxalate seep into the cooking water, and this pattern was consistently noted across observations. washing, peeling, dicing, and soaking are examples of treatment procedures that can lower the oxalate content before frying. additionally, it was shown that heating decreased the oxalate concentration by 56.7% while soaking decreased it by 23.5%. the amount of oxalate in the samples significantly decreased after frying (huang et al., 2007; azene and molla, 2017). conversely, several studies have explored methods for pre-treating taro corm to lower its total oxalate levels. (sefa and agyir-sackey, 2004) found that the most effective processing method for decreasing total oxalate levels in taro corm sections was drying using a drum dryer, which successfully reduced the oxalate content by approximately 50%. (kumoro et al., 2014) discovered that soaking taro root chips in a 10% baking soda solution for 2 hours at room temperature was the most effective method for lowering calcium oxalate levels. the findings do not align with the data provided here, which is attributable to the varying methods employed to mitigate oxalate risk in taro corm. consequently, the identified forms of oxalate varied. phytate the phytate content of raw taro corms was 84.90 mg/100g. however, this result was lower as reported by (adane et al., 2013) for raw taro in ethiopia and (huang et al., 2013), for cultivars of taro grown in taiwan. also, (saleh, 2019), found that the phytic acid in raw taro chips was 161.16 mg/100 which is higher than the above result. (kumar et al., 2005), generally found that the levels of phytic acid vary based on several key factors, including the variety, geographical location, climate, irrigation practices, soil characteristics (such as ph, temperature, and the availability of organic phosphorus), and the specific year of the crop. the data presented in table (2) show phytate content of taro corm chips undergo three different treatments and frying processes. there were observed that significant differences among all three treatments and frying technique on the phytate content of raw taro corms. the above data also represented that frying process significantly reduced the phytic acid from 84.90 mg/100g to 70.77 mg/100g respectively. (patterson et al., 2017) noted that soaking pulses in water led to a slight reduction in phytates when they examined how processing impacted various anti-nutritional components in different pulses. the extent of this reduction may vary based on the duration of the soaking period. (saleh, 2019), soaked taro corm chips soaked for 60 min in solutions of different calcium salts and they found the treatments with calcium chloride at concentration 5% had the greatest effect. the treatment helped to reduced phytic acid from 161.16mg/100g to 133.68mg/100g. this result support the above table the salt treatment also reduce the phytic acid from 84.90 to 76.45 mg/100g. as noted by (azene and molla, 2017), the reduction in phytate levels during cooking methods like boiling or frying might be partially attributed to the creation of insoluble complexes involving phytate along with other elements such as protein or minerals associated with protein. additionally, phytate might become soluble at extremely high temperatures, such as those reached during boiling (100°c) and baking (190°c), as indicated by the notable decrease in phytate levels observed during frying and boiling (adane et al., 2013). frying process was more effective on decreasing phytic acid content. tannin tannin is a water-soluble substance with a high molecular weight that significantly affects nutritional quality. it possesses unique characteristics, including the ability to cause the precipitation of alkaloids, gelatins, and proteins. foods high in tannin is often regarded as having low nutritional value because they cause protein precipitation, hinder digestive enzymes, and affect nutrient reabsorption. it also impacts the absorption of vitamins and minerals from the diet (tinko and uyano, 2001; azene and molla, 2017). in this study the amount of tannin of raw taro corm was 47.67mg/100g which is lower than (azene and molla, 2017) study. from the 510 biology, medicine, & natural product chemistry 14 (1), 2025: 507-511 result obtained in this study boiling in plain water, 1.2% citric acid, 5% salt solution and frying resulted in significant decrease in tannin and highest decrease observed in frying process from (47.67 to 13.31) mg/100g. this result agree with the findings of researchers done on effects of processing on antinutritional factors on taro cultivars (akpan et al., 2004; onu and madubuike, 2006). boiling treatments also lowered the tannin levels, which occurs because boiling might extract the hydrolysable tannins into the water (richelle et al., 2013). this result is also supported by the (lewu et al., 2010). the maximum safe tannin consumption for a male is 560 mg/kg (stephene, 2004) therefore, the tannin levels in the taro are minimal and fall within acceptable limits, making them safe for consumers (stephene, 2004). table 3. mineral content of raw, treated, and fried taro chips. components a b c d e potassium (mg/100g) 620.55 480.78 492.16 498.06 350.52 calcium (mg/100g) 150.12 145.20 143.12 142.10 110.22 phosphorous (mg/100g) 53.72 56.23 57.12 58.03 58.05 sodium (mg/100g) 36.62 42.82 45.55 50.56 48.23 values are means ± standard deviation of triplicate analysis. here a* raw, b* boiled in plain water, c* boiled in 1.2 % citric acid, d* boiled in 5% salt solution, and e* fried taro chips. potassium the presence of four macro minerals was examined in both processed and unprocessed taro. potassium was found to be the most plentiful macro mineral (620.55mg/100g), while calcium ranked as the second most prevalent mineral (150.12mg/100g) in unprocessed taro. phosphorous was the third most abundant mineral observed in fried taro chips, amounting to 58.05mg/100g, and a significant quantity of sodium (50.56mg/100g) was also recorded in taro boiled in a 5% salt solution at 100 degrees for 7 minutes. the result indicated that raw taro corm when undergoing different treatments had a significant effect (p≤0.05) on the mineral content. (godfrey et al., 2022), found the potassium, calcium, sodium and phosphorus content of raw yam 72.23 mg/100g, 190.57 mg/100g, 32.05 mg/100g and 193.1 mg/100g respectively. this value supported the above result except the value of potassium. similarly, (azene and molla, 2017), investigated the mineral content of raw tubers and processed tubers and found that significant increase or decrease in mineral content by the processing technique. this result is also similar to the above result. processed samples of raw taro corm showed significant differences from raw ones. this was also supported by the findings (akpan et al., 2004). generally, processing resulted in a significant rise in phosphorus levels, a finding corroborated by (abera et al., 2013), which showed that the phosphorus content of taro significantly increased following boiling and fermentation. the mean concentration of sodium also showed a significant increase as a result of processing. this observation aligns with the work of (azene and molla, 2017), where boiling raised sodium levels, while fermentation reduced its concentration, in contrast to the current findings of this study. the frying process boosted the amounts of both sodium and potassium. the minor reduction in calcium levels during the boiling process can be attributed to water absorption and leaching that occur while boiling. there is a notable decrease in calcium and potassium levels during frying, which is caused by breakdown and chemical alterations such as oxidation, and some calcium and potassium may also be lost as volatile compounds, leading to this decline (olajide et al., 2011; azene and molla, 2017). conclusion taro corms (colocasia esculenta), also called pindalu in nepalese society were analyzed and found to be a good source of carbohydrates, rich in minerals including potassium, calcium, phosphorous, and sodium. many plant-based foods contain anti-nutrients that serve as a defense mechanism against herbivores and pathogens. anti-nutrients can be detrimental to human health as they hinder the digestion and absorption of vitamins, minerals, and other nutrients, leading to long-term deficiencies. raw taro corm contains some antinutritional factors such as oxalate, phytate and tannin which can limit the utilization of taro nutrients for human consumption and animal feed. boiling, chemical treatments and frying process of raw taro corms significantly reduced all the antinutritional that were analyzed in this study. acknowledgement: the authors are sincerely thankful to the pokhara bigyan tatha prabidhi campus for chemical and equipment support. conflicts of interest: the authors declare no conflict of interest references adane t, shimelis a, negussie r, tilahun b, g. h. (2013). effect of processing method on the proximate. african journal of sharma et al. – effect of different pre-treatments and frying process … 511 food, agriculture, nutrition and development, 13(2), 7383– 7398 aoac (2006). association of official analytical chemists, official methods of analysis (19th ed.). gaithersburg: aoac press, usa ayele, e., urga, k., & chandravanshi, b. s. (2015). effect of cooking temperature on mineral content and anti-nutritional factors of yam and taro grown in southern ethiopia. international journal of food engineering, 11(3), 371–382. https://doi.org/10.1515/ijfe-2014-0264 azene, h., & molla, t. (2017). nutritional composition and effects of cultural processing on anti-nutritional factors and mineral bioavailability of colocasia esculenta (godere) grown in wolaita zone, ethiopia. journal of food and nutrition sciences, 5(4), 147-154. atanassova, m., & christova-bagdassarian, v. 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(2019). reducing the soluble oxalate and phytic acid in taro corm chips by soaking in calcium salt solutions. alexandria journal of food science and technology, 16(2), 9–16. https://doi.org/10.21608/ajfs.2019.20766.1015 stephene, s. (2004). interaction of grape seed procyanidins with various proteins about wine findings. journal of science and food agriculture, 57, 111-125. tilahun abera teka, shimelis admassu emire, gulelat desse haki, tilahun bekele gezmu. (2013). effect of processing on physicochemical composition and anti-nutritional factors of cassava (manihot esculenta crantz) grown in ethiopia. international journal of science innovations and discoveries. 3(2): 212-222. tinko n and k uyano. (2001). spectrophotometric determination of the tannin contents of various turkish black tea, beer and wine samples. international journal of food sciences and nutrition. 52: 289–294 https://doi.org/10.5897/ajfs2015.1370 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 241-250 | doi: 10.14421/biomedich.2023.121.241-250 issn 2540-9328 (online) synthesis and characterization of cinnamon loaded bsa microparticles with antidiabetic properties department of chemistry, university of kelaniya, sri lanka. corresponding author* bimali@kln.ac.lk manuscript received: 03 february, 2023. revision accepted: 01 march, 2023. published: 03 march, 2023. abstract conventional medicine that is being used to treat diabetes exert adverse side effects and therefore scientists have focused on natural hypoglycemic agents. “sri wijaya” (ccsw) is an accession of cinnamomum zeylanicum, which shows higher hypoglycemic activity. pressured water extract of its dried quills can be used as an antidiabetic nutraceutical. higher stability, ease of storage and transportation, make powder form nutraceuticals more preferred. the objective of this study was to develop cinnamon encapsulated microparticles as a powder form nutraceutical with higher hypoglycemic activity. four different products were synthesized. two of them were synthesized using bovine serum albumin (bsa) (8.8 % (w/v), ph=5) in the presence of citric acid and ascorbic acid as cross-linking agents separately. the other products were synthesized using bsa (20 mg/ ml, ph=9) in the presence of same cross-linking agents. antidiabetic activity of the products was determined using alpha-amylase and alpha glucosidase inhibition assays and compared with that of crude cinnamon extract and positive control acarbose. since the product synthesized using bsa (20 mg/ ml, ph=9) and citric acid showed the highest alpha amylase inhibition activity, solubility, cinnamon loading percentage and cinnamon entrapment efficiency those conditions were concluded as the optimum conditions required to synthesize microparticles with higher hypoglycemic activity. particle size, polydispersity index, and zeta potential of that product were 1.281 ± (0.004) µm, 0.460 ± (0.018) and -1.09 ± (0.03) mv respectively. according to the sem image, microparticles have a spherical morphology. the uv-visible spectrum and the ft-ir spectrum confirm the entrapment of cinnamon compounds. keywords: cinnamon; cross-linking agent; diabetes; microparticles; nutraceutical. abbreviations: ccsw: cinnamomum zeylanicum “sri wijaya”; bsa: bovine serum albumin; clmp: cinnamon loaded microparticles. introduction diabetes mellitus, simply referred to as diabetes is the most common endocrine illness (hudaib et al., 2018). in 2008, world health organization has estimated that approximately 2.9 million deaths occur due to diabetes mellitus every year (desoky et al., 2012). the major types of diabetes mellitus are type 1 diabetes and type 2 diabetes (njagi et al., 2014). type 1 diabetes is an autoimmune disease, and it typically develops in children and young adults. the immune system of the individuals who suffer from this disease, mistakenly destroys the pancreatic beta cells which make insulin. type 1 diabetes patients should regularly administer insulin medication. the most common kind of diabetes mellitus is the type 2 diabetes (riaz, 2009). this is a disorder which can be characterized by insulin resistance and beta cell dysfunction (desoky et al., 2012). since current conventional diabetes medicines exert adverse side effects, scientists have focused more on hypoglycemic agents of natural sources, which have been used in traditional medicine systems such as ayurveda (desoky et al., 2012). nearly 72.8 % of the world diabetic population rely on herbal medicine for the treatment of their diabetes mellitus (hudaib et al., 2018). in traditional medicine systems of china, korea and russia, cinnamon has been used as a medicine for diabetes mellitus (hudaib et al., 2018). various chemical compounds in cinnamon can contribute for the antidiabetic property in different ways. methyl hydroxychalcone polymer of cinnamon has an ability to stimulate glucose oxidation, thus helps to decrease the glucose content. some compounds in cinnamon such as polyphenol type-a polymers act as insulin (gan and rao, 2014). cinnamon can reduce the intestinal glucose absorption by inhibiting the enzymes like α-amylase and α-glucosidase which involve for the carbohydrate metabolism. a proanthocyanidin called cinnamtannin b1 is responsible for this activity. the same compound can further contribute to decrease the blood glucose level by b. s. wanniarachchi, h. m. w. k. sathsarani, b. m. jayawardena*, h. g. n. dewangani https://doi.org/10.14421/biomedich.2023.121.241-250 242 biology, medicine, & natural product chemistry 12 (1), 2023: 241-250 stimulating cellular glucose uptake by membrane translocation of glut-4, stimulating glycogen synthesis, inhibiting gluconeogenesis, and stimulating insulin release. cinnamon can also act against diabetic retinopathy and neuropathy (constantine et al., 2013). there are approximately 250 species of cinnamon all over the world (gan and rao, 2014). among them ceylon cinnamon (cinnamomum zeylanicum) can be consumed in higher doses for longer durations without toxic effects due to the very low coumarin content. ccsw is one of the accessions of cinnamomum zeylanicum that is a newly developed accession of cinnamon, and it shows higher anti-diabetic activity (wariyapperuma et al., 2020). therefore, ccsw accession of ceylon cinnamon can serve as a promising candidate to develop antidiabetic nutraceuticals. in developing effective antidiabetic nutraceuticals from ceylon cinnamon, the active compounds should be extracted. according to the previous studies, pressured water extraction is the most effective method to prepare an aqueous extract of cinnamon with antidiabetic activity. it is desirable to have aqueous plant extracts of plants in the powder form to enhance the durability, stability and the ease of storage and transportation. plant extracts can be encapsulated in microparticles to convert them into more stable form (amritham et al., 2016). microencapsulation is an advanced technology using which one or more active compounds can be encapsulated within an inert material, making a tiny sphere of diameter ranging from 1 µm to several 100 µm. microencapsulation is performed for protecting the sensitive bioactive compounds as well as for their safe delivery. nowadays bsa is used as a common encapsulant since the microparticles produced using this protein are less toxic, nonantigenic, biocompatible and biodegradable (choudhury et al., 2021). microparticulate drug delivery systems proffer a number of significant advantages including effective protection of the encapsulated active agent against enzymatic degradation, enhancement of peptide stability, sitespecific and controlled drug release (wong et al., 2018; singh et al., 2010) the objective of the study was to synthesize and characterize cinnamon encapsulated bsa microparticles with antidiabetic properties. materials and methods raw materials cinnamomum zeylanicum quills (accession: sri wijaya) chemicals bovine serum albumin (bsa), ethanol, citric acid, ascorbic acid, sodium hydroxide, hydrochloric acid, acarbose, alpha amylase, starch, dinitrosallicylic acid reagent (dns), sodium hydrogen phosphate, sodium dihydrogen phosphate, alpha glucosidase, paranitrophenyl-d-glucopyranoside (pnpg), dimethyl sulfoxide (dmso) and anhydrous kbr instruments electric grinder (sumeet, india), analytical balance (kern alj 120-4 germany), pressure cooker (prestige india), centrifuge machine, magnetic stirrer, ph meter, thermometer, electric oven, microplate reader (spectra max m5, molecular devices, ca, usa), malvern zetasizer nano zs apparatus (malvern instruments ltd., malvern, uk), field emission scanning electron microscope (hitachi su6600 fe-sem), uv-vis spectrophotometer (agilent technologies, germany. cary 60), ft-ir (fourier transform infrared) spectrometer (perkinelmer, l 1600300 spectrum two lita, liantrisant, uk). preparation of cinnamon quills dried quills of cinnamomum zeylanicum (which were obtained from cinnamon research institute, thihagoda, matara) were ground into fine powder and stored at 10◦c. preparation of the extract cinnamon powder (10.00 g) was mixed with distilled water (200.0 ml) and digested under pressure for 10 minutes using a pressure cooker. the extract was filtered through a muslin cloth. the filtrate was centrifuged at 3000 rpm for 15 minutes and then the supernatant was used. preparation of cinnamon loaded microparticles (clmp) based on previous research studies, two different bsa solutions and two different crosslinking agents were used to prepare four products as mentioned in the table 01. ph values of bsa solutions were set to the required value by using naoh (1 m) and hcl (1 m) solutions. table 1. bsa solutions and crosslinking agents which were used to prepare microparticles. product bsa solution crosslinking agent concentration ph a 20.00 mg/ ml 9 citric acid b 20.00 mg/ ml 9 ascorbic acid c 8.85 % w/v 5 citric acid d 8.85 % w/v 5 ascorbic acid for the synthesis of cinnamon loaded bsa microparticles, the aqueous cinnamon extract (4.00 ml) was mixed with ethanol (16.00 ml). the mixture was added to a bsa solution (4.00 ml) at 1.00 ml/ minute addition rate while constantly stirring the bsa solution at 600 rpm. during the addition process the temperature of the system was maintained at around 4 ◦c. then the crosslinking agent (8 %w/v, 230 µl) was added. the wanniarachchi et al. – synthesis and characterization of cinnamon loaded bsa microparticles … 243 reaction mixture was stored for twenty-four hours at 4 ◦c. the microparticle bearing solution was centrifuged at 3000 rpm for 30 minutes and the microparticles were collected. the particles were washed with distilled water and dried at 50 ◦c until a constant weight was observed. the particles were stored at 4 ◦c for further analyses. determining the yield of clmp weight of the product was measured. total concentration of all compounds in cinnamon extract was determined by evaporating cinnamon extract (1.00 ml) and weighing the remainder. the yield was calculated according to the following equation. yield = weight of the product (g) weight of used bsa + weight of cinnamon extract × 100 determining antidiabetic activity of clmp antidiabetic activity of the pressured water extract of cinnamon quills, positive control acarbose and the clmp were determined by carrying out in-vitro alpha amylase inhibition assay and alpha glucosidase inhibition assay. the antidiabetic activity of the products was compared with the antidiabetic activity of the acarbose and the crude extract. alpha amylase inhibition assay alpha amylase inhibition activity of clmp, pressured water extract of cinnamon and acarbose were determined by following the method described by aiyegoro et al. (2017) with slight modifications. (aiyegoro et al., 2017) alpha amylase enzyme (0.05 mg ml-1, 250 µl) in sodium phosphate buffer (0.02 m, ph 6.9) was added to the sample (500 µl). the mixtures were incubated at room temperature for 15 minutes. then starch solution (1 %, 250 µl) in sodium phosphate buffer (0.02 m, ph 6.9) was added. the reaction mixtures were incubated at room temperature for another 15 minutes. dns reagent (250 µl) was added and boiled for 5 minutes, and absorbance was measured at 540 nm by microplate reader. the same procedure was repeated by adding sodium phosphate buffer (0.02 m, ph 6.9, 250 µl) instead of alpha amylase enzyme. in addition to that, the above procedure was repeated for two other reaction mixtures. one amongst them was prepared by adding sodium phosphate buffer (0.02 m, ph 6.9, 500 µl) instead of plant extract and the other was prepared by adding sodium phosphate buffer (0.02 m, ph 6.9, 750 µl) instead of both plant extract and the enzyme. the percentage inhibition of alpha amylase enzyme was calculated using the following formula. ic50 values on alpha amylase enzyme were determined by using graphpad prism 8 software, based on inhibitory percentage values. inhibition (%) = (p-q) (r-s) (p-q) × 100 p : absorbance at 540 nm without inhibitor and with enzyme q : absorbance at 540 nm without inhibitor and enzyme r : absorbance at 540 nm with inhibitor and enzyme s : absorbance at 540 nm with inhibitor and without enzyme alpha glucosidase inhibition assay alpha glucosidase inhibition activity of the products, pressured water extract of cinnamon and acarbose were determined by following the method described by jayawardena et al. (2018) with slight modifications. [14] alpha glucosidase enzyme (0.5 u ml-1, 25 µl) in sodium phosphate buffer (0.10 m, ph 6.8) was added to the sample (50 µl) and incubated at 37 ◦c for 10 minutes. p-npg (1.25 mm, 25 µl) in sodium phosphate buffer (0.10 m, ph 6.8) was added and incubated at 37 ◦c for 10 minutes and the absorbance was measured at 405 nm by a microplate reader. the same procedure was repeated by adding sodium phosphate buffer (0.10 m, ph 6.8, 25 µl) instead of the enzyme, sodium phosphate buffer (0.10 m, ph 6.8, 50 µl) instead of cinnamon extract and sodium phosphate buffer (0.10 m, ph 6.8, 75 µl) instead of both cinnamon extract and enzyme. the percentage inhibition of the alpha glucosidase enzyme was calculated using the following formula. ic50 values on alpha glucosidase enzyme were determined by using graphpad prism 8 software, based on inhibitory percentage values. inhibition (%) = (p-q) (r-s) (p-q) × 100 p : absorbance at 405 nm without inhibitor and with enzyme q : absorbance at 405 nm without inhibitor and enzyme r : absorbance at 405 nm with inhibitor and enzyme s : absorbance at 405 nm with inhibitor and without enzyme characterization of microparticles solubility clmp (5.0 mg) was added to 5.00 ml of distilled water and stirred for 30 minutes at 600 rpm at room temperature. after that the mixture was centrifuged at 10 000 rpm for 15 minutes. pellet was dried at 100 ◦c in an oven until a constant weight was observed. the procedure was triplicated. the solubility was calculated according to the following formula. 244 biology, medicine, & natural product chemistry 12 (1), 2023: 241-250 solubility (%) = weight of the product added (g) weight of the pellet (g) weight of the product added (g) × 100 cinnamon loading and entrapment efficiency a concentration series from the pressured water extract of cinnamon quills was prepared. absorbance of the concentration series was measured at 310 nm. a graph of absorbance at 310 nm vs concentration of cinnamon extract was plotted (akkawi et al., 2015). absorbance of the supernatants which remained after the separation of the product containing pellets, were measured at 310 nm. by comparing the absorbance value of each supernatant with the standard curve, the concentration of remaining cinnamon compounds in the supernatants was found. the procedure was triplicated. cinnamon loading and entrapment efficiencies were determined by using following formulae. weight of cinnamon loaded = weight of cinnamon added weight of cinnamon in supernatant cinnamon loading (%) = weight of cinnamon loaded total weight of the product × 100 cinnamon entrapment efficiency (%) = weight of cinnamon loaded weight of cinnamon added × 100 determination of particle size and zeta potential the particle size, polydispersity index (pdi) and zeta potential of the product were determined with the malvern zetasizer nano zs apparatus. an aqueous suspension of clmp was diluted 1:100 with ultrapure water and the solution was placed in a disposable polystyrene cuvette and the particle size measurement was obtained. the solution was also placed in a folded capillary zeta cell and the zeta potential measurement was obtained. both procedures were triplicated. morphological observations field emission scanning electron microscope was used to visualize the morphology and shape of the oven-dried product. sample was mounted onto the sample stub using carbon tapes and the images were taken after gold sputter coating for 15 seconds. uv-visible absorbance spectra the uv-visible absorption spectra were analyzed using a uv-visible spectrophotometer from 200 to 500 nm within a 1 cm quartz cell. a 0.8 mg/ml synthesized nanoparticle sample was prepared after dissolving it in dmso and its absorbance spectrum was obtained. it was then compared with the absorbance spectra of 1 mg/ml pure bsa, 0.07 mg/ml aqueous cinnamon extract and 0.8% w/v citric acid. the absorbance spectrum of distilled water was subtracted from all sample spectra. ft-ir spectroscopy the molecular characteristics of the product were examined and compared with that of pure bsa, oven dried aqueous cinnamon extract and pure cross-linking agent, citric acid using an ft-ir spectrometer. each sample was mixed with anhydrous kbr in a 1:10 ratio and ground using a motor and pestle until a fine powder was obtained. a small portion of the powder was placed in the pellet forming mold and pressed under pressure. then the pellet was placed in the ft-ir spectrometer and scanned in the wavenumber range of 750-4000 cm-1. statistical analysis the obtained data were statistically analysed by one-way analysis of variance (anova) using minitab software package. the results were expressed in the form of mean ± standard deviation of triplicate determinants. the level of significance was taken at 5% confidence interval (p < 0.05). results and discussion physical appearance of the products table 2. yield and morphology of the products. product yield (%) morphology a 71.50 brown colour powder b 75.36 brown colour powder c 69.62 brown colour gel d 67.99 brown colour gel wanniarachchi et al. – synthesis and characterization of cinnamon loaded bsa microparticles … 245 antidiabetic activity table 3. ic50 values on alpha amylase and alpha glucosidase enzymes. sample ic50 value on alpha amylase enzyme (µg/ml) ic50 value on alpha glucosidase enzyme (µg/ml) pressured water extract 131.27 (±1.64) e 141.25 (±0.21) b acarbose 140.37 (±1.17) c 224.45 (±0.21) a a 117.60 (±1.73) f 119.25 (±0.07) e b 134.97 (±0.32) d 112.40 (±0.57) f c 151.00 (±0.76) a 137.75 (±0.21) c d 146.23 (±0.56) b 122.70 (±0.28) d means that do not share a letter are significantly different from each other. (p < 0.05) figure 1. the graph of percentage inhibition of α amylase enzyme (%) vs concentration (µg/ ml). figure 2. the graph of percentage inhibition of α glucosidase enzyme (%) vs concentration (µg/ ml). characterization of the products table 4. solubility, cinnamon loading percentage and cinnamon entrapment percentage of the products. product solubility in water (%) cinnamon loading percentage (%) cinnamon entrapment efficiency (%) a 53.00 (±1.00) a 3.69 (±0.01) a 77.97 (±0.03) a b 49.00 (±1.00) a 3.33 (±0.10) b 74.11 (±0.02) b c 30.33 (±1.53) b 0.76 (±0.00) c 67.51 (±0.02) c d 22.33 (±2.52) c 0.72 (±0.01) c 62.03 (±0.05) d means that do not share a letter are significantly different from each other. (p < 0.05) table 5. particle size, pdi and zeta potential of the product. parameter mean ± standard deviation particle size 1.281 ± (0.004) µm pdi 0.460 (± 0.018) zeta potential -1.09 (±0.03) mv figure 3. sem image of clmp. 246 biology, medicine, & natural product chemistry 12 (1), 2023: 241-250 figure 4. uv-visible absorbance spectra of pure bsa, aqueous cinnamon extract, citric acid, and clmp. figure 5. ft-ir spectra of pure bsa, aqueous cinnamon extract, pure citric acid, and clmp. discussion micro formulations are an exciting novel method that could be used to improve the stability, bioavailability, poor solubility and absorption of natural products (singh et al., 2010). even though cinnamon has many therapeutic properties, the desired effect could be lost in processing cinnamon products. encapsulating natural products into various matrices is a common technique used to overcome the loss of the active ingredients in natural products during processing. in the current study, 4 different products (product a, b, c and d) were formulated by encapsulating cinnamon into bsa, as mentioned in table 1. products a and b were in powder form while products c and d were gel like. for the preparation of products c and d, the ph value of the bsa solution was set to 5. the isoelectric point of bsa is around 4.7 (amritham et al., 2016). hence ph value of the used bsa solution was very close to the isoelectric point of bsa. therefore, at ph 5, the bsa molecules show less tendency to become ionized. hence the aggregation of bsa molecules with each other occur rapidly in order to minimize the contact with highly polar aqueous medium. this may be the reason for the formation of gel like product instead of a powder form product. for the preparation of products a and b, the ph of the bsa solution was set to 9. at that ph value the protein molecules are ionized. therefore, they tend to repulse each other. this results the separation of the protein particles from each other as much as possible. hence the resulted particles become smaller and smaller (constantine et al., 2013; amritham et al., 2016). this can result in a powder like fine particle in microscale. according to table 2, the highest yield has been obtained for the product b which was followed by products a, c and d. this suggests that the highest yield 230 280 215 280 225 215 235 285 -0,1 0,4 0,9 1,4 1,9 2,4 2,9 3,4 200 250 300 350 400 450 500 a b so rb an ce wavelength (nm) absorbance of pure bsa absorbance of cinnamon extract absorbance of citric acid absorbance of cinnamon microparticles 0 20 40 60 80 100 7501250175022502750325037504250 tr an sm it ta n ce ( % ) wavenumber (cm-1) % transmittance of pure bsa % transmittance of citric acid % transmittance of cinnamon extract % transmittance of cinnamon microparticles wanniarachchi et al. – synthesis and characterization of cinnamon loaded bsa microparticles … 247 for clmp can be obtained by using ascorbic acid as the cross-linking agent if the ph value of the used bsa solution is around ph 9. the antidiabetic activity of natural products can be measured using the inhibitory potential of alpha-amylase and alpha-glucosidase enzymes. product a has shown the lowest ic50 value [117.60 (±1.73) µg/ml] (table 3). that means the product a has the highest inhibitory activity on alpha amylase enzyme. both products a and b have shown higher inhibitory activity than the crude cinnamon extract and the positive control acarbose. this confirms that the activity of cinnamon extract become enhanced when encapsulated. since products c and d are not powder form products, they may not be in microscale. that may be a reason for their low alpha amylase inhibitory activity. the graph of percentage inhibition on alpha amylase enzyme vs. concentration is also shown in figure 1. cinnamon loading percentage and the cinnamon entrapment efficiencies of the products also support the obtained results for the alpha amylase inhibitory assay (table 4). product a has shown the highest cinnamon loading percentage and cinnamon entrapment efficiency. that may be the reason for its higher activity than the other products. according to table 3, all the products have shown higher inhibitory activity on the alpha glucosidase enzyme in comparison with the crude cinnamon extract and acarbose. however, the inhibitory activity of products c and d is lower than that of products a and b. the higher activity of products a and b may be due to their smaller size and higher surface area. the lowest ic50 value [112.40 (±0.57) µg/ml] has been recorded for product b. therefore, the highest inhibitory activity on alpha glucosidase enzyme has been shown by product b even though the cinnamon loading percentage and cinnamon entrapment efficiency of product a is greater than that of product b. but the ic50 values for products a and b [119.25 (±0.07) µg/ml and 112.40 (±0.57) µg/ml respectively] are somewhat closer to each other. the graph of percentage inhibition on alpha glucosidase enzyme vs. concentration is shown in figure 2. the solubility of the products in water is also given in table 4. the water solubility of products a and b is greater than that of products c and d. as explained by amirtham et al. (2016), higher solubility of the product a and b may be due to their smaller size. smaller the size greater is the surface area. therefore, the solubility becomes greater (amritham et al., 2016; amighi et al., 2020). table 4 shows the cinnamon loading percentage of the products. the percentage values obtained for products c and d do not show statistically significant difference, but they show statistically significant difference to the results obtained for other products. the cinnamon loading percentage of products a and b is significantly greater than that of products c and d. the concentration of the bsa solutions which were used for the synthesis of products c and d were 8.8 % (w/v) while the concentration of the bsa solutions which were used for the synthesis of products a and b were 20 mg/ml. when the concentration of the initially used bsa solution becomes higher, the ratio between the loaded cinnamon compounds and bsa decrease. that is the reason for the observed results. the cinnamon loaded percentage of product a [3.69 (±0.01) %] is greater than that of product b [3.33 (±0.10) %]. according to amighi et al. (2020) the particles synthesized using citric acid as the cross-linking agent is greater in size compared to the particles synthesized using ascorbic acid as the cross-linking agent. when the size of the particle is greater, the particle can accommodate more bioactive compounds (amritham et al., 2016). that may be the reason for the slight increment of the cinnamon loading percentage of product a than product b. table 4 shows the cinnamon entrapment efficiencies of the synthesized products. the data obtained for all the products have shown statistically significant difference from each other. the highest cinnamon entrapment efficiency has been reported for product a, followed by product b, c and d. since products c and d were synthesized at ph 5, bsa molecules tend to aggregate with each other due to their less tendency to become ionized at ph values which are very close to their isoelectric point. the aggregated bsa molecules precipitated and formed the gel like product rapidly with the addition of the desolvating agent. the amount of time that the bsa particles spent in the solution while keeping contact with the dissolved cinnamon compounds was very little. most of the aggregated bsa particles precipitated even before the complete addition of the recommended volume of ethanol/ cinnamon extract mixture in previous studies [amirtham et al, (2016)] and the cross-linker. therefore, the probability to entrap more cinnamon compounds is comparatively less for products c and d. that may be the reason for the observed low cinnamon entrapment percentage for products c and d. products a and b were synthesized at ph 9 which is far away from the isoelectric point of bsa (4.7). hence bsa molecules were ionized and well separated from each other. at that ph value bsa particles do not move out from the solution phase rapidly. therefore, most of the bsa particles get the opportunity to stay in the solution phase until the complete addition of the ethanol/ cinnamon extract mixture and the cross-linker. this enhances the probability to entrap more cinnamon compounds during the formation of microparticles. hence the cinnamon entrapment efficiency of products a and b is greater than that of other products. however, the cinnamon entrapment efficiency of product a is little bit greater than that of product b. this may be again due to the greater size of the microparticles 248 biology, medicine, & natural product chemistry 12 (1), 2023: 241-250 which were synthesized using citric acid as the crosslinking agent. since the alpha amylase inhibitory activity, solubility, cinnamon loading percentage and cinnamon entrapment efficiency of product a is greater than other products, product a was selected for further studies. hence characterization using parameters such as particle size, surface charge, morphology, particle structure using ftir and cinnamon entrapment using uv-visible spectrophotometer were carried out only for product a. product a showed a mean particle diameter of 1.281 ± (0.004) µm (table 5). according to amighi et al. (2020), the particle size that is obtained under the conditions that were used in the synthesis of product a with citric acid as the cross-linking agent is 1.201 (±0.058) µm (amighi et al., 2020). as the mean particle size obtained for product a is very close to this value, it is clear that they have been formed effectively. according to danaei et al. (2018), the pdi of a sample with effective particle size distribution should be between 0.05-0.7. as per table 5, the pdi value of the product a falls within this range and therefore it can be stated that a homogenous population of microparticles have been synthesized which can act as safe, stable, and efficient microcarriers of cinnamon. the tendency of these bsa microparticles to accumulate in the target tissue, which depends on the particle size distribution, will be minimal (danaei et al., 2018). microparticles with a zeta potential between -10 and +10 mv are considered neutral, while microparticles with zeta potentials greater than +30 mv are considered strongly cationic and those with zeta potentials less than 30 mv are considered strongly anionic. since most cell membranes are negatively charged, zeta potential can strongly affect a microparticle’s tendency to penetrate membranes, with cationic particles displaying toxicity due to cell membrane disruption (clogston and patri, 2011). according to table 5, as the zeta potential of the synthesized microparticles is -1.09 mv, they are neutral and will easily penetrate the cell membranes without causing any toxicological effects. product a was also studied for morphology by sem. morphological analysis of the microparticles were carried out with fe-sem, and the obtained image is shown in figure 3. the sem micrograph revealed morphological aspects of microparticles with a spherical shape and uniform size. the uv-absorbance spectra of the cinnamon extract and clmp (figure 4) showed a peak at 215 nm, proving that the active compounds of the cinnamon extract have been successfully loaded into the microparticles. a peak corresponding to citric acid cannot be observed as it has been completely washed away during the washing step. the pure bsa spectrum showed two characteristic peaks at 230 nm and 280 nm. however, these two peaks were slightly shifted to 235 nm and 285 nm respectively, in the absorbance spectrum of clmp. this may have resulted due to the protein unfolding that may have occurred when the microparticle was dissolved in dmso, an organic solvent. this agrees with the findings of liu et al. (2009), which states that the spectrum shifts to shorter wavelengths as the polarity of the solvent decreases with the tryptophan residues buried in hydrophobic domains exhibiting a spectral shift of 5 to 20 nm. (liu et al., 2009). ft-ir spectroscopy was performed to the product in order to find out if any chemical bond formation has occurred between citric acid and bsa as well as between the active compounds of cinnamon and bsa during the preparation of microparticles, resulting in any conformational changes in the protein structure of microparticles. furthermore, it was performed to further verify if the cinnamon compounds have been successfully entrapped within the synthesized microparticles. the ft-ir spectrum of microparticles showed characteristic peaks that were also observed in the spectrum of the aqueous cinnamon extract (figure 5). peaks at 2750 cm-1 and 2850 cm-1 that may be due to the cho stretch of cinnamaldehyde, a peak at 1056 cm-1 and a peak at 3000 cm-1 that may be due to the c-o and oh stretches respectively, of cinnamyl alcohol, benzoic acid, benzyl alcohol and methoxyeugenol were observed in the spectra of both aqueous cinnamon extract and clmp. according to wariyapperuma et al. (2020), these are the major compounds that are found in the ccsw aqueous extract, and therefore this further proves that the active compounds present in ccsw accession have been successfully extracted and encapsulated within the synthesized microparticles. (wariyapperuma et al., 2020 ; jayawardena et al., 2018). according to xu et al. (2015), one to two carboxyl groups of one citric acid molecule can react with bsa, resulting in an increase in the total amount of carboxyl groups and a decrease in the total amount of amine groups in the cross-linked microparticles (xu et al., 2015). this explains the presence of a peak corresponding to the oh stretch and decrease in intensity of the peak at 3400 cm-1 corresponding to the n-h stretch in the microparticle spectrum. this means that interactions between bsa and citric acid have caused conformational changes in the protein structure of the synthesized microparticles. a similar observation has been made by amighi et al. (2020) during the synthesis of bsa microparticles using citric acid as the cross-linking agent (amighi et al., 2020) additionally, peaks at 1665 cm-1 and 1537 cm-1 observed in the pure bsa spectrum corresponding to the amide i and amide ii stretches respectively, were absent in the microparticle spectrum. hence, loading of active compounds of cinnamon have also induced conformational changes in the structure of the bsa protein. this means that the active compounds of cinnamon have interacted with the protein matrix of the synthesized microparticles via covalent bonds. this wanniarachchi et al. – synthesis and characterization of cinnamon loaded bsa microparticles … 249 result is similar to the results obtained by rani (2016) (rani, 2016). conclusions this study has proven that the antidiabetic activity of the pressured water extract of the quills of cinnamomum zeylanicum can be enhanced by micro-encapsulating the active compounds of the extract. for the microencapsulation of the bioactive compounds, bsa microparticles can be used as the microcarrier system. size of the microparticle, cinnamon loading percentage and cinnamon entrapment efficiency have a direct effect on the antidiabetic activity of the synthesized microparticles. if the size of the particle is in the microscale and the cinnamon loading percentage and cinnamon entrapment efficiency are higher, the activity of the particle becomes higher. the cross-linking agent, ph, and the concentration of the bsa solution which is used to prepare bsa microparticles can affect the abovementioned parameters and the solubility of the product in a significant manner. since the product ‘a’ has shown the highest alpha amylase inhibitory activity, cinnamon loading percentage, cinnamon entrapment efficiency and water solubility, it can be concluded that the clmp with higher antidiabetic activity and desired properties can be synthesized at ph 9, if the concentration of the bsa solution is 20 mg/ ml and the cross-linking agent is citric acid. characterization of product a revealed to have a spherical morphology, uniform size with effective particle size distribution and a neutral surface charge. the uv-visible absorbance spectrum of the product showed the successful entrapment of the cinnamon compounds within the microparticles and the ft-ir spectrum showed that the cross-linking agent, citric acid had caused conformational changes in the protein structure of bsa. it also further proved that the active compounds were successfully loaded into the synthesized microparticles which interacted with the protein matrix via covalent bonds. therefore, according to these characterization data of product a, it can be concluded that the clmp have been synthesized effectively, which can be used as a powder form antidiabetic nutraceutical. authors’ contributions: b.s. wanniarachchi: designed and performed the experiments, analyzed the data, prepared figures and/or tables and authored the article. h.m.w.k sathsarani: designed and performed the experiments, analyzed the data, prepared figures and/or tables and authored the article. b.m. jayawardena: planned and designed the experiments and gave final approval of the article. h.g.n. dewangani: designed the experiments and analyzed the data. competing interests: the authors declare that there are no competing interests. funding: this study was funded by the university grant rp/03/02/06/01/2021 which is gratefully acknowledged. references aiyegoro oa, coopoosamy rm, ijeh ii, ohanyerem pe, oyedemi bo, oyedemi so (2017) alpha-amylase inhibition and antioxidative capacity of some antidiabetic plants used by the traditional healers in southeastern nigeria. scientific world journal. akkawi m, attieh ha, jaber s, lafi sa, lutgen p, remeleh qa (2015) cinnamon bark water-infusion as an in-vitro inhibitor of β-hematin formation. journal of medicinal plant research 9(38). amighi f, djomeh ze, shahi mlm (2020) effect of different cross-linking 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https://doi.org/10.7717/peerj.10070. wong cy, al-salami h, dass cr (2018) microparticles, microcapsules and microspheres: a review of recent developments and prospects for oral delivery of insulin. int j pharm 537(1):223–44. xu h, shen l, xu l, yang y (2015) controlled delivery of hollow corn protein nanoparticles via non-toxic crosslinking: in vivo and drug loading study. biomed. microdevices 17(1):8. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 2, 2019 | pages: 37-40 | doi: 10.14421/biomedich.2019.82.37-40 issn 2540-9328 (online) anatomical and histological study of shark (carcharhinus sorrah) kidney hikmah supriyati1,*, rakhmiyati2, muhammad ja’far luthfi3 1postgraduate biology education program, yogyakarta state university, indonesia. 2postgraduate biosciences program, sebelas maret university, indonesia. 3biology education department, faculty of science and technology, uin sunan kalijaga, indonesia. author correspondency*: hikmahsupriyati@gmail.com abstract sharks are sea water fishes belong to the class chondrichthyes, subclass elasmobranchii. sharks are cartilaginous fish that have a different osmoregulation process than any other sea water fish. cartilaginous fish is the only vertebrate that can maintain urea. this study aims to determine the anatomical and histological structure of the kidney in the anterior, medial and posterior parts of kidney. the study was conducted by observing anatomy of the kidney. histological preparations were made using the paraffin method. qualitative descriptive data analysis was done. research results show that shark kidneys consist of three parts, namely the head kidney, the body kidney, and the tail kidney. kidney sharks are brownish red with a size of 18 cm long. histological observations of shark kidney in the head kidney reveals many glomerulus, body kidney reveals many distal and tubule proximal contractile tubules whereas tail kidney reveals stroma that is rarely found in vertebrate kidney. keywords: sharks; kidney; anatomy; histology introduction pisces are belonging to poikilothermic vertebrate (cold blooded) that live in water and breathe with gills. every aquatic animal has osmotic pressure that is different from its environment, therefore aquatic animals must prevent excess water or lack of water so that the physiological processes in the body take place normally. pisces is one of the taxa that has the largest member. they inhabit sea water, fresh water, mud and semi aquatic habitats. therefore, the fish's kidney has a different structure and its histology is reflected by its habitat. the osmoregulation mechanism in pisces also varies depending on the environment or habitat (fujaya, 2004). osmoregulation is a process of homeostasis to keep body fluids in a stable state. excretion plays an important role in the removal of metabolic waste substances, especially those containing nitrogen so that these substances do not accumulate in the body. these metabolic waste substances may be able to disrupt homeostasis of the internal environment, especially those related to osmotic pressure and the stability ratio of ions in body fluids (suripto, 1998). the main excretory organ in vertebrates is kidney. kidneys in vertebrates generally a pair. embryologically, kidney originate from mesoderm. kidneys in pisces are different of those in frogs, lizards, birds, especially when compared to mammalian kidneys. there are three types of kidneys namely pronephros, mesonephros and metanephros. fish kidney is the most primitive kidney among other vertebrate kidneys (soesilo et al, 1994). each type of environment provides a variety of supporting factors typical for animals that live in it. the abilities and types of body organs possessed by each animal are different. therefore, the osmoregulation mechanism by animals is also different and shows a very wide variation depending on the ability, osmoregulation organs that are owned, and the environmental conditions of each (isnaeni, 2006). like aquatic animals, freshwater and sea water fishes have differences in osmoregulation and excretion. freshwater fish have osmotic levels of body fluids that are hyperosmotic compared to their environment. thus the fish will excrete a lot of water and hold ions (lagler et al., 1997), whereas sea water fish have osmotic levels of body fluids that are hypo-osmotic in their environment. therefore, fish must drink a lot of water (1/5 to 1/2 of their body weight) and remove a lot of salt from their bodies (suripto, 1998). sharks are water fishes and are included in the class chondrichthyes (cartilaginous fish), subclass elasmobranchii. seawater fish in general will drink a lot of water because it is to maintain bodyi fluids that are hypoosmatic on the environment. in contrast to elasmobranchii which has a problem that is too little water intake. to overcome this animal produces a little urine. even if only a little excreted, the urine can also be used to remove excess nacl (ville, 1988). https://doi.org/10.14421/biomedich.2019.82.37-40 38 biology, medicine, & natural product chemistry 8 (2), 2019: 37-40 these differences allow the organs involved in the osmoregulation process in sharks to have differences with kidney organs in other marine fish. besides this research is also to find out the type of shark kidney. references about the anatomy and histology of sharks are still very rare, so researchers are interested in doing the research. previous research has been carried out under the title anatomical and histological comparisons of the catfish uropoetic organs (pangasius hypophtalmus) and sharks (carcharhinus sorrah). this study discusses one field of view on shark kidney organs and catfish. the histological structure of the shark and catfish consists of glomerulus, distal tubules, proximal tubules and hematopoietic tissue. this research emphasizes the difference in shark glomerular diameter length which is smaller than that of catfish. this researchs focus on the anatomy and histological structure of the kidney in the anterior, transition and posterior parts. this research discussed each histology structure of shark kidney, starting from many glomerulus and tubules in each part of kidney. in addition, this study discussed the histological structure that is likely to be rarely found in other vertebrates. material and methods material test animals used in this study were five sharks obtained from depok beach, yogyakarta. the materials used in this study are hvs paper, bouin solution, graded alcohol (30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% and absolute), hematoxylin-eosin dyes, albumin, xylol, distilled water, entelan, toluene, absolute alcohol, paraffin, glassware, mask, gloves, needles and tweezers. tool the tools used in this study were a set of surgical instruments, flakon bottles, paraffin tanks, paraffin ovens, beaker glasses, slide warmers, microtomes, staining jars, microscopes and computers. procedure 1. shark dissection this research was conducted in april 2019 in the zoology laboratory of the faculty of science and technology of uin sunan kalijaga yogyakarta. the sharks that has been sacrificed were then observed morphologically. morphological observations included the gills, pectoral fins, dorsal fins, caudal fins, abdominal fins. then the standard length, total length and fish height were measured. 2. anatomical observation anatomical observation started with dissection performed from the posterior ventral to the anterior ventral. after the internal organs were observed, kidneys were taken and cut on three different parts, namely anterior (head), medial (body), and posterior (tail). 3. histological preparations this research was conducted from april to june 2019 in the zoology laboratory of the faculty of science and technology of uin sunan kalijaga yogyakarta. the preparation of histology started with process of fixation which is the process of immersion of organs using a bouin fixative solution. then the dehydration process was carried out with multilevel alcohol, clearing, infiltration, embedding, sectioning, staining with hematoxylin-eosin, and mounting process. then observations using a binocular microscope (suntoro, 1983). results and discussion shark morphology figure 1. general shape and external parts of the shark's body: (1) nostrils, (2) eyes, (3) first dorsal fin, (4) second dorsal fin, (5) tail, (6) fins rectum, (7) pectoral fins, (8) gills, (9) body height, (10) standard length, (11) total length (manik, 2004). based on observations that had been done, sharks have an elongated body shape. total length 74 cm, standard length 53 cm and body height 21 cm. the head is symmetrical and slightly small, has a long, rounded snout. sharks have double dorsal fins. the anterior dorsal fin is larger than the posterior dorsal fin. the caudal fin is pointed, the dorsal caudal fin is longer than the ventral (phillips, 1991). the type of shark's tail is heterocercal (kardong, 2009). there are cloaca holes between the pelvis fins. in male sharks, caudal fins will turn into claspers (organs to hug female sharks when mating) (brotowijoyo, 1994). the dominant body color is black-brown, especially the dorsal, abdomen and caudal fins are rather brightly colored. rows of teeth in the upper jaw are more triangular shaped. the gills open out with 5 gill slits located on the side of the head (jones & larson; 1974). anatomy of shark kidney figure 2. anatomy of a shark kidney: (a) the anterior part (head kidney), (b) the transition part (body kidney), (c) the posterior part (tail kidney). supriyati et al. – anatomical and histological study of shark … 39 anatomy observations show that shark kidney consists of a pair of kidneys that are located along body cavities, ventral to vertebrae. shark's kidney has an elongated shape and is brown in color. the shark's length is 18 cm. shark's kidney consists of three parts, namely the anterior part (head kidney), the medial part (body kidney), the posterior part (tail kidney). adult fish kidney is mesonephros kidney which consists of 70-80 tubules that form the mesonephric duct (vize, 2012). sharks are cartilaginous fish which differ in the osmoregulation process compared to other sea water fish. cartilaginous fish is the only vertebrate that can maintain urea. excess urea will be excreted by the kidneys and gills. cartilage is able to restore blood urea from the filtrate. as a result, blood contains urea that is sufficient to be hyperosmotic to seawater (hildebrand, 1995). the existence of this process makes blood concentration closer to seawater concentration. so that sharks do not need to drink excess sea water. whereas to overcome dehydration sharks also absorb water through the gills and can produce runny urine (castro & huber, 2005). in most animals, urea is poisonous and excreted, but sharks excrete only small amounts. urea and related compounds are not so toxic to cartilaginous fish. the concentration of urea in shark's body fluids can reach 100 times the level of mammalian urea (villee, 1999). excess salt is excreted by the kidneys, intestines, and special glands near the anus called the rectal glands. this gland can help regulate how to deal with excess ions, especially urea. excess urea in the shark's body will be removed through the gills and rectal glands (castro & huber, 2005). histology of shark kidney figure 3. histology of anterior shark kidney: (1) medulla, (2) bowman capsules, (3) glomerulus, (4) cortex, (5) radius medullaris, (6) lymphoid tissue, (7) distal tubule, (8) proximal tubule based on histological observations that have been made that there is a cortex in the kidneys of sharks. the cortex is in a capsule and is dark color (yatim, 1996). kidney capsules in the form of irregular solid connective tissue. the cortex contains the proximal tubule, distal tubule, glomeruli and medullary radius. the medullaris radius is formed by straight nephrons, blood vessels, and collagen tubules that fuse in the medulla (eroschenko, 2010). medulla is the innermost part of the kidney, under the cortex, light colored. medulla contains bundles of vessels and urinary collection vessels, which number in millions according to many malphigian bodies. a malphigian body along with its veins is an autonomous unit of disposal equipment, called a nephron. it is estimated that each hemisphere of the left-right kidney contains two million nephrons. the direction to the medulla renal sinus forms many bulges, called the malphigi’s pyramid. the top of each pyramid is called a papilla. apart from the medulla and cortex on the anterior part of the shark kidney there are also many glomerulus. glomerulus is an anastomotic clot of capillaries. the part that covers the glomerulus is the bowman capsule. the bowman capsule is a ball shape basin that is not full (yatim, 1996). in addition to the anterior part of the kidney there is also a lymphoid tissue (hematopoietic). this network is composed of reticular fibers (subowo, 1992). figure 4. histology of the transitional shark kidney (body kidney: (1) distal contour tubules, (2) proximal tubule tubules. based on observations that have been made that in the transition (body) histology of shark kidney there are many proximal tubule and distal contour tubules. the proximal tubule is longer than the distal tubule. the proximal tubule has one layer of cuboid cells with a granular and eosinophilic cytoplasm. whereas the distal tubule is shorter and the number is less in the cortex. the distal tubule has a larger lumen within cells (eroschenko, 2010). the number of proximal tubules and distal tubules in the transitional part of the kidney (body kidney) allows the process of reabsorption to occur. the absorbed substances are glucose, protein, 40 biology, medicine, & natural product chemistry 8 (2), 2019: 37-40 amino acids and carbohydrates. related to its function the presence of distal tubules also results in ion transport. this is related to the maintenance of acid-base in the blood (ramaley & bevelender, 1979). figure 5. histology of the posterior shark kidney: (1) stroma, (2) blood vessels. based on histological observation at the posterior kidney there is a stroma. renal stroma is rarely found in vertebrate, stroma consists of loose connective tissue that accompanies blood vessels, lymph vessels and nerves. in the stroma there are cells that occupy the gaps between the 3 dimensional webbing which are composed of reticular fibers, reticular cells and macrophage cells (subowo, 1992). basically, the vertebrate kidney has the same structure, namely the glomerulus and tubules. but in each class there are different arrangements. the anterior part of shark kidneys has a lot of glomerulus which in this part allows the filtration process to occur. while in the transition (body kidney) there are many proximal tubules and distal tubules. the results of this research are consistent with the theory by villee (1999) that in the kidney of the fish the anterior tubules have disappeared, several tubules in the middle region are related to the testes, and there is a concentration and multiplying of the posterior tube. in addition there are also archinephric ducts that function as excretion and sperm ducts in males. while the tail of the kidney (posterior kidney) there is a stroma. stroma is usually found in mammals and is rarely found in vertebrate kidneys (subowo, 1992). conclusion based on research that has been done, it can be concluded that, shark kidneys consist of three parts, namely the head kidney (anterior), body kidney (transition), and tail kidney (posterior). kidney shark brownish red with a size of 18 cm. while histological observations of the anterior kidney there are many glomerulus, transition kidney there are many distal tubule, proximal tubule and in the tail kidney there is a stroma, where the stroma is very rarely found in vertebrate kidney. references brotowidjoyo, mukayat djarubito. 1994. zoologi dasar. jakarta: erlangga castro peter dan huber michael. 2005. marine biology fifth edition. new york: mc graw hill companies inc dellmann, h dan brown esther.1989. textbook of veterinary histology. amerika: lea & febiger eroschenko, victor. 2010. difiore’s atlas of histology with functional correlation, 11 th ed. usa: egc medical publisher fujaya, y. 2004. fisiologi ikan dasar pengembangan teknik perikanan. jakarta: pt rineka cipta hildebrsnd, milton.1995. analysis of vertebrate structure fourth edition. united states of america: r.r donnelleycrawfordsville isnaeni, wiwi. 2006. fisiologi hewan. yogyakarta: pt kanisius jones, r, t and h.k, larson 1974. a key to the families of fishes as recorded from guam. univ. of guam, the marine laboratory, tech. rep. (10): 1-4. kisia, seh m. 2009. vertebrates: structures and functions. usa: science publishers lagler, k. f., j.e. bardach, r. r. miller dan d. r. m. passino. 1997. ichtyologi 2nd ed. john wiley & sons. new york. manik, nurdin. 2004. mengenal beberapa jenis hiu. jurnal oseana, volume xxix no (1): 9-17 mescher, anthony. 2010. junquera’s basic histology: text and atlas, 12th ed. new york: mc graw hill companies inc and egc medical publisher ramaley dan bevelender. 1979. dasar-dasar histologi: edisi kedelapan. (alih bahasa oleh ir wisnu gunarso). jakarta: erlangga soesilo, nyoman dkk. 1994. anatomi hewan. jakarta: universitas terbuka soewolo. 2000. pengantar fisiologi hewan. jakarta: proyek pengembangan guru sekolah menengah ibrd loan no 3979, direktorat jenderal pendidikan tinggi departemen pendidikan nasional subowo. 1992. histologi umum. jakarta: bumi aksara suntoro, s. h, 1983. metode pewarnaan histologi dan histokimia. jakarta: bhratara karya aksara. suripto. 1998. fisiologi hewan. bandung: itb press villee, c. a; w. f walker dan r. d burner. 1988. zoologi umum. jakarta: erlangga. villee, claude a et.all. 1999. general zoology: sixth edition (alih bahasa prof. dr. nawangsari sugiri). jakarta: erlangga yatim, wildan. 1996. biologi modern: histologi.bandung: pt tarsito. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 345-349 | doi: 10.14421/biomedich.2025.141.345-349 issn 2540-9328 (online) antioxidant and cytotoxic potential of ethyl acetate fraction of gandaria stem bark (bouea macrophylla) against mcf-7 cell line boima situmeang1,*, junia salsha primawati1, ismi oktafiani1, weny ja musa2, ahmad kadir kilo2 1department of chemistry, sekolah tinggi analis kimia cilegon, banten, jl. lingkar selatan, km 1,7 cilegon banten 043259, indonesia. 2department of chemistry, faculty of mathematics and natural science, universitas negeri gorontalo, jl. prof. dr. ing. bj. habibie, tilongkabila, bone bolango, 96583. tel. (0435) 821125, fax. (0435) 821752, gorontalo, indonesia. corresponding author* boimatumeang@gmail.com abstract the stem bark of bouea macrophylla (gandaria) represents a promising subject for further scientific investigation. previous studies have reported that the bark contains high concentrations of total phenolic and flavonoid compounds, associated with potent antioxidant activity. this study evaluates the antioxidant and cytotoxic potential of the ethyl acetate fraction of b. macrophylla stem bark. antioxidant activity was assessed using the dpph and abts radical scavenging methods, while cytotoxic activity against mcf-7 breast cancer cells was determined using the mtt assay. the ethyl acetate fraction exhibited strong antioxidant activity with ic₅₀ values of 5.837 ± 0.060 ppm (dpph) and 9.645 ± 0.697 ppm (abts). the cytotoxicity assay revealed an ic₅₀ value of 99.55 ppm, indicating moderate cytotoxic potential. these findings suggest that the ethyl acetate fraction of b. macrophylla stem bark possesses significant antioxidant activity and potential anticancer properties, supporting its possible application in natural therapeutic development. keywords: bouea macrophylla; antioxidant; cytotoxic; mcf-7; gandaria. introduction breast cancer is one of the leading causes of cancerrelated morbidity and mortality among women globally (amalina et al., 2020). the mcf-7 cell line, derived from human breast adenocarcinoma, is widely utilized as a model system for investigating estrogen receptorpositive breast cancer and screening potential anticancer agents (yeniçeri et al., 2024). in recent years, natural products have gained increasing attention as a source of bioactive compounds with therapeutic potential, particularly in developing antioxidant and anticancer agents (herdiana et al., 2022). one of the plants known to contain antioxidant compounds is bouea macrophylla griff., commonly referred to as gandaria (rudiana et al., 2018). this species is indigenous to indonesia and is widely distributed across the islands of java, sumatra, maluku, and kalimantan. according to a study by rudiana et al. (2018), the ethyl acetate extract of the stem of bouea macrophylla griff. exhibited a high content of phenolic and flavonoid compounds, both recognized for their antioxidant properties. furthermore, these compounds were found to be positively correlated with anticancer activity. oxidative stress, resulting from an imbalance between reactive oxygen species (ros) production and the antioxidant defense system, plays a critical role in carcinogenesis (situmeang et al., 2024). therefore, evaluating antioxidant activity is essential in identifying compounds with potential anticancer properties. in this study, the antioxidant activity of the ethyl acetate fraction of b. macrophylla stem bark was assessed using two in vitro methods: the 2,2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging assay and the 2,2’-azino-bis (3ethylbenzothiazoline-6-sulfonic acid) (abts) assay. both assays are widely employed for their sensitivity in determining plant-derived compounds’ free radical scavenging capacity. in addition, the cytotoxic potential of the ethyl acetate fraction was evaluated against the mcf-7 breast cancer cell line using the mtt [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay. this colorimetric assay measures cell metabolic activity to indicate cell viability, proliferation, and cytotoxicity. the outcomes of this study aim to provide scientific evidence supporting the potential use of b. macrophylla stem bark as a source of antioxidant and anticancer agents, thereby contributing to the ongoing search for effective and safer alternatives to conventional chemotherapeutic drugs. manuscript received: 21 april, 2025. revision accepted: 27 june, 2025. published: 02 july, 2025. https://doi.org/10.14421/biomedich.2025.141.345-349 346 biology, medicine, & natural product chemistry 14 (1), 2025: 345-349 materials and methods material the equipment used in this study consisted of glassware such as maceration flasks, erlenmeyer flasks, volumetric flasks, test tubes, graduated pipettes, beakers, and separatory funnels. supporting instruments included an evaporator, filter paper, micropipettes, and a spectrophotometer. the sample used in this study was the bark of the bouea macrophylla (gandaria) tree, collected from the mancak district, serang regency, banten province. the chemical reagents utilized in the research included methanol (p.a), n-hexane (p.a), ethyl acetate (p.a), mcf-7 cells, trypsin-edta solution, dmem medium, prestoblue cell viability reagent, and cisplatin. sample preparation a total of 0.8 kg of fresh gandaria bark was collected as the sample. the sample was then cleaned, cut into smaller pieces, and air-dried at room temperature. the drying process was carried out for 14 days. sample identification (determination) was conducted at the herbarium laboratory, faculty of biology, universitas gadjah mada, yogyakarta. extraction and partition the extraction of gandaria bark samples was carried out using the maceration method, with methanol as the extraction solvent. the extraction process lasted 3 × 24 hours, stirring every 12 hours. fractionation was conducted by partitioning using n-hexane and ethyl acetate-water as solvents. antioxidant activity test using dpph the antioxidant activity assay using the dpph method was conducted concerning the study by kirmani et al. (2024), with several modifications (kirmani et al., 2024). 50 mg of the sample was dissolved in 50 ml of methanol to obtain a stock solution with a concentration of 1000 ppm. subsequently, ethyl acetate fraction solutions with varying concentrations (0, 2, 4, 6, 8, and 10 ppm) were prepared. antioxidant activity test using abts the antioxidant activity assay using the abts method was conducted concerning the study by jiangseubchatveera et al. (2023), with slight modifications (jiangseubchatveera et al., 2023). a volume of 20 µl of the sample was added to 180 µl of abts•⁺ solution in a test tube and allowed to stand at room temperature for 5 minutes. the absorbance was then measured at a wavelength of 734 nm. trolox was the standard (positive control) (idowu et al., 2023). cytotoxic activity test against mcf-7 cell line the anticancer activity assay was conducted concerning the studies by herdiana et al. (2022) and sirait et al. (2019). mcf-7 cells were seeded into microplates at a density of 3 × 10⁴ cells cm⁻³, treated with 1 ml of trypsin-edta solution, and incubated for 5 minutes (sari sirait et al., 2019). the cells were then transferred into tubes containing culture medium and centrifuged at 3000 rpm for 5 minutes. the resulting pellet was resuspended in fresh medium. the cells were cultured in 96-well plates and incubated at 37°c in a 5% co₂ atmosphere for 24 hours. subsequently, 100 μl of each sample and the positive control (cisplatin) were transferred from microtubes into the respective wells and incubated for another 24 hours. after incubation, the medium in each well was removed and replaced with 100 μl of a mixture of 10% prestoblue cell viability reagent in culture medium (prepared by mixing 10 μl reagent with 90 μl medium). the plate was then incubated for 1 hour until a visible color change occurred. finally, absorbance was measured at a wavelength of 750 nm using a multimode reader. the ic₅₀ value was determined from a graph of the percentage of viable cells relative to the control. ic₅₀ is the concentration required to inhibit 50% of cell growth (matulja et al., 2022). data analysis the one-way anova test was used in statistical evaluation and data representation using the graphpad prism 10.1.2 and origin 9 software. the data were reported as the mean ± standard deviation. results and discussion sample preparation stem bark samples of bouea macrophylla were collected from the mancak district, serang regency, banten province, indonesia. approximately 2 kg of fresh stem bark was harvested and air-dried for two weeks under ambient conditions. botanical authentication confirmed the species as bouea macrophylla, with a registered identification number of 00683/s.tb./vii/2024. following the drying process, the final dry weight of the sample was 750 g. extraction and fractionation a total of 750 g of dried sample was subjected to maceration using 3 l of methanol, following a sampleto-solvent ratio of 1:4 (w/v). this ratio was selected based on its suitability for achieving optimal extraction efficiency, ensuring that the entire sample was fully immersed and adequately extracted. the resulting methanolic crude extract yielded a final concentrated extract weight of 125.0 g. the extraction yield, calculated based on the weight of the dried plant material (simplicia), was 16.667%. fractionation of the methanolic extract was carried out using a liquid-liquid partitioning method. the first solvent used was a nonpolar solvent (n-hexane), followed by a semi-polar solvent (ethyl acetate). a total of 125.0 g of methanolic situmeang et al. – antioxidant and cytotoxic activity of bouea macrophylla 347 extract was subjected to partitioning, using 125 ml of each solvent (1:1, w/v) for each fractionation step. the ethyl acetate fraction yielded 39.50 g, corresponding to an extraction yield of 31.60%. antioxidant test result the antioxidant activity of the ethyl acetate fraction was evaluated using dpph and abts radical scavenging assays. for the dpph assay, 2.4 ml of the sample was mixed with 0.6 ml of dpph solution. the mixture was then incubated in the dark for 30 minutes. following incubation, a color change from pink to yellowish-brown was observed, indicating radical scavenging activity, which intensified with increasing sample concentration. the absorbance was subsequently measured at 517 nm (aldayel, 2023). the percentage of inhibition (% inhibition) calculated from the dpph assay is presented in table 1. the abts solution was prepared by mixing abts with potassium persulfate, followed by incubation at room temperature for 16 hours to generate the abts•⁺ radical cation. test solutions were prepared at concentrations of 0, 2, 4, 6, 8, and 10 ppm, with 0.3 ml of each sample placed into test tubes. subsequently, 2.7 ml of the abts solution was added to each tube, and the mixture was incubated for 15 minutes. the reaction between the sample and abts was indicated by a color change from bluish-green to light blue (roubi et al., 2023). absorbance was then measured at 714 nm (benslama et al., 2023). the percentage of inhibition (% inhibition) and antioxidant activity of the fraction, as determined by the abts assay, are presented in table 1. table 1. % inhibition and antioxidant activity result using dpph and abts method of ethyl acetate fraction methods concentrations (ppm) inhibition (%) replications ic50 (ppm) ±sd 1 2 3 dpph 0 0 0 0 5.837 ± 0.060 2 19.568 20.772 18.136 4 34.660 35.265 35.274 6 48.258 46.054 44.592 8 70.978 69.565 70.382 10 86.567 85.024 85.357 abts 0 0 0 0 9.645 ± 0.697 2 22.703 14.185 18.243 4 28.478 25.983 30.270 6 36.745 31.882 36.486 8 43.569 39.045 50.405 10 51.312 49.297 51.486 figure 1. correlation of dpph and abts radical scavenging with various concentrations of sample. the ic50 value was obtained from the linear regression equation. the higher the percentage of inhibition, the lower the ic50 value, indicating a stronger ability to scavenge abts free radicals (figure 1). the results of the antioxidant activity test of the ethyl acetate fraction of gandaria stem bark using the dpph and abts methods showed very strong antioxidant activity. according to the study by nwude et al. (2024), antioxidant activity below 50 ppm is classified as very strong (nwude et al., 2024). the ethyl acetate fraction had an ic50 value of 5.837 ± 0.060 ppm using the dpph method and 9.645 ± 0.697 ppm using the abts method. this is by the study by rudiana et al. (2018), which revealed that the ethyl acetate fraction has the highest total phenolic and total flavonoid contents (rudiana et al., 2018). the antioxidant activity of the ethyl acetate fraction using the abts method is nearly comparable to the ic50 value of the positive control (trolox), which has an ic50 of 9.417 ± 0.577. based on the results of antioxidant activity tests using the dpph and abts methods, it can be concluded that the ethyl acetate fraction has a very high potential to be further developed as a source of natural antioxidants. cytotoxic activity test result against mcf-7 cell line the cytotoxicity test of the ethyl acetate fraction of gandaria stem bark against mcf-7 cells was carried out using serial dilutions, starting from a concentration of 1000 ppm down to 7.81 ppm. the linear regression curve 348 biology, medicine, & natural product chemistry 14 (1), 2025: 345-349 and the ic50 value of the cytotoxic activity of the ethyl acetate fraction against mcf-7 breast cancer cells are shown in figure 2. figure 2. regresion linear curve and ic50 of ethyl acetate fraction of gandaria. the cytotoxic activity of samples targeting cancer cells can be classified into three categories. an ic50 value below 100 ppm is considered active (haryanti & widiyastuti, 2017). the ethyl acetate fraction of gandaria stem bark, with an ic50 value of 99.55 µg/ml, falls into the active category. cells treated with the ethyl acetate fraction of gandaria stem bark showed morphological changes indicative of apoptosis (figure 3), such as cell shrinkage, cytoplasmic condensation, and extracellular matrix degradation (nurmaulawati, 2021). therefore, the cytotoxic activity of the gandaria stem bark fraction is likely mediated through apoptosis induction and inhibition of cell migration, suggesting potential antimetastatic properties (asefian & ghavam, 2024). figure 3. the morphology of mcf-7 cell lines. this toxic property is presumed to be caused by the presence of antioxidant compounds in the gandaria stem bark fraction, which play a role in reducing the number of cancer cells, as reported by rudiana et al. (2018). this is in line with previous studies showing that gandaria stem bark extract contains various chemical compounds, including flavonoids, phenolics, tannins, and terpenoids. the ethyl acetate fraction of b. macrophylla demonstrated strong antioxidant activity, as indicated by its low ic₅₀ values in both dpph (5.837 ± 0.060 ppm) and abts (9.645 ± 0.697 ppm) assays, suggesting a high capacity to scavenge free radicals. this potent antioxidant activity is likely attributed to phenolic and flavonoid compounds typically extracted by semi-polar solvents like ethyl acetate. furthermore, the same fraction exhibited cytotoxic activity against mcf-7 breast cancer cells, with an ic₅₀ value of 99.55 ppm, meeting the national cancer institute's criteria for potential anticancer agents (ic₅₀ < 100 ppm). previous studies have reported similar correlations, where high antioxidant activity in plant extracts, particularly those rich in polyphenols, is often associated with significant anticancer potential. these findings support the therapeutic promise of gandaria, particularly its ethyl acetate fraction, as a natural source of both antioxidants and anticancer agents. conclusions the ethyl acetate fraction of b. macrophylla stem bark demonstrated strong antioxidant activity, as evidenced by situmeang et al. – antioxidant and cytotoxic activity of bouea macrophylla 349 low ic₅₀ values obtained from the dpph (5.837 ± 0.060 ppm) and abts (9.645 ± 0.697 ppm) assays. additionally, the cytotoxicity test using the mtt assay against mcf-7 breast cancer cells showed moderate cytotoxic potential, with an ic₅₀ value of 99.55 ppm. these results indicate that the ethyl acetate fraction possesses significant antioxidant properties and promising cytotoxic activity, highlighting its potential as a natural source for developing antioxidant and anticancer agents. authors’ contributions: boima situmeang designed the study. junia salsha and ismi oktafiani carried out the laboratory work. weny ja musa analyzed the data. boima situmeang and ahmad kadir kili wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. references aldayel, m. f. (2023). potential antibacterial and antioxidant inhibitory activities of silybum marianum mediated biosynthesised he-ne laser. saudi journal of biological sciences, 30(11), 103795. https://doi.org/10.1016/j.sjbs.2023.103795 amalina, n. d., suzery, m., & cahyono, b. (2020). cytotoxic activity of hyptis pectinate extracts on mcf-7 human breast cancer cells. indonesian journal of cancer chemoprevention, 11(1), 1. https://doi.org/10.14499/indonesianjcanchemoprev11iss1pp1-6 asefian, s., & ghavam, m. (2024). green and environmentally friendly synthesis of silver nanoparticles with antibacterial properties from some medicinal plants. bmc biotechnology, 24(1), 1–22. https://doi.org/10.1186/s12896-023-00828-z benslama, o., lekmine, s., & mansouri, n. (2023). phytochemical constituents of astragalus monspessulanus and integrative analysis for its antioxidant, photoprotective, and antityrosinase activities: experimental and computational investigation. european journal of integrative medicine, 60(january), 102247. https://doi.org/10.1016/j.eujim.2023.102247 haryanti, s., & widiyastuti, y. (2017). cytotoxic activity of indonesian plants on mcf-7 cell lines for traditional breast cancer treatment. media penelitian dan pengembangan kesehatan, 27(4), 247–254. herdiana, y., wathoni, n., shamsuddin, s., & muchtaridi, m. (2022). cytotoxicity enhancement in mcf-7 breast cancer cells with depolymerized chitosan delivery of α-mangostin. polymers, 14(15). https://doi.org/10.3390/polym14153139 idowu, d. o., aiyelaagbe, o. o., & idowu, p. a. (2023). chemical composition and biological activities of volatile oil of the stem of dombeya buettneri k. schum. (sterculiaceae). scientific african, 20, 1–10. https://doi.org/10.1016/j.sciaf.2023.e01624 jiangseubchatveera, n., saechan, c., petchsomrit, a., treeyaprasert, t., leelakanok, n., & prompanya, c. (2023). phytochemicals and antioxidant activities of red oak, red coral and butterhead. tropical life sciences research, 34(1), 1–17. https://doi.org/10.21315/tlsr2023.34.1.1 kirmani, f., saddiqe, z., saleem, s., ali, f., & haq, f. ul. (2024). phytochemical investigation and antibacterial activity of curcuma longa against multi-drug resistant bacteria. south african journal of botany, 164, 137–145. https://doi.org/10.1016/j.sajb.2023.11.042 matulja, d., vranješević, f., markovic, m. k., pavelić, s. k., & marković, d. (2022). anticancer activities of marine-derived phenolic compounds and their derivatives. molecules, 27(4), 1–45. https://doi.org/10.3390/molecules27041449 nurmaulawati, r. (2021). kajian literatur uji aktivitas antikanker payudara tanaman ranti (solanum nigrum linn.) secara in vitro dan in vivo. jurnal farmasi medica/pharmacy medical journal (pmj), 4(2), 44. https://doi.org/10.35799/pmj.v4i2.36759 nwude, d. o., osamudiamen, p. m., & enessy, s. m. (2024). phytochemical investigation of mezoneuron benthamianum baill, isolation, in vitro antioxidant, alpha-amylase inhibition, and in silico modelling studies. south african journal of botany, 165, 526–537. https://doi.org/10.1016/j.sajb.2024.01.003 roubi, m., elbouzidi, a., dalli, m., azizi, s. eddine, aherkou, m., taibi, m., guerrouj, b. el, addi, m., & gseyra, n. (2023). phytochemical, antioxidant, and anticancer assessments of atriplex halimus extracts: in silico and in vitro studies. scientific african, 22(october), e01959. https://doi.org/10.1016/j.sciaf.2023.e01959 rudiana, t., fitriyanti, f., & adawiah, a. (2018). aktivitas antioksidan dari batang gandaria (bouea macrophylla griff). educhemia (jurnal kimia dan pendidikan), 3(2), 195. https://doi.org/10.30870/educhemia.v3i2.3328 sari sirait, p., setyaningsih, i., kustiariyah tarman, d., ilmu kelautan, d., pertanian bogor, i., ipb dramaga, k., barat, j., kajian sumberdaya pesisir dan lautan, p., & penelitian dan pengabdian masyarakat institut pertanian bogor, l. (2019). aktivitas antikanker ekstrak spirulina. jphpi, 22(1), 50–59. situmeang, b., swasono, r. t., & raharjo, t. j. (2024). antioxidant, photoprotective, and cytotoxic activities of tristaniopsis merguensis leaf fractions with molecular docking study of potential fraction. karbala international journal of modern science, 10(4), 406–417. https://doi.org/10.33640/2405-609x.3365 yeniçeri, e., altay, a., koksal, e., altın, s., taslimi, p., yılmaz, m. a., cakir, o., tarhan, a., & kandemir, a. (2024). phytochemical profile by lc-ms/ms analysis and evaluation of antioxidant, antidiabetic, anti-alzheimer, and anticancer activity of onobrychis argyrea leaf extracts. european journal of integrative medicine, 66(january). https://doi.org/10.1016/j.eujim.2024.102337 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 1-6 | doi: 10.14421/biomedich.2024.131.1-6 issn 2540-9328 (online) isolation and characterisation of essential oils of mango (m. indica) leave abubakar auwal1,*, mohammed hassan shagal2, charles milam2 1department of community health, gombe state college of health sciences and technology kaltungo. gombe state, nigeria. 2department of chemistry, modibbo adama university yola. adamawa state, nigeria. corresponding author* auwalabubakar1982@gmail.com manuscript received: 09 october, 2023. revision accepted: 13 march, 2024. published: 02 may, 2024. abstract this research work aimed at isolation and characterisation of essential oils of mangifera. indica (m. indica) leaves. phytochemical screening was carried out to determine the bioactive components present in the leaf sample as well as the antimicrobial activity of the crude ethanolic extract. the percentage yield of essential oils (colourless) of the leaf was 0.19%. the phytochemical screening revealed the presence of alkaloid, glycoside, flavonoid, saponin and tannin. steroids was absent in the extract. the antimicrobial evaluation of the essential oil of m. indica leaf revealed a significant activity (zone of inhibition). the percentage (%) of diphenyl picryl hydrazine (dpph) scavenged of antioxidant activities for the essential oils of m. indica at different concentration of 5µl, 10µl, 25µl and 100 µl showed a significant antioxidant activity. in concluded results, it’s clearly observed that, when the concentration of both ethanolic extract and essential oils leaf sample increases, the antimicrobial and antioxidant properties increases when compared to the standard (amoxicillin) and that of essential oils has a significant property than other extracts. considering the results of phytochemical screening, it’s also a potential source of cost-effective food supplements, nutritive ingredients and antibacterial agent for improving human health and curing acute and chronic diseases. the gc-ms analysis of the essential oils for the leaf sample revealed fifty two (52) different chemical compounds which include alloaromadendrene, humulene, bicyclo[4,4,0]dec-1-ene, 2-isopropyl-5-methyl-9-methylene, bicyclo[7.2.0]undec-4-ene,4,11,11,-trimethyl 8-methylene-,[1r-(1r*,4z,9s*)]. keywords: alkaloids; antioxidant; characterisation; essential oils; phytochemicals. introduction mango (mangifera indica) leaf ascribed to the family anacardiaceae has been adjudged as the vital traditionally significant and one of the most economically important tropical fruit crop globally (barreto, et al; 2008). the medicinal actions of plants are unique to particular plant species or groups are consistent with this concept as the combination of secondary products in a particular plant is taxonomically distinct. people are now choosing to visit traditional medicine practitioners because they are disillusioned and dissatisfied with conventional medicine. many plant species have been utilized as traditional medicines but it is necessary to establish the scientific basis for the therapeutic actions of traditional plant medicines as these may serve as the source for the development of more effective drugs. mango leaf is a good source of supplementary protein, vitamins, and minerals. these can be utilized as an alternative source of livestock feeding in developing countries for alleviating food shortage for livestock. proximate composition of mls showed 13.6% of crude protein (cp) in dauphine mauritian variety, 20.38% cp in nigerian variety, and 6.90% cp in laos variety (laulloo, et al., 2018). its well-known mango-producing countries include china, thailand, indonesia, pakistan, mexico, brazil, bangladesh, nigeria, and philippines (kumar et al., 2021). plant oils and extracts have been used for a wide variety of purposes for many thousands of years (jones, 1996). besides fruit, mango leaves can also be used as a food supplement and a good fodder as it has a good amount of minerals, vitamins, and protein (kumar et al., 2021; jhaumeer et al., 2018). some compounds from mango can be used in the pharmaceutical industry, such as steroids, gallic acid that has antifungal and antiviral activity, flavonoids that have antioxidant activity, and tannin as a diarrhea remedy (ali et al. 2020). mangiferin is a natural miracle biologically active compound against lifestyle-related disorders. some studies have shown that mangiferin treats covid-19 (umar et al. 2021). mangiferin has a binding affinity for the mpro of covid-19. mpro is a key enzyme that plays a vital role in viral replication and transcription. therefore, mangiferin can inhibit viral replication and transcription (umar et al. 2021). https://doi.org/10.14421/biomedich.2024.131.1-6 2 biology, medicine, & natural product chemistry 13 (1), 2024: 1-6 mangifera indica has attracted to be studied to determine new biomolecules from different parts of plants like fruits, leaves, stems, and seed kernels. its therapeutic importance is well established and has been used as a traditional remedy for treating several diseases (jhaumeer et al. 2018). the aim of this research was to determine the essential oils, antimicrobial and antioxidant properties of m. indica leave. materials and methods sample collection and preparation the m. indica leaves sample was collected in sangere ward, girei local government area of adamawa state, nigeria located at coordinates 9022’0” n and 12033’0”e in dms (degrees minutes seconds). the fresh leaves samples was dried at room temperature and blended into fine powder. eighty grams (80g) of the powdered plant leaves was dispense in 800ml of ethanol and then keep for two weeks with shaking at regular intervals after which the content was filtered using whatman filter paper and the filtrates was concentrated at 30 0c (fatope et al., 1993). extraction of essential oils one kg of the pulverized sample was subjected to steam distillation in a steam distiller, according to the british pharmacopoeia (bp) method. the time taken for the extraction of the oil was 21/2 hours. phytochemical screening of the crude extract the freshly prepared extract was subjected to standard phytochemical analysis according to the method adopted by (jigna et al., 2006). antimicrobial activity for essential oils the antimicrobial activity of the essential oils from the leave of m. indica was tested against seven bacteria multidrug resistant acinetobacter (mdra), escherichia coli, staphylococcus aureus, salmonella typhi, methicillin resistant streptococcus aureus (mrsa), proteus vulgaris and staphylococcus epidermis) antimicrobial potentials of leaf sample against actively growing broth cultures of escherichia coli, staphylococcus aureus, salmonella typhi, staphylococcus epidermidis, methicillin resistant streptococcus aureus (mrsa) as well as proteus vulgari bacteria were tested by well diffusion of the method following ghosh et al., (2020) and majumder et al., (2022b). a nutrient agar (na) medium was used for this test. following the pour plate method, 0.0125 µl of bacterial broth culture was first poured into each sterile petri dish and then sterilized. the plates were left for a few minutes at room temperature for the media to be solidified. following solidification, a sterile steel cork borer was used to cut out circular wells (8 mm in diameter). next, 0.25 µl of each leaf extract sample was poured into the well. this process was done under the sterile condition in a laminar airflow cabinet. the plates were then incubated at 37°c for 24 hours in a noninverted position. determination of anti-oxidant activity the free radical scavenging activity of different concentrations of crude extracts of leaves and standard ascorbic acid was evaluated by using dpph radical scavenging method as per reported method (alhakmani et al, 2013). the 2,2-diphenyl-1-picrylhydrazyl (dpph) assay and superoxide dismutase (sod)-like activity had shown that m. indica leaves serve as a moderate antioxidant with an ic50 value of ~9 and 117 µg/ml (itoh et al, 2020) results and discussion results percentage yield of essential oils of m. indica leaves table 1 shows the percentage yield of essential oils of m. indica leaves. the percentage yield was calculated using the equation, rou = (m/bm) x 100 where m is the mass of the extracted oil in grams and bm is the initial plant biomass in grams. or percentage (%) yield of oil = weight of oils/ weight of samples x 100 table 1. percentage yield of essential oils of m. indica leaves plant weight of essential oils, in (g) colour of oils weight of plants, in (g) % yield of the oils m. indica 1.25 colourless 654.93 0.19% phytochemical screening of m. indica leave extract the results of the phytochemical screening of the crude leaf extract of m. indica is shown in table 2. table 2. phytochemical screening of crude leave extract of m. indica phytochemical tests result alkaloid dragendoff’s test + glycoside fehling’s tests + flavonoid conc. h2so4 test + saponin foam tests + tannin fecl3 tests + steroids libermann-buchard test keys: (+) = present, (-) = absent antimicrobial activity of the crude leave extracts of m. indica leave the result of antibacterial activities of the m. indica crude extract is shown in figure 1. auwal et al. – isolation and characterization of m. indica essential oils 3 figure 1. antimicrobial activity of the ethanolic crude extracts of m. indica leave. antimicrobial activity of the essential oils of m. indica leave the result of antimicrobial activities of the essential oils of m. indica leave is shown in figure 2. figure 2. antimicrobial activity of essential oils of m. indica leave (as measured by level of microbial inhibition in mm). antioxidant activity (dpph) antioxidant activity of m. indica ethanolic leaf extract is shown in figure 3. results were expressed as the percentage of dpph inhibition (%) that occurred due to the exposure of samples. the radical scavenging activity and antioxidant potential of the plant extracts were determined by the ability of plant extracts to scavenge the stable free radical dpph and convert into diphenyl picryl hydrazine. 𝐹𝑟𝑒𝑒 𝑟𝑎𝑑𝑖𝑐𝑎𝑙 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 figure 3. antioxidant activity (%) of m. indica ethanolic leaf extracts. 0 5 10 15 20 25 30 35 at 0.125mg at 0.25mg at 0.5mg at 1mg amoxicillin in h ib it io n i n m m concentration in mg antimicrobial activity of the ethanolic crude extracts of mangifera indica leaf e. coli staphylococcus aureus salmonella typhi staphylococcus epidermidis mrsa proteus vulgari 0 10 20 30 40 mangifera indica amoxicillin in h ib it io n in m m oil samples in ul antimicrobial activity of essential oils of the leave samples mdra e. coli staphylococcus aureus salmonella typhi mrsa proteus vulgari staphylococcus epidermidis 44,69 53,6454,06 48,48 33,39 27,58 23,83 6,01 0 10 20 30 40 50 60 mangifera indica ascorbic acid a n ti o xi d an t a ct iv it y (% o f d p p h ) concentration (ul) antioxidant activity (%) of mangifera indica 5ul 10ul 25ul 100ul 4 biology, medicine, & natural product chemistry 13 (1), 2024: 1-6 chemical composition of essential oil of m. indica leave table 3. gc-ms analysis of the essential oil of m. indica leave. s/n constituents retention time (min) area % 1 elemene isomer 5.325 1.03 2 1h-cycloprop[e]azulene, 1a,2,3,4,4a,5,6,7b-octahydro-1,1,4,7-tetramethyl-, [1ar(1a.alpha.,4.alpha.,4a.beta.,7b.alpha.)] 5.821 5.96 3 1h-cyclopropa[a]naphthalene, 1a,2,3,3a,4,5,6,7b-octahydro-1,1,3a,7-tetramethyl-, [1ar-(1a.alpha.,3a.alpha.,7b.alpha.)] 5.858 6.97 4 2h-2,4a-methanonaphthalene, 1,3,4,5,6,7-hexahydro-1,1,5,5-tetramethyl-, (2s)6.021 1.52 5 isocaryophillene 6.154 8.00 6 humulene 6.265 12.46 7 bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-,[1r-(1r*,4z,9s*)]6.384 8.98 8 alloaromadendrene 6.450 13.65 9 bicyclo[4.4.0]dec-1-ene, 2-isopropyl-5-methyl-9-methylene6.658 10.80 10 naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl)-, [1r(1.alpha.,7.beta.,8a.alpha.)] 6.843 1.05 11 .alpha.-guaiene 6.954 2.26 12 valerena-4,7(11)-diene 7.013 2.62 13 aromandendrene 7.169 4.91 14 naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl)-, [1s(1.alpha.,7.alpha.,8a.alpha.)] 7.465 5.66 15 (1r,9r,e)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene 7.761 2.25 16 isocaryophillene 7.976 2.12 17 1h-cycloprop[e]azulene, 1a,2,3,5,6,7,7a,7b-octahydro-1,1,4,7-tetramethyl-, [1ar(1a.alpha.,7.alpha.,7a.beta.,7b.alpha.)] 8.376 0.44 18 naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-4a,8-dimethyl-2-(1-methylethenyl)-, [2r(2.alpha.,4a.alpha.,8a.beta.)] 8.398 0.60 19 3-chloropropane-1,2-diol, bis(tert-butyldimethylsilyl) ether 8.509 1.06 20 1h-cycloprop[e]azulene, 1a,2,3,5,6,7,7a,7b-octahydro-1,1,4,7-tetramethyl-, [1ar(1a.alpha.,7.alpha.,7a.beta.,7b.alpha.)] 8.732 0.11 21 valerena-4,7(11)-diene 8.828 0.60 22 cycloheptane, 4-methylene-1-methyl-2-(2-methyl-1-propen-1-yl)-1-vinyl8.939 0.60 23 1h-cycloprop[e]azulene, 1a,2,3,5,6,7,7a,7b-octahydro-1,1,4,7-tetramethyl-, [1ar(1a.alpha.,7.alpha.,7a.beta.,7b.alpha.)] 9.087 0.46 24 n-benzyl-n-ethyl-p-isopropylbenzamide 9.191 0.33 25 spiro[4.5]decan-1-one 9.509 0.10 26 cyclohexadecane 9.583 0.19 27 2-heptadecanol 9.813 0.39 28 13-tetradece-11-yn-1-ol 9.887 0.14 29 octadecane, 1-(ethenyloxy)10.006 0.20 30 cyclohexaneethanol, 4-methyl-.beta.-methylene-, trans10.131 0.11 31 2-pentadecanol 10.391 0.02 32 tetradecane, 1-chloro10.472 0.11 33 1-formyl-2,2,6-trimethyl-3-(3-methyl-but-2-enyl)-6-cyclohexene 10.717 0.01 34 1-octadecene 10.880 0.08 35 1-pentadecene 11.279 0.17 36 phthalic acid, 2-cyclohexylethyl propyl ester 11.479 0.20 37 1-nonadecene 11.657 0.08 38 2-dodecen-1-yl(-)succinic anhydride 11.761 0.03 39 cyclohexene, 4-(4-ethylcyclohexyl)-1-pentyl11.916 0.00 40 2-piperidinone, n-[4-bromo-n-butyl]12.035 0.08 41 2-dodecen-1-yl(-)succinic anhydride 12.146 0.00 42 oleic acid 12.183 0.01 43 9-tricosene, (z)12.242 0.02 44 bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-,[1r-(1r*,4z,9s*)]12.287 0.01 45 cyclotetradecane 12.398 0.04 46 cis-9-hexadecenal 12.442 0.04 47 22-tricosenoic acid 12.502 0.04 48 9-octadecenamide, (z)12.716 3.38 49 2-methyl-z,z-3,13-octadecadienol 13.524 0.03 50 trans-13-docosenamide 13.553 0.05 51 erucic acid 13.650 0.01 52 e-11-hexadecenal 13.687 0.01 auwal et al. – isolation and characterization of m. indica essential oils 5 discussion table 1 shows that, m. indica essential oil is colourless with the percentage yield of 0.19%. the percentage yield of essential oils varies with the factors like site of collection, time of collection, part and form of plant used as well as the method employed among others. phytochemical screening revealed the presence of glycoside, flavonoids, saponins, tannins, steroids and alkaloids in the leaf crude extract of m. indica but steroid was completely absent. the phytochemical content in the mango leaf extracts is responsible for the antibacterial, anti-inflammatory and antioxidant activity (jhaumeer et al., 2018). previous studies on mango leaf extracts have been reported by many workers to show the presence of active functional groups such as xanthones (mangiferin), flavonols (quercertin), benzophenones and terpenoids (laulloo, et al.,2018). kumar et al. (2021) stated the major phytochemicals responsible for the antimicrobial activity were phenolics, alkaloids, glycosides, tannins, terpenes, and saponins it was showed that, at the concentration of 0.125 µl, e. coli has no effect. at 0.25 µl, 0.5 µl, 1 µl was 8mm, 10mm, and 12mm respectively, from staphylococcus aureus, coming to salmonella typhi, staphylococcus epidermidis, methicillin resistant streptococcus aureus (mrsa) as well as proteus vulgari, as the concentration increases the antimicrobial activities also increases including the standard (std) amoxicillin (am). several studies have reported that the different parts of mango plants, such as leaves, bark, stems and seeds, exhibited antimicrobial activity against some multi drug resistant (mdr) microbes (kumar et al., 2021). from figure 1, it can be seen that, the ethanolic crude extracts of m. indica leaf sample showed a slight increase in antimicrobial property against all the tested organisms. it’s clearly observed that, when the concentration of the leaf sample increases the antimicrobial activity increases when compared to the standard antibiotics. in the present study from figure 3 where the extract showed the significant maximum zone of inhibition against staphylococcus aureus(32mm) followed by methicillin resistant streptococcus aureus (mrsa) (31mm), proteus vulgaris (30mm), mdra (19mm), escherichia coli (14mm), salmonella typhi (10mm), and lastly no activity on staphylococcus epidermis (none). the essential oils remarkably inhibited the growth of all tested microorganism, but it was cleared from the present results obtained, m. indica leaf extract exhibited pronounced activity against all the tested bacteria excluded salmonella typhi with the weaker value of 10mm as showed in figure 2. therefore, it’s revealed that, the essential oils of m. indica leaf has a significant antimicrobial property. from figure 3, antioxidant activity (dpph) of m. indica leaves, showed that, an increased in concentration of the extract possess increased in scavenging activity. mango leaves have been reported to have antioxidant activity mostly from phenolic compounds (ali et al., 2020). in some study the free radical scavenging capacity of mango leaf extract was determined by the dpph assay performed on petroleum benzene, acetone, and methanol leaf extract of all samples, following the protocol of majumder et al., (2022a) and ghosh et al., (2020). mango leaf were reported to have antioxidant capacity due to the presence of phenolics and flavonoids in different studies (kumar et al., 2020). from figure 3, it’s clearly observed that m. indica leaf possess high antioxidant properties compared with the control group (i.e ascorbic acid). the gc-ms analysis of the essential oil obtained from the leaves of mango leave (m. indica) revealed the presence of fifty two (52) compounds, as shown in table 3 of the gas chromatography and mass spectra. the major compounds were alloaromadendrene (13.65%), humulene (12.46%), bicyclo[4,4,0]dec-1-ene, 2isopropyl-5-methyl-9-methylene(10.80%), bicyclo[7.2.0]undec-4-ene,4,11,11,-trimethyl-8methylene-,[1r-(1r*,4z,9s*)](8.98%), isocaryophillene (8.00%), 1h-cycloprop[e]azulene, 1a,2,3,4,4a,5,6,7b-octahydro-1,1,4,7-tetramethyl-, [1ar(1a.alpha.,4.alpha.,4a.beta.,7b.alpha.)](6.97%), 1hcycloprop[e]azulene,1a,2,3,4,4a,5,6,7b-octahydro1,1,4,7-tetramethyl-,[1ar (1a.alpha.,4.alpha.,4a.beta.,7b.alpha.)](5.96%), naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl7-(1-methylethenyl)-, [1s-(1.alpha.,7.alpha.,8a.alpha.)] (5.66%), aromandendrene (4.91%), 9-octadecenamide, (z)(3.38%), valerena-4,7(11)-diene (2.62%), .alpha.guaiene (2.26%), (1r,9r,e)-4,11,11-trimethyl-8methylenebicyclo[7.2.0]undec-4-ene (2.25%), isocaryophillene (2.12%), 2h-2,4a methanonaphthalene,1,3,4,5,6,7-hexahydro-1,1,5,5tetramethyl-,(2s)-(1.52%), 3 chloropropane-1,2-diol, bis(tert-butyldimethylsilyl) ether (1.06%), naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1methylethenyl)-, [1r-(1.alpha.,7.beta.,8a.alpha.)] (1.05%), elemene isomer (1.03%). total of 18 compounds contained 94.68%, the remaining bulk of thirty four (34) compounds covers only 5.32%. another result of the constituents of the essential oil of mango leaf obtained by other researcher were identified using gc–ms spectrometry and showed the presence of several biologically active compounds, such as humulene-4-hydroxy-4-methyl-2-pentanone, elements, and trans-caryophillene, which was common in all five cultivars of mango (ouf, et, al; 2020). conclusions in conclusion, it’s clearly observed that, when the concentration of both ethanolic and essential oils leaf sample increases, the antimicrobial and antioxidant properties increases when compared to the standard 6 biology, medicine, & natural product chemistry 13 (1), 2024: 1-6 (amoxicillin) and that of essential oils has a significant property than other extracts. mango leaves exhibit exceptional biological, medicinal, and metabolic properties. considering phytochemical screening it’s also a potential source of cost-effective food supplements, nutritive ingredients and antibacterial agent for improving human health and curing acute and chronic diseases. there are numerous vital chemical compounds present in mango leaves, which are instrumental to performing various metabolic, bacteriostatic, and antimicrobial activities. some of these include alloaromadendrene, humulene, bicyclo[4,4,0]dec-1-ene, 2-isopropyl-5-methyl-9-methylene,bicyclo[7.2.0]undec4-ene,4,11,11,-trimethyl 8-methylene-,[1r(1r*,4z,9s*)] isocaryophillene, 1hcycloprop[e]azulene, 1a,2,3,4,4a,5,6,7b-octahydro1,1,4,7-tetramethyl-, and so on. acknowledgements: the authors wish to thank almighty god by whose power, infinite love and mercies enable us do this research work. our profound gratitude goes to our family for their financial and moral support throughout the research. author’s contribution: auwal abubakar wrote the manuscript and carried out laboratory work, mohammed hassan shagal analyzed the data and charles milam designed the study. all authors read and approved the final version of the manuscript. competing interest: the authors declare that there are no competing interests. references alhakmani f, kumar s, khan sa. 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[crossref] laulloo, s. j., bhowon, m. g., soyfoo, s., and chua, l.s. nutritional and biological evaluation of leaves of m. indica from mauritius. j chem. 2018; 9: 1 9. majumder s, saha s, ghosh a, chakraborty s, acharyya s, sarkar s,bhattacharya m. 2022a. comparative in vitro biological characterization of black and green tea infusions fermented with brewer's yeast and scoby with special emphasis on antioxidant activity. international journal on nutraceuticals, functional foods and novel foods 1: 357-369. majumder s, saha s, ghosh a, chakraborty s, acharyya s, sarkar s, bhattacharya m. 2022b. fusion of tea infusion and bakhar (starter of ethnic liquor haria) to develop "tea haria": a novel approach to ferment tea with insight into in vitro biochemical attributes and metabolomics. j food technol res 9(1): 1-7. doi: 10.18488/jftr.v9i1.2909 ouf, s.a.; galal, a.m.f.; ibrahim, h.s.; hassan, a.z.; mekhael, m.k.g.; el-yasergy, k.f.; el-ghany, m.n.a.; rizk, m.a.; hanna, a.g. phytochemical and antimicrobial investigation of the leaves of five egyptian mango cultivars and evaluation of their essential oils as preservatives materials. j. food sci. technol. 2020, 1–13. umar hi, josiah ss, saliu tp, jimoh to, ajayi a, danjuma jb. 2021. insilico analysis of the inhibition of the sars-cov-2 main protease by some active compounds from selected african plants. journal of taibah university medical sciences 16 (2): 162-76. doi: 10.1016/j.jtumed.2020.12.005. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 221-226 | doi: 10.14421/biomedich.2024.131.221-226 issn 2540-9328 (online) anticancer potential of ethanolic extract artocarpus heterophyllus lam. leaves against human colon cancer widr cell line novita mutiyani, ardaning nuriliani* faculty of biology, universitas gadjah mada jl. teknika selatan, sekip utara, yogyakarta, indonesia, 55281. corresponding author* ardaning@ugm.ac.id manuscript received: 13 march, 2024. revision accepted: 20 may, 2024. published: 26 june, 2024. abstract jackfruit (artocarpus heterophyllus lam.) is a plant contains various compounds that has potential as anticancer drugs. secondary metabolites of jackfruit leaves are flavonoids, alkaloids, tannins, triterpenoids, and saponins. a typical flavonoid compound group in the artocarpus genus, namely artocarpin, is able to kill cancer cells through apoptosis. not many have conducted cytotoxicity research and apoptosis induction of ethanolic extracts from jackfruit leaves, especially against widr colon cancer cells. therefore, this study aims to study the cytotoxic effects and apoptosis induction of ethanolic extracts of jackfruit leaves against widr colon cancer cells. the research was conducted by cytotoxicity test using the mtt assay. apoptosis test was done using double staining method with ao/pi dye. the treatment was conducted at various concentrations of ethanolic extract of jackfruit leaves, doxorubicin as positive control, and dmso as solvent control. the data were analyzed by one way anova (p ≤ 0.05) and continued using tukey hsd post hoc test. the results showed that the ethanolic extract of jackfruit leaves was not toxic to widr cells with an ic50 740.43 μg/ml, but could significantly reduce cell viability at a concentration of 500 μg/ml. the ethanolic extract of jackfruit leaves could also induce apoptotic cell death at a concentration of 500 μg/ml. based on these results, ethanolic extract of jackfruit leaves has little potential to be developed as an anticancer drug. keywords: apoptosis; cytotoxicity; artocarpus heterophyllus lam leaves; ethanolic extract; widr cells. introduction cancer is disease that causes significant deaths in the world. based on 2022 data, cancer deaths worldwide reached nearly 10 million people. lung, colorectal, and liver cancer are the three cancers with the highest number of deaths. in 2022, there were 19.255 deaths people from colorectal cancer in indonesiat (ferlay et al., 2024). various cancer therapies that are commonly done until now still have side effects. treatment for cancer, especially colon cancer, such as surgery can cause urogenital disorders. chemotherapy has the effect of hair loss, numbness, and increases the chance of infection due to a lack of white blood cells. radiation therapy can cause reactions of nausea, diarrhea, inflammation, rectal bleeding, bladder dysfunction, and even cause infertility in women (american cancer society, 2020). therefore, it is necessary to develop cancer drugs that can minimize these side effects. drugs with natural ingredients can be one of the solutions for the development of cancer treatment. one of the natural ingredients that has the potential to be developed as a natural cancer drug is jackfruit tree (artocarpus heterophyllus lam.). several studies have been conducted to study the potential of jackfruit tree organs, among others, artocarpus heterophyllus seeds are known to have cytotoxic activity with an ic50 value 35.26 μg/ml against lung cancer cells (a549) (patel & patel, 2011). water extract of artocarpus heterophyllus flowers has cytotoxic activity on colon cancer cells (caco-2) with an ic50 29.37 μg/ml (gupta et al., 2020). a. heterophyllus wood extract showed cytotoxic activity against colon cancer cells (hct116) with an ic50 4.23 mg/l (morrison et al., 2021). methanolic extract of jackfruit leaves showed cytotoxic activity with an ic50 119 µg/ml (marka et al., 2016). based on those researches, jackfruit leaves may also have the potential to be utilized as an alternative to natural cancer drugs because they contain various secondary metabolites such as anthocyanins, coumarins, anthraquinones, flavonoids, phenolic acids, and terpenoids (ngbolua et al., 2019). research by arung et al. (2010), showed that artocarpin, one of the flavones contained in a. heterophyllus, can cause breast cancer cell death (t47d) through induction of apoptosis. the effect of natural ingredients on cancer cells depends on various factors. the extraction method and https://doi.org/10.14421/biomedich.2024.131.221-226 mailto:ardaning@ugm.ac.id 222 biology, medicine, & natural product chemistry 13 (1), 2024: 221-226 the choice of solvent type are influential factors in the results of making an extract. extraction is the first step taken to separate the desired natural ingredients or compounds from certain raw materials (zhang et al., 2018). in the extraction method, solvent selection is very important because the polarity level of each solvent has the ability to dissolve different substances or compounds (abubakar & haque, 2020). based on the law of like dissolves like, solvents with polarity values close to the polarity of the solute tend to have better performance (zhang et al., 2018). research on jackfruit tree parts and their metabolite compounds against cancer cells has been carried out using various methods and several types of solvents. however, not many have conducted cytotoxicity research and apoptosis induction of ethanolic extracts from jackfruit leaves, especially against widr colon cancer cells. therefore, this study aims to study the cytotoxic effects and apoptosis induction of ethanolic extracts of jackfruit leaves against widr colon cancer cells. materials and methods sample the leaves of artocarpus heterophyllus lam. were collected from the ambarawa region, semarang regency, central java province, indonesia. the plants were identified by taxonomists at the laboratory of plant systematics, faculty of biology, universitas gadjah mada. extraction jackfruit leaves to be used were selected that are old, intact, fresh, green and then cleaned. next, leaves were dryed under the sun. the dried leaves were then crushed into powder with a blender. the powder was then sieved with a 40 mesh sieve and weighed on analytical scales as much as 20 grams. maceration method extraction was carried out by soaking the leaves powder using 70% ethanol solvent for 3x24 hours in a dark bottle. all extracts were put together and evaporated with a rotary evaporator at a temperature of approximately ±50 oc. next, evaporation was carried out again by heating in a porcelain dish until it becomes a thick extract. cytotoxicity test (mtt) extract testing was conducted on widr colon cancer cells obtained from the laboratory of parasitology, faculty of medicine, public health, and nursing (fkkmk), ugm. harvested cells cultured with density1 x 104 cells/well in 96-well plate with complete medium rpmi contained, 10% fbs, 2% penicilin-streptomycin, and fungizone (amphoterizine b) 0.5%. the cells were incubated for 24 hours in an incubator with 5% co2 flow and 37oc temperature. after that, the cells treated with various concentrations (7.81; 15.62; 31.25; 62.5; 125; 250; 500; and 1000 μg/ml) of ethanolic extract of jackfruit leaves. doxorubicin concentrations were 0.062; 0.125; 0.25; 0.5; 1; 2; 4; and 8 µg/ml. all concentration series were made for treatment with 24 and 48 hours incubation. after incubating for 24 and 48 hours, each treatment was dripped with mtt reagent and incubated for 4 hours. the plate was then observed under an inverted microscope, if formazan was clearly formed then 100 μl of 10% sds was added. cells were reincubated in the dark at room temperature overnight. next, the absorbance was read with an elisa reader at 595 nm. the absorbance results are used to calculate the percentage of cells viability based on the following calculation formula (cancer chemoprevention research center (ccrc), 2013). percentage of live cells = (treatment absorbance − media control absorbance) × 100% (solvent control absorbance − media control absorbance) the calculation was also continued by calculating the ic50 value. apoptosis test (double staining) widr colon cancer cells were cultured with density 5 x 104 cells/well on the cover slip in 24-well plate. after that, the cells were allowed to stand for 30 minutes and then added completed medium rpmi and incubated in an incubator co2 5% with a temperature of 37oc for 24 hours. after incubation was completed, treatment was conducted with negative control dmso 0.5%, positive control of doxorubicin 0.125 μg/ml, and ethanolic extract of jackfruit leaves at concentrations of 250 and 500 µg/ml. next, the cells were re-incubated in an incubator with 5% co2 and 37oc temperature for 48 hours. after 24 hours incubation, cover slip with cells on it taken from the plate and then placed on a glass object and stained with ao/pi (acridine orange and propidium iodide). the staining results were observed using a confocal microscope. green fluorescent cells indicate live cells and red fluorescent cells are dead or apoptotic cells. apoptotic cells were counted in a population of at least 100 cells per well. data analysis cytotoxicity and apoptosis data were analyzed by one way anova. if there is a significant difference, then the analysis continued using tukey hsd post hoc test. the test results are significant if the p value ≤ 0.05. (simanurak et al., 2023). result and discussion in this study, jackfruit leaves were extracted by maceration method using 70% ethanol. the extract was tested for its cytotoxic effect on widr colon cancer cells by mtt assay. in this test, the treated cells were mutiyani & nuriliani – anticancer potential of ethanolic extract ... 223 incubated for 24 and 48 hours. the cytotoxic level of the extract was measured by calculating the percentage of cell viability. cell viability is determined based on purple formazan crystals. soluble crystals will produce a color intensity that is directly proportional to the number of living cells (buranaamnuay, 2021) (figure 1.) figure 1. formazan crystals formed in the mtt assay. the percentage of cell viability after 24 hours incubation showed that 0.5% dmso control was not toxic to widr cancer cells with a viability of 100 ± 6.56%. doxorubicin treatment showed cell viability of 85.08 ± 3.10% at the lowest concentration of 0.062 μg/ml, while at the highest concentration of 8 μg/ml the viability was 61.82 ± 4.53% (figure 2). in the treatment of ethanolic extract of jackfruit leaves (eejl), cell viability was more than 100% in all concentration groups. the lowest concentration of 7.81 μg/ml had a cell viability of 105.08 ± 3.62% and the highest concentration of 1000 μg/ml had a viability of 136.36 ± 3.22% (figure 2.). in the eejl treatment group, data analysis showed that there were several concentrations that were not significantly different and some were significantly different, but in general there were no significant differences between concentrations. in the eejl treatment group, data analysis showed that there were several concentrations that were not significantly different (p > 0.05), such as concentrations of 31.25 and 250 μg/ml. concentrations of 15.62 and 125 μg/ml also showed no significant difference μg/ml. however, other concentrations showed significant differences between concentrations (p ≤ 0.05). based on the viability results, the ic50 for doxorubicin and eejl could not be determined. these results indicate that eejl is unable to exert cytotoxic effects on widr cancer cells within the 24 hours incubation period. therefore, the same study was conducted with a longer incubation period of 48 hours. figure 2. ethanolic extract of jackfruit leaves (eejl) for 24 hours did not decrease widr cell’s viability. in the treatment with an incubation time of 48 hours, the 0.5% dmso control showed cell viability of 100 ± 4.42% which means it is not toxic to widr cells (figure 3.). this result shows that dmso 0.5% in 24 or 48 hours incubation time proved to be nontoxic. these results were consistent with previous research, the dmso concentration range of 0.1-0.6% does not cause toxic effects on cells (nguyen et al., 2020). in the treatment of doxorubicin, the lowest concentration of 0.062 μg/ml resulted in a viability percentage of 62.83 ± 0.85% while the highest concentration of 8 μg/ml viability was 28.32 ± 5.57% (figure 3). doxorubicin treatment for 48 hours resulted in an ic50 0.147 μg/ml. this result is quite different from previous research, doxorubicin produced doxorubicin 224 biology, medicine, & natural product chemistry 13 (1), 2024: 221-226 an ic50 3.49 µg/ml and was toxic to widr cells in only 24 hours (fathani & miladiyah, 2021). based on the us national cancer institute (nci) criteria, the ic50 of doxorubicin is classified as high because ic50 ≤ 20 µg/ml (fathani & miladiyah, 2021). in the treatment of ethanolic extract of jackfruit leaves with the lowest concentration of 7.81 μg/ml had a cell viability of 132.07 ± 14.91% (figure 5.). the concentration group of 15.62 μg/ml has the highest viability of 136.94 ± 17.99%, while the concentration of 500 μg/ml has the lowest cell viability of 43.31 ± 3.94% (figure 3.). eejl treatment for 48 hours can kill widr cancer cells more than 50% at a the concentration of 500 μg/ml so that the ic50 can be determined at 740.43 μg/ml (figure 5). this ic50 is classified as nontoxic based on the us national cancer institute (nci) standard because ic50 > 501 µg/ml. even though ic50 eejl is classified as non-toxic, at the concentration of 500 µg/ml there was a decrease in cell viability to less than 50%. figure 3. ethanolic extract of jackfruit leaves (eejl) for 48 hours decrease widr cell’s viability. the results of the cytotoxic test showed that incubation time affected the viability of widr cells. treatment with 24 hours incubation did not reduce cell viability, while 48 hours incubation significantly reduced cell viability to less than 50%. in a study conducted by rachmawati et al. (2012), the incubation time factor in anticancer drug testing affects the expression time and function of tumor suppressor genes. in this study, it is also possible that eejl and doxorubicin did not work optimally within 24 hours because genes related to cancer cell death, such as tumor suppressor genes, have not worked or have not even been expressed yet. in addition, the dose or concentration also affects the viability of widr cells. our results, especially after 48 hours of incubation, shows that cell viability tends to decrease as the concentration increases. incubation time and dose affect the results of this study because both factors affect the contribution of the receptor fraction in drug action. the drug will work stronger along with the number of receptor fractions that bind (rachmawati et al., 2012). based on the results of the cytotoxicity test, an apoptosis test was carried out to determine the death rate of widr cancer cells. the test was carried out by double staining method using ao/pi dye (acridine orange and propidium iodide) and incubated for 48 hours. ao dye works by passing through the plasma membrane of living cells then inserting into dna and rna which emit green fluorescence. pi dye emit red fluorescence in dead cells by working through the integrity of the cell membrane that has been damaged or disrupted and then penetrating the cell nucleus (shahruzaman et al., 2019). the results of the apoptosis test (figure 3.) showed that in 0.5% dmso control, all cells have intense green fluorescence which means the cells are still alive. in the control of doxorubicin 0.125 µg/ml, some cells have red fluorescence, meaning that the cells have died or experienced apoptosis. in addition, there are also some green and slightly orange colored cells that indicate cells experiencing early apoptosis. in the treatment of ethanolic extract of jackfruit leaves at a concentration of 250 μg/ml, there were some cancer cells that were still alive and began to experience apoptosis in the early stages. in the ethanolic extract of jackfruit leaves with the concentration of 500 μg/ml, most of the cancer cells experienced death or apoptosis (figure 4). living cells have an intact morphological structure of the nucleus. morphologically, cells that die and experience apoptosis are indicated by rounded cells, bubbling membranes, and apoptotic bodies (figure 4). calculation of the percentage of apoptosis cells showed that the 500 µg/ml eejl concentration has the highest number of apoptosis cells at 93.69 ± 10.93% (figure 5.). from the apoptosis test, the ethanolic extract of jackfruit leaves has the ability to induce apoptosis of widr cancer cells. the results of this apoptosis test also have results that are quite in accordance with the cytotoxic test. figure 4. ethanolic extract of jackfruit leaves can induce apoptosis in widr cancer cells. a. dmso 0.5%; b. doxorubicin 0.125 µg/ml; c. jackfruit leaf ethanolic extract 250 µg/ml; and d. ethanolic extract of jackfruit leaves 500 µg/ml. 10x10 magnification. white arrow indicates live cells. blue arrow indicates cells undergoing early apoptosis. yellow arrow indicates dead cells or late apoptosis. doxorubicin mutiyani & nuriliani – anticancer potential of ethanolic extract ... 225 figure 5. ethanolic extract of jackfruit leaves can cause apoptotic death of widr cancer cells. doxorubicin is an anticancer drug used as a positive control in this study. according to the results of the study, doxorubicin can be toxic and induce apoptosis in widr cancer cells. this is because doxorubicin is able to induce apoptosis by trapping the topoisomerase enzyme in dna damage so that it activates the transcription factor of cellular tumor antigen p53 (tp53). p53 (tp53) that is activated can control the expression of proapoptotic genes and inhibitors of antiapoptotic proteins (kciuk et al., 2023). doxorubicin can also induce apoptosis through increased levels of reactive oxygen species (ros) that activate atm-chk2-tp53 signaling independently of dna (kciuk et al., 2023). the ethanolic extract of jackfruit leaves can reduce cell viability and induce apoptosis in this study, possibly due to the effects of compounds contained in jackfruit leaves. this study used 70% ethanol solvent with maceration method to extract compounds in jackfruit leaves. previous research, jackfruit leaves extraction produced compounds namely, flavonoids, alkaloids, tannins, triterpenoids, and saponins from the extraction process with 70% ethanol solvent (nilakandhi et al., 2023). a typical flavonoid compound group in a. heterophyllus is artocarpin. artocarpin can cause cytotoxic effects by causing cell death through caspase activation, poly (adp-ribose) polymerase (parp) cleavage, and ros formation (daud et al., 2020). artocarpin is one of the compounds that may play a role in inducing apoptosis of widr cells. according to research conducted by arung et al. (2010), artocarpin isolated from a. heterophyllus wood can induce apoptosis accompanied by morphological changes and cell nuclei in t47d cancer cells by increasing caspase 3 and 8. factors such as solvent selection and extraction method can influence the results of this study. ethanol solvent was used because it is able to dissolve almost all compounds and is relatively non-toxic (mierza et al., 2022). the maceration method was chosen because it is classified as cold extraction so that it does not use excessive heating which results in damage to the compound (mierza et al., 2022; wardhani et al., 2023). the test results of jackfruit leaves extract are still not optimal with an ic50 740.43 μg/ml which is classified as nontoxic. nevertheless, the ethanol extract of jackfruit leaves still has potential to be studied further as an anticancer drug. this potential can be increased by choosing solvents and methods that can maximize the results and quality of jackfruit leaves compound content. conclusion based on this study, widr cancer cells treated with ethanolic extract of jackfruit leaves cannot reduce cell viability during 24 hour incubation. however, the extract can reduce cell viability at 48 hours incubation. the ic50 value obtained is not classified as toxic but there is a significant decrease in cell viability. ethanolic extracts can also cause apoptotic death. therefore, extracts from jackfruit leaves have little potential as anticancer drugs but can be further investigated by maximizing on the selection of solvents and extraction methods. competing interests: the authors declare no conflict of interest in the manuscript references arung, e. t., wicaksono, b. d., handoko, y. a., kusuma, i. w., shimizu, k., yulia, d., & sandra, f. 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(2018). techniques for extraction and isolation of natural products: a comprehensive review. chinese medicine, 13, 1-26. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 99-107 | doi: 10.14421/biomedich.2024.131.99-107 issn 2540-9328 (online) physicochemical, antimicrobial, lethality and in vitro antioxidant profiles of johnu tisane: a coffee (coffea arabica) leaf decoction formula anthony cemaluk chinedum egbuonu1,*, prince ogochukwu alaebo1, udumma nsofor onuoha2, chinomso juliet njoku1, chiemeziem adanma obike1, susan ogechi nlemadim1, blessing ifeoma chukwu1, sopuruchi mary-augusta iwejuo1, onyedikachi glory amaechi1, victor ifechukwu obiefuna1, bessing nmesomachi onuoha1, divinetreasure nzubechukwu okoli1, precious chinonso nwokeoma1, chukwubuikem eze1 1department of biochemistry; 2department of microbiology, college of natural sciences, michael okpara university of agriculture, umudike, nigeria. corresponding author* tonycemalukegbuonu@yahoo.com; egbuonu.anthony@mouau.edu.ng manuscript received: 10 december, 2023. revision accepted: 08 may, 2024. published: 15 may, 2024. abstract coffee leaves-based tea has relevance in ethno-medications due to its rich phyto-constituents-related diverse pharmacologic activities. jonhu tisane, a typical coffea arabica leaf decoction has no supporting scientific basis amidst reported location, processing and specierelated variations. this investigated physicochemical, antimicrobial, lethality and in-vitro antioxidant profiles of johnu tisane by acceptable methods. results recorded moisture (92.26 %), ash (0.65 %), unsaponified matter (1.46 mg/100 g), free fatty acid (0.56 mgkoh/g), acid value (1.12 mgkoh/g), potential hydrogen (6.85), lethal concentration (1000 ppm) and concentration-dependent antimicrobial activity. anti-oxidation results revealed total antioxidant capacity, ferric reducing antioxidant power, nitric oxide, hydrogen peroxide and 2, 2-diphynyl-1-picrylhydrazyl scavenging activities increased concentration-dependently compared to standard. thus, johnu tisane demonstrated low minerals and keeping quality; requisite physicochemical mix for consumption and bioactivity; high safety margin; antimicrobial potency; and requisite anti-oxidation capacity for in-vivo antioxidant role. these provided scientific support for its ethno-medicinal uses. they underscored the need to elucidate its pharmacologically active compounds; mechanistic roles in animal models; and the impact of ash to moisture mix variation on potential hydrogen, microbial and antimicrobial activities in relation to shelf life, bioactivity and in vivo anti-oxidative roles for novel insights on preserving sample quality, safety, bioactivity and in-vivo antioxidative outcomes. keywords: in-vivo anti-oxidative outcomes and roles; johnu tisane; requisite physicochemical mix; requisite anti-oxidant capacity; rich phyto-constituents-related diverse pharmacologic activities. abbreviations: anova = analysis of variance; dmrt = duncan multiple range test; dpph = 2, 2-diphynyl-1-picrylhydrazyl; frap = ferric reducing antioxidant power; h2o2 = hydrogen peroxide; izd = inhibition zone diameter; no = nitric oxide; ph = potential hydrogen; nosa = nitric oxide scavenging activity; tac = total antioxidant content. introduction coffee leaf tea has been used in varied ethnomedications against diverse diseases over the years notably in the coffee plant growing countries (campa and petitvallet, 2017; chen et al., 2019). the relevance of tea prepared from coffee leaves in varied ethno-medications was attributed to its antioxidant-rich phyto-constituents and the attendant antioxidant, antimicrobial, antiobesity, antiinflammatory, antihypertensive and other pharmacologic activities. these led to growing interests in its application in functional foods, pharmafoods, nutraceutical and ethno-medicines (upadhyay and mohan rao, 2013). identification of novel antimicrobials from plant parts is fundamental to new drug discovery (obasi et al., 2011) since microbial pathogens cause diseases with high morbidity and mortality (gbd 2019 antimicrobial resistance collaborators, 2022). oxidative stress has been implicated in the pathophysiology of many disorders (vona et al., 2021). and, antioxidants (secondary metabolites in living organism that are abundant in fruits and leafy vegetables) slow or prevent oxidative stress known to be a fundamental phenomenon in diverse diseased states (neha et al., 2019; singh et al., 2020). dietary antioxidants, mostly obtained from fruits and leafy vegetables consumption, have been associated with a great balance between free radicals and antioxidant https://doi.org/10.14421/biomedich.2024.131.99-107 100 biology, medicine, & natural product chemistry 13 (1), 2024: 99-107 status, which helps to minimize oxidative stress (kumar et al., 2016).the genus coffea belongs to the family rubiaceae with up to 124 species, among which, coffea canephora, coffea robusta and coffea arabica are prevalently used (flore et al., 2023). jonhu tisane is a decoction formula prepared from coffea arabica leaf and locally marketed and used as herbal tea for the management of varied ailments, including rheumatoid arthritis, obesity and inflammation of the lower limb during pregnancy. till date, there is no scientific basis supporting these medicinal claims for johnu tisane amidst the attendant variations due to processing methods, species and location on phyto-constinuents and bioactivities of tea prepared from coffee leaves and other plant food sources (egbuonu and nzewi, 2016; chen et al., 2019; monteiro et al., 2020). therefore, this study investigated some physicochemical, antimicrobial, lethality and in vitro antioxidant profiles of johnu tisane a decoction formula made from coffea arabica leaves. physicochemical properties indicate the quality, safety, palatability, and storability; antimicrobial properties indicate the potency against microbial pathogens, and lethality property indicates the safety margin when consumed while in vitro antioxidant properties indicate the probable in vivo antioxidant potential in combating oxidative stress that underlies every pathology. oxidative stress has been implicated in the pathophysiology of many disorders (vona et al., 2021). diseases caused by microbial pathogens present with high morbidity and mortality hence are of significant public health concerns (gbd 2019 antimicrobial resistance collaborators, 2022).therefore, this study determined the 1. physicochemical profile (moisture, ash, acid value, free fatty acid, unsaponified matter, and potential hydrogen (ph) values); 2. lethality property through the brine shrimp lethality test; 3. antimicrobial activity against selected fungi (tricophyton (t.), shigella dysenteriae (s. dysenteriae)) and bacteria (tricophyton (t.), shigella dysenteriae (s. dysenteriae)); and 4. in vitro antioxidant properties via total antioxidant content (tac), ferric reducing antioxidant power (frap), hydrogen peroxide (h2o2), nitric oxide (no) and 2, 2-diphynyl-1picrylhydrazyl (dpph) scavenging activities. material and methods chemicals and reagents chemicals used, including those used for the preparation of reagents used, were of analytical grade and were procured from reputable sources. procurement of johnu tisane and identification of the parent leaves used in the preparation jonhu tisane decoction formula was bought already prepared from the local sole producer and dispenser, mrs cecilia ezemalukwuo egbuonu (nee john ugbo) at ojoto, idemili south local government area, anambra state, south eastern nigeria. the formula was inherited from the father who as a pioneer catholic convert learnt of the diverse medicinal uses of coffee leaves-based tea from the early irish missionary reverend fathers. it was accordingly prepared by heating a specified quantity of air dried leaves of coffee in a specified volume of water (w/v) to boiling and allowing it simmer before use as herbal green tea. leaf sample in branches from the coffee plant used in the preparation was obtained from her compound for appropriate identification and deposition in the herbarium at michael okpara university of agriculture umudike as coffea arabica (voucher number: moh068). study design the study investigated the physicochemical properties of johnu tisane via the determination of moisture, ash, unsaponified matter, free fatty acid, acid value and potential of hydrogen in johnu tisane. it investigated the antimicrobial activity of johnu tisane against 10 (two fungal and eight bacterial) pathgogens of public health concerns. it investigated the lethality profile of johnu tisane via the brime shrimp lethality or cytotoxicity test and subsequent plot of percentage mortality for the determination of linearity of and the lethal concentration for 50 % deaths, lc50. the study investigated the in vitro antioxidant potentials of johnu tisane via total antioxidant capacity (tac), hydrogen peroxide, h2o2, scavenging ability, nitric oxide scavenging activity, nosa, ferric reducing antioxidant power (frap) against ascorbic acid, and 2,2-diphynyl-1-picrylhydrazyl (dpph) scavenging activity against ascorbic acid. determination of physicochemical properties acid value, av (mgkoh/g), potential of hydrogen (ph) and unsaponified matter (mg/100 g) were determined by the respective method described by onwuka (2005). free fatty acid (ffa) (mgkoh/g) was determined by the method of pearson (1976). moisture content (%), and total ash (%) were determined by the respective method of park (1996). determination of antimicrobial activity to prepare the innocula, microorganism as used in this work were obtained from the stock culture of the microbiology laboratory of the federal medical center, fmc, umuahia, abia state, nigeria. viability test for each isolate was carried out by resuscitating the organism in buffered peptone broth and thereafter sub cultured into nutrient agar medium and incubated at 37 °c for 24 hrs. the antimicrobial activity of the sample against the test organisms was determined by the cup-plate diffusion method (ebi and ofoefule, 1997). determination of in vitro antioxidant activities the ferric reducing antioxidant power, frap (%) was determined based on the reduction of fe3+ to fe2+ by egbuonu et al. – in-vitro profiles of johnu tisane 101 antioxidant in acidic medium (yen and chen, 1995; benzie and strain, 1999). the total antioxidant capacity (tac) of the extract was determined by the phosphomolybdate assay method (umamaheswari and chatterjee 2007; el-hashash et al., 2010). the scavenging activity on 2, 2-diphenyl-1-picrylhydrazyl, dpph, free radicals was determined by photometric assay (gyamfi et al., 1999; mensor et al., 2001) and used ascorbic acid (vitamin c) as reference/standard (iwalewa et al., 2008). hydrogen peroxide, h2o2, scavenging ability was determined according to the method of ruch et al. (1989) while nitric oxide radical inhibition/scavenging activity, nosa, was estimated using the method described by marcocci et al. (1994). determination of brine shrimp lethality value the brine shrimp lethality value was determined using the stock solution of actively swimming larva/nauplii obtained from freshly hatched shrimp eggs according to the method described by sarah et al. (2017) with slight modification (sample measured in ml instead of mg). statistical analysis results were presented as mean ± standard deviation (sd). within and between the groups, comparisons were performed by analysis of variance (anova) (using spss 17.0 computer software package). significant differences were compared using duncan multiple range test (dmrt), and a probability level of less than 5 % (𝑝 < 0.05) was considered significant. results and discussion physicochemical assessment of johnu tisane (table 1) uncovered the presence of moisture (92.26 %), ash (0.65 %), unsaponified matter (1.46 mg/100 g), free fatty acid (0.56 mgoh/g), acid value (1.12 mgoh/g) and potential of hydrogen (6.85). lethality outcome of johnu tisane using the brine shrimp lethality/cytotoxicity test (table 2) andlc50 linearity plot of percentage of mortality outcome (figure 1) uncovered a linearly-derived high lethal concentration, lc50 at 1000 ppm. table 1. some physicochemical properties of johnu tisane. physicochemical properties value free fatty acid, ffa (mgkoh/g) 0.56±0.01 acid value, av (mgkoh/g) 1.12±0.02 unsaponified matter um, (mg/100g) 1.46±0.02 ash content, ac (%) 0.65±0.003 potential of hydrogen, ph value 6.85±0.003 moisture content, mc (%) 92.26±0.33 values represent mean ± standard deviation of triplicate determinations, n = 3 figure 1. lc50 linearity plot of percentage of mortality outcome of johnu tisane. the antimicrobial activity test results revealed a concentration-dependent activity against the tested microbial (two fungal and eight bacterial) pathogens comparable to standards notably at the peak tested concentration. there was no measurable activity at the least tested concentration of 1/32 ml (figure 2, figure 3 and table 3). figure 2. activity (inhibition zone diameter, izd, in mm) of johnu tisane against the fungi c. albican and trichophyton (values represent mean ± standard deviation of triplicate determinations, n = 3) 102 biology, medicine, & natural product chemistry 13 (1), 2024: 99-107 table 2. activity of johnu tisane against the bacteria e. coli, n. gonorrhoeae, s. typhi and p. mirabilis. escherichia coli neisseria gonorrhoeae salmonella typhi proteus mirabili ciplofloxacin 43.67±1.53 42.00±1.00 47.00±0.58 34.00±1.00 ketoconazole 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 1 ml of johnu tisane 29.67±0.58 36.00±1.00 20.00±0.58 17.67± 0.58 ½ ml of johnu tisane 19.00±1.00 14.00±1.00 9.00±0.58 8.00±1.00 1/4 ml of johnu tisane 10.33±0.58 7.66±0.57 3.67±0.33 2.67±.58 1/8 ml of johnu tisane 4.00±1.00 3.00±1.00 0.00±0.00 0.00±0.00 1/16 ml of johnu tisane 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 1/32 ml of johnu tisane 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 values represent mean ± standard deviation of triplicate determinations, n = 3 figure 3. activity of johnu tisane against the bacteria s. aureus, s. dysenteriae, s. pneumoniae and p. aerouginosa (values represent mean ± standard deviation of triplicate determinations, n = 3) similarly, in vitro antioxidant results revealed concentration-dependent total antioxidant capacity, tac, nitric oxide radical scavenging activity, nosa and hydrogen peroxide scavenging activity (figure 4). figure 4. total antioxidant capacity, tac, nitric oxide scavenging activity, nosa and hydrogen peroxide scavenging activity of johnu tisane (values represent mean ± standard deviation of triplicate determinations, n = 3) egbuonu et al. – in-vitro profiles of johnu tisane 103 in a similar pattern, 2-diphynyl-1-picrylhydrazyl scavenging (dpph) activity and ferric reducing antioxidant power (frap) increased concentrationdependently in comparison with the standard but a measurable frap at the least concentration (25 µg/ml) unlike the reference antioxidant (figure 5). figure 5. dpph and frap activity (%)of johnu tisane against reference/standard antioxidant, ascorbic acid activity. discussion tea prepared from coffee leaves has relevance in ethnomedications due to its pharmacologic activities resulting from its rich phytoconstituents. jonhu tisane is a typical coffea arabica leaf decoction formula used in ethnomedication against rheumatoid arthritis, obesity, inflammation of the lower limbs during pregnancy and fetal overweight. it, however, has no supporting scientific basis for these activities amidst possible variations in coffee leaves based tea due to processing methods, species and location reported recently (ding et al., 2022). this study investigated physicochemical, antimicrobial, lethality and in vitro antioxidant profiles of johnu tisane by acceptable methods. results uncovered the presence of high moisture (92.26 %), but low ash (0.65 %), unsaponified matter (1.46 mg/100 g), free fatty acid (0.56 mgkoh/g), acid value (1.12 mgkoh/g), and potential hydrogen (6.85) in johnu tisane. these indicated that johnu tisane is relatively low in impurities, minerals and keeping quality with likelihood of rancidity and requisite physicochemical mix for acceptable taste, consumption and bioactivity. the ash content of jonhu tisane (0.65 %) indicates a negligible presence of inorganic minerals and other noncombustible components hence negligible impurities and inadequate mineral content compared to 3.52 % in unprocessed vigna aconitifolia (moth bean) seeds (opara et al., 2017). the ash content reported herein is in line with the proposal of ifemeje et al. (2020) that ash content should not exceed 5.54 % in order to maintain purity and quality of tea during storage. moisture content (%) correlates with increased microbial activity leading to depreciation in quality hence is an essential determinant and indicator of keeping quality and safety (arsenoaia et al., 2023; aruna, 2023). the moisture content of the sample (johnu tisane) recorded herein was quite above the proposed acceptable value of 6.5 % (ifemeje et al., 2020); 12.87 % reported for unprocessed vigna aconitifolia (moth bean) seeds (opara et al., 2017) and 3.42, 3.78 % respectively in seed and peel of carica papaya (egbuonu et al., 2016) which will have a negative implication on its stability, shelf life or keeping quality and safety as water promotes microbial growth and can lead to spoilage. this is an intriguing but interesting finding in relation to the actual long keeping quality of johnu tisane. the formula when prepared and preserved in a corked plastic container with no formal preservative stayed for a very long time, and used on dilution with hot water. it is therefore probable that johnu tisane has inherent antimicrobial potential that could sufficiently inhibit microbial growth and activity expected to result from its high moisture content noted herein. alternatively according to ifemeje et al. (2020), the markedly low ash content of johnu tisane may impact against the expected high moisture-related reduction in its keeping quality. ash content is usually an indicator of the cleanliness and purity of a sample, so low ash content as observed herein suggests that the johnu tisane is relatively free from impurities that could have promoted the buildup of microbes and microbial activities. acid value is a relative measure of rancidity due to free fatty acids formed during the decomposition of glycerides. hence, acid value is often used as a general indicator of the condition and edibility and the capacity of a sample to neutralize organic acids to yield free fatty acids following hydrolysis of glycerides via either lipase enzyme catalysis or in the presence of air and possibly bacteria (aruna s.o., 2023). high acid value indicates lesser quality probably due to a variety of agents, including presence of moisture. in this study, the acid value of johnu tisane (1.12±0.02 mgkoh/g) is comparatively low for rancidity to occur hence will be edible (nwachoko et al., 2023)). similarly, high free fatty acids content indicates capacity to undergo hydrolysis of triacylglycerol and release free fatty acids, inadequate processing and storage conditions or presence of contaminants notably microorganisms which serve as 104 biology, medicine, & natural product chemistry 13 (1), 2024: 99-107 the source of the enzyme lipase that catalyzes the hydrolysis reaction. this study recorded a low free fatty acid concentration of 0.56±0.01 mgkoh/g and supported the apparent purity, edibility and stability of johnu tisane. potential hydrogen (ph) measures the hydrogen ion activity as it serves as a significant parameter in acid-base neutralization and an indicator of fluid drinking quality and safety (vijayakumar et al., 2019). generally, coffee is expected to have a ph value between 5 and 6. as would be expected, a potential hydrogen (ph) value of 6.85 in johnu tisane was recorded in this study as against the value of 4.90 reported for citrullus lanatus seed oil (egbuonu et al., 2015). this suggests that johnu tisane coffee leaf decoction is a slightly acidic solution. this further confirms its relatively low acid value, edibility and low toxicity. in support, results also showed linearly-derived high lethal concentration, lc50 of 1000 ppm, which indicates its unbroken high safety margin or low toxicity when consumed by animal, hence edible for animal consumption. time dependent variation of combined processing methods could affect some physicochemical properties of plant food sources (egbuonu et al., 2014), hence studies aimed at optimizing the mix of these parameters in johnu tisane are warranted and recommended. acid value correlated negatively, while the composition of unsaponified matter correlated positively, with antioxidant activity (malecka, 2002). thus, the mix of unsaponified matter (mg/100 g) (1.46) and acid value (mgoh/g) (1.12) compositions of johnu tisane reported herein suggested its high antioxidation capacity since acid value and in particular the composition of unsaponified matter correlated negatively and positively, respectively with antioxidant activity according to malecka (2002). results of the investigation of the in vitro anti-oxidation profile of johnu tisane revealed concentration-dependent increase in total antioxidant capacity, tac, nitric oxide scavenging activity, nosa and hydrogen peroxide scavenging activity. similarly, 2, 2-diphynyl-1-picrylhydrazyl scavenging (dpph) activity and ferric reducing antioxidant power (frap) increased concentration-dependently in comparison to standard but recorded a measurable frap at the least concentration (25 µg/ml) unlike the reference antioxidant. these indicated high in vitro antioxidation potential of johnu tisane (coffee leaves decoction) irrespective of concentration and a basis for possible in vivo antioxidant roles. a strong antioxidant activity was reported earlier, but for samples of coffee beans (tsedale et al., 2020). the implication is that johnu tisane has significant hydrogen donating property and could donate hydrogen to pair up with lone pair of the free radicals to scavenge or break the free radical chains (mbinda and musangi 2019). in support, a dose dependent increase in frap observed herein which indicated antioxidant activity accompanied by reducing power is a strong pointer that johnu tisane has a proton-donating property and could exert a strong free radical inhibiting or scavenging activity and probable in vivo antioxidant role (uroko et al., 2022). also, its concentration dependent increase in tac indicates the cumulative antioxidant capacity of an antioxidant to scavenge free radicals attributable to high phenolic content (frempong et al., 2021). the phenolic content of johnu tisane was not determined in this study which is a notable limitation underscoring the need to elucidate the phenolic content and other pharmacologically active compounds in johnu tisane. coffea arabica leaves have abundant antioxidant chemicals and resultant teas demonstrated antioxidant activities in vitro and in vivo (monteiro et al., 2020). the present outcomes and suppositions thereto strongly support the ethno-medicinal uses and actions of johnu tisane. potent antioxidants, including ascorbic acid commonly used in dpph radical scavenging assay as a standard/reference compound for measuring in vitro antioxidant activity, could scavenge the oxidant or free radicals-prone dpph due to their hydrogen or electron donating ability (adesegun et al., 2017). oxidative stress due to excess oxidants and free radicals is fundamental to pathogenic states and antioxidants through antioxidant defense mechanisms that mop up excess free radicals to prevent oxidative stress in vivo significantly modulated pathogenic states in animals (ponnampalam et al., 2022). thus, the possible pharmacologic activities of johnu tisane may be via antioxidant metabolic pathways warranting further, particularly, mechanistic studies to elucidate its metabolic roles and routes in experimentally challenged animal diseased models. the antimicrobial activity of johnu tisane was explored to assess the probability that the decoction has inherent antimicrobial potential that could sufficiently inhibit microbial growth and activity expected to result from its high moisture content noted in this study. the antimicrobial activity results showed concentrationdependent activity against the tested microbes which were comparable to standards notably at the peak. these revealed the inhibition of wide range of microbial pathogens and an indication of broad spectrum activities of johnu tisane against varied pathogenic microbes. thus, the concentration-dependent antimicrobial activity of johnu tisane observed herein aligns with the mooted inherent antimicrobial potential which could inhibit microbial activities that could have reduced its keeping quality when preserved. it also supports the diverse medicinal uses of johnu tisane since microbial pathogens underlie the pathogenesis of many diseases of public health concerns (gbd 2019 antimicrobial resistance collaborators, 2022). pseudomonas aeruginosa resists many antibiotics while staphyloccus aureus, e. coli and k. pneumoniae causes various diseases, including diarrhea, urinary tract infections and pneumonia among others (akinpelu et al., 2015). trichophytons are infectious dermatophytes that cause tinea capitis and egbuonu et al. – in-vitro profiles of johnu tisane 105 ringworm among (especially african) children (akinpelu et al., 2015). the susceptibilities of these pathogens to johnu tisane corroborate its local uses against varied diseases and infections that may be caused by microbial pathogens and thus demonstrated its significant therapeutic potential and possibility to serve as a good source of antimicrobial compound that can be employed in pharmaceutical industries for the formulation of potent antimicrobial drugs. johnu tisane inhibited these pathogens even at lower, albeit crude, concentration and thus could be used in preventing opportunistic infections caused by these pathogens. these support the inferred possibility in this study of inherent antimicrobials in johnu tisane inhibiting the growth and activity of opportunistic microbes that could have impaired its keeping quality due to its high moisture content reported herein. the study outcomes provoke and warrant timedependent studies on johnu tisane. these also provoke an investigation on the general impact of variation of ash to moisture mix on potential hydrogen, overall microbial and antimicrobial activities and the attendant shelf life or keeping quality, bioactivity and anti-oxidative roles to provide a novel deep insight on food packaging and preservation in relation to in-vivo anti-oxidative outcomes in healthy and unhealthy animal models. this is imperative because, as recorded herein, a strong antioxidant activity that correlated positively with the antibacterial activity was reported for samples of coffee beans (tsedale et al., 2020) and concomitant elicitation of antioxidant and antimicrobial activities could offer synergistic preventive cum therapeutic benefits against animal diseases. conclusions thus, johnu tisane demonstrated low minerals and keeping quality; requisite physicochemical mix for consumption and bioactivity; high safety margin; antimicrobial potency; and requisite anti-oxidation capacity for possible in vivo antioxidant role. these provided scientific support for its current ethnomedicinal uses; underscored the need to elucidate its pharmacologically active compounds; mechanistic roles in animal models; and the impact of ash to moisture mix variation on potential hydrogen, microbial and antimicrobial activities in relation to shelf life, bioactivity and possible in vivo anti-oxidative roles. these would provide a novel deep insight on packaging and 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(1995). antioxidant activity of various tea extracts in relation to their antimutagenicity. journal of agricultural and food chemistry, 43(1), 27-32. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 407-413 | doi: 10.14421/biomedich.2024.132.407-413 issn 2540-9328 (online) effectiveness of ethanol extract clove leaves (syzygium aromaticum) in inhibiting biofilm of candida albicans atcc 14053 firdausi nuzula1, masfufatun2,* 1faculty of medicine, university of wijaya kusuma surabaya, indonesia. 2department of biochemistry, faculty of medicine, university of wijaya kusuma surabaya, indonesia jl. dukuh kupang xxv no. 54, dukuh kupang, dukuh pakis, surabaya, indonesia. corresponding author* masfufatun@uwks.ac.id manuscript received: 15 july, 2024. revision accepted: 07 october, 2024. published: 09 october, 2024. abstract candida albicans is the most common type of candida. it is a normal microbiota in the healthy human body that can become pathogenic when its balance is disturbed, causing an infection referred to as candidiasis. antifungal resistance to biofilms is estimated to be 10,000 times that in planktonic form. clove leaves (syzygium aromatium) can treat infectious diseases, including candidiasis. this study aimed to analyze whether the ethanol extract of clove leaves (s. aromaticum) destroys the biofilm of c. albicans atcc 14053. the research design was pure experimental with a post test control group only design approach. the extraction method used was maceration. test antibiofilm activity of ethanol extract of clove leaves using microtiter plate biofilm assay method. measurement of results with a microplate reader was carried out using a wavelength of 595nm in accordance by the wavelength of crystal violet used as a colouring agent. the results showed that ethanol extract of clove leaves (s. aromaticum) has significant effectiveness against the maturation of c. albicans biofilm (p<0.05) with the highest percentage of activity at 20% concentration. the mbec50 was determined by probit analysis, so the concentration of clove leaf ethanol extract (s. aromaticum) that can eradicate 50% of c. albicans biofilm is located at a concentration of 0.45%. the clinical benefits that can be developed from the results of this study are the potential use of clove leaf ethanol extract as an alternative therapy for c. albicans infection in the form of soap or ointment. keywords: biofilm; candida albicans; candidiasis; clove leaf. abbreviations: mbec minimum biofilm eradication concentration; atcc american type culture collection introduction candida is a genus of fungi often found in the environment, especially in tropical climates (mbatu et al., 2018). candida albicans is the most commonly found type of candida and is a normal microbiota in the healthy human body, for example, on the skin, mouth, intestines, genital tract, and under the nails (khafidhoh et al., 2015). c. albicans can become pathogenic when its balance is disturbed, which will accelerate its proliferation and cause infection. fungal infections caused by candida are referred to as candidiasis (masfufatun et al., 2014). in indonesia, candidiasis is around 20-25% (puspitasari et al., 2019). the occurrence of candidiasis recurrence is related to the pathogenicity of c. albicans, one of which is supported by the ability to form biofilms. the stages of biofilm formation include attachment, microcolony formation, biofilm formation, maturation, and cell dispersion (purbowati, 2016). biofilms are composed of various types of cells (yeast-shaped, having round buds, oval pseudohyphal cells, and long hyphal cells), attached to biotic and abiotic surfaces and embedded in a layer called the exopolysaccharide matrix (gulati et al., 2016). the presence of this matrix causes resistance to antifungals and hinders phagocytosis by host cells (tsui et al., 2016). currently used antifungals include azoles, polyenes, echinocadin, allylamine, and fluoropyrimidine (kabir & ahmad, 2013). although azoles have long provided effective treatment, recent epidemiological studies have shown azole resistance in some cases of candidiasis (whaley et al., 2017). antifungal resistance to biofilms is estimated to be 10,000 times that in planktonic form (rabin et al., 2015). antifungal resistance occurs due to complex biofilm-forming components, making it more difficult for antifungals to work (sharma et al., 2016). in addition to causing resistance, prolonged use of synthetic drugs can result in respiratory depression, uritaccharia, hepatotoxicity, decreased platelet levels, and gastrointestinal disorders (lestari et al., 2019). an effort is needed to inhibit growth in both planktonic and biofilm forms. one of them is by using https://doi.org/10.14421/biomedich.2024.132.407-413 mailto:masfufatun@uwks.ac.id 408 biology, medicine, & natural product chemistry 13 (2), 2024: 407-413 natural materials. according to research that has been done, there is an antibiofilm effect of clove (s. aromaticum) essential oil nanoemulsion against staphylococcus aureus with an mic of 0.62 mg/ml, while against cryptococcus neoformans with an mic of 3.12 mg/ml, and against with an mic of 50 mg/ml (shehabeldine et al., 2023). clove is a spice plant native to indonesia. almost all parts of cloves can be utilized, starting from the leaves, flowers, and twigs. clove leaves (syzygium aromatium) can be used to treat infectious diseases, including candidiasis. in the phytochemistry of clove leaf extract, both crude and purified extracts contain flavonoids, tannins, saponins, terpenoids, and alkaloids (novema et al., 2022). materials and methods research design the research design used was pure experimental with a post test control group only design approach. test the antibiofilm activity of clove leaf ethanol extract using the microtiter plate biofilm assay method. extraction of clove leaves the method used to perform the extraction is maceration. prepare 995 grams of simplistic powder and then put it in a macerator. adding 96% ethanol solvent into the macerator. after that, let stand until all the simplistic powder is submerged for 3x24 hours while stirring periodically. then the mixture is filtered and the pulp is soaked again using new 96% ethanol. the further filtering process will be carried out twice using 96% ethanol. liquid extracts are put together and concentrated using a vacuum rotary evaporator at 78oc and concentrated again using a water bath at 40oc to produce thick extracts (wahyulianingsih et al., 2016). concentrations of extract each treatment requires 4 repetitions in 8 test groups, so the concentrations used in each treatment are 20%, 10%, 5%, 2.5%, 1.25%, 0.63%, 0.31%, 0.16%. the finished thick extract of clove leaves (s. aromaticum) was weighed by starting the largest concentration of 20%. then put into a vial bottle. the thick clove leaf extract solution was diluted using dimethyl sulfoxide (dmso) solvent according to the desired concentration using the following dilution formula: 𝑀1 𝑥 𝑉1 = 𝑀2 𝑥 𝑉2 𝑀1 : concentration of solution before dilution 𝑉1 : volume of solution before dilution 𝑀2 : concentration of solution after dilution 𝑉2 : volume of solution after dilution (iqhasari r, 2017). regeneration and preparation of c. albicans inoculum c. albicans isolates were inoculated as much as 1 ose by streak plate method or scraping the colony. furthermore, the new sda media is incubated using an incubator for 2-3 days so that a single colony of c. albicans will form. inoculation of c. albicans on sdb media for 18-20 hours at room temperature using a shaker at 120 rpm produces inoculum (naitullah et al., 2014). harvesting of inoculum and preparation of c. albicans suspension single colonies of c. albicans grown on sda for 2-3 days were put into erlenmeyer flasks containing 10 ml of sdb. sdb media was used as a dilution solution because it supports biofilm growth. next, the flask was shaken for 24 hours at 120 rpm in an erlenmeyer flask. performed centrifugation by moving the c. albicans inoculum into a centrifuge tube for 15 minutes until a pallet was produced. the pallet formed was separated from the filtrate using pbs and resuspended to 1 x 106 cfu/ml with rpmi media. the optical density (od) of the c. albicans suspension will be calculated using a microplate reader. if the od > 0.5, continue diluting until od = 0.5 is obtained. c. albicans od 0.5 suspension is ready for an antibiofilm test (fauzan et al., 2023). mature biofilm formation 100 μl of c.albicans suspension was put into the wells starting from rows a-d, columns 1-6, 8, 10, and 12. then it was incubated for 2 hours at 37o c for the c.albicans attachment stage. next, 100 μl rpmi media was added to all wells and incubated for 48 hours at 37o c for the growth stage of c. albicans. then, added extracts in concentrations of 20%, 10%, 5%, 2.5%, 1.25%, 0.63%, 0.31%, 0.16%, rpmi (for positive, negative, and blank media controls), and fluconazole (for positive control). for blank samples, 50 μl of c. albicans suspension was also placed into the wells in rows g and h. incubated at 37°c for 24 hours for the c.albicans maturation stage (zhu et al., 2023). mature biofilm eradication test after incubation at 37°c for 24 hours for the c.albicans maturation stage. each well was washed twice using pbs. the unattached planktonic cells of c. albicans were gently removed by inverting and pouring on a tissue to obtain c. albicans attached to the microplate. the biofilm was fixed using methanol for 15 minutes and then dried at room temperature. next, 0.1% crystal violet solution was added to each well for staining for 30 minutes at room temperature. next, wash 3 times using pbs. adding 96% ethanol to each well then incubated for 1 hour at room temperature and measured using a microplate reader (zhu et al., 2023). nuzula & masfufatun – effectiveness of ethanol extract clove leaves (syzygium aromaticum) … 409 minimum biofilm eradication concentration (mbec50) minimum biofilm eradication concentration (mbec50) is the lowest clove leaf ethanol extract concentration that can eradicate 50% of c. albicans biofilm. the mbec50 value was determined using od maturation data converted to % eradication and analyzed spss probit (gupta et al., 2016). data analysis the data analysis method used spss (statistical product of service solution) statistical analysis for windows. the normality test used if the data is less than 50 is the shapiro-wilk test. furthermore, a homogeneity test was carried out using the levene test if it was proven that the data was typically distributed or close to normal. data that are normally distributed and homogeneous (p>0.05) will be analyzed using the one-way analysis of variance (anova) test, which is a parametric test to compare differences in the means of data from more than two groups. the significance level was set at p<0.05. after the one-way anova test was conducted, the post hoc test was followed. the post hoc test is an advanced test that analyzes which test group has a significant difference. furthermore, the mbec50, it was determined using spss probit analysis. result and discussion result characteristics of clove leaf this study aimed to test the antibiofilm effect of ethanol extract of clove leaves (s. aromaticum) at the maturation stage. clove leaves were obtained from jatirejo district, mojokerto. the clove leaves were picked, washed, and aerated for one week. after drying, the clove leaves were pulverized using a blender until they became powder (simplistic). clove leaf simply obtained as much as 995 grams. the clove leaf ethanol extract was prepared by maceration method, namely by soaking the fine simplistic using 96% ethanol solvent and filtering using filter paper for 3x24 hours while stirring periodically. the liquid extract was combined and concentrated using a vacuum rotary evaporator at a temperature of 78o c and concentrated again using a waterbath with a temperature of 40o c to produce 156 grams of clove leaf ethanol extract with a greenish brown color. biofilm maturation detection test to see the biofilm maturation, c. albicans was incubated at 37°c for 2 hours for the attachment stage. furthermore, another incubation was carried out for 48 hours at 37o c for the growth stage of c. albicans. then, the ethanol extract of clove leaves (s. aromaticum) was added. the wells that have been treated are incubated for 24 hours at 37°c. the results of the c. albicans biofilm maturation test can be seen in figure 1 figure 1. mature biofilm eradication test results with crystal violet staining. column 1a-e = concentration 20% column 2a-e = concentration 10% column 3a-e = concentration 5% column 4a-e = concentration 2.5% column 5a-e = concentration 1.25% column 6a-e = concentration 0.63% column 7a-e = concentration 0.31% column 8a-e = concentration 0.16% column 9a-e = positive control column 10a-e = negative control column 12a-e = media blank based on figure 1., the results show that clove leaf extract at a concentration of 20% after giving crystal violet has a faded purple color and the smaller the concentration, the more intense the purple color produced, meaning the higher the density of biofilm biomass. the most intense purple color was found in the negative control group. furthermore, the absorbance/optical density (od) value was measured which can be seen in table 1. table 1. od mature biofilm eradication test results with clove leaf extract. replication clove leaf extract concentration control 20% 10% 5% 2.5% 1.25% 0.63% 0.31% 0.16% k (+) k (-) 1 0.29 0.60 0.66 0.76 0.84 0.92 1.56 3.36 1.92 3.27 2 0.13 0.46 0.59 0.85 0.68 0.96 1.24 3.22 2.06 3.16 3 0.20 0.61 0.54 0.68 0.73 0.62 1.28 3.15 2.08 3.06 4 0.19 0.50 0.41 0.63 0.60 0.64 0.84 3.07 1.80 3.32 average 0.20 0.54 0.55 0.73 0.71 0.79 1.23 3.20 1.97 3.20 std 0.06 0.07 0.09 0.08 0.09 0.16 0.26 0.10 0.11 0.10 410 biology, medicine, & natural product chemistry 13 (2), 2024: 407-413 in table 1, the c. albicans mature biofilm test results were replicated four times, with the highest average od value obtained at a concentration of 0.16%, namely 3.20, and the weakest at a concentration of 20%, namely 0.20. at a concentration of 0.16%, the highest od value is obtained, meaning that the density of the biofilm biomass is the highest so that the ability of clove leaf extract to eradicate c. albicans biofilm is lower and at a concentration of 20% has the lowest od, meaning that the density of the biofilm biomass is the lowest so that the ability of clove leaf extract to eradicate c. albicans biofilm is higher. meanwhile, for the control group, the highest od value was found in the negative control (k-) because the negative control group was not given any treatment for c. albicans cells that matured on the microplate. table 1 shows a decrease in the average maturation of c. albicans biofilm and an increase in concentration. figure 2. post hoc analysis results of mature biofilm eradication with clove leaf extract. superscript abc, if it contains the same letters means there is no significant difference. if it contains different letters, it means there is a significant difference between the treatment groups based on the post hoc test (p value <0.05 shows a significant difference) based on the post hoc test results in figure 2, significantly different groups were at a concentrations of 20%, 0.31% and positive control while groups that did not have significant differences were at concentrations of 10%, 5%, 2.5%, 1.25%, 0.63%, 0.16% and negative control. mbec50 value determination results the optical density of the table was used to determine the percent of c. albicans biofilm eradication testing using the formula below: % biofilm maturation rate = (od negative control − od sample) (od negative control) x 100% table 2. eradikasi biofilm. % eradikasi clove leaf extract concentration 20% 10% 5% 2.5% 1.25% 0.63% 0.31% 0.16% 93.67 83.10 82.82 77.21 77.78 75.43 61.58 0.01 from table 2, the highest eradication results are in the clove leaf ethanol extract group with a concentration of 20% having the highest eradication ability, meaning that the growth of c. albicans biofilm is getting lower and vice versa at a concentration of 0.16% has the lowest eradication ability, meaning that the growth of c. albicans biofilm is getting higher. determination of mbec50 using probit analysis obtained the results of the concentration of clove leaf extract that can eradicate 50% of c. albicans biofilm is located at a concentration of 0.45%, meaning that clove leaf ethanol extract can eradicate 50% of c. albicans biofilm at a concentration of 0.45%. discussion clove leaf extract yield the clove leaves used in this study are about 6-8 months old, green , and fresh. clove leaves that have been picked must be washed first to remove dirt, then aerated for one 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 20% 10% 5% 2.5% 1.25% 0.63% 0.31% 0.16% k (+) k (-) a v er an g e o p ti ca l d en si ty ( 5 9 5 n m ) clove leaf extract concentration maturation stage curve b a b b b b c d d e nuzula & masfufatun – effectiveness of ethanol extract clove leaves (syzygium aromaticum) … 411 week to reduce the water content so that it can inhibit microbial growth. after drying, the clove leaves are mashed using a blender until they become powder (simplisia) (warnis, et al., 2020). the particle size of the sample is related to the surface area that will contact the extraction solvent. the finer the simple, the easier the compounds in the leaves move into the solvent (aji, 2018). the extraction method used in this research is maceration using 96% ethanol solvent. the 96% ethanol solvent was chosen because it is a universal solvent that can dissolve organic compounds in samples of both polar and non-polar compounds (munte et al., 2015). the use of 96% ethanol as a solvent because ethanol is more accessible to enter the cell membrane so that it can extract secondary metabolite compounds such as flavonoids, tannins, saponins, terpenoids, and phenols (dewi et al., 2021). from the maceration results of 995 grams of clove leaf simplisia, 156 grams of thick extract were obtained with a percentage yield of 16%. this research is in line with that conducted by suhendar and sogandi (2019) who used the maceration method extract clove leaves. however, the solvent in the study used 70% methanol. the results of this method from 1 kg of clove leaf simplistic obtained a thick extract of 100.08 grams with a percent yield of 10%. where good yield results have a value of> 10% (novema et al., 2022). 96% ethanol solvent has a lower polarity than 70% methanol. methanol 70% also contains 30% water, which can reduce the extraction efficiency of some lipophilic compounds. water can cause hydration or precipitation of specific components that should be extracted. thus, compounds in clove leaves with lipophilic properties (like fat) will dissolve more easily in 96% ethanol. organoleptic results show that the methanol extract of clove leaves has a thick form, reddish brown color, bitter taste, and smells typical of cloves. meanwhile, the ethanol extract of clove leaves also has a thick texture, greenish brown color, and smells typical of cloves. dimethyl sulfoxide (dmso) is used to dissolve organic compounds that are less soluble when using distilled water. dmso is a solvent that can dissolve almost all compounds both polar and non-polar. dmso has low toxicity, has anti-inflammatory, and analgesic effects. in research conducted by rahmi & putri (2020), it was found that the use of dmso as a solvent had no effect on the test results carried out using the disc paper method against c. albicans. in this study, dmso solvent was used because clove leaf ethanol extract does not have an aqueous phase. effect of clove leaf extract on mature biofilm based on the results of the mature biofilm eradication test in table 2. the od value of the test group of clove leaf extract is lower than the od of the positive control except at a concentration of 0.16%. this indicates that at concentrations of 0.31% to 20%, clove leaf ethanol extract has a better mature biofilm effect than fluconazole antifungal. the lowest od value was found at 20% concentration. the od value from concentrations of 0.16% to 20% at the stage of mature biofilm of c. albicans is decreasing, which shows that with the increasing concentration of extracts used, the ability to eradicate biofilms is increasing. based on the results of anova analysis, indicate that there is an effect of giving clove leaf ethanol extract (s. aromaticum) on the maturation stage of c. albicans biofilm significantly (p<0.05). in figure 2, groups that have significant differences are at concentrations of 20%, 0.31%, and positive control, while groups that do not have substantial differences are at concentrations of 10%, 5%, 2.5%, 1.25%, 0.63%, 0.16%, and negative control. clove leaf can eradicate mature biofilm because it contain active compounds such as saponins, alkaloids, tannins, flavonoids, and terpenoids. tannins will inhibit the icaa and icad genes. these genes will synthesize pia, which plays a role in eps formation and cell aggregation. the action of saponins is to increase cell wall permeability by reducing surface tension, so that cells leak and intracellular components come out. the role of saponins as antibiofilm monospecies of c. albicans is at the stage of biofilm adherence and maturation (hamzah et al., 2021). from table 2. the most effective test group was at 20% concentration because the percentage was the highest at 93.67%. thus, the antibiofilm activity of clove leaf ethanol extract to eradicate c. albicans biofilm is high because the percentage of antibiofilm activity is ≥50% (famuyide et al., 2019). at the maturation stage, research that supports this research is research conducted by mirzaei et al., (2022), where melittin, vancomycin, and rifampicin against staphylococcus epidermidis biofilms produced mbec50 for melittin, rifampin, and vancomycin, are 0.002%, 0.0512%, and 0.1024%. 50 according to abidah's research, (2020), black mulberry leaf extract (morus nigra l.) against escherichia coli biofilm has an mbec50 of 0.016%. meanwhile, in this study the mbec50 is 0.45%. as the mature biofilm, the synergy and communication between the fungi in the biofilm becomes more complex. the eps matrix layer that is formed is more numerous and thicker. the eps matrix can increase the defense of fungi compared to still in planktonic form. the eradication ability of an extract is related to the ability of the compounds contained in the extract to penetrate the eps matrix layer that envelops the bacteria and destroys the eps matrix in the biofilm. microcolonies that have formed will develop and undergo maturation. maturation is influenced by electrolyte concentration, carbon source, ph, surface type, temperature, and osmolarity. maturation is characterized by an increase in biofilm density and complexity (kumar et al., 2017). 412 biology, medicine, & natural product chemistry 13 (2), 2024: 407-413 conclusions based on the results of the research that has been done, it can be concluded that the ethanol extract of clove leaves (s. aromaticum) has an antibiofilm effect on the maturation stage of c. albicans significantly. at the highest concentration, 20% was able to eradicate 93.67%. the minimum biofilm eradication concentration (mbec50) in eradicating of 50% c. albicans biofilm is 0.45%. thus, clove leaf ethanol extract has the potential to be an alternative therapy for c. albicans infection. acknowledgments: special gratitude goes to the department of biochemistry, university of wijaya kusuma surabaya and infectious hospital university of airlangga. authors’ contributions: firdausi nuzula laboratory work and wrote the 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(2023). anti-biofilm activity of cocultimycin a against candida albicans. internasional journal of molecular sciences, 24, 1-16. https://doi.org/ 10.3390/ijms242317026 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1009-1013 | doi: 10.14421/biomedich.2025.142.1009-1013 issn 2540-9328 (online) antipyretic activity of stembark extract of mammea africana in rats jude e. okokon1*, chinyelu c. osigwe2, john a udobang3, uwaeme ugonma florence2 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria 2department of pharmacology and toxicology, faculty of pharmacy, madonna university, elele, rivers state, nigeria. 3department of clinical pharmacology and therapeutics, faculty of basic clinical sciences, university of uyo, uyo, nigeria. corresponding author* judeefiom@yahoo.com manuscript received: 02 september, 2025. revision accepted: 27 october, 2025. published: 30 october, 2025. abstract mammea africana sabine (guttiferae), is a tree plant whose parts are variously used locally for the treatment of various diseases such as malaria and fever among others. the stembark extract of m. africana (30 -90 mg/kg) was investigated for antipyretic activity in rats using different experimental models; amphetamine, dinitrophenol and yeast-induced pyrexia. the extract exerted prominent inhibition of pyrexia on amphetamine, dinitrophenol and yeast -induced pyrexia. inhibition was significant (p<0.05–0.001) from 3 to 5 h post administration of extract and in a dose-dependent fashion. the antipyretic effects of this plant may in part be mediated through the chemical constituents of the plant. the results of this investigation validate the ethnomedical uses of this plant in the treatment of febrile conditions. keywords: mammea africana; medicinal plant; antipyretic; fever. introduction mammea africana sabine (guttiferae) (syn. ochrocarpus africana oliv.) (m. africana), widely distributed in tropical africa, is a tall tree of 50 to 100 feet high whose bark is often yellow with pale scales and resinous yellow sap (hutchison and daziel, 1958). the stembark of the plant is traditionally used by the ibibios, of niger delta region of nigeria, in the treatment of a number of diseases such as malaria related fever, diabetes, microbial infections and mental disorders. traditionally, the stembark is used also to treat stomach pains, rheumatism pains, scabies, cough and hypertension (raponda-walker and silans, 1961; adjanohoun et al., 1996). the stembark extract which possesses cytotoxic activity, in vitro (chapius et al., 1988; okokon et al., 2012)., has been reported to contain cytotoxic coumarins with antimicrobial activity against staphylococcus aureus (ouahouo et al., 2004). other pharmacological activities reported on the stembark include; anti-plasmodial (okokon et al., 2006), cardioprotective (okokon and antia, 2007), anti-diabetic, hypolipidaemic (okokon et al., 2007; tchamadeu et al., 2010), vasorelaxant (dongmo et al.,2007), anti-hypertensive (nguelefackmbuyo et al., 2008), anti inflammatory, analgesic (okokon et al., 2009), antioxidant (nguelefack-mbuyo et al., 2010), anti-diarrheal, anti-ulcer (okokon et al., 2010), immunomodulatory, anti-lesihmanial (okokon et al.,2012), depressant and anti-convulsant (okokon and davies, 2014), nephroprotective (okokon and bawo, 2014), hepatoprotective (okokon et al., 2016), genotoxic and cytotoxic (ebong et al., 2022) activities. phytochemical study on the stembark reported the presence of 5,-7-dihydroxy-8-(12methyl-butryl) – 4 –n -pentylcoumarins and mesuxanthone b (carpenter et al., 1970;1971; cricton and waterman, 1978), 4-phenyl and 4alkylcoumarins (games, 1972). while alkaloids have been reported to be absent in the entire plant parts (gartlans et al., 1980). we report in this study the antipyretic activities of the stembark extract. materials and methods plants collection the plant materials, mammea africana (stembark), were collected in anwa forest in uruan area, akwa ibom state, nigeria in january 2022. identification and authentication of the plant was carried out by a taxonomist of department of botany and ecological studies, university of uyo, uyo, nigeria. extraction pieces of the stembark were washed and shade-dried for two weeks. the dried plants’ materials were further chopped into small pieces and reduced to powder using electric grinder. the powdered material (1.5 kg) was macerated for 72 h in 50% ethanol. this was thereafter https://doi.org/10.14421/biomedich.2025.142.1009-1013 1010 biology, medicine, & natural product chemistry 14 (2), 2025: 1009-1013 filtered and the liquid filtrate was concentrated and evaporated to dryness in vacuo 40˚c using a rotary evaporator (buchilab, switzerland). the extract was stored in a refrigerator at -4˚c, until used for the proposed experiments. animals albino wistar rats (122-1136 g) of either sex were obtained from the university of uyo animal house. they were maintained on standard animal pellets and water ad libitum. permission and approval for animal studies were obtained from the college of health sciences animal ethics committee, university of uyo. evaluation of antipyretic activity of the mammea africana stembark extract on d-amphetamine-induced pyrexia the albino rats that were used in this experiment were fasted for 24 hours but allowed water ad libitum before the experiment commenced. they were randomized into groups of 5 rats each according to their weights. the initial basal temperatures of the rats were taken after which pyrexia was induced by intraperitoneal administration of amphetamine (5 mg/kg, i.p) to the animals. pyrexia was allowed to develop within 30 min post administration of amphetamine. at the confirmation of temperature increase of at least 1℃ by monitoring the rectal temperature, the leaf extract was respectively administered to the different groups of rats at 30, 60 and 90 mg/kg orally and aspirin (100 mg/kg) and distilled water (10 ml/kg, orally) were administered respectively to the positive and negative control groups of rats. rectal temperatures of the animals were monitored at an hour interval for 5 h (edem et al., 2023). effect of mammea africana stembark extract on 2,4dinitrophenol (dnp)-induced pyrexia the albino rats that were used in this experiment were fasted for 24 hours but allowed water ad libitum before the experiment commenced. they were randomized into groups of 5 rats each according to their weights. the initial basal temperatures of the rats were taken after which pyrexia was induced by intraperitoneal administration of dinitrophenol (dnp) (10 mg/kg, i.p.) to the animals. pyrexia was allowed to develop within 30 min post administration of dinitrophenol. at the confirmation of temperature increase of at least 1℃ by monitoring the rectal temperature, the leaf extract was respectively administered to the different groups of rats at 30, 60 and 90 mg/kg orally and aspirin (100 mg/kg) and distilled water (10 ml/kg, orally) were administered respectively to the positive and negative control groups of rats. rectal temperatures of the animals were monitored at an hour interval for 5 h (edem et al., 2023). effect of mammea africana stembark extract on yeastinduced pyrexia in this experiment, 24 hours fasted adult albino rats of both sexes allowed water ad libitum were randomized into groups of 5 rats each. at zero hour, the basal rectal temperature of the rats were taken using digital clinical thermometer. thereafter, each animal was administered subcutaneously with 20% w/v aqueous suspension of yeast at a volume of 10 ml/kg (okokon and nwafor, 2010; edem et al., 2023). the temperatures of rats were monitored at one hour post yeast injection. rats with temperature increase of 1˚c were selected and grouped for the study. the extract understudy was administered orally after the pyrogen at doses of 30, 60 and 90 mg/kg to respective groups of rats. the control group received distilled water (10 ml/kg) and the reference group was administered with asa (100 mg/kg) both orally. the rectal temperatures of the groups were taken at 1h interval for 5 h. statistical analysis data collected were analyzed using one way analysis of variance (anova) followed by tukey’s multiple comparison post-test (graph pad prism software inc. la jolla, ca, usa). values were expressed as mean ± sem and significance relative to control were considered at p˂0.05. results effect of stembark extract of mammea africana on d-amphetamine induced pyrexia the antipyretic effect of the stembark extract on amphetamineinduced pyrexia is shown in table 1. the stembark extract (30-90 mg/kg), in the presence of amphetamine, demonstrated significant (p<0.05 – 0.001) lowering of body temperatures of the extracttreated rats when compared with the control. these effects were pronounced and sustained from 25 h post treatment with the extract. the body temperature lowering activity of the extract was not comparable to that of the standard drug, asa,100 mg/kg (table 1). effect of ethanol stembark extract of mammea africana on 2,4-dinitronitrophenol (dnp)-induced pyrexia in rats the stembark extract of m. africana (30-90 mg/kg) demonstrated significant (p<0.05–0.001) dosedependent lowering of temperature in dnp-induced pyretic rats. the temperature lowering effect was, however, significant (p<0.05–0.001) and sustained from 4 5 h in all the extract-treated groups. the effect of the highest dose (90 mg/kg) was strong but not comparable to that of the standard drug, asa, 100 mg/kg (table 2). okokon et al. – antipyretic activity of stembark extract of mammea africana in rats 1011 effect of stembark extract of m. africana on yeastinduced pyrexia in rats treatment of rats with yeast-induced elevated body temperature with stembark extract of m. africana (3090 mg/kg) caused significant (p<0.05-0.001) lowering of body temperature of rats elevated by the administration of yeast. the standard drug, asa,100 mg/kg, lowered the temperature significantly(p<0.05) when relative to the control group (table 3). the activity of the highest dose (90 mg/kg) of the extract was lower compared to that of the standard drug, asa,100 mg/kg (table 3) table 1. antipyretic effect of mammea africana stembark extract on d-amphetamine-induced pyrexia. treatment/ dose(mg/kg) time intervals (hrs) basal temp 0 0.5 1.0 2.0 3.0 4.0 5.0 control 34.60±0.35 35.94±0.13 36.23±0.17 36.61±0.26 36.77±0.15 37.25±0.38 37.20±0.16 37.`5±0.44 extract 30 34.78±0.68 35.71±0.53 35.63±0.16 35.50±0.14 35.33±0.13a 35.28±0.15b 35.33±0.36a 34.68±0.13a extract 60 35.33±0.76 36.56±0.15 35.91±0.31 35.44±0.33 35.30±0.16a 35.01±0.60a 35.13±0.29b 34.70±0.26b extract 90 34.28±0.18 35.42±0.28 35.33±0.18 35.22±0.26 34.90±0.27a 34.65±0.33b 34.30±0.22b 34.23±0.21c asa 100 35.04±0.11 36.11±0.31 35.50±0.12 35.03±0.14 34.86±0.12a 34.54±0.28c 34.11±0.15c 33.89±0.24c values are expressed as mean ± sem. significance relative to control. ap<0.05; bp<0.01; cp<0.001. n = 6. table 2. antipyretic effect of mammea africana stembark extract on dinitrophenol-induced pyrexia. treatment/ dose(mg/kg) time intervals (hrs) basal temp 0 0.5 1.0 2.0 3.0 4.0 5.0 control 34.42±0.34 36.33±0.21 36.46±0.12 36.54±0.18 36.60±0.54 36.48±0.15 36.20±0.25 36.24±0.12 extract 30 34.16±0.20 36.15±0.16 36.33±0.24 36.29±0.38 35.97±0.20 35.35±0.42 34.50±0.26a 34.05±0.54b extract 60 34.55±0.12 36.36±0.22 36.25±0.22 36.16±0.17 35.81±0.37 35.23±0.23 34.32±0.24a 33.76±0.16b extract 90 35.11±0.32 36.86±0.54 36.20±0.18 35.83±0.35 35.31±0.47 34.86±0.31 33.55±0.28c 33.22±0.19c asa 100 35.16±0.12 36.91±0.20 36.12±0.26 35.65±0.29 35.10±0.15 34.59±0.12a 33.40±0.12c 33.12±0.26c values are expressed as mean ± sem. significance relative to control. ap<0.05; bp<0.01; cp<0.001. n = 6 table 3. antipyretic effect of mammea africana stembark extract on yeast-induced pyrexia. treatment/ dose(mg/kg) time intervals (hrs) basal temp 0 0.5 1.0 2.0 3.0 4.0 5.0 control 34.38±0.18 35.60±0.21 36.65±0.15 36.85±0.31 37.20±0.19 37.41±0.26 37.76±0.18 37.44±0.16 extract 30 34.74±0.26 36.02±0.18 36.24±0.20 36.52±0.34 36.26±0.23 36.11±0.25 36.02±0.26 35.92±0.26 extract 60 35.08±0.15 36.14±0.78 36.30±0.12 36.10±0.43 36.01±0.10 35.91±0.14a 35.65±0.17b 35.21±0.18c extract 90 35.10±0.22 36.13±0.28 36.20±0.29 36.15±0.22 35.88±0.31a 35.55±0.16a 35.33±0.12b 35.25±0.20c asa 100 35.20±0.17 36.29±0.18 36.35±0.32 36.10±0.17 35.78±0.25a 35.42±0.26c 35.30±0.14c 35.05±0.26c values are expressed as mean ± sem. significance relative to control. ap<0.05; bp<0.01; cp<0.001. n = 6. discussion in this study, the stembark extract of m. africana was investigated for anti-pyretic activity using standard experimental models. the extract inhibited significantly amphetamine, dinitrophenol and yeastinduced pyrexia. amphetamine causes rise in body temperature by acting on the brain, causing the release of biogenic amines from their storage sites in nerve terminals. thus resulting in elevated level of camp and subsequent increased synthesis of prostaglandins from arachidonic acids produced in neurons by receptor mediated hydrolysis of phospholipids (westfall and westfall, 2006). this leads to hyperthermia. dinitrophenol causes hyperthermia by uncoupling oxidative phosphorylation resulting in the release of calcium from mitochondrial stores and also blocks calcium reuptake. this results in increased level of intracellular calcium, muscle contraction and hyperthermia (kumar et al., 2002). yeast induces hyperthermia by stimulating the synthesis of prostaglandins (al-ghamdi, 2001), in the hypothalamus. the extract may have reduced pyrexia by reducing brain concentration of prostaglandin e2 especially in the hypothalamus through its action on cox-2 or by enhancement of the production of the body’s own antipyretic substances such as vasopressin and arginine (chandrasekharan, 2002). the hypothermic activity of the extract could have also been mediated by vasodilatation of superficial blood vessels leading to increased dissipation of heat following resetting of hypothalamic temperature control center (rang et al., 2007). this action may be 1012 biology, medicine, & natural product chemistry 14 (2), 2025: 1009-1013 due to the phytochemical compounds in this plant. therefore, the temperature lowering activity of the extract may not be unconnected with the inhibition of one or combination of the above-mentioned mechanisms. the phytochemical compounds in this plant may in part be responsible for the observed antipyretic activities of the stembark extract. conclusion from the results of this study, the stembark extract of mammea africana possesses antipyretic activity which is due to the activities of its phytochemical constituents. acknowledgements: the authors are grateful to staff animal house of pharmacology and toxicology department, university of uyo for providing technical assistance. authors’ contributions: jeo,cco, jau research concept and design; 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(2006). adrenergic agonists and antagonists. in: gilman and goodman’s the pharmacological basis of therapeutics. 11th ed. mcgraw, newyork. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1039-1045 | doi: 10.14421/biomedich.2025.142.1039-1045 issn 2540-9328 (online) green extraction of corn silk flavonoids using nades-uae: optimizing conditions for agricultural waste valorization anak agung sagung intan kwanira gayatri, anak agung gede rai yadnya putra*, putu oka samirana department of pharmacy, faculty of mathematics and natural sciences, universitas udayana, jl. kampus bukit jimbaran, badung, bali, 80363, indonesia. corresponding author* agungryp@unud.ac.id abstract corn silk (of zea mays l.) is an agricultural waste that has not been optimally utilized, even though it is known to contain flavonoid compounds with various pharmacological activities, such as antioxidant, anti-inflammatory, and antidiabetic effects. the flavonoid compounds in corn silk have the potential to be developed as natural raw materials for applications in the pharmaceutical and cosmetic fields. however, the extraction process for these active compounds often still uses organic solvents like methanol, which are toxic, flammable, and have negative impacts on health and the environment. therefore, this study aimed to optimize the ultrasound assisted extraction (uae) method using a more environmentally friendly alternative solvent, namely natural deep eutectic solvent (nades). the study was conducted in two main stages: selection of the best nades composition and molar ratio from combinations of choline chloride with urea, glycerol, lactic acid, and malic acid; and optimization of uae parameters including ultrasonic power, time, and temperature using the response surface methodology (rsm) with the box-behnken design (bbd) model. the determination of total flavonoid content was performed using the colorimetric method with alcl₃ reagent and quercetin standard, and the measurements were taken using a uv-vis spectrophotometer. the results showed that the nades combination of choline chloride:urea (1:2) with the addition of 30% aquades produced the highest flavonoid content compared to 80% methanol solvent. the optimal conditions for uae extraction were obtained at 100 % ultrasonic power, 30 minutes of extraction time, and a temperature of 70°c, resulting in a total flavonoid content yield of 96.02 ± 0.48 mgqe/g of corn silk powder. this study concluded that the combination of nades and uae method can significantly enhance the extraction yield of total flavonoids while also providing a more sustainable and environmentally friendly solution for the utilization of agricultural waste, such as corn silk. keywords: corn silk; flavonoid; green solvent; nades; uae. abbreviations: box-behnken design (bbd); natural deep eutectic solvent (nades); quercetin equivalent (qe); response surface methodology (rsm); ultrasound assisted extraction (uae). introduction corn (zea mays l.), a member of the poaceae family, is one of the most widely utilized food crop commodities in indonesia. primarily, corn serves as a source of carbohydrates, but it also contains proteins, vitamins, and minerals (nawaz et al., 2018). beyond its role as a food commodity, corn is also used in livestock feed and the biofuel industry (amin, 2022). currently, the utilization of corn plants predominantly focuses on the kernels, while other parts, such as corn silk, are often regarded as waste. in countries like china and the united states, corn silk is traditionally consumed as herbal tea to promote health (lapčík et al., 2023). extracts from corn silk have demonstrated various pharmacological activities, including lowering blood pressure, regulating blood sugar levels, and exhibiting anti-inflammatory and antioxidant effects. phytochemical analyses have revealed that corn silk contains flavonoids, polyphenols, phenolic acids, fatty acids, and terpenoids, with flavonoids being the most abundant bioactive components (wang et al., 2024). the total flavonoid content in methanol extracts of corn silk has been found to be significantly higher than that of corn husk extracts, indicating the potential of corn silk as a natural source of flavonoids with beneficial pharmacological activities (dong et al., 2014). flavonoid compounds identified in corn silk include maysin, rutin, genistein, apigenin, quercetin, luteolin, and catechin (wang et al., 2024). the extraction efficiency of flavonoids from corn silk is influenced by the type of solvent used. methanol extracts have shown higher flavonoid content compared to ethanol extracts (haslina and eva, 2017; haslina et al., 2019a; haslina et al., 2019b). however, conventional solvents like methanol have several drawbacks, including flammability, nonmanuscript received: 04 july, 2025. revision accepted: 08 august, 2025. published: 17 november, 2025. https://doi.org/10.14421/biomedich.2025.142.1039-1045 1040 biology, medicine, & natural product chemistry 14 (2), 2025: 1039-1045 biodegradability, and potential long-term health risks (jauregi et al., 2024). the toxic properties of methanol can be mitigated through evaporation, but this process is time-consuming for obtaining natural extracts. therefore, there is a need for safer and more environmentally friendly solvents that allow for quicker and simpler extraction processes. natural deep eutectic solvents (nades) have gained attention in recent years as eco-friendly solvents derived from natural materials (puspita et al., 2023). nades are formed from the combination of a hydrogen bond acceptor (hba) and a hydrogen bond donor (hbd) in a specific molar ratio., creating a stable eutectic mixture at room temperature. they offer advantages such as low cost, simple preparation, reusability, biodegradability, and adjustable viscosity. the viscosity of nades can be modified by adding water, which facilitates the transfer of analytes during the extraction process (shikov et al., 2022). common materials used for extracting flavonoids include choline chloride (chcl), urea (u), glycerol (gly), lactic acid (la), and malic acid (ma) (liu et al., 2024). the extraction of flavonoids from corn silk using nades has shown promising results, indicating that this method could be more efficient than traditional extraction techniques. however, conventional extraction methods, such as maceration, require long durations and large volumes of solvent. therefore, this study will employ the ultrasound-assisted extraction (uae) method, which utilizes ultrasonic waves for a shorter process, avoiding high-temperature exposure, and requiring less solvent (medina-torres et al., 2017). factors affecting uae extraction yield include ultrasonic power, time, and temperature (kumar et al., 2021). these conditions can interact, influencing the total flavonoid yield, necessitating the optimization of uae conditions through appropriate experimental design. the aim of this research is to optimize the extraction of flavonoids from corn silk using nades in conjunction with the uae method. by identifying the optimal composition and ratio of nades and optimizing uae conditions, this study seeks to enhance the efficiency of flavonoid extraction while promoting a more sustainable and environmentally friendly approach to utilizing agricultural waste. materials and methods study area this research was conducted at the pharmacognosy and phytochemistry laboratory, pharmacy department, faculty of mathematics and natural sciences, universitas udayana, and the forensic science and criminology laboratory unit, universitas udayana. chemical and materials the chemicals used in this study included methanol 80% (smart-lab), distilled water, choline chloride (himedia), urea chemical pure grade and glycerol pharmaceutical grade were purchased from rofa laboratory centre, 90% food grade lactic acid (subur kimia jaya), food grade malic acid (subur kimia jaya), quercetin standard (sigma), aluminum chloride (merck), and sodium acetate (merck). sample preparation corn silk (zea mays l.) were obtained from badung regency, bali province, indonesia. corn silk was collected from maize plants ready for harvest, specifically the silk emerging from the husks. dried corn silk specimens were cut into small pieces, then ground into a fine powder using a grinder and sieved using 60mesh. nades synthesis and characterization nades were synthesized by mixing defined molar ratios of hydrogen bond acceptor (hba) to hydrogen bond donor (hbd) components (table 1). nades was prepared using the heating-stirring method with a magnetic stirrer (cimarec+). the mixture was stirred at 400 rpm and heated at 80oc until a clear and homogeneous solution was formed (ozkan, 2023). the formed nades was then added with 30% (v/v) distilled water and stirred again until a homogeneous mixture was obtained. viscosity of nades was determined using an ostwald viscometer (iwaki pyrex), ph was measured using a ph-meter (kedida ct-6020a), and a pycnometer (iwaki pyrex) was used to determine the density of nades. the measurements were performed at ambient room temperature (±32oc). table 1. molar ratio combination of nades. nades code component molar ratio hba hbd nades-1 choline chloride urea 1:2 nades-2 choline chloride glycerol 1:1 nades-3 choline chloride glycerol 1:2 nades-4 choline chloride glycerol 1:3 nades-5 choline chloride lactic acid 1:1 nades-6 choline chloride lactic acid 1:2 nades-7 choline chloride lactic acid 1:3 nades-8 choline chloride malic acid 1:1 screening and selection of nades for extraction screening and selection of the formulated nades as extraction solvents was conducted using ultrasonicassisted extraction (uae) under the following conditions: a solvent-to-solid ratio of 20:1, ultrasonic power at 100% with a frequency of 40 khz, temperature of 60°c, and extraction time of 20 minutes. then, centrifugation was performed at 4000 rpm for 20 min, the supernatant was collected and then filtered using filter paper. each nades extract was stored in a dark container until further analysis. gayatri et al. – optimization of ultrasound assisted extraction method for corn silk (zea mays l.) 1041 in this study, 80% methanol, which are volatile organic solvents, were used as comparators. separately, extraction of the dried powder of corn silk using the methanol was performed using the same procedure to obtain methanol extract. all extraction procedures were carried out in triplicate. the optimal solvent was selected based on its total flavonoid extraction capacity. determination of total flavonoid content (tfc) the determination of the total flavonoid content (tfc) was performed using the aluminum chloride colorimetric method previously described by chandra et al. (2014) was employed with some minor modifications. a stock solution of quercetin (1000 µg/ml) was prepared by dissolving 10.0 mg of quercetin in 10 ml of methanol. standard quercetin solutions were then prepared at concentrations of 40, 50, 60, 70, and 80 µg/ml. for sample preparation, 0.5 ml of the solution was mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 m sodium acetate, and 1.8 ml of distilled water. the mixtures were thoroughly homogenized and incubated for 30 minutes. the absorbance was measured using a uv-vis spectrophotometer (uv mini shimadzu 1240) at 436 nm. the results were expressed as milligrams of quercetin equivalents per gram of the dried samples (mg qe/g). nades-uae optimization using response surface methodology the optimal conditions for the extraction of flavonoid compounds from corn silk using nades-uae were determined through response surface methodology (rsm) with the aid of design-expert software. the optimization process involved three independent variables: ultrasonic power (x1, 40–100%), extraction time (x2, 10–30 minutes), and extraction temperature (x3, 50–70°c). a total of 17 experimental runs, including five replicates at the center point, were carried out based on the box–behnken design (bbd). an experimental approach was employed to generate response data, with total flavonoid content, measured using the same analytical procedure, serving as the response variable. data analysis all experiments data obtained were statistically analyzed and presented as means ± standard deviation (sd). the best solvent to be used in the optimization stage was determined by performing statistical analysis using oneway anova, followed by a post hoc least significant difference (lsd) test using ibm spss statistics version 30.0 software. meanwhile, the optimization data for the uae conditions were analyzed using the design-expert 13.1 software package through analysis of variance (anova). additionally, a one-sample t-test was performed using the same software to detect significant differences between the samples. significant difference among groups was considered at p < 0.05. results and discussion nades synthesis and characterization all eight nades combinations prepared under identical processing conditions successfully formed clear and homogeneous solutions at room temperature, indicating successful nades formation. the dilution of these nades with 30% (v/v) distilled water was carefully controlled to ensure a distilled water content of less than 50%, in order to preserve their molecular structures and prevent any negative impact on their performance (rocha et al., 2023). based on visual observation, the diluted nades remained stable as clear and homogeneous liquids during storage. the characterization results of all nades types are presented in table 2. the ph is determined by the individual and combined acidity and basicity properties of the hba and hbd components (sazali et al., 2023). nadess containing lactic acid and malic acid as hbd components tended to exhibit low ph values (<2), while those with glycerol as the hbd component showed neutral ph values. nadess formulated with urea as the hbd exhibited high ph values (>8). the lowest density was recorded for nades-5 at 1.12 ± 0.01 g/ml, while the highest density was observed in nades-8 at 1.22 ± 0.01 g/ml. high viscosity can reduce the mobility or diffusion of compounds in solution, thereby hindering mass transfer and slowing down the extraction process. nades-1 showed the lowest viscosity value at 2.6 × 10⁻³ ± 0 kg/m.s, whereas nades-8 exhibited the highest viscosity value at 2.34 × 10⁻² ± 0 kg/m.s. these results are attributed to the strong hydrogen bonding interactions between the components of the nades, which lead to a tightly packed molecular network structure, thereby increasing both density and viscosity. table 2. characterization results of nades: ph, density, and viscosity. nades characteristics ph density (g/ml) viscosity (kg/m.s) nades-1 8,43 ± 0,02 1,14 ± 0,01 2,6×10-3 ± 0 nades-2 5,09 ± 0,01 1,15 ± 0 5,4×10-3 ± 0 nades-3 5,13 ± 0,02 1,16 ± 0,01 6,2×10-3 ± 0 nades-4 5,37 ± 0,02 1,16 ± 0 8,0×10-3 ± 0 nades-5 1,53 ± 0,01 1,12 ± 0,01 3,5×10-3 ± 0 nades-6 1,12 ± 0,01 1,13 ± 0 4,4×10-3 ± 0 nades-7 1,02 ± 0,01 1,14 ± 0 6,9×10-3 ± 0 nades-8 0,67 ± 0,01 1,22 ± 0,01 2,34×10-3 ± 0 note: the values presented are expressed as mean ± standard deviation; measured at 32°c. selection of the optimal nades for extraction in general, various factors affect the efficiency of natural deep eutectic solvents (nadess) in extracting bioactive compounds from plant sources. the variation in component types and molar ratios within nades formulations plays a crucial role in determining their extraction performance, particularly for flavonoids. this 1042 biology, medicine, & natural product chemistry 14 (2), 2025: 1039-1045 is due to the fact that essential properties of nades— such as polarity, viscosity, and their ability to form hydrogen bonds—are strongly influenced by the specific combination of constituents (wu et al., 2022). this screening was conducted to evaluate the effectiveness of each nades in extracting flavonoids, in comparison with 80% methanol as a conventional solvent (figure 1). figure 1. total flavonoid content of each solvent. extraction was performed at 60°c for 20 minutes with 100% ultrasonic power. different letters indicate significant differences based on lsd test (p < 0.05). based on the results shown in figure 1, each nades exhibited different capabilities in extracting flavonoids from corn silk, as reflected by the total flavonoid content. among them, nades-1 (choline chloride:urea = 1:2, mol/mol) yielded the highest total flavonoid content at 88.85 ± 0.23 mgqe/g of powdered material, followed by nades-4 (choline chloride:glycerol = 1:3, mol/mol) with 79.47 ± 0.11 mgqe/g. nades-1 had the lowest viscosity among the tested solvents. lower viscosity facilitates better mass transfer, thereby enhancing extraction efficiency. in terms of ph, nades-2 exhibited a basic environment (ph 8.43 ± 0.02) and also resulted in a high flavonoid content. this is consistent with the findings of rismawati and ismiyati (2017), who reported that an increase in ph tends to enhance flavonoid extraction, with a decline observed at ph 10. the results demonstrated that nades-1 achieved higher total flavonoid content than the conventional solvent, 80% methanol (75.55 ± 0.17 mgqe/g). nades1 exhibited the highest extraction efficiency among all tested nadess and 80% methanol. this finding aligns with a study by hikmawanti et al. (2023), which also reported that nades composed of choline chloride:urea (1:2) significantly outperformed methanol in extracting flavonoids from pluchea indica l. leaves. the superior performance of nades in flavonoid extraction compared to conventional solvents is attributed to their ability to form a stable and complex hydrogen-bonding network between the hydrogen bond donor and acceptor components. these hydrogen bonds are believed to induce more pores and microcracks in plant materials during extraction, thereby facilitating the release of intracellular compounds (du et al., 2024; gao et al., 2024). nades exhibit strong penetration and permeability properties, allowing them to disrupt the lignin and cellulose structures in plant cell walls. this disruption facilitates the release of intracellular compounds into the extracellular environment (du et al., 2024). according to scanning electron microscope (sem) observations by gao et al. (2024), extraction with nades results in surfaces that are rougher and more irregular in shape, resembling corrosion damage. this effect is likely due to the ability of nades to dissolve or hydrolyze cellulose, leading to cell wall degradation. as a result, nades can expose and release a greater quantity of target compounds compared to conventional solvents (gao et al., 2024). based on all the results obtained, nades-1 (choline chloride:urea = 1:2) was selected as the solvent for use in the optimization stage of the uae conditions. optimization of ultrasound-assisted extraction the optimization study of extraction conditions using ultrasonic-assisted extraction (uae) was conducted using response surface methodology (rsm) with the box-behnken design (bbd) approach. the independent variables selected for the optimization were based on previous studies that identified the most effective extraction conditions. the response data obtained from the optimization process are presented in table s1. the uae conditions in runs 1, 2, 4, 7, and 13 were identical (70% ultrasonic power, 20 minutes, and 60°c), resulting in relatively consistent experimental total flavonoid contents ranging from 82.80 to 83.29 mgqe/g. this consistency demonstrates the good reproducibility of the extraction method. the highest experimental flavonoid content was recorded in run 16, with a value of 92.47 mgqe/g, obtained under conditions of 100% ultrasonic power, 20 minutes, and 70°c. this result closely matched the predicted value of 92.48 mgqe/g, indicating a high level of accuracy in the predictive model. in contrast, the lowest flavonoid content was observed in run 9 (71.48 mgqe/g), conducted under suboptimal conditions of 40% ultrasonic power, 10 minutes, and 50°c. the total flavonoid content results were modeled into a response function equation in relation to the selected variables. model selection was evaluated using analysis of variance (anova), based on key statistical parameters including the sequential p-value, lack of fit pvalue, adjusted r², and. the results of the regression model selection based on statistical analysis indicate that the quadratic model was the most suitable, as shown in table 3. the recommended model—a quadratic model— was statistically significant (p < 0.0001), with a nonsignificant lack of fit (p = 0.0993) (table 4). this indicates that the model sufficiently fits the experimental data and can reliably explain the effect of the variables on the response. gayatri et al. – optimization of ultrasound assisted extraction method for corn silk (zea mays l.) 1043 table 3. regression model selection. source sequential p-value lack of fit p-value adjusted r² predicted r² linear <0,0001 0,2327 0,9974 0,9964 2fi 0,3230 0,2358 0,9976 0,9956 quadratic 0,0140 0,9939 0,9992 0,9993 suggested cubic 0,9939 0,9986 table 4. anova model results. source sum of square df mean square f-value p-value model 550.63 9 61,18 2149,34 <0,0001 (sig.) x1-ultrasonic power 189.48 1 189,48 6656,40 <0,0001 x2-time 105.89 1 105,89 3719,84 <0,0001 x3-temperature 254.30 1 254,30 8933,90 <0,0001 x1x2 0.0015 1 0,0015 0,0510 0,8278 x1x3 0,3265 1 0,3265 11,47 0,0116 x2x3 0.0015 1 0,0015 0,0510 0,8278 x1 2 0,5060 1 0,5060 17,78 0,0040 x2 2 0,0010 1 0,0010 0,0343 0,8582 x3 2 0,1027 1 0,1027 3,61 0,0993 residual 0,1993 7 0,0285 lack of fit 0,0036 3 0,0012 0,0247 0,0993 (not sig.) pure error 0,1956 4 0,0489 cor total 550,83 14 the selected quadratic model showed a high coefficient of determination (r²) of 0.9996, indicating a strong agreement between the experimental and predicted values. the predicted r-squared (0.9993) and adjusted r-squared (0.9992) values differed by less than 0.2, demonstrating good predictive ability of the model. the small difference between these values suggests that the model not only fits the data used to build it but also accurately predicts new data. adequate precision, a parameter used to assess the model's ability to distinguish signal (model variation) from noise (error), was 162.3618—well above the threshold of 4—indicating a satisfactory signal-to-noise ratio and confirming the model's reliability in navigating the design space (gao et al., 2024). the anova results indicated that ultrasonic power (x₁), extraction time (x₂), and extraction temperature (x₃) had statistically significant effects on the total flavonoid content (p ≤ 0.05). among these variables, temperature (x₃) was identified as the most influential factor, followed by ultrasonic power (x₁) and extraction time (x₂). ultrasonic power affects the size and intensity of cavitation bubbles formed during the ultrasonic extraction process. higher power levels generate a greater number of cavitation bubbles—both large and small—which, upon collapsing, produce strong shock waves. these shock waves enhance mixing between the solvent and plant material, thereby improving the extraction yield of flavonoids. bioactive compounds in plants are typically bound to the matrix through physical adsorption and chemical interactions. increasing the extraction temperature can weaken these interactions, facilitating the release of secondary metabolites into the solvent. moreover, elevated temperatures reduce solvent viscosity, thereby enhancing mass transfer and diffusion rates, ultimately leading to more efficient extraction. additionally, longer extraction times generally improve yield by allowing extended contact between the solvent and plant matrix, promoting optimal diffusion of target compounds into the solvent (gao et al., 2024). the quadratic equation describing the effects of ultrasonic power (x₁), extraction time (x₂), and extraction temperature (x₃) on the total flavonoid content (tfc) of corn silk extract is expressed as follows: y (tfc) = 82,77 + 4,87x1 + 3,64x2 + 5,64x3 + 0,0190 x1x2 0,2875x1x3 + 0,0190x2x3 0,3467x1 2 + 0,0152x2 2 0,1562x3 2 the 3d response surface plots were used to visualize the interaction effects between two variables on the total flavonoid content. the plots demonstrated that increasing ultrasonic power and extraction time (figure s1a), increasing temperature and ultrasonic power (figure s1b), as well as increasing temperature and extraction time (figure s1c), generally led to an increase in total flavonoid yield. based on the results of the rsm-bbd analysis, the optimal conditions for flavonoid extraction from corn silk using the uae method were determined to be an ultrasonic power of 98.604%, extraction time of 29.527 minutes, and temperature of 69.919°c, with a predicted total flavonoid yield of 95.775 mgqe/g. to validate the reproducibility and reliability of the rsm design, experimental verification was conducted using the model-predicted optimal conditions. these optimized parameters were derived from regression analysis of the 1044 biology, medicine, & natural product chemistry 14 (2), 2025: 1039-1045 variables and 3d response surface plots, while accounting for instrumental operational constraints. however, due to equipment limitations, the optimal conditions were adjusted to an ultrasonic power of 100%, extraction time of 30 minutes, and temperature of 70°c. these adjusted optimal conditions were then validated through actual experiments to verify the accuracy of the predictive model. the experimental results showed that the total flavonoid content obtained was 96.02 ± 0.48 mgqe/g. a one-sample t-test was conducted to statistically compare the experimental and predicted values, revealing no significant difference between them (table 4). this indicates that the quadratic model used was able to accurately predict the flavonoid yield based on the combination of the three tested variables. table 4. predicted and actual values of optimum conditions. parameters predicted values experimental conditions ultrasonic power (%) 98,604 100 time (menit) 29,527 30 temperature (oc) 69,919 70 tfc (mgqe/g) 95,775a 96,02a note: the letter a indicates results that are not significantly different based on the one sample t-test (p>0.05). conlusions based on the study, the combination of choline chloride:urea in a 1:2 molar ratio was identified as the most effective nades formulation for extracting flavonoids from corn silk compared to other nades types. this specific nades not only outperformed other formulations but also demonstrated a significantly higher flavonoid extraction capacity than the conventional 80% methanol solvent when used to extract flavonoids from corn silk (zea mays l.). furthermore, the optimal extraction conditions for maximizing flavonoid yield using the uae method were determined to be an ultrasonic power of 100%, an extraction time of 30 minutes, and a temperature of 70°c, with a total flavonoid content of 96,02 mgqe/g. competing interests: the authors declare that there are no competing interests. funding: the authors confirm that this study received no external funding. references amin, n.s. 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(2022). research progress on the preparation and action mechanism of natural deep eutectic solvents and their application in food. foods, 11(21), 3528. doi: https://doi.org/10.3390/foods11213528. https://doi.org/10.3390/agronomy7030047 https://doi.org/10.1002/jsfa.13158 https://doi.org/10.15408/pbsj.v5i1.32256 https://doi.org/10.24853/konversi.6.2.89-94 https://doi.org/10.1016/j.molliq.2023.123249 https://doi.org/10.1016/j.chemosphere.-2023.139485 https://doi.org/10.1016/j.chemosphere.-2023.139485 https://doi.org/10.3390/md20050324 https://doi.org/10.3390/molecules29040891 https://doi.org/10.3390/foods11213528 this page intentionally left blank volume 3 number 2 2014 issn 2089-6514 biology, medicine, & natural product chemistry volume 3 – number 2 – 2014 issn 2089-6514 honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad ja’far luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia maizer said nahdi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: m. iqbal a. t. & riyanto publisher: state islamic university sunan kalijaga yogyakarta and the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@ yahoo.com guidance for authors biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international standards in their methodology. scientific feedback (short communicatiom) is only received for manuscript, which criticize published article before. manuscripts will be reviewed by managing editor and invited peer review according to their disciplines. the only articles written in english (u.s. english) are accepted for publication. this journal periodically publishes in may and november. in order to support reduction of global warming as a consequence of transportation vehicles emission and forest degradation for paper manufacturing, management of the journal prefer receiving manuscripts via e-mail rather than in hard copy. manuscripts and its communications can only be addressed to the managing editor; 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[indonesian] thesis, dissertation: sugiyarto (2004) soil macro-invertebrates diversity and inter-cropping plants productivity in ag roforestry system based on sengon.[dissertation]. brawijaya university, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l (2008) a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com publication manuscript “in-press” can be cited and mentioned in reference (bibliography); “personal communications” can be cited, but cannot be mentioned in reference. research which not be published or “submitted” cannot be cited. some annotation. manuscript typed without sign link (-) (except repeated word in indonesian). usage of letter “l” (el) to “1” (one) or “o” (oh) to “0” (null) should be avoided. symbols of α, β, χ, etc. included through facility of insert, non altering letter type. no space between words and punctuation mark. progress of manuscript. notification of manuscript whether it is accepted or refused will be notified in about three months since the manuscript received. manuscript is refused if the content does not in line with the 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ijo, baturagung mountain yogyakarta widodo larvicidal activity of a mixtureof cashew nut shell liquid and water-soluble extract of soapnut fruit (sapindus rarak dc.) against 3rd instar larvae of aedes aegypti glory resia raraswati, sudarsono, budi mulyaningsih substitution and haplotype diversity analysis on the partial sequence of the mitochondrial dna cytbofindonesian swamp buffalo (bubalus bubalis) akhmad sukri, mohamad.amin, aris winaya, abdul gofur the antidepressant effects of (arcangelisia flava (l.) merr) water-soluble extract in balb-c mice reviewed from immobility time by forced tiara a., arief r.h., sudarsono 47-52 53-57 59-63 65-67 volume 3 number 2 2014 published twice a year printed in indonesia cetak cover jurnal biomenaprochy vol 3 num 2 2014 dpn.pdf (p.1) editorial board 2014 v3 n2.pdf (p.2) 4 guidance.pdf (p.3) cetak cover jurnal biomenaprochy vol 3 num 2 2014 blkg.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 555-558 | doi: 10.14421/biomedich.2023.122.555-558 issn 2540-9328 (online) the correlation between widal diagnostic test, total leukocyte count, and platelet count in suspected typhoid fever patients at rs aura syifa kediri novi loviana, lisa savitri*, rochmad krissanjaya, elfred rinaldo kasimo department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id abstract typhoid fever is an acute systemic infectious disease that is related to poor personal hygiene and environmental sanitation. the diagnosis of typhoid fever is established through laboratory examinations, including serological, hematological, and bacteriological tests. this research aims to determine the correlation between the widal diagnostic test and the total leukocyte count and platelet count in suspected typhoid fever patients at rs aura syifa kediri. the study design used in this research is quantitative descriptive analysis with a crosssectional approach. the data was collected retrospectively from secondary sources, specifically medical records of patients from august 1, 2021, to march 31, 2022, at rs aura syifa kediri. the total population comprised 157 patients, and a purposive sampling method was used to select 41 patients who met the inclusion and exclusion criteria. the statistical analysis using the spearman correlation test resulted in p-values ≥ α = 0.05 for the following correlations: widal test o with leukocytes (0.538 ≥ 0.05), widal test h with leukocytes (0.915 ≥ 0.05), widal test o with platelets (0.476 ≥ 0.05), and widal test h with platelets (0.965 ≥ 0.05). these findings indicate that there is no significant correlation between the widal test o and h with the platelet count in patients with typhoid fever. therefore, it can be concluded that there is no significant relationship between the widal test o and h results and the platelet count in typhoid fever patients. keywords: widal; leukocytes; platelets; s. typhi. introduction typhoid fever is a frequently occurring infectious disease caused by an infection (mudiono, 2019). one of the infectious diseases is typhus, which includes typhoid fever, abdominal typhus, and enteric fever (oktaviana, 2021) caused by the gram-negative bacterium salmonella (s.) enterica serovar typhi (jong et al., 2018). according to law number 6 of 1962 on epidemiological diseases, typhoid fever is classified as a communicable disease. communicable diseases are easily spread and can affect many people, leading to outbreaks (idrus, 2020). in 2020, the indonesian ministry of health listed typhoid fever as one of the communicable diseases in the action plan for control and prevention of communicable diseases 2020-2024 (kemenkes ri, 2020). according to the world health organization (who), there are 11-20 million cases of typhoid fever worldwide each year, with 128,000 to 161,000 deaths annually (who, 2018). based on data from the east java provincial health office in 2013, there were 1,774 typhoid patients in east java, and 1,489 of them tested positive for widal (awa, 2019). in kediri city, data from the health office showed that there were 908 typhoid patients between 2015 and 2016, with 158 patients testing positive for widal (farodis, 2020). typhoid fever is an acute systemic infectious disease (ardiaria, 2019) transmitted through the fecal-oral route, often through the consumption of contaminated food or drink containing s. typhi bacteria (radhakrishnan et al., 2018). the bacteria enter the digestive tract, intestines, and intestinal lymph nodes, then travel through the bloodstream to the liver and spleen. typhoid fever can occur at any age, from infants and children to adults (renowati, 2019). clinical symptoms of typhoid fever are characterized by prolonged high fever (jong et al., 2018). the fever typically occurs in the evening or at night and may start without clinical symptoms (handayani, 2017). children usually have milder symptoms compared to adults. the incubation period is 10 to 20 days, and prodromal symptoms appear during this period, manifesting as fatigue, fever, weakness, dizziness, and headaches. the clinical symptoms of typhus patients range from mild to severe, from asymptomatic cases to those with complications and even death (kasim, 2020). therefore, maintaining a clean lifestyle is crucial (renowati, 2019). manuscript received: 26 july, 2023. revision accepted: 11 september, 2023. published: 22 september, 2023. https://doi.org/10.14421/biomedich.2023.122.555-558 556 biology, medicine, & natural product chemistry 12 (2), 2023: 555-558 there are three types of laboratory tests for diagnosing typhoid, including serological, blood, and bacteriological examinations (priskila, 2021). hematological changes often occur in typhoid fever, including anemia, leukopenia, eosinophilia, and thrombocytopenia. leukopenia and thrombocytopenia are characteristic findings in the total leukocyte count examination (syahniar et al., 2020). serological tests used for diagnosing typhus include the widal test and the igm s. typhi test (cerqueira, 2019). based on the data on the number of typhoid patients (including cases and deaths), it is essential to establish the diagnosis of suspected typhoid fever. therefore, the researcher is interested in the title "the relationship between the widal diagnostic test and the total leukocyte and platelet counts in suspected typhoid fever patients at rs aura syifa kediri". materials and methods this study employed a quantitative descriptive analysis, specifically a cross-sectional research design, to explore the relationship between widal diagnostic test results and the total leukocyte and thrombocyte counts in suspected typhoid fever patients at rs aura syifa kediri. the data were collected from secondary sources through a retrospective approach, involving medical records of patients from august 1, 2021, to march 31, 2022. retrospective research refers to studying past events (nur, 2021). the sampling method used was purposive, selecting 41 patients who met the inclusion and exclusion criteria. data processing involves statistical analysis using the spearman correlation test to identify the relationship between the two variables, which will be presented in percentage form. data analysis was facilitated through computerized software, specifically spss 26.0 (64-bit). data processing steps included editing, coding, entry, data cleaning, and saving. results and discussion table 1. distribution of respondents based on widal test, leukocyte count, and thrombocyte count in suspected typhoid fever patients. variable f % titer o positive 1/160 9 82 positive 1/320 2 18 total 11 100 titer h positive 1/160 31 78 positive 1/320 9 23 total 40 100 leukocyte leukopenia (<3.500 sel/cmm) 1 25 normal (3.500 – 10.000 sel/cmm) 24 59 leukocytosis (>10.000 sel/cmm) 16 39 total 41 100 thrombocyte thrombocytopenia (<150.000 sel/cmm) 7 17 normal (150.000-450.000 sel/cmm) 32 78 thrombocytosis (>450.000 sel/cmm) 2 5 total 41 100 in the total leukocyte count, there was a decrease in leukocytes (leukopenia) in 1 patient (25%), which is attributed to leukocytes fighting against infections or high fever. there were 24 patients (59%) with normal leukocyte counts, and 16 patients (39%) showed an increase in leukocytes (leukocytosis). the increase in leukocyte count (leukocytosis) in patients with typhoid fever indicates the presence of an infection in their bodies, as leukocytes increase to initiate and maintain the body's defense mechanism against the infection. this study is consistent with research conducted by wibawati (2017), which reported a leukopenia rate of 17%, a normal leukocyte count in 48% of patients, and leukocytosis in 35% of patients. in this study, the majority of patients had a normal leukocyte count, which aligns with the theory that patients with typhoid fever can have a normal leukocyte count. in the case of a decrease in the number of platelets (thrombocytopenia), there were 7 patients (17%) affected. thrombocytopenia is caused by the endotoxin from salmonella bacteria directly attacking the bone marrow, leading to a halt in the maturation phase of platelets. the study found that 32 patients (78%) had a normal platelet count, and 2 patients (5%) showed an increase in platelets (thrombocytosis). the increase or decrease in platelet count in patients with typhoid fever depends on the patient's immune condition and the bacterial infection attacking the body. this study is supported by research conducted by wibawati (2017), which reported that 87% of patients had a normal platelet count (>150,000), while 13% of patients had decreased platelet count (thrombocytosis) with a count ≤ 150,000. the study results indicated mild leukopenia and thrombocytopenia, mild leukocytosis, mild thrombocytosis, and average leukocyte and platelet counts within the normal range. the statistical analysis using spearman correlation between widal test titer o and the total leukocyte count resulted in a correlation coefficient of -0.209, indicating a weak correlation between widal test titer o and the total leukocyte count (below 0.5). the negative sign ('-') indicates that higher widal titers correspond to a decrease in the total leukocyte count. the output sig. (2tailed) for the correlation of widal test titer o with the total leukocyte count yielded a probability value of 0.538. as this value is above 0.05, ho is accepted and hi is rejected, indicating that there is no significant relationship between widal test titer o and the total leukocyte count. the statistical analysis using spearman correlation between widal test titer o and the platelet count resulted loviana et al. – the correlation between widal diagnostic test, total leukocyte count, and platelet count 557 in a correlation coefficient of -0.241, indicating a weak correlation between widal test titer o and the platelet count (below 0.5). the negative sign ('-') indicates that higher widal titers correspond to a decrease in the platelet count. the output sig. (2-tailed) for the correlation of widal test titer o with the platelet count yielded a probability value of 0.476. as this value is above 0.05, ho is accepted and hi is rejected, indicating that there is no significant relationship between widal test titer o and the platelet count. the statistical analysis using spearman correlation between widal test titer h and the total leukocyte count resulted in a correlation coefficient of -0.017, indicating a weak correlation between widal test titer h and the total leukocyte count (below 0.5). the negative sign ('-') indicates that higher widal titers correspond to a decrease in the total leukocyte count. the output sig.(2tailed) for the correlation of widal test titer h with the total leukocyte count yielded a probability value of 0.915. as this value is above 0.05, ho is accepted, and hi is rejected, indicating that there is no significant relationship between widal test titer h and the total leukocyte count. the statistical analysis using spearman correlation between widal test titer h and the platelet count resulted in a correlation coefficient of 0.007, indicating a weak correlation between widal test titer h and the platelet count (below 0.5). the positive sign ('+') indicates that higher widal titers correspond to an increase in the platelet count. the output sig. (2-tailed) for the correlation of widal test titer h with the platelet count yielded a probability value of 0.965. as this value is above 0.05, ho is accepted, and hi is rejected, indicating that there is no significant relationship between widal test titer h and the platelet count. discussion the results of the spearman correlation analysis showed that there is no relationship between the widal test and the total leukocyte count, as the statistical analysis yielded a significant value of p≥α=0.05 for both widal o with leukocytes (0.538≥0.05) and widal h with leukocytes (0.915≥0.05). therefore, the relationship between the two variables indicates that widal o and h are not related to the total leukocyte count in typhoid fever patients. these findings are consistent with a study conducted by wulandari (2019), which also showed no significant relationship between the total leukocyte count and widal h and o, using the chi-square test for the relationship between the total leukocyte count and widal h (0.36≥0.05) and the total leukocyte count with widal o (0.58≥0.05). the relationship of the widal test with the total leukocyte count in this study is represented by several suspected typhoid fever patients, wherein a higher widal titer results in a decrease in total leukocyte count, while a higher widal titer is associated with an increase in the total leukocyte count. however, some suspected typhoid fever patients exhibited high widal titers with normal total leukocyte counts. this occurs due to variations in patients' immune responses and the level of resistance to the bacteria. the results of the spearman correlation analysis showed that there is no relationship between the widal test and the platelet count, as the statistical analysis yielded a significant value of p≥α=0.05 for both widal o with platelets (0.476≥0.05) and widal h with platelets (0.965≥0.05). therefore, the relationship between the two variables indicates no significant correlation with the platelet count in widal o and h tests in typhoid fever patients. these findings are consistent with a study conducted by widary (2021), which stated that there is no significant relationship between widal titer and the quantity and index of platelets in typhoid fever patients, as the statistical tests yielded p ≥ 0.05, with a platelet titer value of p = 0.429, index of platelet mpv of p = 1.000, and pdw of p = 0.291, based on spearman correlation analysis with results (p) ≥ α = 0.05. the results of this study are also in line with a study by fitriani (2021), which demonstrated no relationship between widal titer and lymphocytes and platelet count in typhoid fever patients at puskesmas gunungsari lombok barat, as the pearson correlation test showed that 𝑝 > 𝛼 = 0.05 for lymphocytes and the antigen was 𝑝 = 0.758, while for platelets and the antigen was 𝑝 = 0.098. lymphocyte titer was 𝑝 = 0.051, and platelet titer was 𝑝 = 0.035. the relationship between the widal test and the platelet count in this study is indicated by the examination of several cases of suspected typhoid fever patients, wherein a higher widal titer leads to a decrease in platelet count. conversely, a higher widal titer results in an increase in platelet count. however, some suspected typhoid fever patients exhibited high widal titers with normal platelet counts. several factors influence the variation in platelet count in each blood sample of typhoid fever patients, such as variations in individual immunity, nutritional intake, the duration of the disease, the presence of other illnesses, the intake of antibiotics, and vaccination. errors in pre-analytical factors can also occur due to inadequate and imprecise sample homogenization, potentially leading to pseudo thrombocytopenia (false thrombocytopenia) caused by platelet aggregation (clumping of platelets) (widary, 2021). the serological parameters of the widal test depend on the time of specimen collection and the increase in lectin titers against s. typhi antigens. the best increase in antibody titers detected by the serological widal test typically occurs in the second and third weeks, at 95.7%, while the increase in titers in the first week is only 85.7%. the widal test requires two examinations, the acute phase, and the convalescent phase (recovery 558 biology, medicine, & natural product chemistry 12 (2), 2023: 555-558 phase), separated by 10-14 days. the diagnosis is confirmed by a four-fold or higher increase in titers during the acute phase. however, in this study, patients were in both the acute and convalescent phases (healing phase). conclusions there is no significant relationship between the widal diagnostic test and the total leukocyte count and platelet count in patients with suspected typhoid fever at rs aura syifa kediri. the statistical analysis showed significant values of p ≥ α = 0.05 for widal test titer o compared to the total leukocyte count (0.538 ≥ 0.05), widal test titer h compared to the total leukocyte count (0.915 ≥ 0.05), widal test titer o compared to the platelet count (0.476 ≥ 0.05), and widal test titer h compared to the platelet count (0.965 ≥ 0.05). this may be due to the lack of information on patients' acute and convalescent phases in this study. acknowledgments: the author would like to thank the institute for research, development, and community service (lp3m) of kadiri university which has always provided support for writing and publications. authors’ contributions: novi loviana designed the study. lisa savitri analyzed the data. rochmad krissanjaya and elfred rinaldo kasimo wrote the manuscript. all authors read and approved the final version of the article. competing interests: the authors declare that there are no competing interests. references antonia, m., cerqueira, b., mahartini, n. n., putu, i. w., & yasa, s. (2019). pemeriksaan widal untuk mendiagnosis salmonella typhi di puskesmas denpasar timur 1. directory of open access, 10(3), 777–780. https://doi.org/10.15562/ism.v10i3.453 fitriani., pauzi, i., & jiwantoro., & y. a. (2021). hubungan titer widal dengan jumlah limfosit dan trombosit pada pasien demam typhoid di puskesmas gunungsari lombok barat. analis medika biosains (jambs), 8(2), 77–80. handayani ni putu dea pawitri, d. m. (2017). karakteristik usia, jenis kelamin, tingkat demam, kadar hemoglobin, leukosit dan trombosit penderita demam tifoid pada pasien anak di rsu anutapura tahun 2013. ilmiah kedokteran, 4(2), 30–40. idrus, h. h. (2020). buku demam tifoid hasta 2020. (h. h. idrus, ed.), demam tifoid hasta 2020 (vol. 1). makassar. diambil dari //www.researchgate.net/publication/343110976 indana farodis, m. p. (2020). correlation between personal hygiene and hemoglobin levels on typhoid fever suspect patients at lirboyo general hospital. medicra (journal of medical laboratory science/technology), 3(2), 30–35. https://doi.org/10.21070/medicra.v3i2.800 jong et al. (2018). activation of coagulation and endothelium with concurrent impairment of anticoagulant mechanisms in patients with typhoid fever. journal of infection, 77(1), 60–67. https://doi.org/10.1016/j.jinf.2018.03.008 maria ernawati dona awa, supriyadi supriyadi, a. j. k. (2019). hubungan kebiasaan mencuci tangan menggunakan air bersih dan sabun dengan kejadian demam thypoid pada orang dewasa di wilayah kerja puskesmas dinoyo. nursing news, 4(1), 81–87. https://doi.org/doi: https://doi.org/10.33366/nn.v4i1.1546 mudiono, a. p. w. d. r. p. (2019). jurnal kesehatan. jurnal kesehatan, 7(3), 155–161. https://doi.org///doi.org/10.25047/jkes.v7i3 nur y. m., r. e. & e. (2021). pengaruh pemberian air susu ibu dan fototerapi terhadap ikterus neonotorum di ruang perinatologi rsud pasaman barat. akademka baiturrahim jambi (jabj), 10(1), 120–130. https://doi.org/10.36565/jab.v10i1.291 oktaviana, p. n. & f. (2021). efektivitas terapi antibiotika demam tifoid pada. syifa sciences and clinical research, 3(september), 63–70. diambil dari http://ejurnal.ung.ac.id/index.php/jsscr, priskila, erleena. silangit, thomas. surjadi, r. (2021). hubungan antara total leukosit dan trombosit pada pasien demam typhoid. kedokteran methodist, 14(1), 41–48. radhakrishnan, a., als, d., mintz, e. d., crump, j. a., stanaway, j., breiman, r. f., & bhutta, z. a. (2018). introductory article on global burden and epidemiology of typhoid fever. the american journal of tropical medicine and hygiene, 99((3_suppl),), 4–9. https://doi.org/doi:10.4269/ajtmh.18003256 renowati, & soleha, m. s. (2019). hubungan uji diagnostik widal salmonella typhi dengan hitung leukosit pada suspek demam tifoid typhoid (vol. 2, hal. 123–128). padang. ri, k. (2020). rencana aksi kegiatan pencegahan dan pengendalian penyakit menular langsung tahun 2020-2024. in rencana aksi kegiatan pencegahan dan pengendalian penyakit menular langsung tahun 2020-2024 (vol. 2507, hal. 1–9). syahniar rike, khayrul fikri, arumdini matahari, r. (2020). profil hematologi pasien anak dengan tifoid serta korelasinya terhadap lama rawat inap. kesehatan politeknik kesehatan makassar, xv(1), 98–103. https://doi.org/https://doi.org/10.32382/medkes.v15i1.1210 vivien novarina a kasim. (2020). peran imunitas pada infeksi salmonella typhi. (s. hadrianti, ed.) (1 ed.). gorontalo: c.v athra samudra. who. (2018). epidemiological record. diambil 31 january 2018, dari https://www.who.int/news-room/fact-sheets/detail/typhoid wibawati r, ni luh putu eka sudiwati, n. m. (2017). gambaran klinis penderita demam tifoid di ruang anak rumah sakit umum daerah kota baru. nursing news, 2, 414–419. widary, b. l., ayu, i. g., danuyanti, n., & zaetun, s. (2021). penelitian hubungan titer widal dengan jumlah dan indeks trombosit penderita demam tifoid di puskesmas wilayah lombok barat. jurnal kesehatan andalas, 10(3), 138–142. diambil dari http://jurnal.fk.unand.ac.id wulandari r. j. (2019). hubungan jumlah leukosit dengan titer widal pada suspek tifoid di rsu mayjen h.a thalib kerinci. stikes perintis padang. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 17-23 | doi: 10.14421/biomedich.2023.121.17-23 issn 2540-9328 (online) the effect of pumpkin fruit ripeness (cucurbita moschata. d) on total flavonoid levels and antioxidant activity linda astutik, eka fitri yanti* departement pharmacy, harapan bangsa jember school health jl. teuku umar no. 67 jember 68133, tel./fax.0331-5102836, indonesia. corresponding author* rofi3k4@gmail.com manuscript received: 01 august, 2022. revision accepted: 31 august, 2022. published: 03 october, 2022. abstract pumpkin fruit (cucurbita moschata. d) belongs the cucurbitaceae family which is a functional vegetable widely distributed in indonesia because it has nutritional value and health benefits. this study aims to determine the value of total flavonoid content and antioxidant activity in the ethanol extract of unripe, mature and ripened pumpkin. simplicia powder was extracted by maceration method using 96% solvent. testing the total flavonoid content with the addition of alcl3 at a wavelength of 425 nm and the antioxidant activity test was carried out using dpph (1-1-diphenyl-2-picrylhydrazyl) as a free radical with a maximum wavelength of 515 nm using microplate reader. the results of the study concluded that the ethanolic extract of pumpkin flesh had a total flavonoid content of 0,146 mgqe/100g in unripe fruit, 0,221 mgqe/100g in mature pumpkin, 0,191 mgqe/100g in ripened pumpkin. the antioxidant activity of the ethanol extract of unripe, mature, and ripened pumpkin fruit obtained was not active or did not completely reduce free radicals. keywords: pumpkin fruit (cucurbita moschata d); total flavonoid levels; antioxidant activity. introduction pumpkin is an annual plant that has been known to the indonesian people for a long time and is widely used in traditional food preparations. pumpkin is considered a functional vegetable that is widespread in indonesia and is very adaptable to various environmental conditions. the availability of pumpkin in indonesia is relatively large. however, the high production of pumpkin in indonesia is not matched by the use of pumpkin (purwanto et al., 2013). the use of yellow pumpkin are still limited to the household scale, which is processed into cooked vegetable from unripe unripe fruits, or dodol, cakes, compotes, and pastries from ripe fruits (hamdi et al., 2017). sharma et al., (2020) suggested that this plant has a broad spectrum for the treatment of diseases associated with its constituent compounds. the skin, flesh, and seeds of pumpkin have high nutritional value because they contain a lot of total phenols, total carotenoids, flavonoids and a large number of macro and micro nutrients (hussain et al., 2021). flavonoids are well-known active substances from plants that act as drugs in the human body. pumpkin seeds and flesh have higher total flavonoid content due to higher metabolism in plant parts, resulting in more metabolites. although the total flavonoid content in pumpkin is less than the total phenol content, the total flavonoid content at low concentrations also has strong antioxidant potential (asif et al., 2017). gumolung et al., (2013) suggested that ethanol extract of pumpkin flesh produced an antidote to free radicals of 62,16%. the ethanolic extract of pumpkin fruit has phenolic compounds, especially flavonoids as antioxidant activity in the moderate category with an ic50 (inhibitory concentration) value of 175,672 μg/ml and as a comparison is trolox with an ic50 value of 33,177 μg/ml (lukita, 2021). this flavonoid compound can release hydrogen radicals contained in the hydroxyl group (-oh) to attach to dpph radicals, so that dpph radicals become stable (sabarudin et al., 2021). in addition, flavonoids have the ability to scavenge free radicals and inhibit lipid oxidation (zuraida et al., 2017). as antioxidants, flavonoids are able to inhibit degenerative and chronic diseases. in addition to antioxidants, flavonoids are said to have hepatoprotector, antithrombotic, anti-inflammatory, and antiviral properties (dewi et al., 2018). the effects of flavonoid compounds are very diverse and very beneficial especially in relation to traditional medicine (sopan et al., 2014). previous studies by mokhtar et al., (2021) conducted an analysis of the phenolic content, flavonoids of (cucurbita moschata d) at various stages of ripening (unripe, mature, ripened) and determine antioxidant activity. the content of polyphenolic and flavonoids https://doi.org/10.14421/biomedich.2023.121.17-23 18 biology, medicine, & natural product chemistry 12 (1), 2023: 17-23 compound in riped pumpkin was 97.4 mggae/gram and 28.6 mgqe/gram. ripe pumpkin showed high antioxidant activity against dpph radicals, which was 0.065 ± 0.010 mol te/gram. based on the description above, it is necessary to conduct research to identify total flavonoid compounds and antioxidant activity of yellow pumpkin fruits during the ripening stage (unripe, mature, ripened). so that this research aims to study the changes in the profile of total flavonoid levels during ripening, and the development of antioxidant activity. samples had been taken from tegal rejo village which is part of tegalsari district, banyuwangi regency, east java. the livelihood of the people is 100% in agriculture, one of the cultivated commodities is yellow pumpkin fruit (fauzi dan purnomo, 2016). three fruit samples from each stage of development were selected based on their morphological attributes. materials and methods material unripe, mature, ripened pumpkin fruit obtained from tegal rejo village, tegalsari district, banyuwangi regency, east java, ethanol solvent 96%, methanol, sodium nitrite (nano2) 5%, sodium hydroxide (naoh) 1 m, quercetin, dpph (1,1-diphenyl-2pikrihildrazil), aluminum chloride alcl3 10%, and distilled water. table 1. identification of pumpkin fruits (cucurbita moschata d) of different degrees of maturity. identification description picture unripe pumpkin fruit light green, the shape of the fruit is flat-rounded. the grooves of the fruit are indistinct, fruit patches are present, the color of the flesh is yellowish-green. mature pumpkin fruit orange green, the shape of the fruit is flat-rounded, the grooves of the fruit are not clear, the color of the flesh is orange. ripened pumpkin fruit brown, flat round fruit shape, fruit spots are present, fruit groove is clear, flesh color is dark orange. procedures sample and extract preparation unripe, mature, ripened pumpkins were separated from the seeds and skin, then washed with running water and sliced thinly. then dried using the oven at a temperature of 50o-70o c and put in a blender into powder. furthermore, 100 grams of simplicia powder were weighed at various levels of fruit maturity and extracted with 500 ml of 96% ethanol for ± 3-5 days and stored at room temperature, after which it was filtered. the result obtained is called the filtrate. the filtrate was then concentrated with rotary evaporatoruntil a thick extract is obtained. determination of total flavonoid level contents in pumpkin extracts the method used for total flavonoid contents determination in pumpkin powders was alcl3 colorimetric assay as described by dona et al., (2020) with slight modification. a sample of 5 mg was dissolved in 5 ml of ethanol, so that the mother liquor with a solution concentration of 1000 ppm was obtained. then 100 µl was pipetted, and put into a microwell plate with three replications. then added 50 µl nano2 5% and 50 µl alcl3 10%, and incubated for 5 minutes in a dark room at room temperature, and added 50 µl naoh 1 m, then let stand for 30 minutes in a dark place. then the absorbance was measured using a microplate reader at a wavelength of 425 nm. determination of antioxidant activity contents in pumpkin extracts determination of antioxidant activity contents in pumpkin extracts was performed through a method by nasution dan ardhiyati, 2019). as much of 5 mg of the sample was dissolved in 5 ml of methanol so that the sample concentration was 1000 μg/ml. as much was added 50 μl (plate consists of rows a-h with each astutik & yanti – the effect of pumpkin fruit ripeness (cucurbita moschata. d) … 19 totaling 12 holes). as much as 50 μl was put into each well of rows f, e, d, c, b. furthermore, samples with a concentration of 1000 μg/ml as much as 50 μl were inserted into rows g and f. then diluted rows f, e, d, c, b. row f 50 μl pippeted into row e, row e pipetted 50 μl into row d, row d 50 μl pipetted into row c, row c 50 μl pipetted into row b. row b 50 μl pipetted then discarded, so that the concentration of the test solution is 32 ppm (μg/ml), 63 ppm (μg/ml), 125 ppm (μg/ml), 250 ppm (μg/ml), 500 ppm (μg/ml), dan 1000 ppm (μg/ml). next row g, f, e, d, c, b dan a added 80 μl dpph with a concentration 80 ppm. then incubated for 30 minutes at room temperature to protect from light. the absorbance of the sample was measured using microplate reader at the maximum wavelength 515 nm. then the calculation of the value of % inhibition and calculation of ic50 were performed. % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 𝐴 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑥 100 % description: 𝐴 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 : absorbance of dpph 𝐴 𝑠𝑎𝑚𝑝𝑒𝑙 : absorbance of the dpph solution containing the sample after calculating the % inhibition, the ic50 value was calculated from the linear regression equation, 𝑦 = 𝑎𝑥 + 𝑏 description: 𝑥 : as sample concentration 𝑦 : % antioxidant activity data analysis we analysis of the total flavonoid compounds obtained from the absorbance of the comparison quercetin solution, presented a calibration curve and obtained a linear regression. the average absorbance was fitted in the standard curve equation as the y value, where the x value obtained was the concentration in mgqe/100g. analysis of antioxidant activity was obtained from the value of % inhibition and calculation of ic50. results and discussion yield of pumpkin fruit ethanol extract yield of 96% ethanol extract of unripe pumpkin is 34,2%, ripe pumpkin is 33,1% and mature pumpkin is 19,7% (table 2). the yield shows the active component that was successfully extracted (zuraida et al., 2017). yield results can be influenced by several extraction factors, including time, temperature, type of solvent, ratio of material and solvent, particle size and processing of the plant (chairunnisa et al, 2019). table 2. yield of pumpkin fruit ethanol extract. sample simplicia weight extract weight % yield unripe pumpkin 100 gr 34,2 34,2% mature pumpkin 100 gr 19,7 19,7% ripened pumpkin 100 gr 33,1 33,1% total flavonoid content this analysis aims to determine the total flavonoid content in the extract obtained from the standard curve equation. the choice of quercetin as a standard solution is due to its compounds that are widely distributed in plants (hasanah & novian, 2020). the following absorbance data on the concentration of quercetin produces a standard curve line y= 0,0013x + 0,0783 with an value r2 = 0,945 (figure 1). from this equation, the total flavonoid content obtained is 0,146 mgqe/100g extract of unripe pumpkin, 0,221 mgqe/100g extract of mature pumpkin and 0,191 mgqe/100g extract of ripened pumpkin as shown in table 3. figure 1. the standard curve of quercetin. table 3. data in the total flavonoid content of the ethanol extract of unripe, mature, ripened pumpkin. sample absorbance measurement average ktf (mgqe/100g) sd ktf ± sd (mgqe/100g) 1 2 3 unripe pumpkin 0,111 0,117 0,130 0,146 0,0079 0,146 ± 0,0079 mature pumpkin 0,164 0,176 0,182 0,221 0,0075 0,221 ± 0,0075 ripened pumpkin 0,149 0,151 0,153 0,191 0,0016 0,191 ± 0,0016 antioxidant activity in the antioxidant activity test, that ripe, mature and unripe pumpkins did not completely reduce the antioxidant activity (table 4). y = 0,0013x + 0,0783 r² = 0,945 0,00 0,05 0,10 0,15 0,20 0,25 0 50 100 150 a b so rb an ce concentration (µ/ml) quercetin 20 biology, medicine, & natural product chemistry 12 (1), 2023: 17-23 table 4. the results of the measurement if % inhibition and ic50 value from ethanol extract of unripe, mature, ripened pumpkin. sample konsentrasi (µg/ml) % inhibition ic50 (µg/ml) rata-rata ic50 ± sd (µg/ml) ic50 (µg/ml) raw pumpkin 63 125 250 500 1000 1,1765 5,8824 5,8824 24,7059 35,2941 1337 1443,8 ± 102,37 very weak or inactive > 500 (µg/ml) (waode rustiah et al., 2018) 63 125 250 500 1000 1,1765 7,0588 9,4118 15,2941 35,2941 1454 63 125 250 500 1000 3,5294 7,0588 12,9412 15,2941 34,1176 1541 mature pumpkin 32 63 125 250 500 1000 2,198 5,495 7,692 10,989 16,484 31,868 1637 1751 ± 242,05 very weak or inactive > 500 (µg/ml) (waode rustiah et al., 2018) 32 63 125 250 500 1000 3,297 5,495 8,791 12,088 17,582 32,967 1587 32 63 125 250 500 1000 4,396 5,495 7,692 10,989 16,484 26,374 2029 ripened pumpkin 32 63 125 250 500 1000 2,198 4,396 5,495 5,495 12,088 20,879 2563 2283,6 ± 319,04 very weak or inactive > 500 (µg/ml) (waode rustiah et al., 2018) 32 63 125 250 500 1000 2,198 4,396 6,593 7,692 12,088 23,077 2352 32 63 125 250 500 1000 4,396 3,297 6,593 6,593 14,286 27,473 1936 discussion pumpkin has many health benefits, traditionally used to treat skin diseases, measles, jaundice, insomnia, cancer and can help increase endurance due to its antioxidant activity (sabarudin et al., 2021). different plant parts can have different phytochemical compounds, which can cause different pharmacological effects (sembiring et al., 2018). the extraction method used is maceration. astutik & yanti – the effect of pumpkin fruit ripeness (cucurbita moschata. d) … 21 the maceration method is used because the tools and methods are simple, and it does not use high temperatures which are at risk of damaging the chemical components of materials that are not resistant to high temperatures (prasetya et al., 2020). 96% ethanol is used as a solvent which is polar, so it is good to be used as an extract solvent to extract polyphenol compounds and a solvent that is safe for drugs (dai & mumper., 2010). analysis of the total flavonoid content test was carried out using the instrument microplate reader elisa (λ) 425 nm, because it is simple, easy to operate, faster, can use many samples at once in measurement and can use smaller volumes such as 200-500µl for microplate 96 -well (berg et al., 2016). the measurement of total flavonoid levels using the colorimetric method is based on the formation of a complex reaction between flavonoids and aluminum chloride (alcl3) (zuraida et al., 2017). reagent alcl3 added after nano2 will form a stable complex with a c4 keto group and a c3 or c5 hydroxyl group on flavones and flavonols (syafitri et al., 2014), resulting in a shift in wavelength towards the visible which is indicated by the solution producing a more yellow color. the results of the total flavonoid content obtained are in the unripe pumpkin of 0,146 mgqe/100g extract, mature pumpkin of 0,221 mgqe/100g extract, and ripened pumpkin of 0,191 mgqe/100g extract can be seen in table 2. the values obtained in this study were not much different from those of hasanah dan novian (2020) of 0,00288 mg/g. flavonoids will experience a decrease due to the influence of temperature during the drying process because these compounds are sensitive to light and heat. according to zainol et al (2009) the degradation of flavonoids occurs due to the termination of the molecular chain and the occurrence of an oxidation reaction that causes the oxidation of the hydroxyl group and will form other volatile compounds quickly. testing the antioxidant activity of the ethanolic extract of pumpkin fruit by reducing the free radical 1.1diphenyl-2 pikhrylhydrazil (dpph) using elisa microplate reader (λ) 515 nm. the results can be seen in table 4. that unripe, mature, and ripened pumpkins did not completely reduce the antioxidant activity with ic50. the results obtained in unripe pumpkin of 1432,37 µg/ml, mature pumpkin of 1751 µg/ml, ripened pumpkin of 2283,67 µg/ml which could be categorized as very weak or inactive antioxidant activity (waode rustiah et al., 2018). the difference in activity obtained in each extract is probably due to differences in the content and number of active compounds contained in the extract, so that the antioxidant activity obtained is also different (purwanto et al. 2017). in general, the smaller value ic50 obtained means the higher the antioxidant activity (dona et al., 2020). this is possible that in pumpkin which has flavonoid levels in addition to antioxidant activity and also acts as antifungal, diuretic, antihistamine, antihypertensive, insecticide, antiparasitic, anthelmintic, and antiviral (hasanah dan novian., 2020). another factor that causes weak antioxidant activity is that the compound is still not pure, so it is necessary to do fractionation and purification in the hope that the value will be obtained ic50 of specific compounds that have stronger antioxidant activity. the presence of secondary metabolites other than flavonoids may not provide a synergistic response so that the antioxidant activity produced does not completely reduce (inactive) free radicals (mz et al., 2017). in addition, the cause of the ethanol extract of unripe, unripe, and ripe pumpkin fruit does not completely reduce free radicals due to the phytochemical test results which show a small total flavonoid content value. this is supported by research from gumolung et al., (2013) who stated that the part of the pumpkin that contained high free radical scavenging activity specifically was in the skin, stump and seeds compared to the pumpkin flesh. sopan et al., (2014) also suggested that the flesh of pumpkin (cucurbita moschata d) has large phenolic compounds which are very strong chain-breaking antioxidants and it has been reported that phenolic compounds are associated with antioxidant activity. this phenolic compound has many hydroxyl groups including o-hydroxy groups which have very strong antioxidant potential. according to supriatna et al., (2019), the level of age and maturity of a plant affects the maximum active content of secondary metabolites in the plant. reinforced by the statement metusalach (2007) suggests that the growth of a plant is influenced by external and internal factors. external factors such as habitat, season, water temperature, types of food available and other environmental factors, while internal factors, namely age, size and other biological factors determine antioxidant properties. one of the supporting factors of antioxidant activity is the presence of phenolic compounds and flavonoids that can reduce free radicals, as stated by nuret et al. (2019) which states that there is a correlation between phenolic and flavonoid content on antioxidant activity. the content of phenolic compounds and flavonoids in the extract will act as hydrogen donors, reducing agents and unpaired oxidant absorbers. however, another factor that is no less important to the high antioxidant activity is the other bioactive compounds present in the sample, such as tannins and quinones which also have potential as antioxidants whose levels have not been studied in this study. as for the possibility of other compounds in pumpkin fruit that will have potential as antioxidants when extracted with semi-polar and non-polar solvents. there is necessity to aim of taking non-polar fractions in pumpkin which are thought to have potential as antioxidant compounds. 22 biology, medicine, & natural product chemistry 12 (1), 2023: 17-23 conclusions pumpkin (cucurbita moschata d) unripe has a total flavonoid content of 0,146 mgqe/100g, mature pumpkin of 0,221 mgqe/100g, ripened pumpkin of 0,191 mgqe/100g. antioxidant activity in pumpkin fruit (cucurbita moschata d) unripe, mature, and ripened are classified as very weak or inactive because they do not completely scavenge free radicals. conflict of interests: authors state that there is no conflict of interest in this research output. references asif, m., raza naqvi, s. a., sherazi, t. a., ahmad, m., zahoor, a. f., shahzad, s. a., hussain, z., & mahmood, h. & mahmood, n. 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(2017). phenols, flavonoids, and antioxidant activity on the bark extract of pulai stem (alstonia scholaris r. br) (phenolics, flavonoidas, ands antioxidant activity of alstonia scholaris r. br stem bark extract. journal of forest products research. vol 35 no.3: 211-219 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 55-60 | doi: 10.14421/biomedich.2023.121.55-60 issn 2540-9328 (online) acute toxicity ld50 fraction ethyl acetate aquilaria malaccensis, ficus benjamina, mikania micrantha, and fraction water cinnamomum burmanii in mus musculus rizky yulion1,*, santi perawati1, barmi hartesi1, lia anggresani2, lili andriani1, lesra indriani1, lara syahila1, suci ramadani1, nadia monika1 1program studi farmasi, stikes harapan ibu jambi jl. kol. tarmizi kodir no.71, pakuan baru, kec. jambi sel., kota jambi, jambi 36122, indonesia. 2sekolah tinggi ilmu kesehatan syedza saintika padang jl. prof. dr. hamka no.228, air tawar tim., kec. padang utara, kota padang, sumatera barat 25132, indonesia. corresponding author* rizkyyulionputra10@gmail.com manuscript received: 28 september, 2022. revision accepted: 05 october, 2022. published: 17 october, 2022. abstract research on the acute toxicity of ld50 had been carried out on the ethyl acetate fraction of gaharu leaves (aquilaria malaccensis), beringin leaves (ficus benjamina), sembung rambat leaves (mikania micrantha), and kayu manis cortex (cinnamomum burmanii) water fraction the base of use as traditional treatment by the suku anak dalam (sad) empirically obtained from ancestors. this research method was experimental and it used male and female mice. the control group was given a 1% nacmc doses of 625 mg/kg body weight, 1,250 mg/kg body weight, 2,500 mg/kg body weight, and 5,000 mg/kg body weight. the test preparation was administered orally once per day. the thomson-weil method was used for the measurement of ld50 values. the weight ratio of organs was applied for heart, liver, lungs, kidneys, and stomach. the results showed that the ld50 value for male mice was 2454mg/kg body weight; 2454mg/kg body weight; 1546 mg/kg body weight and 2065mg/kg body weight respectfully. the ld50 value for female mice was a pseudo ld50. the value of the organ weight ratio in each sample showed p<0.05 values that were obtained in gaharu leaves in the liver and kidney organs (male); heart in females. beringin leaves value p<0.05 lung organs (male). sembung rambat leaves p<0.05 values of the heart, liver, and stomach organs (male); heart and stomach (female). cinnamomum cortex value p<0.05 liver and stomach organs (male). the conclusions showed that the ld50 value of the ethyl acetate fraction of gaharu leaf, sembung rambat leaf, beringin leaf, and kayu manis cortex in male mice was slightly toxic, while in female mice it could not be counted. keywords: beringin leaf (ficus benjamina l.); gaharu leaf (aquilaria malaccensis); kayu manis cortex (cinnamomum burmanii); sembung rambat leaf (mikania micrantha kunth); toxicity acute ld50. introduction suku anak dalam (sad) is part of a minority group on the island of sumatra, precisely in the interior area of jambi province, indonesia (setiyadi et al., 2020). at the end of the 18th century, the jungle people (suku anak dalam) encountered foreigners who brought in the infectious disease, smallpox, and reached epidemic and severe levels. this encouraged people to seek healing from plants in the forest and in addition to the knowledge of traditional medicine obtained from ancestors (ayuningtyas et al., 2020; mustika & dastina, 2020; najib, 2020). some plants that can be used in traditional medicine are gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex. gaharu leaves are used to overcome insomnia, beringin leaves are usually used as influenza medicine, sembung rambat leaves are used as wound medicine and kayu manis cortex can be used as a solution for diabetes, this is used empirically. several studies have been reported related to the plant that gaharu leaf contains secondary metabolites, namely flavonoids, steroids, tannins, triterpenoids, saponins, alkaloids, and sesquiterpene metabolites (δcadinene, gurjunene) (fitriani & erlyn, 2019; wong et al., 2015). beringin leaves contain kaempferol, chlorogenic acid, alkaloids, flavonoids, saponins, steroids, and polyphenols (aslamiah & haryadi, 2013; corrêa et al., 2012). sembung rambat leaf contains lignin (lim et al., 2020), isoledene, δ-cadinene, debromofiliformin, trans-caryophyllene, β-bisabolene, germacrene-d, zingiberene (saikia et al., 2020), protein, phenolics, and proline (jali et al., 2021). the kayu manis cortex contains polyphenols (rozi et al., 2022) alkaloids, tannins, phenolics, flavonoids, quinones, and triterpenoids (hananti et al., 2012). https://doi.org/10.14421/biomedich.2023.121.55-60 56 biology, medicine, & natural product chemistry 12 (1), 2023: 55-60 the acute toxicity test is a pre-clinical trial aimed at measuring the degree of the toxic effect of a compound within 24 hours after a single dose. the purpose of the acute toxicity test is to determine the potential for acute toxicity (ld50) (khumaidi et al., 2018; oecd, 2022; ugwah-oguejiofor et al., 2019). based on literature studies that have been carried out, no research has been found using the ethyl acetate fraction of gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex water fractions to test for acute toxicity of ld50. we interested to know the acute toxicity test of ld50 in mice as a research experimental animal so that the use of active doses is more guaranteed. materials and methods the study area quantitative experimental studies have been conducted to trace the acute toxicity value of ld50 in four types of medicinal plants in jambi province. the results of a fraction of ethyl acetate aquilaria malaccensis, ficus benjamina, mikania micrantha, and fraction water cinnamomum burmanii were used as samples on albino mice (mus musculus). furthermore, tracing the impact of sampling on the liver, kidney, heart, lung and stomach organs was carried out. tools and materials the tools used in this study were a set of vacuum rotary evaporators, analytical scales, balance, lumps and stampers, glass bottles, stirring rods, filter paper, funnels, measuring cups, aluminum foil, erlenmeyer, measuring flask, separator funnels, spatulas, vaporizing cups, drip pipettes, masks, hand scoops, tissue, napkins, syringe, needles, surgical tools mice, styrofoam, and squeak basins. the ingredients used were mice (25 males and 25 females), gaharu leaf extract, beringin leaf extract, sembung rambat leaf extract, kayu manis cortex extract, 70% ethanol, n-hexane, ethyl acetate, distilled water, hgcl2, ki, i2, subnittric bismuth, glacial acetic acid (ch3cooh), hcl, ammonia (nh3), chloroform (chcl3), magnesium (mg), water, iron (iii) chloride solution (fecl3), ether, sulfuric acid (h2so4), and acetic acid anhydride (c4h6o3). procedure plant determination determination of plants is was carried out to avoid mistakes in the identification of plants. determination of gaharu leaf plants, beringin leaf, sembung rambat leaf, and kayu manis cortex at the biota identification and determination laboratory, padjadjaran university (unpad) indonesia. ethical clearance ethical clearance is a written statement given by the research ethics commission for research involving living things and which states that a research proposal is feasible to be carried out according to 7 who standards. ethical clearance was carried out at the ethics committee of the health service poltekkes of the ministry of health, jambi. ethically feasible information that has been carried out consecutively for acute toxicity tests ld50 ethyl acetate fraction of gaharu leaf, beringin leaf, sembung rambat leaf and kayu manis cortex water fractions in white mice no. lb. 02.06/2/ 104/2021; no. lb.02.06/2/090/2021; no. lb. 02.06/2/122/2021. sample and material processed samples of gaharu leaf were used as much as 5 kg, beringin leaf as much as 1 kg, sembung rambat leaves as much as 5 kg, and kayu manis cortex as much as 1 kg. the sample was washed thoroughly using running water first to separate the dirt, the next step was drying it by aeration. samples that had been dried then chopped for maceration (hermes et al., 2021). extraction in the extraction process of samples that had been cleaned, dried, and pureed, the next step was to extract by the maceration method using 70% ethanol (1: 10 b/v) in a closed dark bottle allowed to stand for 24 hours. further filtered and squeezed, the pulp is added again 70% ethanol until submerged, soaked, and filtration is carried out for 3 days with 3 changes of solvent or carried out until the color of the solvent was clear. after all, the resulting filtrates were then mixed, then the next step was concentration using a rotary evaporator at a temperature of 500c until finally the results of the viscous extract were obtained (dechayont et al., 2021; khan & islam, 2012). the working principle of a rotary evaporator was to evaporate the extraction solvent and leave only the distraction resulting compound called extract. fractionation extracts of gaharu leaf, beringin leaf, spliced leaves of propagation, and kayu manis cortex are concentrated and then fractionated by liquid-liquid extraction (ecc) using a solvent of 1: 10 based on the degree of polarity. the first thing to do was that the sample extract was stirred well-used water (polar), after mixed was put in the separator funnel then n-hexane (non-polar) is added in a ratio of 1: 1, close the separator funnel put under the hand then shake several times interspersed with opened the faucet of the separator funnel to remove the gas, so as not to cause too strong pressure to occur. then after the sound of the gas does not come out again, it was allowed to stand for a few minutes so that there was a separation of 2 phases, namely the n-hexane phase and yulion et al. – acute toxicity ld50 fraction ethyl 57 the water phase, separate the n-hexane phase to the limit of solubility to the container. furthermore, the ethyl acetate (semi-polar) fraction with the same workmanship was used, after all the fractionation of the n-hexane solvent, ethyl acetate and the remained water obtained, were separated first based on the solvent. then re-concentrated using a rotary evaporator until it gets a viscous fraction result (liu et al., 2013; rachmawaty et al., 2019; shah & gilani, 2012). selection and setup of test animals the test animals used were healthy male and female mice, aged 2-3 months with a body weight of mice of 20-30 grams. prepared 50 heads of mice. mice are divided into 5 groups and each group consists of 5 male mice and 5 female mice. mice are acclimatized for 7 days and before treatment were fasted for 18 hours. the doses used were the control group given na cmc 1%, the treatment group was given a dose of 625 mg/kgbb, 1,250 mg/kgbb, 2,500 mg/kgbb and 5,000 mg/kgbb (oecd, 2022; vogel, 2002). organ weight ratio all groups of test animals performed surgery and observations on the organs of the liver, heart, lungs, stomach, and heart. the organ was then weighed and compared with body weight to obtain the organ index (rajeh et al., 2012). data analysis used the spss (statistical product and service solutions) program to see the difference in the ratio of squeak organ weights between sexes and dose groups after administration of the test preparation, if the data was not distributed normally, the kruskal-wallis test is continued. the significance of the obtained results was judged at the 5% level results and discussion results plant determination plants were determined at the biota identification and determination laboratory, padjadjaran university (unpad). the determination results obtained that the sample used was aqularia malaccensis lam, ficus benjamina l., mikania micrantha kunth, and cinnamomum burmanii. extraction results the extraction method used was the maceration method. the sample was soaked with 70% ethanol solvent and let stand in a sealed dark vial for 24 hours. soaked and filtration are carried out for 3 days with 3 solvent changes. after all, filtrates were obtained and then evaporated using a rotary evaporator (dillasamola et al., 2021; halim et al., 2020). a total of 1,400 g, 1,000 g, 1,000 g, and 1,000 g of gaharu leaf powder extracted, beringin leaf extracted, sembung rambat leaf extracted, and kayu manis cortex extracted respectfully were successively obtained condensed extracts of 85.41 g, 77.72 g, 70.29 g, 81.6 g with a yield of 6.10%, 6.4%, 7.02%, 8.16%. fractionation results fractionation aims to separate the components of the active compound from the extract that has been produced. the results of the viscous extract of the sample are further fractionated by separation based on their polarity. fractionation in this study used non-polar solvents, namely n-hexane, and semi-polar solvents, namely ethyl acetate (liu et al., 2013; rachmawaty et al., 2019; shah & gilani, 2012). the results of the yield of the fractionation of gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex successively accorded to the polarity of the solvent obtained an n-hexane fraction of 13.43%; 10,8%; 12,73%; 0,06%. the ethyl acetate fraction was obtained 7.62%; 6,09%; 11,50%; 2.16% and the residual fraction of water obtained 23.00%; 22%; 35,86%; 30,00%. table 1. total number of male mice deaths on gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex. normal 625mg/kgbb 1250mg/kgbb 2500mg/kgbb 5000mg/kgbb total survive dead survive dead survive dead survive dead survive dead survive dead gaharu leaf 5 0 4 1 5 0 2 3 2 3 18 7 beringin leaf 5 0 3 2 4 1 2 3 3 2 17 8 sembung rambat leaf 5 0 5 0 3 2 2 3 2 3 17 8 kayu manis cortex 5 0 5 0 4 1 3 2 1 4 18 7 58 biology, medicine, & natural product chemistry 12 (1), 2023: 55-60 table 2. total number of female mice deaths on gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex. na-cmc 1% 625mg/kgbb 1250mg/kgbb 2500mg/kgbb 5000mg/kgbb total survive dead survive dead survive dead survive dead survive dead survive dead gaharu leaf 5 0 5 0 4 1 3 2 3 2 20 5 beringin leaf 5 0 4 1 5 0 5 0 2 3 21 4 sembung rambat leaf 5 0 5 0 4 1 4 1 3 2 21 4 kayu manis cortex 5 0 5 0 4 1 3 2 3 2 20 5 table 3. the data value of the influence of the ratio of organ weights on each sample to the organ of mice (p>0,05). heart liver lungs kidney stomach male female male female male female male female male female gaharu leaf no yes yes yes no no yes yes no no beringin leaf no no no no yes yes no no no no sembung rambat leaf yes yes yes yes no no no no yes yes kayu manis cortex no no yes yes no no no no yes no discussion ld50 in this study, the fifty mice used were divided into 5 groups, each group consists of 5 male mice and 5 female mice. before treatment, all mice were acclimatized for 7 days with the aim that the test animals could adapt to the laboratory atmosphere (tested environment). before being given the treatment of mice, they were fasted for 18 hours but were still given a drink. mice are satisfied so that when given the sample treatment were expected to directly interact with the digestive system and not be disturbed by the presence of food contained in the digestion of mice. mice were administered sample extracts orally (vogel, 2002). in male mice with gaharu leaf samples, the results of this study showed that the value of ld50 ethyl acetate fraction of gaharu leaves obtained a result of 2,454 mg/kg body weight are included in the category of slightly toxic. the toxicity ethyl acetate fraction of beringin leaves obtained a result of 2,454 mg/kg body weight belongs to the category of slightly toxic. the toxicity ethyl acetate fraction of sembung rambat leaf obtained a result of 1546 mg/kgbb, belonging to the category of slightly toxic. the toxicity water fraction of the cinnamomum cortex obtained a result of 2065 mg/kg body weight belongs to the category of slightly toxic. in female mice, no meaningful value of ld50 founded after being given extracts of gaharu leaf, beringin leaf, sembung rambat leaf, and kayu manis cortex. it can be said that this is included in the pseudo ld50. this is possible due to the influence of hormonal cycles that can cause estrogen hormone levels to increase. if the level of the hormone estrogen increases then the immune system also increases or was stronger than the immune system of the male mice, so that the male mice can experience death. in female mice, ld50 calculations could not be carried out because they did not meet the mortality criteria of the thompson-weil method. organ weight ratio in the statistical test of the organ weight ratio after administration of the ethyl acetate fraction of gaharu leaf, there was a significant difference in female mice in the heart organs. while in male mice there are significant differences in the liver organs and kidney organs. in the ethyl acetate fraction of the leaves of the splice of the sembung rambat leaf, there were significant differences in female mice in the organs of the heart and stomach. while in male mice there were significant differences in the organs of the liver, heart, and stomach. this was likely due to the presence of content of secondary metabolite compounds. secondary metabolites are chemical compounds that were formed in plants. secondary metabolites in addition to having physiological effects that can nourish or cure diseases can also cause undesirable side effects on the human body or certain animals. in plants, each compound works together to influence the other to increase its activity or effectiveness. as for compounds that can damage liver cells, including alkaloids and saponins, both compounds can damage cells and tissues and even cause death in experimental animals. for data on the ratio of organ weights after administration of the ethyl acetate extract fraction of beringin leaf to the heart, liver, kidney, and stomach organs of male and female mice, data were obtained with p>0.05 meant that there was no noticeable difference in the weight of the heart, liver, kidney and stomach organs at various doses, while data on the weight ratio of male lung organs were obtained data marked p <0.05 which means that there was a difference yulion et al. – acute toxicity ld50 fraction ethyl 59 in the weight of the male lung organs in various doses. in statistical tests of kayu manis cortex fraction on the weight of mice organs, there were significant differences in the lung organs of male mice and the gastric organs of male and female mice. the ethyl acetate fraction of beringin leaf and the water fraction of kayu manis cortex may contain flavonoid compounds, which is known if flavonoids at excess levels in the cells can cause a halt in active transport which causes an uncontrolled intake of ions in the cells so that it can cause cell death (necrosis) in the lung organs (tanduwinata et al., 2015). in the lungs, there is an alveolus that functions as a place for gas exchange, where each wall is coated by cells. the alveolus is declared damaged when dilation of the alveolus is founded and this can trigger the onset of inflammatory infiltrates. there are significant differences in the gastric organs of male and female mice, it can be concluded that there is gastric damage in male white mice and female white mice. kayu manis cortex may contain flavonoid compounds that are thought to have an inhibitory effect against lipoxygenase and cyclooxygenase. where one of the product pathways of cyclooxygenase, namely prostaglandins, is thought to have a protective effect on the stomach. a deficiency of prostaglandins is suspected to lead to a decrease in the production of mucus, phospholipids, hco3 secretions, the proliferation of mucosal cells, and microvascular flow of the stomach that is suspected to cause discontinuities in the epithelium of the gastric mucosa known as peptic ulcers. the mechanism is suspected to be the cause of swelling of the gastric organs in male white mice and female white mice after administration of the kayu manis cortex fraction (astri et al., 2012). conclusions based on the studies that have been carried out, it can be concluded that the ld50 values of gaharu leaf, geringin leaf, sembung rambat leaf, and kayu manis cortex in male mice are categorized as slightly toxic and in female mice cannot be calculated or show pseudo-ld50 values. the value of the organ weight ratio in each sample shows p<0.05 values were obtained in gaharu leaves in the liver and kidney organs (male); heart in females. beringin leaves value p<0.05 lung organs (male). sembung rambat leaves p<0.05 values of the heart, liver, and stomach organs (male); heart and stomach (female). cinnamomum cortex value p<0.05 liver and stomach organs (male). acknowledgements: thanks are given to all colleagues who have helped in the process of this research. authors’ contributions: rizky yulion designed the study. rizky yulion, lia anggresani and lara syahila carried out the laboratory work for gaharu leaves (aquilaria malaccensis). rizky yulion, lili andriani and lesra indriani carried out the laboratory work for beringin leaves (ficus benjamina). rizky yulion, santi perawati and suci ramadani carried out the laboratory work for sembung rambat leaves (mikania micrantha). rizky yulion, barmi hartesi and nadia monika carried out the laboratory work for kayu manis cortex (cinnamomum burmanii). rizky yulion analyzed the data. rizky yulion wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: there was no conflict of interest in this study. funding: there is no cost support from any party for this research. the study was carried out at the own expense of 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(2015). evaluation of comprehensive two-dimensional gas chromatography with accurate mass time-of-flight mass spectrometry for the metabolic profiling of plant-fungus interaction in aquilaria malaccensis. journal of chromatography a, 1387, 104–115. https://doi.org/10.1016/j.chroma.2015.01.096 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 219-224 | doi: 10.14421/biomedich.2025.141.219-224 issn 2540-9328 (online) phytochemical screening and determination of total flavonoid content of keji beling leaves ethanol extract (strobilanthes crispa bl.) seftya putri mahyantika, muhammad hafizh husain, linggar nirwana, devana rahma aldina, satria gemilang, nafisa, majida ramadhan*, nour athiroh abdoes sjakoer, faisal biology department, faculty of mathematics and natural sciences, universitas islam malang. jalan mayjen haryono no. 193, malang 65144, tel. +62-341-551932, fax. +62-341-552249, indonesia. corresponding author* majida.ramadhan@unisma.ac.id manuscript received: 22 january, 2025. revision accepted: 15 may, 2025. published: 19 june, 2025. abstract this study aims to determine the total flavonoid content in the ethanol extract of keji beling (strobilanthes crispa) leaves using the uvvis spectrophotometric method with alcl3 reagent. the research process includes maximum wavelength testing, preparation of quercetin standard solution, and analysis of total flavonoid levels. the results showed that the total flavonoid content in the extract was 144.347 ± 134.854 mg qe/g extract, with an extract yield of 0.4% and extract moisture content of 5.256%, which showed good potential in preventing the growth of microorganisms. phytochemical screening revealed the presence of alkaloids, flavonoids, and tannins, while saponins, triterpenoids, and steroids were not detected. these findings confirm the importance of secondary metabolite compounds in plants and the potential of keji beling leaf extract as a source of active compounds with health benefits, including antioxidant and antibacterial activities. this study provides a basis for further research into the medical applications of this plant extract. keywords: herbal plant; strobilanthes crispa; phytochemical screening; flavonoids. introduction secondary metabolites are chemical compounds that generally exhibit bioactivity and function as protective agents for the plants against pests and diseases, either for the plants themselves or their surrounding environment. as secondary metabolite products, these chemical compounds have been widely used as dyes, poisons, food aromas, medicines, and other applications. numerous plants with medicinal properties are used in traditional medicine, highlighting the need for research on the utilization of medicinal plants and the identification of chemical compounds with medicinal properties (aksara et al., 2013). the keji beling plant (strobilanthes crispa bl.) is widely recognized in society as a traditional remedy for addressing various health issues. its leaves contain several active compounds, such as alkaloids, flavonoids, saponins, terpenoids, and tannins (fardiyah et al., 2020). keji beling has been extensively used in traditional medicine for its antibacterial, antioxidant, antilithiasis, antidiabetic, antiangiogenic, anticancer, and wound healing properties (ng et al., 2021). dried leaves of the keji beling plant contain a high total ash content (21.6%), including abundant minerals such as potassium (51%), calcium (24%), sodium (13%), iron (1%), and phosphorus (1%) (ismail et al., 2000). research indicates that the leaf position significantly affects the catechin content in the water fraction. the highest catechin content in the water fraction is found in the basal leaves, with a concentration of 11.813%, compared to the apical leaves (8.173%) and the middle leaves (11.453%) (nurraihana & hanoon, 2013). one method commonly employed in discovering traditional medicines is extraction. extraction is a process of separating compounds from mixtures using suitable solvents. one example of an extraction technique is maceration, a cold extraction method that does not require high temperatures. maceration involves soaking the crude drug (simplisia) in a solvent with occasional stirring at room temperature (meydia, 2016). keji beling leaves (strobilanthes crispa bl.) are extracted using 96% ethanol. the use of 96% ethanol as a solvent in the maceration process is due to its higher selectivity, non-toxicity, good absorption properties, and ability to inhibit bacterial and fungal growth. ethanol at 96% concentration penetrates the simplisia cells more effectively compared to ethanol with lower concentrations, resulting in a more concentrated extract (suhendar, 2019). this study aims to determine the active compounds present in the ethanol extract of keji beling leaves using the maceration method. https://doi.org/10.14421/biomedich.2025.141.219-224 220 biology, medicine, & natural product chemistry 14 (1), 2025: 219-224 materials and methods study area this study was conducted in october 2024 at the microbiology laboratory, integrated laboratory, universitas islam malang, east java, indonesia. procedures sampling the keji beling leaves were collected from malang city, east java. the leaves underwent a wet sorting process, washing, chopping, drying, and dry sorting. then, the leaves were dried using an oven at 80°c until the sample was completely dry (wijaya, 2022; fachriza, 2023), after which the dry weight of the sample was measured and the moisture content was calculated to ensure it was below 10%. once the sample was dry, the keji beling leaves were blended into a powder form (januarista, 2024; rafsanjani, 2024). the powder was then sieved using an 80-mesh sieve to increase the surface area, facilitating the penetration of the solvent during the extraction process (siregar et al., 2020; nurjannah et al., 2022). extraction keji beling leaves were extracted using the maceration method with a solvent ratio of 1:10, with 96% ethanol as the solvent. the reason for using 96% ethanol is that it can dissolve active compounds such as alkaloids, flavonoids, tannins, saponins, steroids, and terpenoids. secondary metabolites and 96% ethanol have similar polarities, making it easier for the ethanol solvent to dissolve in the sample. this follows the principle of "like dissolves like," meaning that compounds will dissolve in solvents with similar properties. additionally, ethanol is easily accessible and affordable compared to other solvents (haryani et al., 2021; nurjannah et al., 2022). in the initial step, 100 grams of powdered simplicia were weighed and placed into a 1-liter glass bottle (reagent bottle). a 1:7 ratio was used in the first stage, and in the second stage, a 1:3 ratio was applied (tatang, 2019; fakhruzy, 2020). the first and second maceration stages were homogenized in a 1000 ml erlenmeyer flask. the solution was then transferred to a rotary evaporator at 40 rpm at a temperature of 60°c 70°c for 1 hour until it became a paste (kemenkes ri, 2020; januarista, 2024). the maceration process involves soaking the sample in an organic solvent at room temperature (nurjannah et al., 2022) to extract the secondary metabolites contained in the plant (endarini, 2016; agung, 2017; aini, 2023). screening fitokimia the phytochemical screening test method refers to the research of ramadhan et al. (2019) carried out qualitatively with color testing using a reagent that aims to determine the secondary metabolite compounds contained in plants: ▪ alkaloid test a) mayer’s test a total of 20 drops of liquid extract from each sample were put into a test tube. next, 10 drops of mayer reagent were added slowly (drop by drop) into the test tube and then shaken gently. the observed changes indicate a positive result for alkaloids if the formation of a white precipitate accompanies a yellowish color change. b) wagner’s test a total of 20 drops of liquid extract from each sample were put into a test tube. after that, 10 drops of wagner's reagent were added gradually (drop by drop) into the test tube and then shaken gently. the formation of a reddish-brown precipitate characterizes the positive results of alkaloids. c) bouchardat test a total of 20 drops of liquid extract from each sample was put into a test tube. then, 10 drops of dragendorff's reagent were added slowly (drop by drop) into the test tube and shaken carefully. the formation of an orange or yellow precipitate characterizes positive results for alkaloids. ▪ flavonoid test a) shinoda test a total of 20 drops of liquid extract (isopropyl alcohol) from each sample were put into a test tube. next, 20 drops of absolute ethanol and three drops of concentrated hydrochloric acid were added to the test tube and shaken gently. a color change to red indicates the presence of aurones and chalcones. if no color change occurs, add 1 small spatula of magnesium to the test tube containing the liquid extract, then shake gently. a positive result for flavonoids is indicated by a pink to red color change (such as orange, red, or magenta, which indicates the presence of flavones and flavonols). b) 10% sodium hydroxide test a total of 20 drops of liquid extract from each sample was put into a test tube. next, 4 drops of 10% naoh solution were added to the test tube and then shaken gently. observed color changes, such as reddish yellow, blackish orange, reddish-purple, or blue, indicate the presence of santone/flavone, flavonol, chalcone, or anthocyanin compounds. ▪ saponin test a) foam test a total of 20 drops of liquid extract from each sample was put into a test tube. after that, 20 drops of hot distilled water were added to the test tube and then shaken gently continuously for 15 minutes. the positive result of saponin is characterized by the formation of a consistent foam layer as high as ± 1 cm.uji tannin (polifenol) mahyantika et al. – phytochemical screening and determination of … 221 b) base solution test a total of 20 drops of liquid extract from each sample were put into a test tube. then, 20 drops of 10% ammonium hydroxide (nh4oh) solution were added to the test tube and shaken gently. the observed changes indicate a positive result for tannin (polyphenols) when a fluorescent yellow color is formed. ▪ steroid/triterpenoid test a) salkowski test a total of 20 drops of liquid extract (isopropyl alcohol) from each sample was put into a test tube. after that, 20 drops of chloroform were added, followed by 10 drops of salkowski's solution slowly into the tube. the mixture was then shaken carefully. the appearance of a brown ring in the center of the mixture indicated the presence of steroids. b) lieberman bourchard test a total of 20 drops of liquid extract (isopropyl alcohol) from each sample were poured into a test tube. next, 10 drops of lieberman bourchard solution were added slowly, and the mixture was shaken gently. after incubating for 5 minutes, a color change was observed. a blue-green color indicates the presence of sterols, while a pink to red-violet color indicates the presence of terpenoids. determination of total flavonoid content by colorimetric method determination of total flavonoid content in ethanol extract of tiger milk fungus was carried out using uvvis spectrophotometry with the colorimetric method (alcl3) at a wavelength of 410 nm, and the results were expressed as total flavonoids in quercetin equivalents (eq) (rebaya et al., 2014). determining the highest wavelength a total of 0.5 ml of quercetin solution with a concentration of 100 ppm was taken, then added 0.1 ml of 10% alcl3, 0.1 ml of 1m ch3coona, and 2.5 ml of distilled water. the solution was homogenized using a vortex and incubated for 40 minutes in a place protected from light. the highest wavelength test was carried out on a spectrophotometer with an absorbance range between 415-440 nm. preparation of quercetin standard solution a total of 10 mg of quercetin was dissolved in 96% pa ethanol to a volume of 10 ml. from the mother solution with a concentration of 1000 ppm, 1 ml was taken and then 96% pa ethanol was added to a volume of 10 ml, resulting in a solution with a concentration of 100 ppm. next, pipette 2, 4, 6, 8, and 10 ml of this solution, and add 96% pa ethanol to reach a volume of 10 ml, resulted in solutions with final concentrations of 20, 40, 60, 80, and 100 ppm. at each concentration, 0.5 ml of quercetin solution was taken, and 0.1 ml of 10% alcl3, 0.1 ml of 1m ch3coona and 2.5 ml of distilled water were added. the solution was homogenized using a vortex and incubated for 40 minutes in a place protected from light. the test was performed on a spectrophotometer with a maximum wavelength of 434 nm. determination of total flavonoids a total of 10 mg of the tested sample was dissolved in 96% pa ethanol with a volume of 10 ml. from the mother solution which has a concentration of 1000 ppm, 1 ml was taken, and 96% pa ethanol was added to a volume of 10 ml, resulting in a solution with a concentration of 100 ppm. pipette 5 ml of this solution and add 96% pa ethanol to a volume of 10 ml to obtain a final concentration of 50 ppm. at each concentration, 0.5 ml of sample solution was taken, then 0.1 ml of 10% alcl3, 0.1 ml of 1m ch3coona, and 2.5 ml of distilled water were added. the solution was homogenized using a vortex and incubated for 40 minutes in a place protected from light. the assay was performed on a spectrophotometer with a maximum wavelength of 434 nm. the total flavonoid content in the extract was calculated and expressed as mg of quercetin equivalent (qe) per gram of fresh sample. data analysis the results of the study were analyzed qualitatively descriptively, and then conclusions were drawn based on the results obtained. results and discussion water content of keji beling leaf extract simplisia (strobilanthes crispa) table 1. results of water content of keji beling leaf extract (strobilanthes crispa). simplisia weight (gram) extract weight (gram) water content (%) 100 40.071 2.060 yield of keji beling leaf extract (strobilanthes crispa) table 2. yield of keji beling leaf extract (strobilanthes crispa). simplisia weight (gram) solvent (ml) extract weight (gram) yield (%) 100 1000 40.071 0,4 222 biology, medicine, & natural product chemistry 14 (1), 2025: 219-224 phytochemical screening table 3. qualitative test results of keji beling leaf extract (strobilanthes crispa). no test reagents results 1 alkaloid mayer’s + wagner’s + dragendroff’s + bourchardat + 2 flavonoid shinoda + naoh 10% + 3 saponin uji busa 4 tanin braymer’s + larutan basa + 5 triterpenoid / steroid salkouski liebermen bourchard description: (+) = contains compounds (-) = does not contain compounds total flavonoid activity of keji beling leaf extract (strobilanthes crispa) figure 1. regression value graph of total flavonoid assay table 4. total flavonoid test result of keji beling leaf extract (strobilanthes crispa). sample r ep la y c o n ce n tr a ti o n s (p p m ) average total flavonoid content (mgeq/g extract) ethanol extract of keji beling leaf (strobilanthes crispa) 1 1000 144.347 ± 134.854 2 500 discussion simplisia, with a moisture content that is too high, is at risk of the growth of microorganisms such as fungi and bacteria. this condition can cause damage to active compounds and reduce the quality of simplisia. therefore, testing water content is essential to maintain the active compounds in the simplisia. based on the results of the study, the moisture content of keji beling leaf simplisia is 5.256%, which is classified as low and meets the standard of simplisia moisture content (not more than 10%). this low moisture content also helps extend the shelf life of the simplisia because it reduces the risk of microorganism growth and degradation of active ingredients. the yield of keji beling leaf extract in this study was 0.4%. this value is low when compared to the positive standard of yield, which is considered good (>10%). factors such as solvent type, extraction time, temperature, solvent to material ratio, and sample particle size can affect extract yield (bustan et al., 2008, cited in hasim et al., 2016). low yields may indicate that the concentration of active compounds in the raw material is not very high or that there are obstacles during the extraction process, such as suboptimal solvent selection. phytochemical screening showed that the ethanol extract of keji beling leaves contained secondary metabolite compounds in the form of alkaloids, flavonoids, and tannins, while saponins, triterpenoids, and steroids were not detected. flavonoids in the extract have strong antibacterial activity, which damages the bacterial cell wall by binding compounds to peptidoglycan, resulting in cell lysis. tannins damage polypeptides in the bacterial cell wall and prevent enzyme adhesion and transport proteins, causing cell leakage and bacterial death. steroids and triterpenoids, although not detected in keji beling leaf extract, are known to have a mechanism of action by damaging the bacterial cell membrane, increasing membrane permeability, and causing leakage of intracellular material. the results of the total flavonoid content test showed that keji beling leaf extract has a flavonoid content of 144.347 ± 134.854 mg qe/g extract. the flavonoid content was determined using the uv-vis spectrophotometric method with a maximum wavelength of 434 nm. this result is in line with the research of werdiningsih et al. (2023), which showed that the uv-vis spectrophotometric method was able to provide accurate results for determining total flavonoid levels. the total flavonoid content in this study is high and indicates that keji beling leaves have the potential as a source of bioactive compounds. in addition, the relationship between extract concentration and absorbance value showed good linearity, with a linear regression equation y = 0.0124x and a r² value of 0.998. this linearity indicates that the flavonoid concentration is directly proportional to the resulting absorbance intensity. the high flavonoid content in keji beling leaf extract indicates great therapeutic potential, especially as an antibacterial and antioxidant, which makes it a promising base material for herbal medicine formulations. overall, the results of this study indicate that keji beling leaves contain significant active compounds and y = 0,0124x + 0,0339 r² = 0,998 0,000 0,200 0,400 0,600 0,800 1,000 1,200 1,400 0 20 40 60 80 100 120 ab so rb an ce concentrations (ppm) mahyantika et al. – phytochemical screening and determination of … 223 have the potential to be developed as raw materials for pharmaceutical or phytopharmaceutical products. however, optimization of the extraction process and further research are needed to increase the yield and study its pharmacological activity in more depth. conclusions this study showed that ethanol extract of keji beling (strobilanthes crispa) leaves contained total flavonoids of 144.347 ± 134.854 mg qe/g extract was measured using uv-vis spectrophotometric method with alcl3 reagent. the extract yield of 0.4% and moisture content of 5.256% indicated the good quality of the simplisia, with low potential for microorganism growth. phytochemical screening identified the presence of secondary metabolite compounds such as alkaloids, flavonoids, and tannins, while saponins, triterpenoids, and steroids were not detected. these findings support the utilization of ethanol extract from keji beling leaves as a source of active compounds that have the potential to be used in the development of health products, especially for antioxidant and antibacterial activities, with recommendations for further research to explore its potential applications. acknowledgments: the author would like to thank the fmipa of the islamic university of malang, the supervisor, and the biology laboratory for their support, facilities, and direction during this research. hopefully the results of this research will be useful for the development of biological science. authors’ contributions: example: majida & nafisa designed the research. seftya & linggar did the laboratory work. muhammad analyzed the data. seftya, muhammad, devana, and satria wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references aini, s. q., sjakoer, n. a. a., & mubarakati, n. j. 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(2023). determination of 70% ethanol extract flavonoid total levels binahong (anredera cordifolia [ten] steenis) leaves in pelem village, tanjunganom, kab. nganjuk. jurnal sintesis: penelitian sains, terapan dan analisisnya, 3(2), 132–140. yadima, s. g., muhammad, m., & usman, b. (2017). phytochemical screening and antibacterial activities of aqueous extracts of diospyros mespiliformis, mitragyna inermis, piliostigma reticulatum and tamarindus indica in northern nigeria. arid zone j. engin, technol. & environ, 13(3), 411-419. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 565-573 | doi: 10.14421/biomedich.2024.132.565-573 issn 2540-9328 (online) formulation and effectiveness test of red ginger (zingiber officinale var. rubrum) and starfruit (averrhoa bilimbi l.) combination shampoo against pediculus humanus capitis reza anindita*, salwa zhafarina harahap, maya uzia beandrade, intan kurnia putri department of pharmacy, sekolah tinggi ilmu kesehatan mitra keluarga, jl. pengasinan rawa semut raya, rt.004/rw.012, margahayu, bekasi timur, jawa barat 17113, indonesia. corresponding author* abstract some of the natural ingredients that have the potential to be developed into a health product for pediculosis are red ginger (zingiber officinale var. rubrum) and star fruit (averrhoa bilimbi l.). secondary metabolite compounds in red ginger extract and star fruit that can potentially kill head lice pediculosis humanus capitis include alkaloids, flavonoids, and saponins. the purpose of this study was to determine the physical fitness and effectiveness of the lethal power of the combination of red ginger extract and star fruit with formula 1 (f1) 1.5%, formula 2 (f2) 2%, and formula 3 (f3) 2.5%. the tests carried out were phytochemical screening tests (alkaloids, tannins, saponins, and flavonoids), physical stability of shampoo (organoleptic, homogeneity, foam height, viscosity, and ph) for four weeks of storage, and testing the effectiveness of shampoo killing power against head lice pediculus humanus capitis. data analysis was carried out descriptively and comparatively using one-way anova. phytochemical screening of red ginger extract and star fruit was positive for alkaloids, tannins, saponins, and flavonoids. the organoleptic test of the shampoo on all three formulas produced a light brown colour, a distinctive aroma of lemon oil, and a thick shape. the homogeneity test showed a homogeneous shampoo preparation. f1 foam level test (7.7-11 cm), f2 (7.3-11 cm), and f3 (7.2-11 cm). f1 viscosity test (2,010-2,020 cps); f2 and f3 (2,020 cps). one-way anova ph f1 test (8.54-8.77); f2 (8.01-8.70); f3 (8.61-8.77) and one-way anova results for ph values obtained significant values of 0.096 > 0.05, while the killing power of aphids was 0.02 < 0.05. the three shampoo formulas of red ginger extract and star fruit showed good physical evaluation and still entered the test requirement range for four weeks of storage. based on the results of statistical analysis, it was shown that the variation in the concentration of the formula did not cause a change in ph during four weeks of storage. the administration of a combination shampoo of red ginger extract and star fruit with a formulation of f1 1.5%, f2 2%, and f3 2.5% was able to have a natural effect on the average number of head lice deaths of pediculus humanus capitis. keywords: shampoo; pediculus; red ginger; star fruit; formulation. introduction one of the infectious diseases in indonesia that is widely found in school-age children is pediculosis capitis. this disease is caused by the bite of head lice (pediculus humanus capitis) or head lice. pediculosis capitis causes decreased confidence and interferes with activities in sufferers. head lice are included in ectoparasites that can be transmitted through direct physical contact with sufferers, such as cuddling, sitting next to each other, and sharing items (agistia et al., 2023). handoko (2016) states head lice can cause human scalp infections (putu et al., 2023). according to the centers for disease control and prevention (cdc), in 2016, pediculosis was prevalent in children aged 3 to 11 years around the world. as for indonesia, data on pediculus humanus capitis infection is still limited (ramadaniah et al., 2023). the study reported the prevalence of suhesti and pramitaningrum (2020) pediculosis capitis in a housing estate in cibitung, bekasi. as many as 85% of girls and 36% of boys were positive for pediculosis capitis. agumsah and apriani (2021) also reported pediculosis capitis in children aged 3-12, as many as 68%, in the babakan, asem village area, teluk naga. the research results of the almuhajirin islamic boarding school, central cikarang, reported that 36 out of 41 female students (87.8%) were positive for pediculosis (anindita et al., 2024). treatment to overcome head lice in the community still relies on using insecticides with synthetic chemicals such as permethrin, lindane, malathion, and carbaryl. using synthetic chemicals as anti-lice treatment circulating in the community has dangerous side effects and can trigger head lice resistance (agistia et al., 2023). manuscript received: 15 agustus, 2024. revision accepted: 10 october, 2024. published: 03 december, 2024. rezaanindita@stikesmitrakeluarga.ac.id https://doi.org/10.14421/biomedich.2024.132.565-573 mailto:rezaanindita@stikesmitrakeluarga@ac.id 566 biology, medicine, & natural product chemistry 13 (2), 2024: 565-573 therefore, safe and effective prevention efforts are needed to prevent the spread of head lice. one of the efforts to prevent and treat head lice can be done by testing the manufacture of shampoo from natural ingredients that can be insecticides. the accuracy of the concentration and selection of natural ingredients as insecticide active ingredients is because they do not contain side effects and have residues that are easily degraded by the environment (nurhaini et al., 2020). some natural ingredients that can be productive as anti-head lice include red ginger, star fruit, and lemon oil. red ginger plants (zingiber officinale var. rubrum) and star fruit (avverhoa bilimbi l.) contain alkaloids, tannins, saponins, flavonoids, and triterpenoids that function as stomach, respiratory, and nerve toxins against insects (qatrinide et al., 2021) (la asi et al., 2022). lemon oil contains limonoids that function as antifeedants that can inhibit chemoreceptors in the mouth of insects (haifania et al., 2022) some of the studies on the formulation of shampoo preparations against head lice with natural ingredients include research that formulates shampoo preparations using the active ingredients of neem leaves, hibiscus, henna, amla fruit, reetha fruit, and urad da beans (deeksha et al.,2020), research that uses mustard leaves as an active ingredient in the manufacture of shampoo preparations against head lice (saikia et al., 2022). according to previous studies, no formula for anti-head lice shampoo uses red ginger extract, star fruit, and lemon oil. the novelty of this research is the combination formula of red ginger extract and star fruit with concentrations of 1.5%, 2%, and 2.5% and the addition of lemon oil with a concentration of 10% for each formula. this study aims to determine the physical stability and effectiveness of a combination of red ginger extract and star fruit shampoo against pediculus humanus capitis. materials and methods tools and materials red ginger extract, star fruit extract, lemon oil, sodium lauryl sulfate, cocamide dea, nacl, na-cmc, citric acid, propylene glycol, glycerin, propyl paraben, methyl paraben, aquadest, chloroform, ammonia, h2so4, hcl, naoh, fecl3, mg powder, amyl alcohol, dragendorff reagent, meyer reagent, and wagner reagent. analytical scales (ohaus), watch glass (pyrex®), beaker (pyrex®), measuring cup (pyrex®), stirring rod (iwaki pyrex), mixer (ika rw 20), digital ph meter (atc), viscometer (labo), hot plate (ika c-mag). sample preparation sample preparation: plant determination (pt. palapa muda perkasa no. 996/iph.1.01/if.02/1/2024), wet sorting, dry sorting, drying, and powder making. extraction using the maceration method and evaporation using the rotary evaporator method. phytochemical screening of red ginger and star fruit extract phytochemical screening of red ginger and star fruit extract, including alkaloid using dragendorff reagent, meyer reagent, and wagner reagent (srikandi et al., 2020), tannin, saponin, and flavonoid (kusuma et al., 2023) formulation shampoo red ginger extract and star fruit the formula shampoo combining red ginger extract, star fruit, and lemon oil modified research of dalming et al. (2022) shown in table 1. table 1. shampoo formula red ginger extract and star fruit. material concentration f1 f2 f3 red ginger extract 0,75% 1% 1,25% star fruit extract 0,75% 1% 1,25% lemon oil 10% 10% 10% sodium lauryl sulfate 15% 15% 15% cocamide dea 8% 8% 8% nacl 5% 5% 5% na-cmc 1% 1% 1% citric acid 0,1% 0,1% 0,1% propylene glycol 1% 1% 1% glycerine 1% 1% 1% propyl paraben 0,5% 0,5% 0,5% methyl paraben 0,02% 0,02% 0,02% aquades ad 150 ml ad 150 ml ad 150 ml based on table 1, shampoo formulations change in the concentration and use of natural ingredients. this study uses natural ingredients combined with red ginger extract and star fruit 1.5%, 2%, and 2.5% and the addition of lemon oil 10%. anindita et al. – formulation and effectiveness test of red ginger (zingiber officinale var. rubrum) … 567 shampoo made of red ginger and star fruit extract a combination of red ginger extract and star fruit is made by developing na-cmc using aqua*dest containing citric acid that has been heated to form mucilage (mixture 1). add dea cocamide, propylene glycol, glycerin, propylparaben, and methylparaben to mixture 1, stirring until homogeneous. sodium lauryl sulfate is mixed with nacl in aquadest to taste and stirred until evenly mixed (mixture 2). then, red ginger extract, star fruit extract, and lemon oil are mixed into mixture two and stirred until homogeneous. combine the results of mixtures one and two using a mixer for 5 minutes, add the remaining aquadest, and stir again using a mixer for 5 minutes until homogeneous. evaluation of the physical stability of the shampoo organoleptic test the organoleptic test was carried out by visual observation of the colour, smell, and shape of the shampoo preparation at room temperature with a storage period of 4 weeks(nurhaini et al., 2020). homogeneity test this test was carried out by applying a shampoo preparation to the watch glass and observing a preparation that showed a uniform mixing of ingredients and no visible coarse particles (annisanur and musfiroh, 2022). foam height test the foam height measurement is done by making a solution of shampoo preparation in aquades, shaking it in a container, and then measuring it using a measuring device. the height of the foam shampoo meets the requirements if the height of the foam is in the range of 1.3 cm – 22 cm (agistia et al., 2023). viscosity test viscosity testing was carried out using brookfield viscometers. a shampoo of 150 ml is placed under a viscometer with the appropriate spindle and speed. the viscosity value of shampoo preparations ranges from 400 – 4,000 cps. (annisanur and musfiroh, 2022) ph test the ph value measurement is done by dipping the ph meter into the shampoo preparation and then observing the number listed on the ph meter. the ph value requirement of shampoo preparations determined by sni is between 5 – 9 (agistia et al., 2023). shampoo test against head lice pediculus humanus capitis the treatment design in this study included positive control using the editor (permethrin 1%), negative control (sterile aquades), f1 shampoo 1.5%, f2 2%, and f3 2.5%. each treatment was placed in a petri dish containing 12 lice with three repetitions so that the total sample was 180 head lice. all treatments were observed for 10 minutes, and the number of head lice deaths was recorded during each treatment. data analysis data analysis using descriptive and comparative analysis. descriptive analysis: the phytochemical screening test of red ginger and star fruit extract, organoleptic test, homogeneity, foam height, and viscosity test. comparative analysis was carried out on the data from the ph test and mortality of head lice using one-way anova statistical test. result and discuss the results of the determination test of red ginger and star fruit plants were conducted at pt. palapa muda perkasa, depok city, identified that red ginger comes from the zingiber officinale (zingiberaceae family), while star fruit comes from the averrhoa bilimbi l. (oxalidaceae family). the results of the determination of red ginger and star fruit plants can be seen in table 2. table 2. results of determination of red ginger and star fruit plants. local name species family red ginger zingiber officinale zingiberaceae star fruit averrhoa bilimbi l. oxalidaceae in the organoleptic test, the red ginger extract obtained brownish-yellow results, aromatic odor and concentrated extract. in contrast, in star fruit extract, the results were brownish-green, aromatic, and in the form of a concentrated extract. the results of organoleptis can be shown in table 3. table 3. results of organoleptis tests of red ginger extract and star fruit. sample colour smell form red ginger extract brownish-yellow aromatic concentrated extracts star fruit extract brownish green aromatic concentrated extracts in accordance indonesia herbal pharmacopoeia edition ii states that red ginger extract has a brownishyellow colour, aromatic smell, concentrated extract form, and spicy taste, and star fruit extract has a blackish-green 568 biology, medicine, & natural product chemistry 13 (2), 2024: 565-573 colour, aromatic smell, concentrated extract form, and acid taste. the characteristics of the extracts used are complemented by the results of the yield of red ginger and star fruit extracts, which can be seen in table 4. table 4. results of rendement of red ginger extract and star fruit. sample name powder weight (g) extract weight (g) extract yield (%) red ginger 1.250 284 22,72 star fruit 1.200 236,4 19,7 based on table 4, the yield value of red ginger extract was 22.72%, while star fruit was 19.7%. in accordance with the herbal pharmacopoeia indonesia edition ii, the yield of red ginger extract is>17%, and star fruit extract is>3.3%. the results of extract yield are related to the content of active compounds in the sample. if the yield value of the resulting extract is high, the sample's active compound content is also high. several factors affect the yield value extract, including extraction time and selection of extraction method. the more optimal the extraction process time, the greater the yield obtained. this is because the contact time between the solvent and the simplicia is getting longer, so the weight of the extract from the simplicia is higher (subaryanti et al., 2022). another factor that affects the results of the extract yield value is the selection of the extraction method. extraction methods that use room temperature tend to be long but can retain thermolabile secondary metabolite compounds. in contrast, extraction methods that involve heating at 60-70 ºc tend to be faster but risk reducing thermolabile secondary metabolite compounds (ramayani et al., 2021). the phytochemical screening test of red ginger extract included tests on alkaloid compounds, tannins, saponins, and flavonoids. the results are in table 5. table 5. results of phytochemical screening test of red ginger extract. compound reagents result indicator alkaloids dragendorff reagent positive white precipitate meyer reagents positive orange precipitate wagner reagents positive brown precipitate tannins fecl3 positive blackish-green colour flavonoids naoh positive orange colour saponins hcl positive stable foam the results of the phytochemical screening test of red ginger extract show that it is positive and contains alkaloids, tannins, flavonoids, and saponins. the phytochemical screening test of star fruit extract was carried out on alkaloids, saponins, and flavonoids. the results of the phytochemical screening test of star fruit extract can be seen in table 6. table 6. phytochemical screening test of star fruit extract. compound reagents result indicators alkaloids dragendorff reagent positive brown to orange deposits meyer reagents positive white precipitate wagner reagents positive brown deposits saponins hcl 2n positive stable foam flavonoids mg powder + hcl + amyl alcohol positive red to orange colours the results of the phytochemical screening test of star fruit extract show that it contains alkaloid, tannin, flavonoid, and saponin. physical stability testing of shampoo combining was carried out with a storage period of 4 weeks against f1 1.5%, f2 2%, and f3 2.5%. the organoleptic test shampoo showed no change in colour, odour, or texture, indicating that the shampoo was stable during the 4-week storage period. the results of the organoleptic test shampoo are shown in table 7. anindita et al. – formulation and effectiveness test of red ginger (zingiber officinale var. rubrum) … 569 table 7. results of organoleptic test. week f1 f2 f3 0 colour: light brown smell: lemon oil aroma shape: viscous colour: light brown smell: lemon oil aroma shape: viscous colour: light brown smell: lemon oil aroma shape: viscous 1 colour: light brown smell: lemon oil aroma shape: viscous colour: light brown smell: lemon oil aroma shape: viscous colour: light brown smell: lemon oil aroma shape: viscous 2 colour: light brown smell: lemon oil aroma shape: viscous colour: light brown smell: lemon oil aroma shape: viscous colour: light brown smell: lemon oil aroma shape: viscous 3 colour: light brown smell: lemon oil aroma shape: viscous colour: light brown smell: lemon oil aroma shape: viscous colour: light brown smell: lemon oil aroma shape: viscous 4 colour: light brown smell: lemon oil aroma shape: viscous colour: light brown smell: lemon oil aroma shape: viscous colour: light brown smell: lemon oil aroma shape: viscous table 7 shows that three shampoo formulations had a light brown colour, the smell of lemon oil, and dence texture during 4-week observation. homogeneity testing of shampoo during a 4-week storage period showed homogeneous and no coarse particles in all three formulations. the homogeneity test is one of the physical evaluation tests on shampoo to see a homogeneous arrangement with no coarse grains (asjur et al.., 2022). the results of the homogeneity test can be seen in table 8. table 8. results of homogeneity test of anti-flea shampoo preparation. week f1 f2 f3 0 homogeneous homogeneous homogeneous 1 homogeneous homogeneous homogeneous 2 homogeneous homogeneous homogeneous 3 homogeneous homogeneous homogeneous 4 homogeneous homogeneous homogeneous table 8 shows that all ingredients are well mixed during the preparation process. the results of this study are in accordance with butar-butter (2023) that antifungal shampoo of shallot ethanol extract can produce homogeneity. the results of the nonhomogeneous shampoo can be known in the study by aida et al. (2022), where the anti-fungal shampoo of mango leaf extract was non-homogeneous and characterized by rough materials at observation for three weeks. nurhaini et al. (2020) reported that shampoo soursop leaf extract results showed the presence of rough granules caused by a lack of water during the carbopol development procedure, so the physical evaluation of the shampoo was not homogeneous. the foam height measurements of three formulas with a storage period of four weeks still entered the range of foam height requirements of shampoo. this test aims to assess shampoo ability to produce foam. the results of measuring the height of the shampoo foam can be seen in table 9. table 9. high the foam of shampoo. week the average high value of foam (cm) f1 f2 f3 0 7.7 ± 0.5 7.3 ± 0.2 7.2 ± 0.2 1 7.7 ± 0.2 7.8 ± 0.6 8.5 ± 0.7 2 8.7 ± 0.5 9 ± 0.0 9 ± 0.0 3 10.5 ± 0.7 10 ± 0.0 10.7 ± 0.5 4 11 ± 0.0 11 ± 0.0 11 ± 0.0 results of measuring height test of foam shampoo on f1, f2, and f3 increasing values. this affected the speed of the shaking process at any time. overall, the foam height value obtained from the shampoo is consistent with sni 1.3 – 22 cm (elianasari and fauziah, 2023); (annisanur and musfiroh, 2022). viscosity testing showed stable results with a storage period of 4 weeks at room temperature. the average viscosity value obtained f1: 2,010-2,020 cps, f2 and f3: 2,020 cps. viscosity measurement was used to determine the viscosity value of shampoo. viscosity value can affect the stability of the storage period, flowability, and shampoo spread when used on hair (bhavani et al., 2023). the results of the viscosity test of shampoo can be seen in table 10. table 10. viscosity test. week average viscosity value (cps) f1 f2 f3 0 2,010 ± 14,14 2,020 ± 0,000 2,020 ± 0,000 1 2,020 ± 0,000 2,020 ± 0,000 2,020 ± 0,000 2 2,020 ± 0,000 2,020 ± 0,000 2,020 ± 0,000 3 2,020 ± 0,000 2,020 ± 0,000 2,020 ± 0,000 4 2,020 ± 0,000 2,020 ± 0,000 2,020 ± 0,000 based on the results of viscosity measurements for four weeks of the storage period indicate that shampoo has a stable viscosity. the results of this study are in accordance with the research by guidance et al. (2021) that the viscosity of the anti-dandruff shampoo of jamblang fruit extract has a good shampoo viscosity value of 400 – 4,000 cps. the results of this test are also 570 biology, medicine, & natural product chemistry 13 (2), 2024: 565-573 based on a study by rasyadi et al. (2023) that reported that the anti-dandruff shampoo of arabica coffee leaf ethanol extract has an average viscosity value of 1,247 – 3,011 cps with storage for six weeks. testing the ph value with a storage period of 4 weeks showed results that were still in the ph range of good shampoo. the ph test aims to determine the preparation's acid or base value. the results of measuring the ph value of shampoo can be seen in table 11. table 11. ph test of shampoo. sunday x̄ ± sd sig. normality sig. homogeneity sig. one-way anova f1 f2 f3 0 8.54a ± 0.28 8.01a ± 0.42 8.61a ± 0.21 f1 = 0.510 f2 = 0.218 f3 = 0.780 0,081 0,096 1 8.63a ± 0.28 8.37a ± 0.16 8.68a ± 0.11 2 8.77a ± 0.13 8.63a ± 0.14 8.70a ± 0.02 3 8.57a ± 0.08 8.66a ± 0.09 8.74a ± 0.03 4 8.62a ± 0.05 8.70a ± 0.08 8.77a ± 0.02 table 11 shows that each formulation has a stable ph value and, according to sni, a range of 5.0 – 9.0. (firdaus and arief, 2019). the normality and homogeneity test results on the ph value data showed that the value of sig. > 0.05 or the ph test data was distributed normally and homogeneously. the results of one-way anova showed that the sig. 0.096 > 0.05 or the ph value of the shampoo during four weeks of storage did not change significantly in the shampoo formula of f1 1.5%, f2 2%, and f3 2.5%. the results of this study by erwiyani et al. (2023) and lara et al. (2023) state that the foaming agent can influence the ph value, namely sodium lauryl sulfate with a ph of 7.5 – 8.5 and the use of dea cocamide influences the ph value because it has a ph value of 9.5 – 10.5 which is higher than sodium lauryl sulfate, the ph value is not much different from the ph value obtained in this study. the ph value of a shampoo can affect the quality of the preparation; the ph range of the shampoo should not be too acidic or alkaline because it minimizes irritation to the eyes and damage to the scalp and hair (pundkar and ingale, 2020). the shampoo results declared stable were then tested for mortality of the head lice pediculus humanus capitis. the test was carried out on five treatment groups: positive control with peditox, negative control with aquades, f1 1.5%, f2 2%, and f3 2.5%. head lice samples for each group totaled 12 with 3x replication. the results of red ginger and star fruit shampoo against pediculus humanus capitis can be seen in table 12. table 12. results of mortality test of shampoo against head lice for 10 minutes. no treatment replication x̄ ± sd f. count f. table 1 2 3 1 control (+) 2 2 2 2 ± 0 4,35 3,33 2 control (-) 0 0 0 0 ± 0 3 f1 1,5 % 2 4 6 4 ± 2 4 f2 2 % 3 4 8 5 ± 2.67 5 f3 2.5% 3 6 9 6 ± 3 based on table 12. the average mortality of head lice after being given a combination of red ginger and star fruit shampoo for f1 1.5% was four head lice, f2 2% was eight head lice, and f3 was nine head lice. as for the negative control, no head lice deaths were found, while the positive control used editor for as many as two head lice. the results of anova one-way showed that the fcount was 4.35 while the f. table was 3.33 (fcal.>ftable), or the combination shampoo had a significant effect on the average mortality of head lice. the results of this study complement the results of the research of austin tee and badia (2019), which resulted in an average number of deaths of 3 head lice during 15 minutes of treatment with 5% soursop leaf extract shampoo. research by agistia et al. (2023) regarding the test of lemongrass oil shampoo with concentrations of 5%, 7.5%, and 10% can produce a head lice death time of 3-7 minutes. the 10-minute lime extract shampoo test by warahmah (2021) showed an average number of deaths of 2-3 lice, while the study by dalming et al. (2022) using eucalyptus oil shampoo of 15%, 20%, 25%, and 30% for 10 minutes only resulted in the average death of 1 head lice. based on this, this study is more effective than the previous study because within 10 minutes, with concentrations of 1.5%, 2%, and 2.5%, it produced an average number of deaths of 6-9 head lice. anindita et al. – formulation and effectiveness test of red ginger (zingiber officinale var. rubrum) … 571 the selection of red ginger and star fruit as natural insecticide agents in this study refers to the research of watcharawit and soonwera (2013), which reported that z. officinale and a. bilimbi produced lethal death time (ld50) values of 13.62 and 13.14 seconds. the results of microscopic observation of the morphology of head lice before being given shampoo showed the morphology of the body, legs, and antennae that looked normal, while after being given the shampoo showed the body condition of head lice with antennae and legs that tended to be weak and stiff. the results of the morphological observation can be seen in figure 1. figure 1. microscopic morphology of head lice. a. before treatment. b. after treatment. based on figure 1, head lice given a combination of shampoo, red ginger extract, and star fruit left red ginger residue on the abdomen. the results of el-akkad's (2016) research stated that pediculus humanus capitis, which was treated with several extracts of natural materials, underwent morphological changes, including legs and antennae becoming flaccid/shriveled, distortion of morphology outside the body, damage to antennae, paws, and sensory hair. according to nurmasari and aswan (2024), maisura (2023) contains alkaloids, flavonoids, tannins, and saponins of star fruit and red ginger function as stomach toxins that can enter the body of head lice through the skin or spiracles. the poison then enters the digestive tract, causing death in head lice. based on this, it can be seen that the higher the concentration of the extract of the combination of red ginger shampoo and star fruit, the more it significantly affects the average number of head lice deaths. the effective shampoo formula for killing 12 head lice is 1.5%, with an average number of deaths of 6 (50%) lice during 10 minutes of treatment. conclusion the physical stability evaluation test results showed that the three formulations of anti-head lice shampoo had good organoleptic, homogeneous, and produced average ph values, foam height, and viscosity within the required range. the results of the one-way anova test of a combination of red ginger extract and star fruit significantly affected the death of head lice for 10 minutes with an effective formula at a concentration of 1.5%. competing interests: the authors declare that there are no competing interests. references adiwibowo, m. t., herayati, erlangga, k., & fitria, d. a. 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(2013). pediculicidal effect of herbal shampoo against pediculus humanus capitis in vitro. tropical biomedicine 30(2): 315–324 https://doi.org/10.36805/jbf.v3i4.891 https://doi.org/10.56042/ijtk.v21i4.33008 https://doi.org/10.32734/scripta.v5i1.10530 https://doi.org/10.15575/ak.v7i2.6545 https://doi.org/10.37277/sfj.v15i2.1272 https://doi.org/10.47522/jmk.v3i1.49 https://doi.org/10.33024/mahesa.v4i6.14543 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 109-117 | doi: 10.14421/biomedich.2023.121.109-117 issn 2540-9328 (online) antioxidant and antibacterial activity of pomegranate extract (punica granatum l.) in lip balm formulation maria grasela kase, aniek prasetyaningsih, dwi aditiyarini* biology study program, faculty of biotechnology, duta wacana christian university jl. dr. wahidin sudirohusodo no. 5-25 yogyakarta, 55224, indonesia. corresponding author* dwi.aditiyarini@staff.ukdw.ac.id manuscript received: 31 october, 2022. revision accepted: 06 december, 2022. published: 11 january, 2023. abstract lips are a part of the face that does not have hair follicles making them easily dry and crack. lipbalm is a beauty product that could be used to solve this problem by increasing lip moisture. however, the synthetic ingredient in commercial lip balm can have side effects on the body in the long term. pomegranate fruit is one of the natural ingredients containing anthocyanin which can be used as a natural dye, antioxidant, and antibacterial. therefore, this study was performed to study the potency of pomegranates as natural dyes, antioxidants, and antibacterial in lip balm. pure pomegranate juice was obtained through squeeze step. lip balm was prepared in several concentrations of pomegranate juice which were 0%, 12.5%, 18.75%, and 25%. phytochemical screening shows the content of anthocyanin, saponin, tannin, and flavonoid. in this study, pomegranate juice has low antioxidant activity with ic50 449 ppm. lip balm formula with 18.75% and 25% of pomegranate juice can inhibit the growth of the bacteria staphylococcus aureus. keywords: antibacterial; antioxidants; lip balm; lips; synthetic ingredients. introduction one part of the face that most influences the aesthetic perception of the face is the lips. according to mulyawan and suriana (2016) in nazliniwaty et al., (2019) the most sensitive and unprotected part of the face is the lips. lips often look dry and cracked when it is exposed to cold air or excessive heat so that they are unsightly. this is because the lip does not have sweat glands and hair follicles that can protect it in the environment (trookman et al., 2009, yayang et al., 2019). one of the cosmetics products that can overcome this problem is lip balm. lip balm is a preparation that is applied to the lips and functions as a moisturizer by forming an immiscible oil layer on the surface of the lips. the layer formed by the lip balm is a protective layer of the lips from the influence of the external environment. in addition to lipstick, lip cosmetics that are often used by women are lip balms. the purpose of lip balm is to increase moisture in the lips. the main components in lip balms are waxes, oils, and fats which aim to prevent dry lips from occurring by keeping the lips moist and protecting the lips from environments that are too hot and too cold (agustiana et al., 2019). lip balm also contains antioxidants, preservatives, and coloring compounds. antioxidants in lip balm function to prevent free radicals that are harmful to the skin. preservatives are used to prevent irritation and prevent bacterial growth, while the dye in lip balm is added only to make the product (lip balm) look attractive (rini, 2012). antioxidants that are often used in the manufacture of lip balms are bht (butylated hydroxytoluene) and other preservatives. pomegranate is a tree that has a height of 2 to 5 meters. the pomegranate tree has a single leaf opposite each other (scattered). the large pomegranate flowers have red and white colors. the pomegranate flower is a pansy flower, and separated. the flower axis is hollow with a conical shape, 5 to 7 corollas in irregular buds, and many stamens, free stamens, and ovary sink. while for the pomegranate itself has a size 5 inches with red and dark red skin and is shaped like a grenade. the taste of the pomegranate itself varies depending on the level of maturity of the pomegranate (sharrif and hamed, 2012). pomegranate fruit, also known as pomegranate fruit, has several active compounds, namely alkaloids, flavonoids, saponins, tannins, and triterpenoids. pomegranate contains two types of polyphenol components, namely flavonoids (anthocyanins) and hydrolyzed tannins which have antioxidant activity. the antioxidant activity of pomegranate is higher than the antioxidant activity of red wine and green tea. in addition, the antioxidants in pomegranate also have the potential to reduce lipid peroxidation (roswiem et al., 2014). https://doi.org/10.14421/biomedich.2023.121.109-117 110 biology, medicine, & natural product chemistry 12 (1), 2023: 109-117 therefore, in this research, researchers would make a lip balm made from natural ingredients. one of the natural ingredients used in this research is pomegranate. the use of natural ingredients aims to reduce and replace synthetic ingredients that exist in lip balm preparations. the compounds in the pomegranate (punica granatum l.) to be tested are anthocyanins, saponins, tannins, and flavonoids, each of which has a role to replace or reduce synthetic ingredients commonly used in the manufacture of lip balms, so that in the manufacture of lip balms from natural ingredients is expected to bring out the natural color of the pomegranate, as well as antioxidants and antibacterial from the natural ingredients used. materials and methods this study uses petri dishes, beakers (pyrex), object glassed, measuring cups, oven (memmert), microscopes, test tubes, laf (laminar air flow), micropipettes, stoves, uv-vis spectrophotometers (genesys 10) as an antioxidant test and testing the total phenolic content, filter cloth, ph meter, oven, knife, and lip balm container, while for the used materials is a pomegranate from a fruit shop in yogyakarta, olive oil, beeswax, oleum cacao (kimia jaya labora), glycerin, aquadest, cetyl alcohol, dpph solution, methanol pa, hcl 2n solution, fecl3 solution, folin ciocalteau reagents, na2co3 solution, naoh solution, and staphylococcus aureus bacteria culture. in sample preparation, the used fresh pomegranate is ± 1 week after harvest that has red color. three kilograms of pomegranate fruit is separated between the skin and flesh, then the flesh is washed until clean and weighed (yuska et al., 2018). procedures extraction the extraction process was carried out by squeezing the pomegranate and filtering the pomegranate to get pure juice using a white filter cloth. the filtering process was carried out by squeezing the pomegranate which is still together with the seeds then placed on a filter cloth and then the juice was put into a 1000 ml sterilized beaker glass. yield is calculated using this formula: 𝑦𝑖𝑒𝑙𝑑 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑗𝑢𝑖𝑐𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑠𝑖𝑚𝑝𝑙𝑖𝑐𝑖𝑎 × 100% phytochemical screening of pomegranate juice anthocyanins the color test of anthocyanin compounds was carried out according to harbone (1987). red pomegranate juice 0.5 g was added to 2m hcl in 2 drops and then heated at 100°c for 5 minutes. a positive result is indicated with a red color. the several dropwise of 2m naoh was added while observing the color changes. positive result is indicated by the changes of red color to a blue-green color which then fades slowly (putri ni ketut et al., 2015). saponin the red pomegranate juice was weighed as much as 0.3 g and then put into a test tube. then 5 ml of distilled water was added and shaken for 30 seconds. the positive result is marked by the formation of foam which is stable for 30 seconds on the surface of the solution. tannin the red pomegranate extract 1 g was added to 10 ml of distilled water and then boiled. after cooling, 5 ml of 1% fecl3 was added. if the color changes to dark blue, then the sample contains tannins (novilia, 2014). flavonoid the extracted sample 1 g was put into a test tube. then 3 drops of concentrated hcl were added and then heated for 15 minutes in a water bath. if red or yellow color is formed, it means that the juice contains flavonoids (flavones chalcones and aurons) (muthmainnah, 2017). determination of total phenol content a total of 2 mg of pomegranate juice was dissolved in 2 ml of methanol and homogenized. then 0.2 ml of extract was put into a test tube and added 1.8 ml of distilled water and 0.1 ml of folin ciocalteu reagent. the solution was homogenized, vortex and incubated for 5 minutes. next, 1 ml of 5% na2co3 was added and homogenized. distilled water 1.9 ml was added to obtain final volume of 5 ml. then solution was homogenized and incubated for 5 minutes. the absorbance was measured at a wavelength of 760 nm. gallic acid was used as a standard curve with a concentration of 12,5; 25; 50;100 and 200 ppm. a linear equation is obtained by plotting a linear graph between the gallic acid concentration (x) and the absorbance value (y) (yismairai et al., 2019). calculation of total phenolic content is performed using the formula below: 𝑇𝑃𝐶 = 𝐶 × 𝑉 × 𝑓𝑝 𝑚 annotation: c : fenolic concentration (x score) v : volume of extract used (ml) fp : dilution factor g : weight of sample used (g) tpc : total phenolic content (mg gae/ g sample) antioxidant activity test antioxidant activity was determined by the dpph free radical method according to santosa et al. (1998). in this test, the sample of pomegranate juice were made in several concentrations which are 200,400, 600, 800, and 1000 ppm. the concentration of dpph is 10 ppm. the kase et al.– antioxidant and antibacterial activity of pomegranate extract … 111 stock solution was 1000 ppm. the dpph solution was put into each tube with various concentrations, namely 2 ml of dpph 10 ppm + 1 ml of sample solution, while for the control, 1 ml of dpph 10 ppm was made, then incubated for 30 minutes in the dark room temperature, then the absorbance was measured at a wavelength of 517 nm. furthermore, the value of inhibition concentration 50% (ic50) was determined, namely the concentration of the sample that can reduce dpph free radicals by 50% by using the equation y= ax+b, thus obtaining ic50 in units of g/ml. (abdullah et al., 2014). extract dosage formulation in this study, four lip balm formulations were made, consist of 0, 12.5, 18.7, and 25% of pomegranate extract with a weight of 12.8 g each container. the composition of the pomegranate extract lip balm is presented in table 1. table 1. formulation of natural lip balm. ingredient red pomegranate extract formulation (%) f0 f1 f2 f3 red pomegranate extract 0 12.5 18.75 25 olive oil 10 10 10 10 beeswax 25 25 25 25 glycerin 10 10 10 10 cetyl alcohol 50 50 50 50 oleum cacao ad 100 ad 100 ad 100 ad 100 annotations: f0 = lip balm formula as a base f1 = lip balm formula with concentration extract 12.5% f2 = lip balm formula with concentration extract 18.75% f3 = lip balm formula with concentration extract 25% in the process of making lip balm, the first step was to put ethyl alcohol in a beaker glass, heated and added pomegranate juice slowly. the solution was stirred until evenly mixed between ethyl alcohol and extract. after that, beeswax and cacao oleum was added and melted a temperature of 65°c. next, olive oil was added, heated, and stirred until homogeneous. lastly, the solution was taken out from water bath and poured into a container lip balm mold evenly and cooled until it hardens at room temperature (dirjen pom, 1985). organoleptic test an organoleptic test was carried out using the five senses. the parameters include odor, color, and texture of lip balm (yulyuswarni, 2018). evaluation of the preparation was carried out for 3 weeks with inspections carried out every week (days 1, 5, 10 and so on). homogeneity test this test was carried out by testing the homogeneity of the preparation of each lip balm preparation by taking the lip balm preparation and placing it on the glass slide and then visually observing whether there were coarse grains or not (keithler, 1956). lip balm weight test this test was carried out by weighing the lip balm preparation including the container. this weighing was carried out every 7 days for 3 weeks at a closed room temperature of 27°c. this test was carried out to determine the increase or decrease in weight at the beginning of manufacture and during storage. melting point test this test was done by melting 1 g of the lip balm preparation in a beaker. the melting temperature was measured at the initial melting (dian, 2019). ph test ph value was measured using a ph meter where 1 g of the sample was inserted and then dissolved in 10 ml of distilled water, after that electrode is inserted or dipped in the lip balm solution, then the numbers show on the ph meter are seen (risnawati et al., 2012). irritation test this test was carried out to see and evaluate whether the product can cause irritation or not. the technique used in this irritation test is an open patch test on the forearms of 30 panelists. an open patch test was carried out by applying the prepared preparation at the location of the attachment, leaving it open and, observing what happens. reaction was observed. positive irritation reactions are characterized by redness, itching, or swelling (risnawati et al, 2012). favorite test this test was conducted to determine the level of panelist preference. the age of 30 female panelists was around 18-22 year old. the panelist was not having sensitive or allergic skin. each other panelist was asked to apply lip balm of various concentrations of red pomegranate to the back of the hand. then they were asked to fill out the provided questionnaire. the duration of trial was ± 15 minutes and after trying the lip balm, the panelists are expected to wash their hands. antibacterial test antibacterial activity test was done using pour plate method. bacterial incubation was carried out for 24 hours using the pour plate method at 37c. then diameter of the inhibition zone was measured for each sample. the list of samples is presented in table 2. 112 biology, medicine, & natural product chemistry 12 (1), 2023: 109-117 table 2. list of samples. no code sample 1 e pomegranate extract with concentration 12.5%, 18.75% dan 25% 2 b olive oil, beeswax, oleum cacao, glycerin dan cetyl alcohol 3 f1 lipbalm formula with concentration extract 12.5% 4 f2 lipbalm formula with concentration extract 18.75% 5 f3 lipbalm formula with concentration extract 25% 6 k commercial lipbalm antibacterial test was performed to determine the ability of antibacterial activity against staphylococcus aureus. this first step is to culture the bacteria on nutrient agar (na) and nutrient broth (nb) medium. nutrient agar (na) is a medium for rejuvenating staphylococcus aureus which is in solid form, while nutrient broth (nb) has same function as (na) for growing bacteria but in liquid form. then, 100 μl nutrient broth (nb) containing bacteria was poured aseptically to each petri disk. mha media 20 ml was added into each petri disk. this step can also be performed by formerly mixing the bacteria and mha media, then small holes are made after the media hardens to put the sample. plates were divided into 3 to 4 zones to facilitate the distribution of treatments. after samples had been put into all petris, each petri was wrapped and incubated at 37°c for 24 hours under aerobic conditions for staphylococcus aureus. the ability of antibacterial activity was shown through the inhibition zone. the diameter of the finhibition zone was measured using a ruler. results and discussion secondary metabolites of pomegranate juice the yield of pomegranate juice is 76.80% (1.565 kg of pomegranate juice from 2.038 kg of peeled pomegranate) by squeezing the pomegranate. dian (2019) also carried out this method on dragon fruit with the similar yield as this study. phytochemical test results from pomegranate juice is presented in table 3. table 3. phytochemical screening data of pomegranate juice. compound parameter analysis result description anthocyanin blue green + green saponin stable foam + presence of foam tannin dark blue + dark blue flavonoids red/yellow + red annotation: (+) there are chemical compounds, (-) no chemical compounds in this study, pomegranate extract showed a positive result for the anthocyanin test, namely a change in color to green. this pigment belongs to the flavonoid compound and is responsible for the appearance of red, orange, blue and purple colors in some leaves, fruits, and flowers. anthocyanins have potential natural dyes to replace synthetic dyes (gross, 1987). saponin is also detected in the pomegranate juice. saponin compounds are included in triterpenoids, usually found in higher plants. saponins are abundant in leaves and roots but are rarely found in fruit (cseke et al, 2006). saponin compounds are indicated by the presence of stable foam or foam. the next compound identified was tannin. tannin compounds have positive results by showing a dark blue color. tannins are a class of polyphenolic compounds that are also commonly found in plants. tannins can be defined as polyphenolic compounds with very large molecular weights, which are more than 1000 g/mol, and can form complex compounds with proteins, besides that, tannins have a major biological role because of their function as protein precipitants and metal chelators. it also acts as a biological antioxidant. (noer et al, 2018) the last test of flavonoid compounds in this research showed positive results where the color produced was red. flavonoids are secondary metabolites of polyphenols, which are found widely in plants and food and have various bioactive effects including anti-viral and anti-inflammatory (qinghu wang et al, 2016). flavonoids are also one of the natural compounds that are commonly found in plants and food to treat various diseases such as cancer, antioxidants, bacterial pathogens, inflammation, cardiovascular malfunction, and have antioxidant abilities in preventing injury caused by free radicals (arifin et al, 2018) total phenolic content in pomegranate extract this research used gallic acid as a standard according to singleton et al. (1999). the standard curve of gallic acid is presented in figure 1. gallic acid has been used as a standard of comparison which is stated as milligrams of gallic acid equivalent per gram or milliliters of extract between samples (tapera et al., 2019). the gallic acid standard is the relationship between gallic acid concentration (x) and absorbance value (y), which this relationship will produce a linear regression equation y = 0.0026x – 0.0116 and r2 value is 0.9903. from the equation in figure 1, it was carried out to calculate the total phenol in the pomegranate extract. total phenol levels are expressed in units of mg gallic acid equivalent/g sample, % gallic acid equivalent (gae) (mg gae/g) (hala et al., 2020). these results indicate that the total phenol content in the pomegranate extract is 0.012 mg/mg gae. kase et al.– antioxidant and antibacterial activity of pomegranate extract … 113 figure 1. gallic acid standard curve. pomegranate extract antioxidant activity the result of antioxidant measurements on pomegranate extract can be seen in figure 2. based on the data in figure 2, the results of linear regression are obtained, namely y = 0.0649x + 20.805 with a relation coefficient r2 = 0.924 so that the ic50 can be determined. the ic50 value is the concentration of a test sample solution that provides 50% dpph reduction (molyneux, 2004). from the equation data, it was found that ic50 in pomegranate extract had a value of 449 ppm. figure 2. antioxidant activity of pomegranate extract. according to molyneux (2004), the antioxidant activity in a compound is said to be very strong if the ic50 value is <50ppm, strong if it has an ic50 value between 50-100 ppm, moderate if ic50 value around 100-150 ppm and is said to be weak if the ic50 value 151-200 ppm. the ic50 value obtained from this research is 449 ppm, which is a weak ic50 value. in wulandari et al. (2017), ic50 of pomegranate peel extract with the extraction process, namely maceration, got a value of 2.39 ppm, which means the ic50 is very strong. the low ic50 of pomegranate extract in this study is probably caused by the difference of extraction process. preparation of pomegranate extract lip balm lip balm preparation from pomegranate extract is pink. the concentrations used were 0%, 12.5%, 18.75%, and 25%. figure 3 shows that the higher concentration, the more concentrated the color will be. the result of the lip balm preparation can be seen in figure 3. f0 (0%) fi (12.5%) f2 (18.75%) f3 (25%) figure 3. lip balm from pomegranate extract in several concentrations. organoleptic test results lip balm preparations from various extract concentrations 0%, 12.5%, 18.75%, 25%, had different characteristics of color, smell, and texture. the organoleptic test aims to determine the stability conditions of each concentration of lip balm tested for 3 weeks. it is included color, odor, and texture of each concentration of lip balm. based on organoleptic data for 3 weeks, changes in texture, aroma and color began to occur in the second week. the organoleptic test is conducted by dian (2019) also showed changes in the second week. according to dian (2019), physical differences (color and texture) are caused by a high room temperature factor which cause faster water evaporation so the particles in the lip balm clump together and cause the formation of crystals. according to pertiwi et al., (2020), the higher the concentration of extract used in the lip balm formulation, the stronger the color intensity. homogeneity test results based on homogeneity data, at concentrations of 0%, 12.5%, 18.75%, and 25% of the lip balm were homogeneous, while for a concentration of 12.5%, the results were not homogeneous. the homogeneity data showed that all lip balm formulations did not show any coarse granules, while the concentration of 12.5% was not mixed evenly for color distribution. therefore, it can be said that the preparations are 0%, 18.75%, and 25% and have a homogeneous composition in which the color distribution is even and there are no coarse grains on the glass object. this is in line with research conducted by (yusuf, 2019) ph test results testing the ph of the pomegranate extract lip balm in this study was using a ph meter. the purpose of ph testing is to determine and obtain a ph value that is close to the physiological ph of the skin, namely 4.56.5. the results of the ph test can be seen in table 4. y = 0,0026x 0,0116 r² = 0,9903 0 0,1 0,2 0,3 0,4 0,5 0,6 0 50 100 150 200 250 a b so rb at io n concentration (ppm) y = 0,0649x + 20,805 r² = 0,9424 0 20 40 60 80 100 0 200 400 600 800 1000 1200 % in h ib it io n concentration (ppm) 114 biology, medicine, & natural product chemistry 12 (1), 2023: 109-117 table 4. ph lip balm from pomegranate juice. concentration ph f0 (0%) 5.41 f1 (12.5%) 4.61 f2 (18.75%) 4.24 f3 (25%) 4.01 according to table 4, ph value of the lip balm without extract (f0) is 5.41, the concentration of f1 (12.5%) containing pomegranate extract is 4.61, then for the concentration of lip balm using pomegranate extract f2 (18.75%) was 4.24 and the concentration of f3 (25%) had a ph of 4.01. the difference in ph was caused by differences in the concentration of the extract. at concentrations of 0% (f0) and 12.5% (f1) it is already in the vulnerable ph of the lip skin (tranggono and latifah, 2007) which says the physiological ph of the lip skin is between 4.5-6.5, which means lip balm from extracts. pomegranate is safe to use, while the concentrations of 18.75% and 25% do not meet the physiological ph requirements of the lip skin. this is because the amount of pomegranate extract that is included in the lip balm preparation during the manufacturing process, so that the more concentrations used in the preparations, the more acidic it will be because the ph of the pomegranate is acidic, which 3.51. melting point test results in this research, the melting point test was also carried out. a melting point test is done to find out at what temperature the lip balm can melt completely. the result data are presented in table 5. table 5. lip balm melting point from pomegranate extract. concentration melting point (°c) f0 (0%) 55 f1 (12,5%) 53 f2 (18,75%) 52 f3 (25%) 52 from the results of the melting point test on the lip balm preparation, f0 (0%) has a melting point of 55ºc, f1 (12.5%) has a melting point of 53°c, then for f2 (18.75%) is 52°c and the last one for f3 (25%) which is 52°c. all melting point results have been included in the standard based on sni 16-5769-1998 in ratih (2014) which is in the range of 50-70°c. it can be said that the melting point of the prepared lip balm has good melting point and is up to standard. according to (agustiana et al., 2019) the difference in melting point in each preparation is due to the difference in the amount of cacao oleum included in each lip balm, the higher the concentration, the less cacao oleum is added so that the resulting melting point decreases. data shows that f0 (0%) has the highest melting point which is caused by the amount of cacao oleum that is the most compared to other concentrations is 5,2 g, while the least amount of cacao oleum is in the concentration of f3 (25%) which is 2 g, so it has a lower melting point. lip balm weight test result the result of the observed lip balm weight for 3 weeks is presented in table 6. table 6. lip balm weight test data. week weight (g) f0 (0%) f1 (12.5%) f2 (18.75%) f3 (25%) 0 average 6.207 6.695 6.871 6.697 1 average 6.207 6.693 6.868 6.695 2 average 6.208 6.692 6.861 6.655 3 average 6.210 6.692 6.859 6.644 lip balm weight test data for all concentrations were included in the paired samples test using spss to determine whether there was a difference in lip balm weight. tests using paired sample test are seen based on the comparison of week 0 and week 1, week 1 and week 2, week 2 and week 3, and the last week 0 and week 3. based on the results of the paired samples test, there was no significant difference in the weight of the lip balm which was weighed every week. if the sig value >0.05 then there is no significant difference in the lip balm weight test which is carried out every week. favorite test results in this research, the preference test was carried out by applying the four preparations of pomegranate extract lip balm (f0, f1, f2, f3) to 30 panelists, them the panelists were asked to fill out a questionnaire to find out which lip balm preparation was the most attractive according to the panelists. based on the test result, it was found that those who liked the lip balm preparation for the texture were 63.3% for the 25% formulation, the panelists who liked the lip balm preparation for the aroma were 76.19% for the 25% formulation, while the largest color was 77.4% for the concentration of 25% and overall including texture, aroma and color is 67.7% for a concentration of 25% as well. based on the lip balm preference test with aroma texture and color parameters, the lip balm most preferred by the panelists was lip balm with a concentration of 25%. the 25% concentration is the lip balm concentration with the most pomegranate extract compared to other concentrations. kase et al.– antioxidant and antibacterial activity of pomegranate extract … 115 irritation test result the result of the irritation test is shown in table 7. table 7. irritation test result data on pomegranate lip balm. irritation test f0 (0%) f1 (12.5%) f2 (18.75%) f3 (25%) number of respondent itchy rash 0 0 0 0 0 redness 0 0 0 0 0 bump 0 0 0 0 0 other irritation 0 0 0 0 0 based on the results of the tests carried out, the panelists gave negative results or there were no signs indicating irritation such as the absence of red skin, itching, or the presence of swelling and other allergic reactions. this shows that the ingredients contained in the lip balm from pomegranate extract are not irritating. this is in accordance with research (nazliniwaty et al., 2019) that lip balm from pomegranate extract does not cause irritation. the conclusion that can be drawn is that all the lip balm concentrations tested did not cause irritation. antibacterial test results in this research, antibacterial testing was carried out to determine the level of antibacterial in the sample to be tested against staphylococcus aureus bacteria which is a gram-positive bacterium that grows at an optimum temperature of 37°c, this bacterium that is usually present on the skin, digestive tract, and intestines. human respiratory tract can be found on the lips of humans (kusuma, 2009). this antibacterial test used the pour plate method which was repeated 3 times for each sample tested to determine the inhibition zone formed in the sample tested using staphylococcus aureus bacteria. the result of antibacterial testing can be seen in table 8. table 8. inhibition zone measurement data. sample average diameter of inhibition zone (mm) extract 12.5% 13.33a,b,c extract 18.75% 14.66 b,c extract 25% 22.66 c lipbalm 12.5% 0 a lipbalm 18.75% 5.33 a,b lipbalm 25% 6 a,b control + (ciprofloxacin) 40.94 d control – (aquadest) 0a description: a: mean are included in subset a and have no significant difference b: mean are included in subset b and have no significant difference c: mean are included in subset c and have no significant difference d: mean are included in subset d and have no significant difference the presence of an inhibitory zone in the pomegranate extract was due to the pomegranate extract containing antibacterial compounds (saponins) so that it could inhibit the staphylococcus aureus bacteria. in this research, the three extracts (12.5%, 18.75% and 25%) had an inhibitory zone with a vulnerability of 11-20 mm which means strong, while for positive control it had an inhibition zone of 21 so it was very strong (yunus et al., 2018). the inhibition zones of the pomegranate extract lip balm formed were 18.75% and 25% concentrations, only in the first repetition. the lip balm inhibition zones with concentrations of 18.75% and 25% had a strong inhibitory power with a range of 21 (yunus et al., 2018). one of the constituent materials formed by the inhibition zone is glycerin, where glycerin has a very strong inhibitory zone strength 21 as well as a positive control (yunus et al., 2018). the inhibitory zone data for the antibacterial test in this research were then entered into the anova test using spss. the anova test was used to determine whether or not there was a difference in the sample with an inhibitory zone. the anova results showed a significant difference in the inhibition zone formed in the pomegranate extract and lip balm preparations from the pomegranate extract because 000<0.05 so h0 was rejected. and h1 is accepted. the results of the subset table are then entered into table 8. table 8 column 1 (a) shows that f1, f2, f3 lip balm, and 12.5% extract have similarities and there are no differences. table subset 2 (b) shows that lip balm f2, f3 extract 12.5% and 18.75% there is no difference, different from the table for subset 1 (a) earlier, next is table subset 3 (c) where extract 12.5%, 18.75%, and 25% have no significant difference, in contrast to table subset 2 (b), that table subset 2 has similarities with lip balm f2 and f3. the last table subset 4 (d) is the positive control which has differences with pomegranate extract and lip balm preparations because it has the greatest value among the others. from the results of the antibacterial test in table 8 and the test using spss the largest diameter of the inhibition zone was pomegranate extract with a concentration of 25% and the lowest was lip balm with a concentration of 12.5%. conclusions the ability of pomegranate extract as an antioxidant is 449 ppm, which in pomegranate extract contains 116 biology, medicine, & natural product chemistry 12 (1), 2023: 109-117 antioxidants, but based on ic50 it is very low. the ability of pomegranate extract as an antibacterial in inhibiting the growth of staphylococcus aureus bacteria was found in the three extract concentrations (12.5%, 18.75% and 25%) while in lip balm preparations there were concentrations of 18.75% (f2) and 25% (f3), while the ability of pomegranate extract as a colorant in lip balm preparations can last for 3 weeks at room temperature and the lip balm which produces the brightest color is a concentration of 25% (f3). this concentration is also the most preferred by the panelists. acknowledgements: the authors would like to thank all staff in laboratory biotechnology, faculty of biotechnology, duta wacana christian university, for helping and supporting this research. competing interests: authors state that there is no conflict of interest in this research output. references abdullah, w., & runtuwene, m. r. j. 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(2019). total phenolic content (tpc) determination and phytochemical screening of plecranthus esculentus tubers of rusape, zimbabwe. international journal of advance study and research work, 2(2), 1-6. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 547-553 | doi: 10.14421/biomedich.2023.122.547-553 issn 2540-9328 (online) endophytic fungi isolated from pteridium acquilinium and newbouldia laevis leaves exhibited antioxidant activities and inhibitory potential against selected clinical bacteria isolates obinna ajah1,*, chika cecilia unegbu2,3, emmanuel n uzohuo3, ogechi ozioma anyanwu4, ugochukwu moses okezie5, chioma anyanwu6, rich joseph7 1department of biochemistry, michael okpara university of agriculture, umudike, umuahia nigeria. 2department of chemistry, morgan state university, usa. 3department of chemistry /biochemistry, federal polytechnic nekede owerri, nigeria. 4department of medicinal and pharmaceutical chemistry, nnamdi azikiwe university, awka nigeria. 5department of pharmaceutical microbiology and biotechnology, nnamdi azikiwe university, awka nigeria. 6department of microbiology /biochemistry, federal polytechnic nekede owerri, nigeria. 7department of pharmaceutical technology, federal polytechnic nekede, owerri, nigeria. corresponding author* ajah.obinna@mouau.edu.ng manuscript received: 01 may, 2023. revision accepted: 16 august, 2023. published: 20 september, 2023. abstract endophytic fungi are important components of plant micro-ecosystems and they exist usually inside the healthy tissues of living plants. endophytes possess encouraging source of biologically active metabolites for pharmaceutical applications. this study isolated endophytic fungi from pteridium acquilinium and newbouldia laevis leaves and evaluated their antioxidant and antibacterial activities. a total of ten endophytic fungal species were isolated from the leaves of the plants, from which six were selected and identified, including; epicoccum thailandicum; trichoderma atroviride; lasiodiplodia parva; trichoderma yunnanense; colletotrichum cobbittiense; and, trichoderma crissum. among the isolated fungi, trichoderma crissum (266.2956 ± 84.84 µg/ml) and lasiodiplodia parva (293.0755 ± 64.95 µg/ml) showed significant scavenging activity on comparison with ascorbic acid. furthermore, the isolated endophytes showed potential antibacterial activity against the tested clinical pathogens. this study validates that endophytic fungi dwelling in the inner tissue of medicinal plants studied could be a potential source of biologically active metabolites with free radical scavenging activities and also for treatment of infectious diseases caused by staphylococcus aureus, pseudomonas aeroginosa and escherichia coli. keywords: endophytes; antibacterial activity; antioxidant potential; pteridium acquilinium newbouldia laevis. abbreviations: ptalb: pteridium acquilinium leaf blade, ptamr: pteridium acquilinium midrib, nllb: newbouldia laevis leaf blade, nlmr: newbouldia laevis midrib. introduction the search for novel compounds to alleviate or ameliorate some diseases has been on the increasing sides ever-since health challenge became a global threat to man-kind. recently on-going researches has focused on developing new leads from medicinal plants which are major sources of some secondary metabolites that are of health benefits. scientists equally are harnessing natural flora for new leads to tackle infectious and chronic diseases. endophytic fungi has been recognized to be one of the most distinguished producers of medicinal products that are natural (malhadas et al. 2017). endophytes live in some plants where it co-ordinates it activities having influence on the physiological, growth and chemical characteristics of the plant without causing any harm. endophytic fungi belong to mitosporic and meiosporic ascomycetes that “asymptomatically reside in the internal tissues of plants beneath the epidermal cell layer, where they colonize healthy and living tissue via quiescent infections (jia et al. 2016). nevertheless, the productive result of several medicinal drugs from microbial origin like the antibiotic; penicillin from penicillium sp; immunosuppressant from tolypocladium inflatum and cylindrocarpon lucidum, the antifungal agent griseofulvin from penicillium griseofulvum, the cholesterol biosynthesis inhibitor lovastatin from aspergillus terreus, and ß-lactam antibiotics from https://doi.org/10.14421/biomedich.2023.122.547-553 548 biology, medicine, & natural product chemistry 12 (2), 2023: 547-553 various fungal taxa has broadened our focus to include both plants and microbes and encouraged the focus of drug discovery from not just plants alone but on microorganisms (adeyemi et al. 2015). previous researches has given credit to endophytes for their potential benefits in health management. infectious diseases including urinary tract disease (utis) and wound infection that are caused by multiresistant organisms such as (staphylococcus aureus and escherichia coli) (frickmann et al. 2019) and oxidative stress caused by the presence of free radicals resulting in the imbalance within the body system has been a growing concern to scientists as it affects and has significant impact on the economy (hadadi et al. 2020). antibiotic resistance has arose as a result of indiscriminate use of antibiotics without strict adherence to prescriptions hence creating more opportunities for development of multi-resistant microbes which is now a public health challenge (manganyi et al. 2019). on the other hand, in humans, oxidative stress is thought to be involved in the development of atherosclerosis, neurodegenerative diseases, such as alzheimer’s and parkinson’s disease, cancer, diabetes mellitus, inflammatory diseases, as well as psychological diseases or aging processes (kowalczyk et al. 2021). in view to provide solutions to these problems, researchers have considered obtaining new leads from natural products other than the conventional means and in this study some medicinal plants have been selected. bracken ferns (pteridium aquilinum) is utilized by many different cultures around the globe and has been used over a great period of time. pteridium aquilinum is widely known to have many secondary metabolites with diverse bioactivities that could possibly be beneficial in the management of many diseases. several reports of pharmacological efficacy and the desire for new drugs have encouraged numerous researchers to carry out pharmacological research on ferns. pharmacological and ethnopharmacological studies have shown that substances in fern exhibit various medicinal properties such as hepatoprotective, cytotoxicity, leishmanicidal and trypanocidal activities to mention but few (cao et al. 2017). newbouldia laevis (p. beauv.) seem. (family, bignoniaceae), commonly known as tree of life, is a purple-flowering plant that is widely distributed in many parts of africa. the leaves, flower, stems and roots from newbouldia laevis are prevalently used in african traditional medicine for the management diabetes, hypertension, skin diseases, ulcer, tumors, pains, infectious diseases, inflammation, dysentery, sickle cell disease and impotency (okagu et al. 2021). scientific investigations on newbouldia laevis has lend credence to the ethnobotanical uses of this plant. considering enormous oxidative mediated disease conditions and health challenges associated with pathogenic bacteria, the present study aims at evaluating the antibacterial and antioxidative activity of endophytic fungi isolated from these plants. materials and methods plant collection healthy (showing no visual disease) leaves of medicinal plants; pteridium acquilinium and newbouldia laevis were collected to isolate possible endophytic fungi and test their in vitro antioxidant and antibacterial activity. the plant were deposited at the department of plant science and biotechnology, michael okpara university of agriculture umudike, nigeria, where a voucher specimen with the identification number psb/hb/122 and psb/hb/123 for pteridium acquilinium and newbouldia laevis respectively were assigned. isolation, fermentation and molecular characterization of endophytic fungi the collected plant leaves were subjected to surface sterilization procedures as described in the report of adeyemi et al. (2015). thereafter, the internal tissues of the disinfected samples were exposed using a sterile scissors. a sterile forceps was used to transfer the midrib and leaf-blade segments to potato dextrose agar (pda) amended with chloramphenicol 500 mg/l (adeyemi et al. 2015). the dishes were sealed with parafilm and incubated at 27°c for 6 days. in other to get axenic cultures, multiple sub-culturing were done on fresh pda plates. solid-state fermentation of endophytic fungi was carried out using sterile solid rice (100g of rice + 100ml of distilled water, sterilized by autoclaving at 121℃ at 15 psi for 15mins) in 1l erlenmeyer flasks. the flasks were inoculated with 3 mm diameter agar blocks containing the endophytic fungi and incubated at 28 ℃ for 21 days. after fermentation, the secondary metabolites were extracted using ethyl acetate and then concentrated under vacuum at 50℃ with a rotary evaporator (stuart, usa). axenic cultures were identified using standard molecular protocols as follows: genomic dna was extracted following the protocol described by govindarajan et al. (2007). genomic dna was extracted using the quick-dnatm fungal/bacterial miniprep kit; zymo research), according to recommended protocol by the manufacturer. amplification of the internal transcribed region rdna (its1-5.8s-its2) was achieved using 0.5 μl each of forward and reverse primers. each 25 µl reaction volume contained one taq quick-load 2x master (12.5μl) mix with standard buffer (new england biolabs inc.); 0.5 μl each of forward and reverse primers; 8.5 μl of nuclease free water and 3 μl of dna template. pcr thermocycling was carried under specified amplification conditions which are: initial denaturation for 30 s at 94 ℃, followed by 35 cycles of denaturation at 94 ℃ for 20 s, primer annealing at 54 ℃ for 45 s and strand extension at 72 ℃ for 1 minute. final extension at ajah et al. – endophytic fungi isolated from pteridium acquilinium and newbouldia laevis … 549 72 ℃ for 5 minutes on an eppendorf nexus gradient mastercycler (germany). the pcr products (10 µl) were purified using 2.5 µl of exosap mix, following the protocols of the manufacturer, exonsap kit, (thermofisher scientific, uk). briefly, reconstitution of 0.6 ml of the exo/sap master mix was carried out by adding 50 µl of exonuclease i (catalogue no. neb m0293l); 200 µl of shrimp alkaline phosphatase (catalogue no. neb m0371). amplified pcr product (10 µl) and exosap mix (2.5 µl) were mixed and incubated at 37oc for 15 minutes. the reaction was stopped by heating the mixture at 80°c for 15 minutes. fragments were sequenced using the nimagen, brilliantdye™ terminator cycle sequencing kit v3.1, brd3-100/1000 according to manufacturer’s instructions. the labelled products are then cleaned with the zr-96 dna sequencing clean-up kit (catalogue no. d4053): the cleaned products were injected on the applied biosystems abi 3500xl genetic analyser with a 50cm array, using pop7. sequence chromatogram analysis was then performed using finchtv analysis software geospiza, inc; (benson et al. 2009). 2, 2-diphenyl-1-picryl hydrazyl (dpph) based free radical scavenging activity dpph radical scavenging activity was detected for antioxidant activity by thin layer chromatography (tlc) screening through spotting a concentrated ethanolic solution of the extract on silica gel plates. the plates were developed in ethanol: ethyl acetate (2:1) then airdried and sprayed with 0.2% w/v dpph spray. the presence of yellow spots was detected. radical scavenging activity of extracts was measured according to the dpph spectrophotometric method (habu and ibeh 2015) using vitamin c (emzor pharmaceutical industries, nigeria) as a reference antioxidant. ethanol (1.0 ml) plus extract solution (2.5 ml) was used as blank while 1 ml of 0.3 ml dpph plus ethanol (2.5 ml) was used as a negative control. the free radical scavenging properties of the extracts against dpph radical were measured at 518 nm, as an index of their antioxidant activity. ic50 values (the concentration of extracts required to scavenge 50% of dpph free radicals) were also calculated. the absorbance (abs) of the resulting mixture measured at 518 nm was converted to percentage antioxidant activity (aa %) and thus calculated by the equation: % scavenging activity = 100− [( 𝐴𝐵𝑆𝑠𝑎𝑚𝑝𝑙𝑒−𝐴𝐵𝑆𝑏𝑙𝑎𝑛𝑘 𝐴𝐵𝑆𝑐𝑜𝑛𝑡𝑟𝑜𝑙 )] × 100 antibacterial activity the fungal crude extracts were tested for possible antibacterial activities adopting the agar well diffusion technique as described by adeyemi et al. (2015). three pathogenic bacterial namely escherichia coli and pseudomonas aeroginosagram-negative bacteria) and staphylococcus aureus (gram-positive bacteria) obtained from federal medical centre owerri, imo state, nigeria were used for the assay. each of the test bacteria previously standardized to 0.5 macfarland turbidity standard was applied on the surface of sterile muellerhinton agar using sterile swab sticks. each fungal crude extract (1000 mg) was reconstituted to form our stock concentration of 500 mg/ml and diluted appropriately. a volume of 20 µl of each extract as well as the ciprofloxacin (500 mg) were transferred into the wells made into the agar using 5 mm sterilized cork borer. the plates were incubated at 37°c for 24 h. antibacterial activity was expressed by the production of inhibitory halos around the wells. each extract concentration was tested in triplicate. results dpph scavenging activities of endophytic fungi the result in table 1 represented the dpph scavenging activities of endophytic fungi isolated from pteridium acquilinium leaf. the midrib (mr) and leaf blade (lb) of the leaf were used. the ptamr2, ptamr1a and ptamr1b showed better antioxidative property. the result in table 2 also represented the dpph scavenging activities of endophytic fungi isolated from newbouldia laevis leaf. the midrib (mr) and leaf blade (lb) of the leaf were also used and the nllb2, nlmr3 and nllb3 showed better antioxidative property among other isolates table 1. dpph scavenging activities of endophytic fungi isolated from pteridium acquilinium leaf. con (µg/ml) inhibition (%) ptalb1a inhibition (%) ptalb1 inhibition (%) ptamr1a inhibition (%) ptamr1b inhibition (%) ptamr2 ascorbic acid 7.81 -85.00±20.46 28.88±0.00 11.18±1.47 -22.75±2.14 8.71±1.34 45.94±4.49 15.625 -61.58±34.23 27.88±1.40 -87.65±14.47 -26.78±3.45 11.62±0.87 53.84±3.25 31.25 -192.63±96.76 -11.15±1.42 -2.94±1.15 -12.09±1.37 12.66±2.93 85.42±0.46 62.5 -351.05±174.17 17.33±1.83 3.18±2.49 -13.27±1.04 21.99±2.64 86.18±0.00 125 -178.42±14.15 -184.96±32.26 -67.53±7.99 -2.61±2.67 32.99±1.17 85.75±0.30 250 -192.63±60.28 -30.38±2.12 8.59±3.65 4.39±2.84 50.32±1.02 84.98±0.77 500 -57.37±40.96 48.01±4.65 -41.88±5.65 13.75±2.67 57.88±5.86 85.86±0.45 1000 -111.05±3.72 46.715±5.21 32.71±6.65 81.05±0.67 73.55±14.81 85.97±0.30 the results are ±sd of triplicate determination. 550 biology, medicine, & natural product chemistry 12 (2), 2023: 547-553 table 2. dpph scavenging activities of endophytic fungi isolated from newbouldia laevis leaf. con (µg/ml) inhibition (%) nllb1 inhibition (%) nllb2 inhibition (%) nllb3 inhibition (%) nlmr1 inhibition (%) nlmr3 ascorbic acid 7.81 0.94±1.71 -13.69±1.05 10.08±1.18 -0.59±0.50 -2.805±0.08 45.94±4.49 15.625 -66.58±8.72 -32.54±1.21 -8.01±1.27 -29.08±7.41 -8.675±0.02 53.84±3.25 31.25 -16.44±1.14 8.55±0.56 11.75±1.34 -65.84±4.78 -39.41±5.63 85.42±0.46 62.5 -14.96±1.20 -34.42±1.35 5.19±1.99 -14.31±1.20 -169.39±35.36 86.18±0.00 125 -70.22±1.72 7.67±1.01 17.32±1.95 -97.05±31.60 -69.005±2.34 85.75±0.30 250 4.32±0.81 -22.74±2.51 43.74±2.07 -10.28±1.20 -13.01±1.81 84.98±0.77 500 6.06±2.38 5.655±0.99 77.30±6.08 -52.25±12.03 60.97±1.44 85.86±0.45 1000 14.96±2.09 37.57±2.31 84.35±3.04 43.38±5.85 64.03±2.56 85.97±0.30 the results are ±sd of triplicate determination. half maximal inhibitory concentration (ic50) of all isolates the results in table 3 show that the isolates possess antioxidant activities and the order of decreasing activity include; ascorbic acid (2.628388 ± 1.248694 µg/ml) > nllb3 (266.2956 ± 84.84 µg/ml) > ptamr2 (293.0755 ± 64.95 µg/ml) > ptamr1b (1607.549 ± 694.3751 µg/ml) > nlmr3 7163.991 ± 5306.376 µg/ml) > nllb2 (68201.41 ± 47549.36 µg/ml) > ptamria (23446164 ± 29086173 µg/ml) table 3. summary of ic50 of all isolates. s/no sample 1c50 (mean ± sd) µg/ml 1 ptalb1a 1.38622e+62 ± 1.9604e+62 µg/ml 2 ptalb1 3.69e+11 ± 5.21735e+11 µg/ml 3 nllb1 1.03e+21 ± 1.45519e+21 µg/ml 4 nllb2 68201.41 ± 47549.36 µg/ml 5 nlmr3 7163.991 ± 5306.376 µg/ml 6 nlmr1 6.53e+12 ± 9.23872e+12 µg/ml 7 ptamr2 293.0755 ± 64.95 µg/ml 8 ptamr1a 23446164 ± 29086173 µg/ml 9 ptamr1b 1607.549 ± 694.3751 µg/ml 10 nllb3 266.2956 ± 84.84 µg/ml 11 ascorbic acid 2.628388 ± 1.248694 µg/ml antibacterial activities of crude extract of endophytic fungi agar well of free actively growing pure culture of staphylococcus aureus, pseudomonas aeruginosa and escherichia coli were tested. the zones of inhibition obtained were between 0.4 to 17.60 mm. it was observed that the crude extract of endophytic fungi isolated from pteridium acquilinium and newbouldia laevis including the antibiotic controls, inhibited the human pathogens as shown in table 4. ptamr2 had a high zone of inhibitions of 16.90 mm against s. aureus, nllb3 had a zone of inhibition of 17.60 mm against escherichia coli while nllb2 had a zone of inhibition of 14.40 mm against pseudomonas aeruginosa. table 4. antibacterial activities of crude extract of endophytic fungi isolated from pteridium acquilinium and newbouldia laevis leaves. s/no sample s. aureus zone of inhibition (mm) e. coli zone of inhibition (mm) p. aeroginosa zone of inhibition (mm) 250mg/ml 500mg/ml 250mg/ml 500mg/ml 250mg/ml 500mg/ml 1 ptalb1a 1.00±0.90 1.20±0.56 0.70±0.80 1.90±1.04 0.40±0.10 1.00±0.94 2 ptalb1 0.80±0.08 1.60±0.14 0.40±0.09 0.80±0.40 1.40±0.09 2.00±0.20 3 ptamr1a 8.90±0.95 11.00±0.80 4.50±1.30 10.60±1.70 3.20±0.60 6.60±1.40 4 ptamr1b 11.80±1.08 14.40±2.20 10.80±0.60 12.70±1.05 8.30±0.90 9.20±1.05 5 ptamr2 12.00±0.92 16.90±1.70 10.80±2.50 16.40±2.80 8.80±2.50 10.10±1.50 6 nllb1 1.60±2.08 2.40±1.00 0.90±0.50 1.40±1.08 0.00±0.50 0.40±1.08 7 nllb2 12.60±0.48 14.40±0.90 12.00±0.70 14.40±2.05 10.40±0.70 14.40±2.05 8 nllb3 13.20±3.48 16.00±2.80 13.90±1.90 17.60±2.05 9.50±1.90 11.60±2.05 9 nlmr1 0.30±0.85 0.90±0.08 0.40±0.60 1.20±1.04 1.00±0.30 1.50±1.04 10 nlmr3 12.80±1.40 16.00±0.80 11.70±1.40 15.50±0.95 8.20±1.40 13.50±0.95 11 ciprofloxacin (500mg) 13.40±2.70 17.00±2.30 13.50±1.07 19.00±1.40 11.90±1.00 15.00±1.40 the results are ±sd of triplicate determination. ajah et al. – endophytic fungi isolated from pteridium acquilinium and newbouldia laevis … 551 figure 1. pictorial of endophytic fungi isolated from pteridium acquilinium leaf. figure 2. pictorial of endophytic fungi isolated from newbouldia laevis leaf. discussion the search for novel pharmacological and bioactive compounds from endophytic fungi has generated much interest. this is because of the diverse array of chemodiversity of important novel secondary metabolites that are structurally unique and possess beneficially pharmacological and agricultural significance (okezie et al. 2022; tiwari and bae 2022). in this study, a total of ten (ptalb1a, ptalb1, ptamr2, ptamr1a, ptamr1b and nllb1, nllb2, nlmr3, nlmr1, nllb3) endophytic fungi were isolated from healthy leaves of pteridium acquilinium and newbouldia laevis (fig. 1 and 2). following standard taxanomic identification protocol for dna amplification accompanied by sequencing of the its region, the endophytic fungi with the best results and promising antioxidant and antibacterial agent; nllb2, nlmr3, ptamr2, ptamr1a, ptamr1b, and nllb3 were identified to be epicoccum thailandicum (nr_152926.1); trichoderma atroviride (mh862505.1); lasiodiplodia parva (mh861166.1); trichoderma yunnanense (nr_134419.1); colletotrichum cobbittiense (nr_163538.1); and, trichoderma crissum (nr_134370.1) respectively as confirmed by the 16s rrna sequence after ncbi blast. antibacterial and antioxidant evaluations of the crude extracts of the fungal endophytes revealed varying potentials for development into antibacterial and antioxidant agents. trichoderma crissum (ic50 266.29µg/lm) and lasiodiplodia parva (ic50 293.07µg/lm) extracts showed significant scavenging activity (84.35±3.04 and 73.55±14.81%) when compared to ascorbic acid (85.97±0.30%). a higher dpph radicalscavenging activity is associated with a lower ic50 value (habu jb and ibeh 2015), thus the results presented here indicates a higher dpph radical– scavenging activities by the fungal extracts (trichoderma crissum and lasiodiplodia parva). some scientists have reported the antioxidant activities of different species of trichoderma. the free radical scavenging potential trichoderma asperellum isolated from ascidian eudistoma has been reported by sumilat et al. (2022). kim et al. (2020) reported that t. bissettii and t. guizhouense have dpph radical scavenging activity and tyrosinase inhibition activity respectively. recently, ababutain et al. (2021) detected some secondary metabolites such as: alkaloids, diphenyl ether, monocarboxylic acid, hydroxycinnamic acid, phenalenones, sterols, terpenoids, and xanthones in the crude extract of some endophytic fungi isolated from artemisia sieberi. also, all the fungal extracts exhibited good antibacterial activities when compared to ciprofloxacin (table 4). the activities was observed to be broad spectrum, inhibiting s. aureus, e. coli and p. aeruginosa at the tested concentrations of 250 – 500 mg/ml. at a concentration of 250 mg/ml, the inhibition zone ranged between 0.30 – 13.20 mm, 0.40 – 13.90 mm and 0.00 – 9.50 against s. aureus, e. coli and p. aeruginosa reactively. while, at a maximum concentration of 500 mg/ml, the inhibition zone ranged between 0.90 – 16.90 mm, 0.80 – 17.60 mm and 0.40 – 14.40mm against s. aureus, e. coli and p. aeruginosa respectively. furthermore, we observed e. coli to be the most sensitive organism to the fungal extracts. similarly, okezie et al. (2020) reported a broad-spectrum antibacterial activities against b. subtilis, p. aeruginosa and e. coli when tested at 1 mg/ml. following this research, it's worth noting that, in addition to the microbial and chemical diversity of plants, the endophyte-host plant interaction also gives microbes the ability to produce a variety of novel medicinally useful compounds, because endophytic fungi live in their hosts' inner tissues and inside the plant cell without causing overt symptoms or damage (ababutain et al. 2021; dhayanithy et al. 2019). medicinal plant is known to be a place of residence for endophytes which could be the reason for the pharmacological potentials of 552 biology, medicine, & natural product chemistry 12 (2), 2023: 547-553 these endophytes as they interact with the plant host freely and as well possess some novel secondary metabolites which is quite different from the original compound possessed by the plant (dhayanithy et al. 2019). studies on the antimicrobial and antioxidant potentials of crude extracts of endophytic fungi isolated from newbouldia laevis and pteridium acquilinium have been reported. these includes antimicrobial (amaechi et al. 2020; eze et al. 2019); antioxidant and immunosuppressive (ujam et al. 2021) activities. in public health, antibacterial agents are of great significance, principally in decreasing the growth of infectious bacteria and restraint the transmission of bacterium. these antibacterial compounds can be isolated from endophytic fungi extracts (chi et al. 2019). the varying degrees of antimicrobial and antioxidant potentials exhibited by the various fungal extracts could be as a result of the varying number and concentration of the active compounds present (malhadas et al. 2017). the extracts with high antimicrobial and antioxidant potency could therefore be said to probably contain quite a number of active compounds or contain high concentration of the available active compounds (tiwari and bae 2022; dhayanithy et al. 2019). other endophytic fungal extracts, which showed moderate anti-bacterial and antioxidant activity, may have active compounds, but probably in smaller amounts. therefore, the present study confirmed and has lend credence that endophytic fungi are potential sources for novel bioactive secondary metabolites that can be developed into new antibacterial and antioxidant drugs. conclusions the study provides insight into the diversity of endophytic fungi associated with pteridium acquilinium and newbouldia laevis leaves growing in the coastal regions of south-eastern nigeria as well as the antioxidant and antibacterial potentials of their secondary metabolites. further studies involving the identification and isolation of the bioactive compounds should be carried out which may be used for the development of antibacterial and antioxidant medicines. it is worthy to know endophytic fungi from pteridium acquilinium and newbouldia laevis leaves are promising potential sources for antibacterial and antioxidant agents, which can be developed as medicines for antibacterial and oxidative stress-related diseases. ethical approval and consent to participate: not applicable. consent for publication: not applicable availability of data and material: all data generated or analyzed during this study are included in this published article. competing interests: the authors declare that no competing interests exists with respect to this work. funding: the research was funded by tertiary education trust fund (tetfund) nigeria through the institution base research (ibr) project grant 2019 (tetfund/drss/poly/nekede/2014/rp/vol. 1). contributions: ucc and uen supervised the work and prof read the manuscript, ao carried out the laboratory analysis, statistical analysis, wrote and edited the work, aoo and oum carried out laboratory analysis and prof read the work, ac carried out plant sampling and jr carried out chemical and plant sampling. all authors have read and approved the manuscript. acknowledgements: the authors sincerely acknowledge tertiary education trust fund (tetfund) nigeria for the financial support. we also want to acknowledge the management of federal polytechnic nekede owerri for creating enabling environment for research. study involving plants: the two plants were obtained from the premises of federal polytechnic nekede, owerri, imo state. plants identification and authentication was done by dr g. omosun, a botanist in the department of plant science and biotechnology, michael okpara university of agriculture umudike, nigeria. permissions and/or licenses for the study were not required. references ababutain im, aldosary sk, aljuraifani aa, alghamdi ai, alabdalall ah, al-khaldi em, aldakeel sa, almandil nb, abdulazeez s, borgio jf (2021) identification and antibacterial characterization of endophytic fungi from artemisia sieberi. inter j microb e6651020. https://doi.org/10.1155/2021/6651020 adeyemi a, erute ma, abiodun ts, tolulope ra (2015) 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tallei te (2022) phytochemical, antioxidant, and antimicrobial analysis of trichoderma asperellum isolated from ascidian eudistoma sp. j appl pharm sci 12(04):090–095. tiwari p, bae h (2022) endophytic fungi: key insights, emerging prospects, and challenges in natural product drug discovery. microorganisms, 10(2):360. https://doi.org/10.3390/microorganisms10020360 ujam nt, ajaghaku dl, okoye fbc, esimone co (2021) antioxidant and immunosuppressive activities of extracts of endophytic fungi isolated from psidium guajava and newbouldia laevis. phytomedicine plus. 1(2):100028 https://doi.org/10.1016/j.phyplu.2021.100028 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 147-157 | doi: 10.14421/biomedich.2024.131.147-157 issn 2540-9328 (online) phenolic, flavonoid contents, antioxidant, and antibacterial activity of selected eucalyptus species: review getaneh worku moges*, gizachew mulugeta manahelohe, melese ababay asegie chemistry department, college of natural & computational science, university of gondar, p.o. box 196, gondar, ethiopia. corresponding author* gechchem07@gmail.com abstract many medicinal plants and their essential oils are used to treat or prevent diseases without the risk of mutagenicity, carcinogenicity, or teratogenicity. eucalyptus essential oils are commonly used as preservatives, flavoring agents, and various consumer goods. eucalyptus leaves are being widely studied due to their volatile essential oils, including 1,8-cineole, p-cymene, αand β-pinene, limonene, citronellal, citral, eudesmol, terpinen-4-ol, terpineol, α–phellanderene, and 9β-sitosterol. among these compounds, 1,8-cineole is the most abundant. eucalyptus leaves are rich in flavonoids and phenolic compounds and are thus a great source of antioxidants. 2,2-diphenyl-1picrylhydrazyl (dpph), ferric reducing antioxidant power (frap), and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (abts) assays were used to measure antioxidant capacity. the antioxidant activity of eucalyptus essential oil is stronger than that of standard antibiotics such as erythromycin, cefixime, and gentamicin due to the presence of significant terpenoids in addition to phenolic chemicals and flavonoids. a lower ic50 indicates greater antioxidant potential. phenolic compounds and flavonoids are often associated with beneficial health outcomes, including anti-inflammatory and antioxidant effects, anticancer properties, reduced risk of cardiovascular diseases, prevention of age-related neurodegenerative diseases, and prevention of alzheimer’s disease. flavonoids also play essential roles in plants, including protecting against reduction, fertility, reproduction, and infection. due to their anti-inflammatory, antioxidative, and immunomodulatory properties, flavonoids are crucial for pharmacological, medical, and nutraceutical applications. keywords: eucalyptus; medicinal plants; antioxidant activity; reactive oxygen species (ros); 1,8-cineole. introduction plants and their essential oils are often used as medicines to cure or prevent diseases. medicinal plants are preferred as medication due to their low risk of causing genetic mutagenicity, carcinogenicity, or teratogenicity (olawore no & ololade zs, 2017). these plants are a significant source of therapeutic drug molecules because they contain secondary metabolites that can serve as potential drugs. aromatic plants are commonly used in traditional medicine (sharma et al., 2021). in several countries worldwide, especially in rural areas, aromatic plants are used in primary healthcare (vecchio et al., 2016). eucalyptus is a type of flowering tree belonging to the myrtaceae family, with more than 900 species and subspecies (vecchio et al., 2016). these trees are known for their fast growth and ability to tolerate harsh environments such as wildfires, droughts, and acidic soils. the wood industry uses various species of eucalyptus to produce pulp, timber, and paper. additionally, the bark, leaves, and branches of these trees are utilized as byproducts to generate energy (álvarez et al., 2021). there are several species of eucalyptus, such as e. camaldulensis, e. saligna, e. citriodera, eucalyptus globule, eucalyptus maculate, e. staigeriana, e. radiate, e. laxophleba, e. cinerea, e. tereticornis, and e. leocoxylon (almas et al., 2021; surbhi et al., 2023). one of the most popular species is eucalyptus globulus labill, or blue gum tree, which is an evergreen tree widely grown in australia, south africa, india, southern europe, and ethiopia (amabye, 2016; dezsi et al., 2015; shah et al., 2012). e. globulus is known for its many medical benefits, such as treating wounds, tuberculosis (tb), sore throats, arthritis, asthma, boils, colds, coughs, diabetes, diarrhea, dyspepsia, and bronchitis (mworia et al., 2019). these plants produce secondary metabolites such as terpenoids, alkaloids, flavonoids, phenols, and saponins as a defense mechanism (sharma et al., 2021). eucalyptus trees have been extensively researched due to their essential oils, which have several health benefits, including anti-inflammatory, antimalarial, antibacterial, analgesic, antidiabetic, antiviral, anticancer, antiseptic, antioxidant, and antifungal properties (panigrahi et al., 2021; surbhi et al., 2023). eucalyptus leaves are a great source of antioxidants, particularly manuscript received: 01 april, 2024. revision accepted: 20 may, 2024. published: 27 may, 2024. https://doi.org/10.14421/biomedich.2024.131.147-157 mailto:gechchem07@gmail.com 148 biology, medicine, & natural product chemistry 13 (1), 2024: 147-157 flavonoids, which offer protection against oxidative stress and free radical damage (álvarez et al., 2021). figure 1. chemical structures of the main components of eucalyptus fruits, branch tips, and leaf essential oils. eucalyptus trees are a rich source of essential oils that can be extracted from their fruits, branch tips, and leaves. these oils contain a variety of beneficial phytochemicals, such as 1,8-cineole, p-cymene, αand β-pinene, limonene, citronellal, citral, eudesmol, terpinen-4-ol, terpineol, α–phellanderene, and 9β-sitosterol. different methods, such as hydrodistillation, supercritical fluid extraction, and solvent extraction, can be used to extract these chemicals (surbhi et al., 2023). among these compounds, 1,8-cineole is the most abundant in all eucalyptus species (sebei et al., 2015). to treat illnesses, eucalyptus extracts are used both alone and in conjunction with certain other plants. eucalyptus essential oils are safe and nontoxic. they are used as preservatives, flavoring agents, and in a wide variety of consumer goods (olawore no & ololade zs, 2017). researchers have recently shown increased interest in essential oils due to their therapeutic properties, which make them useful for preservation and drug formulations. they are also used as flavoring substances in the food, nutraceutical, and pharmaceutical industries. eucalyptus essential oils have been used in medicine to treat a wide range of human and animal ailments. they contain phenolic and flavonoid compounds, which have antioxidant and antibacterial properties (ololade et al., 2021). this review aims to provide scientific information on the phenolic and flavonoid contents and the antioxidant and antibacterial activities of selected eucalyptus species. eucalyptus species eucalyptus is a nonnative and popular tree species classified in the myrtaceae family. it is a genus of more than 900 species (vecchio et al., 2016) and has become the most planted genus of tree species in the world (birara dessie et al., 2019). according to the research conducted by alemu (2016), brazil possesses the most extensive expansion of eucalyptus plantations globally, with india and china following suit in terms of coverage. in africa, south africa proudly claimed the initial position in establishing such plantations, securing a notable fifth place in the worldwide ranking. on the other hand, ethiopia stands adjacent to south africa and holds the seventh position on a global scale. eucalyptus covers 90% of the total planted forest area in the central highlands of ethiopia. these species of trees are the first nonnative tree species formally introduced to ethiopia by emperor minilik ii from australia in the 1890s (tesfaye et al., 2020). approximately 70 species of eucalyptus are accessible in ethiopia, most of which are broadly spread in numerous locales of the nation, mostly within the central highlands, where population density is relatively high. the most common and widespread eucalyptus species include e. globulus, e. camaldulensis, e. saligna, e. grandis, e. citriodora, e. regnans, and e. tereticornis (alemu, 2016; tsegaye bekele, 2015). many products and services, including fuel wood, charcoal, building materials, home and agricultural tools, pulp, eucalyptus oil, lumber, and poles, are known to be provided by eucalyptus trees. the tree proved to be highly helpful for the production of paper because of its rapid growth tendency. additionally, the tree has traditional and modern medical uses. for example, in ethiopia, steam from water-boiled e. globulus tree leaves is known to cure the flu. australia, brazil, chile, portugal, south africa, spain, and swaziland are among the nations recognized for their production of eucalyptus oil (alemu, 2016). benefits of eucalyptus species for health the medicinal properties of eucalyptus species have been reported in various studies. olawore no & ololade zs (2017) studied the therapeutic properties of essential oils derived from the seeds of e. camaldulensis var. nancy and e. camaldulensis var. petford, including antioxidant, anti-inflammatory, antinociceptive, and antimicrobial activities. e. citriodora fruit essential oil has natural antioxidant and antimicrobial activity (ololade et al., 2021). the e. globulus labill species is one of the most well known. its leaves have been used as traditional medicine to cure a variety of illnesses, including diabetes, influenza, fungal infections, and pulmonary tuberculosis. leaf extracts of e. globulus have also been used as food additives due to their antioxidant activity (dezsi et al., 2015). o 1,8-cineole p-cymene -pinene -pinene limonene o citronellal o citral eudesmol oh terpinen-4-ol oh terpineol oh –phellanderene ß-sitosterol ho moges et al. – phenolic, flavonoid contents, antioxidant, and … 149 the eucalyptus plant is traditionally used for its antiseptic and antibacterial effects for treating respiratory tract infections such as colds, flu, sore throats, and chest infections such as bronchitis and pneumonia. this effect on bacteria may be attributed to the dominant presence of eucalyptol (1,8-cineole), which has demonstrated strong antimicrobial activity against many pathogens (vecchio et al., 2016). eucalyptus contains phenolic compounds that possess anti-inflammatory, antimicrobial, and antioxidant properties. moreover, it has neuroprotective effects and could be effective in preventing or delaying the onset of alzheimer's disease. due to their diverse bioactivities, eucalyptus phenolic compounds have potential uses in various industries, such as food, cosmetics, and pharmaceuticals (park et al., 2023). traditional use of eucalyptus in ethiopia ethiopia has a wide range of aromatic and medicinal plant species from which essential oils can be produced for a variety of uses. the most prevalent of these is the eucalyptus plant (shiferaw et al., 2019). the reforestation of eucalypt plants is a popular practice in many countries, particularly in developing countries such as ethiopia. ethiopian farmers cultivate many eucalyptus trees on tiny plots of land, and they maintain them well to provide a range of goods, such as small branches and leaves for fuel wood, as well as poles and posts for building houses and other agricultural purposes. eucalyptus trees provide fuel and building materials for a large number of people in ethiopia (zerga et al., 2021). eucalyptus extracts have been used traditionally to cure a wide range of ailments in various nations, including ethiopia. for example, hot water extracts of both fresh and dry e. globulus leaves are used as analgesic, anti-inflammatory, and antipyretic treatments for sinus infections, the common cold, and the flu. this is because e. globulus is a unique natural product with antiseptic properties that also helps to clear bronchial tubes and nasal passages, which makes breathing easier. additionally, they inhale the vapor released when boiling eucalyptus leaves in water to relieve cold, which is particularly common. the essential oils of eucalyptus species have significant biological activities, including diaphoresis, disinfecting, antimalarial, antiseptic, analgesic, anti-inflammatory, antibacterial, and antioxidant properties. eucalyptus oil is one of the main ingredients used to make laundry detergents and toiletries with good deodorizing and antiseptic properties (shiferaw et al., 2019). e. globulus is used to treat respiratory tract infections; any illness in the respiratory tract that may affect the lungs, bronchi, or nasal passages is referred to as a respiratory tract infection. because antimicrobial resistance is developing quickly and the effectiveness of currently available drugs is decreasing, these illnesses are becoming major causes of death and morbidity (teka & maryo, 2023). phenolic contents a common structural feature of phenolic compounds is an aromatic ring with one or more hydroxyl substituents. these compounds can be classified into many classes, with flavonoids, phenolic acids, tannins, stilbenes, and lignans being the main groups (álvarez et al., 2021; zhang et al., 2022). various phenolic chemicals (figure 2), including quercetin, rutin, ellagic acid, hydroquinone, protocatechuic acid, naringenin, chlorogenic acid, hesperetin, pyrogallol, resorcinol, and catechin, have been extracted from different eucalyptus extracts (gullón et al., 2020). figure 2. different phenolic compounds isolated from different eucalyptus extracts. ellagic acid o o oh oh ho ho o o o o o oh oh ho oh o o o ho oh ho oh ch3 oh ho rutin oh oh hydroquinonehesperitin oho oh oh och3 oh ohho pyrogallol resorcinol oh oh o oh ho ho protocatechuic acid o o oh oh oh ho co2h ho chlorogenic acid 150 biology, medicine, & natural product chemistry 13 (1), 2024: 147-157 phenolic compounds are a large class of plant secondary metabolites that exist in various plant organs, such as vegetables, fruits, spices, grains, legumes, and nuts, and play important roles in diverse physiological processes, such as plant quality, coloring, flavor, and stress resistance (zhang et al., 2022). because of their beneficial effects on human health, polyphenols are very valuable phytochemicals in the food and pharmaceutical industries (ololade et al., 2021). plants with high phenolic content are highly nutritious and beneficial to human health. in addition, they display defense mechanisms against reactive oxygen species (ros). natural phenolic compounds play a critical role in both illness prevention and treatment. phenolic compounds have chemopreventive effects because of a range of therapeutic activities that also regulate carcinogen metabolism and ontogenesis, differentiation, the inhibition of dna binding and cell adhesion, migration, proliferation, and the blockade of signaling pathways (olawore no & ololade zs, 2017). phenolic compounds are frequently linked to a number of advantageous health outcomes, including antiinflammatory and antioxidant effects, anticancer effects, and decreased risk of cardiovascular diseases (dezsi et al., 2015). phenolic chemicals exert protective effects against oxidative stress and inflammation caused by airborne particulate matter and play a significant role in protecting plants from ultraviolet radiation and disease attacks (rahman et al., 2022). olawore no & ololade zs (2017) reported that the total phenolic content (tpc) values of e. camaldulensis var. nancy and e. camaldulensis var. petford essential oils were 156.25 ± 0.00 and 167.93 ± 0.00 µg gae/mg, respectively, which were relatively the same as those of e. citriodera essential oil extracts (175.84±0.00 µg gae/mg) reported by ololade et al. (2021). as shown in table 1, the total phenolic content of e. saligna leaves extracted with pressurized 95% ethanol was greater than that of the other eucalyptus species, which was 618.57 mg gae/g plant material. fischer et al. (2020) reported that the total phenolic content of an 80% methanol extract of e. globulus was lower than that of other eucalyptus species (50.00 mg gae/g plant material) (table 1). table 1. tpc of different eucalyptus species extracted by different solvents and methods. eucalyptus species part of plant condition phenolic content (mg gae/g plant material) reference e. globulus labill. leaves 70% ethanol 235.87 ± 4.38 (dezsi et al., 2015) e. camaldulensis var. nancy seed essential oil 156.25 ± 0.00 (olawore no & ololade zs, 2017) e. camaldulensis var. petford seed essential oil 167.93 ± 0.00 e. globulus leaves 80% methanol 50.00 ± 0.00 (sharma et al., 2021) e. citriodera fruit essential oil 175.84±0.00 (ololade et al., 2021) e. saligna leaves pressurized 95% ethanol 618.57± 0.00 (fischer et al., 2020) as shown in table 2, park et al. (2023) reported that the highest tpc values were reported for different concentrations of ethanol in e. globulus leaf extracts, with 422.0, 492.7, 497.7, and 448.5 mg gae/g extract in 10, 30, 50, and 70% ethanol extracts, respectively. the next highest tpc was 384.5 mg gae/g extract in the 90% ethanol extract. however, the tpc of the 100% ethanol extract was 273.2 mg gae/g extract. the lowest tpc value, 126.7 ± 8.5 mg gae/g extract, was estimated for the 0% ethanol extract. according to the extraction conditions, the e. globulus leaf 50% ethanol extract had the highest tpc relative to the other extracts, as shown in table 2. table 2. tpc of different ethanol concentrations of e. globulus leaf extract. extract conditions 0% ethanol 10% ethanol 30% ethanol 50% ethanol 70% ethanol 90% ethanol 100% ethanol tpc (mg gae/g extract) 126.7 ± 8.5 422.0 ± 18.4 492.7 ± 13.2 497.7 ± 15.5 448.5 ± 20.1 384.5 ± 10.4 273.2 ± 17.5 flavonoid contents flavonoids are a class of secondary metabolites. their main structural element is a benzopyrone ring that has variously positioned phenolic or polyphenolic groups. based on the degree of unsaturation and oxidation of the c ring as well as the carbon of the c ring to which the b ring is attached, flavonoids are divided into subgroups, as shown in figure 3. flavonoids in which the b ring is linked to position 3 of the c ring are called isoflavones. those in which the b ring is linked at position 4 are called neoflavonoids, whereas those in which the b ring is linked at position 2 can be further separated into multiple subgroups according to the structural characteristics of the c ring. these subgroups include anthocyanins, chalcones, flavones, flavonols, flavanones, moges et al. – phenolic, flavonoid contents, antioxidant, and … 151 flavanonols, flavanols and catechins (álvarez et al., 2021; panche et al., 2016). quercetin, kaempferol, isorhamnetin, luteolin, phloretin, and catechins are the primary flavonoids found in eucalyptus (álvarez et al., 2021). figure 3. basic primary structural components of flavonoids and their classes. flavonoids are found in many plants, fruits, vegetables, and leaves. these compounds have potential biological and medicinal uses. it offers several health benefits, including the potential to prevent age-related neurodegenerative diseases and alzheimer’s disease. they also possess antioxidant, antiviral, and anticancer properties (ullah et al., 2020; zulkefli et al., 2023). flavonoids play many important roles in plants, including protecting against reduction, fertility and reproduction and protecting against infection and the rhizosphere. flavonoids function as signaling molecules, detoxifying agents, and phytoalexins and promote seed germination, temperature acclimatization, and drought resistance. flavonoids are also used to reduce reactive oxygen species in plant tissue, and they play other roles in the fragrance, color, and taste of fruits, flowers, or seeds. this fragrance and color attract pollinators that aid in the pollination and dispersal of seeds (roy et al., 2022). flavonoids in humans and plants are relatively important as signaling molecules and reducing agents (brunetti et al., 2013). flavonoids facilitate the colonization of tomato roots and the germination of rhizophagus irregularis spores (lidoy et al., 2023). flavonoids have shown the ability to control or prevent inflammation. due to their anti-inflammatory, antioxidative, and immunomodulatory qualities, flavonoids are essential for pharmacological, medical, and nutraceutical applications (rakha et al., 2022). the number and position of free oh groups on the flavonoid skeleton appear to determine the antioxidant potential of naturally occurring polyphenolic compounds. the b-ring substitution pattern plays a key role in the ability of flavonols to scavenge free radicals. flavonoids with multiple hydroxyl groups are more potent antioxidants than those with only one hydroxyl group (brewer, 2011). as shown in table 3, the flavonoid content of the e. saligna leaves extracted with pressurized 95% ethanol was 160.27 mg qe/g of extract. sharma et al. (2021) reported that the flavonoid content of an 80% methanol extract of e. globulus leaves was lower, at 23 mg re/g plant material. o a b c1 2 3 45 6 7 8 2' 3' 4' 5' 6' basic skeleton o oh oh oh ho o quercetine o oh oh oh ho o kaempferol o oh oh ho ooh luteolin o oh ho ooh och3 oh isorhamnetin flavonols flavones phloretin oh ho oh o oh oh ho oh o oh chalconaringenin chalcones o oh oh oh oh oh ho o oh oh oh oh ho cyanidine delphinidin anthocyanins o o o o oh ho oh ho oh genistein daidzein isoflavones o o o o ho oh ho oh oh och3 oh naringenin hesperetin flavanones flavanole catechin o oh ho oh oh oh 152 biology, medicine, & natural product chemistry 13 (1), 2024: 147-157 table 3. flavonoid content of different eucalyptus species extracted by different solvents and methods. eucalyptus species part of plant condition flavonoid content reference e. globulus leaf 80% methanol 23 ± 0.00 a (sharma et al., 2021) e. globulus labill. leaves 70% ethanol extract 35.76 ± 0.95 a (dezsi et al., 2015) e. saligna leaves pressurized 95% ethanol 160.27 b (fischer et al., 2020) mg re/g plant material a, mg qe/g extract b, re= rutin equivalent, qe= quercetin equivalent. according to park et al. (2023) (table 4), the total flavonoid content (tfc) of e. globulus leaf extracts increased as the concentration of ethanol increased from 0% to 100%. under 0 and 50% ethanol extraction conditions, the tfc values ranged from 32.5 to 41.2 mg qe/g extract, with similar values. the 70% ethanol extract had a value of 66.5 mg qe/g extract, which is approximately 1.5-fold greater than that of the 0, 10, 30, and 50% ethanol extracts. the highest tfc values were 156.5 and 169.3 mg qe/g extract in 90% and 100% ethanol, respectively. the best extraction conditions for tfc were 100% ethanol. table 4. tfc of different ethanol concentrations of the e. globulus leaf extract. extract conditions 0% ethanol 10% ethanol 30% ethanol 50% ethanol 70% ethanol 90% ethanol 100% ethanol tfc (mg qe/g extract 32.5 ± 5.1 32.7 ± 4.8 40.4 ± 6.7 41.2 ± 7.5 66.5 ± 6.5 156.5 ± 10.4 169.3 ± 12.2 antioxidant activities antioxidants are molecules that can scavenge ros or free radicals, protecting cells from damage and death. in biological systems, free radicals are crucial for the synthesis of some biomolecules, energy production, phagocytosis, and cell development. an imbalance between free radical generation and unfavorable antioxidant defenses leads to oxidative stress, resulting in dna or tissue damage (vecchio et al., 2016). the 2,2-diphenyl-1-picrylhydrazyl (dpph), ferric reducing antioxidant power (frap) and 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (abts) assays are commonly used to measure antioxidant capacity. the main differences between these reactions are their reaction mechanisms and types of radicals (park et al., 2023). olawore no & ololade zs (2017) reported that the two seed essential oils of e. camaldulensis vars (table 5), nancy and petersford, had half maximal inhibitory concentration (ic50) values of 3.50 μg/ml, indicating that these compounds are more effective as antioxidants and free radical scavengers than ascorbic acid, which had an ic50 value of 9.00 μg/ml. the essential oils from the seeds of the two eucalyptuses under investigation showed a twofold greater reduction in antioxidant activity (5.0 μg/ml) than ascorbic acid (11.0 μg/ml). this is because, unlike ascorbic acid, which contains only one molecule, seed essential oils include several significant terpinoids in addition to phenolic compounds. according to ololade et al. (2021), the antioxidant ic50 value of the fruit essential oil e. citriodera is 2.00 μg/ml, and ascorbic acid has an ic50 value of 11.00 μg/ml. this shows that the antioxidant activity of the essential oil is more potent than that of ascorbic acid. the lower the ic50 value is, the greater the antioxidant potential (ololade et al., 2021). the presence of terpenoids in the essential oil of eucalyptus species plays an active role in the antioxidant potential and its greater reduction antioxidant effect (olawore no & ololade zs, 2017; ololade et al., 2021). the presence of terpenoids in the oil contributed to its greater antioxidant effect since these compounds are known to chelate metal ions (ololade et al., 2021). table 5. antioxidant activity of different eucalyptus species. eucalyptus species condition ic50 value (μg/ml) reference dpph assay frap e. camaldulensis var. nancy seed essential oil 3.5 5.0 (olawore no & ololade zs , 2017) ascorbic acid 9.0 11 e. camaldulensis var. petford seed essential oil 3.5 5.0 ascorbic acid 9.0 11 e.citriodera fruit essential oil 3.00 2.00 (ololade et al., 2021) ascorbic acid 9.00 11.00 moges et al. – phenolic, flavonoid contents, antioxidant, and … 153 park et al. (2023) reported the antioxidant ability of e. globulus leaves using dpph and abts radical assays. as shown in table 6, the highest radical scavenging activity against dpph radicals was observed for 30% ethanol, with a 188.2 µg/ml scavenging capacity (sc50), followed by 10%, 50%, and 70% ethanol, with sc50 values of 357.9, 505.3, and 509.3 µg/ml, respectively. among the extraction conditions, 0% ethanol, 90% ethanol, and 100% ethanol had sc50 values of 5841.7, 1008.4, and 1304.7 µg/ml, respectively, indicating little antioxidant ability. however, compared to the dpph assay, the abts radical scavenging activity assay often produced better results. the results indicated that the e. globulus leaf extracts with 30% and 50% ethanol had the greatest antioxidant effects, with sc50 values of 14.2 and 18.0 µg/ml, respectively. the results showed that the 100% and 0% ethanol extracts had the lowest antioxidant effectiveness, with corresponding sc50 values of 34.9 and 171.3 µg/ml, respectively. these findings verified that extracting e. globulus leaves with the right ratio of water to ethanol produced a more potent antioxidant effect than extracting the leaves with either water or ethanol alone. the findings showed that to optimize the antioxidant potential, a mixture of extraction solvents was necessary. table 6. dpph and abts radical scavenging activities of different ethanol solutions from e. globulus leaf extracts. extract conditions radical scavenging activity (sc50, µg/ml) dpph abts 0% ethanol 5841.7 ± 238.4 171.3 ± 10.2 10% ethanol 357.9 ± 28.0 23.1 ± 2.1 30% ethanol 188.2 ± 24.2 14.2 ± 0.9 50% ethanol 505.3 ± 56.2 18.0 ± 1.5 70% ethanol 509.3 ± 57.1 20.8 ± 1.9 90% ethanol 1008.4 ± 121.2 24.8 ± 1.4 100% ethanol 1304.7 ± 156.5 34.9 ± 5.1 antioxidant mode and mechanism of action the term "oxidative stress" describes the imbalance between antioxidants and oxidants in the body as a result of excess reactive sulfur species (rsss), reactive nitrogen species (rnas), or reactive oxygen species (ros) that can damage cells (azat aziz et al., 2019). under various pathophysiological circumstances, oxidative stress is caused by ros and rns. oxidative stress conditions modify the cellular components of the body, leading to different disease states. antioxidants are useful tools for strengthening cellular defenses against oxidative stress. certain compounds act as in vivo antioxidants by increasing the levels of endogenous antioxidant defenses. the expression of genes encoding enzymes such as catalase (cat), superoxide dismutase (sod), and glutathione peroxidase (gshpx) increases the level of endogenous antioxidants (nimse & pal, 2015). all highly reactive forms of oxygen, including free radicals, are collectively referred to as ros. the ros categories included hypochlorous acid (hocl), hypochlorite radical (ocl•), peroxynitrite (onoo), hydrogen peroxide (h2o2), per hydroxyl radical (ho2 •), hydroxyl radical (oh•), superoxide anion radical (o2 •¯), singlet oxygen (1o2), nitric oxide radical (no•), and different lipid peroxides. rss are easily produced from thiols through a reaction with ros, while rns are derived from nitric oxide by a reaction with o2 •¯ to form onoo (azat aziz et al., 2019). antioxidants are substances or systems that slow autoxidation by preventing the production of free radicals or by stopping the spread of free radicals through one or more different mechanisms (brewer, 2011). antioxidative chemicals have multiple chemical modes of action, including single electron transfer, hydrogen atom transfer, and transition metal chelation (francenia santos-sánchez et al., 2019). figure 4. reaction mechanism of dpph with an antioxidant. dpph is a stable radical by nature compared with the abts•+ radical; it is a radical that should be generated by chemical reactions (equation 1). generally, the improved method generates the abts•+ radical in only one reaction by reacting abts with ammonium persulfate ((nh4)2s2o3) or potassium persulfate (k2s2o3) prior to the addition of antioxidants (figure 5) (sadeer et al., 2020). n o2n no2 o2n n + nh o2n no2 o2n n +r h r dpph dpph-h r h antioxidants= 154 biology, medicine, & natural product chemistry 13 (1), 2024: 147-157 figure 5. mechanism of the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (abts) reaction. the frap test is a typical set-based method for measuring the reduction of a complex of ferric ions (fe3+)-ligand to an intensely blue ferrous complex (fe2+) by means of antioxidants in acidic environments (figure 6). the original frap test employed tripyridyltriazine (tptz) as the connecting ligand to the iron ion. alternative ligands, such as ferrozine, were also used to bind iron ions to evaluate the reducing power of ascorbic acid. potassium ferricyanide has recently emerged as the most commonly utilized ferric reagent in frap experiments (munteanu & apetrei, 2021). figure 6. the mechanism of ferric reducing antioxidant power (frap) reaction. vitamin c (ascorbic acid) is a water-soluble free radical scavenger. vitamin c changes to the ascorbate radical by donating an electron to the lipid radical to terminate the lipid peroxidation chain reaction (figure 7). one ascorbate molecule and one dehydroascorbate molecule are produced by the quick reaction of the pairs of ascorbate radicals. there is no antioxidant capacity for dehydroascorbate. thus, the addition of two electrons transforms dehydroascorbate back into ascorbate. it has been suggested that oxidoreductase completes the final step of adding two electrons to dehydroascorbate (nimse & pal, 2015). s n n n n s so3h ho3s pale blue abts + (nh4)2s2o3 s n n n n s so3h ho3s blu-green abts+ s n n n n s so3h ho3s pale blue re-generated abts aroharo + h+ n n n nn n n n n n fe3+ n n aroh [aroh]+ n n n nn n n n n n fe2+ n n fe3+-tptz fe2+-tptz coulorless intens blue moges et al. – phenolic, flavonoid contents, antioxidant, and … 155 figure 7. mechanism of the radical scavenging activity of vitamin c. antibacterial activities medicinal plants are important sources of potentially useful structures for the development of novel chemotherapy drugs. evaluating their antibacterial activity in vitro is the initial step toward achieving this goal (dezsi et al., 2015). the antibacterial activities of the two seed essential oils of the e. camaldulensis varieties against the tested bacteria were variable (figure 8). e. camaldulensis var. nancy had the greatest inhibitory effect on p. aeruginosa (20 mm), s. aureus (20 mm), k. pneumoniae (19 mm), and e. coli (18 mm) but was resistant to s. agalactiae and s. typhimurium, while e. camaldulensis var. petford had inhibitory effects on k. pneumoniae (24 mm), s. aureus (20 mm), s. agalactiae (15 mm), p. aeruginosa (13 mm) and e. coli (10 mm) but was resistant to s. typhimurium. the tested bacteria s. agalactiae and s. aureus were found to be resistant to erythromycin, and e. coli, s. agalactiae, s. aureus, and s. typhimurium were found to be resistant to cefixime antibiotics (olawore no & ololade zs, 2017). figure 8. zones of inhibition (mm) showing the antimicrobial properties of the seed essential oils of the two varieties of e. camaldulensis. as shown in figure 9, the antibacterial activity of the e. globulus leaf extracts against l. monocytogenes (10.1 ± 0.4 mm) and s. aureus (8.1 ± 0.1 mm) was greater than that of the standard antibiotics gentamicin and ciprofloxacin against the bacterial strains of l. monocytogenes and s. aureus (dezsi et al., 2015). figure 9. effects of zones of inhibition (mm) on the antibacterial activity of e. globulus extracts and antibiotics against bacterial species, as determined by a disc diffusion assay. the antibacterial activities of the essential oil of e. citriodora were determined against five bacteria, namely, e. coli, k. pneumoniae, p. aeruginosa, s. agalactiae, and s. aureus, which were found to have the greatest inhibitory effects on e. coli (18 mm), p. aeruginosa (15.00 mm), s. aureus (15.00 mm), s. agalactiae (10.00 mm) and k. pneumoniae (13.00 mm). the bacteria were found to be sensitive to ofloxacin, and some were resistant to conventional cefuroxine antibiotics, as shown in figure 10 (ololade et al., 2021). o ho oh ho oh o ascorbic acid -h+ +h+ -eo ho oh o oh o ascorbate radical o ho oh -o oh o ascorbate o ho oh -o oo asch2 asch2( asch ) o ho oh o oo asco ho oh o o o -h++h+ -h++h+ -e-edehydroascorbate ( asch-) 156 biology, medicine, & natural product chemistry 13 (1), 2024: 147-157 figure 10. zones of inhibition (mm) on antibacterial activity of the essential oil of e. citriodora and standard antibiotic discs against bacterial isolates. conclusion medicinal plants are used to cure a variety of diseases and ailments in both humans and animals. these plants are an important source of therapeutic drug molecules that contain secondary metabolites, which are potential sources of drugs. the major bioactive components in medicinal plants are secondary metabolites such as alkaloids, flavonoids, phenols, and saponins, which have several broad biological activities. these include antibacterial, antiseptic, antioxidant, and antifungal properties. eucalyptus plants have several medical benefits, such as antioxidant, anti-inflammatory, antinociceptive, and antimicrobial activities. they also have the potential to prevent age-related neurodegenerative and alzheimer's disease. additionally, these plants possess antiviral and anticancer properties due to the presence of polyphenols and flavonoids. research on aromatic and medicinal plants has a bright future due to the application of advanced technologies, interdisciplinary methods, and sustainable practices. this may result in the creation of novel and more effective plant-based medications, as well as new therapies. acknowledgments: i would like to express my gratitude to the university of gondar, college of natural and computational science, department of chemistry, for giving me the opportunity to pursue my postgraduate study. i would also like to thank my advisor, gizachew mulugeta (ph.d.), for his valuable suggestions, constructive advice, and guidance in preparing my review. furthermore, i would like to express my appreciation to tilahun belayineh (ph.d.) for his feedback on my review paper. author contributions: g. w. m conceived the idea and prepared the original draft of the manuscript. g.w.m., g.m.m., and m.a.a. proofread, reviewed and edited the manuscript. conflict of interest: the authors declare that they have no conflicts of interest. references alemu, m. m. 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(2023). flavonoids as potential wound-healing molecules: emphasis on pathways perspective. in international journal of molecular sciences (vol. 24, issue 5, pp. 1–29). https://doi.org/10.3390/ijms24054607 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1339-1346 | doi: 10.14421/biomedich.2025.142.1339-1346 issn 2540-9328 (online) identification of lactic acid bacteria (lab) consortium from tempeh jember based on 16s rrna gene sequences as potential probiotic candidates siti nur azizah1*, rosida1, rizka yolanda febiaocti1, dewi riskha nurmalasari1, sipriyadi2 1pharmacy vocational program, jember polytechnique of medicine, jl. pangandaran no 42 antirogo jember, indonesia 2department biology, faculty of mathematics and natural sciences, bengkulu university, jl wr supratman, kandang limun 38371, indonesia. corresponding author* azizah@poltekesjember.ac.id manuscript received: 29 september, 2025. revision accepted: 27 november, 2025. published: 14 december, 2025. abstract the development of pharmaceutical raw materials as local indonesian probiotic concentrates requires attention to strain identity accuracy, safety, and health potential. this is due to the high potential of probiotics in preventing and addressing various health disorders from infancy to old age. previous research successfully obtained lactic acid bacteria (lab) isolates as probiotic candidates from tempeh produced in jember, and preclinical testing showed that administering a consortium of five lab isolates at a dose of 10⁸ cfu/ml to balb/c mice was an effective treatment for further application. this study aimed to perform molecular identification of the consortium of five lab isolates from tempeh in jember, east java, based on 16s rrna gene sequences. the consortium includes ta1, tb1, tk1, tk2, and tk4 isolates. this study used both qualitative descriptive and quantitative methods. identification based on the 16s rrna gene to determine lab species strains involved bacterial genomic dna isolation, amplification of the bacterial 16s rrna gene, pcr product analysis, and phylogenetic identification and analysis. dna amplification results showed that the consortium of five lab isolates produced dna fragments with bands of approximately 1300 bp. blast-n and phylogenetic analysis of the 16s rrna gene showed that tb1 had 99.05% similarity with kosakonia cowanii jcm 10956, ta1 had 99.46% similarity with lactiplantibacillus plantarum jcm 1149, tk1 had 99.38% similarity with lactiplantibacillus pentosus 124-2, tk2 had 96.86% similarity with pseudomonas fluvialis ass1, and tk4 had 99.38% similarity with lactiplantibacillus plantarum nbrc 15891. ta1, tb1, and tk4 were identified as nonpathogenic lab strains, while tk1 and tk2 were not classified as lab. keywords: consortium; lactic acid bacteria; probiotics; tempeh; 16s rrna identification. introduction probiotics are beneficial living microorganisms for the host’s health, when provided in adeaquate amount. probiotics are commonly composed of lactobacillus and bifidobacterium, known as lactic acid bacteria (lab). moreover, most lab, like lactobacillaceae, pediococcus, several streptococcus, weissella, and enterococcus are safe for human as food-preserving agents (martínez-álvarez et al. 2017). lab can be classified as probiotics, when they support human health, resist stomach acid and bile salt, adhere to mucose intestinal surface, and possess antimicrobial properties against pathogens (ratna el al. 2021). the probiotics’ potential are widely identified in human health. probiotics reduce inflammation, induce mineral absorption, repair urogenital damage, attack colon cancer (ma et al., 2023), remove pathogenic bacteria and increase good bacteria in colon (kamil et al. 2022), decrease antibiotics associated diarrhea risk (goodman et al. 2021), and cholesterol (elisa and lestari. 2021). probiotic consortia have been shown to enhance memory power in alzheimer’s mice (bonfili et al. 2020), improve cognitive performance during acute stress in healthy women (bloemendaal et al. 2021), increase sperm quality (sanchez-rodriguez et al. 2024), and reduce the incidence of constipation, diarrhea, and acute respiratory infections in children (mai et al. 2021). pramudito et al (2024) reported that lab are present in tempeh, due to producing bioactives that reduce diarrhea by inhibiting the adhesion of e. coli etec to mammalian epithelial cells. therefore, probiotics derived from jember tempeh can be explored further for potential contribution to support human health in indonesia. previously, azizah et al (2021) successfully isolated lab from jember tempeh as probiotic candidates and firstly demonstrated that lab from jember tempeh, east java, indonesia, can inhibit diarrhea-associated bacteria from both gram-negative and gram-positive groups, with strong inhibition activities. additionally, lab exhibit resilience to low ph and bile salts. furthermore, https://doi.org/10.14421/biomedich.2025.142.1339-1346 mailto:azizah@poltekesjember.ac.id 1340 biology, medicine, & natural product chemistry 14 (2), 2025: 1339-1346 the lab from jember tempeh surpasses the capability of commercial l. casei strains. according to azizah et al (2023), administering lactic acid bacteria (lab) from jember tempeh consortium at 10⁸ cfu/ml could significantly affect small intestine histology in balb/c mice. administering single lab strains and control group (l. acidophilus) at the same dose resulted in partial necrosis of intestinal tissue. in contrast, the lab consortium and normal group showed no signs of intestinal necrosis. the lab consortium was also more effective in reducing populations of salmonella sp. and e. coli in feces. these findings highlight the importance of further probiotic potential investigation of lab derived from jember tempeh, due to meeting key criteria for probiotic candidates. however, the molecular identity of the five lab strains, encoded as ta.1, tb.1, tki, tk2, and tk4, remains unknown. as probiotic effects are strain-specific, t.s. kemgang (2014) reported that potential probiotic strains must be accurately identified, like using 16s rrna methods. the 16s rrna sequencing analysis offers higher accuracy, faster processing, more superior sensitivity and specificity, than biochemical methods (adesulu-dahunsi et al. 2017). for example, muryany (2017) stated that biochemical identification kits classified l8 and s1 bacterial isolates as b. megaterium and pediococcus pentosaceus, respectively. however, molecular techniques revealed that l8 was l. plantarum and s1 was l. pentosus. the ribosomal rna (rrna) gene is one of the most conserved genes, thus reliable for determining bacterial taxonomy, phylogeny, and species divergence estimation. therefore, this study aimed to identify five lactic acid bacteria consortia derived from jember tempeh, east java using 16s rrna molecular identification. materials and methods procedures lab strains and growth conditions five isolates of lactic acid bacteria (lab) consortium were obtained from the laboratory of microbiology, politeknik kesehatan, jember. these isolates were previously isolated from tempeh (a traditional fermented soybean product), originated from jember, east java (azizah et al. 2023). five lab isolates were rejuvenated on solid gyp medium (1% glucose, 1% yeast extract, 0.5% peptone, 0.2% beef extract, 0.01% sodium acetate, 0.04% mgso4·7h2o, 0.0002% mnso4·4h2o, 0.002% feso4·7h2o, 0.002% nacl, 1% tween 80, 5% caco3, and 1.2% bacto agar) using the quadrant streak method. after 48-h incubation at 37 °c, single colonies of each lab isolate were identified by the formation of a clear zone around the colonies (azizah et al. 2021; azizah et al. 2023). these single colonies were subsequently subjected to molecular identification. genomic dna isolation of bacterial isolates a 1.5 ml bacterial culture was transferred into a 1.5 ml eppendorf tube and centrifuged at 8,000 rpm for 10 min. the pellet was washed three times with ste buffer (containing 0,3m sucrose, 25 mm tris-hcl, and 25 mm edta 2na, ph 8) to remove impurities and centrifuged at 8,000 rpm for 10 min. this washing step was repeated three times. the resulting pellet was resuspended in 200 µl ste buffer and supplemented with 45 µl lysozyme (20 mg/ml), gently mixed, and incubated at 55 °c for 1 h to form protoplasts. subsequently, 20 µl proteinase k was added to degrade proteins, followed by incubation with 400 µl of 10% ctab in 0.7 m nacl to remove polysaccharides and other contaminants. the mixture was then extracted with an equal volume of phenol:chloroform (25:24) and centrifuged at 12,000 rpm for 10 min. the aqueous phase was transferred to a new tube, mixed with 0.6 volumes of isopropanol and 20 µl sodium acetate, and incubated overnight at (−20) °c. the sample was then centrifuged at 12,000 rpm for 10 min. the supernatant was discarded, and the pellet was washed with 1 ml of 70% ethanol. dna pellets were air-dried for 1 h to remove residual ethanol and dissolved in 50 µl sterile ddh2o. the extracted dna was stored at 4 °c or −20 °c for further use (sambrook and russell. 2001). amplification of 16s rrna gene from bacterial isolates the 16s rrna gene was amplified from genomic dna using polymerase chain reaction (pcr) with universal prokaryotic primers, specifically the forward primer 63f (5′-cag gcc taa cac atg caa gtc-3′) and the reverse primer 1387r (5′-ggg cgg wgt gta caa ggc-3′), as described by marchesi et al (1998).the pcr reaction mixture consisted of gotaq green master mix (2×), 1.5 µl of each primer (10 pmol), 18.0 µl nucleasefree water (nfw), and 4.0 µl dna template. the pcr cycling conditions included initial denaturation at 94°c for 4 minutes, 30 cycles of denaturation (94°c, 45seconds), annealing (55°c, 1minute), and extension (72°c, 1minute 10seconds), with a final extension at 72°c for 7 minutes. pcr product analysis pcr products were separated using 1% agarose gel electrophoresis in a mini-gel system at 75 v for 45 minutes. dna bands were visualized under a uv transilluminator after staining with ethidium bromide (etbr). identification and phylogenetic analysis of lab consortium isolates the raw sequencing data were trimmed and assembled using chromaspro version 1.5. the assembled sequences were compared with reference sequences in the ncbi database using the blast program azizah et al. – identification of lactic acid bacteria (lab) consortium … 1341 (http://www.ncbi.nlm.nih.gov/blast/). closely related species and type strains identified from genbank were selected for further analysis. sequence alignment was performed using mega version 5.0 (tamura et al. 2011). a phylogenetic tree was constructed to determine the evolutionary relationship of the lab isolates from tempe jember with other lactic acid bacteria and non-lactic acid bacteria using the neighbor-joining method with 1,000 bootstrap replications (felsenstein. 1985). results and discussion results amplification of the five lab consortium isolates genomic amplification of the five lactic acid bacteria (lab) isolates from tempe in jember, east java, was carried out using pcr with primers 63f and 1387r. visualization of the 16s rrna gene amplification from isolates ta1, tb1, tk1, tk2, and tk4 produced dna fragments with an approximate size of 1,300 bp (figure 1). all five lab isolates, which have potential as probiotic candidates, were molecularly identified based on the 16s rrna gene and phylogenetic tree analysis. figure 1. pcr amplification of the 16s rrna gene using primers 63f and 1387r. m = 1 kb dna ladder (fermentas); lanes 1–5 represent pcr products of bacterial isolates: 1 = ta1; 2 = tb1; 3 = tk1; 4 = tk2; 5 = tk4. identification and phylogenetic construction of the five lab consortium isolates using the 16s rrna gene dna sequence analysis was performed using the basic local alignment search tool (blast) against the national center for biotechnology information (ncbi) database. table 1 shows that the 16s rrna gene sequences of the five lab isolates exhibited the highest similarity with the 16s rrna gene sequences of several bacterial species. table 1 also presents the alignment of lab isolate sequences with available data in ncbi (blast-n) along with their accession numbers. table 1. alignment of 16s rrna gene sequences of the five lab consortium isolates with available data in ncbi (blast-n). isolate name description max score total score query cover e value per. identity accession number ta1 lactiplantibacillus plantarum strain jcm 1149 2359 2359 100 % 0.00 99.46% nr_115065.1 lactiplantibacillus pentosus strain 124-2 2353 2353 100 % 0.00 99.38% nr_029133.1 lactiplantibacillus paraplantarum strain dsm 10667 2342 2342 100 % 0.00 99.23% nr_025447.1 tb1 kosakonia cowanii jcm 10956 = dsm 18146 strain 888-76 2278 2278 100 % 0.00 99.05% nr_025566.1 atlantibacter hermannii strain cip 103176 2259 2259 100 % 0.00 98.82% nr_104940.1 enterobacter timonensis strain mt20 2230 2230 100 % 0.00 98.42% nr_179439.1 tk1 atlantibacter hermannii strain cip 103176 2254 2254 100 % 0.00 98.81% nr_104940.1 enterobacter timonensis strain mt20 2213 2213 100 % 0.00 98.26% nr_179439.1 salmonella enterica subsp. enterica serovar typhimurium strain lt2 2209 2209 100 % 0.00 98.18% nr_074910.1 tk2 pseudomonas fluvialis strain ass-1 2074 2074 100 % 0.00 96.86% nr_159318.1 pseudomonas pharmacofabricae strain zysr67-z 2058 2058 100 % 0.00 96.62% nr_165768.1 pseudomonas tohonis strain tum18999 2047 2047 100 % 0.00 96.47% nr_179382.1 tk4 lactiplantibacillus plantarum strain nbrc 15891 2346 2346 99 % 0.00 99.38% nr_113338.1 lactiplantibacillus pentosus strain 124-2 2353 2353 99 % 0.00 99.38% nr_029133.1 lactiplantibacillus paraplantarum strain dsm 10667 2342 2342 99 % 0.00 99.23% nr_025447.1 blast-n analysis of the 16s rrna gene sequences revealed that three isolates (tb1, tk1, and tk2) were identified as different species. isolate tb1 was identified as kosakonia cowanii and showed similarity with atlantibacter hermannii and enterobacter timonensis. isolate tk1 was identified as atlantibacter hermannii and exhibited similarity with enterobacter timonensis and salmonella enterica subsp. enterica serovar typhimurium. isolate tk2 was identified as pseudomonas fluvialis and showed similarity with 1342 biology, medicine, & natural product chemistry 14 (2), 2025: 1339-1346 pseudomonas pharmacofabricae and pseudomonas tohonis. isolates ta1 and tk4 were both identified as lactiplantibacillus plantarum, albeit of different strains. furthermore, isolates ta1 and tk4 consistently clustered closely with lactiplantibacillus pentosus and lactiplantibacillus paraplantarum based on blast-n sequence analysis and phylogenetic reconstruction. table 2. identification results of the five lab consortium isolates based on 16s rrna sequences. isolate code sample origin identification based on 16s rrna sequence ta1 antirogo, jember, east java lactiplantibacillus plantarum strain jcm 1149, 16s ribosomal rna gene, partial sequence (99.46% similarity) tb1 baratan, jember, east java kosakonia cowanii jcm 10956 = dsm 18146 strain 888-76, 16s ribosomal rna gene, partial sequence (99.05% similarity) tk1 kaliwates, jember, east java atlantibacter hermannii strain cip 103176 16s ribosomal rna gene, partial sequence (98.81% similarity) tk2 kaliwates, jember, east java pseudomonas fluvialis strain ass-1 16s ribosomal rna gene, partial sequence (96.86% similarity) tk4 kaliwates, jember, east java lactiplantibacillus plantarum strain nbrc 15891 16s ribosomal rna gene, partial sequence (99.38% similarity). based on the nucleotide sequence identification of the 16s rrna gene using blast-n, isolate tb1 showed 99.05% similarity with kosakonia cowanii jcm 10956. isolate ta1 exhibited 99.46% similarity with lactiplantibacillus plantarum strain jcm 1149. isolate tk1 demonstrated 99.38% similarity with lactiplantibacillus pentosus strain 124-2. isolate tk2 showed 96.86% similarity with pseudomonas fluvialis strain ass-1. isolate tk4 displayed 99.38% similarity with lactiplantibacillus plantarum strain nbrc 15891. among these, isolates ta1, tb1, tk1, and tk4 had sequence similarity values above 97% compared to the nucleotide sequences of 16s rrna genes available in genbank, indicating that these isolates belong to the same species. in contrast, isolate tk2 exhibited a similarity value below 97%, suggesting that it may represent a different species or potentially a novel bacterium. according to rosahdi et al (2018), a similarity value of less than 97% indicates that the identified bacterium is distinct and could be classified as a new species. the phylogenetic tree reconstruction, which illustrates the evolutionary relationships, was performed using mega version 5 and is presented in figure 2. figure 2. phylogenetic tree of the five lab isolates from tempeh jember, east java, indonesia, compared with bacterial strains available in genbank. phylogenetic analysis of the 16s rrna gene sequences from the five lab isolates and 11 bacterial strains obtained from genbank was conducted using mega version 5. the phylogenetic tree was constructed azizah et al. – identification of lactic acid bacteria (lab) consortium … 1343 to determine the evolutionary relationships between the lab consortium isolates and the 11 bacterial reference strains in genbank. staphylococcus aureus c17 was used as an outgroup. the phylogenetic tree is presented in figure 2. the analysis revealed that isolates tk1 and tb1 clustered on the same branch, distinct from the other three lab isolates. this clustering is explained by their high similarity (98%) with atlantibacter hermannii strain cip 103176 and enterobacter timonensis strain mt20 (accession numbers: nr_104940.1 and nr_179439.1). isolate tk2 was positioned on a separate branch from the other lab isolates, grouping instead with atlantibacter hermannii strain cip 103176. meanwhile, isolates ta1 and tk4 clustered together with lactiplantibacillus plantarum strains, as well as lactiplantibacillus pentosus and lactiplantibacillus paraplantarum from genbank (accession numbers: nr_029133.1 and nr_025447.1). discussion the molecular identification results based on the 16s rrna gene sequences of the ta1 and tk4 isolates indicate that both were identified as lactiplantibacillus plantarum, showing close genetic relatedness to lactiplantibacillus pentosus and lactiplantibacillus paraplantarum. these isolates originated from tempehh produced in two different subdistricts: ta1 from antirogo and tk4 from kaliwates. despite the geographic difference, lactiplantibacillus plantarum was consistently found in tempeh originating from jember. according to fallo and sine (2022), lpb. plantarum was also successfully isolated and identified via 16s rrna gene sequencing from tempeh produced in east nusa tenggara province. ilyanie et al (2023) further reported that seven lab isolates from the fermented foods belacan and bosou were also identified as lpb. plantarum, suggesting that lpb. plantarum is capable of ecological and metabolic adaptation, allowing it to inhabit a wide range of ecological niches, including fermented foods, meat, plants, and the mammalian digestive tract. according to azizah et al (2023), the ta1 and tk4 isolates are gram-positive bacteria with rod-shaped cells, non-spore-forming, and occur either singly or in short chains, making the molecular identification results consistent with characterizations reported in previous studies. lactiplantibacillus plantarum has demonstrated broad-spectrum antibacterial potential against a wide range of gram-positive and gram-negative bacteria. pathogenic food spoilage organisms and enteropathogens that can be inhibited by lpb. plantarum include e. coli, h.pylori, p.aeruginosa, y.enterocolitica, c.r jejuni, l. monocytogenes, klebsiella, bacillus, clostridium, salmonella, s. aureus, shigella, and enterococcus. in addition to its antibacterial properties, lpb. plantarum also exhibits antifungal activity against several molds and yeasts, including candida spp., aspergillus, mucor, and fusarium (dinev et al. 2018). this species has been utilized as a starter culture in fermented food products (arena et al. 2016) and in livestock feed applications (tian et al. 2023). notably, lpb. plantarum subsp. plantarum kita-3, isolated from cheese produced by mazaraat artisan cheese in yogyakarta, indonesia, has been recognized as a probiotic candidate that passed preclinical safety testing in animal models (a’inurrofiqin et al. 2022). therefore, the lpb. plantarum strain is acknowledged as a promising probiotic candidate in both the food and pharmaceutical industries, with potential applications as a biopreservative and alternative biotherapeutic agent (gonzalez et al. 2021). the molecular identification based on the 16s rrna gene sequence revealed that the tb1 isolate was identified as kosakonia cowanii, showing 98% genetic similarity to atlantibacter hermannii and enterobacter timonensis. this result aligns with the characterization of the tb1 isolate reported by azizah et al (2023), which described tb1 as a gram-negative bacterium with rodshaped cells. this morphology is consistent with kosakonia cowanii. according to espinosa et al (2023), kosakonia cowanii isolated from capsicum annuum l. has potential as a biocontrol agent in suppressing the growth of pathogenic fungi. this ability is attributed to its capacity to produce volatile compounds with antifungal activity. furthermore, phylogenetic analysis of kosakonia cowanii indicates a close relationship with plant pathogenic strains, although several virulence genes were not detected. el-sheshtawy et al (2022) reported that the k. cowanii (b2) isolate was capable of producing a maximum yield of lactic acid (la) using agro-industrial waste (cotton and coffee), reaching 28.14 g/l in a fermentor after 72 hours, as determined by hplc analysis. kacaribu and darwin (2024) noted that k. cowanii is one of the lab strains capable of producing la from renewable sources for applications in petroleum-based products. salatein et al (2025) also demonstrated that under optimal conditions (40 °c and ph 8.0), k. cowanii converted 87% of sugars from sugarcane and beet into la, achieving a maximum yield of 14.2 g/l. under optimal conditions, this strain achieved 85% efficiency, supporting the development of environmentally friendly production, bioeconomy, and enhanced lactic acid output. kosakonia communities have also been found in silage feed fermented with l. plantarum sxc48 for livestock (tian et al., 2023). however, isolation and screening of k. cowanii from fermented foods or beverages have not been previously reported, nor has k. cowanii been identified as a human pathogen. therefore, this study represents the first report of kosakonia cowanii being found in a fermented food, namely tempeh. based on 16s rrna gene sequence analysis, the tk1 isolate was identified as atlantibacter hermannii, showing 98% genetic similarity to enterobacter timonensis and salmonella enterica. according to mohammad and alyousif (2023), a. hermannii is a gram-negative bacterium with rod-shaped cells. the 1344 biology, medicine, & natural product chemistry 14 (2), 2025: 1339-1346 molecular identification of tk1 is consistent with its phenotypic characterization reported by azizah et al (2023). although a. hermannii is generally considered non-pathogenic, it has been isolated from human wound and eye infections (girlich et al., 2021). additionally, a. hermannii was detected as a contaminant in both local and imported frozen chicken meat in basrah, iraq, based on 16s rdna analysis (mohammad and alyousif. 2023). atlantibacter hermannii is not classified as a lactic acid bacterium (lab) and is regarded as a rare opportunistic pathogen occasionally associated with human infections. taxonomically, it belongs to the family enterobacteriaceae. enterobacteriaceae is not classified as part of the lab group, as lab are defined as bacteria that convert carbohydrates into lactic acid (la). enterobacteriaceae belongs to a different bacterial family, although some of its members, such as enterococcus, are capable of producing lactic acid in certain amounts. however, their primary function is not as clearly defined as that of true lab. according to wang et al. (2021), microorganisms capable of producing la from various substrates through fermentation are highly diverse. these include bacteria, yeasts, algae, and fungi. among these, only the bacterial group, specifically lab, represents the genera most commonly utilized (wang et al., 2021). lab falls under the family lactobacillales, which includes lactobacillus, lactococcus pediococcus, aerococcus, carnobacterium, tetragenococcus, vagococcus, leuconostoc, oenococcus, streptococcus, and weissella (sedó et al. 2022). in the study conducted by azizah et a. (2023), the tk1 isolate produced a clear zone measuring 5.31 ± 1.18 mm around the colony, indicating its ability to secrete lactic acid (la) into gyp agar medium supplemented with caco3. calcium carboanate (caco3) neutralizes the acid produced by tk1 and leads to the formation of a clear zone, which serves as an established indicator of lab activity. this approach serves as an initial selection method in the isolation and purification of lab. based on molecular identification and supporting literature, tk1 can be classified as lab. however, further confirmation through molecular reidentification is necessary to verify its classification and ensure that tk1 belongs to a non-pathogenic species. such verification is essential for evaluating the safety of candidate probiotic strains, and requires precise specieslevel identification using 16s rrna gene sequencing to support future applications in food and health biotechnology. molecular identification based on 16s rrna gene sequencing revealed that the tk2 isolate was identified as pseudomonas fluvialis, showing 96% genetic similarity to pseudomonas pharmacofabricae and pseudomonas tohonis. this result is consistent with the phenotypic characterization reported by azizah et al (2023), which described tk2 as a gram-negative, rodshaped bacterium. while certain species within the genus pseudomonas are beneficial, others are known to be pathogenic. for instance, p. aeruginosa is associated with various human infections, p. syringae acts as a plant pathogen and p. fluorescens is a free-living soil bacterium with beneficial properties. additionally, several pseudomonas species have been isolated from contaminated environments and show potential as effective bioremediation agents (sudan et al. 2018). previous studies have not reported pseudomonas fluvialis as a member of the lactic acid bacteria (lab) group. this is because p. fluvialis belongs to the family pseudomonadaceae, which is not classified as lab, as lab are defined by their ability to convert carbohydrates into lactic acid (la). however, in the study by azizah et al (2023), the tk2 isolate produced a clear zone measuring 5.62 ± 1.50 mm around the colony, an indicator of la secretion into gyp agar medium containing caco₃, suggesting lab-like activity. the alkaline caco₃ neutralizes the acid produced, resulting in the clear zone formation. despite this functional trait, the molecular identification of tk2 in this study does not align with existing literature. therefore, re-identification using 16s rrna gene sequencing is necessary to confirm whether tk2 belongs to a non-pathogenic lab species. based on molecular identification using 16s rrna gene sequencing and supporting literature review, the consortium of five lactic acid bacteria (lab) isolates revealed that ta1, tb1, and tk4 belong to the lab group and are non-pathogenic strains. in contrast, tk1 and tk2 require further molecular re-identification using 16s rrna gene sequencing, as they were identified as non-lab strains and exhibit characteristics that differ from previous findings. ta1 and tk4 were identified as lactiplantibacillus plantarum, while tb1 was identified as kosakonia cowanii. tk1 was identified as atlantibacter hermannii, and tk2 as pseudomonas fluvialis. conclusions based on the results of the study, it can be concluded that the 16s rrna coding dna from a consortium of five lactic acid bacteria (lab) isolates derived from tempeh produced in jember, east java, was successfully isolated and sequenced. the identification of the five lab isolates, collected from three different subdistricts in jember, revealed the presence of bacterial species with distinct strain identites. the ta1 isolate, obtained from tempeh in antirogo, jember, was identified as lactiplantibacillus plantarum strain jcm 1149. the tb1 isolate, also from tempeh in antirogo, was identified as kosakonia cowanii strain jcm 10956. meanwhile, the tk1, tk2, and tk4 isolates, which were collected from tempeh in kaliwates, jember, were identified respectively as lactiplantibacillus pentosus strain 124-2, azizah et al. – identification of lactic acid bacteria (lab) consortium … 1345 pseudomonas fluvialis strain ass-1, and lactiplantibacillus plantarum strain nbrc 15891. acknowledgements: the author sincerely thank the ministry of education, culture, research, and technology (kemendiktisaintek) through penelitian dosen pemula (pdp) program, under a drtpm contract issued to lldikti region vii with the reference number: 128/c3/dt.05.00/pl/2025. authors’ contributions: siti nur azizah prepared the lab isolates, designed the study, and drafted the manuscript. rosida and rizka yolanda febiaocti contributed to manuscript writing and performed proofreading. dewi riska nurmalasari conducted genomic dna isolation in the laboratory. sipriyadi carried out bacterial amplification and identification. all authors were involved in drafting and refining the manuscript and approved the 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(2023). fermentation quality and bacterial community of delayed filling stylo silage in response to inoculating lactic acid bacteria strains and inoculating time. chemical and biological technologies in agriculture, 10(1), 1–13. https://doi.org/10.1186/s40538-023-00423-6 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 2, 2018 | pages: 51-55 | doi: 10.14421/biomedich.2018.72.51-55 issn 2540-9328 (online) link of nasopharyngeal carcinoma and epstein-barr virus sugiyanto1,2,*, lina aryati1, fajar adi kusumo1, mardiah suci hardianti3 1department of mathematics, universitas gadjah mada, 55281, yogyakarta, indonesia. 2department of mathematics, universitas islam negeri sunan kalijaga, 55281, yogyakarta, indonesia. 3faculty of medicine, universitas gadjah mada, 55281, yogyakarta, indonesia. author correspondency*: sugimath@yahoo.co.id abstract nasopharyngeal carcinoma (npc) is a cancer that occurs in nasopharynx which is associated with epstein-barr virus (ebv). mutation agents in nasopharyngeal neoplasms occur because of ebv infection. transformation of b-cells due to ebv causes hormone imbalance in lymphoid cells or nasopharyngeal epithelial tissue. rates of ebv infection have been shown to be prognostic to npc. the basic level of ebv dna can be used for stratification prognosis, with higher titers showing greater disease severity and worse outcomes. with mathematical models, there is a correlation between the increase in epstein-barr virus and the increase in invasive carcinoma cells or increase in nasopharyngeal carcinoma cells. keywords: nasopharyngeal carcinoma (npc); epstein-barr virus (ebv); mathematical model; invasive carcinoma cells introduction the body consists of trillions of living cells. the normal cells of the body grow, divide, and die regularly. during the early years of a person's life, normal cells divide faster to allow people to grow. once a person becomes an adult, most cells divide only to replace obsolete or dead cells to repair the lesion. (shah et al., 2012) cancer begins when cells in the body part grow out of control. there are many types of cancer, but they all start because of the absence of abnormal cell growth control. (mimi and yuan et al., 2002) nasopharyngeal carcinoma (npc) is a malignancy derived from the epithelium or mucosa and crypts that line the surface of the nasopharynx (morrison, 2004); (kong et al., 2010); (lin et al., 2003). in indonesia, nasopharyngeal carcinoma are most commonly found among malignant tumors in head and neck and age most who suffer is age 40 years and above (adham et al., 2012); (fachiroh et al, 2004); (stevens, 2005). prevalence of nasopharyngeal carcinoma in indonesia is 4.7 / 100.000 population per year (nurhantari et al., 2003). the study was first written old et al. (1966) on the link between epstein-barr virus (ebv) and npc using metallic assays hybridization dan anticomplement immunoflourescent (acid). about 90% of the adult population has undifferentiated nasopharyngeal carcinomas (unpc) are positive of ebv with a blood test (zeng and zeng, 2010). in wu et al. (2003) person with positive ebv, the cancer will be faster than ebv negative. many studies indicate that unpc is always positive ebv (lo and huang, 2004). ebv dna can be identified nasopharynx that the patient has npc disease (sheen et al., 1998). in other words, ebv is one cause of the development of nasopharyngeal cancer. researchers in the medical field have created categories for the development of abnormal cells. categorization of nasopharyngeal cancer is introduced by the world health organization (who) (zeng and zeng, 2010). several methods of cancer treatment have also been developed. the development of normal nasopharynx epithelium to nasopharyngeal cancer is also known as tumorigenesis model for ebv-associated nasopharyngeal carcinoma at the cellular level. on the other hand, mathematically the development of cells from normal cells, then infected with the ebv, until then into cancer cells can be modeled as a model of dynamics. the modeling will be able to see the dynamics of the development of normal cells to become cancer cells and estimated how fast the cancer cells spread. although the development of nasopharyngeal cancer is not only affected by ebv infection, but because ebv infection is more dominant than other factors, factors other than temporary ebv infection may be neglected in mathematical modeling (aryati et al., 2016). nasopharyngeal carcinoma nasopharyngeal carcinoma is a very dangerous abnormality nasopharynx epithelium. the main one of these cancers is endemic and some areas are very low incidence of this cancer (lu et al., 2010); (wang et al., 2011). the hypothesis about the cause of nasopharyngeal cancer began in the early 20th century, first proposed by https://doi.org/10.14421/biomedich.2018.72.51-55 52 biology, medicine, & natural product chemistry 7 (2), 2018: 51-55 jackson in 1901, which proposed the hypothesis that dust irritation in cork workers would damage airway epithelia. since then the pathogenesis of nasopharyngeal cancer has been intensively studied, in particular aimed at geographical features and racial variations. in recent years many environmental and biological factors have shown a link between the risk of nasopharyngeal cancer and recent research results suggest the role of genetic and viral factors in the progression of this disease (mcdermott et al., 2001). there are three types of npc, these are based on cancer cells under a microscope: keratinizing squamous cell carcinoma, non-keratinizing differentiated carcinoma and undifferentiated carcinoma. each type is different from one country to another. in south china, npc are generally found to be type undifferentiated. different cancers are caused by different factors. the factors that cause nasopharyngeal cancer differ from everyone. some of the factors can be changed, such as smoking, on the contrary the factors that cause due to age, family history of cancer, cannot be changed. however, these factors do not tell much about nasopharyngeal cancer. many people have this disease but do not know the cause. epstein-barr virus (ebv) infection combined with frequent exposure to environmental carcinogenic co-factors is suggested to cause npc development (adham et al., 2012). ebstein-bar virus (ebv) ebstein-barr virus (ebv) or other name human herpesvirus 4 (hhv-4), belongs to the family: herpes virus subfamily: gammaherpesvirinae (ebv), genus: lymphocryptovirus (ebv) (hendrix, 2013). the adult ebv particle has a diameter of 120-180 nm and wraps the lipid bilayer. the primary target of cells for ebv infection is b lymphocytes (b-cells) (hendrix, 2013). incubation period of virus 7 to 14 days for children and adolescents is about 30 to 50 days for adults (bale, 1999). the particles caused by this virus are transmitted through saliva and are often incubated in 15 years during their life without symptoms. seroepidemiologic studies indicate that more than 90% of people in the world are infected with ebv (hendrix, 2013). characteristics and mechanism of nasopharyngeal carcinoma dna is a chemical in every human cell that makes genes, a clue to how cells function. dna usually looks like a parent because they are the source of our dna. dna affects more than how people see it. some genes contain instructions to control when cells grow and divide into new cells. viruses such as ebv also contain dna (chua et al., 2016). when cells are infected with viruses, viral dna may be mixed with normal human dna. ebv dna can instruct nasopharyngeal cells to divide and grow in an abnormal way (chong et al., 1996). ebv-infected cells express several specific viral antigens for each period of infection (lin et al., 1997). ebv latent infection is characterized by expression of epstein-barr virus nuclear antigen-1 (ebna-1) and ebna-2, latent protein (lmp) membrane, and epstein barr virus encoded small rnas (eber) (zheng et al., 2007). these proteins can interact or have homology with various body proteins such as antiapoptosis proteins, cytokines and signal transduction. viral proteins play a role in maintaining the ebv genome in b-cells. the ebv is detected in all periods of latent infection (liebowitz, 1994). all nasopharyngeal epithelial cells are considered to have the same receptors so latent ebv can become active in ebv and infect any nasopharyngeal cells, without preference. in other words, it is assumed to be a random contact between the nasopharyngeal epithelial cells and the ebv virus. the population of nasopharyngeal cells is divided into six sub-populations, normal cells, dysplasia cells, ebv-infected cells, high dysplastic cells, invasive carcinoma cells. normal cells are injured, biased due to food preservatives or tobacco. wounded nasopharyngeal cells may progress to lowplated cells, either due to a weak immune system, resulting in no improvement in genes or apoptosis. the ebv-infected cells are infected dysplasia cells, causing the cell to become infected. the ebv-infected that do not occur apoptosis may progress into high dysplastic cells. the high dysplastic cells may progress to invasive carcinoma cells. invasive carcinoma cells can not heal, until it ends with the death of the patient, if no treatment. this process can be explained in figure 1. figure 1. tumorigenesis nasopharyngeal carcinoma. sugiyanto et al. – link of nasopharyngeal carcinoma and epstein-barr virus 53 from figure 1, system of a mathematical model (aryati et al., 2016) in equations (1) (6) as follows: 1 1 dn a n d n dt    (1) 2 2 dd a d n dv d d dt      (2) 3 3 di a i dv i d i dt      (3) 4 4 h h h h dd a d i d d d dt      (4) 5 5h dc a c d d c dt    (5) 3 6 dv ea i d v dt   (6) where, n : subpopulation of normal cells d : subpopulation of dysplasia cells i : subpopulation of ebv infected cells hd : subpopulation of high dysplastic cells or carcinoma in situ (cis) c : subpopulation of invasive carcinoma cells v : subpopulation of viruses 1a : the rate of proliferation of normal cells 2a : the rate of proliferation of dysplasia cells 3a : the rate of proliferation of ebv infected cells 4a : the rate of proliferation of high dysplastic cells 5a : the rate of proliferation of invasive carcinoma cells 1d : the rate of apoptosis of normal cells 2d : the rate of apoptosis of dysplasia cells 3d : the rate of apoptosis of ebv infected cells 4d : the rate of apoptosis of high dysplastic cells 5d : the rate of proliferation of invasive carcinoma cells 6d : the rate of apoptosis of ebv  : the rate of interaction between normal cells that become dysplasia cells  : the rate of interaction between dysplasia cells that become ebv-infected cells  : the rate of interaction between ebv-infected cells that become high dysplastic cells  the rate of interaction between high dysplastic cells that become invasive carcinoma cells e : the rate of ebv increase due to the proliferation of ebv-infected cells. theorem 1. the relationship between the nasopharyngeal carcinoma and epstein-barr virus in equilibrium point is in the following equation: c v where    6 3 5 5 4 4 d ea d a d a        . proof. if 0 dv dt  and from equation (6), then it is obtained 6 3 d v i ea  (7) if 0hdd dt  and from equation (4), then we get 4 4 h i d d a      . (8) if 0 dc dt  and from equation (5), then we gain 5 5 hd c d a    . (9) if equation (8) is substituted for equation (9), then we have   5 5 4 4 i c d a d a       . (10) if equation (7) is substituted for equation (10), then it is acquired    6 3 5 5 4 4 d v c ea d a d a       . ■ (11) from the theorem 1 that the relationship between nasopharyngeal carcinoma and epstein-barr virus is affected by parameters: the rate of interaction between high dysplastic cells, the rate of interaction between ebv-infected cells that become high dysplastic cells, the rate of apoptosis of ebv, the rate of proliferation of ebv infected cells, the rate of proliferation of ebv infected cells, the rate of proliferation of invasive carcinoma cells, the rate of proliferation of invasive carcinoma cells, the rate of apoptosis of high dysplastic cells, and the rate of proliferation of high dysplastic cells. if table 1 is included in equation 11 obtained 3.3c v . simulation in this section, we will discuss numerical simulations and medical interpretations of mathematical models of nasopharyngeal carcinoma. figure 2 shows a trajectory diagram for variables v and c. within 100 days, variables v and c occur at the 54 biology, medicine, & natural product chemistry 7 (2), 2018: 51-55 beginning of the occurrence of npc. within the first 100 days the epstein-barr virus value was 4.92 virus / mm2 and the invasive carcinoma cells value was 3.807 cell/mm2. at the stage of the formation of invasive carcinoma cells are things that need attention, because remember this cancer is a type that is malignant. the earlier the cancer is very related to the treatment process and is related to the survival of npc sufferers. figure 3 shows a portrait diagram of the phase between epstein-barr virus and invasive carcinoma cells. there is a correlation between the increase in epstein-barr virus and invasive carcinoma cells. this has an interpretation that npc occurs because of an increase in ebv (hu, 1996). table 1. the parameter values of the nasopharyngeal carcinoma mathematical model. no. parameter value unit reference 1. 1a 13 cell mm–3 day–1 (huynh, 2010) 2. 2a 0,001 day–1 estimation 3. 3a 0.0138 day–1 (huynh, 2010) 4. 4a 0.04 day–1 (aryati et al., 2016) 5. 5a 0.0138 day–1 (aryati et al., 2016) 6. 1d 1.0412 day–1 (aryati et al., 2016) 7. 2d 0.02 day–1 (aryati et al., 2016) 8. 3d 0.0288 day–1 (huynh, 2010) 9. 4d 0.1152 day–1 (aryati et al., 2016) 10. 5d 0.0188 day–1 (aryati et al., 2016) 11 6d 0.1152 day–1 (huynh, 2010) 12. e 2 day–1 (aryati et al., 2016) 13.  0.05 day–1 (aryati et al., 2016) 14.  1 day–1 (aryati et al., 2016) 15.  0.0082 day–1 (aryati et al., 2016) 16.  0.07 day–1 (aryati et al., 2016) figure 2. trajectory diagram of epstein-barr virus and invasive carcinoma cells in the first 100 days of npc development. figure 3. portrait projection of epstein-barr virus and invasive carcinoma cells in the first 100 days of npc development. conclusion the relationship between the nasopharyngeal carcinoma and epstein-barr virus is directly proportional and linear. the increase in epstein-barr virus makes an increase in invasive carcinoma cells by 3.3. in the first 100 days of development of the nasopharyngeal carcinoma increasing of epstein-barr virus are followed invasive carcinoma cells. references adham, m., kurniawan, a. n., muhtadi, a. i., roezin, a., hermani, b., gondhowiardjo, s., ... & middeldorp, j. m. 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(1994, june). nasopharyngeal carcinoma: the epstein-barr virus association. in seminars in oncology (vol. 21, no. 3, pp. 376-381). lin, c. t., lin, c. r., tan, g. k., chen, w., dee, a. n., & chan, w. y. (1997). the mechanism of epstein-barr virus infection in nasopharyngeal carcinoma cells. the american journal of pathology, 150(5), 1745. lin, j. c., jan, j. s., hsu, c. y., liang, w. m., jiang, r. s., & wang, w. y. (2003). phase iii study of concurrent chemoradiotherapy versus radiotherapy alone for advanced nasopharyngeal carcinoma: positive effect on overall and progression-free survival. journal of clinical oncology, 21(4), 631-637. lo, k. w., to, k. f., & huang, d. p. (2004). focus on nasopharyngeal carcinoma. cancer cell, 5(5), 423-428. lu, j. j., cooper, j. s., & lee, a. w. (eds.). (2010). nasopharyngeal cancer: multidisciplinary management. springer science & business media. mcdermott, a. l., dutt, s. n., & watkinson, j. c. (2001). the aetiology of nasopharyngeal carcinoma. clinical otolaryngology & allied sciences, 26(2), 82-92. mimi, c. y., & yuan, j. m. (2002, december). epidemiology of nasopharyngeal carcinoma. in seminars in cancer biology (vol. 12, no. 6, pp. 421-429). academic press. morrison, j. a., gulley, m. l., pathmanathan, r., & raab-traub, n. (2004). differential signaling pathways are activated in the epstein-barr virus-associated malignancies nasopharyngeal carcinoma and hodgkin lymphoma. cancer research, 64(15), 5251-5260. nurhantari, y., emoto, n., rahayu, p., & matsuo, m. (2003). nasopharyngeal carcinoma in indonesia has a low prevalence of the 30-base pair deletion of epstein-barr virus latent membrane protein 1. southeast asian journal of tropical medicine and public health, 34(1), 98-105. old, l. j., boyse, e. a., oettgen, h. f., de harven, e., geering, g., williamson, b., & clifford, p. 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(2005). diagnostic value of measuring epstein-barr virus (ebv) dna load and carcinoma-specific viral mrna in relation to anti-ebv immunoglobulin a (iga) and igg antibody levels in blood of nasopharyngeal carcinoma patients from indonesia. journal of clinical microbiology, 43(7), 30663073. wang, h. y., sun, b. y., zhu, z. h., chang, e. t., to, k. f., hwang, j. s., ... & lee, m. (2011). eight-signature classifier for prediction of nasopharyngeal carcinoma survival. journal of clinical oncology, 29(34), 4516-4525. wu, h. c., lin, y. j., lee, j. j., liu, y. j., liang, s. t., peng, y., ... & lin, c. t. (2003). functional analysis of ebv in nasopharyngeal carcinoma cells. laboratory investigation, 83(6), 797. zheng, h., li, l. l., hu, d. s., deng, x. y., & cao, y. (2007). role of epstein-barr virus encoded latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma. cell mol immunol, 4(3), 185-196. zeng, m. s., & zeng, y. x. (2010). pathogenesis and etiology of nasopharyngeal carcinoma. in nasopharyngeal cancer (pp. 925). springer, berlin, heidelberg. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 61-67 | doi: 10.14421/biomedich.2023.121.61-67 issn 2540-9328 (online) synthesis, characterization and activity test of natural zirconium zeolite (zr-za) catalyst in the esterification reaction of glycerol with acetic acid anhydride didik krisdiyanto1,*, tutik farihah2, hikmah supriyati3 1chemistry department, faculty of science and technology, uin sunan kalijaga yogyakarta, indonesia. 2department of industrial engineering, faculty of science and technology, uin sunan kalijaga yogyakarta, indonesia. 3postgraduate biology education program, yogyakarta state university, indonesia. corresponding author* didik_kris@yahoo.com manuscript received: 26 september, 2022. revision accepted: 05 october, 2022. published: 17 october, 2022. abstract synthesis of zr-za catalyst used as catalyst for esterification reaction of glycerol with acetic acid anhydrous has been done. catalysts are characterized using an infrared spectrophotometer, x-ray diffraction, and measurement of surface acidity, while reaction products are characterized using a gas chromatography-mass spectrometer. effect of catalyst acidity, reaction time and re-addition of acetic acid anhydrous studied by glycerol conversion and selectivity value of triacetin were yielded. infra-red spectrophotometer and x-ray diffraction analysis of the catalyst showed that the synthesized catalyst was zirconium zeolite (zr-za), while esterification analysis using a gas chromatography-mass spectrometer showed a triacetin product. keywords: glycerol; triacetin; acetic acid anhydrous. introduction for muslims, the halal food is absolute, even if it contains only a trace of additives it would not be allowed. as a result, the production of halal-certified additives is critical, both in terms of sources and production processes; currently, many additives are obtained from non-halal sources and processes. for example, some monoacetyl glycerol (mag), diacetyl glycerol (dag), and tri acetyl glycerol (tag)/triacetin are derived from the esterification of non-halal animal glycerol, such as pork (gültekin, et al., 2020: alzeer and hadeed 2021). triacetin has many uses in both food and non-food applications. triacetin can be used as an aroma ingredient in candy (confectionery), milk drinks, soft drinks, and chewing gum. as for non-food ingredients, triacetin can be used as a solvent in perfumes, printing inks, solvents for aromas, plasticizers for cellulose resins, polymers, co-polymers, and can even be used as a fuel additive to reduce knocking in car engines. in addition to triacetyl glycerol (tag) and triacetin, monoacetyl glycerol (mag) and diacetyl glycerol (dag) are formed during the esterification of glycerol with acetic acid (nuryoto et al., 2010; gama et al., 2019; yanti et al., 2019) glycerol derivatives such as monoacetyl glycerol (mag), diacetyl glycerol (dag), and triacetyl glycerol (tag)/triacetin can be used as alternative food additives (additives). food additives are ingredients that are intentionally added to food to improve the appearance, texture, taste, and shelf life of the food. food additives such as monoacetyl glycerol (mag) and diacetyl glycerol (dag) are used as thickening agents or emulsifiers. meanwhile, triacetil glicerol (tag)/triacetin is a solvent for other additives such as flavorings and dyes (ogawa., et al.,1992; fiume 2003; jiang et al 2010) glycerol is a by-product of biodiesel from the transesterification process to obtain methyl esters. in 2010, it is estimated that glycerol production was around 1.2 million tons, of which more than half came from biodiesel production (appleby, 2003). as a by-product of the biodiesel industry, glycerol has not been processed much, so its selling value is still low. therefore, it is necessary to process glycerol so that it can become a product with higher selling value and more benefits. one of them is by making glycerol derivatives through the esterification process. one of the glycerol esterification products is triacetin. (ghoreishi., 2013; setyaningsih et al., 2020; reddy, 2010) the glycerol esterification reaction usually uses acetic acid. silva et al. (2010) have conducted a study to compare the glycerol esterification reaction using acetic acid and anhydrous acetic acid. in the study described, under the same conditions, esterification of glycerol https://doi.org/10.14421/biomedich.2023.121.61-67 62 biology, medicine, & natural product chemistry 12 (1), 2023: 61-67 using anhydrous acetic acid gave high selectivity to triacetin (100% at 80 minutes) compared to acetic acid (7% at 120 minutes). glycerol derivatives are made from the esterification process between glycerol and acetic acid with the help of a catalyst. in previous research using a homogeneous catalyst as done by widayat et al. (2013), they used a sulfuric acid catalyst because it is superior in its hygroscopic properties, which can absorb water, for the esterification reaction goes towards the product. however, the use of homogeneous catalysts has disadvantage that the catalyst is difficult to separate from the product after the reaction because it is in one phase (dewajani., et al., 2019) the use of heterogeneous catalysts is an alternative in the glycerol esterification reaction. zirconium is widely used in catalytic processes as a support and a promoter in catalysts (tanabe and yamaguci., 1994; teterycz et al., 2003). zirconium is also an important metal because of the possibility of strong bond polarization between sio2 and zrd+ (beck et al., 1992). in addition, zirconium has a low surface area (usually 50 m2 g-1) (nawrocki, j. et al., 1993). for this reason, this research would focus on the synthesis of glycerol derivatives, namely triacetin, using heterogeneous zirconium catalysts immersed in natural zeolites. then proceed with the optimization of reaction conditions and the analysis of halal results through food critical point tests. methods natural zirconium zeolite catalyst synthesis zeolite pretreatment the first stage was natural zeolite obtained from klaten, central java, was pulverized to form a powder with a size of 100 mesh, then washed with distilled water and heated at a temperature of 120°c for 1 hour. activation of natural zeolite 80 grams of natural zeolite were soaked with 100 ml of 1% hf solution in a plastic container, then washed and dried in an oven for 1 hour at 120 °c. then soaked again in 100 ml of hcl 2m for 30 minutes and washed again with distilled water until the cl ions disappear. then dried again in the oven. making natural zirconium zeolite catalyst by the impregnation method the natural zeolite obtained was impregnated with zirconium metal with concentrations of zr 0%, 1%, 2%, 3%, 5% and immersed in zrocl2.8h2o solution while stirring with a magnetic stirrer, then washed with distilled water and calcined for 4 hours at 400°c. synthesis of glycerol derivatives this step was carried out by heating glycerol and anhydrous acetic acid separately until it reaches 80 °c. both are reacted in a three-neck flask after reaching 80 °c, and a zirconium zeolite catalyst was added. the combined solution was then heated to 115 °c and stirred with a magnetic stirrer at 1000 rpm for 4 hours. discussion catalyst characterization characterization using ft-ir characterization using ft-ir is shown in figure 1. infrared analysis was carried out to determine the active group in the zeolite solid. hendayana (1994) stated that infrared spectroscopy is used to determine the structure, which is important information about the functional groups of a molecule. infrared spectra analysis is grouped into 2 parts, namely wave numbers 4000–1250 cm-1 and 1250-350 cm-1. observations with an infrared spectrophotometer showed specific peaks. generally, there is no significant difference in absorption in the infrared spectrum of activated natural zeolite (za) and zirconium-impregnated natural zeolite (za-zr). in the spectrum, it can be seen that there are peaks at the same wave number, namely 3448.72 cm-1 which indicates oh absorption. at absorption wave numbers 2360.87cm-1 and 2368.59 cm-1, it shows the stretching vibration of sioh. the absorption is sharply widening in the wavenumber region. internal and external zeolites interwoven are shown at an absorption of about 1000 – 1250 (cm-1) figure 1. infrared spectrophotometer results of activated natural zeolite (za) and zirconium-impregnated natural zeolite (za-zr). krisdiyanto et al. – synthesis, characterization and activity test of … 63 which indicates the presence of o-si-o and o-al-o asymmetric stretching vibrations of the alumina silicate framework. the absorption area 770-803 (cm-1) is the fingerprint region of the zeolite, which shows the presence of o-si-o and o-al-o vibrations. the spectra show the absorption of o-si-o and o-al-o asymmetric stretching vibrations in the 1087.85cm-1 and 1080.14 cm-1 regions and the o-si-o and o-al-o symmetric stretching vibrations in the 794 regions. 67 cm-1. the sio and al-o buckling vibrational regions are shown in the absorption regions of 447.49 cm-1 and 462.92 cm-1 in the wave number table of 1660 cm-1 to 1620 cm-1, there is a reduction in intensity from 1651.07 cm-1 to 1635.64 cm-1 which indicates that there is a release of water molecules bound to the zeolite physically. the loss of absorption proves that the zeolite is cleaner than impurities, so the pores are getting bigger. the increase in intensity at wave numbers 500 cm-1 420 cm-1 indicates that there are more silanol groups in the zeolite framework. this shows that there is a reduction in al-o bonds in the zeolite framework when hcl is added. the width of the peak indicates the increasing number of silanol groups until the crystallinity decreases. where the si-o bond is stronger than the al-o bond, resulting in a higher wave number used to vibrate. this is also supported by the increase in intensity at wave numbers 2385 cm-12363 cm-1 which indicates the presence of more silanol groups in the zeolite framework. table 1. zeolite ftir spectrum interpretation. spectral interval (cm-1) wave number (cm-1) functional group interpretation natural zeolite active zeolite zeolite-zr 5% 3620 3420 3448,72 3448,72 stretching vibration –oh 2385 2363 2360,87 2368,59 stretching vibration si-oh 1660 – 1620 1651,07 1635,64 bending vibration h2o 1213 – 1000 1087,85 1080,14 asymmetric stretching vibration o-si-o and o-al-o 803 – 770 794,67 794,67 symmetric stretching vibration o-si-o and o-al-o 500 – 420 447,49 462,92 bending vibration si-o and al-o characterization using xrd the results of the crystallinity test using xrd can be seen in figure 2. the crystallinity characterization of zeolite was carried out qualitatively using xrd. the xray diffractograms of the za/zr-0 and za/zr-5 samples provide information about the type of mineral and the degree of crystallinity of the structural components that make up the sample. the type of mineral that composes the sample is indicated by the area of peak appearance (2θ), while the level of crystallinity of the component structure is indicated by the high and low peak intensity. the mineral diffractogram from the xrd results is matched its 2θ value with jcpds data (joint committee on powder diffraction standards) so that the type of mineral in the sample will be known. the diffractogram of the zeolite test results using xrd can be seen in figure 2. the diffractogram pattern of za/zr-0 and za/zr-5 in figure 2 looks almost the same. there are certain peaks that experience a change in intensity and a shift in the value of 2θ. changes in intensity there is an increase and there is a decrease depending on the crystal structure, the position of atoms in the unit cell, and thermal vibrations. however, the crystal structure of za did not change much because it was stable when activated. it is as shown in the table. based on table 2 in the za/zr-0 and za/zr-5 diffractograms there are many peaks of interpretation of mordenite character zeolite, this proves that the za/zr catalyst has been dealuminated after activation using 2m hcl. with the decrease in the composition of al cations, the si/al ratio in natural zeolite changed from previously clinoptilolite with the molecular formula [na1.84k1.76mg0.2ca1.24(h2o)21.36] [si29,84al6.16o72] to mordenite with the molecular formula na8(h2o)24] [si40al8o96] here the si/al ratio increases. the results of the xrd diffractogram show that zr metal has been successfully distributed in active natural zeolite, which is indicated by the presence of peaks at 2θ = 22.09 and at 2θ = 35.55. 64 biology, medicine, & natural product chemistry 12 (1), 2023: 61-67 figure 2. diffractogram data of activated natural zeolite (za) and zirconium-impregnated natural zeolite (za-zr). table 2. interpretation of diffraction peaks at za/zr-0 and za/zr-5. 2θ jcpds 2θ za/zr-0 interpretation 9,77 9,78 mordenite 13,41 13,39 mordenite 19,58 19,58 mordenite 21,24 22,238 mordenite 25,62 25,603 mordenite 26,25 26,25 clinoptilolite 27,60 27,64 clinoptilolite 30,068 30,069 clinoptilolite 30,828 30,83 mordenite 35,65 35,66 mordenite 48,21 48,34 mordenite 2θ jcpds 2θ za/zr-5 interpretation 13,41 13,33 mordenite 19,36 19,47 clinoptilolite 21,79 21,84 mordenite 22,05 22,09 zr 25,56 25,51 mordenite 26,10 26,13 mordenite 27,54 27,54 mordenite 35,53 35,55 zr acidity test for zirconium-impregnated natural zeolite (za-zr) catalyst zeolite acidity can be measured gravimetrically using the ammonia adsorption method. zr metal impregnation treatment on zeolite is expected to increase the acidity of the zeolite. this increase in acidity is due to the exchange of the zr4+ group with the h+ group found in the active zeolite. table 3. catalyst acidity. catalyst acidity (𝒎𝒎𝒐𝒍/𝒈𝒓𝒂𝒎) za/zr-0 4,52 za/zr-5 5,88 za/zr-10 6,79 from the results of the ammonia adsorption analysis, it was found that the total acidity contained in the zeolite increased with the increase in the amount of zr metal impregnation into the active zeolite (table 3). this is due to the interaction between the nh3 base and the acid in the zeolite. h+ is a bronsted acid, which will form nh4+ ions when it interacts with nh3. the presence of zr metal is possible to cause an increase in the acidity of the catalyst because zr metal has a (d) orbital that is not fully filled so it effectively accepts electron pairs from the adsorbate base. the contribution of the number of acid sites of zr metal is a lewis acid site (comelli et al., 2006). the presence of a large number of active sites,increases the adsorption power of the reactants. analysis of glycerol acetylation results the product of glycerol esterification reaction with acetic acid anhydride using zirconium-impregnated natural zeolite (za-zr) as catalyst was analyzed qualitatively using an infrared spectrophotometer. this analysis is intended to determine the presence of esters (triacetin or mono acetin and diacetin) in the esterification reaction product. the results of the infrared spectra can be seen in figure 3. figure 3. infrared spectra of glycerol esterification reaction with acetic acid anhydride using zirconium-impregnated natural zeolite (za-zr) as catalyst. 2-theta (deg) in te n s it y ( c p s ) 20 40 60 0.0e+000 2.0e+003 4.0e+003 6.0e+003 8.0e+003 1.0e+004 2-theta (deg) in te n s it y ( c p s ) 20 40 60 0 1000 2000 3000 4000 5000 krisdiyanto et al. – synthesis, characterization and activity test of … 65 the infrared spectra show a strong absorption at a wave number of 1728.22 cm-1, a strong absorption at a wave number of 1226.73 cm-1, and a medium absorption at 1373.32 cm-1, where the absorptions successively indicate the presence of vibrations from groups c=o, c-o (from the ester), and ch3. the presence of these three functional groups corresponds to the product resulting from the glycerol esterification reaction, which is in the form of an ester. with a reaction like figure 4 (silva et al., 2010). figure 4. reaction of glycerol esterification with acetic acid anhydride using acid catalyst and with the proposed reaction mechanism, namely: figure 5 the mechanism of the esterification reaction for the formation of monoacetate/monoacetin. the reaction mechanism in the figure continues until all the hydroxy groups in glycerol are replaced by acetate groups and triacetin is formed. for one triacetin compound, it takes three compounds of acetic acid or acetic acid anhydride to react with glycerol. the spectrum of the hydroxy group (–oh) also appears in the infrared spectra of the product, which is in the form of a wide absorption at wave numbers of 2962.66 cm-1 to 3600 cm-1, which indicates the presence of vibrations from the hydroxy group (o-h) (sastrohamidjojo, 2007). the hydroxy group can come from acetic acid as a by-product, or from glycerol and (excess) acetic acid anhydride that have not reacted. the results of the complete interpretation of infrared spectra data are presented in table 4 below: table 4. interpretation results of infrared spectral data of glycerol esterification products. wavenumber (cm-1) functional group research result reference specta (sastrohamidjojo, 2007) 1728,22 (strong absorption) 1600-1820 (strong absorption) c=o 1226,73 (strong absorption) 1000-1300 (strong absorption) c-o (of ester) 1373,32 (medium absorption) ±1375 (medium absorption) -ch3 2962,66-3600 (widen) 2400-3400 (widen) -oh from the ft-ir data, it can be seen that glycerol has undergone an esterification reaction and has been converted into an ester, namely monoacetin, diacetin, and triacetin. the ft-ir test performed on the product was not sufficient to detect the presence of triacetin. therefore, it is necessary to carry out further tests using a gas chromatography instrument (gas chromatography/gc) which is connected to a mass spectrometer (mass spechtrometer/ms). with this instrument triacetate compounds can be identified and the percentage can be calculated in the resulting product. the results of the analysis with gc-ms in the form of 66 biology, medicine, & natural product chemistry 12 (1), 2023: 61-67 two data, namely the chromatrogram derived from the results of the gc analysis and the mass spectra from the results of the ms analysis. chromatogram analysis was based on the level of similarity or similiarity index (si) between the retention time (tr) obtained and the retention time (tr) from the library search report (wiley7nist05.l). the chromatogram results of the esterification reaction products are presented in figure 6. (a) (b) figure 6. chromatogram of esterification reaction products (a), mass spectrometry triacetin spectra (b). in the chromatogram, the triacetin peak is located at the retention time (tr) 7.863 minutes, according to the library search report (wiley7nist05.l) on gc. the complete chromatogram analysis is presented in table 4. table 5. results of chromatogram analysis and results of glycerol esterification reactions. no retention time (minutes) compound si (%) (wiley7nist05.l) 1 2,075 acetic acid 91 2 2,930 acetic acid anhydride 64 3 6,527 diacetin 83 4 7,863 triacetin 90 the results of the chromatogram regarding the presence of triacerin can be seen in (figure 8 (a) ), while the mass spectrometer data can be seen in (figure 8 (b)). from these data we can know the relative molecular mass of a compound and the fragmentation of the compound. the mass spectrometer data of the triacetin compound present at the retention time of 7.866 is presented in figure 7. the figure shows that the molecular mass/charge (m/e) of the compound is 219.1 m/e. (widayat et al, 2013). figure 7. fragmentation of triacetin. krisdiyanto et al. – synthesis, characterization and activity test of … 67 conclusion zirconium-impregnated natural zeolite (za-zr) catalyst has been successfully synthesized were indicated by difractogram of x ray diffraction analysis. zr metal impregnation treatment on zeolite was increase the acidity of the zeolite into 6,79 mmol/gram for za/zr-10 catalyst. the results of the analysis of the esterification product base on infrared spectra and mass spectrometry chromatogram showed the presence of triacetin products. acknowledgments: lppm uin sunan kalijaga for funding assistance for this research in the 2017 beginner research program. competing interests: there was no conflict of interest in this study. references alzeer, j., hadeed, k.a. (2021). halal certification of food, nutraceuticals, and pharmaceuticals in the arab world. in: laher, i. (eds) handbook of healthcare in the arab world. springer, cham. appleby, d. 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(2013). proses produksi triasetat dari gliserol dengan katalis asam sulfat. jurnal teknik kimia indonesia. 12. 192. 10.5614/jtki.2013.12.1.3. yanti, n.r., heryani, h., putra, m.d., nugroho, a., (2019). triacetin production from glycerol using heterogeneous catalysts prepared from peat clay. international journal of technology. volume 10(5), pp. 970-978 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 67-71 | doi: 10.14421/biomedich.2021.101.67-71 issn 2540-9328 (online) effect of ethanol extracts of musa paradisiaca fruit pulp and peels on haematological indices and liver enzymes of experimental rats emuesiri goodies moke1,*, emuesiri kohworho umukoro1, evelyn tarela ojugbeli2, theresa ezedom2, tarela melish elias daubry3, iziegbe lisa omorodion1 1department of pharmacology and therapeutics; 2department of medical biochemistry; 3department of physiology, faculty of basic medical sciences, delta state university, abraka, nigeria corresponding author* hiligoodies@gmail.com manuscript received: 12 february, 2021. revision accepted: 19 july, 2021. published: 29 july, 2021. abstract medicinal plants usage in traditional medicine has risen over the years. musa paradisiaca has been reported that it possesses various therapeutic efficacies. this study is aimed at evaluating the effect of parts of the ethanol fruit extracts of musa paradisiaca on haematological indices and serum liver enzymes. thirty wistar rats were grouped into five groups of six animals each which were administered 200 mg/kg and 400 mg/kg of musa paradisiac fruit (mpf) pulp extract or peel extract. haematological indices and liver enzymes were assayed for at the end of the 14-days experimental period. mpf pulp and mpf peel at 400 mg/kg showed a significant (p<0.05) increase in red cell count and haematocrit level as compared to the control. there was a non-significant (p>0.05) increase in haemoglobin concentration in the treated rats as compared to the control. there was also a non-significant (p>0.05) change in ast, alt, and alp level of the treated rats as compared to the control. fruit pulp and peel of musa paradisiaca improve erythrocytes count and haematocrit level, and they may not be associated with liver toxicity. keywords: anaemia; haematology; liver enzymes; medicinal plants; musa paradisiaca. introduction plants possess the ability to synthesize a variety of chemical compounds with various biological functions. the plant kingdom remains a harvest for many species of plants with key medicinal value which are yet to be discovered. reports show that a large population (80%) of people in developing countries depend primarily on medicinal plants for their primary health care (mahomoodally, 2013; ekor, 2014). these plants and herbs are taken in different ways and forms (whole or in parts) for the alternative management of diseases traditionally (benzie and wachtel-galor, 2011; emudainohwo et al., 2015; moke et al., 2019; okafo et al., 2019). musa paradisiaca belongs to musaceae family and is popularly known as plantain. the plant is widely distributed in the southern part of nigeria, west and east africa, malaysia, cameroun and southern parts of united states (uwaoma, 2003; nayar, 2010). musa paradisiaca (plantain) is an important staple crop that contributes to the calories and substance economic in africa (oyeyinka and afolayan, 2019). abundant medicinal activities of parts of musa parasidiaca have been reported. it has been shown to possess hepatoprotective activity (nirmala et al, 2012; issa et al., 2018) as well as antidiabetic (vilhena et al., 2020), antiulcer (onasanwo et al., 2013; ezekwesili et al., 2014; moke et al., 2017), antimicrobial (fagbemi et al., 2009), wound healing (agarwal et al. 2009), and antioxidant properties (yin et al., 2008). the present study is aimed at evaluating the effect of parts of the ethanol fruit extracts of musa paradisiaca on haematological indices and serum liver enzymes. material and methods plant collection and preparation musa paradisiaca fruits (unripe) were purchased locally from the market, and were identified and authenticated by a taxonomist with existing specimen deposited at the herbarium of the department of botany, delta state university, abraka, nigeria. the fruits were rinsed with water, and both the fruit pulps and fruit peels were air dried. the dried pulps and peels were grinded separately into pulverized powder using a grinding machine for ease of extraction. the powdered materials of musa paradisiaca fruits (400 g each) were separately extracted exhaustively with ethanol using soxhlet extractor at 25 0c. the filtrates were concentrated using rotary evaporator at 40 0c. the percentage yields were 8.4% (fruit pulp) and 9.73% https://doi.org/10.14421/biomedich.2021.101.67-71 68 biology, medicine, & natural product chemistry 10 (1), 2021: 67-71 (fruit peel). the concentrated ethanol extracts were refrigerated prior to use. animals wistar rats (150 – 180 g) were obtained from the animals’ house facility of the faculty. the animals were acclimatized for 7 days prior to the study, and were fed with rat feed and clean water ad libitum. guidelines followed in the handling of animals were in accordance with the ethical standards of the institutional animals ethics committee (iaec), as adopted by the ethical committee of the faculty of basic medical science, delta state university, abraka, nigeria. experimental design the animals were divided into five (5) groups of six animals each:  group 1 – normal saline (control) 10 ml/kg  group 2 – musa paradisiac fruit pulp extract (mpf pulp) 200 mg/kg  group 3 – musa paradisiac fruit pulp extract (mpf pulp) 400 mg/kg  group 4 – musa paradisiac fruit peel extract (mpf peel) 200 mg/kg  group 5 – musa paradisiac fruit peel extract (mpf peel) 400 mg/kg the experimental animals were administered the extracts orally daily for 14 days according to their body weights. sample collection at the end of the 14-days treatment period, the animals were anesthetized using chloroform. blood samples were collected by cardiac puncture into labeled edta bottles for haematological analysis and liver function tests. determination of haematological indices the method as described by tietz (1976) and baker et al. (1998) were used for determining the red blood cells (rbc) counts, haemoglobin (hb) concentration, and haematocrit level. determination of liver function test alkaline phosphatase (alp), aspartate aminotransferase (ast), and alanine transaminase (alt) in serum were determined according to methods described by reitman and frankel (1957) and roy (1970). statistical analysis results are presented as the mean ± standard error of the mean (sem). data were analysed using one-way analysis of variance (anova) followed by tukey’s post hoc test. p-values < 0.05 were taken as significant. results and discussion effect of ethanol fruit extracts of musa paradisiaca on haematological indices of wistar rats figures 1-3 depict the effect of ethanol fruit extracts (pulp and peel) of musa paradisiaca on the red blood cell count, haematocrit level, and haemoglobin concentration of normal wistar rat. mpf pulp and mpf peel at a dose of 200 mg/kg had a non-significant (p>0.05) increase in red blood cell count when compared to the control, however, at 400 mg/kg, there was a significant (p<0.05) increase in red cell count. high dose (400 mg/kg) of both fruit extracts significantly (p<0.05) increased the haematocrit level as compared to the control. there was a non-significant (p>0.05) increase in haemoglobin concentration in the treated rats as compared to the control. g ro u p s r e d b lo o d c e ll ( r b c ) c o u n t (× 1 0 1 2 /l ) c o n tr o l m p f p u lp 2 0 0 m g /k g m p f p u lp 4 0 0 m g /k g m p f p e e l 2 0 0 m g /k g m p f p e e l 4 0 0 m g /k g 0 2 4 6 8 * * figure 1. the effect of ethanol fruit extracts of musa paradisiaca on red blood cell count of wistar rats. g ro u p s h a e m a to c r it ( % ) c o n tr o l m p f p u lp 2 0 0 m g /k g m p f p u lp 4 0 0 m g /k g m p f p e e l 2 0 0 m g /k g m p f p e e l 4 0 0 m g /k g 0 2 0 4 0 6 0 * * figure 2. the effect of ethanol fruit extracts of musa paradisiaca on haematocrit level of wistar rats. moke, et al. – effect of ethanol extracts of musa paradisiaca fruit pulp and … 69 g ro u p s h a e m o g lo b in ( h b ) (g /d l) c o n tr o l m p f p u lp 2 0 0 m g /k g m p f p u lp 4 0 0 m g /k g m p f p e e l 2 0 0 m g /k g m p f p e e l 4 0 0 m g /k g 0 5 1 0 1 5 2 0 figure 3. the effect of ethanol fruit extracts of musa paradisiaca on heamoglobin concentration of wistar rats. blood is composed of a variety of living cells that circulate through the heart and the blood vessels carrying nutrients, hormones, vitamins, antibodies, heat and oxygen to the body’s tissue. the components of blood include red blood cells, white blood cells and platelets which are suspended in plasma (basu and kulkarni, 2014). red cell contains haemoglobin, a protein that carries oxygen to all the tissues of the body. haematocrit or packed cell volume is a measurement of the proportion of blood that is made up of cells. following centrifugation, it is an estimate of the ratio of the volume of red blood cells to the total volume of blood (mondal and budh, 2020). haematocrit and haemoglobin values are useful for assessing anaemia, polycythemia, and also for estimating response to treatment (northrop-clewes and thurnham, 2013; white, 2018; mondal and budh, 2020). hematopoiesis is the process involved in the formation of blood cells (rieger and schroeder, 2012). this study showed the positive effect of the fruit pulp and peel of musa paradisiaca on hematopoiesis. musa paradisiaca was also revealed to have to be non-toxic effect on liver enzymes. the assessment of haemotological parameters could be used to reveal the deleterious effect of foreign compounds including plant extract on the blood constituent of animals. they can also be used to determine possible alteration in the levels of biomolecules, metabolic products, as well as histomorphology of the organs (magalhães et al., 2008). following the administration of the extracts, there was an increase in red cells count and haematocrit level, which was significant at a higher dose of 400 mg/kg as compared to the control group. mpf pulp had as better increase in red cell count when compared with mpf peel, whereas mpf peel showed a much effect in increasing the haemotocrit level as compared to mpf pulp. the results also revealed an increase in haemoglobin concentration (figures 1-3). these increments indicate that m. paradisiaca contains phytochemicals that stimulate the synthesis of erythrocytes possibly by stimulating erythropoietin formation and secretion. erythropoietin is a glycoprotein hormone which stimulates stem cells in the bone marrow to produce red blood cells (ohlsson and aher, 2009). effect of ethanol fruit extracts of musa paradisiaca on serum liver enzymes of wistar rats figure 4 shows the effect of ethanol fruit extracts (pulp and peel) of musa paradisiaca on the liver enzymes of normal wistar rat. there was a non-significant (p>0.05) change in ast, alt, and alp level of the treated rats as compared to the control. l iv e r e n z y m e s ( iu /l ) a s t a l t a l p 0 2 0 4 0 6 0 c o n tro l m p f p u lp 2 0 0 m g /k g m p f p u lp 4 0 0 m g /k g m p f p e e l 2 0 0 m g /k g m p f p e e l 4 0 0 m g /k g figure 4. the effect of ethanol fruit extracts of musa paradisiaca on liver enzymes of wistar rats. an assessment of the effect of ethanol fruit extracts of musa paradisiaca on liver enzymes of wistar rats showed a non-significant change in serum aspartate aminotransferase (ast), alanine aminotransferase (alt) and serum alkaline phosphatase (alp) levels of the treated rats as compared to the control (figure 4). serum ast, alt, and alp, which are cytoplasmic enzymes released into circulation after cellular damage are useful enzymes biomarkers in predicting liver damage (ramaiah 2011; zhao et al., 2018). alt and ast are largely used in the assessment of liver damage by drugs or any other hepatotoxins, while alp is a marker enzyme for the plasma membrane and endoplasmic reticulum (giannini et al., 2005; mcgill, 2016). the observed non-significant differences in the liver enzymes are an indication that both fruit pulp and peels extracts of musa paradisiaca are non-toxic to the hepatic cells, thus, suggesting that musa paradisiaca fruit extracts may not possess hepatotoxic effects, perhaps, a protective effect by stabilization of plasma membrane thereby preserving the structural integrity of the cell (pari and murugen, 2004). this is corroborated by the findings of iweala et al (2011) which reported significantly reduced liver enzymes level with the consumption of a musa paradisiaca-supplemented diet by wistar rats. the hepatoprotective properties of musa 70 biology, medicine, & natural product chemistry 10 (1), 2021: 67-71 paradisiaca against experimentally induced hepatotoxic models have also been reported (nirmala et al., 2012) conclusion this study evaluated the effect of ethanol fruit extracts of musa paradisiaca on haematological indices and serum liver enzymes. fruit pulp and peel of musa paradisiaca improve erythrocytes count and haematocrit level, and they may not be associated with liver toxicity. fruits of musa paradisiaca can be used as food therapy in raising red cells synthesis in anaemic conditions. conflict of interest: the authors declare that there are no conflicts of interest concerning the publication of this article. references agarwal pk, singh a, gaurav k, goel s, khanna hd, goel rk (2009). evaluation of wound healing activity of extracts of plantain banana (musa sapientum var. paradisiaca) in rats. indian j exp biol 47: 322-40. baker fi, silverton re, pallister cj (1998). baker and silvertons introduction to medical laboratory technology, 7th edition, bounty press ltd, nigeria, 339-373. basu d, kulkarni r (2014). overview of blood components and their preparation. indian j anaesth 58(5): 529-37. benzie iff, wachtel-galor s (2011). herbal medicine: biomolecular and clinical aspects. 2nd edition, crc press/taylor & francis, boca raton (fl). ekor m (2014). the growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. front pharmacol 4:177. emudainohwo jot, erhirhie eo, moke eg, edje ke (2015). a comprehensive review on ethno-medicine, phytochemistry and ethnopharmacology of chrysophyllum albidum. journal of advances in medical and pharmaceutical sciences 3(4): 147-154 ezekwesili cn, ghasi s, adindu cs, mefoh nc (2014). evaluation of the anti-ulcer property of aqueous extract of unripe musa paradisiaca linn. peel in wistar rats. african journal of pharmacy and pharmacology 8(39): 1006-1011. fagbemi jf, ugoji e, adenipekun t, adelowotan o (2009). evaluation of the antimicrobial properties of unripe banana (musa sapientum l.), lemon grass (cymbopogon citratus s.) and turmeric (curcuma longa l.) on pathogens afr j biotechnol 8(7): 1176-1182. giannini eg, testa r, savarino v (2005). liver enzyme alteration: a guide for clinicians. cmaj 172(3): 367-79. issa mt, agbon an, balogun su, mahdi o, bobbo ka, ayegbusi fo (2018). hepatoprotective effect of methanol fruit pulp extract of musa paradisiaca on carbon tetrachlorideinduced liver toxicity in wistar rats. j exp clin anat 17: 1-7 iweala eej, obichi ic, omotosho oe (2011). biochemical and histological responses of hepatotoxic rats fed musa paradisiaca l. supplemented diet. int j pharmacol 7(4): 471477. magalhães p, appell h, duarite j (2008). involvement of advanced gyration and production pathogenesis of diabetes complication: the protective role of regular physical activity. eur rev aging phys act 5: 17-29. mahomoodally mf (2013). traditional medicines in africa: an appraisal of ten potent african medicinal plants. evid based complement alternat med, 617459. mcgill mr (2016). the past and present of serum aminotransferases and the future of liver injury biomarkers. excli j 15: 817-828. moke eg, anachuna kk, edje ke, ojezele mo (2019). hepatoprotective effect of methanol seed extract of citrus tangerina on paracetamol-induced hepatotoxicity in wistar rats. niger j nat prod med, 2019, 23: 83-87. moke eg, omorodion li, akpoguma ha, imere p, ahante e (2017). anti-ulcerogenic activity of aqueous extract of unripe fruit of musa sapientum linn in combination with vitamin c on ulcer induced models in experimental rats. eur j med plants 19(2): 1-6 mondal h, budh dp (2020). hematocrit [updated 2020 jul 10]. in: statpearls [internet]. treasure island (fl): statpearls publishing; 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(2018). comparison of the hepatoprotective effects of four endemic cirsium species extracts from taiwan on ccl4-induced acute liver damage in c57bl/6 mice. int j mol sci 19(1329): 1-18. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 235-238 | doi: 10.14421/biomedich.2024.131.235-238 issn 2540-9328 (online) the study of biosurfactant stability and the effect on lipase activity mieke alvionita1,*, rukman hertadi2, rahmat rizki fazli3, andi alfira ratna faradisa dewi1, tasyang oktavia rose1 1department of chemistry, faculty of mathematics and natural sciences, state university of malang, indonesia. 2bandung institute of technology, jalan ganesa no 10 coblong, west java, indonesia. 3faculty of pedagogy and education, syiah kuala university. jalan syech abdurrauf no 4 23111 banda aceh, aceh, indonesia. corresponding author* mieke.alvio.fmipa@um.ac.id abstract lipase is one of hydrolase enzyme that catalyzed hydrolysis reaction of triacylglycerols into free fatty acids and monoglycerides or glycerol. these biocatalysts are widely used in several industries, namely food and pharmaceutical industry. the activity of lipase can increase significantly if the substrate forms an emulsion. since biosurfactant has been known to have emulsification characteristic, the effect of biosurfactant addition into lipase is necessary to be investigated. it is the first report that evaluate the effect of microbial surfactant on lipase activity. the purpose of this research is to analyze the stability of biosurfactant emulsion under various conditions, such as salinity and ph as well as their potential to enhance lipase activity. biosurfactant used was halomonas elongata bk-ag18 from the collection of biochemistry research group itb, bandung, indonesia. it was found that after the addition of nacl, there was no significant decrease in the emulsification activity of the biosurfactant. the emulsification index (ie24) of biosurfactant with several nacl concentrations (2, 5, 10, 15, 20, and 25%) was obtained around 50%. stability test of biosurfactant at ph range 4-10 showed the highest ie24 of biosurfactant was obtained at ph 6. the effect of biosurfactants on lipase hydrolysis activity is also discussed in this article. lipase hydrolysis activity was tested using p-nitrophenyl palmitate substrate. the highest lipase activity was obtained after the addition of 70% biosurfactant (v/v) at 0.026 units. this study shows that biosurfactant from h. elongata bk-ag18 has the potential to increase lipase activity. keywords: biosurfactans; bledug kuwu; h. elongata; lipase activity; stability. introduction lipase is a type of water-soluble hydrolase enzyme that has the ability to hydrolyse triacylglycerols into free fatty acids and monoglycerides or glycerol (yadav, et al., 2017). this ability is utilised in the food and pharmaceutical industry, biodiesel, and biomederation (melani, tambourgi and silveira, 2020). guerrand (2017) reported that the dairy industry used lipase to break down milk fat and give cheese its unique flavour. lipase can also modify fats and oils to produce biodiesel (silveira, tardioli and farinas, 2016), while in the pharmaceutical industry lipase can synthesise chiral intermediate compounds to produce polixatel as an anticancer drugs (fukaya, et al., 2016). lipase can be isolated from plants, animals, fungi and bacteria. when bacteria produce lipase, intermediates (by-products) are also produced, namely surfactants and known as biosurfactants. there are two types of surfactants, synthetic (artificial) and derived from living things called biosurfactants. biosurfactants are surface-active amphiphilic compounds that have surfactant properties, consisting of hydrophilic and hydrophobic parts (xi, ping and alikhani, 2021). the two substances function to reduce surface tension and interference between two immiscible substances (sharma and oberoi, 2017) by forming interfacial micelles (roy, 2017). in addition, biosurfactants can assist lipase to bind to the oil so that lipase can easily degrade the long chains of tag. biosurfactants are preferred over synthetic surfactants because of their environmental friendliness, low toxicity, and biodegradability (santos, et al., 2016), whereas synthetic surfactants have properties that are opposite to surfactants derived from living things. markande, et al. (2021) explained that biosurfactants are widely used environmentally (biomederation, wastewater treatment, soil health, biofilm); industrial sector (food, textile, chemical, petroleum); agricultural sector as antifitogenic, animal manure management; health as antitumor, antimicrobial, antioxidant, antiinflammatory. many studies on biosurfactants isolated from microbes have been done in the past including ochrobactrum intermedium (zarinviarsagh, ebrahimipour and sadeghi, 2017), serratia (oliveira, et al., 2021), burkholderia sp. (carvalho-gonçalves and manuscript received: 04 april, 2024. revision accepted: 14 june, 2024. published: 28 june, 2024. https://doi.org/10.14421/biomedich.2024.131.235-238 236 biology, medicine, & natural product chemistry 13 (1), 2024: 235-238 gorlach-lira, 2018). it was reported by spreb, et al. (2017) that the correlation of emulsification activity of aspergillus strains with the biosurfactants produced was very high. rahayu, et al. (2019) also reported linear lipase activity with biosurfactant activity isolated from keratinolytic bacteria. lipase activity can increase significantly if the substrate forms an emulsion, so lipase activity depends on hydrophobic and hydrophilic regions. therefore, lipase is defined as a carboxylesterase that reacts with the substrate to form an emulsion. it is known that biosurfactants have emulsification activity, therefore this study aims to determine whether lipase activity decreases or increases due to the addition of biosurfactants. materials and methods study area bledug kuwu is a crater mud located in semarang, central java province, indonesia. sampling was conducted in sample point 7°07'04"s 111°07'17"e. procedures biosurfactant stability and emulsification index (ie24) the stability of biosurfactant was conducted by performing biosurfactant emulsification test on various concentrations of nacl and buffer with ph range 4-10. nacl solution was made with various concentrations (2, 5, 10, 15, 20, and 25% (b/v)) then 2 ml of nacl solution was put into a test tube, added to each tube 2 ml oil and 2 ml biosurfactant. homogenised using a vortex for 2 minutes. after 24 hours, the ie24 value was determined. glycine buffer was prepared with a ph range of 4-10. the ie24 value in the ph range of 4-10 was carried out in the same way as the emulsification test at various nacl concentrations. lipase activity test the standard curve of p-nitrophenol was prepared with a concentration variation of (0, 10, 20, 30, 40, 50, 60, 70) 120 μg/ml. afterwards, biosurfactant solution (cmc = 275 mg/l) with concentrations of 0%, 10%, 30%, 50%, 70%, and 100% of the total volume of glycine buffer was prepared. lipase activity test was carried out by preparing substrate solution (ph = 7.0) for blank, sample, and control made in a centrifuge tube by: 960 μl buffer and 100 μl ethanol (blank); 950 μl buffer, 100 μl ethanol, 10 μl pnpp (sample); 950 μl buffer, 100 μl ethanol, 10 μl pnpp (control). to determine the occurrence of the enzymatic reaction, each substrate solution was mixed and added with the mixture as follows: 900 μl of substrate solution and 300 μl of soluble buffer (blank); 900 μl of substrate solution, 300 μl of crude porcine extract, biosurfactant solution of various concentrations (0, 10, 30, 50, 70, 100%) (sample); 900 μl of substrate solution and 300 μl of crude porcine extract that had been boiled at 80°c for 30 minutes. if it was homogeneous, the incubation process was continued for 15 minutes in a 70°c incubator. afterwards, centrifuged at 12000 rpm for 10 minutes. the absorbance of blank, sample, and control was measured at a wavelength of 405 nm in duplo. data analysis ie24 value was determined by the following equation. he stands for height of emulsion layer and hs for total height of the solution and emulsion mixture 𝐼𝐸24 = ℎ𝑒/ℎ𝑠 × 100% results and discussion results stability of biosurfactant in nacl solution figure 1. emulsification activity of biosurfactants at various nacl concentrations. stability of biosurfactant at ph change figure 2. emulsification activity of biosurfactants at various ph. effect of biosurfactants on lipase activity figure 3. lipase activity after the addition of biosurfactant (v/v) alvionita et al. – the study of biosurfactant stability 237 discussion the stability of biosurfactant in certain temperature, ph, and salinity range determines the application of the biosurfactant. in this study, only the influence of salinity and ph conditions on the stability of biosurfactant was conducted. the stability of biosurfactant produced by h. elongata bk-ag18 in this study was measured by looking at its emulsification activity (ie24) in various nacl levels, specifically 2, 5, 10, 15, 20 and 25%. biosurfactant is said to have low emulsification activity if it has a low ie24 value, and vice versa. the ie24 value of biosurfactant at several salt concentrations is shown in figure 1. the emulsification index of biosurfactant without nacl addition has a percentage of 53.9%. interestingly, the higher concentration of nacl added did not cause a significant decrease in biosurfactant activity. this is shown from the ie24 value of 45.3% at 25% nacl concentration. the stability of biosurfactants at various salt concentrations was proven by several researchers. the findings by purwasena, et al. (2019) stated that salinity does not significantly affect the stability of biosurfactants. in addition, the emulsification activity of biosurfactants also did not change drastically when adding nacl up to 10% (saikia, et al. 2012). according to bognolo, g. (1999) biosurfactants have a better ionic strength tolerance than synthetic surfactants that deactivate at 2-3% salt concentration. this ability of biosurfactants is needed especially for microbial enhanced oil recovery (meor) and bioremediation of oil spills in oceans that have extreme salinity conditions (prieto, et al., 2008). figure 2 shows the effect of ph value between 4-10 on the emulsification activity of biosurfactant from h. elongata bk-ag18. starting from ph 4-6, there was a slight increase in biosurfactant emulsification activity until it reached its peak where almost 50% emulsification activity at ph 6. at ph 7-9, there was a slight decrease, followed by a very significant decrease at ph 10 where only 34.4% emulsification activity occurred. according to ahmad, et al., (2021), the emulsification activity of biosurfactants decreases at high ph levels. different from the study conducted by zambir, et al. (2017), where biosurfactant from streptomyces sp. r1 bacteria showed relatively the same stability over a wide ph range (2-12) as proven by the relatively constant surface tension value at that ph. this study investigates the effect of biosurfactant addition on lipase activity using the p-npp method. so far, there have been many studies on the effect of surfactant addition on lipase activity. however, this study is the first to use crude biosurfactant from h. elongata bk-ag18 to test its effect on lipase activity from burkholderia sp. surfactants and lipase can interact with each other, either in solution or at the interface. several types of surfactant interactions with lipase enzymes can form micelle aggregates in solution or compete with lipase in the adsorption process. at low concentrations, surfactants can form complexes with enzymes, yet at high concentrations surfactants can also cause unfolding of the enzyme structure. the type and concentration of surfactant significantly determines the consequences of its interaction with lipase (delorme, 2011). this shows that surface active agents can have both positive and negative effects on the lipase itself. therefore, the effect of biosurfactants on lipase is very interesting to study. in this study, it has been discovered that biosurfactants have the ability to emulsify palm oil up to 50%. the activity of emulsifying insoluble substrates such as oil or fat may affect the interaction between the substrate and lipase. based on figure 3, lipase activity without the addition of biosurfactant showed a value of 0.023 units. after the addition of 10% biosurfactant, there was a slight increase in lipase until it reached its peak (0.026 units) as a result of the addition of 70% biosurfactant. a decrease in lipase activity began to occur after the addition of 90% biosurfactant and the lowest activity (0.022 units) was shown in the addition of 100% biosurfactant. this shows that crude biosurfactant at certain concentrations can increase lipase activity despite the increase is not very significant. some previous studies proved that surfactant can have a positive effect on lipase activity. a study conducted by oliveira, et al. (2017) proved that the addition of triton x-100 surfactant slightly increased the catalytic activity of lipase from rhizomucor miehei (rml). conclusions therefore, from this study can be conclude that the emulsification activity of biosurfactant from h. elongata bk-ag18 was not significantly decreased by the addition of nacl concentration up to 25%. this indicates that the biosurfactant has good stability in high salinity conditions. in contrast, ph has a more significant effect on emulsification activity than salt concentration. in this study, the biosurfactant was most stable at ph 6. in addition, this study also showed that the addition of biosurfactant at different concentrations showed different effects on lipase activity. the best lipase activity was shown by the addition of 70% biosurfactant. acknowledgements: this research is supported by funding from pnbp universitas negeri malang 2021 with contract number: 5.3.523/un32.14.1/2021. authors’ contributions: rukman hertadi designed the study. mieke alvionita carried out the laboratory work & analyzed data. mieke alvionita, rahmad rizki fazli, andi alfira ratna faradisa dewi, and tasyang oktavia rose wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. 238 biology, medicine, & natural product chemistry 13 (1), 2024: 235-238 funding: this research is supported by funding from pnbp universitas negeri malang 2021 with contract number: 5.3.523/un32.14.1/2021. references ahmad, z. et al. 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(2017). lipase and biosurfactant from ochrobactrum intermedium strain mzv101 isolated by washing powder for detergent application. lipids in health and disease. 16(1), p. 177. doi:10.1186/s12944-017-0565-8 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1331-1338 | doi: 10.14421/biomedich.2025.142.1331-1338 issn 2540-9328 (online) utilization of qr code technology in population management and identification of cattle in sindue district, central sulawesi yudi mujayin*, hauris, fitrah fakhirah, alda pratiwi faculty of animal husbandry and fisheries, tadulako university. jl. soekarno hatta no.km. 9, tondo, kec. mantikulore, kota palu, sulawesi tengah 94148, indonesia. corresponding author* yuditangahu23@gmail.com manuscript received: 07 october, 2025. revision accepted: 29 november, 2025. published: 14 december, 2025. abstract the implementation of a qr barcode-based recording system in sindue district aimed to improve livestock management efficiency. however, digital transformation in rural livestock management faces challenges such as a lack of technological understanding and limited smartphone access. the objective of this study was to analyze livestock farmers' perceptions of the recording system and digital transformation. the method used in this study was a survey with structured interviews (questionnaires) with 66 sample livestock farmers. data analysis used descriptive statistics and correlation. the results showed that farmers' perceptions of qr barcode technology fell into the "quite agree" category, with a total score of 398. the knowledge subvariable showed limited understanding of qr barcode technology (63.8% did not understand), but awareness of its benefits received a high response (83.3% agreed). the socio-cultural aspect received a very positive response (score 195), while the constraints subvariable received a low response (score 106). farmers support the implementation of a digital qr barcode recording system, although implementation remains hampered by limited technical understanding. furthermore, correlation analysis shows a significant relationship between the recording system and digital technology (p < 0.05), with the strongest relationship being between sociocultural aspects and constraints (r = 0.750). keywords: digital transformation; perception; qr barcode; recording. introduction the digitalization of cattle recording systems using qr barcodes represents a crucial innovation for improving the efficiency of data management related to cattle production and reproduction (mishra et al. 2022; džermeikaitė et al. 2023). in sindue district, this program was initiated in 2022 through livestock extension officers, encompassing the recording of animal identity, health history, and ownership (mawar & nurapiah 2015). however, the implementation of this digital transformation at the farmer level still faces several challenges, such as low digital literacy among farmers and limited smartphone infrastructure (holzinger et al. 2022; yuan & sun 2024). farmers’ perceptions as end-users play a key role in determining the program’s sustainability and are influenced by various factors, including knowledge, socio-cultural aspects, and practical constraints (ponce et al. 2016). accurate information regarding livestock productivity, health, and reproduction is essential for enabling farmers to make informed decisions and for providing a basis for government monitoring and policy formulation (garmendia-lemus et al. 2024; schillings et al. 2024). to enhance the efficiency of recording livestock production, reproduction, and health data, the government has initiated a digitalization program using qr barcodes to improve data accuracy and accessibility (hernandez et al. 2023). the technology employed to accelerate this transformation utilizes qr barcodes attached as eartags on cattle ears, which can be accessed through smartphones (alsuhibany 2025). although the initial adoption rate was low and progressed slowly due to farmers’ limited understanding, continuous outreach and training by livestock extension officers have gradually increased its implementation. however, the success of this program is strongly influenced by farmers’ perceptions of the technology (paul & naikar 2024). factors such as age, education level, and farming experience can affect the degree of acceptance of digital technologies. perception is an individual’s process of interpreting an object based on experience, knowledge, and social environment (bhattacharya & singla 2024)). in the livestock sector, perception plays a crucial role in determining the acceptance of new technologies such as digital recording systems. age, education, and farming experience are key determinants of response maturity (rostan 2025). https://doi.org/10.14421/biomedich.2025.142.1331-1338 1332 biology, medicine, & natural product chemistry 14 (2), 2025: 1331-1338 the qr barcode stores livestock data in a digital format that can be accessed via smartphones. this technology enhances recording accuracy, reduces manual errors, and facilitates the tracking of livestock health history (siew et al. 2023). a study by morais et al. (2019) demonstrated that its implementation in a teaching farm increased data storage efficiency by up to 40%. the qr barcode is a two-dimensional technology capable of storing large amounts of data that can be easily accessed using smartphones. its application in livestock management includes animal identification, health recording, and production history tracking (neethirajan et al. 2017). a recording system refers to the process of documenting livestock data, including identification, production, reproduction, and health. proper recording can improve productivity and management efficiency (ajibola et al. 2020). therefore, this study was conducted to analyze farmers’ perceptions of the implementation of a qr barcode-based recording system in sindue district and the transformation of livestock recording practices through digital technology adoption. materials and methods study area this study was conducted in sindue district, donggala regency, central sulawesi, indonesia. sindue district is characterized by a predominantly agricultural landscape with mixed farming systems, where cattle rearing represents one of the main sources of livelihood. the area has a tropical climate with an average temperature ranging between 25–33°c and annual rainfall of approximately 2,000–2,500 mm. the study locations were selected based on the concentration of smallholder cattle farmers who had participated in the qr barcodebased livestock recording program initiated by local livestock extension officers since 2022. procedures data collection data were collected from january to april 2025 using a descriptive survey method. primary data were obtained through structured interviews and questionnaires distributed to cattle farmers participating in the qr barcode recording program. the questionnaire was designed to assess farmers’ perceptions of the technology in terms of usefulness, ease of use, accuracy, and accessibility. additional data were gathered through field observations to verify the implementation of qr barcode tagging on cattle eartags and its functionality using smartphones. sampling technique a purposive sampling technique was employed to select respondents who had been involved in the digital livestock recording program. a total of 50 cattle farmers from different villages within sindue district were selected as respondents. selection criteria included ownership of cattle registered in the program and willingness to participate in interviews. instruments and validation the research instruments consisted of structured questionnaires containing both closed and open-ended questions. the questionnaire’s validity was tested through expert judgment involving livestock extension officers and academics from the faculty of animal science, universitas tadulako. reliability testing was conducted using cronbach’s alpha, with a coefficient value above 0.70 considered acceptable for internal consistency. data analysis quantitative data obtained from questionnaires were analyzed using descriptive statistics (mean, percentage, and frequency distribution) to summarize farmers’ demographic characteristics and perceptions. the perception scores were categorized into three levels: positive, neutral, and negative. to identify relationships between perception and farmer characteristics (e.g., age, education, and farming experience), a chi-square test and spearman’s rank correlation were applied using spss version 26. qualitative data from interviews and observations were analyzed thematically to complement and explain the quantitative findings. results and discussion farmer age the classification of farmers based on age in sindue district, donggala regency, is presented in table 1. table 1. classification of farmers by age in sindue district. age (years) number of farmers percentage (%) 0-14 0 15-63 63 95.45 >64 3 4.55 total 66 100 based on table 1, it can be seen that the highest percentage (93.33%) falls within the age range of 15–63 years. this indicates that the farmers in sindue district, donggala regency, belong to the adult (productive) age category, suggesting that they are more mature in making decisions. emishyants et al. (2020) stated that a person’s ability and maturity in thinking are also influenced by age. education level the characteristics of farmers based on their level of education are presented in table 2. mujayin et al – utilization of qr code technology in population management … 1333 table 2. classification of farmers by education level in sindue district. education level number of farmers percentage (%) elementary school 11 16.7 junior high school 17 25.75 senior high school 36 54.55 bachelor’s degree 2 3.0 total 66 100 based on table 2, it is known that the majority of farmers (54.55%) have completed senior high school education. this indicates that the farmers’ education level is relatively adequate, enabling them to absorb information and apply new innovations. education is an important factor for farmers, particularly in sindue district. however, there are still farmers with a low level of education (elementary school), which often causes difficulties in accessing information and the latest technology, thereby hindering the development of their farming businesses. this is in line with phiri et al. (2019), who stated that a low level of education makes it difficult for farmers to obtain information necessary for developing their enterprises. education is closely related to farmers’ attitudes and mindsets in decision-making. livestock farming experience the characteristics of farmers based on their livestock farming experience are presented in table 3. table 3. classification of farmers by livestock farming experience in sindue district farming experience (years) number of farmers percentage (%) 1-10 24 36.36 11-20 35 53.03 21-25 7 10.61 total 66 100 based on table 3, the classification of farmers according to their livestock farming experience in sindue district, donggala regency, shows that out of 66 surveyed farmers, the majority (53.03%) have between 11–20 years of experience. this indicates that most farmers fall into the experienced category. the longer the livestock farming experience, the more opportunities farmers have to learn from experience, making it easier for them to adopt technological innovations related to their cattle farming practices. manevska-tasevska et al. (2013) stated that longer farming experience allows farmers to develop useful skills that help them achieve more optimal results. knowledge the characteristics of farmers based on their livestock farming experience are presented in table 4. table 4. classification of farmers by livestock farming experience in sindue district. indicator response category score frecuency of farmers total percentage (%) farmers’ understanding of qr barcode highly understand 3 16 48 24.25 understand 2 35 70 53.03 do not understand 1 15 15 22.72 subtotal 45 135 100 farmers’ understanding of the benefits of using qr barcode highly understand 3 45 135 68.19 understand 2 21 42 31.81 do not understand 1 0 0 0 subtotal 66 177 100 based on table 4, the results indicate that farmers in sindue district, donggala regency, “agree” in terms of their knowledge of qr barcode technology. this finding suggests that the majority of farmers have a fairly good understanding of the qr barcode concept. this can be attributed to the fact that most farmers have a senior high school education level, which is sufficient to adapt to technological advances in the livestock sector. it also demonstrates the importance of education as a foundation for accessing and utilizing modern technology. however, some farmers expressed lower levels of agreement or limited understanding regarding how to use qr barcodes. one influencing factor is farmers’ dependence on agricultural extension officers to perform qr barcode scanning. as a result, those who are less familiar with the technology often do not take the initiative to learn how to use the qr barcode (ear tag) independently. in line with the study by andriati (2022) on cattle recording using qr and barcode technology in a project-based learning model at the faculty of animal science, university of mataram, it was shown that cattle data recorded using qr and barcode systems at the teaching farm were well-stored and maintained in the livestock database via google drive. this storage method made data retrieval easier when needed and was more reliable compared to paper-based records, which are easily damaged. thus, using google drive provides an effective solution for long-term data storage. this finding indicates the potential to expand educational programs that could help improve farmers’ understanding of qr barcode utilization. research by 1334 biology, medicine, & natural product chemistry 14 (2), 2025: 1331-1338 rahman et al. (2021) supports this, stating that the application of information technology such as qr barcode systems can enhance efficiency in livestock management. therefore, it is essential to provide better educational access so that farmers can optimally utilize this technology. regarding the second indicator farmers’ understanding of the benefits of using qr barcode technology the results show that most farmers “strongly agree.” this is a very positive outcome, indicating that nearly all farmers believe in the advantages of using qr barcodes. one of the main benefits is the use of ear tags, which help farmers identify vaccinated cattle. with this system, the risk of damage or loss of livestock data can be minimized. farmers also feel that extension programs on ear tag installation provide tangible benefits. research by susanty et al. (2024) supports this, showing that the use of qr code technology in agricultural marketing can increase transparency and efficiency in product distribution a concept that is also relevant to livestock management. socio-cultural aspects farmers’ perceptions of the implementation of the digital cattle recording system using qr barcode technology in sindue district, donggala regency, under the sociocultural sub-variable, are presented in table 5. table 5. classification of farmers by livestock farming experience in sindue district. indicator response category score frecuency of farmers total percentage (%) farmers’ agreement with the livestock recording system program (qr barcode) strongly agree 3 54 162 81.81 agree 2 12 24 18.19 disagree 1 0 0 0 subtotal 66 186 100 level of trust in new technology strongly trust 3 46 138 69.69 trust 2 18 36 27.28 do not trust 1 2 2 3.03 subtotal 66 176 100 based on the table 5 show that the majority of farmers have a positive perception of both indicators studied. out of 66 farmers, most “strongly agree” with the implementation of the livestock recording system using qr barcode technology. this reflects a high level of confidence among farmers in the benefits of the system for improving efficiency and data accuracy in livestock management. similarly, for the second indicator, the results show that farmers have a high level of trust in new technology, with most respondents “strongly agreeing” that they trust these innovations. this attitude demonstrates farmers’ openness to change and their belief that technology can provide significant benefits to their livestock practices. the productive age range of the farmers facilitates their acceptance of qr barcode technology, as they can easily perceive its advantages. additionally, the farmers’ moderate experience levels contribute to their readiness to adopt new technologies. according to chedid et al. (2022), factors influencing attitude formation include knowledge, work experience, culture, mass media, educational institutions, and individual emotional factors. this suggests that farmers’ attitudes and behaviors play an important role in the adoption of new technologies and that their knowledge and experience influence their ability to accept and apply innovations. overall, the findings of this study confirm that digital transformation has been progressing well, as farmers exhibit strong positive attitudes toward the qr barcode recording system and new technology (kubáňová et al. 2022). the high percentage of agreement indicates the potential for successful implementation of this program in improving livestock management practices (delgado-demera et al. 2024). however, further steps are still needed to strengthen technology adoption and ensure that farmers receive adequate training to maximize the benefits of this system. constraints to identify farmers perceptions of the implementation of the digital cattle recording system using qr barcode technology in sindue district, donggala regency, the constraints sub-variable is presented in table 6. mujayin et al – utilization of qr code technology in population management … 1335 table 6. farmers perceptions of the constraints sub-variable in sindue district. indicator response category score frecuency of farmers total percentage (%) challenges faced by farmers in using qr barcode (ear tag) very challenging 3 0 0 0 challenging 2 25 50 37.88 not challenging 1 41 41 62.12 subtotal 66 91 100 level of difficulty experienced in accessing livestock data without a smartphone very agree 3 38 114 57.58 agree 2 19 38 28.78 disagree 1 9 9 13.64 subtotal 66 161 100 based on table 6, the results show that no farmers strongly agreed that they faced challenges in using the qr barcode. this indicates that the technology has been relatively well accepted by farmers. the majority of farmers disagreed that they experienced difficulties in using qr barcodes, and only a small number reported facing challenges, which were not considered significant. these findings are consistent with rahman et al. (2021), who stated that the adoption of new technology in livestock management tends to proceed smoothly when supported by adequate training and education. although these results reflect a good level of acceptance of qr barcode use, attention should still be given to farmers who reported challenges. field observations revealed that difficulties often relate to ear tags attached to the cattle’s ears. in some cases, the ear tags fell off, requiring farmers to wait for extension officers to reinstall them in their original positions. therefore, it is important to identify and address these technical issues to optimize the use of qr barcode technology in livestock practices (susanty et al. 2024). for the second indicator, the majority of farmers strongly agreed that they experienced difficulties accessing livestock data without a smartphone. this shows that smartphones are considered essential tools in livestock management. the findings align with drafor (2016), who found that access to information technology can assist farmers in decision-making and market access. overall, the total score for the constraints sub-variable falls under the “disagree” category, indicating that most farmers did not experience major issues when using the qr barcode system. for the second indicator, the majority of farmers strongly agreed that they experienced difficulties accessing livestock data without a smartphone. this indicates that smartphones are considered essential tools in livestock management. the findings of this study are consistent with previous literature, which shows that the adoption of technology in the agricultural and livestock sectors can enhance productivity and efficiency. a study by drafor (2016) revealed that access to information technology can assist farmers in decision-making and market access. based on table 6 above, it can be seen that the total score for the constraints sub-variable as a whole falls under the “disagree” category. this indicates that only a few farmers encountered problems or obstacles when using the qr barcode system. overall perception the overall perception assessment of farmers in sindue district, donggala regency, can be seen in table 7. table 7. classification of farmers by education level in sindue district. variable sub-variable score category farmers perception knowledge 310 agree socio-cultural 362 strongly agree constraints 252 disagree based on table 7, it can be seen that the overall perception of farmers falls under the “agree” category with a total score of 924. this assessment includes knowledge indicators related to farmers’ understanding of qr barcodes and their perceived benefits, which obtained a total score of 310, indicating the category “strongly agree.” this implies that the majority of farmers possess good knowledge of qr barcode technology, although a small number still feel less confident in using it. this finding highlights the need for more intensive educational programs to further improve farmers’ understanding and technical skills in using digital technology. the socio-cultural sub-variable recorded a total score of 362, categorized as “agree.” this figure reflects a strong positive attitude from farmers toward new technology and the recording system. most farmers believe that the use of this technology can improve 1336 biology, medicine, & natural product chemistry 14 (2), 2025: 1331-1338 efficiency and accuracy in livestock data recording. their open attitude toward innovation shows the potential for successful implementation of technology in the field. the constraints sub-variable obtained a total score of 252, categorized as “disagree.” this result shows that most farmers did not experience major difficulties in using the qr barcode system. although some farmers faced minor issues, the overall level of acceptance toward this technology remains high. however, attention should still be given to farmers who experience challenges, particularly related to accessing data via smartphones. overall, these findings demonstrate that farmers in sindue district generally have a positive perception of the implementation of the qr barcode-based recording system. their socio-cultural attitude toward technology is supportive, and their level of knowledge facilitates the adoption of new technology. the relatively low level of constraints indicates good acceptance of the system. therefore, to maximize the benefits of this technology, further steps such as education and training are necessary to strengthen farmers’ digital competence. therefore, to maximize the benefits of this system, further steps are needed in terms of education and training for farmers. by improving their knowledge and skills, farmers are expected to utilize this technology optimally, which will ultimately have a positive impact on productivity and efficiency in livestock farm management. this is supported by a study conducted by kentjonowaty & masyitoh (2025) on farmers perceptions of extension workers performance in developing cattle waste processing technology in kaligondo village, which reported that among the respondents, 16 farmers (53.33%) stated that the assistance provided by the extension workers was “fairly good,” while 14 farmers (46.67%) stated that it was “good.” relationship between the cattle recording system and digital technology based on the research and data analysis, the results of the correlation analysis for these components are presented in table 8. table 8. relationship between the cattle recording system and digital technology in sindue district. variable farmers knowledge socio-cultural constraints farmers 1 0.600 (p = 0.010) 0.500 (p = 0.020) 0.550 (p = 0.015) knowledge 0.600 (p = 0.010) 1 0.650 (p = 0.005) 0.700 (p = 0.003) socio-cultural 0.500 (p = 0.020) 0.650 (p = 0.005) 1 0.750 (p = 0.001) constraints 0.550 (p = 0.015) 0.700 (p = 0.003) 0.750 (p = 0.001) 1 based on the pearson correlation analysis presented in table 8, the results show that all relationships among variables have significant correlation levels at a 95% confidence interval (p < 0.05). the relationship between farmers and knowledge has a positive correlation of 0.600, which means that the greater the number of farmers, the higher the level of knowledge obtained. this finding is consistent with the study by adenuga & jack (2025), who stated that higher participation in group discussions increases farmers’ knowledge. furthermore, the relationships between farmers and the socio-cultural and constraints variables show positive correlations of 0.500 and 0.550, respectively. these results indicate that the number of farmers contributes to the improvement of socio-cultural aspects and the challenges identified. research by besser et al. (2017) also supports these findings, showing that social interactions within farming communities have a positive influence on farmers’ understanding of cultural values and the challenges they face. discussion the integration of qr barcode technology into livestock management represents a significant advancement in digitalizing agricultural practices in rural areas (manevska-tasevska 2013). the findings of this study highlight the readiness of farmers in sindue district, donggala regency, to adopt digital tools for livestock identification and data management. this readiness reflects not only their openness to innovation but also the broader transformation occurring in indonesia’s agricultural sector, where traditional practices are gradually shifting toward data-driven systems. the use of qr barcode technology provides several key advantages for livestock management. it enhances the accuracy and efficiency of data recording, minimizes human error, and ensures long-term data storage and accessibility through digital platforms (ajibola et al. 2020). these benefits align with the observations of siew et al. (2023), who emphasized that the integration of qrbased systems in animal husbandry supports better record-keeping and simplifies access to livestock data compared to manual paper-based systems. furthermore, the ability to access data in real time facilitates improved monitoring, decision-making, and traceability factors increasingly important in modern livestock production and trade. education emerges as a critical determinant in the successful adoption of this technology. farmers with higher levels of education tend to have greater digital literacy and a better understanding of the advantages offered by qr-based systems (paul & naikar 2024). this mujayin et al – utilization of qr code technology in population management … 1337 supports the argument of rostan et al. (2025), who stated that education enhances a farmer’s ability to absorb, evaluate, and apply information for innovation. the implication is that educational programs and extension services play a pivotal role in accelerating technology adoption, particularly among farmers with limited formal education. targeted training initiatives are therefore essential to ensure equitable participation and understanding among all farming groups. another important consideration is the social and cultural environment in which farmers operate. the positive socio-cultural perception identified in this study indicates that community dynamics in sindue district are conducive to collective learning and technology diffusion. farmers often exchange knowledge through informal networks, peer discussions, and collaboration with extension officers. this finding aligns with rahman et al. (2021) and hernandez et al. (2023), who emphasized that social cohesion strengthens the diffusion of technological innovations in rural communities. social capital trust, cooperation, and shared experience serves as an enabling factor that reduces skepticism toward new technologies and fosters a sense of shared responsibility in their implementation. although technological acceptance among farmers is generally high, a few constraints remain, particularly regarding accessibility and infrastructure (mishra et al. 2022). limited smartphone ownership and unstable internet connectivity in rural areas can hinder full utilization of qr barcode systems. these challenges echo the observations of schillings et al. (2024), who argued that digital agriculture can only be effective when supported by adequate infrastructure and access to information technology. addressing these barriers requires coordinated action from local governments, telecommunication providers, and agricultural institutions to improve connectivity and ensure that all farmers benefit from digital advancements. the strong relationships among knowledge, sociocultural factors, and constraints identified in this study suggest that technology adoption is not merely a technical process but a social one (džermeikaitė et al. 2023). as holzinger et al. (2022) noted, the success of technological innovation depends on how well it integrates with users’ existing practices, values, and social systems. when farmers perceive technology as compatible with their daily routines and cultural norms, they are more likely to adopt it sustainably. therefore, promoting qr barcode systems should be accompanied by community-based learning approaches that emphasize local relevance and practical benefits rather than purely technical explanations (ponce et al. 2016). from a development perspective, the adoption of qr barcode technology supports indonesia’s broader agricultural modernization goals (siew et al. 2023). by digitizing livestock data, farmers and institutions can establish more transparent, traceable, and data-driven production systems. this contributes to improved biosecurity, disease control, and resource management key components of sustainable livestock farming. moreover, integrating technology into rural livestock systems aligns with the national agenda for “smart farming,” which seeks to enhance productivity while ensuring environmental and economic sustainability (phiri et al. 2019; andriati 2022). conclusions the adoption of qr barcode technology in livestock management in sindue district had been well received by farmers, showing their readiness to embrace digital innovation. supported by adequate education, farming experience, and strong community engagement, farmers demonstrated positive attitudes toward technology use. although minor challenges such as limited smartphone access and unstable internet connectivity remain, these do not significantly hinder adoption. strengthening education, training, and collaboration among stakeholders is essential to ensure sustainable implementation and improve efficiency, accuracy, and traceability in livestock management. acknowledgements: the authors gratefully acknowledge tadulako university for its valuable support in the completion of this research. authors’ contributions: yudi mujayin designed the study. fitrah fakhirah analyzed the data. hauris & alda pratiwi wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. funding: this research received no specific grant or funding from any public, commercial, or not-for-profit organization. references adenuga, a. h., & jack, c. 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(2024). practices, challenges, and future of digital transformation in smallholder agriculture: insights from a literature review. agriculture; basel, 14(12). biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 133-136 | doi: 10.14421/biomedich.2022.112.133-136 issn 2540-9328 (online) the triterpenes of kageneckia oblonga jorge tapia-merino1,*, lily arrue2, josé san martín3, orlando muñoz4 1universidad bernardo o´higgins, facultad de salud, departamento de ciencias químicas y biológicas, general gana 1702, santiago, chile. 2centro de investigación de estudios avanzados del maule (cieam) vicerrectoría de investigación y postgrado, universidad católica del maule, chile. 3instituto de ciencias biológicas, universidad de talca, talca, chile. 4departamento de química, facultad de ciencias, universidad de chile, casilla 653, santiago, chile. corresponding author* jorgeat@uc.cl manuscript received: 27 june, 2022. revision accepted: 22 july, 2022. published: 01 august, 2022. abstract three known triterpenes were isolated from leaf extracts of kageneckia oblonga by conventional chromatographic methods: ursolic acid, benthamic acid and a third called kc-iii. the structure of kc-iii was determined by rmn spectroscopy, ft-it and hr-ms. the compound was identified as fern-7-en-3β-ol (motiol), not previously reported in kageneckia. keywords: kageneckia oblonga; motiol; triterpene derivative; hopane. introduction kageneckia oblonga ruiz & pav (known as “bollén”) is an evergreen tree native to chile. it is found from the northern part of coquimbo to malleco in north-central and central chile, in dry or semi-humid soils below 1800 m elevation in the lower part of the andes range. its wood is hard and resistant, due to which the natives used it to construct tools.gay, 2010) . leaves and roots of k. oblonga are used in ethnomedicine; infusions or powders are used to treat fevers, renal and hepatic lesions and digestive problems. these medicines should be used in moderation, since an excess may cause poisoning. its use as a medicinal plant has decreased due to this property. its seeds are sometimes used to treat the “evil eye” (berguecio, nicolás garcía. pagliotti, 2008; gay, 2010; mélica. muñoz & barrera, 1981). previous studies of k. oblonga have reported triterpenes and cyanogenic glucosides, ursolic acid and benthamic acid (cassels et al., 1973); cucurbitacins and a new cucurbitane triterpene (23,24 dihidrocucurbitacin f). biological and toxicological trials of cucurbitacins isolated from different extracts of the tree showed high toxicity in dichloromethane and methanol extracts. biological activity trials to test anti-inflammatory, antipyretic and analgesic effects showed that the cucurbitacins found are partly responsible for these biological effects (delporte et al., 2002; o. muñoz et al., 2002). a screening of 31 chilean medicinal plants evaluated for anti-trypanosoma cruzi activity found that the methanol extract of showed significant inhibition in the mtt trial: mtt ic50 = 35.7 ± 40 ug/ml. these trials were performed with t. cruzi trypomastigotes (o. muñoz et al., 2013). the compound fern-7-en-3β-ol was previously isolated from several plant species, including ainsliaea yunnanensis, hibiscus cannabinus, rhododendron macrocepalum, rhodendron linearifolium and scorzonera latifolia, among others (acikara et al., 2014; ageta & ageta., 1984; nakamura et al., 1965; seca et al., 2000). the compound kc-iii has some important pharmacological properties. it has been reported that it has anti-cancer agonistic properties against the cell line thp-1 (ic50 of 1.75 µm) used to study leukemia (li et al., 2016). the presence of this triterpene in k. oblonga reinforces the biological effects of this tree. material and methods experimental section samples of k. oblonga were collected in april, 2017 near the las trancas bridge in the quebrada del cepillo sector, road g-546, 8 km from the laguna aculeo, province of maipo, metropolitan region, chile (33°50'02.3"s 71°01'17.2"w). the plants (4.2 kg) were identified by dr. josé san martin (u. of talca). a herbarium specimen was stored in the natural products laboratory of the science faculty of the university of chile, identified as n°0017-017. the leaves (used for extraction) were removed from the branches and dried for three weeks in the shade. then they were ground in an electric grinder with a 1.5 mm grill. the powdered material (2.6 kg) was placed in 5l glass recipients and degreased with hexane (3*4l), after which a sufficient https://doi.org/10.14421/biomedich.2022.112.133-136 134 biology, medicine, & natural product chemistry 11 (2), 2022: 133-136 volume of dcm was added to cover the plant material (3*3 l). the mixture was percolated with sporadic agitation for 72 hours. the extract was then filtered and concentrated by distilling to dryness under reduced pressure. this procedure was repeated three times, obtaining a total of 18g of dcm extract. extraction and isolation the total h-dcm extract was placed on a silica gel 60 chromatographic column (merck). dichloromethane was used as the mobile phase; 35 fractions were obtained. chromatographic analysis of the fractions revealed a mixture of the triterpenes ursolic acid and benthamic acid. these compounds were identified by tlc, using commercial standards and estimating their fusion points. the remaining fractions were concentrated in a rotatory evaporator, dried with anhydrous na2so4, filtered and concentrated to a yellow residue. this fraction was purified by crystallization, dissolving the sample in methanol and then cooling to 0 °c. the result was 0.14g of white kc-iii crystals. analysis of the sample some of the separation and purification steps were performed in the department of phytochemistry and bioactive natural products university of geneva unige(suiza). luhplc was performed on an acquity i-class plus uplc system hyphenated with an acquity photodiode array (pda) detector (waters). the separation was performed in an acquity beh c18 uplc column (50 mm × 2.1 mm i.d.; 1.7 μm, waters), using a generic gradient (mecn and h2o both containing 0.1% formic acid) of 5% to 98% mecn in 4 min, followed by a washing step with 98% mecn for 2 min. after the washing step, the column was equilibrated with 5% mecn for 2 min before the next injection. the flow rate was set to 0.6 ml/min, the temperature to 40 °c, and the injection volume was 1 μl. hrms: hrms spectra were obtained on a q exactive focus hybrid quadripole-orbitrap mass spectrometer (thermo scientific, waltham, ma, usa) using electrospray in positive or negative mode. the spray voltage was set at 3.5 kv or 2.5 kv; the sheath gas flow rate (n2), 50 units; the capillary temperature, 320 °c; the s lens rf level, 50 and the probe heater temperature, 425 °c. spectroscopy and complementary spectroscopic analyses of 1h-rmn, 13c-rmn, cosy, hmqc and hmbc were performed in a bruker avance 400 mhz nuclear magnetic resonance spectrometry at 25 ºc. the kc-iii sample was dissolved in deuterated chloroform. tetramethylsilane was used as internal standard. the spectra were processed using the mestre nova 9.0 program. the ft-ir spectrum was obtained from a bruker ft-ir perkin-elmer 1310 spectrometer in kbr disks recorded from 500-4000cm-1. these last rmn and ft-ir analyses were performed in the instrumentation unit of the pontificia universidad católica de chile. results compound kc-iii was identified as fern7-en-3β-ol (motiol) by spectroscopic comparison and mp: 210-213 °c. (ageta & ageta., 1984; nakamura et al., 1965; seca et al., 2000). figure 1. kc-iii structure, fern7-en-3β-ol (motiol). fern-7-en-3β-ol (motiol): translucent yellow crystals, apparently rectangular in shape. yield: 0.14g, 0.004% of dried leaf. mp: 210-213 °c. ir: 3500 cm-1, 2941 cm1,2852 cm-1,2361 cm-1,1469 cm-1,1386 cm-1. esims: 514.25 m/z, [ m+ch3cn+ hcooh] +; 498.26 m/z, [ m-ch3 + ch3cn + hcooh] +; 404.20 m/z, [ m -oh ch3cn] +; 227.10 m/z y 167.01 m/z [ fragmentation d ring] +. 1h-rmn: δ 5.37 (d, j = 3.6hz, 1h, h-7), 3.23 (1h, h-3), 2.34 (m, 1h, h-9), 2.17 (m, 1h, h-6a), 2.00 (m, 1h, h-6b), 1.82 (m, 1h, h-20), 1.73 (m, 1h,h-16a), 1.69 (m, 4h, h-1,2), 1.58 (m, 1h, h-11a), 1.54 (m, 1h, h-11b), 1.50 (m, 1h, h-16), 1.48 (m, 1h, h-18), 1.46 (m, 1h, h-15), 1.43(m, 1h, h-22), 1.40(m, 1h, h-19a), 1.35 (m, 1h, h-12a), 1.33 (m, 1h, h-5), 1.32 (m, 1h, h12b), 1.25 (m, h, h-19b) 1.24 (m, h, h-20), 1.06 (m, 1h,h-2), 0.99 (s, 3h, h-26), 0.96 (s, 4h, h-24,h-21), 0.91 (s, 3h, h-30), 0.89 (s, 3h, h-27), 0.85 (s, 3h, h23), 0.83 (d, j = 6.7 hz3h, h-29), 0.74 (s, 3h, h-25), 0.73 (s, 3h, h-28).13c-rmn: δ 145.29 (c-8), 116.28 (c-7), 79.41 (c-3), 59.71 (c-21), 54.26 (c-18), 50.89 (c-5), 48.04 (c-9), 42.99 (c-17), 41.66 (c-14), 39.10 (c-4), 36.95 (c-2); 36.41 (c-16), 36.21 (c-13), 35.48 (c-10), 32.47 (c-12), 30.82 (c-22), 30.44 (c-15), 28.37 (c-20), 27.85 (c-1), 27.67 (c-24), 24.29 (c-6), 24.15 (c-26), 23.14 (c-29), 22.26 (c-30), 21.20 (c-27), 20.15 (c-19), 16.22 (c-11), 14.76 (c-23), 14.19 (c-28), 13.01 (c-25). tapia-merino et al. – the triterpenes of kageneckia oblonga 135 discussion the ir spectrum of compound kc-iii allowed the rapid assignment of the signal at 3500 cm-1, assigned to an alcohol. the absorbances between 2350 and 2450 cm-1 were assigned to an alicyclic skeleton, with possible presence of a double bond, and the proton of neighboring hydrogens to oh. complementary analysis with reported spectroscopic data for this kind of structure allowed deducing that kc-iii should have a hopane skeleton. the mass spectrum signals showed that ion 514.25 m/z could be assigned to the sum of the masses of the sample, acetonitrile and formic acid [m+ ch3cn+ hcooh]+. the 498.26 m/z and 496.24 m/z fragments correspond to the mentioned adduct, but with a loss of 15 uma, typical of the loss of a methyl group [mch3+ ch3cn+ hcooh]+ for 498.26m/z. by the 18 m/z difference from 514.25m/z it may be inferred that the signal at 496.24 m/z is due to dehydration, [m+ ch3cn+ hcoohh2o]+. ion 404.20 m/z was assigned to the mass of the molecule, with loss of the hydroxyl at c-3 and isopropyl in c-21 forming two new double bonds in their respective carbons that underwent the elimination and addition of acetonitrile [m-oh-58]+. in consequence, compound kc-iii has the molecular formula c30h50o. the c-nmr showed 30 signals, including eight methyl groups, nine methylene groups, seven methine groups and four quaternary carbons, all deduced from the dep 135 experiments. the high-field signals of the h-nmr spectrum showed six singletons, assignable to tertiary methyl groups and two secondary methyl groups, assigned as doublets. the δ 5.37ppm signal (1h, q, j = 3.3 hz) is typical of double bonds, ratifying the ir signal at 3050 cm-1 and 1639 cm-1, while the h-3 axial singleton at δ 3.23 ppm is displaced to the low field due to the presence of the 3 β-oh group in c-3. the analysis of the bi-dimensional spectra and the conclusions of the previous paragraphs allow assigning the double bond to the b ring, and the connectivity shown in figure 2. the interactions of the hydrogens (cosy) show the correlations of the h-22 with the hydrogens of the h-29 and h-30 methyl groups; the interaction of h-22 with h-21. both interactions describe the union of the isopropanol group in the c-21 carbon, which coincides with the fractioning of the ms of 167 m/z, corresponding to a retro diels-alder ion of the e ring after losing a methyl group. the hmbc spectrum (s12) shows evidence of the union of the a-b, b-c, c-d and d-e rings by the interactions of the c-3 and h-5 atoms; h-5 with c-9, h9 with c-12; h-12 with c-18 and h-18 with c-21, respectively. the position of the geminal group formed by c-23 and c-24 in the a ring is inferred by the interactions with c-5 and h-3 with the methyl substituents in c-4. the position of c-24 between the union of the a-b rings is deduced from the interactions of c-5 and c-9. c-26 and c-27 in the union of the c-d rings is interpreted by the interaction of its hydrogens with c-13, c-14, c-15 and c-18. the c-28 methyl is assigned as the substituent in c-17 by the interactions of h-28 with the four carbons that surround c-17. the interaction of c-29 and c-30 and the interaction of its ch3 hydrogens with c-21 reaffirm the isopropyl structure in the molecule. figure 2. hmbc coupling of the kc-iii structure. conclusion the triterpene fern-7-en-3β-ol (motiol) is reported for the first time in the genus kageneckia, and thus in k. oblonga. the presence of motiol in the leaves of k. oblonga along with the presence of other substituted triterpenes and cucurbitacins would explain some of the medicinal properties of this tree. the toxicity of its 136 biology, medicine, & natural product chemistry 11 (2), 2022: 133-136 extracts recommends against its medicinal use. however, this native chilean tree has potential uses as an antiparasite, anti-inflammatory and anti-carcinogen. thus pharmacological in vitro, in vivo, pre-clinical and clinical trials should be continued, to produce the bases for a specific and sure phytopharmaceutical for the population, which self-medicates with crude extracts. acknowledgments: we warmly thank professor dr. philippe christen and dr. sylvian creton for advice and assistance with spectroscopic measurements. competing interests: the authors declare that there are no competing interests. references acikara, b., glu, g. s. c., dall’acqua, s., özbek, h., ka, j. c., žemlič ka, m., & šmejkal, k. (2014). bioassay-guided isolation of the antinociceptive compounds motiol and βsitosterol from scorzonera latifolia root extract. pharmazie, 69(9), 711–714. https://doi.org/10.1691/ph.2014.3920 ageta, h., & ageta., t. (1984). ercaceous constituyents: seventeen triterpenoids isolated from the buds of rhododendron macrocepalum. chem. pharm. bull., 32, 369– 372. https://doi.org/10.1061/͑asce͒1090-02412͑0031͒29:11͑2 ͒ berguecio, nicolás garcía. pagliotti, c. o. (2008). arboles nativos chilenos. enersis s.s. http://fundacionphilippi.cl/sites/default/files/arboles-nativosenersis.pdf cassels, b., urzúa, a., cortez, m., & gabardino, juan. (1973). triterpenoid constituents of kageneckia oblonga. phytochemistry, 12, 3009. delporte, c., muñoz, o., rojas, j., ferrándiz, m., payá, m., erazo, s., negrete, r., maldonado, s., san feliciano, a., & backhouse, n. (2002). pharmaco-toxicological study of kageneckia oblonga, rosaceae. zeitschrift fur naturforschung section c journal of biosciences. https://doi.org/10.1515/znc-2002-1-218 gay, c. (2010). historia física y política de chile. botánica, tomo vii. biblioteca fundamentos de la constrcción de chile. li, j., zhang, b., liu, h., zhang, x., shang, x., & zhao, c. (2016). triterpenoids from ainsliaea yunnanensis franch. and their biological activities. molecules, 21(11), 1–7. https://doi.org/10.3390/molecules21111481 muñoz, mélica., & barrera, elizabeth. (1981). el uso medicinal y alimenticio de plantas nativas y naturalizadas en chile. museo nacional de historia natural. publicacion ocacional, 33, 63. muñoz, o., maya, j., ferreira, j., & christen, p. (2013). medicinal plants of chile: evaluation of their antitrypanosoma cruzi activity. zeitschrift fur naturforschung c, 198–202. https://doi.org/10.5560/znc.2013.68c0198 muñoz, o., ravelo, a., & gonza, a. (2002). cucurbitacin f in seeds of kageneckia angustifolia (rosaceae). zeitschrift fur naturforschung, 24–26. nakamura, s., yamada, t., wada, h., & inoue., y. (1965). the structures of five new triterpenoids obtained from rohodendron linearifolium. tetrahedron letters, 6(24), 2017– 2022. seca, a., silva, a., silvestre, a., cavaleiro, j., domingues, f., & neto, c. (2000). chemical composition of the light petroleum extract of hibiscus cannabinus bark and core. phytochemical analysis, 11(6), 345–350. https://doi.org/10.1002/10991565(200011/12)11:6<345::aid-pca540>3.0.co;2-t biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 601-608 | doi: 10.14421/biomedich.2024.132.601-608 issn 2540-9328 (online) comparison of interleukin-1-511 c/t polymorphism in schizophrenia of batak and minangkabau ethnicities in pematang siantar raysha awlia wijaya1, edy fachrial2,*, i nyoman ehrich lister3, bayu ariatama4 1magister of biomedical science student, faculty of medicine, dentistry, and health science, universitas prima indonesia. 2,3faculty of medicine, dentistry, and health science, universitas prima indonesia. 4mental health polyclinic of dr. djasamen saragih general hospital pematang siantar, indonesia. corresponding author* fachrial_edy@yahoo.co.id abstract this research is an overview of a comparative study on the interleukin-1β-511 c/t polymorphism in individuals with schizophrenia of batak and minangkabau ethnicities in pematang siantar, indonesia. the study aims to investigate genetic differences among these ethnic groups to better understand susceptibility to schizophrenia, which is influenced by multiple factors including genetics. the research uses a comparative categorical analytic approach with a cross-sectional method and involves dna isolation and polymerase chain reactionrestriction fragment length polymorphism (pcr-rflp) examinations. based on the results, significant demographic differences between the batak and minangkabau ethnic groups, particularly in gender distribution, age, onset of illness, duration of illness, and panss scores. notably, it finds a significant difference in the occurrence of the c allele and the t allele between the two ethnic groups, with the c allele being more prevalent in the minangkabau group and the t allele more so in the batak group. this suggests that the il-1β -511 c/t polymorphism may play a differential role in the susceptibility to schizophrenia among these ethnicities, indicating a potential for ethnicspecific risk factors or protective elements. keywords: polymorphism; schizophrenia; batak; minangkabau; ethnicities. abbreviations: odgj: mental disorder; ods: people with schizophrenia. introduction mental disorders remain a serious issue to date, with approximately 35 million people experiencing depression and 21 million suffering from schizophrenia in 2016. there has been an increase in the number of individuals affected by mental disorders, reaching 7 per thousand households, estimating around 450 thousand individuals with severe mental disorders (odgj). according to the indonesian law no. 18 of 2014, odgj are individuals experiencing disturbances in behavior, thoughts, and feelings manifested in symptoms and/or significant behavioral changes that can cause suffering and impair their ability to function as humans (nma, 2021). schizophrenia is a severe mental disorder characterized by profound disturbances in thinking processes, affecting language, perception, and selfawareness. the disease is marked by the presence of two or more symptoms, including delusions, hallucinations, disorganized speech, catatonic behavior, and negative symptoms. schizophrenia typically affects over 21 million people worldwide and it has been estimated that approximately 7 individuals per 1000 will develop schizophrenia during their lifetime (orrico-sánchez et al., 2020). schizophrenia has been identified as one of the major health disorders worldwide. this mental disorder has significant effects on the global population, impacting over 21 million individuals, with 50% of sufferers not receiving appropriate treatment, and 90% of those untreated occurring in developing countries. the causes of this disease are multifactorial, including genetic, neurodevelopmental, social, immunological, and degenerative factors in the central nervous system (kahn, 2020). genes play a significant role in schizophrenia, as schizophrenia patients exhibit rare gene variations resulting from gene segment deletions or duplications. many genes have been selected as indicators extensively associated with schizophrenia (damanik et al., 2020). ven and susser (van der ven & susser, 2023) and chung et.al (chung et al., 2023) has examined recent trends in the incidence of psychotic disorders, demographic characteristics, and comorbid psychiatric and medical conditions among six racial or ethnic groups. the theory suggests that individuals genetically predisposed to schizophrenia when exposed to infections manuscript received: 25 june, 2024. revision accepted: 04 december, 2024. published: 31 december, 2024. https://doi.org/10.14421/biomedich.2024.132.601-608 602 biology, medicine, & natural product chemistry 13 (2), 2024: 601-608 during pregnancy, disrupt normal brain development, mediated through the immune response to these infections (llorca-bofí et al., 2024). the main candidates implicated in these infections are cytokines associated with inflammation (called proinflammatory cytokines). cytokines are soluble polypeptide signaling proteins involved in initiating and sustaining immune responses and serve as important mediators of crosstalk between the brain and the immune system. infections will activate proinflammatory cytokines (along with other immune factors), such as tumor necrosis factor (tnf)-α, interleukin (il)-1β, and il-6, which play crucial roles in the early defense against infection and the initiation and/or development of inflammation (dinarello, 2018). furthermore, fetal exposure to proinflammatory cytokines has been associated with lesions in fetal brain white matter and developmental abnormalities, such as periventricular leukomalacia, cerebral palsy, and mental retardation (amin et al., 2020). white matter abnormalities and premorbid motor and cognitive disturbances are often found in the course of schizophrenia, indicating that fetal exposure to inflammation can cause residual neurodevelopmental symptoms associated with schizophrenia even in the absence of genetic vulnerability to schizophrenia. cytokines also play a complex role in schizophrenia, either caused by or resulting from other neuropathological processes. in the brain, cytokines may be involved in regulating several neurotransmitters, such as serotonin, noradrenaline, dopamine, and glutamate (lesh et al., 2018). cytokine interactions with dopamine and glutamate are known to be relevant to the pathophysiology of schizophrenia. several studies measuring levels of proinflammatory cytokines, such as interleukin (il)-1, -6, and tnf-α, in peripheral blood or cerebrospinal fluid of schizophrenia patients have shown dysregulation of these cytokines in schizophrenia. il-1β is an agonistic proinflammatory cytokine on the interleukin-1 receptor. il-1β is involved in the development of the nervous system, where it can have neurotoxic effects and act as a protective agent for the nervous system (liu et al., 2021; reale et al., 2021). the gene encoding interleukin-1β (il-1β) is located on chromosome 2q14. il-1β has also been proposed to modulate synaptic plasticity underlying learning and memory in the adult nervous system, with inhibitory or enhancing effects depending on the concentration where it is produced (damanik et al., 2020). some association studies in caucasian samples suggest that genetic variability in il-1β may increase the risk of schizophrenia, particularly the -511 c/t variant. such as the study conducted on 356 individuals from 89 families affected by schizophrenia, found an association between the -511 t allele in the il-1β gene promoter region with psychosis, indicating a possible role of this gene in vulnerability to schizophrenia spectrum disorders (harrison, 2015). according to lesh (lesh et al., 2018) on 533 schizophrenia patients in japan showed the first evidence that il-1β gene polymorphism is associated with vulnerability to schizophrenia in the japanese population. the population of north sumatra province, according to ethnic groups, consists of indigenous north sumatran residents, indigenous immigrant residents, and foreigners. the batak ethnic is one of the indigenous north sumatran residents. based on studies by tampubolon in 2015 and puspasari in 2019 on the characteristics of inpatients with schizophrenia at prof. dr. m. ildrem medan psychiatric hospital, it is known that the highest proportion of schizophrenia patients belongs to the batak ethnic. pro-inflammatory studies related to ethnicity have also been conducted in indonesia, especially in north sumatra. some studies include the levels of tumor necrosis factor‐ α and its relationship with cognitive function in schizophrenia in the malayan-mongoloid ethnic group by amin et al (amin et al., 2020). another study on the relationship between inf-γ+874 a/t gene polymorphism and schizophrenia in batak ods (people with schizophrenia) with control groups by damanik et al (damanik et al., 2020) and a study by saragih et al (saragih et al., 2021)on the difference in the frequency of appearance of g allele and c allele variant –g174c il-6 in the batak ods group with the control group. west sumatra ranks fourth highest in the prevalence of schizophrenia in indonesia. in north sumatra, the minangkabau ethnic is one of the indigenous immigrant residents. the minangkabau ethnic ranks second as the largest minority ethnic group in medan city after the chinese ethnic group. minangkabau society values life highly. in line with the view of life being beneficial to their families and communities, work is highly regarded. work is a necessity for minang society. wealth can shield a family from shame. there are many customary events like weddings that require significant costs. therefore, hard work is highly prioritized. this may be related to triggering factors for schizophrenia in the minangkabau ethnic. individual genetic differences among different ethnic groups can determine who is susceptible to schizophrenia or not, and identifying these variations can help provide personalized care to patients. personalized care also involves prevention and treatment for patients, based on their genetic makeup and environment. genome-wide association studies (gwas) have proven to be a useful technique in associating variations in the genome with specific diseases using statistical procedures (falola et al., 2017) the presence of schizophrenia patients is considered disruptive and even hazardous. research on interleukin1β –511 c/t polymorphism in individuals with schizophrenia (ods) is still very limited, and the research has only been conducted in a few countries with varying results. meanwhile, in indonesia, particularly in wijaya et al. – polymorphism in schizophrenia of ethnicities in pematang siantar 603 north sumatra, there has been no previous research comparing interleukin-1β –511 c/t polymorphism in ods of batak and minangkabau ethnicities. based on the above background, the researcher is interested in investigating whether there are differences in interleukin1β –511 c/t polymorphism in individuals with schizophrenia of batak and minangkabau ethnicities in pematang siantar. materials and methods this study is comparative categorical analytic research with non-paired one-time measurement comparing the groups of individuals with schizophrenia of batak and minangkabau ethnicities using a cross-sectional method. the research was conducted at the mental health polyclinic of dr. djasamen saragih regional general hospital in pematang siantar. the target population in this study is individuals with schizophrenia. the accessible population consists of patients who seek treatment at the mental health polyclinic of dr. djasamen saragih regional general hospital in pematang siantar from december 2023 to january 2024. the research sample comprises schizophrenia patients who seek treatment at the mental health polyclinic of dr. djasamen saragih regional general hospital in pematang siantar from december 2023 to january 2024. the sampling method used is non-probability sampling, specifically consecutive sampling, where all subjects who come and meet the selection criteria are included in this study until the required number of subjects is met. demographic characterization the demographic characteristics analyzed in the groups of individuals with schizophrenia of batak and minangkabau ethnicities are gender, age, age of onset, duration of illness, and total panss score following method by opler et.al (opler et al., 2024). in the healthy control group, the demographic characteristics analyzed are gender and age. inclusion and exclusion criteria the inclusion criteria include: ▪ individuals with schizophrenia of batak ethnicity diagnosed based on ppdgj-iii criteria. ▪ individuals with schizophrenia of minangkabau ethnicity are diagnosed based on ppdgj-iii criteria. ▪ having panss scores ranging from 80 to 120 and age between 18 to 45 years old. ▪ two generations of first-degree family members are of batak ethnicity. ▪ two generations of first-degree family members are of minangkabau ethnicity. ▪ cooperative and willing to be interviewed the exclusion criteria include: ▪ having a history of other psychiatric disorders. ▪ having a history of neurological diseases, endocrine disorders, and autoimmune diseases and having a history of alcohol and other addictive substance use (except nicotine and caffeine). sample the sampling technique used in this study is consecutive sampling, where all subjects who come and meet the selection criteria are included in the study until the required number of subjects is met. sample size calculation for comparing the interleukin-1β – 511c/t polymorphism in batak and minangkabau ethnicities with a non-paired categorical-comparative 2x2 table. sample size calculation is determined by establishing the effect size (p1-p2). this study requires 97 individuals with schizophrenia batak and minangkabau ethnicity respectively. data collection was carried out by the researcher and assisted by the team, preceded by screening using inclusion and exclusion criteria. individuals who met the inclusion criteria and did not have exclusion criteria were explained about the aims and objectives of the research. then the selected subjects were asked to sign an agreement/informed consent to take part in the research. subjects diagnosed with schizophrenia using the ppdgj iii diagnostic criteria were assessed using the panss interview where a panss score between 80-120 indicated that people with schizophrenia were cooperative enough to take part in the research. after that, blood is drawn from the subject and then laboratory examinations, statistical analysis, and preparation of results reports are carried out. dna isolation technique blood samples were taken in the amount of 5 cc from the anterior cubital vein. the blood will be put in vacutaner which contains ethylenediamine tetraacetic acid (edta) and stored at 4-8oc until dna isolation is carried out. the method used for dna isolation is salting out. a total of 2 ml of a peripheral blood sample containing edta was included falcon tube, then 6 ml red blood cells (rbc) lysis solution (blood ratio: rbc = 1:3), rbc lysis solution containing 199mm edta, 100 mm khco3, and 1.45 nh4cl. the tube was homogenized by turning it upside down and incubated at room temperature 27oc for 10 minutes. next, the tube was inverted again before centrifugation was carried out at a speed of 1500 rotations per minute(rpm) for 10 minutes at room temperature to obtain a white supernatant and pellet (precipitate), then supernatant thrown away. the above procedure is repeated 3 times until you get a pellet that is free from red blood cells. after that, the formed pellets are added to the cell lysis solution (cls) containing 10 mm tris-hcl, 0.25 mm edta, and 20% sds. 1.334 ml of cls was added and carried out up and down with a transfer pipette slowly until the mixture became homogeneous. the mixture was incubated deeply in water bath at a temperature of 37oc for 30-60 minutes. next, the mixture 604 biology, medicine, & natural product chemistry 13 (2), 2024: 601-608 is added protein precipitation (pp) (5 m ammonium acetate) as much as 867 μl and carried out vortex enough. the mixture was centrifuged again at 3000 rpm for 15 minutes at a temperature 4oc is thus obtained supernatant and brown pellets on the tube walls. the supernatant was transferred into a new tube containing 767 μl of cold isopropanol solution, then the tube was turned back and forth 25-30 times until dna strands were seen floating in the isopropanol solution. next, the dna in isopropanol was incubated overnight at 20oc. after overnight incubation, centrifugation was carried out at 3000 rpm for 5 minutes. the supernatant is then thrown away. next, 867 μl of cold 70% ethanol was added for washing and carried out inverted, then centrifuged again at 3000 rpm for 5 minutes at a temperature 4oc until obtained supernatant and dna pellets. dna pellets were dried for 2 hours at room temperature or 1 hour at 37°c. drying is carried out by placing the tube in an inclined position on the base tissue dry. the dna pellet was dissolved by adding 300 μl of te solution (10mm trishcl, 0.25 edta) and then incubated for 2 hours at 370c inside the water bath. next, the dna was transferred to a 1.5 ml eppendorf tube and stored at -200c, and the dna was ready for analysis for the next stage. detection of interleukin -1β -511 c/t polymorphism genomic dna is extracted from samples that have undergone freezing using standard methods. the single base polymorphism at position -511 in the interleukin-1β promoter region was read by pcr. for each allele, the pcr reaction was carried out on template dna with a pair of oligonucleotide primer, namely 5' tggcattgatctggttcatc 3' (forward) and 5' gtttaggaatcttcccactt 3' (reverse). pcr cycling conditions were denaturation at 94°c for 5 minutes followed by 30 cycles at 94°c for 30 seconds, 55°c for 30 seconds, and 72°c for 7 minutes. the pcr product was stored at 37°c for 1 night with 10 units of ava 1 restriction enzyme for il-1β and will produce products at 190 bp + 114 bp (cc) and 304 bp (tt). data analysis data normality tests will be carried out using the kolmogorov-smirnov test for a sample size of more than 50. if both variables are normally distributed and the x2 condition is met, a chi-square test will be carried out. after that, the odds ratio (or) value will be calculated. an or value of more than one is a risk factor, an or equal to one or above one indicates that the independent variable is not a risk factor, and an or of less than one indicates that the independent variable is a protective factor. data processing and analysis is carried out with the statistical software spss ver 21. results and discussion gender is a categorical variable and will be presented in a frequency distribution. this variable with the phenotype of the ods group and the control group of the batak ethnic and the minangkabau ethnic was tested using an unpaired comparative hypothesis 2x2 table with conditions chi-square fulfilled so the test used chi-square. table 1 shows the demographic characteristics of the ods (people with schizophrenia) group of the batak and minangkabau ethnicities. the batak ods group with the most subjects was 63 women (52.5%) and 57 men (47.5%). the median age was 31 years with a minimum value of 19 years and a maximum value of 44. the median of disease onset was 30 years with a minimum value of 17 years and a maximum value of 44 years. the median duration of illness was 10 years with a minimum value of 2 years and a maximum value of 20 years. the median panns score is 101 with a minimum score of 82 and a maximum score of 120. table 1. demographic characteristics of batak ods and minangkabau ods. gender ethnic group batak (n=120) minangkabau (n=120) p value man 57 (47.5) 63(52.5) woman 63 (52.5) 57(47.5) 0.001* age (years) 31 (19-41) 30(19-44) onset 30 (17-44) 29.5(19-44) long time of illness 10(2-20) 10(1-20) <0.001** total score panss 101 (82-120) 99.5(86-110) *chi-square with continuity correction **mann_whitney u ods = people with schizophrenia in the ods group of the minangkabau ethnicities, the majority of subjects were 63 men (52.5%) and 57 women (47.5%). the median age is 30 years with a minimum value of 19 years and a maximum value of 44 years. the median disease onset was 29.5 years with a minimum value of 19 years and a maximum value of 44 years. the median duration of illness was 10 years with a minimum value of 2 years and a maximum value of 20 years. and wijaya et al. – polymorphism in schizophrenia of ethnicities in pematang siantar 605 the median panns score is 99.5 with a minimum score of 86 and a maximum score of 110. the results of the analysis show that there are significant differences between the two ethnic groups in terms of gender and value =0,000. there were also significant differences in age, onset, duration of illness, and total panss scores in the two ethnic groups value =0,000. in terms of demographic characteristics, the largest sample was women in the ods group of the batak ethnic and men in the ods group of the minangkabau ethnic, namely 63 people. there were significant differences in gender between the two groups, and significant differences were found in age, onset, duration of illness, and total panss scores in the two ethnic ods groups. the median age in the ods group of the batak ethnic was 31 years, with a minimum age of 19 years, and a maximum value of 44 years, while the age in the ods group of the minangkabau ethnic was found to be a median of 30 years, with a minimum of 19 years and a maximum value of 44 years. charlson et al. (charlson et al., 2018) stated that no gender differences were observed in global prevalence and around 70.8% of schizophrenia disorders were found in those aged 25-54 years with the highest prevalence in their 40s and decreasing in older age groups. the median onset in the ods group of the batak ethnic is 30 years, with a minimum value of 17 years and a maximum of 44 years. in the ods group of the minangkabau ethnic, the median onset was 29 years with a minimum of 19 years and a maximum of 44 years. the median duration of illness in the ods group of the batak ethnic is 10 years with a minimum of 2 years and a maximum of 20 years, and in the ods group of the minangkabau ethnic the median duration of illness is 10 years with a minimum of 1 year and a maximum of 20 years. schizophrenia can occur in all age groups, where schizophrenia disorder is characterized by a pro-moral stage that lasts for several years and causes social consequences. the onset of schizophrenia shows a sharp increase reaching a peak at the age of 15 to 25 years in men, while in women the onset is at the age of 15 to 30 years at the first peak and the second peak at the age of 44 to 49 years (charlson et.al., 2018). the most common initial onset of this disease is 15-30 years of age and is a chronic disease that causes disruption to the patient and family and has a major social and economic impact. the median total panss score in the ods group of the batak ethnic is 101 with a minimum score of 82 and a maximum score of 120, while in the ods group of the minangkabau ethnic the median panss score is 99.5 with a minimum score of 86 and a maximum of 110. the total panss score was divided into 3 parts, namely low (<75), medium (75-94), and high (>95), in patients with a medium total panss score, compared to a low panss total score (purnama et al., 2023). schizophrenia is not a common mental disorder, affecting 20 million people worldwide. schizophrenia is a complex mental disorder that generally occurs earlier in men and is influenced by genes with mild to moderate effects and non-genetic risks such as environmental and psychological factors that change the chemical structure of the brain (nasution et al., 2023). differences between the il-1β -511 c/t variant allele in ods of the batak and the minangkabau ethnicities il-1β -511 c/t variant, the allele consists of two, namely the c allele and the t allele. the allele variable is a categorical variable, where the data is presented in a frequency distribution. the relationship between the il1β -511 c/t gene allele and schizophrenia is expressed odds ratio/or obtained from the risk estimate chi-square test (table 2). batak ods group, the frequency of occurrence of the c allele is 98 times (40.8%) and the t allele is 142 times (59.2%). for the ods group of the minangkabau ethnic, the frequency of occurrence of the c allele was 127 times (52.9%) and the t allele 113 times (47.1%). the p-value was found to be 0.008 with the or being 0.614 with a 95% confidence interval between 0.428 to 0.881 (table 2). table 2. differences between the il-1β -511 c/t variant allele in ods of the batak and the minangkabau ethnicities. allele ethnic group p value or (95% ci) batak (n=120) minangkabau (n=120) c 98(40.8) 127(52.9) 0.008 0.614(0.428-0.881) t 142(59.2) 113(47.1) *ods= people with schizophrenia based on the research results, it was found that the frequency of occurrence of the c allele in the ods group of the batak ethnic was 40.8% and the t allele was 59.2%. meanwhile, in the ods group of the minagkabau ethnic, the frequency of occurrence of the c allele was 52.9% and the t allele was 47.1%. the occurrence of the c allele in the ods group of the minangkabau ethnic is 29 times more than in the ods group of the batak ethnic. furthermore, the occurrence of the t allele in the ods group of the batak ethnic was 29 times more than in the ods group of the minangkabau ethnic. furthermore, based on chi-square analysis, the p-value = 0.008 was obtained, indicating that there was an insignificant relationship between the allele and 606 biology, medicine, & natural product chemistry 13 (2), 2024: 601-608 schizophrenia. the or value obtained was 0.614 with a 95% ci between 0.428-0.881. this shows that in the ods of the batak and minangkabau ethnic, the t allele was found to have a 0.008 times probability of experiencing schizophrenia compared to individuals who had the c allele. the or value was below 1, so the t allele was a protective factor that caused schizophrenia. zanardini et.al's research stated that the proportion of the c allele appeared significantly more frequently in the ods group than in healthy controls with a p value of 0.047. furthermore, sasayama et.al stated that male ods was at an average age of 43.3 years and female ods was at an average age of 44.8 years, finding that the proportion of the c allele was more frequent in the healthy control ods group. the significant difference in the il-1β -511 c/t polymorphism can be a risk factor for schizophrenia in the minangkabau community compared to the batak community. risk factors for schizophrenia start from developmental processes, neurodegenerative and neurotransmitter disorders to autoimmune processes or infections (dinarello, 2018). cytokines and other growth factors are associated with the genesis of schizophrenia or pathology and it is found that the central nervous system (cns) contains and utilizes the receptors. cytokines are polypeptides and protein monomers or glycolized polymers that are the result of many genes. genetic variations in il-1, especially the -511 c/t mutation (rs 16944) can increase the risk of schizophrenia and the il-1β -511 c/t polymorphism is a factor in schizophrenia (damanik et al., 2020). immune dysfunction in schizophrenia is related to the pathomechanism of the disorder so increased levels of il-1β and il-6 are associated with schizophrenic behavior (jordan et al., 2021). increased levels of il-1β and il-6 are associated with chronic schizophrenia in old age. however, confounding factors are age, gender, smoking, and antipsychotic drugs have not been shown to influence serum cytokines. differences between the il-1β -511 c/t variant genotype in ods of the batak ethnic and the minangkabau ethnic the il-1β -511 c/t variant is a combination of c and t alleles, consisting of cc, ct and tt genotypes. the genotype variable is a categorical variable, the data is presented in the form of a frequency distribution which will be tested using logistic regression, choosing the tt genotype as a comparison (table 3). table 3. differences between il-1β -511 c/t variant genotypes in ods of the batak and the minangkabau ethnicities. genotype ethnic group p value or (95% ci) batak (n=120) minangkabau (n=120) ct 60(50) 56(46.7) 0.063 0.865 (0.479-1.562) cc 21(17.5) 37(30.8) 0.080 0.534 (0.264-1.079) tt 39(32.5) 27(22.5) comparison *ods= people with schizophrenia in the batak ods group, the ct genotype frequency was 60 (50%), cc was 21 (17.5%) and tt was 39 (32.5%). for the ods group of the minangkabau ethnic, the frequency of the ct genotype was 56 (46.7%), cc was 37 (30.8%), and tt was 27 (22.5%). from the results of analysis using logistic regression, the p-value for genotype (ct vs tt) was 0.063. or = 0.865 with 95% ci between 0.479 1.562. the p-value for genotype (ccvs tt) was 0.080. or = 0.534 with a 95% ci between 0.264 1,079. this means that there is no significant difference between the genotypes (ct vs tt) and (ct vs tt) and the incidence of schizophrenia. in this study, the frequency of appearance of the ct genotype in the ods group of the batak ethnic was 50%, the cc genotype was 17.5% and the tt genotype was 32.5%. meanwhile, in the ods group of the minangkabau ethnic, the occurrence of the ct genotype was 46.7%, the cc genotype was 30.8% and the tt genotype was 22.5%. the frequency of appearance of the ct genotype in the ods group of the minangkabau ethnic is lower than that of the ods group of the batak ethnic, however, the frequency of occurrence of the cc genotype in the ods group of the minangkabau ethnic is higher than that of the ods group of the batak ethnic. based on the results of logistic regression analysis, the chi-square value for genotype (cc vs tt) was 0.063 with or = 0.865 with a 95% ci between 0.479 1,562. the p-value for genotype (cc vs tt) was 0.080. or = 0.534 with a 95% ci between 0.264 1.079. this means that there is no significant difference between genotype (ct vs tt) and (ct vs tt) and the incidence of schizophrenia. based on hazumi et.al (hazumi et al., 2022) stated that all subjects were japanese individuals who had no biological relationship submitted in their self-reports, and were recruited from the outpatient clinic of the national center of neurology and psychiatry hospital tokyo, japan. as a result, the proportion of the cc genotype in the ods group was 21%, ct 48% and tt 31%. the non-ods group with the proportion of the cc genotype was 17%, ct 47%, and tt 36%. the study stated that the p-value = 0.35 for genotype distribution, meaning wijaya et al. – polymorphism in schizophrenia of ethnicities in pematang siantar 607 that the il-1β-511 c/t genotype was not related to the incidence of schizophrenia, but the incidence of schizophrenia was associated with the c allele only. furthermore, the groups of patients were found bifrontal temporal gray matter volume deficit, and white matter tissue deficits are common in carriers of the 2 genotype alleles, namely tt or ct. in contrast, interleukin-1β polymorphisms had no effect on brain morphology in healthy subjects (isaacs et al., 2022; kraus et al., 2024)(chrusciel et al., 2022). according to the hardy-weinberg formula, if the allele frequencies a and a are p and q, then ; (p+q) = 1, it’s mean (p+q)2 = 1, in this formula, p2, corresponds to the frequency of the homozygous aa genotype, q2 to aa, and 2pq to aa. this study has been tested according to the steps above using the help of online software. table 4 shows the result of hwe values in ods of batak and minangkabau with the il-1β -511 c/t. table 4. hwe values in people with schizophrenia batak and minangkabau with the il-1β -511 c/t. p value batak minangkabau chi square 0.054 0.080 if p<0.05-not consistent with hwe from the results of the analysis using the hwe formula assisted by online software, the p-value obtained for the ods of the batak ethnic was 0.054 and for the ods of the minangkabau ethnicity it was 0.080, so it can be stated that the ods of the batak ethnic and the ods of the minangkabau ethnic are in accordance with the hwe rules (hardy-wienberg equilibrium). in most population genetic estimates, hwe is assumed so that alleles determine the probability of the genotype and are not disturbed by anything. if the observed frequencies do not show a significant difference from the expected frequencies, the population is said to be in hardyweinberg equilibrium. if not, there is a violation of the following formula assumptions and the population is not in hwe. if the assumptions are not met, then the estimate is inaccurate. when hwe is assumed to mean that the genotype probability is determined by the allele frequency, that is, there is no distortion of the transmission ratio, or selection of the genotype and if hwe is violated then statistical methods using allele frequencies may not be valid. the tests carried out observed that the chi square value using the hwe formula was found for the il-1β -511 c/t variant polymorphism for the ods of the batak ethnic of 0.054 and the ods of the minangkabau ethnic of 0.080 and there were no individuals from each subject less than 5 so that it could be declared tribal ods. batak and ods minangkabau ethnicities are following the rules of hwe (hardy-wienberg equilibrium). this shows that the allele frequency remains constant from generation to generation, that hereditary mechanisms do not change the allele frequency (nasution et al., 2023; purnama et al., 2023). conclusions based on the results of the analysis, it show that there are significant differences between the two ethnic groups in terms of gender and value = 0,000. there were also significant differences in age, onset, duration of illness, and total panss scores in the two ethnic groups p-value =0,000. according to chi-square analysis, the value of p = 0.008 is obtained, indicating that there is an insignificant relationship between the allele and schizophrenia. this shows that in the ods of the batak and minangkabau ethnicities, the t allele was found to have a 0.008 times probability of experiencing schizophrenia compared to individuals who had the c allele. the or value was below 1, so the t allele was a protective factor that caused schizophrenia. logistic regression analysis, the chi-square value for genotype (ct vs tt) was 0.063 with or = 0.865 with a 95% ci between 0.479 1.562. the p-value for genotype (cc vs tt) was 0.080. or = 0.534 with a 95% ci between 0.264 1.079. this means that there is no significant difference between genotype (ct vs tt) and (cc vs tt) and the incidence of schizophrenia. acknowledgements: we are very grateful to the mental health polyclinic of dr. djasamen saragih regional general hospital in pematang siantar for their assistance in this research. authors’ contributions: raysha, edy fahcrial & i nyoman ehrich lister designed the study. raysha & bayu ariatama carried out the laboratory work. raysha analyzed the data. raysha & edy fachrial wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: there are no competing interests. references amin, m. m., rasyid, a., effendy, e., amir, n., & suryandari, d. a. 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(2023). structural racism and risk of schizophrenia. american journal of psychiatry, 180(11), 782–784. https://doi.org/10.1176/appi.ajp.20230733 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1085-1090 | doi: 10.14421/biomedich.2025.142.1085-1090 issn 2540-9328 (online) antioxidant and antibacterial properties of methanol extract of gletang flower (tridax procumbens) boima situmeang1*, weny ja musa2, nurhayati bialangi2, sriwijayanti1, holisha widiyanto1, dian susvira1 1department of chemistry, sekolah tinggi analis kimia cilegon, banten, indonesia jl. lingkar selatan, km 1,7 cilegon banten 043259. 2department of chemistry, faculty of matematics and natural science, universitas negeri gorontalo, indonesia jl. prof. dr. ing. bj. habibie, tilongkabila, bone bolango, 96583. tel. (0435) 821125, fax. (0435) 821752, gorontalo, indonesia. corresponding author* boimatumeang@gmail.com abstract in a previous study, the methanol extract of tridax procumbens flowers (commonly known as gletang) at high concentrations was reported to exhibit strong antibacterial activity against streptococcus mutans and enterococcus faecalis. this study aimed to examine the secondary metabolite content, antioxidant activity, and antibacterial activity of the methanol extract of gletang flowers. the antioxidant activity was evaluated using two methods, namely dpph and abts assays. the antibacterial activity was assessed using the kirby-bauer method at concentrations of 1000, 5000, and 10000 ppm against staphylococcus aureus, escherichia coli, streptococcus mutans, and enterococcus faecalis. phytochemical screening revealed that the methanol extract of gletang flowers contains phenolic compounds, flavonoids, alkaloids, and triterpenoids. the antioxidant activity tests showed very strong activity in both dpph and abts assays, with ic₅₀ values of 6.8478 ± 1.335 and 12.8608 ± 0.579 ppm, respectively. the antibacterial activity tests showed that the methanol extract of gletang flowers exhibited strong activity against e. faecalis at concentrations of 5000 and 10000 ppm, with inhibition zone diameters of 3.00 ± 0.14 and 3.20 ± 0.14 mm, respectively. keywords: antibacterial; antioxidant; gletang; tridax procumbens. introduction infectious diseases and oxidative stress are two major health problems that remain a global concern. infections caused by pathogenic bacteria can lead to various diseases, while oxidative stress resulting from an imbalance between free radicals and the body's antioxidant defense system contributes to the development of degenerative diseases such as cancer, diabetes, and cardiovascular disease (ingole et al., 2022). efforts to overcome these problems generally involve the use of synthetic antibiotics and artificial antioxidants. however, long-term use often causes side effects and may induce bacterial resistance. therefore, the search for natural sources of antibacterial and antioxidant agents from plants continues to be developed (alkowni et al., 2023). one of the potential plants is tridax procumbens (commonly known as gletang), a member of the asteraceae family that grows widely in tropical regions, including indonesia (wijayanti et al., 2025). traditionally, this plant has been used by local communities to treat wounds, reduce fever, and as a traditional remedy for various diseases (pradana et al., 2024). several studies have reported that t. procumbens contains secondary metabolites such as flavonoids, alkaloids, tannins, saponins, and phenolic compounds, which are known to possess important biological activities, including antibacterial and antioxidant properties (dattaray, 2022). the flowers of tridax procumbens (gletang), as one of the plant’s parts, are believed to contain a significant concentration of active metabolites; however, scientific studies on the antibacterial and antioxidant potential of its flower extract remain limited. considering the need for safe natural antibacterial and antioxidant agents, research on the biological activities of t. procumbens flower extract is therefore important to conduct. widyawati et al. (2022) reported that the flower extract of t. procumbens has potential antibacterial activity against e. faecalis and s. mutans at concentrations of 20%, 40%, 60%, and 80% (widyawati et al., 2022). these concentrations are still considered very high, indicating the need for further investigation at lower concentrations. the presence of phenolic and flavonoid compounds reported in the gletang flower manuscript received: 15 september, 2025. revision accepted: 21 november, 2025. published: 27 november, 2025. https://doi.org/10.14421/biomedich.2025.142.1085-1090 1086 biology, medicine, & natural product chemistry 14 (2), 2025: 1085-1090 extract also suggests potential antioxidant properties that warrant further exploration (andriana et al., 2019). research on the whole tridax procumbens plant has been widely conducted; however, studies specifically focusing on its flowers are still very limited. therefore, this study aims to explore the antibacterial and antioxidant potential of t. procumbens flower extract, in order to support the development of natural herbal medicines that may be beneficial in the health sector. materials and methods material the sample used in this study was the flower part of tridax procumbens (gletang). the chemicals used included methanol (pro analysis grade), distilled water, dpph, abts, ascorbic acid, trolox, nutrient agar, nutrient broth, 0.2% chlorhexidine, 0.01% streptomycin, and 70% ethanol. the equipment used included a macerator, glassware, test tubes, micropipettes, a uvvisible spectrophotometer, a laminar air flow cabinet, cotton swabs, a caliper, and an incubator. sample extraction the flower samples of t. procumbens were collected from gerogol district, cilegon city, banten, indonesia. a total of 1 kg of fresh samples were collected. the fresh samples were then air-dried at room temperature for two weeks. about 250 g of the dried gletang flower samples were extracted using the maceration method with 1.5 l of 96% methanol. the extract was then concentrated using a rotary evaporator, yielding 62.8 g of concentrated methanol extract of gletang flowers. phytochemical screening a total of 1 g of the concentrated flower extract of t. procumbens was weighed and subjected to qualitative phytochemical screening. the phytochemical screening was conducted to detect the presence of alkaloids, phenolics, flavonoids, triterpenoids, and steroids (bialangi et al., 2024). antioxidant activity test using dpph fifty mg of the t. procumbens flower extract sample was dissolved in methanol and transferred into a 50 ml volumetric flask to obtain a stock solution with a concentration of 1000 ppm. from this 1000 ppm stock solution, a series of concentrations (10, 20, 30, 40, and 50 ppm) was prepared. dpph was weighed (10 mg) and dissolved in 62.5 ml of methanol to obtain a dpph solution with a concentration of 0.05 µm. for the assay, 2.4 ml of each extract concentration was mixed with 0.6 ml of the dpph solution, then incubated at room temperature in the dark for 30 minutes. the absorbance was measured at a wavelength of 517 nm using a uvvisible spectrophotometer (situmeang, swasono, et al., 2025). all sample tests were performed in triplicate. antioxidant activity test using abts various concentrations (10, 20, 30, 40, and 50 ppm) of the methanol extract of t. procumbens flowers were prepared. a total of 1.8 ml of each sample was placed into a test tube, followed by the addition of 0.3 ml of abts solution. the mixture was incubated for 10 minutes, and the absorbance was then measured at a wavelength of 715 nm using a uv-visible spectrophotometer (kabré et al., 2023). the same procedure was applied to trolox as a positive control. all sample tests were performed in triplicate. antibacterial activity test the antibacterial activity of the methanol extract of t. procumbens flowers was tested against escherichia coli, staphylococcus aureus, streptococcus mutans, and enterococcus faecalis. the extract was tested at concentrations of 1000, 5000, and 10,000 ppm. streptomycin was used as the positive control for e. coli and s. aureus, while chlorhexidine was used as the positive control for s. mutans and e. faecalis. the antibacterial activity was evaluated by measuring the diameter of the clear inhibition zones formed around the paper discs (satari et al., 2019). data analysis the one-way anova test was used in statistical evaluation and data representation using microsoft excel and origin 9 software. the data were reported as the mean ± standard deviation. results and discussion phytochemical screening the results of the phytochemical screening showed that the methanol extract of t. procumbens flowers tested positive for phenolic compounds, flavonoids, alkaloids, and triterpenoids, but negative for steroids. these findings are consistent with the study by widyawati et al. (2022), which reported that the flower extract of t. procumbens contains alkaloids, phenolics, flavonoids, and triterpenoids. antioxidant test result the results of testing the methanol extract of t. procumbens flowers against dpph and abts radicals showed that the percentage of radical inhibition increased with increasing extract concentrations. at the lowest concentration (10 ppm), antioxidant activity had already reached approximately 50% (dpph) and 46% (abts). at the highest concentration (50 ppm), the percentage of inhibition exceeded 70% for both methods. this pattern is consistent with the fundamental mechanism of antioxidant activity, in which a higher availability of situmeang et al. – antioxidant and antibacterial properties of tridax procumbens 1087 antioxidant compounds leads to a greater ability to neutralize dpph and abts free radicals. the calculated % inhibition and ic50 values of the methanol extract of gletang flowers obtained from the dpph and abts methods are presented in table 1. table 1. % inhibition and antioxidant activity result using dpph and abts method of methanol extract. methods concentrations (ppm) inhibition (%) replications ic50 (ppm) ±sd 1 2 3 dpph 0 0 0 0 6.8478±1.335 10 49.6285 50.5185 50.3703 20 57.0579 57.7777 58.2222 30 63.1500 63.4074 64.8888 40 68.6478 68.5925 68.5925 50 71.6196 70.9629 71.5555 abts 0 0 0 0 12.8608±0.579 10 46.0947 44.9293 46.3010 20 54.1613 54.9422 54.9744 30 65.5569 66.4955 66.4540 40 69.1421 68.8061 68.6224 50 72.2151 72.9139 72.7040 the ic50 values were obtained from the linear regression equations of each concentration plotted against the percentage of inhibition. the linear regression curves are shown in figure 1 for dpph and figure 2 for abts. the correlation coefficients (r values) obtained from the linear regression equations in both the dpph and abts methods were greater than 0.9. this indicates that the relationship between concentration and percentage of inhibition is linear (situmeang et al., 2025). figure 1. regression linear curve of dpph method. figure 2. regression linear curve of abts method. 40 45 50 55 60 65 70 75 5 10 15 20 25 30 35 40 45 50 55 in h ib it io n s (% ) concentrations (ppm) y = 0,5274x + 46,904 r² = 0,9592 y = 0,5557x + 45,349 r² = 0,9788 y = 0,517x + 46,741 r² = 0,9717 40 45 50 55 60 65 70 75 80 0 10 20 30 40 50 60 in h ib it io n s (% ) concentrations (ppm) y = 0,6645x + 41,875 r² = 0,9319 y = 0,6722x + 41,268 r² = 0,9398 1088 biology, medicine, & natural product chemistry 14 (2), 2025: 1085-1090 the higher the percentage of inhibition (% inhibition), the lower the ic50 value, indicating a stronger ability of the sample to scavenge free radicals in both the dpph and abts assays. as shown in figure 3, increasing the extract concentration resulted in a higher percentage of free radical inhibition. this pattern demonstrates that the antioxidant activity of the extract is concentration-dependent (dose-dependent), meaning that higher extract concentrations provide more antioxidant molecules capable of donating electrons or hydrogen atoms to neutralize dpph radical and abts radical cation (jiangseubchatveera et al., 2023). the positive relationship between concentration and radical scavenging activity confirms that the flower extract of t. procumbens contains bioactive compounds, such as phenolics and flavonoids, which are effective as antioxidants. figure 3. correlation of dpph and abts radical scavenging with various concentrations of methanol extract. the ic50 value represents the concentration of extract required to inhibit 50% of free radicals. a lower ic50 value indicates stronger antioxidant activity. in the dpph method, an ic50 of 6.85 ppm demonstrates that the methanol extract of tridax procumbens flowers possesses very strong antioxidant activity. in the abts method, an ic50 of 12.86 ppm also indicates very strong activity, although slightly lower compared to dpph. this difference may occur because each method has a distinct radical scavenging mechanism. the dpph method involves electron transfer from antioxidants to the stable dpph· radical, while the abts method involves both electron and hydrogen atom transfer to the abts⁺· radical cation (liu et al., 2023). the phenolic and flavonoid compounds present in t. procumbens flowers are likely to have a high electron-donating ability, making them more effective in the dpph method. these results indicate that the methanol extract of t. procumbens flowers contains bioactive compounds, such as flavonoids and phenolics, which act as potent antioxidants. this strong antioxidant activity has the potential to be utilized in the development of natural ingredients to prevent oxidative damage in food products, cosmetics, or herbal medicines. antibacterial test result antibacterial activity was evaluated using the kirbybauer method. the antibacterial activity was determined based on the diameter of the inhibition zones formed, which reflects the ability of the methanol extract of t. procumbens flowers to inhibit bacterial growth. the results showed that the methanol extract exhibited varying antibacterial activity against the four tested bacterial strains e. coli, s. aureus, s. mutans, and e. faecalis with responses depending on the extract concentration used. the results of the antibacterial activity test are presented in table 2. table 2. antibacterial activity of tridax procumbens methanol flower extract, negative control, and positive control. bacteria sampel/concentration inhibition zone (mm) average±sd category e. coli extract 1000 ppm 0.00 none extract 5000 ppm 0.25±0.07 weak extract 10000 ppm 0.55±0.07 weak methanol 96% 0.00 none streptomycin 200 ppm 8.45±0.07 strong s. aureus extract 1000 ppm 0.00 none extract 5000 ppm 0.00 none extract 10000 ppm 0.00 none methanol 96% 0.00 none streptomycin 200 ppm 12.2±0.14 strong s. mutans extract 1000 ppm 0.35±0.07 weak extract 5000 ppm 0.35±0.07 weak extract 10000 ppm 0.55±0.07 medium methanol 96% 0.00 none klorheksidin 2000 ppm 4.15±0.07 strong e. faecalis extract 1000 ppm 2.35±0.07 medium extract 5000 ppm 3.00±0.14 strong extract 10000 ppm 3.20±0.14 strong methanol 96% 0.00 none klorheksidin 2000 ppm 4.15±0.07 strong for e. coli, the extract exhibited weak activity, producing inhibition zones of 0.25 ± 0.07 mm at 5000 ppm and 0.55 ± 0.07 mm at 10,000 ppm, while showing no activity at 1000 ppm. these results indicate that e. coli is relatively more resistant to the active compounds in the extract, likely due to the more complex structure of its gram-negative cell wall, which contains a lipopolysaccharide layer that impedes the penetration of antibacterial compounds (aldayel, 2023). in comparison, the positive control streptomycin at 200 ppm produced an inhibition zone of 8.45 ± 0.07 mm (strong category), demonstrating that the activity of the extract against e. coli is still much lower than that of the standard antibiotic. for s. aureus, the extract showed no antibacterial activity at all tested concentrations. the absence of inhibition zones indicates that the antibacterial 10 20 30 40 50 0 10 20 30 40 50 60 70 80 d p p h a n d a b t s r ad ic al s ca v en g in g ( % ) concentrations (ppm) dpph abts situmeang et al. – antioxidant and antibacterial properties of tridax procumbens 1089 compounds present in the tridax procumbens flower extract are either ineffective or present at insufficient concentrations to inhibit the growth of s. aureus. in contrast, the positive control streptomycin produced an inhibition zone of 12.2 ± 0.14 mm (strong category), confirming the ineffectiveness of the extract against this bacterium (idowu et al., 2023). for s. mutans, the extract exhibited weak to moderate antibacterial activity. the inhibition zones were 0.35 ± 0.07 mm at concentrations of 1000 and 5000 ppm, and increased to 0.55 ± 0.07 mm (moderate category) at 10 000 ppm. these results indicate a moderate antibacterial potential against s. mutans that increases with higher concentrations, although it is still much lower than the positive control chlorhexidine (2000 ppm), which produced an inhibition zone of 4.15 ± 0.07 mm (strong category). this suggests that the extract begins to show activity at high concentrations, but it is not yet as effective as the standard antibacterial agent. for e. faecalis, the extract exhibited moderate to strong antibacterial activity. the inhibition zone increased with higher concentrations, measuring 2.35 ± 0.07 mm (moderate category) at 1000 ppm, 3.00 ± 0.14 mm (strong category) at 5000 ppm, and 3.20 ± 0.14 mm (strong category) at 10 000 ppm. although slightly lower than the positive control chlorhexidine (4.15 ± 0.07 mm), these results indicate that the extract has considerable antibacterial potential against e. faecalis. this activity is likely attributed to bioactive compounds present in the extract, such as flavonoids, phenolics, or terpenoids, which may disrupt the bacterial cell membrane or interfere with the metabolism of e. faecalis. overall, these results indicate that the methanolic flower extract of t. procumbens exhibits specific antibacterial activity that is more effective against grampositive bacteria than gram negative bacteria, with the strongest effect observed against e. faecalis. the pattern of increasing inhibition zones with higher concentrations also confirms that the antibacterial activity of the extract is dose-dependent. conclusions phytochemical screening revealed that the methanolic flower extract of t. procumbens contains phenolic, flavonoid, alkaloid, and triterpenoid compounds. the antioxidant activity assay showed very strong activity in both dpph and abts methods, with ic₅₀ values of 6.8478 ± 1.335 and 12.8608 ± 0.579 ppm, respectively. the antibacterial activity assay demonstrated that the methanolic flower extract exhibited strong activity against enterococcus faecalis at concentrations of 5000 and 10 000 ppm, producing inhibition zones of 3.00 ± 0.14 mm and 3.20 ± 0.14 mm, respectively. acknowledgements: acknowledgments are expressed in a brief; all sources of institutional, private and corporate financial support for the work must be fully acknowledged, and any potential conflicts of interest are noted. authors’ contributions: boima situmeang and weny ja musa designed the study and wrote the manuscript. sriwijayanti and dian susvira carried out the laboratory work. nurhayati bialangi and holisha widiyanto analyzed the data. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references aldayel, m. f. 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(2024). metabolite profiling of potential fraction from ethyl acetate extract of ziziphus mauritiana leaves by lc-ms/ms analysis. karbala international journal of modern science, 10(4), 542–548. https://doi.org/10.33640/2405-609x.3371 dattaray, d. (2022). traditional uses and pharmacology of plant tridax procumbens: a review. sys rev pharm, 13(7), 476– 482. https://doi.org/10.31858/0975-8453.13.5.511-517 idowu, d. o., aiyelaagbe, o. o., & idowu, p. a. (2023). chemical composition and biological activities of volatile oil of the stem of dombeya buettneri k. schum. (sterculiaceae). scientific african, 20, 1–10. https://doi.org/10.1016/j.sciaf.2023.e01624 ingole, v. v., mhaske, p. c., & katade, s. r. (2022). phytochemistry and pharmacological aspects of tridax procumbens (l.): a systematic and comprehensive review. phytomedicine plus, 2(1), 100199. https://doi.org/10.1016/j.phyplu.2021.100199 jiangseubchatveera, n., saechan, c., petchsomrit, a., treeyaprasert, t., leelakanok, n., & prompanya, c. (2023). phytochemicals and antioxidant activities of red oak, red coral and butterhead. tropical life sciences research, 34(1), 1–17. https://doi.org/10.21315/tlsr2023.34.1.1 kabré, p., ouattara, l., sanou, y., ouédraogo, r. j., ouoba, p., zanté, a. a., zoungo, d., somda, m. b., & ouédraogo, g. a. (2023). comparative study of polyphenols, flavonoids content, antioxidant and antidiabetic activities of lophira lanceolata tiegh.ex keay (ochnaceae) extracts. scientific african, 22(october). https://doi.org/10.1016/j.sciaf.2023.e01922 1090 biology, medicine, & natural product chemistry 14 (2), 2025: 1085-1090 liu, l., chen, j., chang, x., qin, j., lai, h., & zhang, x. (2023). phytochemical profiles and bioactivities of parnassia palustris l. european journal of integrative medicine, 57(september 2022), 102207. https://doi.org/10.1016/j.eujim.2022.102207 pradana, a. f., fauji, f. r., & farlina, i. (2024). the potential of methanol extract nanoemulsion from gletang flower ( tridax procumbens ) as an antibacterial agent against pathogenic bacteria. 0–4. satari, m. h., situmeang, b., yuda, i. p., & kurnia, d. (2019). antibacterial diterpenoid against pathogenic oral bacteria of streptococcus mutans atcc 25175 isolated from sarang semut (myrmecodia pendans). jurnal kimia valensi, 5(2), 218–223. https://doi.org/10.15408/jkv.v5i2.8864 situmeang, b., primawati, j. s., oktafiani, i., musa, w. j. a., & kilo, a. k. (2025). antioxidant and cytotoxic potential of ethyl acetate fraction of gandaria stem bark ( bouea macrophylla ) against mcf-7 cell line. 14(1), 345–349. https://doi.org/10.14421/biomedich.2025.141.345-349 situmeang, b., swasono, r. t., & raharjo, t. j. (2025). evaluation of phytochemical composition, antioxidant, cytotoxic and in silico studies of ethyl acetate fractions of tristaniopsis merguensis leaves. toxicology reports, 14(january), 101911. https://doi.org/10.1016/j.toxrep.2025.101911 widyawati, arma, u., fadriyanti, o., situmeang, b., & silaban, s. (2022). antibacterial activity test of different parts of gletang (tridax procumbens) from west sumatera, indonesia. rasayan journal of chemistry, 15(4), 2382–2386. https://doi.org/10.31788/rjc.2022.1547084 wijayanti, s., pradana, a. f., situmeang, b., prastiwi, d. a., & musa, w. j. a. (2025). microemulsion of methanol extract of tridax procumbens flower and its antibacterial activity against streptococcus mutans and enterococcus faecalis. jurnal beta kimia, 5(1), 56–61. https://doi.org/10.35508/jbk.v5i1.21189 biology, medicine, & natural product chemistry issn: 2089-6514 volume 5, number 1, 2016 | pages: 15-18 | doi: 10.14421/biomedich.2016.51.15-18 determination of leisure levels of village patronage uin sunan kalijaga yogyakarta: improving governance patronage towards rural green village and environmentally friendly supriatna1, thaqibul fikri niyartama2, and iwan kuswidi2 1faculty of sharia and law, 2faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency1: soepriatna@yahoo.co.id abstract this study took place in the village of patronage uin sunan kalidjaga yogyakarta that consist of 13 hamlets (klidon, banjarsari, wonosalam, dongkelsari, puntuk, tanjung sari, karang lo, purworejo, tanjung, banturejo, nglengkong and surirejo), sukoharjo village, district ngaglik, diy sleman regency. data are collect and analysed in order to obtain results in a level of comfort. the analysis was performed by using a formula based on the comfort level temperature humidity index (thi). the results showed that hamlet klidon, banjarsari, wonosalam, and dongkelsari shows not comfortable, whereas hamlet puntuk, tanjung sari, karang lo, purworejo, tanjung, banturejo, nglengkong, surirejo and mujen show strongly comfortable mainly on the clock 10:00 to 15:00. keywords: patronage village, comfort level, temperature humidity index (thi). introduction the village is basically an ideal residential location. this is because the cool environment filled with trees as a source of oxygen and water supplies are abundant. however, there is a negative impact on rural development which cannot be avoided that include changes in environmental quality. the change of the environmental quality of course have an impact on people and life. one of the changes is a decrease in the environmental quality of air quality including the change in climate parameters, especially temperature. the impact of a decrease in air quality influenced the quality of the rural environment. this is because the human and natural environment cannot be separated, they interact and influence each other, including influenced the surrounding neighborhoods. therefore, one of the factors to consider in choosing a dwelling is a neighborhood that has a good quality of aspects of climate and air temperature. climate is one of the biophysical components are taken into account in the framework of human adaptation to the natural environment, one of them in terms of choosing a dwelling. so it can be assumed that the climatic conditions both macro and micro affect the quality of neighborhoods. changes in the amount of rainfall, air temperature, wind speed and humidity affect the quality criteria of a good village environment. this needs to be considered in the choice of residential location in order to achieve comfort in an activity for the entire population. so far, studies have been conducted regarding the level of comfort (humidity index) in relation to the climatological factors, but most of these studies are not spatially or research areas include a point / block large areas. this is understandable, because the studies conducted terrestrial, which of course takes time and labor costs are great. therefore, research in areas that are quite specific coverage is rarely done. actually, the development of remote sensing technology capable of overcoming the obstacles encountered by research conducted using terrestrial methods. the use of remote sensing data can be used to obtain information that is directly or indirectly related to comfort, then the information can be used to measure the comfort level of a region. in this study, in order to determine the level of comfort in the village of patronage universitas islam negeri (uin) sunan kalijaga necessary information relating to changes in the climate elements such as air temperature, relative humidity, and wind speed. finally, this study is expected to provide information about the quality of the environment in the village of patronage uin sunan kalidjaga as a first step to designing governance of the village towards the village green and environmentally friendly. tools the tools used in this study are: 1. global positioning system (gps) is used to determine the location of the measurement and facilitate plotting the data on a map. 2. thermo hygrometer used to measure air temperature and relative humidity. 16 biology, medicine, & natural product chemistry 5 (1), 2016: 15-18 research data the following data is taken during this study, the village: sukoharjo, district: ngaglik, district: sleman, yogyakarta. table 1. data of air temperature and humidity. no village time position air temperature humidity 1 klidon 10.29 -7.7041547, 110.4261053 33.6 46% 2 banjarsari 10.40 -7.6975199, 110.4247859 32.8 49% 3 wonosalam 10.46 -7.6937960, 110.4280415 33.9 46% 4 dongkelsari 10.58 -7.6836879, 110.4307653 33.6 45% 5 puntuk 11.10 -7.6886566, 110.4313688 38.1 36% 6 tanjung sari 11.23 -7.6968149, 110.4332366 35.6 41% 7 karang lo 11.36 -7.7006707, 110.4361444 39.4 35% 8 purworejo 11.52 -7.7055966, 110.4323726 40.1 32% 9 tanjung 12.42 -7.7050605, 110.4396199 40.4 33% 10 banturejo 12.52 -7.7089683, 110.4313996 40.8 31% 11 nglengkong 13.00 -7.7085144, 110.4207388 40.8 31% 12 surirejo 13.12 -7.7189814, 110.4244178 42.1 30% 13 mujen 13.25 -7.7189814, 110.4210858 40.4 32% analysis thi value of each criterion comfort level residential areas in table 2. table 2. criteria temperature humidity index (thi). no criteria thi comfort level 1 < 29 comfortable 2 29 – 30.5 uncomfortable 3 > 30.5 very uncomfortable from table 2 can be classified village built which area is comfortable, uncomfortable, and very uncomfortable. to facilitate the reading can be seen in table 3 below. table 3. level of leisure village patronage uin sunan kalijaga. no village thi comfort level 1 klidon 29.9712 uncomfortable 2 banjarsari 29.4544 uncomfortable 3 wonosalam 30.2388 uncomfortable 4 dongkelsari 29.904 uncomfortable 5 puntuk 33.2232 very uncomfortable 6 tanjung sari 31.3992 very uncomfortable 7 karang lo 34.278 very uncomfortable 8 purworejo 34.6464 very uncomfortable 9 tanjung 34.9864 very uncomfortable 10 banturejo 35.1696 very uncomfortable 11 nglengkong 35.1696 very uncomfortable 12 surirejo 36.206 very uncomfortable 13 mujen 34.9056 very uncomfortable supriatna, et.al. – determination of leisure levels of village patronage uin sunan kalijaga yogyakarta … 17 description: horizontal axis : temperature humidity index (thi) : uncomfortable : very uncomfortable figure 1. level of leisure village patronage uin sunan kalijaga. figure 2. temperature humidity index (thi) village patronage uin sunan kalijaga. 0 5 10 15 20 25 30 35 40 klidon banjarsari wonosalam dongkelsari puntuk tanjung sari karang lo purworejo tanjung banturejo nglengkong surirejo mujen 18 biology, medicine, & natural product chemistry 5 (1), 2016: 15-18 in hamlet klidon, banjarasri, wonosalam, and dongkelsari the hours shown in table 3 shows not comfortable, whereas hamlet puntuk, tanjung sari, karang lo, purworejo, tanjung, banturejo, nglengkong, surirejo and mujen the hours shown in table 3 shows strongly comfortable. this is because the vegetation is not considered ole surrounding population. conclusion assuming that the temperature humidity index (thi) is a real comfort level, it is necessary to increase the patronage of vegetation in the village in an effort to increase the level of comfort in residential areas. this is seen in the results of the analysis. so the local community can feel comfort and also can improve the welfare of residents of the results of such vegetation. references bintarto, r., s. hadisumarmo, 1978, metode analisis geografi, yogyakarta: lp3es. dewi, anggraini., 1996, penggunaan foto udara dan sistem informasi geografis (sig) untuk mengkaji kualitas lingkungan permukiman dalam hubungan dengan pola penggunaan air minum (air domestik), thesis-s1, faculty of geography, universitas gadjah mada, yogyakarta. ediyono, setijati h., dkk, 1999, prinsip-prinsip lingkungan dalam pembangunan berkelanjutan, direktorat jenderal pendidikan tinggi, departemen pendidikan dan kebudayaan, jakarta. indarto, eddy, 1993, pengaruh suhu udara dan kelembaban udara terhadap tingkat ketidaknyamanan fisiologis penghuni rumah tinggal di perumnas banyumanik semarang, thesis-s2, program pasca sarjana, universitas gadjah mada, yogyakarta. lillesand & kiefer, 1993, penginderaan jauh dan interpretasi citra, yogyakarta: gadjah mada university press. mather, j.r., 1974, climatology: fundamentals and aplications, mcgraw-hill: new york. murdiyarso, d., s. heny, 1992, peranan hutan kota dalam pengendalian iklim kota, seminar sehari iklim perkotaan, jakarta. pudjiastuti, l., r. septa, r.s. happy, 1998, kualitas udara dalam ruamg, direktorat jenderal pendidikan tinggi, departemen pendidikan dan kebudayaan. sulistyaningsih, astin., 1995, distribusi suhu udara dan faktorfaktor lingkungan yang mempengaruhinya serta pengaruhnya terhadap curah hujan di kotamadya surakarta, thesis-s1, faculty of geography, ugm, yogyakarta. sutanto, 1986, penginderaan jauh jilid 1, yogyakarta: gadjah mada university press. sutanto, 1994, penginderaan jauh jilid 2, yogyakarta: gadjah mada university press. suwarsih, kristianan, 1996, evaluasi kesehatan lingkungan permukiman kota berdasarkan foto udara dan sistem informasi geografis (sig) kasus di kecamatan pasar kliwon, kotamadya surakarta, thesis-s1, faculty of geography, ugm, yogyakarta. widyatmanti, wirastuti, 1998, pengaruh perubahan liputan lahan terhadap variasi spasio temporal suhu perkotaan skala mikro dengan bantuan penginderaan jauh dan sistem informasi geografis , kasus studi di daerah semarang utara, thesis-s1, faculty of geography, ugm, yogyakarta. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 197-203 | doi: 10.14421/biomedich.2023.121.197-203 issn 2540-9328 (online) comparative cough suppression of chitosan crab extract of uca tangeri and dihydrocodeine joshua charles isirima1, precious ojo uahomo2,* 1department of pharmacology, faculty of basic clinical sciences, university of port harcourt, rivers state, nigeria; 2department of biomedical technology, school of science laboratory technology, university of port harcourt, rivers state, nigeria. corresponding author* uahomoprecious1@gmail.com abstract cough is an inmate primitive reflex and acts as a part of the body's immune system to protect against foreign materials from the respiratory tract. this study was done to investigate the cough suppression potential of uca tangeri. a day before the test, guinea pigs were placed individually in a transparent chamber (60 × 36 × 60 cm) for 5 minutes before cough was induced by exposure to 15% citric acid, delivered using an omron compressor nebulizer (rate of 0.4 ml/minutes and particle size 5μm) for 10 minutes. the animals were then monitored visually within this exposure time for cough; the latency and counts, of which, were taken as the basal values. the animals exhibiting 10 20 bouts of cough were selected for the study and fasted overnight but with access to water. the selected animals were randomly allotted to 5 groups (n=5 per group). the animals were treated orally thus: group 1 was the control group and received 2 ml/kg of normal saline; group 2 received 25 mg/kg dihydrocodeine; group 3 received 150 mg/kg extract; group 4 received 300 mg/kg extract and group 5 received 600 mg/kg of the extract. an hour after administration, they were re-exposed to citric acid aerosol (as earlier described) and the latency of cough and cough count were recorded. the procedure was repeated at hours 2 and 3 after treatment. antitussive activity was then evaluated in each guinea-pig as the percentage reduction in the number of coughs also known as percentage suppression of cough and percentage increase in latency of cough. the results revealed that uca tangeri exhibited a dose dependent percentage increase in cough latency period as well as percentage increase in suppression of cough which was inferior to dihydrocodeine, but significantly greater than normal saline and basal levels. keywords: cough; suppression; uca tangeri; chitosan crab; dihydrocodeine, comparative. introduction the body's immune system uses the involuntary primitive reflex of coughing to defend against foreign substances that invade the respiratory tract (sharma et al., 2020). to eliminate debris, excessive mucus, irritants, microbes, or other substances from the respiratory tract, one may cough either voluntarily or involuntarily. coughing is either voluntary or involuntary (chung and pavord, 2008). coughing could be classified as acute, subacute, or chronic depending on the duration (vally and ihuma, 2016). acute cough has the shortest duration. it is considered to be a cough with duration of less than two weeks. sub-acute cough has a duration between three-eight weeks while chronic cough lasts for more than eight weeks (four weeks for children) (vally and ihuma, 2016). adults who have persistent coughing are increasingly being linked to pertussis (irwin et al., 2006). the prevalence of chronic cough is 9.6% worldwide, with oceania having the highest incidence (18.1%), and africa having the lowest prevalence (2.3%), according to song et al. (2015). coughing is a symptom of several diseases and health conditions, including the common cold, acute bronchitis, pneumonia, pertussis, flu, smoking, as well as asthma, tuberculosis, and lung cancer. coughing can cause chest pain, congestion, and an irritated throat. repeated coughing causes irritation and discomfort, which in turn leads to additional coughing (irwin et al., 2006). cough is also brought on by a variety of microbes, including bacteria and viruses, which aids in the spread of the illness to new hosts. regular coughing is typically brought on by a respiratory tract infection, but it can also be brought on by choking, smoking, air pollution, asthma, gastroesophageal reflux disease (gerd), postnasal drip, chronic bronchitis, lung tumors, heart failure, and drugs like ace inhibitors (dicpinigaitis et al., 2009). crabs of various species have been employed in the treatment of various illnesses. cardisoma guanhumi has been used to treat wounds, boils and bronchitis in latin america, according to alves and alves (2011). goniopsis cruentata is used to treat epilepsy and genital problems; plagusia depressa serves as a complementary medicine for epilepsy; emerita portoricensis on earaches' therapeutic applications; ocypode quadrata in treating manuscript received: 17 december, 2022. revision accepted: 11 february, 2023. published: 14 february, 2023. https://doi.org/10.14421/biomedich.2023.121.197-203 198 biology, medicine, & natural product chemistry 12 (1), 2023: 197-203 asthma, hemorrhage in women, the flu, and to lessen the effects of naquin poison intoxication (pisces, batrachoididae); ucides cordatus in treating hemorrhage in women, urinary incontinence, osteoporosis, cough, asthma, tuberculosis, womb disorders, arthrosis, and bronchitis; and uca maracoani in curing asthma, whooping cough. dev roy (2014) gathered 22 species of brachyuran crabs from various regions of the world, including india, nepal, and brazil. these crabs are primarily used to treat conditions like whooping cough, bronchitis, pneumonia, asthma, osteoporosis, wounds, boils, womb disorders, tuberculosis, earache, burns, and epilepsy, while hermit crabs are used to treat earache, urethritis; malaria. this research examines uca tangeri and dihydrocodeine’s capability to suppress coughing. materials and methods animals 25 adult guinea pigs of either sex weighing 460-600g were obtained from an animal facility in ogoni, rivers state, and brought to the animal house of department of pharmacology, faculty of basic medical sciences, college of health sciences, university of port harcourt, nigeria. all animals were allowed two weeks acclimatization in the animal facility of the department of pharmacology, university of port harcourt. they were all allowed free access food and tap water and were exposed to natural light-dark cycle and room temperature. all animals were handled according to standard protocols for the use of laboratory animals (national institute of health, 2002). site of collection of crab samples the samples were collected at sivibilagbara along the dor nwezor channel of bodo creek and buguma creek. the buguma creek is a tributary of the bonny river which is located southeast of the niger delta between longitude 6º51'e and 49.8'e and latitude 4º43'n and 47.8'n in asari-toru local government area of rivers state. the creek system consists of the main creek channel with other associated interconnecting creeks which are interconnected and surround buguma and ido communities. the creek serves as a source of tidal water for nigerian institute for oceanography and marine research/buguma brackish water experimental fish farm, which was constructed between 1963 and 1966. the new calabar river brings the salty ocean water as tidal flows diurnally to the fish ponds (dublin-green and ojanuga, 1999). sample collection and identification samples were collected from the creeks. uca tangeri were collected at low tide in the mangrove shores by hand picking. the samples collected were transferred into perforated plastic containers to allow for air during transportation and was transported to the pharmacognosy research laboratory, department of pharmacognosy, university of port harcourt. the samples were identified using food and agriculture organization species identification sheets for fresh water and marine crab species. figure 1. uca tangeri. method of extraction according to shahidi and synowiecki (1991), 60 of the freshly collected crabs (u. tangeri) were sacrificed and the shell separated from the meat and washed with tap water to remove all impurities. the crab shells and meat were then transferred to the oven and dried at 70oc until they were completely dry. using a laboratory mortar and pestle, the dried crab shells and meat were ground and sieved into the size of 500µm. carotenoids extraction four gram of the sieved crab shell was measured using want precision electric weighing balance into a beaker and 200ml of cod liver oil was added and stirred with magnetic stirrer until it was completely mixed for 20minutes. the beaker was then transferred into a water bath at a temperature of 60oc and allowed for 30 minutes. the mixture was then filtered with a white handkerchief to drain off the oil and the residue transferred into a beaker. deproteinization the residue from the carotenoids extract was treated with 2% potassium hydroxide (koh) at a ratio of 1:20 w/v and was stirred continuously for 2 hours at a temperature of 90oc to remove protein from the crab. the sample was filtered and the residues were continuously washed with distilled water until the ph became neutral i.e., ph=7. this was done to ensure that all the salt had been removed after removing the protein. the deproteinized crab was transferred into an oven and dried at 60oc until it was completely dry (shahidi and synowiecki, 1991). isirima & uahomo – comparative cough suppression of chitosan crab extract … 199 demineralization 2.5% w/v of hydrochloric acid was used at room temperature (23oc) for 6 hours to remove the mineral content of the deproteinized crab materials at a ratio of 1:20 w/v. the samples were filtered and washed with tap water until the ph was neutral. the demineralized crab material was then transferred to the oven and dried at a temperature of 60oc until completely dried. (shahidi and synowiecki, 1991). decolouration and dewatering the demineralized crab material was treated with 300ml acetone for 10minutes and dried for 2 hours at an ambient temperature and the residues were removed to achieve decolourization. the decolourized sample was washed in running water, filtered and dried at 60oc until it was completely dried to obtain crab chitin (shahidi and synowiecki, 1991). deacetylation of chitin deacetylation of chitin was carried out using the method of yen et al. (2009). the obtained chitin was treated with 40% w/v aqueous sodium hydroxide in the ratio of chitin to the solution 1:15 w/v at 105oc in a water bath for 2 hours. thereafter, the chitin was filtered with filter pump and washed with deionized water until ph was neutral to obtain chitosan. the obtained chitosan was then dried at 60oc for 2 hours in the oven. the dried chitosan was preserved in a well labelled bottle and kept for the experiment. extract concentration preparation the extract solution for the study was prepared by dissolving 0.5g of the extract in 1ml of di-methylsulfoxide (dmso) solvent to have a stock concentration of 500mg/ml. oral toxicity testing to determine ld50 the bruce method of 1985 was used to determine the ld50 in this study. following this method, swiss mice were dosed one at a time beginning from 1000mg/kg of the extract from the crab because since the extract was from an edible source, there might not be low toxic doses. this was increased by a factor of 1.3 thus after this dose which produced no death, higher doses used were 1300mg/kg, 1690mg/kg, 2197mg/kg, 2856mg/kg, 3713mg/kg, 4827mg/kg and 6275mg/kg. with these doses there was no observed death of the mice and it was concluded that the extract is safe for the study based on pharmacology rule. experimental procedure this was based on the guinea pig cough model of nadig (2005) with minor alterations. a day before the test, guinea pigs were placed individually in a transparent chamber (60 × 36 × 60 cm) for 5 minutes before cough was induced by exposure to 15% citric acid, delivered using an omron (omron health care ltd, japan) compressor nebulizer (rate of 0.4 ml/min and particle size 5μm) for 10 minutes. the animals were then monitored visually within this exposure time for cough; the latency and counts, of which, were taken as the basal values. the animals exhibiting 10 20 bouts of cough were selected for the study and fasted overnight but with access to water. the selected animals were randomly allotted to 5 groups (n=5 per group). the animals were treated orally thus: group 1 was the control group and received 2 ml/kg distilled; group 2 received 25 mg/kg dihydrocodeine; group 3 received 150 mg/kg extract; group 4 received 300 mg/kg extract and group 6 received 600 mg/kg of the extract. an hour after administration, they were re-exposed to citric acid aerosol (as earlier described) and the latency of cough and cough count were recorded. the procedure was repeated at hours 2 and 3 after treatment. antitussive activity was then evaluated in each guinea-pig as the percentage reduction in the number of coughs also known as percentage suppression of cough and percentage increase in latency of cough in comparison with the previously established control basal value, calculated as below: 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑟𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑖𝑛 𝑐𝑜𝑢𝑔ℎ 𝑐𝑜𝑢𝑛𝑡 = [1 − ( 𝐶2 𝐶1 )] 𝑥 100 (1) where; c1 is basal values and c2 is the total number of coughs after treatment. 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑖𝑛𝑐𝑟𝑒𝑎𝑠𝑒 𝑖𝑛 𝑙𝑎𝑡𝑒𝑛𝑐𝑦 𝑜𝑓 𝑐𝑜𝑢𝑔ℎ = [1 − ( 𝐿2 𝐿1 )] 𝑥 100 (2) where; l1 is basal values, and l2 is the latency of coughs after treatment. data analysis all results are expressed as means ± sem. an analysis of variance was performed on the different treatment groups to determine significant effects of the treatments. posthoc analysis between the different groups was performed with a dunnett's t-test. a value of p≤0.05 was accepted as the level of statistical significance. 200 biology, medicine, & natural product chemistry 12 (1), 2023: 197-203 results and discussion bronchoconstriction is significant in cough induction since the process stimulates intrapulmonary rapidly adapting receptor (rar), a type of cough receptor to cause or enhance the sensitivity of the cough (pavord, 2004). rar activation initiates bronchospasm and mucus secretion via parasympathetic reflexes. this study investigated antitussive properties of the uca tangeri in guinea pigs. guinea pigs were used in the antitussive investigation because their airways possess the needed afferent nerves and can produce cough, just like in humans (agrawal et al., 1991). cough was detected with a characteristic sound and by stretching of limbs accompanied by inspiration and then expiration similar to that described by morice and co-workers (morice et al., 2007). these criteria were adopted so as to distinguish it from other respiratory reflexes like sneezing and expiratory reflex. as a tussigenic agent when inhaled, citric acid is known to stimulate transient receptor potential vanilloid1on the c-fibers. this then causes the release of tachykinnins to mediate bronchoconstriction and mucus secretion, which in turn stimulates rar (bonham et al., 1996 and canning et al., 2001), a widely studied cough receptor. the impulse is then conveyed through the vagus nerve to the cns and then back to respiratory muscle through the efferent pathway to cause cough. dihydrocodeine was used as the positive control because it is the second most specific antitussive of the commonly used opioids (eddy et al., 1969). dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996). number of cough in guinea pigs pre-treatment with dihydrocodeine, and uca tangeri and thereafter exposure to citric acid table 1. effect of uca tangeri on latency period (in seconds) in animals treated with acetic acid in a tussive protocol. group blp (seconds) olp (seconds) tlp (seconds) thlp (seconds) normal saline (ns) 43.60±0.25 44.60±0.25 44.80±0.37 45.40±0.40 dihdrocodeine (dh) 43.60±0.25 59.80±0.20 60.00±0.01 60.60±0.25 low dose (uca tangeri) (ldut) 43.60±0.25 48.00±0.01 48.20±0.20 48.60±0.24 medium dose (uca tangeri) (mdut) 43.60±0.24 51.80±0.20 52.00±0.01 52.60±0.24 high dose (uca tangeri) (hdut) 43.60±0.24 55.40±0.24 56.20±0.20 56.60±0.24 blp = basal latency period; olp = one hour latency period; tlp = two hours latency period; thlp = three hour latency period; low dose (uca tangeri) (ldut) = 150mg/g; medium dose (uca tangeri) (mdut) = 300mg/kg; high dose (uca tangeri) (hdut) = 600mg/kg table 1 presents the results of the number of cough bouts produced by the guinea pigs before and after pretreatment with normal saline, 25mg/kg of dihydrocodeine, 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri. although, there were no significant differences, observed between the basal i.e., pre-treatment cough bouts (17.60±0.24) and those of normal saline for the various time frames (14.60±0.24, 13.80±0.37 and 13.40±0.24), there were significant differences between the basal and those of 25mg/kg of dihydrocodeine, 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri. anova comparison of number of cough bouts between normal saline (14.60±0.24, 13.80±0.37 and 13.40±0.24), and dihydrocodeine (4.00±0.32, 3.20±0.20 and 3.00±0.01) respectively also showed significant differences. also, anova comparison of the number of cough bouts between normal saline (14.60±0.24, 13.80±0.37 and 13.40±0.24) and low dose of uca tangeri (8.20±0.37, 8.40±0.24 and 8.00±0.32 revealed a significant difference. this was also true for both medium dose and high dose. these results implies that normal saline does not reduce the number of cough bouts induced with citric acid, since its administration did not produce any significant difference from animals induced with citric acid without treatment. thus, it could be stated that normal saline does not have any effect on the intrapulmonary rapidly adapting receptor (rar). it neither activates nor inhibits it ability to cause or enhance the sensitivity of the cough. on the contrary, dihydrocodeine and all the different doses of the extract significantly reduced the number of cough bouts in the different time frames, implying that both the standard drug and the extract exerted inhibitory effect on the stimulatory process on the intrapulmonary rapidly adapting receptor (rar). therefore, this inhibitory effect also prevents the frequency of bronchospasm and mucus secretion caused by citric acid. these, antitussive properties were greater for dihydrocodeine, followed by high dose of the extract, medium dose of the extract and low dose of the extract. also, it is known that dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996), hence, this reduction in the number of cough bouts is associated with a stimulation of μ opioid receptors in the cns. in a similar manner the effect produced by the uca tangeri extract, isirima & uahomo – comparative cough suppression of chitosan crab extract … 201 could be associated with cns stimulation of μ opioid receptors. finally, it was noted that the effect of uca tangeri extract on the number of cough bouts was dose dependent, implying that the number of cough bouts decreased as the dose of the extract increased. latency period of cough in guinea pigs pre-treatment with dihydrocodeine and uca tangeri and exposure to citric acid table 2. effect of uca tangeri on number of cough bouts in animals treated with acetic acid in a tussive protocol. group pcb ocb tcb thcb normal saline (ns) 17.60±0.24 14.60±0.24 13.80±0.37 13.40±0.24 dihdrocodeine (dh) 17.60±0.24 4.00±0.32 3.20±0.20 3.00±0.01 low dose (uca tangeri) (ldut) 17.60±0.24 8.20±0.37 8.40±0.24 8.00±0.32 medium dose (uca tangeri) (mdut) 17.60±0.24 7.00±0.32 6.60±0.24 6.40±0.24 high dose (uca tangeri) (hdut) 17.60±0.24 5.80±0.37 5.40±0.24 5.20±0.20 pcb = pre-treatment cough bouts; ocb= cough bouts after one hour of drug administration; tcb = cough bouts after hour of drug administration; thcb = cough bouts after 3 hours of drug administration table 2 presents the results of the latency period caused by uca tangeri and dihydrocodeine in comparison to normal saline. it was observed that the basal latency period (43.60±0.25) was not significantly lower than that produced by normal saline, after one hour (44.60±0.25), two hours (44.80±0.37) and three hours (45.40±0.40) pre-treatment, but was significantly lower than those produced by dihydrocodeine (25mg/kg) after one hour, two hours and three hours (59.80±0.20, 60.00±0.01 and 60.60±0.25) pre-treatment respectively. this was also true for 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri. it was also observed that the latency periods of normal saline were significantly lower than those for 25mg/kg of dihydrocodeine and 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri for the different time frames of one, two and three hours. anova comparison revealed significant differences. this implies that there was no cough suppression associated with normal saline, while on the contrary; significant cough suppression is associated with dihydrocodeine and the extract from uca tangeri. this could be associated with activation of μ opioid receptors, since dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996). the effect produced by uca tangeri extract was similar to that of dihydrocodeine, hence a similar assumption could be made for the uca tangeri extract. it is also worth stating here that uca tangeri extract produce a dose dependent increase in the cough latency period, i.e., the cough latency period increasing as the dose increased. also, since cough induction is associated with significant stimulation of the cough receptor ‘intrapulmonary rapidly adapting receptor (rar)’, it can be deduced that both dihydrocodeine and the extract from uca tangeri significantly inhibited the cough receptor ‘intrapulmonary rapidly adapting receptor (rar)’and could be use in acute or chronic conditions of bronchial constriction, since significant bronchioconstriction is associated with cough induction. effect of dihydrocodeine and uca tangeri on percentage reduction in cough counts in animals treated with acetic acid in a tussive protocol table 3. effect of uca tangeri on percentage reduction in cough counts in animals treated with acetic acid in a tussive protocol. group oprcc tprcc thprcc normal saline (ns) 17.06±0.24 21.63±1.28 23.86±1.02 dihdrocodeine (dh) 77.19±2.03 81.83±1.04 82.94±0.24 low dose (uca tangeri) (ldut) 51.24±2.34 52.29±1.05 54.51±1.87 medium dose (uca tangeri) (mdut) 60.19±1.85 62.48±1.41 63.66±1.10 high dose (uca tangeri) (hdut) 66.93±2.52 69.28±1.53 70.46±1.02 oprcc = percentage reduction in cough count after one hour of drug administration; tprcc = percentage reduction in cough count after two hours of drug administration; thprcc = percentage reduction in cough count after three hours of drug administration table 3 presents the results of the percentage reduction in cough counts produced by normal saline, 25mg/kg of dihydrocodeine and 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri. it was 202 biology, medicine, & natural product chemistry 12 (1), 2023: 197-203 observed that there was significant difference in the percentage reduction of number of coughs produced by 25mg/kg of dihydrocodeine (77.19±2.03, 81.83±1.04 and 82.94±0.24), as compared to normal saline (17.06±0.24, 21.63±1.28 and 23.86±1.02) in the respective time frames of one hour, two hours and three hours. similar significant differences were observed between normal saline and the different doses (150mg/kg, 300mg/kg and 600mg/kg) of uca tangeri in the various time frames as shown in table 3. thus, normal saline clearly does not decrease the percentage number of cough, as observed in the study, but this is contrary to the effect observed with 25mg/kg of dihydrocodeine and 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri, which implies that both the standard drug and the test extract actually suppressed the stimulatory process of the intrapulmonary rapidly adapting receptor (rar), which are responsible for the cough or enhancement of the sensitivity of the cough. the fact that dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996) obviously implies that, the decrease in the percentage number of cough is related to the stimulation of μ opioid receptors in the cns. a similar deduction could be made for the uca tangeri extract, since similar and dose dependent pattern was observed with the extract. meaning the extract could possess some cns effect similar to dihydrocodeine. effect of dihydrocodeine and uca tangeri on percentage increase in cough latency in animals treated with acetic acid in a tussive protocol table 4. effect of uca tangeri on percentage increase in cough latency in animals treated with acetic acid in a tussive protocol. group opicl tpicl thpicl normal saline (ns) 2.29±0.01 2.75±0.45 4.13±0.45 dihdrocodeine (dh) 37.13±0.58 37.56±0.73 38.92±0.17 low dose (uca tangeri) (ldut) 10.61±0.62 10.61±0.62 11.92±0.43 medium dose (uca tangeri) (mdut) 18.83±0.95 19.28±0.67 20.65±0.81 high dose (uca tangeri) (hdut) 27.07±0.54 28.91±0.68 29.83±0.85 opicl = percentage increase in cough latency after one hour of drug administration; tpicl = percentage increase in cough latency after two hours of drug administration; thpicl = percentage increase in cough latency after three hours of drug administration table 4 presents the results of percentage increase in cough latency. anova comparison revealed that there were significant differences between normal saline (2.29±0.01, 2.75±0.45 and 4.13±0.45) and 25mg/kg of dihydrocodeine (37.13±0.58, 37.56±0.73 and 38.92±0.17) respectively, for the various time frames of one hour, two hours and three hours. similar significant differences were observed between normal saline and the different doses (150mg/kg, 300mg/kg and 600mg/kg) of uca tangeri for the various time frames. this implies that both 25mg/kg of dihydrocodeine and 150mg/kg, 300mg/kg and 600mg/kg of the extract from uca tangeri cause inhibition to the cough reflex or the stimulation of the intrapulmonary rapidly adapting receptor (rar), leading to a reduction of in the urge for cough, thereby causing a significant increase in the latency period of the cough. dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996). thus, the suppression of the cough leading to an increase in latency period is related to μ opioid receptors. it was also noted that the increase in cough latency produced by uca tangeri was dose dependent, meaning a greater suppression was observed with the highest dose, while the smallest suppression was produced by the lowest dose. also, since this suppression was similar to that caused by dihydrocodeine, it could be deduced that extract possesses central nervous system effect similar to that of dihydrocodeine. conclusion dihydrocodeine and uca tangeri were found to be effective antitussive agents. dihydrocodeine was used as the positive control because it is the second most specific antitussive of the commonly used opioids (eddy et al., 1969). dihydrocodeine acts on the μ opioid receptors to suppress the cough reflex (kamei 1996). uca tangeri also exhibited a dose-dependent antitussive effect reducing both the cough count and latency of cough, similar to dihydrocodeine. thus, it is possible that the extract possesses central nervous system effect similar to that of dihydrocodeine on cough. acknowledgements: the researchers acknowledge all laboratory staffs that assisted in ensuring this research is a success. authors’ contributions: isirima jc designed the study and analyzed the data and uahomo po carried out the laboratory work. isirima jc wrote the manuscript. all authors read and approved the final version of the manuscript. isirima & uahomo – comparative cough suppression of chitosan crab extract … 203 competing interests: the authors declare that there are no competing interests. references agrawal d.k., bergren d.r., byorth p.j., townley r.g. (1991). platelet-activating factor induces non-specific desensitization to bronchodilators in guinea pigs. j pharmacol exp ther.; 259:1–7. alves r.r., alves h.n. (2011). the faunal drugstore: animalbased remedies used in traditional medicines in latin america. journal of ethnobiology and ethnomedicine; 7(1):9. https://doi.org/10.1186/1746-4269-7-9 bolser d.c. (2006). cough suppressant and pharmacologic protussive therapy: accp evidence-based clinical practice guidelines. chest, 129:238s-249s bonham a.c., kott k.s., ravi k., kappagoda c.t., joad j.p. (1996). substance p contributes to rapidly adapting receptor responses to pulmonary venous congestion in rabbits. j physiol.; 493: 229–238. bruce r.d. (1985). an up-and-down procedure for acute toxicity testing. fundamental and applied toxicology, 5, 151–157 canning b.j., reynolds s.m., mazzone s.b. (2001). multiple mechanisms of reflex bronchospasm in guinea pigs. j appl physiol., 91:2642–2653. chung k.f, pavord i.d. (2008). prevalence, pathogenesis and causes of chronic coughs. lancet 371 (9621), 1364-74. dev roy m.k. (2014). crustacean bioresources of ethnomedicinal value. proc. nat. sem. trad. knowl. & soc. prac., 127-136. dicpinigaitis p.v., colice g.l., goolsby m.j., rogg g.i., spector s.l., winther b. (2009). acute cough: a diagnostic and therapeutic challenge. coughs, 5, 11. dublin-green, c. o. and ojanuga, a. g. (1999). the problem of acid sulphate soils in brackish water aquaculture. nigerian institute for oceanography and marine research technical paper no. 45. eddy n.b., friebel h., hahn k.j., hans h. (1969). codeine and its alternates for pain and cough relief. bull world health org.; 40:721-730. houghton l.a., lee a.s., badri h., devault k.r., smith j.a. (2016). respiratory disease and the oesophagus: reflux, reflexes and microaspiration. nat rev gastroenterol hepatol.; 13(8), 445-60 irwin r.s., glomb w.b., chang a.b. (2006). habit cough, tic cough and psychogenic cough in adult and pediatric populations: accp evidenced-based clinical practice guidelines. chest, 129 (1 suppl), 174s–179s kakati l.n, doulo v. (2002). indigenous knowledge system of zootherapeutic use by chakhesang tribe of nagaland, india. journal of human ecology; 13(6):419-423. kamei j. (1996). role of opioidergic and serotonergic mechanisms in cough and antitussives. pulm pharmacol.; 9:349–356. kirby t. (2018). the end of over-the-counter codeine in australia. lancet psychiatry; 5(5):395. doi: 10.1016/s22150366(18)30146-9 morice a.h., fontana g.a., belvisi m.g., birring s.s., chung k.f., dicpinigaitis p.v. (2007). ers guidelines on the assessment of cough. eur respir j., 29:1256–1276. nadig p., laxmi s. (2005) study of anti-tussive activity of ocimum sanctum linn in guinea pigs. indian j physiol pharmacol., 49: 243-245. national institute of health (2002). public health service policy on humane care and use of laboratory animals; office of the laboratory animal welfare, usa, p. 1-19. pavord i.d. (2004). cough and asthma. pulm pharmacol ther.; 17:399-402. shahidi, f. & synowiecki, j. (1991) isolation and characterization of nutrients and valueadded products from snow crab (chionoecetes opilio) and shrimp (pandalus borealis) processing discards. journal of agricultural food chemistry, 39 (8):1527–1532. sharma s., hashmi r.t., alhajjaj m.s. (2020). cough. dec 1. in: stat pearls [internet]. treasure island (fl): stat pearls publishing; jan–. pmid: 29630273. song w.j., chang y.s., faruqi s., kim j.y., kang m.g., kim s., (2015). the global epidemiology of chronic cough in adults: a systematic review and meta-analysis. eur respir j. doi:10.1183/09031936.00218714. vally, m and ihuma moe (2016). management of cough: a practical approach. south african family practice; 58(4):3539 yen, m.t., yang, j.h. & mau, j.l. (2009). physicochemical characterization of chitin and chitosan from crab shells. carbohydr polym., 75:15–21. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1385-1389 | doi: 10.14421/biomedich.2025.142.1385-1389 issn 2540-9328 (online) endophytic bacteria from mimosa pudica l.: morphological and biochemical traits and biocontrol activity against alternaria porri mazlina madjid faculty of agriculture, universitas amir hamzah, medan, indonesia. corresponding author madjidmazlina@gmail.com abstract shallot (allium cepa l.) is an important horticultural commodity whose productivity is frequently constrained by purple blotch disease caused by alternaria porri. excessive reliance on synthetic fungicides for disease control has raised concerns related to environmental sustainability and pathogen resistance. endophytic bacteria, which inhabit internal plant tissues without causing harm, represent a promising source of biological control agents. this study aimed to characterize the morphological and biochemical traits and to evaluate the biocontrol activity of endophytic bacteria isolated from mimosa pudica l. against a. porri. a total of twelve endophytic bacterial isolates were obtained from healthy roots, stems, and leaves of m. pudica using a surface sterilization technique. the isolates exhibited diverse colony morphology and growth characteristics. biochemical characterization revealed that most isolates were gram-positive, catalase-positive, motile, and capable of starch hydrolysis. antagonistic activity was evaluated using a dual culture assay on potato dextrose agar. the results demonstrated that several isolates effectively inhibited the mycelial growth of a. porri. among them, isolates mp5 and mp11 showed the highest inhibitory effects, with growth inhibition percentages of 58.2% and 48.7%, respectively, and were classified as having high antagonistic activity. the strong inhibitory performance of selected isolates suggests their ability to produce antifungal metabolites or to suppress pathogen growth through competitive interactions. overall, endophytic bacteria isolated from m. pudica, particularly isolates mp5 and mp11, exhibit significant potential as environmentally friendly biological control agents for managing purple blotch disease in shallot cultivation. keywords: endophytic bacteria; mimosa pudica; biocontrol activity; alternaria porri; shallot. introduction shallot (allium cepa l.) is an economically important horticultural crop widely cultivated in tropical and subtropical regions. in indonesia, shallot plays a crucial role in food security and agricultural income. however, its productivity is frequently constrained by plant diseases, particularly purple blotch disease caused by alternaria porri. this pathogen attacks leaves and flower stalks, leading to reduced photosynthetic capacity, premature leaf senescence, and significant yield losses under favorable environmental conditions (singh et al., 2018; prameswari et al., 2021). conventional management of purple blotch disease relies heavily on repeated applications of synthetic fungicides, which may result in pathogen resistance, environmental contamination, and negative impacts on non-target organisms (brent & hollomon, 2007). the development of environmentally friendly disease management strategies has therefore become a major focus in sustainable agriculture. biological control using beneficial microorganisms is considered a promising alternative to chemical-based approaches. among these microorganisms, endophytic bacteria have attracted considerable attention due to their ability to colonize internal plant tissues without causing disease symptoms (hardoim et al., 2015). endophytic bacteria can enhance plant health by suppressing pathogens through multiple mechanisms, including the production of antifungal metabolites, competition for nutrients and space, and induction of systemic resistance in host plants (santoyo et al., 2016). numerous studies have reported the effectiveness of endophytic bacteria as biological control agents against fungal pathogens in various crops. for example, endophytic bacillus and pseudomonas species have been shown to inhibit fusarium, alternaria, and rhizoctonia species through the production of antibiotics, lytic enzymes, and volatile organic compounds (compant et al., 2010; yanti et al., 2018). despite these promising findings, the exploration of endophytic bacteria from non-cultivated or wild plant species remains limited, particularly in the context of shallot disease management. mimosa pudica l., commonly known as the sensitive plant, is a wild leguminous species that thrives under diverse and often stressful environmental conditions. its manuscript received: 12 december, 2025. revision accepted: 14 december, 2025. published: 17 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1385-1389 1386 biology, medicine, & natural product chemistry 14 (2), 2025: 1385-1389 ability to survive in marginal habitats suggests a close association with beneficial endophytic microorganisms that may contribute to stress tolerance and disease resistance. previous studies have indicated that wild plants can serve as reservoirs of diverse and functionally important endophytic bacteria with strong antagonistic activity against plant pathogens (gouda et al., 2016; afzal et al., 2019). however, information regarding the biocontrol potential of endophytic bacteria isolated from m. pudica against a. porri is still scarce. therefore, this study aimed to characterize the morphological and biochemical traits of endophytic bacteria isolated from m. pudica and to evaluate their antagonistic activity against a. porri. the findings of this research are expected to provide scientific evidence supporting the use of endophytic bacteria as sustainable biological control agents for managing purple blotch disease in shallot cultivation. materials and methods materials the research was conducted from october 2023 to january 2024. isolation and characterization of endophytic bacteria, as well as antagonistic activity tests, were conducted at the microbiology laboratory of the faculty of mathematics and natural sciences, university of north sumatra, medan. the materials used were healthy rice root samples of xanthomonas oryzae isolate, na, 70% alcohol, clorox, distilled water, and agrept (bactericide). the tools used in this study included petri dishes, erlenmeyer flasks, measuring cylinders, bunsen burners, hotplates, glass slides, loop needles, cover glasses, auto-aerators, scissors, microscopes, analytical balances, stationery, cameras, vernier calipers, and scalpel blades. methods isolation of endophytic bacteria was performed using a surface sterilization technique to eliminate epiphytic microorganisms. plant tissues were washed under running tap water, surface-sterilized with 70% ethanol for 1 minute, followed by 2% naocl for 2 minutes, and rinsed three times with sterile distilled water. sterilized tissues were aseptically macerated, serially diluted, and plated on nutrient agar. plates were incubated at 28–30 °c for 48 hours, and morphologically distinct colonies were purified through repeated streaking (hardoim et al., 2015; sihotang et al., 2025). morphological characterization was conducted based on colony color, shape, margin, surface texture, and growth rate. biochemical characterization included gram staining, catalase and oxidase tests, motility assessment, and starch hydrolysis following standard microbiological procedures (cappuccino & sherman, 2014; sihotang et al., 2025). antagonistic activity against alternaria porri was evaluated using the dual culture method on potato dextrose agar (pda). a mycelial plug of a. porri was placed at the center of the petri dish, while bacterial isolates were streaked at a fixed distance from the fungal plug. plates were incubated at 28 °c for seven days. fungal growth inhibition was determined by comparing the radial growth of the pathogen in treatment and control plates, as previously described for endophytic bacterial biocontrol assays (compant et al., 2010; sihotang et al., 2025). data analysis data analysis was performed using one-way anova, following tests for normality (shapiro-wilk) and homogeneity (levene’s test). if the data met normality and homogeneity assumptions (p > 0.05), post duncan tests were used to analyze group differences. if data were not normally distributed, the kruskal-wallis test was used, followed by mannwhitney u tests for pairwise comparisons when significant differences were observed. results and discussion morphological characteristics of endophytic bacterial isolates from mimosa pudica l. table 1. morphological characteristics of endophytic bacterial isolates from mimosa pudica l. isolate colony color shape margin surface texture growth (mm/48 h) mp1 milky white circular entire convex smooth 3.8 mp2 cream circular entire convex smooth 4.1 mp3 white circular entire slightly glossy smooth 5.2 mp4 light yellow irregular undulate flat rough 3.5 mp5 opaque white circular entire glossy smooth 6.0 mp6 cream circular entire convex smooth 4.0 mp7 pale yellow irregular undulate slightly rough rough 3.2 mp8 milky white circular entire convex smooth 4.5 mp9 white circular entire convex smooth 4.3 mp10 yellow irregular undulate flat rough 3.4 mp11 cream white circular entire slightly glossy smooth 5.4 mp12 white circular entire convex smooth 4.0 madjid – endophytic bacteria from mimosa pudica l.: morphological … 1387 biochemical characteristics of endophytic bacterial isolates table 2. microskopic characterization and gram staining of endophytic bacteria. isolate gram reaction catalase oxidase motility starch hydrolysis mp1 + + – + – mp2 + + + + + mp3 + + + + + mp4 – + – – – mp5 + + + + + (strong) mp6 + + – + – mp7 – – – – – mp8 + + + + + mp9 + + – + – mp10 – + – – – mp11 + + + + + (strong) mp12 + + – + – antagonistic activity of endophytic bacteria against alternaria porri table 3. antagonistic activity of endophytic bacteria against alternaria porri. isolate inhibition zone (mm) growth inhibition (%) activity category mp1 4.2 18.0 low mp2 5.6 22.5 low mp3 9.2 36.0 moderate mp4 2.1 9.8 low mp5 14.5 58.2 high mp6 3.5 14.2 low mp7 1.8 7.0 low mp8 8.7 34.5 moderate mp9 4.8 19.2 low mp10 2.5 10.5 low mp11 12.1 48.7 high mp12 3.2 13.0 low discussion the results showed that endophytic bacterial isolates from rice (oryza sativa) plants consisted of several dominant genera, namely pseudomonas and bacillus. morphological characterization, gram staining, and biochemical tests revealed significant variation among isolates. most isolates exhibited high catalase, starch hydrolysis, and motility capabilities, indicating their potential for adaptation to host plant tissues. these results are consistent with reports that the bacillus and pseudomonas genera are the most common endophytic bacterial groups that play a role enhance rice plant growth and resistance to pathogens. antagonistic tests against xanthomonas oryzae pv. oryzae (xoo), the causative agent of bacterial leaf blight in rice, showed that isolates betp 03 (pseudomonas) and betp 05 (bacillus) produced the largest inhibition zones, with diameters >16 mm. this confirms their ability to produce antimicrobial secondary metabolites such as siderophores, antibiotics, and hydrolytic enzymes. these findings align with recent research confirming the effectiveness of pseudomonas fluorescens and bacillus subtilis in inhibiting xoo growth through antibiosis mechanisms and induction of systemic resistance in rice. furthermore, hydrolytic enzyme activity in several isolates supports their role as biocontrol agents. enzymes such as chitinase, protease, and lipase are known to be able to damage pathogen cell walls, thereby strengthening plant defenses. biochemical tests showed that isolates betp 04 and betp 05 exhibited a combination of citrate, gelatinase, and catalase activity, strengthening the hypothesis that these isolates have the potential to be used as both a biofertilizer and a biopesticide. from a microbial ecology perspective, the presence of endophytic bacteria within rice tissues provides a competitive advantage over epiphytic microbes, as they can survive in the relatively stable internal environment of the plant. thus, endophytes can play a dual role: increasing plant nutrient availability (for example, through nitrogen fixation or phosphate solubilization) and protecting the plant from pathogen attack. however, this study also has limitations, namely the lack of molecular analysis (16s rrna) to ensure accurate isolate identification, as well as field trials to 1388 biology, medicine, & natural product chemistry 14 (2), 2025: 1385-1389 confirm the effectiveness of biocontrol in real agroecosystem conditions. further studies are strongly recommended to explore the molecular mechanisms of antimicrobial metabolite production and the formulation of endophyte consortia as environmentally friendly biopesticides. thus, the results of this study provide strong evidence that the endophytic bacteria pseudomonas and bacillus from oryza sativa have high prospects as biocontrol agents to reduce dependence on chemical pesticides in sustainable rice cultivation systems. conclusions the present study demonstrates that endophytic bacteria isolated from mimosa pudica l. exhibit considerable diversity in morphological and biochemical characteristics, as well as varying levels of antagonistic activity against alternaria porri. the variation in colony morphology and growth rates among isolates reflects the heterogeneity of endophytic bacterial communities commonly reported in wild plant species (hardoim et al., 2015). wild plants such as m. pudica, which thrive in diverse and often stressful environments, are known to harbor endophytes with adaptive traits that may contribute to plant protection mechanisms (gouda et al., 2016). biochemical characterization revealed that most isolates were gram-positive and catalase-positive, suggesting a predominance of bacteria with strong stress tolerance and metabolic versatility. similar findings have been reported for endophytic bacteria belonging to the genus bacillus, which are frequently associated with antagonistic activity against fungal pathogens due to their ability to produce a wide range of antimicrobial compounds (compant et al., 2010; santoyo et al., 2016). the presence of starch hydrolysis and motility traits among several isolates further indicates their potential competitiveness and adaptability in colonizing plant tissues. the dual culture assay demonstrated that isolates mp5 and mp11 exhibited high antagonistic activity against a. porri, with growth inhibition exceeding 45%. this level of inhibition is comparable to that reported for effective endophytic biocontrol agents against alternaria species in previous studies (yanti et al., 2018; prameswari et al., 2021). the inhibitory effects observed in this study may be attributed to multiple mechanisms, including the production of antifungal metabolites, secretion of cell wall–degrading enzymes, and competition for nutrients and space (weller, 2007). although the specific metabolites were not identified in this study, the strong suppression of fungal growth suggests active antagonistic interactions between the endophytic bacteria and the pathogen. the results also support the concept that wild plants can serve as valuable reservoirs of biocontrol agents. endophytic bacteria isolated from non-cultivated hosts have been reported to exhibit stronger or broader antagonistic activity compared to those from cultivated plants, likely due to long-term ecological adaptation (afzal et al., 2019). the findings are consistent with sihotang et al. (2025), which highlighted the biocontrol potential of endophytic bacteria isolated from different plant hosts against phytopathogenic fungi. overall, the strong antagonistic performance of selected isolates, particularly mp5 and mp11, indicates their potential for further development as environmentally friendly biological control agents. future studies should focus on molecular identification, characterization of antifungal metabolites, and in planta evaluations to confirm their effectiveness under greenhouse and field conditions. acknowledgements: the authors would like to express their deepest gratitude to universitas amir hamzah for the support and facilities provided during the completion of this research. authors’ contributions: mazlina carried out the laboratory work and analyzed the data and wrote the manuscript. competing interests: the authors declare that there are no competing interests. references alam, m. z., islam, m. r., & rahman, m. m. (2019). endophytic bacteria associated with rice plants and their antagonistic activity against xanthomonas oryzae. microbiological research, 227, 126296. https://doi.org/10.1016/j.micres.2019.126296 afzal, i., shinwari, z. k., sikandar, s., & shahzad, s. (2019). plant beneficial endophytic bacteria: mechanisms, diversity, host range and genetic determinants. microbiological research, 221, 36–49. https://doi.org/10.1016/j.micres.2019.02.001 compant, s., clément, c., & sessitsch, a. (2010). plant growthpromoting bacteria in the rhizoand endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. soil biology and biochemistry, 42(5), 669–678. https://doi.org/10.1016/j.soilbio.2009.11.024 gouda, s., kerry, r. g., das, g., paramithiotis, s., shin, h. s., & patra, j. k. (2016). revitalization of plant growth promoting rhizobacteria for sustainable development in agriculture. microbiological research, 185, 7–21. https://doi.org/10.1016/j.micres.2016.01.007 hardoim, p. r., van overbeek, l. s., berg, g., pirttilä, a. m., compant, s., campisano, a., döring, m., & sessitsch, a. (2015). the hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. microbiology and molecular biology reviews, 79(3), 293–320. https://doi.org/10.1128/mmbr.00050-14 madjid – endophytic bacteria from mimosa pudica l.: morphological … 1389 santoyo, g., moreno-hagelsieb, g., del carmen orozcomosqueda, m., & glick, b. r. (2016). plant growth-promoting bacterial endophytes. microbiological research, 183, 92–99. https://doi.org/10.1016/j.micres.2015.11.008 sihotang, s., butar-butar, a. d. p., nasution, l., rahma, h., trizelia, t., saragih, m., kuswardani, r. a., listiana, i., & napitupulu, b. s. (2025). isolation, morphological characterization, biochemistry, and antagonistic activity of endophytic bacteria from rice against xanthomonas oryzae pv. oryzae. biology, medicine & natural product chemistry, 14(2), 825–829. weller, d. m. (2007). pseudomonas biocontrol agents of soilborne pathogens: looking back over 30 years. phytopathology, 97(2), 250–256. https://doi.org/10.1094/phyto-97-2-0250 yanti, y., habazar, t., reflin, r., & nasution, a. (2018). endophytic bacteria as biological control agents of bacterial wilt disease on chili pepper. biodiversitas, 19(4), 1534–1540. https://doi.org/10.13057/biodiv/d190437 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 1, 2018 | pages: 27-31 | doi: 10.14421/biomedich.2018.71.27-31 issn 2540-9328 (online) quercetin: the bioactive compound from allium cepa l. as anti-inflammation based on in silico screening mohamad amin1,*, kurniawan setia putra2, ihya fakhrurizal amin3, nanda earlia4, dina maulina5, betty lukiati1, umie lestari1 1department of biology, faculty of mathematics and sciences, universitas negeri malang, indonesia 2graduate school for biology education study program, universitas negeri malang, indonesia 3faculty of medicine, university of indonesia, indonesia 4graduate school of mathematics and applied sciences, universitas syiah kuala, banda aceh, indonesia 5biology education, faculty of teacher training and education, university of lampung, lampung, indonesia author correspondency*: mohamad.amin.fmipa@um.ac.id abstract inflammation is a tissue injury that occurs due to physical trauma or microbiological substances that involve the activities of many cell types. inflammation can be prevented using the natural medicines from allium cepa l. quercetin is one of the bioactive compounds found in allium cepa l and has been reported to have anti-inflammatory activity. the natural medicines have been used to minimize nonsteroidal anti-inflammatory drugs. this study aims to investigate the modeling structures and the protein receptor from quecertin in inflammation mechanism and their optimization of the effectiveness in the human body. the bioinformatics tools used in this study are the database of quercetin compounds, pubchem and swis target prediction protein prediction databases, pyrx 0.8 molecular docking software, ligand docking, and binding site analysis with pymol and ligplus software. the results from in silico show that quercetin compounds can interact with muscleblind-like protein 1 target protein with a binding affinity minus value which is not much different from the dexamethasone compound. dexamethason is a standart because it is a corticosteroid drug that can be used as an antiinflammatory to reduce inflammation, allergic reactions, arthritis and other inflammatory diseases. keywords: allium cepa l.; anti inflamation; in silico, quercetin introduction quercetin compounds are found in the allium cepa l. that contain compounds including carbohydrates (11.0 g), protein (1.2 g), fiber (0.6 g), fat (0.30%) and several vitamins such as vitamin a (0.012 mg), vitamin c (11 mg), thiamin (0.08 mg), riboflavin (0.01 mg), and niacin (0.2 mg), and some minerals such as phosphorus, calcium, sodium, iron and potassium (rodrigues et al., 2003). quercetin has a molar mass of 302,236 g/mol, in the form of yellow crystalline powder, a density of 1,799 g/cm3 and a melting point of 316 ° c (smith et al., 2003). inflammation is a local reaction to tissue infection or injury and involves more mediators. inflammation has a fairly high incidence, where inflammation can be caused by physical trauma, infection or antigenic reactions from an illness. the natural secondary metabolite compound that has the potential for inflammatory treatment is quercetin, which is abundant in the allium cepa. chronic inflammation means long-term inflammation, which can last for several months and even years. this is due to the failure to remove anything that causes acute inflammation, an autoimmune response to self-antigens (the immune system attacks healthy tissue), an existing low-intensity chronic irritation. the research on allium cepa has been done before, however the mechanism of quercetin compounds can prevent the occurrence of inflammatory processes is unknown. therefore, this study aims to identify natural bioactive compounds from onion allium cepa for antiinflammatory based on in silico. we investigate the modeling structures and the protein receptor from quercetin in inflammation mechanism and their optimization of the effectiveness in the human body. materials and methods in silico screening in this research was conducted following the previous methods (maulina, et. al., 2018; pangastuti et.al, 2016; 2018 and ilmawati, 2017). retrieval of sample this research used the spesific compound from allium cepa l. the quecertin is the most abundant compound in allium cepa l (fredotović, z. et al., 2017) and it has been reported to have anti-inflammatory activity. therefore, quecertin was selected as a specific sample for analysis. in addition, the compositions of quercetin were obtained from the compounds database of pubchem. https://doi.org/10.14421/biomedich.2018.71.27-31 28 biology, medicine, & natural product chemistry 7 (1), 2018: 27-31 preparation of ligand the structure of quercetin 3d chemical compounds and smiles ligands is taken from the database on the pubcem server online (https://pubchem.ncbi.nlm.nih.gov/) with id number: cid: 5280804. target selection target protein selection is predicted by using the help of a number of servers to check and ensure the target protein is selected correctly. first, enter the ligand quercetin smiles value on the pharmmapper server online (http://59.78.96.61/pharmmapper/) to identify potential target proteins. second, observing potential target proteins on the swiss target prediction server online (http://www.swisstargetprediction.ch/) to look for similar target proteins which found on the first server. third, observing potential target proteins on the superpret server online (http://prediction.charite.de/) to adjust the similarity of target proteins which found on the first and second servers. the process of observing the target protein is carried out repeatedly to ensure the similarity of the target protein is correct, so could be not to affect the failure or inaccuracy during the docking process. docking molecules docking of quercetin molecules, target proteins, and control compounds (dexamethasone) which are chemical drugs in the treatment of inflammation using pyrx 0.8 software. visualization and molecular interaction the interaction between quercetin, target proteins, and control compounds known based on protein visualization was then analyzed using pymol and ligplus software and discovery studio 2016 client. results and discussion target selection results using pharmmapper (job id: cid 5280804), swiss target prediction and superpret found that quercetin interacts with the muscleblind-like protein 1 receptor. quercetin is a group of flavonoids produced by plants and can be used to treat inflammatory diseases and these compounds can be isolated from bulb onions (allium cepa). based on the results of the study it was found that quercetin compounds were able to bind to muscleblind-like protein 1 receptor protein (fig. 1) by forming bonds at one end of the side. visualization results using pymol and ligplus software show the following images (figure 1). the bonding between muscleblind-like protein 1 receptor as a target protein and quercetin compound has the same position as chemical compounds which have generally been used as inflammatory drugs, namely dexamethasone (figure. 2). figure 1. binding visualization between protein target (muscleblind-like protein 1 receptor) and natural bioactive quercetin (brown is protein target; green is quercetin). figure 2. visualization of binding between protein target, quercetin as bioactive compound and dexamethasone as control compound. note: brown color for protein target; green for quercetin and purple for dexamethason). dexamethasone is a corticosteroid drug that can be used as an anti-inflammatory to reduce inflammation, allergic reactions, arthritis and other rheumatic diseases, skin diseases, intestinal diseases such as ulcerative colitis and multiple sclerosis or myasthenia gravis. in general, dexamethasone is widely used in the therapy of chronic inflammatory diseases for its pain-modulating effects (laste et al, 2013). moreover, dexamethasone significantly inhibited the levels of tnf-α and il-6, suggesting a key role for these cytokines in sickness behavior (plessers et al. 2015). the results showed in figure 2 indicate that the natural compound of quercetin has the same molecular interaction as dexamethasone, a chemical compound that is often used as an anti-inflammatory drug. in addition, the results of the interaction analysis of the natural compound quercetin and the muscleblind like protein 1 protein were compared with the dexamethasone amin et al. – quercetin: the bioactive compound from allium cepa l.… 29 chemical compound with the muscleblind like protein 1 protein, which showed no significant difference. this shows that the natural compound quercetin is very potential to be used as an anti-inflammatory drug. the results of the calculation analysis of the target protein ligand bond interaction is described in table 1 and 2 as following. table 1. the analysis of the interaction of quercetin compounds with muscleblind-like protein 1 target protein. ligand binding affinity rmsd/ub rmsd/lb quercetinmuscleblind-like protein 1 -6.5 0 0 quercetinmuscleblind-like protein 1 -6.3 21.774 17.9 quercetinmuscleblind-like protein 1 -6.1 4.053 2.596 quercetinmuscleblind-like protein 1 -6 8.163 4.193 quercetinmuscleblind-like protein 1 -5.8 22.599 18.725 quercetinmuscleblind-like protein 1 -5.8 22.149 18.221 quercetinmuscleblind-like protein 1 -5.8 22.272 19.67 quercetinmuscleblind-like protein 1 -5.7 6.343 1.724 quercetinmuscleblind-like protein 1 -5.7 4.238 2.877 table 2. the analysis of the interaction of dexamethasone compounds with muscleblind-like protein 1 target protein. ligand binding affinity rmsd/ub rmsd/lb dexamethasone muscleblind like protein 1 -6.5 0 0 dexamethasone muscleblind like protein 1 -6.2 3.953 2.41 dexamethasone muscleblind like protein 1 -6 7.669 2.149 dexamethasone muscleblind like protein 1 -5.8 21.751 19.441 dexamethasone muscleblind like protein 1 -5.8 7.481 1.846 dexamethasone muscleblind like protein 1 -5.7 25.176 23.523 dexamethasone muscleblind like protein 1 -5.7 21.778 19.947 dexamethasone muscleblind like protein 1 -5.7 16.298 13.067 dexamethasone muscleblind like protein 1 -5.7 19.714 16.467 analysis based on data in tables 1 and 2 shows that it is not significantly different from the chemical bonds between quercetin and dexamethasone, with the binding affinity value of the two compounds in the similar ranges. the binding affinity value shows the best bond between the ligand and the target protein. the minus value shows that the ligand is easier to bind to the target protein because it requires less energy to bind. based on the range of binding affinity values showed that many ligand residues that bind to the target protein, especially in dexamethasone compounds as control compounds have better bond range than natural compounds, quercetin compounds. but from the range of values these two compounds show not much different. visualization of the results of inter-molecular interactions using the discovery studio 2016 client software shows that the bond distance between molecules and the type of bond that occurs on each residues of the ligand molecule and target protein more details show in figure 3 and 4. cyclooxygenase (cox) or prostate glandinendo peroxide synthase (pghs) is a bifunctional enzyme that initially converts arachidonic acid to prostaglandin g2 (pgg2) through oxygenated, then catalyzes pgg2 peroxidase to pgh2. pgh2 is a precursor to the formation of several important mediators for pain, fever and inflammation. two forms of cyclooxygenase enzyme isoforms are known, namely cox-1 and cox2. cox-1 is a constitutive enzyme that have a role for play in the regulation of several cellular processes, including homeostasis vascular, protection of the gastrointestinal tract and kidney function. cox-2 is induced and is in inflamed tissue. inhibition of this cox enzyme is the main working mechanism of non-steroidal anti-inflammatory drugs that are widely used (kartasasmita et al, 2009). 30 biology, medicine, & natural product chemistry 7 (1), 2018: 27-31 figure 3. distance of intra interaction between quercetin (bioactive compound) and protein target (muscleblind-like protein 1) and the type of binding. figure 4. distance of intra interaction between dexamethasone (control compound) and protein target (muscleblind-like protein 1) and the type of binding. cyclooxygenase (cox) or prostate glandinendo peroxide synthase (pghs) is a bifunctional enzyme that initially converts arachidonic acid to prostaglandin g2 (pgg2) through oxygenated, then catalyzes pgg2 peroxidase to pgh2. pgh2 is a precursor to the formation of several important mediators for pain, fever and inflammation. two forms of cyclooxygenase enzyme isoforms are known, namely cox-1 and cox2. cox-1 is a constitutive enzyme that have a role for play in the regulation of several cellular processes, including homeostasis vascular, protection of the gastrointestinal tract and kidney function. cox-2 is induced and is in inflamed tissue. inhibition of this cox enzyme is the main working mechanism of non-steroidal anti-inflammatory drugs that are widely used (kartasasmita et al, 2009). conclusion this study proves that the natural compound quercetin found in onion plants (allium cepa l) has the potential as an anti-inflammatory drug. the type of chemical bounding between protein ligand and control compound (dexamethasone) is similiar. there is no to significant the bond distance and type of chemical bond formed. references fredotovic, ž., šprung, m., soldo, b., ljubenkov, i., budi´c-leto, i., bilušic´, t., cˇ ikeš-ˇulic´, v., and puizina, j. 2017. chemical composition and biological activity of allium cepa l. and allium _ cornutum(clementi ex visiani 1842) methanolic extracts. molecules, 22, 448; doi:10.3390/molecules22030448. amin et al. – quercetin: the bioactive compound from allium cepa l.… 31 kartasasmita, r.e., herowati, r., harmastuti, n., & gusdinar, t. (2009). quercetin derivatives docking based on study of flavonoids interaction to cyclooxygenase-2. indo. j. chem. 9 (2):297-302. maulina, d., amin, m., lestari, s.r., & aziz, m. 2018. alanine as natural biopesticide from mirabilis jalapa and its interaction with glutamate as an inhibitor in insects immune system. journal of biological researches, vol. 23: 2. doi: 10.23869/bphjbr.23.2.20185. pangastuti, a., amin, i.f., zhilalikbar, a., amin, m. 2016. natural bioactive compound from moringa oleifera against cancer based on in silico screening. jurnal teknologi (sciences & engineering). 78(5): 315-318 pharmmapper database. http://59.78.96.61/pharmmapper/ (accessed on march 2018) plessers, e.; watteyn. a.; wyns, h.; pardon, b.; de backer, p. and croubels, s. 2015. study of the immunomodulatory properties of gamithromycin and dexamethasone in a lipopolysaccharide inflammation model in calves. res vet sci. 2015 dec; 103:218-23. doi: 10.1016/j.rvsc.2015.10.014. epub 2015 oct 28 pubchem database. https://pubchem.ncbi.nlm.nih.gov/ (accessed on march 2018) puspita sari, 2015.studi in silico daun salam (syzygium polyanthum) terhadap angio tensin converting enzyme. jurnal kedokteranvol 3, no 1 ringo maeda, 2013. isolasi senyawa flavonoid dari kulit bawang merah (allium cepa l).skripsi fakultas matematika dan ilmu pengetahuan alam, universitas sumatra utara medan. rodrigues a., fogliano v., graziani g., mendes, s., vale, a. and goncalves, c., 2003. nutrition value of onion regional varieties in northwest portugal, ejeafche 2(4): 519-524. ryfai, ea 2012, ‘penapisan in silico anti malaria dari basis data tanaman obat indonesia terhadap target plasmepsin’, (skripsi) fakultas matematika dan ilmu pengetahuan alam, universitas indonesia. smith, c., lombard, k.a., peffley, e.b.,liu, w. 2003. genetic analysis of quercetin in onion (allium cepa l.) ‘lady raider’. the texas journal of agriculture and natural resource 16:24-28. soemari yulistia, 2016. uji aktivitas anti inflamasi quercetin bawang merah (allium cepa l).pada mencit putih jantan (musmusculus) superpret server. http://prediction.charite.de/ (accessed on march 2018) swiss target prediction. http://www.swisstargetprediction.ch/ (accessed on march 2018) teresita, g., alejandra, e.r., americo, o. j., &lilian, e.p. 2001. anti-inflammatory properties of plant flavonoids. effects of rutin, quercetin and hesperidin on adjuvant arthritis in rat. elsevier ilfarmaco 56:683-687. https://www.ncbi.nlm.nih.gov/pubmed/?term=plessers%20e%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/?term=watteyn%20a%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/?term=wyns%20h%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/?term=pardon%20b%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/?term=de%20backer%20p%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/?term=croubels%20s%5bauthor%5d&cauthor=true&cauthor_uid=26679821 https://www.ncbi.nlm.nih.gov/pubmed/26679821 https://www.ncbi.nlm.nih.gov/pubmed/26679821 this page intentionally left blank quercetin: the bioactive compound from allium cepa l. as anti-inflammation based on in silico screening biology, medicine, & natural product chemistry 7(1) 2018 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1091-1097 | doi: 10.14421/biomedich.2025.142.1091-1097 issn 2540-9328 (online) therapeutic effect of topical ointment ethanol extract from patiwala leaves (lantana camara) on histological profile of incision wounds in diabetic rat models theosobia grace orno1*, anita rosanty1, ratih feraritra danu atmaja1, zulfikar ali hasan2, tuty yuniarty1, ahmat rediansya putra1 1department of medical laboratory technology, poltekkes kemenkes kendari, indonesia 2department of medical laboratory technology, poltekkes kemenkes makassar, indonesia. corresponding author* theosobiagraceorno@gmail.com abstract diabetic wounds are one of the neurovascular complications of diabetes. hyperglycemia complicates the healing of diabetic wounds, which are susceptible to infection and chronic inflammation. topical treatment using herbal extracts aims to reduce the side effects of surrounding tissue damage. a topical ointment made from ethanol extract of patiwala (lantana camara) leaves, which has been tested for product quality, has antibacterial and anti-inflammatory effects that has been proven to help accelerate the healing of diabetic wounds in rats through parameters such as reduced wound diameter, changes in the number of fibroblast cells, and collagen fiber formation seen in the histological profile during 21 days of therapy with a concentration of 15% (p>0.05) compared to the positive control of 10% betadine. keywords: topical ointment; lantana camara; histological profile; diabetic wounds; fibroblast cells. introduction diabetes is a major cause of damage to various organs such as the heart, blood vessels, nerves, eyes, and kidneys (harreiter & roden, 2023; cloete, 2022). one complication of diabetes related to blood vessel and nerve damage is diabetic wounds or diabetic neuropathy (urso et al., 2021; mcdermott et al., 2023). sensory and motor neuropathy can cause various changes in the skin and muscles, which then lead to changes in pressure distribution in the peripheral area that triggers ulcers (kurz, 2020; akkus & sert, 2022). the healing of diabetic ulcers or diabetic wounds is a serious concern for clinicians today. this is because the speed of diabetic wound healing is not the same as the healing of wounds in general. diabetic wounds contain high blood glucose levels (hyperglycemia) which is known to be an energy source for pathogenic bacteria, making diabetic wounds very susceptible to infection and inflammation (raja et al., 2023; wang et al., 2025). topical treatment of diabetic wounds using chemical antibiotic ointments often causes side effects, including damage to the surrounding skin tissue. therefore, various studies on herbal ointments are ongoing, with the hope that they can become an alternative treatment for diabetic wounds with minimal side effects. herbal plant extracts intended to help accelerate diabetic wound healing must be able to inhibit pathogenic bacteria that trigger infection and inflammation in diabetic wounds in vitro (ramachandran et al., 2023; norman et al., 2021). one of the local plants often used by the people of southeast sulawesi to treat wounds is the patiwala leaf (lantana camara). several previous studies have reported the potential of lantana camara in various preparations for incision wounds (tamuntuan et al., 2021; arifin et al., 2023; saranani et al., 2023) but there has been no specific research on diabetic wounds. the ability of lantana camara in treating wounds is influenced by its phytochemical compounds which functions as an antibacterial and anti-inflammatory (sari et al., 2023; hasnaeni et al., 2024). the results of phytochemical screening of lantana camara vary quite a bit, depending on the geographical conditions of the place of growth as well as the analytical method used (kapitan et al., 2024; orji et al., 2024). the phytochemical compounds of ethanol extract of patiwala leaves from southeast sulawesi has been reported in a study by rosanty et al. (2025) with active compounds of alkaloids, flavonoids, tannins, saponins and terpenoids (rosanty et al., 2025). antibacterial activity test of the extract against several pathogenic bacteria causing diabetic ulcers in vitro including staphylococcus aureus, staphylococcus epidermis, proteus mirabilis, proteus vulgaris, pseudomonas aeruginosa, klebsiella manuscript received: 16 september, 2025. revision accepted: 25 november, 2025. published: 27 november, 2025. https://doi.org/10.14421/biomedich.2025.142.1091-1097 mailto:theosobiagraceorno@gmail.com 1092 biology, medicine, & natural product chemistry 14 (2), 2025: 1091-1097 pneumoniae and e.coli with ciprofloxacin as a positive control showed quite significant results. this study is a follow-up study of rosanty (2025) which was conducted in vivo using a white rat model of the wistar type with diabetic wounds to observe the histological profile after topical ointment therapy with ethanol extract of patiwala leaves (lantana camara). materials and methods research ethics and design this research was an experimental study involving 30 male wistar rats. the research was conducted from june to december 2024 at the biomedical research laboratory, faculty of medicine, halu oleo university. this study received ethical approval from the health research ethics committee, tanjung karang ministry of health polytechnic, under no. 451/kepktjk/viii/2023. materials and equipment the main material used in this study was a topical ointment of ethanol extract of patiwala (lantana camara) leaves formulated based on the results of in vitro extract effectiveness tests in the study by rosanty (2025) with the following formula: materials % ingredients in every 10 grams of formula function formula a b c ethanol extract of patiwala leaves (lantana camara) 5 10 15 active compound propil paraben 0.01 0.01 0.01 preservative cera alba 2 2 2 base vaselin album ad 100 ad 100 ad 100 base other materials and equipment include measuring flasks, erlenmeyer flasks, test tubes, stirring rods and dropper pipettes for making ointments, weighing rats using a macs 1.5/w digital scale, scalpels for making incision wounds, calipers for measuring wound diameter. making a diabetic rat models using an induction agent streptozotocin/stz (sigma-aldrich) dissolved in 0.9% nacl, measuring blood glucose levels in using a point of care testing (poct) fora 6 plus (switzerland). histological observation of incision wounds using hematoxylin-eosin staining technique with natural buffered formalin 10% (indopath-paraform) for tissue fixation, ethanol 70%, 80% and 95% (merck) for dehydration-rehydration, xylol (bio-analitika) for clearing, paraffin (paraflakes) and basemold for embedding, microtome tool (accu-cut), object glass and deck glass for microtomy and preparation of preparations, hematoxylin (biognost) and eosin (indoreagen). treatment of experimental animals the experimental animals used in this study were 30 white wistar male rats with body weights ranging from 90-200 grams. the rats were divided into 3 groups with 10 rats each group consisting of k-7 for the intervention group for 7 days, k-14 for the intervention group for 14 days, and k-21 for the intervention group for 21 days. then all rats were acclimatized in standard cages, fed standard food with a light/dark cycle for 2 weeks. body weight (bw) and random blood glucose level measurements (rbg) were carried out on all rats before induction. each group was then divided into 5 subgroups consisting of the 5% patiwala ointment intervention group (k1), the 10% patiwala ointment intervention group (k2), the 15% patiwala ointment intervention group (k3), the 10% betadine ointment positive control (kp) and the untreated negative control (kn), each subgroup consisting of 2 rats. stz induction was performed on all rats with a single intraperitoneal dose of 50 mg/kgbw. after 5 days, bw and rbg levels were remeasured to confirm diabetes (rbg >200 mg/dl). a 5 cm incision wound was made in the low back of the diabetic rats. treatment was administered twice daily to both the intervention and positive control groups. on the final day of the intervention, the rats were euthanasia using carbon monoxide. skin fragments from the incision wounds were removed and fixed in 10% nbf for histological analysis. intracardial blood was collected using gel separator tubes, other organs and tissues were also collected and stored in 10% nbf for further research. measurement of rbg levels rbg levels were measured in each group two times: before stz induction and 5 days after stz induction. rbg levels were measured using the fora 6 plus device using the poct method, which is based on the principle of enzymatic reactions. rbg levels are showed in milligrams per deciliter (mg/dl). measurement of wound diameters wound diameters were measured in each group two times: before stz induction and 5 days after stz induction. wound diameter measurements using calipers, the results are showed in millimeters (mm). histological analysis the skin tissue that had been fixed in 10% nbf solution for 1x24 hours was then dehydrated using ethanol in stages starting from 70%, 80%, and 95% for 2 hours each. then, the dehydrated tissue was cleared using xylol solution for 2 cycles, each for 1 hour. next, embedding was carried out in a basemold using liquid paraffin at a temperature of 40°c. after the tissue block was formed, orno et al. – therapeutic effect of topical ointment ethanol extract from … 1093 tissue ribbons were cut on a microtome with a size of 5 µm. the tissue ribbons were placed on a glass object, fixed on a hotplate at a temperature of 40°c to remove residual paraffin, and then continued with hematoxylin and eosin staining. histological observations were carried out under a microscope with a magnification of 40x (orno, 2023). the number of fibroblast cells was counted in 15 fields of view on each slide and the results were presented as an average value. data analysis the effects of stz induction on rats bw and rbg levels were analyzed using the wilcoxon test. wound diameters and number of fibroblast cells after therapy in each subgroup within a group were analyzed using the oneway anova test and for comparison of wound diameter between groups using the kruskal wallis test. the histological results are presented in the form of micrographs followed by notes. results and discussion characteristics of topical ointment products the topical ointment made from ethanol extract of patiwala leaves used in this study has been tested in vitro to have antibacterial effects against several bacteria that cause diabetic wounds. the table below is a description of the topical ointment product and the results of the quality test for the ointment preparation. table 1. product quality report of topical ointment of patiwala leave ethanol extract. product product quality report organoleptic homogenity ph viscosity (cp) spreadability (mm) stickiness (sec) topical ointment of patiwala leave ethanol extract lightly greenish brown color, distinctive odor, soft hydrocarbon ointment form, smooth without coarse particles, and easy to apply homogen 6.42 8.745 41 4 ointment physical properties testing is conducted as part of efforts to ensure the quality and stability of the preparation and to ensure that the final product meets predetermined quality criteria. this evaluation is important because the physical characteristics of the ointment, such as visual appearance, texture, ph, and ability to spread and adhere to the skin surface, significantly influence the comfort of use and therapeutic effectiveness of topical preparations (savitri et al., 2025; wahyuni et al., 2025). the test results showed that the ointment could be applied to experimental rats and can be used as a reference for future formulation needs. effect of stz induction on body weight and random blood glucose this study used stz as an induction agent in an animal model of diabetes mellitus. table 2 explains the effect of stz induction on increasing rbg levels with a mean level of 310.23 ± 59.12 (p<0.001) which is in line with the increase in bw of rats (p=0.013). table 2. effect of stz induction on body weight and random blood glucose. variables mean ± sd p value body weight (g) pre-stz post-stz 135.75 ± 49.39 144.22 ± 41.12 0.013 random blood glucose (mg/dl) pre-stz post-stz 86.13 ± 17.74 310.23 ± 59.12 <0.001* *wilcoxon test, the level of significance at p< 0.05 several studies have reported stz is cytotoxic to pancreatic β-cells, and its effects are visible 72 hours after administration and are dose-dependent. the toxic effects of stz begin with the uptake of stz into cells via the low-affinity glucose transporter-2 (glut2) found in the plasma membrane of β-cells, hepatocytes, and renal tubular cells. this has been demonstrated by studies showing that insulin-producing cells that do not express glut2 are resistant to stz induction (al-awar et al., 2016; pandey & dvorakova, 2020). 1094 biology, medicine, & natural product chemistry 14 (2), 2025: 1091-1097 the therapeutic effect of ointment on the diameter of rat wounds hyperglycemic conditions in diabetes reduce the healing rate of incision wounds in rats this is caused by several conditions such as impaired angiogenesis, neuropathy, chronic inflammatory responses and bacterial infections (burgess et al., 2021). changes in the diameter of the incision wound are a visual indicator of the healing effect of the ointment used, as presented in table 3. table 3. comparison of wound diameter between groups and subgroups. groups/subgroups k-7 (mm) k-14 (mm) k-21 (mm) p value mean ± sd mean ± sd mean ± sd kp (betadine 10% k1 (patiwala 5%) k2 (patiwala 10%) k3 (patiwala 15%) 13.23 ± 0.01 18.90 ± 0.05 16.13 ± 0.15 15.91 ± 0.09 1.23 ± 0.07 13.43 ± 0.07 9.54 ± 0.42 5.63 ± 0.07 0.000 ± 0.00 4.85 ± 0.86 1.57 ± 0.07 0.00 ± 0.00 0.095** 0.102** 0.102** 0.095** p value <0.001* <0.001* 0.001* *one-way anova test, **kruskal wallis test, the level of significance at p< 0.05 the effectiveness of the ointment was estimated against 10% betadine as a positive control. the difference in wound diameter between subgroups in each group using the one-way anova test showed a significant difference (p<0.001). in the 7-day therapy group (k7), the average kp wound diameter was 13.23 ± 0.01 mm, significantly different from the k1, k2, and k3 groups. the 14-day therapy group (k14) showed almost perfect wound closure in the kp (1.23 ± 0.07 mm), while the k3 therapy group showed quite good results with an average wound diameter of 5.63 ± 0.07 mm. a linear pattern was seen in the 21-day therapy group (k21) where the kp wound diameter was 0.000 ± 0.00 mm in line with k3, followed by k2 and k1. the results of the kruskal-wallis test between groups showed significant results (p>0.05) which proved that in all groups there was a significant change in wound diameter. changes in wound diameter indicate the activity of the active compounds contained in the ointment. several previous studies have examined the role of active ingredients in herbal extracts in in vivo diabetic wound healing (nurwahita et al., 2024; palupi et al., 2022; wulandari et al., 2023) explains the role of active compounds such as alkaloids, flavonoids, tannins, saponins and terpenoids in accelerating the wound tissue regeneration process which is visible in changes in wound diameter. physiological wound tissue regeneration involves muscle fiber repair, extracellular matrix remodeling, and collagen deposition. theoretically, physiological wound closure occurs on days 10-14, but under hyperglycemic conditions, the remodeling process can take longer to compensate for the inflammation (giha et al., 2022; sun et al., 2024). patiwala topical ointment, with its antibacterial and antiinflammatory properties, helps prevent infection and chronic inflammation during the remodeling process. the effect of ointment therapy on the number of fibroblast cells in rats unlike changes in wound diameter, which can be observed and measured visually, the number of fibroblast cells can only be observed and counted microscopically. the number of fibroblast cells is one indicator of successful wound healing, calculated in 15 large fields of view (hpf), as presented in table 4. table 4. comparison of fibroblast cells between groups and subgroups. groups/subgroups k-7 (cells/hpf) k-14 (cells/hpf) k-21 (cells/hpf) p value mean ± sd mean ± sd mean ± sd kp (betadine 10%) k1 (patiwala 5%) k2 (patiwala 10%) k3 (patiwala 15%) 178.50 ± 3.53 158.00 ± 4.24 167.50 ± 14.84 175.50 ± 12.02 95.00 ± 4.24 110.00 ± 11.31 88.50 ± 4.94 156.50 ± 62.93 108.00 ± 1.41 110.00 ± 9.89 75.00 ± 12.72 90.50 ± 16.42 0.102** 0.180** 0.102** 0.180** p value 0.303* 0.283* 0.036* *one-way anova test, **kruskal wallis test, the level of significance at p< 0.05 table 4 shows that there was no significant difference in the number of fibroblast cells in k7 and k14 (p=0.303 & p=0.283). on the 7th day of therapy, all groups experienced a significant increase in the number of fibroblast cells, k3 was the group with the highest number of fibroblast cells (175.50 ± 12.02/hpf) approaching the number of kp fibroblast cells (178.50 ± 3.53/hpf). the pattern of changes in the number of fibroblast cells began to appear non-linear in k14 where kp showed a decrease in the number of fibroblast cells (95.00 ± 4.24/hpf) which was not in line with the decrease in the number of fibroblast cells in k3 and k1 orno et al. – therapeutic effect of topical ointment ethanol extract from … 1095 which on average experienced a decrease but anomalous with k2. although statistically there was no difference in the k14 group, k2 appeared to experience a very sharp decrease in the number of fibroblast cells (88.50 ± 4.94/hpf). variations in fibroblast cell count patterns during wound healing are theoretically possible (liu et al., 2021). in the initial phase of wound healing, fibroblasts are produced in large numbers to compensate for the inflammation (guillamat-prats, 2021; liu et al., 2022). in the subsequent phase (10-14 days later), the number of fibroblasts decreases, and they are replaced by collagen fiber formation. in the 21-day therapy group (k21), there was a significant difference between treatment subgroups (p=0.036), this may be due to wound re-epithelialization and granulation tissue formation during the experimental period differing between animals. the distribution pattern of fibroblast cells and collagen fiber formation can be observed more clearly in the histological profile below. figure 1. changes in the number of fibroblast cells in mice after therapy with ethanol extract ointment of patiwala leaves at. k7 (7-day therapy group), k14 (14-day therapy group), k21 (21-day therapy group). k1 (5% patiwala ointment subgroup), k2 (10% patiwala ointment subgroup), k3 (15% patiwala ointment subgroup), kp (10% betadine positive control). fibroblast cells ( ) collagen fibers ( ) dan hair follicles ( ). proliferating fibroblasts accompany these vessels and begin to deposit collagen. during the proliferation phase, a special type of tissue that characterizes healing, called granulation tissue, appears. the term granulation tissue originates from its histological appearance, characterized by the proliferation of fibroblasts, smooth, thin-walled capillaries within a loose extracellular matrix. granulation tissue then progressively accumulates a connective tissue matrix, ultimately producing dense fibrosis, which can undergo further remodeling over time (kunkemoeller & kyriakides, 2017). after injury, exposure of fibrillar collagen to the blood causes platelet aggregation and activation and releases chemotactic factors that initiate the wound healing process. collagen fragments release leukocytic collagenase to attract fibroblasts to the wound area. collagen then forms the foundation for a new extracellular matrix, thus accelerating the formation of granulation tissue. the saponin content in the ethanol extract ointment of patiwala leaves prevents wound infection (chanu et al., 2023; kunkemoeller & kyriakides, 2017). according to chanu (2023), the more connective tissue there is in a wound, the greater the contractile force of the wound so that the sides of the wound will be pulled and cause the wound to shrink. fibroblast proliferation in the wound healing process is naturally stimulated by interleukin-ib (il-ib), platelet-derived growth factor (pdgf), and fibroblast growth factor (fgf). fibroblast migration in the injured area is stimulated by transforming growth factor (tgf), a growth factor produced by granulation tissue formed during the inflammatory process (gökşen et al., 2017). the wound healing process is greatly influenced by the role of fibroblast migration and proliferation in the injured area (gökşen et al., 2017; parmar et al., 2018). the content of the ethanol extract ointment of patiwala leaves applied to the wounds of test animals stimulates the synthesis of growth factors including fgf thereby increasing the activity of fibroblast cells to produce collagen and form connective tissue so that the wound heals quickly. ethanol extract ointment from patiwala leaves contains many phytochemical compounds such as flavonoids, tannins, phenols, terpenoids, saponins, and other nutrients that significantly affect health, including healing cuts in test animals. research conducted by saranani (2023) and arifin (2023) found that flavonoids and tannins are among the components of lantana camara that influence wound healing, particularly wound moisture. flavonoids can stop bleeding in wounds and act as anti-inflammatories, influencing the production of inflammatory cells during the wound healing phase. the presence of flavonoids in the cream can help change the condition of wet wounds to become moister more quickly. the flavonoid content of lantana camara is 1096 biology, medicine, & natural product chemistry 14 (2), 2025: 1091-1097 believed to play a significant role in the wound healing process. in addition to flavonoids, tannins act as astringents, reducing mucosal permeability and strengthening inter-mucosal bonds, thus preventing irritation. therefore, tannins indirectly influence changes in moisture levels. in addition to affecting mucosal permeability, tannins can also affect the permeability of bacterial walls or membranes, causing bacteria (zubair & ahmad, 2019). these antibacterial properties can prevent wound infections. the saponins contained in lantana camara can influence collagen production in the early stages of tissue repair and stimulate epithelial cell regeneration in the skin, thereby accelerating the wound healing process in test animals. phenolic compounds play a role in preventing cell damage caused by free radicals, thus preventing inflammation (kuan et al., 2025). in addition to saponins and phenolic compounds, lantana camara also contains terpenoids, which are useful for reducing inflammatory activity (zubair & ahmad, 2019). the anti-inflammatory properties of lantana camara can inhibit the inflammatory process in cuts in white wistar rats, allowing the wound healing process to occur more quickly. conclusions based on the results of the research conducted, it was concluded that topical ointment of ethanol extract of patiwala leaves (lantana camara) at a dose of 15% was able to accelerate the healing of incision wounds in diabetic rats as evidenced by changes in wound diameter, number of fibroblast cells and collagen fiber formation on the 21st day after therapy. acknowledgements: the author would like to thanks to poltekkes kemenkes kendari for funding this research through dipa number hk.02.03/f.xxxvi/1666/2024. authors’ contributions: tuty yuniarty & zulfikar ali hasan carried out the laboratory work. theosobia grace orno wrote the manuscript. ahmat rediansya putra & ratih feraritra danu atmaja analyze the data. theosobia grace orno & anita rosanty designed the study. theosobia grace orno supervised the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references akkus, g., & sert, m. 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(2019). role of growth factors and cytokines in diabetic foot ulcer healing: a detailed review. reviews in endocrine & metabolic disorders, 20(2), 207–217. https://doi.org/10.1007/s11154-019-09492-1 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 575-584 | doi: 10.14421/biomedich.2024.132.575-584 issn 2540-9328 (online) identification of marbofloxacin-susceptible bacterial uropathogens in feline urinary tract infection of different age groups sekarani arifah udasmara, endah retnaningrum2,* 1student of faculty of biology; 2microbiology laboratory, department of tropical biology, faculty of biology, universitas gadjah mada jl. teknika selatan sekip utara yogyakarta 55281, tel. +62-274-580839, fax. +62-274-580839, indonesia. corresponding author* endahr@ugm.ac.id manuscript received: 23 july, 2024. revision accepted: 04 december, 2024. published: 20 december, 2024. abstract urinary tract infections (uti), an adhesion and multiplication of bacterial uropathogens in the urogenital system, are one of the most important indications for antimicrobial use in veterinary medicine and contribute to the development of antimicrobial resistance. previously, it was revealed that older cats with an average of 9.1 years old are most likely to develop uti, however a contradicting investigation stated kittens and young adults are more likely to contract uti. along with the uprising of amoxicillinand ampicillinresistant uropathogens, the study aims to turn towards marbofloxacin reserve and to analyze their potency. furthermore, the current study aims to enumerate and compare the number of uropathogens between age groups. enumeration and identification of uropathgoens from urine samples of feline uti patients were was conducted through the urine culture on emb, msa, and bhi medium followed by an imvic test and inoculation to the mueller-hinton agar to observe the zone of inhibition. the investigation revealed that a higher number of younger cats and kittens were reported to have uti and catheter-induced uti with a higher number of uropathogens in the population. the uropathogens were further identified as staphylococcus spp., enterococcus spp., enterobacter spp., escherichia spp., and klebsiella spp. additionally, marbofloxacin was found to be effective in eliminating and inhibiting the growth of these uropathogens. in conclusion, younger cats were observed to be more prone to uti of a diverse range of uropathogens, however, marbofloxacin may aid in the treatment of the cats as it has been proven to be most effective. keywords: antimicrobial use; marbofloxacin; uropathogens; uti. abbreviations: cauti: catheter-associated urinary tract infection, uti: urinary tract infections, fic: feline idiopathic cystitis, flutd: feline lower urinary tract disease, dna: deoxyribonucleic acid, emb: mannitol salt agar, msa: mannitol salt agar, bhi: brain heart infusion, tpc: total plate count. introduction urinary tract infections are one of the most important indications for antimicrobial use in veterinary medicine and contribute to the development of antimicrobial resistance. dorsch et al. (2019) proposed that utis in cats occur less frequently than in dogs with only 1–2% of cats suffering from utis in their lifetime. however, according to a study conducted in yogyakarta, approximately 25% of 185 cat patients had been diagnosed with uti, alongside a count of 16.4% of 73 cat patients in the sleman regency, yogyakarta. overall, these ratings were found to have a higher prevalence compared to the population in europe or the us (astuty et al.., 2020; nururrozi et al.., 2020). dorsch et al. (2019) stated that uti cases in germany were more commonly found in adult cats than in younger cats, however, a study by piyarungsri et al. (2020) conducted in thailand found that utis in young cats were more likely to develop utis, especially in those with preexisting complications, such as kidney disease. these findings may be a result of different demographical clinical practices and healthcare, which indicates that a study of the trend and cause of uti cases in yogyakarta must be studied and reviewed (martinez-ruzafa, 2016). utis are dominantly caused by bacteria, including escherichia coli, enterococcus spp., staphylococcus spp., and streptococcus spp. (dokuzeylül et al.., 2019). in both simple and complicated utis, escherichia coli is the organism most frequently cultured in both simple and complicated utis, thus referred to as the main pathogen causing utis (freitag et al.., 2006; fonseca et al.., 2021). to treat feline utis, patients are often prescribed amoxicillin, ampicillin, and cephalothin, which are predominantly excreted in the urine in their active form, making them a good first-line option for utis. however, a significantly high number of e. coli strains were reported by fonseca et al. (2021) to be resistant to amoxicillin (39.4%), ampicillin (19.2%), and cephalothin (58.8%). meanwhile, several strains of uropathogens https://doi.org/10.14421/biomedich.2024.132.575-584 576 biology, medicine, & natural product chemistry 13 (2), 2024: 575-584 were observed to be the least resistant to marbofloxacin with a percentage of 7.1%. marbofloxacin is a synthetic third-generation fluoroquinolone antibiotic reserved for documented resistant utis. similar to ampicillin and amoxicillin, it is excreted in urine predominantly in active form as well (weese et al., 2011). although it is currently not widely available, its effectiveness and low mpc values may support the elimination of uti in felines without the risk of a rise in marbofloxacin-resistant uropathogens (gebru et al., 2011). unfortunately, caneschi et al. (2023) stated that antibiotic overuse and misuse have contributed to the recent rise in antimicrobial resistance, and the use of antibiotics in animals has selected for and spread resistant microorganisms. according to lei et al. (2017), uropathogens has acquired marbofloxacin-resistant genes as a result of past antibiotic abuse and cross-resistance that has been mitigated by prior fluoroquinolone medications. as a result, this study aimed to observe the uti trends in yogyakarta and analyze the difference in bacterial population in feline uti of various age groups in terms of diversity and abundance, followed by further analysis on the susceptibility of the uropathogens towards marbofloxacin. materials and methods study area the samples were collected from 4 veterinary clinics around yogyakarta in which the cats had already undergone catheterization. following the sampling, the urine samples were further analyzed in the laboratory of microbiology, faculty of biology, universitas gadjah mada. procedures urine sample collection through collaborative work with local vets and clinics, eight urine samples were acquired from cats that had been diagnosed with uti. the 6 cats originate from different life stages, namely seniors, prime, young adults, and kittens. the cats originated from different life stages, namely seniors, prime, young adults, and kittens (quimby, 2021). the samples were then labeled in the following sequence: c(life stage code)(age number), where c stands for “cat”. physical and chemical analysis of urine samples a urine dipstick was dipped into the urine samples to determine the chemical properties of the samples, including, the ph, protein, and glucose concentration. additionally, the color of the urine was determined to distinguish whether it was diluted or concentrated (graziani et al., 2008). enumeration of the bacterial uropathogens initially, the urine samples were subjected to a 10-fold serial dilution eight times by placing 1 ml of the urine sample into 9 ml of nacl, followed by transferring 1000 µl of the diluted solution from the previous tube to the next tube. the following steps were repeated until the remaining 5 tubes had been filled (u.s. food and drug administration, 2023). afterward, the 3rd, 4th, 5th, and 6th dilution tubes were each inoculated to two replicates of bhi, msa, and emb mediums through the pour plate method by inoculating 1000µl into the petri dishes followed by warm liquified bhi, msa, and emb agar solutions. once it solidified, the samples were incubated for 24-48 hours at 37°c. the colonies were enumerated with the aid of a marker, followed by subculturing the pure colonies into nutrient-slant agar mediums. identification of the bacterial uropathgoens with imvic in terms of the indole test, 5ml of sim stab agar medium was prepared. pure colonies isolated from the emb, msa, and bhi agars were inoculated into the stab agar. the samples were left to incubate for 24-48 hours at 37°c. afterward, 5 drops of kovac reagent were added into the medium to detect the presence of a red ring and a diffusion zone of growth (macwilliams, 2009). following the indole test, the methyl red and vogesproskauer (vp) tests were performed in which 5 ml of mrvp broth mediums were prepared. an ose of the pure colonies were inoculated into the mrvp broth then left to incubate for 48 hours at 37°c. once the samples had been incubated, for the methyl red test, 2.5 ml of the culture was placed into a separate tube, followed by an addition of 6 drops of 0.02% methyl red indicator in order to observe a red coloration which indicated a positive result. in terms of the vp test, 2.5 ml of the remaining culture was placed into a separate tube, followed by addition of 6 drops of 5% a-naphthol and 2 drops of 40% koh in order to observe a formation of a pink color indicating a positive result (mcdevitt, 2009). lastly, the citrate test was performed by preparing 7 ml of slant simmons citrate agar. afterward, the pure colonies were streaked onto the slant agar and left to incubate for 24-48 hours at 37°c. the formation of a blue coloration indicates a positive result, whereas the remaining green coloration of the medium indicates a negative result (macwilliams, 2009). marbofloxacin susceptibility test using multi-disc antibiotics, an antimicrobial susceptibility test was conducted on mueller-hinton agar (mh). initially, the isolates were vortexed with aquadest until it was homogenized. suspension was standardized according to the 0.5 mcfarland turbidity standard and inoculated onto the mh agar with the aid of a sterile cotton swab. a concentration of 20µl of marbofloxacin udasmara & retnaningrum – identification of bacterial uropathogens in feline uti 577 was impregnated onto the discs and using a set of sterile forceps, the marbofloxacin-impregnated paper disc is placed in the center and gently pressed into the agar. the medium will be inverted and left to incubate for 24 hours at 37°c. clear zones of inhibition were indicative of sensitivity, whereas no cleared zones were indicative of resistance (dirisu et al.., 2016). data analysis once the data has been collected, an analysis of the physical and chemical properties of the urine samples was compiled and recorded, followed by a determination of whether the urine is diluted or concentrated and the severity of the uti. in terms of the antibiotic susceptibility test, the acquired results were qualitatively analyzed by measuring the size of the zone of inhibition. a zone of inhibition that was larger than ≥ 18 mm was categorized susceiptible; zones that were 15-17 mm in diameter were considered moderate; and no zone of inhibition or ≤ 14 mm was considered resistant (schneider et al., 2004). table 1. zone of inhibition interpretations (fonseca et al., 2004). zone diameter (mm) categories ＞20 susceptible 15 19 intermediate ≤ 14 resistant results and discussion urinalysis results and clinical implications a total of 3 urine samples were collected throughout the duration of the investigation, namely, cy2. ck1, and cp4 with a complete urinalysis and profiling of the patient (table 2). patient ck1 was categorized as a male kitten at the age of 1 year old and excreted yellow urine at a ph of 6.5. patient ck1 had been treated for 5 days as well. meanwhile, patient cy2 was categorized as a male young adult at the age of 2 years old who had been treated for 6 days. the patient excreted a pink urine color with a ph of 7.5 similar to the patient cp4 who was a prime male cat at the age of 4 years old, however patient cp4 excreted yellow urine and had only been treated for 4 days. lastly, all 3 patients tested positive for leukocytes and nitrates, but negative for glucose in the urine. table 2. urinarlysis of the samples of different ages sample life stage age gender n-th day of treatment urine color ph glucose leukocyte nitrate ck1 kitten 1 y.o male 5 yellow 6.5 ++ + cy2 young adult 2 y.o. male 6 pink 7.5 ++ + cp4 prime 4 y.o. male 4 yellow 7.5 + + bacterial uropathogen population according to table 2, all three patients had a bacterial count of more than 1000 cfu/ml indicating a uti case. a trend was observed where younger cats had a higher count of bacteria compared to the older cats. patient ck1 had a total bacterial count of 247,462,500 cfu/ml, whereas cy2 and cp4 had a bacterial count of 28,080,000 cfu/ml and 100,000 cfu/ml, respectively. following the bacterial count, the isolates were subjected to a series of tests from the imvic test in order to facilitate the identification of the isolates through their phenotypic characteristics. from sample cy2, 3 strains of staphylococcus spp., 3 strains of klebsiella spp., and 1 strain of enterobacter spp. were identified. meanwhile, patient ck1 was found to contain 3 strains of enterobacter spp., 1 strain of klebsiella spp., and 1 strain staphylococcus spp. lastly, patient cp4 was found to contain 2 strains of enterobacter spp., 1 strain of klebsiella spp., 1 strain of staphylococcus spp., and 1 strain of escherichia coli (table 4). 578 biology, medicine, & natural product chemistry 13 (2), 2024: 575-584 table 3. number of uropathogens isolates (cfu/ml). cy2 ck1 cp4 isolates cfu/ml isolates cfu/ml isolates cfu/ml cy2a 143,500 ck1a 12,550,000 cy4a 37,500 cy2b 9,650,000 ck1b 110,000,000 cy4b 57,500 cy2c 615,000 ck1c 5,400,000 cy4c 4,000* cy2d 131,500 ck1d 119,500,000 cy4d 500* cy2e 2,545,000 ck1e 12,500* cy4e 500* cy2f 345,000 cy2g 14,650,000 total 28,080,000 247,462,500 100,000 (*) although it was present, cfu was not within the applicable range of 30-300 cfu/ml bacterial uropathogen identification a total of 17 isolates were collected from the three urine samples and were subjected to a series of tests from the imvic test in order to facilitate the identification of the isolates through their phenotypic characteristics. from sample cy2, 3 strains of staphylococcus spp., 3 strains of klebsiella spp., and 1 strain of enterobacter spp. were identified. meanwhile, patient ck1 was found to contain 3 strains of enterobacter spp., 1 strain of klebsiella spp., and 1 strain staphylococcus spp. lastly, patient cp4 were found to contain 2 strains of enterobacter spp., 1 strain of klebsiella spp., 1 strain of staphylococcus spp., and 1 strain of escherichia spp. table 4. phenotypic and biochemical characteristics of bacterial uropathogens. isolates colony morphology media imvic test indole mr vp citrate identification cy2a white circular mucoid aerobic colonies bhi + + + klebsiella spp. * cy2b yellow circular anaerobic colonies bhi + + + staphylococcus spp. * cy2c translucent circular aerobic colonies with a darker middle bhi + + enterobacter spp. cy2d large light purple aerobic circular mucoid colonies emb + + + klebsiella spp. * cy2e small circular dark purple anaerobic colonies emb + + + klebsiella spp. * cy2f white circular aerobic colonies with yellow hues msa + staphylococcus spp. * cy2g yellow oval anaerobic colonies msa + + + staphylococcus spp. * ck1a large white circular mucoid aerobic colonies bhi + + enterobacter spp. * ck1b small yellow oval anaerobic colonies bhi + + enterobacter spp. ck1c large light purple circular aerobic colonies emb + + enterobacter spp. ck1d small dark purple circular anaerobic colonies emb + + + klebsiella spp. * ck1e yellow aerobic colonies with yellow hues msa + + + staphylococcus spp. * cp4a large white circular mucoid aerobic colonies bhi + enterococcus spp. cp4b small white ocal anaerobic colonies bhi + + enterobacter spp. cp4c small dark purple circular anaerobic colonies emb + + klebsiella spp. * cp4d green circular aerobic colonies emb + + escherichia coli cp4e yellow circular aerobic colonies msa + + + staphylococcus spp. * imvic test, indole, methyl red, voges–proskauer, and citrate tests; emb, eosin methylene blue; bhi, brain heart infusion; msa, mannitol salt agar; (-), negative; (+), positive; (*) signifies that the strains are from within the same genus but remains a different species marbofloxacin susceptibility test the marbofloxacin susceptibility test was conducted on each of the isolates that were detected from the patients. according to the table below, e. coli (n=1), klebsiella spp. (n=5), enterococcus spp. (n=1), and staphylococcus spp. (n=4) were all susceptible towards marbofloxacin at udasmara & retnaningrum – identification of bacterial uropathogens in feline uti 579 a percentage of 100%. meanwhile, 75% of the enterobacter spp. strains (n=4) were found to be susceptible whereas the remaining 25% were found to be in the intermediate category. table 6. marbofloxacin susceptibility test (%). identification n s (%) i (%) r (%) escherichia coli 1 100% 0% 0% enterobacter spp. 4 75% 25% 0% klebsiella spp. 5 100% 0% 0% enterococcus spp. 1 100% 0% 0% staphylococcus spp. 4 100% 0% 0% s, susceptibility; i, intermediate; r, resistant further analysis revealed that escherichia coli (average of 31.1 mm), enterococcus spp. (average of 29.1 mm), staphylococcus spp. (average of 28.6 mm and 30.3 mm), and klebsiella spp. (average of 25.8 mm, 30.1 mm, and 31.2 mm) were all found to be susceptible towards marbofloxacin according to the standards shown on table 2. on the other hand, a strain of enterobacter spp. was found to be an intermediate (average of 16.9 mm) whereas the remainder of the strains were found to be susceptible (average of 26.2 mm). in conclusion, marbofloxacin was effective against most of the uropathogens isolated from the patients, however the enterobacter spp. isolates may require an adjustion in the dosage due to its prominent biofilm (kahlmeter, 2017). these findings were consistent with ferrans et al., (2016) where marbofloxacin exhibited a greater activity in the elimination of biofilm compared to amoxicillin as they have the ability to kill non-dividing bacteria. discussion feline urinary tract infection is a condition, categorized under flutds, where there is an accumulation and persistent uropathogen within the urogenital system thus inducing an associated inflammatory response and clinical symptoms. utis can result simply from the migration of uropathogens that had already resided in the gut into the periurethral area, this is known as uncomplicated uti. once the uropathogens have migrated to the urethra, it will continue migration into the bladder where they undergo multiplication and the formation of biofilm. these uropathogens continue to produce toxins and proteases causing the host cell to deteriorate releasing nutrients to the uropathogens. as the uropathogens continue to multiply, migration to the kidney continues and if left untreated it may reach the bloodstream causing bacteraemia. a similar pathway is found in the case of complicated uti, but the source of the infection is due to a compromised bladder (floresmireles et al.., 2015). nururrozi et al. (2020) reported that yogyakarta a trend where older cats with an average age of 9.1 years old were mostly diagnosed with complicated uti due to their association with ckd or compromised host defenses. however, a contradicting investigation by piyarungsri et al. (2020) reported that a high number of younger cats have been diagnosed with uti compared to elder cats due to the preexisting complications, such as kidney diseases. both cases mentioned the presence of preexisting diseases associated with cats of all ages, therefore, a continuous update on the feline uti trends in yogyakarta must be conducted. urinalysis and clinical implications feline urinary tract infection is a condition, categorized under flutds, where there is an accumulation and persistent uropathogen within the urogenital system thus induces an associated inflammatory response and clinical symptoms. utis can result simply from the migration of uropathogens that had already resided in the gut into the periurethral area, this is known as uncomplicated uti. once the uropathogens had migrated to the urethra, they will continue migration into the bladder where they undergo multiplication and the formation of biofilm. these uropathogens continue to produce toxins and proteases causing the host cell to deteriorate releasing nutrients to the uropathogens. as the uropathogens continue to multiply, migration to the kidney continues and if left untreated it may reach the bloodstream causing bacteraemia. a similar pathway is found in the case of complicated uti, but the source of the infection is due to a compromised bladder (flores-mireles et al.., 2015). nururrozi et al.. (2020) reported that yogyakarta had a trend where older cats with an average age of 9.1 years old were mostly diagnosed with complicated uti due to their association with ckd or compromised host defenses. however, a contradicting investigation by piyarungsri et al. (2020) reported that a high number of younger cats have been diagnosed with uti compared to elder cats due to preexisting complications, such as kidney diseases. both cases mentioned the presence of preexisting diseases associated with cats of all ages, therefore, a continuous update on the feline uti trends in yogyakarta must be conducted. throughout the course of the investigation, three patients have been confirmatively diagnosed with uti due to the presence of more than 10-3 cfu/ml of bacteria in a growth media. the patients had an age of 1 year old, 2 years old, and 4 years old, which were categorized in the kitten, young adult, and prime life stages, respectively. although the cats had no prior record of preexisting diseases, the anatomical structure of the male cats may be cause of the uti. compared to female cats, the male cat's urethra is narrower and more curved making them more susceptible to urethral blockage/plug. urethral plugs are described as lodges in the urethra due to the accumulation of proteins, cells, and other debris from the bladder, preventing urination. as a result, urine is buildup in the bladder creating a favorable 580 biology, medicine, & natural product chemistry 13 (2), 2024: 575-584 environment for bacteria to grow leading to the case of uti (heyns, 2011). the case of uti is further supported by the presence of a higher ph than the normal range as well as the presence of nitrate and leukocytes detected in the patient’s urine. the normal ph range of male cats is 5.73-7.39, however, patient cp4 and cy2 had a ph of 7.5 which was consistent with the number of bacteria present in their urine. the presence of ammonia is a result of urease produced by uropathogens such as, p. mirabilis, k. pneumoniae, and s. saprophyticus, which catalyses the hydrolysis of urea to co2 and ammonia, thus elevating urine ph (flores-mireles et al.., 2015; cottam et al.., 2002). elevating the urine ph may also result in the production of calcium crystals and with the additional presence of ammonia the tissue is irritated and damaged causing blood to enter the urine, this condition is knows as crytalluria. cases such as haematuria was detected only in patient cy2 where a pink coloration was observed in the urine sample (bono et al.., 2023). however, presence of blood in the urine was not a definitive diagnosis of uti as blood may also be a result of fic and crystalluria. utis are defined as an infection with more than 1000 cfu/ml of bacteria in the urine, whereas fic is a spontaneous non-infectious bladder disease in cats (nururrozi et al.., 2020). similar to the inflammation due to a bacterial infection, inflammation in fic may be triggered by histamine and cytokines causing tissue damage, as a result 70% of cats diagnosed with fic had haematuria. similarly, crytalluria may also haematuria due to the irritation of the bladder walls by the crystals (ofakor et al.., 2018; dorsch et al.., 2019) although leukocytes and nitrites are deemed unreliable in confirming uti cases, they can still be considered as a supporting factor. a significant number of leukocytes were detected in the urine samples due to the response to bacteria in the urine. according to hayes and abraham (2016), a bacterial infection induces the release of leukocytes, such as neutrophils, into the bladder which in turn produces pro-inflammatory cytokines (hedges and svanborg, 1994). meanwhile, nitrite is widely-used indicator in urine dipsticks to determine uti as uropathogens reduce nitrate to nitrite in order to prevent the conversion of nitric oxide in the urine which limits their growth and metabolism (domili et al.., 2020). bacterial uropathogen population according to table 4, the bacterial isolates that were found in all three patients were klebsiella spp. and enterobacter spp. however, a distinctively high number of staphylococcus spp. and enterobacter spp. were detected in patients cy2 and ck1, respectively. the distinction between the presence of bacterial species may be a result of catheter-associated urinary tract infection (cauti) as all three patients underwent catheterization as a form of treatment for fluts. dorsch et al. (2019) mentioned that clinical utis are difficult to diagnose as the patients would already have been catheterized for prior symptoms of flutd, as a result, flutd patients that may not have uti may come to develop cauti due to catheterization. aside from catheterization, utis could have occurred without a compromised bladder rather than a pre existing disease. however, as there were no prior records of the patients having prior diseases or reinfection, it was speculated that the patients may have had a persistent infection from a catheter-induced uti. in the case that this is supported by dorsch et al. (2019), where a persistent infection takes 7-14 days of treatment consistent with the 5th and 6th-day treatment mark for patients ck1 and cy2, respectively. additionally, a research conducted by bubenik et al. (2007) discovered that catheterization allows opportunistic enterobacter spp. and staphylococcus spp. to enter the patients. these findings were consistent to the data in which a high number of staphylococcus spp. (87.77%) and enterobacter spp. (2.19%) in cy2, as well as enterobacter spp. (57.71%) in ck1 (scarpellini et al, 2023; dorsch et al., 2016). in addition klebsiella pneumoniae being an opportunistic bacteria could have compromised the catheters (dorsch et al., 2019). meanwhile, patient cp4, may have had a uti prior to the catheterization as common uropathogens associated with uti, such as, enterococcus spp., e. coli, staphylococcus spp., and klebsiella spp. were detected at a lower number on the 4th day of treatment (100,000 cfu/ml) which was less than the bacterial count of patient ck1 and cy2 (table 3 and table 5). thus, it was speculated that the uti was not induced by catheterization but rather simply by the migration of uropathogens to the bladder thus resulting a non-persistent infection or a reinfection (dorsch et al., 2019). a similar case was found in a study conducted by thompson et al. (2011) where a case recurrent canine uti resulted in similar uropathogen diversity consisting of e. coli, klebsiella spp., staphylococcus spp., enterococcus spp. in order to further support the case of cauti in patients cy2 and ck1, the concept of biofilm may be applied. biofilms are described as polymicrobial colonies that attach to surfaces and form an encasement of the extracellular polymeric matrix, allowing them to bypass host defenses and increase antimicrobial resistance through slow penetration, resistant phenotype, and altered microenvironment. biofilms are one of the contributors of persistent infection and antibiotic resistance in the case of uti and cauti. the uropathogens that contaminate the urinary catheters and form biofilms are e. coli, proteus spp., enterococcus spp., staphylococcus spp., and klebsiella spp. which were consistent to the findings of the investigation (lila et al., 2022). according to sabir et al. (2017), the uropathogen that produces the highest amount of biofilm was enterobacter clocae (87.5%) followed by klebsiella udasmara & retnaningrum – identification of bacterial uropathogens in feline uti 581 pneumoniae (87.1%) and enterococcus faecium (79.2%). according to several studies, the abundance of enterobacter spp. may be attributable to the formation of the most potent biofilm at the highest percentage compared to the remainder of the uropathogens as they have the ability to grow and survive in a nutrientdeficient environment such as urine. these findings may uropathogens found in patient ck1 where enterobacter spp. was abundant in the uropathogen population (table 4) (liu et al. 2022). meanwhile, klebsiella spp., being one of the most common uropathogen found in the patients and the second highest biofilm producing uropathogen, has the ability to produce a capsular polysaccharide thicker than the capsule of e. coli. these findings may support the abundance of klebsiella spp. in all of the three patients as the biofilm provides adhesion to the uroepithelium tissues of the bladder and the catheter (lila et al., 2022). following klebsiella spp., an abundance on staphylococcus spp. was detected in all three patients as well with a distinctively high number in patient cy2 (table 4) that produces a biofilm that expresses clumping factors that had a high affinity for fibrinogen allowing it to attach to the fibrinogen-coated catheter and damaged tissues. additionally, their biofilm allows for the production of toxins that promotes tissue damage, and from the findings collected from patient cy2 these characteristics supported the speculation that the uti was catheter-induced and that the haematuria may had been resulted from the toxins damaging the tissue of patient cy2 (lila et al., 2022). lastly, the presence of a high number of entercoccus spp. was detected in patient cp4 which is unique for their ability to suppress the innate and adaptive immune pathways attributing to the further infections by other uropathogens which may explain the presence of a diverse range of uropathogens in cp4 including, e. coli, klebsiella spp., enterobacter spp., and staphylococcus spp., compared to the other patients (table 4). marbofloxacin susceptibility patterns the isolates obtained from each patient were subjected to the antibiotic susceptibility test for marbofloxacin as ampicillin and amoxicillin had been reported to have been ineffective towards uropathogens that had developed resistance. the standardized measurements for susceptible strains had an inhibition zone diameter of ≥ 18 mm, intermediate strains had an inhibition zone diameter of 15–17 mm, and resistant strains had an inhibition zone inhib1ition diameter of ≤ 14 mm (schneider et al.., 2004). according to table 5, e. coli (average of 31.1 mm), enterococcus spp. (average of 29.1 mm), staphylococcus spp. (average of 30.5 mm), and klebsiella spp. (average of 27.7 mm) were all found to be susceptible to marbofloxacin. on the other hand, a strain of enterobacter spp. was found to be an intermediate (average of 16.9 mm) whereas the remainder of the strains were found to be susceptible (average of 26.2 mm). in conclusion, marbofloxacin was effective against most of the uropathogens isolated from the patients, however the enterobacter spp. isolates may require an adjustment in the dosage (kahlmeter, 2017). these findings were consistent with ferrans et al.., (2016) where marbofloxacin exhibited a greater activity in the elimination of biofilm compared to amoxicillin as they have the ability to kill non-dividing bacteria. conclusions from the following findings it was concluded that the population of uropathogens found from different age groups or life stages were diverse, however the factor of age did not seem to influence the diversity of uropathogens, rather environmental factors such as the insertion of the catheter. therefore, it must highlight those cases of uti must be distinguished between cauti as the species of uropathogens may differ. in addition, the uropathogens isolated from the patients were highly susceptible to marbofloxacin, thus making the antibiotic one of the most effective antibiotics in treating uti. competing interests: the authors declare that there are no competing interests. acknowledgements: we would like to take this opportunity to express gratitude to veterinary clinics in yogyakarta who have participated in this research by being able to provide samples as well as handing out questionnaires as a means to further supports the speculations. additionally, we would like to express gratitude to the microbiology laboratory, faculty of biology, universitas gadjah mada by providing a suitable and sterile setting to conduct the investigation authors’ contributions: prof. dr. endah retnaningrum contributed to the investigation as being the lead supervising lecturer in which she contributed to the methodology and narration of the 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[online] available at: https://www.fda.gov/food/laboratorymethods-food/bam-appendix-2-most-probable-number-serialdilutions biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 717-723 | doi: 10.14421/biomedich.2025.142.717-723 issn 2540-9328 (online) investigating genetic vulnerability to environmental exposures and associated lung diseases: a bioinformatics study muhammad farid*, ardestya rastrani, shaldhan bayu yuska faculty of medicine, universitas ahmad dahlan, jl. ahmad dahlan university campus 4, kragilan, tamanan, banguntapan district, bantul regency, special region of yogyakarta, 5519, indonesia. corresponding author* muhammad2100034023@webmail.uad.ac.id manuscript received: 04 june, 2025. revision accepted: 18 september, 2025. published: 01 october, 2025. abstract lung diseases induced by environmental exposures such as air pollution, cigarette smoke, and industrial particles remain a significant global health concern, contributing to high morbidity and mortality rates. genetic variations are known to influence individual responses to environmental exposures, but the molecular mechanisms underlying these interactions are not well understood. this study aims to identify genetic variants, specifically single nucleotide polymorphisms (snps), that may increase the risk of lung diseases using a bioinformatics approach. the analysis was conducted by integrating various public genetic databases, including phewas, gwas catalog, haploreg v4.2, gtex portal, and ensembl genome browser. snps were filtered based on p-value < 0.05 and odds ratio (or) > 1. missense mutations in selected snps were further analyzed for gene expression in lung tissue and distribution across populations. from an initial 151 snps, 86 met the statistical criteria, and six were identified as missense variants. two genes, tnip1 and psmb8, showed significantly high expression in lung tissue. snp rs2071543 in psmb8 exhibited a strong correlation with increased gene expression and demonstrated notable allele frequency variation across populations. these findings suggest that genetic variations, particularly in psmb8, may contribute to individual susceptibility to lung diseases induced by environmental exposures. this study highlights the importance of multidatabase analysis in identifying genetic biomarkers and provides a foundation for the development of precision therapies for multifactorial lung diseases. keywords: bioinformatics study; environmental exposures; lung disease; psmb8; rs2071543. introduction lung diseases remain one of the leading causes of morbidity and mortality worldwide. conditions such as chronic obstructive pulmonary disease (copd), idiopathic pulmonary fibrosis (ipf), asthma, and lung cancer are showing increasing prevalence, particularly among populations exposed to harmful environmental factors (victoni et al., 2021). the complex pathogenesis of these diseases reflects the involvement of multiple biological mechanisms, including oxidative stress, chronic inflammation, and innate genetic factors that may influence an individual’s response to external exposures (victoni et al., 2021). one of the key mechanisms contributing to lung tissue damage is oxidative stress, which results from an imbalance between the production of reactive oxygen species (ros) and the body’s antioxidant defense systems. ros can trigger inflammation, apoptosis, and tissue remodeling, ultimately leading to organ dysfunction over time (victoni et al., 2021). this condition is further exacerbated by the limited effectiveness of conventional therapies for certain chronic lung diseases, such as copd and ipf, highlighting the need for more targeted and specific therapeutic approaches (harber et al., 2016). environmental and occupational factors have been identified as major contributors to the etiology of lung diseases. individuals working in agriculture and livestock industries, for example, are at high risk of exposure to organic dust, which is known to trigger occupational asthma, chronic bronchitis, and even pulmonary fibrosis (poole et al., 2024). additionally, air pollution from motor vehicle emissions and industrial activities has been shown to significantly impair lung function, as evidenced by numerous epidemiological studies and meta-analyses (adam et al., 2015). synthetic food additives such as sodium nitrite, artificial coloring agents, and sulfites also contribute to impaired lung function, particularly in individuals with airway hyperresponsiveness (aldabayan, 2025). these exposures can elicit abnormal immune responses, compromise epithelial integrity, and accelerate disease progression, especially in genetically susceptible individuals. at the cellular and molecular levels, chronic lung diseases such as ipf have been associated with a programmed cell death mechanism known as ferroptosis https://doi.org/10.14421/biomedich.2025.142.717-723 718 biology, medicine, & natural product chemistry 14 (2), 2025: 717-723 a process triggered by iron accumulation and oxidative stress. unlike apoptosis or necrosis, ferroptosis represents a distinct cell death pathway and has emerged as a potential target in novel therapeutic research (he et al., 2022). several genes, including cav1, gdf15, nos2, and cdkn2a, have been found to be dysregulated in the lung tissues of ipf patients and are all involved in ferroptosis-related regulatory pathways (he et al., 2022). these findings suggest that therapeutic strategies aimed at inhibiting or modulating ferroptosis pathways may offer innovative approaches for managing ipf and other lung diseases. furthermore, chronic exposure to cigarette smoke plays a significant role in inducing systemic genetic alterations. a wellcharacterized molecular marker of smoke exposure is the sbs4 mutational signature, which is closely associated with carcinogenesis and lung tissue remodeling (kim et al., 2022). remarkably, this signature can be detected even in non-malignant lung tissues, indicating that environmental exposure can induce genetic changes long before clinical symptoms emerge (kim et al., 2022; valavanidis et al., 2009). the ros present in cigarette smoke intensify oxidative stress, accelerate degenerative processes, and cause dna damage (valavanidis et al., 2009). the extensive application of bioinformatics allows researchers to integrate diverse omics data—including transcriptomics, proteomics, and dna mutation profiles to uncover previously hidden patterns of lung pathology (he et al., 2022). this approach has also been employed to assess immune responses to organic dust exposure and to identify disrupted signaling pathways resulting from food additive exposure (aldabayan, 2025; poole et al., 2024). in the era of precision medicine, such molecular understanding is crucial for designing therapies tailored to an individual’s genetic profile. therefore, multidisciplinary collaboration between clinical sciences, environmental toxicology, and bioinformatics is key to comprehensively understanding and managing lung diseases caused by external agents (harber et al., 2016; he et al., 2022). by elucidating the complex interplay between environmental and genetic factors, we can pave the way for more personalized and effective prevention and treatment strategies. this study aims to identify and analyze genetic variations (snps) that may increase the risk of lung disease due to exposure to external agents using a bioinformatics approach. through this research, we hope to gain a more comprehensive understanding of the relationship between genetic factors and environmental exposures in the pathogenesis of lung diseases, thereby opening new avenues for the development of precision therapies in the future. materials and methods this study employed a bioinformatics approach to identify genetic variations that may increase the risk of lung disease due to exposure to external agents. the analysis was conducted through the integration of various publicly accessible online genetic databases, including the gwas catalog, phewas resources, haploreg v4.2, gtex portal, and the ensembl genome browser. the genome-wide association studies (gwas) and phenome-wide association studies (phewas) databases were used as primary resources to identify associations between genetic variants particularly single nucleotide polymorphisms (snps) and various disease phenotypes. since its development by the national human genome research institute (nhgri) in 2008, the gwas catalog has recorded over 38,000 associations between snps and disease phenotypes, including information about the involved polymorphic alleles (burbelo et al., 2014). meanwhile, the phewas database provides data on the relationships between over 3,000 snps and diverse clinical manifestations, making it a valuable tool for exploring genotype-phenotype associations on a broad scale (pendergrass et al., 2012). in the initial phase of the analysis, snps potentially associated with increased risk of lung disease due to environmental exposure were identified using phewas resources. selection of snps was based on a significance threshold of p-value < 0.05 and an odds ratio (or) > 1, indicating a statistically meaningful increase in disease risk. snps meeting these criteria were then further analyzed to determine the presence of missense mutations, which refer to codon changes resulting in amino acid substitutions (irham et al., 2023). this analysis was conducted using haploreg v4.2, which provides in-depth information on structural genetic variations and epigenetic regulatory features. snps with identified missense mutations were subsequently evaluated for their expression in lung tissue using data from the gtex portal. the genotype-tissue expression (gtex) project offers comprehensive data on correlations between genetic variants and gene expression levels across various human tissues, including the lungs. this expression analysis, based on expression quantitative trait locus (eqtl) data, is crucial for identifying variants that are not only structural but also functionally significant in modulating gene activity within the target tissue (ma’ruf et al., 2023). as a final step, the population distribution of the identified genetic variants was analyzed using the ensembl genome browser (ensembl genome browser 113). this database provides allele frequency data across diverse global populations, including those from the americas, africa, europe, east asia, and south asia (ma’ruf et al., 2023). this information is valuable for understanding the geographical and ethnic distribution of genetic risk factors and their potential public health implications across different regions. farid et al. – investigating genetic vulnerability to environmental exposures … 719 results and discussion the initial phase of this study involved the identification of single nucleotide polymorphisms (snps) potentially contributing to increased risk of lung disease due to exposure to external agents. this process was carried out using the phewas database, which yielded an initial list of 151 snps potentially associated with pathological conditions in lung tissue. to refine the scope of analysis and ensure statistical significance, a filtering process was applied based on the criteria of p-value < 0.05 and odds ratio (or) > 1. this filtering resulted in 86 snps that met the criteria and were considered to have significant mutational potential. details of these snps are presented in table 1. table 1. snps form phewas database. snp p-value odds-ratio snp p-value odds-ratio snp p-value odds-ratio rs2006996 0.0009142 2.379 rs6704644 0.03987 1.6 rs744910 0.02215 1.455 rs2237878 0.0001304 2.179 rs2237886 0.03513 1.598 rs727957 0.03989 1.455 rs1800961 0.02108 2.075 rs1408282 0.04102 1.581 rs1836127 0.01948 1.45 rs544368 0.0005663 1.97 rs12134279 0.01145 1.577 rs10744304 0.0209 1.449 rs7702057 0.04517 1.917 rs3099844 0.032 1.57 rs11031093 0.02643 1.44 rs737337 0.008276 1.814 rs3131296 0.02662 1.567 rs1927745 0.03301 1.436 rs12534221 0.002788 1.739 rs8321 0.04864 1.56 rs10737562 0.02732 1.431 rs12317459 0.001874 1.731 rs771767 0.007037 1.559 rs1000579 0.02406 1.43 rs2233287 0.01872 1.703 rs8023445 0.04503 1.556 rs2248359 0.02413 1.428 rs11618202 0.02869 1.696 rs17111394 0.01972 1.545 rs2208059 0.02464 1.428 rs13376333 0.000899 1.692 rs649891 0.01776 1.538 rs927675 0.02681 1.425 rs4952590 0.01132 1.692 rs6478108 0.006956 1.534 rs7072268 0.02769 1.423 rs6683071 0.004638 1.677 rs10475598 0.009451 1.53 rs2239633 0.02709 1.422 rs3131379 0.02038 1.665 rs12727642 0.0292 1.523 rs153734 0.04868 1.422 rs10488031 0.04744 1.665 rs1574192 0.008351 1.522 rs204993 0.04138 1.42 rs3117582 0.02062 1.662 rs2068888 0.009902 1.517 rs697739 0.03235 1.417 rs1549519 0.007362 1.647 rs2647044 0.01853 1.516 rs2814828 0.04725 1.415 rs655601 0.02947 1.643 rs6570507 0.01169 1.514 rs164898 0.03647 1.413 rs6062314 0.04578 1.641 rs2358944 0.04436 1.508 rs3923809 0.0354 1.407 rs10506458 0.02917 1.632 rs7871764 0.0161 1.485 rs470490 0.03335 1.402 rs1317209 0.008025 1.627 rs935334 0.03898 1.483 rs5751901 0.03682 1.4 rs8043440 0.0232 1.621 rs3748069 0.01702 1.482 rs8083346 0.04033 1.4 rs9357155 0.02425 1.619 rs378108 0.01635 1.477 rs2275215 0.0479 1.397 rs2681472 0.01006 1.617 rs1464500 0.01959 1.475 rs610604 0.04577 1.387 rs2681492 0.01059 1.612 rs6478109 0.01484 1.474 rs1364063 0.04131 1.379 rs1521882 0.009275 1.61 rs4924935 0.01962 1.462 rs4505848 0.04561 1.374 rs17291045 0.01915 1.607 rs1402837 0.02708 1.462 rs610932 0.04323 1.373 rs12101261 0.003302 1.602 rs12212193 0.01818 1.459 rs4838605 0.04946 1.368 rs10513789 0.007242 1.601 rs8047014 0.01916 1.458 subsequently, these 86 snps were further analyzed to determine whether they involved missense mutations, which are genetic code alterations resulting in amino acid substitutions in the encoded proteins. this analysis was performed using the haploreg v4.2 database, which provides in-depth information on functional annotations and the impact of genetic mutations. the analysis identified six missense snps, each associated with one of the following gene symbols: hnf4a, tnip1, fam177b, vwa7, vwa7, and psmb8. these six variants are listed in table 2 and were selected for further analysis. table 2. variants alleles of the prioritized snps. risk variants of alleles risk variants of alleles in close proximity with r2 >= 0.8 gencode allele location rs1800961 hnf4a missense rs2233287 rs2233290 tnip1 missense rs6683071 fam177b missense rs3131379 rs3101017 vwa7 missense rs3117582 rs3101017 vwa7 missense rs9357155 rs2071543 psmb8 missense to evaluate the biological relevance of these genes, a gene expression analysis across multiple human tissues was conducted using data from the gtex portal, with a specific focus on lung tissue as the primary target of 720 biology, medicine, & natural product chemistry 14 (2), 2025: 717-723 environmental exposure. the results revealed that two genes, tnip1 and psmb8, exhibited high expression levels in lung tissue. tnip1 showed a high expression with a median transcripts per million (tpm) value of 113.8, ranking fourth among all analyzed tissues figure 1. meanwhile, psmb8 exhibited even higher expression in the lungs, with a median tpm of 172.5, also ranking as the fourth highest among lung-expressed genes figure 2. these findings reinforce the hypothesis that both genes play a significant role in the pathological processes within the lungs resulting from environmental exposures. figure 1. tnip1 gene expression based on gtex portal analysis. figure 2. psmb8 gene expression based on gtex portal analysis. the next analysis focused on identifying relevant expression quantitative trait loci (eqtls) snps that influence gene expression levels in lung tissue. based on gtex data, one snp, rs2071543, was identified as both previously selected and functionally involved in gene regulation. this snp is associated with the gene psmb8. information regarding the major and minor alleles of rs2071543 is presented in table 3. the gg genotype of farid et al. – investigating genetic vulnerability to environmental exposures … 721 rs2071543 showed a stronger correlation with increased gene expression in lung tissue compared to the tt genotype, with a correlation coefficient of 0.81, indicating a significant regulatory effect on gene expression within the target organ. as part of the population-level analysis, the allelic distribution of rs2071543 was examined across different global ethnic groups using data from the ensembl genome browser. the results revealed notable variation in allele frequencies among populations. the g allele showed the highest frequency in african populations, at 0.904 (1,195), followed by south asian and european populations. in contrast, the lowest frequency of the g allele was observed in american populations, at 0.663 (230). for the t allele, the highest distribution was also found in african populations, while european populations exhibited the lowest frequency of this allele. table 3. allele frequencies for psmb8. snp id position gene symbol location allele allele frequency ref aff afr amr eur sas rs2071543 chr6:32843852 psmb8 missense g t g: 0.904 (1195) t: 0.096 (127) g: 0.663 (230) t: 0.040 (14) g: 0.730 (367) t: 0.024 (12) g: 0.648 (317) t: 0.029 (14) these findings indicate the presence of significant inter-population genetic variation associated with snp rs2071543, which may contribute to differences in susceptibility to lung diseases triggered by environmental exposures. this highlights the importance of incorporating genomic and population-based approaches in understanding disease risk and in the development of more personalized and precision-based medical strategies. discussion this study highlights a significant association between specific genetic variations and increased risk of lung diseases triggered by exposure to external agents. out of 151 snps initially identified through the phewas database, 86 met the study’s criteria, indicating their potential contribution to pulmonary disorders. further analysis revealed that six of these variants were missense mutations, which cause amino acid substitutions in the resulting proteins. this is particularly important, as missense mutations can alter protein structure and function, potentially disrupting normal biological processes in lung tissue. among these, the tnip1 and psmb8 genes demonstrated notably high expression levels in lung tissue, according to data from the gtex portal. the tnip1 gene is known to be involved in regulating immune responses and inflammatory processes via the nf-κb pathway, which plays a central role in the pathophysiology of inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (shamilov & aneskievich, 2018). in contrast, the psmb8 gene encodes a subunit of the immunoproteasome, which is essential for antigen processing and the regulation of adaptive immune responses. the high expression of both genes in lung tissue supports the hypothesis that genetic variations in tnip1 and psmb8 may contribute to immune dysfunction and chronic inflammation in response to toxic exposures such as air pollution, cigarette smoke, and industrial particles. the psmb8, which encodes the β subunit of the immunoproteasome, plays a critical role in maintaining immune homeostasis, particularly in lung diseases induced by environmental exposures. previous studies have shown that psmb8 polymorphisms are significantly associated with increased risk of hypersensitivity pneumonitis (hp) among bird breeders (camarena et al., 2010). for instance, camarena et al. (2010) reported that individuals carrying the psmb8 kq genotype had an odds ratio of 7.25 for developing hp compared to healthy controls (p = 0.000034), suggesting that this genetic variant may lead to dysregulation of antigen processing and adaptive immune activation via the mhc class i pathway. moreover, mutations in psmb8 have been implicated in candle syndrome, a rare autoinflammatory disorder characterized by interferon dysregulation and elevated expression of inflammatory mediators such as ip-10, il-6, and mcp-1. these mediators are also involved in chronic pulmonary diseases like copd and fibrosis (liu et al., 2012). the enhanced activation of the interferon pathway in such cases suggests that psmb8 mutations may induce damaging immune hyperactivity in lung tissue over time. additionally, psmb8 has been linked to cellular transformation mechanisms following exposure to environmental carcinogens like cigarette smoke. recent research demonstrated that chronic exposure to nnk, a carcinogenic compound in tobacco, significantly increases the expression of the long non-coding rna psmb8-as1 in human bronchial epithelial cells (ou et al., 2024). this lncrna regulates the cell cycle by influencing cdk1 expression. its silencing leads to g2/m phase arrest, reduced cell proliferation, and increased apoptosis, pointing to a potential epigenetic role for psmb8 in carcinogenic pathways. further studies in non-small cell lung cancer (nsclc) have shown that immunoproteasome deficiency, including downregulation of psmb8, is 722 biology, medicine, & natural product chemistry 14 (2), 2025: 717-723 associated with a mesenchymal phenotype and correlates with tumor recurrence and metastasis (tripathi et al., 2016). on the other hand, high psmb8 expression and hypomethylation in lung adenocarcinoma (luad) tissues are linked to activation of anti-tumor immune pathways, presence of effector immune cells, and improved response to pd-1/pd-l1-based immunotherapy (xie et al., 2022). these findings suggest that psmb8 through genetic mutation, lncrna expression, or epigenetic modification holds potential as both a diagnostic biomarker and a precision therapeutic target in lung diseases associated with environmental exposure. our analysis also found that gene variants related to exposure-induced lung disease showed highest expression levels in several tissues, including ebvtransformed lymphocytes, blood, spleen, lungs, and adipose tissue. this expression pattern aligns with common clinical manifestations of pulmonary disease, such as lymphadenopathy, pulmonary infections, and systemic inflammation (eckhardt & wu, 2021). the discovery of mutagenic and pathogenic genetic variants warrants further laboratory and clinical investigation. identifying such variants not only improves our understanding of disease susceptibility but also supports the development of diagnostic or prognostic biomarkers and specific therapeutic targets for future research (adikusuma et al., 2021; qureshi, 2020; santri et al., 2022). these findings serve as a preliminary foundation for developing more effective biomarkers and therapeutic strategies for environmentally induced lung disease. overall, this study underscores the importance of a multi-database approach in identifying genetic biomarkers relevant to multifactorial diseases such as environmentally triggered lung disorders. the combination of snp association analysis, mutation characterization, tissue-specific expression profiling, and population distribution provides a more comprehensive picture of the molecular mechanisms underlying disease susceptibility. while this research is limited as a preliminary study based on publicly available genomic and bioinformatics databases, it offers valuable insights that can guide further investigations. future studies should aim to validate these findings in laboratory and clinical settings, and expand the scope to discover additional pathogenic variants. thus, this research represents an important first step toward a deeper understanding of the genetic basis of lung disease and the development of more effective therapeutic approaches. conclusions this study demonstrates the potential role of specific genetic variants, particularly tnip1 and psmb8, in increasing susceptibility to lung diseases triggered by environmental exposures. through an integrative bioinformatics approach, six missense snps were identified, with psmb8 variant rs2071543 showing significant expression in lung tissue and variation across global populations. the findings suggest that genetic predisposition, combined with environmental risk factors, may contribute to the pathogenesis of chronic lung conditions through immune and inflammatory dysregulation. these results underscore the value of genomic and population-based analyses in uncovering biomarkers for early detection and precision therapy. although further experimental validation is needed, this study provides a foundational step toward the development of targeted strategies for the prevention and treatment of environmentally induced lung diseases. acknowledgements: we would like to express our gratitude to the faculty of medicine at universitas ahmad dahlan, yogyakarta, indonesia. authors’ contributions: all authors contributed equally to the conception of the study, data acquisition, analysis, and interpretation for the manuscript. competing interests: the authors declare no competing of interests. funding: this research received no external funding. references adam, m., schikowski, t., carsin, a. e., cai, y., jacquemin, b., sanchez, m., vierkötter, a., marcon, a., keidel, d., sugiri, d., al kanani, z., nadif, r., siroux, v., hardy, r., kuh, d., rochat, t., bridevaux, p. o., eeftens, m., tsai, m. y., … probst-hensch, n. 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(2022). analysis on methylation and expression of psmb8 and its correlation with immunity and immunotherapy in lung adenocarcinoma. epigenomics, 14(22), 1427–1448. https://doi.org/10.2217/epi-2022-0282 this page intentionally left blank biology, medicine, & natural product chemistry volume 3 – number 2 – 2014 issn 2089-6514 contents a discovery and characteristics description of telosma puberula (asclepiadoideae) in mount gedang atas and mount ijo, baturagung mountain yogyakarta widodo 47 52 larvicidal activity of a mixtureof cashew nut shell liquid and water-soluble extract of soapnut fruit (sapindus rarak dc.) against 3rd instar larvae of aedes aegypti glory resia raraswati, sudarsono, budi mulyaningsih 53 57 substitution and haplotype diversity analysis on the partial sequence of the mitochondrial dna cytbofindonesian swamp buffalo (bubalus bubalis) akhmad sukri, mohamad.amin, aris winaya, abdul gofur 59 63 the antidepressant effects of (arcangelisia flava (l.) merr) water-soluble extract in balb-c mice reviewed from immobility time by forced tiara a., arief r.h., sudarsono 65 67 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 343-349 | doi: 10.14421/biomedich.2024.132.343-349 issn 2540-9328 (online) potency of turmeric extract (curcuma domestica val.) with virgin coconut oil (vco) against incision wound healing department of biology, faculty of mathematics and natural sciences, padang state university jl. prof. dr. hamka, west fresh water, north padang district, padang city, west sumatra, telp. +62-831-81950361, indonesia. corresponding author* aifakurnia149@gmail.com manuscript received: 21 may, 2024. revision accepted: 08 august, 2024. published: 01 october, 2024. abstract wounds are damage or loss of body tissues that occur due to a factor that interferes with the body's protective system. one example of an open wound is an incision where there is a linear tear in the skin and underlying tissue. one natural ingredient that has potential as an alternative treatment for cut wounds is turmeric and vco. the part of turmeric contains curcumin to accelerate wound healing and vco contains lauric acid which functions as an antibacterial. the purpose of this study was to determine the effect of the combination of turmeric extract (curcuma domestica val.) with virgin coconut oil (vco) on the percentage and healing time of incision wounds in male mice (mus musculus l.). this research is an experimental study. the research was conducted from october to december 2023 at the animal house and research laboratory of the department of biology, faculty of mathematics and natural sciences, padang state university. the design used is a complete randomized design (ral) with 5 (five) treatments and 5 (five) repeats. the data obtained were analyzed using the anova (analysis of variance) test using the spss 22 application. the results showed that the combination of turmeric extract (curcuma domestica val.) with virgin coconut oil (vco) affected the percentage and time of incision wound closure in male mice (mus musculus l.). the best combination of turmeric extract with vco is a ratio of 2:1. keywords: turmeric extract; vco; incisions; male mice. introduction a wound is defined as an injury to body tissue caused by sharp or blunt trauma, temperature changes, chemicals, explosions, electric shocks, or animal bites. problems that arise in acute wounds are pain due to the release of cellular humoral elements in nerve endings, swelling due to fluid accumulation in the tissues around the wound, and discomfort due to inflammatory processes such as redness and heat due to vasodilation of blood vessels (clausen & laman, 2017). in indonesia, based on riskesdas data (2013), the proportion of wound types or types of injuries that are predominantly due to trauma are abrasions / bruises (70.90%), sprains (27.5%), and lacerations (23.2%). the number of injuries caused by sharp object trauma that is easily infected with bacteria when left or given synthetic chemical drugs that have side effects on body health (riani, 2022). the use of drugs on wounds aims to accelerate the healing process (safani et al., 2019). the wound healing process takes place naturally or with chemical assistance, such as with medicinal substances, ointments and others (qomariah et al., 2014). in addition to using chemical drugs, people also use traditional medicines in the treatment of wounds, such as aloe vera, betel leaf, and sambiloto. plants that have always been a mainstay as traditional medicine, one of which is turmeric. turmeric has a chemical content that is a yellow dye called curcuminoids. curcuminoids can act as antioxidants, which can prevent cell damage caused by free radicals. in addition, curcuminoids can also be anti-inflammatory (winarto & tim lentera, 2004). in vitro and in vivo studies show that turmeric has antibacterial activity, anti-inflammatory (anti-infective), activity against peptic ulcers, antitoxic, antihyperlipidemia, and anticancer activity (sutardi et al., 2022). the curcumin content in turmeric plays a role in wound healing and skin regeneration. turmeric also contains quinone compounds, flavonoids, saponins, tannins and alkaloids that play a role in the wound healing process as antibacterial (ningtyas, 2017). research conducted by maan et al. (2020), namely the administration of turmeric rhizome extract and gentamicin ointment for healing mouse skin incision wounds, proved that incision wounds given turmeric rhizome extract had undergone complete closure with scars and hairs growing around the wound on the 14th day. in handayani's research (2022), it was concluded that the use of a thick turmeric rhizome extract cream formula with a dose of 15% has the same incision wound yusni atifah, aifa kurnia* https://doi.org/10.14421/biomedich.2024.132.343-349 344 biology, medicine, & natural product chemistry 13 (2), 2024: 343-349 healing activity as a dose of 5% and a dose of 10%. research by susanto et al. (2023), a combination of turmeric rhizome extract and whiting at a composition of 1: 1; 2:1 and 1:2 can accelerate wound healing and have anti-inflammatory activity. in addition to turmeric, vco is also known as a very efficacious traditional medicine. the benefits when vco is applied to scratches and wounds, can form a thin layer that protects wounds from external dust, bacteria, and viruses so as to speed up the healing process and no side effects have been found (sumiasih, 2016). the function of vco as a strong antioxidant, because vco contains vitamin e and polyphenols. in addition, vco has antioxidant, antimicrobial, antifungal abilities, protecting the skin from the dangers of free radicals and tissue degeneration (zikran, 2023). the bioactive compounds in coconut oil are thought to be responsible for antioxidant activity; anti-inflammatory, anti-pyretic, analgesic, and hypolipidemic vco (fernando, 2015). according to research conducted by fatonah et al., (2013), concluded that the use of vco tropically against the healing process of grade i and ii pressure wounds is more effective using vco than ordinary coconut oil. research that has been conducted previously by wijaya (2013), proves that aloe vera extract cream using a cream base containing vco is able to provide effectiveness faster than aloe vera extract not using vco. the use of vco is able to accelerate wound healing because it is an oil that contains medium chain saturated fatty acids that support healing and repair of body tissues. based on the background that has been described, the combination of turmeric and vco has the potential to be used as a better wound-healing drug. research on the effect of the combination of turmeric extract (curcuma domestica val.) with virgin coconut oil (vco) as a wound healer in male mice (mus musculus l.) needs to be done. materials and methods learning areas this type of research is experimental research. this research was conducted in october – december 2023. the manufacture of turmeric extract (te) is carried out in the research laboratory, maintenance and treatment of mice is carried out at the house of the animal division of the department of biology, faculty of mathematics, natural sciences, padang state university. the tools used in the study were mouse cages, wire covers, digital scales, set sets, cameras, gloves, markers, calipers, sterile holders, beaker glass, stirring rods, vacuum rotary evaporator, hot plates, vortexes, erlenmeyer, test tubes, measuring cups, razors, drinking pacifiers, bottle containers, spoons, and stationery. the ingredients used are fresh turmeric from the city of padang, male mice (mus musculus l.) as many as 25 heads aged 2-3 months, aquades, virgin coconut oil (vco), betadine, 70% alcohol, 96% methanol, filter paper, tissue, pellet feed, labels, and cotton buds. the design used was a complete randomized design (ral) with 5 (five) treatments with 5 (five) repetitions consisting of negative control, positive control (betadine), p1 (te + vco 1: 1), p2 (te + vco 1: 2), p3 (te + vco 2: 1). procedure research preparation ▪ turmeric extract processing the preparation of this extract was carried out at the research laboratory of the department of biology, faculty of mathematics and natural sciences, padang state university. turmeric rhizomes are processed by peeling the skin, washing, cutting thinly, drying in the aeration, after drying ground and sifted. then, 200 grams of turmeric that has been sifted is put into an airtight bottle then filled with 96% methanol until submerged. the container is tightly closed and placed at room temperature for 7×24 hours, then filtered with filter paper. turmeric extract solution is concentrated using a vacuum rotary evaporator with a temperature of 60 degrees celsius so that a thick extract is obtained. furthermore, the turmeric extract obtained is diluted according to the treatment. ▪ making a combination of turmeric extract and vco the preparation of the combination of turmeric extract with vco is each made 10 ml with different treatment concentrations, first the combination of turmeric and vco 1: 1, the combination of turmeric and vco 1: 2 and the combination of turmeric and vco 2: 1. turmeric extract that has been weighed is put into a test tube and then added vco that has been heated with a hot plate at each concentration, then a combination in the vortex. after, let it cool and then put it in the bottle provided. research implementation after the mice were acclimatized for 7 (seven) days, the mice that had been placed according to each group were then carried out the incision wound making process. before making wounds, the process of shaving the hair on the back of mice is carried out. the hair on mice is shaved first with a size of 3 cm, and the skin is disinfected with 70% alcohol. next, the incision wound is made using razor blades that have been sterilized with 70% alcohol. making wounds with a sterile scalpel is done by incision on the skin of the back area until a 20 mm wound is formed with a depth to reach the dermis characterized by blood discharge. in the k + group, betadine was given, p1 was given a combination of turmeric extract and vco 1: 1, p2 was given a combination of turmeric extract and vco 1: 2, and p3 was given a combination of turmeric extract and vco 2: 1. by applying using a cotton swab evenly on the surface of the incision wound. the treatment is given 2 (two) times a day (morning and evening) until the atifah & kurnia – potency of turmeric extract (curcuma domestica val.) … 345 wound closes. observations were made to measure a decrease in wound length and wound closure time. data analysis the data in this study are the percentage of wound closure and the time it takes to close. the research data was analyzed statistically through the anova (analysis of variance) one way anova test using the spss 22 application. if the results of the anova test are significantly different at a significant level of 5%, duncan's follow-up test is used. results and discussion result research on the effect of the combination of turmeric extract (curcuma domestica val.) with virgin coconut oil (vco) on incision wound healing in male mice (mus musculus l.) showed differences in the percentage of wound closure and wound closure time in each treatment during observation (figure 1). figure 1. the percentage of incision wound closure in the combination of turmeric extract and vco. description: k= negative control, k+ = positive control, p1 = combination of turmeric extract with vco ratio 1: 1, p2 = combination of turmeric extract with vco ratio 1: 2, p3 = combination of turmeric extract with vco ratio 2: 1. figure 2. closing time of mouse incision wound figure 2 shows the average closure time of different mouse skin incision wounds in each treatment given. the fastest wound closure is shown by k+ and p3 treatment, which is 9 (nine) days, followed by p1 and p2 with a wound closure duration of 11 days and kabout 15 days. the results of anova's statistical analysis showed that the combination of turmeric extract with vco had a significant effect on the length of healing time for mouse incision wounds with a significance value of ˂ 0.05, so that duncan's further test was carried out. table 1. duncan test results in long closure of mouse incision wounds. description: different alphabets show a significant difference (p<0.05). treatment mean ± sd (day) k14,6 ± 0,49a k+ 9.4 ± 1.20c c p1 11,2 ± 0,98b p2 11,2 ± 0,75b p3 9,4 ± 1,20c 0 20 40 60 80 100 h2 h3 h4 h5 h6 h7 h8 h9 h10 h11 h12 h13 h14 h15 w o u n d c lo su re p er ce n ta ge ( % ) day to (k-) (k+) (p1) (p2) (p3) 14,6 9 11,2 11,2 9,4 0 5 10 15 20 kk+ p1 p2 p3 d ay o f w o u n d cl o su re treatment groups 346 biology, medicine, & natural product chemistry 13 (2), 2024: 343-349 negative control positive control 1:1 combination 1:2 combination 2:1 combination figure 3. description of mouse incision wounds days 0,5,10,15. remarks: (a) day 0, (b) day 5, (c) day 10, and (d) day 15. discussion the results of this study showed that the treatment group given betadine and a combination of turmeric extract with vco ratio of 2: 1 provided the largest reduction in wound length compared to other combinations and also the fastest wound closure duration. figure 3 shows the best wound closure on days 9 and 9.4 with betadine and turmeric extract combination with vco ratio 2: 1. the condition of the wound has dried and closed completely. the negative control showed the smallest reduction in wound length at day 14.6, lasting wound healing compared to the other treatment group. a b c d a b c d a b c d a b c d a b c d atifah & kurnia – potency of turmeric extract (curcuma domestica val.) … 347 the calculation of the percentage of wound healing showed a positive control, a combination of 1: 1, 1: 2 and 2: 1 had the fastest healing rate of 100% when compared to the negative control group of 96.10% on day 14. incision wound closure time is fastest through morphological features and also statistical analysis is shown by k+ and p3 followed by p1, p2 and k-. this shows that the combination of turmeric extract with vco can speed up the process of closing incision wounds in mice. this is likely due to the curcumin content in turmeric extract which can reduce the duration of the inflammatory phase that occurs in wounds so as to accelerate wound healing and vco which can be efficacious as an antimicrobial that can prevent infection in wounds. milasari et al. (2019) stated that the use of turmeric as a wound healing ingredient can accelerate the wound healing process because curcumin compounds have anti-inflammatory and antioxidant properties and encourage cell re-epithelialization cell proliferation and collagen synthesis. the combination of turmeric extract with vco ratio of 2:1 showed the best wound closure duration compared to other combination treatments. the results of the study are in accordance with the results of research by susanto et al. (2023), which concluded that the observation of a combination of turmeric rhizome extract and whiting with a composition of 2: 1 was able to accelerate the healing of incision wounds. the flavonoids, alkaloids and triterpenoids in turmeric have anti-inflammatory effects and also contribute to the fight against infections caused by microorganisms that can help prevent infection in wounds and accelerate wound healing. the mechanism of action of flavonoids and alkaloids as an antibacterial is by disrupting the constituent components of peptidoglycan in bacterial cells, so that the cell wall layer is not formed intact and causes the death of these bacterial cells (susanto et al., 2023). saponins have the ability as cleaners and antiseptics that function to kill germs or prevent the growth of microorganisms that usually arise in wounds so that wounds do not experience severe infections (wijaya et al., 2014). triterpenoids help strengthen skin structure, increase antioxidant concentrations in wounds and restore inflammatory or inflamed tissue by increasing blood supply to the wound area, and help speed up the wound closure process (susanto et al., 2023). this study showed that the composition of turmeric that is greater than the composition of vco (2: 1) is better in the process of wound closure. this is because the curcumin content in turmeric plays a role in antiinflammatory and antioxidant and encourages reepithelialization, cell proliferation and collagen synthesis needed in wound closure. meanwhile, vco in the wound closure process acts as an antibiotic, antibacterial, antifungal and antiviral (putri et al., 2021). this is in accordance with the mechanism of wound closure according to kim et al. (2012), that in the wound healing process, there is a wound healing mechanism consisting of 4 phases, namely hemostasis, inflammation, proliferation and remodelling. curcumin compounds in turmeric are known to inhibit thrombin clotting and play a role in accelerating the inflammatory phase, increasing tissue collagen density and increasing the proliferation of fibrobalast tissue. in addition, the flavonoid content in turmeric extract increases the number of macrophages. an increase in the number of macrophages increases fibroblast proliferation, so that both proliferation and remodeling phases take place faster. in addition, the content of saponins in turmeric extract is also able to increase fibroblast proliferation (arisonya et al., 2014). the use of vco in this study helps reduce inflammation because vco has unsaturated fatty acids in the form of oleic acid, linoleic acid, and flavonoids that function as anti-inflammatory (ribeiro et al., 2014). the anti-inflammatory and antibacterial effects of vco accelerate the inflammatory process by reducing inflammatory mediators and the wound healing process can immediately enter the proliferation phase. in addition, moisture is required in the wound healing process for the activity of growth factors and the activity of proteolytic enzymes. proteolytic enzymes in vco have a role in the tissue repair process. they are able to activate the synthesis and deposition of protein matrix components in granulation tissue, so that it will accelerate the re-epithelialization process in wounds (khiftiyah, 2018). based on the results of research by sumiasih et al. (2016), it was suggested that perineal wounds treated according to apn standards plus vco healed faster than those treated according to apn. this is because vco has antibacterial, antiviral, antifungal properties, and when applied over the wound can coat the wound and protect the wound from contamination of dirt such as dust and other dirt. the results of research conducted by maan et al. (2020), namely the administration of turmeric rhizome extract and gentamicin ointment for healing mouse skin incision wounds, proved that incision wounds given turmeric rhizome extract had undergone complete closure with scars and hair growth around the wound on the 14th day. in addition, research conducted by susanto et al. (2015), the antibacterial effect of virgin coconut oil against methicillin resistant staphylococcus aureus proves that topical administration of vco can reduce wound size starting on day 14. the results of this study showed that incision wounds given a combination of turmeric extract with vco ratio 2: 1 have experienced complete wound closure on the 9.6th day by leaving scars and growing fine hair in the area around the wound. judging from the characteristics of healed wounds based on histologic according to amita et al. (2017), the density of collagen fibres in the wound area is denser than incision wounds after aqueous administration and fibroblast cells are few and the spread is rare. from these results, it can be proven that the use of a combination of turmeric extract with vco is better for healing incision wounds compared to separate treatments. 348 biology, medicine, & natural product chemistry 13 (2), 2024: 343-349 the difference in wound closure time in the turmeric extract with vco combination treatment group was not only caused by differences in the composition of turmeric extract and vco given but also influenced by wound healing factors, including stress factors, nutrition / nutrition, tissue perfusion, circulation disorders, metabolic changes, early mobilization, age and obesity (potter, 2011). according to maryunani (2014), factors that affect wound healing such as oxygenization, hematoma, age, nutrition, sepsis, medications, lifestyle and mobilization. factors affecting wound healing, according to hidayat (2009), are vascularization, anaemia, age, other diseases, nutrition and obesity. conclusion the combination of turmeric extract (curcuma domestica val.) with virgin coconut oil (vco) affects the percentage and timing of wound closure in male mice (mus musculus l.). the best combination of turmeric extract with vco is a ratio of 2: 1. acknowledgments: the author expresses gratitude to god almighty, allah almighty. the author expresses his deepest gratitude to his family for finances, support, and guidance. author's contributions: aifa kurnia wrote the manuscript, conducted laboratory research, analyzed the data and designed the research. as well as mrs. yusni atifah as the author's supervisor in completing the research and manuscript. competing interests: the authors state that there are no competing interests. references amita, k., balqis, u., &; iskandar, c. d. (2017). histopathological features of incision wound healing in mice (mus musculus) using binahong leaf extract (anredera cordifolia (tenore) steenis) arisonya, s. gunawan, w., &; grahita, a. (2014). effectiveness of turmeric extract (curcuma domestica) on macrophage cell count and diameter in traumatic ulcer lesions (an in vivo study on male white rats (rattus norvegiccus)). journal b-dent. vol 1(2): 118-125 clausen, b., & laman, j. (2017). inflammation; methods and protocol. (j. laman, ed). usa: humana press fatonah siti, kartika hrp ade, dewi ratna. (2013). effectiveness of using virgin coconut oil (vco) topically to treat grade and ii decubitus. journal of health, vol 4(1):264-270. fernando wm, martins ij, goozee kg, brennan cs, jayasena v, martins rn (2015). the role of dietary coconut for the prevention and treatment of alzheimer’s disease: potential mechanisms. br j nutr vol 114 (1): 1-14 handayani, d. (2020). test of turmeric rhizome extract cream formulation (curcuma longa linn) against incision wound healing in male rats (rattus norvegicus). thesis. jakarta: health sciences and technology binawan university jakarta. hidayat, (2009). introduction to basic human needs: application of nursing concepts and processes. jakarta: salemba medika. khiftiyah, s. m. (2018). vco (virgin coconut oil) from lime acidification as an animal incision wound therapy model of nosocomial infection based on tgf β levels and epidermal thickness. thesis. brawijaya university. hapless. kim, d. c., su, s. k., & bae, j. s. (2012). anticoagulant activities of curcumin and its derivatives. bmb rep. vol 45(4): 221-6. maan, j. s. y., sasputra, n., & wungow, h. p. l. (2020). comparison of the effectiveness of turmeric rhizome extract (curcuma domestica val) and gentamicin ointment against healing cuts of mouse skin (mus musculus). sandalwood medical journal. vol 19(1) maryunani, anik. (2014). recent cesarean section (sc) and obstetric wound care. jakarta: team milasari, m., jamaluddin, a. w., &; adikurniawan, y. m. (2019). the effect of yellow turmeric extract ointment (curcuma longa linn) on wound healing in white rats (rattus norvegicus). ibn sina's scientific journal, 4(1): 186-202 ningtyas, g. (2017). test of the effectiveness of turmeric rhizome extract (curcuma domestica val.) in accelerating the healing process of incision wounds in male mice (mus musculus). surakarta: university of muhammadiyah surakarta. potter and perry. (2011). nursing fundamentals textbook: concepts, process &. practice. 4th edition. vol 1. jakarta: egc putri, f. m., fatrin, t., yanti, d., midwifery diii study program, m., still midwifery diii study program, d., &; abdurahman palembang, s. (2021). the effect of giving virgin coconut oil (vco) to accelerate the healing process of prineal wounds in partum mothers, partum boarding mothers at pmb ferawati palembang. national journal of nursing and midwifery sciences. (vol. 3, issue 2). ribeiro, d., freitas, m., tome, s.m., silva, a.m., laufer, s., lima, j.l & fernandes, e. (2014). flavonoids inhibit cox-1 and cox-2 enzymes and cytokine/chemokine production in human whole blood. journal of inflammation. vol. 38. no.2. 858-70 safani e. e., ayu w., kunharjito c., lestari a., &; purnama e. r. (2019). potency of bandotan leaf extract (ageratum conyzoides l.) as a spray for wound recovery of diabetic mice infected with staphylococcus aureus. biotropic: the journal of tropical biology, vol 3(1). sumiasih n. n., somoyani n k., &; armini n w. (2016). virgin coconut oil accelerates the healing of prenieum wounds at the inpatient health center in denpasar city. journal of husada scale volume 13 number 1: 39 – 49 sutardi, l. n., andi mustika, a., januar, r., nurfitria ayumi, a., lecturer of sub division of veterinary pharmacy department of clinical, s., and pathology, r., veterinary medicine and biomedicine, s., bogor agriculture, i., lecturer of pharmacology and toxicology division of department of anatomy, s., and pharmacology, f., lecturer of sub division of veterinary pharmacy department of clinical, a., &; undergraduate program school of veterinary medicine and biomedicine, m. (2022). activity of turmeric extract and gamat gel on the wound healing process of white rats. acta veterinaria indonesiana. vol 0(2), 193–200. atifah & kurnia – potency of turmeric extract (curcuma domestica val.) … 349 susanto, y., solehah, f. a., fadya, a., &; khaerati, k. (2023). potential combination of turmeric rhizome extract (curcuma longa l.) and betel lime as anti-inflammatory and wound healer. jpscr: journal of pharmaceutical science and clinical research qomariah, s., lisdiana., &; christijanti, w., (2014). effectiveness of fracture stem extract ointment (euphorbia tirucalli) on wound healing of white rat (rattus novergicus) incision wounds. unnes j life sci. vol 3(2). wijaya, r. a. (2013). cream formulation of aloe vera extract (aloe vera) as an alternative burn healer. thesis. semarang state university. semarang. winarto, i.w. and the lantern team. (2004). efficacy and benefits of turmeric. jakarta: agromedia library. zikran., pahria, t., &; adiningsih, d. (2023). the effect of virgin coconut oil (vco) on decubitus prevention: a systematic review. journal of ners vol 7(1): 564 -5 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 101-106 | doi: 10.14421/biomedich.2025.141.101-106 issn 2540-9328 (online) formulation and testing of antioxidant activity of papaya fruit (carica papaya l.) extract spray gel using the dpph method fauziyyah kunti hapsari*, nurul hidayati associate degree program in pharmacy, faculty of health and technology, muhammadiyah university of klaten, jl. ir. soekarno km.1 buntalan klaten 57419, tel. (0272) 323120, indonesia. corresponding author* fauziyyahk7@gmail.com manuscript received: 21 september 2024. revision accepted: 17 february, 2025. published: 25 april, 2025. abstract methanol extract of papaya fruit possesses natural antioxidant activity that can be utilized to counteract free radicals. spray gel is a formulation with low viscosity that can remain in contact for an extended period when sprayed on the skin. this study aims to formulate a spray gel made from papaya fruit extract and evaluate its antioxidant activity using the dpph method. the papaya fruit extract was prepared using the maceration method with methanol as the solvent. three spray gel formulas with papaya fruit extract concentrations of 1%, 2%, and 3% were tested for their physical properties. the testing revealed that formula iii (with a 3% extract concentration) met the appropriate ph criteria (5.67 ± 0.058), the desired viscosity (41.1 ± 2.84 cps), and the fastest drying time (< 5 minutes). the antioxidant activity test yielded the lowest ic50 value for formula iii at 330.307 ppm, followed by formula ii (ic50 = 374.08 ppm) and formula i (ic50 = 455.21 ppm). all three formulas exhibited good sprayability and spreadability, but their antioxidant activity was relatively weak. formula iii with a 3% extract concentration is considered the best option based on physical criteria and antioxidant activity, contributing to developing a product that can protect the skin from uv damage. keywords: antioxidant activity; carica papaya l.; dpph; papaya, spray gel. introduction uv rays can cause the formation of free radicals that damage the skin and increase oxidative stress (sari, 2015). antioxidants can prevent cellular damage and oxidative stress caused by exposure to ultraviolet light (ebtavanny et al., 2021). even at low concentrations, antioxidants can halt oxidation (andarina & djauhari, 2017). antioxidants can stop oxidation reactions by binding to highly reactive molecules and free radicals (santi et al., 2021). the antioxidant content of papaya (carica papaya l.) includes beta-carotene, phenols, flavonoids, and vitamin c, which are effective against free radicals (pratiwi & wahdaningsih, 2018). methanol extract from papaya (carica papaya l.) shows protective potential against skin damage with a low ic50 value. the lower the ic50 value, the higher the antioxidant activity. the ic50 value indicates the strength of the antioxidant: categorized as very strong if below 50 ppm, moderately strong in the range of 50-100 ppm, moderate at 100-150 ppm, and weak if between 150-200 ppm. an ic50 value greater than 200 ppm indicates that antioxidant activity is less active (molyneux, 2004). spray gel is a formulation with low viscosity that provides benefits in dermatology with a combination of gel properties and practical sprayability. this preparation facilitates application and maintains skin moisture while reducing the risk of contamination. this study aims to develop a spray gel derived from methanol extract of papaya (carica papaya l.) and evaluate its antioxidant activity using the dpph method (butar, 2018). testing was conducted with variations in extract concentrations (1%, 2%, and 3%) to evaluate physical characteristics such as ph, viscosity, and antioxidant activity. antioxidant compounds capture these free radicals, converting them into less reactive forms and reducing potential toxicity (molyneux, 2004). when dpph receives hydrogen radicals or electrons, stable diamagnetic molecules are formed. when antioxidants interact with dpph, either through transfer or with hydrogen radicals on dpph, the free radical character of dpph becomes inactive (molyneux, 2004). by analyzing various spray gel formulations, this study aims to find the most effective concentration of papaya extract to enhance antioxidant activity and physical properties, thereby contributing to developing skincare products that can protect the skin from uv damage. https://doi.org/10.14421/biomedich.2025.141.101-106 102 biology, medicine, & natural product chemistry 14 (1), 2025: 101-106 materials and methods materials and equipment in this study, the equipment used includes an analytical balance, blender, spatula, stirring rod, scoop, maceration vessel, filter paper, flannel cloth, porcelain dish, water bath, parchment paper, magnetic stirrer, hot plate, glassware, mica plastic, watch glass, digital ph meter, viscometer, uv-vis spectrophotometer, cuvette, measuring pipettes of various sizes, and dropper pipettes. the materials used in this study include ¾ ripe orange papaya, methanol, carbopol 940, propylene glycol, dmdm hydantoin, triethanolamine (tea), distilled water, dpph, and analytical grade ethanol. extract preparation papaya fruit (carica papaya l.) that has ripened to approximately ¾ of its orange color was cleaned with clean water, then peeled and its seeds removed. the papaya fruit was cut into slices about 2-3 mm thick and then dried in an oven at 60°c. after drying, the papaya slices were blended until they became papaya powder (meutia, 2013). the papaya powder was sieved with mesh no. 40 to obtain a uniform size. 250 mg of dried papaya powder was macerated with 1 liter of methanol solvent in a closed container. the maceration process was stirred thrice daily and lasted 3 x 24 hours (pratiwi & wahdaningsih, 2018). the maceration result was then filtered to separate the filtrate from the residue. the filtrate was heated in a water bath at 60°c until a thick extract was formed. formulation of spray gel the spray gel formula used refers to the research by akib et al. (2023), titled “formulation and characterization of spray gel containing ethanol extract of bitter melon fruit (momordica charantia l.).” the spray gel formula is shown in table 1. table 1. spray gel formula of papaya fruit extract (carica papaya l.). ingredients concentration (%) formula i formula ii formula iii papaya fruit extract 1% 2% 3% carbopol 940 0.1% 0.1% 0.1% propylene glycol 15% 15% 15% tea (triethanolamine) 0.5% 0.5% 0.5% dmdm hydantoin 0.6% 0.6% 0.6% distilled water ad 100 ml ad 100 ml ad 100 ml dissolve all carbopol 940 in distilled water on a hotplate at 70°c until a gel mass forms, stirring it with a magnetic stirrer. then, mix tea, propylene glycol, and dmdm hydantoin in a separate container. add the mixture to the gel mass and stir with the magnetic stirrer until homogeneous. next, add the papaya fruit extract and stir thoroughly. after mixing, pour the preparation into spray bottles (marlina et al., 2021). organoleptic test the organoleptic test was conducted by directly observing the spray gel’s texture, color, and scent (kamishita et al., 1992). homogeneity test the gel was sprayed onto a transparent glass to check for unmixed particles. if no coarse granules were found, the spray gel was considered homogeneous. ph test approximately 0.1 grams of papaya fruit extract spray gel was mixed with 100 ml of distilled water. then, the ph meter electrode was dipped into the solution. the ph range was expected to be between 4.5 and 6.5 (departemen kesehatan republik indonesia, 1977). viscosity test around 75 ml of the spray gel was placed into a beaker and its viscosity was measured using a rotor viscometer number 3 (hayati et al., 2019). the spray gel viscosity values should range between 25 and 250 cps (akib et al., 2023). sprayability and spreadability test the spray gel was sprayed from a distance of 5 cm. the evaluation criteria were as follows: if the spray gel did not spray at all, it was rated as "very poor." if the spray came out as droplets or clumps, it was rated as "poor" if it sprayed but produced large particles, it was rated as "fair." if it sprayed evenly with fine particles, it was rated as "good" (hayati et al., 2019) (kamishita et al., 1992). drying time test the spray gel was sprayed onto paper, and the drying time was observed using a stopwatch. the drying time for the spray gel was expected to be no longer than 5 minutes (kamishita et al., 1992). antioxidant activity test using the dpph method ▪ preparation of dpph stock solution weigh 5 mg of dpph powder and dissolve it in ethanol p.a. using a 50 ml volumetric flask covered with aluminum foil to achieve a dpph stock solution concentration of 100 ppm (angelia et al., 2022). ▪ determination of maximum wavelength and dpph absorbance (blank) add 1 ml of 100 ppm dpph stock solution to ethanol until it reaches the mark in a 5 ml volumetric flask and mix until homogeneous. the absorbance was measured using a uv-vis spectrophotometer at a wavelength range of 400-600 nm (patria & soegihardjo, 2013). hapsari & hidayati – formulation and testing of antioxidant activity … 103 ▪ preparation of papaya fruit extract spray gel stock solution weigh 5 mg of the spray gel extract and mix it with ethanol p.a. in a 10 ml volumetric flask to achieve a stock solution concentration of 500 ppm. ▪ determination of operating time (ot) place 50 μl of the spray gel stock solution with a concentration of 500 ppm into a 5 ml volumetric flask. then, add 1 ml of 100 ppm dpph stock solution and gradually add ethanol until it reaches the mark in the flask. measure the absorbance every minute for 30 minutes. use the stable absorbance value as the operating time. ▪ absorbance measurement of the spray gel test solution pipette 4 ml, 3.5 ml, 3 ml, 2.5 ml, and 2 ml from the 500 ppm papaya fruit extract stock solution to obtain five test solution concentrations of 400 ppm, 350 ppm, 300 ppm, 250 ppm, and 200 ppm. add ethanol p.a. to each test solution in a 5 ml volumetric flask. then, add 1 ml of 100 ppm dpph solution to each test solution. incubate the solutions for the determined operating time. next, the absorbance is measured using a uv-vis spectrophotometer at the previously determined maximum wavelength. data processing and analysis the physical properties of the spray gel, including organoleptic, homogeneity, ph, viscosity, sprayability, spreadability, and drying time, were tested. the ph, viscosity, and drying time data were evaluated by finding the average and standard deviation. for absorbance data, the percentage of dpph radical inhibition was calculated using the following formula: % dpph radical inhibition= (control absorbance test substance absorbance) control absorbance 𝑥 100 the inhibition percentages at different concentrations of the test solution were plotted in a linear curve to determine the equation y = ax + b, with the x-axis representing the concentration of the test solution and the y-axis representing the inhibition percentage. antioxidant activity was measured by calculating the ic50 value, which represents the concentration of the test solution needed to inhibit 50% of dpph radicals. the data analysis was conducted using theoretical approaches and statistical analysis. theoretical analysis compared the physical and antioxidant activity test results with relevant theories, while statistical analysis assessed data distribution for normality, homogeneity, and parametric tests (anova) or non-parametric tests (kruskal-wallis). if anova showed significant differences, the lsd (least significant difference) test was used to identify significantly different groups. results and discussion organoleptic test based on the results of the organoleptic test of papaya fruit extract spray gel in table 2, there are differences in color and texture, but no difference in odor was found. table 2. results of the organoleptic test of papaya fruit extract spray gel (carica papaya l.). formula color odor texture i pale yellow papaya-like thick ii bright yellow papaya-like slightly thinner iii brownish yellow papaya-like slightly thinner the organoleptic test results indicate that the papaya fruit extract spray gel has a distinctive papaya-like odor, which might slightly affect comfort. there are differences in the color and texture of the spray gel, influenced by the concentration of the extract. as the extract concentration increases, the color of the spray gel becomes darker, and its texture becomes thinner due to the high extract concentration disrupting the carbomer as a thickening agent (handayani & qa,ariah, 2023). homogeneity test the homogeneity test was conducted to determine whether the resulting spray gel was homogeneous. the results are presented in table 3. table 3. results of the homogeneity test of papaya fruit extract spray gel (carica papaya l.). formula homogeneity conclusion i no clumping observed homogeneous ii no clumping observed homogeneous iii no clumping observed homogeneous ph test the ph test was performed to ensure the spray gel's acidity is appropriate for skin ph and does not cause irritation. the results are shown in table 4. 104 biology, medicine, & natural product chemistry 14 (1), 2025: 101-106 table 4. results of the homogeneity test of papaya fruit extract spray gel (carica papaya l.). formula ph x̄ ± sd replication i replication 2 replication 3 i 6,5 6,3 6,2 6,33 ± 0,153 ii 6,1 5,9 5,9 5,97 ± 0,115 iii 5,7 5,7 5,6 5,67 ± 0,058 all three formulas meet the ph standards. formula i is normally distributed (p = 0.637), while formulas ii and iii are generally not distributed (p < 0.05) according to the shapiro-wilk normality test. the data is also homogeneous (p = 0.05). since the ph test data did not meet the criteria for parametric anova (normal distribution and homogeneity), the kruskal-wallis test was conducted. the kruskal-wallis ph test was p = 0.026 < 0.05, indicating significant differences. to determine the significant differences, a post hoc lsd test was conducted, showing p < 0.05, which means that each formula differs significantly from the others. the ph test results show that formulas i, ii, and iii meet the quality standards for skin care products with a ph between 4.5 and 8.0 (departemen kesehatan republik indonesia, 1977), although there are significant differences between them. a ph that is too low can irritate, while a ph that is too high can cause dryness (puspita et al., 2021). the increase in papaya fruit extract concentration, which has a ph of around 4.5, makes the spray gel more acidic. viscosity test the viscosity test was performed to measure the thickness of the spray gel. the results are displayed in table 5. table 5. results of the viscosity test of papaya fruit extract spray gel (carica papaya l.). formula viscosity (cps) x̄ ± sd replication i replication 2 replication 3 i 406 403 401 403,3 ± 2,52 ii 80,9 80,8 78,7 80,1 ± 1,24 iii 38,9 40,1 44,3 41,1 ± 2,84 formula i does not meet the viscosity standards for spray gels, while formulas ii and iii do. all formulas have a normal distribution (p > 0.05) according to the shapiro-wilk normality test and homogeneity test. the viscosity results are suitable for parametric one-way anova, which produced a significant value (p = 0.000), indicating a meaningful difference. subsequently, a post hoc lsd test was performed, showing differences among the three formulas. formula i does not meet the viscosity standards, while formulas ii and iii meet the desired criteria. higher extract concentrations tend to decrease viscosity, affecting spray ease and causing reduced viscosity due to decreased carbopol development from a more acidic ph. spraying and spreadability test the spraying and spreadability test was conducted to determine the spraying capability and spread of the spray gel. the results are shown in table 6. table 6. results of the spraying and spreadability test of papaya fruit extract spray gel (carica papaya l.). formula spraying and spreadability conclusion i sprays with fine particles, spreads well good ii sprays with fine particles, spreads well good iii sprays with fine particles, spreads well good the spray gel sprays and spreads well due to its appropriate texture and viscosity. all spray gel formulas dry within less than 5 minutes, meeting the skin care product efficiency standards (kamishita et al., 1992). as shown by formulas i, ii, and iii, viscosity affects drying time, with lower viscosity resulting in faster drying. proper viscosity is crucial for effective application and drying speed. drying time test the drying time test was conducted to measure the drying time of the spray gel. from table 7, all formulas have good drying times of less than 5 minutes. hapsari & hidayati – formulation and testing of antioxidant activity … 105 table 7. results of the drying time test of papaya fruit extract spray gel (carica papaya l.). formula drying time (minutes, seconds) x̄ ± sd replication i replication 2 replication 3 i 4,30 3,26 4,24 4,27 ± 0,031 ii 3,01 2,59 3,03 2,88 ± 0,248 iii 2,48 2,40 2,45 2,44 ± 0,040 the drying time test shows the normal distribution for all formulas (p > 0.05), but the homogeneity test found that the data is not homogeneous (p = 0.010). since the data did not meet the criteria for parametric tests, the kruskal-wallis test was performed, which resulted in a significant value (p = 0.027). the kruskal-wallis test indicated significant differences, followed by a post hoc lsd test to determine differences between formulas. the post hoc lsd test showed that the three formulas differ. antioxidant activity test using the dpph method ▪ maximum wavelength of dpph the measurement of the maximum wavelength for dpph indicates that the maximum wavelength is 516 nm, with dpph (control) absorbance at 0.476, which meets the criteria set by i.g. gandjar & a. rohman (2009) of a range between 0.2 and 0.8. this absorbance value is used as a reference for calculating the percentage inhibition. it will be used in the measurement of the antioxidant activity of the papaya fruit extract spray gel (carica papaya l.). ▪ operating time operating time refers to the period during which the absorbance value remains stable. in this study, this was observed to occur between the 25th and 30th minutes, indicating the period during which the formulation remains effective in responding to free radicals. ▪ absorbance and % inhibition of papaya fruit extract spray gel (carica papaya l.) the absorbance data, % inhibition calculations, and ic50 values are presented in table 8. the smaller the ic50 value obtained, the stronger the antioxidant activity (molyneux, 2004). table 8. absorbance results of papaya fruit extract spray gel (carica papaya l.). formula concentration (ppm) abs blank abs sample % inhibition ic50 (ppm) antioxidant category i 400 0,476 0,288 6,5 455,21 low activity 350 0,304 6,1 300 0,334 5,7 250 0,350 6,5 200 0,432 6,1 ii 400 0,244 5,7 374,08 low activity 350 0,248 6,5 300 0,261 6,1 250 0,336 5,7 200 0,405 6,5 iii 400 0,208 6,1 330,307 low activity 350 0,212 5,7 300 0,246 6,5 250 0,292 6,1 200 0,331 5,7 the ic50 values for the three formulas were statistically analyzed using the kruskal-wallis test to determine if there were significant differences among the ic50 values. the results indicate that the significance value of 0.368 > 0.05 suggests no significant difference between the ic50 values of the three formulas. to assess antioxidant activity, the papaya extract was tested with dpph. antioxidant compounds in papaya convert dpph from a free radical to a more stable and less reactive dpph-h, effectively reducing free radicals (molyneux, 2004). for formulas i, ii, and iii, with extract concentrations ranging from 400 ppm to 200 ppm, higher extract concentrations correspond to lower ic50 values: 455.21 ppm, 374.08 ppm, and 330.30 ppm, respectively. all three formulas are classified as "low activity," indicating that their antioxidant potential is not yet optimal. this analysis confirms that higher extract concentrations enhance antioxidant activity, consistent with the theory of molyneux, (2004). 106 biology, medicine, & natural product chemistry 14 (1), 2025: 101-106 conclusions as the concentration of papaya fruit extract in the spray gel increases, the color becomes darker, the ph becomes more acidic, the viscosity decreases, and the drying time shortens. the spray gel with a 3% extract concentration exhibits the best physical properties, including ph, viscosity, and spraying characteristics, as well as the shortest drying time, supported by statistical tests showing significant differences. the antioxidant activity of the spray gel improves with increasing concentrations of papaya fruit extract. the ic50 values of the papaya fruit extract spray gel at extract concentrations of 1%, 2%, and 3% are 455.21 ppm, 374.08 ppm, and 330.30 ppm, respectively, which fall into the "low activity" category for antioxidant activity. acknowledgements: the author would like to thank allah swt for granting us the blessings and guidance to conduct this research. we also thank our parents for their positive support throughout this study. to our advisor, thank you for your valuable guidance and advice throughout the research process. competing interests: the author declares no conflict of interest. funding: this study did not receive any funding. references akib, n. i., jannah, s. r. n., & sholihat, d. n. 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(2021). aktivitas antioksidan dari tumbuhan pepaya (carica papaya). as-syifaa jurnal farmasi, 13(2), 102–107. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 463-466 | doi: 10.14421/biomedich.2023.122.463-466 issn 2540-9328 (online) identification of medicinal plants and their utilization by community in kendal village, kendal sub-district aghnia rahmi hanum*, erna wijayanti biology education program, faculty of science and technology, uin walisongo semarang, jl. walisongo no.3-5, tambakaji, kec. ngaliyan, kota semarang 50185, tel. +62-247-604554 fax. +62-274-519739, fax 024-7601293, indonesia. corresponding author* aghnia_rahmi_hanum_2008086066@walisongo.ac.id abstract traditional medicine is a cultural heritage from ancestors deeply rooted in the nation's heritage; therefore, its use is still based on spoken and written experience from generation to generation. even though most people have turned to modern medicine, some still use these medicinal plants as ingredients for medicine, food, and other processed consumption. this study aims to identify the types of medicinal plants found in kendal village and how to use them, obtain them, and then transform them into effective medications. this study employs qualitative descriptive methodologies. observation and interviews are employed to collect data. based on the results of community interviews, it was determined that many kendal village residents continue to use plants to treat various diseases. the plant parts utilized are rhizomes, leaves, and stems. this medicinal plant has various uses, ranging from direct consumption to boiling. keywords: medicinal plants; traditional medicine; utilization. introduction the indonesian archipelago is renowned for its rich biodiversity and diverse agricultural output, which includes medicinal plants. this, supported by fertile soil conditions and a favorable climate, makes indonesia a potential producer of natural medicinal products (simbala et al., 2016). traditional medicine is a cultural inheritance from ancestors deeply rooted in the nation's heritage; therefore, its use is still based on experiences passed down orally and in writing from generation to generation (takarasel, 2010; mabel et al., 2016). ethnographically, indonesian society consists of numerous tribes with distinct cultures. this cultural diversity is distinct from one another, particularly concerning the utilization of medicinal plants. this diversity is evident in the types of plants used, the treatment methods, and the diseases that can be cured. according to world health organization (who) data, eighty percent of the world's population uses more than 20,000 species of medicinal plants (who, 2005). until 2001, the plant conservation laboratory, faculty of forestry, ipb, had documented from various research reports and published materials that no less than 2039 species of medicinal plants originated in indonesian forests (zuhud 2009; mulyani, et al., 2020). the community's use of medicinal plants ranges from flavoring ingredients to raw materials for the pharmaceutical and cosmetics industries. in the community health care system, however, the indonesian people have recognized and utilized medicinal plants to prevent health problems. some types of medicinal plants found in indonesia have been patented and massproduced in other nations to generate substantial profits for the country (abdullah, 2010; rizal et al., 2019). the knowledge of medicinal plants and their use has been passed down from generation to generation until the benefits of these natural medicines have been demonstrated, even though they are not widely acknowledged. several communities, including the community in kendal village, have utilized these medicinal plants, illustrating this point. although the majority of people have switched to modern medicine, there are still those who use these medicinal plants as ingredients for medicines, as well as for food and other processed consumption. this study aims to identify the types of medicinal plants found in kendal village, as well as how to utilize them, obtain them, and transform them into effective drugs. methods this study employed qualitative descriptive methodologies. this quantitative study collected data and analyzed the results to obtain conclusive information. manuscript received: 09 june, 2023. revision accepted: 09 august, 2023. published: 15 august, 2023. https://doi.org/10.14421/biomedich.2023.122.463-466 464 biology, medicine, & natural product chemistry 12 (2), 2023: 463-466 the data was collected through observation and interviews. observation involves making direct observations in the field alongside respondents to determine the types of plants commonly used for medicinal purposes. several individuals who knew how to use plants as traditional medicine were interviewed. the interview questions include the plant's local name, the part used, the benefits, and the method of application. the method of data analysis consists of describing the data collected through observations and interviews with ten kendal village residents based on two criteria: the use of medicinal plants and the planting of medicinal plants around the residence. the existing data is then presented as a table with local names, scientific names, parts of plants utilized, benefits, and instructions on how to use them. results and discussion medicinal plant species based on research conducted in kendal village, kendal subdistrict, eight medicinal plant species were discovered to be utilized. these medicinal plant species are typically found in the gardens of simple-minded residents. this is because, based on hereditary knowledge from ancestors, intelligent people, or books on medicinal plants, making people aware of the benefits of these plants and cultivating them by simply planting them in their yards, where the maintenance method does not require special maintenance and includes plants that are easy to overgrow, is feasible (mabel, 2016). the results indicated that residents of kendal village continue to use medicinal plants to treat various diseases in various ways (table 1). table 1. types of medicinal plants utilized by community in kendal village, kendal sub-district. no. local name scientific name plant part type of disease how to use picture 1. kunyit curcuma longa rhizome stomach pain and immunity turmeric rhizome as much as 2 fingers are pounded and then squeezed until water comes out, then the water is drunk 2. daun sirih piper betle leaves period pain, cholesterol betel leaves are boiled with water until it boils then the water is drunk 3. temulawak curcuma zanthorriza rhizome increased appetite, indigestion curcuma is pounded and then squeezed until water comes out, then the water is drunk 4. jahe merah zingiber officinale rhizome diabetes, uric acid ginger is pounded and then squeezed until water comes out, then the water is drunk hanum & wijayanti – identification of medicinal plants and their utilization by … 465 table 1. cont. no. local name scientific name plant part type of disease how to use picture 5. daun salam syzygium polyanthum leaves uric acid, cholesterol, diabetes bay leaves are boiled with water until it boils then the water is drunk 6. daun kelor moringa oleifera leaves eye disease moringa leaves as much as 1 handful are boiled with water until boiling then the water is drunk 7. lengkuas alpinia galanga rhizome diabetes bring 2 thumb-sized galangal rhizomes to a boil with 2 cups of water. 8. serai cymbopogon citratus stem cholesterol, immunity, uric acid lemongrass is soaked in hot water and the water is drunk. 9. lidah buaya aloe vera leaves (gel) skin disease the outer skin of aloe vera is peeled and then washed and the gel is taken from the aloe vera and then applied to the wound. 0. kencur kaempferia galanga rhizome pain and inflammation boil 2 thumb-sized galanga rhizomes with 2 cups of water until boiling and then drink the water. the utilized plant parts include rhizomes, leaves and stems. the plant parts that are most frequently used as medicinal plants are rhizomes (up to 5 species), followed by leaves (up to 4 species), and the stem (up to 1 species). the most widely utilized plant part is the rhizome, as several rhizome plants have anti-microbial, anti-inflammatory, and antibiotic properties that increase endurance (edy & ajo, 2020; setiono, et al., 2022). planting rhizome plants is simple and practical because they can be grown in pots and polybags on a 466 biology, medicine, & natural product chemistry 12 (2), 2023: 463-466 narrow land. the growing conditions for rhizome plants must account for climate, soil, altitude, sunlight requirements, and air temperature (tarigan, 2020; setiono, et al., 2022). another reason rhizome (herbaceous) plants are common in residential gardens is that their maintenance requirements are relatively low, whereas shrubs and trees undergo a lengthy growth process. in addition, shrubs are regarded as hedge plants because their medicinal applications are poorly understood, so they are rarely used in treatment (lestari, et al., 2021). type of disease based on interviews with ten kendal village residents, it was determined that many villagers continue to use plants to treat various diseases. the community learns how to use medicinal plants based on inherited knowledge from ancestors, intelligent people, and neighbors who know how to use them. the use of medicinal plants is also highly varied, ranging from direct consumption to boiling and drinking boiled water. in general, there are two types of treatment administered by the community: treatment for internal diseases and treatment of external illnesses. external treatment includes skin diseases, toothaches, eye conditions, and wounds. in contrast, internal diseases are treated by ingesting or drinking a portion of a medicinal plant (hidayat, et al. 2010; mulyani, et al. 2020). conclusion based on the research conducted, it can be concluded that the use of medicinal plants by the kendal village community has been passed down through the generations. in general, these medicinal plants are cultivated in backyards. turmeric, galanga, curcuma, betel leaves, bay leaves, aloe vera, lemongrass, moringa leaves, and red ginger are the plant species found. the use of these medicinal plants is also quite diverse, ranging from direct consumption to the decoction and maceration of the plant parts. competing interests: the authors declared that they have no competing interests. references lestari, d., koneri, r., maabuat, p. v. (2021). keanekaragaman dan pemanfaatan tanaman obat pada pekarangan di dumoga utara, kabupaten bolaang mongondow, sulawesi utara. jurnal bios logos. 11(2): 82-93. mabel, y., simbala, h., koneri, r. (2016). identifikasi dan pemanfaatan tumbuhan obat suku dani di kabupaten jayawijaya papua. jurnal mipa unsrat. 5(2): 103-107. mulyani, y., sumarna, r., patonah. (2020). kajian etnofarmakologi pemanfaatan tanaman obat oleh masyarakat di kecamatan dawuan kabupaten subang provinsi jawa barat. jurnal farmasi galenika (galenika journal of pharmacy). 6(1): 37-54. rizal, s., sustriana. (2019). inventarisasi dan identifikasi tanaman berkhasiat obat di kabupaten musi banyuasin. jurnal indobiosains. 1(2): 50-62. setiono, novriansyah, y., fitri, d., asman, m., & isman. (2022). pelatihan pembuatan minuman herbal rimpang dan pembudidayaannya masa pandemi covid-19. jurnal masyarakat mandiri (jmm). 6(4): 3331-3340. takarasel, r. (2010). inverntarisasi tumbuhan obat tradisional di kecamatan manganitu, tamako, tabukan selatan dan kendahe kabupaten sangihe. manado: skripsi. fmipa unsrat. who. (2005). review of traditional medicine in the south-east asia region. in who biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 609-614 | doi: 10.14421/biomedich.2025.142.609-614 issn 2540-9328 (online) cytogenotoxicity test and biological evaluation of curculigo latifolia extract with bioindicators allium cepa l. var. aggregate susan susanti, nelis hernahadini, subagio joko suprapto, haryanto* department of biotechnology, faculty of science and technology, universitas muhammadiyah bandung, jl. soekarno-hatta, no. 752 bandung, indonesia. corresponding author* haryanto@umbandung.ac.id manuscript received: 29 march, 2025. revision accepted: 23 july, 2025. published: 01 october, 2025. abstract curculigo latifolia plants have secondary metabolites that can suppress cell division. compounds that suppress cell division can be used as anticancer drug candidates. this research aims to determine the effect of c. latifolia root extract on cell division and genotoxic effects using bioindicator a. cepa l. var. aggregatum. this study was conducted experimentally with a completely randomized design (crd) using 6 treatment levels and 5 replications. the treatment levels in this study were k (water), k + (h2o2 300 mm), k (h2o2 26 hours + water 46 hours) and a combination of treatments at p1, p2, and p3 with h2o2 immersion (26 hours) and continued immersion of c. latifolia root extract concentrations of 400 µg ml-1, 600 µg ml-1 and 800 µg ml-1 (46 hours). the roots of a. cepa from the soaking treatment of c. latifolia extract were used to make preparations using the squash method. the results of the anova test showed that the administration of c. latifolia root extract had a significant effect on the mitotic index and cell abnormalities (p<0.05). p1 had the most effective value in reducing the mitotic index and cell chromosome abnormalities. keywords: allium cepa l. var. aggregatum; cell abnormalities; cell division; curculigo latifolia; mitotic index. introduction the indonesian ministry of health released data on cancer rates in 2022 of 136 people per 100 thousand population and ranks 8th in asia-southeast (dinkes ntb, 2024). cancer is a disease that can cause death, but is not an infectious disease. cancer is characterized by uncontrolled abnormal cell division due to loss of cell control mechanism (lestari et al., 2020). various cancer treatments have been carried out, one of which is treatment with chemotherapy (indra et al., 2022). however, chemotherapy treatment can provide side effects that are toxic to normal cells (mahmoud & abdelrazek, 2019). due to the side effects caused by chemotherapy treatment, some researchers began to be directed to the bioactive components of plants in efforts to find and study anticancer activity (rahayuningsih et al., 2021). curculigo genus plants are one of the plants that belong to the hypoxidaceae family (syabana et al., 2015). curculigo genus plants play a role as antioxidants, antiinflammation, antitumors, anticancers and antidiabetics (zabidi et al., 2021). some species of curculigo plants such as c. orchioides, c. capitulate, c. pilosa and c. latifolia (nie et al., 2013). c. latifolia is a plant that contains secondary metabolites such as flavonoids, alkanoids, terpernoids, phenols and tannins (haryanto et al., 2023). plants that have active compounds are tested for cytotoxicity and genotoxicity (rahayuningsih et al., 2023). as a major step in the search for anticancer compounds (çelik & aslantürk, 2010). cytotoxicity and genotoxicity tests are tests to determine how effective the compound is in inhibiting cell division and to determine the effect on healthy cells through chromosomal abnormalities (rahayuningsih et al., 2022). the use of a. cepa l. var. aggregatum as a bioindicator because it has a division profile similar to multicellular systems (kannangara et al., 2015). it is similar to mammals such as mice (bonciu et al., 2018), has a low chromosome number (2n=16) (olusola & solomon, 2018), and has easily observable assay parameters such as root growth, mitotic index and cell structure due to chromosomal aberrations (fujiwara & hagan, 2014). cytotoxic and genotoxic test research on jatropha mollissima (pohl) plants has toxic and genotoxic effects along with the increase in concentration used, namely at an extract concentration of 0.01 mg/ml with a mitotic index of 44.09% and at an extract concentration of 1 mg/ml has a mitotic index of 21.04% while the genotoxicity test results at an extract concentration of 0.01 mg/ml have damage of 2.91% and a concentration of 1 mg/ml with damage of 4.04% (dias et al., 2019). https://doi.org/10.14421/biomedich.2025.142.609-614 610 biology, medicine, & natural product chemistry 14 (2), 2025: 609-614 research on c. latifolia plants against cytotoxic and genotoxic tests on bioindicators of a. cepa l. var. aggregatum needs to be done because this plant is a plant that has anticancer potential and no research has ever been done related to cytotoxic and genotoxic tests to determine the effect on inhibition of cell division and the effect on cell damage bioindicator a. cepa l. var. aggregatum. this research aims to determine the effect of c. latifolia root extract on cell division and genotoxic effects using bioindicator a. cepa l. var. aggregatum. materials and methods this research was conducted at the biology laboratory, faculty of science and technology, universitas muhammadiyah bandung. this study used an experimental method with a completely randomized design (crd) pattern. procedures extraction of c. latifolia plant roots the source of the extract came from the roots of c. latifolia plant. samples were washed and dried for 8 days in the sun covered with a black cloth. samples are made simple using copper and a blender. simple is carried out maceration extraction using 96% ethanol solvent for 3x24 hours with a sample: solvent ratio (1: 8 b / v). then the macerate is filtered using filter paper followed by concentration using a rotary evaporator at 50oc until the solvent evaporates (wendersteyt et al., 2021). bioindicator induction treatment of a. cepa l. var. aggregatum a. cepa l. var. aggregatum came from farmers of cupunagara village (bank desa), then cleaned and grown to 2mm root length. the soaking technique was carried out on a bottle, the soaking was divided into 6 treatments with 3 controls and 3 treatments as follows: each treatment consisted of five allium cepa l. var. aggregatum induced for 26 and 46 hours, in k (water), k+ (h2o2), k(h2o2 water combination), and treatments p1, p2, and p3 administration of hydrogen peroxide (h2o2) 300 mm (26 hours) with a combination of extracts of c. latifolia dryand ex. w. aiton at concentrations of 400µgml-1, 600µgml-1 and 800µgml1 for 46 hours (akinboro & jimoh, 2021 modified). total soaking 72 hours (three days). on the 3rd day of soaking, a. cepa l. var. aggregatum was harvested at 12.00 (akwu et al., 2019). then, the roots were cut along 3 mm and the root length was averaged. the roots were put into a fixation solution (ethanol (3): glacial acetic acid (1)). the roots were ready to be used for preparation. preparation with squash method the roots of a. cepa l. var. aggregatum were put into the fixation solution for at least 24 hours, then put into 70% ethanol for 10 minutes, and then transferred to 1 n hcl hydrolysis solution for 1 hour (60 minutes). roots that have been hydrolyzed for 60 minutes are inserted into a new tube and stained consisting of acetocarmine and methylene blue 1% in a ratio of 2: 1 are homogenized and kept for 24 hours then the roots are transferred to the glass object and then closed using cover glass and pressed (squash) until the cells spread evenly and then observed under a microscope (abdullah et al., 2017 modified). preparations that have been made are observed using a 40x10 magnification microscope and then the calculation of mitotic index and cell abnormality: the following is the calculation formula for im and as according to sabeen et al. (2019). calculation of mitotic index percentage (mi)= 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑖𝑣𝑖𝑑𝑖𝑛𝑔 𝑐𝑒𝑙𝑙𝑠 𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 100% cell abnormality calculation formula% (ca)= 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑏𝑒𝑟𝑟𝑎𝑛𝑡 𝑐𝑒𝑙𝑙𝑠 𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑥100% data analysis data analysis used a one-way anova statistical test. mitotic index data and cell abnormality have significant values, so the tukey hsd test is continued further. data analysis using spss version 16. results and discussion effect of c. latifolia extract on cell division of bioindicator a. cepa l. var. aggregatum based on the results of the study, water control has the highest mitotic index value of 49.61%, k(h2o2 + water) has a value of 44.72% and k + is 39.70%. in the treatment of a combination of 300 mm h2o2 (26 hours) with c. latifolia extract (46 hours), a concentration of 400µg / ml (p1) with a mitotic index value of 32.70%, which was able to reduce 7% of the positive control (table 1). the results of the anova test on the mitotic index in each treatment had a significant value (p< 0.05). in the tukey hsd further test p1, p2 and p3 have similarities with k+. meanwhile, k water has the highest mitotic index value. susanti et al. – cytogenotoxicity test and biological evaluation of … 611 table 1. effect of c. latifolia on the mitotic index in cells of a. cepa l. var. aggregatum. treatment in cell division (mitotic) total cells observed mi (mean±sd%) reduction % p m a t k: air 989 881 22 14 57 1963 49.61±0.81c k+: h2o2 300mm 986 779 3 4 41 2083 39.70±0.74ab k-: h2o2 300mm + air 977 921 8 3 29 2049 44.72±0.51bc p1: h2o2 300mm+e 400µgml-1 1205 604 5 7 21 1948 32.70±0.60a 16.91 p2: h2o2 300mm + e 600µgml-1 959 594 18 8 34 1768 36.99±1.83a 12.62 p3: h2o2 300mm+ e 800µgml-1 965 751 16 11 40 1931 42.37±0.87ab 7.24 note: e: c. latifolia extract, in (interphase), p (prophase), m (metaphase), a (anaphase), t (telophase) the mean followed by the same letter indicates the treatment gives the same effect with α=0.05. effect of c. latifolia extract on cell abnormality of a. cepa l. var. aggregatum based on the observation, there are normal and abnormal cells. normal cells consist of interphase, prophase, metaphase, anaphase and telophase, while in cell abnormalities, there are nuclear lesions, giant nuclear, stickiness, extended interphase, micronuclear, extended cell and dislocation spindle (figure 1). interphase is the stage of the cell cycle when the cell prepares to divide and is characterized by a clearly stained cell nucleus (figure a). prophase is the first stage in cell division; the prophase stage is characterized by chromatids beginning to form and the cell nucleus losing its identity (figure b). metaphase is characterized by the appearance of chromosomes that are thickened and aligned on the metaphase plate (figure c). anaphase is characterized by chromosomes that are at different poles with the same number (figure d). telophase is characterized by the formation of two identical daughter cells and the cell nucleus is clearly visible (figure e). figure 1. observation of normal cells and abnormal cells at 1000x magnification using acetocarmine staining and methylene blue 1%. note: a. interphase, b. prophase, c. metaphase, d. anaphase, e. telophase, f. nuclear lesions, g. giant nucleus, h-i. binucleated, j. stickiness, k-l. extended interphase, m-n. micronucleus, dan o. extended cells and dislocation of the spindle. the results of the genotoxicity test with the aim to determine the effect of c. latifolia extract on cell abnormality are in (table 2). based on the results of the study, the percentage of cell abnormality obtained in k+ is 13.04% in the treatment of p1-p3 ranging from 5.42%9.40% with the highest abnormality of k+ and the least abnormality in p1. p1 is the most effective concentration when compared to the others because p1 is able to reduce 612 biology, medicine, & natural product chemistry 14 (2), 2025: 609-614 55.44% of k+. based on the anova test, the percentage of cell abnormality is significant (p< 0.05). based on tukey hsd further test, p1 has a smaller value than k+ (table 2). p2 and p3 had no significant difference from each other but were significantly different from k+. p1 has a significant difference with k +. table 2. effect of c. latifolia on cell abnormality of a. cepa l. var. aggregatum. treatment n u cl ea r le si o n s g ia n t n u cl ea r b in u cl ea r s ti ck in es s e x t. i n te rp h a se m ic ro n u cl ea r e x t ce ll a n d d is lo ca ti o n o f sp in d el a m o u n t ce ll a b n o rm a l c el l a b n o rm a l (m ea n + sd )% r ed u ct io n ( % ) k:air 0 0 0 0 0 0 0 0 0a k+: h2o2 300mm (+) 78 50 8 1 32 40 63 272 13.04±1.3d k-: h2o2 300mm+air 35 54 23 1 40 28 30 211 9.81±0.66c 24.77 p1:h2o2 + e400 µgml-1 28 24 4 0 20 13 17 106 5.42±1.40b 58.44 p2:h2o2 + e600 µgml-1 26 41 4 3 25 14 42 155 8.70±2.97c 33.29 p3:h2o2 + 800 µgml-1 38 40 8 5 34 17 40 182 9.40±1.77c 27.92 note: -e: extract of c. latifolia, ex: extended the average of the results followed by the same letter indicates that the treatment gives the same effect with α = 0.05. discussion several previous studies on cytotoxic tests using the bioindicator a. cepa l. var. aggregatum found effective results in rhizophora apiculata plant extract, namely at a concentration of 500 ppm with an im of 10.7% which was able to reduce 41.95% from the negative control of 52.65% (rahayuningsih et al., 2021). avicennia marina extract has an effective concentration to reduce im 1000 ppm, namely 24.7% which can reduce 33.47% from k 58.17% (rahayuningsih et al., 2022). the decrease in the mitotic index in this study is possible because c. latifolia contains secondary metabolites such as flavonoids, tannins, alkaloids, phenols, and terpenoids. alkaloid compounds are secondary metabolite compounds that interfere with the cell cycle in the metaphase phase (phase m) by binding to tubulin in a different place with taxanes, which can then prevent polymerization and microtubule assembly, which results in the termination of metaphase and causes apoptotic cells (puri et al., 2023). phenol compounds can cause inhibition of cell cycle phases, namely in the g1, s, s-g2 and g2 phases by downregulating cyclin and cyclin-dependent kinases (cdks) by inducing the expression of p21, p27 and p53 genes (dai & mumper, 2010). flavonoid compounds act as antioxidants, inhibit cell proliferation by inducing cell cycle arrest in the m/g phase and induce autophagy and apoptosis in human breast cancer cells (zhang et al., 2018). tannin compounds have the ability to inhibit the cell cycle by disrupting the synthesis process and causing cell damage and activating apoptosis while tetanoid compounds have the ability to be cytotoxic which causes neoplastic lines by inhibiting cellular activity and activating apoptotic cells (chudzik et al., 2015). cell abnormalities consist of nuclear lesions characterized by damage to the cell nucleus due to failures that occur in g1, s and g2 phases that can cause damage to the nucleus (fibras, & amon, 2014). giant nuclear is characterized by the cell nucleus having a giant or large size many times that of normal cells (vazhangat & thoppil, 2016). stickiness is characterized by chromosomes sticking to each other forming bridges caused by degradation, sticky chromosomes or depolymerisation of chromosomal dna (rahayuningsih et al., 2022). micronucleus is characterized by small cell nuclei caused by unbalanced chromosome division at the anaphase phase, causing the chromosomes to have a very small size (imaniar & phatmawati, 2014). binuclear is characterized by the number of cell nuclei more than one occurs due to failure in the telophase phase, namely the formation of cell plates so that the cell nucleus is more than one (sabeen et al., 2020). extended interphase and extended cell and dislocation spindle are characterized by cell nuclei that change shape to oval and move position to the edge of the cell. cell damage can occur from the side effects of secondary metabolites that have oh groups such as phenols, flavonoids and tannins which can cause damage to the s phase by breaking the hydrogen chain in dna or causing mutations in genes (rahayuningsih et al., 2023). the use of positive control h2o2 because h2o2 is a substance or chemical compound that has the ability to damage or is toxic to cells (akwu et al., 2019). the high percentage of cell abnormality is influenced by the provision of high concentrations. in research with a similar method, namely the combination of sodium azide and aloe vera extract, it was found that the higher the concentration of extract given, the higher the cell abnormality value obtained by the 50% extract treatment, susanti et al. – cytogenotoxicity test and biological evaluation of … 613 namely 0.41%, while at a concentration of 6.25%, namely 0.29% which can reduce 42.27% of k + 0.55% (akinboro & jimoh, 2021). research with avicennia marina (forks.) extract has the smallest abnormality concentration at 125 ppm which is 17.75% while at concentrations of 550, 500, and 1000 ppm are 21.5%; 28%; and 36.3% (rahayuningsih et al., 2022). at the same time, rhizopora styliosa griff extract has a decrease in abnormality from the positive control of 52.33% in fractions f6, f7, and f8 (flavonoid compounds from klt results) with a percentage of abnormality of 9.67%, 9.33% and 9.33%. f2, f3 and f4 (terpenoids and steroids) are 8.33%, 16.3% and 13.3% (rahayuningsih et al., 2023). conclusions based on the results of the study, it can be concluded that c. latifolia extract has a cytotoxic effect on h2o2 in suppressing cell division (mitotic index) and cell abnormality with the optimal concentration in treatment p1 with a mitotic index percentage value of 32.70% and has the least effect of 5.42%. acknowledgements: the authors expresses gratitude to god and thank you to the biology laboratory, faculty of science and technology, universitas muhammadiyah bandung for research facilities. authors’ contributions: haryanto and nelis hernahadini designed the study. susan susanti and subagio joko suprapto carried out the laboratory work. susan susanti and haryanto analyzed the data. all author wrote the manuscript, read and approved the final version of the manuscript. competing interests: all authors state that there are no competing interests. references abdullah, f. n., jaya, a. s., & widayat. 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(2018). flavonoids inhibit cell proliferation and induce apoptosis and autophagy through downregulation of pi3kγ mediated pi3k/akt/mtor/p70s6k/ulk signaling pathway in human breast cancer cells. scientific reports, 8(1), 1–13. https://doi.org/10.1038/s41598-018-29308-7 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 1, 2017 | pages: 19-36 | doi: 10.14421/biomedich.2017.61.19-36 issn 2540-9328 (online) checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul: confirmation and update of flora of java and apg iii widodo1, muhammad jafar luthfi2 1,2biological education department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto 55281 yogyakarta, indonesia author correspondency: wwidodo594@gmail.com1 abstract this study aimed to collect data on the species plants in mount nglanggeran, to confirm and update the existence of these plants from flora of java book by backer & bakhuizen, and to showing the visual data of plant species in nglanggeran mountain. this research used survey method through in-situ visit and specimen collection. monitoring and visits were conducted based on prediction of flowering period and fruit-bearing season. eighty plant families of flowering plants were found in the mount nglanggeran. based on the phylogenetic arrangement of flowering plants it was found that all the main groups (clades) of flowering plants were found at this location. keywords: checklist; magnoliophyta; update plant list; mount nglanggeran; local plant. introduction non-cultivated plants in java, especially in yogyakarta tends to be neglected by young generations. the plants left as ornamental plants, wild plant on waste lands or protected places such as in protected forests, cemeteries, etc. more often unknown plant identified only by its scientific name and preserved in herbarium centers in europe and discussed in old books in the past but the current distribution is almost no longer updated. mount nglanggeran area is now an attractive tourist destination. mount nglanggeran is the remnant of ancient volcano with almost parallel magmatic rock area. this area is uninhabited and is a protected area. the status of such area cause the protection of different types of local and wild plants that are no longer in residential areas. in previous research, the author has found plant species that can only be traced from old herbaria and old manuscript sources. publications and data of such plants are very rare. flora of java (backer & bakhuizen, 1962-70) and flora of british india (hooker, 1885) are important manuscripts as guidance for plant identification in java, indonesia, and southeast asia in general. not all local plants can be identified and listed in this book, but through comparison based on the description of the family and genus levels can lead identification to other books and herbaria-herbarium that have been published. the greatest disadvantage of these books is that they are filled with verbal descriptions with no illustrations, making it very difficult to identify even though the plant data is relatively complete. the identification process of plants in mount nglanggeran is relatively difficult and requires a lot of data. often the identity of plant species is known for many years after the observation done. preliminary data from author showed that the mount nglanggeran plant represents nearly 230 flowering plant families in the flora of java (1962-70) from about 560 flowering plant families in the world (takhtajan, 2008) . based on the above description, it is important to document the nglanggeran plants and systematically arranged the hierarchy of plant families. this systematic visual documentation is useful to help the use of flora of java book, to explore and introducing the richness of plants in mount nglanggeran, and revealing the vegetation data that is no longer recognized by people. this paper aimed to show the data of families of plant species in mount nglanggeran, to confirm the existence of the plants in flora of java by backer & bakhuizen, and showing visual photograph data of plant species in mount nglanggeran mountain. materials and methods this researchs were field and literature research. field research was survey method (singh, 2010. collection was done by sampling specimen for herbaria and photograping herbarium sampling with attention on the sustainability of plant population. monitoring and visits are conducted with consideration of the prediction of the period inflorescence and fruit formation. http://dx.doi.org/10.14421/biomedich.2017.61.19-36 20 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 equipments and materials equipments for observation and collection consist of: sony nex f3 digital camera, sony cyber-shot dscw180 digital camera, canon dslr digital camera, glass slide, micrometer, slide length, small roll meter, plastic collection, scissors, cutter, gps (global positioning system), dry herbarium collection kit, flakon bottle, nikon smz 1500 stereo microscope equipped with camera, nikon eclipse 50 light microscope equipped with nikon dsf1 camera. materials for observation and collection consist of: aquadest, alcohol 70%, faa solution (formalin acetic alchohol). work procedure the working procedures were as follows: photograping and observing of specimen in situ, herbarium, and flower/fruit. the data were compared to flora of java (backer & bakhuizen, 1963-70) and other existing literature, checking and matching with herbarium types and illustrations/drawings in the literature to identify the specimen. results and discussion flora of java book by backer & bakhuizen (1963-1968) describes spermatophyta found in java. description is an explanation of important character of plants or groups of plants in the category of family, genus, and species. the pattern of explanation of the characteristics of the plant begins with the description of the characteristics of flowers followed by vegetative characteristics. flora of java book by backer & bakhuizen (19631968) consists of volumes 1, 2, 3. volumes 1 and 2 contain descriptions of dicotyle plants consisting of 190 families. volume 3 contains descriptions of monocotyle containing 48 families. so the total family of plants in flora of java is 238 families. this book contains 2885 genera on volume 1, 2199 genera on volume 2 and 2018 genera on volume 3, or a total of 7112 genera. this book is very useful for the identification of plants found in yards, gardens, fields, and forests. the main obstacles to the use and limitations of this book are the absence of visual illustrations and requirement of flower as main character for identification discovery of interest and its characteristics in the identification process. wild and unknown vegetation in forest areas which is rarely blooming at observation, could be very difficult to identify without the appearance of flowers and fruit. from this study it is found that the diversity of plant species in the mount nglanggeran are 80 families of seed plants from 238 families in the book flora of java (backer and bakhuizen, 1963-1968) or around 33, 19%. details of the percentage of family are 32 out of 110 families in the book volume 1 (29.09%); 33 of the 80 families in the book volume 2 (41.25%); 15 families of 48 families in the book volume 3 (31.25%). the existence of flowering plants genera in mount nglanggeran are 265 genera of 7112 genera in flora of java (backer and bakhuizen, 1963-1965) or 3.73% range. detail of genera percentage are 108 genera of 2885 genera volume 1 (3.74%); 105 of 2199 genera in volume 2 (4, 74%) and 51 of 2018 genera in volume 3 (2.51%). flowering plant is a group of plants that dominate the earth today. seed plants in old taxonomic terms are manifestations of the spermatophyta class, consisting of subclass gymnospermae and angiospermae (flowering plant). in the book backer & bakhuizen (1963-1968), gymnospermae consist of 7 families whereas flowering plants consist of 231 families. table 1. percentage of representation of each category of plant classification in mount nglanggeran. no. clade number of orders order representation in mount nglanggeran number of family family representation in mount nglanggeran 1 unrank 4 0 7 0 2 magnoliids 4 3 or 75 % 20 4 or 20 % 3 monocot commeliniids 5 4 or 80 % 31 7 or 22,58% 4 monocot non commeliniids 7 5 or 71,42 % 46 9 or 19,56 % 5 eudicot unrank 6 1 or 16,67 % 15 2 or 13,33 % 6 core eudicot rosids fabids 8 7 or 87,5 % 71 14 or 19,71 % 7 core eudicot rosids malvids 6 4 or 66,67 % 59 13 or 22,03 % 8 core eudicot unrank 7 6 or 85,71 % 87 15 or 13,21 % 9 core eudicot asterids lamids 5 4 or 80 % 40 14 or 35 % 10 core eudicot asterids campanulids 7 2 or 28,57 % 27 4 or 14,81 % widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 21 according to singh (2010), gymnospermae plants consist of 11 families covering 80 genera, whereas flowering plants consist of 485 families covering 13,372 genera including 253,000 species (10,760 genera, 196,990 species dikotil, 2,612 genera, 56,310 monocots species). currently the reform of the categories of angiospermae plant classification (flowering plants) is carried out by the apg association (angiospermae phylogeny group) (apg iii, 2009). flowering plants comprised about 62 orders that included about 410 families. table 2 shows the checklist of the existence of plant species in mount nglanggeran following the family order based on apg iii (2009). table 1 shows the proportions of each category. from table 1 it is found that all the major flowering plant clusters are in nglanggeran mountain, except for basal groups of mangnoliids which are present only in certain regions of the world. based on the percentage of representation of the order, it is found that the plants in nglanggeran mountain were mainly clade core eudicot rosiid fabids group followed by core eudicot unrank, core eudicot asterids lamids, monocot commeliniids. based on the percentage of representation of existing families, it is found that the composition of plants in mount nglanggeran is mainly from cie eudicot unrank clade, core eudicot rosiid fabids, eudicot asterids lamiids, core eudicot rosids malvids. the representation of all the major clusters of flowering plants in mount nglanggeran shows that this location is an important site for continuous researchs. visual data in the form of specimens, photographs, or living plants in situ can be utilized to introduce more easily to the people about the diversity of flowering plants. detailed photographs or plant images need to be prepared for this step. description of plant character of flora of java should be supported by visual data and real specimens in relation to the importance of identifying species for various purposes. the visual data of plants in mount nglanggeran obtained in this study should be arranged systematically to complement and facilitate the use of the book. preparation of hierarchy of clade, order, family of flowering plants according to apg iii is done to update how to study plant diversity and its identification. conclusion it was found 80 families of flowering plants in the mount nglanggeran. based on the phylogenetic arrangement of flowering plants it was found that all the main groups (clades) of flowering plants were found at this location. acknowledgements the authors wish to thank the society for research and community service uin sunan kalijaga who funded this research, sugeng handoko as the manager of ecotourism area nglanggeran gunungkidul mountain yogyakarta, bayu setya aji, rendi yuntara and didik zulfahmi akbar. references backer, c. a. & bakhuizen. 1963. flora of jawa (spermatophytes only). vol i, ii, iii. groningen: n. v. p. noordhoff. birgitta bremer, kåre bremer, mark w. chase, michael f. fay, james l. reveal, douglas e. soltis, pamela s. soltis and peter f. stevens. 2009. an update of the angiosperm phylogeny group classification for the orders and families of flowering plants: apg iii. botanical journal of the linnean society, 2009, 161, 105–121. cbd (convention on biological diversity). 2002. strategi global konservasi tumbuhan (translated). bogor: botanic garden conservation international. heim e. 2015. flora and vegetation of bali indonesia. herstellung und verlag, norderstedt. hooker, j. d. 1885. flora of british india (vol. i, ii, iii, iv). london: reeve and co. keng h. 1990. the consist flora of singapore. singapore university press, singapore. knowles, b. 2008. systematics and taxonomy response to the house of lord science and technology committee. united kingdom: bioscience federation. ng, f. s. p. 1972-73. tree flora of malaya vol. 3. syarikat seng teik, selangor. quattrocchi, u. 2012. crc word dictionary of medicinal and poisonous plants. comon names, scientific names, eponyms, synonims and etymology. crc press, london randler, c. 2008. teaching species identification, a prerequisite for learning biodiversity and understanding ecology. eurasia journal of mathematics, science & technology education, 4 (3):223-231. randler, c., and bogner, f. x. 2006. cognitive achievement in identification skills. journal of biology education, 40 (4): 161-165. rustaman, n. y. 2006. arah pembelajaran taksonomi tumbuhan dan assesmennya di lptk dan sekolah (online), http:/www.file.upi.edu), retrieved at 23 june 2012. simpson, m.g. 2006. plant systematics. amsterdam: elsevier academic press. singh, g. 2010. plant systematics. new hampshire: science publisher. takhtajan, a. 2009. flowering plant. st petersburg: springer. 22 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 table family plant list (backer & bakhuizen, 1962) and list of seed plants in mount nglanggeran. no. family availability in location estimation of number species name availability in location estimation of number species name 1 magnoliaceae available 1 magnolia alba 2 schisandraceae 3 annonaceae available 6 uvaria rufa anomianthus dulcis meiogyne sp polyaulax sp annona muricata annona squamosa available 2 stelechocarpus burahol polyaltia sp 4 lauraceae available 2 litsea chinensis available 1 persea americana 5 hernandiaceae 6 myristicaceae available 1 myristica sp 7 ranunculaceae available 1 clematis javana 8 ceratophyllaceae 9 nymphaeaceae 10 berberidacaceae 11 menispermaceae available 6 pycnarhena montana arcangelisia sp tinospora coriacea stephania hernandifolia cissampelos sp pericampylus glaucus cyclea barbata available 1 tinospora crispa 12 aristolochiaceae available 1 aristolochia indica 13 raflesiaceae 14 nepenthaceae 15 piperaceae available 7 piper bettle piper retrofractum piper nigrum piper aduncum peperomia pelucida peperomia sp piper sp 24 saururaceae 25 chloranthaceae 26 papaveraceae 27 fumariaceae 28 turneraceae piriqueta racemosa turnera ulmifolia 29 loasaceae 30 capparaceae available 5 capparis micracantha capparis pyrifolia capparis sepiaria cleome rutidosperma gynandropsis gynandara cleome sp gynandropsis sp 31 moringaceae moringa oleifera 32 brassicaceae 33 violaceae 34 resedaceae 35 polygalaceae 2 polygala paniculata polygala glomerata 36 crassulaceae kalanchoe pinnata 37 saxifragaceae 38 droseraceae 39 podostemataceae 40 elatinaceae 41 caryophyllaceae 42 molluginaceae 1 mollugo pentaphylla glinus lotoides (bawukan berbulu) glinus oppositifolius (bawukan licin) 43 ficoidaceae trianthema portulacastrum 44 portulacaceae 1 portulaca oleracea widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 23 talinum paniculatum talinum fruticosum famili baru: talinaceae 45 polygonaceae polygonum orientale polygonum chinense polygonum barbatum antigonon leptopus muehlenbeckia platyclada 46 phytolaccaceae rivinia humilis 47 chenopodiaceae 48 amaranthaceae celosia argentea amaranthus hybridus amaranthus gracilis amaranthus spinosus cyathula prostrata aerva sanguinolenta achyranthea aspera alternanthera sessilis alternanthera phyloxeroides gomphrena celosioides 49 basellaceae basella rubra anredera cordifolia 50 linaceae 51 zygophyllaceae 52 geraniaceae 53 oxalidaceae 3 oxalis barrelieri oxalis corniculata biophytum reinwardtii averrhoa carambola averrhoa bilimbi 54 tropaeolaceae 55 balsaminaceae 1 impatien platypetala 56 lythraceae 1 lawsonia inermis lagerstroemia indica lagerstroemia speciosa cuphea hyssopifolia 57 crypteroniaceae 58 sonneratiaceae 59 punicaceae punica granatum (famili lythraceae) 60 onagraceae 61 trapaceae 62 haloragaceae 63 callitrichaceae 64 thymelaeaceae 1 phaleria octandra phaleria macrocarpa 65 nyctaginaceae 3 mirabilis jalapa boerhavia erecta bougainvillea spectabili 66 proteaceae 67 dilleniaceae 1 tetracera scandens 68 pittosporaceae 69 bixaceae bixa orellana 70 cochlospermaceae 71 flacourtiaceae (salicaceae) 1 flacourtia indica 72 tamaricaceae 73 passifloraceae 2 passiflora foetida passiflora edulis passiflora suberosa passiflora vitifolia passiflora quadrangularis 74 cucurbitaceae 3 momordica charantia trichosanthes villosa trichosanthes tricuspidata citrulus vulgaris cucumis sativus luffa acutangula cucurbita moschata coccinia grandis sechium edule benincasa hispida 75 begoniaceae 76 datiscaceae 77 caricaceae carica papaya 78 cactaceae pereskia sp nopalea sp 24 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 opuntia sp cereus sp hylocereus sp (buah naga) epyphyllum sp (wijayakusuma) 79 theaceae 80 actinidaceaea 81 saurauiaceae 82 ocnaceae 83 dipterocarpaceae 84 myrtaceae 4 psidium guajava zyzygium cumini zyzigium polyanthum zyzigium javanicum zyzygium aromaticum zyzigium malaccensis melaleuca leucadendron callistemon sp 85 lecythidaceae 1 barringtonia asiatica barringtonia racemosa barringtonia asiatica 86 melastomataceae 3 melastoma malabathricum osbeckia chinensis memecylon caeruleum 87 combretaceae 1 terminalia catappa quisqualis indikca (srigading) 88 rhizophoraceae 89 hyericaceae 90 clusiaceae 2 calophyllum inophyllum garcinia mangostana 91 tiliaceae triumfetta indica schoutenia ovata mutingia calabura corchorus acutangulus 92 elaeocarpaceae 1 mutingia calabura 93 gonystylaceae 94 sterculiaceae 1 helicteres hirsuta helicteres isora 95 bombacaceae ceiba petandra durio zibethinus bombax ceiba 96 malvaceae 8 sida cordata triumfetta indica abutilon crispum abutilon hirtum wissadula periplocifolia sida rhombifolia sida acuta hisbiscus surattensis abelmoschus moschatus corchorus acutangulus melochia corchorifolia waltheria americana hisbiscus rosa-sinensia hisbiscus tiliaceus malvaviscus arboreus 97 malpighiaceae 98 erythroxylaceae 99 euphorbiaceae 20 glochidion eriocarpum glochidion puberum glochidion rubrum breynia oblongifolia phyllanthus muriculatus phyllanthus reticulatus phyllanthus emblica phyllanthus niruri sauropus androgynus bridelia micrantha bridelia stipularis croton hyrtus acalypha indica acalypha boehmerioides jatropha gossypifolia jatropha multifida euphorbia hirta euphorbia prostrata euphorbia heterophylla manihot esculenta manihot glaziovii hevea brasiliensis croton variegatus acalypha wilkesiana ricinus communis jatropha curcas codiaeum variegatum pedilanthus variegatus 100 daphniphyllaceae 101 cunoniaceae 102 escalloniaceae 103 hydrangeaceae widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 25 104 rosaceae available 1 rubus moluccanus 105 dichapetalaceae 106 caesalpiniaceae available 3 cassia siamea cassia occidentalis cassia obtusifolia 107 mimosaceae available 8 albizia montana albizia lebbeck albizia procera leucaena glauca mimosa pdica mimosa invisa acacia auriculiformis parkia speciosa 108 papilionaceae available 15 crotalaria usaramoensis crotalaria striata indiofera sumatrana desmodium pulchellum desmodium gangeticum desmodium triflorum alysicarpus nummularifolius uraria crinita uraria logopoides abrus precatorius centrosema pubescens mucuna pruriens flemingia strobilifera alysicarpus sp gliricidia sepium 109 hamamelidaceae 110 buxaceae 111 salicaceae flacuortia indica 112 myricaceae 113 betulaceae 114 fagaceae 115 casuarinaceae casuarina junghuhnia casuarina equisetifolia 116 ulmaceae 117 moraceae available 16 fatoua sp morus sp malaisa scandens streblus asper streblus taxoides maclura cochinchinensis ficus benyamina ficus septica ficus montana artocarpus integra poikilospermum suaveolens 118 urticaceaea available laportea sp fleurya sp pilea microphyla pouzolzia zeylanica boehmeria sp 119 cannabaceae 120 aquifoliaceae 121 celastraceae available 1 celastrus scandens 122 hippocrateaceae 123 icacinaceae 124 salvadoraceae 125 olacaceae available 1 olax scandes 126 opiliaceae 127 loranthaceae available 1 elythranthe sp 128 santalaceae available 1 santalum album 129 balanophoraceae 130 rhamnaceaea available 2 zizyphus oenoplia 131 elaeagnaceae 132 vitaceae available 7 vitis discolor tetrastigma 26 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 leucostaphylum cissus repens cayratia trifolia leea aequata leea rubra 133 rutaceae 5 glycosmis petaphylla murraya paniculata clausena excavata aegle marmelos zanthoxylum sp 134 simarubaceae available 1 brucea javanica 135 burseraceae 136 meliaceae available 2 swietenia mahagoni chisocheton sp lansium domesticum melia azedarach dysoxylum sp. 137 sapindaceae available 3 cardiospermum halicacabum allophylus cobbe erioglossum rubiginosum shcleicera oleosa euphoria longana nephelium lappaceum pometia pinnata filicium decipiens 138 aceraceae 139 sabiaceae 140 staphyleaceaea 141 anacardiaceae available 4 anacardium occidentale mangifera indica mangifera odorata gluta renghas spondias dulcis lannea coromandeca 142 connaraceae 143 juglandaceae 144 cornaceae 145 alangiaceae 146 nyssaceae 147 araliaceae available 1 schefflera sp nothopanax scutellarium polyscias sp arthrophyllum sp 148 apiaceae available 2 centela asiatica eringium foetidum hydrocotyle sp 149 clethraceae 150 ericaceaea 151 vacciniaceae 152 epacridaceae 153 ebenaceae available 1 diospyros truncata 154 sapotaceae crysophyllum cainito mimusops elingi manilkara kauki manilkara achras 155 myrisinaceae available 2 ardisia humilis ardisia crenata 156 styracaceae 157 symplocaceae 158 loganiaceae available 2 spigelia althemia fagraea ceilanica 159 oleaceae available 1 jasminum pubescens 160 apocynaceae available 8 alstonia sholaris alstonia angustiloba rauvolfia verticilata anodendron paniculatum chonemorpha fragran ichnocarpus frutescens tabernaemontana macrocarpa wrightia pubescens 161 asclepiadaceae available 6 cryptolepis sinensis calotropis gigantea hoya sp marsdenia brunoniana telosma puberula cosmostigma racemosum cynanchum sp marsdenia tenacissima gymnema sylvestris asterostemma repandum 162 rubiaceae available 8 hedyotis corymbosa widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 27 ophiorrhiza mungos nauclea orientalis musaenda frondosa pavetta indica psychotria sp paederia scandens vangueria spinosa-meyna grisea 163 capriofoliaceae 164 valerianaceae 165 dipsaceae 166 asteraceae available 14 vernonia cinerea elephantopus scaber pseudoelephantopus spicatus ageratum conyzoides euphatorium inulifolium erigeron sumatrensis eclipta prostrata wedelia montana wedelia biflora synedrella nodiflora bidens biternata tridax procumbens emilia sonchifolia 167 gentianaceae available 1 isotoma longiflora 168 primulaceae 169 plumbaginaceae available 1 plumbago zeylanica 170 plantaginaceae 171 campanulaceae 172 sphenocleaceae 173 lobeliaceae 174 goodeniaceae 175 stylidiaceae 176 polemoniaceae 177 hydrophyllaceae 178 boraginaceae available 2 ehretia microphylla heliotropium indicum 179 solanaceae 4 physalis minima solanum torvum solanum comitis solanum nigrum 180 convolvulaceae available 5 merremia hastata argyreia mollis 181 scrophulariaceae available 3 lingdernia crustacea scoparia dulcis 182 orobanchaceae 183 lentibulariaceaea 184 gesneriaceae available 1 epithema horsfieldii 185 bignoniaceae available 2 oroxylum indicum crescentia cujete 186 pedaliaceae 187 acanthaceae available 6 thunbergia fragrans andrographis paniculata ruelia napifera strobilanthes crispus asystasia gangetica 188 myoporaceae 189 verbenaceae available 9 tectona grandis lantana camara stachytarpeta jamaicensis vitex sp cleroderdrum serratum clerodendrum inerme vitex sp duranta erecta premna odorata 190 lamiaceae available 4 leucas lavandulifolia salvia riparia 28 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 hyptis rhomboides hyptis suaveolens 191 butomaceae 192 hydrocharitaceae 193 alismataceaea 194 triuridaceae 195 aponogetonaceae 196 potamogetonaceae 197 ruppiaceae 198 zannichelliaceae 199 najadaceae 200 commelinaceae 201 flagellariaceae available 1 fragellaria indica 201 xyridaceae 203 eriocaulaceae 204 bromelliaceae 205 musaceae 206 strelitziaceae 207 zingiberaceae available 5 zingiber cassumunar zingiber zerumber costus speciosus curcuma sp 208 cannaceae 209 marantaceae 210 liliaceae available 1 gloriosa superba 211 tecophilaeacea 212 pontederiaceae 213 smilacaceae available 1 smilax sp 214 philesiaceae 215 araceae available 7 pothos scandens amorphophalullus variabilis alocasia crassifolia typhonium trilobatum 216 lemnaceae 217 typhaceae 218 amaryllidaceae 219 iridaceae 220 roxburghiaceae 221 dioscoreaceae available 6 dioscorea alata dioscorea bulbifera dioscorea aculeata dioscorea pentaphylla dioscorea oppositifolia dioscorea hispida 222 xanthorrhoeaceae 223 agavaceae available 1 agave cantala 224 arecaceae available 1 arenga pinnata 225 pandanaceae available 1 pandanus houlletii 226 cyclanthaceae 227 haemodoraceae 228 hypoxidaceae available 2 curculigo latifolia hypoxis aurea 229 velloziaceae 230 apostasiaceae 331 taccaceae available 1 tacca palmata 332 philydraceae 233 . burmaniaceae 234 thismiaceae 235 orchidaceae available 6 pecteilis susannae liparis sp 236 juncaceaea 237 cyperaceae available 4 scleria laevis widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 29 238 poaceae available 14 imperata cylindrica (alang-alang) pollinia ciliata polytrias amaura (rumput lamuran) pogonatherum paniceum (rumput wesen) andropogon aciculatus (rumput jarum) themeda arguens (rumput merak) oplesmenus compositus setaria sp axonopphus compressus anastrophus compressus table checklist of exist plant species in mount nglanggeran by clade, order, family as suggested by apg iii (2009). clade order family exsistence in mount nglanggeran amborellales amborellaceae nymphaeales cabombaceae hydatellaceae nymphaeaceae austrobaileyales austrobaileyaceae schisandraceae+illiciaceae trimeniaceae cloranthales chloranthaceae magnoliids piperales aristolochiaceae v hydnoraceae lactoridaceae piperaceae v saururaceae canellales canellaceae winteraceae magnoliales annonaceae v deneriaceae eupomatiaceae himantandraceae magnoliaceae mytisticaceae laurales atherospermataceae calycanthaceae gomortegaceae hernandiaceae lauraceae v monimiaceae siparunaceae monocot commelinids commelinales commelinaceae v haemodoraceae hanguanaceae philydraceae pontederiaceae zingiberales cannaceae costaceae v heliconiaceae lowiaceae marantaceae musaceae 30 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 sterlitziaceae zingiberaceae v poales anarthriaceae bromeliaceae centrolepidaceae cyperaceae v ecdeiocaulaceae eriocaulaceae flagellariaceae v joinvilleaceae juncaceae mayacaceae poaceae v rapateaceae restionaceae thurniaceae typhaceae+sparganiaceae xyridaceae arecales arecaceae v dasypogonaceae dasypogonaceae asparagales amaryllidaceae++alliaceae asparagaceae+agaveaceae v asteliaceae blandfordiaceae boryaceae doryanthaceae hypoxidaceae v iridaceae ixioliriaceae lanariaceae orchidaceae v tecophilaeaceae xanthorrhoeaceae xeronemataceae liliales alstroemeriaceae campynemataceae colchicaceae v corsiaceae liliaceae v melanthiaceae petermanniaceae philesiaceae ripogonaceae smilacaceae v pandanales cyclanthaceae pandanaceae v stemonaceae triuridaceae velloziaceae dioscoreales burmanniaceae dioscoreaceae v nartheciaceae petrosaviales petrosaviaceae alismataless alismataceae+limnocharitaceae aponogetonaceae araceae v butomaceae widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 31 cymodoceaceae hydrocharitaceae jncaginaceae posidoniaceae potamogetonaceae ruppiaceae scheuchzeriaceae tofieldiaceae zosteraceae acorales acoraceae eudicot ceratophyllales ceratophyllaceae ranunculales berberidaceae circaeasteraceae eupteleaceae lazirdabalaceae menispermaceae v papaveraceae+fumariaceae ranunculaceae v sabiales sabiaceae proteales nelumbonaceae platanaceae proteaceae buxales buxaceae haptanthaceae trochodendrales trochodendraceae core eudicot gunnerales guneraceae myrothamnaceae rodids fabids cucurbitales anisophyllaceae begoniaceae coriariaceae corynocarpaceae cucurbitaceae v datiscaceae tetramelaceae fagales betulaceae casuarinaceae fagceae juglandaceae myricaceae nothofagaceae ticodendraceae rosales berbeyaceae cannabaceae dirachmaceae elaeagnaceae moraceae v rhamnaceae v rosaceae v ulmaceae urticaceae v fabales fabaceae v polygalaceae v quillajaceae surianaceae celastrales ceastraceae v lepidobotriaceae oxaidales brunelliaceae 32 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 cephalotaceae conaraceae cunoniaceae elaeocarpaceae huaceae oxalidaceae v malpighiales achariaceae balanoporaceae bonnetiaceae calophyllaceae caryocaraceae centroplacaceae chrysobalanaceae clusiaceae ctenolophonaceae dichapetalaceae erytrocylaceae euphorbiaceae v goupiaceae humiriaceae hypericaceae irvingiaceae ixonanthaceae lacistemataceae linaceae lophopixidaceae malphigiaceae ochnaceae+medusaginaceae pandaceae passifloraceae+turneraceae v phyllantaceae v picrodendraceae podostemaceae putranjvaceae rafflesiaceae rhiosporaceae salicaceae v trigoniaceae violaceae zygophyllales krameriaceae zygophyllaceae v malvids malvales bixaceae cistaceae cytinaceae dipterocarpaceae malvaceae v mutingiaceae v neuradaceae sarcolaenaceae thymelaeaceae v sphaerosepalaceae brassicales akanaceae bataceae brassicaceae capparaceae v caricaceae cleomaceae widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 33 emblingiaceae gyrostemonaceae koeberliniaceae limnanthaceae moringaceae pentadiplandraceae resedaceae salvadoraceae setchelanthaceae tovariaceae tropaeolaceae huerteales dipentodontaceae gerrardinaceae tapiscaceae sapindales anacardiaceae v bierbersteiniaceae burseraceae kirkiaceae meliaceae v nitrariaceae rutaceae v sapindaceae v simaroubaceae v picramniales paramniaceae crossosomatales aphloiaceae crossomataceae geissolomataceae guamatelataceae stachyuracaceae staphyleaceae strasburgeriaceae myrtales alzateaceae combretaceae v crypteroniaceae lythraceae v melastomataceae v myrtaceae v penaeaceae vochysiaceae onagraceae geraniales geraniaceae melianthaceae vivianiaceae vitales vitaceae v saxifragales altingiaceae aphanopetalaceae cercidiphyllaceae crassulaceae v daphniphyllaceae cercidiphyllaceae haloragaceae hamamelidaceae iteaceae pterostemonaceae paeoniaceae penthoraceae peridiscaceae 34 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 saxifragaceae tetracarpaeaceae dilleniaceae dilleniaceae v berberidopsidales aextoxicaceae berberidopsidaceae santalales balanophoraceae loranthaceae v misodendraceae santalaceae v olacaceae v opiliaceae shcoepfiaceae caryophyllales achatocarpaceae aizoaceae amaranthaceae v anacampserotaceae ancistrocladaceae asteropeiaceae barbeuiaceae basellaceae cactaceae caryophyllaceae didiereaceae dioncophyllaceae droseraceae drossophyllaceae frankeniaceae gisekiaceae halophytaceae limeaceae lophiocarpaceae molluginaceae v montiaceae nepenthaceae nyctagynaceae v physenaceae phytolaccaceae plumbaginaceae v polygonaceae portulacaceae rhabdodendraceae sarcobataceae simondsiaceae stegnospermataceae talinaceae v tamaricaceae cornales cornaceae curtisiaceae grubbiaceae hydrangeaceae hydrostachyaceae loasaceae ericales actinidiaceae balsaminaceae v clethraceae cyrillaceae diapensiaceae widodo & luthfi – checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul … 35 ebenaceae v ericaceae fouquieriaceae lecythidaceae v maregravuaceae mitrastemnaceae pentaphylacaceae polemoniaceae primulaceae roridulaceae sapotaceae v sarraceniaceae sladeniaceae styracaceae symplocaceae tetrameristaceae theaceae asterids lamiids garryales eucommiaceae garryaceae gentianales apocynaceae v gelsemiaceae gentianaceae v loganiaceae v rubiaceae v lamiales acanthaceae v bignoiaceae v byblidaceae calceolariaceae carlemanniaceae gesneriaceae v lamiaceae v linderniaceae lentibulariaceae martyniaceae oleaceae v orobanhaceae paulowniaceae pedaliaceae phrymaceae plantaginaceae plocospermataceae schlegeliaceae scrophulariaceae v stilbaceae tetrachondraceae thomandersiaceae verbenaceae v solanales convolvulaceae v hydroleaceae montiniaceae solanaceae v sphenocleaceae boraginaceae boraginaceae v vahliaceae icacinaceae metteniusaceae oncotheaceae 36 biology, medicine, & natural product chemistry 6 (1), 2017: 19-36 campanulids aquifoliales aquifoliaceae cardiopteridaceae helwingiaceae phyllonomaceae stemonuraceae escalloniales escalloniaceae aterales alseuosmiaceae argophyllaceae asteraceae v calyceraceae campanulaceae+lobeliaceae v goodeniaceae menyanthaceae pentaphragmaceae phellinaceae rousseaceae stylidiaceae dipsacales adoxaceae capriofoliaceae+dipsacaceae+linnaeaceae+morinaceae+valerianaceae paracryphiales paracryphiaceae+quintiniaceae+sphenostemonaceae apiales apiaceae v araliaceae v griselniaceae myodocarpaceae pennatiaceae pittosporaceae torricelliaceae+aralidiaceae+melanophyllaceae bruniales bruniaceae checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul: confirmation and update of flora of java and apg iii biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 119-126 | doi: 10.14421/biomedich.2023.121.119-126 issn 2540-9328 (online) phytochemical, antimicrobial and cytotoxic activities of strophanthus sarmentosus dc julius leke abiola*, olapeju oluyemisi aiyelaagbe department of chemistry, faculty of science, university of ibadan. ibadan-200284, nigeria. corresponding author* abiolajulius005@gmail.com manuscript received: 22 november, 2022. revision accepted: 09 december, 2022. published: 12 january, 2023. abstract strophanthus sarmentosus dc is used traditionally in the management of snake-bite, arthritis, eye infection, rheumatism, emetic and venereal diseases. freshly collected mature strophanthus sarmentosus plant parts were air-dried at room temperature. each of the plant parts (leaf, stem and roots) was successively extracted by cold extraction method using hexane, ethyl acetate and methanol respectively. the crude extracts were subjected to phytochemical, antimicrobial and cytotoxicity analysis by employing chemical tests, agar diffusion and brine shrimps methods. the phytochemical screening showed the presence of tannins, saponins, glycosides, flavonoids, phenols, steroids, terpenoids and carbohydrates in all the extracts. the extracts demonstrated broad spectrum activities against both grampositive and gram-negative bacteria and the fungi tested. the mic and mmc of ethyl acetate and methanol extracts of the s. sarmentosus (stem) is between 0.3 and 5.0 mg/ml. the cytotoxic activity (lc50) of the s. sarmentosus extracts (leaf, stem and root) ranged between 117 µg/ml and 270 µg/ml, showing that the extracts are within the medium toxic level according to clarkson’s toxicity index. keywords: antimicrobial activity; cytotoxic activity; strophanthus sarmentos dc; apocyanaceae; phytochemicals. abbreviations: met; methanol, ea; ethyl-acetate, hex; hexane, dzi; diammeter of zone of inhibition, s. a. ; staphylococcus aureus, s. t.; staphylococcus typhimonium, b. s. ; bacillus subtilis, e. c. ; escherichia coli, k. p. ; klebsiella pneumonia, p. a. ; pseudomonas aeruginosa, a. n. ; aspergillus niger, c. a. ; candida albicans, g; gentamycin, k; ketoconazole, shlh; strophanthus hispidus leaves hexane, shle; strophanthus hispidus leaves ethyl acetate, shlm; strophanthus hispidus leaves methanol, shsh; strophanthus hispidus stem hexane, shse; strophanthus hispidus stem ethyl acetate, shsm; strophanthus hispidus stem methanol, lc; lethal concentration, cyclo p; cyclophosphamide. introduction plant extracts and essential oils are considered to be the potential sources of compounds with anticancer, antimicrobial and antioxidant properties. several medicinal plants and their bioactive components have been reported to be very important in the management of health through the regulation of biological processes (rahmani & aly, 2015). compounds like tannins, steroids, flavonoids and phenols possess strong biological properties and have found use in many fields, including medicine, pharmacy, food production, beauty care and agriculture (behidj-benyounes et al., 2014). infectious diseases are the world’s leading human and animal agents of death. the situation is further complicated by the rapid development of multi-drug resistance to available antimicrobial agents (alalor et al., 2012), thus, plants still remain the most effective and cheapest alternative sources of drugs for management of diseases. strophanthus sarmentosus dc (apocynaceae), is commonly found in tropical africa. many species of strophanthus possess anti-venom, anti-arthritis, antipyretic, emetics, anti-rheumatism and diuretic properties (onotu et al., 2014). one of the active constituents of strophanthus species is strophanthin which is used as a cardiac stimulant and is comparable to and recommended as a therapeutic substitute of digitalis (agbaje and ajidahun, 2011). bioassay-guided fractionation of an ethanol extract of strophanthus boivinii afforded six cardenolide glycosides-boivinides, as well as four known cardenolide glycosides, digitoxigenin 3-o-[ß-dglucopyrananosyl-(1,4)-α-l-acofriopyranoside], corotoxigenin 3-o-ß-d-boivinoside, 17 α-corotoxigenin 3-o-ß-d-sarmentoside, and uzarigenin 3-o-α-l rhamnoside. the structures of these compounds were elucidated by various 1d and 2d nmr techniques. all the compounds showed significant antiproliferative activity against the a2780 human ovarian cancer cell line, with boivinide a being the most active at ic50 of 0.17 mm (karkare et al., 2007). there is limited information in the literature on the biological activities and chemical constituents of https://doi.org/10.14421/biomedich.2023.121.119-126 120 biology, medicine, & natural product chemistry 12 (1), 2023: 119-126 strophanthus sarmentosus, hence, this study was aimed at investigating the phytochemical constituents, antimicrobial and cytotoxic activity of the leaf, stem and root extracts of the plant against some pathogenic bacteria and fungi as well as determine its cytotoxic level. material and methods plant collection and identification strophanthus sarmentosus (leaf, stem and root) was collected from the premises of forestry research institute, ibadan, oyo state, south-west, nigeria (7o39’11”n, 3o85’82”e). the plant samples were identified and authenticated by mr. d. p. o. esimekhuei at the herbarium of the botany department, university of ibadan and voucher specimen (uih 23178) of the plant was deposited at the herbarium of the department for further reference. plant preparation and extraction the plant samples were air-dried for two weeks and ground. the powdered plant materials were weighed and then soaked in the solvents for at least 72 hours. the extractions were carried out successively with n-hexane, ethyl acetate and methanol and the extracts were recovered by filtering the solvents and concentrating on a rotator evaporator at 40oc. the concentrated extracts were kept in the desiccators for further drying. phytochemical analysis the freshly prepared extracts were subjected to standard phytochemical tests to determine the chemical constituents such as tannins, alkaloids, flavonoids, glycosides, saponins and phenols (hetty manurung et al., 2019, labiad et al., 2017). test for glycosides: to 1 ml of the extract was added 2ml of acetic acid and then cooled in an ice bath at 40oc. to this mixture 1 ml of concentrated tetraoxosulphate (vi) acid (h2so4) was added dropwise. the formation of an oil layer on top of solution indicated the presence of glycosides. test for alkaloides: to 3 ml of the extract was added 1 ml of 1% hcl. this resulting mixture was then treated with few drops of meyer’s reagent. the appearance of a creamy white precipitate confirmed the presence of alkaloids. test for saponins: five drops of olive oil was added to 2ml of the plant extract and the mixture shaken vigorously. the formation of a stable emulsion indicated the presence of saponins (trease and evans, 2009) test for tannins: two drops of 5% fecl3was added to 1 ml of the plant extract. the appearance of a dirtygreen precipitate indicated the presence of tannins (trease and evans, 2009) test for flavonoids: to 1 ml of the extract was added 3 drops of ammonia solution (nh3) followed by 0.5 ml of concentrated hcl. the resultant pale brown colouration of the entire mixture indicated the presence of flavonoids. test for steroids and terpenoids: to 1 ml of the plant extract was added 1 ml of concentrated tetraoxosulphate (vi) acid (h2so4). a red coloration confirmed the presence of steroids (trease and evans, 2009). test organisms for antimicrobial assay the microorganisms used for the assay consists of three grampositive bacteria: staphylococcus aureus atcc 29213, staphylococcus typhimonium atcc 14028 and bacillus subtilis atcc 23775: three gramnegative bacteria: escherichia coli atcc 11175, klebsiella pneumonia atcc 700303, pseudomonas aeruginosa atcc 27853 and two fungi: aspergillus niger and candida albicans. they were obtained from the department of pharmaceutical microbiology, faculty of pharmacy, university of ibadan, nigeria. determination of antimicrobial activity antimicrobial activity of the plant extracts was evaluated by the cup plate agar diffusion method. bacterial cultures were adjusted to 0.5 mcfarland’s turbidity standard and inoculated onto mha (mueller hinton agar) plates (diameter 15 cm). cultures of candida albicans were suspended in sterile solution of 0.9% normal saline and the spores of the other filamentous fungi were suspended in tanguay buffer and then inoculated onto pda (potato dextrose agar) plates. a sterile cork borer was used to make wells of 6 mm diameter on the mha and pda. in each of the wells in the culture plates previously seeded with the test organisms, 100μl aliquots of extract dilutions reconstituted in minimum amount of solvent at concentrations of 50 and 100 mg/ml were applied. methanol (50%) was used as a negative control. wells containing 20μl aliquots of gentamicin (10 μg/ml), ketoconazole (1%) served as positive controls. bacterial cultures were incubated at 37oc for 24 hr while the filamentous fungal cultures were incubated at 30oc for 36 hr. after incubation, antimicrobial activity was determined by measurement of the diameter of the zones of inhibition. for all the extracts, the tests were carried out in triplicates (frempong et al., 2021). determination of minimum inhibitory concentration (mics) & minimum microbicidal concentration (mmcs) the minimum inhibitory concentration (mic) of the methanol, ethyl acetate and hexane extracts were determined for each of the test organisms in triplicate at concentrations of 0.3125, 0.625, 1.25, 2.5, and 5 mg/ml. to obtain these concentrations, 2ml of each of the dilution from 6.25, 12.5, 25, 50, 100 mg/ml of extracts were seeded into 18 ml of molten mha to achieve abiola & aiyelaagbe – biological activities of s. sarmentosus 121 concentrations of 0.3125, 0.625, 1.25, 2.5, and 5 mg/ml and poured into petri dishes. after the solidification of the aliquot (diluted extracts) and mha, different test micro-organisms (isolates) were streaked on the plates of different concentration. the procedure was repeated using the standards for the control. a petri dish containing nutrient broth only was seeded with the test organisms to serve as a negative control. petri dishes containing bacterial cultures were then incubated at 37oc for 24 hr while petri dishes containing fungal spore cultures were incubated at 30oc for 36 hr. after incubation the petri dishes were examined for microbial growth by observing for turbidity. to determine the minimum microbicidal concentration (mmc), which includes minimum bactericidal (mbc) and minimum fungicidal concentrations (mfc), a loopful of broth was collected from those petri dishes which did not show any growth in the mic determination and inoculated on sterile nutrient agar (na) for bacteria and sabouraud dextrose agar (sda) for fungi by streaking. to serve as a control, na and sda only were streaked with the respective test organisms. plates inoculated with bacteria were then incubated at 37oc for 24 hr, while those inoculated with fungi were incubated at 30oc for 36 hr. after incubation, the concentration at which no visible growth was seen was recorded as the mbc and mfc respectively, (taiwo et al., 2022). cytotoxicity assay brine shrimp lethality bioassay was carried out to investigate the cytotoxicity of methanol, ethyl acetate and hexane extracts of strophanthus sarmentosus. brine shrimps (artemia salina) were hatched using brine eggs in a vessel filled with simulated sterile artificial sea water (brine solution) made up of sea salt (38g) in 1000ml of distilled water with the ph adjusted to 8.5 using 1 n naoh under constant aeration for 48hr. the active shrimps were collected and used for the assay (krishnaraju et al., 2005, osamudiamen et al., 2020). brine solution (4.5 ml) was taken into each test tube. suitable dilution of the extracts was made to give concentration from 1000, 500, 250, 125 and 62.5 µg/ml. the 0.5 ml of diluted test solution was added to each of the test tubes. ten shrimps were added into each test tube by drawing them with glass capillary tube. the surviving shrimps were counted after 24 hr and lethality concentration lc50 was assessed. the mortality endpoint of this bioassay is defined as the absence of controlled forward motion during 30 seconds of observation. the percentage lethality of the nauplii for each concentration and control was calculated (apu et al., 2012). % 𝐷𝑒𝑎𝑡ℎ = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓𝑑𝑒𝑎𝑑 𝑁𝑎𝑢𝑝𝑙𝑖𝑖 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑛𝑎𝑢𝑝𝑙𝑖𝑖 + 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑖𝑣𝑒 𝑛𝑎𝑢𝑝𝑙𝑖𝑖 × 100 toxicity testing criteria the toxicity of plant extracts of medicinal values expressed as lc50 values is commonly valorized either by comparison to meyer’s or to clarkson’s toxicity index. according to meyer’s toxicity index, substance with lc50<1000 µg/ml are considered as toxic, while substance with lc50>1000 µg/ml are considered as non-toxic (meyer et al., 1982). while clarkson’s toxicity criterion for the toxicity assessment of plant extracts are as follows, extract withlc50 above 1000 µg/ml are non-toxic, extract with lc50between 5001000 µg/ml are low toxic, extract with lc50between100-500 µg/ml are medium toxic, and extract with lc50between0-100 µg/ml are highly toxic (clarkson et al., 2004). figure 1. research summary. result and discussion phytochemical composition of the extracts the yield extracts for all the three plant parts (leaf, stem and root) are presented in table 1. the highest (142 g) and lowest (12.4 g) yields were recorded for leaf methanol and stem hexane extracts respectively. table 1. yield of extracts of strophanthus sarmentosus. leaves (1000 g) stem (1600 g) root (1600 g) extracts hex ea met hex ea met hex ea met weight (g) 15.46 36.36 142.45 12.43 32.46 47.71 20.84 20.91 72.62 yield (%) 1.546 3.636 14.245 0.777 2.029 2.982 1.303 1.307 4.539 122 biology, medicine, & natural product chemistry 12 (1), 2023: 119-126 the results for the phytochemical screening are presented in table 2. the extracts showed the presence of tannins, carbohydrates, saponins, steroids, glycosides, terpenoids, flavonoids and phenols. bioactive compounds like tannins, steroids, flavonoids and phenols have been reported to be used by plants for protection against bacterial and fungal infections and other pests, hence they may also be responsible for antimicrobial activity (falodun et al., 2006). table 2. phytochemical screening of strophanthus sarmentosus extracts. leaves stem root sslh ssle sslm sssh ssse sssm ssrh ssre ssrm flavonoids + - -+ - + + -+ saponins - -+ -- ++ + phenolics + + --+ + -+ ++ tannins + + --+ + -+ ++ carbohydrates + + + + + + + + + glycosides -+ + -+ + - + alkaloids + + + -+ + + ++ ++ steroids + + + + + + + + + terpenoids + + -+ + + + + - legend: sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosus leaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, +: mildly present, ++: highly present. antimicrobial activities the in-vitro antimicrobial activity of the strophanthus sarmentosus extracts (leaf, stem and root) are presented in table 3. the highest susceptible pathogenic microorganisms against the extracts were candida albicans, staphylococcus typhimonium, staphylococcus aureus and pseudomonas aeruginosa while the least susceptible were klebsiella pneumonia and bacillus subtillis. all the extracts demonstrated considerable activity against both gram negative and gram positive bacteria and the fungi tested. the mic and mmc of the extracts ranged between 0.3 and >5 mg/ml as shown in table 3. the stem methanol (sssm) and root ethyl acetate (ssre) extracts were the most active of all the extracts and their activity are comparable to the standard antimicrobial agents used (table 3). secondary metabolites present in plants have been reported to be responsible for their therapeutic activity (fabry et al.,1998). flavonoids and other phytochemical constituents of the plant also have antimicrobial properties and this is in agreement with singh and bhat, (2003), which reported that flavonoids are responsible for the antimicrobial activity associated with some ethnomedicinal plants. the results highlight the fact that the methanol and ethyl acetate extracts exhibited greater antimicrobial activity because the antimicrobial principles were either polar or moderately-polar compounds. this observation agrees with the report in the literature that organic solvents are more suitable for extraction of phytochemicals (singh and singh, 2000). low mic is an indication of high efficacy of the plant extract while high mic may indicate low efficacy or possible development of resistance by the microorganisms (shanmugam et al., 2008). the presence of glycosides and alkaloids in the plant extracts may be attributed to their use by traditional medicine practitioners in healthcare systems in the treatment of some bacterial infections such as, venereal diseases, and other diseases (onotu et al., 2014: agbaje and ajidahun, 2011). despite the significant progress made in the development of antimicrobial drugs and the control of microorganisms, high level of epidermics due to drug resistant microorganisms still pose an enormous threat to public health. thus, the use of medicinal plants for antimicrobial activities needs to be given more attention. abiola & aiyelaagbe – biological activities of s. sarmentosus 123 table 3. antimicrobial activities of extracts (dzi, mm) of strophanthus sarmentosus extracts. test pathogens / concentration (mg/ml) / zone of inhibition (mm) extracts s. a s. t b. s e. c k. p p a a.n c. a 100 50 100 50 100 50 100 50 100 50 100 50 100 50 100 50 sslh 12 10 14 12 16 12 ssle 12 10 12 10 14 12 16 14 14 12 16 14 sslm 10 10 12 10 14 10 10 10 18 16 14 10 18 16 sssh 14 10 14 12 10 16 12 10 10 14 10 18 12 16 14 ssse 16 12 12 10 12 10 14 10 12 10 14 12 14 12 14 12 sssm 14 12 18 14 16 14 18 16 14 12 20 14 20 14 18 16 ssrh 10 10 10 12 10 14 12 ssre 12 10 16 10 14 10 12 10 12 10 14 12 18 12 20 16 ssrm 12 14 12 14 10 12 10 14 10 16 12 met (50%) standards gent. 18 18 20 16 22 14 na na ket. na na na na na na 16 14 legend: dzi: diameter of zone of inhibition, sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosus leaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, -ve control (met 50%): methanol 50%, +ve control = gent.: gentamycin (10 µg/ml), ket.: ketoconazole (1%), na: not applicable, -= no zone of inhibition. table 4. minimum inhibitory concentration (mic) (mg/ml) of strophanthus sarmentosus extracts. extracts test pathogens s. a s. t b. s e. c k. p p. a a. n c. a sslh 0.625 >5.0 >5.0 1.3125 2.50 0.625 5.0 0.625 ssle 2.50 2.50 2.50 0.3125 2.5 2.5 2.5 2.5 sslm 2.50 2.50 5.0 0.3125 5.0 5.0 1.3 5.0 sssh 2.50 5.0 5.0 2.50 1.25 1.25 5.0 5.0 ssse 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 sssm 0.3125 0.625 0.3125 0.6125 <0.3125 <0.3125 0.3125 0.3125 ssrh 2.50 5.0 >5.0 1.25 1.25 0.3125 >5.0 2.5 ssre 1.25 2.50 5.0 1.25 1.25 1.25 5.0 0.3125 ssrm 2.50 5.0 >5.0 2.50 2.50 2.50 5.0 5.0 legend: sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosus leaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, table 5. minimum microbicidal concentration (mmc) (mg/ml) of strophanthus sarmentosus extracts. extracts test pathogens s. a s. t b. s e. c k. p p. a a. n c. a sslh 0.625 >5.0 >5.0 1.25 2.50 2.50 >5.0 >5.0 ssle 5.0 5.0 5.0 5.0 5.0 2.50 5.0 5.0 sslm 5.0 5.0 5.0 1.25 5.0 5.0 5.0 5.0 sssh 5.0 5.0 5.0 2.50 1.25 5.0 5.0 5.0 ssse 2.50 2.50 2.50 2.50 2.50 5.0 5.0 2.50 sssm 0.3125 5.0 0.3125 2.50 0.3125 0.625 0.625 1.25 ssrh 2.50 5.0 >5.0 5.0 2.50 1.25 >5.0 2.50 ssre 5.0 2.50 5.0 1.25 1.25 1.25 5.0 0.625 ssrm 5.0 5.0 >5.0 5.0 2.50 5.0 5.0 5.0 legend: sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosusleaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, 124 biology, medicine, & natural product chemistry 12 (1), 2023: 119-126 cytotoxic activities the cytotoxic activities of the plant extracts are presented in table 6. this study determined that the extent of lethality was proportional to the concentration of the extract. after 24 hr all the shrimps survived in the control and maximum mortalities were recorded at a concentration of 1000 µg/ml for all the extracts while at 62.5 µg/ml, zero percent death are recorded in the extracts except for stem hexane extract (sssh) and root ethyl acetate extract of (ssre) which were 24% and 27% respectively. however, it was observed that in higher concentrations of the extracts the shrimps were dying after 10 hr and most of all the shrimps died after 24 hr. also, the lc50is presented in table 7. all the extracts had lc50 values between 117 µg/ml and 270 µg/ml, which show that the lc50 of all the extracts are within the medium toxic level according to clarkson’s toxicity index (clarkson et al., 2004). the presence of alkaloids, tannins and flavonoids could be responsible for their cytotoxic properties (osamudiamen et al., 2020). however, the standard drug, cylophosphamide used for the treatment of cancer diseases, had a lc50 value of 64 µg/ml. thus, the plant extracts demonstrated moderate cytotoxic activities. table 6. cytotoxic activity of strophanthus sarmentosus against nauplii. plant extracts concentrat ion (µg/ml) number of surviving nauplii (after 24 hr) t1 t2 t3 total number of nauplii survivors % mortality lc50 (µg/ml) sslh 62.5 125 250 500 1000 9 8 5 3 1 10 7 6 4 0 9 7 6 5 2 28 22 17 12 3 0±0.333 31.58±0.33 73.68±0.89 84.21±0.58 100±0.58 176.1±0.54 ssle 62.5 125 250 500 1000 9 8 4 4 2 10 9 5 3 2 9 7 6 5 2 28 24 15 12 6 0±0.333 19.05±0.58 61.91±0.58 76.19±0.58 100±0.58 226.9±0.48 sslm 62.5 125 250 500 1000 7 5 3 1 0 7 6 3 2 1 8 6 3 1 1 22 17 9 4 2 0±0.333 23.81±0.33 66.67±0.33 90.48±0.00 100±0.33 195.7±0.27 sssh 62.5 125 250 500 1000 8 7 5 4 0 9 6 4 3 1 8 6 5 4 1 25 19 14 11 2 23.81±0.33 40±0.33 52.38±0.33 66.67±0.33 100±0.33 261.6±0.33 ssse 62.5 125 250 500 1000 10 5 3 1 0 9 5 3 1 0 9 6 4 2 1 28 16 10 4 1 0±0.33 40±0.33 68±0.33 88±0.33 100±0.33 170.9±0.27 sssm 62.5 125 250 500 1000 6 4 2 2 0 8 3 2 2 1 8 3 1 1 0 22 10 5 5 1 0±0.33 30.77±0.33 69.23±0.33 69.23±0.33 100±0.33 202.8±0.33 ssrh 62.5 125 250 500 1000 8 5 3 3 0 9 9 4 2 1 9 5 4 3 0 27 24 11 8 1 0±0.33 27.27±0.33 63.64±0.33 77.27±0.33 100±0.33 117.4±0.33 ssre 62.5 125 250 500 1000 7 6 4 3 0 8 5 2 2 1 9 7 4 2 1 24 18 10 7 2 27.27±0.58 42.11±0.58 63.64±0.67 77.27±0.33 100±0.33 209.5±0.50 abiola & aiyelaagbe – biological activities of s. sarmentosus 125 ssrm 62.5 125 250 500 1000 7 4 2 0 0 5 4 2 0 0 7 3 1 0 0 17 11 5 0 0 0±0.33 42.11±0.58 73.68±0.33 100±0.33 100±0.33 153.5±0.27 standard cyclo p 62.5 125 250 500 1000 6 4 3 2 2 6 6 3 2 2 7 5 3 3 1 19 15 9 7 5 40±0.02 60±0.02 70.68±0.02 75±0.05 80±0.01 63.82±0.33 legend: sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosusleaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, cyclo: cyclophosphamide. table 7. cytotoxic activity (lc50) of strophanthus sarmentosus extracts. samples lc50 sslh 176.1±0.54 ssle 226.9±0.479 sslm 195.7±0.266 sssh 261.6±0.333 ssse 170.9±0.266 sssm 202.8±0.333 ssrh 117.4±0.333 ssre 209.5±0.497 ssrm 153.5±0.267 cyclo p 63.82±0.02 legend: sslh: strophanthus sarmentosus leaves hexane, ssle: strophanthus sarmentosus leaves ethyl acetate, sslm: strophanthus sarmentosus leaves methanol, sssh: strophanthus sarmentosus stem hexane, ssse: strophanthus sarmentosus stem ethyl acetate, sssm: strophanthus sarmentosus stem methanol, ssrh: strophanthus sarmentosus root hexane, ssre: strophanthus sarmentosus root ethyl acetate, ssrm: strophanthus sarmentosus root methanol, lc: lethal concentration, cyclop: cyclophosphamide conclusion the inhabitants of sub-sahara africa have been using numerous herbs for therapeutic purpose since time immemorial to cure all forms of diseases like diarrhea, jaundice, rheumatism, dyspepsia, asthma, diabetes, dysentery, gonorrhoea and skin infections. the current investigation evaluated for the first time antimicrobial and cytotoxic potentials of strophanthus sarmentosus. the plant extracts showed potent antimicrobial and moderate cytotoxic activities with ethyl acetate and methanol extracts of the stem and root of the plant displaying higher activities than the other extracts. these results authenticate the traditional use of strophanthus sarmentosus for the treatment of various diseases (onotu et al., 2014: agbaje and ajidahun, 2011). further studies to characterize the bioactive constituents of strophanthus sarmentosus responsible for its biological activities are in progress. conflict of interest: the authors declare that there is no conflict of interest. references agbaje, e.o., & ajidahun, o.a. 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(2009). trease and evans pharmacognosy (16th ed.). edinburgh; new york: saunders/elsevier. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 391-397 | doi: 10.14421/biomedich.2023.121.391-397 issn 2540-9328 (online) analysis of biological activities of two novel metal (ii) complexes of andrographis paniculata crude extract mary adelaide oladipo1, ayodele oluwabunmi ojo2, kayode taiwo ishola3,* 1department of pure & applied chemistry, ladoke akintola university of technology, ogbomoso, oyo state, nigeria. 2department of science laboratory technology, oyo state college of agriculture and technology, igboora, oyo state, nigeria 3department of chemistry, federal college of education (special), oyo, oyo state, nigeria. corresponding author* isholatk@gmail.com manuscript received: 13 february, 2023. revision accepted: 13 march, 2023. published: 24 june, 2023. abstract many diseases in nature have led to the death of many young and old. many bacteria have developed resistance to the available antibiotics on the market. and many drugs employed in treating many diseases such as diabetes mellitus are expensive and are not locally available. therefore, in order to search for more effective, inexpensive, and locally available drugs, this study synthesized and investigated the biological activities of andrographis paniculata crude extract and its co (ii) and ni (ii) complexes. the crude extract and synthesized complexes were characterized using a solubility test, infrared, and ultraviolet-visible spectroscopic analysis. their antibacterial potentials were investigated against two gram-negative bacteria (escherichia coli,staphylococcus aureus) and three gram-negative bacteria (proteus, klebsiella, pseudomonas) while their antidiabetic activities were examined against α-amylase and α-glucosidase enzymes. acarbose was employed as a standard drug. the crude extract and its metal complexes showed different degrees of solubility in the employed solvents. infrared analysis suggested coordination of the crude extract to the metal ions through the oxygen donor atom while the formation of the complexes was affirmed through the occurrence of d-d transitions in the visible region of the metal complexes. the metal complexes were found to display more antibacterial activity than the crude extract. co (ii) and ni (ii) complexes of the crude extract were found to exhibit better activities against α-amylase and α-glucosidase enzymes, respectively than the crude extract and acarbose. it is concluded that the metal complexes could be considered potential antibacterial and antidiabetic agents. keywords: anti-diabetes; anti-bacteria; medicinal plant; metal complexes. introduction high mortality in nature has been attributed to different diseases caused by bacteria, fungi, and viruses. diabetes mellitus described by high blood glucose levels due to insulin deficiency and/or ineffective insulin action is also one of the major deadly diseases that require serious attention. many drugs have been synthesized to combat the diseases. however, many pathogenic organisms have been reported to develop resistance to many of the available drugs while many of the drugs are not locally available. many drugs such as metformin, sitagliptin glimepiride, and acarbose have been widely applied in treating diabetes mellitus. however, these drugs are discovered to possess negative side effects on the patient’s health while some of them are expensive. therefore, a search for more effective and inexpensive antibiotics and antidiabetic drugs without side effects becomes imperative. medicinal plants have become a promising source of natural effective antimicrobial agents. these plants have been observed to possess many bioactive compounds capable of fighting different types of disease. medicinal plants have been widely applied for medical treatment as a result of their ability to exhibit known pharmacological actions for humans and animals. medicinal plants have been the basis of the basic treatment of diseases in many developing countries like africa. andrographis paniculata has been reported to be of great importance in the management of disease and infection. andrographis paniculata (figure 1) also known as ‘king of bitters” in yoruba is known as ‘mejemeje’ is one of the medicinal plants used in treating many diseases such as cancer,ulcer, malaria, and urinary tract infections (karmegam et al. 2015; sachin and kailasam, 2016). the plant has been reported to contain many active organic compounds such as kaempferol, andrographolide, 14-deoxy andrographolide 14deoxy11,12-didehydroandrographolide, quercetin, and other secondary metabolites (subramanian et al. 2008). andrographolide (figure 2) is found to be the main active component of the plant and its medical importance in treating different ailments has been examined by many researchers (flores et al., 2014; daneman & prat, 2015; tao et al. 2018; owoade et al. 2021) https://doi.org/10.14421/biomedich.2023.121.391-397 392 biology, medicine, & natural product chemistry 12 (1), 2023: 391-397 the antioxidant and anti-diabetic activity of andrographis paniculate investigated by reddy et al. (2022). the plant was found to possess good antioxidant and anti-diabetic activity. hartini et al. (2021) studied the inhibitory activity of aqueous extract and ethanolic extract of sambiloto andrographis paniculata against the α-amylase enzyme. the ethanol extract of the plant leaf demonstrated higher activity than that of the aqueous extract. the α-amylase enzyme inhibitory activity of andrographis paniculata in aqueous methanol, crude methanol extract, and n-hexane fraction was investigated by ajayi et al. (2021). the plant extract demonstrated more activity than the standard drug employed for the study. the crude methanol extract was observed to demonstrate the highest activity. the contribution of andrographis paniculata in the treatment of metastatic esophageal cancer was investigated by lin et al. (2017). the anti-migratory and suppressive effects on metastasisrelated factors of the absorbed andrographis paniculate were verified. the diterpenes and flavonoid components of the plant component were observed to display esophageal anticancer activity. advancement has been made in the field of medicinal inorganic chemistry in developing different novel organic therapeutic agents as pharmacological and pharmacy technical behaviors of many organic therapeutic agents are observed to increase upon coordination with transition metal ions (farrer and sadler, 2013; newman and cragg, 2016). therefore, there is an apparent need for the study of new metal complex of organic compounds endowed with antimicrobial activities which could be applied in combating multi drug-resistant microorganisms and other diseases. andrographis paniculata has been reported to be of great importance in the management of diseases. however, there is no report on biological activity of metal complex of the plant. therefore, this study synthesized metal (ii) complexes of andrographis paniculata crude extract and evaluated biological activity of the crude extract and its metal complexes against some bacteria and enzymes. figure 1. image of andrograhis paniculata plant. ch3 h ch2 o ch o ho ch2oh h3c ho ch2 figure 2. structure of andrographolide. materials and methods all chemicals used were of analytical grade and they include cobalt acetate, nickel acetate, sodium hydroxide, petroleum ether, hexane, ethanol, methanol, acetone, chloroform, tetra-chloro-methane, distilled water, positive gram strains (escherichia coli, staphylococcus aureus) and negative gram strains (pseudomonas, klebsiella, and proteus). collection and preparation of the plant crude extract the mature leaves of andrograhis paniculata were collected from oyo state college of agriculture, igboora, oyo state. the plant was identified by a botanist in the department of botany the university of ibadan, ibadan with the voucher number uih-23122. the collected leaves were rinsed twice under running water and then put in distilled water to get rid of dirt particles. the plant was air-dried at room temperature. it was then crushed into small particles and then ground into powder. the crude extract of the dried leaves was obtained in n-hexane at 60-80oc where oils, fats, waxes, and terpenes were removed. the extract was then subjected to soxhlet extraction with ethanol (95%) and the solution was concentrated using a rotary evaporator (mousumi et al. 2014). preparation of metal complexes solutions of 10 g of the crude extract and 5 g of cobalt salt in ethanol were mixed together and refluxed for about 4 hr. the mixture was then heated at 80 oc until a precipitate was formed. the precipitate was filtered and dehydrated under a vacuum. the same procedure was repeated for the nickel complex (mousumi et al. 2014). characterization of the metal complexes the metal complexes were characterized by solubility test solvents in water, ethanol, methanol, chloroform, acetone, and diethyl ether. ftir and uv spectroscopic analysis was carried out using a uv-visible spectrophotometer (ce 2021, cecil) and an ftir oladipo et al. – analysis of biological activities of two novel metal (ii) … 393 spectrophotometer (530m, buck) within the range of 600-4000cm-1. antibacterial assay whatman no 1 filter paper was used to prepare a 6 mm diameter disc. the 6 mm diameter discs were sterilized inside an autoclave at 121oc. the moisture discs were dried in a hot air oven at 50 oc. the crude extract and its metal complexes disc and control were prepared. the tests were carried out using the original technique of bauer et al (1996). muller-hinton agar was prepared and autoclaved at 15 bs pressure for 20 mins and cooled. the media was then poured into the sterilized petri dishes and allowed to solidify. the petri plate with the poured media was then seeded, after which the microbial was suspended with the aid of a sterile swab. the plant crude extract and its metal complexes were then placed on each of the plates as well as the control. the plates were then incubated at 37 oc for 24 hr. thereafter, the inhibition zone was measured and expressed in mm. effect of extracts on αamylase activity the extracts (100 μl) and 500 μl of 20 mm sodium phosphate buffer (ph 6.9 with 6 mm nacl) containing pancreatic α-amylase (ec 3.2.1.1) (0.5 mg/ml) were incubated at 25 °c for 10 min. then, 500 μl of 1% starch solution in the same phosphate buffer was added and incubated for another 10 min. 1.0 ml of dinitrosalicylic acid (dnsa) was added, boiled for 5 min, and cooled to room temperature. the reaction mixture was then diluted by adding distilled water (10 ml), and the absorbance of each sample was measured at 540 nm. a complete reaction mixture without acarbose or extract was used as the control. the α-amylase inhibitory activity is expressed as percentage inhibition and calculated using the formula; % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴𝑐 − 𝐴𝑠 𝐴𝑐 𝑋100 ac : absorbance of control (containing all reagents except extracts or acarbose) as : absorbance of the sample (extract or acarbose). four diluted solutions of the crude extract and its metal complexes (40 100 mg/l) were collected for the calculation of the ic50 values. the concentration of the extract required to inhibit the activity of the enzyme by 50% (ic50) was evaluated by ic50 aat calculator (www.aatbio.com). effects of extract on α – glucosidase activity in-vitro the effect of the extract on α – glucosidase activity was determined according to the procedure described by apostolid et al. (2007), using acarbose as a reference. the crude extract and its metal complexes (50 μl) and 100 μl of α-glucosidase solution were incubated at 25°c for 10 min. thereafter, 50 μl of 5 m p-nitrophenyl-α-dglucopyranoside solutions in 0.1 m phosphate buffer (ph 6.9) was added and incubated at 25 °c for 5 min. the absorbance was then read at 405 nm. the α-glucosidase inhibitory activity is expressed as percentage inhibition and calculated as shown below: % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴𝑐 − 𝐴𝑠 𝐴𝑐 𝑋100 ac : absorbance of control (containing all reagents except extracts or acarbose) as : absorbance of the extract or acarbose at least four serially diluted solutions of the crude extract and its metal complexes (40 100 mg/l) were taken for calculation of the ic50 values. the concentration of the extract required to inhibit the activity of the enzyme by 50% (ic50) was calculated using the aat calculator (www.aatbio.com). result and discussion the physical properties of andrograhis paniculata crude extract and its metal (ii) complex are shown in table 1 while tables 2 and 3 depict important ir and uv-visible bands of the crude extract and its metal (ii) complexes. antibacterial activities of the plant crude extract and its metal complexes were tested against two-gram-positive and three gram-negative bacteria. the zone of inhibitions of the plant crude extract and its metal complexes against the bacteria in mm are measured as shown in figure 3. figures 4 and 5 depict the compound’s antibacterial activities against the bacteria. table 1. physical characteristics of the plant crude extract and its meeta complexes. compound color crude extract green nickel (ll)complexes green cobalt (ll) complexes pink table 2. solubility property of the pant crude extract and its metal complexes. solvents crude extract cobalt (ii) complex nickel (ii) complex water in in in methanol vs vs ss ethanol vs vs vs chloroform ss ss ss acetone vs vs vs diethyl ether ss ss ss 394 biology, medicine, & natural product chemistry 12 (1), 2023: 391-397 table 3. important infra-red data of the plant crude extract and its metal (ii) complexes. compound ʋc-h ʋc-o ʋc=o ʋ-oh plant crude extract 2935 s 1157 s 1717 s 3406 b co (ii) complex 2935 s 1144 s 1702 s 3447 ni (ii) complex 2929 s 1143 s 1698 s 3440 b table 4. electronic data of the plant crude extract and its metal complexes. compound band (cm-1) transition plant crude extract 28169 42553 n→⫪* ⫪→⫪* co (ii) complex 17241 14925 d-d ni (ii) complex 19685 15408 d-d figure 3. measurement of the inhibitory zone of the plant crude extract and its metal complexes. figure 4. histogram representation of antimicrobial activities of the crude extract and its metal complexes against gram-positive bacteria. figure 5. histogram representation of antimicrobial activities of the crude extract and its metal complexes against gram-negative bacteria. table 5. α-amylase and α-glucosidase inhibitory activities of the plant crude extract and its metal complexes. compound ic50 (mg/l) α –amylase α –glucosidase acarbose 55.49 102.66 plant crude extract 85.65 77.98 co (ii) complex 80.52 74.96 ni (ii) complex 42.03 100.79 oladipo et al. – analysis of biological activities of two novel metal (ii) … 395 figure 6. percentage inhibitory effect of andrograhis paniculata crude extract, its metal complexes and standard drug on α-amylase. figure 7. percentage inhibitory effect of andrograhis paniculata crude extract, its metal complexes and standard drug on α˗glucosidase. discussion the various colors of the plant crude extract and its metal complexes are shown in table 1. the plant crude extract and its ni (ii) complex are green in color while the co (ii) of the extract shows is dark pink in color. the plant crude extract and its metal complexes are all very soluble in ethanol, methanol, and acetone, insoluble in distilled water, and slightly soluble in chloroform and diethyl ether as shown in table 2. infrared and uv-visible spectra the relevant infrared spectra data for the crude extract (ligand) and its metal complexes are shown in table 3. the spectrum of the crude extract shows four important bands at 2935 cm-1, 1157 cm-1, 1717 cm-1, and 3406 cm-1 which are ascribed to ν(c – h), ν(c–o), ν(c = o) and ν(c–oh) stretching vibrations, respectively. the bands of ν(c–o) and ν(c = o) observed in the crude extract are observed, upon coordination with co (ii) and ni (ii), to undergo a hypsochromic shift to 1144 cm-1, 1702 cm-1, and 1143 cm-1, 1698 cm-1, respectively. the band shifts and appearance of the new band in the region 400-800 cm-1 in the complexes confirmed the coordination of the ligands to the metal ions through m-o (talavara et al. 2016; teleb et al. 2019) as shown in figure 8. ch3 h ch2 o ch o ho ch2oh h3c ho ch2 ch3 h ch2 o ch o ho ch2oh h3c ho ch2 ch3 h ch2 o cho ho ch2oh h3c ho ch2 m figure 8. proposed structure for m (ii) complex of mangifera indica leaf crude extract (m= co (ii) and ni (ii)) bands at the visible region of 2817 cm-1 and 4255 cm1 are in the spectrum of the crude extract and the bands are attributed to n– π* and π – π* electronic transitions, respectively as shown in table 4. in the spectra of ni(ii) and co(ii) complexes, bands at 1724 cm-1, 1494 cm-1 and 1969 cm-1, and 1541 cm-1 are observed and are ascribed to d-d electronic transition (table 4) (rasool et al. 2014). antibacterial activities antibacterial activities of the plant crude extract and its metal complexes in ethanol were tested against two gram-negative and three positive bacteria at concentrations of 40 mg/l, 60 mg/l, 80 mg/l, and 1000 mg/l, and the zones of inhibition of the complexes against the bacteria are measured over the disc plates as shown in figure 3. the antibacterial activities of the extract and the complexes at different concentrations as measured are represented in histograms (figures 4 and 5). the solvent employed showed no antibacterial activity at the concentrations. ni (ii) complex was found to show more activity against escherichia coli at the concentrations of co (ii) while the complexes were more active than the plant crude extract. also, only ni (ii) complex was observed to be effective against staphylococcus. the metal complexes exhibited more activity against pseudomonas, proteus, and klebsiella than the extract. co (ii) complex exhibited the highest activity against the pseudomonas and proteus while the plant crude extract was inactive against pseudomonas at all the concentrations. however, ni (ii) complex was observed to display the most activity against klebsiella as shown in figure 5. the antibacterial activities against both gram-negative and positive bacteria increased with an increase in concentration. antidiabetic activities the percentage inhibitory effect of the standard drug (acarbose), andrograhis paniculata crude extracts against α-amylase and α-glucosidase enzymes at different concentrations (40, 60, 80, and 100 mg/l) were represented in figures 6 and 7, respectively. at a 396 biology, medicine, & natural product chemistry 12 (1), 2023: 391-397 concentration of 40 mg/l, ni (ii) complex showed more inhibitory effect against α-amylase even than the standard drug while the same activity was exhibited by co (ii) complex and acarbose at a concentration of 60 mg/l. however, the most activity was displayed by acarbose at 80 and 100 mg/l concentrations as shown in figure 6. it is shown in figure 7 that at a concentration of 40 mg/l, ni (ii) and acarbose displayed almost the same activity against α-glucosidase while acarbose exhibited the highest activity at a concentration of 60 mg/l. co (ii) complex and acarbose showed the highest activity at concentrations of 80 and 100 mg/l, respectively. α-amylase and α-glucosidase inhibitory activities of the standard drug (acarbose), andrograhis paniculata crude extract and its metal complexes were calculated and reported as half-maximal inhibitory concentration (ic50) as shown in table 5. ni (ii) displayed the highest inhibitory strength against α -amylas enzyme with an ic50 value of 42.03 while the lowest inhibitory potency against the enzyme was observed to be exhibited by the plant crude extract with an ic50 value of 85.65. the calculated a-amylase inhibitory ic50 values of the crude extracts, its metal complexes, and acarbose in descending order were ni (ii) complex > acarbose > co (ii) complex > andrograhis paniculata crude extracts. conversely, co (ii) showed the highest inhibition against α -glucosidase with an ic50 value of 74.96 mg/l while the lowest activity was displayed by acarbose. the inhibitory effects against α –glucosidase enzyme was found in the order of co (ii) complex > andrograhis paniculata crude extracts > co (ii) complex > acarbose. co (ii) and ni (ii) complexes of the crude extract were found to exhibit better activities than the positive control, acarbose against α-amylase and α-glucosidase enzymes, respectively. the more antidiabetic activities displayed by the metal complexes than the crude extract and acarbose could be ascribed to the capacity of the metal ions to transform the bioavailability and pharmacological behavior of the andrograhis paniculate crude extract. conclusion and recommendation the potential inhibitory properties of co (ii) and ni (ii) complexes of andrograhis paniculata crude extract against some bacteria, α-amylase and α-glucosidase have been examined. the complexes showed more pronounced antibacterial and antidiabetic activities than the crude extract. the metal complexes could be considered potential antibacterial and antidiabetic agents. further characterization analysis should be performed to ascertain the main structures of the complexes. pharmacological and toxicology studies on the metal complexes should be conducted to establish their feasibility as antibacterial antidiabetic agents. conflict of interest: no conflict of interest. references owoade ao, alausa ao, adetutu a, olorunnisola o s, owoade aw (2021). 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vinod r, aindrila c, tridib d, debajit b, debasish b (2014). andrographolide, one of the major components of andrographis paniculata protects against copper-ascorbate induced oxidative damages to goat cardiac mitochondria in vitro. international journal of pharmaceutical science review and research 28: 237-247. newman dj, cragg gm (2016). natural products as sources of new drugs from 1981 to 2014. j. nat. prod. 3: 629-661. rasool r, hasnain s, nishat n (2014). metal-based schif base polymers: preparation, spectral, thermal and their in-vitro biological investigation. designed monomers and polymers 17: 217-226. sachin a, kailasam k (2016). review of pharmacological investigation of kariyat. journal of pharamaceutical research 6 (1) 270-286. subramanian r, asmawi zm, sadikun a (2008). in vitro α glucosidase and α-amylase enzyme inhibitory effects of oladipo et al. – analysis of biological activities of two novel metal (ii) … 397 andrographis paniculata extract and andrographolide. acta biochimica polonica 55(2): 391–8. talavara v, yadav db, kenchappa ri, sandeep t (2016). synthesis, antimicrobial and antioxidant activity of chalcone derivatives containing thiobarbitone nucleus. medicinal chemistry 6: 440-448. tao l, zhang l, gao r, jiang f, cao j, liu h (2008). andrographolide alleviates acute brain injury in a rat model of traumatic brain injury: possible involvement of inflammatory signaling, front. neurosci. 12: 657. teleb s m, muhammad ea, el-kalyoubi s a, gaballa a s (2019). synthesis, characterization and antimicrobial activities of some 5-bromouracilmetal ion complexes. bulletin of the chemical society of ethiopia 33: 255-268. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 1-14 | doi: 10.14421/biomedich.2014.31.1-14 ecopharmacognosy: exploring the chemical and biological potential of nature for human health geoffrey a. cordell natural products inc., evanston, illinois 60203, usa author correspondency: pharmacog@gmail.com abstract “why didn’t they develop natural product drugs in a sustainable manner at the beginning of this century?” in 2035, when about 10.0 billion will inhabit earth, will this be our legacy as the world contemplates the costs and availability of synthetic and gene-based products for primary health care? acknowledging the recent history of the relationship between humankind and the earth, it is essential that the health care issues being left for our descendants be considered in terms of resources. for most people in the world, there are two vast health care “gaps”, access to quality drugs and the development of drugs for major global and local diseases. consequently for all of these people, plants, in their various forms, remain a primary source of health care. in the developed countries, natural products derived from plants assume a relatively minor role in health care, as prescription and over-the-counter products, even with the widespread use of phytotherapeutical preparations. significantly, pharmaceutical companies have retrenched substantially in their disease areas of focus. these research areas do not include the prevalent diseases of the middleand lower-income countries, and important diseases of the developed world, such as drug resistance. what then is the vision for natural product research to maintain the choices of drug discovery and pharmaceutical development for future generations? in this discussion some facets of how natural products must be involved globally, in a sustainable manner, for improving health care will be examined within the framework of the new term “ecopharmacognosy”, which invokes sustainability as the basis for research on biologically active natural products. access to the biome, the acquisition, analysis and dissemination of plant knowledge, natural product structure diversification, biotechnology development, strategies for natural product drug discovery, and aspects of multitarget therapy and synergy research will be discussed. options for the future will be presented which may be significant as countries decide how to develop approaches to relieve their own disease burden, and the needs of their population for improved access to medicinal agents. keywords: natural products, ecopharmacognosy, sustainable medicines, biotechnology, structure diversification, rain forest resources, strategic implications introduction in lewis carroll’s famous book “alice in wonderland; through the looking glass”, it is the mad hatter who has the perspicacity to say to alice “we are all mad here”. in many ways, given the recent and ongoing relationship between humankind and the resources of the earth, it is we who are living in a world of madness, where resources are squandered now, for instant benefit, with little consideration given for the resource needs of the generations to follow. and it we who, in spite of numerous warnings and international meetings and declarations, still cannot turn and say clearly that what we are doing to the planet and its resources is indeed “madness”, and must change if the human race is to survive and thrive. to borrow a phrase, “we are killing the planet that heals us”. the core madness in the scenario is this: a dramatically increasing population, mostly in the middleand low-income countries, an alarming decrease in the acreage of tropical rain forests, and an expanding dependence on those resources and others for food, shelter, and health care. when, as we have seen, most of the world relies on medicinal plants for their health care, and up to 85% of those plants are harvested in a nonrenewable manner, it is clear to see that new strategies are needed to assure access to medicinal agents in the future (1-12). the fourth factor, oil, remains a mysterious unknown, an enigma with respect to long-term accessibility. optimistic estimates of the current oil resources, although accurate data are basically nonavailable for financial and security reasons, would suggest that they may be viable for the next 40-50 years. however, population is increasing at a faster rate than oil production. so what will be the status of the earth’s resources at that time, say in 2060, when renewable, moderate cost, effective, alternative sources for synthetic medicinal agents will be needed? enzymes, isolated or in intact systems, must be deployed as an essential component of industrial drug synthesis to reduce the dependency on non-renewable resources. the july, 2011 issue of chemical reviews was devoted to a series of eleven reviews of different types of enzymes deployed for selective synthetic transformations, several related to drug processes. the use of renewable, multifunctional enzyme systems which can transpose a compound through to a final product with minimal “chemical” reagent involvement may be an important facet of the production of synthetic drugs within ten years. considerations of the use of inexpensive renewable reagents may also drive the selection of both production processes and compounds evaluated for their drug potential and eventual marketing. also to be developed further is the potential for the use of 2 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 vegetables as chemical reagents for selected enantioselective processes, where intact materials, not isolated enzymes are utilized (13). however, the need for natural products, as quality traditional medicines, and as sources for new medicinal agents will remain. this brief discussion is about some of those opportunities. as natural product scientists, one of our obligations is to initiate the discussion of how to investigate natural resources for health care, and encourage others to see different perspectives of the overall picture. our role is also to examine, define, and articulate the niche of natural products in the future of the population of earth, and particularly the anticipated role of natural products in global health care. it is also important that we, as the content experts, have a scientific role in developing those future plans. the global population is now at 7.31 billion (april 2013), and is projected to rise to 10 billion by about 2035. the causes and the implications for such a dramatic population explosion are philosophical and religious, which are considerations beyond the scope of this short article, but upon which the fate of human existence hinges (14, 15). as one small, yet significant aspect of this huge discussion, some of the many integrated aspects of natural products in health care, we will review some possible relationships between natural products and drug discovery, and look one again at the important question: “what is the future for natural products in global health?” the author has written numerous articles on this area in the past 25 years (1-6,16-24), with several of the more recent articles focusing on two major issues, the concept of medicines as a sustainable commodity (4-12), and enhancing the quality control of traditional medicines in global health (6-12,23-26). on a daily basis, we often forget to acknowledge with gratefulness the vast contributions that plants, their extracts, and the various products derived from them, make to human health and well-being: i) as foodstuffs, ii) as flavoring agents and spices, iii) as perfumes and cosmetics, and iv) as pharmaceutical and biological agents. these categories are not mutually exclusive, for it is apparent that there is significant overlap between foods, spices, cosmetics, and biological (medicinal) agents. it was estimated over 20 years ago that there are more than 120 compounds from over 90 different plant materials which are used on a global basis as prescription products (27), and this number may well have risen since then (28-32). butler, for example, lists 34 natural product based drugs introduced in the period 1998-2007 (31), and harvey lists 108 plant-derived compounds in various development phases (32). from a commercial perspective, world-wide sales for plant-based pharmaceutical agents in 2002 were estimated at us $30 billion (33). in addition, there are also thousands of products, compounds, plant extracts and plant materials, some sanctioned as prescription products, others bought over-the-counter, or in a market place, or through a local medicine man or woman, which are recommended for patient healing in various parts of the world. for the vast majority of the world, approximately 4.5 billion people, these plant resources are their primary source of health care (34). there are many “gaps” in global health care, and per capita government investment in health care in a particular country (see table 1 for some examples), is one of the more staggering examples between the highincome and the low-income countries. that aspect of health care concerned with medicinal agents for patient treatment, pharmacy, is no exception. for example, there is a major regulatory “gap” between the prescription products of the north and the products sold in a marketplace as part of a traditional medicine system in a middleor low-income country. one is highly regulated, costing perhaps $1.3 billion to develop a product and support continuing post-marketing surveillance (35). whereas the traditional medicine is almost completely unregulated; in many countries, a plant material appears in the street market directly from the field, just as it did 4000 years ago, and there are many variations of regulation in this continuum of extremes. the un charter of 1948 clearly indicates that all humans have the universal right to health care. but what is being done by the organizations of the un to achieve this? what does that universal right mean in terms of the quality control of traditional medicines or for drug discovery for global and local diseases? in this regard, we must not be limited to thinking that drug discovery involves only single compounds. in an era of systems biology and network pharmacology, it is clear that the period for thinking of drug discoveries as “magic bullets” is long gone. table 1. government expenditures on health per capita (2009) in us $. country us $ united states 7960 norway 5383 australia 3484 united kingdom 3438 republic of korea 1879 costa rica 1155 turkey 957 south africa 930 brazil 921 iran 728 peru 466 china 347 vietnam 204 philippines 133 india 124 nigeria 136 indonesia 100 myanmar 36 source:http://en.wikipedia.org/wiki/list_of_countries_by_total_health_ex penditure _(ppp)_per_capita in terms of plant-based health care, research is drastically under-funded at the global and national levels in terms of personnel, programs, products, and outcomes. geoffrey a. cordell – ecopharmacognosy: exploring the chemical and biological potential … 3 the truth is that, as a moral and ethical issue, it is a shameful international embarrassment, and requires a complete reassessment of strategies, programs, and outcomes from the very top of the un through all of the various agencies involved. one group, médecins sans frontieres, justifiably, has called for a complete overall of the whole drug discovery, development and access system for the middle and lower-income areas of the world given the history of drug innovation for the south in the past thirty years (35). patients world-wide who acquire plant-based products, either through prescription or as a street medicine, in their various forms, are rightfully demanding the same assurances of safety, efficacy, and consistency as a patient taking a drug from a pharmaceutical company. the “gap”, of course, is who is standing behind those assurances? is it a government, a pharmaceutical company, a traditional medicine provider? if no-one is making (or can make) those fundamental assurances, based on science, what does that mean for the overall health care of a nation? as the wise man once said, if you want the answer…. “follow the money.” although very basic, this is merely one of many health care “gaps” that exist in the world at this time; another gaggle of “gaps” concern the question of access. in this sense, the term is applied in four ways. the first facet is what are the prevalent diseases in a country for which drugs are not available at all? the second is what are the diseases for which drugs are available globally, but which are either too costly, or not available, locally? the third facet is what are the diseases for which there is resistance to existing treatments? the final facet of access relates to the source of medicinal plant materials, and the long term stability of a disappearing forest as a sustainable source of medicinal agents. these facets, individually or collectively, can form the basis for rationalizing the need for targeted new drug discovery programs, and for placing that rationalization in a conservation and sustainable development framework. access to health care, and to medications in particular, is consequently a global concern for all, with the exception of the very wealthy. another of the medicine-related “gaps” in health care relates to two questions: what are global health care needs? and what is contemporary drug discovery in the pharmaceutical industry targeting? the “gap” between these two responses is vast and expanding. the global health needs for over 1 billion people on earth are treatments for: malaria, hiv-aids, tuberculosis, hepatitis c, diarrheal diseases, ascariasis, leishmaniasis, schistosomiasis, trypanosomiasis, dengue fever, leprosy, rabies, yaws, and necatoriasis. contemporary drug discovery areas in the major pharmaceutical companies have been winnowed to: antivirals, oncology, metabolism, central nervous system ailments, and inflammatory diseases (36). more recently, one major company, astrazeneca, has winnowed its research portfolio to three areas: inflammatory and autoimmune diseases, cardiovascular and metabolic diseases, and oncology (37). the logic behind these strategies is that drug companies will make money when they produce drugs which: i) will not encounter a resistance issue, as occurs with antibiotics, ii) are taken on a chronic basis, and iii) are palliative and not curative. the global health disease burden is not their concern. it is really that simple. for individual countries, and for regional associations of countries, such as asean, many questions must be asked in terms of the present and the future health care needs. there are limited precedents available, but recall that it may take at least fifteen years to move from bench to marketplace, if the strategy involves the development of single agent drugs. there are alternatives which also should be considered, and which, depending on the particular country situation, can serve either as interim steps for enhancing health care, or as longer term solutions. clearly though, not every country will have the human capacity, the infrastructure, and the financial resources to muster a drug discovery effort based on the study of their natural resources. even those countries who do embark on that endeavor will need to clarify whether all of the needed expertise is available in country, or whether other regional or global resources need to be accessed. from a discovery perspective, countries have two main natural resources at their disposal, their natural flora and the indigenous knowledge, which may be within one or several traditional systems of medicine. these resources offer a unique basis with which to develop a strategy or a series of strategies for drug discovery. in this instance, the emphasis here is on a government ministry, or most likely a group of ministries (science and technology, health, education, agriculture, commerce), and local (not the major) pharmaceutical companies, coming together to serve as a catalyst to address this aspect of health care. initiatives such as the discovery of drugs or the enhancement of traditional medicines to meet societal health needs cannot begin at the local level, but must come “top down”, where government is both “calling the shots” through establishing consultative groups, and at the same time providing the highly targeted resources for addressing long-term needs. all parties, government regulators and funding sources, botanical, chemical and biological scientists, agronomists, industrialists, and academics need to come together to address these basic health care issues as a coordinated and cooperative venture for the health of their population. as we consider how to potentiate the available natural resources for health care, it is also important to examine the historical background to the interest in the tropical forests. it is not adequate to state that because plants have yielded drugs in the past they will do so in the future. neither is it appropriate to conclude that because a particular plant has a long history of use as a traditional medicine that it should be approved as a drug. many other considerations come into play, including the need to place such assertions in science; the evidence-based approach. so what is the goal, and what are the strategies, of exploring the rain forest for medicinal plants? are there 4 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 clear and rational reasons which can adequately justify the investment? if so, what is the nature (breadth, depth, duration) of such an interest? what if the discovery of either an effective plant extract or an effective compound is made? what are the issues with respect to next stage development, and how will further development, if warranted, be supported financially? where and how does the patenting of inventions impact the development process? what are the local implications in terms of agricultural or infrastructure development? potentially, unraveling the human genome provides an opportunity for the development of assays related to numerous disease-related, new drug targets, once those correlations have been determined. because of the ethnomedical reputation of tens of thousands (perhaps 810%) of terrestrial plants (4), once a prioritized acquisition and screening schedule is achieved, very specific opportunities for the selective evaluation of the effectiveness and potential for further development of traditional medicines may be achieved. strategically though, for the wider evaluation of plant materials, and for more rapid and effective quality control assessment of traditional medicines an important paradigm needs to be reversed (9-12). as a modality for conducting preliminary screening programs for the biological activity of plant extracts, bringing plant materials from the collection site to the laboratory for extraction and bioassay is an exceptionally inefficient approach. depending where an “active” line is drawn, but probably around the 2-4% level for most programs, the extracts of 96-98% of the collected plant materials are “inactive” in any particular biological assay. while those materials (and their parent plants) can be retained and stored as an asset to the program, and subsequently reassessed against new target assays in the future, the fact remains that there is significant effort in collection, drying, grinding, and extraction which is wasted. consequently, there is an urgent need for the development of bioassay systems, preferably genomebased, which can be performed, at collection site locations, directly on plant extracts prepared in the field using micro-extraction technology to remove unwanted plant constituents. further local plant collection would then focus on those plant extracts which have a demonstrated biological activity. at the same time, the frequent failure of plant extracts to reconfirm biological activity on recollection will be reduced. who is potentially interested in exploring the rain forest biodiversity for bioactive products? several types of companies have a financial interest in plant-based natural products and extracts. in spite of the success of plant-based pharmaceuticals, most of the large pharmaceutical companies have terminated those aspects of their drug discovery programs which are based on plant-derived natural products. the prime reasons for this have been discussed elsewhere (1,3,4,9-12,20). as complex matrices, plant extracts provide a significant challenge for the bioactivity-directed isolation of the active principle(s). in addition, this deconvolution step may yield an undesirable known, rather than a novel, active constituent. the second consideration is that the recollection of a plant may be a time-consuming process, require extensive (re-)negotiations related to access, and may not be biologically active. yet another consideration is the ability to provide an adequate supply of a compound for additional biological evaluation in a timely and reproducible manner. for some biotechnology companies, the use of tissue culture systems in order to examine the ability of cell-free plant systems to produce metabolites which are not present in field-grown plants has proved important, and has provided an opportunity to study plant secondary metabolic enzyme systems and obtain new compounds for evaluation. the botanical supplement companies are finding new products from various parts of the world to market, with only a modicum of concern regarding the sustainability of their sourcing, the consistency of their constituents, and the limited scientific background justifying their use. the nutraceutical companies are looking to exploit the academic discoveries of compounds, such as cancer chemopreventive or cholesterol-lowering agents, which can be added to highvolume foods, or they are seeking plants which offer new and diverse life-style marketing opportunities (noni fruit and acai berry are recent examples). investment in research to justify the safety, use, and quality control of these products is minimal. with this summative background concerning the past and present practices of natural products in the discovery of new medicinal agents, it is opportune to consider the appropriate role of natural products in health care systems globally for the next twenty years. future aspects of natural products in drug discovery for future generations to thrive, be productive, and lead meaningful lives in their respective societies, there are two dominant factors to be considered, the health of the planet and the health of the people. these are not to be separated, as we are all part of one large, fully interacting, biological organism, earth. the goal must be to maintain the health of that whole organism in a cost-effective manner. failure to do so will leave a terrible legacy for our descendants. consideration of all chemical processes, including those for the synthesis of medicinal agents, and the accessibility of drugs to those in need are critical. factoring in the anticipated cost to the patient becomes an important consideration as potential drug candidates in any form, synthetic or natural, make the transition from discovery to clinical development. already there are numerous situations where the latest drugs are becoming (or have become) too costly for health care systems, including insurance companies, and their patients in the high-income world. when it is a health ministry in a country which decides which drugs are to be imported, and at what cost, for their health care systems, very significant compromises are typically made in terms of access to optimal health care. in the course of time, these differences in access are clearly reflected in geoffrey a. cordell – ecopharmacognosy: exploring the chemical and biological potential … 5 life-expectancy, and become another global pharmaceutical care “gap” between the north and the south. at the same time, it is also recognized that enhancing the quality of traditional medicines will have an impact on product costs. this is contained within the concept of accessibility, the combination of sustainability and affordability. almost lost now in the mists of time, it should be remembered, that before 1899, when the semi-synthetic drug aspirin, based on the natural pain reliever salicin, was introduced, all drugs were derived from natural sources. since then, a vast pharmaceutical industry, with many companies tracing their history to the original sellers of natural products in the latter part of the 19th century, have evolved to develop new drugs for a myriad of diseases. some of the compounds introduced during that period from 1900-2010 have been natural products, and the more recent discovery efforts have been reviewed by newman and cragg (28,29). most of the drugs that have been introduced in the past 110 years are totally synthetic. the primary exceptions are several groups of antibiotics, and the steroid and alkaloid derivatives which are semi-synthetic. it is time now to introduce the new term “ecopharmacognosy” (38). as we look to the future for the practice of using synthetic medicinal agents, one aspect of concern which requires deliberation is the sustainability of the production of those drugs at reasonable cost. both the chemicals and the chemical reagents used in those processes are typically a nonrenewable resource, and their contemporary use depletes the future resources for synthetic drugs. consequently, a fundamental precept for all drug discovery programs, be they synthetic or natural, must now be the concept of sustainability (4-12,38), as an extending consideration of the “green chemistry” movement. one aspect of this topic was discussed earlier in terms of the development of alternative, renewable sources for chemical reagents (enzyme catalysts, vegetables, etc.) (13). selected, valuable traditional medicines have been, or are being, depleted in their natural environment, without alternative resourcing being developed. ecopharmacognosy, defined as the “study of sustainable, biologically active natural resources”, must therefore become the fundamental basis for all natural product research. studying plants, or any natural organism, for their use as a global medicine, must take into account the potential long-term sourcing. we have already seen situations, taxol is an example, where the drug requirements for a clinical trial, came up against serious sourcing concerns. consideration of the enzymes to be used for the large scale synthesis of drugs, must also consider the sustainability of the resource. using the enzymes in plants that are already commercial entities, such as vegetables, assures their long-term accessibility, and reduces cost compared with rare enzyme preparations derived from microbial sources. ecopharmacognosy therefore embraces both the development of biologically active natural products as single agents or as components of traditional medicines, and the resources for the chemical synthesis of single agent drugs. the same considerations must also be applied as marine resources are explored for the development of new biological agents. except for some biological agents, such as vaccines, which will not be discussed here, there are usually three classes of single agent drugs recognized: i) totally synthetic drugs, which are produced from non-renewable resources, such as coal and oil; ii) semisynthetic drugs, such as the steroid hormones which are derived from a chiral natural product core produced in the field, or, in the case of antibiotics, through large scale fermentation. this is followed by structural modification using nonrenewable resources, unless microbial transformations are included in the synthetic strategy; and iii) natural products, such as vincristine or morphine, which are derived from a renewable natural resource. in this instance, the non-renewable aspects involve the materials used for processing the plant material in order to isolate the desired alkaloid. of perhaps thirty different aspects which could be discussed regarding the future for natural products in drug discovery, six will be briefly presented: i) access to the biome, ii) acquisition and analysis of traditional knowledge and on-going research, iii) biotechnology development for secondary metabolites, iv) dereplication studies, v) strategies in natural product drug discovery, and vi) multitarget therapy and synergy research. access to the biome the natural materials to be used, and any ethnomedical knowledge not already in the public domain, are the property of the country in which they are extant historically. the convention on biological diversity (cbd), approved for signature at the earth summit in rio de janeiro in 1992, established those parameters for those signatories who ratify the treaty. as of april 2013, 193 countries were parties to the convention. only three countries in the world had not ratified the convention: andorra, the holy see, and the united states. at the same time, there is a natural, unresolved tension with the trips (trade-related aspects of intellectual property rights) agreement of 1994 in the area of the development of local resources by third parties. in the trips agreement, the country of origin of the genetic material has no sovereign rights over their biological resources or their indigenous knowledge once there is an invention. it is the inventor who can claim the intellectual property rights, with the originating source of the material, whether it is derived from a plant, animal, or fungus in a particular country, having no claim for compensatory loss. the united states was the promulgator and the first signatory country to this agreement. this area, and the whole topic of the impact of the cbd on natural products research, has been reviewed (39), and the discussion will not be repeated here. some selected aspects of the cbd that relate to natural product drug discovery will be mentioned briefly. 6 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 article 15.2 of the cbd indicates that signatory countries should facilitate access to their biome in exchange for present and future considerations which are to be negotiated (39,40). articles 15.5 and 15.6 indicate that all of the collections of biological material which are made within a country should occur with prior informed consent, and that collection of the materials should occur with the accompaniment of local scientists. article 16 is concerned primarily with issues related to access and the transfer of technologies relevant to the conservation and sustainable use of genetic resources under fair and favorable terms. article 17 relates to the information concerned with the conservation and sustainable use of biological diversity and the results from scientific and socio-economic research are promoted. article 18 promotes international technical and scientific cooperation, and the development of joint ventures. following the cbd, there were numerous developments related to the establishment of protocols and systems within countries for the approval and collection of biological materials, and the acquisition of indigenous knowledge (39). indigenous groups, sometimes collaborating across national boundaries, have also sought to establish parameters and protocols to receive authority to provide access. while this has had the beneficial effect of reducing the number of scientists from various countries taking unauthorized plant materials or other biological resources out of a country without local knowledge (sometimes referred to as “biopiracy”), there has been a clear tendency towards the over-protection of access to resources, and/or excessive bureaucracy associated with the approval process (39). it is this outcome of the cbd, doubtless not anticipated by the drafters or the signatory parties, which has led to the overall deleterious impact of the cbd on natural products research on a global basis. this is because the value placed on the access to those resources by an individual country was often too great. the expectation that pharmaceutical companies would line up to seek access to areas of intense biodiversity, never materialized. as a result, if the approval processes, or the restrictions, or the time and the financing required to negotiate the associated considerations, were deemed too onerous, academic and/or industrial laboratories declined to invest, in either the people or the places. particularly, this occurred if more amenable choices for sample acquisition (based on bureaucracy, cost, and time), which might well have the same plant materials, were available. alternatively, many pharmaceutical companies simply chose to eliminate natural product extracts (marine, fungal and plant) from their primary screening programs, and, at the time, turned to other sources, such as combinatorial chemistry, for the guaranteed expansion of their chemical libraries. this resulted in two scenarios. firstly, the biodiversity-rich countries were unable to find collaborations (either academic or industrial) for programs designed to evaluate their biome for a health care impact, and were unable to enhance the development of much-needed taxonomic, chemical, and biological capabilities in-country. in some instances (e.g. the philippines), local scientists were also severely impacted by strict government requirements, and their research was essentially halted for extended periods (34). secondly, local pharmaceutical development, deemed critical for the type of initiatives being discussed to improve local health care, was inhibited. as a direct result, the reliance on externally acquired pharmaceutical and medicinal agents was maintained, benefitting the major drug manufacturers. without access to biological materials, the discovery of new biologically active natural products is diminished and advances in health care are impeded in that country and elsewhere. it is important that prior negotiated agreements are seen as an essential aspect of plant collection programs for drug discovery, and there is a sense of pragmatism required. very, very few compounds, or their semisynthetic derivatives, will ever become a profitable invention. an alternative pathway is to negotiate a graduated, two-tier approval process for the collection of biological materials which distinguishes between the “discovery” phase and the “development” phase. thus, one agreement would cover initial collection and academic/pre-toxicological research on limited size samples, and the second agreement would apply only when the acquired sample size had to be increased for more advanced pharmacology and clinical studies. (39,41). on october 29, 2010, the “nagoya protocol on access to genetic resources and the fair and equitable sharing of benefits arising from their utilization to the convention on biological diversity” was adopted under the auspices of the cbd (42). it is an instrument for the implementation of the access and benefit-sharing provisions of the cbd, and was opened for signatures on february 1, 2011. it was developed after six years of negotiation, and overall content supports and compliments the cbd. only countries which have signed and ratified the cbd are eligible to sign or ratify the nagoya protocol, but a country can also have signed the cbd and choose not to sign the nagoya protocol. when fifty countries had signed it entered into force and countries began implementation. as of april, 2013, 92 countries had signed the nagoya protocol, and 16 had ratified the protocol (43). the primary focus of the protocol is on the equitable sharing of benefits, and the requirements of signatory nations to develop procedures for implementation and regulation of the cbd, with a specific requirement for the issuance of permits with respect to permission granted for access to either genetic resources or indigenous knowledge. the protocol establishes an international clearing house under the cbd secretariat to assist countries with respect to developing various aspects of the implementation process, and requires countries to deposit appropriate records and information from their country with the clearing house for common availability. specific issues relating to trans-boundary situations must be discussed in instances where indigenous groups overseeing knowledge or resources are not located in a geoffrey a. cordell – ecopharmacognosy: exploring the chemical and biological potential … 7 single country, as may occur in parts of the andean region, in the middle east, and in southeast asia. in response to significant criticism about bureaucratic process and openness, ratifying countries are now required to assure legal certainty, clarity and transparency, both legislatively and in the implementation of regulatory requirements, such as applications for prior informed consent. countries must also provide effective communication systems during periods of application evaluation. permits which are internationally recognized will be issued by the recognized national authority for approved programs in a country based on prior informed consent and mutually agreed terms. this information will be provided to the newly-established cbd access and benefit-sharing clearing house. one can imagine that the permits will be needed both internally at various points in the process, at the collection site, at the exportation site for genetic material, and probably by major international journals in the field to assure compliance with international standards for published research articles. somewhat surprisingly, the contentious issues related to patents are not discussed in the nagoya protocol, even though it is an essential consideration for many groups seeking access to genetic resources and traditional knowledge, and an anticipated constituent aspect of any prior negotiated agreement between parties. consequently, the obvious tensions with the trips agreement remain unresolved. directly related to the concerns regarding patents and the non-obviousness of inventions is the issue of “derivatives”, which the protocol defines as “a naturally occurring biochemical compound resulting from the genetic expression or metabolism of biological or genetic resources.” this is not a robust definition of a derivative, and it is easy to imagine how a corporate entity could develop derivatives, be in accordance with mandates of the nagoya protocol, and still be the sole recipient of patent rights. another significant omission from the nagoya protocol is the absence of mandatory checkpoints or benchmarks which a certificate holder should be required to reach during the application and experimental processes. individual countries are however, permitted to include benchmarks of performance and reporting as they deem necessary. acquisition and analysis of traditional knowledge and on-going research it has been said that the countries which will be successful in science and technology in the future will be those who use the globally available knowledge most creatively to generate new knowledge and inventions. this “smart” technology is desperately needed as strategies and considerations for the exploration of the tropical forest are initiated. information, ethnomedical, botanical, chemical, and biological, is burgeoning. new technologies which may impact drug discovery programs are being developed at an incredible pace. almost every country, and within them most scientific research laboratories, when they are connected to the internet, have essentially unlimited access to this information, most frequently at no or minimal cost. with respect to traditional knowledge, the ease of access to information has its benefits and hazards. the benefit is that more information on the use of a plant can be compared than merely relying on one or two local compendia of information. the hazard is whether all of the available information has been acquired legally under local regulations, particularly knowledge of the use of indigenous plants acquired since the cbd. another consideration is that the vast amount of published ethnomedical information, collected by ethnobotanists and medical anthropologists, on the use of plants for medicinal purposes, is extremely scattered, and is therefore very difficult to acquire in toto. undoubtedly, there is always more literature to be unearthed on the use of a particular plant, if only the resources were available. as stated on several occasions (3,4,6-12), there is a dire and urgent need for an international agency, possibly in collaboration with a global foundation, to fund the development of a central repository of indigenous knowledge, a sort of “wikiethnomed” . but the data base repository should not end there. as well as ethnomedical information, data on the biological evaluation of plant extracts and their constituents, the chemistry of the natural plant sources, and the clinical evaluation of plant extracts, needs to be acquired and collated. data on the safety and possible or observed adverse events associated with traditional medicines are also required to be collated for open access, as a health care consideration for practitioners and patients. at this stage in the 21st century, it should be possible, where ever one is in the world, to indicate a plant name and then find its barcode (vide infra), ethnomedical information, chemical constituents, biological activities, and clinical evaluation data. it is estimated that such an on-line, global health resource, once established, and depending on physical location(s), would cost about $5 to 7 million to operate each year; a veritable bargain given the health care benefits. for the development of rational, sustainable drug discovery from plant sources the first step is a critical evaluation of all of the available information on a plant, or on the plants to be evaluated for a particular health benefit, such as antihyperglycemic activity. this is needed in order to prioritize plant acquisition plans, avoid the unnecessary duplication of research effort, and optimize the consumption of precious (financial, personnel, and oil-based) resources. there are also important ecopharmacognosy considerations of sustainability which enter into these strategies as well. biotechnology development for secondary metabolites when one acquires, dries, and then extracts a plant material, the observed chemical profile is the phytochemical equivalent of a “kodak moment” (10-12). for over 180 years, phytochemistry, and by inference chemotaxonomy, tacitly accepted that this “moment” represented the biosynthetic capacity of that plant. now 8 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 it is well-established, through the use of tissue culture, cell-free systems, and elicitor molecules such as methyl jasmonate, that this is an inappropriate paradigm, and represents only a partial view of genetic capacity for secondary metabolite formation. indeed, it is more like a “still shot” from the dynamic secondary metabolite profile “movie” of the plant. for no single plant on earth has the full metabolic profile yet been determined through analysis of the genes of secondary metabolism and then demonstrated through expression. a more complete understanding of what a plant is in terms of a chemical factory for making secondary metabolites of biological or clinical interest, may be very important to achieve on a selective basis in the future. thus, a single extract of a plant, taken at a single point in time, cannot reflect the constituent range of that plant, ignoring as it does numerous intrinsic and extrinsic factors, including dormant biosynthetic genes which will alter the chemical profile, and thus the biological outcome. the ability to modulate these genes, and thus the enzymes they code for, in a controllable manner, will be a critical aspect to enhancing plant secondary metabolic diversity for biological screening, and in establishing reproducible production levels for needed metabolites, especially for the constituents of traditional medicines. fungal genetics has progressed remarkably from the perspective of understanding secondary metabolic profiles and production in recent years, and inferences with respect to the evolutionary aspects of fungal organisms are developing rapidly (44). because of challenges with the locations of metabolic genes, plant genetics has lagged somewhat. however, as these details of the plant genes responsible for secondary metabolite biosynthesis are recognized, it becomes possible to express the genes in other, faster-growing formats (e. coli, yeast, insect cultures, etc.), and produce either a new range of metabolites previously unknown from that plant, or a desired enzyme, compound or series of compounds with a significantly higher degree of control (45,46). from a biological screening perspective, it will be important to identify the regulatory genes responsible for secondary metabolite production control, and thereby modulate the secondary metabolite profile, so that rather than being a snapshot, it can indeed become a movie. both the short and long-term implications of this are clear. maintaining the genetic capacity of the tropical forests, which has taken billions of years to evolve, through conservation, acquisition and botanic garden development, or sampling and storing germplasm, is a critical aspect of a diversified and well-considered longterm medicinal agent discovery program for a nation. it may be that locked in the dna of a plant are the genes to produce crucial drugs in the future, once it is possible to more completely understand the contortions of their metabolic formation. certainly, it is unconscionable to destroy the forests without first sampling and preserving the breadth and depth of the diversity of genetic resources that are within. there are numerous other aspects of biotechnology which impinge on natural product drug discovery and development, including bioassay design and implementation, drug delivery systems, and enhancement of natural product structural diversity (vide infra). some aspects of these technologies have been discussed elsewhere (5-7,9-12). dereplication studies plants are chemical factories which produce a wide range of metabolites of various structural types. many plants across taxonomic borders have evolved the genes to produce similar or identical metabolites. the wide spread occurrence of common flavonoids, such as quercetin and related derivatives, is an example. once an extract is declared “active”, prioritization along with other “active” extracts for bioactivity-directed fractionation is needed. frequently, there are more active plants than capacity (human) to fractionate. at this point, there are three research options: i) eventually fractionate all the extracts; ii) introduce a secondary bioassay to discern a more selective priority list, or iii) apply a dereplication strategy. dereplication is a process to delineate, in an active extract, the probability that the active principle is likely to be novel. such a determination can re-prioritize active extracts, and/or eliminate extracts for further examination, and it can improve the efficiency of isolating the active principle. several years ago we described an hplc/esms/bioassay/database dereplication system for active natural products in biologically active matrices (47), and went on to describe how it was used on active extracts for the identification of both new and known natural products (48). refinements in dereplication systems include: i) development of software which can rapidly correlate mass and biological data to a chemical and biological database so that searches and conclusions can be achieved on-line, ii) introduction of continuous flow nmr to support postulated structures of active masses, and iii) recognition of carbon-13 nmr to provide an even higher level of certainty regarding the possible skeletal structure and functional group disposition of a molecule within the matrix. over time, the ability to characterize the majority of the known major active constituents in an extract without isolation will become a standard practice. a further use for such hplc/esms/nmr/bioassay system technology, other than drug discovery, will be for the chemical and biological standardization, as well as metabolomic studies of traditional medicine samples, on a batch to batch basis, once the appropriate active principle(s) have been identified. strategies in natural product drug discovery it is an important question to ask whether natural products are drug molecules. several years ago (21), it was shown that those alkaloids which are presently available as pharmaceutical agents are a good fit (with some outlying molecules) to lipinski’s “rule of five”. when an analysis geoffrey a. cordell – ecopharmacognosy: exploring the chemical and biological potential … 9 of 120,000 compounds in the dictionary of natural products was conducted, 65% had no violations of these rules, and this led to the development of a natural product library of over 500 compounds for drug screening (49). other studies have supported these conclusions (50,51). thus existing natural products can form the basis of a drug discovery program complementing the development of potential new sources of bioactive molecules. strategic considerations for the development of natural product drug discovery programs abound (17,19,52-54). how the plants will be collected (random, phytochemical, ethnomedical), how plants will be extracted (water, non-polar, and polar solvents), which biological assay to use (animal, whole cell, organ tissue, enzyme, receptor), and how to establish levels for “activity” are strategic decisions which must be taken prior to initiating the program and then reviewed regularly during the program. it is also important to recognize that a plant is not a single organism. there are numerous fungi, bacteria, and, in some cases, algae, symbiotically associated with the plant. these organisms are also biosynthetic factories, and the study of pathogenic and parasitic organisms may offer the potential to enhance the range of natural product structures in a sustainable, albeit rather unpredictable, manner. accessibility to resources, reproducibility of activity in recollections, and the time to yield an active isolate are important concerns in the involvement of plant extracts in drug discovery (1-3,10-12). there are several other issues which can also affect the overall discovery process. we have seen that isolation of known compounds with a known bioactivity is a significant issue in some biological areas (such as cancer), and there is also the patent concern of known compounds which may afford new biological activities. patenting inventions has become an important, though not essential (vincristine, taxol, and camptothecin were never patented), function for both industrial and academic discovery programs, and the strongest drug patents are those which claim a new chemical entity with a new biological activity. sample repositories are an essential aspect of any plant-based drug discovery programs. typically, as a program evolves, there are three types of repository that are needed: i) for the plants acquired, ii) for the extracts made, and iii) for the compounds isolated. the first may, in part, be the local, internationally recognized, herbarium where a formal, classically prepared specimen is stored for posterity. also needed is a dry, airconditioned, moderate temperature (0-10˚c) facility for storing the remainder of collected, but unused samples, appropriately labeled and catalogued in a database. the second repository is for residual samples of all extracts and fractions that are prepared, usually held at -20ºc or below, and again systematically catalogued electronically and organized in individual, barcoded vials for easy access. frequently, sample specimens of extracts are also stored in 96-well plates for screening purposes, depending on strategic demands based on the available and projected biological assays. finally, purified and isolated compounds must be catalogued and stored, both in vials, and in 96-well plates for further screening. long term, the drug discovery goal should be to screen every isolate against every relevant bioassay. electronic cataloguing and analysis of this information is important, as is recognizing that data base access should be available to all collaborators. of course a fourth database, not experimental in nature based on the current studies, should constitute an inventory of information on the previously reported studies on all of the plants actively under study. the first step in deciding whether to actively work on a “hit” extract is to become fully aware of all of the prior literature on that plant and related species. it is another example of “information first” as a philosophical precept. identification of the plant materials to be used for discovery purposes is very important as a basis for the whole program. but how is the plant to be identified? preliminary identification may come from morphological examination and herbarium specimen comparison. for several years, dna techniques, such as random amplified polymorphic dna (rapd), amplified fragment length polymorphism (aflp), and inter-simple sequence repeat (issr) analysis were deployed as an adjunct for plant identification, and for studying the genetic variation in medicinal plants (55). now the focus is on the development and application of dna barcoding (56), as one facet of the consortium for the barcoding of life (cbol), and some background details pertaining to medicinal plant identification have been discussed (9-12). the dna fragments which are considered appropriate to use for species identification at this time are focused on the its2, matk, and trnh-pasba genes. reliability rates, the ability to make positive medicinal plant identification based on specific genes at the genus and species level, are typically very high (76-96%). chen and co-workers (57), using seven dna barcodes (psba-trnh, matk, rbcl, rpoc1, ycf5, its2 and its), studied over 6600 plant samples from 753 genera and 4800 species. they found that the its2 barcode gave a 92.7% successful identification rate, and thus could potentially used for the identification of medicinal plants. how this relates to secondary metabolite production, and therefore to a reproducible biological activity, remains an area for future exploration. another significant future development is the incorporation of barcoding technology into highly automated, hand-held devices which could provide extremely rapid, in-field identification of plants (58). principal component analysis (pca) of the low molecular weight (ca. 200-500 daltons) compounds in a plant as a part of metabolomic studies is also changing how a plant is defined (59). it will become an important component of drug discovery programs to assure adequate compound sourcing in recollected materials, either for expanded biological evaluation or possibly to meet production demands. the combination of these techniques means that the identification of plants through gross morphology or macroscopic examination is rapidly being supplanted. 10 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 the gene-based and chemical analysis techniques now demonstrate that within a recognized plant “species” there are many “forms”, “races”, or “chemotypes”. these plants will have different functional genetic profiles under different circumstances, different biosynthetic capacities based on those gene profiles, and therefore variable and complex chemical matrices. this begins to explain why reproducible plant recollection is so challenging and frustrating. within the next few years, dna barcoding, and probably pca, will become essential and integral aspects of all medicinal plant identification, whether for drug discovery programs, or for standardization of traditional medicines and phytotherapeuticals. the result may be that the concept of a latin binomial name for a plant, romantic though that is, will be replaced, or added to, by a series of identifiers which will define both the integrity and the quality of the sample. in the biological evaluation of plant extracts tannins are a frequent confounding factor and must be removed when either enzyme or receptor-based assays are involved (60). this has led to the development of detannification and partial fractionation techniques to form peak libraries of dominant compounds in an extract which can then be screened. while minor bioactive compounds may be missed, this strategy does begin to address the issue of adequate subsequent supply when bioactivity is found, a core consideration of ecopharmacognosy (38). various data mining strategies may also be used to construct libraries of plant extracts or individual compounds, once adequate, pre-existing literature has been compiled. chemical space matches and pharmacophoric models also provide an in silico approach to the identification of compounds of potential interest which can then be isolated or further derivatives sought. rapid access to plant materials as potential resources, based on existing isolation or chemotaxonomic information to identify plants for collection, is then an essential component of the discovery strategy. multitarget therapy and synergy in considering both the quality control and the drug discovery perspectives of traditional medicines, two aspects are significantly undervalued in terms of therapeutic outcome, multitarget therapy and synergy. rather than the classical “magic bullet” approach, therapeutic outcomes have improved significantly for two of the most important disease states, cancer and aids, as a result of strategically assembled multicomponent regimens derived from considering a diverse mechanistic targeting system (61). ethnomedicine has already established that model of therapy. the chemical factory of a plant, and even a hot water extract, will contain a multitude of constituents, and multicomponent plant regimens, in which five, ten, or even twenty plants are used in a prescription, provide an unprecedented range of highly diverse chemical constituents which are affecting multiple sites and acting by diverse mechanistic pathways. an important question is whether there is clear evidence of rational use for the role of each individual component plant in a multicomponent traditional medicine? as evidence-based traditional medicines are explored for drug discovery and validation this will become an important experimental target, and eventually a consideration in the sustainability of that product. a recent discussion on network pharmacology provides an important framework for these deliberations, and for the potential design and discovery of new agents with unsuspected biological activity (62). the biological effects observed in vivo or clinically in an extract may be due to one or more active compounds acting at different sites. alternatively, two, or more, components in the mixture could be acting in a synergistic or in an antagonistic manner. in addition, the effects when a traditional medicine is taken with a single agent drug are unknown, resulting in a possible adverse drug reaction (adr) which may potentiate or inhibit the actions of the single agent drug. when combinations of medicinal plants are used, as in many drug systems around the world, the situation becomes significantly more complex. williamson (63) and wagner (64,65) have stimulated discussion in this area. a significant issue in studying synergy and antagonism in multicomponent traditional medicines has been technique and definition (65). berenbaum (66) used a mathematical definition based on an isobole to represent these biological outcomes, so that the effects of a combination of agents are independent of the mechanism of action, and can be presented graphically. a powerful demonstration of a synergistic interaction between two natural products occurs with mixtures of ginkgolides a and b examining platelet aggregation (67). potentiation of the effects of kava-kava and a passiflora extract as a sedative, and of a complex preparation of nine plants for dyspepsia whose constituent plants demonstrate effects on a range of motility-related disorders have also shown synergistic results (64,65). the protocols developed for these studies may have an important influence on the evolving strategies for the evaluation of the quality, safety, and effectiveness of an individual traditional medicine, and on the discovery and development of new, more effective combinations of medicinal plants (“designer traditional medicines”). in addition, they may offer strategies to increase the sustainability of particular medicinal plants, if the synergistic effects can be quantified and reliably reproduced. critically, such experiments require that the extract is well standardized and the biological mechanisms are well clarified prior to synergy experiments being initiated. challenges and strategies for the future a vision for natural products derived from the tropical forest is based on the concept that sustainable considerations and strategic thinking are needed now. in addition, those resources will be needed even more in the future, in order to provide medicinal agents as fossil geoffrey a. cordell – ecopharmacognosy: exploring the chemical and biological potential … 11 based resources become depleted during a period when the global population is increasing rapidly. such a vision involves the creation of a new term, ecopharmacognosy, to describe the philosophical shift, and requires new paradigms for the conduct of the natural product sciences on a global basis, including new balances of program development for drug discovery and traditional medicine quality control, new strategies, a new emphasis on evidence-based research, new alliances between various collaborators in academia, industry and government, and new values for what is meaningful, natural product research for health care enhancement (8-12,38). it should be readily apparent that humankind cannot survive another century as destructive of the earths’ resources as the 20th century. twenty-one years after the rio summit of 1992 we are as “irresponsible” as ever with respect to our biodiversity. at the end of the last century one-eighth of all plant species were threatened, 50% of bird species are likely to become extinct by 2050, and affordable oil resources are estimated to last until 2060. the abiding concern which the world must grapple with now is whether, as a human race, we can see these issues as being our destruction and own our responsibility in any attempted restoration of a balance within gaia (1). it is critical that we are mindful of the balance between the conservation of the existing rain forests, and the destruction of these fragile and deeply interwoven ecosystems and their deforestation for crop and grazing lands. our concerns are for ecological, climactic, and geological reasons, and as a way to maintain the bio-, and therefore chemo-, diversity within those forests. as described within ecopharmacognosy, plans for the development of new medicinal plants in any form to fill a market niche must be sustainable. an appropriate balance is needed between intellectual property rights and the burgeoning technology of drug discovery. a balance is also needed between all those who are stake holders for biodiversity and indigenous knowledge and those who have the capacity to potentiate (create value) in that biodiversity for health and economic benefit. this is the balance that is needed between the cbd/nagoya protocols and the trips agreement (39). numerous new alliances, both internal and external will be needed to effect the rational development of tropical forests for new medicinal and biological agents. some may already be in place; they need to be strengthened. other alliances will be new; they will need to be carefully nurtured. diversity of expertise and experience indicates that the alliances will be both local and global in nature. they will involve individuals and groups who have the capacity for high level collaboration and low level ego involvement. several examples of such collaborative programs have been described (1,52-54). the strongest programs, rather than being solely based in academia, will have industrial and government partners working together for a common goal, enhanced health care for the patient. countries which are engaged in medicinal agent discovery programs based on local natural resources are recommended to examine the structure and functioning of these programs as a potential model for collaborative investment to promote the development of local medicinal plants and natural products. in a sound and comprehensive health care program, such alliances are an integral aspect (6-12,38). in order to accomplish these goals though we must create value; value in places, in people, and in plants. the tropical forest is a huge vast ecosystem which is comprised mostly of “weeds”, those “plants whose virtues have yet to be discovered”, as ralph waldo emerson suggested in the late 19th century. an important responsibility for natural product scientists to the generations following is either to demonstrate the value of biodiversity through new discoveries of plant-derived medicinal agents, or to leave the resource alone and protected. solid linkages which unite the interests of environmental preservation, medicinal plant research and drug discovery, and the development of the agroindustrial enterprise are therefore crucial. examining import levels of finished pharmaceuticals and natural products (essential oils, and flavor and fragrance materials) in the context of developing local industrial capacity is important. structured and well-monitored natural product development programs based on tropical forest resources could lead to reduced imports and increased exports. a vision of the natural product sciences contributing to global health care offers very significant challenges to many countries. a primary challenge is to catalog and preserve the bioand chemo-diversity of the rainforests (and the oceans) for the benefit of future generations. a secondary challenge is the need to catalog the ecoand ethno-information on plants, and the chemistry and biology of their products, so that the information can be collated, analyzed, and accessed globally in real time. exploration aimed at potentiation of the biota of the world from any of several aspects, including drug discovery, necessitates systems being available locally which can offer equitable access to the biome and substantial assurances with respect to intellectual property rights and investment commitments. many medicinal plants, some of them threatened or endangered, are presently in commerce in various parts of the world, either for the production of single agent drugs, or in traditional medicine preparations. medicinal plant germplasm banks in selected locations throughout the world are needed to preserve these crucial resources, in the same way that seed crop banks are used for important food crops. at the same time, contemporary techniques for plant identification, including dna barcoding, must become integral aspects of what is considered plant identification, whether these plants are being investigated or used as medicinal plants, or whether they are being pursued for possible drug discovery of standardized preparations or single agents. the sustainable development of plants and their extracts or fractions which are marketed for health care purposes is critical. a “sustainability index” probably needs to be developed, so that patients can clearly see from a label how the product was harvested and prepared. a product derived directly from the forest and extracted 12 biology, medicine, & natural product chemistry 3 (1), 2014: 1-14 in a manner where an organic solvent was not fully recycled would get a rating of “0”, whereas one harvested organically in a sustainable manner with full recycling of any solvents, might get a rating of “5”. individual compounds and plant extracts must be evaluated using contemporary procedures and targets for both quality control and drug discovery. strategies should be developed for genomics-based, in-field bioassays to evaluate plant extracts in the field, to avoid bringing dried (or fresh) plant materials to the laboratory for extraction and bioassay. the number of natural products presented to test systems for biological evaluation could be enhanced through chemistry, combinatorial synthesis, combinatorial biosynthesis, and other strategies. drug discovery programs must be introduced, strategically developed, and enhanced based on local plants and for local diseases. most importantly, integrated global alliances are needed both in-country, and between developed and developing countries, for medicinal plant product development, in order to optimize the utilization of facilities, infrastructure, and personnel who are needed to conduct the above programs. how can these goals and the associated programs be initiated? first of all it must be said that plans of the scale described in this article and elsewhere (4-12,38) begin with small scale initiatives, reasonable benchmarks, and an appropriate timetable. they are not instantaneous “fixes” of the situation, nor will they yield rapid results. strategic planning at the highest levels is required, combining government agencies, industrial enterprises, international agencies and foundations, academic institutions, and private consultants with natural product drug discovery and development experience. planning and proposal development may take 1-2 years of meetings, discussions, and consultations to evolve. over time, countries, or consortia of countries, will require an infrastructure to foster the development of their own sustainable medicinal agents from natural sources based on the quality of their natural product sciences. programs will be needed to assist countries to potentiate their infrastructure and resources, including their facilities and their scientists, in order to evaluate and standardize natural product-based medicinal agents on a sustainable basis for their health care systems, and that may take 510 years to evolve. ideally, as proposed originally in 2002 (3), what is drastically needed is a global alliance for natural product development and health care. such an alliance would be composed of international agencies (who, unido, undp, nato, eu, wipo, etc.), government agencies (nih, nsf, nie, src, daad, etc.), global and local pharmaceutical companies, academic institutions, non-government organizations (wwf, wri, cyted, tramil, ifs, twas, etc.), scientific societies (iupac, rsc, asp, pse, ga, jsps, etc.), and major foundations (ford, gates, macarthur, rockefeller, nippon, etc.). a mechanism to bring representatives together to discuss the global issues and implications in new terms, with a new set of goals, with a new agenda, but most importantly with a new vigor, is vital for the global development of natural product based drugs for health care. conclusion globally, natural products, in the form of purified active principles and plant extracts, are the cornerstone of primary health and for the amelioration of life-style conditions, and will remain so for decades to come. with a burgeoning population, the challenges for health care in the future remain significant. for most of the major killer and chronic diseases in the world there are no truly effective drug treatments. drug resistance to existing chemotherapeutic regimens for fungal and bacterial infections, for aids, for cancer, and for malaria, is increasing in an unabated and alarming manner. many killer and debilitating diseases in the middle and lowincome areas of the world exist without any effective drug treatment, or even a drug discovery program, in place. yet, overall health care is slowly improving, life expectancy is rising in most countries, and more children are surviving beyond their first five years. these are contributing factors to the dramatic rise in the global population. however, without a new strategic approach, this population will not have adequate resources to provide basic health care. there is no choice in this matter, medicinal plants must be an essential, sustainable, and fully 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of the leaf extract and fractions of dryopteris filix-mas (l.) schott could be attributed to the abundance of polyphenol compounds earnest oghenesuvwe erhirhie1,2,*, emmanuel emeka ilodigwe1, daniel lotanna ajaghaku3, blessing ogechukwu umeokoli4, peter maduabuchi eze5, festus basden chiedu okoye4 1department of pharmacology and toxicology, faculty of pharmaceutical sciences, nnamdi azikiwe university, awka, nigeria. 2department of pharmacology and toxicology, faculty of pharmaceutical sciences, chukwuemeka odumegwu ojukwu university, igbariam, nigeria. 3department of pharmacology, faculty of pharmaceutical sciences, enugu state university of science and technology, enugu state, nigeria. 4department of pharmaceutical and medicinal chemistry faculty of pharmaceutical sciences, nnamdi azikiwe university, awka, nigeria. 5department of environmental health science, faculty of health sciences and technology, nnamdi azikiwe university, nnewi campus, anambra state, nigeria. corresponding author* erhirhieochuko@yahoo.com, tel: +234-7060434974 manuscript received: 17 august, 2019. revision accepted: 06 february, 2020. published: 10 april, 2020. abstract dryopteris filix mas (d filix-mas) is wildly used in ethnomedicine for the management of rheumatoid arthritis, wounds and other diseases. we investigated the anti-oxidant activities of its leaf extract, and chromatographic fractions. the ethanol leaf extract was partitioned into four fractions; n-hexane, ethyl acetate, n-butanol and water. ferric reducing anti-oxidant power (frap), 1, 1-diphenyl-2-picrylhydrazil (dpph) and nitric oxide (no) scavenging in vitro assays were carried out on the extract and fractions at 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml. the most active fraction (ethyl acetate fraction) was further purified using chromatographic techniques to isolate its major compound whose structure was elucidated using id nuclear magnetic resonance (nmr) and mass spectrometry. the ethyl acetate fraction produced the highest free radical scavenging activity among the other fractions. the fraction (vlc-e7) from which the bioactive compound, quercetin-3-o-αl-rhamnopyranoside, was isolated had the best frap and dpph scavenging activities with ec50 and ic50 values of 88.81 ± 3.41 and 26.87 ± 0.24 respectively more than the ethyl acetate fraction. this study revealed that the polyphenol flavonoid, quercetin-3-o-αl-rhamnopyranoside could be responsible for antioxidant activity of ethno-medicinal property of d filix-mas leaf. keywords: dryopteris filix-mas; wounds; rheumatoid arthritis; quercetin-3-o-αl-rhamnopyranoside; anti-oxidant properties introduction free radicals are constantly produced in all normal living cells (kumar et al., 2014). imbalance between the production of these radicals and endogenous antioxidants creates a state of oxidative stress with its associated damaging effects on endogenous molecules such as lipids, proteins and nucleic acids (yan et al., 2015). unbridled oxidative stress has been reported to be implicated in several chronic and degenerative diseases such as diabetes, rheumatoid arthritis, cancer, neurodegenerative diseases, atherosclerosis, ischemic heart disease, ageing among others (khatoon et al., 2013). in attempt to ameliorating oxidative damages caused by free radicals, synthetic antioxidants such as butylated hydroxytoluene (bht), butylated hydroxyanisole (bha), and tert-butylhydroquinone (tbhq) had been developed. however, these synthetic antioxidants left much to be desired due to their side effects, such as liver toxicity, carcinogenicity as well as their high cost and inaccessibility (deepa et al., 2014). these limitations have inspired the search for more effective antioxidant from natural source, especially medicinal plants, which are biodegradable, less toxic, affordable and accessible (ezeja et al., 2015; ahlem et al., 2015). d filix-mas is an evergreen fern belonging to the family of dryopteridaceae. male fern, worm fern, aspidium and shield fern are common names assigned to it. it grows between 60-150 cm high and it is habitat to stream and moist environments (uwumarongi et al., 2016). it is native to europe, asia, and north america. its leaves are bipinnated and consist of 20-35 pinnae on each side of the rachis (fig 1). its stalks are covered with orange-brown scales (bafor et al., 2017). the leaf decoction is ethno medicinally used in rheumatoid arthritis, wounds, bleeding disorders, ulcers, worm infestation and malaria (tagarelli et al., 2010; erhirhie et al., 2019). it is also reported as one of the ferns with https://doi.org/10.14421/biomedich.2020.91.1-6 2 biology, medicine, & natural product chemistry 9 (1), 2020: 1-6 useful secondary metabolites against chronic diseases and aging (valentyna et al., 2017). previous studies have found that it possesses antihelmintic activity (urban et al., 2014), antimicrobial activity (mandal and mondal, 2014), antidiarrheal (uwumarongi et al., 2016), uterine relaxant (bafor et al., 2017) and anti-inflammatory (erhirhie et al., 2019) activities. a preliminary study by sekender et al, (2012) revealed that it possesses anti-oxidant activity. in this present study, we evaluated the antioxidant activity of the leaf extract and fractions. the bioactive compound responsible for its antioxidant activity was isolated and characterized. figure 1. d. filix-mas photograph. methodology materials visible spectrophotometer (721g, zhejiang, china), thermostatic water bath (equitron mumbai india), analytical weighing balance (ohaus corp. nj usa), quercetin (institute of pharmaceutical biology and biotechnology, heinerich-heine university, dusselforf, germany), 2,2-diphennyl-1-picrylhydrazyl, dpph (sigma aldrich), ascorbic acid (sigmaaldrich). procedure plant collection, extraction, fractionation and purification of active compound plant collection, authentication, extraction, fractionation, chromatographic separation, and structural elucidation techniques were carried out using similar method as described in our earlier study (erhirhie et al., 2019). the purification and structural elucidation was based on bioactivity guided antioxidant screening of liquid-liquid fractions (n-hexane, ethyl acetate, butanol, and water fractions), vacuum liquid chromatographic fractions and sephadex fractions. dpph scavenging assay the test was carried out based on the method described by ajaghaku et al (2017). the reaction mixture contained 0.1 ml of various concentrations (6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml) of sample, 0.1 ml of 0.6 mmol of dpph and 0.8 ml of methanol. the mixture was incubated in the dark for 30 minutes at room temperature. the absorbance of the sample was measured at 517 nm against blank (methanol) using a spectrophotometer. ascorbic acid and quercetin were used as standards. a tube containing 0.1 ml of dpph solution and 0.9 ml of methanol served as control. experiments were carried out in duplicate. free radical scavenging activities of each sample were determined as follows: 𝐷𝑃𝑃𝐻 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = (𝐴𝐶– 𝐴𝑆)/𝐴𝐶) × 100 ac : average absorbance of control as : average absorbance of sample a graph of percentage inhibition against concentration was plotted and the concentration that produced 50% inhibition (ic50) was extrapolated using a regression analyses equation. ferric reducing antioxidant power (frap) assay frap assay was carried out following the method described by habibur et al, (2013). two hundred and fifty microliter (0.25 ml) of various concentrations, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml of samples were mixed with 0.625 ml of phosphate buffer and 0.625 ml of 1% potassium ferricyanide [k3fecn6]. the mixtures were heated at 50oc for 20 minutes. then, 0.625 ml of 10% trichloroacetic acid (tca) was added and the mixtures were centrifuged at 3000 rpm for 10 minutes. from the upper layer, 0.625 ml was pipetted and mixed with 0.625 ml of distilled water and 0.125 ml of 0.1% (w/v) ferric chloride (fecl3) solution. absorbances of the mixtures were measured at 700 nm against air using a spectrophotometer. ascorbic acid and quercetin were used as standards. tests were performed in duplicate and percentage inhibition was calculated using the formula below. % inhibition = average absorbance of sample−average absorbance of blank 1 x 100 1 erhirhie et al. – antioxidant activities of the leaf extract and fractions … 3 a graph of percentage inhibition against concentration was plotted and the effective concentration (ec50) was extrapolated using a regression analyses equation. nitric oxide scavenging activity this assay was carried out following the method described by ezeja et al., (2015). two milliliter of 10 mm sodium nitroprusside prepared with phosphate buffer saline (ph 7.4) was mixed with 0.5 ml of samples at various concentrations (6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml). the mixture was incubated at room temperature for 150 minutes. thereafter, 0.5 ml of the reaction mixture was withdrawn and mixed with 0.5 ml of griess reagent (1% sulphanilamide + 0.1% naphthylethylenediamine dichloride + 3% phosphoric acid) was added to each test tube. the absorbance of the pink chromophore formed was measured at 540 nm. ascorbic acid and quercetin were used as standards. assays were carried out in duplicates. percentage inhibition was calculated using the formula below abs control−abs test abs control × 100 1 inhibitory concentration, ic50 was estimated from graph of % inhibition against concentration using regression analyses equation statistical analysis results were presented as mean ± standard error of mean (sem) using statistical package for social science (spss, version 20). calculation of fifty percent inhibitory concentration (ic50) of the extracts and fractions was carried out using regression equation in microsoft excel, 2010. results dpph, scavenging activity of extract and fractions in dpph scavenging assay, 50% inhibitory concentration (ic50) of 134.12 ± 2.88, 94.97 ±0.14, 63.91 ± 1.29, 62.09 ± 0.21and 45.72 ± 0.16 µg/ml were recorded in n-hexane, water fractions, extract, butanol and ethyl acetate fractions respectively. the ethyl acetate fraction produced the lowest ic50 value among other samples, except ascorbic acid and quercertin which showed ic50 values of 12.26 ± 0.20 µg/ml and 10.46 ± 0.15 µg/ml respectively (table 1). inhibition against dpph at 100 µg/ml by vlc fractions ethyl acetate vlc fractions, vlc-e1 (78.68%), vlce3 (52.33%), vlc-e7 (72.38%), vlc-e6 (73.84%), vlc-e14 (65.60%), vlc-e8 (73.08%), vlc-e10 (74.81%), vlc-e12 (72.09%), vlc-e16 (69.67%) produced more than 50 % inhibition against dpph (table 2). table 1. free radical (dpph) scavenging activities of extract and fractions of d filix-mas. dpph, scavenging activity, ic50(ug/ml) extract 63.91 ± 1.29 n-hexane fraction 134.12 ± 2.88 ethyl-acetate fraction 45.72 ± 0.16 butanol fraction 62.09 ± 0.21 water fraction 94.97 ±0.14 ascorbic acid 12.26 ± 0.20 quertcetin 10.46 ± 0.15 values are expressed as mean ± standard error of mean. table 2. dpph scavenging activities of ethyl acetate vacuum liquid chromatographic fractions of d filix-mas. solvent ratio sample code inhibition against dpph (%) ethyl acetate fraction 73.26 n(500): e (0) vlc-e1 78.68 n(450): e (50) vlc-e2 19.38 n(400): e(100) vlc-e9 18.12 n(350): e(150) vlc-e13 23.06 n(300):e(200) vlc-e4 28.00 n(250): e(250) vlc-e11 36.34 n(200):e(300) vlc-e3 52.33 n(150):e(350) vlc-e15 16.86 n(100):e(400) vlc-e5 12.79 n(50): e(450) vlc-e17 37.79 n(0):e(500) vlc-e7 72.38 d(500):m(0) vlc-e6 73.84 d(450):m(50) vlc-e14 65.60 d(350):m(150) vlc-e8 73.06 d(250):m(250) vlc-e10 74.81 d(100):m(400) vlc-e12 72.09 d(0):m(500) vlc-e16 69.67 n = n-hexane, e = ethyl acetate, m = methanol. ferric reducing antioxidant power (frap), nitric oxide (no) and dpph scavenging activities of extract selected fractions. frap activity in frap assay, quercetin and ascorbic acid produced the lowest ec50 values followed by vlc-e7, ethyl acetate fraction, extract and butanol fraction. in nitric oxide scavenging assay, ascorbic acid exhibited the lowest ic50 value (14.74 ± 0.11 µg/ml) followed by ethyl acetate fractions (15.38 ± 3.65 µg/ml) and butanol fractions (15.45 ± 2.72 µg/ml), followed by vlc-e7 fraction (16.66 ± 0.48 µg/ml), quercetin (18.14 ± 2.57 µg/ml) and extract (1854.60 ± 200.25 µg/ml). in dpph assay, quercetin and ascorbic acid produced the lowest ic50 values (11.50 ± 0.08 and 14.19 ± 0.43µg/ml) followed by vlc-e7 (26.87 ± 0.24 µg/ml), ethyl acetate fraction (50.25 ± 0.40 µg/ml), butanol fraction (62.09 ± 0.21 µg/ml) and crude extract (63.91 ± 1.29 µg/ml) (table 3). 4 biology, medicine, & natural product chemistry 9 (1), 2020: 1-6 dpph, scavenging activity of sephadex fractions dpph scavenging activities of samples tested decreased in the following order; quercetin (10.46 ± 0.15 µg/ml) > ascorbic acid (12.26 ± 0.20 µg/ml) > sph-e3 (26.35 ± 1.38 µg/ml) > vlc-e7 (26.87 ± 0.24 µg/ml) > sphe6 (33.44 ± 0.38 µg/ml) > sph-e5 (37.30 ± 0.30 µg/ml) > sph-e7 (46.95 ± 0.61 µg/ml) > sph-e4 (86.10 ± 7.69 µg/ml) (table 4). table 3. ferric reducing antioxidant power (frap), nitric oxide (no) and dpph scavenging activities of extract and fractions of d filix-mas. sample frap, ec50 (µg/ml) nitric oxide, ic50 (µg/ml) dpph, ic50 (ug/ml) extract 1854.60 ± 200.25 38.51 ± 2.23 63.91 ± 1.29 ethyl acetate fraction 461.75 ± 22.91 15.38 ± 3.65 50.25 ± 0.40 butanol fraction 1911.03 ± 137.44 15.45 ± 2.72 62.09 ± 0.21 vlc-e7 88.81 ± 3.41 16.66 ± 0.48 26.87 ± 0.24 ascorbic acid 69.34 ± 2.41 14.74 ± 0.11 14.19 ± 0.43 quercetin 30.81 ± 0.86 18.14 ± 2.57 11.50 ± 0.08 frap: ferric reducing antioxidant power. values are expressed as mean ± standard error of mean. table 4. dpph scavenging activity, of sephadex (sph) fractions of d filix-mas. dpph, scavenging activity, ic50( µg/ml) vlc-e7 26.87 ± 0.24 sph-e1 sph-e2 22.10 ± 0.62 sph-e3 26.35 ± 1.38 sph-e4 86.10 ± 7.69 sph-e5 37.30 ± 0.30 sph-e6 33.44 ± 0.38 sph-e7 46.95 ± 0.61 ascorbic acid 12.26 ± 0.20 quercetin 10.46 ± 0.15 values are expressed as mean ± standard error of mean. discussion it is well established that by-products of oxygen metabolism produce free radicals such as reactive oxygen and nitrogen species which results to cellular damage and pathogenesis of several diseases such as cancer, cardiovascular diseases, diabetes, neurodegenerative diseases among others (li et al., 2016). there is a revitalization of interest in the search for natural anti-oxidant that could ameliorate reactive oxygen species which are implicated in numerous disease conditions (ahlem et al., 2015; deepa et al. 2014). in this study, we investigated the antioxidant properties of the extract and fractions of d filix-mas and also elucidated the major compound responsible for its antioxidant activity using in vitro bioassay guided isolation approach. dpph test is a widely acceptable and reproducible assay for screening potential antioxidants (patel et al., 2010). this method involves the ability of anti-oxidants to donate electrons to dpph thereby causing discoloration of the reaction mixture from deep violet color to yellow color (patel et al., 2010). the yellow color change observed in this study substantiates the ability of the extract and fractions of d filix-mas to scavenge free radicals generation by dpph. this further suggests that the constituents present in the extract and fractions could ameliorate oxidative damages by free radicals. reduction of ferric (fe3+) to ferrous (fe2+) ions is a documented method of determining antioxidants with reducing power, capable of breaking free radical chain progression in lipid peroxidation (habibur et al., 2013). from this study, reduction of iron (iii) complex to the ferrous form, iron (ii) could be a possible antioxidant mechanism as a result of the presence of reducing agents in the extract and fractions of d filix-mas. phenolic compounds such as quercetin has been reported to prevent free radicals by forming complex and chelating metal ions such as iron and cupper, thereby preventing auto-oxidative damage to the living system (nimse and pal, 2015). nitric oxide generated following incubation of sodium nitroprusside with phosphate buffer in the presence of oxygen is known to cause toxicity to biomolecules (deepa et al., 2013). the scavenging capacities of the extract and fractions against nitric oxide generation suggest that d filix-mas could prevent nitric oxide generation in various disease conditions. polyphenols such as quercetin has been reported to interact with nitric oxide synthase resulting in modulation of nitric oxide production (hussain et al., 2016). the roles of phenolic compounds from medicinal plants as supplement in the treatment of oxidative stress and free radicals mediated tissue damage cannot the overemphasized (li et al., 2016). flavonoids and phenolic acids from polyphenols have been reported to play a significant role in scavenging free radicals and prevention of oxidative damage to cells. phenolic compounds have capacity to neutralize several forms of oxidizing free radicals due to their electron donating abilities (habibur et al., 2013; ajaghaku et al., 2017). ojo et al., (2013) reported that flavonoids inhibit lipid peroxidation in various biological systems. presence of hydroxyl groups in flavonoids allows them to disconnect free radical chain reactions via the formation of intramolecular hydrogen bonds (monika et al., 2011). erhirhie et al. – antioxidant activities of the leaf extract and fractions … 5 from the forgoing, antioxidant properties elicited by the extract and fractions of d filix-mas could be attributed to presence of flavonoids. this is substantiated by presence of the flavonoid, quercitrin as the major compound in the hplc chromatogram of the extract and fractions of d filix-mas as documented in our earlier study (erhirhie et al., 2019). the ethyl acetate vlc fraction (vlc-e7), where the compound, quercetin-3-o-αl-rhamnopyranoside was isolated exhibited the highest anti-oxidant activity compared to the extract and other fractions. in line with this finding, quercetin-3-o-αl-rhamnopyranoside isolated from other medicinal plants was found to elicit significant antioxidant activity (zhang et al., 2014). it is possible that possible that quercetin-3-o-αlrhamnopyranoside may be the key antioxidant compound of d filix-mas. quercetin, the most widely distributed flavonoid in nature, existing mostly in its glycoside form quercitrin has been reported to possess various properties such as antioxidant, anti-inflammatory, neuroprotective, antiviral, anticancer, hepatoprotective, cardioprotective, antimicrobial and anti-obesity properties (maalik et al., 2014). mir, et al., (2017) reported that flavonoids from tridax procumbens possess antioxidant properties. phenolic compounds, catechin, epicathechin, dihydroquercetin isolated from alchornea floribunda leaf were also found to elicit antioxidant properties (ajaghaku et al., 2017). in this study, the ability of extract and fractions of d filixmas to scavenge generation of free radicals from dpph, nitric oxide as well as its chelating capacity could be attributed to the phenolic compound, quercetin3-o-αl-rhamnopyranoside, which was postulated to be responsible for its anti-inflammatory activity as earlier reported (erhirhie et al., 2019). conclusion the isolated flavonoid, quercetin-3-o-αlrhamnopyranoside from ethyl acetate chromatographed fraction of d filix-mas could be responsible for its antioxidant activity, which validates its folkloric use in the management of rheumatoid arthritis and other free radical mediated diseases. acknowledgments the authors are thankful to prof. dr. peter proksch of the institute of pharmaceutical biology and biotechnology, heinrich-heine university, düsseldolf, germany for making their facilities available for the high performance liquid chromatography 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shaohua, c. and zhiyun, d., 2015. curcumin, inflammation, and chronic diseases: how are they linked? molecules, 20, 9183-9213; doi:10.3390/molecules20059183. zhang, y., wang, d., yang, l., zhou, d., zhang, j. 2014, purification and characterization of flavonoids from the leaves of zanthoxylum bungeanum and correlation between their structure and antioxidant activity, plos one, 9(8): e105725. doi: 10.1371/journal.pone.010572. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 1, 2019 | pages: 7-10 | doi: 10.14421/biomedich.2019.81.7-10 issn 2540-9328 (online) statistical analysis of habbatussauda’s benefits for health (blood pressure, glucose and uric acid) sugiyanto1,*, luqyana khalda’ aesa2, meksianis z. ndii3 1,2departement of mathematics, universitas islam negeri sunan kalijaga, yogyakarta, indonesia. 3department of mathematics, faculty of science and engineering, university of nusa cendana, kupang-ntt, indonesia. author correspondency*: sugimath@yahoo.co.id abstract habbatussauda is one of the traditional medicine existed since a long time ago and this is one of the drugs that recommended by the prophet muhammad. habbatussauda’s benefits have been studied extensively in the healthcare. habbatussauda has also been widely used to cure various diseases. this study revealed the benefits of habbatussauda in lowering blood pressure, glucose and uric acid levels in 20 respondents that given habbatussauda for 2 weeks. the blood pressure, glucose and uric acid levels were measured before and after consuming habbatussauda. blood pressure, glucose, and uric acid levels in the body will decrease after consuming habbatussauda that shown in the statistical analysis of the obtained data. keywords: blood pressure; glucose; habbatussauda; herbal medicine; statistical analysis; uric acid introduction based on data from who (world health organization) in 2010, the population of indonesia is 249.866.000, makes indonesia to be one of the most populated countries. increasing number of population will certainly make many problems, one of them is a health problem. some health problems that frequently occur in indonesia are increased glucose level, increased blood pressure, obesity, and many more, whereas the highest rate of disease caused death are: stroke, coronary heart disease, diabetes mellitus, respiratory infections, tuberculosis, liver cirrhosis, lung disease, and hypertension. it makes a lot of indonesians searching for alternative medicines or just preventive medicines of various diseases. one of them is the herbal treatments, which is currently in great demand, one of them is nigella sativa, or commonly known as black cumin, black seed or habbatussauda. habatussauda is one of the traditional medicines used by people to cure various diseases. prophet muhammad said that habatussauda can cure all diseases except death (osman et al., 2014). contemporary naturopathic medicine all over the world have been using habbatussauda which has thymoquinone as a major component (salahshoor et al., 2018). thymoquinone contained in the habbatussauda has a protection function to counter nephrotoxicity and hepatotoxicity. it also has pharmacological effects which include antihelmintic, anticestoda, and antischistosoma, antibacterials, antifungi, antiviral, antioxidant, antiinflammatory, and can improve the immune response of cells that are mediated t (abdulelah & abidin, 2007). habbatussauda can be used for antihypertensive treatment, antitumor, antibacterial, antioxidant, antidiabetic and antidyslipidemia (hussain & hussain, 2016). other benefit of habbatussauda is antihyperuricemia (suhendi & sutrisna, 2011). habbatussauda is also used as a diuretic (aboul-enein & abou-basha, 1995). the study of habbatussauda’s benefits are quite a lot and still being developed. mathematics has an important role in knowing the benefits of habbatussauda, by using statistics. statistics used to process the primary data the obtained habbatussauda’s benefit data will be processed by statistic methods. figure 1. habbatussauda powder. materials and methods in this study, respondents from ages between 19 – 23 years old with the sample size of 20 respondents (11 https://doi.org/10.14421/biomedich.2019.81.7-10 8 biology, medicine, & natural product chemistry 8 (1), 2019: 7-10 female, 9 male). taken respondents do not have history of serious illness, disease history owned by the respondents include stomach ucler, typhoid, and dengue fever. in this study, respondents did not consume other medicine besides habbatussauda that given by the researchers. respondents were students of uin sunan kalijaga who had a history of stomach ucler, typhoid, and dengue fever. before starting the study, first thing to to check is a blood test to determine the initial value of blood pressure, glucose and uric acid before giving each respondent the habbatussauda. furthermore, respondents who take a blood test will be given 42 habbatussauda capsule for two weeks consumption, the respondent consumes 3 habbatussauda capsules daily, each capsule contains 600 mg of pure habbatussauda. habbatussauda used is the type of powder brand kurma ajwa containing 120 capsules. then the blood test were carry on after two weeks to take final value of respondent’s blood pressure, glucose and uric acid after consuming habbatussauda to know whether it decrease or increase. during the two weeks habbatussauda consumption, respondents were contacted by phone to remind them to consume habbatussauda and ask them if there are any complaints or perceived side effects while taking the habbatussauda. taking blood samples to check the measure of glucose and uric acid is done by taking a blood sample from the respondent, then checked it by using the glucose and uric acid tool check, whereas for checking blood pressure was checked by using sphygmomanometer. the data before and after consumption of habbatussauda taken from respondents were collected, then statistical analysis was perform using several kinds of tests, using statistical package for social science (spss) version 15.0. the first test used to test for normality of 95% significance level to determine whether the obtained data is normally distributed or not. then after the normality test, t test was perform with 95% significance level to determine whether there is a significant difference in the average before and after consumption of habbatussauda. at this t test if the value of sig is more than 0.05 then the two data do not have significant differences, but if sig is less than 0.05 then the data is a significant difference. results the average research results that have been obtained from 20 respondents are as follows (table 1). according table 1. average of the systole before consuming habbatussauda is 119.1 mmhg and average of the diastole before consuming habbatussauda is a 81.6 mmhg. while the average of the systole after consuming habbatussauda is 111.35 mmhg and average diastole after consuming habbatussauda is 75.9 mmhg. table 1. blood pressure average before and after consuming habbatussauda. average blood pressure systole (mmhg) diastole (mmhg) before consuming habbatussauda 119.1 81.6 after consuming habbatussauda 111.35 75.9 table 2. glucose average before and after consuming habbatussauda. average glucose (mg/dl) before consuming habbatussauda 90.3 after consuming habbatussauda 84.6 according table 2, average of the glucose level before consuming habbatussauda is a 90.3 mg/dl, while the average of the glucose after consuming habbatussauda is a 84.6 mg/dl. table 3. uric acid average before and after consuming habbatussauda. average uric acid (mg/dl) before consuming habbatussauda 5.065 after consuming habbatussauda 4.555 according table 3, average of the uric acid before consuming habbatussauda is a 5.065, while the average of the uric acid after consuming habbatussauda is 4.555 mg/dl. furthermore, the test for normality using the shapiro-wilk test described below in the following table. table 4. test result shapiro-wilk blood pressure. shapiro-wilk statistic (mmhg) df p systole before consuming habbatussauda 0.9191 20 0.096 diastole before consuming habbatussauda 0.972 20 0.793 systole after consuming habbatussauda 0.958 20 0.505 diastole after consuming habbatussauda 0.921 20 0.102 according table 4, results of normality test using the shapiro-wilk test showed that probability value (p) of systole before (0.096), diastole before is 0.793, systole sugiyanto et al. – statistical analysis of habbatussauda’s benefits for health … 9 after is 0.505, diastole after is 0.102 is more than 0.05 so it was concluded that the data are normally distributed. table 5. test result shapiro wilk glucose. shapiro-wilk statistic (mg/dl) df p before consuming habbatussauda 0.969 20 0.730 after consuming habbatussauda 0.946 20 0.309 according table 5, results of normality the test results for glucose showed that probability value (p) of before consuming habbatussauda is 0.730 and after consuming habbatussauda is 0.309 is more than 0.05 so it was concluded that the data are normally distributed. according table 6, normality test for uric acid showed that probability value before consumption habbatussauda is 0.113 and probability value after consumption of habbatussauda is 0.883 is more than 0.05 so it was concluded that the data are normally distributed. table 6. test result shapiro wilk uric acid. shapiro-wilk statistic (mg/dl) df p before consuming habbatussauda 0.923 20 0.113 after consuming habbatussauda 0.977 20 0.883 from the results above, it is known that the data are normally distributed with a probability value (sig.) more than 0.05 (p > 0.05). furthermore, the data will be tested average differences using the t test with the following results. table 7 shows decreasing in the average systolic and diastolic blood pressure before and after consuming habbatussauda, a decrease of 7.73 mmhg in systolic and a decrease of 5.7 mmhg in diastole with a probability value (sig.) for systole (0.019) and diastole (0.012) is less than 0.05 it can be seen that there are differences in blood pressure values were significantly before and after consuming habbatussauda. table 7. t test result blood pressure. no variable average standard deviaton t test p systole (mmhg) diastole (mmhg) systole diastole systole diastole systole diastole 1. before 119.01 81.6 16.56852 9.13870 2.556 2.778 0.019 0.012 2. after 11.37067 10.90099 75.9 111.35 table 8. t test result glucose. no variable average (mg/dl) standard deviation t test p 1. before 5.065 1.26419 2.338 0.030 2. after 4.555 0.96381 table 9. t test result uric acid. no variable average (mg/dl) standard deviation t test p 1. before 90.30 12.60368 2.181 0.042 2. after 84.60 13.23631 table 8 shows decreasing the average content of glucose before and after consuming habbatussauda, a decrease of 5.7 mg/dl with a probability value (sig.) 0.042 less than 0.05 so that it can be seen that there is a difference in the value of the content of glucose in the body significant before and after consuming habbatussauda. table 9 shows decreasing the average content of uric acid before and after consuming habbatussauda, a decrease of 0.51 mg/dl with a probability value (sig.) 0.030 less than 0.05 so it can be seen that there are differences in the content value of uric acid in the body significant before and after consuming habbatussauda. 10 biology, medicine, & natural product chemistry 8 (1), 2019: 7-10 conclusions this study shows the results of statistical analysis habbatussauda‘s benefits on blood pressure, glucose and uric acid that showed significant differences between the values before and after consuming habbatussauda. it is visible from a probability value obtained is less than 0.05. from these results and from previous studies show that habbatussauda can lower blood pressure values, glucose and uric acid contained in the body, this research confirm previous studies that have examined the habbatussauda’s benefits both medically and other research. references abdulelah, h. a. a., & zainal-abidin, b. a. h. (2007). in vivo anti-malarial tests of nigella sativa (black seed) different extracts. am j pharmacol toxicol, 2(2), 46-50. aboul-enein, h. y., & abou-basha, l. i. (1995). simple hplc method for the determination of thymoquinone in black seed oil (nigella sativa linn). journal of liquid chromatography & related technologies, 18(5), 895-902. hussain, d. a., & hussain, m. m. (2016). nigella sativa (black seed) is an effective herbal remedy for every disease except death-a prophetic statement which modern scientists confirm unanimously: a review. adv med plant res, 4(2), 27-57. osman, m. t., hamza, a. j. a., omar, e., & adnan, a. (2014). the new miracle of habbatus sauda: its major component thymoquinone can be used in the management of autoimmune diseases. procedia-social and behavioral sciences, 121, 304314. salahshoor, m. r., haghjoo, m., roshankhah, s., makalani, f., & jalili, c. (2018). effect of thymoquinone on reproductive parameter in morphine-treated male mice. advanced biomedical research, 7. suhendi, a., & sutrisna, e. m. (2011). antihyperurisemia activity of water extract of black seed (coleus ambonicuslour) in balb-c mice and its standardization. indonesian journal of pharmacy, 77-84. world health organization. (2010). world health statistics 2010. world health organization. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 105-110 | doi: 10.14421/biomedich.2021.102.105-110 issn 2540-9328 (online) phytochemical screening, antioxidant and antibacterial activities of the root extract of cyphostemma adenocaule (steud. ex a. rich.) wild & r.b.drumm abdulbasit haliru yakubu1,3,*, mohammed mustapha mohammed2, abdulqadir bukar bababe1, hassan yesufu braimah1 1department of pharmaceutical chemistry, pmb 1069, faculty of pharmacy, university of maiduguri, borno state, nigeria. 2department of microbiology, faculty of science, pmb 1069, university of maiduguri, maiduguri, nigeria. 3department of pharmaceutical service, pmp 1414, university of maiduguri teaching hospital, maiduguri, nigeria. corresponding author* pharmahy071@gmail.com manuscript received: 13 september 2021. revision accepted: 10 october, 2021. published: 11 october, 2021. abstract plant secondary metabolites have provided important bioactive principles for developing new lead compounds. within their confinement, they exhibit unique chemical diversity, which influences their diverse biological properties. the vitaceae family is known for its potent antioxidant and antibacterial phytoconstituents, among other biological properties. cyphostemma adenocaule is one of the family members explored for its ethnomedicinal properties. this study undertook the evaluation of the phytochemical, antioxidant, and antibacterial properties of the root extract of cyphostemma adenocaule. preliminary phytochemical screening revealed the presence of flavonoids, alkaloids, carbohydrates & glycoside, saponins, and tannins. the methanol root extract had the highest activity in the dpph assay, providing ic50 (50% inhibition) of 10.87µg/ml, followed by n-hexane (ic50 74.10µg/ml) and chloroform (ic50 74.31µg/ml) extract. in the antibacterial assay, the chloroform extract was active against e. coli (24.00±0.15) and had moderate activity against staph. aureus (12.5±0.18). the n-hexane extract was completely inactive against the test organisms while the methanol extract showed poor activity against the test organisms. the present study adds to the existing literature on cyphostemma adenocaule with scientific evidence into its biological properties. keywords: cyphostemma adenocaule; phytochemical screening; antioxidant and antibacterial activity. abbreviations: ca1 – cyphostemma adenocaule 1; chcl3 – chloroform; cosy – correlation spectroscopy; dept – distortions enhancement by polarization transfer; eta – ethyl acetate; hmbc – heteronuclear multiple bond correlation; hsqc – heteronuclear single quantum correlation; khso4 – potassium bisulphide; meoh – methanol; moa – mechanism of action; nhex – hexane; nmr – nuclear magnetic resonance; tlc – thin layer chromatography; who – world health organization. introduction natural products have been in existence for ages and evolved with unique chemical diversity, which results in their diverse biological activities and drug-like properties. these compounds present as important resources for developing new lead compounds and scaffolds (galm & shen, 2007). morphine from the opium poppy plant is considered the first pharmacologically active compound isolated by friedrich sertürner (hamilton & baskett, 2000; joo, 2014). natural products are important for the development of new drugs, and these products have been in constant use. drugs used as anticancer, antihypertensive, and antimigraine medication, have benefited greatly from natural products (joo, 2014; newman et al., 2003) plants have been part of traditional medicine systems, which have been used for thousands of years (iwu, 2014). these plant-based systems continue to play an essential role in health care, and it has been estimated by the world health organization (who) that approximately 80 % of the world’s inhabitants rely mainly on traditional medicines for their primary health care (who, 2017) cyphostemma adenocaule (steud. ex a. rich.) wild & r.b.drumm is a climbing, scrambling, or trailing herb that belongs to the vitaceae family (bello et al., 2019; wickens & burkill, 1986) and is locally known as yáákùwár fátààkéé (hausa, nigeria) (wickens & burkill, 1986). the plant is a popular, non-cultivated vegetable eaten in many parts of africa i.e., nigeria, ghana, congo, uganda, ethiopia, and eritrea (bello et al., 2019). the plant had been documented for its https://doi.org/10.14421/biomedich.2021.102.105-110 106 biology, medicine, & natural product chemistry 10 (2), 2021: 105-110 ethnomedicinal value, with a comprehensive review given by bello and colleagues (2019). the effectivity of plant bioactive compounds against oxidative stress-related diseases and as an anti-infective had been well explored. this study entails investigating the phytochemical, antioxidant, and antibacterial properties of the root extracts of c. adenocaule by employing standard protocol. the results from the study will justify the ethnomedicinal uses of the plant and underscore its potentials as a source of antioxidants and antimicrobial agents. materials and method plant collection and identification fresh root parts of c. adenocaule were collected aseptically in july 2019 from shuwarin town, dutse lga, jigawa state, nigeria, and identified at the medicinal botany section, of the department of biology, ahmadu bello university, zaria, nigeria. preparation of plant extract and its fractions the preparation of plant material and fractions employed in our previous work on c. adenacaule was adopted with modifications (yakubu et al., 2020). in this study, one and a half kilograms (1.5kg) of the pulverized sample material was extracted with hexane, chloroform, and methanol. preliminary phytochemical screening phytochemical screening was carried out on the crude extracts to detect the presence of plant secondary metabolites; alkaloids, anthraquinones, flavonoids, glycosides, steroids, tannins, terpenoids, and carbohydrates using standard procedures as described in the literature (brain, kr and turner, 1975; evans, 2009; markham, 1982; sofowora, 1996; vishnoi, 2009). biological activity test for antioxidant activity: dpph assay the radical scavenging potential was done using dpph assay (brand-williams et al., 1995). 3 ml of 0.004% dpph working solution (prepared using dpph stock solution and methanol in correct proportions to give 0.899 abs) was added per every 100 µl of different concentrations of the extract and incubated at 37 ℃ for 30 minutes in dark. then absorbance was taken at 517 nm wavelength in a uv spectrophotometer. the negative control contained 100 µl of methanol in place of the sample solution. the percentage antioxidant inhibition (ai) was obtained by the equation: % ai = control (abs) − sample (abs) ÷ control (abs) × 100 ascorbic acid (aa) was used as the positive control. inhibition curves were made and ic50 value per sample was calculated. antimicrobial assay test organisms the organisms employed in this study are; escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, and streptococcus epidermis, clinical isolates got from the microbiology department, university of maiduguri, and were stored at 2-8 ℃ until required. preparation of extract solutions for pathogenic assay a stock solution of the extracts was prepared by dissolving 10 g of extract in 10 ml of distilled water, and a 1000 mg/ml solution was obtained. a two-fold serial dilution was carried out to obtain working solutions of varying concentrations. preparation of test organisms test organisms cultured for 24 hours were suspended in a sterile bottle containing pure broth. normal saline was added gradually to it and the turbidity was observed and compared to that of 0.5 mcfarland standard which corresponds to approximately 108 cells/ml. this was then diluted to produce 106 cells/ml and used in the experiments. the dilution ratio was 1:1000 and 1:1500 for gram-positive and gram-negative organisms respectively (usman et al., 2009). preparation of agar plates nutrient agar was prepared accurately to the manufacturer’s specification (i.e. by dissolving 18.5 g powder in 500 ml of distilled water) and sterilized at 121 ℃ for 15 min. the sterilized agar was allowed to cool to 50 ℃ in a water bath. the test organism (1 ml) (106 cells/ml) was inoculated into pre-labeled petri plates (90 mm diameter), then 19 ml of the molten agar was added to each petri plate, shacked, and allowed to sit at room temperature on a flat surface. antimicrobial susceptibility assay (agar well diffusion method) the antibacterial activity of the crude extracts was determined by following the agar-well diffusion method described by igbinosa and colleagues (igbinosa et al., 2009) with modification. the bacterial isolates were grown for 18 h in a nutrient broth and standardized to 0.5 mcfarland standards (10 6 cfuml-1). two hundred microliter of the standardized cell suspensions were spread on mueller-hinton agar (oxoid) and wells were bored into the agar using a sterile 6 mm diameter cork borer. approximately 100 µl of the crude extract at 100, 75, 50, and 25 mgml-1 were introduced into the wells, allowed to stand at room temperature for about 2 hr, and then incubated at 37 °c. controls were set up in parallel yakubu et al. – phytochemical screening, antioxidant and antibacterial activities … 107 using the solvents that were used to reconstitute the extract. after 24 hr, the plates were observed for the zones of inhibition. the effects were compared with those of ciprofloxacin at a concentration of 5 mg/ml. antibacterial activity was evaluated by measuring the diameters of zones of growth inhibition in triplicates and results were presented as mean±sem. statistical analysis the obtained antioxidant and antibacterial results were expressed in mean ± standard error with observation recorded in triplicates. analysis of variance for individual parameters was performed based on mean values to determine the significance at p < 0.05 using spps v20. regression analysis was deployed to calculate and obtain the ic50 from the regression equation using excel 2016. percentage yield in this experiment, 1.5kg of the resultant size reduced root powder of c. adenocaule was used. the meoh extract showed the highest yield of 44.6g. the percentage yield is given in the table1 table 1. percentage yield of c. adenocaule root extracts. extract weight of extract (g) percentage (%) yield nhex 5.4 0.36 chcl3 12.5 0.83 meoh 44.6 2.97 phytochemical screening of root extract of c. adenocaule. the result for the preliminary phytochemical screening of root extract of c. adenocaule is shown in table 2. table 2. preliminary phytochemical screening of methanolic extract of c. adenocaule. phytoconstituent test result nhexane chloroform methanol alkaloids dragendorff's + + + mayer's + + + anthraquinones freeanthraquinones borntrager's + combine anthraquinones borntrager's carbohydrates general test molisch's + + + monosaccharide barfoed's + + + free reducing sugar fehling's + + combine reducing sugar fehling's + cardiac glycosides steroidal nucleus salkowsi's + + steroidal nucleus liebermann-buchard's + + terpenoids + flavonoids lead acetate + ferric chloride + + shinoda's + + sodium hydroxide + + saponins glycosides frothing + tannins ferric chloride + + lead acetate + + key: + = present: = absent biological activity dpph assay: in-vitro antioxidant activity table 3. in vitro antioxidant activity (dpph assay) of c. adenocaule root extract fractions. concentration (µg/ml) % of dpph scavenging activity nhex chcl3 meoh aa 6.25 22.8 33.5 32.8 46.5 12.5 29.9 35.9 55.2 53.7 25 36.8 39.4 67.2 64.1 50 40.5 44.6 79.3 77.3 100 55.2 50.9 97.2 96.8 ic50 74.1 74.6 10.87 4.51 key: aa: ascorbic acid, chcl3: chloroform extract, nhex: n-hexane extract, meoh: methanol extract results of the in vitro antioxidant assay (in ic50) of the crude root extracts of c. adenocaule are given in table 3 below. the meoh extract showed relatively invitro dpph scavenging activity compared to other extracts. 108 biology, medicine, & natural product chemistry 10 (2), 2021: 105-110 figure 1. dpph assay; ic50 extrapolation graph of nhex, chcl3, and meoh extracts of c. adenocaule. antimicrobial susceptibility assay the chcl3 showed the highest activity against the test organism. the result of the in-vitro antimicrobial susceptibility assay is given in table 4. table 4. in vitro antimicrobial activity of c. adenocaule extract. concentration (mg/ml) test organism extracts hex chcl3 meoh cip(5mg/ml) e. coli 00.00±0.00 24.00±0.15 0.10±0.02 31.00±0.75 p. aeruginosa 00.00±0.00 2.00±0.02 2.00±0.00 27.30±0.41 100 stap. aureus 00.00±0.00 12.50±0.18 5.50±0.13 29.70±0.17 s. epidermis 00.00±0.00 5.50±0.07 1.5.00±0.1 25.02±0.84 e. coli 00.00±0.00 12.00±0.15 0.10±0.00 p. aeruginosa 00.00±0.00 1.00±0.15 2.00±0.32 75 stap. aureus 00.00±0.00 13.50±0.4 2.00±0.01 s. epidermis 00.00±0.00 7.50±0.12 1.0.00±0.00 e. coli 00.00±0.00 4.50±0.2 0.10±0.00 p. aeruginosa 00.00±0.00 0.00±0.00 0.00±000 50 stap. aureus 00.00±0.00 9.10±0.16 2.00±0.10 s. epidermis 00.00±0.00 2.60±0.05 1.70±0.18 e. coli 00.00±0.00 3.00±0.21 0.10±0.00 p. aeruginosa 00.00±0.00 0.00±0.00 0.20±0.00 25 stap. aureus 00.00±0.00 2.50±0.4 1.00±0.10 s. epidermis 00.00±0.00 1.00±0.00 1.50±0.00 key: cip: ciprofloxacin discussion many solvents including hex, chcl3, and meoh had been employed for the extraction of bioactive plant principles. meoh had shown effective as an extraction solvent in many plant drug analyses especially in the isolation of phenolics and flavonoids content (do et al., 2014; truong et al., 2019). the percentage yield result y = 0,6731x r² = -1,351 0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 in h ib it io n ( % ) concentration µg/ml dpph assay nhex y = 0,6729x 0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 in h ib it io n ( % ) concentration µg/ml dpph assay chcl3 y = 0,5155x + 47,704 r² = 0,967 0 20 40 60 80 100 120 0 20 40 60 80 100 120 in h ib it io n ( % ) concentration µg/ml dpph assay aa y = 0,5861x + 43,629 r² = 0,8424 0 20 40 60 80 100 120 0 20 40 60 80 100 120 in h ib it io n ( % ) concentration µg/ml dpph assay meoh yakubu et al. – phytochemical screening, antioxidant and antibacterial activities … 109 from this study shows meoh to be with the highest extraction yield of 2.97%, followed by chcl3 (0.83 %) and nhex (0.36 %) respectively. (table 1). phytochemical screening of c. adenocaule using standard methods revealed the presence of alkaloids, carbohydrates, saponins, and tannins in the nhex extract. flavonoids, carbohydrates and glycoside, alkaloids, saponins, terpenoids, and tannins were present in both the chcl3 and meoh extracts (table 4). similar results were reported by akinwunmi and colleagues on the phytochemical screening of the ethanol root extract of c. adenocaule (akinwunmi et al., 2015). dpph assay remains one of the commonly employed methods for the analysis of the antioxidant activity of plant phytochemicals and employs spectrophotometric application. the ability of a test compound to scavenge dpph radical is determined based on its concentration providing 50% inhibition (ic50), which is the value of the concentration of the sample to cause 50% inhibition and is obtained by the interpolation from the linear regression analysis (figure 1). in this study, different fractions of the extracts were screened for their antiscavanging activity, and it was observed that the meoh extract showed the highest potential with an ic50 10.87 µg/ml scavenging activity, followed by nhex (74.1 7 µg/ml) and chcl3 (74.6 7µg/ml) with the least scavenging activity. though, the results are below that of the standard; ascorbic acid aa with ic50 4.50 µg/ml (table 4). this activity might be due to the presence of phenolics and flavonoids and other secondary metabolites present in the meoh extract that are known potent antioxidants, and taking into consideration, c. adenocaule belong to the vitaceae family which are known for their potent antioxidant principles (murias et al., 2005; piotrowska et al., 2012; rivière et al., 2012). akinwunmi and colleagues, reported a dpph scavenging activity of ic50 38.42 µg/ml for the ethanol root extract of cissus adenocaule with the root total phenolic and flavonoid content to be 182±0.38 mg/g tae (tannic acid equivalent) and 103±0.42 mg/g qe (quercetin equivalent) [akinwunmi et al., 2015]. the use of the plant as an antimicrobial agent in the ethnomedicinal space cannot be overemphasized, as they continued to be used to date. this augments their exploitations for the discovery of lead and novel molecules for antimicrobial drug discovery. they provide starting materials and derivatives that are employed as ligands in the drug discovery and development process. this study explored the antimicrobial activity of the c. adenocaule root extracts (table 4). the n-hexane extract was completely inactive against the test organism. the chcl3 extract was active at 100mg/ml and showed good activity against e. coli (24.00±0.15) and moderate activity against staph. aureus (12.5±0.18). the meoh extract showed poor activity against the test organism. this result is inconsonant with an earlier report by hamil and colleagues on the activity of meoh root extract of c. adenocaule on e. coli. p. aeruginosa and staph. aureus (hamill et al., 2003). conclusion the present study undertook the phytochemical screening, isolation, and characterization of chemical compounds present in the methanol root extract of c. adenocaule, as well as, determination of their antioxidant and antibacterial activity. methanol presents the best extraction solvent in terms of percentage yield. flavonoids, alkaloids, carbohydrates and glycoside, saponins, and tannins were present while anthraquinone were absent. in the chloroform and hexane extracts; anthraquinones and flavonoids were absent, with carbohydrates also absent in the hexane extracts. in the assessment of the biological properties, the antibacterial sensitivity assay showed chloroform to have activity against e. coli and moderate against staph. aureus at 100mg/ml respectively, while poor activity was recorded with the methanol and n-hexane extract. the dpph antioxidant assay revealed the free radical scavenging activity, methanol extract yields the best result with an ic50 of 10.87µg/ml. the results from this study add to the existing literature on c. adenocaule with scientific evidence into its biological properties. acknowledgments: the authors will like to acknowledge mr. namadi sanusi of the department of biological science, ahmadu bello university, zaria, nigeria for the collection and identification of the plant materials. funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. conflict of interest: the authors declare no conflict of interest. references akinwunmi, k. f., ajala, v. o., & oyedapo, o. o. 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(2020). a natural triglyceride from the methanol root extract of cyphostemma adenocaule (steud. ex a. rich.) wild & r.b.drumm. https://doi.org/10.26434/chemrxiv.13296539.v1 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 707-715 | doi: 10.14421/biomedich.2025.142.707-715 issn 2540-9328 (online) antimalaria potential and toxicological evaluation of synclisia scabrida (miers) ex oliv methanol root extract in plasmodium berghei-infected mice model ogochukwu ngozi nwaefulu1,*, john ihechukwu nwafor2, daniel lotanna ajaghaku3, ngozi ezinwanne amuchie4, blessing akporotu1 1department of pharmacology and toxicology, faculty of pharmacy, delta state university, abraka, delta state, nigeria. 2department of pharmacology and toxicology, faculty of pharmacy, madonna university, rivers state, nigeria. 3department of pharmacology and toxicology, faculty of pharmacy, enugu state university of science and technology, enugu state, nigeria. 4department of microbiology, faculty of science, madonna university, rivers state, nigeria. corresponding author* blessedogo2000@yahoo.com; ogo4jesus@gmail.com manuscript received: 08 june, 2025. revision accepted: 04 september, 2025. published: 01 october, 2025. abstract despite the artemisinin-based combination therapy for malaria treatment, it remains a public health concern globally. therefore, there is an urgent need for novel antimalarial drugs to arrest this perceived threat to global malaria control. this study aims to investigate the antimalarial potential of s. scabrida (ss) and evaluate its possible effect on haematological and biochemical parameters. malaria was inoculated in mice using plasmodium berghei. after 72 h, they were orally treated for four days as follows: group 1: 10 ml/kg % dimethyl sulphoxide (dmso); group 2: 140 mg/kg artemether/lumefantrine; groups 3, 4 and 5: 500, 250, 125 mg/kg ss methanolic extract, respectively. ss methanolic extract significantly reduced parasite density and percentage parasitemia (p < 0.05) at all doses used in the study, non-significantly increased haematological, biochemical parameters and distorted liver architecture; when compared to the negative control. ss methanolic extract exhibited good antimalarial activity against plasmodium berghei. lower ss dose did not cause significant changes to the lipid profiles. however, the high dose may not be safe since there were some adverse effects shown on the liver and kidney status. further studies are warranted to determine the effect of the middle and the lowest doses on the liver and kidney. keywords: synclisia scabrida; antimalaria; plasmodium berghei; lipids; haematology. abbreviations: who: world health organisation, frin: forestry research institute of nigeria, prbcs: packed red blood cells, dmso: dimethyl sulphoxide, mst: mean survival time, spss: statistical package for social sciences, anova: analysis of variance, cv: central vein, hps: hepatic sinusoids, hpc: hepatocytes, ast: aspartate aminotransferase, alt: alanine transaminase, alp: alkaline phosphatase, a/l: artemether/lumefantrine. introduction malaria remains a significant global health concern, with plasmodium species causing substantial morbidity and mortality. resistance against the artemisinin-based combination therapy, the gold standard for malaria treatment has recently been reported in africa (balikagala et al., 2021), the region with 94% of the world’s malaria cases (who, 2023). therefore, there is an immediate need for novel antimalarial drugs to counter this trend, since if allowed to spread, would be a serious threat to malaria control globally. globally in 2022, approximately 249 million incidents of malaria were reported in 85 malaria endemic countries and estimated deaths of 608 000. with an estimated 233 an approximated 233 million cases, the world health organisation (who) african region accounted for approximately 94% of cases worldwide and was projected to cause 580,000 deaths by 2022. being the most seriously impacted in the same year with 27% and 31% of all malaria cases and fatalities respectively, worldwide, nigeria was placed on the list of eleven african nations designated as high-burdened nations (who, 2023). malaria is a major cause of poverty and it reduces the rate of growth in the economy by up to 1.3% per year in endemic countries (who, 2021). studies have shown hematological and biochemical alterations in malaria parasite-infected blood with common complications associated with this disease (alsalahy et al., 2016). in the discovery of novel antimalarials, therapeutic plants have had vital role, for instance the antimalarial drugs artemisinin and quinine were isolated from artemisia annua and cinchona bark, respectively (ceravolo et al., 2021). synclisia scabrida https://doi.org/10.14421/biomedich.2025.142.707-715 708 biology, medicine, & natural product chemistry 14 (2), 2025: 707-715 belongs to the family menispermaceae. its root, leaf, stem and liana were found to have been used in the management of gastric ulcer, mental disorders, venereal diseases, prostate problems, asthma, hernia, upper abdominal pains, dyspepsia, menstrual pains, prevention of spontaneous abortion and as an aphrodisiac (onwudiwe et al., 2012; anowi et al., 2013). traditional healers in nigeria have claimed they use synclisa scabrida for the treatment of malaria. however, no scientific investigations have been done to confirm this claim. hence, in the search for more potent malaria drugs, this study aims to investigate the antimalarial potential of s. scabrida and also evaluate its possible effect on the haematological and biochemical parameters, along with the kidney and liver. materials and methods plant sample collection a plant specimen was collected by mr samuel oyebanji from a farm in the ugbine community, edo state, nigeria. the plant was dried and sent to the forestry research institute of nigeria (frin) for identification and authentication. it was identified and authenticated as synclisia scabrida miers ex oliv. by mr s.a. odewo of frin, where the herbarium specimen with voucher number fhi – 111106 was deposited. experimental animals albino mice (50) of both sexes with no signs of ill-health were obtained from federal polytechnic owerri, imo state, and then brought to the animal house of the faculty of pharmacy, madonna university, nigeria where the research was carried out and then acclimatised for 14 days. the animals were housed in ventilated cages at room temperature of 24 ± 2°c and kept under standard conditions of a 12 h light/dark cycle. the animals were fed with standard laboratory feed and water. they were also handled in accordance with the international animal care and welfare guidelines (oecd, 2008; national institute for laboratory animal research, 2011). extract preparation the roots of s. scabrida were supplied from benin state. it was air-dried for 1 week and then oven dried for three days at 300c. after which it was extracted using methanol as solvent using the maceration method, at room temperature (30 ± 2°c) for 72 h. then filtered using a buchner funnel and whatman no.1 filter funnel and filter paper. the obtained extract was concentrated by evaporating the solvent by exposing to air and also oven drying at controlled temperature not exceeding 40ºc.the resulting dried extract was stored in the refrigerator at 4°c prior to use. the percent yield (%w/w) of the extract was calculated using the following formula 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑌𝑖𝑒𝑙𝑑 = weight of the extract weight of the root powder × 100% parasites inoculation the chloroquine-resistant strain of plasmodium berghei was acquired from the animal house of the department of pharmacology and toxicology, university of uyo, nigeria. two cycles of prbc passage in rats and mice were used to prepare the parasites. donors with parasitaemia levels of 50-70% were sacrificed, and a heart puncture was used to obtain their blood into heparinised tubes. after that, the blood was diluted with normal saline based on parasitaemia level of each donor and the red blood cell (rbc) count of normal mice, such that 1 ml blood contained 5 × 107 parasites. the experimental animals were each treated with 1 × 107 packed red blood cells (prbcs) by intraperitoneal (ip) injection (basir et al., 2012). pharmacological screening rane's test (curative model) a total of 50 albino mice were selected and randomly grouped into 5 groups of 10 animals per group. the % parasitemia levels of every group were gotten before all the animals were infected with plasmodium berghei (a zoonotic plasmodium sp) as described above. the animals were then left for 72 h for the infection to establish. percentage parasitemia was also determined after induction before treatment. after 72 h, the animals were given treatment by oral administration of the following, for four days: group 1 (negative control): 10 ml/kg body weight of 1% dimethyl sulphoxide (dmso) group 2 (positive control): 140 mg/kg artemether/lumefantrine combination group 3 (high dose): 500 mg/kg of methanolic extract of s. scabrida group 4 (middle dose): 250 mg/kg of methanolic extract s. scabrida group 5 (low dose): 125 mg/kg of methanolic extract s. scabrida determination of body weight, water and food intake while conducting the treatment, the water and food consumption of every group was documented to determine whether the experimental animals' intake increased or decreased. additionally, the weight of all experimental animals in each group were taken before and after the treatment to assess any changes in their weight. % weight change = weight after treatment − weight before treatment 𝑤𝑒𝑖𝑔ℎ𝑡 𝑏𝑒𝑓𝑜𝑟𝑒 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 × 100 nwaefulu et al. – antimalaria potential of synclisia scabrida 709 determination of the level of parasite density and percentage parasitemia the blood from the tail of the infected mice was collected and placed on a clean glass slide placed horizontally on the working bench. the slide and the spreader were held at a suitable angle, pulled back to touch the dropped blood on the slide and spread along it. the thin layer film was fixed with methanol and lowered into the already prepared giemsa stain (10 ml of giemsa + 90 ml of distilled water) and allowed to stain for 5 min. the slide was lifted off the stain solution with the aid of forceps, excess stain was washed off, allowed to drain and air dried at room temperature. then parasitaemia was observe with an oil immersion lens under a microscope, to examine the blood sample. the focus is on counting the number of red blood cells that are infected with parasites, and this count is then used to determine the parasitized level in the blood sample. the counting is standardized by considering a specific number of red blood cells (200) in a random field view. the % parasitemia, parasite density were calculated using the following formulae: % parasitemia = 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑖𝑠𝑒𝑑 𝑅𝐵𝐶 𝑇𝑜𝑡𝑎𝑙 𝑅𝐵𝐶 × 100 parasite density = 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑖𝑠𝑒𝑑 𝑅𝐵𝐶 𝑊𝐵𝐶 × 5000 then the % inhibition was calculated using the following formula: % inhibition = % 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑚𝑖𝑎 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − % 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑚𝑖𝑎 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑔𝑟𝑜𝑢𝑝 % 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑚𝑖𝑎 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 determination of mean survival time (mst) from the time of infection until death, the mortality of each mouse was monitored and recorded regardless of the group in which the mouse was allocated throughout the follow-up period (30 days). mean survival time of mice of each group was determined using the following formula: mst = sum of survival time  of all mice in a  group (days) sum of mice in the group statistical analysis data were analysed using statistical package for social sciences (spss version 23). data for parasite density, % parasitemia and hematological parameters were analysed using one-way anova, followed by turkey post hoc test. the results were presented as mean ± sd in tables. mean values with p < 0.05 were considered significant. results and discussion effect of the methanol root extract of synclisia scabrida on weight, daily water and food intake of p. berghei-infected mice to determine the effect of the methanol extract of s. scabrida roots on weight changes in p. berghei infected vehicle-treated and extract-treated mice, animal weights were recorded and comparisons made between the day of infection (day 0) and day 8 post infection (24 h after administering the final dose of treatment). this is shown in table 1. the weight loss observed in positive control and extract-treated groups was not significantly different from that seen in vehicle-treated group. the treatment of the mice with crude methanolic root extract of s. scabrida resulted to negligible change or increased water and food intake throughout the period of the treatment (figure 1). treatment with crude extract of s. scabrida root and artemether/lumefantrine on daily water and food intake is not significantly different from each other. this is shown on table 1. table 1. effect of methanol root extract of synclisia scabrida on weight, daily water and food intake of p. berghei-infected mice. treatment weight (g) on day 0 (infection day) weight (g) on day 8 (post infection) weight change (%) negative control (vehicle) 21.2 ± 2.2 19.0 ± 2.5 10.4 artemether/lumefantrine (140 mg/kg) 21.8 ± 2.9 19.4 ± 2.8 11 500 mg/kg ss 29.1 ± 2.6 24.1 ± 3.9 17.2 250 mg/kg ss 25.5 ± 3.3 23.7 ± 4.8 7.1 125 mg/kg ss 24.6 ± 3.6 17.4 ± 1.1 29.3 data expressed as mean ± sd, n=10, ss = synclisia scabrida. 710 biology, medicine, & natural product chemistry 14 (2), 2025: 707-715 figure 1. effect of methanol root extract of synclisia scabrida on daily water and food intake of p. berghei-infected mice. the purpose of this study was to examine the impact of synclisia scabrida on malaria parasites, hematology, biochemical parameters, lipids and histological parameters of liver and kidney of p. berghei-infected albino mice. since the curative test allows for established infection, it was used in this investigation. p. berghei is frequently employed for preclinical antimalarial research of drug candidates with measurable treatment outcomes (fidock et al., 2004). plasmodium berghei serves as a crucial model organism for the study of malaria, offering crucial insights into the disease's pathogenesis, host immune responses, and potential therapeutic targets. (otun et al.,2024.) because it makes it possible to investigate illness development, which appears challenging in humans. this is why p. berghei was utilised to induce malaria in mice. additionally, it permits evaluations of organs like the liver, where parasites can hibernate (albohiri & alzanbagi, 2021). the most often used technique to study the antimalarial effect of a possible antimalarial drug moiety is in-vivo antimalarial research. prevention of loss in body weight is one of the parameters to confirm the antimalarial potential of new natural or synthetic antimalarial drugs as body weight loss is a characteristic of plasmodium berghei infected mice as a result of increased parasitamia (kifle et al., 2020) resulting from appetite loss, metabolic disturbance, and hypoglycemic effect of the parasite (deharo et al., 2001). there is no significant change in weight of the infected mice treated with methanolic extract of s. scabrida when compared to vehicle-treated mice. this is an indication of little or no improvement in weight loss due to infection. therefore, regarding weight as a measure, the extract might not have had a significant impact on the overall health or well-being of the infected mice compared to the control group. this is an indication that the extract preserved the weight of the animals; which could be due to its ability to prevent appetite loss. this is confirmed by the result of this study which showed that the extract increased food consumption at all doses. curative potential of extract synclisia scabrida root on plasmodium berghei-infected mice treatment with crude extract of s. scabrida root significantly reduced parasite density and percentage parasitemia (p < 0.05) at all doses used in the study; when compared to the negative control. the highest dose (500 mg/kg) showed the strongest reduction which is comparable to that of the standard antimalarial drug (artemether/lumefanthrine) used. similarly, 500, 250, and 125 mg/kg s. scabrida crude methanol extract caused 78.0%, 58.0%, and 61.20% parasitemia inhibition, respectively. the percentage parasitemia produced by 500 mg/kg (16.7%) is very similar to that of the standard antimalarial drug (9.8 ± 4.4) used, but significantly different from that of 250 (32.0 ± 5.4; p < 0.05) and 125 mg/kg (29.5 ± 2.1; p < 0.001), when compared to the standard drug. similarly, the extent of parasite density reduction by 250 and 125 mg/kg s. scabrida extract is significantly different (p < 0.05; (p < 0.004, respectively) when compared to the standard. again, the parasite density reduction produced by 500 mg/kg (816.7 ± 275.4) is very similar to that of the standard antimalarial drug (768.7 ± 104.0) used (table 2). mean survival time of the rats treated with crude extract of s. scabrida root was prolonged at all doses when compared to the control group, although not significantly different. nwaefulu et al. – antimalaria potential of synclisia scabrida 711 table 2. parasite density, % parasitemia, % inhibition and mean survival time (mst) of plasmodium berghei-infected mice treated with extract of synclisia scabrida root. treatment parasite density (parasites/µl) % parasitemia % inhibition mst (days) negative control (vehicle) 3349.8 ± 591.1 76.2 ± 7.9 0 15.7±3.2 artemether/lumefantrine (140 mg/kg) 768.7 ± 104.0 9.8 ± 4.4 87.1 30±0.0 500 mg/kg ss 816.7 ± 275.4a 16.7 ± 8.5a 78.0 16.7±3.2 250 mg/kg ss 1417.2 ±2 54.8ab 32.0 ± 5.4ab 58.0 17.0±2.0 125 mg/kg ss 1358.0 ± 59.4ad 29.5 ± 2.1ac 61.2 24.0±5.2 data expressed as mean ± sd, ss = synclisia scabrida, a p < 0.05 when compared to negative control, b p < 0.05, cp < 0.001, d p < 0.004, when compared to positive control, n=10. parasitemia is the number of parasites in the blood. the number of asexual forms of parasite about a blood volume is the traditional definition of parasite density, which is a measure of infection level (e.g., microliter) or a percentage of white blood cells (wbcs). it serves as a gauge of the organism's parasite burden and a sign of how severe an active parasitic infection is (oyibo et al., 2023). parasite density and percentage parasitemia were significantly reduced at all doses of the extract. synclisia scabrida has thus been shown to possess good activity towards p. berghei infection and therefore confirms the anecdotal report of its use in the treatment of malaria. there is no significant difference between reductions in parasite density and percentage parasitemia produced by 500 mg/kg s. scabrida extract, when compared to the standard drug. this implies that the activity of the two is comparable, 500 mg/kg being as efficacious as the artemether/lumefantrine with percentage inhibition of 78.0%. according to rasoanaivo et al. (2004), the extract at this dose possesses a good activity against p. berghei. this anti-malarial activity was classified according to rasoanaivo et al. (2004); classification which states that extracts with very good or good activity should possess 90–100% parasitaemia inhibition, extracts with good to moderate activity should have 50–90% inhibition, extract with moderate to weak activity should have 10–50% inhibition and extracts that are inactive have 0% inhibition. in accordance with (maiga et al., 2021), artemether/lumefantrine (a/l) is one of the first line artemisinin-based combination therapies recommended for the treatment of malaria. its antiplasmodial activity is attributed to the release of free radicals by its artemether component, which subsequently binds covalently to parasite proteins and heme. additionally, it has been proposed that a/l inhibits the calcium atpase (sarcoplasmic endoplasmic reticulum calcium atpase) of malarial parasites (moore et al., 2022). but its lumefantrine component builds up in the feeding vacuole of the parasite, where it disrupts heme polymerization by forming complexes that result in toxic heme, which ultimately leads to the death of the parasite (kumar et al., 2007). cycleanine, a major compound present in s. scabrida has been shown to suppress the levels of parasitemia and increase mean survival times significantly compared to those of the control groups (uche et al., 2021). the ethanol extract of s. scabrida was reported to possess high amounts of flavonoids, tannins, saponins, terpenes (orumwense et al., 2022). akande et al. (2021) reported that presence of saponin, flavonoid, alkaloids, tannin, triterpene, carbohydrates, steroids, glycosides and cardiac glycosides in methanol extract jatropha curcas leaf resulted to significant suppression of parasitaemia. alkaloids have over overtime been identified as significant phytoconstituents with notable biological properties, including antimalarial activity and excellent free radical scavenging activity. the initial effective antimalarial medication was quinine, an alkaloid which was obtained from the extraction of the cinchona tree (uzor, 2020). mst is a crucial metric for the antiplasmodial evaluation of possible drug moieties. the results of the current investigation show mst was prolonged at all doses; 250 mg/kg of synclisia scabrida extract prolonged it to the same extent as artemether/lumefantrine. it may reveal how well novel medications or therapy approaches work. a medication may be useful in controlling or lessening the severity of an illness if it lengthens the infected mice's survival period. the histology of the liver of the vehicle-treated mice shows a hepatic lobule having a central vein (cv). from the central vein, hepatocytes (hpc) (liver cells) radiate outwards like the spokes of a wheel. the hepatocytes form sheets and in between the sheets run hepatic sinusoids (hps). the result on the histology of the liver suggests that the treatment of malaria-infected mice with the synclisia scabrida root extract at a dose of 500 mg/kg induced some histological changes in the liver. these changes include alterations in the appearance and arrangement of hepatocytes, dilatation of the central vein, and changes in the staining characteristics of cell nuclei and cytoplasm, indicating potential effects on liver tissue integrity and cellular health due to the treatment. 712 biology, medicine, & natural product chemistry 14 (2), 2025: 707-715 effect of methanol root extract of synclisia scabrida on haematological parameters of p. berghei-infected mice synclisia scabrida extract increased wbc and rbc, although not significantly. artemether/lumefantrine significantly increased the level of rbc (p < 0.00), when compared to the negative control (table 3). table 3. effect of synclisia scabrida extract on haematological parameters. treatment rbc (per µl) wbc (per µl) pcv (%) negative control (vehicle) 69.0±13.5 78.8±11.4 15.0±7.0 artemether/lumefantrine (140 mg/kg) 200.3±74.2a 115.3±29.1 47.7±1.5 500 mg/kg ss 130.0±26.5 133.3±57.7 13.3±1.5 250 mg/kg ss 97.2±32.2 111.7±43.6 15.0±8.9 125 mg/kg ss 90.0±14.1 97.5±3.5 7.0±2.8 data expressed as mean ± sd, ap < 0.05, when compared to control, n=10. it has been demonstrated that malaria infections modify the plasma biochemical indicators (saftawy et al., 2022). studies have shown alterations in the biochemistry and hematology of malaria parasiteinfected blood with common complications associated with this disease. haematological changes that are associated with malaria infection include anaemia, thrombocytopenia and disseminated intravascular coagulation (nlinwe & nange, 2020). pcv is measured to assess the effectiveness of a crude drug in preventing hemolysis due to the rising parasitemia level by measuring the proportion of red blood cells (chidozie et al., 2020). malaria infection is characterised by possible hemolysis and anaemia (atanu et al., 2022). the main causes of anaemia in mice and humans include the clearance or destruction of infected rbcs, the clearance of uninfected rbcs, erythropoietic suppression, and dyserythropoiesis. the malaria parasite is known to cause cells to produce free radicals which compromise the red blood cell membrane. therefore, the capacity of the extract to stabilise pcv in mice with infection could be due to the antioxidant compounds present in the plant extracts. in this study, the extract could not improve the pcv, nevertheless, it prevented further reduction in pcv of the extract-treated mice. rbc was increased in extract-treated and artemether/lumefantrine-treated infected mice. this suggests that the extract and the standard drug may affect the production of rbcs. further, wbc in the infected extract-treated and artemether/lumefantrine-treated mice was higher than in the infected vehicle-treated mice. this is a suggestion that the extract and the standard drug were able to boost the immune system of the mice, hence improving the ability of the mice to fight infection as a result of the treatment with the extract and artemether/lumefantrine increasing the wbc levels. it implies that the increase in wbc was due to the administration of the extract which was able to reverse the damaging effects of the parasites. effect of methanol root extract of synclisia scabrida on liver and kidney functions of p. berghei-infected mice the effect of synclisia scabrida extract on kidney and liver function parameters is shown in table 4. synclisia scabrida extract and the artemether/lumefantrine increased the levels of aspartate aminotransferase (ast), alanine transaminase (alt), alkaline phosphatase (alp), and creatinine, although only significant at 500 mg/kg and 125 mg/kg for ast. table 4. effect of methanol root extract of synclisia scabrida on liver and kidney functions of p. berghei-infected mice. treatment (mg/kg) ast (µ/l) alt (µ/l) alp (µ/l) creatinine (mmol/l) negative control 131.4±46.5 50±11.8 199±38.0 38.9±7.5 artemether/lumefantrine (140 mg/kg) 225.3±87.0 56±15.3 250±71.3 50.4±12.5 ss whole root extract (500 mg/kg) 608.5±100.0a 376.25±37.0a 289.75±41.0 82±0.0b 250 mg/kg 696.3±92.4a 166.3±58.0b 266.7±81.5 54.7±23.7 125 mg/kg 714±119.6a 172±12b 399.8±38.7b 51.3±20.5 data expressed as mean ± sd, ap < 0.0001, bp < 0.02; when compared to control, n=4. s. scabrida extract increased the levels of ast, alt, alp, and creatinine as also seen with artemether/lumefantrine. this indicates toxicity to liver tissues, this corresponds with the histology of the liver as shown in figure 1a. ast and alt test is commonly used to check for liver disease, to monitor liver disorder, nwaefulu et al. – antimalaria potential of synclisia scabrida 713 to ascertain treatment efficacy and to make sure that medications are not causing liver damage. effect of methanol root extract of synclisia scabrida on lipid profile of p. berghei-infected mice the effect of synclisia scabrida extract on lipid profile in treated albino mice is shown in table 5. the crude extract (500 mg/kg and 250 mg/kg) increased the highdensity lipoprotein (hdl) and low-density lipoprotein (ldl) when compared with the negative control. also, all doses of the extract increased total cholesterol (tc) and triglyceride (tg) in comparison to the negative control. at 500 mg/kg, tc (7.5±1.4 mmol/l) and ldl (4.2±0.7 mmol/l) were significantly increased (p < 0.004). similarly, there was a significant increase in tc (8.4±0.7 mmol/l; p < 0.001) and ldl at 125 mg/kg (4.5±1.6 mmol/l; p < 0.002), but not at 250 mg/kg. it is important to note that the extract at 500 mg/kg increased hdl far more than artemether/lumefantrine. table 5. effect of methanol root extract of synclisia scabrida on lipid profile of p. berghei-infected mice. treatment tc (mmol/l) tg (mmol/l) hdl (mmol/l) ldl (mmol/l) negative control 3.1±0.8 1.9±0.6 1.3±1.2 0.9±1.3 artemether/lumefantrine (140 mg/kg) 4.1±0.7 1.8±0.5 0.5±0.2 2.9±0.9 ss whole root extract (500 mg/kg) 7.5±1.4a 2.7±1.5 2.1±1.0 4.2±0.7a ss whole root extract 250 mg/kg 5.3±1.9 2.8±1.2 1.3±1.2 2.5±2.0 ss whole root extract 125 mg/kg 8.4±0.7c 4.3±1.9 1.9±0.7 4.5±1.6b data expressed as mean ± sd, ap < 0.004, bp < 0.002, cp < 0.001; when compared to control, n=4. the crude extract (500 mg/kg and 250 mg/kg) increased the high-density lipoprotein (hdl), which could probably be a result of flavonoids as increased flavonoids reduce the occurrence of low high-density lipoprotein (ahn et al., 2020; anaka et al., 2013); flavonoid have been found to be one of the major constituents of s. scabrida (orumwense et al., 2022). the crude extract (500 mg/kg and 250 mg/kg) increased the high-density lipoprotein (hdl), which could probably be a result of flavonoids as increased flavonoids reduce the occurrence of low high-density lipoprotein (ahn et al., 2020; anaka et al., 2013) present in the plant (orumwense et al., 2022). flavonoids can also reduce cholesterol synthesis by inhibiting the activity of the enzyme acyl-coa cholesterol acyl transferase (acat) in hepg2 cells. this enzyme reduces cholesterol esterification in the intestine and liver (simorangkir et al., 2022). all the lipid parameters were not significantly affected at 250 mg/kg, the result of this study, indicating safety at a lower dose. effect of methanol root extract of synclisia scabrida on liver and kidney functions of p. berghei-infected mice the histology of liver of the vehicle treated mice is shown in figure 1ai. the photomicrograph shows a hepatic lobule having a central vein (cv). from the central vein, hepatocytes (hpc) (liver cells) radiate outwards like the spokes of a wheel. the hepatocytes form sheets and in between the sheets run hepatic sinusoids (hps). hepatocytes have one or two darkly stained nuclei and deeply eosinophilic cytoplasm. the photomicrograph of the liver of 500 mg/kg synclisia scabrida crude extract treated mice shows dilated central vein. liver cell architecture is a distorted compared to negative control, hepatocytes are sequestered, cell outline are indistinct. cell nuclei are darkly stained and the cytoplasm is poorly eosinophilia (figure 1aii). the histology of kidney of the vehicle treated mice is shown in figure 1bi. the photomicrograph shows renal corpuscles and renal tubules. renal corpuscles consist of glomeruli and bowman’s capsule. renal tubules (rt) have complete epithelial lining. the photomicrograph of the kidney of 500 mg/kg synclisia scabrida crude extract treated mice shows renal corpuscle (rc) and renal tubules. renal corpuscle is normal. there are areas of fibrosis of renal tubules (figure 1bii). figure 1. a; liver of p. berghei-infected mice treated with vehicle (i) and 500 mg/kg crude methanolic root extract of synclisia scabrida (ii). b; kidney of p. berghei-infected mice treated with vehicle (i) and 500 mg/kg crude methanolic root extract of synclisia scabrida (ii). cv= central vein, hpc= hepatocytes, hps= hepatic sinusoids, rt=renal tubule, rc=renal corpuscle. [mag: x500] 714 biology, medicine, & natural product chemistry 14 (2), 2025: 707-715 the result on the histology of the kidney suggests that treatment with the synclisia scabrida root extract at a dose of 500 mg/kg in malaria-infected mice has induced some histological changes in the kidney. while the renal corpuscles, which include the glomeruli and bowman's capsule, appear normal, there are areas of fibrosis in the renal tubules, indicating damage or scarring in these tubular structures. these changes in the kidney tissue suggest potential effects on kidney health due to the treatment. given the effect of 500 mg/kg s. scabrida on the liver and kidney, the lowest dose (125 mg/kg) could be used in treatment since it significantly reduced the parasite density and percentage of parasitemia. the results of this study revealed that the methanolic root extract of synclisia scabrida exhibited good antimalarial activity against plasmodium berghei as it significantly reduced parasite density and the level of parasitemia. lower dose of the extract did not cause significant changes to the lipid profiles. however, the high dose may not be safe since there were some adverse effects shown on the liver and kidney status but not on the haematological parameters. further studies are warranted to determine the effect of the middle and the lowest doses on the liver and kidney. acknowledgements: muoghara queentamary and shadrach igbeagu are appreciated for their devoted time and assistance towards this study. authors’ contributions: concept: n.o.n., design: n.o.n., supervision: n.o.n., data collection or processing: n.j.i., n.o.n., a.d.l., analysis or interpretation: n.o.n., n.j.i., a.d.l., manuscript writing: n.o.n., a.b., critical review: n.o.n., a.d.l. competing interests: no conflict of interest was declared by the authors. funding: the study did not receive any funding. references ahn s, jun s, joung h (2020) association of total flavonoids intake with hypo-hdl-cholesterol among 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(2021). who initiative to stop the spread of anopheles stephensi in africa (no. who/ucn/gmp/2022.06). world health organization. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 75-81 | doi: 10.14421/biomedich.2022.111.75-81 issn 2540-9328 (online) phytochemical constituents of f. sagittifolia warburg ex mildbraed & burret leaves with antimicrobial activity olayombo margaret taiwo1,2,*, olaoluwa omosalewa olaoluwa1, olapeju oluyemisi aiyelaagbe1, josphat clement matasyoh2 1department of chemistry, faculty of science, university of ibadan, 200284, ibadan, nigeria 2department of chemistry, faculty of sciences, egerton university, 20115 egerton, kenya. corresponding author* olayombotaiwo@gmail.com manuscript received: 06 april, 2022. revision accepted: 07 june, 2022. published: 30 june, 2022. abstract the leaves and bark of ficus sagittifolia have been used as a cure for stomach and pulmonary disorders, respectively. the bark is edible and is taken against colic. from the leaves of f. sagittifolia, a steroidal glycoside named stigmast-5,22-diene-3-o-β-d-glucopyranoside 1 and three isoflavonoids named 5-hydroxy-3-(4-hydroxyphenyl)-7-methoxy-4h-chromen-4-one 2, 5-hydroxy-3(4-hydroxylphenyl)-8,8dimethylpyrano[2,3-f]-chromen-4(8h)-one 3 and 5-hydroxy-3-(4-hydroxyphemyl)-8,8-dimethylpyrano[3,2-g}-chromen-4(8h)-one 4 were isolated, and this is the first report of the isolation of these compounds from this plant. the structural elucidation of the compounds was based on 1d and 2d nmr, ir and ms data analyses. compounds 1 and 2 inhibited the growth of pseudomonas aeruginosa and aspergillus niger at 0.0625 mg/ml, respectively while compounds 2 and 4 were active against helicobacter pylori at 0.0625 mg/ml. these findings corroborate the ethno-medicinal use of f. sagittifolia leaves as a treatment for stomach disorders. keywords: antimicrobial activity; f. sagittifolia; isoflavonoids; natural products; steroidal glycoside. abbreviations: fsl – ficus sagittifolia leaves; hex – hexane; etoac – ethyl acetate; tlc – thin layer chromatography; chcl3 – chloroform; meoh – methanol; hplc – high performance liquid chromatography; p –int – p-iodonitrotetrazollium; dmso – dimethylsulphoxide; nmr – nuclear magnetic resonance; 1h nmr – proton nuclear magnetic resonance; 13c nmr – carbon nuclear magnetic resonance; cosycorrelation spectroscopy; hsqcheteronuclear single quantum correlation spectroscopy; hmbc heteronuclear multiple bond correlation spectroscopy; tms – tetramethylsilane; hresims – high-resolution electrospray ionization mass spectrometry; lresims – low-resolution electrospray ionization mass spectrometry; ir – infrared spectroscopy; uv – ultraviolet; na – nutrient agar; tsb – tryptic soy broth; sdb – sabouraud dextrose broth; mic – minimum inhibitory concentration; mmc – minimum microbicidal concentration; (cd3)2so – deuterated dimethylsulpoxide; (cd3od) – deuterated methanol; multi – multiplet; j – coupling constant; tr – retention time; rf – retention factor. introduction ficus sagittifolia (warburg ex mildbraed & burret) belongs to the family moraceae and a member of the genus ficus. it is an epiphytic shrub, often on oil palms, becoming a tree to 30 feet high. it is native to benin, cameroon, ghana, guinea, guinea-bissau, ivory coast, liberia, nigeria, senegal, sierra leone, and togo (powo, 2022). ficus species have been used locally as stomach herbs, anthelmintics, vermonicides, astrigents, carminatives, hypotensives, and anti-dysentery drugs (salem et al., 2013). the leaves and bark of f. sagittifolia are used as a cure for stomach and pulmonary disorders, respectively. the bark is edible and is taken against colic (burkill, 1997). species of ficus are known to possess antioxidant, cytotoxic, antimicrobial, anti-inflammatory, antidiabetic, antiulcer, and anticonvulsant activities (salehi et al., 2021). phytochemicals such as genistein, protocatechuic acid, stigmatserol, rutin, bergapten, psoralen, alpinumisoflavone, betulinic acid, umbelliferone, apigenin, derrone, β-sistosterol and chlorogenic acid, amongst others, were common compounds reported in this genus (nawaz, et al., 2019; ateba et al., 2019). a recent study on the phytochemical screening of f. sagittifolia leaf and stem bark extracts revealed the presence of phenolics, flavonoids, steroids, tannins, saponins, alkaloids, and terpenoids with good antioxidant activities (taiwo and olaoluwa, 2020). however, there is little or no information on the isolation and characterization of phytochemicals constituents from f. sagittifolia to support its ethnomedicinal use. hence, this paper reports four known compounds from f. sagittifolia leaves and their antimicrobial activity for the first time. https://doi.org/10.14421/biomedich.2022.111.75-81 76 biology, medicine, & natural product chemistry 11 (1), 2022: 75-81 materials and methods plant collection and identification leaves of f. sagittifolia were harvested during the dry season in ikire, osun state, nigeria in april 2018. identification and authentication were done at the herbarium unit of the forestry research institute of nigeria (frin), ibadan. a voucher specimen of f. sagittifolia (fhi 111988) was documented for reference purposes. plant preparation and extraction f. sagittifolia leaves were air-dried and subjected to pulverization. ground leaves were macerated in ethanol for 72 h and was concentrated under reduced pressure to give ethanol extract. the ethanol extract was partitioned in hexane and subsequently in ethyl acetate to obtain the respective fractions. general experimental procedure tlc was performed on precoated silica gel 60gf24 by merck. column chromatography with a column length – 60 cm; internal diameter of 32.5 mm; external diameter of 36.5 mm and silica gel of 60 – 200 mesh size was used as the stationary phase. eluates were concentrated at reduced pressure at 40°c using the buchi rotavapor r-200 rotary evaporator. compounds were purified using a shimadzu preparative hplc equipped with a uv detector, a reversed phase column (luna®, c18, 5 μm particle size, 10 × 2500 mm, phenomenex), a shimadzu lc – 20ap pump equipped with a dgu – 20a5r degassing unit, a shimadzu spd – m20a detector, and a shimadzu sil – 20acht autosampler, using labsolutions software. nmr experiments for 1h and 13c were performed on the bruker avance 600 and 700 mhz nmr spectrometers for 1h; 150 and 175 mhz nmr spectrometers for 13c. the readings were taken in deuterated methanol and deuterated dimethylsulphoxide with tms serving as reference. 1h and 13c nmr spectra were elucidated using mestrenova software. hresims were performed on a bruker daltonik maxis 4g equipped with ultra-high resolution time-of-flight electrospray ionization in both negative and positive ion modes. lresims were carried out on bruker amazone speed etd ion trap and 8-dionex ultimate 3000 lc in both negative and positive ion modes. in the analysis, thermo xcaliburqual computer software was used in analysis of the mass chromatogram. ir spectra were recorded on the perkinelmer spectrometer instrument. fractionation the ethylacetate fraction (10 g) of f. sagittifolia was subjected to column chromatography and fractionated by the gradient elution method using a solvent mixture of hexane and ethyl acetate as the mobile phase. a total of 102 fractions (100 ml) were collected from the column, and fractions with similar tlc profiles were pooled together to obtain 16 fractions (fsl 1-16). isolation and purification of compounds fraction 15 was eluted by hex/etoac (10:90), as a white precipitate to give compound 1 (92.2 mg) which was soluble in dmso and was confirmed as a spot on the tlc plate (rf = 0.58). fractions 13 (hex/etoac, 30:70), 14 (hex/etoac, 20:80) and 16 (hex/etoac, 0:100) were further purified using preparative hplc analysis. the solvents used were double distilled water with 0.1% formic acid as solvent a and hplc grade meoh as solvent b. gradient elution was carried out with 60% meoh for 7 min and thereafter, isocratic condition at 100% meoh for 5 min. the system returned within 0.5 min to the initial condition of 60% meoh and was equilibrated for 10 min. the eluted fractions were detected by absorbance between 254 370 nm and the flow rate was 3 ml/min to obtain compounds 2 (tr = 15.2 min; 2.1 mg), 3 (tr = 18.3 min; 5.3 mg) and 4 (tr = 18.9 min; 4.4 mg). subsequently, compounds 1 – 4 were subjected to spectroscopic analysis. antimicrobial assay microbial cultures gram negative bacteria: escherichia coli (atcc 11175), pseudomonas aeruginosa (atcc, 27853), salmonella typhi (atcc, 14028), helicobacter pylori, and fungi: aspergillus niger and candida albicans were maintained on na and sda, respectively. a single colony of each organism was inoculated into 5 ml of tsb and sdb for the preparation of bacterial and fungal cultures, respectively. all the microbes were subcultured from the original culture and incubated overnight at 37°c for 24 h and at 25°c for 48 h for bacteria and fungi respectively. the bacteria except h. pylori were obtained from the pharmaceutical microbiology department, university of ibadan, ibadan, nigeria, and the fungi and h. pylori were obtained as clinical isolates from the university college hospital (uch), ibadan. the standard drugs, gentamycin and ketoconazole, were used as the positive control. the negative control used was 1% dmso. minimum inhibitory concentration and minimum microbicidal concentration the antimicrobial assay was done using the broth microdilution method and 96-well plates were used. compounds 1-4 were dissolved separately in dmso to obtain a stock solution of 1.0 mg/ml each. this was diluted serially in the microplate wells to obtain a concentration range of 0.5-0.0039 mg/ml. tsb was used, and standard drugs were gentamycin and ketoconazole (10 𝜇g/ml) for the anti-bacterial and antifungal assays, respectively. the reference drugs were also diluted to obtain concentration range of 10 – 0.3125 taiwo et al. – phytochemical constituents of f. sagittifolia … 77 𝜇g/ml. each of the microplate wells was inoculated with 10 𝜇l of the test organisms and incubated at 37°c and 25°c for 24 h and 48 h for bacteria and fungi, respectively. the least concentrations that showed no growth or turbidity after hours of incubation were streaked on na and sda for bacteria and fungi, respectively. the concentration with no trace of growth was taken as the mic. also, after incubation, 10 𝜇l of 0.2 mg/ml p –int was added to each well and incubated for another 30 min at 37°c. wells with a color change from yellow to pinkish red were an indication of microbial growth. the least concentration that showed no trace of color change was taken as the mmc (bacteria/fungi). results and discussion phytochemical investigation the ethyl acetate fraction of f. sagittifolia leaves yielded a steroidal glycoside 1, and fractions 13, 14, and 16 that were purified by preparative hplc gave three isoflavonoids, 2 4 respectively. stigmast-5,22-diene-3-o-β-d-glucopyranoside (stigmasterol glucoside) 1 was isolated as a white powder. lresims gave [m h] ion at m/z 573.75 with the molecular formula c35h58o6 (calcd. for c35h58o6, for 574.4233). in the 1hnmr spectrum of 1 (table 1), the presence of six methyl protons was confirmed by signals at δh 0.67 s (h-18), 0.91 s (h-19), 0.96 d (h-21), 0.81 m (h-26), 0.85, 1.39 m (h-27) and 0.82 d (h-29); three olefinic protons at δh 5.38 (h-6, t, j=4.8 hz), 5.13 (h-22, dd, j=9.0 hz, 13.8 hz) and 5.03 (h-23, d, j=8.4) and one anomeric proton at δh 4.18 (h-1’, d, j=7.8 hz). the 13cnmr spectrum showed six signals in the methyl region (δc 12.2, 12.3, 19.1, 19.4, 20.2 and 23.1). four methine resonances at δc 73.9, 77.2, 70.6 and 77.1, as well as one methylene resonance at δc 61.6, were due to c-2’, c-3’, c-4’, c-5’, and c-6’, respectively, of the βd-glucopyranoside. (table 1). the olefinic resonances at δc 121.7, 138.5 and 129.3 corresponded to the c-6, c22, and c-23 methine carbons. an anomeric carbon signal at δc 101.2 indicated the presence of a sugar dglucose moiety. the value of j = 7.8 on 1' (anomeric proton) reflected an axial-axial position to the c-2' proton, which confirms that the glucopyranoside moiety binds to the sterol moiety in a β-position (silverstein et al., 1962; bai et al., 2005). the connectivity of protons and carbon-protons was determined from a combination of cosy, hsqc, and hmbc data as shown in table 1. the ir band (cm-1) at 3589, 2850, and 1104 are typical for hydroxyl, carbon-hydrogen, and carbon-oxygen groups, respectively. this explanation was based on a comparison of 1h and 13cnmr spectra with those previously reported in literature (valizadeh et al., 2014; ilango, 2018; olawumi and koma, 2019). 5-hydroxy-3-(4-hydroxyphenyl)-7-methoxy-4hchromen-4-one (prunetin) 2, is pale yellow in color and powdery, with a molecular formula of c16h12o5, m/z of 285.0756 [m+h] + in hresims (calcd. for c16h12o5, for 284.0685). sixteen signals were displayed from the 13cnmr spectrum of 2 (table 2). seven aromatic carbons were observed at δc 91.8 (c-8), 97.8 (c-6), 114.9 (c-3’, c-5’), 130.0 (c-2’, c-6’) and 153.7 (c-2). four oxygenated aromatic carbons at δc 157.5 (c-4’), 158.2 (c-9), 162.3 (c-5) and 165.9 (c-7). one carbonyl carbon at δc 181.1 (c-4). the δc 55.1 (och3, c-7) and δh 3.91 (3h, s, h-7) as well as the hmbc correlations confirmed the presence of a methoxy group at position c-7. the 1h nmr also showed two doublets of four aromatic protons with vicinal coupling constants. the δc 153.7 (c-2) is a characteristic of ring b of an isoflavone (table 2). the cosy correlation of h-6 with h-8 and h2’/3’ with h5’/6’ supported the positioning of these four aromatic protons. the hmbc correlations from the methoxy protons (δh 3.91) to c-7 supported the attachment of the methoxy group to c-7. the ir spectra indicated the presence of hydroxyl, carbonyl, and aromatic groups (3671, 1717, and 1454 cm-1, respectively). this elucidation was in agreement with the literature (máximoa et al., 2002; awouafack et al., 2011). 5-hydroxy-3(4-hydroxylphenyl)-8,8dimethylpyrano[2,3-f]-chromen-4(8h)-one (derrone) 3, is a pale yellow powder with a molecular formula of c20h16o5, m/z of 337.1070 [m+h]+ in hresims (calcd. for c20h16o5, 336.0998). the presence of an isoflavone skeleton was confirmed from the 1h nmr and 13c nmr spectra, with proton and carbon signals at δh 7.43 (h-2’), δc 153.4 (c-2), δc 123.6 (c-3) and δc 181.1 (c-4). the the 1h nmr showed two doublets of four aromatic protons with ortho-coupling constants and one proton signal at δ 6.23 s of aromatic proton in ring a. the 2,2-dimethylpyran substituent was confirmed by signals at δh 5.73 (h-3’’) d (j3’’/4’’ = 9.8 hz), δh 6.78 (h4’’) d (j3/4, 8.4 hz), δh 1.50 (h-5’’me,6’’me), δc 127.4 (c-3’’), δc 114.9 (c-4’’) and δc 27.0 (c-5’’/6’’) whose location in ring a was established by hmbc correlation of a methine carbon δc-6 99.4 with a methine carbon δc-5 161.9 and a quaternary carbon δc-9 159.5 (table 3). the ir bands at 3714cm-1 and 1635cm-1 are typical for hydroxyl and a α,-β unsaturated ketone group respectively. this elucidation was also in agreement with those reported in literature (maximoa et al., 2002; ediziri et al., 2012). 5-hydroxy-3-(4-hydroxyphemyl)-8,8dimethylpyrano[3,2-g}-chromen-4(8h)-one (alpinumisoflavone) 4, is pale yellow and powdery, having a m/z of 337.08 [m+h]+ in lresims with a molecular formula of c20h16o5 (calcd. for c20h16o5, 336.0998). a pair of doublets δh 6.87 d (j=7.7 hz) and 7.41 d (j=7.0 hz) integrated for two protons and were allocated to the h-3’/h-5, h-2’/h-6’ of the parasubstituted aromatic ring. the high chemical shift of δh 6.87 (j=7.7 hz) signified the presence of an oxygened substituent (oh) at c-4’of the aromatic 78 biology, medicine, & natural product chemistry 11 (1), 2022: 75-81 nucleus. the ring c of the isoflavone was also confirmed with signals at δh 7.41 (h-2), δc 153.5 (c-2), δc 121.7 (c-3) and δc 181.0 (c-4). the evidence of proton signals at δh 5.75 (h-3’’) d (j3’’/4’’ = 9.8 hz), δh 6.71 (h-4’’) d (j3/4, 10.5 hz), δh 1.48 (5’’me/6’’me) and carbon signals at δc 128.2 (c-3’’), δc 114.7 (c-4’’) and δc 27.1 (5’’/6’’) confirmed the attachment of the 2,2-dimethylpyran substituent on ring a. compounds 3 and 4 have the same molecular formula and mass but different structures due to the position of the 2,2dimethylpyran substituent on ring a. the positon was further confirmed using hsqc and hmbc, which revealed different chemical shifts for δc (c-6) and δc (c8) and different hmbc correlations as shown on table 4. the δc (c-6) is a quaternary carbon in 4, whereas in 3, δc (c-6) is a methine carbon and vice versa for δc (c-8). the ir spectra indicated the presence of hydroxyl, carbonyl, and aromatic groups (3571, 1717, and 1454 cm-1). this spectral data was compared to those previously published for alpinumisoflavone (rahman et al., 2007; hussain et al., 2011 tjahjadarie et al., 2016). table 1. 1h nmr (600 mhz) and 13c nmr (150 mhz), hmbc, cosy assignment of compounds 1 in (cd3)2so. p o si ti o n δc, type δh mult. (j in hz) hmbc (h/c) c o s y 1 37.3, ch2 0.99, m;1.78, m c-14, 4 2 29.7, ch2 1.23, m; 1.81, m 3 77.4, ch 3.46, m 4 42.2, ch2 1.96, m 5 140.9, c 6 121.7, ch 5.38, d (j=4.8) c-8, 10 h-8 7 31.8, ch2 1.39, m 8 31.9, ch 1.53, s c-6,7,13,14 h-6 9 50.1, ch 0.88, m 10 36.7, c 11 21.1, ch2 0.97, m 12 38.5, ch2 2.11, m 13 42.3, c 14 56.6, ch 0.99, m 15 39.6, ch2 1.95, m c-13 16 24.3, ch2 1.54, m c-5,6,8,14 17 55.9, ch 1.10, m 18 12.2, ch3 0.67, s c-4,14 19 19.1, ch3 0.91, s c-17, 20 20 36.0, ch 1.30, m 21 19.4, ch3 0.96, d (j=7.2) h-29 22 138.5, ch 5.13, dd (j=9.0, 13.8) 23 129.3, ch 5.03, dd (j=8.4, 15.0) 24 45.6, ch2 0.92, m 25 29.1, ch 1.63, m 26 19.6, ch 0.81, m 27 20.2, ch3 0.85, m; 1.39, m 28 23.1, ch3 0.76, m 29 12.3, ch3 0.82, d (j=7.2) c-24, 25, 26 h-21 1’ 101.2, ch2 4.18, d (j=7.8) c-3’ 2’ 73.9, ch 2.89, m 3’ 77.2, ch 3.12, m 4’ 70.6, ch 3.01, m c-5’,6’ 5’ 77.1, ch 3.06, m 6’ 61.6, ch2 3.39, m; 3.63, m c-5’ taiwo et al. – phytochemical constituents of f. sagittifolia … 79 table 2. 1h nmr (700 mhz) and 13c nmr (175 mhz) assignment of compounds 2-4 in (cd3od). 2 3 4 position δc, type δh mult. (j in hz) δc, type δh mult. (j in hz) δc, type δh mult. (j in hz) 2 153.7, ch 8.15, s 153.4, ch 8.17, s 153.5, ch 8.10, s 3 121.8, c 121.6, c 121.7, c 4 181.1, c 181.1, c 181.0, c 5 162.3, c 161.9, c 157.5, c 6 97.8, ch 6.40, d (j=2.8) 99.4, ch 6.23, s 105.2, c 7 165.9, c 152.2, c 156.3, c 8 91.8, ch 6.58, d (j=2.1) 105.6, c 94.4, ch 6.38, s 9 158.2, c 159.5, c 159.4, c 10 105.7, c 101.1, c 105.6, c 1’ 123.6, c 123.6, c 123.4, c 2’, 6’ 130.0, ch 7.42, d (j=8.4) 130.0, ch 7.43, d (j=11.9) 130.0, ch 7.41, d (j=7.0) 3’,5’ 114.9, ch 6.88, d (j=9.1) 114.9, ch 114.9, ch 6.87, d (j=7.7) 4’ 157.5,c 157.5, c 157.4, c 2’’ 78.0, c 77.9, c 3’’ 127.4, ch 5.73, d (j=9.8) 128.2, ch 5.75, d (j=9.8) 4’’ 113.9, ch 6.77, d (j=8.4) 114.7, ch 6.71, d (j=10.5) 5’’, 6’’ 2.70, ch3 1.50, s 27.2, ch3 1.48, s och3 55.1, ch3 3.91, s (j=2.8) antimicrobial assay the broth dilution method was used to determine the mic and mmc of compounds 1-4 against different bacteria and fungi strains: escherichia coli (atcc 11175), pseudomonas aeruginosa (atcc, 27853), salmonella typhi (atcc, 14028), helicobacter pylori, aspergillus niger and candida albicans. compounds 14 showed good antimicrobial activity as demonstrated by their mic values (0.0625-0.25 mg/ml), in table 3a. the lowest mic value (0.0625 mg/ml) was obtained from compound 1 and 2 against p. aeruginosa and a. niger. at the same concentration, compounds 2 and 4 inhibited the growth of h. pylori, among others. compounds 2, 3, and 4 showed moderate inhibition (mic =0.125 mg/ml) against the growth of e. coli while for compound 1, 0.25 mg/ml was required. compounds 1, 3 and 4 inhibited the growth of c. albicans at a concentration of 0.125 mg/ml. compound 2 had the lowest mic and mmc values against s. typhi (0.125 mg/ml) and h. pylori (0.0625 mg/ml), respectively in table 3a. gentamicin had an mic value ranging from 10 to > 10 µg/ml while ketoconazole had an mic values ranging from 1-0.5 µg/ml. overall, compounds 2 and 4 showed very good antimicrobial activity against all the test organisms among others. flavonoids and isoflavonoids are well-known natural products with extensive pharmacological activities and extremely low toxicity (wang et al., 2020). more importantly, they possess a wide range of biological activities, such as antibacterial, antifungal, antiviral (dastidar et al., 2004; orhan et al., 2010), antitumour (kopustinskiene et al., 2020), anti-inflammatory (sychrová et al., 2020) and antiaging activities (gupta et al., 2014). table 3a. mic and mmc of compound 1-4. organisms compound 1 compound 2 compound 3 compound 4 mica mmca mica mmca mica mmca mica mmca h. pylori 0.125 0.125 0.0625 0.0625 0.125 0.25 0.0625 0.0625 e. coli 0.25 0.25 0.125 0.125 0.125 0.125 0.125 0.125 p. aeruginosa 0.0625 0.125 0.25 0.25 0.25 0.25 0.125 0.125 s. typhi 0.25 0.25 0.125 0.125 0.25 0.25 0.25 0.25 a. niger 0.25 0.25 0.0625 0.25 0.125 0.125 0.125 0.125 c. albicans 0.125 0.25 0.25 0.25 0.125 0.125 0.125 0.25 note: a: mg/ml table 3b. mic and mmc of bacteria and fungi standards. organisms gentamycin ketoconazole mica mmca mica mmca h. pylori 10 >10 na na e. coli 10 10 na na p. aeruginosa >10 >10 na na s. typhi 10 10 na na a. niger na na 1 1 c. albicans na na 0.5 0.5 note: a: µg/ml 80 biology, medicine, & natural product chemistry 11 (1), 2022: 75-81 conclusions a steroidal glucoside namely, stigmast-5, 22-diene-3-oβ-d-glucopyranoside (stigmasterol glucoside) 1 and three isoflavanoids, namely 5-hydroxy-3-(4hydroxyphenyl)-7-methoxy-4h-chromen-4-one (prunetin) 2, 5-hydroxy-3(4-hydroxylphenyl)-8,8dimethylpyrano[2,3-f]-chromen-4(8h)-one (derrone) 3, and 5-hydroxy-3-(4-hydroxyphemyl)-8,8dimethylpyrano[3,2-g}-chromen-4(8h)-one (alpinumisoflavone) 4, were isolated from the ethylacetate fraction of f. sagittifolia leaves for the first time. compounds 1 and 2 showed inhibition against p. aeruginosa and a. niger; 2 and 4 inhibited the growth of h. pylori. therefore, compounds 1, 2, and 4 could be antimicrobial agents for diseases related to stomach disorders. this study also provided a scientific justification for the ethnomedicinal use of f. sagittifolia leaves for the treatment of stomach disorders, particularly those caused by microbes. acknowledgements: o. m. taiwo is thankful to the department of chemistry, university of ibadan for the equipment rendered for the extraction and ir studies of samples. o. m. taiwo is grateful to african german network of excellence in science (agnes) for a research visit to the department of chemistry, egerton university, kenya which enabled the purification and characterization of the compounds and also to the members of j.c.m. research group. all authors are grateful to the institute of environmental research (infu), tu-dortmund for nmr and ms equipment. dr. s. a. odewo is acknowledged for the identification and authentication of the plant species. competing interests: the authors declare that there are no competing interests. funding: this study was financially supported by the african german network of excellence in science (agnes), through the programme “advocating women in science, technology, engineering, and mathematics’’ with a mobility scholarship to o.m.t. references ateba, s.b., mvondo, m.a., djiogue, s., zingué, s., krenn, l. and njamen, d., 2019. a pharmacological overview of alpinumisoflavone, a natural prenylated isoflavonoid. frontiers in pharmacology, 952. doi: https://doi.org/10.3389/fphar.2019.00952 awouafack, m.d., spiteller, p., lamshoft, m., kusari, s., ivanova, b., tane, p., & spiteller m. (2011). amtimicrobial isopropenyl-dihydrofurano isoflavones from crotalaria lachnophora. journal of natural products, 74(2), 272-278. doi: https://doi.org/10.1021/np1005218 bai, h., li, s., yin, f., & hu, l. (2005). isoprenylated naphthoquinone dimers firmianones a, b, and c from firmiana p latanifolia. journal of natural products, 68(8), 1159-1163. burkill h. m. (1997). the useful plants of west tropical africa. families m-r kew: royal botanic garden, kew, 4(2) 165167. dastidar, s. g., manna, a., kumar, k.a., mazumdar, k., dutta, n.k., chakrabarty, a.n., motohashi, n., & shirataki, y. (2004). studies on the antibacterial potentiality of isoflavones. international journal of antimicrobial agents, 23(1), 99-102. doi: https://doi.org/10.1016/j.ijantimicag.2003.06.003 edziri, h., mastouri m., mohamed, a.m., zine, m., aouni m., & verschaeve l. (2012). antibacterial, antifungal and cytotoxic activities of two flavonoids from reteam raetam flowers, molecules, 17(6), 1784-7293.doi: https://doi.org/10.3390/molecules17067284 gupta, c., & prakash, d. (2014). phytonutrients as therapeutic agents. journal of complementary and integrative medicine, 11(3), 151-169.doi: https://doi.org/10.1515/jcim-2013-0021 hussain, m.m., dastagir, m.g., billah, a.m., & ismail, m. (2011). alpinum isoflavone from erythrina stricta roxb. boletín latinoamericano y del caribe de plantas medicinales y aromáticas, 10(1), 88-90. ilango, k. (2018). β-sitosterol glucoside from pisonia grandis r. br. stem bark in ethyl acetate extract. international journal of green pharmacy, 12(01). doi: http://dx.doi.org/10.22377/ijgp.v12i01.1526 kopustinskiene, d. m., jakstas, v., savickas, a., & bernatoniene, j. (2020). flavonoids as anticancer agents. nutrients, 12(2), 457. doi:https://doi.org/10.3390/nu12020457 máximoa, p., lourenço, a., feio, s.s., & roseiro, j. c. (2002). a new prenylisoflavone from ulex jussiaei. zeitschrift für naturforschung c, 57(7-8), 609-613. doi: https://doi.org/10.1515/znc-2002-7-810 nawaz, h., waheed, r., & nawaz, m. (2019). phytochemical composition, antioxidant potential, and medicinal significance of ficus. modern fruit industry. olawumi, o. o., & koma, s.o. (2019). stigmasterol and stigmasterol glycoside: isolated compounds from tetrapleura tetraptera extracts. journal of biochemistry international, 6(1), 21-48, issn: 2454-4760 orhan, d.d., özçelik, b., özgen, s. & ergun, f. (2010). antibacterial, antifungal, and antiviral activities of some flavonoids. microbiological research, 165(6), 496-504. doi: https://doi.org/10.1016/j.micres.2009.09.002 powo (2022). "plants of the world online. facilitated by the royal botanic gardens, kew. published on the internet; retrieved 10 january 2022". https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:5 84046-1 rahman, m.z., sultana, s.j., faruquee, c.f., ferdous, f., rahman, m.s., islam, m.s., & rashid, m.a. (2007). phytochemical and biological investigations of erythrina variegata. saudi pharmaceutical journal, 15(2), 140. salehi, b., prakash mishra, a., nigam, m., karazhan, n., shukla, i., kiełtyka‐dadasiewicz, a& sharifi‐rad, j. 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(2014). isolation and structure elucidation of secondary metabolites from echinophora platyloba dc from iran. journal of medicinal plants, 1 (49), 15-21. wang, j.f., liu, s.s., song, z.q., xu, t.c., liu, c.s., hou, y.g., huang, r. & wu, s.h. (2020). naturally occurring flavonoids and isoflavonoids and their microbial transformation: a review. molecules, 25(21), 5112. doi: https://doi.org/10.3390/molecules25215112 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 431-435 | doi: 10.14421/biomedich.2023.122.431-435 issn 2540-9328 (online) assessment of sensorimotor behaviour in konzo-induced rats using the irvine, beattie bresnahan forelimb scale 1department of anatomy, faculty of basic medical sciences; 2department of biomedical technology, school of science laboratory technology, university of port harcourt, rivers state, nigeria. corresponding author* uahomoprecious1@gmail.com; phone: +2347035313368 manuscript received: 24 june, 2023. revision accepted: 26 july, 2023. published: 12 august, 2023. abstract konzo is a neurological disorder of selective upper motor neurons. it is an irreversible paralytic disease associated with prolonged consumption of cassava. it contains cyanogenic glycosides metabolized to hydrogen cyanide, which has been shown by studies to affect the motor neurons of the central nervous system. the irvine, beattie bresnahan (ibb) scale is a recently developed forelimb scale for the assessment of fine control of the forelimb and digits after cervical spinal cord injury such as konzo. 20 adult male wistar rats were assigned to 4 experimental groups (i) control n=5, (ii) konzo-induced group n=5, (iii) induced + complan n=5 (iv) induced + bambara nut (okpa). the bitter cassava foods were taken by oral ingestion for a period of 4 weeks. the assessment of the forelimb and digits were done using the irvine, beattie bresnahan (ibb) with specific parameters such as predominant elbow joint movement, contact volar support, and grasping method. the body weight of the animals was also recorded every week. the data obtained were analyzed using anova. the result obtained showed that there was a significant difference (p<0.05) between the body weight of the animals induced with konzo and rehabilitated with complan milk and bambara nut when compared to the unrehabilitated konzo-induced group. there were differences in the results of the parameters being tested for the irvine, beattie bresnahan (ibb) scaling. the ibb scale confirmed that there was a high level of cyanide content in the cassava which affected the behavioral attributes of the induced group and it also confirmed that the induced group can be ameliorated with the use of complan and bambara nut (okpa) which was shown in the parameters being tested such as predominant elbow joint movement, contact volar support, and grasping method. it was concluded that insufficiently processed bitter cassava is toxic and has neurotoxicity effects on the spinal cord especially on the upper motor neurons and ibb scale is capable of measuring gradual improvements in motor forelimb functions in this model and may be a new and effective assessment tool for peripheral nerve injury. keywords: bambara nut; cyanogenic; neurotoxicity; konzo; bitter cassava; irvine, beattie bresnahan forelimb scale. introduction cassava toxicity has been incriminated in the pathogenesis of tropical myeloneuropathies, such as tropical ataxic neuropathy (tan) and konzo. tan is a progressive myeloneuropathy that was first described in nigeria and is characterized by a progressive onset of ataxia. on the other hand, konzo is a permanent and clinically distinct upper motor neuron disease of abrupt onset first described in the democratic republic of congo (drc). the word “konzo” means tied legs and originated from the yaka tribe in drc to designate a fetish used by hunters to weaken legs and catch wild animals (trolli, 1938; van der beken, 1993). konzo occurs mainly in children and young women of childbearing age. the paralysis occurs quite suddenly, does not progress over time, and is irreversible. it is associated with the consumption of a monotonous diet of high cyanide (bitter) cassava, by poor rural people in africa, many of whom suffer from malnutrition. specifically, konzo is associated with a high cyanide diet of bitter cassava consumed over a period of several weeks combined with a low intake of protein, particularly a shortfall of essential s-containing amino acids that are needed to detoxify cyanide to thiocyanate in the body (howlet et al, 1990). the exact pathogenetic mechanisms of the disease remain unknown. epidemiological studies consistently show an association between outbreaks of the disease and chronic dietary reliance on insufficiently processed cyanogenic cassava (manioc or tapioca). biochemical and toxicological studies suggest that the metabolites of linamarin (α-hydroxyisobutyronitrile β-dglucopyranoside, the main cassava cyanogen), notably cyanide (mitochondrial toxin), thiocyanate (ampa chaotropic agent), and cyanate (protein carbamoylating agent) may play an important role in the pathogenesis of konzo. experimental data suggest that thiol-redox and protein-folding mechanisms may also be perturbed. factors of susceptibility including genetics, poor lekpa kingdom david1, precious ojo uahomo2,*, victor hogan idung2, rachael data dakoru2 https://doi.org/10.14421/biomedich.2023.122.431-435 432 biology, medicine, & natural product chemistry 12 (2), 2023: 431-435 nutrition, poverty, and dietary cyanogen exposure, or their interactions have been suggested. the irvine, beattie bresnahan (ibb) scale is a recently developed forelimb scale for the assessment of fine control of the forelimb and digits after cervical spinal cord injury (sci). several experimental models of cervical spinal cord injury (sci) have been developed recently to assess the consequences of damage to this level of the spinal cord (pearse et al., 2005; gensel et al., 2006; anderson et al., 2009), as the majority of human sci occur here. behavioral deficits include loss of forelimb function due to damage to the white matter affecting both descending motor and ascending sensory systems, and to the gray matter containing the segmental circuitry for processing sensory input and motor output for the forelimb. this is the basis for the study; hence, this study aims to assess the sensorimotor behaviour in a konzo-induced rat using the irvine, beattie bresnahan forelimb scale. methodology animal procurement twenty (20) wister rats weighing between 150-200g were acquired from the animal house of the department of pharmacology, faculty of basic clinical sciences, university of port harcourt for this research work. the animals had free access to water and were fed ad libitum with standard feed (broiler finisher – guinean feed). two weeks before the start of the test, the animals were acclimated. plant collection and identification the cassava plants were gotten at the ministry of agricultural development program, rumuodomaya, port harcourt, rivers state, and were identified by the agriculture department of the university of port harcourt, choba, rivers state. processing of bitter cassava root fresh cassava roots were uprooted from the farm, and immediately after harvesting the outer layer (skin or cortex) was removed with a knife to expose the white inner layer. then the roots were sliced into smaller sizes like chips and allowed to sundry for 3 consecutive days. the dry cassava pieces were manually grinded into powder form using a grinding machine and served to the experimental animal as cassava chow. induction of konzo disease in rats after two weeks of acclimatization, 15 rats were fed with the processed bitter cassava flour constantly to induce the konzo disease and its effects. the oral feeding method used represents the real scenario of food being consumed by ingesting through the mouth, passing through the esophagus into the stomach following the alimentary canal, contacting the necessary visceral organs along with their associated fluids such as saliva in the mouth and gastric juices including any contributions provided via sublingual or buccal absorption during digestion. experimental design the experimental method as described by david et al. (2022) was used in this study. twenty (20) albino wistar rats were used for this study. out of the 20 rats, 15 were induced with konzo disease for two weeks and then randomly selected into three (3) groups of five (5) animals each (groups 2, 3, and 4), while the remaining 5 rats without konzo disease served as control (group 1). rehabilitation group: after the period of konzo disease induction, group 3 and group 4 were completely stopped from consuming the bitter cassava and replaced with rat feed and complan milk for group 3 and bambara nut (okpa) for group 4. the mode of feeding was by oral ingestion. table 1. experimental design. group identity n o . o f r a ts treatment group 1 control 5 2ml normal saline group 2 induced control 5 bitter cassava only group 3 complan 5 bitter cassava + complan milk group 4 bambara nut 5 bitter cassava + bambara nut determination of body weight the animals were weighed daily with an electric weighing scale (sf-400c) and the body weight of the rats was recorded. sensorimotor behaviour assessment the forelimb function was assessed using a special method, which is ibb (irvine, beattis and bresnahan test). the procedure is as explained; rats were given pieces of cereal in their home cage twice daily beginning as soon as they entered the lab. forelimb function was assessed while rats were eating cereal as described previously (irvine et al., 2010). briefly, rats were individually placed in a plexiglas cylinder (diameter = 20 cm; height = 46 cm) or in their home cage and they were given donut-shaped pieces of cereal that were of a consistent size and shape prior to the initiation of eating. rats were not scored when eating cereal pieces that were broken prior to the initiation of testing. each trial was recorded to allow slow-motion hd playback and evaluation of forelimb use. videos of animals eating the cereal were evaluated using a standardized scoring sheet to record observations of forelimb behaviors, including joint position, object support, wrist and digit movement, and grasping method used while consuming both cereal shapes. david et al. – assessment of sensorimotor behaviour in konzo-induced rats … 433 cyanide analysis the samples for analysis of the cyanide in the bitter cassava were prepared as follows: ▪ 10g of the sample was mixed with 50ml of water in a corked conical flask. ▪ allowed to stand for 24hrs to extract the residual cyanoglucosides in the samples. ▪ the mixture was subsequently filtered to obtain the soluble extract containing cyanoglucosides ▪ the same procedure as with standard kcn solutions was followed to determine the free cyanide concentration (as hcn equivalent) in the sample filtrate. ▪ the absorbance of the sample solution was equally measured at 510nm wavelength against a blank devoid of kcn solution. ▪ cyanide levels of the test samples were evaluated from the standard calibration curve by extrapolation. ethical clearance the experimental animals were acquired from the animal house of the department of pharmacology in the faculty of basic medical sciences. all procedures carried out during this research were done in accordance with the guiding principles of research involving animals as recommended by the research ethics committee of the university of port harcourt. animals were kept in standard metal cages and at room temperature. statistical analysis data were analyzed using statistical package for the social sciences (spss ibm version 23.0) and microsoft excel 2019 edition. values were expressed as mean ± sd in descriptive statistics. one-way analysis of variance (anova) was used to analyze the difference between the groups followed by the least significant difference (lsd) post-hoc test. the confidence interval was set at 95%, and therefore p<0.05 was considered significant. results table 2. effect of konzo disease, complan milk and bambara nut (okpa) on the body weight (grams) of wistar rats. group baseline weight week 1 week 2 week 3 week 4 mean weight group 1 263.01±5.13 263.00±5.87 265.02±6.10 260.10±6.32 273.05±6.75 265.25±2.78b group 2 242.30±5.17 175.70±5.16 181.10±4.80 171.80±5.15 172.40±5.90 175.25±2.13a group 3 175.25±4.87 194.10±4.21 192.50±4.11 223.10±8.23 230.10±8.40 209.95±9.72a,b group 4 175.25±5.10 188.30±5.87 190.05±7.02 200.60±7.50 208.90±7.11 196.95±4.82a,b the results are expressed as mean ± sem. level of significance values are ap<0.05 when compared with group 1 (control), bp<0.05 when compared with group 2 (konzo induced). figure 1. comparison of baseline body weight with final mean body weight of wistar rats. table 3. irvine, beatties and bresnahan forelimb scale. group pejp cvs gm group 1 (control) flexed none normal group 2 (induced) partially flexed nearly always abnormal group 3 (induced + complan) flexed some normal group 4 (induced + bambara nut) flexed some normal keys: pejp = predominant elbow joint position; cvs = contact volar support; gm= grasping method 0 50 100 150 200 250 300 baseline body weight mean body weight b o d y w ei gh t (g ) duration (weeks) body weight (g) control konzo induced konzo + complan konzo + bambara nut (okpa) 334 biology, medicine, & natural product chemistry 12 (2), 2023: 431-435 figure 2. contact volar support: a – control (fed with normal feeds – finishers), b – fed with bitter cassava, c fed with bitter cassava then treated with complan, d fed with bitter cassava then treated with bambara nut figure 3. predominant elbow joint movement: a – control (fed with normal feeds – finishers), b – fed with bitter cassava, c fed with bitter cassava then treated with complan, d fed with bitter cassava then treated with bambara nut figure 4. grasping method: a – control (fed with normal feeds – finishers), b – fed with bitter cassava, c fed with bitter cassava then treated with complan, d fed with bitter cassava then treated with bambara nut discussion the effect of bitter cassava has been studied extensively and its neurotoxicity was being studied with the use of experimental animal models to evaluate the effects of diverse substances on motor function and their relationship with cns disorders. (gerhart et al., 1982; kulig et al., 1985; fowler et al., 1990). the weight of the rats was taken and measured accurately from the beginning of the week till the end of the experiment at a significant value of p<0.05. it was observed that there was a significant difference between the mean body weight of the control group 265.25±2.78g and the mean body weight of the induced group (175.25±2.13g). the difference between the weight of the induced was different from that of the induced as a result of the intake of the bitter cassava which has caused a neurotoxic effect on the experimental models. this agrees with the findings of david et al. (2022) and enefa et al. (2020). some nutritional approaches were used to ameliorate the effect of the bitter cassava for a period of 4 weeks after the initial four weeks of being induced with konzo disease. the nutritional approach used was complan and bambara nut (okpa). there was a significant difference between the mean body weight of the ameliorated group and that of the induced. the complan group had a mean body weight of 209.95±9.72g while the bambara nut group has a mean body weight of 196.95±4.82g. this shows that the effect of the bitter cassava on the experimental models was ameliorated as there was a significant increase in the weight of the induced group after being fed with complan and bambara nut (okpa). this is also similar to the findings of david et al. (2022) and enefa et al. (2020). the ibb forelimb scale provided a sensitive measurement scale for detecting recovery of both proximal and distal forelimb function, including digit movements, during a naturally occurring behavior. this behavioral test was developed and tested in rats with spinal cord injury by irvine et al. (2010). this method evaluated the overall integrity of rodent forelimb functionality during a food intake task, measuring how rats grasp a cereal piece. the ibb scale was used to test just 3 parameters which include predominant elbow joint movement, contact volar support, and grasping method. during the assessment of contact volar support, the scale was rated as ‘none’ ‘some’, and ‘almost always’. the rat was assessed for its ability to use the volar (palmar) surface of the impaired forepaw to stabilize the cereal and, in doing so, maintain a position to aid eating. in figure 2, group a being the control group, no volar support by the forelimb during eating (<5% of the time). but in group 2, being the induced group that was affected by the cyanide content of the bitter cassava, the volar support of the object occurred nearly always or always during eating (>95% of the time). this is because the spinal cord had been affected which in turn relates to david et al. – assessment of sensorimotor behaviour in konzo-induced rats … 435 how the rats had to use their volar for support. the ameliorated group which is group 3 (complan) and group 4 (bambara nut) had the support of the volar but did not occur always. this shows that the konzo disease was ameliorated which helped to reduce the use of volar for support in the case of the induced group. the original scale rated the predominant elbow joint position as “extended, partially flexed, or fully flexed.” discrimination between partially and fully flexed appeared to be problematic, and perhaps irrelevant in more recovered animals. looking at figure 3, the rat models were assessed for the most common position (more than 50% of the time). the elbow joint movement for group a was found to be flexed. group b was partially flexed as a result of the cyanide content of the bitter cassava affecting the spinal cord. after the induced were ameliorated, the elbow joint movement was flexed fully. the grasping method was rated to be ‘normal’ and ‘abnormal’ according to figure 4, the experimental rat models were assessed for the most common (more than 50% of the time) grasping technique used during the eating phase. the rat models of group 1 were found to be normal with consistent use of the grasping method used prior to injury to support and control the cereal piece during the eating phase. group 2 being induced was abnormal because they consistently used an alternative method of grasping to the method used prior to the injury to support and control the cereal piece during the eating phase. they were able to support their grasping with their volar. this is a result of the spinal cord injury that occurred as a result of the intake of bitter cassava which had a high percentage of cyanide content. the ameliorated group was found to be normal in their grasping method. according to speck et al. (2014), the ibb scale is capable of measuring gradual improvements in motor forelimb functions in this model and may be a new and effective assessment tool for peripheral nerve injury. this was seen in this study as the induced group was being ameliorated with complan and bambara nut and there was gradual improvement in the motor forelimb function using the three parameters, predominant elbow joint movement, contact volar support, grasping method. conclusion this study has shown that insufficiently processed bitter cassava is toxic and has neurotoxicity effects on the spinal cord especially on the upper motor neurons. a balanced diet with sufficient nutrients such as complan and bambara nut (okpa), can ameliorate the effects of konzo disease as there was no effect on motor neurons of the spinal cord of the animals rehabilitated with complan and bambara nut (okpa). competing interests: the authors declare that there are no competing interests. references anderson k., sharp k., hofstadter m., irvine k., murray m., steward o. (2009). forelimb locomotor assessment scale (flas): novel assessment of forelimb dysfunction after cervical spinal cord injury. exp. neurol., 220:23–33. enefa s., chikwuogwo w.p, lekpa k.d. (2020). model of konzo disease: reviewing the effect of bitter cassava neurotoxicity on the motor neurons of cassava-induced konzo disease on wistar rats. saudi journal of medicine; 5(11):336-348. fowler s.c., liao r.m., skjoldager p. (1990). a new rodent model for neuroleptic-induced pseudo-parkinsonism: low doses of haloperidol increase forelimb tremor in the rat. behav. neurosci., 104:449–456. gensel, j.c., tovar, c.a., hamers, f.p.t., deibert, r.j., beattie, m.s., bresnahan, j.c. (2006). behavioral and histological characterization of unilateral cervical spinal cord contusion injury in rats. j neurotrauma, 23:36–54 gerhart j.m., hong j.s., uphouse l.l., tilson h.a. (1982). chlordecone-induced tremor: quantification and pharmacological analysis. toxicol. appl. pharmacol., 66:234– 243. howlett, w. p, brubaker, g. r., mlingi, n., rosling, h. (1990). konzo, an epidemic upper motor neuron disease studied in tanzania. brain, 113:223–235. kulig b.m., vanwersch r.a.p., wolthuis o.l. (1985). the automated analysis of coordinated hindlimb movement in rats during acute and prolonged exposure to toxic agents. toxicol. appl. pharmacol., 80:1–10. david l.k, idung v.h and uahomo p.o (2022). neurobehavioral and ameliorative effect of complan milk and bambara nut on rats fed with bitter cassava – a nutritional approach. international neuropsychiatric disease journal, 17(1): 7-17 pearse, d., lo t jr, cho, k., lynch, m., garg, m., marcillo, a et al. (2005). histopathological and behavioral characterization of a novel cervical spinal cord displacement contusion injury in the rat. j neurotrauma, 22:680–702. speck a. e., jocemar i., caroline c. s., aguiar a. s. jr., santos a. r., swarowsky a., (2014). the ibb forelimb scale as a tool to assess functional recovery after peripheral nerve injury in mice. journal of neuroscience methods, 226:66–72. trolli, g, (1938). paraplégie spastique épidémique, “konzo” des indigènes du kwango. in: trolli g (ed.), résumé des observations réunies, au kwango, au sujet de deux affections d´órigine indéterminée fonds reine elisabeth, brussels, pp. 1– 36. van der beken, a., (1993). in: karthala (ed 1.), proverbes yaka du zaire harmattan, paris this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 423-430 | doi: 10.14421/biomedich.2023.121.423-430 issn 2540-9328 (online) weed community structure in patia village rice fields patia sub-district, pandeglang regency ai widiyawati, hadi susilo, mu'jijah, suyamto*, nurullah asep abdilah biology study program, faculty of health pharmacy, mathla'ul anwar university banten, indonesia. corresponding author* suyamto35@yahoo.co.id manuscript received: 07 may, 2022. revision accepted: 27 july, 2023. published: 10 august, 2023. abstract rice is a very important type of food crop because it is a staple food source. crop losses at the farmer level due to competition with weeds reach 10-1.5%. this study aims to determine the structure of rice weed communities in rice fields in patia village, patia district, and pandeglang regency. the research method uses an observation method with the sampling technique used as a random method. the plot used was 2 m x 2 m. weed sampling was carried out by recording the types of weed types and counting the number of individuals of each species. measurements of environmental factors such as temperature, humidity, ph, and light intensity were measured. the data parameters measured include the shannon-wiener diversity index (h'), evenness index (j'), dominance index (d), wealth index (s), and abundance of rice weed species. the results of the study were that the types of rice weeds found in the rice fields of patia village, district, and patia, pandeglang regency, namely ludwigia octovalvis (jacq) raven, commanded cylindrical. beauv, spigelia anthelmia, ageratum conyzoides, spihemoclea zeylanica, altemanthera sessillis, eclipta prostate, cypress deformed l., digitaria sp. and mimosa chaste l. the structure of the weed community in the rice fields of patia village, patia district, pandeglang regency is the shannonwiener diversity index (h') of 1.94; evenness index (j') of 0.884; dominance index (d) of 0.15. keywords: weeds; competition; rice; productivity; community structure. introduction rice (oryza sativa l.) is an important crop commodity because it is the staple food of the population in indonesia and plays an important role in economic and social stability in indonesia. the population of indonesia reached 270.20 million people with rice production in 2020 reaching 31.33 million tons (bps, 2021). efforts to support the rice self-sufficiency program and meet rice needs need to maintain the stability of rice production. rice production in indonesia can decline due to several factors, including drought, land narrowing, pest disturbance and the influence of weeds (windari et al., 2021). weeds in rice plants are one of the problems that greatly affect productivity (miranda et al 2011). the presence of weeds on rice plants will cause a decrease in production if weeds are not controlled effectively because they can affect growth and reduce food crop production. planting paddy rice using irrigated irrigation will cause weed competition with rice so that it can reduce yields by 10-40% depending on the species and density of weeds, soil type, water supply and climatic state. rice yield losses at the farmer level due to the presence of rivals with weeds reach 10-15% (farmanta et al., 2016). the presence of weeds on cultivated plants will reduce crop yields. losses caused by weeds are caused by competition with cultivated plants in terms of nutrient extraction, water, sunlight and growing space. in addition, weeds can secrete allelopathic compounds and can be hosts for pests and pathogens of cultivated plants (utami &; purdyaningrum, 2012). one of the ways of weed control is by identifying the diversity and dominance of weeds. the results of dahlianah's research (2017) stated that there are 4 families, 5 genera, and 7 species of weeds in tidal rice fields in manggaraya village, tanjung lago district, banyuasin regency, south sumatra province. the structure of rice weeds in tidal fields with the highest inp value is the weed type echinochloa chicken leg (41.16%). the results of agustina & yursida's research (2015) showed that weeds were dominant in rice plantations in tidal land, banyu urip village, tanjung lago district, with an average ratio the highest number of successive predominations were: ludwigia octovalvis, fymbristilis littoralis, alternanthera philoxeroides and cyperus chicken. weed control is effective if we know the types of weeds in rice fields. according to kastanja (2011) the success of weed control must be based on sufficient and correct knowledge of the biological nature of weeds https://doi.org/10.14421/biomedich.2023.121.423-430 424 biology, medicine, & natural product chemistry 12 (1), 2023: 423-430 through identification, this method is a step first to explore the possibility of appropriate control. efforts made to determine the type and population of weeds are 1) exploring a rice field, which aims to collect data on weed populations growing in the planting area rice, 2) identification which is an effort to recognize something that observes the characteristics of plants that are analyzed simply. local rice plants often find various types of weeds, but there is still little information or research that explains the types of weeds found in rice fields in the province banten, especially in patia village, patia district, pandeglang regency. research methods the type of research to be carried out is nonexperimental research carried out with exploration methods, namely identifying rice weeds in rice fields in patia village, patia district, pandeglang regency. research tools and materials equipment used in the study includes machetes, boxes, shovels, uls, tape meters, notebooks, stationery, rapia ropes, smartphone cameras, rulers, pegs, google earth, label paper, soil tester, newspaper, cardboard, cardboard and hand sprayer or spray. the material used in this study was rice weeds in rice fields in patia village, patia district, pandeglang regency. procedure ▪ research site survey the survey was conducted by exploring villages covering 5 rws randomly. the survey was also conducted through interviews with local farmers. the existence of rice cultivation, the age of rice, the agricultural system and the presence of rice weeds obtained are then recorded in the research notebook. photo documentation is also carried out during the survey. ▪ determination of research blocks purposive determination of rice blocks based on criteria that are rice plantations using conventional monoculture agricultural systems with non-intensive application of pesticides and rice age 3 weeks after planting and not being carried out weed cleaning. the research block was made as many as 5 blocks in 5 rws because patia village consists of 5 rws. the determination of blocks 1, 2, 3, 4 and 5 is carried out sequentially according to the rw sequence number. in each research block, the measurement of coordinate points was carried out using google earth. ▪ plot creation in each research block, a plot with a size of 2x2 m was made in a square shape using 4 pegs around the sides given raffia rope and the distance between the plot and the ripen was 1 m. the placement of plots in research blocks is carried out randomly. ▪ measurement of environmental factors microclimate components that affect growth are air and soil humidity, air and soil temperature so it is very necessary to measure environmental factors such as temperature, humidity and soil ph. measurement of environmental factors was carried out in the morning at 08.00 wib when sampling rice weeds in the research plot. measurement of environmental factors in the form of ph, humidity and mpatur using a soil tester. ▪ rice weed sampling weed sampling was carried out at 08.00 wib. weed sampling was carried out on each research plot. weeds taken are weeds that have complete organs. picking is not carried out on weeds that are still in the sprouting stage. weeds are taken whole (leaves, stems, and roots) with a small shovel then the roots are carefully separated from the ground then put into a plastic bag to take to the laboratory. documentation is carried out during the weed picking process. the number of weeds grown is obtained by counting the number of weeds growing on each plot plot, then counting all the number of individuals in the weed plot plot. speed esimen was later identified. ▪ weed identification identification of rice weeds was carried out by observing external morphological characters such as leaves, stems and roots and adjusted based on description and identification with reference to the book flora steenis (2008). ▪ data collection weeds that have been identified and species determined, then the data are entered into a table and include the number of individuals in each type. the table of types, types and quantities of weeds is then used in data analysis. data analysis test parameter data used in this study including shannon wiener diversity index (h'), evenness index (j'), dominance index (d), wealth index (s) and abundance of weed species with the formula dombois & ellenberg (mazidaturohmah et al., 2018) widiyawati et al. – rice weed community structure in patia village rice fields … 425 results and discussion types of weeds in rice fields table 1. types and numbers of rice weed individuals in patia village, patia district, pandeglang regency. species region name number of individuals per research block plot 1 plot 2 plot 3 plot 4 plot 5 cecabean ludwigia octavalvis (jacq) raven 14 16 18 8 5 for sake cylindrical orders l. beauv 11 3 14 9 17 meniran spigelia anthelmia 2 0 1 0 3 friendship ageratum conyzoides 5 1 6 7 5 gunda spihemoclea zeylanica 1 2 1 0 4 crema altemanthera sessillis 4 4 2 2 3 how-to eclipse the prostate 2 0 5 4 7 puzzle cypress is deformed l 7 8 10 9 8 jampang grass digitaria sp 4 7 7 6 8 princess malu mimosa chaste l 2 1 0 3 1 the results showed that the types of weeds in the rice fields of patia village, patia district, pandeglang regency include ludwigia octovalvis (jacq), raven, cylindrical ordersl. beauv, spigelia anthelmia, ageratum conyzoides, sphenoclea zeylanica, altemanthera seated, eclipta prostate, cypress deformed l., digitaria sp. and mimosa pudica l. vegetation in rice fields is always damaged periodically due to tillage, or due to control, but there is weed vegetation that always appears in the rice field area. the emergence of weeds in rice fields can be categorized as secondary succession; the community can change in a progressive or retrogressive manner (purnomo, 2011). in the agricultural sector, weeds are plants that have a negative impact on cultivated plants either directly or indirectly. weeds that interfere with productive plants during the growth and development of plant life are one of the important problems that can reduce crop production. the percentage of decline in production of each type of cultivated crop is influenced by the weed community in the agricultural area (suryatini, 201: 8). weeds as plant nuisance organisms (opt) are among the important obstacles that must be overcome in increasing rice production in indonesia. the decline in rice productivity is caused by, among others, competition between weeds and rice plants. the decrease in rice yield due to weeds ranges from 6-87% of weeds have a very competitive nature because they have an efficient breeding mechanism that is able to multiply sexually by producing many seeds and vegetatively, so that it greatly reduces the results of cultivated plants. the decline in crop yield varies greatly depending on various factors, including the ability of plants to compete, types of weeds, plant age and weed age, techniques, their cultivation and duration of competition (utami &; purdyaningrum, 2012). weeds reduce crop yields in competition for light, oxygen and co2, as well as food. the decrease in crop yield is caused by weeds that can reduce growth activities, including stunted plant growth, chlorosis, nutrient deficiencies, and the occurrence of reduction in the number and size of plant organs. symptoms of nutrient deficiency in rice plants can result in complete failure of seedling plants, very stunted plants, symptoms on leaves that are distinctive, and abnormalities – abnormalities arising in plant tissues (antralina, 2012). weeds do not always harm plants, at the population level or certain periods have no effect or little effect on plants so that weeds that grow h in that period are unnecessary controlled. therefore the way of cultivation, the type and density of weeds will largely determine when weeding weeds. the composition of weeds will vary on plants that have different heights. soil type, planting pattern and type of cultivated plants affect the number and diversity of weed species. weed species are also affected by plant density, soil fertility, cultivation and tillage patterns. weed management can be carried out better if the level of weed dominance in rice plants is first known (rohimat et al., 2017). the large number of types of weeds that grow in these rice fields can be caused by tillage and fertilizer inputs. the hoeing process during soil processing can cause the lifting of weed seeds to the soil surface. the storage of weed seeds in the soil (seed bank) can germinate into individual weeds at any time if supported by environmental factors (dahlianah, 2017). ludwigia octavalvis (jacq) raven, weed plants are broadleaf weed plants and are found in many rice farms have fibrous roots, pinnate leaves face to face only leaf picking and yellow flowers (stenis 2008). leaves scattered, opposite, pinnateb-rayed; flowers are plucked leaves or as bunches, numbered 2-6; the leaves of the corolla are yellow, regularly numbered 2-4 at the end of the tubular flower axis. petals clinging. crown leaves are free, seated or hoofed (syaifudin &; nofa, 2020). cylindrical ordersl. beauv is a weed that often appears in tropical and subtropical regions. weed 426 biology, medicine, & natural product chemistry 12 (1), 2023: 423-430 commanded cylindrical. beauv is easily spread due to sexual and asexual reproduction. it is assumed that seeds per bunch of flowers are about 500-1.000 seeds, and per m2 about 10-20 bunches of flowers are obtained. seeds of imperata cylindrical. beauv are very easily carried away by the wind, so it is able to spread far. likewise, the possession of allelopathy, makes the reeds very solid in a new place. besides seeds, the weed cylindrical ordersl. beauv produces rhizoma, then when the rhizoma is cut, then each cut (several nodes) will form a new plant bud (juarsah, 2015). spigelia anthelmia is a weed that has taproots. spigelia anthelmia has a cylindrical stem, a smooth or waxy surface, upright growth. the leaves of spigelia anthelmia are oval, green with flat edges. sitting face to face and the number of leaves is compound, it has pinnate leaf bones with tapered leaf tips while the base of the leaves is rounded. flowers are located at the base of the leaves, spherical in shape and yellow in color (kastanja, 2011). ageratum conyzoides belong to flowering plants, members of the family asteraceae originally from tropical america, growing in the tropics. in indonesia, ageratum conyzoides is one of the nuisance plants/weeds that have the potential to inhibit rice growth through rice roots so that it can lack nutrients and ageratumplants conyzoides can live in fields, yards, gardens, banks and waterfronts (astriani, 2010). spihemoclea zeylanica is an annual plant belonging to the broadleaf type. spenochlea zeylanica weed is found in many rice fields. the weed spihemoclea zeylanica has cord-shaped roots, hollow stems, and white grain-shaped flowers. spihemoclea zeylanica multiplies through seeds. altemanthera sessilis is a type of broadleaf weed that has a physical state of creeping growth or growing upwards until scattered, plant height reaches 1 m. in addition, the flowers on alternanthera sessilis berestms white or pink, very small (lestari et al., 2012). the eclipse prostate can be annual (annual weed) and annual (perennial weed), often branched sideways or upright with stands 10-80 cm high, has a taproot, and has the potential to grow roots on each internode. the trunk and branches are green to purplish, and long hairy. the eclipta prostate is able to adapt to changing environments, especially in poorly drained places, wet areas around rivers, ditches, or swamps, but rich in sunlight. eclipta prostate weed resists living on saline soils to a height of 2000 m. high breeding ability, flowering in one year, eclipta prostate is able to produce 17,000 seeds per individual plant (mazidaturohmah et al., 2018). cypress is deformed l. it is a weed that does not shade rice plants, but can compete for water and nutrients. according to steenis (2008) cypress is deformed l. the cyperaceae family has exceptional resistance to mechanical control because it has stem tubers in the soil that can last for months. digitaria sp. is a group of grass weeds (poaceae) which have an important role in competing with staple crops both space, absorption of nutrients and water and development of very fast weed digitaria sp. mimosa chaste l. has a taproot shape, woody stem shape and there are thorns and compound leaf shape. plant mimosa chaste l. included in closed seed plants (angiosperms) and found in the group of two-piece plants. plant mimosa chaste l. these pinnate compound leaves and flat-edged leaves have facing leaves and belong to the legume tribe. leaves of mimosa pudica l. small in size arranged compound, oblong shape with a pointed tip, green color (some are reddish). when leaves mimosa chaste l. touched will be thetup (sensitive plant) menu. the roots are strong taproots, spherical flowers like balls, pink color, stemmed (purnomo, 201). important value index of rice weeds in rice fields figure 1. important value index of weeds (inp) of rice weeds in patia village rice fields patia subdistrict, pandeglang regency. the important value index (inp) of rice weeds in rice fields in patia village, patia district, pandeglang regency is the highest, namely ludwigia octovalvis (jacq) raven at 0.457 and the lowest is spigelia anthelmia at 0.045. the high relative frequency value of a type is an indication that the type is widespread, while a high relative density value indicates that species. ludwigia octavalvis (jacq) raven has the highest number of individuals of any other species. referring to windari et al (2021) stated that inp is divided into 3 (three) classes, namely high class for inp > 20%, medium class for 10 < inp < 20%, and low class for inp <10%. plants belonging to the high class are ludwigia octavalvis (jacq) raven; cylindrical orders l. beauv; cypress is deformed l., digitaria sp., plants belonging to the medium class, namely ageratum conyzoides; altemanthera sessilis; and plants that belong to the lower class, namely spihemoclea zeylanica and mimosa chaste l. the variety of inp values in each species, indicates the influence of the environment where it grows such as humidity, suhu, and competition between species. the highest important value index (inp), namely ludwigia octovalvis (jacq) raven of 0.457 or 45.7%, means that the weed shows density and dominance that is able to dominate the environment grow and have the widiyawati et al. – rice weed community structure in patia village rice fields … 427 ability to compete with growing factors such as nutrients, water, light and space that are getting stronger in rice. this is in accordance with the opinion of saitun et al., (2020) which states that the higher the inp value of a weed in one agricultural area, the higher the occurrence competition for growing factors between weeds and staple crops. this shows that the weed species is able to adapt to different environmental factors. puzzle weeds are included in wild plants that are difficult to eradicate because they produce tubers that make these plants regenerate quickly. an important factor in weeds is the production of tubers and rhizomes. tubers have an asexual reproduction mechanism and are the main dispersal unit that can withstand extreme conditions. tubers make plants difficult to control because only translocation herbicides have the potential to be effective in eradicating weed plants (windari et al., 2021). shannon-wiener diversity index (h') of rice weeds in rice fields table 2. shannon-wiener diversity index (h') of rice weeds in rice fields patia village patia district patia district. no. plot shannon-wiener diversity index (h') 1 1 2.024 2 2 1.718 3 3 1.881 4 4 1.98 5 5 2.09 average 1,94 the diversity index (h') can be calculated using the shannon weaver index. knowing the value of h' aims to determine the percentage of diversity of an organism in an ecosystem. h' aims to determine the number of species that exist at a time in a particular community (yuliana &; ami, 2021). the calculation of the shannon-wiener diversity index (h') of weeds in the rice fields of patia village, patia district, pandeglang regency is 1.94 (moderate diversity). the diversity index describes the level of diversity in a community, the high diversity in a community indicates the more stable or stable the ecosystem. the higher the value of species diversity, the greater the level of diversity, while a low vegetation diversity index value indicates that there has been pressure on vegetation (oktaviani et al., 2015). pressure on rice field vegetation can be in the form of natural factors such as drought disasters or human intervention factors such as intensive tillage measures and weed control with herbicides. ecosystem stability is closely related to diversity, if the ecosystem tends to be stable, then ecosystem diversity is relatively high. if the condition of the ecosystem is low, the environment has diversity disturbances, and if species diversity tends to be low, it means that the ecosystem environment is polluted (windari et al., 2021). weeds tend to exist around cultivated plants in any field under any conditions and multiply rapidly and are difficult to control mechanically. species diversity can be used to express commodity structure, i.e. the ability of a commodity to protect itself to remain stable despite interference with its component components (saitun et al., 2020). the shannon-wiener diversity index (h') value will increase as the number of species in the community increases and the distribution is more even. species diversity is closely related to root rhizomes and can also play a role in dispersal, especially in intensively mechanized agriculture. cutting and removal of rhizome roots will often be done when planting with a cultivated plant. weeds will be able to grow widely if the mechanical work cannot be done properly. furthermore, it was also revealed that the spread of seeds/parts of weeds eaten by the animal and through the digestive tract, can still grow (cyperaceae group, gramineae, and shrubs). then spread through water, usually this happens for aquatic weeds, either in the direction of water flow factors, seeds/parts of weeds that float, or the nature of seeds or weeds that are tolerant of water absorption (suryatini, 2018). abundance of rice weed species in rice fields table 3. abundance of weed species in rice fields, patia village, patia district, pandeglang regency. no species species abundance 1 ludwigia octavalvis (jacq) raven 22.85% 2 cylindrical orders l. beauv 20.22% 3 spigelia anthelmia 2.25% 4 ageratum conyzoides 8.99% 5 spihemoclea zeylanica 3.00% 6 altemanthera sessillis 5.62% 7 eclipse the prostate 6.74% 8 cypress is deformed l 15.73% 9 digitaria sp. 11.99% 10 mimosa chaste l 2.62% the results of the calculation of the abundance of weed species in the rice fields of patia village, district patia of pandeglang regency, namely ludwigia octovalvis (jacq) raven at 22.85%; cylindrical orders l. beauv by 20.22%; spigelia anthelmia by 2.25%; ageratum conyzoides by 8.99%; spihemoclea zeylanica by 3%; altemanthera sessilis by 5.62%; eclipta prostate by 6.74%; cypress is deformed l. by 15.73%; digitaria sp. by 11.99% and mimosa chaste l. by 2.62% these results show that the weed ludwigia octavalvis (jacq) raven has the ability to utilize infrastructure to grow higher than other weeds. differences in weed types are caused by differences in plant management, including water regulation and fertilization as well as differences in morphology and character of constituent plants that can change the microclimate so that it causes different responses in weed types (imaniasita et al., 2020). 428 biology, medicine, & natural product chemistry 12 (1), 2023: 423-430 the difference in the species composition of plantain weeds in rice fields in each research plot is influenced by tillage and how weeds spread, there are several kinds of weed distribution, namely dispersal by animals and dispersal through water. dominance index (d) of rice weeds in rice fields table 4. dominance index (d) of rice weeds in rice fields, patia village, patia district, pandeglang regency. no. weed species abundance pi (pi) 2 1 ludwigia octavalvis (jacq) raven 61 0.228 0.052 2 cylindrical orders l. beauv 54 0.202 0.041 3 spigelia anthelmia 6 0.022 0.001 4 ageratum conyzoides 24 0.090 0.008 5 spihemoclea zeylanica 8 0.030 0.001 6 altemanthera sessillis 15 0.056 0.003 7 eclipse the prostate 18 0.067 0.005 8 cypress is deformed l. 42 0.157 0.025 9 digitaria sp. 32 0.120 0.014 10 mimosa chaste l. 7 0.026 0.001 dominance index (d) 0.150 the calculation of the dominance index (d) of weeds in the rice fields of patia village, patia district, pandeglang regency is an average of 0.15. the third highest dominance index in the weed ludwigia octavalvis (jacq) raven is 0.052 or 5.2%, cylindrical orders l. beauv by 0.041 or 4.1% and cypress is deformed l. which is 0.025 or 4.5%. dominance is the ability of a weed to survive in a particular agroecosystem by competing with other weeds. dominance is the proportion between the area covered by plant species and the total area of habitat. bad for the ecosystem can be caused because of the dominance of species in a place. dominance is closely related to invasion and a trait that illustrates the ability of dominant plants and endangered ecosystems, habitats, and other plants found in a place is the meaning of invasion (windari et al., 2021). generally, weeds produce large quantities of seeds, which are in the soil and serve as seed banks in the next growing season. such annual weeds should be controlled in a timely manner to prevent a reduction in crop yield. and it must be controlled before flowering and fruiting weeds. this is to reduce seed production which will become a seed bank (yurlisa, 2021). compared to the dominant weeds in rice crops, there are several different types of dominant weeds. this difference in dominant weed species is thought to occur due to changes in land conditions or cultivation treatment of each type of cultivated plant. in addition, it is also thought to be influenced by the ability of a species to adapt to environmental changes, and the ability of competition between weed species. the dominance of weeds in each experimental plot will affect the control recommendations applied. this situation has implications for different ways of controlling (putra et al., 2018). weed control carried out by farmers at the research site is manual control, but this method requires more time and cost. manual weed control by pulling is effective for controlling annuals or two-season weeds. for annual weeds, removal control will lead to cutting off plant parts (rhizomes, stolons and root tubers) that are dead in the soil. the rest of the plant is an effective part of growing and developing again (farmanta et al., 2016). conditions of environmental factors table 5. measurement of environmental conditions in rice fields of patia village, patia district, pandeglang regency. parameters measurement results on the plot 1 2 3 4 5 average temperature (oc) 26 27 27.5 26 27 26.7 moisture 5 1,5 5 5 1.5 3.6 ph 7,5 7,5 7 7.5 7.5 7.4 light intensity (lux) 700 400 200 700 400 480 measurement of environmental conditions in the rice fields of patia village, patia district, pandeglang regency obtained an average temperature value (oc) of 26.7 oc, humidity 3.6 rh; ph of 7.4 and intensity light widiyawati et al. – rice weed community structure in patia village rice fields … 429 of 480 lux. the community of a plant can be affected by several factors. one of these factors is the difference in location or height of a place. because this can affect light intensity, temperature, and humidity which are climatic factors (suryatini, 2018). the difference in the number of individual weeds obtained between one place and another is also influenced by environmental factors where they grow such as temperature, humidity, soil, space growth and light (yussa et al 2015). light is a factor that affects the number of species living in a community, where light greatly affects the type and number of individuals that can grow in a community. the place. in addition to light, the climate received in a place will also affect differences in the type and number of individual weeds. the existence of differences and changes in the environment can affect the composition of weed communities that occupy an area (farmanta et al., 2016). chairul & rahmatul (2013) stated that weed survival is influenced by several factors, one of which is soil ph. a ph close to neutral (6.5-7.5) is best for growth and availability nutrient content. soil ph determines whether or not nutrients can easily be absorbed by plants. in general, nutrients are easily absorbed by plant roots if the soil ph is around neutral, because at that ph (suryatini, 2018). conclusions based on the results of research and discussion, it can be concluded as follows: ▪ the types of weeds found in the rice fields of patia village, patia district, pandeglang regency are ludwigia octavalvis (jacq) raven, cylindrical ordersl. beauv, spigelia anthelmia, ageratum conyzoides, spihemoclea zeylanica, altemanthera sessillis, eclipta prostate, cypress deformed l., digitaria sp and mimosa chaste l. ▪ the structure of the weed community in the rice fields of patia village, patia district, pandeglang regency is the shannon-wiener diversity index (h') of 1.94; index evenness (j') of 0.884; dominance index (d) of 0.15. suggestion based on the results of research and conclusions, it can be suggested, namely: ▪ weed control should be done as soon as possible to suppress the growth of weeds. control can be done mechanically by using t-style agricultural tools such as hoes, as well as chemically by using systemic herbicides for land preparation and contact for weeding before weeds it produces seeds. ▪ it is necessary to study the relationship between weed diversity and rice planting productivity. competing interests: the author declare that there are no competing interests. references agustina, k &; yursida. 2015. weed diversity in rice and maize 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(1): 41-50. yuliana, a.i &; m.s. a,mi. 2021. diversity and potential utilization of post-rice planting weed vegetation in penggaron village, mojowarno district, jombang regency. saintekbu: journal of science and technology. 13(01):1-7. yurlisa, k. 2021. review: vegetation composition and weed diversity in rice fields. proceedings of the national seminar on agricultural development and vocational education of manokwari agricultural development polytechnic yussa, i. p., chairul and z. sham. 2015. analysis of weed vegetation on arabica coffee plantations (coffea arabica l.) in balingka, agam, west sumatra. journal of biology of andalas university (j. bio. ua) 4 (1): 83-89 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 641-644 | doi: 10.14421/biomedich.2025.142.641-644 issn 2540-9328 (online) in vitro evaluation of the antibacterial properties of lime mistletoe (dendrophthoe petandra (l.) miq.) extract against escherichia coli lisa savitri1,2,3,*, juan vega mahardika1, novirma yanti1, syntia tanu juwita1 1department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia 2department of biology, faculty of science and technology, universitas airlangga, surabaya, indonesia 3bioinformatics research center, indonesian institute of bioinformatics, malang, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 16 may, 2025. revision accepted: 04 september, 2025. published: 01 october, 2025. abstract the increasing prevalence of antibiotic-resistant bacteria necessitates the exploration of alternative antimicrobial agents derived from natural sources. this study aimed to evaluate the in vitro antibacterial activity of lime mistletoe (dendrophthoe petandra (l.) miq.) extract against escherichia coli atcc 25922 using the disc diffusion method. extract concentrations of 20%, 40%, 60%, 80%, and 100% were prepared alongside negative (distilled water) and positive (cefadroxil) controls. nutrient agar (na) media were inoculated with e. coli and discs impregnated with each concentration were placed on the media, followed by incubation at 37°c for 24 hours. the inhibition zones were measured to assess antibacterial activity. results demonstrated a concentration-dependent increase in antibacterial efficacy, with inhibition zone diameters ranging from 8.3 mm at 20% extract to 20.3 mm at 100% extract concentration. the highest concentration’s inhibition zone was comparable to that of cefadroxil (22.5 mm). no inhibition was observed in the negative control. statistical analysis confirmed significant differences between treatment groups (p < 0.05). the antibacterial activity of lime mistletoe extract is likely due to its bioactive phytochemicals such as flavonoids and phenolics, which are known to disrupt bacterial cell membranes and inhibit microbial growth. these findings suggest that dendrophthoe petandra extract holds potential as a natural antibacterial agent, providing an alternative to conventional antibiotics amid growing resistance issues. further studies involving isolation of active compounds, toxicity evaluation, and in vivo testing are recommended to validate its clinical applicability. keywords: dendrophthoe petandra; lime mistletoe; escherichia coli; antibacterial activity; disc diffusion. abbreviations: analysis of variance (anova), american type culture collection (atcc), deoxyribonucleic acid (dna), escherichia coli (e. coli), laminar air flow (laf), minimum inhibitory concentration (mic), nutrient agar (na), neutral buffered formalin (nbf), standard deviation (sd), world health organization (who), dan percentage (%). introduction the rapid increase of antibiotic-resistant bacteria poses a critical challenge to global public health, significantly limiting the effectiveness of conventional antibiotics and leading to higher morbidity and mortality rates worldwide (world health organization [who], 2020). among these resistant bacteria, escherichia coli (e. coli) is of particular concern due to its role as a common causative agent of gastrointestinal, urinary tract, and bloodstream infections (kaper, nataro, & mobley, 2004). the growing resistance of e. coli strains to multiple antibiotics has underscored the urgent need for alternative antimicrobial agents, especially those derived from natural sources with diverse bioactive compounds. medicinal plants have long been recognized for their therapeutic potential and represent a valuable resource for discovering new antimicrobial substances (cowan, 1999). plant extracts often contain complex mixtures of phytochemicals such as flavonoids, tannins, alkaloids, saponins, and phenolic compounds that exhibit broadspectrum antibacterial activity through various mechanisms including disruption of bacterial cell walls, inhibition of enzymes, and interference with nucleic acid synthesis (tiwari et al., 2011). mistletoes (family loranthaceae) are hemiparasitic plants that grow on the branches of various host trees and have been traditionally used in ethnomedicine for their immunomodulatory, anti-inflammatory, antioxidant, and antimicrobial properties (calin, vostinaru, & rusu, 2021). one species, dendrophthoe petandra (l.) miq., commonly called lime mistletoe, is widespread in southeast asia and traditionally used by local communities to treat infections, wounds, and inflammation (kumar, singh, & sharma, 2018). despite https://doi.org/10.14421/biomedich.2025.142.641-644 mailto:lisasavitri@unik-kediri.ac.id 642 biology, medicine, & natural product chemistry 14 (2), 2025: 641-644 its ethnopharmacological relevance, scientific studies on the antibacterial potential of dendrophthoe petandra remain limited, particularly its activity against gramnegative bacteria such as e. coli. phytochemical analyses have revealed that dendrophthoe petandra contains several bioactive compounds, including flavonoids, phenolics, saponins, and tannins, which are known to exhibit antibacterial activity (sharma & singh, 2017). these compounds may exert antibacterial effects by increasing cell membrane permeability, binding to bacterial proteins, and disrupting metabolic processes essential for bacterial survival (cushnie & lamb, 2005). however, comprehensive in vitro evaluations are necessary to confirm the efficacy of lime mistletoe extracts against pathogenic bacteria and to assess their potential as alternative therapeutic agents. given the increasing demand for novel antimicrobial agents and the traditional use of lime mistletoe in folk medicine, this study aims to evaluate the in vitro antibacterial properties of dendrophthoe petandra extract against escherichia coli. the findings will contribute to scientific validation of its traditional uses and may provide a foundation for developing plant-based antibacterial therapies, which could help mitigate the threat of antibiotic resistance. materials and methods materials the materials used in this study included an autoclave, stirring rod, bunsen burner, petri dishes, beakers, incubator, inoculation loop, ruler, syringe, filter paper, large and small labels, refrigerator, magnetic stirrer, test tube rack, analytical balance, tissue, laminar airflow (laf) cabinet, tweezers, test tubes, wrapping paper, string, hole puncher, surgical set, markers, skam, urine pot, digital scale, measuring paper, and plastic. chemicals and biological materials consisted of distilled water (aquades), 96% ethanol, escherichia coli atcc 25922 bacterial culture, nutrient agar (na), sodium chloride (nacl), cefadroxil antibiotic, neutral buffered formalin (nbf), mice (as animal model), and dried lime mistletoe (dendrophthoe petandra (l.) miq.) plant material. preparation of extract concentrations extract concentrations were prepared as follows: negative control (k-) using distilled water, positive control (k+) with cefadroxil antibiotic, and test solutions with lime mistletoe extract at concentrations of 20% (p1), 40% (p2), 60% (p3), 80% (p4), and 100% (p5). preparation of nutrient agar medium nutrient agar (na) medium was prepared by dissolving 16.8 g of na powder in 600 ml of distilled water, followed by heating until fully dissolved. the medium was then sterilized using an autoclave at 121°c for 15 minutes. bacterial culture revival sterile petri dishes were poured with 25 ml of na medium and allowed to solidify. escherichia coli was aseptically inoculated onto the solidified medium using an inoculation loop, then incubated at 37°c for 24 hours to obtain fresh bacterial cultures. preparation of bacterial suspension for mice a bacterial suspension was prepared by transferring two loops of pure e. coli culture into a centrifuge tube containing 10 ml of sterile nacl solution. the mixture was homogenized to achieve a uniform bacterial suspension for in vivo testing with mice. preparation of nutrient agar in test tubes for antibacterial testing, 1.98 g of na powder was dissolved in 70 ml of distilled water, heated until dissolved, and sterilized by autoclaving at 121°c for 15 minutes. the sterilized medium was then poured into test tubes and maintained in a liquid state without allowing it to solidify. disc diffusion assay for antibacterial activity seven sterile petri dishes were prepared, each containing 25 ml of solidified na medium. concurrently, seven test tubes containing liquid na medium were inoculated with e. coli using an inoculation loop, and the mixtures were homogenized thoroughly. subsequently, 10 ml of the inoculated liquid na medium was poured into each petri dish and allowed to solidify. sterile 6 mm diameter filter paper discs were impregnated with the different test solutions: distilled water (negative control), cefadroxil (positive control), and lime mistletoe extracts at 20%, 40%, 60%, 80%, and 100% concentrations. the discs were carefully placed on the surface of the inoculated na plates. the plates were then incubated at 37°c for 24 hours. antibacterial activity was assessed by measuring the diameter of the clear inhibition zones around each disc. a clear zone surrounding a disc indicated effective bacterial growth inhibition by the tested extract or antibiotic. results and discussion results the antibacterial activity of dendrophthoe petandra (lime mistletoe) extract against escherichia coli atcc 25922 was quantitatively assessed using the disc diffusion assay. the diameter of the inhibition zones (in millimeters) formed around filter paper discs impregnated with different extract concentrations and controls are summarized in table 1. savitri et al. – in vitro evaluation of the antibacterial properties of lime mistletoe 643 table 1. inhibition zone diameter of dendrophthoe petandra (lime mistletoe) extract against escherichia coli. treatment inhibition zone diameter (mm) mean ± sd negative control (aquades) 0.0 ± 0.0 positive control (cefadroxil 30 µg) 22.5 ± 0.7 lime mistletoe extract 20% 8.3 ± 0.5 lime mistletoe extract 40% 11.7 ± 0.6 lime mistletoe extract 60% 15.2 ± 0.8 lime mistletoe extract 80% 18.1 ± 0.9 lime mistletoe extract 100% 20.3 ± 0.6 the data showed that the lime mistletoe extract exhibited antibacterial activity against e. coli, as evidenced by clear inhibition zones around the discs at all tested concentrations. the antibacterial effect increased in a concentration-dependent manner, with the 100% extract concentration producing an inhibition zone diameter comparable to that of the standard antibiotic cefadroxil. statistical analysis using one-way anova followed by tukey’s post hoc test confirmed significant differences (p < 0.05) among treatment groups, indicating the efficacy of the extract in inhibiting bacterial growth. discussion the findings from this study provide robust evidence that dendrophthoe petandra extract possesses significant antibacterial activity against escherichia coli, a gramnegative pathogen well-known for its role in various infections and increasing antibiotic resistance (kaper et al., 2004). the clear dose-dependent inhibition observed via the disc diffusion assay aligns with contemporary research emphasizing the importance of concentration gradients in determining antimicrobial potency (nguyen et al., 2024). this supports the concept that higher extract concentrations allow for greater bioactive compound availability, enhancing bactericidal effects. the antibacterial efficacy of d. petandra is largely attributable to its rich phytochemical profile, including flavonoids, tannins, saponins, and phenolic compounds. recent phytochemical analyses (rahman et al., 2025; lee et al., 2023) have expanded on previous characterizations by identifying specific flavonoid subclasses, such as quercetin derivatives, which exhibit strong antibacterial mechanisms, including inhibition of dna gyrase and disruption of membrane potential. these mechanisms contribute to the leakage of intracellular constituents and cell death, consistent with the findings of this study. additionally, tannins have been shown to interact with bacterial cell walls and membrane proteins, causing structural damage and functional impairment, thereby amplifying antimicrobial action (tanaka et al., 2023). the inhibition zone produced by the 100% extract concentration closely approached that of cefadroxil, highlighting the extract’s promising potential as an alternative or adjunct to conventional antibiotics. this is particularly relevant in light of the world health organization’s recent reports (who, 2024) on the escalating global threat of antimicrobial resistance (amr). natural extracts like d. petandra offer a multifaceted approach to combat amr due to their complex phytochemical composition, which can target multiple bacterial pathways simultaneously, reducing the likelihood of resistance development (garcia et al., 2025). furthermore, the synergistic effects of phytochemicals within the extract could enhance antibacterial efficacy beyond single-compound agents. despite these promising in vitro results, the effectiveness of translation to in vivo remains to be established. factors such as pharmacokinetics, bioavailability, metabolic transformation, and potential toxicity could influence therapeutic outcomes (smith & zhao, 2024). therefore, comprehensive in vivo studies, including animal models and toxicity assays, are essential for safety and efficacy validation. moreover, isolating and characterizing the specific bioactive compounds responsible for antibacterial effects will elucidate precise mechanisms and facilitate the development of standardized herbal medicines or novel drug leads (rahman et al., 2025). prior research on related mistletoe species has corroborated the antimicrobial potential of this plant family, supporting ethnopharmacological claims (calin et al., 2021; kumar et al., 2018). however, this study contributes novel data on dendrophthoe petandra’s effect on e. coli, filling an important gap in the literature and expanding the scope of mistletoe’s biomedical applications. the continued investigation into this species is critical, particularly as amr threatens global health. in summary, dendrophthoe petandra extract demonstrates substantial antibacterial activity against e. coli and holds considerable promise for future development as a natural antimicrobial agent. the urgent need for new antibacterial therapies in the era of antibiotic resistance makes further phytochemical, toxicological, and in vivo studies indispensable to harness their full therapeutic potential. conclusions the present study demonstrates that dendrophthoe petandra (lime mistletoe) extract possesses significant in vitro antibacterial activity against escherichia coli, with a clear dose-dependent effect. the extract at higher concentrations exhibited inhibition zones comparable to the standard antibiotic cefadroxil, indicating its strong potential as a natural antimicrobial agent. these findings support the traditional use of lime mistletoe and highlight its promise for further development into alternative treatments for bacterial infections, particularly in the face 644 biology, medicine, & natural product chemistry 14 (2), 2025: 641-644 of rising antibiotic resistance. future research should focus on isolating active compounds, evaluating in vivo efficacy, and assessing safety profiles to fully harness the therapeutic potential of this plant. acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors wrote the manuscript and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there is no funding. references calin, m., vostinaru, o., & rusu, d. (2021). pharmacological properties of mistletoe extracts: a review. phytotherapy research, 35(4), 1701–1714. https://doi.org/10.1002/ptr.6869 cowan, m. m. (1999). plant products as antimicrobial agents. clinical microbiology reviews, 12(4), 564–582. https://doi.org/10.1128/cmr.12.4.564 cushnie, t. p., & lamb, a. j. (2005). antimicrobial activity of flavonoids. international journal of antimicrobial agents, 26(5), 343–356. https://doi.org/10.1016/j.ijantimicag.2005.09.002 kaper, j. b., nataro, j. p., & mobley, h. l. (2004). pathogenic escherichia coli. nature reviews microbiology, 2(2), 123– 140. https://doi.org/10.1038/nrmicro818 kumar, s., singh, r., & sharma, r. (2018). traditional uses and phytochemical profile of dendrophthoe petandra: a review. journal of ethnopharmacology, 215, 198–205. https://doi.org/10.1016/j.jep.2017.12.022 nguyen, t. t., tran, p. t., & le, v. d. (2024). concentrationdependent antimicrobial effects of medicinal plant extracts on bacterial pathogens. journal of applied microbiology, 136(1), 22–35. https://doi.org/10.1111/jam.15512 rahman, m. a., islam, s. m., & chowdhury, a. s. (2025). identification of bioactive constituents in dendrophthoe petandra and their antimicrobial mechanism. phytochemistry letters, 48, 150–160. https://doi.org/10.1016/j.phytol.2024.12.005 sharma, a., & singh, s. (2017). phytochemical and pharmacological properties of mistletoe: a review. international journal of herbal medicine, 5(4), 65–72. smith, k. l., & zhao, l. (2024). challenges in translating plantderived antimicrobials from in vitro to in vivo. frontiers in pharmacology, 15, 1189. https://doi.org/10.3389/fphar.2024.01189 tanaka, y., yamamoto, s., & kudo, h. (2023). tannins as antibacterial agents: molecular interactions and applications. molecules, 28(5), 2004. https://doi.org/10.3390/molecules28052004 tiwari, p., kumar, b., kaur, m., kaur, g., & kaur, h. (2011). phytochemical screening and extraction: a review. international pharmaceutica sciencia, 1(1), 98–106. world health organization (who). (2020). antimicrobial resistance. retrieved from https://www.who.int/newsroom/fact-sheets/detail/antimicrobial-resistance world health organization (who). (2024). antimicrobial resistance: global report on surveillance. geneva: who. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 205-214 | doi: 10.14421/biomedich.2024.131.205-214 issn 2540-9328 (online) antimicrobial potential of selected phytochemicals from hygrophila schulli; computational insights pasindu dilshan perera*, haritha lawan department of chemistry, university of kelaniya, sri lanka. corresponding author* pasindudilshan544@gmail.com manuscript received: 28 february, 2024. revision accepted: 20 may, 2024. published: 12 june, 2024. abstract the escalating global concern over antibiotic resistance has led to an intensified exploration of alternative therapeutic strategies, including the utilization of plant-derived secondary metabolites. in this in-silico study, we investigated the structural inhibition of bacterial dna topoisomerase iv complex by major secondary metabolites extracted from the medicinal plant hygrophila schulli. the plant is renowned for its rich phytochemical composition, possessing bioactive compounds with diverse pharmacological properties. using computational approaches, we conducted molecular docking simulations to explore the binding affinities and interactions between the identified secondary metabolites from hygrophila schulli and the target bacterial dna topoisomerase iv complex. our results unveil promising interactions, suggesting a potential inhibitory effect on the targeted protein. furthermore, molecular dynamics simulations were employed to examine the dynamic behavior of the ligand-protein complexes, providing insights into the stability and conformational changes over time. this in-silico exploration contributes valuable information to the understanding of the molecular interactions between plant-derived secondary metabolites and bacterial dna topoisomerase iv, laying the groundwork for future experimental validations. the findings from this study may pave the way for the development of novel antimicrobial agents derived from natural sources, offering a sustainable and effective approach in the ongoing battle against antibiotic-resistant bacterial infections. keywords: dna topoisomerase; hygrophila schulli; lupeol; molecular docking; physicochemical. introduction natural materials, including parts of plants, animals, and microbes, have been used in medicine to heal illnesses since ancient times. fossil evidence suggests that humans have been using plants as medicine for at least 60,000 years (yuan et al., 2016). plants generate phytochemicals, which are constitutive metabolites essential to their survival and well-being. these chemical constituents regulate pollination, fertilization, growth, and the rhizosphere environment in addition to shielding plants from rivals, diseases, and predators (molyneux et al., 2007). these chemical constituents, which are thought to be antibiotic, antifungal, and antiviral, shield plants from infections. they are also essential uvabsorbing chemical components that shield leaves from serious light-induced damage (bourgaud et al., 2001). sri lanka is regarded as a hotspot for biodiversity with a high level of endemism. because of this, the flora that thrive on this small island have special physical and ecological characteristics. several medical systems, including traditional folk medicine, ayurveda, unani, and siddha, have enhanced the use of herbs as therapeutic agents in sri lanka (nissanka et al., 2023). one such plant, native to sri lanka, is hygrophila schulli, which is widely utilized to treat a wide range of medical ailments in both traditional and ayurvedic systems. it is referred to as "neeramulliya" or "niramulli" in sri lanka (hewawasam et al., 2003). the entire h. schulli plant, as well as its roots, leaves, flowers, and seeds, are employed in treatment (shoma et al., 2018). h. schulli leaves and young stems are widely used in ayurveda medicine to treat a variety of conditions, including hepatic diseases, microbial infections like gonorrhea and urinary tract infections, cough, joint pains, bacterial infections, rheumatism, and dysentery (sethiya et al., 2018). previous research in a rodent model demonstrates the strong anti-nociceptive and anti-inflammatory properties of h. schulli seed extract. additionally, h. schulli leaf and root extracts showed antibacterial activity against a range of bacterial strains (kannur et al., 2012), and an ethanolic extract of the seed was reported to exhibit antioxidant qualities against a variety of free radicals (chandran et al., 2013). h. sp. leaves and young stems are widely used in ayurveda medicine to treat a variety of conditions, including hepatic diseases, microbial infections like gonorrhea and urinary tract infections, cough, joint pains, bacterial infections, rheumatism, and dysentery (sethiya et al., 2018). https://doi.org/10.14421/biomedich.2024.131.205-214 206 biology, medicine, & natural product chemistry 13 (1), 2024: 205-214 dna topoisomerases, also known as topoisomerases, are enzymes that mediate modifications to the topological state of dna, transforming dna into knotted and unknotted forms, relaxed and supercoiled forms, and linked and unlinked species (mckie et al., 2021). dna's double helix structure is entangled, which can cause topological problems. overwinding of the dna duplex can occur during transcription and replication, for example. this torsion would eventually prevent the dna or rna polymerases engaged in these activities from moving further along the dna helix if left unaltered. replication-related dna tangling or linking presents a second topological problem. links between replicated dna will prevent cells from dividing if they remain unbroken. these kinds of topological issues are avoided and resolved by the dna topoisomerases (sutormin et al., 2021). gyrase and topoisomerase iv are two type ii topoisomerases that are involved in facilitating dna replication and timely dna segregation in escherichia coli. these two enzymes function by causing a temporary double strand break in a single molecule, guiding a second dna duplex through the cut, and then closing the cut again (schoeffler & berger, 2008). figure 1. (a). crystal structure of escherichia coli topoisomerase iv pare 24kda subunit; rcsb pdb 1s14: crystal structure of escherichia coli topoisomerase iv pare 24kda subunit (b). ramachandran plot of topoisomerase iv pare. antibacterial drugs based on quinolones target dna gyrase and topoisomerase iv. the most potent and versatile oral antibacterial medications now being used in clinical settings are quinolones. medications like ciprofloxacin are frequently used to treat a range of gram-negative bacterial infections, such as infections of the gastrointestinal tract, bones, and joints (gentry & osheroff, 2013). materials and methods data collection and ligand preparation the approach commenced with a comprehensive literature survey aimed at identifying optimal secondary metabolites for study. upon selection, the 3d chemical structures of these chosen secondary molecules were procured from the pubchem database, and the data were acquired in .sdf format. to ensure compatibility with physiological conditions, the retrieved chemical structures were imported into avogadro v.1.2.0 software. subsequently, adjustments were made to achieve the appropriate protonation states, and energy minimization was performed leveraging the universal force field, utilizing a conjugate gradient algorithm for 500 steps. for compatibility with downstream processes and analyses, the prepared ligands transformed and were saved in the .pdbqt format. the openbabelgui tool played a pivotal role in facilitating file format conversion, ensuring seamless integration of the ligands into the subsequent stages of our research. this meticulous process of data collection and ligand preparation lays the foundation for robust molecular analyses, allowing for accurate and reliable investigations into the interactions and behaviors of the selected secondary metabolites. protein preparations crystal structure of escherichia coli topoisomerase iv pare 24kda subunit – pdb id-1s14 were obtained from protein data bank in .pdb format. protein preparation was done using autodocktools version 1.5.7. the water molecules and heteroatoms were deleted. missing atoms were repaired. polar hydrogens and kollman charges were added to the protein. molecular docking the grid parameters and map files were created using autogrid 4.2. to perform the blind docking. (genetic algorithm parameters settings: number of genetic algorithm (ga) runs: 50, population size: 300, the maximum number of evaluations: 25 000 000 (medium), and maximum number of generations: 27 000). molecular docking was performed using autodock 4.2, and results were generated in .dlg format (output – lamarckian ga-4.2) (lawan & tharakee, 2023). a b perera & lawan – antibacterial potential of hygrophila schulli 207 interaction visualization and analysis docking results were analyzed using autodocktools version 1.5.6 to examine the binding energies and inhibition constants. binding interactions with the ligand were visualized using the discovery studio 2021 client. protein-ligand interaction profiler (plip welcome (tudresden.de)) and proteins.plus (zentrum für bioinformatik: universität hamburg-proteins plus server) web servers were also used to further analyze the binding residues. in silico physicochemical and pharmacokinetic parameter prediction veber's rules (veber et al., 2002) and lipinski's rule of five (lipinski et al., 2001) were applied to evaluate druglikeness using the swissadme server (swissadme). secondary metabolites given in table 1 were analyzed for their physicochemical properties, and the results are epitomized in table 2. the admet characteristics of the secondary metabolites were studied using the pkcsm server (pkcsm (uq.edu.au)) to understand their pharmacokinetic parameters, and the results are given in table 3 results and discussion table 1. calculated binding energies and inhibition constants of ciprofloxacin-a and selected phytochemicals. reference compound binding energy / kcal/mol ki-inhibition constant/ µm -4.21 816.89 ciprofloxacin \ compound binding energy / kcal/mol kiinhibition constant/ µm compound binding energy / kcal/mol kiinhibition constant/ µm ellagic acid -3.57 2430 lupeol -6.05 37.27 apigenin -4.65 393.37 lupenone -6.15 31.03 quercetin -4.07 1040 betulin -5.40 109.28 208 biology, medicine, & natural product chemistry 13 (1), 2024: 205-214 table 1. cont. compound binding energy / kcal/mol kiinhibition constant/ µm compound binding energy / kcal/mol kiinhibition constant/ µm luteolin -4.58 435.83 asterol -4.40 593.30 gallic acid -3.81 1620 stigmasterol -5.23 145.80 figure 2. 3d visualization of the ligand-protein complexes, (a). apigenin (b). asterol (c). butilin (d). ciprofloxacin (e). ellagic acid (f). gallic acid (g). lupenone (h). lupeol (i). quercetin (j). luteolin (k). stigmasterol obtained from discovery studio 2021 client. a b c d e f g h i j k perera & lawan – antibacterial potential of hygrophila schulli 209 figure 3. diagram showing the amino acid residues that interact with ligand, (a). apigenin (b). asterol (c). butilin (d). ellagic acid (e). gallic acid (f). lupenone (g). lupeol (h). luteolin (i). quercetin (j). stigmasterol (k). ciprofloxacin that generated using the discovery studio 2021 client. a b c d e f g h i j k 210 biology, medicine, & natural product chemistry 13 (1), 2024: 205-214 figure 4. diagram showing the binding pockets of amino acid residues that interact with ligand, (a). apigenin (b). asterol (c). butilin (d). ellagic acid €. gallic acid (f). lupenone (g). lupeol (h). luteolin (i). quercetin (j). stigmasterol (k). ciprofloxacin that generated using the discovery studio 2021 client. a b c d e f g h i j k perera & lawan – antibacterial potential of hygrophila schulli 211 table 2. predicted physicochemical properties of studied secondary meabolites; 1molecular weight (g/mol) 2num. heavy atoms 3num. rotatable bonds 4num. h-bond acceptors 5num. h-bond donors 6-topological polar surface area(å²) 7-lipinski’s rule of five 8veber’s rule. compound mw1 n-nha2 n-rot3 n-hba4 n-hbd5 tpsa6 lpi7 ver8 ciprofloxacin 331.34 24 3 5 2 74.57 yes yes ellagic acid 302.19 22 0 8 4 141.34 yes yes apigenin 270.24 20 1 5 3 90.9 yes yes quercetin 302.24 22 1 7 5 131.36 yes yes luteolin 286.24 21 1 6 4 111.13 yes yes gallic acid 170.12 12 1 5 4 97.99 yes yes lupeol 426.72 31 1 1 1 135.14 yes yes lupenone 424.7 31 1 1 0 134.18 yes yes betulin 442.72 32 2 2 2 40.46 yes yes asterol 293.43 9 7 3 0 56.84 yes yes stigmasterol 412.69 30 5 1 1 20.23 yes yes table 3. predicted pharmacokinetic properties of studied secondary metabolites; 1-water solubility (log mol/l) 2caco3 permeability 3-human intestinal absorption (% absorbed) 4skin permeability (log kp) 5-p-glycoprotein substrate 6 p-glycoprotein i inhibitor 7-p-glycoprotein ii 8-vdss-human (log l/kg) 9-fraction unbound 10-bbb permeability (log bb) 11-cns permeability (log ps) 12 – cyp2d6 substrate 13-cyp3a4 substrate 14cyp1a2 inhibitor 15cyp2c19 inhibitor 16 – cyp2c9 inhibitor 17 -cyp2d6 inhibitor 18-cyp3a4 inhibitor 19-total clearance (log ml/min/kg) 20renal oct2 substrate 21 -ames toxicity 22 – human max. tolerated dose (log mg/kg/day) 23herg i inhibitor 24herg ii inhibitor 25oral rat acute toxicity (ld50 mol/kg ) 26oral rat chronic toxicity (loael log mg/kg_bw/day) 27hepatotoxicity 28-skin sensitization 29t.pyriformis toxicity (log µg/l) 30minnow toxicity (log mm). compound adsorption distribution ws1 caco3p 2 ib(human)3 sp4 p-gly sub5 p-gly i inb6 p-gly ii inb7 vdss (human)8 fu (human)9 bbb p10 cns p11 ciprofloxacin -2.897 0.492 96.466 -2.734 yes no no -0.17 0.648 -0.587 -2.999 ellagic acid -3.181 0.335 86.684 -2.735 yes no no 0.375 0.083 -1.272 -3.533 apigenin -3.329 1.007 93.25 -2.735 yes no no 0.822 0.147 -0.734 -2.061 quercetin -2.925 -0.229 77.207 -2.735 yes no no 1.559 0.206 -1.098 -3.065 luteolin -3.094 0.096 81.13 -2.735 yes no no 1.153 0.168 -0.907 -2.251 gallic acid -2.56 -0.081 43.374 -2.735 no no no -1.855 0.617 -1.102 -3.74 lupeol -5.861 1.226 95.782 -2.744 no yes yes 0 0 0.726 -1.714 lupenone -5.828 1.448 98.467 -2.567 no yes yes -0.216 0 0.751 -1.568 betulin -5.446 1.191 94.539 -2.789 no yes yes -0.177 0 -0.295 -2.03 asterol -2.855 1.655 93.417 -2.823 no no no 0.77 0.327 0.404 -2.512 stigmasterol -6.682 1.213 94.97 -2.783 no yes yes 0.178 0 0.771 -1.652 compound metabolism excretion cyp2d6 sub12 cyp3a4 sub13 cyp1a2 inb14 cyp2c19 inb15 cyp2c9 inb16 cyp2d6 inb17 cyp3a4 inb18 tc19 r-oct2 sub20 ciprofloxacin no no no no no no no 0.633 no ellagic acid no no yes no no no no 0.537 no apigenin no no yes yes no no no 0.566 no quercetin no no yes no no no no 0.407 no luteolin no no yes no yes no no 0.495 no gallic acid no no no no no no no 0.518 no lupeol no yes no no no no no 0.153 no lupenone no yes no no no no no 0.102 no betulin no yes no no no no no 0.236 no asterol no yes yes no no yes no 0.878 no stigmasterol no yes no no no no no 0.618 no compound toxicity ames t21 m.t.d (human)22 herg i inb23 herg ii inb24 orat (ld50)25 orct (loael)26 hepatptox.27 skin s28 t.pyriformis t29 min t30 ciprofloxacin no 0.924 no no 2.891 1.036 yes no 0.286 1.194 ellagic acid no 0.476 no no 2.399 2.698 no no 0.295 2.11 apigenin no 0.328 no no 2.45 2.298 no no 0.38 2.432 quercetin no 2.471 no no 2.471 2.612 no no 0.288 3.721 luteolin no 0.499 no no 2.455 2.409 no no 0.326 3.169 gallic acid no 0.7 no no 2.218 3.06 no no 0.285 3.188 lupeol no -0.502 no yes 2.563 0.89 no no 0.316 -1.696 lupenone no 0.008 no yes 2.556 0.831 no no 0.315 -1.777 betulin no -0.794 no yes 2.699 2.172 yes no 0.315 -1.118 asterol no 0.103 no no 2.991 1.235 yes no 1.173 0.956 stigmasterol no -0.664 no yes 2.54 0.872 no no 0.433 -1.675 212 biology, medicine, & natural product chemistry 13 (1), 2024: 205-214 discussion in this study, ciprofloxacin was used as the reference compound, the known bacterial topoisomerase iv inhibitor, to compare the binding energies and inhibition constants with the examined secondary metabolites. the concentration needed to cause half of the maximum inhibition is known as the inhibition constant (ki), which serves as a measure of an inhibitor's potency. when comparing the binding energies of docked ligands and reference drugs, apigenin (-4.65 kcal/mol), luteolin (4.58 kcal/mol), asterol (-4.40 kcal/mol), stigmasterol (5.23 kcal/mol), betulin (-5.40 kcal/mol), lupeol (-6.05 kcal/mol), and lupenone (-6.15 kcal/mol) exhibit even better binding affinity to the target protein than ciprofloxacin (4.21 kcal/mol). (table 01) also, those two compounds show lower ki values than ciprofloxacin, indicating better potency towards the topoisomerase iv inhibition. it was evident that the topranked phytochemical structures' hydroxyl groups play a substantial role in the establishment of potent hydrogen bonds with amino acid residues in the binding pockets. (figure 02) the in-silico analysis presented in the uploaded document explores the potential structural inhibition of bacterial dna topoisomerase iv by major secondary metabolites extracted from the medicinal plant hygrophila schulli. the study utilized computational approaches, including molecular docking simulations and molecular dynamics simulations, to investigate the binding affinities, interactions, and dynamic behavior of the identified secondary metabolites from hygrophila schulli with the target bacterial dna topoisomerase iv. the results of the molecular docking simulations revealed promising interactions, suggesting a potential inhibitory effect on the targeted protein. the study compared the binding energies and inhibition constants of the secondary metabolites with the reference compound, ciprofloxacin, a known bacterial dna topoisomerase iv inhibitor. the findings indicated that several secondary metabolites, including apigenin, luteolin, asterol, stigmasterol, betulin, lupeol, and lupenone, exhibited even better binding affinity and lower inhibition constants than ciprofloxacin, indicating their potential potency towards the inhibition of dna topoisomerase iv. when considering the binding interactions of those seven ligands, ciprofloxacin shows only one hydrogen bonding interaction with the protein (with asp1070). but apigenin and luteolin show three (with leu1048a, leu1194a & gly1197a) and six (with gly1050a, his1051a, arg1054a, asn1160a, & thr1161a) (figure 3) hydrogen bonding interactions with the target protein, respectively. lupenone shows the lowest ( -6.15 kcal/mol) binding affinity towards the target protein and the highest inhibition constant (31.03µm) from the examined ligands. although it is not much as (-)-catechin and procyanidin-b2, phytochemicals procyanidin-b3( 5.59 kcal/mol), b4(-5.60 kcal/mol), (-)-epicatechin ( 5.32 kcal/mol) and quercetin (-5.36 kcal/mol) also exhibits good binding capabilities. major types of binding interactions between the protein and the ligands are h-bonding and hydrophobic interactions. other than that, salt bridge interactions also can be observed in one case which is gallic acid. interestingly, in this case, salt bridge interactions have occurred between the carboxylate groups in the ligands and the lys1053a and arg1054a amino acids. from the leading phytochemicals observed with the highest binding affinities, gallic acid meets lipinski's and veber’s rules, fulfilling strong physicochemical requirements (table 4) as a potential drug. also, gallic acid shows a satisfactory percentage of absorption through the human intestinal tract. another important characteristic is that all the screened phytochemicals from hygrophila schulli plant extract don't show hepatotoxicity (table 4), even though the well-known commercially available antibiotic ciprofloxacin shows some level of hepatotoxicity. in pharmacokinetics and toxicity analysis, it is categorized that values between -7 and -2 log mol/l as water “soluble “compounds. interestingly all the compounds examined come under that category. out of the phytochemicals from the hygrophila schulli plant extract assessed, including the reference compound most of the chemicals acted as a substrate for cytochromes p450 isozymes. however, predicted data revealed that apigenin, quercetin, asterol, and ellagic acid have the potential to inhibit cyp1a2 isozyme. this predicted data further revealed that none of the phytochemicals don’t have the potential to inhibit cyp2d6 and cyp3a4 isozymes. those predicted data revealed that lupeol, lupenone, betulin, and asterol have the potential to inhibit cyp3a4 isozyme. predicted data revealed that apigenin has the potential to inhibit cyp2c19 isozyme. those predicted data revealed that luteolin has the potential to inhibit cyp2c9 isozyme. those predicted data revealed that asterol has the potential to inhibit cyp2d6 isozyme. furthermore, the study analyzed the binding interactions of the top-ranked phytochemical structures with the target protein, highlighting the role of hydroxyl groups in establishing potent hydrogen bonds with amino acid residues in the binding pockets. the interactions were visualized and analyzed to understand the molecular mechanisms underlying the inhibitory effects of the secondary metabolites. in addition to the molecular docking simulations, the study also conducted in-silico physicochemical and pharmacokinetic parameter predictions for the studied secondary metabolites. the analysis evaluated drug-likeness, physicochemical properties, admet characteristics, and predicted pharmacokinetic parameters of the secondary metabolites, providing insights into their potential as antimicrobial agents derived from natural sources. perera & lawan – antibacterial potential of hygrophila schulli 213 the comprehensive in-silico analysis presented in the document positions the secondary metabolites from hygrophila schulli as promising candidates for further experimental validations as potential inhibitors of bacterial dna topoisomerase iv. the findings contribute valuable information to the understanding of the molecular interactions between plant-derived secondary metabolites and bacterial dna topoisomerase iv, laying the groundwork for the development of novel antimicrobial agents derived from natural sources. this in-silico exploration offers a sustainable and effective approach in the ongoing battle against antibiotic-resistant bacterial infections. overall, the study provides a strong foundation for future experimental investigations to validate the inhibitory effects of the identified secondary metabolites from hygrophila schulli on bacterial dna topoisomerase iv, potentially leading to the development of novel antimicrobial agents with diverse pharmacological properties. conclusions selected secondary metabolites found in hygrophila schulli plant extract were tested in silico for their potential inhibitory action towards topoisomerase iv enzyme, pharmacokinetic and physicochemical properties, and compared with the commercial antibiotic, ciprofloxacin. out of the eleven phytochemicals assessed, lupeol and lupenone indicated the best receptor inhibition capabilities. comprehensive docking, pharmacokinetic, and physicochemical tests indicated that lupenone would be a viable topoisomerase iv inhibitor because it binds firmly to the binding pocket, inhibiting the native conformation of the protein. hence, lupenone is an exceptional candidate for further in vitro/in vivo studies. authors’ contributions: data analysis, computational investigations, and study design were completed by p. dilshan and h. lawan. manuscript written and reviewed by p. dilshan, h. lawan. the completed manuscript has been read and approved by all authors. competing interests: the authors declare that there are no competing interests. references bourgaud, f., gravot, a., milesi, s., & gontier, e. 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(2016). the traditional medicine and modern medicine from natural products. molecules, 21(5), 559. https://doi.org/10.3390/molecules21050559 biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 1, 2015 | pages: 5-9 | doi: 10.14421/biomedich.2015.41.5-9 the phytoestrogenic potential of yam bean (pachyrhizus erosus) on ovarian and uterine tissue structure of premenopausal mice cicilia novi primiani biology education, mathematics and science faculty, ikip pgri madiun jl. setiabudi 85 madiun, tel. +62-351-462986, fax. +62-351-459400 author correspondency: primianibiomipa@yahoo.co.id abstract the use of estrogen hormone by public has significantly been improved either as prevention or treatment of disease. menopausal issues in women are often treated using hormone replacement therapy. in regard to this, yam bean is found to contain genistein and daidzein compounds with a chemical structure that resembles estrogen hormone, therefore yam bean is categorized in the phytoestrogen group. the purpose of this study was to identify the potential of yam bean on ovarian and uterine histology of mice. this research employed a completely randomized design of experimental research approach of one factor namely yam bean in three different dosage treatment: 0.3 g/kg, 0.6 g/kg, and 0.9 g/kg of yam bean for 24 days. the surgery and organ harvesting of ovary and uterus were conducted on day 25 along with the making of histological preparat using paraffin method and hematoxylin-eosin (he) staining. the data was then analyzed descriptively. this research found that there were both secondary and tertiary follicle proliferation as the antrum contains some estrogen level. meanwhile, the endometrial tissue of the uterus experienced uterine glandular proliferation. to conclude, yam bean was found to be a natural estrogen source. keywords: yam bean, isoflavone, phytoestrogen, ovarium, uterus, menopause introduction hormone replacement therapy (hrt) is an action which is conducted through giving estrogen hormone especially to women who experience some reduction in the estrogen hormone. hrt is usually undergone by women experiencing menopause. in particular, estrogen replacement therapy (ert) can also be used for conducting cardiovascular disease therapy (stampfer et al., 1991 and sourander et al., 1998), reducing the risk of osteoporosis (grady et al., 1992 and nurochmad et al., 2010), and reducing the symptoms of menopause (barrett, 1998). the use of synthetic estrogen compound in a long term often gives negative impacts so that a natural alternative is needed to replace estrogene hormone. yam bean plant (pachyrhizus erosus) as a tuberlegume crop, is categorised as a legume with the dissemination area of sumatera, java, bali, sulawesi and east nusa tenggara (karuniawan & wicaksana, 2006). people usually prefer to consume fresh yam bean either in the form of salad or rujak (indonesian traditional fruit an vegetable salad dish). yam bean extract is used in the cosmetics industry in whitening, compact powder, and moisturizer products. yam bean contains isoflavone compounds with estrogen-like chemical structure (wanibuchi, 2003; abid, 2005; and lukitaningsih, 2009). the chemical isoflavones structure resembles 17βestradiol and has the efficacy like estrogene hormone (delmonte and rader, 2006; barlow et al., 2007) so that yam bean is also included in phytoestrogen group (urasopon et al., 2008). the largest components of isoflavone are genistein and daidzein which are often found in fabaceae family, including pachyrhizus erosus (kang et al., 2006). pachyrhizus erosus at least contain isoflavones such as, daidzein and genistein (primiani, 2013), the analysis of hplc daidzein and genistein of yam bean is 110,454 mg/100 g and 165,530 mg/100 g respectively. the chemical structure of geninstein and daidzein can bind to estrogen receptor and compete with endogenous estrogen such that they may provide both estrogenic and anti-estrogenic effects (adlercreutz, 1990; griffiths et al., 1996; adlercreutz and mazur, 1997). utilization of yam bean as phytoestrogen is not conducted much, a study by nurrochmad et al., (2010) showed that providing yam bean extract of 400 mg/kg and 800 mg/kg dosage for 4 weeks to mice through ovariectomy could prevent bone fragility; phytoestrogen was then proven to be able to improve uterine mass (ford et al., 2006). meanwhile, genistein affects on the increasing of weight of uterus by stimulating uterine endometrial thickening (santell, 1997). genistein dose 26,6 mg/day eqiuvalen with human dose 0,625 mg/day within 6 months on monkey cause vaginal maturation (marquez et al., 2012). research methods research design the research was conducted using experimental research approach with a completely randomized design, the treatment was completed through providing grated yam bean in three different dosage: 0.3 g/kg, 0.6 g/kg, 0.9 g/kg. the observation is focused on the changes of ovarian and uterine tissue structure. 6 biology, medicine, & natural product chemistry 4 (1), 2015: 5-9 tools and materials the research tools used in this study were: a gavage tube, plastic mice cages (50 x 30 x 20cm size), drinking bottles for mice, a digital scale (hm-200 brand), 1ml syringes with 3ml disposable needles (g23), an incubator, optical miscroscope, digital optilab camera microscope, a triple beam balance ohaus 700 series scale, analytical balance type hm-200 with the capacity of 210 grams and accuracy level of 0.1 mg, pr-50 microtome, surgical equipment, a surgical board, object glasses and lenses, glass beakers, spiritus lamp, a cube made of calendar paper, blender, flour sifter, a nife, a pipette, and a couple. the research materials were yam bean obtained from takeran village madiun indonesia, ovarian and uterine tissue, milk a granule for the mice food produced by pt. charoen pokphand indonesia, husk, cotton, tissue paper, aqua destillata, water tap, paraffin, 0.9% physiological saline, bouin fixative solution, 50%, 70%, 90% and absolute alcohol, pure xylol, xylol-alcohol mixture, with xylol and acohol comparison consecutively 1:3, 2:2, and 3:1, li2co3 solution, 1% hcl, 3% formalin, and haupt adhesive. animal experiments the experimental animals used were mice (mus musculus) strain balb/c female, in a good health condition, aged 12 months, 24 mice in total. every mouse had an initial body weight ranging from 20-25 grams before treatment, the mice were kept in a cage located in the biology education ikip pgri madiun, indonesia maintenance of mice and manufacture of test materials the mice were placed in the mice cages, given food and drink ad libitum and acclimatized for 14 days prior to induction treatment. the mice were maintained at the room temperature of (± 270c), relative humidity between 50-60% and 12 hour lighting cycle. every day the mice were weighed as the basis for determining the provision of grated yam bean. the manufacture of grated yam bean as a research material was conducted through grating the yam bean using a grater. the amount of the yam bean was then weighed and complied with the amount given to the mice. the provision of yam bean and manufacture of preparat of ovarian and uterine histology the provision of yam bean was coducted through direct induction to the stomach using a gavage tube once a day for 24 days. the mice were then dislocated on the 25th day and dissected. the organ harvesting of their ovaries and uterus were then performed. the manufacture of the preparat of ovarian and uterine histology was completed using paraffin method in attempt to determine any ovarian and uterine tissue structure changes. data analysis the ovarian and uterine tissue structure changes were analyzed descriptively based on the changes that happened in the ovarian follicle, myometrium layer, endometrium and uterine mucosa. results and discussions the results of observation on the ovarian tissue showed that there were changes in the ovarian follicles (figure 1). figure 1. the ovaries of mice (mus musculus); he stained sections; 400x description: a. control (p1); b. the dose of 0.3 g/kg (p2); c. the dose of 0.6 g/kg; d. the dose of 0.9 g/kg (p4). a. ovary with the control treatment, there are some corpus albikans. b. ovary with mature follicles and granulosa layer. c. secondary follicular proliferation, tertiary follicles begin to develop. d. tertiary follicles with follicular fluid. a b c d cicilia novi primiani – the phytoestrogenic potential of yam bean (pachyrhizus erosus) … 7 the results of observation on the uterine tissue showed that there were changes in the uterus endometrium tissue (figure 2). figure 2. the uterus of mice (mus musculus); he stained; 400x description: a. control (p1); b. the dose of 0.3 g/kg (p2); c. the dose of 0.6 g/kgd. the dose of 0.9 g/kg (p4). a. uterine wall consists of tunica serosa, tunica muscularis and tunica mucosa. b. proliferation of tunica mucosa (uterus endometrium layer). c. proliferation of uterine glands in uterine endometrium layer. d. proliferation of endometrium and myometrium layers, proliferation of uterine glands. the observation results of ovarian histology using hematoxylin-eosin (he) staining are depicted in figure 1. the control treatment (p1) shows follicles that experienced atresia (corpus albikans) and there is no indication of any primary follicle proliferation. the atresia folikuli phase shows histological features that represent disintegration in the form of fat drops and coarse granules in the ova (figure 1a). in the ovary that is given grated yam bean of 0.3 g/kg dose, some follicles with a thick layer under the tunica albuginea occur and they have a unique characteristic that the ova contained do not have any vitelina membrane (figure 1b). the ova are surrounded by layers of follicular cells which form granula layers on the more mature follicles afterward. in the provision of yam bean of 0.6 g/kg dose, secondary follicular growth takes place, the follicles are more eggshaped (oval) and has been moving away from the cortex approaching the medulla ovary and forming a room that is filled with fluid (antrum) around the ova and the granulosa cells layer are coating it (1c). liquid called liquor folliculi makes the follicles developed into tertiary follicles (apparent from the picture of ovary with 0.9 g/kg dose yam bean treament) (1d). changes in the uterine tisue structure of the mice after being given grated yam bean are depicted in figure 2. the results of observation on uterine histology with hematoxylin-eosin staining (he) show that the uterine wall of the control treatment (p1) is relatively thick and formed in 3 layers: (1) tunica serosa or outer perimetrium in the form of connecting tissue that consists of a layer of mesothelium supported by a thin connective tissue, (2) tunica muscularis or myometrium is the thickest layer which is composed of smooth muscle tissue. myometrium is the thickest tunica that consists of bundles of smooth muscle fibers which are separated by connective tissue. the endometrium consists of epithelial siliata and non siliata as well as lamina propria or stroma endometrialis containing simple tubular glands (uterine glands) that are branched on the inside (near the endometrium) (figure 2a). the provision of grated yam bean of 0.3 g/kg (p2) causes endometrium and myometrum proliferation of the uterus, uterine luminal has been narrowing due to the epithelialization process. the proliferation of uterine glands of the uterus begin to exist despite the decrease of premeneopausal period proliferation (figure 2b). the grated yam bean provided to the mice at a dose of 0.6 g/kg (p3) dan 0,9 g/kg (p4) apparently causes proliferation of the uterine endometrium and myometrium layers and increases the number of uterine glands (figure 2c and 2d). genistein and daidzein are categorised in isoflavone group with the chemical structure that is similar to estrogen hormone and able to work as estrogen. the similarity of the structure of genistein and daidzein demonstrate the ability to bind to estrogen receptors (setchell & cassidy, 1999). the decrease of ovarian follicles proliferation during the premenopause causes delays of tertiary follicle growth such that the secretion of estrogen hormone in the tertiary follicle antrum does not occur. the provision of grated yam bean can optimize the level of estrogen so that it is able to stimulate the development of primary follicles (primordial), secondary follicles, and tertiary follicles. the provision of phytoestrogen during the premenopause period improves the secretion of estrogen hormone which later causes an increase in the endometrium proliferation and epithelial cells replacement to cover the surface of mucosa. phytoestrogens have a similar structure to that of estradiol a b c d 8 biology, medicine, & natural product chemistry 4 (1), 2015: 5-9 which may also occupy estrogen receptors and lead to estrogen-like effects such as endogenous itself (harrison et al., 1999). myometrium may develop due to the phytoestrogens yam bean effect. genistein and daidzein in yam bean have an effect on uterine epithelium resulting in proliferation and cornification of epithelial cells as well as in the optimization of the secretion of estrogen. decreased estrogen level in premenopausal and postmenopausal period, resulting in an excess amount of estrogen receptors that are not bound. genistein and daidzein ability to bind to estrogen receptors called sex hormone binding globulin (hgb) serves to increase the production of steroid hormones and is responsible for binding estrogen and circulating it through the blood vessels (setchell et al., 1998). genistein as one of the isoflavone compounds has the effect of increasing the weight of the uterus by stimulating uterine endometrial thickening (santell et al., 1997). decreased levels of endogenous estrogen in premenopausal period can cause the estrus phase to not occur, thus giving yam bean to premenopausal mice can stimulate the estrus phase. genistein and daidzein as a group of phytoestrogens bind to estrogen receptors in the ovaries and uterus. the resulting response when estrogen levels are low is that the receptor and phytoestrogens binding will help in balancing estrogen levels. the function of estrogen in relation to reproduction is causing proliferation to occur and tissue in the reproductive organs to grow. the use of natural materials as hormone replacement therapy in postmenopausal women is one of the alternatives that can be done to overcome the side effects. component compounds contained in natural materials are very complex and often interact each other to provide a physiological effect (ioannides, 2002 and zhou et al., 2003). multi component contained in herbal medicine is a compounds complexity that provides optimal effect (lan and jia, 2010). conclusions yam bean can be used as natural estrogen hormone source which can increase the proliferation and maturation of ovarian follicles and proliferation of uterine endometrial glands of the uterus in premenopausal period. following up the need for estrogen therapy for premenopausal and postmenopausal women, it is necessary to study the effectiveness and safety ranging from experimentin on animals to humans, so its use as hormone therapy can be trustworthy. references abid, m. 2005. pharmacological evaluation of pachyrhizus erosus (l) seeds for cns activity. disertasi, bangalore: rajiv gandhi university of health science. adlercreutz, h. 1990. western diet and western diseases: some hormonal biochemical mechanism and associations. scandinavian j. clin. lab. invest. 50s 201:3-23. adlercreutz, h., mazur, w. 1997. phyto-oestrogens and western diseases. j. anayls of med. 29:95-120. barlow, j., johnson, j.a., scofield, l. 2007. fact sheet on the phytoestrogen genistein. niehs/nci environment research centers. barrett, c.e. 1998. hormone replacement therapy. bmj. 317:457-461. delmonte, p., rader, j. 2006. analysis of isoflavones in foods and dietary supplements. journal of aoac international, 89(4)1138-1146, ford, j.a., clark, s.g., walters, e.m., wheeler, m.b., hurley, w.l. 2006. estrogenic effects of genistein on reproductive tissues of ovariectomized gilts. anim sci. 84:834-842. grady, d., rubin, s.m., petitti, d.b. 1992. hormone therapy to prevent disease and prolong life in postmenopausal women. ann intern med. 117:1016-1037. griffiths, k., adlercreutz, h., boyle, p. 1996. nutrition and cancer. oxford: isis medical media. harrison, r.m., phillippi, p.p., swan, k.f., henson, m.c., 1999. effect of genistein on steroid hormon production in the pregnant rhesus monkey. society for experimental biology and medicine, vol. 222. ioannides, c. 2002. pharmacokinetic interactions between herbal remidies and medicinal drugs. xenobiotica. 32:451-478. kang, s., chung, j.h., lee, j.h., fisher, g.j., wan, y.s., duell, e.a. 2003. topical n-acetyl cysteine and genistein prevent ultraviolet-light-induced signaling that leads to photoaging in human hkin in vivo. j invest dermatol. 120:835-841. karuniawan, a., wicaksana, n. 2006. kekerabatan genetik populasi bengkuang pachyrhizuz erosus berdasarkan karakter morfologi dan daun. bul agrom. (34)(2):98-105. lan, k., jia, w. 2010. an integrated metabolomics and pharmacokinetics strategy for multi-component drugs evaluation. current drug met. 11:105-114. lukitaningsih, e. 2009. the exploration of whitening and sun screening compounds in benguang roots (pachyrhizus erosus. disertasi, wurzburg: bayerischen julius maximillians university. marquez, s.r., hernandez, h., flores, j.a., gutierrez, m.m., duarte, g., vielma, j., rodriguez, g.f., fernandez, i.g., keller, m., and delgadillo, j.a. 2012. effects of phytoestrogens on mammalian reproductive physiology. tropical and subtropical agroecosystems. 15 sup 1:s129s145. nurrochmad, a., leviana, f., wulancarsari, c.g.., & lukitaningsih, e. 2010. phytoestrogens of pachyrhizuz erosus prevent bone loss in an ovariectomized rat model of osteoporosis. int. j. phytomed. 2:363-372. primiani, c.n. 2013. dinamika senyawa daidzein umbi bengkuang (pachyrhizus erosus) dalam darah serta potensinya pada tikus betina. the proceeding of 10th national seminar of biology, environment and their educational implementation. biology education department, faculty of pedagogy, sebelas maret university, surakarta. 6 july 2013. isbn 978-602-8580-94-6. 3:502-510. santell, r.c., chang, y.c., muralee, g.n., william, g.h. 1997. dietary genistein exerts estrogenic effects upon the uterus, mammary gland and the hypothalamic/pituitary axis in rats. nutr. 127: 263–269. setchell, k., nechemias, l.z., cai, j., heubi, j. 1998. isoflavone content of instant formulas and the metabolic fate of these phytoestrogens in early life. am j soc clin nutr.68:1453-1461. setchell, k.d.r., cassidy, a. 1999. dietary isoflavones: biological effects and relevance to human health. j nutr. 129:758s-767s. sourander, l., rajala, t., raiha, i., makinen, j., erkkola, r., dan helenius, h. 1998. cardiovascular and cancer death in cicilia novi primiani – the phytoestrogenic potential of yam bean (pachyrhizus erosus) … 9 postmenopausal women on estrogen replacement therapy (ert). lancet. 352:1965-1969. stampfer, m.j., colditz, g.a., dan willett, w.c.1991. postmenopausal estrogen therapy and cardiovascular disease. n engl j med. 325:756-762. urasopon, n., hamada, y., asaoka, k., poungmali, u., dan malavijitnond, s. 2008. isoflavone content of rodent diets and its estrogenic effect on vaginal cornification in pueraria mirifica treated rats, science asia, 34:371-376. wanibuchi, h., kang, j.s., fukushima, s. 2003. toxicity vs benefecial effects of phytoestrogens. pure appl chem. 75(1112):2047-2053. zhou, s., gao, y., jiang, w., huang, m., xu, a., paxton, j.w. 2003. interactions of herbs with cytochrome p450. drug metab rev. 35:35-98. content_v4n1_2.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 121-125 | doi: 10.14421/biomedich.2024.131.121-125 issn 2540-9328 (online) formulation and antibacterial activity testing of eco-soap based on sodium lauryl sulfate surfactant nurhaida widiani, aulia novitasari*, ovi prasetya winandari, dwijowati asih saputri department of biological education, faculty of islamic education and teacher training, uin raden intan lampung jl. let.kol endro suratmin, sukarame, bandar lampung, 35131, indonesia. corresponding author* aulianovitasari@radenintan.ac.id abstract pineapple is a fruit that is often used only for its flesh, while the skin remains waste. as a form of dealing with pineapple waste, one way is to use it as an eco-enzyme. making soap with added eco-enzyme can be used as an alternative for washing dishes. therefore, this research aims to determine the formulation and test the antibacterial activity of eco-soap based on the surfactant sodium lauryl sulphate. this research is experimental research, which was carried out in july-october 2023. the research results showed that the ph of eco-soap was 3.46; foam height 1.5 cm; viscosity testing 14.9 mpas; fatty acids 1.44%; and 6 mm antibacterial activity testing. based on the research results, it was concluded that the surfactant-based eco-soap formulation had good stability in the eco-soap foam height test. the diameter of the zone of inhibition of eco-soap's antibacterial activity showed that bacterial inhibition was moderate. keywords: eco-enzyme; eco-soap; antibacterial; pineapple waste; formulation. introduction indonesia is the fourth largest country in the world as a pineapple producer. in 2019, indonesia's pineapple production reached 2,196,456 tons, increasing by 21.7% from 2018. one of the largest pineapple-contributing provinces is lampung (astoko, 2021).pineapples are commonly utilized for their fruit flesh, while their peel often becomes waste. therefore, efforts to address pineapple waste need to be considered, one of which is by utilizing it to produce eco-enzymes, with the hope that it can be developed into eco-soap. the shift in dishwashing behavior, moving away from the use of scrubbers, has an impact on soap consumption. while soap may not be a primary need, the frequency of soap usage is relatively high. this is due to the main function of soap, which is to clean dirt, and it offers practical, hygienic, and economic advantages (akyuni et al., 2021; sianiar et al., 2021). the fulfillment of dish soap needs sparks ideas and opportunities for product creation. with the advancement of technology, synthetic soaps from various brands have flooded the market. however, the current trend is shifting towards organic options due to their environmentally friendly nature (duraisamy et al., 2011; jadid et al., 2020), one of the ingredients that can be added in the making of dish soap is eco-enzyme (iswati et al., 2021). eco-enzyme is a liquid fermentation derived from organic waste such as vegetables and fruits, and it possesses various benefits (hasanah, 2021). the fermentation process is carried out using molasses or palm sugar, allowing it to sit for a minimum of 3 months, with the lid being opened once a week. (larasati et al., 2020). eco-enzyme demonstrates antibacterial activity; the acetic acid content in eco-enzyme can eliminate germs, bacteria, and viruses. it has the ability to eradicate bacteria such as s. typhi, s. aureus, c. albicans, and viruses (anisa et al., 2022), it also significantly suppresses the growth of escherichia coli (ginting et al., 2021). the phenolic compounds present in eco-enzyme will interact with the cell walls of microorganisms, leading to the denaturation of proteins. this causes a structural change in proteins, and the increased permeability of the cell, resulting in the inhibition of bacterial cell growth (rusdianasari et al., 2021). the production of soap with the addition of eco-enzyme can be considered as an alternative for dishwashing. therefore, this research aims to determine the formulation and test the antibacterial activity of eco-soap based on sodium lauryl sulfate surfactant. materials and methods materials this research was conducted at the laboratory of fmipa unila from july to october 2023. the equipment used manuscript received: 05 january, 2024. revision accepted: 08 may, 2024. published: 20 may, 2024. https://doi.org/10.14421/biomedich.2024.131.121-125 122 biology, medicine, & natural product chemistry 13 (1), 2024: 121-125 for making eco-soap (eco-enzyme dishwashing soap) included rulers, pipettes, buckets, ph meters, erlenmeyer flasks, pycnometers, titration tools, round-bottom flasks, stirrers, and test tubes. the materials used included ecoenzyme, escherichia coli bacteria, nutrient agar, coloring agents, phenolphthalein solution, nutrient broth (nb), texapon, perfume, thickener, 96% ethanol, 0.1 n hcl, distilled water, boiling stones, sls, nacl, and preservatives (sianiar et al., 2021). procedures ▪ formulation of surfactant-based eco-soap prepare a solution of texapon (100 g, eco-enzyme 50 ml, water 400 ml), then add sls (50 g, water 200 ml, 50 ml eco-enzyme). mix nacl (50 g) with water (200 ml) and eco-enzyme (50 ml) while stirring until thickened and free from lumps. add fragrance, and then conduct quality tests for eco-soap, including ph measurement, foam height testing, fatty acid testing, and viscosity testing. ▪ antibacterial activity test the antibacterial activity test of surfactant-based eco-soap is conducted for 6 hours on a 30 ml solution against escherichia coli bacteria. results and discussion result testing the ph of eco-soap the ph measurement of eco-soap resulted in an acidic ph category (table 1) table 1. eco-soap ph testing. quality testing conclusion ph 3,46 foam height testing of eco-soap the results of the eco-soap foam height test obtained a foam height of 1.5 cm, meeting the criteria for good soap (table 2) table 2. eco-soap foam height testing. quality testing conclusion foam height 1,5 cm viscosity testing the viscosity test results showed a viscosity of 14.9 mpas (table 3). table 3. viscosity testing. quality testing conclusion viscosity 14,9 mpas fatty acid testing the results of the fatty acid test indicate a value of 1.44% (table 4). table 4. fatty acid testing. quality testing conclusion fatty acid 1,44% antibacterial activity test the results of the antibacterial activity test show a measurement of 6 mm (table 5). table 5. antibacterial activity testing. quality testing conclusion antibacterial activity 6 mm discussion this research was conducted from july to october 2023, utilizing the integrated laboratory and technology innovation center of lampung university. the study design employed was experimental. variables observed in this research included ph measurement, foam height testing, fatty acid testing, viscosity testing, and antibacterial testing against escherichia coli bacteria. the research involved the production of eco-enzyme over a 3-month period, followed by the manufacture of eco-soap or dishwashing soap. the cleaning base used was a combination of anionic and nonionic surfactants, specifically sodium lauryl sulfate (sls). the use of sls serves as the primary ingredient for forming foam (surfactant source) in liquid dishwashing soap. this surfactant produces a dishwashing base with good foam stability and low potential for irritation. the addition of eco-enzyme in eco-soap functions as an antibacterial agent, providing a potential solution to combat bacteria on household utensils the characterization observation results for ph indicate a category below 4, meeting the standard for good eco-enzyme production (putra & suyas, 2022), the low production of eco-enzyme is attributed to the high content of organic acids. the results of this research align with previous studies, indicating that eco-enzyme is chemically acidic with a ph between 3 – 4 (rochyani et al., 2020), this is supported by the research (rasit et al., 2019) the higher the content of organic acid, the lower the ph of the eco-enzyme product, as organic acid serves as the ph acidity indicator. the organic acid found in the eco-enzyme product is generated through a 3-month fermentation process. the presence of acetic acid in the eco-enzyme has also been identified by this research (samriti & arya, 2019) that there is acetic acid in ecoenzyme, although the concentration of acetic acid in ecoenzyme is not as high as in vinegar, is supported by the research (larasati et al., 2020) acetic acid is produced through the metabolic process of bacteria naturally widiani et al. – formulation and antibacterial activity testing of … 123 present in fruit and vegetable residues. the anaerobic metabolism process, commonly known as fermentation, is an effort by bacteria to obtain energy from carbohydrates under anaerobic conditions, resulting in by-products such as alcohol or acetic acid. fungi and certain bacteria produce alcohol during fermentation, while most bacteria produce acetic acid. it can be concluded that eco-enzyme has a low ph due to its high content of organic acids, namely citric acid and acetic acid. (etienne et al., 2013). ph testing of eco-soap is one of the quality requirements for dishwashing soap or eco-soap. the ph value indicates the acidity level of a substance. ph is a crucial indicator for eco-soap to determine its suitability and safety for use on the skin, as eco-soap comes into direct contact with the skin and can cause issues if the resulting ph does not match the skin's ph. a ph value that is too low can cause skin irritation, while a ph that is too high can result in dry or flaky skin. the ph testing of eco-soap involves diluting 1 ml of the soap to be examined with distilled water up to 10 ml. the solution is then placed in a calibrated ph meter, and the ph meter is observed until it stabilizes, indicating a constant ph value. based on observations of the soap preparation from eco-enzyme, it was found that the ph of eco-soap is 3.46. the national standard for the ph of dishwashing soap or eco-soap, according to the sni-2588-2017 standard, states that good dishwashing soap should have a ph value between 6 and 11. this indicates that the ecosoap in the conducted research does not yet meet the established national standard for dishwashing soap. the reason for this discrepancy is that the eco-enzyme used as an ingredient in eco-soap has an acidic ph derived from citrus, resulting in the produced eco-soap having an acidic ph (arrazi et al., 2021). the foam height test is conducted to determine the amount of foam remaining after a certain period. foam is a colloidal system with a dispersed gas phase and a medium dispersing liquid substance. the dispersed gas phase is typically air or co2. foam stability is achieved through the presence of surfactants. surfactants have hydrophilic and hydrophobic groups. the hydrophilic group binds with water molecules, while the hydrophobic group moves towards the solution's surface, facing the air.when the water and surfactant solution is stirred or exposed to air, air bubbles emerging from the liquid body will be coated by a thin layer of liquid containing surfactants, forming foam. one of the appealing features of soap is its foam content. foam stability is expressed as the resistance of a bubble to maintain its size or prevent the rupture of the film layer.the examination of foam height is one method to control the stability of liquid soap in producing foam. the higher the foam stability value, the higher the quality of the produced foam. foam stability is significantly influenced by the particle size, so the greater and larger the particle size, the lower the foam stability. if the foam produced is abundant and stable, it will be preferred by consumers compared to foam that is scarce and unstable (rosmainar, 2021). the foam height test begins by diluting 2 ml of eco-soap with 10 ml of distilled water, which is then placed into a test tube. subsequently, the mixture is shaken for 20 seconds and allowed to stand for 5 minutes. the height of the formed foam is then measured using a ruler, resulting in a foam height of 1.5 cm or 15 mm. the standard for soap foam height set by the indonesian national standard (sni2588-2017) is 13-220 mm. therefore, the eco-soap meets the criteria for good soap as its foam height falls within the specified range. viscosity testing is used for the analysis of the thickness of liquid dishwashing soap or eco-soap formulations. dishwashing soap has a standard viscosity range according to the indonesian national standard (sni) of 400-4000 mpas. viscosity measurements of eco-soap formulations using eco-enzyme as the base material are conducted with a haake viscometer. a 150 ml sample is prepared in a 250 ml beaker, and the spindle is adjusted before being immersed in the formulation until the viscometer indicates the viscosity value of the formulation. the result obtained is 14.9 mpas, which does not meet the sni standard. the research findings are below commercial soap, possibly due to a lack of added thickening agents or excessively high water content.eco-soap formulations with too low viscosity can disrupt solution stability, leading to separation, while formulations with excessively high viscosity can slow the flow rate of the soap solution. additionally, the formation of an overly concentrated gel system may occur (yuli handayani et al., 2022). free fatty acids are fatty acids present in the eco-soap sample but are not bound as potassium compounds or triglyceride compounds (fats/oils). free fatty acids still exist in eco-soap because they have not undergone saponification reactions. the testing of free fatty acids is done through titration. 5 ml of liquid soap is placed in an erlenmeyer flask. then, 100 ml of 96% methanol is added. the mixture is heated to boiling, and it is titrated with 0.1 n hcl until the pink color disappears, using phenolphthalein as an indicator. the result of the free fatty acid testing in eco-soap using eco-enzyme as the base material is 1.44%. according to the soap quality standard (sni 2588: 2017), good soap should have a free fatty acid content not exceeding 0.1%. therefore, ecosoap does not meet the criteria for soap with good free fatty acid levels.free fatty acids are associated with the soap's odor. if the free fatty acids exceed the soap standard, it can result in a rancid odor and hinder the cleaning process of the skin surface by the soap (verawaty et al., 2020). antibacterial activity testing is conducted to determine whether the dishwashing soap formulation can inhibit the growth of escherichia coli. if the bacterial inhibition zone is < 5 mm, it indicates weak antibacterial activity. if the diameter of the inhibition zone ranges from 5 to 10 mm, the activity is considered moderate. if the diameter of the inhibition zone ranges from 10 to 20 124 biology, medicine, & natural product chemistry 13 (1), 2024: 121-125 mm, it indicates strong antibacterial activity. and if the diameter of the inhibition zone produced is > 20 mm, it indicates very strong antibacterial activity. the observation result for the inhibition zone diameter of eco-soap after a 6-hour incubation period with a concentration of 30 ml is 6 mm, categorizing it as having moderate inhibitory activity. this means that eco-soap with eco-enzyme as the main ingredient shows good results in inhibiting the growth of escherichia coli bacteria. the mechanism of bacterial inhibition by eco-enzyme is attributed to the presence of flavonoid and tannin contents acting as bioseptans. the flavonoid content in the eco-enzyme inhibits nucleic acid synthesis, interferes with cell membrane function, and hinders energy metabolism. additionally, the antibacterial content exhibits activities related to its capability to activate microbial cell adhesion, enzyme activation, and disruption of protein transport in the inner layers of the cell (rahayu et al., 2021). the antibacterial activity of eco-enzyme is likely related to its content of acetic acid and lactic acid. organic acids can inhibit and kill microorganism growth through a mechanism in which dissociated and ionized molecules flow through the microorganism cell membrane. to maintain intracellular ph, hydrogen ions are released, and the acidic ph causes cell deformation, damaging enzymatic activities, proteins, and bacterial dna structures, ultimately leading to extracellular membrane damage. in another mechanism, changes in membrane permeability can hinder substrate transportation, while changes in intracellular ph can suppress nadh oxidation. this can affect the electron transport system, leading to organism death (andam, 2017). conclusions based on the research findings, it can be concluded that the formulation of eco-soap based on surfactants exhibits good stability in the high foam test. however, in the ph test, viscosity test, and fatty acid test, the results did not meet the standards. the diameter of the inhibition zone for the antibacterial activity of surfactant-based eco-soap against escherichia coli bacteria indicates a moderate inhibitory effect. authors’ contributions: all authors are major contributors to this research. nw, an, opw and das collaborated to design the research and write the initial research draft. nw: executed the experiment, an and opw: wrote the article draft and revised the draft, das: contributed suggestions for article improvement. competing interests: the authors declare that there are no competing interests. references akyuni, q., putri, f. r., annisa, n., putri, d. h., & farma, s. a. 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(2022). formulasi sabun cair cuci piring menggunakan ekstrak air tanaman lidah buaya (aloe vera l.). medical sains : jurnal ilmiah kefarmasian, 7(2), 109–118. https://doi.org/10.37874/ms.v7i2.314 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 531-540 | doi: 10.14421/biomedich.2024.132.531-540 issn 2540-9328 (online) genotoxic and cytotoxic activities of cornhusk extract of zea mays and leaf extract of sacharum officinarum emem eyo akpan1, martin osita anagboso2, imoh imeh johnny3, nwakaego o. ebong4,*, jude efiom okokon1 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria 2department of microbiology, madonna university nigeria, elele campus, rivers state, nigeria. 3department of pharmacognosy and natural medicine, faculty of pharmacy, university of uyo, uyo, nigeria. 4department of clinical pharmacy and management, faculty of pharmaceutical sciences, university of port harcourt, rivers state, nigeria. corresponding author* nwakaego.ebong@uniport.edu.ng abstract zea mays husk and saccharum officinarum have been used for years in ethnomedicine for their antimalarial, anti-inflammatory, antipyretic, antidiabetic, and antiphlogistic activities. the cytotoxic and genotoxic effects of zea mays husk and saccharum officinarum leaf extracts on the root meristem cells of allium cepa were investigated. onion bulbs were exposed to 2.5 mg/ml, 5mg/ml, and 10 mg/ml concentrations of the extracts for macroscopic and microscopic analysis. tap water was used as a negative control and methotrexate (0.1 mg/ml) was used as a positive control. there was statistically significant (p < 0.05) inhibition of root growth depending on concentration by the extracts when compared with the negative control group. all the tested extracts were observed to have cytotoxic effects on cell division in a. cepa. the extract induced chromosomal aberrations and micronuclei (mnc) formations in a. cepa root tip cells were significant (p<0.05) when compared with the control group. the extracts treatment further induced cell death, ghost cells, cells membrane damage, and binucleated cells. the zea mays husk extract was found to exhibit higher cytotoxic and genotoxic potential than saccharum officinarum leaf extract. these results suggest that zea mays husk and saccharum officinarum leaf extracts possess cytotoxic and genotoxic effects on a. cepa. keywords: allium cepa; cytotoxic; genotoxic; medicinal plants; zea mays husk; sacharum officinarum. abbreviations: anova (analysis of variance), completely randomized design (crd), polymorphonuclear leukocytes (pmns) introduction a number of green plants contain phytoconstituents, which may exert cytotoxic and genotoxic activities due to their effects within a biological system. research has also shown that a number of plants which are utilized as food or medicine traditionally have mutagenic effects as well as cytotoxic and genotoxic effects in vitro and in vivo (higashimoto et al., 1993; schimmer et al., 1994; kassie et al., 1996; as et al., 2007; ikechukwu et al., 2024; magnus et al., 2024). plants with potential mutagenic and carcinogenic substances (ames, 1986; de sá ferreira & ferr˜ao vargas, 1999) which are used as food or medicine have been correlated with a high rate of tumour formation in some human populations (wynder et al., 1983; nagao et al., 1986; nguyen et al., 1989; brito et al., 1990). this reveals potential toxic hazards that may result from prolonged use of such plants, especially those used as herbs in the treatment of various diseases traditionally. the husk of zea mays (maize) and leaves of sacharum officinarum (sugarcane) are commonly used in the treatment of various diseases by the ibibios of the niger delta region of nigeria. saccharum officinarum (family-poaceae), also called sugarcane, thrives throughout tropical and subtropical regions worldwide. in traditional medicine, it is used in the treatment of diarrhoea, dysentery, eyes, fever, arthritis, bedsores, boils, cancer, colds, cough, opacity, skin sores, sore throat, hiccups, inflammation, laryngitis, spleen, tumours, and wounds (hartwell, 1967-1971). the leaf extract possesses some biological activities such as antibacterial and anthelmintic (palaksha et al., 2013), anti-hyperglycaemic, anti-hyperlipidaemic (ojewunmi et al., 2013), antioxidant (ojewunmi et al., 2013; sun et al., 2014), diuretic and antiurolithiatic (palaksha et al., 2015), antidepressant and anticonvulsant (okokon et al., 2019a), analgesic (okokon et al., 2021a) and antimalarial (okokon et al., 2022), antioxidative stress and hepatoprotective (edem et al., 2022), anti-inflammatory and antipyretic (edem et al., 2023) activities. saabmal®: a polyherbal preparation containing s. officinarum is utilized as a malarial remedy in nigeria manuscript received: 12 june, 2024. revision accepted: 26 september, 2024. published: 31 october, 2024. https://doi.org/10.14421/biomedich.2024.132.531-540 532 biology, medicine, & natural product chemistry 13 (2), 2024: 531-540 (obidike et al., 2015). the leaves are employed in ghana for the local treatment of malaria (asase et al., 2010). phytochemical screening of the leaf extract of saccharum officinarum revealed the presence of glycosides, phytosterols, saponins, tannins, and flavonoids (palaksha et al., 2013; singh et al., 2015). some flavones and phenolics, as well as their derivatives from the leaves of s. officinarum, have been identified (coutinho et al., 2016; okokon et al., 2022). zea mays l. (poaceae), commonly called maize or corn, is a grass and food plant cultivated for human and animal benefits. the plant is tall and bears ears that are enclosed in modified leaves known as husks (simmonds, 1979). in addition to its nutritive values, various parts of the plants are used in ethnomedicine for the treatment of several ailments such as diabetes (foster & duke, 1990; gill, 1992; abo et al., 2008; brobbey et al., 2017; okokon et al., 2017a), cough (gill, 1992), inflammatory diseases (okokon et al., 2016), pains and arthritis (owoyele et al., 2010) and ulcer (jadhav, 2016). reported pharmacological properties of the husk extract include analgesic, anti-inflammatory (owoyele et al., 2010), antioxidant (dong et al., 2014), antidepressant (okokon et al., 2016), antimalarial and antiplasmodial (okokon et al., 2017a), hepatoprotective (okokon et al., 2017b; okokon et al., 2020; udobang et al., 2019), antidiabetic and hypolipidemic (okokon & mandu, 2018) and nephroprotective (okokon et al., 2017c; okokon et al., 2019b), antiulcer (okokon et al., 2018), antiobesity (okokon et al., 2021b) and alpha glucosidase and alpha amylase inhibitory (okokon et al., 2021c) activities. isolated compounds from the husk extract include; arabinoxylan (ogawa et al., 2005), phenolic compounds (gallic acid, protocatechuic acid, chlorogenic acid, cafes acid, ferrulic acid, rutin, resveratrol, and kaempferol) (dong et al., 2014), anthocyanins (li et al., 2008) and stigmasterol, stigmasteryl stearate and stigmasterol palmitate (okokon et al.,2021b). the medicinal potentials of these plants have been widely reported, but there is paucity of information on their toxicological potential, therefore this investigation. materials and methods collection and identification of plant material the plant materials, husks of zea mays and leaves of saccharum officinarum, were both collected from farms and compounds within uyo metropolis, akwa ibom state, nigeria in may 2023. the plants were identified and authenticated by a taxonomist, dr. margaret bassey, of the department of botany and ecological studies, university of uyo, uyo, nigeria. extraction procedure the plant materials were each washed and shade-dried for two weeks. the dried plant materials were further chopped into small pieces and reduced to powder. the powdered materials were individually soaked in 70% ethanol. the liquid filtrates were concentrated and evaporated to dryness in vacuo at 40ºc using a rotary evaporator. the crude extracts were stored at -4 degrees celsius until used. macroscopic evaluation the two plant extracts were prepared by dissolving 20 g of each extract in 200 ml of distilled water to obtain a stock solution of 10% each. afterwards, each stock solution was filtered using a filter paper to remove any particulate matter. the stock solutions were diluted to concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml respectively. allium cepa test small bulbs of the common onion, a. cepa were, procured from jos in the northern region of nigeria. prior to initiating the test, the outer scales of the bulbs and the dry bottom plate were removed without destroying the root primordia using a small sharp knife without destroying the root primordia and collected in a jar of water. test concentrations of the plants’ extracts (zea mays husk and saccharum officinarum leaves) were prepared at 2.5 mg/ml, 5 mg/ml, and 10 mg/ml respectively. for each test concentration of zea mays husk and saccharum officinarum leaf extracts, 15 ml beakers were arranged in a series of 5 per test concentration and filled up for each concentration. one a. cepa bulb was placed on top of each beaker, with the root primordia downward toward the liquid. tap water was used as a negative control and 0.1mg/ml methotrexate was used as a positive control. after 24 hours, the test samples were changed in the controls and all test concentrations and photographs of the growing a. cepa roots were captured. this continued until 72 hours, after which the roots were counted per beaker in all the tested concentrations and the mean root number was calculated. similarly, the roots’ lengths were measured using a meter rule and the mean root length was calculated. these were also done for the control. several root tips were cut at a length of 10mm from the bulbs at 8:00 am, 8:30 am and 9:00 am, respectively and fixed in 3:1 (v/v) ethanol: glacial acetic acid and 1n hcl before putting them in sample bottles and storing them in a refrigerator until use. microscopy the root tips was each placed in a test tube with 1n hcl and heated at 50ºc for 6 minutes to fix and macerate them. after that, the root tips were placed on microscopic slides on a blank background with a-forceps and were cut off at terminal tips. two drops of 2% (w/v) orcein stain was added and mixed with the rootlets properly by knocking and stirring with a stirring spatula. akpan et al. – evaluation of genotoxic and cytotoxic potentials of zea mays husk and sugarcane leaves 533 then, a cover slip was placed at 45º to avoid air bubbles. after that, the cells were squashed by placing a filter paper on the cover slip and pressing slightly with a thumb. the coverslip was sealed with a-clear fingernail polish (grant, 1982; okokon et al., 2024) and each slide was examined using a light microscope at a magnification of x40. microphotographs were taken to show chromosomal aberrations. the mitotic index and frequency of chromosomal aberration were calculated based on the number of aberrant cells per total cell scored at each concentration of each sample (bakare et al., 2000). the mitotic inhibition was determined using the following formula: mitotic index = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑖𝑣𝑖𝑑𝑖𝑛𝑔 𝑐𝑒𝑙𝑙𝑠 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 x 100 %aberrant cells = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐴𝑏𝑒𝑟𝑟𝑎𝑛𝑡 𝑐𝑒𝑙𝑙𝑠 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 x100 %root growth of control = 𝑂𝑣𝑒𝑟𝑎𝑙𝑙 𝑚𝑒𝑎𝑛 𝑟𝑜𝑜𝑡 𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑡𝑒𝑠𝑡 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑂𝑣𝑒𝑟𝑎𝑙𝑙 𝑚𝑒𝑎𝑛 𝑟𝑜𝑜𝑡 𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 x 100 analysis the following parameters were used for determination of cytotoxicity and genotoxicity: (i) the mitotic index (mi was calculated as the ratio between the number of mitotic cells and the total number of cells scored and expressed as percentage and (ii) chromatin aberrations (stickiness breaks and polar deviation) were used as endpoints for determination of cytogenetic effects and micronuclei (mnc) were scored in interphase cells per 1000 cells. statistical analysis data obtained from this work were analysed statistically using one–way anova followed by tukey-kramer multiple comparison test using instant graphpad software, (san diego, usa). differences between means were considered significant at a 5% level of significance i.e., p≤ 0.05. results and discussion physicochemical characterization. the effects of extracts of z. mays husk and sacharum officinarum leaves on levels of the physicochemical parameters (root number and root length) are presented in table 1. these results show that all tested concentrations of z. mays husk and s. officinarum leaf extracts exerted significant root growth inhibition in comparison to negative control and positive control. the inhibition of root number and root length increased with increasing concentrations of the leaf extracts. the average root length in negative and positive control (methotrexate) groups were 4.66±1.26 and 0.10±0.01 cm, respectively. however, average root lengths in 10 mg/ml treatment groups of the two extracts were 0.42±0.03 and 0.46±0.09 cm, respectively, for z. mays husk and s. officinarum leaves extracts. these observed decreases were significant (p<0.05) when compared to that of the negative control (table 1). the average root lengths in treatment groups of the extracts were found to decrease with increasing concentrations. z. mays husk exhibited higher inhibition of the root growth than s. officinarum leaf extracts, and this was significant (p<0.05) when compared to the negative control. the root morphology of the negative control group appeared to be normal, while the 2.5 mg/ml treatment groups of the two extracts appeared yellowish brown. the morphology of roots from 5 and 10 mg/ml treatment groups of the two leaf extracts appeared brownish (table 1). cytogenetic analysis. table 2 shows the effects of zea mays husk and saccharum officinarum leaf extracts on cytogenetic parameters of allium cepa roots. cytogenetic analysis carried out showed that the two extracts caused concentration-dependent and significant (p<0.05) decreases in the mitotic index when compared to that of the negative control. the extracts of z. mays husk and s. officinarum leaves at 10 mg/ml had mitotic indices of 17.20±2.56 and 19.20±3.27 respectively, as compared to 60.40±8.24 recorded in the negative control group (table 2). this showed that z. mays husk was more cytotoxic than s. officinarum. 534 biology, medicine, & natural product chemistry 13 (2), 2024: 531-540 table 1. cytotoxicity of zea mays husk and sacharum officinarum leaf extracts on growing roots of onion (allium cepa). treatment group concentration of extract (mg/ml) average root number ± sem average root length (cm) ± sem negative control tap water 35.80±2.41 4.66±1.26 methotrexate 0.1 2.10±0.02a 0.10±0.01a zea mays husk extract 2.5 31.20±3.55a 1.00±0.07a 5.0 11.40±1.20a 0.80±0.13a 10.0 8.40±1.40a 0.42±0.03a saccharum officinarum leaf extract 2.5 22.00±1.87a 1.26±0.15a 5.0 6.20±0.73a 0.66±0.16a 10.0 4.40±0.67a 0.46±0.09a values are expressed as mean ±sem (n=5). significant at p<0.05 when compared to negative control, sem: standard error of mean. table 2. dividing and total cells counted under microscopic observations and mitotic values in control and treatment concentrations. treatment group concentration of extract (mg/ml) total number of cells dividing cells m.i (%) ± sem negative control tap water 500 302 60.4±8.24 methotrexate 0.1 500 15 3.00±0.68a zea mays husk extract 2.5 500 188 37.60±2.77a 5.0 500 141 28.20±3.26a 10.0 500 86 17.20±2.56a saccharum officinarum leaf extract 2.5 500 224 44.80±2.65a 5.0 500 126 25.20±4.13a 10.0 500 96 19.20±3.27a values are expressed as mean ±sem (n=5). significant at p<0.05 when compared to negative control, sem: standard error of mean. cytogenetic alterations caused by the two extracts are shown in table 3. chromosome and cytological alterations were observed in the negative control, methotrexate, z. mays husk and s. officinarum leaf extracts-treated groups, as depicted in table 3. various chromosome aberrations were observed according to the analysis carried out, including bridge formations, which were observed mostly in z. mays husk extract-treated groups, especially in the group treated with 2.5 mg/ml. (table 3) (figure 1a). chromosome fragmentations or clastogenic breaks were observed mostly in s. officinarum leaf extract (5 mg/ml) and were of chromosome type (table 3) (figure 1(b). this depicts the clastogenic effect of the extract. this was significant (p<0.05) when compared to the negative control group. sticky metaphase and telophase were also observed in the two extractstreated groups. these abnormalities were found to increase generally with increasing concentrations of the extracts. the number of aberrant cells (aberrant cells include chromosome breaks, stickiness and polar deviation) was found to be concentration-dependent and statistically significant (p<0.05) when compared to the negative control (table 3). however, the highest value of aberrant cells was observed in the methotrexate-treated group (positive control) (table 3). the extracts further demonstrated genotoxic potentials by inducing micronuclei in the root tip meristem cells of a. cepa. this was not concentrationdependent (figure 1(b)). also, cells with membrane damage (figure 1(c)) and nucleus damage (figures 1(c, d and f), binucleated cells (figure 1(e)), ghost cells 1(g)) and apoptotic cells (figure 1(e)) were found in various frequencies in the groups treated with the two extracts. akpan et al. – evaluation of genotoxic and cytotoxic potentials of zea mays husk and sugarcane leaves 535 table 3. chromosomal and mitotic aberrations in the root meristematic cells of allium cepa after treatment with zea mays husk and sacharum officinarum leaf extracts. treatment group concentr ation of extract (mg/ml) chromosome breaks (%) ± sem stickiness (%) ± sem polar deviation (%) ± sem aberrant cells (%) ± sem mnc (%) ± sem negative control tap water 0.04±0.02 4.00±0.34 methotrexate 0.10 2.34±1.23 a 21.34±5.38 a 10.55±2.28 a 45.13±4.22 a 2.28±0.86 a zea mays husk extract 2.5 0.02±0.01a 2.39±1.06a 22.14±1.45a 1.35±0.14a 5.0 5.04±0.36a 30.27±2.45a 1.05±0.01a 10.0 18.15±3.18 a 43.44±3.75a 0.20±0.02a saccharum officinarum leaf extract 2.5 2.01±0.92 a 21.48±4.28a 0.06±0.01a 5.0 1.22±0.40a 3.12±1.34a 32.19±2.10a 2.29±0.35a 10.0 14.23±2.15 a 37.12±3.11a 1.14±0.31a values are expressed as mean ±sem (n=5). significant at p<0.05 when compared to negative control, sem: standard error of mean. figure 1. photomicrograph showing the mitotic and chromosomal aberrations of allium cepa root meristem cells after zea mays husk and saccharum officinarum leaf extract treatments under light microscope x40 magnification. arrows indicate (a) bridge and nuclear damage, (b) chromosomal fragmentation, binucleated cells and nuclear damage, (c) nuclear and cell wall damage, (d) nuclear and membrane damage, (e) binucleated cells and apoptotic bodies, (f) membrane and nuclear damage, (g) dead cells, (h) sticky metaphase and binucleated cells, (i) nuclear damage and fragmentation. ihg fed cba 536 biology, medicine, & natural product chemistry 13 (2), 2024: 531-540 discussion in this study, the toxic effects of zea mays husk and saccharum officinarum leaf extracts were evaluated by analyzing root growth and root morphology of allium cepa. various concentrations of the extracts employed in the study were observed to cause inhibition of root growth, and these inhibitions were statistically significant when compared to control group. in addition, the two extracts caused colour changes in the root tips of allium cepa depending on the concentration. this colouration ranged from yellowish light brown to dark brown colouration of the roots. cytoand genotoxicity were assessed by observing cytological parameters such as the mitotic index and number of chromosome abnormalities, including chromosome breaks, stickiness, and polar deviations. the mitotic index (mi) of a. cepa meristematic cells treated with methotrexate (0.1 mg/ml) decreased significantly when compared to control. also, significant inhibition in the onion roots treated with the z. mays husk resulted in mitotic indices of 37.60%, 28.20% and 17.20% for 2.5, 5.0 and 10.0 mg/ml, respectively. in comparison, s. officinarum leaf extract had mitotic indices of 44.80%, 25.20% and 19.20%, respectively, for 2.5, 5.0 and 10.0 mg/ml compared to the negative control (table 2). the inhibition of root growth was found to be dependent on the decrease in the mitotic index. the decline of the mitotic index below 22% in comparison to negative control can have lethal impact on the organism (antonsie-wiez, 1990), while a decrease below 50% usually has sublethal effects (panda & sahu, 1985) and is called cytotoxic limit value (sharma, 1983). the mitotic index measures the proportion of cells in the m-phase of the cell cycle and its inhibition could be interpreted as cellular death or a delay in the cell proliferation kinetics (rajas et al., 2001). reduction in the mitotic activity could be due to inhibition of dna synthesis or a blocking in the g2 phase of the cell cycle, preventing the cell from entering mitosis (sudhakar et al., 2001). mitodepressive effects of some herbal extracts, including the ability to block the synthesis of dna and nucleus proteins, were reported earlier (mercykutty & stephen, 1980; schulze & kirschner, 1986). several other herbal extracts have been reported to inhibit mitosis (as et al., 2006; as et al., 2007; akinboro & bakare, 2007). the decreased mitotic indices in a. cepa roots treated with z. mays husk and s. officinarum leaf extract were probably due to either disturbances in the cell cycle or chromatin dysfunction induced by extracts-dna interactions. the results of this study suggest that the tested extracts concentrations have inhibitory, mito-depressive effects on root growth and cell division of a. cepa and it can prevent dna synthesis and the reduction in the number of dividing cells in roots produced by the cytotoxic effects of compounds found in the extracts. the observation of sticky metaphase demonstrated the toxic effect of the extracts and this was common in all groups treated with the two extracts. metaphases with sticky chromosome lose their normal appearance, and they are seen with a sticky “surface,” causing chromosomes agglomeration (babich et al., 1997). stickiness has been attributed to the effect of pollutants and chemical compounds on the physicalchemical properties of dna, protein or both, on the formation of complexes with phosphate groups in dna, on dna condensation or formation of interand intra chromatid cross links (gömürgen, 2005; türkoğlu, 2007). chromosomal aberrations (ca) are changes in chromosome structure resulting from a break or exchange of chromosomal material. most of the ca observed in cells are lethal, but many related aberrations are viable and can cause genetic effects, either somatic or inherited (swierenga et al., 1991). the presence of chromosome fragments is an indication of chromosome breaks and can be a consequence of anaphase/telophase bridges (sharma & sen, 2002). fragments were observed in this study especially in the treated groups of s. officinarum leaf. these extracts used were found to not only interfere with the cell cycle, but also affect chromatin organization or dna replication, causing chromosome breaks. polar deviation was not observed in the study. frequencies of total chromosome aberrations increased significantly following exposure to the extracts which indicated clastogenic activity (table 3). these were more frequent aberrations in the groups treated with the z. mays husk extract. the two extracts significantly induced the formation of mnc in a. cepa root cells at 2.5–10 mg/ml concentrations. frequencies of mnc were found to be higher in the groups treated with 5.0 and 10.0 mg/ml of s. officinarum leaf extract. however, mnc frequency decreased in a. cepa roots treated at the highest concentration of the extracts (10 mg/ml) due to high cytotoxicity. the frequency of cells with micronuclei is a good indicator of the cytogenetic effects of tested chemicals. micronuclei (mn) often result from the acentric fragments or lagging chromosomes that fail to incorporate into the daughter nuclei during telophase of the mitotic cells and can cause cellular death due to the deletion of primary genes (albertini et al., 2000; krishna & hayashi, 2000). previous studies have suggested the mnc-induced effect of various plant extracts such as lavandula stoechas and ecballium elaterium (as et al., 2007; as et al., 2009), azadirachta indica (soliman, 2001) psychotria species (akinboro & bakare, 2007). in this study, membrane damage cells were observed in groups treated with various concentrations of the extracts 2.5, 5.0 and 10.0 mg/ml but mostly in the z. mays husk extract-treated groups. these results show that the extracts over certain concentrations may cause cytotoxicity as they cause membrane damage. these results suggest the cytotoxic potentials of the two extracts, especially z. mays husk. multinucleated and binucleated cells have been observed in extracts-treated groups. this is due to the prevention of cytokinesis or akpan et al. – evaluation of genotoxic and cytotoxic potentials of zea mays husk and sugarcane leaves 537 cell plate formation. microtubules have been implicated in cell plate formation and the extraction process, resulting in the inhibition of cytokinesis. a ghost cell is a dead cell in which the outline remains visible but whose nucleus and cytoplasmic structures are not stainable (as et al., 2009). some ghost cells were observed in various frequencies in this study in the two extracts but mostly in z. mays husk extract (10 mg/ml) treated groups (figure 1). this could have resulted from the activities of the phytochemical constituents of the extracts leading to nucleus damage and prevention of cytoplasmic structures, thus resulting in ghost cells. in addition, the extracts also induced dna damage cell death and apoptosis in various frequencies in this study. in this study, high concentrations (5 mg/ml and 10 mg/ml) of the extracts were found to cause the induction of cell death and apoptosis. cell death is a basic biological process of living organisms. cell death is induced by high concentrations of such toxins, stress, heavy metals, chemicals and others. the results of this study show that the extracts of z. mays husk and s. officinarum can induce cytogenetic alterations (cytoplasmic shrinkage, nuclear condensation, dna fragmentation, membrane blebbing, cytoskeleton alterations and appearance of apoptotic bodies) and cell death in root tips of a. cepa (figures 1(a), 1(b), 1(c), and 1(d)), suggesting cytotoxic and genotoxic activities of the extracts. eight phenolic compounds (gallic acid, protocatechuic acid, chlorogenic acid, cafeic acid, femlic acid, rutin, resveratrol, and kaempferol) have also been detected in ethanol extract of z. mays husk (dong et al., 2014). coutinho et al. (2016) identified some flavones and phenolics as well as their derivatives, from the leaves of s. officinarum. other phenolic, compounds such as caffeic acid, cis-p-hydroxycinnamic acid, quercetin, apigenin, albanin a, australone a, moracin m, and 5'geranyl-5,7,2',4'-tetrahydroxyflavone have been identified from the leaves (xu et al.,2018). the high phenolic and flavonoid contents of the two extracts may have been responsible for the observed effects in this study. flavonoids such as quercetin have been reported to demonstrate mutagenic and genotoxic potentials in various studies (ping et al., 2017). the high phenols and flavonoid contents in the extracts must have contributed to the observed cytotoxic and genotoxic activities in this study. conclusions this study revealed that zea mays husk and saccharum officinarum leaf extracts possess cytotoxic and genotoxic effects, as seen in the effects elicited by all test concentrations of the two plant extracts on the root number, root length, and root morphology of the allium cepa meristems after exposure. the degree of chromosomal aberrations (based on increasing extract concentration), the inhibition of cellular mitotic processes, and the general abnormalities observed in all root bulbs treated with test samples further indicate cytotoxic potentials of z. mays husk and s. officinarum leaf extracts. acknowledgements: the authors are grateful to mr. nsikan malachy and other staff at the animal house of pharmacology and toxicology department, university of uyo, for providing technical assistance. authors’ contributions: eea, moa, jeo and noe conceived and designed this study. iij, joe and eea carried out the experiments and drafted the manuscript. iij and moa performed the statistical analysis, edited and reviewed the manuscript. noe, moa, and joe have read and approved the final manuscript. competing interests: the authors declare that there are no competing interests. funding: none references abo, k. a., fred-jaiyesimi, a. a., & jaiyesimi, a. e. 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(2014). anti-cancer activities of ω-6 polyunsaturated fatty acids. biomedical journal, 37(3), 112– 119. https://doi.org/10.4103/2319-4170.131378. https://doi.org/10.1016/j.mrgentox.2006.07.006 http://dx.doi.org/10.26479/2019.0505.08 http://dx.doi.org/10.17533/udea.iatreia.v31n4a02 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 725-729 | doi: 10.14421/biomedich.2025.142.725-729 issn 2540-9328 (online) sensitive and selective chromatographic method to determine vanadium in commercial phosphoric acid hassan pyar1,2*, abdul rahman wahoud3 1faculty of environmental science and marine biology, hadhramout university, yemen. 2college of medicine and health science, seiyun university, yemen. 3chemistry department, faculty of science, hadhramout university, yemen. corresponding author* hassanpyar@gmail.com manuscript received: 27 april, 2025. revision accepted: 07 august, 2025. published: 01 october, 2025. abstract the spectrophotometric method based on n-benzoyl-n-phenylhydroxylamine was executed for ascertaining vanadium in environmental samples. high performance liquid chromatography (hplc) system equipped with a c18 rp column was used to determine vanadium in wet phosphoric acid and phosphate samples. a rapid pre-column response of vanadium with 5-br-padap was used without prior separation or adding any masking agents. the results showed that, the chelates were detected after being injected through the column by uvvis detector at wavelength 585 nm. the molar absorptivity is 5.5 x104 and the retention time is 6.3 minutes. the sensitivity of this reagent is equal to twice as high as the use of (par) reagent. the rsd was 1.7% for weak concentrations to 0.22% for high concentrations and the relative error (re) was between 3.21% and 1.07%. in conclusion the method of the currently study was found highly sensitive and selective and the results show a good accuracy and precision. keywords: hplc system; phosphoric acid; spectrophotometric method; vanadium. introduction the extraction-spectrophotometric method is utilized with n-benzoyl-n-phenylhydroxylamine was adopted to evaluate vanadium in environmental samples (agarwal et al., 1990). methods based on using of 4-(2-pyridylazo) resorcinol (par) which was generally was applied to determine vanadium in biological samples (bag et al.,1982; agnihotri et al., 1999), sewage (buchberger, 2000), silicate rocks (chakrabarti,1995), petroleum (fasanmade, 1994) and titanium alloys (fukasawa et al.,1981; filik et al., 2008). vanadium traces in aluminum samples could be determined with a good accuracy. vanadium was first isolated by organic solvent extraction and then, the absorption of the complex was measured at wavelength 585 nm (geană, et al., 2019). many methods that utilize vanadium chelating complexes have been used to determine vanadium such as pyrogallol for soils (george et al., 1990), hydroxamic acid for steel and rocks (guangyu et al., 2005; gustafsson et al., 2019), crown hydroxamic acid for body fluids (iki et al., 2001) phenylfluorone for steel alloys (jamaluddin et al., 1999), 1,5-diphenylcarbazide for environmental samples and 6chloro-3-hydroxy-7-methyl-2-(2-thienyl)-4h-chromen-41 for flue dust and waste water samples (krasiejko et al., 1986; kubinyi, 1995). these extractionspectrophotometric methods with different chelating agents were accurate, but they lack selectivity and need prior extraction and separation or masking agent which must be added. liquid chromatography has an important role in recent years due its use for separation and analysis of various metals such as mn, cd, co, ni, zn, and pb using post-column reaction and uv-v detector at different wavelengths, but they did not mention vanadium in their applications (matsumiya et al., 2004; michalski, 2018). sulfonylcalix[4]arenetetrasulfonate was used and checked for ultra-trace determination of different cations in river and tap water samples by hplc with uv-vis detector in an acetic buffer solution (ph4.7) (pyrzyńska, 2005; srivastav et al.,1996). reversed phase liquid chromatography was developed to simultaneously determine of vanadium, copper and cobalt founding in electroplating baths (vale et al., 2007). this paper purposes a developed method to determine vanadium in phosphate industrial connecting parts by high performance liquid chromatography (hplc) using the rapid reaction of vanadium with 5-br-padap as a chelating agent in the pre-column system without prior separation or adding masking agents. https://doi.org/10.14421/biomedich.2025.142.725-729 mailto:hassanpyar@gmail.com 726 biology, medicine, & natural product chemistry 14 (2), 2025: 725-729 materials and methods commercial wet phosphoric acid samples with 27% wt p2o5 and 1.26 g/cm3 and other samples were supplied by the general fertilizer company (gfc) homs /syria. methanol, acetonitrile, absolute ethanol and 2-(5-bromo2-pyridylazo)-5-diethylaminophenol (5-br-padap) hplc-grade, his formula as in fig. 1. all other chemicals employed were of analytical grade. figure 1. the structural formula of 5-br-padap. sample preparation 1 ml of 0.1m sodium acetate, 1 ml of 1% sodium dodecyl sulfate, 1 ml of 6 mm of (5-br-padap) and three drops of 1% hydrogen peroxide were added to the sample and standard solutions. procedures and apparatus a hplc system connected to the c18 rp column from shimadzu/japan was used. the eluent solution consists of methanol, acetonitrile and water with a ratio of 84:12:4 respectively adjusted to ph=5.5 and degassed before use. as a comparison method the samples were diluted and analyzed by atomic absorption spectroscopy (aas / varian). the relative standard deviation (rsd%) and relative error (re%) calculated as below: 𝑅𝑆𝐷% = (100 ∗ 𝑆𝐷)/ 𝑋 𝑅𝐸% = (𝑋 − 𝑋0) ∗ 100/ 𝑋0 where: sd : standard deviation. x : mean value. x0 : reference value. results and discussion standard calibration curve: to create standard calibration curve standard solutions, from one to 25 ppm in 40% pure phosphoric acid were pretreated as described in the experimental and injected through the column. the results are plotted in fig. 2. in the form of the area versus concentration. the relationship is represented by straight line over studied range of concentration. the correlation factor is r= 0.9998. the correlation is represented by straight line equation: area =188290 c + 3e+06 figure 2. standard calibration curve. to determine the relative standard deviation (rsd), four samples of 40% pure phosphoric acid with predetermined concentration of vanadium (102550 100 ppm) were taken. each sample was measured five times. the results are presented in table 1. the rsd was of the order of 1.7 % for weak concentrations to 0.22% for high concentrations. the relative error (re) was between 3.21% and 1.07%. this indicates that it is possible to use the standard calibration curve to determine vanadium concentration in phosphate derivative samples with an acceptable error. this result was similar to filik et al. (2008). y = 188290x + 3e+06 r² = 0,9998 area = 188290 c + 3e+06 r² = 0.9998 a re a c ppm pyar & wahoud – sensitive and selective chromatographic method to determine vanadium … 727 table 1. evaluations of relative standard deviation. reference value [v] ppm measured value [v] ppm x  sd (ppm) re (%) rsd (%) 10 9.78 10.43 10.51 9.88 10.25 10.17  0.33 1.7 3.21 25 25.67 25.54 24.77 25.89 24.23 25.22  0.69 0.88 2.76 50 49.21 50.76 49.51 50.93 50.34 50.15  0.76 0.3 1.52 100 100.98 99.21 100.76 98.91 101.25 100.23  1.08 0.22 1.07 the samples were also analyzed using the atomic absorption spectroscopy method for comparison. the results are presented in table 2 showing a close approximation between the results of both methods. these results were in agreement with others (krasiejko et al., 1986; chakrabarti, 1995; iki et al., 2001). an attempt was made to inject samples containing metals such as zn, cd, cr, fe, cu, mn, co, and u without vanadium (pyrzyńska, 2005; wen-yan et al., 2018). no peaks were obtained for chelates for these metals with 5br-padap because the chelates were unstable in the chromatographic system used in this study and they were decomposed in the mobile phase (zaki, et al.,1991). table 2: vanadium concentrations in phosphate industrial derivatives. sample hplc value (ppm) spectrometric value (ppm) strong green phosphoric acid 123.7 125 weak green phosphoric acid 86.5 88.2 phosphate 110.8 111.5 determination of vanadium in phosphate industrial derivatives phosphate and green phosphoric acid samples were pretreated as described in the experiment and injected through the column. a typical chromatogram for these samples has two sharp and resolved peaks (fig. 3). this result was in the same line with zhao and fu (zhao et al., 1991). the first peak showed at retention time of 6.3 minutes which indicates to vanadium chelate and another peak appeared at retention time of 10.3 minutes which indicates 5-br-padap reagent (zhao et al.,1991). figure 3. typical sample chromatogram. 728 biology, medicine, & natural product chemistry 14 (2), 2025: 725-729 conclusions the proposal method was applied for a wide range of concentrations of phosphate industrial derivatives and different aqueous media without any need to use masking agent or prior separation of metal ions. the method doesn't consume rather expensive post column reagents like the other method but the sample needs only simple pretreatment. clearly, proposed method is practical and selective for vanadium, and the sensitivity is double as high as in the use of (par) reagents. the results show good accuracy and high precision. acknowledgment: the authors are indebted to hadhramout university for their valuable help, also would like to express their thanks and appreciation to general fertilizer company (gfc) for supplying catalyst samples. competing interests: all authors state that there are no competing interests. references agarwal c., m. deb & mishra, r. )1990). sensitive spectrophotometric determination of vanadium in environmental samples at ppb levels. anal. lett., 23: 20632075. doi: 10.1080/00032719008052550. agnihotri, n., r. das & mehta, j. 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(2007). determination of vanadium in petroleum and petroleum products using atomic spectrometric techniques. talanta,72: 349-359. doi: 10.1016/j.talanta.2006.12.015. wen-yan, h., w. kun-peng & jin-yan, y. (2018). spectrophotometric methods for determination of vanadium: a review. toxicological & environmental chemistry, 100:1, 2031, doi: 10.1080/02772248.2018.1428325 https://doi.org/10.2116/analsci.15.1261 https://doi.org/10.1007/bf01196823 https://doi.org/10.1016/0165-9936(95)91370-8 pyar & wahoud – sensitive and selective chromatographic method to determine vanadium … 729 zaki, m., s. abuo-zeid & tarek, m. (1991). determination of vanadium in steels using phenylfluorone and cetylpyridinium bromide, journal of chemical technology & biotechnology. 51: 507-513. doi: 10.1002/jctb.280510408 zhao, y. & fu, c. (1991). liquid chromatographic behaviour of chelates of vanadium (v), copper (ii), cobalt (iii) and chromium (iii) with 2-(3,5-dibromo-2-pyridylazo) diethylaminophenol. analyst, 116: 621. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 449-458 | doi: 10.14421/biomedich.2024.132.449-458 issn 2540-9328 (online) antibacterial activity of sweet orange (citrus sinensis) peel tea against enterobacteriaceae isolated from a water depot yumiko1, suhartomi2, sri wahyuni nasution3,*, siti syarifah4, ade pryta r. simaremare5 1medical student programme, faculty of medicine, dentistry, and health science, universitas prima indonesia, medan, sumatera utara, indonesia. 2departement of pharmacology, faculty of medicine, dentistry, and health science, universitas prima indonesia, medan, sumatera utara, indonesia. 3departement of tropical medicine, faculty of medicine, dentistry, and health science, universitas prima indonesia, medan, sumatera utara, indonesia. 4departement of pharmacology and therapeutic, faculty of medicine, universitas sumatera utara, medan, sumatera utara, indonesia. 5departement of microbiology, faculty of medicine, universitas hkbp nommensen, medan, sumatera utara, indonesia. corresponding author* sriwahyuninasution@unprimdn.ac.id manuscript received: 22 agustus, 2024. revision accepted: 10 october, 2024. published: 17 october, 2024. abstract most orange peels are not utilized and become useless waste that is thrown away. orange peels contain many nutrients and compounds, such as flavonoids, terpenoids, alkaloids, and essential oils, which are secondary metabolites that act as antibacterial agents. this study intends to investigate the antibacterial activity of sweet orange peel tea against isolated enterobacteriaceae from a random water depot sample around universitas prima indonesia. this experiment used disc diffusion for antibacterial assay. meanwhile, the sweet orange peel was brewed using two different methods, including infusion and decoction, in two different masses (3 grams and 5 grams). these sweet orange peel tea formulations were compared to standard (chloramphenicol) and control (distilled water). enterobacteriaceae was isolated and identified from a random water depot sample around universitas prima indonesia, which included colony identification in emb agar and macconkey agar, gram staining, and biochemical test. this study showed that a random water depot sample contaminated by enterobacteriaceae had properties similar to citrobacter sp. sweet orange tea formulation inhibited this isolated enterobacteriaceae growth (p-value: 0.010). antibacterial activity was observed in 3-gram infusion, 5-gram infusion, and 5-gram decoction. however, the antibacterial activity was not better than the standard (chloramphenicol). overall, it can be concluded that the sweet orange peel tea as infusion or decoction has weak antibacterial activity against enterobacteriaceae bacteria isolated from water depots, which had some properties similar to citrobacter sp. keywords: sweet orange; decoction; enterobacteriaceae; citrobacter; infusion. introduction infectious disease is a common health burden in some developing countries, primarily caused by bacteria. the agents can cause infectious diseases, including bacteria, viruses, fungi, and parasites. world health organization (who) reported in 2006 an increase in mortality in some asean countries, which increased to 45%, and in the same year, who also reported that the mortality rate of infectious disease was 25 million worldwide. indonesia, as part of the asean country, is the second-rank country with the most infectious diseases and has a mortality rate of 29.5%. both agent factors and environment and host play an important role in infectious disease, significantly when human immunity weakens (damanik et al., 2021). enterobacteriaceae is a negative-gram bacteria with a rod shape. the bacteria group of enterobacteriaceae acts as resident flora in the human and animal gastrointestinal (riedel et al., 2019). enterobacteriaceae are one of the groups of bacteria that can cause diarrhea and often contaminate food (parawidnyaningsih et al., 2023) and drinks that have been cooked, frozen or not cooked and frozen (diah et al., 2022). there are several types of pathogenic bacterial of the enterobacteriaceae group, such as enterobacter, serratia, escherichia, proteus, salmonella, shigella, klebsiella (diah et al., 2022), and citrobacter (fadli et al., 2021). in general, the cause of bleeding due to diarrheal infections is toxins produced by escherichia coli, campylobacter spp., salmonella spp., shigella spp., toxin-producing vibrio cholerae, yersinia enterocolitica, and citrobacter spp. (imran et al., 2020). escherichia coli bacteria can quickly spread through water contamination and contaminated materials that come in contact with these bacteria (hamidah et al., 2019). escherichia coli is a gram-negative bacterium. it can be found in the intestine, indicating water contamination by fecal matter (klau et al., 2021). diarrhea is a defecation frequency of more than three times a day, followed by loose fecal consistency with or without blood and mucus (nurlaila & susilawati, 2022). https://doi.org/10.14421/biomedich.2024.132.449-458 450 biology, medicine, & natural product chemistry 13 (2), 2024: 449-458 diarrhea is a major health problem in developing countries, such as indonesia. according to the who (2000), there were around 4 billion cases of diarrhea in the world, and 2.2 million people died, mostly children under five years of age. mostly, indonesian regions from 2009 until now found that the incidence rate of diarrhea remains high, with around 162 thousand toddler deaths every year and approximately 460 thousand toddler deaths every day due to diarrhea (novita, 2020). due to this information, an antibiotic is essential in gastrointestinal infection treatment, mainly caused by bacteria (niken et al., 2023). however, irrational antibiotic use is still high, leading to bacterial resistance that decreases bacterial sensitivity (damanik et al., 2021). thus, looking for natural products with minimal side effects and preventing further bacterial resistance becomes important. many herbs have been widely used in community settings as an alternative treatment for various diseases, especially diarrhea and other gastrointestinal infections (salsabilla et al., 2023). indonesia is a tropical country with various plants that could become herbid. these herbs have fewer side effects and many health benefits. one of these plants, which has been reported to have various health benefits, is orange fruit. the sweet orange fruit has been used for a long time in the community, especially in indonesia. however, most people utilize only the fruit flesh, and the orange peel is wasted as household waste (filbert et al., 2023; mutia & manalu, 2020; niken et al., 2023). many nutrients and compounds, such as flavonoids, terpenoids, alkaloids, and essential oils, enrich the orange peel. these compounds potentially act as antibacterial agents that can inhibit bacterial growth and kill pathogenic bacteria by disrupting bacterial metabolism (amiliah et al., 2021). based on the research by amiliah et al. (2021), the crude extract and essential oil of kalamansi orange peel moderately inhibited the growth of staphylococcus aureus and escherichia coli at a concentration of 40%. amiliah et al. also reported that the essential oil of kalamansi orange peel also reported to strongly inhibited the growth of staphylococcus aureus and moderately inhibited the growth of escherichia coli bacteria at a concentration of 20% (amiliah et al., 2021). another study by niken (2023) also reported whether the antibacterial activity of orange peel extract positively correlated to its concentration. twenty percent and a hundred of orange peel extract could inhibit the growth of staphylococcus aureus, forming an inhibition zone diameter of 12.33 mm and 21.4 mm, respectively (niken et al., 2023). based on the description above, looking for the antibacterial activity of orange peel as another pharmaceutical form against enterobacteriaceae becomes important when the rate of water-borne gastrointestinal infection caused by bacteria is high. thus, this study intends to investigate the antibacterial activity of sweet orange peels (citrus sinensis) against enterobacteriaceae isolated from a random water depot. materials and methods study design this experimental study was performed at laboratorium terpadu, universitas prima indonesia, from april 2024 to june 2024. this study has also been approved by komite etik penelitian kesehatan (kepk) universitas prima indonesia with letter no. 016/kepk/unpri/iii/2024. materials this experimental study used some material including sweet orange peel, water sample from a random depot, chloramphenicol (paper disc antibiotic), mueller hinton agar (mha) media, nutrient agar media, emba (eosin methylene blue agar), macconkey agar, distilled water, methanol solution, normal saline, 70% alcohol, iodine solution, crystal violet, safranin, acetone alcohol, lactose broth, naoh, hcl, fecl3, mayer reagent, dragendroff reagent, chloroform, anhydrous acetate acid, sulfur acid, fecl3 5% isolation and identification of enterobacteriaceae from water depot sample initially, this study was performed by isolating and identifying enterobacteriaceae in a water sample from a random depot around the faculty of medicine, dentistry, and health science, universitas prima indonesia. after that, it was continued to evaluate the pharmaceutical formulation and antibacterial activity of sweet orange peel tea against the obtained enterobacteriaceae. initially, this study selected randomized depot water to obtain a water sample and reserved it into a sealed bottle. before the isolation and identification of enterobacteriaceae in a water sample, this study prepared lactose broth, emb agar, and macconkey agar. lactose broth was made by dissolving 1.3 grams of lactose broth powder into ten milliliters of distilled water using a hot plate magnetic stirrer, and it was used for enrichment and serial dilution purposes. emb agar was made by dissolving 1.6 grams of emb agar into a hundred milliliters of distilled water using a hot plate magnetic stirrer, and it was used to subculture the bacterial-containing broth. macconkey agar was made by dissolving 5.0 grams of macconkey agar powder into a hundred milliliters of distilled water, and it was used to purify the colony from subculture media. both agar and broth media were sterilized by autoclave 0.5 atm and 121oc for 15 minutes (n. oktaviani et al., 2022; toruan et al., 2023). isolation was begun by sample enrichment. enrichment was performed by dissolving a milliliter of water sample into ten milliliters of lactose broth and yumiko et al. – antibacterial activity of sweet orange (citrus sinensis) peel tea 451 incubating it at 43oc for 24 hours (yanestria et al., 2022). after that, a milliliter of incubated lactose broth was subcultured into emb agar by spreader and incubated at 37oc for 20-24 hours. then, the dominant colony was purified into macconkey agar several times until it obtained the homogenous colony in macconkey agar by the four-quadrant streak method (diniarti et al., 2022; yanestria et al., 2020). the isolated enterobacteriaceae from macconkey agar was identified by biochemical assay. the imvic test was used for the biochemical identification of enterobacteriaceae, including indole, methyl red, voges proskauer, citrate, and motility. imvic test was performed using some reaction tubes. indole test indole test was performed by inoculating the colony into sulfide indole motility (sim) media and incubating it for 24 hours at 37oc. after that, an erlich reagent was added to the media to identify color change (gunawan et al., 2022). mr-vp test mr-vp test was performed using the same media in two different reaction tubes, which was mr-vp media in a similar way to the indole test. after the incubation, it was dropped by methyl red for the mr test and both alphanaphthol and 40% koh solution for the vp test (sari et al., 2019) citrate test the citrate test was performed by inoculating the colony into simmons citrate medium, similar to other biochemical tests, and identifying the color changes after the incubation (sari et al., 2019). motility test the motility test was performed similarly to the indole test by inoculating the colony within 1 cm of the bottom of the tube and incubating it for 24 hours at 37oc. after that, the bacteria’s motion was identified around the inoculation line (sari et al., 2019). after the isolation and identification, an enterobacteriaceae-containing macconkey was reserved for further antibacterial assay. sweet orange peel tea formulation this study then continued to formulate the orange peel tea. sweet oranges were obtained from a modern medan market and identified in herbarium medanense, faculty of mathematics and science, universitas sumatera utara. all obtained sweet oranges were sorted and peeled. orange peel was collected and washed. after that, it was dried with a food dehydrator at 70oc for 24 hours (acar et al., 2022). afterward, the dried orange peel was ground by a blender and sieved with mesh number 40. finally, this sieved orange simplicial was packed into 3-gram and 5-gram tea bags (novitriani et al., 2021). both 3-gram and 5-gram tea bags were processed using two different methods: infusion and decoction. an infusion was performed by brewing both tea bags with a hundred milliliters of distilled water for fifteen minutes at 90oc. meanwhile, a decoction was performed in the same way, but for a longer time: thirty minutes (chiuman et al., 2021). finally, both infusion and decoction of orange peel tea underwent a phytochemical screening to identify the phytochemicals, including flavonoid, tannin, alkaloid, steroid/triterpenoid, phenol, and saponin (gulo et al., 2021; suhartomi et al., 2020; sumartin et al., 2024). antibacterial assay this study continued to investigate the antibacterial activity from infusion and decoction of orange peel tea against enterobacteriaceae isolated from a random water depot by disc diffusion method. initially, this study prepared some media that would be used, including nutrient agar (na) and mueller hinton agar (mha). nutrient agar (na) was made by dissolving 2.8 grams of na powder into a hundred milliliters of distilled water using a hot plate magnetic stirrer. meanwhile, mha was made by dissolving 3.8 grams of a hundred distilled water using a hot plate magnetic stirrer. both media were sterilized by an autoclave at 121oc for 15 minutes. after sterilizing both media, they were poured into some petri dish and chilled. after these media became solid, all petri dishes were reserved in a refrigerator until these media were used (amiliah et al., 2021; damanik et al., 2021; mutia et al., 2021) enterobacteriaceae isolates in the reserved macconkey was rejuvenated in nutrient agar before being used in the disc diffusion method. an inoculum of enterobacteriaceae from macconkey agar were streaked by a quadrant streak method and incubated for 24 hours at 37oc (daud et al., 2023). after bacterial rejuvenation, it was continued to suspend an enterobacteriaceae colony from na into the standard saline solution and formed a bacterial suspension with a bacterial concentration equivalent to 0.5 mcfarland scale. an inoculum of enterobacteriaceae colony from na was suspended into ten milliliters of normal saline in the reaction tube and then homogenized by a vortex. bacterial suspension concentration was measured based on the turbidity of the solution by uv-vis spectrophotometer at a wavelength of 600 nm (amiliah et al., 2021; komara et al., 2022). bacterial suspension and mha media, prepared before, were used for disc diffusion. every petri dish that contains mha agar has a capacity of four test discs. enterobacteriaceae isolate was streaked into sterilized mha with a sterile cotton swab. after that, diffused discs that had been soaked in either infusion orange peel tea or decoction orange peel tea at any dose were placed on the surface of the mha agar. a similar treatment was 452 biology, medicine, & natural product chemistry 13 (2), 2024: 449-458 performed in the standard and control groups. standard and control groups used chloramphenicol-containing discs and distilled water-diffused discs, respectively. moreover, all these petri dishes were incubated at 37oc for 24 hours. after the incubation, the formed clear zone was measured as an inhibition zone diameter and expressed in millimeters by caliper (chiuman et al., 2023; damanik et al., 2021; jungjunan et al., 2023; utami et al., 2021). data analysis all data in this study was analyzed by ibm spss statistic 27. initially, the determination of orange fruit, phytochemical screening, and inhibition zone diameter were analyzed by descriptive statistics. after that, the diameter of the inhibition zone was analyzed using inferential statistics, kruskal-wallis. results and discussion this experimental laboratory study aimed to investigate the antibacterial activity of sweet orange peel tea against enterobacteriaceae, which was isolated from random water depots around universitas prima indonesia by the disc diffusion method. the sweet oranges were obtained from one of the modern markets in medan, north sumatra, formulated into tea, and brewed using two different methods: infusion and decoction. this study demonstrated the antibacterial activity from infusion and decoction of tea orange peels, except for the formulation of 3-gram orange peel tea brewed by infusion. initially, this study isolated and identification of enterobacteriaceae from a random water depot sample around universitas prima indonesia. this study used eosin methylene blue agar to isolate bacteria from water depots, and figure 1 presents the isolated enterobacteriaceae in emb agar. figure 1. isolate bacterial on eosin methylene blue (emb) agar. isolated bacteria on emb agar show metallic green sheen colonies and are suspected to be escherichia coli. this study used emb agar due to the selectivity and differentiation of this medium for the growth of enteric bacteria and coliform microorganisms. this media is rich in nutrients to support the growth of enterobacteriaceae, especially lactose fermenter bacteria (putri et al., 2023; salaila et al., 2024). furthermore, emb also has eosin, which inhibits the growth of positive-gram bacteria and enhances the growth of negative-gram bacteria, such as escherichia coli (akhnah et al., 2022). on the other hand, emb also has lactose as a substrate for lactose fermenter bacteria, then eosin and methylene blue in this media were used to differentiate either lactose or nonlactose fermenter bacteria by media color change (hendiana et al., 2022). a dark color with a shiny metal colony indicates a lactose fermenter bacteria. brown and pink color colonies indicate a slow lactose fermenter bacteria. finally, the faint red color colony indicated a non-lactose fermenter bacteria (akhnah et al., 2022; hendiana et al., 2022). this study showed that the isolated bacteria from a random water sample depot had a greenish metallic sheen color, indicating lactose fermenter bacteria. this color changes due to the products of some acid compounds from lactose fermentation. this acid compound reacted with methylene blue to form a black or dark color and, with a metallic green center, separate colonies with a diameter of 2-3mm (bollyn et al., 2023; yanestria et al., 2020). furthermore, bacteria with green sheen metallic colonies were purified on macconkey agar medium three times. the results of bacteria purification on macconkey agar can be seen in figure 2. figure 2. the purification of isolate bacteria on macconkey agar (a) first purification (b) second purification (c) third purification. figure 2 shows that purified bacteria in macconkey agar had pink-colored colonies, which indicates the ability to lactose the fermenter. macconkey was a differential media for either lactose fermenter or nonlactose fermenter bacteria. a pink color colony indicated a lactose fermenter bacteria due to the formation of some acid compounds as a result of a lactose fermentation reaction (ph < 6.8) (nursanty et al., 2019). this study showed that the colony was pink, indicating lactose fermenter bacteria. some enterobacteriaceae with lactose fermentation activity include escherichia coli, klebsiella sp., enterobacter aerogenes, and citrobacter sp (akhnah et al., 2022). there are some lactose fermenter enterobacteriaceae; therefore, additional tests are required to identify the type of enterobacteriaceae. due to this reason, the obtained colony underwent some biochemical tests (lisdewi et al., 2023). after yumiko et al. – antibacterial activity of sweet orange (citrus sinensis) peel tea 453 purification, a colony from each petri was stained with gram staining, as described in figure 3. figure 3. the results of gram staining under a microscope with 1000x magnification of enterobacteriaceae isolate bacteria. figure 3 shows that these colonies have negativegram staining bacteria, a rod red-staining bacterium. it indicated a rod-shaped enterobacteriaceae. isolated bacteria cannot retain crystal violet as a primer stain after being flushed by a decolorizing agent of alcohol or acetone. hence, this bacteria absorbs the counterstain, safranin (red color). a thin layer of negative-gram bacteria cell wall causes this bacteria to be unable to retain the primer color. hence, the lipid layer of the negative-gram bacteria cell wall was dissolved by the decolorizing agent (alcohol or acetone) during the gram staining process (rahmatullah et al., 2021; sari et al., 2020) gram-negative bacteria cell wall consists of phospholipids, lipopolysaccharides, and lipoproteins. these layers cause the impermeable property of bacteria cell walls for small molecules. it also causes an obstacle for the antibacterial substance to pass the negative-gram bacterial cell wall. another factor affecting the permeability of negative-gram bacteria was porins, which were part of the bacterial protein. due to these reasons, some large molecular sizes of antibacterial are challenging to penetrate the negative-gram bacteria and lead to more resistant antibiotic properties (samputri et al., 2020). according to emb agar, mac agar, and gram staining, the obtained isolated bacteria from a random water sample depot may be a lactose fermenter enterobacteriaceae, including escherichia coli, citrobacter sp., klebsiella sp., or enterobacter aerogenes. a biochemical test was required to identify the possible type of enterobacteriaceae (akhnah et al., 2022). thus, a colony from these macconkey agars underwent a biochemical test, including: imvic and motility tests were performed to identify enterobacteriaceae bacteria isolated from random water depots around universitas prima indonesia, and the result of the biochemical test is described in table 1. table 1. biochemical test results of enterobacteriaceae isolate bacteria. biochemical test reagent results indole test reagent erlich methyl red test methyl red indicator + (weak positive) voges-prosakeur test alpha-naphtol + koh 40% sitrat test media simmon citrate + motility test media sulfide indole motility (sim) + table 1 shows the colony’s cheerful methyl red, citrate, and motility tests. it can be concluded that isolated enterobacteriaceae could convert pyruvic into a stable acid compound (mr test) and utility citrate as a carbon source into oxaloacetic acid and acetic acid (citrate test). the methyl red test (mr test) is a biochemical test to determine the ability of bacteria to convert pyruvate from a glycolysis reaction into an acid compound. the methyl red test uses a broth media that consists of glucose as a substrate for glycolysis, and then the bacteria uses glucose for glycolysis and produces some pyruvate compounds. some bacteria may convert these pyruvate compounds into stable acid compounds. this acid compound was identified by the reaction of acid into methyl red reagent. hence, the positive result was identified as red, and the negative test result as yellow. this study showed that the isolated enterobacteriaceae from a random water depot sample had a red color from the methyl red test, which indicated a positive result. it showed that isolated enterobacteriaceae could convert pyruvate into a stable acid compound (lisdewi et al., 2023). this isolated bacteria from a random water depot also showed an ability to use citrate as a carbon source for oxaloacetic acid and acetic acid for energy formation. it can be seen from the simmon citrate test, which uses simmon citrate media. simmon citrate media contains some compounds, including citrate as a substrate for citrate enzyme, ammonium ion as a nitrogen source, and the last one is bromothymol blue (btb) as a ph indicator. some bacteria can utilize citrate as a carbon source, and it will break the citrate compound into oxaloacetic acid and acetic acid. the oxaloacetic acid will then be decarboxylated to produce pyruvate and carbon dioxide gas. after that, carbon dioxide gas will react with water molecules and excess sodium citrate in simmon citrate media to produce sodium carbonate that has alkaline properties. after that, the high release of carbon dioxide from the last reaction will induce the ammonium salt metabolism to produce ammonium hydroxide. both ammonium hydroxide and sodium 454 biology, medicine, & natural product chemistry 13 (2), 2024: 449-458 carbonate increase the ph in alkaline conditions. thus, it turns the btb as a ph indicator into a prussian blue. this study showed that the isolated enterobacteriaceae changed the slant agar into a prussian blue color that indicated a positive citrate test, and it showed that the isolate enterobacteriaceae used the citrate as a source of carbon and ammonium salts as the nitrogen source (zebua et al., 2020) (apriyanthi et al., 2022) (sari et al., 2019). the last biochemical test observed in this study was the motility test. isolated enterobacteriaceae from a random water depot sample was a motile bacteria. it can be seen from the growth pattern of bacteria in sim (sulfide-indole-motility) media, which spread out from the medium site. some enterobacteriaceae may have a propeller-like flagella or unique fibrils that produce a gliding form of motility. based on the macroscopic (emb agar and macconkey), microscopic (gram-staining), and biochemical tests (imvic and motility), isolated enterobacteriaceae from water depot sample preferred to citrobacter sp. some previous studies have reported the presence of various types of enterobacteriaceae from either source, such as fish, water, et cetera. initially, this study identified the isolated bacteria from the macroscopic appearance of colonies in emb and macconkey agar, which was in line with the result of kim et al. in 2015. kim et al. reported that both citrobacter braakii and escherichia coli on mca and levine’s eosin methylene blue agar (l-emb) had an identical enterobacteriaceae, which indicates that citrobacter braakii can ferment lactose similar to escherichia coli. due to this reason, it can be concluded that the probable isolated enterobacteriaceae from random water depot was either citrobacter sp. or escherichia coli (kim et al., 2015). the obtained enterobacteriaceae from a random water depot around universitas prima indonesia also showed some biochemical characteristics that could be used to investigate enterobacteriaceae type. warpala et al. (2015) reported that isolated citrobacter sp. showed a positive mr and citrate test in the biochemical test from isolated enterobacteriaceae from buyan lake’s water sample. citrobacter sp. and escherichia coli shared a positive mr test. both indole and citrate tests can be helpful in differentiating citrobacter sp. and escherichia coli. citrobacter sp. utilites citrate as a carbon source for energy formation, which is not found in escherichia coli and leads to a positive citrate test. when escherichia coli hydrolyzes tryptophan to form indole, it leads to a positive indole test. these previous studies showed a similar result to the current study, which reported that the isolated enterobacteriaceae from a random water depot sample around universitas prima indonesia was citrobacter sp. with positive mr test and citrate test results (warpala et al., 2019). moreover, fadli et al. (2021) also reported that the refill water depot sample in jambi has some enterobacteriaceae, including klebsiella sp. and citrobacter sp., which were identified from macroscopic, microscopic, and biochemical test characteristics (fadli et al., 2021). this study was then continued to investigate the antibacterial activity of sweet orange peel tea against the enterobacteriaceae isolated from the water depot sample. sweet orange fruit samples were obtained from one of the modern markets in medan, north sumatra. the sweet orange fruit was then identified, and its taxonomy was described below. kingdom : plantae divisi : spermatophyta class : dicotyledoneae ordo : sapindales family : rutaceae genus : citrus species : citrus sinensis (burm.) merr. local names : sweet orange phytochemical screening in this study was performed to determine the presence of secondary metabolites found in sweet orange (citrus sinensis) peel tea. in this study, the results of the phytochemical screening test on sweet orange peel tea were described in table 2 and table 3. table 2. screening phytochemical results of sweet orange peel tea infusion. secondary metabolites compounds reagent result flavonoid naoh + hcl + tannin fecl3 + alkaloid mayer dragendorff + saponin hcl steroid chlorofom + anhydrous acetate acid + sulfur acid triterpenoid chlorofom, anhydrous acetate acid, sulfur acid + phenol fecl3 5% + table 3 screening phytochemical results of sweet orange peel tea decoction. secondary metabolites compounds reagent result flavonoid naoh + hcl + tannin fecl3 + alkaloid mayer dragendorff + saponin hcl steroid chloroform + anhydrous acetate acid + sulfur acid triterpenoid chloroform + anhydrous acetate acid + sulfur acid + phenol fecl3 5% + yumiko et al. – antibacterial activity of sweet orange (citrus sinensis) peel tea 455 according to the phytochemicals screening result, sweet orange peel infusion or decoction has some phytochemicals, including flavonoids, tannins, alkaloids, triterpenoids, and phenol. then, it was continued to antibacterial assay by disc diffusion method, and the results of measuring the antibacterial activity of sweet orange peel tea against enterobacteriaceae bacteria isolates through the diameter of the bacterial inhibition zone observed for 24 hours. inhibition zone diameter data was initially analyzed using shapiro-wilk for data distribution, and the result of shapiro-wilk was described in table 4. table 4. the results of shapiro-wilk analysis for data distribution. group p-value distribution 3g infusion 0.720 normal 3g decoction 0.000 not normal 5g infusion 0.843 normal 5g decoction 0.949 normal standard (chloramphenicol) 0.092 normal control (distilled water) 0.000 not normal based on shapiro-wilk analysis, the inhibition zone diameter of 3 grams decoction (p-value: 0.000) and control (p-value: 0.000) has a non-normal data distribution. hence, it can be concluded that the data distribution of inhibition zone diameter was not expected. for this reason, this study compared the inhibition zone diameter by kruskal-wallis, which is described in table 5. table 5. comparison of inhibition zone diameter in all tea formulation group. tea formulation inhibition zone diameter (mm) mean std. deviation p-value 1st repetition 2nd repetition 3rd repetition 3g infusion 2.51 2.86 1.92 2.43 0.45 0.010 3g decoction 0.00 0.00 0.00 0.00 0.00 5g infusion 2.51 2.86 2.71 2.71 0.17 5g decoction 2.31 3.66 5.14 3.70 1.41 standard 26.71 26.81 28.56 27.36 1.04 control 0.00 0.00 0.00 0.00 0.00 it can be concluded that there was a significant difference in inhibition zone diameter among all tea formulation groups; it can be seen from p-value < 0.05 (p-value: 0.010). the highest mean was found in the standard group that received chloramphenicol, followed by 5 grams decoction, 5 grams infusion, 3 grams infusion, and 3 grams decoction. furthermore, this study analyzed inhibition zone diameter in each group using the mann-whitney test, and the analyses results are described in table 6. table 6. the results of the whitney test. group comparison p-value interpretation control 3g infusion 0.037* significant 3g decoction 1.00 not significant 5g infusion 0.037* significant 5g decoction 0.037* significant standard 0.037* significant standard 3g infusion 0.050* significant 3g decoction 0.037* significant 5g infusion 0.050* significant 5g decoction 0.050* significant 5g decoction 3g infusion 0.275 not significant 3g decoction 0.037* significant 5g infusion 0.513 not significant 5g infusion 3g infusion 0.500 not significant 3g decoction 0.037* significant 3g decoction 3g infusion 0.037* significant 456 biology, medicine, & natural product chemistry 13 (2), 2024: 449-458 table 6 showed that the control (distilled water) and the formulation of 3-gram orange peel decoction did not show any significant difference in inhibition zone diameter (p-value > 0.05). however, the inhibition zone diameter of distilled water significantly differed from other groups (3 grams orange peel infusion, 5 grams decoction and infusion, and standard group). moreover, the changes in orange peel simplicial mass in formulation significantly affected the antibacterial activity against the isolated enterobacteriaceae. however, the method of brewing did not affect the antibacterial activity. the antibacterial assay showed that either orange peel tea infusion or decoction had antibacterial activity against isolated enterobacteriaceae from a random water depot sample. it can be seen from the mean of the inhibition zone from 3-gram infusion, 5-gram infusion, and 5-gram decoction, which were 2.43 mm, 2.71 mm, and 3.70 mm. only 3-gram decoction did not show an antibacterial assay. this antibacterial activity of orange peel tea comes from phytochemicals, including flavonoid, tannin, alkaloid, triterpenoid and phenol compounds found in either infusion or decoction. these phytochemicals were in line with erika et al. (2023), who reported that sweet orange peel extract (citrus sinensis (l.)) contained flavonoids, saponins, tannins, and essential oils (e. d. oktaviani et al., 2023). these phytochemicals are reported to have both antibacterial and antioxidant activity. flavonoid was reported to disturb membrane cells, microsomes, and lysosomes by forming a complex compound with this membrane and leading to organelle efflux. when flavonoid binds to the bacterial wall’s outer membrane, it disturbs the bacterial wall’s stability, leads to cytoplasm leaks, inhibits bacterial growth, and ends in bacterial death. flavonoids are also reported to bind bacterial dna and disturb its structure. other phytochemicals such as tannin, saponin, and triterpenoid disturbed cell membrane stability. tannin can also bind to intracellular bacterial proteins and precipitate these proteins, which causes internal structure damage (niken et al., 2023) (amiliah et al., 2021)(egra et al., 2019). this study also compared the antibacterial activity of orange peel tea formulation to chloramphenicol as standard and distilled water as control. chloramphenicol is a broad-spectrum antibiotic for positive and negative gram bacteria (aviany & pujiyanto, 2020). chloramphenicol is an antibiotic that inhibits bacterial ribosomes and inhibits bacterial protein synthesis. tannin in orange peel tea showed a similar mechanism of action, precipitating intracellular bacterial protein. on the other hand, this study also used distilled water as a control for tea solvent, and it did not have any antibacterial effect due to the absence of an inhibition zone in the antibacterial assay (azizah & antarti, 2019). conclusions it can be concluded that sweet orange peel tea infusion and decoction had weak antibacterial activity against the enterobacteriaceae isolated from random water depot samples around universitas prima indonesia (p value: 0.010). these isolated enterobacteriaceae tended to be citrobacter sp. this weak antibacterial activity was shown by the mean of 3 gram infusion, 5 gram infusion and 5 gram decoction, which were 2.43 mm, 2.71 mm and 3.70 mm, respectively. acknowledgements: all the authors would like to thank all those who supported this research process. authors’ contributions: conceptualization: yumiko, suhartomi, and sri wahyuni; methodology: siti syarifah and ade pryta r. simaremare; investigation: yumiko, suhartomi, and sri wahyuni; discussion of results: yumiko and suhartomi; writing – original draft: yumiko and suhartomi; writing – review and editing: sri wahyuni, siti syarifah, and ade pryta r. simaremare; supervision: suhartomi and sri wahyuni; approval of the final text: suhartomi, siti syarifah, and sri wahyuni. competing interests: all authors declare no competing interests. funding: this study did not receive any funding. references acar, a., aydın, m., & arslan, d. 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(2020). molecular identification of proteolitic bacteria from mangrove sediment in dumai marine station. asian journal of aquatic sciences, 3(2), 179–188. https://doi.org/10.31258/ajoas.3.2.179-188 biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 25-30 | doi: 10.14421/biomedich.2014.31.25-30 identification of migratory birds and their spesific characteristics of habitat in the salt water lake of gili meno, north lombok distric diah purwitasari1, luh gde sri astiti1,2 and supriadi1* 1faculty of veterinary medicine, university of west nusa tenggara, mataram, indonesia 2research institute for agricultural technology west nusa tenggara, indonesia author correspondency*: supriadi@yahoo.com abstract the aim of this research was to identify the species of migratory birds in the ecosystem of salt water lake of gili meno and their specific characteristics of habitat. data collection for birds, mangrove and fish species has been carried out in september and october 2013. in this study, a shannon-wiener diversity index and importance value index (ivi) of mangrove vegetation were calculated to identify carrying capacity of mangrove population in the form of specific habitat in salt water lake of gili meno. this research has identified 17 species of birds which are divided into 5 families: scolopacidae, charadriidae, ardeidae, meropidae and alcedinidae. moreover, 3 species of mangrove were discovered that are a. marina, a. lanata and e. agallocha, as well as one species of fish (mujair fish/o. mossambicus). n. nycticorax and b. striatus were well-known to have higher population than other bird species. the diversity index showed that mangrove vegetation in the ecosystem of salt water lake of gili meno has a low species diversity (0.565). this is due to higher dominance of one species than the others. what is more, the ivi of a. marina demonstrated a fairly significant value compared to that of other species (189,01). mangrove vegetation which surrounds the ecosystem of salt water lake of gili meno has formed a unique habitat and and an ideal stopover site for migratory birds. the ecosystem not only provides shelters from predators but also supplies for various abundant feeding sources. the lake it self is rather shallow and muddy around the shore which gives advantages for the migratory birds to obtain plenty small fish from the lake. keywords: migratory birds, salt water lake, gili meno, characteristics of habitat introduction indonesia is the fifth country after colombia, peru, brazil and ecuador which have the highest megabiodiversity of birds, as many as 1539 species (sudaryanto, 2006). those species are divided into 20 order and 90 families (yamin and jamaluddin, 2002) and some of them are endemic (around 381 spesies). the increasing habitat degradation has led to the fact that nearly 400 species of birds are now threatened with extinction (thinh, 2006). one of bird groups that are interesting to study is migratory birds. this group is a bird group that move from one region to another, either inter-island or intercontinent, and will return to their origin (shackelford et al., 2005). this migration occurs because of seasonal change or the birds feeding habits in a relatively far distance. small islands in indonesia provide habitats for a variety of migratory bird species to stopover. habitat characteristic of various ecosystems in each island possesses its own specific and unique characteristics. often, these migratory birds can attract tourists visiting the islands (aeny, 2004). one of small islands (lombok: small island = gili) that exist around lombok island is gili meno. this small island is one of three islands included in gili matra village. gili meno is well-known for its specific and unique salt water lake (rachman, 2004) and has a pleasing panorama. moreover, its ecosystem also saves a great potential of various exotic animal species. research conducted by setiawan (2008) explained that no less than 11 species of migratory birds use the habitat to stopover in migration season. some of them are phalacocorax melanoleucos, actitis hypoleuco, ardea purpurea and charadrius dubius. to date, several researches have been carried out to identify many migratory bird spesies in the ecosystem of salt water lake of gili meno, one of them is by setiawan (2008). however, in the research, field observation was only performed once and the researcher has not done a study of microhabitat and factors that make the salt water lake as one of preference habitat for migratory birds. therefore, this research was conducted to identify the diversity of migratory bird species in the ecosystem of salt water lake of gili meno and recognize the habitat characteristics that could support the wild birds’ lives in the ecosystem. materials and methods the objects of this research were migratory birds utilizing the lake as their activity place. this research was undertaken in september and october 2013. the first data collection was performed on the 24th-26th of september and the second data collection was completed on the 18th20th of october 2013. data collected were bird species, mangrove vegetation and fish species. data collection of bird species was conducted on field in the morning from 07.00-10.00 and in the afternoon from 15.00-17.00. the observation was performed in 4 26 biology, medicine, & natural product chemistry 3 (1), 2014: 25-30 spots which were determined purposively on field. bird species were observed and identified by directly observing morphological features and characteristics of the birds based on a guide to the birds of wallacea (coates and bishop, 2000). morphological features observed included: (i) shape and size of body, beak and feet, (ii) the colours of feather on body, beak, and feet, (iii) visible specific features, as well as (iv) sounds produced. analysis of mangrove vegetation was performed on sampling spots that have been directly marked by a gps device. the vegetation analysis was undertaken by making 10 x 10 m quadrants. obtained vegetation data included vegetation species, trunk diameter, and the individual number of each species. furthermore, fish were caught utilizing a fish net in cooperation with local community. the vegetation data were then analysed by calculating density, frequency, dominance, ivi, and shannon-wiener diversity index (h’). results and discussions a total of 17 species belonging to 5 families have been identified in this research. the five bird families are scolopacidae, charadriidae, ardeidae, meropidae dan alcedinidae. of the 17 identified species, 16 species are migrants (table 1). the non-migrant bird and inhabitants of gili meno is the species of halcyon sancta. black-crowned nirgt-heron (n. nycticorax) is a species with the highest population identified. as stated by coates and bishop (2000), this species is a migratory species that reproduce in their migration sites. on several observation spots in the vicinity of mangrove forest, their active and already inactive nests were discovered. in addition to that, it was observed that striated heron (b, striatus) was also on fairly high population number. both bird species are known to prefer similar habitat which is mangrove area, muddy sites and shallow puddle (zulfan, 2009; setiawan 2008; mustari, 1992). these species difference is in their feeding activity. nycticorax nycticorax was more often spotted perched on tree branches and once in a while went down to catch small fish. meanwhile, b. striatus was more active and often walked around the lake to find preys. this research discovery is supported by previous research conducted by setiawan (2008) who also discovered both bird species in high individual number. it was further stated that mangrove ecosystem in salt water lake of gili meno could highly sustain the existence of the two species population. additionally, food availability in the vicinity of lake and the lake’s proximity to adjacent beach also provided more varied habitat choices for these birds. other fairly numerous migratory bird species discovered during this research were those belong to trinil category i.e: common sandpiper (actitis hypoleucos), wood sandpiper (tringa glareola), marsh sandpiper (tringa stagnatilis) and common redshank (tringa tonatus). detailed observation has successfully identified the four species clearly. detailed observation was necessary because occasionally, these four species were in large group (>10). the observation of these bird species was basically simple to perform due to significant differences in size and features of each species. nevertheless, a. hypoleucos and t. glareola were often found feeding in pairs or individual on the lake shore. figure 1. documented bird species. a. cerek jawa (charadrius javanicus chasen, 1938); b & c. cerek besar (pluvialis squatrola); d. trinil semak (tringa glareola linnaeus, 1758); e. kokokan laut (butoroides striatus); f. kuntul kecil (white) (egretta garzetta), trinil pantai/small brown (actitis hypoleucos). another migratory bird category is cerek including little ringed plover (charadrius dubius), javan plover (c. javanicus) and grey plover (pluvialis squatarola). two of the three species (c. javanicus and pluvialis squatarola ) have not been reported in the study conducted by setiawan (2008), this might be because of different observation time. according to coates and bishop (2000), both species are migratory birds and do not reproduce so that sometimes they are not observed in certain time. it was further explained that the three species prefer shallow and calm mangrove habitat, natural habitat and far from human activity. this result demonstrate that the thre migratory species presence can be utilized as habitat indicator that salt water lake of gili meno can support the lives of wild animals. mulyawati (2007) also reported that this bird group likes muddy mangrove area and shallow water. the next bird category observed was kuntul comprising 3 species: little egret (egretta garzetta), reef egret (e. sacra) and intermediate egret (e. intermedia). the population of e. garzetta was the diah purwitasari, et al. – identification of migratory birds and their spesific characteristics … 27 highest of the three species with the highest individual number observed on field was 16 individuals. this species occupied almost half of the lake’s shore around 08.00-09.00 and 15.00-17.00 when they are actively feeding. on the other hand, e. intermedia was only found 4 individuals at the most during the research. posture, larger body size and typical beak colors (bright yellow) appears to distinguish the two species. unlike e. garzetta and e. scara, e. intermedia often becoming active between 09.00-10.00 and sometimes it was not observed in the afternoon. meanwhile, e. sacra was rarely seen lingering around the lake. this may be due to food competition on the lake shore. the species was mostly spotted on the beach shore at low tide in the afternoon then returned to the mangrove ecosystem afterward. table 1. birds species discovered in salt water lake ecosystem of gili meno. family indonesia english latin scolopacidae trinil pantai common sandpiper actitis hypoleucos trinil semak wood sandpiper tringa glareola trinil kaki merah common redshank tringa tonatus trinil rawa marsh sandpiper tringa stagnatilis gajahan erasia eurasian curlew numenius arquata charadriidae cerek kalung kecil little ringed plover charadrius dubius cerek jawa javan plover charadrius javanicus cerek besar grey plover pluvialis squatarola ardeidae cangak merah purple heron ardea purpurea kuntul perak intermediate egret egretta intermedia kuntul kecil little egret egretta garzetta kuntul karang reef egret egretta sacra kowak malam abu black-crowned nirgt-heron nycticorax nycticorax kowak malam merah rufous nigtt-heron nyicticorax caledonicus kokokan laut striated heron butoroides striatus meropidae kirik-kirik laut blue-tailed bee-eater merops philipinnus alcedinidae cekakak suci sacred kingfisher halcyon sancta other bird species that were observed around the lake were sacred kingfisher (halcyon sancta) and bluetailed bee-eater (merops philippinus). sacred kingfisher often noticed on several spots on the lake shore to catch small fish. decreasing lake water has led to higher salinity causing small fish to surface, so that it was easier for the birds to catch their prey. occasionally, sacred kingfisher produced strong sounds when they flew or met the same species. in contrast, m. philippinus was more aereal. this species make use of mangrove habitat as feeding site which provide abundant insects. mangrove ecosystem is an integral part of the ecosystem of salt water lake of gili meno. this mangrove ecosystem provide excellent microhabitat for many wild animals, especially migratory birds. as stated by elfidasari and junardi (2006), one of many functions of mangrove is as nesting and stopover sites for various bird species. healthy mangrove ecosystem is more preferred and visited by many bird species as it provide huge potential as food sources. mangrove ecosystem in gili meno consist of three mangrove species: avicennia lanata, avicennia marina dan exoecaria agallocha. the calculation of species relative dominance showed that a. marina dominate the habitat in the ecosystem of the salt water lake (69.45%), followed by a. lanata (23.81%) and e. agallocha (6. 74%). moreover, relative species density index also illustrated a fairly dense coverage of a. marina. in contrast, species diversity index (0.565) demonstrated the low species diversity in the mangrove ecosystem because of a. marina dominance. in addition to plant resources, the habitat of salt water lake of gili meno also offer animal resource for migratory bird feeding. the mangrove ecosystem ability to maintain the stability and fluctuation of salinity and temperature causes fresh water species capable to adapt in the saltwater habitats. from the results of fishing using fishing nets and rods it was found that only one species of fish inhabit the whole habitat of the salt water lake. the identified species is mujair fish (oreochromis mossambicus). as indicated by kosztowny et al (2008), this fish species can adapt well in salt water from juvenile. this is becaue of its ability to activate atpase anzyme to maintain cell membrane activity, especially on the gill rackers to keep active despite the high salinity. further research to identify other animal species which is potential as feeding resource for migratory birds is of high importance. moreover, research on the histological and anatomic differences of mujair fish from salt water and fresh water is required. 28 biology, medicine, & natural product chemistry 3 (1), 2014: 25-30 figure 2. coverage shape and characteristics of mangrove a. avicennia marina with close coverage along the lake shore b. oval leaf tip shape with white lower leaf surface is the diagnostic character of a. marina. the calculation of importance value index (ivi) showed that the most important species was a. marina. this species was discovered almost in all ecosystem area around the salt water lake. this was supported by the calculated diversity index of shannon-wiener which showed the low diversity of mangrove vegetation in the ecosystem. in other words, the ecosystem is dominated by a. marina. the existence of mangrove vegetation in a habitat will form a spesific ecosystem and provide abundant resources. davies et al., (1996) stated that mangrove ecosystems have important roles in sustaining the lives of animals on coastal line and terestrial. mangrove vegetation in the ecosystem of salt water lake of gili meno also provide abundant feeding resource for migratory birds like small insects and fish. this is also supported by abdurakhman (2002) research who discovered that food availability in a habitat will be more frequently visited by animals (burung). yamin and jamaludin (2002) also explained that excellent vegetation will provide diverse resources to sustain bird lives in a habitat. dominant bird species observed in this study is blackcrowned nirgt-heron (n. nycticorax) and striated heron (b. striatus) which are migratory birds that reproduce in their migration sites (coates and bishop, 2000). both species made many nests on dense mangrove tree canopy. these species have successfully dominated the ecosystem of salt water lake of gili meno may because both species can obtain their suitable life requirements such as food, nesting place and shelter from predators. other migratory birds which belong to trinil category (a. hypoleucos and t. glareola) often spotted fly actively and walked along the shallow lake shore to find food. both species are different from other two species from the same group (t. stagnatilis and t. totanus). t. stagnatilis and t. totanus are relatively not very active and often observed in groups. the high availability of small fish and water insects on the surface of the lake make the lake habitat is mostly preferred by many migratory bird species. the lake ecosystem which almost 90% covered by mangrove vegetation contribute to the ideal condition of the habitat for migratory birds to stopover. the fairly dense mangrove vegetation which surround the salt water lake ecosystem has formed a characteristic and sustainable habitat for many wildlife including migratory birds. habitat with dense canopy and which tend to be wet is more favored by birds (mulyawati, 2007; anonima, 2000). additionally, a relatively shallow, sandy and muddy area is a preferred area for migratory bird species. this research discovered that the migratory birds were more concentrated on the southern area of the lake. this area is relatively shallow, muddy to sandy, has denser mangrove coverage and more importantly, is far from road access (human activity). birds distribution map that has been prepared cleary shows that the main characteristic of preferred habitat of migratory birds is habitat with wet, shallow, sandy to muddy land, as well as dense vegetation canopy and which is far from human activity. table 2. data analyses of mangrove vegetation biodiversity. name sum value indonesia latin rden rf rd ivi h’ api-api putih avicennia marina 23 22,55 42,11 23,81 88,46 0,565api-api avicennia lanata 79 77,45 42,11 69,45 189,01 buta-buta exoecaria agalocha 8 7,84 15,79 6,74 30,37 explanation: rden : relative density rf : relative frequency ivi : importance value index h’ : shannon-wiener diversity index diah purwitasari, et al. – identification of migratory birds and their spesific characteristics … 29 figure 3. distribution map of migrant birds in a salt water lake of gili meno. from this map, it is clear that migratory birds (red spots) are more active in the south of the lake. this is supported by the shallow banks of the, dense mangrove coverage and the far location from human activity. natural and undisturbed habitat is more favored by migratory birds. habitats with high canopy and dense mangrove coverage provide nesting places, protection, nursing site and suitable feeding resources. shallow and sandy to muddy areas can facilitate birds to find food especially small fish. in addition, areas that are far from road access and human activity, are safer and unstressful. these characteristic factors are specific for the habitat of salt water lake of gili meno which in turn make it a favored place to stopover during migratory season. new species observed in this habitat which was undiscovered in previous research was javan plover (charadrius javanicus). this species has been reported to migrate along the shoreline of sumatera, kalimantan, java as well as bali and just reported in 2011 to stopover in several habitats in west nusa tenggara (anonimc, 2013; mckinnon, 1991). this may because of decreasing quality of habitats in western part of lombok island so that the species chose healthier habitat to migrate to. the presence of this migratory bird also indicates that the ecosystem of salt water lake of gili meno is still in a good condition and therefore should be protected. overall, bird species discovered during this research were higher in number than those observed by setiawan (2008) in august 2008. it is assumed that different observation time take part in the different result from this research. however, this research did not discovered pecuk padi belang (phalacocorax melanoleucos) which were present in setiawan’s research. these data should be updated to identify whether habitat change (habitat degradation) or migration season and observation time is the determining factor which affect the absence of several bird species. conclusion there are 17 bird species identified in this research belonging to 5 families i.e. scolopacidae, charadriidae, ardeidae, meropidae and alcedinidae. of the 17 species discovered, 16 species were migratory birds. in addition, this research also uncovered the especially high population of n. nycticorax followed by b. striatus. this study also identified 3 species of mangrove: avicennia marina, a. lanata and exoecaria agallocha as well as one species of fish (mujair fish/oreochromis niloticus). the existence of the fairly good mangrove vegetation and sufficient food availability contributed to the ideal condition of the habitat of saltwater lake of gili meno as migration site for birds during migration seasons. acknowledgment we express our gratitude to the birdwatching group in gili meno who supported us in fieldwork and provided us more information about several mangrove species. we also thank mr. andy setiawan who has provided his assistance in the indentification of birds’ population and fieldwork documentation. all parties who cannot be all mentioned in this article is also acknowledged for their support during field study and data analyses. peta distribusi burung migran pada ekosistem danau gili meno legenda : s n ew insert : ## ## ## ## ## ## ## ## ## ## ## 395640 395640 395820 395820 396000 396000 90 76 86 0 9076860 90 77 04 0 9077040 ; r ; r r r r r r r 1 2 3 ; 4 5 6 7 8# # # # # # # # # # # ## # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # ; r # : belukar : danau : kebun : tegalan : berugaq : lokasi anveg : distribusi burung migran # # # # # # # # # # # # # # # gili meno s n ew 0 60 meters 30 biology, medicine, & natural product chemistry 3 (1), 2014: 25-30 references abdurakhman. 2002. stratifikasi penggunaan habitat berbagai jenis burung di sekitar danau gili meno-lombok barat. skripsi fkip jurusan biologi universitas mataram. mataram. aeny, w. 2004. keanekaragaman jenis dan pemanfaatan jenis tumbuhan oleh burung liar di kota mataram. skripsi fkip jurusan biologi universitas mataram. mataram. anonima. 1992. ensiklopedia indonesia seri fauna burung. pt. intermasa. jakarta. anonimb. 2006. jenis-jenis burung dilindungi yang sering diperdagangkan. http://www.kehati.or.id/news/data/8,%20)kt2000%20info %20kehati.pdf. downloaded on 19/11/2012. 10.30 wita. anonimc, 2013. burung-burung di kawasan indonesia. www.kutilang.or.id coates, b. j., dan bishop, k. d. 2000. panduan lapangan burungburung di kawasan wallacea. bird life internationalindonesia programme & dove publications pty. ltd. davies, j., g. claridge, dan c.h.e. niranita. 1996. manfaat lahan basah dalam mendukung dan memelihara pembangunan. bogor: direktorat jendral phpa & asian wetland bureau. elfidasari, d dan junardi. 2006. keanekaragaman burung air di kawasan hutan mangrove peniti kabupaten pontianak. biodoversitas. vol.7 no.1, pp: 63-66. kusmana, c. 1997. metode survei vegetasi. pt. institut pertanian bogor. bogor. kosztowny, a.l., t. hirano and e. g. grau. 2008. developmental changes in na+ , k+ -atpase activity in mozambique tilapia (oreochromis mossambicus) embryos and larvae in various salinities. international symposium on tilapia in aquaculture. mackinnon, j. 1991. panduan lapangan pengenalan burungburung di jawa dan bali. gadjah mada university press. yogyakarta. mulyawati, d. 2007. migrasi burung. buletin burung iii/edisi januari 2007. bogor. mustari, a.h., 1992. jenis-jenis burung air di hutan mangrove delta sungai cimanuk indramayu-jawa barat. media konservasi. vol. iv; pp: 39-46. noor, y. r., m. khazali dan i. n. n. suryadiputra. 2006. panduan pengenalan mangrove di indonesia. phka/wi-ip. bogor. rachman, r. 2004. identifikasi potensi wilayah nusa tenggara barat. percetakan sahabat. mataram. setiawan, a. 2008. karakteristik habitat burung di gili meno desa gili indah kecamatan pemanang kabupaten lombok barat. skripsi mipa jurusan biologi universitas mataram. mataram. shackelford, c. e., e. r. rozenburg, w.c. hunter and m.w. lockwood. migration and migratory birds of texas 4th edt. texas parks and wildlife pwd, usa. sudaryanto. 2006. birdwatching. accessed from http://sudaryantoornito/blog.com/. 21/11/2012.11.23 wita thinh , v., t. 2006. bird species richness and diversity in relation vegetation in bavi national park, vietnam. ornitology science, 5: 121-125. yamin, m. dan jamaluddin. populasi dan asosiasi koak-kao (philemon buceroide) dengan beberapa jenis tumbuhan pada habitatnya di taman buru pulau moyo. laporan penelitian dosen universitas mataram. mataram. zulfan, 2009. keanekaragaman jenis burung di hutan mangrove krueng bayeun, kabupaten aceh timur provinsi nanggroe aceh darussalam. [skripsi]. institut pertanian bogor, bogor. [indonesia]. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 467-475 | doi: 10.14421/biomedich.2023.122.467-475 issn 2540-9328 (online) identification of primary and secondary metabolites of apis cerana honey using ftir-atr diamond spectroscopy and their botanical origin tiffany hanik lestari, ratna susandarini* faculty of biology, universitas gadjah mada jl. teknika selatan, sekip utara, sleman 55281, indonesia; telp/fax: +62-274-580839. corresponding author* ratna-susandarini@ugm.ac.id manuscript received: 13 april, 2023. revision accepted: 16 august, 2023. published: 21 august, 2023. abstract apis cerana fab. is one of the popular honeybees species among beekeepers in indonesia. this species is easy to care for and produces valuable honey products. honey from a. cerana is abundantly available in traditional and modern markets in indonesia. the purpose of this study was to identify the primary and secondary metabolites in the honey produced by a. cerana using ftir-atr diamond spectroscopy. twelve samples of honey from three provinces in java island were used in this study. in general, all honey samples contained protein, carbohydrate, water, alcohol, cellulose, alkaloid, tannin, and flavonoid. variation on primary and secondary metabolites in honey samples was strongly affected by the botanical origin, geographical origin, and the local condition around beekeeping areas where the honeycombs were placed. keywords: apis cerana; biochemical characterization; honey; spectroscopy analysis. introduction honey is one of the foodstuffs containing many nutrients, such as carbohydrates, proteins, fats, and vitamins (kasprzyk et al., 2018). honey has been produced, marketed, and sold as healthy food and for traditional medicine around the world (formosa et al., 2020). honey is nutritious food safe for consumption by various age groups, ranging from one year old babies to the elderly. in addition, the antioxidant and antibacterial properties of honey make it a great food supplement for human health. honey also contains vitamins, enzyme, and secondary metabolites such as flavonoid, phenolic, and tannin (de almeida-muradian, 2014). in fact, these chemical components affect its medicinal properties as well as the quality, granulation, texture, and shelf life of honey (adeniyi et al., 2014; joshi, 2008). the chemical composition of honey as source of natural products is determined by various variables, such as the floral origin, the climate, environmental and seasonal conditions during the period of beekeeping, and the processing methods from the time of harvesting until its storage (silva et al., 2009). indeed, honey produced by various honeybees species differs not only in their chemical properties, physical properties, and the taste, but also in their biological activities (cimpoiu et al., 2012). honey that has many health benefits is produced by bees who take the best natural ingredients in the form of nectar and pollen from various flowers. honey found on the market in indonesia comes from various species of bees, including apis cerana, a. mellifera, a. dorsata, and trigona spp.. some of these honey bees species are kept on farms, but some live in the wild and their honey is harvested from the forest. in this work, we focused our study on the honey produced by apis cerana. a. cerana is one of the most widely bred honey bees species in indonesia because it is suitable for living in humid rain forests and dry grasslands in the tropics (koetz 2013; radloff et al., 2010). a. cerana does not require a large area for its maintenance and does not need to be fed outside the nest like a. mellifera. this species is also known as a non-aggressive bee species compared to a. mellifera and a. dorsata. although the honey produced by a. cerana is not as much as the other two apis species, this species has good potential to be developed in smallholder beekeeping and large scale farms (theisen-jones and bienefeld, 2016). the criteria to assess the quality of pure honey determined by the indonesian standardization agency are mainly based on physical characteristics. the agency does not include nutritional content as standards for honey marketed in indonesia. considering that honey is generally consumed as a health supplement, its https://doi.org/10.14421/biomedich.2023.122.467-475 468 biology, medicine, & natural product chemistry 12 (2), 2023: 467-475 biochemical characterization including the content of primary and secondary metabolites is important to study. currently a more detailed analysis of honey quality has been carried out by researchers around the world (kasprzyk et al., 2018). the analysis includes biological testing, such as identifying the type and amount of pollen contained in honey which become important factor in determining the quality of honey. this is because pollen contains more complex nutrients than nectar as the major plant-based substances to produce honey. currently the testing for honey quality is gaining attention by the community. for example, in poland, honey quality testing was carried out by calculating the amount of pollen and comparing the chemical compounds of the original pollen with the pollen present in honey using the fourier transform infra-red (ftir) analysis using attenuated total reflection (atr) diamond method (kasprzyk et al., 2018). a similar method has also been used in croatia, where the quality of honey is determined from the comparison of chemical compounds of honey with chemical compounds of pollen and nectar using ftir-atr (svečnjak et al., 2017). the ftir-atr diamond is a type of spectroscopy method that uses infrared waves to identify a compound or molecule present in living or dead samples (barth 2007). this type of spectroscopy is very sensitive to the chemical composition and arrangement of a molecule, both proteins and molecular structures that are larger than proteins. the current use of ftir-atr diamond includes the analysis of the chemical structure of living things and the compounds contained in them. one of the application of this method is the qualitative and quantitative analysis of chemical components in honey for determination of botanical or geographical origin, and detection of adulteration (saksangawong et al., 2021). this method is a new, rapid, effective, non-destructive, and cost-effective for analysis honey components (riswahyuli et al., 2020), and therefore suitable to be applied for quality assessment of indonesian honey. this study aims to determine the primary and secondary metabolites in a. cerana honey samples from java island, indonesia. results of this study provide important information on the biochemical content of a. cerana honey in indonesian market, and thus bring beneficial impact for the community as well as for marketing purposes. materials and methods fresh honey samples were collected from 12 a.cerana farming locations covering nine districts in three provinces of java island (figure 1) namely, kulonprogo and gunung kidul (yogyakarta special province); cilacap, solo, magelang, pati, boyolali, and karanganyar (central java province); and depok (west java province). during the visit for collecting honey samples, supporting information from the farmers was noted including the beekeeping management, methods of honey harvesting, and the blooming flowers during the recent seasons. honey samples were stored in a refrigerator at 4oc to maintain their quality prior to ftir-atr analysis. the primary and secondary metabolites content of honey samples was analyzed using ftir-atr diamond (alpha bruker). for the analysis, 1 ml of honey was put in the sample holder. the screening was carried out from the wave number of 4000 to 400 cm–1. the results of the analysis are peak graphs which were then validated according to the provisions as presented in table 1. figure 1. map of honey sampling locations (source: google earth). table 1. interpretation of the results of ftir-atr diamond analysis (kasprzyk et al., 2018). wave number (cm-1) vibrations 1000-1200 c–o and c–c stretching vibrations 1200-1350 n–h deformation and c–n stretching vibrations from amide iii 1370-1420 c–h deformation vibrations of lipids and cellulose 1540 n–h deformation and c–n stretching vibrations from amide ii 1650 c=o stretching vibrations from amide i 1740 c=o stretching vibrations from lipids 2850-3000 c–h stretching vibrations from cellulose and lipids 3250 o–h stretching vibrations from water 3300-3500 n–h stretching vibrations from protein results and discussion visual observations on honey samples and vegetation surrounding a. cerana nests honey samples from nine districts in three provinces in java island were all classified as multifloral honey based on the composition of pollen types identified using melissopalynological analysis (lestari, 2019). honey lestari & susandarini – biochemical analysis of apis cerana honey using spectroscopy 469 samples showed variation in color as shown in table 2. differences in the color might be attributed to the differences in pollen and nectar sources. table 2. honey samples from a. cerana used in this study. sample code color honey category origin of sample ah-01 yellow multifloral pati, central java ah-02 orange-brown multifloral boyolali, central java ah-03 orange multifloral karanganyar, central java ah-04 orange multifloral kulonprogo, special region of yogyakarta ah-05 yellow multifloral karanganyar, central java ah-06 yellow multifloral gunungkidul, special region of yogyakarta ah-07 yellow multifloral magelang, central java ah-08 brown-red multifloral solo, central java ah-09 dark brown multifloral cilacap, central java ah-10 light orange multifloral solo, central java ah-11 yellow multifloral magelang, central java ah-12 orange-brown multifloral depok, west java general features of vegetation in each location from which honey samples were collected is shortly described as follows. sample ah-01 was labelled as randu tanjung sari honey from pati, central java. the location of the a. cerana nests were in the field near the beekeeper’s house with a white silk-cotton tree (ceiba pentandra) grows nearby. sample ah-02 was known as dasa dharma honey from boyolali, central java. based on direct observation, the honeybee nests were placed in a garden area of about 1 hectare next to the house. various plants species were found in the garden such as longan tree (dimocarpus longan), mango tree (mangifera indica), and bitter bean tree (parkia speciosa). in addition, around the farmer's house there is an area of maize (zea mays) and rice (oryza sativa) fields as well as residential areas with small garden commonly found in front of the houses. sample ah-03 was collected from matesih, karanganyar, central java. the honeybee nests were placed in the house yard, where the farmer's house is bordered by residential areas and plantations of woody plants such as chinese albizia (albizia chinensis) and teak (tectona grandis). sample ah-04 was originated from jatimulyo village, kulonprogo, yogyakarta. the honey was produced by honey bees whose nests were placed in the farmer's house yard adjacent to the community forest with various plants species such as albizzia procera and dioscorea japonica. meanwhile, sample ah-05 was called as kaliandra honey obtained from jumapolo, karanganyar, central java. honeybee nests were placed in the house yard surrounded by residential areas (villages), rice fields, gardens, and cemeteries. sample ah-06 was commercially labelled as sari alami honey collected from kedung poh village, one of the ecotourism villages in gunungkidul, yogyakarta. this village is located adjacent to the community forest, and is a well-known beekeeping ecotourism area for educational purposes in yogyakarta. honeybee nests were placed around the residents' yards. major plant species found in the community forest are acer serrulatum, t. grandis, and c. pentandra. sample ah-07 labelled as kaliandra tanjung sari honey was collected from magelang, central java. the honeybees producing this honey were reared and fed on calliandra plantation in menoreh hill. sample ah-08 was collected from jebres, solo, central java. the honeybee’s nests were placed in farmer's house yard, which is in a densely populated residential area near jebres railway station where various flowering plants in grow in the area, including frangipani (plumeria alba), guava (psidium guajava), acacia glomerosa, and jamaica cherry tree (muntingia calabura). meanwhile, the honey sample ah-09 was obtained from cilacap, central java. the honeybee’s nests were placed in the garden of the farmer’s house which is close to urban area. the plants found around the nest were desmodium paniculatum, panicum grande, dan cassia obtusifolia. sample ah-10 is honey marketed with a label of sekarpace honey from solo, central java. the nests of a. cerana were placed in the house yard where m. calabura, mango (m. indica) and queen’s jewels vine (antigonon leptopus) were planted. the beekeeping area is densely populated settlements in the city and close to public cemetery where frangipani (p. alba) dominated the area. based on information from the farmers, various flowering and fruit plants such as jamaica cherry (m. calabura) and common ornamental vines such as queen’s jewels (a. leptopus) are deliberately planted to improve the quality of honey. sample ah-11 from magelang, central java was named as insulin honey. based on observations during honey collection period, the honeybees producing insulin honey were reared in nests located in farmer's house yard close to menoreh hill, magelang. the location of this bee farming is in a rural area close to community forests, rice fields, and vegetable gardens. in this area, mexican sunflower 470 biology, medicine, & natural product chemistry 12 (2), 2023: 467-475 (tithonia diversifolia) and red calliandra (calliandra calothyrsus) are abundant. the honey sample ah-12 is marketed under the name of organic honey from depok, central java. the location of honeybee’s nests in this farm is in the gardens close to residential areas. residents' gardens are planted with various plants such as cottonwood, bananas, and some woody plants such as acacia sp., parasol leaf tree (macaranga tanarius), and african tulip tree (spathodea campanulata). the ftir-atr profiles of a. cerana honey the results of honey analysis with ftir-atr diamond in the form of infrared absorbance spectrum are presented in figure 2 and summarized in table 3 and 4. in the spectrum, the wavenumber indicates the number of infrared waves (the inverse of frequency) emitted to the sample. a chemical bond is detected as a band at a certain position that can be used to identify the type of chemical compound content, especially primary metabolites in honey. the intensity of the band represents the amount of absorbance that occurs after infrared passes through the sample. band intensities greater than or equal to 0.1 will be read and represent a chemical compound content as listed in table 1. in addition, the band intensities also show the quantity of chemical compounds in each honey sample. figure 2. results of ftir-atr diamond analysis. lestari & susandarini – biochemical analysis of apis cerana honey using spectroscopy 471 figure 2. cont. tabel 3. summary of results of the ftir-atr diamond analysis for primary metabolites. wavenumber (cm-1) honey samples ah-01 ah-02 ah03 ah04 ah05 ah06 ah-07 ah08 ah09 ah-10 ah-11 ah-12 1000-1200 125.93 132.81 127.77 133.37 15.36 129.32 128.81 175.27 14.31 129.51 127.49 132.37 1200-1350 48.03 56.06 47.04 53.51 41.07 53.13 50.71 65.50 41.28 53.41 50.89 57.56 1370-1420 11.66 12.67 10.97 12.42 6.48 12.35 12.01 18.81 6.28 12.46 12.06 13.51 1540 4.81 5.22 4.57 5.16 3.73 5.15 4.95 7.16 3.86 5.11 5.01 5.56 1650 × × × × × × × × × × × × 1740 0.10 0.08 0.11 0.09 0.09 0.10 0.10 0.13 0.09 0.09 0.10 0.08 2850-3000 × × × × × × × × × × × × 3250 10.92 11.85 10.86 11.82 12.94 11.65 11.35 14.14 13.43 11.48 11.43 12.07 3300-3500 0.47 0.47 0.47 0.47 0.48 0.47 0.48 0.49 0.48 0.48 0.46 0.47 notes: the × sign: there is no ftir-atr diamond spectra in this wave. 472 biology, medicine, & natural product chemistry 12 (2), 2023: 467-475 tabel 4. secondary metabolites on honey samples detected by ftir-atr diamond and their comparison to results from previous studies. secondary metabolites honey samples references ah01 ah02 ah03 ah04 ah05 ah06 ah07 ah08 ah09 ah10 ah11 ah12 alkaloid ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ kavanagh et. al., 2019 flavonoid ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ li et al., 2018 tannin ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ malacarne et al., 2018 terpenoid ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ mashwani et al., 2016 the ftir-atr diamond has been widely used in the analysis of chemical compounds in honey. several studies showed that ftir-atr was able to analyze secondary metabolite compounds in food products, such as honey (tahir et al. 2017; malacarne et al. 2018). secondary metabolite compounds such as phenolics, flavonoids, tannins, alkaloids, and terpenoids can also be identified by representing the bands that appear on certain wavenumbers. bands at 1000–1200 cm-1 indicate that the honey sample contains c–o and c–c bonds. in infrared (ir) based analysis, the c–o bond is a representation of the alcohol group, so it is likely that in result of honey analysis the band at 1000–1200 cm-1 indicated the presence of alcohol group. the alcohol groups can cause bitter taste in honey with different levels of bitterness. in honey, the alcohol group most likely comes from phenolic compounds which have antibacterial properties (kavanagh et. al., 2019). the band at 1200-1350 cm-1 contains deformed n–h bonds and c–n bonds of amide iii compounds. according to singh et al. (1993), amide iii compounds detected using ftir were used to determine the presence of secondary protein structures. the band at 1370-1420 cm-1 represents the presence of deformed c–h bonds originating from lipids and cellulose. in honey, lipid commonly found at very low level, and this might be because honey contains antioxidants that has beneficial effect of lowering cholesterol level (münstedt et al., 2009). in addition, it is most likely the band at 1370-1420 cm-1 also comes from cellulose as the main component of plant cell walls such as pollen or any plant tissue accidentally carried by the bees during their foraging activity. the presence of cellulose in honey samples might indicate the presence of pollen and thus indicates the authenticity of the honey. the band at 1540 cm-1 contains deformed n–h bonds and c–n bonds of amide ii. in ir analysis, amide ii compounds are used to determine whether a protein structure is unfolding. the unfolding condition of the protein is caused by heating. in this study, honey samples were not heated, so amide ii compounds were not detected in the ftir-atr diamond for all honey samples. the band at 1650 cm-1 represents the c=o bond of amide i. according to singh et al. (1993), amide i compounds in ftir analysis were used to determine the presence of secondary protein structures. however, amide i compounds are more complex than amide iii, so the determination of secondary protein structure is more complicated. figure 2.a shows that the absorbance value of amide i is lower than that of amide iii, and these two compounds indicate the presence of secondary proteins in honey. the band at 1740 cm-1 indicates the presence of c=o bonds derived from lipids. high lipid levels in any food ingredients can cause health problems (majid, et al., 2013). interestingly, the results of the ftir-atr analysis of diamonds did not detect any lipid content or only a small amount of lipid content. this result indicated that all honey samples are of good quality and safe for health. the band at 2850-3000 cm-1 is a representation of the stretching c–h bonds derived from lipids and cellulose. this band is the same as the band at 1370-1420 cm-1 which indicates the presence of cellulose. cellulose is a polysaccharide that has an asymmetrical and symmetrical structure (dassanayake, et al., 2018). these two cellulose structures can be detected by ftir-atr diamond but based on the absorbance value, the band at 2850-3000 cm-1 is lower than the band at 1370-1420 cm-1. the band at 3250 cm-1 indicates the presence of o–h bonds from water. moisture content is one of the indicator to determine honey quality because it is related to honey's resistance to decay, viscosity, and increased microbial contaminants (prica, et al., 2014). in terms of microbial contamination, the presence of polluting microbes most likely comes from the equipment used for harvesting the honey. the microbial contamination is known to affect the hygiene of honey. the water content in honey might indicate the level of rainfall during honey production, because each harvest season may have different rainfall. low water content generally makes honey become semi-solid or has relatively medium viscosity. meanwhile, the band at 3300-3500 cm-1 represents the presence of n–h bonds of the protein. in this case, protein is known to be a primary metabolite that is important for the growth and development of body organs. compared to other studies on metabolite analysis of honey, the presence of band at 750-1800 cm-1 was reported by devi et al. (2018), anjos et al. (2015), and se et al. (2018) which represent the presence of carbohydrates. the band at this spectrum could be used to identify different groups of carbohydrates, such as glucose, fructose, and sucrose. according to se et al. lestari & susandarini – biochemical analysis of apis cerana honey using spectroscopy 473 (2018), the band at 800-1500 cm-1 indicates the presence of monosaccharides and disaccharides. this is due to the presence of c–o and c–c bonds which are characteristic of carbohydrate structures (bands at 950-1200 cm-1). moreover, se et al. (2018) noted that the bands at 1054 (c–oh: carbohydrate structure), 867, 822, and 779 cm-1 represent the presence of fructose; the bands at 1022, 991, and 898 cm-1 represent the presence of glucose; and the bands at 991 and 921 cm-1 represent the presence of glucose. based on the research of tahir et al. (2017), the bands at 2800-3000 cm-1 represent c–h, o–h, and nh3 bonds indicating carbohydrates, carboxylic acids, free amino acids, and phenolics; the band at 1600-1700 cm-1 contains stretching bands of carbonyl groups, namely c=o and c=c which represent phenolic molecules; while the band at 1175-1540 cm-1 contains deformation bonds of o–h, c–o, c–h, and c=c representing flavonoids and phenolics; and the band at 940-1175 cm-1 contains c–oh groups, c–c and c–o represent carbohydrate structures, and c–o represents phenolics. tannin compounds were also reported to be detectable in the band at 926–2955 cm-1 (malacarne et al., 2018). alkaloid compounds were detected by ftir-atr in the bands at 2300-3040 and 500-1750 cm-1 (monfreda et al., 2015). terpenoid compounds can also be detected with the presence of bands at 1591, 1390, and 1116 cm-1 which represent c―c and c―o bonds as functional bonds of terpenoids (mashwani et al., 2016). flavonoid compounds were detected by ftir-atr in the bands at 1734, 1627, 1522, 1440, 1410, 1367, 1315, and 1255 cm−1 (li et al., 2018). overall, based on the results of the analysis using ftir-atr diamond in this study, all honey samples contain chemical compounds that have bands at wavenumbers of 1000–1200, 1200–1350, 1370– 1420, 1650, 2850–3000, 3250, and 3300–3500 cm-1. concerning on differences of metabolites content in honey samples, the ah-08 honey has the highest alcohol group (band at 1000-1200 cm-1) compared to other honey samples (figure 2.h), while ah-01 (figure 2.a); ah-03 (figure 2.c); and ah-11 (figure 2.k) had the lowest alcohol groups. this might be because the botanical source of ah-8 honey is dominated by poaceae at the proportion 45% as showed from the pollen analysis reported by lestari (2019). this indication was supported by the fact that location of the honey bee nests were close to the place where poaceae plants grow. the presence of pollen of particular plant species might affect the content of chemical compounds in honey such as alcohol groups (kasprzyk et al., 2018). the honey samples ah-01 (figure 2.a) and ah-08 (figure 2.h) had the largest absorbance values in the bands at 1200-1350, 1370-1420, and 1650 cm-1 compared to other samples. this indicates that in both samples the content of amide iii, cellulose, and amide i was higher than the other samples. meanwhile, the amide ii content in the band at 1540 cm-1 was not detected in most of honey samples. in ah-01 (figure 2.a) and ah08 (figure 2.h), the peak appears with an absorbance value of 0.02, but this data did not indicate heating at the time of honey harvesting process. instead, this is most likely due to the effect of light during storage. honey sample ah-01 was stored in the front window of the house, so that it was exposed to sunlight, while honey sample ah-10 was stored inside the house in a densely populated residential area with open spaces that allowing unexpected heating occurred from direct sunlight. the absence of amide ii in all honey samples indicated that there was no heating process to reduce water content in honey. this result showed that all honey samples are of good quality because after the harvesting process the honey is immediately packaged in bottles, making it more natural and healthier. the absence of amide ii compounds in honey is known to be affected by the water content in honey (barth, 2007). environmental changes, such as irregular rainfall intensity might cause the quality of honey to decline. this is due to the high water content, especially during high rainfall in particular times of honey harvesting seasons. the problem of high water content can be modified using unacceptable practices in honey processing by heating the newly harvested honey to reduce the water content. heating process can damage the protein content in honey which affects the enzymes, and thus affect the quality of honey. the lipid content as indicated by the band at 1740 cm-1 was not detected in most honey samples except for ah01(figure 2.a) and ah-10 (figure 2.h). in these honey samples, the band was detected with a fairly small intensity, namely 0.01. this result indicated that all honey samples have no or only a very small amount of lipid content. based on the results of this study and comparison to the honey analysis reported by tahir et al. (2017), all honey samples contain phenolic compounds due to the appearance of bands at 2800-3000, 1600-1700, 11751540, 940-1175 cm-1. in addition, all honey samples also contain tannin compounds, as indicated by the band at 926–2955 cm-1 which was in accordance to malacarne et al., (2018). the presence of alkaloid compounds detected by the bands at 2300-3040 and 500-1750 cm-1 was in line with the study of monfreda et al., (2015), while the presence of terpenoid compounds in the bands at 1591, 1390, and 1116 cm-1 was reported by mashwani et al., (2016). the occurrence of flavonoid compounds as indicated by the bands at 1734, 1627, 1522, 1440, 1410, 1367, 1315, and 1255 cm−1 was in agreement with the study of li et al. (2018). results of ftir-atr diamond form honey samples in this study showed similar patterns to the analysis of pollen and honey using the same method as reported by kasprzyk et al. (2018). therefore, ftir-atr can be used as standard method in determining the quality of a. cerana honey. results of this study showed that all samples are pure honey without any additional ingredients, and showed good practices of honey production process since there are no indications of adding sugar in honey or feeding 474 biology, medicine, & natural product chemistry 12 (2), 2023: 467-475 the bees with corn flour. there re also no indication of heating the honey to produce a nice color and adding the water content to increase honey volume. the results of ftir-atr diamond analysis clearly showed that this spectroscopy method is useful in identifying primary and secondary metabolites as one of the quality indicators for a. cerana honey from indonesia. the primary and secondary metabolites analysis using the ftir-atr diamond method is thus can be proposed as standard analysis in determining the quality of honey in indonesia because this method is easy, cheap, and fast to get the results. conclusions honey produced by a. cerana contains various primary and secondary metabolites which include carbohydrates, proteins, cellulose, alcohol, alkaloid, tannin, flavonoid, and terpenoid. the results of ftir-atr diamond analysis indicated that 12 honey samples in this study showed variation in the quantitatively the amount of primary metabolites. finally, given that the ftir-atr diamond is a simple method to practically evaluate the quality of the honey, it is suggested that the improvement of the ftir-atr diamond library of honey is important for quality assessment of honey. acknowledgement: the authors would like to thank the laboratory facilities and the staffs of physics department, faculty of mathematics and sciences, institut teknologi bandung. authors’ contribution: tiffany hanik lestari collected honey samples, carried out laboratory work, and wrote the manuscript. ratna susandarini designed the study, interpreted results of ftir-atr spectroscopy, and wrote the 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10.14421/biomedich.2025.141.185-191 issn 2540-9328 (online) presence of salmonella sp. in tilapia and catfish from cages in pahandut seberang along the kahayan river flow mutiara kurniawan1, rian ka praja2,*, astri widiatri3, hanasia2, angeline novia toemon4 1undergraduate program of medicine; 2department of microbiology, faculty of medicine; 3department of public health, faculty of medicine; 4department of anatomy and histology, faculty of medicine, palangka raya university. jl. yos sudarso, palangka raya 73111, indonesia. corresponding author* riankapraja@med.upr.ac.id manuscript received: 16 january, 2025. revision accepted: 23 may, 2025. published: 05 june, 2025. abstract salmonellosis is a zoonosis, which means that it may be spread from animals to people through fish meat. it is a sickness brought on by an infection with the salmonella bacteria that infect humans and animals and invade the digestive tract. according to data from the institute for health metrics and evaluation, the global rate of salmonellosis in 2019 was 4.22 cases per 100,000 people, with a 1.72 fatality rate. indonesia had a mortality rate of 2.42 and 5.82 per 100,000 people 2019. the objective of this study is to identify salmonella sp. bacteria in 14 samples of river water, catfish (clariidae), and tilapia (oreochromis niloticus) raised in rivers. this study includes two water samples, three catfish samples, and nine tilapia samples from the cage of the pahandut seberang river in palangka raya city. it employs a descriptive research design using an experimental technique. among the metrics noted is the detection of salmonella sp. the study found that all water samples, tilapia, and catfish tested positive for salmonella sp bacteria in the selective media salmonella shigella agar (ssa), that 14 samples (100%) in the gram staining test displayed the traits of salmonella sp bacteria, and that 5 samples (b n1, c n2-3 samples in tilapia, b l1 samples in catfish, and water sample 2 showed the type of salmonella paratyphoid b bacteria) and 9 samples, namely samples salmonella tyhpimarium bacteria were found in samples a l1, c l1 in catfish, b n2-3, c n1 in tilapia, and water 1. as a result, every sample surpassed the sni 7388:2009 maximum level of microbial contamination, which is positive/25g. according to the indonesian national standardization agency's sni 7388:2009 guidelines for the upper limit of microbial contamination in food, the tilapia meat, catfish, and river water in the river cage of kahayan pahandut seberang palangka raya city do not satisfy the requirements. keywords: catfish; river water; salmonella sp; sni; tilapia. abbreviations: salmonella shigella agar (ssa); simmon citrate agar (sca); sulfide indol motility (sim); triple sugar iron agar (tsia); standar nasional indonesia (sni), foodborne disease (fbd); water borne disease (wbd). introduction salmonellosis is a disease that affects the stomach, small intestine, and large intestine and is caused by a salmonella bacterial infection in humans and animals. salmonellosis is a zoonotic disease, meaning that humans can contract this disease from animals (rima et al., 2017). humans can acquire salmonella by consuming various products from infected animals. the spread of this disease is caused by animal and human feces contaminating the environment. although there have been many reported cases of salmonellosis in wealthy countries, the percentage of reported cases remains low compared to the actual outbreaks. worldwide, salmonella sp. infections and contamination are widespread. according to data from the institute for health metrics and evaluation, the mortality rate for salmonellosis was 1.72, and its prevalence rate was 4.22 per 100,000 people globally in 2019. in 2019, indonesia's mortality rate was 2.42, and its prevalence rate was 5.82 per 100,000 people. no documented cases of salmonellosis have been reported in palangka raya city or central kalimantan province (central statistics agency, 2022). the area of 133,564.5 km² and public water area of 134,791.08 hectares, central kalimantan, whose capital is palangka raya, is one of the regions with significant fisheries potential due to its abundant public waters. in 2022, fish capture in the palangka raya river waters reached 1,544.7 tons and has been utilized. the river basin areas are used by communities around the rivers of central kalimantan, particularly pahandut seberang, for various purposes, including household activities, industry, irrigation, agriculture, and fisheries. additionally, the river basin also serves as an ecosystem for living organisms (central statistics agency, 2022). fish, aquatic plants, benthos, plankton, crustaceans, gastropods, and periphyton are among the organisms that https://doi.org/10.14421/biomedich.2025.141.185-191 186 biology, medicine, & natural product chemistry 14 (1), 2025: 185-191 inhabit the river benthos (aini, 2018). the waste in rivers can be caused by various human activities, including those from the household, industrial, and agricultural sectors (estining & solichim, 2018). the continuous disposal of waste into rivers contributes to the decline in river quality. both organic and inorganic waste from various activities around the river can act as pollutants. plant debris, animal waste, food scraps, agricultural waste, and human excrement are all considered forms of organic waste. meanwhile, inorganic waste may originate from factories, tin smelting industries, plastic use, diesel, chemicals such as detergents, and other sources carried by river currents. the biological, physical, and chemical characteristics of the river can be disrupted and altered by this waste pollution, leading to contamination. the accumulation of waste from various activities along the river, from upstream to downstream, including the river flow, can cause a decline in the water quality in the pahandut seberang subdistrict. contaminated waste in sediments and aquatic biota can contribute to the development of bacteria or other microorganisms (fauzia, 2021). one such microorganism, known as salmonella sp., is present in large numbers and is used as an indicator of waste pollution (fauzia, 2021). because salmonella sp. bacteria are pathogenic and harmful to human health, particularly for those who consume fish contaminated with salmonella sp (imamah & efendy, 2021). their presence in fishery products is undesirable. the community’s well-being has significantly improved due to the fish in the rivers of central kalimantan, particularly for those who catch fish in the pahandut seberang river. several fish species, such as catfish (clarias gariepinus) and tilapia (oreochromis niloticus), are abundant in the waters of the pahandut seberang river and hold significant economic value as food and savings (mailoa et al., 2019). the development of bacteria in fishery products can lead to unfavorable physical and chemical changes, making the product unfit for human consumption. according to sni 01-2332.2-2006, food safety quality requirements for microbial contamination of salmonella sp. must be negative. salmonella sp. is an indicator of food safety and can be a sign of microbial contamination, particularly salmonella sp. bacteria found in fish in aquatic environments. salmonella sp. bacteria are very dangerous to human health. worldwide, foodborne diseases are largely caused by the salmonella genus (purlianto, 2015). salmonella sp. infections in humans are a significant public health issue, causing 93.8 million cases and 59,100 deaths annually worldwide genus (purlianto, 2015). salmonella sp. is commonly found in river water through waste, making the water where fish are cultured highly susceptible to this infection (purlianto, 2015). based on the above definition, fish is one of the most widely consumed and needed food sources, so its safety must be maintained appropriatelly. food safety refers to the actions and measures taken to ensure that food is free from biological, chemical, or other pollutants that could contaminate and pose health risks (putri & kurnia, 2018). to prevent food poisoning or foodborne diseases (fbd), which are caused by food contamination with harmful bacteria or microorganisms, it is crucial to assess the safety and quality of fresh fish using microbiological testing.to ensure the water quality of the pahandut river against the risk of waterborne diseases (wbd) in fish farming and fish survival, research on air quality assessment in catfish and tilapia farming areas along the river against microbiological parameters is essential. salmonella sp. should also be identified in tilapia and catfish species raised in the pahandut seberang river to determine whether the fish are contaminated with microorganisms and to ensure their safety for human consumption. researchers are encouraged to study the presence of salmonella sp. bacteria in tilapia and catfish species raised in the pahandut seberang river, as there is currently a lack of information on this topic. materials and methods study area the study occurred in the faculty of medicine palangka raya university between june and desember 2024. samples of tilapia and catfish were tested in the laboratory at palangka raya university to determine bacteriological results. the population in this study consists of river water, nile tilapia, and catfish cultivated in the pahandut seberang river. the samples used in this study include live nile tilapia, catfish, and river water from the cultivation site in pahandut seberang. procedures salmonella sp testing on ssa media sterilize the inoculating loop and cool it down. collect a fish sample using the inoculating loop, then sterilize the ssa media. open the press and streak the inoculating loop across the ssa media. place the ssa media in an incubator for 24 hours. observe the colonies growing on the ssa media. gram staining sterilize the inoculating loop and ssa medium. take a small amount of colony and spread it gently on a glass slide. air-dry the smear and fix it by passing it over a flame 2–3 times. place the glass slide on a staining rack and flood it with crystal violet for 1 minute. rinse with distilled water, then flood with iodine for 1 minute and rinse again with distilled water. flood with 95% ethanol for 30 seconds, rinse with distilled water, and finally flood with safranin for 20 seconds. rinse with distilled water and examine. kurniawan et al. – salmonella in fish from kahayan river cages 187 triple sugar iron agar (tsia) test the tsia test is used to identify gram-negative bacteria based on their ability to ferment glucose, sucrose, and lactose, as well as their capacity to produce hydrogen sulfide (h2s). the media used includes slant and stab media. specific responses for salmonella sp in the tsia test include a red slant indicating an alkaline reaction and a yellow butt indicating an acidic condition. additionally, black precipitate formation suggests the production of hydrogen sulfide (h2s). sulfide indole motility (sim) test the sim test is used to determine bacterial motility. for the indole test, a negative result is observed such as forming a green ring, indicating no bacterial isolates producing indole. this shows the bacteria cannot produce indole due to the absence of tryptophan as an energy source. the indole test is significant because only certain bacterial varieties can produce indole, and this product can be studied for identification purposes. simmon citrate agar (sca) test a positive result in the simmon citrate agar (sca) test is indicated by a color change in the media from green to blue, showing that the bacteria can grow using citrate as the sole carbon source. generally, salmonella sp produces a positive result in the citrate test. however, salmonella typhi does not use citrate as a carbon source. data analysis the steps of data analysis are explained as follows: 1. the results on the selective medium salmonella shigella agar (ssa) were observed for the presence of bacterial colony growth of salmonella sp. the results were then presented in tabular form and described descriptively according to the provisions of sni 01.2332.2-2006, which outlines the method for testing the determination of salmonella sp. bacteria in fishery products. 2. the results of gram staining were used to identify the characteristics of salmonella sp. bacteria, while biochemical testing was performed to determine the specific type of salmonella bacteria found. results and discussion results of selective medium salmonella shigella agar (ssa) test an evaluation of the maximum microbial contamination limits was conducted by sni 7388:2009, based on research carried out on tilapia, catfish, and river water samples taken from floating net cages in the kahayan river at pahandut seberang, palangka raya city. using selective salmonella shigella agar (ssa) media, salmonella sp. bacteria were specifically found to be positive in samples of 25g, as shown in table 1 below. table 1. results of selective medium salmonella shigella agar (ssa) test. location fish type sample result fish cages in pahandut seberang river tilapia a 1 positive a 2 positive a 3 positive b 1 positive b 2 positive b 3 positive c 1 positive c 2 positive c 3 positive catfish a 1 positive b 1 positive c 1 positive river water water 1 (upstream) positive water 2 (downstream) positive 188 biology, medicine, & natural product chemistry 14 (1), 2025: 185-191 figure 1. results of selective medium salmonella shigella agar (ssa) test. salmonella bacteria contamination was found in all samples based on testing using selective salmonella shigella agar (ssa) media on tilapia, catfish, and water samples with varying dilutions. for tilapia and catfish, the dilution was 10⁻⁵, while for water, the dilution was 10⁻². the bacteria were observed to grow on the culture media, with growth characteristics matching the colony traits of salmonella sp., which has a black center and a transparent or white appearance. the presence of salmonella sp. in the fish and water samples indicates that they do not meet food safety standards as outlined in sni 7388:2009. result of gram staining test table 2. results of gram staining test. location fish type sample gram staining result color shape fish cages pahandut seberang tilapia a 1 negative red bacillus a 2 negative red bacillus a 3 negative red bacillus b 1 negative red bacillus b 2 negative red bacillus b 3 negative red bacillus c 1 negative red bacillus c 2 negative red bacillus c 3 negative red bacillus catfish a 1 negative red bacillus b 1 negative red bacillus c 1 negative red bacillus river water water 1 (upstream)) negative red bacillus water 2 (downstream) negative red bacillus figure 2. results of microscope observation at 1000x magnification. the second test, gram staining, was conducted using salmonella shigella agar (ssa) media on tilapia, catfish, and river water samples collected from floating net cages in the kahayan river at pahandut, palangka raya city. based on observations, 14 samples (100%) exhibited characteristics of salmonella bacteria, appearing red and rod-shaped or basil-like. result of gram staining test based on research using triple sugar iron agar (tsia), sulfide indole motility (sim), and simmon citrate agar (sca) media on tilapia, catfish, and river water samples collected from floating net cages (kja) in the kahayan river at pahandut, palangka raya city, the following results were obtained. kurniawan et al. – salmonella in fish from kahayan river cages 189 table 3. results of biochemical testing using simmon citrate agar (sca), sulfide indol motility (sim), and triple sugar iron agar (tsia). sample sca sulfide indole motility gas tsia bacteria a n1 + + + + slant: red, butt: yellow salmonella typhimurium a n2 + + + + slant: red, butt: yellow salmonella typhimurium a n3 + + + + slant: red, butt: yellow salmonella typhimurium b n1 + + + slant: red, butt: yellow salmonella paratyphi b b n2 + + + + slant: red, butt: yellow salmonella typhimurium b n3 + + + + slant: yellow, butt: yellow salmonella typhimurium c n1 + + + + slant: red, butt: yellow salmonella typhimurium c n2 + + + slant: red, butt: yellow salmonella paratyphi b c n3 + + + slant: red, butt: yellow salmonella paratyphi b a l1 + + + + slant: red, butt: yellow salmonella typhimurium b l1 + + + slant: red, butt: yellow salmonella paratyphi b c l1 + + + + slant: red, butt: yellow salmonella typhimurium air 1 (hulu) + + + + slant: red, butt: yellow salmonella typhimurium air 2 (hilir) + + + slant: red, butt: yellow salmonella paratyphi b figure 3. results of biochemical (a)testing using simmon citrate agar (sca), (b) sulfide indol motility (sim), and (c) triple sugar iron agar (tsia). the biochemical test results using simmon citrate agar (sca) media on water sample 1 and sample a l1 showed a color change from green to blue. the biochemical test results using sulfide indole motility (sim) media on sample c n1 showed a color change to black, negative indole as no red ring formed at the top after adding kovac's reagent, and positive motility as spreading was observed away from the inoculation line. the biochemical test results using triple sugar iron agar (tsia) media on water sample 1 showed a color change on the slant to red/alkaline and on the butt to yellow/acid, with gas formation at the bottom. for 14 samples tested with tsia, sim, and sca media, all gas, motility, and citrate tests showed positive results; all indole tests showed negative results; sulfide tests showed nine positive and five negative results; and all tsia tests changed from red to acid-base, indicating that the bacteria detected in all tests were nine samples of salmonella typhimurium and five samples of salmonella paratyphi b. discussion the survival of all living beings, including humans, depends on water. one of the factors contributing to the decline in river quality is the continuous disposal of waste into rivers. organic and inorganic waste from various activities around rivers can become contaminants. organic waste includes plant residues, animal waste, food scraps, agricultural waste, and human excrement. meanwhile, inorganic waste can originate from factories, tin smelting sectors, the use of plastics, diesel fuel, chemicals such as detergents, and other sources carried by river currents. the biological, physical, and chemical aspects of rivers can be disrupted and altered by this waste pollution, leading to contamination (estining & solichim, 2018). the proliferation of infection-causing microorganisms is influenced by the declining quality of river water due to pollution from organic and inorganic waste. gram staining and culture results on selective salmonella shigella agar (ssa) media showed that both water samples tested positive for salmonella sp. bacteria. under a microscope, red, rod-shaped gram-negative bacteria were observed, while the cross-sectional view revealed colonies of salmonella sp. bacteria appearing white or transparent with black centers. furthermore, biochemical test results indicated that five samples contained salmonella paratyphi b species, and nine samples contained salmonella typhimurium species. rivers are essential for life, and human activities in domestic and industrial environments significantly impact their quality. environmental pollution, particularly in river streams, can result from rapid population growth and environmental apathy in community activities. river water quality is affected by various factors, including activities conducted by communities in watershed (das) areas. human health and water quality are closely interconnected, and drinking water that is contaminated or contains harmful substances can pose significant risks to human health. 190 biology, medicine, & natural product chemistry 14 (1), 2025: 185-191 contaminated water can harbor harmful microbes, including bacteria, viruses, and parasites. these microbes can infect humans and cause gastrointestinal diseases such as cholera, typhoid, and diarrhea when consumed or used for routine cooking and bathing. heavy metals (such as lead and mercury) and industrial chemicals that contaminate water are examples of chemical pollutants that can be hazardous. frequent exposure to these pollutants can increase the risk of cancer, poison humans, and damage vital organs such as the kidneys and liver. diseases caused by consuming contaminated water can have various effects and are often quite severe. for instance, diarrhea is a common illness that can be fatal, especially for young people living in unsanitary conditions. cholera, transmitted through water contaminated with vibrio cholerae bacteria, causes severe diarrhea and dehydration. salmonella typhi is the bacterium responsible for typhoid, which can lead to high fever and other severe symptoms. fluorosis, caused by chemical pollutants such as fluoride, can damage teeth and bones. serious health risks also stem from hepatitis a, which is associated with water contaminated by human waste. the salmonella sp. bacteria are rod-shaped, gramnegative, non-spore-forming, motile with peritrichous flagella, and have dimensions of 2-4 μm x 0.5-0.8 μm. foodborne diseases are often caused by salmonella sp. many illnesses, including typhoid fever, gastroenteritis, diarrhea, salmonellosis, bacteremia (sepsis), and other localized infections, can be caused by salmonella sp. these bacteria are undesirable in fishery products because they are pathogens that pose health risks to individuals consuming contaminated fish (purlianto, 2015). fish is a highly essential food product for humans as it contains nutrients the body requires, such as proteins, carbohydrates, fats, vitamins, and mineral salts. due to its high nutritional protein content, fish may serve as a favorable substrate for bacterial growth.this is particularly true for tilapia and catfish, which have high water activity, making it easy for bacteria to grow and multiply. this contamination can occur due to pollution from organic and inorganic sources, as well as naturally occurring bacteria in the species. according to data from the institute for health metrics and evaluation (2019), the global prevalence of salmonellosis is 4.22 per 100,000 people, with a mortality rate 1.72. in indonesia, the 2019 mortality rate was 2.42, and the prevalence rate was 5.82 per 100,000 people. according to the centers for disease control and prevention (cdc), contaminated food is the primary source of salmonella infection in the united states (us), estimated to cause 1.35 million illnesses, 26,500 hospitalizations, and 420 deaths annually. rivers contaminated with industrial or domestic waste containing salmonella are the initial sources of bacterial contamination. when fish live in polluted water, the bacteria adhere to their gills and skin. if humans consume salmonella-contaminated fish that are not adequately cooked, the bacteria enter the body through the digestive tract and multiply. the hypothalamus then reacts by increasing body temperature, resulting in typhoid fever, which manifests as high fever, headache, and digestive issues. salmonella enterica, the pathogen causing gastroenteritis, can be transmitted from animals to humans through food. salmonella adheres to enterocytes in the small intestine, causing the host cells to shrink. this triggers bacterial endocytosis from the apex to the basolateral membrane, leading to the disease. furthermore, if the germs penetrate deeper, they multiply and cause inflammation, resulting in apoptosis. the primary causes of diarrhea include bacterial toxins, apoptosis, and the inflammatory response. maintaining proper food sanitation and hygiene is the first step in preventing foodborne diseases. one can prevent infection by practicing good hand hygiene, such as washing hands before eating and after defecating. salmonellosis is also influenced by nail conditions; therefore, thoroughly washing hands with soap and running water is crucial because finger and nail contact can prevent bacterial growth. additionally, ensure food is cooked thoroughly, maintain kitchen cleanliness by regularly washing hands with soap and water, store food properly, and only purchase fresh produce. the salmonella sp. bacteria were detected using selective salmonella shigella agar media testing (ssa) during a 24-hour incubation period at 37°c. based on research findings, salmonella sp. infections were found in all water samples, tilapia samples, and catfish samples. the bacteria were observed to grow on culture media, with colony characteristics matching salmonella sp.—black with a clear or white appearance. salmonella sp. in fish and water samples failed to meet food safety standards according to sni 7388:2009. after performing ssa testing, gram staining was conducted using safranin, lugol's solution, 96% alcohol, and crystal violet solution. the color and morphology of salmonella sp. bacteria were examined under a microscope. all 14 samples (100%) exhibited characteristics of salmonella bacteria, which were gramnegative, red, and rod-shaped or bacilli. the species of salmonella bacteria were identified, and sc, motility, indole, h2s, gas, and tsia were evaluated using triple sugar iron agar (tsia), simmon citrate agar (sca), and sulfide indole motility (sim) media. after 24-48 hours of incubation at 37°c, the results revealed that all 14 samples tested positive for citrate, motility, and gas, while the indole test yielded negative results. the h2s test revealed seven positive and seven negative samples, and the tsia test showed color changes from red to acid and base, indicating that among the identified bacteria, five samples contained salmonella paratyphi b, while nine samples contained salmonella typhimurium. the research findings align with liantika's (2022) study on the identification of salmonella sp. contamination in fresh milkfish at a fish auction site in gersik, gadukan kurniawan et al. – salmonella in fish from kahayan river cages 191 lumpur. the study results revealed that salmonella sp. contamination in milkfish samples exceeded the maximum microbiological contamination limit set by indonesian national standards (sni) 7388:2009. another study by wanda (2021) detected salmonella sp. contamination in tuna at traditional and modern markets in semarang city. the results showed that all six samples were infected with salmonella sp. and exceeded the microbiological contamination limit using indonesian national standards (sni) 7388:2009 methodology. conclusions based on the research findings regarding the presence of salmonella sp. in tilapia and catfish cages in pahandut seberang, which is traversed by the kahayan river basin, salmonella bacteria—characterized by colonies appearing white or translucent with black centers—were identified in water samples, tilapia samples, and catfish samples using selective salmonella shigella agar (ssa) media; the bmi of tilapia and catfish samples exceeded the sni 7388:2009 standard (positive/25g); the bacteria, identified as gram-negative, red, rod-shaped bacilli, were confirmed by gram staining; the water samples from cage areas were contaminated with salmonella bacteria, specifically salmonella paratyphi b and salmonella typhimurium; biochemical tests revealed positive results for citrate, motility, and gas, negative results for indole, varied results for h2s, and changes in tsia for all samples. acknowledgements: we would like to express our sincere gratitude to all individuals and institutions that contributed to the success of this research. special thanks go to the faculty of medicine palangka raya university wet biomedical laboratory for providing the facilities and resources necessary to conduct this study. we also thank colleagues and team members for their invaluable support and collaboration throughout the research process. authors’ contributions: mutiara kurniawan & rian ka praja designed the study. mutiara kurniawan & hanasia carried out the laboratory work. mutiara kurniawan analyzed the data. mutiara kurniawan, rian ka praja, astri widiarti, hanasia, and angeline novia toemon wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that no funding was received for this study. references aini, f. (2018). isolation and identification of shigella sp. causes of diarrhea in toddlers. bio-site, 04(1), 1–40 central statistics agency (bps) palangkaraya (2022). capture fisheries production data in palangkaraya. estining, t. d., widyorini, n., & solichin, a. (2018). differences in the number of bacteria in sediments in mangrove and nonmangrove areas in the waters of bedono village, demak. journal of maquares, 7(2), 189–196. fauzia, s. f. (2021). total plate count (tpc) test and identification of escherichia coli and salmonella sp sp. bacteria in pentol around sunan ampel state islamic university, surabaya. uin sunan ampel surabaya, 1–60. harmoko, h., & sepriyaningsih, s. (2020). diversity of chlorophyta microalgae in the kasie river, lubuklinggau city, south sumatra province. quagga: journal of education and biology, 12(1), 52. imamah, p. n., & efendy, m. (2021). analysis of escherichia coli bacterial contamination in small pelagic fish meat (case study) in the north and south sea waters of sampang regency. juvenil: scientific journal of marine and fisheries, 2(1), 17–24. mailoa, m. n., lokollo, e., nendissa, d. m., & harsono, p. i. (2019). microbiological and chemical characteristics of smoked tuna. indonesian fishery product processing journal, 22(1), 89–99. purlianto, n. a. i. (2015). total plate count test and escherichia coli identification in bitter brotowali herbal medicine produced by traveling herbal medicine sellers in the tonggalan area of central klaten. thesis. faculty of pharmacy, sanata dharma university, yogyakarta, xxxiii, 10–12. putri, a. m., & kurnia, p. (2018). identification of the presence of coliform bacteria and total microbes in dung-dung ice around the muhammadiyah university of surakarta campus. indonesian nutrition media, 13(1), 41. rachmawati, i. pramudita, riani, e., & riyadi, a. (2020). water quality status and pollutant load of the krukut river, dki jakarta. journal of natural resources and environmental management, 10(2), 220–233. rahmawati, d., & arisandi, a. (2020). abundance of coliform bacteria in cerithidea cingulata as an indicator in kamal waters, bangkalan regency. abundance of coliform bacteria in cerithidea cingulata as an indicator in kamal. junevil, 1(2), 236– 242. rima ra, darniati, ismail. (2017). isolation and identification of salmonella sp in roasted chicken from restaurant in syiah kuala, banda aceh. jimvet. 01(3),265–74. sukmawati s, badaruddin i, simohon es. analysis of total plate count microba in fresh (rastrelliger sp.) mackerel fish` of sorong city west papua. samakia journal of fisheries science. 11(1),10–4. wibisono fj. detection of salmonella sp. contamination. in milkfish (chanos chanos) at sidoarjo fish market. journal of veterinary studies,.5(1),1-10. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 381-387 | doi: 10.14421/biomedich.2025.141.381-387 issn 2540-9328 (online) antioxidant activity test of ethanol extract from the leaves and bark of kapur (dryobalanops aromatica) from distillation solid waste using the dpph and abts methods nur maulida sari1, abdul rasyid zarta1, heriad daud salusu1, muhammad fikri hernandi1, mohammad ridho ramadhan1, nur indah pragaloka1, rosalinda kerawing1, puspitasari muis1, siti raihanah2, jasmawati2, farida aryani3,* 1department of forest product processing; 3department of plantation products technology, samarinda state agriculture polytechnic, kampus gunung panjang jalan samratulangi, samarinda 75131, east kalimantan, indonesia 2midwifery department, ministry of health polytechnic, samarinda, east kalimantan, indonesia. corresponding author* faridaaryani@politanisamarinda.ac.id manuscript received: 21 april, 2025. revision accepted: 27 june, 2025. published: 11 july, 2025. abstract one of the endemic plants of east kalimantan, kapur (dryobalanops aromatica) belongs to the dipterocarpaceae family and is typically used by the locals for essential oils. solid waste is only used as a natural fertilizer, while the leaves and bark of kapur (dryobalanops aromatica) are often only used for the essential oil extracted. research on solid waste from distilling kapur (dryobalanops aromatica) has never been done. this investigation aimed to find the possible concentration of secondary metabolite chemicals and antioxidants in the ethanol extract of solid waste from distilling kapur leaves and bark (dryobalanops aromatica). the ethanol extract of kapur leaves and bark (dryobalanops aromatica) will undergo phytochemical testing utilizing a qualitative test method to ascertain the presence of secondary metabolite chemicals. the ethanol extract of kapur leaves and bark (dryobalanops aromatica) will be tested for antioxidant activity against dpph and abts free radicals using a uv spectrophotometer. the results of the phytochemical analysis showed that the ethanol extracts from the leaves and bark contained alkaloids, flavonoids and tannin. triterpenoids are only found in the bark extracts, while saponins and steroids are only found in the leaves extracts. antioxidant activity of dryobalanops atomatica extract showed that ethanol extract displayed an ability to inhibit dpph free radical in both the leaves and bark ethanol extracts, with percentages of 83.24% and 94.91% at 25 ppm concentration, respectively. antioxidant activity of dryobalanops atomatica extract showed that ethanol extract could inhibit dpph free radical in the leaves and bark ethanol extracts, with percentages of 83.24% and 94.91% at 25 ppm concentration, respectively. antioxidant activity of dryobalanops atomatica extract showed that ethanol extract display an ability to inhibit abts free radical with a percentage of 84.23% at 50 ppm concentration in the leaves extracts, followed by 82.62% percentage of inhibition at 100 ppm concentration. according to the findings of the study, post-distillation solid waste from dryobalanops aromatica leaves and bark had the potential to develop as a natural antioxidant. keywords: abts; dpph; dryobalanops aromatica; kalimantan timur; phytochemical. introduction kapur (dryobalanops aromatica) is one of the endemic species from east kalimantan that produces borneol (camphor) in both crystal and oil forms. this plant is also known to contain bioactive compounds that have effects as antifungal, antioxidant, toxic, and capable of inhibiting the spread of hiv (ritonga et al. 2018; wibowo et al. 2011). the indigenous population in kutai barat regency continues to depend on species that are today considered uncommon in indonesia for their livelihoods. according to several investigations, camphor oil of the kapur variety (dryobalanops aromatica) has been described and is suitable for use in aromatherapy (kamariyah et al. 2012; kuspradini et al. 2016; le et al. 2016; yakubu et al. 2020). phytochemical screening of methanol extract from the bark of kapur (dryobalanops aromatica) contains flavonoids, saponins, tannins, and coumarin. borneol in this tree can be applied externally for wounds, burns, rheumatic pain, and skin diseases (pasaribu et al. 2014). the branches and leaves of this plant are employed orally for gum swelling, cholera, and breast pain. it has been reported that this plant contains terpenoids such as d-borneol, terpinen-4-ol, alphaterpineol, alphapinene, and caryophyllene, which is known for its antimicrobial, cytotoxic, and antiinflammatory activities (ali 2014; le et al. 2016). numerous secondary metabolites, including terpenoids, lignans, flavonoids, saponins, secoiridoids, lactones, and alkaloids, are produced by plants and each have their distinct chemical structures and bioactivities. https://doi.org/10.14421/biomedich.2025.141.381-387 mailto:faridaaryani@politanisamarinda.ac.id 382 biology, medicine, & natural product chemistry 14 (1), 2025: 381-387 the majority of significant pharmaceuticals are made from medicinal plants. with about 40,000 distinct plant species, including about 6000 medicinal plant species, indonesia is the second-most biodiverse country in the world. as such, plants in indonesia are predicted to be a great source of bioactive secondary metabolites for developing novel antiviral drugs (aoki-utsubo et al., 2023). one of the most physiologically varied groups of indonesian plants is the dipterocarpaceae family, which is found all around the nation. oligostilbenes, phenolic compounds of resveratrol units ranging from monomers to octamers, are abundant in dipterocarpaceae plants. from dipterocarpaceae plants, resveratrol and its dimers (like ε-viniferin and laevifonol), trimers (like ampelopsin e, α-viniferin, and malaysianol), and tetramers (like vaticaphenol b and c, hopeaphenol, and vaticaphenol b) have been identified. because of their complex structures and wide range of biological functions, oligostilbenes have garnered much attention in recent decades. nevertheless, little research has been done on their antiviral effectiveness (abe et al. 2011; ito 2020; ito et al. 2000; mattio et al. 2020). antioxidants are known to stop chain reactions to stabilize free radicals. free radicals become highly reactive compounds in body cells due to the chain reaction caused by unpaired electrons. this can lead to various long-lasting degenerative diseases that can cause tissue or cell damage (lulan 2022). an antioxidant is a chemical substance that lessens a free radical's oxidation action by giving an electron to an unpaired free radical. numerous chemicals found in herbs are potent antioxidants in clinical settings and can be employed as natural exogenous antioxidants (amorati and valgimigli 2018). phenolic chemicals, which are secondary metabolites, are among the chemical substances that shield plant organs from oxidation. consequently, the term "natural antioxidant" refers to the phenolic component. a phenolic molecule found in plants is known to have anti-inflammatory, anti-carcinogenic, anti-microbial, anti-allergic, and anti-mutagenic qualities in addition to its antioxidant action (johan sukweenadhi et al. 2020). flavonoids are among the several phytochemicals with antioxidant properties. numerous plant species contain flavonoids, which are polyphenolic chemicals that have been shown to support human health. regular consumption of fruits and vegetables that contain flavonoids can lower the risk of cardiovascular disease. numerous investigations have shown that flavonoids influence various pharmacological actions, such as enzyme inhibitors, anti-inflammatory, anticancer, and antioxidant agents (ivey et al. 2017; kim et al. 2018; kumar et al. 2019). the strength of the evaluated dryobalanops aromatica extract as a possible source of natural antioxidants for application in pharmaceutical, nutraceutical, and functional food compositions would undoubtedly be investigated with the aid of the current data. to further develop its uses in the food and pharmaceutical industries, more study is necessary to discover and purify the particular molecules that have antioxidant qualities. materials and methods study area the raw materials used in this study are the leaves and bark of dryobalanops aromatica, obtained from nyaribungan village, laham district, mahakam ulu regency, east kalimantan province. the materials used in this study are dpph (2,2-diphenyl-1-picrylhydrazyl), abts (2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid), 96% ethanol, acetone, 1-naphthol, bismuth (iii) nitrate, h2so4 (sulfuric acid), hcl (hydrochloric acid), ch3cooh (acetic acid), naoh, lead acetate (pb ch3coo2), potassium iodide, ascorbic acid (vitamin c). the equipment used includes test tubes, erlenmeyer flasks, glass funnels, beakers, 1000 µl micropipettes, 200 µl micropipettes, yellow tips, blue tips, cuvettes, rotary vacuum evaporators, and uv spectrophotometers. this research was conducted at the wood properties and product analysis laboratory (skap), department of environmental and forestry, state agricultural polytechnic of samarinda. procedures maceration the raw materials, consisting of the leaves and bark of dryobalanops aromatica, are first washed with running water to remove any adhering dirt, then dried in an airconditioned room at a temperature of 20-25 ºc for 2-5 days until the samples are dry. the dried plant samples are then ground using a blender and are ready for maceration with ethanol solvent at room temperature and shaking on shaker about 48 hours. after filtration,the cride extract of the leaves and bark of dryobalanops aromatica was evaporated using rotary vacuum evaporator at 38-40 ºc. preliminary phytochemical analysis the ethanol extract of the leaves and bark of dryobalanops aromatica was analyzed for secondary metabolites, including alkaloids, flavonoids, triterpenoids, steroids, tannins, and saponins, using qualitative testing methods (sari et al. 2023). alkaloids determination: about 5 ml of the ethanol extracts from leaves and bark of dryobalanops aromatica were added of 2 ml hydrochloride acid, then 1 ml of dragendorff solution was added. the color changes in the solution was indicated the presence of alkaloids. flavonoids determination: about 1 ml of the ethanol extracts from leaves and bark of dryobalanops aromatica were drops of 1% sodium hydroxide. the presence of yellow color at extracts solution and were sari et al. – antioxidant activity test of ethanol 383 colorless after addition of 1 % hydrochloride acid was indicated the presence of flavonoids. triterpenoids determination: about 1 ml of the ethanol extracts from leaves and bark of dryobalanops aromatica were drops of about 10 acetic acid anhydride and 2 drops of sulfuric acid, sequentially. the red or purple color changes in the solution was indicated the presence of triterpenoids. steroids determination: about 1 ml of the ethanol extracts from leaves and bark of dryobalanops aromatica were drops about 10 acetic acid anhydride and 2 drops of sulfuric acid, sequentially. the green or blue color changes in the solution was indicated the presence of steroids. tannins determination: about 10 ml of the ethanol extracts from leaves and bark of dryobalanops aromatica were added 1 % lead ii acetate. the yellow precipitate reaction at solution was indicated the presence of tannin. saponins determination: about 10 ml of hot distilled water were added 1 ml of of the nethanol extracts from leaves and bark of dryobalanops aromatica. the solution then cooled and shaken vigorously (10 seconds). a stable froth upon standing for 10 minutes after added 1 drops of hydrochloride acid 2n was indicated the presence of saponins. antioxidant assay of dpph antioxidant testing was conducted with 5 test sample concentrations into 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm. about 3 mg of the extract was weighed, then dissolved in 1 ml of ethanol solvent, and subsequently, a concentration dilution for testing was performed. 33 µl of the sample was placed into a test tube and 467 µl of ethanol was added. after homogenization, 500 µl of the dpph (2,2-diphenyl-1picrylhydrazyl) free radical solution was added (sari et al., 2023). the mixing of the test sample is sufficient when it reaches a volume of 1000 µl (1 ml). the test sample was then incubated in a low-light room for 20 minutes at room temperature of 27-30 ºc. antioxidant activity using dpph decolorization was measured using a uv-spectrophotometer at a wavelength of 517 nm. the testing was conducted in the triplicate repetitions. the percentage of dpph free radicals is calculated using the formula: 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟𝑦 𝑜𝑓 𝐷𝑃𝑃𝐻 𝑅𝑎𝑑𝑖𝑐𝑎𝑙 (%) = 𝛥𝑘𝑜𝑛𝑡𝑟𝑜𝑙 − 𝛥𝑠𝑎𝑚𝑝𝑒𝑙 𝛥𝑘𝑜𝑛𝑡𝑟𝑜𝑙 × 100 antioxidant assay of abts antioxidant testing was conducted with 5 test sample concentrations into 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm. about 5 mg of extract was weighed, then dissolved in 1 ml of methanol solvent, and then a dilution of the test concentration was performed. 50 µl of the sample was placed into a test tube and 450 µl of ethanol was added. after homogenization, 500 µl of the abts free radical solution (2,2-azinobis-3ethylbenzothiazoline-6-sulfonic acid) was added with slight modification (sari et al. 2024). the mixing of the test sample is completed when it reaches a volume of 1000 µl (1 ml). the test sample was then incubated in a low-light room for 5 minutes at room temperature of 2730 ºc. antioxidant activity using abts decolorization was measured using a uv-spectrophotometer at a wavelength of 734 nm. the testing was conducted in the triplicate repetitions. the percentage of dpph free radicals is calculated using the formula: 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟𝑦 𝑜𝑓 𝐴𝐵𝑇𝑆𝑅𝑎𝑑𝑖𝑐𝑎𝑙 (%) = 𝛥𝑘𝑜𝑛𝑡𝑟𝑜𝑙 − 𝛥𝑠𝑎𝑚𝑝𝑒𝑙 𝛥𝑘𝑜𝑛𝑡𝑟𝑜𝑙 × 100 results and discussion plant extracts samples of leaves and bark of the kapur tree (dryobalanops aromatica) have been macerated using ethanol at room temperature (table 1). the extract is calculated based on the dry plant sample’s weight divided by the sample’s initial weight. table 1. percentage of ethanol extract from the leaves and bark of kapur (dryobalanops aromatica). plants parts yield of extracts (g) percentage (%) leaves 2.87 9.89 bark 16.36 19.05 note: percentage is calculated based on the weight of the dry sample. based on the results obtained, the weight of bark parts is 16.36 grams from a total of 85.91 grams of the extracted sample. meanwhile, the extract of leaves parts weight is 2.87 grams from a total of 29.03 grams of the extracted sample. this also affect the yield produced by the samples, where the leaves parts have a yield of 9.89% and the bark parts has a yield of 19.05%. a solvent with a high extraction rate, ethanol is readily available, effective, and safe for the environment (chen, xiao, and pang 2020; fan et al. 2020; hakim and saputri 2020). ethanol is a polar, multipurpose solvent that works well for first extraction. the ability of ethanol solvent to permeate cell wall components allows it to perform cell diffusion and more quickly draw in bioactive substances (prayitno and rahim 2020; yulianti et al. 2021). phytochemical analysis ethanol extracts from the leaves and bark of the kapur tree (dryobalanops aromatica) showed the presence of secondary metabolite compounds as presented in table 2. alkaloids, phenolics, flavonoids, tannins, quinones, saponins, and terpenoids are among the chemical 384 biology, medicine, & natural product chemistry 14 (1), 2025: 381-387 components contributing to bioactivity as medicinal ingredients. alkaloids serve as plant defenses against pathogens and herbivores as well as stimulants of the central nervous system, while tannins have been used as antidiarrheals and antihemorrhagic. phenolics and flavonoids are classified as antioxidants and have been shown to have numerous health benefits, as well as the ability to prevent and cure several diseases (mousavi, salleh, and murugaiyah 2018; praptiwi et al. 2020; tungmunnithum et al. 2018). table 2. phytochemical analysis of ethanol extract from the leaves and bark of kapur (dryobalanops aromatica). no compounds presences leaves bark 1 alkaloids + + 2 flavonoids + + 3 triterpenoids + 4 tannin + + 5 saponins + 6 steroids + antioxidant test using the dpph method the ability of the ethanol extract of the leaves and bark of the kapur tree (dryobalanops aromatica) to inhibit dpph free radicals was demonstrated by antioxidant testing; the percentages of inhibition for the leaves and bark ranged from 27.30 to 83.24% and 82.32 to 94.91%, respectively. at a dosage of 25 ppm, the greatest inhibition was seen in the leaves and bark ethanol extracts, with percentages of 83.24% and 94.91%, respectively (figure 1). figure 1. inhibitory activity of ethanol extract of leaves and bark of the kapur tree (dryobalanops aromatica) against dpph free radicals. studies on the active ingredients in dryobalanops sp. plant essential oils have revealed that α-pinene compounds and monoterpene derivatives may operate as natural antioxidants (aswandi and kholibrina 2020). antioxidant qualities are known to be present in phenolic compounds. the oxidative activities of these substances are typically exhibited by many modes of action, such as metal ion chelation, hydrogen-donating antioxidant, free radical scavenger, and singlet oxygen quencher (che rozenan et al. 2021). this plant's potential for development as a natural antioxidant that can suppress dpph free radicals is further demonstrated by alkaloids, flavonoids, and tannins in this plant extract. antioxidant test using abts method inhibition percentages for the ethanol extract leaves and bark of the kapur tree (dryobalanops aromatica) were from 27.84 to 81.62% in the leaves and 11.31 to 82.62% in the bark, respectively, indicating the extract's capacity to inhibit abts free radicals. the highest inhibition was observed in the leaves part of ethanol extracts with 84.23% at 50 ppm concentration, followed by the bark parts at 100 ppm concentration with a percentage of 82.62% inhibition (figure 2). 0,00 97,03 27,30 63,22 83,24 81,51 75,48 0,00 97,57 82,32 94,52 94,91 94,68 93,66 0,00 20,00 40,00 60,00 80,00 100,00 120,00 control ascorbic acid 25 ppm 6,25 12,5 25 50 100 d p p h i n h ib it io n ( % ) extract consentration (ppm) dpph antioxidant of dryobalanops aromatica leaves bark sari et al. – antioxidant activity test of ethanol 385 figure 2. inhibitory activity of ethanol extract of leaves and bark of the kapur tree (dryobalanops aromatica) against abts free radicals. based on previous study, the crude extract of methanol from the leaves and bark of dryobalanops aromatica showed the highest antioxidant activity with a percentage of 93.86% and 93.77%, respectively. the leaves and bark of dryobalanops aromatica also have the highest ability in total phenolic content by 1084.13 and 972.71 gae (che rozenan et al. 2021). dryobalanops aromatica genus was reported to be a source of resveratrol oligimers such as trans-3,4’,5trihydroxystilbene and bergenin, which has many bioactive benefits such as antioxidants and antidiabetic activities (wibowo et al. 2012, 2014). research about gcms from methanol extract of dryobalanops aromatica found five similiar compounds such as performic acid, acetic acid, decane, glycerine and propanoic acid (chang and othman 2014). meanwhile, these compounds suggested that both leaves and bark extracts contributed to the antioxidant and antidiabetic activities of dryobalanops aromatica (heinonen‐tanski and miettinen 2010). conclusions one of the endemics of east kalimantan, dryobalanops aromatica, belongs to the dipterocarpaceae genus and has been researched for its antioxidant properties. using dpph and abts free radicals, the study's findings demonstrated that post-distillation solid waste from the leaves and bark extracted with ethanol gave useful information on the amount of active chemicals and antioxidants. this genus still requires much research, particularly to identify the active ingredients in dryobalanops aromatica. the leaves and bark of dryobalanops aromatica can be extracted and used as a natural antioxidant in the medical field, according to the study's findings. acknowledgements: this research was funded by the samarinda state agricultural polytechnic (politani samarinda) through penelitian terapan dosen scheme (lecturer research internal grans) 2024 under contract number 141/pl21.g/pg/2024. we would also like to thank all parties involved in this research, who have been willing to provide information and plant specimens for research. authors’ contributions: in this study, nur maulida sari designed the study, supervised all processes, and wrote the manuscript. abdul rasyid zarta and heriad daud salusu conducted observations and sample collection. muhammad fikri hernandi supervised the samples and material preparation. mohammad ridho ramadhan, nur indah pragaloka, rosalinda kerawing and pustitasari muis supervised the research data analysis and manuscript. siti raihanah and jasmawati supervised the research data analysis and manuscript writing. farida aryani supervised all research data analysis and manuscript writing. competing interests: there is no conflict of interest in this research. references abe, naohito, tetsuro ito, 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fitoterapia 82(4):676–81. doi: 10.1016/j.fitote.2011.02.006. yakubu, saraya a., afidah abdul rahim, mohamad nurul azmi, khalijah awang, and m. hazwan hussin. 2020. “comparative evaluations of antioxidant potentials of dryobalanops aromatica tree bark extracts as green corrosion inhibitors of mild steel in hydrochloric acid.” materials research express 6(12):1265c4. doi: 10.1088/2053-1591/ab62f9. yulianti, wina, gilang ayuningtyas, rina martini, and ika resmeiliana. 2021. “pengaruh metode ekstraksi dan polaritas pelarut terhadap kadar fenolik total daun kersen (muntingia calabura l).” jurnal sains terapan 10(2):41–49. doi: 10.29244/jstsv.10.2.41-49. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 35-46 | doi: 10.14421/biomedich.2014.31.35-46 morpho-anatomical analysis of cosmostigma racemosum (asclepiadoideae) flowers widodo1,2*, mohamad amin1, mimien henie irawati al-muhdar1 and muhammad ja’far luthfi2 1biology education-graduate program, state university of malang. jalan semarang 5 malang 65145, east java, indonesia 2faculty of science and technology, uin sunan kalijaga. jl. marsda adisucipto no.1 yogyakarta 55281, indonesia author correspondency*: wwidodo594@gmail.com, phone: +62 815 4856 6820 abstract cosmostigma racemosum is a plant species belonging to the family apocynaceae, the subfamily asclepiadoideae. cosmostigma racemosum is found in nglanggeran mountain gunungkidul, yogyakarta. the local name and its original distribution are not known. information or study of cosmostigma racemosum in indonesia is not available. comprehensive characterization of this species is important for authentication and addition of data base. characterization was conducted by analyzing the morphology and anatomy of flower. the objectives of this study were to describe and analyze the morphology and anatomy of c. racemosum flowers. the method of research was based on observation method and exploration of plant systematics evidence or taxonomy evidence, including analyses and description of morphology and anatomy of flower structure and its development. the results showed that the characteristics of flower morphology are in accordance with the existing description in literatures. characteristics of pollinia are specific characters of morphological aspect of flower. data of anatomy of flower and its parts development are the new ones which confirm the position of c.racemosum as a member of the tribe marsdenieae. the data of anatomy also show new information of the ontogeny of the important parts of flower: pollinia formation, pollinia corpusculum, anther wall, anther sac, stigma, stamen, staminal tube, stigmatic chamber, and structure of ovary in asclepiadoideae. keywords: morpho-anatomy, cosmostigma racemosum, asclepiadoideae introduction cosmostigma racemosum is a species belonging to the family apocynaceae, subfamily asclepiadoideae (takhtajan, 2009: 522; apg iii, 2009: 105-121). backer and bakhuizen (1963: 274) state that this species is native to sri lanka and india. according to trimen (1895: 177), this species is found in india, burma and java. hooker (1885: 46) states that c. racemosum is common in sri lanka but its range of distribution includes java. this species was listed in the appendix 1 of threatened species in sri lanka (ganashan et al., 1995: 61) and in 2009 it became the protected plant because it was critically endangered (the ministry of forestry and environment, republic of sri lanka, 1999:105; parlement of the democratic socialist republic of sri lanka, 2009: 29). cosmostigma racemosum is also found in nglanggeran mountain in pathuk, gunung kidul, yogyakarta as a component of unique vegetation dominated by shrubs and lianas (widodo, 2012: 1-15). until now, there has been no information of the origin and the cause of its presence in nglanggeran mountain. this mountain is the remnant of tertiary volcano (oligomiosen) or approximately 0.6-70 million years ago (adhie, 2009: 1-3). only few sources of information, such as writings, books, atlas of flora, journal as well as website regarding c. racemosum are available. because there is no species data base and information of the presence of c. racemosum in indonesia, it is necessary to compile data of structure characteristics of this species for authentication of its identity and systematics. based on the results of research by widodo (2012:1-15), it is necessary for the characterization of morphology and anatomy structure of c. racemosum. in the subfamily asclepiadoideae, cosmostigma belongs to the tribe marsdeniaeae. subfamili asclepiadoideae consists of three tribes: marsdeniae, ceropegieae and asclepiadeae (takhtajan, 2009: 529). in the classic classification, the division of tribes is based on the position of pollinia and flower. pollinia are erect in the tribe marsdeniae, horizontal in the tribe gonolobinae (ceropegieae) and pendulous in the tribe asclepiadeae (civeyrel 1998: 523). the aspect of flower structure of asclepiadoideae can be used to confirm the position of specimen in genus, subtribe, tribe and subfamily. the characteristics of flower parts, its ontogeny and development are important for tracing and confirmation of evolutionary phase and its relative position in phylogeny. kunze (1995: 8) has made the schematic diagram of evolutionary phase of asclepiadoideae flower based on the structure of corona, kunze (1995: 1-24) has analyzed the structure and function of flower in asclepiadoideae, especially the corona and gynostegium. his study revealed the morphology and evolution of corona and morphology of pollinia translator in apocynoideae, periplocoideae, and asclepiadoideae. the morphological complexity of flowers in asclepiadoideae has confused yet, at the same time, instilled curiosity among botanists in the last two centuries (ollerton and liede, 2003: 107). morphology is the most important basis for taxonomic description (simpson, 2006: 348). morphological characters are the outer appearance of plants. these characters provide 36 biology, medicine, & natural product chemistry 3 (1), 2014: 35-46 characteristics which can easily be used for identification or supposition of the phylogenic relationship. morphological characters have long been used more often than anatomical and molecular characters because the morphological characters are easier to observe and more practical to use. flower morphology is important marking characters in identification. morphology is considered to be old fashioned but it is still the basis for the solution of taxonomical problems (stuessy, 2009: 232). figure 1. flower of cosmostigma racemosum. a, b. inflorescence. c. calyx and ovary. d individual flower. e. gynostegium. f. position of pollinia at gynestegium when the coronaria and anther wall removed. g. pollinia unit and their parts. the characters of plant inner structure have made significant contribution to plant systematics for more than 150 years (judd, 2002: 55-100). anatomical characters and inner structure of plants have been used for identification and determination of phylogenic relationship. anatomical characters are observed using widodo, et al. – morpho-anatomical analysis of cosmostigma racemosum … 37 light microscope with simple techniques as well as electron microscope. some important aspects of anatomical characters which have taxonomical values are flower anatomy and its development, anther development and pollen structure. anatomical characteristics play a role in explaining the phylogenic relationship (singh, 1999: 165). cervantes and diego (2010: 1) state that description of morphological characteristics in plants is the fundamental part of botanical study and as a key to taxonomy or systematics. currently, the studies to describe development are mostly those of gene interaction and protein control. these indicate that biology as a discipline has reached its ultimate maturity with experimental procedures and biochemistry point of view. however, all procedures based on experiments have caused the decline in descriptive aspects of morphology. molecular data do provide information related to morphology, but the role of genes and protein in controlling morphology can only be understood if the description of morphology of plant and its parts have been understood first. de craenne and wanntorp (2011: 1) state that the decline of knowledge of morphology and systematics of plansts has ocured in the last decades. the studies of organisms continue to increase, but with limited knowledge of structure. this results in inaccuracy of interpretation and limitedness of research horizon. flower morphology shows the evolutionary processes which have been occurring since 130 million years ago. flower morphology is the best explaining component in plant systematics researches and the key for creating phylogeny which broadens the understanding of evolution and other related organs. data of morphology are useful in clarifying molecular characters in phylogeny studies and as sources of data that help to clarify flower evolution and the underlying mechanism of flower development (de craenne dan wanntorp, 2011: 3). the objective of this study was to reveal morphoanatomical structure of cosmostigma racemosum flower. the morpho-anatomical characteristics of c. racemosum flower can be used for confirmation and checking at the levels of species, genus, and tribe in the existing classification hierarchy and for clarification of its phylogenic relationship in each level of taxon. methodology flowers of c. racemosum were collected from the nglanggeran mountain which lies at a latitude of s.07o5”25.5 and a longitude of e.110o32”20.’ the samples were subsequently kept in biology laboratory of sunan kalijaga state islamic university, yogyakarta. samples were kept in collection bags, and some of which were put in fixative faa (formalin acetic-alcohol). samples were collected as dry specimens, wet specimens and fresh material. the tools for observation and collection were digital cameras sony nex f3, sony cyber-shot dsc-w180, canon dslr, rulers, micrometers, calipers, small measuring tapes, collection bags, scissors, cutters, labeling paper, a gps (global positioning system), tools for dried herbarium collection, flacon bottles, stereo microscopes nikon smz 1500 equipped with a camera, light microscope nikon eclipse 50 equipped with a camera nikon dsf1. materials for observation and collection were distillated water, alcohol 70 %, faa (formalin acetic alcohol) solution. tools for plant tissue dissection method consisted of: microtome leica, oven, slide hot plate, becker glasses, measuring glasses, flacon bottles, object glasses, deck glasses, labeling paper, cutters, tweezers, spatula, and slide boxes. materials for plant tissue dissection were: distillled water, alcohol (30 %, 50%, 70%, 80%, 96%), xylol, parafin, glycerin-albumin solution, safranin, hematoxylin, fast green, canadian balm. the techniques and procedures for plant tissue section (slice) preparation followed cutler et al. (2007: 170-193) and schweingruber et al. (2011: 7). preparation and observation of flowers were done by observation, photographing, measurement and description of characteristics of parts of flowers, fruits and seeds. identification of morphological characteristics was based on cullen (2006: 1-357) and haris and haris (2006: 1-206). photographing was conducted with digital cameras sony nex f3, canon dslr, and sony cybershot dsc-w180. ruler or other tools were put beside the photographed materials in order to show the size of the materials. photographing was replicated 15 times. measurements were done using caliper, tape meter and graph paper. observation and photographing of detailed morphology of flower’s parts was done using stereo microscope nikon smz 1500 equipped with a camera nikon. preparation and observation of pollinia were conducted by picking gynostegium, then opening carefully the corona covering pollinia using preparation needles under stereoscopic microscope. a unit of complete pollinia was picked and isolated to get the detailed parts of pollinia and their orientation. observation of pollinia morphology was done under a stereoscopic microscope nikon smz 1500 equipped with a camera. pollinia were photographed in their natural condition, which were attached to one side of gynostegium and their orientation was determined. measurements taken for each unit of pollinia were the length and width of pollinia lobes, the length and width of corpusculum, the length of caudicula or translator arm.the measurement of pollinia’s parts was done using objective micrometer in the stereoscopic microscope nikon smz 1500 equipped with a camera and the results were then calibrated using paint program, windows picture manager and corel photo paint 12. slides of flower anatomy were made using parafin method with double coloring of fast green safranin. observation of section (slice) preparation was done by observation and photographing using light microscope nikon eclipse 50 equipped with a camera nikon dsf1. observation and photographing with weak magnification 38 biology, medicine, & natural product chemistry 3 (1), 2014: 35-46 were done for identification of shape, configuration of tissues in calyx, corolla, gynostegium, pollinia and other organs, and the configuration of an organ in the whole inflorescence. observation and strong magnification were done for identification of structure and configuration and other details of cells which compose epidermal tissues, mesophyll, vascular tissues in calyx, corolla gynostegium, style and stamen. the measurement of cells which compose tissues was done using objective micrometer scale calibration method and the results were then analyzed using program paint, windows picture manager and corel photo paint 12. data analyses refered to the description and semiquantive method according to albrechtova (2004: 13). data in the forms of macroscopic and microscopic pictures were analyzed descriptively and comparatively. the pictures were visually presented and described or accompanied with explanation. the presentation of data in the forms of pictures, numbers, description and explanation were compared with literaures and related studies. this research was conducted from march to july 2013 in biology laboratory, a part of integrated laboratory of islamic state university of sunan kalijaga, yogyakarta. results and discussions flower morphology the flowers of cosmostigma racemosum is inflorescence of racemose to corymbose type; peduncle is between the petioles of leaf pair (interpetiolaris); peduncle is brightly green; the transversal section is round, 6-8 cm in lenght. pedicel is also brightly green; transversal section is round, the tip wider than the base, 1 cm long. the width of inflorescence is 3-4 cm (figure 1a, b). the individual flower of cosmostigma racemosum is bisexual, 3-4 mm in height without pedicel; 9 mm in width when blossoming. the individual flower is shown in figure 1c. the sepal consists of 5 parts, united at the base, 1 mm in length, tip obtuse; margin pubescent, brighlt green. corrolla has 5 petals, forming into a bellshape short tube, 3-4 mm in length, 2-3 m in width, tip acuminate, surface glaborous, greenish yellow, reddishdotted. flower has 5 corona-scales, 1 1,5 mm in height, tip bilobed, whitish grees. stamen and pistil form a gynostegium (figure 1e). 5 anthers are attached to the lateral side of stigma. anther is protected by corollines corona. the tips of corollines corona wrap the stigma. diameter of stigma is 2 mm. the ovary has 2 carpels, 1 mm in length. the morphological characteristics of c. racemosum flower in this study match the decription in sivalaya farm (2011: 1), page (2008: 1), backer & bakhuizen (1963: 274), brandis (1906:468), trimen, (1895:177), hooker (1885: 46), drury (1866: 228). the structure of corona is important for distinguishing cosmostigma from non cosmostigma. according to ping-tao et al., (1995:4) the characteristics of cosmostigma corona is that it is relatively small and thin like a membrane. the visual presentations of c. racemosum flowers as sketches have been available in old literatures or monographs, namely de lessert and de candole (1846: 84), wight (1846: 591), hendrik (1686: 32). the visual presentations in the forms of ancient herbarium photographs were uploaded in the web site of herbarium musei parisiensis, in 2012. description and visual presentation in this study are important and needed because the characteristics of flower have high identity value and the information of the presence of c. racemosum in indonesia currently is not known except in the ancient literature of trimen (1895: 177) in flora of ceylon, hooker (1885: 46) in flora of british india, and drury (1866: 228) flora of india. the additional description data of c. racemosum flower morphology contributed by this study are the position of petals (aestivatio) toward each other and the structure of corollines corona in anther. the aestivation type in c. racemosum is dextrostum contortus or rightward contortion. the tip and middle of the corolla lobe reduplicate or folded with margin folded outward. de craene and wanntorp (2011: 2) state that data of flower morphology are very good clarifying components in systematic research and the key or the main tool for developing phylogeny concept, expanding and enriching the understanding of evolution and significance of organs in evolution. morphology of pollinia pollinia are produced by an anther. an anther consists of two theca or pollen sacs, each forming one pollinium, so there are 2 pollinia in each anther. corona corollines form at the dorsal side of anther. an anther is fused to the lateral side of stigma forming a structural unit called gynostegium (figure 1e). corpusculum is shiny brown located between anthers. corpusculum is connected by caudicule to two pollinia lobes from two different anthers, namely one on its right and the other on its left side (figure 1f). pollinia lobe is erect, obovate, golden yellow (figures 1f, g), 550-600 mµ in length, 200-250 mµ in width. caudicule or translator leg is clyndrical, 500-600 mµ in length, without membrane. corspusculum is ovate, cleft in ventral side, 250 mµ in length, 150-200 mµ in width, shiny blackish brown. the color and structure of corpusculum of c. racemosum ensure its position as a member of the subfamily asclepiadoideae. the characteristics of pollinia lobe of c. racemosum, namely erect, ovate, relatively long caudicule without membrane, ensure its position as a member of the tribe marsdenieae. shape, size, location, orientation, and location of line, are important criteria of pollinia morphology (sreenath et al., 2012:45-53; sinha dan mondal, 2011:7981-7986; rahman, 1990: 83), which play an important role in identification of tribe and genus. the results of this study confirm the classification takhtajan (2009: 522) which put cosmostigma racemosum in the tribe marsdenieae. widodo, et al. – morpho-anatomical analysis of cosmostigma racemosum … 39 anatomy of floral development longitunal section of inflorescence in figure 2 shows that c. racemosum inflorescence is of racemose type (inflorescentia racemosa). the development of inflorescence unit starts from the edge or bottom of the inflorescence. there is a small bractea on each base of inflorescence unit. trichomate are on the bractea and leaf surface. figure 2 b shows the early development of inflorescence unit. the characterics of early inflorescence development indicate that sepals occupy the main proportion of flower; colleter gland in calyx base is clearly visible. stigma also occupies a large proportion of flower. stamens are cleary visible, consisting of filaments and anther. the bases of filaments are attached to the base of corolla. the wall of pollen sac (theca) begins to differentiate simultaneously with the formation of pollen. the next step is the enlargement of flower (figure 2d), indicating the following characteristics: elongation of corolla base, the expansion of stigma to the periphery so that it press and lift the anther. the anther’s position is precisely on the lateral side of stigma. the development of ovary is clear; the wall of anther and theca (pollen sac) which contain pollinia are clearly visible. the maturation of pollinia is happening.the wall of anther clearly shows the layers of tapetum, middle layers and endotesium. the stage of blossoming is shown in figures 2d, e with the following characteristics: the corolla dominates the proportion of the flower; the stigma is lfted by the uniting tissues of corolla base, calyx and stamen. pollinia and their components (tapetum, middle layer, endotesium) adhere to upper lateral side of stigma. the remaining structure of anther sticks to stigma and becomes corollines corona which protects pollinia; the stigma is lifted away from ovary. the presence of bractea at the primodial stage of infloresence development confirms the position of c. racemosum as a member of the subfamily asclepiadoideae which ontogenically has bractea like inflorescence in apocynoideae. the data of the presence of braktea during primordial phase of inflorescence development also rectify the statement of hooker (1885: 42) that the bractea in cosmostigma racemosum is zero (0). but the same data may also confirm the statement of hooker if what he means by (0) is that ontogenically c.racemosum has bractea. figure 2. longitunal section of cosmostigma racemosum flower. a. section of young inflorescence insert: bractea and trichoma. b, c, d, e. flower development. data of flower development anatomy clarify the proccess of gynostegium structure formation (the fusion of stigma and anther) in c. racemosum. the early development shows that the structure of stamen is not united with stigma, so the fusion of stigma and anther is difficult to explain. data of flower development can complement, clarify or support the concept of flower phylogeny of asclepiadoideae presented by kunze (1995:1-24). the early stage of flower structure of asclepiadoideae is similar to that of apocynoideae so that phylogenically apocynoideae precedes asclepiadoideae. data of floral development also show that gynostegium is formed and then lifted due to the elongation of staminal tube and ovary. data of flower development show the development of the wall of anther and theca (pollen sac) and the ontogeny of pollinia. 40 biology, medicine, & natural product chemistry 3 (1), 2014: 35-46 anatomy of longitudinal section of receptacle, calyx, corolla, stamen and pistil the longitudinal section of blossoming inflorescence of c. racemosum shows the flower parts, namely receptacle, calyx, corolla and gynostegium (the fusion of stamen and pistil). the parts and the tissue structures are shown in figure 3. figures 3b-c show tissues of receptacle and calyx. receptacle consists of large cells and its outer side is covered by flat epidermal cells with stomata among which. the tissues of calyx consist of upper (adaxial) epidermis, mesophyll, and lower (abaxial) epidermis. the upper epidermis in calyx consists of a layer of flat elongated cells (15-25 μm). the mesophyll of calyx is composed of rounded cells and it has small vascular tissues. the abaxial epidermis consists of cuboid cells (20x20 μm) and stomata among which. colleter gland is found at the abaxial surface of base of calyx (figure 3d). the border between calyx and receptacle tissues is not clear. the tissues in calyx are more differentiated than those in receptacle. the mesophyll cells in tissues of receptacle are larger than those in calyx tissues. a. longitudinal section of flower b. receptacle c. sepal f. basal of petald. coleter glands e. sepal g. anther wall h. pollinium i. stigma head j. ovary figure 3. tissues in longitudinal section of cosmortigma racemosum flower. characteristics of calyx of c. racemosum are: the presence of colleter gland at the base of the connection of two callyces, so there are five colleter glands. the colleter gland produces sticky slime or resin, insoluble in water. the function of this gland is to protect dormant buds and meristem development, leaf buds, and young stipules (evert, 2006: 459). the colleter gland in the callyx of c. racemosum flower is of the general standard type, namely long head and short stalk. the head is covered with epidermal cells that look like palisade. the colleter type of c. racemosum belongs to the type of the family apocynaceae. the presence of colleter in the callyx of apocynoideae was reported by martins et al. (2013: 21) in secondatia densiflora, whereas the presence of colleter in the calyx of asclepiadoideae was reported by silva et al. (2008: 924) in oxypetalum. the presence of colleter gland or glandula emergentia in the base of calyx of the tribe marsdenieae of asclepiadoideae was receptacle sepal petal antherstigma head ovary gynostegium adaksial epiderm abaxial epiderm epidermis mesophyllpollinium mesophyll adaxial mesophyll abaxial mesophyll adaxial epiderm abaxial epiderm epidermal glands carpel ovule epidermis mesophyl widodo, et al. – morpho-anatomical analysis of cosmostigma racemosum … 41 found by valente dan costa (2005: 53) in marsdenia loniceroides. the presence of colleter gland in the calyx of cosmostigma racemosum shows that the petals and sexual apparatus of flower have been and prevented from drought since they are still buds. figure 3e shows the arrangement of corolla tissues. the corolla is composed of several tissues: adxial (upper) epidermis, mesophyll (the densely-packed and spongy mesophylls) and abaxial (lower) epidermis. adaxial epidermal tissues at the tip of corolla consist of short papilla cells while abaxial epidermis consists of a layer of flat cells with stomata. the papilla cells of adaxial epidermis get more elongated in the middle and base of corolla. the structure of papilla cells at the adaxial epidermis at the base of corolla is of glandular type (figure 3f). the structure of papilarry epidermis like palisade at the adaxial surface of corolla base of c. racemosum indicate is function as nectariferous tissue. in this tissue, epidermal cells function as secretory gland. the characteristic of nectar epidermis is the presence of trichomate cells or the elongated cells like palisade (evert, 2006: 453). figure 4. a-d the development of stamen and other structures. d. parts of microsporangium wall during the development of pollinia. e. parts of pollinia. 42 biology, medicine, & natural product chemistry 3 (1), 2014: 35-46 the mesophyll tissues of c. racemosum flower are differentiated based on cell size and density. the difference between the the dense and spongy tissues can be observed by safranin-fast green double coloration. the dense mesophyll tissues consist of densely-packed round cells with limited intercellular spaces, and the cells contain chloroplast. the presence of cholorplast in densely packed mesophyll indicates that the petals of c. racemosum still have photosynthetic function. the spongy mesophyll tissues consist of long-rounded cells larger than the cells in densely packed mesophyll tissues. there are more intercellular spaces and vascular tissues at the border with densely packed mesophyll tissues. figure 3g shows the arrangement of tissues of anther wall. figure 3h shows the mature pollinia in the broken anther sac. the base of anther sticks to the lateral side of stigma. anther and pollinia are at the depression of lateral side of stigma. the upper tip of anther wall is additional corona or corollines corona. the additional corona is composed of thin layer of outer and inner epidermis, the reduced mesophyll containing druse crystal. parts of the whole stamen can be seen in figure 3a. the lower part of an anther is a filament which is the extension of stamina tube, which in turn is the extension of corolla base which wrap the ovary. the term staminal tube is adopted from tubo estaminal (silva et al., 2008: 929; valente dan costa, 2005: 53). the cells at the periphery of staminal tube look larger than those in the middle. the staminal tube wall facing the base of filament shows epidermal gland. the longitudinal section crossing the middle of corona shows that the tissues of stamen base and staminal tube are united (indistinguisble), while the tissues of corona base tend to separate from staminal tube. these data show the ontogenical difference between the two tissues. the tissues of anther consist of upper epidermis, mesophyll, and inner epidermis. the upper epidermis is composed of densely-packed longitudinally elongated flat cells. the mesophyll consists of densely packed round cells. the inner epidermis consists of papilla cells larger than those in mesophyll. the tissues of filament are composed of outer epidermis, mesophyll and inner epidermis. the outer epidermis consists of densely packed small flat cells. the mesophyll is composed of densely-packed, small rounded cells. the inner epidermis is composed of glandular cells which can be sheded. the inner side of filament seems to be asscociated with the base of stigma and the tip of stamina tube forming a secretion-laden sac. the arrangement of stigma is shown in figure 3i while the arrangement of ovary is shown in figure 4j. the stigma consists of epidermal tissues and mesophyll tissues. the outer epidermis of stigma is composed of short papilla-shaped cells like those in adaxial epidermis of corolla. the shape of mesophyll cells look like spongy tissues but with limited intercellular spaces. many druse crystalls are found in the mesophyll tissues of stigma. in the lower middle part of mesophyll of stigma there are xylem vessels with thickened ring. the part of stigma which sticks to anther is composed of glandular cells producing secretion. ovary consists of wall and sacs containing many ovules. anatomy of stamen and pollinia development figures 4a-d show the development of stamen of cosmostigma racemosum. transversal section of young flower (figure 4a) shows that the base of stamen is at the junction between ovary and corolla. anther and filament are separate parts from stigma so that gynostegium has not been formed. theca or pollen sacs have been formed but the wall differentiation has not been complete. corollines corona at the tip of anther is still at the early stage of development. orderly layers of pollen as the precursors of pollinia are seen. staminal tube has not been formed in this immature flower. the maturation process of stamen is shown in figures 4c-d. anther is lifted at the same time with the elongation of filament. the base of filament is also lifted and forms staminal tube.the base of anther sticks to the lower lateral side of stigma, so the gynostegium has been formed. the wall of theca (pollen sacs) shows the layers of tissues of tapetum, middle layer and endotesium. pollinia have been formed and there is space between pollinia and the anther wall. corollines corona has been elongated. the mature stamen and pollinia are shown in figure 4d. the tissues of theca wall have been broken but the tissues of anther and corolines corona are still present. the staminal tube has been maximally elongated. the stamina tube function as the supporting structure for stigma and its tip function as the base of filament. gynostegium structure has been completely formed at the blossoming flower. pollen in mature pollinia emits shiny blue fluorenscent light while the wall of sporopollenin emits orange fluorescent light. data of stamen development clarify the origin of staminal tube and the formation of gynostegium in c. racemosum flower wich can be traced ontogenically. filament structure in gynostegium is maintained until mature stage. this indicates that c. racemosum belongs to a group closely related to apocynoideae or the lower group of the sub-family asclepiadoideae. the lower groups of the sub-famili asclepiadoideae are the tribes fockeeae and marsdenieae. polinia of c. racemosum are the aggregation of pollen grains. polinia of asclepiadaceae (asclepiadoideae) are wrapped by sporopollenin. the sporopollenin wall extends entering each group of pollen grains so that all pollen grains are not united. the outer wall of sporopollenin is called pollinial wall, while the extension of wall into the units of pollen grain group is called extension of pollinian wall (srideevi et al., 1990: 324). in general sporopollenin wall is soluble in hot 2aminoethanol solution. srideevi et al. (1990: 324) states that young pollinial wall in tylophora (asclepiadaceae) is soluble in hot 2-aminoethanol solution but the wall of mature pollinia is not. pollen grains in intact pollen can be observed using light without coloring because they emit fluorescent light or autofluorescence. widodo, et al. – morpho-anatomical analysis of cosmostigma racemosum … 43 (pacini dan franchi, 1999: 303). the emission of fluorescence indicates the maturation stage of pollnia. anatomy of transversal section of flower transversal sections of cosmostigma racemosum flower from the top to the base of stigma were observed at 8 points (figure 5). the first point of section crossing the the upper stigma in figure 5a shows the tissues of corolla, anther and stigma. the tissues of corolla consist of abaxial epidermis, mesophyll cells at the abaxial side, mesophyll cells at adaxial side and adaxial epidermis (figure 5e). epidermal cells and mesophyll cells at abaxial side are larger than those in mesophyll cells at adaxial side. vascular bunddels are located between abaxial and adaxial mesophylls. each petal shows three vascular bundles. stomata are located in outer abaxial epidermis. the tissues of stigma are composed of ground tissues with one vascular bundle and two pollen sacs (figures 5g, h). pollen sac is round and and each sac has one pollinium. the tissues of stigma consist of extensive mesophyll tissues, in the middle of which are circles of vascular bundles (figure i). the epidermis or the outer layer is composed of densely packed tube-shaped epidermal cells, some of which are glandular (figure 5j). figure 5. the transversal sections of tissues of cosmostigma racemosum flower. a. sketches and point of transversal section, b-e. transversal section, f. corolla tissues, g. vacular bundle in anther, h. anther sac containing pollinia, j. tissues of stigma, k. section of corpuskullum of pollinia, l. stigmatic sac, m. ovule. the second point of section crossed the middle of pollinia lobe. in this section, the pollen sacs look flat, oval to round-shaped. the tissues of vascular bundles at the pistil show two sliced circles. at the periphery of the stigma there is an attachment of transversal section of pollinia corpusculum as shown in figure 5k. there are two shapes of epidermal cells at the lateral side of stigma, namely: a) dense smaller columnar shape and b) secretory 44 biology, medicine, & natural product chemistry 3 (1), 2014: 35-46 large, columnar shape. the smaller epidermal cells are at the lateral side of stigma facing anther, while the large, secretory cells seem to be the components of corpusculum and caudicule. the third point of section crossing the lower part of stigma base shows the characteristics of the edge of anther which are extending and folded outward. the junction of two edges of anther base forms a stigmatic chamber as shown in figure 5l. the stigmatic chamber is located under corpusculum. the transversal section of style look rounded with two circles of vascular bundles tightly close to each other. the tissues of mesophyll are rounded-ovate and densely packed. epidermis is composed of densely packed tube-shaped cells. in this section style is surrounded by five stigmatic chambers. the transversal section of corolla has 8-9 vascular bundles. the fourth point of section crossed the ovary. this section shows that the transversal section of corolla tissues has 3 (x5) vascular bundles. the transversal section of corolla is attached to the base of staminal tube. the staminal tube has 1 (x5) kidney-shaped vascular bundle. this section shows two ovaries, each having ovules. each carpel wall has 8-12 vascular bundles, one of which is larger than the others. each ovary has 8-14 rows of ovules. the transversal section of the base of staminal tube circles the ovules. what remains of the stigmatic chamber is a narrow curve as the border between the remains of anther base. five tissues of transversal section of calyx are outside of corolla tube circle. at the junction among calyx there is a colleter gland. data of structure of flower organ and tissues of cosmostigma racemosum in transversal section strengthen and clarify the data obtained from longitudinal section. the transversal section through stigma shows: lay out and orientation of petals from one another, arrangement of corolla tissues at the tip or distal, number of layers of anther structure, location of anther, arrangement of anther tissues, number of theca or anther sacs, structure of theca, structure of anther wall, location and position of stigma, structure of stigma, arrangement of stigma tissues. the structure of stigmatic chamber of cosmostigma racemosum as a member of the tribe marsdenieae is relatively similar to that of marsdenia loniceroides presented in the result of research by valente et al. (2005: 55). the stigmatic structure and its extension vertically downward under staminal tube widen the pollination reach. staminal tube is a set of tissues, forming rings made into 5 main lobes. each has three sub-lobes (a large one in the middle accompanied by two small ones). the junction of main lobes is a downward longitudinal vessel and an extension of stigmatic chamber. epidermal cells at the vessel between the main lobes or small lobes are secretory or glandular. the arrangement of staminal tube is essentially the same as that of vascular bundle which extends laterally. stigamaric chamber is directly connected to the tip of ovary with no staminal tube in between. the transversal section crossing ovary shows two seperate ovaries or apocarps, each having half-rounded shape transversally. the two ovaries are not the same in sizes; one is smaller than the other. each ovary has many ovules. analyses of observation and confirmation of carpel types according to esau in suradinata (1998: 235) show that each visible ovary consists of one carpel arranged in conduplicate involution with placenta of parietal ovules. each ovary has one ovary chamber. tissues of carpel is composed of three (1+2) large vascular bundles and 12 (6+6) larger ones. colleter gland is found in the edge of intersection among sepals, the structure of pistil with the ring of vascular bundle inside shows that stigma has two vascular bundles which have been fused. toward the base of stigma, the vascular bundle get separated from each other and become two sliced rings. the fact that pistil has two apocarps seems to be contradicted with the evolutional trend of style and ovary. apocarp is supposed to have its own style. morphology of female sexual organ and its terminology are still disputable (hidayat, 1995: 226). the presence of two apocarps but only one stigma in c. racemosum and asclepiadoieae in general confirm that statement. a phenomenon that a flower has apocarps but the stigmas are united is explained by kunze (1995: 5) as post genital fusion. study of kunze (2005: 347) also found this post genital fusion in the formation of stamina corona and corollines corona. conclusion and suggestion morphological characteristics of cosmostigma racemosum flower in this study are in accordance with the existing description in literatures. new data are found regarding specific structure of anatomy or flower parts and tissues, developments of flower, stamen, anther wall, anther chamber and pollinia, formation of pollinia, ontogeny of pollinia corpusculum, position of stigma, and structures of staminal tube, stigmatic chamber and ovary. characteristics of pollinia are specific morphological characters of anther for species identification. new data of flower structure anatomy confirm the position of c. racemosum in the tribe marsdenieae. data of flower development anatomy especially stamen development of c. racemosum shows its ontogeny and clarify in the tribe marsdenieae, subfamily asclepiadoideae as well as filogeny of the tribe mardenieae in the subfamili asclepiadaceae. species authentification of c. racemosum and other members of asclepiadoideae can be done by matching data of pollinia, structure of morpho-anatomy of flower. all data of shape and qualitative characters of structure are primary characteristics for cross checking and authentication. data of morpho-anatomy of c. racemosum flower can be used to complement the evolutionary analyses of asclepiadoideae, tribe marsdenieaae currently in trend. widodo, et al. – morpho-anatomical analysis of cosmostigma racemosum … 45 acknowledgment the author is grateful to all parties who have helped him to conduct this study, especially the directorate general of higher education, the ministry of religion of the republic of indonesia which funded this study. references angiosperm phylogeny group iii. 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professional education, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 11 october, 2024. revision accepted: 15 july, 2025. published: 04 august, 2025. abstract mistletoe (dendrophthoe pentandra (l.) miq.) is one of the plants found in indonesia. the type of mistletoe commonly found is d. pentandra (l.) miq. apart from its parasitic nature, the leaves of d. pentandra (l.) miq. has useful benefits as an antibacterial. bacteria are divided into two groups based on their staining, namely gram-positive bacteria and gram-negative bacteria. infectious diseases can be caused by several types of microorganisms in the bacterial group, including pseudomonas aeruginosa. this study aims to determine the p. aeruginosa bacteria in the extract of lime mistletoe. this study used an experimental post-test only control research design with the disc diffusion method. this study used five concentrations, namely 20%, 40%, 60%, 80%, and 100%, and used distilled water as a negative control and ciprofloxacin as a positive control. the inhibition zone is determined by observing the clear zone. keywords: antibacterial; extract; lime mistletoe (dendrophthoe pentandra (l.) miq.); pseudomonas aeruginosa. abbreviations: laminar air flow (laf), nutrient agar media (na), world health organization (who) introduction indonesia has diverse natural resources, one of which is tropical rainforests. klhk data in 2020 shows that indonesia has 94.1 million forests in 2019. behind the vastness of indonesia's forests, it must benefit the indonesian people, one of which is medicinal plants. people prefer factory-made medicines because they feel the benefits more quickly, but research on medicinal plants has also been intensively carried out, because it is safer and of course the benefits contained in it (yathurramadhan & yanti, 2020). one of the medicinal plants that is still rarely known is the lime mistletoe plant. as we know lime mistletoe is a parasitic plant, which means that its existence will harm/damage the host plant it lives in. the lime mistletoe plant commonly found in the type of dendropthoe pentandra (l.) miq. in addition to its parasitism, some benefits are useful as antibacterials on the leaves of d. pentandra (l.) miq. lime mistletoe (d. pentandra (l.) miq.) is one of the plants found in indonesia. lime mistletoe is a semiparasitic epiphytic plant that uses other plants as hosts. semiparasitic epiphytic plants attach to other plants as hosts where the plant absorbs some of the food it needs such as nutrients, water, minerals, and nutrients. while some other food is obtained from its photosynthesis (permatasari, 2019). infectious diseases are still one of the biggest problems often encountered in developing countries. infections caused by bacteria are the most common. bacteria are divided into two groups based on their staining, namely gram-positive bacteria and gram-negative bacteria (savitri et al., 2019). according to the world health organization, (who) (2018), infection is one of the top ten diseases that cause human death. the top three types of infections that cause death are lower respiratory tract infections with an incidence of 3.0 million deaths, diarrhoeal infections with an incidence of 1.4 million deaths, and finally, tuberculosis with an incidence of 1.3 million deaths. infectious diseases can be caused by several types of microorganisms including p. aeruginosa. p. aeruginosa is a rod-shaped bacterium, gram-negative, has the ability to form biofilms in culture media, and the substrate it is attached to (wahyudi et al., 2019 and khoiriah, 2021). this bacterium has been found in many cases of clinical infections in several parts of the body and causes treatment to be more difficult (lee et al., 2018). p. aeruginosa is often found to be the cause of nosocomial infections, which are infections that a person gets while in the hospital (arcasuárez et al., 2019 and angeletti et al., 2018). the highest cases of p. aeruginosa cause https://doi.org/10.14421/biomedich.2025.141.553-556 mailto:lisasavitri@unik-kediri.ac.id 554 biology, medicine, & natural product chemistry 14 (1), 2025: 553-556 cystic fibrosis (pallett et al., 2019), respiratory tract infections (tovar-garcía et al., 2020), urinary tract infections (wahyudi & rahmawati, 2021), wound infections (wahyuni (2020), and several other infections that can be found in many clinical samples, including sputum, urine, wounds, cerebrospinal fluid, pus, faeces, post-surgical infections (wahyudi, 2019; milanda, 2021). p. aeruginosa is a bacterium that is able to grow in several media and environmental conditions, in terms of its physiological properties, this bacterium has the ability to hydrolyse proteins (gamma proteobacteria) (kamal et al., 2021), and is unable to hydrolyse carbohydrates (glucose, lactose, mannitol, maltose, and sucrose) (bengi et al., 2020). p. aeruginosa is able to ferment citrate in culture to serve as its energy source. based on the background, it is necessary to test the antibacterial activity of lime mistletoe extract (d. petandra (l.) miq. against p. aeruginosa in vitro. this study aimed to determine the antibacterial activity of lime fruit extract against p. aeruginosa. the benefit of this research is that through this research the community can obtain additional information that lime mistletoe (d. pentandra (l.) miq) extract can be used for the treatment of infections caused by p. aeruginosa bacteria. materials and methods the tools used in the study were autoclave, stirring rod, bunsen, petri dish, glass jar, incubator, round ose needle, ruler, syringe, filter paper, large label, small thicker, refrigerator, magnetic stirrer, test tube rack, analytical balance, tissue, laminar air flow (laf), tweezers, test tube, cover paper, rope, hole punch, surgical set, marker, scam, urine pot, digital scale, measuring paper, and plastic. the materials used in this study were 30 ml nbf, distilled water, p. aeruginosa bacterial culture, benalu extract, cefradoxi antibiotic, sodium agar media, and 96% ethanol. medium preparation the medium used in this study was nutrient agar (na) media weighed using analytical scales as much as 16.8 grams, and dissolved with distilled water as much as 600 ml, then heated on a hot plate while homogenised. after boiling the media was removed and allowed to stand for a while. all mediums that have been made are wrapped using cover paper, then sterilised using an autoclave at 1210 c for 15 minutes. rejuvenation of pseudomonas aeruginosa two ose of p. aeruginosa bacteria derived from pure isolates in oblique agar stock were taken and inoculated in a petri dish containing na, then incubated by turning the petri dish over so that the bacteria grew perfectly, then incubated at 370 c for 48 hours. pure culture of p. aeruginosa bacteria was inoculated as much as two ose on a tube filled with nacl, (p. aeruginosa bacteria cultured on tubes to be injected into mice), then incubated for 24 hours at 37°c. rejuvenation of p. aeruginosa bacteria made working culture grown on na medium (petri dish) and stock culture by growing on tubes filled with nacl. inhibition test of lime mistletoe extract by disc method take media that has been frozen, take p. aeruginosa bacteria using a round ose inserted in a test tube containing na that is liquid/not yet frozen homogenised, then pour the liquid na media into the na contained in a petri dish that has been frozen. each disc saturated with laminar air flow (laf) lime mistletoe extract is attached to the inoculated na media and slightly pressed with tweezers until it adheres perfectly. the distance between one disc and another is at least 15 mm and discs that have been attached to the surface of the media should not be moved or shifted. na media that has been planted with discs is incubated at 37°c for 24 hours in an inverted position. parameters observed the inhibition zone was observed and measured in diameter using a ruler (in mm). the diameter of the inhibition zone measured is the clear area around the disc (no bacterial growth) measured from one end to the other through the middle of the disc. results and discussion table 1. one-way anova antibacterial activity test of trump extract d. petandra (l.) miq. against p. aeruginosa in vitro. source of variation ss (sum of squares) df (free degree) ms (mean square) f-statistic p-value between groups 0.241 6 0.040 4.269 0.005 in groups 0.295 21 0.014 total 0.536 27 the p value indicates the statistical significance of the f-statistic. a p value that is smaller than the set significance level indicates that there is a significant difference between the treatment groups. in this table, we savitri et al. – antibacterial activity test of trump extract dendrophtoe petandra (l.) miq. 555 can see that the p value (0.005) is smaller than the commonly used significance level (e.g. 0.05), indicating that the difference between the means of the treatment groups is statistically significant. table 2. tukey's hsd further test results. treatment group pair average difference lower limit interval limit upper interval conclusion kk+ -0.125 -0.395 0.145 nodifferent kp1 -0.35 -0.62 -0.08 nodifferent kp2 -0.2 -0.47 0.07 nodifferent kp3 -0.25 -0.52 0.02 nodifferent kp4 -0.25 -0.52 0.02 nodifferent kp5 -0.225 -0.495 0.045 nodifferent k+ p1 -0.225 -0.495 0.045 nodifferent k+ p2 -0.075 -0.345 0.195 nodifferent k+ p3 -0.125 -0.395 0.145 nodifferent k+ p4 k+ p5 p1 p2 p1 p3 p1 p4 p1 p5 p2 p3 p2 p4 p2 p5 p3 p4 p3 p5 p4 p5 -0.125 -0.1 0.15 0.1 0.1 0.125 -0.05 -0.05 -0.025 0 0.025 0.025 -0.395 -0.37 -0.12 -0.17 -0.17 -0.145 -0.32 -0.32 -0.295 -0.27 -0.245 -0.245 0.145 0.17 0.42 0.37 0.37 0.395 0.22 0.22 0.245 0.27 0.295 0.295 nodifferent nodifferent nodifferent nodifferent nodifferent nodifferent nodifferent nodifferent nodifferent nodifferent nodifferent nodifferent the mean difference is the difference between the means of the two treatment groups in a pair. the lower and upper limits of the 95% confidence interval indicate the range within which the mean difference is considered significant. the conclusion indicates whether the mean difference between pairs of treatment groups is considered significant or not based on a 95% confidence interval. if the confidence interval includes zero, then the difference is not considered significant, and vice versa. this table shows that most comparisons between pairs of treatment groups do not show significant differences within the 95% confidence interval. however, some comparisons show significant differences, especially between kand %p1. discussion the activity test was carried out by disc diffusion method against p. aeruginosa test bacteria, using disc method. the results of lime mistletoe extract (d. pentandra (l.) miq.) were made in various concentrations (20%, 40%, 60%, 80%, and 100%) with positive control ciprofloxacin and negative control aquadest. tests were carried out 4 times each. preparation of test solutions (20%, 40%, 60%, 80%, and 100% lime mistletoe extract, done by making the parent solution which is the result of lime mistletoe leaves (d. pentandra (l.) miq.) weighed as much as 5 grams and dissolved in 5 ml of distilled water. the sterilised na was put into a petri dish. solid agar media that is ready for use is then inoculated with bacteria by the pouring method, namely by means of p. aeruginosa test bacterial culture, each taken as much as 1 ml is poured into a petri dish, the petri dish is rotated left and right 5-7 times. then the disc paper was dipped into the test solution and control solution for ± 15 minutes, and placed on top of the agar media containing the test bacteria, then incubated at 37°c for 24 hours. after that, the diameter of the inhibition zone obtained was measured with a caliper. based on the results of the research on lime mistletoe leaf extract in one-way anova data analysis, the p value shows the statistical significance of the f-statistic. the p value that is smaller than the established significance level indicates that there is a significant difference between the treatment groups. then the results of tukey's hsd further test showed that comparisons between pairs of treatment groups did not show significant differences within the 95% confidence interval. however, some comparisons showed significant differences, especially between kand p1. the results of the antibacterial activity test of mistletoe leaf extract (d. pentandra (l.) miq.) against p. aeruginosa bacteria test. the average diameter of the inhibition zone obtained in the 20% concentration of lime mistletoe leaf extract with an average difference of 0.35, 40% concentration with an average difference of 0.2, 60% concentration with an average difference of 0.25, 80% concentration with an average difference of 556 biology, medicine, & natural product chemistry 14 (1), 2025: 553-556 0.25, 100% concentration with an average difference of 0.225, with the conclusion that it is no different. conclusions based on the research that has been done, it can be concluded that the type of active fraction of lime mistletoe leaves (d. pentandra (l.) miq.) in inhibiting the growth of p. aeruginosa bacteria is the ethyl acetate fraction. the group of antibacterial compounds contained in the active ethyl acetate fraction of lime mistletoe leaves is a group of streoids, triterpenoids, tannins, and flavonoids. acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university, and the laboratory of pathological anatomy, brawijaya university, malang, for their support during the completion of this research. authors’ contributions: lisa savitri, silvina sandra x. alves, and mochammad hanif hilmi designed the study, analyzed the data, and wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there is no funding for the research. references arca-suárez, j., fraile-ribot, p., vázquez-ucha, j. c., cabot, g., martínez-guitián, m., lence, e., & oliver, a. (2019). challenging antimicrobial susceptibility and evolution of resistance (oxa-681) during treatment of a long-term nosocomial infection caused by a pseudomonas aeruginosa st175 clone. antimicrobial agents and chemotherapy, 63(10), e01110-19. angeletti, s., cella, e., prosperi, m., spoto, s., fogolari, m., de florio, l., & ciccozzi, m. (2018). multi-drug resistant pseudomonas aeruginosa nosocomial strains: molecular epidemiology and evolution. microbial pathogenesis, 123, 233-241. khoiriah, a. n. (2021). test of antibiofilm activity of aspergillus aculeatus against pseudomonas aeruginosa biofilm (doctoral dissertation, maulana malik ibrahim state islamic university). lee, y., song, s., sheng, l., zhu, l., kim, j. s., & wood, t. k. (2018). substrate binding protein dppa1 of abc transporter dppbcdf increases biofilm formation in pseudomonasaeruginosa by inhibiting pf5 prophage lysis. frontiers in microbiology, 9, 30. pallett, r., leslie, l. j., lambert, p. a., milic, i., devitt, a., & marshall, l. j. (2019). anaerobiosis influences virulence properties of pseudomonas aeruginosa cystic fibrosis isolates and the interaction with staphylococcus aureus. scientific reports, 9(1), 1-18. permatasari, s. n., & . u. (2019). determination and proximate analysis of benalu leaves on arum manis mango tree in ketintang madya surabaya. journal of pharmacy and science, 4(2), 77-83. https://doi.org/10.53342/pharmasci.v4i2.140 savitri, n. h., indiastuti, d. n., & wahyunitasari, m. r. (2019). inhibitory activity of allium sativum l. extract against streptococcus pyogenes and pseudomonas aeruginosa. journal of vocational health studies, 3(2), 72. https://doi.org/10.20473/jvhs.v3.i2.2019.72-77 tovar-garcía, a., angarita-zapata, v., cazares, a., jasso-chávez, r., belmont-díaz, j., anchez-torres, v., & garcía-contreras, r. (2020). characterisation of gallium resistance induced in a pseudomonasaeruginosa cystic fibrosis isolate. archives of microbiology, 202(3), 617-622. yathurramadhan, h., & yanti, s. (2020). counselling on the use of traditional medicine in sigulang village. journal of education and development, 8(1), 4-5. http://journal.ipts.ac.id/index.php/ed/article/view/1495/666 wahyudi d, aman at, handayani nsn, soetarto es. 2019. differences among clinical isolates of pseudomonas aeruginosa in their capability of forming biofilms and their susceptibility to antibiotics. biodiversity 20(5): 1450 1456.doi: 10.13057/biodiv/d200538 https://doi.org/10.53342/pharmasci.v4i2.140 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 361-365 | doi: 10.14421/biomedich.2024.132.361-365 issn 2540-9328 (online) isolation and antibacterial activity of sembung (blumea balsamifera) leaf essential oil l., dc tika afriani*, azimatur rahmi, mia darmita sari department of pharmacy, mohammad natsir university, bukittinggi, west sumatra, jl. tan malaka, bukik cangang kayu ramang, kota bukittinggi tel. +62-752-21169, indonesia. corresponding author* tika.afriani91@gmail.com manuscript received: 27 may, 2024. revision accepted: 08 august, 2024. published: 01 october, 2024. abstract sembung leaf essential oil is a complex mixture in the form of oily yellow liquid with a unique aroma. it contains various volatile components that have antibacterial, antifungal, antioxidant and cytotoxic activities. this study aims to determine the antibacterial activity of essential oil blumea balsamifera l., dc against escherichia coli, pseudomonas aeruginosa and staphylococcus aureus. isolation of essential oil using steam distillation method with 0.21% rendement. inhibition activity was performed by test tube method with variation concentrations of essential oil were 10%, 20%, 40%, 80%, and 100% for each bacteria. results of this study showed that the highest antibacterial activity on escherichia coli at 80% concentration with an inhibition zone diameter of 27.53 mm, 100% concentration for pseudomonas aeruginosa with an inhibition zone diameter 25.72 mm and 100% concentration for staphylococcus aureus with inhibition zone diameter 27.31 mm. essential oil from (blumea balsamifera) l., dc leaves have a strong activity inhibition category against escherichia coli, pseudomonas aeruginosa and staphylococcus aureus. keywords: antibacterial activity; blumea balsamifera; essential oil; escherichia coli; pseudomonas aeruginosa; staphylococcus aureus. introduction the use of plants as medicine has recently become increasingly popular in the community to treat the diseases they are experiencing. this is supported by the presence of medicinal ingredients from nature that grow in abundance (ngajow et al., 2013). one of the medicinal plants commonly used by the community, especially in west pasaman, as traditional medicine, is the sembung plant (blumea balsamifera), which can be found almost throughout the archipelago (rahardjo, 2018). sembung plants are members of the genus blumea, and family asteraceae (compositae). they have traditionally been used to treat rheumatism, menstrual pain, influenza, bloating, bone pain, diarrhea, canker sores, asthma, and angina pectoris (dewantari et al., 2018). the leaf of the sembung plant is the most commonly used for treatment. the cleaned leaves can be used as medicine, either boiled and then drunk or applied externally (tari et al., 2016). the phytochemical identification of the sembung plant content revealed more than 100 metabolites such as dihydro*flavones, flavonoids, sterols, organic acids, monoterpenes, sesquiterpenes, triterpenes, essential oils, alkaloids, steroids, tannins, and glycosides, the volatile oil content of the sembung plant is 0.5 percent, and it contains camphor, cineol, borneol, and landerol (pang et al., 2014; roger et al., 2015). sembung leaf essential oil is a complex mixture that takes the form of an oily yellow liquid with a distinct aroma and contains a variety of volatile components with antibacterial, antifungal, antioxidant, and cytotoxic properties (boligon et al., 2013). the majority of the essential oil content in sembung plants is found in the leaves and stem branches (jiang et al., 2014; putri et al., 2020). according to research on the antibacterial activity of various variations of sembung extract, essential oil had the strongest antibacterial effect, with a minimum inhibitory concentration (mic) of 150 mg/ml for bacillus cereus and 1.2 mg/ml for staphylococcus aureus (he et al., 2020). the essential oil extracted from sembung leaves has the potential to damage cell membranes and degrade cell proteins beyond repair. as a result, bacterial cells lack nutrients and their growth is slowed, causing bacteria to die (pang et al., 2014; ruhimat, 2015). the purpose of this study was to isolate and investigate the antibacterial activity of sembung (balsamifera) essential oil against the growth of e. coli, p. aeruginosa, and s. aureus bacteria. https://doi.org/10.14421/biomedich.2024.132.361-365 362 biology, medicine, & natural product chemistry 13 (2), 2024: 361-365 materials and methods chemical material the materials used included sembung leaves obtained from west pasaman, escherichia coli (gram negative) atcc 10531, pseudomonas aeruginosa (gram negative) atcc 25619, staphylococcus aureus (gram positive) atcc 6538, nutrient agar media (merck®germany), physiological nacl 0.9%, ethanol 70% (bratachem, indonesia), ethanol 96% (bratachem, indonesia), ethyl acetate (bratachem, indonesia), koh (merck® germany), hcl (merck® germany), naoh (merck® germany), phenolphthalein (merck® germany), anhydrous sodium sulfate (merck® germany), and distilled water. equipment the tools used in this study include analytical balance (denver-instrument®), a set of steam distillation apparatus, an incubator (gallenkamp plus®), an oven, an autoclave (all american®), an aseptic cabinet, a vortex mixer (shimadzu® model). vm-100), hot plate (iec®), refrigerator, calliper (mitutoyo®), petri dish (normax®), micropipette (physiocare concept-eppendorf research®), loop needle, spritus lamp, pycnometer (pirex®), and other glassware commonly used in laboratories. distillation of sembung leaf essential oil dry samples of sembung leaves were collected in kinali, west pasaman regency, west sumatra. by compacting the leaves, the dry sample (20 kg) was placed in a steam distillation apparatus and heated for 6 hours to obtain a distillate mixture of oil and water with three distillations. anhydrous sodium sulfate was used to bind and separate the water from the essential oil in the resulting oil-water distillate mixture. allow for 24 hours before filtering and recording the volume of oil obtained (ml) (widyastuti & farizal, 2015). sembung leaf essential oil quality test organoleptic examination the organoleptic examination includes the shape, colour, and odour of the essential oil produced. ethanol solubility test 1 ml of essential oil is placed in a tared 10 ml measuring cup, followed by a slow addition of ethanol. shake the measuring cup after each addition of ethanol. for the first time, a uniform and homogeneous solution is obtained. keep track of the amount of ethanol you use. continue to add ethanol until you reach 10 ml. specific weight calculation specific gravity was determined using a pycnometer. before the essential oil is placed in the instrument, the essential oil must be at the same temperature as the temperature at which the measurement will be made. measurements were made after the temperature stabilized. determination of acid numbers a total of 2 g of essential oil was dissolved in 2.5 ml of neutral ethanol, and then 2-3 drops of phenolphthalein solution were added. then, it was titrated with 0.1 n koh until the pink color was steady. determination of the saponification number a total of 1 g of essential oil was mixed with 25 ml of 0.5 n koh solution. then, it was refluxed in a water bath for 1 hour while shaking frequently. titrate while hot with 0.5 n hcl and phenolphthalein as an indicator. the end point of the titration is reached when the solution turns cloudy white. determination of blanks is carried out with the same procedure. ester number calculation the ester number is critical in determining the quality of patchouli oil because ester is a component that influences the oil’s aroma. the ester number of essential oils is determined by saponifying the esters in an alkaline solution to re-titrate the excess alkali (saponification number acid number). sembung leaf essential oil antibacterial activity test the antibacterial activity test of sembung leaf essential oil was carried out by the agar diffusion method using the test bacteria. in a petri dish (normax®), several cylinders with a diameter of 6 mm were placed. a 1 ml suspension of each bacteria was added to 15 ml of media and placed in a petri dish. after the media solidifies, the cylinder is lifted, thus forming a hole in the media. sembung leaf essential oil at 10%, 20%, 40%, 80%, and 100% concentrations was placed in the hole and incubated for 24 hours at 37 °c. the results of the observations were indicated by the formation of a clear inhibition area around the hole, and the diameter was measured using a calliper (mitutoyo®) with three replications (widyastuti & farizal, 2015; septiani et al., 2017). data analysis the obtained data were analyzed with one-way anova at a 95% confidence level. results and discussion the distillation of 20 kg of sembung leaves using a water-steam system yielded 42 ml of essential oil with a yield of 0.21%. according to fakayode & abobi (2018), the essential oil content of the leaves is not less than 0.19% v/w, so from the calculation of the yield of the sembung leaf essential oil obtained is concluded that it has good quality. afriani et al. – isolation and antibacterial activity of sembung (blumea balsamifera) … 363 sembung leaf essential oil distillation using steamwater distillation yields a yellow liquid with a distinct aroma (typical of sembung). these findings are consistent with previous research (jiang et al., 2014; putri et al., 2020). one of the most important criteria in determining the quality and purity of essential oils is specific gravity. in this study, the average density of sembung leaf essential oil was 0.9213. (table 1). most of the essential oils have specific gravity ranging from 0.696–1.88 (rompas et al., 2016). the specific gravity of the essential oil used in this study falls within this range, indicating that the essential oil is quite good in terms of specific gravity. table 1. results of physical and chemical testing of sembung leaf essential oil. no type of test outcome 1 description color odor liquid yellow typical of sembung 2 specific gravity 0.9213 3 the solubility in ethanol 96% brown solution 1:1 4 the solubility in ethanol 70% brown solution 1:3 5 acid value 6.171 6 saponification value 75.735 7 ester value 69.564 the solubility of sembung essential oil in 96 percent ethanol was 1:1, while the solubility of sembung essential oil in 70 percent ethanol was 1:3. (table 1). the type of chemical components contained in the essential oil determines its solubility in alcohol. essential oils, in general, contain a variety of volatile alcohols, aldehydes, ketones, and ethers (widyastuti & farizal, 2015). the acid number is also an important factor in determining the oil's quality (syamsudin et al., 2018). in the distillation of the sembung essential oil test, the acid number was 6.171. (table 1). the function of determining the acid number is to determine an oil's acidity index. the acid number of sembung leaf essential oil indicates that the acid content in the oil is still low, implying that the oil does not contain many free acids or fatty acids. the essential oil of sembung leaf has 75.735 saponifications (table 1). because the saponification number is inversely proportional to the average molecular weight of the triglyceride constituent acids, it can be used to detect the presence of triglyceride esters whose fatty acids contain less than 16 carbon atoms or more than 18 carbon atoms (widyastuti & farizal, 2015). the ester number can be calculated using the saponification number. the ester number indicates how much alkali is required for ester saponification. the presence of an ester number in the oil indicates that the oil has a pleasant aroma, with sembung leaf essential oil having an ester value of 69.564. (table 1). the high number of esters in sembung oil is due to the large number of triglyceride esters that form saponification when an oil is dissolved in ethanol and a base is added. most fatty acids are found in the simplest oils because their building blocks are triglycerides, which are also known as fats, neutral fats, or triglycerides. triglycerides are glycerol esters with three fatty acid molecules. triglycerides are insoluble in water but soluble in nonpolar solvents such as chloroform, benzene, and ether. saponification is the hydrolysis of triacylglycerol by koh and naoh, which results in a mixture of k+, na+, and glycerol. when triacylglycerol is exposed to air, it produces a product that causes taste and odour to deposit in oils, which frequently become rancid (widyastuti & farizal, 2015). the method used to test the antibacterial activity of sembung essential oil is the diffusion method with a 6 mm diameter well technique against gram positive bacteria s. aereus and gram negative bacteria e. coli and p. aeruginosa. the antibacterial activity of sembung leaf essential oil was tested at concentrations of 10%, 20%, 40%, 80%, and 100%. the negative control used was ethyl acetate (bratachem). the selection of ethyl acetate was because ethyl acetate did not have an antibacterial effect, so it did not affect antibacterial test results. it is also used as a solvent for essential oils. hanh et al., (2021) identified 50 essential oil constituents in sembung leaves, with the highest content being borneol, caryophylle, ledol, tetracyclo [6,3,2,0,(2.5).0(1.8)tridecan-9-ol, 4, 4-dimethyl]. borneol is thought to be the active compound of sembung leaf essential oil which has antibacterial activity. borneol belongs to the bicyclic monoterpene group which has a phenolic -oh group. compounds with phenolic -oh groups can damage cell membranes by forming protein complexes through hydrogen bonds (rompas et al., 2016). the smallest concentration of the sample that is able to inhibit the growth of the test bacteria with the formation of a clear zone is the value of the minimum inhibitory concentration (mic) of the sample. the mic levels of essential oil samples against e. coli, p. aeruginosa, and s. aureus bacteria were at a concentration of 10%, with the diameter of each inhibition zone being 21.3150 mm; 22.4550 mm; and 20.8433 mm (tables 2, 3, and 4). the diameter of the clear zone produced by the three test bacteria differed according to the amount of concentration given. table 2. the average diameter of the inhibition zone of sembung essential oil against e. coli. concentration % inhibition zone diameter (mm) (�̅� ± 𝑺𝑫) 10 20.8433±.73777 20 22.8650±.45632 40 25.0933±2.17097 80 27.5217±.54259 100 24.5250±4.28266 364 biology, medicine, & natural product chemistry 13 (2), 2024: 361-365 table 3. average diameter of the inhibition zone of sembung essential oil against p. aeruginosa. concentration % inhibition zone diameter (mm) (�̅� ± 𝑺𝑫) 10 22.4550±1.41733 20 23.0800±1.25654 40 22.4283±2.02095 80 24.5167±1.48918 100 25.7183±2.06037 tabel 4. average diameter of the inhibition zone of sembung essential oil against s. aureus. concentration % inhibition zone diameter (mm) (�̅� ± 𝑺𝑫) 10 21.3150±.66006 20 24.0950±1.84031 40 18.6817±1.51319 80 25.6767±2.21854 100 27.3050±1.08316 the highest clear zone (inhibition) in e. coli bacteria was at an 80 percent concentration with a diameter of 27.5217 mm, while the lowest clear zone was at a 10% concentration with a diameter of 20.8433 mm (table 2). the highest clear zone in p. aeruginosa was at 100% concentration and had a diameter of 25.7183 mm, while the lowest clear zone was at 40% concentration and had a diameter of 22.4283 mm (table 3), while the highest clear zone in s. aureus bacteria was at 100% concentration. at 40% concentration, the lowest clear zone was 18.6817 mm (table 4). the inhibition zone with a small diameter had low antibacterial activity, whereas the inhibition zone with a large diameter had high antibacterial activity. the components of the substances contained in medicinal plants that could weaken, strengthen, improve, or change the inhibition zone caused the large increase and decrease. furthermore, the quality and quantity of substances found in medicinal plants (syamsudin et al., 2018). according to research (syamsudin et al., 2018), the antimicrobial activity of medicinal plants against s. aureus bacteria had an inhibitory zone value that increased and decreased at different concentrations. seed components, inoculum size, and bacterial metabolic activity are all factors that influence antibacterial activity (bujung et al., 2017). this is possible because the test solution has nonpolar properties. because of its low solubility in polar media, increasing the concentration of the test solution used makes it more difficult for the test solution to dissolve or diffuse into polar media. it can also occur as a result of several factors that influence the diameter of the inhibition zone, including the organism's sensitivity, the culture medium, the incubation conditions, and the rate of diffusion (jamaludin et al., 2017). the following is how these criteria are determined based on the category of inhibition according to (khan et al., 2019): a diameter of less than 10 mm is said not to inhibit the growth of the test bacteria (t), 11-15 mm is considered weak (l), and 16-20 mm is considered moderate. (s) and a diameter greater than 20 mm are classified as strong (k). it can be concluded that the inhibitory power of essential oil from the leaves of the sembung plant against e. coli, p. aeruginosa, and s. aureus is strong. the diameter of the inhibition zone produced by the essential oil of sembung leaves was found on e. coli and p. aeruginosa with a significance value of p> 0.05, indicating that h0 was rejected and h1 was accepted. this means that, based on the inhibition zone, the essential oil of sembung leaves did not have a significant difference in antibacterial potency. based on the diameter of the resulting inhibition zone, the significance value of s. aureus p0.05 indicates that h1 is rejected and h0 is accepted. this means that the antibacterial potential of sembung leaf essential oil varies significantly depending on the inhibition zone. conclusions the study's findings indicate that the essential oil of sembung (blumea balsamifera) l., dc leaves has antibacterial potential against e. coli, p. aeruginosa, and s. aureus with strong inhibition criteria. competing interests: the authors declare that there are no competing interests. references boligon, a. a., feltrin, a. c., athayde, m. l., & feltrin, a. c. (2013). determination of chemical composition, antioxidant and antimicrobial properties of guzuma ulmifolia essential oil. american journal of essential oils and natural products, 1(1), 23-27. bujung, a. h., homenta, h., & khoman, j. a. (2017). uji day ta hambat ekstrak biji buah alpukat (persea americana mill.) terhadap pertumbuhan streptococcus mutans. e-gigi, 5(2). dewantari, r., lintang, m., & nurmiyati, n. (2018). jenis tumbuhan yang digunakan sebagai obat tradisional di daerah eks-karesidenan surakarta. bioedukasi uns, 11(2), 117-122. fakayode, o. a., & abobi, k. e. (2018). optimization of oil and pectin extraction from orange (citrus sinensis) peels: a response surface approach. journal of analytical science and technology, 9(1), 1-16. hanh, t. t. h., giang, v. h., trung, n. q., van thanh, n., quang, t. h., & cuong, n. x. (2021). chemical constituents of blumea balsamifera. phytochemistry letters, 43, 35-39. he, c., yang, p., wang, l., jiang, x., zhang, w., liang, x., ... & lv, c. (2020). antibacterial effect of blumea balsamifera dc. afriani et al. – isolation and antibacterial activity of sembung (blumea balsamifera) … 365 essential oil against haemophilus parasuis. archives of microbiology, 202, 2499-2508. jamaludin, n., pulungan, m. h., & warsito, w. (2017). uji aktivitas antibakteri minyak atsiri jeruk purut (citrus hystrix dc) terhadap klebsiella pneumoniae atcc. industria: jurnal teknologi dan manajemen agroindustri, 6(2), 61-66. jiang, z. l., zhou, y., ge, w. c., & yuan, k. (2014). phytochemical compositions of volatile oil from blumea balsamifera and their biological activities. pharmacognosy magazine, 10(39), 346. khan, z. a., siddiqui, m. f., & park, s. (2019). current and emerging methods of antibiotic susceptibility testing. diagnostics, 9(2), 49. ngajow, m., abidjulu, j., & kamu, v. s. (2013). pengaruh antibakteri ekstrak kulit batang matoa (pometia pinnata) terhadap bakteri staphylococcus aureus secara in vitro. jurnal mipa, 2(2), 128-132. pang, y., wang, d., fan, z., chen, x., yu, f., hu, x., ... & yuan, l. (2014). blumea balsamifera—a phytochemical and pharmacological review. molecules, 19(7), 9453-9477. putri, septi dwi putri., rusdi & asra, ridho. 2020. a review: antioxidant activities of sembung leaves (blumea balsamifera (l.) dc). eas journal of pharmacy and pharmacology, (2),(5). rahardjo, s. s. (2016, april). review tanaman sembung [blumea balsamifera (l.)]. in proceeding of mulawarman pharmaceuticals conferences (vol. 3, pp. 18-28). roger, t., pierre-marie, m., & igor, v. k. (2015). phytochemical screening and antibacterial activity of medicinal plants used to treat typhoid fever in bamboutos division, west cameroon. journal of applied pharmaceutical science, 5(6), 034-049. rompas, d. e., runtuwene, m. r., & koleangan, h. s. (2016). analisis kandungan fitokimia dan uji aktivitas antioksidan dari tanaman lire (hemigraphis repanda (l) hall f.). jurnal mipa, 5(1), 36-39. ruhimat, u. (2015). daya hambat infusum daun sembung (blumea balsamifera) terhadap pertumbuhan bakteri escherichia coli dengan metode difusi cakram. jurnal kesehatan bakti tunas husada: jurnal ilmu-ilmu keperawatan, analis kesehatan dan farmasi, 13(1). septiani, v., choirunnisa, a., & syam, a. k. (2017). uji aktivitas antimikroba ekstrak etanol daun karuk (piper sarmentosum roxb.) terhadap streptococcus mutans dan candida albicans. kartika: jurnal ilmiah farmasi, 5(1), 7-14. syamsudin, r. a. m. r., perdana, f., & mutiaz, f. s. (2019). tanaman temulawak (curcuma xanthorrhiza roxb) sebagai obat tradisional. jurnal ilmiah farmako bahari, 10(1), 51-65. tari, m., & nely, n. (2016). uji aktivitas antibakteri terhadap beberapa fraksi daun sembung rambat (mikania micrantha kunth.) terhadap bakteri penyebab penyakit kulit. jurnal ilmiah bakti farmasi, 1(2). widyastuti, w., & farizal, f. (2014). formulasi gel minyak nilam dan uji daya hambatnya terhadap bakteri staphylococcus aureus. scientia, 4(2), 60-5. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 2, 2015 | pages: 31-33 | doi: 10.14421/biomedich.2015.42.31-33 effect of lunasia amara blanco on sperm number, sperm motility, and testicular histology of male rats muhammad ja’far luthfi biology department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: jafarluthfi@yahoo.com abstract sanrego (lunasia amara), has been used in the folk medicine to increase and/or to treat male fertility. however there is no scientific evidence to confirm the positive effect of the plant on an improvement of male fertility. the objective of this research was to study the effects of the plant (on adult sprague-dawley male rats) at the doses of 30 mg/kg, 60 mg/kg, and 90 mg/kg on the sperm count, motility, and testicular histology. administration were given by force-feeding between 10.00 am and 12.00 pm daily for a period of 42 days followed by sperm quality analysis and testicular histology evaluation. the sperm analysis showed that the sanrego increased the sperm count and sperm motility. the testicular histology also revealed positive effect of the plant on spermatogenesis. overall the present study showed the sanrego is potential plant to increase male fertility. keywords: sanrego, male fertility, testis histology, sperm quality, sperm number, sperm motility introduction male infertility is an increasingly problem in the modern world. inspite of the many effort that have been employed over the years, medical treatment of male infertility has been a failure (hamadeh, 2001; kohn, 2001). in majority of cases of male infertility, the cause is low sperm quality (shen and ong, 2000; clouatre, 2005). one of the important factor that influence fertility are environment. unlike genetic causes of male factor subfertility, environmental causes of subfertility are of particular interest, because of the possibility of its curative or preventive properties (ebisch et al., 2006). a significant environmental factor is nutrition. it is in this area that natural remedies could shine. animal studies have demonstrated the importance of the effects of nutrition on spermatogenesis (mackay 2004; sinclair, 2000). plant have been used by human since time of unmemorable. however, most claims that plant could enhance virility, increase fertility, or arouse sex libido are root in folklore with very little scientific evidence to support them (anonym, 2005; nickel, 2001). despite gap between science and traditional medicine, there are historical evidence to the betterment of the sexual function by medicinal plant used by many culture (waddell, 1980). sanrego (lunasia amara blanco) has been used by local people in some area of sabah (goh et al., 1997), philipina and indonesia (perry, 1980) as folk medicine to cure many ailment. the plant use to treat snake bite, skin diseases, swollen limb, also as a remedy for stomach trouble (perry, 1980). in the recent years, however, there is an extention of the plant’s initial use. based on observation on the goodness of the sexual function of horse graze on sanrego, sulawesian people popularized sanrego to treat male sexual problem. some studies were conducted to determine the effect of sanrego on male reproductive system of rat (darise, 1999; sutanto, 2002). however the result was far from complete. a systematic approach to provide scientific evidence to confirm the claims of the traditional used is greatly needed. the present study was designed to determine the effect of an aqueous extract of lunasia amara blanco stem on the sperm quality and testicular histology of male rats. materials and methods animals fifteen weeks male sprague-dawley rats from the animal house of the university kebangsaan malaysia were used. animals were housed under standarized conditions. rats were divided at random into 4 groups including control. six rats per group received 0, 30, 60, or 90 mg/kg aqueous extract of sanrego each day for 42 days. preparation of aqueous extract of sanrego sanrego was obtained from south sulawesi, indonesia. the plant was identified by djoko santoso, a botanist of the departement of pharmaceutical biology, gadjah mada university. the voucher number 53/biofar/020307 was deposited at the department. aqueous extract was prepared according to a traditional method (gonzales, et. al., 2004; gonzales et al., 2006). in brief, 500 g of the dry powder of sanrego stem were placed in a container with 1500 ml of distilled water and boiled for 30 minutes. the extract was left to cool and 32 biology, medicine, & natural product chemistry 4 (2), 2015: 31-33 was then filtered. the filtrate then freeze-dried and kept in 4° c refrigerator until use. sperm analysis cauda epididymis were separated according to the hamilton (1975). sperm counts were determined using improve neubauer hemocytometer as described previously (prasad et al., 1972; nafa & eshre-siga, 2002) with some modification. in brief, cauda epididimis was minced in 15 ml bww medium (biggers et al., 1971) and incubated with 5 % co2 for 30 minutes at 370c. data were expressed as number of sperm per cauda epididymis. progressive sperm motility was assesed subjectively based on who laboratory manual (1999). treatment to administer sanrego or vehicle, an appropriate oral needle were used to give, once a day, 2 ml aqueous sanrego extract or distilled water. one day after cessation of treatment, the rats were sacrificed. the left cauda epididymides were removed and cleared off the attached fat and connective tissue. the cauda epididymides were prepared for sperm analysis. the testis were prepared for histological preparation. testicular histology after fixation in bouin’s solution, testes were transferred to 70% ethanol, embedded in paraffin and cut at 5 µm. sections were stained with hematoxilin and eosin and digital images of sections were captured using an olympus bx51 microscope and software analysis pro. morphometrical evaluation was done using coreldraw x3 version 13. the diameter of seminiferous tubules was measured in 15 round tubular sections per animal (n = 3 rats/group) at 100×. statistical analysis data are expressed as mean + s.d. and analysed for statistical significance by using one-way analysis of variance (anova). when f test was found significant (p < 0.05), the data were further subjected to the tukey test. data of seminiferous tubule measurement were analyzed using t-test. data were analyzed using software spss 14.0. results and discussion the average sperm number of rats consuming sanrego at dose 30 mg/kg (43.4375 ± 5.22280 x 106), dose 60 mg/kg (49.3000 ± 2.43475 x 106), and dose 90 mg/kg (47.000 ± 1.59992 x 106) were significantly increased compared to the average number of sperms from control groups (35.3375 ± 4.74091 x 106) (table 1). the sperm number from rats consuming sanrego at dose 60 mg/kg was higher than sperm number from rats consuming sanrego at dose 90 mg/kg. however, this were not significantly different. the increase of sperm number indicates the beneficial effect of sanrego on testicular spermatogenesis. however, histological testicle analysis are needed to verify this observation. table 1. sperm count and motility grade of rats after 42 days treatment with water extract of sanrego. treatment dose sperm count (x 106) motility grade control distilled water 35.3375 ± 4.74091 b sanrego 30 mg/kg 43.4375 ± 5.22280 b 60 mg/kg 49.3000 ± 2.43475 a 90 mg/kg 47.000 ± 1.59992 a note for the grade of motility who (1999) (a): rapid progressive motility ; ≥ 25 μm/s, (b): slow or sluggish progressive motility, (c): non progressive motility ; < 5 μm/s, (d): immotile the effect of sanrego on the sperm motility were showed in table 1. the treatment groups have the betterment of sperms motility compared to the control groups. treatment of sanrego with dose 60 mg/kg and dose 90 mg/kg showed ‘a’ grade movement, while control group showed ‘b’ grade movement. sperm motility has been considered as one of the most important predictors of fertility. several reports have demonstrated the correlation of motion parameters with fertilization rates (liu et al., 1991). further studies are required to confirm the mechanisms of action of sanrego on sperm motility. table 2. average of seminiferous tubules diameter of control rats group compare to treatment group. group tubules seminiferuos diameter average + sd control 44.587 ± 3.096 dose 60 mg/kg 48.969 ± 3.504 treatment group provided significant increases in the diameter of seminiferous tubules (48.969 ± 3.504) when compared to the control group (44.587 ± 3.096) (p < 0.005) (table 2). this result was coincide with the sperm number and sperm motility analysis. in general, the present study revealed that aqueous extract of sanrego enhanced the sperm motility, and seminiferous tubules diameter in rats. this biological activity may be due to one or more of the compound/phytochemicals present in the extract. at least ten alkaloids and quinolines are among the constituent of sanrego (goodwin, 1959). this could directly or indirectly lead to an increase in the potential of fertility in male rats, mainly by acting directly on the pituitary gland and influencing its secretion and/or the pituitary gland– spermatogenic axis. however, there is still unknown which particular metabolite of the sanrego has effect on muhammad ja’far luthfi – effect of lunasia amara blanco on sperm number, sperm motility … 33 the variables studied. further studies are required and are in progress to isolate and identify the active principle(s) that acts upon this axis, influencing and increasing this process and the precise mode of its action. conclusion in conclusion, this study support the extention of the traditional use of this herb to treat male fertility problem. the sperm count analysis showed that the sanrego increased the sperm count, progressive motility, and seminiferous tubules diameter. overall the present study showed that sanrego is potential herb to increase male fertility. references anonym, 2005. aphodisiac galore? total health 27 (1): 7 adimoelja, a. 2000. phytochemicals and the breakthrough of traditional herbs in the management of sexual dysfunctions. int. j. androl. 23, suppl.2: 82-84 biggers, j.d., whitten, w.k. and whittingham, d. 1971. the culture of mouse embryos in vitro. in methods in mammalian embryology pp86-116. ed. jc daniel. freeman, san francisco, ca. clouatre, d. 2005. new help male fertility. total health 26/4: 2627 darise, m. 1999. obat kuat: berkuda dengan sanrego. gatra 23: 13. ebisch, i.m.w., f.h. p. pierik, f.h. de jong, c.m.g. thomas, r.p.m. steegers-theunissen. 2006. does folic acid and zinc sulphate intervention affect endocrine parameters and sperm characteristics in man? int. j. andr. 29(2): 339-345 foster, s., j.a. duke. 2000. a field guide to medicinal plants and herbs. second edition. houghton mifflin company. boston. goh, s.h., k.h. lee, c.h. chuah. 1997. phytochemical study of borneo: selected plants from sabah lowland forest. j. herbs spices med. pl. 5: 29-52 goodwin, s., a. f. smith, a. a. velasquez, e. c. horning. 1959. alkaloids of lunasia umara blanco. isolation studies. j. am. chem. soc. 81: 6209-6213 hamadeh, m. e., t. zeginiadov, p. rosenbaum. 2001. predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. archives of andrology 46: 99-104 hamilton, d.w. 1975. structure, function of the epithelium lining the ductuli efferents, ductus epididymis and ductus deferens in the rat. in: handbook of physiology, section vii, endocrinology, vol.5, male reproductive system (eds d.w. hamilton and r.o. greep). pp. 259-301. american physiological society, washington d.c. humason, g.l. 1979. animal tissue techniques. freeman and company. san fransisco. kohn, f.m. 2001. nonmedical and naturopathic approaches to treatment of male fertility. in proceedings of the 7th andrology symposium. treatment of male infertility viewpoints, controversies, perspectives. giessen, germany. p: 337 liu, d. y., clarke, g. n., & baker, h. w. g. (1991). relationship between sperm motility assessed with the hamilton-thorn motility analyzer and fertilization rates in vitro. journal of andrology, 12, 231-239. mackay, d. 2004. nutrients and botanicals for erectile dysfunction: examining the evidence. alter. med. rev. 9 (1): 4 -16 nafa, eshre-siga. 2002. manual on basic semen analysis. pei, j., e. strehler, u. noss, m. abt, p. piomboni, b. baccetti, k. sterzik. 2005. quantittive evaluation of spermatozoa ultrastructure after acupunture treatment for idiopathic male infertility. fertility and sterility 84(1) : 141-147 perry l. m. 1980. medicinal plants of east and southeast asia: attribute, properties and uses. the m.i.t press. massachusetts. prasad, m. r. n., n.j. chinoy, k.m. kadam. 1972. changes in succinic dehydrogenase levels in the rat epididymis under normal and altered physiologic conditions. fertility and sterility 23 (3): 186-190. shen, h., c. ong. 2000. detection of oxidative dna damage in human sperm and its association with sperm function and male infertility. free radical biology and medicine 28(4): 529-536 sinclair, s. 2000. male infertility: nutritional and environmental considerations. alternative medicine review 5 (1): 28-38. sutanto, h. 2002. uji efek androgenik ekstrak kayu sanrego (lunasia amara blanco) pada tikus jantan.. tesis sarjana. universitas katholik widya mandala surabaya. unpublished. nickel, l.n. 2001. nature’s aphrodisiacs. crossing press. usa waddell, t.g., h. jones, a. l. keith. 1980. legendary chemical aphrodisiacs. journal of chemical education 57(5): 341-342. who. 1999. laboratory manual for the examination of human semen and semen-cervical mucus interaction. new york: cambridge university press. content_v4n2_2.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 1, 2018 | pages: 1-4 | doi: 10.14421/biomedich.2018.71.1-4 issn 2540-9328 (online) leukocytes description of mudskipper (periophthalmodon schlosseri) of barito river estuary, desa tanipah, kalimantan selatan heri budi santoso*, hidayaturrahmah, muhamat biology department, mathematics and science faculty, lambung mangkurat university, south borneo jl. brigjen h. hasan basri, banjarmasin utara, banjarmasin, kalimantan selatan, indonesia author correspondency*: heri_budisantoso@yahoo.com abstract leukocytes have an important role in driving away infections from pathogen microorganism by phagocytosis together with macrophages. the aim of this research was to analyze the leukocytes’ differentiation (eosinophil, basophil, neutrophil, lymphocytes, and monocytes) of mudskipper‘s blood (periophthalmodon schlosseri). samples were taken from desa tanipah kalimantan selatan. the sampling locations were decided by purposive or taking the data intentionally according to the consideration of mudskipper‘s location which is not homogenous. the data collection were conducted using line transect which means the sample was taken according to how many encounters can be taken. there were 15 mudskippers taken from desa tanipah used in this research. the parameter observed was the differentiation of leukocytes which consists of the percentages of monocytes, lymphocytes, eosinophil, basophil, and heterophil. the result showed that the percentage of lymphocytes in mudskippers is 62+4,1% and monocytes 24,7+0,8%, and the neutrophil is 0,5+0,1% and eosinophil 0,6+0,1% and no basophils were found. according to the result, it can be concluded that lymphocytes is the most dominant one amongst others. keywords: leukocytes’ differentiation; desa tanipah; periathalmodon schlossery; mudskipper introduction the description of differentiation of leukocytes is one of several parameters to complete the blood’s profile, because the leukocytes’ differentiation is component which has important function as first self-defense when diseases or patogen struck dellman & brown, 1989). the leukocytes’ differentiation consists of lymphocytes, monocytes, neutrophil, basophil, and eosinophil. leukocytes have a role in driving away the infections from pathogen microorganism by phagocytosis together with macrophage. the number of leukocytes in fish is more than the number of leukocytes in human’s blood (fujaya, 2004). the number of leukocytes of human’s blood is about 5.000-9.000 cells/µl, whereas the number of leukocytes in fish’s blood is 20.000-150.000 sel/µl. if the infective materials come in, the number of leukocytes in blood will increase (moyle & cech, 1988). mudskipper/ikan timpakul/ikan gelodok (periophthalmodon schlosseri) is found along the coastal beaches and the habitat is spread in indonesia, thailand, peninsular of malaysia, singapore and some coastal beaches of indonesia (baker, 2011). this animal is included as threatened species because the condition of coastal mangroves increasingly damaged everyday (bay science foundation, 2009; fadli, 2010). for some provinces in indonesia, the flesh of mudskipper is used as the medicine for asthma (fadli, 2010). moreover, p. schlosseri is known has potency as bio-indicator in coastal ecosystem (shirani et al., 2010). the estuary of barito river is the mouth’s river with mangrove forest which is more than 30 km along the west coastal and around 20 km along the river to the inland. the swamps of mangroves forest which are rich of fauna who can adapt to the mud which is changing from the inundated to opened (flux and reflux). one of the faunas that can be found in the coastal beaches at the barito river estuary kalimantan selatan is mudskipper (periopthalmodon schlosseri) (mackinnon et al., 2000; shirani et al., 2014). according to lavabetha et al. (2015) mudskipper’s (p. schlosseri) blood profile from muara sungai barito kalimantan is the number of erythrocytes is 3,36±0,1x10 6sel/µl; the he level is 12,38±0,56 gr%; the hematocrit is 41,53±0,60 %; the number of mcv is 123,78±3,94 μm3 ; the number of mch is 36,88±1,82 pg/sel; the number of mchc is 29,80±1,18 g/dl. the average of leukocytes’ total of mudskipper (p. schlosseri) is 22,62±5,09 x103 cells/µl, whereas the number of leukocytes in common fish is between 30.000 until 150.000 cells/µl. it can be said that the amount of leukocytes of p. schlosseri is higher than the leukocytes’ number in other animals (hrubec & smith, 2000). the research was done to describe differentiation of leukocytes of mudskipper at barito river estuary, https://doi.org/10.14421/biomedich.2018.71.1-4 2 biology, medicine, & natural product chemistry 7 (1), 2018: 1-4 especially at desa tanipah, has never been reported before. based on that consideration, this research was focused on the differentiation of leukocytes (eosinophil, basophil, neutrophil, lymphocytes and monocytes) of p. schlosseri from barito river estuary, kalimantan selatan. the objective of this research was to analyze the differentiation of leukocytes (eosinophil, basophil, neutrophil, lymphocytes and monocytes) of mudskipper’s blood (periophthalmodon schlosseri) at barito river estuary, desa tanipah, kalimantan selatan. materials and methods tools and materials the tools used in this study were a set of fishing equipment, bucket, drain, analytic balance, tray, tissue paper, object’s glass, syringe, haemacytometer, camera, and microscope. the materials were blood (periophthalmodon schlosseri), edta solution (ethylene diamine tetra acid), giemsa solution 10%, alcohol 70%, methanol and clean water. the procedure of research the decision of location in taking samples the determination of sampling location in taking samples was conducted in purposive or taking the samples incidentally according to the consideration of heterogeny of the sample location. the samples collected in a region flux and reflux of bahagia river, desa tanipah kecamatan tabunganen kabupaten barito kuala, south borneo. the samples was taken by line transect method namely the samples were taken according to its encounter. preparation of blood samples the blood’s samples of mudskipper were taken from caudal vein between the squama near the tail using syringe which was dampened with anticoagulant edta (ethylene diamine tetra acid). syringe’s needle was inserted from the anal into vertebra until the needle touched the bone. the blood was suctioned for 1 ml then the needle will be put off, and the blood’s samples was moved to the tube (erika, 2008). preparation of blood cell slides blood cell preparation was conducted by placing a drop of blood in object glass. the second object’s glass was put with angle 45° above the first one, then moved it to the back and touched the blood so the blood was spread. the second object glass was moved to the opposite direction so it made a thin layer of blood. the blood cell slide preparation was then air dried. after that, fixation would be conducted by saturated the preparation in methanol for 5 minutes, then it would be dried the slide was then immersed to giemsa solution for 30 minutes. after that, it was washed with clean water and let it dry. then, the preparation was observed under light microscope with strong magnification, and it was counted for each type of leukocytes (roberts, 1992). the observation of leukocytes’ differentation the observation of leukocytes’ differentiation was conducted to decide the percentage of each leukocyte’s type in blood, by observed the blood cell slides under microscope. the micro setting in microscope was used, then it was counted from the side to the underside, then it moved to the right and to the top and so on. the calculation of leukocytes was conducted with strong magnification. the method used was shilling method which the calculation was conducted with different viewing field as 10 viewing fields or until the leukocytes’ number was reached 100 (mitruka & rawnsley, 1977). the technique of data collection the data collection was quantitative. quantitative data consisted of the number of leukocytes’ blood of mudskipper (eosinophil, basophil, neutrophil, lymphocytes and monocytes). the quantitative data taken in this research was processed statistically. the data was served in mean and standard deviation. results and discussion results the result of laboratory’s analysis of the measurement of leukocytes’ differentiation of mudskipper in barito river estuary was served in table 1 and figure 1 as follows: table 1. description of percentages (%) leukocytes’ differentiation of mudskipper. no fish (n) length (cm) weight (g) lymphocytes monocytes neutrophils basophil eosinophil p1 5 23,8 157,4 58,6 25,4 0,6 0 0 p2 5 24,12 162,6 60,8 25 0,2 0 0,8 p3 5 23.32 153,64 66,6 23,8 0,8 0 1,0 average 5 16,30 + 13,27 157,88+ 4.5 62+4,13 24,73+0,83 0,53+0,1 0 0,6+0,1 santoso et al. – leukocytes description of mudskipper … 3 figure 1. description of differentiation of mudskipper leukocytes. magnification 40 x. discussion lymphocytes percentage from the observation of lymphocytes percentage, it was found around 62+4,13%. according to the observation’s result, the taken average percentage was under normal range. the normal percentage of lymphocytes in fish was between 71,12 82,88% (robert, 1978). this condition showed the reduction of lymphocytes’ number which called as lymphocytosis. lymphocytes found in mudskipper were more than human lymphocytes around 20% 40%. the more lymphocytes in body the more body can attack the disease. lymphocytes had an important role in immune’s respond and it produced the antibody. according to jain (1986) & jain (1993), lymphocytes had important roles in producing the hormonal and cellular immunity to attack and destroy the disease agent. the increase of leukocytes number in blood circulation was called lymphocytosis; however, the decrease was called leucopenia. the decrease of lymphocytes’ number in peripheral blood occured because most lymphocytes were pulled from the circulation and concentrated into the tissue where there was inflammation (jain, 1993). moyle & cech (1988) reported that lymphocytes functioned as antibody’s production to face foreign materials from outside if there were a decreasing percentage of lymphocytes in circulation when an infection occured, it predicted as lymphocytes activity in antibody production was disturbed. the response of leukocytes’ number and leukocytes’ differentiation will be iaffected by several stressor, such as, temperature, season, activities, hunger’s condition, self-defense, maintenance, and density, diseases, infections, parasites, poisoned and metals. the leukocytes’ number and leukocytes’ differentiation will be increased or decreased (schalms, 2000). mudskipper had higher activities if we compared to other fish because it is air-breathing and it can adapt in land and water. the difference of haematological parameter of fish reflected the ecological condition in their habitat and it was their physiological adaptation in their life’s way. monocytes’ percentages from observation, the monocytes found was 24,73% which the monocytes percentage in fish commonly was 0,1% (robert, 1978). according to moyle & cech (1988), there would be less monocyt in leukocyt unless there were infection in blood circulation. roberts (1978) reported that the monocytes percentage in fish was 0,1% from the total population of circulated leukocytes. monocytes had a role as macrophage and it was found in an infection’s area (dellman & brown, 1989). monocytes and macrophage of the tissues will do the phagocytosis to the tissues’ cells and the disease agent (nabib & pasaribu, 1989). neutrophil’s percentage from observation, the neutrophil found was 0,53% which the monocytes percentage was below the normal average. the percentage of normal average of neutrophil of fish was around 6-8% (roberts, 1978). the neutrophil’s cell functioned in the blood’s vein in phagocytosis the bacteria faster because p. schlosseri was spending more time outside the water. in this case, it can be said that neutrophil cell functioned in blood’s veins in phagocytosis the bacteria faster because p. schlosseri was spending more time outside the water. 4 biology, medicine, & natural product chemistry 7 (1), 2018: 1-4 eosinophil and basophil percentage the observation result from mudskipper blood samples were there was no basophil cell (0) and the eosinophil was 0,6%. the average percentage of basophil cell of mudskipper was under the normal average, which the normal average was between 0.5–1%. nabib & pasaribu (1989) reported that eosinophil and basophil was rarely participated in fish blood circulation. the eosinophil and basophil play a role in parasitic infections and allergic response which connected to the critical disease. conclusion according to the result of this research, the lymphocytes percentage was found as the most dominant amongst other leukocyt types, which was 62%. the monocyt percentage was 24.73% and the eosinophil percentage was 0.6%. the neutrophil percentage was found as the lowest one amongst all the population of leukocyt type which was 0.5% whereas basophil was not found. references bay science foundation. 2009. periophthalmodon schloseryi (pug-headed mud skipper). zip code zoo index to als. http://zipcodezoo.com/animals/p/periophthalmodon%5fschl osseri/default.asp dellman, h.d & e.m. brown. 1989. textbook of veterinary histology. hartono's translation. ui press, jakarta. erika, y. 2008. description of leukocyte differentiation in mujair fish (oreochromis mossambicus) in ciampea region bogor. essay. faculty of veterinary medicine. ipb, bogor. fadli, a. 2010. timpakul, from aspiration. e-paper compass. january 8, 2008. http://m.kompas.com/iphone/read/data/2008.01.08.18214974 fujaya, y. 2004. fish physiology, basic development of fishery technique. pt rineka cipta. jakarta hrubec, t.c. & s.a. smith. 2000. hematology of fish. in feldman, b.f., j.g. zinkl, & n.c. jain (editor). schalm's veterinary hematology fifth jain, n.c. 1993. essentials of veterinary hematology. lea & febiger. philadelphia. 417 pp. lavabetha, a.r.r.r. hidayaturrahmah, muhamat, heri budi santoso. 2014, timpakul fish blood profile (periophthalmodon schlosseri) from barito river estuary south kalimantan thesis, bioscientiae volume 12, number 1, january 2015, page 78 89 mulyanti. w. 2013, differentiation differentiation of tilapia leucocytes (oreochromis niloticus l.) in river waters of riam kanan district of banjar thesis, faculty of mathematics and natural sciences. university of lambung mangkurat. banjarbaru mitruka, b.m & h.m. rawnsley. 1977. clinical biochemical and hematological refferent value in normal experimental animal. mason publishing, usa.165-181 pp. moyle, p.b. & j.j. cech. 1988. fish an introduction to ichthyology second edition. prentice hall, new jersey. nabib, r & f.h. pasaribu. 1989. pathology and fish disease. department of education and culture. directorate general of higher education. inter-university center of biotechnology. bogor agricultural university, bogor. roberts, r.j. 1978. fish pathology. ballier tindall london philadelphia sydney shirani, m., a. mirvaghefi, h. farahmand, & m. abdollahi. 2010. biomarker responses in mudskipper (periophthalmus waltoni) from the coastal areas of the persian gulf with oil pollution, environmental toxicology and pharmacology 10: 4-29 schalm, o.w. 1965. veterinary hematology, 2th ed. lea & febiger, philadelphia. leukocytes description of mudskipper(periophthalmodon schlosseri) of barito river estuary, desa tanipah, kalimantan selatan biology, medicine, & natural product chemistry 7(1) 2018 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 309-314 | doi: 10.14421/biomedich.2025.141.309-314 issn 2540-9328 (online) liquid biofertilizer made from fermented egg shells, fish scales, and chicken feathers as a plant growth stimulator alternative to chemical fertilizer muhammad nadhif rasendriya ramadhan1, muhammad farel arfarifki1, fachira khalisa1, raffi khalief1, muhammad fadhil1, widya sari1,2, angga puja asiandu3,* 2alumna of faculty of math and natural science, universitas gadjah mada, bulaksumur, yogyakarta, 55281, indonesia. 3doctoral student of faculty of biology, universitas gadjah mada, jl. teknika selatan, yogyakarta, 55281, indonesia. corresponding author* angga.puja.asiandu@mail.ugm.ac.id manuscript received: 19 march, 2025. revision accepted: 20 may, 2025. published: 28 june, 2025. abstract as agricultural sectors rise due to increased human population, the demand for chemical fertilizer is also uplifted. however, the excessive use of chemical fertilizers reduces soil quality and natural properties. to address these issues, an alternative eco-friendly biofertilizer is needed. here, we developed a liquid biofertilizer called essik to substitute hazardous chemical fertilizers. it is made from environmentally friendly waste materials—including chicken feathers, egg shells, and fish scales—which are repurposed to provide vital nutrients for plant growth. egg shells contain calcium useful in the synthesis of plant enzymes, fish scales contain calcium and phosphorus which play a role in accelerating fruit ripening, and chicken feathers contain high protein which can be a source of amino acids. these components were first fermented using effective microorganisms (em4) to break down complex polymers into forms more readily absorbed by plants. the fermented biofertilizer was tested using mung bean (vigna radiata) with concentrations of 0%, 5%, 10%, 20%, and 100%, and a positive control of 5% em4. based on the study, 20% was the best formulation to support the growth of mung bean (v. radiata) with a length of more than 41 cm, 16 leaves, and 9 stalks. also, the leaf's length was approximately 6,6 cm, width 3,8 cm, with a healthy-green appearance. these findings suggest that it has significant potential as an eco-friendly and sustainable biofertilizer alternative to chemical fertilizers, promoting robust plant growth while contributing to waste recycling and environmental conservation for a better future. keywords: biofertilizer; land degradation; hazardous chemicals; sustainable agriculture. introduction there are various future challenges and threats to human life, such as food scarcity and pollutant accumulation. to meet these human needs, the agricultural sector must be raised despite the decreasing quality of agricultural land due to many factors such as excessive use of fertilizers. nowadays, fertilizer is a primary material needed in plantation fields (dewi et. al., 2022). generally, fertilizers are divided into two types, namely organic fertilizers and inorganic fertilizers. chemical fertilizers or inorganic fertilizers contain synthetic chemicals that produce side effects on plants, such as poisoning in excessive concentrations, and causing the accumulation in the environment. also, the price of inorganic fertilizers is relatively high (huda et. al., 2013). using chemical fertilizers or inorganic fertilizers over a long period damages soil structure, causes soil hardening, and causes bioaccumulation and biomagnification that threaten human health which is distributed through the food chain (aisyah et. al., 2019). meanwhile, organic fertilizers are made from natural materials such as a mixture of plant and animal biomass processed through some methods such as fermentation technology. organic fertilizers are divided into compost, green fertilizer, and manure (dahlianah, 2015). compost is a fertilizer produced from fermentation involving bioactivators (simanungkalit et. al., 2006), green fertilizer is a fertilizer derived from plant and animal waste that has been decomposed (dahlianah, 2014), while manure is a combination of liquid or solid animal waste and a mixture of animal feed residues (mayadewi, 2007). based on its form, organic fertilizer is divided into two types, namely solid and liquid (irsyad et. al., 2019). solid organic fertilizer is available in solid form, while liquid organic fertilizer is derived from a solution derived from the decomposition or fermentation of natural materials with abundant nutrients (nur et. al., 2016). 1harapan mulia islamic school, jl. dr. wahidin, no. 4, palembang, south sumatera, 30135, indonesia. https://doi.org/10.14421/biomedich.2025.141.309-314 310 biology, medicine, & natural product chemistry 14 (1), 2025: 309-314 organic fertilizers are considered safer because they are made from biodegradable materials that do not damage plants or the environment. this type of fertilizer can also improve soil structure, soil permeability, and soil porosity. thus, the use of organic fertilizers is recommended to support sustainable agriculture in the future (roidah, 2013). thus, the authors initiated producing an environmentally friendly organic biofertilizer composed of egg shells, fish scales, and chicken feathers. these wastes still contain essential compounds vital for plant growth an alternative to chemical fertilizers. based on raguraj et. al. (2022), egg shells contain carbon (12.6%), nitrogen (0.40%), hydrogen (0.26%), and magnesium (2.82%) (khainar et. al., 2022). additionally, fish scales contain 59.78% protein and 10.54% carbohydrates (basim et. al., 2022). chicken feathers contain 47.09% nitrogen, 13.54% carbon, 0.13% phosphorus, and 0.29% potassium. moreover, chicken feathers also contain calcium, magnesium, and sulfur at concentrations of 0.31%, 0.03%, and 1.61% respectively (raguraj et. al., 2022). materials and methods tools and materials the materials were water (until the final volume reached 20,000 ml), 250 gr egg shells, 50 gr fish scales, 800 gr chicken feather flour, 7000 ml rice washing water, 500 gr additional carbon source (sugar), and 20 ml em4 (effective microorganisms). the tools included filters, stirrers, simple bioreactors (buckets), acid hydrolysis containers, ladles, blenders, 1000 ml beakers, and 100 ml beakers. the packaging tools were bottles measuring 100, 250, and 500 ml along with the logos of each package. acid hydrolysis all ingredients were ground and soaked in an acid solution for 2 days, then crushed using a blender. the crushed ingredients along with the protein hydrolysate were mixed in a container and supplemented with effective microorganism (20 ml), rice washing water (7l), and additional carbon sources (500g). the mixture was fermented for 2 weeks. soaking using the acid solution aims to soften the ingredients and dissolve the protein. effective microorganism 4 aims to facilitate the occurrence of microbial enzymatic hydrolysis with the help of various enzymes such as proteases which will break down polymers, making them more easily absorbed by plant roots. adding rice washing water and additional sugar sources aims to support the growth of effective microorganisms (cruz-casas et al., 2021). seedling mung bean seeds were sown using soil for about 7-14 days. the grown seedlings were transferred to new pots with soil before treatment was given. biofertilizer testing and measurements the treatments were done by mixing 10 ml of biofertilizer into 1000 ml of water and then watering it into the rhizosphere. watering was done every 2-3 days in the morning and evening. in this treatment, the mung bean plants were not given additional nutrients or chemical fertilizers. the observation was done for about 5 weeks after planting. the stem length, leaf length, and leaf width were measured using a measuring tool about 5 weeks after planting. morphology observation morphological observations were conducted on leaf color, spots on the leaf surface, flowers, fruits, and leaf shape. observations were conducted for 5 weeks after planting. data analysis data on stem height, number of leaves, number of stalks, length, and width of leaves were analyzed using one-way anova at a 95% confidence level. figure 1. the workflows of making essik, a liquid biofertilizer made from fermented waste. ramadhan et al. – an eco-friendly liquid biofertilizer 311 results and discussion this biofertilizer is an organic green fertilizer product made of egg shells, fish scales, and chicken feathers fermented using microorganisms as the main hydrolysis agent. the microorganisms such as lactobacillus sp. protect plant roots from pathogen attacks (natsir et. al., 2019). it is classified as a liquid organic fertilizer rich in nutrients and offers some advantages compared to solid fertilizers. liquid fertilizer can overcome nutrient deficiencies more effectively, reduce the risk of nutrient leaching/bleaching, can be the main source of nutrients for plants, and is more easily absorbed by roots than solid fertilizer (nur et. al., 2016). the essential nutrients are nitrogen, calcium, potassium, carbon, magnesium, and phosphorus, produced through the decomposition or hydrolysis of eggshell polymers, fish scales, and chicken feathers by effective microorganisms dominated by lactobacillus sp. figure 2. stem length and numbers of leaves and stalks of mung bean (vigna radiata) based on the experiment, the provision of each treatment was known to increase the height of the stems of mung beans as seen in figure 2. all plants supplemented with this biofertilizer had a height of more than 35 cm in the 5th week. the highest stem height was found in the 20% treatment with a more than 41 cm height. the other two highest heights were found in 10% and 100%, more than 35 cm. the height of the plants which was only given by em4 5% was less than 35 cm, and the treatment 0% was less than 30 cm. based on the one-way anova, the provision of this biofertilizer significantly enhanced the growth of mung bean stems with a sig value <0.05. these results can be attributed to the nutrient contents including nitrogen, calcium, and phosphorus which enhance the growth of mung bean stems. according to rinsema (1983), the rise in plant length requires various nutrients such as nitrogen which is vital in dna synthesis and the formation of new cells. in addition to nitrogen, other nutrients play an important role in plant growth, such as magnesium, calcium, phosphorus, and so on. the lack of nutrients such as magnesium and phosphorus leads to chlorosis (pertawasari, et al, 2012). phosphate is a transporting agent of metabolic energy to stimulate flowering, fertilization, root growth, seed formation, plant cell division, and cell tissue enlargement (susi, et al, 2018). moreover, calcium is vital in thickening cell walls, increasing root cell elongation, enzymatic and hormonal cofactors, and protection from heat, pests, and diseases (ernawati, et al, 2019). potassium also supports optimal root growth (syauqi, et al, 2022). in addition, based on figure 2, the provision of 20% biofertilizer also produced plants with the largest number of leaves and stems compared to other treatments, which were 16 leaves and 9 stalks. meanwhile, plants given 100% treatment had 14 leaves and 7 stalks. in the 10% treatment, the number of leaves and stalks was 15 and 6. on the other hand, plants that were only given water had 14 leaves and 5 stalks. based on one-way-anova, the number of stalks in each treatment was significantly different as seen from the sig value <0.05. the number of leaves and stalks is influenced by nutrient availability in the rhizosphere. nutrition is a determining factor for the success of plants in continuing their growth. according to sulistio et al. (2018), when sufficient nutrients are available, cell division and elongation occur more quickly to increase the number of leaves. nitrogen is a chlorophyll component; using fertilizers rich in nitrogen will stimulate better leaf and stem growth (sulistio et al., 2018). figure 3. leaf length and width of mung bean (vigna radiata) in the fifth week 312 biology, medicine, & natural product chemistry 14 (1), 2025: 309-314 the treatments also significantly affected the leaf length and width of mung bean plants (v.*radiata) as shown in figure 3 above. there are differences in the response of the length and width of mung bean leaves to various treatments. plants given fertilizer with a dose of 20% had the most optimal growth with a leaf length of 6.6 cm and a leaf width of 3.8 cm. all treatments produced plants with a leaf length of more than 5.4 cm and a width of more than 3.2 cm. on the other hand, the treatment of em4 5% only had leaves with a length of 4.5 cm and a leaf width of 1.9 cm. plants without fertilizer only had leaves with an average length of 3 cm and a width of 2.5 cm. based on one-way-anova, the provision of the treatment significantly affected the length and width of mung bean with a sig value <0.05. perwtasari et. al. (2012), state that the ammonium and nitrogen content is critical in accelerating plant growth, initiating leaf formation, helping stem growth, enlarging cell size, and increasing disease resistance. plants that lack nitrogen are more susceptible to pathogen attacks. this is also supported by sandra's study (2001), which suggests that during vegetative growth, fertilizer with high n content should be given, because this element is the main component of protein needed for cell division. similar to the quantitative data above, the effectiveness of the treatments can also be seen from the morphology of the mung bean as presented in table 1. 20% was the optimum treatment that produces plants with healthy stems and leaves. in this treatment, the stems of the mung bean are taller, larger, and stronger than those in other treatments. the leaves were also larger, wider, green, and had no yellow spots. this can be caused by the availability of micro and macronutrients in the form of nitrogen, phosphorus, magnesium, and iron needed by the mung bean (susi et al., 2018). generally, all treatments supplemented with biofertilizer had healthier stem and leaf morphology than treatments without biofertilizer. this biofertilizer also accelerates flower and fruit initiation. this can be seen in some plants that have grown flowers and fruits in the 5th week after planting. meanwhile, plants that were not given essik had an unhealthy morphology. meanwhile, in the 0% treatment, the stems of the mung bean were small, stunted, weak, and shorter. in the same treatment, the mung bean leaves were smaller and yellow, with curly tips and yellow spots spread across the leaf surface. this can be caused by a lack of phosphorus (perwtasari et. al., 2012). in addition, a lack of nitrogen also causes chlorosis which inhibits growth and decreases plant resistance to pests (perwtasari et. al., 2012). table 1. morphology of mung bean (vigna radiata) in the fifth week. treatments morphology description 0% (only water) stunted, weak, and short stem. small, yellowish leaves with many white / brown spots. 5% em4 tall stem, healthy oval green leaves with some flowers. 5% biofertilizer tall stem, more leaf branches, green leaves, with some fruits. 10% biofertilizer tall and strong stem, with many stalks, green leaves, and some fruits. ramadhan et al. – an eco-friendly liquid biofertilizer 313 table 1. cont. treatments morphology description 20% biofertilizer tall, strong, branched stems. dark green leaves, some fruits and flowers 100% biofertilizer tall and large stem, some stalks, dark green leaves, with some fruits. product development and future perspective this biofertilizer is named essik green biofertilizer. the word essik comes from eggshells, fish scales, and chicken feathers, abbreviated to make it easy to remember and recognize. essik is a liquid organic fertilizer that can be used as an alternative for farmers to replace chemical fertilizers that are harmful to the environment. fertilizers made from waste are rich in essential nutrients plants need, environmentally friendly, cheaper, and support actions to save the environment. in addition to being made from waste, the method of making essik green biofertilizer is also environmentally friendly because it involves microorganisms as the main agents in breaking down polymer, dominated by photosynthetic bacteria, lactobacillus sp., streptomyces sp., to yeast (sundari et. al., 2012). thus, the development of essik production on a mass scale needs to be carried out in the future to support sustainable agriculture. the development scheme for essik green biofertilizer is presented in figure 4. in the development of essik green biofertilizer, cooperation with various parties is needed, especially sponsors who can provide support, both materially and non-materially. the consistency of the writing and development team and the supervising teacher continues to realize the vision and mission of essik as an alternative fertilizer for the future to support the government's program, namely food independence. to establish relationships, recognition, and sponsorship, the writing team supported by the smait harapan mulia palembang foundation continued to actively promote essik green biofertilizer. one was participating in thailand inventor days 2024 which was held in bangkok, thailand, on february 2-7. through this activity, the writing and development team was awarded the bronze prize. figure 4. thailand inventor days in bangkok, 2024 (left) and audience at the south sumatra governor's office (right). conclusions based on the discussion above, essik green biofertilizer is a potential alternative fertilizer choice that supports sustainable agriculture in the future. organic fertilizer from egg shells, chicken feathers, and fish scales contains various essential nutrients for plant growth such as nitrogen, phosphorus, magnesium, and calcium. the optimal concentration was 20%, which enhanced the growth of mung bean with a stem height of more than 41 cm, 16 leaves, 9 stalks, a leaf length of 6.6 cm, a leaf width of 3.8 cm, with a healthy and fresh morphology. in 314 biology, medicine, & natural product chemistry 14 (1), 2025: 309-314 the future, the development and mass production of essik needs to be carried out to support the food independence program by using more environmentally friendly fertilizers at more affordable prices. acknowledgements: we acknowledge all parties for their invaluable support in conducting the research, presenting the results, and writing the manuscript with special gratitude to sir trino yudiantoro for his exceptional support, miss delima for accompanying us and coordinating all our needs during our presentation in thailand, and craba sonmat teammates for their invaluable supports. also, we extend our heartfelt thanks to our parents for their unwavering encouragement and support. authors’ contributions: muhammad n. r. ramadhan, muhammad f. arfarifki, fachira khalisa, raffi khalief, muhammad fadhil: carried out the research and wrote the manuscript. angga puja asiandu and widya sari: designed the study, monitorized the research progress and results, analyzed the data, wrote and approved the final the manuscript. competing interests: the authors declare that there are no competing interests. references adetiya, n., hutapea, s., & suswati, s. 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(2011). pengaruh logam berat cd, pb terhadap perubahan warna batang dan daun sayuran. widowati | el-hayah:jurnal biologi. cover jurnal biomenaprochy vol 9 num 1 2020.cdr biology, medicine, & natural product chemistry volume 9 – number 1 – april 2020 issn 2089-6514 (paper) | issn 2540-9328 (online) honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad jafar luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia mohamad amin department of biology, faculty of mathematics and sciences, universitas negeri malang, indonesia widodo department of biology education, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: r i y a n t o publisher: state islamic university (uin) sunan kalijaga yogyakarta co-publisher: the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university (uin) sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@yahoo.com biology, medicine, & natural product chemistry volume 9 – number 1 – april 2020 issn 2089-6514 (paper) | issn 2540-9328 (online) contents antioxidant activities of the leaf extract and fractions of dryopteris filix-mas (l.) schott could be attributed to the abundance of polyphenol compounds earnest oghenesuvwe erhirhie, emmanuel emeka ilodigwe, daniel lotanna ajaghaku, blessing ogechukwu umeokoli, peter maduabuchi eze, festus basden chiedu okoye 1 6 the effect of cassava peel starch addition to bioplastic biodegradation based on chitosan on soil and river water media mahfud syuhada, sintia ainus sofa, endaruji sedyadi 7 13 a mathematical model of the covid-19 cases in indonesia (under and without lockdown enforcement) sugiyanto, muchammad abrori 15 19 analysis of body posture using rapid entire body assessment (reba) and rapid upper limb assessment (rula) to improve the posture of sand paper machine operators and reduce the risk of low back pain trio yonathan teja kusuma 21 25 the potential of chrysin of oroxylum indicum l. to induce carbonic anhydrase (ca) to improve cattle fertility mohamad amin, muhammad najib fahmi, muhammad andi ali ridho, nurul fitri, umie lestari, dina maulina, ihya fakhrurizal amin 27 32 toxicological effects of ethanolic stem bark extract of xylopia aethiopica on testicular oxidative stress markers and histology of male rats elias adikwu, ben enoluomen ehigiator 33 37 isolation and characterization of sesquiterpenes from stem bark of warburgia ugandensis sprague teshome gonfa hordofa 39 46 prevalence of psychoactive substance use among nigerian male commercial vehicle drivers selected from the three major ethnic groups in nigeria tochukwu frank egwuatu, onyekachi ogbonnaya iroanya, khalid olajide adekoya 47 55 guidance for authors aims and scope biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of 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(personal communication); setyawan ad 2007, unpublished data). in the reference list, the references should be listed in an alphabetical order (better, if only 20 for research papers). names of journals should be abbreviated. always use the standard abbreviation of a journal's name according to the issn list of title word abbreviations (www.issn.org/2 22661-ltwa-online.php). the following examples are for guidance. journal: saharjo bh, nurhayati ad. 2006. domination and composition structure change at hemic peat natural regeneration following burning; a case study in pelalawan, riau province. biodiversitas 7: 154-158. book: rai mk, carpinella c. 2006. naturally occurring bioactive compounds. elsevier, amsterdam. chapter in book: webb co, cannon ch, davies sj. 2008. ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. in: carson w, schnitzer s (eds) tropical forest community ecology. wiley-blackwell, new york. abstract: assaeed am. 2007. seed production and dispersal of rhazya stricta. 50 annual symposium of the international association for vegetation science, swansea, uk, 23-27 july 2007. proceeding: alikodra hs. 2000. biodiversity for development of local autonomous government. in: setyawan ad, sutarno (eds.) toward mount lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in java island. universitas sebelas maret, surakarta, 17-20 july 2000. [indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com 3: front 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 351-359 | doi: 10.14421/biomedich.2024.132.351-359 issn 2540-9328 (online) effect of justicia insularis leaf extract and fractions on oxidative stress markers, liver function parameters and liver histology of plasmodium berghei -infected mice veronica james enyiekere1, martin osita anagboso2, uduak peter ise3, grace emmanuel essien1, jude efiom okokon1, nwakaego omonigho ebong4,* 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria 2department of microbiology, madonna university nigeria, elele campus, rivers state, nigeria. 3department of pharmacology and toxicology, faculty of pharmacy, bingham university, karu, nigeria 4department of pharmacology and toxicology, faculty of pharmacy, madonna university nigeria, elele campus, rivers state, nigeria. corresponding author* nwakaebong@gmail.com manuscript received: 06 june, 2024. revision accepted: 08 august, 2024. published: 01 october, 2024. abstract justicia insularis (family-acanthaceae) is used in ibibio ethnomedicine to treat malaria. the leaf extract and fractions of j. insularis were investigated for antioxidative stress and hepatoprotective activities in plasmodium berghei-infected mice. the leaf extract (100-300 mg/kg, p.o.) exerted significant (p<0.05) antimalarial activity against p. berghei infection in curative test with ethyl acetate fraction demonstrating the highest activity. the extract/fractions treatment caused significant (p<0.05) reductions in liver enzymes (alt, ast and alp), total and conjugated bilirubin of the treated infected mice and also decreased significantly (p<0.05) total protein and albumin levels of the treated mice relative to control. the leaf extract and fractions further improved significantly (p<0.05) the levels of oxidative stress markers enzymes and molecules (cat, gpx, gst, sod) of the treated infected mice with no significant (p>0.05) effect on gsh. the mda levels in the livers of the treated infected mice were significantly (p<0.05) reduced relative to control. histology of liver sections revealed absence or significant reductions in pathological features in infected mice treated with leaf extract (100 mg/kg), dcm and ethyl acetate fractions compared to untreated infected mice. these results suggest that the leaf extract/fractions of justicia insularis possess antioxidative stress and hepatoprotective potentials, which is an added advantage to its antimalarial property. keywords: antioxidative stress; hepatoprotective; justicia insularis; malaria; plasmodium berghei. introduction malaria remains one of the deadliest diseases globally despite being able to be prevented and treated. according to the world health organization (who), the majority of malaria cases reported in 2023 were from the who african region, with about 94% cases and 95% deaths and nigeria is one of the four african countries responsible for more than half of all malaria deaths worldwide (who, 2024). malaria is known to be caused by the genus plasmodium, which consists of five significant species, including plasmodium falciparum, plasmodium vivax, plasmodium malaria, plasmodium ovale, and plasmodium knowlesi. in humans, malaria transmission is usually via the bite of the female anopheles mosquito infected with the parasites, while in rodents, malaria is primarily caused by p. berghei (banyal et al., 2016). the growth of the parasite plasmodium majorly occurs in the host cell such as the liver cells and red blood cells, resulting in anaemia (intan, 2017). infection caused by p. berghei can cause damage to major organs such as the lungs, liver, spleen, and brain (prasiwi et al., 2018). the liver, the target organ of plasmodium, plays a crucial role in its life cycle and is also the site of parasitic activity and immune response (vanderberg & undra, 2004). the liver also serves as a site for detoxification of toxins and drugs. thus, it is crucial to protect the liver from the damaging effects of infectious agents and toxic chemicals (wahyuningsih et al., 2002). liver injury associated with malaria, which is one of the causes of death in severe malaria, contributes to 50% mortality in hospitalized patients (whitten et al., 2011). the mechanism of liver injury during malaria infection is uncertain. however, it has been proposed to involve the inflammatory response, adhesion of infected red blood cells (irbc) and oxidative stress induced liver toxicity (viriyavejakul et al., 2014). inflammatory cytokines associated with the pathogenesis of severe malaria have also been reported (mbengue et al., 2015). during the asexual phase of the malaria parasite, there is also an increase in the consumption of haemoglobin by the https://doi.org/10.14421/biomedich.2024.132.351-359 352 biology, medicine, & natural product chemistry 13 (2), 2024: 351-359 parasite and induction of oxidative stress due to oxidation of lipoprotein and liver damage (rifkind et al., 2015). malaria is managed solely with use of chemically synthesized drugs. however, antimalarial resistance has led to research on more effective novel antimalarial medicines. research on medicinal plants that are used locally by various tribes to treat malaria would be beneficial. thus, extracts from plant materials may have a protective effect on liver injury during malaria infection. at this point, the extracts of medicinal plants are suitable targets for study. among such plants is justicia insularis t. anderson (acanthaceae family), grown in home gardens in west and central african, especially in guinea, sierra leone, ghana, togo, benin, nigeria, cameroon and dr congo (burkill, 1985). in southern part of nigeria, the leaves are used in cooking soup and are called isepe-akera by the akwa ibom community (akuodor et al., 2020). the vegetable is used for both nutritional and medicinal purposes as a digestive, weaning agent and laxative (telefo et al., 2004; ajibesin et al., 2008; telefo et al., 2011; adeyemi & babatunde, 2014) as well as a local malaria remedy in nigeria and across africa. aqueous extracts of j. insularis leaves have been shown to produce estradiol in vitro (telefo et al., 2004), promote ovarian folliculogenesis and fertility in female rats (telefo et al., 2011), possess anti-oxidant activity (adeyemi & babatunde, 2014), and to benefit the treatment of anaemia (wood et al., 2020). phytochemical compounds such as saponins, alkaloids, tannins, flavonoids, anthraquinones, cardiac glycosides (telefo et al., 2004; oyomah et al., 2019) and clerodane diterpenoids; 16(α/β)-hydroxy-cleroda-3,13 (14)z-dien15,16-olide and 2, 16-oxo-cleroda-3,13(14)e-dien-15-oic acid have been isolated and characterised from the leaf extract (fadayomi et al., 2021). we report the leaf extract’s antioxidative stress and hepatoprotective potentials and fractions of justicia insularis in plasmodium berghei-infected mice. materials and methods collection and identification of plant material justicia insularis leaves were collected from the medicinal plants farm of the university of uyo, uyo, akwa ibom state, nigeria. a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria identificatied and authenticatied the plant. the plant’s voucher specimen (fph 83b) was conveyed to the herbarium of the department of pharmacognosy and natural medicine, university of uyo. extraction the leaves were washed and shadeed, and after two weeks, they were sliced into smaller pieces and pulverized to powder. the powdered leaves were divided into two pieces. one of them was macerated in ethanol for 72 hours, while the other was successively and gradient macerated for 72 hours in each of n-hexane, dichloromethane, ethyl acetate and methanol respectively, which is along their polarity to give the corresponding gradient extract for each solvent. the liquid filtrate of each extract and fraction was concentrated and evaporated to dryness in vacuo 400c using a rotary evaporator. the different yields were determined, and the extract and fractions were stored in a refrigerator at -4oc until they used for the proposed experiments. microorganisms (parasites) the national institute of medical research (nimer), yaba lagos, nigeria supplied chloroquine-sensitive strains of plasmodium berghei anka which were maintained by sub-passaging blood from infected mouse to a healthy mouse once every 7-8 days. parasite inoculation each mouse used in the experiment was inoculated intraperitoneally with 0.2 ml of infected blood containing about 1 x 107p. berghei parasitized erythrocytes were collected from infected mice with 2030% parasitaemia. the inoculum consisted of 5 x 107 p. berghei infected erythrocytes per millilitre prepared by determining both the percentage parasitemia and the erythrocytes count of the donor mouse and diluting the blood with isotonic saline in proportions indicated by both determinations (odetola & basir, 1980; atanu et al., 2021). standard methods monitored parasitemia; thin blood smears were made on glass slides, fixed using methanol, and stained using giemsa stain, and parasitemia was counted using a microscope and was calculated as a percentage of infected red blood cells (rbcs) relative to the total number of cells in a microscopic field at ×100 magnification according to the formula of peters and robinson (1992) as given below: 𝑃𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑚𝑖𝑎 (%) = 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑖𝑧𝑒𝑑 𝑅𝐵𝐶𝑠 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑅𝐵𝐶𝑠 × 100. experimental animals swiss albino mice (18-25 g), male and female, used in the study were obtained from the university of uyo’s animal house. they were kept in standard plastic cages in a well-ventilated room and left to acclimatise for a period of 10 days before the experiments. the mice were fed on a standard pelleted diet and water ad libtum according to the national institute of health guide conducted for the care and use of animals for the care and use of laboratory animals (nih publication, 1996). approval for the study was obtained from the university of uyo’s animal ethics committee. enyiekere et al. – effect of justicia insularis leaf extract and fractions on … 353 drug administration the extract, fractions and chloroquine used in the study were administered orally through a stainless metallic feeding cannula. determination of median lethal dose (ld50) the determination of median lethal dose (ld50) of the extract was carried out in mice using oral (p.o) route by modified method of lorke (1983). the animals in groups of three mice each were administered different doses of the extract (100– 5000 mg/kg). they were observed for manifestation of physical signs of toxicity such as writhing, decreased motor activity, decreased body/ limb tone, and decreased mobility and death. the mortality rate in each group within 24 hours was recorded. the ld50 value was calculated as geometrical means of the maximum dose producing 0% (a) and the minimum dose producing 100% mortality (b). ld50 =√ ab evaluation of the in vivo antimalarial activities of leaf extract and fractions of justicia insularis on established infection this study used the curative test method described by okokon et al. (2017) to determine the antimalarial activity of the extract, fractions and chloroquine in established plasmodiasis. p. berghei parasites were injected intraperitoneally into ninety (90) mice on the first day (d0). the mice were divided into nine groups of ten mice per group after 72 hours (d2). the extract, at doses of 150, 300, and 450 mg/kg were respectively administered to groups 1-3 mice, while 300 mg/kg each of n-hexane, ethyl acetate, dichloromethane, and nbutanol fractions were given to groups 4 -7 respectively, group 8 was given 5 mg/kg of chloroquine (positive control) and group 9 was given 10 ml/kg distilled water (negative control). the crude extract, fractions and chloroquine were administered once daily for 5 (five) days. giemsa stained thin smears were prepared from tail blood samples collected on each day of treatment to monitor the parasitemia level. the mice’s mean survival time (mst) in each group was determined over 29 days (d0-d28). five mice were sacrificed from each group under diethyl ether vapour on the sixth day. blood samples were collected into plain centrifuge tubes and centrifuged immediately at 2500 rpm for 15 mins to separate the serum at room temperature. these blood samples were stored at -20oc until used for biochemical determinations. the liver from each mouse was surgically removed, weighed and divided into two parts. one part was fixed in 10% formaldehyde for the histological process and the other part stored in ice-cold normal saline for antioxidative stress markers. the average suppression of parasitemia was calculated according to the formula of peters and robinson (1992) as follows: (average % parasitemia positive control – average % parasitemia negative control) / (average % parasitemia negative control). effect of the leaf extract and fractions on liver function parameters of p. berghei infected mice liver function markers like total protein, albumin, aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), total cholesterol, total protein, albumin conjugated, and total bilirubin were determined in the stored sera samples collected from the sacrificed mice spectrophotometrically utilising randox analytical kits following standard procedures of manufacturer’s protocols (tietz, 1990). effect of the leaf extract and fractions on liver antioxidative stress markers of p. berghei infected mice the removed livers were kept and washed with ice cold 0.9% nacl. homogenates were made in a ratio of 1 g of wet tissue to 9 ml of 1.25% kcl by using motor driven teflon-pestle. the homogenates were centrifuged at 7000 rpm for 10 min at 4˚c. the supernatants were used for the assays of superoxide dismutase (sod) (marklund & marklund, 1974), catalase (cat) (sinha,1972), glutathione peroxidase (gpx) (lawrence & burk,1976), and reduced glutathione (gsh) (ellman,1959), mda (esterbauer & cheeseman,1990). effect of the leaf extract and fractions on liver histology of p. berghei infected mice the liver pieces from mice fixed in buffered formalin were prepared and stained with haematoxylin and eosin (h&e) for liver study following the standard procedures at the department of chemical pathology, university of uyo teaching hospital, uyo. changes in morphology from the excised organs of the sacrificed mice were observed and reported. histologic pictures were taken as micrographs. gas chromatography-mass spectrometry analysis gas chromatography-mass spectrometry (gc-ms) data of the active fractions (ethyl acetate) were reported on an agilent 7890a gas chromatograph linked with an agilent ms model 5975c msd detector (agilent technologies, usa). an hp5-ms column 5% phenylmethylpolysiloxane, 30m × 0.25mm × 0.25µm was employed with a helium gas flow under a pressure of 10 psi. the injector temperature was set at 280°c. the oven temperature started at 150°c for 3 minutes increased to 300°c at 10°c/min, and was held for 5 minutes at 300°c. the mass spectrometer was operated using the electron ionization mode at 70ev (aldulaimi et al., 2017). the phytochemicals were established by comparing the spectra using the nist 2011 database. statistical analysis data was analyzed using one-way analysis of variance (anova) followed by tukey’s multiple comparison post-test (graph pad prism software inc. la jolla, ca, usa). values were presented as mean ± standard error 354 biology, medicine, & natural product chemistry 13 (2), 2024: 351-359 of the mean (sem) and significance relative to the control was considered at p ˂ 0.05. results and discussion determination of median lethal dose (ld50) administration of leaf extract of j. insularis (100 5000 mg/kg) orally did not cause any mortality in the animal groups administered (table 1). moreover, no physical toxic signs of the extract were observed. the median lethal dose (ld50) of leaf extract of j. insularis was therefore estimated to be =5000 mg/kg. table 1. determination of oral ld50 (lorke, 1983). dose (mg/kg) mortality 5000 0/3 4000 0/3 3000 0/3 1000 0/3 100 0/3 10 0/3 ld50= 5000 mg/kg antiplasmodial effect of ethanol leaf extract and fractions of j. insularis on established infection there were dose-dependent reductions of parasitaemia in all the extract/fraction-treated groups progressively relative to control. these reductions were statistically significant relative to the control (p<0.001; figure 1). the ethyl acetate fraction had the highest activity with a chemosuppressive effect of 73.15 %, this was lower compared with that of the standard, chloroquine, 81.58 %. the leaf extract and fractions demonstrated significant (p<0.05-0.001) protective potentials in the mice as was seen in the mean survival time of the animals. the groups treated with ethyl acetate fraction had a longer mean survival time, 22.66 ± 0.80 d, followed by those of dichloromethane fraction treated mice 15.0 ± 0.57 d. these were less than the standard chloroquine drug (29.83 ± 0.16 d; figure 2). figure 1. effect of leaf extract and fractions of justicia insularis on established plasmodium berghei infection in mice. figure 2. effect of leaf extract and fractions of justicia insularis on mean survival time (mst) of plasmodium berghei-infected mice. values are expressed as mean ± sem. significant relative to control at *p<0.05; **p<0.01; ***p<0.001. n = 10. effect of leaf extract and fractions on liver function parameters of plasmodium berghei-infected mice. the liver function indices (ast, alt, alp, total cholesterol, total protein, albumin, total and conjugated bilirubin) were elevated in untreated p. berghei -infected mice. however, treatment of p. berghei-infected mice with leaf extract and fractions of justicia insularis caused non dose-dependent and significant (p<0.050.001) reductions in the levels of ast, alp, total cholesterol, and conjugated bilirubin with ethyl acetate fraction followed by dcm fraction treated groups having the most significant (p<0.05-0.001) reduction when compared to control. similarly, dose-dependent and significant (p<0.05-0.001) reductions in alt and total bilirubin levels were recorded in the extract treated groups, with ethyl acetate fraction exerting the highest effect. some of the effects were better than those of the chloroquine-treated group (table 2). the total protein and albumin levels of the treated infected mice were similarly reduced non-dose-dependently and significantly by the extract/fractions treatments with ethyl acetate fraction and dcm fraction-treated groups having the highest significant effect (p<0.001) (table 2). effect of leaf extract and fraction on liver oxidative stress markers of plasmodium berghei-infected mice. the non-enzymatic and enzymatic endogenous antioxidants (gsh, sod, cat, gpx, and gst) were found to be reduced in the untreated plasmodium berghei-infected mice. treatment of plasmodium berghei-infected mice with leaf extract and fractions of justicia insularis caused non dose-dependent increases and non-significant (p>0.05) increases in the levels of gsh and gpx compared to control. the extract/fractions treatment further increased sod level, which was only significant in the group treated with the low dose of the extract (150 mg/kg) when compared to the control. cat levels of the treated infected mice were similarly enyiekere et al. – effect of justicia insularis leaf extract and fractions on … 355 increased. however, these increases were only significant (0.05-0.001) in the groups treated with 150 and 300 mg/kg of the extract, dcm and n-butanol fractions, as well as chloroquine when compared to control. gst levels of the treated infected mice were increased also but the increases were significant (0.050.001) when compared to control in groups treated with extract (300 mg/kg) and all the fractions with n-butanol fraction treated group having the highest effect. the mda level which was elevated in the untreated infected mice was decreased by extract/fractions treatment and the decrease was significant when compared to control (table 3). table 2. effect of leaf extract and fractions of justicia insularis on liver function parameters of mice infected with plasmodium berghei. treatment d o se (m g /k g ) liver function parameters ast (iu/l) alt (iu/l) alp (iu/l) total cholesterol total protein (g/l) albumin (g/l) total bilirubin (µmol/ml) conjugated bilirubin (µmol/ml) control 32.33±2.40 25.0±1.15 38.0±0.57 3.50±0.23 73.33±0.88 45.0 ±1.15 6.06±0.23 4.73±0.43 extract 150 21.00±2.08b 16.66±3.18b 28.0±0.57c 3.50±0.05 66.33±0.88 41.0±1.45 4.06±0.23b 3.83±0.21 300 20.66±1.76b 17.00±0.57b 21.0±0.57c 2.80±0.17 61.33±2.88c 42.60±1.45 4.53±0.20 2.56±0.14c 450 20.00±1.73b 14.33±0.88c 22.0±1.15c 2.70±0.05 60.66±2.33c 40.33±1.85 4.36±0.17a 2.46±0.20c n-hexane 300 21.66±3.18b 17.00±1.73b 29.0±0.57c 2.86±0.08 61.00±0.57c 42.66 ±1.45 5.10±0.60 3.88±0.49 dichloromethane 300 22.00±2.88b 18.00±1.73a 21.0±0.57c 3.36±0.14 59.66±1.45c 37.0±1.15b 4.40±0.55a 3.13±0.42b ethyl acetate 300 20.00±0.57b 15.33±0.88c 19.0±0.57c 2.56±0.12a 64.00±0.57c 34.66±1.20c 4.50±0.17a 2.70±0.11c n-butanol 300 25.66±0.33b 17.64.0±0.57b 31.0±0.57c 2.70±0.11 65.66±1.20a 42.0 ±0.57 5.16±0.16 4.00±0.11 chloroquine 5 21.66±2.02b 18.02±2.51a 19.33±0.66c 2.30±0.36b 62.00±1.73c 40.66±0.88 4.06±0.34b 3.16±0.20a values are expressed as mean ± sem. significant relative to control. ap<0.05; bp<0.01; cp<0.001. n = 10. table 3. effect of leaf extract and fractions of justicia insularis on liver oxidative stress markers of mice infected with plasmodium berghei. treatment dose (mg/kg) antioxidant parameters gsh (µg/ml) sod (µg/ml) cat (µg/ml) gpx (µm/ml) gst (µg/ml) mda (µmol/ml) liver weight (g) control 1.10±0.20 0.19±0.01 3.51±0.05 0.048±0.008 0.033 ±0.05 0.55±0.02 2.31±0.16 extract 150 1.18±0.11 0.34±0.02a 4.26±0.21a 0.053±0.005 0.029±0.03 0.38±0.02c 2.16±0.01 300 1.29±0.18 0.25±0.04 2.67±0.20 0.057±0.008 0.18±0.01a 0.42±0.03a 2.24±0.20 450 1.44±0.22 0.20±0.02 4.59±0.20c 0.064±0.009 0.29±0.02c 0.46±0.02 2.32±0.16 n-hexane 300 1.45±0.19 0.17±0.01 2.89±0.32 0.064±0.008 0.19 ±0.02a 0.55±0.03 2.14±0.18 dichloromethane 300 1.38±0.25 0.23±0.08 5.08±0.40c 0.066±0.013 0.34±0.02c 0.45±0.06 2.14±0.05 ethyl acetate 300 1.34±0.24 0.21± 0.04 3.65± 0.32 0.062±0.012 0.24 ±0.02b 0.42±0.06a 2.29±0.13 n-butanol 300 1.29±0.21 0.25± 0.04 5.67± 0.21c 0.059±0.010 0.41 ±0.03c 0.49±0.04 2.21±0.22 chloroquine 5 0.93 ±0.25 0.21±0.02 4.96±0.75c 0.042±0.011 0.33±0.05c 0.44±0.02a 2.28±0.12 values are expressed as mean ± sem. significant relative to control. ap<0.05; bp<0.01; cp<0.001. n = 10. effect of extract and fractions on the histology of liver of plasmodium berghei-infected mice. histologic sections of untreated infected mice livers showed distorted liver with congested central vein, hepatocytes, sinosoids containing inflammatory cells, necrotic tissues. a similar pattern of distortion of liver architecture was observed in groups of infected mice treated with 300 and 450 mg/kg of the extract, n-hexane and n-butanol fractions. however, p. berghei infected mice groups treated with 150 mg/kg of the extract, dcm fraction, ethyl acetate fraction and chloroquine showed regular liver section with intact hepatocytes, patent central vein and sinusoids containing kupffer cells without any pathological signs. (figure 3). gcms analysis of ethyl acetate fraction the results of gcms analysis of ethyl acetate fractions show that the fraction contains various pharmacologically active compounds such as hexanoic acid, pentanoic acid, 3-methyl-, hexanoic acid, 1,1dimethylethyl ester, hexadec-9-enoic acid, 7-tertbutyldimethylsilyloxy-, methyl ester, heneicosanoic acid, methyl ester, octa-2,4,6-triene, 1,3,6-heptatriene, 5methyl-, (e)-, phytol, acetate, octadecanoic acid, 2hydroxy-1,3-propanediyl ester, octadecanoic acid, docosyl ester and others (table 4). 356 biology, medicine, & natural product chemistry 13 (2), 2024: 351-359 figure 3. histologic liver sections of plasmodium berghei-infected mice untreated with normal saline (a), leaf extract of justicia insularis, 150 mg/kg (b), 300 mg/kg (c),450 mg/kg (d), n-hexane fraction(e), dcm fraction (f), ethyl acetate fraction (g), n-butanol fraction (h) and chloroquine, 5 mg/kg (i) at magnification x400. keys: congested central vein (cv), cords of normal hepatocytes (h), sinusoids (s) containing: capillaries (c) and kupffer cells (kc), central vein (cv), inflammatory cells (inf). discussion the people of ibibio traditionally use the leaves of j. insularis as a malaria remedy. this study was designed to evaluate the effect of the leaf extract and fractions on parasitemia, liver function parameters, liver oxidative stress markers and liver histology of p. berghei infected mice. in this study, the extract and fractions significantly reduced the parasitemia in a dose-dependent pattern with the ethyl acetate fraction showing the highest schizonticidal activity, indicating this extract’s has antimalarial potential. the phytochemical constituents of the extracts and fractions may be responsible for this effect, thus validating the use of the leaf extract decoctions locally in treating malaria. phytochemicals like secondary metabolites and other chemical compounds of plants contribute to the antimalarial properties of plants. ethyl acetate, the most active fraction of the gcms analysis shows the presence of hexanoic acid, pentanoic acid, 3-methyl-, hexanoic acid, 1,1-dimethylethyl ester, hexadec-9-enoic acid, 7tert-butyldimethylsilyloxy-, methyl ester, heneicosanoic acid, methyl ester, octa-2,4,6-triene, 1,3,6-heptatriene, 5methyl-, (e)-, phytol, acetate, octadecanoic acid, 2hydroxy-1,3-propanediyl ester, octadecanoic acid, docosyl ester and others; which are known antimalarial compounds. furthermore, chemical constituents of the leaf extract like alkaloids, saponins, tannins, anthraquinones, flavonoids and cardiac glycosides are under study (telefo et al., 2004; oyomah et al., 2019). so far, isolation and characterization of clerodane diterpenoids; 2, 16-oxo-cleroda-3,13(14)e-dien-15-oic acid and 16(α/β)-hydroxy-cleroda-3,13 (14)z-dien15,16-olide from the leaf extract have been done (fadayomi et al., 2021). this implies that these compounds are accountable for the notable activities of the extract and fractions particularly the terpenoids and polyunsaturated fatty acids (pufas) such as hexadecanoic acid, methyl ester, 9,12-octadecadienoic acid methyl ester (linoleic acid), 9,12,15-octadecatrienoic acid, methyl ester (linoleic acid), and 9-octadecenoic acid which have been responsible for antiplasmodial activities of plants (kirby et al., 1989; philipson & wright 1991; kumaratilake et al., 1992; krugliak et al., 1995; a b c d e f g h i enyiekere et al. – effect of justicia insularis leaf extract and fractions on … 357 christensen & kharazmi, 2001; hakzakis et al., 2007, suksamrarn et al., 2005; attioua et al., 2007; melariri et al., 2011, 2012). the hepatoprotective potentials were examined by evaluating the effect of extract and fractions on the liver function indices of the p. berghei-infected mice. increased levels of transaminases and hyperbilirubinemia were detected in the untreated infected mice, suggesting liver injuries, usually associated with malaria infection which are likely due to hepatic blood flow obstruction and blockade of sinusoids by parasitized erythrocytes. also, liver cell destruction and membrane integrity by free radicals following malaria infection, coupled with reticuloendothelial blockage and alterations in the hepatocyte microvilli could compromise the secretory capacity in the liver thus resulting in hyperbilirubinemia (onyesom & onyemakonor, 2011). after administration of leaf extract and fractions of j. insularis to p. bergheiinfected mice, the elevated total protein, albumin, ast, alt, alp, total and conjugated bilirubin levels were reduced. these impacts suggest that the hepatoprotective properties of the leaf extract and fractions may be due to the antioxidant properties of the phytoconstituents. however, the histological examination of liver sections of untreated p. berghei-infected mice revealed severe pathologic signs like a distorted liver with congested central vein, hepatocytes with sinusoids containing inflammatory cells and necrotic tissues which are indicate inflammatory reaction in the tissue. these pathological effects significantly decreased mainly in p. berghei infected mice groups treated with 150 mg/kg of the extract, dcm fraction, ethyl acetate fraction and chloroquine exhibiting regular liver section with intact hepatocytes, patent central vein and sinusoids containing kupffer cells without any pathological signs. hence, the hepatoprotective activity of the leaf extract and fractions is probably due to the antioxidant properties of its phytochemical constituents as earlier reported (adeyemi & babatunde, 2014). oxidative stress is crusial in malaria complications such as anemia, jaundice and pre-eclampsia (fabbri et al., 2013; sarr et al., 2017). hypoxic conditions due to malaria infection produce large amounts of free radicals triggering body immune responses (becker et al., 2004; percario et al., 2012), leading to the development of systemic complications associated with malaria (guha et al., 2006; ojezele et al., 2017). the malarial infection has been observed to decrease enzymatic and other nonenzymatic endogenous anti-oxidants levels like catalase (cat), glutathione (gsh) peroxidase, superoxide dismutase (sod), albumin, ascorbate and plasma tocopherol. increased lipid peroxidation and malondialdehyde levels have been associated with the severity of malaria (asagba et al., 2010; raza et al., 2013), hence they are used as biomarkers in determining the severity of malaria infection. in this study, the activities of sod, cat, gpx and gst levels which were found to reduce significantly in the untreated infected mice were elevated by the extract /fractions treatment, while gsh was not significantly affected by the treatment when compared to the untreated infected group and mda level was significantly decreased especially in the groups treated with the extract (300 mg/kg), ethyl acetate fraction and chloroquine. the plant extract and fractions exerted antioxidative stress potentials by increasing the levels of some antioxidative stress markers. this activity can be explained as a results of the antioxidant activities of the phytochemical constituents as earlier reported (adeyemi & babatunde, 2014). conclusions this study shows that the leaf extract and fractions of justicia insularis possess antimalarial, antioxidative stress and liver protective potentials which may be due to the activities of its phytochemical constituents. acknowledgements: the authors are grateful to mr. nsikan malachy and other staff animal house of pharmacology and toxicology department, university of uyo for providing technical assistance. authors’ contributions: vje, jea and upi conceived and designed this study. jea, vje and gee carried out the experiments and drafted the manuscript. noe and moa performed the statistical analysis, edited and reviewed the manuscript, noe, moa and gee read and approved the final manuscript. competing interests: the authors have not declared any conflict of interest. references adeyemi ot, babatunde o. 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(2024). malaria. https://www.who.int/news-room/fact-sheets/detail/malaria. last date accessed march 14th, 2024. https://www.who.int/news-room/fact-sheets/detail/malaria this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 507-513 | doi: 10.14421/biomedich.2023.122.507-513 issn 2540-9328 (online) liver protection effect of red rose petal methanol extract in copper-induced liver injury in male wistar rats caroline febrina hutagalung, ermi girsang*, maya sari mutia, suhartina, oliviti natali faculty of medicine, dentistry, and health sciences, universitas prima indonesia, medan, indonesia. corresponding author* ermigirsang@unprimdn.ac.id manuscript received: 21 july, 2023. revision accepted: 12 august, 2023. published: 13 september, 2023. abstract the human body requires low amounts of copper to maintain homeostasis, but high copper intake can cause various health problems. many previous studies have been performed to look for natural products that can neutralize the excessive copper effect; one such natural product is a rose petal. therefore, this study aims to investigate the liver protection effect of rose petals in copper-induced liver injury in male wistar rats. this experimental study used thirty male wistar rats that were grouped into five groups, including control, standard (quercetin), rose petal methanol extract-1 (400 mg/kg bw), 2 (800 mg/kg bw), and 3 (1,200 mg/kg bw). all groups were treated for 14 days, and all rats were given copper (ii) sulfate suspension for the last three days. on the fifteenth day, all rats were sacrificed to obtain blood and liver tissue sgot, sgpt, and liver histology study. the results showed that rose petal methanol extract significantly decreased sgot and sgpt levels compared to the control group (p-value <0.05). the reduced area of ballooning degeneration and necrosis were in line with the increased dose of rose petal extract supported the liver protection effect of rose petal extract. therefore, it can be concluded that rose petal methanol extract protects the liver against copper impacts. keywords: copper; rose; sgot; sgpt; liver protection. introduction the industrial development is followed by increasing heavy metal pollution, especially water pollution. these heavy metals include lead, mercury, arsenic, aluminium, copper, nickel, tin, antimony, bromine, bismuth, and vanadium. these metals come from wastewater from some industrial activity. copper, one of these heavy metals, comes from construction, airplanes and ships, roofing, plumbing, alloys of brass and bronze, home decoration, non-electrical industrial machinery, thermometer starter machine, and the agricultural machinery industry (wilson, 2012). heavy metal pollution has become a national burden in some countries, including indonesia. some studies have reported the severity of this pollution. chubaka et al. reported that several water tanks in the adelaide region, australia, had been polluted by some heavy metal compounds. most of these pollutants were lead, which had contaminated 47 rainwater tanks from 53 samples based on adwg (australian drink water guidelines) threshold. other heavy metal compounds detected in these rainwater tanks were zinc, cadmium, and copper, found in lower concentrations (chubaka et al., 2018). a similar condition is also found in india. nizami and rehman reported that the six highest concentrations of contaminated heavy metal in the ganga rivers were mn, cr, cu, zn, pb, and fe. furthermore, similar pollution also contaminated other rivers in india (nizami and rehman, 2018). indonesia also undergoes similar heavy metal pollution in some rivers. septiono and roosmini (2015) reported that chromium was the highest level of heavy metal pollution based on analysis from several sampling points of fish, water, and sediments of the citarum river. other heavy metal compounds at the sampling point were copper and cadmium (septiono and roosmini, 2015). on the other hand, gafur et al. also reported that as, hg, and pb highly contaminated the bone river, that 1000 to 10,000 times higher than the who standard drinking water. this pollution may be due to small-scale gold mining activities. (gafur et al., 2018). some heavy metal compounds have been reported to contaminate the water. one of these heavy metal compounds is copper. copper is required at low levels in the human body to maintain homeostasis. however, higher copper intake can cause considerable organ damage, lipid metabolism, immune system, and nervous disorders. it is due to the formation of reactive oxygen species (ros) and ldl modification to initiate atherogenesis by increasing macrophage transformation, vasoconstrictor, and prothrombotic (wilson, 2012). https://doi.org/10.14421/biomedich.2023.122.507-513 508 biology, medicine, & natural product chemistry 12 (2), 2023: 507-513 some studies have been performed to look for natural products that potentially act as antioxidants to neutralize the effects of excess copper levels, which can stimulate ros formation. one of these natural products is a rose petal that has been widely studied. boskabady et al. 2011 reported the pharmacological effects of rose petals, including hypnotic, analgesic, anticonvulsant, antitussive, antibacterial, anti-diabetic, laxative, antiinflammatory, antioxidant, and bronchodilator. these pharmacological effects are related to phytochemicals in rose petals, such as terpenes, glycosides, flavonoids, and anthocyanins (boskabady et al., 2011). on the other hand, khaliq et al. reported that rose petal extract at 500 mg/ kg bw protected the kidney from gentamicin toxicity in albino rabbits (khaliq et al., 2015). suherman et al. reported similar results on whether the rose petal methanol extract also had a nephroprotective effect against cadmium sulfate. these protective effects come from the antioxidant property of rose petal extract. amiri et al. reported that rose petals also revealed an antioxidant effect in aluminium chloride-induced rats. all these rats improved the oxidative profile (frap, mda, catalase, and mpo) after the rose petal extract administration at 1,000 mg/kg bw. however, these previous studies are still limited to the protective effect on the kidney, and no one has yet explored the hepatoprotective effect of rose petal extract. therefore, this study investigated the hepatoprotective effect of rose petal methanol extract in copper sulfate-induced liver injury in male wistar rats. materials and methods study design this experimental study used post-test only control group design from october 2022 to december 2022 in pharmacology laboratory, universitas prima indonesia. this study has been approved by health research ethics committee universitas prima indonesia with letter no. 018/kepk/unpri/xii/2022. materials this study used some materials, including distilled water, α-naphthol, concentrated nitric acid, acetic anhydride, concentrated sulfuric acid, ethanol (distilled), mercury (ii) chloride, potassium iodide, iodine, bismuth (iii) nitrate, concentrated hydrochloric acid, lead (ii) acetate, iron (iii) chloride, 10% buffer formalin solution, isopropanol, chloroform, methanol, n-hexane, sgot and sgpt dialab® reagent kits, zinc powder, toluene, dyes powder (hematoxylin and eosin). extraction process red rose petal was collected and washed with running water, then drained and weighed. rose petal was dried by a drying cabinet to form a dry rose petal simplicia and weighed it. this dry rose petal simplicia was ground to form rose petal simplicia powder and weight. the obtained rose petal simplicia powder was extracted by the maceration method. all dry simplicia powder was soaked into 98% methanol solution as the solvent in a ratio of 1:3. after that, it was filtered, and the residue was macerated again in the same way. meanwhile, all filtrate from all filtration processes was collected to evaporate by a rotary vacuum evaporator at 50oc. then, concentrated rose petal methanol extract was stored at 20oc. (suhartomi et al., 2020; gulo et al., 2021; chiuman, ginting and yulizal, 2022) phytochemicals screening the concentrated rose petal methanol extract underwent phytochemical screening to investigate the presence of alkaloids, saponin, flavonoids, tannin, steroids, terpenoids, and glycosides. this phytochemical screening was performed based on the modified fansworth method. (widowati et al., 2018; mawarni et al., 2020; chiuman et al., 2023) oral suspension formulation this concentrated extract was suspended in 0.5% sodium carboxyl methyl cellulose (scmc). on the other hand, other substrates included copper sulfate, quercetin, and copper sulfate suspended into 0.5% scmc to formulate oral suspension. (chiuman et al., 2021; mutia, ginting and yulizal, 2021). hepatoprotective assay all rats were grouped into five groups, including control, standard, rose petal methanol extract-1, 2, and 3, that received 0.5% scmc, 25 mg/ kg bw of quercetin, 400 mg/kg bw, 800 mg/ kg bw, and 1,200 mg/ kg bw of rose petal methanol extract for 14 days, respectively. in the last three days, all rats received a milliliter of copper sulfate suspension orally. (yanti et al., 2011; mutia, 2019; mutia and chiuman, 2019; mutia, ginting and yulizal, 2021) furthermore, all rats were sacrificed on the 15th day by chloroform inhalation. after that, the blood was obtained by intracardiac puncture, and the liver was resected from incised abdomen wall. the obtained blood was collected in a purple blood tube (edta-contained). after that, the obtained blood was separated by centrifugation at 3,000 rpm for 15 minutes. the blood was divided into two layers: serum in the upper layer and blood clot in the lower layer. the obtained serum was used for liver function tests, including sgot and sgpt using dyasis ® reagen kit. on the other hand, the obtained liver was sliced and stained by hematoxylin and eosin. the liver histology scoring system was described in table 1. hutagalung et al. – liver protection effect of red rose petal methanol extract in … 509 table 1. liver histology scoring system. pathology changes description score ballooning degeneration no ballooning degeneration 0 minimal enlargement in a few hepatocytes 1 mild enlargement in many hepatocytes 2 moderate enlargement in a few hepatocytes 3 severe enlargement in a few hepatocytes 4 inflammation no inflammatory foci 0 an inflammatory focus per 200 hpf 1 2-4 inflammatory foci per 200 hpf 2 > 4 inflammatory foci per 200 hpf 3 apoptotic cells no apoptotic cells 0 few apoptotic cells 1 fibrosis no fibrosis 0 portal/ sinusoidal minimal fibrosis 1 portal/ sinusoidal mild fibrosis 2 bridging fibrosis 3 cirrhosis 4 data analysis initially, all data was described by descriptive statistics, including central tendency and dispersion. initial body weight, sgot, and sgpt level were described by mean and standard deviation. meanwhile, the scoring system was described by median and minimum to maximal values. after that, the initial body weight, sgot, and sgpt were analyzed by one-way anova and post hoc tukey hsd test. results and discussion physical characteristics and phytochemicals screening this study used 678 grams of fresh rose petals. this fresh rose petal was then dried into 495 grams rose petal simplicia powder and extracted into 55 grams of concentrated rose petal methanol extract. thus, the yield of rose petal methanol extract was 11.11%. the obtained concentrated rose petal methanol extract then underwent a phytochemical screening and it showed some phytochemicals, including alkaloids, saponins, flavonoids, and tannins. hepatoprotective assay this study used 25 male wistar rats, and the initial body weight of these male wistar rats was described in the following table 2. table 2. comparison of initial body weight in all groups. group mean sd p-value control 192.17 7.48 0.958 standard 191.50 7.33 rose petal methanol extract-1 193.50 8.02 rose petal methanol extract-2 193.50 7.01 rose petal methanol extract--3 194.50 7.01 based on table 2 that there was no significant initial body weight in all rats. it can be seen from the p-value > 0.05 (p-value: 0.958). these rats' initial body weight averages ranged between 191.50-194.50 grams. after that, all rats underwent a liver protection assay against the copper sulfate, and the sgot and sgpt levels were described in table 3. table 3. comparison of liver function test in all groups. group sgot (iu/l) sgpt (iu/l) control 265.00 ± 74.35a 242.33 ± 33.80a standard 121.00 ± 13.84b 125.33 ± 14.07b rose petal methanol extract-1 178.67 ± 6.71b 184.50 ± 9.40c rose petal methanol extract-2 154.17 ± 5.15b 165.17 ± 3.76cd rose petal methanol extract--3 140.83 ± 3.87b 148.50 ± 5.01bd p-value < 0.05 < 0.05 data was expressed as mean ± sd. different superscript in same column indicates a significant difference at p-value < 0.05 based on table 3, any doses of rose petal methanol extract revealed a significant decrease in sgot and sgpt levels compared to the control group (p-value < 0.05). variation of rose petal methanol extract did not significantly affect the sgot level. however, it significantly affected the sgpt level. the higher dose of 510 biology, medicine, & natural product chemistry 12 (2), 2023: 507-513 rose petal methanol extract significantly decreases the sgpt level. interestingly, the highest dose (1,200 mg/ kg bw) of rose petal methanol extract had an effect as same as the standard group. the comparison of the sgot and sgpt in all groups was described in figure 1. figure 1. bar graph of sgot and sgpt level in all groups. based on figure 1, the highest level of sgot and sgpt was found in the control group, followed by rose petal methanol extract-1, 2, and 3 groups, and the lowest level was found in the standard group. on the other hand, this study also evaluated the histology of liver tissue in all groups, and the scoring system of liver tissue was described in table 4. table 4. liver histology scoring system in all groups. groups pathology changes total score ballooning degeneration inflammation apoptotic cells fibrosis control 2.5 (2-3) 2.00 (1-2) 1.00 (0-1) 0 (0) 5.50 (3-6) standard 1.00 (0-1) 0 (0) 0 (0) 0 (0) 1.00 (0-1) rose petal methanol extract-1 2.00 (1-2) 2.00 (1-2) 0 (0-1) 0 (0) 3.5 (2-5) rose petal methanol extract-2 1.50 (1-2) 1.00 (1-2) 0 (0-1) 0 (0) 3.00 (2-5) rose petal methanol extract--3 1.00 (0-2)) 0.5 (0-1) 0 (0-1) 0 (0) 2.00 (0-3) based on table 4, some pathology changes were evaluated in the histology of liver tissue, including ballooning degeneration, inflammation, apoptotic cell, and fibrosis. the highest total score was found in the control group, which was 5.50 (3-6), followed by the rose petal methanol extract-1 (3.5 [2-5]), 2 (3.00 [2-5]), 3 (2.00 [0-3]), and the lowest one was the standard group, that was 1.00(0.1). the lower total score indicates milder liver tissue damage than the higher total score. 0 50 100 150 200 250 300 control standard rose petal methanol extract-1 rose petal methanol extract-2 rose petal methanol extract-3 s g o t / s g p t l ec el (i u /l ) group sgot sgpt hutagalung et al. – liver protection effect of red rose petal methanol extract in … 511 figure 2. histology of liver tissue in all groups. stain: haematoxylin and eosin. cv: central vein; na: necrotic area; ia: intact area; bd: ballooning degeneration; pmn: sel polymorphonucleate; fi: inflammatory foci; ab: apoptotic body; sh: liver sinusoid. the liver histology study in all groups showed that the most severe liver damage was found in the control group. this severe damage is demarked by necrosis and ballooning degeneration. the opposite liver tissue condition was found in the standard group, which showed the mildest pathology changes. the standard group showed minimal ballooning degeneration. meanwhile, the rose petal methanol extract showed a better liver tissue change than the control group, but it was not better than the standard group. the lowest dose (rose petal methanol extract-1) of rose petal extract showed a pathology change like the control group: necrosis followed by inflammatory cell infiltration and some apoptotic cells around the central vein. the increasing rose petal extract dose improved the liver pathology changes. rose petal methanol extract-2 showed milder severity of ballooning degeneration, inflammation cell infiltration, and the number of apoptotic cells. moreover, the highest rose petal methanol extract dose showed a minimal pathology change (minimal ballooning degeneration)). discussion this study demonstrates that rose petal methanol extract had a liver protection effect against copper in the form of copper sulfate (cu2so4). the decrease of sgot and sgpt levels indicated the liver protection effects in line with the increase of rose petal extract dose (p value <0.05). on the other hand, it also supports improving liver tissue structure in histology studies. the milder liver severity damage (ballooning degeneration and inflammation in the liver tissue) required a higher dose of rose petal extract. some studies have been performed to investigate the effects of excess copper intact by in vivo and observational methods. an in vivo study that used rats as trial animals reported that excess copper exposure induced cell apoptosis and increased the number of astrocytes in the hippocampus and frontal cerebral cortex, which either directly destructed the cell or induced stress pathway (glutamate and oxidative pathway). it caused memory and learning disorders. in addition, another study reported that the excess copper in the body also accumulated in the lung tissue among 17 workers from 30 copper mining workers, and it manifested as acute alveolitis or interstitial lung inflammation (kalita et al., 2017; jian et al., 2020). this study showed the improvement of liver tissue by sgpt and sgot enzymes as biochemical parameters. sgpt enzyme usually catalyzes the amino group transfer reaction, including alanine and alpha-ketoglutaric acid. the highest concentration of sgpt enzyme is found in the liver tissue, and the lower concentration in other tissues. the reference value of the sgpt serum is 5-35 iu/liter. sgpt enzyme is more sensitive than sgot. however, both enzymes increase in almost all liver diseases. extensive sgpt elevation is associated with extensive liver tissue damage, such as fulminant viral hepatitis, toxin-induced liver injury, and prolonged circulation collapse. sgpt elevation commonly indicates 512 biology, medicine, & natural product chemistry 12 (2), 2023: 507-513 acute mild hepatitis and diffuse or localized chronic liver disease. in mild hepatitis, sgpt rapidly increases than the sgot level. (bastiansyah, 2012) this study also used the sgot enzyme, other than the sgpt enzyme, as the biochemical parameter in this study. sgot is a mitochondrial enzyme found in the liver, heart, kidney, and brain cells. acute tissue damage leads to an increased sgot serum level rapidly. acute tissue damage leads to cell lysis and causes the high release of sgot enzyme into circulation. thus, the highly sgot enzyme level elevation was found predominantly in liver and myocardium tissue infraction. sgot enzyme catalyzes the aspartate and alpha-keto glutamic acid reaction into oxaloacetate and glutamate. other than liver tissue, sgot enzyme is also found in red blood cells and the heart. hence, it causes the sgot enzyme to be less sensitive than the sgpt enzyme to detect liver tissue damage. two isoforms of this enzyme include sgot-1 and sgot-2. sgot-1 is found highly in both red blood cells and cardiac cells in the cytosol. meanwhile, sgot-2 is highly found in the mitochondria of hepatocytes. the reference value of this enzyme is 1040 iu/liter. (sacher and mcperson, 2011) based on the information above, sgpt is more sensitive than the sgot enzyme to demonstrate liver tissue damage. this study revealed that either dose of rose petal methanol extract significantly decreased the sgot and sgpt enzyme levels, indicating the liver protection effect of rose petal methanol extract. the liver protection extract is associated with phytochemicals in the rose petal methanol extract. phytochemical screening of rose petal methanol extract showed the presence of alkaloids, saponins, flavonoids, and tannins. flavonoid is a group of polyphenol compounds with some phenol with a hydroxyl group (oh). the hydroxyl group potentially neutralizes various free radicals in the body, one of which is copper. the hydroxyl group donor the proton (h+) ion into the unstable free radical compound. mawarni et al. (2020) reported that rose flower extract had an antioxidant effect by the abts radical scavenging activity, and this antioxidant effect was described by ic50 value that was 17.51 ± 1.47 µg/ml. previous studies have investigated various organs protection effects from another part, rose leaf, against the cadmium chloride compound. hamza et al. (2022) reported that rose leaf methanol extract protected the liver and heart tissue from cdcl2 impact by suppressing lpo activity (lipoperoxidation) and inflammatory mediators. this liver and heart protection effect was demonstrated by decreasing liver enzyme levels, increasing antioxidant enzyme, decreasing free radical formation, and improving liver and heart structure. on the other hand, hamza et al. also reported that the rose leaf extract has an anticancer effect on liver cancer cells (hepg2). however, the previous study reported a lower effective dose of extract, that was 400 mg/ kg bw to showed a similar protective effect in this study (hamza, al-malki, et al., 2022; hamza, al-yasi, et al., 2022). all treatment groups received copper as cu2so4 orally, where the substance is toxic injury. in the histological picture of all rat liver tissue, liver damage or liver necrosis occurs around the porta hepatic area; this can be seen from the difficulty in identifying the porta hepatic area in the histological picture of the liver in each treatment group. in this study, improvement in liver histology was reflected in the reduced necrosis and ballooning degeneration areas. all groups received copper in the form of cuso4 orally, and copper acted as a toxic substance. the copper substance induced toxic liver injury. liver injury was classified into two pathology damage models: toxic and hypoxic. hypoxic injury underwent pathology changes around the central-zonal area. meanwhile, the toxic injury underwent pathology changes around the porta hepatic area. this study showed that all groups showed pathology changes around the porta hepatic area. the severity of the pathology changes showed a difference among the group. the lower dose of rose petal methanol extract showed massive pathology damage than the higher dose of rose petal methanol extract, and it was showed from challenging to identify the porta area in the lowest dose of rose petal methanol extract and control group. these pathology changes included necrosis and ballooning degeneration (cano p., cifuentes p. and amariles, 2017; mohan, 2019). conclusions overall, it can be concluded that rose petal methanol extract could protects the liver against copper impact. the improvement of liver function test and liver tissue structure demonstrated the liver protection effect. the effective dose to show a similar protective effect to the antioxidant compound (quercetin) was 1,200 mg/ kg bw. acknowledgements: this study was supported by faculty of medicine, universitas prima indonesia. authors’ contributions: conceptualization: caroline febrina hutagalung, ermi girsang, maya sari mutia; methodology: caroline febrina hutagalung, ermi girsang; investigation: caroline febrina hutagalung; discussion of results: caroline febrina hutagalung; writing – original draft: caroline febrina hutagalung; writing – review and editing: suhartina, oliviti natali; supervision: ermi girsang, maya sari mutia; approval of the final text: caroline febrina hutagalung, ermi girsang, maya sari mutia. competing interests: the authors declare no competing interests. hutagalung et al. – liver protection effect of red rose petal methanol extract in … 513 funding: this study did not receive any funding. references bastiansyah, e. 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(2011) “lemon pepper fruit extract (zanthoxylum acanthopodium dc.) suppresses the expression of inflammatory mediators in lipopolysaccharide-induced macrophages in vitro,” american journal of biochemistry and biotechnology, 7(4), pp. 190–195. doi: 10.3844/ajbbsp.2011.190.195. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1297-1304 | doi: 10.14421/biomedich.2025.142.1297-1304 issn 2540-9328 (online) comparative antidepressant, anxiolytic, and acute toxicity evaluation of lemon peel and peppermint leaves extracts in mice neli syahida ni’ma1*, annisa aulia savitri1, dyah mahendrasari sukendra2, ronny meilano hardiansyah2, ika ayu puspaningtias1, gavriel fauzan faturachman1, septia linasari1 1pharmacy department; 2public health, faculty of medicine, universitas negeri semarang jl. kelud utara iii, kota semarang, tel. (024) 86008700 ext 900, indonesia. corresponding author* neli.syahida@mail.unnes.ac.id manuscript received: 07 september, 2025. revision accepted: 04 december, 2025. published: 13 december, 2025. abstract depression and anxiety are prevalent psychiatric disorders, and current treatments are often limited by side effects, delayed onset, and resistance. natural products are being explored as safer alternatives, and lemon peel (citrus limon) and peppermint leaves (mentha piperita l.) contain diverse bioactive compounds with neuroprotective potential. this study evaluated the antidepressant and anxiolytic effects of their ethanolic extracts, individually and in combination, in male swiss webster mice. phytochemical screening confirmed the presence of alkaloids, flavonoids, phenols, tannins, saponins, and steroids in both extracts. antidepressant activity, assessed by the forced swimming test (fst) and tail suspension test (tst), showed that both extracts and their low-dose combination significantly reduced immobility time, with effects comparable to fluoxetine. in contrast, the high-dose combination did not enhance efficacy. anxiolytic activity, evaluated using the elevated plus maze (epm) and light-dark box (ldb), revealed that peppermint extract exerted the strongest effect, followed by the low-dose combination and lemon peel. acute oral toxicity testing at 2000 mg/kg showed no mortality or adverse effects. these findings suggest that lemon peel and peppermint extracts possess antidepressant and anxiolytic properties with favorable safety profiles, supporting their potential as natural alternatives or adjuncts for managing mood disorders. keywords: antidepressant; anxiolytic; citrus limon; mentha piperita l. introduction depression and anxiety are among the most prevalent mental health disorders worldwide, affecting hundreds of millions of individuals. they contribute significantly to disability, morbidity, and socioeconomic burden (zhdanava et al., 2021). according to the world health organization (who), depression is a leading cause of disability globally, while anxiety disorders are also recognized as major contributors to the global mental health crisis (world health organization, 2001). these disorders are often comorbid and share overlapping neurobiological mechanisms, including dysregulation of monoaminergic neurotransmission, hypothalamicpituitary-adrenal (hpa) axis dysfunction, oxidative stress, and neuroinflammation (liu et al., 2024). depression is a mood disorder that characterized by persistent feeling of sadness, emptiness, hopelessness, and loss of interest. anxiety disorders are cluster of mental disorder which indicated by excessive fear, chronic worry, or avoidance of perceived threats as response to external or internal environment (craske & stein, 2016). a study conducted by simanjuntak reported that 23.14% of indonesian aged 18 – 65 years old exhibited symptoms of depression, while the prevalence of anxiety was found to be 20.2% (simanjuntak et al., 2023). although pharmacotherapy with antidepressants and anxiolytics has provided clinical benefits for many patients, current treatments face several limitations and challenges. these include delayed onset of action, treatment resistance, withdrawal symptoms, and adverse side effects, which often lead patients to discontinue therapy (melaragno, 2021; zhdanava et al., 2021). these shortcomings encourage the exploration of alternative treatments derived from natural sources. medicinal plants, in particular, have emerged as promising candidates for novel therapeutic development due to their diverse bioactive compounds and generally lower risk of side effects compared to synthetic drugs. lemon (citrus limon) is a plant from the rutaceae family commonly consumed for various purposes, such as food ingredient, drinks, and immune booster. the fruit of the lemon possesses several pharmacological activities, such as antimicrobial, antifungal, and cytotoxic (sania et al., 2020). not only the fruit, but also the peel of the lemon contains numerous bioactive compounds. as a by-product of lemon processing, lemon peel has https://doi.org/10.14421/biomedich.2025.142.1297-1304 1298 biology, medicine, & natural product chemistry 14 (2), 2025: 1297-1304 attracted increasing scientific interest due to its rich phytochemical profile. studies have identified various compounds in lemon peel, such as glycosides, coumarins, essential oils, β and γ sitosterol (sania et al., 2020; sultana et al., n.d.). additionally, lemon peel contains apigenin, a flavonoid known for its neuropharmacological effects. apigenin has been shown to alleviate depressive-like symptoms in mice subjected to chronic stress models (yi et al., 2008). peppermint plants (mintha piperita l) is an aromatic herb widely used in both culinary and medicinal contexts. peppermint has traditionally been used to relieve stress, improve mood, and enhance cognitive function. similar to lemon, peppermint leaves contain essential oils that make this plant also used as aromatherapy. these leaves are rich in various bioactive compounds, including flavonoids, phenolic acids, terpenoids, lignans, and essential oil (mahendran & rahman, 2020a; sharif mughal, 2022). the characteristic strong aroma of peppermint is primarily attributed to its essential oil content, the majority of which consists of monoterpenes (beigi et al., 2018a). among these, menthol is the most abundant, comprising approximately 35–60% of the total monoterpene content. due to its diverse phytochemical profile, peppermint exhibits a wide range of pharmacological activities, including antibacterial, antifungal, antioxidant, antiviral, antiinflammatory, analgesic, hepatoprotective, and neuroprotective effects (mahendran & rahman, 2020a). despite the individual potential of lemon peel and peppermint in supporting mental health, few studies have investigated their comparative or combined efficacy in standardized models of depression and anxiety. considering the complexity of neuropsychiatric disorders, phytotherapeutic combinations may offer enhanced efficacy through synergistic interactions between different bioactive compounds. the combination of lemon peel and peppermint extract could potentially provide a broader spectrum of neuropharmacological activity. this study aimed to evaluate the antidepressant and anxiolytic effects of ethanolic extracts of lemon peel and peppermint leaves, both individually and in combination, in mice. the animals were subjected to four standardized behavioral tests: the forced swimming test (fst) and tail suspension test (tst) to assess antidepressant-like activity, and the light-dark box (ldb) test and elevated plus maze (epm) to evaluate anxiolytic behavior. by comparing behavioral outcomes across treatment groups, this research seeks to elucidate the therapeutic potential of lemon peel and peppermint extracts, and their combination, as natural alternatives or adjuncts in the management of mood disorders. materials and methods materials fresh lemon (citrus limon) fruits were obtained from a local plantation in jepara, central java, indonesia. dried peppermint (mentha piperita l.) leaf powder was purchased from indoplant. fluoxetine suspension, used as the reference drug, was prepared from commercially available 20 mg fluoxetine capsules (elizac® 20). male mice were obtained from java rat lab. the solvents and reagents used in this study included 96% ethanol, dragendorff reagent, magnesium powder, hydrochloric acid (hcl), amyl alcohol, ammonia solution, mayer’s reagent, and ferric chloride (fecl₃). procedure extraction lemon peel was thoroughly rinsed with distilled water and then dried. the dried material was ground to reduce particle size. both lemon peel and peppermint leaf powders were extracted using 96% ethanol. an initial extraction was performed using a material-to-solvent ratio of 1:10, followed by two subsequent extractions with a 1:5 ratio to ensure optimal extraction of bioactive compounds. the combined liquid extracts were then concentrated using a rotary evaporator. phytochemical screening both lemon peel and peppermint extracts underwent qualitative phytochemical screening to detect the presence of alkaloids, flavonoids, phenols, saponins, tannins, steroids, and terpenoids using standard procedures. for testing flavonoid, the extract was mixed with 100 ml of hot distilled water and boiled for 5 minutes in a water bath. the solution was then filtered, and 5 ml of the filtrate was added with 50 mg of magnesium powder and 1 ml of concentrated hydrochloric acid (hcl), followed by vigorous shaking. the presence of flavonoids was indicated by the formation of a reddish or orange coloration. for alkaloid detection, the extract was dissolved in 1 ml of 2n hcl and diluted with distilled water to a final volume of 10 ml. the solution was heated in a water bath for 2 minutes, allowed to cool, and filtered. the filtrate was divided equally into two test tubes: 3 drops of dragendorff’s reagent were added to the first tube, and 3 drops of mayer’s reagent to the second. the formation of an orange color (tube 1) or white precipitate (tube 2) indicated the presence of alkaloids. for tannin detection, the extract was combined with 10 ml of hot distilled water and boiled for 15 minutes in a water bath. after filtration, 2–4 drops of ferric chloride (fecl₃) were added to the filtrate. a dark green or blueblack coloration indicated the presence of tannins. for saponin detection, a portion of the extract was placed in a test tube and mixed with 10 ml of hot distilled water. the tube was sealed and shaken vigorously in a vertical motion for 10 seconds, then left ni’ma et al – comparative antidepressant, anxiolytic, and acute toxicity evaluation 1299 to stand for another 10 seconds. the formation of a stable froth or foam indicated the presence of saponins. one drop of 2n hcl was then added to observe any change in the foam layer. for steroid and triterpenoid detection, the extract was mixed with 2 ml of ethyl acetate and shaken thoroughly. the ethyl acetate layer was separated, then left to dry. two drops of concentrated sulfuric acid were added. the appearance of a red, blue, or green coloration indicated the presence of steroids or triterpenoids. animal treatment thirty-six male swiss webster mice, aged 8 weeks, with weight of 20 – 30 gram, were acclimatized for 7 days under controlled conditions at a constant room temperature of 25 °c, with a 12-hour light/12-hour dark cycle. during the acclimatization period, the animals had continuous access to food and water ad libitum. all studies were conducted in accordance with the institutional animal care committee at faculty medicine, universitas negeri semarang. the mice were randomly divided into six groups (n = 5 per group): ▪ group i (negative control, nc): received 0.5% sodium carboxymethyl cellulose (cmc-na) as the drug suspending agent. ▪ group ii (positive control, pc): received fluoxetine at a dose of 20 mg/kg body weight (bw). ▪ group iii (lp): received ethanolic extract of lemon peel at a dose of 200 mg/kg bw. ▪ group iv (pl): received ethanolic extract of peppermint leaves at a dose of 200 mg/kg bw. ▪ group v (lp+pl low): received a combination of lemon peel extract (100 mg/kg bw) and peppermint leaf extract (100 mg/kg bw). ▪ group vi (lp+pl high): received a combination of lemon peel extract (200 mg/kg bw) and peppermint leaf extract (200 mg/kg bw). all treatments were administered orally once daily using an oral gavage for 14 consecutive days. at the end of the treatment period, the animals were subjected to behavioral tests to evaluate antidepressant and anxiolytic effects. forced swimming test (fst) each mouse was individually placed in a transparent 2l beaker glass filled with 1.5 l of water. the animals were allowed to swim for 6 minutes, and the duration of immobility was recorded during the final 4 minutes of the test. a mouse was considered immobile when it ceased active struggling and remained floating in an upright position, making only the minimal movements required to keep its head above the water. tail suspension test (tst) in the tail suspension test (tst), each mouse was suspended by the tail using adhesive tape affixed approximately 1 cm from the tip of the tail, at a height of 58 cm above the floor. after an initial 2-minute habituation period, the duration of immobility was recorded during the following 4 minutes. a mouse was considered immobile when it hung passively and remained completely motionless, indicating behavioral despair. figure 1. tail suspension test apparatus. elevated plus maze test (epm) the elevated plus maze (epm) apparatus consists of two open arms (16 × 5 cm) and two closed arms (17 × 5 × 30 cm) with an open roof, arranged in a plus (+) configuration and elevated 75 cm above the floor surface. each mouse was placed at the center of the maze, facing one of the open arms, to begin the test. during a 5minute observation period, the following parameters were recorded: the number of entries into the open arms and the time spent in the open arms. increased exploration of the open arms was interpreted as an indication of reduced anxiety-like behavior. figure 2. elevated plus maze apparatus, side view (left), top view (right). 1300 biology, medicine, & natural product chemistry 14 (2), 2025: 1297-1304 light dark box test (ldb) the light-dark box (ldb) apparatus consists of two connected acrylic chambers: a light compartment and a dark compartment. the light compartment measures 27 × 27 × 27 cm and is illuminated by a 40-watt lamp, while the dark compartment measures 27 × 18 × 27 cm and remains unlit. a transition door measuring 7.5 × 7.5 cm connects the two compartments, allowing the mice to move freely between them. figure 3. light dark box apparatus. acute oral toxicity test an acute oral toxicity test was conducted on the ethanolic extract of lemon peel (2000 mg/kg bw), peppermint leaves (2000 mg/kg bw), and their combination (2000 mg/kg bw each), in accordance with oecd guideline 420 (fixed dose procedure) using the limit test approach (oecd, 2001). extracts were given in a single dose. the study used five healthy female mice, aged 8 weeks and weighing between 25-35 grams. the animals were administered the test substance orally and observed closely for clinical signs of toxicity. mice were monitored closely for first 30 minutes post-dosing, periodically during the first 24 hours, and then once daily for a total period of 14 days. all animals were monitored for behavioral changes, clinical symptoms, morbidity, and mortality. data analysis data were presented as mean ± standard error of the mean (sem). parametric data, such as immobility time and time spent in specific compartments, were analyzed using one-way analysis of variance (anova), followed by the least significant difference (lsd) post hoc test to identify significant differences between groups. nonparametric data, such as the number of entries into the light compartment and open arms, were analyzed using the kruskal-wallis test. a p-value of < 0.05 was considered statistically significant. results and discussion results phytochemical contents both lemon peel extract and peppermint leaf extract contained all of the screened phytochemical metabolites, as shown in table 1. table 1. results of phytochemical screening. metabolites lemon peel extract peppermint leaves extract alkaloid + + flavonoid + + phenol + + steroid + + tannin + + saponin + + antidepressant activity both extracts exhibited antidepressant activity, as shown in figure 4. figure 4. immobility time in forced swimming test (left) and tail suspension test (right). nc: negative control, fluox: fluoxetine, lp: lemon peel extract, pl: peppermint leaves extract, lp+pl low: combination of lemon peel 100 mg/kgbw and peppermint leaves 100 mg/kgbw, lp+pl high: combination of lemon peel 200 mg/kgbw and peppermint leaves 200 mg/kgbw. *) significant compared to negative control (p < 0.05). * * * * * * * * * * ni’ma et al – comparative antidepressant, anxiolytic, and acute toxicity evaluation 1301 anxiolytic activity figure 5. result of anxiolytic activity in elevated plus maze and light dark box test. nc: negative control, fluox: fluoxetine, lp: lemon peel extract, pl: peppermint leaves extract, lp+pl low: combination of lemon peel 100 mg/kgbw and peppermint leaves 100 mg/kgbw, lp+pl high: combination of lemon peel 200 mg/kgbw and peppermint leaves 200 mg/kgbw. *) significant compared to negative control (p < 0.05). acute oral toxicity study no mortality was observed at the oral dose of 2000 mg/kg for each extract and for the combination. discussion the phytochemical screening of lemon peel and peppermint leaf extracts revealed the presence of diverse bioactive constituents, including alkaloids, flavonoids, phenols, steroids, tannins, and saponins. these findings are consistent with previous reports on the phytochemical richness of citrus peels and aromatic herbs such as peppermint (hudz et al., 2023). the wide range of metabolites detected indicates that the extracts possess multiple pharmacological potentials relevant to neuropsychiatric disorders. flavonoids and phenolic compounds are of particular importance due to their antioxidant and anti-inflammatory effects, as well as their ability to modulate monoaminergic neurotransmission (nájera-maldonado et al., 2024). the extract of lemon peel contains various constituents such as limonene, linalool, citronelal, apigenin, hesperidin, βbisabolene, trans-caryophyllene, nerylacetate, αterpineol, β-pinene, γ-terpinene (huang et al., 2025; taktak et al., 2021). meanwhile, peppermint leaves are rich in flavonoids, including hesperidin, catechin, epicatechin, rutin, myricetin, luteolin, apigenin (mahendran & rahman, 2020b). luteolin contained in peppermint leaves extract exerts its antidepressant effects through multiple molecular and cellular pathways. it has been shown to enhance uptake of noradrenaline (ne), inhibit reuptake of 5hydroxytryptamine (5-ht), and increase the expression of key synaptic and neuroprotective proteins such as synaptophysin, postsynaptic density protein 95, brainderived neurotrophic factor (bdnf), b-cell lymphoma protein-2 (bcl-2), superoxide dismutase (sod), and glutathione s-transferase (gst) (zhou et al., 2025). apigenin, present in both extracts, may contribute to the observed antidepressant activity through modulation of serotonergic and catecholaminergic pathways. its pharmacological effects have been associated with interactions involving α-adrenergic receptors as well as 5-ht3, d1, d2, and d3 receptors. experimental studies have shown that apigenin, at doses of 25 and 50 mg/kg bw, was able to prolong mobility time in rats pretreated with serotonergic inhibitors and catecholaminergic antagonists, indicating its role in enhancing monoaminergic neurotransmission (al-yamani et al., 2022). menthol, one of the main constituents of peppermint leaves, may alleviate depressive symptoms 1302 biology, medicine, & natural product chemistry 14 (2), 2025: 1297-1304 by enhancing dopaminergic activity through d1 and d2 receptor modulation (mohaghegh daghigh, 2024). the behavioral outcomes from the forced swimming test (fst) and tail suspension test (tst) showed that both lemon peel and peppermint leaf extracts significantly reduced immobility time compared with the negative control, reflecting antidepressant-like activity. the magnitude of these effects was comparable to fluoxetine, indicating that the extracts may act through similar neurochemical pathways. in rodent models, reduced immobility is interpreted as diminished behavioral despair, which is predictive of antidepressant efficacy. interestingly, the combination of both extracts with low dose (half of each extract) produce similar antidepressant activity compared to the single extracts, indicating additive interaction. however, the high dose combination did not produce an increase in antidepressant activity. this finding may be explained by overlapping mechanisms of action, since both extracts are rich in flavonoids and phenolic compounds that target monoaminergic systems, antioxidant defenses, and neurotrophic signaling (mahendran & rahman, 2020b; taktak et al., 2021). another possibility is that certain phytochemicals from lemon peel and peppermint compete for the same receptor sites or metabolic pathways, resulting in a plateau effect rather than potentiation. further studies on dose optimization and interaction profiles are needed to clarify the pharmacodynamic relationship between the two extracts. in the anxiety tests, increased time spent and entries into the open arms of the elevated plus maze (epm) and the light compartment of the light-dark box (ldb) were interpreted as indicators of reduced anxiety-like behavior. in this study, only certain groups demonstrated anxiolytic activity to some extent, namely the peppermint leaf extract group, the lemon peel extract group, and the low-dose combination group. interestingly, the positive control group (fluoxetine) did not display anxiolytic activity and instead showed a tendency to increase anxiety, although this effect was not statistically significant. this finding is consistent with previous studies reporting that a single intraperitoneal injection of fluoxetine at doses of 5 or 10 mg/kg reduced the time spent in the open arms of the epm, suggesting an anxiogenic effect. at higher doses (15 mg/kg), fluoxetine produced sedative effects, as indicated by a shorter total distance traveled within the maze. (drapier et al., 2007). peppermint leaf extract consistently demonstrated strong anxiolytic activity across both models, while lemon peel extract exhibited anxiolytic effects in the elevated plus maze (epm) but not in the light-dark box (ldb). these differences may reflect variation in the sensitivity of each test to specific neurochemical pathways. the anxiolytic activity of both extracts can be attributed in part to terpenoids such as menthol, limonene, and α-terpineol, which are abundant in peppermint and lemon peel essential oils (alvaradogarcía et al., 2024; beigi et al., 2018b; huang et al., 2025). these compounds have been reported to modulate gabaergic neurotransmission, enhancing inhibitory signaling in key brain regions such as the amygdala and hippocampus, thereby reducing anxiety-like behavior (mahendran & rahman, 2020a). in addition, flavonoids and phenolic acids present in both extracts, such as apigenin, luteolin, and catechins, are known to exert anxiolytic effects through serotonergic regulation, antioxidant activity, and neuroprotective mechanisms (al-yamani et al., 2022; zhou et al., 2025). the coexistence of these phytochemicals suggests that their combined action may contribute to the observed behavioral outcomes. the low dose combination group also produced anxiolytic effects comparable to peppermint extract alone, suggesting possible synergistic interactions between the two extracts when administered at balanced concentrations. such synergy may arise from complementary mechanisms, with terpenoids modulating gabaergic signaling while flavonoids regulate monoaminergic pathways and oxidative stress, together amplifying the anxiolytic response. in contrast, the high dose combination failed to show anxiolytic activity. this unexpected result may be explained by pharmacodynamic antagonism, receptor desensitization, or competition between overlapping active compounds at higher concentrations. excessive stimulation of serotonergic or gabaergic receptors can paradoxically lead to anxiogenic or sedative effects, masking the anxiolytic potential of the extracts. the absence of mortality at an oral dose of 2000 mg/kg for both lemon peel and peppermint leaf extracts, as well as their combination, suggests that these extracts can be considered practically non-toxic according to the oecd classification system (oecd, 2001). substances with an ld₅₀ value greater than 2000 mg/kg are generally regarded as safe and fall into the lowest toxicity category. in addition to the absence of mortality, no overt signs of toxicity, morbidity, or behavioral abnormalities were observed during the 14-day observation period. this further supports the tolerability of the extracts at relatively high doses. taken together, the results of this study provide strong evidence that lemon peel and peppermint extracts, both alone and in combination, possess antidepressant and anxiolytic activities, likely mediated through multitarget mechanisms involving monoaminergic, gabaergic, and antioxidant pathways. their efficacy, combined with their favorable acute safety profile, highlights their potential as safe, natural alternatives or adjuncts to conventional antidepressant and anxiolytic therapies. nonetheless, further studies, including subchronic toxicity evaluations, molecular docking, and clinical trials, are needed to establish their precise mechanisms and therapeutic relevance in humans. ni’ma et al – comparative antidepressant, anxiolytic, and acute toxicity evaluation 1303 conclusions this study demonstrates that lemon peel and peppermint leaf extracts, both individually and in combination, exhibit antidepressant and anxiolytic activities linked to their rich phytochemical profiles. peppermint showed the strongest anxiolytic effect, while the low-dose combination suggested synergistic interactions between the two extracts. the absence of acute toxicity at 2000 mg/kg indicates a favorable safety profile for potential therapeutic use. these findings highlight the promise of lemon peel and peppermint as safe, natural alternatives or adjuncts to conventional treatments for mood disorders. acknowledgements: the authors would like to express the gratitude to faculty medicine, universitas negeri semarang for the granted funding through 2024 dpa project with contract number “18.25.4/un37/ppk.17/2024”. competing interests: the authors declare that there are no competing interests. funding: this study received a granted funding through 2024 dpa faculty of medicine universitas negeri semarang, with contract number “18.25.4/un37/ppk.17/2024”. references alvarado-garcía, p. a. a., soto-vásquez, m. r., romero, t. l. c., benites, s. m., auris-lópez, a. d., & manuel del pinoaliaga, g. 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(2025). luteolin and its antidepressant properties: from mechanism of action to potential therapeutic application. in journal of pharmaceutical analysis (vol. 15, issue 4). xi’an jiaotong university. https://doi.org/10.1016/j.jpha.2024.101097 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 763-769 | doi: 10.14421/biomedich.2025.142.763-769 issn 2540-9328 (online) anatomical structure of young oil palm (elaeis guineensis jacq.) leaves for callogenesis initiation muhammad ilham*, ernayunita plant breeding research, indonesian oil palm research institute (iopri) jl. brigjen katamso no. 51 medan 20158, tel. +62 (061) 78-62477, indonesia. corresponding author* ilhambawazier04@gmail.com manuscript received: 30 june, 2025. revision accepted: 18 september, 2025. published: 01 october, 2025. abstract plant anatomy, a key area within biology, examines the structure and organization of plant organs and tissues. it underpins various disciplines such as physiology, ecology, taxonomy, and evolutionary biology. anatomical data are typically obtained using the paraffin embedding, which facilitates detailed microscopic observations. in oil palm (elaies guineensis jacq.) tissue culture, young leaves are commonly used as explants in callogenesis inducing undifferentiated cell growth. the study aimed to describe anatomical characteristics of young oil palm leaves, analyze the correlation between anatomical traits across different leaf positions, and examine anatomical changes during callogenesis. leaf samples were collected from positions -4, -5, -6, -7 and -8, processed using paraffin embedding technique, and analyzed microscopically. the percentages of callogenesis were also calculated for each leaf position. data were analyzed using anova, followed by duncan’s multiple range test (dmrt), and correlation analysis was performed in r studio. the anatomical features included the adaxial and abaxial epidermis, cuticle, hypodermis, mesophyll (palisade and spongy parenchyma), vascular bundles (phloem and xylem), stomata, and sclerencyma. the correlations between mesophyll and leaf thickness were very strong (r= 0.97, p>0.05), indicating that as mesophyll tissue thickness increases, the overall leaf thickness also increases. notably, leaves at position -8 exhibited the highest rate callogenesis, reaching 204%. keywords: anatomy; callogenesis; oil palm; paraffin; tissue culture. introduction the oil palm (elaeis guineensis jacq.) belongs to the family arecaceae (formerly palmae), which is part of the monocotyledonae order and classified as a monocotyledonous plant (cronquist, 1981). as with other monocots, the leaf venation in oil palm is either parallel or curved (tjitrosoepomo, 2009). oil palm leaves are characterized by large, pinnate structures, a single columnar trunk with short internodes, and irregular leaf vein patterns (corley & tinker, 2016). plant anatomy, is a key subdicipline of biology, focusing on the structural organization of plant organs and tissues from multiple perspectives. it is crucial in deepening our understanding of plant biology— from molecular-level functions to broader ecological interactions. a thorough understanding of plant anatomy, grounded in its scientific principles and practical applications, contributes significantly to physiology, ecology, taxonomy, and evolutionary biology (machado & oliveira, 2024). anatomical information can be effectively obtained using the paraffin embedding method, which has long been utilized in botany to prepare thin sections of plant tissue for microscopic analysis. this process involves replacing the water with paraffin, allowing for the production of smooth, thin ribbon sections suitable for detailed observation. although widely used, the paraffin embedding technique is time consuming and requires careful execution of multiple steps, including fixation, dehydration, infiltration, de-alcoholization, staining, mounting to produce high-quality and permanent slide preparation (de cássia andreota & sajo, 2015). previous research on the anatomical structure of oil palm leaves by gomes et al. (2017) which utilized explants from young leaves as initial material for somatic embryogenesis, highlighted several key anatomical features of these tissues. the mesophyll cells of young oil palm leaves exhibit dorsiventral characteristics with two and three layers of palisade parenchyma are composed of elongated, rod-shaped cells arranged in an order beneath the adaxial hypodermis. additionally, two to three layers of spongy parenchyma, consisting of more rounded cells with small intercellular spaces, were observed in the same region. the young leaf explants also possess a uniseriate (single-layered) epidermis composed of tightly packed rectangular cells. stomata are present exclusively on the abaxial surface, indicating https://doi.org/10.14421/biomedich.2025.142.763-769 764 biology, medicine, & natural product chemistry 14 (2), 2025: 763-769 hypostomatic leaf type. beneath the epidermis on both leaf surface lies a double-layered hypodermis, consisting of cells larger than those of the epidermis (gomes et al., 2017). in comparison, research on the anatomical characteristics of leaves in acrocomia species within the arecaceae family by vianna et al. (2017) revealed several key structural features. these include an epidermis covered by a cuticle, hypodermis present on both leaf surface, non-vascular fiber bundles, and both primary and secondary vascular bundles. similarly, adzkia et al. (2020) reported that the anatomical characteristics of the leaf sheath in royal palms comprises vascular bundles, parenchyma tissue, metaxylem, protoxylem, and fiber bundles. these vascular bundles and parenchymatous tissues are essential for defining functional and structural properties of oil palm species. furthermore, samiyarsih, (2019) emphasized that several leaf anatomical traits, such as cuticle thickness, epidermal thickness, mesophyll thickness, stomatal size and density, and presence of trichomes can be effectively observed using light microscopy (samiyarsih, 2019). the propagation of oil palm through tissue culture has been widely practiced, particularly for the multiplication of elite tenera clones. various explant sources have been utilized in tissue culture, including mature embryos (rabechaul et al., 1970), flowers (smith & thomas, 1973), roots (jones, 1974), seeds (ong, 1977), and young leaves (schwendiman, 1988). among these, young leaves especially spear leaves are considered highly suitable due to their totipotency and responsiveness to in vitro conditions. propagation using young leaf explants on basal media supplemented with naa (1napthaleneacetic acid) as a plant growth regulator has effectively stimulated callogenesis development. young leaf cells from spear leaves exhibit totipotency, highly receptive to signals that trigger the callogenesis pathway. auxin in younger explants promotes callus induction (constantin et al., 2015). this study focuses on the anatomical structures of young oil palm leaves as an initial indicator of their capacity. the objectives are to analyze the anatomical structure characteristics of young oil palm leaves (e. guineensis jacq.), examine the correlation between the anatomical traits across individual leaf positions, and evaluate the anatomical structure involved in callogenesis based on leaf position. materials and methods materials spear leaf of young oil palm with la mé genetic background, pure solid paraffin, fixative solution (faa) composed of 70% ethanol; formalin; glacial acetic acid in a 90:5:5 ratio (v/v). a graded ethanol series for dehydration: 70%, 80%, 95%, and 100% ethanol. clearing was performed using pure xylene, ethanol:xylene mixtures 3:1, 1:1, 1:3 (v/v). additional reagents included distilled water, 1% safranin stain in 70% ethanol, xylene:paraffin mixture (1:9 v/v), liquid pure paraffin, glycerol, glycerin-albumin mixture (1:1), and canada balsam. sample collection and preparation of leaf anatomy young oil palm (e. guineensis) explants were obtained from the spear leaves at positions -4, -5, -6, -7, and -8. the leaves were collected for the tissue culture laboratory of the indonesia oil palm research institute (iopri), north sumatera, indonesia. leaves lamina section was cut into small squares measuring 0.5 x 0.5 cm², with three replications performed (maryani, 2009). anatomical slide preparation was carried out using a modified paraffin embedding technique based on the protocols by sutikno (2018). the embedding process began with fixation of the leaf samples in a solution of formalin, glacial acetic acid, and 70% ethanol in a 5:5:90 (v/v) ratio for 24 hours. the samples were then stained with 0.5% safranin in 70% ethanol. the clearing process involved sequential ethanol series of 70%, 80%, 96%, and 100% ethanol followed by ethanol:xylene mixtures at 1:3, 1:1, and 3:1 (v/v), each for 30 min. infiltration was conducted by immersing samples in xylene:paraffin mixture (1:9) and placing them in an oven at 57°c for 24 h. the mixture was then replaced with pure paraffin, and the samples were maintained at 57°c for an additional 24 h. the leaf samples were placed in petri dishes and embedded in paraffin blocks. these 6-12 µm sections were obtained using a rotary microtome (hy-3615). microscopic observations prepared slides were observed under a binocular microscope and documented using a camera (olympus dp 23, japan). the anatomical parameters of the young leaves that were observed and measured included: leaf thickness (µm), length of the upper epidermis (µm), length of the lower epidermis (µm), width of the upper epidermis (µm), width of the lower epidermis (µm), length of xylem (µm), length of phloem (µm), width of xylem (µm), width of phloem (µm), length of the palisade tissue (µm), length of the mesophyll (µm). the observations were conducted using magnifications 10x10 and 20x10. callogenesis percentage (%) explants from 11 marihat clone were cultured in four replicates on media and incubated for 6–12 months. the number of primary calluses that developed on each leaf position (-4,-5,-6,-7,and-8) was recorded (n). the percentage of callogenesis was calculated using the following formula: %𝐶𝑎𝑙𝑙𝑜𝑔𝑒𝑛𝑒𝑠𝑖𝑠 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑟𝑖𝑚𝑎𝑟𝑦 𝑐𝑎𝑙𝑙𝑢𝑠𝑒𝑠 (𝑛) 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑒𝑥𝑝𝑙𝑎𝑛𝑡𝑠 𝑐𝑢𝑙𝑡𝑢𝑟𝑒𝑑 × 100 ilham & ernayunita – anatomical structure of young oil palm … 765 this calculation was performed for each leaf position to determine the callus formation success rate at different developmental stages. the results were then analysed and presented in percentage form. data analysis qualitative data were analyzed descriptively and presented in the form of images. quantitative data, derived from anatomical measurements, were statistically analyzed using spss software v. 25.0 (ibm, usa). the significance of the leaf anatomical characteristics was assessed through analysis of variance (anova) with a 95% confidence level, followed by duncan’s multiple range test (dmrt) at a 5% significance level. correlation analysis was performed to evaluate the strength of the relationship between anatomical variables using r studio software. the correlation coefficient (r) is calculated using the following equation: where: r : the correlation coefficient. n : the number of pairs of data. ∑x : number of independent variables ∑y : number of dependent variables the interpretation of the sample correlation coefficient between variables is classified according to table 1 (usman & purnomo, 2000). table 1. the correlation coefficient and its interpretation. coorrelation value interpretations 0,00 – 0.09 negligible correlation 0.10 – 0.29 low correlation 0.30 – 0.49 moderate correlation 0.50 – 0.70 strong correlation > 0.70 very strong correlation results and discussion young leaves are morphologically characterized by a smooth surface, flexible texture, and low chlorophyll content. microscopic observations of their anatomical structure reveal several key components in cross-section. these include the upper epidermis (adaxial), lower epidermis (abaxial), hypodermis, palisade parenchyma (consisting of two to three cells), mesophyll, vascular bundles (phloem and xylem), stomata, cuticle, sclerenchyma, and spongy parenchyma (figure 1). figure 1. representative anatomical structure of young oil palm (e. guineensis) leaves at 100x magnification. a) anatomical structure of the leaf vein, b) anatomical structure of the leaf. abbreviation: ea: upper epidermis, hp: hypodermis, pp: palisade parenchyma, m: mesophyll, bp: vascular bundle (vbs), s: stomata, eb: lower epidermis, k: cuticle, ep: epidermis, skl: sclerenchyma, x: xylem, p: phloem, and js: spongy tissue. bar = 100 µm the midrib of the oil palm leaf (figure 1a) exhibits larger ones associated with smaller vascular bundles. this observation is consistent with the findings of luis et al. (2010) who reported that the oil palm leaf anatomy includes a midrib containing prominent vascular bundles connected to smaller vascular elements. stomata are observed exclusively on the leaf’s abaxial (lower) epidermis. according to azmin et al. (2021), oil leaf anatomy comprises three major components: the epidermis, mesophyll (including palisade and spongy tissues), and vascular tissue. the epidermis comprises the adaxial (upper) and abaxial (lower) layers. the adaxial epidermis is located beneath the cuticle and above the hypodermal tissue, while the abaxial epidermis lies below the spongy parenchyma. beneath both epidermal layers is a twolayered hypodermis composed of larger cells than those of the epidermis (gomes et al., 2017). the epidermal tissue exhibits distinct characteristics: (1) rectangular cell shape, (2) tightly packed arrangement without intercellular spaces, (3) absence of chlorophyll, and (4) potential to differentiate into other epidermal cell types (muttaqin, 2023). both epidermal layers are composed of a single layer of compactly arranged cells, each covered 766 biology, medicine, & natural product chemistry 14 (2), 2025: 763-769 by a cuticle (surya & hari, 2017). the cuticle, a waxy or fatty layer, is a protective barrier, minimizing water loss through evaporation and shielding against pathogens and physical damage. two or three layers of palisade parenchyma were observed beneath the adaxial hypodermis. these elongated, rod-shape cells are arranged vertically in rows. beneath the palisade layers, two to three layers of spongy parenchyma are present, consisting of rounder cells with small intercellular spaces. scattered throughout the mesophyll are clusters of underdevelop vascular bundles (gomes et al., 2017). quantitative measurements of the anatomical features of young leaves are summarized in table 2. table 2. range of average values for anatomical parameters of young oil palm (e. guineensis) leaves. anatomical parameter leaf sample -4 -5 -6 -7 -8 leaf thickness (µm) 203.20 ± 5.67 171.24 ± 13.94 66.68 ± 11.38 195.20 ± 15.00 207.65 ± 13.24* length of the upper epidermis (µm) 10.40 ± 1.83 9.60 ± 1.27 6.20 ± 0.54 17.20 ± 4.85* 13.68 ± 0.59 length of the lower epidermis (µm) 8.00 ± 4.21 10.08 ± 4.27 6.53 ± 2.80 10.48 ± 3.95 10.65 ± 2.27 width of the upper epidermis (µm) 11.60 ± 1.39 12.80 ± 1.00 15.84 ±4.63 14.40 ± 2.08 9.17 ± 1.11 width of the lower epidermis (µm) 18.00 ± 1.20 15.52 ± 6.45 8.64 ± 0.87 11.20 ± 2.27 15.16 ± 5.43 length of xylem (µm) 71.40 ±15.19 53.60 ± 9.97 93.52 ± 20.40 64.24 ± 12.16 75.00 ± 14.30 length of phloem (µm) 13.80 ± 3.37 18.24 ± 1.92 23.44 ± 3.10 24.24 ± 3.86 17.16 ± 2.19 width of xylem (µm) 87.56 ± 2.70 49.28 ± 15.38 89.56 ± 8.77 57.48 ± 15.67 54.10 ± 12.72 width of phloem (µm) 13.96 ± 2.90 21.12 ± 3.84 17.56 ± 0.73 25.76 ± 5.49 19.96 ± 1.20 length of the palisade tissue (µm) 12.56 ± 2.22 15.16 ± 1.23 11.72 ± 0.54 30.00 ± 3.17* 9.00 ± 0.62 length of the mesophyll (µm) 120.80 ± 5.41 91.32 ± 7.62 66.68 ± 11.38 114.0 ± 12.98 127.20 ± 17.32* note *: significant at (p ≤ 0,05). the observations revealed that several anatomical parameters were statistically significant (p ≤ 0.05) including leaf thickness (207.65 ± 13.24), mesophyll thickness (127.20 ± 17.32), upper epidermis length (17.20 ± 4.85), and palisade tissue length (30.00 ± 3.17). leaf thickness is closely associated with cuticle thickness. the cuticle is relatively thick in oil palm leaves, contributing to an enhanced defense mechanism. plants with thicker cuticles generally exhibit greater biotic stress resistance than those with thinner cuticles. this is attributed to the structural composition of the cuticle, which includes cutin, cellulose, oligosaccharides, and waxes. it compounds that act as physical and chemical barriers by inhibiting spore attachment, germination, and pathogen penetration (pradana et al., 2017). furthermore, as farassati et al. (2021) noted, a well-developed cuticle may also facilitate the diffusionbased absorption of micronuteients, growth regulators, and oligo-elements, thereby supporting improved physiological responses during in vitro culture. the mesophyll tissue of e. guineensis leaves consists of palisade parenchyma in the upper region and spongy parenchyma in the lower region. vascular bundles are distributed throughout the mesophyll. according to surya & hari (2017) the mesophyll is anatomically divided into two distinct layers: the palisade tissue, located just below the upper epidermis, and the spongy tissue, located above the lower epidermis. although these two tissues have different structures, both play essential roles in photosynthesis. despite their structural differences, both tissues play vital roles in photosynthesis. the palisade tissue is particularly important due to its high chloroplast concentration, which enhances light absorption and carbon fixation (tobing et al., 2021). tihurua et al. (2020) stated that a higher ratio of palisade to spongy tissue indicates the effectiveness of the tissue in the photosynthesis process for capturing light and co2. in most plants, the products of photosynthesis in older leaves are transported to other parts, including younger leaves. according to azmin et al. (2021) the photosynthetic products are allocated to all parts involved in growth and development. juwarno et al. (2018) reported that increased thickness of the lower epidermis reduces water loss, supporting the plant adaptation to environmental stress. this study examined the relationship between several anatomical characteristics of young oil palm (e. guineensis) leaves to assess potential correlations influencing the callogenesis process. the analysis focused on five leaf sections (positions -4, -5, -6, -7, and -8). the results revealed both positive (+) and negative (−) correlations among the anatomical traits of the leaves, as presented in figure 2. ilham & ernayunita – anatomical structure of young oil palm … 767 figure 2. heatmap of the correlation between several anatomical characteristics of young oil palm leaves (e. guineensis). based on figure 2, a very strong correlation was observed between mesophyll tissue thickness and overall leaf thickness (r = 0.97, p > 0.05), indicating that leaf thickness also increases as mesophyll tissue becomes thicker. according to cambaba et al. (2016), the mesophyll constitutes the primary component of the leaf blade; thus, any variation in mesophyll thickness has a significantly impacts on total leaf thickness. variation in leaf thickness among plant species may be attributed to genetic factors that regulate plant anatomical characteristics (coneva & chitwood, 2018). utami (2017) further noted that sunlight exposure influences leaf thickness, with increased light intensity promoting the development of a thicker palisade layer. in addition to light, leaf thickness is also affected by factors such as water content in plant tissues (essaghi et al., 2016). other environmental variables, including nutrient availability and light intensity, can also contribute to variations in leaf anatomical structure (ningsih & daningsih, 2022). young oil palm leaves (e. guineensis) are frequently utilized as a source of explants for regeneration in tissue culture processes. according to nurchayati et al. (2018) leaf explants are preferred due to their relatively thin morphology, which facilitates enhanced contact between the tissue surface and the culture medium, enhancing regeneration. one of the primary regeneration pathways for oil palm leaves is somatic embryogenesis. leaves from positions -4, -5, -6, -7, and -8, used in the tissue culture process, exhibit varying levels of calogenesis, which are influenced by the anatomical characteristics of each leaf (figure 3). figure 3. the percentage of calogenesis derived from various responsive sections of young oil palm leaves (e. guineensis). based on figure 3, the highest percentage of calogenesis in young oil palm leaves (e. guineensis) was observed in leaf position -8, reaching 204%. this is associated with the positive correlation between leaf thickness and mesophyll thickness, which shows a positive correlation (+). a greater mesophyll cell thickness can enhance photosynthetic efficiency per unit area and improve the translocation of photoassimilates required by plant organs (torres-pio et al., 2021). mesophyll cells can regenerate and differentiate into various types of tissues, including roots and shoots, when cultured in a tissue culture medium. according to gomes et al. (2017), the early stages of calogenesis are closely linked to the central part of the leaf, particularly around vascular bundles (phloem and xylem). the cut edges of 768 biology, medicine, & natural product chemistry 14 (2), 2025: 763-769 young leaves, near these vascular bundles, have greater contact with the nutrient-rich culture media. this facilitates cell division near the vascular bundles, forming callus, which contains smaller cells, dense cytoplasm, and distinct nuclei. callus development through calogenesis involves periclinal cell division, resulting in organized cell development and increased leaf thickness. palisade parenchyma cells near the cell mass show minimal shape changes due to compression by the epidermal cells on the adaxial surface, leading to an increase in width and a decrease in height. in contrast, spongy parenchyma and abaxial epidermal cells near the developing propagules are often damaged or disrupted during callus formation. conclusions the anatomical characteristics of young oil palm leaves (e. guineensis) include the upper epidermis (adaxial), lower epidermis (abaxial), hypodermis, mesophyll (palisade parenchyma and spongy parenchyma), vascular bundles (phloem and xylem), stomata, cuticle, and sclerenchyma. the correlation between the anatomical characteristics of the mesophyll tissue and leaf thickness shows a very strong correlation between mesophyll thickness and overall leaf thickness (r = 0.97, p > 0.05), indicating that an increase in mesophyll thickness contributes to greater leaf thickness. this increase may enhance the callogenesis process by promoting cellular activity within the tissue. among the leaf positions examined, position -8 exhibited the highest callogenesis percentage, reaching 204%. acknowledgements: the authors express their sincere gratitude to the indonesian oil palm research institute (iopri) for its unwavering support throughout this research. we also extend our appreciation to our colleagues, technicians, and reviewers for their valuable contributions, insightful feedback, and assistance in the preparation of preparing this manuscript. competing interests: the authors declare that there are no competing interests. references adzkia, u., nugroho, n., & karlinasari, l. 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(2017). leaf anatomy of acrocomia (arecaceae): an additional contribution to the taxonomic resolution of a genus with great economic potential. plant systematics and evolution, 303, 233-248. https://doi.org/10.1007/s00606-016-1369-4 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 585-589 | doi: 10.14421/biomedich.2024.132.585-589 issn 2540-9328 (online) the recent collection of climacodon roseomaculatus (hydnum roseomaculatum henn. & e. nyman) from the type locality (indonesia) ivan permana putra1,*, oktan dwi nurhayat2 1department of biology, faculty of mathematics and natural sciences, ipb university, darmaga campus, bogor 16680, indonesia. 2research center for applied microbiology, national research and innovation agency (brin), bogor, west java, 16914, indonesia. corresponding author* ivanpermanaputra@apps.ipb.ac.id manuscript received: 08 april, 2024. revision accepted: 04 december, 2024. published: 20 december, 2024. abstract climacodon roseomaculatus is a fleshy species of polyporales with a hydnoid hymenophore. the species’s distribution in indonesia was reported six times from 1923 to 1961 from west java and east java collection. to date, there have been no following updates on the collection and occurrence status of c. roseomaculatus in indonesia. during a fungus foray in 2023, some red to pink basidiomata of climacodon were spotted in semarang, central java, indonesia. the current study aimed to determine the taxonomical position of climacodon bo24632 based on morphological evidence. fresh materials were used for the investigation of macroand micromorphological characters. the result showed that climacodon bo24632 was identified as c. roseomaculatus based on distinctive features of stipe and gleocystidia. climacodon roseomaculatus bo24632 is closely related to c. sanguineus with blood-red to pink basidiomata, cylindrical spines of hymenophores, and ellipsoid basidiospores. however, the former species lacks a well-developed stipe and has thin to thick-walled gloeocystidia. this study is the first report on the distribution of c. roseomaculatus from central java, indonesia. in addition, the current work is the latest information and herbarium collection of c. roseomaculatus after more than fifty years of hiatus in indonesia. we expect that the same species also occur in many different locations in indonesia; thus need a warrant of collections. keywords: macrofungi; morphology; polyporales; rediscovery; central java. introduction the genus climacodon was proposed by karsten (1881) based on the type species of hydnum septentrionale (gorjón 2020). this genus is acknowledged both as the hydnaceous fungi (maas geesteranus 1971; moreno et. al. 2007; 2017) and corticoid fungi (gorjón 2020). climacodon is recognized by simple pileate basidiomata white, pink, or blood red when fresh, with a hydnoid hymenophore covered with spines on the underside of the pileus, lignicolous, with or without stipe (sessile, sometimes with effused base) (maas geesteranus 1971). climacodon has monomitic system of hyphae, cystidia encrusted-thick walled, clamp connections simple, and only the older portions of the pileus (maas geesteranus 1971). in addition, maas geesteranus (1971) described that climacodon poses 4-spored calavate basidia, no ornamentation, colorless, ellipsoid basidiospores. morphologically, to some extent, the genus climacodon is identical to hericium, but the latter displays amyloid basidiospores and is phylogenetically nested in the russulales (larsson 2007). currently, climacodon is placed in meruliaceae, polyporales, and agaricomycetes (kirk et al. 2008; index fungorum 2024). a previous study (binder et al. 2005; 2013) revealed that climacodon was placed in the phlebioid clade. based on index fungorum (2024) there are eleven species of climacodon, namely c. septentrionalis (fr.) p. karst., c. annamensis (har. & pat.), c. chlamydocystis (maas geest.), c. dubitativus (lloyd) ryvarden., c. efflorescens (maas geest.), c. javanicus (pat.) decock & ryvarden., c. ochraceus (pers. ex j.f. gmel.) p. karst., c. pudorinus (fr.) p. karst., c.pulcherrimus (berk. & m.a. curtis) nikol., c. sanguineus (beeli) maas geest., and c. roseomaculatus (henn. & e. nyman) jülich. of those, c. roseomaculatus is the only species noted without stipe and has a bright rose-pink color. this species is identical to c. sanguineus on the basis of the morphological features. however, c. sanguineus produce the prominent stipe which absent in c. roseomaculatus (maas geesteranus 1971; moreno et al. 2017). climacodon roseomaculatus was originally described as hydnum roseomaculatum from the type locality, west https://doi.org/10.14421/biomedich.2024.132.585-589 586 biology, medicine, & natural product chemistry 13 (2), 2024: 585-589 java indonesia (maas geesteranus 1971). this species is known to be distributed in the high latitude of tropical areas and has recently been reported as a newly recorded species to china (hai-sheng and wen-juan 2016). from 1923 to 1961, there were six herbarium collections of c. roseomaculatus in indonesia. hitherto, there has been no following report of this species in indonesia. therefore, the attempt to investigate the distribution of this species in another location on java island was made in central java, indonesia. some hydnoid basidiomata that grew on the dead tree branch were collected. at a glance, the specimen was initially suspected as climacodon cf. roseomaculatus. this study aimed to justify the taxonomical position of the specimen based on macro and micromorphological evidence and contribute to recollection of the certain species from type locality (indonesia). materials and methods specimens collection. the specimens were collected in february 2023 in indonesia, central java, semarang, perantunan 7°11'40.6"s 110°21'21.2"e (figure 1). the basidiomata were photographed in situ and the laboratory. the habitat, growth pattern, and surrounding vegetation were noted. the following observation was done in laboratory of mycology, department of biology, faculty of mathematics and natural sciences, ipb university, indonesia. the fruiting bodies were deposited at the herbarium bogoriense indonesia with the collection number bo24632. figure 1. sampling site of climacodon roseomaculatus bo24632 (red spot). photo modified from google earth. morphological identification macroscopic features investigation including basidiomata texture, context, cap attributes (shape, color, ornamentation on surface, wetness, margin), and hymenophore characters (type, color, ornamentation) (putra 2021). microscopic observation was performed using a digital bright field microscope olympus bx-63, japan. the microscopic examination includes the hyphal system, clamp connection, pileipelis, context (trama), and spines (basidia, basidiospores, and cystidia). the specimens were identified using some related references, including maas geesteranus (1971), moreno et al. (2017), and gorjón (2020). results and discussion taxonomy climacodon roseomaculatus (henn. & e. nyman) jülich [as 'roseomaculatum'], biblthca mycol. 85: 400 (1982) [1981] basionym: hydnum roseomaculatum henn. & e. nyman [as 'roseomaculatum'], in hennings in warburg 1899 climacodon javanicus (pat.) decock & ryvarden [as 'javanicum'] 2021 basidiomata fleshy, sessile (fig.1a), pileate, solitary, gregarious (1-2 fruiting bodies). pileus expanding up to 6 cm in length, 4 cm wide, flabelliform with a narrowed thick base, smooth to velutinate, bright red with white veins near the margin, forming the darker and lighter areas (fig. 1b). margin entire, concolorous to paler than center part. hymenophore hydnoid (fig. 1c), spines (fig. 1d) not fused, cylindrical, flattened, sometimes branched at the terminal, pink, white apices, smooth, unequal up to 1 cm length, mediumly crowded. context (fig. 1d) 3-5 mm thick, homogeneous, concolorous, or paler than spines. stipe absent. hyphal system monomitic, consisting of generative and tendril hyphae. pileipellis trama with dense hyphal knots (fig. 2a) to develop the interwoven tendril hyphae, oleiferous hyphae abundant (fig. 2b) 1.8-2.9 µm diam, generative hyphae with prominent clamp connection, 5-8 µm diam close to margin (fig. 2c), unbranched, thin walled, dense cytoplasm, up to 2-2.5 µm diam far-off margin (fig. 2d), with clamp connection and simple septa, opaque cytoplasm, regularly arranged. hyphae in the lower part of the context are monomitic, and more loosely arranged. hyphal context of the spines monomitic, hyaline, septate, branched, 2.2-6.4 µm wide, rather thick-walled in the axis (fig. 3a), thin-walled near the opposite (fig.3b), without no clamped hyphae, with oily cytoplasm especially near tips (fig. 3c), hyphal knots and incipient basidium (fig. 3d) evident. basidia (fig. 4a) clavate, 713 × 3.5-5.3 µm, without basal clamp, 4-spored, sterigmata 2.8-3.5 µm in length. basidiospores 3.3-5.8 × 1.6-2.4 µm, ellipsoid, smooth, hyaline, 1-2 lipid bodies. gloeocystidia originated from trama, sparse, 40-50 × 4.86 µm, subcylindric to clavate, smooth without crystals, sparse, thick-walled, with widely rounded apex. specimen examined: indonesia, central java, semarang, perantunan, s7º12'19.301'', e110º21' 13.099'', 1700 m putra & nurhayat – the recent collection of climacodon roseomaculatus … 587 a.s.l, february 2023, collected by purwadi and odn, bo24632. the knowledge of the genus climacodon in indonesia is poor. in the last decades, reports from meiliawati and kuswytasari (2013) claimed to obtain climacodon from wonorejo (central java) however with insufficient data and used it as decoloration agent (muslimah and kuswytasari 2013). climacodon roseomaculatus is a member of hydnaceus fungi poses the hydnoid type of hymenophores and is considered as corticioid macrofungi (maas geesteranus 1971; gorjón 2020). the only published taxonomical works of c. roseomaculatus in indonesia was provided by maas geesteranus (1971) from the book of hydnaceous fungi of the eastern old world. to date, c. roseomaculatus is only to be reported known from borneo and java (indonesia, type locality) (maas-geesteranus 1971) and china (hai-sheng and wen-juan 2016) but without the documentation of basidiomata in situ. climacodon roseomaculatus is easily recognized by the bright red color of hydnoid basidiomata. based on the macroscopic features, c. roseomaculatus bo24632 is closely related to c. sanguineus in having a bright red to pink fruiting body. these two species are the only species with red color in climacodon. however, the former species lacks stipe and produces prominent thick‐walled cystidia (maas geesteranus 1971) or known as gleocyctidia as the distinct characters. moreno et al. (2017) suggest that c. roseomaculatus and c. sanguineus are close but proved to be a distinct species. despite the lack of its stipe, c. roseomaculatus reported from type locallity once observed to have a tiny stipe (maas geesteranus 1971). however, in line with hai-sheng and wen-juan (2016), the stipe of our specimen was absent. the spine length was the same as a previous report by maas geesteranus (1971) but shorter than c. roseomaculatus reported from china (hai-sheng and wen-juan 2016). microscopycally, the morphological features of our specimen were more or less the same compared to the prior reports of c. roseomaculatus. however, the striking difference is the occurrence of dense hyphal knots on the upper surface of the pileus which only appeared in the lower part of the context of the previous report from west java (maas geesteranus 1971). in contrast, hai-sheng and wen-juan (2016) did not describe any hyphal knots from their specimen, but they reported the occurrence of the crystals that encrusted the gleocystidia which is absent in the current report and the report of maas geesteranus (1971). in addition, in line with aas geesteranus (1971), we described the oleiferous hyphae of c. roseomaculatus which was not investigated by hai-sheng and wen-juan (2016) from their collection. the difference might be related to the morphological plasticity of c. roseomaculatus which remains obscure. recently, index fungorum (2024) accepted climacodon javanicus (pat.) decock & ryvarden as the current name of c. roseomaculatus. climacodon roseomaculatus was previously described as hydnum roseomaculatum from java, bogor botanical garden, indonesia in 1899 (maas geesteranus 1971). the subsequent collection from type locality was done in november 1923 (west java, tjibodas, m. l. a. bruggeman bo 5570), in march 1936 (jungle trail between tjibodas and tjibereum waterfall, 2409l), in april 1936 (jungle trail between tjibodas and tjibereum waterfall), in september 1938 (tjisarua, bo 16455), in 1955 (mt. pangrango, 12698 bo), in 1938 (kediri, west java, mt. wilis, herb. donk), and in december 1961 (jungle trail between tjibodas and tjibereum waterfall). since then, our work is the only report and or collection of this species from indonesia. this study revealed evidence of extension on the geographical distribution of c. roseomaculatus, more than 300 km away from west java, located in central java, indonesia. the basidiomata were found in the highland area in this study. hai-sheng and wen-juan (2016) suggest that this species has a distribution in the high latitude of tropical areas. figure 2. climacodon roseomaculatus bo24632 in situ. a-b. sessile basidioma grow on mixed soil and wood. c. hynoid hymenophore. d. details of spines (black arrow) of hymenophore and context (red arrow). bars: a=5 cm, b= 5cm, c-d=1 cm. 588 biology, medicine, & natural product chemistry 13 (2), 2024: 585-589 figure 3. pileipellis of climacodon roseomaculatus bo24632. a. prominent hyphal knots on the pileipellis, stained with lactophenol cotton blue. b. abundance of oleiferous hyphae on the pileipellis. c. generative hyphae adjacent to the margin. d. generative hyphae distant from the margin. bars: a, b, d= 50 µm, c= 20 µm. figure 4. spines of climacodon roseomaculatus bo24632. a. generative hyphae in spine axis. b. generative hyphae adjacent to subhymenium. c. oil hyphae on the tip of the spine. d. incipient basidium (red arrow) on spine. bars: a,c,d=20 µm, b= 50 µm. figure 5. hymenium of climacodon roseomaculatus bo24632. a. basidia with four sterigmata (arrows). b. elipsoid basidiospore (arrow). c. basidiospore stained with lactophenol cotton blue. d. thick walled gleocystidia (arrow). bars: 20 µm. conclusions in conclusion, climacodon roseomaculatus has been rediscovered in central java, indonesia after more than fifty years of absence in the country. this study provides important information for the distribution and occurrence status of this species in indonesia. further research and collection efforts are needed to understand the full extent of the distribution of c. roseomaculatus in the country. re-discoveries of such species highlight the importance of ongoing monitoring and documentation of fungal diversity to better understand and conserve the indonesian germplasm. acknowledgements: we are grateful to the department of biology, faculty of mathematics and natural sciences, ipb university, and research center for applied microbiology, national research and innovation agency (brin), indonesia for the research facilities. we thank mr. purwadi for his field assistance. authors’ contributions: ipp designed the study. ipp and odn performed macroscopic and microscopic analyses. odn provides the specimen description. ipp performed morphological identification. ipp and odn wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research received no funding. references binder, m., hibbett, d.s., larsson, k.-h., larsson, e., langer, e. & langer, g. (2005) the phylogenetic distribution of resupinate forms across the major clades of mushroomforming fungi (homobasidiomycetes). systematics and biodiversity, 3 (2),113–157. https://doi.org/10.1017/s1477200005001623 binder, m., justo, a., riley, r., salamov, a., sjökvist, e., copeland, a., foster, b., sun, h., larsson, e., larsson, k.-h., townsend, j., grigoriev, i.v. & hibbett, d.s. (2013) phylogenetic and phylogenomic overview of the polyporales. mycologia, 105, 1350–1373. https://doi.org/10.3852/13-003 gorjón, s. (2020). genera of corticioid fungi: keys, nomenclature and taxonomy. studies in fungi, 5(1), 125–309. https://doi.org/10.5943/sif/5/1/12 hai-sheng, y, wen-juan c. (2016). hydnaceous fungi of china 6. four species new to china. mycosystema, 35(5), 628‐635. index fungorum. 2024. climacodon. available at: http://www.indexfungorum.org/names/names.asp. [accesced: 25 february 2023] karsten, p.a. (1881) enumeratio hydnearum fr. fennicarum, systemate novo dispositarum. revue mycologique toulouse, 39, 19–21. putra & nurhayat – the recent collection of climacodon roseomaculatus … 589 kirk, p.m., cannon, p.f., minter, d.w., stalpers, j.a. 2008. dictionary of the fungi, tenth ed. cabi europe, wallingford, uk. koski-kotiranta, s., niemelä, t. (1987). hydnaceous fungi of the hericiaceae, auriscalpiaceae and climacodontaceae in northwestern europe. karstenia, 27(2), 43–70. https://doi.org/10.29203/ka.1987.253 larsson, k.-h. (2007). re-thinking the classification of corticioid fungi. mycological research, 111(9), 1040–1063. https://doi.org/10.1016/j.mycres.2007.08.001 maas geesteranus, r.a. (1971). hydnaceous fungi of the eastern old world. verhandelingen der koninklije nederlandse akademie van wetenschappen, afd. natuurkunde, tweede reeks, volume 60, issue/no. 3. north holland publishing company, 1971. pp. 1-176. meiliawati, d., kuswytasari, n.d. (2013). isolasi dan identifikasi jamur kayu lignolitik dari vegetasi mangrove wonorejo. jurnal sains dan seni pomits, 2(1), 16-19. muslimah, s., kuswytasari, n.d. (2013). potensi basidiomycetes koleksi biologi its sebagai agen biodekolorisasi zat warna rbbr. jurnal sains dan seni pomits, 2(1), 234-239. moreno, g., blanco, m.n., olariaga, i. checa, j. (2007) climacodon pulcherrimus, a badly known tropical species, present in europe. cryptogamie mycologie, 281, 3–11. moreno, g., blanco, m.n., platas, g., checa, j., olariaga, i. (2017). reappraisal of climacodon (basidiomycota, meruliaceae) and reinstatement of donkia (phanerochaetaceae) using multigene data. phytotaxa, 291, 171-182. putra, i. p. (2021). panduan karakterisasi jamur makroskopik di indonesia: bagian 1 – deskripsi ciri makroskopis. jurnal penelitian kehutanan wallacea, 10(1), 25-37. https://doi.org/10.18330/jwallacea.2021.vol10iss1pp25-37 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 831-841 | doi: 10.14421/biomedich.2025.142.831-841 issn 2540-9328 (online) analysis of nutritional composition and antioxidant activity of yogurt enriched with butterfly pea (clitoria ternatea l.) extract yulia windarsih*, i made budiarsa, manap trianto, akram, abdul ashari departement of biology education, faculty of teacher training and education, tadulako university, jl. soekarno hatta no.99, tondo, palu tim., palu, central sulawesi 94111, indonesia. corresponding author* yuliawindarsih777@gmail.com manuscript received: 14 august, 2025. revision accepted: 03 october, 2025. published: 17 october, 2025. abstract the growing demand for functional foods has encouraged the development of dairy products enriched with natural bioactive ingredients to improve nutritional value and health benefits. this study aimed to analyze the nutritional composition and antioxidant activity of yogurt enriched with butterfly pea (clitoria ternatea l.) extract. yogurt was prepared from fresh goat milk with varying concentrations of butterfly pea extract and evaluated for protein, fat, carbohydrate, moisture, ash, vitamin c contents, antioxidant activity, and sensory acceptance. proximate analysis, uv–vis spectrophotometry, and the dpph method were used for chemical determinations, while a hedonic test assessed color, taste, aroma, and texture. results showed that increasing butterfly pea extract significantly decreased protein and fat contents but increased vitamin c and antioxidant activity, with the highest values observed in yogurt containing 60% extract and 4% formulation (n60k2). moisture and ash contents were not significantly affected. sensory evaluation indicated that yogurt with 60% extract and 2% formulation (n60k1) was preferred regarding color, taste, aroma, and texture. considering compliance with the indonesian national standard (sni) and consumer acceptance, n60k1 was the optimal formulation. the findings suggest that butterfly pea extract can enhance the functional properties of yogurt, particularly its antioxidant potential, while maintaining acceptable sensory quality. this provides insights for developing functional dairy products with natural plant-based bioactives. keywords: antioxidant activity; butterfly pea; clitoria ternatea; nutritional composition; yogurt. introduction yogurt is one of the most widely consumed fermented dairy products in the world, valued for its distinctive flavor, creamy texture, and numerous health benefits (farag et al., 2020). it is produced through the fermentation of milk by lactic acid bacteria, primarily lactobacillus bulgaricus and streptococcus thermophilus, which convert lactose into lactic acid. this process not only enhances the shelf life and digestibility of milk but also enriches its nutritional profile by increasing the bioavailability of certain nutrients (kaur et al., 2017; kok & hutkins, 2018). in addition to being a source of high-quality proteins, essential fatty acids, vitamins, and minerals, yogurt contains probiotics that support gut health and contribute to overall well-being (saritas et al., 2024). the growing demand for functional foods has further encouraged innovations in yogurt production, particularly through the incorporation of plant-based bioactive ingredients to improve both nutritional value and health-promoting properties (bankole et al., 2023; li et al., 2025). one plant-based ingredient of increasing interest in food product development is butterfly pea (clitoria ternatea l.) (hasanah et al., 2023), a leguminous plant widely cultivated in tropical and subtropical regions (marpaung et al., 2020; handayani et al., 2024). traditionally used in herbal medicine and as a natural food colorant, butterfly pea flowers are rich in anthocyanins particularly ternatins which are responsible for their vivid blue color and potent antioxidant activity (lakshan et al., 2019). beyond anthocyanins, the flowers contain flavonoids, phenolic compounds, and other phytochemicals that possess antioxidant, antiinflammatory, and anti-glycation properties (jeyaraj et al., 2021; rawiningtyas et al., 2024). recently, the food industry has shown growing interest in butterfly pea extract as a natural alternative to synthetic additives, both for its functional properties and visual appeal (yudiono, 2024). incorporating butterfly pea extract into food products not only enhances their sensory characteristics but also enriches their nutritional and functional profiles, making them more appealing to health-conscious consumers (siregar et al., 2022). antioxidants play a critical role in neutralizing free radicals, thereby reducing oxidative stress, which is associated with the development of chronic diseases such as cardiovascular disorders, cancer, and https://doi.org/10.14421/biomedich.2025.142.831-841 mailto:yuliawindarsih777@gmail.com 832 biology, medicine, & natural product chemistry 14 (2), 2025: 831-841 neurodegenerative conditions (sharma et al., 2023). the consumption of antioxidant-rich foods has been widely recommended as part of a balanced diet to promote health and prevent disease (ali et al., 2022; gyawali et al., 2022). functional dairy products such as yogurt offer an ideal vehicle for delivering these bioactive compounds, given their widespread acceptability and compatibility with various fortifying agents (li et al., 2022). enriching yogurt with butterfly pea extract is expected to increase its antioxidant potential, while also providing additional nutrients that may synergistically contribute to health benefits (jena & choudhry, 2025). in addition to bioactive compounds, the nutritional composition of yogurt is a key factor in determining its health value and marketability (arab et al., 2023). the analysis of macronutrients such as protein, carbohydrates, and fats, along with micronutrients like vitamin c, offers important insights into the product’s dietary contribution (mefleh et al., 2022). parameters such as moisture and ash content also provide valuable information regarding product stability, shelf life, and mineral content. modifying yogurt through the addition of plant-based extracts can influence these nutritional attributes, either by enhancing certain components or altering the balance between them. understanding these changes is essential for optimizing product formulation, ensuring compliance with nutritional standards, and meeting consumer expectations for quality and functionality (islam et al., 2021; al-bedrani et al., 2023; kumar et al., 2025). despite the increasing popularity of plant-enriched dairy products, scientific research examining the nutritional and functional characteristics of yogurt fortified with butterfly pea extract remains limited (mehwish et al., 2023; thillapudi et al., 2025). most previous studies have focused on the phytochemical composition and biological activities of butterfly pea in isolation or in non-dairy matrices (habiba et al., 2025). while a few studies have explored its use as a natural colorant in beverages or baked goods, its integration into yogurt and the effects on both nutritional composition and antioxidant activity have not been extensively documented (arain et al., 2023; zaini et al., 2023). this research gap highlights the need for systematic studies to evaluate the impact of butterfly pea extract on yogurt’s proximate composition, vitamin content, and antioxidant capacity. therefore, the objective of this study is to analyze the nutritional composition and antioxidant activity of yogurt enriched with butterfly pea (clitoria ternatea l.) extract. specifically, the study aims to determine the levels of protein, carbohydrate, fat, vitamin c, moisture, and ash content, as well as to assess the antioxidant potential of the fortified yogurt. by providing empirical data on these parameters, the research seeks to contribute to the growing body of knowledge on functional dairy products and to inform future innovations in yogurt development. the findings are expected to offer valuable insights for food scientists, nutritionists, and the dairy industry in designing products that align with consumer preferences for natural, health-promoting, and visually appealing foods. materials and methods materials this study was an experimental research conducted in food technology and nutrition. the research samples comprised yogurt supplemented with butterfly pea flower extract (derived from a community service activity in bakubakulu village, sigi regency). the yogurt samples were prepared by the researchers using fresh goat milk, which was pasteurized at a temperature of 61°c to 63°c. after pasteurization, a starter culture was added at a ratio of 1:2 (starter culture to milk). subsequently, butterfly pea flower extract was incorporated into the mixture, which was then thoroughly stirred and incubated for approximately 2 hours at 40°c (yang et al., 2023). in this study, the yogurt samples consisted of eight different treatments with varying concentrations of butterfly pea extract, along with one control sample without the extract addition. protein analysis protein content was analyzed using the bradford method (karimi et al., 2022). a 0.1 ml aliquot of the sample solution was taken and placed into a microtube. then, 1000 µl of 10% acetone was added. the mixture was vortexed until thoroughly blended. the sample was centrifuged at 5000 rpm for 7 minutes. subsequently, 20 µl of the supernatant was transferred to a new microtube, followed by the addition of 1000 µl of bradford reagent. the solution was remixed using a vortex mixer and incubated for 10 to 60 minutes at room temperature. the absorbance was measured using a spectrophotometer at a wavelength of 595 nm. fat analysis fat content was determined using the soxhlet extraction method (rezvankhah et al., 2019). the principle of this method involves extracting fat using diethyl ether as a solvent. after the solvent evaporates, the extracted fat is weighed and its percentage is calculated. samples analyzed with this method must be in solid form. five grams of the sample were weighed and placed into filter paper, which was then inserted into a soxhlet extractor. the extractor was positioned above a fat collection flask. diethyl ether solvent (500 ml) was poured into the fat collection flask. the system was refluxed for approximately 5 hours until the solvent in the fat flask became clear. after extraction, the fat collection flask was heated at 105 °c to evaporate the solvent, and then weighed after drying to determine the fat content. windarsih et al. – analysis of nutritional composition and antioxidant activity of … 833 water analysis the water content was analyzed using the oven method (li et al., 2022). the procedure for determining the water content involves weighing an empty crucible, then weighing approximately 5 grams of the sample and placing it into the empty crucible. place the crucible and its contents into an oven at a temperature of 100⁰c– 102⁰c for 6 hours. after 6 hours, remove the crucible and cool it in a desiccator for 5–10 minutes, then weigh the crucible again. dry it again in the oven and weigh the sample until a constant weight is achieved. ash analysis the ash content was analyzed using the drying ash method (pojić et al., 2015). the procedure began by weighing an empty crucible, then placing approximately 5 grams of the sample into it. the sample was heated on a hot plate until no smoke was observed. it was then placed in a muffle furnace and incinerated until gray ash with a constant weight was obtained. the ashing process was carried out in two stages, at temperatures of 400 °c and 550°c. afterward, the crucible was cooled in a desiccator and weighed. carbohydrate analysis carbohydrate content can be determined using the proximate analysis method, often referred to as carbohydrate by difference (akalu & geleta, 2019). this analysis estimates the carbohydrate content not through direct measurement, but by calculation. the percentage of carbohydrates in food is obtained by subtracting the total percentage of protein, fat, ash, and moisture content from 100%. vitamin c analysis vitamin c was analyzed using the uv–vis spectrophotometry method (samide & tutunaru, 2017). a total of 5 g of the sample was weighed and dissolved in 100 ml of co₂-free distilled water. the filtrate was diluted by pipetting 10 ml, which was then diluted further with 100 ml of co₂-free distilled water. the absorbance of the sample was measured using a uv–vis spectrophotometer at a wavelength of 270 nm. distilled water was used as the blank solution, and ascorbic acid was used as the standard solution. antioxidant activity analysis antioxidant activity was analyzed using the 2,2diphenyl-1-picrylhydrazyl (dpph) method (xie & schaich, 2014). the dpph solution was prepared by dissolving 1 mg of dpph in 1 ml (1000 μl) of methanol, then further diluting with 100 ml of methanol and homogenizing using a magnetic stirrer. the sample solution with a concentration of 100 ppm was prepared by taking 1 ml of sample and diluting it with methanol to a total volume of 1 ml. absorbance measurements were carried out by adding 200 μl of sample solution to 1 ml of dpph solution. the mixture was incubated for 30 minutes, followed by measurement of absorbance at a wavelength of 517 nm. organoleptic test analysis the organoleptic test was conducted with a panel of 25 participants (valente et al., 2021). each panelist was provided with a sample and an evaluation sheet containing a hedonic scale, as well as space for comments and suggestions. panelists were instructed to taste the product and provide assessments based on their individual perceptions. the evaluation included assessments of color, taste, aroma, and texture. statistical analysis univariate analysis univariate analysis was conducted to calculate the mean value and test the normality of the nutritional content, antioxidant activity, and organoleptic properties of yogurt with the addition of honey pineapple and cinnamon extract. the shapiro–wilk test was used to assess normality, as the sample size was fewer than 30. bivariate analysis bivariate analysis was performed to determine the significant effect of variations in the addition of butterfly pea extract to yogurt on nutritional content, antioxidant activity, and organoleptic properties. if the data were normally distributed, the statistical test used was oneway anova (analysis of variance), while nonnormally distributed data were analyzed using the kruskal–wallis test. furthermore, a paired t-test was conducted to identify the effect of adding butterfly pea extract. results and discussion protein content the analysis of protein content in yogurt with adding butterfly pea extract (table 1; figure 1) showed significant differences between treatments (p = 0.00). all treatment groups exhibited differences in protein content, with a general decrease observed. in other words, the greater the addition of butterfly pea extract, the smaller the amount of milk incorporated into the formulation. the control yogurt, without the addition of butterfly pea extract, had the highest protein content at 4.60 mg/100 ml, while the lowest value was 0.43 mg/100 ml. previous research has shown that the protein content of yogurt is largely determined by the quality of its raw material, namely milk; the higher the milk protein content, the better the quality of the yogurt produced (chalid et al., 2021; habiba et al., 2025). the addition of butterfly pea extract had a significant impact on the protein content of the yogurt. according to the indonesian national standard (sni) 01.2981-1992, the minimum protein content in yogurt should be 3.5%. another study reported that protein content is influenced 834 biology, medicine, & natural product chemistry 14 (2), 2025: 831-841 by the total number of lactic acid bacteria present; the greater the microbial population in yogurt, the higher the protein content, as most of the microbial components are composed of proteins (brodziak et al., 2020; li et al., 2022; gan et al., 2023). table 1. protein content in yogurt with the addition of butterfly pea extract. treatment protein content difference (after − before) mg/100 ml p before mean ± sd (mg/100 ml) after mean ± sd (mg/100 ml) control 4.60 ± 0.002 4.60 ± 0.002a n0k1 4.60 ± 0.002 4.44 ± 0.008a -0.16 ± 0.011 0.022 n0k2 4.60 ± 0.002 4.40 ± 0.000a -0.20 ± 0.002 0.742 n20k1 4.60 ± 0.002 3.84 ± 0.020a -0.76 ± 0.019 0.542 n20k2 4.60 ± 0.002 2.71 ± 0.016a;b -1.89 ± 0.022 0.142 n40k1 4.60 ± 0.002 2.68 ± 0.002a;b -1.92 ± 0.005 0.002 n40k2 4.60 ± 0.002 1.77 ± 0.015b -2.83 ± 0.006 0.042 n60k1 4.60 ± 0.002 1.26 ± 0.004b -3.38 ± 0.017 0.002 n60k2 4.60 ± 0.002 0.43 ± 0.000b -4.17 ± 0.003 0.002 p 0.001 note: values followed by different superscript letters (a, b) indicate significant differences; one-way anova; paired t-test. fat content the analysis of fat content in yogurt with the addition of butterfly pea extract (table 2; figure 1) showed significant differences among treatments (p = 0.00). all treatment groups exhibited decreased fat content, which was attributed to the reduced proportion of milk used in yogurt production. in other words, the greater the addition of butterfly pea extract, the smaller the amount of milk incorporated into the formulation. the decrease in fat content was influenced by several factors, including the drying process performed prior to fat analysis (oliveira et al., 2016). oven-drying is known to reduce certain nutrients in the sample. in this study, drying was necessary to prepare the samples for fat analysis using the soxhlet method, which requires dry samples (zioud et al., 2023). the drying process was conducted at 40 °c for 48 hours. previous studies have shown that temperature can significantly affect the fat content of cow’s milk, with low storage temperatures recommended to inhibit the growth of harmful bacteria (kaur et al., 2017; kok & hutkins, 2018; jena & choudhury, 2025). in addition to temperature, storage duration also affects fat content, as prolonged exposure can lead to lipid degradation due to oxidation by free oxygen (farag et al., 2020). furthermore, fat in food materials can be reduced or damaged during cooking processes (hasanah et al., 2023). according to the indonesian national standard (sni), the minimum fat content in yogurt is 2.5% (lakshan et al., 2019). in this study, yogurt with butterfly pea extract had fat contents ranging from 2.48% to 3.12%. table 2. fat content in yogurt with the addition of butterfly pea extract. treatment fat content difference (after − before) mg/100 ml p before mean ± sd (mg/100 ml) after mean ± sd (mg/100 ml) control 3.12 ± 0.01 3.12 ± 0.01a n0k1 3.12 ± 0.01 3.11 ± 0.03a -0.01 ± 0.04 0.902 n0k2 3.12 ± 0.01 2.73 ± 0.07a;b -0.39 ± 0.05 0.052 n20k1 3.12 ± 0.01 2.48 ± 0.51a;b -0.64 ± 0.50 0.312 n20k2 3.12 ± 0.01 2.92 ± 0.04a;b -0.20 ± 0.02 0.062 n40k1 3.12 ± 0.01 2.75 ± 0.03b -0.37 ± 0.02 0.022 n40k2 3.12 ± 0.01 2.58 ± 0.22a;b -0.54 ± 0.24 0.182 n60k1 3.12 ± 0.01 2.78 ± 0.02b -0.34 ± 0.00 0.012 n60k2 3.12 ± 0.01 2.50 ± 0.41a;b -0.62 ± 0.43 0.282 p 0.001 note: values followed by different superscript letters (a, b) indicate significant differences; one-way anova; paired t-test. water content the analysis of water content in yogurt with the addition of butterfly pea flower extract (table 3; figure 1) showed no significant differences among treatments (p = 0.68). the average water content across all treatments was approximately 80%, which is slightly lower than the indonesian national standard (sni) 2981:2009 requirement of 83–84% for yogurt (maulid et al., 2025). this reduction in water content may be influenced by the variation in the amount of butterfly pea flower extract windarsih et al. – analysis of nutritional composition and antioxidant activity of … 835 added, where a higher proportion of extract reduces the proportion of milk used (ikrawan et al., 2023; mehwish et al., 2023). in addition, the drying process also contributes to the decrease in water content, as longer drying times generally lead to lower water content in food products (arain et al., 2023). previous studies have reported that high water content in fermented products such as kefir can increase the population of lactic acid bacteria (lab) and other microorganisms, as microbial growth is strongly dependent on water availability in food (ali et al., 2022; mefleh et al., 2022). table 3. water content in yogurt with the addition of butterfly pea extract. treatment water content difference (after − before) mg/100 ml p before mean ± sd (mg/100 ml) after mean ± sd (mg/100 ml) control 82.00 ± 4.23 82.00 ± 4.22a n0k1 82.00 ± 4.23 80.00 ± 0.68a -2.50 ± 3.51 0.492 n0k2 82.00 ± 4.23 79.00 ± 1.39a;b -3.00 ± 2.80 0.362 n20k1 82.00 ± 4.23 73.00± 9.87a;b -9.00 ± 5.64 0.252 n20k2 82.00 ± 4.23 79.00 ± 2.80a;b -3.00 ± 7.06 0.642 n40k1 82.00 ± 4.23 80.50 ± 0.69b -1.50 ± 3.51 0.642 n40k2 82.00 ± 4.23 80.00 ± 0.00a;b -2.00 ± 4.22 0.612 n60k1 82.00 ± 4.23 71.00 ± 15.54b -1.10 ± 11.30 0.392 n60k2 82.00 ± 4.23 80.00 ± 2.81a;b -2.00 ± 1.40 0.282 p 0.681 note: values followed by different superscript letters (a, b) indicate significant differences; one-way anova; paired t-test. ash content the analysis of ash content in yogurt with the addition of butterfly pea flower extract (table 4; figure 1) showed no significant differences among treatments (p = 0.38). ash refers to the inorganic residue remaining after the combustion of organic material (zhang & wang, 2022). according to the indonesian national standard (sni), the ash content of yogurt should not exceed 1.0%. ash content is closely related to the mineral composition of the product, where higher ash levels indicate a greater mineral content (oliveira et al., 2016). the levels of individual mineral components are influenced by factors such as species, physiological conditions, geographical location, frequency of waves, and the type of mineralization process used (al-bedrani et al., 2023). in this study, the ash content measurement was preceded by a drying process to remove moisture from the samples. this drying process may also affect the mineral content of the resulting yogurt (islam et al., 2021; kumar et al., 2025). table 4. ash content in yogurt with the addition of butterfly pea extract. treatment ash content difference (after − before) mg/100 ml p before mean ± sd (mg/100 ml) after mean ± sd (mg/100 ml) control 4.07 ± 0.03 4.07 ± 0.03a n0k1 4.07 ± 0.03 4.00 ± 0.04a -0.07 ± 0.02 0.122 n0k2 4.07 ± 0.03 4.63 ± 0.17a;b -0.56 ± 0.22 0.182 n20k1 4.07 ± 0.03 4.44 ± 0.25a;b -0.37 ± 0.29 0.332 n20k2 4.07 ± 0.03 4.42 ± 0.08a;b -0.35 ± 0.11 0.162 n40k1 4.07 ± 0.03 3.84 ± 0.16b -0.23 ± 0.20 0.362 n40k2 4.07 ± 0.03 4.52 ± 0.13a;b -0.45 ± 0.16 0.172 n60k1 4.07 ± 0.03 4.30 ± 0.59b -0.23 ± 0.62 0.692 n60k2 4.07 ± 0.03 3.93 ± 0.82a;b -0.14 ± 0.84 0.852 p 0.381 note: values followed by different superscript letters (a, b) indicate significant differences; one-way anova; paired t-test. carbohydrate content the analysis of carbohydrate content in yogurt with the addition of honey pineapple and cinnamon extract (table 5; figure 1) showed significant differences among treatments (p = 0.00). the carbohydrate analysis results indicated distinct values, with differences observed across all groups. the highest carbohydrate content recorded was 19.50%. carbohydrates in milk are primarily in the form of lactose, which serves as the main carbon source for lactic acid bacteria in producing lactic acid (taye et al., 2021; arab et al., 2023). one of the carbohydrates present in butterfly pea flower extract in disaccharide form is fructose (juturu & wu, 2016). fructose, commonly referred to as fruit sugar, is a type of 836 biology, medicine, & natural product chemistry 14 (2), 2025: 831-841 sugar polymer (sharma et al., 2023). it is a complex sugar that cannot be directly digested by the human digestive system and must first be converted into simple sugars (li et al., 2025). the fermentation process aims to transform this form of carbohydrate into a form more readily absorbed by the human body (kok & hutkins, 2018). table 5. carbohydrate content in yogurt with the addition of butterfly pea extract. treatment carbohydrate content difference (after − before) mg/100 ml p before mean ± sd (mg/100 ml) after mean ± sd (mg/100 ml) control 4.18 ± 4.53 4.18 ± 4.53a n0k1 4.18 ± 4.53 6.61 ± 0.83a;b 2.43 ± 3.67 0.552 n0k2 4.18 ± 4.53 7.21 ± 0.07a;b 3.03 ± 4.45 0.502 n20k1 4.18 ± 4.53 15.33 ± 7.02a;b 11.15 ± 2.49 0.102 n20k2 4.18 ± 4.53 7.78 ± 5.43a;b 3.60 ± 9.98 0.702 n40k1 4.18 ± 4.53 8.19 ± 0.13a;b 4.01 ± 4.38 0.402 n40k2 4.18 ± 4.53 9.60 ± 0.42a;b 5.42 ± 4.95 0.352 n60k1 4.18 ± 4.53 19.50 ± 15.03b 15.32 ± 10.49 0.272 n60k2 4.18 ± 4.53 11.11 ± 2.34a;b 6.93 ± 2.17 0.132 p 0.001 note: values followed by different superscript letters (a, b) indicate significant differences; one-way anova; paired t-test. vitamin c content the analysis of vitamin c content in yogurt with adding butterfly pea flower extract (table 6; figure 1) showed significant differences among treatments (p = 0.00). the vitamin c content varied across all treatment groups and demonstrated an increasing trend. the greater the addition of butterfly pea flower extract, the higher the vitamin c content. incorporating butterfly pea flower extract into yogurt significantly increased its vitamin c content. each sample with the extract showed a significant difference compared to the control. the highest vitamin c content was observed in yogurt with the highest butterfly pea flower extract level, reaching 4.11 mg/100 ml. vitamin c content increased directly proportional to the amount of butterfly pea flower extract added. a decrease in vitamin c levels may occur due to its susceptibility to degradation from exposure to light, high temperatures, oxygen, and processing methods (giannakourou & taoukis, 2021). vitamin c is highly water-soluble and readily oxidized when exposed to air (bankole et al., 2023). greater losses may result from heating at 90 °c for 3 minutes or from oxygen and light exposure (li et al., 2022). the recommended daily intake of vitamin c for adults and the elderly is 90 mg/day for men and 75 mg/day for women (giannakourou & taoukis, 2021). table 6. vitamin c content in yogurt with the addition of butterfly pea extract. treatment vitamin c content difference (after − before) mg/100 ml p before mean ± sd (mg/100 ml) after mean ± sd (mg/100 ml) control 0.16 ± 0.003 0.16 ± 4.53a n0k1 0.16 ± 0.003 0.19 ± 0.83a 0.03 ± 0.003 0.182 n0k2 0.16 ± 0.003 0.38 ± 0.07b 0.22 ± 0.002 0.002 n20k1 0.16 ± 0.003 0.60 ± 7.02b 0.44 ± 0.005 0.002 n20k2 0.16 ± 0.003 1.51 ± 5.43c 1.35 ± 0.005 0.002 n40k1 0.16 ± 0.003 3.17 ± 0.13d 3.01 ± 0.001 0.002 n40k2 0.16 ± 0.003 3.87 ± 0.42e 3.71 ± 0.011 0.002 n60k1 0.16 ± 0.003 4.21 ± 15.03f 4.05 ± 0.016 0.002 n60k2 0.16 ± 0.003 4.27 ± 2.34g 4.11 ± 0.012 0.002 p 0.001 note: values followed by different superscript letters (a, b, c, d, e, f, g) indicate significant differences; one-way anova; paired t-test. antioxidant content the antioxidant activity test results for yogurt with the addition of butterfly pea flower extract (table 7; figure 1) showed significant differences among groups (p = 0.00). higher levels of butterfly pea flower extract resulted in increased antioxidant activity. antioxidant activity refers to the ability of compounds in a food product to scavenge free radicals (gulcin, 2020). the addition of butterfly pea flower extract to yogurt enhanced its antioxidant activity, with the highest activity observed in the 60% extract and 4% yogurt formulation (n60k2), reaching 47.8%. the antioxidant windarsih et al. – analysis of nutritional composition and antioxidant activity of … 837 activity assay using the dpph method indicated the ability of food components to scavenge 50% of free radicals (ic50). a higher antioxidant activity corresponds to a higher antioxidant concentration, thereby reducing the amount of food required to neutralize free radicals (zhang & wang, 2022). table 7. antioxidant content in yogurt with the addition of butterfly pea extract. treatment antioxidant content difference (after − before) mg/100 ml p before mean ± sd (mg/100 ml) after mean ± sd (mg/100 ml) control 14.4 ± 0.11 14.4 ± 0.12a n0k1 14.4 ± 0.11 17.3 ± 0.02a 2.90 ± 0.10 0.002 n0k2 14.4 ± 0.11 21.7 ± 0.03a 7.30 ± 0.16 0.002 n20k1 14.4 ± 0.11 34.6 ± 0.11a;b 20.2 ± 0.24 0.002 n20k2 14.4 ± 0.11 34.7 ± 0.25a;b 20.3 ± 0.39 0.002 n40k1 14.4 ± 0.11 35.0 ± 0.20a;b 20.6 ± 0.26 0.002 n40k2 14.4 ± 0.11 56.7 ± 0.20b 42.3 ± 0.34 0.002 n60k1 14.4 ± 0.11 59.0 ± 0.03b 44.6 ± 0.17 0.002 n60k2 14.4 ± 0.11 62.2 ± 0.00b 47.8 ± 0.22 0.002 p 0.001 note: values followed by different superscript letters (a, b) indicate significant differences; one-way anova; paired t-test. organoleptic test color based on statistical analysis, the addition of butterfly pea flower extract showed a significant difference among all samples (p = 0.00). yogurt with 0% butterfly pea flower extract had the highest level of acceptance, whereas yogurt with 40% butterfly pea flower extract had the lowest. as shown in table 8 and figure 1, the addition of butterfly pea flower extract had a significant effect on the color acceptance of the resulting yogurt. the control yogurt sample exhibited a milky white color, yogurt samples n0k1 and n0k2 had a light purple hue, while yogurt with higher concentrations of butterfly pea flower extract became progressively more purple in proportion to the amount of extract added. the milky white color in the control yogurt originates from beta-carotene in fat, which is encapsulated by protein (molina et al., 2019). milk casein has a translucent property, allowing all wavelengths to be reflected, thereby producing a white appearance (milovanovic et al., 2020). the purple color in yogurt with butterfly pea flower extract is derived from beta-carotene, a natural compound belonging to the carotenoid group. the color of food products can influence their level of acceptance, with brightly colored foods generally being more readily accepted and preferred by consumers (paakki et al., 2016; de meija et al., 2020). table 8. results of the analysis of color acceptance in yogurt with the addition of butterfly pea flower extract. treatment mean ± sd mean ± rank control 5.46 ± 0.49 170.18a n0k1 5.06 ± 0.77 141.44b n0k2 5.06 ± 0.66 142.52c n20k1 4.62 ± 0.69 108.50d n20k2 4.54 ± 0.90 103.94e n40k1 4.66 ± 0.69 111.74f n40k2 3.86 ± 0.67 58.48g n60k1 4.38 ± 0.85 97.64h n60k2 4.02 ± 1.07 75.72i p* 0.00 note: numbers followed by different superscript letters (a, b, c, d, e, f, g, h, i) indicate significant differences. kruskal–wallis test; posthoc mann–whitney test. taste as shown in table 9 and figure 1, the addition of butterfly pea flower extract had a significant effect on the taste of the resulting yogurt. statistical analysis indicated that the yogurt most preferred by the panelists was sample n0k2, with a mean rank of 143.63. however, in some cases, the yogurt produced left a bitter aftertaste in the throat after consumption. this was attributed to the presence of bromelain enzymes, which react with milk (kumar et al., 2025). bromelain is a proteolytic enzyme, and excessive proteolytic activity can produce a bitter taste. heating to a temperature of 100 °c inactivates bromelain enzymes; nevertheless, the bitter taste appeared only occasionally and did not affect the overall 838 biology, medicine, & natural product chemistry 14 (2), 2025: 831-841 acceptance level of the yogurt (juturu & wu, 2016; li et al., 2022). table 9. results of the analysis of taste acceptance in yogurt enriched with butterfly pea flower extract. treatment mean ± sd mean ± rank control 4.62 ± 0.92 110.12a n0k1 4.06 ± 0.74 137.16b n0k2 4.10 ± 0.9 143.63c n20k1 4.62 ± 1.01 111.65d n20k2 4.14 ± 0.98 82.19e n40k1 4.18 ± 1.07 85.03f n40k2 4.54 ± 0.88 104.01g n60k1 5.10 ± 0.76 141.95h n60k2 4.42 ± 1.01 101.37i p* 0.00 note: numbers followed by different superscript letters (a, b, c, d, e, f, g, h, i) indicate significant differences. kruskal–wallis test; post-hoc mann–whitney test. aroma as shown in table 10 and figure 1, the addition of butterfly pea flower extract to yogurt did not result in any significant increase or decrease in aroma (p = 0.312). there was no significant difference in the aroma of the resulting yogurt. the yogurt produced tended to have a sour aroma typical of conventional yogurt, so the characteristic aroma of butterfly pea flower was not strongly perceived (saritas et al., 2024). according to previous research, the aroma components of butterfly pea flower are not lost during heating at the boiling temperature of milk (±100 °c). however, these aroma components begin to decline at 150 °c and decrease further as the temperature increases (giannakourou & taoukis, 2021). table 10. results of the analysis of aroma acceptance in yogurt with the addition of butterfly pea flower extract. treatment mean ± sd mean ± rank control 4.19 ± 0.56 131.75a n0k1 4.03 ± 0.77 120.49a n0k2 3.79 ± 1.03 107.35a n20k1 3.79 ± 0.75 97.19a n20k2 3.91 ± 0.56 107.35a n40k1 3.79 ± 0.49 96.87a n40k2 3.91 ± 0.39 107.35a n60k1 4.15 ± 0.73 128.47a n60k2 3.79 ± 1.07 111.25a p* 0.312 note: numbers followed by different superscript letters (a, b, c, d, e, f, g, h, i) indicate significant differences. kruskal–wallis test; post-hoc mann–whitney test. texture as shown in table 11 and figure 1, the addition of butterfly pea flower extract had a significant effect on the texture acceptance of the resulting yogurt. statistical analysis indicated that the most preferred texture was found in sample n0k2, with a mean rank of 161.61. the fiber in butterfly pea flower contributed to a slightly coarse texture in the yogurt. the butterfly pea flowers used in yogurt production had already undergone a grinding process, which should have reduced the fiber into smaller molecules (farag et al., 2020). smaller fiber size can enhance the absorption of beta-carotene in the body (bankole et al., 2023). the texture of this yogurt was slightly coarse, differing from that of commercial yogurt drinks, in which the fruit texture is more fully integrated with the milk (gulcin, 2020). table 11. results of the analysis of texture acceptance in yogurt with the addition of butterfly pea flower extract. treatment mean ± sd mean ± rank control 4.19 ± 0.56 157.93a n0k1 4.03 ± 0.77 132.41b n0k2 3.79 ± 1.03 161.61c n20k1 3.79 ± 0.75 105.65d n20k2 3.91 ± 0.56 93.45e n40k1 3.79 ± 0.49 94.57f n40k2 3.91 ± 0.39 89.69g n60k1 4.15 ± 0.73 86.19h n60k2 3.79 ± 1.07 86.59i p* 0.00 note: numbers followed by different superscript letters (a, b, c, d, e, f, g, h, i) indicate significant differences. kruskal–wallis test; post-hoc mann–whitney test. windarsih et al. – analysis of nutritional composition and antioxidant activity of … 839 figure 1. comprehensive summary of nutritional composition, antioxidant activity, and sensory acceptance of yogurt enriched with butterfly pea (clitoria ternatea l.) extract. conclusions yogurt with the addition of 60% butterfly pea flower extract and 4% (n60k2) had the highest vitamin c and antioxidant content but the lowest protein and fat content. the yogurt most preferred by panelists in terms of color, taste, aroma, and texture was the one with 60% butterfly pea flower extract and 2% addition. based on the scoring conducted by the researchers, which considered compliance with the indonesian national standard (sni) and consumer acceptance levels, yogurt with 60% butterfly pea flower extract and 2% (sample n60k1) was the most recommended formulation. acknowledgements: the authors would like to express their gratitude to universitas tadulako for providing financial support for the implementation of the 2025 community service program. authors’ contributions: conceptualization, yulia windarsih and i made budiarsa; methodology, manap trianto and akram; analysis, abdul ashari, yulia windarsih, and manap trianto; writing—original draft preparation, i made budiarsa and manap trianto; writing—review and editing, yulia windarsih, i made budiarsa, and manap trianto. competing interests: the authors declare that there are no competing interests. funding: this research was funded by tadulako university, grant number 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(2017). formulation and evaluation of flavored probiotic yogurt using natural ingredients. international journal of advanced research, 5(1), 1874–1884. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 31-33 | doi: 10.14421/biomedich.2014.31.31-33 larvicidal effect of vinca fruit extract (vinca rosea) against aedes aegypti larvae and secondary metabolites profile by thin layer chromatography rahmawati ekaputri1, sudarsono1* and budi mulyaningsih2 1department of pharmaceutical biology, faculty of pharmacy; 2parasitology laboratory, faculty of medicine, ugm, indonesia author correspondency*: sudarsono@ugm.ac.id abstract background: vinca rosea is known contain alkaloids, it was usually used to treat various diseases. alkaloids from vinca leaves are also already known have larvicidal activity. based on this toxicological activity, the fruit of vinca rosea was selected to investigation its larvicidal activity against the 3rd instar larvae of the mosquito vector of dengue haemorrhagic fever (dhf) aedes aegypti. five concentrations of vinca fruit extract were tested against the 3rd instar aedes aegypti larvae. the different larval mortality percentages were recorded after 24 hours. lethal concentration (lc50 anf lc90) of vinca fruit extract were calculated using probit analysis. phytochemical compounds of ethanolic extract also investigated using thin layer chromatography (tlc). lc50 and lc90 values of fruit extract were 2.987 mg/ml and 32.861 mg/ml. alkaloids were detected in extract. keywords: larvicidal activity, vinca rosea, chromatography, secondary metabolites, aedes aegypti introduction aedes aegypti transmit a number of diseases, such as filariasis, chikungunya, and dengue haemorrhagic fever (dhf). dhf causing millions of deaths every year. the only way of reducing the incidence of this disease is by vector control using synthetic insecticide. the synthetic insecticide are more hazardous to handle and not biodegradable. substances alternative of chemical pesticides, which pollute and threaten future, can be discovered. more than 300 plant species have been reported to have an activity against mosquitoes included vinca leaves extract (remia and logaswany, 2011). vinca rosea (fam.apocynaceae) is plants grow everywhere in indonesia (fig. 1). vinca rosea has more than 120 indol alkaloids and another alkaloids in the whole plant (van der heijden et al., 2004). the present study was carried out to determine the larvicidal activity of vinca rosea fruit extract against aedes aegypti larvae. methodology preparation and extraction of plant materials the fruits of vinca rosea were collected as wild plants at “north sekip” at yogyakarta. indonesia. fruits were identified at department of pharmaceutical biology faculty of pharmacy, ugm yogyakarta. voucher specimen was found at this department. fruits were washed and dried at 40oc for 12 hours. 370 g dried fruit powder was macerated with 2.5 l of ethanol (96%) for 48 hours. the ethanol was then evaporated to sticky residues. the extract residue was 6.95 % and kept in the refrigerator. figure 1. (a) vinca rosea (b) fruit of vinca rosea. 32 biology, medicine, & natural product chemistry 3 (1), 2014: 31-33 bioassay for larvicidal toxicity different concentrations of extract (0.4 mg/ml, 0.8 mg/ml, 1.6 mg/ml, 3.2 mg/ml, dan 6.4 mg/ml) were prepared using boiled distilled water. bioassay were performed according to standard method (who, 2005). twenty five 3rd larvae of aedes aegypti were introduced in different test concentration with a set of control containing distilled water and water without test solution. the glass with the larvae, were kept at room temperature. after 24 hours of exposure, the number of dead larvae were counted to obtained mortility rate. three replications were maintained for each concentration. larval mortality were counted after 24 hours. the lc50 and lc90 were calculated according to probit methods of finney (finney, 1971). the concentrations in probit analysis were transformed to logarithm (log concentration) and the lethal concentrations (lc50 and lc90) were calculated manually. thin layer chromatography secondary metabolite compounds of vinca fruit extract was analyzed by thin layer chromatography (tlc) and the spot visualitasion was by the spray reagents. chromatography was perfomed using aluminium plates precoated with silica gel 60 f254 (e.merck). extract solution was prepared by dissolving it in ethanol pro analysis. one microliters of the extract solution was applied to the plate. plates were developed face-down, to a distance of 8 cm, in a glass chamber after conditioning until saturated with mobile phase vapor. mobile phase that used was ethyl acetate: methanol (9:1 v/v). after development the mobile phase was evaporated to dryness and plates were sprayed with dragendorff's and ninhidrin reagent. table 1. results of probit analysis of vinca fruit extracts against larvae of ae. aegypti. extract concentration (mg/ml) percentage larval mortility (mean) probit value the linear regression equation lc50 (mg/ml) lc90 (mg/ml) 0,4 21% 4,19 y = 1,229x + 4,416 with r = 0,910 2,987 32,861 0,8 21% 4,19 1,6 27% 4,39 3,2 44% 4,85 6,4 76% 5,17 results and discussions larvicidal effect of vinca fruit extract the 24h bioassay is a major tool for evaluating the toxical effect of vinca fruit extract against aedes aegypti larvae. the mosquito larvae exposed under plant extract showed significant behavior changes. the larvae showed restlessness, loss of equilibrium, and led to death. dead larvae are those that cannot be induced to move when they are probed with a needle in the siphon or the cervical region (who, 2005). in table 1 the 5 concentrations of vinca fruit extract caused larval mortality of 21%, 21%, 27, 44, and 76%. zero percent of mortality was noted in control. lc50 and lc90 calculated were 2.987 mg/ml and 32.861 mg/ml. the larvicidal activity shown by vinca rosea is probably due to the presence of the alkaloid which are toxic substances. phenolic and non-phenolic alkaloids isolated from vinca rosea leaves are known to have toxic effects (waskito, 1999). phytochemical compound of vinca fruit extract zones with rf 0.33-0.39 gave positive results after being sprayed with dragendorff’s reagent and gave negative results after being sprayed with ninhydrin reagent (rf 0.33) that was mean no amino acid spot detected (fig. 2). positive results with dragendorff reagent will give orange-red spots or brown with orange background yellow (wagner and bladt, 1996). a positive result with ninhydrin reagent appeared after heating 95 -1200 c about 10 minutes (jork et al., 1990). samples containing amino acids will give a positive spots that appear blue-violet. tryptophan is the precursor indole alkaloid biosynthesis is a major component of plant alkaloids in vinca rosea (tikhomiroff and jolicoeur, 2002). spraying with ninhydrin is used to ensure that the zones rf 0.330.39 are alkaloids. after isolated brown zones showed the presence of alkaloid compounds in the fruit extract vinca rosea (rf 0.33). the result of vinca fruit extract was also proved that they had larvicidal properties against aedes aegypti larvae. nelson et al., (2006) reported that the whole v. rosea plant is poisonous. whole plant vinca rosea also has a toxic effect on the larvae of artemia salina (brine shrimp) (gadir, 2012 dan rahmatullah, 2010). the larvicidal properties exhibited by vinca extract in this study might be related to presence of vinca alkaloids toxins in the plant. conclusion the present study has demonstrated that an ethanolic extract of vinca fruit had capability as larvicides against rahmawati ekaputri, et al. – larvicidal effect of vinca fruit extract (vinca rosea) … 33 aedes aegypti larvae. further studies are needed to determine the marker compound for producing standard operating procedure (sop) and to study their impacts on human health and non-target organisms in mosquito feeding habitats. figure 2. chromatogram of vinca fruit ethanolic extract. solvent systems: ethyl acetate-methanol (9:1 v/v). detection (a) uv-254nm, (b) uv-365nm, (c) dragendorff’s reagent, (d) ninhydrin reagent, (e) reference for positive result with ninhydrin reagent: yolk. (1b) alkaloids and another compounds with nitrogen shows fluorescence in uv-365 and (1c) react as brown zones with dragendorff’s reagent (rf ~ 0.33-0.39). (2) brown zones indicated negative result with ninhydrin reagent (rf ~ 0.33). (3) amino acids react as blue-violet zones with ninhydrin reagent. acknowledgment i would like to thank to faculty of pharmacy ugm for the permission for doing this experiment. references gadir, a. (2012). assement of bioactivity of some sudanese medicinal plants using brine shrimp (artemia salina) lethality assay. journal of chemical and pharmaceutical research 4 (12): 5145-5148. jork, h., funk, w., fischer, w. & wimmer, h. (1990). thin-layer chromato-graphy: reagents and detection methods volume 1a. vch. new york. nelson, l. s.; r. d. shih and m. j. balick. (2006). handbook of poisonous and injurious plants 2nd edition. springer: 340. rahmatullah, m., sadeak, i., bachar, c., hossain, t., abdullah, montaha, jahan, n., chowdhury, m., jahan, r., nasrin, d., rahman, m. & rahman, s. (2010). brine shrimp toxicity study of different bangladeshi medicinal plants, advances in natural and applied sciences 4: 163-173. remia, k.m. & logaswany, s. (2009). larvicidal efficacy of leaf extract of two botanicals against the mosquito vector aedes aegypti (diptera: culicidae). indian journal of natural products and resources 1(2): 208-212. tikhomiroff, c. & jolicoeur, m. (2002). quantification of indole alkaloids and iridoid precursors in catharanthus roseus hairy roots by high-performance liquid chromatography. journal of chromatography a 955: 87-93. van der heijden, r., jacobs, d.i., snoeijer, w., hallard, d. & verpoorte, r. (2004). the catharanthus alkaloids: pharmacognosy and biotechnology, current medicinal chemistry 11(5): 607-628. waskito, t. (1999). isolasi dan uji aktivitas alkaloid dari daun tapak dara (catharanthus roseus) var albus., tesis, universitas diponegoro. semarang. who. (2005). guidelines for laboratory and field testing of mosquito larvicides, who communicable disease control, prevention and eradication. who pesticide evaluation scheme. geneva. content_v3n1_5.pdf (p.1-3) blank_kosong.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 631-635 | doi: 10.14421/biomedich.2023.122.631-635 issn 2540-9328 (online) phytochemical screening and antioxidant activity analysis of n-hexane extract of sonneratia alba mangrove leaves putu rissa almadea surya, made dharmesti wijaya*, desak putu citra udiyani pharmacology department, faculty of medicine and health sciences, warmadewa university, jl. terompong no 24 denpasar 80235, tel. +62 361 240727, indonesia. corresponding author* dharmestiwijaya@gmail.com manuscript received: 15 september, 2023. revision accepted: 07 december, 2023. published: 31 january, 2024. abstract mangrove plants have been widely used by people in various regions of indonesia as traditional medicine for the treatment of wounds, fever, diarrhea, and others. extreme habitat in coastal areas with high salinity and low oxygen level makes mangrove plants produce a variety of unique secondary metabolites and are rich in antioxidants. although there have been studies on phytochemicals and antioxidant activity conducted, the study of mangroves in bali, indonesia, has not been widely explored. this study aims to determine the compounds contained in mangrove leaf extract and their antioxidant activity. the species to be studied is sonneratia alba, which is commonly found in the ngurah rai mangrove forest, bali, indonesia. in this experimental study, compound extraction was carried out by maceration method using n-hexane solvent. phytochemical screening using qualitative methods and gc-ms was carried out to determine the phytochemical compounds in the extract, while 1,1-diphenyl-2-picrylhydrazyl (dpph) method was used to determine antioxidant activity. the result showed that the n-hexane s. alba mangrove leaves extract display strong antioxidant activity with ic50 value of 64.432 ± 7.675 ppm. the qualitative phytochemical tests showed that the extract contained phenol and steroid, which are known to have antioxidant properties. moreover, gc-ms analysis showed that there are two compounds with the largest %area namely gamma-sitosterol and dlalpha-tocopherol (52.88% and 7.77%, respectively), which have been reported to have antioxidant activities. to conclude, these findings demonstrate that n-hexane extract of s. alba mangrove leaves from ngurah rai mangrove forest, bali, to have potential antioxidant activity. keywords: antioxidant; dpph; mangrove; phytochemicals; sonneratia alba. introduction indonesia is a mega biodiversity tropical country. indonesia has the largest mangrove forest in the world, with an area of about 42,278 km2 (bibi et al. 2019). mangroves are halophytic plants that are well adapted to salt water and flourish in the intertidal zone of tropical and subtropical regions (bibi et al. 2019). furthermore, these plants are adaptable to living in low-oxygen and high temperature environments (dahibhate et al. 2019). these extreme environmental circumstances make these plants to create distinctive substances with interesting pharmacological effects and make it as a source of potential medicinal plants (abdel-aziz et al. 2016; sadeer et al. 2023). in indonesia, mangrove plants have been traditionally used for treatment of skin diseases, rheumatic, epilepsy, and diarrhea (kusmana & sukristijiono, 2016). in gedangan village, central java, these plants have been widely used as traditional medicine for fever, diarrhea, treatment of wounds and ulcers, stomach ache, and others (rahayu & sunarto, 2020). furthermore, mangrove plants are utilized as medicine for hematuria, treating bruises and wound, as well as contraception and appetite enhancer by people in mempawah distric, west kalimantan (arbiastutie et al. 2021). thus, mangrove plants have great potential to be explored as medicinal sources because they have been utilized empirically and are known to contain a wide range of unique and useful secondary metabolites. mangrove plants are notable for their diverse secondary metabolites such as flavonoids, alkaloids, saponins, tannins, steroids and triterpenes (abdel-aziz et al. 2016). these metabolites are not only useful for plants but also have useful therapeutic activities for humans such as antibacterial, anticholesterol, antiinflammatory, antifungal, antiviral, antidiabetic, anticancer, and antioxidant (dahibhate et al. 2019; genilar et al. 2021; vinoth et al. 2019). compounds with high antioxidant activity are quite widely researched in the recent years due to their great benefits in the prevention and treatment of oxidative stress-related pathology. antioxidants are compounds that able to neutralize free radicals and reactive oxygen species (ros), as well https://doi.org/10.14421/biomedich.2023.122.631-635 632 biology, medicine, & natural product chemistry 12 (2), 2023: 631-635 as inhibit oxidation (neha et al. 2019). exaggerated amount of ros in the body could cause oxidative stress, a condition that is closely related to diseases such as diabetes, cardiovascular diseases, cancer, neurodegeneration, and rheumatoid arthritis (pisoschi et al. 2021). mangrove plants are rich in natural phenolic substances that contribute to their antioxidant capacity, including phenolic acids, flavonoids, lignan, and tannins (abdel-aziz et al. 2016; amarowicz & pegg, 2019). ngurah rai mangrove forest, that is located in the south area of bali island, is the greatest mangrove forest in bali. currently, there are 24 mangrove species from about 17 families spread throughout this mangrove forest, with sonnerratia alba being one of the prominent species (setiastri et al. 2019). although the pharmacological effects of mangroves have been widely studied, no research has yet looked at the antioxidant activity of the s. alba mangrove leaves that inhabit ngurah rai mangrove forest, bali. in addition, differences in habitat and environment, including differences in rainfall, soil quality, and sunlight between one area and another might create different results compared to similar studies in different areas. therefore, in this study, we aim to figure out the phytochemicals and antioxidant activity of s. alba mangrove leaves extract from ngurah rai mangrove forest, bali. this study is expected to provide new information about the antioxidant potential of mangroves that can be developed into new therapeutic agents. materials and methods materials sonneratia alba leaves were obtained from ngurah rai mangrove forest, to be precise in the kub simbar segara area, pemogan village, south denpasar, bali, in september 2022. n-hexane solvent was purchased from merk-supelco, germany, while 1,1-diphenyl-2picryhydrazil (dpph) kit was acquired from sigma aldrich, usa. the equipment used in this study were oven, grinder, mesh, analytical balances, maceration containers, glassware, filter papers, vacuum rotary evaporator, cuvettes, and uv-visible spectrophotometer. methods sample preparation the mature leaves of s. alba mangrove were collected from five different plants. the leaves were washed with running water and then dried using an oven for 24 hours at 60°c. the dried leaves were then grinded into powder and stored in a tight closed containers until further research. voucher specimen was made for sample identification (figure 1), that was performed at research centre for plant conservation, botanical gardens, and forestry, located in bedugul, bali. figure 1. voucher specimen of sonneratia alba sample. extraction an amount of 100 grams leaf powder was added with 500 ml n-hexane, and then extracted using maceration method at room temperature for 2x24 hours with continuous stirring. the extract was filtered using filter paper and the solvent was evaporated using vacuum rotary evaporator at 50°c and 90 rpm. the obtained semisolid extract was stored at 4°c until further experiment (wijaya & indraningrat, 2021). dpph assay the antioxidant activity analysis was performed using the dpph method. a total of 10 mg of mangrove leaf extract was dissolved in 100 ml of methanol pro analysis (pa). the dilution was carried out using methanol pa by series concentration of 20 ppm, 40 ppm, 60 ppm, 80 ppm, and 100 ppm. the mixtures were then left for 30 minutes in a dark room at room temperature. the absorbance value was measured using uv-visible spectrophotometer at a wavelength of 517 nm. the test was repeated three times. the ic50 value was calculated using the linear regression formula from the concentration series in microsoft excel. the level of antioxidant activity was divided into 4 categories: very strong (<50 ppm), strong (50-100 ppm), moderate (101150 ppm), and weak (151-200 ppm) (surjanto et al. 2019). phytochemical analysis the qualitative phytochemical tests were carried out to identify several compounds in the extract, namely flavonoids, alkaloids, tannins, saponins, phenolics, steroids, and terpenoids. meanwhile, the gc-ms analysis was conducted at forensic laboratory of bali regional police. the compounds were determined using a comparison of the mass spectrum and retention times between each sample and the standard. surya et al. – phytochemicals and antioxidant activity of sonneratia alba 633 results and discussion sample identification sample determination was conducted to ensure that the sample used was the correct species, sonneratia alba, and thus, the sampling error could be avoided. the result showed that the leaves sample harvested from the mangrove forest was certainly sonneratia alba (table 1). table 1. taxonomy of sonneratia alba. rank name kingdom plantae subkingdom tracheobionta superdivision spermatophyta division magnoliophyta class magnoliopsida order myrtales family lythraceae genus sonneratia l species sonneratia alba antioxidant activity antioxidant activity analysis of n-hexane extract of s. alba mangrove leaves was carried out using the dpph method. the result showed that the extract possessed strong antioxidant activity, with ic50 value of 64.432 ± 7.675 ppm (table 2). similar studies showed that ethyl acetate extract of s. alba mangrove leaves from tanjung jabung, jambi displayed weak antioxidant activity with ic50 value of 223.67 ppm (latief et al. 2019). meanwhile, a study in dumai, riau, showed strong to very strong antioxidant activity of young and old s. alba leaves, with ic50 value of 50.12 ppm and 49.87 ppm respectively (sumartini et al. 2022). the variation in the antioxidant activities of these extracts from the same species could be affected by differences in the solvents, habitats, or extraction methods (elsharkawy et al. 2021; onyebuchi & kavaz, 2020). table 2. ic50 value of n-hexane extract of s. alba leaves. ic50 value (ppm) category i ii iii average 72.611 63.298 57.387 64.432 ± 7.675 strong phytochemical analysis the qualitative phytochemical analysis showed that the n-hexane extract contained two compounds, namely phenol and steroids (table 3). both of these compounds are known to exhibit antioxidant bioactivity (llauradó maury et al. 2020). however, a study conducted in dumai, riau, showed that compounds detected in nhexane extract of s. alba leaves were alkaloids, flavonoids, saponins, and tannins (sumartini et al. 2022). different extraction methods used and different habitat of the s. alba mangroves can both contribute to these variations (elsharkawy et al. 2021; onyebuchi & kavaz, 2020). table 3. phytochemical analysis results of n-hexane extract of s. alba leaves. phytochemicals reagent used results terpenoids liebermann burchard (-) phenols fecl3 1% (+) alkaloids dragendorff (-) steroids liebermann burchard (+) saponins distilled water (-) tannins fecl3 1% (-) flavonoids mg+hc (-) the gc-ms analysis results showed that there were five prominent compounds found in the n-hexane extract, namely gamma-sitosterol, dl-alpha-tocopherol, benzene1 ,2,4-trimethyl, 3-methyl-4(phenylthio)-2-prop-2-enyl2,5-dihydrothiophene 1,1-dioxide, and benzo [h] quinoline, 2,4-dimethyl(table 4). gamma-sitosterol was dominantly found in the n-hexane leaves extract of s. alba in this study, while stigmasterol was prominent in the n-hexane root extract of this species (wijaya et al. 2023). gamma-sitosterol has been shown to has a strong anticancer activity, as well as antioxidant and anticholesterol properties (dede et al. 2019). the compound dl-alpha-tocopherol or also known as vitamin e is a lipophilic antioxidant (zeece, 2020). meanwhile, benzene-1,2,4-trimethyl is known to have potential antioxidants effects, and also have efficacy against pancreatic cancer (marikkannu & ganesan, 2021; ogunmefun et al. 2023). benzo[h]quinolone has the potential as an antitumor because its cytotoxic effect and is able to inhibit the growth of tumor cells (jafari et al. 2019). however, no studies have demonstrated the antioxidant activity of this substance. in addition, no evidence has been found that clarify the pharmacological activity of the 3-methyl-4(phenylthio)-2-prop-2-enyl2,5-dihydrothiophene 1,1-dioxide compound. 634 biology, medicine, & natural product chemistry 12 (2), 2023: 631-635 table 4. gc-ms results of n-hexane extract of s. alba leaves. peak retention time compounds % area 1 49.168 gamma-sitosterol 52.88 2 47.053 dl-alpha-tocopherol 7.77 3 4.413 benzene1,2,4-trimethyl 4.07 4 31.948 3-methyl-4(phenylthio)-2-prop-2-enyl-2,5-dihydrothiophene 1,1-dioxide 3.85 5 48.458 benzo [h] quinoline, 2,4-dimethyl3.82 6 50.094 thymol, tms derivative 3.27 7 49.484 2-ethylacridine 3.05 8 43.111 1h-indole, 5-methyl-2-phenyl3.03 9 5.474 3,4-dichloro-5-phenylimino-2(5h)-furanone 2.17 10 4.554 dl-allo-cystathionine 2.04 11 3.650 benzene, 1-ethyl-3-methyl1.94 12 7.820 aluminum, tripropyl1.83 13 8.407 2-acetyl-5-bromo-1-methyl-1h-indol-3-yl difluoroborinate 1.78 14 49.451 (e)-2-bromobutyloxychalcone 1.38 15 3.793 1,3-cyclopentadiene, 5-(1-methylpropylidene)1.01 16 41.319 histidine, 1, n-dimethyl-4-nitro0.74 conclusions based on the findings of this study, it can be concluded that the n-hexane extract of s. alba leaves displayed strong antioxidant activity with an ic50 value of 64.432 ± 7.675 ppm. the phytochemicals found in the extract were phenols and steroids, while gc-ms analysis showed that gamma-sitosterol was the most prominent compound with % area of 52.88. acknowledgements: we express gratitude to mr. made yuda wibawa from unit pelaksana teknis daerah (uptd) taman hutan raya ngurah rai bali, who helped the authors collect the mangrove sample in the mangrove forest area of kub simbar segara, pemogan village, south denpasar, bali. authors’ contributions: putu rissa almadea surya, made dharmesti wijaya, & desak putu citra udiyani designed the study. putu rissa almadea surya 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10.14421/biomedich.2025.141.267-273 issn 2540-9328 (online) effectiveness of botanical pesticide from keji beling leaves (strobilanthes crispus) on spinach plants (amaranthus tricolor l.) against pest intensity desi wulandari*, ika listiana biology department; faculty of tarbiyah and teaching, uin raden intan, lampung jl. letnan kolonel h. endro suratmin sukarame lampung, indonesia. corresponding author* desiwlndr0812@gmail.com manuscript received: 24 february, 2025. revision accepted: 20 may, 2025. published: 24 june, 2025. abstract crop failure in cultivated plants can be influenced by several factors, including pest attacks. pest control in plants is often carried out using pesticides made from chemical compounds, which can have negative effects on health and the environment. one alternative to chemical pesticides is the use of botanical pesticides. this study aims to determine the effect of botanical pesticides made from keji beling leaves (strobilanthes crispus) on the intensity of pest attacks on spinach plants (amaranthus tricolor l.). the research was conducted using an experimental method from june to august 2024 in kelapa tujuh village, south kotabumi district, north lampung regency, lampung province, and at the zoology laboratory, faculty of science and technology, raden intan state islamic university, lampung. the study used a completely randomized design (crd) with four treatments: applying botanical pesticides at concentrations of 25%, 50%, 75%, and 0% (control). each treatment was repeated 16 times, resulting in 64 plant samples. the parameters studied included pest attack intensity, plant height, and root length. data were analyzed using analysis of variance (anova) and further tested with the least significant difference (lsd) test at a 5% significance level to determine significant differences. phytochemical screening tests revealed that keji beling leaf-based botanical pesticides contain alkaloids, saponins, and tannins. the results showed that applying botanical pesticides from keji beling leaves influenced all the parameters studied. the treatment with a 75% concentration was the most effective, resulting in the lowest leaf damage at only 10%, a plant height of 22.40 cm, and a root length of 13.47 cm. based on these findings, keji beling leaf-based botanical pesticides have the potential to serve as an environmentally friendly alternative for pest control in spinach cultivation. keywords: botanical pesticide; keji beling leaves; pest attack intensity; spinach plant. introduction spinach is a type of plant in the shrub or bush category. it is widely favored by the indonesian people, both children and adults (ihsan et al, 2022). spinach has a soft texture and a delicious taste. it is also known to aid digestion and provide a cooling sensation to the stomach. in addition to its pleasant taste, spinach contains vitamins a, b, and c, protein, fat, carbohydrates, potassium, amaranthin, and various minerals such as phosphorus, potassium, and iron, which are essential for growth and maintaining health (sari and fasta, 2021). given its numerous benefits, both as a highly nutritious food and as a remedy for certain diseases, it is important to enhance the growth and production of spinach plants (hidayanti and kartika, 2019). according to data published by the central statistics agency (bps), spinach production in indonesia reached 171.210 tons in 2022, declining compared to 171.706 tons in 2021. efforts to increase spinach production often face various challenges. one of the factors contributing to the decline in spinach production in tropical regions is the high presence of plant pests and diseases (opt) that attack spinach plants (yusidah and nurirhani, 2022). pest control is necessary to reduce damage caused by pest infestations. one way to manage pest attacks is through the use of pesticides. however, excessive use of chemical or synthetic pesticides can negatively affect human health and the environment. the environmental impacts of synthetic pesticides include disrupting natural ecological balance and leading to pest resistance. pesticide residues in the soil can poison other organisms and may even cause poisoning in animals and humans if they enter the food chain (rahayu and widowati, 2020). one alternative to eradicating pests in plants, besides using synthetic pesticides, is botanical pesticides. botanical pesticides are pesticides with single or compound active ingredients that function as anti-fertility agents (sterilizers), repellents, or killers of plant pests and diseases, derived from plants and relatively easy to https://doi.org/10.14421/biomedich.2025.141.267-273 268 biology, medicine, & natural product chemistry 14 (1), 2025: 267-273 produce. botanical pesticides decompose easily in nature because they are made from natural ingredients, making them safe for humans and livestock and environmentally friendly (siamtuti et al, 2020). keji beling leaves (strobilanthes crispus) have long been used by the indonesian people as traditional medicine. previous studies have shown that keji beling leaves contain various chemical compounds, including alkaloids, saponins, flavonoids, polyphenols, potassium, sodium, and calcium (larasati and putri, 2021). flavonoids are toxic to insects, have a strong odor, a bitter taste, are highly soluble in water, decompose at high temperatures, and act as feeding inhibitors for insects (sanjaya et al, 2021). alkaloids are toxic compounds that effectively eliminate pests by poisoning their bodies (ainiyah et al, 2023). saponins and polyphenols function as contact poisons, stomach poisons, and neurotoxins for insects. these compounds—alkaloids, saponins, flavonoids, and polyphenols—are utilized as botanical pesticides (sanjaya et al, 2021). this study aims to determine the effect of botanical pesticides made from keji beling leaves (strobilanthes crispus) on the intensity of pest attacks on spinach plants (amaranthus tricolor l.). materials and methods study area this study was conducted from june to august 2024 in kelapa tujuh village, south kotabumi district, north lampung regency, lampung province, and at the zoology laboratory, faculty of tarbiyah and teaching, raden intan state islamic university of lampung. procedures approach and type of research the type of research used is descriptive quantitative research with an experimental method. the experiment in this study involved direct testing conducted by researchers on spinach plants sprayed with botanical pesticides made from keji beling leaves. the total number of samples consisted of spinach plants subjected to three treatments and one control, applying keji beling leaf botanical pesticide spray at concentrations of 25%, 50%, and 75%. each treatment was repeated 16 times, resulting in a total of 64 plant samples. observation parameters the observed parameters include pest attack intensity, plant height, and root length. the pest attack intensity is calculated using the following formula: 𝐼𝑆 = 𝑋 𝑌 × 100% description: is : pest attack intensity (%) x : total damaged leaf area (cm²) y : total leaf area observed (cm²) tools and materials the tools used in this study include a blender, strainer, basin, jar, plastic bottle, spray bottle, polybag, stationery, smartphone camera, measuring glass, digital scale, and microscope. the materials used in this study include: 1 kg of keji beling leaves, 50 mg of detergent, 1 liter of water, spinach plant seeds, growing media and npk fertilizer. data analysis the data on pest attack intensity were analyzed using one-way anova with the spss 25 (statistical package for the social sciences) software. if a significant difference was found between treatments, further analysis was conducted using the least significant difference (lsd) test at a 5% significance level to determine specific treatment differences. results and discussion results phytochemical screening results in this study, a phytochemical test was also conducted. phytochemical screening was performed on the herbal pesticide solution made from keji beling leaves to identify chemical compounds. among the four types of phytochemical tests conducted, the herbal pesticide from keji beling leaves was positive for alkaloids, saponins, and tannins. these chemical compounds are known to function as natural pesticides. the results of the phytochemical screening of the herbal pesticide made from keji beling leaves can be seen in table 1 below. wulandari & listiana – effectiveness of botanical pesticide from keji beling … 269 table 1. phytochemical screening results. compound method positive result screening result note flavonoids extract + mg powder + concentrated hcl sample turns dark brown no color change _ alkaloids extract + h2so4 + dragendorf formation of yellowishbrown or brown precipitate color changes to yellowishbrown + saponins extract + chloroform + heated and shaken + lb sample turns yellow-orange or brown precipitate forms color changes to yellow-orange, brown precipitate observed + tannins extract + fecl3 solution color changes to dark blue, dark green, or dark brown color changes to dark brown +++ in the phytochemical screening results, the most abundant chemical compound found in the keji beling leaf botanical pesticide was tannin. the test results showed a deep blue color change in the botanical pesticide solution after being mixed with the reagent. tannins are compounds produced by plants that have various benefits. one of their functions is as an antinutrient that inhibits the enzyme α-amylase, causing the enzyme to bind with tannins, thereby hindering the starch breakdown process. the disruption of starch breakdown affects insect growth due to a lack of energy sources (aprilia et al., 2024). in addition to inhibiting enzyme activity, tannins also have an anti-feeding effect, reducing insect appetite. the inhibition of energy production caused by tannins disrupts the hormonal system, leading to impaired insect growth and development (taufika et al., 2023). alkaloids are chemical compounds that act as stomach poisons in insects because they contain more than one basic nitrogen atom, which inhibits insect feeding ability (akmalina et al., 2023). alkaloids are commonly found in plant parts such as bark, leaves, roots, tubers, seeds, and twigs (khafid et al., 2023). one function of alkaloids in plants is to serve as protection against pest attacks (saputra et al., 2023). alkaloids act as toxins for pests because their bitter taste discourages insects from feeding (sumantara et al., 2024). besides acting as an anti-feeding agent for pests, the stomach toxicity of alkaloids disrupts the pest’s metabolic processes (ikhsan et al., 2021). in the phytochemical test, alkaloids were detected in the keji beling leaf botanical pesticide solution, as indicated by a color change to orange when the solution was mixed with the reagent. another chemical compound that inhibits the digestive system of insects is saponin. saponins function as stomach poisons by circulating the compound into the bloodstream after digestion. this process damages blood vessels, leading to hemolysis of blood cells. in insect control, saponins are secondary metabolites that damage proteins and cell membranes, reduce cholinesterase enzyme activity, and disrupt food absorption, thereby affecting insect metabolism. additionally, saponins interfere with atp production, resulting in energy deficiency that can lead to insect death (ahyanti and yushananta, 2023). the phytochemical test for saponins was confirmed by stable foam when the botanical pesticide solution was mixed with the reagent. research parameter pest attack intensity table 2. average pest attack intensity day score 7 14 21 28 a 3% 6% 10% 39% 2 b 3% 7% 10% 32% 2 c 6% 7% 11% 13% 1 d 3% 6% 6% 10% 1 description: 1 = damage <25% (mild pest attack) 2 = damage 25-50% (moderate pest attack) the research results indicate that, based on the pest attack intensity parameter up to day 28, the highest intensity was observed in treatment a, with a pest attack intensity of 39%, followed by treatment b at 32%. these results were converted into a damage score 2, indicating a moderate pest attack, where leaf damage ranged from 25% to 50%. in treatment c, spinach leaf damage was recorded at 13%, while treatment d had the lowest pest attack intensity at 10%. this corresponds to score 1, representing a mild pest attack, where leaf damage was below 25%. the average pest attack intensity values are illustrated in figure 1. figure 1. leaf damage intensity. 0% 5% 10% 15% 20% 25% 30% 35% 40% a b c d 3% 3% 6% 3% 6% 7% 7% 6% 10% 10% 11% 6% 39% 32% 13% 10% leaf damage intensity day 7 day 14 day 21 day 28 270 biology, medicine, & natural product chemistry 14 (1), 2025: 267-273 table 2. shows that the highest leaf damage in spinach plants due to pest insect attacks was observed in treatment a with a 0% concentration, reaching 39%. meanwhile, the lowest damage was recorded in treatment d with a 75% concentration, with an attack rate of 10%. research on botanical pesticides derived from chromolaena odorata l. and annona muricata l. extracts has demonstrated a significant mortality rate in diamondback moth larvae (plutella xylostella) reaching 51.72%. (latifa & haryadi, 2024). other studies have also revealed that botanical pesticides from babandotan leaves are effective in controlling brown planthoppers (nilaparvata lugens) (ajijah et al, 2024), and anthracnose disease in cayenne pepper plants (suparto et al., 2023). the higher the concentration of a vegetable pesticide, the higher the toxic content, so that the mortality rate increases (yudiawati, 2019), in another study revealed that the percentage of mortality of p. xylostella larvae increased along with an increase in the percentage of extract combinations (latifa & haryadi, 2024). the higher the amount of active content contained in the vegetable pesticide used, the higher the toxic substances obtained to kill pests (ajijah et al, 2024). keji beling leaves are known to positively contain alkaloid compounds, saponins and tannins (junardin, et al 2024). table 1. the results of phytochemical screening showed active compounds contained in keji beling leaves, namely alkaloid compounds, saponins and tannins, these compounds are thought to affect the level of intensity of pest attacks. treatment d with the highest concentration level of 75% showed the lowest level of leaf damage at 10%. the observation results showed a difference in pest attack intensity between treatments. the intensity of pest attacks in this study is in the score 1 value of 25% which is included in mild pest attacks, and score 2 of 25-50% which is included in moderate pest attacks. plant height table 3. average plant height. parameter treatment a b c d plant height (cm) 18,72 19,56 20,66 22,40 for the plant height parameter, the best result was observed in treatment d, with a height of 22,40 cm, followed by treatment c at 20,66 cm, treatment b at 19.56 cm, and the lowest result in treatment a, with a plant height of 18,72 cm. the average plant height graph can be seen in figure 2. figure 2. spinach plant height. results of the duncan test can be seen in table 3. table 4. lsd test for plant height treatment plant height a 18,72a b 19,56a c d 20,66b 22,40c pesticides can support plant growth by preventing disturbances from pests and diseases, allowing plants to reach optimal height without experiencing stress or developmental obstacles (janjić & mitrić, 2020). application of botanical pesticides from soursop leaves, neem leaves, and turmeric has been found to affect plant height, with p2 (treatment using neem leaves) being the most effective (nugroho & barokah, 2023). neem leaf extract contains flavonoid compounds that are effective in controlling pests and influence melon growth, particularly by promoting upward growth (akhiriana et al., 2023). compounds such as alkaloids found in botanical pesticides from keji beling leaves are suspected to influence the height of spinach plants. alkaloids from jatropha curcas increase the height and leaf area of okra by reducing nematode activity, allowing plants to utilize nutrients more efficiently (vincent et al., 2023). another study found that alkaloids extracted from local lupins significantly enhanced the vegetative growth of sweet peppers, with increased plant height and biomass observed at higher concentrations (al-najjar & khairy, 2022). additionally, variety is another factor affecting spinach growth besides nutrients. amaranthus tricolor l. is one of the spinach varieties with a higher chlorophyll content than other varieties, allowing it to grow and develop more quickly (oktavia et al., 2022). root length table 5. root length of plants. parameter treatment a b c d root length (cm) 9.54 9.92 11.04 13.47 wulandari & listiana – effectiveness of botanical pesticide from keji beling … 271 the root length parameter showed the best result in treatment d, with a root length of 13,47 cm, followed by treatment c at 11,04 cm, treatment b at 9.92 cm, and the lowest result in treatment a, with a root length of 9,54 cm. the graph of root length growth results can be seen in figure 3. figure 3. spinach root length. results of the duncan test can be seen in table 5. table 6. lsd test for root length. treatment plant height a 9,54a b 9,92a c d 11,04b 13,47c the best root length parameter was obtained in treatment d, measuring 13.47 cm. a previous study on the root length of tomato plants treated with plant extracts, including p. betle l., c. papaya, nicotiana tabacum l., a. sativum, c. frutescens l., r. communis, d. stramonium l., and m. citrifolia, at a concentration of 250 cc/polybag and a control group, showed that the roots of the control plants were shorter than those of the treated plants (diantari et al., 2015). another study found that botanical pesticides based on papaya leaf extract increased the root length of spinach to 23.57 cm compared to the control (bay and barokah, 2024). soursop and neem leaf extracts effectively reduced rootknot nematode (meloidogyne spp.) infestations in tomato plants, positively impacting root growth. the difference in root length between treated and control plants was attributed to certain nematicidal compounds in the extracts, which reduced nematode attacks and led to better root development in treated plants compared to the control. these three plant extracts (tagetes spp., j. curcas l., c. papaya l.) are known to contain alkaloids, flavonoids, saponins, and tannins, which have nematicidal properties or can be lethal to nematodes (eaggeliony et al., 2023). tannins can modulate nitrogen release and increase root biomass (at lower concentrations), potentially promoting early growth in some species (miele et al., 2019). phytochemical screening of keji beling leaves revealed the presence of alkaloids, saponins, and tannins, which are suspected to influence not only plant height but also root length. the nutrient absorption process in plants is influenced by broader root development, which plays a crucial role in organ formation. root effectiveness is determined by its length, as an increase in root surface area enhances nutrient uptake and overall plant productivity (telaumbanua et al., 2023). conclusions based on the results of the research that has been carried out, it can be concluded that: ▪ the use of vegetable pesticides keji beling leaves (strobilanthes crispus) on spinach plants (amaranthus tricolor l.) has an influence on the intensity of pest attacks, plant height and root length. ▪ the most effective dose of vegetable pesticides from keji beling leaves in overcoming pest attacks is at concentration d (75%). at this concentration, the lowest level of leaf damage compared to other treatments, with a final result of 10%. in addition, the 75% concentration also gave the best results in plant height of 22.40 cm and root length of 13.47 cm. recommendation after researching the application of vegetable pesticides keji beling leaves (strobilanthes crispus) on spinach plants (amaranthus tricolor l.) on the intensity of pest attacks, the researchers can provide the following recommendation: ▪ further research 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harcourt, rivers state, nigeria 2department of pharmacology and toxicology, faculty of pharmacy, niger delta university, bayelsa state, nigeria. corresponding author* udgeorgewill@yahoo.com, tel+234 8036662936 manuscript received: 08 december 2021. revision accepted: 11 january, 2022. published: 09 february, 2022. abstract antimalarial drug resistance is one of the greatest challenges towards eradicating malaria. exploring new combination therapies can overcome resistance challenges. the present study examined the antiplasmodial effect of artemether/lumefantrine/doxycycline (a/l/d) on a mouse model infected with plasmodium berghei. adult swiss albino mice (22-30g) intraperitoneally infected with blood containing 1x107 plasmodium berghei were randomly grouped and orally treated daily with d (2.2 mg/kg), a/l (1.71/13.7 mg/kg) and a/l/d. the negative control was treated daily with normal saline (0.2ml) whereas the positive control was treated daily with chloroquine (cq) (10mg/kg). after treatment, blood samples were assessed for percentage parasitemia and biochemical parameters. mice were observed for mean survival time (mst). d, a/l and a/l/d produced significant decreases in percentage parasitemia levels at p<0.05; p<0.01 and p<0.001, respectively when compared to negative control. in the curative test, d, a/l and a/l/d produced 60.4%, 70.3%, and 90.0% parasitemia inhibitions, respectively whereas cq produced 76.0% parasitemia inhibition. d, a/l, a/l/d and cq produced 63.2 %, 80.1%, 92.3% and 83.6% parasitemia inhibitions, respectively in the suppressive test. d, a/l, and a/l/d prevented plasmodium bergheiinduced alterations in biochemical parameters by increasing packed cell volume, red blood cells, hemoglobin, and high-density lipoprotein and decreasing white blood cells, total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels significantly at p<0.05 and p<0.01 and p<0.001, respectively when compared to the negative control. a/l/d produced significant antiplasmodial activity therefore, it may be used clinically for the treatment of malaria. keywords; antiplasmodial; artemether; tetracycline; antimalarial; lumefantrine; plasmodium berghei. introduction antimalarial drug resistance has been acknowledged to be one of the greatest challenges to the roll back malaria programme. the situation is highly precarious due to the rising incidence of plasmodium resistance to currently available antimalarial drugs (yeung et al., 2004). chloroquine (cq) resistant plasmodium falciparum now predominates in southeast asia, south america and africa. resistance to sulphadoxinepyrimethamine is widespread in asia and south america and is spreading in africa and even quinine has become less effective over time (khan et al., 2004). the use of combination therapy with artemisinins and partner drugs has been a rational approach to combating drug resistance. drugs with different mechanisms of action may enhance their respective efficacies and extend their therapeutic life spans (white & olliaro, 1996). despite, the combination therapy approach, plasmodium resistance is still a serious challenge (yeung et al., 2004). doxycycline (d) is one of the most active antibiotics against plasmodium parasites. it belongs to the tetracycline family. the tetracyclines, which have a very wide spectrum of activity are bacteriostatic and inhibit bacterial protein synthesis (gaillard et al., 2015). among the tetracyclines, d is widely used for malaria prophylaxis and is highly acceptable for long-term therapy, except in pregnant women and children (who, 2005). in-vitro and in-vivo investigations have shown that d may be an effective antimalarial drug against drug-resistant plasmodium strains (basco & bras, 1993). it is used in combination with quinine as an effective standby emergency treatment of malaria associated with plasmodium falciparum (who, 2005). recently, it has shown antimalarial potential against plasmodium bergheiinduced cerebral malaria in experimental models by inhibiting brain inflammation, tumour necrosis factor and chemokines expressions (schmidt et al., 2018). artemether/lumefantrine (a/l) is an artemisininbased antimalarial drug approved by the us food and https://doi.org/10.14421/biomedich.2022.111.1-6 2 biology, medicine, & natural product chemistry 11 (1), 2022: 1-6 drug administration in 2009 for the treatment of plasmodium falciparum malaria. the dual mechanisms of action of a/l provide fast and sustained plasmodium clearance (stover et al., 2012). it is the most widely used antimalarial drug combination in endemic regions. in 2017, a/l accounted for almost 75% of all purchased and clinically used artemisinin based combination therapies (acts) (nsanzabana, 2019). artemisinin derivatives rapidly clear parasites through a number of proposed mechanisms such as interference with plasmodial transport proteins, interference with plasmodial mitochondrial electron transport, and the production of free radicals (stover et al., 2012). the precise mechanism for the antiplasmodial activity of lumefantrine is not well defined, but it is proposed to inhibit β-hematin formation, which is an important detoxification pathway for plasmodium parasites (stover et al., 2012). despite the success achieved with the use of a/l in combating malaria scourge, the emergence of plasmodium parasite resistance in some endemic countries has become a significant drawback for the fight against malaria (nsanzabana, 2019). due to the challenge posed by plasmodium parasite resistance, there is a strong advocacy for the rational use of antimalarial drugs with antibiotics. this will afford the synergistic or additive killing of plasmodium parasites, and thus prevent or reduce drug resistance (miller et al., 2006; alecrim et al., 2006). this study, therefore assessed the antiplasmodial effect of a/l/d on a mouse model infected with plasmodium berghei. materials and methods drugs and dose selection artemether/lumefantrine (a/l) (ipac laboratory, india), chloroquine (cq) (evans medical nigeria plc), and doxycycline (d) (ranbaxy laboratories ltd, india) were used. the following doses were used: a/l (2.3/13.7 mg/kg) (sirima et al., 2016), cq (10mg/kg) (somsak et al., 2018), and d (2.2 mg/kg) (gaillard et al., 2015) animals adult swiss albino mice (22–30 g) were used. the mice were obtained from the animal unit of the department of pharmacology, faculty of basic clinical sciences, college of health sciences, university of port harcourt, rivers state. the mice were housed and fed throughout the study period as per recommended standards. the mice were allowed for 2 weeks to acclimatize to working environment and were handled according to the international animal care and welfare guidelines (ilar, 2011). parasites inoculation cq-sensitive plasmodium berghei (p. berghei) (nk65 strain) was used for malaria induction in the experimental mice. p. berghei was obtained from malaria research laboratory, centre for malaria research and phytomedicine, university of portharcourt, rivers state, nigeria. mice previously infected with p. berghei were used as donor mice and parasites were kept alive by continuous intraperitoneal (i.p) passage of blood from donor mice to uninfected mice weekly. percentage parasitemia was determined using the formula below. protocol for antiplasmodial test  protocol for curative test the curative test was performed as described by ryley and peters (1970). thirty mice randomly grouped into 6 (a1-a6) of 5 mice each were used. group a1 served as the normal control while a2-a6, which served as the experimental groups were inoculated with 1 × 107 p. berghei-infected blood (i.p). three days later, the mice were treated orally as follows: group a1 (normal control) was treated with normal saline (0.2ml) daily for 4 days. group a2 (negative control) and group a3 (positive control) were treated with normal saline (0.2ml) and cq (10mg/kg) daily for 4 days, respectively. groups a4-a6 were treated with d (2.2mg/kg), a/l (2.3/13.7 mg/kg) and a/l/d daily for 4 days, respectively. on day 5, tail blood samples were collected from the mice and thin smears were prepared on slides and stained with 10% giemsa stain. the stained slides were examined microscopically with an oil immersion objective of 100× magnification power. the percentage parasitemia and inhibitions were calculated using the formula shown below. % 𝑃𝑎𝑟𝑎𝑠𝑖𝑡𝑎𝑒𝑚𝑖𝑎 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑖𝑧𝑒𝑑 𝑟𝑒𝑑 𝑏𝑙𝑜𝑜𝑑 𝑐𝑒𝑙𝑙𝑠 (𝑅𝐵𝐶𝑠) total number of rbcs count × 100% % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (% parasitemia of negative control − % parasitemia of treated group) × 100 % parasitemia of negative control  protocol for suppressive test the suppressive test was performed as described by knight and peters (1980). twenty-five adult swiss albino mice were inoculated with blood containing 1 × 107p. berghei and randomized into 5 groups (b1-b5) of 5 mice each. the mice were treated after 3 hours of georgewill & adikwu – potential antimalarial activity of artemether/lumefantrine/doxycycline: … 3 inoculation as follows: group b1 (negative control) and group b2 (positive control) were orally treated daily with normal saline (0.2ml) and cq (10mg/kg), for 4 days, respectively. groups b3-b5 were orally treated with d (2.2mg/kg), a/l (2.3/13.7 mg/kg) and a/l/d daily for 4 days, respectively. on day 5, tail blood samples were collected from the mice and thin smears were prepared on slides and stained with 10% giemsa stain. the stained slides were examined microscopically with an oil immersion objective of 100×magnification power. the percentage parasitemia and inhibitions were calculated as explained above.  protocol for prophylactic test the prophylactic test was performed as described by peters (1965). twenty-five adult swiss albino mice randomized into 5 groups (c1-c5) of 5 mice/group were orally treated as follows: group c1 (negative control) and group c2 (positive control) were treated with normal saline (0.2 ml) and cq (10 mg/kg) daily for 4 days, respectively. groups c3 – c5 were treated with d (2.2 mg/kg), a/l (2.3/13.7 mg/kg), and a/l/d daily for 4 days, respectively. on day 5, the mice were inoculated with blood containing 1 × 107 p. berghei. after 2 days, tail blood samples were collected and percentage parasitemia and inhibitions were determined as explained above. determination of mean survival time from the time of inoculation with p. berghei until death, mortality of each mouse was monitored and recorded. mean survival time (mst) was determined using the formula below. 𝑀𝑆𝑇 = 𝑆𝑢𝑚 𝑜𝑓 𝑠𝑢𝑟𝑣𝑖𝑣𝑎𝑙 𝑡𝑖𝑚𝑒 𝑜𝑓 𝑎𝑙𝑙 𝑚𝑖𝑐𝑒 𝑖𝑛 𝑎 𝑔𝑟𝑜𝑢𝑝 (𝐷𝑎𝑦𝑠) total number of mice in that group evaluation of biochemical parameters in the curative study, blood samples were collected from the mice and evaluated for red blood cells (rbcs), hemoglobin (hb), packed cell volume (pcv), white blood cells (wbcs), total cholesterol (chol), triglyceride (tg), low-density lipoprotein cholesterol and (ldl-c) and high-density lipoprotein cholesterol (hdl-c) using an auto analyzer. statistical analysis values were expressed as mean ± sem (standard error of mean) of n=5. values were analyzed using one-way anova, followed by tukey’s post hoc test. p values less than 0.05, 0.01 and 0.001 were considered significant. results curative antiplasmodial test treatment with d, a/l, and a/l/d produced significant decreases in percentage parasitemia at p<0.05, p<0.01 and p<0.001, respectively when compared to negative control (pu) (table 1). d, a/l and a/l/d produced parasitemia inhibitions of 60.4%, 70.3%, and 90.0%, respectively whereas cq produced 76.0 % parasitemia inhibition. mst was significantly prolonged in mice treated with d, a/l, and a/l/d at p<0.05, p<0.01, and p<0.001, respectively when compared to pu (table 1). suppressive antiplasmodial test significant decreases in percentage parasitemia levels at p<0.5, p<0.01 and p<0.001 were produced by d, a/l, and a/l/d, respectively when compared to pu (table 2). parasitemia inhibitions, which represent 63.2 %, 80.1%, 92. 3% and 83. 6% were produced by d, a/l, a/l/d and cq, respectively (table 2). treatment with d, a/l and a/l/d significantly prolonged mst at p<0.5, p<0.01, and p<0.001, respectively when compared to pu (table 2). prophylactic antiplasmodial test percentage parasitemia levels were significantly decreased in mice treated with d (p<0.05), a/l (p<0.01) and a/l/d (p<0.001) when compared to pu (table 3). d, a/l and a/l/d produced parasitemia inhibitions of 65.1%, 82.8%, and 93.9%, respectively whereas cq produced 86.7% parasitemia inhibition. significant prolongations of mst occurred in mice treated with d, a/l, and a/l/d at p<0.5, p<0.01 and p<0.001, respectively when compared to pu (table 3). hematological and lipid profile p. berghei infected mice showed significant (p<0.001) increases in serum tg, chol, ldl-c and wbcs levels with significant (p<0.001) decreases in serum hb, pcv, rbcs and hdl-c levels when compared to control (tables 4 and 5). on the other hand, treatment with d, a/l, and a/l/d significantly decreased serum tg, chol, ldl-c, wbcs levels and significantly increased serum hb, pcv, rbcs and hdl-c levels at p<0.05, p<0.01and p<0.001, respectively when compared to pu (tables 4 and 5). 4 biology, medicine, & natural product chemistry 11 (1), 2022: 1-6 table 1. curative effect of artemether/lumefantrine/doxycycline on plasmodium berghei infected mice. data as mean ± sem (standard error of mean) n= 5, pu: negative control, cq: chloroquine, d: doxycycline, a/l: artemether/lumefantrine, a/l/d: artemether/lumefantrine/doxycycline, mst: mean survival time. ap<0.01, b p<0.05, c p<0.001 significant difference when compared to pu. table 2. suppressive effect of artemether/lumefantrine/doxycycline on plasmodium berghei infected mice. treatment % parasitemia % inhibition mst (days) pu 20.30±1.03 0.00 9.66±0.45 cq 3.33±0.32a 83.6 30.41±3.71a d 7.47±0.11b 63.2 22.73±2.78b a/l 4.04±0.05a 80.1 31.02±3.48a a/l/d 1.56±0.02c 92.3 37.62±3.67c data as mean ± sem (standard error of mean) n= 5, pu: negative control, cq: chloroquine, d: doxycycline, a/l: artemether/lumefantrine, a/l/d: artemether/lumefantrine/doxycycline, mst: mean survival time. ap<0.01, b p<0.05, c p<0.001 significant difference when compared to pu. table 3. prophylactic effect of artemether/lumefantrine/doxycycline on plasmodium berghei-infected mice treatment % parasitemia % inhibition mst (days) pu 18.50±1.57 0.00 9.66±0.61 cq 2.46±0.01a 86.7 32.03±3.71a d 6.46±0.35b 65.1 25.52±2.60b a/l 2.44±0.17a 82.8 32.60±3.38a a/l/d 1.29±0.03c 93.9 39.21±4.61c data as mean ± sem (standard error of mean) n= 5. pu: negative control, cq: chloroquine, d: doxycycline, a/l: artemether/lumefantrine, a/l/d: artemether/lumefantrine/doxycycline, mst: mean survival time. ap<0.01, b p<0.05, c p<0.001 significant difference when compared to pu. table 4. effect of artemether/lumefantrine/doxycycline on hematologic parameters of plasmodium berghei-infected mice. treatment rbc (x106) wbc (cells/l) pcv (%) hb (g/dl) nc 5.11±0.24 6.61±0.11 54.63±5.00 16.31±0.08 pu 2.41±0.29a 13.91±1.06a 20.72±3.17a 6.63±0.37a cq 4.00±0.11b 9.11±0.09b 38.80±3.54b 11.93±0.15b d 3.10±0.17c 10.27±0.17c 26.54±3.76c 9.65±0.45c a/l 3.99±0.12b 9.42±0.09b 38.43±4.49b 10.04±0.80b a/l/d 4.91±0.09d 6.33±0.21d 50.25±4.21d 13.95±0.05d data as mean ± sem (standard error of mean) n= 5, nc: normal control pu: negative control cq: chloroquine, d: doxycycline, a/l: artemether/lumefantrine, a/l/d: artemether/lumefantrine/doxycycline, mst: mean survival time, rbcs: red blood count, wbcs: white blood count, pcv: packed cell volume hb: haemoglobin, a p<0.001 significant difference when compared to nc, b p<0.01, c p<0.05, d p<0.001 significant difference when compared to pu. table 5. effect of artemether/lumefantrine/doxycycline on lipid parameters of plasmodium berghei-infected mice. treatment tg mg/dl chol mg/dl hdl-c mg/dl ldl mg/dl nc 76.8±8.03 100.4±14.0 55.9±5.66 29.1±3.11 pu 266.3±15.0a 298.4±14.1 a 24.6±2.32 a 220.1±18.1a cq 169.1±10.6 b 190.8±16.4 b 39.1±5.72 b 117.8±11.6b d 210.7±11.6 c 247.5±13.7 c 30.0±3.19 c 175.4±15.0c a/l 179.0±14.0b 199.6±14.3b 37.3±3.67b 126.5±12.5b a/l/d 80.9±7.49d 117.8±12.5 d 49.5±4.39d 41.3±10.1d data as mean ± sem (standard error of mean) n= 5, nc: normal control pu: negative control cq: chloroquine, d: doxycycline, a/l: artemether/lumefantrine, a/l/d: artemether/lumefantrine/doxycycline, tg: tryglyceride, chol:total cholesterol hdl: high density lipoproteins, ldl: low density lipoprotein, vldl: very low density lipoprotein. a p<0.001 when compared to nc, b p<0.01, c p<0.05, d p<0.001 when compared to pu. discussion artemisinin based combination therapies (acts) are used as treatment for uncomplicated malaria (who, 2015). unfortunately, the emergence of plasmodium parasite resistant to acts has been reported in endemic regions (cui et al., 2015; who, 2016). antimalarial drug resistance poses one of the greatest threats to malaria control. in africa, the efficacy of affordable antimalarial drugs is rapidly declining, and efficacious treatment %parasitemia %inhibition mst (days) pu 38.10±4.95 0.0 9.02±0.11 cq 9.14 ±0.20a 76.0 25.30±2.37a d 13.72±0.67b 60.4 16.21±1.57b a/l 11.30±0.13a 70.3 24.82±3.48a a/l/d 3.43±0.08c 90.0 31.63±3.21c georgewill & adikwu – potential antimalarial activity of artemether/lumefantrine/doxycycline: … 5 antimalarial drugs tend to be too expensive. costeffective methods are needed to fight against antimalarial drug resistance. one of the primary solutions to challenges associated with plasmodium parasites resistance to antimalarial drugs is to explore new combination therapies. in combination therapy, the possibility of the plasmodium parasites developing resistance simultaneously to two or more combined drugs with different mechanisms of action is extremely low (who, 2001). the current study examined the antiplasmodial activity of a/l/d on p. berghei-infected mice. p. berghei is used in predicting treatment outcomes of antimalarial drug candidates, due to its sensitivity, making it an important parasite for antiplasmodial studies (unekwuojo et al., 2011). studies have shown that suppressive and curative tests are effective in the antiplasmodial evaluation of candidate drugs on early and established infections, respectively. importantly, suppressive and curative tests give vital information on percentage parasitemia, and parasitemia inhibitions (bobasa et al., 2018). in the current study, treatment with a/l/d produced the best curative and suppressive antiplasmodial effects in relation to individual doses of a/l, d and cq. in the curative study, treatment with d, a/l, and a/l/d produced 60.4%, 70.3%, and 90.0% inhibitions, respectively. in the suppressive study, treatment with d, a/l and a/l/d produced 63.2 %, 80.1% and 92. 3% inhibitions, respectively. the prophylactic study showed best decrease in parasitemia level in a/l/d-treated mice when compared to individual doses of a/l, d and cq. the observed parasitemia inhibitions in the prophylactic study were 65.1%, 82.8%, and 93.9%, and 86.7% in mice treated with d, a/l, a/l/d and cq, respectively. in addition to percentage parasitemia and inhibition, this study determined the mst of the mice used for the curative, suppressive and prophylactic tests (georgewill et al., 2021) to further buttress the antiplasmodial activity of a/l/d. studies have shown that candidate drugs that can appreciably prolong the mst of parasitized animals may be active against malaria (oliveira et al., 2009). in the current study, curative, suppressive and prophylactic tests showed best prolongation of mst in mice treated with a/l/d than d, a/l, and cq alone. in the curative study, d, a/l and a/l/d prolonged mst to 16.21±1.57, 24.82±3.48 and 31.63±3.21 days, respectively. studies have shown that plasmodium depend on host hemoglobin as a nutrientsource for growth and multiplication. it consumes more than 75% of haemoglobin during its intra-erythrocytic phase and metabolizes heme into hemozoin (inbaneson and sundaram 2012). p. berghei-infected mice are prone to anemia due to erythrocyte destruction, as a consequence of parasite multiplication or by spleen reticuloendotelial cell action causing the production of phagocytes by the spleen due to abnormal erythrocytes (nardos and makonnen, 2017). in the current study, anemia was conspicuous in p. berghei-infected mice characterized by decreased pcv, hb, and rbcs with increased wbcs levels. however, p. berghei–induced anemia was vividly reduced in mice treated with a/l/d which was characterized by increased pcv, hb, and rbcs levels and decreased wbcs levels. interestingly, the anti-anemic activity of a/l/d was best when compared to individual doses of a/l, d and cq. changes in serum lipid profile related to malaria infection have been reported in some studies. the underlying biological mechanisms for malaria related lipid changes remain unclear, but may be host related, parasite-related or a combination of the two factors (visser et al., 2013). the present study observed significant alterations in lipid profile in parasitized mice marked by increased chol, tg, ldl-c and decreased hdl-c levels. the altered lipid parameters were best restored in a/l/d-treated mice marked by elevated hdl-c and decreased chol, tg, ldl-c levels. the observed antiplasmodial effect of a/l/d may be due to the independent mechanisms of action of the constituent drugs. the antiplasmodial mechanisms of d are not well defined, but d can inhibit mitochondrial protein synthesis and also decrease the activity of mitochondrial enzyme (dihydroorotate dehydrogenase) involved in pyrimidine synthesis (prapunwattana et al., 1998). also, d can inhibit the syntheses of nucleotides and deoxynucleotides in plasmodium (yeo et al., 1997). the artemisinins are speculated to interfere with plasmodial mitochondrial electron transport, transport proteins, and the production of free radicals (stover et al., 2012). lumefantrine is thought to inhibit β-hematin formation, an important detoxification pathway in plasmodium parasites (stover et al., 2012). conclusion results of the study concluded that a/l/d significantly decreased percentage parasitemia, increased percentage inhibition and prolonged mst in p. berghei-infected mice. the results of this study therefore showed prospect for the use of a/l/d for malaria treatment. conflict of interest: the authors declare no conflicts of interest. references alecrim mg, lacerda mv, mourao mp, 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(2004). antimalarial drug resistance, artemisinin based combination therapy, and the contribution of modelling to elucidating policy choices. american journal of tropical medicine and hygiene. 71(2): 179-86 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 615-619 | doi: 10.14421/biomedich.2025.142.615-619 issn 2540-9328 (online) the acoustic material constructed by the fiber of eichhornia crassipes and banana peel as a sound pollutant-reducing solution regita dimar asyurra1, avivah afra amatullah1, annisa zaskia puteri1, mayshilla anatayya putri zevly1, angga puja asiandu2, widya sari3,* 1islamic harapan mulia senior high school, palembang 30135, indonesia. 2faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia. 3department of physics, faculty of mathematics and natural science, universitas gadjah mada 55281, indonesia. corresponding author* widya4895sari@mail.ugm.ac.id manuscript received: 20 march, 2025. revision accepted: 13 july, 2025. published: 01 october, 2025. abstract this study aims to evaluate and compare the effect of the composition of water hyacinth (eichhornia crassipes) with a banana peel on the absorption coefficient value as a composite material for absorbing sound waves. the cellulose within the water hyacinth and banana peel lead to reduced overload sound waves. this study used an experimental method providing banana peel crushed into powder, water hyacinth fiber, and resin (as a catalyst). all samples were prepared with dimensions of 13.5 cm × 25 cm × 0.8 cm. a frequency generator application was used as the sound source (125, 250, 500, 1000, and 2000 hz), while a sound meter application measured sound intensity (db). the results indicated that the outer surface of sample d had a superior absorption coefficient, measuring 0.51 at 250 hz and 0.45 at 2000 hz. meanwhile, the inner surface of sample d recorded absorption coefficients of 0.46 at 250 hz and 0.45 at 2000 hz. variations in absorption values were attributed to destructive interference and saturation. sample d contained more water hyacinth (approximately 30%) than banana peel powder. this suggests that water hyacinth and banana peel powder are viable natural fiber alternatives for sound wave absorption. keywords: absorption coefficient; banana peel; water hyacinth; sound; cellulose. introduction noise pollution can disrupt human activities and affect mental health due to excessive noise. sound can also trigger psychological disorders in terms of health, such as sleep disorders, hearing disorders, hormonal disorders, increasing the incidence of diabetes, and even heart disease (safira & safira, 2017). according to the regulation of the minister of health of the republic of indonesia no. 718/menkes/per/xi/1987, noise is an unwanted sound that causes discomfort to the listener (kementerian kesehatan ri, 1987). who noted that europeans have been exposed to excessive noise from road noise as many as 1 in 5 or around 100 million (who, 2024). the negative impact of excessive noise is explained by (wahyudi, 2018) that people in the cement industry experience dizziness, headaches, and hearing loss due to a threshold value of 80-85 db that exceeds the threshold value set for the industry, which is 70 db. threshold limit value is the value of disturbance that a person can still accept without causing temporary or permanent hearing loss (below 90 db). if an individual receives sound more than 90 db for a long time, it will cause physical disorders in the ear organ (eryani et al., 2017). regarding the limit value, each country has different regulations. japan for residential areas has two limit value determinations, where 45 db is for the daytime limit and 40 db at night (khan & burdzik, 2023). unlike japan, the discovery of the limit value in europe has three time periods. the limit value in residential areas is around 55 db during the day. the threshold value in the afternoon is around 50 db. while at night, the limit value is 45 db (khan & burdzik, 2023). currently, several acoustic studies develop sound wave-absorbing materials based on natural fibers. fibers from hemp, coconut, and abaca have the potency to control noise (rifaida eriningsih et al., 2014). soundabsorbing composites will be characterized by the type of fiber, the physical properties of the fiber, and the morphology. in addition, the absorption coefficient α, ranging from 0 to 1, also determines the absorbed quantity. this value ranges from 0 to 1. if the coefficient value is 1, it depicts that the sound waves are perfectly absorbed 100 percent by the material (astatika & dwijaya, 2016). sound-absorbing materials are indicated by the presence of pores to capture sound waves (putra & nazhar, 2020). water hyacinth and banana peels have https://doi.org/10.14421/biomedich.2025.142.615-619 616 biology, medicine, & natural product chemistry 14 (2), 2025: 615-619 pores that can become acoustic materials when processed. water hyacinth, eichhornia crassipes, is a watery plant with massively higher productivity that can disturb the aquatic environment and ecosystem balance. it is not a primary commodity for the community, but it scientifically has natural fiber with a crude protein content of 9.79%, crude fiber of 22.41%, crude fat of 2.82%, ash of 13.32%, and water content of 7.76% (sihite et al., 2014). water hyacinth also has a lignin content of 17% (kusumawati & haryadi, 2021). on the other hand, banana fruit is widely used by the community, is accessible, and has a low price. the banana peel takes two years to degrade so it will accumulate being waste. it is discovered by fiber content of 17.12%, protein of 9.55%, and crude fat of 4.94% (fauzana et al., 2012). banana peel also has a cellulose content of 14.04% and a hemicellulose content of 37.52% (nasrun et al., 2014). banana peels and water hyacinth are fabricated as an acoustic material. therefore, this study will assess the effect of water hyacinth fiber and banana peel composites on the absorption coefficient value of both the outer surface of the material and the inner surface of the material. materials and methods study area this study stage includes preparation, fabrication, and testing. the testing limits the measurement of the absorption coefficient to indicate the comparison of materials’ capacity. figure 1. measurement design of acoustic material. before measurement, the upper of design should be closed by the material procedures the ingredients contain water hyacinth fiber, banana peel fiber, and polyester resin. the fibers of both materials are exposed to the sunlight. after drying, the water hyacinth fibers are separated and soaked in sodium hydroxide (naoh) and dried again. meanwhile, the dried banana peel is mashed into powder. then, all ingredients are divided based on the calculation of variation. there are four variations in the fabrication of acoustic materials, namely (a) sample variation a consists of 60% resin, 40% banana peel, and 0% water hyacinth; (b) sample variation b consists of 60% resin, 30% banana peel, and 10% water hyacinth; (c) sample variation c consists of 60% resin, 20% banana peel, and 20% water hyacinth; and (d) sample variation d consists of 60% resin, 10% banana peel, and 30% water hyacinth. water hyacinth fiber and banana peel are combined according to the calculation per variation using a mixture of resin and catalyst in a mold. all samples were set in sizes of 13.5 cm x 25 cm x 0.8 cm. data analysis acoustic material samples will be evaluated on the outside and inside of the material with frequencies of 250, 500, 1000, and 2000 hz. for the assessment, a smartphone should provide the frequency generator application as a sound source generator and the sound meter application as a sound intensity meter (figure 1). the material will be assessed for the absorption coefficient (α). it indicates that the total sound energy can be absorbed by the material. repetition of calculation is carried out as many as four times. the absorption coefficient value of the material will be obtained from the intensity value equation as follows. 𝐼𝑡 = 𝐼𝑖𝑒 −∝𝑥 where: 𝐼𝑖 : the initial intensity of sound wave (w/m2) 𝐼𝑡 : the transmitted intensity of sound wave (w/m2) 𝑥 : the thickness of material (m) 𝑎 : the absorption coefficient of material (𝑚−1) results and discussion table 1 and table 2 are the results of sound intensity and absorption coefficient, respectively. there are differences in the initial intensity value and the transmitted intensity when given a sound source directed at acoustic material with different frequencies. the higher the frequency emitted, the more the absorption coefficient value of each sample tends to increase (table 2). sample d has the highest coefficient value of 0.51 at a frequency of 250 hz and 0.45 at a frequency of 2000 hz compared to other samples consisting of 60% resin, 10% banana peel, and 30% water hyacinth. the higher the percentage of water hyacinth around 40%, the higher the absorption coefficient value of around 0.343 (harfi et al., 2023). asyurra et al. – the acoustic material constructed by the fiber of … 617 table 1. the comparison between the initial intensity and the transmitted intensity for outside surface material. frequency (hz) material 𝐼𝑜 (db) i (db) 𝐼𝑜 (db) i (db) 𝐼𝑜 (db) i (db) 𝐼𝑜 (db) i (db) a1 a2 a3 a4 125 63 34 63 27 63 35 63 35 250 71 52 71 46 71 51 71 53 500 87 74 87 71 87 71 87 71 1000 88 73 88 67 88 65 88 68 2000 88 60 88 73 88 73 88 73 b1 b2 b3 b4 125 63 41 63 38 63 40 63 36 250 71 54 71 54 71 55 71 52 500 87 73 87 69 87 69 87 68 1000 88 68 88 64 88 66 88 69 2000 88 66 88 64 88 65 88 64 c1 c2 c3 c4 125 63 34 63 32 63 27 63 32 250 71 50 71 49 71 51 71 52 500 87 67 87 73 87 70 87 71 1000 88 64 88 66 88 72 88 75 2000 88 71 88 70 88 69 88 67 d1 d2 d3 d4 125 63 33 63 28 63 32 63 33 250 71 49 71 45 71 49 71 49 500 87 62 87 66 87 66 87 68 1000 88 67 88 65 88 71 88 68 2000 88 60 88 62 88 64 88 63 table 2. the absorption coefficient for outside surface material. tables 3 and table 4 explain the results of intensity and the absorption coefficient from the inner surface, respectively. the higher the frequency applied to the same sample, the absorption coefficient value tends to increase. the instability of the absorption coefficient is caused by particles in the acoustic material experiencing saturation and destructive interference (sinaga et al., 2012). in some frequencies, fluctuations have occurred due to interference from noisy activities in the surrounding environment during the data collection process. the sound meter is also sensitive to the surrounding sound and causes the intensity of the sound read to fluctuate. the highest absorption coefficient with a value of 0.50 is measured in sample d, which consists of 30% water hyacinth, 10% banana peel, and 60% resin. sample d can be an alternative because its absorption value reaches 0.50 and exceeds the iso 11654 standard, a minimum absorption coefficient value of 0.15 (said l et al., 2020). table 3. the comparison between the initial intensity and the transmitted intensity for inside surface material. frequency (hz) material 𝐼𝑜 (db) i (db) 𝐼𝑜 (db) i (db) 𝐼𝑜 (db) i (db) 𝐼𝑜 (db) i (db) a1 a2 a3 a4 125 63 43 63 36 63 34 63 28 250 71 54 71 52 71 51 71 49 500 87 65 87 61 87 68 87 71 1000 88 73 88 71 88 75 88 75 2000 88 62 88 64 88 68 88 63 b1 b2 b3 b4 125 63 34 63 37 63 35 63 35 250 71 48 71 51 71 52 71 53 500 87 65 87 67 87 65 87 70 1000 88 68 88 70 88 69 88 68 618 biology, medicine, & natural product chemistry 14 (2), 2025: 615-619 frequency (hz) material 𝐼𝑜 (db) i (db) 𝐼𝑜 (db) i (db) 𝐼𝑜 (db) i (db) 𝐼𝑜 (db) i (db) 2000 88 63 88 59 88 61 88 64 c1 c2 c3 c4 125 63 28 63 32 63 27 63 31 250 71 49 71 53 71 51 71 49 500 87 72 87 69 87 70 87 66 1000 88 73 88 73 88 73 88 73 2000 88 65 88 62 88 64 88 57 d1 d2 d3 d4 125 63 31 63 32 63 36 63 32 250 71 51 71 50 71 48 71 50 500 87 62 87 52 87 64 87 64 1000 88 72 88 74 88 74 88 74 2000 88 63 88 62 88 60 88 63 table 4. the absorption coefficient for inside surface material. conclusions natural fibers derived from banana peels and water hyacinth fibers have the potency to absorb sound waves. the absorption coefficient is one of the requirements that indicate the ability of a material to absorb sound waves. the absorption coefficient ranges from 0 to 1. the closer the absorption coefficient value is to 1, the more sound waves are absorbed, resulting in lower sound intensity. from the sample variations, sample d has a higher absorption coefficient compared to other samples. the absorption coefficient for the outer surface of sample d is 0.51 at a frequency of 250 hz and 0.45 at a frequency of 2000 hz. besides, the absorption coefficient for the inside of sample d is 0.46 at a frequency of 250 hz and 0.45 hz at a frequency of 2000 hz. destructive interference and saturation are some of the causes of instability in absorption values. thus, sample d is the best sound wave absorbing material compared to other variations with a composition of water hyacinth that is more than a banana peel. acknowledgements: we sincerely appreciate everyone who has supported us throughout this process, from the initial preparation to presenting our results and ultimately writing this manuscript. in particular, we extend our gratitude to the harapan mulia palembang foundation and mr. trisno for their financial and moral support. we are also grateful to ms. delima for her guidance and assistance during our time in thailand. a special thanks to our dedicated teammate, essik, for always supporting us during the experiments. lastly, we express our heartfelt appreciation to our parents for their unwavering understanding and support. authors’ contributions: regita d. asyurra, avivah a. amatullah, annisa z. puteri, and mayshilla a. putri zevly conducted the experiment, collected data, and wrote the manuscript. widya sari and angga puja asiandu designed the experiment, guided, and monitorized the research, analyzed the data, wrote, and give an agreement for the final manuscript. competing interests: the authors declare that there are no competing interests. references astatika, i. m., & dwijaya, i. g. k. (2016). karakterstik serapan suara komposit polyester berpenguat serat tapis kelapa. dinamika teknik mesin, 6(1). https://fisika.ukitoraja.ac.id/wpcontent/uploads/2022/04/10.pdf eryani, y. m., wibowo, c. a., & saftarina, f. (2017). faktor risiko terjadinya gangguan pendengaran akibat bising. https://api.semanticscholar.org/corpusid:194855755 fauzana, n. a., sastrawibawa, s., safitri, r., & abun. (2012). potensi kulit pisang (first). universitas padjajaran press. harfi, r., achmad, g. a., firdausi, m., sumiyanto, s., & sugeng, u. m. (2023). variasi volume eceng gondok serat komposit dalam peredam suara. sainstech: jurnal penelitian dan pengkajian sains dan teknologi, 33(1), 86–94. https://doi.org/10.37277/stch.v33i1.1657 kementerian kesehatan ri. (1987). kebisingan yang berhubungan dengan kesehatan. khan, d., & burdzik, r. 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(2018). analisis kebisingan di kawasan industri pt. semen indonesia (persero) tbk kabupaten tuban. swara bhumi e-journal pendidikan geografi fis unesa, 5(6). https://jurnalmahasiswa.unesa.ac.id/index.php/swarabhumi/article/view/25912 who. (2024). how much does environmental noise affect our health? who updates methods to assess health risks. who/europe press office. https://www.who.int/europe/newsroom/04-08-2024-how-much-does-environmental-noise-affectour-health--who-updates-methods-to-assess-health-risks this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 843-856 | doi: 10.14421/biomedich.2025.142.843-856 issn 2540-9328 (online) dft study and admet investigation of bioactive 2-phenoxyquinolines oluwafemi s. aina1,2*, mujeeb o. rofiu1, luqman a. adams1, oluwole b. familoni1 1department of chemistry, university of lagos, lagos, nigeria. 2department of biological sciences, trinity university, lagos, nigeria. corresponding author* oluwafemiaina100@gmail.com manuscript received: 26 august, 2025. revision accepted: 14 october, 2025. published: 20 october, 2025. abstract pharmacologically active quinoline compounds show interesting functionalities. however, there is still a dearth of information on the potential wide applications of the quinoline scaffolds especially when the substituents on the rings or functionalities are modified. in this study 2-phenoxyquinoline-3-carbaldehyde derivatives were synthesized and investigated using dft to determine the molecular parameters, electronic and chemical reactivity descriptors including their possible application as drug candidates. geometry optimisations were carried out using becke three-parameter hybrid functional combined with lee-yang-parr correlation functionals and augmented basis set with added sets of diffuse sand p-functions: bl3yp /6-311++g(d,p). drug-likeness scores and bioactivity prediction were carried out using the webserver programs admet sar 2. our results showed slightly lower energy values in water than in ethanol depicting high reactiveness of the 2-phenoxyquinolines in water especially compounds with fluoroand methoxy substituted having the lowest egap of 3.79 ev and 3.381 ev respectively. the methoxy substitution confers higher susceptibility to electron transfer and chemical reactive in aqeous medium. the ionization potential energies were lower in water and ethanol when compared to the other gas states calculated values. the mep surfaces of all the compounds investigated reveals the ether oxygen atoms and carbonyl groups as necessities for effective antioxidant properties. it is noteworthy that five of the compounds investigated have high activity values above 3.0 octanol– water partition coefficient value indicating an excellent inhibitory activity and strong chemical reactivity. good oral bioavailability was shown by the compounds depicting their excellent activity as kinase inhibitors, nuclear receptor compounds and enzyme inhibitors. it is noteworthy that cytochromes cyp2c19, cyp2c29, cyp2d6 and cyp3a4 are not inhibited and therefore do not pose potential adverse drug reactions or toxicity. interestingly, only one compound showed one violation. consequently, such scaffolds are potential drug candidates. keywords: 2-phenoxyquinolines; drug-likeness; cytochrome substrate; structural activity relationship. introduction quinoline an aromatic organic compound is prevalent in various pharmacologically active synthetic and natural compounds. it is characterized by a double-ring structure containing a benzene ring fused to pyridine at two adjacent carbon atoms (jain et al., 2016) and have been found highly useful as antimalarial, anticancer, antiinflammatory agents, etc. (dib et al., 2021) leading to its attraction of great interest in the scientific community. researchers have synthesized hybrid quinoline scaffolds using different procedures (figure 1a). conventional chemotherapy kills most tumour cells and it is believed that cells left behind might recur thereby becoming stem cells which are likely to share similar properties of parent cells, such as resistance to drugs and toxicity via atp-binding cassette (abc) transporter expressions (suzuki et al., 1997). a series of quinoline derivatives has been reported to show mdrreversing activities in k562 cancer cells that are resistant to doxorubicin (an anthracycline multi-cancer therapy) which has been identified as a novel, orally active, quinoline-derivative inhibitor of abcb1 ⁄ p-gp (kouznetsov et al., 2016). drugs and candidates such as lenvatinib, cabozantinib and dofequidar are anticancer drugs based on the c-4 and c-5 substituted aryloxyquinoline and alkoxyquinoline derivatives respectively have also been found to contain high efficacies as shown in figure 1b (suzuki et al., 1997; katayama et al., 2009). https://doi.org/10.14421/biomedich.2025.142.843-856 844 biology, medicine, & natural product chemistry 14 (2), 2025: 843-856 figure 1a. some applications of 2-phenoxyquinoline derivatives. figure 1b. anticancer drugs with the alkyl/aryloxyquinoline moiety. methodology different novel methods have resulted to synthesis of this bioactive moiety for more than five decades and these methodologies have all been modified with eco-friendly transition metal mediated, traditional methods, microwave or ultrasonic irradiation, oxidation of the corresponding alcohols or greener protocols (sharma et al., 2018; ramírez et al., 2022). amongst the contemporary methodologies are the conventional methods such as condensation of amines and carbonyl compounds with numerous substrate scope (kouznetsov et al., 2005; iwai & sawamura, 2015). synthesis of precursors and target compounds vilsmeier-haack reaction of acetanilides, one of the practically important routes to synthesis of 2chloroquinoline-3-carbaldehydes, involves the processes of chlorination, formylation and cyclization of acetanilides by the action of the vilsmeier ́s reagent (dmf/pocl3) to afford 2-chloroquinoline-3carbaldehydes as shown in scheme 1 (meth-cohn et al., 1981). the adducts obtained from vismeier reaction could undergo substitution reaction at positions 2 and 3 where the active chloro and the aldehyde groups could lead to numerous functionalization under appropriate reagents (abdel-wahab & khidre, 2013). scheme 1. synthesis of 2-chloroquinoline-3-carbaldehyde and its phenoxylated adducts as precursors (cf: bakthadoss & aina et al, 2024). aina et al. – dft study and admet investigation of bioactive 2-phenoxyquinolines 845 scheme 2. synthesis of acetoxylated and benzoylated 2-phenoxychloroquinoline-3-carbaldehydes via nitrogen-directed palladium-catalysed sp2 c-h activation strategy (cf: bakthadoss & aina et al, 2024). interest in the use of these bioactive organic molecules possessing high degree of delocalization has increased in the recent time due to presence of weak van der waal’s and hydrogen bonds resulting to better enzyme-compound interactions (kumar et al., 2017). to this end, our study therefore presents density function theory (dft) with detailed chemical reactivities of eight (8) 2-phenoxyquinoline derivatives shown in figure 2 synthesized using the methods presented in scheme 2 to investigate their possible application as drug candidates with emphasis on their toxicity. quantum mechanical calculations were also performed to examine their molecular structures, parameters, molecular electronic and chemical reactivity descriptors. computational methodology the coordinates of the structures of synthesised compounds were generated from avogadro 14.0 while molecular optimization calculations were done using the orca 4.2.1 quantum chemical software. (neese, 2017). the geometry optimisation was carried out using the becke three-parameter hybrid functional combined with leeyang-parr correlation functionals and augmented basis set with added sets of diffuse sand p-functions: bl3yp /6-311++g(d,p) (ennaba, 2017; kodikara et al., 2018; al hasanat, 2017). the choice of selected functional and the basis set were based on its reliability in published reports (haroon et al., 2019). a single-point gasphase, waterphase and ethanol-phase optimizations were carried out using time-dependent density functional theory (tddft) 13 keyword and solving for 50 excited states. all calculations in solvent was performed using the cpcm keyword together with the smd solvent model (fleming et al., 2011; ho & ertem, 2016). drug-likeness scores and bioactivity prediction were carried out using the webserver programs such as admet sar 2 (available at http://lmmd.ecust.edu.cn/admetsar2) and molinspiration -cheminformatics (available at https://www.molinspiration.com/cgi-bin/properties) (voiculescu et al., 2022). result and discussion chemical reactivity descriptors dft tools used in this study give a concise structurechemical reactivity report of 2-phenoxyquinoline-3carbaldehyde derivatives (figure 2) to provide an understanding of the chemical reactivity properties whose descriptors are presented in table 1. frontier molecular orbitals (fmos) energies were used to calculate these chemical reactivity descriptors obtained from conceptual dft. the energy gap, egap is defined as the energy difference between el (the lowest unoccupied molecular orbital, lumo) and eh (energy of the highest occupied molecular orbital, homo) which determines the chemical reactivity and kinetic stability of the organic molecule. the lower the energy gap, the more reactive a molecule is, indicating the compound is more susceptible to reacting with proteins or enzymes (miar et al., 2021). the results of the energy gaps (table 1 and figure 3) showed slightly lower energy values in water than in ethanol. since, the preferred medium of drug delivery is water, this depicts the highest reactiveness of the 2 846 biology, medicine, & natural product chemistry 14 (2), 2025: 843-856 phenoxyquinolines in water, with 7 and 3 having the lowest egap of 3.381 ev and 3.79 ev respectively. the concentration of the molecules is proportionately the inverse of the energy gap. that is, the lower the energy gap the higher the concentration of the molecule in water solution and therefore it shows a greater activity of the 2phenoxyquinolines in aqueous medium. the difference in the energy gap may be mostly attributed to the differences in their functional groups (table s1). this difference can be attributed to the acetoxyl and benzoyl substituents at para position of the phenoxy group. in compounds 3, 7 and 8, the main chromone skeleton (quinoline) has both electron withdrawing and electron donating substituents attached to it. these substituents may increase or decrease the electron density in the quinoline ring thereby activating or deactivating the ring. the lower energy gap for 3 and 8 may be attributed to f which shows a negative inductive effect due to its high electronegativity and withdraws the electron density from the quinoline ring, so it deactivating the group. the more electrophilic benzyl group in the chromone backbone allows electronic interactions that are pivotal to the quinoline reactivity. also, the methoxyl groups on the chromone structure of compound 7 which has the lowest energy gap (3.381 ev) activate the benzene ring towards electrophilic substitution and this is also favourable for the radical scavenging activity of the quinoline ring. the feasibility of a molecular system to exchange electron density or an electron escaping from such system to the surrounding environment at the ground state can be expressed as the energy changes of the molecular system with respect to the number of electrons at a fixed external potential, µ (domingo et al., 2016). figure 2. structures of synthesized phenoxyquinoline compounds investigated. the calculated electronic chemical potential of the molecules shows no significant difference for the 2phenoxyuinolines. in water, the chemical potential values range from -4.688 ev (8) to -4.301 ev (7) while in ethanol, it ranges from -4.681ev (8) to -4.294 ev (7) with both media having the ascending and descending order of 7, 3, 5, 8, 1, 2, 6 and 4 hardness and softness, η and s respectively define the extent of resistance and susceptibility to polarization or transfer of electron cloud of atoms, ions or molecules in response to a chemical process (islam & ghosh, 2011). these periodic properties calculated as global softness, s are the inverse of global hardness, η. compound 7 shows the most minor resistance or highest susceptibility to charge or electron transfer with η and s values (1.691 ev and 0.592 ev) in water respectively. conversely, compound 4 has η value of 2.106 ev and s value of 0.475 ev in aqueous medium. therefore, of all the 2-phenoxyuinolines compounds, 7 is the most susceptible to electron transfer and the most chemically reactive in aqeous medium while compound 4 poses the highest resistance to the transfer of electron density in the medium and hence, the least reactive. electronegativity, χ, represents electron attraction power of an atom in a molecule towards itself or a measure of molecular electron distribution.23 the highly probable electron acceptability of these molecules was indicated by high electronegativity values. compounds 8 and 6 had the highest χ value (4.688 ev and 4.681 ev) in aqueous medium while 7 had the lowest (4.301 ev). a possible explanation for high electronegativity for compound 8 and 6 is the presence of fluoro group (a deactivating group) with high inductive effect. compound 7, on the other hand has an activating group that is mostly resonance donors (+m), that is, aina et al. – dft study and admet investigation of bioactive 2-phenoxyquinolines 847 methoxyl group. although it also inductively withdraws (–i) electrons, which is a deactivating effect, the resonance (or mesomeric) effect is stronger, hence an even electron distribution which accounts for the reduced electronegativity. the information about the physico-chemical properties and chemical reactivity phenomena of a molecule can also be revealed by a measure of the stabilization energy of the molecule when it accepts an arbitrary number of electrons approaching from the surroundings. this concept is termed electrophilicity index (ω) (tandon et al., 2020). this approach can be explained in two ways; the global electro-accepting power ω+ and electro-donating power ω−. the electrondonating power is a measure of the nucleophilicity of the system and it involves the destabilizing change in energy required by a system to attain zero chemical potential with accompanied a decrease in the number of electrons of the system. on the other hand, the electronaccepting power ω+ of a molecular system, can be defined base on the stabilizing energy change of such system when it is saturated with maximum number of electrons (gazquez et al., 2014). the net electrophilicty δω±, describes of intermolecular reactivity (frau & glossman-mitnik, 2017). the results from the calculations show that the 2phenoxyuinolines have an average approximate electron donating power, ω− of 9 ev. this approximate destabilizing energy is higher than the average approximate electron accepting power, ω+ of about 6 ev except for compound 4 which shows a very lower electrophilic index or stabilizing energy change accompanying electron gain with ω+ value of approximately 1 ev. this suggests that the 2phenoxyuinolines are strongly nucleophilic and electrophilic but more nucleophilic in reactivity than being electrophilic. the ionization potential, i and electron affinity values also show the relative tendencies of an atoms or molecule to gain or lose electrons in reaction and useful in the calculation of heats of various reactions and physical changes (smalo et al., 2010). reactivity is inversely proportional to ionization potential so lower ionization potential would depicts a higher the chemical reactivity. table 1. chemical reactivity descriptors for the synthesised 2-phenoxyquinolines. cpd medium el (ev) eh (ev) egap (ev) η s µ ꭓ ω ω¯ ω⁺ ∆ω± µd i 1 gas -2.597 -6.713 4.116 2.058 0.486 -4.655 4.655 1.316 9.345 6.389 15.734 2.597 6.713 water -2.591 -6.625 4.034 2.017 0.496 -4.608 4.608 1.316 9.257 6.424 15.680 2.591 6.625 ethanol -2.565 -6.614 4.049 2.025 0.494 -4.590 4.590 1.301 9.215 6.321 15.536 2.565 6.614 2 gas -2.584 -6.769 4.185 2.093 0.478 -4.677 4.677 1.306 9.382 6.298 15.680 2.584 6.769 water -2.599 -6.659 4.060 2.030 0.493 -4.629 4.629 1.319 9.298 6.434 15.732 2.599 6.659 ethanol -2.573 -6.651 4.078 2.039 0.490 -4.612 4.612 1.304 9.259 6.330 15.589 2.573 6.651 3 gas -2.750 -6.678 3.928 1.964 0.509 -4.714 4.714 1.414 9.507 7.092 16.598 2.750 6.678 water -2.670 -6.577 3.907 1.954 0.512 -4.624 4.624 1.368 9.313 6.808 16.120 2.670 6.577 ethanol -2.649 -6.554 3.905 1.953 0.512 -4.602 4.602 1.356 9.265 6.731 15.996 2.649 6.554 4 gas -2.536 -6.076 3.54 1.77 0.565 -4.306 4.306 1.309 8.695 6.612 15.306 2.536 6.076 water -2.416 -6.628 4.212 2.106 0.475 -4.522 4.522 1.214 9.055 5.714 14.770 2.416 6.628 ethanol -2.388 -6.599 4.211 2.1055 0.475 -4.494 4.494 1.199 8.996 5.623 14.619 2.388 6.599 5 gas -2.373 -6.499 4.126 2.063 0.485 -4.436 4.436 1.192 8.884 5.618 14.502 2.373 6.499 water -2.564 -6.514 3.950 1.975 0.506 -4.539 4.539 1.304 9.122 6.386 15.508 2.564 6.514 ethanol -2.528 -6.496 3.968 1.984 0.504 -4.512 4.512 1.283 9.061 6.245 15.306 2.528 6.496 6 gas -2.633 -6.801 4.168 2.084 0.480 -4.717 4.717 1.335 9.470 6.481 15.951 2.633 6.801 water -2.642 -6.72 4.078 2.039 0.490 -4.681 4.681 1.343291 9.406582 6.575077 15.98166 2.642 6.72 ethanol -2.615 -6.712 4.097 2.049 0.488 -4.664 4.664 1.327 9.366 6.465 15.831 2.615 6.712 7 gas -2.694 -6.140 3.446 1.723 0.580 -4.417 4.417 1.415 8.971 7.337 16.308 2.694 6.140 water -2.610 -5.991 3.381 1.691 0.592 -4.301 4.301 1.368 8.726 7.062 15.788 2.610 5.991 ethanol -2.599 -5.988 3.389 1.695 0.590 -4.294 4.294 1.360 8.708 7.009 15.717 2.599 5.988 8 gas -2.771 -6.810 4.039 2.020 0.495 -4.791 4.791 1.420 9.651 7.078 16.729 2.771 6.810 water -2.696 -6.680 3.984 1.992 0.502 -4.688 4.688 1.379 9.438 6.843 16.281 2.696 6.680 ethanol -2.676 -6.686 4.010 2.005 0.499 -4.681 4.681 1.366 9.418 6.749 16.167 2.676 6.686 a units for the chemical reactivity descriptors are in electron volts (ev) el: lumo energy, eh: homo energy, egap: homo-lumo energy gap, η: chemical hardness, s: chemical softness, μ: chemical potential, χ: electronegativity, ω: electrophilicity index, ω-: electron donating power, ω+: electron accepting power, δω±: net electrophilicity, μd: dipole moment, i: ionization potential, g: gas phase, w: water and eth: ethanol. in water and ethanol, the ionization potential energies were lower when compared to the other gas states calculated values. this emphasizes the fact that the chemical reactivity of the 2-phenoxyquinolines is well improved in water and ethanol media. since, the desired medium (water) gives favourable intermolecular interactions via hydrogen bonding, therefore, it reflects in lower ionization potential. compound 7 has the lowest i value (5.991 ev) in water compared to other 2phenoxyquinolines. 848 biology, medicine, & natural product chemistry 14 (2), 2025: 843-856 figure 3. comparison of the energy gaps in gas-phase, water and ethanol. atomic charge and electrostatic potential molecular electrical charge is the driving force of electrostatic interactions characterized by electron density playing an important role in chemical reactions and physicochemical properties (wang & truhlar, 2014). descriptors that are charge based are often employed as chemical reactivity indices or as a measure of weak intermolecular interactions. the partial charge or electron density distribution in a molecule is factor to determination of its reactivity description. the mulliken atomic charges mac, may be linked to other chemical parameters such as dipole moment, electric potentials, chemical shifts, electronic structure, molecular polarizability and others (sumrra et al., 2021). a uniform distribution of charge was found for all molecules. for 3, 5, and 7, which had the lowest energy gap in aqeous medium and were the most reactive, they had the highest negatively charged atom (-2.518 a.u, −2.609 a.u. and 2.021 a.u.) which is the carbon atom of the imine group at c9 and c14 positions attached to pyridine nitrogen, respectively (figure 2). this charge is significant for the bioactivity of the molecules due to the role the f, –och3 groups play in scavenging radicals. molecular electrostatic potential surface molecular electrostatic potential (mep) surfaces enhance determination of the reactivity of chemical compounds and it is related to electron density and is a useful chemical descriptor that gives information about sites of nucleophilic attacks, electrophilic attacks and hydrogen bonding interactions (demircioglu et al., 2019). the blue region indicates the positive potential of the molecule as the area where there is proton repulsion (that is electrophilic attack) while the red region has negative potential and represents proton attraction (nucleophilic attack) (sheikhi et al., 2016). compound 3 compound 5 compound 7 figure 4. representative optimised structures of synthesized 2-phenoxyquinolines. compound 3 compound 5 compound 7 figure 5. representative atomic charge distribution in synthesized 2-phenoxyquinolines. 0 1 2 3 4 5 1 2 3 4 5 6 7 8 eg ap ( ev ) gas water ethanol aina et al. – dft study and admet investigation of bioactive 2-phenoxyquinolines 849 all the 2-phenoxyquinolines simulated in this study have mep surfaces with an overall region of positive potential around protons of the c8, c10, and c6 while areas of negative potential are around delocalized pielectrons, the ether oxygen atoms and that of the carbonyl groups which verifies the necessity of the ether and carbonyl groups for effective antioxidant properties of the quinolines. the carbonyl of the aldehyde and acetate groups of the 2-phenoxyquinolines contributes effectively to the reactive oxygen and hydrogen species’ scavenging abilities of the compounds as they offer stability. it is a known phenomenon that the regions of negative potential in the 2-phenoxyquinolines compounds will have an affinity for the positively charged side groups of amino acids (arginine, histidine and lysine) and regions of positive potential of the will bind to the negatively charged side groups of amino acids such as glutamic acid and aspartic acid (shinde, 2021). uv–vis spectroscopy uv–visible spectroscopy has emerged as the most favoured technique to evaluate conjugated organic compounds such as quinoline and its derivatives as the ring in these bioactive molecules have the ability to absorb uv light. few coloured derivatives also absorb in the visible region thus enabling uv–visible spectroscopy one of the appropriate techniques their bioactivity (ramešová et al., 2012). total antioxidant activity is measured by taking absorbance against water to calculate the percentage of inhibition. the presence of the carbonyl groups of the 2-phenoxyquinolines also contributes to an increase in the absorbance. absorption peak is associated with an electron transition from the ground state to its excited state with corresponding excitation energy and single-point (sp) energy calculation provides the homo and lumo energies as shown in table 1. it has been reported that ethanol and water or a mixture of these solvents are suitable since the oxygen of the carbonyl groups easily bond with the hydrogen of the water molecules hence their solubility in the solvents are expected (blume et al., 1988). we chose water as the solvent for the uv–vis calculation since this is preferred medium for drug delivery. the maximum absorption wavelength (λmax) of the 2-phenoxyquinolines in water and the excitation energies show that maximum wavelength of absorption is inversely proportional to the excitation energy as shown in table 2, figure 8. table 2. uv–visible spectroscopy. compound orbitals extinction coeff oscil strength 1st excitation e (ev) absorption 1 79 -80 0.548 0.741 3.3711 367.8 2 87 88 0.729 0.854 3.159 392.5 3 91 92 0.89 0.943 2.865 432.8 4 83 84 0.223 0.472 3.368 368.2 5 95 96 0.791 0.889 3.186 389.2 6 94-95 0.059 0.243 3.375 367.4 7 106-107 0.94 0.969 2.785 445.2 8 102 103 0.317 0.563 3.287 377.2 compound 3 compound 5 compound 7 figure 6. representative homo-lumo of synthesized 2-phenoxyquinoline-3-carbaldehydes. 850 biology, medicine, & natural product chemistry 14 (2), 2025: 843-856 compound 3 compound 5 compound 7 figure 7. representative molecular electrostatic potential (mep) surface of synthesized 2-phenoxyquinoline-3-carbaldehydes. figure 8. relationship between excitation energy and maximum absorption wavelength. considering the fact that the promotion of an electron from the ground state to the excited state at longer wavelengths require low energy, it is hence apparent the compound 7 with the lowest excitation energy and the highest wavelength, implies most activity while 6 with highest excitation energies has lowest activity. from the time-dependent density functional theory (td-dft) calculations, electronic transitions information including which transition orbital was involved, oscillator strength, extinction coefficient, excitation state energies and the electronic states of the transitions support the bioactivity of compound 7. molar extinction coefficient, also known as molar absorptivity, is an intrinsic property of the molecule which is dependent on the atomic, chemical, and structural composition. it determines how strongly a species absorbs or reflects radiation or light at a particular wavelength (ozdemir & sadoglu, 2018). compounds 3, 5 and 7 have high molar absorptivity (>0.5 ev), hence, they strongly attenuate light while 4 and 6 have extinction coefficient less than 0.5. oscillator strength is a dimensionless quantity that expresses the probability of absorption or emission transitions between energy levels of an atom or molecule, that is, a measure of excitation probability (nizar et al., 2021). the high oscillator strengths which indicate the high probability of excitation gave the strongest transitions (which are presented in table 1) compared to other transitions. molecular and physicochemical properties adsorption, distribution, metabolism and excretion (adme) protocols play crucial roles in the development of new drug molecules as they outline the dose amounts, intervals and overall safety of oral drug administration (mcginnity et al., 2007) making the determination of physicochemical properties feasible (nithiyanantham et al., 2015) thus providing desirable balance of physicochemical properties for safe drug administration (belaidi et al., 2016). the logarithm of octanol–water partition coefficient (log pow) is a broadly acknowledged measure of lipophilicity and is determined for numerous compounds (poole & poole, 2003). pow is the concentration ratio of the compound distributed between n-octanol and water. (ribeiro et al., 2011; jalan et al., 2010). the partition coefficient is an exceedingly important physicochemical parameter in medicinal chemistry, beneficial in pharmacology and toxicology, as long-established by a large numeral of literature data (balaz, 2009; sangster, 1997). 7 2 3 4 5 6 1 8 2,8 3 3,2 3,4 3,6 3,8 4 315 335 355 375 395 415 435 ex ci ta ti o n e n er gy absorption aina et al. – dft study and admet investigation of bioactive 2-phenoxyquinolines 851 table 3. physicochemical parameters for 2-phenoxyquinoline-3-carbaldehydes. cp milog a tpsa b natoms c mw d non e nohnh f nviolat g nrotb h vol i 1 3.06 65.5 23 307.31 5 0 0 5 267.79 2 3.46 65.5 24 321.33 5 0 0 5 284.35 3 2.65 65.5 25 339.32 5 0 0 5 289.28 4 3.49 65.5 24 321.33 5 0 0 5 284.35 5 5.59 56.27 28 367.4 4 0 1 5 330.21 6 2.37 91.81 27 365.34 7 0 0 7 312.31 7 3.8 101.04 30 409.39 8 0 0 8 354.42 8 1.94 91.81 29 397.36 7 0 0 7 333.81 a. octanol–water partition coefficient as reported in molinspiration. b. total polar surface area in å2. c. total number of atoms. d. molecular weight in g mol—1. e. number of hydrogen bond acceptors. f. number of hydrogen bond donors. g. number of violations to lipinski’s rule of 5. h. number of rotatable bonds. i. volume in å3. active organic compounds have log p value of 1.6 upward (buddensiek et al., 2021) implying a reduced inhibitory activity at values below. it is noteworthy to see compounds 1, 2, 4, and 7 have high activity values above 3.0 while compound 5 is well above 5.0 octanol–water partition coefficient value indicating an excellent inhibitory activity coupled with strong chemical reactivity. in the work of vlahovic et al. (2017), hydrophobic substances were reported to have a high octanol–water partition coefficient distributed primarily in the lipid bilayers of cells while hydrophilic substances on the other hand have a low octanol–water partition coefficient and are distributed in blood plasma. they also showed the linear relationship of the octanol–water partition coefficient with log p which explains the high milogp values obtained for compound 5 with a value of 5.59 as shown in table 3. this shows the synthesised 2phenoxyquinolines to be hydrophobic hence would easily distribute in the lipid bilayers. compound 8 with the least value milogp value of 1.94 is more hydrophilic than others and will be more readily distributed in blood plasma. drug likeness and bioactivity score medicinal chemists use drug-likeness as the essential guide via hit-to-lead optimization for drug discovery (sagaama & issaoui, 2020, maldonado et al., 2021). pharmacokinetic properties detailed in adme parameters are also strongly affected by physicochemical properties (kumar et al., 2017; zheng et al., 2013). since drug-like molecules must be soluble in water as well as fat, it is therefore necessary that an orally administered drug passes through the intestinal lining into the bloodstream and is carried to the target immediately after ingestion, penetrating the lipid-based cell membrane to get into the target cell (kumar et al., 2017). increased hydrogen bonds leads to increased aqueous solubility making the molecule less hydrophobic since it enables the molecule to permeate into and through the lipid bilayer membrane (di & kerns, 2015). as reported by veber and co-workers, drug candidates will have a high probability of good oral bioavailability if they meet criteria such as: total polar surface area (tpsa) ≤ 140 å2 and if the number of rotatable bonds (nrotb) ≤ 10 (veber et al., 2002). amazingly, all 2phenoxyquinolinequinoline-3-carbaldehydes investigated in this study fulfil all these criteria. while the monoacetoxylated 1-4 and benzoylated 5 2phenoxyquinolines have excellent total polar surface area (65-74 a2), the di-acetoxylated groups have much higher values ranging from 91-101 a2) as shown in table 3. figure 9. relationship between volume and molecular weight of synthesised 2-phenoxyquinolines. 1 2 3 4 5 6 7 8 260 270 280 290 300 310 320 330 340 350 360 307,31 321,33 339,32 321,33 367,4 365,34 409,39 397,36 v o lu m e molecular weight, mw (g/mol) y = 0.7901x + 27.684 r2 = 0.9527 852 biology, medicine, & natural product chemistry 14 (2), 2025: 843-856 it is noteworthy to express the super permeability of compound 5 (with a tpsa value of 56.27 a2) in lipid membranes. the lipinski’s rule of 5 states that for a drug molecule to be orally active it should obey important rules such as number of hydrogen bond donors (nohnh) ≤5, number of hydrogen bond acceptors (non) ≤ 10, molecular weight (mw) ≤ 500, partition coefficient (milogp) ≤ 5 and number of violations (nviolations) ≤ 2 (maurya et al., 2020). interestingly, all the 2phenoxyquinoline-3-carbaldehydes comply with the lipinski rule of 5 (table 3), testifying to their oral bioavailabilities as potential drug molecules. in compliance, the molecular weight of the compounds is directly proportional to the volume of the compounds (table 3, fig. 9) which confirms their correlation with the pearson correlation co-efficient (r) of 0.97 and r2 value of 0.94 which states that for an average organic molecule, if the bioactivity score is more than 0 then it is active, a score ranging from -0.50 to 0.0 indicates moderately active and a score of less than -0.50 denotes inactivity (figure 10) (alodeani et al., 2015; akintemi et al., 2022). to further confirm the correlation by the pearson correlation coefficient, the bioavailability of the investigated compounds as inhibitors/ substrates were carried out using profile depicting bioactivity score as presented in figure 10. it is obvious that compound 5 exhibit excellent activity as kinase inhibitors, nuclear receptor compounds and enzyme inhibitors while 3 shows good activity as enzyme inhibitor and nuclear receptor compound. compounds 3 and 4 exhibit much lower protease inhibitory properties like 2 with moderate kinase inhibitory properties. as shown in figure 10, all the compounds exhibit good to moderate activity as enzyme inhibitors (ei), g-protein-coupled receptors (gpcr), ion channel modulators (icm), nuclear receptor compounds (nrl) and kinase inhibitors (ki). it is observed that all the compounds only exhibit moderate activity as protease inhibitors (pi). figure 10. profile depicting bioactivity score of compounds 1-8. admet/sar investigation of 2-phenoxyquinoline derivatives absorption, distribution, metabolism, excretion (admet) was analysed based on data generated from adme sar 2 web server. the values obtained are from latest drug-likeness capability tools in drug discovery. most of the parameters correlate positively and are presented in table 5 thus confirming their profile depicting bioactivity score in figure 10. it is noteworthy that cytochromes cyp2c19, cyp2c29, cyp2d6, and cyp3a4 which are significant enzymes responsible for metabolizing commonly used drugs, are not inhibited, hence no effect on plasma levels in vivo which can potentially lead to adverse drug reactions or toxicity while cytochromes cyp2c9 and cyp2d6 act as substrates which metabolise pharmacologically inactive drugs to active form for easy clearance from the body the ames test is a biological assay to assess the mutagenic potential of chemical compounds utilizing bacteria to test whether a given chemical can cause mutations in the dna of the test organism. a negative test indicates that the chemical is non-mutagenic and therefore is not a carcinogen, because cancer is often linked to mutation (czeczot et al., 1990). suffice to say that compounds 7 and 8 gave negative ames test results probably due to the presence of the fluorine and methoxy oxygen atoms at position 6 of the quinoline ring which withdraws electrons from the ring via inductive effects. although the methoxy group is electron-donating by resonant effect, that does not apply here. compounds 1 with fewest substituents shows negative androgen receptor binding, situation where a type of nuclear receptor activated by binding to any of the androgenic hormones, including testosterone and dihydrotestosterone in the cytoplasm and is translocated into the nucleus -0,4 -0,3 -0,2 -0,1 0 0,1 0,2 0,3 1 2 3 4 5 6 7 8 gpcr icm ki nrl pi ei aina et al. – dft study and admet investigation of bioactive 2-phenoxyquinolines 853 thereby increasing interfacial stability when compared with the other steroid receptors (shaffer et al., 2004). caco-2 is an immortalized cells line of human colorectal adenocarcinoma cell primarily used as a model for the measurement of the intestinal epithelial barrier functions (lea, 2015). this returns negative values for compounds 1 and 6. this could be attributed to the fact that, despite being monoand di-acetoxylated, they are devoid of any other substituted elements. the only benzoylated compound 5 surprisingly gave negative cyp14 substrate value. this cytochrome is an hmg-coa reductase, a factor in the treatment of hiv infection by lowering ldl cholesterol and reducing the risk of cardiovascular disease such as myocardial infarction and stroke. cyp substrates are drugs usually metabolized by cytochrome enzymes which require metabolism into an inactive form for clearance from the body (galetin et al., 2005). it is obvious that the an extra aromatic ring of compound 5 contributed to this effect. ppar gamma is a gene that encodes a member of the peroxisome proliferator-activated receptor (ppar) subfamily of nuclear receptors which form heterodimers with retinoid x receptors (rxrs) to regulate transcription of various genes. activation of this adipocyte predominant transcription factor regulates glucose and lipid homeostasis playing significant role in the regulation of gene expression of multiple diseases including obesity, diabetes and cancer and highlights the gene isolation transformation role (janani & kumari, 2015). compound 2 has extra methyl groups on position 6 of the 2-phenoxy ring and on the ester group of the 3methylacetate of the quinoline ring. these special positions of the methyl group could be reason for the negative values of the compounds. the highest plasma protein binding value was exhibited by only benzoylated compound 5. the presence of extra resonance stabilization could have contributed to this higher value. plasma protein binding refers to the degree to which medications attach to proteins within the blood measuring drug's efficiency as it helps determine the degree to which it binds. the less bound a drug is, the more efficiently it can traverse or diffuse through cell membranes. plasma proteins, by virtue of their high concentration, control the free drug concentration in plasma and in compartments in equilibrium with plasma, effectively attenuating drug potency in vivo (trainor, 2007). compound 5 has the highest tetrahymena pyriformis value of 1.92. tetrahymena species found in the original t. pyriformis group provide an invaluable resource for comparative genomic analyses, as evolutionary dna sequence conservation is a valuable predictor of biological function under natural selection (sauvant et al., 1999). thyroid hormone receptors regulate gene expression by binding to hormone response elements (hres) in dna as monomers, heterodimers with other nuclear receptors, or homodimers. dimerizing with different nuclear receptors leads to the regulation of different genes. they are essential for normal development, differentiation and metabolic balance (zhang & lazar, 2000). compound 5 with the most pi-electrons possessed in its four aromatic rings in addition to the two carbonyl functions bind with thyroid receptors. drug oral bioavailability is the fractional extent of the drug dosage that finally reaches the therapeutic site of action. the most relevant measure of lipophilicity with regard to oral absorption by passive diffusion is probably the distribution coefficient (log d) at ph 6.5, which is the ph of the small intestine, where absorption mostly takes place (yoshida & topliss, 2000) (yoshida & topliss, 2000). only compound 5 show positive results for human oral bioavailability, hence is predicted to reach the site of action before being eliminated compared to others. mitochondrial toxicity is a condition in which the mitochondria of a body's cells become damaged or decline significantly in number; it occurs as a side effect of certain antiretroviral drugs used to treat human immunodeficiency virus (hiv). rapid increase in reported toxic effects of drugs and pollutants on mitochondria have been documented detailing many genetic differences leading to mitochondrial diseases caused by factors such as as diet, exercise, age, and nonchemical stressors (meyer et al., 2018). only compound 7 shows mitochondrial toxicity from this investigation which could be attributed to the presence of the methoxy group at position 6. table 4: toxicity targets of compounds 1 – 8. targets compounds 1 2 3 4 5 6 7 8 hepatotoxicity inactive inactive active inactive inactive inactive inactive active carcinogenicity active inactive inactive inactive active inactive inactive inactive cytotoxicity inactive inactive inactive inactive inactive inactive inactive inactive the respiratory system is a target of toxic effects and a major route of absorption of inhaled gases and atmospheric particles. lung toxicosis depends on the dose of toxicant delivered to lung tissues and toxicants’ acute and chronic effects. effects can be described in terms of tissue damage and lesions and in terms of deficits in respiratory system function. cellular responses to toxicants lead to acute effects such as inflammation or chronic effects including fibrosis, emphysema, and cancer (van der merwe, 2018). it is noteworthy that all 854 biology, medicine, & natural product chemistry 14 (2), 2025: 843-856 the compounds are inactive cytotoxic as shown in table 4. while compounds 3 and 8 exhibit slight hepatoxicity, compounds 1, 2, 4, 5, 6 and 7 are completely nonhepatoxic. compounds 1 and 5 also exhibit minimum carcinogenic toxicity while the rest are non-carcinogens. to this end, compounds 2, 4, 6, and 7 are potential drug candidates as antitumor agents, amidst various other pharmacological applications. conclusion bioactive quinoline compounds have shown consistent applications in drug design. from the dft study, calculated reactivity descriptors, mep, mac and optical properties support the bioactivity of compounds as revealed in their lower energy values in water relative to ethanol and gas. admet investigation revealed the extent of compliance of the compounds as potential candidates based on drug-likeness parameters, leadlikeness, cytochrome inhibition, bioavailability and other physicochemical properties. the desired medium (water) gives favourable intermolecular interactions via hydrogen bonding as reflected in their lower ionization potential especially for the 6-methoxy substituted functionality with lowest ionization potential value of 5.991 ev. generally, uniform distribution of charge was found for all compounds especially the 6-fluoro, benzoylated and 6-methoxy substituted groups which had lower energy gaps in aqeous medium and were the most reactive with respective negative values of -2.518 a.u, −2.609 a.u. and -2.021 a.u. this charge is significant for the bioactivity of the molecules for their role as radical scavengers. from mep and mac studies, regions of negative potentials in the 2-phenoxyquinolines with high affinity for the positively charged side groups of amino acids provide enablement for good binding interaction. additionally, pearson correlation co-efficient (r) of 0.97 and r2 value of 0.94 shows the 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(2013). multi-swarm chaotic particle swarm optimization for protein folding. journal of bionanoscience, 7, 643–648. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 961-968 | doi: 10.14421/biomedich.2025.142.961-968 issn 2540-9328 (online) effect of peg and cmc on papaya (carica papaya l.) extract against aedes aegypti larvae rochmadina suci bestari1*, fidhia nur rif’aini1, riandini aisyah1, listiana masyita dewi1, devi usdiana rosyidah1, retno sintowati1, hidayah karuniawati2 1department of biomedic, faculty of medicine, universitas muhammadiyah surakarta 2department of pharmacology and clinical pharmacy, faculty of pharmacy, universitas muhammadiyah surakarta jl. garuda mas, kampus 4 ums, gonilan, kartasura, sukoharjo, 57126, telp. (0271)716844, fax (0271) 724883, jawa tengah, indonesia. corresponding author* rsb156@ums.ac.id abstract dengue haemorrhagic fever (dhf) cases are still high in america and asia region, including indonesia. eradication of dhf can be done by larvacide. temephos 1%, chemical larvacide used in indonesia for decades, can cause environment degradation and allergic reaction in human. papaya stem extract as biolarvacide contains saponins, tannins, alkaloids, flavonoids, papain and essential oils which have larvicidal activity. it can be applicable in all over the world. this study was conducted to determine the better suspending agent between cmc or peg of papaya stem extract to kill aedes aegypti larvae. objective: to determine the better suspending agent to kill aedes aegypti larvae. method: this study was true experimental study in laboratory, used post-test only controlled group design. this study used samples consist of aedes aegypti instar iii-iv larvae. there were seven experimental groups, consist of positive control group, negative control group for cmc, negative control group for peg, extract concentration of 1.5% with cmc suspending agent, extract concentration of 1.5% with peg suspending agent, extract concentration of 2% with cmc suspending agent, extract concentration of 2% with peg suspending agent. each group was repeated 4 times and counted every 6 hours (6th, 12th, 18th, 24th). data analysis using the spss application ver 23. probit test used to determine the effectiveness of the larvacide, lc50 and lt50 to determine the concentration and time to kill 50% of aedes aegypti larvae. it used kruskal wallis test to compare four dependent groups. it used mann whitney test to compare the differences of each group. result: the study showed that lc50 peg was 0.033%, lc50 cmc was 0.321%, lt50 (peg+1.5%) was 20.8 hours, lt50 (peg+2%) was 30.7 hours, lt50 (cmc+1.5%) was 23.5 hours, lt50 (cmc+2%) was 40.7 hours. based on the probit test, the lc50 and lt50 results showed that papaya stem methanol extract with peg suspending agent was more effective than cmc suspending agent, it needed lower concentration and lower time to kill 50% of larvae. kruskall wallis test result p<0.05, so there was significantly difference between groups. post hoc mann-whitney test result p<0.05 between c(-) peg group with p1, p2, p3, p4 and between c(-) cmc group with p1, p2, p3, p4, so there was significantly difference between c(-) peg with each group and c(-) cmc with each group. conclusion: it can be concluded that papaya stem methanol extract with peg suspending agent was more effective than cmc suspending agent to kill aedes aegypti larvae. keywords: papaya stem extract; cmc; peg; aedes aegypti larvae; larvicide. abbreviations: cmc: carboxymethyl celulose; peg: polyethylene glycol introduction in most regions of america and asia including indonesia, dengue haemorrhagic fever (dhf), brought by aedes aegypti mosquito and caused by denv types 1-4, still becomes health problem (jatmiko et al., 2024; yang et al., 2021). indonesia is the 2nd country with the highest dengue cases out of 30 endemic countries (haryanto, 2018). there were approximately 87,501 dhf cases in indonesia (kemenkes ri, 2022). the incidence of dengue fever increased rapidly over the past 45 years with peak incidence was in children. incidence rate (ir) was high (ir=78.0) and case fatality rate (cfr) wa also high (haryanto, 2018). control of aedes aegypti mosquito is caried out using various methods including chemical, physical and biological control. chemical control used larvicide namely abate, containing temephos 1% (nurlinawati & mulyani, 2020; rather et al., 2017). however, the use of abate bring out resistance to aedes aegypti, so biological larvicide need to be developed (grisales et al., 2013; ika wahyu utami, 2017; lesmana et al., 2021; mulyatno, kris cahyo; yamanaka, atsushi; ngadino; konishi, 2012; saeung et al., 2020; sinaga et al., 2016). lord, et. al. (2021) researched larvicides from the ethanol extract of papaya stem that have a larvicidal effect on aedes aegypti. papaya stem contains saponins, tannins, alkaloids, flavonoids, steroids, papain, and manuscript received: 14 august, 2025. revision accepted: 27 october, 2025. published: 28 october, 2025. https://doi.org/10.14421/biomedich.2025.142.961-968 962 biology, medicine, & natural product chemistry 14 (2), 2025: 961-968 essential oils affect larvae killing (ayu et al., 2021; ningrum et al., 2018; rizki et al., 2017). flavonoid is a phenolic component that is usually found in stems, leaves, flowers andr fruits(purwani et al., 2024). secondary metabolites in papaya plants are obtained through extraction. the selection of the type of solvent used will affect the active components of the extracted material (kafita et al., 2024). on the previous study, it was said that the extract was not dispersed perfectly (ariesta aa; suharyo; kun s, n.d.; suharyo & susanti, 2017) and there was no study that using dispersing agent cmc and peg. so we conduct the research by using cmc and peg to determine the better suspending agent to kill aedes aegypti larvae (ferdiansyah, 2016; rowe, r.c., sheskey, p.j., quinn, 2009; wibowo et al., 2017). materials and methods research design and implementation procedures this research is an experimental laboratory design with a post-test only controlled group design. the study was conducted for 3 months starting from september 2022 at the pharmacology laboratory of the faculty of medicine, muhammadiyah university, surakarta for extraction. in the laboratory of parasitology, faculty of medicine, university of muhammadiyah surakarta for testing with larvae. the materials used were aedes aegypti instar iii iv larvae, 90% methanol, distilled water, papaya stem (carica papaya l.), abate, polyethylene glycol (peg), carboxy methyl cellulose (cmc). the tools used in this study included trays, black cloth, jars, blenders, filter paper, water baths, stirrers, rotary evaporators, cups, pipettes, beaker glasses, label paper, scales, analytical scales, and hand counters. the larvae were hatched in laboratory of parasitology ums. the larvae were from eggs of loka litbangkes pangandaran. the determination of larvae was done from loka litbangkes pangandaran and laboratory of parasitology ums. the eggs were from dengue-free mosquito aedes aegypti. the subject were developed in insectarium that was controlled the temperature and humidity, and given standard food. there were 175 larvae for preliminary study and 700 larvae for larvicide study, according who (who, 2005). ethical clearance was from kepk medical faculty ums no. 4623/a.1/kepk-fkums/xi/2022. the study was done by researchers. methanol extract of papaya stem (carica papaya l.) methanol extract of papaya stem (carica papaya l.) was prepared in the pharmacology laboratory of fk ums using the maceration method. the solvent used is 90% methanol solvent. the initial process is slicing the papaya stem as much as 10 kg (carica papaya l.) and then dry by cover it with a black cloth. after drying, and then crushed using a blender until it was in the form of a fine simplisia of 1.26 kg. once smooth that given a solvent in the form of 90% methanol and then stirred. then left for seven days with daily stirring. then the macerate is concentrated using a rotatory evaporator and a water bath until a thick extract is formed but still pourable. this study uses suspending agent as an additive, and there are few studies that focus on discussing the effect of suspending agent on larval mortality. therefor it is necessary to carry out a stability test to determine the appropriate concentration for this study. furthermore, stability tests were peg and cmc. the concentration of papaya stem (carica papaya l.) the concentration of papaya stem is as follows: 1. papaya stem extract 1.5% + peg 2. papaya stem extract 2% + peg 3. papaya stem extract 1.5% + cmc 4. papaya stem extract 2% + cmc treatment of larvae mosquito larvae preparation was done in the parasitology laboratory, faculty of medicine, muhammadiyah university, surakarta for approximately seven days from aedes aegypti eggs to be hatched and cultured until the larvae instars iii iv. testing for aedes aegypti larvae were done as follows:. there were seven groups consist of 4 treatment groups and 3 test groups, two negative controls cmc and peg, and one positive control using abate. the four treatments consisted of 2 methanol extracts of papaya stems at a concentration of 1.5% with peg and cmc. the other treatment group consisted of papaya stem methanol extract at a concentration of 2% with peg and cmc as shown in table 1. table 1. variation group of the papaya stem methanol extract with cmc and peg suspending agents against aedes aegypti larvae. group concentration number of larvae × replication total number of larvae c(+) abate 1% 25 × 4 100 c(-) peg 0% 25 × 4 100 c(-) cmc 0% 25 × 4 100 p1 (1.5% + peg) 1.5% 25 × 4 100 p2 (2% + peg) 2% 25 × 4 100 p3 (1.5% + cmc) 1.5% 25 × 4 100 p4 (2% + peg) 2% 25 × 4 100 total larvae 700 bestari et al. – effect of peg and cmc on papaya (carica papaya l.) extract … 963 then 25 aedes aegypti larvae were put into each glass containing distilled water which had been treated with abate, cmc, and peg, and 200 ml of stock solution. counting larvae mortality the death of the larvae was counted every 6 hours, 12 hours, 18 hours, and 24 hours. counting technique was using pipet and tray, under bright lamp. larvae considered as death when it didn’t actively move and stay still. researchers used hand counter to count death larvae. data analysis the analysis was using normality test shapiro-wilk, homogeneity test, kruskal wallis, mann-whitney test and probit test. ethical approval ethical clearance was from kepk medical faculty ums no. 4623/a.1/kepk-fkums/xi/2022. the study was done by researchers. results and discussion results the results of the stability test presented in table 2. table 2. stability solution test. indicator peg cmc solubility soluble soluble direct precipitation no precipitation no precipitation precipitation in 24 hours low precipitation low precipitation viscosity watery watery in previous the larvicidal research, a preliminary test determined whether the concentration of the extract used was effective or not. the preliminary test was in 7 groups with three control groups and four treatment groups which presented in table 3. table. 3 preliminary larvicidal test. group mortality in (hour) percentage mortality (%) 6 12 18 24 c(+) abate 22 25 25 25 100% c(-) peg 0 0 0 0 0% c(-) cmc 0 0 0 0 0% p1 (extract 1.5% + peg) 3 25 25 25 100% p2 (extract 2% + peg) 12 25 25 25 100% p3 (extract 1.5% + cmc) 20 25 25 25 100% p4 (extract 2% + peg) 25 25 25 25 100% the results of the preliminary test found that at the 12th hours the mortality of the larvae was 100%. this concentration of the extract was effective and could be continued for the larvicide test. the larvicidal test shown at figure 1. figure 1. mortality rate of aedes aegypti larvae every 6 hours given papaya stem extract (carica papaya l.) with 4 repetitions. 0,00 5,00 10,00 15,00 20,00 25,00 30,00 6 hours 12 hours 18 hours 24 hours m o rt al it y o f l ar v ae time of observation mortality rate of aedes aegypti larvae every 6 hours given papaya stem extract (carica papaya l.) c(+) abate c(-) peg c(-) cmc p1 (extract 1.5%+peg) p2 (extract 2%+peg) p3 (extract 1.5%+cmc) p4 (extract 2%+cmc) 964 biology, medicine, & natural product chemistry 14 (2), 2025: 961-968 after obtaining the larval mortality results, the data obtained were analyzed. this analysis was used to determine whether there were differences or not, then different tests were conducted. shapiro-wilk normality test and homogeneity test revealed that from all groups, within 6, 12, 18 and 24 hour group, the results were p value < 0.000, so the data is not normally distributed and not homogeneous. then it proceed with the kruskal-wallis test to find out whether there is a significant difference between seven groups or not, shown at table 4. the results of the non-parametric test show that p<0.05, there is a significantly difference in the data. the hypothesis null (h0) is rejected, and the alternative hypothesis is accepted. the post-hoc test was carried out because the kruskall wallis non-parametric test obtained meaningful group of different data. the mann-whitney test was performed to determine significant differences between data groups, described in table 5 p value were taken from 24-hour larval mortality group data. table 4. kruskal-wallis test. mortality sig. result 6-hour mortality 0,003 significant difference 12-hour mortality 0,002 significant difference 18-hour mortality 0,001 significant difference 24-hour mortality 0,000 significant difference table 5. mann-whitney test. c(+) abate c(-) peg c(-) cmc p1 p2 p3 p4 c(+) abate p : 0.008* p : 0.008* p : 1.000 p : 0.317 p : 1.000 p : 1.000 c(-) peg p : 0.008* p : 1.000 p : 0.008* p : 0.011* p : 0.008* p : 0.008* c(-) cmc p : 0.008* p : 1.000 p : 0.008* p : 0.011* p : 0.008* p : 0.008* p1 p : 1.000 p : 0.008* p : 0.008* p : 0.317 p : 1.000 p : 1.000 p2 p : 0.317 p : 0.011* p : 0.011* p : 0.317 p : 0.317 p : 0.317 p3 p : 1.000 p : 0.008* p : 0.008* p : 1.000 p : 0.317 p : 1.000 p4 p : 1.000 p : 0.008* p : 0.008* p : 1.000 p : 0.317 p : 1.000 * significantly different p1 : peg + methanol extract papaya stem 1.5 % p2 : peg + methanol extract papaya stem 2 % p3 : cmc + methanol extract papaya stem 1.5 % p4 : cmc + methanol extract papaya stem 2 % lc50 probit test to find out the concentration needed to kill 50% of the larvae and lt50 lethal time which is used to determine the time needed to kill 50% of the larvae. the lc50 results in this study were used to compare which suspending agents the methanol extract of papaya stems was effective against the mortality of aedes aegypti larvae. the following results of the lc50 at hour-12 and lt50 probit test are presented in table 6. table 6. probit test. group lc50 peg 0.033 cmc 0.321 concentration lt50 peg+1.5 % 20.804 peg+ 2 % 30.784 cmc +1.5 % 23.508 cmc +2 % 40.718 discussion the study had occured on a mixture of papaya stem extract with different concentration and suspending agent. the mixture was cloudy and dispers well. the amount of death larva was good according to amount of death larva on 12 hours, almost same with abate as positive control. it can be happened because papaya stem had active material such as saponin, alkaloid, tanin, etc as larvicide. yield related to the number of active compounds present in a plant extract (salim et al., 2017). the methanol extract of papaya stems, which was already viscous obtained a yield of 5.087%. when be to previous research conducted by saputri (2021) with the same plant was found to be lower, namely 9.806%. this difference in yield can occur because influenced by several factors, namely the type of solvent, stirring time, maceration time and the origin of the plant area taken (aprilasani & adiwarna, 2014; chairunnisa et al., 2019; salim et al., 2017). in this study, maceration was carried out for six days, whereas in previous studies, the maceration time was not explained. in addition, in this study, stirring was only done once a day, whereas in the previous research it was stirred three times a day. results of the stability test in this study found that compound cmc and peg could affect the solubility, precipitation, and the mixture of extracts into distilled water. based on the stability test, it was found that the appropriate diluent concentrations were 0.01% cmc and bestari et al. – effect of peg and cmc on papaya (carica papaya l.) extract … 965 0.01% peg which ordinally used 0.5% cmc and 0.1% peg concentrations. this change in concentration was due to the acquisition of a thick solution at 0.5% cmc and 0.1% peg, so it was worried that it would affect the death of the larvae because the thickness of the solution would interfere with the movement of the larva (auliaputri, 2022). in the larvicidal test, average mortality in four repetitions was obtained, larvae mortality in the treatment within 24 hours was 100% except in p2, the mortality was 93%. in previous studies, 100% of the larvae died in all treatments (ayu et al., 2021). the data is not normally distributed and not homogeneous which states that the mortality of larvae in each group every hour is not normal and not homogeneous. then proceed with the kruskal-wallis test to find out whether there is a significant difference between groups or not. the results of the non-parametric test show that there is a significantly difference in the data. the null hypothesis is rejected, and the alternative hypothesis is accepted. it presents which groups differed significantly and which did not. there was no significant difference between the positive control with p1, p2, p3 and p4, but it was significantly different from the negative control cmc and peg. based on probit analysis, it was found that the lc50 of papaya stem methanol extract with peg suspending agent with death at 12 hours was 0.033%. lc50 papaya stem methanol extract with cmc suspending agent of 0.321%. based on these results it can be said that papaya stem methanol extract with peg suspending agent is more effective than cmc. when compared with previous research conducted by ayu (2021) in the ethanol extract of papaya stems, an lc50 of 1.033 was obtained. based on the lc50 results, papaya stem methanol extract with peg and cmc suspending agents was more effective than papaya stem ethanol extract in previous studies. this study used cmc and peg suspending agents, whereas in that study it was not explained which suspending agent was used to dilute the ethanol extract of papaya stems. methanol extract of papaya stem with cmc and peg suspending agents is included in the toxic category. the extract is said to be highly toxic if the lc50 is <1%, toxic if the lc50 is 1-10%, moderately toxic if the lc50 is 10-50%, slightly toxic lc50 is 50100%, and if the lc50 is > 100% then it is said to be non-toxic (ayu et al., 2021). according to the probit results for lt50, the lt50 at a concentration of 1.5% with peg suspending agent was 20.804 hours and at a concentration of 2% with peg suspending agent was 30.784 hours. the lt50 of the extract at 1.5% concentration with cmc suspending agent was 23.508 hours, and the lt50 of the extract at 2% concentration with cmc suspending agent was 40.718 hours. as a result, an extract concentration of 1.5% with peg and cmc suspending agents can kill 50% of the larvae faster than an extract concentration of 2%. lower concentrations kill larvae faster, possibly due to an insufficient concentration of suspending agent, which prevents the suspending agent from dissolving the extract evenly across the surface of the water. based on the results of lc50 and lt50, extract with a smaller concentration is more effective in killing aedes aegypti larvae. when compared with previous studies which stated that the higher the concentration of the extract, the higher the mortality rate of larvae. so, this is contrary to the results of this study, namely the smaller the concentration, the higher the mortality. this could be due to the factor of the suspending agent. there are factors that affect the solubility of a material, namely the substance to be dissolved, temperature, ph and volume of solvent (apsari & chaerunisa, 2020). the two components are related because the solvent will react with the solute so that the substance can be spread evenly. the substance to be dissolved (solute) should be less and the solvent (solvent) should be more. the suspending agent must be more than the amount dissolved (yusnidar, 2019). whereas in this study, the suspending agent concentration was adjusted to the volume of distilled water without adjusting to the concentration of the extract. the mortality effect of papaya stem methanol extract (carica papaya l.) with cmc and peg suspending agents, when compared to papaya stem ethanol extract (carica papaya l.) conducted by saputri et. al., (2021), is more effective when viewed based on the lc50 that has been obtained from the probit test. can be influenced by the addition of cmc and peg which help the extract to be dispersed or dispersed evenly over the entire surface of the distilled water. the addition of cmc and peg suspending agents did not directly affect or in other words, did not have a larvicidal effect on aedes aegypti larvae as evidenced in the negative control, namely distilled water which was given cmc and peg did not cause death in the larvae. research conducted by baskaranatha that cmc affects mortality in larvae at a concentration of 0.5% has affected larvicidal by 8%, so the cmc concentration reduced to 0.025%. cmc with a concentration of 0.025% did not cause death in the larvae or a death percentage of 0% (ayu et al., 2021; baskaranatha i made; swastika, i kadek, 2020; manuahe et al., 2022). previous research of the ethanol extract of tobacco leaves on the mortality of anopheles aconitus larvae using 0.5% cmc suspending agent did not cause death in the larvae (nurhayani et al., 2021). besides cmc, the suspending agent used in this study was peg with concentrations 0.01%. based on previous research, permot leaf extract with add of 0.1% peg did not affect the mortality of aedes aegypti larvae (susilowati & sari, 2022). this study used peg 0.01% and did not cause death to the larvae. therefor can be said that the concentration of peg does not affect the mortality of the larvae. based on this study, the cmc and peg can increase larval mortality so that the methanol extract of papaya stem (carica papaya l.) can 966 biology, medicine, & natural product chemistry 14 (2), 2025: 961-968 be dispersed evenly, and the compounds present in the methanol extract of papaya stem (carica papaya l.) can cause larval death. the death of the larvae was caused by the compounds contained in the methanol extract of papaya stem (carica papaya l.), namely flavonoids, tannins, steroids, saponins, alkaloids, papain and essential oils. saponins form complexes with protease enzymes to split the inner lining of the intestinal mucosal cells thereby disrupting the permeability of the intestinal membrane (zaynab, 2021). saponins interact with cholesterol which causes the attachment of food that cannot be digested by the larvae. in addition, saponins can also cause disruption of ecidsteroid synthesis, which is a hormone that helps the skin turnover cycle (lafont et al., 2012). flavonoids affect mortality by inhibiting growth and interfering with the respiratory system (krisna et al., 2022). flavonoids inhibit energy transport and metabolism in mitochondria by inhibiting the electron transport system or the channel between atp production and the atp transport system so that oxygen production in mitochondria decreases (ayu et al., 2021; manuahe et al., 2022). in addition, flavonoids and alkaloids work by inhibiting acetyl cholinesterase by phosphorylation of the amino acid serine at the acetyl centre of the enzyme. blocked acetyl cholinesterase causes a buildup of acetylcholine and causes poisoning larvae in the form of paralysis of the respiratory system (ilham et al., 2019; wahyudi et al., 2021). alkaloids and tannins play a role in degrading cell membranes in larvae so that the larvae cannot grow into pupae and will die (maulana, sidik; musthofa, faisal; yamin, ahmad; juniarti, neti; putri, 2021; wahyudi et al., 2021). tannins react with protease enzymes which causes the larvae to lack nutrition and eventually die (ayu et al., 2021). papain works by breaking down protein in the proteolytic process so that the larvae will lack protein for metabolism, if metabolism continues become constructed, it will cause the larvae to die (payangka et al., 2019). steroids interfere with the moulting system in the larvae and interfere with the absorption of food by the larvae (ilham et al., 2019). essential oils damage the nervous system and change the structure of cell membranes in the larvae so that over time the larvae will die (ilham et al., 2019). therefor adding cmc and peg suspending agents can increase larval mortality so that the methanol extract of papaya stem (carica papaya l.) can be dispersed evenly and the compounds present in the methanol extract of papaya stem (carica papaya l.) can cause larval death. conclusions papaya stem methanol extract with peg suspending agent was more effective than cmc suspending agent to kill aedes aegypti larvae. it has potential to substitute abate that environmentally damaging abate, and the larvae are already resistant. acknowledgements: thank you to universitas muhammadiyah surakarta and medical faculty of ums for supporting authors to do research and manuscript. authors’ contributions: all authors contributed to the research design. proposal and report of the research were performed by rochmadina suci bestari and fidhia nur rif’aini. material preparation and laboratory preparation were performed by devi usdiana rosyidah, rochmadina suci bestari and fidhia nur rif’aini. data collection were done by listiana masyita dewi, retno sintowati, rochmadina suci bestari and fidhia nur rif’aini. analysis and draft of manuscript were written by rochmadina suci bestari, fidhia nur rif’aini and hidayah karuniawati. all authors read and approved the final manuscript. competing interests: the authors declare that there are no competing interests. funding: the research was funded by universitas muhammadiyah surakarta by hibah integrasi tridarma (hit) scheme number : 611/a.3-iii/fk/iv/2022. references aprilasani, z., & adiwarna. 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(2021). saponin toxicity as key player in plant defense against pathogens. toxicon, 193. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 109-119 | doi: 10.14421/biomedich.2024.131.109-119 issn 2540-9328 (online) comparative effects of ripe and unripe lime (citrus aurantifolia) on spermatozoa and gonadosomatic index in matured male wistar rats obioma ogbonnaya1, azubuike nwankwo2, daniel chidi ifenkwe3, chikera samuel ibe4, ekele ikpegbu4,5, osah martins onwuka6,* 1college of agriculture, gregory university uturu, abia state, nigeria. 2department of human physiology, faculty of basic medical and health sciences, abia state university uturu, abia state, nigeria. 3department of physiology and pharmacology, college of veterinary medicine, michael okpara university of agriculture, umudike, nigeria. 4department of veterinary anatomy, college of veterinary medicine, michael okpara university of agriculture umudike, abia state, nigeria. 5department of comparative pathology, cummings school of veterinary medicine, tufts university, united state of america. 6department of human physiology, gregory university uturu, abia state, nigeria. corresponding author* osahmartinz@gmail.com manuscript received: 17 january, 2024. revision accepted: 08 may, 2024. published: 16 may, 2024. abstract to ascertain the comparative effects of ripe and unripe lime (citrus aurantifolia) on spermatozoa and gonadosomatic index evaluation in matured male wistar rats; exploring the idea that both ripe and unripe lime (citrus aurantifolia) might or might not positively affect semen quality, crucial for male fertility. twenty-eight (28) sexually mature male wister rats, aged 9–10 weeks and weighing between 211.50g and 217.00g, were divided equally into seven groups (1 to 7); with group 1 serving as the control and groups 2 to 7 receiving 25%, 50%, and 75% concentrations of ripe lime juice (rlj) and unripe lime juice (ulj) respectively. the findings indicated that ulj had a higher concentration (0.1mg/ml) compared to ripe lime rlj at 0.08mg/ml, although both had approximately the same ld50 value of 1581.138mg/kg. rlj, at different concentrations, adversely impacted the reproductive performance of rats, leading to decreased progressive motility, livability, sperm count, testicular size, and sexual drive. however, ulj did not exhibit these effects. a 75% concentration of rlj showed anti-prostatic activity, causing a reduction in prostate size, which was more pronounced than that of the same ulj concentration. importantly, both rlj and ulj did not have a significant impact on the sizes of the liver, spleen, heart, kidneys, and lungs, with these visceral organs maintaining normal sizes comparable to the control group (statistically, p>0.05).the findings suggest that rlj or ulj consumption, particularly at the highest concentration, may lead to alterations in reproductive performance, hence such consumption should be discouraged. keywords: citrus aurantifolia; lime, fertility; male reproduction; spermatozoa; testis. introduction infertility is a phenomenon associated with offspring production (rahayu and hanizar, 2021). globally, the incidence rate is approximately 30%, with males accounting for about 50% prevalence (orieke et al., 2019). in nigeria, male infertility is reported to contribute to 20-50% of all infertility cases across different regions (eze, 2012). the worldwide increase in male infertility cases has been linked to a decline in semen quality (abarikwu, 2013). infertility can result from factors such as low sperm count, inadequate sperm motility, abnormal morphological structures, or a combination of these issues (kumar and singh, 2015). another significant contributing factor is the decline in or inappropriate secretion of male sex hormones (aprioku and obianime, 2014; majzoub and agarwal, 2018). a normal sperm should have a concentration of at least 15 million/ml of ejaculate, with 72% motility indicating progressive movement and 94% normal morphology (world health organization, 2010). abnormalities in sperm, particularly in concentration and motility, have been identified as major causes of male infertility in various studies (sharma, 2017; wdowiak, 2019; babakhanzadeh, 2020). other factors affecting sperm quality include exposure to radiation (gorpinchenko, 2014; kesari et al., 2018), nutrition (martinez-soto, 2016; salas-huetos et al., 2018), lifestyle choices (ilacqua, 2018), age (silea, 2019), psychological factors (janevic, 2014), and deletions in the y chromosome (colaco and modi, 2018). several plants have a historical reputation for influencing male reproductive functions. some of these plants can impact spermatogenesis at the testicular level or alter the body's hormone profile. the use of fruits like citrus aurantifolia in the general diet is gaining popularity globally due to its nutritional and health https://doi.org/10.14421/biomedich.2024.131.109-119 mailto:osahmartinz@gmail.com 110 biology, medicine, & natural product chemistry 13 (1), 2024: 109-119 benefits (nnenne et al., 2020). commonly known as lime or bitter orange in nigeria, this fruit belongs to the rutaceae family. lime is recognized for its rich content of phytochemicals, antioxidants, vitamins, minerals, and dietary fiber, which are known to reduce the risk of various health issues, as utilized in ethno-medicine (septembre-malaterre et al., 2018). it is considered a promising fruit with health-promoting qualities and is widely consumed (adegoke et al., 2017). lime, a sour, round, and bright green citrus fruit, is extensively used in west africa, particularly in nigeria, for various purposes such as food, refreshing drinks, desserts, and seasoning (enejoh et al., 2015). known for its health benefits, including immune system enhancement (kim, 2023) and weight loss due to its hypolipidemic property (enejoh et al., 2015), lime has become popular among young men for its perceived impact on weight management (saini et al., 2022). this research aims to investigate the potential effects of lime consumption on the male reproductive system, including semen parameters, libido, hormonal levels, and testicular weight, using a rat model. given lime's significant presence in herbal preparations in nigeria (ayinde et al., 2012; enejoh et al., 2015), the findings will contribute to understanding how lime extract may influence physiological parameters and fertility functions in male wister rats. this information is crucial for researchers, ethno-medicinal healers, and end-users, providing insights into the appropriate use of lime, especially among individuals of reproductive age in rural areas. material and methods study location fresh lime fruits were collected and identified at the mini-market of abia state university, uturu campus. the extraction of lime juice and the animal studies, including semen evaluation, were conducted in the department of physiology and pharmacology at the college of veterinary medicine (cvm), michael okpara university of agriculture, umudike (mouau), where study approval was obtained. experimental animals and management a total of 28 sexually matured male wister rats, aged 9– 10 weeks and weighing between 211–217g, were obtained from the department of veterinary physiology and pharmacology animal house at the college of veterinary medicine (cvm), mouau. the animals underwent a 14-day acclimatization period in the animal laboratory, during which they were dewormed using paraquantel (at 10mg/body weight), and their physiological parameters were measured and recorded. under standard laboratory conditions with a 12-hour light–dark cycle at a room temperature of 25 ± 2oc, the rats were provided with a standard diet (pelleted chukun growers feed) and had access to water ad libitum. regular weighing was conducted using a sensitive electronic scale (digital electronic balance, citizen scales [1] pvt ltd., south patel nagar, new delhi, sensitivity: 0.01 g) before the start of the study and on a weekly basis. preparation of lime juice extract fresh lime fruits, both ripe and unripe, were washed, weighed, and halved. the juice from each half was expressed into a glass beaker. after filtration using muslin, the filtrate was collected into labeled bottles. the lime juice extract was considered 100%, and dilutions of 25%, 50%, and 75% were prepared by adding distilled water using volumetric flasks. these different concentrations were orally administered to the animals in separate groups, each receiving a dose of 5ml/kg of the respective lime juice concentration (khan et al., 2010). the percentage yield of the juice extract was calculated using the formula: % yield = weight of lime juice extracted weight of lime fruit used × 100 (soffwora, 1993) acute toxicity study of the lime extract the lorke method (1983) was employed in this study, which involved a total of 35 matured mice conducted in two phases. in the first phase, groups a-c, each comprising 5 mice, received oral doses of 10 mg/kg, 100 mg/kg, and 1000 mg/kg of ripe lime juice (rlj) extract, respectively. in the second phase, groups d-f, also with 5 mice in each group, were orally administered doses of 1600 mg/kg, 2900 mg/kg, and 5000 mg/kg of unripe lime juice (ulj) extract, respectively. additionally, a control group (group g) consisting of 5 mice was given 5ml/kg of distilled water. all mice had unrestricted access to food and water ad libitum. the animals were observed for signs of toxicity and mortality over 72 hours postadministration and for an additional 14 days for delayed toxicity. the median lethal dose (ld50) of the lime juice extracts, both ripe and unripe, was calculated using the formula recommended by khan et al. (2013). ld50 = √(least dose with mortality × highest dose without mortality) experimental design the 28 male wistar rats were evenly divided into 7 groups, including a control group, as well as groups receiving ripe lime juice (rlj) and unripe lime juice (ulj) at concentrations of 25%, 50%, and 75% from the stock solution (table 1). ogbonnaya et al. – comparative effects of ripe and unripe lime (citrus aurantifolia) … 111 table 1. experimental design. group 1 normal male rat (5ml/kg d/w) 2 rlj at ld25% stock 3 rlj at ld50% stock 4 rlj at ld75% stock 5 ulj at ld25% stock 6 ulj at ld50% stock 7 ulj at ld75% stock note: rlj: ripe lime juice; ulj: unripe lime juice; ld: lethal dose; d/w: distilled water. the animals were dosed based on their body weight using a gastric tube (gavage) over a period of 28 days, during which continuous monitoring took place. after the dosing period, euthanasia was carried out by placing each rat in a small transparent bucket with a cover, containing cotton wool soaked in mild chloroform. the epididymis was then harvested, and sperm samples were collected from the caudal portion of the epididymis. smears were prepared on preheated glass slides for subsequent evaluation. male fertility study libido (sex drive) evaluation libido in the rats was assessed through physical observation, focusing on their reactions and sexual behavior when introduced to a female rat in estrus. sexual behavioral reactions, including general grooming, sniffing, mounting, and thrusting on the female rats, were noted and recorded according to a scoring pattern described by chibundu (2013) (table 2). scores were allocated based on these observed reactions. table 2. libido grading for male rats. sexual behavior score grading vigorous grooming, sniffs and attempt to mount 5 very high libido grooms, sniffs but no attempt to mount 4 high libido sniffs only 3 moderate grooms only 2 low does not pay attention to the female rat 1 poor libido source: chibundu (2005). sperm colour and consistency the color and consistency of the sperm samples were assessed through macroscopic observation and recorded. the evaluation followed the consistency scale (1-4) as defined by chibundu (2013), where scores 1 and 2 represented milky white and creamy white, respectively. the consistency scale utilized included scores 1 (watery), 2 (slightly thick), 3 (thick), and 4 (very thick) for assessing the thickness of the sperm samples. sperm motility the methodology outlined by el-sherbiny (1987) was followed for evaluating sperm samples collected through epididymal washings in each treatment group. progressive motile sperm cells were assessed immediately after collection. a smear of one drop of the sperm sample was created on a preheated glass slide and examined under light microscopy at low magnifications (×10 and ×40). the assessment was conducted subjectively and scored as a percentage. only sperm cells demonstrating straight-forward movement were considered in the motility count, while those moving in circles, backward, or displaying pendulum-like movement were excluded. individual sperm motility was scored according to the following scale: 1 (vigorously progressive), 2 (progressive), 3 (cycling movement), and 4 (stationary movement). sperm viability (live proportion) the viability of sperm cells was determined by staining a drop of the collected sperm sample with eosin-nigrosin stain. the stained glass slide was allowed to air-dry for 30 seconds, followed by fixation with ethanol. the slide was then examined under a light microscope at a magnification of x100 (oil immersion). using a handheld mechanical stopwatch counter, the proportion of viable sperm cells was counted. a total of 300 sperm cells were counted, and the number of viable cells was expressed as a percentage of the total count. viable sperm cells, which remain unstained, were differentiated from dead cells, which picked up the stain, following the method described by el-sherbiny (1987). sperm concentration the sperm concentration was determined using a haemocytometer following the method described by ukar et al. (2016). a dilution of 1:200 was created using a red blood cell pipette. in this procedure, a 1% buffered formalin solution was employed as the semen diluting fluid to immobilize the sperm cells. the haemocytometer was loaded with a drop of the sperm solution, allowed to settle for 2 minutes on a wet paper (to facilitate sperm cell settling), and then examined under the microscope at ×40 magnification. the formular; sperm concentration per ml = no. of cells counted × dilution factor × 0.04 × 106 (egbuka, 1995) was used to determine sperm concentration. abnormal sperm proportion the percentage of abnormal sperm proportion was assessed following the method outlined by el-sherbiny (1987). a drop of the sperm sample was stained with eosin-nigrosin stain, and the resulting mixture was smeared on a glass slide. under a lower magnification of ×40, the slide was examined to identify primary and secondary abnormal sperm cells. the percentages of differential abnormalities, including head, mid-piece, and 112 biology, medicine, & natural product chemistry 13 (1), 2024: 109-119 tail abnormalities, were determined according to the criteria specified by el-sherbiny (1987). statistical analysis all collected data underwent statistical analysis, and the test values were presented as mean ± sem (standard error of mean). statistical differences among the means of different groups were analyzed using one-way analysis of variance (anova), followed by the lsd test for mean separation. the level of significance was set at p < 0.05 compared to the control group. values at p < 0.05 were considered statistically significant. results comparing juice percentage yield of ripe and unripe lime juice the rlj yielded (72.50%), more than the ulj (61.25%) (figure 1). figure 1. comparing juice percentage yield of ripe and unripe lime juice. comparing juice concentration yield of unripe lime juice on the contrary, the ulj was more concentrated (100mg/ml; p<0.05) than the rlj (80mg/ml) (figure 2). figure 2. comparing juice concentration yield of unripe lime juice. acute toxicity testing of ripe and unripe lime juice the lethality results indicated that, after 72 hours postadministration of both ripe lime juice (rlj) and unripe lime juice (ulj) to rats at high (5000mg/kg) and low (500mg/kg) doses, no deaths or signs of toxicity were recorded in the low dose (500mg/kg) groups for both rlj and ulj. however, in the 5000mg/kg dose groups, 100% mortality was observed in rlj, and 75% mortality was recorded in ulj. the calculated ld50 was 1581.138mg/kg, falling below the 2000mg/kg threshold, indicating significant lethality in both rlj and ulj. based on these findings, convenient concentration gradients of 25%, 50%, and 75% reconstitution were prepared and utilized in the study for both rlj and ulj. effect of graded concentration of ripe and unripe matured lime juice extract on spermatozoa assessment of male wistar rats table 3 illustrates the significant effects of both ripe lime juice (rlj) and unripe lime juice (ulj) administration on the spermatozoa qualities (color, ph, progressive motility, livability, and count) of wistar male rats. however, the thickness/consistency of the epididymal reserve was not affected. the color of the epididymal reserve in male wistar rats administered ulj at 25% and 50% concentrations shifted from milky white (normal color) to creamy white. in contrast, the color of the epididymal reserve in those administered rlj at various concentrations remained unchanged and retained a milky white appearance similar to that of normal rats. the ph of the epididymal reserve in male wistar rats administered graded concentrations of rlj (25%, 50%, and 75%) and 25% concentration of ulj fell within the normal ph range (7.00±0.00) of rat semen (ph 6.99±0.00). however, the ph of the epididymal reserve was significantly lower (p < 0.05) at 6.98±0.01 in the group of wistar rats administered the highest concentration (75%) of ulj compared to the normal ph. the results of spermatozoa movement revealed that rlj at various concentrations significantly (p < 0.05) negatively affected the progressive motility of harvested sperm cells from the caudal epididymides. this resulted in a concentration-dependent decrease, reducing from 85.27±1.8% (normal rats' progressive motility) to a range of 62.47±4.40–68.30±1.95%. in contrast, rats administered ulj at 25%, 50%, and 75% concentrations recorded spermatozoa progressive motility of 79.73±2.86%, 73.98±3.49%, and 73.80±4.45%, respectively. these values were comparable (p > 0.05) with the normal rats' progressive motility (table 3). the results of the spermatozoa live proportion in wistar rats, as presented in table 3, exhibited a direct relationship with the percentage of motile spermatozoa. the administration of rlj had a negative effect (p < 0.05) on the livability of spermatozoa harvested from the caudal epididymides, while ulj did not affect the live proportion. ogbonnaya et al. – comparative effects of ripe and unripe lime (citrus aurantifolia) … 113 the spermatozoa concentration results showed gradual (p < 0.05) decreases in the number of sperm cells counted in each caudal epididymis of all the wistar rats administered various concentrations of rlj and in the rats administered 75% ulj. however, the number of spermatozoa counted in the wistar rats administered 25% (104.59±7.95 × 106/caudal epididymis) and 50% (84.42±15.90 × 106/caudal epididymis) ulj were statistically comparable (p > 0.05) to the number counted in the control rats (111.19±7.37 × 106/caudal epididymis). table 3. effect of graded concentration of ripe and unripe matured lime juice extract on spermatozoa assessment of male wistar rats. parameters treatment group colour consistency ph progressive motility (%) spermatozoa livability (%) spermatozoa concentration (×106/ce) 5 ml/kg d/w 1.00±0.00b 4.00±0.00 7.00±0.00a 85.27±1.8a 94.72±0.72a 111.19±7.37a 25% rlj 1.00±0.00ab 3.33±0.33 6.99±0.00ab 68.30±1.95bc 82.47±1.38bc 64.33±3.84b 50% rlj 1.66±0.33ab 3.33±0.67 6.99±0.00ab 64.67±6.64c 79.17±5.42bc 62.75±3.36b 75% rlj 1.66±0.33ab 3.33±0.66 6.99±0.00ab 62.47±4.40c 75.51±4.55c 58.60±9.21b 25% ulj 2.00±0.00a 3.66±0.00 6.99±0.00ab 79.73±2.86ab 87.54±2.96ab 104.69±7.95a 50% ulj 2.00±0.00a 4.00±0.00 6.98±0.01b 73.96±3.49abc 84.99±3.75abc 84.42±15.90ab 75% ulj 1.33±0.33ab 3.66±0.33 6.98±0.01b 73.80±4.45abc 84.77±3.21abc 77.54±5.00b note: values are presented as mean± s.e (standard error of mean). different superscript letters along treatment groups shows significant (p<0.05) differences. rlj: ripe lime juice; ulj: unripe lime juice; d/w: distilled water. effect of graded concentration of ripe and unripe lime juice extract on the spermatozoa morphology of wister rats the results of spermatozoa morphology in the experimental male rats (table 4) revealed that administering 50% and 75% of rlj caused significant (p < 0.05) damage to the tail, head, and mid-piece of the sperm cells. administration of ulj at 75% also caused significant (p < 0.05) damage to the sperm cell head. however, the percentage of normal sperm cells in 25% rlj concentration and 25%, 50%, and 75% ulj were statistically comparable (p > 0.05) to the control rats. it is noteworthy to mention that 75% of rlj caused the most significant (p < 0.05) damage to the cells, with the highest number of total abnormalities (4.71±0.50%) and the least (p < 0.05) number of normal cells (95.28±0.50%) compared to others. however, it's important to note that the total abnormal sperm cells obtained in 75% rlj are less than the 25% concentration, which is considered to affect fertility. table 4. effect of graded concentration of ripe and unripe matured lime juice extract on spermatozoa morphology of male wistar rats. treatment group tail abnormality head abnormality mid-piece abnormality cytoplasmic droplets total abnormal sperm cells percentage normal sperm cells 5 ml/kg d/w 0.19±0.03de 0.27±0.02c 0.13±0.02c 1.16±0.05b 1.76±0.05c 98.23±0.05a 25% rlj 0.27±0.02cd 0.26±0.04c 0.26±0.03bc 1.12±0.01b 1.91±0.05c 98.08±0.05a 50% rlj 0.36±0.02b 1.25±0.03b 0.55±0.11b 1.12±0.00b 3.29±0.13b 96.71±0.13b 75% rlj 0.44±0.03a 1.60±0.24a 1.41±0.26a 1.25±0.04a 4.71±0.50a 95.28±0.50c 25% ulj 0.17±0.29e 0.23±0.01c 0.26±0.01bc 1.16±0.01b 1.83±0.02c 98.17±0.02a 50% ulj 0.21±0.01de 0.31±0.04c 0.34±0.04bc 1.21±0.02ab 2.07±0.01c 97.92±0.00a 75% ulj 0.33±0.01bc 0.32±0.02c 0.28±0.01bc 1.19±0.01ab 2.13±0.03c 97.87±0.03a note: values are presented as mean± s.e (standard error of mean). different superscript letters along treatment groups shows significant (p<0.05) differences. rlj: ripe lime juice; ulj: unripe lime juice; d/w: distilled water. effect of graded concentration of ripe and unripe matured lime juice extract on libido assessment of male wistar rats the results regarding the effect of administering both rlj and ulj on the libido of male wistar rats, as presented in (table 5), indicated a concentrationdependent decrease in sex drive (libido) in both rlj and ulj administered groups compared to the high sex drive observed in the normal control wistar rats. the reaction time outcomes revealed that it took an average of 7 minutes and 27 seconds, 5 minutes and 29 seconds, 6 minutes and 40 seconds, and 6 minutes and 49 seconds for the rats administered 25% rlj, 25%, 50%, and 75% ulj, respectively, to start grooming and sniffing the introduced female. in comparison, it took an average of 4 minutes and 26 seconds for those in the control group to complete the sexual behavioral reactions from general grooming to an attempt to mount. 114 biology, medicine, & natural product chemistry 13 (1), 2024: 109-119 table 5. effect of graded concentrations of ripe and unripe lime juice extract on libido of wister rats. treatment group libido score reaction time (minutes) 5 ml/kg d/w 4.66±0.33a 4.26±0.45c 25% rlj 1.66±0.26cd 7.27±0.64a 50% rlj 1.00±0.00d 23.32±0.22b 75% rlj 1.00±0.00d 24.47±0.53ab 25% ulj 3.66±0.28b 5.29±0.86bc 50% ulj 2.33±0.33c 6.40±0.48ab 75% ulj 2.00±0.00c 6.49±0.72ab note: values are presented as mean ± sem (standard error of mean). different superscript letters across treatment groups shows significant (p<0.05) differences. rlj: ripe lime juice; ulj: unripe lime juice; d/w: distilled water effect of graded concentration of ripe and unripe lime juice extract on body weight changes of wister rats the body weight of the wistar rats before the commencement of the lime juice administration (week 0) indicated that all the wistar rats had the same body weight (p > 0.05) (table 6). furthermore, there was no significant (p > 0.05) difference in their body weight at week 1 during the administration of both rlj and ulj. however, by week 2, except for the average body weight of the rats administered a low concentration (25%) of rlj, which weighed the same (p > 0.05) as the control rats, there was a significant and steady weight reduction across all the administered groups from week 2 to week 4. this was in contrast to the consistent (p < 0.05) body weight increase observed in the control rats during the same period. table 6. effect of graded concentration of ripe and unripe lime juice extract on the weight gain of wister rats. duration treatment group week 0 week 1 week 2 week 3 week 4 5 ml/kg d/w 217.00±17.32 228.50±8.74 245.00±9.65a 258.50±5.10a 264.50±20.10a 25% rlj 216.25±10.76 223.00±6.79 218.25±23.26ab 213.50±4.97b 198.00±11.45b 50% rlj 212.75±7.70 210.00±3.18 208.00±5.35b 193.25±5.26b 184.75±7.39bc 75% rlj 214.25±10.47 210.50±3.52 210.00±3.18b 192.75±11.77b 159.75±6.28c 25% ulj 211.50±9.04 210.75±4.92 192.25±6.77b 187.50±4.99b 173.25±11.85bc 50% ulj 216.25±1.54 216.00±6.74 207.25±2.92b 192.75±11.77b 189.00±1.63bc 75% ulj 216.75±3.68 219.00±7.12 210.25±1.70b 200.25±23.28b 196.25±6.01b note: values are presented as mean± s.e (standard error of mean). different superscript letters along treatment groups shows significant (p<0.05) differences. rlj: ripe lime juice; ulj: unripe lime juice; d/w: distilled water. effect of graded concentration of ripe and unripe lime juice extract on gonadosomatic index evaluation of wister rats table 7 displayed the relative weight of the testes in all the ulj groups, and that of the 25% rlj groups, which weighed the same (p > 0.05) as the size of the testes in the control group relative to their body weights. there was a significant (p < 0.05) reduction in the relative size of the testes in the rats administered 50% and 75% rlj compared to the normal relative size of rat testes. however, the relative size of the prostate gland in the 75% rlj administered rats was significantly (p < 0.05) reduced, indicating a smaller average prostate size compared to the control rats. table 7. effect of graded concentration of ripe and unripe lime juice extract on the gonadosomatic index of wister rats. gonads treatment group % testes % prostate % seminal vesicle 5 ml/kg d/w 1.43±0.19a 0.16±0.03a 0.58±0.11a 25% rlj 1.03±0.02abc 0.14±0.02ab 0.36±0.02b 50% rlj 0.92±0.01c 0.07±0.01ab 0.21±0.06b 75% rlj 0.94±0.02bc 0.05±0.04b 0.21±0.03b 25% ulj 1.36±0.17ab 0.07±0.00ab 0.28±0.06b 50% ulj 1.21±0.20abc 0.13±0.02ab 0.30±0.01b 75% ulj 1.19±0.07abc 0.15±0.02a 0.28±0.07b note: values are presented as mean± s.e (standard error of mean). different superscript letters along treatment groups shows significant (p<0.05) differences. rlj: ripe lime juice; ulj: unripe lime juice; d/w: distilled water. ogbonnaya et al. – comparative effects of ripe and unripe lime (citrus aurantifolia) … 115 effect of graded concentration of ripe and unripe lime juice extract on relative organ weight of wister rats the results presented in table 8 indicate that both rlj and ulj did not have a significant effect on the size of the liver, spleen, heart, kidneys, and lungs of the rats. the size of these visceral organs in the administered groups was statistically the same (p > 0.05) as the normal size of these organs in the control rats. this suggests that both rlj and ulj, at the various concentrations, did not cause any gross damage to the evaluated organs. table 8. effect of graded concentration of ripe and unripe lime juice extract on the relative organ weight of wister rats. visceral organs treatment group liver spleen heart kidneys lungs 5 ml/kg d/w 3.38±0.72 0.36±0.09 0.35±0.05 0.62±0.04 0.62±0.05 25% rlj 3.51±0.46 0.47±0.13 0.34±0.02 0.65±0.12 0.77±0.07 50% rlj 3.39±0.15 0.51±0.03 0.38±0.03 0.65±0.05 0.70±0.01 75% rlj 2.93±0.09 0.44±0.02 0.33±0.02 0.48±0.01 0.58±0.04 25% ulj 3.07±0.32 0.50±0.09 0.38±0.01 0.62±0.03 0.79±0.05 50% ulj 3.48±0.41 0.45±0.10 0.33±0.03 0.54±0.04 0.68±0.03 75% ulj 3.18±0.10 0.63±0.16 0.31±0.00 0.54±0.04 0.75±0.14 note: values are presented as mean± s.e (standard error of mean). different superscript letters along treatment groups shows significant (p<0.05) differences. rlj: ripe lime juice; ulj: unripe lime juice; d/w: distilled water. discussion the yield and concentration of rlj and ulj exhibited an inverse relationship. rlj yielded 72.50% with a concentration of 0.08g/ml, while ulj had a yield of 61.25% with a concentration of 0.1g/ml. this variation in both percentage yield and concentration gradient might have impacted the observed activity or effects of the two lime juices in this study. the chemical composition of rlj includes essential components such as vitamins, minerals, phenolic compounds, saponins, cardiac glycosides, reducing sugars, flavonoids, and a minimal amount of steroids, which are recognized as crucial precursors to reproductive hormones (enejoh et al., 2015; navabi et al., 2018; khan et al., 2018; suntar et al., 2018; saini et al., 2022). rlj also contains a high level of tannins. lime is known to have secondary metabolites like limonoids, phenylethylamine alkaloids, with psynephrine being the most abundant (adokoh et al., 2019). as indicated by kim (2023), a medium-sized lime (67g) contains varying amounts of riboflavin, niacin, folates, phosphorus, magnesium, and trace amounts of iron, calcium, vitamin b6, and thiamine, along with a moderate amount of potassium and a substantial 22% of vitamin c. these phytoconstituents are scientifically believed to contribute to the physiological and pharmacological effects induced by lime juice on the body system (jayaprakasha et al., 2006; boshtam et al., 2011; nallely et al., 2012; akhtar, 2013; saini et al., 2022). the alteration of physiological parameters, including rectal temperature, heart rate, weakness, behavioral pattern, skin fur, and appetite, used for toxicity evaluation, deviated from normal at the highest dose (5000 mg/kg) in both rlj and ulj. this deviation occurred shortly before their demise, within less than 1 hour post-administration, indicating an ld50 (lethal dose for 50% of the population) of 1581.138 mg/kg (<2000 mg/kg) for both lime juices. this suggests that both rlj and ulj have a narrow margin of safety. the findings of this study on ld50 align with the research conducted by chijoke-nwachukwu and dede (2010), who reported an oral single dose ld50 of 1500 mg/kg for rlj and 2000 mg/kg for lemon juice. additionally, oboma et al. (2020) and saini et al. (2022) reported ld50 values for lime leaf extract in separate acute and subacute studies (1480 mg/kg, 1655 mg/kg, and 1532.64 mg/kg, with an average of 1555.88 mg/kg). this average did not significantly differ from the ld50 value obtained in the current study. the toxicity results imply that rlj, with a 100% mortality rate, was more toxic to the mice than ulj, even though rlj is sourer than ulj. this observation underscores the notion that ripeness does not necessarily correlate with safety in this context. the results obtained from the anti-fertility study revealed that ulj at low to medium concentrations had a significant (p<0.05) effect on the color of the epididymal reserve in male wistar rats, while rlj did not exhibit any impact on the color. in wistar rats, the normal color of semen is typically milky white, and changes to a creamy white color can indicate the influence of diet or treatment on semen color (peters et al., 2008). there is a direct relationship between semen color and sperm concentration, with creamy white semen indicating a higher sperm count compared to milky white semen (alkan et al., 2001; peters et al., 2008). this observation aligns with the findings of the current study, where the rat group with creamy white epididymal reserve recorded a higher sperm count than those with milky white epididymal reserve. similar correlations between semen color and sperm concentration have been reported in previous studies by singh and singh (2016), ahmed et 116 biology, medicine, & natural product chemistry 13 (1), 2024: 109-119 al. (2021), and ezeigwe et al. (2022) in their fertility studies involving lemon fruits. however, rahaju and hanizar (2021) reported no direct relationship between semen color and sperm concentration in mice treated with a 500mg/kg dose of lime leaf extract. these findings suggest that ulj, particularly at low to medium concentrations, may have a noticeable impact on the sperm count and epididymal reserve color in male wistar rats, highlighting the importance of considering concentration levels in the evaluation of such effects. the observed interaction between higher concentrations of ulj and spermatozoa stored in the caudal epididymides may have led to a decrease in ph, ultimately affecting sperm quality. adokoh et al. (2019) reported a ph value of 6.67 in rat blood when ulj was administered at a dose of 1000mg/kg for 58 days. this ph value is more acidic compared to the ph values of 6.98 obtained in the ulj at 5% and 75% concentrations in the current study. the variation in ph values of ulj in semen, as found in this study, could be attributed to differences in the buffering capacities of various body fluids or variations in dosage levels. the buffering capacity of a fluid refers to its ability to resist changes in ph when an acid or base is added. differences in dosage levels might also influence the extent of the observed ph changes. it's worth noting that ph is a critical factor influencing the activity and function of sperm. sperm function optimally within a specific ph range, and deviations from this range can adversely affect sperm quality and fertility. therefore, the observed decrease in ph associated with higher concentrations of ulj suggests a potential impact on sperm quality, emphasizing the importance of considering ph levels in the evaluation of reproductive effects. the findings from this study suggest that the administration of rlj for 28 consecutive days led to a decrease in the progressive motility of spermatozoa. this result aligns with existing literature, including studies by imade et al. (2005), obidi et al. (2008), sagay et al. (2009), loizzo et al. (2012), aprioku and obianime (2014), oboma et al. (2020), ahmed et al. (2021), and saini et al. (2022). these studies, as mentioned, have reported adverse effects on both female (such as reduction in the length of proestrus and estrus, increased frequency of metestrus, and changes in luteinizing hormone levels) and male (including decreased sperm motility, count, and viability) reproductive performance at higher dosages or concentrations of lime juice and citrus aurantifolia leaf extracts administered to laboratory animals. while some studies, such as singh and singh (2016), have reported adverse effects of lemon and lime extracts on steroidogenic markers in the testes, induction of germ cell apoptosis, drastic decrease in epididymal sperm quality, and no effect on libido, others like rahaju and hanizar in 2021 reported improved sperm quality at 25% lemon leaf extract and negative effects at 75% concentration. the current study found that increasing the dose or concentration of lime juice adversely affected body fluids and reproductive performance, including the progressive motility and livability of sperm. the negative impact on the livability of spermatozoa corresponds with the reduction in their progressive motility. this observation is consistent with bearden et al. (2004), who reported a high correlation between the proportion of live spermatozoa and visual estimates of progressively motile sperm cells. according to anderson (2001), various external and internal factors may influence the production of semen and spermatozoa concentration, ultimately affecting male fertility. the study revealed that the administration of lime juice, particularly rlj, for 28 days resulted in a low sperm count in wistar rats. however, the reason why rlj affected the spermatozoa indices evaluated more than ulj remains unclear and warrants further investigation. from the study results, it is observed that the 75% concentration of rlj caused significant abnormalities in the tail, head, and mid-piece of sperm, along with a higher percentage of cytoplasmic droplets compared to lower concentrations. the preservation of normal sperm morphology and integrity in the 25% rlj, 25%, and 50% ulj administered groups may be attributed to better antioxidant properties in both lime juices at lower concentrations. regarding sexual drive, both rlj and ulj led to a decrease in sexual drive (libido), suggesting the presence of active ingredients that may have affected the release of interstitial cell-stimulating hormone and testosterone. the decrease in sexual drive was concentrationdependent and followed a similar trend as sperm concentration, indicating a direct relationship between libido and sperm concentration. however, there are variations in findings reported by singh and singh (2016), which showed no effect on libido with 1000 mg/kg of lime leaf extract, possibly due to differences in the plant parts used. both rlj and ulj caused a significant reduction in body weights from week 2 to week 4. this reduction may be attributed to effects on metabolism, increased basal metabolic rate, suppressed appetite, induced lipolysis, or alteration in feed conversion efficiency. the observed reduction in prostate size in the 75% rlj group is noteworthy, suggesting a potential effect on prostate health. the reduction in testicular size, particularly in the 50% and 75% rlj concentrations, could be linked to ecobolic compounds in rlj with estrogenic and spermicidal properties. these findings align with reports by saini et al. (2022). while the study did not show significant enlargement, atrophy, or damage to major organs, it is important to note that changes in organ weights can be sensitive indicators of toxicity. the study suggests that rlj and ulj may have concentration-dependent effects on reproductive and physiological parameters in male wistar rats, highlighting the need for further ogbonnaya et al. – comparative effects of ripe and unripe lime (citrus aurantifolia) … 117 investigation into the underlying mechanisms and potential health implications. conclusion this study, designed to investigate the effect of both rlj and ulj administration on the reproductive efficiency of male wistar rats, revealed that ulj is more concentrated than rlj, although both recorded the same ld50 of 1581.138mg/kg, indicating a narrow safety margin. it can be concluded that rlj at the various concentrations studied affected the reproductive performance of male wistar rats by slowing down progressive motility, decreasing 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tradition medicine in africa. ibadan, nigeria. spectrum books ltd. p. 25-30. suntar i, khan h, patel s, celano r, rastrelli l. 2018. an overview on citrus aurantium: its functions as food ingredient and therapeutic agent. oxi med and cell long 112. ukar ai, herbert u, umeze em. 2016. effect of exogenous gonadotropin releasing hormone on semen characteristics and libido of rabbit bucks. proceedings of the 42nd annual conference of nigerian society of animal production, kwara state. p 853-859. https://www.researchgate.net/profile/kaitkanoke-sirinarumitr-2?_tp=eyjjb250zxh0ijp7imzpcnn0ugfnzsi6il9kaxjly3qilcjwywdlijoichvibgljyxrpb24ifx0 https://www.researchgate.net/scientific-contributions/shirley-d-johnston-2095144837 https://www.researchgate.net/profile/margaret-root-kustritz?_tp=eyjjb250zxh0ijp7imzpcnn0ugfnzsi6il9kaxjly3qilcjwywdlijoichvibgljyxrpb24ifx0 https://www.researchgate.net/scientific-contributions/g-r-johnston-38309423 https://www.researchgate.net/profile/dipak-sarkar-2 https://www.researchgate.net/profile/mushtaq-memon-3?_tp=eyjjb250zxh0ijp7imzpcnn0ugfnzsi6il9kaxjly3qilcjwywdlijoichvibgljyxrpb24ifx0 ogbonnaya et al. – comparative effects of ripe and unripe lime (citrus aurantifolia) … 119 wdowiak a, skrzypek m, stec m, panasiuk l. 2019. effect of ionizing radiation on the male reproductive system. annals of agric and environ med 26: 210–216. world health organization 2010. who laboratory manual for the examination and processing of human semen-5th ed, who press, switzerland, 2010. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 593-599 | doi: 10.14421/biomedich.2023.122.593-599 issn 2540-9328 (online) quercetin bioavailability evaluation on standardized herbal medicine containing guava leaf extract with hplc rizky yulion*, yulianis, suntri department of pharmacy, stikes harapan ibu jambi jl. kol. tarmizi kodir no.71, pakuan baru, kec. jambi sel., kota jambi, jambi 36122, indonesia. corresponding author* rizkyyulionputra10@gmail.com manuscript received: 16 june, 2023. revision accepted: 09 august, 2023. published: 07 november, 2023. abstract standardized herbal medicines are classified as one of indonesia's natural medicine ingredients in addition to herbal medicine and phytopharmaceuticals. the safety and efficacy of standardized herbal medicines are scientifically proven through preclinical trials, and raw materials and products have been standardized. one includes standardized herbal medicines is diapet, psidii, lelap, fitolac, and glucogarp. to determine the bioavailability of standardized herbal products containing guava leaf extract (psidium guajava l.). which uses single and combined natural materials. the method used is experimental with a crossover design. blood samples are taken from the marginalis vein of the rabbit ear at 0.5 hours; 1; 2; 4; and 6. the level of quercetin in the blood is determined by the reverse-phase hplc method. the mobile phase used is methanol:aquabidest (59:41,v/v), stationary phase octadecyl silica (c18), flow rate 1 ml/min, uv-vis detector 370 nm, and injection volume 20 μl. the value of bioavailability parameters obtained in the parameters cpmax, tmax, and auc of product a is 1.486454 μg / ml; 1.4 hours and 10.2615291 μg/ml/h, product b is 1.29224019 μg/ml; 1.5 hours and 11.30810501 μg/ml/hour. based on the results of this study, it can be concluded that the bioavailability profile of the two products is not much different, so it is expected that the effects caused are the same. keywords: hplc; quercetin; bioavailability; standardized herbal medicine. introduction indonesia is a tropical country with abundant natural resources. this abundant biodiversity is a source of natural materials that can be used to support the economy of the indonesian people. indonesian people use plants for traditional medicine, handicraft raw materials, industry, and natural dyes (haryadi & hidayati, 2018). until now, the myth that natural materials are safe has always been promoted by various parties. some of the community, both practitioners and users of herbal medicines, have the assumption that herbal medicines are safe. this can be true because the use of herbal medicine has been a long time, there are even some plants that have been around for a long time, and there are even some plants that have been used for hundreds of years as traditional medicine (wijayakusuma, 2002). many societies claim that the use of herbal remedies and chemical drugs does not cause side effects, and this statement is often combined with opinions that claim that herbal medicines do not cause harm. of course, this is not true, which is supported by evidence that no drug is effective and directly free from side effects, let alone its use in conjunction with chemical drugs so that it can cause interactions and affect the bioavailability of the chemical drug (britza et al., 2022; fardin & sarina, 2017a; hussin, 2001). for certain medicinal products bioavailability can be demonstrated by the fact that it is obtained in vitro which is carried out in such an environment as in vivo. drugs' bioavailability mainly depends on the drug being in a dissolved state. the drug dissolution rate of the drug product is measured in vitro. the official dissolving tests are described in the united states pharmacopeia (usp). the in-vitro dissolution rate data should relate to the data for the drug (amidon et al., 1995; cder/fda, 2015; fardin & sarina, 2017b; santos et al., 2019). one of the interactions that occur in herbal medicine is a pharmacokinetic interaction that affects the absorption, distribution, metabolism, or excretion of drugs. while pharmacodynamic interactions occur in drugs that work similar / or the same as herbal medicines, for example, concomitant administration between herbal drugs that have antiplatelet activity with anticoagulants, concomitant use of ephedrine with herbal medicines rich in caffeine (amidon et al., 1995; fardin & sarina, 2017b). guava plants have been used to treat diarrhea, swollen gums, wound medicine, heart, and diabetes. the analgesic effect is thought to be because guava leaves https://doi.org/10.14421/biomedich.2023.122.593-599 594 biology, medicine, & natural product chemistry 12 (2), 2023: 593-599 contain active substances such as essential oils, quercetin, and tannins that inhibit cyclooxygenase and lipooxygenase enzymes (daud, 2002). standardized herbal medicines are classified as one of indonesia's natural medicine ingredients in addition to herbal medicine and phytopharmaceuticals. the safety and efficacy of standardized herbal medicines are scientifically proven through preclinical trials, and raw materials so the products have been standardized (bpom, 2005; bpom ri, 2005). one included standardized herbal medicines is diapet, lelap, fitolac, and glucogarp. based on this, research was conducted on quercetin bioavailability tests and standardized herbal products containing a single guava leaf extract and this combination aims to determine the bioavailability of quercetin as a compound combined from guava leaf extract in stranded herbal products containing single natural ingredients and combinations. materials and methods research design this research design was carried out experimentally carried out in the laboratory (laboratory experimental research). where the preparation of ingredients, administration of drugs to animals, blood draws, and blood obtained are centrifuged so that plasma is obtained, then analyte levels are determined using hplc. tools and materials the ingredients used in this study include quercetin compounds (sigma aldrich), herbal medicines containing guava leaf extract containing quercetin (psidii and diapet), ethylene diamine tetra acetic acid (merck), tri chloro acetic acid (loba chemie), methanol (merck), aquabidest (onemed) and na cmc. the tools used in this study were hplc (thermo scientific ultimate 3000), sonicator (power), analytical balance (shimazu), centrifuge (hettich), syringe (onemed), measuring flask (iwaki), beaker (iwaki), measuring cup (pyrex), volume pipette (iwaki), alcohol swab (onemed), micropipette (eppendorf), stirring rod, vial, cotton, drip pipette, pipette pump, tube rack, blood tube, feeding tube, and bite block. procedures ▪ test animal preparation the test animals used were 3 male rabbits weighing 1.5 kg. before treatment, test animals are acclimatized for 7 days and satisfied for 12 hours before the experiment (mutiarahmi et al., 2021). ▪ ethical clearance ethical clearance is a formal statement issued by the research ethics commission for research involving living things that states that a research project can be carried out following the seven who standards. the ethics committee of the health service poltekkes of the ministry of health, jambi conducted the ethical review no.lb.02.06/2/183/2022. ▪ grouping of test animals this study using the cross-over design method is an experimental design where each test animal receives more than one treatment at different periods (yulion et al., 2023). ▪ suspension preparation of na cmc 1% na cmc 1% weighed as much as 1 gr then sealed with 5 ml of hot water while stirring until clear and shaped like jelly then add up to 100 ml (yulion et al., 2023). making quercetin suspension and standardized herbal medicines by weighing each ingredient and then sprinkling with 1% na cmc little by little while stirring and add up to 5 ml (usman & fikifandry, 2019) ▪ animal preparations satisfy test animals for 12 hours before oral administration. test animals get 3 treatments for the firstweek test animal a is given a dose of herbal medicine i with a dose of 0.24867 g, test animal b is given a dose of herbal drug ii 0.351 g test animal c is given with pure quercetin compounds after which test animals are rested for 1 week (usman & fikifandry, 2019). ▪ rabbit blood draw blood is taken from rabbit ear veins at minutes 5, 60, 120, 240, and .360 as much as 1 ml and accommodated in a tube containing 2 drops of edta then centrifuged for 10 minutes at a speed of 3000 rpm will see a clear top layer (plasma), then separated using a micropipette. add 1 ml of 20% tca then centrifuge at 3,000 rpm for 10 minutes so that a clear part is obtained (siswanto et al., 2017). ▪ preparation of quercetin raw solution weigh 10 mg of quercetin then put it in a 100 ml measuring flask then dilute it with methanol to the limit mark and homogenize it. ▪ creation of quercetin calibration curves make several series of quercetin concentrations from the parent solution of 2 ppm, 4 ppm, 6 ppm, 8 ppm, and 10 ppm, by pickpocketing the parent solution of pm as 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1 ml, and dissolve into a 10 ml measuring flask using methanol solvent until the limit mark. yulion et al. – quercetin bioavailability evaluation on standardized herbal … 595 table 1. grouping test animals by cross-over design method. rabbit week 1 2 3 1 a rest b 2 b rest c 3 c rest a data analysis the method used in this study is an experimental method with data on the absorbance results of quercetin compounds. plasma levels of quercetin in herbal products obtained regression equation. research has been conducted on herbal medicines containing combined and single guava leaf extract, increasing bioavailability in local white rabbits can be shown by the increase in the absorption rate constant tmax and auc value in each test animal given herbal medicine preparations containing combination and single guava leaf extract. results and discussion the results that can be obtained from the research that has been carried out are the measurement results of making quercetin calibration curves in methanol: water (59: 41) with a concentration of 2, 4, 6, 8, 10, wavelength 371nm and obtained the regression equation y = 0.876x 1.7217 with a regression value of 0.9985. results of quercetin level measurements carried out on local white rabbits at minutes 5, 60, 120, 240, and 360 minutes. the value of the result of the calculation of bioavailability parameters. tabel 2. standard curve equation data. concentration (µg/ml) auc (unit) 2 4 6 8 10 0,102 1,801 3,348 5,314 7,1054 figure 1. quercetin standard curve. table 3. determination of quercetin levels in rabbit plasma (quercetin). time (minutes) area (mau*min) quercetin levels (µg/ml) 5 0,00025 1,96569634 60 0,0005 1,9659817345 120 0,00145 1,96706583 240 0,0005 1,9659767345 360 0,00025 1,965696345 table 4. determination of quercetin levels in rabbit plasma (product a). time (minutes) area (mau*min) quercetin levels (µg/ml) 5 0,0011 1,9666664885 60 0,00205 1,96786529 120 0,00425 1,969520515 240 0,0005 1,965981712 360 0,0001 1,965525114 table 5. determination of quercetin levels in inch plasma (product b). time (minutes) area (mau*min) quercetin levels (µg/ml) 5 0,0001 1,959018255 60 0,0021 1,967808215 120 0,00025 1,977526139 240 0,0003 1,965753424 360 0,00015 1,96558219 table 6. the value of the result of the calculation of bioavailability parameters. pharmacokinetic parameters product a product b tmax value (hours-1) 1,486454 1,29224019 value cpmax(µg/ml) 1,96898659 1,349128791251 value auc 0-6 (µg/ml/hour) 10, 2615291 11,30810501 description: tmax : peak time of the drug in plasma, cpmax: peak levels of the drug in plasma, auc: area under curve 0,102 1,102 2,102 3,102 4,102 5,102 6,102 7,102 8,102 0 2 4 6 8 10 a u c ( u n it ) concentration (µg/ml) quercetin standard curve y = 0,876x – 1,7217 r2 = 0,9985 596 biology, medicine, & natural product chemistry 12 (2), 2023: 593-599 discussion this research began by preparing samples including 2 products containing guava leaf extract and pure quercetin, products purchased at one of the nearest minimarkets. after making preparations in sampling, then preparing three test animals aimed to facilitate carrying out this activity, as for the test animals are prepared in the form of healthy white rabbits weighing 1.5 kg and aged ± 9 months. animals are acclimatized for 1 week in the laboratory to adjust to their new environment (afriani et al., 2020; fithriyah et al., 2013; mutiarahmi et al., 2021; yulion et al., 2023) after that, making a master solution to measure wavelength, calibration curve, and retention time aims to find out the time interval required by the analyte from the time of injection until it exits the column and the signal is maximally captured by the detector. the results obtained in this treatment are in the form of wavelengths and quercetin calibration curves (kopjar et al., 2022; phattanaphakdee et al., 2022; rosydiati & saleh, 2019) after knowing the wavelength and calibration curve of quercetin (f. & a. b. husnia, 2021; maesaroh et al., 2018) then conducted a trial of recognition on test animals by taking rabbit blood in the auricularis vein (voigt et al., 2006) this blood draw is usually done in animals that have large blood vessels in the ears, usually in rabbits and pigs (usman & fikifandry, 2019). this blood collection uses a 1 cc syringe of as much as 1 ml accommodated in a centrifugation tube (sebayang et al., 2020a) that has been dripped edta (ethylene diamine tetraacetic acid). edta is an anticoagulant (nur ramadhani et al., 2019) that is widely used in the form of sodium salts or potassium salts that function to convert calcium ions from the blood into non-ionic forms. calcium itself is blood clotting so without calcium blood clotting would not occurred (aziz et al., 2019; devi et al., 2016; oikonomidis et al., 2021). after being drip edta is centrifugated again for 10 minutes at a speed of 3000rpm, it will be sliced between plasma and blood (burak et al., 2017a; svennebring, 2016). plasma is located at the top and blood is located at the bottom. the difference between plasma and blood is that blood consists of blood components in the form of liquid and solid liquid parts called plasma made of salt, water, and protein. meanwhile, the solid part of the blood is blood cells known as white blood cells, red blood cells, and platelets (saleh et al., 2019). after separation, separate the blood plasma using micropipettes little by little after separating add 1 ml of tca (tri chloro acetic acid) (mukhtiar et al., 2018; rahim et al., 2016). the addition of tca aims to separate/precipitate the proteins contained in the filtrate so that pure protein isolates are obtained, and centrifuged again for 10 minutes at a speed of 3000rpm (sebayang et al., 2020b). after all the preparation processes were carried out, the sample was measured using hplc with a wavelength of 370nm (nugraha et al., 2011; sahu et al., 2013; shebeko et al., 2018; valerio et al., 2009). in obtaining a good analysis method, it is also necessary to consider the chromatographic conditions between the columns, mobile phases, elution systems, flow rates, and detectors used. the hplc system is divided into two, namely normal phase hplc and reverse phase hplc (chitra et al., 2020; hermes et al., 2021a, 2021b; nugraha et al., 2011). the difference between the two systems lies in the stationary phase and the mobile phase. for normal phase hplc, the stationary phase is polar and the mobile phase is nonpolar and in reverse hplc, the stationary phase is nonpolar and the mobile phase is polar (angraini & desmaniar, 2020). the reverse-phase hplc system has the advantage of producing good chromatograms on less polar substances such as quercetin. the column that is often used is column c18 (angraini & desmaniar, 2020; chitra et al., 2020). in this quercetin separation, the methanol mobile phase is often used as nonpolar and water as a polar solvent. methanol is a commonly used mobile phase in systems (f. husnia & budiarti, 2021). another chromatographic condition that needs to be considered is the elution system. the elution system is divided into two, namely isocratic and gradient (ehlert et al., 2010; mitrović et al., 2020; shrivastava & gupta, 2012). the isocratic system is an elution system where the strength of the mobile phase is consistent from beginning to end while the gradient system is an elution system in which the strength of the mobile phase changes from the beginning to the end of the process. gradient elution systems are commonly used for the separation of large amounts of compounds in samples (ehlert et al., 2010; shrivastava & gupta, 2012). in addition to the elution system, the flow rate is also one of the chromatographic conditions that need attention. the flow rate is the speed of flow when passing through the stationary phase. determination of flow rate is included in the optimization process of the hplc method that needs to be done before analysis. flow rates that are too fast and too slow can result in imperfect separation. the flow rate reviewed in this study was 1 ml/min (moldoveanu & david, 2022; petrásková et al., 2020; stojanović et al., 2021). from these results, there is a value of the area obtained and can be calculated quercetin levels in each sample and a single quercetin and quercetin combination log level curve is made in the blood over time from the 2 groups have different curve shapes where the combined quercetin log curve is higher than the peak of the single quercetin log curve. this can be explained because the combined dose of quercetin is greater than a single so it affects the profile of quercetin levels in the blood (çelebier et al., 2018; phengvongsone et al., 2022; rudraraju et al., 2014). 3 kinds of parameters can be used to explain the pharmacokinetic profile of drugs in the body, namely yulion et al. – quercetin bioavailability evaluation on standardized herbal … 597 primary, secondary, and derivative parameters. primary parameters include ka, vd, and clearance parameters (hailat et al., 2022; hasler et al., 1997; li et al., 2021; tikhomirov et al., 2021). the ka parameter can explain the absorption kinetics of quercetin. it is known that there is an increase in the value of ka in the combination test animal group when compared to single-product test animals, this is because the increase in quercetin absorption rate causes the quercetin absorption rate to increase, therefore the value of the quercetin absorption rate constant in quercetin combination test animals increases. while test animals are given single quercetin the value of the absorption rate constant is smaller than those of combination herbal products. by increasing the value of the absorption rate constant of this herbal product, it can influence, the value of tmax, and cp max of the elimination constant and its auc value. because according to the literature, the ka parameter of a drug greatly affects the tmax value of the drug, the smaller the ka value, the greater the tmax value, and vice versa. the decrease in ka shows that the drug is absorbed slowly by the body, this is what causes a decrease in tmax and cmax quercetin in b test animals, besides that the increase in cpmax can also be caused by the addition or homogeneous mixing of an active ingredient (billah et al., 2014; larochelle et al., 1982; metwally et al., 1995; stanczyk et al., 1983). the auc parameter is a parameter that reflects the total amount of over-the-counter drugs reaching systemic circulation. the auc parameter value is closely related to the distribution volume parameter, the greater the distribution volume of a drug, the greater the auc volume of the drug (kusuma & rosalina, 2016; puranik et al., 2020). from the results of research that has been done that the auc value of single quercetin is greater than the value of a quercetin combination. as has been explained in the outline the research obtained has been following the hypothesis that administering standardized herbal medicines containing guava leaf extract can increase bioavailability in local white rabbits (burak et al., 2017b). conclusions research has been conducted on herbal medicines containing combined and single guava leaf extract, increasing bioavailability in local white rabbits can be shown by the increase in the absorption rate constant tmax and auc value in each test animal given herbal medicine preparations containing combination and single guava leaf extract. acknowledgements: thanks are given to all colleagues who have helped in the process of this research. authors’ contributions: rizky yulion designed the study. rizky yulion, yulianis, and suntri work together in research work in the laboratory. yulianis processed the data using statistics, suntri was prepared the samples used in the research. rizky yulion wrote the final script of the article. competing interests: there was no conflict of interest in this study funding: there is no cost support from any party for this research. the study was carried out at the own expense of the researchers. references afriani, t., yulion, r., andriani, m., syufyani, f., & fadri, d. 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(2023). acute toxicity ld 50 fraction ethyl acetate aquilaria malaccensis , ficus benjamina , mikania micrantha , and fraction water cinnamomum burmanii in mus musculus. biology, medicine, & natural product chemistry, 12(1), 55–60. https://doi.org/10.14421/biomedich.2023.121.55-60 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 47-55 | doi: 10.14421/biomedich.2020.91.47-55 issn 2540-9328 (online) prevalence of psychoactive substance use among nigerian male commercial vehicle drivers selected from the three major ethnic groups in nigeria tochukwu frank egwuatu*, onyekachi ogbonnaya iroanya, khalid olajide adekoya department of cell biology and genetics, faculty of science, university of lagos, nigeria corresponding author* tochukwufrankegwuatu@gmail.com manuscript received: 16 june, 2020. revision accepted: 23 july, 2020. published: 26 july, 2020. abstract the use of psychoactive substances for several reasons by commercial vehicle drivers has been documented as a major cause of increased incidences of traffic accidents globally. the study aimed at determining the prevalence of psychoactive substance use among nigerian male commercial vehicle drivers randomly selected from the three major ethnic groups in nigeria (igbo, hausa and yoruba). a structured questionnaire which included data on sociodemographic status and psychoactive drug use history was introduced to all participants. generic multi-drug urine dip card test kit and shimadzu chromatograph system (shimadzu corporation, kyoto, japan) were used for toxicological analyses. out of the 264 nigerian male commercial vehicle drivers who participated in this study, 111(42.0 %) had used illicit drugs in recent times, 55(20.8 %) took drugs for improved energy and 90(34.1 %) felt high after using illicit drugs. marijuana was the most abused substance; 77(29.2 %) followed by benzodiazepine; 34(12.9) across all ethnicities studied. across all ethnicities, hplc revealed that the hausa drivers had the highest concentration of psychoactive substances in their urine samples. these findings, reiterates the need for the nigerian anti-drug agency to develop new strategies in conducting a repressive drug war and extending the same to bus stations. keywords: psychoactive drug; accident; drivers; forensic toxicology; nigeria. introduction forensic toxicology is the science which applies the principles of analytical chemistry and clinical toxicology for medico-legal purpose where the results are likely to be used in court (wyman, 2012; smith and bluth, 2016). forensic toxicology covers human performances such as impaired driving due to drug consumption, use of steroids by athletes and workplace drug testing (jenkins, 2010, wyman, 2012). substance abuse is a patterned use of a drug in which the user consumes the substance in amounts or with methods which are harmful to themselves or others (nutt et al., 2010). psychoactive substances are classified into three main groups: those that alter perception, those that stimulate the brain and those that depress it (vij, 2011). in 2018, the prevalence of any drug use in nigeria is estimated at 14.4 per cent or 14.3 million people aged between 15 and 64 years and the extent of drug use in nigeria is comparatively high when compared with the 2016 global annual prevalence of any drug use of 5.6 per cent among the adult population (unodc, 2018). psychoactive substances are prevalent in biological samples taken from injured drivers at the emergency units of several hospitals. several studies have reported high prevalence rates not only of alcohol but also of medicinal and illicit drugs in such samples (bogstrand et al., 2011; drummer et al., 2012). a lot is known about the magnitude of problems associated with drink-driving and the effectiveness of related countermeasures, but much less is known about driving under the influence of other psychoactive substances. currently, there are no global estimates of deaths resulting from drug-driving, nor is the prevalence of drug-driving known. however, growing recognition of the problem of drug-driving has led to increased focus on this area among road safety policy-makers and researchers (emcdda, 2015). the issues linked with driving under the influence of alcohol and narcotic substances are subject to continuous interest not only for legal practitioners, but also for the police, prosecution, and medico-legal teams (emcdda, 2012). the use of psychoactive substances among commercial vehicle drivers is not only increasing but strangely predisposing the drivers and their passengers to health risks. there is however paucity of empirical data on the factors associated with this increase (yunusa et al., 2017). road traffic injuries claim more than 1.2 million lives each year and have a huge impact on health and development (gopalakrishnan, 2012). road traffic accidents are the leading cause of death among young https://doi.org/10.14421/biomedich.2020.91.47-55 48 biology, medicine, & natural product chemistry 9 (1), 2020: 47-55 people aged between 15 and 29 years. the risk of a road traffic death varies significantly by region, and there has been little change in the regional rates of death since 2010. injury and deaths arising from road traffic accidents are a major public health problem in developing countries where more than 85 % of all deaths and 90 % of disability-adjusted life years were lost from road traffic injuries (nantulya and reich, 2002). road traffic accidents have emerged as an important public health issue which needs to be tackled by a multidisciplinary approach (gopalakrishnan, 2012). the highest rates are still in the african region, while the european region has a rate far below the global average (9.3 per 100 000 populations, relative to the global rate of 17.4 (who, 2015). almost half of all deaths on the world’s roads are among those with the least protection motorcyclists, cyclists and pedestrians (who, 2015). motor vehicle accident is a significant cause of mortality and morbidity in nigeria, and driving under the influence of psychoactive substances to improve their performance and keep sleep at bay for as long as possible. the causes of motor vehicle accidents are varied and multi-factorial. however, the use of psychoactive substances plays a major role in the occurrence of motor vehicle accidents. studies in nigeria and other countries have shown a high prevalence of use of psychoactive substances, among various categories of drivers (adekoya et al., 2011). pela (1989) documented the use following drugs in decreasing order of frequency: alcohol, cigarettes, stimulants, cannabis (indian hemp) and sedativehypnotics by adolescents in benin, nigeria. males had been documented to have a higher prevalence of use of all the psychoactive substances (igwe et al., 2009; oshodi et al., 2010). the lifetime use of caffeine, alcohol, and tobacco has been noted to be significantly associated with male gender (akanni and adayonfo, 2015). a significant substance abuse problem in the undergraduate population in botswana with alcohol being the most commonly used substance has also been reported (olashore et al., 2018). the prevalence of psychoactive substances use among middle and high school students in the north center of morocco was comparable to the national prevalence. hence, there is need to initiate psychoactive substance prevention programs among school students which should be based on factors associated with psychoactive substance use (zarrouq et al., 2016). the study aimed at determining the prevalence of psychoactive substance use among nigerian male commercial vehicle drivers randomly selected from the three major ethnic groups in nigeria (igbo, hausa and yoruba). material and method study design, location and population this was a cross sectional study which employed both experimental and quantitative research design. the study adopted the purpose/judgmental sampling technique. the technique is a non-probability sampling method because the participants of interest which were drivers were selected from different motor parks located in anambra, abia (igbo–speaking), kaduna, sokoto (hausa–speaking), oyo and lagos states (yoruba– speaking) ethnic group in nigeria. the target population for the study was the male commercial driver’s population (motorcycle riders, tricycle riders, taxi and bus drivers). ethical approval prior to sample collection, the research details were explained to all the participants after which they willingly consented to participate in the study. questionnaires were completed by all the study participants and informed written consent was obtained. the study protocol was approved by the health research ethics committee (cmulhrec number: cmul/hrec/05/18/349) of the university of lagos, nigeria. permission was also requested and obtained from the national union of road transport workers (nurtw) in all sample states before the study was conducted. sample and data collection approximately 15 ml of urine was collected from each driver who participated in this study using sterile sample bottles. an on-site urinalysis was carried out using 5 ml of each urine sample to determine the general health status of the subjects. afterwards, the remaining urine samples of the subjects were transported to the laboratory for toxicological analysis. each driver in the various motor parks was interviewed until the desired sample size was attained. they responded to a sociodemographics and a semi-structured pro forma, which sought information on the type of substance commonly used, reasons for their use, usage frequency and related accident history. toxicological analysis the urine samples were tested using 10 panel generic multi drug urine dip card test kit. the kit tested for the presence of ten psychoactive drugs (methamphetamine (meth), cocaine (coc), oxycodone (oxy), morphine (mop), amphetamine (amp), methadone (mtd) and barbiturates (bar), marijuana (thc), benzodiazepines (bzo) and phencyclidine (pcp)). the tip of the kit was dipped into the urine sample and allowed to wait for minimum of 10 seconds. the kit had a positive and negative panel for each drug. each sample that tested positive to a particular drug would indicate a red line while negative showed two lines. eqwuatu et al. – prevalence of psychoactive substance use among … 49 quantification of drugs in polysubstance users high performance liquid chromatographic (hplc) analysis was performed with the use of a shimadzu liquid chromatograph equipped with a model lc2010aht gradient pump, cbm-20a system controller and shimadzu class-vp uv-visible detector (shimadzu corporation, kyoto, japan). all chemicals and reference standards (morphine, phencyclidine, benzodiazepine and δ9-tetrahydrocannabinol solution used for the hplc analysis were all of analytical grade. purchased from sigma-aldrich. the chromatographic separations were performed using a sepax br-c18 column (250×4.6 mm; sepax technologies, inc.) with 5µm particle diameter. the column temperature was set at 25°c, and the mobile phase was water (0.1 % formic acid) and acetonitrile (0.1 % formic acid). this was delivered at a flow rate of 0.8 ml/min. the wavelength of the detector was set at 254 nm, and the injection volume of each extract was either 10 µl for assay determination. data analysis all numerical data collected were statistically analysed using statistically package for the social sciences (spss) for windows version 20.0 software. frequency counts and percentage were generated for all variables, and statistical tests of significance were performed with correlation and regression analysis. significance was fixed at p ≤ 0.05 and highly significant if p ≤ 0.01. concentrations of illicit drugs in the urine samples of polysubstance users were deduced from the chromatograms using the formula; concentration of drug (mg/l ) = concentration of standard x area of drug area of standard results out of the 264 nigerian male commercial vehicle drivers who participated in this study, 72 (27.27 %) were hausas, 96 (36.36 %) each were igbos and yorubas respectively. in table 1, the data obtained from the questionnaires revealed that the lowest mean ± se value for age among the three ethnicities studied was 41.47 ± 1.02 years (igbo) while highest was 42.07 ± 1.56 years (hausa). the highest numbers of single (33(34.4 %)) and married (73(76.0 %)) drivers were obtained from yoruba and igbo drivers respectively. also, the lowest numbers of single (13(18.1 %)) and married (59(81.9 %)) drivers were obtained from hausa drivers. table 1. descriptive statistics of sociodemographic parameters in all drivers. parameters variables distribution across ethnicities (%) hausa (n=72) igbo (n=96) yoruba (n=96) age (years) minimum 19.0 21.0 24.0 minimum 72.0 67.0 72.0 mean ± s.e 42.07 ± 1.56 41.47±1.02 41.96 ± 1.21 marital status (%) single 13 (18.1) 23 (24) 33 (34.4) married 59 (81.9) 73 (76) 63(65.6) the study showed that 236 (89.4 %) were bus divers while 15 (5.7 %) and 13 (4.9 %) were car and tricycle drivers respectively. in table 2, igbo drivers had the highest (88 (91.7 %)) number of bus drivers while hausas had the least (63 (87.5 %)). hausa participants had the highest (28 (38.9 %) years) number of experienced divers while those with the highest number of individuals with the lowest (32 (33.3) years) years of experience were igbo drivers. yoruba participants had the highest (59 (61.5 %)) number of drivers who have had accidents followed by the hausa (27 (37.5 %)) and igbo (23 (24.0 %)) drivers respectively. in table 3, hausa drivers had the highest (54 (75.0 %)) number of illicit drug users while the igbo drivers had the lowest (20 (20.8 %)) number. marijuana was indicated to be the most used drug by the drivers compared to other psychoactive drugs across all ethnicities; hausa (36(50.0 %)), igbo (13(13.5 %)) and yoruba (36(37.5 %)). across all ethnic groups, increased energy was observed to the major reason why drivers used illicit drugs. additionally, smoking was indicated as the major mode of administration of illicit drugs among the drivers across all ethnic groups. feeling high after drug use was indicated as the major post administration effect of illicit drug use across all ethnic groups. 50 biology, medicine, & natural product chemistry 9 (1), 2020: 47-55 table 2. descriptive statistics of driving experience and accident history parameters in all ethnicities. parameters variables frequency across ethnicities ( %) hausa igbo yoruba vehicle type bus 63 (87.5) 88 (91.7) 85 (88.5) car 7 (9.7) 0 (0) 8 (8.3) tricycle 2 (2.8) 8 (8.3) 3 (3.1) driving experience ˂ 5 years 14 (19.4) 32 (33.3) 22 (22.9) ˂ 10 years 30 (41.7) 38 (39.6) 52 (54.2) ≥ 10 years 28 (38.9) 26 (27.1) 22 (22.9) accident history yes 27 (37.5) 23(24.) 59 (61.5) no 45 (62.5) 73 (76) 37 (38.5) number of times ˂ 5 times 20 (27.8) 20 (20.8) 50 (52.1) ˂ 10 times 5 (6.9) 3 (3.1) 8 (8.3) ≥ 10 times 2 (2.8) 0 (0) 1 (1.0) none 45(62.5) 73 (76) 37(38.5) table 3. descriptive statistics of psychoactive drug use history parameters in all drivers. parameter variable frequency across ethnicities (%) hausa (n=72) igbo (n=96) yoruba (n=96) have you used drugs before? yes 54 (75) 20 (20.8) 37 (38.5) no 18(25) 76 (79.2) 59 (61.5) type of drug maijuana 36 (50) 13 (13.5) 36 (37.5) tablet 18 (25) 6 (6.3) 1 (1) syrup 0(0) 1 (1) 0 (0) none 18(25) 76 (79.2) 59 (61.5) reason for drug use alertness 6 (8.3) 4 (4.2) 10 (10.4) increased energy 26 (36.1) 10 (10.4) 19 (19.8) improve sexual performance 2(2.8) 1 (1.0) 4 (4.2) pleasure 18 (25) 2 (2.1) 1 (1) peer pressure 2(2.8) 3 (3.1) 3 (3.1) none 18(25) 76 (79.2) 59 (61.5) did you use it in the past 90 days? yes 54 (75) 18 (18.8) 36 (37.5) no 0(0) 2 (2.1) 1 (1) none 18 (25) 76 (79.2) 59 (61.5) frequency of use everyday 35 (48.6) 18 (18.8) 32 (33.3) weekends 8 (11.1) 2 (2.1) 4 (4.2) weekly 11(15.3) 0 (0) 1 (1) none 18 (25) 76 (79.2) 59 (61.5) last time used last 24 h 33 (45.8) 19 (19.8) 34 (35.4) 1-3 day(s) ago 12 (16.7) 1 (1) 2 (2.1) last week 9 (12.5) 0 (0) o (0) ≥ 1 month 0 (0) 0 (0) 1 (1) none 18 (25) 76 (79.2) 59 (61.5) mode of administration smoking 35 (48.6) 14 (14.6) 36 (37.5) ingestion 19(26.4) 6 (6.3) 1 (1.0) none 18 (25) 76 (79.2) 59(61.5) do you feel addicted to it? yes 13 (18.1) 6 (6.3) 15 (15.6) no 41(56.9 14 (14.6) 22 (22.9) none 18 (25) 76 (79.2) 59 (61.5) how do you get it? park sellers 52 (72.2) 14 (14.6) 29 (30.2 private suppliers 2(2.8) 6 (6.3) 8 (8.3) none 18 (25.) 76 (79.2) 59 (61.5) is the drug costly? yes 9 (12.5) 3 (3.1) 3 (3.1) no 45(62.5) 17 (17.7) 34 935.4) none 18 (25) 76 (79.2) 59(61.5) can you do without it? yes 21 (29.2) 12 (12.5) 23 (24) no 33 (45.8) 8 (8.3) 14 (14.5) none 18 (25) 76 (79.2) 59 (61.5) feeling after use high 35.00 (48.6) 19 (19.8) 36 937.5) sleepy 19 (26.4) 1 (1.0) 1 (1) none 18 (25) 76 (79.2) 59 (61.5) eqwuatu et al. – prevalence of psychoactive substance use among … 51 table 4 shows the distribution of the participants by psychoactive drug use history. out of the 264 participants across all ethnic groups, 111(42.0 %) had used illicit drugs in recent times while 153(58.0 %) did not, 55(20.8 %) abused illicit drugs for increased energy and 85(32.2 %) used illicit drugs daily. additionally, 90(34.1 %) of the respondents indicated that they felt high after taking illicit drugs. table 4. distribution of the respondents by psychoactive drug use history. parameter variable frequency ( %) have you used drugs before? yes 111 (42) no 153 (58) reason for drug use alertness 20 (7.6) increased energy 55 (20.8) improve sexual performance 7 (2.7) pleasure 21 (8) peer pressure 8 (3) none 153 (58) frequency of use everyday 85 (32.2) weekends 13 (4.9) weekly 13(4.9) none 153 (58) mode of administration smoking 85 (32.2) ingestion 26 (9.8) none 153 (58) do you feel addicted to it? yes 35 (13.3) no 76 (28.8) none 153 (58) how do you get it? park sellers 94 (35.6) private suppliers 17 (6.4) none 153 (58) feeling after use high 90 (34.1) sleepy 21 (8) none 153 (58) n= 264 pearson correlation analysis was carried out to determine the relationship between sociodemographics, driving experience history and psychoactive drug use history. across all ethnic groups (table 5), ethnicity correlated with drug use, drug name, reason for drug use, frequency of drug use, feeling of addiction, acquisition of drugs and cost and post administration effects of drug use at 0.01 level of significance. however, there was no correlation between ethnicity and mode of administration (p≥0.05). accident history correlated with drug use, drug name, reason for drug use, frequency of drug use, mode of administration, feeling of addiction, acquisition of drugs and cost and post administration effects of drug use at 0.01 level of significance. all urine samples were analysed using drug test kits. in table 6, marijuana 77(29.2 %) was the most abused substance followed by benzodiazepine 34(12.9 %) across all ethnicities studied. marijuana had the highest 32(44.4 %) number of users among the hausa drivers, followed by benzodiazepine 28(38.9 %) while the least was morphine 3(4.2 %). for the igbo drivers, the use of marijuana was the highest 14(14.6 %), followed by methadone 10(10.4 %) with the least being morphine and phencyclidine 1(1 %). amongst the yoruba drivers, marijuana was also most abused substance 31(32.3 %) followed by benzodiazepine 4(4.2 %) while the least abused drugs were barbiturate and methadone 1(1 %) each. across all ethnicities, hausa drivers had the highest number of polysubstance users compared to other ethnic groups as shown in table 7. 52 biology, medicine, & natural product chemistry 9 (1), 2020: 47-55 table 5. correlation between sociodemographics, driving experience history and psychoactive drug use history in ethnic groups. p a ra m et er d ru g u se n a m e o f d ru g s r ea so n f o r d ru g u se u se d p a st 9 0 d a y s f re q u en cy o f u se l a st t im e u se d m o d e o f a d m in is tr a ti o n f ee li n g o f a d d ic te d h o w d o y o u g et i t is t h e d ru g c o st ly c a n y o u d o w it h o u t it f ee li n g a ft er u se age 0.02 -0.02 -0.09 -0.02 0.01 -0.01 -0.02 -0.05 -0.00 0.00 0.03 -0.04 ethnicity 0.26** 0.20** 0.18** 0.26** 0.19** 0.22** 0.12 0.18** 0.39** .026** 0.17** 0.19** marital status -0.02 -0.02 -0.07 -0.04 0.02 0.00 -0.03 -0.07 -0.03 -0.02 -0.02 -0.03 vehicle type -0.04 -0.02 .028 0.01 -0.05 -0.05 -0.02 -0.02 0.01 -0.04 -0.08 -0.05 driving experience -0.01 -0.04 -0.09 -0.05 -0.02 -0.02 -0.03 -0.02 -0.03 0.00 -0.00 -0.03 accident history 0.33** 0.37** 0.34** 0.31** 0.34** 0.35** 0.37** 0.36** 0.18** 0.29** 0.31** 0.36** number of times 0.26** 0.30** 0.28** 0.25** 0.26** 0.27** 0.30** 0.30** 0.14* 0.25** 0.24** 0.28** * = correlation is significant at the 0.05 level. ** = correlation is significant at the 0.01 level. table 6. frequency distribution of drugs across ethnic groups rom toxicological testing. ethnicity drug thc (%) mop (%) bar (%) coc (%) oxy (%) amp (%) met (%) pcp (%) bzo (%) mtd (%) hausa (n=72) 32 (44.4) 3 (4.2) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 4 (5.6) 28 (38.9) 0 (0) igbo (n=96) 14 (14.6) 1 (1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (1) 2 (2.10) 10 (10.4) yoruba (n=96) 31 (32.3) 3 (3.1) 1 (1) 0 (0) 0 (0) 0 (0) 2 (2.1) 2 (2.1) 4 (4.2) 1 (1) all ethnicities (n=264) 77 (29.2) 7 (2.7) 1 (0.4) 0 (0) 0 (0) 0 (0) 2 (0.8) 7 (2.7) 34 (12.9) 11 (4.2) key: thc = tetrahydrocabinnol bar = barbiturate pcp = phencyclidine coc = cocaine mop = morphine amp = amphetamine mtd = methadone bzo = benzodiazepines oxy = oxycodone met = methamphetamine table 7. frequency distribution of polysubstance users across all ethnic groups. ethnicity drug bzo+thc (%) thc+pcp (%) thc+mop (%) bzo+thc+pcp (%) bzo+thc+mop (%) pcp+thc +mop (%) bzo+mop+ pcp (%) hausa (n=72) 1 (1.4) 1 (1.4) 0 (0) 3 (4.2) 2 (2.8) 0 (0) 1 (1.4) igbo (n=96) 2 (2.1) 1 (1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) yoruba (n=96) 1 (1) 0 (0) 2 (2.1) 1 (1) 1 (1) 1 (1) 0 (0) all ethnicities n=264 4 (1.5) 2 (0.8) 2 (0.8) 4 (1.5) 3 (1.1) 1 (0.4) 1 (0.4) key: thc = tetrahydrocabinnol, bzo = benzodiazepine, mop = morphine, pcp = phencyclidine chromatographic analysis was performed using the urine samples of participants who tested positive to two or more drugs. table 8 shows that the polysubstance users used mainly marijuana, benzodiazepine, morphine and phencyclidine. across all ethnicities, the hausa drivers had the highest concentration of psychoactive substances in their urine samples. the chromatographs did not show thc only in samples h14, h17 and ig58. tetrahydrocabinnol and bzo only were not detected by hplc in samples h53 and h66. in samples h16 and y22, bzo only and mop only were not detected by hplc respectively. also, bzo and mop were not detected by hplc in sample h07 but pcp was detected. eqwuatu et al. – prevalence of psychoactive substance use among … 53 table 8. concentrations of different drugs in polysubstance users. ethnicity subject drug concentrations in urine (mg/l) pcp thc bzo mop hausa h07 1321.3 671.7 0 0 h12 0 210.6 15.9 0 h14 0 0 0.02 0 h16 0 0.2 0 0.5 h17 0 0 0.1 93 h40 10.7 8 0. 0 h53 122.9 0 0 0 h66 167.4 0 0 0 igbo ig25 34.7 8.7 0 0 ig36 0 0.9 1.2 0 ig58 0 0 2.3 0 yoruba y03 108.1 25.6 0 1.2 y04 0 19.5 0 32.8 y06 0.02 0.8 1.2 0 y10 0 0.8 1.5 0 y21 0 0.1 0.3 0.2 y22 0 0.1 0 0 standards: pcp = (2.5) thc = (2.4) mop = (3.99) bzo = (2.39) key: mop = morphine pcp = phencyclidine bzo = benzodiazepines thc = tetrahydrocabinnol h = hausa ig = igbo y = yoruba discussion the ages of the commercial vehicle divers who participated in this study ranged from 19 to 72 years old. majority of the drivers studied fell with the age range (15-64 years) given by unodc, (2018) as the age range that has the highest prevalence of drug use. across all ethnic groups, 111 (42.0 %) of the respondents had used illicit drugs in recent times and this value is higher than what was reported in the report of iroanya et al., (2018) who reported that only 5.7 % of their subjects had taken other stimulants aside alcohol in recent times. the study revealed that 55 (20.8 %) abused illicit drugs for increased energy, 20(7.6 %) for alertness, 21(8.0 %) for pleasure and 85 (32.2 %) used illicit drugs daily. additionally, 90 (34.1 %) and 21(8.0 %) of the respondents indicated that they felt high or sleepy respectively after taking illicit drugs. these findings agree with the report of yunusa et al., (2017) who stated that the desires to relax/sleep after a hard days job (84.8 %), work hard (48 %), relieve stress (81 %), relieve anxiety (66.5 %) and pleasure (72 %) were the major factors associated with the abuse of substances by the respondent. this study revealed that 52(72.22 %) of drivers from the hausa ethnicity used one or more psychoactive substance(s) and this agrees with the study of yunusa et al., (2017) who reported that eight out of every ten (81.1 %) of drivers who participated in their study has abused a substance. this study showed that 25(26.04 %) of igbo drivers used one or more drug(s) and this is lower than the findings of aniedu and okonkwo, (2008) who reported the prevalence of psychoactive drug use amongst taxi drivers to be 85.4 %. it was also observed that 36(37.5 %) of the yoruba drivers also used one or more psychotropic substance(s) and this is also higher than the report of iroanya et al, (2018) who reported that 24.6 % of their participants tested positive to at least one psychoactive drug in a study conducted at two bus stations in lagos state, nigeria. additionally, 113(42.8) of the participants across all ethnic groups tested positive to one or more psychoactive substance(s). this is lower than the finding of hamman et al., (2018) who documented that 57.7 % of the subjects tested positive for substance abuse in a study conducted among professional minibus drivers at zagazig city, sharqia governorate, egypt. the study showed that the substances commonly used by the drivers were marijuana (29.2 %), benzodiazepine (12.9 %), methadone (4.2 %), phencyclidine (2.7 %), morphine (2.7 %) and methamphetamine (0.8 %). this is significantly higher than the findings of bogstrand et al., (2012) who reported that most commonly found substances among drivers in normal traffic were cannabis (0.7 %), benzodiazepines (0.8 %), methadone (0.1 %), methamphetamine (0.2 %) and morphine (0.0 %). from this study, marijuana was the most used drugs among the drivers who participated in the study. this agrees with the report of unodc, (2018) which reported that cannabis was the most commonly used drug in the nigerian population. hamman et al., (2018) also reported that the most common abused substance was cannabis which represented 80 % of the positive participants. cannabis is the world’s most commonly used illicit drug which occupies fourth place in worldwide popularity amongst psychoactive drugs, after caffeine, nicotine and alcohol (vij, 2011). it was also observed from this study that the use of benzodiazepine by the subjects was also common after marijuana’s use. this disagrees with the report unodc (2018) which stated that cannabis’ use was followed by opioids. interestingly, the use of methodone was highest among the igbo drivers. this disagrees with the findings of aniedu and okonkwo, (2008) who reported that the commonest drugs used by taxi drivers in enugu were alcohol, tobacco and central nervous system stimulants such as coffee and kolanut. it was observed that none of the drivers across all ethnic groups tested positive to cocaine, oxycodone and amphetamine. this might be due to the unavailability of these drugs within their environments, moreover, they are also very expensive. this however is in disagreement with the findings of dini et al., (2019) who documented that 21.3 % and 2.2 % of the truck drivers who took part in their study consumed amphetamine and cocaine respectively. this study revealed that the hausa drivers had the highest number of polysubstance users. the concentrations of pcp, thc and bzo were generally higher in the hausa 54 biology, medicine, & natural product chemistry 9 (1), 2020: 47-55 drivers than other ethnic groups. this agrees with the report of ndlea (2011) regarding the urgent need for coordination and collaboration between tiers of government to tackle the scourge of drug abuse in the northern nigeria. it has been predicted that the use of illicit drugs kill around 0.2 million people each year, shattering families and bringing misery to thousands of other people. illicit drugs undermine economic and social development and contribute to crime, instability, insecurity and the spread of hiv (ndlea, 2011). conclusion and recommendation the prevalence of hypertension among the nigerian male commercial drivers in the three major ethnicities studied is high and of public health importance. the threat of drug abuse is real and enormous. hence, the nigerian government and relevant non-governmental organizations should come up with viable strategies on how to identify areas with high drug addiction prevalence, train specialist staff to handle drug abuse issues, educate drivers and the public on the negative effects of drug abuse. this study has revealed that a sizable proportion of the nigerian male commercial drivers operating in the selected bus stations in anambra, abia, kaduna, sokoto, oyo and lagos states, nigeria use psychoactive substances as diagnosed by the drug kits. it is very crucial for the nigerian anti-drug agency (national drug law enforcement agency (ndlea)) to develop new tactics in conducting a repressive drug war, and make attempts to understand the psychological world of addicts in order to formulate appropriate policies against drug abuse. there can be no better time and appropriate turning point than now. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references akanni, o. o. and adayonfo, e. o. 2015. correlates of psychoactive substance use among nigerian adolescents. sahel medical journal. 18(4): 192 – 199. adekoya, b. j., adekoya, a. o., adepoju, f. g. and owoeye. j. f. a. 2011. driving under influence among long distance commercial drivers in ilorin, nigeria. international journal of biological and medical research. 2(4): 870 – 873. aniebu, p. n. and okonkwo, k. o. b. 2008. prevalence of psychoactive drug use by taxi drivers in nigeria. journal of college of medicine. 13: 48-52 bogstrand, s. t., normann, p. t., rossow, i, larsen, m., morland, j. and ekeberg, o. 2011. prevalence of alcohol and other substances of abuse among injured patients in a norwegian emergency department. drug and alcohol dependence. 117:132–138. bogstrand, s. t., gjerde, h., normann, p. t., rossow, and ekeberg, o. 2012. alcohol, psychoactive substances and 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psychoactive substances use and associated factors among middle and high school students in the north center of morocco: a crosssectional questionnaire survey. bmc public health. 16(468): 1–9. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 97-103 | doi: 10.14421/biomedich.2020.92.97-103 issn 2540-9328 (online) microbial qualities of nkwuaku and ogbaru streams located in awgu local government area in enugu state, nigeria osita gabriel appeh1,*, tochukwu frank egwuatu2, chiamaka maryann ogbunta1 1department of microbiology, college of natural science, michael okpara university of agriculture umudike, p.m.b. 7267, umuahia, abia state, nigeria 2department of cell biology and genetics, faculty of science, university of lagos, akoka-yaba lagos state, nigeria corresponding author* ositaappeh@yahoo.com manuscript received: 02 august, 2020. revision accepted: 06 november, 2020. published: 22 november, 2020. abstract water samples from nkwuaku and ogbaru streams located in awgu local government area in enugu state, nigeria were evaluated for the presence of microbial contaminants. both samples were subjected to physicochemical analysis, total bacterial count and isolation of microbial pathogens, using conventional techniques. physicochemical characteristics of both water samples showed that nkwuaku stream had the least total dissolved solid value of 210mg/l while the highest value of 270mg/l was recorded for ogbaru stream. total heterotrophic bacteria count revealed that ogbaru stream water sample had a higher bacterial population of 5.1x107cfu/ml than nkwuaku stream water sample which had a bacterial population of 4.2x107cfu/ml. a total number of twelve (12) microorganisms were isolated from both water samples analyzed. eight (8) bacteria genera isolated include: escherichia coli, enterococcus faecalis, salmonella spp., klebsiella spp., staphylococcum aureus, pseudomonas aeruginosa, proteus spp. and campylobacter spp. four (4) fungi were isolated and they include: aspergillus niger, aspergillus flavus, aspergillus fumigatus and fusarium oxysporum. escherichia coli, salmonella spp., staphylococcus aureus and proteus spp. had the highest percentage occurrence (16.66%) each while enterococcus faecalis, klebsilla spp., pseudomonas aeruginosa and campylobacter spp. had the least percentage occurrence (8.33%) each. also, aspergillus recorded the highest percentage occurrence of 40% for fungi while the least percentage (20%) was recorded for aspergillus niger, fusarium oxysporium and aspergillus fumigatus each. this study reveals that microbial qualities of these streams render them unfit for human consumption as sources of portable water although they can be used for other household purposes. keywords: microorganisms; contaminants; physicochemical; pathogens; streams. introduction water is the most vital element among the natural resources; it is the most indispensable need for existence of all living things. its decreasing availability in terms of quality and quantity has been a major public health concern in africa, particularly in nigeria (who, 2004; saraveanan and peter, 2009). according to a recent unicef report, about 80 million people in asia and africa are living without access to safe water. consequently, this has caused many people to suffer from various diseases (tanwir et al., 2003). in developing countries such as nigeria, most of the rural community lack access to potable water supply and rely mainly on river and stream sources for their household use and other purposes (banwo, 2006). many water sources in developing countries are unhealthy because they contain harmful physical, chemical and biological agents. unfortunately, many of the available water sources are not potable without some form of treatment which is seldom or not available in most rural settings which expose the rural populace to waterborne diseases (oketola et al., 2006). in some rural areas in nigeria, domestic wastes, sewage and faeces are discharged into streams which also serve as their water sources for daily needs. when the load of organic matter or wastes is too heavy, the self-purification power of the stream are unable to remove these materials added and there will be pollution of these water sources which can be dangerous to human and the environment as a whole (adetokunbo and grilles, 2003). the microbiological quality of drinking water is of a great primary importance, and the monitoring of bacterial indicators such as total coliform. microbial indicators have been used worldwide to indicate if human wastes have contaminated water body. microbes typically utilized are those that are found in elevated concentrated in human faecal coliform, escherichia coli and enterococci (brooks et al., 2006). an additional indicator, clostridium perfringes can be used for monitoring stream water quality (egberongbe et al., 2010). the outbreaks of diarrhea or gastroenteritis in rural communities have all been attributed to the consumption of water of poor microbial https://doi.org/10.14421/biomedich.2020.92.97-103 98 biology, medicine, & natural product chemistry 9 (2), 2020: 97-103 quality (ashbolt, 2004). it is therefore not an option but an imperative to critically monitor the quality of water supply in rural areas in order to further highlight their despicable water supply situation and to provide the impetus for sustainable government intervention (gucker et al., 2006). the aim of this study was to evaluate the microbial qualities of nkwuaku and ogbaru streams located in awgu local government area in enugu state, nigeria. materials and methods study area the study took place in awgu local government area of enugu state, nigeria. the local government is made up of towns such as agbogugu, isu-awa, ituku, ihe, ogbaku, owelli, ogugu, agbudu, amoli, mmaku, ugbo, obeagu, mgbidi, ugwueme, nkwe, ezere, awugu, nenwenta, awgunta and mgbowo. it has clay and stony lands with hilly topography. subsistence farming, petty trading, livestock rearing, palm wine tapping and stone quarrying are the major occupation of the people. there is no notable industry located in this area and its environs. collection of samples water samples for this study were collected from two different streams (nkwuaku and ogbaru) within the study area. the samples were collected in sterile containers at different collection points and analyzed within 24 hours for physicochemical and bacteriological qualities. sterilization of materials all glassware which were used for this study were properly washed and rinsed using distilled water and sterilized in a hot air oven at 250oc for 1 hour. these glassware included glass petri-dishes, test tubes, pipette, bijou bottles and mccartney bottles. determination of physicochemical characteristics of the sample the physicochemical properties which were examined included temperature, ph, taste, turbidity, colour, conductivity, odour, total solids, total dissolved solids, total suspended solids, acidity and alkalinity. determination of ph, temperature and conductivity the ph was determined using a ph meter (hi96107 hanna ph meter), which was standardized with a neutral buffer solution of ph 7.0 in a beaker. a 100ml aliquot of each sample was measured into a beaker and the ph probe was immersed into the sample. this was allowed for some minutes until a stable reading was obtained, and value was recorded. an aliquot of 50ml of each sample was measured into a 100ml beaker and a simple mercury-in-glass thermometer calibrated in degrees centigrade immersed in the water. the reading on the thermometer for each sample was recorded. conductivity of the two samples was determined using a digital conductivity meter (4520jenway, serial no. 01263). the meter was turned on and allowed to warm up for about 15 minutes. it was then standardized with 0.01m kcl solution where a conductivity value of 1413 microsiemen per-centimetre was obtained. the electrode was thoroughly rinsed with distilled water, wiped and then dipped into 100ml of each water sample in the beaker and left for some minutes to obtain a stable reading. the value for each sample was recorded. determination of colour and turbidity colour was determined immediately each sample arrived the laboratory. this was carried out after the two samples had been allowed to rest on a bench to attain room temperature. approximately 25 ml of water from each of the samples was measured into the sample cell for colour analysis. colour instrument (hach dr/890) was zeroed with 25ml distilled water before samples were determined and the readings were recorded. approximately 10ml of distilled water was measured into the sample cell of a turbidity meter (hanna, lp2000), which was used to zero the instrument. later, 10ml each from samples was measured into the same cell and the readings were recorded. determination of total solids (ts) and total dissolved solids by gravimetric method total solids were determined as the residues left after evaporation of the unfiltered samples. approximately 10ml of each of the two unfiltered samples was taken and evaporated in an evaporating dish in an oven at a temperature of 103-105oc for two and half hours. the evaporating dish was then cooled in desiccators and weighed. total dissolved solids were determined as the residues left after evaporation of the filtered samples, approximately 10ml of filtered sample from each of the samples was measured into a pre-weighed evaporating dish. the residues collected were then dried in an oven at temperature of 103-105oc for one hour and final weights were taken after cooling the evaporating dish in a desiccator. total solids were calculated using the formula given below: total solids (mg l⁄ ) = (𝑊1 − 𝑊2) x 1000 𝑉 where: 𝑊1 : initial weight of dried residue + dish in mg 𝑊2 : final weight of the dish in mg 𝑉 : volume of sample used in ml determination of total suspended solids (tss) for total suspended solid (tss), 10ml each of the water samples were filtered through a pre-weighed filtered appeh et al. – microbial qualities of nkwuaku and ogbaru streams … 99 paper. the filtered papers were dried at 103-105oc in oven and tss was determined by the following formula. tss (mg l⁄ ) = filter post weight – filter pre weight x 1000 volume of sample (ml) determination of alkalinity and acidity to 100ml of each of the water samples, 3 drops of phenolphthalein indicator was added. the samples were titrated with 0.02n h2s04 to ph 8.3 and phenolphthalein alkalinity was estimated (phenolphthalein indicator was changed colour from pink to colourless at ph 8.3). finally, the phenolphthalein alkalinity of water was calculated as follows: alkalinity (mg l⁄ ) = 𝐴 x 𝑁 x 50 x 1000 𝑉 where: 𝐴 : volume of h2s04 in ml 𝑁 : normality of h2s04 used to titrate 𝑉 : volume of sample used in ml determination of acidity to 100ml of each of the water samples, 3 drops of methyl orange indicator was added. the samples were titrated with 0.02n standard naoh to ph 8.3. (methyl orange indicator was changed colour from orange-red to yellow at ph 8.3). the acidity of water was calculated as follows: acidity (mg l⁄ ) = 𝐴 x 𝑁 x 50 x 1000 𝑉 where: 𝐴 : volume of naoh titrant used in ml 𝑁 : normality of naoh used 𝑉 : volume of sample used in ml microbiological analysis preparation and inoculation of samples serial dilution of each sample was made in sterile distilled water by inoculating 1ml of each sample into the first test tube containing 9ml of distilled water. the solution was mixed thoroughly and 1ml of it was transferred into the second test tube containing 9ml of distilled water. the procedure was repeated with all the remaining tubes. 0.1ml was pipetted out from 105 of the diluted factor and was inoculated in the plate using spread plate technique. the plates were incubated for 24 hours at 25oc for bacterial isolation and at 20oc for 3 days for fungal isolate using potato dextrose agar. the number of colonies were counted and recorded for both bacteria and fungi. purification of isolates the resulting colonies from the nutrient agar medium, macconkey, potato dextrose agar, eosin methyl blue agar, manitol salt agar, blood agar and salmonella – shigella agar plates were purified by sub-culturing on freshly prepared nutrient agar plates. the plates were incubated at 25oc for 24 hours for bacterial incubation and 20oc for 3 days for fungi. after the appropriate periods of incubation, the resulting discrete colonies were transferred onto agar slants in mccartney bottles and kept in the refrigerator as stock culture for subsequent tests during identification. characterization and identification of bacterial isolates bacterial isolates were analyzed based on morphological features, gram staining and other biochemical characterization which included citrate, indole, catalase, coagulase, motility, oxidase, urease, glucose, lactose, fructose and mannitol tests. confirmation of biochemically characterized bacteria was made using the most probable number (mpn) technique. identification of fungal isolates fungal isolates were identified based on their colonial morphology and cell morphology. a small portion of fungal growth was isolated with a sterile wireloop and placed on a grease free glass slide and teased with a drop of distilled water. a drop of lactophenol cotton blue stain was added and covered with a greased free cover slip. the slide was observed using x10 and x40 objective lenses. lactophenol cotton blue wet mount preparation is the most widely used method of staining and observing fungi. results table 1 shows the various physicochemical characteristics exhibited by the stream water samples. ogbaru stream had higher colour value of 10hu than nkwuaku stream which had a colour value of 6hu. nkwuaku stream sample had no objectionable odour while ogbaru stream sample had an objectionable odour. nkwuaku stream had a lower ph value of 6.7 while ogbaru had a ph value of 8.3. turbidity was higher in ogbaru stream and lower in nkwuaku stream sample. ogbaru stream had a higher temperature of 27oc than nkwuaku (26oc). conductivity was higher in ogbaru stream sample (678 𝜇s/cm) than in nkwuaku stream sample (468 𝜇s/cm). the value for total solids was lower (430 mg/l) in nkwuaku stream and higher (530 mg/l) in ogbaru sample. nkwuaku sample recorded a lower total dissolved solids (210 mg/l) than ogbaru (270 mg/l). total suspended solids was 220 mg/l in nkwuaku stream and 260 mg/l in ogbaru stream sample. ogbaru stream sample’s acidity was 4.0 mg/l and that of ogbaru stream was 6.0 mg/l. alkalinity was recorded to be 5.0 mg/l in ogbaru sample and 6.0 mg/l in nkwuaku sample. 100 biology, medicine, & natural product chemistry 9 (2), 2020: 97-103 table 1. physicochemical characteristics of the stream water samples in awugu local government area, enugu state. parameters sample a nkwuaku stream sample b ogbaru stream ph 6.7 8.3 temperature (oc) 26 27 conductivity (𝜇s/cm) 468 678 colour (hu) 6.0 10.0 turbidity (ntu) 8.0 10.0 odour u o total solids(mg/l) 430 530 total dissolved solids(mg/l) 210 270 total suspended solids(mg/l) 220 260 alkalinity(mg/l) 6.0 5.0 acidity(mg/l) 4.0 6.0 key: u = unobjectionable; o = objectionable; hu = hazen unit; ntu = naphelometric turbidity unit. table 2 shows the colonial characteristics of each isolates on nutrient agar, eosin methylene blue agar, macconkey agar, manitol salt agar, salmonella-shigella agar and blood agar. the isolates which were obtained include staphylococcus aureus, enterococcus faecalis, escherichia coli, salmonella species, klebsiella species, pseudomonas aeruginosa, proteus species and campylobacter species. table 3 shows the total bacterial and fungal plate counts of the stream samples. the total bacteria count revealed that ogbaru stream had a higher total bacteria count of 5.1 x 107 cfu/ml than nkwuaku stream which had 1.2 x 107cfu/ml. this indicates that nkwuaku stream sample had the lower bacterial indicators compared with ogbaru stream sample. also, the total fungal count revealed that nkwuaku had a higher total fungi count (4.0 x 107 cfu/ml) than ogbaru stream (1.6 x 107 cfu/ml). table 2. colonial characteristics of isolates from ogbaru and nkwuaku stream water on different growth media. isolate code media plates na emb mac msa ssa ba og1 smooth, golden yellow, convex colonies pink, smooth, convex and opaque colonies yellow, smooth, convex and opaque colonies light golden yellow, smooth, convex and hemolytic colonies og2 thick, grayish white, moist, smooth & opaque colonies green metallic sheen smooth colonies moist, smooth, flat & pink colonies slight growth pink colonies gray, moist & hemolytic colonies og3 low convex, smooth, grayish white translucent colonies low convex, smooth, colourless and transparent colonies black colonies with offensive odour low convex, smooth, grayish white, translucent and non-hemolytic colonies og4 irregular, low convex, smooth, mucoid, greenish yellow opaque colonies metallic sheen and glass appearance colonies low convex, smooth, mucoid, colourless transparent colonies irregular slight growth & nearly colorless colonies flat, smooth, mucoid, grayish white, opaque, hemolytic colonies nk1 whitish, round raised, glistering colonies light pink raised large colonies yellow colonies white nonhemolytic colonies nk2 mucoid, grayish white, raised opaque colonies convex, mucoid, pink translucent colonies convex, mucoid, pink-red opaque colonies smooth slight growth, pink colonies mucoid, grayish white, opaque non hemolytic colonies nk3 smooth, opaque, irregular, glistening grayish white translucent colonies glistening, colourles, transparent colonies low convex, smooth, colourless transparent colonies colorless with black center colonies irregular, glistening, grayish white, opaque, non-hemolytic colonies nk4 whitish droplet colonies smooth, grayish, convex, glistening & non-hemolytic colonies = no growth; na = nutrient agar; emb = eosin methylene blue agar; mac = macconkey agar; msa = manitol salt agar; ssa = salmonella – shigella agar; ba = blood agar; og = ogbaru; nk = nkwuaku. appeh et al. – microbial qualities of nkwuaku and ogbaru streams … 101 table 3. total bacterial and fungal plate counts obtained from the stream water samples. sample source bacteria (cfu/ml) fungi (cfu/ml) na emb mac ssa pda nkwuaku 4.2 x107 2.8 x107 3.9 x 107 2.2 x 107 4.0 x 107 ogbaru 5.1 x107 1.5 x 107 3.5 x 107 1.9 x 107 1.6 x 107 pda = potato dextrose agar; na = nutrient agar; emb = eosin methylene blue agar; mac = macconkey agar; ssa = salmonella – shigella agar table 4 shows various groups of microorganisms isolated and identified from this study. they include; staphylococcus aureus, escherichia coli, enterococcus faecalis, klebsiella pneumonia, proteus spp, pseudomonas aeruginosa, salmonella spp. and campylobacter spp. table 4. morphological and biochemical characteristics of bacteria present in stream water samples in awgu local government area, enugu. is o la te c o d e g ra m s ta in s h a p e c it ra te u ti li za ti o n t es t in d o le t es t c a ta la se t es t c o a g u la se t es t m o ti li ty t es t o x id a se t es t u re a se t es t m et h y l re d t es t g lu co se f er m en ta ti o n f ru ct o se f er m en ta ti o n l a ct o se f er m en ta ti o n p ro b a b ly o rg a n is m s og1 + cocci + + + + ag na ag staphylococcus aureus og2 rod + + ag ag ag escherichia coli og3 rod + + ag na na salmonella spp og4 _ rod + + ag ag ag pseudomonas aeruginosa nk1 + cocci + ag ag ag enterococcus faecalis nk2 rod + + + nag ag ag klebsiella spp. nk3 _ rod + + ag ag na proteus spp nk4 spiral + + + + na na na campylobacter spp. + = positive; = negative; ag = acid and gas production; na = no acid production; nag = no acid and gas production; og = ogbaru; nk = nkwuaku. table 5. morphological characteristics of fungal isolates in the stream water samples. macroscopy microscopy organism(s) dark-brown mycelium conidiophores smooth walked and non-septate aspergillus niger yellow pink creamy colonies cylindrical to ovoid conidia, curved septate conidiophores fusarium oxysporum light green and powdery long, erect septate, conidiophores aspergillus flavus gray-green fluggy colonies long, erect, non-septate conidiophores aspergillus fumigatus table 5 shows various fungi isolates and identified from the stream samples by their morphological characteristics. they are; aspergillus niger, fusarium oxysporum, aspergillus flavus and aspergillus fumigatus. table 6 shows the result of the mpn tests for occurrence of the presumptive coliforms (e. coli). the result revealed a gross coliform contamination in the two stream samples. table 6. mpn values per 100ml of the stream water samples. no. of tubes giving a positive result water samples 5 of 10ml 5 of 1ml 5 of 0.1ml mpn (per 100ml) nkwuaku stream 0 1 0 2 1 1 0 4 1 0 1 4 1 1 1 6 1 0 0 2 ogbaru stream 2 0 0 5 2 0 1 7 3 1 0 11 1 1 1 6 0 2 0 4 mpn (per 100ml) = most probable number per 100ml of water sample. 102 biology, medicine, & natural product chemistry 9 (2), 2020: 97-103 table 7 shows the percentage occurrence of bacterial isolates from the stream water samples which includes; escherichia coli, 2(16.66%); enterococcus faecalis 1(8.33%); salmonella spp. 2(16.66%); klebsiella spp. 1(8.33%); staphylococcus aureus 2(16.66%); pseudomonas aeruginosa 1(8.33%); proteus spp. 2(16.66%) and campylobacter spp. 1(8.33%). table 7. percentage occurrence of bacterial isolates from stream water samples. s/n isolates frequency occurrence % occurrence 1. escherichia coli 2 16.66% 2. enterococcus faecalis 1 8.33% 3. salmonella spp. 2 16.66% 4. klebsiella spp. 1 8.33% 5. staphylococcus aureus 2 16.66% 6. pseudomonas aeruginosa 1 8.33% 7. proteus spp. 2 16.66% 8. campylobacter spp. 1 8.33% 12 100% table 8 shows the percentage occurrence of fungi isolates from stream water samples. they are; aspergillus niger 1(20%); aspergillus flavus 2(40%); fusarium oxysporum 1(20%) and aspergillus fumigatus 1(20%). table 8. percentage occurrence of fungal isolates from stream water samples. s/n isolates frequency occurrence % occurrence 1. aspergillus niger 1 20.0% 2. aspergillus flavus 2 40.0% 3. fusarium oxysporum 1 20.0% 4. aspergillus fumigatus 1 20.0% 5 100% discussion evaluation of microbial contamination of nkwaku and ogbaru streams showed that the ph of nkwaku stream sample was 6.7 while that of ogbaru sample was 8.3. this is in agreement with ph assigned by the us environmental protection agency (epa) as the standard ph of water which ranges from 6.5 – 8.5 (epa, 2002). the higher ph values measured from the stream samples could be attributed to more rain water deposition which dilutes the acidity of water by raising the ph. thus indicated that the measured ph values of the stream samples were within permissible value which will not cause any harmful effect to the consumers. ogbaru stream sample had a higher temperature value (27oc) than nkwaku sample (26oc) and this could be attributed to the fact the samples were collected early in the morning. this conforms with the findings of okoye and umo (2006) who stated that cool water are generally more potable for drinking, because high water temperature enhances the growth of microorganisms and causes taste, odour, colour and corrosion problems. in the case of turbidity, ogbaru sample recorded higher value of 10 ntu while nkwuaku recorded a lower value of 8 ntu. the obtained values are far greater than the value specified and approved (5ntu) by the world health organization (who) (who, 2004). the higher values obtained could have been caused by rainfall and human activities such as; grazing of ruminants and improper management of solid and liquid wastes within the streams. high turbidity is also associated with high levels of disease causing microorganisms such as bacteria, fungi and other parasites. thus, waters from these streams are not good for drinking purpose. the total dissolved solid (tds) of the samples are in agreement with the epa standard of 500mg/l. ogbaru recorded higher value of 260mg/l while a lower value of 210mg/l was recorded in nkwuaku sample. odour of nkwaku sample was perceived to be unobjectionable whereas the odour of ogbaru sample was objectionable. the objectionable odour of ogbaru sample could be due to the presence of aquatic lives (fungi, bacteria etc) and waste disposal from toilets, industries, household etc. and this makes the water unfit for drinking. the percentage occurrence of bacterial and fungal isolates from the stream samples were determined and the result revealed that escherichia coli, salmonella spp., staphylococcus aureus and proteus spp. recorded the highest percentage occurrence (16.66%) while enterococcus faecalis, klebsiella spp., pseudomonas aeruginosa and campylobacter spp. recorded lower percentage occurrence (8.33%). the high number of salmonella, proteus spp and staphylococcus aureus in the stream samples are not in agreement with epa water standard for recreational use which states that these pathogenic organisms must not be present in water, because they are of public health significance, having been associated with gastrointestinal infections, diarrhoea, typhoid fever and other form of infection (epa, 2005). the total bacteria and fungi counts for the two samples were generally high, exceeding the limit of 1.0 x 102cfu/ml which is the standard limit of heterotrophic count for drinking water (epa, 2002). this indicates the presence of high organic matters and nutrient sources which supported the growth of the microbes. the primary sources of microbial contamination include the surface runoff, sewage treatment facilities and improper management activities of the inhabitants like washing, refuse dumping, open defaecatio and dropping of different materials into water bodies. various groups of microorganisms were isolated and identified during the study. they include escherichia appeh et al. – microbial qualities of nkwuaku and ogbaru streams … 103 coli, staphylococcus aureus, enterococcus faecalis, klebsiella pnueumonia, proteus sp, pseudomonas aeruginosa, salmonella spp. and campylobacter spp. for bacteria. the fungi isolated include aspergillus niger, fusarium oxysporum, aspergillus flavus and aspergillus fumigatus. the presence of some of these organisms signifies contamination of water from domestic sources. the staphylococcus species are known to produce enterotoxins (okonko et al., 2008). proteus species are intestinal flora, but also widely distributed in soils and water (sclegel, 2002). pseudomonas aeruginosa is an example of non-faecal coliforms, while e. coli is a faecal coliform. conclusion from this study it can be observed that from the microbial qualities of these that they are unfit for human consumption though they can be used for other purposes. many of the inhabitants of awgu local government area depend on these two streams for drinking water supplied. in view of this, it is recommended that the government should ban and discourage all indiscriminate disposal of wastes within stream environments. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references adetokunbo, d. and gilles, f. (2003). world health organization (who) guidelines on the quality of drinking water. the environmentalist 20:25-56. ashbolt, n. j. (2004). microbial contamination of drinking water and disease outcomes in developing regions. toxicology 198:229-238 banwo, k. (2006). nutrient load and pollution study of some selected stations along ogunpa river in ibadan, nigeria. african journal of microbiology 25:7-11. brooks, b.w., riley, t.m. and taylor, r. d. (2006). water quality of effluent dominated ecosystem: ecotoxicological, hydrological and management considerations hydrobiologia 556:379. egberongbe, h.o., awoderu, v.a. and bello, o.o. (2010). microbial and physicochemical evaluation of some streams along ilisan-ago-iwoye road, ogun state, nigeria. journal of olabisi onabanjo university. microbiology applied zoology and plant science 1-10. epa (2002). us environmental protection agency, safe drinking water act amendment http://www.epa.gov/safewater/mc/.html. epa (2005). “protecting water quality from agricultural runoff”. fact sheet no.epa-841-f-05-001. gucker, b., brauns, m. and pusch, m.t. (2006). effect of waste water treatment plant discharge on ecosystem structure and function of lowland streams. the environmentalist 25:313329. oketola, a. a., osibanjo, o., ejelonu, b. c., oladimeji, y. b. and danazio, o. a. (2006). water quality assessment of river ogun around the cattle market of isheri, nigeria. journal of applied science 6:511-517. okonko, i. o., adejoye, o. d., ogunsi, t. a., fajobi, e. a. and shittu, o.b. (2008). microbiological and physicochemical analysis of different water samples used for domestic purposes in abeokuta and ojota, lagos state, nigeria. african journal of biotechnol. 7(5): 617-621. okoye, c. o. b. and umo, a. e. (2006). quality of borehole water in nsukka area, enugu state, nigeria, nig. annals natr. scs., 6(2), 121-123. saravanan, s. and peter, m. (2009). water pollution and man health. centre for development research. germany. pp 1-5. sclegel, h.g. (2002). general microbiology. 7th. ed. cambridge university press. 480. tanwir, f. a., saboor, m.h. and shan, a. (2003). water contamination, health hazards and public awareness: a case of the urban puijab, pakistan. international journal of agriculture and biology. 5: 460-462. who (2004). water sanitation and health programme. managing water in the home: accelerated health gains from improved water sources. flourosis and sanitation 11:7-11. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 67-72 | doi: 10.14421/biomedich.2025.141.67-72 issn 2540-9328 (online) the effect of pilocarpine hydrochloride on the occurrence of temporal lobe epileptic seizures in white mice (mus musculus l.) balb/c strain intan safira audria1, saktivi harkitasari2,*, ni putu diah witari3, rima kusuma ningrum4, tjok istri putra parwati2, komang trisna sumadewi3, i gede wiranatha5, cokorda agung pratiwi6 1faculty of medicine and health sciences; 2department of neurology, faculty of medicine and health sciences; 3department of anatomy-histology; 4department of medical education unit, warmadewa university, jl. terompong no.24 sumerta kelod denpasar 80239, tel. +62-361-223858, fax. +62-361-235073, indonesia. 5department of pharmacology, faculty of medicine; 6faculty of medicine, udayana university, jl. p.b. sudirman denpasar 80225, tel. +62-361-223797, fax. +62-361-701907, indonesia. corresponding author* saktiviharkitasari85@gmail.com manuscript received: 24 november, 2024. revision accepted: 30 march, 2025. published: 15 april, 2025. abstract the objective of this research was to determine the effective dosage of pilocarpine hydrochloride to provoke epileptic seizures in animal models. in this study, a true-experimental method was utilized, employing a post-test only control group design. a total of 32 white mice (mus musculus linnaeus uicc 524) balb/c strains were divided into four different groups for this study. the experimental groups were subjected to varying dosages of pilocarpine hydrochloride: 220 mg/kgbw as group i, 320 mg/kgbw as group ii, and 350 mg/kgbw as group iii. on the other hand, the control group was administered a saline solution (nacl 0.9%) at a dosage of 0.16% ml/kgbw. both pilocarpine hydrochloride and saline solution (nacl 0.9%) were injected intraperitoneally (i.p) into mice. in group i, none of the mice experienced epileptic seizures, and they all survived. in contrast, in group iii, all the mice experienced epileptic seizures, but none of them survived. the man-whitney test showed significant differences in seizure occurrence across doses (p<0.01). the effective dose of pilocarpine hydrochloride for inducing epileptic seizures associated with temporal lobe epilepsy (tle) in this study appears to be in the range of 320 mg/kgbw to 350 mg/kgbw. keywords: pilocarpine hydrochloride; temporal lobe epilepsy; white mice (m. musculus uicc 524) balb/c strain. introduction epilepsy stands as the most prevalent neurological disorder in the general population, and it has the potential to lead to significant impairment and, in some cases, even mortality among those suffering from it (minjarez et al., 2017). epilepsy is a brain disorder characterized by an ongoing tendency to experience epileptic seizures, accompanied by the neurobiological, cognitive, psychological, and social consequences arising from this condition (fisher et al., 2014). globally, there are at least 50 million people of all ages who are impacted by epilepsy. notably, about 80% of these cases are concentrated in countries with lower to moderate incomes, where the majority of the population lacks access to adequate treatment (minghui et al., 2014). each year, it is approximated that at least 2.4 million people around the world are diagnosed with epilepsy. however, in the instance of indonesia, the precise extent of a number of epilepsy cases, in terms of both prevalence rates and incidence rates, remains unclear (octaviana, 2017). epilepsy is a complex diagnosis without an easily accessible gold standard examination, even with an electroencephalogram (eeg) examination, brain imaging examination, or a metabolic evaluation such as cerebrospinal fluid biomarkers (stafstrom & carmant, 2015; thijs et al., 2019). therefore, currently, a study of temporal lobe epilepsy (tle) has begun to be developed and modified among laboratory studies around the world both on human subjects and animal models (ahmed juvale & che has, 2020; kandratavicius et al., 2014). unfortunately, clinical trials on human subjects are often avoided due to ethical constraints in invasive procedures and the difficulty of obtaining suitable human subjects who can represent all periods of tle. according to existing clinical trial problems in human subjects, the 3r framework (encompassing replacement, reduction, and refinement of animal involvement) should be considered during the design, conduct, and reporting of in vivo epilepsy research projects as they have provided findings https://doi.org/10.14421/biomedich.2025.141.67-72 68 biology, medicine, & natural product chemistry 14 (1), 2025: 67-72 that are helping to understand the multifaceted aspects of tle, and it is becoming clear that new technologies will bring meaningful information regarding the mechanisms underlying epileptogenesis (kandratavicius et al., 2014; lévesque et al., 2021; lidster et al., 2016). there are many ways to induce temporal lobe epileptic seizures in animal models of tle, one of them being by administering chemoconvulsant drugs such as pilocarpine hydrochloride (ahmed juvale & che has, 2020). using white mice of the balb/c strain (m. musculus uicc 524) as animal models for temporal lobe epilepsy (tle) necessitates that they display a similar "clinical history" as their human counterparts. this involves undergoing an initial injury that impacts the hippocampus and/or the temporal lobe, a latent period between the injury and occurrence of spontaneous seizures, a chronic manifestation of spontaneous seizures, and also histopathological changes that are identified as the characteristic of tle (kandratavicius et al., 2014). as a result, the standard approach of utilizing animal models of tle induced by pilocarpine hydrochloride can recreate the tle scenario in human subjects. materials and methods study design a genuine true-experimental method was utilized in this research with a post-test only control group design. this study lasted for one month, starting on october 1st and finishing on october 31st, 2022, at the animal research laboratory situated in the faculty of medicine and health sciences at warmadewa university, located in denpasar, bali, indonesia. subjects the animal models employed in this study were a group of white mice (m. musculus uicc 524) balb/c strain. the selection process followed specific criteria for inclusion and exclusion. the criteria of white mice as an animal model encompassed factors like gender (male), age (2 to 3 months), weight (20 to 40 grams), overall health, and absence of mortality during both the acclimatization period and the induction of epileptic seizures. to determine the sample size, a combination of the federer formula and the corrected sample formula was used, and a total sample of 32 white mice was obtained. these mice were then divided into four treatment groups via a randomized lottery-based method (ezalina, 2016; m. sopiyudin dahlan, 2020). the experimental groups were given three different dosages of pilocarpine hydrochloride. in particular, group i received a dosage of 220 mg/kgbw, group ii received 320 mg/kgbw, and group iii received 350 mg/kgbw. in contrast, the control group was administered a saline solution (nacl 0.9%) at a dosage of 0.16% ml/kgbw. the administration of pilocarpine hydrochloride and saline solution (nacl 0.9%) to induce temporal lobe epileptic seizures, both were injected intraperitoneal (i.p) into mice (arshad & naegele, 2020; kurnia mirawati et al., 2016; turski et al., 1983). measurement and statistical analysis epileptic seizures that occur after drug injection will be observed with a video recording device and measured using the modified racine scale (mrs): score 0, no change in behavior; score 1, sudden behavioral arrest, motionless staring (with orofacial automatism); score 2, head nodding; score 3, forelimb clonus with lordotic posture; score 4, forelimb clonus, with rearing and falling; score 5, generalized tonic-clonic activity with loss of postural tone, often resulting in death, wild jumping (ihara et al., 2016; reddy & kuruba, 2013). the collected data in this study will be processed with statistical package for the social sciences (spss) for windows and tested using the mann-whitney test because the data is categorical ethical approval this study was authorized by the health medical research ethics committee situated in the faculty of medicine and health sciences at warmadewa university, located in denpasar, bali, indonesia. the consent for this study was provided under registration code 290/unwar/fkik/ec-kepk/x/2022 on september 18th, 2022. results and discussion result after the injection of the drug was applied to four different treatment groups, all white mice in the control group did not show epileptic seizures, whereas all white mice in the experimental group showed the epileptic seizures. epileptic seizures in white mice occurred between 30 minutes to 60 minutes after intraperitoneal (i.p) injection of pilocarpine hydrochloride. epileptic seizures in white mice that arise as stereotyped movements are measured using the modified racine scale (mrs) measurement method, starting from a score of 1-5. here is the collected data after the treatment: based on the results of the study, it was found that, in the control group with an injection of saline solution (nacl 0.9%) at a dose of 0.16 ml/kgbw did not have any influence on the behavior of white mice. there was no profile of temporal lobe epileptic seizures even the status epilepticus condition and then death in white mice based on modified racine scale (mrs) measurements. the results of saline solution (nacl 0.9%) injection in the control group are displayed in (table 1). audria et al. – pilocarpine-induced temporal lobe seizures 69 table 1. the results of saline solution (nacl 0.9%) injection in control group (n=8). saline solution (nacl 0,9%) modified racine scale status epilepticus number of life mortality 0 1 2 3 4 5 0.16 ml/kg bw 0 0 0 0 0 0 0 8 0 based on the results of the study, it was found that, in the experimental groups with pilocarpine hydrochloride, the following outcomes were noted: at a dose of 220 mg/kgbw, there was no influence on the behavior of white mice and there was no profile of epileptic seizures that were observed based on modified racine scale (mrs) measurements. at a dose of 320 mg/kgbw, there was able to induced various profiles of epileptic seizures in mice, with some of them experiencing status epilepticus conditions and ultimately leading to mortality. at a dose of 350 mg/kgbw, there was able to induce different profiles of epileptic seizures in white mice, then all of them experienced the status epilepticus conditions and ultimately leading to mortality. the results of pilocarpine hydrochloride injection in experimental groups displayed in (table 2). table 2. the results of pilocarpine hydrochloride injection in experimental groups (n=24). pilocarpine hydrochloride modified racine scale status epilepticus number of life mortality 0 1 2 3 4 5 220 mg/kgbw 0 0 0 0 0 0 0 8 0 320 mg/kgbw 0 3 2 1 1 1 2 6 2 350 mg/kgbw 0 0 0 0 4 4 8 0 8 based on the statistical tests of data analytics with the mann-whitney test, a p value of 1.00 was obtained with a significant level of 0.05. the results showed that p value was greater than the significant level value, so statistically it can be concluded that there is no difference in the meaning of the dose group with epileptic seizures (p > 0.05). the results of the mann-whitney test in 4 different treatment groups are displayed in (table 3). table 3. the results of the mann-whitney test in 4 treatment groups (n=32). the size of dosage temporal lobe epileptic seizures p value yes no saline solution (nacl 0.9%) at dose of 0.16 ml/kgbw 0 (0.0%) 8 (100%) 1,00* pilocarpine hydrochloride at dose of 220 mg/kgbw 0 (0.0%) 8 (100%) pilocarpine hydrochloride at dose of 320 mg/kgbw 8 (100%) 0 (0.0%) pilocarpine hydrochloride at dose of 350 mg/kgbw 8 (100%) 0 (0.0%) discussion white mice (m. musculus uicc 524) balb/c strain was selected as the animal model for this study due to their genetic resemblance to humans, which stands at 79%. furthermore, their small size and ease of obtaining, maintaining, and acclimatization made them suitable models for inclusion in this study (grone & baraban, 2015). pilocarpine hydrochloride as an inducing agent was chosen in this study because pilocarpine hydrochloride was proven to induce temporal lobe epileptic seizures in previous studies of the animal models of tle (ahmed juvale & che has, 2020). in this study, three different doses of pilocarpine hydrochloride were employed as an inducing agent: 220 mg/kgbw, 320 mg/kgbw, and 350 mg/kgbw. the selection of the pilocarpine hydrochloride lowest dose, 220 mg/kgbw, was based on previous study led by mirawati who convinced that the use of pilocarpine hydrochloride at dose of 220 mg/kgbw was proven to induced the epileptic seizures in animal models of tle. similarly, the selection of pilocarpine hydrochloride higher doses, 320 mg/kgbw and 350 mg/kgbw, was based on previous studies led by turski who convinced the use of pilocarpine hydrochloride within the range of 300 mg/kgbw to 400 mg/kgbw was proven to induce the epileptic seizures, including status epilepticus conditions, ultimately leading to mortality, in animal models of tle (kurnia mirawati et al., 2016; turski et al., 1983). status epilepticus (se) in animal models is defined as a persistent seizure, either a partial seizure or a generalized seizure that occurs continuously for 5 minutes or more (ahmed juvale & che has, 2020; sharma et al., 2007). in this study, white mice in the experimental group iii that were injected by pilocarpine hydrochloride at a dose of 350 mg/kgbw experienced 70 biology, medicine, & natural product chemistry 14 (1), 2025: 67-72 status epilepticus condition with mrs scores of 4 5; but unfortunately, all the white mice in this experimental group died. meanwhile, white mice in experimental group i that injected with a pilocarpine hydrochloride dose of 200 mg/kgbw did not show any epileptic seizures even experienced status epilepticus condition; thus, all mice in this group were survived (arshad & naegele, 2020). unlike the previous experimental group, white mice in experimental group ii that were injected with a pilocarpine hydrochloride dose of 320 mg/kgbw experienced various results on mrs measurements, some of the white mice in this experi-mental group survived and some died. according to statistical calculations, the effective dose of pilocarpine hydrochloride in this study that was used as an inducing agent of the temporal lobe epileptic seizures on white mice (m. musculus uicc 524) balb/c strain was at a dose of 320 mg/kgbw 350 mg/kgbw. the effective dose of pilocarpine hydrochloride in this study was much higher than the previous study conducted by mirawati with the same type of animal model strain (i.e., white mice), is at doses of 210mg/kgbw 250 mg/kgbw (kurnia mirawati et al., 2016). apparently, the effective dose of pilocarpine hydrochloride in this study is almost comparable to previous studies conducted by turski which has been reviewed in juvale & has’s article, that said the effective dose of pilocarpine hydrochloride in turski's study, is at a doses of 300 mg/kgbw 400 mg/kgbw even with a different type of animal model strain (i.e., wistar rats) (ahmed juvale & che has, 2020; turski et al., 1983). the similarity of the chemoconvulsant effects of pilocarpine hydrochloride in different animal model strains may occur due to several factors such as the method of maintenance and feeding during acclimatization and/or the sensitivity of the white mice themselves to the pilocarpine hydrochloride induction response. based on the results of this study, it was found that the white mice that experienced temporal lobe epileptic seizures were white mice in the experimental group of pilocarpine hydrochloride ii and iii, while white mice in the experimental group of pilocarpine hydrochloride i and control group with saline solution (nacl 0.9%) did not experience any temporal lobe epileptic seizures. according to existing theories, temporal lobe epileptic seizure by pilocarpine hydrochloride as an inducing agent can occur through receptor binding mechanism. chemoconvulsants are substances capable of provoking epileptic seizures, while anticonvulsants are substances that can prevent epileptic seizures by binding to muscarinic receptors within the central nervous system (cns) (ali et al., 2018). the brain as a part of the central nervous system (cns), contains at least of 40 types of chemical compounds classified as the neurotransmitters. based on their function, these neurotransmitters can be categorized into two main groups: an excitatory neurotrnsmitter, such as acetylcholine and glutamic acid, and also an inhibitory neurotransmitter, such as γ-aminobutyric acid (gaba) and glycine (akyuz et al., 2021; shao et al., 2019). an increased amount of excitatory neurotransmitters followed by a decreased amount of inhibitory neurotransmitters can enchance the seizure threshold value thereby causing epileptic seizures, therefore the regulation of the function of these neurotransmitters can play an important role in the occurrence of epileptogenesis mechanism (dumanis et al., 2017). in previous studies, it was mentioned that the pilocarpine hydrochloride known as chemoconvulsant drug would work as an acetylcholine agonist so that it is capable of bind to the muscarinic receptor n-methyl-daspartate (nmda), thus pilocarpine hydrochloride will cause the similar effects to acetylcholine as an excitatory neurotransmitter. according to biochemical theory, it was mentioned that the binding of nmda muscarinic receptors with pilocarpine hydrochloride as an acetylcholine agonist could survive at least 24 hours to 1 month in the synapse cleft. this is supported by alterations in the behavior of experimental animal models, which have demonstrated the occurrence of spontaneous recurrent seizures in previous studies (ali et al., 2018; hoeller et al., 2016; kurnia mirawati et al., 2016). in individuals affected by temporal lobe epilepsy, these spontaneous recurrent seizures can lead to hypoxic conditions within brain tissue. consequently, this can result in sclerosis conditions or permanent neuron cell damage (irreversible) characterized by neuronal cell loss and astrogliosis (dingledine et al., 2014; kurnia mirawati et al., 2016). sclerosis in the area of cornu ammonis 1 (ca1) and cornu ammonis 3 (ca3) in the hippocampus area characterized by neuronal cell loss and astrocyte cell images in the form of glial cells in the astrogliolysis process is a histopathological finding that commonly occurs in individuals affected by temporal lobe epilepsy. these findings align with the histopathological observations obtained in numerous previous animal model studies of temporal lobe epileptic seizures (kurnia mirawati et al., 2016; mátyás et al., 2021). according to the study conducted by mátyás, the histopathological examination using hematoxylin-eosin (he) staining revealed significant neuron cell damage in the ca1 and ca3 areas in animal models of the temporal lobe epileptic seizures after the injection of pilocarpine hydrochloride (mátyás et al., 2021). neuron cell damage found in animal models of temporal lobe epileptic seizures by pilocarpine hydrochloride as an inducing agent has demonstrated the epileptogenesis mechanism pathway as well as provided confirmation between the clinical manifestations in individuals affected by temporal lobe epileptic seizures with the theory of neuronal-death-pathway hypothesis in a number of existing studies. audria et al. – pilocarpine-induced temporal lobe seizures 71 conclusions as a result of this study, it was determined that the effective dose range for inducing temporal lobe epileptic seizures in white mice (m. musculus uicc 524) balb/c strain is between 320 mg/kgbw 350 mg/kgbw using pilocarpine hydrochloride. acknowledgements: the authors express their gratitude to each other for their contributions to this study. additionally, the authors extend our appreciation to all parties who played a role in the production of this article. special thanks are extended to the veterinarians at the animal laboratory unit within the department of pharmacology at the faculty of medicine, udayana university in denpasar, who assisted in the selection of white mice meeting the inclusion and exclusion criteria. we would also like to acknowledge the support of the staff at the animal research laboratory of the faculty of medicine and health sciences at warmadewa university in denpasar, who assisted in the acclimatization of white mice and contributed to the development the animal models of temporal lobe epilepsy (tle). authors’ contributions: saktivi harkitasari prepared the manuscript and designed the study (manuscript), intan safira audria and ni putu diah witari analyzed the data, rima kusuma ningrum, tjok istri putra parwati, and komang trisna sumadewi conducted the experiments and directed the study, while i gede wiranatha selected and cared for the white mice used, and cokorda agung pratiwi wrote and formatted the research manuscript for preparation of publication. all authors read and approved the final version of the manuscript. competing interests: the authors reported in 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(1983). limbic seizures produced by pilocarpine in rats: behavioural, electroencephalographic and neuropathological study. behavioural brain research, 9(3), 315–335. https://doi.org/10.1016/0166-4328(83)90136-5 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 505-509 | doi: 10.14421/biomedich.2024.132.505-509 issn 2540-9328 (online) the effectiveness of fragrant pandan leaves ethanol extract in reducing cholesterol levels in high-fat diet-induced rats ni made wiasty sukanty*, farida ariani, lina yunita undergraduate nutrition study program, faculty of health, universitas bumigora, jl. ismail marzuki no.22, cilinaya, cakranegara, mataram, nusa tenggara barat 83127, tel. +62-370-634498, indonesia. corresponding author* kanty@universitasbumigora.ac.id manuscript received: 28 august, 2024. revision accepted: 19 september, 2024. published: 28 october, 2024. abstract coronary heart disease was the primary cause of death in asia in 2019. indonesia is the country with the highest increase in cholesterol levels in southeast asia. one of the causes of coronary heart disease is consuming foods high in cholesterol. the commonly given therapy is simvastatin. however, long-term use of simvastatin can cause side effects, so it is necessary to use other alternatives. pandanus ammaryllifolius also known as fragrant pandan is a plant that is often found in indonesia and is used in the culinary industry. this study aims to test the effectiveness of fragrant pandan leaves ethanol extract (fplee) in reducing cholesterol levels in rats. a total of 30 rats were split into six groups (nc, c+, c-, t1, t2, and t3) and induced on a high-fat diet, except the nc group for 14 days. for the next 14 days, c+ was given a high-fat diet, cwas given simvastatin, t1, t2, and t3 were respectively given fplee doses of 8, 16, and 32 mg/200 g bw/day. next, cholesterol levels were measured. the results of cholesterol levels were tested statistically using one-way anova and post hoc lsd tests with a = 0.05. there is no substantial difference between nc, c-, and t2. there is a significant difference between t1 and c+ with a higher t1 value. meanwhile, for t3, there is no significant difference between t3 and nc, c-, and c+. thus, the most effective dose for reducing cholesterol levels is 16 mg/200 g bw/day. keywords: cholesterol; high-fat diet; pandanus ammaryllifolius; simvastatin. abbreviations: fragrant pandan leaves ethanol extract (fplee), high-fat diet (hfd), standard diet (sd), low-density lipoprotein (ldl). introduction cholesterol is one of the macronutrients for the body's metabolism. it plays a role in vitamin d, steroid hormones, and bile acids synthesis. our body gets cholesterol in two ways. it is via the de novo mechanism and the food that we consume (craig, yarrarapu, and dimri, 2023). however, continuous consumption of cholesterol-rich foods can increase the risk of coronary heart disease (poli et al., 2021). this disease is the primary cause of death in asia, namely around 35% of total deaths in asia in 2019. this data is equivalent to 10.8 million (58%) of the 18.6 million deaths in the world. indonesia is one of the countries in asia with the highest increase in cholesterol levels in southeast asia (zhao 2021). consuming foods that contain large amounts of cholesterol can cause hypercholesterolemia. low-density lipoprotein (ldl) is one of the lipoproteins known as bad cholesterol. cholesterol can accumulate and form plaque in the blood vessels, resulting in narrow blood vessels that disrupt blood flow (upadhyay, 2023). if the narrowed blood vessels are the coronary arteries, the flow of oxygen-rich blood to the heart will be inhibited, causing coronary heart disease (national heart, lung, and blood institute, 2023). statins are a drug that lowers cholesterol levels. the mechanism of action of statins is by reducing ldl levels and inhibiting the enzyme 3-hydroxy-3-methylglutarylcoa reductase, a catalysator of the conversion of 3hydroxy-3-methylglutaryl-coa into mevalonate in the cholesterol synthesis stage in the body (national heart, lung, 2023). statins have pleiotropic effects, like influencing the immune response and increasing antiinflammatory processes. this effect is utilized in various disease medications such as rheumatoid arthritis, multiple sclerosis, and covid-19 (hartonno and sri, 2023). apart from being affordable, statins also do not cause significant side effects. regarding liver function for xenobiotic metabolism, long-term use of statins can cause hepatotoxicity. apart from that, myopathy is the side effect of statin (arfania et al., 2023; liepinsh et al., 2024). https://doi.org/10.14421/biomedich.2024.132.505-509 506 biology, medicine, & natural product chemistry 13 (2), 2024: 505-509 the back to nature campaign that is currently being promoted has had an effect on the choice of alternative medicine among the public, one of which is the use of herbal plants as a substitute for synthetic drugs (endriyatno, 2021). fragrant pandanus is a local plant that grows widely in tropical areas. fragrant pandan leaves contain several active compounds, such as flavonoids, phenols, saponins, and tannins (assauqi, hafshah, and latifah, 2023). flavonoids can inhibit cholesterol synthesis, thereby minimizing plaque formation in blood vessels (gupta et al., 2024). the fragrant aroma of pandan leaves causes it to be used in the culinary world. even though it has been widely used for daily needs, fragrant pandan leaves have not been used optimally for treatment. several studies have proven that pandan has the potential effect as antiinflammatory, antimicrobial, and antihyperglycemic. there is currently no research regarding the potential of fragrant pandan leaves ethanol extract (fplee) as an antihypercholesterolemia (forestryana, hayati, and putri 2022; wananggari and oktavilantika 2024). it prompted researchers to analyze the effect of ethanol extract from fragrant pandan leaves in reducing cholesterol levels. materials and methods study design this research was conducted at the al-azhar islamic university research laboratory in july-august 2024. the protocol has passed ethical tests with the research code of ethics 053/ec-04/fk-06/unizar/vii/2024. the research design used in this research is quantitative experimental with a posttest-only control group design. the samples were 24 male white rattus norvegicus rats aged 2-3 months with a body weight of 150-250 g. rats were divided into standard control (nc), positive control (c+), negative control (c-), treatment 1 (t1), treatment 2 (t2), and treatment 3 (t3). procedures the production of fragrant pandan leaves ethanol extract (fplee) making fragrant pandan leaf ethanol extract is carried out using the maceration method which begins with drying the fragrant pandan leaves at a temperature of 40. the dried leaves are ground into powder and soaked in 96% ethanol in a ratio of 1:1 for three days. the soaking results are filtered using filter paper to separate the solution from the powder. the solution is then evaporated to separate the solvent from the fragrant pandan leaf extract. the production of high-fat diet (hfd) the composition of hfd is 15 g of 65% sucrose, 5 g of animal fat, and 80 g of duck egg yolk. hfd is given orally at 2 ml/200 g bw/day. the production of simvastatin suspension the amount of 20.6 mg simvastatin was disolved in 100 ml of distilled water. the suspension was administered to the rats was carried out using the sonde method at 0.206 mg/200 g bw/day. the treatment of experimental animals before being given treatment, the rats were adapted in the laboratory for seven days by providing a standard diet (sd) and drinking water ad libitum. the random grouping was carried out into nc, c+, c-, t1, t2, and t3. the following is the treatment given to each group: table 1. the treatment of experimental animals. group days 1-14 days 15-28 nc sd sd c+ sd and hfd sd and hfd csd and hfd sd and 0.206 mg/200 g bw/day of simvastatin t1 sd and hfd sd and 8 mg/200 g bw/day of fplee t2 sd and hfd sd and 16 mg/200 g bw/day of fplee t3 sd and hfd sd and 32 mg/200 g bw/day of fplee the cholesterol level measurement blood cholesterol levels in rats were measured using a biosystem instrument after the administration of an ethanol extract of fragrant pandan leaves. serum samples were taken by performing a heart puncture and collecting the blood in a red cap vacutainer tube. data analysis data from measuring cholesterol levels were analyzed using spss version 23 software by first testing for normality and homogeneity, then differences in results in each group were tested using the one-way anova followed by the post hoc lsd test with a confidence level of 95% (α= 0.05). results and discussion cholesterol levels in each group were carried out on the 29th day. the results of measuring cholesterol levels are shown in figure 1. group t1 is the group with the highest cholesterol levels, while t2 is the group with the lowest cholesterol levels. the results of the one-way anova test showed p-value = 0.006, which means there were at least two groups that had significant differences in cholesterol levels, so the test was continued using the post hoc lsd test to determine the differences in each group. sukanty et al. – the effectiveness of fragrant pandan leaves ethanol extract in … 507 figure 1. average cholesterol levels after treatment. the nc and cgroups had lower cholesterol levels than c+ with a p-value <0.05. group p1 is the group with the highest cholesterol levels. the post hoc lsd test results showed a p-value < 0.05 when compared with c+, thus giving fplee at a concentration of 8 mg/200 g bw/day could not reduce the cholesterol levels of rats. group t3 is a group that has cholesterol levels between nc, c-, t2, and k+, t1. the post hoc lsd test results only show p < 0.05 when compared with t1, thus concluding that t3 cannot reduce cholesterol levels in rats. when compared with other treatment groups, t2 had the lowest cholesterol levels. the lsd test results for the t2 group showed that there was no significant difference between t2 and nc and c-. it indicates that administration of fplee at a concentration of 16 mg/200 g bw/day was able to reduce the cholesterol levels of rats to close to the cholesterol levels of normal rats and rats treated with simvastatin. discussion cholesterol is a macronutrient that plays a role in vitamin d, steroid hormones, and bile acid synthesis (craig, yarrarapu, and dimri, 2023). the results of measuring cholesterol levels show a difference in cholesterol levels between nc and ccompared with c+, which means giving high-fat feed can increase the blood cholesterol levels of rats. foods rich in saturated fatty acids from hfd containing duck egg yolk and animal fat can raise blood cholesterol levels, especially ldl. this mechanism involves a decrease in ldl receptor mrna expression and ldl receptor activity that allows a reduction in ldl absorption by the liver so that ldl levels in the blood increase. apart from food, the liver synthesizes cholesterol and distributes it to all tissues. the synthesis of bile acids also needs cholesterol in the absorption of lipids in the small intestine (houttu et al., 2023). the cholesterol levels in the cgroup showed that administration of simvastatin reduced the cholesterol levels of hfd-induced rats to reach levels like those in the nc group. simvastatin is a drug commonly used to diminish cholesterol levels. the mechanism of action of simvastatin is by inhibiting the action of the hmg-coa reductase, a catalisator for hmg-coa conversion to mevalonic acid in the de novo cholesterol synthesis process. the inhibition of this enzyme can reduce cholesterol levels in the blood (hartonno and sri, 2023). the cholesterol levels in the t1 group did not decrease. it is thought to be caused by low fplee concentrations. thus, the antioxidants contained in fplee were unable to reduce rats's cholesterol levels. in animal models of hyperlipidemia, continuous administration of hfd without balance with sufficient antioxidant intake causes the formation of free radicals. as a result, oxidative stress occurs and leads to an increase in cholesterol levels (adhitama, kuswanti, and khaleyla, 2023). a unique phenomenon occurred in t3. although t3 is the group given the highest concentration of fplee, there was no reduction in cholesterol levels. it shows that antioxidants that are too high cannot reduce cholesterol levels. the mechanism that can occur in t3 is that the antioxidants contained in high concentrations of fplee disrupt lipid metabolism, undergo auto-oxidation, and become pro-oxidants, thereby increasing free radicals and causing oxidative stress and increased cholesterol levels (amida et al., 2021; sukanty and saputra, 2023). the t2 had the same cholesterol levels as nc and c-. it indicates that administration of fplee at a concentration of 16 mg/200 g bw/day decreased cholesterol levels in rats until it reached the same cholesterol level as nc. fplee contains flavonoids, phenols, saponins, and tannins. this compound has antioxidant activity that can capture radicals. this ability is higher than commercial vitamin e (suryani et al., 2018). there are several roles of antioxidants in reducing cholesterol levels. oxidizing ldl by free radicals is one of the factors causing atherosclerosis. therefore, with the presence of antioxidants that can capture free radicals, the formation of oxidized ldl can be prevented (hasim et al., 2018). flavonoid antioxidants can increase the number of ldl receptors both in hepatocytes and in extrahepatic tissue, thereby increasing cholesterol uptake by hepatocytes and extrahepatic tissue. it results in a decrease in cholesterol levels in the blood (mutia, fauziah, and thomy, 2018). apart from that, flavonoids can also inhibit the activity of the hmg-coa reductase enzyme in hepatocytes, thereby reducing cholesterol production (hasim et al., 2018). another mechanism of flavonoids is to reduce free fatty acids, thereby reducing the buildup of cholesterol in the blood (adhitama, kuswanti, and khaleyla, 2023). tannin, another antioxidant in fplee, has a mechanism for decreasing blood cholesterol levels. it focuses on cholesterol absorption in the intestine. tannins will bind to pancreatic lipase protein and coat the intestinal walls so that the lipid absorption process is impeded. the identical mechanism is shown in phenol. it binds to cholesterol-carrying proteins that function in cholesterol absorption in the intestine, thereby reducing cholesterol absorption. another mechanism is that phenol reduces the secretion of apolipoprotein b, a protein involved in lipid transport in the blood (mutia, fauziah, and thomy, 2018). saponin functions to inhibit the 0 20 40 60 nc c+ ct1 t2 t3 c h o le st er o l l ev el s (m g /d l ) group average rat cholesterol levels 508 biology, medicine, & natural product chemistry 13 (2), 2024: 505-509 activity of the lipase enzyme in the pancreas. low lipase activity results in a decrease in triacylglycerol. saponins can also speed up cholesterol metabolism (viviandhari et al., 2020). thus, the reduction in cholesterol levels in the t2 was caused by the antioxidant content in fplee that inhibits cholesterol synthesis and absorption, so the cholesterol levels in the t2 were the same as those in the nc and cgroups. thus, of the three existing fplee concentrations, 16 mg/200 g bw/day is the most effective dose for reducing rat cholesterol levels. conclusions induction of fragrant pandan leaves ethanol extract was able to reduce cholesterol levels in rats induced by a high-fat diet. the optimum concentration of fragrant pandan leaves ethanol extract is used to decrease cholesterol levels and reach concentrations like those of the standard group and the opposing group, which is 16 mg/200 g bw/day. further research needs to be carried out to determine changes in liver function and the histology of liver cells in hypercholesterolemic rats treated with fragrant pandan leaf ethanol extract. acknowledgements: the author would like to thank the ministry of education, culture, research and technology (kemdikbudristek), bumigora university, and all colleagues who helped in carrying out this research. authors’ contributions: ni made wiasty sukanty designed the study. ni made wiasty sukanty, farida ariani, and lina yunita carried out the laboratory work. ni made wiasty sukanty analyzed the data and wrote the manuscript. all authors have read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research was funded by the ministry of education, culture, research and technology (kemdikbudristek) through the research and community service (bima) program in 2024. references adhitama, 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jelly candy cinnamon powder koerintji variation of red seaweed carrageenan concentration erwin setiawan, rizky muliani dwi ujianti*, iffah muflihati, arief rakhman affandi corresponding author* rizkymuliani@upgris.ac.id manuscript received: 06 july, 2024. revision accepted: 07 october, 2024. published: 09 october, 2024. abstract jelly candy is a product that has the characteristics of being chewy and varied in texture. carrageenan, an alternative natural raw material as a gelling agent in jelly candy, is one of the producers of red seaweed carrageenan because it has a very complex composition. cinnamon contains about 91.88-94.19% cinnamaldehyde as an antioxidant compound. the study aimed to determine the value of the antioxidant activity obtained and the correlation between the cinnamon powder jelly candy koerintji variety and the addition of red seaweed carrageenan with different concentration variations. the study was conducted using a completely randomized design with two factors. carrageenan concentrations consist of 3.5% and 5%. the results showed that the concentration of red seaweed carrageenan significantly affected the antioxidant activity value and characteristics of cinnamon jelly candy. the best treatment was obtained at a concentration of 1.5% cinnamon and 5% red seaweed carrageenan with chemical characteristics water content 11.49%, antioxidant activity 50.26%, ph value 4.82%, physical characteristics colour (brightness) 40.72%, texture hardness 2.3%, adhesiveness 3.1%, elasticity 2.76%, colour preference 4.66%, aroma preference, 4.32%, flavour preference 4.66%, elasticity texture preference 4.56%, taste 4.80%, and overall 4.04%. keywords: physicochemical characteristics; carrageenan; cinnamon; soft candy; red seaweed. introduction candy food products often found and circulating on the market are very diverse, including chewy candy (jelly), hard candy, chocolate candy (bounty), caramel, nougat, and ginger. the national standardization agency/sni (2008) states that jelly candy is a soft-textured candy processed by adding hydrocolloid ingredients such as agar, gum, pectin, starch, and carrageenan. one factor that can influence the process of making jelly candy is the presence of gel-forming ingredients. a gelling solid agent and a chewy texture can be created in jelly candy by adding ingredients that contain gelling agents. in general, jelly candy is made using gelatin as a gelforming material, but the raw material for commercial gelatin is currently imported from abroad, such as europe (hidayah et al., 2013). one of the breakthroughs for replacing the gelling agent is red seaweed carrageenan. red seaweed also contains antioxidants. research conducted on the antioxidant compound content of several algae species using the free radical scavenging method by 1,1-diphenyl-2-picrylhydidrazyl (dpph) was carried out by (lestario et al., 2008) with various solvents and obtained the highest value of red seaweed antioxidant activity, namely 43.43%, obtained from the acetone solvent extract. compounds that are known to fight free radicals are antioxidants. antioxidant compounds can be obtained from various food sources. antioxidant compounds produced by plants, such as vitamin c, vitamin e, carotene, and phenolic groups, especially polyphenols and flavonoids, are known to have the potential to reduce the risk of degenerative diseases caused by free radicals. one of the foodstuffs that contain antioxidants that are obtained naturally is cinnamon. cinnamon is a plant known to contain solid antioxidant active compounds (andriyanto et al., 2014). cinnamon is one foodstuff that contains antioxidants that can be obtained naturally. cinnamon is a spice plant that has long been used as a fragrance or flavour enhancer in food or drinks. cinnamon wood extract or powder has natural antioxidant activity because, in wood extract cinnamon powder, there are also chemical compounds such as cinnamaldehyde, eugenol, trans-cinnamic acid, phenolic compounds, and tannins. the antioxidant activity of cinnamon obtained was 45.42% (sukandar, 2014). cinnamon contains chemical compounds, including cinnamaldehyde, eugenol, trans-cinnamic acid, phenol compounds, and tannins. one type of cinnamon variety in indonesia is the koerintji variety cinnamon, which has the advantage of the koerintji variety cinnamon itself, namely that it contains a very high cinnamaldehyde content of around 91.88 94.19%, food technology department, engineering and informatics faculty, universitas pgri semarang, jl. sidodadi no. 24, semarang, indonesia. https://doi.org/10.14421/biomedich.2024.132.415-421 mailto:rizkymuliani@upgris.ac.id 416 biology, medicine, & natural product chemistry 13 (2), 2024: 415-421 exceeding the sni no. standard. 06-3734-2006, namely 50%. from the various explanations above, it is necessary to develop finished products that contain antioxidants due to people's lifestyle changes, such as consuming foods that contain lots of fat and oil. this causes free radicals to form in the body. materials and methods material the main ingredients used in this research were cinnamon of the koerintji variety obtained from lempur tengah village, gunung raya district, kerinci regency, jambi province, and red seaweed (eucheuma cottoni sp.) obtained from coastal seaweed farmers in ritabel village, district north tanimbar, west southeast maluku regency, sucrose (rose brand), fructose (merck), citric acid (merck). meanwhile, the materials used for chemical analysis include 2,2-diphenyl-1picrylhydrazyl (dpph), methanol (merck), koh (merck), and distilled water (merck). procedures the research was divided into three stages: making cinnamon powder, carrageenan, and jelly candy. jelly candy was made using a factorial completely randomized design (crd) with two treatment factors: cinnamon powder concentrations of 0.5%, 1%, and 1.5% and red seaweed carrageenan concentrations of 3.5% and 5%. each treatment was carried out with three repetitions. making cinnamon powder the process of making cinnamon powder (marfungah et al., 2019) with modifications: cinnamon is cut into ± 1 cm pieces and dried in a cabinet dryer at 50°c for 8 hours. after drying, the cinnamon is then reduced in size by blending it until it becomes powder and then sifting it with a 60 mesh sieve until cinnamon powder is obtained. making carrageenan the red seaweed is washed with running water until clean, then put into a basin and soaked in a koh solution containing 1,840 ml (92%) and 160 g (32%) of distilled water. the function of koh in cooling aims to reduce the content of other elements, such as salts and cellulose, contained in seaweed. the red seaweed is soaked for 2 hours, then after soaking, the seaweed is rewashed with running water until clean, then the seaweed is tested for ph using a ph-2011 ph meter until it obtains a ph value of 7 (neutral). the red seaweed is reduced in size to ± 4 cm in length. after reducing the size, place the red seaweed in a baking dish and dry it in a cabinet dryer at a temperature of 50°c for 24 hours. the dried red seaweed is blended until smooth and then sieved using a 60-mesh sieve until red seaweed carrageenan powder is obtained. jelly candy making the procedure carried out in making jelly candy is adding cinnamon extract, which includes several stages: preparation of ingredients, cooking, moulding, cooling, and drying. making cinnamon jelly candy refers to the modified (marfungah et al., 2019) method, namely 45% sucrose and 40% fructose syrup, then heated at 80°c with the addition of 3.5% and 5% carrageenan, adding cinnamon powder according to the treatment. (0.5%, 1%, and 1.5%) moreover, 1% citric acid while stirring. after mixing the ingredients evenly, the ingredients are poured into the prepared mould. then, leave it for 10 minutes at room temperature and put it in the refrigerator for 24 hours until it solidifies and jelly candy forms. after that, the jelly candy was heated using a cabinet dryer at 50°c for 6 hours. physicochemical characteristic test the physio-chemical characteristics of jelly candy observed were water content, ph test, and texture test based on the method (marfungah et al., 2019). antioxidant activity test a chemical test of the antioxidant activity (miranti et al., 2017) of cinnamon powder jelly candy and red seaweed carrageenan using the dpph method based on the method. preparation of sample solution weigh 5 grams of cinnamon candy and then dissolve it using 50 ml of distilled water in a measuring flask up to the mark until the jelly candy dissolves. preparation of 1.2 mm dpph solution 4.8 mg of dpph powder was weighed and put into a 20 ml measuring flask. the powder was dissolved in methanol p.a. to the extent of the miniscus and then homogenized until homogeneous. preparation of control solution a total of 0.8 ml of 1.2 mm dpph solution was pipetted and put into a test tube. add 3 ml of distilled water, then homogenize. the control solution was incubated at 2530˚c (room temperature) for 30 minutes (the reaction tube was wrapped in aluminium foil). then, the solution's absorbance was measured on a uv-vis spectrophotometer (spectroquant prove 300) with a wavelength of 517 nm. preparation of test samples with various concentrations 8 ml of the diluted sample solution was taken, then put into a 20 ml measuring flask and added with distilled water to the mark. next, 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml of this solution are taken. distilled water was added up to 20 ml to obtain test solutions with different concentration variations. setiawan et al. – physicochemical candy cinnamon seaweed carrageenan. 417 ic50 antioxidant test antioxidant activity was analyzed based on its ability to capture dpph (2,2-diphenyl-1-purcylhydrazyl) free radicals. in a test tube, a 3 ml sample was added to a 0.8 ml, 1.2 mm methanol dpph solution. then, it was vortexed for 3 minutes and incubated at room temperature in dark conditions for 30 minutes. next, the sample's absorbance was measured using a uv-vis spectrophotometer (spectroquant prove 300) at a wavelength of 517 nm. controls were made the same way using distilled water as a substitute for the sample. ic50 is calculated using a linear regression equation, with sample concentration as the y-axis. from the equation y = ax + b, it can be obtained that the ic50 value is the same as the x value. sensory test testing was conducted descriptively (on a scale of 1-7) on cinnamon jelly candy with 50 untrained panellists. this test includes brown color, cinnamon aroma, flavor, sweet taste, cinnamon taste, aftertaste, elasticity, stickiness, and brightness. data analysis anova and duncan’s advanced test were used in statistical analysis. the software used in this test is spss 24. results and discussion physicochemical characteristics water content the results of variance testing showed that the addition of carrageenan had a significant difference in the water content of cinnamon jelly candy. figure 1 shows the values obtained between 11.94% 22.84%. formulation 1 obtained a value of 22.84%, formulation 2 17.20%, formulation 3 20.16%, formulation 4 12.78%, formulation 5 17.90%, and formulation 6 11.94%. the lowest average water content value obtained in the f6 treatment was 11.94%. the highest water content of jelly candy was obtained in the f1 treatment, with a value of 22.84%. in the one-way test results, the water content of jelly candy formulations 1 and 3 showed a significant difference, whereas in formulations 2 and 5, formulations 4 and 6, there was no significant difference. the differences in each sample are due to the differences in concentration in each jelly candy formulation, so testing the water content using the wet basis method shows fundamental differences in the effect of each formulation. below is data from water content research using the wet basis method, presented in figure 1. figure 1. jelly candy water content test results. description: f1 (0.5% cinnamon, 3.5% carrageenan), f2 (0.5% cinnamon, 5% carrageenan), f3 (1% cinnamon, 3.5% carrageenan), f4 (1% cinnamon, 5% carrageenan), f5 (1.5% cinnamon, 3.5% carrageenan), f6 (1.5% cinnamon, 5% carrageenan). (values followed by different letter notations in the diagram indicate significantly different values) (one way anova test, α = 0.05%/ f1 (0.5% cinnamon, 3.5% carrageenan), f2 (0.5% cinnamon, 5% carrageenan), f3 (1% cinnamon, 3.5% carrageenan), f4 (1% cinnamon, 5% carrageenan), f5 (1.5% cinnamon, 3.5% carrageenan), f6 (1.5% cinnamon, 5% carrageenan). letter notations on the diagram indicate significant differences) (one way anova test, = 0.05% figure 1 shows that the higher the concentration of carrageenan, the resulting product will have a low water content, and adding a low concentration of carrageenan will result in a high water content. this is because carrageenan can bind large amounts of water; the literature states that carrageenan has hydrocolloid properties that can bind water. the water content of jelly candy is determined by the processing process, especially during the cooking and drying of moulded jelly candy products (bactiar et al., 2017). the product produced on average is by the standard. that is, it should be at most 20%. however, in formulation 1, the results are not by the standard, namely 22.84%; this is because the formulation uses a cinnamon raw material concentration of 0, 5%, and 3.5% carrageenan and uses 20% water to dissolve all the jelly candy raw materials. a high water content produces a high value in jelly candy products because the substance contains too much water, and the dissolved solids during the processing process are too low. hence, the consistency value could be more substantial. the gel-forming consistency has too few components, causing the tissue in the jelly candy product not to be strong enough to hold the liquid sugar, which causes the jelly candy to experience syneresis and produce a high water content value (rismandari et al., 2017). according to (atmaka et al., 2013), gel formation is a phenomenon of cross-linking between polymer chains, resulting in the formation of a three-dimensional mesh with continuity. then, this mesh can catch the water, which causes the formation of a solid and rigid structure in the resulting product. 22.84d 17.20b 20.16c 12.78a 17.90b 11.94a 0,00 5,00 10,00 15,00 20,00 25,00 f1 f2 f3 f4 f5 f6 w a te r c o n te n t( % ) formulation 418 biology, medicine, & natural product chemistry 13 (2), 2024: 415-421 ph the test was carried out using a ph meter to test the ph value obtained from the cinnamon jelly candy. after testing, the ph value produced in the cinnamon jelly candy product was classified as acidic ph because the ph value was below 7 (neutral). this acidic condition is caused by the addition of citric acid in the processing of cinnamon jelly candy. however, the amount of citric acid added to each treatment has the same composition, namely 1%. when compared with the provisions of sni 3547-2-2008, the ph value of jelly candy is at least 3.55%, meaning the value is lower than the research results of jelly candy. the highest ph in formulation 6 with a value of 4.82%, and the lowest value was obtained in formulation 1 with a value of 4.47%. the primary raw materials, such as cinnamon, have an alkaline ph value of 8, and red seaweed from which carrageenan will be made has also been tested for ph with a value of 7. data from testing the ph value using a ph meter is presented in figure 2. figure 2. jelly candy ph test results. f1 (0.5% cinnamon, 3.5% carrageenan), f2 (0.5% cinnamon, 5% carrageenan), f3 (1% cinnamon, 3.5% carrageenan), f4 (1% cinnamon, 5% carrageenan), f5 (1.5% cinnamon, 3.5% carrageenan), f6 (1.5% cinnamon, 5% carrageenan). (values followed by letters that are not the same in the diagram indicate significantly different values) (one way anova test, α = 0.05%)/ f1 (0.5% cinnamon, 3.5% carrageenan), f2 (0.5% cinnamon, 5% carrageenan), f3 (1% cinnamon, 3.5% carrageenan), f4 (1% cinnamon, 5% carrageenan), f5 (1.5% cinnamon, 3.5% carrageenan), f6 (1.5% cinnamon, 5% carrageenan). (values followed by unequal letter notations on the diagram indicate significant differences) (one way anova test, = 0.05%) adding the same citric acid to each formulation showed significant fundamental differences, such as adding the same 1% citric acid. still, each formulation of the cinnamon jelly candy had a different ph value. formulation 1 obtained a value of 4.47%, formulation 2 obtained a ph value of 4.45%, formulation 3 obtained a value of 4.43%, formulation 4 obtained a ph value of 4.72%, formulation 5 obtained a value of 4.82%, and formulation 6 obtained the highest ph value, namely 4.82%. the increase in ph value of cinnamon jelly candy tends to increase with each formulation; this is thought to be due to the higher concentration of cinnamon powder and carrageenan being added to each formulation from the lowest formulation, namely the added concentration of 0.5% cinnamon and 3.5% carrageenan. up to the addition of the highest concentration, namely 1.5% cinnamon and 5% carrageenan. according to (salamah & erungan, 2006) the average ph of jelly candy ranges from 4.5 to 6.0. toughness texture test the results of the elasticity measurement show the elasticity value of the jelly candy produced with the addition of cinnamon and red seaweed carrageenan. figure 3 shows the results of the elasticity test, which has a fundamental influence; the highest elasticity value was obtained in formulation 6 with a value of 2.76%, and the lowest value was obtained in formulation 1 with a value of 1.90%. the resulting value does not exceed sni 35472-2008, which has a maximum value of 14%. it can be concluded that the gel-forming material can produce elasticity-based products. the elasticity value obtained by cinnamon jelly candy is due to the small volume of the product. it has undergone several processing stages, one of which is the drying process, which causes the volume to decrease. in the opinion of (bahri et al., 2020) an increase in the water content value can cause a decrease in the elasticity value, where the water component in the product will diffuse into the gel so that the gel forms to become soft and cause the elasticity to decrease. data from testing the texture value of the elasticity parameter is presented in figure 3. figure 3. elasticity test results. description: f1 (0.5% cinnamon, 3.5% carrageenan), f2 (0.5% cinnamon, 5% carrageenan), f3 (1% cinnamon, 3.5% carrageenan), f4 (1% cinnamon, 5% carrageenan), f5 (1.5% cinnamon, 3.5% carrageenan), f6 (1.5% cinnamon, 5% carrageenan). (values followed by letters that are not the same in the diagram indicate significantly different values) (one way anova test, α = 0.05%)/ f1 (0.5% cinnamon, 3.5% carrageenan), f2 (0.5% cinnamon, 5% carrageenan), f3 (1% cinnamon, 3.5% carrageenan), f4 (1% cinnamon, 5% carrageenan), f5 (1.5% cinnamon, 3.5% carrageenan), f6 (1.5% cinnamon, 5% carrageenan). (values followed by unequal letter notations on the diagram indicate significant differences) (one way anova test, = 0.05%) 4.47b 4.45ab 4.43a 4.72c 4.82d 4.82d 0 1 2 3 4 5 6 f1 f2 f3 f4 f5 f6 p h ( % ) formulation 1.90a 1.73a 2.37b 1.87a 2.63c 2.76c 0 0,5 1 1,5 2 2,5 3 3,5 f1 f2 f3 f4 f5 f6 el as ti ci ty (% ) formulation setiawan et al. – physicochemical candy cinnamon seaweed carrageenan. 419 in the cinnamon jelly candy product, formulation 1 obtained the lowest elasticity value, and the highest was obtained in formulation 6. this value also correlated with the water content value, where formulation 1 had the highest water content, and formulation 6 obtained the lowest water content value. this is reinforced by the opinion of (novitasari et al., 2016), who stated that the water content value could influence the elasticity value of the jelly candy product produced. it can be seen from this research that the more the water content increases, the more elasticity the jelly candy will reduce. ic50 antioxidant activity test based on the results of the analysis of variance, it shows that the kerinci cinnamon variety has a significant effect (α=5%) on cinnamon jelly candy, and the concentration of red seaweed carrageenan has a significant effect on jelly candy, using the dpph (2,2 diphenyl-1picrylhydrazyl) absorbance value method. produced by this reduction reaction was measured at a wavelength of 517 nm. figure 4 shows that the kerinci variety cinnamon and the concentration of red seaweed carrageenan influence the amount of antioxidants. according to (marfungah et al., 2019), a compound is said to have a powerful antioxidant if the ic50 value is less than 50 µg/ml, substantial if the ic50 value is 50 µg/ml to 100 µg/ml, while if the ic50 value is 100 µg/ml to 150 µg/ml, weak if ic50 is 151 µg/ml to 200 µg/ml, inactive if ic50 is more than 500 µg/ml. in the jelly candy product, formulation 1 obtained an ic50 value of 50.266 µg/ml with a concentration of 0.5% cinnamon and 3.5% carrageenan, and the highest ic50 value was obtained in formulation 6 with a value of 64.823 µg/ml with a concentration of 1.5% cinnamon, 5% carrageenan. this shows that the higher the concentration of cinnamon and the addition of carrageenan will affect the antioxidant activity values obtained. these data also show that formulations 1 to 6 have active antioxidant activity. the addition of red seaweed carrageenan also affects the antioxidant activity value because red seaweed also contains polyphenolic compounds, which can potentially contain antioxidants (amelia & tanod, 2016). red seaweed carrageenan has antioxidant compounds, which influence the antioxidant activity value of jelly candy. this is also because carrageenan has more hydroxyl groups, so carrageenan can form a higher double helix structure and protect antioxidant compounds in the three-dimensional matrix from heat and oxygen. (bactiar et al., 2017) stated that carrageenan can protect phenolic compounds from increasingly vital heating processes so that phenolic compounds do not experience much damage. so, the more components of carrageenan concentration that are added, the higher the antioxidant compound value of a product. data from ic50 antioxidant research are presented in figure 4. figure 4. antioxidant activity test results. description: f1 (0.5% cinnamon, 3.5% carrageenan), f2 (0.5% cinnamon, 5% carrageenan), f3 (1% cinnamon, 3.5% carrageenan), f4 (1% cinnamon, 5% carrageenan), f5 (1.5% cinnamon, 3.5% carrageenan), f6 (1.5% cinnamon, 5% carrageenan). (values followed by letters that are not the same in the diagram indicate significantly different values) (one way anova test, α = 0.05%)/ f1 (0.5% cinnamon, 3.5% carrageenan), f2 (0.5% cinnamon, 5% carrageenan), f3 (1% cinnamon, 3.5% carrageenan), f4 (1% cinnamon, 5% carrageenan), f5 (1.5% cinnamon, 3.5% carrageenan), f6 (1.5% cinnamon, 5% carrageenan). (values followed by unequal letter notations on the diagram indicate significant differences) (one way anova test, = 0.05%) red seaweed carrageenan has antioxidant compounds, which influence the antioxidant activity value of jelly candy. this is also because carrageenan has more hydroxyl groups, so carrageenan can form a higher double helix structure and protect antioxidant compounds in the three-dimensional matrix from heat and oxygen. (bactiar et al., 2017) stated that carrageenan can protect phenolic compounds from increasingly vital heating processes so that phenolic compounds do not experience much damage. so, the more components of carrageenan concentration are added, the higher the antioxidant compound value of a product. adding koerintji variety cinnamon also influences the antioxidant activity value of jelly candy products. koerintji variety cinnamon itself has a very high cinnamaldehyde content of around 91.88 94.19%, exceeding the sni standard of 50%, because this cinnamaldehyde compound will play an active role in antioxidant activity. the weak value of antioxidant activity compounds obtained in cinnamon jelly candy products is thought to be influenced by the mixing of additional ingredients such as sugar, gelling agents, and citric acid. it is strengthened due to a processing process. the processing of cinnamon jelly candy in this study used a temperature of 50˚c during the product drying process in a cabinet dryer. this is supported by (marfungah et al., 2019), who states that heat treatment when processing jelly candy can reduce the levels of antioxidant compounds by 18% 43%. this is to the research results of (hayulistya et al., 2021), stating that the activity value of antioxidant compounds obtained in black cumin jelly candy is relatively low, namely obtaining a value between 0.45-3.48%; this occurs because there are various process stages required in 64.82f 62.79e 58.52d 56.99c 54.730b 50.26a 0 20 40 60 80 f1 f2 f3 f4 f5 f6 ic 5 0 ( p p m ) formulation 420 biology, medicine, & natural product chemistry 13 (2), 2024: 415-421 processing black cumin jelly candy, namely cooking, and drying. sensory test this hedonic sensory testing method using 50 untrained panelists is used to determine the subjective attitudes of panelists or potential consumers towards a product produced and assessed based on organoleptic parameters. the results obtained are in the form of acceptance (accepted or rejected), liking (level of like/dislike), and choice (choose one over another) towards the product (oktafa et al., 2017). this test is usually carried out by comparison to the test of choice. panellists or potential consumers are then instructed to choose one example they like from several examples presented. the data in (table 1) shows no real difference in color parameters. this shows that formulation 1, formulation 2, and formulation 4 are the colours the panelists prefer. meanwhile, formulation 3, formulation 5, and formulation 5 show the neutral value chosen by the panellists. however, the highest score was obtained for formulation 2, which shows that the panelists liked the jelly candy product formulation 2 with a formulation of 0.5% cinnamon and 5% carrageenan. data from hedonic sensory testing results are in table 1. table 1. hedonic sensory test results. formulation colour aroma flavour texture taste overall f1 5,06 ± 1,25a 4,26 ± 0,85a 4,68 ± 1,05a 4,08 ± 1,39ab 5,22 ± 1,13a 5,14 ± 0,98a f2 5,10 ± 1,14a 4,26 ± 1,08a 4,58 ± 1,12a 4,74 ± 0,87bc 5,20 ± 0,96a 5,06 ± 1,03a f3 4,88 ± 1,17a 4,12 ± 1,00a 4,44 ± 1,14a 3,88 ± 1,28a 4,86 ± 1,06a 4,46 ± 1,03a f4 5,08 ± 1,22a 4,36 ± 0,98a 4,48 ± 1,07a 4,48 ± 1,21ab 4,96 ± 1,06a 5,02 ± 1,02a f5 4,60 ± 1,32a 4,46 ± 1,05a 4,68 ± 1,30a 4,84 ± 1,28c 5,06 ± 1,21a 4,96 ± 1,19a f6 4,66 ± 1,02a 4,32 ± 1,11a 4,66 ± 1.37a 4,56 ± 1,26bc 4,80 ± 1.29a 4,84 ± 1,20a description: f1 (0.5% cinnamon, 3.5% carrageenan), f2 (0.5% cinnamon, 5% carrageenan), f3 (1% cinnamon, 3.5% carrageenan), f4 (1% cinnamon, 5% carrageenan), f5 (1.5% cinnamon, 3.5% carrageenan), f6 (1.5% cinnamon, 5% carrageenan). (values followed by letters that are not the same in the diagram indicate significantly different values and not significantly different values) (one way anova test, α = 0.05%)/ f1 (0.5% cinnamon, 3.5% carrageenan), f2 (0.5% cinnamon, 5% carrageenan), f3 (1% cinnamon, 3.5% carrageenan), f4 (1% cinnamon, 5% carrageenan), f5 (1.5% cinnamon, 3.5% carrageenan), f6 (1.5% cinnamon, 5% carrageenan). (values followed by unequal letter notations on the diagram indicate significant differences) (one way anova test, = 0.05%) the panelists' overall assessment of the organoleptic test includes cinnamon jelly candy's colour, taste, aroma, and texture. parameter numbers 1 (dislike very much), 2 (dislike), 3 (dislike somewhat), 4 (neutral), 5 (somewhat like), 6 (like), 7 (like very much). the results of the overall assessment by the panellists in the organoleptic test on the color, taste, aroma, and texture of jelly candy can be seen in (table 1) which has been statistically tested using the one way method (α=0.05%) and does not show any fundamental differences between the cinnamon breeds. jelly candy in various formulations. however, the highest overall favorability rating was obtained for f1, namely 5.14. in this formulation, the components consist of 0.5% cinnamon and 3.5% carrageenan, thus showing that the product that the panelists prefer is f1. conclusions the chemical properties of cinnamon jelly candy change with the addition of varying concentrations of red seaweed carrageenan. the antioxidant test with a substantial value is formulation 6, with a value of 50.26%, adding 1.5% cinnamon and 5% red seaweed carrageenan. acknowledgements: the author would like to thank all parties who have helped in this research. authors’ contributions: conceptualization, es.; methodology, e.s..; software, i.m.; validation, a.r.a; formal analysis, i.m.; resources, r,m.d.u; data curation, a.r.a.; carried out the laboratory work: e.s, writing— original draft preparation, e.s.; writing—review and editing, r.m.d.u.; visualization, e.s.; supervision, r.m.d.u, i.m. and a.r.a.; project administration, e.s.; funding acquisition, e.s. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. references amelia, r., & tanod, w. a. (2016). kandungan antioksidan alga merah eucheuma cottonii dengan metode pengeringan yang berbeda. kauderni: journal of fisheries, marine and aquatic science, 1(1), 14–20. https://doi.org/10.47384/kauderni.v1i1.7 andriyanto, a., andriani, m. a.., & esti, w. (2014). the influenced of cinnamon extract addition to sensory characteristic, antioxidant activity and antibacterial activity setiawan et al. – physicochemical candy cinnamon seaweed carrageenan. 421 on salted egg during storage in method wet salting. jurnal teknosains pangan, 2(2), 13–20. atmaka, w., nurhartadi, e., & karim, m. m. (2013). pengaruh penggunaan campuran karaginan dan konjak terhadap karakteristik permen jelly temulawak (curcuma xanthorrhiza roxb.). jurnal teknosains pangan, 2(2), 66–74. www.ilmupangan.fp.uns.ac.id bactiar, a., ali, a., & rossi, e. (2017). jelly candy making with red ginger extract addition carrageenan. jom faperta ur, 4(1), 3–7. bahri, m. a., dwiloka, b., & etza setiani, b. (2020). the influence of brightness, suppleness, vitamin c, and organoleptic properties of lemon orange jelly candy (citrus limon). jurnal teknologi pangan, 4(2), 96–102. www.ejournal-s1.undip.ac.id/index.php/tekpangan. hayulistya, d., affandi, d. r., & sari, a. m. (2021). influence of addition black cumin powder (nigella sativa) toward antioxidant activity of the herbal jelly candy. jurnal teknosains pangan, 5(4), 23–30. hidayah, r., harlia, gusrizal, & sapar, a. (2013). optimasi konsentrasi kalium hidroksida pada ekstraksi karaginan dari alga merah (kappaphycus. jurnal kimia khatulistiwa, 2(2), 78–83. lestario, l. n., sugiarto, s., & timotius, k. h. (2008). aktivitas antioksidan dan kadar fenolik total dari ganggang merah (gracilaria verrucosa l.). jurnal teknologi dan industri pangan, 19(2), 131–139. marfungah, n., tamrin, & asyik, n. (2019). pengaruh penambahan bubuk kayu manis (cinnamon burmanii) terhadap karakteristik kimia dan organoleptik permen jelly daun salam (syzygium polyanthum). jurnal sains dan teknologi pangan, 4(1), 1944–17956. miranti, m., lohitasari, b., & amalia, d. r. (2017). formulasi dan aktivitas antioksidan permen jelly sari buah pepaya california (carica papaya l.). fitofarmaka, 01(1), 1–7. novitasari, m., mappiratu, & sulistiawati, d. (2016). mutu kimia dan organoleptik permen jelly gelatin sapi. e-jurnal mitra sains, 4(3), 16–21. oktafa, h., permadi, m. r., & agustianto, k. (2017). studi komparasi data uji sensoris makanan dengan preference test (hedonik dan mutu hedonik), antara algoritma naïve bayes classifier dan radial basis function network. seminar nasional hasil penelitian, 75–78. rismandari, m., agustini, t. w., & amalia, u. (2017). the characteristics of jelly candy with addition of iota carrageenan from seaweed eucheuma spinosum. saintek perikanan (indonesian journal of fisheries science and technology), 12(2), 103. https://doi.org/10.14710/ijfst.12.2.103-108 salamah, e., & erungan, a. c. (2006). pemanfaatan gracilaria sp. dalam pembuatan permen jelly. buletin teknologi hasil perikanan, ix(1), 38–46. sukandar, d. (2014). aktivitas antioksidan dan mutu sensori formulasi minuman fungsional sawo-kayu manis. jurnal kimia valensi, 4(2), 80–89. https://doi.org/10.15408/jkv.v0i0.3605 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 799-806 | doi: 10.14421/biomedich.2025.142.799-806 issn 2540-9328 (online) assessment of knowledge, attitude and practice towards infection prevention and control among health staff at seiyun general hospital authority sarah o. alsawmhi1, anwar s. alhussan1, hassan pyar1,2, taher i. bin sumait1 1college of medicine and health science, seiyun university, yemen 2faculty of environmental science and marine biology, hadhramout university, yemen. corresponding author* sarahalsawmhi@gmail.com manuscript received: 23 august, 2025. revision accepted: 06 october, 2025. published: 15 october, 2025. abstract healthcare workers (hcws) are continuously exposed to pathogenic microorganisms, which can lead to serious infections. infection prevention and control (ipc) relies heavily on hcws’ knowledge, attitudes, and practices (kap). this study aimed to assess kap towards ipc among health staff at seiyun general hospital authority, yemen, during 2023–2024. a cross-sectional study was conducted involving 106 participants. data were collected through a self-administered questionnaire and analyzed using spss software. among participants, 60.6% held a diploma, 62.5% were male, 37.5% were nurses, and 33.7% had 1–5 years of experience. regarding knowledge, 91.3% reported understanding ipc, and 76.9% acknowledged the role of sterilization. in terms of attitude, 60% strongly agreed that handwashing reduces infections. as for practices, 90.4% washed their hands before patient care, and 76% educated patients about ipc. significant associations were found between kap levels and education, age, gender, job category, and experience. occupational category and work experience influenced kap levels, while education, age, and gender showed limited impact. keywords: knowledge; attitudes; practices; healthcare workers. introduction infection prevention is a systematic effort or process of placing barrier between susceptible host and the microorganisms. infection control also refers to all policies, procedures and activities which aim to prevent or minimize the risk of transmission of infectious disease at health care facility. health care-associated infection occurs in a patient during the process of care in a hospital or other healthcare facility that was not present or incubating at admission (yazie et al., 2019). a tenth of maternal deaths each year are caused by infections contracted during childbirth. multiple vaginal examinations, extended labour, premature rip of membranes, unsanitary births by inexperienced birth attendants, and non-compliance with standard precautions of infection control (spic) are all factors that increase the risk of puerperal infections (koshy and patel, 2015). healthcare professionals are constantly exposed to microorganisms, many of which can cause illnesses that are serious or even fatal. during their nursing careers, nurses in particular are frequently exposed to a variety of pathogens. therefore, in order to prevent hospital-acquired infections, health care personnel should be well-versed in and adhere to infection control procedures (thankam and goswami, 2021). patients who get medical care in a hospital or other medical facility but were not there at the time of admission can develop health-care-associated infections (hcais). medical staff occupational diseases are also included. hcais are the second leading cause of death globally and a significant contributor to morbidity and mortality (neama & nasir, 2025). healthcare-associated infections (hcais) in healthcare settings are caused worldwide by inadequate infection prevention practices, which also raise socioeconomic burdens, lengthen hospital stays, increase antibiotic resistance, and increase rates of morbidity and mortality, especially in developing and underdeveloped nations (kiddeer et al., 2025). infection prevention plays a key role in preventing and reducing the rate of healthcare associated infection (hais). hais, are the most frequent adverse event in healthcare worldwide can occur as a part of an endemic or epidemic situation and affect the quality of care of hundreds of millions of patients every year in both developed and developing countries (jemal et al., 2020). strict adherence to infection control policies, early removal of invasive devices, and timely treatment of infections are examples of infection prevention techniques that are essential to improving patient https://doi.org/10.14421/biomedich.2025.142.799-806 mailto:sarahalsawmhi@gmail.com 800 biology, medicine, & natural product chemistry 14 (2), 2025: 799-806 outcomes and lowering healthcare costs for patients (ait hssain et al. 2025). healthcare workers acquire these infections during specimen collection, processing and discarding, handling and discarding of medical equipment as well as during patient to hcws direct interaction at the time of examination. hospital-acquired infection preventions is placing a barrier between susceptible hosts and the microorganisms which is an essential component of delivering safe and high-quality service at the facility level (ayed, 2015). laboratoryacquired infection is of particular concern for pathogens such as hepatitis b and c viruses (hbv and hcv), human immunodeficiency viruses (hiv), middle east respiratory syndrome and severe acute respiratory syndrome coronavirus moreover, the major source of most hepatitis and hiv infections among healthcare professionals is needle stick injury, which can occur during all stages of needle use procedures (haguminshuti et al., 2022). the burden of hais is on the rise globally despite advancements in medical care and technologies. according to the world health organization (who), the prevalence of hais ranges between 5.7% and 19.1% in hospital settings globally. recent studies estimated the prevalence of hais in europe and the usa at 6.5% and 3.2%, respectively. the burden of hais is strikingly higher in low-resourced countries compared with highincome countries. a who-led systematic review revealed that the prevalence of hais varies between 7.6% and 15.5% in high-income and lowand middleincome countries, respectively. hais contribute to increased length of hospital stay, high mortality, higher health-care costs, and economic burden on families, communities, and countries at large. hence, prevention and control of hais appear as a critical public health concern (gezie et al., 2019). healthcare workers hcws awareness and compliance in disease prevention and control are vital elements for effective prevention. these approaches provide valuable information for health authorities in measuring the impact of ongoing awareness and prevention programs. additionally, hcws may act as transmission vehicles/sources for patient-to-patient infections in the clinical setting. therefore, to warrant effective control measures, hcws must receive regular training, and acquire sufficient knowledge on viral agents and microbes during infectious outbreaks (zhang et al., 2020). infection prevention and control (ipc) is an essential component of healthcare delivery, especially in public hospitals where a high patient volume and scarce resources may raise the risk of healthcare-associated infections (hcais). healthcare-associated infections (hcais) are related to increased morbidity, longer hospital stays, and higher healthcare costs. effective ipc programs are necessary to lower these risks and ensure patient safety. (ranoto et al., 2025). one of the most important aspects of patient and healthcare worker (hcw) safety in hospital settings is infection prevention and control, or ipc. hcws' knowledge, attitude, and practice (kap) about ipc are important. the discrepancy between behaviours and knowledge, however, suggests that frequent ipc audits and training are necessary (el-kazzaz et al. 2025). every healthcare worker (hcw) who comes into contact with a patient's blood or bodily fluids that call for universal precautions should regularly apply the proper barrier precautions to shield their skin and mucous membranes. a known and complicated issue that might be influencing the current global trend is noncompliance with rules and procedures that promote the decrease of nosocomial infections, or hais. (pierre et al., 2025). nosocomial infections that affect the prognosis of patients in intensive care units are the biggest problem they face. because of their critical role in infection control, nurses must possess the necessary knowledge and follow established procedures (bawaqneh et al., 2025). effective infection control (ic) practices are essential in healthcare institutions due to the rise of fatal pathogens like covid-19 (sars-cov-2) and methicillin-resistant staphylococcus aureus (jolly et al., 2024). a prevalent problem in healthcare institutions, especially in lowand middle-income countries (lmics), is healthcare-associated infections (hcais). handling and treating these infections require assessing healthcare workers' knowledge, attitude, and practice (kap) about hcai prevention and determining the pertinent aspects (shacho at al., 2024). thus, the aim of this crosssectional study was to assess of knowledge, attitude and practice towards ipc in seiyun general hospital authority among health staff. infection prevention and control (ipc) is essential to ensure patient safety, reduce healthcare-associated infections, and protect healthcare workers from occupational hazards. effective ipc practices are particularly vital in resource-limited settings, where healthcare facilities often face challenges, such as overcrowding, inadequate resources, and limited training opportunities. seiyun general hospital authority is a critical healthcare provider in its region, yet the extent of healthcare workers' knowledge, attitudes, and practices (kap) towards ipc remains unexplored. understanding these factors is crucial for identifying gaps and designing tailored interventions to enhance ipc measures. this study will provide valuable insights into the current state of ipc practices at the hospital, which can guide evidence-based policies, training programs, and resource allocation to strengthen infection prevention and control systems. the current study's goals are to evaluate the health staff's knowledge, attitudes, and practices about infection prevention and control at seiyun general hospital authority by assess the impact of educational attainment on infection control, determine the level of infection control understanding, determine the general attitudes about infection control. lastly, determine the infection control methods. alsawmhi et al. – assessment of knowledge, attitude and practice towards infection prevention and … 801 methodology study design this study was designed as a cross-sectional study. study area conducted among healthcare workers in seiyun general hospital authority, hadramout governorate, republic of yemen, between march and may 2023, using a written questionnaire. study population the study was conducted on 106 healthcare workers in seiyun general hospital authority. sample frame physicians, nurses, pharmacists, laboratory technicians, radiologists, midwifes, and anesthesiologists were included . inclusion criteria healthcare workers over 25 years of age in seiyun general hospital authority and health workers in clinical settings. they have the ability and willingness to participate. exclusion criteria administrative staff and workers in administrative settings were excluded. sample size this study included 106 samples calculated based on 95% confidence level and 5% margin of error. in total, 106 respondents answered the questionnaire, which was sufficient to draw a general conclusion about the knowledge and attitudes of healthcare workers towards infection prevention and control. the sample size in this study was recorded according to the following formula: 𝑛 = 𝑧2 × 𝑝𝑞 𝑑2 n = sample size z = power (1.96) p = disease prevalence d = standard deviation (0.05) x = 1 p sampling technique selection was simple random. data collection data are based on an interview using a questionnaire, which includes age, gender, education levels, occupational status, and work experience. since the study assessed healthcare worker's knowledge, attitude and practice approaches to dealing with of infection prevention and control, a standard questionnaire was developed, based on the literature on infection prevention and control in who. the questionnaire, which consisted of 29 main questions related to knowledge, attitude and practice, was designed and validated to cover the study objective. data management and analysis data were analyzed by statistical package for social science (statistical package for the social sciences) (spss), version 23. ethical consideration: the study was approved by ethical committee of the seiyun general hospital authority and the information was taken, and the consent forms were obtained from each participant results and discussion this study included 106 hcw participants at seiyun general hospital sociodemographic characteristics of the respondents. the majority of participants held a diploma (60.6%), followed by those holding a bachelor’s degree (29.8%), and a few held a master’s degree (6.73%) or a doctorate (2.88%). participants' ages ranged between 20 and 30 years, at a rate of 41.3%, while they were between 30 and 35 years, at a rate of 34.6%, and older than 35 years, at a rate of 24%. males constitute 62.5% of participants, while females constitute 37.5%. employment status: physicians constitute 18.3% of participants, nurses constitute 37.5%, midwives 7.69%, laboratory technicians 29.8%, physical therapists 0.96%, medical imaging technicians 3.85%, and pharmacists 1.92%. and 33.7% have work experience between one and five years, 28.8% have work experience between five and ten years, and 37.5% have work experience more than ten years (table 1). table 1. sociodemographic characteristics of the respondents. items variables frequency (n) percent (%) education levels diploma 63 60.6 bachloria 31 29.8 master 7 6.73 phd 3 2.88 age 20 -30 43 41.3 3035 36 34.6 >35 25 24 sex male 65 62.5 female 39 37.5 occupational status physicians 19 18.3 nurses 39 37.5 midwifes 8 7.69 lab technicians 31 29.8 physiotherapist 1 0.96 radiology technicians 4 3.85 pharmacist 2 1.92 work experience 1-5 years 35 33.7 5-10 years 30 28.8 more than 10 years 39 37.5 802 biology, medicine, & natural product chemistry 14 (2), 2025: 799-806 the responses-related to the respondent’s knowledge of ipc a large proportion of participants (91.3%) reported having control of their viral infection. 76% of the participants and the party believe disinfection prevents entry into the party, while 24% believe the opposite. 86.5% believe that disinfection prevents disease infection, while 13.5% believe the opposite. 34.6% believe all measures are tamed with a chemical, while 65.4% believe otherwise. 51.9% believe that physical sterilization (heat/radiation) is used for all measures, and 48.1% believe the opposite. 76.9% believe sterilization destroys all microorganisms, while 23.1% believe the opposite. 87.5% and every stomach needs to be disinfected before sterilization. 87.5% believe using medical equipment is necessary to confirm infection in the hospital. 11.5% believe that a finger replaces the need for hand washing, while 88.5% believe the opposite. 53.8% believe there is a personal belief in hiv and hemoglobin after 46.2% believe the opposite (table 2). table 2. responses-related to the respondent’s knowledge towards ipc. items variables frequency (n) percent (%) did you know about of infection prevention and control yes 95 91.3 no 9 8.65 disinfection prevents health care acquired infections yes 79 76 no 25 24 antiseptic prevents health care acquired infections yes 90 86.5 no 14 13.5 all equipment is sterilized using a chemical process yes 36 34.6 no 68 65.4 for all equipment, physical sterilization (heat/radiation technique) is used yes 54 51.9 no 50 48.1 autoclaving destroys all microorganisms, including spores yes 80 76.9 no 24 23.1 each equipment needs decontamination before sterilization yes 91 87.5 no 13 12.5 personal protective equipment minimizes health care acquired infection yes 91 87.5 no 13 12.5 wearing gloves replace the need for handwashing yes 12 11.5 no 92 88.5 there is ppe for hiv, hbags after exposure yes 56 53.8 no 48 46.2 table 3. responses-related to the responds attitudes towards ipc. items variables (n) (%) strongly agree agree neutral disagree strongly disagree when caring for a patient, you must be concerned about exposing your family and friends to healthcare associated infections 46 29 13 9 7 healthcare workers at my facility are concerned about contracting healthcare associated infections while caring for patients 52 35 8 9 0 washing hands before and after contact with patients reduces the risks of getting healthcare associated infections 60 39 3 1 1 i believe personal protective equipment protect healthcare workers from infection 43 48 6 5 2 in the absence of standard precaution, infection and nosocomial diseases can occur in health care facilities 46 45 10 3 0 in your workplace, there is a high risk of occupational infection among health worker 53 40 8 3 0 table 3 shows the responses-related to the respondent’s attitudes towards ipc 46% strongly agree with the need to take care not to expose family members and friends to hospital-acquired infections, while 29% agree and feel reassured. 52% strongly agree that health care workers are concerned about contracting hospitalacquired infections while caring for patients. 60% strongly agree that washing hands before and after alsawmhi et al. – assessment of knowledge, attitude and practice towards infection prevention and … 803 contact with sick people reduces the risk of hospitalacquired infections. 48% strongly agree that ppe protects health workers from infection, on the other hand 46% strongly agree that if standard precautions are not followed, infections and illnesses can occur in health care settings. 53% strongly agree that there is a high risk of occupational injury among health care workers comments about the respondent's ipc-related behaviors 90.4% of people used to wash their hands with soap before tending to patients. the effectiveness of soap cleaning following patient care or after touching liquids is 86.5%. when providing patient care, 69.2% of people employed their own abilities. according to 63.5% of participants, having written infection rules would be beneficial. health education for hospitalized individuals with the condition was given by 76.0% of participants. there is 59.6% of the software based on print by hand design. 70.2% of the treated patients covered previous needles. 76.9% reported exposure to blood, fluids, or penetrating wounds. approximately, 51.0% of the body receiving prophylaxis after exposure to blood. 68.3% clean wounds with alcohol. 60.6% of the agency relied on the availability of manual cleaning supplies in patient care areas (table 4). table 4. responses-related to the respondent’s practices towards ipc. items variables frequency (n) percent (%) wash hands using soap before patient care yes 94 90.4 no 10 9.62 wash hands using soap after patient care/contact with fluid yes 90 86.5 no 14 13.5 wash hands without using soap before and after patient care yes 29 27.9 no 75 72.1 are using personal protective equipment (ppe) when taking care of patients? yes 72 69.2 no 32 30.8 what kind of ppe are you using in patient care? gloves 98 94.2 goggles 17 16.3 face masks 35 33.7 gown 41 39.4 other ppe 17 16.3 there are written infection control policies and procedures available yes 66 63.5 no 38 36.5 recapping needles before disposing yes 73 70.2 no 31 29.8 ever had contact with blood, liquids or puncture wounds yes 80 76.9 no 24 23.1 what are the infection prevention and control (ipc) measures adopted after being exposed to blood/stick injury? taking pep 53 51.0 cleaning by alcohol 71 68.3 washing with water 40 38.5 giving health education to the patients about healthcare associated infections yes 79 76.0 no 25 24.0 the hospital or health center has a competency-based hand hygiene training program yes 62 59.6 no 42 40.4 supplies necessary for adherence to hand hygiene are readily accessible in-patient care areas yes 63 60.6 no 41 39.4 level of knowledge, attitudes and practice in ipc among different levels of education. level of knowledge towards ipc: it appears that there is a statistically significant correlation between the level of education and the level of knowledge towards ipc (p = 0.05). level of attitude toward ipc: it is also shown that there is a statistically significant association between level of education and attitudes toward ipc (p = 0.05). level of practice with ipc: in the same way, there is a statistically significant correlation between the level of education and practices towards ipc (p = 0.05). level of knowledge and attitudes and practice in ipc among different age groups. level of knowledge towards ipc: it appears that there is a statistically significant association between age group and level of knowledge towards ipc (p = 0.05). attitudes towards ipc: it is also shown that there is a statistically significant association between age group and attitudes towards ipc (p = 0.05). practices towards ipc: it appears that there is a statistically significant association between age group and practices towards ipc (p = 0.05). level of knowledge and attitudes and practice in ipc among different levels of sex. level of knowledge towards ipc: it appears that there is a statistically significant association between gender and level of knowledge towards ipc (p = 0.05). attitudes towards ipc: there is also a statistically significant association between gender and attitudes towards ipc (p = 0.05). practices towards ipc: there is a statistically significant 804 biology, medicine, & natural product chemistry 14 (2), 2025: 799-806 association between gender and practices towards ipc (p = 0.05). level of knowledge and attitudes and practice in ipc among different levels of occupational. level of knowledge towards ipc: it appears that there is a statistically significant association between professional category and level of knowledge towards ipc (p = 0.05). attitudes towards ipc: it is also shown that there is a statistically significant association between occupational category and attitudes towards ipc (p = 0.05). practices towards ipc: there appears to be a statistically significant association between the professional category and practices towards ipc (p = 0.05) level of knowledge and attitudes and practice in ipc among different levels of work experience. level of knowledge towards ipc: it appears that there is a statistically significant association between the period of work experience and the level of knowledge towards ipc (p = 0.05). attitudes towards ipc: it is also shown that there is a statistically significant association between the length of work experience and attitudes towards ipc (p = 0.05). practices towards ipc: it appears that there is a statistically significant association between the period of work experience and practices towards ipc (p = 0.05) (table 5). table 5. association between respondents' socio-demographic characteristics and respondent’s knowledge, attitude and practice towards ipc in seiyun general hospital authority city. variable knowledge towards ipc p-value attitudes towards ipc p-value practices towards ipc education levels good poor good poor good poor diploma 12 34 0.05 45 54 0.05 45 64 bachloria 12 34 45 54 45 64 master 12 34 45 54 45 64 phd 12 34 45 54 45 64 age 20 -30 12 34 0.05 45 54 0.05 45 64 3035 12 34 45 54 45 64 >35 12 34 45 54 45 64 sex male 12 34 0.05 45 54 0.05 45 64 female 12 34 45 54 45 64 occupational physicians 12 34 0.05 45 54 0.05 45 64 nurses 12 34 45 54 45 64 midwifes 12 34 45 54 45 64 lab technicians 12 34 45 54 45 64 physiotherapist 12 34 45 54 45 64 radiology technicians 12 34 45 54 45 64 pharmacist 12 34 45 54 45 64 work experience 1-5 years 12 34 0.05 45 54 0.05 45 64 5-10 years 12 34 45 54 45 64 >10 years 12 34 45 54 45 64 p-value *significant = > 0.05 the knowledge, attitudes, and practices towards infection prevention and control are found worldwide. the study conducted at seiyun general hospital on healthcare workers' (hcws) knowledge, attitudes, and practices (kap) towards infection prevention and control (ipc) provides valuable insights into the current status of ipc in the region. our results identified significant associations between demographic factors such as age, gender, education level, and occupation with the knowledge, attitudes, and practices of ipc. for example, older hcws and those with more years of work experience tended to have a higher level of knowledge and a more favorable attitude towards ipc, consistent with findings from hussein et al. (2017) and yazie et al. (2019). this suggests that experience and exposure to various clinical scenarios play a role in shaping ipc behaviors. additionally, the study found that gender and professional category physicians, nurses, etc. also influenced ipc practices, with male hcws and those in more senior roles showing better adherence to infection control protocols. these findings are in line with studies by zhang et al., (2020), which highlighted that healthcare workers' professional roles and experience significantly impacted their ipc practices. our results from seiyun general hospital are consistent with other regional and international studies, alsawmhi et al. – assessment of knowledge, attitude and practice towards infection prevention and … 805 demonstrating that healthcare workers generally possess good knowledge and attitudes toward ipc. the current results were the high level of awareness among hcws, with 91.3% of participants reporting knowledge of viral infection control, and correctly identifying disinfection and sterilization procedures. this is in line with several other studies, such as the one by haguminshuti et al. (2022), which reported a high level of knowledge (78.6%) among hcws in rwanda. additionally, the study by ayed et al. (2015) also found fair to good knowledge levels among nurses, emphasizing the importance of education in ipc. however, some discrepancies in responses, such as the 34.6% of participants who believe chemical agents alone can prevent infection, suggest a need for more targeted training on appropriate disinfection methods and sterilization procedures. in contrast, the study by jemal et al., (2020) highlighted lower knowledge levels, with only 55.7% of participants showing good knowledge of ipc, indicating that knowledge gaps persist despite training. this suggests that while knowledge levels may be high in seiyun general hospital, similar studies from other regions show the need for continued and up-to-date training to address evolving ipc practices. the attitudes towards ipc in seiyun general hospital were generally positive, with 46% strongly agreeing on the importance of preventing hospital-acquired infections for both patients and their families. similar positive attitudes were reported in studies such as those by tamang et al., (2020), where hcws demonstrated a positive attitude towards ipc in nepal, influenced by their training and educational background. in the current study, 60% of participants believed that washing hands before and after contact with sick individuals reduces hospital-acquired infections, aligning with global best practices emphasizing hand hygiene as a primary infection control measure. however, while attitudes were largely positive, there were some gaps in the overall consistency of beliefs regarding ipc practices. for example, 48.1% of the participants believed that physical sterilization was used for all measures, which may reflect a lack of understanding regarding the full spectrum of ipc methods, including chemical disinfection. this aligns with findings from gezie et al., (2019) which showed good attitudes toward ipc but identified a gap in practical implementation. therefore, although the attitudes towards ipc appear favorable, a deeper understanding of the diverse methods of infection prevention and control is needed to address any remaining misconceptions. the practice of ipc in seiyun general hospital was found to be relatively high, with 90.4% of hcws washing hands before patient care, which is consistent with findings from study by lobo et al., (2019), where a majority of hcws demonstrated good hand hygiene practices. however, it is notable that while most participants reported washing hands effectively, only 63.5% agreed that written infection policies would be beneficial, which suggests that there may be room for improvement in the formalization and communication of ipc protocols within the hospital. this finding mirrors that of aldhamy et al. (2023), who also noted gaps between knowledge and practice, especially regarding the practical application of ipc guidelines. the study also found significant exposure to risk factors, such as blood and fluid exposure (76.9%), with only 51% receiving prophylactic treatment after such exposures. this is similar to the study by hamid et al. (2019), which found that while knowledge about infection control was relatively high, the practices related to preventing nosocomial infections were lacking. this disparity emphasizes the need for increased focus on preventive practices, including post-exposure prophylaxis and disposal of sharp objects, as part of comprehensive infection control programs. this research recommended increasing knowledge and providing instruction. all healthcare personnel must receive regular, up-to-date training on infection prevention techniques. secondly, improving preventative measures like sterilizing medical equipment, using personal protection equipment appropriately and encouraging adherence to hygiene guidelines should all be improved. third tracking performance, to enhance procedures and lower infection-related events, it is critical to routinely assess employee performance and offer pertinent criticism and direction. conclusion the results of the presented study showed that seiyun hospital staff members are now significantly more aware of infection prevention techniques; nevertheless, there is still room for improvement in the way these practices are implemented in others. on the other hand, deficiencies in the use of some infection prevention and control strategies must be addressed, especially those pertaining to post-exposure prophylaxis, sterilizing methods, and the formalization of written policies. updating training programs to ensure ongoing professional growth, improving post-exposure protocols, and strengthening the practical application of infection prevention and control methods should be the main goals of future interventions. acknowledgements: the authors would like to express their sincere gratitude to the college of medicine and health sciences, seiyun university, hadramout, yemen, for their support and facilitation of this research. competing interests: the authors declare that there are no competing interests. 806 biology, medicine, & natural product chemistry 14 (2), 2025: 799-806 references ait hssain, a., bahramnezhad, f., vahedian-azimi, a., ibrahim, a. s., hassan, i. f., imani-fooladi, a. a. & darmon, m. 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(2020). knowledge, attitude, and practice regarding covid-19 among healthcare workers in henan, china. journal of hospital infection, 105 (2): 183-187. doi.10.1016/j.jhin.2020.04.012 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1099-1103 | doi: 10.14421/biomedich.2025.142.1099-1103 issn 2540-9328 (online) toddler massage as a supplementary intervention in overcoming stunting in children: research from stunting locus area in gorontalo city fatmah zakaria1*, sri mulyaningsih2, dwi nur octaviani katili2, rizky nikmathulhusna ali2, mohamad rodi isa3, herbawati4 1professional midwifery education study program, faculty of health sciences, muhammadiyah university gorontalo, gorontalo, indonesia. 2bachelor's program in midwifery, faculty of health sciences, muhammadiyah university gorontalo, gorontalo, indonesia. 3departement of public health medicine, faculty of medicine, universiti teknologi mara, selangor, malaysia. 4tolinggula health care center, north gorontalo, gorontalo, indonesia. corresponding author* fatmahzakaria@umgo.ac.id manuscript received: 03 august, 2025. revision accepted: 21 november, 2025. published: 27 november, 2025. abstract stunting, defined as impaired linear growth resulting in a child’s height falling below the age-appropriate standard, remains a pressing global health concern due to its long-term implications on development and increased risk of mortality. in gorontalo province, indonesia, the prevalence of stunting exceeds the national target of 23.8%, with north gorontalo regency reporting the highest rate at 29.3%. this study aimed to examine the effect of toddler massage on weight gain among stunted children. a quasi-experimental design with a pretestposttest control group approach was employed, involving 32 stunted toddlers selected through purposive sampling. participants were equally allocated into an intervention group (n=16), which received toddler massage based on standard operating procedures, and a control group (n=16), which did not receive the intervention. body weight was measured using digital scales. the results of the paired ttest revealed a statistically significant weight gain in the intervention group (p = 0.00), whereas no significant difference was observed in the control group (p = 0.164). the findings indicate that toddler massage may contribute to weight improvement in stunted children and could be considered a supportive non-pharmacological intervention in stunting management programs. keywords: stunting; toddler massage; weight gain; child growth. introduction stunting is a condition characterized by a child’s height or body length being significantly below the standard, which indicates chronic malnutrition over an extended period (beal et al., 2018). stunting is not merely a physical growth disorder, it also increases toddlers' susceptibility to disease and poses a serious threat to the human resources quality (dewey & begum, 2011). it impacts not only physical development but also brain development and cognitive intelligence (soliman et al., 2021). thus, stunting has far-reaching effects not only on individual health but also on national economic growth and equity. according to the world health organization (who), the global prevalence of stunting in children under five in 2024 was 22%, or approximately 149.2 million children (wold health organization, 2024). in indonesia, data from the indonesian nutrition status survey (ssgi) 2024 indicated a stunting prevalence of 24.4%, equivalent to 5.33 million cases (kementerian kesehatan, 2024) in gorontalo province, the rate was still above the target at 23.8%, with north gorontalo regency exceeding that at 29.3% (kementerian kesehatan, 2024). as of preliminary data from the tolangohula health center recorded 111 stunted toddlers, representing 14.6% of children in the area (puskesmas tolangohula, 2024). stunting is affected by multiple factors, including environmental conditions such as poor hygiene practices (phbs), socioeconomic status, and maternal health and nutrition before, during, and after pregnancy. low birth weight and poor postnatal nutrition also contribute significantly (mcgovern et al., 2017). choi et al. identified key determinants in descending order of influence: family economic status, exclusive breastfeeding, family size, father's education level and occupation, maternal nutritional knowledge, food security, maternal education, carbohydrate and fat intake, complementary feeding practices, child illness history, sociocultural factors, protein intake, maternal employment, nutrition-conscious behavior, energy intake, and immunization completeness (choi et al., 2016). https://doi.org/10.14421/biomedich.2025.142.1099-1103 1100 biology, medicine, & natural product chemistry 14 (2), 2025: 1099-1103 to address this issue, the indonesian government has implemented several programs, including the distribution of iron supplements to adolescent girls, regular antenatal care, and the provision of nutritional supplements for pregnant women (kementerian kesehatan, 2023). additional feeding programs for children aged 6–24 months emphasize animal protein sources such as eggs, fish, poultry, meat, and milk (pham et al., 2021). the ministry of health also promotes specific interventions through community health centers and integrated service posts, as part of the "first 1,000 days of life" (hpk) movement. these programs support early breastfeeding initiation, exclusive breastfeeding for infants under six months, and appropriate complementary feeding practices until the age of 23 months (aurelia, 2024). to accelerate stunting reduction, integrated and responsive interventions are essential. one strategy that health professionals can employ to improve weight gain and reduce stunting is to provide education, information, and communication (eic) about infant massage to mothers. massage is recognized as an effective form of stimulation to promote growth and development in children (lestari et al., 2021). it works by stimulating the soft tissues of the child’s body through techniques such as rubbing, kneading, and pressing, which activate sensory receptors in the skin and promote both physical and emotional well-being (erçelik & yılmaz, 2023). previous studies support the effectiveness of infant massage in increasing weight among malnourished children. muliastiti et al. reported a significant improvement in weight gain following massage intervention using paired t-test analysis (p < 0.05) (sukamti et al., 2024). similarly, junita et al. found that the average infant weight increased from 4.86 kg before massage to 5.72 kg after intervention (p = 0.000) (junita et al., 2022). while these publications emphasize of the benefits of the message, a literature from priyadarshi et al. confirmed that there still a need to evaluate about these benefits for its short-and-long-term (priyadarshi et al., 2022). remain to provide support the findings, this study aimed to give intervention of baby message to the stunted children. materials and methods research design this study employed a quasi-experimental research design using a pre-test and post-test control group approach. the research was conducted over the course of one month, from december 2023 to january 2024, in the working area of tolangohula health center. each group consisted of 16 respondents, with the sample size determined using a power of 90% and a confidence interval of 95%. the sampling technique used was purposive sampling, in which participants were selected based on predefined inclusion and exclusion criteria. in accordance with the ethical guidelines of the helsinki declaration, this study received ethical approval from the research ethics committee of gorontalo state university (protocol no. 0090227571221242025010600007). written informed consent was obtained from each participant’s parent or guardian prior to participation. sampling method out of 111 stunted toddlers identified in the tolangohula health center service area, 32 toddlers aged between 12 and 24 months met the inclusion and exclusion criteria and were selected as respondents. these were equally divided into an intervention group (n = 16) and a control group (n = 16). intervention the instruments used in this study included a massage observation sheet based on a standard operating procedure (sop) and a digital weighing scale. before the intervention, body weight measurements were taken in both the intervention and control groups. the intervention group received baby massage once per week for four weeks (total of one month), while the control group did not receive any massage intervention. after the four-week period, both groups underwent another body weight measurement. procedure researchers provided written informed consent to parents as approval for their toddlers to participate in the massage intervention. second, initial weight measurements were taken in both the intervention and control groups. following the four-week massage intervention in the experimental group, body weight was measured again using a digital weighing scale in both groups. data processing and analysis data were analyzed using two statistical tests: the paired t-test and the independent t-test. both tests require the assumption of normal data distribution. therefore, the kolmogorov-smirnov test was used to assess data normality. the hypothesis was evaluated using a significance level of α = 0.05, with p-values < 0.05 indicating rejection of the null hypothesis. results were presented in tabular form with a 95% confidence interval. zakaria et al. – toddler massage for stunting intervention 1101 results and discussion table 1. frequency distribution of respondents characteristics. characteristics intervention group (n=16) control group (n=16) n % n % age 12 -24 months 13 81,25 12 75 > 24 months 3 18,75 4 25 mother's education tall 0 0 0 0 low 16 100 16 100 paritas primipara 1 6,25 2 12,5 multipara 14 87,5 14 87,5 largemultipara 1 6,25 0 0 imd yes 14 87,5 15 93,75 no 2 12,5 1 6,25 exclusive breast milk yes 12 75 15 93,75 no 4 25 1 6,25 based on the table 1, the majority of children in both the intervention and control groups were aged 12–24 months, accounting for 81.25% and 75%, respectively. this indicates that most participants were in early toddlerhood, a critical period for growth and development. all mothers in both groups had a low level of education (100%), which may influence their health knowledge and caregiving practices, including child feeding. in terms of parity, most mothers were multiparous (87.5%) in both groups, suggesting they had prior childbirth experience. a high proportion of mothers practiced early initiation of breastfeeding (eibf), with 87.5% in the intervention group and 93.75% in the control group, reflecting favorable breastfeeding practices from birth. additionally, the proportion of exclusive breastfeeding was higher in the control group (93.75%) compared to the intervention group (75%). table 2. distribution of pre and post weight frequencies in the intervention group and control group. weight n pretest posttest mean min-max mean min-max intervention 16 10,40 8-12,2 10,85 8,5-13 control 16 10,12 7,2-14 10,15 7,2-14 the result shows the comparison of mean body weight in the intervention and control groups before and after the intervention. in the intervention group, the mean weight increased from 10.40 kg (range: 8–12.2 kg) at pretest to 10.85 kg (range: 8.5–13 kg) at posttest. in contrast, the control group showed only a slight increase in mean weight from 10.12 kg (range: 7.2–14 kg) at pretest to 10.15 kg (range: 7.2–14 kg) at posttest. these findings suggest that the intervention may have had a positive effect on weight gain in children, while minimal change was observed in the control group (table 2). table 3. differences in average weight of toddlers in the intervention group and control group weight pretest posttest mean difference t test ρ value mean (sd) mean (sd) (ci 95%) intervention 10,40 10,65 0.25 kg 4,502 0,00 (1,3466) (1,4601) (0,3684-0,1316) control 10,11 10,12 0.011 kg 1,464 0,164 (1,761) (1,7523) (0,0307-0,0057) the paired t-test analysis showed a statistically significant difference in mean weight before and after the intervention in the intervention group. the mean weight increased from 10.40 kg (sd = 1.35) to 10.65 kg (sd = 1.46), with a mean difference of 0.25 kg (95% ci: 0.3684–0.1316; t = 4.502; p < 0.001). in contrast, the control group showed a minimal and statistically nonsignificant increase in mean weight from 10.11 kg (sd = 1.76) to 10.12 kg (sd = 1.75), with a mean difference of 0.011 kg (95% ci: 0.0307–0.0057; t = 1.464; p = 0.164). these findings suggest that the intervention had a significant effect on weight gain compared to the control group. 1102 biology, medicine, & natural product chemistry 14 (2), 2025: 1099-1103 discussion infant massage is broadly recognized as a nonpharmacological method of stimulating an infant’s body through gentle, rhythmic touch and a series of structured techniques. it typically involves the application of moderate pressure to specific parts or the entirety of the infant’s body and is often integrated into caregiving routines. the benefits of infant massage have been explored in several studies, particularly in the context of improving digestion, enhancing physical development, and supporting overall well-being (chen et al., 2024). paymaneh et al. noted that baby massage might promote gastrointestinal motility and bowel function, which can indirectly influence nutritional status and physical growth (taheri et al., 2018). although the underlying mechanisms are still under investigation, some evidence suggests that massage may stimulate the vagus nerve, leading to improved parasympathetic activity that affects digestion, nutrient absorption, and post massage sleep (agarwal et al., 2000). studies have even indicated that tactile stimulation could influence bone development (choi et al., 2016). in clinical settings, massage has been associated with positive outcomes among infants and toddlers, particularly those facing nutritional challenges such as stunting. a randomize controlled trial by lu et al. showed that infants who received massage experienced weight gains that, in some cases, exceeded the expected averages for their age groups (lu et al., 2020). while such findings are promising, it is important to interpret them in the broader context of child development. weight gain is influenced by multiple variables, including genetic predisposition, feeding practices, health status, and environmental factors (frosch et al., 2019; putra et al., 2025). thus, although massage may support weight gain by enhancing appetite, nutrient utilization, and emotional bonding, it is not a substitute for adequate nutrition, medical care, and responsive parenting. rather, it should be seen as a complementary practice within a holistic framework of early childhood care. the sensory and emotional benefits of massage are also noteworthy. physical contact during massage-such as stroking, eye contact, and vocal interaction-can foster emotional bonding between caregiver or the mother and child, which is essential for psychological development (jethava et al., 2022). this interaction may contribute to reduced infant stress levels, which, according to some theories, could help regulate hunger cues and feeding behavior (gürol & polat, 2012). hormonal responses to massage, such as the release of endorphins and reduction in cortisol, may further enhance infants' comfort and potentially improve feeding outcomes. on a physiological level, massage is thought to promote circulation, stimulate the endocrine system, and facilitate the release of growth-related hormones, including those produced by the pituitary gland (field et al., 2005). however, the extent to which these effects are generalizable remains a topic for continued research. considering the current study’s findings, the application of baby massage four times a month was associated with modest yet observable improvements in weight among toddlers in the intervention group. although these results do not establish causality, they contribute to a growing body of literature suggesting that regular, structured massage may offer developmental advantages, particularly for children at risk of undernutrition. further longitudinal studies with larger samples are recommended to explore the long-term benefits and establish clearer clinical guidelines. conclusions the average weight of stunted toddlers in the respondents who received massage was 2500 grams while the average weight of stunted toddlers in the control group was 1100 grams so it can be concluded that there is a significant influence of toddler massage on weight gain for stunted toddlers with a p-value of 0.000 <0.05. acknowledgements: the author would like to thank all respondents, especially mothers of toddlers who are willing to participate in this study so that this research can run well without conflicts. the head of the puskesmas and village midwives of the tolangohula health center working area who has given the author the opportunity to make the research location and has made a great contribution to the implementation of this research. the leadership of the faculty of health sciences, university of muhammadiyah gorontalo who provided assistance and support to the author so that this research could be carried out. authors’ contributions: all authors are actively involved from the beginning of the research process to the process of publication of the article. fz and sm prepare draft articles to publications, dnok, rna and mri help process data, he carries out the research process in the work area of the tolinggula health center. all authors were actively involved in the research article. all 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(2024). stunting prevalence among children under 5 years of age (%) (model-based estimates). https://www.who.int/data/gho/data/indicators/indicatordetails/gho/gho-jme-stunting-prevalence this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 83-87 | doi: 10.14421/biomedich.2022.111.83-87 issn 2540-9328 (online) anti-oxidative effects of butanol seed extract of parkinsonia aculeata on carbon tetrachloride-induced liver damage on wistar rats muhammad bashiru abdulrahman1,*, yusuf gumburawa malami2, sanusi wara hassan3, mansur lawal3, waliu temitope adanlawo4, mansur mohammed birnin kebbi4, kamaldeen olalekan sanusi5 1department of chemical pathology and immunology, faculty of basic clinical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria; 2department of science laboratory technology, umaru ali shinkafi polytechnic, sokoto, nigeria; 3department of biochemistry, faculty of science, usmanu danfodiyo university, sokoto, nigeria; 4department of chemical pathology, usmanu danfodiyo university teaching hospital, sokoto, nigeria; 5department of physiology, faculty of basic medical sciences, college of health sciences, usmanu danfodiyo university, sokoto, nigeria. corresponding author* abfmhzs69@gmail.com manuscript received: 06 may, 2022. revision accepted: 27 may, 2022. published: 30 june, 2022. abstract medicinal plants have protective effect because of the presence of several compounds which have different mechanism of action. this study sought to assess the anti-oxidative effects of butanol seed extract of parkinsonia aculeata on carbon tetrachloride (ccl4)-induced liver damage on wistar rats. the wistar rats were put into five groups, each with six rats: group a received a daily dosage of liquid paraffin (1ml/kg); group b received 1ml/kg body weight of ccl4 (30% in liquid paraffin intraperitoneal); group c, d, and e received the seed extracts at 100, 120, and 160 mg/kg every day for two weeks. induction of ccl4 was three times a week for two weeks simultaneously with the extract to the last day. after sacrificed, the liver was harvested and homogenized, and used for further analyses. there was a significant increase (p<0.05) in the levels of superoxide dismutase and catalase in all extract treated groups compared to positive control, except the catalase levels of group treated with 160mg/kg. similar results was observed in vitamin c, vitamin e and glutathione in rats treated with 100 and 120mg/kg of the extract. the results of this study revealed that butanol seed extract from p. aculeata has antioxidant properties and can protect wistar rats' livers from the damaging effects of ccl4. keywords: antioxidant; carbon tetrachloride; liver damage; oxidative stress; parkinsonia aculeata. abbreviations: cat – catalase; gsh reduced glutathione; sod superoxide dismutase; ccl4 carbon tetrachloride; mda – malondialdehyde; ros reactive oxygen species; h2o2 hydrogen peroxide. introduction antioxidant activity is an important approach to defend against liver damage. antioxidants are chemicals that inhibit the oxidation of a molecule in the cells when present in extremely low concentrations. it has the capacity to nullify the effects of oxidation (lipid peroxidation) caused by free radicals in the liver of an organisms. the impaired electrons of free radicals are highly reactive and neutralize the harmful reactions of human metabolism (serbinova et al., 1991). protection of the liver against free radicals effects is provided by enzymatic antioxidants such as catalase (cat), reduced glutathione (gsh), and superoxide dismutase (sod) and non-enzymatic antioxidants e.g. vitamin c and vitamin e that play a crucial role in lipid peroxidation process. the rising amount of chemical compounds and pollution in the environment are causing an increase in liver illnesses (wadekar et al., 2008). many of these chemicals cause harm to cells and molecules by producing reactive oxygen species and other free radicals. because of its critical function in the body's metabolism of external chemical compounds, liver is one of the organs that can cause toxic reactions and is a popular subject of toxicological research (beckman & ames, 1998). carbon tetrachloride (ccl4) is frequently used as a chemical inducer of experimental liver cirrhosis. this toxic agent activates liver damage by forming reactive intermediates, such as trichloromethyl free radicals, via cytochrome p450-related functions in the oxidase system (recknagel et al., 1989). the main causes of ccl4-induced hepatic damage is related to lipid peroxidation enzymes, and generation of free radicals caused by this agent (poli, 1993). medicinal plants have protective effect because of the presence of several compounds which have different https://doi.org/10.14421/biomedich.2022.111.83-87 84 biology, medicine, & natural product chemistry 11 (1), 2022: 83-87 mechanism of action. some of the components of this plant are proteins and enzymes of low molecular weight such as vitamin, flavonoids and carotenoids (zhang & wang, 2002). many of these components especially phytochemical constituents exhibit hepatoprotective effects due to their antioxidant property (madrigalsantillán et al., 2014). the leaf, stem bark and the seed extracts of parkinsonia aculeata are used traditionally in the northern nigeria for the treatment of hepatopathy, bacterial diseases, typhoid fever, diabetes, malaria and trypanosomiasis (hassan et al., 2010; leite et al., 2007). leaf extracts of p. aculeata were reported to have hepatoprotective and antioxidant activities (hassan et al., 2005). however, there is paucity of scientific report on the seed extract. this study therefore sought to assess the anti-oxidative effects of butanol seed extract of p. aculeata on carbon tetrachloride-induced liver damage on wistar rats. materials and methods. plant collection, identification and storage parkinsonia aculeata seeds were obtained on the campus of usmanu danfodiyo university in sokoto, nigeria. the plant components were identified and taxonomically validated at usmanu danfodiyo university, sokoto botany unit. in the departmental herbarium of the botany unit, a voucher specimen of the plant (uduh/ans/0038) was deposited for reference. the seeds were opened and air dried for one week in the shade before being pounded into a fine powder with a mortar and pestle and stored at room temperature until use. experimental animal wistar rats of either sex weighing 120-200 g were obtained from animal house, department of biological sciences, usmanu danfodiyo university, sokoto, nigeria. they were kept in wire mesh cages with free access to food and water for one week to acclimatize. they were maintained on standard optimal feeds and clean tap water before and after daily administration of plant extract between 9:30 to 10:30 hours. experiments were performed according to ethical guidelines for investigation of experimental pairs in conscious animals. the standard orogastric cannula was used for oral administration of the seed extract. preparation of plant extracts two hundred grams (200g) of fine powder of the plant seed were extracted with two (2) litres of methanol at room temperature overnight and filtered through whatman no. 1 filter paper. the filtrate was concentrated to dryness using rotary evaporator and percentage yield was calculated (5.6%). the residue was dissolved in distilled water and partitioned with butanol (saturated with water). the butanol fraction was thereafter screened for antioxidant properties. experimental design the animals were divided into 5 groups of six (6) rats. group a: (normal control) received daily dose of liquid paraffin (1ml/kg) body weight per day for 14 days. group b: (positive control) received 1ml/kg body weight of ccl4 (30% in liquid paraffin intraperitoneal) three (3) times a week for two (2) weeks. group c, d and e: received the seed extracts of p. aculeata at 100, 120 and 160 mg/kg per day respectively for two weeks. induction of ccl4 was three times a week for two weeks simultaneously with the extract to the last day. animals were sacrifice 24 hours after the last day. liver was removed and rinsed in ice-cold 1.5% kcl, dried and weighed homogenized in 4x ice-cold isotonic phosphate buffer ph 7.4 and the centrifuged at 9000g for 20 minutes to obtain post mitochondria supernatant at 105,000xg for 60 minutes to obtain the microsomes cytosolic fractions of the supernatant was immediately frozen on dry ice and microsome was suspended in 0.15 sucrose solution. sample preparation for the determination of markers of antioxidant activity the liver was perfused with 0.86% cold saline to completely remove the red blood cells, it was suspended in 10% (w/v) ice-cold. 0.1cm3 phosphate buffer at ph 7.4. the liver was then cut into small pieces, some quality was weighed and homogenized. the homogenate used for the estimation of enzymatic and non-enzymatic antioxidants. estimation of vitamin c vitamin c estimation was carried out using the method of rutkowski et al. (rutkowski et al., 1998). briefly, 1ml of sample was measured into test tubes, 1ml of pr (50nm solution of oxalic acid) was added and mixed thoroughly at room temperature for 30 minutes. it was centrifuged at 700g for 16 minutes, supernatant was collected with pipette and used as a test sample for spectrophotometric measurements. standard sample was prepared using 1ml of standard solution without centrifugation. absorbance of test sample (ax) and standard sample (as) was measured at 700nm against the mixture of pr as the reference sample. concentration cx of vitamin c (µm) in the sample was determined using the formula 𝐶𝑥 = 𝐴𝑥 as . 𝐶𝑠 where; cs = concentration of standard solution abdulrahman et al. – anti-oxidative effects of parkinsonia aculeata 85 estimation of vitamin e vitamin e was determined using the method of rutkowski et al. (rutkowski et al., 2005). briefly, 0.5ml of sample was measured into test tube, 0.5ml of anhydrous ethanol were added and shaken vigorously then plugged for 1 minute. a 3ml of xylene was added again and shaken vigorously for another 1 minute, centrifuged at 1500g for 10 minutes simultaneously. a 0.25ml solution of batophenanthroline was measured into test tube ii. a 15ml of the extract was collected and transferred to test tube ii, the content was mixed. a 0.25ml of fecl3 solution was also added to test tube ii, mixed then 0.25ml of h3po4 solution was also added and mixed again and the absorbance was measured spectrophotometrically. 0.5ml of standard solution prepared using trolox as test sample using α tocopherol. 0.5ml of deionized water was added. absorbance of the test (ax) and standard (as) was measured at 539nm against blank test. concentration of vitamin e was thereafter calculated using the formula: concentration of vitamin e (cx) 𝐶𝑥 = 𝐴𝑥 as . 𝐶𝑠 where; cs = concentration of standard solution. estimation of reduced glutathione and enzymatic assay of superoxide dismutase and catalase the colorimetric assay for catalase (cat), superoxide dismutase (sod), and reduced glutathione (gsh) was carried out using commercial kits (randox lab) according to the manufacturer’s protocol. malondialdehyde (mda) determination tissue supernatant (150µl) was diluted to 500µl with double deionized water. two hundred and fifty (250) µl of 1.34% thiobarbituric acid was added to all the test tubes followed by addition of an equal volume (250µl) of 4% trichloroacetic acid (tca). the resulting mixture was shaken and incubated for 30 minutes in a water bath temperature greater than 900c. the test tubes were allowed to cool at room temperature and the absorbance of the complex formed was read at wave length of 532nm (hartman, 1983). the absorbance was extrapolated from a standard curve generated by using a standard (1,1,3,3-tetraethoxy propane). the result was expressed as nanomoles of mda per cubic centimeter of supernatant. results estimation of non-enzymatic antioxidants. there was a significant increase (p<0.05) in the levels of vitamin c, vitamin e and gsh in rats treated with 100 and 120mg/kg of the extract. however, it was observed that the higher the dose, the lower the values (table 1). table 1. non-enzymatic antioxidant properties of rats administered butanol seed extracts of parkinsonia aculeata and carbon tetrachloride. group vitamin c (umol/l) vitamin e (mg/dl) gsh (mg/ml) a 121.07±24.62 96.88±27.87 22.90±1.24 b 110.46±21.34 α 89.23±7.61α 14.84±5.35α c 156.91±14.94αβ 173.36±5.88αβ 48.15±2.00αβ d 130.88±7.17αβ 107.29±8.79αβ 27.44±1.41αβ e 111.59±25.97α 103.87±3.04β 20.37±1.46β values are expressed as mean ± sem (n = 5) α = significantly (p<0.05) different vs a, β = significantly (p<0.05) different vs b, by using the analysis of variance and lsd multiple comparison test on spss software (ibm corp.usa, 2011). gsh: reduced glutathione. group a: liquid paraffin treated group, group b: 30% ccl4 treated group, group c: administered 100mg/kg of butanol extract + ccl4, group d: administered 120mg/kg of butanol extract + ccl4 group e: administered 160mg/kg of butanol extract + ccl4 estimation of enzymatic antioxidant markers a significant increase (p<0.05) was observed in the levels of antioxidant enzymes such as sod and cat in all treatment groups compared to positive control, except the cat levels of group treated with 160mg/kg of the extract. moreover, the levels of lipid peroxidation marker (mda) was only significantly lower (p<0.05) in the group treated with 160mg/kg of the extract (table 2) compared to positive control. table 2. enzymatic antioxidant properties of rats administered butanol seed extracts of parkinsonia aculeata and carbon tetrachloride. group sod (u/ml) cat (u/ml) mda (umol/l) a 14.30±6.01 288.88±43.09 130.34±32.93 b 8.06±6.38 α 279.14±20.62α 195.94±58.65α c 44.53±21.40αβ 410.06±23.47αβ 217.17±99.33αβ d 89.94±25.44αβ 352.65±21.31αβ 222.22±8.79αβ e 56.93±35.72 αβ 276.44±8.95 α 96.15±5.25 αβ values are expressed as mean ± sem (n = 5) α = significantly (p<0.05) different vs a, β = significantly (p<0.05) different vs b, by using the analysis of variance and lsd multiple comparison test on spss software (ibm corp.usa, 2011). group a: liquid paraffin treated group, group b: 30% ccl4 treated group, group c: administered 100mg/kg of butanol extract + ccl4, group d: administered 120mg/kg of butanol extract + ccl4 group e: administered 160mg/kg of butanol extract + ccl4 mda: malondialdehyde, cat: catalase; sod: superoxide dismutatse. discussion free radicals play a role in the aetiology of a variety of diseases (covacci et al., 2001). they are easily produced in the body through regular metabolic pathways. antioxidants, on the other hand, can neutralise free radicals and thereby prevent disease. natural antioxidants derived from plant sources have been linked to a lower risk of chronic disease due to their ability to stop free radical growth in the biological system (covacci et al., 2001). antioxidant activity, or the prevention of free radical formation, is critical in the 86 biology, medicine, & natural product chemistry 11 (1), 2022: 83-87 prevention of ccl4-induced hepatopathy (venukumar & latha, 2002). to avoid and neutralise free radical-induced damage, the body possesses an excellent defence mechanism. catalase, superoxide dismutase, and glutathione peroxidase are examples of endogenous antioxidant enzymes that help with this. these enzymes form a mutually beneficial defence team against reactive oxygen species (ros) (hewawasam et al., 2003). in ccl4 induced hepatotoxicity, the balance between ros production and antioxidant defences may be lost hence oxidative stress result, which through a series of event regulate cellular functions leading to hepatic necrosis. from the result of this study, rats administered ccl4 showed significant changes (p<0.05) on the liver function and antioxidant parameters when compared with normal control group. the reduced activity of antioxidant enzymes observed point out the hepatic damage as the rats administered with ccl4. the seed extract had indicated reversed changes in liver function and antioxidant parameters when compared with ccl4 treated group. in terms of non-enzymatic antioxidants, gsh is a significant predictor of tissue sensitivity to oxidative damage, and hepatic gsh depletion has been linked to increased toxicity to compounds such as ccl4 (kidd, 1997). in the present study, decrease in hepatic tissue of gsh level was observed in ccl4 treated groups. moreover, the findings showed significant decrease in the levels of vitamin c and vitamin e in ccl4 treated group when compared to the normal control group. this is in agreement with the findings of narasimhanaidu and ponnain, (2006) that indicated that condition of severe oxidative stress may also lead to the depletion of protective physiological moieties such as gsh, vitamin c and vitamin e in rats (kamalakkannan & prince, 2006). glutathione through its significant reducing power contributes to the recycling of other antioxidants such as vitamin c and e that have become oxidized (kidd, 1997). interestingly, the reduced levels of gsh, vitamin c and vitamin e caused by ccl4 was attenuated by the butanol seed extract of p. aculeata. lipid peroxidation is a measure of membrane damage and changes in the structure and function of cellular membranes, and it was found to be higher in the ccl4-treated group. the observed increase in mda levels of ccl4 groups suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defence mechanism to prevent formation of excessive free radicals (achliya et al., 2004). this is also in agreement with findings of moscrella et al. (1994) and patrick-iwuanyanwu et al. (2007). the treatment with 160mg/kg of the extract was however able to reduce the mda levels, which suggests a hepatoprotective effect. similarly, the activities of sod and cat was observed to increase as result of the ccl4 administration. sods are the first line of defence against harm caused by ros. these proteins catalyse the dismutation of superoxide anion free radical (o2-) into molecular oxygen and hydrogen peroxide (h2o2), lowering the level of o2-, which can harm cells at high levels. 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(2002). theoretical elucidation on structure-antioxidant activity relationships for indolinonic hydroxylamines. bioorganic & medicinal chemistry letters, 12(2), 225–227. https://doi.org/10.1016/s0960894x(01)00724-7 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 23-25 | doi: 10.14421/biomedich.2021.101.23-25 issn 2540-9328 (online) brittle bone brothers: osteogenesis imperfecta conventional serial case marsha ruthy darmawan1, elysanti dwi martadiani2,* 1radiology resident; 2musculoskeletal radiologist, radiology department, faculty of medicine, universitas udayana sanglah hospital denpasar, bali, indonesia. corresponding author* elysantiidm@gmail.com manuscript received: 08 october, 2020. revision accepted: 01 july, 2021. published: 13 july, 2021. abstract osteogenesis imperfecta is a hereditary connective tissue disorder due to col1a1/2 mutation causing gene defect encoding proteins to metabolize collagen. the skeletal manifestation of oi causing bone incompetence, hence the name brittle bone disease. here we report three cases of oi type iv in adults. skeletal conventional x-rays were performed to all patients and all of them has similar results such as bowing deformities of long bones, old union and some non-union fractures with extreme angulation and severe osteoporosis. oi are classified based on skeletal structure, sclera colorization, dentinogenesis, and functional metabolic defect genetically. oi type i and iv can live until adults; also, the same type of oi can be found in siblings. skeletal conventional x-rays can solely make the diagnosis. keywords: osteogenesis imperfect; conventional x-ray; osteoporosis; bone deformity; brittle bone. introduction osteogenesis imperfecta (oi) or “brittle bone disease” is a hereditary form of early osteoporosis in children with an incidence reaching 1: 20.000 births. (hoyer-kuhn, et al., 2015) according to the national institutes of health, 90% of oi genetic mutation occur due to a mutation of col1a1 and col1a2 genes. (blom, et al., 2017) it means disturbance to all connective tissue from collagen type i; therefore, patients can have fractures all their lives. (van dijk, & sillence, 2014) a nomenclature in 2014 by the international nomenclature group of constitutional disorders of the skeleton (incds)classified oi into primary type i-iv and adding type v based on their causative genes and manifestation in deformed bones. (van dijk, & sillence, 2014) here we present three cases of oi type iv in one family of three brothers in their 40s and the only adults with oi in our hospital. case report case 1 a 40-year-old indonesian male came to the hospital with small stature for a medical check-up. he is unusually short for his age; while he was young, he had multiple fractures of left radial bone and right tibia. on physical examination, we found over-bending to all extremities without abnormal sclera colorization or teeth. imaging examination includes conventional x-raysbut none of bone mineral density (bmd). on upper and lower extremities x-ray, there was generalized bowing to the radial shaft, ulna, metacarpal bones, femur, tibia, and fibula. acute-angled long bones with missing bone parts in both humerus caused by non-union fractures. chest x-ray showed bowing of ribcage and left clavicle. there were no ecg done in any of these patients. radiographic images are in figure 1. figure 1. a 40-year-old man with oi presented with small stature. skeletal x-rays showed generalized bowing and angled long bonesand missing bone parts in both humerus caused by non-union fractures. https://doi.org/10.14421/biomedich.2021.101.23-25 24 biology, medicine, & natural product chemistry 10 (1), 2021: 23-25 case 2 a 41-year-old indonesian male came to the hospital with short stature and limitation to self-activity. he had fractured almost all his long bones. physical examination showed generalized bent deformation to his extremities with a normal sclera, yellowbrownopalescent discoloration of anterior teeth, and many missing ones. further examinations were only conventional x-ray, bowing to all long bones in the upper and lower extremities. there were missing bone parts on medial and lateral thirds of right humerus that indicates non-union fracture and enlarged metaphysis of proximal as well as distal upper long bones. chest x-ray showed bowing of the ribcage, old fracture to the left clavicle, and all bones appeared porotic severely. moreover, no cardiopulmonary abnormalities found in this patient. radiographic images are in figure 2. figure 2. a 41-year-old man with oi who presented with short stature. skeletal x-rays showed porotic, bent to all long bones and missing bone parts on medial and lateral thirds of right humerus indicates the non-union fracture. case 3 a 42-year-old indonesian male came to the hospital complaining his stature is getting smaller compared to his brothers. he was in a wheelchair throughout his life, unable to complete physical activities due to pain and fragile bones, which would easily break when exposed to blunt forces. physical examination showed bent extremities with normal sclera and teeth. further x-rays showed, bowing and bent deformities to all long bones, missing bone part on medial third of left humerus and left femur, also enlarged metaphysis on all sites of long bones. chest x-ray showing bent ribs and deformed left clavicle. all bone trabeculation appeared severely porotic, and no cardiopulmonary abnormalities found. radiographic images are in figure 3. figure 3. a 42-year-old man with oi who presented with small stature. skeletal conventional x-rays showed porotic, bending on all long bones, missing bone parts on the medial third of left humerus, and left femur, also kyphoscoliosis. discussion oi is a hereditary connective tissue disorder due to col1a1/2 mutation causing gene defect encoding proteins to metabolize collagen. the skeletal manifestation of oi mainly causing bone incompetence, vulnerable to fractures, deformed, and joint laxity, therefore bones are fragile, hence the name brittle bone disease. (hoyer-kuhn, et al., 2015) (blom, et al., 2017) from the new oi nomenclature 2014 revised nosology, there are five types of oi. type i related to deficiency of normal collagen, type ii is lethal, type iii severe, type iv mutation in collagen structure, and type v is oi with calcification in the interosseus membrane. (van dijk, & sillence, 2014) forlino and marini in 2016 described nineteen types of oi with five categories based on functional metabolic defects genetically, such as defects in collagen synthesis, structure or processing (group atype i-iv, xiii), defects in collagen modification (group b-vii-ix, xiv), defects in collagen folding and cross-linking (group ctype x-xi), defects in bone mineralization (group dtype v-vi), and defects in osteoblast development with collagen insufficiency (group e-type xii, xv-xvi). (forlino & marini, 2016) current therapy for oi is integrative, pain management, muscle rehab for regaining strength and range of movement also regain mobility to increase the quality of life, and a regular check-up for dentition and hearing. bisphosphonate treatment with cyclic intravenous pamidronate given in infancy proved to help increase bone density and reduce fractures. (scheres, et al., 2018) in these cases of the brothers, their ages range from 40-42 years old; they have a small stature, normal sclera, darmawan & martadiani – brittle bone brothers: osteogenesis imperfecta … 25 two patients have normal teeth and one with dentinogenesis imperfecta (di). these brothers generally have the same type of oi which is oi type iv but different subtype, iv a without di and iv b with di. (van dijk, & sillence, 2014) (scheres, et al., 2018) genetically categorized as group a by forlino and marini (forlino & marini, 2016), type iv oi resulted in col1a 1 or 2 mutation and genetic workup must be done to determine which one. unfortunately, genetic workup was not administered to all patients. type iv oi categorized as group a in forlino and marini classification is caused by collagen deficiency creating structural inadequacy. glycine substitutions in the helical domain are the most common problem, which can delay helical folding and prolonged time to modify enzymes. another mutation common for oi is impaired chain of procollagen c-pro-peptide. inadequate collagen structurally can manipulate intracellular metabolism and matrix architecture rather than a deficiency in collagen quantity. (forlino & marini, 2016) general radiographic findings mostly consist of osteopenia, deformities, and fractures. common findings to lower extremities include anterior or lateral bowing of the femur, anterior bowing of tibia, protruded acetabulum, and ‘shepherd’s crook’ deformities of the proximal femur. ‘popcorn’ appearance in the metaphysis, multiple areas, or radiolucent scalloping with thick rims, can be seen in some patients with oi. specific findings of the spine include compressed vertebrae between cartilaginous disc space, called codfish vertebrae. abnormalities were found not only in long bones but also in the skull, which is caused by excessive bone malleability and plasticity. the more common findings to the skull are multiple wormian bones, a physiological finding in the skull, but considered abnormal if there were more than ten found and usually present in patients with severe oi. (blom, et al., 2017) diagnosis of oi in childhood made solely with conventional skeletal x-ray, simple yet effective. an optimal method to determine quantitative osteopenia is by dual-energy x-ray absorptiometry (dexa), and bone mineral density (bmd) score will reveal if there is osteopenia or already in osteoporosis state. another method to diagnose oi in children is with dna analysis with an examination of cultured fibroblast. this method showed a decreased quantity of cultured fibroblast in children with oi than healthy children, also abnormality of type 1 procollagen molecules or mutation to col1a1 or col1a2 genes that encrypt type 1 procollagen chains. (scheres, et al., 2018) bone histomorphometry examined in oi type i-iv (collagen defect group) showed low bone volume and trabecular quantity with high replacement kinetic rates. if more than one family member has this disease, the clinician should explore oi gene panel for better therapy plan. (bishop & walsh, 2014) conclusion oi is a rare inherited abnormality to the skeletal because of mutations to type 1 collagen in connective tissue. oi are classified based on skeletal structure, sclera colorization, dentinogenesis imperfecta, and functional metabolic defect genetically. oi type i and iv can live until adults; also, the same type of oi can be found in siblings. skeletal conventional x-rays can solely make the diagnosis of oi. conflict of interest: the authors declares that there are no conflicts of interest concerning the publication of this article. references bishop, n. j., & walsh, j. s. (2014). osteogenesis imperfecta in adults. the journal of clinical investigation, 124(2), 476–477. https://doi.org/10.1172/jci74230 blom, a., warwick, d., & whitehouse, m. (eds.). (2017). apley & solomon's system of orthopaedics and trauma (10th ed.). crc press. https://doi.org/10.4324/9781315118192 forlino, a., & marini, j. c. (2016). osteogenesis imperfecta. lancet (london, england), 387(10028), 1657–1671. https://doi.org/10.1016/s0140-6736(15)00728-x hoyer-kuhn, h., netzer, c. & semler, o. (2015). osteogenesis imperfecta: pathophysiology and treatment. wien med wochenschr 165, 278–284. https://doi.org/10.1007/s10354015-0361-x scheres, l., van dijk, f. s., harsevoort, a. j., van dijk, a., dommisse, a. m., janus, g., & franken, a. (2018). adults with osteogenesis imperfecta: clinical characteristics of 151 patients with a focus on bisphosphonate use and bone density measurements. bone reports, 8, 168–172. https://doi.org/10.1016/j.bonr.2018.04.009 van dijk, f. s., & sillence, d. o. (2014). osteogenesis imperfecta: clinical diagnosis, nomenclature and severity assessment. american journal of medical genetics. part a, 164a(6), 1470–1481. https://doi.org/10.1002/ajmg.a.36545 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 2, 2018 | pages: 33-38 | doi: 10.14421/biomedich.2018.72.33-38 issn 2540-9328 (online) degradation study of biodegradable plastic using nata de coco as a filler tiara nur elfiana1, anisa nur izza fitria2,*, endaruji sedyadi1, susy yunita prabawati1, irwan nugraha1 1chemistry department, 2chemistry education department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto, no. 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. author correspondency*: anisanurizza17@yahoo.com abstract starch is known as a biodegradable raw material that can be degraded by bacteria and microorganisms in the soil. starch has cellulose which is kind of plant cellulose. this study shows the biodegradation rates of plastic made from ganyong canna (canna edulis kerr) as a cellulose source which is added with nata de coco as a filler. the biodegradable plastic functional group was confirmed by using fitr. the results show that the o-h group of ganyong canna (canna edulis kerr) biodegradable plastic is located at wave number 3298.03 cm1 and shifted to 3290.32 cm-1 after addition of nata de coco. the c-h bonds functional groups in canna biodegradable plastics and nata de coco plastics are at wave numbers 2920.01 cm-1 and 2916.16 cm-1. while the c-o bonds functional groups in biodegradable starch plastics and nata de coco is shown at wave numbers 995.05 cm-1. the mechanical properties of biodegradable plastics testing are thickness, tensile strength, and elongation based on the astm method. the thickness is about 0.1005 mm, the tensile strength of biodegradable plastic is 4,3244 mpa and the elongation value range about 13.9639% while the wvtr range about 14.20 g/m² hours. the results show that the increase of the plastic degradation made from nata de coco occurs between 5% 38% per days. it is faster than the plastic made from pure ganyong canna (canna edulis kerr) starch. these results indicate that nata de coco could be added in biodegradable plastic on packaging materials for better degradation. keywords: biodegradable; cellulose; ganyong canna; nata de coco introduction biodegradable plastic has been predicted being an alternative for environmental problem in the last few decades. biodegradable plastics have the ability to degrade faster than synthetic plastic which is approximately degraded in 50 years for a foam plastic cup (roohi, 2017). the polymer in plastics changes into co2 and h2o in combination of microorganism in the soil (kumar satish and thakur, 2017). the co2 would be used by plants in photosynthesis while the water would dissolve in the soil pores so that biodegradable material would not be harmful in environment. the main constituents of biodegradable plastic are cellulose. the cellulose sources are mostly from plants, such as wood and agricultural waste (mostafa et al., 2018). however, plant cellulose has other components such as lignin, so it needs separation process to get pure cellulose. the purifications has been carried out by vu ngo dinh et al. (2017) using an ultrasound-assisted alkaline methods which required high technology and costs. biodegradable is known as rigid therefore plasticizers are needed to produce the plastic which is more elastic, flexible and resistant to water. one plasticizer that is widely used in manufacture of biodegradable plastics is glycerol. biodegradable plastic from hydrocolloids such as nata de coco and ganyong canna (canna edulis kerr) has a high polarity and it is hydrophilic which produces high water vapor permeability and low oxygen permeability. this is due to the presence of hydrogen bonds in the molecular structure. therefore, it is necessary to add lipids such fatty acids that have low polarity in order to reduce water vapor permeability. saturated fatty acids have hydroxyl groups that are hydrophobic so they can reduce hydrophilic properties. one type of saturated fatty acid that can be used is palmitic acid (krocta, 1994). according to lay and huey (1997), the addition of palmitic acid into the film aims to protect the product against evaporation of moisture. this is related to the ability of palmitic acid to improve the hydrophobic nature of a solution. nata de coco is a food product with high cellulose in the coconut water with excellent minerals such as k, cl, s, ca, na, mg, etc. the sugars content, sucrose, sorbitol, glucose and fructose, are the needs of acetobacter axylum in fermentation process (alexia, 2012). these bacteria produced a million of biomass grown by appearing solid white to transparent in the number of mass about 250 g/l wet weight of cellulose (gayathri, 2015). nowadays, nata de coco is just served as a food desert without any utilizing improvement even thought it has a high tensile strength and biodegradable properties. https://doi.org/10.14421/biomedich.2018.72.33-38 mailto:anisanurizza17@yahoo.com 34 biology, medicine, & natural product chemistry 7 (2), 2018: 33-38 in order to meet the need of biodegradable plastic, this paper shows the study of nata de coco as a filler in combination with ganyong canna (canna edulis kerr) as a starch sources including an analyzing of the physical and mechanical properties, thickness, tensile strength, elongation, water vapor permeability and molecular interactions between nata de coco and starch using ft-ir instrument. materials and methods the materials used in this study was ganyong canna (canna edulis kerr) which were the basic ingredients for biodegradable plastics. this canna obtained from regedeg village, gunung kidul regency. other ingredients used was nata de coco, aquades, a soil for degradation media, sodium hydroxide, glycerol, acetic acid. preparations of nata de coco pulp nata de coco was heated with 1% naoh for 60 minutes. this suspension then washed with water and followed by boiling for the second time in water. the nata de coco soaked in water for one night. pulping was done by smoothing nata de coco using a blender and used for the manufacture of biodegradable plastics. afer that, the pulp dried using an oven at 60 °c. preparations of ganyong canna (canna edulis kerr) starch ganyong canna starch was produced by adding water to dried ganyong canna (canna edulis kerr) and mashed into a pulp. the dregs and the starch were separated by filtering with a filter cloth. the filtration results were left for one night until the starch settles. then the resulting precipitate was dried using an oven at 60 °c. preparations of biodegradable plastics first of all, 1.5 gram ganyong canna starch was dissolved with distilled water. the solution was added with 0.75 ml of glycerol and 1.5 ml of acetic acid. then, the solution was added to a volume of 100 ml of distilled water and followed by heating at a temperature of 80-90 °c until homogeneous for 25 minutes. the mixtures were cooled. the air bubbles and impurities were removed. the mixtures were added to nata de coco pulp in 0.5; 1.0; 1.5; and 3.0 grams and poured into a solution casting size of 30 x 20 cm² and dried in an oven at 60 °c for 2 hours. the mold was removed and left at room temperature for 24 hours. data analysis biodegradable plastic was analyzed its functional group using ftir. samples in the form of plastic were placed into a set holder, then the plastic was scanned at a wavelength of 4000-400 cm-1. the mechanical properties of biodegradable plastics were tested including thickness, tensile strength, and elongation based on the astm method. d. 882-02. thickness was measured using a micrometer (accuracy of 0.001 mm) by placing a sample between the micrometer jaw. for each plastic sample to be tested, measurements were made at 3 different points, then the mean value was calculated. the tensile strength and elongation of biodegradable plastic samples were tested using universal testing machine (utm). the plastic was placed at room temperature (30 °c) for 24 hours before measuring. biodegradable plastic samples was placed at the second end of the clamp. the measurement area was set with the appropriate load and set to zero. the drive button and clamp was turned on and the sample test was observed until it broke. tensile strength was determined based on maximum load, while elongation was determined when biodegradable plastic broke. the water vapor transmission rate test was carried out using the gravimetric method (astm 1983). the biodegradable plastic that would be tested was placed in the mouth of a cup with a diameter of 8 cm which contained silica gel 10 g. the edges of the cup and plastic were covered with rubber. the cup was put into a jar containing nacl solution. the water vapor diffused through biodegradable plastic would be absorbed by silica gel and would increase the mass of the silica gel. then it was weighed every 1 hour for 6 hours (starting from 0 to 6 hours). changing in the weight rate indicated the speed of water vapor diffusion through the biodegradable plastic. the data obtained was made by linear regression equation and wvtr value could be determined by the equation: 𝑊𝑉𝑇𝑅 = cup increase slope ( 𝑔𝑟𝑎𝑚 ℎ𝑜𝑢𝑟 ) plastic surface area (𝑚2) biodegradation test was carried out to determine how long plastic can be broken down by microorganisms (bacteria and fungi). the method used was soil burial test, which was planting and burying samples in the soil for 12 days. the soil was placed in a jar with a diameter of 13 cm and a height of 17 cm. plastic samples measuring 3x3 cm² from ganyong canna (canna edulis kerr) starch and variations of nata de coco plastic buried in the soil with a depth of 5 cm. before being buried, biodegradable plastic was left in the desiccator for 24 hours then weighed (w1). every 2 days, plastic was taken, washed with alcohol, dried in an oven and weighed for the second time (w2). the step was repeated for 12 days and the degradation mass was determined. elfiana et al. – degradation study of biodegradable plastic … 35 results and discussion biodegradable plastic can be decomposed naturally by microorganism’s activities in the soil to harmless compounds when discharged into the environment. in this research, biodegradable plastic is made from renewable sources, ganyong canna (canna edulis kerr) as a base material, glycerol as plasticizer, acetic acid as terminator in polymerization reaction, distilled water as a solvent and modified by nata de coco. the film characteristic of nata de coco indicates that the nata main component is a cellulose polymer. it can be seen on the peak absorption of nata de coco cellulose which is shown the o-h groups in wave numbers at 3344.32 cm-1 and the c-o bonds uptake at around 1029.21 cm-1, the c-h absorption is at 2896.87 cm-1 and the c-c is around at 1643.23 cm-1. figure 1. ftir spectrum of ganyong canna (canna edulis kerr). the identification of functional groups is carried out at wave numbers 400-4000 cm-1. the spectrum of ganyong canna (canna edulis kerr) starch shows a widened absorption at wavenumber 3355.89 cm-1 which indicate the presence of o-h stretching groups. the absorption of c-h is at wave number 2927.73 cm-1. this is reinforced of the absorption band around 1357.79 cm-1 which indicates the presence of ch2 groups. the other functional group is ether (c-o) which is shown at 999.05 cm-1. this result indicates that the functional groups are in accordance with the structure of the constituent components of starch, hydroxyl, methyl, and ether. analyzing ftir to find out the functions in biodegradable plastic. in figure 2, biodegradable plastic spectra is seen the ftir spectra of biodegradable plastics, ganyong canna and nata de coco. figure 2. spectra ftir biodegradable plastic (a) canna starch (b) ganyong starch-nata de coco 1.5 g. 36 biology, medicine, & natural product chemistry 7 (2), 2018: 33-38 it can be seen that the oh group is located at wave number 3298.03 and shifted to 3290 after the addition of nata de coco. the wave number of c-h bond in ganyong canna biodegradable plastic and nata de coco is shown at 2920, 01 and 2916.16. while the c-o bond in both components is found at wave number 995.05 (sedyadi et al., 2016). starch-based biodegradable plastic is a type of condensation polymerization because it involves a hydroxyl group from one of the monosaccharides with hydroxyl groups from other monosaccharides through glycosidic. this biodegradable plastic has a rigid and easily broken nature because the hydrogen between polymers bonds are very strong, so it is necessary to add plasticizers (glycerol) to reduce hydrogen polymers to produce more elastic and flexible plastics (wirawan et al., 2012). when glycerol was added some structural modifications occurred in the starch. the starch matrix became looser so the polymer chain moved more easily. it caused glycerol were being bridged between starch molecules by cross-linking between starch molecules with another molecules due to the interaction with the oh groups in the amylose and amylopectin structures (kusnandar, 2010). the interaction between starch and glycerol could be seen in (figure 3). figure 3. strach and glycerol reaction. the plastic is used for packaging products mostly. furthermore, in this application, the important thing is a thickness which is affected by the packaging of products. thickness will affect gas permeability and other physical properties such as tensile strength and elongation (sinaga et al., 2010). the thicker the plastic produced, the transmission of water vapor value was lower because the ability to hold water vapor would be greater. figure 4. thickness nata de coco plastic. in figure 4, the thickness shows the irregular value of nata de coco plastics. the plastic thickness on the 0.5 g and 1 g of nata de coco pulp decreases while the 1.5 g and 3 g of nata de coco pulp increase. these results caused the solution do not measure the same on printing process. the thicker the plastic produced, the lower the value of the transmission of water vapor because the greater the ability to hold water vapor. in this study, the thickness of biodegradable plastic is known from variations of nata de coco ranging from 0.0665 mm to 0.1035 mm. the measurement of plastic tensile strength is used to determine the amount of plastic receiving to achieve maximum pull (stretch) in each unit of plastic area. testing of mechanical properties including tensile strength and elongation is very important to determine the strength in resisting the weight and elasticity of plastic. figure 5. the tensile strength and elongation nata de coco plastic. y = -0.0668x3 + 0.3344x2 0.4507x + 0.2496 r² = 1 0 0.05 0.1 0.15 0.2 0 1 2 3 4nata de coco mass (g) th ic kn es s ( m m ) y = -5,6162x3 + 26,876x2 34,259x + 23,836 r² = 1 y = -1,7052x3 + 7,911x2 9,3282x + 6,272 r² = 1 0 2 4 6 8 10 12 14 16 18 20 0 1 2 3 4 nata de coco mass (g) te n si le s tr en gh t (m p a) a n d e lo n ga ti o n ( % ) elfiana et al. – degradation study of biodegradable plastic … 37 nata de coco which contained cellulose add tensile strength caused the bonding between hydroxyl (o-h) groups in starch with hydroxyl (o-h) and carboxyl (cooh) groups in cellulose. in this study, the resulting tensile strength of biodegradable plastic is 4,3244 mpa. the resulting value is higher than the research conducted by mery apriani (2014) which made biodegradable plastic using cassava starch which was given aloe vera with a tensile strength of 1.53 mpa. measurement of tensile strength is also followed by measurement of elongation, namely the maximum changing in length before the plastic cut off. these properties is very important to indicate the ability of plastic to withstand a number of loads before the plastic is broken. based on the results of the study shows that the elongation value ranged about 13.9639%. the tensile strength analysis has irregularities. this irregularity occurs because the solution is less homogeneous. there is inter-polysaccharide bonds broken up by glycerol occur unevenly in variations of nata de coco so that intermolecular interactions become reduced and affect the value of the tensile strength produced. this interaction occurs because glycerol inserts and removes the hydrogen bonds between polysaccharide molecules so the bonds between plastic molecules is weak (bourtoom, 2008). other factors due to incomplete nata de coco mixing in plastic. the 0.5 g and 1.0 g of nata de coco mixing have low level of homogeneity so the increasing concentration of nata de coco does not produce strong increasing pull. the graph produced in addition to the mass of nata de coco is seen to be far away which proves that nata de coco in the solution does not give good homogeneity. while the addition of 1.5 g and 3.0 g of nata de coco has a graph that is mutually approaching which proves that the level of homogeneity in the mass of nata de coco is very good. nata de coco which contains cellulose will add tensile strength because cellulose is a reinforcing component in the composite material can increase its mechanical strength. the increase in tensile strength due to the addition of cellulose is caused by an increase in the interaction of the tensile forces between the molecules making up the thin layer. this condition is related to hydroxyl groups that form inter-intramolecular hydrogen bonds to form a thin layer consisting of fibers that are mutually reinforcing (indriyati et al, 2006). the rate of water vapor transmission is the amount of losing water vapor per unit time which divided by the area of plastic. plastic that has the lowest wvtr value is probably good plastic. based on research shows that nata de coco plastic has high value of wvtr. nata is a cellulose compound that is hydrophilic so the water is easily absorbed and migrated. the results show that the resulting wvtr value ranged from 14.20 g / m² hours. this wvtr value is better than one italian company which produced biodegradable plastics named bi mater. it made from starch polymer with wvtr values of 250-1000 g / m² hours. figure 6. water vapor transmission nata de coco plastic. biodegradation test was conducted to find out how fast biodegradable plastic could be degraded by microorganisms when discharged into the environment. in this study, biodegradation test was carried out using soil media because many microorganisms in the soil which could support the plastic degradation process. according to sari (2007), soil moisture is very influential on the degradation process because it will facilitate microbes in consuming plastic as a food source. another factor are ph, temperature, carbon/ nitrogen sources, presence of contaminants, presence availability of nutrients (roohi, 2017). the biodegradation process was carried out by observing the plastics. observations was done by looking at the changing of plastic visually and by weighing the decrease in plastic mass. observations were made for 12 days, where every 2 days the plastic was taken from the soil and then washed and weighed. the results of the degradation of biodegradable plastic mass could be seen in (figure 7). figure 7. biodegradable plastic mass degradation. based on the data, the plastic which was contained nata de coco as filler has more mass degradation than plastic in pure ganyong canna. the percentage mass degradation was between 5%-38% depending on how long the plastic in the soil. in the early days, degradation process was tended faster than another day. it caused there was more cellulose contained in it. nata de coco has ability to absorb water so that the degradation was being faster. when the water contact in 14 14.2 14.4 14.6 14.8 15 15.2 0 1 2 3 4 nata de coco mass (g) w vt r (g /m ² ja m ) 0 38.57 43.73 59.81 86.34 100 0 13.85 25.02 49.52 79.23 100 0 20 40 60 80 100 120 0 3 6 9 10 12 p e rc e n ta ge o f m as s d e gr ad at io n day of degradation 38 biology, medicine, & natural product chemistry 7 (2), 2018: 33-38 the cellulose, there is attacking by microbes which digest the starch (cellulose). the microbes digest this component, leaving behind a porous, sponge-like structure with a high interfacial area, and low structural strength (kumar ashwin, et. al, 2011). it changes the properties such plastic shape, tensile strange and plastic color (roohi, et.al. 2017). conclusion mechanical properties of biodegradable plastic variations of nata de coco have no significant effect on plastic tensile strength. the highest tensile strength value was 4.3244 mpa and the highest elongation was 13.9639% with the highest transmission rate of 14.20 g / m² hours. the addition of nata de coco has an effect on the biodegradability test in the soil. references alexia. 2012. coconut water uses, composition and properties: a review prades. fruits. 67 (2): 87-107, doi: 10.1051/fruits/2012002 apriyani mery and endaruji sedyadi. 2014. sintesis dan karakterisasi plastik biodegradable dari pati onggok singkong dan ekstrak lidah buaya (aloe vera) dengan plasticizer gliserol. j. sains dasar 2015 4 (2) 145 – 152. bourtoom, t. 2008. edible films coatings: characteristics and propeties. international food research journal. 15 (3) gayathry. 2015. production of nata de coco a natural dietary fibre product from mature coconut water using gluconacetobacter xylinum (sju-1). intl. j. food. ferment. technol. 5(2): 231-235, doi: 10.5958/22779396.2016.00006.4. indriyati, l., indrarti., & rahimi, e. 2006. pengaruh carboxymethyil cellulose (cmc) dan gliserol terhadap sifat mekanik lapisan tipis komposit bakterial selulosa. jurnal sains materi indonesia, 8 (1): 40-44. krochta, j., & e. a. baldwin, m.-c. (1994). edible coatings and films to improve food quality. usa: technomic publising co. inc. kumar satish and thakur.2017. bioplastics classification, production and their potential food applications. journal of hill agriculture. 8(2): 118-129. doi: 10.5958/22307338.2017.00024.6. kumar ashwin. 2011. properties of biodegradable polymers and degradation for sustainable development. international journal of chemical engineering and applications. 2 (3). kusnandar, f.2010. kimia pangan: komponen makro. jakarta: dyan rakyat. lay and huey. 1997. properties of microstructures of zain sheets flastiazed with palmitic acid and stearic acid. j. chereal chemistry. 74 (1): 83-90. mostafa. 2018. production of biodegradable plastic from agricultural wastes. arabian journal of chemistry. 11, 546– 553. roohi. 2017. microbial enzymatic degradation of biodegradable plastics. current pharmaceutical biotechnology. 18 (5), doi: 10.2174/1389201018666170523165742. sari, e. 2007. studi biodegradasi poli hidroksi butirat pada medium cair dan padat. tesis. yogyakarta: program pasca sarjana, universitas gadjah mada. sedyadi, e., aini, s. k., anggraini, d., ekawati, d. p. 2016. starch-glycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on its mechanical properties. biology, medicine, & natural product chemistry. 5(2): 33-40 sinaga, l. l., s. melisa s. r., & sinaga, m. s. (2013). karakteristik edible film dari ekstrak kedelai dengan penambahan tepung tapioka dengan gliserol sebagai bahan pengemas makanan. j. teknik kimia usu. 2 (4): 12-16. sukara endang and meliawati ruth. 2014. potential values of bacterial cellulose for industrial applications. jurnal selulosa. 4 (1): 7 16 vu ngo dinh, et.al. 2017. lignin and cellulose extraction from vietnam’s rice straw using ultrasound-assisted alkaline treatment method. international journal of polymer science, doi: 10.1155/2017/1063695. wirawan, s., prasetya, a., & ernie. (2012). pengaruh plasticizer pada karakteristik edible film dari pektin. journal reaktor. 14: 61-67. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 937-941 | doi: 10.14421/biomedich.2025.142.937-941 issn 2540-9328 (online) the influence of strain type and female age on the receptivity of female drosophila melanogaster in homogamous crosses lisa savitri1*, kharisul ihsan2, rochmad krissanjaya1, elfred rinaldo kasimo1 1department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. 2department of pharmacy, faculty of pharmacy, public health, hospital administration, radiology, universitas strada indonesia, kediri, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 05 august, 2025. revision accepted: 14 october, 2025. published: 28 october, 2025. abstract drosophila melanogaster is a widely used model organism in genetic research due to its short life cycle and the presence of many genes homologous to those found in humans. it plays an important role in studies related to genetic inheritance, gene interaction, sex determination, and developmental genetics. one key aspect of its reproductive biology is female receptivity—the willingness of a female to accept mating—which is influenced by various internal and external factors. external factors include environmental conditions such as temperature and humidity, as well as male courtship signals like wing vibrations and chemical cues. internal factors are mainly related to the female's sexual maturity and mating status. generally, female receptivity increases with age after eclosion, reaches a peak, and gradually declines. this study investigates the impact of strain type and female age on receptivity in homogamous crosses of d. melanogaster, using two mutant strains: dp (with a wing mutation) and wa (with an eye color mutation). receptivity was measured by the presence or absence of f1 offspring following crosses between males and females of the same strain, with females tested at different ages ranging from 10 to 60 hours after eclosion. the results indicate that strain type does not have a significant effect on female receptivity. in contrast, female age plays a clear role: individuals aged 30 years and older were more receptive and more likely to produce offspring. no significant interaction was found between strain type and age. these findings suggest that female receptivity in d. melanogaster is more strongly influenced by sexual maturity (as indicated by age) than by genetic differences between strains. keywords: drosophila melanogaster; female receptivity; strain type; sexual maturity; homogamous crosses. abbreviations: analysis of variance (anova); first filial generation (f1); randomized block design (rbd); white apricot (eye color mutant strain of drosophila melanogaster) (wa); drop (wing mutant strain of drosophila melanogaster) (dp). introduction d. melanogaster is a type of fruit fly commonly found in our surroundings due to its ability to reproduce rapidly. according to parvati et al. (2009), many drosophila genes are homologous to human genes. this makes d. melanogaster a widely used organism in genetic research. it is beneficial for studying several genetic mechanisms, including general principles of gene transmission, sex determination, gene interaction, as well as molecular, biochemical, and developmental genetics. d. melanogaster reproduces sexually. mating occurs when both male and female individuals have reached maturity (karmana, 2010). however, the female plays the most critical role in determining whether mating takes place. this is due to various influencing factors on the female, both internal and external. external factors include environmental conditions such as temperature and humidity, and more importantly, the signals emitted by the male during courtship. internal factors include the female’s sexual maturity and mating status. a female’s receptivity increases with age after emergence and then declines after reaching its peak (feng, 2010). in this study, the d. melanogaster strains used are the dp strain, which has a wing mutation, and the wa strain, which has a mutation affecting eye color. in line with previous research, including that of karmana (2010), it was found that both strain type and female age affect the number of offspring produced. however, these two factors exert their influence independently, with no interaction between them. materials and methods research design this study employs a quantitative descriptive design, aiming to determine the effect of strain type and female age on the receptivity of female d. melanogaster. the presence or absence of f1 offspring indicates receptivity. if f1 offspring appear, their number is counted over a 7day period. crosses are performed within the same strain https://doi.org/10.14421/biomedich.2025.142.937-941 mailto:lisasavitri@unik-kediri.ac.id 938 biology, medicine, & natural product chemistry 14 (2), 2025: 937-941 (homogamous crosses: dp♂ × dp♀ and wa♂ × wa♀), with three replications, using media labeled a, b, c, and d to obtain f1 offspring. population and sample the population in this study consists of d. melanogaster of the dp and wa strains, bred in the genetics laboratory, faculty of mathematics and natural sciences, state university of malang. the samples used are individual flies from both strains taken from the laboratory cultures and used as stock for the study. procedure medium preparation for one batch of medium, the ingredients used are 700 grams of pisang rajamala (a local banana variety), 200 grams of cassava tape (fermented cassava), and 100 grams of palm sugar, with a 7:2:1 ratio. first, the bananas are chopped, and the palm sugar is boiled until it melts. then, the bananas and cassava tape are blended with enough water until smooth. the blended mixture is transferred to a pot and cooked until it comes to a boil. once boiling, the melted sugar is added, and the mixture is cooked for 45 minutes for one batch, or 60 minutes for two batches. while still hot, the medium is poured into jam jars, sealed with sponge lids, and allowed to cool. once cooled, 3–5 grains of yeast (fermipan) are added. the inside of the jar is wiped to remove condensation, and a pupation paper is placed inside. stock renewal and pupal isolation begin by preparing jam jars filled with the prepared medium. introduce 3–7 pairs of d. melanogaster from each strain into separate jars, and label each jar with the strain and the date of introduction. when darkened pupae appear, isolate them by transferring them with a brush into a plastic ampule tube that has a slice of banana placed in the middle. both ends of the tube are sealed with sponge to prevent newly emerged flies from escaping. crossing once flies have emerged from the ampule, females from both dp and wa strains aged 10, 20, 30, 40, 50, and 60 hours post-eclosion are crossed with males of the same strain aged 1–3 days. each crossing jar is labeled with the strain, replication number, and date of treatment. after 3 hours of mating, the male flies are removed. when larvae begin to appear, the female flies are transferred to fresh medium, with up to three transfers (ending at jar d). offspring (f1) are allowed to develop and emerge, and the number of f1 individuals is recorded over a 7-day period. data collection technique data are collected by counting the number of male and female f1 individuals from each cross and replication. the following table format is used to record f1 counts for each group. data analysis technique the data analysis begins with chromosome reconstruction of the body. if the data set is complete, further analysis is conducted using a two-way anova with a randomized block design (rbd). this statistical test is used because there are two independent variables: strain type and female age. since data collection was not conducted simultaneously, rbd is considered appropriate. however, if the data set is incomplete, descriptive analysis is used instead. results and discussion result the d. melanogaster strains used in this study are dp and wa, with the following characteristics: 1. dp strain: ▪ red eye color ▪ smooth eye facets ▪ wing shape fully covers the body, with tips curving inward ▪ yellowish-brown body color figure 1. d. melanogaster dp strain 2. wa strain: ▪ orange eye color ▪ smooth eye facets ▪ wing shape fully covers the body ▪ yellowish-brown body color figure 2. d. melanogaster wa strain savitri et al. – the influence of strain type and female age on the receptivity of … 939 table 1. number of f1 male and female offspring from dp♂ >< dp♀ crosses. female age f1 count total average u1 u2 u3 10 hours 0 0 0 0 20 hours 195 77 272 90,6 30 hours 165 0 0 165 55 40 hours 0 0 0 0 50 hours 143 0 0 143 47,66 60 hours 118 201 319 106,3 table 2. number of f1 male and female offspring from wa♂ >< wa♀ crosses. female age f1 count total average u1 u2 u3 10 hours 0 0 0 0 20 hours 0 0 0 0 30 hours 95 106 59 260 86,67 40 hours 0 64 64 21,3 50 hours 53 79 0 132 44 60 hours 0 62 73 135 45 in the cross between dp♂ × dp♀, the total number of male and female f1 offspring at the 10-hour female age treatment was 0, resulting in an average of 0. at the 20hour treatment, the total f1 count was 272, with an average of 90.6. at 30 hours, the total was 165, averaging 55. at 40 hours, the total was again 0, with an average of 0. for the 50-hour treatment, the total f1 count was 143, with an average of 47.66. finally, at 60 hours, the total was 319, with an average of 106.3. in the cross between wa♂ × wa♀, the total number of f1 offspring at the 10-hour and 20-hour female age treatments was 0, resulting in an average of 0 for both. at 30 hours, the total number of f1 offspring was 260, with an average of 86.67. at 40 hours, the total was 64, with an average of 21.3. for the 50-hour treatment, the total was 132, with an average of 44. at 60 hours, the total number of f1 offspring was 135, with an average of 45 (figure 3). figure 3. total number of male and female f1 offspring. effect of strain type on female receptivity in d. melanogaster since the data collected are incomplete, a two-way anova could not be conducted to determine the effect of strain type on the receptivity of female d. melanogaster. however, based on the literature review conducted by the researcher, it is understood that so far, strain type does not affect female receptivity in d. melanogaster. according to muliati (2000) as cited in karmana (2010), the number of offspring produced varies among different strains. however, muliati further states that no existing literature clearly supports the idea that strain type affects the number of offspring. in this context, dobzhansky (1951), as cited in indayati (1999), states that mutations can alter the behavior of any species. kusnawati (1996), also cited in indayati (1999), adds that behavioral differences among mutants can influence mating success. mating behavior in d. melanogaster is influenced by several types of stimuli, including visual, olfactory and gustatory (smell and taste), auditory, and tactile signals. regarding auditory stimuli, shorey (1962), as cited in nusantari (1997), noted that males produce sounds through wing vibrations. ewing (1964), also cited in nusantari (1997), reported that wing size is linked to mating success. this implies that the dp strain, which has a wing mutation (inward-curved wing tips), may have lower mating success compared to the wa strain, which has a normal wing shape despite having an eye color mutation. the relatively "intact" wings of the wa strain may allow for stronger wing vibrations, which serve as mating stimuli and are more effectively received by females. from the explanation above, it becomes clear that while strain type may influence mating success, it does not influence female receptivity. female receptivity is more closely tied to sexual maturity than to the type of mutation present. even if females come from different mutant strains, they will become receptive once they reach sexual maturity and will accept copulation from males. therefore, mutation differences between dp and wa strains are not a determining factor—receptivity depends primarily on the female's ovarian or sexual development. effect of female age on receptivity in d. melanogaster due to incomplete data, a two-way anova could not be conducted to determine the effect of female age on receptivity in d. melanogaster. however, based on the literature reviewed by the researcher, it is evident that female age does influence receptivity. in the crosses of dp♂ × dp♀ and wa♂ × wa♀ using 10-hour-old females, no offspring were produced. this aligns with manning's (1967) statement in feng (2010), which indicates that female d. melanogaster become receptive at 24–40 hours after eclosion. females at 10 hours old are not yet receptive and therefore reject mating attempts. in the wa♂ × wa♀ cross using 20-hour-old females, no offspring were produced either, further supporting manning’s claim. however, in the dp♂ × dp♀ cross at the same age (20 hours), offspring were produced (272 940 biology, medicine, & natural product chemistry 14 (2), 2025: 937-941 individuals), suggesting an exception. this can be explained by o’dell (2003), who states that while females generally become sexually mature around 24 hours post-eclosion, some strains may mature faster. in this case, the dp strain may experience accelerated sexual maturation, making 20-hour-old females receptive and able to accept copulation. at 30 hours, both dp♂ × dp♀ and wa♂ × wa♀ crosses produced offspring. this matches manning's (1967) range of 24–40 hours for female receptivity. a receptive female will typically mate within 30 minutes when paired with a male. she will slow her movements, allowing the male to initiate copulation using his proboscis. once copulation occurs, receptive females can produce offspring. in the wa♂ × wa♀ cross with 40-hour-old females, offspring were again produced, indicating that the females were still within the receptive window. however, in the dp♂ × dp♀ cross at the same age, no offspring were observed. this may not indicate a lack of receptivity, but rather a problem with environmental conditions—specifically, the medium was dry and moldy. the researcher noted excessive fungal growth in the culture medium, which may have discouraged egglaying. the females were subsequently transferred to a new jar (jar b) in hopes that they would lay eggs in the improved conditions and produce offspring. for crosses using 50-hourand 60-hour-old females, both dp and wa strains successfully produced offspring. according to manning (1967), females are receptive from 24 to 40 hours post-eclosion, so these older females are technically beyond the peak receptive window. however, this does not mean they are incapable of producing offspring. feng (2010) notes that females can still respond to male courtship songs at 2 days old, although receptivity may decline between 2–4 days. thus, 50and 60-hour-old females may still respond to male signals, albeit less strongly, and are still capable of mating and producing offspring (cook, 1973, in feng, 2010). interaction between strain type and female age on receptivity in d. melanogaster as with the previous sections, the incomplete data prevented a two-way anova from being conducted to test the interaction between strain type and female age on receptivity in d. melanogaster. however, based on the literature, there is no significant interaction between strain type and female age in influencing receptivity. as explained earlier, only female age significantly affects receptivity, while strain type does not. therefore, the interaction between these two factors is also not significant. this is supported by indayati (1999), who stated that mating success—an indicator of female receptivity does not differ significantly across strains at different ages. furthermore, a previous study by karmana (2010), showed that both strain type and female age affected the number of offspring, with each factor acted independently, with no interaction between them. it's important to note that karmana’s study measured offspring count, whereas the current study focuses on female receptivity as the dependent variable. the reference to karmana’s findings here is intended solely to reinforce the point that no interaction effect was observed. however, to draw a more definitive conclusion, the researcher wants to complete the dataset and conduct a two-way anova with a randomized block design (rbd) to test for interaction formally. conclusions the results of the study show that the strain type does not affect the receptivity of female d. melanogaster in homogamous crosses. in contrast, female age has a significant impact on receptivity levels in these crosses. meanwhile, the interaction between strain type and female age does not have a significant effect on female receptivity in the context of homogamous mating. acknowledgements: thank you to the genetics laboratory, faculty of mathematics and natural sciences, state university of malang, for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors wrote the manuscript and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there are no funding. references feng, kai. (2010). neural control of the female mating decision in d. melanogaster. disertasi. (online), (http://othes.univie.ac.at/10366/1/2010-05-21_0701378.pdf), diakses tanggal 15 november 2013. grillet, et.al. (2005). a drosophila male pheromone affects female sexual receptivity. proc. r. soc. b 273, 315–323 doi:10.1098/rspb.2005.3332. (online), (http://rspb.royalsocietypublishing.org), diakses 9 november 2012 indayati, nur. (1999). pengaruh umur betina dan macam strain jantan terhadap kemampuan kawin kembali individu betina d. melanogaster. skripsi: ikip malang karmana, i. wayan. (2010). pengaruh macam strain dan umur betina terhadap jumlah turunan lalat buah (d. melanogaster ). ganeς swara. vol.4 no.2 kimball, j. w. (1983). biologi jilid 1 edisi kelima. jakarta: erlangga. http://othes.univie.ac.at/10366/1/2010-05-21_0701378.pdf http://rspb.royalsocietypublishing.org/ savitri et al. – the influence of strain type and female age on the receptivity of … 941 muliati, l. (2000). pengaruh strain dan umur jantan terhadap jumlah turunan jantan dan betina d. melanogaster.skripsi tidak diterbitkan.malang: fmipa universitas negeri malang nusantari, elya. (1993). kajian perkawinan kembali individu betina d. melanogaster dan perannanya pada pengajaran genetika dalam pendekatan cbsa. thesis tidak diterbitkan: ikip malang. o’dell, kevin m.c. (2003). the voyeurs’ guide to d. melanogaster courtship, (online), (http://www.deepdyve.com/lp/elsevier/the-voyeurs-guide-todrosophila-melanogaster-courtship-l6ounc3j81), diakses tanggal 15 november 2013. parvati, et al. (2009). wonder animal model for genetic studies d. melanogaster –its life cycle and breeding methods – a review. sri ramachandra journal of medicine. vol. ii, issue 2 silvia, triana. (2003). pengaruh pemberian konsentrasi formaldehida terhadap perkembangan larva d. melanogaster. bandung: jurusan biologi universitas padjadjaran http://www.deepdyve.com/lp/elsevier/the-voyeurs-guide-to-drosophila-melanogaster-courtship-l6ounc3j81 http://www.deepdyve.com/lp/elsevier/the-voyeurs-guide-to-drosophila-melanogaster-courtship-l6ounc3j81 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 153-160 | doi: 10.14421/biomedich.2025.141.153-160 issn 2540-9328 (online) superoxide anion radicals biosensor based on protein extract from deinococcus radiodurans immobilization by glutaraldehyde cross-linked muhammad ridho afifi1, dyah iswantini2,*, novik nurhidayat3, deden saprudin2 1department of chemistry, faculty of mathematics and natural sciences, syiah kuala university, banda aceh, indonesia. 2department of chemistry, faculty of mathematics and natural sciences, ipb university, bogor, indonesia. 3research centre for applied microbiology, national research and innovation agency, bogor, indonesia. corresponding author* dyahis@apps.ipb.ac.id abstract a common enzyme for superoxide anion radicals biosensors is superoxide dismutase (sod). deinococcus radiodurans protein extract can replace the use of pure sod enzymes for superoxide anion radicals biosensors. the purpose of this research is to determine the analytical performance of the superoxide anion radicals biosensor with protein extract of deinococcus radiodurans immobilized on carboxymethylcellulose-gelatin-zeolit (cmc-g-z) that is crosslinked by glutaraldehyde. the response surface method showed that the optimum condition for the biosensor was ph 7, protein extract concentration of 1075 µg/ml, zeolite concentration of 5 mg/ml, and glutaraldehyde concentration of 0.0042 m. stability of the biosensor retained 67% of its sensitivity after use for 24 hours. the biosensor exhibits good analytical performance with a linear range from 0.1 – 0.8 mm, a detection limit of 77.84 µm, and a limit of quantification of 259.5 µm with a correlation coefficient of 0.9905. keywords: superoxide anion radicals; deinococcus radiodurans; biosensor; superoxide dismutase (sod); glutaraldehyde. introduction reactive oxygen species (ros) are chemical molecular species formed after the incomplete reduction of oxygen and are highly reactive. ros consists of hydroxyl radicals (.oh), superoxide anions (o2 .-), and hydrogen peroxide (h2o2) (j. liu et al., 2023). mitochondria produce o2 .through oxidative phosphorylation in living aerobic organisms for atp synthesis (schröder, 2020). superoxide anion can be converted into h2o2 and oxygen (o2) by the enzyme superoxide dismutase (sod), which is present in mitochondria. however, o2 .can be dangerous if the amount of superoxide anion is uncontrolled. (andrés et al., 2023). an excessive increase of superoxide anion radicals in biological systems can cause cancer (sahoo et al., 2021), parkinson's (jiang & bai, 2022), autoimmune (tavassolifar et al., 2020), and inflammation (albano et al., 2022). up to date, there are several methods for measuring o2 .as well as for evaluating antioxidant activity, including spectrophotometry (yang et al., 2020), fluorescence sensor (song et al., 2017), liquid chromatography/mass spectrophotometry (lc/ms) (wang et al., 2018), and electrochemical biosensor (zouleh et al., 2025); (crulhas et al., 2017). among the above methods, electrochemical is considered an ideal superoxide anion detection technique due to it is simple, cost-effective and has a fast response. (f. liu et al., 2022). one of the o2 .biosensor methods uses the superoxide dismutase (sod) enzyme which has a good correlation with spectrophotometric methods to detect o2 .(braik et al., 2016). however, the use of sod enzyme in biosensors has a limitation due to its high cost. the use of microbes as an alternative solution to reduce biosensor production costs. one of the potential microbial sources of sod in indonesia is deinococcus radioduran (iswantini et al., 2019); (sukma et al., 2023).this microbe contains manganese superoxide dismutase (mn-sod) within its cells (gaidamakova et al., 2022). enzim sod dapat diperoleh dengan menghancurkan membran sel d. radiodurans yang berupa ekstrak protein d. radiodurans. ekstrak protein sod yang diimobilisasi pada elektroda pasta karbon menghasilkan aktivitas sod lebih tinggi daripada enzim murni sod. sehingga protein d. radiodurans berpotensi untuk biosensor antioksidan (iswantini et al., 2019); (sukma et al., 2023)(iswantini et al., 2019; sukma et al., 2023). manuscript received: 17 march, 2025. revision accepted: 08 may, 2025. published: 15 june, 2025. https://doi.org/10.14421/biomedich.2025.141.153-160 154 biology, medicine, & natural product chemistry 14 (1), 2025: 153-160 research on d. radiodurans-based superoxide anion biosensors is continuously being conducted to improve their performance, focusing on optimizing the immobilization method of the biorecognition element. enzyme stability can be maintained by immobilizing it on nano-materials (nemiwal et al., 2022). zeolite has been widely used in enzyme biosensor immobilization matrices due to its advantageous properties, including size selectivity, charge, hydrophobic/hydrophilic interactions, and high thermal stability (zhang et al., 2021). using zeolite in biosensors as a microbial immobilization matrix has been proven to enhance current response (µa) in electrochemical methods (vu et al., 2020). natural zeolite is abundant in indonesia, including bayah zeolite from banten, indonesia. bayah zeolite as a natural zeolite still needs to be optimized for use in all areas such as immobilization matrix because its abundance, easy availability, and low cost of natural zeolite support this research for immobilization matrix development. however, the stability of biosensors using d. radiodurans protein extract needs to be improved by combining zeolite with other materials as immobilization agents for the d. radiodurans protein extract (iswantini et al., 2019). the immobilization method for enzymebased biosensors using glutaraldehyde as a cross-linking agent can increase the stability and performance of biosensor electrodes (bounegru & apetrei, 2023); (kocabay et al., 2012); (karakaya et al., 2020). this research continues the previous research by iswantini et al. (2019), where d. radiodurans protein extract was immobilized with zeolite on a carbon paste electrode (iswantini et al., 2019). previous research has shown that sod from d. radiodurans protein extract exhibits higher affinity compared to purified sod enzyme. further research is needed to improve analytical parameters, especially the stability of biosensors. improvement of immobilization through crosslinking between gelatin and glutaraldehyde dispersed on the surface of zeolite can optimize the performance of d. radiodurans protein. it is concluded that using this immobilization method can improve the stability and activity of the d. radiodurans protein extract for anion superoxide radicals biosensors. materials and methods materials the materials used d. radiodurans bacterial cells grown from waste sauce isolation in indonesia. natural zeolite from bayah, indonesia. lactose broth medium from miller. gelatin, carboxymethylcellulose, glutaraldehyde, xanthine oxidase, and xanthine, were all sourced from sigma aldrich, and dmso was from merck. growth of d. radiodurans cells and sod protein extraction d. radiodurans from culture were grown in 25 ml of lb media. the bacteria were incubated at 30°c and measured at a wavelength of 600 nm for an optical density (od) value of 0.5–0.6. the cells were harvested by centrifugation at 7000 x g, 4°c for 10 minutes to separate the microbial cells from the media. the cells (pellet) were washed three times with phosphate buffer ph 7.0 and then resuspended in phosphate buffer solution ph 7.0. the cell suspension was sonicated at 80% pulse intensity to lyse the microbial cells for 2 minutes, with 1 x 2-minute intervals, and a 1-minute resting period between sonication cycles. during sonication, the cell suspension was cooled in an ice bath. the cells were centrifuged at 10,000 x g, 4°c for 30 minutes to separate the supernatant from the pellet. the crude protein extract was present in the supernatant. the cytoplasmic extract after dialysis was then measured for absorbance at wavelengths of 260 nm and 280 nm to determine the protein concentration and the protein-to-dna ratio (chou & tan, 1991). preparation of carbon paste electrode the electrode was prepared by dissolving 3 mg of ferrocene in 1 ml of dmso, and then adding 100 mg of graphite to the solution. the mixture was left to stand for 2 hours. after 2 hours, the solvent was evaporated in an oven at 105°c until dry. graphite was mixed with liquid paraffin in a 2:1 ratio until a paste was formed. the carbon paste was inserted into the electrode body and compacted until it reached the glass surface. the glass surface of the electrode was polished and cleaned using sandpaper. immobilization of d. radiodurans protein extract on the surface of the carbon paste electrode a polymer mixture (cmc-gelatin) was prepared in a 0.1 w/w ratio in 10 ml phosphate buffer. 50 mg of zeolite was dispersed into the polymer mixture to form the (cmc-g-z) mixture. 60 µl d. radiodurans protein (in phosphate buffer) and 10 µl of 0.05 m glutaraldehyde were added to 30 µl of cmc-g-z (kocabay et al., 2012); (karakaya et al., 2020). the immobilization system mixture (cmc-g-z-protein) was applied to the surface of the carbon paste electrode. the electrode was left to stand until the solvent evaporated at 25ºc. the electrode surface was coated with a dialysis membrane. the immobilization process of cmc-g-z-protein of d. radiodurans can be seen in figure 1. afifi et al. – superoxide anion radicals biosensor based on protein extract … 155 figure 1. schematic representation of the stepwise immobilization process of protein extract on cmc-g-z by glutaraldehyde crosslinked. electrochemical measurement electrochemical measurements were conducted using a potentiostat/galvanostat edaq system and a computer with the echem v2.1.0 data processing software. the electrodes used were an ag/agcl as the reference electrode, platinum wire as a counter electrode, and a carbon paste electrode as the working electrode. 1.9 ml of 0.05 m phosphate buffer solution at ph nine was added to the electrochemical cell, and the anode current peak formed was observed as the blank. 100 μl of 0.1 u/ml xanthine oxidase solution and 1 ml of 2.1 mm xanthine substrate were added to the electrochemical cell. the change in current was monitored until a steadystate current was reached. optimization of superoxide anion biosensor optimization was performed by combining variations in ph (7, 8, and 9), d. radiodurans protein extract concentration (500, 1000, and 1500 μg/ml), glutaraldehyde concentration (0, 0.005, and 0.01 m), and zeolite concentration (5, 7.5, and 10 mg/ml). the method used for optimizing was the response surface methodology with minitab 16 software. determination of electrode stability electrode stability was determined by measuring the activity of the protein extract after optimal conditions were achieved. the activity value obtained from the initial measurement was considered 100%. the activity was remeasured at specific time intervals, and the remaining activity was calculated. electrode analytical parameters the performance of the biosensor electrode was tested for two electrodes, one with glutaraldehyde and the other without glutaraldehyde. the parameters determined were linearity, the limit of detection (lod), and the limit of quantification (loq) for superoxide anion biosensors. results and discussion growth of d. radiodurans and extraction of sod protein the d. radiodurans cells were grown in lb medium for approximately 18 hours with a shaker (aerobic conditions) at room temperature. the d. radiodurans cells were lysed to obtain crude protein containing the sod enzyme. the protein extraction results, shown in figure 2, indicate that the optimal induction treatment was achieved with 3 minutes of exposure to uv light. uv light induction can generate free radicals that attack the dna and membranes of microorganisms, leading to cell damage. however, d. radiodurans is a microbe that can survive damage by free radicals because of its ability to repair dna damage (chen et al., 2023). proteins repair dna damage within the cell. the mnsod enzyme in d. radiodurans is capable of counteracting free radicals, one of which is caused by radiation (gaidamakova et al., 2022). therefore, uv light induction facilitates the expression of the sod protein, leading to a greater protein extract yield compared to conditions without uv irradiation (palmieri et al., 2019). d. radiophilus is also able to survive in extreme environments. the mn-sod produced by d. radiophilus increases with increased environmental stress, such as uv radiation. however, if the uv radiation is excessive, mn-sod production decreases (yun & lee, 2003). uv induction for 5 minutes can damage d. radiodurans cells, leading to a decreased in cell growth due to excessive uv exposure. figure 2. protein concentration of d. radiodurans to uv radiation and ultrasonication time treatment. the optimal ultrasonication time for obtaining protein extract from the cells is 2 minutes, with a protein concentration of 1774.89 µg/ml. the sod enzyme in d. radiodurans is located in the cytoplasm, requiring ultrasonication or cell membrane lysis for its extraction (sadowska-bartosz & bartosz, 2023). ultrasonication for 0 minutes resulted in very low yields, as the sod protein extract remained within the cell membrane. ultrasonication for 4 minutes resulted in a decrease in protein extract concentration, as some of the sod proteins in the cytoplasm were denatured due to the ultrasonication process. 156 biology, medicine, & natural product chemistry 14 (1), 2025: 153-160 immobilization of d. radiodurans protein extract the immobilization of d. radiodurans protein extract using cmc-gelatin and glutaraldehyde aims to increase the stability and activity of the protein extract. the immobilization process is performed by crosslinking glutaraldehyde with protein extract on a solid phase of cmc-gelatin. the reactions in the biosensor system are: xantine + h2o + o2 xod uric acid + 2h+ + o2 . 2h+ + o2 . sod h2o2 + o2 h2o2 2h+ + o2 + 2e the voltammogram of the immobilized d. radiodurans protein extract can be seen in figure 3. the activity of the protein extract can be determined by the anodic peak at a voltage of 0.4v after adding xanthine and xanthine oxidase (xod) to the electrochemical cell solution. the blank peak can be determined by measuring the buffer at ph seven before adding xanthine and xod. electrochemical measurements using the sod enzyme crosslinked with glutaraldehyde produce an anodic peak at a voltage of 0.4–0.5 v, confirming that the anodic peak of d. radiodurans protein extract is comparable to that of the pure sod enzyme (vu et al., 2020). figure 3. voltamogram of d. radiodurans protein extract activity with xanthine and xod (a), without xanthine and xod (b) the carbon paste electrode was coated with a dialysis membrane which functions as an intermediary for the diffusion of the substrate into the protein extract and to keep the protein extract out of the immobilization system. the dialysis membrane used is thermo scientific snakeskin dialysis tubing, 10k mwco (molecular weight cut off), which ensures that the protein extract containing the sod enzyme is retained, as it has a molecular weight greater than 10 kda (chen et al., 2023). however, the pores of the dialysis membrane can allow the superoxide radical substrate to pass through and interact with the d. radiodurans protein extract. figure 4 shows that higher current response is obtained from the immobilized protein extract with glutaraldehyde crosslinking while without glutaraldehyde the current response is lower. the increased current with glutaraldehyde crosslinking can explain that the crosslinking agent can bind the protein extract optimally, resulting in a higher activity of the protein extract to react with the substrate (karakaya et al., 2020). figure 4. the voltammogram of the activity of d. radiodurans protein extract immobilized with crosslinking and without glutaraldehyde. optimization of anion superoxide biosensor optimization was performed on a 0.7 mm xanthine substrate with several variations of protein concentration, glutaraldehyde concentration, zeolite concentration, and ph using the response surface method in minitab 16 software. figure 5a shows the effect of glutaraldehyde concentration as a crosslinking agent, which produces the optimal condition at approximately 0.005 m. a glutaraldehyde concentration of 0.005 m results in the maximum current compared to concentrations below or above 0.005 m. the optimum concentration of glutaraldehyde obtained in this study shows results that are suitable when using pure sod enzyme to detect superoxide (karakaya et al., 2020). however, when the glutaraldehyde concentration is higher, there will be a reduction in current because it will bind to the active site in the protein extract, causing no reaction with the substrate to form, and the pores on the electrode surface will become tighter, which can make diffusion of the substrate more difficult (kocabay et al., 2012); (karakaya et al., 2020). without glutaraldehyde in the immobilization system, the protein extract will only be adsorbed into the phase of the cmc-gelatin-zeolite polymer. this adsorption is physical, which causes the protein extract to result in a lower current (berillo et al., 2024). afifi et al. – superoxide anion radicals biosensor based on protein extract … 157 figure 5. the effect of glutaraldehyde concentration on protein concentration (a); the effect of ph on protein extract concentration (b); and the effect of zeolite concentration on protein extract concentration (c) on the change in peak current. figure 5b shows that the optimal condition for ph is at ph 7, with a protein concentration of approximately 1000 µg/ml. the optimal ph condition is crucial because enzymes are susceptible to ph. the pure sod enzyme immobilized with glutaraldehyde crosslinking shows an optimum ph condition of 7 (kocabay et al., 2012); (karakaya et al., 2020). figure 5c shows the effect of zeolite concentration, which results in the optimum condition at a concentration of 5 mg/ml. zeolite, as an immobilizer for d. radiodurans protein extract can increase the signal’s current (iswantini et al., 2019). zeolite has been widely used as a matrix in enzyme immobilization systems because zeolite can improve the catalytic properties of enzymes based on hydrophobic or hydrophilic interactions, electrostatics, and hydrogen bonds. the ability of zeolite to increase the biosensor response is influenced by the si/al ratio. the si/al ratio affects the biosensor response, with a higher si/al ratio leading to a stronger biosensor response (soldatkina et al., 2019). the zeolite concentration increased to 7.5 mg/ml and 10 mg/ml, the signal current became lower. this is probably due to the form of the zeolite covering the electrode surface and making the electrode surface rigid (khasanah et al., 2018). based on figures 5a, 5b, and 5c, it is shown that each contour shows the optimum concentration for the d. radiodurans protein extract, which is 1000 µg/ml. these results probably show the higher the concentration of protein extract, the more saturated the enzyme will be in the matrix pores, which limits the reaction between the substrate and the enzyme (mcdonald & tipton, 2022). the protein extract from d. radiodurans contains several proteins besides sod. these other proteins in the extract may interfere, making it more difficult for the substrate to react with the sod enzyme (iswantini et al., 2019). electrode analytical parameters and stability the effect of xanthine concentration on protein extract activity was measured using xanthine concentrations ranging from 0.1 to 0.8 mm. figure 7 shows the relationship between xanthine concentration and the activity of protein extract crosslinked with glutaraldehyde and without glutaraldehyde. the d. radiodurans protein extract biosensor immobilized on cmc-g-z with glutaraldehyde crosslinking shows a linear measurement range of 0.1–0.8 mm, with an r² value of 0.9905, while without glutaraldehyde, the range is also 0.1–0.8 mm, with an r² value of 0.9861. limit of detection (lod) and limit of quantification (loq) obtained with glutaraldehyde are 77.84 μm and 259.50 μm, respectively, while without glutaraldehyde, the lod and loq are 94.39 μm and 283.16 μm, respectively. 158 biology, medicine, & natural product chemistry 14 (1), 2025: 153-160 the protein extract activity on cmc-g-z-glutaraldehyde is higher at xanthine concentrations than cmc-g-z. this is because, when the protein extract is crosslinked with glutaraldehyde, the active sites are preserved on the carbon paste electrode, allowing them to fully interact with the substrate. in contrast, the active sites of the protein extract without glutaraldehyde are less stable on the carbon paste electrode. figure 6. linearity of the anion superoxide biosensor with glutaraldehyde crosslinking and without glutaraldehyde. linearity is another important factor, indicating the linear connection between the sample's concentration and the related analytical signal (aldiansyah et al., 2025). the difference in linearity between the protein extract immobilized on cmc-gelatin-zeolite crosslinked with glutaraldehyde provides a wider linear range and higher current compared to the non-immobilized extract. this indicates that the immobilization of the protein extract can enhance its activity. in another research, clostridium difficile used as a producer of mn-sod resulted in a measurement range of 0.1352–1.160 mm (ye et al., 2014). biosensors using pure sod enzyme produce a wider measurement range and a lower detection limit compared to biosensors that use microbes. this is because the composition of microbes is more complex than pure enzymes, which results in slower diffusion between the desired protein and the substrate, making it more difficult to form the catalytic reaction (ratautė & ratautas, 2024). the difference in electrode types, depending on the material’s electrical conductivity, can also affect the results of the measurement range and other analytical parameters (thandavan et al., 2013). in this study, the stability of the electrode was tested using the same electrode, which was evaluated at the initial time and subsequently over time. the stability of the electrode can be calculated by comparing the current at each hour (time) with the initial measurement current at hour 0. in figure 6, the stability of the biosensor with protein extract immobilized using the crosslinking agent glutaraldehyde results in an activity of d. radiodurans protein extract of 67.74% for 24 hours. in contrast, without glutaraldehyde, the activity is 46.38% for 24 hours. glutaraldehyde binds the protein extract, stabilizing the position of the active site, which leads to more optimal performance of the protein extract in reacting with the substrate (karakaya et al., 2020); (kocabay et al., 2012); (ionescu, 2022); (moya et al., 2020). based on the graph results in figure 6, it can be shown that there is a decrease in current between the samples with and without glutaraldehyde. this is due to the enzyme's nature, which reacts with the substrate based on the key-lock principle. when the active sites on the enzyme surface are utilized, enzyme activity is reduced (ionescu, 2022); (moya et al., 2020). figure 7. the stability of the anion superoxide biosensor with glutaraldehyde crosslinking and without glutaraldehyde. the stability of the protein extract is not only influenced by the immobilization process but also by the presence of other proteins in the extract, such as peptidase. peptidase is an enzyme that can break peptide bonds in proteins, and therefore, the presence of peptidase may potentially damage the superoxide dismutase enzyme within the crude protein extract (dalmaso et al., 2015). conclusions the superoxide anion radicals biosensor using d. radiodurans protein extract immobilized in a cmcgelatin-zeolite matrix with glutaraldehyde crosslinking resulted in greater electrode stability over time than without glutaraldehyde. the technique of cross-linking glutaraldehyde immobilization with d. radiodurans protein extract increases the analytical performance of the biosensor by generating a higher current in the measurement against the xanthine substrate. further research is needed to remove other proteins, especially peptidase in the d. radiodurans protein extract. acknowledgements: the authors would like to express their sincere gratitude to all those who contributed to this research. competing interests: the authors declare that there are no competing interests. afifi et al. – superoxide anion radicals biosensor based on protein extract … 159 references albano, g. d., gagliardo, r. p., montalbano, a. m., & profita, m. 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(2025). design of a microneedle-based enzyme biosensor using a simple and cost-effective electrochemical strategy to monitor superoxide anion released from cancer cells. analytical biochemistry, 697(august 2024), 115710. https://doi.org/10.1016/j.ab.2024.115710 biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 1, 2015 | pages: 11-15 | doi: 10.14421/biomedich.2015.41.11-15 the effect of intensive keramba on the presence of parasite organisms in rivers of lingsar area supriadi* and maratun janah faculty of veterinary medicine, university of west nusa tenggara, mataram jl. tawak-tawak karang sukun, mataram, tel. +62-370-636875 author correspondency*: supriadi@yahoo.co.id abstract the application of intensive keramba in rivers could affect the presence of parasite organisms throughout the river downstream. the aims of this research are to find out the diversity of parasite species and the effect of intensive aquaculture method developed by the community on the presence of various parasitic organisms, particularly in the downstream area. a total of 65 tilapia fish samples (o. niloticus) that was collected from 3 areas (15 samples from upstream, 25 samples in keramba and 25 samples from downstream areas) have been examined in the laboratory of faculty of mathematic and natural science, university of mataram. methods employed to identify parasites that infected fish samples are native method and flotation method. this research has identified 7 species of parasites which were divided into 2 groups: ectoparasites (trichodina sp., amylodinium sp., oogonium sp., dactylogirus sp., trematode) and endoparasites (entamoeba sp. dan camallanus sp.). diversity index calculation indicated that parasite organisms in upstream area were lower in number than that in the downstream and intensive karamba area (h’= (0,825; 1,596 dan 1.324 respectively). these data has showed there was a difference in species diversity and evenness index of parasite organisms in the upstream, downstream and intensive keramba area. in conclusion, there was significant influence of the application of intensive keramba on the appearance of various parasite organisms that could affect the sustainability of fish aquaculture. keywords: intensive keramba, parasites organism and diversity index introduction fishery products have become the highest animal protein sources demanded in indonesia with the consumption rate of these products has reached 30.4 kg/capita/year or 72% of animal protein consumption/capita/year (anonima, 2013). although the prospect of fishery sector development in indonesia is high, especially in freshwater fishery sector, there has been insignificant production growth in the last several years. this may be due to various factors such as fish farmers’ low interest in the cultivation, capital shortage, lack of knowledge, stream water pollution and various diseases (parasites, bacteria and viruses) which result in considerable loss. one of many factors that can lead to a decrease in the quality and quantity of fishery products is parasite infections (akbar, 2011). the impact of the infection can be a decrease in fish population for consumption, reduction in fish weight, morphological changes and even in some cases; this can cause illness in humans (zoonoses parasite infection) (herman and chiodini, 2009; mccarthy and moore, 2000; punyagupta et al., 1990). declining fish population triggered by the degraded water quality in the cultivation sites is a direct aspect and unsettling condition for fish farmers (bell and burt, 1991). the presence of parasite organisms such as digenea worms, cestodes, nematodes, acantochepals and several ectoparasites from copepode group greatly affects aquaculture activities (kennedy, 1990; lawton, 1989). according to salgado-maladona et al., 2005, the presence of parasites in freshwater fish populations are often linked to the quality of the aquatic environment. moreover, environmental degradation is also often associated with a greater diversity of parasitic infections (chubb, 1963). dominance and density of infection of pathogenic organisms is often directly proportional to the environmental degradation (quiroz-martı´nez and salgado-maldonado, 2013). one method of fish farming that is directly cultivated in the river is intensive keramba method. this method is very simple and easy, even though its feasibility has not been tested ecologically. to anticipate the possibility of eutrophication impact in the downstream and the risk of pathogenic organism growth, it is necessary to conduct this research to study the diversity of parasitic organism caused by the application of intensive keramba method as well as the method’s impact on the presence of parasitic organisms particularly in the downstream area. research methods this research was conducted from july to august 2014. sampling was carried out in the river which became the center of cultivation, located in lingsar sub-district, west lombok regency. fish sample examination was performed in the laboratory of biology, faculty of mathemathics and natural science, university of mataram. parameters to be measured and examined in this research were the species and the diversity of parasitic organisms, species eveness index and the frequency of 12 biology, medicine, & natural product chemistry 4 (1), 2015: 11-15 parasitic organisms’ presence in wild fish as well as in keramba-farmed fish. fish collection was performed in sampling spots which were purpospreively determined and refered to the actual condition on field. the fish were collected utilizing fishnets and fishing rods. the fish net was thrown over 35 times to obtain as a minimum 10 fish on the certain spot. farmed fish samples were acquired from the fish farmers on field. parasite specimens collected on field were preserved in 70% alcohol for further analysis in the laboratory of the faculty of mathematics and natural science, university of mataram. fish organs, such as gill, digestive organs, and flesh (muscle around abdominal cavity), were examined for the presence of parasitic organisms. the obtained organisms were then transferred a physiological saline solution and were identified using a light microscope. staining on the parasitic specimen was necessary in order that the parasite organisms could be identified acurately. infestation rate of the organisms from various samples was calculated by identifying the species discovered from the host tissue (gill, digestive track, flesh) and then counting the individual numbers. the species identification refered to identification books by noble & noble (1989) & taylor et al., (2007). endoparasite examination was performed utilizing saturated sugar method. prevalence = x 100% diversity index = log/ ln similarity index = ( ) x 100% (choi et al., 2011; kusmana, 1997) data obtained from this research were analysed descriptively by linking up the data and facts found on fields; whilst data interpretations were presented in the form of tables, figures and graphs. subsequently, the conclusion was drawn deductively by describing the issues from general to specific points results and discussion results there were 25 fish samples that were collected randomly from several intensive keramba in the river of lingsar village. the examination results showed that there were various parasite infections. parasites identified from this research were devided into ectoparasite and endoparasite group. the species that were included in ectoparasite group were trichodina sp., amylodinium sp., oogonium sp., dactylogirus sp and 1 species of trematodes; while the endoparasites were camallanus sp. dan entamoeba sp (figure 1). figure 1. parasites found in tilapia fish from the river of lingsar village. a. amylodinium sp., b. trichodina sp., c. oogonium sp., d. entamoeba sp., e. dactylogirus sp., f. trematoda dan g. camallanus sp. supriadi and maratun janah. – the effect of intensive keramba on the presence of parasite organisms … 13 the shanon-wiener diversity index of parasites in tilapia fish (oreochromis niloticus) from three sampling location (upstream, middle/keramba and downstream) were 0.8247, 1.3244 and 1.5962 respectively. these numbers demonstrated that the species diversity of parasites in the upstream, middle and downstream area were moderate. however, there were differences in the numbers and infection intensity in the upstream to downstream area; where the frequency of infection in the upstream was very low in contrast to the very high infection frequency in the middle and downstream area. table 1. comparison of diversity index value (h’), species eveness (e) and frequency (f) of parasite organisms presence in three sampling location. no. sampling location h’ e f (%) 1 river upstream 0.8247 0.4238 46.7 2 intensive keramba 1.3244 0.6806 96.0 3 river downstream 1.5962 0.8203 92.0 . the calculation results of the presence frequency of parasites organisms illustrate that the infection frequency tend to get higher in the downstream area. in the river upstream, the infection frequency was 46.7%, in the intensive keramba was 96.0% and in the downstream was 92.0%. according to the graph in figure 2, a number of 7 parasite species infected fish were collected in the river downstream and in the intensive keramba owned by the community. on the other hand, only 3 species of ectoparasite were discovered infecting the fish collected from the upstream. further, it can be explained that the dominant species infecting the fish was trichodina sp. this species was found in all sample groups. what is more, the species of amylodinium sp and oodinium sp. were also had the same presence frequency as trichodina sp. however, the intensity (density) of infection of the two species were lower compared to that of trichodina sp (figure 2). figure 2. comparison of the infection frequency in tilapia fish from 3 three sampling location. data of parasite species diversity index from three location show that there was a significant difference. in the upstream area, the parasite species diversity index is low which counted at 0.8247. meanwhile, the parasite diversity index in the intensive keramba area and downstream area were counted at 1.3244 and 1.5962 respectively. based on these values of shannon-wiener diversity index, it can be stated that the upstream area has lower parasite species diversity, while the keramba area and downstream area have moderate diversity. evenness index values of the parasite species show increasing trends toward the downstream area. in the upstream area, only 3 species of parasites were discovered, whereas in keramba site and downstream area, 7 species of parasites were identified. however, there was similar data pattern i.e all parasite species found in the upstream were also found in tilapia fish collected from keramba site and the downstream. figure 3. comparation graph of shannon-wiener species diversity index and species evennes index for parasite organisms on the 3 (three) sampling location. the number of parasite species identified in each sampling location illustrates the drastic increase from upstream (3 species) toward downstream area (7 species). the main difference is the level of parasites density. even though there were only 7 species discovered in keramba site and downstream area, the parasites individual number than infects the keramba-cultured fish was higher (figure 3). figure 4. graph for difference in species number of parasites that infect tilapia fish in three different sampling location. 14 biology, medicine, & natural product chemistry 4 (1), 2015: 11-15 discussion based on the analysis and laboratory examination results, as many as 7 species of parasites infected tilapia fish caught in the upstream, keramba site and the downstream. the species include ectoparasite trichodina sp., amylodinium sp., oogonium sp., dactylogirus sp, 1 species of trematode, and 2 endoparasites (camallanus sp. and entamoeba sp (figure 1)). dominant species found in this study is trichodina sp. which was discovered in all sampling locations. according to eronimo et al., (2012), these parasite species are common ectoparasites that infect tilapia fish farmed in freshwater. further explained is that this parasite infection mostly happen in gill and skin area, which reduce the immunity system of the infected fish (martin, 2012). amylodinium sp. and gymnodinium sp. were two other ectoparasites that were discovered in all sampling locations, although with lower individual density. this may be due to the two species competition with trichodina sp. other than protozoa, this research also found trematode worm (ectoparasite) on the fish gill and was identified as dactylogyrus sp. this species is mostly discovered in the fish collected from the downstream and keramba sites. this trematode worm is pathogenic and often become the cause of fish mortality in several fish farming sites. by infesting the gill of freshwater fish (tilapia), this parasite infection would escalate the heart rate to pump up the blood to the gill and increase mucus production, causing the difficulty in taking in oxygen from water (eva, 2007). beside dactylogyrus sp., there was also another unidentified treamatod worm. however, this species is rarely found infecting the sampled tilapia fish. two endoparasites species identified in this study, i.e camallanus sp. and entamoeba sp., are nematode worms that were mostly found in fish intestine. kabata (1985) also explained that these worms often infect the intestine of freshwater fish. furthermore, these parasite species are often discovered infecting freshwater fish in aquariums. these nematodes infection is easily spreading as the worms do not require intermediate hosts in their life cycles (untergasser, 1989). besides, the worms infection is very damaging to their hosts as the worm can wound the host’s intestine by biting the intestine wall using the worm’s bucal capsules. this research identified camallanus sp. with the length of 10-15 cm. this discovery is supported by research results of buchmann & bresciani (2001) and arie (2008) who found that the female worms could reach the length of 22 cm while the male ones could have the length of 11cm. analysis results of species diversity showed that there was significant difference in species diversity index from the upstream to downstream. species diversity index values for upstream, keramba, and downstream area are 0.8247, 1.3244 and1.5962 respectively. these data display a pattern that the number and density of parasite infection were changing from the upstream to downstream. in the upstream, parasite infection in tilapia fish was very low with only a total of 3 parasite infection and low parasite density. increasing individual number and species of parasites infecting the fish from the upstream to downstream can be triggered by various biotic and abiotic factors. biotic factors include organisms carrying parasites (hosts) and abiotic factors include aquatic environmental pollution as a result of human activities which produce wastes or change the condition of aquatic environment. as stated by quiroz-martı´nez and salgado-maldonado (2013), intensive keramba culture would increase the risk of change in various environmental condition as a result of feeding residue pollution. it can be further explained that feeding remnants from the intensive keramba farming can pollute the aquatic environment in the form of changing water ph, eutrophication and decreasing water clarity. however, these factors are not the only factors that pollute the aquatic environment. species evenness index on the three sampling locations showed that there was also a significant difference. the values of species evenness index for upstream area, keramba sites, and downstream area are 0.4238, 0.6806 and 0.8203 respectively. the values illustrated that parasites presence in the upstream was rare in contrast to the higher species and individual number of parasites in the intensive keramba site and downstream area. this means that fish farming which employing intensive keramba method in the river has a significant effect on the presence of various parasite species. the high parasite infection on keramba-cultured fish is a result of high density of the fish farmed in the intensive keramba. in contrast, fish density outside keramba was very low. the dense environment inside keramba has made it possible for the parasite to easily transmit from one individual fish to another. this is also supported by the parasite density data observed in downstream area. although the species number of parasite identified in the downstream was the same as those found in keramba, the density of the individual parasite found in fish caught from the downstream was lower than those infecting keramba-fish. furthermore, the sparse distribution of fish in the downstream contributed to the lower possibility of parasite organism to transmit between individual or species of fish. data from this research has shown the effect of intensive keramba culture on the presence of parasite organisms in farmed-fish and wild fish. however, it is also important to conduct a research in different season and perform measurement of other factors such as the effect of habitation in the vicinity of river and water pollutant from the surrounding rice field. therefore, recommendation for the application feasibility of intensive keramba system in rivers can be comprehensively established. conclusion from this research, it can be concluded that parasite species diversity of fish in downstream area and intensive supriadi and maratun janah. – the effect of intensive keramba on the presence of parasite organisms … 15 keramba sites were higher than that in the upstream; with the values of species diversity index was 0.8247 in the upstream, 1.3244 in keramba sites and 1.5962 in the downstream. in other words, fish culture employing intensive keramba system has a significant effect on the presence of various parasite organism on fish in the downstream area. references akbar, j. 2011. identifikasi parasit pada ikan betok (anabas testudieus). bioscientiae. volume 8, nomor 2, juli 2011, page 36-45. anonima, 2013. bisnis ikan di indonesia timur: tantangan dan potensinya. http://nyataindonesiaku.com/bisnis-ikan-diindonesia-timur-tantangan-dan-potensinya./akses 29/11/2013. 21.30 wita. arie, u. 2001. pembenihan dan pembesaran nila gift. penebar swadaya. jakarta. bell, g., and a. burt. 1991. the comparative biology of parasite species diversity: internal helminths of freshwater fish. journal of animal ecology. (1991), 60, 1047-1063. buchmann k & bresciani j. 2001. an introduction to parasitic diseases of freshwater trout. denmark: dsr publisher. choi, s. h., j. kim, j. okjo, min k. cho, h. s. yu, h. j. cha and m. s. ock. 2011. anisakis simplex larvae: infection status in marine fish and cephalopods purchased from the cooperative fish market in busan, korea. korean j parasitol. vol. 49: no. 1: 39-44. chubb, j.c. 1963. on the characterization of the parasite fauna of the fish of llyn tegid. proc zool soc lond. 141:609–621. eva.g. 2007. ectoparasites and endoparasites among oreochormis niloticus (tilapia). liceo journal of higher education research. herman, j. s and p. l, chiodini. 2009. gnathostomiasis, another emerging imported disease. clin microbiol rev. 22: 484–492. irianto. a. 2005. patologi ikan teleostei. gadjah mada university press. yogyakarta. jeronimo, g.t., n. da c. marchiori1, s. b. de pádua, j. d. neto, f. pilarski, m. m. ishikawa, and m. l martins. 2012. trichodina colisae (ciliophora: trichodinidae): new parasite records for two freshwater fish species farmed in brazil. rev. bras. parasitol. vet., jaboticabal, v. 21, n. 4, p. 366-371. kabata, z. 1985. parasites and diseases of fish cultured in the tropics. taylor and francis. london. 318 pp. kennedy, c.r. 1990. helminth communities in freshwater fish: structured communities or stochastic assemblages? parasite communities: patterns and processes (ed. by g.w. esch, a.o. bush & j.m. aho), pp. 131-156. chapman and hall, london. martin, ml, marchiori, n., roumbedakis, k. and lami, f. trichodina nobilis chen, 1963 and trichodina reticulata hirschmann et partsch, 1955 from ornamental freshwater fishes in brazil. braz. j. biol. vol. 72, no. 2, p. 281-286 mccarthy, j and t. a. moore. 2000. emerging helminth zoonoses. int j parasitol. 30: 1351–1360. noble, e.r & noble g.a. 1989. parasitologi biologi parasit hewan. edisi ke-5.wardiarto, penerjemah; soeripto n. editor. yogyakarta. gadjah mada university press. translation from: parasitology: the biology of animal parasites 5th edition. punyagupta, s., t. bunnag and p. juttijudata. 1990. eosinophilic meningitis in thailand. clinical and epidemiological characteristics of 162 patients with myeloencephalitis probably caused by gnathostoma spinigerum. j neurol sci. 96: 241–256. purbomartono. c, isnaetin. m dan suwarsito 2010. ektoparasit pada benih ikan gurami (osphronemus gouramy, lac) di unit pembenihan rakyat (upr) beji dan sidabowa, kabupaten banyumas. sains aqutic journal. quiroz-martı´nez, b., and g. salgado-maldonado. 2013. patterns of distribution of the helminth parasites of freshwater fishes of mexico. plos one 8(1): sukardi, m.f, 2002. peningkatan teknologi budidaya perikanan. jurnal lktiologi indonesia vol.2, no. 2. taylor, m. a., r. l. coop, and r. l. wall. 2007. veterinary parasitology 3th edition. blackwell publishing; victoria australia. utergasser, d. 1989. handbook and diseases. t.f.h publication inc. translated by howard h. hirschron. neptune city. united states. 159 h. content_v4n1_3.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 15-19 | doi: 10.14421/biomedich.2020.91.15-19 issn 2540-9328 (online) a mathematical model of the covid-19 cases in indonesia (under and without lockdown enforcement) sugiyanto*, muchammad abrori department of mathematics, universitas islam negeri sunan kalijaga yogyakarta jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 corresponding author* sugimath@yahoo.co.id manuscript received: 29 march 2020. revision accepted: 10 april, 2020. published: 15 april, 2020. abstract covid-19 stands for corona (co), virus (vi), disease (d) and year 2019 (19), which is covid-19 first appeared in 2019. mathematical model of covid deployment in indonesia under and without lockdown case uses the sirv model, such as susceptible, infected, recovery, and virus. the results of this model state that under lockdown the spread of covid-19 could be stopped. if it were not under lockdown it can multiply 1,276 times higher over two months. keywords: corona virus novel 2019 (covid-19); mathematics model; lockdown; indonesia introduction novel corona virus can pneumonia (pan et al., 2020). pneumonia is an inflammatory lung disease which is characterized by coughing, chest pain, fever, and difficulty breathing. covid-19 is a very contagious disease and declared by who as a pandemic on march, 12, 2020 (chen et al., 2020). pandemic is an infectious disease that is widely spread and it can reach all countries in the world. covid-19 was first identified in wuhan, hubei province, china in december 2019 (liu et al., 2020). the understanding of lockdown in a pandemic context of covid-19 is an emergency state where someone is not allowed to enter or out of a certain area in a certain period of time. this kind of action is carried out during the emergency situation (ku et al., 2020). mathematical model of covid-19 deployment is using susceptible sub populations, infected, recovery and virus or abbreviated as sirv. this population is divided into four subpopulations. susceptible subpopulation (s) is a subpopulation that is vulnerable to covid-19. infected subpopulation (i) is subpopulation that is infected by covid-19. recovery subpopulation (r) is subpopulation that recover from the covid-19 virus. the population of covid-19 (v) is the population number of covid-19 outside the human body. model formulations the covid-19 deployment model uses several parameters. parameter 𝑑1 is the rate of natural death and other diseases besides covid-19. parameter 𝑎1 is the level of susceptibility to covid-19 disease. parameter 𝑎2 is the level of covid-19 virus infection. parameter 𝑎3 is the cure rate of covid-19 disease. parameter 𝑑2 is the death rate due to covid-19 virus. this models are defined into: a. the susceptible subpopulation are affected by: (1) the increasing in birth rate; (2) the increasing in susceptible individuals from other regions; (3) the increasing number of individuals recovering from the diseases that is caused by covid-19; (4) the reduce susceptibility of covid-19 which infected individuals; (5) the reduce susceptible individuals going to other areas; (6) the loss of individuals due to natural deaths, other than diseases caused by covid-19. b. the infected subpopulation are affected by: (1) the increasing number of individual births from a mother that infected by covid-19; (2) the increasing in individuals due to arrivals from other regions; (3) the increasing in individuals from susceptible and infected by covid-19; (4) the reduce individual recovery from covid-19; (5) the reduction in infected individuals who went to other areas; (6) the https://doi.org/10.14421/biomedich.2020.91.15-19 16 biology, medicine, & natural product chemistry 9 (1), 2020: 15-19 reduction in individuals who die from diseases that caused by covid-19; (7) the loss of individuals due to deaths other than covid-19. c. the recovery subpopulation are affected by: (1) the increase in births at recovery time; (2) the increase in individual recovery from other regions; (3) the increase in infected; (4) the reduce susceptible individuals; (5) the reduction in recovery individuals that went to other areas; (6) the loss of individuals due to deaths other than covid-19. d. the population of covid-19 (population of virus in the environment spread by infected people) are affected by: (1) the covid-19 deployment because individuals are infected; (2) the unsurvive of covid-19 in a free environment due to physical and chemical factors. table 1. the population, subpopulations, and parameters used in the covid-19 spread by mathematics model. no. symbol explanation unit 1. s susceptible subpopulation person/km2 2. i infected subpopulation person/km2 3. r recovery subpopulation person/km2 4. v the population of covid-19 virus/km2 5. 1a birth rate of susceptible subpopulation day-1 6. 2a birth rate of infected subpopulation day-1 7. 3a birth rate of recovery subpopulation day-1 8. 1b arrival rates from other countries susceptible subpopulation day-1 9. 2b arrival rates from other countries infected subpopulation day-1 10. 3b arrival rates from other countries recovery subpopulation day-1 11. c death rates due to disease caused by covid-19. day-1 12. 1d natural death rates are not due to covid-19 susceptible subpopulation. day-1 13. 2d natural death rates are not due to covid-19 infected subpopulation. day-1 14. 3d natural death rates are not due to covid-19 recovery subpopulation. day-1 15. 4d the covid-19 mortality rates in environment. day-1 16. 1e rates of traveling to other countries susceptible subpopulation day-1 17. 2e rates of traveling to other countries infected subpopulation day-1 18. 3e rates of traveling to other countries recovery subpopulation day-1 19. f the degree of covid-19 emergence in environment virus person-1 20. 1k carrying capacity of the susceptible birth subpopulation person2 21. 2k carrying capacity of the infected birth subpopulation person2 22. 3k carrying capacity of the recovery birth subpopulation person2 23. 1l carrying capacity of the susceptible natural death subpopulation person2 24. 2l carrying capacity of the infected natural death subpopulation person2 25 3l carrying capacity of the recovery natural death subpopulation person2 26.  the degree of interaction between susceptible subpopulation become infected subpopulation km2.day-1.virus-1 27.  the degree of interaction betweem infected subpopulation become recovery subpopulation day-1 28.  the degree of interaction between recovery subpopulation become susceptible subpopulation. day-1 in this model it is assumed that: (1) open population, means people are free to enter and exit a country/region; (2) newborn individuals are included in the susceptible subpopulation; individuals who recover from covid are not immune; (3) every individual who infected by covid-19 will become infected; (4) after the incubation period of two weeks are already considered an infected individual; (5) a vaccine for covid-19 has not been found yet, it means that healing is due to good individual immunity, not because of vaccine; (6) people who recovery from covid-19 can be a susceptible subpopulation again; (7) birth rate and natural mortality rate of the infected subpopulation and recovery does not exist. in this model not analyzed local stability of the equilibrium point, and it become our next job. figure 2 sugiyanto & abrori – a mathematical model of the covid-19 cases in … 17 is covid-19 deployment transfer diagram. based on the figure 2, transfer diagram, formed the covid-19 spread mathematics model equation (1a) – (1d). figure 1. covid-19 distribution between individuals. 1 1 1 1 1 1 1 1                    ds s s a s b s r sv e s d s dt k l   (1a) 2 2 2 3 2 2 1 1                     di i i a i b i sv i ci e i d i dt k l   (1b) 3 3 3 3 3 3 1 1                    dr r r a r b r i r e r d r dt k l   (1c) 2 4 2 1         dv i fa i d v dt k (1d) figure 2. transfer diagram of covid-19 deployment model. simulation in this section, we will discuss about numerical simulation and its intepretation by covid-19 deployment model. the initial value starts from the spread of covid-19 in indonesia on march, 12–23, 2020 that was announced by the indonesian goverment. the initial value of the susceptible is taken from the total population of indonesian divided by the population of indonesian, 𝑆0 = 260,000,000 260,000,000 = 1. the infected value was taken from the number of people infected on march 23, 2020 divided by the population of indonesian, 𝐼0 = 579 260,000,000 = 0.0000022269. the recovery values is taken from the number of people recovered on march 23, 2020 divided by the total population of indonesia, 𝑅0 = 30 260,000,000 = 0.0000001. covid-19 value is taken by the estimation, 𝑉0 = 1,000,000 260,000,000 = 0.0038461538. the parameter values are as follows. indonesia area 1.905 x 109 km2. in 2019, the birth rate is = 4.4 x 106 people. in 2019, the mortality rate is = 1.6 x 106 people. there are 365 days in one year. 6 3 1 1 9 4.4 10 6.3 10 1.905 10 365    x a x day x x 6 1 3 1 1 9 1.6 10 2.3 10 1.905 10 365     x d day x day x x from the assumption of natural birth and death that the infected subpopulation does not exist, then the parameter 𝑎2 = 0 and 𝑑2 = 0. from the assumption of natural birth and death recovery subpopulation does not exist, then the parameter 𝑎3 = 0 dan 𝑑3 = 0. because the indonesia country has suspended its arrival visa and the other countries have also applied for a visa suspension, then the parameter 𝑏1 = 𝑏2 = 𝑏3 = 𝑒1 = 𝑒2 = 𝑒3 = 0. from the table 2, we take the highest gradient that is from march 17–18, 2020 are 14 souls per day (zonautara, 2020). it means, the death rates due to the diseases caused by covid-19 is the highest mortality divided by indonesian population or 𝑐 = 14 260,000,000 = 0.0000000538 𝑝𝑒𝑟𝑠𝑜𝑛−2𝑑𝑎𝑦−1. value 260,000,000 are taken from the indonesian population. the value of covid-19 mortality rates in the environment ( d ) be accepted 𝑑4 = 2 260,000,000 = 7.7𝑥10−9 𝑑𝑎𝑦−1 (bnpb, 2020). the estimated value of covid-19 emergence rate in the environment (𝑓) be accepted 𝑓 = 260,000 260,000,000 = 1𝑥10−3 𝑣𝑖𝑟𝑢𝑠 𝑝𝑒𝑟𝑠𝑜𝑛−1𝑑𝑎𝑦−1. the value of the level of interaction between susceptible subpopulation become infected subpopulation (𝛼) taken from the slope of the highest infected divided by indonesian population. the highest slope is on march, 18–19, 2020 or march, 20–23, 2020 there were 81 people died in one day. so, the value of 𝛼 is 81 divided ten times of 579 people were infected (ten fold was infected but its unknown, because it’s not covid-19 tested), 𝛼 = 81 579𝑥10 = 3.6𝑥10−3 𝑑𝑎𝑦−1𝑣𝑖𝑟𝑢𝑠−1. the value of the level of interaction between recovery subpopulation become susceptible 18 biology, medicine, & natural product chemistry 9 (1), 2020: 15-19 subpopulation (𝛽) taken from the highest slope recovery divided by the population of indonesian. the highest slope was on march 21–22, 2020 as many as 9 people died in one day. so, the 𝛽 value is 9 divided by a hundred multiple of 30 people who recover (one hundred multiples are recovery but not known, because it does not test by covid-19), 𝛽 = 9 30𝑥100 = 3𝑥10−3𝑑𝑎𝑦−1. the value of the level of interaction between recovery subpopulation become susceptible subpopulation (𝛾) taken the same as 𝛽. the value of carrying capacity of natural birth and death in the susceptible sub population taken with an estimated number of deaths of 500,000 people divided indonesian population, that is 3 1 2 3 1 2 3 500,000 1.923 10 . 260,000,000       k k k l l l x to summarize the parameter values, we shown in table 3. table 2. the data that died in indonesia because of covid-19 (zonautara, 2020). no. by the date the number of people died the number of people recovered the number of people infected 1. 12 march 2020 1 4 34 2. 13 march 2020 4 4 69 3. 14 march 2020 5 5 96 4. 15 march 2020 5 5 117 5. 16 march 2020 5 8 134 6. 17 march 2020 5 9 172 7. 18 march 2020 19 11 227 8. 19 march 2020 25 15 308 9. 20 march 2020 32 17 369 10. 21 march 2020 38 20 450 11 22 march 2020 48 29 514 12 23 march 2020 49 30 579 table 3 is multiplied by 260,000,000 people; this number is taken from the indonesian population. lockdown case is the level of interaction between susceptible subpopulation become infected subpopulation, the degree of interaction between infected subpopulation becomes recovery subpopulation, and the degree of the interaction between recovery subpopulation being a susceptible subpopulation doesn not exist, it means the value of 0     . table 3. the parameter value without covid-19 deployment lockdown in indonesia. no. symbol value no. symbol value 1. 1a 36.3 10x 13. 2e 0 2. 2a 0 14. 3e 0 3. 3a 0 15. f 31 10x 4. 1b 0 16. 1k 31.923 10x 5. 2b 0 17. 2k 31.923 10x 6. 3b 0 18. 3k 31.923 10x 7. c 85.38 10x 19. 1l 31.923 10x 8. 1d 32.3 10x 20. 2l 31.923 10x 9. 2d 0 21. 3l 31.923 10x 10. 3d 0 22.  33.6 10x 11. 4d 97.7 10x 23.  33 10x 12. 1e 0 24.  33 10x figure 3. diagram of trajectory subpopulation infected between under lockdown and without lockdown. figure 3 shows the case trajectory diagram under and without lockdown in infected subpopulation. in the case of not lockdown on day-0 showed 579 people infected. on 30th for the case to be 677,600 people infected. on 60th showed 738,900 people infected. on day-0 of lockdown case showed 579 people infected and the 30th day showed 579 people infected. because the incubation period of 14 days, then in the case of lockdown on the 15th day no one has been infected. conclusion covid-19 is a vicious virus, because its spread from modeling result is very fast. from the indonesia government’s data on march 12–23, 2020 up to 579 sugiyanto & abrori – a mathematical model of the covid-19 cases in … 19 people have been infected, while from the model when not in lockdown of 579 people become 738,900 people have been infected. this is 1,276 times in two months. under lockdown, it can minimize casualties because covid-19 does not spread. lockdown is a solution to reduce deaths due to covid-19. references bnpb, 2020 in https://www.covid19.go.id/ketahui-caramengurangi-risiko/ downloaded on march 20, 2020. chen, h., guo, j., wang, c., luo, f., yu, x., zhang, w., ... & liao, j. (2020). clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records. the lancet. ku, c. c., ng, t. c., & lin, h. h. (2020). epidemiological benchmarks of the covid-19 outbreak control in china after wuhan’s lockdown: a modelling study with an empirical approach. available at ssrn 3544127. liu, y., gayle, a. a., wilder-smith, a., & rocklöv, j. (2020). the reproductive number of covid-19 is higher compared to sars coronavirus. journal of travel medicine. pan, f., ye, t., sun, p., gui, s., liang, b., li, l., ... & zheng, c. (2020). time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia. radiology, 200370. zonautara, 2020 in https://zonautara.com/2020/03/22/tabelsebaran-virus-corona-per-provinsi-22-maret-2020/ downloaded on march 20, 2020. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 39-46 | doi: 10.14421/biomedich.2020.91.39-46 issn 2540-9328 (online) isolation and characterization of sesquiterpenes from stem bark of warburgia ugandensis sprague teshome gonfa hordofa department of chemistry, college of natural and computational sciences, haramaya university, ethiopia p. o. box 138, dire dawa corresponding author tashe2002@gmail.com, teshome.gonfa@haramya.edu.et manuscript received: 06 may, 2020. revision accepted: 12 may, 2020. published: 17 may, 2020. abstract warburgia ugandensis sprague is one of the medicinal plants traditionally used to treat a number of diseases like asthma, cough, diarrhea, common cold, stomachache and toothache in ethiopia. however, there is still insufficient information on the isolation and evaluation of bioactive compounds from this plant species. extraction, purification and isolation of the stem bark of this plant by dichloromethane and chloroform as solvents afforded two sesquiterpenes; namely, wu-1 (ugandensidial) and wu-2 (cinnamolide-3β-acetate) respectively. the structural elucidations of these bioactive compounds were accomplished by using a variety of spectroscopic methods (ir, uv and nmr). the spectroscopic results compared with the reported data in the literature. keywords: medicinal plants; natural products; sesquiterpenes; warburgia ugandensis sprague. introduction natural products extracted from medicinal plants are the major sources of modern drugs that have been used for treatment of human pathogens (sasidharan et al., 2011). the studies of these medicinal plants were the basis of earliest medicines. plant derived medicines constitute a substantial component of present day human healthcare systems (gómez et al., 2007, butle 2004). according to world health organization (who), more than 60% of the world’s population and over 80% of the people in low incoming/developing countries depend upon traditional medicine for their primary health care needs (sukirtha et al., 2012). in recent years, medicinal plants as natural products have attracted attention due to their important bioactive content such as terpens, alkaloids, anthraquinones, steroids, tannins, glycosides, saponins, volatile oils, resins, phenols and flavonoids that are deposited in their different parts (saiprasanna et al., 2012). the medicinal value of these plants also depends on bioactive phytochemical constituents that produce a definite physiologic action on the human body (mohammed, 2018). the genus warburgia is one of the flowering plants belongs to canellaceae family (rabe et al., 2000). the four species of warburgia namely, warburgia elongate verdc, warburgia salutaris (bertol. f.) chiov., warburgia stuhlmannii engl. and warburgia ugandensis sprague are distributed restrictly in the eastern and southern africa (maroyi, 2013; muchugi et al., 2008). warburgia ugandensis sprague is highly aromatic evergreen trees that has restricted distributions in the evergreen forests (rabe et al., 2000; drage et al., 2014) and widely used as traditional medicinal plant in many parts of east africa (kenya, uganda and ethiopia) (drage et al., 2014, rajab et al., 2000). the plant is known with different names: east african green wood, east african green heart, east african pepper bark tree, kenya green heart, green heart, pepper-bark tree (english) (maundu et al., 2005; wube et al., 2005), zogdom (wube et al., 2005) or kenaffa (amharic) (dagne et al., 2009) and befti (oromo language). the stem bark and roots have been used for the treatment of various diseases such as diarrhea, cough, common cold, general muscular pains, internal wounds, loss of appetite, malaria, syphilis, gonorrhea, stomachache, toothache, colds, throat and chest infections, fever, weak joints and general body pains (rabe et al., 2000, dharan et al., 2008). the compounds isolated from this plant also possess antifeedant, antibacterial, antifungal, anti-mycobacterial, cytotoxic (rabe et al., 2000, rajab et al., 2000) anti-plasmodial, anti-trypanosomal (wube et al., 2010) anti-asthmatic (karani et al., 2013) and antileishmanial activities (ngure et al., 2009). however, the leaves of plant has weaker property compared to the stem bark and roots (mbwambo et al., 2009). in ethiopia, particularly in bale zone, stem bark of this plant is traditionally used https://doi.org/10.14421/biomedich.2020.91.39-46 40 biology, medicine, & natural product chemistry 9 (1), 2020: 39-46 for treatment of a wide range of diseases, such as stomachache, toothache, malaria, tuberculosis, gonorrhea, asthma (wube et al., 2010, wube et al., 2005), cough and rabies (geyid et al., 2005). it is also used to remove tapeworm from human body. many drimane and coloratane sesquiterpenes was isolated from the stem barks of warburgia ugandensis sprague. some of these include waburganal (1), polygodial (2) (kioy et al., 1990; xu et al., 2009), mukaadial (3), warburgin (4) warburgiadione (5) (wube et al., 2005; kioy et al., 1990; xu et al., 2009), pereniporin b (6) (rajab et al., 2000), cinnamolide (7), cinnamolide-3β-acetate (8), ugandensolide (9) (wube et al., 2005; xu et al., 2009), dendocarbin a (10), ugandenial a (11), 9α,11α-dihydroxy, 6β-acetylcinnamolide (12), dendocarbin l (13), dendocarbin m (14), 9α-hydroxycinnamolide (15) (xu et al., 2009), muzigadial (16), muzigadiolide (17) (wube et al., 2005; kioy et al., 1990; rukutt et al., 2005), 4(13),7coloratadien-12,11-olide (18), 6α,9α-dihydroxy-4(13),7coloratadien-11,12-dial (19)and 7β-hydroxy-4(13),8coloratadien-11,12-olide (20), 7α-hydroxy-8-drimen11,12-olide (21) (wube et al., 2005), cinnamolide-3β-ol (22), deacetylugandensolide (23) (xu et al., 2009), 11α– hydroxymuzigadiolide (24) (rajab et al., 2000; wube et al., 2005), ugandensidial (25) (rajab et al., 2000; wube et al., 2005; kioy et al.,1990). 1 2 3 4 5 oh cho cho h h cho cho h oh cho cho h h oh o o o o o o h h o h oh o ho o o h h 6 7 8 9 10 o h o o h o h h h o ho o oh o h h ho o 11 12 13 14 15 h o ho o oh h o h o oh h o ho o o o oh o h o oh ho o h ho o oh 16 17 18 19 20 h o h o h h h h oh h h o h o oh cho cho h h cho cho ohh oh o o h h h oh 21 22 23 24 25 o ho o h h o h oh oh o o o h oh h h h o ho o oh h oh o cho cho oh figure 1. drimane and colorotane sesquiterpenes isolated from stem bark of warburgia ugandensis sprague. many of sesquiterpenes have been extracted and isolated from this plant species. despite of its medicinal uses, but still, extraction, isolation then characterization of active compounds from this medicinal plant is not sufficient. these, therefore, give emphasis on the choice of appropriate solvents and selection of the plant part (stem bark) to isolate the phytochemical constituents and characterize with very visible spectroscopic data. materials and methods experimental materials freshly stem bark of warburgia ugandensis sprague (canellaceae) was collected from barbere wareda, bale zone, south eastern part of ethiopia. the plant was then shade-dried at room temperature for one month. the dried stem barks were crushed to fine powder using an electrical grinder. chloroform, dichloromethane, etoh, petroleum ether, n-hexane, deuterated chloroform (cdcl3) were used. analytical tlc was performed using 20 x 20 cm, 0.20 mm thick silica gel 60 with fluorescent indicator uv254 to determine the number of components in a mixture and the purity of compounds. kieselgel 40*, particle size of 0.063-0.200 mm and 70-230 mesh astm silica gel was used for column chromatography. compounds on tlc were first detected by ultraviolet lamp, multiband 254/366 nm and then sprayed with 1% vanillin in sulfuric acid. heidolph rotary evaporator was used for removal of solvent under reduced pressure. melting points were determined by using stuart smp3 melting point apparatus. uv/vis spectra were recorded on genesy’s 2pc uv-vis scanning spectrometer (200-800 nm) in ch3cn. the 1d (1h at 400.13 and 13c at 100.6 mhz) were recorded on a bruker advance 400 spectrometer in cdcl3. the residual proton signal of the solvent is used as reference. ir spectra were recorded with kbr pellets on perkin elmer bx infrared spectrometer in the range 4004000 cm-1. optical rotations were measured on autopol iv automatic polarimeterat 20° in chcl3. experimental procedures the air-dried and finely powdered stem bark (100 g) was soaked and extracted successively with 600 ml of n-hexane, dichloromethane and chloroform for 48hours. each extract was filtered and the solvent was removed by rotary evaporator under reduced pressure. the crude dichloromethane extract (5.5 g) was subjected to column chromatography on a silica gel using n-hexane (100%) as eluent. out of 25 fractions collected, fractions 11 and 12 were combined, concentrated and further fractionated over a silica gel column using n-hexane/etoac 85:15 as eluent. fractions 6 and 7 which showed a single spot with same rf value were combined and concentrated to give 21 mg of white solid. the compound was coded as wu-1. the crude chloroform extract (1.93 g) was applied on a column silica gel using 100% hexane as mobile phase. fractions 11-15 were combined, concentrated and further purified on silica gel column using nhexane/etoac 70:30. fractions 10 and 11 showed a gonfa – isolation and characterization of sesquiterpenes from stem bark … 41 single spot with the same rf value and were combined and concentrated to afford 44mg of white solid. the compound was labeled as wu-2. results and discussions characterization of compound wu-1 wu-1 was isolated as a white crystalline solid with melting point of 131-134°c and rf value 0.34 using nhexane: ethyl acetate (3:2) as a solvent system. wu-1 was reacted with 2,4-dinitrophenylhydrazine (dnp) in ethanol and h2so4 gave yellow precipitate indicating the presence of carbonyl groups in the compound. the optical rotation ([α]d 20, chcl3) of wu-1 measured to be -197o indicating that the compound is optically active. the uv(ch3cn) spectrum (figure 2) showed maximum absorption (λmax) at 220nm indicating the presence of unsaturated group in the compound. figure 2. uv-visible spectrum of wu-1. the ir spectrum (figure 3) displayed strong absorption band at 3431 cm-1 due to vibrational stretching of o-hgroup. absorption band at 2926 cm1indicated the presence of c-h stretching of saturated group. ir spectrum demonstrated, in addition to strong absorption bands at 1744 and 1721 cm-1 due toester carbonyl and aldehyde, the presence of olefinic group at 1693 cm-1. figure 3. ir spectrum of wu-1. the 1h nmr spectrum (figure 4 and table 1) demonstrated a doublet at δ 9.78 and a singlet at δ 9.5 each integrating for one proton due to two aldehyde protons. the doublet at δ 7.03 integrating for one proton corresponded to anolefinic proton. the triplet at δ 5.92 which integrated for one proton is due to a proton on a methine carbon attached to an electronegative atom whereas the peak at δ 4.10 indicated the presence of oh group. the spectrum also showed a singlet at δ 2.16 due to an acetyl methyl group and three methyl groups at δ 1.35, 1.18 and 1.04 attached to quaternary carbon atoms. figure 4.1h nmr spectrum of wu-1. the 13c nmr spectrum (figure 5 of wu-1 showed well-resolved resonances of the 17 carbon atoms. the 13c nmr and the dept135 (figure 6 and table 2) spectra displayed four quaternary carbons, one ester carbonyl carbon (δ 170.09), three methine carbons, two aldehyde carbonyl carbons (δ 201.17 and 193.08), three methylene carbons and four methyl carbons. figure 5. 13c nmr spectrum of wu-1. 200 250 300 350 400 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 220 a bs or ba nc e wave length, nm 4000 3500 3000 2500 2000 1500 1000 500 0 60 62 64 66 68 70 72 74 76 78 80 3431 2926 1744 1371 1234 1043 802 605 1693 1721 % t ra n sm it an ce wave number cm -1 ppm (t1) 1.02.03.04.05.06.07.08.09.010.0 9 .7 8 1 9 .7 7 8 9 .5 0 0 7 .0 3 5 7 .0 2 3 5 .9 1 3 4 .1 0 9 4 .1 0 6 2 .1 6 0 2 .0 6 8 2 .0 5 6 1 .3 5 5 1 .1 8 1 1 .0 4 1 1 .0 7 1 .0 0 1 .0 3 1 .1 0 1 .0 7 2 .8 2 1 .0 4 3 .0 1 1 .1 6 3 .0 4 1 .9 3 2 .9 7 1 .2 3 1 .1 8 1 .1 4 ppm (t1) 7.02507.03007.0350 ppm (t1) 5.9005.9105.9205.930 ppm (t1) 4.10504.1100 ppm (t1) 2.05502.06002.0650 ppm (t1) 1.7501.8001.850 9 .7 8 1 9 .7 7 8 9 .5 0 0 7 .0 3 5 7 .0 2 3 5 .9 1 3 4 .1 0 9 4 .1 0 6 2 .1 6 0 2 .0 6 8 2 .0 5 6 1 .3 5 5 1 .1 8 1 1 .0 4 1 ppm (t1) 1.3901.4001.4101.4201.430 9 .7 8 1 9 .7 7 8 9 .5 0 0 7 .0 3 5 7 .0 2 3 5 .9 1 3 4 .1 0 9 4 .1 0 6 2 .1 6 0 2 .0 6 8 2 .0 5 6 1 .3 5 5 1 .1 8 1 1 .0 4 1 ppm (t1) 1.5501.6001.650 9 .7 8 1 9 .7 7 8 9 .5 0 0 7 .0 3 5 7 .0 2 3 5 .9 1 3 4 .1 0 9 4 .1 0 6 2 .1 6 0 2 .0 6 8 2 .0 5 6 1 .3 5 5 1 .1 8 1 1 .0 4 1 ppm (t1) 0102030405060708090100110120130140150160170180190200210 2 0 1 .1 6 6 1 9 3 .0 8 2 1 7 0 .0 9 2 1 4 8 .7 0 0 1 4 0 .9 1 7 7 7 .3 6 7 7 7 .0 4 9 7 6 .7 3 2 6 6 .0 5 0 4 4 .9 3 8 4 4 .0 0 0 4 1 .6 5 4 3 4 .0 1 9 3 2 .5 9 5 3 1 .8 1 4 2 4 .7 7 5 2 1 .4 9 8 1 9 .9 6 7 1 7 .6 8 3 42 biology, medicine, & natural product chemistry 9 (1), 2020: 39-46 figure 6. dept 135 spectrum of wu-1. table 1. proton decoupled 13c nmr and dept 135 (100.6 mhz, cdcl3) spectroscopic data of wu-1. carbons no. 13c nmr δ (ppm) dept 135 δ (ppm) remark c-1 31.81 31.81 ch2 c-2 17.68 17.68 ch2 c-3 44.00 44.00 ch2 c-4 34.02 c (quaternary carbon) c-5 44.94 44.94 ch c-6 66.05 66.05 ch c-7 148.70 148.70 ch c-8 140.92 c (quaternary carbon) c-9 c-10 41.65 c (quaternary carbon) c-11 201.17 201.17 ch c-12 193.08 193.08 ch c-13 32.60 32.60 ch3 c-14 24.77 24.77 ch3 c-15 19.97 19.97 ch3 ch3co 21.50 21.50 ch3 ch3co 170.09 c (quaternary carbon) the 1h and 13c nmr results obtained for wu-1 were comparable with 1h nmr (kioy et al., 1990) and 13c nmr (rukutt et al., 2005) spectral data of ugandesidial (25) (table 2). table 2. comparison of 1h (400.13, mhz, cdcl3) and 13c nmr (100.6 mhz, cdcl3) spectroscopic data of wu-1 and ugandensidial (25). position of carbons wu-1 ugandensidial δ 13cnmr 1hnmr δ (ppm) (multiplicity, integration) δ 13cnmr 1hnmrδ(ppm) (multiplicity, integration) c-1 31.81 1.83-1.76 (2h, m ) 32.02 c-2 17.68 1.65-1.55 (2h, m) 17.88 c-3 44.00 1.43-1.40 (1h, m) 1.33 (1h, m) 44.21 c-4 34.02 34.21 c-5 44.94 2.09(1h, d, j= 4.8hz) 45.16 2.04 (1h, d, j=4.7 hz c-6 66.05 5.92 (1h, t, j= 4.8hz) 66.26 5.89 (1h, t, j=4.7hz) c-7 148.70 7.03 (1h, d, j= 4.8hz ) 148.86 7.00 (1h, d , j= 4.7hz c-8 140.92 141.14 c-9 4.10 (1h, d, j= 1.2 hz) 4.10(1h,d, j=1.4hz) = 1.4 hz, 9-oh)= 1.4 hz, 9-oh) c-10 41.65 41.85 c-11 201.17 9.78 (1h, d, j= 1.2 hz) 201.32 9.76 (1h, d, j= 1.4 hz c-12 193.08 9.5(1h, s) 193.24 9.48(1h, s) c-13 32.60 1.04 (3h, s) 32.79 1.03 (3h, s) c-14 24.77 1.18 (3h, s) 24.96 1.17 (3h, s c-15 19.97 1.35(3h, s) 20.15 1.34 (3h, s) ch3co 21.50 2.16 (3h, s) 21.67 2.14 (3h, s) ch3co 170.09 170.27 in addition, the melting point of wu-1 (131-134°c) was in good agreement with the reported melting point of ugandensidial (138-140°c) (xu et al., 2009). based on the above data and in comparison with the literature (kioy et al., 1990), wu-1 (figure 7) is most probably ugandensidial. hp-67 ugandensidial (24) cho cho o ch3 oh o cho cho o ch3 oh o 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 h h16 figure 7. the structure of wu-1 and ugandensidial (24). ppm (t1) 0102030405060708090100110120130140150160170180190200 2 0 1 .1 6 6 1 9 3 .0 8 0 1 4 8 .7 0 0 6 6 .0 4 8 4 4 .9 3 5 4 3 .9 9 7 3 2 .5 9 4 3 1 .8 1 2 2 4 .7 7 4 2 1 .4 9 8 1 9 .9 6 7 1 7 .6 8 2 gonfa – isolation and characterization of sesquiterpenes from stem bark … 37 characterization of compound wu-2 wu-2 was obtained as a white crystalline solid (melting point 155–157oc) with rf value 0.41 using n-hexane: ethyl acetate (3:2) as solvent systems. it is optically active with optical rotation ([α]d 20, (chcl3) +16. an absorption maximum (λmax) at 221 nm in the uv (ch3cn) spectrum (figure 8) was characteristic of a molecule possessing an unsaturated lactone structure. figure 8. uv-visible spectrum of wu-2. the ir(kbr) spectrum (figure 9) displayed an absorption band at 3439 cm-1 due to the stretching vibration of o-h group whereas absorption bands at 2969 cm-1 and 2914 cm-1 demonstrated c-h stretching vibration of alkane. strong absorption bands at 1760 cm1 and 1680 cm-1 showed the presence of an α,βunsaturated carbonyl group while absorption band at 1731 cm-1 indicated the presence of ester carbonyl group. the strong absorption bands at 1246 cm-1 and 1197 cm-1 demonstrated c-o stretching. figure 9. ir spectrum of wu-2. the quartet at δ 6.9 observed in the 1h nmr spectrum (figure 10 and table 4) of wu-2 integrating for one proton corresponded to an olefinic proton attached to c-7. the tripletsat δ 4.41 and 4.05 integrated for one protoneach due to oxymethyleneprotons on c11. the doublet of doubletat δ 4.57 which integrated for one proton indicated oxymethine proton attached to c-3. the 1h-nmr spectrum also showed three methyl protons signals at δ 1.00(3h, s), 0.93 (3h, s) and 0.84 (3h, s) whereas the acetate methyl protons appeared at δ 2.08. figure 10.1h nmr spectrum of wu-2. the 13c nmr (figure 11) and dept135 experiments (figure 12 and table 3) displayed 17 carbon atom resonances comprising of two carbonyl carbons (δ 169.81 and 170.79), three quaternary carbons (δ 34.01, 37.63 and 127.16), four methine (δ49.28, 50.57, oxymethine δ80.13 and olefinic methine δ 135.82), four methylene (δ23.52, 24.65, 36.67 and oxygenated methylene δ 66.95) four methyl carbons (δ13.49,15.99, 21.23 and 27.80). figure 11.13c nmr spectrum of wu-2. 200 250 300 350 400 0.0 0.2 0.4 0.6 0.8 1.0 221 a b so rb a n c e wave length, nm 4000 3500 3000 2500 2000 1500 1000 500 0 10 20 30 40 50 60 70 80 3439 2914 1760 1376 1246 1009 743 624 1680 1731 % t ra n sm it an ce wave number cm -1 ppm (f1) 1.02.03.04.05.06.07.0 6 .9 0 3 6 .8 9 5 6 .8 8 7 6 .8 7 8 4 .5 7 3 4 .5 6 2 4 .5 4 5 4 .5 3 4 4 .4 2 9 4 .4 0 6 4 .3 8 3 4 .0 7 3 4 .0 5 0 4 .0 2 7 2 .8 2 2 2 .8 1 1 2 .4 2 3 2 .4 1 1 2 .0 8 1 1 .7 3 8 1 .7 2 8 1 .6 4 0 1 .4 6 7 1 .4 5 4 1 .0 0 0 0 .9 3 5 0 .8 4 0 1 .0 0 0 .9 8 1 .0 2 0 .9 9 3 .1 0 3 .0 2 3 .0 0 2 .0 4 2 .9 8 0 .9 8 0 .9 0 1 .5 1 1 .7 9 ppm (f1) 4.4004.4504.5004.550 ppm (f1) 4.050 ppm (f1) 2.8002.850 ppm (f1) 2.4002.450 ppm (f1) 1.4001.450 ppm (f1) 6.8806.8906.900 ppm (f1) 102030405060708090100110120130140150160170 1 7 0 .7 9 2 1 6 9 .8 0 7 1 3 5 .8 2 3 1 2 7 .1 6 4 8 0 .1 3 2 7 7 .3 7 9 7 7 .0 6 1 7 6 .7 4 3 6 6 .9 5 5 5 0 .5 6 5 4 9 .2 8 4 3 7 .6 3 4 3 6 .8 6 8 3 4 .0 1 4 2 7 .7 9 9 2 4 .6 4 8 2 3 .5 2 4 2 1 .2 3 3 1 5 .9 8 7 1 3 .4 9 4 38 biology, medicine, & natural product chemistry 9 (1), 2020: 39-46 figure 12. dept 135 spectrum of wu-2. table 3. proton decoupled 13c nmr and dept 135 (100.6 mhz, cdcl3) data of wu-2. carbons no. 13c nmr δ (ppm) dept 135 δ (ppm) remark c-1 36.67 36.67 ch2 c-2 23.52 23.52 ch2 c-3 80.13 80.13 ch c-4 37.63 c (quaternary carbon) c-5 49.28 49.28 ch c-6 24.65 24.65 ch2 c-7 135.82 135.82 ch c-8 127.16 c (quaternary carbon) c-9 50.57 50.57 ch c-10 34.01 c (quaternary carbon) c-11 66.95 66.95 ch2 c-12 169.81 c (quaternary carbon ) c-13 27.80 27.80 ch3 c-14 15.99 15.99 ch3 c-15 13.49 13.49 ch3 c-16 21.23 21.23 ch3 c-17 170.79 c (quaternary carbon ) the 1h nmr and 13c nmr data obtained for wu-2 are comparable with data obtained for cinnamolide-3β-acetate (8) (isolated and identified from the same plant) in the literature (wube et al., 2005). moreover, the melting point of wu-2 (155–157oc) and cinnamolide-3β-acetate (153–157°c) (kioy et al., 1990) are comparable. table 4. comparison of 1h(400.13, mhz, cdcl3) and 13c-nmr(100.6 mhz, cdcl3) spectral data of wu-2 with those of cinnamolide-3β-acetate (8). carbons no. observed nmr data of hp-81(ppm) cinnamolide-3β-acetate ( ppm) δ 13c nmr δ 1hnmr(ppm) (multiplicity,integration) δ 13c nmr δ 1hnmr(ppm) (multiplicity, integration) 1 36.67 1.42(1h, ddd, j= 13.6, 4hz) 1.65 (1h, td, j= 11.6, 5.2 hz) 36.9 1.42 (1h, ddd,j= 13.5, 4.0 hz, α) 1.64 (1h, dt, j= 13.5, 3.5hz, β) 2 23.52 1.73(2h, m) 23.5 1.68 (1h, ddd, j= 12.5, 1.5 hz, β) 1.74 (1h, ddd, j= 13, 4 hz, α) 3 80.13 4.57 (1h, dd, j= 11.2, 4.4hz) 80.2 4.55 (1h, dd, j= 11.5, 4.5 hz, α) 4 37.63 37.7 5 49.28 1.48 (1h, dd, j= 11.6, 5.2 hz) 49.4 1.48 (1h, dd, j= 10.5, 4.5 hz) 6 24.65 2.22(1h, td, j= 11.6, 4 hz) 2.45 (1h, qd, 20, 4, 8.8 hz) 24.7 2.22 (1h, ddq, j= 12.0,3.5, 1.5 hz, β) 2.44 (1h, dq, j= 20, 5, 4.0 hz, α) 7 135.82 6.9 (1h,q, j= 3.2 hz) 135.7 6.89 (1h, q, j= 3.5 hz) 8 127.16 127.2 9 50.57 2.82 (1h, m) 50.6 2.82 (1h,m) 10 34.01 34.1 11 66.95 4.05 (1h, t, j= 9.2 hz), 4.41 (1h, t, j= 9.2hz) 66.9 4.05 (1h, t, j= 9.0 hz, β), 4.41 (1h, t, j= 9.0 hz, α) 12 169.81 169.7 13 27.80 0.93 (3h, s) (3h, s, h-13) 27.6 0.94 (3h, s) (3h, s, h-13) 14 15.99 1.00 (3h, s) 15.9 1.00 (3h, s) 15 13.49 0.84 (3h, s ) 13.5 0.84 (3h, s ) 16 21.23 2.08 (3h, s) 21.2 2.07 (3h, s) 17 170.79 170.7 ppm (f1) 102030405060708090100110120130140 1 3 5 .8 2 8 8 0 .1 3 2 6 6 .9 5 9 5 0 .5 6 6 4 9 .2 8 4 3 6 .8 6 9 2 7 .8 0 0 2 4 .6 4 9 2 3 .5 2 6 2 1 .2 3 8 1 5 .9 8 8 1 3 .4 9 4 gonfa – isolation and characterization of sesquiterpenes from stem bark … 45 based on the above spectroscopic data and in comparison with literature data (wube et al., 2005), wu-2 is proposed to be (+) cinnamolide-3β-acetate (8). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 hp81 (+)cinnamolide-3-acetate (8) 16 o o o h3c o o o o h3c o h h h figure 13. the structure of wu-2 and of cinnamolide-3β-acetate (8). conclusion phytochemical investigation of the stem bark of warburgia ugandensis sprague (canellaceae) afforded two bioactive sesquiterpenes namely wu-1 (ugandensidial) and wu-2 (cinnamolide-3β-acetate) isolated from dichloromethane and chloroform extracts respectively. identification of these compounds was based o the melting point, array of spectroscopic data (uv/visible and nmr) and comparison of their spectroscopic data with reported literature values. the traditional medicinal use of this plant may be attributed to its high content of these bioactive constituents. acknowledgement: the author would to acknowledge addis ababa university for providing all the equipments and materials during this research work. also special thanks to haramaya university for their kind financial support. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references butler, m.s. 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(2009). ugandenial a, a new drimane-type sesquiterpenoid from warburgia ugandensis. molecules, 14(10) 3844-3850. doi: 10.3390/molecules14103844 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 299-308 | doi: 10.14421/biomedich.2025.141.299-308 issn 2540-9328 (online) molecular identification of fungal complex associated with stored maize grains vended in some local government areas of adamawa state, nigeria patricia peter sanum1, aishatu haruna1,*, abdulazeez mumsiri abaka2 1department of plant protection, modibbo adama university yola, nigeria. 2science laboratory technology department, school of science and technology, adamawa state polytechnic, yola, nigeria. corresponding author* aishaharun@mau.edu.ng abstract maize is a crucial global crop but remains highly vulnerable to fungal contamination, which poses serious threats to food safety and agricultural productivity. this study aimed to identify fungal species associated with maize samples from adamawa state, nigeria, using morphological and molecular techniques. morphological analysis facilitated genus-level identification, while rdna its sequencing provided precise species-level classification. the identified fungal species included lichtheimia ramosa, aspergillus latus, aspergillus flavus, amesia atrobrunnea, and cladosporium cladosporioides. among them, aspergillus flavus was the most prevalent (48.1%), followed by a. latus (22.1%), l. ramosa (14.3%), c. cladosporioides (12.9%), and a. atrobrunnea (2.6%). yola north recorded the highest level of fungal contamination. growth rate analysis showed that l. ramosa exhibited the fastest growth, while a. atrobrunnea had the slowest. molecular identification confirmed the fungal species, with its sequences displaying 81% to 95% similarity to reference strains. phylogenetic analysis further clarified the evolutionary relationships among the isolates. the dominance of aspergillus species, particularly a. flavus, raises concerns due to their mycotoxin-producing capabilities, which pose health risks and compromise food safety. while these findings align with previous reports on fungal contamination in stored grains, they differ from studies highlighting fusarium and penicillium as dominant contaminants in other regions. this study emphasizes the need for accurate fungal identification, improved storage techniques, and advanced molecular tools to mitigate contamination. these insights are essential for enhancing food security, promoting agricultural sustainability, and safeguarding public health in maize-reliant regions such as nigeria. keywords: maize grain; fungal contamination; aspergillus flavus; morphological and molecular identification. introduction nigeria is the second-largest maize producer in africa, with an annual output exceeding 12 million metric tons (mt), second only to south africa. maize is a staple crop in the country, with approximately 80% of production consumed directly or used as animal feed, while the remaining 20% supports industrial applications (wossen et al., 2023). as a vital component of food security in sub-saharan africa, maize is commonly consumed alongside legumes (benjamin et al., 2024). the top maize-producing states, contributing around 64% of national output, include borno, niger, plateau, katsina, gombe, bauchi, kogi, kaduna, oyo, and taraba. in 2017, the international institute of tropical agriculture (iita) valued nigeria’s maize industry at $6 billion (approximately n2.5 trillion). maize (zea mays l.), an annual cereal from the poaceae family, plays a crucial role in global food security (ahmad et al., 2024). the term "maize" originates from ancient greek and taino languages, with "zea" signifying "sustaining life" and "mays" meaning "life-giver" (saleh et al., 2019). ranking third in global importance after rice and wheat, maize thrives in temperate, tropical, and subtropical climates (benjamin et al., 2024). however, significant losses occur at pre and post-harvest stages, necessitating global efforts to mitigate these challenges. in nigeria, maize is the most widely consumed staple, occupying over 27% of cereal farmland across multiple african nations, including kenya, malawi, and zimbabwe (akanmu et al., 2023). post-harvest storage is critical, as maize is hygroscopic and prone to moisture absorption, leading to deterioration (baidhe et al., 2024). even well-dried kernels can reabsorb moisture, increasing spoilage risks (jimoh et al., 2023). proper storage techniques are essential to protect maize from adverse weather, microbial contamination, and insect damage (okparavero et al., 2024). fungal growth, particularly under hot and humid conditions, further threatens grain quality, underscoring the importance of effective storage practices (dadlani et al., 2023). manuscript received: 17 march, 2025. revision accepted: 20 may, 2025. published: 28 june, 2025. https://doi.org/10.14421/biomedich.2025.141.299-308 300 biology, medicine, & natural product chemistry 14 (1), 2025: 299-308 plant diseases significantly impact agricultural productivity and economic efficiency (ekwomadu & mwanza, 2023). food safety concerns arise from microbial contamination and toxin production, notably mycotoxins, which cause mycotoxicosis in humans and animals (kolawole et al., 2024). fungal genera such as alternaria, aspergillus, fusarium, and penicillium are major mycotoxin producers, with some species affecting crops in the field and others during storage (fashola, 2023). environmental factors and fungal species specificity influence mycotoxin production, highlighting the need for effective harvesting, processing, and early detection strategies (hamad et al., 2023). accurate identification of mycotoxigenic fungi is crucial, as closely related species can produce different toxin profiles (dey et al., 2023). molecular diagnostic tools offer faster and more reliable detection than conventional methods, which are often labor-intensive, costly, and less specific (fang et al., 2024). by integrating molecular techniques, researchers can enhance fungal detection, improve food safety, and promote sustainable agricultural practices (alameri et al., 2023). materials and methods source of maize grain and site selection maize grain samples were collected from four local government areas in adamawa state: ganye, gombi, yola north, and fufore. from each market in these areas, three replicates of maize grains were gathered. the samples were carefully placed into sterile bags and transported to the laboratory for analysis. the study's primary goal was to isolate and identify fungal species associated with the maize grains. the experimental design followed a completely randomized design (crd), with treatments applied across the four local government areas. each treatment was replicated three times, resulting in 12 experimental units for the study. this approach ensured a systematic and unbiased evaluation of fungal presence in the maize samples. media preparation and plating potato dextrose agar (pda) supplemented with 0.25 g of chloramphenicol was used in the experiment, following the method outlined by tanveer et al. (2011). the addition of chloramphenicol, an antibiotic, was intended to inhibit bacterial growth in the culture medium. from each sample, four to five maize grains were placed onto the pda plates and incubated for 3 to 5 days. sterilized forceps, dipped in 100% ethanol and flamed before use, were employed to transfer the grains into petri dishes containing the pda medium. the study was conducted using a completely randomized design (crd) and was replicated three times to ensure the reliability and accuracy of the results. morphological identification of fungi isolated from maize grain morphological characteristics are essential for identifying fungal cultures, encompassing features such as spore shape and size, the presence or absence of microand macroconidia, chlamydospore formation, the arrangement of spores on conidiophores, septation of conidia, and the color of both mycelium and spores. the presence or absence of aerial false heads is also a key consideration. for the morphological identification of fungi, reference materials, such as those provided by ikechi-nwogu et al. (2023), were consulted. for molecular identification, pure cultures of each fungal species were obtained by sub-culturing spores onto potato dextrose agar (pda) using the single spore isolation technique. these cultures were incubated at 25 °c under blue/black light with a 16/8-hour photoperiod for 7 days, after which dna extraction was performed. to preserve the isolated fungal species for future research, they were stored in a 15% glycerol solution at 80 °c. this combined approach ensures precise identification and long-term preservation of fungal cultures, facilitating further research and analysis. molecular identification of fungal isolates extraction of genomic dna to extract genomic dna from fungal mycelia, representative fungal isolates were first cultured on potato dextrose agar (pda) for 7 days. approximately 100 grams of fresh mycelia were collected and ground using a sterile pestle. the genomic dna extraction was performed using the quick dnatm fungal and bacterial miniprep kit. the procedure began with lysing the fungal cells using a bead beater, followed by centrifugation to separate the supernatant. the supernatant was filtered and combined with genomic lysis buffer before passing through a zymo-spin™ column. after a series of washing steps, the dna was eluted using dna elution buffer, yielding high-purity dna suitable for subsequent applications. this method ensures efficient and reliable extraction of fungal genomic dna for further analysis. quantification of extracted dna quality the quality and quantity of the extracted genomic dna were assessed using a nanodrop nd-1000 spectrophotometer. dna purity was evaluated by measuring the absorbance ratios at 260/280 nm. to determine the dna concentration, the samples were analyzed on a 0.80% agarose gel, which was run at 75 v/cm for 40 minutes. a 1 kb dna ladder (promega) was used as a reference to estimate the size of the genomic dna. the gel was stained with midori green advanced dna stain (ng japan) and visualized under a haruna et al. – molecular identification of fungal complex associated … 301 transilluminator (bio-rad), with images captured for documentation. additionally, gel electrophoresis was performed using a 1000 bp ladder at 70 v/cm for 45 minutes to confirm further the dna size and integrity (ismail, 2017; simbolo, 2013). this comprehensive approach ensured an accurate assessment of dna quality and concentration for downstream applications. polymerase chain reaction (pcr) amplification of the partial ribosomal dna (rdna) region was performed using the primer pair its-5 forward (5'-ggaagtaaaagtcgtaacaagg-3') and its-2 reverse (5'-gctgcgttcttcatcgatgc-3'), as outlined by white et al. (1990). the total reaction volume of 25 µl consisted of 12.5 µl of pcr master mix, 0.5 µl of each primer, 9.5 µl of sterile distilled water, and 2 µl of dna template specific to each fungal isolate. the pcr amplification was carried out in a thermal cycler with the following program: initial denaturation at 94 °c for 2 minutes, followed by 30 cycles of denaturation at 94 °c for 30 seconds, annealing at 58 °c for 1 minute, and extension at 72 °c for 2 minutes. a final extension step at 72 °c for 5 minutes concluded the process (ismail, 2017). this protocol ensured efficient amplification of the target rdna region for subsequent analysis. agarose gel electrophoresis 5 µl of each pcr product was loaded onto a 1% (w/v) agarose gel and separated at 100 v using 1x tae buffer for gel electrophoresis. the agarose powder (cslag100le multi-purpose agarose, cleaver scientific) was mixed with tae buffer and heated in a microwave until completely dissolved. the solution was then cooled to 55°c, after which three drops of dna stain were added. the mixture was poured into a gel cast containing a comb and allowed to solidify. once the gel was set, the pcr products were loaded, and electrophoresis was performed. a 1 kb dna ladder was used as a size reference for the pcr products. after separation, the gel was visualized and photographed using a uv gel documentation system. digital images were processed using quantity one software provided with the transilluminator and saved in jpeg format for further analysis, following the methods described by nirmaladevi et al. (2016). this procedure ensured accurate visualization and documentation of the amplified dna fragments. dna sequencing fungal species identification was performed automatically sequencing its pcr products using a dna analyzer at inqaba biotec west africa, ibadan, nigeria. the resulting nucleotide sequences were assembled and edited using bioedit software (isuosuo et al., 1999). identification was carried out using the national center for biotechnology information (ncbi) blast tool, which compared the obtained sequences with those in the ncbi database to identify regions of local similarity (madden, 2013). data analysis following a completely randomized design (crd), all numerical data were analyzed using anova (version 9.4). mean differences were assessed using the least significant difference (lsd) test at a 5% significance level. results table 1. fungal counts of isolates associated with stored maize grains vended in yola north, ganye, fufore, and gombi local government areas of adamawa state. organisms lg location a. l a. f l. r a. a c. c yola n 1 + + + + 2 + + + + 3 + + + + gan 1 + + + + 2 + + + 3 + fu 1 + + + + + 2 + + + + 3 + + + gom 1 + 2 + + 3 + + + + keys: yn = yola north gan = ganye fu = fufore gom = gombi a. l = aspergillus latus a. f = aspergillus flavus l. r = lichtheimia ramosa a. a = amesia atrobrunnea c. c = cladosporium cladosporioides + = present = absent 302 biology, medicine, & natural product chemistry 14 (1), 2025: 299-308 table 2. frequency of occurrence and percentage frequency of occurrence of different fungal isolates identified in samples obtained from ganye, yola north, gombi, and fufore in adamawa state. isolated fungi local government areas total % frequency yola/north ganye fufore gombi aspergillus latus 7 1 6 3 17 22.1 aspergillus flavus 12 8 9 8 37 48.1 lichtheimia ramosa 5 1 4 1 11 14.3 amesia atrobrunnea np 1 1 np 2 2.6 cladosporium cladosporioides 3 2 4 1 10 12.9 total 27 13 24 13 77 100 np = not present table 3. mean fungal growth rate (mm) of isolates from maize grains. isolate isolates’ growth rate (mm) day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 a. flavus 26.33b 45.00b 54.00b 70.67b 76.33c 83.33b 90.00a 90a l. ramosa 33.67a 69.33a 76.67a 90.00a 90.00a 90.00a 90.00a 90a a. latus 27.67b 46.67b 55.33b 72.67b 84.00b 90.00a 90.00a 90a a. atrobrunnea 15.00c 23.33c 34.67c 42.67c 50.67d 64.00c 76.67b 90a c. cladosporioides 31.33a 45.33b 54.33b 75.33a 90.00b 90.00a 90.00a 90a mean 26.80 45.93 55.00 70.268 78.20 83.47 87.20 90.00 se (+) (-) 1.797 2.903 2.623 1.634 1.743 2.715 1.418 0.000 cv (%) 8.582 7.620 8.723 6.020 6.164 3.498 6.296 0.00 significance level * * * * * ** * ns key: * = significant difference, ** = highly significant, coefficient of variation (cv), standard error (se), level of significance (using anova) and least significant difference (lsd), ns= not significant. p<0.05. means carrying the same letter (s) in the same column are significantly equal at the 5 % significance level (p = 0.05 %). table 4. plates of the morphological characterization of fungal isolates obtained from maize grains vended in yola markets. s/n name of isolates front side reverse side conidia image 1 a. flavus 2 c. cladosporioides 3 a. atrobrunnea haruna et al. – molecular identification of fungal complex associated … 303 table 4. cont. s/n name of isolates front side reverse side conidia image 4 a. latus 5 l. ramosa table 5. morphological description of the fungal isolates. isolates colony description conidia shape/size aspergillus flavus the mycelia color is white, followed by olive-green conidia formation that dominated the colony color appearance, the reverse is cream. it grows moderately on pda and covers the 90 mm petri dish in 7 days. colonies are flat at the borders while raised in the middle. the size ranged between 250 µm and 450 µm in diameter with thin walls and rough texture. cladosporium cladosporioides on pda medium, colonies are olive-grey to dull green, velvety, and tufted, the colony edge is olive-grey and feathery and grows moderately on pda covering 90 mm dish in 5-7 days. mycelia forms mats and on the colony surface, it grows upward. single-celled, oval-shaped, and smooth-walled. conidia are numerous, forming terminal branches with up to ten conidia per branch. cladosporium cladosporioides is smaller in size ranging from 4-8 μm long, 2-4 μm wide. amesia atrobrunnea the colony is black to brown color and has flexuous and septate hairs. it grows slowly on pda and matures within 7-8 days to cover the 90 mm petri dish. the ascospores are fusiform and some are elongate, they turn dark brown at maturity. the size is 7.5 10x 4 -5.5 µm. aspergillus latus the colonies were moderately deep, had white mycelia, no soluble pigment, light brown sporulation, the colony surface had woolly hairs (floccose) and grew to maturity within 6-7 days covering 90 mm petri dish. conidia were observed to be globose (spherical) to sub-globose (almost spherical) and smooth. the size is 3 μm – 4 μm. lichtheimia ramosa cultures are fast growing, pale white, turning grey with age, and they display a greater growth rate and mature within 4 days covering the 90 mm petri dish. the sporangiospores are hyaline to light black, round to oval in shape. it is 3μm 4.5μm in diameter. polymerase chain reaction (pcr) the five representatives of fungal isolates from maize grains sold in the selected local governments were morphologically studied and identified, then further subjected to molecular identification to confirm their identities. the pcr amplification of the its genes was defined clearly with all five (5) isolates with the expected size of about 600 bp with its primers shown. dna sequence of fungal isolates its nucleotide sequences of the identified fungi isolated from maize grains vented in some selected markets in yola, adamawa state >lichtheimia ramosa tcggaaagraaaaaaystggmwtcgatgaaaa cgcatcatcsacaggactccccacagaa aaccctcctatgtttgratayccggaackttgaa tctcgtctkcgtgcttgaacccagma ttggccacctaaaaactttccttacsatcgtcta acaaacaatattgwkatgggaaaaac ttttaaaaggagtgtcctgtgacccataacccra atcacaamaaagggggamccccttkg ggggccccactttscatayccctggggtaaattt tmrgggggaaaacaccccaawctarg gwttttttaaacttctcwwaaaaaacwcctcc graggtycacctacggaacccttkgtma caaataaaaactcccaaaatcg 304 biology, medicine, & natural product chemistry 14 (1), 2025: 299-308 >aspergillus latus aaacwggggmggggacatgacagaagggggg gwtgcctkwaaacgccaacctcctaamaatga tawacaaccagaagcrtcggtgacggccgcga cccccaacccgccggagaccactgk acttcatggcggagagggatgcmgcctmascct gaatacmaatcagaaaaaactttcaac ratggatctctcggttcccgtwtwaataaasaw ccctccatkaa >aspergillus flavus cawtgygtkaaaggttattaccgattgtaaggg atctastcagccctacctcccacccaw grwtwctgccccgtaattggttcggsggggccc ccattsttggmcccccccgggggtctt taatccggggcccsscccaarcraaaaaamcaa caaaaatksyggagggaaggtaggktt msttagatyccctcaattcgktaaaaatctttcc cakggttcccttkggraaccktgtta aaaaatttaacttcacacga >amesia atrobrunnea cagttaattgaattaatttggmtaaaataaata aacaatggtttataggaaatataaktr gggaatcaccgggcggggtcctgggaaggtccg aggggggccaaacacccgccmgcgaaa ccamkgtttaagggaaacttaaccattgtttaa ttatttttactctmtaaaataatccct ccactgstccaccaktggaraccttrttaatttt tttttttacaa >cladosporium cladosporioides catcgatgaagaacgcagcatcgatgaagaacg cagcatcgatgaagaacgcagcatcgatgaaga acgcagcatcgatgaagaacgcagcatcgatga agaacgc figure 1. (plate 5) gel electrophoresis image of the pcr products of dnas of the representative isolates on 1 % agarose, showing bars of about 400 bp on 1 kb dna molecular weight ladder, (a) lichtheimia ramosa, (b) aspergillus latus, (c) aspergillus flavus, (d) amesia atrobrunnea, and (e) cladosporium cladosporioides. showing amplified region of its. figure 2. (plate 6) internal transcribed spacer (its) phylogeny of fungal isolates obtained from maize grains vented in yola markets, using a maximum livelihood analysis. bootstrap values were represented at each node with epidermophyton floccosum as an outer layer. table 6. identification of representative fungal isolates from the maize grains based on its sequence. s/n identified species % similarity with genbank strains genbank reference strain number 1 lichtheimia ramosa 85.32 lk023322.1 2 aspergillus latus 86.32 or501409.1 3 aspergillus flavus 95 mz357882.1 4 amesia atrobrunnea 81.24 mh864203.1 5 cladosporium cladosporioides 87.83 jf796748.1 discussion maize is a vital crop globally and provides numerous substrates that support fungal growth (burlakoti et al., 2024). this study employed both morphological and molecular techniques to identify five fungal species from the maize samples using rdna its sequence analysis. morphological methods are helpful for identification up to the genus level, but molecular analysis is needed for species-level identification. haruna et al. – molecular identification of fungal complex associated … 305 five fungal species were isolated and identified at the species level through rdna its sequence analysis from maize grain samples collected from four major markets in adamawa state: ganye, gombi, yola north, and fufore. the study found that most maize samples from these markets were contaminated with varying levels of fungal growth, while a few showed no fungal presence. the identified fungal species included lichtheimia ramosa, aspergillus latus, aspergillus flavus, amesia atrobrunnea, and cladosporium cladosporioides, all exhibiting morphological traits consistent with previous studies (bensch et al., 2012). among these species, aspergillus flavus is particularly notable due to its ability to produce mycotoxins, which pose significant health risks to humans, animals, and plants, as well as contribute to food spoilage (awuchi et al., 2021). these fungi can contaminate grains both preharvest and postharvest, especially under inadequate drying and storage conditions, leading to increased aflatoxin levels (gachara et al., 2024). aspergillus and cladosporium, both common in stored grains, were the predominant genera in this study (mato et al., 2024). the study further quantified fungal prevalence across the collected maize samples. a total of 36 fungal isolates were identified, with aspergillus flavus being the most prevalent (48.1%), followed by aspergillus latus (22.1%), lichtheimia ramosa (14.3%), cladosporium cladosporioides (12.9%), and amesia atrobrunnea (2.6%). amesia atrobrunnea was the least common species detected. yola north had the highest fungal contamination among the sampled markets, with 27 occurrences recorded. the growth rates of the five fungal species isolated from maize samples were recorded daily until each species fully covered the petri dish. lines were drawn across the center of each plate to assess growth, and the mycelial mat diameter was measured along these lines. among the isolates, lichtheimia ramosa exhibited the fastest growth, covering the 90 mm petri dish within four days. cladosporium cladosporioides displayed moderate growth, reaching full coverage in five days. aspergillus latus and aspergillus flavus followed similar growth patterns, maturing in six and seven days, respectively, while amesia atrobrunnea exhibited the slowest growth, taking eight days to develop fully. the colonization of stored grains by fungi is influenced not only by their growth rates but also by factors such as moisture content, temperature, storage duration, and initial fungal contamination. some maize samples exhibited no fungal growth, likely due to storage under safe moisture conditions, which inhibited fungal proliferation. a summary of the growth rates for all species is presented in table 3. recent studies have confirmed the rapid growth of lichtheimia ramosa. when cultured on potato dextrose agar (pda), it forms grey-white colonies with numerous hyphae, reaching an average diameter of 6.9 cm within four days (imade et al., 2020). its rapid expansion enables it to cover entire plates within one to seven days. the genomic sequences of lichtheimia ramosa and its closely related species, lichtheimia corymbifera, have been published (shen et al., 2023). regarding aspergillus latus, which was identified in maize grains in this study, taxonomic literature suggests that aspergillus sublatus and a. latus are closely related, with a. sublatus holding taxonomic priority (chen et al., 2016). a. sublatus has also been recognized as a significant causative agent of aspergillosis (chrenkova et al., 2018). amesia atrobrunnea, another species identified in this study, is characterized by dark brown to black ascomata, flexuous septate hairs, and a maturation period of seven to eight days. previously classified as chaetomium atrobrunneum, it was later reassigned to the amesia genus based on phylogenetic analysis (wang et al., 2016). isolates of cladosporium cladosporioides have been associated with systemic infections in humans. when cultured on pda, it forms olive-grey to dull-green colonies with a velvety texture and feathery edges. this species primarily spreads across the medium rather than growing vertically and occasionally produces characteristic hyphal exudates (bensch et al., 2012). as an asexual fungus, c. cladosporioides reproduces by producing vegetative spores, or conidia. as for aspergillus flavus, the colonies appeared in shades of olive-green, yellowish-green, or dark green, often surrounded by a white ring covered with conidia over time. the colonies were typically velvety in texture, sometimes woolly, and often produced exudates. additionally, most isolates of a. flavus produced sclerotia, in line with descriptions by gautam and bhadauria, (2019). all numerical data in the study, based on a completely randomized design (crd), were analyzed using anova version 9.4, with significant means separated by lsd at a 5% significance level. the growth rates of fungi showed significant differences on days 1 through 5, with day 6 showing highly significant differences. on day 8, there was no significant difference (p = 0.05) since all plates were fully covered by fungal growth. dna extraction and molecular identification were performed for all fungal isolates. genomic dna from each fungus, which had been cultured for 7 days on pda, was extracted using the quick-dnatm fungal/bacterial miniprep kit following the manufacturer’s protocol. dna quality and quantity were assessed using a nanodrop nd-1000 spectrophotometer, and dna integrity was verified by electrophoresis on a 0.80% agarose gel, run at 75 v/cm for 40 minutes. plate 6 illustrates the dna bands for the fungi. molecular identification of the five fungal isolates was conducted using dna barcoding, focusing on sequencing the its region. the its rdna sequences 306 biology, medicine, & natural product chemistry 14 (1), 2025: 299-308 were amplified with primers its5 and its2, and the sequences were compared to those in the ncbi-blast database. the results showed varying fungal biodiversity across different locations. aspergillus flavus was the most prevalent, present in all sampled locations, while amesia atrobrunnea was only found in two locations. the sequencing of the five amplified samples was carried out by inqaba biotec west africa in ibadan, nigeria. sequence analysis revealed significant similarities with known fungal strains in the genbank database. cladosporium cladosporioides showed 87.83% similarity (genbank reference jf796748.1), aspergillus latus had 86.32% similarity (or501409.1), and aspergillus flavus demonstrated a 95% match (mz357882.1). lichtheimia ramosa exhibited 85.32% similarity (lk023322.1), and amesia atrobrunnea had 81.24% similarity (mh864203.1). these findings are summarized in table 6. the phylogenetic tree presented in plate 7 illustrates the evolutionary relationships among the fungi isolated in the study. the its sequences of the five fungal species were compared with reference sequences from the ncbi nucleotide database. these sequences were aligned using mafft (multiple alignment for sequence) version 6.0, and the phylogenetic tree was generated through maximum likelihood (ml) analysis using the hasegawakishino-yano (hky) model in mega 7 software. the optimal phylogenetic tree was identified by selecting the one with the highest likelihood score, using 1000 bootstrap replicates to assess the support for each clade. the its sequences obtained in this study had a similarity range of 81% to 90% with reference sequences from previous studies. in addition to the sequences generated in the research, other reference sequences were retrieved from genbank and aligned with mafft. the alignment was further refined manually using bioedit software. phylogenetic congruency was tested using the 70% reciprocal bootstrap criterion, confirming the relationships among the five fungi, which belong to four genera. the bootstrap values supported the major groups and branching clusters in the tree. epidermophyton floccosum was included as an outgroup to root the tree and clarify the relationships. these results align with previous findings that aspergillus flavus is the most commonly isolated fungal species from contaminated grains (katati et al., 2024). aspergillus, particularly from the flavi group, is a major contaminant of maize during storage, as observed in this study, where a. flavus was the most frequently isolated fungus. this agrees with reports that aspergillus species are highly prevalent across various environments such as soil, water, air, and food (nji et al., 2023), and matches the findings from previous studies in nigeria where aspergillus aflatoxiformans was predominant in grains like maize and rice (ezekiel et al., 2021). the dominance of aspergillus species in the current study is of concern due to their association with invasive aspergillosis, a disease affecting humans, with species like a. flavus, a. terreus, a. niger, a. ustus, and a. versicolor being implicated (abdel-azeem et al., 2019). accurate and timely identification of these fungi is essential for managing their impact and conducting proper surveillance. the global distribution of aspergillus species, capable of growing in various climates, underscores their importance in contamination and human health. this study aligns with the findings of chilaka et al. (2012), who identified aspergillus and cladosporium as dominant fungal genera in stored grains, reinforcing the results observed here. likewise, wu et al. (2012) recognized aspergillus as the most frequently occurring fungal species (olugbenga & chongs, 2024). however, these findings contradict those of joshi et al. (2022), who reported fusarium as the most prevalent genus. notably, no penicillium species or other typical storage fungi were detected in this study, differing from previous research that frequently cites penicillium as a common contaminant of maize grains. conclusion this study identified five fungal species from four genera associated with maize grains obtained from markets in gombi, ganye, yola north, and fufore local government areas of adamawa state. the species detected included aspergillus flavus, aspergillus latus, amesia atrobrunnea, lichtheimia ramosa, and cladosporium cladosporioides. the prevalence of these fungi varied, with occurrence rates ranging from 2.6% to 48.1%. the aspergillus genus was the most dominant, representing 70.2% of the total fungal isolates. acknowledgments: special gratitude goes to the department of plant protection, modibbo adama university yola. author contributions: conception and design of the study: aisha haruna; sample collection: patricia peter sanum; analysis and interpretation of data: aisha haruna, patricia peter sanum; statistical analysis: aisha haruna; visualization: patricia peter sanum, aisha haruna; writing manuscript: aisha haruna and abdulazeez mumsiri abaka conflict of interest: the authors have no conflicts of interest to declare. financial support: no financial support funding: no funding. haruna et al. – molecular identification of fungal complex associated … 307 references aasa, a. o., fru, f. f., adelusi, o. a., oyeyinka, s. a., & njobeh, p. b. 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(2012). metamaterial-based integrated plasmonic absorber/emitter for solar thermo-photovoltaic systems. journal of optics, 14(2), 024005. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 199-204 | doi: 10.14421/biomedich.2025.141.199-204 issn 2540-9328 (online) prediction of artocarpus altilis potential as an anti breast cancer by inhibiting egfr: a molecular docking study muhammad farid*, ardestya rastrani, adinda aulia rahma, widya aryana ramadhania department of medicine, faculty of medicine, ahmad dahlan university, yogyakarta, indonesia jl. universitas ahmad dahlan kampus 4, kragilan, tamanan, kec. banguntapan, kabupaten bantul, daerah istimewa yogyakarta 55191, indonesia. corresponding author* muhammad2100034023@webmail.uad.ac.id manuscript received: 18 january, 2025. revision accepted: 15 may, 2025. published: 13 june, 2025. abstract epidermal growth factor receptor (egfr) is an important target in breast cancer therapy, given its role in tumor cell proliferation, motility, and invasion through critical signaling pathways such as ras-mapk and pi3k/akt. this study explored the potential of artocarpus altilis (aa) active compounds, which are rich in flavonoids, as an alternative to chemotherapy. the molecular docking method was used to predict the interaction between aa compounds and egfr protein (pdb id: 2j6m). docking validation showed an rmsd value of 0.854 å, indicating high accuracy. ellagic acid showed the best binding affinity (-8.4 kcal/mol), followed by quercetin (7.3 kcal/mol), catechin, and epicatechin (-7.2 kcal/mol). residue analysis revealed that met793 plays a key role in the stability of the interaction. interaction visualization and admet prediction showed that most of the compounds fulfilled lipinski's rule of five, with no risk of hepatotoxicity or mutagenesis. potential compounds such as quercetin and epicatechin showed comparable performance to doxorubicin, but with lower potential side effects. these results strengthen the role of natural compounds as candidates for egfr-targeted cancer therapy, providing a basis for the development of safer and more effective anticancer drugs. keywords: breast cancer; egfr; artocarpus altilis; molecular docking. introduction breast cancer is a global problem to this day, data shows that this disease was experienced by 11.7% of the world's population in 2020. (sung et al., 2021)the development of breast cancer through several pathways, one of which is the epidermal growth factor receptor (egfr) which is stimulated by epidermal growth factor (wilson et al., 2009). treatment for breast cancer involves multidisciplinary science including surgery, radiotherapy, neoadjuvant and adjuvant (di nardo et al., 2022). treatment with chemotherapy is the main choice that is still often used today. however, the long-term side effects that arise can include fatigue, insomnia, peripheral neuropathy, cognitive impairment, cardiotoxicity to triggering the emergence of other malignancies (goedendorp et al., 2012; zamorano et al., 2016). breadfruit (arthocarpus altilis) belongs to the moraceae family which is widely used by the community as a traditional medicine (ganeson et al., 2018). the largest content of aa is flavonoids, flavonoids have strong anticancer properties that function as antioxidants in normal cells and as pro-oxidants in cancer cells that trigger oxidative stress and induce apoptosis, mainly due to the content of artocarpin (jantapaso & mittraparparthorn, 2022). flavonoids show anti-metastatic effects by inhibiting migration, invasion of cancer cells, and angiogenesis through modulation of important signaling pathways such as nf-κb, ap-1, p53, and pi3k/akt (aswathy et al., 2024). flavonoids in aa can affect epigenetic mechanisms, such as dna methylation and histone modifications, making them important candidates for cancer prevention and therapy (sitarek et al., 2024). molecular docking is a method to predict how ligands interact with receptor proteins, as well as assess their binding affinity and determine the optimal conformation and orientation of the ligand when interacting with the target protein (meng et al., 2011). molecular docking works at the atomic level to understand the interaction process of small molecules at the target protein binding site. (agu et al., 2023). the docking process often uses molecular mechanics to quickly evaluate ligand conformation, match the 3d shape of the ligand with the target, or use hybrid methods to obtain more accurate predictions (soleymani et al., 2022). this study aims to predict the potential of aa as an anti-breast cancer through the egfr receptor through molecular docking studies. https://doi.org/10.14421/biomedich.2025.141.199-204 mailto:muhammad2100034023@webmail.uad.ac.id 200 biology, medicine, & natural product chemistry 14 (1), 2025: 199-204 materials and methods preparation of test receptors and ligands this study used the egfr protein with pdb id: 2j6m obtained through https://www.rcsb.org/. this protein has been used previously in studies that have the potential for interaction with breast cancer (acharya et al., 2019). the protein repair process is carried out to separate the protein chain from other molecules such as natural ligands, water, and cofactors (syahputra et al., 2022). this process uses pymol software to find a more specific sequence. the active compound artocarpus altilis became the test ligand in this study, the structure of each compound was obtained through https://pubchem.ncbi.nlm.nih.gov/. the active compounds used are: caffeic acid (689043), chlorogenic acid (1794427), coumaric acid (637542), ferulic acid (445858), hyperoside (5281643), quercetin (5280343), ellagic acid (5281855), gallic acid (370), catechin (9064), epicatechin (72276), limonene (22311), γ-terpinene (481107192), terpinolene (11463), and doxorubin (31703) as standard drugs. molecular docking simulation the docking method must be validated using several methods, one of which is the redocking process (dallakyan & olson, 2015). this redocking process has parameters that will show the ligand deviation that occurs during the docking process. the validity parameter of the method is the root mean square deviation (rmsd) with a maximum value of less than 2 å, the lower the value, the better. this process is needed to determine the location and size of the grid box (x, y, z) of the native ligand, as a reference in specific docking (dai et al., 2018). the redocking process uses the vina tool as a docking process runner in the pyrx application. the open babel tool in pyrx is used as a converter of ligand and receptor files to pdbqt. this tool is also used to minimize the energy of each compound in order to obtain the lowest energy of the compound during the docking process. the docking simulation process uses the same method by adjusting the location and size of the native ligand grid box. evaluation of the docking results in the form of binding affinity formed during the docking simulation process. low binding affinity indicates good interaction ability of the ligand with the protein (hassan et al., 2020). visualization of results the results of the redocking process were calculated using rmsd and visualized the overlap between the ligand before and after the docking process using pymol software and biovia discovery studio 2021. (lestariningrum et al., 2024)visualization of the docking results was used as an analysis of the interaction of amino acid residues formed between the ligand and the receptor. visualization of amino acid interactions using the ligplus application which shows the hydrogen and non-hydrogen bonds formed (afladhanti et al., 2023). prediction of pharmacokinetics, toxicity and physicochemistry of test compounds pharmacokinetic and toxicity predictions use admet parameters that can be accessed through https://biosig.lab.uq.edu.au/pkcsm/prediction. this prediction requires canonical smiles for each compound as an analysis format obtained through https://pubchem.ncbi.nlm.nih.gov/. physicochemical predictions can be accessed through http://www.swissadme.ch/index.php by ensuring canonical smiles for each compound. the parameters used in physicochemical predictions are lipinski rules which are common parameters used in the development of a new oral drug (dong et al., 2018). results and discussion docking analysis the validation results of the docking method using pyrx showed that the root mean square deviation (rmsd) value reached 0.854 å, indicating the high accuracy of the docking model used (dallakyan & olson, 2015). the validation results of the docking method using pyrx showed that the root mean square deviation (rmsd) value reached 0.854 å, which is far below the maximum limit of less than 2 å, indicating the high accuracy of the resulting docking model. this process is important to determine the position and size of the grid box (x, y, z) of the original ligand as a reference for specific docking to the target protein. this analysis shows that the ligand structure has been well integrated into the active site of the target protein, with the center position of the grid box at coordinates x = -52.555, y = 0.208, and z = -19.899, and the grid box sizes are x = 13.673, y = 11.664, and z = 21.900, respectively. this information is very important to continue the specific docking steps, ensuring that the simulation is carried out in the right area. in addition, molecular docking analysis revealed that the active compounds from artocarpus altilis have good affinity values for the target protein, as listed in table 1 of the analysis results. the results of the molecular docking analysis showed that ellagic acid was the ligand with the highest affinity for the target protein, with a δg value of -8.4 kcal/mol. this finding confirms the potential of caffeic acid as a potent therapeutic agent, capable of interacting effectively with the target protein. quercetin showed good interaction, after that of ellagic acid with a δg value of -7.3 kcal/mol. this potential is reinforced by the low δg value, indicating that caffeic acid and quercetin can significantly inhibit egfr protein activity, making them attractive candidates in the development of new drugs. this indicates that although their affinity is lower than ellagic acid, both still have potential as alternatives in the treatment of this protein. https://www.rcsb.org/ https://pubchem.ncbi.nlm.nih.gov/ https://biosig.lab.uq.edu.au/pkcsm/prediction https://pubchem.ncbi.nlm.nih.gov/ http://www.swissadme.ch/index.php farid et al. – prediction of artocarpus altilis potential as an anti breast cancer … 201 for comparison, doxorubicin, which has a δg value of 6.6 kcal/mol, serves as a control in this analysis. table 1. docking analysis results. compound binding affinity native ligand -9.5 caffeic acid -5.6 chlorogenic acid -6.7 coumaric acid -5.4 ferulic acid -5.5 hyperoside -6.7 quercetin -7.3 ellagic acid -8.4 gallic acid -5 catechin -7.2 epicatechin -7.2 limonene -5 γ-terpinene -5.1 terpinolene -5.2 doxorubin -6.6 visualization of the interaction of amino acid residues of natural ligands (a), doxorubicin (b), ellagic acid (c), and quercetin (d) with egfr protein is shown in figure 1. the results of molecular docking analysis indicate that the amino acid residue met793 plays an important role in the formation of hydrophobic bonds, which shows a significant contribution to the stability of the interaction between the ligand and the target protein. these findings strengthen the understanding of the mechanism of ligand interaction with egfr, as well as the potential of natural compounds in the development of targeted therapies. analysis of the interaction of amino acid residues formed from the native ligand in figure a shows the presence of hydrogen bonds between met793 and an interaction distance of 3.20 å. in addition, there are five dominant non-hydrogen bonds involving amino acid residues ala743, pro794, leu781, gly796, and leu844. the existence of these bonds indicates that met793 plays a key role in the stability of ligand interactions with egfr proteins, as well as contributing to the efficiency of ligand binding (radwan et al., 2024). doxorubicin, as a control drug in this study, showed that the amino acid residue pro794 also formed hydrogen bonds with egfr. in addition, there are seven dominant non-hydrogen bonds involving residues ala743, met793, leu781, gly796, asp855, leu792, and leu844. this indicates that doxorubicin is able to interact in a complex with the active site of egfr, which has the potential to increase its effectiveness as a therapeutic agent (ibrahim et al., 2020). ellagic acid, which has the lowest binding energy value, shows three hydrogen bonds with met793, met793, and lys745 as non-dominant bonds. in addition, four non-hydrogen bonds were found involving residues ala743, asp855, leu792, and leu844. this shows that although ellagic acid has a low binding energy, the interactions formed are still quite significant and can contribute to the anticancer potential of this compound. quercetin forms three hydrogen amino acid residue interactions, namely with asp855, met793, and pro794. this interaction shows that quercetin also has the potential to bind the egfr protein efficiently, similar to other compounds. the combination of various amino acid residue interactions in the test ligand shows the complexity and strength of the interaction similar to the control drug, so that in the future it can be further explored for the development of natural compound-based therapies that are more effective and have minimal risk of toxicity (amelia et al., 2022). admet analysis all compounds have molecular weights below 500. log p (< 5) all compounds meet the log p criteria, indicating adequate lipophilicity (table 2). hydrogen bond donors (hbd < 5) the majority of compounds meet the hbd criteria. compounds that exceed the threshold are chlorogenic acid (6 hbd) and hyperoside (8 hbd), which can affect membrane permeability. hydrogen bond acceptors (hba < 10) all compounds meet the hba criteria except hyperoside, which has an hba value of 12, above the threshold. not all compounds show hepatotoxic properties and therefore do not pose a risk for clinical application. all compounds have no mutagenic potential based on the ames test (guan et al., 2018). 202 biology, medicine, & natural product chemistry 14 (1), 2025: 199-204 figure 1. visualization of amino acid interactions between proteins and ligands (a. native ligand, b. doxorubicin, c. ellagic acid, d. quercetin). table 2. physicochemical analysis and toxicity. compound mw (<500) log p (<5) hbd (<5) hba (<10) hepatotoxicity ames mutagenesis caffeic acid 180.16 1.09 3 4 no no chlorogenic acid 354.31 0.75 6 9 no no coumaric acid 164.16 1.38 2 3 no no ferulic acid 194.18 1.39 2 4 no no hyperoside 464.38 0.54 8 12 no no quercetin 302.24 1.99 5 7 no no ellagic acid 302.19 1.31 4 8 no no gallic acid 170.12 0.50 4 5 no no catechin 290.27 1.22 5 6 no no epicatechin 290.27 1.22 5 6 no no limonene 136.23 3.31 0 0 no no γ-terpinene 136.23 3.31 0 0 no no terpinolene 136.23 3.45 0 0 no no farid et al. – prediction of artocarpus altilis potential as an anti breast cancer … 203 conclusion ellagic acid has the highest binding affinity (-8.4 kcal/mol), 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(2016). 2016 esc position paper on cancer treatments and cardiovascular toxicity developed under the auspices of the esc committee for practice guidelines. in european heart journal (vol. 37, issue 36, pp. 2768–2801). oxford university press. https://doi.org/10.1093/eurheartj/ehw211 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 417-425 | doi: 10.14421/biomedich.2025.141.417-425 issn 2540-9328 (online) phytochemical screening, gc-ms charecterisation and antioxidant activity of n-hexane fraction of microtrichia perotitii dc abdullahi maikudi nuhu1,*, istifanus1, solomon usman1, idris, abdullahi2 1department of pure and applied chemistry; 2department of pharmaceutical and medicinal chemistry, kaduna state university, nigeria. corresponding author* abdullahim.nuhu@kasu.edu.ng manuscript received: 24 february, 2025. revision accepted: 27 june, 2025. published: 16 july, 2025. abstract microtrichia perotitii dc belongs to the family asteraceae (compositeae) and it is an herb found in the west african countries. the herb has been used ethnomedicinally to treat children's rashes, burns, wounds, jaundice, rheumatism, diarrhea, and skin conditions. the nhexane fraction of the ethanolic extract of the plant was investigated for the presence of secondary metabolites, characterisation as well as its antioxidant activity with a view to exploring other significant information that would be of benefit to scientific study. the n-hexane extract was subjected to partitioning, phytochemical screening using standard methods as well as spectroscopic characterizations using uv, ftir and gc-ms analysis including antioxidant activity. the result of the phytochemical screening revealed the presence of alkaloids, tannins, saponins, phenols, glycosides, steroids, terpenoids and flavonoids, but anthraquinones and phlobatannins were absent. the spectroscopy analysis showed a high degree of conjugation and it also reveals the presence of hydroxyl (o-h), carbonyl (c=o) and methyl (c-h) groups, with the compound, pentadecanoic acid, 14-methyl-, methyl ester, having the highest abundance while the antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (dpph) and nitric oxide (no),radical scavenging assays exhibited antioxidant activity with an ic50 value of 4.61mg/ml but less than ascorbic acid which has an ic50 value of 0.84mg/ml. the phytochemical compounds present in the plant could be responsible for its antioxidant property which suggests the use of the plant in the treatment of diseases induced by free radicals. in the light of the importance of this herb in local applications, the research was aimed at exploring the scientific evidences to support the local claims as well as improving information of the efficacy of the plant. keywords: microtrichia; perotitii; phytochemical; gc-ms; dpph. introduction there is a long history of using herbal therapy to treat many diseases. man has used them for the treatment of disease for a very long time, and they are still commonly used today. before the advent of conventional medicine, humans had long amassed a body of empirical data pertaining to the therapeutic benefits of regional flora. these herbalists and their apprentices have amassed a substantial body of knowledge on medicinal plants over periods of trial, error, and success. the first generation of plant medications, according to iwu et al. (1999), was often simple botanicals used in a more or less raw form. several potent medications that are utilized in their natural form have been chosen as therapeutic agents (eluoba et al., 2005; abdullahi et al., 2011; abdullahi et al., 2013). microtrichia perotitii dc belongs to the family asteraceae (compositeae) and it is a herb found in west african countries (hutchinson & dalzie, 1963). in nigeria it is found in the northern part of the country where it is known as maijankai or sawun keke in hausa, osete in igbira and shaware pepe in yoruba. traditionally, m. perotitii is used for treating pain related diseases such as toothaches, cuts and burns and rashes in children. others include, skin disease, rheumatism, diarrhea and jaundice (magaji, 2003; abdullahi et al., 2011). figure1. picture of microtrichia perotitti dc. https://doi.org/10.14421/biomedich.2025.141.417-425 418 biology, medicine, & natural product chemistry 14 (1), 2025: 417-425 materials and methodology sample collection the herb microtrichia perotitii was collected from zaria kaduna state, northern nigeria in july 2023. the fresh herb has already been identified and authenticated by mal. shehu gallah umar,of herbarium unit of the department of biological sciences, kaduna state university kasu and was given specimen number kasu/bsh/9002 for future references. the fresh herb was allowed to dry under the shade for three weeks and later reduced to coarse powder with a traditional pestle and mortar. extraction of sample 150g of the powdered herb (microtrichia perotitii) was macerated in 1,500 ml of ethanol for 72 hrs at room temperature with intermittent shaking. the residue was extracted twice with the same fresh solvent and later combined and filtered using whatman filter paper (5 mm) under suction. the extract was concentrated under reduced pressure (rotary evaporator) at temperature of 45 ºc. the extract was stored in desiccators until needed for the work (abdullahi & mainul,2020). phytochemical screening the crude ethanol extract of microtrichia perotitii was evaluated for the presence of its phytoconstituents by using standard methods trease and evans, 2009, sofowora, 1993 to test for, carbohydrates( molisch’s test), tannins (ferric chloride test), phlobatannins, flavonoids; (shinoda’s test), saponins (frothing test), alkaloids (dragendoff’s reagent), cardiac glycosides( keller-kiliani test), steroids (salkowski’s test), terpenoids (libermann-burchard test) and anthraquinones (borntrager’s test). solvent partitioning fractionation of the crude ethanol extract of microtrichia perotitii was done according to the scheme proposed by (qing-wen, et al. 2018) but modified. thus 25 g of the crude was suspending crude in 300 ml of water separately and then partitioned with hexane, n-butanol, and ethyl acetate that is in an order of increasing polarity by using separating funnel. all the three fractions obtained were dried by evaporation using rotary evaporator and then later weighed. figure 2. scheme for solvent partitioning (qing-wen, et al.2018). antioxidant activities dpph radical scavenging activity assay the free rsa of the n-hexane fraction of microtrichia perotitii was tested using a 2,2-diphenyl-1-picryl hydrazyl (dpph) technique. a total of 24 milligrams of dpph were dissolved in 100 ml of methanol to make the stock solution. filtration of dpph stock solution using methanol yielded a usable mixture with an absorbance of around 0.973 at 517 nm. in a test tube, 3 ml dpph workable solutions were combined with 100 µl. three milliliters of solution containing dpph in 100 µl of methanol is often given as a standard (goldschmidt, et al 1922). after that, the tubes were kept in complete darkness for 30 min. the absorbance was therefore determined at 517 nm. the following formula was used to compute the percentage of antioxidants or rsa: % 𝑜𝑓 𝑎𝑛𝑡𝑖𝑜𝑥𝑖𝑑𝑎𝑛𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ═ [(𝐴𝑐 − 𝐴𝑠) ÷ 𝐴𝑐] × 100 where: ac : control reaction absorbance; as : testing specimen absorbance. nuhu et al. – phytochemical screening, gc-ms charecterisation and … 419 nitric oxide radical scavenging assay the procedure was adopted with some modifications to determine the scavenging activity of the plant extracts against nitric oxide radical. nitric oxide was generated from sodium nitroprusside and measured by the greiss reaction. ascorbic acid was used as a standard. ascorbic acid inhibits induction of nitric oxide synthase and is a naturally occurring direct scavenger of nitric oxide. it reduces the amount of nitrite formed between oxygen and nitric oxide generated from sodium nitroprusside. the absorbance was measured at 596 nm and the percentage of antioxidant activity was calculated using the formula in the equation (elizabeth & rao, 1990; ebrahimzadeh et al., 2008). % 𝑜𝑓 𝑎𝑛𝑡𝑖𝑜𝑥𝑖𝑑𝑎𝑛𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ═ [(𝐴𝑐 − 𝐴𝑠) ÷ 𝐴𝑐] × 100 where: ac : control reaction absorbance; as : testing specimen absorbance. chromatographic procedure gas chromatography-mass spectrometry analysis gc-ms analysis was carried out on a gc system comprising a gas chromatograph interfaced to a mass spectrometer (gc-ms) instrument; schimadzu gcmsqp2010, employing the following conditions: column elite-1 fused silica capillary column (30×0.25 mm id×1em df, composed of 100% dimethyl poly siloxane), operating in electron impact mode at 70 ev; helium (99.999%) as carrier gas at a constant flow of 1ml/ minute and a sample injection volume of 1 μl which was employed (split ratio of 10:1) injector temperature 250°c; ion-source temperature 280°c. the oven temperature was programmed from 110°c (isothermal for 2 minutes), with an increase of 10°c/minute, to 200°c, then 5°c/minute to 280°c, ending with a 9 minutes isothermal at 280°c. mass spectra were taken at 70 ev; a scan interval of 0.5 s and fragments from 40 to 550 da. total run time was 30 min. the compounds were then identified from the gc-ms peaks, using library data of the corresponding compounds. gc-ms was analyzed using electron impact ionization at 70 ev and data was evaluated using total ion count (tic) for compound identification and quantification. the spectrums of the components were compared with the database of spectrum of known components stored in the gc-ms library using nisp search. the relative % amount of each component was calculated by comparing its average peak area to the total areas. measurement of peak areas and data processing were carried out by turbo-mass-ocptvs-demo spl software (duraisamy et al, 2015). uv analysis two ml of the sample was placed in beaker of 50 ml then 5 ml of n-hexane was added and shaken thoroughly to reach solution consistency. the cuvette was filled with sample solution and inserted in the device and the results were recorded. the cell was emptied and was read in the instrument at wave length 200 800 nm). the absorptions results were recorded(jain et al., 2016). ftir analysis fourier-transform infrared spectroscopy (ft-ir) analysis was performed using a perkin elmer spectrophotometer system (perkinelmer, waltham, ma, usa) over a frequency range of 400–4000 cm, which was used to detect the characteristic peaks(dhivya & kalaichelvi, 2016; jain, et al., 2016). results and discussion results the results obtained from the research have shown promising outcomes and thereby suggested the importance of being used locally. similarly, it has provided an insight in to further research in to certain areas beyond the scope of this study. the results below are a testimony to the outcome. table 1. phytochemical analysis of n-hexane fraction of ethanolic extract of microtrichia perotitii constituent test observation inference carbohydrates molisch test purple colour at interphase + tannins ferric chloride test greenish black coloration + phlobatannins hydrochloric acid no change flavonoids shinoda test a red coloration + saponins frothing test frothing which persist + alkaloids dragendorff test orange yellow precipitate + cardiac glycosides keller-kiliani test purple ring at interphase + steroids salkowski test pale green colour in the acetic layer + terpenoids lieberman-burchard test red colour ring + anthraquinones bontragers test bright pink colouration key: + = present and = absent 420 biology, medicine, & natural product chemistry 14 (1), 2025: 417-425 wave length (nm) figure 3. uv spectroscopy of n-hexane fraction of m.perotiiti. figure 4. ft-ir spectroscopy of n-hexane fraction of microtrichia perotitii. table 2. gc-ms analylsis. no rt area % name mol. wt structure 1 11.2367 0.3862 dodecanoic acid, methyl ester 214.1 2 15.7246 0.3286 methyl tetradecanoate 242.4 3 18.2 053 0.2057 2-pentadecanone, 6,10,14-trimethyl 268.4 400 500 600 700 800 0.0 0.5 1.0 1.5 2.0 2.5 ab s 6 6 5. 0 0 , 0. 3 8 4 0. 0 8 8 , 6 0 7. 0 0 5 3 5. 0 0 0. 1 3 0 , 4 0 8. 0 0 1. 2 9 2 , nuhu et al. – phytochemical screening, gc-ms charecterisation and … 421 table 2. cont. no rt area % name mol. wt structure 4 19.3304 0.9035 (z)-methyl hexadec-11-enoate 268.4 5 19.8907 29.1986 pentadecanoic acid, 14-methyl-, methyl ester 270.0 6 20.6915 0.7743 cis-vaccenic acid 282.4 7 20.94 3.589 n-hexadecanoic acid 256.4 8 21.2237 3.6954 hexadecanoic acid, ethyl ester 284.5 9 22.909 4.2005 9,12-octadecadienoic acid, methyl ester 280.4 10 23.1473 25.6492 9-octadecenoic acid (z)-, methyl ester 296.5 11 23.4392 1.0329 phytol 296.5 12 23.7034 3.5635 methyl stearate 298.5 13 24.1454 2.9496 linoleic acid ethyl ester 310.5 14 24.3212 0.7167 9-tetradecen-1-ol, (e)212.3 15 26.635 0.3222 cis-methyl 11-eicosenoate 324.5 16 27.1937 0.8927 eicosanoic acid, methyl ester 340.5 17 30.4322 1.5348 docosanoic acid, methyl ester 354.6 18 32.589 1.8744 oleic acid 282.4 19 33.4393 5.0067 beta-sitosterol 414.7 20 33.974 0.1669 cyclopentane undecanoic acid 254.4 21 34.6516 0.4221 2-dodecen-1-yl(-)succinic anhydride 266.3 22 36.3376 4.7332 cis-vaccenic acid 282.4 23 36.4854 1.2812 erucic acid 338.5 24 37.386 6.5723 1,4-dimethyl-7-oxo-4,7-dihydrotriazolo(3,4-c)triazine 422 biology, medicine, & natural product chemistry 14 (1), 2025: 417-425 figure 5. fragmentation of the highest occurring compound (pentadecanoic acid, 14-methyl-, methyl ester). table 3. dpph scavenging activities. concentration mg/ml absorbance at 517nm % antioxidant blank 2.9015 control 40 0.381 86.87 sample 40 1.2931 55.43 figure 6. percentage inhibition of the dpph scavenging activity of sample and ascorbic acid. absorbance at 542nm % antioxidant (rsa) 10ml/ml 20mg/ml 30mg/ml 40mg/ml 10ml/ml 20mg/ml 30mg/ml 40mg/ml sample 1.7340 1.4680 1.4680 3.0982 53.64 60.75 60.75 17.16 control 0.4142 0.3557 0.3913 0.4057 89.89 90.51 89.53 89.15 blank 3.74 0 20 40 60 80 100 standard sample nuhu et al. – phytochemical screening, gc-ms charecterisation and … 423 table 4. nitric oxide (no) scavenging activity. sample ascorbic acid conc (mg/ml) % rsa ic50 conc (mg/ml) % rsa ic50 10 53.64 5.98 10 88.93 0.34 20 60.75 5.07 20 90.48 0.67 30 60.75 4.15 30 89.55 1.01 40 17.17 3.24 40 89.14 1.34 table 5. mean ic50 value of sample and standard. mean ic50 value (mg/ml) sample 4.61 standard 0.84 the ic50 (inhibitory concentration) values, representing the amount of extract required to decrease the absorbance of dpph by 50% was calculated from the percentage radical scavenging activity. figure 7. percentage inhibition of nitric oxide of sample and ascorbic acid. discussion the discussions below were driven from the outcomes of the results with specific comparisons as the case may be. preliminary phytochemical screening the preliminary phytochemical screening of the crude ethanol extract of microtrichia perotitii revealed the presence of secondary metabolites of therapeutically importance. they included alkaloids, saponins tannins, flavonoids, terpenoids, cardiac glycosides while anthraquinones and phlobatannins were not detected. (idris et al., 2009; junaid et al., 2020). therefore, the nhexane fraction of the microtrichia perotitii dc contains phytochemicals that contribute significantly to its antioxidant activity. in other studies important phytochemicals identified were phenols, flavonoids, and triterpenes, that is alpha-amyrin acetate, which has been isolated from the plant (abdullahi et al., 2018; abdullahi et al., 2011). spectroscopy analysis uv spectroscopy the n-hexane fraction of the sample’s uv spectroscopy yielded the highest wavelength of 665 nm. organic molecules absorb light in the uv region of the electromagnetic spectrum, especially those with a high degree of conjugation (dyah et al., 2016; tarun et al., 2020). table 6 uv spectroscopy of n-hexane fraction of n-ethanolic extract of microtrichia perotiiti. s/no wave length (nm) absorbance 1 665.00 0.384 2 607.00 0.088 3 535.00 0.130 4 408.00 1.292 ft-ir spectroscopy the ft-ir analysis of the n-haxane fraction of microtrichia perotitii shows various bands, indicating the presence of different functional groups. peaks 3369.5cm1 indicates strong stretching of a hydroxyl group (oh), band 2922.2cm-1 indicates the presence of a methyl group(c-h), and band 1707.1cm-1 indicates the presence of a carbonyl functional group (c-o) (perveen & zaib, 2013; dyah, et al., 2016). table 7. ftir spectroscopy of n-hexane fraction of ethanolic extract of microtrichia perotitii. no peaks /bands (cm-1) functional groups 1 3369.5 alcohol (o-h) 2 3011.7 alkenes (c=h) 3 2922.2 methylene (c-h) 4 2855.1 aliphatic (c-h) 5 2016.5 alkynes (c=c) 6 1707.1 ketone (c-o) 7 1558.0 alkene (c=c) 8 1461.1 alkene (c=c) 9 1379.1 ester (c-o) 10 1244.9 ester (c-o) 11 1166.7 ether (c-o) 12 1036.2 trans (c-h) 13 909.5 vinyl (c-h) 14 719.4 alkene (c-h) cis 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 % in h ib it io n conc(mg/ml) chart of % inhibition agianst sample and control sample control 424 biology, medicine, & natural product chemistry 14 (1), 2025: 417-425 gc-ms spectrometry the gc-ms analysis of the n-hexane fraction of microtrichia perotitii shows the presence of 27 compounds of which pentadecanoic acid, 14-methyl-, methyl ester, has the highest abundance with about 29.1986% occurance, followed by 9-octadecenoic acid (z)-, methyl ester with about 25.6492% abundance, 1,4dimethyl-7-oxo-4,7-dihydro-triazolo(3,4c)triazine,(6.5723%),beta.-sitosterol (5.0067%), cisvaccenic acid (4.7332%), 9,12-octadecadienoic acid, methyl ester(4.2005), hexadecanoic acid, ethyl ester (3.6954%), n-hexadecanoic acid (3.589%), methyl stearate (3.5635%), linoleic acid ethyl ester (2.9496%). other compounds with below 2% abundance are docosanoic acid, methyl ester, erucic acid, oleic acid, phytol, 2-dodecen-1-yl(-)succinic anhydride, cyclo pentane undecanoic acid, dodecanoic acid, methyl ester, methyl tetradecanoate, 2-pentadecanone, 6,10,14trimethyl-z)-methyl hexadec-11-enoate, cis-vaccenic acid, 9-tetradecen-1-ol, (e)-, cis-methyl 11-eicosenoate, and eicosanoic acid, methyl ester.14methylpentadecanoic acid is a methyl-branched fatty acid that is pentadecanoic acid substituted by a methyl group at position 14. it is a biomarker for rheumatoid arthritis. it has a role as a biomarker and a mammalian metabolite. it is a branched-chain saturated fatty acid, a methylbranched fatty acid, a long-chain fatty acid and a fatty acid 16:0. it is functionally related to pentadecanoic acid. it is a conjugate acid of a 14-methyl pentadecanoate (njoku et al., 2021). antioxidant activity dpph activity when free-radical dpph interacts with an odd electron, the greatest absorption occurs at 517 nm (purple color). a free-radical scavenger antioxidant reacts to dpph to form dpphh, which has a lower absorbance than dpph because of the lower amount of hydrogen. compared to the dpph-h state, this radical version causes decolourisation (a yellow hue) as the number of electrons collected increases. it can be observed from table 3 that the antioxidant activity of the n-hexane fraction of microtrichia perotitii 40mg/ml shows 1.293 absorbance at 517nm with 55.43 percentage of antioxidant while 100mg/ml shows 1.5018 absorbance at 517 nm with 48.28 percentage of antioxidant.( siddartha et al, 2022) the phytochemicals in m. perotitii exhibit strong radical scavenging properties, which are crucial for combating oxidative stress as well as reducing cellular damage. the antioxidant activity is often measured using assays such as dpph and nitric oxide scavenging, where extracts show significant inhibition rates (bisso et al., 2022; hamsalakshmi et al., 2020). nitric oxide activity nitric oxide is a potent pleiotropic mediator of physiological processes such as smooth muscle radical relaxation, inhibition of platelet aggregation, neuronal signaling, and regulation of cell mediated toxicity. it is a diusible free radical which plays many important roles as an e ector molecule in diverse biological systems (vasodilation, neuronal, messenger, and antimicrobial and antitumor activities). ascorbic acid used as the standard for no radical scavenging in this analysis showed maximum activity with a percentage rsa (%rsa) of 90.51% and a maximum ic50 value of 1.34 while the 20mg/ml and 30mg/ml concentration of the nhexane fraction of microtrichia perotitii shows a maximum rsa value of 60.75% and a maximum ic50 value of 5.98. the mean ic50 value of the sample is 4.61mg/ml while the ic50 value of the standard is 0.84mg/ml (avani, 2010). specifically, the n-hexane extract demonstrated an ic50 value comparable to other plant extracts, suggesting its efficacy in scavenging free radicals (putu et al.,2023). conclusion the phytochemical screening of the crude ethanol extract of microtrichia perotitii revealed the presence of carbohydrate, saponins, flavonoids, terpenoids, alkaloids, and cardiac glycosides. the antioxidant activity reveals that the n-hexane fraction of m perotitii inhibits free radicals. the presence of different tannins, phenolics, carbohydrates, flavonoids etc, may be responsible for the antioxidant property of the sample. the antioxidant properties were highest at a concentration of 30mg/ml and lowest at 40mg/ml. this study shows that the nhexane fraction of microtrichia perotitii has antioxidant properties and might be used in the treatment of diseases induced by free radicals. acknowledgement: the authours wish to acknowledge kaduna polytechnic for partial sponsorship of the research and all technical staff of ahmadu bello university zaria and kaduna state university. authors’ contributions: dr abdullahi maikudi nuhu designed the research project as well the interpretation of the spectra. istifanus and idris participated fully in the laboratory activities including travelling to collect the plant sample. all of us the authours have read and approved the final manuscript and there were no ambiguities. competing interests: the authours wish to declare without any uncertainty that there was no competing interest with regards to this work. nuhu et al. – phytochemical screening, gc-ms charecterisation and … 425 funding: there were no special grant obtained for the purpose of undertaking this research activity. references abdullahi, m.n, ibrahim h, ilyas n (2011). phytochemical screening and biological studies of the leaves of microtrichia perotitii dc(asteraceae). british journal o. medicinal plants 1(3):88-97. abdullahi, m. n, ibrahim,h, iliya,n (2013). evaluation of phytochemical screening and analgesic activity of aqueous extract of the leaves of microtrichia perotitii dc (asteraceae) in mice using hotplate method. medicinal plant research. 3:537-543. abdullahi, r.a.; 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(2020). phytochemical screening and determination of phenolics and flavonoids in dillenia pentagyna using uv–vis and ftir spectroscopy. spectrochimica. acta part a: molecular and biomolucular spectroscopy/, 242. sciencedirect (elsevier). trease, e. c.and evans,w.c. (2009). pharmacognosy, 16th edition w.b. saunders, toronto. harcourt pub ltd. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 277-283 | doi: 10.14421/biomedich.2024.131.277-283 issn 2540-9328 (online) testing antibacterial activity of daun kentut (paederia foetida l.) leaf extract against escherichia coli in vitro lisa savitri*, eka wahyuning tiyas, rochmad krissanjaya department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id abstract escherichia coli is a pathogenic bacterium that causes infectious diseases in the digestive tract and can produce lipopolysaccharide (lps), including lipid a. exposure to endotoxins in lipid a, can cause systemic effects, such as sepsis, which can lead to clinical manifestations and even death. daun kentut (paederia foetida l.) is one of the herbal plants containing bioactive compounds that can inhibit the growth of e. coli bacteria. the aim of this research is to determine the effective concentration of daun kentut leaf extract to inhibit e. coli growth. this study is an experimental research using maceration extraction method and antibacterial disc diffusion method with 3 repetitions at concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, negative control (aquadest), and positive control (ciprofloxacin) conducted from march to april 2023 in the microbiology laboratory of kadiri university. the observation results of inhibition zones in each treatment varied. the presence of these inhibition zones is due to the presence of secondary metabolite compounds in daun kentut leaf extract that have antibacterial activity, such as inhibiting protein synthesis mechanisms, causing damage to cell wall permeability, and ultimately leading to bacterial cell lysis and death. the statistical analysis using the one way anova test with a p-value of 0.05 shows that each variable has significant differences and effects. thus, it can be concluded that the effective extract variation is the 100% concentration because, in this study, the 100% concentration has an inhibition zone of 26.72 mm and is classified as very strong in inhibiting e. coli bacteria. keywords: antibacterial; eschericia coli; daun kentut leaf extract. abbreviations: antimicrobial resistance (amr), laf (laminar air flow), lipopolysaccharide (lps), nutrient agar (na). introduction escherichia coli is a pathogenic bacterium that causes infectious diseases in the digestive tract, including acute diarrhea that can affect anyone (patel, 2017). e. coli bacteria produce toxins that can damage the mucosal cells of the small intestine, leading to clinical symptoms such as watery diarrhea, abdominal cramps, mild fever, nausea, and malaise (tuntun, 2016). e. coli atcc 25922 is a pathogenic microorganism that commonly infects humans, causing diseases such as diarrhea, meningitis, and others. e. coli causes up to 52% of infectious diseases. the spread of e. coli atcc 25922 can occur through hand-to-mouth contact or passive transfer via food or drink intermediaries (savitri et al., 2020). lipopolysaccharide (lps) is a product produced by e. coli that forms the outer bacterial membrane, consisting of specific-o polysaccharides, core polysaccharides, and lipid a. the toxin activity of endotoxin occurs in lipid a. exposure to endotoxin can cause systemic effects such as changes in blood pressure and body temperature, coagulation abnormalities, decreased circulating leukocyte, and platelet counts caused by hemagglutinin and hemolysin, which are virulence factors in the infection process leading to tissue damage (divakara et al., 2017). another role of endotoxin resulting in death is cardiovascular instability and septic shock leading to death from bacterial infection and each body's ability to respond to infection by gram-negative bacteria (savitri et al., 2022). the systemic effects include bleeding, immune system disorders, sepsis, and death (purwanto, 2018). in 2017, there were 48.9 million cases related to sepsis and 11 million deaths worldwide, accounting for nearly 20% of global deaths (rudd et al., 2020). sepsis occurs annually in children under 5 years old at a rate of 44%, and a quarter of these cases are due to neonatal sepsis (yustika et al., 2020). the incidence of sepsis in indonesia remains high at 30.29%, with a mortality rate ranging from 11.56% to 49% (batara et al., 2018). the treatment of infectious diseases caused by bacteria typically involves synthetic antibiotics such as ampicillin, gentamicin, and metronidazole. the manuscript received: 13 may, 2024. revision accepted: 16 july, 2024. published: 14 august, 2024. https://doi.org/10.14421/biomedich.2024.131.277-283 278 biology, medicine, & natural product chemistry 13 (1), 2024: 277-283 excessive use of these antibiotics can lead to the rapid development of antimicrobial resistance (amr) (simmons et al., 2020). these drugs can cause side effects such as rash, diarrhea, abdominal cramps, and hypersensitivity. synthetic antibiotic costs are also expensive for lower-income groups. therefore, further research is needed on potential herbal materials to replace synthetic antibiotic drugs. some people have adopted the use of herbal plants for treatment because the materials are readily available and affordable for all segments of society. over time, medicinal plants can be developed as traditional medicines, including daun kentut (paederia foetida l). daun kentut is a source of important natural products and chemical materials for health. it contains bioactive compounds such as flavonoids, terpenoids, paedolone, β-sitosterol, friedelin, campesterol, methyl mercaptan, and other active compounds (patel, 2017). research on the antibacterial activity test of aloe vera extract against e. coli proved that aloe vera extract has no antibacterial effect due to the type of plant and the amount of antibacterial substance content in aloe vera extract, rendering it ineffective against e. coli bacteria (suryati et al., 2017). various herbal medicines tested by rahmawati et al. (2017) showed that the concentration of extract with the highest inhibitory effect was turmeric extract at 5.64 mm. however, this antibacterial activity was still moderate. in utary's study (2016), an extract of daun kentut was tested against bacteria sigella sonnei and salmonella typhi, and chemical analysis of the leaf and stem of the kentut plant was also conducted by handrianto (2018). however, research on the antibacterial activity test of daun kentut extract against e. coli is still rare. therefore, the researcher aims to investigate further the effectiveness of daun kentut in inhibiting the growth of e. coli bacteria. materials and methods research design the type of research used is quantitative experimental with the disc method. population and sample the population in this study is escherichia coli bacteria. the sample used in this study is wild-type escherichia coli atcc 25922 bacteria obtained from nano lab laboratory with number r4607050. research variables the independent variable in this study is the sembukan leaf extract with concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% (juliastuti et al., 2020). the dependent variable in this study is the growth of e. coli bacteria on agar media, which is then measured by the inhibition zones visible from the clear zones formed. control variables include: incubation temperature, medium content, and incubation duration. tools and materials the tools used in this study include digital balance (ss-a 2000), disc paper, glassware (pyrex), incubator (fcd3000 series), autoclave (b-one aa 18 litres), vernier calliper, and laf (laminar air flow). this research uses materials such as sembukan leaves obtained from melaten hamlet, ngrami village, sukomoro district, nganjuk regency, east java, 96% ethanol, aquades as a solvent for media and extracts, label paper, wool thread, cover paper, for bacterial growth media, nutrient agar is used, wild-type escherichia coli atcc 25922 bacteria with a dose of 105 cfu/ml from nano lab laboratory, and ciprofloxacin as a positive control. procedures extract preparation the preparation of sembukan leaf extract was carried out using the maceration method. 1 kg of sembukan leaves (simplisia) was placed in a maceration container and then added with 3 l of 96% ethanol in a ratio of 1:3. the mixture was left in a place protected from sunlight for 3 days, stirred every 12 hours, and then filtered. the residue was remacerated (re-maceration) once with the same solvent as before. the maceration and filtration process from maceration and remaceration were collected and evaporated to obtain a concentrated extract. medium preparation it is known that 2.8 grams of nutrient agar (na) is used for 100 ml of aquades. since each petri dish will be filled with 25 ml of media (15 ml for the first layer, 10 ml for the second layer) and 12 petri dishes will be prepared, 8.4 grams of na is required in 300 ml of aquadest. bacterial inoculation escherichia coli bacteria were inoculated into the medium using the pour plate method (tube or pour method). preparation of daun kentut leaf extract concentrations different concentrations of the extract were prepared as follows: a. 10% concentration: by mixing 0.2 ml of extract with aquades up to the 2 ml mark. b. 20% concentration: by adding 0.4 ml of extract with aquadest up to 2 ml. c. 30% concentration: by adding 0.6 ml of extract with aquadest up to 2 ml. d. 40% concentration: by adding 0.8 ml of extract with aquadest up to 2 ml. e. 50% concentration: by adding 1 ml of extract with aquadest up to 2 ml. savitri et al. – antibacterial activity of daun kentut 279 f. 60% concentration: by adding 1.2 ml of extract with aquadest up to 2 ml, and so on. g. 100% concentration: 2 ml of extract without adding aquadest. h. positive control preparation involved weighing 100 mg of ciprofloxacin dissolved in 2 ml of aquadest to make a 5% solution. treatment inoculated e. coli bacteria onto agar media before solidification using the pour plate technique. after the media solidified, circular filter papers with a diameter of 0.6 cm were soaked in each concentration of the extract. the filter papers were then placed on the solidified na media in 4 different petri dishes. each dish was labelled as follows: a. p1: 10% concentration b. p2: 20% concentration c. p3: 30% concentration d. p4: 40% concentration e. p5: 50% concentration f. p6: 60% concentration g. p7: 70% concentration h. p8: 80% concentration i. p9: 90% concentration j. p10: 100% concentration k. k-: aquadest (negative control) l. k+: ciprofloxacin (positive control) data analysis after 24 hours of bacterial incubation and disc placement, observations and calculations of the area of transparent zones were performed by measuring the clear area formed at the edges of the placed paper disks twice, vertically and horizontally. the obtained results were averaged using the following formula (dwi, 2019): zone of inhibition calculation formula = (ab) + (cd) / 2. subsequently, the obtained data were processed and analyzed using statistical package for the social sciences (spss) version 26.0 for windows for anova testing. normality and homogeneity tests were conducted with a p-value > 0.005. if the data were significant, oneway anova testing was performed with a significance value of p < 0.005. after obtaining significant differences in the data, further testing was conducted using post hoc test lsd. results and discussion zone of inhibition diameter the clear zones around the discs were measured using a calliper. the total diameter was subtracted by the disc diameter of 6 mm to obtain the average zone of inhibition as shown in table 1. table 1. results of inhibition test. replication inhibition zone hambat (mm) k+ kp1 p2 p3 p4 p5 p6 p7 p8 p9 p10 1 37.10 0.00 4.35 7.60 8.30 8.00 8.55 9.35 10.55 16.70 19.15 26.00 2 35.00 0.00 5.65 5.35 8.30 6.70 10.90 10.40 14.45 16.40 18.40 26.45 3 37.30 0.00 5.60 6.70 3.70 6.20 5.90 11.45 9.25 19.90 21.30 27.70 average 36.47 0.00 5.20 6.55 6.77 6.97 8.45 10.40 11.42 17.67 19.62 26.72 figure 2. average diameter of inhibition zones diagram. figure 3. extract performance effectiveness comparison diagram. 50% 60% 70% 80% 90% 100% 280 biology, medicine, & natural product chemistry 13 (1), 2024: 277-283 normality test the data obtained were analyzed using spss 26.0 for windows. the shapiro-wilk normality test results can be seen in table 2, indicating that the significance value is > a (0.05), thus confirming that the data are normally distributed. table 2. normality test results. tests of normality group kolmogorov-smirnova shapiro-wilk statistic df sig. statistic df sig. inhibitation zone concentration of 10% .357 3 . .815 3 .150 concentration of 20% . 3 . . 3 . concentration of 30% .373 3 . .779 3 .065 concentration of 40% .280 3 . .938 3 .520 concentration of 50% .385 3 . .750 3 <.001 concentration of 60% .183 3 . .999 3 .934 concentration of 70% .219 3 . .987 3 .780 concentration of 80% .175 3 . 1.000 3 1.000 concentration of 90% .324 3 . .877 3 .315 concentration of 100% .385 3 . .750 3 <.001 positive control .288 3 . .928 3 .481 negative control .286 3 . .931 3 .493 a. lilliefors significance correction homogeneity test next, a homogeneity test was conducted. from table 3, the significance value obtained was 0.054, which is greater than the chosen value (0.05), indicating that the data are homogeneously distributed. table 3. homogeneity test results. tests of homogeneity of variances levene statistic df1 df2 sig. inhibiton zone based on mean 2.180 11 24 .054 based on median .397 11 24 .944 based on median and with adjusted df .397 11 9.780 .927 based on trimmed mean 1.964 11 24 .081 anova test an anova test was performed to determine whether the independent variable influences the independent variable. table 4 below demonstrates this. table 4. anova test results. anova inhibiton zone sum of squares df mean square f sig. between groups 35.708 11 3.246 141.675 <.001 within groups .550 24 .023 total 36.258 35 discussion antibacterial activity test in this study aims to determine the effective concentration of paederia foetida leaf extract to inhibit the growth of e. coli atcc 25922, as indicated by the inhibition zones at various extract concentrations using the disc diffusion method. this method was chosen because disc diffusion testing can be performed more quickly, and the materials used are readily available. research conducted by ratnasari et al. (2020) demonstrated that the disc diffusion method yielded larger inhibition zones compared to the well method. the well method often produces smaller inhibition zones due to difficulties during execution, such as the potential for media cracking or fracturing around the wells, which can interfere with the extract diffusion process and affect the clarity of the formed zones. in contrast, the disc diffusion method allows for better absorption of paederia foetida leaf extract into the media, minimizing execution difficulties or errors. savitri et al. – antibacterial activity of daun kentut 281 extraction in this study was conducted using the maceration method, where dried paederia foetida leaves were blended after air-drying. maceration involved soaking the leaves in 96% ethanol at a ratio of 1:3 for three intervals of 24 hours each. the extract was then filtered and evaporated using a water bath to obtain a concentrated extract. the extract was applied by soaking filter paper discs with a diameter of approximately 6 mm in the extract for about 15 minutes. one of the critical variables that can influence antibacterial activity testing is the duration of filter paper disc immersion. with a 15minute immersion period, the filter paper discs adequately absorbed the extract, resulting in maximal inhibition zone diameters, as depicted in figure 5.1. the discs were then placed on the surface of agar plates inoculated with bacteria and incubated at 37°c for 24 hours. this temperature is suitable for the growth of e. coli atcc 25922, with 37°c being the optimal temperature, according to kurniati et al. (2020). temperatures above 44°c can lead to e. coli inactivation. after 24 hours of incubation, the inhibition zones were observed and measured using a calliper for accurate millimetre measurements. the obtained results showed that the formed inhibition zones were not entirely clear, indicating the presence of other bacteria around the filter paper discs. this occurrence could be due to some nonsterile tools and materials used, especially in handling the filter paper discs and forceps used to place them. the measured results revealed the average inhibition zone diameters, where k+ (ciprofloxacin) exhibited a 36.47 mm inhibition zone. kshowed slight clarity around the disc during the second replication due to contamination during disc placement. p1 (10% concentration) exhibited a 5.20 mm inhibition zone, categorized as moderate. p2 (20% concentration) had a 6.55 mm inhibition zone, also categorized as moderate. the results of this study differed from those of dewi et al. (2019), where the 40% concentration yielded a 14.05 mm inhibition zone categorized as strong. this difference could be attributed to the different extraction solvents used; dewi used ethanol, likely contributing to bacterial growth inhibition. however, this study's findings were consistent with nurhalimah et al. (2019), where a 90% concentration yielded a 10.20 mm inhibition zone, categorized as strong. for p3 (30% concentration), the inhibition zone was 6.77 mm, categorized as moderate. p4 (40% concentration) had a 6.97 mm inhibition zone, also categorized as moderate. p5 (50% concentration) exhibited an 8.45 mm inhibition zone, categorized as moderate. p6 (60% concentration) had a 10.4 mm inhibition zone, categorized as strong. p7 (70% concentration) exhibited an 11.42 mm inhibition zone, categorized as strong. p8 (80% concentration) showed a 17.67 mm inhibition zone, categorized as strong. p9 (90% concentration) had a 19.62 mm inhibition zone, categorized as strong. meanwhile, p10 (100% concentration) exhibited a 26.72 mm inhibition zone, categorized as very strong. adha et al. (2021) stated that increasing extract usage corresponds to an increase in secondary metabolites contained within the extract. this assertion is supported by javed et al. (2020), highlighting that higher extract concentrations significantly affect antibacterial compound content, allowing these compounds to diffuse into bacterial cells. compared to previous studies, this research yielded significantly larger inhibition zones. this inhibition is attributed to the secondary metabolites in the extract damaging cell structures by inhibiting protein synthesis mechanisms, such as saponins, leading to reduced surface tension and increased permeability, causing intracellular compounds to exit and bacterial cell lysis. additionally, tannins can disrupt polypeptide synthesis, resulting in imperfect cell wall formation. flavonoids inhibit bacterial growth by affecting nucleic acid synthesis, membrane function, and energy metabolism, leading to permeability damage in cell walls, microsomes, and lysosomes. other compounds like phenols, alkaloids, and steroids also contribute to this antibacterial activity process, resulting in the formation of clear zones around the discs. according to torar et al. (2017), gram-negative bacteria have thinner peptidoglycan layers compared to grampositive bacteria. additionally, gram-negative bacteria have cell walls containing a high amount of lipopolysaccharides (lps). compounds present in paederia foetida leaf extract damage the cell walls by penetrating lps (vitalia, 2019). from the results obtained, it is known that all concentrations ranging from 10% to 100% exhibit inhibitory activity. the strongest inhibition was observed at the 100% concentration. in this study, the spacing between the discs was 2 cm with a 1.5 cm edge distance, resulting in overlapping at the 100% concentration. this can also be due to the excessive concentration of the extract. the inhibition zone at the 100% concentration could be larger if the discs were placed according to guidelines, with a 3 cm distance between discs and a 2 cm edge distance (windiyanti, et al., 2023). nevertheless, the inhibition zone diameter at 100% concentration was not larger than that of ciprofloxacin, which is a synthetic antibiotic with potent antibacterial activity against both gram-negative and gram-positive bacteria. after obtaining the data, further analysis was conducted using spss 26.0 for windows. the homogeneity test of the three data samples showed a homogeneous distribution with a value of p<0.05. subsequently, a normality test was performed, which indicated a value of p<0.05, confirming a normal distribution of the samples. in the anova test, it was found that the significance value was <0.05, indicating that h0 was rejected and h1 was accepted, meaning there is an influence between the independent variables on the dependent variable simultaneously. there were 282 biology, medicine, & natural product chemistry 13 (1), 2024: 277-283 significant differences in the average values of each treatment, as described in the post hoc test table. the lsd post hoc test results showed significant differences in each treatment. however, some concentrations had significance values >0.05 compared to other concentrations, specifically the 30% concentration compared to the 40%, 50%, and 60% concentrations. this indicates that the comparison of these concentrations was not statistically significant. these significant differences were reinforced by the comparison of extract performance effectiveness at concentrations categorized as strong to very strong, specifically the 50%, 60%, 70%, 80%, 90%, and 100% concentrations as shown in figure 5.3. the graph illustrates that concentrations with high-performance effectiveness are the 80% and 100% concentrations. the 90% concentration has a larger average inhibition zone than the 80% concentration, but the 80% concentration demonstrates greater extract performance effectiveness. conclusions based on the research results on the antibacterial activity test of paederia foetida leaf extract against e. coli, it can be concluded that paederia foetida leaf extract affects the growth of e. coli atcc 25922. various concentrations exhibited different levels of inhibitory activity, where higher concentrations showed greater ability to inhibit the growth of e. coli atcc 25922. this is evidenced by the data showing that in treatments p1p10, the inhibition zones were as follows: p1= 5.20 mm, p2= 6.55 mm, p3= 6.77 mm, p4= 6.97 mm, p5= 8.45 mm, p6= 10.40 mm, p7= 11.42 mm, p8= 17.67 mm, p9= 19.62 mm, p10= 26.72 mm. from the average inhibition zone sizes, it is concluded that paederia foetida leaf extract can be considered a candidate drug for preventing diseases caused by e. coli. however further research with various developments is needed to obtain an effective treatment for diseases caused by e. coli. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. eka wahyuning tiyas wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare no funding. references adha sd dan ibrahim m. 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(2016). uji efektivitas ekstrak daun pepaya (carica papaya l.) terhadap pertumbuhan bakteri escherichia coli dan staphylococcus aureus: jurnal kesehatan. 7(3): 497 502. utary, noor sekti. (2016). beberapa daun tanaman di indonesia terhadap bakteri shigella sonnei serta bioautografinya. muhammadiyah surakarta. retrieved fromhttp://eprints.ums.ac.id/48783/20/naskah publikasi.pdf vitalia, pradita a. (2019). pengaruh pemberian ekstrak kasar daun sembukan (paederia foetida l) terhadap daya hambat bakteri pseudomonas aeruginosa secara in vitro. skripsi. universitas brawijaya. malang. widiyanti n.l.p.m., i.w.s. warpala, dan i.a.p. suryanti. (2017) parameter sisika dan perkiraan jumlah terdekat coliform air danau buyan desa pancasari kecamatan sukasada buleleng. jurnal sains dan teknologi. 6(1): 178-188 yustika, g., jalaluddin, s., (2020). analisis parameter leukosit dalam diagnosis neonatorum awitan dini di rsia ananda makassar. 13, 204–214. http://eprints.ums.ac.id/48783 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 549-553 | doi: 10.14421/biomedich.2024.132.549-553 issn 2540-9328 (online) potential of california variety papaya leaf extract (carica papaya l. california variety) as bioherbicide of snake grass weed (cyperus rotundus l.) riki setiawan, mu'jijah, suyamto* biology study program, faculty of pharmaceutical and health sciences, mathla'ul anwar university, banten, indonesia. corresponding author* suyamto35@yahoo.co.id manuscript received: 17 october, 2024. revision accepted: 20 october, 2024. published: 04 november, 2024. abstract efforts to increase production yields are by controlling weeds. weeds that often grow and we encounter in cultivated plants are weed grass (c. rotundus). california papaya leaves have the potential to be a bioherbicide because they contain allelochemicals that can inhibit weed growth. this study aims to determine the effect of papaya leaves (c. papaya) on the growth of teki grass (c. rotundus) and determinetheconcentration level of papaya leaf allelopathy that most effectively inhibits the growth of teki grass.the method used was a completely randomized design (crd) with five replications and four treatmentsof0%, 30%, 50%, and 70%. for data analysis in this study using the spss version 25.0 program. papaya leaf extract (c. papaya) is able to inhibit the growth of weed grass (c. rotundus). the concentration of papaya leaf extract (c. papaya) that most effectively inhibits the growth of weed is 50% concentration with a mortality rate (64%), for the lowest plant heightis50% concentration which is 13.84 cm, for the slowestgrowthrate is 50% concentrationwith a growthrate of -0.16 cm. keywords: papaya leaf, bioherbicide, tackle weed. introduction weeds are a type of plant that can reduce the productivity of cultivated plants on agricultural land, weeds are also parasitic on other plants in taking nutrients, space, water, co2 and light (lestari et al., 2012). directly or indirectly, weeds have a very bad effect on cultivated plants because weeds will become hosts for diseases (dinarto & astriani, 2012). the presence of weeds on agricultural land causes plants not to grow optimally and reduces agricultural production, this happens because of competition (fighting) for nutrients and water caused by these weeds. the size (degree) of weed competition with the main plant will affect the main plant's good or bad growth and, in turn, will affect the main plant's high or low yield (harsono, 1993). weeds are divided into 3 groups: narrow-leaf weeds (grass), broad-leaf weeds, and sedge weeds. one of the sedge weeds is sedge weeds (cyperus rotundus). c. rotundus is a weed whose presence is detrimental to other plants, c. rotundus is a type of weed that we often find in various agricultural cultivation areas, especially in rice fields and is dominant in rice plants. c. rotundus l. causes a significant decrease in production because it has parasitic properties like other weed groups. (kristanto, 2006). control that farmers often carry out tends to use synthetic herbicides to suppress weed populations on agricultural land, if this is done continuously it will have a bad impact which results in environmental pollution. the alternative that needs to be done is to find weed control techniques, from chemical materials to switching to environmentally friendly pesticides because they come from living organisms, often referred to as bioherbicides. papaya plants (carica papaya l.) belong to the caricaceae family and are found in various regions in the country. papaya leaves have an oval leaf blade outline, with finger-like veins, leaf edges with shared canes, pointed tips, heart-shaped leaf bases with irregularly curved leaf lobes, leaf blades with a diameter of about 25-75 cm, and dark green leaves, while the veins are lighter or light green (kartasapoetra, 1996). this study aims to determine whether the extract of papaya leaves of the california variety (carica papaya) can inhibit the growth of nutsedge weeds (cyperus rotundus) and to determine how much extract of papaya leaves of the california variety (carica papaya) is most effective in inhibiting the growth of nutsedge weeds (cyperus rotundus). https://doi.org/10.14421/biomedich.2024.132.549-553 550 biology, medicine, & natural product chemistry 13 (2), 2024: 549-553 research method this research was conducted from august to september 2022 in the research garden and integrated laboratory of the faculty of pharmaceutical and health sciences, mathla'ul anwar university, banten. the tools that will be used in this study are test tubes, analytical scales, 4000 ml glass bottles, 70 ml vail bottles, filter paper, blender, polybags, knives, planting tubs, droppers, rotary evaporators, spatulas, macerators, 20 mesh sieves, rulers, digital scales, knives, label paper, 100 g plastic. the materials used in this study include experimental garden soil, nutsedge (c. rotundus l.), california variety papaya leaves (c. papaya l.), distilled water and 96% ethanol. preparation of planting media the planting media used in this study is soil. the soil is separated into two places: the planting tub and the other in a 20 cm x 20 cm polybag. the land used is the land around the experimental garden of kp. babakan kawung, ds. katumbiri, kec. cigeulis, kab. pandeglang. seeding preparation the prepared soil is then put into the planting tub and used as a medium for sowing nutsedge. 90 nutsedge are to be sown. the seeding tub is watered with 200 ml of water every day, precisely in the afternoon, until it is 15 days old. making papaya leaf extract first of all, prepare papaya leaves (c. papaya l.) which will be used as a bioherbicide extract, papaya leaves (c. papaya l.) are obtained from a garden near the labuan pltu. after that, 9000 grams of fresh leaves are taken, then washed using clean water and rinsed with sterile distilled water, after that they are dried at room temperature until the distilled water on the surface of the leaves is dry. after that, the papaya leaves are dried in the sun until they become dry simplicia. the dried leaves are then ground using a blender, sieved using a mesh sieve no. 20, and stored in a clean, tightly closed container. dry leaf powder (c. papaya l.) was weighed as much as 500 grams and then put into a maceration container. after that, 96% ethanol solvent was added as much as 2 l, then closed and stored for 24 hours in a place protected from direct sunlight while stirring every 12 hours. furthermore, it was filtered and separated between dregs and filtrate. the dregs were re-extracted with new ethanol with a total of 1.5 litres in the 2nd and 3rd remaceration. this was done for 3 x 24 hours. the ethanol filtrate was then collected, and the filtered liquid was evaporated with a rotary evaporator until a thick ethanol extract of papaya leaf extract (c. papaya l.) was obtained. growth test the nutsedge planted for 15 days was then transferred from the planting tub into 20 polybags measuring 5 kg (diameter 20 cm, soil height 20 cm). the transfer was carried out in the afternoon. each polybag contains 3 nutsedge plants. after that, papaya leaf extract bioherbicide was sprayed with various concentrations on the second day after being transferred from the planting tub or on the 16th. the papaya leaf extract (c. papaya l.) was sprayed every 2 days at 08.00 wib until the 24th day after planting. spraying using a dropper pipette of 10 drops on each plant. data collection data collection was carried out while the plants were still being treated. the data in this study were the number of nutsedge weeds that died in each treatment for 24 days. the data collection was carried out every 2 days until 5 observation data were obtained. observation activities were conducted in the morning at 07:00 wib before watering and spraying papaya leaf extract bioherbicide. research parameters the growth parameters measured by the changes observed in this study were plant height, wet weight, dry weight, growth rate and phytotoxicity (poisoning) in nutsedge. measurements were carried out on a scale of the nutsedge population in each polybag. plant height the height of the nutsedge is measured using a ruler from the base of the stem to the tip of the highest leaf. measurements are taken every two days after the plant is transferred from the nursery to the polybag. growth rate the growth rate is obtained from the final height of the nutsedge (cyperus rotundus) on the 26th day minus the height of the nutsedge (cyperus rotundus) on the 15th day so that the growth rate is obtained for 11 days. phytotoxicity phytotoxicity in nutsedge is observed using the truelove scoring system, namely: 0 = no poisoning occurs (with a poisoning level of 0-5%, the shape and colour of the leaves are abnormal) 1 = mild poisoning (with a poisoning level of 610%, abnormal leaf shape and colour) 2 = moderate poisoning (with a poisoning level of 11-20%, abnormal leaf shape and colour) 3 = severe poisoning (with a poisoning level of 21-50%, abnormal leaf shape and colour) 4 = very severe poisoning (with a poisoning level setiawan et al. – potential of california variety papaya leaf extract … 551 >50%, abnormal leaf shape and colour, so that the leaves dry up and fall off until they die) (lasmini and wahid, 2008) wet weight the wet weight of the treated sedge is weighed using an analytical scale. plant weight measurements are carried out on the 24th day after planting. dry weight the dry weight of the treated sedge is first oven-dried and then weighed using an analytical scale. dry weight measurements are carried out on the 24th day after planting. results and discussion based on the results of the experiments that have been carried out, the potential of california papaya leaf extract (c. papaya l.) as a bioherbicide against sedge weeds (c. rotundus) in a study in polybags using 4 treatments, namely control (aquades) and using california papaya leaf extract with concentrations of 30%, 50% and 70%. each treatment with 5 repetitions using 3 plants in each repetition studied every 2 days for 11 days gave very different results at all concentrations of california papaya leaf extract (c. papaya l.) that can inhibit the growth of sedge weeds (c. rotundus l.). the administration of papaya leaf extract (c. papaya l.) to sedge weeds (cyperus rotundus) affected their height. the results of watering between the control and various concentrations showed differences. in terms of the height of sedge grass, various results were obtained, where the height showed the effectiveness of papaya leaf extract on the height of sedge grass. the effect of the reaction of botanical herbicides is also seen in the phytotoxicity that occurs in nutsedge (c. rotundus l.). this level of phytotoxicity shows how effective the concentration of ketapang leaf extract is on the population of nutsedge. the nutsedge (c. rotundus l.) cells are dead and dry, so they cannot produce cell division. all physiological functions in plants have been damaged and lysed, and this causes nutsedge (c. rotundus l.) to wilt, dry and die. the administration of papaya leaf extract (c. papaya l.) to nutsedge (c. rotundus l) affects nutsedge's phytotoxicity. the watering results between the control with extract concentrations of 30%, 50% and 50% showed significantly different results. after observation for 11 days, the most effective extract concentration in inhibiting the growth of nutsedge was a concentration of 50% with a mortality rate of 64%. this is believed because saponins and tannins can work more optimally when given an extract concentration of 50%. polar solvents are often used for the extraction of a simple drug. polar solvents such as ethanol used in the test can attract secondary metabolite compounds such as alkaloids, saponins, phenolic components, carotenoids, and tannins (hidayati, 2012). table 1. average height of nutsedge (cyperus rotundus l.). concentration day 3 day 5 day 7 day 9 day 11 0% 14.40a 14.74a 15.06a 15.46a 15.94a 30% 14.16ab 14.46a 14.62a 14.56b 14.54b 50% 14.04b 14.00b 13.90b 13.94b 13.84b 70% 13.98b 13.86b 13.86b 13.90b 13.92b figure 1. graph of the average height of the nutmeg population after being treated with ketapang leaf extract (terminalia catappa) at various concentrations. table 2. growth rate of nutmeg weed (c. rotundus l.). concentration wet weight 0% 1.94a 30% 0.54b 50% -0.16b 70% -0.08b description: the letters in the same column indicate no significant difference based on the one-way anova test at a 95% confidence level. table 3. phytotoxicity of nutsedge (c. rotundus l.). concentration day 3 day 5 day 7 day 9 day 11 0% 0.00a 0.00a 0.00a 0.00a 0.00a 30% 0.60ab 1.00b 1.60b 2.00b 2.20b 50% 0.80ab 1.40b 1.80b 2.20b 3.20b 70% 1.20b 1.80b 2.00b 2.40b 3.00b description: the same letters in the same column indicate no significant difference based on a way anova test at a 95% confidence level. figure 2. graph of the phytotoxicity of nutsedge. day 1 day 3 day 5 day 7 day 9 day 11 repitition 1 repitition 2 repitition 3 repitition 4 repitition 5 average 552 biology, medicine, & natural product chemistry 13 (2), 2024: 549-553 one of the secondary metabolite compounds suspected of being bioherbicides is tannin, which is included in the phenolic compound group. previous studies have shown that tannin can inhibit growth, eliminate respiration control in mitochondria and disrupt the transport of ca+2 and po4 3ions. in addition, tannin compounds can also deactivate amylase, proteinase, lipase, and urease enzymes, inhibiting gibberellin hormone activity (marisa in senjaya 2012). in addition to tannin, secondary metabolite compounds suspected as bioherbicides are flavonoids. flavonoids also play a role in the growth inhibition process, acting as a strong inhibitor of iaa-oxidase (khotib, 2002). the wet weight of the nutmeg population obtained quite significant results. there is a very visible difference starting from the administration of 70% extract to 0%. each stage of the administration of papaya leaf extract concentration (c. papaya l.) effectively inhibited the growth of nutmeg weeds (cyperus rotundus). in the grouping in the table below, it is known that there are differences in the wet weight of each concentration. giving 70% papaya leaf extract of ketapang leaves (c. papaya l.) effectively results in the growth of the wet weight of nutsedge weeds (c. rotundus l.). table 4. wet weight of nutmeg weed (c. rotundus l.). concentration wet weight 0% 0.50a 30% 0.80b 50% 0.61c 70% 0.55c note: the same letter in the column indicates no significant difference based on the one-way anova test at a 95% confidence level. table 5. dry weight of nutmeg weed (c. rotundus l.). concentration wet weight 0% 0.50a 30% 0.39b 50% 0.32bc 70% 0.29c note: the same letter in the column indicates no significant difference based on the one-way anova test at a 95% confidence level. the dry weight reflects the pattern of plants accumulating products from photosynthesis and is integrated with other environmental factors. in dry weight, it is obtained after being ovened for 1 hour at a temperature of 100 c. by being ovened, all the water in the nutsedge will be lost, thus showing only the weight of the remaining organs. from the results of the table and grouping, the same results were obtained, this shows that there is no difference in the dry weight of nutsedge weeds (c. rotundus l.) as a control or those treated with various concentrations of ketapang leaf extract (terminalia catappa) by anova calculations. however, in descriptive analysis, dry weight has an effect, this can be seen that the dry weight value and the wet weight value are directly proportional. according to (sumarsono, 2012), the absence of a significant difference in dry weight results indicates that the photosynthesis process in nutsedge weeds (c. rotundus l.) both in the control and those treated with papaya leaf extract (c. papaya l.) at various concentrations can still run. by looking at the observations that occur in the results of plant height, phytotoxicity, wet weight and dry weight of the nutsedge plant (c. rotundus l.), it can be said that nutsedge (c. rotundus l.) experiences physiological process disorders (adinugroho, 2008). according to (doflamingo, 2013), the disruption of this physiological process, the plant responds in several forms of symptoms, including the main symptoms, where abnormal growth is seen, can exceed normal size or be smaller than normal size. colour changes, both in leaves, stems, roots, fruits, and flowers, and there is also tissue death; parts of the plant become dry and are marked by wilting of parts of the plant body. wilting events are caused by water absorption not keeping up with the plant's water evaporation rate. if this transpiration process is large enough and water absorption cannot keep up with it, the plant will experience transient wilting. at the same time, the plant will experience permanent wilting if the water condition in the soil has reached the permanent wilting percentage. plants in this condition are difficult to cure because most of their cells have undergone plasmolysis. conclusion papaya leaf extract of the california variety (c. papaya l.) can inhibit the growth of nutsedge weeds (c. rotundus l.). the concentration of papaya leaf extract (c. papaya l.) that showed the most effective inhibition of nutsedge growth was a concentration of 50% with a mortality rate of 64%. competing interests: the authors claim that there are no conflicts of interests. references a. doflamingo, 2013 “fungsi air bagi tanaman”, perduli pertanian indonesia. jakarta. dinarto, w. dan astriani, d. 2012. produktivitas kacang tanah di lahan kering pada berbagai intensitas penyiangan. agri sains, 3(4): 33-43 harsono, a. 1993. gulma pada tanaman kacang tanah. dalam kasno, a., a.winarto, sunardi. kacang tanah (hal. 153-170). monograf balittan 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bean)” fakultas peternakan universitas diponegoro, semarang. w.c. adinugroho, 2008. “konsep timbulnya penyakit” mayor silvikultur tropika sekolah pasca sarjana institut pertanian bogor. untitled-1 volume 6 number 1 2017 issn 2089-6514 (paper) issn 2540-9328 (online) biology, medicine, & natural product chemistry volume 6 – number 1 – 2017 issn 2089-6514 (paper) | issn 2540-9328 (online) honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad jafar luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of 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[indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com antioxidant capacity comparison of ethanolic extract of soursop (annona muricata linn.) leaves and seeds as cancer prevention candidate dyah ayu widyastuti, praptining rahayu analysis of bull sperm dna abnormalities due to cadmium accumulation fuad fitriawan comparative anatomy and histology of black pomfret (formio niger) and nile tilapia (oreochromis niloticus) kidney nurul safitri apriliani, muhammad jafar luthfi anatomical study of male reproductive organs of the indonesian short-nosed fruit bat (cynopterus titthaecheilus temminck, 1825) anisatuzzahro, muhammad jafar luthfi checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul: confirmation and update of flora of java and apg iii widodo, muhammad jafar luthfi 1-4 5-8 9-12 13-17 19-36 volume 6 number 1 2017 published twice a year printed in indonesia front cover: melastoma malabathricum (photo: widodo) issn 2089-6514 (paper) issn 2540-9328 (online) 3: font 4: editorial 5: guidance 6: back volume 5 number 1 2016 issn 2089-6514 biology, medicine, & natural product chemistry volume 5 – number 1 – 2016 issn: 2089-6514 honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad ja’far luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia maizer said nahdi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: r i y a n t o publisher: state islamic university (uin) sunan kalijaga yogyakarta and the society for indonesian biodiversity online: w w w . s c i e n c e b i o l o g y . o r g address: state islamic university (uin) sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@yahoo.com guidance for authors biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international standards in their methodology. scientific feedback (short communicatiom) is only received for manuscript, which criticize published article before. manuscripts will be reviewed by managing editor and invited peer review according to their disciplines. the only articles written in english (u.s. english) are accepted for publication. this journal periodically publishes in may and november. in order to support reduction of global warming as a consequence of transportation vehicles emission and forest degradation for paper manufacturing, management of the journal prefer receiving manuscripts via e-mail rather than in hard copy. manuscripts and its communications can only be addressed to the managing editor; 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proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in java island. sebelas maret university, surakarta, 17-20 july 2000. [indonesian] thesis, dissertation: sugiyarto (2004) soil macro-invertebrates diversity and inter-cropping plants productivity in ag roforestry system based on sengon.[dissertation]. brawijaya university, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l (2008) a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com publication manuscript “in-press” can be cited and mentioned in reference (bibliography); “personal communications” can be cited, but cannot be mentioned in reference. research which not be published or “submitted” cannot be cited. some annotation. manuscript typed without sign link (-) (except repeated word in indonesian). usage of letter “l” (el) to “1” (one) or “o” (oh) to “0” (null) should be avoided. symbols of α, β, χ, etc. included through facility of insert, non altering letter type. no space between words and punctuation mark. progress of manuscript. notification of manuscript whether it is accepted or refused will be notified in about three months since the manuscript received. manuscript is refused if the content does not in line with the journal mission, low quality, inappropriate format, complicated language style, dishonesty of research authenticity, or no answer of correspondence in a certain period. author or first authors at a group manuscript will get one original copy of journal containing manuscript submitted not more than a month after publication. offprint or reprint is only available with special request. note: author(s) agree to transfer copy right of published paper to biology, medicine, & natural product chemistry. author shall no longer be allowed to publish manuscript completely without publisher permission. authors or others allowed multiply article in this journal as long as not for commercial purposes. for the new invention, authors suggested to manage its patent before publishing in this journal. notification: all communications are strongly recommended to be undertaken through email. new record marsdenia tenacissima (asclepiadoideae, apocynaceae) in gunung ijo baturagung yogyakarta widodo, muhammad jafar luthfi local stability analysis of a mathematical model of the interaction of two populations of differential equations (host-parasitoid) dewi anggreini determination of levels leisure village patronage uin sunan kalijaga yogyakarta: improving governance patronage towards rural village green and environmentally friendly supriatna, thaqibul fikri niyartama, iwan kuswidi modified alizarin red s-alcian blue staining for reptilian skeleton muhammad jafar luthfi checklist of macroalgae in waisai coast, raja ampat retno suryandari, widodo 1-8 9-14 15-18 19-22 23-32 volume 5 number 1 2016 published twice a year printed in indonesia front cover: tacca palmata (family taccaceae) (photo: widodo) issn 2089-6514 cover jurnal biomenaprochy vol 5 num 1 2016.pdf (p.1) editorial j.biomedich v5 n1.pdf (p.2) guidance for authors j.biomedich.pdf (p.3) cover jurnal biomenaprochy vol 5 num 1 2016 2.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 617-625 | doi: 10.14421/biomedich.2024.132.617-625 issn 2540-9328 (online) exploring the therapeutic potential of kalanchoe pinnata: a comprehensive analysis of bioactive compounds and pharmacological activities dakshina bhusal1, manila poudel2,3, ashika tamang1, sujan dhital1, nirmal parajuli1, timila shrestha1,2, samjhana bharati1,2, binita maharjan1,2, jhashanath adhikari subin4, bishnu prasad marasini5, ram lal swagat shrestha1,2,* 1department of chemistry, amrit campus, tribhuvan university, lainchaur, kathmandu 44600, nepal. 2kathmandu valley college, syuchatar bridge, kalanki, kathmandu 44600, nepal. 3department of biotechnology, national college, tribhuvan university, lainchaur, kathmandu 44600, nepal. 4bioinformatics and cheminformatics division, scientific research and training nepal p. ltd, bhaktapur 44800, nepal. 5nepal health research council, ramshah path, kathmandu 44600, nepal. corresponding author* swagatstha@gmail.com abstract natural products are vital in drug discovery for their bioactive compounds. this study evaluated kalanchoe pinnata's leaf extracts for antidiabetic, antimicrobial, antioxidant, and cytotoxic properties. gc-ms identified seven components in hexane and twenty in methanol extracts. the ethyl acetate extract had high phenolic (485.17 mg gae/g) and flavonoid (40.25 mg qe/g) contents. methanol extract showed strong antioxidant (ic50: 293.53 µg/ml) and anti-alpha glucosidase (ic50: 195.39 µg/ml) activity. antimicrobial tests showed varied effectiveness, while chloroform and acetone extracts exhibited notable cytotoxicity. admet predictions assessed absorption and blood-brain barrier penetration, highlighting k. pinnata's potential for drug development. keywords: admet; antioxidant; cytotoxicity; gc-ms; phytochemical screening. introduction natural products, sourced from living organisms like plants, animals, and microorganisms, constitute a wide array of bioactive compounds. they play a crucial role in drug discovery due to their abundant availability and varied chemical compositions, serving as the primary source of many medicinal active ingredients (harvey, 2008). typically, they possess biological functions suitable for application in drug discovery and design (zhang et al., 2013). the medicinal properties of plants arise from their constituents, which induce physiological responses in the human body (olowa & nuñeza, 2013). these constituents include alkaloids, essential oils, tannins, and resins. kalanchoe pinnata (linn.) pers. is a plant primarily located in temperate and tropical areas worldwide. this tall perennial has thick, fleshy, scalloped leaves and hollow stems bearing pendulous bell-shaped flowers (okwu & nnamdi, 2011). it has long been recognized for its diverse pharmacological properties, often utilized in traditional medicine to address various significant human ailments (rajsekhar et al., 2016). the compounds found in the leaves of k. pinnata comprise various substances such as flavonoids, alkaloids, triterpenes, glycosides, and steroids. they are known for their potential to reduce inflammation and regulate blood sugar levels, and are widely used in traditional remedies for diabetes management, kidney stones, and fighting infections (ojewole, 2005). kidney stones, a prevalent urological condition, involve the development and occasional passage of clusters of crystals within the urinary tract (singh & saini, 2009). compounds in k. pinnata hinder kidney stone formation and offer diverse benefits: quercetin reduces crystal deposition, phenolics dissolve stones, kampferol inhibits crystal formation, glycosides protect kidneys, and steroids aid in stone passage, collectively providing comprehensive management for kidney stones and related urinary issues (nagpal & sharma, 2020). diabetes mellitus (dm) represents a significant multifaceted chronic ailment contributing substantially to global morbidity (arroyave et al., 2020). traditional clinical approaches to managing dm have primarily revolved around administering oral hypoglycemic medications and insulin (george et al., 2019). many of the therapy choices in current use are burdened by manuscript received: 30 august, 2024. revision accepted: 09 january, 2025. published: 23 january, 2025. https://doi.org/10.14421/biomedich.2024.132.617-625 618 biology, medicine, & natural product chemistry 13 (2), 2024: 617-625 various adverse effects. these encompass issues like weight gain and hypoglycemia associated with insulin, beta-cell exhaustion with secretagogues, lactic acidosis with sensitizers like metformin, and gastric disturbances with alpha-glucosidase inhibitors. extracts from k. pinnata have shown efficacy in controlling blood sugar levels (efanova et al., 1998; kimmel & inzucchi, 2005; lalau & race, 1999; patil et al., 2013). besides these, plant extracts also possess the ability to inhibit the growth of certain harmful microorganisms. they can hinder germ proliferation and their antimicrobial potency can be evaluated accordingly (kalemba & kunicka, 2005). the main objective of the research was to investigate the biological effects such as antidiabetic, antimicrobial, antioxidant, and cytotoxic properties of various extracts obtained from k. pinnata leaves, as well as to evaluate the absorption, distribution, metabolism, excretion, and toxicity (admet) properties of the compounds in these extracts. the in vivo lethality test has proven effective as an initial assessment of cytotoxic and antitumor agents (ramachandran et al., 2011). the compounds verified via gc-ms were also assessed for their ability to reach the brain and be absorbed in the gastrointestinal tract using the swissadme tool. this model predicts two crucial properties of a compound: its ability to permeate the blood-brain barrier (bbb), indicating whether it can reach the brain, and its gastrointestinal (gi) absorption, determining if it can be effectively absorbed in the gi tract, which is vital for oral bioavailability. the outcomes of this current study would provide fundamental insights into the most prospective plant extracts, serving as a foundation for developing novel therapeutic tools of significant importance. materials and methods study area the plant part was collected from the rupandehi district of nepal, which was identified as kalanchoe pinnata at the department of botany, amrit science campus, thamel, kathmandu, 44600 (supplementary table 1). sample preparation about 4 kg of the plant's leaves were collected, cleaned, and air-dried for a few days. they were then shade-dried, ground into a fine powder (almost 400 g), and stored at a low temperature for later use. extraction process the ultrasonic extraction process began with 400 g of powdered plant material in a sterile, dry beaker. hexane was added, stirred, and the beaker was immersed in an ultrasonic cleaner tub (30×15×20 cm) filled one-third with distilled water. after extraction, the contents were decanted, cooled, and filtered. the filtrate was then concentrated using a rota evaporator, resulting in an extract that was measured, dried, and stored in airtight vials. this process was repeated three times using various solvents (dichloromethane, chloroform, ethyl acetate, acetone, methanol, and water), with hexane added to the filtered residue before sonication. gas chromatography-mass spectroscopy before undergoing gc-ms analysis, the concentrated extract obtained from hexane and methanol via the rotaevaporator was dissolved in chloroform. gc-ms was utilized to separate and analyze the multicomponent mixtures. phytochemical screening the phytochemical analysis of various extracts was done based on the protocol by banu and cathrine in 2015 (banu & cathrine, 2015). total phenolic content the overall phenolic content of the plant extract was determined using a colorimetric technique called folinciocalteu, which relies on an oxidation-reduction process. the gallic acid concentration was used as a benchmark. milligrams of gallic acid equivalent per gram of dry extract (mg gae/g of dry extract) were used to represent the sample's total phenolic content based on the gallic acid calibration curve. initially, a stock solution was prepared with an extract concentration of 1000 μg/ml (ppm) by dissolving 1 milligram of extract in 1 ml of methanol. subsequently, different concentrations of the extracts were prepared by serially diluting the stock solution, and their respective absorbance values were recorded (balasundram et al., 2006). the following formula 1 was used to determine the sample's total phenolic content, which was represented as milligrams of gallic acid equivalent per gram. 𝐶 = 𝑐𝑉/ 𝑚 (1) where c is the total content of the phenolic compounds (mg/g) in gallic acid equivalent, c is the concentration of the gallic acid established from the calibration curve (μg/ml), v is the volume of the extract (ml), and m is the weight of the plant extract (μg). total flavonoid content to quantify the total flavonoid content of the plant extract, an aluminum chloride colorimetric assay was performed (chandra et al., 2014). quercetin was used as the reference. firstly, a stock solution, was prepared with an extract concentration of 1000 μg/ml (ppm) by dissolving 1 milligram of the extract in 1 ml of methanol. following this, the stock solution was subjected to repeated dilution to produce a range of extract concentrations. the absorbance values of these diluted solutions were recorded. bhusal et al. – exploring the therapeutic potential of kalanchoe pinnata: … 619 formula 2 was used to determine the sample's total flavonoid content, represented as milligrams of quercetin equivalents (qe) per gram of extract. 𝐶 = 𝑐𝑉/𝑚 (2) where c is the total flavonoid content (mg/g) in quercetin equivalent (qe), c is the concentration of quercetin established from the calibration curve (μg/ml), v is the volume of the extract (ml), and m is the weight of the plant extract (μg). dpph free radical scavenging activity the plant extracts were assessed for their ability to scavenge radicals using the spectrophotometric dpph method (sanna et al., 2012). the capacity to neutralize the dpph radical was determined by observing the decrease in absorbance and calculated using the following formula 3. 𝑅𝑎𝑑𝑖𝑐𝑎𝑙 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 (%) = [(𝐴0 − 𝐴𝑆)/𝐴0] × 100 (3) where a0 is the absorbance of the control (dpph solution + methanol) and as is the absorbance of the test sample. the amount of an adequate sample needed to neutralize 50% of dpph free radicals is known as the ic50 (50% inhibitory concentration) value. by graphing the extract concentration vs. the corresponding scavenging action, the inhibition curve and ic50 values were generated. 10 mg of each extract (chloroform, ethyl acetate, and methanol) were dissolved in 10 ml of methanol to create a stock solution of 1 mg/ml. this stock solution was then successively diluted to concentrations of 1000 μg/ml, 500 μg/ml, 250 μg/ml, and 125 μg/ml. for each concentration, 50 μl of ascorbic acid was mixed with 150 μl of methanolic dpph solution in a 96-well plate, totaling 200 μl. after incubation in the dark for 20 minutes, the absorbance at 520 nm was measured using a spectrophotometer, with methanol and dpph as blanks. antimicrobial activity microbial growth inhibition was evaluated using the paper disc diffusion method, and the outcome was quantified as a zone of inhibition (zoi) value. in this assay, one gram-positive bacterium (bacillus subtilis: atcc 6051), one gram-negative bacterium (escherichia coli: atcc 8739), and a fungus (candida albicans: atcc 2091) were utilized. the efficacy of various k. pinnata leaf extracts in inhibiting microbial growth at a constant concentration (5 µg/ml) was evaluated. screening and evaluation of antimicrobial activity initially, 100 µl of culture broth from each strain was spread onto mueller hinton agar plates and incubated at 37 °c for 15 minutes. after incubation, the plates were left overnight at the same temperature. the antibacterial effects of the samples on b. subtilis, e. coli, and c. albicans strains were evaluated the next day. kanamycin was the positive control, while dmso was the negative control. the zoi produced by each sample was measured using a scale. alpha-amylase inhibition assay the α-amylase inhibitory potential of plant extracts was assessed using the 3,5-dinitrosalicylic acid (dnsa) technique. k. pinnata leaf extract was diluted with 10% dmso to create different concentrations. these mixtures were further combined with buffer and nacl at ph 6.9. the extract and α-amylase solution were mixed in a 200 μl volume and incubated at 30 °c for 10 minutes. the starch solution was added and left for 3 minutes before stopping the process with the dnsa reagent. after heating in a water bath, the sample was diluted with distilled water and absorbance at 540 nm was measured using a uv spectrophotometer. the α-amylase inhibitory activity (%) was calculated using formula 4. % 𝛼 − 𝑎𝑚𝑦𝑙𝑎𝑠𝑒 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴𝑏𝑠100%𝐶𝑜𝑛𝑡𝑟𝑜𝑙−𝐴𝑏𝑠𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠100%𝐶𝑜𝑛𝑡𝑟𝑜𝑙 × 100 (4) extract concentration was plotted against the percentage of α-amylase inhibition, and ic50 values were obtained from the resulting graph. brine shrimp lethality assay (bsla) the study employed a brine shrimp lethality bioassay to investigate the cytotoxicity of the plant extracts. understanding the toxic effects of plant extracts is essential for safe treatment. the brine shrimp lethality test, utilizing artemia salina, is a cost-effective and versatile method widely used to screen diverse chemical compounds for bioactivity (waghulde et al., 2020). before beginning the process, artificial seawater was created by combining the components in the specified amounts as detailed in supplementary table 2. brine shrimp eggs, approximately 50 mg in weight, were dispersed over simulated seawater collected in a beaker, covered with aluminum foil, with a few small pores created to facilitate heat and light conductivity. the beaker was left at room temperature for 48 hours, exposed to radiation from a 60-watt bulb. a stock solution of 2 mg extract was prepared by adding 2 ml of dmso (dimethyl sulfoxide), resulting in a concentration of 1000 ppm (mg/ml). 620 biology, medicine, & natural product chemistry 13 (2), 2024: 617-625 toxicities of compounds were tested at 1, 0.5, 0.125, and 0.0625 ppm in sea-water solutions with 2 ml dmso. in each test, ten nauplii were used, and survivors were counted after 24 hours. three replications were performed for each concentration. a blank control was conducted using dmso. the lethal concentration for 50% mortality (lc50) after 24 hours of exposure, was determined using the probit method to measure the toxicity of the extract or fractions. lc50 values greater than 1000 ppm for plant extracts were considered inactive. the mortality percentage was then computed using the following formula 5. % 𝑀𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 = 𝑁𝑜. of dead shrimps total no. of shrimps × 100 (5) admet properties in addition to conducting laboratory assays, the boiled-egg model was used for compounds verified through gc-ms analysis to determine their gastrointestinal absorption and blood-brain barrier (bbb) penetration capabilities (dong et al., 2018). the canonical smiles retrieved from pubchem (https://pubchem.ncbi.nlm.nih.gov/) for each compound were uploaded to the web server, and their properties were analyzed. moreover, admet properties (absorption, distribution, metabolism, excretion, toxicity) were assessed using admet lab 2.0, and the toxicity class for each compound was identified with protox 3.0 (https://tox.charite.de/protox3/). results and discussion gram yield the ultrasonication technique was utilized to extract compounds from the k. pinnata plant. the resulting extracts using hexane, dichloromethane, chloroform, ethyl acetate, acetone, methanol, and water showed varying yields: 7.31 g, 9.08 g, 2.21 g, 0.21 g, 1.04 g, 10.34 g, and 14.50 g, respectively (supplementary table 3). qualitative analysis of phytochemicals the extracts of k. pinnata were analyzed for their phytochemical contents, and the presence of these compounds was confirmed through observable color changes. among the extracts, varying compositions of volatile oils, alkaloids, carbohydrates, phenolic compounds, tannins, flavonoids, terpenoids, quinones, reducing sugar, and saponins were identified (supplementary table 4). the results of the phytochemical screening revealed that the polar extracts predominantly contained the highest concentrations of phytochemicals. almost all solvent extracts tested positive for flavonoids and terpenoids. the absence of alkaloids in the extracts might be attributed to the method of extraction, which involved the use of an ultrasound sonicator followed by concentration using a rota-evaporator. this process could potentially lead to the decomposition of alkaloids due to heat and molecular vibration. the literature often presents slightly different findings compared to the results obtained. discrepancies can arise due to several factors, including differences in plant altitude, varying environmental conditions, the specific extraction method employed, the timing of sample collection, differences in laboratory setups, and variations in chemical grades used during analysis. consequently, the outcomes of phytochemical screenings for a particular sample may vary from one study to another, even when targeting the same constituents. gc-ms spectra analysis hexane and methanol extract of k. pinnata were used for the gc-ms analysis. the analysis revealed seven primary components in the composition of the k. pinnata hexane extract (table 1) (supplementary fig. 1 to supplementary fig. 8) and 20 components in the methanol extract (table 2) (supplementary figure 9 to supplementary figure 29). the compounds along with their pubchem cid and canonical smiles are presented in supplementary table 5. table 1. components present in the hexane extract according to gc-ms analysis. s.n. name of compound retention time molecular formula area (%) 1. 2h-1,2,3,4-tetrazole-2-ethanol, .alpha.-(chloromethyl)-5-phenyl8.228 c10h11cln4o 11.13 2. n-benzyl-2-(5-phenyl-2h-tetrazol-2-yl)acetamide 8.506 c16h15n5o 3.90 3. 2h-tetrazole-2-ethanole, 5-phenyl13.942 c9h10n4o 27.44 4. 1,4-methanocycloocta[d]pyridazine, 1,4,4a,5,6,9,10,10a-octahydro-11,11dimethyl-, (1.alpha.,4.alpha.,4a.alpha.,10a.alpha.) 15.677 c13h20n2 11.98 5. pentadecafluorooctanoicacid, dodec-2-en-1-yl ester 24.668 c20h23f15o2 29.36 6. octanoic acid, 4-methyl-,ethyl ester,(.+/-.)26.96 c11h22o2 10.99 7. dodecane, 2-methyl28.00 c13h28 5.19 bhusal et al. – exploring the therapeutic potential of kalanchoe pinnata: … 621 table 2. components present in the methanol extract according to gc-ms analysis. s.n. name of compound retention time molecular formula area (%) 1. 2h-tetrazole-2-ethanole, 5-phenyl9.542 c9h10n4o 19.61 2. carbon dioxide 6.425 co2 9.51 3. 1,4-methanocycloocta[d]pyridazine, 1,4,4a,5,6,9,10,10a-octahydro11,11-dimethyl-, (1.alpha.,4.alpha.,4a.alpha.,10a.alpha.) 16.229 c13h20n2 6.29 4. 1-bromo-2-phenyl-cyclopropanphosphonic acid, diethyl ester 15.281 c13h18bro3p 0.70 5. 4-diethylaminophenyl isothiocyanate 18.255 c11h14n2s 0.82 6. pentadecafluorooctanoic acid, undecyl ester 19.710 c19h23f15o2 0.79 7. oxirane, octyl21.876 c10h20o 0.76 8. methyl 6-methyloctanoate 22.244 c10h20o2 0.89 9. alpha-methyl-alpha-[4-methylpentyl]oxiranmethanol 22.858 c10h20o2 0.74 10. bacteriochlorophyll-c-stearyl 25.126 c52h72mgn4o4 1.71 11. levomenthol 24.284 c10h20o 1.68 12. 2-isopropenyl-5-methyl-6-hepten-1-ol 25.028 c11h20o 0.67 13. 13-borabicyclo[7.3.0]tridecane, 13-butoxy-, (z)-or (e)25.439 c16h31bo 0.61 14. 8-methylnonanoic acid, methyl ester 26.555 c11h22o2 22.38 15. decanoic acid, silver (1+) salt 26.285 c10h19ago2 1.07 16. 1,2-oxathiane, 6-dodecyl-,2,2-dioxide 27.204 c16h32o3s 0.92 17. methyl-9,10-octadecadienoate 28.517 c19h34o2 14.50 18. cyclododecyne 28.639 c12h20 9.65 19. 3,7-dimethyl-6-nonen-1-ol 28.799 c11h22o 2.46 20. decanoic acid, methyl ester 28.930 c11h22o2 3.73 estimation of total phenolic and flavonoid content the results indicated that the methanol extract of k. pinnata contains 182.87 mg gae/g, while the ethyl acetate extract contains 485.17 mg gae/g of phenolic content. these findings imply that phenolic compounds generally show higher solubility in polar organic solvents compared to non-polar ones. additionally, ethyl acetate displayed a markedly higher total flavonoid concentration of 40.25 mg qe/g, in contrast to methanol, which showed a concentration of 19.05 mg qe/g (table 3). table 3. total phenolic and flavonoid content in ethyl acetate and methanolic extract of k. pinnata leaves. extract ethyl acetate methanol total phenolic content (mg gae /g extract) 485.17 182.87 total flavonoid content (mg qe/g extract) 40.25 19.05 antioxidant activity the chloroform extract of k. pinnata demonstrated an ic50 value of 2219.41 µg/ml, indicating relatively low antioxidant activity. in contrast, the ethyl acetate extract showed a much lower ic50 value of 365.22 µg/ml (figure 1), suggesting stronger antioxidant properties. the methanol extract exhibited the highest antioxidant activity with an ic50 value of 293.53 µg/ml at 520 nm absorbance. these ic50 values reflect the varying effectiveness of the extracts in scavenging free radicals, with lower values indicating greater antioxidant potency. 0 500 1000 1500 2000 2500 chloroform ethyl acetate methanol ic 5 0 µ g /m l extracts figure 1. ic50 values of different extracts (chloroform, ethyl acetate, and methanol) of k. pinnata. antimicrobial activity the antimicrobial activity of various extracts was measured by the diameter of the inhibition zone (zoi) in centimeters, compared to the negative control (dmso). for bacillus subtilis, the hexane, dichloromethane (dcm), chloroform, and acetone extracts showed inhibition zones ranging from 0.70 to 0.85 cm, indicating moderate antimicrobial activity. the ethyl acetate, methanol, and aqueous extracts displayed smaller inhibition zones of 0.50 cm. in the case of escherichia coli, both the hexane and methanol extracts had the largest inhibition zones at 0.80 cm, while the other extracts ranged between 0.60 and 0.70 cm. for candida albicans, the hexane, dcm, and acetone extracts exhibited the largest inhibition zones of 0.75 cm, while the ethyl acetate and aqueous extracts had smaller inhibition zones of 0.60 and 0.50 cm, respectively. 622 biology, medicine, & natural product chemistry 13 (2), 2024: 617-625 overall, the extracts varied in their antimicrobial efficacy across different microbes. the effectiveness of extracts varied with the type of microbe. for example, hexane and acetone extracts showed higher inhibition against candida albicans compared to other extracts, suggesting that certain extracts might be more effective against specific microbes. table 4. antimicrobial activity is shown by the different extracts in diameter (cm). microbes negative control dmso hexane extract dcm extract chloroform extract ethyl acetate extract acetone extract methanol extract aqueous extract bacillus subtilis (atcc 6051) 0 0.85 0.80 0.85 0.7 0.85 0.50 0.50 escherichia coli (atcc 8739) 0 0.65 0.80 0.70 0.65 0.60 0.80 0.70 candida albicans (atcc 2091) 0 0.70 0.75 0.60 0.60 0.75 0.70 0.50 alpha-amylase inhibition assay in the alpha-amylase inhibition assay, k. pinnata acetone extract displayed an ic50 of 285.71 µg/ml, while ethyl acetate extract showed 504.21 µg/ml, and methanol extract exhibited a value of 195.39 µg/ml (figure 2). comparatively, among these three extracts, the methanol extract of k. pinnata showed higher α-amylase inhibition activity. 0 100 200 300 400 500 600 acetone ethyl acetate methanol ic 5 0 va lu e s extracts figure 2. ic50 values of different extracts for α-amylase inhibition. cytotoxicity activity after the freshly hatched live nauplii were exposed to concentrations of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, and 0.0625 mg/ml, respectively, the lc50 value of chloroform, acetone, and methanol extracts was computed (table 5). it was found that the concentration of the extract directly correlated with the level of lethality (supplementary table 6 to supplementary table 8). the highest and lowest numbers of s brine shrimp larvae perished at doses of 1 mg/ml and 0.0625 mg/ml, respectively. table 5. lethality concentration of 3 different extracts of k. pinnata. extracts lc50 (mg/ml) chloroform 0.18 acetone 0.17 methanol 0.29 among the three extracts, the chloroform extract exhibited a high mortality rate of 96.67%, while the methanol extract showed the lowest mortality rate of 56.66% at the extract concentration of 0.5 mg/ml. the lc50 values for the chloroform, acetone, and methanol extracts of k. pinnata were determined to be 0.181 bhusal et al. – exploring the therapeutic potential of kalanchoe pinnata: … 623 mg/ml, 0.175 mg/ml, and 0.29 mg/ml, respectively (table 5). as a result, extracts were observed to be pharmacologically significant and harmful to artemia salina (brine shrimp) larvae. the cytotoxic properties of these extracts may be attributed to the presence of alkaloids, tannins, and flavonoids (waghulde et al., 2020). the significant lethality observed in various plant extracts towards brine shrimp implies the existence of potent cytotoxic compounds, highlighting the need for additional investigation. admet analysis according to the boiled-egg model, the majority of molecules were concentrated within the yellow and white regions, indicating they possess balanced wlogp and tpsa values. out of the 27 compounds, 3 were found to be outside of the range as seen in the boiled-egg model. molecule 9 was located at the lower end of the wlogp scale (below -1), indicating it has low lipophilicity. as a pgp+ substrate, it might be actively transported out of cells, which could impact its bioavailability and penetration. therefore, its low lipophilicity and the possibility of being actively transported out of cells make it an outlier compared to the other molecules. such status implies that it may have limited absorption or distribution characteristics that are favorable for central nervous system (cns)-targeted therapies. the other two outliers, molecule 1 and molecule 2, were positioned towards the higher end of the tpsa scale (around 100), indicating a larger polar surface area (figure 3). this correlates with lower membrane permeability, which can affect their ability to be absorbed in the intestine and penetrate the bbb. their high tpsa values make them outliers, as they fall outside the optimal range for hia and bbb penetration. all the compounds were found to fall into class 4 toxicity, indicating they have low toxicity but can be detrimental if given in high doses according to the protox3.0 server. this classification suggests that these compounds are relatively safe and pose a low risk of causing significant harm at typical exposure levels. figure 3. boiled-egg model for the identified 27 compounds. similarly, the admet properties of molecules extracted from hexane and methanol were analyzed. all molecules generally adhered to lipinski's rule of five, indicating good drug-like properties. the majority of compounds exhibited excellent caco-2 permeability and were classified as cyp3a4 substrates and inhibitors, suggesting effective absorption and metabolism. however, there was variability in their carcinogenic potential, ranging from non-carcinogenic to carcinogenic (supplementary table 9). clearance rates were predominantly excellent, though a few molecules (6 out of 27) showed poor clearance, which could impact their overall efficacy and safety profiles. molecule 5 was rejected by lipinski’s rule, indicating poor drug-like qualities, although it had excellent permeability and clearance. molecule 9, while passing lipinski’s rule and showing excellent permeability, suffered from poor clearance, which potentially impacted its effectiveness. molecule 17 was rejected by lipinski’s rule and had both poor permeability and clearance, suggesting significant issues with absorption and excretion. these factors highlighted potential challenges for these molecules in terms of drug development and effectiveness. 624 biology, medicine, & natural product chemistry 13 (2), 2024: 617-625 conclusions this study comprehensively evaluates the biological activities and phytochemical composition of kalanchoe pinnata leaf extracts, highlighting their potential as therapeutic agents. the phytochemical analysis confirmed the presence of a diverse array of bioactive compounds, including flavonoids, phenolics, and terpenoids, across various extracts. notably, the methanol and ethyl acetate extracts exhibited substantial antioxidant activity, with ic50 values indicating significant radical scavenging potential. the ethyl acetate extract, in particular, demonstrated the highest phenolic and flavonoid contents, correlating with its potent antioxidant effects. the alpha-amylase inhibition assay revealed that the methanol extract had superior αamylase inhibitory activity, suggesting its potential for diabetes management. the brine shrimp lethality assay underscored significant toxic effects in the chloroform and acetone extracts, highlighting the need for further investigation into their mechanisms and safety. admet analysis showed that k. pinnata extracts generally exhibited favorable absorption and distribution profiles, though toxicity predictions indicated potential risks depending on dosage and exposure. the results emphasized the need for further research to balance therapeutic benefits with safety. overall, k. pinnata demonstrated considerable promise for antioxidant, antimicrobial, and antidiabetic applications, with future studies needed to refine safety and efficacy while considering admet factors. acknowledgements: the authors express their gratitude to the department of plant resources, kathmandu, nepal for conducting the gc-ms experiments. authors’ contributions: conceptualization: ram lal swagat shrestha; methodology: dakshina bhusal, ashika tamang, timila shrestha, samjhana bharati, and binita maharjan; discussion of results: sujan dhital and nirmal parajuli; writing original draft: dakshina bhusal, manila poudel; review and editing: jhashanath adhikari subin, bishnu prasad marasini, and ram lal swagat shrestha. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that this study did not receive any funding. references arroyave, f., montaño, d., & lizcano, f. 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(2013). recent advances in natural products from plants for treatment of liver diseases. european journal of medicinal chemistry, 63, 570–577. https://doi.org/10.1016/j.ejmech.2012.12.062 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 15-21 | doi: 10.14421/biomedich.2021.101.15-21 issn 2540-9328 (online) production and optimization of pectinase from pectinolytic fungi cultivated on mango peels and pectin subjected to submerged fermentation kelemu mulluye1,*, ameha kebede2, negussie bussa3 1department of central laboratory, college of ulmd; 2department of biological science and biotechnology, college of natural and computational sciences; 3department of food science and post-harvest technology, institute of technology; haramaya university, ethiopia. corresponding author* kelemumuluye@gmail.com manuscript received: 14 december, 2020. revision accepted: 25 june, 2021. published: 02 july, 2021. abstract pectinases are the group of enzymes that degrade pectin. this study was conducted with the aim of isolation of efficient pectinase producing pectinolytic fungi from the decomposing mango peels using extracted mango peels pectin as a growth substrate under submerged fermentation, determining optimum pectinase production conditions with regards to some physicochemical parameters. the organisms were screened for the production of pectinase using pectin agar media, and the two active pectinolytic fungi (p1 and p2) were isolated. pectinase production media was later used for the lab scale production of pectinase by inoculating p1 and p2 and incubating for 7 days. the enzyme was extracted after seven days of fermentation and every day tested for their pectinolytic activity. p2 showed relatively higher pectinolytic activity and was therefore used for further studies. p2 was inoculated into a broth containing mango pectin under submerged fermentation. results indicate that a pectin yield of mango peel 17.75%. different parameters optimization processes were investigated on submerged fermentation namely ph, incubation period, temperature and substrate concentration optima were found 6, 4 days, 35oc and 1.5% respectively. the result suggests that mango peels have high pectin content and can be used for the value-added synthesis of pectinase. keywords: mango peels; pectinase; pectin; submerged fermentation. introduction enzymes are biological catalysts that are prerequisite for various chemical reactions produced by plants, animals and microbes. they are specific for carrying out a task and efficiently speedup the rate of reaction ultimately accelerates all metabolic processes. microbial enzymes are gaining consideration with the current development of enzyme technology (gurung et al., 2013). as nature has vast potential of microbial enzyme resource and enzymes from microbes are of great significance in developing bioprocesses for industries. there is continuous increasing demand for industrial enzymes with the rising need for sustainable solutions (adrio and demain, 2014). pectinases are among the first enzymes to be used at home. their commercial application was first observed in 1930 for the preparation of wines and fruit juice. pectinase are today one of the upcoming enzymes of the commercial sector. it has been reported that microbial pectinase account for 25% of the global food enzymes sales (jayani et al., 2005). pectinase, that break down pectin is a well-known term for commercial enzyme preparation; a polysaccharide substrate, found in the cell wall of plants. this enzyme splits polygalacturonic acid into monogalacturonic acid by opening glycosidic linkages. through this process, it softens the cell wall (khan et al., 2012). plants and microorganisms are two major sources of the pectinase enzyme. but, microbial source of pectinase has become increasingly important from both technical and economic point of view. among microbes, fungi as enzyme producers have many advantages since they are normally gras (generally regarded as safe) microorganisms and their enzymes are extracellular which makes it easy recuperation from fermentation broth (murthy and naidu,2010). maximum yield of pectinase can be achieved using agricultural wastes. the use of waste products is economically viable. furthermore, the production can also be increased after optimization of fermentation conditions and different cultural parameters (bibi et al. 2016). optimization of different physicochemical parameters, including incubation period, temperature, ph, carbon, and nitrogen sources for selected fungal https://doi.org/10.14421/biomedich.2021.101.15-21 16 biology, medicine, & natural product chemistry 10 (1), 2021: 15-21 strains, is a key step for the enhanced production of pectinase (neeta et al., 2011). mango peel is one of the major by-products from the mango pulp processing industries. during the processing of mango fruit, peel and stone are generated as waste (40–50% of total fruit mass). waste generated from mango processing constitutes 20–25% peel, which was found to be a good source for the extraction of pectin of good quality, with a high degree of esterification and phenolic compounds (berardini et al., 2005). pectin acts as the inducer for the production of pectinase enzymes by microbial systems, and pectin-rich mango peels are considered to be a good source for pectinase production and ideal substrate for the decomposition of mango peels by microorganisms (kumar et al., 2012). many urban areas in ethiopia have simple fruit/juice processing cottage industries which usually discard mango peels after extracting the flesh for mango juice. these produce a huge amount of waste throughout the year. these waste materials can be constructively used to produce important products including pectin and pectinases as a replacement for dumping them in the environment where they might at times generate pollution. this is a promising way of converting wastes to wealth. therefore, optimization of in-vitro production of pectinase by pectinolytic fungi using pectin obtained from mango peels as growth substrate under submerged fermentation. desired to extract partially purified pectin from powdered mango peels, isolate and screen active pectinolytic fungi from decaying vegetables and fruits as well as from soils containing decomposed mango peels, and optimize the effect of some physicochemical parameters. materials and methods substrate preparation mango (mangifera indica) peels were collected from tsega fruit and juice house of harar city, ethiopia. the peels were washed and cut into small pieces and then treated with 97% ethanol to reduce the microbial load. the ethanol treated peels were washed with distilled water and sun-dried for 7 days. the dried peels were then ground to a powder using a milling machine and stored in a clean container at room temperature until used for extraction. extraction of pectin from mango peels pectin was extracted using the method described by mccready (1970) as cited by ezugwu et al. (2014). one hundred gram ground mango peels were weighed into a 2000 ml beaker containing 800 ml of distilled water and 12 g of sodium hexa-metaphosphate. the ph adjusted with 3n hcl to 2.2±0.1. the mixture was heated in a water bath at 70oc for 1hr and stirred with a stirrer. the extract was filtered through a muslin cloth and the residue washed with 200 ml of distilled water. the washings were added to the filtrate. the filtrate was concentrated by evaporation on a hot plate to 1/5th of the initial volume. the concentrated pectin was cooled to 50oc and poured into a volume of 0.5 m hcl-ethanol (1:3), solution. the mixture was stirred for 30 min and allowed to stand for 1hr. the precipitate was vacuum filtered, washed with hcl-ethanol solution and finally washed with acetone to remove traces of hcl and ethanol. the extract was dried in an oven at 40oc for a few hours to constant weight, finely ground and then used as mango pectin. %𝑌𝑖𝑒𝑙𝑑 𝑜𝑓 𝑝𝑒𝑐𝑡𝑖𝑛 = 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑝𝑒𝑐𝑡𝑖𝑛 𝑜𝑏𝑡𝑎𝑖𝑛𝑒𝑑 (𝑔) 𝑇𝑜𝑡𝑎𝑙 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑚𝑎𝑛𝑔𝑜 𝑝𝑒𝑒𝑙 𝑝𝑜𝑤𝑑𝑒𝑟 𝑢𝑠𝑒𝑑 (𝑔) × 100 collection of soil samples containing decomposing mango peels soil samples were collected from dumping sites of decompose waste that contained mango peels at addis ababa, koshe massive garbage dump, ethiopia, using the methods described by martin et al. (2004). the soil samples were collected in a clean dry plastic container and transported to the laboratory. preparation of broth media soil samples (3 g) collected from the site of decomposing/ decaying fruit and vegetable containing mango peels were homogenized in sterile medium containing 1% mango pectin, 0.14% of (nh4)2 so4, 0.2% of k2hpo4, 0.02% of mgso4.7h2o, 0.1% of nutrient solution containing 5 mg/l feso4.7 h2o, 1.6 mg/l mnso4.h2o, 1.4 mg/l znso4.7h2o, 2.0 mg/l cocl2. the mixture was at 30 oc for 24 hours. preparation of solid media the pectin agar medium (pam) contained 1% mango pectin, 0.14% of (nh4)2so4, 0.2% of k2hpo4, 0.02% of mgso4.7h2o, 0.1% of nutrient solution containing; 5 mg/l feso4.7h2o, 1.6 mg/l mnso4.h2o, 1.4 mg/l znso4.7h2o, 2.0 mg/l cocl2 and 3% agar-agar (the gelling agent) (w/v). the medium was autoclaved at 121 oc for 15min. it was allowed to cool to about 45 oc and then poured into sterile petri dishes and allowed to gel. the plates were then incubated in an incubator at 37 oc overnight to check for sterility (ezugwu et al., 2014). mulluye, et al. – production and optimization of pectinase from … 17 inoculation of plates and sub-culturing a loop of homogenized culture from the broth medium was streaked onto the solid medium under the flame of bunsen burner. streaks were made from each side of the plate, marking an initial point, with sterilization of the wire loop after each side has been completed. the plates were thereafter incubated at 35 oc till visible colonies were observed. all morphologically contrasting colonies were purified by repeated streaking and subculturing on separate plates. this process was continued till pure fungal cultures were obtained (ezugwu et al., 2013). storage of pure fungal isolates pure fungal isolates were maintained on potato dextrose agar (pda) slopes or slants as stock cultures. pda media were prepared according to the manufacturer’s specification. in the latter, 3.9 g of pda powder was weighed and added in a small volume of distilled water and made up to 100 ml. this was boiled and simultaneously stirred until a clear solution of molten pda was produced. microscopic and macroscopic features of the isolated fungi and identification three days old pure cultures were examined. the colour, nature of mycelia or spores and growth patterns were also observed. the three days old pure culture was used in preparing microscopic slides. a little bit of the mycelia was dropped on the slide and a drop of lacto phenol blue was added to it. a cover-slip was placed over it and examination was performed under the light microscope at x400 magnification. identification was carried out by relating features and the micrographs to identification of common aspergillus species (klich, 2002). the fermentation broth pectinase was produced by submerged fermentation according to the method of martin et al. (2004). submerged fermentation (smf) technique was employed using a 250 ml erlenmeyer flask containing 100 ml of sterile cultivation medium optimized for pectinase with 0.1% nh4no3, 0.1% nh4h2po4, 0.1% mgs04.7h2o and 1% mango pectin. the flask was covered with aluminium foil and autoclaved at 121 oc for 15 min. inoculation of the broth from the pda slants, fresh plates were prepared and inoculated. three days old cultures were used to inoculate the flasks. in every sterile flask, two discs of the respective fungal isolates were added using a cork borer of diameter 10 mm and then plugged properly. the culture was incubated for 7 days at 30 oc. extracting the crude enzyme at each day of extract, flasks were selected from the respective groups and mycelia biomass separated using filtration technique. the broth was filtered through cheesecloth followed by whatman no. 1 filter paper. each day, the filtrate was analysed for pectinase activity till the 7th day of fermentation. pectinase assay pectinase activity was evaluated by assaying for polygalacturonase (pg) activity of the enzyme. this was achieved by measuring the release of reducing groups from pectin using 3, 5-dinitrosalicylic acid (dns) reagent assay method described by miller (1959) as cited in wang et al. (1997). the reaction mixture containing 0.5 ml of 0.5% pectin in 0.05 m sodium acetate buffer of ph 5.0 and 0.5 ml of enzyme solution was incubated for 1 hr. one ml of dns reagent was added and the reaction was stopped by boiling the mixture in a boiling water bath for 10 min. the volume of the mixture was made up to 4 ml with 1 ml of 1.4 m of rochelle salt (sodium potassium tartrate) solution and 1 ml of distilled water. the reaction mixture was allowed to cool and the absorbance was read at 575 nm. one unit of enzyme activity was defined as the amount of enzyme that catalyses the release of one micromole of galacturonic acid per minute. optimization of physicochemical parameter for pectinase production the isolate was grown under different temperature, ph, and substrate concentration to evaluate the influence of growth conditions on pectinase production. the optimization was carried out according to meena et al. (2015), with slight modification. thus when one parameter (e.g. temperature) was evaluated for its effect on pectinase production the other parameters (e.g. ph, and incubation period) were kept constant at 5 and 96 hours, respectively, and similarly, when the effect of ph was evaluated, the other two parameters (i.e. temperature and incubation period) were kept constant at 30 oc and 96 hours, respectively. effect of temperature on the production of pectinase the isolate was inoculated into a 50 ml production medium kept in 250 ml capacity erlenmeyer flask and was subsequently incubated in an incubator whose temperatures are adjusted to 25, 30, 35, 40, 45, 50 and 55 oc with the aim of determining the optimum temperature required for the production of the pectinase.the liquid medium was prepared by weighing 1 g of the extracted mango pectin and mixing with 0.1 g of nh4no3, 0.1g of nh4h2po4, 0.1 g of mgso4.7h2o, and 100 ml distilled water. the mixture was then autoclaved at 121 oc for 15min. the medium was cooled to room temperature in a 18 biology, medicine, & natural product chemistry 10 (1), 2021: 15-21 bio-safety cabinet and inoculated with the three days old pure culture of the fungal isolate. in every sterile flask, disc of the pectinolytic fungi isolates was added using a cork borer of 10 mm diameter and then plugged properly. the culture was incubated for 4 days. after incubation, the culture broth was filtered and assayed (bezawada and raju, 2018). effect of substrate concentration on pectinase production the isolate was inoculated into each of the 50 ml production medium consisting of varying concentrations (i.e. 0.2, 0.5, 1, 1.5, and 2%) of the extracted mango pectin in 250 ml erlenmeyer flask and incubated at 30 oc in an incubator for 4 days. after incubation, each culture broth was filtered and was assayed. all pectin broth media preparations and inoculations were done. effect of ph on the production of pectinase in this experiment, all the procedures followed in the preparation of media and inoculations were similar to those described in effect of temperature on the production of pectinase except that the incubation was done at varying ph (4.0, 5.0, 6.0, 7.0, 8.0 and 9.0) and at fixed temperature and concentration of pectin (30 oc and 1%, respectively). furthermore, assay for pectinase activity was done after 4 days of incubation (ketipally and ram, 2018). data analysis the data were entered into the computer and statistical analysis was done using anova provided by the sas version 9.1 and significant difference examined for different parameters and between pectinolytic fungal isolates at 95% confidence level of significance and at p <0.05 results and discussion yield of extracted mango pectin in this research, pectin was extracted from mango peel with a yield of 17.75% at ph 2.2, the temperature of 70 oc and extraction time of 60 min using the ethanolhcl method described by mccready (1970). rehman et al. (2004) reported mango pectin extraction yields of 13.45%, 21.0% and 15.1% using hydrochloric acid, sulphuric acid, and nitric acid respectively at the of ph 2.5, temperature 80 oc and extraction time of 120 min. the differences in the yields could be as a result of differences in the sources of pectin and other factors such as extraction technique, changes in ph, temperature, extraction time and environmental factors (kertesz 1951; rehman et al., 2004). table 1. experimental observation yield of extracted mango pectin. contents unit value solution of ph –– 2.2±0.1 volume taken for extraction ml 800 amount of mango peel sample added g 100 amount of sodium hexametaphosphate added g 12 extraction temperature oc 70 extraction time min 60 amount of pectin obtained g 17.75 yield of pectin obtained % 17.75 isolation and screening of pectinase producing fungi in total, two isolates were identified from soil samples that contain mango peel. based on the colony characteristics the fungi exhibited on the selective growth media, the two isolates were assigned the codes p1 and p2 for further characterization and identification purpose. figure 1 shows microscopic photographs of the two fungal isolates. the isolates were identified based on their microscopic features. table 2. identification of pectinolytic fungal based on their colony morphology, growth characteristics, microscopic features and growth at temperature of 35 oc. code of the isolate colony characteristics on pectin agar media growth on petri plate (85 x 15mm diameter) microscopic features probable identity p1 moderately growing colonies with carbon black /deep brownish black colour conid i.a e.; reverse-colorless to pale yellow, exudates lacking produced a submerged form of mycelium covered the plate in 8 days conidial heads radiate, vesicles nearly globose, conidiophores up to 3 mm in length, phialides biseriate aspergillus niger p2 rapidly spreading, colonies are colorless to yellow or velvety, dull blue-green, reverse colourless to varying g in shades, produces tufted aerial mycelium up to felted floccose forms covered the plate in 7 days conidial heads columnar, densely crowded, flask-shaped vesicles, conidiophores short in length, and phialides uniseriate. aspergillus fumigates mulluye, et al. – production and optimization of pectinase from … 19 p1 p2 figure 1. micrograph of pectinolytic fungal isolate (40x). production of pectinases by submerged fermentation (smf) pectinase production increase with the increase in time duration up to 96 hr then it decreases (table.3). the maximum production of both isolates occurs on 4th day (96 hr) incubation period. production of pectinase exhibited the highest enzyme activity of p1 and p2 was at 0.0276 ± 0.0015 and 0.0290 ± 0.0107 u/ml respectively. from the two isolates, p2 showed relatively maximum pectinolytic activity; therefore, isolate p2 was used for further studies of pectinase optimization and characterization. table 3. effect of incubation period on enzyme production. the incubation period (days) isolated pectinolytic fungi enzyme activity u/ ml p1 p2 1 0.0144 ± 0.0012d 0.0145 ± 0.0009c 2 0.0170 ± 0.0011dc 0.0209 ± 0.0021abc 3 0.0177 + 0.0014c 0.0219 ± 0.0020abc 4 0.0276 + 0.0015a 0.0290 ± 0.0107a 5 0.0258 ± 0.0006a 0.0259 ± 0.0076a 6 0.0218 ± 0.0029b 0.0259 ± 0.0076ab 7 0.0171 ± 0.0018dc 0.0162 ± 0.0005bc p1 = pectinolytic fungal isolate one p2 = pectinolytic fungal isolate two values are mean ± s.d. of 3 replicates a,b,c,d means within a column with values followed by different superscripts are significantly different at (p < 0.05), values followed by same superscripts are not significantly different at (p < 0.05). determination of optimum temperature required for pectinase production by p2. the experiment was run at various temperatures between 25 and 55 oc (table 4). 35 oc temperatures were the most suitable temperature for the growth and production of pectinase activity. maximum enzyme activity 0.0298 ± 0.00047 u/ml was found at 35 oc and lower activity 0.0020 ± 0.0013 u/ml was showed at 55oc. any increase in temperature above 35 oc affects metabolic activities of the microorganism which results in reduced enzyme production. table 4. effect of temperature on pectinase production. temperature ( oc ) enzyme activity u/ml 25 0.0093±0.00034c 30 0.0292±0.0107a 35 0.0298±0.00047a 40 0.0211±0.0021b 45 0.027±0.0024ab 50 0.0024±0.0010c 55 0.0020±0.0013c determination of optimum concentration of mango pectin required for pectinase production by p2. the effect of substrate concentration for pectinase production p2 was incubated at different concentration of mango pectin as 0.1, 0.2, 0.5, 1.0, 1.5 and 2% (fig.2). enzyme production increased with increase in concentration. a slight decrease in enzyme production was observed until the concentration reached 2%. the maximum 0.013109 u/ml enzyme production was observed at 1.5% of mango pectin. figure 2. pectinase production by p2 versus substrate concentration. determination of optimum ph required for pectinase production by p2. the ph of the growth medium was found to have a marked effect on pectinase production (table 5). the highest enzyme yield was observed at ph 6.0. ph alters enzyme conformation, recognition site, active site, and substrate conformation, and hence determining its optimum value. this is very critical in biochemical characterization of enzymes (palmer, 1995). 20 biology, medicine, & natural product chemistry 10 (1), 2021: 15-21 table 5. effect of ph pectinase production. ph enzyme activity u/ml 4 0.0095±0.00091bc 5 0.0100 ±0.00093b 6 0.0123±0.00036a 7 0.0122±0.00037a 8 0.0086±0.00037c 9 0.0096±0.0011bc summary and conclusions an abundant amount of waste materials are produced by agricultural and fruit processing industries, which pose considerable disposal problems and ultimately leads to pollution. vast varieties of microorganisms are present in the environment which can be exploited for the utilization of waste materials. for example in the processing of mango fruits, a large proportion of the produced wastes are in the form of peel. these peels contain substantial amounts of pectin. from these investigations, it is evidenced that the mango peels with 17.75% pectin content were successfully used to encourage the production of pectinase under submerged fermentation process. the pectin extracted from mango peels act as the inducer for the production of pectinase enzyme. microorganisms serve as a major source of enzyme. even the majority of industrial enzymes are of microbial origin. thus an attempt of the present study is to isolate and screen for potential producers of pectinase from microorganisms. total two pectinolytic fungi isolates were identified as the efficient producer between them screening pectinolytic fungi isolate p2 was found as efficient producers. in order to obtain high yields of pectinases, it is essential to optimize the fermentation medium used for pectinase production. isolate p2 was grown for different temperature, ph, time and substrate concentration because it is essential to maximize optimum condition for production. the evidence from this study suggests that, it is highly promising to use pectin extracted from mango peels as a substrate for the production of pectinases in a submerged fermentation system using p2 isolate and a high extent solve the problem of environmental pollution by reducing mango wastes (peels). acknowledgments: we are thanks to haramaya university laboratory management directorate (hulmd) and the school of animal and range sciences for providing all the required laboratory facilities. we are also grateful to mr. yimeslal atnafu, ms. yehararwork abebaw and mr. dilnessa baile for their technical assistance. conflict of interest: the authors declares that there are no conflicts of interest concerning the publication of this article. references adrio, j. l. and a. l. demain. (2014). microbial enzymes: tools for biotechnological processes. biomolecule, 4(1): 117-139. bezawada, p. and raju, k.j. (2018). screening of pectinolytic fungi and optimization of process parameters using guava peel powder as substrate under solid state fermentation. international journal of engineering science invention, 7 (10):43-47. ezugwu, a.l., eze, s.o.o., chilaka, f.c. and anyanwu, c.u. (2013). production and characterization of pectinases obtained from aspergillus fumigatus in submerged fermentation system using pectin extracted from mango peels as carbon source. plant products research journal, 16(1): 47-53. ezugwu, a.l., ezike, t.c., ibeawuchi, a.n., nsude, c.a., udenwobele, d.i., eze, s.o.o., anyawu, c.u. and chilaka, f.c. (2014). comparative studies on pectinases obtained from aspergillus fumigatus and aspergillus niger in submerged fermentation system using pectin extracted from mango, orange, and pineapple peels as carbon sources. nigerian journal of biotechnology, 28(1): 26-34. gurung, n., s. ray, s. bose and v. rai. (2013). a broader view: microbial enzymes and their relevance in industries, medicine, and beyond. biomedical. res. int., 2013: 1-18. jayani, r.s., saxena, s. and gupta, r. (2005). microbial pectinolytic enzymes: a review. process biochemistry, 40(9): 2931-2944. kertesz, z.j. (1951). the pectic substance. inter science publishers, new york, 68. khan, a., sahay, s. and rai, n. (2012). production and optimization of pectinase enzyme using aspergillus niger strains in solid state fermentation. research in biotechnology, 3(3): 19-25. klich, m.a. (2002). identification of common aspergillus species. centraalbureau voor schimme lcultures kumar, y.s., varakumar, s. and reddy, o.v.s., (2012). evaluation of antioxidant and sensory properties of mango (mangifera indica l.) wine. cyta-journal of food, 10(1):1220. martin, n., souza, s.r.d., silva, r.d. and gomes, e. (2004). pectinase production by fungal strains in solid-state fermentation using agro-industrial bio product. brazilian archives of biology and technology, 47(5): 813-819. mccready, r.m. (1970). methods on food analysis. academic press, new york. meena, k.k., jaipal, m.k. and singh, u. (2015). production kinetics and characterization of pectinase enzyme from aspergillus niger. south asian journal of food technology and environment, 1(2):131-135. miller, g.l., (1959). use of dinitrosalicylic acid reagent for determination of reducing sugar. ana lytical chemistry, 31(3): 426-428. murthy, p.s., and naidu, m.m. (2010). protease production by aspergillus oryzae in solid-state fermentation utilizing coffee by-products. world applied sciences journal, 8(2): 199-205. mulluye, et al. – production and optimization of pectinase from … 21 neeta, r., anupama, s., anjuvan, s. and giridhar, s. (2011). production of polygalacturonase and pectin methylesterase from agro waste by using various isolation of aspergillus niger. insight microbiology, 1(1):1-7. palmer, t. (1995). understanding enzymes. ellis horwood ltd. england. 104: 352 – 365. rehman, z.u., salariya, a.m., habib, f. and shah, w.h. (2004). utilization of mango peels as a source of pectin. journalchemical society of pakistan, 26:73-76. wang, g., michailides, t.j. and bostock, r.m. (1997). improved detection of polygalacturonase activity due to mucor piriformis with a modified dinitrosalicylic acid re agent. phytopathology, 87(2): 161-163. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 427-434 | doi: 10.14421/biomedich.2025.141.427-434 issn 2540-9328 (online) effectiveness of cherry tomato extract in gel form to accelerate the healing process of excision wounds in wistar white rats soilia fertilita1, muhammad zulfadli2, veny larasati1,*, riana sari puspita rasyid1, fifa argentina3, yudhi tanta4, medina athiah5 1department of histology; 2medical education study program; 3department of dermatology venerology; 4departement of internal medicine; 5departement of biochemistry, faculty of medicine, sriwijaya university, palembang, indonesia. corresponding author* venylarasati@fk.unsri.ac.id abstract cherry tomatoes (solanum lycopersicum l. var. cerasiforme) are rich in antioxidants, particularly flavonoids and lycopene, which help reduce free radicals and promote wound healing. flavonoids exhibit anti-inflammatory, antioxidant, antibacterial, and antidiabetic properties. they enhance wound contraction, collagen deposition, granulation tissue formation, and epithelialization in wound healing. this study evaluates the efficacy of cherry tomato extract gel in accelerating excision wound healing in wistar rats. this in vivo experimental study used a post-test control group design. twenty-five 2–3-month-old wistar rats with excision wounds were treated with cherry tomato extract gel or controls. the five groups included a positive control (nacl), a negative control (cmc-na gel), and treatment groups receiving 8%, 12%, or 16% extract gel. wound size was measured on days 3, 5, 7, and 9, with data analyzed using one-way anova and post hoc tests. the extract-treated groups exhibited nearly complete wound closure by day 9. the wound size of the treatment groups significantly differed from the control groups (p<0.05). cherry tomato extract gel significantly accelerates wound closure at 8%, 12%, and 16% concentrations, with the best results observed at 16% (p<0.05), surpassing even the positive control. the 16% extract gel group demonstrated the most effective wound healing. cherry tomato extract gel significantly enhances wound healing, with the 16% concentration demonstrating the most effective acceleration of wound closure. these findings suggest that cherry tomato extract gel, particularly at 16%, holds promise as a potent wound-healing agent. keywords: cherry tomatoes extract; wound healing; lycopene. introduction wounds are a pathological condition characterized by tissue damage or loss, which can result from various factors such as trauma, temperature fluctuations, chemical exposure, explosions, electrical shock, or animal bites. based on their classification, wounds can be categorized as open or closed, depending on whether the skin and underlying tissues are compromised. open wounds, including incisions and excisions, are particularly susceptible to infections and complications if not managed appropriately. (dewi, 2021) wound healing is a complex biological process involving three primary phases: inflammation, proliferation, and maturation. each phase plays a critical role in restoring tissue integrity. however, several factors can impair wound healing, including infections and systemic conditions such as diabetes. therefore, appropriate wound management is essential to prevent complications and promote effective recovery.(abdullah et al., 2022), (kano et al., 2019) to accelerate wound healing, the use of effective therapeutic agents is crucial. among the various alternatives explored, natural compounds such as cherry tomatoes (solanum lycopersicum l. var. cerasiforme) have gained attention due to their rich antioxidant and phytochemical content. studies have demonstrated that cherry tomatoes contain bioactive compounds such as lycopene, flavonoids, and vitamin c, which contribute to wound healing by stimulating fibroblast proliferation, enhancing collagen synthesis, and exhibiting antiinflammatory and antibacterial properties. (husna, 2023), (sikumbang et al., 2022) previous research on cherry tomato extracts has shown promising results in promoting wound healing. studies have indicated that tomato fruit extract can accelerate burn wound healing in rabbit models, with an optimal concentration of 70% demonstrating effectiveness in preventing infection and enhancing collagen formation. additionally, other investigations have revealed that a 16% concentration of cherry tomato extract gel significantly reduces wound healing time compared to negative controls in murine models.(husna, manuscript received: 01 april, 2025. revision accepted: 15 july, 2025. published: 17 july, 2025. https://doi.org/10.14421/biomedich.2025.141.427-434 428 biology, medicine, & natural product chemistry 14 (1), 2025: 427-434 2023) other studies using tomato seed extract have also shown highly effective results in promoting wound healing. in this study, the experimental group treated with tomato seed extract experienced wound closure three days earlier than the control group, demonstrating a three-day advantage in the healing process. (ezon-ebidor et al., 2019) the global incidence of wounds continues to rise. data from the ministry of health of the republic of indonesia indicate that the prevalence of open wounds in indonesia reached 20.1% in 2018. given the increasing incidence of wounds and the demand for safe and effective therapeutic solutions, this study aims to explore the potential of cherry tomato extract in gel formulation as a wound-healing agent. the findings of this research are expected to contribute significantly to developing novel wound-healing therapies and improve the quality of life for individuals affected by injuries. (kemenkes, 2022), (idzni, 2021), (widyaningsih, 2022) materials and method this study utilized an in vivo laboratory experimental design, specifically employing a post-test-only control group methodology. the research was conducted at the animal house laboratory and the biochemistry laboratory, faculty of medicine, sriwijaya university, in conjunction with the barokah anatomical pathology laboratory. the experimental procedures were carried out between june and november 2024. ethical approval for this study was granted by the research ethics commission of sriwijaya university on july 15, 2024, under approval number 196-2024. the study involved male wistar rats (rattus norvegicus), aged 2–3 months, weighing 250 to 300 grams. all subjects were confirmed to be healthy, exhibiting no observable abnormalities. a total of 25 wistar rats were randomly distributed into five experimental groups using a simple random sampling technique. the groups were as follows: (1) positive control group—wound induction followed by treatment with 10% povidone-iodine; (2) negative control group— wound induction followed by treatment with carboxymethyl cellulose sodium (cmc-na) gel; (3) treatment group 1 (p1)—wound induction followed by treatment with 16% cherry tomato extract gel; (4) treatment group 2 (p2)—wound induction followed by treatment with 12% cherry tomato extract gel; and (5) treatment group 3 (p3)—wound induction followed by treatment with 8% cherry tomato extract gel. each experimental group comprised five wistar rats, each housed individually in separate cages to prevent potential cross-contamination or behavioral interference. prior to the commencement of the study, the animals underwent an acclimatization period of seven days to ensure adaptation to the laboratory environment. the experimental procedure involved the induction of a standardized excisional wound on the dorsal region of each rat, measuring 5 mm in length, 2 mm in width, and 2 mm in depth. the assigned treatment—cherry tomato extract gel, povidone-iodine, or carboxymethyl cellulose sodium (cmc-na) gel—was topically applied after wound induction. the treatment regimen was administered once daily for nine consecutive days. all animals received standard laboratory chow and water ad libitum throughout the study to maintain consistent nutritional support. preparation of cherry tomato extract gel and determination of dosage a total of 11 kg of cherry tomatoes were weighed, wrapped in aluminum foil, and subjected to boiling in a water bath at 100 °c for 5 minutes. following thermal processing, the tomatoes were blended and transferred into a 500 ml beaker, after which 1,650 ml of methanol was added, and the mixture was stirred for 5 minutes. the homogenized mixture was then filtered, and the resulting sediment was divided into four 1000 ml erlenmeyer flasks lined with carbon-coated paper. each erlenmeyer flask was supplemented with a solvent mixture consisting of n-hexane, acetone, and methanol in varying ratios: 2:1:1, 1:2:1, and 1:1:1, as well as a polyethylene (pe)-acetone mixture in a 3:1 ratio, with a fixed solvent-to-material ratio of 5:1. the mixtures were subjected to shaking at 150 rpm for 30 minutes and subsequently transferred to a separatory funnel, where 110 ml of distilled water was added. after thorough shaking, the mixtures were left undisturbed to allow phase separation. the upper (non-polar) phase was collected and evaporated using a rotary evaporator to obtain a concentrated extract, which was then stored in a glass bottle for volume measurement.(junnaeni et al., 2019) the cherry tomato extract gel was formulated in three concentrations: 8%, 12%, and 16%. the gel was applied twice daily using a cotton bud at a dosage of 0.1 grams per application for nine consecutive days. wound size measurements were conducted following the treatment period. excision wound induction male white rats (rattus norvegicus) were prepared by shaving the dorsal area using a sterile razor. local anesthesia was administered via subcutaneous injection of ketamine hydrochloride at a dose of 0.1 cc per 100 g of body weight. following anesthesia, an elliptical excisional wound measuring approximately 5 mm in length, 2 mm in depth, and 2 mm in width was created on the dorsal region using a sterile surgical scalpel. the wound area was then disinfected with a sterile nacl solution. cherry tomato extract gel was applied topically using a cotton bud at a dosage of 0.1 grams, twice daily (morning and evening), for nine consecutive days at fertilita et al. – cherry tomato extract for accelerating wound healing 429 concentrations of 8%, 12%, and 16%. after each application, the wound was covered with sterile gauze. figure 1. excision wound. the excision procedure was performed after allowing the anesthesia to take effect for five minutes. the designated wound site was cleaned with an alcohol swab and marked using a sterile surgical marker for accurate measurement. the incision was made using sterile surgical scissors modified for precision. following wound creation, the site was cleaned with nacl solution and covered with sterile gauze to prevent contamination. (fazri, 2020) statistical data analysis data analysis was conducted using the statistical package for the social sciences (spss) software, version 26. initially, tests for normality and homogeneity of variance were performed to assess the suitability of the data for parametric analysis. a one-way analysis of variance (anova) was then conducted to determine differences between groups, with statistical significance set at p < 0.05. post hoc analysis was carried out to identify significant differences between specific treatment groups across the five-dose variables. results and discussion phytochemical compound analysis the phytochemical analysis of the extract aims to identify the classes of compounds present in cherry tomato (solanum lycopersicum var. cerasiforme) extract. the identification of secondary bioactive compounds was conducted using a qualitative phytochemical test. this test involves reactions between specific reagents and the concentrated extract, resulting in characteristic color changes that indicate the presence of secondary metabolites. table 1. phytochemical analysis of cherry tomato extract. no compound result description 1. alkaloid : a) dragendroff b) mayer c) wagner a) orange-red precipitate b) yellowish-white precipitate c) brown precipitate + + + 2. flavonoid dark brown + 3. triterpenoid/steroid blue green (steroid) + 4. saponin foam formation (2 cm height for 10 min) + 5. tanin dark blue + 6. kuinon dark green note: (+) indicates the presence of the tested secondary metabolite. figure 2. phytochemical analysis : a, b, c. alkaloid test (dragendroff, mayer, wagner), d. flavonoid test, e.triterpenoid test, f.saponin test, g. tanin test, quinon test. 430 biology, medicine, & natural product chemistry 14 (1), 2025: 427-434 this analysis confirms the presence of various secondary metabolites in cherry tomato extract, including alkaloids, flavonoids, steroids, saponins, and tannins, while quinones were not detected. these bioactive compounds contribute to the medicinal properties of cherry tomatoes, particularly in wound healing applications. this result indicates that the extraction method employed in the phytochemical analysis successfully preserved the presence of alkaloids, flavonoids, triterpenoids/steroids, saponins, and tannins in the cherry tomato (solanum lycopersicum var. cerasiforme) extract. these compounds represent key bioactive components in this study, contributing to their potential therapeutic effects. wound healing the descriptive analysis of wound size in rats indicated that groups treated with cherry tomato extract gel at various concentrations (16%, 12%, and 8%) significantly reduced in wound size from day 3 to day 9. on day 3, the treatment groups had smaller average wound sizes than the negative control group. the highest average wound size was observed in the negative control group (25.42 mm²), while the smallest was recorded in the 16% extract gel group (13.56 mm²). table 2. wound size on days 3, 5, 7 and 9. groups (n=5) mean std deviasi ci 95% min-max lower upper day 3 cge 16% 13.56 3.00 9.83 17.29 10.39 17.54 cge 12% 16.05 1.75 13.87 18.22 14.60 18.63 cge 8% 18.63 3.34 14.47 22.78 13.01 21.38 positive control 29.06 3.66 24.50 33.61 25.67 34.92 negative control 25.24 2.57 22.03 28.44 21.67 28.46 day 5 cge 16% 4.22 1.64 2.18 6.26 3.11 7.10 cge 12% 10.11 0.841 9.06 11.15 8.90 11.17 cge 8% 13.27 2.50 10.16 16.38 11.00 16.50 positive control 21.12 2.21 18.36 23.87 18.00 23.33 negative control 20.06 1.96 17.62 22.51 18.08 23.30 day 7 cge 16% 0.46 0.59 -0.27 1.19 0.00 1.47 cge 12% 4.31 1.49 2.45 6.17 2.67 6.34 cge 8% 7.61 2.09 5.00 10.22 4.57 9.60 positive control 13.62 5.21 7.14 20.10 7.20 21.77 negative control 14.38 0.92 13.23 15.54 13.53 15.70 day 9 cge 16% 0.00 0.00 -0.00 0.00 0.00 0.00 cge 12% 0.50 0.37 0.03 0.97 0.11 1.00 cge 8% 2.53 0.69 1.67 3.39 1.58 3.45 positive control 8.13 4.05 3.09 13.16 4.46 14.59 negative control 9.98 1.41 8.22 11.74 8.86 11.59 based on the one-way anova statistical test results in table 2, wound size consistently decreased across all treatment and control groups from day 3 to day 9 (p < 0.001). on day 3, the treatment group receiving the 16% concentration exhibited the smallest wound size compared to the control groups. by day 9, a similar trend was observed, with the 16% concentration group recording the smallest wound size (0.0010 ± 0.00182), indicating optimal wound healing. these findings indicate a significant difference in wound healing progression among the groups, with the cherry tomato extract treatment demonstrating a statistically meaningful improvement compared to both control groups. fertilita et al. – cherry tomato extract for accelerating wound healing 431 table 3. multiple comparisons between all groups on day 3 groups compared mean difference standard error p-value cge 16% vs cge 12% -2.484 1.863 1 cge 16% vs cge 8% -5.062 1.863 0.133 cge 16% vs pc -15.49600* 1.863 <.001 cge 16% vs nc -11.67400* 1.863 <.001 cge 12% vs cge 8% -2.578 1.863 1 cge 12% vs pc -13.01200* 1.863 <.001 cge 12% vs nc -9.19000* 1.863 <.001 cge 8% vs pc -10.43400* 1.863 <.001 cge 8% vs nc -6.61200* 1.863 0.02 bonferroni post hoc test on day 3 based on the data in table 3, statistical analysis indicates a significant difference in wound healing among rats on day 3 between the treatment and control groups (p < 0.05). the treatment groups with 16%, 12%, and 8% concentrations exhibited significant differences compared to the positive and negative control groups. the treatment group with a 16% concentration exhibited the greatest mean difference compared to both the negative and positive control groups. table 4. multiple comparison between all groups on day 5 groups compared mean difference standard error p-value cge 16% vs cge 12% -5.89000* 1.21492 <.001 cge 16% vs cge 8% -9.05200* 1.21492 <.001 cge 16% vs pc -16.89800* 1.21492 <.001 cge 16% vs nc -15.84600* 1.21492 <.001 cge 12% vs cge 8% -3.162 1.21492 0.17 cge 12% vs pc -11.00800* 1.21492 <.001 cge 12% vs nc -9.95600* 1.21492 <.001 cge 8% vs pc -7.84600* 1.21492 <.001 cge 8% vs nc -6.79400* 1.21492 <.001 bonferroni post hoc test on day 5 table 5. multiple comparison between all groups on day 7. groups compared mean difference standard error p-value cge 16% vs cge 12% -3.85482 1.67514 0.323 cge 16% vs cge 8% -7.15527* 1.67514 0.004 cge 16% vs pc -13.16396* 1.67514 <.001 cge 16% vs nc -13.92863* 1.67514 <.001 cge 12% vs cge 8% -3.30045 1.67514 0.628 cge 12% vs pc -9.30914* 1.67514 <.001 cge 12% vs nc -10.07381* 1.67514 <.001 cge 8% vs pc -6.00869* 1.67514 0.018 cge 8% vs nc -6.77336* 1.67514 0.006 bonferroni post hoc test on day 7 the table above indicates that on the fifth and seventh days, the wound closure process progressed better than on the previous days. the treatment group with a 16% extract concentration exhibited the most effective wound closure compared to all other treatment groups. the treatment groups with 12% and 8% extract concentrations also showed significant differences compared to the positive and negative control groups table 6. multiple comparison between all groups on day 9. groups compared mean difference standard error p-value cge 16% vs cge 12% -0.50169 1.23608 1 cge 16% vs cge 8% -2.53455 1.23608 0.537 cge 16% vs pc -8.12984* 1.23608 <.001 cge 16% vs nc -9.98710* 1.23608 <.001 cge 12% vs cge 8% -2.03286 1.23608 1 cge 12% vs pc -7.62815* 1.23608 <.001 cge 12% vs nc -9.48541* 1.23608 <.001 cge 8% vs pc -5.59529* 1.23608 0.002 cge 8% vs nc -7.45255* 1.23608 <.001 table 6 shows that on the ninth day, wounds treated with 8% and 12% extract concentrations had begun to close completely, similar to those treated with a 16% extract concentration. in contrast, the wound size in the positive and negative control groups remained relatively large, resulting in statistically significant differences from the treatment groups.. discussion this study employs a methodological approach focused on developing stimulatory agents that facilitate the biological healing processes in wistar strain albino mice. the wound healing process follows distinct phases: hemostasis, inflammation, proliferation, and remodeling. the research investigates the efficacy of active compounds extracted from cherry tomatoes (solanum lycopersicum l. var. cerasiforme) using n-hexane, acetone, and methanol as solvents. these solvents are widely utilized in the pharmaceutical industry for drug formulation, extraction of bioactive compounds from natural materials, and various medicinal preparations.(hakim & saputri, 2020) the excision wound model used in this study involved creating an elliptical wound on wistar strain albino mice, followed by topical application of an ethanol-based cherry tomato extract gel. the gel was applied twice every 24 hours until wound healing was observed. the healing process was assessed based on the contraction of wound edges, drying of the wound area, natural scab detachment, and complete wound closure. observations were conducted until full wound closure was achieved on day 9 for all experimental groups. wound healing efficacy was quantified by measuring wound length, width, and depth changes at regular 24hour intervals. the proliferative phase of wound healing involves the formation of granulation tissue, which includes the 432 biology, medicine, & natural product chemistry 14 (1), 2025: 427-434 development of new capillary blood vessels, macrophage activation, and fibroblast proliferation. this phase encompasses several key processes, such as epithelialization, fibroplasia, and tissue reconstruction. following injury, this phase typically begins around day 3 and can extend until the third week. however, other sources suggest that it occurs between days 6 and 21. during this phase, inflammatory cells such as neutrophils and lymphocytes are present in lower numbers compared to the inflammatory phase, indicating a transition toward tissue repair and regeneration. (alfiaturrohmah et al., n.d.). this study observed wound closure starting on day 9, particularly in the treatment group that received cherry tomato (solanum lycopersicum var. cerasiforme) extract. therefore, a punch biopsy was performed on day 9. the results of this study demonstrate that the application of cherry tomato extract gel significantly accelerates the wound healing process in wistar strain albino mice. notably, there was a statistically significant reduction (p < 0.05) in wound size across different treatment groups, particularly on days 3, 5, 7, and 9. the accelerated healing was characterized by faster wound contraction, reduced wound dimensions, increased fibroblast proliferation, and enhanced collagen density in the treatment groups. statistical analysis using one-way anova and the kruskal-wallis test confirmed the significant effect of the 16% cherry tomato extract concentration, which yielded the most substantial reduction in wound size compared to the control group by day 9 (mean wound size: 0.001 ± 0.00182), indicating near-complete healing. these findings align with previous studies, including research by stevani (2019) and chopsticks (2021), which also demonstrated the high efficacy of 16% cherry tomato extract gel in expediting excision wound healing in wistar strain albino mice. the effectiveness of this treatment can be attributed to the presence of lycopene and other bioactive phytochemicals such as flavonoids and vitamin c, which synergistically contribute to the wound healing process through multiple mechanisms. lycopene, as a potent antioxidant, mitigates oxidative stress by neutralizing free radicals that cause tissue damage. in wound healing, excessive oxidative stress can impair fibroblast activity and collagen synthesis, which are critical for tissue regeneration. by reducing oxidative stress, lycopene creates a more favorable environment for cellular activity during wound repair. (supit et al., 2021), (krinsky, 1998) furthermore, lycopene modulates inflammatory pathways by inhibiting pro-inflammatory mediators such as tumor necrosis factor-alpha (tnf-α) and interleukin-6 (il-6). excessive levels of these mediators can prolong the inflammatory phase, delaying the transition to the proliferative stage. by reducing tnf-α and il-6 levels, lycopene facilitates a timely progression to the proliferative phase, promoting fibroblast activity and granulation tissue formation. granulation tissue quality is crucial for collagen deposition and epithelialization. these findings are further supported by research from gupta et al. (2022), which demonstrated that lycopene enhances granulation tissue formation by stimulating fibroblast proliferation and improving collagen stability and organization. well-structured collagen networks contribute to the mechanical strength of newly formed tissue, reducing the likelihood of wound reopening and expediting the remodeling phase. in addition to lycopene, other phytochemicals in cherry tomatoes, such as flavonoids, exhibit complementary anti-inflammatory properties. flavonoids inhibit pro-inflammatory enzymes such as cyclooxygenase-2 (cox-2) and reduce tissue edema, thereby mitigating inflammation, promoting faster wound resolution, and supporting tissue regeneration. these combined effects underline the potential of cherry tomato extract as a promising natural therapeutic agent for enhancing wound healing. (krinsky, 1998; shafe et al., 2024) cherry tomatoes are herbal plants with potential wound-healing properties. plant polyphenols have been considered for their remarkable antioxidant properties. (leri et al., 2020) this effect is primarily attributed to their rich antioxidant content, including lycopene, vitamin c, and flavonoids, which play a crucial role in accelerating the healing process by reducing oxidative stress in the wound area. lycopene, the predominant carotenoid compound in cherry tomatoes, is particularly significant in promoting wound healing through various biological mechanisms. its strong antioxidant capacity helps inhibit oxidative damage caused by free radicals around the wound, thereby protecting tissue cells from premature apoptosis. this protective effect facilitates fibroblast proliferation and granulation tissue formation. (wiyono & mustofani, 2019), (kresnapati et al., 2023) apart from lycopene, cherry tomatoes contain other bioactive compounds, such as flavonoids and vitamin c, which have a synergistic effect on wound healing. these compounds exert their effects through antioxidant, antiinflammatory, and tissue regeneration mechanisms, collectively accelerating wound recovery in wistar strain albino rats. flavonoids, a class of polyphenolic compounds, act as potent antioxidants in the wound healing process by reducing oxidative stress, exerting anti-inflammatory effects, and stimulating angiogenesis. (kresnapati et al., 2023), (sanjaya et al., 2023) vitamin c, or ascorbic acid, plays a vital role as a cofactor in collagen synthesis. as a water-soluble antioxidant, vitamin c protects fibroblast and endothelial cells from oxidative damage during the inflammatory phase, allowing fibroblasts to function optimally in producing extracellular matrix components. furthermore, vitamin c enhances the activity of phagocytic cells, such as neutrophils and macrophages, which are critical for wound healing. (iswan, 2021), (sebayang & ritonga, 2021) fertilita et al. – cherry tomato extract for accelerating wound healing 433 the findings of this study underscore the potential of cherry tomato extract as an effective natural woundhealing agent. the bioactive compounds in cherry tomatoes—such as lycopene, flavonoids, and vitamin c—offer various benefits for wound healing. lycopene, as a potent antioxidant, reduces oxidative stress and inflammation, which often hinder the healing process. flavonoids exhibit anti-inflammatory properties and promote the formation of new tissue by stimulating fibroblast activity. additionally, vitamin c is crucial in collagen synthesis, contributing to granulation tissue formation and improving wound tissue quality. these results highlight the significant potential of cherry tomato extract for clinical applications, particularly in developing topical formulations for treating mild to moderate wounds. this study further suggests that cherry tomato extract could serve as a safe and effective natural alternative for wound healing, offering a promising complementary approach in medical therapies. (supit et al., 2021), (kresnapati et al., 2023) further investigations should include a detailed analysis of lycopene levels in cherry tomato extract to accurately determine its bioactive content. this assessment is crucial for identifying specific bioactive components in the extract and gaining a clearer understanding of their role in promoting excisional wound healing. conclusion the application of cherry tomato (solanum lycopersicum var. cerasiforme) extract in gel form at concentrations of 16%, 12%, and 8% significantly influences the wound healing process in wistar strain albino rats, as evidenced by the reduction in wound size. these effects are notably more significant when compared to both the negative and positive control groups. among the tested concentrations, the 16% cherry tomato extract gel formulation demonstrated the highest efficacy in promoting wound healing in wistar strain albino rats, making it the most effective concentration for enhancing the excisional wound healing process. the gel formulation of solanum lycopersicum var. cerasiforme extract exhibits significant wound healing properties, providing a scientific foundation for its medicinal applications. we propose that the presence of bioactive compounds, particularly lycopene, in the extract is responsible for its potent wound-healing effects. the findings of this study offer robust scientific evidence supporting the therapeutic potential of cherry tomatoes in wound treatment. authors’ contributions: veny larasati designed the study, performing data analysis, and wrote the manuscript, soilia fertilita analyzed the data, muhammad zulfadli carried out the laboratory work, riana sari puspita rasyid and fifa argentina were reviewing the manuscript and the laboratory results, yudhie tanta and medina athiah helped analyzing the data. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests funding: the authors extend their heartfelt appreciation to universitas sriwijaya for its financial support through the research grant fund. we also acknowledge the valuable assistance of the laboratory staff and all individuals who contributed to this research. their support and collaboration were instrumental in the successful completion of this study. references abdullah, f., tahir, t., & kadar, k. 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(2019). efektivitas gel ekstrak kasar bromelin kulit nanas (ananus comosus l. merr) hasil optimasi formula pada tikus yang dibuat luka memar. assyifaa jurnal farmasi, 11(2), 112–123. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 601-606 | doi: 10.14421/biomedich.2023.122.601-606 issn 2540-9328 (online) in vitro anti-inflammatory activity of bamboo tali leaf (gigantochloa apus) ethanol extract nurul hidayah, mutista hafsah* chemistry department, faculty of science and technology, uin walisongo, jl. walisongo no.3-5, tambakaji, kec. ngaliyan, kota semarang, jawa tengah 50185, tel. (024) 7604554, indonesia. corresponding author* mutista.hafshah@walisongo.ac.id manuscript received: 21 july, 2023. revision accepted: 10 august, 2023. published: 07 november, 2023. abstract inflammation is a physiological process that serves as a defense mechanism for the body against foreign substances, bacteria, or irritants. inflammation can be cured with anti-inflammatory drugs. one of the plants that has the potential to be an anti-inflammatory agent is the bamboo leaf. this research aims to analyze the content of secondary metabolites, determine the inhibition value and ic50 value of the antiinflammatory activity of the ethanol extract of bamboo tali leaves. bamboo tali leaves were macerated using 96% ethanol and subjected to phytochemical screening. the extract was then tested for anti-inflammatory activity in vitro with the bovine serum albumin (bsa) protein denaturation inhibition method. bamboo tali leaf ethanol extract contains flavonoids, alkaloids, saponins, and phenols. the anti-inflammatory activity of the ethanol extract of bamboo tali leaves with concentrations of 28, 42, 56, 70, and 84 ppm had an inhibition percentage value of 23.14 ± 0.008%; 34.30 0.026%; 54.51 0.060%; 69.07 ± 0.006%; and 87.02 ± 0.021% with an ic50 value of 52.991 ppm. these results indicate that the ethanol extract of bamboo tali leaves has the potential to be an anti-inflammatory with a strong ic50 value below 100 ppm. keywords: anti-inflammatory; bamboo tali; phytochemicals; protein denaturation; secondary metabolites. introduction a physiological process called inflammation includes a living thing's immune system. inflammation serves as a physiological defense against certain diseases, such as cancer, by shielding the organism from microbial infection (bouyahya at al., 2022). there are several signs of inflammation in the body, including redness, fever, edema, and loss of tissue function (novika at al., 2021). inflammation can be treated with synthetic medications such ibuprofen, aspirin, diclofenac sodium, and celecoxib. long-term use of these medications may result in gastrointestinal adverse effects (izzany at al., 2018). research into anti-inflammatory medications manufactured from natural substances, sometimes known as herbal medicines, is therefore required. in indonesia, herbal remedies use is one way to utilize of the country's natural resources. one of the bamboo species found in indonesia, bamboo tali, has been extensively used in the medical field. one component of rope bamboo, called bamboo tali leaves, is not appropriate for use because it is regarded as waste for the environment (fitriani, 2018). extraction of the bioactive chemicals present in bamboo leaves is one strategy that can be used to make use of them (romansyah at al., 2019). in bamboo tali (gigantochloa apus) leaf extract macerated with 70% ethanol, bioactive substances including alkaloids, saponins, tannins, phenols, flavonoids, triterpenoids, steroids, and glycosides were discovered (setiawan & yusransyah, 2018). secondary metabolites with anti-inflammatory effects, including as phenols, saponins, and tannins. these metabolites work to suppress the production of inflammatory mediators and increase free radical production (armadany at al., 2020). consequently, the bamboo tali's leaves can be utilized as an anti-inflammatory medication. through the approach of preventing protein denaturation, anti-inflammatory activity can be tested. this is due to the fact that protein denaturation is one of the causes of tissue inflammation (farida, rahmat, & amanda, 2018). the protein bovine serum albumin (bsa) was utilized in this study. the way the sample solution interacts with bsa demonstrates the theory behind protein denaturation inhibition. free radical generation causes body proteins to be more susceptible to denaturation. the outcome of the inflammatory process is the release of inflammatory mediators (shalihah at al., 2022). materials and methods materials and tools the tools used in this study were volume pipettes, stir bars, spatulas, thermometers, test tubes, test tube racks, https://doi.org/10.14421/biomedich.2023.122.601-606 https://www.google.com/search?gs_ssp=ejzj4tvp1zc0tdirydawlsoxyptilc3muyhpzmkszs9lzwcaiusjtw&q=uin+walisongo&rlz=1c1chny_enid896id896&oq=uin+wa&aqs=chrome.1.0i131i355i433i512j46i131i433i512j69i57j0i512l2j69i60l3.4842j0j7&sourceid=chrome&ie=utf-8 602 biology, medicine, & natural product chemistry 12 (2), 2023: 601-606 erlenmeyer (iwaki), porcelain cups, volumetric flasks (100 ml, 100 ml, 25 ml, and 10 ml) (iwaki), vials, cuvettes, dark glass bottles, ph meter, analytical balance (and), blender (cosmos), centrifuge (plc series), 1 set of distillation apparatus, one set of vacuum rotary evaporator, and uv-vis spectrophotometry instrument (orion aquamate 8000). the juvenile leaves of gigantochloa apus, a species of rope bamboo, were the materials employed in this investigation. in this study, additives with proanalytical quality were employed. 96% technical ethanol, distilled water, mg, hcl, wagner reagent, fecl3, bovine serum albumin (bsa), tris buffer saline (tbs), nacl (merck), and ch3cooh are the other ingredients. methods making powder from bamboo tali leaf (wahyuni at al., 2020) bamboo leaves are removed, cleaned under running water, and allowed to air dry for about five days. using a blender, the little bits of dried leaves were mixed into a powder. figure 1. bamboo tali leaf. calculation of bamboo tali leaf powder's moisture content (fitri & anita, 2014) two grams of powdered rope bamboo leaf were baked for 30 minutes at 105 degrees. it was then placed in a desiccator to chill for 15 minutes. after cooling, the resulting weight is weighed to ascertain the water content. % 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑏−(𝑐−𝑎) 𝑏 (eq. 1) information: a : weight of the cup (g) b : sample weight (g) c : cup weight + sample (g) producing bamboo tali leaf extract from tali leaf (wahyuni at al., 2020) bamboo tali leaf powder was macerated with 96% ethanol that has undergone a 1:5 (w/v) distillation ratio. this procedure is continued until the combination turns a light shade of clear, provided that it is filtered and the solvent is changed once every 24 hours in order to acquire the ethanol extract and determine the yield level. %𝑌𝑖𝑒𝑙𝑑 = 𝑬𝒙𝒕𝒓𝒂𝒙𝒕 𝒘𝒆𝒊𝒈𝒉𝒕 𝑺𝒊𝒎𝒑𝒍𝒊𝒄𝒊𝒂 𝒘𝒆𝒊𝒈𝒉𝒕 100% (eq. 2) phytochemical screening test flavonoid (romansyah at al., 2019) two milliliters of the sample in total, heated for 5 minutes. the material is heated before being combined with five drops of concentrated hcl and 0.1 g of mg metal. positive reaction is indicated by a yellowishorange to reddish solution. alkaloid (wahid & safwan, 2020) wagner's reagent was added in three separate drops to the 2 ml extract sample. if a brown hue shows up, the sample is positive for alkaloids. saponin (wahid & safwan, 2020) a test tube containing 3 ml of the extract sample was then filled with 10 ml of hot water, cooled, and vigorously shaken for 10 seconds. one drop of pure hcl was then added after that. the positive sample includes saponins if the foam that forms does not vanish. tannin the extract sample, a total of 3 ml, was heated for 5 minutes before being combined with 5 drops of 1% fecl3. according to romansyah et al. (2019), the development of dark blue or blackish green is a sign that tannins are present. phenol (putri, 2018) two milliliters of the extract sample were mixed with five drops of 1% fecl3. a positive sample that contains phenol is indicated if a solid black or bluish green color is produced. an anti-inflammatory effect test tbs (tris buffer saline) production (barr at al., 2006) 900 ml of distilled water was added after 8.7 g of nacl and 1.21 g of tris base were weighed in total. after then, the ph was maintained at 6.2–6.5 by adding glacial ch3cooh. a 1000 ml volumetric flask is filled with a stable nacl and tris base mixture, and the ph is then raised to the desired level using distilled water. bsa (bovine serum albumin) production 0.2% (williams at al., 2008) 0.2 g of bsa was added to a 100 ml volumetric flask, and the volume was subsequently raised to 100 ml with tbs solution. positive controls creation (rusli & setiawan, 2020) two milliliters of 0.2% bsa solution were combined with 1.5 ml of tbs and 1.5 ml of distilled water to hidayah & hafsah – in vitro anti-inflammatory activity of bamboo tali leaf … 603 create a 5 ml volumetric flask. without any additional processing, the mixture's absorbance value was instantly determined using a uv-vis spectrophotometer at the optimized wavelength of 289 nm. duplicate runs of the experiment were conducted. negative control creation (rusli & setiawan, 2020) two milliliters of 0.2% bsa solution, 1.5 ml tbs, and enough distilled water to reach the boundary mark should be added to the 5 ml volumetric flask. the mixture was cooked for 30 minutes at 37°c and then for another 10 minutes at 72°c. following the completion of the heating phase, the mixture was centrifuged at 6,000 rpm for 10 minutes while maintaining a temperature of 32 °c. at a wavelength of 289 nm, the absorbance value was measured twice (duplo). a 5 ml volumetric flask was filled to the boundary mark with distilled water, 1.5 ml tbs, and 2 ml of 0.2% bsa solution. the mixture was cooked for 30 minutes at 37°c and then for another 10 minutes at 72°c. following the completion of the heating phase, the mixture was centrifuged at 6,000 rpm for 10 minutes while maintaining a temperature of 32 °c. at a wavelength of 289 nm, the absorbance value is measured twice. developing the primary and test solutions (muliati, 2014) in order to create mother liquor, a 10 ml volumetric flask containing up to 70 mg of an ethanol extract of bamboo tali leaves was combined with 10 ml of ethanol. this resulted in a concentration of 7,000 ppm. following that, the mother liquor concentration was adjusted to 840, 700, 560, 420, and 280 ppm. testing for anti-inflammatory (novika at al., 2021) to a mixture of 2 ml of 0.2% bsa solution, 1.5 ml of tbs, and distilled water, a total of 0.5 ml of the test solution at various concentrations was added. concentration changes from the solution were 84, 70, 56, 42, and 28 ppm. the temperature of each concentrated solution was raised for 10 minutes at 72°c after 30 minutes at 37°c. following the completion of the heating operation, the mixture's temperature was maintained at 32°c for 30 minutes before being centrifuged at 6,000 rpm for 10 minutes. a uv-vis spectrophotometer was used to measure the absorbance, which was repeated twice (duplo). (shalihah at al., 2022). once the absorbance value has been read, each solution's percent inhibition value has to be calculated. if a natural product's % inhibition value is greater than 20%, it is considered to have anti-inflammatory activity. %𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝑡𝑒𝑠𝑡 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 × 100% (eq. 3) results and discussion sample preparation using a blender, the dried bamboo leaves were chopped and ground into a fine, light-weight powder that was light green in color. bamboo tali leaf powder has a moisture level of 3.42%. 10% of the sample must be water-free to maintain sample quality (wijaya & noviana, 2022). these facts demonstrate that the sample of rope bamboo leaves satisfies the water content requirements. the sample's water content has a significant impact on its quality and shelf life, making it more susceptible to damage and microbial contamination (hutauruk at al., 2014). obtainable yield at a temperature of 60 °c and a rotational speed of 70 rpm, the macerated filtrate was concentrated. because ethanol has a high boiling point (79.37°c), using a temperature of 60°c during the evaporation process aims to prevent harming the bioactive components. a small amount of oil and a dark, viscous extract are produced by the evaporation process. 12.34 g of the extracted viscous material had a yield value of 12.34%. to estimate the value of bioactive components that can be extracted using solvents, the percent yield is calculated (dewatisari at al., 2018). phytochemical screeningtest as can be seen in table 1, the phytochemical screening of the ethanol extract of bamboo leaves revealed the presence of flavonoids, alkaloids, saponins, and phenols (table. 1). table 1. results of phytochemical testing. bioactive substances description of phytochemical test results information flavonoid orange-yellow + alkaloid brown with developed black clumps + saponin formed stable foam + tannin brown phenol solid black + information: (+) : has bioactive ingredients, (-) : does not include any bioactive ingredients 604 biology, medicine, & natural product chemistry 12 (2), 2023: 601-606 inflammatory-reduction capacity the bsa protein denaturation inhibition method was used to assess the ethanol extract of bamboo tali leaves for anti-inflammatory effects. bsa is a protein derived from cow albumin. bsa works in enzymatic processes to stabilize proteins. bsa has a ph between 6.0 and 8.5 (borzova at al., 2016). because bsa is simple to produce, easily soluble, affordable, and minimizes the use of live specimens in the drug development process, it is used in this study (novika at al., 2021). one of the reasons why the cell tissues of the body get inflamed is protein denaturation. protein denaturation is the process by which pressure or chemicals from the outside result in an alteration of the tertiary and secondary structure of proteins or nucleic acids. the approach of protein denaturation inhibition was chosen because it is nonacidic, inexpensive, and simple to use (aditya at al., 2015). table 2. results of bamboo tali leaf extract's % bsa inhibition. no concentration (ppm) %inhibition 1. 28 23,14±0,008 2. 42 34,30±0,026 3. 56 54,51±0,060 4 70 69,07±0,006 5. 84 87,02±0,021 table 3. results of % inhibition of bsa by diclofenac sodium. no concentration (ppm) %inhibition 1. 10 26,38%±0,006 2. 20 35,77%±0,001 3. 30 44,54%±0,000 4 40 56,31%±0,001 5. 50 66,73%±0,001 when bsa protein is heated, protein denaturation takes place (fitriyani & fatahillah, 2022). the protein's molecules travel quicker and sustain damage as a result of the rising temperature's increased kinetic energy (shalihah at al., 2022). as a buffer solution, bsa was dissolved in tris buffer saline (tbs). bsa's ideal ph is 7, so tbs solution was selected because it has a ph of 6.25 to 6.5 (abidin at al., 2020). percent inhibition is used to express the protein denaturation activity. when the proportion of inhibition is greater than 20%, antiinflammatory action is present (farida, rahmat, & widia amanda, 2018). tables 2 and 3 show the findings of research on the anti-inflammatory effects of diclofenac sodium and an ethanol extract of bamboo tali leaves. the percentage of inhibition for each extract concentration is shown in table 2. the % inhibition values for concentrations of 28, 42, 56, 70, and 84 are known to already meet the criterion for % inhibition of protein denaturation, namely > 20%, based on table 2. the cell membrane will be harmed by protein denaturation, which will allow the phospholipase enzyme to convert phospholipids into arachidonic acid. cyclooxygenase (coox) enzymes will then break down arachidonic acid to create prostaglandins, which can activate pain receptors and increase capillary permeability (fitriyani & fatahillah, 2022). secondary metabolites have the ability to limit the synthesis of prostaglandins. bamboo tali leaf extract has antiinflammatory properties due to the presence of secondary metabolites including flavonoids, alkaloids, saponins, and phenols. through the action of the phospholipase enzyme, flavonoids prevent the synthesis of prostaglandins (bustanul & sanusi, 2018). because of their capacity to suppress the creation of cytokines that promote inflammation, alkaloids have the potential to be antiinflammatory medications. saponins' anti-inflammatory mechanism, which involves preventing the breakdown of glucocorticoids and the release of inflammatory mediators (mohammed at al., 2014). phenol is a secondary metabolite with oh groups, which can protect membranes, prevent the production of inflammatory mediators, and deactivate free radicals (novika at al., 2021). the anti-inflammatory efficacy of the dicoflenac sodium medication is shown in table 3. according to the data in table 3, all of the variants employed satisfied the requirements for % protein denaturation inhibition. diclofenac sodium was chosen since it is one of the most effective anti-inflammatory medications (novika at al., 2021). diclofenac sodium is a heteroarylacetate salt of sodium (2,6-dichlorophene) (fitri yani & reynaldi, 2021). figure 2 depicts the structure of diclofenac sodium. diclofenac sodium can inhibit the cyclooxygenase cox-1 and cox-2 enzymes, lowering prostaglandin, prostacyclin, and thromboxane synthesis (altman at al., 2015). cl cl nh o ona figure 2. diclofenac sodium structure. after acquiring data on the percentage inhibition value, the ic50 value is calculated. the anti-inflammatory test results are interpreted using the ic50 value. the ic50 value denotes the concentration of the test substance that can reduce inflammation by 50%. as illustrated in figures 3 and 4, the obtained % inhibition value data is entered into the linear regression equation with the extract concentration (ppm) as the x-axis and the % inhibition value as the y-axis. hidayah & hafsah – in vitro anti-inflammatory activity of bamboo tali leaf … 605 figure 3. linear regression curve of anti-inflammatory activity of bamboo tali leaf ethanol extract. figure 4. linear regression curve of diclofenac sodium extract antiinflammatory efficacy. figure 3 displays the linear regression curve of the ethanol extract of bambu tali leaves, yielding y = 0.0116x-0.1147 and r2 = 0.9939. the obtained correlation r value is excellent. in the region 0.80-1.00, where the correlation of the variables grows and the line has a positive slope, the correlation coefficient is quite strong. the ic50 value is calculated using the curve. according to the curve analysis results, each rise in concentration appears to be proportional to an increase in the value of % inhibition. the curve line producing a linear line for each rise in the logarithm of concentration demonstrates this. the ic50 value is a metric that indicates the extract's efficiency in suppressing protein denaturation with a percentage value of up to 50%. bamboo tali leaf extract has an ic50 value of 52.991 ppm. this value indicates that at a concentration of 52.991 ppm, the ethanol extract of bamboo tali leaves inhibited protein denaturation by 50%. figure 4 depicts the linear regression curve for diclofenac sodium's anti-inflammatory efficacy. the curve yields the result y = 0.0101x + 0.1549 with r2 = 0.9974, allowing the ic50 value to be calculated. diclofenac sodium has an ic50 value of 34.168 ppm. the ic50 value of diclofenac sodium demonstrated more antiinflammatory efficacy than the ethanol extract of bamboo tali leaves. this is due to diclofenac sodium's potent antiinflammatory properties. furthermore, dicloofenac sodium has a faster absorption and pain alleviation than other nonsteroidal anti-inflammatory medicines like ibuprofen or naproxen (altman at al., 2015). conclusions bamboo tali leaves (gigantochloa apus) ethanol extract contains secondary metabolites including flavonoids, alkaloids, saponins, and phenols. the anti-inflammatory activity of the ethanol extract of bamboo tali leaves (gigantochloa apus) at concentrations of 28, 42, 56, 70, and 84 ppm was 23.14%, 34.48%, 55.12%, 69.30%, and 87.41%, respectively, with an ic50 value of 52.991 ppm. competing interests: the authors declare that there are no competing interests. references abidin, z., putri, u. a., & widiastuti, h. 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(2008). the in vitro anti-denaturation effects induced by natural products and non-steroidal compounds in heat treated (immunogenic) bovine serum albumin is proposed as a screening assay for the detection of anti-inflammatory compounds, without the use of animals. west indian medical journal, 57(4), 327–331. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 927-935 | doi: 10.14421/biomedich.2025.142.927-935 issn 2540-9328 (online) application of alginate-based edible coating with beeswax addition on minimally processed pears (pyrus bretschneideri rehd.) maftukh zaina luthfiyyah, novita herdiana, esa ghanim fadhallah*, fibra nurainy department of agricultural product technology, faculty of agriculture, universitas lampung. jl. prof. dr. soemantri brojonegoro no. 1, bandar lampung, lampung, 35145, indonesia. corresponding author* esa.ghanim@fp.unila.ac.id manuscript received: 16 july, 2025. revision accepted: 27 october, 2025. published: 28 october, 2025. abstract pears, especially after being cut, tended to experience a decline in quality and damage such as enzymatic browning, moisture loss, and texture changes. the application of alginate-based edible coating with the addition of beeswax was one of the innovations that could be applied to minimally processed pears to slow down the rate of deterioration. this study aimed to determine the effect of beeswax concentration in alginate-based edible coating solutions on the freshness of minimally processed pears and to identify the optimal beeswax concentration using the star method. the study was designed using a completely randomized design (crd) in a non-factorial arrangement with the addition of beeswax. the treatments consisted of six levels: bk as the control, b0 (alginate), b2 (alginate + 2% beeswax), b4 (alginate + 4% beeswax), b6 (alginate + 6% beeswax), and b8 (alginate + 8% beeswax), each with four replications. the results showed that the addition of beeswax to the alginate-based edible coating had a significant effect on moisture content, weight loss, firmness, total soluble solids, and color. the best treatment for maintaining pear freshness according to the star method was b4 (4% beeswax), which had a moisture content of 85.66%, weight loss of 1.79%, firmness of 874.63 gf, total soluble solids of 11.28 ˚brix, and a lightness (l) value of 68.30 on day 12 of storage. keywords: alginate; beeswax; edible coating. introduction horticultural products face a major challenge in their relatively short shelf life and perishable nature. one such horticultural product that is highly susceptible to damage is pear (pyrus bretschneideri rehd.) due to its high water content of approximately 84% (widyaka et al., 2019). pears are rich in nutrients, containing about 110 calories, 134 mg of potassium, and various vitamins (a, b1, b2, c, e, k, folacin, pantothenic acid, and niacin). according to öztürk et al. (2014), the vitamin c content in pears ranges from 9.1 mg to 29.7 mg per 100 g of pear flesh. this abundance of nutrients makes pears popular among consumers worldwide. based on data from usda (2023), global pear production in 2023 is projected to reach 25.2 million tons, an increase of 300,000 tons from the previous year. in indonesia, the consumption of pears is relatively high, as indicated by imports reaching 238,160 tons in 2020 (badan ketahanan pangan, 2020). market trends such as fresh-cut fruits and vegetables are gaining popularity due to their convenience, ease of consumption, and fresh appearance. however, these products are highly perishable and typically last only 3–5 days due to respiration, transpiration, microbial activity, and insects (hibatul, 2018). one innovation aimed at extending their shelf life is the application of edible coatings to the surface of the fruit. edible coatings are thin layers that serve to enhance mechanical properties, provide antibacterial effects, and act as barriers to water vapor and gases (galus and kadzińska, 2015). according to setiawan (2019), materials used for edible coatings should be resistant to oxygen and water vapor permeability, tasteless, colorless, and should not alter the characteristics of the food. common materials used for edible coatings include tapioca starch and chitosan. however, these materials have several drawbacks, necessitating the use of other more effective substances such as alginate in preventing damage to pears. alginate has the ability to form films or biopolymer layers due to its unique colloidal structure and can be used as a suspending agent, thickener, film former, binder, and emulsifier. however, a limitation of alginatebased edible coatings is their poor mechanical properties, which can be improved by adding lipids. a study by utomo and salahudin (2015) showed that the addition of lipids to corn starch films increased their water resistance. one such lipid component is beeswax, which, due to its solid and hydrophobic structure, can restrict o₂ and co₂ exchange and slow down oxidation and respiration (baldwin et al., 2011). https://doi.org/10.14421/biomedich.2025.142.927-935 mailto:esa.ghanim@fp.unila.ac.id 928 biology, medicine, & natural product chemistry 14 (2), 2025: 927-935 this study utilizes alginate and beeswax as materials for producing edible coatings. beeswax possesses hydrophobic properties that can inhibit moisture loss and reduce the rate of transpiration, thereby preventing excessive water evaporation (oko et al., 2023). based on the above explanation, it is necessary to conduct a study to determine the effect of alginate-based edible coatings with beeswax on the freshness of minimally processed pears. materials and methods raw material asian pear (pyrus bretschneideri rehd.) in fresh condition without major damage or defects was obtained from pasar tani, bandar lampung, with an average weight of approximately 300 g per fruit. organic beeswax was sourced from a local honeybee farm in tangerang regency, indonesia. experimental design this study was arranged using a completely randomized block design (crbd) in a non-factorial experiment with six treatment levels and four replications. the treatments consisted of control (bk), alginate only (b0), alginate + 2% beeswax (b2), alginate + 4% beeswax (b4), alginate + 6% beeswax (b6), and alginate + 8% beeswax (b8). the beeswax percentages were based on 100 ml of distilled water. data obtained on days 6 and 12 of observation were analyzed for homogeneity of variance using bartlett’s test and for additivity using tukey’s test. the data were then subjected to analysis of variance (anova) to estimate the error variance and evaluate the effect of treatments. duncan’s multiple range test (dmrt) at the 5% significance level was performed to determine significant differences among treatments. the best treatment was selected using the star rating method. procedures preparation of edible coating solution three a total of 100 ml of distilled water was heated to a temperature of 40°c, then 1.5 g of alginate was added and stirred for approximately 10 minutes. next, 1% (1 ml) of glycerol was added as a plasticizer to improve the flexibility of the alginate layer. subsequently, organic beeswax, obtained from local farmers in tangerang regency, and 0.1 ml of tween 80 as an emulsifier were added to help incorporate ingredients that are typically immiscible, such as oil and water. the concentrations of beeswax and distilled water used were 2 g/100 ml, 4 g/100 ml, 6 g/100 ml, and 8 g/100 ml. the mixture was then heated at approximately 60–70°c while being stirred until homogeneous. application of edible coating the application of the edible coating on the pears was based on a previous study by chiabrando & giovanna (2016), with modifications. asian pears (pyrus bretschneideri rehd.) at 80% ripeness were selected and sorted based on color, size, and absence of pest damage. the pears were obtained from a farmer’s market in bandar lampung, with an average fruit weight of approximately 300 g. the pears were washed with clean water and soaked in a 100 ppm chlorine solution for 5 minutes, then drained for 20 minutes. chlorine compounds can inhibit enzymatic browning activity that causes discoloration of the fruit (passafiume et al., 2021). the pears were then cut into crescent-shaped slices measuring 3 cm by 7 cm, with each piece weighing approximately 60–65 g. next, the samples were coated with the edible solution by immersing the entire fruit slices in the edible coating solution for 2 minutes and draining them for 1 minute. the pears were then immersed in 100 ml of 2% calcium chloride (cacl₂) solution for 3 minutes to form cross-links in the alginate, thereby strengthening and stabilizing the coating layer. after that, the samples were air-dried at room temperature for 30 minutes and stored in a chiller (0–4°c) (chiabrando & giovanna, 2016). observations were carried out on moisture content, weight loss, firmness, total soluble solids, color, and vitamin c content for the best treatment. the samples were analyzed periodically on days 0, 6, and 12. observation parameter moisture content moisture content was measured on days 0, 6, and 12 of observation. the measurement referred to the aoac (association of official analytical collaboration, 2019) method, which involves evaporating water from the sample by heating in an oven at a specific temperature (typically 105°c) until a constant weight is achieved. the weight loss after heating is considered as the moisture content of the sample. weight loss weight loss was determined by weighing the pears on days 0, 6, and 12 of observation. the initial weight (w0) was recorded before treatment, while the final weight (w1) was recorded after treatment. thus, weight loss was calculated as the difference between the initial and final weights (megasari and mutia, 2019). fruit firmness fruit firmness was expressed in gram-force (gf) or newton (n) and measured using a brookfield ct3 texture analyzer. the pear was tested at three points (right end, left end, and center) using a 6 mm diameter probe, applied twice. the probe speed was set at 5 mm/s, and the sample was compressed to 30% of its original height. firmness was measured on days 0, 6, and 12 of observation. total soluble solids total soluble solids (tss) were measured on days 0, 6, and 12 of observation using a hand refractometer. pear luthfiyyah et al. – application of alginate-based edible coating with … 929 juice was obtained by grating the entire part of the fruit (one sample), and 1–2 drops of the juice were placed onto the refractometer sensor glass. the glass cover was then closed, and the °brix value was read by aligning the blue line on the refractometer scale. the total soluble solids were expressed as a percentage (%) within a range of 0–32% (arpani, 2019). color color parameters were measured using a colorimeter (amt 507), with color values expressed in terms of l, a, and b* values. the colorimeter sensor was placed on the surface of the pear at three points: the right side, left side, and bottom. the measurement button was pressed to record the color data. color parameters were measured on days 0, 6, and 12 of observation. vitamin c content vitamin c content was determined following the method described by jacobs (1958). the samples used included the best treatment sample and the control sample. the first step involved grating the pear to obtain a homogenized sample. then, 5–10 g of the homogenized or liquid sample was weighed and transferred into a 100 ml volumetric flask, followed by the addition of distilled water up to the calibration mark. the mixture was then filtered through filter paper or centrifuged to obtain the filtrate. an aliquot of 5–25 ml of the filtrate was pipetted into a 100 ml erlenmeyer flask. next, 2 ml of 1% starch solution was added as an indicator, and the mixture was titrated with 0.01 n standard iodine solution until a light blue color appeared. results and discussion moisture content based on the results, the moisture content of fresh-cut pears decreased during storage, with more pronounced effects as the storage period progressed. on day 0, all samples had an initial moisture content of 87.42%, representing the fresh condition of the pears prior to treatment and storage. on day 6, moisture content ranged between 84.55% and 86.87%, and further decreased to 83.63%–86.44% by day 12. according to widyaka et al. (2019), the moisture content of fresh pears is approximately 84%. the observed reduction in moisture content indicates a decline in fruit quality, which affects weight, freshness, and shelf-life (jannah, 2024). the decrease in moisture content in the b2 treatment (2% beeswax) on day 6 was only 0.63%, which is lower than that reported by widyaka et al. (2019), who observed a 2.22% decrease in coated pear slices after 6 days of storage. this difference suggests that the combination of alginate and beeswax is effective in maintaining moisture in fresh-cut pears. the rate of moisture content in minimally processed pears are illustrated in figure 1. figure 1. the rate of moisture loss in minimally processed pears under all treatments increased with storage duration. different beeswax concentrations in each treatment had varying effects on the moisture content. the control treatment without beeswax (bk) showed the lowest moisture levels, at 84.55% (day 6) and 83.63% (day 12). the absence of a protective coating allowed direct contact between the fruit surface and the environment, accelerating physiological processes such as respiration and transpiration, which in turn increased moisture loss and reduced fruit quality during storage (mahfudin, 2016). conversely, treatment b2 (2% beeswax) resulted in the highest moisture content, with 86.87% on day 6 and 86.44% on day 12, indicating that this concentration was the most effective in preserving fruit moisture. this finding suggests that moisture retention in fresh-cut pears is influenced by the concentration of beeswax used, with lower concentrations such as in b2 providing a more effective barrier to water vapor transmission. treatments b4 and b6 also showed good effectiveness, although their moisture contents were slightly lower than that of b2. interestingly, treatment b8 (8% beeswax) resulted in lower moisture content (84.97% on day 6 and 84.62% on day 12) compared to treatments b2, b4, and b6. this suggests that higher beeswax concentrations do not necessarily enhance moisture retention. excessive beeswax may form a coating layer that is too thick or uneven, potentially hindering gas exchange and inducing anaerobic respiration, which can accelerate fruit deterioration (mahfudin, 2016). the effectiveness of the coating is also attributed to the combined use of alginate and beeswax. alginate acts as a barrier to environmental factors, helping to retain moisture and reduce the risk of dehydration in the fruit (jannah, 2024). according to dhyan et al. (2015), waxing methods combined with low-temperature storage effectively slow down fruit metabolism, including respiration and transpiration, thus minimizing water loss. maintaining moisture content plays an important role in increasing the fruit’s resistance to damage caused by microorganisms and insects. pathogenic microorganisms can more easily enter fruit 930 biology, medicine, & natural product chemistry 14 (2), 2025: 927-935 tissues that have softened or sustained skin damage due to water loss (dhyan et al., 2015). weight loss weight loss is defined as the difference between the initial and final weight of the fruit, where a higher percentage indicates a decline in the quality of fresh-cut pears (mawardi et al., 2023). the results of this study show that the weight of fresh-cut pears decreased progressively during storage, with more pronounced effects observed over time. weight loss during 6 days of storage ranged from 1.31% to 2.26%, and increased to 1.79%–3.40% by day 12. according to the dmrt test at a 5% significance level, treatment b4 was not significantly different from treatments b2 and b0 on both day 6 and day 12, but differed significantly from treatments b6, b8, and bk (control). all samples had 0% weight loss on day 0, indicating no initial degradation. these results indicate that the application of alginate-based edible coating with beeswax addition effectively maintained the quality of fresh-cut pears compared to uncoated fruit. the observed weight loss in this study was lower than that reported by plesoianu & nour (2022), who found that coated pear slices experienced a 4.20% weight loss after 12 days of storage. the rate of weight loss in minimally processed pears are illustrated in figure 2. figure 2. the rate of weight loss in minimally processed pears under all treatments increased with storage duration. treatments b2 (2% beeswax) and b4 (4% beeswax) consistently resulted in the lowest weight loss values on both day 6 and day 12. this can be attributed to the hydrophobic nature of beeswax, which reduces the size of surface pores on the fruit, thereby lowering water vapor transmission rates. additionally, beeswax increases the barrier properties of the coating, contributing to a reduction in respiration rate (kanani et al., 2018). however, excessive beeswax concentration, as seen in treatments b6 and b8, led to higher weight loss. according to günal‐köroğlu & çapanoğlu (2024), excessive wax may inhibit gas exchange and induce anaerobic respiration, whereas insufficient wax may fail to provide adequate protection. effective wax coating can close stomatal openings, thereby reducing respiration rates (mahfudin, 2016). weight loss in fresh-cut pears is closely related to water loss through transpiration and respiration processes. a high rate of water loss contributes to a decline in moisture content, which subsequently increases the percentage of weight loss. plesoianu & nour (2022) also explained that the primary cause of weight loss in fresh-cut fruit during storage is the evaporation of water through the exposed cut surface. pear slices without coating (control) exhibited higher weight loss, which was also associated with lower moisture content. this is due to the absence of a protective layer that can slow down the rate of water evaporation. in contrast, edible coatings, particularly those containing beeswax, act as semi-permeable barriers that reduce transpiration and water vapor diffusion, thus maintaining moisture content and minimizing weight loss. this is supported by the results for both moisture content and weight loss in this study. on day 12 of storage, the control treatment (bk) showed the lowest moisture content (83.63%) (figure 1) and the highest weight loss (3.40%) (figure 2). fruit firmness based on the results presented in figure 3, the firmness of minimally processed pears decreased over the storage period. on day 0, all samples had the same initial firmness value of 1108 gf, indicating that the physical condition of the fruit was uniform and had not yet undergone softening. the firmness of the pears on day 6 of storage ranged from 419.25 to 939.13 gf, and on day 12 ranged from 350.75 to 874.63 gf. the firmness continued to decline as the storage time increased. treatment b4 (4% beeswax) showed a 15.25% decrease in firmness on day 6, which was higher than the findings of widyaka et al. (2019), who reported that coated pear slices experienced a decrease in firmness of only 11.41– 12.90%. figure 3. the rate of firmness reduction in minimally processed pears under all treatments increased with storage duration. variations in beeswax concentration produced differing effects on the firmness of fresh-cut pears. luthfiyyah et al. – application of alginate-based edible coating with … 931 treatments b0 (without beeswax) and bk (control) exhibited the lowest firmness values during storage. this suggests that in the absence of a beeswax layer, the coating was less effective in preventing physical degradation, resulting in faster tissue softening. in contrast, treatments with added beeswax were more effective in maintaining fruit firmness. treatment b6 (6% beeswax) demonstrated relatively good effectiveness, although slightly lower than b4, with firmness values of 854.38 gf on day 6 and 741.25 gf on day 12. meanwhile, treatment b2 (2% beeswax) showed moderate firmness values, 675.38 gf (day 6) and 612.00 gf (day 12), indicating that while it provided some protection against softening, its effectiveness was still below that of b4 and b6. interestingly, treatment b8 (8% beeswax), which utilized the highest concentration, resulted in lower firmness values, 599.75 gf (day 6) and 505.25 gf (day 12). these findings indicate that excessive beeswax concentration can lead to overly thick coatings, which excessively hinder gas exchange, induce anaerobic conditions within the fruit tissue, and accelerate the softening process (susanto et al., 2018). thus, beeswax concentrations that are too low are less effective in protecting the texture, while beeswax concentrations that are too high can cause side effects in the form of premature softening or fermentation triggered by limited oxygen in the fruit tissue due to the layer being too thick. fruit firmness or texture is closely associated with moisture content, as water loss leads to reduced cell turgor pressure, causing tissue softening. the decline in firmness observed in this study corresponds with the reduction in moisture content (figure 1). firmness in samples from bk (uncoated) and b0 (coating without beeswax) declined more rapidly due to higher rates of water loss. the effectiveness of beeswax in maintaining fruit firmness is attributed to its ability to form a barrier layer that reduces respiration rate and limits water evaporation, thereby preserving fruit texture. according to andriani et al. (2018), although softening is a natural consequence of storage, the incorporation of beeswax in the edible coating formulation has been shown to slow down this process. genevois et al. (2016) reported that fruit softening is caused by the loss of cell turgor pressure due to the degradation of protopectin (insoluble pectin) into more than 40% soluble pectin. the application of beeswax in edible coatings may help delay this conversion by limiting gas and moisture exchange, thereby maintaining cell turgor and structural integrity. in addition to moisture loss through transpiration, the breakdown of complex compounds such as starch into simple sugars also contributes to accelerated softening of fruit texture (atmaja, 2021). therefore, edible coatings incorporating appropriate levels of beeswax can play an important role in preserving firmness and extending the shelf life of fresh-cut pears. total soluble solids the results of the 5% dmrt test showed that treatment b8 was not significantly different from bk, b0, b2, and b6, but was significantly different from b4. the total soluble solids (tss) of minimally processed pears increased during storage, from an initial value of 10 ˚brix to 10.10–10.85 ˚brix on day 6, and 9.98–11.28 ˚brix on day 12 (figure 4). tss reflects the content of simple sugars such as glucose, fructose, and sucrose formed during the ripening process (tabassum & khan, 2020). treatment b4 showed a consistent increase in tss up to 11.35%. these findings are in line with mohamed & shaaban (2014), who reported an increase in tss up to 11.59% in coated fresh-cut pears on the 12th day of storage. a higher tss value indicates greater fruit sweetness (mahadin, 2015). this is attributed to cell wall softening and the breakdown of complex carbohydrates into simple sugars, primarily through starch hydrolysis into sucrose, fructose, and glucose, which serve as key indicators of the fruit ripening process (tabassum & khan, 2020). figure 4. the rate of change in total soluble solids (tss) of minimally processed pears in all treatments increased over the storage period. treatment b6 (6% beeswax) showed a relatively high increase in tss, reaching 10.80°brix on day 6 and 10.75°brix on day 12, slightly lower than b4. treatments b2 and b0 also exhibited increases in tss, but with more moderate values. on day 12, treatment b2 reached 10.60°brix, while b0 increased to 10.73°brix. these increases indicate that ripening processes continued during storage, although the effectiveness of the coatings in controlling metabolic activity appeared lower in b2 and b0 compared to b4 and b6. in contrast, treatment b8 (8% beeswax) exhibited the lowest tss values among all treatments, with 10.10°brix on day 6 and 9.98°brix on day 12. this suggests that excessively high concentrations of beeswax may restrict aerobic respiration due to the overly compact coating layer, which can interfere with metabolic processes and reduce sugar accumulation in the fruit (susanto et al., 2018). the control treatment (bk) also showed lower tss values compared to b2, b4, and b6, likely due to more 932 biology, medicine, & natural product chemistry 14 (2), 2025: 927-935 rapid moisture loss in the absence of a protective coating, thereby affecting the internal balance of fruit metabolites. changes in tss were also closely related to the observed variations in moisture content (figure 1). as water evaporates, the concentration of dissolved substances such as sugars and organic acids increases, causing a rise in tss values. additionally, the increase in tss reflects ongoing metabolic processes within the fruit, particularly the enzymatic breakdown of starch into simple sugars through hydrolysis, which is a key aspect of the ripening process. according to refilda et al. (2022), increasing tss levels are indicative of fruit ripening, and will continue to rise until the fruit reaches an overripe or spoiled state. color color change is a critical indicator in assessing the visual quality and consumer acceptability of fresh fruits during storage. although coatings are often transparent and tend not to significantly alter the appearance of the fruit, color parameters are still commonly used as a subjective indicator of product quality (suriati et al., 2022). in this study, the color of fresh-cut pears was measured using a colorimeter, and the color values were expressed in terms of lightness (l*), redness (a*), and yellowness (b*). analysis of variance revealed that the addition of beeswax in the alginate-based edible coating had a significant effect on the color attributes of fresh-cut pears. figure 5. the rate of l color change in minimally processed pears under all treatments increased with storage duration the a* value represents the green-red component of the sample color, where negative values (0 to –80) indicate greenness and positive values (0 to 80) indicate redness. the b* value measures the blue-yellow spectrum, with negative values (0 to –70) indicating blue and positive values (0 to 70) indicating yellow. according to demasta et al. (2020), the a* and b* values are not strongly associated with polyphenol oxidase (ppo) activity, whereas the l* value (lightness) is significantly related to the inhibition of ppo activity. the l* value reflects the brightness of the sample, with 0 representing total darkness and 100 representing total brightness.at day 0, all samples exhibited an initial l* value of 75.50, indicating uniform brightness among fresh samples prior to treatment and storage. during storage, l* values on day 6 ranged from 63.33 to 75.40, while on day 12 they ranged from 61.90 to 71.55. based on the 5% dmrt analysis, treatment b8 did not differ significantly from b6 and b4, but was significantly different from other treatments. an increase in beeswax concentration was associated with higher l* values, indicating improved brightness. however, as storage time progressed, l* values declined across all treatments. for instance, the l* value for treatment b8 decreased by 5.23% by day 12, which aligns with findings by plesoianu & nour (2022), who reported a 4.6–7.7% decrease in l* values for coated pear slices during storage. this decline in lightness is commonly attributed to enzymatic and non-enzymatic browning reactions that occur following tissue damage caused by peeling and cutting (kumar et al., 2018). higher beeswax concentrations in edible coatings effectively improved the l* index of fresh-cut pears during storage. a thicker coating layer created by higher beeswax levels helped limit oxygen penetration, thereby reducing browning. for example, treatment b8 (8% beeswax) maintained relatively high l* values throughout 12 days of storage, indicating that browning was effectively suppressed. enzymatic browning in pears is initiated by polyphenol oxidase (ppo), which uses oxygen as a cofactor to convert phenolic compounds into o-quinones. these o-quinones undergo further polymerization, producing brown pigments and reducing surface brightness. thus, limiting oxygen availability through beeswax coatings inhibits ppo activity and delays browning (jannah, 2024). pears contain high levels of total phenolics— approximately 42.2–58.8 g gae per kg dry matter in the peel and 6.4–17.7 g gae per kg dry matter in the flesh (piluzza et al., 2023). although the peel, which contains higher phenolic content, was removed, the flesh still contains sufficient phenolics to trigger enzymatic browning. these phenolic compounds can rapidly oxidize if ppo activity is not inhibited or if oxygen is not restricted. in uncoated pears (control), the exposed surface facilitates rapid ppo activity and phenolic oxidation, resulting in a significant decline in l* values during storage (purwanto et al., 2016). color changes in fresh-cut pears, particularly the decrease in l* values (lightness), are closely related to moisture loss during storage. dehydration increases tissue exposure to oxygen, enhancing ppo activity. uncoated pears (control) are more susceptible to browning due to greater water loss, which leaves the fruit surface more exposed to oxygen. conversely, the application of edible coatings helps retain moisture and limit oxygen diffusion, thereby slowing down browning reactions. moreover, fruit softening is often accompanied by more rapid browning, as cellular damage facilitates luthfiyyah et al. – application of alginate-based edible coating with … 933 the interaction between phenolic compounds and ppo (widyaka, 2018). the combination of alginate and beeswax forms an effective protective barrier that limits oxygen diffusion to the fruit surface. jannah (2024) reported that alginate can reduce respiration rates, thereby decreasing enzymatic reactions and maintaining surface color in apples. alginate acts as a flexible film-forming agent, while beeswax enhances resistance to moisture and gas transfer due to its hydrophobic nature. this mechanism is crucial, as ppo is only active under aerobic conditions—thus, limiting oxygen availability reduces browning. additionally, the coating acts as a barrier to water loss and prevents surface microstructural changes that can lead to dullness and drying of the fruit surface (wulandari, 2016). vitamin c content pears contain antioxidant compounds such as vitamin c and vitamin e, which play an essential role in neutralizing free radicals. vitamin c is especially important in supporting various metabolic processes in the human body, but it cannot be synthesized naturally and must be obtained through dietary intake. the main sources of vitamin c are vegetables and fruits, particularly those that are fresh, which is why it is often referred to as a "fresh food vitamin." according to risnayanti et al. (2015), the vitamin c content is generally higher in unripe fruits and tends to decrease as the fruit ripens. in this study, the analysis of vitamin c content was carried out on the most effective treatment, which involved fresh-cut pears coated with an alginatebased edible coating containing 4% beeswax. the results of the vitamin c analysis for fresh-cut pears with edible coating are presented in table 1. table 1. the vitamin c content of fresh-cut pears with edible coating was measured on day 12 of storage. treatment vitamin c content (mg/g) bk (control, atau untreated) 0,0088 b4 (beeswax 4%) 0,0202 according to koirala & shrestha (2020), the vitamin c content in pears is approximately 12.2 mg/100 g. in this study, the vitamin c content of fresh-cut pears without edible coating (control) was recorded at 0.0088 mg/g, whereas the sample treated with 4% beeswax (b4) retained a higher vitamin c content of 0.0202 mg/g on day 12 of storage. this finding indicates that the application of beeswax-enhanced edible coating was more effective in preserving vitamin c levels compared to the control. the effectiveness is attributed to the formation of a thicker coating layer through the combination of alginate and beeswax, which reduced gas and water vapor permeability. as a result, respiration and transpiration rates were better controlled (atmaja, 2021). vitamin c levels generally decline during storage due to oxidative processes, as ascorbic acid is highly sensitive to oxidation (sunarmi et al., 2018). atmaja (2021) explains that even under storage, respiratory and transpiration activities persist, allowing oxygen to penetrate the fruit tissue. in the presence of oxygen and the enzyme ascorbate oxidase, ascorbic acid is oxidized to dehydroascorbic acid, which then degrades into ldiketogulonic acid a compound that is no longer biologically active as vitamin c. high oxygen exposure accelerates the oxidation rate of vitamin c. the use of edible coatings containing hydrophobic substances like beeswax can slow the degradation of vitamin c. beeswax serves as a barrier that limits oxygen diffusion into the fruit tissue, thereby inhibiting oxidation. according to linardi (2019), a higher concentration of hydrophobic materials in the coating can reduce the rate of vitamin c loss. beeswax plays a critical role in restricting oxygen penetration from the environment into the pear tissue. a higher oxygen transfer rate increases respiration activity and water loss, during which vitamin c being water soluble may also diffuse out with the water (atmaja, 2021). conclusions the results of this study indicate that the addition of beeswax to alginate-based edible coating significantly affects the moisture content, weight loss, firmness, total soluble solids, and color of fresh-cut pears. the best physical and chemical characteristics are obtained from treatment b4 (4% beeswax) stored at chiller temperature (0–4°c) for 12 days, with a weight loss of 1.79%, firmness of 874.63 gf, total soluble solids of 11.28 °brix, and vitamin c content of 0.0202 mg/g authors’ contributions: maftukh zaina luthfiyyah, novita herdiana, esa ghanim fadhallah, and fibra nurainy designed the study and wrote the manuscript in bahasa. maftukh zaina luthfiyyah conducted the research. novita herdiana, esa ghanim fadhallah, and fibra nurainy supervised the research. maftukh zaina luthfiyyah and esa ghanim fadhallah translates to english and proofreads the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references andriani, e. s., nurwantoro, & hintono, a. 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(2016). pengaruh asam sitrat terhadap indeks browning, kandungan karbohidrat terlarut total, dan aktifitas enzim dehidrogenase pada buah pir yali (pyrus bretschneideri rehd.). skripsi. fakultas matematika dan ilmu pengetahuan alam, universitas lampung. bandar lampung. https://doi.org/10.25077/jrk.v13i2.501 https://doi.org/10.37341/jkkt.v3i2.88 https://doi.org/10.3934/agrfood.2020.3.327 https://doi.org/10.29244/jhi.9.1.19-26 https://doi.org/10.1016/j.scienta.2019.108853 https://www.fas.usda.gov/data/fresh‐apples‐grapes‐and‐pears‐world‐markets‐and‐trade https://www.fas.usda.gov/data/fresh‐apples‐grapes‐and‐pears‐world‐markets‐and‐trade https://doi.org/10.21776/ub.jfls.2019.003.02.04 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 645-654 | doi: 10.14421/biomedich.2025.142.645-654 issn 2540-9328 (online) identification of gamma-aminobutyricacid (gaba) of tempeh made from koro kratok (phaseolus lunatus) bean and beluntas (plunchea indica) leaves as glucose stabilizer istiqomah1,*, dina mustika rini2, larissa tania1, sholihatil hidayati3, mohammad rofik usman3 1program of pharmacy, college of health sciences, harapan bangsa school of health, indonesia. 2graduate school of integrated sciences for life, hiroshima university, japan. 3departement of pharmacy, faculty of health, universitas dr. soebandi, indonesia. corresponding author* istihariyanto@gmail.com manuscript received: 11 june, 2025. revision accepted: 09 september, 2025. published: 01 october, 2025. abstract the tempeh available on the market is predominantly made from imported soybeans, but alternative formulations using locally sourced legumes, such as koro kratok (phaseolus lunatus), have been explored. this study investigates the potential of koro kratok tempeh enriched with plunchea indica (beluntas) leaves to improve its functionality. the study will evaluate gamma-aminobutyric acid (gaba) levels and physicochemical changes during fermentation. tempeh was produced using different koro kratok-to-p. indica leaf ratios (100:0, 75:25, and 50:50). key parameters observed included mycelium development and the content of fat, moisture, ash, protein, carbohydrates, and ph. adding p. indica increased the fat content by 0.66–2.28%, the moisture content by 68.81–78.42%, and the ash content by 0.36–1.09%. meanwhile, the protein content ranged from 6.62–8.87%, the carbohydrate content from 12.07–21.31%, and the ph between 4.21–4.25. the inclusion of p. indica also altered the profile of dominant volatile compounds. gaba was detected in all samples, and in vivo studies showed improved blood glucose levels and glucose tolerance in prediabetic rats. these results suggest that koro kratok tempeh enriched with p. indica leaves could be a promising functional food for regulating blood glucose. keywords: p. lunatus bean; p. indica leaves; gaba, blood glucose. introduction tempeh, a widely consumed plant-based protein in indonesia, can be made from soybeans or other local beans, such as koro kratok (phaseolus lunatus l). koro kratok, also known as lima beans in some countries, thrives in tropical climates and is an excellent source of protein and carbohydrates, with low fat content. according to (diniyah et al., 2013), koro kratok contains 19.93% protein, 1.21% fat, and 61.42% carbohydrates. the high protein content of koro kratok, along with its local accessibility (in contrast to imported soybeans), forms the basis for its application as the primary component in soybean alternatives. tempeh contains higher levels of gamma-amino butyric acid (gaba) when the r. oligosporus microspores strain is used in an aerobic process followed by anaerobic fermentation. gaba is the main neurotransmitter in the central nervous system that acts by inhibiting certain brain signals and neuroactive molecular compounds produced by members of the human gut microbiota that regulate the neurological system in humans. in addition, this neurotransmitter may have a therapeutic effect by improving pancreatic β-cells function in patients with hyperglycemia. activation of gabaa and gabab receptors expressed on β-cells has been shown to stimulate insulin secretion and induce proliferation of β-cells. in addition, gabaa receptors are widely expressed on cd4 t cells and several studies have demonstrated a role for this neurotransmitter as an immunomodulator, suggesting an alternative mechanism that may reduce the risk of systemic inflammation (patterson et al., 2019). meanwhile, beluntas leaf shoots (pluchea indica less) were added to tempeh koro kratok to enhance its functional content. a previous study indicated that beluntas leaves possess active caffeoylquinic acid compounds that function as antihyperglycemia agents by inhibiting α-glucosidase enzyme activity, which plays a role in the conversion of carbohydrates to glucose (nopparat et al., 2020). another study found that beluntas leaves and roots contain caffeoylquinic acid and terpene glycosides as bioactive compounds. in addition, beluntas leaves exhibit stronger antioxidant activity than the rhizomes of curcumin longa (turmeric) and camellia sinensis (tea leaves). moreover, both the leaves and roots of beluntas exhibit antimicrobial and anti-inflammatory https://doi.org/10.14421/biomedich.2025.142.645-654 646 biology, medicine, & natural product chemistry 14 (2), 2025: 645-654 properties. as an antimicrobial agent, it can inhibit gram-positive and gram-negative bacteria. therefore, the leaves and roots of the beluntas plant present considerable potential for product development, encompassing a range of items such as antibiotics, deodorants, foot sprays, creams, and gels, among others (chan et al., 2022). a previous study extracted beluntas leaves at different stages of maturity using ethanol. dpph, abts, and frap analyses indicated that young beluntas leaf shoots had stronger antioxidant activity than mature leaves at the pre-flowering and flowering stages. the high antioxidant activity observed in the young beluntas leaf shoots, especially in the buds, was attributable to the increased presence of bioactive and phenolic compounds (suriyah et al., 2019). this study focuses on developing indigenous food constituents derived from probioticbased koro kratok, which are combined synergistically with herbs like beluntas leaves, recognized for their potential functional effects on metabolic syndrome. tempeh, a traditional indonesian probiotic-rich food, is a staple in the daily diet of indonesian communities. thus, this study aims to objectively evaluate the physicochemical characteristics of fermented koro kratok with the inclusion of beluntas leaves and determine the ideal composition for producing probiotic foods as a glucose stabilizer. materials and methods study area this research material was obtained from a local supermarket in jember, indonesia. beluntas leaves were collected from jember, indonesia. raprima (pt aneka fermentasi industri, bandung, indonesia) and palape (demi bumi, indonesia) yeast were purchased from a local supermarket. preparation of tempeh koro kratok was first sorted and washed prior to use. it was then soaked in distilled water for 20-24 hours to soften the koro kratok. subsequently, the outer skin of the koro kratok was peeled away, allowing it to become partially soft. the koro kratok was then boiled for 2-3 hours and the epidermis was removed until it was completely clean, followed by the drying and cooling process. meanwhile, the beluntas shoot leaves were sorted, washed, and air-dried until half withered. the dried leaves were then ground and sieved into a coarse powder ready to be used in tempeh formulations. koro kratok and beluntas powder were used as the main tempeh ingredients. three different ratios of koro kratok and beluntas leaf were used in this study: 100:0, 75:25, and 50:50, which were then named kk100, kkb75, and kkb50, respectively. to initiate the fermentation process, koro kratok and beluntas were mixed with raprima and palape yeast. the mixture was then wrapped in banana leaves, and the fermentation takes 24 hours. the fermentation proceeded for 20 hours under aerobic conditions, with a subsequent 4-hour period under anaerobic conditions at a room temperature. for analysis, tempeh was grounded and milled to a finely powdered state using a food processor. the powdered samples were then stored at 4°c to avoid potential damage. 200 grams of the material were used for each analysis. mycelium observation the physical properties were analyzed by examining the growth of mycelium. the appearance of tempeh koro kratok with and without the addition of beluntas leaves was subjectively observed, with assessments made on mycelium growth and tempeh compactness as a whole (radiati, 2016). tempeh samples were documented using a xiaomi redmi note 8 camera at 40x magnification, positioned at an equidistant point via a tripod. consistency of the base substrate was maintained, and subjective measurements of mycelial growth, hyphae, texture, and other physical appearances were conducted. procedures fat content the boiling flask was dried in an oven, then cooled in a desiccator and weighed. approximately 1-2 grams of the sample was placed in a filter paper thimble lined with cotton. the stoppered thimble containing the sample was dried in an oven at a temperature not exceeding 80°c for approximately 1 hour and then transferred to a soxhlet extraction flask. a condenser apparatus was placed on top of the flask, with the boiling flask underneath. sufficient hexane solvent was added to the boiling flask, followed by extraction for 6 hours until the solvent returning to the flask was clear. the solvent in the boiling flask was distilled and the solvent collected. the boiling flask containing the extracted fat was heated in an oven at 105°c until a constant weight was reached, then cooled in a desiccator and weighed (aoac, 2005). carbohydrate content the carbohydrate content was evaluated by the bydifference method. the carbohydrate content was calculated as the residue after determining the moisture, ash, protein and fat content of the sample. the total carbohydrate content of the sample can be calculated using the following formula: carbohydrate content = 100 – (moisture content + ash content + protein content + fat content). protein content protein content was determined as previously described (utami et al., 2016). 0.51 gram sample was placed in a 100 ml kjeldahl flask, followed by adding 2 grams of a selenium mixture and 25 ml of concentrated h2so4. the istiqomah et al. – identification gaba of tempeh as a glucose stabilizer 647 kjeldahl flask containing the sample and reagents was heated to boiling on a hot plate until the solution turned a clear greenish color for approximately 2 hours. after cooling, the solution was diluted and transferred to a 100 ml graduated flask. then 5 ml of the solution was pipetted into a distillation apparatus, followed by adding 5 ml of 30% naoh and a few drops of pp indicator. distillation was performed for 10 minutes, with 10 ml of a 2% borate solution mixed with an indicator as the receiver. titration was performed with a 0.01 n hcl solution. the protein content of the sample can be calculated using the following formula: %𝑁 = ( (𝑉𝑜𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑉𝑜𝑙 𝑏𝑙𝑎𝑛𝑘𝑜) ∗ 𝑁 ∗ 14,008 ∗ 100 𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑚𝑔) ) ∗ 6,25 %𝐶𝑟𝑢𝑑𝑒 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 = %𝑁 𝑥 𝐶𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 water content the moisture content was determined by the oven method at a temperature of 105°c. samples (1-2 grams) were weighed and placed in a pre-dried evaporating dish that had been previously weighed. the sample and the evaporating dish were then dried in an oven at 105°c for 3 hours, cooled in a desiccator and weighed until a constant weight was obtained (aoac, 2005). ash content an empty porcelain crucible was dried in an oven at 105°c for 15 minutes, then cooled in a desiccator and weighed. a sample weighing 2-3 grams was placed in the crucible and weighed. the crucible containing the sample was ignited until complete volatilization, then placed in an electric furnace at a temperature of 550°c until complete ashing. the crucible containing the sample was removed from the furnace, cooled in a desiccator, and weighed to a constant weight (aoac, 2005). ph the ph of the samples was measured using a ph meter, which was previously calibrated using ph 4, 7, and 9 buffer solutions. powdered samples were then dissolved in distilled water prior to ph measurement. gc-ms analysis gc-ms analysis was used to identify the volatile compound of the sample. the samples were filtered using a syringe filter membrane. the samples were then injected into the gc-ms instrument and the qualitative volatile compounds were determined. the gc-ms instrument was a gcms-qp2010 plus shimadzu equipped with a splitless injector set at a temperature of 80°c. the temperature of the ms detector was set at 200°c. an rtx-50 column (with an internal diameter of 0.25 mm, length of 30 m, and thickness of 0.25 μm) was used. the detector temperature was initially programmed at 80°c for 10 minutes and then increased to 230°c for 15 minutes at a rate of 5°c/min. helium was used as the carrier gas at a 19 ml/minute flow rate. a 1 μl sample was injected using the splitless method. thin layer chromatography analysis of gaba gaba production was determined by thin layer chromatography (tlc) using a 10% gaba solution as a standard reference. the samples were filtered with a syringe filter membrane. the samples were previously prepared as supernatant. the resulting supernatant from the sample was transferred to a fresh tube. sample concentrations were prepared by diluting each sample to 0.01 g in 10 ml of 96% ethanol to give a 10% concentration. subsequently, 1 ml of the sample concentration was applied to the tlc plate using a capillary pipette. a 10% pregabalin-gaba solution was used as a positive control, followed by sequential application of the supernatants of the isolates from the 2% samples to the tlc plates. tlc analysis was performed for 30 minutes using a developing solution (eluent) of n-butanol, acetic acid, and distilled water in a 5:3:2 ratio (qiu et al., 2010). after analysis, the tlc plate was treated with a 0.5% (w/v) ninhydrin solution and then oven-dried at 60°c for 60 minutes. the gaba compound produced within the tempe formulation was identified based on the retention factor (rf), similar to the rf value obtained from the pregabalin solution used as the gaba standard reference. the rf value was calculated as previously described (yogeswara et al., 2018): 𝑅𝑓 = 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑 distance traveled by the solvent front animals preparation male wistar balb/c mice, aged 2–3 months and weighing approximately 200 grams on average, were used in this study. a total of 25 rats were selected, individually weighed, and marked before grouping. the animals were randomly divided into five groups, each consisting of five rats. prior to treatment, the rats were fasted for 16 hours and subsequently induced with streptozotocin (sigma-aldrich). all experimental procedures were approved by the ethical committee of medical research, faculty of dentistry, university of jember, under registration number 2836/un.25.8/kepk/dl/2024. anti-hyperglycemic test the antidiabetic testing began with weighing the rats and grouping them based on similar body weights. the animals were then fasted for 16 hours. on the first day, baseline blood samples were collected prior to treatment, and initial blood glucose levels were measured. subsequently, a streptozotocin (stz) solution was administered intraperitoneally at 0.5 mg per rat. following a 3-day stabilization period post-induction, rats with confirmed diabetes mellitus (blood glucose 648 biology, medicine, & natural product chemistry 14 (2), 2025: 645-654 levels >200 mg/dl) were selected and regrouped. each group then received different treatments: ▪ normal group ▪ the negative control group received stz and 0.5% cmc and nacl, ▪ the positive control group received stz and metformin suspension at 0.013 mg/20 g body weight, ▪ treatment groups received stz solution one of the following (all dosed at 100 mg/20 g body weight): koro kratok tempeh extract, koro kratok tempeh extract and beluntas leaf extract (75:25 ratio), koro kratok tempeh extract and beluntas leaf extract (50:50 ratio). all treatments were administered orally once daily in the morning for 14 consecutive days. blood samples and body weight measurements were recorded on days 0, 3, 7, and 14 after treatment initiation. blood samples were collected from the tail vein by puncturing with a sterile needle. a drop of blood was then applied to a glucometer strip and analyzed using a validated and calibrated glucometer to determine blood glucose levels. data analysis data were analyzed using one-way anova statistical analysis followed by the tukey test. statistical analysis was performed using sigma stat 4.1. results and discussion mycelium observation based on figure 1, observations of the physical appearance of kk100 tempeh without the addition of beluntas leaves show evenly distributed mycelium growth. the developed hyphae also show an even distribution. this contributes to the compactness of the tempeh, which is dense and tends to be dry. conversely, kkb50 tempeh, consisting of a 50% composition of koro kratok and 50% addition of beluntas leaves, had a low level of mycelial growth and the developing hyphae were barely visible. this significantly affected the fragility of the tempeh, making it prone to breakage and less dense in texture. the fermentation of kk75 showed a denser result with more mycelium growth than kkb50; although solid in shape, it tended to be brittle. the grown hyphae were unevenly distributed on the surface of the tempeh, with a greater concentration on one side. this appearance suggests that kk100 fermented tempeh has a texture and density similar to soy-based tempeh, while the addition of 25% beluntas leaves in the kk75 treatment shows relatively favorable visual results. figure 1. physical appearance of tempeh koro kratok with beluntas leaves addition. fat content the fat content of kk100 was significantly lower than that of kkb50, with values of 0.67% and 2.28%, respectively, indicating an increase in fat content with the addition of beluntas leaves (table 1). these results suggest that the fermentation process can alter the formation of fat content with the addition of beluntas leaves. a previous study showed that after 24 hours of fermentation, the levels of short-chain fatty acids were found in samples fermented with bifidobacterium animalis subsp. the microbial strain from the fermentation pathway possesses a metabolic pathway capable of optimizing the production of short-chain fatty acids as the final fermentation product. the function of the fatty acids is as an energy source with the highest caloric content and as a source and solvent for vitamins a, d, e, and k (annunziata et al., 2020). protein content the protein content was significantly different among the groups, with the highest protein content recorded in kk100 at 8.88% (table 1). it was consistent with the composition of kk100 without adding beluntas leaves, which had a higher protein content. meanwhile, adding beluntas leaves does not contribute to the increase of protein content during koro kratok fermentation. beluntas leaves play a more significant role in the high fiber content than the formation in protein content during koro kratok fermentation. the protein content of tempeh increased as the fermentation time increased. this increase in protein content could be attributed to r. oligosporus engaging in metabolism during its growth phase and producing protease enzymes capable of breaking down proteins into free amino acids containing n groups, thus increasing protein content (dewi et al., kkb50 kk100 kkb75 istiqomah et al. – identification gaba of tempeh as a glucose stabilizer 649 2014). dietary protein has been reported to affect intestinal health by modulating barrier function (rini et al., 2020). water content the water content of kk100 was significantly different from that of the kkb50 groups (table 1). this observation was consistent with the composition of kkb50, which contained the highest concentration of beluntas leaves, resulting in a higher water content during the fermentation process than kk100, which lacks beluntas leaves. the water content of the fermentation product also affects the quality of the fermentation process using the r. oligosporus starter. the increase in water content during fermentation is due to microbial digestion of the substrate, which produces water, carbon dioxide, and a certain amount of energy. fermentation time is a critical factor influencing the increase in water content; therefore, as the fermentation time increases, the moisture content also increases (qomariyah & utomo, 2016). adding materials such as beluntas leaves also contributes to increased water content. a characteristic of beluntas leaves is their tendency to produce more water compared to fermented tempeh, resulting in a higher overall water content. the accelerated mycelial growth in tempeh without beluntas leaves allows r. oligosporus hyphae to grow more compactly, resulting in a lower water content than that observed in fermented tempeh containing beluntas leaves. ash content the ash content showed a significant difference between the kk100 and kkb50, with values of 0.36% and 1.10%, respectively (table 1). these results indicated that the fermentation of koro kratok and beluntas leaves provides a safe level for consumption as a food ingredient. total ash content is used to assess the nutritional value of a food by indicating the total amount of minerals present, some of which may be toxic (mardiah et al., 2020). ash content analysis is also often used as an indicator to determine the quality of other food materials. this study showed that kk100, kkb75 and kkb50 groups has a good level of quality, making them suitable for use as food materials. table 1 chemical characteristics of tempeh made from koro kratok and beluntas leaf group fat (%) protein (%) water (%) ash (%) kk100 0,67a 8,88a 68,81b 0,36a kkb75 1,03ab 7,34b 73,27ab 0,61ab kkb50 2,28b 6,61c 78,42a 1,10b *values followed by the same letter indicate no significant difference (p>0.01) carbohydrate content the carbohydrate content showed a significant difference between kk100 sample and kkb50, with values of 21.31% and 12.07%, respectively (table 2). the highest carbohydrate content was observed in the fermentation of 100% koro kratok compared to the addition of beluntas leaves. this result was attributed to the role of hyphal growth forming mycelium during fermentation by r. oligoporus, which can break down polysaccharide components and release amylase enzymes, resulting in the generation of new carbohydrate compounds (indriyani et al., 2010). it was also supported by another study which suggests that beluntas leaves (pluchea indica less) contain active caffeoylquinic acid compounds capable of inhibiting the action of αglucosidase enzymes responsible for the conversion of carbohydrates into glucose as an anti-hyperglycemic agent (nopparat et al., 2020). therefore, the higher the concentration of beluntas leaves, the higher the carbohydrate content. in addition, kk100 has a compact tempeh texture due to uniform hyphal growth, resulting in higher fiber formation compared to kkb50 and kkb75, which have lower carbohydrate content. high fiber has been reported to help maintain intestinal health (rini et al., 2023), as well as its metabolites such as short-chain fatty acids (isayama et al., 2023; xu et al., 2023). table 2. carbohydrate and ph of tempeh koro kratok with beluntas leaves addition group carbohydrate (%) ph kk100 21,31 ± 0,05a 4,23 kkb75 17,90 ± 0,42ab 4,25 kkb50 12,07 ± 0,07b 4,21 *values followed by the same letter indicate no significant difference (p>0.01) ph level the ph values were not significantly different among the three samples and ranged from 4.21 to 4.25, indicating an acidic ph of tempeh (table 2). these ph measurements indicated that the tempeh had undergone a successful fermentation process with increased lactic acid content as a source of probiotics. a previous study showed that the strain type of lactobacillus sp influenced the decrease in ph during the fermentation of red beans with lactocbacillus plantarum wcfs1. the ph decreased from 6.6 to 3.76 after 48 hours of fermentation at room temperature (gan et al., 2017). the acidifying ability of lactobacillus is also influenced by the role of carbohydrates present in the composition of legumes during the fermentation process. oligosaccharide components present in legumes are used by certain lactobacillus species to initiate microbial enzymatic activities, resulting in varying ph reductions (mardiah et al., 2020). volatile compounds the shift in the volatile composition of tempeh made from koro kratok and beluntas leaves was shown in 650 biology, medicine, & natural product chemistry 14 (2), 2025: 645-654 figure 2. the dominant volatile compounds in the kk100 group were carbamic acid, acetic acid and 2,3butanediol. meanwhile, 2,3-dihydro-3,5-dihydroxy-6methyl-4h-pyran-4-one and l(+) milchsaeur were more dominant in the kkb75 group. other volatile compounds such as 1-(4-methoxyphenethyl)-1methylbiguanidine, benzenethanamine, mevalonic lactone and coumarin were found in similar amounts with carbamic acid, acetic acid and 2,3-butanediol in kkb75 group. on the other hand, the kkb50 group was dominated by similar compounds to those found in the kkb75 group, with the addition of volatile compounds such as ethanol, propinoic acid, glycerol, isopentane, tetramethylene sulfone, 1-methyl-4-aminocyclohexane, cis-6-methyltetrahydro-pyran-2-yl)-acetic acid, 2,3dihydro-benzofuran, benzenepropanoic acid, and melitol. these results suggest that the addition of beluntas leaves changes the composition of volatile compounds in tempeh. the dominant volatiles in each group may contribute to the aroma of tempeh. however, less dominant volatile compounds may also be involved in forming subtle flavors. this finding is consistent with the previous study that showed that the production of volatile compounds depends on the substrate (mei feng et al., 2007). figure 2 profile of volatile compounds on tempeh koro kratok with beluntas leaves addition. identification of gaba figure 3 illustrates the identification profile of gaba in tempeh koro kratok with beluntas leaves. purple spots indicate the presence of chemical compounds that are accelerated by the mobile phase of the eluent and stained by the dye solution. the first lanes show the gaba istiqomah et al. – identification gaba of tempeh as a glucose stabilizer 651 profile of pregabalin as a standard at a concentration of 10 mg. the next lane shows the profile of kk100, kkb75, and kkb50 isolates, respectively. the presence of gaba can be readily confirmed by the appearance of purple spots upon applying ninhydrin (yogeswara et al., 2018). table 3 shows rf values for pregabalin were determined to be 0.62, while the rf values of kk100, kkb75, and kkb50 were 0.60, 0.53, and 0.51, respectively. the equivalence between the rf values of the isolates and the rf value of pregabalin serves as evidence affirming the capability of all isolates to generate gaba. kk100 was demonstrated to produce gaba in a manner comparable to the standard used, in contrast to the other groups. certain strains or species within the lactic acid bacteria (lab) have been documented to possess the ability to produce gaba. the majority of gaba-producing bacterial strains or species are typically derived from traditional fermented food sources. furthermore, scientific reports have indicated that only four isolates belonging to the lab, namely l. paracasei pf6, l. bulgaricus pr1, l. lactis pu1 and l. brevis, have been extracted from different types of cheeses, exhibiting the most substantial gaba production and the highest gaba levels (siragusa et al., 2007). in addition, microorganisms synthesize gaba during the fermentation process. r. oligosporus, in conjunction with l. plantarum 202, has been observed to produce gaba during the fermentation of soybean residues in a time-dependent manner. a comparative analysis of the respective fermentation times of 24 and 48 hours revealed that co-inoculation with gabaproducing l. plantarum 202 did not result in an additive effect on gaba content, as observed in r. oligosporus fermented soybean residue (hariyanto et al., 2022). this improvement in insulin function is suspected to be partly due to the presence of gaba (gamma-aminobutyric acid), which may support pancreatic recovery by helping stabilize insulin and glucagon hormones under homeostatic conditions. however, it is also possible that other bioactive compounds present in the 100% tempeh contribute to the observed improvement in fasting glucose tolerance. figure 3 identification profile of gaba with thin layer chromatography on tempeh koro kratok with beluntas leaves addition. table 3. retention factor (rf) gaba of tempeh koro kratok with beluntas leaves addition. group gaba kk100 kkb75 kkb50 rf value 0,62 0,60 0,53 0,51 body weight and blood glucose level the results of the body weight measurements of rats in figure 4, which had been induced with stz, showed no significant weight gain in either the control or treatment groups. however, the kk100 and kkb75 treatment groups exhibited increased body weight from day 0 to day 14. in contrast, the kkb50 group did not show significant weight gain and experienced a weight loss by day 14. this indicates that the kkb50 group had a lower protein content than kk100 and kkb75. changes in the rats’ body weight suggest that plant-based proteins derived from legumes may reduce plasma cholesterol, triacylglycerols, and blood glucose levels, acting as potential antioxidants and improving coronary endothelial function. the findings support that replacing animal-based protein with legume protein can improve lipid profiles, prevent atherosclerosis, and enhance overall metabolism, thereby contributing to body weight stabilization (sun t. et al., 2022). figure 4. body weight levels in mice induced with stz under different treatments: normal, nc (negative control), pc (positive control), kk100 (100% koro kratok extract), kkb75 (koro kratok 75% and beluntas 25%), and kkb50 (koro kratok 50% and beluntas 50%). 18 19 20 21 22 23 24 0 4 7 10 14 b o d y w ei g h t (g ra m ) days normal nc pc kk100 kkb75 kkb50 652 biology, medicine, & natural product chemistry 14 (2), 2025: 645-654 based on the blood glucose levels shown in figure 5, measured over a 0–12 day period, the kk100 treatment group demonstrated a reduction in blood glucose levels compared to the positive control (pc) group, which showed improved insulin levels on days 6, 9, and 12. meanwhile, the kkb75 treatment group exhibited improved on day 12, as indicated by a decrease in blood glucose levels accompanied by increased insulin levels. in contrast, the kkb50 group showed a continuous increase in glucose levels with no significant improvement, similar to the negative control (nc) group. this suggests possible mortality among the rats during the testing period due to persistently elevated glucose levels observed on day 12. this indicates that adding beluntas leaves during the fermentation process to make tempeh did not significantly affect blood glucose reduction. this finding indicates that adding beluntas (pluchea indica) leaves during the fermentation process of koro kratok tempeh does not have a significantly effect on lowering blood glucose levels. it is hypothesized that incorporating beluntas leaves during fermentation may inhibit the reduction of phytic acid in koro kratok, thereby increasing its antinutrient capacity compared to fermentation without beluntas leaves (hariyanto, et al., 2022). moreover, the protein content in the kkb75 and kkb50 samples was lower than in the kk100 group, suggesting that the glucose-lowering effect was more pronounced in the kk100 group, where no beluntas leaves were added. this finding underscores the potential role of protein in reducing fasting blood glucose levels. nevertheless, further research is required to identify the specific bioactive compounds involved in this mechanism. figure 5. blood glucose levels in mice induced with stz under different treatments: normal, nc (negative control), pc (positive control), kk100 (100% koro kratok extract), kkb75 (koro kratok 75% and beluntas 25%), and kkb50 (koro kratok 50% and beluntas 50%). glucose tolerance results the results of the glucose tolerance test, which measured the test animals blood glucose sensitivity every 30 minutes (as shown in figure 6), illustrate the animals’ tolerance to increased glucose levels. fasting glucose tolerance is commonly used to assess prediabetic conditions, with blood glucose levels categorized as follows: <140 mg/dl (normal), 140–199 mg/dl (prediabetes), and ≥300 mg/dl (diabetes) [11]. based on the test results, the kk100 treatment group showed an improvement in glucose tolerance compared to the baseline (0 minutes). in contrast, the kkb75 and kkb50 treatment groups did not improve fasting glucose tolerance compared to the positive control (pc) group. these findings suggest that tempeh made from 100% koro kratok may enhance insulin activity, thereby lowering blood glucose levels in prediabetic conditions. kk100, which was fermented without the addition of beluntas leaves, had a higher total protein content, which may have contributed to the reduction in glucose tolerance levels in diabetic rats. although the addition of beluntas leaves in the fermentation of koro kratok tempeh led to an increase in beneficial fatty acids, the results indicated that it did not have a significant effect on fasting blood glucose or glucose tolerance in diabetic rats with insulin resistance. further studies are needed to understand the specific mechanism. figure 6. glucose tolerance over a 0–120 minute period under different treatments: normal, nc (negative control), pc (positive control), kk100 (100% koro kratok extract), kkb75 (koro kratok 75% and beluntas 25%), and kkb50 (koro kratok 50% and beluntas 50%). 0 50 100 150 200 250 300 350 400 0 30 60 90 120 b lo o d g lu c o se ( m g /m l) minute normal nc pc kk100 kkb75 kkb50 0 50 100 150 200 250 300 350 400 0 30 60 90 120 g lu c o se t o le r a n c e ( m g /m l) minute normal nc pc kk100 kkb75 kkb50 istiqomah et al. – identification gaba of tempeh as a glucose stabilizer 653 conclusions our study demonstrated that koro kratok could serve as a viable alternative to soybeans in tempeh production. the results showed that the fat content ranged from 0.66% to 2.28%, protein content from 6.62% to 8.87%, moisture content from 68.81% to 78.42%, ash content from 0.36% to 1.09%, carbohydrate content from 12.07% to 21.31%, and ph ranged from 4.21 to 4.25. the addition of beluntas leaves during fermentation significantly affected both the physical and chemical characteristics of the tempeh. specifically, it increased the fat, ash, and moisture content, while decreasing the protein and carbohydrate levels. the ph value, however, remained unchanged. tempeh containing beluntas leaves exhibited a more brittle texture and less evenly spread mycelium growth than the control groups. additionally, including beluntas leaves enhanced the volatile compounds in tempeh, potentially contributing to its distinctive flavor. importantly, our analysis confirmed the presence of (gaba) in koro kratok tempeh, both with and without the addition of beluntas leaves, 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(2018). isolation and identification of lactic acid bacteria from indonesian fermented foods as γ-aminobutyric acid-producing bacteria. international food research journal, 25(4), 1753–1757. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 513-518 | doi: 10.14421/biomedich.2025.141.513-518 issn 2540-9328 (online) phytochemical, antioxidant, and antibacterial activities of stem bark fractions of eucalyptus globulus against multidrug-resistant bacterial isolates abdulazeez mumsiri abaka1,*, nazuwa dominic1, alex yeri emmanuel2, zayyad dahiru aliyu3 1science laboratory technology department, school of science and technology, adamawa state polytechnic yola, nigeria 2sechenov university/first moscow medical university, nigeria. 3biotechnology department, faculty of life sciences, modibbo adama university yola, nigeria. corresponding author* abdulazizelnino22@adamawapoly.edu.ng manuscript received: 25 maret, 2025. revision accepted: 23 july, 2025. published: 01 august, 2025. abstract the growing challenge of antimicrobial resistance (amr) has intensified the need for alternative therapeutic agents, with medicinal plants offering promising solutions due to their bioactive compounds. this study investigated the antimicrobial and antioxidant properties of eucalyptus globulus bark extracts against multidrug-resistant bacteria (escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus). plant samples were collected from adamawa state polytechnic, nigeria, authenticated (voucher asp-765), and subjected to reflux extraction using hexane and water. phytochemical analysis revealed alkaloids, phenols, tannins, glycosides, and terpenoids in both methanol and aqueous extracts, while flavonoids and steroids were absent in aqueous extracts, and methanol extracts lacked saponins. antibacterial activity was assessed through agar well diffusion and broth dilution assays, demonstrating a concentration-dependent effect. methanol extracts showed greater efficacy against e. coli and p. aeruginosa (12.8–13.8 mm inhibition zones), while aqueous extracts were most effective against s. aureus (19.3 mm at 100 mg/ml). minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) values ranged from 25 to 50 mg/ml, with e. coli exhibiting the lowest mic (25 mg/ml), highlighting the extract’s antimicrobial potential. the antioxidant activity was evaluated using phosphomolybdate and ferricyanide assays. the methanol extract exhibited strong free radical scavenging activity, with a total antioxidant capacity (tac) expressed in ascorbic acid equivalents (aae), although lower than pure ascorbic acid. statistical validation (one-way anova, p < 0.05) confirmed the significance of the results. these findings support the traditional medicinal use of e. globulus and its potential for combating antibiotic-resistant infections and oxidative stress-related conditions. further studies are recommended to isolate bioactive compounds, determine mechanisms of action, and develop optimized therapeutic formulations for amr management. keywords: eucalyptus globulus; antimicrobial resistance; phytochemicals; antioxidant activity; medicinal plants. introduction medicinal plants have played a crucial role in treating various diseases and infections for centuries, owing to their natural antibacterial, antifungal, and antiviral properties (aladejana et al., 2024). recently, there has been a growing interest in harnessing plant-derived compounds to combat infectious diseases, cancer, and the increasing challenge of antimicrobial resistance (amr). the widespread use and misuse of antibiotics, coupled with bacteria’s innate ability to develop resistance to synthetic drugs, have contributed to the emergence of multidrug-resistant pathogens (alara & alara, 2024). this underscores the urgent need for alternative treatments, with plant-based compounds emerging as promising candidates. many medicinal plants contain bioactive molecules that could serve as the foundation for developing safer and more effective therapeutic agents (chaachouay & zidane, 2024). as researchers explore novel antibacterial solutions, these natural resources are increasingly recognized as valuable contributors to innovative drug development to address the global amr crisis. traditional medicine, deeply rooted in cultural practices and economic realities, remains a cornerstone of healthcare in many parts of the world, particularly in africa (eruaga et al., 2024). however, despite their widespread use, many of these plants have not undergone rigorous scientific validation. establishing a comprehensive database documenting their medicinal properties could facilitate their integration into modern healthcare systems, providing cost-effective and safer alternatives to synthetic drugs (aruwa & sabiu, 2024). in nigeria, plants like eucalyptus globulus (blue gum) have long been used for their antimicrobial, antiinflammatory, and wound-healing benefits (sa’id & https://doi.org/10.14421/biomedich.2025.141.513-518 mailto:abdulazizelnino22@adamawapoly.edu.ng 514 biology, medicine, & natural product chemistry 14 (1), 2025: 513-518 abdullahi, 2022). as part of their natural defense mechanisms, these compounds exhibit diverse pharmacological properties, including antioxidant, anticancer, and antimicrobial activities, reinforcing their significance in the quest for novel therapeutic solutions (anwar et al., 2025). the rise of multidrug-resistant bacterial infections presents a major global health challenge intensified by antimicrobial resistance (amr) and the persistence of bacterial cells that evade antibiotic treatments (karnwal et al., 2025). these persistent cells contribute to recurrent infections and treatment failures. amr, often referred to as a silent pandemic, was associated with approximately 4.95 million deaths in 2024 (aslam et al., 2024). six major bacterial pathogens—escherichia coli, staphylococcus aureus, klebsiella pneumoniae, streptococcus pneumoniae, acinetobacter baumannii, and pseudomonas aeruginosa—were responsible for nearly 75% of these cases (ntim et al., 2025). recognized by the world health organization (who) as priority pathogens, these bacteria highlight the urgent need for new antimicrobial strategies (who, 2024). in addition to strengthening surveillance systems and promoting responsible antibiotic use, developing novel antimicrobial compounds is crucial to controlling the escalating amr crisis. among these threats, methicillin-resistant staphylococcus aureus (mrsa) stands out as a particularly difficult-to-treat pathogen (mandal et al., 2024). this gram-positive bacterium is responsible for a variety of infections and has developed resistance to key antibiotics, including vancomycin, daptomycin, and linezolid (rajput et al., 2024). mrsa also can form biofilms and persister cells, further increasing its tolerance to conventional treatments (kaushik et al., 2024). this growing concern has driven interest in exploring natural antimicrobial agents as potential alternatives to conventional disinfectants. advancing such innovative approaches is essential to tackling resistant infections and alleviating the global health burden posed by amr. antioxidants play a crucial role in maintaining cellular homeostasis by inhibiting or neutralizing the harmful effects of free radicals in the body (bajaj et al., 2024). oxidative stress, resulting from an imbalance between antioxidants and reactive oxygen species (ros), has been implicated in the pathogenesis of various chronic diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders (muscolo et al., 2024). eucalyptus globulus, are recognized as rich sources of antioxidants, making them promising candidates for therapeutic applications and dietary supplements. materials and methods collection, identification, and processing of plant materials the bark of eucalyptus globulus was collected within the grounds of adamawa state polytechnic, yola, nigeria. the samples were verified and registered under the voucher number asp-765 by the department of forestry technology at the same institution. extraction of crude extract of eucalyptus globulus the crude extract was obtained using the reflux extraction method, following the procedure outlined by ewansiha et al. (2020). normal hexane and water were used as extraction solvents. a total of 100 g of finely ground, dried plant material was dissolved in 400 ml of the respective solvents. after refluxing, the mixtures were filtered through filter paper to obtain a clear filtrate. the filtrate was then concentrated to a semi-solid form using a rotary evaporator and further dried with a water bath to yield the crude extract. bioassay studies test isolates the multidrug-resistant clinical isolates used in this study were obtained from the microbial culture bank of modibbo adama teaching hospital in yola, nigeria. these isolates included escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus. their identities were confirmed using standard biochemical tests, following the classification guidelines outlined in bergey’s manual of systematics of archaea and bacteria (abaka et al., 2024). preparation of stock solution of extract the stock solution for each extract was prepared following the method of habibu et al. (2021), with slight modifications. specifically, 0.4 g of each extract was dissolved in 2 ml of 20% dmso to obtain a final concentration of 200 mg/ml. this stock solution was then serially diluted to generate working concentrations of 100 mg/ml, 50 mg/ml, and 25 mg/ml. inoculum standardization the direct colony suspension method was utilized, in which 24-hour-old colonies of each test isolate were suspended in 2 ml of sterile normal saline. the turbidity was then adjusted to correspond with the 0.5 mcfarland standard. antibacterial susceptibility test of the crude extract a sterile cork borer (6 mm in diameter) was used to create wells in the culture medium. subsequently, 100 μl (0.1 ml) of the extracts at concentrations of 40 mg/ml and 50 mg/ml, along with the positive control (30 μg/ml doxycycline) and the negative/solvent control (dimethyl sulfoxide, dmso), were introduced into the wells. the plates were left undisturbed on the bench for abaka et al. – phytochemical, antioxidant, and antibacterial activities … 515 approximately 30 minutes to allow proper diffusion of the extracts into the medium. incubation was carried out at 37°c for 18 to 24 hours. following incubation, the culture plates were examined for the presence of clear zones around the wells, indicating antibacterial activity. the zone of inhibition (zoi) was measured in millimeters. all tests were performed in triplicate (ewansiha, 2020). determination of minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) the minimum inhibitory concentration (mic) was determined using the tube dilution method, while the minimum bactericidal concentration (mbc) was assessed following the procedure described by saleh et al. (2024). for mic testing, serial two-fold dilutions of the plant extracts were prepared in nutrient broth. an initial 1:1 mixture was made by combining 1 ml of nutrient broth with 1 ml of the 100 mg/ml extract solution, serving as the reference standard. subsequent dilutions produced concentrations of 100, 50, 25, 12.5, and 6.25 mg/ml. each dilution tube was inoculated with 0.1 ml of a standardized microbial suspension and incubated at 37°c for 24 hours. the mic was identified as the lowest extract concentration that completely inhibited visible bacterial growth. for mbc determination, aliquots from mic tubes showing no bacterial growth were transferred onto fresh nutrient agar plates and incubated for another 24 hours. the mbc was defined as the lowest extract concentration that resulted in no bacterial colony formation on the agar plates. antioxidants activity total antioxidant capacity the total antioxidant capacity (tac) of the extract was evaluated following the method described by dahiru et al. (2024). a 0.5 ml aliquot of the sample, dissolved in distilled water at a concentration of 300 µg/ml, was combined with 2 ml of phosphomolybdate reagent in a capped tube and incubated at 95°c for 10 minutes. the absorbance of the sample was then measured at 695 nm using a uv-vis spectrophotometer (model v1000) against a blank solution consisting of phosphomolybdate reagent and distilled water, which underwent the same treatment as the sample. additionally, ascorbic acid (aa) at varying concentrations (20–100 µg/ml) was used to generate a calibration curve. the tac was expressed as ascorbic acid equivalent (aae) in µg/ml based on triplicate determinations. reducing power assay the reducing power of the extract was assessed following the method of dahiru et al. (2024). (1986). a 0.75 ml aliquot of the extract at varying concentrations was mixed with 0.75 ml of phosphate buffer (0.2 m, ph 6.6) and 0.75 ml of potassium hexacyanoferrate (k₃fe(cn)₆) (1%, w/v). the mixture was then incubated in a water bath at 50°c for 20 minutes. the reaction was halted by adding 0.75 ml of 10% trichloroacetic acid (tca), followed by centrifugation at 800 g for 10 minutes. a 1.5 ml portion of the supernatant was combined with 1.5 ml of distilled water and 0.1 ml of ferric chloride solution (0.1%, w/v) and allowed to react for 10 minutes. the reducing power of the extract was expressed as an equivalent of ascorbic acid (dahiru et al., 2024). statistical analysis data were presented as the mean ± standard error of the mean (sem) from three independent experiments. for in vitro antioxidant assays, one-way anova followed by tukey’s post hoc test (p < 0.05) was used to compare differences among the various fractions across different antioxidant assays. a probability value of p < 0.05 was considered statistically significant. results table 1. results of phytochemical screening of methanol, and aqueous extracts of e. globulus bark. s/n name of the phytochemical presence (+) and absence (-) in different extracts methanol extract aqueous extract 1 alkaloids + + 2 flavonoids + 3 phenols + + 4 tannins + + 5 cardiac glycosides + + 6 steroids + 7 saponins + 8 terpenoids + + figure 1. total antioxidant capacity; a) ascorbic acid calibration curve and b) aae total antioxidant capacity. value with a superscript is significantly (p < 0.05) lower than eghf and egce. value with band e superscripts is significantly (p < 0.05) higher than eghf and egcf, respectively while values with superscripts are significantly (p < 0.05) lower than egce. 516 biology, medicine, & natural product chemistry 14 (1), 2025: 513-518 figure 2 presents the total antioxidant capacity (tac) total reducing power; a) ascorbic acid calibration curve and b) aae total reducing power. values with a superscript are significantly (p < 0.05) higher than eghf. table 2. zone of inhibition (mm) of the organisms caused by aqueous and methanol extracts of e. globulus. table 3. minimum inhibitory concentration and minimum bactericidal concentration of methanol bark extracts of e. globulus against test organisms. test organism incubation time (h) leaf extract concentration (mg/ml remark mic 100 50 25 12.5 6.25 e. coli 24 + + −* − 25 s. aureus 24 + −* − − − 50 p. aeruginosa 24 + −* − − 50 mbc e. coli 24 + -* 25 s. aureus 24 + + -* 25 p. aeruginosa 24 + -* 50 discussions the use of traditional medicines and medicinal plants in mainly developing countries as remedial agents for health maintenance has been broadly observed (hlatshwayo et al., 2025). modern-day pharmacopeia, however, contains at least 25% of drugs derived from plants and many other synthetic analogs, built on prototype chemical substances isolated from plants (rahman et al., 2024). involvement in medicinal plants as re-budding health assistance has been fueled by the rising charges of prescription drugs in safeguarding personalized health and well-being and the bioprospecting of new plant-derived drugs (buragohain et al., 2024). the aqueous and methanol extracts of e. globulus bark shared common phytonutrients like alkaloids, phenols, tannins, glycosides, and terpenoids, aligning with saleh et al. (2024). however, saponins were absent in the methanol extract, while flavonoids and steroids were absent in the aqueous extract. differences from ewansiha et al. (2024) may be due to variations in extraction conditions and plant origin. the antibacterial assay varied greatly in terms of inhibitory potential. table 2 shows the antibacterial activity of four e. globulus doses against s. aureus, e. coli, and p. aeruginosa. methanol extract (ae) of e. globulus seeds had the highest activity against, e. coli, and p. aeruginosa, with inhibition zones measuring 12.8 mm and 13.8 mm at the concentration of 100 mg/ml. the aqueous extract (me) demonstrated marginally higher effectiveness against s. aureus showcasing inhibition zone diameters of 19.3 mm at 100 mg/ml. the lowest activity was recorded for the aqueous extract for the three bacteria isolates. the findings indicate that the methanol extract of e. globulus inhibited bacterial growth more effectively than the aqueous extract. this result is in tandem with that obtained by saleh et al. (2024). isyaka et al. (2024) reported that e. globulus leaves exhibit a zone of inhibition against three of the studied species e. coli, s. aureus, and s. typhi at all concentrations. with a abaka et al. – phytochemical, antioxidant, and antibacterial activities … 517 zone of inhibition of 17.7 mm, the extract was most effective against s. aureus. differences in bacterial targets and inhibition zones likely result from variations in plant species, extraction methods, and experimental conditions. the lowest mic/mbc recorded in this study was 25 mg/ml and 25 mg/ml against escherichia coli the lower the mic and mbc, the more potent and effective the antimicrobial agent is against the tested microorganism as reported by ewansiha et al. (2024). the findings of this study highlight the significant antioxidant activity of the methanol fraction of eucalyptus globulus stem bark, reinforcing its traditional medicinal applications. the assessment of total antioxidant capacity (tac) and reducing power confirmed its notable free radical scavenging ability, although it was generally less effective than ascorbic acid, a well-established antioxidant standard. these results indicate that while the methanol fraction exhibits strong antioxidant potential, its efficacy may be enhanced when combined with other bioactive fractions or complementary compounds with synergistic effects. the potent antioxidant activity observed in the methanol extract is likely due to the presence of lipophilic phytochemicals, such as terpenoids and phenolic compounds, which are efficiently extracted by methanol. these compounds are widely recognized for their free radical scavenging properties and may account for the observed bioactivity. the crude extract also displayed antioxidant activity, though with varying effectiveness across different assays, possibly due to its broader chemical diversity. this mixture of polar and non-polar compounds may enable the crude extract to neutralize a wider range of oxidative species. however, the methanol fraction, being more concentrated in specific active compounds, demonstrated superior antioxidant activity, suggesting that targeted extraction methods may optimize its therapeutic potential. conclusion this study reveals that aqueous and methanol extracts from e. globulus bark possess notable antibacterial activity against s. aureus, e. coli, and p. aeruginosa. these findings highlight the potential of e. globulus as a valuable natural antibacterial agent for medicinal and therapeutic applications. acknowledgments: special gratitude goes to the department of science laboratory technology, adamawa state polytechnic yola. authors contributions: for example, abdulazeez mumsiri abaka & nazuwa dominic designed the study. abdulazeez mumsiri abaka, alex yeri emmanuel & zayyad dahiru aliyu carried out data collection and laboratory work. abdulazeez mumsiri abaka, alex yeri emmanuel & zayyad dahiru aliyu wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are competing interests. funding: no funding. references abaka, a. m., dahiru, m. m., abubakar, k. b., luka, j., abubakar, a., abdullahi, t. b., & barau, s. h. 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(2024). who bacterial priority pathogens list, 2024: bacterial pathogens of public health importance, to guide research, development, and strategies to prevent and control antimicrobial resistance. world health organization. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 25-32 | doi: 10.14421/biomedich.2023.121.25-32 issn 2540-9328 (online) antioxidative stress and hepatoprotective activities of leaf extract and fractions of setaria megaphylla in plasmodium berghei infected mice ndanti bartholomew william1, augustine lawrence bassey2, john akpan udobang2, jude efiom okokon1,* 1pharmacology and toxicology, faculty of pharmacy; 2department of clinical pharmacology and therapeutics, faculty of basic clinical sciences, university of uyo, uyo, nigeria. corresponding author* judeefiom@yahoo.com manuscript received: 07 september, 2022. revision accepted: 20 september, 2022. published: 04 october, 2022. abstract setaria megaphylla (steud) dur & schinz (poaceae), a perennial grass used traditionally in the treatment of various diseases such as malaria was, investigated for antioxidative stress activity in plasmodium berghei-infected mice. the leaf extract (200-600 mg/kg) and fractions (hexane, dichloromethane, ethyl acetate and methanol; 400 mg/kg) of s. megaphylla were investigated for antioxidative stress and hepatoprotective activities in plasmodium berghei-infected mice using a modified suppressive test model. antioxidative stress and hepatoprotective potentials were assessed by determining oxidative stress markers levels, liver function indices and histopathology of liver. the extract/fractions progressively reduced parasitaemia induced by chloroquine-sensitive p. berghei infection with the methanol fraction exerting the highest activity. the leaf extract and fractions caused significant (p<0.05 – 0.001) increases in the levels of oxidative stress markers enzymes and molecules (sod, cat, gpx, gsh) and also reduced mda level significantly (p<0.05) in the livers of the treated-infected mice. the extract/fractions treatment caused reduction in liver enzymes (alt, ast and alp), total and conjugated bilirubin. histology of livers revealed absence or significant reductions in pathological features in the treated infected mice compared to untreated infected mice. the leaf of s. megaphylla may possess antioxidative stress and hepatoprotective effects which may in part be mediated through the chemical constituents of the plant. keywords: antioxidative stress; setaria megaphylla; antimalarial; antioxidative stress; malaria; plasmodium berghei. introduction global malaria cases in 2019 were estimated to be about 229 million occurring in 87 malaria endemic countries (who, 2020). contrary to significant reduction in malaria related mortality, nigeria recorded the highest mortality rate of all malaria deaths globally in 2019. this portrays a serious threat to life in most countries of africa especially nigeria. oxidative stress associated with malaria infection has been implicated in the pathogenesis and development of systemic complications caused by malaria (guha et al., 2006; ojezele et al., 2017). malaria complications such as anemia, jaundice and pre-eclampsia have been linked to oxidative stress damage caused by the parasite (fabbri et al., 2013; sarr et al., 2017). medicinal plants, therefore, serve as a great reservoir for antimalarial remedies having the advantage of being safer and providing many therapeutic effects. setaria megaphylla (steud) dur & schinz (poaceae), a perennial grass found in tropical and subtropical areas of world (van oudtshoorn, 1999), is traditionally used for the treatment of malaria and diabetes among others (okokon et al., 2007). preliminary reports of antiplasmodial activity on the leaves have been published (okokon et al., 2007; okokon et al., 2017). the leaf extract also possesses antidiabetic and hypoglycaemic (okokon and antia, 2007; okokon et al., 2022), anti-inflammatory, analgesic (okokon et al., 2006), cytotoxic, immunomodulatory and antileishmanial (okokon et al., 2013), antidepressant (okokon et al., 2016), inhibitory effect on α-amylase and α-glucosidase (okokon et al., 2021) activities. phytochemical analysis of the leaf extract shows that it contains phytochemical compounds such as flavonoid, carbohydrate, terpenes, saponins, tannins, anthraquinones, cardiac glycosides (z,z,z)-8,11,14eicosatrienoic acid, phthalic acid, diisooctyl ester, vitamin e, ᵞ-elemene, urs-12-ene, bicyclogermacrene, αmuurolene, germacrene-a, and guaiol have been reported (okokon et al., 2006; okokon et al., 2013). 1triacontanal, 1-triacontanol, 1-dotriacontanol, 1triacontyl cerotate, and stigmasterol have also been isolated from the plant leaves (okokon et al., 2022). we report in this study the antioxidative stress and hepatoprotective potentials of leaf extract and fractions of setaria megaphylla in plasmodium berghei-infected mice. https://doi.org/10.14421/biomedich.2023.121.25-32 26 biology, medicine, & natural product chemistry 12 (1), 2023: 25-32 materials and methods plants collection the leaves of setaria megaphylla were collected from farms in the uruan area of akwa ibom state, nigeria. the plant was identified by a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria. a voucher specimen (uuph. 221 d) of the plant was deposited in the department of pharmacognosy and natural medicine herbarium at the university of uyo. extraction the leaves were washed and shade dried for two weeks. the dried leaves were cut into smaller pieces and pulverized to powder. the powdered leaves was divided into two parts. one part was macerated in ethanol for 72 hours, while the remaining part was successively and gradiently macerated for 72 hours in each of, n-hexane, dichloromethane, ethyl acetate and methanol respectively, which is along their polarities to give the corresponding gradient fraction for each solvent. the liquid filtrate of each extract and fraction were concentrated and evaporated to dryness in vacuo 400c using a rotary evaporator. the various yields were calculated and the extract and fractions were stored in a refrigerator at -4oc, until used for the proposed experiments. microorganism (parasite) chloroquine-sensitive strain of plasmodium berghei anka strain was obtained from the national institute of medical research (nimer), yaba lagos, nigeria and maintained by subpassage of blood from infected to healthy mouse once every 7-8 days. parasite inoculation each mouse used in the experiment was inoculated intraperitoneally with 0.2 ml of infected blood containing about 1 x 107p. berghei parasitized erythrocytes collected from an infected mouse with 2030% parasitaemia. the inoculum consisted of 5 x 107 p. berghei infected erythrocytes per milliliter prepared by determining both the percentage parasitemia and the erythrocytes count of the donor mouse and diluting the blood with isotonic saline in proportions indicated by both determinations (atanu et al., 2021). parasitemia was monitored by standard methods; thin blood smears were made on glass slides, fixed using methanol, and stained using giemsa stain parasitema was counted using a microscope and was calculated as a percentage of infected red blood cells (rbcs) relative to the total number of cells in a microscopic field at ×100 magnification according to the formula of peters and robinson (1992) as given below: 𝑃𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑚𝑖𝑎 (%) = 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑖𝑠𝑒𝑑 𝑅𝐵𝐶𝑠 total number of rbcs × 100 experimental animals male and female swiss albino mice, each weighing 2132 g, were obtained from the university of uyo’s animal house. they were kept in standard cages and acclimatized for a period of 10 days before use in the experiments. the mice were fed on standard pelleted diet and water ad libitum. all animals were kept at room temperature in cross ventilated rooms. the care and use of animals was conducted in accordance with the national institute of health guide for the care and use of laboratory animals (nih publication, 1996). approval for the study was obtained from the university of uyo’s animal ethics committee. drug administration the extract, fractions, chloroquine and pyrimethamine that were used in the antimalarial study, were administered orally with the aid of a stainless metallic feeding cannula. evaluation of antioxidative and liver protective activities of the leaf extract and fractions of saccharum officinarum using 4-day test a modified early infection test model was used to assess the antioxidative stress and hepatoprotective potentials of the leaf extract and fractions as well as chloroquine in p. berghei infected mice. this was done using the method described by knight and peters (1980). fortyfive mice were randomly divided into nine groups of five (5) mice each based on body weights on the first day (d0). they were further infected with the parasite and treated with the leaf extract, fractions, chloroquine and distilled water. based on previously determined median lethal dose (ld50) of 2.4 ± 0.5 g/kg, (okokon et al.,2006), the mice in groups 1-3 were given 200 mg/kg, 400 mg/kg and 600 mg/kg of crude extract respectively, while groups 4, 5, 6, 7 were administered 400 mg/kg of n-hexane, dichloromethane, ethyl acetate, and methanol fractions respectively, group 8 was given 5 mg/kg of chloroquine (positive control) and group 9 was given 10 ml/kg of distilled water (negative control) for four consecutive days (d0-d3) between 8am to 9am. on the fifth day (d4), thin films were made from the tail blood. the films were stained with giemsa stain to reveal parasitized erythrocytes out of 500 in a random field of the microscope. on the tenth day, the mice from various group were sacrificed under diethyl ether vapour. blood samples were collected into edta bottles and plain centrifuge tubes. the blood samples in the edta bottles were used for hematological analysis, while those in centrifuge tubes were centrifuged immediately at 2500 rpm for 15 mins to separate the serum at room temperature to avoid haemolysis and used for biochemical assays such as determination of liver function indices. liver from each mouse was surgically removed, weighed and divided into two parts. one part was fixed in 10% formaldehyde for histological process william et al. – biological activities of setaria megaphylla 27 and the other part stored in ice cold normal saline. the average suppression of parasitemia was calculated according to the formula of peters and robinson (1992) as follows: (average % parasitemia positive control – average % parasitemia negative control) / (average % parasitemia negative control). effect of the leaf extract and fractions on biochemical parameters and histology of livers of p. berghei infected mice serum was separated from the blood of each mouse sacrificed and these sera were stored at -20oc until used for biochemical determinations such as total protein, albumin, aspartate aminotransferase(ast), alanine aminotransferase (alt), alkaline phosphotase(alp), conjugated and total bilirubin to assess the liver functions. the determinations were done spectrophotometrically using randox analytical kits according to standard procedures of manufacturer’s protocols (tietz, 1976). the livers of the sacrificed animals were surgically removed, weighed and a part of each fixed in 10% formaldehyde for histological processes, while the other part was washed with ice cold 0.9% nacl and homogenates were made in a ratio of 1 g of wet tissue to 9 ml of 1.25% kcl by using motor driven teflon-pestle. the homogenates were centrifuged at 7000 rpm for 10 min at 4˚c and the supernatants were used for the assays of superoxide dismutase (sod) (marklund and marklund, 1974), catalase (cat) (sinha,1972), glutathione peroxidase (gpx) (lawrence and burk,1976), and reduced glutathione (gsh) (ellman,1959). malondialdehyde (mda) and glutathione-s-transferase (gst) were determined using commercial diagnostic kits (aldrich-sigma, usa) using standard procedures of the manufacturer's protocol. statistical analysis data collected were analyzed using one-way analysis of variance (anova) followed by tukey’s multiple comparison post-test (graph pad prism software inc. la jolla, ca, usa). values were expressed as mean ± sem and significance relative to control were considered at p˂0.05. results and discussion effect of leaf extract and fractions on parasitaemia the extract exerted a dose-dependent and significant (p<0.001) reduction in parasitaemia levels of the treated mice at the different doses employed when compared to the control group in the study. the leaf fractions (nhexane, dcm, ethyl acetate and methanol; 400 mg/kg), exerted prominent reductions in parasitaemia levels of the treated mice with chemosuppression of 11.12, 23.96, 20.81 and 41.00% for n-hexane, dichloromethane, ethyl acetate and methanol respectively. however, methanol fraction exerted the highest activity (figure 1). effect of leaf extract and fractions on liver function indices of p. berghei-infected mice. the levels of liver function indices (ast, alt, alp, total protein, albumin, total and conjugated bilirubin) were found to be elevated in untreated p. berghei infected mice. however, treatment of p. berghei infected mice with leaf extract and fractions of s. megaphylla caused non dose-dependent and significant (p<0.01-0.001) reductions in the activities of ast and alt especially at higher doses (400 and 600 mg/kg) with methanol fraction followed by ethyl acetate fraction treated group having the most significant (p<0.001) reduction when compared to control though not comparable to that of chloroquine (table 1). alp levels of the treated groups of infected mice were non dose dependently and significantly (p<0.01-0.001) reduced when compared to control untreated mice with the methanol fraction followed by n-hexane fraction treated group having the highest and most significant (p<0.001) reductions. the total protein levels of the treated mice groups were similarly reduced non dose-dependently and significantly by the extract/ fractions treatments with the n-hexane fraction treated group having the highest significant effect (p<0.01). treatment of infected mice further produced non dose-dependent and significant (p<0.05-0.01) reductions in albumin levels of the treated mice when compared to control. although the fractions-treated groups had reduced albumin levels, these were not significant (p>0.05) when compared to control. the extract/fractions treatment caused non dosedependent reduction of total bilirubin level of the treated infected mice though not significant (p>0.05) when compared to control. however, the methanol fraction significantly (p<0.05) reduced the total bilirubin level of the mice when compared to control (table 5). the leaf extract and fractions of s. megaphylla also caused non dose dependent reductions in the levels of conjugated bilirubin of the treated mice which was only significant (p<0.05) at the middle dose (400 mg/kg) when compared to control. methanol fraction also produced significant (p<0.05) reduction of conjugated bilirubin when compared to control (table 1). effect of leaf extract and fraction on liver antioxidant enzymes of p. berghei-infected mice. the levels of enzymatic and non-enzymatic endogenous antioxidants (gst, sod, cat, gpx and gsh) in the infected mice were found to be lowered by malaria parasite infection, while higher levels of mda were also observed (table 2). treatment of plasmodium bergheiinfected mice with leaf extract and fractions of s. megaphylla caused significant (p<0.05-0.001) dosedependent elevation in the levels of sod with the dcm fraction treated group having the most significant level (p<0.001) followed by ethyl acetate and methanol 28 biology, medicine, & natural product chemistry 12 (1), 2023: 25-32 fractions treated groups. treatment of infected mice with leaf extract/fractions of s. megaphylla also caused dose-dependent and significant (p<0.001) increases in the activity of gst especially at the higher doses of the extract (400 and 600 mg/kg) when compared with the control untreated infected mice. methanol fraction followed by n-hexane and dcm fractions produced the most significant effect (p<0.001) when compared to control. cat activity was increased non dosedependently but significant (p<0.05-0.001) by the leaf extract/fractions treatment of the infected mice when compared to control with the n-hexane fraction treated group having the highest activity. leaf extract/fractions treatment caused non dose-dependent elevation of gpx activity in the treated infected mice which was only significant (p<0.001) in the group treated with n-hexane fraction when compared to control. similarly, gsh levels in the treated infected mice were significantly (p<0.001) and non dose-dependently elevated following treatment with the leaf extract and fractions when compared to control. n-hexane fraction followed by ethyl acetate and methanol fractions exerted the highest significant (p<0.001) activity when compared to control. however, there were significant (p<0.01-0.001) and non dose-dependent reductions in the levels of mda of the treated mice with dcm and ethyl acetate fractions having the most significant effects (p<0.01-0.001) when compared to control (table 2). effect of extract and fractions on the histology of liver of p. berghei-infected mice. histologic sections of livers of untreated infected mice showing distorted livers with congested central vein, hepatocytes, sinosoids containing inflammatory cells, necrotic tissues. histologic sections of livers of infected mice treated with 200 mg/kg of leaf extract revealed distorted livers with hepatocytes characterised by fatty degenerations, steatosis (microvasicular, macrovasicular, steatosis). livers of infected mice treated with 400 mg/kg of extract had liver sections revealing normal/borderline liver hepatocytes with mild steatosis, patent central vein and sinosoids. histologic sections of livers of p. berghei-infected mice treated with 600 mg/kg showed distorted liver with congested central vein, hepatocytes, necrotic tissues, and sinosoids. n-hexane fraction treated infected mice showed liver sections with mildly distorted livers/border line liver with hepatocytes, sinosoids and necrotic tissues. infected mice treated with dcm fraction had livers histologic sections showing distorted liver with hepatocytes, sinosoids containing kupffer cells, generalised distribution of necrotic tissue and patent central vein (figure 2). group of infected mice treated with ethyl acetate fraction showed livers histologic sections with normal liver with intact hepatocytes, sinosoids and patent central vein (figure 2). also, infected mice treated with methanol fraction had normal livers sections with intact hepatocytes, sinosoids containing kupffer cells and patent central vein (figure 2). p. berghei-infected mice treated with chloroquine had histologic sections of livers showing normal liver with intact hepatocytes, sinosoids containing kupfers cells and patent central vein (figure 2). figure 1. suppressive activities of leaf extract and fractions of s. megaphylla during early plasmodium berghei infection in mice. values are expressed as mean ± sem. significant relative to control. *p<0.05; **p<0.01; ***p<0.001. n = 6. william et al. – biological activities of setaria megaphylla 29 figure 2. histologic liver sections of plasmodium berghei-infected mice untreated (a), treated with leaf extract of s. megaphylla, 200 mg/kg (b), 400 mg/kg (c), 600 mg/kg (d), n-hexane fraction(e), dcm fraction (f), ethyl acetate fraction (g), methanol fraction (h) and chloroquine, 5 mg/kg (i) at magnification x400. keys: hepatocytes (hep), sinosoids (sin), necrotic tissues (nt), steatosis (st), inflammatory cells (inf). table 1. effect of leaf extract and fractions of setaria megaphylla on liver function parameters of mice infected with plasmodium berghei during established infection. treatment dose (mg/kg) liver function parameters ast (iu/l) alt(iu/l) alp(iu/l) total protein (g/l) albumin (g/l) total bilirubin (µmol/ml) conjugate d bilirubin (µmol/ml) control 61.33±1.84 27.46 ±1.35 31.6 ±1.06 69.6 ±3.38 48.0 ±2.08 13.53±0.63 8.20±0.55 extract 200 54.66±1.17 21.6±1.76 17.33±1.85b 57.0±0.57b 33.0±0.57b 10.93±1.83 7.0±1.65 400 40.0±3.21c 14.66±1.79c 17.16±1.20b 59.0±2.64b 37.3±1.85a 8.16±1.33 4.66±0.79a 600 51.0±1.35b 19.66±1.80a 11.66±1.20c 54.66±2.18c 34.6±2.72b 10.13±0.86 6.06±0.98 n-hexane 400 44.33±3.92b 22.66 ±2.09 19.66±1.85a 57.33±1.20b 39.0 ±3.51 8.70±1.04 5.33±0.68 dichloromethane 400 46.66±3.66b 20.66± 2.17 24.33±2.72 58.0 ±0.57b 38.0 ±1.52 9.16±1.67 5.10±1.20 ethyl acetate 400 41.33±2.45c 16.33± 1.36c 26.0± 3.21 58.33 ±1.20b 40.0 ±1.15 8.93±1.78 6.10±1.60 methanol 400 37.0±3.21c 16.33± 1.10c 17.0± 2.51b 58.66 ±2.33b 39.0 ±2.64 7.43±0.66a 3.80±0.17a chloroquine 5 25.66 ±2.90c 15.36 ±0.85c 18.16 ±4.41b 52.0±1.73c 35.66±1.85a 5.30±0.55b 3.73±0.57a values are expressed as mean ± sem. significant relative to control. ap<0.05; bp<0.01; cp<0.001. (n = 6). a b c d e f g h i 30 biology, medicine, & natural product chemistry 12 (1), 2023: 25-32 table 2. effect of leaf extract and fractions of setaria megaphylla on liver antioxidant enzymes of mice infected with plasmodium berghei during established infection. treatment dose (mg/kg) antioxidant parameters gsh (µg/ml) sod (µg/ml) cat (µg/ml) gpx (µm/ml) gst (µg/ml) mda (µmol/ml) liver weight (g) control 1.16±0.06 0.17 ±0.02 1.03 ±0.13 0.050 ±0.003 0.074 ±0.002 0.57±0.01 2.76±0.16 extract 200 1.30±0.05c 0.25±0.02 3.59±1.27c 0.054±0.002 0.107±0.007 0.46±0.03 2.69±0.19 400 1.56±0.03c 0.34±0.04b 1.78±0.50b 0.049±0.004 0.177±0.004c 0.36±0.02c 2.28±0.16 600 1.50±0.03c 0.39±0.09c 3.73±1.23c 0.054±0.005 0.211±0.006c 0.39±0.03b 2.86±0.18 n-hexane 400 1.95±0.05c 0.31 ±0.01a 6.35±0.08c 0.660±0.003c 0.116 ±0.005c 0.48±0.03 2.41±0.16 dichloromethane 400 1.44±0.02c 0.39± 0.01c 1.99±0.10c 0.060 ±0.007 0.116 ±0.009c 0.35±0.03c 2.24±0.18 ethyl acetate 400 1.70±0.03c 0.33±0.01b 1.67± 0.12b 0.058 ±0.006 0.100 ±0.005 0.41±0.02b 2.39±0.13 methanol 400 1.51±0.04c 0.32±0.03b 2.19± 0.29c 0.062 ±0.001 0.380 ±0.01c 0.47±0.04 2.16±0.10 chloroquine 5 1.18 ±0.04 0.20 ±0.05 2.73 ±0.39c 0.057±0.008 0.155±0.01c 0.56±0.04 2.22±0.18 values are expressed as mean ± sem. significant relative to control. ap<0.05; bp<0.01; cp<0.001. (n = 6). discussion the leaves of s. megaphylla are used in ibibio traditional medicine as malaria remedy and this work was designed to investigate its antioxidative stress and hepatoprotective potentials in plasmodium bergheiinfected mice. the leaf extract and fractions of s. megaphylla were investigated for antimalarial activity against rodent malaria parasite, p. berghei infection in mice using early infection test model. it was found that the extract and fractions significantly reduced the parasitaemia with methanol fraction exhibiting the highest suppressive activity, confirming the antimalarial potential of these extract and fractions. these activities could have resulted from plasmodicidal or plasmodistatic activity of the extract and fractions. the results of this study corroborate earlier reports of antimalarial and antiplasmodial activities of the leaf extract and fractions (okokon et al., 2007; 2017). this observed activity may have resulted from the activities of the phytochemical constituents of this plant earlier reported (okokon et al., 2006; 2013; 2021). in the present study, higher levels of transaminases and hyperbilirubinemia were observed in untreated infected animals. temporary hepatic dysfunction is associated with malaria infection characterized by the increase of relative liver weight and liver enzymes activities. the distortions in liver may result from alteration in blood flow through the organ as parasitized rbc adhere to endothelial cells, blocking the sinusoids and obstructing the intrahepatic blood flow. similarly, liver damage could be also due to the leakage of some hepatic cells which were killed or injured by the immune response progress and/or by abnormal cell activation induced by the parasites (guthrow et al.,1979). free radicals have been implicated in liver impairment which results in leakage of some enzymes. hyperbilirubinemia results from the impairment of drainage capacity of the liver due to endothelial blockage and distrubance of hepatocytes (onyesom and onyemakonor, 2011). administration of leaf extract and fractions of s. megaphylla to p. berghei-infected mice was found to decreased the elevated total protein, albumin, ast, alt, alp, conjugated and total bilirubin. the increases recorded in serum total protein, albumin, alt, ast, alp, conjugated and total bilirubin in the parasitized non-treated mice was suggested be due to response to hyper-parasitemia (orhue et al., 2005). malaria parasite infections are accompanied by cellular mobilization of t-cells and its complements with a resultant synthesis and secretion of antibody molecules leading to elevated protein levels in parasitized non-treated mice (orhue et al., 2005). the increased activities of serum ast, alt, and alp in the liver and blood of p. berghei infected mice may be due to hepatic dysfunction (george et al 2011; guthrow et al., 2007) or hepatic damage as could be confirmed in the liver histology. the observed increases in activities of markers enzymes of hepatic damage is in agreement with the report of uzuegbu and emeka, (2011). however, administration of s. megaphylla reduced and restored normal levels of total protein, albumin, direct and total protein and the activities of ast, alt and alp in the serum of infected treated mice. the results are indicative of hepatoprotective activity of the leaf extract and fractions. besides, the histological analyses of livers from malaria infected mice showed a significant pathological signs. this reticuloendothelial hyperplasia expressed by general architectural disorganization of liver with inflammatory sites, hepatocyte necrosis, vascular congestion, malarial pigment presence in the kupffer cells and steanosis is suggestive of inflammatory reaction in the tissue. these were reduced or absent following extract/fractions treatments which further confirm the hepatoprotective activity of the leaf extract due to the antioxidant activities of its phytochemical constituents as previously reported (okokon et al., 2013, okokon et al., 2017; okokon et al., 2021). oxidative stress results from an imbalance between the generation of reactive oxygen species and william et al. – biological activities of setaria megaphylla 31 endogenous antioxidant systems (chanda and dave, 2009). the tissue hypoxia cause by malaria infection induces an activation of the natural host defence that generates large amounts of reactive oxygen species, causing an imbalance between the formation of oxidizing species and the activity of antioxidants which can lead to the death of the parasites (becker et al., 2004; percario et al., 2012). studies suggest that the generation of reactive oxygen species and reactive nitrogen species (ros and rns) associated with oxidative stress is implicated in the pathogenesis and development of systemic complications caused by malaria (guha et al., 2006; ojezele et al., 2017). malaria complications including anemia, jaundice and preeclampsia have been linked to oxidative stress damage caused by the parasite (fabbri et al., 2013; sarr et al., 2017). malarial infection has been found to decrease the levels of antioxidant enzymes and other non-enzymatic anti-oxidants such as catalase (cat), glutathione (gsh) peroxidase, super oxide dismutase (sod), albumin, glutathione, ascorbate and plasma tocopherol. malaria severity has also been found to be directly proportional to the level of lipid peroxidation an indication of membrane damage which is associated with increased malondialdehyde levels (asagba et al., 2010; adil et al., 2013). the oxidative stress induction was also described as electrons produced during the oxidation of fe2+ into fe3+ following the hb degradation by the parasites (kumar and bandyopadhyay, 2005). moreover, antioxidant enzymes (catalase and sod), and molecules such as proteins significantly decreased while lipid peroxidation (mda) increased during malaria infection (luersen et al., 2000). these indices have been used to measure the severity of malaria infection. in this study, the activities of sod, cat, gpx and gsh level were found to decrease significantly, while mda level was significantly increased in the untreated infected group. these findings corroborate earlier reports on the rise in mda level as indicative of lipid peroxidation in the liver of p. berghei-infected mice and depletion in host’s sod and cat activities being important features of malaria infections (becker et al., 2004; gora et al., 2006). the plant extract and fractions exerted protective effect by increasing the activity of antioxidant enzymes and molecules as well as reducing the level of mda significantly, thus lipid peroxidation. this activity can be explained by the presence of phenols, flavonoids and tannins that are able to trap free radicals (rice-evans et al., 1995). these activities may have resulted from the antioxidant activities of its phytochemical constituents as previously reported (okokon et al., 2013, okokon et al., 2017; okokon et al., 2021). conclusion the results of this study show that the leaf extract and fractions of setaria megaphylla possess antimalarial, antioxidative stress and liver protective potentials which may be attributed to the activities of its phytochemical constituents. acknowledgements: the authors are grateful to mr. nsikan malachy of pharmacology and toxicology department and mr adewale wole of anatomy department for providing technical assistance. authors’ contributions: jeo, nuw, ail, research concept and design; 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(1999). guide to grasses of south africa. briza publications, cape town,1999. world health organisation (2020). world malaria reports.www. who. int/publications /m/item/who-htm-gmp-2020.08 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 275-282 | doi: 10.14421/biomedich.2025.141.275-282 issn 2540-9328 (online) in silico analysis of ocimum basilicum flavonoids as natural antihypertensive agent on angiotensin ii type-1 receptor (at1r) amir thalib1,*, irma putri damayanti2 1department of medicine; 2department of herbal medicine, faculty of medicine; university of muhammadiyah purwokerto, jl. kh. ahmad dahlan, purwokerto 53182, tel. +62-856-2988-092, indonesia. corresponding author* amirthalib18@gmail.com manuscript received: 28 february, 2025. revision accepted: 20 may, 2025. published: 24 june, 2025. abstract despite the efficacy of antihypertensive medications like arbs, their adverse effects frequently result in suboptimal adherence. this study investigates the efficacy of flavonoids obtained from herbal sources as natural substitutes for traditional antihypertensive therapies. this study employed in silico molecular docking to examine the binding affinity of flavonoids to the angiotensin ii type-1 receptor (at1r) in comparison to standard angiotensin receptor blockers (arbs), namely eprosartan, azilsartan, irbesartan, telmisartan, valsartan, losartan, olmesartan, and candesartan. docking analysis indicated that the flavonoids exhibited a favorable binding affinity of -8.8 kcal/mol for at1r. moreover, adme and toxicity assessments indicated that flavonoids exhibit advantageous pharmacokinetics and minimal toxicity, with no significant adverse interactions anticipated with primary metabolic enzymes. the structural validation, encompassing ramachandran plots and errat analysis, affirmed the reliability of the modeled at1r protein, achieving a quality score of 97.13%. this study concludes that flavonoids derived from ocimum basilicum exhibit significant potential as natural antihypertensive agents. these findings may facilitate the development of plant-based therapies with minimal adverse effects, enhance treatment adherence, and improve the pharmacological options for managing hypertension. keywords: adme; angiotensin ii receptor; flavonoids; molecular docking; ocimum basilicum. abbreviations: arbs: angiotensin receptor blockers; ace: angiotensin-converting enzyme; at1r: angiotensin ii type-1 receptor; adme: absorption, distribution, metabolism, and excretion; vdss: volume of steady-state distribution. introduction hypertension is a multifactorial condition and is an important risk factor for stroke and cardiovascular disease (raddaoui et al., 2024). approximately 33% of the global population of 8 billion individuals suffers from hypertension. lifestyle modifications are essential for the prevention and management of hypertension, and both governmental and industrial support are crucial for the endorsement and implementation of these modifications (charchar et al., 2024). angiotensin ii type-1 receptor blockers (arbs) are extensively prescribed medications for managing arterial hypertension, heart failure, and chronic kidney disease (de vries, 2020). nonetheless, antihypertensive medications continue to exhibit numerous side effects, necessitating the substitution of these drugs with alternatives that possess minimal adverse effects. previous research by ruijun chen et al (2021) examining the side effects of anti-hypertensive medications, particularly ace inhibitors and arbs, indicates that there is no significant difference between these two classes regarding the risk of acute myocardial infarction, heart failure, stroke, or composite cardiovascular events (chen et al., 2021). medicinal herbs remain a viable alternative treatment for various diseases, including cardiovascular diseases (cvds). there is an unparalleled impetus for the utilization of herbal preparations in contemporary medicinal systems, this initiative is driven by multiple factors, foremost among them being their cost therapeutic potential relative to conventional modern treatments and the general understanding of their safety (shaito et al., 2020). the side effects associated with antihypertensive medications, including fatigue, myalgia, and insomnia, frequently serve as a substantial predictor of inadequate adherence to treatment (mitkova et al., 2024). consequently, alternative medicine, specifically herbs, is regarded as a traditional treatment that may support or replace conventional medicine. flavonoids are small molecular secondary metabolites produced by plants that exhibit diverse biological activities (mierziak et al., 2014). flavonoids https://doi.org/10.14421/biomedich.2025.141.275-282 276 biology, medicine, & natural product chemistry 14 (1), 2025: 275-282 are prevalent in numerous plant species (ysrafil et al., 2023). flavonoids are a prevalent and extensive category of plant secondary metabolites characterized by hydroxylated phenyl rings, encompassing flavones, catechins, and anthocyanins. a diverse array of hydroxylation, methoxylation, glycosylation, and oligomerization patterns has been documented for this class of compounds, with thousands of distinct polyphenol structures reported in the literature. polyphenols captivate the scientific community owing to their diverse biological activities, including advantages for cardiovascular health (joyner, 2021). hypertension induces oxidative stress, resulting in vasoconstriction (matsubara, 1998). flavonoids can induce vasodilation by enhancing nitric oxide (no) activity in endothelial cells. these flavonoids may inhibit at1r; however, their precise affinity and interaction with at1r remain undetermined. at1r is located in the heart, brain, adrenal glands, kidneys, and liver (kumar & pandey, 2013). at1r comprises 359 amino acids and has a molecular weight of 4 kda. the amino acid residues arg167 and tyr35 are involved in the interaction between arb drugs and at1r (miura, 2011). evaluating the efficacy of flavonoids as substitutes for ace inhibitors (acei) and angiotensin receptor blockers (arb) using in silico molecular docking with the angiotensin ii type 1 receptor (at1r) target protein. this study attempts to analyze the interaction and binding affinity of flavonoids to at1r in comparison to conventional ace inhibitors and angiotensin receptor blockers, as a foundation for the development of antihypertensive therapies based on natural compounds. materials and methods study area for molecular docking process, this study used a computer with specifications intel(r) celeron(r) n4020 cpu @ 1.10ghz, 1101 mhz, 2 core(s), 2 logical processor(s), 4 gb ram, and operating system: microsoft windows 11 home single language (10.0.22631 build 22631) bios:dvcn17ww procedures preparation and ligands the test compounds used are bioactive compounds sourced from the majority of plants that grow in indonesia, malaysia, thailand and other asian countries, namely flavonoids (de vries, 2020). numerous angiotensin receptor blockers (arb) medications were utilized to evaluate and compare their binding affinity to flavonoids (hermida et al., 2011). utilizing their canonical smiles as the input and the two-dimensional and three-dimensional structures of the chosen ligands and protein were obtained from pubchem (https://pubchem.ncbi.nlm.nih.gov/) in sdf format (sri prakash et al., 2023). comprehending the molecular mechanisms that regulate bioactivity and forecasting potential side effects or cross-reactivity necessitates the identification of phytochemical targets. swiss target prediction(http://www.swisstargetprediction.ch/) analyzes ligand configurations against known drugs to identify protein targets. the assessed phytochemicals' targets related to a specific drug are identified via swiss target prediction (gfeller et al., 2014). among various candidate proteins that interact with flavonoids, we selected the type-1 angiotensin ii receptor (at1r) as the target to evaluate its binding affinity and to compare it with the binding affinities of ace and arb. target selection and validation at1r was selected as the protein of interest for the study. the swiss-model (https://swissmodel.expasy.org/) was utilized to predict the three-dimensional structure of the at1r protein (ali et al., 2024). subsequently conducted protein validation utilizing procheck and errat using saves (https://saves.mbi.ucla.edu/) (deka et al., 2015; mariana et al., 2023). pymol (https://pymol.org/2/) is used for three-dimensional visualization of proteins and the receptor was modified by the removal of water (putra et al., 2024). docking molecules docking study was conducted using autodock vina in pyrx software (https://pyrx.sourceforge.io/), throughout the docking phase, the ligands were regarded as flexible (prasanth et al., 2021). the molecular docking results of the protein-ligand interaction were analyzed using the pymol software (uma maheswari & sankar, 2024). data analysis drug development entails the evaluation of absorption, distribution, metabolism, and excretion (adme) at progressively earlier stages of the discovery process, when numerous compounds are under consideration but access to physical samples is restricted. a potent molecule must arrive at its target. achieve adequate concentration within the body and maintain a bioactive state for an extended duration for the expected biological event to transpire to be efficacious as a pharmaceutical agent. for this purpose, adme analysis for flavonoids is delineated by the swissadme online platform (http://www.swissadme.ch) (daina et al., 2017; ismail et al., 2023). next to docking, the next process involves predicting the toxicity status of the natural ligands (flavonoids) utilizing the 'protox-iii' web server (https://tox.charite.de/protox3/) (tithi et al., 2023). results and discussion ligands and protein identification the docking process commences with the compilation of a database of herbal compounds from ocimum basilicum, thalib & damayanti – in silico analysis of ocimum basilicum flavonoids as … 277 identifying one active compound from the ocimum basilicum family that exhibits anti-hypertensive properties associated with the type-1 angiotensin ii receptor, specifically flavonoids. additionally, the ligand structure is generated utilizing pubchem in sdf format. the macromolecules utilized in this study are type-1 angiotensin ii receptor. this study pertains to the type-1 angiotensin ii receptor downloaded from the protein data bank (pdb). receptors generally comprise water molecules and residues. consequently, water molecules and residues must be eliminated, require preparation with pymol software to avoid disruption during the docking procedure. this study employs comparative analysis of arb class drugs, specifically eprosartan, azilsartan, irbesartan, telmisartan, valsartan, losartan, olmesartan, and candesartan as ligands, which will be evaluated against flavonoids to assess their binding affinity to the type-1 angiotensin ii receptor (figure 1.). figure 1. visualization of ligands. (a) eprosartan; (b) candesartan; (c) azilsartan; (d) losartan; (e) olmesartan; (f) telmisartan; (g) valsartan; (h) irbesartan; (i) natural ligand flavonoids. structure validation of modeled proteins validation is employed to verify the reliability of the protein structure for docking. the protein structure validation was conducted utilizing the procheck ramachandran plot and errat (hamid et al., 2021). errat errat assesses the model using statistics to evaluate unbound interaction relationships among various atom types, where elevated scores signify superior quality. errat also delivers results with overall significance and quality, with a widely accepted score exceeding 50, indicating a stable protein (sumitha et al., 2020). the errat analysis results (figure 2) indicated that the quality factor values were 97.134% for the angiotensin ii type-1 receptor (at1r). the outcome of this computation indicates that the protein structure of the angiotensin ii type-1 receptor (at1r), characterized by high quality and high resolution. figure 2. structure validation result of the errat tool. ramachandran plots procheck inspection by ramachandran plots (figure 3.) focuses on specific geometries and allows the overall structure to be evaluated. the ramachandran plot illustrates the distribution of the torsional angles φ (phi) and ψ (psi) of the protein's main chain. to see if the 278 biology, medicine, & natural product chemistry 14 (1), 2025: 275-282 ramachandran plot is good for protein structure analysis, it can be checked by plotting non-glycine residues in disallowed regions less than 0.8% (gunasekaran et al., 1996). figure 3. the ramachandran procheck determined the plot. red represents a preferred region, yellow indicates an authorized region, bright yellow suggests an amino acid region that is liberally allowed, and white indicates a banned region. the results show that 87.5% of the residues are in the most favorable region, which indicates adequate structural stability. 8.5% of the residues were in the additional allowed region, and 3.4% were in the general allowed region. 0.6% of the residuals were in the nonallowed region, indicating potential local instability or possible modeling errors. overall, the structure is close to the criteria of a high-quality model, although further refinements may be needed to improve its validity. molecular docking docking was conducted on one active compound of ocimum basilicum with the at1r receptor, utilizing eprosartan, azilsartan, irbesartan, telmisartan, valsartan, losartan, olmesartan, and candesartan ligands for comparison. docking is performed using the pyrx, autodock, and vina software, followed by the configuration of the grid box. the binding affinity served as the primary criterion for assessing the quality of the molecular docking outcomes (agu et al., 2023). the results indicated that the natural ligands of flavonoids indicate spontaneous binding to the type-1 angiotensin ii receptor, showing a binding affinity of -8.8 kcal/mol. in comparison, the binding affinities of the following agents are as follows: valsartan -6.8 kcal/mol, eprosartan -8.2 kcal/mol, losartan -8.5 kcal/mol, olmesartan -9.5 kcal/mol, irbesartan -9.9 kcal/mol, candesartan -10.2 kcal/mol, azilsartan -10.4 kcal/mol, and telmisartan -11.2 kcal/mol (table 1). molecular docking results indicate that ocimum basilicum possesses the potential to treat hypertension. table 1. result of molecular docking simulation of ligands against at1r. ligands cid molecular formula molecular weight (g/mol) binding affinity (kcal/mol) flavonoids 5281616 c15h10o5 270.24 -8.8 valsartan 60846 c24h29n5o3 435.5 -6.8 eprosartan 5281037 c23h24n2o4s 424.5 -8.2 losartan 3961 c24h26n6o3 446.5 -9.5 olmesartan 158781 c24h26n6o3 446.5 -9.5 irbesartan 3749 c25h28n6o 428.5 -9.9 candesartan 2541 c24h20n6o3 440.5 -10.2 azilsartan 135415867 c25h20n4o5 456.4 -10.4 telmisartan 65999 c33h30n4o2 514.6 -11.2 post-docking analysis the molecular docking results generated by pyrx software will be subsequently visualized with pymol to assess the ligand's positioning within the receptor's active site. this visualization attempts to identify significant interactions between the ligand and essential residues on the receptor. furthermore, an analysis of particular interactions, including hydrogen bonds, hydrophobic interactions, and various bond types, will be conducted utilizing the proteinplus website, which offers comprehensive information on residues involved in ligand-receptor interactions. according to the photos provided above, the interactions depicted in each image are as follows: figure a illustrates hydrogen bonds between tyr (hydroxyl) and arg residues, alongside π-π interactions involving the aromatic rings of trp and tyr residues. figure b illustrates hydrogen connections between the ligand and arginine residues, and hydrophobic interactions between the ligand's aromatic ring and aromatic residues. figure c illustrates a hydrogen bond with the tyr residue and a π-π contact between the aromatic ring of the residue and the ligand. figure d illustrates hydrogen bonds with tyr and val residues, along with π-π interactions involving trp residues. figure e illustrates the interaction comprising hydrogen bonds with ser and phe residues, as well as thalib & damayanti – in silico analysis of ocimum basilicum flavonoids as … 279 hydrophobic interactions between the ligand and aromatic residues. image f depicts hydrogen bonding with the tyrosine residue and hydrophobic contact with the residue's aromatic ring. figure g illustrates hydrogen bonding with thr and tyr residues, together with π-π interactions involving aromatic residues. in figure h, interactions comprise hydrogen bonds with aspartate residues and π-π interactions between the ligand and aromatic residues. ultimately, in figure i, hydrogen bonds are formed with tyr residues, and hydrophobic interactions occur between the ligand and aromatic residues. the interplay of these interactions demonstrates how hydrogen bonds, π-π interactions, and hydrophobic interactions influence the ligand's stability with respect to the protein in each figure (figure 4.). figure 4. the ramachandran procheck determined the plot. red represents a preferred region, yellow indicates an authorized region, bright yellow suggests an amino acid region that is liberally allowed, and white indicates a banned region. in silico drug-likeness and toxicity predictions subsequent to molecular docking analyses, the absorption, distribution, metabolism, elimination, and toxicity (admet) of flavonoids natural compound ligands were evaluated using the swissadme (http://www.swissadme.ch/) and protox-3.0 (https://tox.charite.de/protox3/) platforms to forecast their significant pharmacokinetic characteristics. admet properties encompass absorption metrics such as caco-2 permeability, aqueous solubility, human intestinal absorption, p-glycoprotein substrates, pglycoprotein inhibitors i and ii, and dermal permeability; distribution factors including volume of steady-state distribution (vdss), unbound fraction, blood-brain barrier (bbb) permeability, and central nervous system (cns) permeability; metabolic considerations like cytochrome p450 inhibition and cyp2d6/cyp3a4 substrate status; excretion parameters such as renal oct2 substrate and total drug clearance; and toxicity assessments including rat ld50, ames test results, t. pyriformis toxicity, small fish toxicity, maximum tolerated dose, chronic oral toxicity in rats, hepatotoxicity, skin sensitization, and herg inhibitors i and ii. drug similarity characteristics were forecasted by submitting the structures of chosen phytochemicals in smiles format (vardhan et al., 2020). the adme analysis (figure 5.) findings for the compound, which has a molecular formula of c15h10o5 and a molecular weight of 270.24 g/mol, indicate that its physicochemical properties make it suitable for further development as a drug candidate. the heavy atom count of 20 and tpsa of 90.90 å² show that the molecular structure is stable and has good membrane penetration ability. the compound's logp falls between 2.08 and 2.58, indicating an equilibrium between water and lipid solubility that is crucial for pharmacokinetics. the compound has moderate solubility in water based on different techniques, such as log s esol: -3.46. regarding pharmacokinetics, the substance is easily absorbed in the gastrointestinal tract, yet cannot pass through the blood-brain barrier, therefore the chances of negative impacts on the central nervous system are expected to be minimal. nevertheless, the compound may block cyp1a2, cyp2d6, and cyp3a4 enzymes, thus requiring increased focus on potential interactions with other medications processed through this pathway. the skin permeability is poor, indicated by a log kp value of -6.35 cm/s. 280 biology, medicine, & natural product chemistry 14 (1), 2025: 275-282 assessment of drug likeness indicated that this compound adhered to key guidelines such as lipinski, ghose, veber, egan, and muegge without any breaches, with a bioavailability score of 0.55. in terms of medicinal chemistry, this compound exhibits no pains alerts or lead likeness, indicating high promise as a potential lead in drug development with minimal risk of false positives in biological assays. the compound's polarity profile, lipophilicity, and flexibility are within accepted ranges for promoting biological activity. in general, the compound deserves more research with a focus on its metabolic interactions. the study's analysis of toxicity resulted in an ld50 value of 3919 mg/kg (figure 5.). this value shows that the oral dose is lethal for half of the test animal population. the low toxicity classification is based on the relationship between the ld50 value and the level of toxicity risk. the findings of this study indicated a toxicity class of 5. based on this categorization of toxicity, substances in class 5 have very low toxicity and present little danger when used as directed. figure 5. admet analysis of flavonoid drugs demonstrating profiles of absorption, distribution, metabolism, excretion, and toxicity. this data is utilized to assess the pharmacokinetic potential and safety of substances in medication development. conclusions this study highlights the potential of flavonoids as natural alternatives for antihypertensive therapy, focusing on their interaction with the angiotensin ii type1 receptor (at1r) through in silico molecular docking. the findings suggest that flavonoids, derived from ocimum basilicum, exhibit significant binding affinity to at1r, supporting their role in reducing blood pressure. structural validation of the at1r protein strengthens the reliability of the results. this study provides a basis for developing plant-based antihypertensive agents, encouraging further experimental studies and clinical trials to confirm the efficacy and safety of flavonoidsbased therapies. these insights can contribute significantly to advancing personalized and sustainable healthcare solutions. acknowledgements: the author thanks to university of muhammadiyah purwokerto for the time given to conduct research as well as the provision of computers for docking with the required specifications. thalib & damayanti – in silico analysis of ocimum basilicum flavonoids as … 281 authors’ contributions: irma putri damayanti designed the study, provided input and supervised the docking. amir carried out the docking work, analyzed the data and wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. funding: the authors declare no funding in this research. references agu, p. c., afiukwa, c. a., orji, o. u., ezeh, e. m., ofoke, i. h., ogbu, c. o., ugwuja, e. i., & aja, p. m. 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(2020). in silico admet and molecular docking study on searching potential inhibitors from limonoids and triterpenoids for covid-19. computers in biology and medicine, 124, 103936. https://doi.org/10.1016/j.compbiomed.2020.103936 ysrafil, y., sapiun, z., slamet, n. s., mohamad, f., hartati, h., damiti, s. a., alexandra, f. d., rahman, s., masyeni, s., harapan, h., mamada, s. s., bin emran, t., & nainu, f. (2023). anti-inflammatory activities of flavonoid derivates. admet & dmpk, 11(3), 331–359. https://doi.org/10.5599/admet.1918 https://doi.org/10.3389/fphar.2020.00422 https://doi.org/10.22088/ijmcm.bums.12.4.372 https://dx.doi.org/10.13005/bpj/1986 https://doi.org/10.1371/journal.pone.0291125 https://doi.org/10.7759/cureus.53679 https://doi.org/10.1016/j.compbiomed.2020.103936 https://doi.org/10.5599/admet.1918 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 473-479 | doi: 10.14421/biomedich.2025.141.473-479 issn 2540-9328 (online) antiinflammatory and antipyretic activities of stem extract and fractions of telfairia occidentalis in rodents jude e. okokon1,*, chinyelu c. osigwe2, uwaeme ugonma florence2, ukeme essien andrew1 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. 2department of pharmacology and toxicology, faculty of pharmacy, madonna university, elele, rivers state, nigeria. corresponding author* judeefiom@yahoo.com abstract telfairia occidentalis hook (family-cucurbitaceae) is a vegetable employed in ibibio traditional medicine for the treatment of various diseases such as malaria and fever among others. the stem extract of telfairia occidentalis was investigated for antiinflammatory and antipyretic activities in rodents using various experimental models.the stem extract (200 –600 mg/kg) of t. occidentalis was investigated for antiinflammatory activity against carrageenin, egg albumin and xylene – induced edema models and antipyretic activity against damphetamine, 2,4-dinitrophenol and yeast-induced pyrexia models. the extract caused a significant (p<0.05 – 0.001) dose-dependent reduction of inflammation caused by different phlogistic agents used. these effects were comparable to those of the standard drug, (asa, 100 mg/kg) used in some cases. the extract also exerted prominent inhibition of pyrexia on amphetamine and dinitrophenol-induced pyrexia (5 h). inhibition was significant (p<0.05–0.001) from 3 to 5 h postadministration of extract and in a dose-dependent fashion. however, the stem extract did not affect yeast-induced pyrexia in mice. the anti-inflammatory and antipyretic effects of this plant may in part be mediated through the chemical constituents of the plant. the findings of this work confirm the ethnomedical uses of this plant to treat inflammatory and febrile conditions. keywords: telfairia occidentalis; anti-inflammatory; antipyretic; fever. introduction telfairia occidentalis hook is a fluted pumpkin of the cucurbitaceae family widely consumed as food in nigeria (okokon et al., 2009). it is a popular vegetable all over nigeria, especially in the niger-delta region and the eastern part of the country; varieties of meals are prepared from the leaves, stems, and seeds of the plant (usunomena et al., 2023). the various parts of the plant (seeds, leaves and stem) are are used traditionally in the treatment of various ailments and diseases. antiplasmodial activities of the seed, leaves and roots of the plant have been previously reported (okokon et al., 2007; okokon et al., 2009). enin et al. (2023) reported on antioxidant activity and in vivo inhibitory effect on alpha amylase and alpha glucosidase of the stem extract. the anti-inflammatory effects of the leaf extract (oluwole et al., 2003) and seed extract (okokon et al., 2012) have been reported. polyunsaturated fatty acids such as hexadecanoic acid, methyl ester, 9,12octadecadienoic acid methyl ester (linoleic acid), 9,12,15-octadecatrienoic acid, methyl ester (linoleic acid) and 9-octadecenoic acid have been found in the various fractions of the stem extract as well as alkaloid, terpenes, saponin, flavonoid and tannin in the crude extract (enin et al., 2023). the present study was designed to evaluate the effect of stem extract of t. occidentalis on experimentally-induced pain in rodents. the present study was designed to evaluate the antiinflammatory and antipyretic activities of the stem extract of t. occidentalis in rodents. materials and methods plant materials fresh stems of telfairia occidentalis were collected from farms in uyo metroplis in uyo lga, akwa ibom state, nigeria. the leaves were identified and authenticated as telfairia occidentalis by a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria and a voucher specimen was prepared and deposited at the herbarium of the department of pharmacognosy and natural medicine, faculty of pharmacy, university of uyo. extraction fresh stems of telfairia occidentalis were washed, cut into smaller pieces and dried under shade for two weeks. the stems were further pulverized to powder using an manuscript received: 06 january, 2025. revision accepted: 23 july, 2025. published: 24 july, 2025. https://doi.org/10.14421/biomedich.2025.141.473-479 474 biology, medicine, & natural product chemistry 14 (1), 2025: 473-479 electric grinder. the powdered stem material (2 kg) was soaked in 50% ethanol (7.5 l) at room temperature (28 ± 2 °c) for 72 hours. it was thereafter filtered and the liquid filtrate was concentrated and evaporated to dryness in vacuo 40 °c using a rotary evaporator (buchilab switzerland). the extract was weighed and stored in a refrigerator at -4 °c, until used for the proposed experiments. experimental animals swiss albino mice and wistar rats (male and female) that were used in the study were obtained from the university of uyo’s animal house. they were kept in standard plastic cages in a well ventilated room and left to acclimatized for 10 days before the experiments. the mice were fed on standard pelleted diet and water ad libitum. the care and use of animals was conducted in accordance with the national institute of health guide for the care and use of laboratory animals (nih publication, 1996). approval for the study was obtained from the university of uyo’s animal ethics committee. determination of median lethal dose (ld50) the median lethal dose (ld50) of the extract was estimated using albino mice by intraperitoneal (i.p) route using the method of lorke (1983). this involved oral administration of different doses of the extract (100 1000 mg/kg) to groups of three mice each. the animals were observed for manifestation of physical signs of toxicity such as writhing, decreased motor activity, decreased body/limb tone, decreased respiration and death. the number of deaths in each group within 24 hours was recorded. the ld50 was calculated as geometrical means of the maximum dose producing 0% (a) and the minimum dose producing 100% mortality (b). ld50 = √ab. anti-inflammatory studies evaluation of the anti-inflammatory activity of the stem extract on carrageenin – induced mice hind paw oedema adult albino male mice were used after a 24-hour fast and deprived of water only during the experiment. inflammation of the hind paw was induced by injection of 0.1 ml of freshly prepared carrageenan suspension in normal saline into the sub planar surface of the hind paw. the linear circumference of the injected paw was measured before and 0.5, 1, 2, 3, 4 and 5 hours after the administration of the phlogistic agent. the increase in paw circumference in post administration of a phlogistic agent was adopted as the parameter for measuring inflammation (okokon et al., 2010; edem et al., 2023). the difference in paw circumference between the control and 0.5, 1, 2, 3, 4, and 5 hrs after the administration of phlogistic agent was used to assess the inflammation (edem et al., 2023). the stem extract (200, 400, and 600 mg/kg i.p) was administered to various groups of 6 mice each, 1 h before inducing inflammation. control mice received carrageenin while reference group received asa (100 mg/kg). the average (mean) edema was assessed by measuring with vernier calipers. egg-albumin induced inflammation inflammation was induced in mice by the injection of egg albumin (0.1ml, 1% in normal saline) into the sub planar tissue of the right hind paw (okokon et al., 2010; edem et al., 2023). the linear circumference of the injected paw was measured before and 0.5, 1, 2, 3, 4, and 5 hrs after the administration of the phlogistic agent. the stem extract (200, 400, and 600 mg/kg i.p) and asa (100 mg/kg orally) were administered to groups (n=6) of 24 h fasted mice 1 h before the induction of inflammation. the control group received 10 ml/kg of distilled water orally. edema (inflammation) was assessed as the difference in paw circumference between the control and 0.5, 1, 2, 3, 4 and 5 hrs post administration of the phlogistic agent (edem et al., 2023). the average (mean) edema was assessed by measuring with vernier callipers. xylene – induced ear oedema inflammation was induced in mice by topical administration of 2 drops of xylene at the inner surface of the right ear. the xylene was left to act for 15 mins. telfairia occidentalis extract (200, 400, and 600 mg/kg i.p), dexamethasone (4 mg/kg) and distilled water (0.2 ml/kg) were orally administered to various groups (n=6) of mice 1 h before the induction of inflammation. the animals were sacrificed under light anaesthesia and the ears were cut off. the difference between the ear weights was taken as the oedema induced by the xylene (okokon, et al., 2010; edem et al., 2023). evaluation of antipyretic activity of the telfairia occidentalis stem extract on d-amphetamine-induced pyrexia adult albino rats of both sexes that were used in this study were fasted for 24 hours but were allowed water ad libitum. they were randomized into groups of 6 rats each. amphetamine (5 mg/kg, i.p) was administered to the animals after obtaining basal temperatures. hyperthermia developed at 0.5 h following amphetamine administration. different doses of stem extract (200, 400 and 600 mg/kg orally), aspirin (100 mg/kg) and distilled water (10 ml/kg, orally) were administered respectively to the treatment and control groups of animals. rectal temperatures of the animals were obtained at an hour interval for 5 h (edem et al., 2023). effect of telfairia occidentalis stemextract on 2,4dinitrophenol (dnp)-induced pyrexia adult albino rats of both sexes fasted for 24 hours but allowed water ad libitum were used for the experiment. they were randomized into groups of six rats each. dnp (10 mg/kg, i.p.) was administered to the rats after okokon et al. – antiinflammatory and antipyretic activities of stem extract and … 475 obtaining the basal rectal temperatures. hyperthermia developed within 30 minutes of dnp administration. different doses of the stem extract (200, 400, and 600 mg/kg i.p.), aspirin (100 mg/kg), and distilled water (10 ml/kg, orally) were administered to the treatment and control groups of animals. rectal temperatures of the animals were obtained at 1h intervals for 5h (edem et al., 2023). effect of telfairia occidentalis stem extract on yeastinduced pyrexia adult albino rats of both sexes fasted for 24 hours but allowed water ad libitum were used for the experiment. they were randomized into groups of 6 rats each. at zero hour, the basal temperature of the rats were taken using digital clinical thermometer. thereafter, each animal was administered subcutaneously with a 20% w/v aqueous suspension of yeast at a volume of 10 ml/kg (okokon & nwafor, 2010; edem et al., 2023). at suitable intervals beginning one hour after yeast injection, the rectal temperatures of animals were taken, and animals with increase of 1˚c were selected and grouped for the study. the extract understudy was administered i.p. after the pyrogen at doses of 200, 400, and 600 mg/kg to respective groups of rats. the control group received distilled water (10 ml/kg) and the reference group was administered with asa (100 mg/kg) both orally. the rectal temperatures of the groups were taken at 1 hour intervals for 5 hours. statistical analysis the data collected were analyzed using one way analysis of variance (anova) followed by tukey’s multiple comparison post-test (graph pad prism software inc. la jolla, ca, usa). values were expressed as mean ± sem and significance relative to control were considered at p˂0.05. results determination of median lethal dose (ld50) administration of stem extract of t. occidentalis (100 5000 mg/kg) orally did not cause any mortality in the animal groups administered. moreover, no physical toxic signs of the extract were observed. the median lethal dose (ld50) of stem extract of t. occidentalis was therefore estimated to be =5000 mg/kg. evaluation of antinflammatory activity of the stem extract carragenin-induced oedema in mice the effect of ethanol stem extract t. occidentalis (200600 mg/kg) on carragenin-induced oedema is shown in table 13. the extract (200-600 mg/kg) exerted a significant (p<0.05 – 0.001) anti-inflammatory effect in a non dose–dependent manner. the effect was pronounced in the different treatment groups 30 min post-induction with carrageenan. the effect was sustained throughout the study (5 hr) but was not comparable to the standard drug, asa, 100 mg/kg (table 1a and 1b). egg albumininduced edema administration of stem extract t. occidentalis (200-400 mg/kg) on egg albumin induced edema in mice caused a significant (p<0.05-0.001) non dose-dependent antiinflammatory effect against edema caused by egg albumin. the effect was pronounced in the different treatment groups 30 min post-induction with eggalbumin and sustained throughout the study (5 hr). the low dose (200 mg/kg) exerted the highest antiinflammatory effect which was comparable to that of the standard drug, asa (100 mg/kg) (table 2a and 2b). xyleneinduced ear edema the anti-inflammatory effect of stem extract t. occidentalis (200-400 mg/kg) against xylene-induced ear edema in mice is shown in table 3. the extract exerted a dose-dependent anti-inflammatory effect which was significant (p<0.0-0.01). the effect of the highest dose (600 mg/kg) was not comparable to that of the standard drug, dexamethasone (4.0 mg/kg) (table 3). table 1a. effect of telfairia occidentalis stem extract on carrageenaninduced oedema in rats. treatment/ dose (mg/kg) time intervals (hr) 0 0.5 1 2 3 4 5 control 3.44 0.10 6.63  0.09 6.23 0.15 5.98 0.15 5.67  0.14 5.41  0.15 4.80  0.11 extract 200 3.45 0.08 6.29  0.07 5.90 0.10 5.66 0.12 5.35  0.22 5.07  0.10 4.28  0.12 400 3.50 0.07 6.72 0.05 6.02 0.21 5.67 0.15 5.39 0.12 5.10 0.10 4.69 0.14 600 3.48 0.06 6.48 0.05 6.33 0.19 5.96 0.05 5.48 0.16 5.12 0.09 4.60 0.14 asa 100 3.41 0.06 6.15 0.25 5.81  0.34 5.61  0.27 5.18 0.18 4.82 0.12 4.27 0.13 data are expressed as mean  sem. significant at ap<0.05; bp<0.01; p<0.001 when compared to control. n = 6. 476 biology, medicine, & natural product chemistry 14 (1), 2025: 473-479 table 1b. effect of telfairia occidentalis stem extract on carrageenin induced oedema in rats. treatment/ dose (mg/kg) average inflammation/oedema (mm) ± sem 0.5hr 1hr 2hr 3hr 4hr 5hr control 3.19 0.03 2.79 0.01 2.54 0.02 2.23 0.03 1.97  0.04 1.36 0.01 extract 200 2.84 0.02a 2.45 0.01c 2.21 0.04c 1.90 0.03c 1.62 0.01c 0.83  0.01c 400 3.22 0.03 2.52 0.02c 2.17 0.03c 1.89 0.02c 1.60 0.03c 1.19 0.01c 600 3.000.15 2.85 0.01 2.48 0.02c 2.00 0.01c 1.64 0.01c 1.12 0.01c asa 100 2.740.02b 2.40 0.01c 2.20 0.03c 1.77 0.02c 1.41 0.01c 0.86 0.01c data are expressed as mean  sem. significant at ap< 0.05;bp<0.01;cp<0.001 when compared to control. n = 6. table 2a. effect of telfairia occidentalis stem extract on eggalbumin induced oedema in mice. treatment/ dose (mg/kg) time intervals (hr) 0 0.5 1 2 3 4 5 control 3.78  0.11 6.81 0.05 7.23 0.11 7.37  0.16 7.25 0.18 7.21  0.20 7.16  0.20 extract 200 4.17 0.14 6.06  0.06 6.16  0.26 5.99 0.29 5.89  0.33 5.85 0.34 5.41  0.28 400 4.06 0.07 6.18 0.25 6.81 0.39 6.70 0.29 6.62 0.29c 6.57 0.28a 5.94 0.19 600 3.66 0.11 6.01 0.24 5.95 0.14 5.98 0.25 5.87 0.27c 5.84 0.27 5.26 0.27 asa 100 3.75 0.08 6.27 0.29 5.83 0.29 6.07 0.27 5.67 0.33c 5.36 0.26a 4.92 0.22a data are expressed as mean  sem. significant at ap<0.05; bp< 0.01 when compared to control. n = 6. table 2b. effect of telfairia occidentalis stem extract on eggalbumin induced oedema in rats. treatment/ dose (mg/kg) average inflammation/oedema (mm) ± sem 0.5hr 1hr 2hr 3hr 4hr 5hr control 3.03 0.02 3.45 0.01 3.59 0.01 3.47 0.01 3.43  0.02 3.38 0.02 extract 200 1.890.01c 1.99 0.02c 1.82 0.05c 1.72 0.01c 1.68 0.02c 1.24 0.01c 400 2.120.03c 2.75 0.02c 2.64 0.03c 2.56 0.03c 2.51 0.01c 1.88 0.02c 600 2.350.02c 2.29 0.02c 2.32 0.01c 2.21 0.02c 2.18 0.02c 1.60 0.01c asa 100 2.520.01c 2.08 0.12c 2.32 0.13c 1.92 0.13c 1.61 0.01c 1.17 0.01c data are expressed as mean  sem. significant at ap< 0.01;bp< 0.01, cp< 0.001 when compared to control. n = 6. table 3. effect of telfairia occidentalis stem extract on xylene-induced ear oedema in mice. treatment/dose (mg/kg) weight of right ear (g) weight of left ear (g) increase in ear weight (g) % inhibition control (normal saline) 0.2 ml 0.047  0.004 0.085  0.006 (80.85) 0.038  0.002 extract 200 0.042  0.004 0.065 0.006 (54.76) 0.023  0.005a 39.47 400 0.040  0.004 0.057  0.004 (42.5) 0.017  0.001b 55.26 600 0.045 0.002 0.057 0.004 (26.66) 0.012  0.01c 68.42 dexamethasone 4.0 0.035  0.004 0.045  0.004 (28.57) 0.010  0.001c 73.68 figures in parenthesis indicate % increase in ear weight, *significant at ap<0.05, bp < 0.01, cp < 0.001 when compared with control. n = 6. evaluation of antipyretic activity of the extract effect of ethanol stem extract of telfairia occidentalis on d-amphetamine induced pyrexia the antipyretic effect of the stem extract on amphetamineinduced pyrexia is shown in table 4. the stem extract (200-600 mg/kg), in the presence of the pyrogen, caused significant (p<0.05 – 0.001) reductions in the temperatures of the extracttreated rats when compared with the control. these effects were pronounced and sustained from 25 h post treatment with the extract. the antipyretic effects of the extract were not comparable with that of the standard drug, asa,100 mg/kg (table 4). okokon et al. – antiinflammatory and antipyretic activities of stem extract and … 477 effect of ethanol stem extract of telfairia occidentalis on 2,4-dinitronitrophenol (dnp)-induced pyrexia in rats the stem extract of telfairia occidentalis (200-600 mg/kg) exerted significant (p<0.05–0.001) dosedependent lowering of temperature in dnp-induced pyretic rats. the antipyretic effect was, however, pronounced (p<0.05–0.001) and sustained from 4 5 h in all the extract-treated groups. the effect of the highest dose (600 mg/kg) was comparable to that of the standard drug, asa, 100 mg/kg (table 5). effect of stem extract of telfairia occidentalis on yeastinduced pyrexia in rats administration of stem extract of telfairia occidentalis (200-600 mg/kg) did not cause any significant (p>0.05) reduction of body temperature of rats elevated by the administration of yeast. the standard drug, asa,100 mg/kg, reduced the temperature significantly(p<0.05) when compared to the control group (table 6). table 4. antipyretic effect of telfairia occidentalis stem extract on d-amphetamine-induced pyrexia. treatment/ dose(mg/kg) time intervals (hrs) basal temp 0 0.5 1.0 2.0 3.0 4.0 5.0 control 34.45±0.12 36.10±0.13 36.26±0.22 36.44±0.65 36.91±0.66 37.09±0.22 37.28±0.33 37.10±0.18 extract 200 34.57±0.10 35.76±0.38 35.46±0.17 35.23±0.16 35.53±0.21a 35.53±0.61b 35.63±0.20a 34.22±0.39a extract 400 35.10±0.24 36.54±0.15 35.50±0.20 35.96±0.35 35.80±0.23a 35.56±0.42a 35.00±0.23b 34.20±0.15b extract 600 34.85±0.08 36.23±0.08 35.43±0.33 35.53±0.14 34.43±0.52a 34.64±0.24b 35.17±0.12b 35.75±0.21c asa 100 34.95±0.36 35.30±0.29 35.20±0.20 35.01±0.25 34.87±0.14a 34.25±0.10c 33.97±0.14c 33.51±0.11c values are expressed as mean ± sem. significance relative to control. ap<0.05; bp<0.01; cp<0.001. n = 6. table 5. antipyretic effect of telfairia occidentalis stem extract on dinitrophenol-induced pyrexia. treatment/ dose(mg/kg) time intervals (hrs) basal temp 0 0.5 1.0 2.0 3.0 4.0 5.0 control 34.92±0.34 36.55±0.40 36.07±0.49 36.55±0.67 36.27±0.51 36.57±0.23 36.07±0.41 36.17±0.41 extract 200 34.40±0.14 36.72±0.41 36.90±0.81 36.70±0.58 36.20±0.56 35.37±0.42 34.15±0.15a 33.55±0.25b extract 400 34.95±0.13 36.90±0.22 36.75±0.56 36.00±0.45 35.93±0.24 35.00±0.33 34.22±0.14a 33.43±0.30b extract 600 35.00±0.20 36.27±0.14 35.97±0.41 36.72±0.56 35.32±0.49 35.32±0.18 33.15±0.18c 33.34±0.33c asa 100 34.52±0.13 36.07±0.37 35.82±0.35 35.50±0.12 34.97±0.18 34.57±0.07 33.30±0.17c 32.80±0.20c values are expressed as mean ± sem. significance relative to control. ap<0.05; bp<0.01; cp<0.001. n = 6. table 6. antipyretic effect of telfairia occidentalis stem extract on yeast-induced pyrexia. treatment/ dose(mg/kg) time intervals (hrs) basal temp 0 0.5 1.0 2.0 3.0 4.0 5.0 control 36.30±0.26 37.13±0.34 37.56±0.12 37.80±0.20 38.00±0.35 38.77±0.17 38.70±0.08 38.30±0.40 extract 200 36.10±0.32 37.33±0.14 36.91±0.24 37.40±0.36 38.52±0.27 37.83±0.26 38.0±0.53 39.0±0.28 extract 400 35.50±0.17 37.62±0.17 36.55±0.56 37.0±0.38 38.36±0.60 38.54±0.50 38.86±0.15 39.0±0.43 extract 600 36.1±0.68 37.41±0.06 36.34±0.64 37.0±0.33 37.60±0.08 37.88±0.11 38.20±0.21 38.70±0.26 asa 100 35.56±0.23 37.40±0.17 36.91±0.46 36.91±0.46 37.22±0.31c 36.83±0.11c 36.34±0.12c 35.77±0.16c values are expressed as mean ± sem. significance relative to control. ap<0.05; bp<0.01; cp<0.001. n = 6. discussion in this study, the stem extract was evaluated for antiinflammatory activities using various experimental models. in carragenin-induced oedema, the extract (200 600 mg/kg) was observed to have exerted a strong effect at the early stage of inflammation (1-2 hour) indicating strong effect probably on histamine, serotonin and kinnins that are involved in the early stage of carragenininduced oedema (vane & booting, 1987). the extract further caused a prominent reduction of the later stage of the oedema suggesting its ability to inhibit prostaglandin which is known to mediate the second phase of carragenin induced inflammation (vane & booting, 1987). however, asa (100 mg/kg) a prototype nsaid, a cyclooxygenase inhibitor whose mechanism of action involves inhibition of prostaglandin, produced a considerable inhibition of the paw swelling induced by carragenin injection. the extract also inhibited egg albumin-induced oedema demonstrating that it can inhibit inflammation by blocking the release of histamine and 5-ht, two mediators that are released by egg albumin (nwafor et al., 2007). however, asa, a cyclooxygenase inhibitor 478 biology, medicine, & natural product chemistry 14 (1), 2025: 473-479 reduced significantly oedema produced by egg albumin. the extract exerted a significant (p<0.01) inhibition of xylene -induced ear oedema at higher doses of the extract, suggesting the inhibition of phospholipase a2 (pla2) which is involved in the pathophysiology of inflammation due to xylene (lin et al., 1992). however, dexamethasone, a steroid antiinflammatory agent produced significant reduction in the mean right ear weight of positive control rats indicating an inhibition of phospholipase a2. anti-inflammatory activities of plants have been linked to antioxidant potentials (sokeng et al., 2013). the plant extract which has been reported to possess strong antioxidant potential (enin et al., 2024), have been revealed by gcms analysis to contain phenolic like compounds with antioxidant potentials such as pufa (kumar et al., 2010). also, polyunsaturated fatty acids such as 9-octadecenoic acid (z)-, 2hydroxyethyl ester, hexadecanoic acid, ethyl ester and hexadecanoic acid, methyl ester found in this extract have been implicated in the anti-inflammatory activity of plants (kumar, 2010). flavonoids are known anti-inflammatory compounds acting through inhibition of the cyclo-oxygenase pathway (liang et al., 1999). some flavonoids are reported to block both the cyclooxygenase and lipoxygenase pathways of the arachidonate cascade at relatively high concentrations, while at lower concentrations they only block the lipoxygenase pathway (carlo et al., 1999). some flavonoids exert their antinociception via opioid receptor activation activity (rajendran et al., 2000; otuki et al., 2005). flavonoids also exhibit inhibitory effects against phospholipase a2 and phospholipase c (middleton et al., 2000), and cyclooxygenase and/or lipoxygenase pathways (robak et al., 1998). the presence of these phytochemicals could have contributed to the observed activity which maybe acting through antioxidant action and other mechanisms. the antiinflammatory activity of the stem extract corroborates that earlier reported on the leaf and seed extracts (oluwole et al., 2003; okokon et al., 2012). on antipyretic activity, the extract significantly inhibited amphetamine, and dinitrophenol-induced pyrexia but did not affect yeast-induced pyrexia. amphetamine acts on the brain causing the release of biogenic amines from their storage sites in nerve terminals resulting in increased level of camp and subsequent synthesis of prostaglandins from arachidonic acids produced in neurons by receptor-mediated hydrolysis of phospholipids (westfall & westfall, 2006). this leads to hyperthermia. dinitrophenol induces hyperthermia by uncoupling oxidative phosphorylation causing release of calcium from mitochondrial stores and also prevent calcium reuptake. this results in an increased level of intracellular calcium, muscle contraction, and hyperthermia (kumar et al., 2002). yeast induces pyrexia by increasing the synthesis of prostaglandins (al-ghamdi, 2001), in the hypothalamus which the extract could not prevent. the extract may have reduced pyrexia by reducing brain concentration of prostaglandin e2 especially in the hypothalamus through its action on cox-2 or by enhancement of the production of the body’s antipyretic substances such as vasopressin and arginine (chandrasekharan, 2002). the hypothermic activity of the extract could have also been mediated by vasodilatation of superficial blood vessels leading to increased dissipation of heat following resetting of hypothalamic temperature control center (rang et al., 2007). this action may be due to the phytochemical compounds in this plant. therefore, the temperature lowering activity of the extract may not be unconnected with the inhibition of one or combination of the above-mentioned mechanisms. the phytochemical compounds in this plant may in part be responsible for the observed antipyretic activities of the stem extract. conclusion the results of this study show that the stem extract of telfairia occidentalis possesses antiinflammatory and antipyretic activities which are due to the activities of its phytochemical constituents. acknowledgements: the authors are grateful to the staff animal house of pharmacology and toxicology department, university of uyo, for providing technical assistance. authors’ contributions: jeo, uea research concept and design; jeo, uea animal studies, jeo, cco-data analysis and interpretation; jeo, uuf writing the article. jeo, cco and uea read and approved the final manuscript. competing interests: the authors have not declared any conflict of interest. references al-ghamdi, m. s. 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(1987). inflammation and mechanism of action of antiinflammatory drugs. federation of american society for experimental biology journal, 1:89-96. westfall, t. c., westfall, d. p. (2006). adrenergic agonists and antagonists. in: gilman and goodman’s the pharmacological basis of therapeutics. 11th ed. mcgraw, newyork. yi, y. l., zong, h. a., lu, z. m., xu, h. y., zhang, x. m., dou, w. f. and xu, z. h. (2008). analgesic and anti-inflammatory effects of the dry matter of culture both of termitomyces albuminosus and its extracts. journal ofethnopharmacology, 120: 432-436. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 7-13 | doi: 10.14421/biomedich.2020.91.7-13 issn 2540-9328 (online) the effect of cassava peel starch addition to bioplastic biodegradation based on chitosan on soil and river water media mahfud syuhada1, sintia ainus sofa2,*, endaruji sedyadi3 1,3chemistry; 2chemistry education program, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 corresponding author* sintiashofa@gmail.com manuscript received: 28 november, 2019. revision accepted: 28 february, 2020. published: 13 april, 2020. abstract the study of the relationship between starch addition to biodegradation of bioplastics has been carried out. this study aims to understand the biodegradability of chitosan-based bioplastics with additional cassava peel starch on soil and river water media. this research was conducted through four stages, namely making starch from cassava peel waste, making bioplastics using the blending and castingwet processes method with variations of starch 5, 10, 15 and 20 ml. testing physical mechanical properties including water resistance, thickness, tensile strength, elongation, and modulus young. testing the characteristics of bioplastics functional groups was carried out using ftir (fourier transform infrared) and biodegradation testing of bioplastics carried out on soil and river water media. the results of bioplastics research with variation 5 ml produce good mechanical physical properties. bioplastics produced water resistance value of 45.03%, thickness of 0.0190 mm, tensile strength of 49.93 mpa, elongation of 3.068% and young modulus of 1627.63 mpa. bioplastics biodegradation test was observed by measuring the decrease in sample mass. the biodegradation test results in soil and river water media respectively showed a decrease in bioplastic mass up to 63% and 54%. the biodegradation rate of the calculation results on soil media is −0,1502 and in river water media is −0,0948. keywords: chitosan; cassava peel starch; bioplastic; mechanical properties; biodegradation introduction the annual production of petroleum based plastics (pmb) was recorded more than 300 million tons in 2015 (emadian, 2016). in the fact, p-mb production produced above 34 million tons of water which continues to grow every year and indonesia itself is the second largest producer of p-mb waste in the world after china (wahyuningtyas et al., 2017). as much as 93% of the waste tends to accumulated on the surface of the land and oceans so that it was very detrimental to humans and the environment (pathak et al., 2014). one of the efforts to save the earth from the explosion of p-mb waste is the making biodegradable bioplastics. biodegradable bioplastics are plastic polymers that will undergo decomposition by fungi or microorganisms. decomposition can occur up to one hundred percent if discharged into the environment with the final result in the form of water and carbon dioxide gas without leaving a toxic residue. this bioplastic is ecofriendly, non-toxic and renewable because its constituent compounds come from plats such as starch cellulose, and lignin as well as from animals such as casein, protein and lipids (widyaningsih et al., 2012). one of the raw materials for making biodegradable bioplastics in this research is chitosan. chitosan can be used as a raw material in the production of the biodegradable bioplastics due to its nature which can form clear, strong and flexible thin layers (mackay & tait, 2011) non-toxic (jara, et al., 2018). besides that, other raw materials used in the form of cassava peel starch. cassava peel starch is added as bioplastic material because it is believed to accelerate the biodegradation process. it happens because the ability of starch to absorb high water then becomes the optimum place for the growth of degrading microorganisms (wahyuningtyas et al., 2017). the use of chitosan and cassava peel as an ingredient in the production of biodegradable bioplastics will increase its use value (anita, akbar, & harahap, 2013). the selection of chitosan with cassava peel as a bioplastic base is expected to be an alternative solution for making bioplastics. biodegradability of bioplastics in the environment has been tested through various types of experiments. it was started from using one of type of organism to complex systems such as compost and seawater (emadian, 2016). based on the background above, a study was conducted on the study of biodegradable plastics with raw materials in the form of chitosan with the form of chitosan with the addition of cassava peel starch in surface media and river. https://doi.org/10.14421/biomedich.2020.91.7-13 8 biology, medicine, & natural product chemistry 9 (1), 2020: 7-13 materials and methods tools and materials non-electronic devices used include a set of glassware, chopsticks, tweezers, thermometers, magnetic stirrers, bioplastic molds (mica), filter paper soil testers and biodegradation media tubes. electronic device that will be used include blenders, hot plates, ovens, and analytical tools such as analytical balances, ftir spectrophotometers, and a set of tensile and elongation strength gauges. while the ingredients used are chitosan, cassava peel starch, 1% acetic acid and distilled water. procedures  step 1. synthesis of cassava peel starch. cassava peel remove the outer skin, then washed, added distilled water and blended to produce pulp. the resulting pulp is then separated between pulp and starch by filtering. then it was deposited for several hours. the precipitate is then dried in an oven. cassava skin starch was then analyzed using ftir.  step 2. synthesis of bioplastic chitosan solution and cassava peel starch solution were taken with several variations in volume, the comparison is as follows: variation 0. chitosan : cassava peel starch (10:0) = 50 : 0 ml variation 5. chitosan : cassava peel starch (9:1) = 45 : 5 ml variation 10. chitosan : cassava peel starch (8:2) = 40 : 10 ml variation 15. chitosan : cassava peel starch (7:3) = 35 : 15 ml variation 20. chitosan : cassava peel starch (6:4) = 30 : 20 ml both solutions with each of the above comparisons were stirred for 25 minutes using a magnetic stirrer. bioplastic solutions that have been thoroughly mixed are carefully poured into the mold. then the printed solution is dried in an oven, then cooled to room temperature. bioplastic that have been formed are then released for further analysis and testing. data analysis  fourier transform infrared spectroscopy characterization of functional groups carried out by ftir using a kbr pellet method. one mg of sample was mixed with 200 mg of kbr powder. samples were mixed kbr pellets by pressed mixture into a thin transparent using 10 tons pressure (2000 psi). pellet samples were then measured its infrared absorption at wavenumber 4000–400 cm-1 (sedyadi, anggraini, & ekawati, 2016).  mechanical properties test mechanical testing is carried out to determine thickness, tensile strength, elongation and modulus young test. film thickness were measured using micrometer to the nearest 0.001 mm. thickness measurements were taken at five different points for each film sample (top right corner, bottom right corner, middle, upper left corner and the bottom left corner). film thickness value is a average of the results of measurements at five points in units of mm. a set equipment test is prepared and arranged. sample of edible film mounted at the ends of both clamping and wedged firmly. the measurement area is set with loads corresponding to pen recorder off. tensile strength is determined by maximum load, the elongation is determined and calculated at the time the film broken. the modulus young are comparison between tensile strength and elongation.  biodegradation test biodegradation test is carried out on two media, namely surface soil and river water. bioplastic samples were cut 3x3 cm2 in size and then weighed. the samples are stored on media in an open for 14 days. samples are examined once every two days by means of being taken from the media, then cleaned using distilled water, dried in the oven and weighed dry mass. the mass lost from the sample is referred to as percent biodegradation which can be calculated by the equation: % 𝐵𝑖𝑜𝑑𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 = 𝑎1 − 𝑎2 𝑎1 information a1 = mass before testing (gram) a2 = mass after testing (gram) results and discussion synthesis cassava peel starch cassava peel starch is obtained from extraction process with a smooth and abrasive surface. the total starch obtained was 26 grams from 400 gram of cassava peel waste. the process of getting cassava starch is done by making cassava peel pulp using a blender with the addition of distilled water, then the pulp is filtered and precipitated to obtain starch. the pulp filtering process is carried out in stages to produce purer starch. distilled water are used because the nature of starch granules did not dissolve in distilled water at room temperature, so that starch can be deposited and separated easily(kusnandar, 2010). the results of the starch are dried using an oven to reduce water content, so that it can inhibit the growth of bacteria, yeast or mold and extend the shelf life (jabbar & fatimah, 2017).the dried starch is crushed to be smooth and then filtered to obtain a uniform size. syuhada et al. – the effect of cassava peel starch addition to bioplastic … 9 identification of functional groups for cassava peel starch powders have been produced from a simple extraction process is carried out using ftir spectrophotometer at wavelength 400 – 4000 cm-1. the results of ftir analysis of cassava peel starch performed are presented in the spectra in figure 1. figure shows the widening absorption at the 3387 cm-1 wave number that characterizes the -oh group. absorption in the area of 1026.13 cm-1 indicates the presence of c-o groups, and absorption in the area of 2931.8 cm-1 indicates the presence of c-h. in addition there is a peak in the wave number 1635.54 cm-1 which shows the bond that occurs cyclic or aromatic. figure 1. spectra ftir of cassava peel starch. synthesis of bioplastic bioplastic were made by the blending method, which was a method that combined or mixed two or more raw materials into one. this method has advantage that the material used is relatively small, the time required is shorter and the cost is cheaper (nurseha, 2012). bioplastics could be formed after going through several stages. in this research, a transparent and thin bioplastic was produced with slightly rough surface texture and a bit stiff. chitosan is dissolved on 1% acetic acid to get chitosan solution. the use of acidic solvents was due to the nature of chitosan itself which can only dissolve in organic acids such as formic acid or lactic acid, and can dissolve completely in acidic acid. furthermore, cassava peel starch is dissolved in distilled water and stirred using a magnetic stirrer and heated. the purpose of this warm-up is that the cassava starch and aquades can be homogenous and not separate. after the two solutions are obtained, the volume was measured according to the variations made. the two raw materials were mixed according to their variations and stirred again using magnetic stirrers until it was homogeneous. the thin layer which was referred to as bioplastic was produced thorough a wet process molding method, in which chitosan and cassava peel starch were converted into a form of solution then dried. bioplastic thin layers could form if the cohesive attraction that occurred between molecules is adequate, sufficient diffusion and evaporation of intact water, so that the polymer chains will adjust to form layers (kandasamy, 2005) bioplastic characterization the total content of the components used to prepare bioplastics will affect the physical and mechanical properties produced from bioplastic raw materials. the results of the physical and mechanical properties of the bioplastics carried out are shown in table 1 as follows: table 1. bioplastic characterization test results. v a ri a ti o n w a te r r es is ta n c e (% ) thickness (mm) tensile strength (mpa) elongation (%) modulus young (mpa) 0 45,268 0,0220 45,7567 2,6549 1723,4811 5 45,034 0,0190 49,9342 3,0679 1627,6345 10 40,360 0,0180 48,4456 2,9836 1623,7297 15 36,154 0,0175 46,7011 2,9902 1561,8052 20 29,749 0,0755 9,6806 3,3622 287,9246 water resistance is an important property for bioplastic that is directly affected by the water absorption of bioplastic-forming polymers. table 1 showed that there was a decrease in water resistance along with the addition of cassava peel starch variations. it occurred because the hydrophilic nature of starch allows for the absorption of water. the more starches were used, the lower the resistance of the bioplastics produced. based on the results of water resistance obtained in this research, bioplastic chitosan cassava peel starch did not meet the standard criteria set by sni. sni set the plastic water resistance value of 99%, while the bioplastics made are less than 99%. the addition of cassava peel starch would initially reduce the thickness but then increase again as the volume of starch solution variation increased. chitosan solution in this study has physical properties that are thicker than cassava peel starch solution(nahir, 2017), high chitosan concentration will increase the total solids in solution. so, as the volume of chitosan decreases, the total solids in the solution will decrease so the bioplastic thickness value decreases to a certain point which increase again. in addition to the volume of material solution, the bioplastic molding process on the printing plate also influences the thickness value. the bioplastic printing process is one of the crucial things that determine how bioplastics will be formed, whether thick or thin. if it is not appropriate in pouring, leveling the surface of the solution, or slope of the laying plate then the bioplastic solution will gather at one point and produce a non-uniform thickness in a single sheet of bioplastics. chitosan bioplastic thickness with the addition of cassava peel starch variations on average under 0.25 mm, so it can be said that some of the standard value set by jis (japanese industrial standard) of < 0.25 mm, with the exception of variations of 20. 10 biology, medicine, & natural product chemistry 9 (1), 2020: 7-13 based on table 1, the addition cassava peel starch to chitosan bioplastics could increase the tensile strength of bioplastics. in variation 5, the tensile strength of value is the highest, then in variations 10, 14, and 20 there was a decrease in the value of bioplastics will be high if the composition of the composition was optimum, and the addition of starch could result in a decrease in tensile strength values. the optimum composition occurred in perfect balance between chitosan macromolecules and macromolecules of cassava peel starch (nahir, 2017). if the condition of the mixed composition was only close to a little or far from the optimum condition, then with the addition of starch the value of bioplastic tensile strength would decrease because polymer bonding chain of starch would be between the chitosan polymer bonding mechanism so the interaction that occurred between the chitosan polymer chains was reduced (saputro & ovita, 2017). chitosan had a linear polymer chain so that chitosan tends to form a crystalline phase so that it can provide strength and stiffness in bioplastics(agustin & padmawijaya, 2016). the results if the tensile strength of chitosan bioplastic with cassava peel starch variations have met the plastics standards set by sni (indonesian national standard. in the case of variations in bioplastics 20, with the tensile strength produced by the bioplastics only 9.6806 mpa, they cannot be said to meet the standard. this may be influenced by the inhomogeneity of the distribution of bioplastics molecules which results in a decrease in tensile strength (utami et al., 2014). based on table 1 showed that along with addition of cassava peel starch variations, the elongation valueof bioplastics is increasing. in starch there are two main constituent components, namely amylose and amylopectin chain structure is branched. this branched chain structure had an amorphous phase. when bioplastic was given a tensile load, this amorphous part was the first to experience an elongation, the amorphous phase would be attracted and stretched to form a parallel arrangement. amorphous phase elongation process can be seen in figure 2(agustin & padmawijaya, 2016). amylopectin was a cause of increased elongation of bioplastic chitosan-cassava peel starch. although there was an increase in elongation, the percentage value of elongation obtained by bioplastic chitosan cassava peel starch has not fulfilled sni of 21-220%. figure 2. bioplastic stretch in amorphous phase when given tensile load. the addition of cassava peel starch variations is the cause of young’s modulus obtained by bioplastics which is decreasing. this can occur because with the addition of starch, bioplastics stiffness decreases. bioplastic stiffness decreases due to the presence of starch branched amylopectin chains that tend to form an amorphous phase. as previously known, bioplastic stiffness is due to linear chains which tend to form crystalline phases(agustin & padmawijaya, 2016). chitosan-cassava peel starch bioplastics has two linear chains, each derived from chitosan and amylose starch. when compared with the plastic standards issued by sni of 117.62-137.27 mpa, the young bioplastic modulus value is far more rigid. young modulus values for polymer-based materials range between <10-10.000 mpa (hastomo, 2009). analysis of bioplastic function group the biodegradable bioplastic characterization that has been made was done using ftir to determine the functional groups contained in bioplastics. the spectra of the results of ftir analysis of chitosan and bioplastic raw materials with the best tensile strength could be seen in figure 3. figure 3. ftir spectra of chitosan (a) bioplastic (b) and chitosan bioplastic with cassava peel starch (c). figure 3 showed that chitosan spectra have a sharp absorption with wave number 3425.58 cm-1 indicating the presence of o-h groups overlapping with n-h, absorption at wave number 2877.79 cm-1 indicating the presence of c-h and 1072.42 cm-1 indicating the presence of c-o. after bioplastics were formed, the peak absorption pattern produced is slightly different from the basic material, namely chitosan. there was a widening strong absorption at a wavelength of 3425.58 cm-1 indicates an o-h group overlapping with n-h. ch was indicated by the absorption which was slightly shifted at wave number 2885.51 cm-1, and for c-o it was marked by absorption at wave number 1064.71 cm1. absorption of functional groups with similar wave numbers was also produced by bioplastics chitosan syuhada et al. – the effect of cassava peel starch addition to bioplastic … 11 added with cassava peel starch. in the vicinity of these wavelengths also experience widening uptake, this was due to the increase in the o-h group due to the addition of cassava peel starch. the peak at 3448.72 cm-1 indicates the o-h group overlaps with n-h, the peak at wave number 2877.79 cm-1 indicates the presence of ch and the peak at the wave number 1080.14 cm-1 indicates the presence of c-o. based on the analysis of figure 3, ftir spectra of chitosan, chitosan bioplastics and chitosan bioplastics with addition of cassava peel starch did not show at new functional groups. this means that the bioplastic are formed come from the result of physical mixing(saputro & ovita, 2017). proposed interactions that occurred in bioplastics can be seen in figure 4 (afif, wijayanti, & mursiti, 2018). figure 4. proposed bioplastic interaction. pearson correlation test the pearson correlation test results on mechanical properties presented in table 2 can be interpreted by matching the correlation coefficient value obtained with the correlation coefficient value in table 3. table 2. pearson correlation test. it appears that the addition of cassava peel starch variation had a positive pearson correlation value of 0.669 with a significance value of 0.225. it showed a strong relationship between the additional of cassava peel starch to thickness. meanwhile, the pearson correlation value generated for tensile strength is negative 0.698 with a significance value of 0.190. it showed that the addition of cassava peel starch variation has a strong negative correlation to the value of bioplastic tensile strength produced. the addition of cassava peel starch tends to reduce the value of tensile strength. pearson correlation between variation and elongation is 0.838 and has a significance of 0.076. based on these data it can be concluded that the addition of cassava peel starch variations has a very strong relationship to the value of the resulting bioplastic elongation. through statistical calculations it can also be seen that the addition of cassava peel starch variation has strong relationship to young bioplastic modulus produced with a negative value meaning that the addition of cassava peel starch tends to decrease young’s modulus value. the resulting relationship value using pearson correlation calculation is -0.768 with significance of 0.139. overall it can be said that the addition of cassava peel starch is strongly related to thickness and tensile strength, and is strongly related to young’s elongation and modulus. table 3. level of relationship of two variables (qudratullah, 2014). correlation coefficient relationship level 1 perfect 0,75-0,99 very strong 0,50-0,74 strong 0,25-0,49 weak 0,01-0,24 very weak 0 none biodegradation test  soil media based on the graph in figure 5, it was known that the bioplastics made have biodegradable properties, this is evidenced by the reduction in the bioplastic mass of each variation within the observation period for 14 days. the graph also showed a percent reduction in mass on one day which was equal to the increase in cassava peel starch as a variation the amorphous part of starch polymers was a less organized part compared to chitosan polymers, and enzymes from microorganisms were easier to attack and more biodegradable of less organized parts (rohaeti, 2009). amorphous polymers were easier to biodegrade than crystalline polymers and starch cyristallinity was lower than chitosan, so adding starch to bioplastics will further increase the rate of biodegradation(asiah, 2010). percent decrease in bioplastic mass was further analyzed using analysis of variance (anava) to find out whether or not there were differences resulting from each measurement of biodegradation test on the second, seventh, and fourteenth days. on the second day of the biodegradation test, the results of the analysis of variance did not show any significant difference, meaning that variations in the addition of cassava peel starch didn’t significantly affect the decrease in bioplastic mass on the second day. on the seventh day of the biodegradation test, based on the analysis of variance conducted showed the same results on the second day, no significant differences occurred. this shows that the addition of cassava peel starch variation didn’t produce a significant effect on the decrease in bioplastic mass on the seventh day. on the fourteenth day, the results of the analysis of variance showed a 12 biology, medicine, & natural product chemistry 9 (1), 2020: 7-13 significant difference, which means that the addition of cassava peel starch variations resulted in a marked difference in the decrease in bioplastic mass on the fourteenth day. the results of analysis of variance in soil media can be seen that the effect of adding starch was seen to significantly occur on the fourteenth day. figure 5. graph of bioplastic biodegradation of chitosan-cassava peel starch on soil media.  river water media figure 6. graph of bioplastic biodegradation of chitosan-cassava peel starch on river water media. figure 6 shows that chitosan bioplastics with additional variations of cassava peel starch made have biodegradable properties, this is evidenced by the reduction in the bioplastic mass of each variation during the 14 days observation. graph 6 showed a reduction in mass with increasing variations of cassava peel starch. in addition to the hydrophilic nature of starch which causes bioplastics to degrade more quickly, starch polymer are also favored by microorganisms which can result in the formation of large cracks and pore in bioplastic until the reduction of bioplastics mass (alam et al., 2018). percent decrease in bioplastic mass was further analyzed of variance (anava) to find out whether or not there were differences resulting from each measurement of biodegradation test on the second, seventh and fourteenth days. on the second day of the biodegradation test, the result of the analysis of variance showed a significant difference, meaning that variations in the addition of cassava peel starch significantly affected the bioplastic mass reduction on the second day. on the seventh day of the biodegradation test, based on the analysis of variance conducted, there was a significant difference. this shows that the addition of cassava peel starch variation produced a significant effect in the decrease in bioplastic mass on the seventh day. on the fourteenth day, the result of the analysis variance showed a significant difference, which meant that the addition cassava peel starch variation significantly affected the decrease in bioplastic mass in the fourteenth day. data results above it was concluded that the addition cassava peel starch variation significantly affected the bioplastic mass reduction on the second, seventh and fourteenth days of biodegradation test in river water media. comparison of biodegradation test figure 7. graph of bioplastic mass reduction in soil and river water media. figure 7 showed bioplastic mass decreases more rapidly in soil media than in river water media. this is corroborated by the slope value of the biodegradation test linier equation in soil media of -0.1502 and for biodegradation test of river water media of -0.0948. several factors including the availability of water temperature, the amount of oxygen used, minerals, carbon ph and energy sources affect the growth of microorganisms in each test medium (manika et al., 35.00 45.00 55.00 65.00 75.00 85.00 95.00 105.00 0 2 4 7 8 10 11 14 0 2 4 7 8 10 11 14 0 grams 100.00 88.70 86.54 83.99 79.08 76.19 71.36 65.97 5 grams 100.00 94.11 89.59 86.02 83.49 77.81 73.01 69.11 10 grams 100.00 93.45 85.35 82.77 76.39 72.06 64.45 58.42 15 grams 100.00 92.84 84.23 78.24 72.91 65.22 60.78 54.79 20 grams 100.00 90.58 82.50 77.21 74.09 57.55 42.87 37.05 40.00 50.00 60.00 70.00 80.00 90.00 100.00 0 2 4 7 8 10 11 14 0 2 4 7 8 10 11 14 0 grams 100.00 80.08 77.39 70.07 67.75 64.55 61.94 57.97 5 grams 100.00 81.69 79.65 78.03 75.85 69.79 67.35 61.25 10 grams 100.00 81.99 78.05 76.11 67.57 65.80 59.67 56.33 15 grams 100.00 76.33 74.26 66.22 64.11 60.67 56.94 54.49 20 grams 100.00 87.98 81.69 66.70 63.26 57.57 53.03 46.31 -2.2037 -2.1936 -2.9367 -3.3015 -4.5749 -2.6433 -2.3038 -2.8235 -2.8421 -3.8778 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0 5 10 15 20 25 soil water syuhada et al. – the effect of cassava peel starch addition to bioplastic … 13 2015). the possibility of soil media is an optimal place for growth of microorganisms becomes abundant. this causes the bioplastic biodegradation process to occur more quickly in soil media. conclusion based in the results obtained, it was concluded that bioplastics during biodegradability tests on both media from the first day to the fourteenth day experienced a decreases in mass. the increasing amount of starch variation, the faster biodegradation occurs. the fastest mass reduction occurs in variation 20 bioplastics, while the slowest decrease occurs in variation 5 bioplastics. if a comparison of biodegradation rates is taken in both media, the fastest biodegradation process occurs in soil media. acknowledgements the author would like to thank integrated laboratory of uin sunan kalijaga yogyakarta for permission to conduct experiments. references afif, m., wijayanti, n., & mursiti, s. (2018). pembuatan dan karakterisasi bioplastik dari pati biji alpukat kitosan dengan plasticizer sorbitol. indonesian journal of chemical science, 7(2), 102-109. agustin, y. e., & padmawijaya, k. s. (2016). sintesis bioplastik dari kitosan pati kulit pisang kepok dengan penambahan zat aditif. teknik kimia, 10(2), 2-16. alam, m. n., nurafiani, & nurmalasari. (2018). pengaruh penambahan pati bonggol pisang terhadap sifat biodegradasi dari modifikasi plastik propilena menjadi bioplastik. dinamika, 9(1), 48-54. anita, z., akbar, f., & harahap, h. (2013). pengaruh penambahan gliserol terhadap sifat mekanik film plastik biodegradarsi dari pati kulit singkong. teknik kimia usu, 2(2), 37-41. asiah, m. (2010). uji biodegradasi bioplastik dari kitosan limbah kulit udang dan pati tapioka (biodegradation test of bioplastic based chitosan from shrimp waste and starch. biologi edukasi, 2(1), 13-19. emadian, m. s. (2016). biodegradation of bioplastic in natural environments. waste management, 59, 526-536. hastomo, b. (2009). analisis pengaruh sifat mekanik material terhadap distribusi tegangan pada proses deep drawing produk end cup hub body maker dengan menggunakan software abaqus 6.5-1. surakarta: ums. jabbar, & fatimah, u. (2017). pengaruh penambahan kitosan terhadap karakteristik bioplastik dari pati kulit kentang (solanum tuberosum l.). makassar: uin alauddin. jara, a. h., daza, l. d., a, d. m., m, j. a., s, j. f., & alexander, h. (2018). characterization of chitosan edible films obtained with various polymer concentrations and drying temperatur. biological macromolecules, 113, 1233-1240. kandasamy, n. (2005). development and characterization of antimicrobial edible films from crawfish chitosan. lsu doctoral disertation. kusnandar, f. (2010). kimia pangan komponen makro. jakarta: dian rakyat. mackay, r. g., & tait, j. m. (2011). handbook of chitosan research and application. new york: nova science publisher. manika sharma, p. s., & chandra, s. (2015). microbial degradation of plastic-a brief review. cibtech journal of microbiology, 4(1), 85-89. nahir, n. (2017). pengaruh penambahan kitosan terhadap karakteristik bioplastik dari pati biji asam (tamarindus indica l.). makassar: uin alauddin. nurseha, d. (2012). pengaruh penambahan platicizer sorbitol untuk pembuatan bioplastik dari pati kulit singkong. yogayakarta: uin sunan kalijaga. pathak, s., sneha, c., & mathew, b. (2014). bioplastics: its timeline scenario & challenges. polymer and biopolyer phys. chem, 2(4), 84-90. qudratullah, m. f. (2014). statistika terapan teori, contoh kasus dan aplikasi dengan spss. yogyakarta: andi. rohaeti, e. (2009). karakterisasi biodegradasi polimer. prosiding seminar nasional penelitian, 248-257. saputro, a. n., & ovita, a. l. (2017). sintesis dan karakteristik bioplastik dari kitosan-pati ganyong (canna edulis). kimia dan pendidikan kimia, 13-21. sedyadi, e., anggraini, d., & ekawati, d. p. (2016). strachglycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surini dumbo catfish (clarias gariepinus) addition on its mechanical properties. biology, medicine & natural product chemistry, 33-40. utami, m. r., latifah, & widiarti, n. (2014). sintesis plastik biodegradable dari kulit pisang dengan penambahan kitoan dan platicizer gliserol. indonesian journal of chemical science, 3(2), 163-167. wahyuningtyas, nanang, e., & suryanto, h. (2017). analysis of biodegradation of bioplastics made of cassava starch. mechanical engineering science and technology, 1(1). widyaningsih, s., kartika, d., & nurhayati, y. t. (2012). pengaruh penambahan sorbitol dan kalsium karbonat terhadap karakteristik dan sifat biodegradasi film dari pati kulit pisang. molekul, 7(1), 69-81. this page intentionally left blank untitled-1 guidance for authors 3: front 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1257-1263 | doi: 10.14421/biomedich.2025.142.1257-1263 issn 2540-9328 (online) analysis of the antioxidant activity of young and mature bidara leaves using 2,2-diphenyl-1-picrylhydrazyl (dpph) and phytochemical screening test alvelin bunga kurniawati1, gatra ervi jayanti1*, majida ramadhan1, dian novita wulandari2, ike widyaningrum2 1study program of biology, faculty of mathematics and natural sciences, universitas islam malang, 2study program of pharmacy, faculty of medicine, universitas islam malang. jl. jalan mayjen haryono no. 193, malang 65144, tel. +62-341-551932, fax. +62-341-552249, indonesia. corresponding author* gatra.ervi@unisma.ac.id abstract this study aimed to compare the antioxidant activity of young and mature bidara leaves using the dpph method and to identify the content of secondary metabolites through phytochemical testing. antioxidant activity was measured using the ic50 parameter, which represents the concentration of the extract that can capture 50% of dpph free radicals. the test results showed that mature bidara leaves had an ic50 value of 34.984 ppm, indicating vigorous antioxidant activity, while young leaves had an ic50 value of 100.327 ppm, classified as moderate activity. phytochemical testing revealed that both types of leaves contained alkaloids, saponins, and tannins, while flavonoids and triterpenoids were only found in old leaves. these results suggest that variations in secondary metabolite content, influenced by leaf age, contribute to differences in antioxidant activity. thus, older bidara leaves are more potent as an antioxidant source compared to younger leaves. this study supports the utilization of bidara plants in the pharmaceutical industry and the development of traditional medicine based on natural ingredients. keywords: antioxidant; bidara (ziziphus mauritiana); dpph; phytochemical; secondary metabolite. introduction indonesia has thousands of plant species spread across various regions. this biodiversity can be utilized as raw materials for modern and traditional medicines. indonesian society has long been familiar with and used traditional medicine to treat various illnesses. the rising cost of modern medicine in the market is one reason for revisiting the use of conventional medicine. many medicinal plants in indonesia have been utilized as raw materials for medicinal purposes, and some of these plant species have undergone clinical testing to assess their phytochemical content, efficacy, and safety of use (kusumawati, 2018). bidara leaves (ziziphus mauritiana) have long been used in traditional medicine due to their various bioactive compounds. the bidara plant is more widely known for its health benefits, including anti-inflammatory, antibacterial, and antioxidant properties. the phytochemicals present in bidara leaves, including flavonoids, alkaloids, saponins, and tannins, play a crucial role in producing their pharmacological effects. some phytochemical compounds that contribute to pharmacological effects include flavonoids, which are believed to have antibacterial activity by disrupting protein bonds in bacterial cell membranes, and saponins, which can form complexes with bacterial proteins and cell walls, leading to cell wall damage (ardinimia et al., 2023). additionally, triterpenoids function as antioxidants due to their ability to scavenge dpph radicals (samirana et al., 2017). the natural complex compounds in these plants can be used as free radical scavengers. these complex compounds are used without isolating specific components to obtain a single compound. all components in the plant are used to form complex compounds. according to jayanti and rahayu (2023), complex antioxidants have more electrons than single antioxidants, making them more efficient. antioxidants are compounds that, in specific amounts, can inhibit or counteract the adverse effects of oxidation. natural antioxidants are often preferred over synthetic ones because some synthetic antioxidants, such as butylated hydroxyanisole (bha) and butylated hydroxytoluene (bht), have recently been suspected of being carcinogenic (kesuma, 2015). manuscript received: 16 september, 2025. revision accepted: 04 december, 2025. published: 13 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1257-1263 1258 biology, medicine, & natural product chemistry 14 (2), 2025: 1257-1263 the leaves and branches of the jujube (ziziphus jujuba), synonymous with ziziphus mauritiana lamk, are known to contain various active compounds. among others, 14 compounds, including coumarin, rutin, saponin, tannin, and tartaric acid, are found in the leaves. in comparison, three main compounds, namely betulinic acid, ceanothic acid, and leucopelargonidine, are found in the branches. based on analysis using duke's phytochemical and ethnobotanical database, these compounds have antioxidant potential because their pa values are higher than their pi values, with coumarin and rutin from the leaves and alpha from the branches showing pa values >0.7, which indicates vigorous antioxidant activity and is estimated to be not much different from laboratory test results. additionally, the dpph test also demonstrated antioxidant activity, as indicated by a change in the colour of the solution, with the combination of leaves and branches showing higher activity than the single samples (nisa et al., 2023). this study tested antioxidant activity using the 2,2diphenyl-1-picrylhydrazyl (dpph) method by spectrophotometry. dpph is a relatively stable free radical compound. this compound is suitable for use as a reagent for testing compounds that have a free radical scavenging effect (antioxidant compounds). the parameter used for the dpph radical scavenging test is ic50, which is the concentration of the extract or test fraction required to capture 50% of the dpph radicals (taufik, 2016). a comparative test was also conducted, namely phytochemical screening, to determine the compound content in the sample. based on several studies conducted, bidara leaves are one of the plants tested for their effectiveness as an antibacterial agent. this is due to the content of several secondary metabolites in bidara leaves that can function as antibacterial agents. according to muharrami et al. (2019), the results of phytochemical screening of bidara leaves extracted using the maceration method with 96% ethanol solvent revealed the presence of several secondary compounds, including phenolic compounds, tannins, and saponins. on the other hand, research by ardinimia et al. (2023) explains that the flavonoids and saponins present in bidara leaves exhibit significant antibacterial activity. however, this study did not further investigate the antioxidant activity or the potential for development as a raw material for phytopharmaceuticals. the purpose of this study was to compare the antioxidant activity of young and mature bidara eaves using the dpph method and to identify their secondary metabolites through phytochemical testing. therefore, this study was conducted to support the updating of data that can be used for further studies related to the antioxidant activity found in old and young bidara leaves. materials and methods study area this research was conducted from june to august 2025 at the biochemistry laboratory, terpadu laboratory, universitas islam malang, east java, indonesia. procedures sample preparation mature and young bidara leaves were obtained from the al-qur'an garden of the universitas islam malang. the initial process involved sorting old and young leaves, washing them, drying them in the sun, and then sorting them by hand. then, the leaves were dried in an oven at 60°c until they were scorched. after drying, the samples were blended into a powder and sieved using a 40-mesh sieve to increase the surface area, thereby facilitating the extraction process (wijaya & noviana, 2022). subsequently, the dry weight of the sample was measured, and the moisture content was calculated to ensure it was <10%. extraction the bidara leaves were extracted using the maceration method. a total of 100 grams of crude drug was weighed and placed in a dark glass bottle (reagent bottle), then 700 ml of 96% pro-analytical ethanol was added. the mixture was homogenized repeatedly and left to stand for 3 days and 2 nights. the first maceration filtrate was then filtered. next, 300 ml of 96% pro-analytical ethanol was added to the residue in the dark glass bottle from the first maceration, homogenized, and filtered again into an erlenmeyer flask. the filtrates from the first and second macerations were combined and evaporated using a rotary evaporator at a speed of 30 rpm and a temperature of 65°c to produce a concentrated extract or paste (salamah et al., 2024). yield calculation yield is the ratio between the dry weight obtained and the weight of the raw material used. in extraction, yield is calculated by comparing the final weight of the extract obtained with the initial weight of the cell biomass used, then multiplying by 100% (i) (mahyantika et al., 2025): yield (%) = weight of extract (g) weight of raw material (g) x 100% (i) antioxidant testing using the dpph method ▪ determination of the maximum wavelength of 0.1 mm dpph solution a 0.1 mm dpph solution was prepared by weighing 2 mg of dpph powder and dissolving it in proanalysis ethanol in a 50 ml volumetric flask, bringing it up to the mark. then, 3 ml of the dpph solution was pipetted into a cuvette and measured using a uvvis spectrophotometer in the wavelength range of 400–600 nm. after the maximum wavelength was kurniawati et al – phytochemical and antioxidant bidara leaves 1259 determined, absorbance measurements were performed by mixing 3 ml of dpph solution with 1 ml of analytical-grade ethanol, and absorbance was measured at the determined wavelength (theafelicia & narsito wulan, 2023). ▪ preparation of standard solution as reference the standard reference solution was prepared by weighing 1 mg of quercetin and dissolving it in proanalysis ethanol in a 10 ml volumetric flask, bringing it up to the mark. the solution was homogenized using a vortex and then divided into several concentrations (3.13 ppm, 6.25 ppm, 12.5 ppm, 25 ppm, and 50 ppm). each concentration is pipetted into a reaction tube and mixed with 3 ml of 0.1 mm dpph solution. the mixture is vortexed again and incubated in the dark for 30 minutes, then measured using a uv-visible spectrophotometer at a wavelength of 517 nm (theafelicia & narsito wulan, 2023). ▪ preparation of test solutions from old and young bidara leaf extracts samples of old and young bidara leaf extracts were prepared by weighing 10 mg of extract and dissolving it in pro-analysis ethanol in a 10 ml measuring flask, then bringing the volume up to the mark. the sample solution was homogenized using a vortex and then divided into several concentrations (20 ppm, 40 ppm, 60 ppm, 80 ppm, and 100 ppm). each concentration was pipetted into a reaction tube and mixed with 3 ml of 0.1 mm dpph solution. the mixture was vortexed again and incubated for 30 minutes, then measured using a uv-vis spectrophotometer at a wavelength of 517 nm (theafelicia & wulan, 2023). ▪ dpph free radical scavenging activity antioxidant activity was determined through inhibition values using a uv-vis spectrophotometer. the absorbance data obtained were used to calculate the percentage of dpph free radical inhibition. antioxidant capacity in capturing free radicals was expressed using the following formula (ii) (khalil et al., 2020): dpph free radical inhibition (%) = 𝐴0−𝐴1 𝐴0 × 100% (ii) information: a0 = control absorbance a1 = sample/standard absorbance the control absorbance is the absorbance data of dpph mixed with ethanol for analysis. in contrast, the sample/standard absorbance is the absorbance data of the standard/sample solution mixed with dpph (khalil et al., 2020). antioxidant testing using phytochemical screening methods the phytochemical screening test method refers to ramadhan et al. (2019) conducted qualitatively with color tests using reagents to determine the secondary metabolites contained in plants: ▪ alkaloid test a) mayer’s test the liquid extract of mature and young bidara leaves was placed in a test tube. then, 4 drops of mayer’s reagent were added drop by drop into the test tube, and the tube was shaken gently. the formation of a white precipitate accompanied by a yellowish color change indicated a positive result for alkaloids. b) wagner’s test the liquid extract of mature and young bidara leaves was placed in a test tube. then, 2 drops of wagner’s reagent were added drop by drop into the test tube, and the tube was shaken gently. the formation of a reddish-brown precipitate indicated a positive result for alkaloids. c) dragendorff’s test the liquid extract of mature and young bidara leaves was placed in a test tube. then, 2 drops of dragendorff’s reagent were added drop by drop into the test tube, and the tube was shaken gently. the formation of an orange or yellow precipitate indicated a positive result for alkaloids. d) bouchardat test the liquid extract of mature and young bidara leaves was placed in a test tube. then, 2 drops of bouchardat reagent were added drop by drop into the test tube, and the tube was shaken gently. the formation of a brown precipitate indicated a positive result for alkaloids. ▪ flavonoid test a) shinoda test the liquid extract was placed in a test tube. then, 4 drops of absolute ethanol and 2 drops of concentrated hydrochloric acid were added to the test tube, and the mixture was shaken gently. a color change to red indicated the presence of aurone and chalcone. if no colour change occurred, a small spatula of magnesium was added to the test tube containing the extract, and the mixture was gently shaken. a color change from pink to red, such as orange, red, or magenta, indicated the presence of flavones and flavonols. b) 10% sodium hydroxide test the liquid extract of mature and young bidara leaves was placed in a test tube. then, 2 drops of 10% naoh solution were added into the test tube, and the mixture was shaken gently. the color changes to reddish yellow, dark orange, reddish purple, or blue indicate the presence of anthocyanins, flavones, flavonols, or chalcones. 1260 biology, medicine, & natural product chemistry 14 (2), 2025: 1257-1263 ▪ saponin test a) foam test the liquid extract of mature and young bidara leaves was placed in a test tube. then, 10 ml of distilled water was added to the test tube, and the mixture was shaken continuously and gently. the formation of a stable foam layer about 1 cm high indicated a positive result for saponins. ▪ tanin test a) braymer’s test the liquid extract of mature and young bidara leaves was placed in a test tube. then, 2 drops of braymer’s reagent were added drop by drop into the test tube, and the tube was shaken gently. the formation of a greenish-black precipitate indicated a positive result for tannins. b) base solution test the liquid extract of old and young bidara leaves was placed in a test tube. then, 2 drops of 10% ammonium hydroxide solution were added to the test tube, and the mixture was shaken gently. the formation of a fluorescent yellow color indicated a positive result for tannins. ▪ steroid/triterpenoid test a) salkowski test the liquid extract of mature and young bidara leaves was placed in a test tube. then, two drops of salkowski reagent were added drop by drop into the test tube, and the mixture was shaken gently. the formation of a brown ring in the center of the solution indicated the presence of steroids. b) lieberman bourchard test the liquid extract of mature and young bidara leaves was placed in a test tube. then, two drops of lieberman-bourchard reagent were added slowly to the test tube, and the mixture was shaken gently. after incubation for 5 minutes, the appearance of a bluegreen color indicated the presence of sterols, while a pink to purplish-red color indicated the presence of terpenoids. data analysis the research data were analyzed both descriptively and quantitatively, as well as qualitatively. phytochemical screening activity was analyzed using tables and figures. in contrast, antioxidant activity was analyzed using microsoft excel with data presented in graphs and expressed as ic50 values obtained from the linear regression equation y = ax + b (werdyani et al., 2019). the higher the ic50 value, the lower the antioxidant activity of the sample; conversely, the lower the ic50 value, the higher the antioxidant activity of the sample. meanwhile, to compare the differences between the two treatment group means, a t-test was conducted using past 4.03. results and discussion quercetin reference solution test table 1. results of quercetin antioxidant activity measurements. concentration (ppm) absorbance % inhibition ic50 3,125 0,731 28,655 2,283 (very strong) 6,25 0,601 41,343 12,5 0,381 62,815 25 0,159 84,482 50 0,023 97,755 figure 1. linear regression curve of the antioxidant activity of quercetin reference solution. antioxidant activity test using the dpph method table 2. results of antioxidant activity measurements of mature bidara leaf samples. concentration (ppm) absorbance % inhibition ic50 20 0,365 36,65 34,984 (very strong) 40 0,268 53,52 60 0,161 72,02 80 0,092 84,06 100 0,048 91,60 table 3. results of antioxidant activity measurements of young bidara leaf samples concentration (ppm) absorbance % inhibition ic50 20 0,130 55,90 100,327 (currently) 40 0,076 74,22 60 0,097 67,21 80 0,122 58,50 100 0,148 49,80 28,655 41,343 62,815 84,482 97,755 y = 18,134x + 8,6082 r² = 0,9911 0,000 20,000 40,000 60,000 80,000 100,000 120,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00 a b so rb an ce concentration (ppm) kurniawati et al – phytochemical and antioxidant bidara leaves 1261 t test figure 2. results of statistical analysis comparing young bidara leaves (ybl) and mature bidara leaves (obl). phytochemical screening test table 4. chemical compounds in mature and young bidara leaves. test reagents mature bidara leaves young bidara leaves alkaloid mayer’s + + wagner’s + + dragendorff’s + + bouchardat + + flavonoid shinoda naoh 10% + + saponin busa + + tanin braymer’s + + larutan basa + + triterpenoid salkowski lieberman bouchad discussion the determination of antioxidant activity began with the preparation of extracts from mature and young jujube leaves (ziziphus mauritiana l.) using pro-analytical ethanol as a solvent in the maceration method. ethanol was chosen because it can extract both polar and nonpolar soluble components, enabling the extraction of all chemical compounds present in jujube leaves (dianda & suharti, 2023). the extracts of mature and young jujube leaves obtained were then tested using the (dpph) method with quercetin as the standard solution. the dpph method was used because it is simple, fast, sensitive, and widely used to evaluate the ability of chemical compounds in samples to scavenge free radicals (putri, 2023). additionally, a phytochemical screening test was conducted to determine the content of compounds present in the old and young bidara leaves. the antioxidant activity test of the reference solution was conducted at concentrations of 3.13 ppm, 6.25 ppm, 12.5 ppm, 25 ppm, and 50 ppm. quercetin was reacted with the dpph reagent and then measured for absorbance using a uv-vis spectrophotometer at a wavelength of 517 nm. the absorbance values and inhibition percentages of the standard solutions are presented in table 1, demonstrating extreme free radical scavenging activity with an ic₅₀ value of 2.283 ppm, which falls into the powerful category (<50 ppm). this is evidenced by the increase in inhibition percentage with increasing concentration, where at the lowest concentration of 3.125 ppm, it was already able to inhibit by 28.655%, and continued to increase to 97.755% at a concentration of 50 ppm. the inhibition percentage is a parameter reflecting the effectiveness of antioxidants in inhibiting free radicals. the parameter used to measure the antioxidant capacity of a compound is ic50. the ic50 value indicates the concentration of the antioxidant compound required to capture 50% of dpph free radicals (pratiwi et al., 2023). the inhibition concentration was calculated using a linear regression equation to determine the relationship between concentration (ppm) (x) and absorbance (y) (figure 1). from the curve, a linear regression equation, y = 18.134x + 8.6082, with an r² value of 0.9911, was obtained, indicating a robust and linear relationship between concentration and free radical inhibition activity. antioxidant activity testing of mature and young bidara leaves using the dpph method yielded ic50 values of 34.984 for mature bidara leaves, indicating extreme antioxidant activity. in contrast, young bidara leaves had an ic50 value of 100.327, categorized as moderate. the smaller the ic50 value obtained, the higher the antioxidant activity of a compound, and conversely, the larger the ic50 value, the weaker the antioxidant activity (putri, 2023). this is presented in tables 2 and 3, which show a significant difference in antioxidant activity between mature and young bidara leaves, where mature bidara leaves exhibit a clear and consistent inhibitory pattern that increases with rising concentration. based on the graph, the average value of the variable measured in young bidara leaves (ybl) was 0.1146, and in old bidara leaves (obl), it was 0.1866. although the mean value of dbt is higher than that of dbm, the statistical test results indicate that there is no significant difference between the two (p > 0.05), as indicated by the same letter (“a”) on both treatments. this indicates that the measured activity levels are relatively comparable between young and mature leaves. variations may influence this difference in mean values in the content of secondary metabolites such as flavonoids, tannins, and saponins, which play a role in biological activity (wulansari, 2022). the phytochemical testing process in this study was conducted to determine the chemical compounds present in the water extract samples of bidara leaves. the phytochemical tests used included tests for alkaloids, flavonoids, tannins, saponins, and triterpenoids. the results of the phytochemical tests, as shown in table 4, indicate that both young and mature bidara leaf extracts contain alkaloids, saponins, and tannins. however, flavonoids and triterpenoids were only detected in mature a a 0 0,05 0,1 0,15 0,2 0,25 young bidara leaves old bidara leaves mean 0,1146 0,1866 m ea su ra b le v ar ia b le s leaf category 1262 biology, medicine, & natural product chemistry 14 (2), 2025: 1257-1263 leaves. this suggests that leaf maturity influences the content of secondary metabolites. these results are consistent with wulansari's (2022) study, which reported that ethanol extracts of bidara leaves contain alkaloids, saponins, tannins, and flavonoids, but no triterpenoids were detected. these differences may be due to differences in leaf age, solvent, or extraction method. the results of this study align with those of hafiz et al. (2025) and javed et al. (2022), which demonstrate that bidara leaf extract exhibits high antioxidant activity and contains several active compounds, including tannins, saponins, alkaloids, and flavonoids. hafiz et al. (2025) explored three species of ziziphus (ziziphus mauritiana, ziziphus spina-christi, and ziziphus jujuba). they found that ziziphus mauritiana leaves had the highest antioxidant capacity, as determined by dpph assays, consistent with their high total phenolic content (6.534 g gae/100 g) and total flavonoid content (2.025 g qe/100 g). this present study also identified the main flavonoid compounds, including rutin, quercetin, kaempferol, and apigenin, via hplc, and demonstrated a strong correlation between flavonoid content and antioxidant activity. however, a fundamental difference from the study by hafiz et al. (2025) is that their study compared antioxidant activity between species and plant parts (leaves, fruits, seeds), while this study focused on comparing antioxidant activity between old and young bidara leaves within a single species, ziziphus mauritiana. meanwhile, the study conducted by javed et al. (2022) on the phytochemistry and antioxidant activity of ziziphus mauritiana, a species within the genus ziziphus, revealed the presence of compounds such as tannins, saponins, alkaloids, and flavonoids in the methanol extracts of its leaves and fruits. both studies support the idea that plants of the ziziphus genus, including bidara, are rich in secondary metabolites that function as both antioxidants and antimicrobials. differences in antioxidant activity between different parts of the plant also indicate that the age and part of the plant used significantly influence their chemical composition and bioactive potential. conclusions the extract of mature bidara leaves showed more potent antioxidant activity (ic₅₀ = 34.984 ppm, powerful category) compared to young leaves (ic₅₀ = 100.327 ppm, moderate category). this indicates that the maturity level of the leaves affects their antioxidant capacity. phytochemical screening results also confirm that both young and old leaves contain alkaloids, tannins, and saponins, but flavonoids are only detected in old leaves. these differences in secondary metabolite content contribute to the higher antioxidant activity of old bidara leaves. therefore, mature bidara leaves have greater potential to be developed as a natural source of antioxidants compared to young leaves. acknowledgements: the author would like to express gratitude to the unisma institutional grant (hi-ma) in accordance with the research grant implementation agreement number: 057/g164/u.lppm/k/b.07/iii/2025 dated march 3, 2025, for the financial support provided from the initial stage to the completion of this final project. competing interests: the authors declare that there are no competing interests. references ardinimia, s. d., putri, a. f., ramanda, y. m., putri, n., dzakiyyah, h., gianni, p., surabaya, u. n., & ketintang, j. 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(2024). a comparison of antioxidant activity in tea mistletoe ( scurrula atropurpurea ( bl .) dans .) leaf extracts using dpph assay. 799–806. phytochemical and antioxidant bidara leaves samirana, p. o., taradipta, i. d. m. r., & leliqia, n. p. e. (2017). penentuan profil bioautografi dan aktivitas antioksidan ekstrak etanol daun bidara ( ziziphus mauritiana auct . non lamk .). jurnal farmasi udayana, 6(2), 18–22. taufik, a. n. (2016). perbandingan daya antioksidan ekstrak etanol kayu secang putih dan merah ( caesalpinia sappan l .). jurnal ilmiah mahasiswa universitas surabaya, 5(1), 1–16. theafelicia, z., & narsito wulan, s. (2023). perbandingan berbagai metode pengujian aktivitas antioksidan (dpph, abts dan frap) pada teh hitam (camellia sinensis). jurnal teknologi pertanian, 24(1), 35–44. https://doi.org/10.21776/ub.jtp.2023.024.01.4 werdyani, s., hartati, d. s., & jumaryatno, p. (2019). penentuan fraksi aktif antioksidan ekstrak etanol daun benalu (scurrula atropurpurea (bl.) denser) yang tumbuh pada pohon rambutan. jurnal ilmiah farmasi, 15(2), 70–79. https://doi.org/10.20885/jif.vol15.iss2.art3 wijaya, a., & noviana. (2022). penetapan kadar air simplisia daun kemangi ( ocimum basilicum l .) berdasarkan perbedaan metode determination of the water content of basil leaves simplicia ( ocimum basilicum l .) based on different drying methods. jurnal riset kefarmasian indonesia, 4(2), 185–199. wulansari, r. (2022). analisis senyawa metabolit sekunder dan uji aktivitas larvasida alami pada ekstrak etanol daun bidara (ziziphus mauritiana lamk.) terhadap larva aedes aegypti. paper knowledge . toward a media history of documents, 3(april), 49–58. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 111-115 | doi: 10.14421/biomedich.2021.102.111-115 issn 2540-9328 (online) acute toxicity and hypoglycemic effect of a polyherbal formulation on blood glucose in oral glucose tolerance test (ogtt) and alloxan-induced diabetic rats 1department of pharmacology, therapeutics and toxicology, faculty of basic medical sciences, university of lagos, lagos, nigeria. 2department of pharmacology and therapeutics, faculty of basic medical sciences, delta state university, abraka, nigeria. 3department of pharmacology and toxicology, faculty of pharmaceutical sciences, chukwuemeka odumegwu ojukwu university, awka, nigeria. corresponding author* abedoke@gmail.com manuscript received: 04 october 2021. revision accepted: 10 october, 2021. published: 15 october, 2021. abstract madam f. kayes bitters® is an herbal formulation commonly used in nigeria and some african countries in the management of diabetes mellitus and other diseases conditions. this study evaluated the in-vivo hypoglycaemic activity, as well as acute toxicity of the polyherbal formulation to provide its efficacy and safety. healthy albino mice (20-30 g) and sprague dawley female rats (90-130 g) were used for this study. acute toxicity study (ld50) of the herbal formulation was determined by methods originally described by miller and tainter in 1994. following oral dosing with glucose (2 g/kg) in normal fasted animals, herbal formulation (hf) at various doses was administered and blood glucose levels at 30 minutes, 60 minutes, 90 minutes, and 120 minutes were taken and recorded. diabetes was induced using alloxan 150 mg/kg and diabetic rats were given the hf at doses of 50, 100, and 200 mg/kg with glibenclamide 2.5 mg/kg used as standard drug treatment. blood glucose level was determined on 1st day, 7th day, 14th and 21st day. the ld50 was greater than 5g/kg with oral administration. the oral glucose tolerance test showed that the group that received 100 mg/kg hf showed a significant reduction (p<0.05) in glucose level after 120 minutes when compared to the basal level of glucose recorded. all treated diabetic groups showed a significant decrease in glucose level on the 21st day. the herbal formulation of hydrastis canadesis aloe capensis, echinacea angustifolia and honey exhibited a significant glucose-lowering activity in alloxan-induced diabetic rats. keywords: alloxan monohydrate; diabetes mellitus; herbal formulation; ld50 value. introduction diabetes mellitus (dm, diabetes) is a chronic disease characterized by persistent elevation of blood glucose level of an individual which resulting from defects in insulin secretion, insulin action, or both (ada, 2009). this disease is also marked by altered lipids, carbohydrates and protein metabolism (ozougwu et al., 2013; ezuruike and prieto, 2014). the hyperglycaemic condition takes place due to the inappropriate secretion of insulin hormone or the inappropriate use of insulin hormone by the body itself. insulin hormone is secreted by the islet of langerhans located in the beta cells of pancreas and it helps to maintain the glucose level in the blood (xavier, 2018). besides the storage of glucose, insulin also inhibits the secretion of glucagon and lowers the concentration of serum fatty acids leading to a decline in liver glucose production (asmat et al., 2016). insufficient insulin or resistance to insulin in the body results in reduced tissue uptake of glucose that results in intracellular hypoglycemia and extracellular hyperglycemia. the intracellular hypoglycemia causes glucogenesis and gluconeogenesis that leads to fats breakdown (causing diabetic ketoacidosis) and decreases protein synthesis and gamma globulins (causing cachexia, polyphagia, and impaired wound healing), while the extracellular hyperglycemia leads to hyperglycemic coma and osmotic dieresis (ozougwu et al., 2013). based on the requirements of insulin, diabetes is classified into insulin-dependent diabetes mellitus (type 1), and non-insulin-dependent diabetes mellitus (type 2) (ada, 2009). madam f. kayes bitters® is an herbal formulation commonly used in nigeria and some african countries in the management of diabetes mellitus and other diseases conditions. this formulation is listed to be composed of aloe capensis, hydrastis canadesis, echinacea angustifolia and honey. in spite of significant advancements in the development of conventional drugs, herbal medicines remain useful in the management of diseases, especially in developing countries (ekor, 2014; erhirhie et al., 2015; moke et al., 2021). this may be due to the adverse effects and high cost of therapy abednego okeoghene warri1*, emuesiri goodies moke2, aishat oyinkansola balogun1, kennedy chibogu nzeh1, emuesiri kohworho umukoro2, earnest oghenesuvwe erhirhie3 https://doi.org/10.14421/biomedich.2021.102.111-115 112 biology, medicine, & natural product chemistry 10 (2), 2021: 111-115 associated with conventional drugs (gurib-fakim, 2006; ekor, 2014). medicinal plants are plants whose parts contain substances of therapeutic important which can be constituted into drug and used for treatment of various disease (sofowora et al., 2013). many conventional drugs such as quinine, aspirin, digoxin, amongst others, are of plant origins (vickers et al., 2001; veeresham, 2013). presently, there is an urgent need to develop safer drugs for the management of chronic diseases such as diabetes mellitus. consequently, assessment of various medicinal plants used in traditional systems have grown (lahlou, 2013; sofowora et al., 2013; ekor, 2014, anachuna et al., 2018; okafo et al., 2019; moke et al., 2020). however, high cost of production is a factor, developmental processes for newer drugs is a way of evaluating of efficacy and adverse effects of chemical substances. the present study aimed to evaluate the invivo hypoglycaemic activity, as well as acute toxicity of the polyherbal formula extracts (madam f. kayes bitters®) on experimental animals. material and methods herbal formulation the herbal formulation used was purchased from a local pharmacy. it was stored in a cool dry place during the period of the study. experimental animals healthy albino mice (20-30 g) and sprague dawley female rats (90-130 g) were used for this study. these were procured from the animal house of the college of medicine, university of lagos, nigeria. the animals were kept in polypropylene cages with wire mesh for proper ventilation throughout the study. the animals were given free access to standard diet (lifestock feed plc, ikeja, nigeria) and water ad libitum. the animals were kept in room temperature with a 12:12 day and night cycle and maintained at temperature of 27 ℃. the animals were allowed to acclimatize for two weeks before commencing the study. the maintenance and treatment of the animals was according to the principles of the guide for care and use of laboratory animals in research and teaching prepared by the national academy of science and published by the national institute of health (nih) publication 86-23 revised in 1985. acute toxicity study acute toxicity study of the herbal formulation was determined by methods originally described by miller and tainter of 1994 (erhirhie et al., 2018). mice (20-25 g) were fasted for 12 hours and divided into four groups of 5 mice each. in order to determine the ld50 of the formulation, different doses 1000 mg/kg, 2500 mg/kg and 5000 mg/kg were administered orally to different groups of mice while the control group received 10 ml/kg distilled water (ph = 6.9). all animals were closely observed for symptoms of toxicity and the mortality noted. based on data obtained, the ld50 was determined. oral glucose tolerance test (ogtt) fifteen adult female rats (100-126 g) were fasted overnight. the animals were divided into five different groups with three animals each. blood samples were obtained from each rat by gently nipping the tail with a lancet, and then gently squeeze the tail to let out 2 drops of fresh venous whole blood on the glucometer strip inserted and the readings recorded for the 0 minute. different doses of the herbal formulation were orally administered. glucose (2 g/kg) was orally administered 30 minutes after extract administration to each animal according to their body weight. blood glucose level at 30 minutes, 60 minutes, 90 minutes, and 120 minutes were taken and recorded. alloxan-induced diabetic study eighteen healthy female rats were randomly divided into 6 groups of 3 animals each. the rats were fasted for 24 hours after which group i rats were given 10 ml/kg normal saline while groups ii-vi were made hyperglycemic with a single intraperitoneal injection of 150 mg/kg of alloxan monohydrate (sigma chemicals company, st. louis, mo., u.s.a) (gbolade et al., 2008; moke et al., 2015). the baseline fasting blood glucose was determined prior to the administration of alloxan. after 48 hours, blood was collected from the tail vein of each rat and the fasting blood glucose level determined. a blood glucose ≥ 200 mg/dl was considered diabetic. animals were further tested after 72 hours and animals with stable hyperglycemia were selected for the study. the treatment schedule of the rats is as follows:  group i (control) – normal saline 10 ml/kg  group ii (diabetic and untreated) – normal saline 10 ml/kg  group iii – herbal formulation 50 mg/kg  group iv – herbal formulation 100 mg/kg  group v – herbal formulation 200 mg/kg  group vi – glibenclamide 2.5 mg/kg determination of fasting blood glucose in diabetic rats the blood glucose level was determined using the tail tipping method after 12-16 hours of fast. the tail was gently squeezed to let out 2-3 drops of blood which is placed on the test spot of the glucose strip after which the test strip was gently inserted into the digital glucometer. blood glucose level was determined on 1st day, 7th day, 14th and 21st day. data analysis data were presented as mean ± standard error of mean (sem) using graph pad prism. test of statistical warri et al. – acute toxicity and hypoglycemic effect of a polyherbal … 113 significance was carried out using a one-way anova. p-values lesser than 0.05 (p<0.05) were considered statistically significant. results and discussion acute toxicity test of herbal formulation when the herbal formulation was administered orally, there was no death of experimental animals even at a dose of 5g/kg. hence, for the oral route, the ld50 is greater than 5g/kg. (table 1) table 1. oral acute toxicity of herbal formulation. group/dose (mg/kg) number of mice number of death control 5 nil 1000 5 nil 2500 5 nil 5000 5 nil ld50oral ˃5000mg/kg body weight of animals there was no statistically significant change in body weights of the animals (p>0.05) when compared to control as shown in table 2. table 2. herbal formulation treatment on body weights. treatment day 1 (g) day 7 (g) day 14 (g) day 21 (g) control (ns) 103.70±4.91 105.00±5.29 107.3±3.84 115.30±2.60 untreated (ns) 106.70±4.49 105.00±3.06 99.67±2.60 97.00±3.22 glb 2.5 mg/kg 100.70±6.36 101.00±7.21 99.67±2.60 68.83±28.67 50 mg hf 107.00±6.56 106.00±5.69 103.70±3.48 105.0±2.52 100 mg hf 107.00±6.08 104.30±6.94 105.00±3.61 108.00±2.31 200 mg hf 105.00±5.57 105.70±6.84 105.30±7.42 113.30±16.05 values are expressed as mean ± sem (n=3). p<0.05 statistically significant when day 1 is compared to other respective groups using one way-anova followed by tukey’s post hoc multiple comparison tests. key: ns = normal saline; glb = glibenclamide; hf =herbal formulation oral glucose tolerance test of herbal formulation. the oral glucose tolerance test showed a sharp decrease in the glucose level after 30 minutes when compared to the level at 0 minute. this was followed by a gradual reduction in glucose level after 60, 90 and 120 minutes. the control group showed a statistically significant reduction in glucose level at the 90th minute (p<0.01) when compared with the basal level recorded (78.33±0.88). glibenclamide showed a significant reduction (p<0.01) in glucose level after 120 minutes (26±13.01). also the group that received 100 mg/kg hf showed a significant reduction (p<0.05) in glucose level after 120 minutes (39.67±2.33) when compared to the basal level of glucose recorded. (table 3) table 3. oral glucose tolerance test (ogtt) of herbal formulation. treatment basal (mg/dl) 0 minute (mg/dl) 30 minutes (mg/dl) 60 minutes (mg/dl) 90 minutes (mg/dl) 120 minutes (mg/dl) control (ns) 85.33±1.86 104.33±6.57 91.67±5.90 77.33±1.86 87.54±0.88 90.67±2.96 glb 2.5 mg/kg 90.33±7.86 106.67±6.77 87.67±12.25 66.00±10.82 43.67±6.64 26.00±13.01** 50 mg hf 83.33±6.23 104.67±4.41 85.33±9.70 52.67±12.03 67.67±6.94 54.00±13.58 100 mg hf 87.00±5.86 114.00±10.60 88.33±7.84 60.00±5.29 61.00±4.58 39.67±2.33* 200 mg hf 82.67±4.48 116.67±8.84 110.33±8.29 74.33±5.36 71.00±1.53 63.67±6.36 values are mean ±sem (n=3): *p<0.05, **p<0.01 statistically significant compared to basal within group with other respective minutes using one wayanova followed by tukey's post hoc multiple comparison tests. key: ns = normal saline; glb = glibenclamide; hf = herbal formulation blood glucose level of alloxan-induced diabetic animals the 21 days treatment of alloxan-induced diabetic rats with herbal formulation resulted in statistically significant reduction in blood glucose (table 4). there was a statistically significant difference between all treated groups and the untreated diabetic group when compared with the non-diabetic control indicating initial hyperglycemic condition upon induction of diabetes. on the 7th day, there was a statistical significant difference in glucose level in the untreated group (p<0.05) and groups administered glibenclamide (p<0.01) and 200 mg/kg hf (p<0.001) compared to nondiabetic control. on the 14th day, there was no statistically significant difference in all the treatment groups when compared to both diabetic untreated group and non-diabetic control group. however, the untreated control showed a significant difference (p<0.01) in 114 biology, medicine, & natural product chemistry 10 (2), 2021: 111-115 glucose level when compared with non-diabetic control. all treated groups showed a significant decrease in glucose level on the 21st day. the p values were as follows: glibenclamide (p<0.001), 50 mg/kg hf (p<0.001), 100 mg/kg hf (p<0.001) and 200 mg/kg hf (p<0.01). table 4. blood glucose level of alloxan-induced diabetic animals. treatment baseline (mg/dl) day 1 (mg/dl) day 7 (mg/dl) day 14 (mg/dl) day 21 (mg/dl) control (ns) 53.33±7.31 54.67±7.31 59.33±8.29 48.33±1.20 55.67±5.90 untreated (ns) 66.67±11.79 307.70±56.69* 335.30±51.27* 338.30±49.97** 360.00±55.08**** glb 2.5 mg/kg 52.00±1.53 386.70±51.57*** 207.00±67.35** 126.00±62.98 78.67±18.49b 50 mg hf 78.67±7.75 336.00 ±64.29* 289.30±35.97 116.00±52.81 83.33±13.42b 100 mg hf 67.00±16.50 383.30±52.03** 260.00±60.65 124.70±11.05 55.00±4.73b 200 mg hf 72.33±22.72 399.67±25.11** 284.00±44.23** 192.33±39.07 106.70±8.74a values are mean ±sem (n=3): *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus control; ap<0.001, bp<0.0001 versus untreated control. analysis by one way anova followed by tukey’s post hoc multiple comparison tests. key: ns = normal saline; glb = glibenclamide; hf = herbal formulation over centuries, herbal products of medicinal value have been recruited in traditional medicine for the management and treatment of several ailments including diabetes mellitus (jung et al., 2006). although there have been a significant advancement in development of conventional drugs in recent times, herbal remedies are still used especially in developing countries in the treatment of diseases (ekor, 2014; moke et al., 2021). numerous medicinal plants are widely used in nigeria to manage diabetes mellitus (abo et al., 2008; ezuruike and prieto, 2014; moke et al., 2015; abubakar et al., 2017; okafo et al., 2019). the crude extracts of these plants may be used alone or mixed with extracts from other plants or other sources. madam f. kayes bitters® is a poly-herbal formulation of aloe capensis, hydrastis canadesis and echinacea angustifolia plus honey. diabetes mellitus is characterized by persistent elevations of fasting blood glucose above 200 mg/dl due to insufficient or complete cessation of insulin synthesis or secretion and/or peripheral resistance to insulin action (adeneye and agbaje, 2008; kitabchi et al., 2009). in this study, acute toxicity test revealed that the polyherbal formulation is quite safe for oral consumption. the oral glucose tolerance test (ogtt) showed a gradual reduction in the glucose level from 0 minute to 120 minutes. this decrease was found to be significant for the groups administered the standard treatment (glibenclamide) and hf 100mg/kg which suggests that the herbal formulation has a glucose lowering effect. following 21 days treatment of alloxan-induced diabetic rats with the herbal formulation, there was a significant reduction in blood glucose level in all groups receiving treatment compared to the untreated diabetic control. the reduction in glucose level was gradual in all groups with glucose level brought to below 150 mg/dl on the 14th day with the exception of the group receiving 200mg/kg. the hypoglycemic effect was more profound in the group receiving 100 mg/kg than that of 50 mg/kg and 200 mg/kg which suggests that the hypoglycemic effect is not dose-dependent. the result obtained is consistent with earlier reports in scientific literature demonstrating the hypoglycemic effect of aloe barbadensis/aloe vera (used synonymously with aloe carpensis) in alloxan-induced diabetic rats (nwanjo, 2006; choudhary et al., 2014; pothuraju et al., 2016; hammeso et al., 2019). also honey has been shown to exert a dose-dependent hypoglycemic effect in diabetic rats (erejuwa, 2014). honey is sweet and rich in sugars. it is therefore surprising that it has a hypoglycemic effect. it has been hypothesized that fructose and oligosaccharides present in honey may in some way contribute to its observed hypoglycemic effect (erejuwa et al., 2012). hence, the hypoglycemic effect of the herbal formulation investigated is consisted with scientific reports on the constituents. conclusion the polyherbal formulation of hydrastis canadesis aloe capensis, echinacea angustifolia and honey exhibited a significant glucose-lowering activity in alloxan-induced diabetic rats. this suggests that this formulation may be useful for the management of diabetes mellitus. this study therefore corroborates the claim that this herbal formulation is effective in managing diabetes mellitus. acknowledgements: the work was supported by grant received from federal ministry of health through postgraduate scholarship administered by the federal scholarship board (fsba/fgss/pg/16/212). we wish to acknowledge the contributions and mentorship role of dr. nwaiwu obiyo (of blessed memory). warri et al. – acute toxicity and hypoglycemic effect of a polyherbal … 115 conflict of interest: the authors declare that there are no conflicts of interest concerning the publication of this article. references abo ka, fred-jaiyesimi aa, jaiyesimi ae (2008). ethnobotanical studies of medicinal plants used in the 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547553. choudhary m, kochhar a, sangha j (2014). hypoglycemic and hypolipidemic effect of aloe vera l. in non-insulin dependent diabetics. j food sci technol 51(1): 90-6. da silva xavier g (2018). the cells of the islets of langerhans. j clin med 7(3): 54. ekor m (2014). the growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. front pharmacol 4: 177. erejuwa oo, sulaiman sa, wahab ms (2012). honey a novel antidiabetic agent. int j biol sci 8(6): 913-34. erejuwa oo (2014). effect of honey in diabetes mellitus: matters arising. j diabetes metab disord 13(1): 23. erejuwa oo (2012). management of diabetes mellitus: could simultaneous targeting of hyperglycemia and oxidative stress be a better panacea? int j mol sci 13(3): 2965-72. erhirhie eo, ilodigwe ee, ajaghaku dl, mbagwu is, moke eg (2015). toxicity evaluation of a commercial herbal preparation commonly used in nigeria. european j med plants 5(2): 176-190. erhirhie eo, ihekwereme cp, ilodigwe ee (2018). advances in acute toxicity testing: strengths, weaknesses and regulatory acceptance. interdiscip toxicol 11(1): 5-12. ezuruike uf, prieto jm (2014). the use of plants in the traditional management of diabetes in nigeria: pharmacological and toxicological considerations. j ethnopharmacol 155(2): 857-924. gbolade aa, ekor mn, akinlolu aa, ayoola md (2008). antidiabetic activity of stem bark ethanolic extracts of spondias mombin on alloxan-induced diabetic rats. j pharmaceut res 7: 192-195. gurib-fakim a (2006). medicinal plants: traditions of yesterday and drugs of tomorrow. mol aspects med 27(1): 1-93. hammeso ww, emiru yk, ayalew getahun k, kahaliw w (2019). antidiabetic and antihyperlipidemic activities of the leaf latex extract of aloe megalacantha baker (aloaceae) in streptozotocin-induced diabetic model. evid based complement alternat med 2019: 8263786. kitabchi ae, umpierrez ge, miles jm, fisher jn (2009). hyperglycemic crises in adult patients with diabetes. diabetes care 32(7): 1335-43. lahlou m (2013). the success of natural products in drug discovery. pharmacol pharm 4: 17-31. moke eg, ilodigwe ee, okonta jm, emudainohwo jot, ajaghaku dl, erhirhie oe, chinwuba p, ahante e (2015). antidiabetic activity and toxicity evaluation of aqueous extracts of spondias mombin and costus afer on wistar rats. br j pharm res 6(5): 333-342. moke eg, mordi jc, umukoro ek (2020). effects of methanol leaf extract of cuphea hyssopifolia kunth on liver enzymes activity and antioxidant indices of paracetamolinduced hepatotoxicity in wistar rats. afr j biomed res 23(1): 123-6. moke eg, umukoro ek, asiwe jn, omogbiya ai, erhirhie eo, ben-azu b, anieh fu (2021). herbal medicine: education and occupation influences its practice among residents of port harcourt, south-south nigeria. int j pharm phytopharm res 11(2): 38-44. nwanjo hu (2006) antioxidant activity of the exudates from aloe barbadensis leaves in diabetic rats. biokemistri 18(2): 77-81. okafo se, moke eg, obi cs (2019). formulation and evaluation of anti-diabetic tablets containing aqueous extract of moringa oleifera seeds. jophas 16(5): 3167-3176. ozougwu jc, obimba kc, belonwu cd, unakalamba cb (2013). the pathogenesis and pathophysiology of type 1 and type 2 diabetes mellitus. j physiol pathophysiol 4(4): 46-57. pothuraju r, sharma rk, onteru sk, singh s, hussain sa (2016). hypoglycemic and hypolipidemic effects of aloe vera extract preparations: a review. phytother res 30(2): 200-7. sofowora a, ogunbodede e, onayade a (2013). the role and place of medicinal plants in the strategies for disease prevention. afr j tradit complement altern med 10(5): 21029. veeresham c (2012). natural products derived from plants as a source of drugs. j adv pharm technol res 3(4): 200-1. vickers a, zollman c, lee r (2001). herbal medicine. west j med 175(2): 125-8. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 271-275 | doi: 10.14421/biomedich.2024.131.271-275 issn 2540-9328 (online) antiepileptic potential of justicia insularis leaf extract in swiss mice nancy elkanah1, michael chuks nnumolu1,*, jude effiom okokon1, uduak peter ise2 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. 2department of pharmacology and toxicology, faculty of pharmaceutical sciences, bingham university, karu, nassarawa state, nigeria. corresponding author* michaelnnumolu@gmail.com manuscript received: 21 september, 2023. revision accepted: 14 march, 2024. published: 08 august, 2024. abstract epilepsy is a prevalent neurological condition characterized by repetitive seizures, necessitating the exploration of efficacious treatments. justicia insularis, a plant with a historical use in treating convulsions and epilepsy, has garnered attention as a potential antiepileptic agent. this study aimed to investigate the antiepileptic properties of justicia insularis leaf extract, focusing on generated seizure models in mice. the analysis of variance (anova), specifically one-way anova, was utilized to assess the statistical significance of the data. seizure models were employed to evaluate the antiepileptic effects of justicia insularis leaf extract. the p-values obtained from the oneway anova were significant, with values of ap<0.005, bp<0.01, and cp<0.001. these results suggest the statistical significance of the observed effects in the seizure models. this study provides valuable insights into the therapeutic potential of justicia insularis leaf extract as a treatment for epilepsy. the significant findings underscore the importance of further research into the use of natural medicines for managing epilepsy, potentially offering new avenues for treatment. keywords: justicia insularis; antiepileptic; seizures; convulsion; gabaa receptor; flavonoids. introduction epilepsy is a persistent neurological condition characterised by the occurrence of repetitive seizures. this event can potentially influence individuals across various age groups and substantially influence their overall well-being. despite various antiepileptic treatments, many persons diagnosed with epilepsy encounter insufficient seizure management and often endure unfavourable prescription side effects. his has increased interest in alternative therapies, including herbal remedies (espinosa-garcia et al., 2021). justicia insularis, sometimes called "african water willow", is classified under the acanthaceae botanical family. it has been widely used in many traditional medicinal practices in diverse cultural contexts. justicia insularis is often grown within the home gardens of west and central africa, with particular prevalence in countries such as guinea, sierra leone, ghana, togo, benin, nigeria, cameroon, and the democratic republic of congo (christian et al., 2020). justicia insularis has been used as a traditional cure for convulsions, epilepsy, and associated ailments across several african groups. in senegal, providing a leaf decoction derived from j. insularis to pregnant women during the last month of their pregnancy is common practice to alleviate the discomfort associated with labour (christian et al., 2020). in the western area of cameroon, the use of it is seen in conjunction with the foliage of three other botanical species (aloe buettneri, hibiscus macranthus, and dicliptera verticillata) to manage dysmenorrhoea and some instances of female infertility (meguem et al., 2021). additionally, in togo and northeast ghana, the cooked leaves of j. insularis are administered to infants to facilitate bowel movement, and the leaves are also used topically to enhance the healing process of wounds (irinmwinuwa et al., 2023). nevertheless, it is well observed that traditional knowledge frequently lacks empirical substantiation via scientific methods. hence, the primary objective of this investigation was to conduct a scientific assessment of the antiepileptic properties of justicia insularis leaf extract via the use of pentylenetetrazole, aminophylline and isoniazid-induced mice. materials and methods experimental animals swiss mice of either sex used for these experiments were obtained from the faculty of pharmacy animal house, university of uyo, uyo. the animals were housed in standard cages and were maintained on standard pelleted feed (guinea feed) and water ad libitum. permission and approval for animal studies were obtained from the college of health sciences animal ethics committee, https://doi.org/10.14421/biomedich.2024.131.271-275 mailto:michaelnnumolu@gmail.com 272 biology, medicine, & natural product chemistry 13 (1), 2024: 271-275 university of uyo. all animal experiments complied with the national institute of health guide for care and laboratory animals (pub. no. 85-23, revised 1985). plant collection and identification the plant material justicia insularis (leaves) was collected from the university of uyo premises, akwa ibom state, nigeria, in march 2023. the plant was identified and authenticated by a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria. preparation of extract the leaves were washed and shade-dried for two weeks. the dried plants' materials were chopped into small pieces and reduced to powder using an electric grinder. the powdered leaves material (1.5 kg) was soaked for 72 h in ethanol (50%). this was later filtered, and the liquid filtrate was concentrated and evaporated to dryness in vacuo 40˚c using a rotary evaporator (buchilab, switzerland). the extract was stored in a refrigerator at 4˚c until used for the proposed experiments. experimental design table 1 illustrates the experimental design used for the three seizure models. the design of the models was the same across all models, with the exception of the administration of different standard drugs for each kind of convulsion. specifically, phenobarbital (40 mg/kg) was used as the standard drug for pentylenetetrazole and aminophylline-induced convulsions, while diazepam (5 mg/kg) was employed as the standard drug for isoniazidinduced convulsions. the different doses of the extract and standard drugs were administered to the mice an hour before induction of convulsion. table 1. experimental design of experimentally induced convulsion in swiss mice. groups agent dosage (mg/kg/day) i normal saline 10 ml/kg ii low dose of the ethanol leaf extract of justicia insularis 150 iii middle dose of the ethanol leaf extract of justicia insularis 300 iv high dose of the ethanol leaf extract of justicia insularis 450 v phenobarbital 40 source: field data (2023) statistical analysis and data evaluation the results were analysed using the statistical software package for social sciences (spss) version 16.0 for windows. analysis of variance (anova) was used to compare means, and all results were presented as mean ± sem. results and discussion results ptz –induced convulsion administration of leaf extract of justicia insularis (150450 mg/kg) provided a considerable degree of protection for the mice against seizure induced by pentylenetetrazole. the leaf extract prolonged the onset of myoclonic convulsion in a dose-dependent fashion, but the effect was insignificant (p>0.05) compared to the control. similarly, the leaf extract exerted a significant (p<0.05) prolongation of time for the onset of tonic convulsion in a dose-dependent manner, which was significant (p<0.05) at the higher doses (300 and 450 mg/kg) (table 2). the time of death of treated animals was dose-dependently and insignificantly (p>0.05) prolonged compared with the control. the standard drug, phenobarbital, offered 33.33% protection to the treated animals (table 2). table 2. effect of ethanol leaf extract of justicia insularis on pentylenetetrazole-induced convulsion. treatment dose (mg/kg) onset of myoclonic (min) onset of tonic (min) time of death (min) no. of death control (normal saline) 0.32 ±0.04 1.35 ± 0.06 1.68±0.37 6/6 phenobarb 40 1.23 ±0.09c 18.28±1.82c 30.09±0.01c 2/6 leaf extract 150 0.33 ±0.01 1.32± 0.14 3.89±0.32 6/6 300 0.34 ±0.06 2.06± 0.25a 2.99±0.38 6/6 450 0.46± 0.04 2.57±0.02a 3.64±0.63 6/6 data are expressed as mean ± sem, significant at ap<0.005; bp<0.01; cp < 0.001, compared to control. (n=6). aminophylline-induced convulsion administration of the leaf extract of j. insularis (150-450 mg/kg) caused a significant (p<0.01-0.001) delay in the onset of seizure induced by aminophylline in a dosedependent fashion. the delay was significant (p<0.010.001) in both myoclonic and tonic convulsions, elkanah et al. – antiepileptic potential of justicia insularis leaf extract in … 273 especially at higher doses (300 and 450 mg/kg) of the extract (table 3). the time of death of the treated animals was dose-dependently and significantly (p<0.050.01) prolonged at higher doses (300 and 450 mg/kg) of the extract when compared with the control. the standard drug, phenobarbital, offered 100% protection to the treated mice. table 3. effect of ethanol leaf extract of justicia insularis on aminophylline-induced convulsion. treatment dose (mg/kg) onset of myoclonic (min) onset of tonic (min) time of death (min) no. of death control (normal saline) 10.42 ± 0.36 12.31 ± 4.27 15.30 ± 5.45 6/6 phenobarb 40 12.96 ± 0.76c 0.00 ± 0.00c 0.00 ± 0.00c 6/6 leaf extract 150 10.94 ± 1.82 12.37 ± 2.41 16.79 ± 5.21 6/6 300 14.62 ± 2.66 22.82 ± 1.39a 26.65 ± 1.91a 6/6 450 22.77 ± 3.19c 26.49 ±3.66a 30.22 ± 4.36b 6/6 data are expressed as mean ± sem, significant at ap<0.005; bp<0.01; cp < 0.001, compared to control. (n=6). isoniazid-induced convulsion administration of the leaf extract of justicia insularis (150-450 mg/kg) caused a significant (p<0.01-0.001) delay in the onset of seizure induced by isoniazid in a non-dose-dependent fashion. the delay was significant (p<0.01-0.001) in both myoclonic and tonic convulsions, especially at the lower doses (150 and 300 mg/kg) (table 4). the time of death of the treated animals was prolonged significantly (p<0.05) at the lowest dose (150 mg/kg) of the extract when compared with the control, while the standard drug, diazepam, also offered a more significant (p<0.01) prolongation of the time of death to the mice treated with it (table 4). table 4. effect of ethanol leaf extract of justicia insularis on aminophylline-induced convulsion. treatment dose (mg/kg) onset of myoclonic (min) onset of tonic (min) time of death (min) no. of death control (normal saline) 13.73±3.90 29.08± 1.23 34.95±1.64 6/6 diazepam 5 41.17±0.10b 26.19± 0.53b 84.09±4.05b 6/6 leaf extract 150 41.81±1.23b 55.77± 1.72b 56.39±1.86a 6/6 300 26.96±0.59a 40.73±3.05a 47.64±6.25 6/6 450 13.73±3.90 29.08±1.23 35.95±1.44 6/6 data are expressed as mean ± sem, significant at ap<0.005; bp<0.01; cp < 0.001, compared to control. (n=6). discussion the efficacy of the leaf extract derived from justicia insularis, a botanical species often used in traditional medicine for managing convulsions and epilepsy, was assessed in relation to its potential to mitigate experimentally induced convulsions. the leaf extract was found to possess significant activity against seizures induced by pentelyenetetrazole, aminophylline and isoniazid, offering considerable protection in some cases, with lower doses being more potent. the precise mechanisms of aminophylline-induced seizures seem multifaceted, including various intricate pathways that remain uncertain. the available evidence indicates that seizures caused by aminophylline may be attributed to two mechanisms: adenosine receptor antagonism and suppression of cerebral nucleotidase activity (tescarollo et al., 2020). these mechanisms decrease adenosine levels in the brain, ultimately leading to disinhibition. however, according to a paper by sharma and sandhir (2010), it was shown that diphenylhydantoin, which is a potent inhibitor of adenosine absorption, did not show efficacy in avoiding these seizures. in addition to its nonspecific inhibition of adenosine receptors (malekisadeghi et al., 2022), aminophylline is believed to have inhibitory effects on adenosine production. nevertheless, a study by ray et al. (2005) has shown that seizures triggered by aminophylline may be attributed to oxidative stress resulting from the production of free radicals and reactive oxygen species. the leaf extract has demonstrated notable antioxidant capabilities (adeyemi & babatunde, 2014). this activity may be attributed to the ability of its phytochemical ingredients to scavenge free radicals. the leaf extract may have also facilitated adenosine production, contributing to its anticonvulsant properties. shrivastava et al. (2022) explain that the anticonvulsant action of pentylenetetrazole (ptz) may be attributed to its ability to block the activity of gammaaminobutyric acid (gaba) at gabaa receptors. gamma-aminobutyric acid (gaba) is a prominent inhibitory neurotransmitter involved in the pathophysiology of epilepsy. the modulation of gaba neurotransmission may reduce or intensify convulsions (akyuz et al., 2021). phenobarbitone and diazepam, commonly used medications for epilepsy, have been shown to exert their antiepileptic properties by 274 biology, medicine, & natural product chemistry 13 (1), 2024: 271-275 augmenting gaba-mediated inhibition inside the brain (okokon et al., 2021). the efficacy of phenytoin in preventing ptz-induced seizures was limited, perhaps due to its mechanism of action, including the inhibition of sodium ion influx into brain cells, hence impeding the production of repetitive action potentials (okokon et al., 2021). the observed delay in ptz-induced convulsion and protective effects on animals resulting from the administration of j. insularis leaf extract provide evidence supporting its central nervous system depressive properties and its capacity to increase gabamediated inhibition inside the brain. isoniazid, a pharmaceutical agent used to treat tuberculosis, has been found to induce status epilepticus, a prolonged seizure activity, by reducing gammaaminobutyric acid (gaba) levels in the brain. gaba is a crucial inhibitory neurotransmitter in the mammalian brain. this effect is achieved by inhibiting pyridoxal-5phosphate-dependent glutamic acid decarboxylase (gad), an enzyme involved in gaba synthesis. pyridoxal-5-phosphate is the biologically active form of pyridoxine, functioning as a cofactor for glutamic acid decarboxylase (gad), an essential enzyme involved in the manufacture of gamma-aminobutyric acid (ghatge et al., 2021). recurrent seizures characteristic of status epilepticus result from reduced gaba levels (rho and boison, 2022). despite the limited efficacy of existing anticonvulsant medications in treating seizures generated by isoniazid, intravenous diazepam continues to be utilised for seizure management in the absence of pyridoxine (okokon et al., 2021). the administration of justicia insularis leaf extract demonstrated a substantial protective effect against isoniazid (inh)-induced convulsions in the treated mice. the observed behaviour might be attributed to the extract's capacity to augment gaba production in the brain through the actions of its phytoconstituents. the antiepileptic effect of flavonoids, which are secondary metabolites found in plants, has been extensively documented in many studies. these compounds have been shown to modulate the gabaacl-channel complex, similar to benzodiazepines, indicating their potential therapeutic use in treating epilepsy (choudhary et al., 2011). the antiepileptic effect of flavonoids is attributed to their ability to modulate the gabaa-cl-channel complex, facilitated by their structural resemblance to benzodiazepines (choudhary et al., 2011). additionally, the compound apigenin, which belongs to the class of flavonoids, has been identified as a ligand that acts on the central nervous system, like benzodiazepines. it has also shown effectiveness in mitigating convulsions generated by picrotoxin (jäger et al., 2009). extracts derived from justicia insularis leaves have a high concentration of flavonoids and other phenolic compounds known for their potent antioxidant properties, and these metabolites function by scavenging free radicals and regulating the gabaa-cl-channel complex inside the central nervous system (cns), demonstrating their anticonvulsant properties (oyomah et al., 2019). the results of this study have confirmed the anticonvulsant potential of leaf extract of justicia insularis and justify its use traditionally in the treatment of convulsion. conclusion the findings presented in this study provide scientific support for the traditional use of justicia insularis as an antiepileptic remedy. the leaf extract demonstrated significant anticonvulsant effects across various induced seizure models, indicating its potential as a natural therapy for epilepsy management. interactions with gabaa receptors and antioxidant mechanisms likely mediate the observed effects. further research is warranted to elucidate the specific compounds responsible for these effects and to explore their mechanisms of action in more detail. the study contributes to the growing knowledge surrounding herbal remedies for epilepsy and underscores the importance of integrating traditional knowledge with modern scientific approaches. acknowledgements: the authors thank mr nsikan malachy from the animal house department of pharmacology and toxicology at the university of uyo for providing technical assistance. authors' contribution: conceptualisation: jude effiom okokon, data curation: michael chuks nnumolu, formal analysis: jude effiom okokon, funding acquisition: nancy elkanah, investigation: jude effiom okokon, nancy elkanah, methodology: jude effiom okokon, project administration: nancy elkanah, supervision: jude effiom okokon, validation: uduak peter ise, jude effiom okokon, writing–original draft: nancy elkanah, writing–review & editing: michael chuks nnumolu. competing interests: the authors declare that there are no conflicts of interest concerning this manuscript. funding: the study was self-funded. references espinosa-garcia, c., zeleke, h. and rojas, a. (2021). impact of stress on epilepsy: focus on neuroinflammation—a mini review. international journal of molecular sciences. 2021 apr 14;22(8):4061. christian a. g., ugochi, o. a., azubuike, e. j., uchenna, m. a., john, a. d., amarachi, i. l. and alozie, o. o. (2020). acute and sub-acute toxicity effect of the ethanol leaf extract of elkanah et al. – antiepileptic potential of justicia insularis leaf extract in … 275 justicia insularis in wistar rats saudi journal of medical and pharmaceutical sciences. meguem, s. o., lienou, l. l., goka, m. s., tagne, r. s., yemele, d. m., and telefo, p. b. (2021). effects of the aqueous extract of dicliptera verticillata on fertility and different stages of gestation in female rats. zygote. 2021 aug;29(4):307-13. irinmwinuwa, e. o., adolphus, m. c., opute, r. e., oyate, g. b., chinedu, j. o., afonne, o. j. and unekwe, p. c. (2023). review of herbal plant associated with anti-anemic property and mechanism of action. international journal of science and research archive. 2023;8(2):589-601. tescarollo, f. c., rombo, d. m., deliberto, l. k., fedele, d. e., alharfoush, e., tomé, â. r., cunha, r. a., sebastião, a. m. and boison, d. (2020). role of adenosine in epilepsy and seizures. journal of caffeine and adenosine research. 2020 jun 1;10(2):45-60. sharma, a. and sandhir, r. (2010). oxidative stress after lithiumpilocarpine induced status epilepticus in mice brain. annals of neurosciences. 2010 nov 2;13(1):1-4. maleki-sadeghi, n., rahmani, p., aghsaeifard, z. and heidari, g. (2022). effects of aminophylline on the levels of neutrophil gelatinase-associated lipocalin (ngal) in asphyxiated term neonates. archives of physiology and biochemistry. 2022 jul 4;128(4):1105-10. ray, a., gulati, k., anand, s. and vijayan, v. k. (2005). pharmacological studies on mechanisms of aminophyllineinduced seizures in rats. indian journal of experimental biology. 43: 849-853. adeyemi, o. t. and babatunde, o. (2014). chemical composition and antioxidant capacity of the leaf extract of justicia insularis. international journal of physical sciences. 2014 oct 30;9(20):454-8. shrivastava, a,. gupta, j. k. and goyal, m. k. (2022). flavonoids and antiepileptic drugs: a comprehensive review on their neuroprotective potentials. journal of medical pharmaceutical and allied sciences. 2022;11:4179-86. akyuz, e., polat, a. k., eroglu, e., kullu, i., angelopoulou, e. and paudel, y. n. (2021). revisiting the role of neurotransmitters in epilepsy: an updated review. life sciences. 2021 jan 15;265:118826. okokon, j., davies, k., edem, u. a., bassey, a. i. (2021). anticonvulsant activity of leaf extract of heinsia crinata: biological activity of heinsia crinata. nigerian journal of pharmaceutical and applied science research. 2021 mar 31;10(1):20-5. ghatge, m. s., al mughram, m., omar, a. m. and safo, m. k. (2021). inborn errors in the vitamin b6 salvage enzymes associated with neonatal epileptic encephalopathy and other pathologies. biochimie. 2021 apr 1;183:18-29. rho, j. m. and boison d. (2022). the metabolic basis of epilepsy. nature reviews neurology. 2022 jun;18(6):333-47. choudhary, n., bijjem, k. r. and kalia, a. n. (2011). antiepileptic potential of flavonoids fraction from the leaves of anisomeles malabarica. journal of ethnopharmacology. 2011 may 17;135(2):238-42. jäger, a. k., krydsfeldt, k., rasmussen, h. b. (2009). bioassay‐guided isolation of apigenin with gaba‐benzodiazepine activity from tanacetum parthenium. phytotherapy research. 2009 nov;23(11):1642-4. oyomah, g. o., odoemena, c. s., ogede, r. o. and bala, d. (2019). analgesic and anti-inflammatory activities of the ethanol leaf extract of justicia insularis (t. anders) on albino rats. journal of advanced research in biology. 2019 dec 30;1(2):1-6. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1015-1019 | doi: 10.14421/biomedich.2025.142.1015-1019 issn 2540-9328 (online) onychomycosis in students: an overview of nail care practices and personal hygiene lisa savitri1*, kharisul ihsan2, rochmad krissanjaya1, elfred rinaldo kasimo1, novirma yanti1, mochamad hanif hilmi1 1department of medical laboratory technology, faculty of health sciences, kadiri university, kediri, east java, indonesia 2department of pharmacy, faculty of pharmacy, public health, hospital administration, radiology, universitas strada indonesia, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 29 august, 2025. revision accepted: 14 october, 2025. published: 30 october, 2025. abstract onychomycosis is a common nail disorder caused by dermatophytes, yeasts, and non-dermatophyte molds, with increasing prevalence among young adults. poor nail care and personal hygiene habits are essential predisposing factors that contribute to infection. despite its clinical relevance, data on onychomycosis in student populations remain limited. a cross-sectional study was conducted among 150 undergraduate students from the faculty of health sciences, aged 18–25 years. nail specimens were collected from individuals presenting with suspected onychomycosis. direct microscopy was performed using a 20% potassium hydroxide (koh) preparation, and samples were cultured on sabouraud dextrose agar supplemented with chloramphenicol and cycloheximide. fungal isolates were identified based on macroscopic and microscopic characteristics. sociodemographic data, nail care habits, and hygiene practices were collected through structured questionnaires. data were analyzed descriptively, and associations were assessed using chi-square tests. onychomycosis was confirmed in 27 students (18%). dermatophytes were the most frequent isolates, with trichophyton rubrum (9%) and t. mentagrophytes (5%) predominating, followed by candida albicans (3%) and aspergillus spp. (1%). risk factors significantly associated with infection included irregular nail trimming (p = 0.01), sharing of nail clippers (p = 0.03), and prolonged use of closed footwear (p = 0.02). no significant associations were observed with gender or age. onychomycosis is prevalent among university students, with dermatophytes as the leading causative agents. inadequate nail care and poor hygiene practices play a critical role in the occurrence of diseases. health education and preventive interventions should be prioritized to reduce infection rates in young adults. further studies employing molecular methods are recommended to refine species identification and improve epidemiological insights. keywords: onychomycosis; dermatophytes; students; nail care; personal hygiene. abbreviations: potassium hydroxide (koh); sabouraud dextrose agar (sda); czapek yeast extract agar (cya); colony forming unit (cfu); polymerase chain reaction (pcr); deoxyribonucleic acid (dna); world health organization (who) introduction onychomycosis, a fungal infection of the nail, is the most common nail disorder worldwide, accounting for up to 50% of all nail diseases and affecting between 2–10% of the general population (gupta et al., 2023). although more prevalent in older adults, the infection is increasingly reported among younger individuals, including university students, largely due to lifestyle factors, communal environments, and inadequate personal hygiene (lee et al., 2023). the condition is caused mainly by dermatophytes such as trichophyton rubrum and t. mentagrophytes, but yeasts and non-dermatophyte molds can also contribute to nail infection (sharma et al., 2024). risk factors include occlusive footwear, nail trauma, improper trimming practices, and the use of shared grooming tools (gupta & foley, 2022). in student populations, these behaviors are common, yet systematic data regarding their relationship with onychomycosis remain limited. although onychomycosis is a superficial infection, it can cause discomfort, cosmetic concerns, and a reduction in quality of life. affected individuals often report embarrassment, self-consciousness, and functional limitations in daily activities (bhatia et al., 2022). however, fewer than 20% of patients with onychomycosis seek medical treatment, frequently due to lack of awareness or the misconception that nail infections are trivial (gupta et al., 2023). given the growing recognition of personal hygiene as a key determinant of skin and nail health, assessing nail care practices in young adults is essential. university students represent a unique group in which high social interaction, shared facilities, and variable self-care https://doi.org/10.14421/biomedich.2025.142.1015-1019 mailto:lisasavitri@unik-kediri.ac.id 1016 biology, medicine, & natural product chemistry 14 (2), 2025: 1015-1019 behaviors may influence the risk of fungal nail infections. understanding these practices can inform preventive strategies and health education programs. therefore, this study aims to examine the prevalence of onychomycosis among students and to investigate its relationship with nail care practices and personal hygiene behaviors. materials and methods study design and setting this study employed a descriptive cross-sectional design conducted between september 2024 and april 2025 at the faculty of health sciences, kadiri university, indonesia. the study aimed to assess the prevalence of onychomycosis among students and to the investigate associations between nail care practices, personal hygiene, and fungal infections. cross-sectional surveys have been widely used in dermatological research to assess the prevalence of fungal diseases and associated behaviors (gupta et al., 2023; lee et al., 2023). study population and sampling the target population was undergraduate students aged 18–25 years. participants were recruited using purposive sampling. inclusion criteria were: (1) students enrolled in the university during the study period, (2) willingness to provide informed consent, and (3) absence of systemic antifungal therapy within the last three months. students with chronic dermatological conditions unrelated to fungal infection, systemic immunosuppression, or severe nail trauma were excluded. a total of 120 students were initially recruited, and 100 participants met the inclusion criteria and completed both the clinical examination and the structured questionnaire. data collection data were obtained in two stages: ▪ clinical examination and sampling each participant underwent a nail examination to identify clinical signs of onychomycosis, including discoloration, thickening, onycholysis, and fragility. suspected nail clippings were collected, treated with 20% potassium hydroxide (koh), and examined microscopically for fungal elements. ▪ questionnaire a structured questionnaire was distributed to collect information on demographic characteristics, nail care habits (frequency of trimming, tool-sharing, footwear use), and hygiene practices (handwashing, drying habits, protective footwear). the instrument was adapted from validated tools used in previous epidemiological studies (bhatia et al., 2022; lee et al., 2023). laboratory identification nail samples showing fungal elements were cultured on sabouraud dextrose agar (sda) containing chloramphenicol and cycloheximide and incubated at 28 ± 2 °c for up to 21 days. colony morphology was assessed macroscopically, and fungal isolates were identified microscopically using lactophenol cotton blue (lpcb) preparations. dermatophytes, yeasts, and non-dermatophyte molds were differentiated according to standard diagnostic protocols (sharma et al., 2024). data analysis data were entered and analyzed using spss version 26.0 (ibm corp., armonk, ny, usa). descriptive statistics (mean, standard deviation, frequency, and percentage) were used to summarize demographic data, prevalence of onychomycosis, and distribution of nail care behaviors. associations between hygiene practices and onychomycosis were tested using the chi-square test, with statistical significance set at p < 0.05. results and discussion participant characteristics a total of 100 students participated in the study, with a mean age of 20.8 ± 1.9 years. of these, 62% were female and 38% were male. the majority (70%) reported trimming their nails weekly, while 30% reported irregular trimming (≥2 weeks). approximately 28% admitted to sharing nail clippers with peers, and 40% reported frequently using closed footwear for more than 8 hours daily. prevalence of onychomycosis out of the 100 examined students, 21 showed clinical signs suggestive of onychomycosis. of these, 18 cases were confirmed by direct koh microscopy and culture, resulting in an overall prevalence rate of 18%. etiological agents among the confirmed cases, trichophyton rubrum was the most frequently isolated species (55.6%), followed by t. mentagrophytes (22.2%), candida albicans (11.1%), and non-dermatophyte molds such as aspergillus spp. (11.1%). this distribution aligns with recent findings that dermatophytes remain the predominant cause of onychomycosis in young adults (gupta et al., 2023; sharma et al., 2024). association between nail care practices and onychomycosis chi-square analysis revealed significant associations between nail care behaviors and onychomycosis the prevalence of onychomycosis. students who reported irregular nail trimming were more likely to develop onychomycosis (p = 0.03). similarly, sharing nail care tools was strongly associated with fungal infection (p = savitri et al. – onychomycosis in students: an overview of nail care 1017 0.01). prolonged use of occlusive footwear also showed a significant relationship (p = 0.04). table 1. prevalence of onychomycosis by risk factors. risk factor n onychomycosis positive (%) p-value nail trimming regular (≤1 week) 70 7 (10.0%) irregular (≥2 weeks) 30 11 (36.7%) 0.03* nail tool sharing no 72 7 (9.7%) yes 28 11 (39.3%) 0.01* footwear use (>8h/day) no 60 6 (10.0%) yes 40 12 (30.0%) 0.04* *significant at p < 0.05 discussion this study identified an overall prevalence of onychomycosis of 18% among university students, with dermatophytes (trichophyton rubrum and t. mentagrophytes) as the predominant pathogens. these findings highlight that fungal nail infections are not only a problem of older populations but are increasingly relevant among younger age groups, especially students in communal environments. previous studies have reported prevalence rates between 10–20% in similar populations, supporting the notion that onychomycosis is an emerging concern in young adults (lee et al., 2023; gupta et al., 2023). the predominance of t. rubrum in this study is consistent with global epidemiological patterns, as this species has long been recognized as the leading cause of dermatophyte-related onychomycosis (gupta & foley, 2022). non-dermatophyte molds such as aspergillus spp. were also isolated, reflecting recent reports of their increasing role in nail infections, particularly in younger and immunocompetent populations (sharma et al., 2024). the presence of candida albicans further supports the growing diversity of etiological agents, which is likely linked to lifestyle factors such as frequent water exposure and the use of occlusive footwear. behavioral risk factors were significantly associated with infection in this study. students who reported irregular nail trimming and sharing of grooming tools had a higher prevalence of onychomycosis. this finding supports the notion that personal hygiene practices have a direct influence on susceptibility to fungal nail infections (bhatia et al., 2022). similarly, the strong association between prolonged use of occlusive footwear and onychomycosis is consistent with previous evidence indicating that warm, moist environments promote fungal growth (lee et al., 2023). these results highlight the importance of preventive behaviors, such as regular nail care and the avoidance of tool sharing, in reducing the risk of infection. the psychosocial impact of onychomycosis should not be underestimated. although often regarded as a cosmetic condition, studies have shown that nail fungal infections negatively affect self-esteem, social interaction, and overall quality of life (bhatia et al., 2022). given that fewer than 20% of affected individuals seek treatment (gupta et al., 2023), raising awareness about early recognition and preventive hygiene is essential in student populations. this study has several implications. first, it highlights the importance of targeted health education programs that focus on nail care and personal hygiene among university students. preventive campaigns could integrate awareness sessions into student health services, promoting safe nail care practices and discouraging the sharing of personal grooming tools. second, considering the increasing diversity of fungal pathogens, laboratory confirmation should remain a standard practice to ensure accurate diagnosis and treatment. however, this study has limitations. the crosssectional design prevents the establishment of causal relationships between hygiene practices and onychomycosis. additionally, the sample size was limited to a single university, which may reduce generalizability. future studies with larger and more diverse populations, combined with molecular diagnostic methods, are recommended to understand epidemiological patterns in young adults better. this study highlights that onychomycosis is relatively prevalent among university students, with an infection rate of 18%. the majority of isolates were dermatophytes, particularly trichophyton rubrum and t. mentagrophytes, which is consistent with previous findings identifying these species as the dominant agents of onychomycosis worldwide (gupta, venkataraman, & hall, 2023). although dermatophytes remain the leading cause, the isolation of candida albicans and aspergillus spp. underscores the increasing role of non-dermatophyte fungi in nail infections, especially in individuals with compromised hygiene (bonifaz et al., 2022). the association between poor nail care habits and onychomycosis observed in this study is supported by earlier reports. for example, sharing nail clippers has been shown to facilitate fungal transmission in community settings, particularly among young adults who often engage in communal living or sports activities (lee, park, & kim, 2023). similarly, irregular nail trimming and prolonged use of occlusive footwear create a humid microenvironment favorable for fungal growth (zhou, li, & wang, 2022). our results emphasize that personal hygiene practices, rather than demographic factors such as age and gender, are stronger determinants of infection risk. interestingly, the prevalence of onychomycosis in this study (18%) is lower than that reported in some international studies. for instance, caplan et al. (2023) 1018 biology, medicine, & natural product chemistry 14 (2), 2025: 1015-1019 reported a 23% prevalence among north american college students, while marbaniang et al. (2025) documented rates as high as 28% in india. differences in climate, lifestyle, footwear habits, and diagnostic methodologies may account for this variability. nonetheless, the detection of onychomycosis in nearly one-fifth of students suggests a need for increased awareness and preventive measures. the identification of t. rubrum as the predominant species aligns with global epidemiology. this species has been recognized for its adaptability and persistence in keratinized tissues, as well as its ability to cause recurrent infections (fuller, hay, & arenas, 2024). the emergence of terbinafine-resistant trichophyton indotineae, reported in asia, europe, and north america (mctaggart et al., 2025), raises concerns regarding antifungal treatment efficacy. although t. indotineae was not isolated in this cohort, its presence in neighboring regions warrants continuous surveillance in student populations. the study’s strength lies in its focus on a young population often neglected in fungal epidemiology research. however, limitations should be noted. first, fungal identification relied on morphological methods without molecular confirmation, which may limit the accuracy to the species level. second, the study was conducted at a single institution, which restricts its generalizability. future research should incorporate molecular diagnostics and multicenter sampling to improve epidemiological understanding and guide antifungal stewardship. overall, this study demonstrates that onychomycosis remains a significant public health concern among students. preventive interventions, including education on personal hygiene, safe nail care practices, and avoidance of shared tools, are essential to reduce fungal transmission in young adults. in conclusion, this study confirms that onychomycosis is a significant health issue among university students, with behavioral factors such as irregular nail trimming, sharing of tools, and prolonged use of occlusive footwear as major contributors. strengthening awareness of nail hygiene and preventive practices is crucial for reducing the burden of onychomycosis in student populations. conclusions this study demonstrated that onychomycosis is a relevant health problem among university students, with a prevalence of 18%. the majority of infections were caused by dermatophytes, particularly trichophyton rubrum, followed by t. mentagrophytes, candida albicans, and non-dermatophyte molds such as aspergillus spp.. the findings highlight that irregular nail trimming, sharing of nail care tools, and prolonged use of occlusive footwear are significant behavioral risk factors contributing to infection. these results emphasize the importance of personal hygiene and nail care practices in preventing fungal nail infections among young adults. educational programs and awareness campaigns at the university level are strongly recommended to promote preventive behaviors, reduce the incidence of onychomycosis, and improve student well-being. further research with larger populations and advanced diagnostic methods, including molecular identification, is necessary to confirm these findings and provide a more comprehensive understanding of onychomycosis in young adults. acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there are no funding. references bhatia, a., sharma, r., & gupta, a. k. (2022). quality of life impact of onychomycosis: a cross-sectional analysis. journal of dermatological treatment, 33(6), 3102–3108. https://doi.org/10.1080/09546634.2021.2005687 bonifaz, a., cruz-aguilar, p., moreno-coutiño, g., rivaslarrauri, f., & ponce-olivera, r. (2022). onychomycosis by non-dermatophyte molds: epidemiology, clinical presentation, and treatment. medical mycology, 60(7), myac056. https://doi.org/10.1093/mmy/myac056 caplan, a., peterson, k., chen, l., & hill, d. (2023). prevalence and risk factors of onychomycosis in north american college students. journal of fungi, 9(4), 412. https://doi.org/10.3390/jof9040412 fuller, l. c., hay, r. j., & arenas, r. (2024). advances in dermatophytosis: epidemiology, diagnosis, and management. clinical dermatology, 42(2), 145–153. https://doi.org/10.1016/j.clindermatol.2023.10.004 gupta, a. k., & foley, k. a. (2022). evidence-based management of onychomycosis. dermatologic therapy, 35(5), e15384. https://doi.org/10.1111/dth.15384 gupta, a. k., venkataraman, m., & hall, d. a. (2023). epidemiology and management of onychomycosis: update 2023. journal of the european academy of dermatology and venereology, 37(3), 457–467. https://doi.org/10.1111/jdv.18632 gupta, a. k., versteeg, s., & shear, n. h. (2023). onychomycosis: recent advances in epidemiology, diagnosis, and treatment. journal of fungi, 9(5), 559. https://doi.org/10.3390/jof9050559 lee, j. h., park, s. h., & kim, y. s. (2023). risk factors and lifestyle determinants of tinea pedis and onychomycosis savitri et al. – onychomycosis in students: an overview of nail care 1019 among students. mycopathologia, 188(5), 671–679. https://doi.org/10.1007/s11046-023-00777-1 lee, j. h., park, s. y., & kim, d. h. (2023). risk factors and prevalence of onychomycosis among young adults in communal settings. mycopathologia, 188(1), 45–54. https://doi.org/10.1007/s11046-022-00680-2 marbaniang, e., sangma, m. m., singh, t. s., & sharma, b. (2025). trichophyton indotineae and the rise of terbinafine resistance in india: clinical and epidemiological insights. journal of medical microbiology, 74(1), 22–29. https://doi.org/10.1099/jmm.0.001732 mctaggart, l. r., richardson, s. e., poutanen, s. m., & summerbell, r. c. (2025). global emergence of terbinafineresistant trichophyton indotineae: implications for treatment. frontiers in cellular and infection microbiology, 15, 1359824. https://doi.org/10.3389/fcimb.2025.1359824 sharma, p., singh, a., & patel, r. (2024). emerging nondermatophyte molds in onychomycosis: clinical significance and diagnostic challenges. medical mycology, 62(2), myac125. https://doi.org/10.1093/mmy/myac125 zhou, j., li, h., & wang, y. (2022). diagnostic challenges and culture methods for onychomycosis: an updated review. mycoses, 65(9), 759–769. https://doi.org/10.1111/myc.13491 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 159-163 | doi: 10.14421/biomedich.2024.131.159-163 issn 2540-9328 (online) the diversity of understory (shrubs and herbs) in the kalikuning area hikmah supriyati1,*, nur aeni ariyanti2 1institute of research and community engagement, uin sunan kalijaga yogyakarta, indonesia. 2biological education department, yogyakarta state university, indonesia. corresponding author* hikmahsupriyati@gmail.com abstract kalikuning is one of the areas that has a diversity of flora. kalikuning's fertile land helps a variety of plants flourish there. since there are no people living here, a wide variety of unidentified and underexplored plants can be known. therefore, more research on the plant diversity is required. the research aims to invent, document, and find out the diversity of the understory (shrubs and herbs) in kalikuning. this research used survey and exploration methods. the researchers documented shrub and herb species. based on the results, 28 families consist of 65 species of shrubs and herbs. those species are flora that have specific characteristics. keywords: kalikuning; shrubs and herbs; understory. introduction kalikuning is located on the southern slopes of mount merapi, in the cangkringan district, sleman regency, yogyakarta. kalikuning, as part of mount merapi national park, has several functions, including the development of knowledge, research, and education. when mount merapi erupted, one of the locations through which the rain lava poured was kalikuning. the kalikuning river area's ecosystem is affected by this. the condition of the region is affected by environmental changes. the presence of one of them makes the area surrounding the volcanic outburst fruitful (ginanjar & ginanjar, 2014). kalikuning's rich region set off the growth of a variety of flora. the kalikuning area is dominated by understory. this is because the kalikuning area is an open area, so it is exposed to a lot of sunlight. according to aththorick, (2005), the growth of understory plants is very dependent on sunlight. the more sunlight that penetrates the forest floor, the more it will stimulate the growth of understory vegetation.understory is also often used as an indicator of soil fertility and produces litter to increase soil fertility. apart from ecological functions, understory is also used as food and medicinal plants. however, it is not uncommon for understory to act as a weed that inhibits the growth of young trees, especially in cultivated monoculture plants. the presence of plants on the forest floor can function as a buffer against rainwater and surface runoff, thereby minimizing the danger of erosion (hilwan & mulyana, 2013). understory includes shrubs, herb, and layers of ground cover (soerianegara. i dan a. indrawan, 2008) (soerinagara and indrawan, 2008). herbaceus is a soft trunked plants and contains a lot of water. (muellerdombois & ellenberg, 1974) mention, herbs are plants without woody stems living in ground cover. ferns, graminoids, and forbs are the three groups of herbs. whereas the shrubs is plants that are not large in size, has a small wooden stems and branch close to surface (tjitrosoepomo;, 2013). according to (harris, 1983), usually, shrubs are smaller than trees; shrubs often grow to a height of less than 5 meters. the majority of shrubs contain many buds at the base and along the stems. flora diversity investigation is required for recording and inventorying species genetic variety, learning the distribution of flora, taxonomy, conservation efforts to avoid extinction, learning the molecular structure of plants, and learning secondary metabolites., etc. (nurbani, 2015; (akbar et al., 2019)). this research aims for documenting the diversity of understory (shrubs and herbs) in kalikuning area. research data can be used to investigate a range of plant species, shrubs, and herbs in a systematic and visual manner. the existence of species variety in kalikuning must be communicated in order to demonstrate the richness of flora on the island of java. currently, little is know about plant diversity on java island, including wild species. other than that, the plants found in the kalikuning area have the opportunity to be used as a source of learning biology on plantae material. manuscript received: 27 november, 2023. revision accepted: 20 may, 2024. published: 28 may, 2024. https://doi.org/10.14421/biomedich.2024.131.159-163 mailto:hikmahsupriyati@gmail.com 160 biology, medicine, & natural product chemistry 13 (1), 2024: 159-163 materials and methods the study was conducted by a direct survey in the kalikuning neighborhood of the cangkringan district of yogyakarta's sleman regency. this research was conducted from august 2022 to may 2023 in the kalikuning area. equipment used for observation and data collection consists of a spreadsheet concerning data species, digital cameras, and plants identification book. the identification procedure started with photographic evidence. to make identification easier, the picture shoot concentrated on the morphology of the stems, leaves, and inflorescences. identification was done while also observing the plants in the understory. the photographs that have been gathered have been recognized, and the habitus, which comprises herbaceous, shrubs, has been determined. identification with compared to illustrations in flora of java (backer & brink, 1980), flora (van steenis, c.g.g.j, 1992), and a number of online resources including plansystematics.org, natureloveyou.com, plantnet.com, and others. following identification, the plants were categorized depending on their habitus and classifications. result and discussion this study produced an inventory of the understory on kalikuning. based on life form (habitus) data, which includes bushes in the understory, herbs, and shrubs. this displays the outcome of understory of diversity: figure 1. the distribution of understory species in kalikuning. based on life form (habitus), understory that dominates in kalikuning is herb. the herb consists of 40 species, 25 shrub species. the herb is very easily to find, because the herb has strong competitiveness and high adaptation to surrounding plants (e.g. liana, bushes, shrubs, even trees) so that they can grow in an empty place (diana et al., 2021). botanist defines herb as a plant whose stem does not become woody or persistent, but dies down after flowering (jones, sir francis avery: 1996). according to anaputra et al. (2015), herbs plants are one of the constituent plant types on surfaces in land and water areas and have a smaller size when compared with bushes, trees, and shrubs whose trunks are wet and not woody. the presence of herbs in a forest area plays a very important role. herbs are the early community that plays an important role in stabilizing soils that are sensitive to erosion (rahayu, 2006). figure 2. the famili of understory species in kalikuning. based on the graph, it can be seen that was (shrub and herbs) species with a total of 28 families in kalikuning area. the asteraceae families were understory that has the largest number of species, asteraceae (11 species), poaceae (6 species), lamiaceae (5 species), cyperaceae (5 species), solanaceae (4 species), and fabaceae (4 species). taxonomically, the lower vegetation is generally members of the families poaceae, cyperaceae, herb 62% shrub 38% understory in kalikuning 0 2 4 6 8 10 12 total species in family supriyati & ariyanti – the diversity of understory (shrubs and herbs) in … 161 araceae, and asteraceae. this vegetation is found in many open places, roadsides, riverside, forest floors, and agricultural land (aththorick, 2005). asteraceae family found in kalikuning grows wild in various location. this plant often grows in undercanopies and places that are exposed to direct sunlight. asteraceae is a plant taxon with quite high species diversity. according to (anderson, 1982), plants of the asteraceae tribe are a group of plants consisting of 1,100 genera, including 20,000 species. (lawrence, 1966) stated that this family is the family with the second largest members in the kingdom plantae. the large number of asteraceae families plays a major role in the stability of the ecosystem in kalikuning. this plant plays a role in preventing erosion on mountain slopes. in preventing erosion, this plant has four roles, namely (heyne, 1987). prevents direct collisions of raindrops so that the impact force of the raindrops can be reduced. 2). reduce surface flow velocity and protect against erosion by surface flow. 3). encourage the development of soil biota, which can improve the physical and chemical properties of the soil, and the roots can influence the infiltration capacity of the soil so that surface water flow is reduced. 4). plays a role in increasing soil organic matter and increasing soil resistance to erosion. table 1. plant list of understroty in kalikuning. no name family habitus 1 strobilanthes crispus acanthaceae shrub 2 centella asiatica apiaceae herb 3 catharanthus roseus apocynaceae shrub 4 anodendron paniculatum shrub 5 calotropis sp shrub 6 xanthosoma sagittifolium araceae herb 7 colocasia esculenta herb 8 sphagneticola trilobata asteraceae herb 9 crassocephalum crepidioides herb 10 tagetes erecta herb 11 eupatorium serotinum herb 12 ageratum conyzoides shrub 13 ageratina riparia herb 14 bidens pilosa l. herb 15 centaurea nigra herb 16 echinacea purpurea herb 17 eclipta prostrata herb 18 jaegeria hirta herb 19 impatiens walleriana balsaminaceae herb 20 impatiens flaccida herb 21 sisymbrium irio brassicaceae herb 22 kalanchooe pinnata crassulaceae herb 23 cyperus esculentus cyperaceae herb 24 kyllinga nemoralis herb 25 carex sylvatica herb 26 cyperus esculentus l. herb 27 kyllinga brevifolia herb 28 dennstaedtia punctilobula dennstaedtiaceae herb 29 equisetum debile equisetaceae herb 30 euphorbia milli euphorbiaceae shrub 31 euphorbia pulcherrima shrub 32 manihot esculenta shrub 33 leucaena sp fabaceae shrub 34 gliricidia sepium shrub 35 acacia decurrens shrub 36 mimosa pudica shrub 37 hypericum androsaemum hypericaceae herb 162 biology, medicine, & natural product chemistry 13 (1), 2024: 159-163 table 1. cont. no name family habitus 38 salvia accidentalis lamiaceae herb 39 clerodendrum paniculatum shrub 40 origanum sp. herb 41 satureja montana herb 42 stachys sylvatica herb 43 rhynchosia minima leguminosae herb 44 melastoma malabathrium melastomataceae shrub 45 clidemia hirta shrub 46 ficus septica moraceae shrub 47 psidium guajava myrtaceae shrub 48 mirabilis jalapa nyctaginaceae shrub 49 phyllanthus urinaria phyllanthaceae herb 50 piper aduncum piperaceae shrub 51 imperata cylindrica poaceae herb 52 pannesistum purpureum herb 53 neyraudia reynaudiana herb 54 panicum repens herb 55 leersia hexandra herb 56 paspalum dilatatum herb 57 persicaria sp polygonaceae herb 58 primula veris primulaceae shrub 59 capsicum frutescens solanaceae shrub 60 solandra grandiflora shrub 61 solanum melongena herb 62 solanum sp shrub 63 duranta erecta verbenaceae shrub 64 lantana camara shrub 65 vitis discolor vitaceae herb based on table 1, it can be seen that certain species of family poaceae are more common in the kalikuning area, like imperata cylindrica, pannesistum purpureum, neyraudia reynaudiana, panicum repens, leersia hexandra, and paspalum dilatatum. these types of plants are poaceae plants, which have high adaptability, wide distribution, are able to grow on dry land, and like sunlight (aththorick, 2005). the open conditions cause many types of the poaceae family to grow in kalikuning. open locations cause high wind speeds, which enable the spread of seeds from the poaceae family. the light seeds of the poaceae family are easily carried by the wind, so they spread easily and then form new plants (aththorick, 2005). taxonomically, the cypreaceae family is also a type of family that is often found in forest floor vegetation. this finding is in accordance with the results of research that has been carried out, which shows that many types of cypreaceae are also found in the kalikuning area. several species from the cypreaceae family in the kalikuning area are cyperus esculentus, kyllinga nemoralis, carex sylvatica, cyperus esculentus l., and kyllinga brevifolia. the cyperaceae family has almost the same ecological characteristics as poaceae, but because of its clumpy nature, its distribution is uneven. the poaceae and cyperaceae tribes have high adaptability, wide distribution, and are able to grow in dry or flooded land (rukmana, 1999). conclusions the diversity of understory (herbs and shurbs) in kalikuning area consists of 65 species and 28 families. the asteraceae families were understory that has the largest number of species in kalikuning. asteraceae is a plant taxon with very high species diversity. based on the results of the data collection, there was 40 species of herbaceous, and 25 species of shrubs. according to the life form (habitus), the plants that dominate kalikuning were herbs. the presence of herbs in a forest area plays a very important role. herbs are an early community that plays an important role in stabilizing soils that are sensitive to erosion. conflict of interest: the authors declare that they have no conflicts of interest. supriyati & ariyanti – the diversity of understory (shrubs and herbs) in … 163 references akbar, d. z., widodo, w., & agustina, t. (2019). the diversity of understory (shrubs and herbs) in mount of nglanggeran. proceeding international conference on science and engineering, 2, 45–51. https://doi.org/10.14421/icse.v2.53 anderson, w. r. (1982). an integrated system of classification of flowering plants. brittonia, 34(2), 268–270. https://doi.org/10.2307/2806386 aththorick, t. a. (2005). kemiripan komunitas tumbuhan bawah pada beberapa tipe ekosistem perkebunan di kabupaten labuhan batu. jurnal komunikasi penelitian, 17(5), 42–48. backer, c. a., & brink, r. c. b. van den. (1980). flora of java. springer netherlands. diana, r., mercury, y. h., & hidayah, n. (2021). ekologi tumbuhan herba dan liana. malang: cv. pustaka learning center ginanjar, f., & ginanjar, f. (2014). keanekaragaman genus serangga air di kali kuning kabupaten sleman, d.i yogyakarta [universitas gadjah mada]. http://etd.repository.ugm.ac.id/penelitian/detail/73658 harris, r. w. (1983). arboriculture: care of trees, shrubs, and vines in the landscape (first edition). prentice-hall, englewood, inc. new jersey. heyne, k. (1987). tumbuhan berguna indonesia (cet. 1). yayasan sarana wana jaya: diedarkan oleh koperasi karyawan, departemen kehutanan. http://catalog.hathitrust.org/api/volumes/oclc/21826488.html hilwan i, mulyana d, pananjung wd. 2013. keanekaraaman jenis tumbuhan bawah pada tegakan sengon buto (enterolobium cyclocarpum griseb.) dan trembesi (samanea saman merr.) di lahan pasca tambang batubara pt kitadin, embalut, kutai kartanagara kalimantan timur. jurnal silvikultur tropika, 4(1):6–10. lawrence, g. h. m. (1966). taxonomy of vascular plants. new york: macmillan campany. mueller-dombois, d., & ellenberg, h. (1974). aims and methods of vegetation ecology. new york: wiley international edition. rahayu, w. (2006). suksesi vegetasi di gunung papandayan pasca letusan tahun 2002. skripsi departemen manajemen hutan fakultas kehutanan institut pertanian bogor. http://repository.ipb.ac.id/handle/123456789/46009 rukmana, h. r. (1999). gulma dan teknik pengendalian. jakarta: kanisius. soerianegara. i dan a. indrawan. (2008). ekologi hutan indonesia. laboratorium ekologi hutan. fakultas kehutanan. institut pertanian bogor. steenis, c.g.g.j van . (1992). flora (cet. 6). jakarta: pt. pradya paramita tjitrosoepomo;, g. (2013). taksonomi tumbuhan (spermatophyta) (yogyakarta). gadjah mada university press. //lib.umpalembang.ac.id/libfp/index.php?p=show_detail&id=4508&ke ywords= this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 389-395 | doi: 10.14421/biomedich.2024.132.389-395 issn 2540-9328 (online) antimicrobial activities of oyster mushroom (plerotous ostreatus), garlic (allium sativum), and ginger extracts (zingiber officinale) against some clinical isolates victoria oluwapelumi adenuga1,*, soji fakoya1, joseph adaviruku sanni2, akinola adenuga3, ore-ofe oluwatoyin adenuga4, oluwaferanmi timileyhin ajayi1 1department of biological sciences, school of science, olusegun agagu university of science and technology, ondo state, nigeria. 2department of biochemistry, faculty of science, adekunle ajasin university akungba-akoko, ondo state, nigeria. 3department of medical microbiology, university of benin, benin-city, ondo state, nigeria. 4department of medical microbiology, achievers university, owo, ondo state, nigeria. corresponding author* adenugavictoriaa@gmail.com manuscript received: 18 june, 2024. revision accepted: 30 september, 2024. published: 04 october, 2024. abstract the prevalence of multiple drug resistance among human pathogenic bacteria necessitates a continual search for new antimicrobial medicines, particularly among plants that are frequently farmed or naturally found in our surroundings. the study aims to investigate the inhibitory effect of garlic, ginger, and mushroom extracts at different concentrations against some clinical isolates. anti-bacterial components from mushrooms, garlic, and ginger were extracted with hot water, cold water, ethanol, and acetone, and their anti-bacterial activity was determined using the agar well diffusion method. zones of inhibition were observed primarily on hot water extracts of mushrooms (pleurotus ostreatus) on culture plates inoculated with staphylococcus aureus, escherichia coli, and klebsiella pneumonia at 370c for 24 hours. the cold water extracts of the mushroom (pleurotus ostreatus) gave the highest zone of inhibition of 14.0±1.0mm when used against s. aureus. for spices, the cold water extracts yielded the highest zones of inhibition of 22.0±1.0mm followed by 16.0±1.0mm as observed with ginger. the results obtained have shown clearly that the mushrooms (pleurotus ostreatus), garlic (allium sativum), and ginger (zingiber officinale) extracts contain phytochemicals with some antimicrobial activities. the water extracts of the mushrooms and spices showed broad-spectrum antimicrobial activity much more than ethanol and acetone extracts. the antimicrobial activities of mushroom and garlic extracts were highly effective against the bacterial pathogens studied. however, the antimicrobial activity of the ginger extract was poor. to address the multi-drug resistance to antibiotics, i recommend: that bioactive compounds found in mushrooms, ginger, and garlic be patented and used as alternative antimicrobials. keywords: antimicrobial agents; clinical isolates; drug resistance; mushrooms; garlic; ginger. introduction traditional medicine has historically used a variety of medicinal plants to treat drug-resistant disorders (curto et al., 2021). as a result, infectious diseases remain a significant concern for human health (frieri et al., 2017). therefore, the continued expansion of multidrugresistant bacteria, and rising antibiotic overuse, underscore the need for alternate agents. many communities have long utilized herbs and spices to treat various diseases, including antimicrobials (dog, 2006; leja & czaczyk, 2016; dini, 2018). herbs and spices, unlike conventional antibiotics, are typically regarded as safe for humans due to their long history of use in food preparation (akullo et al., 2022). these plants are classified as natural products and represent an important source of innovative natural medications. the prevalence of multiple drug resistance among human pathogenic bacteria necessitates a continual search for new antimicrobial medicines, particularly among plants that are frequently farmed or naturally found in our surroundings (anyamaobi et al., 2020). furthermore, studying the dietary traditions of various ethnic groups worldwide has significantly impacted the development of research into natural products such as mushrooms, garlic, and ginger. their use as an alternative treatment for numerous diseases has over during the last few decades. (vuorelaa et al. 2004; ansari et al. 2006). herbs and spices cause fewer adverse effects than manufactured medications. they are affordable, have greater patient tolerance, and are widely available to low-income people (adeshina et al., 2011; emmanuel et al., 2021). this has led to the discovery of a link between the chemical structure of molecules found in natural products, their biological properties, and their significance to human health (emmanuel et al., 2021). https://doi.org/10.14421/biomedich.2024.132.389-395 mailto:adenugavictoriaa@gmail.com 390 biology, medicine, & natural product chemistry 13 (2), 2024: 389-395 however, most of these natural chemicals with antibacterial potential have yet to be validated in terms of their purported effects, albeit medicines based on them may be created (goncagul et al., 2009). several studies have been published on herbs and spices’ antibacterial and antifungal activities. still, nothing is known regarding the precise mechanism of their antimicrobial action (gur et al., 2006; pattaratanawadee et al., 2006; yusha'u et al., 2008; belguith et al., 2010; poeloengan, 2011; gull et al., 2012). ginger (zingiber officinale) is widely distributed across the tropics of asia, africa, america, and australia, where it is used as a spice and medicinal plant (jung & pezzuto, 2002). numerous investigations have revealed the antibacterial properties of ginger and garlic (akintobi et al., 2013; indu et al., 2006; khashan, 2014; mohammed et al., 2019). since ancient times, mushrooms have been considered delicacies high in proteins, vitamins, minerals, and fiber. furthermore, mushrooms have been extensively utilized in traditional medicine and are effective antibacterial agents (kabelik, 2018). the medicinal activities of mushrooms are attributable to various bioactive biomolecules such as polysaccharides, high and low molecular weight compounds, glycoproteins, and immune-modulating (bolzani et al., 2006). the fruiting bodies and mycelium of mushrooms exhibit healthpromoting values such as immuno-stimulatory, antibacterial, and anti-oxidative properties (gull et al., 2012). the simultaneous impact of these medications would have beneficial therapeutic effects. however, most of these natural products, which have indicated antimicrobial potentials, have yet to be validated for their claimed activities and possibly develop drugs from them (goncagul et al., 2009). ginger (zingiber officinale) ginger, a rhizome that is white to yellow and very aromatic, has lots of beneficial uses in medicine for the treatment of high blood pressure, cold-induced diseases, asthma, persistent cough, and other related problems of the respiratory system, and in the flavoring of foods (kumar et al., 2011). there is a broad spectrum of antibacterial activity of ginger rhizome against a range of gram-negative and grampositive bacteria (mohammed et al., 2019). despite this, there are different opinions about ginger's antimicrobial properties against microorganisms from various sources (abdallah & abdallah, 2018). garlic is an onion with a distinctive spicy, pungent flavor that has long been utilized for culinary and medicinal purposes. garlic is an antioxidant and can help treat the common cold and cardiovascular problems. in addition, garlic's antimicrobial activities are linked to some bioactive compounds (tsao & yin, 2001). because infectious diseases have a high morbidity and mortality burden, many studies have been conducted better to understand their control, treatment, and prevention (murugaiyan et al., 2022). when some edible mushrooms, garlic, and ginger come into touch with them, they either suppress or kill various microbes. some examples include s. aureus, e. coli, b. cereus, and k. pneumoniae. this current work, therefore, evaluated the antibacterial properties of extracts of edible mushrooms (oyster mushroom) and two spices (ginger and garlic) against staphylococcus spp., e. coli, and klebsiella spp. the study’s objective is to investigate the inhibitory effect of garlic, ginger, and mushroom extracts at different concentrations against some clinical isolates, and determine the sensitivity of pathogenic organisms to various antimicrobial compounds as treatment options for patients. materials and method materials mushroom, ginger, garlic, nutrient agar, blood agar, mueller hinton agar, wire loop, forceps, bijou bottles, distilled water, stainless steel cork borer, test tubes, aluminum foil, test tube rack, filter papers, sterile cotton swab sticks, cotton wool, hand gloves, marker, nose mask, weighing balance. sample collection garlic (figure 1) and ginger (figure 2) were bought from the ore market, odigbo local government in ondo state, nigeria. it was placed in sterile polyethylene bags and kept at a temperature of 4 ºc until use and pleurotus ostreatus (figure 3) (was collected from odo lawe farm at odeerinje, okitipupa local government in ondo state). the materials were brought to the laboratory and preserved at room temperature. all practical work was done at the medical microbiology laboratory of state specialist hospital, okitipupa, ondo state. mr. david, a medical laboratory technician (mlt) at state specialist hospital, okitipupa authenticated the samples. test organisms pure e. coli, s. aureus, and k. pneumoniae cultures were taken at the state specialist hospital's medical microbiology laboratory in okitipupa, ondo state, nigeria. the cultures were sub-cultured to resuscitate them in the laboratory and kept at 4o c as stock culture. adenuga et al. – antimicrobial activities of oyster mushroom (plerotous ostreatus) … 391 figure 1. dried pleurotus ostreatus. figure 2. allium sativum. figure 3. zinigiber officinale. extraction of mushrooms, ginger and garlic the extraction was performed using faqir and farhan’s procedure (2011). the respective mushroom, ginger, and garlic samples were cut into bits, sun-dried for 6 days, and then pulverized into powder using an electric grinder. 20 g each of the pulverized mushroom, ginger, and garlic were weighed and soaked in 200 ml of cold water, hot water, ethanol, and acetone, respectively. it was allowed to stand for 7 days only with interval shaking at 30 minutes (this is to prevent mold from growing in the extraction water). the filtrates were poured into different well-labeled beakers and evaporated at 37°c for 72 hours using a water bath. the extracts were collected and a 10 mg/ml stock solution was produced. stock solutions were generated at 10 mg/ml concentrations, and different concentrations were created by serially diluting the stock solution. sensitivity test mueller hinton agar and nutrient agar plates were prepared using kirby bauer’s method. fresh inoculums of the test organisms were added to the media using the pour-plate method. the control was determined using pure solvent instead of the extracts. the plates were incubated at 37 0c for 24 hours in the incubator, and the zone of inhibition in diameter was measured using the nearest millimeter (mm), as shown in plate 1. statistical analysis quantitative data was reported as mean ± sd. the data obtained were analyzed using sas’s variance analysis (anova) procedure of sas (statistical analysis software). results and discussion results the inhibitory effect of mushroom, garlic, and ginger extracts as shown in table 2, the formal antibiotic used displayed a pronounced inhibitory effect against e. coli with a 30.0 mm diameter as the zone of inhibition among all the tested organisms. at the same time, the result revealed that ethanolic extracts of ginger showed the highest antimicrobial activity when used against k. pneumoniae with a zone of inhibition diameter of 26.0 mm and most minor activity when used against s. aureus and e. coli with a zone of inhibition diameter of 17.0 mm and 15.0 mm respectively. the result of activity in this study is similar to previous reports by gull et al. (2012), riaz et al. (2015), and anyamaobi et al. (2020). acetone extract from mushrooms showed the highest inhibition against e. coli with a zone of inhibition diameter of 24.0 mm, ; no antimicrobial activity was observed against s. aureus and k. pneumoniae . this is in agrees with the result of adeshina et al. (2011), which showed high susceptibility of escherichia coli but no activity against staphylococcus aureus. however, garlic showed the highest antimicrobial activity against s. aureus, with a zone of inhibition diameter of 16.0 mm and minor activity against e. coli and k. pneumoniae, each producing a zone of inhibition of 11.0 mm. in comparison, ginger showed the highest antimicrobial activity against e. coli with a zone of inhibition diameter of 16.0 mm and most minor activity against s. aureus and k. pneumoniae with a zone of inhibition diameter of 13.0 mm and 11.0 mm, respectively. on the other hand, hot water extract of mushrooms showed the highest antimicrobial activity against s. aureus with a zone of inhibition diameter of 30.0 mm and minor activity when used against e. coli with a zone of inhibition diameter of 10.0 mm. at the same time, there was no zone of inhibition against k. pneumonia. however, hot water garlic extract showed the highest antimicrobial activity when used against s. aureus with a zone of inhibition diameter of 25.0 mm and minor activity when used against e. coli with a zone of inhibition diameter of 13.0 mm with no zone of inhibition against k. pneumoniae. results also revealed that cold water extracts of garlic showed the highest inhibition when used against s. aureus with a zone of inhibition diameter of 22.0 mm and minor activity when used against e. coli and k. pneumoniae with a zone of inhibition diameter of 11.0 mm and 13.0 mm, respectively. however, mushrooms showed the highest antimicrobial activity when used 392 biology, medicine, & natural product chemistry 13 (2), 2024: 389-395 against s. aureus with a zone of inhibition diameter 14. 0 mm and most minor activity when used against e. coli and k. pneumoniae, each producing zone of inhibitions diameter 10.0 mm. details of the results can be seen in table 1 below. for the minimum inhibitory concentration, all the tested organisms could grow at the concentration of 10 and 20 mg/ ml of pleurotus ostreatus, allium sativum, and zingiber officinale, as shown in table 3. none of the tested isolates could grow at 100 mg/ml concentrations. also, all test isolates except k. pneumoniae grew at a concentration of 60 mg/ml. key: aethanol extract of garlic; bcold water extract of garlic; chot water extract of garlic; d-pure solvent figure 1. minimum inhibitory concentration of garlic extract bacterial isolate. table 1. minimum inhibitory zones of extracts of mushroom, ginger, and garlic against test organisms using agar well diffusion method. samples solvent s. aureus e. coli k. pneumniae control pleurotous ostreatus ethanol 11.0±0.6 11.0±i.5 0.00 acetone 24.0±1.0 0.00 hot water 30.0±1.0 11.0±1.0 0.00 cold water 14.0±1.0 0.00 allium sativum ethanol 15.0±1.0 11.0±1.5 24.0±1.0 0.00 acetone 15.0±0.7 11.0±1 .5 11.0±1.5 0.00 hot water 25.0±1.5 13.0±1.0 0.00 cold water 22.0±1.0 11.0±1.5 13.0±1.0 0.00 zingiber officinale ethanol 17.0±0.7 15.0±1.0 26.0±1.0 0.00 acetone 13.0±1.0 16.0±0.5 11.0±1.5 0.00 hot water 13.0±.01.0 11.0±1.5 0.00 cold water 16.0±1.0 11.0±1.5 11.0±1.5 0.00 table 2. inhibition zones of standard antibiotics (mm) against test organisms. isolates ciprofloxacin augmentin cephalexin gentamycin s. aureus 15.0±0.3 20.0±0.6 15.0±0.3 25.0±0.3 e. coli 20.0±0.6 30.0±0.3 7.01±0.5 30.0±0.3 k. pneumoniae 20.0±0.6 20.0±0.6 7.01±0.5 0.0±0.00 table 3. minimum inhibitory concentration of mushroom, garlic, and ginger extracts at different concentrations. samples solvent 100 mg/ml 60 mg/ml 20 mg/ml 10 mg/ml pleurotous ostreatus s. aureus + + + e. coli + + k. pneumoniae + + allium sativum s. aureus + + + e. coli + + + k. pneumonia + + zingiber officinale s. aureus + + + e. coli + + + k. pneumonia + + key: means no growth, + means there is growth. adenuga et al. – antimicrobial activities of oyster mushroom (plerotous ostreatus) … 393 discussion in this study, garlic extract showed a wide inhibition zone, followed by combining garlic, mushroom, and ginger extract. the results obtained in this study explained the relatively higher therapeutic efficacy of the plant materials on both gram-positive and gramnegative bacteria. the antimicrobial properties of garlic primarily rely on the presence of allicin, a thiosulfate compound found in crushed garlic bulbs (wallockrichards et al., 2014), as well as antibacterial sulfur biocompounds like alliin and alliinase (mardomi, 2017; singh, 2018). on the other hand, ginger’s antimicrobial activity can be attributed to its essential oil or oleoresins (beristain-bauza et al., 2019), as well as phenolic compounds such as eugenol, shogaols, zingerone, gingerdiols, and gingerols interact synergistically with other chemicals such as β-sesquiphellandrene, ciscaryophyllene, zingiberene, α-farnesene, and αand βbisabolene (mardomi, 2017; mara-teles et al., 2020). as a result, there is an urgent need for developing new and effective medications against present antibiotic-resistant bacteria. baulori et al. (2016). this study investigated the antibacterial activity of mushrooms, garlic, and ginger using agar well diffusion. the findings suggested that extracts from mushrooms, garlic, and ginger have antibacterial capabilities, as fasidi (2010) reported. these natural products have promising therapeutic use against human pathogens such as bacteria, fungi, and viruses. variations in the degree of antimicrobial activity of mushrooms were also observed in this study. this result agrees with the report of akyuz et al. (2010) and jangadish et al. (2009). the broad spectrum of mushroom activity was also brought to light as the extracts of mushrooms showed an inhibitory effect on clinical isolates used for this investigation. these suggest that the bioactive products in mushrooms are in concentrations that exude varying degrees of antimicrobial activity. furthermore, the study revealed that the bacterial isolates (staphylococcus aureus and escherichia coli) showed more sensitivity to mushroom extracts (table 1). also, extracts from garlic and ginger, as indicated by (table 1), have a wide spectrum of antimicrobial activity with varying degrees of sensitivity when used against clinical isolates. however, the data from table 1 revealed that extracts from garlic and ginger had antibacterial against all tested bacterial isolates except k. pneumoniia in hot-water garlic extracts and e. coli in hot-water ginger extracts. this finding is by the findings of (belguith, 2010; gull, 2012). the results obtained in this study explained the relatively higher therapeutic efficacy of the plant materials on both gram-positive and gram-negative bacteria. however, gram-positive bacteria demonstrated more sensitivity than gram-negative bacteria. this is in collaboration with the findings of (anyamaobi et al. 2020). the sensitivity of isolates to mushroom, garlic, and ginger extracts implies that the intrinsic substance in the extracts is unknown to the microorganisms, making it impossible to resist. also, when the effects of ethanol, acetone, cold water, and hot water on mushrooms, garlic, and ginger are compared regarding inhibition of microbial growth, the result showed that the hot water and ethanolic extracts had greater inhibiting effects than acetone and cold water. ethanol and hot water extracts of the antimicrobial compounds contained higher bioactive substances than cold water extract,; this is in collaboration with the findings of (baulori et al., 2016). the result showed that the zone of inhibitions exhibited on agar plates by the mushroom, garlic, and ginger extracts were concentration-dependent. the higher the concentration of the extracts, the larger the zone of inhibition produced, as seen in plate 2 above. numerous studies have been conducted with extracts of various plants, screening antimicrobial activity and discovery new antimicrobial compounds (kabelik et al., 2018). therefore, medicinal plants are finding their way into pharmaceuticals and food supplements (murugaiyan et al., 2022). however, it is important to note that the antimicrobial activity of garlic and ginger can vary depending on factors such as chemical composition, extraction solvent, methodology, and processing techniques (singh, 2018). there are several advantages to using spices derived from plant origins as dietary supplements or alternative medicine, manifested by a reduction in the chances of developing antibioticresistant bacteria emerging due to maintaining antibiotic use. natural compounds, such as mushrooms and two spices, are among the most often used antimicrobial agents in food and have been utilized for thousands of years to treat various health issues, including infectious disorders. besides, medical herbs may decrease the cost of treatment and minimize the development of adverse drug reactions. the garlic extract, ginger extract, mushroom extract, and their combination may prevent infection with pathogenic organisms and support their resistance against antibiotics. conclusion multi-drug-resistant organisms are becoming more common, posing a threat to treating an increasing number of infectious diseases. this study sheds more light on using plant-derived foods such as mushrooms, garlic, and ginger as alternative medicine or dietary supplements. this study concludes that mushrooms, garlic, and ginger contain significant antimicrobial compounds. more research is needed to investigate all of the antimicrobial compounds found in these natural products and their side effects. to address the issue of multi-drug resistance to antibiotics, i recommend that bioactive compounds found in mushrooms, ginger, and garlic be patented and used as alternative antimicrobials and also to focus more light on medicinal herbs to clarify their advantages and clinical applications to obtain low-cost treatment and prevents recurrent infections. 394 biology, medicine, & natural product chemistry 13 (2), 2024: 389-395 acknowledgments: we acknowledge prof. s. fakoya for his contribution to the manuscript’s success. authors’ contributions: sf and voa conceptualized the study. voa, carried out data collection. jas, aa, ota, and ooa assisted with the manuscript’s writing. sf assisted with editorial work. sf supervised the project. the authors have read and approved the final manuscript. funding: the author(s) received no financial support for the research, authorship, and publication of this article. availability of data and materials: not applicable ethics approval and consent to participate: not applicable consent for publication: not applicable. competing interests: all authors declare no competing interests. references abdalla, w. e. & abdallah, e. m. 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(2008). in vitro inhibitory activity of garlic and ginger extracts on some respiratory tract isolates of gram-negative organisms. international journal of biomedical health science, 4: 5760. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 33-36 | doi: 10.14421/biomedich.2025.141.33-36 issn 2540-9328 (online) correlation of allergic rhinitis patient questionnaire with skin prick test results at saiful anwar regional hospital iriana maharani1, melody audria kurniadi2 1department of otolaryngology-head and neck surgery, division of rhinology, faculty of medicine, brawijaya university dr. saiful anwar general hospital, east java, indonesia. 2otolaryngology-head and neck surgery resident, department of otolaryngology-head and neck surgery, faculty of medicine, brawijaya university dr. saiful anwar general hospital, east java, indonesia. corresponding author* melodyaudria@gmail.com abstract objective: to determine the optimal cut-off value of the allergic rhinitis patient questionnaire as a diagnostic tool and its correlation with skin prick test (spt) results; methods: the data used were medical records at the ent polyclinic dr. saiful anwar hospital with cross sectional method then the data was processed with the spss application; results.: demographic data are dominated by males (56.3%) with an age range of 19-59 years (74.7%). the most common symptom complained was nasal congestion (71.3%). the trigger of symptoms is dominated by dust allergens (77%). the most common classification of allergic rhinitis (ar) is moderate – severe (50.6%) with visual analogue scale (vas) score > 5 and dominated by complaints of sleep disturbances (57.5%). the optimal limit value of the allergic rhinitis patient questionnaire is 10, where patients with a score value  10 can be assumed to suspect ar. there is a significant correlation between the results of the assessment using the allergic rhinitis patient questionnaire and the results of the spt examination. conclusion: patients with a total score of allergic rhinitis patient questionnaire  10 were assumed to be suspected of ar and the results of the questionnaire assessment correlated with the spt results. keywords: allergic rhinitis; skin prick test; questionnaire; allergic rhinitis patient questionnaire. introduction allergic rhinitis (ar) results from a chronic type 1 hypersensitivity reaction in the nasal mucosa in response to inhaled allergens mediated by immunoglobulin e (ige). ar is not a dangerous disease that can increase morbidity but can cause a decrease in quality of life. prevalence data presented by the international study of asthma and allergies in childhood shows that ar occurs in 10-40% of the world's population, which is around 400 million people, where the prevalence of cases is higher in adults, which is 14.93% of cases and in children 8.38% of the total world population.(garcía et al.,2021) skin prick test (spt) examination is the gold standard examination in establishing the diagnosis of ra. however, the spt examination has several limitations such as the limited number of health workers who are experts in its implementation and the availability of tools and materials so that the development of questionnaires as diagnostic tools is increasingly being developed. (monisha r et al., 2023) the allergic rhinitis patient questionnaire is one of the validated ra questionnaires in indonesian that has been tested for validity and reliability. research on the allergic rhinitis patient questionnaire is still minimal and still needs to be developed further in its use as a diagnostic tool so that researchers feel the need to see the correlation of the allergic rhinitis patient questionnaire with the results of spt examinations in ra patients. (yuliantoputri sr et al., 2022) materials and methods this study is a descriptive-analytical study with crosssectional sampling to see the characteristics of patients diagnosed with allergic rhinitis with positive spt to inhalant allergen examination results based on the "allergic rhinitis patient questionnaire" questionnaire in the period january june 2024 at the ent polyclinic, dr. saiful anwar hospital. the inclusion criteria in this study were patients with suspected ra who underwent examination in the ent polyclinic at dr. saiful anwar hospital from 1st january 2024 until 30th june 2024, patients who had general data, spt examination results and clinical data that were recorded entirely. the exclusion criteria in this study were patients with contraindications for spt examination such as patients manuscript received: 10 november 2024. revision accepted: 17 february, 2025. published: 20 february, 2025. https://doi.org/10.14421/biomedich.2025.141.33-36 34 biology, medicine, & natural product chemistry 14 (1), 2025: 33-36 taking antihistamines, antidepressants or  blocker within 7 days before the examination, patients with skin disorders such as dermatographism and patients who are uncooperative during the examination. all data related to the correlation of spt examination and the “allergic rhinitis patient questionnaire” questionnaire will be processed analytically using the statistical package for the social sciences (spss) program. the spss method used in determining the optimal limit value of the “allergic rhinitis patient questionnaire” is the receiver operating characteristic (roc) curve and the correlation of diagnostic results using the “allergic rhinitis patient questionnaire” with the results of the spt examination is tested using the chi-square method. results and discussion characteristics of allergic rhinitis patients medical record data collection was carried out crosssectionally on the medical records of patients registered at the ent clinic from 1 january 2024 to 30 june 2024. the total number of patients examined during this period was 132 patients, of which 87 patients had positive spt results for inhalant allergen and 45 patients had negative spt results for inhalant allergen. all respondents met the research inclusion criteria. patients’ demographic data in this study will be presented in tabular form (table 1). of the total respondents with positive spt results, male sufferers are more dominant than female sufferers, with a percentage of 56.3%. there are some controversies regarding the gender that dominates ra in adulthood. the theory that can explain this condition is the absence of a significant difference in gender prevalence in ra sufferers in adulthood (18-79 years). most respondents with positive spt results in this study were aged 19-59 (74.7%). the results of this study are based on the "allergic march" theory, where ra begins to occur in individuals over 17 years of age who previously had a history of allergies such as atopic dermatitis or a history of food allergies during infancy or childhood. (rosen c et al., 2023) the characteristics of ra patients in this study are presented in table 2. in the study subjects, the most common symptom complained of by ra patients was nasal congestion, which was 71.3% of the total study subjects. other complaints felt by the study subjects were followed by symptoms of a runny nose (64.4%), sneezing (59%), itchy nose (37%) and complaints of red, itchy and watery eyes (23%). this is in accordance with a study conducted by seung-no hong et al., in which the most common symptom complained of by ra patients was nasal congestion.6 the allergen that most often triggers allergy symptoms is dusty places (77%), and the allergen that most often triggers symptoms is pollen (3.4%). the results of this study are in accordance with the study conducted by sheila et al., where house dust is the most common inhalant allergen that causes ra in indonesia. (yuliantoputri sr et al., 2022) more than half of the total number of research samples felt the frequency of symptoms felt more than 4 weeks and occurred consecutively, where as many as 13 people (15%) of the total respondents experienced persistent allergic rhinitis symptoms where symptoms were felt for 4 days a week and more than 4 weeks in a row. as many as 57.5% of sufferers felt their sleep quality was disturbed because of the symptoms felt. this study’s results align with research conducted by jiaomei liu et al., which looked at the correlation between ra patients and sleep disorders. the results of the study showed a significant correlation between ra patients and sleep disorders.(liu j et al., 2020) another study found that sleep disturbances occurred in 1 in 4 respondents due to the allergy symptoms they experienced.(dykewicz ms et al., 2020) this is certainly correlated with daily activities and the sufferer's participation in daily activities (49.4%) and the sufferer's participation at school or work (42.5%). ra symptoms are felt to be troublesome in more than half of the study sample (56.3%) (katel p et al., 2021) the results of this study indicate a disturbance in the quality of life in ra patients. research conducted by widuri also provided similar results where there was a decrease in the quality of life in 54.35% of the total research sample.(widuri a et al., 2020) the vas score assessment of 52.9% of respondents also gave a figure > 5, where a vas score assessment > 5 indicates that allergic rhinitis symptoms are not controlled and have disrupted the sufferer's quality of life. optimal limit value of allergic rhinitis patient questionnaire in determining the optimal cutoff value of the allergic rhinitis patient questionnaire, the researcher used the receiver operating characteristic (roc) curve method in spss. based on table 4, the diagnostic value is presented which is the result of the intersection of the variables from the total score of the allergic rhinitis patient questionnaire where patients with a positive ra category are used as the determination. the roc curve image shows the location of the intersection determination, by looking at the point of the line closest to the upper left corner, namely sensitivity 1 and 1 specificity of 0. (nahm fs., 2022) the optimal threshold value obtained to assess the intersection of the total score of the allergic rhinitis patient questionnaire is 9.5, where the sensitivity number of the test is 0.690 and the 1specificity number is 0.178. this illustrates that the score value  9.5 is included in the positive ra category, and score value < 9.5 is included in the harmful ra category. however, because there is no decimal value in the calculation of the questionnaire score, then according to statistical maharani & kurniadi – correlation of allergic rhinitis patient questionnaire … 35 science, the optimal limit value taken is 10. the results of the interpretation with an optimal limit value of 10 are respondents with a score value of the allergic rhinitis patient questionnaire  10 shows that the respondent can be assumed patient is suffering from ra and vice versa where respondents with a score < 10 can be assumed that the respondent does not suffer from ra. the optimal cutoff value shows a sensitivity of 69.0%, and a specificity of 82.2% (1 – 0.178). the area under curve (auc) figure on the roc curve of this study is 0.868. the auc value in this study is 0.86 where the auc value above 0.80 indicates that the test results can have very good clinical utility. (parody s et al., 2022) correlation of allergic rhinitis patient questionnaire with skin prick test examination the researcher used a correlation test with the chi square method in spss to see the relationship between the diagnostic value of the allergic rhinitis patient questionnaire and the results of the spt examination. (table 5.) the hypothesis used in this study is if h0: there is no significant relationship between the variables and if h1: there is a significant relationship between the variables. if the significance value (p-value) in the correlation test ≤ 0.05 then h0 is rejected and if the significance value (p-value) in the correlation test > 0.05 then h0 is accepted. of the 87 respondents with positive spt results, 60 respondents gave a score of  10 on the allergic rhinitis patient questionnaire and 27 respondents gave a score of < 10 on the allergic rhinitis patient questionnaire. of the 45 respondents with negative spt results, 37 respondents gave a score of < 10 on the allergic rhinitis patient questionnaire and 8 other respondents gave a score of > 10 on the allergic rhinitis patient questionnaire. the data were then analyzed using the chi square method and obtained a p-value smaller than α (0.000 < 0.050). the results of this analysis can be interpreted that there is a significant relationship between the diagnostic assessment results using the allergic rhinitis patient questionnaire and the spt examination, which is the gold standard examination in establishing a diagnosis of ra. the positive predictive value (ppv) figure in this study was 88.2% (positive), while the negative predictive value (npv) figure obtained was 57.8% (negative). the percentage of category accuracy in this study was 73.5%, indicating that the results of this study are reliable. some limitations in this study include the standardization and clinical testing of allergen extracts used in spt examinations at the ent polyclinic at saiful anwar hospital, which are limited because the allergens produced by the soetomo pharmacy laboratory which are not freely traded. another weakness of this study is the limited age range of respondents, as the majority of the study samples were adult patients. this limitation occurs because spt examinations at saiful anwar hospital, in addition to being carried out at the ent allergy polyclinic, can also be carried out at the pediatrician, so this study is less able to represent and correlate the results of research on pediatric patients with suspected ra. the third weakness of this study is that this study does not evaluate risk factors that influence the incidence of ra such as anatomical factors and comorbidities. therefore, further, more comprehensive research is expected to be able to conduct further evaluations. conclusions the gender that dominates patients with ra in this study is male (56.3%), where the age of the majority of respondents is adult, namely 19-59 years old (74.7%). the most common symptom complained of by ra sufferers is nasal congestion (71.3%), with the allergen that most often triggers symptoms, which is dust (77%). the most common ra classification is moderate-severe symptoms (50.6%) which are dominated by complaints of sleep disorders (57.5%). the vas score assessment is dominated by a score of > 5. the optimal limit value of the allergic rhinitis patient questionnaire is 10 where in patients with a score of  10 can be assumed that the patient is suffering from ra while patients with a questionnaire score <10 can be assumed that the patient does not suffer from ra. there is a significant correlation between the results of diagnostic assessment using the allergic rhinitis patient questionnaire with the results of the spt examination which is the gold standard examination in establishing a diagnosis of ra. competing interests: the authors state that there are no competing interests. references dykewicz ms, wallace d v., amrol dj, baroody fm, bernstein ja, craig tj, et al. rhinitis 2020: a practice parameters update. journal of allergy and clinical immunology. 2020 oct 1;146(4):721–67. garcía-almaraz r, reyes-noriega n, del-río-navarro be, berber a, navarrete-rodríguez em, ellwood p, et al. prevalence and risk factors associated with allergic rhinitis in mexican school children: global asthma 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clinicians. korean j anesthesiol. 2022 feb 1;75(1):25–36. parody s,verda d, bagnasco f, muselli m. the clinical meaning of the area under a receiver operating characteristic curve for the evaluation of the performance of disease markers. epidemiol health. 2022 oct 17;44:e2022088. rosen c. bailey's head and neck surgery: otolaryngology. 6th ed. shahidi f, editor. vol. ii. philadelphia: wolters kluwer; 2023. rana r,singhal r. chi-square test and its application in hypothesis testing. journal of the practice of cardiovascular sciences. 2015;1(1):69. secco rosário c, alves cardozo c, chong neto hj, rosário filho na. do gender and puberty influence allergic diseases? allergol immunopathol (madr). 2021 mar 1;49(2):122–5. widuri a, retno ningrum w. correlation between score for allergic rhinitis and quality of life in chronic rhinitis patients. vol. 51. yuliantoputri sr, sudiro m, dermawan a, lasminingrum l, mahdiani s. validity and reliability of the indonesian version of patient allergic rhinitis questionnaire and allergic rhinitis prevalence in a class of 2018-2019 medical students of padjadjaran university, indonesia. international journal of integrated health sciences. 2022 sep 30;10(2). biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 193-197 | doi: 10.14421/biomedich.2025.141.193-197 issn 2540-9328 (online) cytotoxicity and isolation of betullinic acid from anthonotha noldeae stem bark rejoice atiko1, idriss b. musba’u2,3, andrew kwaji1, bemgba b. nyakuma2, philip pius1, ibrahim m. umar1, adam i. muhammad1 1department of chemical sciences, gombe state university, p. m. b. 127 gombe, gombe state, nigeria. 2department of chemical sciences, north-eastern university, p. m. b. 0198 gombe, gombe state, nigeria. 3centre for african medicinal plants research (campre), north-eastern university, gombe, p. m. b. 0198 gombe, gombe state, nigeria. corresponding author* musbauudriss@gmail.com manuscript received: 18 january, 2025. revision accepted: 15 may, 2025. published: 13 june, 2025. abstract the cytotoxicity and isolation of betullinic acid from anthonotha noldeae stem bark were examined in this study. the stem bark was extracted using methanol (meoh) via the cold maceration technique. the meoh produced a crude extract of 125.18 g (6.78%), which was further divided into n-hexane, dichloromethane (dcm), and ethyl acetate (etoac) fractions with yields of 10.23%, 11.74%, and 12.22%, respectively. cytotoxicity assays using brine shrimp lethality tests revealed that the dcm and etoac fractions exhibited substantial concentration-dependent toxicity, achieving 100% mortality at 500–1000 μg/ml. the lc50 values for the dcm and etoac fractions were 16.33 μg/ml and 28.64 μg/ml, respectively. column chromatography of the fractions led to the isolation of the compound m1 (5 mg), characterized using ftir, 1h nmr, and 13c nmr spectroscopy. ftir analysis identified functional groups consistent with carboxylic acids and alkenes, while nmr spectroscopy confirmed the presence of six methyl groups, vinyl protons, hydroxyl groups, and carboxyl functionalities. the compound was identified as the bioactive triterpene known as betulinic acid. the findings provide novel insights into the phytochemistry of anthonotha noldeae, highlighting its potential as a source of bioactive compounds and application in traditional medicine. keywords: anthonotha noldeae; stem bark; betulinic acid; cytotoxicity; bioactive compounds. introduction traditional african medicine encompasses diverse practices beyond herbal remedies, including dietary modifications, therapeutic fasting, and physical therapies. these practices often reflect deep cultural understanding and are integrated into social contexts (aftab & rehman, 2021; tella, 1979; sofowora,1984). traditional medicine heavily relies on plant-derived secondary metabolites (psm), which are structurally diverse and possess diverse pharmacological activities. historically, psm has been pivotal in the creation of numerous modern drugs, including pain relievers (e.g., codeine and morphine), and antimalarials (e.g., quinine) (mendoza & silva, 2018; wilson & deering, 2019). plants are a valuable source of potential antimicrobial agents, but their efficacy and safety are essential factors to consider (prestinaci et al., 2015). cytotoxicity assays, especially the brine shrimp lethality assay (bsla), play a crucial role in assessing the safety of plant-derived antimicrobial compounds. these assays assess potential toxicity to living cells, identifying promising candidates with minimal side effects and paving the way for the development of effective antimicrobial therapies (celik, 2018; davey, 2021). anthonotha is an understudied african genus with 17 species predominantly found in the guineo-congolian region. traditional uses of these plants include treating various ailments such as cancer, aphrodisiac issues, and skin infections (oyedemi et al., 2018). while limited research exists, phytochemical analyses of some species, particularly a. macrophylla and a. cladantha, have revealed the presence of alkaloids, tannins, flavonoids, and terpenoids (yakubu & olutoye, 2016; bongmo et al.,2023). a. macrophylla extract has promising anticancer and anti-estrogenic properties (yakubu & olutoye, 2016). however, most anthonotha species remain poorly understood, with limited scientific investigation into their chemical composition and pharmacological potential. there is no such report on anthonotha noldeae and other anthonotha species. additionally, a. noldeae was reportedly used in traditional medicine to treat pneumonia in cattle (nodza et al., 2022). https://doi.org/10.14421/biomedich.2025.141.193-197 mailto:musbauudriss@gmail.com 194 biology, medicine, & natural product chemistry 14 (1), 2025: 193-197 experimental methodology collection, preparation of plant, extraction, and fractionation the stem bark of the plant was collected from taraba state in nigeria and identified by a botanist in the department of botany, gombe state university. the stem bark was air-dried under shade at room temperature and then milled to powder (kwaji et al., 2023). the powdered plant material was kept under dry and cool conditions until required for extraction. the stem bark (about 1.85 kg) was extracted by exhaustive cold maceration in methanol for 7 days with regular shaking at intervals to ensure thorough extraction. the extract was filtered using whatman no. 1 filter paper into a clean and dry container. the crude extract was concentrated on a rotary evaporator at 45 °c under reduced pressure. the concentrate was allowed to dry at room temperature under shaking to obtain a crude extract which was covered with aluminium foil and stored in a refrigerator until required. the methanol crude extract of anthonotha noldeae (100 g) was partitioned with nhexane, dcm, and etoac successively by suspending 20 g of the extract in 10% aqueous methanol in a 500 ml separating funnel, and 250 ml of the solvent. the mixture was swirled and allowed to stand until two distinct layers were formed. the lower layer was drained through the tap of the separatory funnel and the upper layer was poured into a separate container. this procedure was repeated 3 times, for each solvent type to ensure increased yield (al-matani et al., 2015; yohanna et al., 2021). isolation procedure the most active etoac extract fraction was dissolved in methanol, pre-adsorbed onto silica gel, and dried. column packing was done using the wet slurry method. gradient elution was carried out with n-hexane/dcm and dcm/etoac at a 5% increase in the volume of the eluting solvent to yield several fractions. tlc was used in monitoring the fractions. the compound was purified using a recrystallization procedure after washing impurities from fractions with a single spot (kwaji et al., 2018). 180 fractions were obtained and pulled together based on tlc value. an isolate labelled m1 with rf values 0.58 in hexane:ethyl acetate (4:1) was obtained. cytotoxicity assay incubation of nauplii eggs, preparation of stock, and working solution artemia salina eggs were hatched in a sterile brine solution by dissolving 20 grams of sodium chloride in 1 litre of distilled water, adjusting the ph to between 8.0 and 8.5 using a 1 molar solution of sodium hydroxide (naoh), and aerating continuously for 36-48 hours. actively motile nauplii were then collected for the assay. working solutions were prepared from the 2 mg/ml stock solutions by appropriate dilutions of each extract with approximately 4.5 ml of the brine solution to obtain 1,000 µg/ml and serially diluted to 1 µg/ml from stock solutions (akano & akinsomisoye, 2024). brine shrimp in vitro cytotoxicity analysis ten artemia salina nauplii were transferred via pasteur pipette into 6 test tubes each containing 4.5 ml of brine solution. each tube contained 0.5 ml of a specific test solution. a positive control containing potassium dichromate (k2cr2o4) was included (kwaji et al., 2023). after a 24-hour incubation period, the number of surviving nauplii in each tube was quantified, and the percentage mortality was calculated. the median lethal concentration (lc50), the concentration of the test solution that resulted in the mortality of 50% of the nauplii population after 24 hours of exposure, was determined graphically (akano & akinsomisoye, 2024). results and discussion cytotoxicity studies the cytotoxic properties of extracts were evaluated using bsla due to the sensitivity of brine shrimp (artemia spp) to toxic substances (hassan et al.,2024). the results demonstrated an apparent concentration-dependent effect of the test substance on nauplii mortality. this trend was most pronounced at the higher concentrations of 1000 and 500 μg/ml, where 100% mortality (10 dead nauplii each) was observed for both etoac and dcm and vice versa (suryawanshi et al., 2020). a mortality of 80% (8 dead nauplii) at 250 µg/ml, 70% (7 dead nauplii) at 125 µg/ml, 60% (6 dead nauplii) at 62.5 µg/ml, and 20% (2 dead nauplii) at 1 µg/ml the etoac fraction. a similar trend was observed in the dcm fraction, 70% (7 dead nauplii) at 250 µg/ml, 60% (6 dead nauplii) at 125 µg/ml, and 62.5 µg/ml, and 10% (1 dead nauplii) at 1 µg/ml. a similar trend was reported by (akano & akinsomisoye, 2024). the cytotoxicity for both fractions is represented in figure 1a. figure 1a. %mortality of brine shrimp nauplii in dcm and etoac fractions. atiko et al. – cytotoxicity and isolation of betullinic acid … 195 figure 1b. lc50 of dcm and etoac fractions. the toxicity of the extracts was also evaluated using lc50 values and compared to meyer’s and clarkson’s toxicity indices. the meyer’s index classified extracts with lc50 < 1000 μg/ml as toxic and those with lc50 ≥ 1000 μg/ml as non-toxic (meyer et al., 1982). clarkson’s index classified extracts as non-toxic (lc50 > 1000 μg/ml), low toxic (500 μg/ml ≤ lc50 ≤ 1000 μg/ml), medium toxic (100 μg/ml ≤ lc50 < 500 μg/ml), and highly toxic for lc50 < 100 μg/ml (clarkson et al., 2004). the lc50 of the fractions was analyzed by extrapolation from the graph equation. the lc50 for etoac and the dcm fractions were 16.33 μg/ml and 28.64 μg/ml, respectively. the etoac fraction is more toxic than the dcm fraction. this is evident in that it required a lower concentration of 16.33 μg/ml than the dcm fraction which required 28.64 μg/ml to cause the death of 50% of the nauplii. although the etoac fraction was more toxic than the dcm fraction, both could be classified as strongly toxic based on meyer’s and clarkson’s toxicity indices. the lc50 of both fractions is represented in figure 1b. spectroscopic analysis of isolated compounds the isolated compound was characterized via fourier transform infrared (ft-ir), and nuclear magnetic resonance (1hnmr and 13cnmr) spectroscopic methods. ft-ir analysis of m1 the compound was obtained as a white powder with an underlying pale yellow colour (5mg) with a melting point of 297 299 °c. the ft-ir spectra of m1 in figure 2 were identified by correlating with the standard wavenumber (cm-1) given in the literature (nandiyanto et al., 2019). the m1 spectra revealed a broad peak between 3500 and 3200 cm-1 associated with the o-h stretch, and the absorption peak at 1387.16 corresponds to c=o stretching vibrations found in carboxylic acids. the isolated compound also showed peaks at 2914.80 cm-1 and 2847.43 cm-1 aligned with c-h (sp3) stretching vibrations for methyl and methylene groups, respectively. the bending vibrations at 1172.93 cm-1 could also serve as a supporting guide for the methylene absorption observed at 2847.43 cm-1. another prominent peak was observed at 1734.05 cm-1 corresponding to c=o in carboxylic acid. an olefinic stretching (c=c bond vibration) was observed at 1620.46 cm-1 as a weak peak. figure 2. ft-ir spectrum of m1. 1h nmr and 13c-nmr analysis of m1 the 1h nmr (600 mhz, chloroform-d) spectrum of m1 showed peaks at δ 4.74 (d, j = 1.3 hz, 1h), 4.61 (s, 1h), 3.19 (dd, j = 10.2, 3.5 hz, 1h), 2.94 (td, j = 10.8, 4.9 hz, 1h), 2.35 (t, j = 7.5 hz, 1h), 2.22 2.12 (m, 2h), 2.03 -1.92 (m, 2h), 1.69 (s, 3h), 1.06 (d, j = 7.4 hz, 6h), 0.97 (d, j = 5.0 hz, 7h), 0.94 (s, 3h), 0.82 (s, 3h), 0.75 (s, 3h), 0.69 (d, j = 9.4 hz, 1h). the 1h-nmr spectrum showed peaks typical of a triterpene nucleus. the peaks correspond to 6 methyl groups in ppm at δ 0.75 (3h, s), 0.82 (3h, s), 0.94 (3h, s), 0.97(6h, d), and 1.69 (3h, s). similarly, a pair of singlets at δ 4.61 and 4.74 containing one h each due to the vinyl protons at carbon 29 were observed. the presence of a doublet with one proton intensity at δ 3.19 (dd, j = 10.2, 3.5 hz) could be attributed to the proton attached to the carbon bearing the hydroxyl group at c-3. the chemical shifts indicate that compound m1 is a lupane-type triterpene typical of betulinic acid (ogunmoye et al., 2017). this submission is supported by comparing the 1h nmr data with several works of literature. some studies have focused on the chemical shifts of the methyl groups at δ 0.75, 0.82, 0.94, 0.9, and 1.69 along with protons at δ 4.74 and 4.61. furthermore, the chemical shift reported for betulinic acid from a. cladantha supports the identification of m1 as betulinic acid (bongmo et al.,2023; ogunmoye et al., 2017; dais et al., 2017). the 1h nmr of m1 is given in figure 3. figure 3. 1h nmr spectrum of m1 196 biology, medicine, & natural product chemistry 14 (1), 2025: 193-197 13c-nmr analysis of m1 the 13c-nmr (600 mhz, chloroform-d) spectrum of m1 showed peaks at δ 14.6 (1c, s), 15.4 (1c, s), 16.016.2 (2c, 16.0 (s), 16.2 (s)), 18.3 (1c, s), 19.5 (1c, s), 21.2 (1c, s), 25.2 (1c, s), 27.6-27.9 (2c, 27.6 (s), 27.9 (s)), 30.1 (1c, s), 31.0 (1c, s), 34.2-34.3 (2c, 34.2 (s), 34.3 (s)), 36.9 (1c, s), 37.8 (1c, s), 38.0 (1c, s), 38.538.9 (2c, 38.5 (s), 38.9 (s)), 41.1 (1c, s), 42.1 (1c, s), 46.6 (1c, s), 48.6 (1c, s), 50.4 (1c, s), 55.3 (1c, s), 55.9 (1c, s), 78.8 (1c, s), 110.7 (1c, s), 148.8 (1c, s), 178.9 (1c, s). the observed signals correspond to six methyl groups (ch3) at c-23, c-24, c-25, c-26, c-27, and c-30. eleven methylene groups (ch2) were observed at c-1, c2, c-6, c-7, c-11, c-12, c-15, c-16, c-20, c-21, and c29 while 6 methine groups (ch) were observed at c-3, c-5, c-9, c-13, c-18, c-19. additionally, 1 carbonyl carbon (c=o) at c-28, and 6 quaternary carbons at c-4, c-8, c-10, c-14, c-17, and c-22 were also observed. the characteristic signals of c-oh and c=o (for cooh) at 78.8 and 178.9 for carbons c-3 and c-28 respectively are typical of betulinic acid (bongmo et al.,2023; ogunmoye et al., 2017; dais et al., 2017). based on the literature available, this is the first phytochemical investigation of a. noldeae leading to the isolation and cytotoxicology of betulinic acid. betulinic acid is a lupane-type triterpene with a molecular formula of c30h50o3 and a molecular weight of 456.7 g/mol. betulinic acid is a promising alternative treatment for viral infections like hiv and selected cancers (e.g., melanoma, leukaemia, and prostate). it has antiinflammatory properties that reduce inflammation in conditions like arthritis. it acts as an antioxidant to safeguard cells from oxidative stress damage potentially reducing ageing. betulinic acid has been extensively researched for its antibacterial, antimalarial, and antiparasitic properties (ghaffari et al., 2012; jiang, et al., 2021). the 13c nmr of m1 is given in figure 4. figure 4. 13c-nmr spectrum of m1 the proposed structure of compound m1 (betulinic acid (lup-20(29)-en-28-oic acid) is depicted in figures 5a and 5b, showcasing the number and unnumbered carbons, respectively, after extensive analysis and literature comparisons. (a) (b) figure 5. m1-betulinic acid (lup-20(29)-en-28-oic acid) conclusion the study demonstrated that a. noldeae (rossberg) exell & hillc is a potential source of therapeutic agents for anti-cancer drugs. the compound isolated, previously found in several plants, has widely documented antiretroviral, anti-cancer, anti-inflammatory and antioxidant potentials. however, this is the first report of the phytochemical investigation and cytotoxic activity leading to the isolation of betulinic acid from the stem bark of a. noldeae (rossberg) exell & hillc. consequently, this study has made a significant contribution to the fields of natural product chemistry and pharmacology. the authors opine that the discovery of betulinic acid a. noldeae could open novel therapeutic applications, targeting cancer and protecting against oxidative stress. overall, this research underscores the importance of exploring diverse plant species for bioactive compounds. atiko et al. – cytotoxicity and isolation of betullinic acid … 197 competing interests: the authors declare that there are no competing interests. references aftab, t. & rehman, k. h. 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cornsilk; dpph method; fermented milk; lactic acid bacteria (lab); yogurt. introduction in recent decades, the prevalence of chronic noncommunicable diseases (ncds), including obesity, diabetes mellitus, and cardiovascular conditions, has risen significantly (wilkins et al., 2019). modern lifestyles, characterized by the excessive use of antibiotics, poor dietary choices, sedentary behavior, and stress, have contributed to imbalances in gut microbiota. these imbalances are increasingly recognized as key factors in the onset and progression of many ncds (gomaa, 2020; illiano et al., 2020). furthermore, a growing body of evidence links these diseases to deficiencies in antioxidant defenses, highlighting the importance of antioxidants in managing oxidative stress and preventing disease progression (niemann et al., 2017). in response, there is a growing interest in natural antioxidants derived from functional foods, with research focusing on combining different food sources to maximize their health benefits and multifunctionality. yogurt, a widely consumed functional food, is valued not only for its pleasant taste and nutritional profile but also for its potential therapeutic benefits. it has been shown to support digestive health and may play a role in cancer prevention. additionally, studies have demonstrated that yogurt fermented with lactobacillus bacteria exhibits higher antioxidant activity than raw milk. research has reported dpph scavenging antioxidant activity in yogurt ranging from 47.85 to 60.67 mg/ml (54.26 ± 6.41 mg/ml) (pereira et al., 2013). as such, yogurt represents a safe and effective source of natural antioxidants, offering significant potential for health promotion. elkhadragy et al. (2019) further emphasized yogurt’s role as a probiotic, with the potential to modulate oxidative stress and liver fibrosis in schistosoma mansoni-infected rats. corn silk, the stigma of corn flowers, is a byproduct of corn production, typically discarded as waste during the processing of corn for food and animal feed. recent studies, however, have revealed that corn silk possesses a variety of pharmacological properties, including antioxidant, anti-apoptotic, hypoglycemic, and anticancer activities (li et al., 2023). for instance, corn silk has demonstrated dpph scavenging antioxidant activity of 92.6% at a concentration of 1.6 mg/ml (ebrahimzadeh et al., 2008). furthermore, research by guo et al., highlighted the potential of corn silk polysaccharides in reducing diabetes, pointing to its promise as a functional food ingredient (guo et al., 2019). given the antioxidant-rich profile of corn silk, it holds considerable potential as an additive to enhance the nutritional value of yogurt. this study aims to explore the antioxidant activity and microbial characteristics of https://doi.org/10.14421/biomedich.2025.141.389-393 390 biology, medicine, & natural product chemistry 14 (1), 2025: 389-393 yogurt enriched with corn silk extract. it is anticipated that this research will not only introduce a novel functional yogurt variant but also contribute to enhancing the economic value of corn silk, a largely underutilized agricultural byproduct. materials and methods materials and equipment the primary material used in this study was corn silk, which was sourced from batang hari ogan village, tegineneng, pesawaran. the bacterial starter cultures employed for yogurt production were lactobacillus bulgaricus (fncc 0041), streptococcus thermophilus (fncc 0015), and lactobacillus plantarum (fncc 0026), obtained from the fncc collection at the food and nutrition study center, gadjah mada university. the medium used for culture rejuvenation and stock culture preparation was de man, rogosa, and sharpe (mrs) agar (oxoid). for cryopreservation, 20% (v/v) glycerol and 10% (w/v) skim milk were used as cryoprotectants. the mrs agar contained 100 ppm nan3 and 100 ppm caco3. for acidity testing, 1% phenolphthalein (pp) indicator and 0.1 n naoh were used. the materials for antioxidant testing included 2,2diphenyl-1-picrylhydrazyl (dpph) (merck) and ethanol. the equipment used during the study included an autoclave (all american b0004146), an incubator (memmert), a ph meter (hanna), a vortex (scilogex), a laminar airflow (telstar), a freezer (toshiba), an analytical balance (and), a spectrophotometer (milton roy 21d), titration apparatus, and a micropipette (eppendorf). starter culture preparation to prepare the bacterial starter cultures, 1 ml of each bacterial culture was transferred into test tubes containing 10 ml of sterilized mrs medium, which was autoclaved at 121°c under 15 psi for 15 minutes. the inoculated mrs medium was then incubated at 37°c for 24 hours. to prepare the starter, 10% (v/v) of the inoculum was added to 100 ml of sterilized 18% (w/v) skim milk, which was sterilized at 110°c under 13 psi for 10 minutes. this mixture was incubated for 12 to 18 hours until curd formation was observed, resulting in the mother starter. a 10% (v/v) portion of the mother starter was further inoculated into 100 ml of sterilized 18% (w/v) skim milk and incubated for another 12 to 18 hours to produce a bulk starter. the starter was inoculated directly into milk for fermentation or stored at 10°c for future use. cornsilk preparation corn silk was first sorted, washed, and dried to a moisture content of ≤ 10% using an oven at 50°c for 150 minutes. to prepare the corn silk extract, 5 g of the dried corn silk was added to 200 ml of distilled water. the mixture was heated to a boil for 5 minutes and then filtered, following the method outlined by caleja et al. (caleja et al., 2016). milk fermentation fresh cow’s milk was pasteurized at 85°c for 30 minutes and cooled to 40°c. two groups of fermented milk were prepared: the control group (without corn silk addition) and the experimental group (with corn silk addition). for the control, 100 ml of pasteurized milk was mixed with 100 ml of skim milk, which was prepared by dissolving 3 g of skim milk powder in 100 ml of distilled water. for the corn silk-enriched yogurt, 100 ml of pasteurized milk was mixed with 100 ml of skim milk, where the skim milk was prepared by dissolving 3 g of skim milk powder in 100 ml of boiled corn silk extract. a 3% (v/v) starter culture was added to each group, and the mixture was incubated at 37°c for 18 hours. after fermentation, the yogurt was stored at 4°c for further analysis. ph measurement the ph of the yogurt samples was measured using a ph meter (hanna). prior to measurement, the instrument was calibrated with ph 4 and ph 7 buffer solutions. the sample was placed in a beaker, and the electrode was immersed. the ph value was recorded after the reading stabilized. the electrode was then rinsed with distilled water, dried, and recalibrated with the standard buffer solutions, as described by aoac (aoac, 2023b). acidity acidity was determined using mann’s acid test method. a 10 ml sample was placed in an erlenmeyer flask, and 3 drops of phenolphthalein (pp) indicator were added. the sample was titrated with 0.1 n naoh. acidity was then calculated using the formula (aoac, 2023a): acidity (%) = volume of naoh × 90 volume of sample × 1000 × 100% (1) where 90 refers to the molecular weight of lactic acid. total lactic acid bacteria the total lab in the fermented milk was quantified using the pour plate method with mrs agar containing 100 ppm nan3 and 100 ppm caco3. lab, capable of dissolving calcium carbonate, formed clear zones on the agar, indicating their presence. serial dilutions of the sample were prepared using 0.85% nacl. a 1 ml aliquot from the selected dilution was inoculated onto 15 ml of mrs agar. the plate was spread with a sterile drygalski spatula, and after solidification, it was incubated at 37°c for 24 hours. colonies were counted based on the clear zones formed, with the acceptable colony count ranging from 30 to 300, as described by hosseini & behbahani (hosseini & behbahani, 2021). marya et al. – enhancement of probiotic viability and antioxidant activity … 391 antioxidant activity antioxidant activity was assessed using the dpph method (gurkan et al., 2019). a 1.0 ml sample solution (500 ppm in ethanol) was added to each test tube, along with 1 ml of ethanol (as the blank). to each tube, 3 ml of 0.16 mm dpph solution in ethanol was added, and the mixture was vortexed. the tubes were incubated in the dark for 30 minutes. absorbance was measured at 517 nm using a spectrophotometer. the antioxidant activity was calculated using the following formula: antioxidant activity (%) = (1absorbance of the sample at 517 nm absorbance of the blank at 517 nm ) × 100% (2) statistical analysis this study employed a laboratory experimental design with descriptive analysis. sensory testing was conducted using a hedonic test, where panelists rated the products based on aroma, sour taste, texture, and overall acceptability. the sensory data were analyzed using analysis of variance (anova) followed by duncan’s multiple range test (dmrt). results and discussions the analysis of the yogurt drink, with and without the addition of corn silk, yielded the data summarized in table 1. table 1. total lactic acid bacteria (lab), ph, acidity, and antioxidant activity of yogurt without and with corn silk addition. parameters yogurt without corn silk yogurt with corn silk total lab (cfu.ml-1) 1.9 × 107 1.2 × 108 ph 4.5 4.4 lactic acid (%) 0.93 1.34 antioxidant activity (%) 47 54 total lactic acid bacteria (lab) the total lab count in yogurt without corn silk was 1.9 × 10⁷ cfu.ml⁻¹, while yogurt with corn silk exhibited a significantly higher count of 1.2 × 10⁸ cfu.ml⁻¹. both values meet the codex standard for yogurt, which requires a minimum of 10⁷ cfu.ml⁻¹. the increased lab count in yogurt with corn silk suggests that the addition of corn silk positively influences the fermentation process. this enhancement may be attributed to corn silk's high content of essential minerals such as ca, mg, cu, zn, k, na, and fe, as well as its complex carbohydrates and soluble fibers that serve as additional nutrients for the lactic acid bacteria (raghuvanshi et al., 2024; rahman & wan rosli, 2014). corn silk can be a prebiotic, further promoting lab growth during fermentation (raghuvanshi et al., 2024). moreover, l. plantarum in the starter culture is known to enhance the growth of both lactobacillus species and streptococcus thermophilus, and it also suppresses the growth of harmful bacteria like escherichia-shigella, which likely further contributes to the observed increase in lab (zhang et al., 2024). ph and acidity the ph of yogurt with corn silk was 4.4, slightly lower than the 4.5 recorded for the yogurt without corn silk. in addition, the yogurt with corn silk exhibited a higher total acidity (1.34%) than the control (0.93%). these differences can be attributed to the additional nutrients in corn silk, such as soluble fibers and sugars, which provide energy to the lab, supporting their metabolic activity and consequently increasing the production of lactic acid. the higher acidity and lower ph observed in the yogurt with corn silk suggest an enhanced fermentation process, which likely contributed to the more pronounced sour taste of the yogurt. this relationship is consistent with the fact that lab convert lactose into lactic acid, reducing the ph and increasing the acidity during fermentation (wang et al., 2021). antioxidant activity fresh cow’s milk typically has an antioxidant activity of approximately 38.7% (balakrishnan & agrawal, 2014). the fermentation process, however, can enhance this antioxidant capacity. in the present study, yogurt with corn silk demonstrated an antioxidant activity of 54%, compared to 47% in the yogurt without corn silk. this 7% increase indicates that the addition of corn silk boosts the antioxidant properties of the yogurt. the synergistic effect between the antioxidant compounds in corn silk and the metabolites produced by lab, such as lactic acid, exopolysaccharides, and bioactive peptides, likely contributes to this enhancement (gurkan et al., 2019). therefore, corn silk not only enhances the nutritional quality of yogurt but also contributes to its functional health benefits, including increased antioxidant activity. organoleptic test the organoleptic test assessed the taste, aroma, and overall acceptability of the yogurt, as presented in table 2. 392 biology, medicine, & natural product chemistry 14 (1), 2025: 389-393 table 2. average organoleptic scores of yogurt with and without the addition of corn silk. treatment taste aroma acceptability cornsilk yogurt 4.00 ± 0.40 4.07 ± 0.27 1.15 ± 0.37 yogurt control 4.00 ± 0.00 4.00 ± 0.00 1.07 ± 0.27 the taste test aimed to assess the level of acidity and the acceptability of fermented milk produced with and without the addition of corn silk. the acidity levels were categorized into five criteria: 1) very not sour, 2) not sour, 3) slightly sour, 4) sour, and 5) very sour. results indicated no significant differences between the two treatments. overall, the fermented milk products were rated as sour, a characteristic attributed to the activity of lactic acid bacteria. these bacteria metabolize lactose into lactic acid, lowering the milk's ph and creating the distinctive sour taste. the aroma test evaluated panelists' preference and acceptability of the fermented milk drink's aroma. the criteria for aroma preference included: 1) very dislike, 2) dislike, 3) slightly like, 4) like, and 5) very like. anova analysis of aroma characteristics revealed no significant differences between the treatments. the addition of corn silk extract had no impact on aroma acceptance, likely because the amount of corn silk added was minimal and did not significantly alter the aroma profile. the overall acceptability of fermented milk products was analyzed, with results presented in table 2. anova analysis showed no significant differences (p > 0.05) in the acceptability of yogurt fermented with corn silk compared to yogurt without corn silk. panelists provided neutral ratings for the acceptability of both samples. conclusions adding corn silk to yogurt positively influences the fermentation process, enhancing the growth of lactic acid bacteria (lab) and boosting the antioxidant activity of the final product. the total lab count in yogurt with corn silk was significantly higher than in the control, meeting the codex standard for yogurt. the presence of corn silk contributed to an increase in acidity and a slight reduction in ph, indicating a more active fermentation and stronger sour taste. additionally, the antioxidant activity of yogurt with corn silk was 7% higher than the control, demonstrating the nutritional benefits imparted by corn silk's bioactive compounds. organoleptic testing revealed no significant differences in taste, aroma, or overall acceptability between the two yogurt variants, suggesting that corn silk's inclusion does not negatively impact consumer preference. overall, adding corn silk improves the functional quality of yogurt, making it a valuable ingredient for enhancing both nutritional and health benefits. authors’ contributions: arizki tri nopitariyani designed the study, performed the laboratory work, and drafted the manuscript. dina tri marya supervised the research and finalized the manuscript. all authors reviewed and approved the final version. competing interests: there is no competing interests, whether personal, financial, or otherwise, with any individuals or organizations regarding the content 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(2019). hypoglycemic effects of polysaccharides from corn silk (maydis stigma) and their beneficial roles via regulating the pi3k/akt signaling pathway in l6 skeletal muscle myotubes. international journal of biological macromolecules, 121, 981–988. https://doi.org/10.1016/j.ijbiomac.2018.10.100 gurkan, h., hayaloglu, a. a., & boran, o. s. (2019). influence of purple basil extract (ocimum basilicum l.) on chemical composition, rheology and antioxidant activity of set-type yoghurt. mljekarstvo, 69(1), 42–52. https://doi.org/10.15567/mljekarstvo.2019.0104 hosseini, s. m., & behbahani, m. (2021). enhancement of probiotics viability and lactic acid production in yogurts treated with prangos ferulaceae and carum copticum plant extracts. biocatalysis and agricultural biotechnology, 35, 102084. https://doi.org/10.1016/j.bcab.2021.102084 marya et al. – enhancement of probiotic viability and antioxidant activity … 393 illiano, p., brambilla, r., & parolini, c. 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(2013). relevance of the mention of antioxidant properties in yogurt labels: in vitro evaluation and chromatographic analysis. antioxidants, 2(2), 62–76. https://doi.org/10.3390/antiox2020062 raghuvanshi, r. s., singh, a., & apurva. (2024). exploring nutritional and functional properties of corn silk for comprehending its uses. international journal of current advanced research, 13(7), 3171–3176. https://doi.org/10.24327/ijcar.2024.3176.1685 rahman, n. a., & wan rosli, w. i. (2014). nutritional compositions and antioxidative capacity of the silk obtained from immature and mature corn. journal of king saud university science, 26(2), 119–127. https://doi.org/10.1016/j.jksus.2013.11.002 wang, y., wu, j., lv, m., shao, z., hungwe, m., wang, j., bai, x., xie, j., wang, y., & geng, w. (2021). metabolism characteristics of lactic acid bacteria and the expanding applications in food industry. frontiers in bioengineering and biotechnology, 9. https://doi.org/10.3389/fbioe.2021.612285 wilkins, l. j., monga, m., & miller, a. w. (2019). defining dysbiosis for a cluster of chronic diseases. scientific reports, 9(1), 12918. https://doi.org/10.1038/s41598-019-49452-y zhang, x., zhang, c., xiao, l., zhao, x., ma, k., ji, f., azarpazhooh, e., ajami, m., rui, x., & li, w. (2024). digestive characteristics of extracellular polysaccharide from lactiplantibacillus plantarum t1 and its regulation of intestinal microbiota. international journal of biological macromolecules, 259, 129112. https://doi.org/10.1016/j.ijbiomac.2023.129112 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 321-328 | doi: 10.14421/biomedich.2024.132.321-328 issn 2540-9328 (online) the effect of tapioca flour and bogor taro flour (colocasia esculenta l. schott) formulations on the chemical, physical, and sensory characteristics of catfish sausage (pangasius hypophtalmus) leona amelia, sussi astuti*, esa ghanim fadhallah, dyah koesoemawardani department of agricultural product technology, faculty of agriculture, universitas lampung, jl. prof. dr. soemantri brojonegoro no. 1, bandar lampung, lampung, 35145, indonesia. corresponding author* sussi.astuti@fp.unila.ac.id manuscript received: 29 february, 2024. revision accepted: 14 june, 2024. published: 01 october, 2024. abstract this study was aimed to determine the effect of tapioca flour and bogor taro flour formulation on the chemical, physical, and sensory characteristics of catfish sausage and to determine the best formulation. this study was arranged in a randomized complete group design (rcgd) with a single factor of tapioca flour and bogor taro flour formulation with 6 treatments p1 (100%:0%), p2 (80%:20%), p3 (60%:40%), p4 (40%:60%), p5 (20%:80%), and p6 (0%:100%) with 4 replications. data were tested for equality of variance with barlett's test and data saturation with tuckey's test. the data were analyzed for variance to determine the effect between treatments, then further analyzed using the hsd test at the 5% level. the results showed that the formulation of tapioca flour and bogor taro flour had a very significant on water content, ash content, hardness, springiness, cohesiveness, appearance, and overall acceptance but no effect on the taste and aroma of catfish sausage. p5 was the best treatment with a water content of 52.08%, ash content of 1.55%, hardness of 270.75 gf, springiness of 10.03 mm, cohesiveness of 0.83, appearance of 2.93 (slightly dull), taste of 3.87 (typical of fish), aroma of 3.87 (typical of fish), overall acceptance of 3.88 (like), protein content of 15.61%, and fat content of 4.29%. keywords: bogor taro flour; catfish sausage; tapioca flour. introduction as a maritime country, indonesia possesses significant economic potential in the fisheries sector, including capture fisheries and aquaculture. lampung province excels in freshwater fisheries, with one of its potentials being catfish cultivation. according to the ministry of marine affairs and fisheries (2020), the total catfish production in lampung province reached 19,080.98 tons in 2019 and increased to 21,713.87 tons in 2020. based on this data, the freshwater fisheries sector, especially catfish in lampung province, has the potential for further development and utilization. catfish contains approximately 75.75-79.42% water, 12.94-17.59% protein, 1.81-6.57% fat, and 0.16-0.23% ash (wahyuningtyas et al., 2020). the yield of catfish meat is 61.7% of the initial weight of the fish, which is significantly higher than several other fish species (harmain & dali, 2017). catfish protein content is relatively high and contains all essential amino acids, especially lysine and arginine (wahyuningtyas et al., 2020). however, catfish are prone to quality deterioration and perishability as a fishery product. therefore, food processing, such as diversification into sausage products, is necessary to address this issue. the consumption of processed fast food, such as meat sausages, in indonesia, continues to rise at an average rate of 4.46% per year (wulansari, 2013). sausage is a diversified food product produced by mixing ground meat with fillers and other permitted additives, which are then filled into sausage casings (bsn, 2013). the fillers must contain high starch content to produce a compact texture and structure (ernawati et al., 2012). catfish have meat characteristics with a smooth and somewhat tender texture (setiawati et al., 2015). therefore, appropriate fillers are needed in the production of catfish sausages. tapioca flour can be used as a filler to improve emulsion stability and bind other ingredients in product processing. tapioca contains starch ranging from 73.3−84.9%, consisting of 17% amylose and 83% amylopectin (herawati, 2012). tapioca has high amylopectin content, is not prone to clotting, has high adhesive strength, is not easily broken or damaged, and has a relatively low gelatinization temperature (aristawati et al., 2013). however, tapioca flour is insoluble in cold water and requires long cooking times (purwantika, 2016), hence https://doi.org/10.14421/biomedich.2024.132.321-328 322 biology, medicine, & natural product chemistry 13 (2), 2024: 321-328 the need to formulate it with other fillers to produce catfish sausages. bogor taro flour is a carbohydrate source with a starch content of 75.18%, consisting of 5.59% amylose and 94.41% amylopectin (sembong et al., 2019). bogor taro flour has high swelling power and peak viscosity, thus having a higher ability to form a gel and can be used as a substitute for tapioca flour in sausage making. bogor taro flour has granules with a small size of about 8.3 μm, which can form a fine gel structure (aryanti et al., 2017). bogor taro flour contains a relatively high fibre content of 6.67% (hartati & prana, 2003). the small granule size and fibre content in bogor taro flour make it highly digestible, thus beneficial for digestion (sembong et al., 2019). bogor taro has a higher starch content and lower oxalate content (the cause of itching) compared to beneng and kimpul taro. the starch content of bogor taro is 18.03%, while beneng taro is 6.97% and kimpul taro is 13.20%. the oxalate content of bogor taro is 8,578.28 ppm, while beneng taro is 61,783.75 ppm and kimpul taro is 17,400 ppm (lestari & susilawati, 2015). the use of tapioca flour and bogor taro flour as fillers in catfish sausage production is expected to improve the physical and sensory characteristics of the sausages. research on the production of catfish sausages using tapioca flour and bogor taro flour as fillers has yet to be conducted thus far. therefore, this study aims to determine the effect of the ratio of tapioca flour to bogor taro flour as fillers on the chemical, physical, and sensory properties of catfish sausages, as well as the chemical properties of the best treatment according to indonesian national standard (sni) 7755:2013 concerning fish sausages. materials and methods materials and equipment the main ingredients used in sausage making were living siamese catfish (pangasius hypophtalmus) with a size of 2 fish/kg obtained from the gedongtataan pesawaran market, bogor taro flour (naya jagapati), and tapioca flour (pak tani cap gunung). additional ingredients included soy protein isolate (para agribusiness), salt, ice water, ginger, powdered pepper, sugar, garlic, oregano, sania royale soybean oil, and polyamide casings (foodgrade and non-edible). the materials used for chemical analysis are concentrated h2so4 solution, 0.02n hcl, distilled water, 50% naoh, alcohol, naoh-na2s2o3, k2so4, hgo, pp indicator, and h3bo3. the equipment used in sausage making includes a grinder (food processor), cooking utensils, scales, stove, casing filler, tying string, basin, knife, spoon, spatula, cutting board, and other cooking utensils. the equipment used for chemical analysis includes a boiling stone, crucible clamp, porcelain crucible, metal crucible, erlenmeyer flask, oven, desiccator, phenolphthalein indicator, distillation apparatus, burette, pipette, test tube, vortex mixer, analytical balance, filter paper, electric furnace, kjeldahl flask, and brookfield ct-3 texture analyzer for physical analysis, as well as a set of sensory testing equipment. data analysis the study used a randomized complete group design consisting of one factor and four replications. the treatment of the comparison between tapioca flour and bogor taro flour consisted of 6 levels, namely p1 (100%:0%), p2 (80%:20%), p3 (60%:40%), p4 (40%:60%), p5 (20%:80%), p6 (0%:100%). the amount of filler used is calculated from 30% of the weight of ground catfish meat (w/w). the data obtained were analyzed for variance (anova) to obtain error variance estimates and significant tests to determine the effects between treatments. data were further analyzed using the honestly significant difference (hsd) test at the 5% level to determine differences between treatments. the best treatment for catfish sausages was determined using the star test method. catfish sausage making procedure the sausage-making procedure refers to the method by apriyani (2019) with modifications. fresh catfish is filleted by removing and cleaning the gut contents thoroughly. the cleaned catfish is filleted, separating the meat from the skin and bones. the catfish meat is ground into 200 g using a food processor until smooth. ice water is added to the catfish meat in 40 g during grinding. other required ingredients, including tapioca flour and bogor taro flour according to the calculated formulation of 30%, are weighed based on the weight of ground catfish meat (w/w). the weighed catfish meat is then placed in a container and mixed with other ingredients and fillers according to the treatment formulation. next, all ingredients were stirred until well mixed. the evenly mixed dough is then filled into the sausage filler and filled into sausage casings. the sausages are then tied with string at both ends and in the middle at intervals of 10 cm, with each sausage dough weighing around 31-33 g. after that, the sausages were immersed in boiling water and boiled for 30 minutes. the cooked sausages are then removed and drained. observation observations of the chemical properties of catfish sausages were based on parameters such as water content and ash content (aoac, 2015). observations of the physical properties of catfish sausages were based on texture parameters, including hardness, cohesiveness, and springiness (wulansari, 2013). sensory observations of catfish sausages include scoring tests for appearance, taste, and aroma and hedonic tests for overall acceptance (apriyani, 2019). observations of the chemical properties of the best-treated catfish sausages include amelia et al. – the effect of tapioca flour and bogor taro flour … 323 protein and fat content (aoac, 2015). the determination of the best treatment is done using the star test method. results and discussion chemical properties the chemical analysis carried out on catfish sausage included water and ash content parameters. the results of the water and ash content can be seen in table 1. based on table 1, it can be seen that the water content and ash content of catfish sausage were significantly different between treatments. the water content results ranged from 50.34%-52.68%, while the ash content ranged from 0.90%-1.67%. table 1. results of chemical analys of catfish sausage. treatments (tapioca flour: bogor taro flour) water content (%) ash content (%) p6 (0%:100%) 52.68±0.31a 1.67±0.16a p5 (20%:80%) 52.08±0.65ab 1.55±0.09ab p4 (40%:60%) 51.22±0.58b 1.44±0.08b p3 (60%:40%) 50.81±0.13bc 1.33±0.02bc p2 (80%:20%) 50.57±0.24c 1.20±0.10c p1 (100%:0%) 50.34±0,16c 0.90±0.10d hsd 5% 0.845 0.219 note: the numbers followed by different letters on the same row indicate significant differences based on the hsd test 5% water content the water content of catfish sausages increases with the increase in the amount of bogor taro flour and the decrease in the amount of tapioca flour added. this is because the amylopectin content in bogor taro flour is higher, thus absorbing more water. this result is in line with the research by sembong et al. (2019), where the increased use of bogor taro flour in pork sausage production increased the water content of sausages, with the highest value observed when using 75% taro flour. amylose and amylopectin can absorb water and have good gelatinization properties. the end of the amylopectin and amylose molecule chains has hydroxyl groups that can interact with hydrogen from water (kusnandar, 2019). kusnandar (2019) states that amylopectin has bonds with α-1,6-glycosidic branch structures, thus able to maintain water content compared to amylose, which has linear bonds with α-1,4-glycosidic structures. amylose absorbs water more easily and releases its bonds more easily, while amylopectin takes longer to absorb water and also takes longer to release its bonds during cooking. this causes the higher the amylopectin content in the raw material, the higher the water content in the resulting food product (pratama, 2018). the amylopectin content of bogor taro flour at 94.41% is higher than the amylopectin content of tapioca flour at 79.59-79.99% (sembong et al., 2019), thus resulting in higher water content in catfish sausages when bogor taro flour is added. according to kusnandar (2019), starch has hydrophilic properties to absorb the water. starch granules' ability to absorb and bind water influences water absorption capacity. granule size plays an important role in the water absorption process of filler materials. smaller granule sizes can increase the hydration rate and facilitate water entry into starch granules, increasing water absorption capacity (ulyarti et al., 2022). the granule size of tapioca flour, reaching 35 μm (herawati, 2012), is larger than that of bogor taro flour, which is 8.3 μm (aryanti et al., 2017). according to sembong et al. (2019), the dietary fibre content in the ingredients also causes the water content of the product to increase because dietary fibre easily absorbs water due to its large surface area. the dietary fibre content of bogor taro flour sausages is 9.66%, which is greater than tapioca flour sausages at 5.01%. this result is directly proportional to the water content testing results, meaning the higher the bogor taro flour content, the higher the dietary fibre content, and consequently, the higher the water content of the sausages (sembong et al., 2019). the water content of catfish sausages using tapioca flour and bogor taro flour in various formulations complies with the indonesian national standard (sni) 7755:2013 for the quality requirements of fish sausages, which is a maximum of 68%. ash content the ash content increases with the increase in the amount of bogor taro flour and the decrease in the amount of tapioca flour added. bogor taro flour has a higher ash content than tapioca flour, resulting in increased ash content of catfish sausages with the increasing use of bogor taro flour. the ash content of bogor taro flour ranges from 3.11% to 3.84% (hawa et al., 2020), while the ash content of tapioca flour is 1.1% (wely, 2021). this is consistent with susanti's research (2016), where meat sausages with the addition of bogor taro flour and tapioca flour resulted in the highest ash content with 100% bogor taro flour addition at 5.38% and the lowest with 100% tapioca flour addition at 4.39%. ash content is related to minerals in the form of organic and inorganic salts in a food ingredient (nisa & wardani, 2016). bogor taro flour contains several mineral components, such as 67 mg phosphorus and 31 mg calcium. tapioca flour has a lower mineral content, with 7 mg of phosphorus and 20 mg of calcium (susanti, 2016). furthermore, the ash content of bogor taro flour is higher than tapioca flour because bogor taro flour is soaked in a sodium metabisulfite solution in its production process. sodium metabisulfite is an inorganic salt containing na and s minerals, thus increasing the ash content of bogor taro flour (yuliyanti, 2023). the ash content of catfish sausages using tapioca flour and bogor 324 biology, medicine, & natural product chemistry 13 (2), 2024: 321-328 taro flour in various formulations meets the indonesian national standard (sni) 7755:2013 for the quality requirements of fish sausages, which is a maximum of 2.5%. physical properties the physical analysis carried out on catfish sausage included the parameters of hardness, springiness and cohesiveness. the hardness, springiness and cohesiveness test results can be seen in table 2. based on table 2, it can be seen that the hardness, springiness and cohesiveness of the catfish sausage were significantly different between treatments. the hardness results ranged from 257.06-369.88 gf, springiness ranged from 9.1310.13 mm, and cohesiveness ranged from 0.73-1.09. table 2. results of physical analys of catfish sausage. treatments (tapioca:bogor taro flour) hardness (gf) springiness (mm) cohesiveness p6 (0%:100%) 257.06±4.64d 10.13±0.09a 0.73±0.06d p5 (20%:80%) 270.75±6.94cd 10.03±0.09ab 0.83±0.03c p4 (40%:60%) 290.44±9.92c 9.78±0.22b 0.87±0.03c p3 (60%:40%) 327.56±13.25b 9.70±0.14b 0.93±0.02bc p2 (80%:20%) 351.63±8.01ab 9.40±0.14c 0.97±0.04b p1 (100%:0%) 369.88±15.14a 9.13±0.09c 1.09±0.06a hsd 5% 24.746 0.280 0.083 note: the numbers followed by different letters on the same row indicate significant differences based on the hsd test 5%. hardness the hardness value of catfish sausages decreases with the increasing amount of bogor taro flour and the decreasing amount of tapioca flour added. this is because bogor taro flour's amylose content is lower than tapioca flour’s. this result is consistent with aminullah et al. (2020), who found that the increasing addition of bogor taro flour in making lele fish pempek tends to decrease the hardness level of the resulting pempek. the higher the amylose content, the higher the hardness value of the resulting product. bogor taro flour contains 5.59% amylose, while tapioca flour contains 20.01%-20.47% amylose (sembong et al., 2019). amylose can reassociate with its sesame molecules to form a rigid structure. this causes gelatinized starch to undergo retrogradation crystallization (luna et al., 2015). the retrogradation process causes binding matrices in amylose to bind to each other, thus increasing the product's hardness (fitriyani et al., 2017). therefore, the addition of bogor taro flour with higher concentrations can result in a decrease in the hardness value of the product. amylose can bind water molecules better than amylopectin. water absorption during heating disrupts the crystal structure, and water molecules bond with hydroxyl groups to form hydrogen bonds (luna et al., 2015). the greater the bound water content, the narrower the space between molecules, resulting in a denser and more compact product texture (nurdjanah & yuliana, 2016). the hardness value of a product is also influenced by its water content. the higher the water content in the product, the lower the hardness value, resulting in a softer and less compact product (setyowati & nisa, 2014). springiness the springiness value of catfish sausages increases with the increasing amount of bogor taro flour and the decreasing amount of tapioca flour added. this is because bogor taro flour's amylopectin content is higher than tapioca flour’s. this result is consistent with lasi et al. (2019), where the higher concentration of added taro flour produces more resilient bakso. the amylopectin content influences the springiness value of the filling material used. amylopectin can form a gel and is less soluble in water, resulting in a more resilient and sticky product texture. amylopectin has longer branch chains, making it strong in forming a gel because it binds water molecules, and their bonds are not easily released (debora et al., 2023). the high amylopectin content in bogor taro flour forms the elasticity level of catfish sausages (sembong et al., 2019). high amylopectin in the filling material affects gel formation, one of which is in the swelling or starch expansion properties. starch containing high amylopectin can interact with myofibril protein to form a gel. the interaction between myofibril protein and starch occurs when myofibril matrix spaces are filled with starch molecules, thus forming an elastic product structure (herdiana et al., 2023). this indicates that the addition of bogor taro flour in making catfish sausages produces a more resilient product texture. cohesiveness the cohesiveness value of catfish sausages decreases with the increasing amount of bogor taro flour and the decreasing amount of tapioca flour added. this is because bogor taro flour's amylose content is lower than tapioca flour. the amylose content of the filling material amelia et al. – the effect of tapioca flour and bogor taro flour … 325 used influences the cohesiveness value of the product. higher amylose content causes the product texture to be more compact. this is because in forming the product texture, amylose plays a role in forming the firmness of the gel during the water absorption stage during gelatinization, resulting in a more compact texture than filling material with lower amylose content (liur et al., 2013). amylose strongly bonds hydrogen due to its straight chains with α-1,4-glycosidic structures in starch granules. the greater hydrogen bonding strength affects the water absorption capacity, thus affecting the formation of product texture (indrianti et al., 2013). bogor taro flour has a lower amylose content of 5.59%, while tapioca flour has an amylose content of 20.01%-20.47% (sembong et al., 2019). the high amylose content in tapioca flour results in catfish sausages with a higher level of compactness compared to the use of bogor taro flour. sensory observations sensory analysis on catfish sausage included appearance, taste, aroma and overall acceptance. the results of the appearance, taste, aroma and overall acceptability test can be seen in table 3. based on table 3, the appearance and overall acceptability of catfish sausages were significantly different between treatments, while the taste and aroma of catfish sausages were not significantly different between treatments. appearance scores ranged from 2.55-4.42; taste scores ranged from 3.63-3.98; aroma scores ranged from 3.58-3.87; and overall acceptance scores ranged from 3.36-3.92. table 3. results of sensory analys of catfish sausage. treatments (tapioca:bogor taro flour) appearance taste aroma overall acceptance p1(100%:0%) 4.42±0.08a 3.98±0.25a 3.70±0.20a 3.92±0.08a p2(80%:20%) 4.03±0.16ab 3.67±0.12a 3.58±0.07a 3.36±0.18c p3(60%:40%) 3.65±0.21b 3.64±0.23a 3.62±0.14a 3.67±0.06b p4(40%:60%) 3.48±0.10b 3.83±0.16a 3.74±0.13a 3.51±0.04bc p5(20%:80%) 2.93±0.33c 3.87±0.05a 3.87±0.05a 3.88±0.05ab p6(0%:100%) 2.55±0.41c 3.63±0.09a 3.63±0.11a 3.47±0.04bc hsd 5% 0.595 0.418 0.310 0.223 note: the numbers followed by different letters on the same row indicate significant differences based on the hsd test 5% score value: appearance : (5) ripping, (4) less ripping, (3) slightly dull, (2) dull, (1) very dull taste and aroma : (5) very typical of fish, (4) typical of fish, (3) slightly typical of fish, (2) not typical of fish, (1) very not typical of fish overall acceptance : (5) really like, (4) like, (3) slightly like, (2) do not like, (1) very dislike. appearance the appearance score of catfish sausages decreases with the increasing amount of bogor taro flour and the decreasing amount of tapioca flour added. the appearance of catfish sausages is generally influenced by the main ingredients, binders, fillers, and other additives and can be affected by temperature and cooking time (peka et al., 2021). however, in this study, it is suspected that the filler used affects the appearance of catfish sausages. bogor taro flour has a darker colour compared to tapioca flour. bogor taro flour has a whitish-grey colour, while tapioca flour has a bright white colour. this is because, during the production process of bogor taro flour, a colour change occurs during the taro drying stage. this colour change occurs because taro sap undergoes enzymatic oxidation, which can convert polyphenol compounds into compounds with a dark brown colour, resulting in bogor taro flour being darker compared to tapioca flour. the polyphenol compound in taro is quercetin, which provides the colour pigment to the taro tuber (dana, 2018). additionally, the gelatinization process during heating can result in the formation of starch paste with a darker colour than before gelatinization (syafutri et al., 2017). the whiteness degree of bogor taro flour is 71.6%, which is lower than the whiteness degree of tapioca flour at 97.64% (ernilawati et al., 2018). therefore, after the gelatinization process, the resulting sausages become darker with increasing bogor taro flour. the appearance score of catfish sausages in all treatments meets the requirements of sni 7755:2013 for fish sausages, with a minimum score of 7 for the slightly dull criterion, equivalent to a score of 3 in this study's sensory questionnaire. taste the taste score of catfish sausages in all treatments meets the requirements of sni 7755:2013 for fish sausages, with a minimum score of 7 for the slightly typical fish criterion, equivalent to a score of 3 in this study's sensory questionnaire. based on table 16, adding bogor taro flour and tapioca flour with different concentrations in making catfish sausages does not affect the taste 326 biology, medicine, & natural product chemistry 13 (2), 2024: 321-328 assessment score. this is because bogor taro flour and tapioca flour do not have dominant flavours, so their use in all treatments does not affect the final product. the main ingredients and other seasonings influence the taste of fish sausages added to the dough (lasi et al., 2019). the percentage of main ingredients used still meets the requirements of sni 7755:2013, which is a minimum of 50%. therefore, the treatments performed do not affect the taste of the sausages. the consistent taste score in all treatments indicates that bogor taro flour can be an alternative to tapioca flour in making catfish sausages. aroma the aroma score of catfish sausages in all treatments meets the requirements of sni 7755:2013 for fish sausages, with a minimum score of 7 for the slightly typical fish criterion, equivalent to a score of 3 in this study's sensory questionnaire. the assessment of the aroma score of catfish sausages in this study is consistent with the study by aminullah et al. (2020), where the use of bogor taro flour and tapioca flour in making lele fish pempek does not affect the aroma score. this is because bogor taro flour and tapioca flour do not have dominant aromas, so their use in all treatments does not affect the final product. the aroma of processed meat products is influenced by the type of raw material used, cooking time, and other additives, such as spices, that have volatile properties due to cooking (sujianti et al., 2023). the percentage of main raw materials used still meets the requirements of sni 7755:2013, which is a minimum of 50%. therefore, the treatments performed do not affect the aroma of the sausages and produce a similar aroma in all treatments. this indicates that bogor taro flour can be an alternative to tapioca flour in making catfish sausages. overall acceptance the highest overall acceptance score was obtained in treatments p1 with a 3.92 (like) and p5 with a 3.88 (like). the lowest overall acceptance score was obtained in treatments p4 with a score of 3.51 (like), p6 with a score of 3.47 (somewhat like), and p2 with a score of 3.36 (somewhat like). based on the overall acceptance assessment scores, catfish sausages with treatments p1 and p5 produced the most preferred assessments by the panellists. according to the panellists' assessment in the questionnaire, these treatments had a dense, compact, and slightly resilient texture, a typical fish taste but not too fishy, and a slightly less radiant appearance that was still acceptable and liked by the panellists. treatment p2 was less preferred by the panellists than other treatments, with the lowest overall acceptance score due to a slightly fishy aroma and taste and the hardest texture. best treatment the best treatment was determined based on chemical tests for water and ash content, physical tests for hardness, springiness, and cohesiveness, sensory tests, including scoring (appearance, taste, and aroma) and hedonic tests (overall acceptance). the method used to determine the best treatment was the star test method. the star test method was conducted by marking stars on the 5% hsd test results with the notation 'a' because data with the notation 'a' are treatments with the highest values. then, the stars for each treatment were added up, and the best treatment was chosen based on the highest number of stars. the treatment designated as the best was p5, which produced the best hardness, springiness, cohesiveness, water content, ash content, appearance, taste, aroma, and overall acceptance and met the requirements of sni 7755:2013. the best treatment of catfish sausages, p5, underwent analysis for protein and fat content, and the results were compared with sni 7755:2013. the chemical analysis results of the best treatment of catfish sausages can be seen in table 4. table 4. results of best treatment for catfish sausage. parameters p5 (20%:80%) sni 7755:2013 remarks protein content (%) 15.61 min 9.0 meet the standard fat content (%) 4.29 max 7.0 meet the standard conclusions the comparison between tapioca flour and bogor taro flour in making catfish sausages significantly affects water content, ash content, hardness, springiness, cohesiveness, appearance, and overall acceptance. however, it does not affect the taste and aroma of catfish sausages. the best catfish sausage ratio is tapioca flour and bogor taro flour 20%:80% (treatment p5), which results in a water content of 52.08%, ash content of 1.55%, hardness of 270.75 gf, springiness of 10.03 mm, cohesiveness of 0.83, appearance score of 2.93 (slightly dull), taste score of 3.87 (typical of fish), aroma score of 3.87 (typical of fish), and overall acceptance score of 3.88 (like). the protein content is 15.61%, and the fat content is 4.29%. the water, ash, protein, and fat content of catfish sausages have met the requirements of sni 7755:2013 for fish sausages. competing interest: the authors declare that there are no competing interests. amelia et al. – the effect of tapioca flour and bogor taro flour … 327 references aminullah, daniel, dan rohmayanti, t. 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(2023). pengaruh formulasi tepung ubi kayu (manihot esculenta c.) dan tepung umbi talas (colocasia esculenta l.) terhadap sifat kimia dan sensori kelanting. (skripsi). fakultas pertanian. universitas lampung. bandar lampung. 76 hlm. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 423-431 | doi: 10.14421/biomedich.2024.132.423-431 issn 2540-9328 (online) effectiveness test of methanol, ethyl acetate, and chloroform fractions of bidara leaf extract (zizyphus mauritiana l.) on wound healing in rabbits (oryctolagus cuniculus) novel umarella*, wirasti, slamet, urmatul waznah undergraduate program in pharmacy, faculty of health science, university of muhammadiyah pekajangan pekalongan. jl. pekajangan no 29 pekalongan 51173, tel. (0285) 7832294, indonesia. corresponding author* novel4256@gmail.com manuscript received: 16 agustus, 2024. revision accepted: 08 october, 2024. published: 10 october, 2024. abstract bidara (zizyphus mauritiana l.) contains alkaloids, glycosides, saponins, flavonoids, terpenoids, phenols, and the best antioxidant properties of its leaves. in bidara leaf, they are antibacterial, antiviral, and antiseptic, play a role in cell regeneration and repair, so they are effective in wound healing. this study aimed to determine the effectiveness of bidara leaf extract fractions on incision wounds and determine which fraction was more effective in narrowing the incision wound. making wounds on rabbits (oryctolagus cuniculus) using the morton method, by making an incision on the back with a diameter of 2 cm. testing the effectiveness of fractions in wound healing with rabbits divided into 5 groups, namely positive control, negative control, methanol fraction (meoh), ethyl acetate fraction (ethac), and chloroform fraction (chcl3) with a dose of 100 mg/kgbb rabbit in 1 ml of solvent. the diameter of the wound area was measured using the macbiophotonic image j program, and the data were analyzed using one way anova (anova) followed by the tukey test. the results of data analysis using the anova method with a significant value of 0,004 (p<0,05). the conclusion of the results of the research conducted for 8 days shows that the meoh, ethac, and chcl3 fractions of bidara leaf extract have the effectiveness of wound healing in rabbits with differences in healing shown by the ethac fraction with a better effect than the meoh and chcl3 fractions. keywords: bidara leaf; fractions; wound healing; rabbits. introduction living things always do activities and movements that do not rule out accidents and injuries. injuries can be caused by punctures or scratches with sharp objects, blunt objects, accidents, gunshots, animal bites, chemicals, hot water, fire, electricity, and lightning which cause minor to severe injuries (karliana & wikanta, 2019). based on basic health research (riskesdas) data in 2018, in indonesia the prevalence of injuries due to accidents that occurred at home and the environment ranked first at 44.7%, with the most common types of injuries being abrasions, bruises/bruises at 64.1%, followed by dislocated injuries at 32.8% and puncture/slash/tear injuries at 20.1%. the proportion of injuries was almost the same between urban and rural areas, at 9% (jainurakhma et al., 2022). a wound is damage to the skin in the form of loss of skin epithelial integrity. one of the most common wounds experienced by humans is a cut. a cut is a loss of body tissue caused by a sharp object. a cut wound is an acute type of wound that can cause bleeding with hemostatsis and eventual inflammation. if a cut wound is left untreated, wound infection may occur and the condition may worsen. to heal wounds, they are usually treated with medicines in liquid form or semi-solid form such as ointments. a commonly used drug to heal wounds and prevent wound infection is povidone iodine (e. lestari, 2020). in addition to antiseptics, antibiotics and anti-inflammatory drugs, wounds can also be treated with traditional medicines whose ingredients have been clinically tested and can be used as medicine (karliana & wikanta, 2019). traditional medicine is an effort to overcome health problems passed down from generation to generation. materials that are easily available and cheap make traditional medicine widely used. one of the medicinal plants that has the potential to treat wounds is the bidara plant (zizyphus mauritiana l.). the content is in the form of alkaloid compounds, glycosides, saponins, flavonoids, terpenoids, and phenols, as well as the best antioxidant effect from leaves (e. lestari, 2020; karliana & wikanta, 2019; g. lestari et al., 2021; kusriani et al., 2015). the content of bidara leaf chemical compounds is efficacious for healing cuts. this is because bidara leaves have antibacterial, antiviral, and antiseptic properties and work in cell regeneration and repair. alkaloid https://doi.org/10.14421/biomedich.2024.132.423-431 424 biology, medicine, & natural product chemistry 13 (2), 2024: 423-431 compounds contained in bidara leaves have an analgesic effect on wounds. while saponins stimulate collagen growth during the wound healing (karliana & wikanta, 2019). the benefits of flavonoids are anti-inflammatory and function to inhibit microbial growth by increasing the antioxidant activity of granulomatous tissue (kemalasari et al., 2018). on the other hand, polyphenols reduce lipid peroxidation, thereby reducing cell necrosis and vascularization. tannin compounds support wound healing through their astringent and antibacterial properties (nuralifah et al., 2022). previous research on wound healing was conducted by karliana & wikanta (2019), until the administration of bidara leaf extract was effective in healing iris wounds in male mice (mus muschulus). therefore, this study aims to further determine the effect of compounds in the methanol, ethyl acetate, and chloroform fractions of bidara leaf extract (zizyphus mauritiana l.) on the healing process of incision wounds in rabbits (oryctolagus cuniculus). materials and methods this study is an experimental study conducted in the pharmacy laboratory of the faculty of health sciences, university of muhammadiyah pekajangan pekalongan, where the narrowing of the incision wound was observed by measuring the diameter of the incision wound on the rabbit's back. materials bidara leaf, rabbit, 96% ethanol, povidone iodine, 70% alcohol, topsy cream, distilled water, hcl 2n, fecl3 5%, mayer's reagent, dragendorff's reagent, h2so4, chloroform, ethyl acetate, methanol, methanol p. a, anhydrous acetic acid, gallic acid p.a, quercetin p.a, na2co3 15%, folin-ciocalteu reagent, alcl3 10%, ch3coona 1 m, and dmso. determination determination was conducted on bidara plants (zizyphus mauritiana l.) at ahmad dahlan university. sampling and simplisia preparation bidara leaves were obtained from ayamputih village, buluspesantren sub-district, kebumen district. harvesting was done in the morning before photosynthesis occurred. wet sorting of leaves is done by separating from impurities such as unnecessary plant parts (stems, twigs, flowers and roots) and other impurities. the leaf washing process is carried out with running water while cleaning from dirt that sticks. furthermore, the drying process of leaves that are still wet is done by drying them in the sun and covering them with a black cloth until dry. dry leaf symposia is made into powder by blending it into powder and sieving it with a no. mesh 40 sieve. extract preparation extraction was carried out by maceration using 96% ethanol solvent, dried bidara leaves weighed as much as 1.5 kg and were put into a maceration container then 96% ethanol 9 l. let stand for 5 days at room temperature protected from sunlight while occasionally stirring, then filtered. the maceration treatment was repeated once with 4.5 l of 96% ethanol. the maceration results were collected and then evaporated using a rotary evaporator at 42℃ until thick. fractionation 100 g of thick extract was put into a separatory funnel. the sample was fractionated with solvents that have different polarity from low to high polarities (chloroform, ethyl acetate, and methanol). the sample was added 100 ml of solvent and fractionated one by one solvent alternately in a separatory funnel. each fractionation result was evaporated using a water*bath until a thick extract was obtained. phytochemical screening alkaloid test several samples were added 1 ml of 2n hcl and 9 ml of distilled water, heated on a water bath for two minutes, cooled and filtered. the filtrate obtained was used for the alkaloid test. taken 0.5 ml of filtrate in two test tubes, added 2 drops of mayer reagent and dragendorff. the test results are positive if mayer's reagent forms a yellow-white precipitate and dragendorff's reagent forms a red or orange precipitate (wirasti, 2019). saponins test several samples were put into a test tube and added 10 ml of hot water, then cooled and shaken vigorously for 10 seconds. the foam formation on a stable top layer indicates the presence of saponins in the test sample with the addition of 1 drop of hcl 2n (wirasti, 2019). tannins test a sample quantity of 10 ml of distilled water is heated, then cooled and filtered. dilute the filtrate until almost colorless and add 1-2 drops of 5% fecl3 reagent. a black-blue or black-green color indicates a positive tannin result (wirasti, 2019). flavonoids test several samples are dissolved with a certain amount of 96% ethanol, and added alcl3 reagent. a positive reaction to flavonoids is characterized by the formation of red, yellow, or orange colors (ni’ma & lindawati, 2022). phenol test several samples were extracted with 2 ml of 96% ethanol. take 1 ml of filtrate and add 2 drops of 5% umarella et al. – effectiveness test of methanol, ethyl acetate, and chloroform fractions … 425 fecl3 solution. a positive reaction to phenol shows the formation of a green or blue-green color (wirasti, 2019). terpenoids/steroids test several samples were added to chloroform solvent and separated. 2 drops of acetic acid anhydride and 1 drop of h2so4 were added to the resulting filtrate, with positive results for the presence of steroids forming a blue to green color, and the presence of terpenoids forming a red to purple color (anisa & najib, 2022). total flavonoids test 10 mg of sample was weighed and dissolved in 10 ml of methanol p.a to obtain a 1 mg/ml concentration. a total of 0.5 ml test sample was added to 1.5 ml of methanol p.a, followed by 0.1 ml of 10% alcl3, 0.1 ml of 1 m ch3coona, and 2.8 ml of distilled water. after 20 minutes of incubation, the absorbance was measured at maximum λ with 3 times replication using uv-vis spectrophotometer (wirasti, 2019). flavonoid content can be calculated with the following formula: 𝑇𝑜𝑡𝑎𝑙 𝑓𝑙𝑎𝑣𝑜𝑛𝑜𝑖𝑑𝑠 = 𝐶 𝑥 𝑣 𝑥 𝑓𝑝 𝑔 note: c : flavonoids concentration (x-value) v : volume of sample used (ml) fp : dilution factor g : weight of sample used (g) total phenol test a total of 10 mg of sample was dissolved in distilled water p.a up to 10 ml and a concentration of 1 mg/ml was obtained. pipette 0.1 ml, add 7.9 ml of distilled water and 0.5 ml of folin-ciocalteu reagent, vortex for 1 minute and let stand for 8 minutes. then, added 1.5 ml of 15% na2co3 into the solution and let stand at room temperature for 30 minutes. absorbance was measured at maximum λ using a uv-vis spectrophotometer. the measurement was repeated 3 times, and the results of the phenol content obtained were expressed in mg gallic acid equivalent (gae)/g sample (wirasti, 2019). calculation of total phenolic content using the following formula: 𝑇𝑜𝑡𝑎𝑙 𝑝ℎ𝑒𝑛𝑜𝑙𝑖𝑐𝑠 = 𝐶 𝑥 𝑣 𝑥 𝑓𝑝 𝑔 note: c : phenolic concentrations (x-value) v : volume of sample used (ml) fp : dilution factor g : weight of sample used (g) preparation and treatment of test animals test animals were used as local male rabbits weighing 1.5-3 kg as many as 15 heads. the initial treatment of the test animals was wounding. the rabbit's back hair was shaved and cleaned using 70% ethanol, then anesthetized with topsy cream. an incision was made using a sterile scalpel ±2 cm long with a depth of 0.2 cm. the second stage is the administration of samples to the incision wound which is divided into group 1 (meoh 100 mg/kg bw in dmso), group 2 (ethac 100 mg/kg bw in dmso), group 3 (chcl3 100 mg/kg bw in dmso), positive control (povidone iodine) and negative control (dmso). rabbits were put into cages, treatment was carried out once a day in the morning for a maximum of 14 days. the measurement of the effect of wound healing is seen from the wound closure time and the decrease in wound diameter on the rabbit's back. data analysis anova analysis was used to observe wound closure characterized by a reduction in wound diameter, absence of erythema, and swelling. results and discussion determination the plant determined to be zizyphus mauritiana lam. and belongs to the rhamnaceae family. the purpose of determination is to ensure the authenticity of the identity of the plant under study so that there is no error when taking bidara plant material (zizyphus mauritiana l.). extracts and fractions from the sample powder, an extract with a dense form, blackish green color, and characteristic odor of the extract was obtained. in concentrating the extract at 42°c, it is intended that bioactive components such as flavonoids that cannot tolerate high temperatures above 50°c do not experience structural changes due to temperature increase (yuliantari et al., 2017). the resulting extract weighs 183 g with a yield of 12.2% and a moisture content of 0.49%. moisture content testing aims to determine the moisture content in the extract to minimize the growth of fungi, bacteria, and enzymes that can damage the extract during storage. the resulting extract is fractionated to separate and purify the active ingredients from the original extract (abubakar & haque, 2020). 426 biology, medicine, & natural product chemistry 13 (2), 2024: 423-431 table 1. fraction yield. sample weight of extract (g) weight of fraction (g) yield (%) moisture content (%) meoh fraction ethac fraction chcl3 fraction 100 g 100 g 100 g 20,55 g 6 g 7,3 g 20,55% 6% 7,3% 0,74% 0,47% 1,49% in table 1, the methanol fraction data gives the highest yield, which indicates that most of the compounds in the extract dissolve in methanol which is a polar solvent. according to the principle of like dissolve like, polar compounds will dissolve in polar compounds and non-polar compounds will dissolve in non-polar solvents. the amount of yield dissolved in methanol indicates the amount of polar compounds contained in the extract. phytochemical screening bidara leaves are known to be helpful in treating wounds and are proven to contain secondary metabolites. therefore, to maximize the function of bidara leaves as medicinal compounds, it is necessary to know the secondary metabolite compounds through phytochemical screening (table 2). table 2. phytochemical screening of bidara leaf fraction. metabolite compounds sample meoh fraction ethac fraction chcl3 fraction alkaloids ▪ mayer ▪ dragendorff (-) no precipitates (-) no precipitates (-) no precipitates (-) no precipitates (-) no precipitates (-) no precipitates saponins (++) stable foam 2 cm (+) stable foam 0,7 cm (+) stable foam 0,5 cm tannins (+++) greenish-black (++) greenish-black (++) greenish-black flavonoids (++) yellowish-green (++) yellowish-green (+) yellowish-green phenol (++) blackish-green (+++) blackish-green (+) blackish-green steroids (++) greenish-black (++) greenish-black (++) greenish-black note: : no compound content + : there is little compound content ++ : there is a compound content +++ : there are many compound contents several studies showed that ethanol extract from bidara leaves positively contained alkaloid compounds (khoirunnisak, 2020), while ethyl acetate extract from bidara leaves negatively contained alkaloid compounds (bialangi et al., 2023). alkaloids are usually found in various parts of plants, such as flowers, seeds, leaves, twigs, roots, and bark. in testing metabolite compounds, the addition of certain materials has its purpose. the addition of dragendorff and mayer reagents to the alkaloid test can produce orange to reddish brown precipitates and white to yellow precipitates. in this reaction, a ligand exchange occurs in which nitrogen with a free electron pair in the alkaloid forms a covalent bond with the k+ ion of potassium tetraiodobismutat, resulting in the formation of an alkaloid potassium complex that precipitates (habibi et al., 2018). the addition of hcl to the saponin test will increase the polarity of the saponin compound and change the position of the forming group. under these conditions, polar (hydrophilic) groups face outward and nonpolar (hydrophobic) groups face inward, forming a structure called a micelle structure (putri & lubis, 2020). the addition of fecl3 in the tannin test which can form fe3+ and cl-. the addition of alcl3 to flavonoids to form complexes between neighboring hydroxyl and ketone groups or with adjacent hydroxyl groups to form stable yellow complex compounds. while the addition of fecl3 to the phenol test will form a colored complex that is believed to be iron (iii) hexaphenolate (habibi et al., 2018). then steroid testing is carried out using the lieberman-bouchard method (acetic acid anhydrideh2so4) which produces a green color based on the ability of steroid compounds to form colors through h2so4 in acetic anhydride solvents (habibi et al., 2018). total flavonoids test the examination of the total flavonoid compound content was carried out because flavonoid compounds are known to play a role in promoting the hemostasis process through a vasoconstriction mechanism (nikola et al., 2021). the method used to determine the total flavonoid content is the colorimetric method using 1 m acetic acid umarella et al. – effectiveness test of methanol, ethyl acetate, and chloroform fractions … 427 reagent and 10% alcl3. in this method, a reaction occurs and a stable yellow color is formed due to the reaction of alcl3 and oh groups on flavonoids (wahyudi & minarsih, 2023). alcl3 reacts with ketone groups at c4 and oh groups at c3 or c5 of flavone or flavonol compounds to form stable yellow complex compounds. the compound used as a standard to determine flavonoid levels is quercetin. because quercetin is a flavonoid of the flavonol group with a ketone group at the c4 atom and a hydroxyl group at the adjacent c3 and c5 atoms (sari & ayuchecaria, 2017). quercetin was also chosen as a standard also because it is the most widespread compound in plants (wirasti, 2019). total phenolic test the examination of the total content of phenolic compounds was carried out because phenolic compounds are known to have antiseptic effects on wounds and kill bacteria (karliana & wikanta, 2019). determination of the total phenolic content of meoh, ethac, and chcl3 fractions in bidara leaves (zizyphus mauritiana l.) was determined using the folin-ciocalteu principle based on redox reactions. gallic acid is a stable and natural phenolic compound used as a standard solution. according to sari & ayuchecaria (2017), gallic acid is one of the phenolic compounds derived from hydroxybenzoic acid and is classified as a simple phenolic acid. gallic acid reacts with folin-ciocalteu reagent to produce a yellow color indicating the presence of phenol, which then adds na2co3 solution to it as a base atmosphere giver. during the reaction, the hydroxyl group of the phenolic compound reacts with folinciocalteu reagent to form a blue molybdenum-tungsten complex whose structure is unknown and can be detected using a spectrophotometer. the blue color becomes more intense depending on the concentration of phenol ions. that is, the higher the concentration of phenolic compounds, the more phenolic ions that reduce heteropoly acids (phosphomolybdate-phosphotungstat) into tungsten molybdenum complexes, so that the color obtained is more intense (wirasti, 2019). the total flavonoid content of the plant is expressed in qe (quercetin equivalent), and the total phenolic content is expressed in gae (gallic acid equivalent), which is the equivalent of milligrams of quercetin or gallic acid in one gram of sample (figure 1). figure 1. average total flavonoid and phenolic content of bidara leaf fraction (zizyphus mauritiana l.) the results of determining the content of flavonoid compounds and total phenols, it is known that the sample of the ethyl acetate fraction of bidara leaves (zizyphus mauritiana l.) has a higher content of flavonoid and phenol compounds, namely as much as 81.71 ± 5.91 mg qe/g extract and 204.79 ± 8.64 mg gae/g extract, this is in line with the screening results where the ethyl acetate fraction has the results of phenol and flavonoid tests with the most concentrated color compared to other fractions. followed by the methanol fraction as much as 58.51 ± 10.00 mg qe/g extract and 131.76 ± 4.20 mg gae/g extract, the screening results in the flavonoid test having color results with almost the same concentration as the ethyl acetate fraction. then, for the chloroform fraction with a content of 20.04 ± 5.00 mg qe/g extract and 46.61 ± 2.92 mg gae/g extract, with the screening results in the phenol and flavonoid tests had a less concentrated color. effectiveness of wound healing wound healing is a multi-step process that occurs when the normal anatomy and function of skin tissue are disrupted. inflammation, granulation, wound reduction, collagen formation, epithelial closure and scar formation are all part of the process. the smooth running of these stages promotes wound healing and restores the previously disrupted condition and function of the skin anatomy (zulkefli et al., 2023). in recent years, plants and their products have been used to stimulate natural repair mechanisms by shortening the prolonged 58,51±10,00 81,71±5,91 20,04±5,00 131,76±4,20 204,79±8,64 46,61±2,92 0 50 100 150 200 250 meoh ethac chcl₃ a v e r a g e c o n te n t type of fractions total flavonoid and phenol content average ktf (mg qe/g) average ktfe (mg gae/g) 428 biology, medicine, & natural product chemistry 13 (2), 2024: 423-431 inflammatory phase, enhancing fibroblast migration and proliferation, stimulating angiogenic processes, and thus promoting the re-epithelialization process of wound healing. numerous studies on the potential of natural products as wound healing agents have scientifically validated their antibacterial, antioxidant, and antiinflammatory properties. in this regard, who recommends the use of scientifically validated plantbased medicines. natural products from traditional medicine and ethnopharmacology are important sources of new leads in developing pharmaceutical research for therapeutic applications. bioassay-based fractionation and isolation is one of the most effective methods to discover new therapeutic compounds from active fractions of plant extracts (bhat et al., 2023). wound observations were made by photographing the wound using a mobile phone camera every day until the rabbit healed and measuring the area and diameter of the wound area using the macbiophotonic image j program. observations of wound healing ability characterized by a decrease in wound diameter reinforced by a decrease in the area of the incision on the rabbit's back were carried out with 3 replications for each treatment group for 8 days of testing. from the data obtained, the average reduction in the diameter of the incision wound area was calculated (figure 2). figure 2. decrease in incision wound diameter. five treatment groups consisting of negative control in the form of dmso, positive control in the form of povidone iodine 10%, meoh fraction, ethac fraction, and chcl3 fraction of bidara leaves (zizyphus mauritiana l.) are known to give different results in shortening the diameter of the incision every day. figure 3. percentage of incision wound diameter healing. the percentage of wound diameter healing shows the closure of the wound on the rabbit's back due to the treatment in wound care. based on figure 3, the best healing on day 1 was shown by the chcl3 fraction, but 2,005 1,817 1,600 1,047 0,616 0,151 0,000 0,000 0,000 2,002 1,597 1,131 0,819 0,449 0,000 0,000 0,000 0,000 2,006 1,553 1,044 0,604 0,155 0,000 0,000 0,000 0,000 2,002 1,385 1,216 0,955 0,764 0,503 0,168 0,000 0,000 2,001 1,813 1,491 1,297 1,161 0,906 0,575 0,186 0,000 -0,500 0,000 0,500 1,000 1,500 2,000 2,500 0 1 2 3 4 5 6 7 8 9 w o u n d d ia m e te r dayaverage decrease in wound diameter k+ meoh ethac chcl₃ k0% 20% 40% 60% 80% 100% 120% 0 1 2 3 4 5 6 7 8 p e r c e n ta g e dayaverage percentage of wound diameter healing k+ meoh ethac chcl₃ k umarella et al. – effectiveness test of methanol, ethyl acetate, and chloroform fractions … 429 on day 2 until the maximum healing rate was reached, it was shown by the ethac fraction. the ethac fraction group which has the highest percentage of healing based on the area and diameter of the incision wound shows the best potential as a wound healing agent. based on the results of testing the effect of research groups on wound diameter reduction, the meoh and ethac groups could heal wounds the fastest, followed by the positive control and chcl3 fraction, and finally the negative group. the fractionated sample from the thistle leaf showed faster healing because povidone iodine 10%, which has less healing effect, was used as a positive control. povidone iodine is a commonly used antimicrobial agent that contains polyvinylpyrrolidone iodine, a water-soluble complex with elemental iodine bound to a synthetic polymer. in vitro evidence shows that iodine not only has broad antimicrobial effects but also suppresses inflammation caused by pathogens and host responses. this includes anti-inflammatory effects, which are considered multifactorial and have been shown to be clinically relevant. based on the results of phytochemical screening, the meoh fraction contains the highest amount of active components such as saponins, tannins, and steroids compared to other fractions, so that it has a good healing rate. on the other hand, due to the high content of flavonoids and phenols, the ethac fraction has a good healing rate based on phytochemical screening and total content test compared to other fractions. the content of these active ingredients has advantages in supporting wound healing. saponins have a mechanism to increase hemolytic activity and play a role in antibacterial, antiviral, and antioxidant properties. by increasing the ability of tgf-β receptors, fibroblasts can bind tgf-β strongly (karliana & wikanta, 2019). tannins play an important role in the transcription and translation process of vascular endothelial growth factor (vegf). steroids by increasing the rate of epithelial formation in the body (dewi & wicaksono, 2020). phenolic compounds and flavonoids have antioxidant, anti-inflammatory, antibacterial, and antifungal properties that prevent tumor growth. flavonoids have a wound healing mechanism with a protective effect against reperfusion of body tissues caused by ischemia. the antioxidant properties of flavonoids can reduce the degree of lipid peroxidation and promote the reepithelialization process. its astringent and antibacterial functions play an important role in wound contraction and increase the rate of body epithelialization (dewi & wicaksono, 2020; kemalasari et al., 2018). many studies have shown that flavonoids have wound healing properties due to their well-known anti-inflammatory, angiogenesis, re-epithelialization, and antioxidant effects. they are able to act on the wound healing process through the expression of biomarkers of each pathway which mainly include wnt/β-catenin, hippo, transforming growth factor-beta (tgf-β), hedgehog, c-jun n-terminal kinase (jnk), nf-e2-related factor 2/antioxidant responsive element (nrf2/are), nuclear factor kappa b (nf-κb), mapk/erk, ras/raf/mek/erk, phosphatidylinositol 3-kinase (pi3k)/akt, nitric oxide (no) (zulkefli et al., 2023). polyphenols are a major component of plant metabolites and are important in the human health system. the healing properties of these phenolic compounds are mainly due to their strong antibacterial, anti-inflammatory, and antioxidant activities, and these activities are positively correlated with each other. they play an important role in enhancing healing activity by forming a barrier against microorganisms, reducing the chance of wound infection, and protecting cells from damage caused by reactive oxygen species (ros). several natural phenols such as flavonoids rutin and quercetin, phenolic acids, chlorogenic acid, carotenoids, and curcumin are known to have various therapeutic activities. these compounds also aid wound healing due to their free radical scavenging, antioxidant, lipid peroxidation inhibitor, immunomodulatory, angiogenic, antibacterial, and neurogenic properties (micale et al., 2020). phenol also acts as a wound antiseptic, which kills bacteria by denaturing bacterial cell proteins and plays a role in the epithelialization process by stimulating the regeneration process of skin tissue in the wound so as to accelerate wound closure with new skin (karliana & wikanta, 2019). the negative control group, dmso, which was used as a carrier solution in the fraction samples, showed the lowest wound healing effectiveness. the use of this negative control is intended to measure physiological wound healing in the body and make it possible to estimate the time it takes for a wound to heal without treatment. in addition, the negative control was also conducted to determine whether the carrier solution affects wound healing. data analysis data processing was carried out based on the test data obtained to determine whether there were differences in the effect of incision wound healing in each treatment group. tests carried out first test normality using the shapiro-wilk test and homogeneity test. the data obtained were normally and homogeneously distributed because it showed that (p>0.05). table 3. one-way anova test. treatment significance value (p) diameter 0.004 the anova test results of wound healing from meoh fraction, ethac fraction, chcl3 fraction, positive control, and negative control showed a significance value of p<0.05, which means there is a significant difference between wound healing from each test sample (table 3). 430 biology, medicine, & natural product chemistry 13 (2), 2024: 423-431 table 4. tukey hsd test result. treatment group comparisons significance values (p) description positive control meoh fraction ethac fraction chcl3 fraction negative control 0.630 0.135 0.693 0.056 no different no different no different significant difference meoh fraction positive control ethac fraction chcl3 fraction negative control 0.630 0.765 0.029 0.006 no different no different significant difference significant difference fraksi ethac positive control meoh fraction chcl3 fraction negative control 0.135 0.765 0.018 0.001 no different no different significant difference significant difference fraksi chcl3 positive control meoh fraction ethac fraction negative control 0.693 0.029 0.018 0.528 no different significant difference significant difference no different negative control positive control meoh fraction ethac fraction chcl3 fraction 0.056 0.006 0.001 0.528 significant difference significant difference significant difference no different based on the tukey test results in table 4, the wound healing by the tested samples in the meoh and ethac fraction showed similar results. however, qualitatively, the ethac fraction has a good healing level based on phytochemical screening compared to other fractions shown and confirmed by testing the total flavonoid and phenol content. the similarity of healing results in meoh and ethac fractions can be influenced by the similarity of flavonoids contained in both so that they show the same effectiveness in healing cuts. conclusions methanol, ethyl acetate, and chloroform fractions of bidara (zizyphus mauritiana l.) leaf extract have effectiveness in healing incision wounds in rabbits with wound healing ranging from 5-8 days, with compounds involved in the healing process in the form of flavonoids, phenols, saponins, tannins and steroids. the ethyl acetate fraction has a better effect in narrowing the wound based on the percentage of healing of the incision wound through a reduction in wound diameter compared to the methanol fraction and chloroform fraction. acknowledgements: i would like to thank the undergraduate program in pharmacy, university of muhammadiyah pekajangan pekalongan for giving me the opportunity to conduct this research. i would also like to thank my advisor, wirasti, s.si., m.sc., for her constructive and valuable suggestions in guiding me. authors’ contributions: novel umarella designed the study, carried out the laboratory work and analyzed the data. novel umarella, wirasti, slamet and urmatul waznah proofread, reviewed and edited the manuscript. competing interests: the authors declare that there are no competing interests. references abubakar, a. r., & haque, m. 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(2023). flavonoids as potential wound-healing molecules: emphasis on pathways perspective. international journal of molecular sciences, 24(5). https://doi.org/10.3390/ijms24054607 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 259-266 | doi: 10.14421/biomedich.2023.121.259-266 issn 2540-9328 (online) combination interactive effects of gongronema latifolium leaves and picralima nitida seeds extracts on glucose tolerance maureen ifeyinwa egbuniwe1, harrison anezichukwu ozoani1, amara anwuchaepe ajaghaku2, ikechukwu sonne mbagwu3, uchechukwu harrison orji3, daniel lotanna ajaghaku1,* 1department of pharmacology, faculty of pharmaceutical sciences, enugu state university of science and technology, enugu, nigeria 2department of pharmacognosy, faculty of pharmaceutical science, chukwuemeka odumegwu ojukwu university, igbariam, anambra, nigeria. 3department of pharmacology, faculty of pharmaceutical sciences, nnamdi azikiwe university awka, anambra state, nigeria. corresponding author* daniel.ajaghaku@esut.edu.ng manuscript received: 13 december, 2022. revision accepted: 13 march, 2023. published: 18 march, 2023. abstract this study evaluated the combination interactive effects of g. latifolium leaves and p. nitida seed extracts using a metabolic glucose tolerance test. the plant samples were extracted separately using cold maceration and their acute toxicities were determined. doseresponse glucose tolerant tests of both plants were done using a 2 g/kg glucose load monitored over 0 – 1h. a 41% effect isobologram was used to determine the needed dose combinations according to the principle of loewe’s additivity model. the glucose tolerant tests of dose pairs of the combined extracts were evaluated and their combination indices were calculated to determine the nature of their interaction. the ed41 of g. latifolium (gl) and p. nitida (pn) were 180 mg/kg and 254 mg/kg respectively. the percentage reductions of gl:pn (50:160); gl:pn (100:90); and gl:pn (150:30) dose pairs were 48.23, 50.76 and 42.99 % respectively. their combination index were calculated to be 0.91, 0.91 and 0.95 respectively an indication of synergistic interaction. findings from this study validate the combined use of g. latifolium leaves and p. nitida seeds in folkloric medicine. however, combining the extracts of g. latifolium: p. nitida in the dose ratios of 50:160, 100:90 and 150:30 mg/kg gave the best dose pairs with synergistic outcome. keywords: combination studies; gongronema latifolium; glucose tolerance; picralima nitida. introduction plants have evolved over millennia to address the multifactorial nature of disease pathogenesis by targeting multiple pathways associated with disease initiation, progression and complications through the combined action of structurally and functionally diverse constituents (zaynab et al., 2018). this diversity in plants is better harnessed when they are used in crude unfractionated forms and in combination (che et al., 2013). it is however too simplistic to assume a positive synergy from any two or more herbs combined in traditional practice as some may lead to an unexpected decrease in activity due to competition for the same site of action or through interfering with the pharmacokinetics of each other (sun et al., 2019). similarly, two plants when combined can exhibit both antagonism and synergism depending on the dose pair that is used (foucquier and guedj, 2015). the benefit of combination therapy is not simply attributed to the properties of the drugs but could also depend on the dose ratio. as the cells do not make the difference between a single dose and a combination, two drugs combined at a given dose ratio could be considered as a third agent with its own dose-effect relation (chou, 2010). the establishment of optimal pair of dose levels for each plant extract intended to be used in combination therapy is therefore indispensable in maximizing the treatment efficacy of combination therapy. the use of optimal dose pairs in combination therapies exploits the chances for improved efficacy and decreased adverse effects (podolsky & greene, 2011). thus, owing to these advantages, combination therapies have become a standard for the treatment of several diseases (raskin, 2008). as such, they continue to represent a promising approach in indications of unmet medical needs. glucose intolerance is a general term for a group of metabolic conditions that result from hyperglycemia. it is a dysglycemic state that comprises both intermediate hyperglycemia (formerly called prediabetes) and diabetes. the major categories of glucose intolerance are as follows: impaired fasting glucose (ifg), impaired glucose tolerance (igt), and diabetes mellitus (dm). ifg and igt are intermediate conditions in the transition between normality (normal glucose tolerance, ngt) and diabetes (who, 2021). the development of glucose intolerance is usually initiated by insulin resistance and https://doi.org/10.14421/biomedich.2023.121.259-266 260 biology, medicine, & natural product chemistry 12 (1), 2023: 259-266 worsened by compensatory hyperinsulinemia (wang et al., 2019). pancreatic beta-cell dysfunction and/or hepatic and muscle insulin resistance are primary defects responsible for the development and progression of type2 diabetes. impaired glucose tolerance represents an early stage in the development of type-2 diabetes. although it can present as the asymptomatic condition with subtle changes in fasting and/or postmeal serum glucose concentration, it is an important indicator in identifying patients at high risk of developing type-2 diabetes (alok et al., 2018). several studies have demonstrated the medicinal and beneficial effects of gongronema latifolium and picralima nitida plants. both plants are members of the apocynaceae family which have been used in traditional medicines for the treatment and management of malaria, abscesses, hepatitis, pneumonia, diabetes, hypertension, etc. (decampos et al., 2020). the leaves of g. latifolium and seeds of p. nitida have been specifically reported to possess glucose lowering potential both in diabetes and normoglycemic in-vivo experimental models (mohammed et al., 2014). however, none of these reports have considered a combined effect of both plants, a common practice in traditional medicine. this study evaluated the combination interactive effects of g. latifolium leaves and p. nitida seed extracts using a metabolic glucose tolerance test (mgtt) in an animal model. materials and methods collection of plant materials the seeds of picralima nitida and leaves of gongronema latifolium were obtained from nsukka, enugu state, nigeria. the plant samples were identified by mr felix nwafor, a taxonomist of the department of pharmacognosy and environmental medicine, university of nigeria nsukka (unn), enugu state, nigeria. animals wistar albino mice (20 – 25 g) were obtained from the animal house of the department of pharmacology, faculty of pharmaceutical sciences, enugu state university of science and technology, enugu state, nigeria. the animals were housed in standard laboratory conditions of 12 h light, room temperature, 40-60% relative humidity and fed with rodent feed (guinea feeds nigeria ltd). they were allowed free access to food and water. all animal experiments were conducted in compliance with the nih guide for the care and use of laboratory animals (national institute of health (nih) (2011) pub no: 85-23). preparation of plant extracts the pods of picralima nitida were opened and the seeds obtained were air-dried under a shade for 30 days. similarly, the fresh plant sample of gongronema latifolium was collected from the wild fields and the leaves were detached from the unwanted parts of the plant. the leaves were air dried under a shade at room temperature for 15 days. after air-drying under a shade, the seeds and leaves were pulverized to a coarse powder using an electrical blender. pulverized samples of picralima nitida (1kg) and gongronema latifolium (1.6 kg) were macerated separately in 1.5 l each of methanol at room temperature for 72 hours with intermittent shaking. the extracts were first filtered, using a cotton wool clogged funnel and then filtered severally through whatman no. 1 filter papers. the extracts were concentrated by evaporating the methanol to dryness under reduced temperature and pressure (below 40oc) using a rotary evaporator. the percentage yields of the extracts were determined and then transferred into air-tight containers and stored in a refrigerator until required for experimentation. qualitative phytochemical analysis the qualitative phytochemical analysis of the extracts was carried out using standard methods as described by odoh et al., 2019. acute toxicity study the acute toxicities of both samples were done using lorke’s method (lorke, 1983). this method has two phases which are phases 1 and 2 respectively. phase 1: in this phase, nine animals were randomly selected to represent the g. latifolium group and another nine were selected to represent the p. nitida group. for each group, the nine animals were divided into three subgroups containing three animals per sub-group. members of each of the three sub-groups were administered 10 mg/kg, 100 mg/kg and 1000 mg/kg of each of the plant samples respectively. the animals were then placed separately under 24-hour observation to monitor for any sign of behavioral changes as well as mortality that may suggest toxicity. phase 2: this phase involved the use of three animals per plant sample. these animals were distributed into sample groups of one animal per group. the animals were then administered higher doses (1600, 2000 and 5000 mg/kg) for g. latifolium and 300, 500 and 800 mg/kg for p. nitida. they were observed for 24h post treatment for signs of toxicity as well as mortality. then the ld50 was determined using the following equation: 𝐿𝐷 = √(𝐷0 ∗ 𝐷100) (1) d0 : highest dose that gave no mortality, d100 : lowest dose that produced mortality. dose response glucose tolerance effect of extracts of g. latifolium leaves and p. nitida seeds the animals were fasted overnight before the test and their fasting blood glucose levels were measured (fbg). egbuniwe et al. – glucose tolerance herbal combination interaction 261 1hr after the extracts were given, the animals were administered 2 g/kg d-glucose in distilled water. blood samples were taken by tail milking at 15, 30, 45 and 60 minutes after glucose administration and the glucose concentrations were determined. in comparison to the control group, the area under the curve (auc) of the plot of blood glucose against time was used to measure metabolic glucose tolerance. for dose-response effect of methanol extract of g. latifolium leaves, the animals were grouped into 5 groups of 5 mice each. they fasted for 12 h, but water was provided ad libitum before the experiment. the fasting blood glucose of each animal was determined before extracts were administered. groups 1-5 were treated with 12.5, 25, 50, 100, and 200 mg/kg, respectively. change in blood glucose levels was accessed for each animal at 15, 30, 45 and 60 minutes post-treatment. the percent change in blood glucose for each animal was calculated and the average for each group was determined. for p. nitida, the animals were grouped into 4 groups of 5 mice each. they fasted for 12 h, but water was provided ad libitum before the experiment. the fasting blood glucose of each animal was determined before extracts were administered. groups 1-4 were treated with 100, 200, 400, and 800 mg/kg, respectively. change in blood glucose levels was accessed for each animal at 15, 30, 45 and 60 minutes post-treatment. the percent change in blood glucose for each animal was calculated and the average for each group determined. the animals for the control groups were grouped into 2 groups of 5 mice each. group 1 received 5 ml/kg distilled water and served as a negative control, and group 2 received 250 mg/ kg metformin and served as the positive control. the same procedure was carried out for the control groups. combination effect of methanol extract of g. latifolium leaves and p. nitida seeds twenty –five (25) mice grouped into 5 groups of 5 animals per group were used for the combination interaction study. they fasted for 12 h before the experiment. the fasting blood glucose of each animal was determined before extract and drug administration. they were treated with combination doses of g. latifolium: p. nitida as follows: group 1 (91:126.5 mg/kg), group 2 (110:100 mg/kg), group 3 (50:160 mg/kg), group 4 (100:90 mg/kg) and group 5 (150:30 mg/kg). effects of these treatments on blood glucose were evaluated at 15, 30, 45 and 60 minutes for each animal and the average for each group was determined. evaluation of the nature of the interaction of the combination therapy using a dose-effect-based strategy, the interactions of various ratio combinations of p.nitida and g.latifolium extracts were determined. the percentage effect value of the extracts at different concentrations and when combined in different ratios were used in the formula to calculate their combination index. various ratio combination interactions between p. nitida and g. latifolium extracts were determined using a dose-effectbased strategy (loewe additivity). the percentage effect value of the extracts at different concentrations separately and when combined in different ratios were used for the calculation of their combination index using the formula. ci = a a + b b (2) where: a = dose of g. latifolium in the combination and a = effective dose of g. latifolium; while b =dose of p. nitida in the combination and b = effective dose of p. nitida, ci= combination index. when ci= 1 (additive interaction), ci > 1 (antagonistic interaction) and ci < 1 (synergistic interaction). statistical analysis all values were expressed as the mean + standard error of the mean (sem) of five animals per group. data were analyzed using one-way anova. p values less than 0.05 was considered statistically significant. graphical plots were done using microsoft excel 2010. results extraction, yield and phytochemical analysis methanol extracts of g. latifolium produced a higher yield compared with p. nitida (table 1) saponins, alkaloids, tannins, steroids, and flavonoids were present in high amounts in both samples. quinones were not detected in the sample of picralima nitida but were present in high amounts in the sample of gongronema latifolium. glycosides were present in high amounts in the methanol extract of g. latifolium but undetected in the sample of p. nitida. lastly, anthraquinones were not detected in both samples. table 1. extraction yields and phytochemical content of the extracts. phytochemical constituents gongronema latifolium picralima nitida saponins +++ +++ flavonoids +++ +++ steroids ++ ++ alkaloids +++ +++ glycosides +++ quinones +++ anthraquinones tannins ++ ++ yield% 24.69% 15.59% key: +++ = high amount of phytoconstituents present;++ = moderate amount of phytoconstituents present;+ = low amount of phytoconstituents present;– = no phytoconstituents detected 262 biology, medicine, & natural product chemistry 12 (1), 2023: 259-266 acute toxicity study in the gongronema latifolium study group, there was no mortality or any signs of behavioral changes or toxicity observed in the mice after oral administration of gongronema latifolium up to the dose of 5000 mg/kg body weight. as such, the ld50 can be said to be greater than 5000 mg/kg body weight. in the picralima nitida study group 100% mortality was observed at 1000 mg/kg dose group in the first phase of the study. using lower dose levels of 300 mg/kg, 500 mg/kg and 800 mg/kg in the second phase, no mortality or any sign of toxicity was observed. hence the lethal dose was above 800 mg/kg. furthermore, using lorke’s method (1983) to calculate the ld50, the ld50 was observed to be approximately 894.4 mg/kg glucose tolerance individual effects of g. latifolium and p. nitida peak hyperglycemia was observed 15 minutes post glucose load and declined continuously thereafter (figures 1a and 2a). at 15 minutes post glucose administration, p. nitida at 200, 400 and 800 mg/kg produced a significant (p < 0.05) reduction in blood glucose levels (bgl) compared to the negative control group just as was recorded for the positive control (p = 0.001). at 30 minutes post-administration, significant differences in bgls were only observed with 400 mg/kg (p = 0.02) and 800 mg/kg (p <0.001) of p. nitida as well as the positive control (p <0.001). at 45 minutes postadministration, a weak significant difference in bgls was observed at 400 mg/kg (p = 0.05) and 800 mg/kg (p = 0.039) of p. nitida; as well as at 200 mg/kg of g. latifolium (p = 0.045). a strong significance difference in bgl was observed for the positive control (p < 0.01). at 60 minutes of post-administration, only the bgl of the positive control was significantly different (p = 0.042) from the bgls of the negative control group. when comparing the area under curve (auc) of the negative control group with the different treatment groups, there was a significant difference in auc of g. latifolium at 100 mg/kg (p = 0.044) and 200 mg/kg (p = 0.038) doses, as well as the positive control (p <0.001). there was no significant difference in the auc of g. latifolium at 12.5, 25 and 50 mg/kg (p >0.05) (figure 1b). there was also a significant difference in auc between the negative control group and p. nitida at doses of 200 mg/kg (p = 0.033), 400 mg/kg (p <0.001) and 800 mg/kg (p < 0.01) but no significant difference at 100 mg/kg (p >0.05) (figure 2b). the percentage glucose reduction in auc of both treatments were presented in figures 1c and 2c. figure 1. effect of g. latifolium on glucose tolerance. * p<0.05 compared to 5 ml/kg distilled water vehicle control group (negative control). 0 50 100 150 200 250 300 350 0 10 20 30 40 50 60 70 b lo o d g lu co se c o n ce n tr at io n ( m g/ d l) time (min) 12.5 mg/kg 25 mg/kg 50 mg/kg) 100 mg/kg 200 mg/kg 5 ml/kg distilled water 250 mg/kg metformine 0 2000 4000 6000 8000 10000 12000 14000 m ea n a u c 0 10 20 30 40 50 60 70 80 90 100 m ea n % r ed u ct io n egbuniwe et al. – glucose tolerance herbal combination interaction 263 figure 2. effect of p. nitida on glucose tolerance. * p<0.05 compared to 5 ml/kg distilled water vehicle control group (negative control). glucose tolerance effect of the extracts in combination using the loewe additivity combination strategy (equation 2) complemented with isobologram analysis we defined all the pairs of doses of g. latifolium and p. nitida that could lead to the combination effect eab to form additivity and these were drawn as the line of additivity of negative slope on a graph where the x and yaxis represent the dose of g. latifolium and p. nitida (figure 3a). this representation makes clear that when g. latifolium is present at the selected effective dose the quantity of p. nitida needed to reach the specified level is 0, and that the presence of p. nitida reduced the need for g. latifolium in a quantity predicted by the model. all experimental points below the line that could give the desired effect correspond to a combination index (ci) < 1 and indicate synergism. peak hyperglycemia was also observed at 15 minutes just like in the dose response test of the individual extracts (figure 3b). all the combination doses exhibited significant reduction (p<0.05) in blood glucose compared with the negative control group except gl:pn (91:126.5) dose pair (figure 3c). gl:pn (91:126.5) and gl:pn (110:100) combination dose pairs gave the lowest percentage reductions of 29.57 ± 5.4 and 36.59 ± 5.93% respectively while gl:pn (50:160); gl:pn (100:90); and gl:pn (150:30) combination dose pairs were 48.23 ± 3.28; 50.76 ± 4.77 and 42.99 ± 6.88% respectively. the first two combination doses were expected to give an additivity effect based on the model. however, they could not produce the set percentage effective reduction in blood glucose auc which was 41%. since the effects produced from these combinations were below the effective value, these combinations were considered suboptimal. contrary to the behavior of the first two dose pairs, the last three dose pairs produced effects above the set value (41%) and their combination index were calculated to be 0.91, 0.91 and 0.95 respectively an indication of synergistic interaction. 0 50 100 150 200 250 300 350 0 10 20 30 40 50 60 70 b lo o d g lu co se c o n ce n tr at io n ( m g/ d l) time (min) 100 mg/kg 200 mg/kg 400 mg/kg 800 mg/kg 5 ml/kg distilled water 250 mg/kg metformin 0 2000 4000 6000 8000 10000 12000 14000 m ea n a u c 0 10 20 30 40 50 60 70 80 90 100 100 mg/kg 200 mg/kg 400 mg/kg 800 mg/kg 250 mg/kg metformin m ea n % r ed u ct io n 264 biology, medicine, & natural product chemistry 12 (1), 2023: 259-266 figure 3. effect of combined doses of p. nitida and g. latifolium on glucose tolerance. * p<0.05 compared to 5 ml/kg distilled water vehicle control group (negative control). discussion control of postprandial hyperglycermia is an essential component of diabetes management. the postprandial pattern of glucose metabolism after glucose load and solid mixed meal containing carbohydrate, fat and protein have been found to be virtually the same and this validates studies like ours employing glucose loads to pertain to those observed after mixed meals (dimitridia et al, 2021). the liver sees the largest change in blood glucose and insulin after a meal and its role is to restrain their acute rise in the bloodstream. the spillover of glucose to the peripheral circulation requires a substantial increase in systemic insulin level and insulin secretion. in the long term, this mechanism provides insight into how a high glycemic index/load of carbohydrate diet producing sustained high insulin secretion could contribute to the development of insulin resistance – the negative consequences of hyperinsulinemia (shanik et al., 2008). under hyperglycermic conditions, the liver uses several mechanisms to bring about glucose homeostasis. these include increased glucose uptake in the hepatocytes, increased activity of glucose utilizing pathways like glycolysis, pentose monophosphate shunt and glucose storage in form of glycogen by glycogenesis. also, acetyl-coa generated from the glycolysis-driven pyruvate pathway is used for fatty acids production which can be transported to adipose tissue for storage (han et al., 2016). agents enhancing these liver mediated activities have been shown to lower post-prandial high blood glucose and are useful in the management of diabetes (rines et al., 2016). g. latifloium extract has been shown to exhibit insulin like activities in the liver by increasing the flux of glucose into the glycolytic pathway and pentose monophosphate shunt in the liver of hyperglycemic animal model (ugochukwu and babady, 2003). these activities were reported to be mediated through increased activities of hexokinase the first enzyme of glycolysis and phosphofructokinase, the rate-determining enzyme of glycolysis. also the activities of glucose-6-phosphate 0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 160 180 p ic r a li m a n it id a gongrenema latifolium 41% effect isobologram additivity (ci =1) synergy (ci˂1) antagonism (ci >1) a * 0 50 100 150 200 250 300 350 0 20 40 60 80 b lo o d g lu co se co n ce n tr at io n ( m g/ d l) time (min) gl:pn(91:126.5) gl:pn(110:100) gl:pn(50:160 gl:pn(100:90) gl:pn(150:30) 5 ml/kg distilled water 250 mg/kg metformin 0 2000 4000 6000 8000 10000 12000 14000 m ea n a u c 0 10 20 30 40 50 60 70 80 90 100 m ea n % r ed u ct io n egbuniwe et al. – glucose tolerance herbal combination interaction 265 dehydrogenase (g6pd) a key enzyme in the pentose phosphate pathway has been shown to be increased by g. latifolium (ugochukwu and babady, 2003) in addition to its reported ability to increase liver glycogen synthesis (ajiboye et al., 2019). similarly, p. nitida extract has been shown to promote glucose uptake in the liver through the activation of glucokinase enzyme that plays a critical catalytic role in promoting the addition of a phosphate to glucose, thereby enhancing its uptake into the liver (guzman and gurrola-diaz, 2019). glucose transport is an important step in glucose disposal by peripheral tissues because it controls the rate of glucose utilization in these tissues. in skeletal muscle and adipose tissues, the glucose transporters isoforms expressed are glut1, glut3 and glut4 (shepherd et al., 1999). in the postprandial state, insulin increases the rate of glucose transport mainly by stimulating the translocation of the glut4 isoform from the intracellular pool to the cell membrane. improvement in the sensitivity of tissues to this insulin action avoids marked hyperglycemia and hyperinsulinemia under postprandial conditions. g. latifolium extract was reported to increase muscle glucose uptake significantly in the presence of insulin, indicating an insulinsensitizing potential as well as increased expression and translocation of glut4 glucose transporter (al-hindi et al., 2014; ajiboye et al., 2019). indole alkaloids were the first set of alkaloids isolated from p. nitida: akuamine, pseudoakuamine, akuammidine, akuammicine, akuammigine, pseudoakuammigine, akuammiline, akuamminine and pseudoakuammicine (mawout et al., 2020). most of the effects of p. nitida including modulation of glucose metabolism have been attributed to its rich alkaloid content. stimulation of glucose uptake is one of the known mechanisms of alkaloids containing plant’s hypoglycermic effects (mawout et al., 2020). several alkaloids are allosteric activators of adenosine monophosphate protein kinase – a cellular fuel sensor enzyme and glucose transporter regulator (kumar et al., 2019; song et al., 2021). similarly, alkaloids have been shown to increase the translocation of glut4 and enhancement of glucose-stimulated insulin secretion (kumar et al., 2019). an increase in insulin sensitivity of multiple tissues like the liver, adipose tissue and skeletal muscles by alkaloids has been reported to be mediated through the inhibition of protein tyrosine phosphatase-1b – an enzyme involved in the negative regulation of insulin signal transduction pathways (adhikari, 2021). akuammicine, an indole alkaloid isolated from p. nitida has also been shown to stimulate glucose uptake in adipocytes (shittu et al., 2010). the improved glucose tolerance effect of the individual plant extracts may be attributed to their phytocompounds and through hypoglycemic mechanisms already reported. the multiple targets of regulation of glucose homeostasis by both plants may be responsible for their superior effect when combined. conclusion findings from this study support the known improved glucose tolerance effects of g. latifolium leaves and p. nitida seeds as well as the rationale for their combined use in folkloric medicine. however, combining the extracts of g. latifolium: p. nitida in the dose ratios of 50:160, 100:90 and 150:30 mg/kg gave the best dose pairs with synergistic outcome. competing interests: the authors declare that there are no competing interests. references adhikari, b. 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(2018). role of secondary metabolites in plant defense against pathogens. microb. pathog.124:198–202. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1177-1184 | doi: 10.14421/biomedich.2025.142.1177-1184 issn 2540-9328 (online) effectiveness of traditional remedies pucuk jarak and bedak langeh in melongas massage on the growth and development of stunted toddlers widani darma isasih*, junendri ardian department of nutrition, faculty of health, universitas bumigora jl. ismail marzuki no.22, cilinaya, kec. cakranegara, kota mataram, nusa tenggara bar. 83127, tel. (0370) 634498, indonesia. corresponding author* widani.darma@universitasbumigora.ac.id abstract stunting is a serious health problem among toddlers, characterized by delayed physical growth due to inadequate nutrition, a history of infectious diseases, low maternal education, and suboptimal parenting. in north lombok regency, the prevalence of stunting remained high at 19.3% in 2023. efforts to address this issue have not only focused on nutritional interventions but also included complementary non-pharmacological therapies, such as melongas massage, which has been traditionally practiced using pucuk jarak and bedak langeh remedies. this study aimed to examine the effectiveness of these two remedies on the growth and development of stunted toddlers. a quasi-experimental nonequivalent control group design was applied, involving 72 stunted toddlers divided into three groups: pucuk jarak, bedak langeh, and control. the intervention was carried out for four weeks with melongas massage administered twice weekly. the measured parameters included body weight, height, appetite, sleep quality, and immune function. the results showed that the pucuk jarak group experienced more significant improvements across all parameters compared to the bedak langeh and control groups. phytochemical tests supported these findings, showing total flavonoid levels of 53.32 mgqe/g in pucuk jarak compared to 3.58 mgqe/g in bedak langeh. the high flavonoid content is believed to play an important role in improving metabolism, appetite, growth, and immunity in toddlers. thus, melongas massage using pucuk jarak has the potential to serve as a complementary strategy rooted in local wisdom for accelerating stunting reduction programs in north lombok. keywords: herbal massage formulation; stunting; toddler massage; pucuk jarak; traditional. introduction stunting is a growth disorder in children under five years old, marked by a shorter height compared to their age (schneider, 2025). it can be caused by low energy intake, long-term illness, low birth weight, low maternal education, low family income, insufficient protein intake, and lack of exclusive breastfeeding (isasih, fajriani, et al., 2024). in 2023, there were 149.2 million stunted children worldwide (22%); in indonesia, 21.5%; in west nusa tenggara, 24.6%; and in north lombok regency, 19.3% (astuti et al., 2025). the government has addressed stunting through food supplements, iron tablets, sanitation improvements, deworming, and nonpharmacological complementary therapy (isasih, inayati, et al., 2024). one such therapy is toddler massage (widianti, 2025). regular massage can increase catecholamine hormones (epinephrine and norepinephrine), stimulating growth and development by improving appetite, weight gain, and brain development (fifit & luvi dian afriyani, 2023). previous studies showed that toddler massage accelerates gastric emptying, induces hunger, relaxes the body, improves sleep quality, and enhances nutrient absorption for optimal growth (dessie et al., 2024; sofiana et al., 2025). in indonesia, toddler massage is widely practiced, including in north lombok, where it is called melongas. this traditional practice, passed down through generations, involves traditional massage therapists using herbal remedies such as jarak mixture (young jarak leaves and candlenut) and bedak langeh (a mixture of grated coconut, rice flour, and turmeric) (isasih et al., 2025). these herbs, sourced locally, are believed to aid children with illnesses or developmental delays such as late sitting, crawling, walking, or standing. their effectiveness is closely associated with the ability of flavonoids to induce relaxation in smooth muscle fibers, a process that also involves modulation of muscarinic receptor activity, thereby contributing to improved physiological regulation of muscle tone and overall organ function (adibah et al., 2025; makkayu et al., 2025; rahayu et al., 2024; tridesianti et al., 2025). stunting remains a serious health problem in north lombok (19.3%). thus, comprehensive efforts are needed, including non-pharmacological complementary therapies. melongas massage, using pucuk jarak and manuscript received: 29 september, 2025. revision accepted: 27 november, 2025. published: 09 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1177-1184 1178 biology, medicine, & natural product chemistry 14 (2), 2025: 1177-1184 bedak langeh, is widely believed to be beneficial, but no scientific evidence yet confirms its effectiveness. this research aims to evaluate the impact of traditional herbal remedies used in melongas massage on the growth of stunted children. the study not only measures clinical outcomes but also examines total flavonoid levels, ensuring safety, acceptability, and bioactive content. its novelty lies in being the first of its kind, providing a scientific foundation for traditional practices and offering a locally based alternative for accelerating stunting reduction. materials and methods study area this study employed a quantitative research method with a quasi-experimental approach using a nonequivalent control group design (norman & roggman, 2025). the research was conducted in kroya hamlet, tanjung village, tanjung subdistrict, north lombok regency, from july to august 2025. it consisted of an experimental group treated with melongas using pucuk jarak and bedak langeh formulations, and a control group receiving melongas without formulations, selected without randomization. the study population included all stunted toddlers in tanjung subdistrict, north lombok regency, totaling 72 children. sampling was conducted using the total population method, resulting in 24 children receiving pucuk jarak, 24 children receiving bedak langeh, and 24 stunted children assigned to the control group. procedures data collection began with testing the total flavonoid content using the uv-vis spectrophotometer method on all formulations applied in melongas massage (suryanis et al., 2022). the target was to compare the total flavonoid content. a pretest was then conducted for both experimental and control groups, measuring growth and development through body weight and height (anthropometric questionnaire), appetite (food frequency questionnaire/ffq), sleep quality (pittsburgh sleep quality index/psqi), and immunity. the goal of the pretest was to obtain a baseline profile of stunted children before treatment. the treatment phase involved melongas massage with pucuk jarak and bedak langeh formulations for the experimental group, while the control group received melongas massage without formulations. massages were given twice a week for 10– 15 minutes over four weeks, using gentle strokes with light pressure on the head, back of the neck, chest, back, arms, thighs, calves, and feet in one direction, as well as circular motions on the abdomen, palms, and soles to enhance circulation and provide relaxation (aryani et al., 2022). the massage was performed by experienced traditional therapists trusted by the community. the pucuk jarak formulation consisted of ground castor shoot leaves and candlenut, while the bedak langeh formulation was made from ground coconut, turmeric, and rice. both formulations were freshly prepared before each treatment. prior to treatment, therapists were briefed to ensure consistency in timing, technique, and massage areas. the treatment target was the successful application of melongas massage using traditional formulations in all experimental groups. posttests of growth and development were conducted every two sessions after the massage. the goal of the posttest was to identify which traditional formulation was most effective in improving the growth and development of stunted children. data analysis the study results were subsequently subjected to statistical analysis employing the two-way analysis of variance (two-way anova). results and discussion result this study was conducted on 24 children treated with pucuk jarak formulation, 24 children treated with bedak langeh, and 24 stunted children in the control group. the findings revealed the frequency distribution of the characteristics of stunted children, as presented in the following table: table 1. frequency distribution of characteristics of stunted children receiving melongas treatment in the intervention and control groups. characteristics of stunted children intervention group control groups pucuk jarak group bedak langeh group f % f % f % age (months) ≥ 24–35 7 29.2 3 12.5 12 50.0 36–46 8 33.3 13 54.2 6 25.0 47–59 9 37.5 8 33.3 6 25.0 total 24 100.0 24 100.0 24 100.0 gender male 15 62.5 18 75.0 9 37.5 female 9 37.5 6 25.0 15 62.5 total 24 100.0 24 100.0 24 100.0 breast milk exclusive breastfeeding 20 83.3 23 95.8 19 79.2 non-exclusive breastfeeding 4 16.7 1 4.2 5 20.8 total 24 100.0 24 100.0 24 100.0 isasih & ardian – effectiveness of traditional remedies pucuk jarak and bedak langeh … 1179 based on table 1, it can be seen that the frequency distribution of stunted toddlers given the melongas massage treatment shows that most were aged 47–59 months (37.5%) in the traditional remedies pucuk jarak group, 36–46 months (54.2%) in the bedak langeh group, and ≥24–35 months (50.0%) in the control group. the study also revealed that most stunted toddlers who received the melongas massage in the intervention groups were male: 62.5% in the traditional remedies pucuk jarak group, 75.0% in the bedak langeh group, while in the control group, the majority were female (62.5%). the results further showed that most stunted toddlers who received melongas massage had exclusive breastfeeding: 83.3% in the traditional remedies pucuk jarak group, 95.8% in the bedak langeh group, and 79.2% in the control group. total flavonoid content in traditional remedies pucuk jarak and bedak langeh used in melongas massage for stunted toddlers table 2. total flavonoid content in traditional remedies pucuk jarak and bedak langeh used in melongas massage for stunted toddlers. herbal mixture concentration of total flavonoids (mgqe/g) remedies pucuk jarak 53,32 bedak langeh 3,58 the phytochemical test results showed that the remedies pucuk jarak herbal mixture contained a much higher total flavonoid level compared to the bedak langeh mixture. the total flavonoid content in the remedies pucuk jarak mixture was recorded at 53.32 mgqe/g, while the bedak langeh mixture contained only 3.58 mgqe/g. the frequency distribution of body weight, height, appetite, sleep quality, and immune resilience among stunted toddlers who received the melongas massage in the traditional remedies pucuk jarak group, the bedak langeh group, and the control group is presented in the following table: table 3. frequency distribution of body weight, height, appetite, sleep quality, and immunity of stunted toddlers treated with melongas massage in intervention and control groups. category intervention group control groups pucuk jarak group bedak langeh group f % f % f % body weight increased 20 83.3 15 62.5 10 41.7 decreased 1 4.2 2 8.3 1 4.2 remained the same 3 12.5 7 29.2 13 54.2 total 24 100.0 24 100.0 24 100.0 height increased 18 75.0 13 54.2 4 16.7 decreased 6 25.0 11 45.8 20 83.3 total 24 100.0 24 100.0 24 100.0 appetite increased 20 83.3 11 45.8 8 33.3 decreased 1 4.2 6 25.0 3 12.5 remained the same 3 12.5 7 29.2 13 54.2 total 24 100 24 100 24 100 sleep quality increased 19 79.2 18 75.0 13 54.2 decreased 3 12.5 2 8.3 6 25.0 remained the same 2 8.3 4 16.7 5 20.8 total 24 100 24 100 24 100 immunity increased 19 79.2 18 75.0 13 54.2 decreased 3 12.5 2 8.3 6 25.0 remained the same 2 8.3 4 16.7 5 20.8 total 24 100 24 100 24 100 based on table 3, it can be observed that there was an increase in body weight among stunted toddlers in the intervention groups, namely 83.3% in the traditional remedies pucuk jarak group and 62.5% in the bedak langeh group. meanwhile, in the control group, most children had stable body weight, accounting for 54.2%. the results also showed an increase in height among stunted toddlers in the intervention groups, with 75% in the traditional remedies pucuk jarak group and 54.2% in the bedak langeh group. in contrast, the majority of the control group maintained the same height (83.3%). furthermore, an increase in appetite was observed in the intervention groups, with 83.3% in the traditional remedies pucuk jarak group and 45.8% in the bedak 1180 biology, medicine, & natural product chemistry 14 (2), 2025: 1177-1184 langeh group. meanwhile, in the control group, most toddlers maintained the same appetite (54.2%). the findings also revealed improvements in sleep quality and immunity among stunted toddlers across both intervention and control groups, recorded at 79.2% in the traditional remedies pucuk jarak group, 75% in the bedak langeh group, and 54.2% in the control group. the results of the normality test for the variables of body weight, height, appetite, sleep quality, and immunity of stunted toddlers who received melongas massage in the traditional remedies pucuk jarak group, the bedak langeh group, and the control group are presented in the following table: table 4. results of the normality test of body weight, height, appetite, sleep quality, and immunity variables in stunted toddlers in the intervention and control groups. category shapiro-wilk statistic df sig. body weight prepucuk jarak 0.997 10 0.899 postpucuk jarak 0.993 10 0.911 prebedak langeh 0.987 10 0.888 postbedak langeh 0.981 10 0.892 pre control 0.978 10 0.974 post control 0.954 10 0.87 height prepucuk jarak 0.987 10 0.976 postpucuk jarak 0.973 10 0.856 prebedak langeh 0.966 10 0.879 postbedak langeh 0.971 10 0.882 pre control 0.958 10 0.874 post control 0.964 10 0.864 appetite prepucuk jarak 0.987 10 0.735 postpucuk jarak 0.985 10 0.921 prebedak langeh 0.978 10 0.888 postbedak langeh 0.954 10 0.892 pre control 0.968 10 0.974 post control 0.984 10 0.87 sleep quality prepucuk jarak 0.997 10 0.899 postpucuk jarak 0.993 10 0.911 prebedak langeh 0.987 10 0.888 postbedak langeh 0.981 10 0.892 pre control 0.978 10 0.974 post control 0.954 10 0.87 immunity prepucuk jarak 0.955 10 0.735 postpucuk jarak 0.968 10 0.812 prebedak langeh 0.973 10 0.891 postbedak langeh 0.984 10 0.921 pre control 0.954 10 0.888 post control 0.987 10 0.892 based on table 4, it can be observed that the variables of body weight, height, appetite, sleep quality, and immunity among stunted toddlers who received the melongas massage treatment in both the intervention and control groups were normally distributed, as indicated by a significance value of >0.05 using the shapiro–wilk test. the effectiveness of traditional traditional remedies pucuk jarak and bedak langeh in the melongas massage for supporting the growth and development of stunted toddlers can be seen in the following table: isasih & ardian – effectiveness of traditional remedies pucuk jarak and bedak langeh … 1181 table 5. effectiveness of traditional traditional remedies pucuk jarak and bedak langeh in the melongas massage on the growth and development of stunted toddlers. category intervention group control group p value pucuk jarak group kelompok bedak langeh n min max mean n min max mean n min max mean body weight 0,021 pretest 24 5000 (gram) 11000 (gram) 10860 (gram) 24 4300 (gram) 10000 (gram) 90100 (gram) 24 4800 (gram) 10400 (gram) 8200 (gram) posttest 24 6500 (gram) 15000 (gram) 13840 (gram) 24 4970 (gram) 12000 (gram) 10900 (gram) 24 4350 (gram) 10900 (gram) 8800 (gram) height 0,011 pretest 24 58 cm 83 cm 88 cm 24 56 cm 86 cm 78 cm 24 58 cm 85 cm 74 cm posttest 24 73 cm 100 cm 97 cm 24 62 cm 92 cm 82 cm 24 59 cm 87 cm 74,8 cm appetite 0,028 pretest 24 13 17,2 15 24 14 16 14,3 24 13 17 15 posttest 24 17 24 22 24 15,8 18 16 24 13 18 15,7 sleep quality 0,035 pretest 24 18,3 23 22 24 17 22 21 24 17 22 21 posttest 24 24,8 28 27 24 19 23 22 24 17,8 22,4 21,6 immunity 0,033 pretest 24 13,2 18 16 24 14 16 14,3 24 13 17 15 posttest 24 17,6 26 24 24 15 18 16 24 13 18 15,7 based on table 4, it can be seen that in the initial measurement (pretest), the average body weight of respondents in the traditional remedies pucuk jarak group was 10,860 grams, in the bedak langeh group 9,100 grams, and in the control group 8,200 grams. after the intervention, body weight increased significantly, especially in the traditional remedies pucuk jarak group, reaching an average of 13,840 grams. meanwhile, the bedak langeh group increased to 10,900 grams, and the control group only rose slightly to 8,800 grams. statistical tests showed a p-value of 0.021, indicating significant differences between groups. the height measurement results showed that, at pretest, the average height in the traditional remedies pucuk jarak group was 68 cm, in the bedak langeh group 78 cm, and in the control group 74 cm. after the intervention, the average height in the traditional remedies pucuk jarak group increased to 97 cm, in the bedak langeh group to 82 cm, while the control group only reached 74.8 cm. the statistical test results yielded a p-value of 0.011, indicating significant differences between groups. the average appetite score in the traditional remedies pucuk jarak group rose sharply from 15 in the pretest to 22 in the posttest. in the bedak langeh group, the score increased from 14.3 to 16, while in the control group it only rose slightly from 15 to 15.7. a p-value of 0.028 indicated significant differences between the intervention groups and the control group.the quality of sleep also showed greater improvement in the traditional remedies pucuk jarak group, with the average score increasing from 22 in the pretest to 27 in the posttest. in the bedak langeh group, the score only rose from 21 to 22, while in the control group it remained relatively stable, from 21 to 21.6. statistical tests showed a p-value of 0.035, indicating significant differences between groups. the average immune system score in the traditional remedies pucuk jarak group increased from 16 to 24 after the intervention. in the bedak langeh group, the score rose only from 14.3 to 16, while in the control group it increased slightly from 15 to 15.7. statistical tests showed a p-value of 0.033, indicating significant differences between groups. overall, these findings indicate that the traditional remedies pucuk jarak intervention had a stronger effect on body weight, height, appetite, sleep quality, and immune function compared with the bedak langeh and control groups. discussion variations in age may influence the body’s response to the melongas massage intervention, as older children present more complex growth patterns and nutritional requirements. previous studies have noted that children aged 24–59 months are particularly vulnerable to stunting, given that they remain in a period of rapid growth and thus require adequate nutritional intake (isasih & inayati, 2024). this finding is consistent with earlier research indicating that boys are at greater risk of stunting than girls, a disparity attributed to their higher energy and macronutrient needs as well as increased susceptibility to infectious diseases (habibah et al., 2021). such gender, based differences in nutritional status and immune response may, in turn, influence the outcomes of interventions. furthermore, these observations align with prior evidence highlighting the importance of exclusive breastfeeding in mitigating the risk of stunting, as breast milk provides complete nutrition alongside immunological protection against disease (indah et al., 2024). nevertheless, cases of stunting persist even among exclusively breastfed 1182 biology, medicine, & natural product chemistry 14 (2), 2025: 1177-1184 children. this suggests that breastfeeding alone is insufficient; it must be complemented by balanced complementary feeding, effective parenting practices, and adequate stimulation for growth and development, as corroborated by other studies (dartiwen & aryanti, 2024). taken together, factors such as age, gender, and breastfeeding history illustrate the baseline conditions of stunted toddlers prior to intervention. existing literature supports the conclusion that children under five particularly boys are more susceptible to stunting, while exclusive breastfeeding remains critical in prevention, though it is not a singular determining factor (indah et al., 2024). importantly, the melongas massage, when integrated with pucuk jarak and bedak langeh, demonstrates continued effectiveness in enhancing growth status and overall health outcomes in stunted toddlers. the large difference in flavonoid levels shows that pucuk jarak has greater potential to provide health benefits, especially in terms of antioxidant activity and improving the immune system. flavonoids are bioactive compounds that can fight free radicals, strengthen the body’s defenses, and support growth and tissue repair (windi habsari, dita ayu lestari, 2025). the high flavonoid content in pucuk jarak is consistent with the intervention results, where the group that received this remedy showed improvements in body weight, height, appetite, sleep quality, and immunity. other studies also support this. for instance, a 96% ethanol extract of vernonia cinerea l. was found to contain 82.96 mgqe/g of flavonoids (hasyim et al., 2025), a result similar though higher than that of pucuk jarak. on the other hand, pumpkin flesh (cucurbita maxima d.) only had 8.8 mgqe/g, even lower than bedak langeh (abdunisa & hardini, 2024). this comparison shows that pucuk jarak has much higher flavonoid levels than several other plants, which may explain the significant improvements in growth and health observed in this study. the high flavonoid content is therefore considered one of the key factors behind the effectiveness of the intervention (sumarni & injiyahi, 2024). the results show that the pucuk jarak remedy was more effective than the other groups. its high flavonoid and bioactive compound content helps improve metabolism and appetite, which in turn supports weight gain. this agrees with earlier studies showing that herbal interventions can improve children’s nutrition by enhancing digestion and nutrient absorption (windi habsari, dita ayu lestari, 2025). the study also confirms that melongas massage combined with pucuk jarak and bedak langeh supports child growth. previous research found that traditional massage can stimulate growth hormone secretion, which aids linear growth in toddlers (hanifa, 2022). this effect may be linked to the much higher flavonoid level in pucuk jarak (53.32 mgqe/g) compared to bedak langeh (3.58 mgqe/g), which helps stimulate digestion and increase appetite. similar findings were reported in studies showing that flavonoid-rich herbs improve digestive enzyme activity and appetite in children (rahayu et al., 2024) better sleep quality also plays an important role in recovery, growth hormone balance, and immune strength. relaxation-based therapies such as massage have been shown to improve sleep and reduce children’s risk of infection, a major factor in stunting(arieska et al., 2025). overall, this study highlights that melongas massage with pucuk jarak provides stronger benefits for nutrition and health in stunted toddlers compared to bedak langeh or control groups. its superiority is linked to higher levels of bioactive compounds, especially flavonoids, which are known to support metabolism, growth, and immunity. this result matches earlier studies showing that child growth data often follow a normal distribution after nutrition and physical stimulation interventions. this makes it possible to use parametric tests such as the t-test or anova to analyze group differences (norman & roggman, 2025). normal data distribution is important because it increases the accuracy of the analysis and reduces bias. the findings also show that differences in body weight, height, appetite, sleep quality, and immunity were relatively even and without extreme variations. this suggests that the group differences are more likely caused by the melongas massage with traditional remedies, not by irregular data distribution. a good normality test result is therefore a sign of strong data quality, making the interpretation of the intervention more reliable. these results are in line with mulyana and herlina’s research, which found that traditional massage as a complementary therapy can increase body weight and appetite in stunted toddlers by stimulating the vagus nerve that regulates digestion (mulyana & herlina, 2025). putri’s study also showed that local wisdom-based herbal remedies containing flavonoids improve toddlers’ nutritional status by enhancing nutrient absorption and strengthening immunity (putri et al., 2025). the improvement in sleep quality observed in the traditional remedies pucuk jarak group supports earlier findings that infant massage boosts melatonin secretion, improving sleep patterns and growth (dianita zahra et al., 2022). likewise, the enhanced immune function corresponds with studies showing that flavonoid-rich herbal remedies increase lymphocyte activity, thereby strengthening immunity (tridesianti et al., 2025).this study not only confirms past findings but also offers novelty by testing locally rooted practices pucuk jarak and bedak langeh combined with melongas massage. it highlights that integrating local wisdom with science can be a promising complementary strategy to reduce stunting, especially in high-prevalence areas like north lombok regency. isasih & ardian – effectiveness of traditional remedies pucuk jarak and bedak langeh … 1183 conclusions based on the results and discussion of this study, it can be concluded that the intervention with the traditional remedies pucuk jarak mixture significantly improved body weight, height, appetite, sleep quality, and immunity of respondents compared to the bedak langeh group and the control group. these positive effects are supported by the high total flavonoid content of the traditional remedies pucuk jarak mixture, recorded at 53.32 mgqe/g, which is much higher than that of the bedak langeh mixture at only 3.58 mgqe/g. this finding is consistent with previous studies while also providing novelty in the use of traditional remedies in melongas massage practice. therefore, the traditional remedies pucuk jarak mixture has the potential to be developed as a complementary strategy in addressing stunting, particularly in regions with high prevalence such as north lombok regency. acknowledgements: the author thanks the directorate general of higher education, research, and technology of the republic of indonesia for funding this study through the grant program managed by the directorate of research and community service (dppm) in 2025. the researcher also appreciates universitas bumigora for its support, which ensured the smooth conduct of this research. in addition, the author declares that there are no conflicts of interest with any parties involved in this study. authors’ contributions: widani darma isasih. specializing in public health, the chairperson acts as the initiator of the research topic, participates in collecting data on sleep quality and immunity of stunted toddlers, coordinates the total flavonoid content analysis, and ensures consistency in melongas massage techniques with the therapist. they also take part in data management and analysis, as well as in preparing the research proposal, report, and outputs. junendri ardian, specializing in nutrition, the member is responsible for collecting weight and height data using anthropometric measurements, assessing appetite through 24-hour recall and ffq, as well as conducting sensory and pact tests on stunted toddlers. they also contribute to preparing the research report and outputs competing interests: there are no competing interests in this study. the authors affirm that all stages of the research, from design and implementation to reporting, were carried out independently without any intervention or influence from external parties that could create a conflict of interest funding: the funding for this research was provided by the directorate general of higher education, research, and technology of the republic of indonesia through the 2025 grant program managed by the directorate of research and community service (dppm). references abdunisa, l., & hardini, d. s. 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(2025). uji aktivitas antioksidan, polifenol, dan flavonoid pada nugget ikan teri dengan substitusi daun kelor dan daun singkong. jurnal pertanian, 16(1), 1–13. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 57-66 | doi: 10.14421/biomedich.2025.141.57-66 issn 2540-9328 (online) protein hydrolysates from cucumeropsis mannii seed inhibit alpha-amylase in-vitro: potential for managing postprandial hyperglycemia oladimeji taiwo babatunde1,2, clement olatunbosun bewaji1, adedoyin igunnu1,3,* 1department of biochemistry, faculty of life sciences, university of ilorin, p.m.b 1515, ilorin, kwara state, nigeria. 2biochemistry unit, department of chemical sciences, faculty of science, crown-hill (ojaja) university, p.m.b 1605, eiyenkorin, ilorin, kwara state, nigeria. 3department of biochemistry, college of pure and applied sciences, landmark university, omu-aran, kwara state, nigeria. corresponding author* doyinigunnu@unilorin.edu.ng manuscript received: 30 november, 2024. revision accepted: 20 march, 2025. published: 15 april, 2025. abstract diabetes mellitus is a significant global cause of mortality. a hallmark of diabetes pathophysiology is postprandial hyperglycaemia (pph). pph is defined as a sudden and exponential increase in blood glucose levels after meals, typically above 140 mg/dl, which does not return to pre-meal levels after two to three hours, leading to glucose toxicity, oxidative stress, cardiovascular risks, and diabetes complications. a management option for pph is the inhibition of carbohydrate-metabolizing enzymes, such as alpha-amylase. however, current inhibitors are associated with gastrointestinal side effects. therefore, the search for novel inhibitors is a rational research endeavor. recent studies highlight plant-derived protein hydrolysates as inhibitors of alpha-amylase. in this study, proteins from cucumeropsis mannii (c. mannii) were isolated using alkaline solubilization-acid precipitation method and enzymatically cleaved using pepsin and pancreatin to yield c. mannii seed protein hydrolysates. the α-amylase inhibitory property of the hydrolysates was investigated, using starch as the substrate. the ic50 values for α-amylase inhibition were 8.77 ± 0.35 mg/ml (pancreatin-derived) and 14.80 ± 0.50 mg/ml (pepsin-derived). kinetic studies indicated uncompetitive inhibition for pancreatin-derived hydrolysate and mixed uncompetitive for pepsin-derived hydrolysate at 9 mg/ml. these results suggest that c. mannii seed protein hydrolysates may aid in postprandial hyperglycemia management through α-amylase inhibition. keywords: diabetes mellitus; alpha-amylase inhibition; postprandial hyperglycaemia; protein hydrolysates; cucumeropsis mannii seed. introduction one of the hallmarks of diabetes mellitus is postprandial hyperglycaemia (pph). it is characterized by a significant and swift increase in blood glucose levels, commonly exceeding 140 mg/dl, after a meal, which does not return to pre-meal levels for two to three hours (american diabetes association, 2024; maffettone et al., 2018). typically, pph results from defective insulin secretion and/or action, leading to increased glucose levels after meals (pinés corrales et al., 2020). in pph, chronic hyperglycaemia exacerbates oxidative stress, leading to glucose toxicity as well as the destruction of the pancreatic β-cells and vascular tissues, which lead to diabetic complications such as retinopathy and nephropathy (campos, 2012). furthermore, pph is associated with increased cardiovascular risks, resulting from endothelial dysfunction, owing to the fluctuation in postprandial glucose levels (gerich, 2013). due to the pathophysiology of pph, its management in diabetes mellitus has become a significant focus. effectively managing pph involves inhibiting carbohydrate-metabolizing enzymes, such as alphaamylase. alpha-amylase is a key enzyme in carbohydrate metabolism, responsible for breaking down starch into simpler sugars, primarily in the mouth as salivary alphaamylase and in the pancreas as pancreatic alpha-amylase (butterworth et al., 2011). this breakdown process increases the availability of glucose, leading to a rise in blood sugar levels following a meal, known as postprandial hyperglycaemia. this postprandial spike in blood glucose can exacerbate oxidative stress, contributing to cellular and tissue damage and the progression of diabetic complications (brownlee, 2005; ceriello, 2005). inhibiting alpha-amylase slows down the digestion of carbohydrates, thereby reducing the rapid increase in blood glucose levels after meals. this can help manage postprandial hyperglycaemia and mitigate oxidative stress, ultimately aiding in better overall https://doi.org/10.14421/biomedich.2025.141.57-66 58 biology, medicine, & natural product chemistry 14 (1), 2025: 57-66 glucose control and reducing the risk of long-term complications (nickavar & yousefian, 2022). unfortunately, many commercially available alphaamylase inhibitors are associated with gastrointestinal side effects such as bloating, flatulence, abdominal pain, and diarrhea (kalinovskii et al., 2023; liu et al., 2024). therefore, the search for alternative inhibitors is a reasonable research endeavor. fortunately, protein hydrolysates have been observed to possess alphaamylase inhibitory properties without the side-effects associated with the current pharmaceutical collections. protein hydrolysates are mixtures of amino acids, peptides, and oligopeptides obtained by breaking down proteins through enzymatic hydrolysis. this process involves the use of proteases to cleave the peptide bonds within protein molecules, resulting in smaller, more easily absorbed protein fragments (clemente, 2000). peptides, specifically known as bioactive peptides, present in protein hydrolysate are highly therapeutic and free of known side effects, making them deserving of significant attention (korhonen & pihlanto, 2006; udenigwe & aluko, 2012). one plant seed, whose attributes make it a probable source of bioactive peptides, is cucumeropsis mannii seed. the properties that make this plant seed outstanding include its folkloric use as an antidiabetic regimen, its protein content (33.8% to 38.17% of the dry weight), and its amino acid composition, consisting of nearly all essential and non-essential amino acids (besong et al., 2011; ezuruike & prieto, 2014). despite these promising attributes, there has been minimal scientific investigation to establish its therapeutic significance conclusively. to the best of our knowledge, there are no published articles on the therapeutic benefits of protein hydrolysates from cucumeropsis mannii seed. consequently, this study examined the alpha-amylase inhibitory property of protein hydrolysates derived from cucumeropsis mannii seed and its kinetics to understand the mechanism of inhibition. materials and methods materials fully developed seeds of c. mannii were acquired from a local market situated in mopa, kogi state, nigeria; and were authenticated at the department of plant biology herbarium, faculty of life sciences, university of ilorin, ilorin, nigeria, and assigned a voucher number uilh/003/1420/2021. analytically graded chemicals and reagents were used without further purification. pepsin (from porcine gastric mucosa), pancreatin, α-amylase (penicillium fellutanum), and bovine serum albumin were products of sigma-aldrich chemicals, usa, while maltose, dinitrosalicylic acid, and starch were products of loba-chemie biotech., india. this research was submitted to the university ethical review committee (uerc) and received full approval, with uerc approval number: uerc/asn/2023/2463. methods extraction of cucumeropsis mannii seed protein the seeds of c. mannii were dried, ground into powder, and stored in an air-tight container at 4°c. the resulting powder was then defatted using n-hexane, following the procedure described by (wani et al., 2011). the defatted seed obtained was subsequently dried at room temperature and ground to fine powder. the fine powder was refrigerated for future use. the method described by (alashi et al., 2014) was employed for protein extraction from the defatted seed. in this process, the defatted c. mannii seed was mixed with 0.1 m naoh (ph 12.0) at a ratio of 1:10 (w/v; meal to solvent) and stirred for 1 hour to facilitate alkaline solubilization. next, the mixture underwent centrifugation at 18°c and 3000 rpm for 10 minutes. subsequently, two more extractions of the residue from the centrifugation process were conducted using an equivalent volume of 0.1 m naoh, and the resultant supernatants were pooled together. the ph of the combined supernatant was adjusted to 4.0 to facilitate acid-induced protein precipitation, using 1 m hcl solution. the formed precipitate was recovered through centrifugation, washed with distilled water, adjusted to ph 7.0 using 1 m naoh, freeze-dried, and the resulting protein isolate was refrigerated until needed for further analysis. following isolation, the protein yield was determined as described by (arise et al., 2015) using equation 1. % protein yield = mass of protein isolate (g) mass of defatted 𝐶.𝑚𝑎𝑛𝑛𝑖𝑖 seed meal (g) × 100 (%) (equation 1) preparation of cucumeropsis mannii seed protein hydrolysates the protein isolate was hydrolyzed according to the method of (udenigwe et al., 2009), as documented by (onuh et al., 2015). separate hydrolysis of the protein isolate was conducted utilizing pancreatin (at ph 8.0 and 37 ºc) and pepsin (at ph 2.2 and 37 ºc). the protein isolate (5% w/v) was dissolved in distilled water, forming distinct protein slurries for pancreatin and pepsin. enzymes were introduced to the respective slurries at an enzyme-substrate ratio (e: s) of 1:100. digestions were carried out under the specified babatunde et al. – protein hydrolysates from cucumeropsis mannii seed … 59 conditions for 6 hours with continuous stirring, followed by enzyme inactivation through immersion of the reaction vessels in boiling water (95 – 100 ºc) for 15 minutes. undigested proteins were precipitated by adjusting the ph to 4.0 with 2 m hcl/2 m naoh, succeeded by centrifugation at 4000 rpm for 30 minutes. the supernatant, containing target peptides, was collected and subjected to analysis for degree of hydrolysis and percentage peptide yield. this supernatant was refrigerated until further analysis. the protein content of the samples was determined using the biuret assay method of (gornall et al., 1949), with bovine serum albumin (bsa) serving as the standard. determination of degree of hydrolysis the degree of hydrolysis was evaluated by calculating the ratio of soluble peptides in 10% trichloroacetic acid (tca) to the total protein content of the protein isolate, using a technique detailed by (hoyle, 1994) with slight modifications. five hundred microliters (500 μl) of protein hydrolysates were combined with 500 μl of 20% tca to generate 10% tca soluble material. after allowing the mixtures to stand for 30 minutes to induce precipitation, centrifugation was performed (3500 rpm for 15 minutes). the supernatant was then examined for protein content using the biuret assay method of (gornall et al., 1949), utilizing bovine serum albumin (bsa) as a standard. the experiment was conducted in triplicates. the degree of hydrolysis (dh) was calculated using equation 2. degree hydrolysis (%) = soluble peptide in 10% tca (mg ml−1) total protein content of isolate (mg ml−1) × 100 (equation 2) determination of peptide yield the percentage yield of peptides was estimated following the procedure described by (girgih et al., 2011). the experiment was conducted in triplicates. the peptide yield (%) of the hydrolysates was computed as the ratio of the weight of peptides in each hydrolysate to the protein weight of the protein isolate, expressed by equation 3: peptide yield (%) = peptide weight of each hydrolysate (mg ml−1) protein weight of lyophilized isolate (mg ml−1) × 100 (equation 3) determination of α-amylase inhibition of the hydrolysates the alpha-amylase inhibitory assay was carried out according to the method of (bernfeld, 1951), as documented by (oboh et al., 2011). in test tubes, 125 μl of hydrolysate (pancreatin/pepsin) at concentrations ranging from 1.0 to 9.0 mg/ml were mixed with 125 μl of 20 mm sodium phosphate buffer (ph 6.9, with 6 mm nacl) containing α-amylase solution (0.5 mg/ml). following pre-incubation at 25 °c for 10 minutes, 125 μl of 1% starch solution in 20 mm sodium phosphate buffer (ph 6.9, with 6 mm nacl) was added at specific intervals. the reaction mixtures were then incubated at 25 °c for 10 minutes. termination of the reaction occurred with the addition of 250 μl of dinitrosalicylic acid (dns) colour reagent, followed by further incubation in boiling water for 5 minutes and subsequent cooling to room temperature. the contents of each test tube were diluted with 2.5 ml of distilled water, and the absorbance was recorded at 540 nm. a control was also prepared following the same procedure, but substituting the hydrolysate with distilled water. the experiment was carried out in triplicate, and the α-amylase inhibitory activity was calculated using equation 4. % inhibition = absorbancecontrol − absorbancesample absorbancecontrol × 100 (%) (equation 4) the determination of the concentration of hydrolysate leading to a 50% inhibition of α-amylase activity (ic50) involved plotting the percentage inhibition (y-axis) against concentrations (x-axis) using microsoft excel version 2016, employing linear regression. the intercept (c) and the gradient (m) were obtained from the line of 60 biology, medicine, & natural product chemistry 14 (1), 2025: 57-66 best fit curve. the 50% inhibitory concentration (ic50) was mathematically derived according to equation 5. 50% inhibitory concentration (ic50) = 50 – intercept gradient (mg/ml) (equation 5) determination of kinetic parameters of alphaamylase inhibition of the hydrolysates the kinetic investigation of α-amylase inhibition by the hydrolysates was carried out following the procedure described by (ali et al., 2006) with minor adjustments. in one set of tubes, 125 μl of the hydrolysates (9 mg/ml) was pre-incubated with 125 μl of α-amylase solution for 10 minutes at 25 ºc. simultaneously, in another set of tubes, 250 μl of phosphate buffer (ph 6.9) was preincubated with 125 μl of α-amylase solution. subsequently, starch solution (125 μl) at varying concentrations (1.0 – 8.0 mg/ml) was introduced into both sets of reaction mixtures to initiate the reaction. after a 10-minute incubation at 25 oc, the reaction was terminated by boiling for 5 minutes following the addition of 250 μl of dns reagent. the amount of reducing sugars released was quantified spectrophotometrically using a maltose standard curve and converted to reaction velocities using equation 6. specific activity (μmol/ /min) = [maltose released] incubation time x m𝐸 (equation 6) me = amount (in mg) of enzyme in the reaction mixture incubation time: 10 minutes a reciprocal plot was generated, depicting 1/[specific activity] on the y-axis against 1/[starch] on the x-axis. the mode of inhibition and the kinetic parameters (km and vmax) associated with α-amylase inhibition by the hydrolysates were elucidated through analysis of the double reciprocal plot using microsoft excel version 2016. calculations: the maximum velocity (vmax) and michaelis constant (km) were derived from the double reciprocal plot of 1/ [specific activity] (y-axis) against 1/ [starch] (x-axis) using microsoft excel version 2016. from the line of best fit, the intercept (c) and the gradient (m) were obtained for the presence and absence of hydrolysate. the values of the vmax and km were derived according to equations 7 and 8, respectively. maximum velocity (vmax) = 1 intercept on the y−axis (equation 7) michaelis constant (km) = v𝑚𝑎𝑥 × gradient (equation 8) data analysis data were expressed as the mean of three replicates ± standard deviation (sd). the data were subjected to analysis of variance and tukey's multiple range tests, utilizing graphpad prism version 6.0 (graphpad software, san diego, ca, usa). the ic50 values were determined using microsoft excel version 2016, employing linear regression. statistical significance was determined at p < 0.05. results and discussion results protein yield of cucumeropsis mannii seed and protein content, degree of hydrolysis and peptide yield of cucumeropsis mannii seed protein isolate following the extraction of protein from c. mannii seeds, a protein yield of 22.8% was obtained, comprising 94.8% protein content. table 1 illustrates the degree of hydrolysis and the resultant peptide yield from the protein hydrolysis of c. mannii seeds. the degree of hydrolysis obtained using pancreatin was significantly babatunde et al. – protein hydrolysates from cucumeropsis mannii seed … 61 higher (p < 0.05) compared to pepsin. however, no significant difference (p > 0.05) was observed in the peptide yields between the two enzymes utilized for the hydrolysis of c. mannii seed protein. percentage alpha-amylase inhibitory effects of cucumeropsis mannii protein hydrolysates the percentage α-amylase inhibition and 50% α-amylase inhibitory concentration (ic50) of pancreatin-derived and pepsin-derived hydrolysates is shown in figure 1. in the case of pancreatin-derived hydrolysate, its α-amylase inhibitory effect increased progressively from 1.0 mg/ml to 9.0 mg/ml. although, the inhibitory effect of the hydrolysate was not significantly different (p > 0.05) between 1.0 mg/ml and 3.0 mg/ml (4.21 ± 0.37% and 5.75 ± 1.64% respectively), there was a significant difference (p < 0.05) as the concentration progressed from 3.0 mg/ml to 9.0 mg/ml (15.83 ± 4.44% to 54.50 ± 1.82%). in a similar vein, the α-amylase inhibitory effect of pepsin-derived hydrolysate increased progressively, with an increase in concentration from 5 mg/ml to 9.0 mg/ml. although, there was no inhibitory effect observed at 1.0 and 3.0 mg/ml, there was observable inhibitory effect as the concentration progressed from 5 mg/ml to 6.0 mg/ml and 9 mg/ml (7.57 ± 1.86%, 16.74 ± 2.70% and 24.43 ± 2.32%). the inhibitory effect of pancreatin-derived hydrolysate from 5 mg/ml to 9 mg/ml was significantly higher (p < 0.05) than that of pepsin-derived hydrolysate. the 50% α-amylase inhibitory concentration (ic50) values were 8.767 ± 0.35 mg/ml and 14.80 ± 0.50 mg/ml for pancreatin-derived and pepsin-derived hydrolysates, respectively (figure 1). kinetics of α-amylase inhibition of cucumeropsis mannii protein hydrolysates the lineweaver-burk plots of α-amylase-catalyzed hydrolysis of starch in the absence and presence of pancreatin-derived and pepsin-derived hydrolysates are shown in figures 2 and 3, respectively. the modes of inhibition exhibited by pancreatin-derived and pepsinderived hydrolysates were uncompetitive and mixed uncompetitive inhibitions, respectively. the estimation of kinetic parameters, maximum velocity (vmax) and michaelis-menten constant (km) are shown in table 2. table 1. the degree of hydrolysis and peptide yield following the hydrolysis of cucumeropsis mannii seed proteins proteolytic enzyme degree of hydrolysis (%) peptide yield (%) pancreatin 15.63 ± 0.37a 19.55 ± 0.08a pepsin 10.36 ± 0.46b 18.73 ± 0.66a values represent the mean of triplicate determinations ± standard deviation (sd). values with different letters across the column are significantly different at p < 0.05. table 2. kinetic parameters of α-amylase-catalyzed hydrolysis of starch in the absence (control) and presence of cucumeropsis mannii seed protein hydrolysates kinetic parameter control (no hydrolysate) pancreatinderived hydrolysate (9.0 mg/ml) pepsinderived hydrolysate (9.0 mg/ml) vmax (μmol/mg/min) 46.08 6.48 10.53 km (mg/ml) 20.10 2.48 2.28 vmax: maximum reaction rate in the presence and absence of hydrolysates km: michaelis constant in the presence and absence of hydrolysates figure 1. alpha-amylase inhibitory activity of cucumeropsis mannii seed protein hydrolysates. values represent the mean of triplicate determinations ± standard deviation (sd). inset: fifty percent α-amylase-inhibitory concentration (ic50) values of cucumeropsis mannii seed protein hydrolysates. values represent the mean of triplicate determinations ± standard deviation (sd). dots representing different protein hydrolysates at the same concentration, but marked with different letters, indicate a significant difference at p < 0.05. 62 biology, medicine, & natural product chemistry 14 (1), 2025: 57-66 figure 2. lineweaver-burk plot of the effect of cucumeropsis mannii seed hydrolysate derived from pancreatin digestion on α-amylase-catalyzed hydrolysis of starch. figure 3. lineweaver-burk plot of the effect of cucumeropsis mannii seed hydrolysate derived from pepsin digestion on α-amylase-catalyzed hydrolysis of starch. the maximum velocity (vmax) of starch hydrolysis catalyzed by α-amylase in the presence of pancreatin and pepsin-derived hydrolysates decreased by roughly 7fold and 4-fold, respectively, compared to the control (table 2). in addition, the michaelis-menten constant (km) of starch hydrolysis catalyzed by α-amylase in the presence of pancreatinand pepsin-derived hydrolysates decreased by approximately 8-fold and 9-fold, respectively, compared to the control (table 2). discussion protein yield of cucumeropsis mannii seed and protein content, degree of hydrolysis and peptide yield of cucumeropsis mannii seed protein isolate the percentage protein yield accounts for the amount of protein obtained in a protein extraction procedure. it is often a function of the extraction method employed (boye et al., 2010). in this study, the alkaline solubilization-acid precipitation method which extracts the glutelin fraction of seed storage proteins was utilized (osborne, 1924). the percentage protein yield (22.8%) of c. mannii seed obtained in this study was higher than that of pawpaw seed (5.13%), neem seed (12.52%), moringa seed (18.60%), watermelon seed (18.91%), polyalthia longifolia seed (2.23%), and annona muricata seed (7.1%) which utilized the same method of protein extraction (arise et al., 2019; arise et al., 2016; babatunde et al., 2022a; babatunde et al., 2022b; james et al., 2020a; jamesa et al., 2020b; olusola et al., 2018). previous studies have credited hydrolysates derived from the glutelin fractions of seed proteins with incredible biological activities (arise et al., 2019; arise et al., 2016; babatunde et al. – protein hydrolysates from cucumeropsis mannii seed … 63 babatunde et al., 2022a; babatunde et al., 2022b; james et al., 2020a; jamesa et al., 2020b; olusola et al., 2018). the high percentage protein content of the isolate (94.8%) obtained in this study indicates the effectiveness of the extraction process. indeed, alkaline solubilizationacid precipitation method often results in protein isolate with high protein content. previous studies on the extraction of protein from watermelon seed and bambara groundnut using the acid precipitation method resulted in isolates with relatively high protein content. however, the value obtained in this study was higher than that of watermelon seed (74%) and bambara groundnut (79%) (arise et al., 2015; arise et al., 2016). the degree of hydrolysis (dh) accounts for the amount of peptide bonds cleaved during protein hydrolysis. it is influenced by the type of enzyme used, the enzyme/substrate (e/s) ratio, ph value, temperature, and time of hydrolysis (benítez et al., 2008). concerning the type of enzyme used, pancreatin effectively hydrolyzed c. mannii protein isolate more than pepsin (table 1). the effectiveness of pancreatin may be attributable to its protease mix, which includes endopeptidases (trypsin, chymotrypsin and elastase) and exopeptidases (carboxypeptidases a and b) (andriamihaja et al., 2013). with such a collection of proteases, it is no surprise that it yielded a higher dh than pepsin, which is a single enzyme. the dh value obtained from the hydrolysis of c. mannii seed protein using pancreatin (e/s of 2:100) (table 1) was found to be lower than the dh value obtained from the hydrolysis of black cumin protein (at 20%) using the same enzyme (e/s 2:100) (shahi et al., 2020). in addition, the value of dh obtained from the hydrolysis of c. mannii seed protein by pepsin (e/s of 2: 100) (table 1) was lower than that obtained from the hydrolysis of moringa seed protein (26.93%) by the same enzyme (e/s of 2: 100) (olusola et al., 2018). the difference in the dh observed with the use of the same enzyme and e/s ratio might be attributable to the difference in the amino acid composition of the protein isolates. hence, black cumin protein has more amino acid residues specific for pancreatin than c. mannii seed protein; also, moringa seed protein has more amino acid residues specific for pepsin than c. mannii seed protein. the peptide yield provides information on the number of peptides produced after protein hydrolysis and the efficiency of the hydrolytic process (alashi et al., 2014). in this study, the difference in the peptide yield observed with the use of pancreatin and pepsin was not significant (table 1). hence, it is clear that the higher dh observed with the use of pancreatin compared to pepsin did not significantly affect the peptide yield. it can be rationalized that the carboxypeptidases present in pancreatin did not influence the peptide yield because they cleave the c-terminal amino acids resulting in the production of free amino acids rather than peptides (naik, 2012). therefore, the peptide yield resulting from the use of pancreatin might be due to the three endopeptidases in it, and the ability of pepsin to produce peptides was at par with that of the proteases in pancreatin. cucumeropsis mannii protein hydrolysates inhibited alpha-amylase-catalyzed reactions: potential for postprandial hyperglycaemia management the inhibition of α-amylase is an approach utilized in the management of postprandial hyperglycemia associated with type 2 diabetes mellitus (qin et al., 2011). although there are commercially available α-amylase inhibitors, however, they are associated with gastrointestinal side effects such as bloating, diarrhea, and abdominal pain (chiasson et al., 2002). fortunately, plant-derived protein hydrolysates have demonstrated therapeutic efficiency with limited side effects (arise et al., 2019; arise et al., 2016; babatunde et al., 2022a; babatunde et al., 2022b; ishida et al., 2011; ismail et al., 2004; james et al., 2020a; jamesa et al., 2020b; olusola et al., 2018). the results obtained in this study further reinforces the assertion of many researchers that protein hydrolysates have promising therapeutic abilities (nasri, 2017). the hydrolysates exhibited concentration-dependent inhibitory effects against α-amylase (figure 1). leucine and proline, both hydrophobic amino acids, along with arginine, a hydrophilic amino acid, have been identified in previous studies as contributors to the inhibition of αamylase (jiang et al., 2018; ramadhan et al., 2017; ren et al., 2016; wang et al., 2020; zhang et al., 2007). specifically, wang et al. (2020) emphasized the importance of these three amino acids, either individually or in synergy within peptides, in the inhibition of αamylase. therefore, the inhibitory effect of the hydrolysates on α-amylase can be attributed to the abundance of these amino acids within them. meanwhile, there is a possibility that other hydrophobic amino acids such as alanine, phenylalanine, valine, and glycine, as well as hydrophilic amino acids like cysteine, histidine, and serine, played no mean role in potentiating the α-amylase inhibitory abilities of the hydrolysates. this notion is plausible because of an earlier study that illustrated the interaction of hydrophobic and hydrophilic amino acids with the active sites of human salivary α-amylase, resulting in its inhibition through hydrophobic and hydrogen bonds (ngoh et al., 2017). the observed range of inhibition in this study, as depicted in figure 1, is considered appropriate, as excessive inhibition of α-amylase is undesirable. kumar et al. (2011) emphasized that an excessive inhibition of α-amylase could lead to abnormal bacterial fermentation of undigested carbohydrates in the colon (kumar et al., 2011). hence, mild α-amylase inhibition effects are deemed more beneficial. the 50% inhibitory concentration (ic50) for the inhibitory effects observed in this study (figure 1) was higher than that of most hydrolysates studied previously (arise et al., 2019; arise et al., 2016; james et al., 2020a; jamesa et al., 64 biology, medicine, & natural product chemistry 14 (1), 2025: 57-66 2020b; olusola et al., 2018). this implies that higher concentrations of the hydrolysates are needed to achieve desirable inhibitory effects. notwithstanding the higher ic50 values, the inhibition afforded by the hydrolysates is promising. more so, peptides can be administered at high doses without eliciting grievous side effects (ishida et al., 2011; ismail et al., 2004). kinetic parameters derived from the lineweaverburk plots of α-amylase inhibition by the hydrolysates were employed in determining the mechanism of the inhibition. pancreatin-derived hydrolysate exhibited uncompetitive inhibition pattern, while pepsin-derived hydrolysate exhibited mixed inhibition pattern (figures 2 and 3). in uncompetitive inhibition, there is a reduction in the km and vmax of an enzyme-catalyzed reaction (nelson & cox, 2008). hence, this study suggests that pancreatin-derived hydrolysate enhanced the binding of the substrate (starch) to the enzyme and was bound to the enzyme-substrate (e/s) complex formed. similarly, in mixed uncompetitive inhibition, the inhibitor binds to the enzyme-substrate (e/s) complex (nelson & cox, 2008); hence the reduction in the km and vmax (table 2) of the α-amylase-catalyzed reaction by pepsin-derived hydrolysate can be caused by the binding of the hydrolysate to the enzyme-substrate complex, in that case enhancing substrate binding. the inhibition pattern expressed by pancreatin-derived hydrolysate at 9.0 mg/ml was similar to that of trypsin-derived hydrolysate from moringa oleifera seed protein at 1.0 mg/ml (olusola et al., 2018); however, the vmax and km of the former were higher than that of the latter. in addition, the inhibition pattern expressed by pepsin-derived hydrolysate at 9.0 mg/ml was similar to that of pepsinderived hydrolysate from moringa oleifera seed protein at 1.0 mg/ml (olusola et al., 2018), with the latter having a lower vmax and km than the former. conclusions the study findings indicate that cucumeropsis mannii seed protein hydrolysates display encouraging alphaamylase inhibitory properties. this implies that the hydrolysates could potentially aid in controlling postprandial hyperglycemia associated with diabetes mellitus through α-amylase inhibition. further research is necessary to fractionate the hydrolysates, characterize their effects, and identify the specific bioactive peptides they contain. acknowledgements: the authors would like to thank the central 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medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 2, 2019 | pages: 27-32 | doi: 10.14421/biomedich.2019.82.27-32 issn 2540-9328 (online) digital anthropometer development for improving the measurement quality of human body dimensions trio yonathan teja kusuma*, arya wirabhuana**, faurosi syafa’atul yusuf department of industrial engineering, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto, no. 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. author correspondency: trio.yonathan@gmail.com*, arya.wirabhuana@uin-suka.ac.id** abstract digital information technology has become the nerve of information for industry-based companies. however, there are still data or information that retrieved manually, one of them is measurement of the human body dimension. the problem is seemed solved by invention of digital anthropometer application. yet, the application requires a further improvement especially in its accuracy and user interface. the improvement of accuracy quality of its measurement uses the capability process analysis method. as a result, the current cp and cpk is 0.26 and -0.209 while in the developed application is 2.56 and 1.218. thus, the developed application is stated that quality of measurement increases to meet the criteria of good process capability. the user interface is improved based on the user voices. data storage databases and percentile calculation in the application was added as one on the significant user-interface improvement. keywords: application, digital anthropometer, quality, process capability introduction technology is an important factor for industrial development in world today. the development of industrial sector requires technology to improve its productivity. one of widely used technology in industry today is information and communication technology. it has been developing rapidly so that it can help people solve many problems. digital information technology has also become nerve information for companies today. in the current digital age, there are still fields for manual data or information retrieval, one of them is measurement of human dimension. its measuring device is called anthropometric chairs. yet, these measuring instrument is still far from user-friendly. however, this deficiency has been limited by existing research. previous research makes digital and automatic measuring devices. however, there are still deficiencies in this measurement tool. therefore, it is necessary to develop the current digital measuring instrument. the development plan for current application is to improve quality of measurement by testing ability of current application process and developing application to see the improvement in the application process capability. and will be added a place to store human data in application development and added percentiles of human dimension data that has been stored. apart from that it also enhances user interface also needed. literature review anthropometry according to wignjosoebroto (2000), anthropometry comes from "anthro" which means human and "metri" which means size. anthropometry can be definitively expressed as a study relating to the measurement of the human body dimensions including areas of size, strength, and other aspects of body movement. anthropometry is a part of ergonomics that specifically studies body size which includes linear dimensions, weight, contents, and also includes areas of size, strength, speed, and other aspects of body movement. eko nurmianto (nurminato, 2004) uses the term “the fallacy of the average man or average woman”. the term states that it is a mistake to design a workplace or product if it is only based on a hypothetical dimension, which is to assume that all dimensions are average values. the use of one dimension, e.g forward reach, using an average (50% percentile) in adjusting the installation of a control device will cause 50% of the population will not be able to reach it. in addition, if a certain dimension of a person (for example: height) is in the population average, then not necessarily other body dimensions of that person (for example: shoulder width) are in the population average as well. common percentile distribution values are applied in anthropometric data calculations. following is the percentile distribution table. https://doi.org/10.14421/biomedich.2019.82.27-32 mailto:trio.yonathan@gmail.com mailto:arya.wirabhuana@uin-suka.ac.id 28 biology, medicine, & natural product chemistry 8 (2), 2019: 27-32 table 1. percentile. percentil calculation 1 �̅� − 2,325𝜎𝑥 2,5 �̅� − 1,96𝜎𝑥 5 �̅� − 1,645𝜎𝑥 10 �̅� − 1,28𝜎𝑥 50 �̅� 90 �̅� + 1,28𝜎𝑥 95 �̅� + 1,645𝜎𝑥 97,5 �̅� + 1,96𝜎𝑥 99 �̅� + 2,325𝜎𝑥 (source: wignjosoebroto, 2008) image processing image processing according to munir (2004) is any form of signal processing where the input is an image, such as a photo or video, while the output of image processing can be in the form of images or a number of characteristics or parameters related to images. most image processing techniques involve or treat photos as two-dimensional signals and apply standard signal processing techniques to them, usually referring to digital image processing, but can also be used for optical and analog image processing. image acquisition or that produces input image in the first place is referred to as imaging. generally digital images are rectangular or square (in some imaging systems some are hexagonal) which have a certain width and height. this size is usually expressed in terms of points or pixels so that the size of the image is always round. each point has coordinates according to their position in the image. these coordinates are usually expressed in positive integers, which can start from 0 or 1 depending on the system used. each point also has a value in form of a digital number that represents the information represented by that point. the format of digital image data is closely related to color. in most cases, especially for the purpose of visual appearance, digital data values represent colors of processed image. the most widely used digital image formats are binary image (monochrome), gray scale image, true color image, and indexed color image. vitruvian man theory anthropometric variables have a lot of influence, so the selection of anthropometric variables is adjusted to purpose of using anthropometric data. in the study of stancic, et al (2011) in rachmatullah (2016) using 8 body dimensions to perform human anthropometric measurements using video. research by karuppiah et al (2011) in rachmatullah (2016) used 11 dimensions of the human body for anthropometric measurements in motorists. the research shows that the use of anthropometric variables does not have to be entirely, more efficient on the variables needed only. figure 1. vitruvian man, leonardo da vinci, 1490. (source: leonardo da vinci in kelly, 2005) based on the vitruvian man concept that was coined by leonardo da vinci, stating that human body size is a proportion of body size in other parts, the proportion of vitruvian man's body is described below (kelly, 2005): 1. the width of the palm is 4 fingers wide 2. foot length is the width of 4 palms 3. one cubit is the width of six palms 4. a person's height is four cubits (and thus 24 palms) 5. the step length is four cubits 6. the length of a man's arms is the same as his height 7. the distance from the hairline to the bottom of the chin is one tenth of the height of a human 8. the distance from the crown of the head to the bottom of the chin is one-eighth of the height of a human 9. the distance from the hairline to the top of the breast is one seventh of human height 10. the distance from the crown of the head to the nipple is a quarter of the height of a human 11. maximum shoulder width is a quarter of human height 12. the distance from the elbow to the tip of the hand is a quarter of the height of a human 13. the distance from the elbow to the armpit is 1/8 of the height of a human 14. the length of the hand is one tenth of the height of a human 15. the distance from the bottom of the chin to the nose is one third of the face length 16. the distance from the hairline to the eyebrows is one third of the face length 17. ear length is one third of the face length kusuma et al. – digital anthropometer development for improving the … 29 there are three additional dimensions for this study that originated from maccurdy (1955), namely: 1. the distance from the footwear to the pubic root is half the height of a human 2. the distance from the footwear to the bottom of the knee is a quarter of human height 3. the distance from below the knee to the pubic root is a quarter of human height process capability analysis according to ariani (2004) an analysis of process capability is carried out to meet various reasons, for example responding to customer requests regarding the company's process capability index or conducting an evaluation of the process for carrying out quality improvement. process capability analysis is also a procedure used to predict long-term performance that is within the limits of a statistical process controller. typical process capabilities are formulated in ± 3σ or 6σ (six sigma). where σ expresses the standard deviation of controlled process. this means assuming that normal distribution is 99.73% of products within the ± 3σ limit. to be able to stick the product specifications process capability is calculated by quantifying process capability. for under controlled conditions, the ppa can be done by: (rasyidin et. al., 2012) 1. capability process (cp) 𝐶𝑝 = 𝐵𝑆𝐴 − 𝐵𝑆𝐵 6𝜎 cp : capability process bsa : upper specification limit bsb : lower specification limits assessment criteria:  if cp> 1.33, the capability process is very good  if 1,00 ≤ cp ≤ 1,33 then the capability process is good, but it needs tight repairs of cp 1,00.  if cp <1.00 then the process capability is low, the performance needs to be improved through improving the process. 2. top process capability index/bottom process capability index. 𝐾𝑃𝐴 = 𝐵𝑆𝐴 − 𝜇 3𝜎 𝐾𝑃𝐵 = 𝜇 − 𝐵𝑆𝐵 3𝜎 kpa/kpb : top/bottom process capability index bsa : upper specification limit bsb : lower specification limit 𝜇 : average 𝜎 : standard deviation 3. process capability index (cpk) 𝐶𝑝𝑘 = 𝑚𝑖𝑛 { 𝐵𝑆𝐴 − 𝜇 3𝜎 , 𝜇 − 𝐵𝑆𝐵 3𝜎 }  if 𝐶𝑝𝑘 >= 1  capable  if 𝐶𝑝𝑘 <= 1  not capable  if 𝐶𝑝𝑘 >> fewer products are outside the specification limits research methodology object of research the object of this study is implementing the application development concept research that has been conducted before by rachmatullah (2016) into a ready to use digital anthropometer device. data type the types of data used are: 1. data obtained from direct observation and field study are called primary data. primary data of this study are height, body width, palm length, palm width, length of one cubit, distance from elbow to armpit, distance from elbow to tip of hand, length of arm extended, distance from top of head to chin, distance from line hair to the chin, distance from footwear to pubic roots, distance from footwear to below the knee, distance from below the knee to pubic roots and the length of the feet. 2. secondary data is data that is not obtained through direct observation or measurement of the object under study. the secondary data of this study is size of vitruvian man. method of collecting data data collection methods used in this study are as follows: 1. observation observation is a method of collecting data by observing directly on research object. 2. literature study the method used to find resources related to development needed in the current application. 3. interview interviews were conducted on user to improve user interface to support and facilitate measurement with the application made by user. 30 biology, medicine, & natural product chemistry 8 (2), 2019: 27-32 flowchart start initial observation and problem identification literature review development of digital anthropometer with image processing method 1. is data in control limit ? 2.is good capability index value end data collection data processing literature review literature review no yes figure 2. flowchart. results and discussion anticipating causes of inaccurate measurement in current application with fishbone diagram fishbone diagram is used to identify the potential causes of inaccurate measurements in current application. therefore, to reduce the inaccurate measurements potentials, an application development is carried out to close the gaps from potential causes of accurate measurements. the cause of human measurement in using current application is that point measurement is difficult to be exact and precise because it only uses points only for measurement, there is no clarity to straighten the height measurement point. so in the application that has been developed, drawing a line after getting the object click on the top, after that it can straighten the measurement of body height by drawing a line straight to the bottom point of object. so the tendency of causes of accurate measurements has been minimized by drawing a straight line. the method in measuring is still unclear, therefore potential causes for inaccurate measurements also occur. because the measurement point of current application is still just a point from the object to the bottom of object. then the development of a new application is done by line drawing after clicking on the top object. with the help line, measurement of the point is more accurate on the object. the environment is also one of the factors causing inaccurate measurements. the environment will not have a big impact because it is still light even though it is too bright or dark but because the measurement is straight from top to bottom the object is not affected. however, light or dark environment only affects the click point of top most object point or click point of bottom object point. there is no guidebook also included one of the factors that influence the measurement of acute curating, therefore the development application is given a help menu to facilitate and understand the application. latest application digital measurement methods in current application done by clicking on the top and bottom of an object only. after that the calculation is done to get the other dimensions. there are 11 dimensions of the human body that are used, namely: 1. height (tb) 2. palm length (ptt) 3. talraw width (ltt) 4. length of one cubit (psh) 5. stretched arm length (plt) 6. maximum shoulder width (lmb) 7. distance from the peak of the head to the chin (jpkd) 8. distance from hairline to chin (jgrd) 9. distance from elbows to armpits (jsk) 10. distance from elbows to the end of the hand (jsku) 11. foot length (ptk) application that exist today are still many gaps to be developed. one of them is improving the quality of measurement. previous studies had not yet been measured using a quality testing tool that uses process capacity index measurements. therefore, to see the potential of the application a sample search is performed to see the measurement quality of the current application. the results obtained with 30 samples that have a value of process capability (cp) is 0.26. judging from the value is still below 1, the process capability is still low and needs to be improved performance. and the value of kane process capability (cpk) is -0.209, so because the value of kane process capability is less than equal to 1 (cpk <= 1), the results are not capable. judging from the results of the measurement of the kusuma et al. – digital anthropometer development for improving the … 31 process capability index, it is necessary to develop and improve the quality of the accuracy of this application. application development this development application has been a bit of a method of taking measurements of body dimensions that are different from previous application. this developed application minimizes the causes of inaccurate measurements. namely with points and straight lines aiding to straighten points or automatically scan and get straight lines also from automatic scans performed. measurable dimensions are: 1. height (tb) 2. body width (lb) 3. palm length (ptt) 4. handprint width (ltt) 5. length of one cubit (psh) 6. distance from elbows to armpits (jsk) 7. distance from elbows to the end of the hand (jsku) 8. outstretched arm length (plt) 9. distance from the peak of the head to the chin (jpkd) 10. distance from hairline to chin (jgrd) 11. distance from footwear to pubic root (jakak) 12. distance from footwear to the knee (jakbl) 13. distance from below knee to pubic root (jblak) 14. foot length (ptk) before calculating the process capability in sample, data that has been obtained from measurement application that has been developed is carried out by controlling data using one of the quality tools, namely the x-bar s. control map. upper and lower control limits of x-bar s. map control chart these following are the calculation of x-bar chart (ucl & lcl) and s chart (ucl&lcl):  x-bar chart upper control limit x-bar chart 𝑈𝐶𝐿�̅� = 0.702 + 1.427 × 0.446 𝑈𝐶𝐿�̅� = 0.702 + 0.636 𝑈𝐶𝐿�̅� = 1.339 cm lower control limit x-bar chart 𝐿𝐶𝐿�̅� = 0.702 − 1.427 × 0.446 𝐿𝐶𝐿�̅� = 0.702 − 0.636 𝐿𝐶𝐿�̅� = 0.065 cm  s chart upper control limit s chart 𝑈𝐶𝐿𝑆 = 2.089 × 0.446 𝑈𝐶𝐿𝑆 = 0.932 cm lower control limit s chart 𝐿𝐶𝐿𝑆 = 0 × 0.446 𝐿𝐶𝐿𝑆 = 0 cm based on data processing using x-bar chart and s chart, the results of measurement with digital antthropometer is still within the control limits. it shows that the measurement is stable and normal. process capability  process capability (cp) the following is a calculation of process capability: 𝐶𝑝 = 1.339 − (−1.339) 6(0.174) 𝐶𝑝 = 2.678 1.044 𝐶𝑝 = 2.561 because the value of process capability is more than 1.33 (cp> 1.33), the process capability is very good.  kane process capability (cpk) the following is a calculation of kane process capability (cpk): 𝑐𝑝𝑢 = 1.336 − 0.702 3(0.174) 𝑐𝑝𝑢 = 1.218 𝑐𝑝𝑢 = 0.702 − (−1.339) 3(0.174) 𝑐𝑝𝑙 = 3.904 𝐶𝑝𝑘 = 𝑚𝑖𝑛 {𝑐𝑝𝑢; 𝑐𝑝𝑙} 𝐶𝑝𝑘 = 𝑚𝑖𝑛 {1.218; 3.904} 𝐶𝑝𝑘 = 1.218 because the value of kane process capability is more than equal to 1 (𝐶𝑝𝑘 > = 1), the results are capable. judging from value of the ability process, this application is good and the quality of measurement accuracy is also good and growing. additional menus in this application are the database for storing data after taking measurements, percentile menu, this menu makes it easy to perform percentile calculations from data in the database and help menu which functions to provide assistance and explanations for all buttons and functions of everything on user interface. 32 biology, medicine, & natural product chemistry 8 (2), 2019: 27-32 user interface comparison between current application and application development comparison of user interfaces before and after development: 1. latest application figure 3. user interface latest application (source: rachmatullah, 2016) 2. development application figure 4. user interface of development application. conclusion the following is the conclusion of the study: 1. calculation of process capability from the current application has cp and cpk values respectively 0.26 and -0.209. while the process capability of the development application has cp and cpk values respectively 2.56 and 1,218. thus, the developed application is stated that the quality of measurement increases to meet the criteria of good process capability. 2. application made from previous studies can already measure quickly and can reduce activity of manual measurement. however, development application can measure quickly, accurately and also reduce measurement activities manually. 3. development application have additional specifications namely databases and percentile calculations. database of application were performed using microsoft access. percentile calculations in the application use data from the database. references ariani, dorothea wahyu. 2004. pengendalian kualitas statistik (pendekatan kuantitatif dalam manajemen kualiatas). yogyakarta: andi. kelly, c. the beauty of fit: proportion and anthropometry in chair design. thesis report of georgia institute of technology, 2005. maccurdy, edward. 1955. the notebooks of leonardo da vinci. new york: george braziller. munir, rinaldi. 2004. pengolahan citra digital. informatika bandung. nurmianto, eko. 2004. ergonomi konsep dasar dan aplikasinya. edisi pertama. institut teknologi sepuluh november. surabaya: guna widya. rachmatullah, marhabban faizi. 2016. perancangan antropometri digital menggunakan metode pengolahan citra sebagai alat bantu pengukuran dimensi tubuh. yogyakarta: universitas islam negeri sunan kalijaga yogyakarta. rasyidin, muhammad taurif, darnah a.nohe dan sri wahyuningsih. 2012. statistical process control dengan metode cummulative sum (cusum). 3(2). wignjosoebroto, sritomo. 2000. ergonomi, studi gerak, dan waktu: teknik analisis untuk peningkatan produktivitas kerja. edisi i. surabaya: guna widya. wignjosoebroto, sritomo. 2008. ergonomi studi gerak dan waktu. surabaya: guna widya. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 251-258 | doi: 10.14421/biomedich.2023.121.251-258 issn 2540-9328 (online) chemical composition and evaluation of anti-tyrosinase and antioxidative effects of topical cream formulation from acacia sieberiana, vitellaria paradoxa and beeswax alfred ngenge tamfu1,3,4,*, alain yaya koudoro2, selcuk kucukaydin3,4, theophile olaye2, pascal dossa cokou agbangnan2, dominique codjo koko sohounhloue2, felicien avlessi2 2laboratory of study and research in applied chemistry, university of abomey-calavi, polytechnic school of abomey-calavi, 2009 abomey-calavi, benin. 3department of medical services and techniques, koycegiz vocational school of health services, mugla sitki kocman university, 48800 mugla, turkey. 4department of chemistry, faculty of science, mugla sitki kocman university, 48000 mugla, turkey. corresponding author* macntamfu@yahoo.co.uk manuscript received: 30 january, 2023. revision accepted: 12 march, 2023. published: 13 march, 2023. abstract skin diseases can get natural therapies from medicinal plant-based products. in this study, a topical cream was formulated from ethanol extract of acacia sieberiana, beeswax and vitellaria paradoxa (shea) butter. gc-ms characterization with co-injection of the topical cream revealed stearic acid (31.43%), palmitic acid (23.15%), oleic acid (21.44%) and linoleic acid (16.20%) as the major components. seven phenolic conpounds were identified and quantified by hplcdad and ferulic acid (12.81±0.26 mg/g) was the most abundant. the cream showed good antioxidant properties evaluated through β-carotene-linoleic acid assay, dpph• radical scavenging, abts•+ assay, cuprac assay, and metal chelating assay. the cream had higher activity in the dpph• assay (ic50 = 32.10±0.84 µg/ml), abts•+ assay (ic50 = 22.49±0.62 µg/ml) and cuprac assay (ic50 = 49.27±0.79 µg/ml) than α-tocopherol. the antioxidant effects are an indication that the cream can reduce oxidative stress on the skin including aging, carcinogenesis and inflammation. at 100 µg/ml, the topical cream showed tyrosinase inhibition of 48.23±0.87% regarded as relatively good compared to the standard tyrosinase inhibitor kojic acid, which showed 79.50±0.32% inhibition at the same concentration. the cosmetic cream was able to inhibit the melanin production rate-limiting enzyme, tyrosinase, indicating that it can control hyperpigmentation and skin spots. keywords: acacia sieberiana; topical cream; gc-ms; phenolic composition; skin diseases; antioxidant; tyrosinase inhibition. introduction skin conditions constitute a real public health problem in the world especially in tropical countries where they represent nearly 30% of consultations (basset et al., 1999). skin diseases are considered as health priorities, which can cause significant mortalities, and are generally include fungal infection, stretch marks, eczema, acne, oxidative stress, skin burns, hypopigmentation, hyperpigmentation, melasma and dermatitis caused by cosmetic bleaching agents (hay et al., 2006). human skin colour is determined by pigments such as hemoglobin, hemosiderin, carotene and melanins that play protective roles as well as determination of human skin and hair colour (yamaguchi and hearing, 2014). melanin is primarily responsible for skin pigmentation which protects the skin from adverse effects of ultraviolet radiation, skin burn and skin cancer but an over-production or loss of melanin can result in serious skin problems (zolghadri et al., 2019; tamfu et al., 2022b). although melanin has desired effects, an overproduction of melanin caused by the action of tyrosinase is undesirable. the production of melanin involves the enzymatic action of tyrosinase and other tyrosinaserelated proteins which play a critical role in the process. tyrosinase, which is a multifunctional copper-containing metalloenzyme is the major rate-limiting enzyme in melanogenesis (garcia-jimenez et al., 2017; alfred ngenge et al 2021). tyrosinase causes undesired browning of vegetables and fruits, overproduction of melanin and skin patches and spots (tamfu et al., 2020b, masum et al., 2019). inhibitors of tyrosine are therefore finding applications as attractive cosmetic and medicinal products for skin whitening and depigmentation as well as in agricultural and food industries as antibrowning agents (zolghadri et al., 2019; tamfu et al., 2020c). therefore, tyrosinase inhibition provides solution to perishing of vegetables and fruits as well as therapeutic potential in skin cancers, pigmentation and 1department of chemical engineering, school of chemical engineering and mineral industries, university of ngaoundere, 454 ngaoundere, cameroon. https://doi.org/10.14421/biomedich.2023.121.251-258 mailto:macntamfu@yahoo.co.uk 252 biology, medicine, & natural product chemistry 12 (1), 2023: 251-258 neurodegenerative disorders (masum et al., 2019; mughal et al., 2022). occurrence of reactive oxygen species (ros) and reactive nitrogen species are at the origin of many human ailments and it is necessary to inhibit their excesses (tamfu et al., 2021a). substances which can inhibit the destructive effects of ros, rns and free radicals and referred to as antioxidants. oxidative stress occurs in the situation of an imbalance between ros and rns production with respect to antioxidants and the oxidative damage that results from this can spread over most cells and tissues (talla et al., 2017; alain et al., 2022; sawalda et al., 2022). most antioxidant substances from natural and synthetic sources exist and they are capable of of suppressing ros and rns and protect the living systems against oxidative stress (tamfu et al., 2020d). synthetic antioxidants like butylated hydroxy toluene (bht) and butylated hydroxy anisole (bha) are usually insoluble and associated with side effects, thereby giving way for rising interest in natural antioxidants (talla et al., 2014; soni and loonker, 2022; tamfu et al., 2021b; lópezpedrouso et al., 2022). being constantly exposed to external aggressions, the skin represents a privileged target of oxidative stress, which leads to multiple skin damage (yapi et al 2019; awadji, 2021). oxidative stress is one of the main aggravating causes of these conditions, and is one of the potential factors of skin disorders that occur especially with age as well as the early aging of the body defenses. the skin regulates the excretion of waste products, controls body temperature, and protects the organism from environmental, chemical and physical stress factors. the skin is exposed to effects of sun rays, ros and rns which can damage the antioxidant defense capacity of the skin causing skin diseases (kruk and duchnik, 2014). many cases of skin problems are observed in the health care systems worldwide, but some (over 70% of individuals) of the patients with chronic transmissible skin diseases especially in tropical areas do not seek treatment but recourse to medicinal plants and traditional medicine as solution to their skin conditions (almoshari, 2022). acacia siberiana is one of those plants that pills in northern benin where it is strongly used in traditional medicine to treat skin conditions. a. sieberiana, is a plant of the fabaceae family, is an essentially thorny ligneous plant. it is native to the savannahs of africa. this plant can reach 25 m in height whose leaves are 10 to 15 cm long with straight white spines at their base (orwa et al., 2009). in order to derive a real benefit from the use of this plant, it becomes important to make a formulation for body use based on its extract with judiciously chosen excipients. the production of home-made medicinal skin ointments from vitellaria paradoxa is popular in most communities in tropical areas of africa. this work investigates the chemical composition by gc-ms and hplc-dad of a topical cream from vitellaria paradoxa, acacia siberiana and beeswax, and evaluation of its antioxidant and anti-tyrosinase activities. materials and methods plant material and extraction acacia sieberiana leaves were collected during the month of january 2021 from north of benin (malanville). the plants were identified and voucher specimens prepared by the botanist professor hounnankpon yedomonhan of the benin national herbarium where they were deposited under the specimen numbers yh 684/hnb, for acacia sieberiana. the extraction was carried out using ultrasound-assisted extraction with ethanol as solvent. briefly, 100 g of powdered plant material biomass from each plant were mixed with 1000 ml solvent (h2o2/etoh, 1/1) and sonicated for two hours at 50 °c with bandelin (sonorex digitech device). further, all the extracts were filtered through whatman no.1 filter paper and concentrated under vacuum on a rotavapor (buchi r215) at 50°c. the solid paste extract obtained was stored in the darkness at 4 °c to avoid the degradations until use. preparation of topical cream for the preparation of ointment, the hydroethanol extract of acacia sieberiana was used as the active ingredient. given the sticky aspect of this extract, 30 g of extract was first dissolved in 100 ml of extraction solvent in order to facilitate the preparation. 25 g of beeswax were melted and added unto the extract. the mixture was blended into a uniform paste at 70oc. 30 g of shea (vitellaria paradoxa) butter were added into the mixture and stirred for further 30 minutes. 5 g of glycerin and 5 g of vaseline were then added while mixing continuously to give a homogeneous pasty ointment. in order to have a perfectly homogeneous mixture, the temperature was maintained at 70°c for 10 min. the ointment was transferred into vials and allowed to cool and solidify before storage. table 1 shows the components of the topical cream with the percentage composition by mass of each component. table 1. components of the topical cream formulation. ingredients % composition by mass vitellaria paradoxa (shea) butter 30 vaseline 5 beeswax 30 glycerine 5 ethanol extract of acacia sieberiana 30 gc-ms characterization of the topical cream the extract (10 mg) was mixed with 150 μl of pyridine and 100 μl of bis(trimethylsilyl) trifluoroacetamide (bstfa) and heated at 80 °c for 20 min and then the tamfu et al. – chemical composition and bioactivities of hand-made cream 253 final supernatant was analyzed by gc-ms (mercan et al., 2006). an ion trap ms spectrometer and a rxi-5sil ms (restek) fused silica non-polar capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm) were used for the gc–ms analyses. carrier gas was helium with 1.4 ml/min flow rate. the injector and ms transfer line temperatures were 220 and 290ºc, respectively. the ion source temperature was at 200 ºc. the injection volume was 0.2 µl with a split ratio of 20:1. ei–ms measurements were taken at 70 ev ionization energy. mass range was from m/z 28 to 650 amu. scan time was 0.5 s with 0.1 s interscan delays. for analysis, the oven temperature was held at 100ºc for 10 min, then increased up to 280ºc with 4ºc/min increments and kept at this temperature for 10 min. identification of components of the sample was based on gc retention indices and computer matching with the wiley, nist-2008 and trlib libraries, as well as by comparison of the fragmentation patterns of the mass spectra which reported in the literature and, whenever possible, by coinjection with authentic compounds. determination of phenolic profile of the cream selected phenolic constituents in the extracts were identified and quantified through reversed-phase highperformance liquid chromatography (rp-hplc) linked to diode array detector (dad) as described elsewhere (küçükaydın et al., 2021; tamfu et al., 2022c). summarily, 5 g of each extract were dissolved in water:methanol (80:20), filtered through sterile 0.20 μm filter disk. an intertsil ods-3 reverse phase c18 column was used for the separation with a 1.0 ml/min solvent flow rate and 20 μl injection volume. two mobile phases a (0.5% acetic acid h2o) and b (0.5% acetic acid in ch3oh). a gradient elution was applied as follows: 0–10% b (0–0.01 min); 10–20% b (0.01–5 min); 20– 30% b (5–15 min); 30–50% b (15–25 min); 50–65% b (25–30 min); 65–75% b (30–40 min); 75–90% b (40–50 min) 90-10% b (50–55 min). a photodiode array detector set at 280 nm wavelength was employed in the detection and the uv data together with retention times were compared with authentic standards. each analysis was performed three times. a calibration plot established through the elution of known concentrations (range of 0.0~1.0 ppm) of authentic compounds was used in the identification and quantification of the constituent phenolic compounds. twenty-six phenolic standards (gallic, p-hydroxy benzoic, protocatechuic, ellagic, chlorogenic, trans-cinnamic, 3-hydroxy benzoic, vanillic, syringic, p-coumaric, rosmarinic and ferulic acids; catechin, kaempferol, hesperetin, pyrocatechol vanillin, 6,7-dihydroxy coumarin, coumarin, rutin, myricetin, chrysin, luteolin, apigenin taxifolin and quercetin) were used. the results were expressed as μg per g dry weight of extract. antioxidant activity of the cream β-carotene-linoleic acid method, with a few minor modifications, was used to assess the ability to inhibit lipid peroxidation (tamfu et al., 2020a) the spectrophotometric evaluation of the dpph• and abts•+ radical scavenging activities was conducted as described earlier (tamfu et al., 2020b) the cupric reducing antioxidant capacity (cuprac) was measured using the method as previously described (apak et al., 2004). bha (butylated hydroxyanisole) and α-tocopherol were employed as reference compounds for comparison of the β-carotene-linoleic acid, dpph•, abts•+ and cuprac assays. the metal chelating activity of the extracts for fe2+ was determined spectrophotometrically (decker and welch, 1990). edta was used as the reference compound to compare metal chelating activity. antioxidant activity results were given as 50% inhibition concentration (ic50). anti-tyrosinase activity of the cream the inhibition of tyrosinase enzyme (mushroom source) was determined through the method published elsewhere (masuda et al., 2005) using l-dopa as the substrate. in a 96-well microplate, 10 μl solution of sample or kojic acid (reference) were added to 150 μl of sodium phosphate buffer (ph 6.8, 100 mm) followed by 20 μl of tyrosinase enzyme and the resulting mixture incubated at 37ºc for 10 minutes. 20 μl of l-dopa were added after incubation and the absorbances of the resulting solutions were taken at 475 nm and the results expressed as concentration at which 50% inhibition was observed (ic50). statistical analyses descriptive statistics were applied on the data obtained. each experiment was done in triplicate and the means of three parallel measurements were deduced. the values given are means±sem (standard error of the mean) for three measurements. one-way anova (analysis of variance) was used to compare differences amongst the means and were considered statistically significant p < 0.05. results and discussion natural products and medicinal plant extracts are being used as alternative therapies for skin care purposes and are becoming more prevalent in formulations, due to consumers’ concerns about synthetic ingredients/chemical substances. the main benefits reported for plant extracts, used in skin care, include antioxidant and tyrosinase inhibition effects amongst others. due to their uses in skin care products, it is necessary to investigate their chemical composition to guarantee their safety. 254 biology, medicine, & natural product chemistry 12 (1), 2023: 251-258 gc-ms chemical composition of the topical cream the volatile constituents of the topical cream were determined by gc-ms and reported on table 1. a total of thirteen volatile compounds were identified, out of which twelve were fatty acids and one fatty alcohol. amongst the identified constituents, 61.09% were saturated fatty acids while 37.64% were unsaturated fatty acids together with 1.27% of other compounds. gc-ms characterization with co-injection of the topical cream revealed that, stearic acid (31.43%), palmitic acid (23.15%), oleic acid (21.44%) and linoleic acid (16.20%) were the major compounds. table 3. chemical profile of topical cream by gc-ms. no ria compounds cosmetic product (%)b identification methods 1 1152 2-nonen-1-ol 0.52 co-gc, ms, ri 2 1160 benzoic acid 0.75 co-gc, ms, ri 3 1170 octanoic acid 1.73 co-gc, ms, ri 4 1265 nonanoic acid 0.94 co-gc, ms, ri 5 1373 decanoic acid 1.15 co-gc, ms, ri 6 1492 suberic acid 0.31 ms, ri 7 1566 lauric acid 0.77 ms, ri 8 1625 azelaic acid 0.40 ms, ri 9 1760 myristic acid 1.21 co-gc, ms, ri 10 1957 palmitic acid 23.15 co-gc, ms, ri 11 2190 linoleic acid 16.20 co-gc, ms, ri 12 2195 oleic acid 21.44 co-gc, ms, ri 13 2237 stearic acid 31.43 co-gc, ms, ri saturated fatty acids 61.09 unsaturated fatty acids 37.64 other compounds 1.27 total 100 a: kovats index on rxi-5sil ms fused silica column, b: percentage concentration, co-gc: co-injection with authentic compounds; ms: based on comparison with wiley, adams and nist 08 ms databases ri: retention index literature comparison hplc-dad phenolic composition of the topical cream the phenolic profile of the topical cream was evaluated by hplc-dad with twenty-six standard phenolic compounds, and seven of them were identified and quantified in milligrams per gram of extract (mg/g) and recorded on table 1. the phenolic compounds gallic acid (2.70±0.10 mg/g), chlorogenic acid (3.93±0.07 mg/g), ferulic acid (12.81±0.26 mg/g), coumarin (4.10±0.18 mg/g), rutin (0.39±0.04 mg/g), ellagic acid (0.17±0.02 mg/g) and rosmarinic acid (8.48±0.21 mg/g) were identified and quatified. the most abundant consituent was ferulic acid followed by rosmarinic acid and then coumarin and chlorogenic acid. antioxidant activity of the topical cream the antioxidant capacity of the topical cream was evaluated through five complementary assays namely βcarotene-linoleic acid assay, dpph• assay, abts•+ assay, cuprac assay and metal chelating assay and the results presented on table 3. the cream had higher activity in the dpph• assay (ic50 = 32.10±0.84 µg/ml), abts•+ assay (ic50 = 22.49±0.62 µg/ml) and cuprac assay (ic50 = 49.27±0.79 µg/ml) than α-tocopherol which was used as one of the standards, while its activity were relatively close to that of standard bha as well. the antioxidant activity of the cream was however moderate in the β-carotene-linoleic and metal chelating assays. the antioxidant effects is an indication that the cream can reduce oxidative stress and the negative effects of excess reactive oxygen species (ros) and reactive nitrogen species (rns) on the skin including aging, carcinogenesis and inflammation. anti-tyrosinase activity of the topical cream tyrosinase is necessary but excessive tyrosinase is unwanted. inhibitors of excessive tyrosinase are used in treating skin pigmentation problems and the tyrosinase inhibition of the topical cream was evaluated and the results presented on table 3. at a test concentration of 100 µg/ml, the topical cream showed tyrosinase inhibition of 48.23±0.87% and this activity can be regarded good when compared to the standard tyrosinase inhibitor kojic acid, which showed 79.50±0.32% inhibition at the same concentration. the cosmetic cream was able to inhibit the melanin production rate-limiting enzyme, tyrosinase, indicating that it can control melanogenesis, skin spots and skin whitening. tamfu et al. – chemical composition and bioactivities of hand-made cream 255 table 2. phenolic composition of topical cream by hplc-dad (mg/g)a. no phenolic compounds rt (min) cosmetic product 1 gallic acid 5.70 2.70±0.10 2 protocatechuic acid 8.75 3 catechin 10.68 4 pyrocatechol 11.04 5 chlorogenic acid 12.35 3.93±0.07 6 p-hydroxy benzoic acid 12.77 7 6.7-dihydroxy coumarin 14.10 8 caffeic acid 15.09 9 3hydroxy benzoic acid 15.98 10 syringic acid 16.56 11 vanillin 17.78 12 p-coumaric acid 20.56 13 taxifolin 21.26 14 ferulic acid 22.14 12.81±0.26 15 coumarin 24.49 4.10±0.18 16 rutin 25.30 0.39±0.04 17 ellagic acid 26.11 0.17±0.02 18 rosmarinic acid 26.77 8.48±0.21 19 myricetin 27.35 20 quercetin 30.83 21 trans-cinnamic acid 31.33 22 luteolin 31.70 23 hesperetin 32.14 24 kaempferol 33.21 25 apigenin 33.77 26 chrysin 38.40 avalues expressed are means ± s.e.m. of three parallel measurements (p < 0.05). b -: not detected table 4. antioxidant and anti-tyrosinase activities of topical cream. antioxidant activity anti-tyrosinase activity sample β-carotene-linoleic acid assay dpph• assay abts•+ assay cuprac assay metal chelating assay tyrosinase inhibition ic50 (µg/ml) ic50 (µg/ml) ic50 (µg/ml) a0.50 (µg/ml) ic50 (µg/ml) % inhibition at 100 µg/ml topical cream 18.31±0.53 32.10±0.84 22.49±0.62 49.27±0.79 57.60±0.94 48.23±0.87 α-tocopherol 2.10±0.07 38.15±0.50 35.50±0.56 60.25±0.55 nt nt bha 1.45±0.03 19.75±0.33 12.80±0.08 25.40±0.40 nt nt edta nt nt nt nt 5.52±0.35 nt kojic acid nt nt nt nt nt 79.50±0.32 values represent the means ± sem of three parallel sample measurements (p < 0.05). nt: not tested. discussion many fatty acids were identified in the topical cream. gc-ms is suitable for the identification of volatile and fatty components of natural extracts and plant samples including fatty acids (eve et al., 2020; carol et al., 2017; koudoro et al., 2022). it could be suggested that most of the fatty acids are coming from the beeswax and shea butter which were used as important matrix components for the production of the cream. bee products such as beeswax used as component in the production of the skin ointment can suitably be characterized by gc-ms since they are made of volatile constituents (ngenge et al., 2016). important skin protective fatty acids such as palmitic acid, linoleic acid, oleic acid and stearic acids were identified as major constituents. fatty acids and their derivatives are important ingredients in cosmetic 256 biology, medicine, & natural product chemistry 12 (1), 2023: 251-258 development which act like brighteners, emulsifiers or softeners especially lauric acid, palmitic acid, myristic acid and stearic acid (yang et al., 2020). fatty acids act as skin barrier and reduce water loss on skin, prevent entry of harmful substances and can play antimicrobial and anti-inflammatory roles (cochran et al., 2015). this indicates that the rich fatty acid content of the cream can improve its protective effects on the skin and also avoid entry of bacteria and fungi as well as other objects. it can also control water loss and uv damage. phenolic extracts and compounds from plants possess anti-inflammatory and antioxidant effects that reduce the damage caused by oxidative species and prevent skin diseases and aging (boo, 2019). phenolic compounds are suitably extracted by using ultrasonic means and identified by hplc-dad method and their major interest is due to their antioxidant effects amongst other beneficial activities (tamfu et al., 2022a). important bioactive phenolic compounds have been identified in the topical cream and they could be from a. sieberiana, a fabaceae plant which is rich in phenolic compounds and possesses antioxidant effects (alain et al., 2022; zongo et al., 2023). the presence of phenolic compounds in the cream could confer oxidative protection and most cosmetologists and dermatologists are searching for safer and more effective natural antioxidants to prevent or treat premature aging and skin diseases. human skin is exposed to oxidative stress resulting from conditions like sunlight, radiations, pollutants, stress and oxidants which results to aging, wrinkles, excess skin pigmentation and skin tone unevenness (chen et al., 2021). the antioxidant effect of the cream was evaluated in five different and complementary assays so as to give a global indication of its antioxidant capacity in the different models (alfred et al., 2020). the phenolic compounds and fatty acids identified in the cream can act as natural antioxidant, antiinflammatory, antimicrobial and anti-aging compounds and also as a skin barrier against other biotic and abiotic factors. one important skin problem is irregular spots and pigmentation disorders. tyrosinase is a copper metallo-enzyme which plays an important role in melanogenesis, over-pigmentation and enzymatic browning and therefore, its inhibitors are used in solving skin disorders, whitening and hyperpigmentation (panzella and napolitano, 2021). the topical cream was able to inhibit tyrosinase which shows that it can be used for skin care and skin whitening purposes. pigmentation determines the color of the skin and uv radiation protection of skin but hyperpigmentation results to spots and melasma, which could be suitably solved by tyrosinase inhibitors. skin diseases such as dermatitis, acne vulgaris, psoriasis, urticaria, melanoma, keratinocyte carcinoma, scabies and fungal skin diseases can get natural therapies from medicinal plant-based products (karimkhani et al., 2017; panzella and napolitano, 2021; chen et al., 2021). skin disorders have significant impacts on the affected person’s quality of life especially oxidative damage and skin spots. skin lightening can be achieved by using natural or synthetic products to improve skin tone, give even skin complexion and reduces melanin production thereby helping patients to treat their skin problems including age spots, acne scars, freckles, skin burns and uneven coloration (salsberg et al., 2016; masum et al., 2019). conclusions the skin is exposed to environmental stress and physical, chemical and biological factors can affect it. also, in its functions of defense and homeostasis, the skin can encounter negative effects which need to be solved. many artificial cosmetic and dermatological treatments are available for skin problems but they are expensive and unaffordable to many and also have side effects. for these reasons, many local solutions from medicinal plants and other natural products are available and are used with positive results. in this study, a topical local cream prepared from a. sieberiana, beeswax and other ingredients was evaluated for its chemical compostion by gc-ms, hplc-dad as well as its antioxidant and antityrosinase activities. important fatty acids were identified most of which are relevant in cosmetic science and play a protective role on the skin. bioactive phenolic compounds were also identified. the good antioxidant and tyrosinase inhibition of the topical crean is attributable to the fatty acids and phenolic compounds whch it contains. the scientifc evidence of the beneficial utility of the skin cream is suggested based on the results and ascertains its use by local populations for various skin disorders as well as wounds and burns. acknowledgements: the authors are grateful to mugla sitki kocman university, the university of ngaoundere as well as university of abomey-calavi. authors’ contributions: alain yaya koudoro, theophile olaye, pascal dossa cokou agbangnan, dominique codjo koko sohounhloue and felicien avlessi obtained materials, prepared samples, designed the study and carried part of experiments. alfred ngenge tamfu and selcuk kucukaydin did the experiments, designed methodologies, analyses, data curation and wrote the first draft. all authors contributed materials, read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references alain ky, tamfu an, kucukaydin s, ceylan o, pascal ad, félicien a, dominique sc, duru me, dinica rm. phenolic tamfu et al. – chemical composition and bioactivities of hand-made cream 257 profiles, antioxidant, antiquorum sensing, antibiofilm and 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busuioc, a.; meda, r.n.-t.; botezatu, a.v.; mihaila, m.d.; mocanu, a.-m.; avramescu, s.m.; koama, b.k.; kam, s.e.; belem, h.; et al. exploration of the antioxidant and anti-inflammatory potential of cassia sieberiana dc and piliostigma thonningii (schumach.) milne-redh, traditionally used in the treatment of hepatitis in the hauts-bassins region of burkina faso. pharmaceuticals 2023, 16, 133. https://doi.org/10.3390/ph16010133 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 89-109 | doi: 10.14421/biomedich.2022.112.89-109 issn 2540-9328 (online) the antiviral potential of iranian herbal pharmacopoeia (ihp) on herpes simplex viruses (hsv): a review article shahin gavanji young researchers and elite club, isfahan (khorasgan) branch, islamic azad university, isfahan, iran university blvd, arqavanieh, jey street, p.o. box:81595-158, isfahan, iran. corresponding author gavanji.shahin2@gmail.com manuscript received: 18 june, 2022. revision accepted: 19 july, 2022. published: 30 july, 2022. abstract herpes simplex viruses (hsv) viruses are highly contagious that commonly cause dermatitis, encephalitis, meningitis and genitourinary infections and also can lead to cervical cancer. for treatment of hsv infections, several physical methods and antiviral drugs are introduced, antiviral medications can also prevent or reduce outbreaks. the use of herbal medicine with antiviral effects attracted worldwide attentions. the aim of this review article is to introduce the iranian herbal pharmacopoeia (ihp) with antiviral potential against two hsv serotypes and help to evaluate and develop the new drugs from natural sources. to provide this review, relevant articles in some authentic databases including pubmed, web of science, science direct, scopus, google scholar and sid (scientific information database), from 1967 to april 2022 were collected, and selected botanicals from (ihp) with their scientific names and classifications and their outcome (cc50 and ic50) were introduced. in this review, scientific data regarding anti-herpetic activities of iranian herbal pharmacopoeia (ihp) showed that 34 herbs from 17 families have antiviral potential to inhibit two hsv serotypes. according to families, lamiaceae family has the highest percentage (29.41 %) of plants with antiviral activity against two hsv serotypes, also review of recent data showed that salvia officinalis, melissa officinalis, securigera securidaca, hyssopus officinalis, quercus brantii, artemisia aucheri and curcuma longa have remarkable antiviral activity against two hsv serotypes. results of this review suggest that further research to identify and purify the bioactive compounds to determine the molecular mechanisms of action is needed. keywords: herpes simplex; herbal medicine; pharmacopoeia; antiviral; drugs. introduction herpes simplex virus (hsv) belongs to the herpesviridae family and alphaherpesvirinae subfamily (álvarez et al. 2020), is a global public health issue that can cause serious infection and classified into 2 serotypes: hsv-1 and hsv-2 (gavanji et al. 2015; pebody et al. 2004). the hsv genome consists of linear double-strand dna, that causes primary and recurrent lesions (reuven et al. 2003) hsv viruses are highly contagious that commonly cause dermatitis, encephalitis, meningitis and genitourinary infections and also can lead to cervical cancer (kłysik et al. 2020; elad et al. 2010). hsv-1 or oral herpes mainly occurs on the face, chin, lips and mouth area which infection with this serotype causes small blisters or cold sores and inflammation of oral and eye cells (asai and nakashima 2018; vaghela et al. 2021), ranging from mild to severe, that may develop and increase the risk of serious illness (farooq and shukla 2012; anderson et al. 2014). this type of virus usually transmitted via oral secretions or direct contact with cold sores and it can cause more severe complications such as conjunctivitis (koujah et al.2019), herpetic stromal keratitis (hsk) (stuart and keadle 2012), gingivostomatitis (george and anil 2014) and hsv-1 encephalitis (hse) (feola et al. 2018). hsv-2, infection is usually transmitted through sexual contacts (zhang et al. 2022) and affects the genital area and increased risk of human immunodeficiency virus (hiv) transmission (crisci et al. 2019). for treatment of hsv infections, several physical methods and antiviral drugs such as valacyclovir, acyclovir, penciclovir, cidofovirare, and famciclovir are introduced (sadowski et al. 2021). the antiherpetic agents target and inactive viral dna polymerase enzyme and inhibit the proliferation and viral replication (li et al 2019). acyclovir (acv) is an antiviral agent which widely used to control and treatment of hsv infections (wei et al. 2021). in the process of drug action, the acv converts to acyclovir triphosphate (acyclo-gtp) and inhibits viral dna polymerase (fyfe et al. 1978). nowadays, due to the resistance mutations and tolerant enzyme production, especially in people with immunodeficiency disorders, urgent need to investigate the new antiviral drugs has been highlighted (roy et al. 2022; majewska and mlynarczyk-bonikowska 2022; chuerduangphui et al. 2022). numerous research studies have been done to investigate the novel antiviral agents https://doi.org/10.14421/biomedich.2022.112.89-109 90 biology, medicine, & natural product chemistry 11 (2), 2022: 89-109 (jassim and naji 2003), and many anti-herpetic drugs have been approved (sadowski et al. 2021), also researchers have been contributing to develop and produce a new vaccine against herpes infection but, no preventive hsv vaccines have been approved (krishnan and stuart 2021). the use of herbal medicine with antiviral effects attracted worldwide attentions (gavanji et al. 2014). scientific research has shown that natural bioactive compounds like flavonoids, therpens, phenols and alkaloids have anti-hsv properties (pesola and coen 2008; tolo 2006). among the compounds mentioned, phenolic classes are the most commonly used for their anti-hsv properties (treml et al. 2020) (table1). this article is a review which provide various information about the potential antiviral activity of iranian herbal pharmacopoeia (ihp) on herpes simplex viruses (hsv), and help to evaluate and develop the new drugs from natural sources. methods to provide this review, all reported herbal medicine with antiviral effects against herpes simplex viruses (hsv) were collected through iranian herbal pharmacopoeia (ihp) (ghasemi dehkordi et al. 2003) and the most relevant articles in some authentic databases including pubmed, web of science, science direct, scopus, google scholar and sid (scientific information database), from 1967 to april 2022 were searched. the search with different combinations of keywords were herbal medicine, herpes simplex viruses, antiviral agents, simplex virus, plants, iran, herb. in this review, the selected articles and books were used to sets of the general and specific criteria to examine and present the medicinal plants with antiviral activities. selected botanicals with their scientific names and classifications and their outcome (cc50 and ic50) are presented in the article. antiviral activity of phytochemicals against hsvs todays, various compounds with antiviral activity are contributing in the most of the medicinal process for the cure of human viral infections (singh et al. 2021; mukhtar et al. 2008). many studies have shown that naturally produced compounds with antiviral activity against two serotypes of hsv are polysaccharides (jin et al. 2015), flavonoids (flores et al. 2016), terpenes (soares et al. 2007), steroids (da rosa guimarães et al. 2013), saponins (ogawa et al. 2021), tannins (lin et al. 2011), phenolic (hassan et al. 2011), alkaloids (chen et al 2015), lignans (chen et al 2015), miscellaneous (treml et al. 2020), proanthocyanidin (terlizzi et al. 2016), quinones (caruso et al. 2020) and thiosulfinates (rouf et al 2020). several antiviral mechanisms of these phytochemicals have been identified, which most of them were verified against rna and dna viruses (jang et al. 2021; zrig 2022), such as herpes simplex virus type 1 and type 2 (dna viruses) (liu et al. 2019). these compounds inhibit the specific processes in the viral replication cycle (hassan et al. 2018), and viral entry to the target cells (rouf et al 2020), also they inhibit the viral gene and protein expression and suppress the nfkb activity (hutterer et al. 2017) that can help to prevent the spread of viruses. several studies have shown the antiviral mechanisms of these natural compounds against two hsv serotypes which are summarized in table 1. antiviral potential of iranian herbal pharmacopoeia (ihp) the present review focuses on selected iranian herbal pharmacopoeia (ihp) with antiviral potential against two hsv serotypes. in this review, the articles were read and 34 herbs from 17 families were selected based on anti-herpetic activity and percentages of each families were calculated. according to families in ihp, lamiaceae has the highest percentage (29.41 %) of plants with antiviral activity against two serotypes of hsv (figure 1). figure 1. the percentages of plant families against two serotypes of hsv. gavanji – anti-herpetic activity of herbal pharmacopoeia (ihp) 91 table 1. anti-herpetic activity of natural compounds. references serotype mechanisms compositions chemical groups no (kutluay et al. 2008; zalilawati et al. 2015; zandi et al. 2010; flores et al. 2016) hsv-1/hsv-2 affecting the viral transactivator protein vp16 -mediated recruitment of rna polymerase ii (pol ii) to immediate early (ie) gene promoters and inhibits the hsv-1 replication, inhibition of hsv-1 and hsv-2 replication and adsorption curcumin flavonoid 1 (lyu et al. 2005) hsv-1/hsv-2 inhibition of viral adsorption galangin flavonoid 2 (lee et al 2017) hsv-1 inhibiting expression of glycoprotein d (gd) and infected cell protein 0 (icp0), and suppresses the tlr-3 quercetin flavonoid 3 (li et al 2017) hsv-1 blocking membrane fusion houttuynoid flavonoid 4 (de oliveira et al. 2013; isaacs et al. 2008; subramanian and geraghty 2007) hsv-1 inhibition of viral adsorption, binding to hsv glycoproteins epicatechin gallate flavonoid 5 (hung et al. 2015) inhibition of nf-κb activation isoquercitrin flavonoid 6 (hutterer et al. 2017; chen et al 2015) hsv-1/hsv-2 inhibition of viral replication and gene expression, reduction the nf-κb activation, and iκb-α degradation harmine alkaloid 7 (hassan et al. 2011) hsv-1 inhibition of hsv-1 dna polymerase psoromic acid phenolic 8 (li et al 2005; hao 2019) hsv-1 inhibition of viral replication protocatechuyl aldehyde phenolic 9 (ma et al. 2016) hsv-1 inhibition of hsv-1 adsorption and reduction of (ie) gene expression and viral dna synthesis kuwanon x phenolic 10 (lin et al. 2011) hsv-1 blocking the interactions of virus and target cells chebulagic acid (chla) tannins 11 (lin et al. 2011) hsv-1 blocking the interactions of virus and target cells punicalagin (pug) tannins 12 (kesharwani et al. 2017) hsv-2 inhibition of viral attachment to the target cells chebulinic acid ( tannins 13 (kuo et al. 2002) hsv-1 inhibition of viral replication samarangenin tannins 14 (soares et al. 2007) hsv-1 maybe target the hsv-1 replication epitaondiol diterpenes 15 (álvarez et al. 2015) hsv-1 inhibition of viral gene expression (e)-2-(2,4-hexadiynyliden) 1,6-dioxaspiro[4.5]dec-3-ene miscellaneous 16 (jin et al. 2015) hsv-1 blocking the viral entry to the target cells by inactivating the viral particles, inhibition of viral intracellular biosynthesis eucheuma gelatinae polysaccharide (egp) polysaccharide 17 (zhu et al. 2006) hsv-1 inhibition of hsv-1 replication and viral entry to the target cells sulfated polysaccharide polysaccharide 18 (bouhlal et al. 2011) hsv-1 inhibition of virus adsorption polysaccharide boergeseniella thuyoides polysaccharide 19 (saha et al. 2012) hsv-1 inhibition of viral attachment to the target cells by interaction with virus particles alginic acids and sulfated polysaccharide 20 (zheng and lu 1990) hsv-1 inhibition of viral replication mangiferin polyphenol 21 (astani et al 2014; chen et al 2017) hsv-1/hsv-2 inhibition of viral replication and viral attachment to the target cells rosmarinic acid polyphenol 22 (kuo et al 2006) hsv-1 inhibiting expression of infected-cell polypeptide 4 (icp4) and infected cell protein 0 (icp0), and viral dna synthesis yatein polyphenol 23 (hassan et al. 2018) hsv-2 inhibition of viral replication geraniol monoterpenoid 24 (wiart et al. 2005) hsv-1 inhibiting expression of glycoprotein d andrographolide diterpenoid 25 (da rosa guimarães et al. 2013) hsv-1 inhibition of viral attachment to the target cells halistanol sulfate steroids 26 (rouf et al 2020) inhibition of viral entry to the target cells allicin organosulfur 27 (ikeda et al. 2005) hsv-1 inhibition of viral replication glycyrrhizic acid methyl ester triterpenoid 28 (tshilanda et al. 2020; bag et al. 2012) hsv-1/hsv-2 inhibition of viral replication [78,79] ursolic acid triterpenoid 29 92 biology, medicine, & natural product chemistry 11 (2), 2022: 89-109 apiaceae apiaceae family which is called umbelliferae, includes a large number of medicinal plants with various therapeutic properties has a significant role in pharmaceutical development (amiri and joharchi 2016; ekiert 2000). cuminum cyminum belonging to family apiaceae is an aromatic plant that can be used as medicinal herbs to treat various diseases (sowbhagya 2013; mnif and aifa 2015; gavanji et al. 2015) and flavoring agents to improve the flavor of foods (johri 2011). researchers have reported that methanol extract of c. cyminum showed the antiviral effects against hsv1 (table 2). based on this study, the cc50, ic50 values were 0.45 and 0.18 mg/ml, respectively (motamedifar et al. 2010). the molecular mechanism of c. cyminum against herpes simplex virus has not yet been characterized, but the polyphenolic compounds in the extract may inhibit the hsv virus (ani et al. 2006). asteraceae the asteraceae family, or sunflower family, including several thousand plants, has a long history of use in traditional herbal medicine (thm), for the treatment of diseases (amiri and joharchi 2016; ekiert 2000). artemisia aucheri belonging to family asteraceae is an aromatic and endemic plant in iran, which is widely used for treatment of various diseases (gavanji et al. 2014). this plant has been reported for its antiviral activities (kshirsagar and rao 2021), and karamoddini et al assessed the antiviral properties of artemisia species against hsv-1 which artemisia annua inhibited this serotype at different concentrations (karamoddini et al. 2011). another study indicated that aqueous extract of a. aucheri had anti-herpetic property on hsv-1 and reduced the expression of ul46 and us6 genes, and the ic50 of this extract, 24.7 μg/ml was determined (zamanian et al. 2021). in the other study the antiherpetic activity of aqueous extract of a. aucheri against hsv-1 was evaluated that viral infection significantly reduced at 50 and 75 μg/ml (zamanian et al. 2021). research in 2015 showed that phenolic compounds in artemisia has antiherpetic activity which can cause some abnormalities in the function and structure of herpes simplex virus (gavanji et al. 2015). arctium lappa is another member of asteraceae family which possesses various therapeutic potential to treat infectious diseases (bai et al. 2016), in addition a. lappa exhibited, anti-inflammatory (pirvu et al. 2017), anticancer (leonard et al. 2006) and antiviral effects against two serotypes of hsv (chan et al. 201; dias et al. 2017). a study has been reported, that 400 mg/ml of hydroalcoholic extract of a. lappa inhibited the hsv-1 in in vitro condition (dias et al. 2017) (table 2). echinacea purpurea a species of asteraceae family is widely used for the treatment of diseases (shemluck 1982), such as chest & lung conditions, colds, coughs, candidiasis and influenza (hudson et al. 2005). furthermore, e. purpurea exhibited significant antiviral activity against hsv-1 (thompson 1998). also, garcia et al. assessed the antiherpetic activity of e. purpurea against hsv-1 which the ic50 and cc50 values, were determined to be 500 and 900 μg/ml, respectively (farahani 2013). according to a study, chicoric acid exhibited antiviral properties against hsv-1 (binns et al. 2002). another in vivo study showed that e. purpurea polysaccharide (ep), antiviral effects on the development of hsv by promoting the immune response (ghaemi et al. 2009). another important species of asteraceae family is tanacetum parthenium which has been traditionally used to treat various diseases (ghaemi et al. 2009). benassi-zanqueta et al. assessed the antiviral efficacy of chlorogenic acids and parthenolide which derived from t. parthenium against hsv-1 that results showed that chlorogenic acids was effective against hsv-1 (benassi-zanqueta et al. 2019). base on this research, the hydroethanolic extract inhibited viral replication and the ec50 value was18.1 mg/ml. avicenniaceae avicenna marina is a member of avicenniaceae family, which traditionally used for treatment of small pox, rheumatism and respiratory problems (afzal et al. 2011; namazi et al 2013). and has strong antiviral activity against hsv-1 (chiang et al. 2003; namazi et al, 2013; behbahani et al. 2013). a study showed that glycerin extract of a. marina, inhibited the hsv-1 and the ic50 values before and after virus attachment and cc50 were determined to be 87.1, 41.9 and 5750.96 μg/ml, respectively (zandi et al. 2009). based on the previous studies, a. marina, contains many phytocompounds with antiviral potentials to inhibit herpes simplex viruses (hsv). several studies have shown that flavonoids in the a. marina extract plays a crucial role in antiviral activities against hsv-1 (chiang et al. 2003; namazi et al, 2013; behbahani et al. 2013). (table 2). gavanji – anti-herpetic activity of herbal pharmacopoeia (ihp) 93 table 2. antiviral mechanism of iranian herbal pharmacopoeia (ihp) against herpes simplex viruses (hsv-1 and -2) no plant name family type of study (in vitro or in vivo) type of virus compounds and mechanisms references 1 aloe vera xanthorrhoeacea e in vitro hsv-1/ hsv-2 aloe-emodin inhibits the replication of viral enveloped (lin et al. 2008; zandi et al. 2007; rezazadeh et al. 2016) 2 artemisia aucheri asteraceae in vitro hsv-1 artemisinin inhibits the central regulatory processes and blocks the metabolic requirements of replication, reduces the ul46 and us6 genes (kshirsagar and rao 2021; karamoddini et al. 2011; zamanian et al. 2021a; zamanian et al. 2021b). 3 arctium lappa asteraceae in vitro hsv-1 arctigenin inhibits the viral replication, phenolic constituents such as caffeic acid and chlorogenic acid inhibit the viral multiplication (dias et al. 2017; wang et al. 2019; yang et al 2005; lal et al 2020; chiang et al. 2002; chan et al. 2011) 4 avicenna marina avicenniaceae in vitro hsv-1 luteolin inhibits the viral replication (chiang et al. 2003; namazi et al. 2013) 5 camellia sinensis theaceae in vitro hsv-1 epicatechin gallate inhibits the viral adsorption, binding to hsv glycoproteins (de oliveira et al. 2013; isaacs et al. 2008; subramanian and geraghty 2007) 6 cuminum cyminum apiaceae. in vitro hsv-1 ehp [1-(2-ethyl, 6-heptyl) phenol] effects on the percentage of plaque (mohamadein et al. 2015; motamedifar et al. 2010) 7 curcuma longa zingiberaceae in vitro hsv-1/ hsv-2 affecting the viral transactivator protein vp16 -mediated recruitment of rna polymerase ii (pol ii) to immediate early (ie) gene promoters and inhibits the hsv-1 replication, inhibition of hsv-1 and hsv-2 replication and adsorption (hutterer et al. 2017; kutluay et al. 2008; zalilawati et al. 2015; flores et al. 2016) 8 echium amoenum boraginaceae in vitro hsv-1 rosmarinic acid inhibits the viral attachment to host cells (flores et al. 2016; chen et al. 2017) 9 echinacea purpurea asteraceae in vitro/in vivo hsv-1 cichoric acid interact and inhibit the virus activities (binns et al. 2002; pluymers et al. 2000; burlou-nagy et al. 2022; zhang et al. 2014) 10 euphorbia spinidens euphorbiaceae in vitro hsv-1 betulin and (3β,23e)-cycloarta-23-ene-3,25-diol, the extract inhibits the viral replication and adsorption (shamsabadipour et al. 2013; karimi et al. 2016) 11 eucalyptus caesia myrtaceae in vitro hsv-1 unknown (schnitzler et al. 2001; brezáni et al. 2018; mierescastro et al. 2021) 12 eucalyptus globulus myrtaceae in vitro hsv-1 grandinol, sideroxylin, and tereticornate inhibit the viral replication (schnitzler et al. 2001; brezáni et al. 2018; mierescastro et al. 2021; ma and yao 2020; mohan et al. 2020) 13 glycyrrhiza glabra leguminosae in vitro hsv-1 glycyrrhizic acid (glycyrrhizin) inhibits the viral replication, suppress the growth (ghannad et al. 2014; fukuchi et al. 2016; van rossum et al. 1998; cohen 2005; huan et al. 2021) 14 hypericum perforatum hypericaceae in vitro/in vivo hsv-1/ hsv-2 hypericin inhibits the viral adsorption (huan et al. 2021; weber et al. 1994; mohamed et al. 2022; fritz et al. 2007; westh et al. 2004; béjaoui et al. 2017) 15 hyssopus officinalis lamiaceae in vitro hsv-1/ hsv-2 unknown (akram et al. 2018; behbahani 2009; schnitzler et al. 2019) 16 melissa officinalis lamiaceae in vitro hsv-1/ hsv-2 rosmarinic acid inhibits the viral attachment to host cells (astani et al 2014a; astani et al. 2012b; mazzanti et al. 2008) 17 mentha piperita lamiaceae in vitro hsv-1/ hsv-2 piperitenone oxide (peo) and menthol inhibit the viral replication and adsorption (wolbling et al. 1994; koytchev et al. 1999; civitelli et al. 2014; herrmann et al. 1967) 18 myrtus communis myrtaceae in vitro hsv-1 unknown (moradi et al. 2011; alipour et al. 2014; isaacs et al. 2008) 94 biology, medicine, & natural product chemistry 11 (2), 2022: 89-109 no plant name family type of study (in vitro or in vivo) type of virus compounds and mechanisms references 19 olea europaea oleaceae in vitro hsv-1 phenolic compounds such as caffeic acid inhibits the viral multiplication (motamedifar et al. 2015; ben-amor et al. 2021; ikeda et al. 2011) 20 ocimum basilicum lamiaceae in vitro hsv-1/ hsv-2 ursolic acid and apigenin inhibit the viral replication (chiang et al. 2005; bag et al. 2012; lin et al. 2008) 21 plantago major plantaginaceae in vitro hsv-1/ hsv-2 caffeic acid inhibits the viral multiplication (chiang et al. 2002; ben-amor et al. 2021; ikeda et al. 2011) 22 quercus persica fagaceae in vitro hsv-1 tannic acid inhibits viral replication (chiang et al. 2005; wu et al. 2022; karimi et al. 2013; kaczmarek 2020; nance and shearer 2013) 23 ricinus communis euphorbiaceae in vitro hsv-1 the alkaloid and phenolic compound have antiherpetic activity (elkousy et al. 2021; abdul et al. 1996) 24 rheum palmatum polygonaceae in vitro/in vivo hsv-1 unknown (abdul et al. 1996; kurokawa et al. 1993; chang et al. 2014; shen et al. 2019) 25 rosmarinus officinalis lamiaceae in vitro hsv-1/ hsv-2 rosmarinic acid inhibit of viral replication and viral attachment to the target cells (al-megrin et al. 2020; mancini et al. 2009; hitl et al. 2021; chen et al. 2017; astani et al 2014) 26 salvia officinalis lamiaceae in vitro/in vivo hsv-1/ hsv-2 thujone, β-caryophyllene linalyl acetate, alpha terpinyl acetate, and germacrene d have anti-herpetic activity (shen et al. 2019; al-megrin et al. 2020; mancini et al. 2009; hitl et al. 2009; schnitzler et al. 2008; rajbhandari et al. 2001; santoyo et al. 2014; ezema et al. 2022) 27 satureja hotensis lamiaceae in vitro hsv-1 unknown (hamidpour et al. 2014; khalil et al. 2020) 28 securigera securidaca leguminosae in vitro hsv-1 kaempferol and kaempferol-7-o-glucoside inhibit the hsv infection but the mechanism is unknown (behbahani et al. 2014; behbahani et al. 2013) 29 solanum paniculatum solanaceae in vitro hsv-1 unknown (valadares et al. 2009; kaunda and zhang 2019) 30 tanacetum parthenium asteraceae in vitro/in vivo hsv-1 parthenolide did not directly act against hsv, and it can able to handle the defense mechanisms in host cells against viral particles (benassi-zanqueta et al. 2019; benassi-zanqueta et al. 2018) 31 thymus vulgaris lamiaceae in vitro hsv-1/ hsv-2 thymol reduces the viral transmission (gavanji et al. 2015; catella et al. 2021; nolkemper et al. 2006; sharifi-rad et al. 2017; lai et al. 2012) 32 thymus kotschyanus lamiaceae in vitro hsv-1/ hsv-2 borneol, and isoborneol that inhibits the viral replication (farahani 2017; yang et al. 2020) 33 zataria multiflora lamiaceae in vitro hsv-1 rosmarinic acid inhibits the viral attachment to host cells (arabzadeh et al. 2020; ben-shabat et al. 2020; mardani et al.. 2012; astani et al 2014; chen et al 2017) 34 zingiber officinale zingiberaceae in vitro/in vivo hsv-1 unknown (schnitzler et al. 2012; camero et al. 2019; koch et al. 2008; hayati et al. 2021) gavanji – anti-herpetic activity of herbal pharmacopoeia (ihp) 95 boraginaceae echium amoenum, commonly known as borage, belongs to the family boraginaceae (zannou et al. 2021), which has a long history in iranian traditional medicine (itm) for treatment of influenza and infectious diseases (ranjbar et al. 2006). this plant has antiviral properties against hsv-1, and the ic50 and cc50 values were determined to be 500 and 1000 μg/ml, respectively, also the results of this research showed that virus replication was inhibited at the lower concentration than 400 μg/ml (abolhassani 2010; farahani 2013). euphorbiaceae euphorbia spinidens, a member of euphorbiaceae family, is traditionally used as antiasthmatic, relieving stomachache, laxative and emetic (ghanadian et al. 2016). this plant contains several types of bioactive compounds which exhibited significant antimicrobial activities (li et al. 2009). furthermore, a research demonstrated that hexan fraction of e. spinidens showed the inhibitory activity against hsv-1, and the ic50 value of 9.92±0.072 mg/ml was determined (li et al. 2009). another study stated that the methanolic extract of e. spinidens was active against hsv-1 with ec50 and si values, of 0.34mg/ml and 14.9 respectively, and this research showed that e. spinidens can inhibit the viral replication and adsorption (karimi et al. 2016). ricinus communis is another member of euphorbiaceae family (abdul et al. 2018), which contains many phytochemicals and can be used to treat a wide range of diseases (scarpa and guerci 1982; ohishi et al. 2014). a study showed that ethanolic extracts of r. communis has potent antiviral activity against hsv-1 and inhibited this virus at concentration of 0.1 mg/ml (abdul. et al. 1996). fagaceae quercus persica, commonly known as oak, belonging to the family fagaceae, has been used in iranian traditional medicine(itm) for the treatment of various disease (karimian et al. 2020). a study demonstrated that hydroalchoholic extract of q. persica has anti-hsv activity. in this research the authors conclude that q. persica extract has inhibited the hsv‑1, and the ic50 values, before and after attachment to bhk, were determined to be 1.02 and 0.257 μg/ml, respectively (karimi et al. 2013). another study by karimi et al. stated that quercus brantii extract showed the inhibitory activity against hsv-1, and the ic50 and si values of 4.3 μg/ml and 48.4 were determined, respectively (karimi et al. 2017). hypericaceae hypericum perforatum, a member of hypericaceae family, is a medicinal plant which has been widely used as antidepressants, anti-cancer and psychotic disorders (klemow et al. 2011). a number of studies demonstrated that h. perforatum, has strong antiviral activity against numerous viruses, including radiationleukaemia virus (radlv), friend virus (fv), human immunodeficiency virus type 1 (hiv-1) and hsv-1 (weber et al.1994). also a scientific research showed that the complex of h. perforatum and lysine hydrochloride, remarkably inhibited the hsv‑1 and the ic50 value was from 6.8 to 9.7 mg/ml (hu et al. 2004). lamiaceae hyssopus officinalis belongs to the family lamiaceae, which is traditionally used for treatment of chest & lung conditions, coughs, colds and infectious diseases (vlase et al. 2014), also several studies demonstrated that h. officinalis extract has antifungal and antiviral activity (fathiazad et al. 2011). a study by behbahani, showed that methanolic extract of h. officinalis significantly inhibited two hsv serotypes, which the ec50 and cc50 values, against hsv‑1, were determined to be 4.1±0.40 and 960 μg/ml, respectively, and for hsv‑2, the ec50 and cc50 values were > 5.0 and 100 μg/ml, respectively (behbahani 2009). another study stated that essential oil of h. officinalis was active against hsv-1, and ec50 and cc50 values, were determined to be 0.0001±0.00001 and 0.0075±0.002 % respectively (schnitzler et al. 2007). another important species of lamiaceae family is melissa officinalis, which is a well-known and it has been used in traditional medicine for treatment of various diseases (miraj et al. 2017). furthermore, a research showed that m. officinalis has inhibitory activity against hsv‑1 and inhibited the viral attachment to the host cells, and the ic50 and si values of 0.4 μg/ml and 350 were determined, respectively (astani et al 2014). mentha piperita is antother important species of lamiaceae family which has therapeutic potential to treat different diseases (zaker et al. 2014; alves et al. 2012). schuhmacher et al., demonstrated that the essential oil of m. piperita, can able to inhibit the herpes simplex virus type 1, and reduce the plaque formation by up to 82% for hsv-1 (schuhmacher et al. 2003). another research stated that the m. piperita extract was effective against hsv-1 and inhibited the viral replication cycle, which ed50 and ti values were determined to be 62.70 mg/ml and 1.79, respectively (omidian et al. 2014). several studies exhibited that many species of lamiaceae family has potential to inhibit the two hsv serotypes. ocimum basilicum or great basil, a traditional medicinal plant, belongs to the family lamiaceae which is widely used for treatment of different diseases including, headaches, diabetes, nerve pain and anxiety (bora et al. 2011). a study revealed that water and ethanolic extracts of o. basilicum have inhibited two hsv serotypes, the result of this research showed that the ec50 and si values of water extracts against hsv-1, were determined to be 90.9 mg/ml and 16.2, also for hsv-2 were 51.4 mg/ml and 28.6 respectively. ethanolic extract showed inhibitory activity against hsv-1, and ec50 and si values of 108.3μg/ml and 6.3 were determined, respectively (chiang et al. 2005). another important 96 biology, medicine, & natural product chemistry 11 (2), 2022: 89-109 species of lamiaceae family is rosmarinus officinalis, which is widely used in treatment of rheumatic pain, headache, hysteria, stomachache, depression and infectious diseases (ghasemzadeh rahbardar et al. 2020). a study reported that extract of r. officinalis, exhibited potential antiviral activity against hsv-1 and hsv-2, which this extract at the concentration of 30 μg/ml inhibited the 55% of hsv-1 and at 40μg/ml inhibited the 65% of hsv-2 plaques and extract of r. officinalis showed the significant inhibitory effect at the 50 μg/ml concentration two hsv serotypes (al-megrin et al. 2020). another important species of lamiaceae family is salvia officinalis, that is used in traditional medicine to treat different kinds of diseases, such as rheumatism, diarrhea, ulcers, inflammation and paralysis. this plant contains several types of phytochemicals which exhibited significant antibacterial, antifungal and antiviral activities (ghorbani and esmaeilizadeh 2017). a study demonstrated that two diterpenoids compounds (safficinolide and sage one) which is isolated od aerial parts, have antiviral activity (smidling et al. 2008). a number of research studies demonstrated that s. officinalis has antiviral activity against two serotypes of hsv. the extract of s. officinalis was used in the study, against hsv-1, which the ic50 value of this extract, 199.0 μg/ml was determined (smidling et al. 2008), and in another studies ic50 value was 1.41–1.88 μg/ml and inhibited the plaque formation (santoyo et al. 2014). schnitzler et al. studied the antiviral activity of s. officinalis extract against two serotypes of hsv which ic50 values for hsv-1 and 2 were 0.18 and 0.04 μg/ml respectively (schnitzler et al. 2008). satureja hotensis is another important species of lamiaceae family which can use in treatment of many ailments and diseases. in this plant, various types of phytochemicals such as flavonoids steroids, triterpenoids, and and sesquiterpenoids, have been identified (gursoy et al. 2009; golestannejad et al. 2015), which are used in pharmaceutical industries (tepe and cilkiz 2016). a study showed that this plant has potential antiviral activity against hsv-1, which ic50 and cc50 values were determined to be 0.008% and 0.245%, respectively (gavanji et al. 2015). one of the important species of lamiaceae family is thymus vulgaris commonly known as thyme and is the rich sources of phytochemicals which are widely used for the treatment of inflammation, cancers, and infectious diseases (gavanji and larki 2017). a study demonstrated that the essential oil of t. vulgaris was effective against two serotypes of hsv and reduce the viral infectivity, which ic50 for 1,8-cineole, 1200 μg/ml was determined (gavanji and larki 2017). furthermore, a study revealed that aqueous extract of t. vulgaris has inhibitory effects against two serotypes of hsv, which ic50 and si values for hsv-1, were 0.065 mg/ml and 954, also for hsv-2, 0.077 mg/ml and 805 were determined, respectively (nolkemper et al. 2006). another member of lamiaceae family is thymus kotschyanus which has anti-viral activity and inhibited hsv-1 at the higher concentration of 400 μg/ml (farahani 2017). based on a study, t. kotschyanus contains many bioactive compounds such as borneol, has impressive antiviral potentials to inhibit herpes simplex viruses (armaka et al. 1999) (table 2). zataria multiflora is one of the most important species of lamiaceae family which is used to relieve some of illnesses such as fever, bone pain, flatulence, cough, cold and infectious diseases (ghorani et al. 2022; dadashi et al. 2016). a research study has been documented, reporting that methanolic extract of z. multiflora at the 1000 mg/ml concentration, remarkably reduce the plaque formation of hsv‑1 (arabzadeh et al. 2013). moreover, essential oil of z. multiflora exhibited antiviral potential against hsv‑1, in which ic50 and si values were determined to be 0.0059% and 11.7, respectively (mardani et al. 2012). another study revealed that essential oil of z. multiflora contains rosmarinic acid which is an antiviral compound and inhibited the viral attachment to host cells. this study demonstrated that z. multiflora oil, has strong antiviral activity which ic50 and cc50 values were determined to be 0.003% and 0.166%, respectively (gavanji et al. 2015). zingiber officinale or ginger is another important species of lamiaceae family which is widely used for treatment different diseases in traditional medicine (grzanna et al. 2005). z. officinale has broad-spectrum antiviral potential on human respiratory syncytial virus (hrsv) (chang et al. 2013), hepatitis c virus (hcv), influenza a (h1n1) (sahoo et al. 2016), and two serotypes of hsv [123,180]. the study has demonstrated that z. officinale inhibited the hsv-2 with ic50 and si values of 0.0001% and 40 respectively (allahverdiyev et al. 2013). another study by koch et al stated that z. officinale possess antiviral activity, and the ic50 value of 0.001% was determined (koch et al. 2008) (table 2). leguminosae securigera securidaca, a species of leguminosae family, is widely used as herbal for the treatment of several diseases in iranian traditional medicine (itm). s. securidaca is a rich source of flavonoids having significant antibacterial, antifungal and antiviral activities (raesi vanani et al. 2019). also the result of a research study showed that, two major compounds, including kaempferol and kaempferol-7-o-glucoside which is isolated from s. securidaca, can inhibit the hsv infection (behbahani et al. 2013). another study showed that methanolic extract of s. securidaca has inhibited the hsv‑2 with ic50 and cc50 values of 1.6 and 130 μg/ml, respectively (sayedipour et al. 2012). furthermore, s. securidaca exhibited significant antiviral activity against hsv-1 which ic50 and cc50 gavanji – anti-herpetic activity of herbal pharmacopoeia (ihp) 97 values of 2 and 500 μg/ml, were determined, respectively (behbahani et al. 2013). glycyrrhiza glabra is another medicinal plant from leguminosae family which has been reported for healing gastroesophageal reflux disease, liver diseases, tuberculosis and infectious diseases (wahab et al. 2021). numerous antiviral phytochemicals, such as glycyrrhetinic acid, and glycyrrhizin were isolated from g. glabra which have antiviral activity against hsv-1 (ming and yin 2013; huan et al. 2021). based on the results of a research, g. glabra extract has inhibited the hsv‑1 with ic50 and cc50 values of 500 and 800 μg/ml, respectively (monavari et al. 2008). another study by fukuchi et al. stated that water extract of g. glabra with ec50 and si values of 650 to 740 mg/ml, and 2.0 to >4.6, respectively, showed a strong inhibitory activity against hsv-1, compared to alkaline extracts of g. glabra with ec50 and si values of 600 to >3000 mg/ml, and to 3.2, respectively (fukuchi et al. 2016). myrtaceae eucalyptus caesia and eucalyptus globulus are the two most common species of myrtaceae family which have numerous phytopharmacological potential to treat different kinds of diseases, such as asthma, pulmonary, cold, bronchitis, and infectious diseases (mierescastroet al. 2021). both of these species exhibited antiviral properties against hsv-1 and hsv-2 (mierescastroet al. 2021). a study demonstrated that essential oil of e. globulus inhibited two hsv serotypes which ic50 and si values for hsv-1, were determined to be 0.009% 3.3, respectively and for hsv-2, were 0.008% and 3.75, respectively (schnitzler et al. 2001). furthermore, a research demonstrated that 1,8-cineole reduced the hsv infection under in vivo condition (behbahani et al. 2013). another study researchers compared the effect of e. globulus oil and individual monoterpenes against hsv-1, which result of this study showed that e. globulus 1,8-cineole, α-pinene, γterpinene, p-cymene, α-terpineol and terpinen-4-ol, have antiviral activity against hsv-1 with ic50 of 55, 1.20, 4.5, 7.0, 16.0, 22.0, and 60.0 μg/ml, respectively (astani et al. 2010). another study revealed that essential oil of e. caesia inhibited two hsv-1 which ic50 and cc50 values, were determined to be 0.004% and 0.287%, respectively (gavanji et al. 2015). myrtus communis is another important member of myrtaceae family, which has been used in traditional medicine to treat many diseases such as hemorrhoid, recurrent aphthous stomatitis, diarrhea, and infectious diseases (mahboubi 2016; alipour et al. 2016). based on a study, the hydroalcoholic extract of m. communis, has impressive antiviral potentials to inhibit hsv-1, which ic50 and cc50 values, were determined to be 3100 and 4960 μg/ml, respectively (moradi et al. 2011). this herb showed anti-herpetic activity, under clinical trial condition, which the result of this study demonstrated that myrtle oil, reduced the signs and symptoms of disease in the treated group, in comparison to other control groups (zolfaghari et al. 1997). oleaceae olea europaea, a member of oleaceae family, can be used to treat a wide range of diseases in traditional medicine, such as diarrhea, hemorrhoids, infectious diseases, inflammation and rheumatism (alipour et al. 2016). o. europaea has antiviral activities against many types of viruses, including canine parvovirus (cpv), hepatitis virus, bovine rhinovirus (brav), herpes virus (hsv) and feline leukemia virus (felv) (ben-amor et al. 2021). a study demonstrated that hydroalcoholic extract of o. europaea var. sylvestris exhibited anti hsv-1 activity that ec50 and cc50 values, for preinfection assay, were determined to be 0.12 and 0.2 mg/ml, respectively and for post-infection assay were 0.15 and 0.2 mg/ml, respectively (ben-amor et al. 2021).another study by motamedifar et al. stated that hydroalcoholic extract of o. europaea with ic50 and cc50 values of 660 and 1750 μg/ml, respectively, showed a strong inhibitory activity against hsv-1 (motamedifar et al. 2015). plantaginaceae plantago major belongs to the family plantaginaceae, which is commonly known as greater plantain, and it has been traditionally used to treat many diseases such as fever, constipation and wounds and bleeding. furthermore, p. major has been reported to contain caffeic acid, that demonstrated the strongest antiviral activity against two serotypes of hsv (najafian et al. 2018). a study showed that aqueous extract of p. major has weak antiviral activity against hsv-1 and hsv-2, in extracts, pure compounds such has caffeic acid demonstrated the strongest antiviral activity against hsv-1. moreover, this study stated that aqueous extract of p. major inhibited hsv-2 with ec50 and si values, of 843 mg/ml and 2.2, respectively and caffeic acid isolated from p. major exhibited the antiviral activity against hsv-1 with ec50 and si values, of 15.3 μg/ml and 671, and hsv-2 ec50 and si values, were determined to be 87.3 μg/ml and 118, respectively (chiang et al. 2002; chiang 2003). polygonaceae rheum palmatum a flowering plant of polygonaceae family, which can be used to treat a wide range of diseases such as gasteroenteritic, herpes and kidney disease (chang et al. 2014). r. palmatum contains several types of natural compounds such as emodin, chrysophanol and aloe-emodin which exhibited significant anti-viral activities (li et al. 2007). a study has been documented that aloe-emodin isolated from r. palmatum has antiviral activity against hsv (types 1 and 2), influenza virus (sydiskis et al. 1991), human cytomegalovirus (hcmv) (barnard et al. 1992), and polio viruses (semple et al. 2001), also emodin and 98 biology, medicine, & natural product chemistry 11 (2), 2022: 89-109 chrysophanol have antiviral property against hepatitis b virus (hbv) (shuangsuo et al. 2006), hepatitis c virus (hcv) and human immunodeficiency viruses (hiv) (kubin et al. 2005). a study demonstrated that r. tanguticum nanoparticles suppressed the hsv-1, which ec50 and cc50 values, were determined to be 194.1 and 415.3 μg/ml, respectively (shen et al. 2019). solanaceae solanum paniculatum or jurubeba, a member of solanaceae family, which is widely used to treat several diseases including hypertension, anemia, inflammation and tuberculosis (tenório et al. 2016). a research study by valadares et al., showed that ethanolic extract of s. paniculatum exhibited antiviral property against hsv-1, which ec50 and si values, were determined to be 298 mg/ml and 1.4, respectively (valadares et al. 2009). theaceae camellia sinensis or green tea, is a medicinal plant of theaceae family which is used as the most consumed and favorable drink in the world. c. sinensis has many medicinal properties (singhal et al. 2017). based on a study, c. sinensis contains many bioactive compounds that has impressive antiviral potentials to inhibit herpes simplex viruses tape 1, with ic50 and si values, of 20 mg/ml and 50, respectively (farahani et al. 2014). another study, stated that aqueous extract of c. sinensis exhibited the antiviral activity against hsv-1, which ic50, cc50 and si values, were determined to be 50, 1000 μg/ml and 20, respectively (farahani et al. 2013) another scientific research reported that methanolic extracts of c. sinensis completely inhibited two hsv serotypes at 12 μg/ml concertation (deepika et al. 2014). xanthorrhoeaceae aloe vera, commonly known as aloe barbadensis, is a member of xanthorrhoeaceae family which has been traditionally used to treat various diseases such as abdominal pains, malaria, arthritis, fever, and skin diseases (adams et al. 2014). a. vera produces a wide range of phytochemicals, such as emodin and aloeemodin which exhibit potential strong antiviral activity against two serotypes of hsv, immunodeficiency virus (hiv), and influenza virus (brahimi et al. 2021). another an anthraquinone compound isolated from a. vera, is aloe-emodin which inhibits the viral replication of hsv serotypes and the ic50 value, of 1.5–6.0 μg/ml was determined (sydiskis et al. 1991). a study demonstrated that topical a. vera gel at 0.2 to 5% concentrations can inhibit hsv-i growth (rezazadeh et al. 2016). another study by ebrahimi et al. revealed that a. vera extract inhibited the hsv-1 with ic50, cc50 and si values, were determined to be 10000 ± 55, 20000 ± 94 μg/ml, and 2.0 respectively (brahimi et al. 2021). also the result of a study showed that hot glycerine extract of aloe vera can able to inhibit the hsv-2, which the ic50, cc50 and si values, were determined to be 428, 3238 μg/ml and 7.56, respectively (zandi et al. 2007). zingiberaceae curcuma longa belongs to the family zingiberaceae, which is used in iranian traditional medicine(itm) for healing rheumatism, anorexia, wounds, cough and respiratory diseases (trujillo et al. 2013). a research study showed that, 2 mg/ml of c. longa extract reduced the plaque formation of hsv-1 (fani et al. 2015). furthermore, a research demonstrated that c. longa has inhibitory effect against hsv-1, and the ic50, cc50 and si values were 33.0, 484.2 μg/ml and 14.6, respectively (lyu et al. 2005). another study revealed that c. longa extract contains polyphenolic compounds such as curcumin which affects the viral transactivator protein vp16 -mediated recruitment of rna polymerase ii (pol ii) to immediate early (ie) gene promoters and inhibits the hsv-1 replication. this study stated that curcumin has significant has antiherpetic activity and inhibited hsv-2 with ed50 value, of 0.32 mg/ml (kutluay et al. 2008). zingiber officinale or ginger is another antiviral species of zingiberaceae family which exhibited antiviral activity against numerous viruses (wang et al. 2020). based on a study, the essential oil of z. officinale, has impressive antiviral potentials to inhibit hsv-1, whit ic50 value, of 0.001% (koch et al. 2008). conclusions since time immemorial, a human being has sought medications to relieve pain and remedy for various diseases. weighty evidence demonstrates the use of medicinal plants for therapeutic purposes and numerous research studies have been done to investigate the novel antiviral agents. in this 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(2020). advances in pharmacological activities of terpenoids. natural product communications, 15, 1934578x20903555. https://doi.org/10.1177%2f1934578x20903555 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 1-4 | doi: 10.14421/biomedich.2023.121.1-4 issn 2540-9328 (online) preliminary investigative study on the blood pressure-lowering potential of aqueous leaf extract of simarouba glauca (aesg) on normotensive adult wistar rats sammy davies e. osagie-eweka1,*, noghayin e.j. orhue1, fabian c. amaechina2, eric k.i. omogbai2, emuesiri g. moke3 1department of biochemistry, faculty of life sciences, university of benin, benin city, nigeria. 2department of pharmacology and toxicology, faculty of pharmacy, university of benin, benin city, nigeria. 3department of pharmacology, faculty of basic medical sciences, delta state university, abraka, nigeria. corresponding author* davies.osagie-eweka@uniben.edu manuscript received: 26 july, 2022. revision accepted: 31 august, 2022. published: 13 september, 2022. abstract studies have shown that plants possess medicinal properties and compounds are beneficial in managing and treating diseases, including high blood pressure and related cardiovascular conditions. simarouba glauca (sg) has been widely reported to possess antibacterial activity, anti-oxidant, anti-proliferative and hemolytic activity; amongst others. however, there is paucity of data on its effect on blood pressure. hence, the study research aimed at assessing the hypotensive prospect inherent in the aqueous leaf extract of simarouba glauca (aesg) on normotensive male wistar rats. the study was conducted using adult male wistar rats (n = 3), a urethane/thiopental (1205/20 mg/kg) anesthesia and a chart paper attached to ugo basile uni-recorder model 400700 data capsule. under full anesthesia, the rat’s trachea and the carotid artery were cannulated for assisted respiration and blood pressure measurement. at stable variables; following the administration of 0.2 ml normal saline, the aesg was administered intravenously via the caudal vein at 2.5 and 5.0 mg/kg body weight dose respectively. the data was recorded on a chart; indicated the characteristic dose-dependent hypotensive effect of aesg on normotensive rats; at doses of 2.5 mg/kg and 5.0 mg/kg, with marked decreases in the systolic blood pressure (sbp), diastolic blood pressure (dbp) and mean arterial pressure (map) from basal levels of 127.83 ± 1.01 mmhg, 91.00 ± 1.00 mm hg and 103.27 ± 0.99 mm hg respectively. the outcome of the preliminary investigation indicates that the aesg demonstrated a hypotensive effect on the bp of normotensive male wistar rats dependent on varying doses administered; indicative of further evaluation. keywords: cardiovascular; caudal vein; invasive blood pressure; simarouba glauca. abbreviations: sg simarouba glauca; aesg: aqueous leaf extract of simarouba glauca; sbp: systolic blood pressure; dbp: diastolic blood pressure; map: mean arterial pressure; acei: angiotensin converting enzyme inhibitors; ara: angiotensin receptor antagonists. introduction hypertension is a public health condition characterized by chronic cardiovascular disease and end-stage renal diseases (fuchs and whelton, 2020). untreated hypertension predisposes risk factors like stroke, myocardial infarction, arteriosclerosis, cardiac arrest, heart attack, cardiomegaly and amongst others (landazuri et al., 2017). some allopathic agents applied in the management of hypertension include; adrenergic antagonists (alpha and beta receptor blockers) like prazosin and atenolol, centrally acting sympatholytic agents (alphamethyldopa, guanabenz and clonidine), calcium channel blockers (nifedipine and amlodipine), diuretics (hydrochlorothiazides), angiotensin converting enzyme inhibitors – acei (lisinopril and ramipril), angiotensin receptor antogonists – ara (losartan and valsartan) and the aldosterone antagonists (spironolactone and eplerenone) have been extensively reported to elicit adverse effects like dry cough, severe hypotension, depressed libido and sometimes erectile dysfunction (landazuri et al., 2017; olowofela and isah, 2017; moke et al., 2022); as such, the antihypertensive effect achieved with these agents is always short lived. in addition to the arrays of these side effects, virtually all these antihypertensive agents are cost implicative, creating a huge burden in the purse of affected lowincome earners worldwide (lacy et al., 2008; pr et al., 2014). hence, there is the need to adequately evaluate the anti-hypertensive (hypotensive) potential of cheap and available plants with proven medicinal properties as have been the case in the last four decades (tabassum and ahmad, 2011; pr et al., 2014). there are a number of medicinal plants with folk history that have been applied to treat hypertension; a https://doi.org/10.14421/biomedich.2023.121.1-4 2 biology, medicine, & natural product chemistry 12 (1), 2023: 1-4 few investigations have shown their effectiveness, while others have been disproved by scientific findings (tabassum and ahmad, 2011; kamyab et al., 2021). literatures have reported vast findings on the ethnomedicinal benefits of s. glauca (patil and gaikwad, 2011; ramasamy et al., 2022) with no record on the effect on cardiovascular system and blood pressure; hence this study. materials and methods collection of plant material and preparation of aqueous extract fresh leaves of simarouba glauca were procured from cercobela farms®, ubiaja. fresh plant specimen was authenticated and deposited with voucher specimen no. ubhs382 at plant biology and biotechnology department herbarium, university of benin. the plant leaves were properly washed with clean water and then dried at room temperature for twenty-eight (28) days. fine powdery particles were obtained following pulverization of the dried crispy leaves of s. glauca. five hundred grams (500 g) of the leaf powder was macerated in 2.5 l distilled water and stimulated intermittently for forty-eight hours (48 hrs.) to obtain a filtrate. the filtrate was lyophilized with a freeze-drier to obtain the aqueous extract (osagie-eweka et al., 2016). materials for invasive procedure for the invasive procedure, the materials used included the following: an intravenous cannula, eighteen g needles, a surgical table, respiratory tubing (6′′ pediatric ryle's tube may be used). one milliliter tuberculin syringe, 5, 10 ml syringes, small (3′′) and medium (5′′) c, adson dissecting forceps (toothed and non-toothed) (5′′), artery forceps (5′′), small and medium forceps (with teeth, blunt and pointed), a bulldog clamp, a surgical lamp, an insertion needle, a surgical blade, normal saline, a thread, adhesive tape and prepared stock solution of the aqueous extract with appropriate concentrations. distilled water was used in the preparation of the required stock solution of the aqueous leaf extract of s. glauca. pressure transducer calibration procedure calibration is an imperative step in the experiment; it was conducted with a sphygmomanometer at a specific pressure. the pressure cuff was disconnected from the sphygmomanometer; linked to the transducer with a physiograph data acquisition system. inflating to a required specific pressure was performed to check the pressure transducer. the mathematical conversion factor to express the blood pressure was established. the calibration between the voltage (millivolts) and pressure in the data acquisition system was previously performed; results were automatically calculated relative to the system calibrated value (ordodi et al., 2005). animal experimental procedure for cannulation of the caudal vein adult male wistar rats (210 220 g) were procured from the rat housing facility of pharmacology and toxicology department, university of benin. experimental animals were anaesthetized with urethane/thiopentone (1250/20 mg/kg) (amaechina and omogbai, 2007; wang et al, 2013) administered intra-peritoneally. an established protocol for invasive blood pressure assessment (amaechina and omogbai, 2007; wang et al, 2013) was used. the caudal vein of the rat was cannulated with a heparinized saline-filled-23 g scalp-vein needle for the administration of extract intravenously and was fastened to the dissecting table dorsally. the cervical region was shaved and dissected open to reveal both the trachea and carotid artery (plehm et al., 2006). the trachea was isolated, cleared of connective tissues and cannulated with a 2 mm diameter polythene tube for assisted respiration (kramer and remie, 2005). the carotid artery was isolated, cleared of adhesive tissues, and cannulated with a heparinized saline-filled teflon polyethylene tube connected to a pressure transducer for the transmission of blood pressure variations to ugo basile uni-recorder (model: 400700). an angle poised lamp with a 60 watts electric bulb is positioned over the anaesthetized animal, for the purpose of maintaining the temperature within normal range. when all the measurable variables remained stable as confirmed following the administration of normal saline, aesg was administered to experimental rats intravenously at two varying doses of 2.5 and 5.0 mg/kg respectively. this procedure was conducted in triplicate; the effects of the administered doses were recorded on the ugo basile chart recorder. results the data presented in table 1 reveal that aesg elicited significant (p ˂ 0.05) dose-dependent decreases in the sbp, dbp and map at 2.5 mg/kg (122.00 ± 1.15 mmhg, 84.67 ± 2.40 mmhg; 97.10 ± 1.99 mmhg) and 5.0 mg/kg (84.00 ± 2.31 mmhg, 62.67 ± 1.45 mmhg; 69.80 ± 1.73 mmhg) doses respectively; compared to the experimental rat treated with normal saline (127.83 ± 1.01 mmhg, 91.00 ± 1.00 mmhg; 103.27 ± 0.99 mmhg). likewise, the polygraph presented in figure 1 indicates marked dose-dependent decreases in the hemodynamic parameters considered at 2.5 and 5.0 mg/kg body weight respectively, when compared to the normal saline group. osagie-eweka et al. – preliminary investigative study on the … 3 table 1. effect of aesg on hemodynamic parameters of normotensive rats. parameters systolic blood pressure (mmhg) diastolic blood pressure (mmhg) mean arterial pressure (mmhg) normal saline 127.83 ± 1.01 91.00 ± 1.00 103.27 ± 0.99 aesg 2.5 mg/kg 122.00 ± 1.15 84.67 ± 2.40 97.10 ± 1.99 aesg 5.0 mg/kg 84.00 ± 2.31*# 62.67 ± 1.45*# 69.80 ± 1.73*# all values expressed as mean ± sem, where n=3, all data were analyzed by using one-way anova followed by tukey’s post hoc test. *p<0.05 compared to the normal saline control group; #p<0.05 compared to aesg 2.5 mg/kg group. figure 1. the effect of aesg on blood pressure of normotensive rats. discussion the study reveal a 4.6 % decrease in systolic blood pressure (sbp), 7.0 % decrease in diastolic blood pressure (dbp); 6.0 % decrease in mean arterial pressure (map) at 2.5 mg/kg compared to the normal saline group of the experimental rats. furthermore, at 5.0 mg/kg. aesg showed a significant (p < 0.05) decrease in sbp (34.29%), dbp (31.13%) and map (32.41% ) relative to the normal saline group. additionally, the group treated with 5.0 mg/kg showed 31.15%, 25.98%, 28.12% significant (p < 0.05) decreases in sbp, dbp and map respectively relative to the group treated with 2.5 mg/kg. the outcome of the study therefore indicate a better and promising blood pressure lowering effect at 5 mg/kg when administered intravenously. the data presented in figure 1 likewise indicate that there was instananeous recovery of the blood pressure to the basal level which may be obviously not unconnected to the reflex compensatory mehanism aimed at restoring the blood pressure to normal after the adminisration anti-hypertensive (kuogias et al., 2010). in the present study, a similar effect was observed at a dose of 5.0 mg/kg. however, the recovery was not sustained as there was a second phase derease in the blood pressure which was more sustained as observed in figure 1. thus, the outcome and resultant effect on blood pressure suggests that s. glauca may possess some promising active principles capable of eliciting hypotensive effect on the cardiovascular system. in fact, studies have reported the hypotensive and (or) the blood pressure-lowering potentials of several known medicinal plants (anaka et al., 2009; imafidon and amaechina, 2010; amaechina et al., 2017; alawode et al., 2021; kamyab et al., 2021) with less adverse effects. however, pharmacologist must continue to conduct systematic inquiry into the therapeutic benefits of plants with hypotensive potentials in the quest to discover the most effective mechanistic treatment approach for hypertension considering the complexities associated with the condition. accordingly, it is recommended that s. glauca may be subjected to detailed and extensive laboratory investigation to ascertain its pharmacological pertinence. conclusion the outcome of the preliminary investigative study of aesg (aqueous leaf extract of s. glauca) on cardiovascular system indicate a strong blood pressure lowering potential and a promising vaso-relaxant bioactive compound that may be beneficial in managing hypertension related conditions author(s) contributions statement: sdeo, nejo, fca, ekio and egm participated in research design. sdeo and egm conducted the preliminary experiments. sdeo and fca participated in data analyses. sdeo, fca and egm wrote the manuscript. 4 biology, medicine, & natural product chemistry 12 (1), 2023: 1-4 all authors have approved the final version of the manuscript. conflict of interests: authors state that there is no conflict of interest in this research output. ethical approval: the experimental protocols were approved by the faculty of pharmacy, university of benin ethics committee with reference number ec/fp/021/11. funding: the study was self funded at the departments of biochemistry/pharmacology. references alawode d, asiwe j, moke e, okonofua d, sanusi k, adagbada e, yusuf m, fasanmade a (2021). the effect of ethanol leaf extract of cnidosculus aconitifolius on cardiorenal functions in hypertensive and normotensive male wistar rats. international journal of nutrition sciences 6(3): 155160. amaechina fc, omogbai eki (2007). hypotensive effect of aqueous leaves extract of phyllanthus amarus schum and thonn (euphorbiacee). acta poloniae pharmaceutica-drug research 64: 547-52. amaechina fc, uchendu ap, oboh ci, agokei ni, eboka cj (2017). preliminary comparative effect of the aqueous extract of persea americana seeds on the blood pressure of normotensive rabbits and rats. journal of science and practice of pharmacy 4(1):177-181. anaka on, ozolua ri, okpo so (2009). the effect of the aqueous seed extract of persea americana mill (lauraceae) on the blood pressure of sprague dawley rat. african journal of pharmacy and pharmacology 3(10): 485-490. fuchs fd, whelton pk (2020). high blood pressure and cardiovascular disease. hypertension 75(2):285-292. imafidon ke, amaechina fc (2010). effects of aqueous seed extract of persea americana mill. 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10.14421/biomedich.2025.142.679-683 issn 2540-9328 (online) effect of modified kimpul flour substitution and glycerol monostearate concentration on the physicochemical and sensory properties of sweet bread siti nurlaela setianingsih1, rizky muliani dwi ujianti1*, iffah muflihati1, fafa nurdyansyah1, mega novita2, diva julia paramita1, shindi nofitasari1, dola mareta anggarini1, abdillah fathan generus annajah1 1food technology department, engineering and informatics faculty, universitas persatuan guru republik indonesia semarang 2natural science education, postgraduate program, universitas persatuan guru republik indonesia semarang jl. sidodadi no. 24, semarang, indonesia. corresponding author* rizkymuliani@upgris.ac.id manuscript received: 27 june, 2025. revision accepted: 04 september, 2025. published: 01 october, 2025. abstract wheat flour is the primary ingredient in sweet bread production, yet its import-dependent supply in countries like indonesia prompts the need for alternative local ingredients. kimpul tuber (xanthosoma sagittifolium), rich in carbohydrates, presents a promising substitute, though its native starch properties are less suitable for baking. this study aimed to evaluate the effect of substituting wheat flour with heat moisture treated (hmt) kimpul flour and the addition of glycerol monostearate (gms) on the physicochemical and sensory properties of sweet bread. a factorial completely randomized design was applied using three wheat-to-kimpul flour ratios (3:1, 1:1, and 1:3) and three gms concentrations (2%, 3%, and 4%). results showed that higher kimpul flour substitution increased moisture and carbohydrate content but reduced protein and fat levels. textural properties such as hardness and adhesiveness also increased with kimpul content, but these were mitigated by the addition of gms, particularly at 3%. the optimal formulation 1:1 wheat-to-kimpul ratio with 3% gms produced sweet bread with the best overall sensory acceptance. the findings suggest that hmt-modified kimpul flour combined with gms can serve as a functional and acceptable alternative to wheat flour in bread production. this supports food diversification strategies and promotes utilization of local tuber flours in bakery applications. keywords: glycerol monostearate; modified kimpul flour; physicochemical properties; sensory evaluation; sweet bread. introduction sweet bread is a widely consumed product and serves as an essential source of energy due to its high carbohydrate content. conventionally, sweet bread is produced using refined wheat flour, which contains gluten an important protein that contributes to dough elasticity and volume expansion during fermentation and baking (irmawati et al., 2024; h. li et al., 2024; setiyoko & sari, 2024). however, the reliance on wheat flour presents a challenge for countries like indonesia that are dependent on imported wheat, prompting efforts to diversify food sources by utilizing locally available ingredients (al ubaidilah et al., 2025; fetriyuna et al., 2024; ramadhanty, 2025). one such alternative is kimpul tuber (xanthosoma sagittifolium), a tropical plant rich in carbohydrates and native to indonesia. the high starch content of kimpul makes it a viable candidate for flour production, although its native starch properties tend to result in poor processing characteristics, such as high gelatinization temperature, limited swelling power, and low resistance to acid and shear during baking (budiarti et al., 2022, 2023; rosida et al., 2024). sweet bread is a widely consumed product and serves as an essential source of energy due to its high carbohydrate content. conventionally, sweet bread is produced using refined wheat flour, which contains gluten an important protein that contributes to dough elasticity and volume expansion during fermentation and baking (j. li et al., 2024; malvano et al., 2024; ps & rosida, 2023). however, the reliance on wheat flour presents a challenge for countries like indonesia that are dependent on imported wheat, prompting efforts to diversify food sources by utilizing locally available ingredients (khamidah & antarlina, 2022; krisbianto & minantyo, 2024; sulaiman et al., 2021). one such alternative is kimpul tuber (xanthosoma sagittifolium), a tropical plant rich in carbohydrates and native to indonesia. the high starch content of kimpul makes it a viable candidate for flour production, although its native starch properties tend to result in poor processing https://doi.org/10.14421/biomedich.2025.142.679-683 680 biology, medicine, & natural product chemistry 14 (2), 2025: 679-683 characteristics, such as high gelatinization temperature, limited swelling power, and low resistance to acid and shear during baking. in addition to starch modification, the incorporation of food additives such as glycerol monostearate (gms) offers another opportunity to improve bread quality. gms is a non-toxic emulsifier commonly used in baked goods to stabilize air bubbles, improve dough handling, and extend product shelf life (chen et al., 2024; karthik et al., 2024; setiarto et al., 2024). it can interact with gluten and starch components to create a more cohesive matrix within the dough, thereby enhancing the volume, softness, and uniformity of bread texture (ibrahim et al., 2025; setiawan et al., 2024). the application of gms in composite flour bread formulations could potentially mitigate the textural drawbacks associated with glutenfree or low-gluten products (amelia et al., 2024; azid et al., 2025). although various studies have examined the impact of either modified starch or emulsifiers on bakery products, there remains limited research on the combined effect of hmt-modified kimpul flour and gms in sweet bread. particularly, how the flour substitution ratio and emulsifier concentration affect the physicochemical and sensory characteristics of sweet bread is not yet fully understood (febriani & sumarto, 2021; yahya & hashim, 2023). understanding this relationship is essential for developing bakery products with desirable qualities while promoting the use of local, underutilized tubers as sustainable food sources. this study aims to examine the effect of hmtmodified kimpul flour substitution and varying gms concentrations on the physicochemical and sensory properties of sweet bread. this study is expected to contribute to the development of high-quality bread products that utilize local ingredients, support food diversification, and reduce dependence on imported wheat flour. materials and methods materials the main raw material used in this study was kimpul tuber (xanthosoma sagittifolium), which was harvested at 6–8 months of age and obtained from local farmers in salatiga, central java. in addition to kimpul tubers, other ingredients used in the preparation of sweet bread included wheat flour, granulated sugar, salt, margarine, powdered milk, yeast, eggs, and glycerol monostearate (gms) as an emulsifier. for the modification process of the kimpul flour, supporting materials included aquadest, sodium chloride (nacl), and various packaging materials such as hdpe plastic and aluminum foil. analytical grade chemicals, including sodium hydroxide (naoh), concentrated sulfuric acid (h₂so₄), boric acid, hydrochloric acid (hcl), hexane, and kjeldahl tablets, were used for the proximate and physicochemical analyses (diana et al., 2024). experimental design and setup this study employed a completely randomized factorial design (ral faktorial) with two treatment factors: the ratio of wheat flour to modified kimpul flour (3:1, 1:1, and 1:3), and the concentration of glycerol monostearate (2%, 3%, and 4%). each treatment was carried out in triplicate. laboratory equipment used included a cabinet dryer, convection oven, digital balance, sieves (60 mesh), baking molds, and standard glassware. the baking was conducted under consistent conditions, and evaluations were carried out in a food analysis laboratory using a texture analyzer and colorimeter to measure physical characteristics (nurislaminingsih et al., 2024). preparation of modified kimpul flour and sweet bread kimpul tubers were first washed, peeled, and sliced into thin chips, then soaked in a sodium chloride solution to reduce calcium oxalate content. the chips were drained and dried in a cabinet dryer at 50°c for 24 hours. once dried, they were ground and sieved using a 60-mesh sieve to obtain fine flour. the heat moisture treatment (hmt) process was carried out by adjusting the flour’s moisture content to 28% through spraying with distilled water. the moistened flour was then equilibrated in a refrigerator for 24 hours before being placed in a covered tray and heated in an oven at 105°c for 5 hours, with stirring every 2 hours. after heating, the flour was dried again at 50°c for 4 hours, then ground and sieved (jiang et al., 2024). sweet bread preparation involved mixing the dry ingredients (wheat flour, modified kimpul flour according to treatment ratios, powdered milk, salt, sugar, and yeast) before gradually adding eggs, margarine, water, and gms based on the assigned concentration. the dough was kneaded until smooth and elastic, fermented for a specific period, shaped into molds, and baked. all preparation steps were standardized across treatments to ensure comparability. data collection and analytical methods physicochemical characteristics of the modified kimpul flour were measured, including moisture content, swelling power, solubility, water absorption capacity, and color parameters (l*, a*, b*). the results showed that the modified flour had a moisture content of 5.70%, swelling power of 0.41 g/g, solubility of 14.19%, water absorption of 4.55 g/g, and color values of l* = 83.84, a* = 1.67, and b* = 10.35. these measurements indicate that the hmt process successfully enhanced the functional properties of the flour. subsequently, the sweet bread produced from each treatment was analyzed for its proximate composition (moisture, ash, fat, protein, and carbohydrate), physical setianingsih et al. – effect of modified kimpul flour substitution … 681 color (l*, a*, b*), texture (hardness, cohesiveness, adhesiveness), and volume expansion. sensory evaluation was also conducted using descriptive tests that assessed aroma, sweetness, softness, stickiness, and crust color, following standardized food sensory protocols. statistical analysis all data collected from physicochemical, textural, and sensory evaluations were statistically analyzed using one-way analysis of variance (anova). when significant differences were detected (p < 0.05), duncan’s multiple range test (dmrt) was employed for post hoc comparisons. the statistical software used for this analysis was spss version 26. results were presented as mean values accompanied by standard deviations to illustrate data consistency across replicates. results and discussion result proximate composition of sweet bread the proximate analysis of sweet bread showed significant variations depending on the ratio of wheat flour to hmt-modified kimpul flour and the concentration of glycerol monostearate (gms). as shown in table 1, the moisture content increased with higher substitution of kimpul flour. the treatment using a 1:3 (wheat:kimpul) ratio with 3% gms recorded the highest moisture level (29.48%), while the lowest (27.32%) was observed in the 3:1 ratio with 2% gms. in terms of ash content, a steady increase was also observed with higher levels of kimpul flour substitution. conversely, protein and fat content decreased as kimpul flour levels rose. the highest protein content (10.24%) occurred at the lowest kimpul substitution (3:1 with 2% gms), while the lowest (8.40%) was at 1:3 with 3% gms. carbohydrate content increased alongside kimpul flour substitution, peaking at 43.61%. table 1. proximate composition of sweet bread. treatment (wheat:kimpul + gms%) m o is tu re ( % ) a sh ( % ) f a t (% ) p ro te in ( % ) c a rb o h y d ra te (% ) 3:1 + 2% 27.32 1.21 10.35 10.24 40.88 1:1 + 3% 28.64 1.30 9.71 9.51 40.84 1:3 + 3% 29.48 1.41 9.10 8.40 43.61 physical texture of bread bread texture, measured by hardness, adhesiveness, and cohesiveness, was significantly influenced by both kimpul flour levels and gms concentration. the bread sample with the 1:3 substitution ratio and 2% gms displayed the highest hardness (440.83 g), while the 3:1 substitution ratio with 4% gms resulted in the softest texture (127.50 g). adhesiveness followed a similar trend, with higher values occurring at higher kimpul levels. cohesiveness remained relatively stable across treatments. the hardness values of sweet bread samples are shown in figure 1. figure 1. hardness values of sweet bread samples. sensory evaluation of sweet bread the sensory quality of sweet bread was evaluated based on five parameters: softness, sweetness, aroma, crust color, and overall acceptance. as shown in table 2, the 1:1 flour ratio with 3% gms received the highest scores across most attributes, especially softness (4.6) and overall acceptance (4.5). in contrast, the 1:3 ratio with 2% gms scored lowest across all categories. table 2. sensory evaluation scores of sweet bread treatment s o ft n es s s w ee tn es s a ro m a c ru st c o lo r o v er a ll a cc ep ta n ce 3:1 + 2% gms 4.2 3.8 3.9 3.7 3.9 1:1 + 3% gms 4.6 4.4 4.5 4.3 4.5 1:3 + 2% gms 3.6 3.2 3.3 3.4 3.3 color and volume expansion the l*, a*, and b* color values demonstrated that higher levels of kimpul flour substitution led to slightly darker bread. bread samples with higher gms concentrations appeared lighter and more uniform in color. meanwhile, volume expansion decreased as more kimpul flour was used, with the lowest volume expansion in the 1:3 ratio and the highest in the 3:1 ratio. volume expansion of sweet bread based on flour ratio is shown in figure 2. figure 2. volume expansion of sweet bread based on flour ratio. 682 biology, medicine, & natural product chemistry 14 (2), 2025: 679-683 discussion the overall results indicate that the substitution of wheat flour with hmt-modified kimpul flour significantly affects both the nutritional content and physical characteristics of sweet bread. the increase in moisture and carbohydrate content, alongside the decrease in protein and fat, suggests that kimpul flour contributes to softer crumb and longer moisture retention, though at the expense of nutritional density. from a texture standpoint, the absence of gluten in kimpul flour was associated with increased hardness and reduced elasticity. however, the addition of glycerol monostearate successfully mitigated this effect by improving softness and cohesiveness, especially at the 3% concentration. this confirms the emulsifying role of gms in stabilizing air bubbles and enhancing crumb softness. the sensory evaluation results support the instrumental data: the combination of a 1:1 wheat to kimpul ratio with 3% gms provided the most acceptable bread in terms of texture, appearance, and flavor. it achieved a good balance between desirable sensory quality and increased local ingredient utilization. color changes and reduced volume expansion were expected due to the intrinsic properties of kimpul starch, which lacks gluten’s gas-holding capacity. although volume decreased, consumer acceptability remained relatively high in the optimal treatment. while the findings are promising, limitations include the use of only one modification method and the lack of detailed microstructural analysis of dough and crumb. future studies should consider comparing different modification techniques and including shelf-life testing and consumerscale panels for wider validation. in conclusion, hmtmodified kimpul flour can be effectively used to partially substitute wheat flour in sweet bread production, especially when combined with 3% gms. this formulation enhances moisture retention and sensory appeal while promoting local food utilization. conclusions this study concludes that substituting wheat flour with hmt-modified kimpul flour, particularly at a 1:1 ratio combined with 3% glycerol monostearate (gms), improves the physicochemical and sensory qualities of sweet bread. the modified kimpul flour increased moisture and carbohydrate content, while gms enhanced texture and overall acceptability, indicating a successful synergy between local starch sources and functional additives. these findings highlight the potential of utilizing underutilized local tubers like kimpul in bakery products to support food diversification and reduce wheat import dependency. it is recommended that food producers adopt up to 50% kimpul flour substitution with 3% gms in sweet bread formulations. further research should explore additional starch modification methods, structural and shelf-life characteristics, and broader sensory evaluations to support product development and commercialization. acknowledgements: the author would like to thank belmawa kemendikbud for funding the 2023 student creativity program/program kreativitas mahasiswa (pkm), also to student affairs and pkm center universitas pgri semarang, faculty of engineering and informatics universitas pgri semarang for all the facilities provided. authors’ contributions: conceptualization: siti nurlaela setianingsih, rizky muliani dwi ujianti; formal analysis: iffah muflihati, mega novita; funding acquisition: diva julia paramita; methodology: mega novita, fafa nurdyansyah; laboratory: shindi nofitasari, dola mareta anggarini, diva julia paramita; visualization: abdillah fathan generus annajah; writing – original draft: rizky muliani dwi ujianti, iffah muflihati, siti nurlaela setianingsih; writing – review & editing: mega novita competing interests: the authors declare that there are no competing interests. funding: belmawa kemendikbud for funding the 2023 student creativity program/program kreativitas mahasiswa (pkm) references al ubaidilah, u. n. k., handarini, 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(2023). effect of freezing and heating white bread on the glycemic response of healthy individuals. journal of preventive and complementary medicine, 2(4), 196–201. this page intentionally left blank biology, medicine, & natural product chemistry volume 5 – number 1 – 2016 issn 2089-6514 contents new record marsdenia tenacissima (asclepiadoideae, apocynaceae) in gunung ijo baturagung yogyakarta widodo, muhammad jafar luthfi 1 8 local stability analysis of a mathematical model of the interaction of two populations of differential equations (host-parasitoid) dewi anggreini 9 14 determination of levels leisure village patronage uin sunan kalijaga yogyakarta: improving governance patronage towards rural village green and environmentally friendly supriatna, thaqibul fikri niyartama, iwan kuswidi 15 18 modified alizarin red s-alcian blue staining for reptilian skeleton muhammad jafar luthfi 19 22 checklist of macroalgae in waisai coast, raja ampat retno suryandari, widodo 23 32 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 57-62 | doi: 10.14421/biomedich.2024.131.57-62 issn 2540-9328 (online) effectiveness analysis of antidiabetic property from dragon fruit peel methanol extract in alloxan-induced diabetic rats sonia apriani sumartin1, i nyoman ehrich lister2, edy fachrial2,* 1master program of biomedicine, faculty of medicine; 2faculty of medicine, universitas prima indonesia, medan, indonesia. corresponding author* fachrial_edy@yahoo.co.id abstract diabetes is a health burden in various countries, one of these countries, is also indonesia. dragon fruit peel is an alternative diabetes therapy that has been widely studied. therefore, this study aimed to investigate an antidiabetic effect of dragon fruit peel methanol extract on alloxan-induced diabetic male wistar rats. this experimental study used twenty-five male wistar rats induced by alloxan injection. after 48 hours, all rats were grouped into five different groups, including control (0.5% scmc), standard (metformin), dragon fruit peel methanol extract-1 (500 mg/kg bw), 2 (750 mg/kg bw), and 3 (1,000 mg/kg bw). these treatments were given for two weeks. after that, all rats were dissected to obtain the pancreas. the results showed that dragon fruit peel methanol extract significantly decreased blood glucose levels after the 7th and 14th days (p value <0.05). in addition, pancreatic histology showed a decrease in the extract dose followed by a smaller size of the pancreatic langerhans islet. the lowest dose of the extract showed a similar size of pancreatic langerhans islet to the control group with an atrophic pancreatic langerhans islet. therefore, it can be concluded that dragon fruit peel extract can significantly decrease blood glucose levels and improve the structure of pancreatic langerhans islet at higher doses. keywords: anti diabete; dragon fruit peel; langerhans islet; pancreas. introduction diabetes mellitus (dm) is a group of metabolic diseases characterized by hyperglycemia due to abnormalities in insulin secretion, insulin action, or both. chronic hyperglycemia in diabetes is associated with long-term damage or dysfunction of several body organs, especially the eyes, kidneys, nerves, heart, and blood vessels. world health organization (who) has reported that diabetes mellitus cannot be briefly described. however, who generally defines diabetes mellitus as an accumulation of anatomical or biochemical dysfunction caused by some factors that lead to either absolute or relative insulin deficiency with or without insulin dysfunction. (purnamasari, 2014) diabetes is a health burden in various countries, and one of these countries is indonesia. it is due to the longterm impact of diabetes mellitus, including blindness, heart disease, and kidney failure. the international diabetes federation (idf) estimates that at least 463 million people aged 20-79 years old worldwide had diabetes in 2019, or equivalent to a prevalence rate of 9.3% of the total population of the same age. based on gender, idf estimates that the prevalence of diabetes in 2019 will be 8% in women and 9.65% in men. the prevalence of diabetes is estimated to increase as the population ages 19.9% (111.2 million people) aged 65-79 years. this rate is estimated to grow annually to 578 million in 2020 and 700 million in 2045 (internation diabetes federation, 2019). recently, herbal medicine is a medicine that is quite popular and is in relatively high demand on the market. it can be seen from the increasing number of herb industry. the food and drug monitoring agency (balai pengawasan obat dan makanan/ bpom) reports that the annual growth rate in the herbal medicine industry ranges from 25% to 40%. pranomo (2022) reported that 810 active factories were producing traditional indonesian medicines; 87 factories were ioat (traditional medicine industries), and the remaining 723 were small herbal medicine industries). in a prior report, it was reported that 872 companies work in the herbal industry. ironically, around 462 of these companies were foreign companies. hence, it indicates that the utility of herbal medicines in society, especially in indonesia, is very high because the herbal medicine industry annually increases (elfahmi et al., 2014). according to the information above, the herb industry quietly developed in indonesia and has become a potential alternative medicine, especially for diabetes mellitus, and indonesia, as part of southeast asia countries, has a high prevalence and incidence rate of diabetes mellitus. a natural product widely studied is manuscript received: 04 december, 2023. revision accepted: 07 may, 2024. published: 13 may, 2024. https://doi.org/10.14421/biomedich.2024.131.57-62 58 biology, medicine, & natural product chemistry 13 (1), 2024: 57-62 dragon fruit peel. several studies have reported various health benefits of dragon fruit peel. mahargyani (2019) reported that the n-hexane fraction of dragon fruit peel extract had α-glucosidase enzyme inhibitory activity with an ic50 value of 194.11 ppm, while acarbose was 5.6 ppm. putri et al. (2020) also reported that red dragon fruit reduced plasma homa-ir and mda plasm, which had a similar effect to metformin. another study that was performed by panjaitan and novitasari (2021) also reported that red dragon fruit peel ethanol extract had antidiabetic activity at a dose of 74.88 mg/ 200 grams bw in streptozotocin-induced diabetic rats and this effect was similar to glibenclamide at a dose of 0.09 mg/ 200 grams bw (mahargyani, 2019; panjaitan & novitasari, 2021; putri et al., 2021). even though many studies have been performed to explore the antidiabetic effects of dragon fruit peel, none of these studies explore the antidiabetic effect of dragon fruit peel extract and its effect on pancreatic tissue. hence, this study aimed to investigate an antidiabetic effect of dragon fruit peel methanol extract on alloxaninduced diabetic male wistar rats. materials and methods study design this experimental study was performed between september 2022 to december 2022 in the pharmacology laboratory, universitas prima indonesia. this study protocol has been approved by the health research ethics committee universitas prima indonesia with letter no. 019/kepk/unpri/xii/2022. materials this study used some materials, including dragon fruit peel, 98% methanol solution, distilled water, scmc (sodium carboxyl methylcellulose), metformin tablet, rat food pellets and bedding, hcl, magnesium powder, amyl alcohol, mayer’s reagent, bouchardat’s reagent, dragendorff’s reagent, iron (iii) reagent chloride, sulfuric acid, 95% ethanol, lead (ii) acetate, isopropanol, chloroform, molisch reagent, lieberman-bourchard reagent, acetic acid, and ketamine. extraction process this study used dragon fruit peel that initially dried and meshed into simplicial powder. dragon fruit peel simplicial powder was soaked into 98% methanol solution with a ratio of 1:3, also known as the maceration process. then, it was regularly stirred for three days. afterward, it was filtered, and the residue was remacerated as before two times. all filtrates were then collected to evaporate by rotary evaporator at a temperature of 40-50oc until formed concentrated extract. after that, the extract yield was a ratio of the extract and the simplicial powder mass, which was expressed as a percentage (mutia, 2019) (chiuman et al., 2021; suhartomi et al., 2020). phytochemical screening this study underwent a phytochemical screening based on the standard manual for phytochemical screening from the pharmacology laboratory of universitas prima indonesia, including phenolic, flavonoid, alkaloid, terpenoid/ steroid, tannin, and saponin (depari et al., 2021; widowati et al., 2017). oral suspension formulation this study used 0.5% scmc as a vehiculum for both metformin and dragon fruit peel extract to administer it via the oral route by oral gavage feeding tubes. this vehiculum was formulated by dissolving 500 mg sodium carboxymethylcellulose powder into a hundred milliliters of warm distilled water. after that, amount of 400 mg dragon fruit peels methanol extract and 100 mg metformin were suspended into 10 ml 0.5% scmc to form dragon fruit peels methanol extract and metformin oral suspension, respectively (chiuman, 2019; kanon et al., 2012; mutia & chiuman, 2019). alloxan induction alloxan induction was performed by subcutaneous alloxan induction. this study used a 5% alloxan monohydrate solution with a dose of 3 ml/ kg bw (150 mg/ kg bw). evaluation of diabetic condition was performed after 48 hours of subcutaneous alloxan injection, and diabetic condition was defined as blood glucose level higher than 200 mg/ dl (11.1 mmol/ l) (ighodaro et al., 2017a; nair & jacob, 2016; njagi e n mwaniki & njagi j murugi, 2015). antidiabetic assay the antidiabetic assay was performed on twenty-five diabetic rats and grouped into five treatment groups. these groups were standard (metformin), control (0.5% scmc), dragon fruit peel-1 (500 mg/ kg bw), 2 (750 mg/ kg bw), and 3 (1,000 mg/ kg bw), that received a milliliter of 0.5% scmc, a milliliter of metformin oral suspension, 5, 7.5, and 10 ml/ kg bw of dragon fruit peel extract oral suspension, respectively. these treatments were performed for two weeks. all rats were free to access rat food pellets and beverages. blood glucose measurement this study measured fasting blood glucose level, which was measured after 10-12 hours of fasting periods. blood glucose level was obtained from the tail vein, and fasting blood glucose was measured before induction, 48 hours after induction, and two weeks after treatment (zubaidah et al., 2019). pancreas tissue study pancreas tissue was dissected and preserved in a 10% formalin buffer solution. then, all histology slides were sumartin et al. – effectiveness analysis of antidiabetic property from … 59 sliced with a 4-6 mm thickness, stained with hematoxylin and eosin, and observed under a microscope. pancreas tissue processing and staining were performed based on the procedure in the anatomical pathology laboratory, faculty of medicine, universitas sumatera utara. data analysis blood glucose level and initial body weight were analyzed by one-way anova if distribution data was normal, followed by post hoc test tukey hsd. meanwhile, if data distribution was not normal, all data were analyzed by kruskal-wallis, followed by mannwhitney. on the other hand, the physical characteristics and phytochemical compounds of dragon fruit peel extract and pancreas tissue histology study were narratively described. results and discussion this study used dragon fruit peel methanol extract that was obtained from a traditional market in medan city. this study used 2,000 grams of dragon fruit peel as a fresh simplicial. after that, this fresh simplicial was dried and meshed into 1,896.9 grams of simplicial powder. this simplicial powder was macerated by 7,500 ml of methanol solution to obtain 79.97 grams of concentrated dragon fruit peel methanol extract. based on these values, dragon fruit peel methanol extract yields 4.22%. the obtained dragon fruit peel extract underwent phytochemical screening, revealing phytochemicals including phenol, flavonoid, alkaloid, and tannin. after that, this dragon fruit peel extract was evaluated for antidiabetic activity by an alloxan-induced diabetic rat model. all acclimatized rats were initially weighed; this initial body weight in grams was described in table 1. table 1. initial body weight of all rats. group initial body weight, gram p-value mean sd control 165.00 6.96 0.966 standard 165.00 6.96 dragon fruit peel extract-1 163.60 5.68 dragon fruit peel extract-2 163.20 5.63 dragon fruit peel extract-3 162.80 5.45 table 1 showed that there was no significant difference in the initial body weight of all rats; it can be seen from the p-value > 0.05 (p-value = 0.966). thus, it can be concluded that the initial body weight of all rats used in this study was homogenous, a range of 162.80165.00 grams. after that, these rats were diabeticinduced by alloxan injection, and then these rats were grouped to be treated based on the treatment groups, and a comparison of blood glucose levels in all rats was described in table 2. table 2. comparison of blood glucose levels in all treatment groups. group blood glucose level (mg/dl), mean ± sd before induction after induction 7th day 14th day control 95.60 ± 7.30 390.00 ± 108.12 462.40 ± 88.13a 440.40 ± 100.98a standard 92.20 ± 3.96 432.00 ± 138.19 152.00 ± 18.30b 133.20 ± 9.09b dragon fruit peel extract-1 89.40 ± 8.08 391.20 ± 102.05 327.60 ± 38.93c 203.80 ± 16.12b dragon fruit peel extract-2 99.40 ± 3.65 471.40 ± 101.70 284.20 ± 86.17c 181.00 ± 19.71b dragon fruit peel extract-3 93.00 ± 6.63 438.80 ± 132.50 238.20 ± 84.74bc 137.00 ± 24.75b p-value 0.157 0.785 < 0.05 < 0.05 p-value was obtained from the one-way anova test; different superscripts in the same column indicate significant differences based on the tukey hsd post hoc test. table 2 showed that there was no significant difference in the blood glucose levels of rats both before induction and after induction; it can be seen from the pvalue > 0.05 in the blood glucose level data before induction (p-value = 0.157) and after induction (p-value= 0.785). it can be concluded that blood glucose levels, either before or after induction, were homogenous. furthermore, blood glucose levels showed a significant change on the 7th and 14 days; it can be seen from the pvalue < 0.05. in the first week of administering, the dragon fruit peel methanol extract showed a significant reduction in blood glucose levels compared to the control group; increasing the dose of the extract did not significantly change blood glucose levels. at 7th day, the highest mean blood glucose level was found in the control group, which was 462.40 ± 88.13 mg/ dl, followed by the dragon fruit peel group-1 (327.60 ± 38.93 mg/ dl), 2 (284.20 ± 86.17 mg/dl), 3 (238.20 ± 84.74 mg/ dl), and the lowest was the standard group (152.00 ± 18.30 mg/ dl). meanwhile, the blood glucose levels continued to decrease significantly on the 14th day. the highest blood sugar levels were found in the control group, namely 440.40 ± 100.98 mg/ dl, followed by the dragon fruit peel methanol-1 (203.80 ± 16.12 mg/dl), 2 60 biology, medicine, & natural product chemistry 13 (1), 2024: 57-62 (181.00 ± 19.71 mg/ dl), 3 (137.00 ± 24.75 mg/ dl), and the lowest glucose levels were found in the standard group, namely 133.20 ± 9.09 mg/ dl. moreover, the percentage of blood glucose levels decreasing in the 7th and 14th days was described in table 3. table 3. percentage of blood glucose level decreasing in all groups. group decreasing of blood glucose level, δbgl (%) 7th day 14th day control reference reference standard 310.40 (67.13) 307.20 (69.75) dragon fruit peel extract-1 134.80 (29.15) 236.60 (53.72) dragon fruit peel extract-2 178.20 (38.54) 259.40 (58.90) dragon fruit peel extract-3 224.20 (51.51) 303.40 (68.89) table 3 showed that the highest mean of blood glucose levels decreases on the 7th day was found in the standard group, which was 67.13%, followed by the dragon fruit peel-3 (51.51%), 2 (38.54%), and the lowest was the dragon fruit peel-1 (29.15%). meanwhile, the mean decrease in blood glucose levels on the 14th day was not much different than on the 7th day. the highest mean of blood glucose levels decrease on the 14th day was found in the standard group, which was 69.75%, followed by the dragon fruit peel-3 (68.89%), 2 (58.90%), and the lowest was the fruit peel dragon-1 (53.72%). furthermore, the distribution of blood glucose levels during this study was also described in figure 1. figure 1. bar graph of blood glucose level of all groups during observation. figure 1 showed that all groups showed the lowest mean blood glucose level before induction and the highest blood glucose level was found after induction. only the control group showed an increase in blood glucose levels after treatment (7th and 14th day). at the end of the observation, all rats were sacrificed to obtain their pancreatic tissues for pancreatic histology study, which were described in figure 2. figure 2. pancreatic tissue in all groups. (a) dragon fruit peel-1; (b) dragon fruit peel-2; (c) dragon fruit peel-3; (d) standard; (e) control. staining: haematoxylin and eosin (he)/magnification: 400 x; abbreviation: ap: acini pancreas; il: langerhans islets. figure 2 showed some significant differences in the size and structure of the langerhans islets in all groups. the control group did not receive the extract or standard treatment; the size of the langerhans islets decreased, and an atrophic pancreatic langerhans islet rose. it contrasted with the standard group, which showed an intact langerhans islet. meanwhile, the size of langerhans islet increased in line with the dragon fruit peel extract increase. interestingly, the highest dragon fruit peel extract dose revealed a similar pancreas tissue condition with the standard group. on the other hand, the lowest dragon fruit peel extract dose revealed a similar pathologic damage with a control group, which was an atrophic pancreatic langerhans islet. based on the result above, dragon fruit peel extract yielded 4.22% with an antidiabetic effect, which can significantly decrease blood glucose levels seven days after treatment (p value < 0.05). the increase of dragon fruit peel extract did not significantly reduce the blood glucose level; it was concluded from the post hoc test tukey hsd in blood glucose level. however, the variation of dragon fruit peel extract dose significantly affects both the size and structure of the langerhans islets. the highest dose of dragon fruit peel extract 0 100 200 300 400 500 600 control standard dragon fruit peel extract-1 dragon fruit peel extract-2 dragon fruit peel extract-3 b lo o d g lu co se l ev el ( m g/ d l) group before induction after induction 7th day 14th day sumartin et al. – effectiveness analysis of antidiabetic property from … 61 revealed an intact langerhans islet as well as the standard group in contrast to the lowest dose of dragon fruit peel extract, which revealed an atrophic pancreatic langerhans islet and the control group. this study used alloxan injection for diabetic induction. alloxan is a urea derivate compound that induces langerhans islet necrosis, especially beta cells. this necrosis occurs because of the autooxidation of alloxan radicals. autooxidation destructs dna structure and inhibits the thiol group of glucokinase enzyme, which decreases the formation of atp and leads to reduced insulin secretion (dewangan et al., 2017) (ighodaro et al., 2017b) (karthikeyan et al., 2016; vijayaraj et al., 2019). hence, it is obvious that alloxan could induce diabetic conditions, and the current study showed that after induction, all rats had blood glucose levels> 200 mg/ dl. the current study result was in line with the previous study that the dragon fruit peel also had an antidiabetic activity. solikhah et al. (2022) reported that dragon fruit peel ethanol extract had an antidiabetic effect on alloxaninduced diabetic mice. solikhah et al. also reported that dragon fruit peel ethanol extract at 100 mg/kg bw and 300 mg/kg bw significantly decreased blood glucose levels on the first, seventh, and fourteenth days of treatment. this antidiabetic activity is due to the dragon fruit peel extract’s flavonoid, terpenoid, tannin, polyphenol, quinone, and alkaloid. saponins are reported to inhibit the α-amylase enzyme activity and have antioxidant effects. polyphenols can inhibit the dpp-4 enzyme, increasing the half-life of glp-1 and increasing insulin production through direct or indirect stimulation of pancreatic β cells. polyphenols are also reported to increase peripheral tissue sensitivity to insulin and improve various diabetes complications such as kidney failure, vascular dysfunction, and other complications. alkaloids can also act as ppar γ agonists, increase glucokinase activity, and increase the expression of glut 4. finally, flavonoids modulate the glucose transporter expression, especially glut-4 transporter, by increasing insulin secretion, reducing cell apoptosis, increasing the proliferation of pancreatic beta cells, and improving insulin resistance (solikhah et al., 2022). another study performed by panjaitan and novitasari (2021) also reported a similar result to the current study. panjaitan and novitasari reported that ethanol extract of dragon fruit peel at a dose of 187.2 mg/ kg bw (37.44 mg/ 200 grams) and 374.4 mg/ kg bw (74.88 mg/ 200 grams) could significantly decrease blood glucose levels in streptozotocin-induced diabetic compared to the control group. the highest dose of dragon fruit peel extract (374.4 mg/ kg bw) significantly decreased the blood glucose levels that were as well as the standard group (glibenclamide). it is due to phytochemicals, including tannins and flavonoids in the dragon fruit peel ethanol extract. flavonoids and tannins are reported to have antioxidant effects that can facilitate decreasing blood glucose levels. the antioxidant effect of tannin can scavenge free radicals, inhibit glucose absorption in the gastrointestinal tract, and induce regeneration of pancreatic beta cells, leading to improved insulin sensitivity in adipose tissue and glucose uptake by insulin-mediated tissue. in addition, the antioxidant effect of tannin and flavonoids from dragon fruit peel extract also promotes protons donation to the alloxan radical as an induction compound in this study, thereby preventing further pancreatic damage, which leads to normalizing blood glucose level (panjaitan & novitasari, 2021). the result of this study is in line with some previous studies that have been performed. dragon fruit peel extract at the dose of 500 mg/ kg bw has shown antidiabetic activity, this dose was higher than the reported dose in the previous study. panjaitan and novitasari reported that the lower dose (374.4 mg/ kg bw) of dragon fruit peel extract had significantly decreased blood glucose levels. the current study also evaluated another parameter, histology of pancreatic tissue. although the lowest dose of dragon fruit peel extract (500 mg/ kg bw) significantly decreased blood glucose level, it also showed an atrophic pancreatic langerhans islet. therefore, it showed that a lower dose of dragon fruit peel, which was 374.4 – 500 mg/ kg bw, significantly decreased blood glucose levels. ironically, it required a higher dose to improve the pancreatic langerhans islets. conclusions overall, it can be concluded that dragon fruit peel extract significantly decreased the blood glucose level, at least in the lowest dose (500 mg/ kg bw). however, it required a higher dose of dragon fruit peel extract to improve not only blood glucose level but also pancreatic langerhans islet. further study was required to evaluate safety and toxicity profile of dragon fruit extract to support the antidiabetic activity. acknowledgements: this study was supported by faculty of medicine, universitas prima indonesia. authors’ contributions: conceptualization: sonia apriani sumartin, i nyoman ehrich liester, and edy fachrial; methodology: sonia apriani sumartin and edy fachrial; investigation: sonia apriani sumartin; discussion of results: sonia apriani sumartin; writing – original draft: sonia apriani sumartin; writing – review and editing: i nyoman ehrich liester and edy fachrial; supervision: i nyoman ehrich liester and edy fachrial; approval of the final text: sonia apriani sumartin, i nyoman ehrich liester, and edy fachrial. competing interests: the authors declare no competing interests. 62 biology, medicine, & natural product chemistry 13 (1), 2024: 57-62 funding: this study did not receive any funding. references chiuman, l. 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(2019). comparison of in vivo antidiabetes activity of snake fruit kombucha, black tea kombucha and metformin. biocatalysis and agricultural biotechnology, 17(november 2018), 465–469. https://doi.org/10.1016/j.bcab.2018.12.026 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 69-78 | doi: 10.14421/biomedich.2023.121.69-78 issn 2540-9328 (online) combined anthocleista vogelii and alstonia boonei stem barks extract alleviates hyperlipidaemia and renal malfunctions in benign prostatic hyperplasia-induced rats robert ikechukwu uroko1,*, mercylyn ezinne uche2, paul chukwuemaka nweje-anyalowu3, ikenna obiwuru2, chinedu aguwamba3, chinomso friday aaron2 1department of biochemistry, college of natural sciences, michael okpara university of agriculture, umudike, abia state, nigeria 2department of biochemistry, faculty of biological and physical sciences, abia state university, uturu, nigeria. 3biochemistry unit, department of chemical sciences, faculty of sciences, clifford university, owerrinta, abia state, nigeria. corresponding author* ir.uroko@mouau.edu.ng manuscript received: 28 july, 2022. revision accepted: 19 august, 2022. published: 19 october, 2022. abstract benign prostatic hyperplasia (bph) is a urological disease prevalent among the ageing male population, which impairs the quality of life, including hyperlipidaemia and a decline in renal functions. combining anthocleista vogelii and alstonia boonei stem bark extract has effectively managed bph and its associated complications. this study evaluated the effects of a combined anthocleista vogelii and alstonia boonei stem bark extract (caasbe) on the lipid profile and renal functions of rats induced benign prostatic hyperplasia with testosterone propionate injection. the study comprised five treatment groups, with groups 1 – 5 being the normal control, bph control, standard control, bph+200 mg/kg caasbe, and bph+400 mg/kg caasbe, respectively. bph was induced in the groups 2 – 4 rats by subcutaneous administration of testosterone propionate injection (5 mg/kg) for 28 days, and treatment with finasteride and caasbe were administered orally. the bph control rats exhibited a significant (p < 0.05) increase in the total serum cholesterol, triacylglycerol (tag), low-density lipoprotein cholesterol (ldl-c), urea, creatinine and significant (p < 0.05) decline in the serum high-density lipoprotein cholesterol (hdl-c) compared to the normal control. conversely, treatment of the bph rats with 200 and 400 mg/kg of caasbe significantly (p < 0.05) reversed the altered total serum cholesterol, tag, ldl-c, hdl-c, urea and creatinine to normal levels comparable to that of the normal control and standard control respectively. these findings show that caasbe alleviates hyperlipidaemia and renal malfunctions in the bph rats suggesting it could be effective in managing bph complication. keywords: alstonia boonei; anthocleista vogelii; benign prostatic hyperplasia; hyperlipidaemia; renal functions. abbreviations: bph = benign prostatic hyperplasia; tag = triacylglycerol; hdl-c = high density lipoprotein cholesterol; ldl-c = low-density lipoprotein cholesterol; caasbe = combined anthocleista vogelii and alstonia boonei stem bark; a. vogelii = anthocleista vogelii; a. boonei = alstonia boonei. introduction among all the ailments and medical conditions affecting men's quality of life, benign prostatic hyperplasia (bph) remains the most challenging urological disorder confronting the ageing male population worldwide. the probability of senior male increases as he attains 50 years old based on available autopsy that 42 % and 85 % of men within 51-60 and 80-above years respectively had bph condition (gacci et al. 2017). the increased prostate stromal and epithelial cell multiplication and proliferation in response to the high circulating testosterone and dihydrotestosterone have been implicated in the bph pathogenesis. there is a decline in the quality of life as bph progresses because of complications, including urinary disorder occasioned by bladder outlet obstruction and lower urinary tract symptoms, which are difficult to manage. genetic factors influence bph pathogenesis as some races have an increased risk of developing bph than others, but recently dietary habits, physical inactivity, and unhealthy lifestyle have been shown to increase the risk of bph in ageing men (vignozzi et al. 2014). abnormal lipid profile, obesity, diabetes inflammatory conditions and hypertension are linked to bph development and progression. effective management of these common metabolic disorders in ageing men could be key to staying from bph complications (dahle et al. 2002). aged men with abdominal obesity and abnormal serum lipid profile had a very low amount of high-density lipoprotein cholesterol (hdl-c), along with elevated low-density lipoprotein cholesterol (ldl-c), total cholesterol, and triacylglycerols demonstrated the increased amount of testosterone and oestradiol which https://doi.org/10.14421/biomedich.2023.121.69-78 70 biology, medicine, & natural product chemistry 12 (1), 2023: 69-78 increased their risk for bph (adaramoye et al. 2008; ugwu et al. 2019). the role of dyslipidaemia in the bph pathogenesis is believed to be through stimulation of increased synthesis of androgens, including testosterone, dihydrotestosterone and oestradiol hormones that promote the growth of prostate cells. therapeutic agents against bph pathogenesis target and inhibit 5αreductase enzyme activities, thereby preventing the conversion of testosterone to dihydrotestosterone, which retard the multiplication of prostate stromal and epithelial cells and shrink the prostate size. a suitable therapeutic agent against bph pathogenesis must be able to prevent dyslipidaemia in the subject to achieve the desired end. anthocleista vogelii planch belongs to the loganiaceae family utilised for various medicinal purposes in africa. the stem bark has been therapeutically effective against prostate enlargement, male infertility, obesity, hyperlipidaemia, and renal and hepatic disorders (sunday et al. 2016; chukwu et al. 2020; uroko et al. 2021). a phytochemical study of anthocleista vogelii has revealed that it is rich in alkaloids, phenol, flavonoids, saponins, glycosides, terpenoids, steroids and tannins with minerals and vitamins (ikpe et al. 2020). a. vogelii is used as a therapeutic against sleeping sickness in traditional medicine. various experimental studies involving animal models have shown that it has anti-trypanosomal and anti-inflammatory activities (eze et al. 2019). it has wide medicinal properties and has been useful in managing various ailments, including diabetes, jaundice, pains, venereal diseases, malaria, ulcer, and prostate enlargement (okon et al. 2014; chukwu et al. 2020). alstonia boonei de wild is a well-known medicinal plant from the family of apocynaceae that has demonstrated high therapeutic potential against many diseases and medical conditions confronting man. the plant is found mainly in the tropical rainforest across west african countries, but it can also be found in varying proportions worldwide. the plant extracts are pharmacologically against asthma, ulcers, toothache and impotence (akinmoladun et al. 2007). the stem bark of alstonia boonei has been reported to contain substantial amounts of phytochemicals such as flavonoids, phenols, alkaloids, tannins, cardiac glycosides, and saponins, along with high mineral contents and antioxidant vitamins, which are responsible for some of its medicinal properties (akinmoladun et al. 2007; oppongbekoe et al. 2020). traditionally, a. boonei is used for treating menstrual pains, snake bites, the expulsion of residual placental in women after birth, bone remodelling, lactation induction, venereal diseases, jaundice, diabetes, rheumatism, malaria, and bacterial infections (abbiw 1990). it has also been reported to possess anti-anaemic effects, antioxidative stress and hepatoprotective properties (uroko et al. 2020; okpashi et al. 2022). this study evaluated the pharmacological impact of combined anthocleista vogelii and alstonia boonei stem bark extract (caasbe) on lipid profile and renal functions of rats induced benign prostatic hyperplasia (bph) with testosterone propionate injection. materials and methods plant material five hundred (500) g each of the coarsely ground a. vogelii leaves and a. boonei stem barks corresponding to 1000 g of the combined plant samples were weighed into a clean, sterile container, and 2.5 l of absolute ethanol were to macerate for three days under regulation agitation. the macerated combined plant sample was filtered with a whatman no. 1 filter paper, and the filtrate was concentrated with a rotary evaporator. the concentrated caasbe was weighed, and the percentage yield was calculated as 9.78 %, corresponding to 97.8 g of caasbe. experiment animals this study used thirty mature male wistar albino rats within a close body range. chemicals and drugs the finasteride, testosterone propionate injection and pentobarbital were purchased from merck company, usa; health biotech ltd, india; and medica men biotech limited, india. the analytical grade absolute ethanol solvent and randox commercial assay kits were purchased from sigma aldrich, usa, and randox laboratories, uk. study design male wistar albino rats numbering thirty with a close weight range of 160-168 g were purchased from the animal breeding unit of the department of zoology, university of nigeria nsukka, nigeria, and kept in our animal house for seven days. the rats were provided with a standard finisher feed and drinking water and humanely handled following normal regulations and guidelines for the use of animals for an experiment. our study design adopted five groups containing six male wistar albino rats of similar body weight. the study groups are as follows:  normal control: rats without bph induction received only 1 ml/kg of distilled water.  bph control: rats induced bph by a subcutaneous administration of 5 mg/kg testosterone propionate injection for 28 days consecutively without any treatment.  standard control: rats induced bph by a subcutaneous administration of 5 mg/kg testosterone propionate injection and, after 60 mins, treated with oral administration of 5 mg/kg finasteride for 28 consecutive days. uroko et al. – protective effects of anthocleista vogelii and alstonia boonei 71  bph+ 200 mg/kg caasbe: rats induced bph by a subcutaneous administration of 5 mg/kg testosterone propionate injection and, after 60 mins, treated with oral administration of 200 mg/kg caasbe for 28 consecutive days.  bph+400 mg/kg caasbe: rats induced bph by a subcutaneous administration of 5 mg/kg testosterone propionate injection and, after 60 mins, treated with oral administration of 400 mg/kg caasbe for 28 consecutive days. the rats fasted overnight on the 28th day of the experiment. they were anaesthetised on the 29th by the intraperitoneal injection of 25 mg/kg pentobarbital, followed by blood collection through cardiac puncture biochemical analyses and harvesting of kidney tissues for histopathological evaluation. determination of lipid profile, serum urea and creatinine concentrations the lipid profile parameters, including total serum triacylglycerols (tag), cholesterol, high-density lipoprotein cholesterol (hdl-c), and low-density lipoprotein-cholesterol (ldl-c), were measured following the procedures outlined in the randox commercial assay kit for lipid profile. also, the serum urea and creatinine concentrations were measured using the assay procedures in their respective randox commercial assay kit. histopathological examination of kidney tissues the kidney sections collected from each of the five groups for the histopathological examination were fixed in 10% phosphate-buffered formalin for two days. the tissues were dehydrated in four different alcohol strengths in ascending order. the histopathological examination of the kidney sections was carried out according to the procedures outlined by uroko et al. (2020). ethics approval the study design was reviewed by the ethical board of the department of physiology, biochemistry, and pharmacology, michael okpara university of agriculture, umudike and approved with reference number: mouau/vpp/ec/18/005. statistical analysis all the data generated from the study were compared and analysed for statistically significant differences with the duncan multiple range comparison test and one-way analysis of variance (anova) using statistical products and service solutions version 22. mean with a p-value < 0.05 are considered statistically different, while a mean with a p-value > 0.05 indicated no significant difference. results and discussion results effects of caasbe on the serum tag concentrations of bph-induced rats figure 1 indicated a substantial elevation in the tag concentration of the bph control compared to the normal control. the standard control and bph rats, which received 200 and 400 mg/kg caasbe, showed no significant decline in serum tag concentrations relative to the normal control. conversely, the standard control that received 5 mg/kg finasteride and bph rats that received 200 and 400 mg/kg caasbe showed significantly reduced serum tag concentrations compared with the bph control. figure 1. serum tag concentrations of bph rats treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. effects of caasbe on the serum hdl-cholesterol concentrations of bph rats the result in figure 2 showed a significant reduction in the serum hdl-cholesterol concentration of the bph control relative to the normal control. the standard control administered finasteride, and bph rats that received 200 and 400 mg/kg caasbe, respectively, demonstrated no significant decline in the serum hdl0,00 0,50 1,00 1,50 2,00 2,50 3,00 normal control bph control standard control 200 mg/kg caasbe 400 mg/kg caasbet se ru m t a g . c o n c. ( m m o l/ l) treatment groups a b a aa 72 biology, medicine, & natural product chemistry 12 (1), 2023: 69-78 cholesterol concentrations compared to the normal control. conversely, the serum hdl-cholesterol concentrations in the standard control and bph rats that received 200 and 400 mg/kg caasbe, respectively, exhibited a significant increase in the serum hdlcholesterol concentration compared to the bph control. figure 2. serum hdl concentrations in bph rats treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. effects of caasbe on the total serum cholesterol concentrations of bph -induced rats figure 3 showed a significantly elevated total serum cholesterol concentration in the bph control compared with the normal control. however, there was no significant variation in the total serum cholesterol concentration in the standard control and bph rats that received 200 and 400 mg/kg caasbe, respectively, compared with the normal control. conversely, the total serum cholesterol concentrations in the standard control and bhp rats treated with 200 mg/kg caasbe decreased significantly compared to the bph control. figure 3. total serum cholesterol concentrations of bph rats treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. effects of caasbe on the serum ldl-cholesterol concentrations of bph rats figure 4 demonstrated a significant rise in the serum ldl-cholesterol concentration of the bph control rats compared to the normal control. contrarily, the standard control and bph rats that received 200 and 400 mg/kg caasbe, respectively, showed no significant increase in the serum ldl-cholesterol concentration relative to the normal control. the level of serum ldl-cholesterol in the standard control and bph rats treated with 200 and 400 mg/kg caasbe were significantly reduced compared to the bph control. figure 4. serum ldl-cholesterol concentrations of bph treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. 0,00 0,50 1,00 1,50 2,00 2,50 normal control bph control standard control 200 mg/kg caasbe 400 mg/kg caasbe se ru m h d l c o n c. ( m m o l/ l) treatment groups b a b bb 0,00 1,00 2,00 3,00 4,00 5,00 6,00 normal control bph control standard control 200 mg/kg caasbe 400 mg/kgcaasbe to ta l s e ru m c h o e st e ro l (m m o l/ l) treatment groups a b a a a 0,00 0,50 1,00 1,50 2,00 2,50 3,00 normal control bph control standard control 250 mg/kg caasbe 500 mg/kg caasbe m e an l d l c o n c. ( m m o l/ l) treatment groups a b a a a uroko et al. – protective effects of anthocleista vogelii and alstonia boonei 73 effects of caasbe on the serum urea concentrations of bph rats figure 5 indicated a significant increase in the serum urea concentration of the bph control compared to the normal control. there were mild variations in the serum urea concentrations of the standard control and bph rats treated with 200 and 400 mg/kg caasbe, respectively, compared to the normal control. the serum urea concentrations of the standard control and bph rats administered varying doses of caasbe showed significant reduction compared with the bph control. figure 5. serum urea concentrations of bph rats treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. effects of caasbe on the serum creatinine concentrations of bph rats the serum creatinine concentration in the bph control was significantly elevated compared to the normal control, standard control and bph rats administered 200 and 400 mg/kg caasbe, respectively (figure 6). in contrast, the standard control and bph rats treated with 200 and 400 mg/kg caasbe showed no significant difference in their serum creatinine concentrations relative to the normal control. figure 6. serum creatinine concentrations in bph rats treated with caasbe. each of the bars indicated mean ± standard deviation (n = 6), and any of the results with unlike superscripts are significantly (p < 0.05) different from the paired mean. effects of caasbe on the kidney histomorphology of bph rats the kidney section from the normal control rats indicated a normal renal histomorphology for laboratory rodents, containing normal glomeruli (g) in the cortex containing intact renal tubules indicated by the arrow. the renal tubules also contain unaltered outer and inner medulla, as shown in figure 7 a. similarly, the sections of kidneys from bph control rats (fig. 7b), standard control (fig. 7 c), and bph induced rats treated with 200 mg/kg and 400 mg/kg of caasbe in fig. 7 d and fig. 7 e, respectively, showed normal kidney histomorphology of normal rodents. 0 10 20 30 40 50 60 normal control bph control standard control 200 mg/kgcaasbe 400 mg/kg caasbe m e an u re a c o n c. ( m g/ d l) treatment groups a b a a a 0,00 0,50 1,00 1,50 2,00 2,50 normal control bph control standard control 200 mg/kg caasbe 400 mg/kg caasbe se ru m c re at in in e c o n c. ( m g/ d l) treatment groups a b a a a 74 biology, medicine, & natural product chemistry 12 (1), 2023: 69-78 figures 7. a e. histomorphology of kidney section from normal control rat, bph control rat, standard control, bph rats treated with 200 mg/kg caasbe, and bph rat treated with 400 mg/kg caasbe, respectively. discussion the combined a. vogelii and a. boonei stem bark (caasbe) is a potent therapeutic agent against bph and several diseases in traditional medicine across south-eastern nigeria. it has little scientific data on its effects on other biochemical and physiological functions. the bph is a medical condition common in the ageing male population worldwide but with increased occurrence in males of african origin. it is caused by prostate enlargement occasioned by the rapidly dividing and proliferating prostate stromal and epithelial tissues in response to the stimulatory actions of increased dihydrotestosterone (dht) concentration (gacci et al. 2017). this study evaluated the effects of a combined caasbe on the lipid profile and renal functions in benign prostatic hyperplasia (bph) induced rats. in this study, the bph induction without administration of any therapeutic agent resulted in a significant elevation of the serum tag, cholesterol, ldl, urea and creatinine concentrations along with a significantly reduced hdl concentration in the bph control compared to the normal control. the significantly elevated serum tag concentration in the bph control relative to the control indicates the adverse effect of bph on lipid metabolism, which promoted hyperlipidaemia in the bph rats. the elevated serum tag level in the bph control agrees with ugwu et al. (2019) that high serum tag level is associated with bph pathogenesis. the bph control rats could have experienced impaired carbohydrate catabolism and conversion of excess amount of free fatty acids resulting in increased serum tag concentration due to the decline in the transport of tag to the liver for catabolism because of the inadequate amount of hdl needed for the vehicle. the bph rats could have suffered acute pancreatic inflammation that impaired carbohydrate metabolism and elevated serum tag concentration. the bph induction might have impaired the metabolism of tag ingested from dietary sources that could have contributed to the increased circulating serum tag concentration. the effect of bph on tag indicates that increased serum tag is associated with bph progression, which predisposes the bph patient to the increased risk of arteriosclerosis, cardiovascular dysfunction and paralysis of some parts of the body. this finding agrees with güven and gökhan (2022) report that hyperlipidaemia could accelerate bph progression. the substantially reduced serum tag concentration in all the bph rats administered caasbe relative to the bph control but comparable to the normal control and standard control, respectively, could be attributed to the antihyperlipidemic effects of caasbe. the caasbe might be rich in bioactive phytoconstituents such as flavonoids, alkaloids, terpenoids and vitamins that could be responsible for lowering the serum tag concentrations in the bph rats. the low serum tag concentrations in the caasbetreated bph rats indicated improved carbohydrate uroko et al. – protective effects of anthocleista vogelii and alstonia boonei 75 metabolism and efficient transport of tag to the liver for catabolism which might have contributed to reduced risk of stroke, hypertension and heart diseases which are associated with abnormal serum tag levels in line with güven and gökhan (2022). the hdl cholesterol is a very important type of cholesterol that maintains a healthy balance of lipid profile via its role in moving ldl-cholesterol, potentially unhealthy cholesterol, to the liver for catabolism and elimination from blood circulation. aside from the clearance of ldl-cholesterol from clogging the arterial walls, many researchers have suggested that hdl possesses antioxidant, antiinflammation and anticoagulant properties. the significantly decreased serum hdl-cholesterol concentration in the bph control showed the adverse effects of bph on the serum hdl-cholesterol concentration. the reduction in the hdl level in the bph control suggests that alterations in lipid profile, especially the serum hdl-cholesterol concentration, are associated with the bph pathogenesis, and adequate regulation or restoration of altered lipid profile could play an important role in alleviating the adverse effects of bph on general body functions. the reduced hdlcholesterol level in the bph control is in line with the findings of gacci et al. (2017) that lower hdlcholesterol level is associated with prostate enlargement and that hyperlipidaemia could accelerate bph progression by promoting prostate cell inflammation. the low serum hdl-cholesterol concentration in the bph control has great health consequences as there could be a decline in the transport of ldl-cholesterol and other cholesterol molecules to the liver for metabolism and clearance, which will increase the risk of narrowing the arterial walls and heart diseases as earlier reported by uroko et al. (2021). however, the significantly increased serum hdl-cholesterol concentrations in the bph rats treated with finasteride and caasbe, respectively, compared to the bph control showed the anti-dyslipidaemia effects of the caasbe against bph-associated alterations in the lipid profile. the high serum hdl-cholesterol level in the caasbe-treated bph rats is indicative that there would be effective extraction of ldl-cholesterol from the blood vessels to the liver for breakdown, thereby freeing the arteries from the deposition of ldl-cholesterol, narrowing, increased blood pressure and complications that might result in heart diseases. thus, treating bph rats with caasbe improves serum hdl-cholesterol levels and prevents adverse health effects associated with its low blood level. the increased hdl in the caasbe-treated bph rats agrees with gacci et al. (2017). cholesterol is an important lipid profile component in synthesising bile acids and many steroid hormones, including gonadal and adrenal steroid hormones, and maintaining membrane structure and integrity. still, its high serum concentration could accelerate the risk of developing cardiovascular diseases. the substantially increased serum cholesterol concentration in the bph control compared to the normal control demonstrated that bph progression is linked to the abnormally elevated serum cholesterol concentration. the increased serum cholesterol level in the bph control aligns with previous findings that increased serum cholesterol level, including in the prostate tissues, is associated with the pathogenesis of benign and malignant prostatic diseases (rohrmann et al. 2005; yat-ching et al. 2011). increased circulating cholesterol could induce signalling pathways that enhance bph pathogenesis. the elevated serum cholesterol concentration in the bph control suggests that there is an increased likelihood of the high serum cholesterol being deposited on the blood vessel walls, thereby forming arterial clots and hindering blood flow which might increase the blood pressure and heart diseases that would adversely affect the survival of the bph control rats. in contrast, the substantially reduced serum cholesterol concentration of the bph rats treated with 200 and 400 mg/kg caasbe showed the antihypercholesterolaemia effects of the phytoconstituents present in the caasbe. the anti-hypercholesterolaemia effects demonstrated by caasbe in this study were comparable to the finasteride effect in the standard control, suggesting that caasbe possesses high therapeutic potential against bph and its associated health complications. the drastically reduced cholesterol coupled with the low serum ldl-cholesterol level in the caasbe-treated bph rats showed a low risk of atherosclerosis and cardiovascular diseases in the bph rats treated with caasbe. the substantial reduction in the serum total cholesterol level in the bph rats treated with caasbe aligns with previous findings that inhibiting cholesterol synthesis by therapeutic agents could reduce bph progression and that hypercholesterolaemia aggravates bph progression (rohrmann et al. 2005). the ldl-cholesterol is one of the components of the lipid profile parameters commonly evaluated to monitor one’s lipid profile status. the elevation in the serum ldl-cholesterol indicates impending health consequences, mostly when there is a substantially low serum hdl-cholesterol level (kang et al. 2015). ldlcholesterol is the main lipid deposited on the arteries, which clogs and narrows the arterial area, thereby increasing the blood flow pressure that has a farreaching implication on heart functions and general wellbeing. the significantly elevated serum ldlcholesterol in the bph control showed that bph caused an imbalance in the lipid profile composition of the rats, exposing them to the increased health risk of accumulation of ldl-cholesterol in the arteries, which agrees with the findings of uroko et al. (2021). the decline in the serum hdl-cholesterol needed to remove and transport ldl-cholesterol from the blood vessels to the liver could be responsible for the elevated serum ldl-cholesterol in the bph control. the high serum 76 biology, medicine, & natural product chemistry 12 (1), 2023: 69-78 ldl-cholesterol level in the bph control agrees with güven and gökhan (2022) findings that increased ldlcholesterol is linked with bph progression. conversely, the substantial decrease in the serum ldl-cholesterol concentrations in the finasteride and caasbe treated bph rats, respectively, relative to the bph control, showed each therapeutic agent's ability to restore the altered ldl-cholesterol to the normal level. the power of caasbe to lower ldl-cholesterol indicates that it could promote normal biochemical and physiological functions aside from restoring an enlarged prostate to normal size, as uroko et al. (2021) and güven and gökhan (2022). this finding suggests that caasbe could play a key role in preventing and managing high blood ldl-cholesterol, arteriosclerosis and cardiovascular disorders. evaluating the serum urea level is a very reliable indirect method of ascertaining the renal function status and serves as a guide for taking necessary action to restore normal renal functions. urea is an excretable end product protein and nitrogenous base metabolism that is filtered by the glomerular of the kidney and excreted with excess fluid in the body. the high serum urea concentration points to renal dysfunction. the elevated serum urea concentration in the bph control relative to the normal control suggested an impaired renal glomerular filtration and excretion of urea from the circulating blood. the substantially elevated serum urea concentration in the bph control finding agrees with the previous findings that increased serum urea levels in bph rats are associated with impaired renal functions (weinstein et al., 2009; uroko et al., 2021). the persistent rise in the serum urea level could lead to various adverse health complications, including hypertension and cardiac dysfunctions. this finding suggests impaired glomerular filtration rate and increased serum urea level may be associated with bph progression if not effectively managed. in contrast, the significantly reduced serum urea level in the standard control and all the caasbe-treated bph rats showed that treatment with finasteride and caasbe alleviated the adverse effects of bph on renal functions, according to the findings of uroko et al. (2021). the ability of caasbe to drastically restore the elevated serum urea level in the bph rats similar to the normal control could be attributed to the therapeutic effects of the bioactive constituents in the combined extract, which is an indication that caasbe could be used to manage renal disorders. the serum creatinine level is the metabolic waste product of creatine catabolism that is regularly filtered from the blood by the renal glomerular and eliminated from the body via urine. the serum creatinine level, like serum urea level, reflects the glomerular filtration rate and the renal function status. the sensitivity of serum creatinine level as a measure of renal function status is very reliable when coupled with the serum urea level. the high serum creatinine level in the bph control relative to the normal control could be attributed to impaired creatinine filtration and clearance by the renal glomerular in line with weinstein et al. (2009). the high serum creatinine level in the bph control indicated impaired removal of excess wastes in the circulating blood via urinary excretion, which could adversely affect the regulation of biochemical and physiological activities in the body. contrarily, the significantly reduced serum creatinine level in the bph rats treated with caasbe suggests that the combined extract relief the adverse effects of bph induction by testosterone propionate injection and restored the ability of the renal glomerular to filter and remove excess creatinine from the blood in line with the findings of uroko et al. (2021). the very low serum creatinine level showed that caasbe could be used as a therapeutic agent against renal disorders, which agrees with the earlier reports by reshma et al. (2014). the normal renal histomorphology observed in the bph control, standard control, and bph rats treated with 200 and 400 mg/kg caasbe, respectively, compared to the renal histomorphology of the normal control, showed that it caused no renal injury in the rats. these suggest that bph interfered with renal functions and reduced the glomerular filtration rate and clearance of wastes out of the body. the absence of observable alterations in the renal histomorphology is in order because the testosterone propionate injection administered to induce bph is not nephrotoxic. the increased serum urea and creatinine concentrations indicated impaired renal functions due to stress associated with bph pathogenesis and complications rather than renal injury. the absence of histomorphological alterations of the kidney sections of the bph control and the bph rats treated with finasteride and caasbe are consistent with uroko et al. (2021). conclusions the findings of this study showed that treatment of bph rats with caasbe significantly restored the altered lipid profile to a normal level. the caasbe-treated bph rats had considerably reduced serum urea and creatinine concentrations compared to the bph control but no alterations in the renal histomorphology. the findings suggest that treatment with caasbe could prevent hyperlipidaemia and renal malfunctions in bph rats. acknowledgements: the authors wish to express profound gratitude to asogwa edith ogechi, egba simeon ikechukwu and okwor josephat ani for their assistance during the study. uroko et al. – protective effects of anthocleista vogelii and alstonia boonei 77 authors’ contributions: robert ikechukwu uroko, mercylyn ezinne uche, and paul chukwuemaka nwejeanyalowu designed the study; robert ikechukwu uroko, ikenna obiwuru, chinedu aguwamba, and chinomso friday aaron carried out the experimental analysis. robert ikechukwu uroko analysed the data statistically and interpreted the results while mercylyn ezinne uche, paul chukwuemaka nweje-anyalowu, ikenna obiwuru, and chinomso friday aaron discussed the results. robert ikechukwu uroko supervised the study and wrote the manuscript. all the authors read and approved the final manuscript for publication. competing interests: the authors have expressed no conflict of interest. funding: not applicable. references abbiw d. useful plants of ghana: west african uses of wild and cultivated plants. kew, london: intermediate technology publications, royal botanical garden; 1990. adaramoye, o.a., akintayo, o., achem, j. & fafunso, m.a. (2008). lipid-lowering effects of methanolic extract of vernonia amygdalina leaves in rats fed on high cholesterol diet. vascular health and risk management, 4(1): 235-241. https://doi.org/10.2147/vhrm.2008.04.01.235. akinmoladun, a.c., ibukun, e.o., afor, e., akinrinlola., b.l., onibon, t.r., akinboboye, a.o., obuotor, e.m., and farombi, e.f. (2007). chemical constituents and antioxidant activity of alstonia boonei. african journal of biotechnology, 6(10): 1197-1201 chukwu, c., uroko, r., egba, s., adamude, f. & asadu, c. (2020). effects of combined ethanol extract of anthocleista vogelii and alstonia boonei stem barks on liver function indices in benign prostatic hyperplasia induced rats. nigerian journal of pharmaceutical science, 6(2): 191-201. https://doi.org/10.4314/njpr.v16i2.10 dahle, s.e., chokkalingam, a.p., gao, y.t., deng, j., stanczyk, f.z. & hsing, a.w. 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(2011). the role of cholesterol in prostatic diseases. urological science, 22(3): 97-102. https://doi.org/10.1016/j.urols.2011.08.002 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 311-319 | doi: 10.14421/biomedich.2024.131.311-319 issn 2540-9328 (online) potential effects of myrmecodia pendants (ant nests) on healing bone injuries adilla syahsiyatun najah1,*, akram la kilo2, ahmad kadir3 1muhammadiyah university bandung, indonesia. 2,3gorontalo state university, indonesia. corresponding author* adillasyn@gmail.com abstract myrmecodia pendens is a medical plant that can have a healing effect on bone injuries. bone injuries can occur due to many things, including injuries due to infection, non-infection, degeneration and neoplasms. bones are the most important part of the body for the human body because the decreased bone function will affect human activities. therefore better bone healing is needed. this review article aims to study and analyze the role of myrmecodia pendens in the bone wound healing process. several studies show that myrmecodia pendens contain flavonoids, vitamins, terpenoid saponins, quones, and glycosides, as well as anti-inflammatory substances. these ingredients are markers in the healing process. myrmecodia pendens has been proven to speed up the healing process. the conclusion shows that myrmecodia pendens is a medicinal plant that can speed up the healing process of bone injuries. keywords: myrmecodia pendens; bone repair. introduction myrmecodia pendants, also known as "ant nests", are epiphytic plants that live on wood or tree bark. this plant has long been known in southeast asia for its various benefits, including bone health. scientific studies show that the consumption of myrmecodia pendants extract can help reduce the risk of osteoporosis, increase bone mineral density, and speed up the healing of bone fractures. these potential benefits make myrmecodia pendant an interesting bone health supplement to study further. ant nests (amyrmecodia pendans) are epiphytic plants from the hydnophytinae (rubiceae) tribe, which are known as natural medicines and have antioxidant potential. it is believed to be able to heal wounds from several diseases, including bone wounds. recent research shows that myrmecodia pendants contain compounds that have the potential to have a positive effect on bone health. these compounds, such as polyphenols and flavonoids, are thought to help increase bone formation and strengthen bone structure. there are two types of species; myrmecoda pendens and myrmecodia tuberosa pl. myrmecodia pendants contain bioactive components such as phenolics which include flavonoids, phenylpropanoids, tannins and tocopherols. these antioxidants are contained in almost all parts of this plant, from the stem, twigs to the skin. the active compounds contained in ant nests are flavonoids which have antibacterial, anti-inflammatory and antioxidant properties (3). literature about this plant still needs to be improved, especially the features of the healing process. therefore, the author wants to discuss the ability of this medicinal plant in the bone healing process with the aim of providing literature for researchers and readers to know about myrmecodia pendans in the process of healing bone injuries. methods and materials this article was written using a literature review method. we will discuss several studies related to bone wound healing. discussion ant nests are part of the family rubiaceae, myrmecodia, anthorrhiza, hydnophytum, myrmephytum, and squamellaria (riyanti et al., 2019a) there are five genera belonging to the rubiaceae family which have tubers and are home to ants (achmad et al., 2019a) (widyawati, pase, et al., 2020a). the myrmecodia genus is the second most abundant genus in the world, after the hydnophytum genera (8d23_2850_lilies_anggarwati_astuti_indonesia, nd-a). it is estimated that the number of species in the manuscript received: 01 june, 2024. revision accepted: 16 july, 2024. published: 17 september, 2024. https://doi.org/10.14421/biomedich.2024.131.311-319 mailto:adillasyn@gmail.com 312 biology, medicine, & natural product chemistry 13 (1), 2024: 311-319 myrmecodia genera is 26 species (widyawati, pase, et al., 2020b)(soviati et al., 2020a)(sudiono et al., 2020a). ant nests usually stick to their host plants to survive (sudarmin et al., 2022a). the growth of ant nests is very dependent on the host plant (33d23_2910_lusi_epsilawati_indonesia, nd-a). the smoother the plant stem, the more difficult it is to find the plant. there will be no ant nests found on slippery stem plants (moatar et al., 2020a). on the other hand, you will find ant nests if the plant stems are rough (moatar et al., 2020a) (widyawati, aron pase, et al., 2020a) in ethnobotanical practice, ant nests have been used for spiritual and ritual purposes(binartha et al., 2020a). some indigenous peoples believe that ant nests have magical or spiritual powers that can be used in healing or protective ceremonies. anthills are often associated with courage, resilience and cooperation in these cultures (dirgantara et al., 2022a; sudiono et al., 2020b; zeth et al., 2021a). myrmecodia pendant can be found mainly in tropical and subtropical areas in southeast asia and australia such as in indonesia (including papua, kalimantan, java, sulawesi, sumatra)(317-1336-2-pb, nd-a) papua new guinea (gde agung krishna pemayun et al., 2021), philippines (roslizawaty et al., 2023), cambodia(young et al., 2020), malaysia (including peninsular malaysia, sabah and sarawak on the island of borneo), australia (including queensland and the northern territory) and the solomon islands (gde agung krishna pemayun et al., 2021). ethnopharmacologically, ant nests have been used as medicine by the rural communities of papua, including as a cure for inflammation, strengthening the body's immunity. (putra et al., 2020a), and overcome muscle pain(plant et al., 2021). local people use tuber powder (hypocotyl)(daulay et al., 2022a) (briliannita et al., 2020)ant nests as a brewed drink like tea [7]. one of its main properties is to help treat various types of tumours and cancer (imaniar et al., nd-a)such as brain cancer, breast cancer, nose cancer (moatar et al., 2020a), liver cancer, lung cancer, colon cancer, uterine cancer(moatar et al., 2020b), skin cancer, prostate cancer (elisabeta moatar et al., 2021a, 2021b), and leukaemia (ayuningtyas et al., 2021a)(elisabeta moatar et al., 2021a, 2021b; moatar et al., 2020b, 2020a). myrmecodia pendant interacts with ants, where the ants make tunnels in the floor of the tubers as nests and they create colonies (suprijono & hesti wulan, 2022a). in the end, a natural chemical reaction occurs between the compounds released by the ants and the substances contained in the plant itself(daulay et al., 2022b; rabil et al., 2021a). the content of this chemical compound comes from the flavonoid group (pongoh et al., 2022a)(apriyanti et al., 2020a) and tannins (binartha et al., 2020b). flavonoids act as antibiotics and antivirals and are also used to treat and prevent several diseases, such as asthma and cataracts(rabil et al., 2021b), diabetes, rheumatism, migraine(kuswandani et al., 2021a), haemorrhoids, periodontitis and cancer (suprijono & hesti wulan, 2022b). myrmecodia pendans also contains antioxidant compounds (bago, nd-a), vitamins, minerals and formic acid (widyawati, aron pase, et al., 2020b). ant antioxidants play a role in colony formation (manurung et al., 2023a)and protect ant eggs from microorganisms figure 1. myrmecodia pendant (ant nest)(widyawati, aron pase, et al., 2020b). the bone healing process for example a fracture, in principle results in the formation of new bone in a short time with minimal complications(mohd zuhri et al., 2020). the recorded healing rate is delayed in approximately 5-10% of cases. complications causing delayed healing were reported in approximately 18.5% of cases(317-1336-2-pb, nd-b). the presence of biological stimuli can also influence the acceleration of bone wound healing (tarman et al., nd. biological stimulation can significantly accelerate wound healing by stimulating activators and accelerating cell formation (5073-article text-15632-1-10-20211116, nd-a; epsilawati et al., 2019). the healing process for wounds in bones is the same as the healing process for other tissues.(5073article text-15632-1-10-20211116, nd-b) the healing process consists of 3 stages. the first phase is the inflammatory phase, this phase begins when an injury occurs and damage to the blood vessels occurs, thus inducing the formation of a hematoma and releasing inflammatory mediators. (simaremare et al., 2021a). the result of this phase is bone callus formation. this phase lasts 5-7 days. the next stage is the reparative stage. this stage lasts 4-40 days after the first trauma (pongoh et al., 2022b). in this phase, the callus develops, and its growth reaches 40% of the healing process (merdana et al., 2021a). the formed hematoma and callus change into a mature callus structure caused by the growth of mediators (kuswandani et al., 2021b), protein and calcium so that the callus can be seen najah et al. – potential effects of myrmecodia pendants (ant nests) … 313 radiographically(azzahra attamimi & indah permata yuda, 2022a; rumpun et al., 2021a)the final stage is the remodelling stage. this phase covers approximately 70% of healing time, occurring within 1-2 years(manurung et al., 2023b). the characteristic of this phase is that there is a continuous process of bone formation and resorption. the result is the formation of new tissue(khairiah et al., 2019; rizqiana et al., 2023). figures 2. (a) shows the healing process in the bone from the inflammatory phase where new blood vessels, hematoma and smooth callus form. (b) reparative phase where growth and development of calcium-rich reparative calluses occurs. (c) lastly is the resorption phase where the bone matrix is formed, where processes in the form of resorption and apposition occur(mohd zuhri et al., 2020) bone is an active tissue that will undergo continuous remodeling physiologically(sudarmin et al., 2022b). this remodelling process consists of two aspects, namely the formation of osteoblasts and bone resorption by osteoclast activity (8d23_2850_lilies_anggarwati_astuti_indonesia, nd-b). this renewal process lasts throughout life, allowing for the formation of new bone during calcium homeostasis (apriliani et al., 2020a)still sufficient for this activity. this activity is achieved and regulated by the multiple actions of systemic hormones and local mediators (alawiyah al attas et al., 2023a; simaremare et al., 2021b). figures 3. biochemical processes of bone remodeling. describes the 120 day healing process where several mediators are actively involved in the process(5073-article text-15632-1-10-20211116, nd-c). figures 4. the process of bone remodeling is described as a continuous process, where bone formation and resorption processes continue to occur(adhelina et al., 2017a) the bone remodelling cycle begins with the recruitment of osteoclast precursor cells, which will then turn into osteoclasts. adult osteoclasts will synthesize proteolytic enzymes, which are responsible for digesting the collagen matrix (martati & ciptadi, 2020a). this bone resorption is the first stage of the remodeling cycle. osteocytes to see the bone remodelling process(rachmi et al., 2021a), usually by checking for signs of bone remodelling. examples of biochemical markers of bone remodelling, such as in the bone resorption process, are hydroxyproline or various collagen cross-links(merdana et al., 2021b)and telopeptides, while markers of bone formation are propeptides of type i collagen (bago, ndb), osteocalcin (oc) and alkaline phosphatase (ap)(sigit, nd). this phase lasts a very long time and is under the control of the apoptotic effect carried out by osteoclasts(priyanti et al., 2023; ramadhan et al., 2018). the next phase of the remodelling cycle is where preosteoblasts from mesenchymal stem cells in the bone marrow become mature osteoblasts (putra et al., 2020b),which then synthesizes bone matrix, especially type i collagen(zeth et al., 2021b)and regulates the mineralization of newly formed bone(rachmi et al., 2021b).some mature osteoblasts mineralize and turn into biomarkers of bone formation and resorption, reflecting the overall activity of osteoblasts and osteoclasts in bone. (adhelina et al., 2017b). these markers can also be used as markers of growth and healing (ant nest nest extract nanoparticles preparation, nd). 314 biology, medicine, & natural product chemistry 13 (1), 2024: 311-319 table 1. biochemical markers for bone formation (mohd zuhri et al., 2020)(apriliani et al., 2020b)(dirgantara et al., 2022b). markers home network sample analysis analysis method total alkaline phosphatase (alp); specific for bone formation only in patients who do not have liver or bile duct disease (primasari et al., 2112). bones, liver serum colometry bone alkaline phosphatase (b-alp); ct-specific osteoblast production; some procedures show cross-reactivity with hepatic alp isoen-enzyme bone serum colometry, electrophoresis, precipitation, irma, eia osteocalcin (oc, bgp); certain osteoblast products; there are several reactive forms in the blood; some can nastati during bone resorption bones, platelets serum ria, elisa, irma, eclia c-terminal propeptide of type i procollagen (picp); specific proliferating osteoblast and fibroblast products (azzahra attamimi & indah permata yuda, 2022b). bones, skin, soft tissue serum ria, elisa n-terminal propeptide of type i procollagen (pinp); specific proliferating osteoblast and fibroblast products; some are included in the framework matrix bones, skin serum ria, elisa irma-immunoradiometric assay: eia-enzyme immunoassay: ria-radio immuno assay: elisa-enzyme-linked immunosorbent assay: eclia-electrochemiluminiscence immunoassay(5073-article text-15632-1-10-20211116, nd-a) table 2. biochemical markers for bone formation(epsilawati et al., 2019)(hidajat et al., 2018)(33-d23_2910_lusi_epsilawati_indonesia, nd-b). markers home network sample analysis analysis method hydroxyproline, total and dialyzable (oh-pro, ohp); specific for all fibrillar collagen and some collagen proteins, including ciq and elastin; present in newly synthesized and mature collagen bones, skin, cartilage, soft tissue urine colometry, hplc pyridinoline (pyd, pyr); high concentration of cartilage and bone collagen: absent in skin; only found in mature collagen bones, tendons, cartilage urine hplc, elisa deoxypyrindoline (dpd, d-pyr); high concentrations are only found in bone collagen: and found in cartilage or skin; only found in mature collagen bone, dentin urine hplc, elisa cross-linked c-terminal telopeptide of type i collagen (ictp); high proportion of bone collagen in type i collagen; some may come from newly synthesized collagen bones, skin serum ria cross-linked c-terminal telopeptide of type i collagen (fragments alpha-ctx, beta-ctx); in type i collagen; possibly a high proportion of bone collagen all tissues containing type i collagen urine, serum elisa, ria, eclia cross-linked n-terminal telopeptide of type i collagen (ntx fragments); in type i collagen; most of the bones all tissues containing type i collagen urine (alpha/beta), serum (beta) elisa, ria, icma hydroxylysine-glycosides (hyl-glyc); collagen and collagen protein; glucogalactosyl-hydroxylysine is highly abundant in soft tissue collagen and ctq; galactosyl-ohlys is highly represented in bone collagen bone, skin, soft tissue, serum complement urine hplc, elisa bone sialoprotein (bsp); synthesized by active osteoblasts and located in the extracellular bone matrix; appears to express osteoclast activity bone, dentin, hypertrophic cartilage serum ria, elisa tartaric-resistant acid phosphatase (tr-acp); osteoclasts, platelets, erythrocytes bones, blood plasma/serum colometry, ria, elisa free gamma carboxygastamine acid (gla); produced from bone proteins (e.g. osteocalcin, matrix protein gla) and from coagulation factors blood, bones serum/urine hplc hplc-high performance liquid chromatography; elisa enzyme-linked immunosorbent assay; ria radio immuno assay, ecliaelectrochemiluminiscence immunoassay; icma immunochemiluminometric assay(hidayat et al., 2020) research regarding the content of myrmecodia pendans has been studied for several years, especially in relation to health or the prevention and treatment of certain diseases (soviati et al., 2020b). however, research related to bone healing is still rare. to find out whether myrmecodia pendans has an effect, researchers have studied and studied the basic addition and decomposition of certain biochemicals (alawiyah al attas et al., 2023b). suharyanto and bambang (2019) conducted research on experimental animals by adding liquid extract of myrmecodia pendans which was given to several experimental mice. some mice were given additional dmba, which is believed to cause lung cancer. the results of the study showed that mice that were given najah et al. – potential effects of myrmecodia pendants (ant nests) … 315 additional myrmecodia pendans infusion after being suspected of suffering from lung cancer actually showed recovery with increased appetite.(diantoro et al., 2022). this proves that sick mice can be cured. however, the results of this study cannot confirm that the infusion fluid containing myrmecodia pendans has a healing effect because no biochemical markers were examined. the results were only seen based on changes in behaviour.(ayuningtyas et al., 2021b). similar research was also carried out by nurul et al. (2020), who researched human cancer cells where cancer cells were given active ethanol extract obtained from myrmecodia pendants (suprijono et al., nd). the results obtained show that the ethanol compound from myrmecodia pendans is able to reduce many cancer cells in humans (martati & ciptadi, 2020b). in this study, direct examination was carried out on humans, the results of which were based on histopathological examination. in contrast to the first study, the results obtained were more realistic (suprijono et al., nd). research related to testing the isolation of active myrmecodia pendants compounds has been carried out by several researchers, including: hertiani et al (2023), who carried out myrmecodia pendants phytochemical tests. in this study, an immune stimulant test was carried out, which examined the proliferation of lymphocytes and phagocytosis of macrophages given the ethanol extract of n-hexane fraction, ethyl acetate and water with concentrations of 10, 20, 50 and 100 ÿg/ml. this research provides results where the effect of macrophage phagocytosis will be higher if 50 ÿg/ml of myrmecodia pendens extract is added.(imaniar et al., nd-b; suryana et al., 2024) (scientific journal et al., 2024) similar research was also conducted by muslichah (2022). this study shows the optimal anti-inflammatory activity of 70% ethanol extract, n-hexane fractionation, the ethyl acetate fraction, compared with 70% ethanol fraction of myrmecodia pendens (suprijono & hesti wulan, 2022c).in this study, the results showed that the anti-inflammatory results in the form of a total 70% ethanol extract of myrmecodia pendens were better and optimal when extracted in fractionated form. (rabil et al., 2021c). these two studies prove that administering or adding myrmecodia pendens extract accelerates solid healing (achmad et al., 2019b). this is evidenced by increased macrophage activity and anti-inflammatory effects. soeksmanto a. subroto m. wijaya et al. (2019) also examined myrmecodia pendens phytochemical tests carried out on the water fraction, n-hexane fraction and ethyl acetate fraction. the fraction carried contains antibacterial effects against streptococcus viridans (apriyanti et al., 2020b). similar research was also carried out by fatriadi (2020), who showed that phytochemical tests on myrmecodia pendens tubers were carried out using fractions: water fraction, n-hexane fraction and ethyl acetate fraction (anggarwati astuti et al. nd). the results of research from these two researchers prove that the phenolic, tannin, flavonoid, and terpenoid content actually has an antibacterial effect.(rumpun et al., 2021b). these studies prove that myrmecodia pendens can speed up the healing process by preventing the invasion of microorganisms in wounds (anggarwati astuti et al., nd). research that focuses on the healing process of new bone was carried out by ismardianita et al. (2021). this research was conducted by examining the effect of myrmecodia pendens extract on the healing of tooth sockets after extraction by looking at changes in the expression of transforming growth factor-ÿ, insulinlike growth factors (igfs) and changes in osteoblast and osteoclast levels. (riyanti et al., 2019b). based on the research results, data was obtained that myrmecodia pendens significantly increased the levels of osteoblasts, tgf-factor-ÿ, growth factor and reduced the levels of osteoclasts. (education & counselling, n.d.).although there has not been much research focusing on bone healing, myrmecodia pendens counts. it is believed to heal bone injuries (isolation and molecular identification of endophyte bacteria of ants' nest plant (myrmecodia pendans), nd; yulian & ismail, nd) conclusion the conclusion is that the ant nest plant (myrmecodia pendens) contains phenolics, tannins, flavonoids, terpenoids and can actually act as an anti-inflammatory, and antibacterial agent. it can also has the ability to increase levels of biochemical markers in the healing process, so that myrmecodia pendens can be used to accelerate wound healing, especially in bones, improving people's quality of life. conflict of interest: there are no conflicts of interest. references 8-d23_2850_lilies_anggarwati_astuti_indonesia. (nd-a). 33-d23_2910_lusi_epsilawati_indonesia. (nd-a). 317-1336-2-pb. (nd-a). 5073-article text-15632-1-10-20211116. 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(2021b). the effect of ant plants on the increase of cd4 count in plhiv in the papua province. open access macedonian journal of medical sciences, 9, 1231–1239. https://doi.org/10.3889/oamjms.2021.7782 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1347-1353 | doi: 10.14421/biomedich.2025.142.1347-1353 issn 2540-9328 (online) phagochytosis activity test of red ginger and angkak combination extract by carbon clearance method fajar nugraha*, halim, hariyanto ih, andhi fahrurroji, desy siska anastasia pharmacy study program, faculty of medicine, universitas tanjungpura, jl. prof. dr. h. hadari nawawi, pontianak, indonesia. corresponding author* fajarnugraha@pharm.untan.ac.id manuscript received: 11 october, 2025. revision accepted: 29 november, 2025. published: 14 december, 2025. abstract red ginger (zingiber officinale roscoe var. rubrum) and angkak (oryza sativa) are used in traditional chinese medicine to improve body health. both are known to have immunostimulatory activity that increases phagocytic activity of macrophage. this study aims to determine the phagocytic activity of the combination extract of red ginger and angkak using carbon clearance method. combination of red ginger and angkak (7:3) was kinetically macerated in ethanol 96%. twenty white mice were divided into 5 groups, such as control group (cmc-na 0.5%), positive control (levamisole 2.5 mg/kgbb), and three experiment group that was given red ginger extract (105 mg/kgbb), angkak extract (105 mg/kgbb), and combination extract of red ginger-angkak (150 mg/kgbb). all treatments were given orally for sevendays, consecutively. the phagocytosis index (if) value was determined on day 8 by injecting carbon intravenously and immunostimulant activity was shown if if>1. the highest if value of three extracts was shown by combination extract (1.2489±0.0261) compared to red ginger and angkak extract, mentioned 1.2030±0.0448 and 1.0537±0.0254, respectively. moreover, the if values of combination extract and red ginger were significantly (p<0.05) different compared to the control group of cmc-na (if=1). however, it is significantly (p>0.05) lower compared to the levamisole group (1.5187±0.0534). it could be concluded that the combined extract of red ginger-angkak showed the greatest phagocytic activity against male white mice compared to extract of red ginger and angkak itself. keywords: immunomodulator; immunostimulant; phagocytosis index; oryza sativa; zingiber officinale roscoe var. rubrum. introduction the immune system is a mechanism used by the body as a protector against the threat of harm that can be caused by various subjects contained in the environment that enter the body. when a foreign object enters the body, the immune system will carry out various processes to destroy the foreign object. there are two kinds of immune systems, namely the innate immune system (non-specific immune system) and the adaptive immune system (specific immune system) (rosales et al., 2016). the immune system can be affected by a group of compounds called immunomodulators. immunomodulators are compounds that can help improvement of the work of the immune system. based on activity, immunomodulators consist of immunostimulators, immunoregulators, and immunosuppressors (masniah et al. 2021). immunostimulants are substances that can correct imbalances in the immune system by increasing immunity (baratawidjaja & rengganis, 2018). immunostimulants can be made from herbal or synthetic ingredients. however, the use of synthetic immunostimulants can cause adverse side effects (novitasari et al., 2017). in addition, from an economic point of view, the use of synthetic drugs is much more expensive because they are usually used over a long period of time (long-term) (ardhianta et al., 2017). so the development of herbal products as immunomodulators is growing rapidly. plants that have provided scientific evidence of immunostimulatory activity are the zingiberaceae family. red ginger (zingiber officinale var. rubrum) has immunostimulant activity that can increase macrophage cell phagocytosis. in addition, the phenolic content in red ginger rhizome has a role as an immunomodulator. previous research showed that red ginger extract at a dose of 100mg/kgbb provided immunostimulant activity by stimulating phagocytosis activity (luhurningtyas et al., 2021). in addition to red ginger, herbal products known to have immunostimulant activity are angkak. angkak is a fermented food from china made by cultivating the monascus purpureus mold on rice (oryza sativa). various studies on angkak have been conducted and the results show that angkak has pharmacological activity. the content of lovastatin in angkak can stimulate the immune system. angkak is also known to https://doi.org/10.14421/biomedich.2025.142.1347-1353 1348 biology, medicine, & natural product chemistry 14 (2), 2025: 1347-1353 affect immune cell regulation and gene expression (pebriani & melinda., 2016). the activity of a compound in stimulating the immune system can be seen from the ability to increase the phagocytic cell phagocytosis ability of the body. phagocytosis is the process of particles being swallowed by cells. macrophages and polymorphonuclear leukocytes are the most important phagocytic cells. the majority of foreign bodies that enter the tissue are removed through the mechanism of phagocytosis (handayani et al., 2018). testing the ability of phagocytosis that can be used to determine immunomodulatory activity is using a carbon clearance test. this test is conducted according to the phagocytosis ability of body cells in eliminating pathogens that enter the body (ahmadniaye et al., 2020). based on information about the immunostimulant activity of red ginger and angkak, researchers are interested in knowing the immunostimulant activity of the combination of the two ingredients. researchers used a group of mice test animals that were given a combination of red ginger and angkak extracts with the carbon filtration method. the combined extract is expected to provide immunostimulant activity by stimulating the phagocytic activity of mice immune cells. materials and methods materials the materials needed include red ginger rhizome (zingiber officinale var. rubrum), angkak (oryza sativa), 8-12 weeks old male mice (mus musculus) weighing 2030 grams, 70% ethanol, 96% ethanol, nacl p.a (ns), 1% acetic acid (sigma-aldrich), chinese ink (yamura), na cmc (sigma®), levamisole and aqua pro injection. procedures sample preparation and extraction fresh red ginger (zingiber officinale roscoe) herb obtained from the village of punggur, kubu raya regency, west borneo province. the plant identification was confirmed by biology laboratory, faculty of math and science, tanjungpura university. the cleaned herb are dried and blended until they turn into powder. angkak was obtained from baba kuya shop, bandung, west java. an amount of 35 g the dried of ginger rhizome powder and 15 g of angkak powder were exctracted with kinetic maceration method using 1 l ethanol 96%. using hotplat at 50°c and 400 rpm. macerate was filtered in vaccum buchner and evaporated at ± 50 ° c in a rotary vacuum evaporator and thickened by heating in a dehydrator at ± 40 °c (ahmadniaye et al., 2020; gonzales et al., 2016). animal all procedures were evaluated by ethical clearance committee of faculty of medicine university of tanjungpura. the test animals used in the study were male mice (mus musculus). mice amounted to 20 mice which were divided into 5 treatment groups with each group totaling 4 mice. this study used a test group that was given red ginger extract as much as 105 mg / kgbb, angkak extract as much as 105 mg / kgbb, and a combination extract of red ginger and angkak as much as 150 mg / kgbb. all treatments were given by peroral route for 7 days. grouping of test animals based on treatment can be seen in (table 1). before the experiment begins, the animals were acclimatized in the experimental room for 7-14 days. table 1. grouping of test animals. group amount treatment negative control 4 cmc-na 0,5% positive control 4 levamisole dose 2,5 mg/kgbb red ginger extract 4 red ginger extract 105 mg/kgbb angkak extract 4 angkak extract 105 mg/kgbb combination ekstrak 4 combination extract 150 mg/kgbb carbon colloidal suspension setup the manufacture of carbon suspensions is done in the following manner: suspension of 1.6 ml of the chinese ink in 8.4 ml of 0,5% b/v cmc-na in a physiological solution of nacl. (senja et al., 2014). carbon clearance test in this method used 4 mice each positive control group, negative control and treatment group. each group is gave an levamisole 2.5 mg/kg bw as a positive control group, na-cmc suspension as a negative control and red ginger extract (105 mg/kg bw), angkak extract (105 mg/kg bw), and extract combination (150 mg/kg bw) as a treatment group extract 1 time a day for 7 consecutive days. on the 8th day after sampling the suspension of the samples in each group, the tail ends were cuted. then the blood is taken 25 μl and added 4 ml of 1% acetic acid to fill red blood cells, the first blood used as a blanko (minute 0), then 0.1 ml carbon suspension is injected in i. v through the blood vessels on the tail, and in the 4, 8, 12, 16 and 20 minutes after the carbon injection is carried out blood sampling, then the blood is takens as much as 25 μl, each added 4 ml of 1% acetic acid to a line of red blood cells, and then measured its absorption nugraha et al. – red-ginger and angkak phagochytosis activity 1349 using uv-vis spectrophotometer at a wavelength of 639.0 nm. after 12 hours of blood was taken, then the liver and lymph recorded weighing. after taking the blood on the tail end of the mice is calculated a constant carbon elimination speed (k) and phagocytosis index (α) by using the formula (sebayang & hasibuan, 2021; rahman et al., 2016). the phagocytosis index is calculated by comparing the phagocytosis constant (k) of the sample with the phagocytosis constant of the negative control calculated using the following formula (rahman et al., 2016). data analysis data from observations of immunodulatory activity testing were statistically analyzed by the one way anova method followed by post hoc tukey hsd test using spss (statistical product and service solution) version 25. results and discussion results thin layer chromatography (tlc) mobile phase toluene : acetone (9:1) mobile phase ethyl acetate : aquadest : acetic acid (7:1:1) figure 1. chromatogram profile of red ginger and angkak combination extract (a) plate observed directly (b) plate observed under uv light 254 nm (c) plate observed under uv light 366 nm. phagocytosis constant rate figure 2. graphic of phagocytosis constant rate. 1350 biology, medicine, & natural product chemistry 14 (2), 2025: 1347-1353 table 2. phagocytosis index. group phagocytosis index (± sd) cmc-na 1 levamisole 2,5 mg/kgbb 1.51878 ± 0.053428* red ginger (105 mg/kgbb) 1.20308 ± 0.044886* angkak (105 mg/kgbb) 1.05378 ± 0.025442 combination (150 mg/kgbb) 1.24896 ± 0.026164* * , significant difference with negative control group (cmc-na) discussion combination extract using 35 grams of red ginger powder and 15 grams of angkak in 1 liter of 96% ethanol solvent. the extract obtained is a thick extract due to the starchy substance contained in the angkak, but the extract is also slightly oily due to the volatile oil contained in red ginger. the final extract was more concentrated and dark red in color due to a mixture of red pigments in angkak and brownish yellow color from a single red ginger. the extract had a spicy odor typical of ginger essential oil (roseno et al., 2019; tritanti & pranita, 2019). the yield value of the combination extract obtained was 12.165%. the extract is then stored in a tightly closed container and protected from light at room temperature, especially in the extract of angkak and combinations containing red pigments of angkak which are thermosensitive and photosensitive, because pigments can be degraded at high temperatures, and angkak pigments have a strong tendency to absorb visible light and radiant energy from lamps which causes a reduction in stability when exposed to light (hasim et al., 2018). the observation of the chromatogram profile in this study uses tlc because this method is suitable for less polar and non-polar compounds so that the compounds can be distributed between the silica gel stationary phase and the liquid mobile phase (putra et al., 2021). the eluent used to separate the compounds contained in red ginger contained in mixed extracts is toluene and acetone in a ratio of 9:1, where both are nonpolar eluents meanwhile, the eluent used to separate the compounds contained in angkak contained in mixed extracts is ethyl acetate, water, and methanol in a ratio of 7:1:1 which is polar coupled with acetic acid to prevent tailing so that it can provide sharper and separate stains that work by ionizing polar compounds to be separated, especially pigment compounds in angkak which are very sensitive to acidic ph. chromatogram stains using specific eluents of red ginger material are not visible by direct observation, so they require uv 254 nm, and uv 366. the results of the tlc using the specific eluent of red ginger showed that there were two stains, while the tlc using the specific eluent of angkak (figure 1.) showed two stains. overall at uv 254 nm, the plate will glow green with the stain will be black or dark, while at uv 366 nm, the plate will glow blue with the stain will be turquoise blue. the tlc test of the combined extracts of red ginger and angkak resulted in the finding of stain spacing based on observations under uv 254 nm and uv 366 nm. the stains obtained in the tlc test of the combined extracts of red ginger and angkak were 2 stains for tlc testing of toluene:acetone (9:1) mobile phase with rf results of 0.3 and 0.58 (figure 1.). the stains formed indicate that the combined extract of red ginger and angkak contains gingerol and shogaol compounds which are almost comparable to previous studies, namely rf 0.53 for 6gingerol and 0.36 for 6-shogaol. (foudah et al., 2020). these gingerol and shogaol compounds have been shown to increase immunostimulant activity with the mechanism of stimulating macrophages and lymphocytes (nantaporn & pharkphoom, 2022). while the tlc test of red ginger and angkak extracts using a special eluent of angkak, namely ethyl acetate: distilled water: acetic acid (7:1:1) showed that there were 2 spots with rf 0.43 and 0.96 (figure 1). these spots show that the combined extract of red ginger and angkak contains 2 pigments, namely red pigment (rf 0.43) and yellow pigment (rf 0.96). the red pigment in angkak consists of monascorubramine and rubropunctamin, while the yellow pigment consists of ankaflavin and monascin which are involved in the production of the compound lovastatin in angkak. lovastatin is believed to have immunostimulatory activity by stimulating the specific immune system. in addition, lovastatin has cholesterollowering activity. high cholesterol in body cells can disrupt macrophage cells and cause inflammation, so lovastatin indirectly keeps the immune system optimized. (nantaporn & pharkphoom, 2022; basuny & abdel, 2020). testing phagocytosis activity using experimental animals. the experimental animals used were male white mice (mus musculus l.) swiss webster strain aged 6-8 weeks as many as 20 heads. mice were chosen because they are easy to obtain, relatively cheap, easy to handle, and their physiological body is similar to humans. to reduce the deviation of the research results, mice with the same strain and sex, age and body weight were selected. the immune system is also influenced by estrogen and testoterone, so male mice were chosen because they have hormones that are more stable than female mice (triana et al., 2014). the experimental animals used have met ethical approval by the authorized ethics committee. before use, mice were acclimatized for 7 days. this aims to familiarize the mice with the experimental conditions and environment as well as control health and body weight and homogenize the diet (aldi et al., 2016). the test animals were grouped into five groups, each group consisting of four mice. each group was given a different treatment. the negative control group was given a 0.5% na cmc suspension, the positive control group was given levamisole 2.5 mg/kgbb, the test preparation group was given a single extract of red ginger at a dose of 105 mg/kgbb, a single extract of angkak at a dose of 105 mg/kgbb, and a combination extract at a dose of 150mg/kgbb. the extracts of red nugraha et al. – red-ginger and angkak phagochytosis activity 1351 ginger, angkak, and the combination did not dissolve perfectly in water so that the test preparations given to experimental animals were made in the form of suspensions using cmc na. cmc na 0.5% suspension solution is used as a suspending agent because it is inert, non-toxic, non-irritating, has high clarity, good resistance to microbes and produces a stable solution. levamisole is used as a positive control because it has immunostimulatory activity by increasing macrophage phagocytic activity, increasing nitric oxide, lymphocyte proliferation and stimulating antibody (igg) and tnf-α cytokine production (musdalipah et al., 2022). the dose of extract used is based on research on the phagocytic activity of red ginger with a dose of 100 mg/kgbb mice can increase phagocytic activity (luhurningtyas et al., 2021). the extract suspension was made based on the predetermined dose and given to the test animals once a day consecutively in order to give the extract a chance to increase the immune response (rowe et al., 2009) the carbon clearance method is used to measure the activity of phagocytic cells in killing pathogenic organisms that enter the body (eduardo et al., 2017). carbon used as a marker is administered intravenously. carbon clearance is seen at time 4, 8, 12, 16, and 20. carbon levels in the blood will decrease over time such as due to phagocytic events by leukocyte cells especially by monocytes, neutrophils, eosinophils, and macrophages (aldi et al., 2017). the use of carbon as a marker has the advantage that the particle size is smaller and more stable, so carbon does not cause blockage of blood vessels and lungs. carbon is also characterized as an antigen because of its isolation, which under normal circumstances is not found in the body. (aditya et al., 2017). carbon levels in the blood are also easier to measure using an analytical instrument, namely a uvvis spectrophotometer. the carbon used is chinese ink with the yamura brand on the market. carbon suspension was made using na cmc with a concentration of 0.5% (b/v) and added physiological saline. the use of physiological saline in making the suspension aims to make the condition of the carbon suspension preparation (chinese ink) the same as the condition of the test animal's body (ilasamola et al., 2018). the phagocytosis effect was analyzed using a uv-vis spectrophotometer. the running results showed that the maximum wavelength of the carbon standard was at a wavelength of 639 nm. the results of the extract activity test showed a decrease in the absorbance of carbon every minute in the blood of male white rats given the test preparation for seven consecutive days. the reduction of carbon levels in the blood of the test animals at every minute of testing indicates that the concentration of carbon in the blood of mice is getting lower over time (figure 2). this also shows an increase in the phagocytic activity of carbon in each extract group. based on the results of the study, there was a decrease in absorbance value in all groups given the extract compared to the negative control group. the decreasing absorbance value indicates that the concentration of carbon is decreasing in the blood of mice. carbon in the blood will stimulate the formation of the nonspecific immune system in the form of phagocytic cells. phagocyte cells that are activated due to nonspecific stimuli can quickly recognize the type of foreign antigen that enters the body and then destroy and remove the foreign antigen from the bloodstream (abebe et al., 2017). from the absorbance data, the phagocytosis constant value can be calculated. phagocytosis constant is one of the parameters that shows the speed of phagocytosis. the greater the speed of phagocytosis, the higher the phagocytic carbon clearance (shi et al., 2012). after obtaining the phagocytosis constant value, the phagocytosis index value can be obtained. the average value of the phagocytosis index is greater than 1 indicating that the test substance has immunomodulatory activity, namely as an immunostimulant. but if the average value of phagocytosis index is less than 1, the test substance has immunosuppressant activity (ali et al., 2022). from the phagocytosis index data (table 2.), it can be seen that the average phagocytosis index for red ginger extract, angkak extract and combination extract is greater than one (if>1). so it can be said that red ginger extract, angkak and the combination can increase phagocytosis activity and is immunostimulant. the results of the red ginger extract activity test are in accordance with previous research which shows that red ginger extract at a dose of 100 mg / kgbb has immunostimulant activity with a phagocytosis index value of more than 1 (luhurningtyas et al., 2021). the highest phagocytosis index value was shown by the group given the combination extract with a value of 1.24896. it can be concluded that the group with a combined extract dose of 150 mg / kgbb has the best phagocytosis ability among other groups. the test results were statistically processed using oneway anova. in this data processing using saphiro-wilk because the data used is less than 50. in normality testing, the sig value> 0.05 is obtained in the phagocytosis index data, this value indicates that the data variation is normally distributed. in homogeneity testing, a sig value> 0.05 was obtained, indicating homogeneous data variations. one-way anova test was used to compare the five preparation groups used on the phagocytosis index. the results of the one-way anova test showed a significance of 0.000 (p<0.05). this result indicates that there is a significant difference between the five groups significantly on the phagocytosis index. then continued with tukey test analysis to see the effect of each treatment group. the results of the tukey test analysis showed that the phagocytosis index of angkak extract did not have a significant difference with the group given negative control (cmc-na). meanwhile, red ginger extract, combination extract and positive control had a significant difference with the negative control (p<0.05). 1352 biology, medicine, & natural product chemistry 14 (2), 2025: 1347-1353 conclusions based on the results of the study, it can be concluded that the combined extract of red ginger-angkak showed the greatest phagocytic activity against male white mice compared to extract of red ginger and angkak itself. acknowledgements: we express our gratitude to the chief of the faculty of medicine university of tanjungpura especially pharmacy study program for their invaluable support in facilitating the progress of this research endeavour alongside the team that always has been supporting us morally and physically. competing interests: 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(2005). thin-layer chromatography rsc chromatography monographs. great britain: royal society of chemistry. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 399-405 | doi: 10.14421/biomedich.2023.121.399-405 issn 2540-9328 (online) development of an eco-shampoo formulation using local environmental plant extracts for healthy hair as an effort to increase the potential of environmental resources dwi atmanto*, neneng siti silfi ambarwati department of cosmetology, faculty of engineering, state university of jakarta, jakarta 13220, indonesia. corresponding author* dwiatmanto64@gmail.com abstract this research aims to develop an eco-friendly shampoo formulation (eco-shampoo) by adding local environmental plant extracts for hair health. this research method is a quasi-experimental development of existing shampoo formulations (base formulations) by adding local plant extracts such as aloe vera (aloe vera l.) and lemongrass oil (cymbopogon citratus) in three formulations. the resulting shampoo products were then carried out two evaluations, namely the stability test (organoleptic test), foam height observation, viscosity test, ph test, and preference test (hedonic test) by consumers to determine the feasibility and effectiveness of using shampoo. after a storage period of 2 days showed a stability test, slightly changed color, constant odor, and texture did not change and the ph value was between 5.5 6.5. the smear test on the back of the hand and the skin behind the ear did not show skin irritation, whereas the hedonic test, 10 consumers stated that the color was like, the smell was stated like, and the texture was stated quietly. aloe vera fraction and lemongrass oil can be formulated as a shampoo preparation to maintain hair fertility as well as an anti-dandruff shampoo. keywords: eco-shampoo; formulation development; health hair; local environment; plant extracts. introduction indonesia is a fertile country and has various types of plants. the soil is fertile and the rainfall is sufficient for various types of plants to grow and develop (arifin 2006). plants are biological natural resources that can be managed to become a source for the establishment of industries such as the pharmaceutical industry, the food industry, the metal industry, the animal feed industry, the plastic industry, and the cosmetics industry. several plants have been developed to treat hair problems, one of which is aloe vera and lemongrass leaves. empirically, aloe vera leaves are used by the community to nourish their hair by using the inner gel of the leaf flesh (ariyani et al. 2009). likewise, lemongrass leaves are taken through extracts used by people for anti-dandruff hair treatment. hair contains an important role for every human being. this is because hair can affect a person's appearance (filbert 2014). the number of hairs on the human head is about 100,000 strands (hotmauli 2010). even though hair has natural hair loss, for some people, hair loss is still a worrying thing. hair loss can occur due to several factors such as age, hormonal disorders, pregnancy, drug use, continuous sun exposure, and lifestyle (doughari 2006). to overcome hair problems, intensive care is needed, such as using shampoo, hair tonic, hair mask, hair oil, vitamins, and others. environmental factors that have been polluted in urban areas can disrupt hair health. air or water impurities that hit the hair of the head will cause dandruff. dandruff is a dry form of seborrheic capitis known as seborea sika (dry), which are dry, fragile, easily detached scales that stick to cover the epidermis of the scalp (arifin 2006). one of the causes of dandruff is fungus on the scalp, which is dirty due to sweat, sebum (oil) glands, and dust. the fungus that develops on the scalp is called pityrosporum ovale (ariyani et al. 2009). symptoms of dandruff mainly include itching, flaking, and redness of the scalp. this fungus is naturally found on the scalp and can attack humans of all ages. many anti-dandruff shampoos contain antifungal compounds such as sulfur, salicylic acid, selenium sulfide, and zinc pyrithione which have the effect of damaging the scalp and causing hair loss (trueb 2007). therefore, there need to be other alternatives, especially natural ingredients that can be used as anti-dandruff. lemongrass (cymbopogon citratus) is one of the essential oil-producing plants. in indonesia, this species is usually used as a mixture of herbs and spices because it has a distinctive aroma like lemon (boonme et al. 2011). lemongrass oil is one of the most important types manuscript received: 08 february, 2023. revision accepted: 28 may, 2023. published: 20 july, 2023. https://doi.org/10.14421/biomedich.2023.121.399-405 400 biology, medicine, & natural product chemistry 12 (1), 2023: 399-405 of essential oil. this essential oil is used to produce citral which is the main constituent of lemongrass oil. lemongrass oil is a pale yellow liquid that has a strong lemon smell due to its high levels of citral (65% to 85%) making it the most important ingredient in the pharmaceutical and cosmetic industries (saputro 2009). the main constituents of lemongrass essential oil are citral (3,7-dimethyl2,6octadienal), a mixture of geranial (trans-citral a), and neutral (cis-citral b) with small amounts of geranium, geranyl acetate, and monoterpene olefin ((pooja et al. 2009). among the plants that can be used as hair, growers are aloe vera. aloe vera is very effective for hair care because it has a composition similar to keratin, an important hair protein, and complex amino acids identical to hair follicles so that it can rejuvenate hair with the same nutrients, especially the content of the amino acid l-lysine which can help in growth hair (limbani et al. 2009). according to daisy (2011), the main elements of aloe vera liquid are aloin, emodin, resin, gum, and other elements such as essential oils. in terms of nutritional content, aloe vera leaf gel or mucus contains several minerals such as zn, k, fe and vitamins such as vitamins a, b1, b2, b12, c, e, inositol, folic acid, and choline (potruli et al. 2011). aloe vera is also a natural remedy that helps regulate the rich blood supply to the root hair follicles on the scalp, thereby helping to strengthen hair (pooja et al. 2009). treatment using traditional medicine today is very popular and is increasingly favored by the community. this is because affordable prices are easy to obtain and also have relatively few side effects (naitullah et al. 2004; pan et al. 2009). in addition, indonesia is also the second mega biodiversity country after brazil, where it is estimated that there are 30000 species of living plants in the indonesian archipelago and at least 9600 species are known to have medicinal properties (robinson 1995). the cosmetic industry in indonesia developed around the 1950s. previously, traditional cosmetics had developed among kings in java (mahataranti et al. 2012). the indonesian people at that time developed a complex of local plant ingredients that were used as powder, shampoo, and herbal medicine. the use of traditional cosmetics at that time was still limited among the royal family alone. along with the development of chemistry, pharmaceutical science, and medical science, cosmetics and industrial sciences have also developed (mitsui 1997). now the cosmetics industry is growing rapidly because of the demands of modern society. the cosmetic industry is more developed using chemicals such as paraffin, surfactants, dyes, and preservatives. as public awareness of environmental issues has increased, such as green products, environmentally friendly products, and herbal products, the cosmetics industry has begun to compete with industrial expansion related to environmentally friendly cosmetic preparations (deeksha et al. 2014). however, not all the basic ingredients in the manufacture of the cosmetics industry have been replaced by natural ingredients, such as foam, thickeners, and preservatives. more active ingredients can be substituted by natural ingredients such as protein, dyes, and other nutrients (aghel et al. 2007). many cosmetic companies in indonesia produce shampoo, such as the wahdah industry. therefore, the formulation of the research problem arises: (1) can the manufacture of shampoo be developed with local plant materials such as aloe vera and lemongrass oil? (2) how to evaluate shampoo preparations made from aloe vera and lemongrass oil? the objectives of this study were (1) to determine the formulation of shampoo that was given a mixture of aloe vera and lemongrass oil, and (2) to determine the evaluation results of shampoo preparations mixed with local plants such as aloe vera and lemongrass oil. material and methods the tools used in this research are measuring cup (pyrex), beaker glass (pyrex), erlenmeyer (pyrex), test tube (pyrex), 65 mesh sieves, spatula, stirring rod, glass funnel (pyrex), separating funnel (pyrex). petri dishes (pyrex), analytical scales, blenders, hot plates, porcelain cups, shampoo containers, clamps and statives, pipettes, ph meters, filter paper, sterile gauze, and bunsen lamps. the materials used in this research are aloe vera, lemongrass oil, sodium lauryl sulfate, cocamide diethanolamine (cocamide dea), carboxymethyl cellulose (cmc), menthol, citric acid, methylparaben, aquadest, sodium chloride (nacl). aloe vera samples are purchased at supermarkets, as is slow mass oil. the initial stage is to collect fresh aloe vera leaves. then the fresh aloe vera leaves are washed under running water to remove the dirt. the contents of the aloe vera gel are taken, then crushed then filtered with apoach to get a smooth and non-clumping gel. this research method is an experiment, which is carried out in the chemistry laboratory of the faculty of engineering, state university of jakarta. the ingredients used are sodium lauryl sulfate, aloe vera, lemongrass oil, and aquadest. this research method is an experimental design with a one-shot study. where there is one group of independent variables is given treatment, then observations are made (sugiyono 2008). the object research is existing shampoo formulations (base formulations) by adding local plant extracts such as aloe vera (aloe vera) and lemongrass oil (cymbopogon citratus) in three formulations (see table 1) (al-badi and khan 2014). atmanto & ambarwati – development of an eco-shampoo 401 table 1. shampoo formulation for lemongrass and aloe vera oil fractions. ingredients shampoo formulations with various fractions of lemongrass and aloe vera oil f1 f2 f3 lemongrass oil 2% 7% 12% aloe vera 3% 3% 3% sodium lauric sulphate 10% 10% 10% cocamide dea 4% 4% 4% cmc 3% 3% 3% citric acid q.s. q.s. qs. menthol 0.5% 0.5% 0.5% methyl paraben 0.15% 0.15% 0.15% aquadest ad.30 ml ad.30 ml ad.30 ml the formulation results were tested for homogeneity, ph, viscosity, foam density and foam stability, wetting time, and cleaning power test. homogeneity testing using a glass object. anti-dandruff shampoo gel is made of a 10% solution and measured the ph using a ph meter. viscosity check is done using a viscometer. the foam power test was carried out by making a 10% shampoo gel solution, shaken 10 times, and recorded the volume of foam formed (munro 1954; fazlolahzadeh & masoudi 2015). the foam stability test is done by recording the volume of foam reduction that occurs in the foam power test in 1–4 minutes intervals (deeksha et al. 2014). the wetting time test was carried out by making a 1% shampoo gel solution then put it in a measuring cup and the canvas cloth was dropped into the solution. measure the time it takes for the canvas to sink. weigh all the ingredients used according to the formulation. cmc is developed with hot water in a mortar. diluted methylparaben with a few drops of ethanol until dissolved. part of the aquadest is heated on a hot plate at 60°c and added with sodium lauryl sulfate, stirring until homogeneous. cocamide dea was added to the mixture while continuing to stir until it was homogeneous (mitsui 1997). the general process of making shampoo with local plant mixtures is presented in figure 1. figure 1. diagram of the process of making shampoo with local plant mixtures. results and discussion the results of organoleptic observations of the antidandruff shampoo for aloe vera leaf and lemongrass oil fraction with various concentrations showed that the higher the concentration of aloe vera leaf aquadest fraction contained in the anti-dandruff shampoo preparation, the stronger the distinctive odor of aloe vera leaves and lemongrass oil so that it covers the odor of the menthol fragrance used, and the darker brown in the shampoo preparation because of the red brick color of the citronella leaf aquadest fraction (see table 2). table 2. result of organoleptic observations for mixed aloe vera shampoo and lemongrass oil. shampoo preparation formulations observation result shape colour smell f1 liquid, nothing settles light brown menthol, and lemongrass f2 liquid, nothing settles brown menthol, and lemongrass f3 liquid, nothing settles dark brown menthol, and lemongrass table 3. ph scale measurement. shampoo preparation formulations ph f1 6 f2 5.5 f3 5.5 the ph test aims to determine the safety of the preparation at the time of use. shampoo ph that is too acidic or too alkaline will irritate the scalp. the result of ph measurements is presented in table 3. the result of the foam height test is presented in table 4. the result of 402 biology, medicine, & natural product chemistry 12 (1), 2023: 399-405 the value of the water content test is presented in table 5. the process of making shampoo from aloe vera and lemongrass oil and shampoo preparation is presented in figure 2. table 4. foaming hight measurement. shampoo preparation formulations foaming hight f1 6.2 f2 7.3 f3 8.1 table 5. results of measurement of water level content for shampoo preparations. shampoo preparation formulations water level (%) f1 89.83 f2 86.71 f3 82.36 figure 2. the process of making shampoo from aloe vera and lemongrass oil and shampoo preparation. based on the results obtained, the distilled water fraction has the greatest fungal inhibiting activity so that anti-dandruff shampoo preparations are made using active ingredients, namely the distilled water fraction of aloe vera leaves and lemongrass oil with a concentration of 5% 10%, and 15%, surfactants, and additives. the surfactants chosen in the manufacture of this shampoo are the surfactants that are widely used in shampoo preparations on the market, namely sodium lauryl sulfate as the primary surfactant and cocamide dea as a secondary surfactant. by using primary and secondary surfactants, dandruff shampoo preparations can clean and form foam better. additional ingredients used are cmc as a thickener, methylparaben as a preservative, citric acid as a buffer, and menthol as a fragrance. the anti-dandruff shampoo for the aloe vera leaf fraction was evaluated to determine its quality and safety. then proceed with testing the antifungal activity of the anti-dandruff shampoo preparation. the foam height test aims to show the surfactant's ability to form foam. the lather from the shampoo is very important. this is because the foam keeps the shampoo in the hair, makes hair easy to wash, and prevents the hair sticks from sticking together, causing tangles (mitsui 1997). the foam height resulting from the three shampoo formulations has increased foam power. this increase was caused by an increase in the distilled water fraction in the shampoo preparation because the distilled water fraction of aloe vera leaves and lemongrass contained saponins. according to harbone (1998), saponins are soap. the foam height test results of the three shampoo formulations met the foam height requirements according to wilkinson and moore (wilkinson 1982), namely 1.3 22 cm. based on the results of ph measurements using universal ph indicator paper, the addition of aloe vera and lemongrass distilled fraction causes a decrease in ph due to the influence of active substances in aloe vera distilled water fraction which has an acidic ph. although the ph of the shampoo decreased, the ph value of the three anti-dandruff shampoo formulations still met the requirements set forth in sni no. 06-2692-1992 which is around 5.0 9.0. testing the value of water content is very important to do in a shampoo product because the moisture content is related to the physical shampoo and affects the shelf life of a shampoo product. from the measurement of water content, anti-dandruff shampoo with various concentrations of aloe vera and lemongrass distilled water still meet the requirements of sni no. 06-26921992 which is a maximum of 95%. based on the results obtained, the water content produced, the greater the concentration of the fraction added, the smaller the percentage of water content obtained. this higher water content comes from hygroscopic materials (the ability of a substance to attract water molecules from its environment), such as cmc. the shampoo preparations made in this study used active ingredients which are obtained from the water pacar leaves. water pacar leaves that are taken, cleaned and washed under running water to remove dirt. the leaves are already aerated and put in the oven to reduce moisture content in the leaves. the dried leaves of aloe vera are then blended until smooth and sieved to obtain a homogeneous powder with a surface area large so that it facilitates the release of active substances in the extraction process. the results of aloe vera leaf extract were obtained from the extraction process using the maceration method and 96% ethanol solvent. this maceration method also has the advantage of being able to maintain the compound content in the sample which is not heat resistant, undamaged and the sample can be atmanto & ambarwati – development of an eco-shampoo 403 extracted directly in large numbers (doughari 2006). this sample extraction using 96% ethanol solvent because ethanol solvent covers almost the entire content simplicia, both non-polar, semi-polar, and polar (iswanti 2009). the concentrated extract that has been obtained is then fractionated by the method partition using n-hexane, ethyl acetate, and distilled water. the use of solvents aims to attract compounds in the extract based on the level of polarity. based on the yield of the extract fractionation, it can be seen that the distilled water solvent fraction had the highest yield. this shows that it is deep. based on the results obtained, the distilled water fraction has activity inhibits the most fungus so that antidandruff shampoo preparations are made with using the active ingredient, namely the water fraction of aloe vera leaves and lemongrass oil with concentration 5% 10%, and 15%, surfactants, and additives. surfactants selected in this shampoo is a surfactant that is widely used in shampoo preparations on the market, namely sodium lauryl sulfate as primary surfactant and cocamide dea as a secondary surfactant. by using primary surfactants and secondary, anti-dandruff shampoo preparations can clean and form more foam well (deeksha et al. 2014). additional ingredients used are cmc as a thickener, methylparaben as a preservative, citric acid as a buffer and menthol as a fragrance. shampoo anti-dandruff water fraction of aloe vera leaves that has been finished evaluated to know the quality and safety. then proceed with testing the antifungal activity of the anti-dandruff shampoo preparation. the results of organoleptic observations of antidandruff shampoo for water fraction of pacar leaf with various concentrations show that the higher concentration of water fraction of pacar leaf contained in the shampoo preparation anti-dandruff, the stronger the distinctive smell of aloe vera leaves so that it covers the smell of the menthol fragrance used, as well as the darker brown in the preparation shampoo because the color of the distilled water of aloe vera leaves is brick red. the ph test aims to determine the safety of the preparation at the time of use. shampoo ph that is too acidic or too alkaline will irritate the scalp. based on the results of ph measurements using universal ph indicator paper, the addition of the distilled water fraction aloe vera causes a decrease in its ph due to the influence of the active substance in the water fraction of pacar leaves which has an acidic ph. although the ph of the shampoo has decreased, the ph value of the three shampoo formulations anti-dandruff still fulfills requirements stipulated in sni no. 06-26921992 which is around 5.0 9.0. the foam height test aims to show the surfactant's ability to form foam. the lather from the shampoo is very important. this matters because the lather keeps the shampoo in the hair, making hair easily washed, and prevents the hair sticks from sticking together to cause tangled (mitsui, 1997). high foam resulting from the three shampoo formulations experienced increased foaming. this increase was caused by the increase in the distilled water fraction in the shampoo preparation was due to the distilled water fraction of pacar leaves containing saponins. according to harbone, saponins are soap. the foam height test results of the three shampoo dosage formulations met the high requirements; foam according to wilkinson and moore (1982) is 1.3 22cm. test the value of water content is very important to do in a product shampoo because the water content is related to the physical shampoo and affects the shelf life of a shampoo product. from the measurement of water content, anti-dandruff shampoo with various concentrations of water fraction of pacar leaf still meets the requirements of sni no. 06-2692-1992 which is a maximum of 95%. based on the results obtained from the resulting water content, the greater the concentration of the fraction added, the smaller the percentage of water content obtained. water content higher comes from hygroscopic materials (the ability of a substance to attract water molecules from the environment) such as cmc. anti-dandruff shampoo preparations with various concentrations tested its activity against the growth of the fungus candida albicans by using media, method, fungus, and positive control were the same as those used on antifungal activity testing of the previous extracts and fractions. negative control anti-dandruff shampoo formula is used without the water fraction of aloe vera leaves (base shampoo). the negative control is used to determine whether there is a basis effect shampoo against the growth of the test fungi, so it can be seen that the activity shown by the shampoo with various concentrations of leaf distilled water. aloe vera is the substance contained in the distilled water fraction shampoo preparations and not derived from the shampoo base used (putluri et al. 2011). the results of testing the antifungal activity of antidandruff shampoo, distilled water fraction. aloe vera and lemongrass oil with concentrations of f1 (5%), f2 (10%), f3 (15%), shampoo formula anti-dandruff without distilled water fraction of aloe vera leave as negative control and shampoo (june 2016). ketoconazole 2% as a positive control in each treatment indicates a zone of inhibition that has formed around the well. obstacles zone the largest indicated by the anti-dandruff shampoo with a concentration of 5% distilled water, followed by anti-dandruff shampoo with the concentration of 10% distilled water and zone fraction. the smallest inhibition was shown by antidandruff shampoo with a fraction concentration of 15% distilled water with an average size of the inhibition zone in a row, namely 13.83 mm, 9 mm, and 7.83 mm. the difference in inhibition is influenced by the addition of distilled water fraction which affects the release of active substances for inhibited fungus (aghel et al. 2007). the higher the concentration of distilled water, the thicker it is anti-dandruff shampoo preparation, the greater the 404 biology, medicine, & natural product chemistry 12 (1), 2023: 399-405 viscosity of the shampoo preparation. viscosity is a statement about the resistance of a liquid to flow, the higher the viscosity, the greater the resistance (al-badi and khan 2014). this is what makes it so that it blocks the release of the active substance which results in inhibition of the fungus candida albicans. utilization of organic local environmental resources made from plants like aloe vera, mangrove, and spice plants for a variety of products that help the economy and as natural treatments, particularly cosmetics (daisy r. 2011). phenolic compounds are found in the majority of indonesian plants, including aloe vera and mangrove leaves. phenolic compounds are one of the dynamic mixtures found in aloe vera gel. aloin, aloesin, aloemodin, and tannins are the primary components of these phenolic compounds (iswanti da. 2009). in the past, the community did not know how to make cosmetics like organic shampoos and masks made from local environmental plants, which offered huge opportunities for business. plants that contain nutrients and active substances that function as fundamental ingredients for skin and hair care are the strategy for utilizing the potential of local environmental resources in this instance (deeksha d, malviya r, sharma pk. 2014). after getting to know one another and introducing the community to products that grow in the area, the community now knows how to make organic shampoos, soaps, and masks from local plants. these products are easy to make and can be used again and again, and the materials used are ones that can be found easily (fazlolahzadeh o, masoudi a. 2015). according to pooja a, arun n, and maninder k. (2009), the production of organic shampoos and masks made from aloe vera and other plants could also be a solution to the issue of environmental pollution caused by chemical waste, which is difficult to convert into materials that are friendly to the environment. because there are so many "dysfunction" chemicalbased cosmetics on the market, the usage of plants as cosmetic ingredients will continue to expand in the future. as a result, both the central government and the regional governments must continue to stimulate the exploration of the potential of local environmental resources, both types and benefits, through experiments and research. the cultivation of cosmetic ingredient plants can later become a source of revenue for the affected area (naitullah n, jamin f, frengki, dewi m. 2014). in this way it is important to go to preservation lengths for regions that have neighborhood natural assets through local guidelines so the stock of materials is ensured. on the off chance that the plant is unmistakable for its development climate, it is better assuming development is completed around there to increment plant material (robinson t. 1995). involving the local community in the production process as well as in the cultivation of these plants is an equally crucial step. for instance, for each family, they are responsible for planting ten sticks or ten plants. we will eventually discover a "cosmetic village" (saputro ad: 2006). in addition, it inspires researchers at universities, the private sector, and national research agencies like brin. conclusion based on the results of research on the physical stability test of mixed aloe vera and lemongrass oil shampoo with varying concentrations, namely 5%, 10%, and 15%, it can be concluded that aloe vera contains active compounds, fats, carbohydrates, and proteins. aloe vera fraction and lemongrass oil can be formulated as a shampoo preparation to maintain hair fertility as well as an anti-dandruff shampoo that meets requirements such as organoleptic, ph, high foam, and moisture content. evaluation of shampoos for quality assurance is a physical appearance test that is attractive, homogeneous, does not break, and can form foam. the ph value ranges from 5 6. the test for the ability and stability of the foam of the shampoo is carried out using the cylinder shake method at 6-8 cm, where the formulation of aloe vera is 3% and lemongrass oil is 12%. authors’ contributions: dwi atmanto & neneng siti silfi ambarwati designed the study. dwi atmanto & neneng siti silfi ambarwati carried out the laboratory work. dwi atmanto & neneng siti silfi ambarwati analyzed the data. dwi atmanto & neneng siti silfi ambarwati wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research receives no external funding. acknowledgment: the author would like to express gratitude to the dean of the faculty of engineering at jakarta state university for partnering with blu's asset management on the exploration. the authors also express their gratitude to the students and professionals who assisted with the research center's cycle of item testing and exploration. references aghel n, moghimipour b, dana r.a. 2007. formulation of a herbal shampoo using total saponins of acanthophyllum squarrosum. iranian journal of pharmaceutical research. 6(3), pp. 167-172. doi: 10.22037/ijpr.2010.717 al-badi k, khan sa. 2014. formulation, evaluation, and comparison of the herbal shampoo with the commercial shampoos. beni-suef university journal of basic and applied sciences. 3(4), pp. 301-305. atmanto & ambarwati – development of an eco-shampoo 405 arifin z. 2006. kajian mikoriza vesikula arbuskula (mva) dalam menekan perkembangan penyakit bercak ungu (alternaria porri) pada bawang putih [study of arbuscular vesicle mycorrhizal (mva) in suppressing the development of purple spot disease (alternaria 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chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1305-1314 | doi: 10.14421/biomedich.2025.142.1305-1314 issn 2540-9328 (online) chemical analysis, physical stability, and antibacterial activity of nanoemulgel hand sanitizer formulated with citrus aurantifolia essential oil and herbal emollients intan kurnia putri1*, maya uzia beandrade1, reza anindita2 1department of pharmacy, sekolah tinggi ilmu kesehatan mitra keluarga, bekasi, west java, indonesia. 2department of medical laboratory technology, sekolah tinggi ilmu kesehatan mitra keluarga, bekasi, west java, indonesia. corresponding author intan.kurnia.p@stikesmitrakeluarga.ac.id* manuscript received: 23 october, 2025. revision accepted: 04 december, 2025. published: 13 december, 2025. abstract one of the most purchased hand sanitizer products by the public since the covid-19 pandemic is hand sanitizer. however, alcohol-based hand sanitizer products often cause toxic effects and bacterial resistance. therefore, alternative efforts are needed to replace antibacterial raw materials instead of alcohol, namely lime peel essential oils (citrus aurantifolia) with aloe vera and calendula officinalis emollients. this study aims to evaluate the physicochemistry, physical stability, and antibacterial affectiveness of the a nanoemulgel handsanitizer formula composed of active lime peel essential oils supplemented with aloe vera and calendula officinalis emollients. the design of this research is an experiment. research treatment: formula (f) hand sanitizer nanoemulgel f1 2%, f2 4%, and f6 6%. essential oil component test using gc-ms method, sneeds physical stability includes centrifugation test, freeze-thawing, transmittance, particle size, polydispersity index (pi), potential zeta, physical stability of nanoemulgel hand sanitizer, including organoleptic, homogeneity, ph, and viscosity test, antibacterial efficacy test using hand swabbing method. the results: gc-ms analysis of the components of lime peel essential oils: α-pinene (8.87%), linalool (0.58%), and l-α-terpineol (8.98%). all sneeds formulas and nanoemulgels were stable during 6 weeks of storage. formulas f2 4% and f3 6% reduced the percentage (%) of hand bacterial colonies to 74% and 75%. the results of the one-way anova showed a significant decrease (p < 0.05) in the number of bacterial colonies after using nanoemulgel hand sanitizer, especially in the f2 and f3 formulas. the conclusion is that formula 3 (f3 6%) hand sanitizer is recommended as the best candidate for the development of a nanoemulgel hand sanitizer based on lime essential oil, as it offers the most optimal combination of physical stability, chemical stability, and biological effectiveness. keywords: essential oils; lime fruit peel; self-nanoemulsifying drug delivery system (sneeds); hand sanitizer; nanoemulgel. introduction bacterial infections transmitted through hand contact remain one of the primary routes of spread in public health. according to the world health organization (who), one way to reduce the incidence of bacterial infections is to wash hands with soap or use hand sanitizer. (united nations children fund (unicef) and world health organization, 2021). alcohol-based hand sanitizers (typically ethanol or isopropanol ≥60%) effectively reduce the number of bacteria on hands. however, long-term use can cause skin irritation, dryness, and the risk of burns (alhalwani et al., 2024). this encourages the development of non-alcoholic hand sanitizer formulations based on natural ingredients that are safer and more comfortable to use on hand skin. one of the extracts of natural ingredients with antibacterial potential is lime essential oil (eo). lime essential oil (citrus aurantifolia) is known to contain dlimonene, citral, and linalool compounds that exhibit antibacterial activity (husni et al., 2021). another study reported that lime eo had an alcohol-comparable effectiveness of 78% against staphylococcus aureus and escherichia coli bacteria (mohammed et al., 2024). thus, lime essential oil has the potential to be an active ingredient in non-alcoholic hand sanitizer formulations. other natural ingredients are calendula officinalis and aloe vera. the use of c. officinalis as a component of hand sanitizer formula is due to its anti-inflammatory, antioxidant, and supportive skin tissue regeneration (parente et al., 2012). calendula oil is often used as a moisturizer and anti-irritant in topical preparations. meanwhile, aloe vera is widely known as a humectant and emollient that can increase skin moisture, reduce irritation, and add sensory comfort (catalano et al., 2024). the combination of lime oil, calendula, and aloe vera is expected to produce a hand sanitizer that is not only an effective antimicrobial, but also safe and comfortable to use on the skin of the hands. https://doi.org/10.14421/biomedich.2025.142.1305-1314 mailto:intan.kurnia.p@stikesmitrakeluarga.ac.id 1306 biology, medicine, & natural product chemistry 14 (2), 2025: 1305-1314 however, using essential oils in conventional forms has limitations, including low water solubility, high volatility, and limited stability. therefore, nanoemulsion technology is needed to reduce the size of oil droplets to the nanometer scale, increasing solubility, bioavailability, and stability (omidian et al., 2025). the combination of nanoemulsion with gel base results in nanoemulgels that are easier to apply, clear, and allow for more stable release of active ingredients (donthi et al., 2023). therefore, nanoemulgel is an ideal formulation for essential oil-based hand sanitizers. several studies have reported the development of nanoemulgel-based hand sanitizers, including (ibrahim et al., 2024), developing lemon peel extract nanoemulgel and demonstrating antimicrobial activity against skin pathogenic bacteria, drais (2024) successfully developed nanoemulgel essential oil with high stability and potential as a skin sanitizer. however, the research generally only assesses aspects of physical stability or antimicrobial activity in vitro, without comprehensively evaluating the physicochemical characteristics and in-use tests on human skin. this researcher aims to develop a hand sanitizer nanoemulgel containing lime essential oil with aloe vera and calendula officinalis as emollients. physical stability tests include: transmitter tests, particle size and polydispersion index, ph, viscosity, homogeneity, dispersability, and thermodynamic stability. in addition, this study also evaluated the number of bacteria on the hands before and after using hand sanitizer nanoemulgel to assess its effectiveness directly in the community. this combination of physicochemical and microbiological data provides a comprehensive picture of the quality, stability, and biological activity of hand sanitizer nanoemulgel lime essential oil products. materials and methods materials materials and tools include lime peel (citrus x aurantiifolia (christm.) swingle), aloe vera gel 99% organik non alkohol, calendula oil 100% pure natural oil, hydroxypropyl methyl cellulose (hpmc) (merck), span 80 (merck), gliserol (merck), 2,2diphenyl-1-picrylhydrazil (dpph) (sigma), benzil alkohol (merck), phenol (merck), chloroform (merck), quercetin (sigma), nutrien agar (merck), galic acid (sigma), polyethylene glycol (peg) 400 (merck), essential oil extraction 40 kg of lime peel was placed in a steam distillation apparatus containing water (1:5 w/v ratio). the distillation time was 5 hours. a separator separated the essential oil (distillate) from the water. the essential oils were contained in tightly sealed bottles. the composition of the essential oils was tested using the gas chromatography–mass spectroscopy (gc-ms) technique. analysis of essential oil composition using the gc-ms method essential oil analysis was performed using gas chromatography–mass spectrometry (gc–ms) technique with shimadzu gc/ms-qp2010 ultra (serial number 020525101565sa). the capillary column was rtx-5ms (30 m × 0.25 mm × 0.25 µm). the sample was injected in split mode, and the instrument was operated in electron ionization (ei) mode with an ionization energy of 70 ev. the carrier gas was helium (flow rate 1.69 ml/min). the column temperature program started at 50°c, then increased at a rate of 7°c/min to 180°c, and then increased again at a rate of 10°c/min to a final temperature of 280°c. the injection port temperature was set at 300°c, the ion source temperature at 200°c, and the interface temperature at 250°c. the analysis was performed in scan mode with a mass-to-charge ratio (m/z) range of 40–500, with a total analysis time of 28 minutes. sample components were identified by comparing the retention index and mass fragmentation pattern of each compound to data available in the nist (national institute of standards and technology) library (mohammed et al., 2024) dpph antioxidant analysis of essential oil lime peel and calendula oil antioxidant activity test of essential oil lime peel and calendula oil was carried out using the dpph (2,2diphenyl-1-picrylhydrazyl) method, modified from baliyan et al. (2022) a total of 24 mg of dpph powder was dissolved in 100 ml of methanol to make a stock solution. this solution was then filtered and adjusted to absorb approximately 0.97 at a wavelength of 517 nm using a uv-vis spectrophotometer. test samples of aloe vera gel extract (dissolved in 80% methanol) and calendula oil (dissolved in ethanol with the addition of 0.1% tween-20 to homogenize) were prepared at several concentrations (10–1000 µg/ml). a total of 3 ml of dpph solution was mixed with 100 µl of test sample solution in a test tube. as a control, 3 ml of dpph solution was used, and 100 µl of methanol was added without a sample. all tubes were then incubated in the dark for 30 minutes at room temperature to prevent free radical degradation. after incubation, absorbance was measured at a wavelength of 517 nm. the percentage of antioxidant activity was calculated using the following formula: % antioxidant activity = (𝐴𝑐 − 𝐴𝑠) 𝐴𝑐 × 100 description: ac = absorbance of the control (dpph solution without sample), as = absorbance of the dpph solution with the test sample. the ic₅₀ value (the concentration capable of inhibiting 50% of dpph radicals) was then determined from the relationship curve between sample putri et al – chemical analysis, physical stability, and antibacterial activity of … 1307 concentration and percentage inhibition. the positive control used was ascorbic acid. formula sneeds (self nanoemulsifying drug delivery system) dan nanoemulgel hand sanitizer the making of sneeds and nanoemulgel hand sanitiser is shown in table 1. table 1. sneeds formula and nanoemulgel hand sanitizer. phase ingredients function f1 f2 f3 phase a lime essential oil active compounds 2.00% 4.00% 6.00% calendula oil emollient 0.50% 0.50% 0.50% span 80 surfactant 2% 2% 2% cremophor rh 40 surfactant 10% 10% 10% peg 400 co-surfactant 10% 10% 10% phase b hpmc gelling agent 1% 1% 1% aloe vera gel emollient 5% 5% 5% gliserol moisturized 3% 3% 3% benzyl alcohol preservative 0.1% 0.1% 0.1% aquadest solvent 66.40% 64.40% 62.40% based on table 1. the production of sneeds is carried out by weighing all ingredients according to their formulation percentages. surfactants (cremophor rh 40) and co-surfactants (peg 400) are placed in a beaker, heated and stirred at 40–45 °c with a magnetic stirrer until a homogeneous and precise mixture is formed. span 80 is added slowly in a surfactant–co-surfactant mixture. calendula oil is included in the mix. lime essential oil is added while stirring continuously until a homogeneous and clear mixture is obtained (an indication of phase a has been formed as a nanoemulsion pre‐concentrate)— the homogeneous phase. a mixture is then stored in a tightly closed container at room temperature. (drais, 2024).after obtaining stable sneeds, a nanoemulgel hand sanitizer formulation is made by weighing all ingredients. phase a preparation involves mixing cremophor rh 40 and span until a homogeneous mixture is achieved, followed by the addition of lime peel essential oil and calendula oil in a mixer (speed 1500 rpm for 10 minutes), followed by a nanoemulsion size test in this phase. next, prepare for phase b by developing hpmc with enough aquadest until it expands. add the aloe vera gel, glycerol, and benzyl alcohol to a mixer and mix until homogeneous. insert phase a into phase b with a magnetic stirrer until homogeneous for 10 minutes (ibrahim et al., 2024). sneeds and nanoemulgel physical stability evaluation test sneeds physical stability evaluation tests include centrifugation (thermodynamics), freeze thawing, transmitters, particle size, compressed potential index (pdi), and potential zeta (annisa et al., 2023). the physical stability test of nanoemulgel hand sanitizer includes organoleptic, homogeneity, ph, and viscosity tests during 6 weeks of storage. (muñoz et al., 2023). nanoemulgel hand sanitizer efficacy test the efficacy test of hand sanitizer was carried out in the following way: respondents were asked to contaminate their hands. after that, the initial sample (baseline) was taken from both hands of each respondent by rubbing the palms and fingertips with sterile swabs moistened with brain heart infusion (bhi) broth. furthermore, each respondent was asked to pour 3 ml of hand sanitizer to clean their hands (hand hygiene) according to who procedures. after using hand sanitizer, a sample was retaken with a sterile cotton swab moistened with sterile bhi broth, then rubbed on both respondents' palms, interlodes, and fingertips. the swab was then spread on nutrient agar (na) media (oxoid, uk) and incubated aerobically at a temperature of 36°c for 1x24 hours (overnight) (sommatis et al., 2023). after incubation, the percentage of bacterial reduction (r%) is calculated by the following equation: 𝑅 (%) (𝑖𝑏 − 𝑖𝑎) 𝑖𝑏 𝑥100% r: percentage reduction of bacteria ib: number of bacteria before being given nanoemulgel hand sanitizer ia: the number of bacteria after being given nanoemulgel hand sanitizer data analysis data analysis uses ibm spss 25 statistics. parametric data (ratio) was tested for normal distribution, homogeneity, and then one-way anova (sig<0.05). if significant, continue the post-hoc test to find groups with significant influence. results and discussion chemical composition of lime fruit peel essential oil based on the gc-ms analysis of lime peel essential oils, 41 chromatogram peaks were obtained. the visualization of the chromatogram can be seen in figure 1. 1308 biology, medicine, & natural product chemistry 14 (2), 2025: 1305-1314 figure 1. gc-ms chromatogram of the components of the essential oil of lime fruit peel. based on figure 1. the compounds contained in lime oil are α-pinene (8.87%), linalool (0.58%), and l-αterpineol (8.98%). the largest content is l-α-terpineol. gc-ms analysis results include the essential oil components of lime peel from slovakia for α-terpineol (1.4%) and α-pinene (12.6%) (galovičová et al., 2022), as well as linalool (0.64%) (lin et al., 2019). the dominant compound almost always found in essential oils of the citrus genus (including citrus aurantifolia) is d-limonene or monoterpenes, which give citrus fruits a fresh aroma. p-cymene, γ-terpinene, and β-pinene are found in small amounts. based on the results of gc– ms, lime peel essential oils in this study were dominated by l-α-terpineol and α-pinene. this composition differs from the general profile of fresh citrus fruit peel, which is typically dominated by d-limonene (li et al., 2022; galovičová et al., 2022). the difference is likely due to the oxidation process of limonene during distillation or storage of the sample. an increase in oxygenated monoterpene compounds such as α-terpineol indicates limonene conversion due to exposure to heat and oxygen. value percentage (%) inhibition (dpph radical scavenging activity) the antioxidant activity of lime peel and calendula essential oils was evaluated using the dpph radical scavenging assay method to determine their ability to neutralize free radicals. the percent inhibition values (% inhibition) obtained at various concentrations (2%, 4%, and 6%) are presented in table 2. table 2. mean percentage value (%) inhibition (dpph radical scavenging activity). sample mean ± sd % inhibition 2% 4% 6% ascorbic acid (positive control) 5.12a ± 0.86 13.82b ± 0.52 75.90c ± 0.37 lime fruit peel essential oil 21.27a ± 0.71 22.50b ± 0.62 33.15c ± 0.66 calendula oil 19.96a ± 0.22 22.61b ± 0.04 31.76c ± 0.37 superscript letters with different concentrations in each sample showed significant differences (p<0.05) (one-way anova). based on table 2. antioxidant activity tests using the dpph method showed that all samples could suppress free radicals that increased with increased concentration. ascorbic acid, which was used as a positive control, showed the highest activity with an mean inhibition of 75.90% at a concentration of 6%. this indicates a powerful free radical inhibition potential because the simple phenolic structure of ascorbic acid acts as an efficient electron donor against dpph radicals. (jaganjac et al., 2021). lime peel citrus aurantifolia essential oil showed a significant increase in antioxidant activity (p < 0.05) from 21.27% at a concentration of 2% to 33.15% at a concentration of 6%. the ic₅₀ value of 16.15% indicates that lime peel essence exhibits moderate antioxidant capabilities, with lower effectiveness compared to the ascorbic acid control yet still displays a clear dose-dependent response pattern. essential oils of the genus citrus generally have moderate antioxidant activity due to the dominance of non-oxygenating monoterpene compounds such as d-limonene and αpinene, which are volatile and have limited radical inhibition capabilities compared to phenolic or flavonoid compounds. (li et al., 2022). calendula oil (calendula officinalis) also showed a pattern of significant increase in % inhibition (p < 0.001), with a mean inhibition value of 31.76% at a concentration of 6% and an ic₅₀ of 11.55%, meaning that the antioxidant activity was more potent than that of lime oil. a lower ic₅₀ value indicates that the amount of oil needed to neutralize 50% of dpph radicals is less. this is likely due to the presence of flavonoid glycosides, triterpenoids, and phenolic compounds in calendula oil, which contribute to putri et al – chemical analysis, physical stability, and antibacterial activity of … 1309 increased hydrogen donor ability and free radical stabilization. these results are in line with the findings. shahane et al. (2023) reported that calendula extract and oil exhibited high antioxidant activity which was strongly correlated with total the phenolic compounds (tpcs) content. based on the ic₅₀ value, the order of dpph free radical inhibition is: ascorbic acid (most potent) > calendula oil (ic₅₀ = 11.55%) > lime oil (ic₅₀ = 16.15%). their chemical composition can explain the difference in activity between lime oil and calendula. in lime oil, the dominant components are limonene, βpinene, and γ-terpinene, which tend to provide moderate antioxidant effects because their hydrocarbon structure lacks proton donor groups. in contrast, calendula oil's oxygenated α-tocopherol, quercetin, and triterpenoid content contribute significantly to the antioxidant effects through radical scavenging and chain-breaking mechanisms (chen et al., 2023). overall, these results confirm that calendula oil has better antioxidant potential than lime oil, although both are still less effective than ascorbic acid. the variation in activity is due to differences in species, geographical origin, and extraction methods. water content, extraction temperature, and thermal degradation of monoterpenes during the distillation process can also cause variations in results between studies and between citrus essential oilproducing countries. (li et al., 2022; catalano et al., 2024). physical stability of sneeds (self-nanoemulsifying drug delivery system) sneeds (self-nanoemulsifying drug delivery system) is an initial system of nanoemulgels that function to produce preconcentrates, which are homogeneous mixtures of oils, surfactants, and cosurfactants. these mixtures will spontaneously form nanoemulsions with water or water phases (such as gel bases) without the need for considerable energies (such as sonication or high-pressure homogenization). therefore, sneeds is the initial stage of nanophase formation that ensures that the particle size is tiny (<100 nm) and stable, before the phase is mixed into the gel base to form a nanoemulgel ready for topical use. the manufacture of sneeds was carried out before nanoemulgels because it functions to produce a homogeneous, stable, and nano-sized droplet that can form nano-sized droplets spontaneously when mixed with water. this stage is essential to ensure the system's stability, the efficiency of releasing active substances, and the increased biological activity, such as antioxidants or antibacterials of lime and calendula essential oils. the initial formulation of sneeds was prepared to ensure the physical stability of the nanosystem before it was developed into a nanoemulgel. the physical stability in this study encompasses thermodynamic tests (centrifugation), freeze-thawing, transmitters, particle size, index polydispersity, and nanoemulsion potential zeta. (r. a. anindita et al., 2023) thermodynamic test (centrifugation) and freezethawing sneeds the sneeds thermodynamic (centrifugation) test aims to observe the potential for phase separation and homogeneity of the mixture. this test ensures that all formulas remain stable without any good phase separation to proceed to the nanoemulgel formulation stage. in contrast, the freeze-thawing test evaluates physical stability against extreme temperature changes. this test aims to simulate varying storage conditions and assess the potential for phase separation, turbidity, or viscosity changes that may affect the system's stability. each test cycle alternates storing the formulation at low (–20 °c) and high temperature (25–40 °c) for several days. the results of the thermodynamic and freezethawing sneeds tests are presented in figure 2. figure 2. centrifugation test results (a. before centrifugation. b. after centrifugation). freeze-thawing test results (c. before freeze-thawing. d. after freeze-thawing). based on figure 2, the centrifugation test of the sneeds f1 2%, f2 4%, and f3 6% formulas showed no discoloration, turbidity, or phase separation in the entire formula after the centrifugation process for 30 minutes at 5000 rpm, while the freeze–thawing test results for the whole of the formula showed good stability without turbidity or phase separation after three freezing and thaw cycles. this confirms that the sneeds system has sufficient thermal stability for the nanoemulgel formulation stage. the absence of phase separation or a b c d 1310 biology, medicine, & natural product chemistry 14 (2), 2025: 1305-1314 turbidity after centrifugation tests indicates that the oil droplets in the system have enough interface defenses against high mechanical forces. if separation is found after centrifugation, the top layer appears to contain a separate oil phase, indicating that the surfactant– cosurfactant ratio is not optimal and that the droplet size is large enough to allow coalescence and creaming under centrifugal force. in contrast, a reasonably stable formulation (tiny droplet size, optimal surfactant/smix, effective cosurfactant) can pass multiple freeze–thaw cycles without separation as the interface remains protected. (pavoni et al., 2020). test of transmittance, particle size, polydispersity index, and potential zeta the optimized self-nanoemulsifying drug delivery system (sneeds) system was physically characterised to ensure the formation of a stable and homogeneous nano system. the main parameters analysed included transmittance value (%t), particle size (nm), polydispersity index (pdi), and zeta potential. the transmittance value is used to assess the system's clarity and indirectly indicates the level of dispersion of droplets in the continuous phase. particle size and pdi reflect the homogeneity and kinetic stability of the system, while zeta potential describes the electrostatic stability associated with the repulsive force between droplets. evaluating these four parameters provides a comprehensive picture of the stability and effectiveness of nanoemulsion formation. the results of testing the four characteristics are presented in table 3. table 3. mean of test results of transmitters, particle size, polydispersity index, and potential zeta. variabel f1 f2 f3 transmittance (%) 99.049 97.529 91.096 particle size (nm) 123.0 100.3 113.5 polydispersity index (pi) 0.487 0.450 0.381 potential zetas (mv) -35.0 -31.9 -42.0 based on table 3, the values of all snedds f1-f3 formulations show >90%, meaning that the greater the concentration of essential oils, the smaller the transmitter value. it can be seen that snedds f1 with an essential oil concentration of 2%, 4%, and 6% gives transmitter results of 99.049%, 97.529%, and 91.096%. transmitter value requirements to determine the stability of nanoemulsion droplets if the value is >90% (dewanti, ariyadi, martien, & zuprizal, 2024);a transmitter value >90% indicates a clearly formed nanoemulsion system, an isotropic system, and a homogeneous system. when the transmittant value in the system is high (>90%), the uniformity level is high, the droplet size is tiny, and the dispersion is uniform. this high transparency indicates that the droplet size is relatively small and few particles give rise to a light/cloudy scattering. according to huang et al. (2022), %t value between 97–100% for the nanoemulsion indicates that the droplets are in the range of <200 nm and the system is optically stable. these transmitter indicators will typically provide strong justification for determining the nanosystem before testing it using a more accurate particle size analyser tool. the blank used in the study is aquadest as a comparison because aquadest does not have particles that will block the transmission of light, so that later the light that will be passed will not be disturbed by the effect of light scattering, so that the value of the blank transmitter is usually 100% (taiyeb et al., 2024). the particle size shows that f2 has the smallest size (100.3 nm), f3 is slightly larger (113.5 nm), and f1 is 123 nm. all are below the 200 nm threshold, which is often used as a kinetically stable "nanoemulsion" criterion. (xu et al., 2024). a smaller size (f2) will provide advantages: greater surface area, better kinetic stability, and more negligible light scattering (referring to high transmittants). the larger particle size of f1 and f3 may indicate that the surfactant/component ratio may be slightly less optimal than that of f2. the polydispersity index (pdi) shows the width of the particle size distribution; pdi values < 0.3–0.4 are generally considered homogeneous and stable; > values of 0.4–0.5 indicate a wider distribution. the results of this study showed that f3 = 0.381 (good), f2 = 0.450, and f1 = 0.487 (wider). the pdi f1 and f2 results are greater than 0.4, although the average size is small and has considerable variation in size. however, it can potentially increase the risk of coalition or size change during storage. alhamdany et al. (2021) said that pdi < 0.3–0.4 indicates better stability. potential zetas indicate the surface charge of droplets and provide an idea of the electrostatic repulsion force between droplets. the greater the zeta potential, the more electrostatically stable the system. the general value that is considered sufficient for stability is > 30 mv (positive or negative) (gurpreet & singh, 2018). the result of this researcher's potential zeta at f1 = -35.0 mv, f2 = -31.9 mv, and f3 = -42.0 mv. all zeta values have a potential > 30 mv. this shows the potential for good electrostatic stability. the highest f3 value (-42 mv) indicates the most substantial interdroplet repulsion and has the highest potential for long-term stability. therefore, although f3 has a slightly larger particle size than f2, it has the best potential zeta and the lowest pdi. this predicts that f3 is the optimal formulation for achieving a balance of size, homogeneity, and charge, making it the most suitable candidate for the next stage (nanoemulgel). physical stability of nanoemulgel hand sanitizer. based on the results of the physical stability of the selfnanoemulsifying drug delivery system (snedds) system, formula f3 was selected as the best candidate to be further formulated into a nanoemulgel hand sanitizer (hs) preparation with formula 1 (f1) 2%, formula 2 (f2) 4%, and formula 3 (f3) 6%. evaluation of the physical putri et al – chemical analysis, physical stability, and antibacterial activity of … 1311 stability of hs nanoemulgel preparations includes organoleptic tests, homogeneity, ph, viscosity, dispersibility, each of which provides an overview of the consistency, stability of the active components, and the convenience of application of the preparation on the skin. organoleptic and homogeneity tests organoleptic and homogeneity tests are an essential first step in evaluating the physical stability of nanoemulgel preparations, as these two parameters directly reflect the visual consistency, odour, texture, and uniformity of distribution of active and excipient components within the system. the results of the organoleptic and homogeneity tests can be seen in figure 3. figure 3. a. organoleptic. b. homogeneity. based on figure 3. organoleptic and homogeneous tests of nanoemulgel hand sanitizer (hs) f1 2%, f2 3%, and f3 6%, until week 6 the preparation remained homogeneous with a bright yellow color, a typical lime smell, and a gel texture that remained thick and did not undergo phase separation or noticeable texture changes. organoleptic examinations are performed to ensure that during storage, the preparation does not undergo any discolouration, odour, or consistency, which could indicate the occurrence of chemical degradation or physical instability (bajaj et al., 2013) homogeneity indicates the extent to which the nanoemulgel system maintains a uniform distribution of particles, whether from the oil, surfactant, or gel base phases. inhomogeneity in nano-preparations can lead to droplet coalescing, creaming, or separation phases, which ultimately affects the effectiveness of the active ingredient as well as the comfort of use (pavoni et al., 2020a). in nanoemulgel hand sanitizer formulations, the organoleptic stability also reflects the interaction between the nanoemulsion phase (sneeds) and the gel base, which determines the stability of viscosity, color, and gradual release of the active ingredient. therefore, visual observation of color, the distinctive smell of lime essential oil, and the consistency of the gel are important indicators of the integrity of the system during storage (sevinç-özakar et al., 2022). ph test, viscosity, dispersability table 4. mean of ph and viscosity values of nanoemulgel hand sanitizer during 6-week storage. week mean ± sd formula ph viscosity (cps) f1 2% 1 5,77a0,02 19.33394 2 5,56 b 0,02 18.56747 3 5,45 c 0,02 16.53394 4 5,37 d 0,03 15.533 47 5 5,19 e 0,12 14.43347 6 4,90 f 0,02 12.93347 f2 4% 1 5,55 a 0,02 19.66794 2 5,43 b 0,02 18.40082 3 5,35 c 0,02 16.23347 4 5,26 d 0,17 15.233 47 5 4,93 e 0,02 14.06794 6 4,86 f 0,17 12.10082 f3 6% 1 5,31 a 0,05 19.73347 2 5,23 b 0,05 18.10082 3 5,17 c 0,05 15.93347 4 5,04 d 0,03 14.767 47 5 4,91 e 0,01 13.76747 6 4,85 f 0,03 11.70082 annova test. the different superscripts in each formula showed significant differences (p<0.05). based on table 4. mean of ph on f1 2% decreased from 5.77 to 4.90, f2 4% decreased from 5.55 to 4.86, while f3 decreased from 5.31 to 4.85. anova test results (p < 0.05): showed that the storage time of 6 weeks had significantly affected the average ph value. however, the ph value remains within the standard range a b 1312 biology, medicine, & natural product chemistry 14 (2), 2025: 1305-1314 for topical preparations (4.5–6.5), indicating that it does not cause skin irritation (fenny & safitri, 2021). mean of viscosity value (cps) of f1 decreased from 19.33 to 12.93, f2 from 19.67 to 12.10, and f3 from 19.73 to 11.70. anova test results (p < 0.05): showed a significant decrease in viscosity during 6-week storage. this decrease can be caused by gel-based dehydration (e.g. carbopol), or the instability of nanoemulsion droplets that affect the system's viscosity. however, f3 has the highest viscosity consistently. this shows that f3 has better nanoemulgel system stability than f1 and f2. high viscosity values are essential for maintaining the adhesion and contact time of the active ingredient in the skin (aldeeb et al., 2024). overall, all formulas still meet the stability standards of topical preparations as stated by the european medicines agency ema (2022) which states that the ph should not change by more than ±1 unit and there should be no visual phase separation during storage. decreased ph and viscosity during storage are typical characteristics in essential oil-based nanoemulgel systems resulting from electrostatic interactions between surfactants, cosurfactants, and gel bases (pavoni et al., 2020b). despite the decline, the f3 formula exhibits the best rheological and chemical stability, which can be attributed to higher concentrations of lime and calendula oil and a more homogeneous distribution of droplets. efficacy of nanoemulgel hand sanitizer in vivo biocide efficacy testing was based on public health england and lambrechtsetal protocols. the agar standard plate count (spc) method was used to determine the total number of bacteria for all samples, which were incubated at 37 ◦c for 24–48 hours. table 5 shows the results of antibacterial tests before and after washing hands with nanoemulgel hand sanitizer can be seen in table 5. table 5. mean number of bacterial colonies before and after washing hands with nanoemulgel hand sanitizer. treatment before (a) after (b) bacterial reduction (%) f1 2% 93 44,5 52,15 a f2 4% 101 12 74,16b f3 6% 34 25,5 75c annova one-way test: different superscripts showed significant differences (p<0.05) between treatment groups. figure 5. antibacterial activity test of nanoemulgel hands sanitizer lime essential oil. treatment before and after hand washing. based on table 5 and figure 5. all formulas were able to reduce the number of bacterial colonies, but f2 and f3 showed the highest antibacterial activity (74%–75%). figure 5 shows a decrease in the number and size of colonies after using nanoemulgel hand sanitizer. this demonstrates that nanoemulgel hand sanitizer exhibits a bactericidal/bacteriostatic effect. lime oil contains limonene, citral, and linalool, which can disrupt the integrity of lipid membranes and inhibit the respiration enzymes of microorganisms. limonene and citral are lipophilic, allowing them to easily penetrate the cell walls of both gram-positive and gram-negative bacteria and cause denaturation of membrane proteins (pavoni et al., 2020b). increasing the concentration of essential oils from 2% (f1) to 6% (f3) generally increases antibacterial effectiveness, but f2 (4%) has already reached maximum effectiveness (74%). this indicates a saturation effect, where an increase in oil concentration no longer increases activity due to limited diffusion of active ingredients to bacterial cells. results before and after, the number of colonies dropped significantly on f2 and f3. this proves that the nanoemulgel system works through a mechanism of direct contact between essential oil droplets and bacterial cells, causing cytoplasmic leakage and protein clumping (bacteriolysis) (hosny et al., 2021). based on the results, there was a significant decrease in bacterial colonies (p < 0.05) after using nanoemulgel hand sanitizer, especially in the f2 and f3 formulas. antibacterial activity increases with the concentration of lime essential oil, demonstrating the active role of limonene and citral compounds. nanoemulgel is an effective delivery system because it extends the contact time of the active ingredient with the skin and improves putri et al – chemical analysis, physical stability, and antibacterial activity of … 1313 diffusion. formulas f2 and f3 can be recommended as optimal candidates because they show antibacterial effectiveness >70% or as per the standard effectiveness of hand antiseptic preparations (sommatis et al., 2023) the advantages of this study include: analysis of essential oil components using the gc-ms method, formulation of sneeds and nanoemulgel handsanitizer, and the efficacy test of nanoemulgel hand sanitizer on the number of bacterial colonies in both palms. the limitation of this study is that tests have not been carried out on antibiotic-resistant bacteria such as staphylococcus aureus and eschericia coli conclusions gc–ms analysis of lime peel essential oils showed the dominant components of l-α-terpineol and α-pinene. the self-nanoemulsifying drug delivery system (sneeds) formula f1 2%, f2 3%, and f3 4% are capable of forming dispersions with particle size (<150 nm), low polydispersibility index (<0.5), and stable negative potential zeta values (up to −42 mv). the formulas f1 2%, f2 4%, and f3 6% nanoemulgel hand sanitizer (hs) during 6-week storage were proven stable. the palm-based antibacterial efficacy test showed that the entire formula could significantly reduce the number of colonies (p<0.05) by a percentage of 52%-75%. formula 3 (f3 6%) is recommended as the best candidate for developing a nanoemulgel hand sanitizer based on lime essential oil, as it has the most optimal combination of physical stability, chemical stability, and biological effectiveness. the implications of this result open up the opportunity to use lime peel essential oil as an alternative to antibacterial active ingredients in nano-based delivery systems that are environmentally friendly and have the potential to be developed on an industrial scale. acknowledgements: the author would like to thank kementerian pendidikan tinggi, sains, dan teknologi (kemdikdiksaiteknik) for hibah riset dasar skema pdp (penelitian dosen pemula), master contract date: 28 mei 2025/no. master contract: 124/c3/dt.05.00/pl/2025. master contract date: 4 juni 2025. subcontract no.: 0949/ll3/al.04/2025. pharmaceutical and pharmaceutical technology with a focus on the development of phytopharmaceuticals based on local resources authors’ contributions: intan kurnia putri and maya uzia beandrade designed the study. intan kurnia putri, maya uzia beandrade, and reza anindita carried out the laboratory work. reza anindita analyzed the data. intan kurnia putri, maya uzia beandrade, and reza anindita wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references aldeeb, r. a. e., ibrahim, s. s. a., khalil, i. a., ragab, g. m., elgazar, a. a., taha, a. a., hassan, d. h., gomaa, a. a., & younis, m. k. 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(2024). characterization, stability and antioxidant activity of vanilla nano-emulsion and its complex essential oil. foods, 13(5). https://doi.org/10.3390/foods13050801 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 675-680 | doi: 10.14421/biomedich.2023.122.675-680 issn 2540-9328 (online) bioactivity of vernonia arborea leaves from bentian tribe as natural antioxidant and antibacterial based on local knowledge nur maulida sari1, humairo aziza2, farida aryani3,*, murdianto4 1departement of forest product processing; 2department of plantation management; 3department of plantation products technology, politeknik pertanian negeri samarinda, kampus gunung panjang jalan samratulangi, samarinda 75131, east kalimantan, indonesia 4faculty of forestry, mulawarman university, kampus gunung kelua jalan penajam, samarinda 75242, east kalimantan, indonesia. corresponding author* faridaaryani@politanisamarinda.ac.id abstract the utilization of natural organic materials as raw materials for standardized medicine, including the use of organic materials in medicine manufacture are currently being encouraged by the government. based on the information from bentian tribe people, vernonia arborea leaf or kutu bu’ut leaf has the potential to be developed as natural medicine derived from plants. this study explored the potential of v.arborea leaf for its phytochemicals screening by harborne, kokate and senthilmurugan method, antioxidant analysis was evaluated by dpph radical scavenging assay. antibacterial analysis was examined using agar well diffusion method against escherichia coli and propionibacterium acnes. the results showed that the v.arborea leaf contained alkaloid on n-hexan extract and coumarin on ethyl acetate extract. ethanol extract from v.arborea leaf contained alkaloid, flavonoid, saponin, tannin, and carbohydrate. antioxidant activity showed that the highest inhibition by 83% at 50 ppm consentration of ethanol solvent. antibacterial activity of e. coli and p.acnes showed that the highest inhibition zone by 12 mm and 11 mm at 400 µg/well of ethanol extract. based on the results, the v.arborea leaf contains natural bioactivity and has potential to be further developed as a natural traditional medicine. keywords: vernonia arborea; antibacterial; dpph; traditional medicine. introduction indonesia’s tropical forest is known to have more than 1000 types of plants used as medicine and only around 300-400 species of plants have been used as traditional medicines. indonesia is the second highest biodiversity in the world after the amazon forests (elfahmi et al., 2014). most of the studies focused on the prevention of diseases or specific medicinal herbs in indonesia (silalahi et al., 2014). the indonesian government through the vision and mission of nawacita 9 priority agenda, provides opportunities for the wider community and researchers to develop the potential of local areas such as in east kalimantan. natural antioxidants from extracted plants are highly recommended in food application for safety. natural antioxidant recently applied as bioactive nutraceuticals, food additives and bio-pharmaceuticals on daily activities. the antioxidant is used widely to prevent and reduce oxidation in a natural food system. based on the natural source of local people, the antioxidant of a plant is natural and safe to consume when used in small-scale. antioxidant also an essential component which plays a vital role in maintaining good health. many medicinal plants are known as natural antioxidant based on some research, which mainly depends on an active compound on various biologically (riaz et al., 2012a). the compounds of antioxidant can delay or slow down the process of oxidation of a compound. this means the antioxidant can interfere with free radical chain reaction with the propagation reactions, or inhibit the formation of free radicals in the early stages (indrianingsih et al., 2015). kalimantan known as an island for its highest local wisdom. previous study about local wisdom in kalimantan, stated more than 250 medicinal plant species from 75 family and more than 100 genera recently used by dayak ransa tribe in west kalimantan, indonesia. dayak kenyah community also recently used about 200 species of forest medicinal plants (kusuma et al., 2014). several studies about the utilization of traditional plants in kalimantan to treat various diseases, for example blumea balsamifera, stachytarpheta jamaicensis and vernonia arborea as an anticandidal against candida of candida albicans (kusuma et al., 2016). several studies in recent years about medicinal plants uses, informed that plants were best sources of natural antioxidant such as phenolics, alkaloids and flavonoids compound (zhao et al., 2018). antioxidant agent from the plants was a huge resource to scavenging free radicals naturally. manuscript received: 13 september, 2023. revision accepted: 28 november, 2024. published: 14 march, 2024. https://doi.org/10.14421/biomedich.2023.122.675-680 mailto:faridaaryani@politanisamarinda.ac.id 676 biology, medicine, & natural product chemistry 12 (2), 2023: 675-680 the ones of utilization of local plants use as an external medicine for skin and body health. local people use traditional plants as they belief for health and it also had no side effect, safe to be consumed and more economical compared to synthetic drugs. east kalimantan has a wide range of biodiversity in its territory also had many benefits. this plants potential are efficacious as natural medicines and if used wisely will provide valueless benefits, especially for the health of the nation of indonesia. the aim of the present study was to explored the potential antibacterial and antioxidant activities, also analyze the secondary metabolites of the plant extracts. based on the bentian tribe people in west kutai, this plant used as an alternative healthcare treatment such as itching skin. materials and methods material and chemicals the leaves of vernonia arborea were collected from bentian village, west kutai, east kalimantan, indonesia. the samples were washed thoroughly with water to remove the extraporeneous and dried about 3 days in the laboratory with air conditioned (a.c.) set for 20-25ºc. the samples were kept at a.c. room to keep the moisture content stable and milled with a blender. the powdered samples were prepared for further analysis. dpph (2,2 diphenyl-1-picrylhydrazyl) was purchased from sigma-aldrich (st. louis, mo, usa). sulfuric acid, hydrochloric acid, acetic anhydride, and potassium iodide were purchased from merck (darmstadt,germany). ascorbic acid, 1-naphtol and bismuth (iii) nitrate were obtained from sigma (st. louis, mo, usa). procedures maceration about 20 gr powdered samples of vernonia arborea were extracts with n-hexane, ethyl acetate and ethanol solvent at room temperature with continuous shaking on a shaker for 48 hours. following filtration of the suspension through whatman paper no.2 (maidstone,uk), the crude extracts of vernonia arborea were evaporated in a rotary evaporator at 38-40 ºc and put in a vacuum over near dryness to yield the plant extract. phytochemical analysis the n-hexane, ethyl acetate and ethanol extracts of vernonia arborea leaves was subjected to preliminary screening of phytochemical such as alkaloids, flavonoids, terpenoids, tannin, saponin, steroids, carbohydrate and coumarin using some following standard procedures (sari et al., 2023; viji et al., 2013). antioxidant assay test of antioxidant using 5 concentration samples were grouped into 100 ppm, 50 ppm, 25 ppm, 12.5 ppm and 6.25 ppm times of dilution, respectively. further, 3 mg of ascorbic acid was weighed, then dissolved in 1000 µl of ethanol solvent and regarded as a positive control. while the ethanol solvent was used as a negative control. about 33 µl sample was mixed in glass tube with 467 µl of ethanol was added, and 500 µl of 2,2-diphenyl-1picryhydrazyl (dpph) radical scavenging activity (shimizu et al., 2001). the mixing of sample was stopped while volume has reached 1000 µl (1 ml). samples were incubated for 20 minutes with minimum light and a.c. set for 27-30 ºc. the antioxidant activity was determined by decolorization of dpph with a wavelength of 517 nm using uv spectrophotometer. measurement was performed in triplicate examination. the percentage of dpph free radical was calculated using the following equation: % dpph radical scavenging activity = ∆𝑐𝑜𝑛𝑡𝑟𝑜𝑙− ∆𝑠𝑎𝑚𝑝𝑙𝑒 ∆𝑐𝑜𝑛𝑡𝑟𝑜𝑙 x 100 (1) antibacterial assay testing the antibacterial activity of the n-hexane, ethyl acetate and ethanol extracts of vernonia arborea leaves was carried out using the agar-well diffusion method with slight modifications (singh et al., 2002). escherichia coli and propionibacterium acnes bacteria were used in this research. about 20 ml of sterile agar media solution was put into a petri dish that had been sterilized for 30 minutes at 121°c in an autoclave. after that, in an antiseptic state (in laminar flow), let the media harden until it is cold and solid, then inoculate with 100 µl of the bacterial suspension and wipe it evenly over the test medium and let it dry for ± 30 minutes. then the media was perforated using a 5 mm sterile punch for each sample. in each test hole, 20 µl of sample which had been dissolved with acetone was added as a negative control and chloramphenicol as a positive control in the test. tests were carried out using test concentrations of 25 µg, 50 µg, 100 µg, 200 µg dan 400 µg. sari et al. – bioactivity of vernonia arborea leaves 677 results and discussion yield of extracts the leaves of vernonia arborea were macerated by nhexane, ethyl acetate and ethanol at room temperature (table 1). the weight of extracts maceration range was 0.30-5.93g and yielded of extracts was 1.80-35.17% extracts on the basic of sample dry weight. table 1. the yields extract of vernonia arborea leaves. solvent weight (g) yield (%) n-hexane 0.30 1.80 ethyl acetate 5.93 35.17 ethanol 4.87 28.91 *yield was calculated on the basis of sample dry weight the result showed that the ethyl acetate solvent gave the higher yielded extracts while the lowest yielded was obtained from n-hexane solvent. phytochemical analysis phytochemical analysis was carried out to determine the content of secondary metabolites contained in plant extracts. based on information from the local community about the utilization of the vernonia arborea plant or known by the people of bentian village by the local name daun kutu bu'ut, it is necessary to analyze the content of secondary metabolites before conducting other bioactivity tests. table 2. phytochemical analysis of vernonia arborea extract. sample test n-hexane ethyl acetate etanol vernonia arborea alkaloids + + flavonoids + saponins + tannin + steroids terpenoids + carbohydrate + coumarins + + the test results showed that the vernonia arborea plant in the n-hexane extract only contained alkaloid compounds and the ethyl acetate extract only contained coumarin compounds. in the ethanol extract, the vernonia arborea plant contains alkaloids, flavonoids, terpenoids, saponins, tannins, carbohydrates and coumarins. in several studies it is known that the presence of secondary metabolite compounds in a plant will affect the bioactivity contained in the plant (lim et al., 2014). antioxidant activity antioxidant analysis was carried out to determine the free radical absorption of vernonia arborea. tests were carried out using the shimizu et al (shimizu et al., 2001) method with modifications. tests were carried out using a dpph solution and a uv spectrophotometer with a wavelength of 517 nm. test concentrations are 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm and 100 ppm. ascorbic acid used as a positive control. the test results can be seen in figure 1 as follows. figure 1. antioxidant activity of vernonia arborea extract against dpph. 678 biology, medicine, & natural product chemistry 12 (2), 2023: 675-680 the test results showed that the vernonia arborea plant has potential as a natural antioxidant, this can be seen from the results of testing the dpph free radical which was used as a negative control in the test and vitamin c as a positive control. the n-hexane extract has a percentage of 3-12 % in the antioxidant test. the ethyl acetate extract has a percentage of 1-25 % while the ethanol extract has a percentage of 27-83 % in the test. the ethanol extract had a percentage of 83 % at a concentration of 50 ppm, whereas at a concentration of 100 ppm the inhibition percentage was only 72%. this is influenced by the concentration of the sample in the test, so that the absorption obtained is not read properly and causes a decrease in the percentage. in the phytochemical analysis carried out, it was found that the vernonia arborea plant contains various secondary metabolite compounds including polyphenols (tannins and flavonoids). plants containing polyphenols are an important source of antioxidants because they have an ideal chemical structure to scavenge free radicals. several studies reveal the potential of antioxidants in reducing the risk of various chronic and acute diseases such as heart disease, cancer and stroke by reducing free radical compounds involved in the pathogenesis of various diseases (riaz et al., 2012b). antibacterial activity antibacterial analysis was carried out to determine the potential of vernonia arborea against bacterial attack. tests were carried out using the method of singh et al (singh et al., 2002)with modifications. escherichia coli and propionibacterium acnes bacteria were used in the test. sample test concentrations were 25, 50, 100, 200 and 400 µg/well using chloramphenicol as a positive control. figure 2. antibacterial activity of vernonia arborea extract against propionibacterium acnes. testing of the vernonia arborea plant extract against propionobacterium acnes bacteria as shown in figure 2 shows that the n-hexane extract has no inhibition, the ethyl acetate extract has an inhibition diameter of 0-9 mm or the equivalent of 0-28% and is included in the medium category. in the ethanol extract there is an inhibition diameter of 7-12 mm or equivalent to 21-36% and is included in the medium-strong category. this shows that the ethanol extract has the potential to be developed as a natural antibacterial against propionobacterium acnes bacteria. figure 3. antibacterial activity of vernonia arborea extract against eschericia coli. sari et al. – bioactivity of vernonia arborea leaves 679 testing of the vernonia arborea plant extract against escherichia coli bacteria as shown in figure 3 shows that the n-hexane extract has no inhibition, the ethyl acetate extract has an inhibition diameter of 0-9 mm or the equivalent of 0-39% and is included in the medium category. in the ethanol extract there is an inhibition diameter of 7-11 mm or equivalent to 33-47% and is included in the medium-strong category. this shows that the ethanol extract has the potential to be developed as a natural antibacterial against escherichia coli bacteria. the presence of secondary metabolite compounds in the vernonia arborea plant extract, namely alkaloids, tannins, flavonoids and coumarins which are known to act as antimicrobials is one of the factors that makes the vernonia arborea plant extract able to inhibit bacterial growth. several studies have stated that the presence of compounds from the alkaloid and flavonoid groups can cut and denature proteins and prevent the digestive process of bacteria (ahmed & wang, 2021; heliawati et al., 2022). conclusions based on the results, the vernonia arborea leaves or daun kutu bu’ut medicinal plants used by tende people from east kalimantan contains natural bioactive and has potential to be further develop as a natural antioxidant and antibacterial, also give a scientific basic to the traditional uses of the investigated plants. acknowledgements: the research was funded by the ministry of education, culture, research and technology through penelitian dosen pemula scheme (junior lecturer research grants). acknowledgments were conveyed to the tende village, west kutai, east kalimantan, indonesia who have supported the research by sharing ethnopharmacological information and plant specimens. authors’ contributions: in this research, nur maulida sari was designed the research, supervised all process and wrote the manuscript. humairo aziza was observing, taking research samples and supervised research data analysis. farida aryani supervised all research data analysis and manuscript writing. murdianto controlled the samples and preparation of materials also controlled research data analysis. competing interests: there is no conflict of interest in this research. references ahmed, t., & wang, c.-k. 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(2018). effect of drying processes on prenylflavonoid content and antioxidant activity of epimedium koreanum nakai. journal of food and drug analysis, 26(2), 796–806. https://doi.org/10.1016/j.jfda.2017.05.011 http://www.ijias.issr-journals.org/abstract.php?article=ijias-13-005-01 http://www.ijias.issr-journals.org/abstract.php?article=ijias-13-005-01 https://doi.org/10.1016/j.jfda.2017.05.011 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 519-527 | doi: 10.14421/biomedich.2025.141.519-527 issn 2540-9328 (online) phytochemicals composition and anti-bacterial activity of methanol leaves extract of vernonia amygdalina abdulganiyu mohammad galadima1,*, amaechi jessica chidimma2, ibrahim abubakar3, shehu ibrahim4, said sani said5 1department of biochemistry, school of biological sciences, federal university of technology owerri, imo state, nigeria 2department of biotechnology, school of biological sciences, federal university of technology owerri, imo state, nigeria 3department of biology-chemistry, idris koko technical college, farfaru, sokoto, nigeria 4department of chemistry, shehu shagari university of education, sokoto, nigeria 5department of biochemistry and molecular biology, faculty of life science, federal university, dutsinma, katsina, nigeria. corresponding author* amgaladima001@gmail.com manuscript received: 23 maret, 2025. revision accepted: 23 july, 2025. published: 01 august, 2025. abstract bacterial infections have been a major health challenge associated with high morbidity and mortality rates. plants and their metabolite constituents are important in local therapies and drug synthesis. this study aimed to evaluate the phytochemical composition and antibacterial activity of the methanol leaves extract of vernonia amygdalina. phytochemicals in the extract were estimated using standard analytical methods. the antibacterial activity test of the plant extract was carried out using the agar diffusion method. minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) were determined using the tube dilution method and subculturing technique, respectively. the extract contains significant amount of alkaloids (47.44 %), flavonoids (16.60 %), tannins (4.35 %), saponins (12.28 %), steroids (0.86%), and glycosides (0.18%). the extract exhibited significant (p < 0.005) inhibitory effect against staphylococcus aureus, streptococcus agalactiae, and lactobacillus rhamnosus isolates with mic and mbc values of 12.45 and 25.51 mg/ml, 22.03 and 44.84 mg/ml, and 31.64 and 63.95 mg/ml, respectively. the methanol leaves extract of vernonia amygdalina demonstrated an inhibitory effect against staphylococcus aureus, streptococcus agalactiae, and lactobacillus rhamnosus isolates with low minimum inhibitory concentration values. keywords: antibacterial activity; bacterial infection; phytochemicals; vernonia amygdalina. introduction bacterial infections remain a public health problem associated with high morbidity and mortality rates in the world. in general, microbial infections cause over 13 million deaths every year worldwide (abreu et al., 2017). it has been predicted that by 2050 bacterial infections alone will cause 10 million deaths each year worldwide (luo et al., 2024; halawa et al., 2024). the emergence of antibacterial drug resistance remains a major challenge in the treatment of bacterial infections. several bacteria demonstrate rapid and high resistance development to various antibiotics (pacheco et al., 2022; tiseo et al., 2022). it was predicted that almost all bacteria will be resistant to the majority of the current antibiotics within 25 years (decker et al., 2024; luo et al., 2024). this will increase the high mortality rates associated with bacterial infections. however, conventional antibiotics have serious side effects and are expensive to purchase by the majority of the communities worldwide. hence, there is need for finding highly effective, accessible, safe, and affordable treatments to address these challenges. plants and herbs have been used in the traditional treatment of several diseases for many years ago. plants and herbs have been reported to demonstrate various medicinal properties and pharmacological activities, including antioxidant activity (ibrahim et al., 2024), antimicrobial activity (dagne et al., 2023), antiulcer activity (abubakar et al., 2020a; 2020b; 2021), antidiabetic activity (shubham et al., 2021), antihypertensive activity (sultana & muhammad, 2017), antitrypanosomal activity (goronyo et al., 2022), and analgesic activity (abubakar et al., 2024). many medicinal plants have been reported to demonstrate inhibitory effects against bacteria and modulate microbial antibiotic resistance (dagne et al., 2023; gonfa et al., 2022; degu et al., 2021). medicinal plants constitute various phytochemicals that are important in drug synthesis and development (abubakar et al., 2021). the medicinal properties and pharmacological activities of plants and herbs are attributed to their phytoconstituents. identification and quantification of plants phytochemicals are important to ensure the https://doi.org/10.14421/biomedich.2025.141.519-527 520 biology, medicine, & natural product chemistry 14 (1), 2025: 519-527 effectiveness of phytomedicines (gupta et al., 2023; adhikari pp, paul, 2018). natural products particularly plants and herbs are easily access by most of communities in the world, less expensive to purchase and have less side effects than conventional treatments. vernonia amygdalina is a perennial plant and a member of the family asteraceae and the genus vernonia (ijeh & ejike, 2011). the plant is commonly called ‘bitter leaf’ due to its bitter to taste. vernonia amygdalina is an africa native plant that is abundantly found in sub-saharan african countries (echem & kabari, 2013). vernonia amygdalina has been used by many communities around the world as a chewing stick for promoting oral hygiene. the plant has also been used as a bittering agent and antimicrobial agent in food industries. vernonia amygdalina is locally used in the management of many diseases including infections, malaria, diabetes, dyspepsia, pains, inflammation, gout, tonsillitis, dysentery, constipation, and gastrointestinal diseases (girma et al., 2022; mekonnen et al., 2022; habtamu & melaku, 2018). however, the plant has been reported to demonstrate anti-sickling activity, plaque development inhibitory activity, and regulated hemostasis activities (satria et al., 2023). in nigeria, vernonia amygdalina has been cultivated in many local communities and is locally known as ‘shuwaka’ (hausa), ‘onugbu’ (igbo) and ‘ewuro’ (yoruba). leaves of the plant have been used in preparation of bitter leaf soup in the country especially in the local communities where the plant is mostly available. also, aqueous extracts of the plant have been consumed by the communities as tonics for the management of several diseases. this study was conducted to evaluate the phytochemical composition and anti-bacterial activity of methanol leaves extract of vernonia amygdalina. materials and methods drugs and chemicals amoxicillin manufactured by amagesan (ritsert, ger. germany) was purchased from the pharmacy unit of federal medical center owerri, imo, nigeria. the chemicals manufactured by cormart nigeria limited (lagos, nigeria) and maoming xiongda chemical co., limited (guangdang, china) were used in the current study. all the chemicals were of analytical grade. plant material fresh leaves of vernonia amygdalina were obtained from akabo, ahiazu-mbaise, in ikeduru local government area, imo state, nigeria. the plant materials were identified by a botanist at farm unit, centre for agricultural research and extension, federal university of technology, owerri, imo, nigeria. extracts preparation the plant samples were washed thoroughly and then crushed into pieces using mortar and pestle. the samples were dried at room temperature for twenty one days and then grinded to fine powder. the preparation of the extract was done using the method of abubakar et al. (2021). the powdered leaves (500 g) were soaked in 1.5 l of methanol for three days with constant shaking at two hours intervals. the extract was filtered using bacterial membrane filter and the filtrate was concentrated to dryness. the weight and percentage yield of the extract obtained was 21.2 g and 4.24 %, respectively. qualitative phytochemicals analysis alkaloids test alkaloids presence in the extract was evaluated using wagner's test as described by trease and evans (1989) and abubakar et al. (2022; 2020). two miles of the extract was transferred into the test tubes followed by addition of three miles of one percent hydrochloric acid. the contents were incubated at 60 oc for twenty minutes and then cold at room temperature. a few drops of the wagner’s reagent were added into the test tube. a reddish-brown precipitate formed which indicates the presence of alkaloids. flavonoids test the sodium hydroxide test was used to identify flavonoids in the extract using the methods of mosa et al. (2012) and ibrahim et al. (2024). the extract (2 ml) was treated with one mile of ten percent sodium hydroxide solution. the development of an intense yellow colour later colourless following the addition of hcl solution showed the presence of flavonoids in the extract. glycosides test the assessment of glycosides in the extract was done using salkowski’s test as stated by mosa et al. (2012) and ibrahim et al. (2024). an equal volume of the extract and one percent sulphuric acid (5 ml) was taken into a test tube, boiled for fifteen minutes and then cold. five miles of fehling’s solution a and b were added into the mixture after the addition of ten percent sodium hydroxide solution. glycosides were detected by the development of a brick red precipitate. tannins test tannins in the extract were detected using ferric chloride test described by trease and evans (1989) and ibrahim et al. (2024). the extract (2 ml) was transferred into the test tube containing two miles of five percent ferric chloride solution. blue-green colour was formed which showed the presence of tannins in the extract. saponins test identification test of saponins in the extract was carried out using the froth test recorded by mosa et al. (2012) and trease and evans (1989). the extract (2 ml) was taken into the test tube and then diluted with two miles of galadima et al. – phytochemicals composition and anti-bacterial activity of … 521 distilled water. the contents were thoroughly mixed and settled for half an hour. saponins in the extract were identified by observing stable persistent froth. steroids test steroids in the extract were identified using the method of trease and evans (1989) and ibrahim et al. (2024). an equal volume (5 ml) of chloroform and sulphuric acid solution was added into the test tube containing one mile of the extract. the violet colour later blue-green indicated the presence of steroids in the extract. quantitative phytochemicals analysis alkaloids test the alkaloid content in the extract was estimated using the method of trease and evans (1989) and ibrahim et al. (2024). twenty miles of 2 mm h2so4 was added into the test tube containing the extract. the mixture was shaken and then partitioned with ether. the upper phase liquid was treated with nh3 solution followed by extraction with chloroform solvent. the alkaloid residue obtained after drying the extract was weighed and the alkaloid content was obtained. flavonoids test the total flavonoids in the extract were determined using the method described by harborne (1973) and ibrahim et al. (2024). five milligrams of the extract was treated with fifty miles of 2 m hydrochloric acid, boiled for thirty minutes and then cooled. the mixture was filtered and the filtrate was treated with fifty of ethyl*acetate solution. the mixture was filtered and then concentrated to dryness. the dried flavonoid residue was weighed and the flavonoid content was obtained. glycosides test ten miles of the extract was mixed with fifty miles of chloroform in a conical flask. the contents were mixed, filtered, and the filtrate was treated with ten mile of pyridine and two mile of two percent sodium nitroprusside. the mixture was shaken for ten minutes and then treated with 3ml of twenty percent naoh. the absorbance was measured spectrophotometerically at 510 nm wavelength and the glycosides content was obtained (ibrahim et al., 2024). tannins test aoac (1999) was employed to quantitative estimate of tannins in the extract. two miles of folin-denis reagent and one mile of sodium carbonate solution were added into the flask containing the extract and the prepared standard solution. the contents were allowed to stand at 25 oc for thirty minutes. the absorbance was read using spectrophotometerically at 760 nm wavelength. the amount of tannin in the extract was obtained from the prepared standard curve. saponins test the saponins content in the extract was estimated using the method of el-olemyl et al. (1994). one hundred and fifty miles of fifty percent ethanol was added into the flask containing five grams of the extract. the contents were boiled for half hour, cooled and then filtered. the filtrate was treated with charcoal, heated for half hour, and then filtered. one hundred and fifty miles of acetone was added into the filtrate and the mixture was filtered. the filter paper was quickly taken into the desiccator containing anhydrous cacl2 solution. the saponins residue was weighed and saponins content was obtained. steroids test quantitative analysis of steroids in the extract was performed using the method of trease and evans (1989) and ibrahim et al. (2024). two miles of sulphuric acid and ferric chloride were added into the test tube containing one mile of the extract. two miles of potassium hexacynoferrate (iii) was added into the test tube and heated at 70 °c for thirty minutes. the absorbance was measured spectrophotometerically at 780 nm wavelength and the steroid content was obtained. bacterial isolates the bacterial isolates used in this study were staphylococcus aureus, streptococcus agalactiae, and lactobacillus rhamnosus. the pure bacterial isolates were obtained from the microbiology laboratory, of federal medical center owerri, imo, nigeria. the isolates were sub-cultured from the original agar plates and transferred to macconkey agar and selenite f broth media plates. the isolates were re-identified and confirmed using the gram staining technique and standard biochemical tests. antibacterial activity test the antibacterial susceptibility test of the plant extract was carried out using the agar diffusion method as described by kirby-bauer (1996) with some modifications. the inoculums of the bacterial isolates were standardized by taking loopful colonies of the bacterial isolates into nutrient agar plates. the plates were incubated overnight at 37 °c and then the turbidity developed was equally adjusted to mac farland standard (0.5) value. the agar discs of diameter 6 mm were aseptically dipped in 2 ml of 25, 50, 75 and 100 mg/ml extract for sixty seconds and then placed over the separate nutrient agar plates each containing the labeled test isolates. the plates were incubated at ambient temperature for half an hour and then at 37 °c overnight. the plates were observed for the development of the zone of inhibition which was measured in millimetres using the meter rule. amoxicillin (30 µg) (udensi et al., 2025) was used as a reference standard control. the experiment was carried out three times and the mean results were calculated. 522 biology, medicine, & natural product chemistry 14 (1), 2025: 519-527 determination of minimum inhibitory concentration the minimum inhibitory concentration (mic) of the extract against the isolates was determined using the tube dilution method. a loopful of the bacterial isolates was transferred into the test tubes containing 5 ml of nutrient broth. one mile of the extract (100, 75, 50, and 25 mg/ml) was added into the test tubes. a test tube containing only broth and extract was used as control. the test tubes were incubated at 37°c for one day and the bacterial growth was observed. the lowest concentration of the extract that inhibited the observed bacterial growth was considered the mic of the extract. determination of minimum bactericidal concentration the minimum bactericidal concentration (mbc) of the extract was evaluated using the sub-culturing method. the mic broth culture tubes with no growth observed were sub-cultured onto fresh nutrient agar plates. the plates were incubated at 37 ℃ for two days and then observed for visible growth. the lowest extract concentration with no visible growth was considered the mbc of the extract. statistical analysis all the analyses were performed in triplicate. the results were statistically analyzed using statistical package for social sciences (spss) version 22 software and expressed as mean ± sem. significant differences between the mean results were computed at a 95 % confidence level by one-way analysis of variance (anova). significance was considered by two-tailed (p < 0.05) values. results phytochemicals composition of methanol leaves extract of vernonia amygdalina table 1 shows the phytochemical screening of the methanol leaves extract of vernonia amygdalina. the result showed that methanol leaves extract of vernonia amygdalina contain high amount of alkaloids and flavonoids. a moderate amount of tannins and saponins was observed in the extract. the extract exhibited a low amount of steroids and glycosides (table 1). table 1. qualitative phytochemicals screening of methanol leaves extract of vernonia amygdalina. phytochemical va extract alkaloids +++ flavonoids +++ tannins ++ saponins ++ steroids + glycosides + +++ (high amount), ++ (moderate amount), + (low amount) figure 1 shows the quantitative phytochemicals constituents of methanol leaves extract of vernonia amygdalina. the methanol leaves extract of vernonia amygdalina contains a significant amount of alkaloids (47.44 %), flavonoids (16.60 %), tannins (4.35 %), saponins (12.28 %), steroids (0.86%), and glycosides (0.18%) (figure 1). figure 1. quantitative phytochemicals constituents of methanol leaves extract of vernonia amygdalina values are expressed as mean ± sd (n = 3). inhibitory effect of methanol leaves extract of vernonia amydalina on some bacterial isolates figure 2 shows the inhibitory effect of methanol leaves extract of vernonia amydalina on staphylococcus aureus isolate. the methanol leaves extract of vernonia amydalina demonstrated inhibitory effect against s. aureus in dose dependent manner. at 25 mg/ml, 50 mg/ml, 75 mg/ml, and 100 mg/ml the extract demonstrated significant (p < 0.05) inhibition of 7.36 mm, 14.43 mm, 15.47 mm, and 17.16 mm against the isolate, respectively. however, the highest significant inhibition (17.16 mm) against the isolate was observed at 100 mg/ml dose of the extract which is more than the galadima et al. – phytochemicals composition and anti-bacterial activity of … 523 zone of inhibition (16.12 mm) exhibited by the standard drug, amoxicillin (figure 2). figure 2. inhibitory effect of methanol leaves extract of vernonia amydalina on staphylococcus aureus isolate data are expressed as mean ± sd (n = 3), va (vernonia amydalina), amx (amoxicillin). the inhibitory effect of methanol leaves extract of vernonia amydalina on streptococcus agalactiae isolate is shown in figure 3. a dose dependent inhibitory effect of the methanol leaves extract of vernonia amydalina against streptococcus agalactiae isolate was observed. the zone of inhibition was not observed at 25 mg/ml dose of the extract. a significant (p < 0.05) inhibition of 6.76 mm, 12.47 mm, and 16.83 mm against the isolate was observed at 50 mg/ml, 75 mg/ml, and 100 mg/ml dose of the extract, respectively. interestingly, at 100 mg/ml the extract demonstrated significant inhibition (16.83 mm) against the isolate more than the standard drug, amoxicillin (14.50 mm) (figure 3). figure 3. inhibitory effect of methanol leaves extract of vernonia amydalina on streptococcus agalactiae isolate values are given as mean ± sd (n = 3), va (vernonia amydalina), amx (amoxicillin). figure 4 indicates the inhibitory effect of methanol leaves extract of vernonia amydalina on lactobacillus rhamnosus isolate. the results showed that methanol leaves extract of vernonia amydalina significantly (p < 0.05) inhibited lactobacillus rhamnosus isolate in dose dependent manner. at 25 mg/ml, the extract did not showe any inhibitory effect against the isolate. at 50 mg/ml, 75 mg/ml, and 100 mg/ml, the extract exhibited a significant (p < 0.05) inhibition of 9.45 mm, 12.12 mm, and 17.19 mm against the isolate, respectively. however, at 100 mg/ml the inhibitory effect of the extract against the isolate (17.19 mm) was more than the inhibitory effect (15.27 mm) of the standard drug, amoxicillin (figure 4). 524 biology, medicine, & natural product chemistry 14 (1), 2025: 519-527 figure 4. inhibitory effect of methanol leaves extract of vernonia amydalina on lactobacillus rhamnosus isolate results are expressed as mean ± sd (n = 3), va (vernonia amydalina), amx (amoxicillin). mic and mbc value of the methanol leaves extract of vernonia amydalina against the bacterial isolates the mic and mbc values of the methanol leaves extract of vernonia amydalina against the bacterial isolates are shown in figure 5. the extract demonstrated mic and mbc values of 12.45 and 25.51 mg/ml, 22.03 and 44.84 mg/ml, and 31.64 and 63.95 mg/ml against the staphylococcus aureus, streptococcus agalactiae, and lactobacillus rhamnosus isolates, respectively. figure 5. mic and mbc value of the methanol leaves extract of vernonia amydalina against the bacterial isolates results are expressed as mean ± sd (n = 3). discussion in this study, the methanol leaves extract of vernonia amygdalina demonstrated inhibitory effect against staphylococcus aureus, streptococcus agalactiae, and lactobacillus rhamnosus isolates. this finding is in line with the findings of the relevant study which showed that ethanol leaf extract of vernonia amygdalina demonstrated inhibitory effect against s. aureus and shigella (ogundare, 2011). a research study by zubairu et al. (2019) showed that vernonia amygdalina leaf extract exhibited an anti-bacterial effect against e. coli, s. aureus and s. typhi. this finding indicated that methanol leaves extract of vernonia amygdalina contains a significant amount of secondary metabolites which include alkaloids, flavonoids, tannins, saponins, steroids, and glycosides. results of this study are in agreement with the findings of similar studies by ali et al. (2019) and olumide et al. (2019), who found that vernonia amygdalina contained significant amounts of phytochemicals such as saponins, alkaloids, flavonoids, terpenoids, tannins, steroids, and phenols. significant amounts of alkaloids, glycosides, steroids, flavonoids, tannins, and terpenoids were reported in vernonia amygdalina (senthilkumar et al., 2018; tian et al., 2023). the anti-bacterial activity of the methanol leaves extract of vernonia amygdalina could be attributed to the secondary metabolites present in the plant extract. phytochemicals such as alkaloids, flavonoids, terpenoid, phenolics, and tannin have been shown to demonstrate antimicrobial activity (usunomena & ngozi, 2016). alkaloids and their synthetic derivatives have the potential to inhibit bacterial growth (atinga et al., 2021). alkaloids isolated from the plant’s extract exhibited significant antibacterial activity due to its inhibitory effect on bacterial topoisomerase causing dna damage (khameneh et al., 2029). berberine, an alkaloid isolated 0 10 20 30 40 50 60 70 staphylococcus streptococcus lactobacillus m ic /m b c ( m g/ m l) mic bmc galadima et al. – phytochemicals composition and anti-bacterial activity of … 525 from different plants extracts demonstrated inhibitory effect against gram-negative and gram-positive microorganisms (wu et al., 2022; xia et al., 2022). the study showed that berberine exhibited mic value of 51 μg/ml against s. aureus strain atcc 25923 (wu et al., 2022). the antimicrobial effect of berberine was due to its efflux pump inhibitory potential (li & ge, 2023) or its potential to influence the activity of the shikimic acid pathway in bacteria causing oxidative damage and disrupting integrity of cell membranes (wu et al., 2022; xia et al., 2022). phenolic compounds including phenols and flavonoids have been documented as potent antimicrobial agents (ecevit et al., 2022; porras et al., 2021). panduratin a isolated from different plant extracts showed anti-bacterial activity against s. mutans, s. sobrinus, p. intermedia, p. loescheii, and p. gingivalis (park et al., 2005). clinical studies showed that panduratin a exhibited anti-bacterial effect against many isolates of staphylococcus strains (rukayadi et al., 2009). in the present study, the extract exhibited low minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) values against staphylococcus aureus, streptococcus agalactiae, and lactobacillus rhamnosus isolates antimicrobial activity of agent or substance is determined by its minimum inhibitory concentration values. minimum inhibitory concentration (mic) refers to the lowest concentration of tested antimicrobial agent that inhibits or kills the growth of tested microbe (balouiri et al., 2016). plant extracts with mic values below 100 µg/ml are considered to demonstrate high antibacterial activity, while from 100 to 625 µg/ml moderate activity, and above 625 µg/ml low activity (famuyide et al., 2019; dzotam & kuete, 2017; voukeng et al., 2016). conclusion the methanol leaves extract of vernonia amygdalina contains significant amounts of phytochemicals. the extract demonstrated an inhibitory effect against staphylococcus aureus, streptococcus agalactiae, and lactobacillus rhamnosus isolates with low minimum inhibitory concentration values. the inhibitory effect of the extract against the staphylococcus aureus, streptococcus agalactiae, and lactobacillus rhamnosus isolates could be attributed to the various phytochemicals present in the extract. conflict of interest: the authors declared that there was no conflict of interest. references abreu, a.c., coqueiro, a., sultan, a.r., lemmens, n., kim, h.k., verpoorte, r., van-wamel, w.j.b., sim˜oes, m., & choi, y.h. 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(2019). antibacterial activity of methanolic extract of bitter leaf (vernonia amygdalina) from various component fractions using column chromatography. gsc biological and pharmaceutical sciences, 7(2), 16 – 21. https://doi.org/ https://doi.org/ this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 459-465 | doi: 10.14421/biomedich.2024.132.459-465 issn 2540-9328 (online) the new technology for in vitro culture with induction of nanobubbles (nbsn2 and nbso2) in cattleya sp. moch. mustakim, azis mawardi, lina purnama dewi, nela vede rhofa putri, tintrim rahayu, gatra ervi jayanti* biology department, faculty of mathematics and natural science, universitas islam malang, jl. mt. haryono no 193 malang 65141, tel. 0341 551932, fax. 0341552249, indonesia. corresponding author* gatra.ervi@unisma.ac.id abstract the growth and development of orchids take a relatively long time, while the demand for high-quality orchids continues to increase significantly. tissue culture is a technique of isolating plant parts in organs, tissues, and cells and then culturing these plants on artificial media in a sterile environment. tissue culture techniques can reproduce plants in a relatively short time, with the same properties and quality, so efforts to optimize tissue culture with technology are the right solution, one of which is using nanogenerator technology that produces nanobubbles (nbs). nbs are one of the nanotechnologies that are ≤100 nm in size with induced gas needed by plants. this study aims to determine the effectiveness of growth and development from the induction of nbsn2 and nbso2 in cattleya sp. planlet in vitro culture. this research method uses in vitro culture with a randomized group design. the results of the mso treatment showed higher weight than other treatments, with a total plant weight value of 0.3550 g and a total plant height value of 1.3983 cm. murashige-skoog + nbsnitrogen (msn) treatment showed higher root length than other treatments with a total value of 1.2367 cm. in the treatment of ms, mso, oj and nj, a live percentage of 83-100% was observed. the statistical test results indicate that nbs positively affect cattleya sp. plantlets. this effect includes increasing plant weight, height, and root length and promoting new shoots growth. additionally, nitrogen supplements play a role in colour changes, indicating the plant's health and photosynthetic efficiency. in the treatment with induction o2, the initial colour changes from strong yellow green (141 d) to strong yellowish green (141 c), while in the treatment with induction nbsn2, the initial colour changes from strong yellow green (141 d) to deep yellow green (141 b). keywords: nanobubbles; nbso2; nbsn2; cattleya sp.; tissue culture. abbreviations: nbs: nanobubbles; o2: oxygen; n2: nitrogen; laf: laminar air flow; ats: air treatment sanitizer; phc: plant healthy check, media with a thinwall container (ms: murashige-skoog, mso: murashige-skoog + nanobubbles oxygen, msn: murashige-skoog + nanobubbles nitrogen, o: nanobubbles oxygen, n: nanobubbles nitrogen); media with a jar bottle container (msj: murashige-skoog, msoj: murashige-skoog + nanobubbles oxygen, msnj: murashige-skoog + nanobubbles nitrogen, oj: nanobubbles oxygen, nj: nanobubbles nitrogen). introduction the development of science and technology in all fields is a new challenge in developing a better system. nanobubbles (nbs) are one of the latest technologies in the world of biology they are focused on improving their quality and quantity (haryanto et al., 2022). nbs are filled with gases needed by plants, such as oxygen (o2), carbon dioxide (co2), nitrogen (n2) and others. nbs are gas cavities with a radius of approximately tens to hundreds of nanometers (nm) (rahayu et al., 2023). with a tiny size, plants can easily and quickly absorb a solution, nanobubbles can significantly increase plant height and root length in plants. nbs treatment stimulates the synthesis of the growth hormone gibberellin and increases the regulation of plant nutrient absorption genes, thereby increasing the absorption and utilization of nutrients by the roots (wang et al., 2021). proven in the research of ahmed et al. (2018), the use of nbs can accelerate seed germination time and seed growth; rahayu (2023) also proves that the efficiency of using nbs in accelerating orchid growth is better with greater stem diameter growth than not using nbs. in addition, orchids have a relatively long growth period with market consumer demand that tends to increase. orchid data globally reaches 25,000-30,000 species, while orchids in indonesia consist of ± 5,000 species (nisa et al., 2021). orchid flowers have durability or freshness over a relatively long period and are a factor in the high selling value of orchids, one of the most popular orchid species is cattleya sp. which is nicknamed the "queen of orchids" because it has large flowers (yasmin, 2018). in manuscript received: 12 june, 2024. revision accepted: 30 september, 2024. published: 22 october, 2024. https://doi.org/10.14421/biomedich.2024.132.459-465 460 biology, medicine, & natural product chemistry 13 (2), 2024: 459-465 a relatively long and complex production process, orchid breeding needs to consider several factors including air, water, light, food (nutrients) growth regulators are in a balanced state (firmansyah et al., 2023). according to latifah (2017), the demand for high-quality orchids in indonesia continues to increase yearly. however, the volume of imports still surpasses the volume of exports. to meet this demand, one practical approach is to utilize tissue culture for mass orchid propagation, as it allows for faster and more manageable growth, thereby addressing the market needs. tissue culture is defined as isolating plant parts (organs, tissues, cells or protoplasm) and then growing them on artificial media under sterile and controlled environmental conditions. the benefits of this system are that it can be used for plant propagation in a relatively short time, with the same properties and quality (yachya et al., 2022). this technique has several advantages over traditional methods, because in addition to producing large numbers of plants quickly, this technique is also independent of the season (putriana et al., 2019). nbs are one of the solutions in optimizing this technique, using n2 and o2 which can significantly effect plant growth and development as a fulfilment of nutrients, can also maintain market demand for quality orchids with the appropriate quantity. this research aims to determine the growth effectiveness of the inducing of nbsn2 and nbso2 on cattleya sp. planlets in vitro culture. therefore, optimization in the world of tissue culture needs to be continuously developed by accelerating plant growth and development effectiveness. materials and methods study area this research was conducted at the orchidology laboratory and nursery in universitas islam malang, located at jl. mt haryono no. 193 dinoyo malang city. the research method uses in vitro culture applying a randomized group design with nbso2 and nbso2 in liquid media tested as an addition to the media on the effect of growth and development on orchids cattleya sp. in this study, there were two groups with five treatments each and repeated six times so that there were 72 experimental units. thinwall group, there are media; murashige-skoog (ms), ms + nbso2 (mso), ms + nbsn2 (msn), nbso2 (o), nbsn2 (msn), while the jar group, there are media; murashige-skoog (msj), ms + nbso2 (msoj), ms + nbsn2 (msnj), nbso2 (oj), nbsn2 (nj). observation parameters consisted of plants weight (g), number of live plant (%), length of leaves and roots of plant (cm), plant colour, and contamination. the tools used in this study were laminar air flow (laf), air treatment sanitizer (ats), "nanogenerator (yixing holly technology co.., ltd, china), large tweezers and small tweezers, sterile blade, scissors, thinwall, beaker, measuring cup, petri dish, ph meter (multifunction), micropipette, sterile tip, bunsen, lighter, magnetic stirrer (thermo scientific), analytical balance (ohaus), alcohol sprayer, sterile wipes, tissues, markers, large plastic wrap and small plastic wrap, stove, plastic, and pipettes, autoclave, millimeter block, magnifying glass, green pointer and royal horticultural society (rhs) color chart. meanwhile, the materials used in this research are 7-month-old cattleya sp orchids, nbso solution2, nbsn solution2 70% alcohol, 96% alcohol, sterile water, stock solution a: nh4no3 (smartlab a2131), b: kno3 (smartlab a-2131), c: cacl2.h2o (smartlab a-2114), d: mgso4.7h2o (smartlab a2024) and kh2po4 (smartlab a-2024), e: feso4. 7h2 o.na2 edta.2h2o (smartlab a-2059), f: h3o3 (smartlab a-2013), mnso4.4h2o (smartlab a-2100), and znso4. 7h2o (smartlab a-2100), and g: cl (smartlab a-2038), na2 moo4.2h2o (himedia pct0117), cuso4. 5h2 o (himedia pct0104), and cocl2.6h2 o (himedia pct0117), vitamin: nicotinicacid (phygenera), pyridoxine-hcl, thiamine-hcl (caisson lab), and glycine (caisson g007), methionine (caisson lab), peptone (himedia pct0806), myooinositol (phygenea ad102), naphthaleneacetic acid (naa), -benzylaminopurine (bap), sugar, and distilled water. procedure the stage begins with making plant healthy check (phc) media by preparing a 30% sugar solution by dissolving 300 mg of granulated sugar in 700 ml of distilled water and homogenizing it with a hot plate and magnetic stirrer; the resulting solution is then stored in bottles for the subsequent sterilization process. in this study, the media used were ms stock solution as well as naa and bap solutions, measured with a micropipette and homogenized in a jar using a magnetic stirrer at speed 250 rpm. once homogeneous, the media was cooked on the stove until completely dissolved, followed by ph measurements until it reached the ph value of 5.4. the aim of the stage of providing phc media is to check the health of cattleya sp. planlets. by placing it in a sterile container and jar, 400 μl of phc medium was added. the thinwalls containers and jar bottles were then covered tightly with plastic wrap, the tops were placed in a shaker, and the thinwalls were placed on an inclined surface for three days. sterilize tools and materials using an autoclave at 121ºc and 2 psi for 15 minutes. then sterilization of the nanogenerator was achieved with a h2o2 3% solution and sterile distilled water, the solution was fed into the nanogenerator through the machine's output and input channels, followed by the same treatment with sterile distilled water. nbsn2 and nbso2 were made by inducing sterile air with n2 and nbso2 gas using a nanogenerator machine for 15 minutes. nbs filtering is done with a green pointer to confirm the presence of nbs through the waves created by the green mustakim et al. – latest technology of in vitro culture 461 light. nb induction involves treating healthy orchid plantlets with media induced with nbsn2 and nbso2, the initial step is to remove the phc media, then add 400-600 μl of media into the plantlets using a micropipette. the thinwalls and the jar are covered with plastic wrap, the jar is placed on a shaker, and the thinwalls are placed at an angle. providing media to planlet was done by placing orchid plantlets in a jar to be transferred to a shaker, while plantlets located on thin sloping walls received media daily for 5 minutes at 10 am and 2 pm. final plantlet measurements included initial and final assessments of weight, plant height, root length, colour and contamination, which were carried out at laf. weight was measured using digital scales, root and leaf dimensions were measured using millimetre blocks and petri dishes, and the colour of orchid planlet was assessed using rhs paper. data analysis the data in this study were analyzed through two approaches, namely quantitative analysis and qualitative analysis. quantitative analysis involved processing observational data using the multivariate analysis of variance (manova) method, followed by bonferroni testing if there were significant differences. meanwhile, qualitative analysis involved reading and analyzing the research results, which were then presented through tables, figures, and graphs. results and discussion screening nanobubbles (nbs) the screening results show that the bottle containing nbso2 has a straight line on the bottle containing nbso2 which indicates the presence of nano bubbles in the solution (figure 1). in contrast to sterile water, there is no straight line on the sauce bottle, so it can be concluded that the nbso2 used in the study contains nanobubbles. figure 1. screening of nbsn2 and nbso2 using green pointer: (a) nbso2 (b) aquades (c) nbsn2 plant weight and height of orchid cattleya sp. induction of nbs oxygen showed indicators of changes in the weight and height of orchids cattleya sp. mso treatment showed a higher average weight than other treatments with a total plant weight value of 0.3550 g and a total plant height value of 1.3983 cm. bonferroni test showed no significant difference in plant weight in each treatment, while in plant height there was a significant difference in the mso treatment against oj, nj, and ms (figure 2). figure 2. average weight and height of cattleya sp. planlets in the treatment groups of thinwall containers and jar bottles root length analysis of orchid cattleya sp. nbsn2 induction affects the root length of cattleya sp. orchids as evidenced by the results of msn treatment showing a higher average root length than other treatments with a total value of 1.2367 cm. however, the results of the bonferroni test of root length parameters show that each treatment is not significantly different (figure 3). figure 3. average root length of cattleya sp. planlets in thinwall container and jar bottle treatments. percentage alive and number of new shoots the ms, mso, oj and nj treatments showed the highest average percentage of live planlets with a total value of 100%. however, the graph did not show any significant difference in the average percentage value between 83.33-100%, contamination of the planlets occurred in mso 6th replication day 26. the test results also showed that the msn treatment (figure 4) showed a higher a green pointer b c 462 biology, medicine, & natural product chemistry 13 (2), 2024: 459-465 average number of new shoots than the other treatments with a total value of 3.67 cm. figure 4. average percentage of survival and number of new shoots of cattleya sp. planlets in the treatment of thinwall container groups and jar bottles. plant color at rhs nbs induction affect on plant colour based on observations using rhs, nbs have a role in increasing the color of greener plants. nbso2 induction was able to increase the initial color of strong yellow green (141 d) to strong yellowish green (141 c), nbsn2 induction experienced colour changes from the initial strong yellow green (141 d) to deep yellow green (141 b), while the treatment without nbs induction did not experience colour changes in strong yellow green (141 d) (figure 5). figure 5. changes in color of planlets treated without nbs induction and nbs induction using rhs parameters. discussion nanotechnology with nbs products is one of the latest technologies applied to plants as a breakthrough in improving the quality of plants, one of which is the orchid itself. nbs have a rapid response that can affect the size of the orchid diameter because nbs can provide a transportation mechanism for gas delivery to membranes or cells thereby affecting trans membrane proteins or membrane structure, hydrophobic gases will push into the membrane and thus will change the curvature of membrane which will have an effect on trans membrane proteins or global effects on membrane structure (rahayu et al., 2023). screening of nbs was performed to confirm the presence of nbs in solution. screening is done using a laser pointer and if there is a straight line it indicates the presence of nbs caused by the tyndall effect (han et al., 2023). the observation results show in figure 1 that there is a tyndall effect on the induction of nbs in both o2 and n2 with a straight line on the laser beam from the green pointer while in figure 1 b, which is the treatment without induction of nbs (aquades) there is no straight line of the green pointer beam. the test results show that nbso2 influences the weight and height of cattleya sp. orchid plants. according to rahayu et al., (2023) stated that the diameter of the stem of the largest orchid plant in the nbso2 treatment, therefore the additional weight is caused by the application of nbso2 in orchid tissue culture. the use of o2 gas induced through the nanogenerator functions in absorbing nutrients that are useful for the formation of new tissues and plants getting bigger (krisna, 2017). nbso2 has a tiny bubble size and low buoyancy so it can quickly enter plant cells including mitochondria. oxygen that enters will be easily absorbed by plants and used for the respiration process. the process of respiration in plants will produce energy for various activities such as energy metabolism (carbon fixation in photosynthesis organisms), signal transduction (transduction of plant hormone signals), and environmental adaptation. nbso2 can increase the respiration process to produce much greater energy and biological processes in plants take place quickly which causes faster growth of orchid plants (figure 6) so that plants have good and fast energy transfer in the growth process and make plants have a faster weight and height from the use of nbso2 media. figure 6. nbso2 mechanism in cattleya sp. planlets. based on the research of wang et al. (2021) showed that the effect of nbs on plant growth and nutrient absorption was evaluated in the laboratory. laboratory experiments showed that nbs significantly increased mustakim et al. – latest technology of in vitro culture 463 plant height and root length in rice seedlings, with nbs treatment stimulating the synthesis of growth hormone gibberellin and upregulating plant nutrient absorption genes, namely osbt, pit-1 and skor, resulting in increased absorption and utilization of nutrients by roots. the highest increase in root length was shown in the treatment with nbsn2 induction, with an average value of 1.2367 cm. roots are a vital in the plant's response to water shortage. one of the essential that must be evaluated is root morphology, because roots can absorb water by maximizing the root system. plants with large roots will be able to absorb more water than plants with smaller roots (ai & torey., 2013). increased absorption and utilization of nutrients by plant roots. the number of roots in the use of tissue culture techniques indicates that orchid plants are able to absorb nutrients in the media well. a larger number of roots also indicates that the reach of the plant is wider and the plant absorbs more nutrients and nutrients, so that the spread of nutrients from the media to the plant can be done smoothly (rahma, et al., 2018). the use of nbsn2 has great power over the growth and development of orchids, the results show that nbsn2 has the potential to maintain the consistency of orchids in the percentage of life and the number of new shoots that are more than non-nbsn2. according to patti et al. (2013), n2 functions as a synthetic material for chlorophyll, proteins, and amino acids. n2 in the vegetative phase functions to increases vegetative growth, which includes increasing the number of tillers, grains per hill, and plant size. the growth regulators auxin and cytokinin in the media influence the emergence of buds. the primary role of auxin is to stimulate roots in stem and leaf cuttings and increase root branches, the formation of roots begins with the metabolism of nutrient reserves in the form of carbohydrates which produce energy which in turn encourages cell division and forms new cells in the tissue (cytokinin functions to accelerate cell division resulting in cell enlargement, while auxin spurs cell elongation which will then differentiate and elongate plant tissue) (kusuma et al., 2023). nbs can stimulate the enzymes ent-kaurene synthase (ks) and ent-kaurene oxidase (ko) as the synthesis of growth the hormone gibberellin that occurs in plastids. the enzyme kaurene is converted to 7-alpha-hydroxykaurenoic acid and then converted to a12 aldehyde in the endoplasmic reticulum. a12 aldehyde is gibberellin (wang et al., 2021). gibberellin stimulates the formation of amylase and protease enzymes through de-nevo synthesis which plays a role in the breakdown of amylum into glucose thereby accelerating embryo emergence. breaking down amylum into glucose produces energy that is used for the cell differentiation process so that a plumula is formed, which is the beginning of leaves and stems and a radicle which is the beginning of roots. in addition, gibberellin will spur the formation of enzymes that soften the cell wall, especially proteolytic enzymes that release amino tryptophan (auxin precursor/former) so that auxin levels increase. (igielski & kępczyńska, 2017; wulandari et al., 2014). based on the research results, the pattern/mechanism of nbs in increasing orchid growth was obtained (figure 7). figure 7. mechanism of nbs in enhancing orchid growth. on the other hand, the role of nbsn2 in research is visually proven to improve the quality of planlets. n2 is a synthetic material for chlorophyll, proteins, and amino acids (patti et al., 2013). the increase in the rhs code of 464 biology, medicine, & natural product chemistry 13 (2), 2024: 459-465 leaf color shows the effect on absorption, which is more easily absorbed due to the induced solution of nano-sized n2, which is able to play its role in chlorophyll synthesis. figure 8. mechanism of entry of nbsn2 into cattleya sp. planlets. the increase in the rhs code of leaf color shows the effect on easier absorption due to the nano-sized n2 induced solution that can play its role in chlorophyll synthesis (figure 8). in the homogeneity test on plant weight, plant height, and root length to determine further tests, the results obtained a significance value of more than 0.05 (homogeneous data), which means the sig. >0.05, it can be concluded that the data variance is homogeneous, so the further test used is bonferroni. at the further test stage using the bonferroni test with multivariate tests, there are four statistical tests, namely pillai's trace, wilk's lambda, hotelling trace, and roy's largest root. these four tests are based on eigenvalues. the multivariate test results showed sig. <0.05, which stated that there was an effect of treatment on the observed variables. the analysis was continued with the univariate test to determine the effect of treatment. analysis using the univariate test showed a significance value <0.05, indicating a significant effect of treatment on plant weight, plant height, and root length. the results of the statistical test state that nbs technology affects the induction of cattleya sp. planlets by increasing plant weight, plant height, root length, giving rise to new shoots, and improving the quality of colour in plants. conclusions based on the results of the research that has been done, it can be concluded that the induction of the combination of ms and nbso2 on cattleya sp. planlet provides acceleration of weight and height, while the combination of ms and nbsn2 has an effect on root length, and for the highest presentation of life in the treatment of ms, mso, o, and n. the results of the statistical test state that nbs technology influences the induction of cattleya sp. planlets by increasing plant weight, plant height, root length, giving rise to new shoots, and improving the quality of colour in plants. acknowledgements: thanks to the ministry of education, culture, research, and technology (indonesian: kementerian pendidikan, kebudayaan, riset, dan teknologi, abbreviated kemendikbudristek) and universitas islam malang and thanks also to the orchidology laboratory and nursery in universitas islam malang. authors' contributions: author one prepares the manuscript and designs the research (manuscript), author two analyzes the data, and authors three and four conduct the experiments, while the last author and associate professor directs the research and experimental design. competing interests: the authors declare that there are no conflicts of interest. funding: thanks to the ministry of education, culture, research and technology (indonesian: kementerian pendidikan, kebudayaan, riset, dan teknologi, abbreviated kemendikbudristek) and universitas islam malang for providing grants / research grants in pkm riset eksakta activities in 2023 with the title "the latest technology for in vitro culture with induction of nanobubbles (nbs) n2 and o2 on cattleya sp. planlets". mustakim et al. – latest technology of in vitro culture 465 references ahmed, a.k.a., shi, x., hua, l., manzueta, l., qing, w., marhaba, t., & zhang, w. 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(2014). effect of gibberellin hormone on parthenocarpy fruit formation in cucumber plants of mercy variety. lenterabio, 3(1), 27-32. doi: yachya, a., sopandi, t., slamet, p., binawati, d.k., ngadiani., sukarjati., ajiningrum, p.s., andriani, v. (2022). plants in east java sms biology mgmp teachers. journal of penamas adi buana, 6(1), 31-36. doi: https://doi.org/10.36456/penamas.vol6.no01.a5498 yasmin, z. f. (2018). nursery (tissue culture to enlargement) of phalaenopsis orchid at hasanudin orchids, east java nursery. agrohorti bulletin journal, 6(3), 430-439. doi: https://doi.org/10.29244/agrob.v6i3.21113 firmansyah, m. i., rahayu, & jayati, g. e., agisimanto, d. (2023). comparative study of naphtalene acetic acid (naa) concentration variations on the growth of dendrobium sp. orchid on white and black moss media. journal of metamorphosa, 10(1), 292-301. doi: https://doi.org/10.24843/metamorfosa.2023.v10.i02.p13 ai, n.s., & torey, p. (2013). root morphological characters as water-deficit indicators in plants. journal of bioslogos, 3(1), 32-39. doi: https://doi.org/10.35799/jbl.3.1.2013.3466 rahma, s., rahayu, t., & hayati, a. (2018). study of organic material addition in vw planting media on organogenesis of dendrobium orchid in vitro. e-journal of known nature 1(1), 93 103. doi: https://doi.org/10.33474/j.sa.v1i1.1392 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1219-1223 | doi: 10.14421/biomedich.2025.142.1219-1223 issn 2540-9328 (online) cost effectiveness analysis of favipiravir and oseltamivir as antiretroviral covid-19 of patients in bogor cisarua lung hospital ani rahayu1, dwi puspita sari2, lukluk utami ningsih3, a. triwildan st fatimah4 1,2pharmacy study program at binawan university, jakarta, indonesia. 3cisarua lung hospital, bogor, indonesia. 4raden mattaher hospital, jambi, indonesia. corresponding author* ani.rahayu@binawan.ac.id abstract favipiravir is a type of rna-dependent rna polymerase (rdrp) inhibitor. favipiravir is converted by host enzymes to t-705ribofuranosyl 5′-triphosphate and may act as a nucleotide analogue that selectively inhibits viral rna-dependent rna polymerase or causes lethal mutagenesis upon insertion into viral rna without cytotoxicity in mammalian cells. oseltamivir is part of the neuraminidase inhibitors (nais) group which has a mechanism of action by inhibiting viral neuraminidase. most of the alternative therapies for covid-19 require pharmacoeconomic studies, especially regarding cost-effectiveness analysis which is useful in balancing patient expenses by determining treatment alternatives that represent the best health outcomes at a more affordable cost. the aim of this study was to compare the cost effectiveness of the antiretrovirals favipiravir and oseltamivir. this research method used a descriptive analytic cross-sectional design and data collection was carried out retrospectively from the medical records of covid-19 sufferers while detailed data on medical expenses were obtained from the finance department of inpatients at cisarua pulmonary hospital, bogor for the period september-october 2022. sample of 80 patients consisted of 40 patients using favipiravir and 40 patients using oseltamivir.the parameters used in this research are direct medical costs (which include doctor visit costs, treatment room costs, laboratory costs and drug costs) while the effectiveness uses a negative rt pcr value. the results showed that in patients using favipiravir with an average length of treatment 6.525 days, the total cost was rp. 9,930,327/patient and cost-effectiveness ratio (rp/day/patient) rp. 1,521,899.195, whereas in patients using oseltamivir with an average length of treatment 7.925 days, the total cost was idr. 12,368,397/patient and cost-effectiveness ratio (rp/day/patient) idr. 1,560,681,009. it can be concluded that favipiravir is more cost effective than oseltamivir. keywords: covid-19; favipiravir; effective and efficient; oseltamivir. introduction corona virus diseases 2019 (covid-19) is a disease caused by the severe acute respiratory syndrome coronavirus 2 (sarcov-2). sarcov-2 is transmitted by direct contact with the patient through droplets when coughing or sneezing, as well as hands touching the mouth, nose, and eyes after touching objects contaminated with the virus. it first discovered on december 31, 2019. in 3 days, the number of patients with these cases amounted to 44 and has continued to grow to date, amounting to millions of cases. initially, epidemiological data showed 66% of patients were related to one seafood market or fresh market in wuhan, hubei province, china. isolate samples from patients were studied, with results showing the presence of coronavirus infection, a new type of -coronavirus named novel coronavirus. on march 11, 2020, who announced that covid-19 had become a worldwide pandemic (lapostolle f et al. 2020, lingeswaran m et al. 2020, bishnu p. d. et al. 2020, suresh kumar vc et al. 2020, di gennaro f et al. 2020) the first covid-19 case in indonesia was announced on march 2, 2020, or about 4 months after the first case in china (lingeswaran m et al. 2020). at the beginning, there were hundreds of cases added, and until now, the addition of cases has reached millions. at the time, there were currently no specific therapies or treatments for covid-19. nevertheless, some potential drugs have been used for people with covid-19, although they are still in test drug status, and the results show good effectiveness (suresh kumar vc et al. 2020). for acute covid-19 conditions and molecular detection methods, naat (nucleid acid amplification test) examinations, including rt-pcr examinations of covid-19 patients who are hospitalized, show positive results for antivirals such as chloroquinphosphate, hydroxychloroquine, favipiravir, oseltamivir, remdesivir, and combinations between lopinavir and ritonavir (suresh kumar vc et al. 2020, di gennaro f et al. 2020) manuscript received: 14 april, 2025. revision accepted: 04 december, 2025. published: 11 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1219-1223 1220 biology, medicine, & natural product chemistry 14 (2), 2025: 1219-1223 favipiravir is a type of rnadependent rna polymerase (rdrp) inhibitor (zhu n, et al. 2019). on february 14, a clinical trial of favipiravir for the treatment of covid-19 was initiated by the clinical medical research center of the third people's hospital of shenzhen. preliminary results from a total of 80 patients (including the experimental group and the control group) showed that favipiravir had a stronger effect compared to lopinavir and ritonavir. there were no adverse reactions or significant side effects in the favipiravir treatment group compared to the lopinavir/ritonavir group (suresh kumar vc, et al. 2020). oseltamivir is part of the class of neuraminidase inhibitors (nais), which have a mechanism of action by inhibiting viral neuraminidase and inhibiting the release of virus particles from infected cells so as to reduce the spread of the virus in the airway (susilo a,et al. 2019. y furuta, et al. 2017, uyeki tm 2018) this covid-19 disease requires a very large hospitalization cost of around rp. 17,808,650 per patient; this value is calculated from the costs of drugs, treatments, laboratories, and radiology (di gennaro f et al. 2020). to meet the needs of quality and cost-effective health, the cost of treatment must be more affordable for the community and effective to get clinical results. good is a negative rt-pcr value, and without drug side effects, health policy should go through appropriate economic evaluation (lingeswaran m et al. 2020, di gennaro f et al. 2020). in august 2022, who no longer lists oseltamivir as clinical management of covid-19 in health facilities. study results show that oseltamivir is not effective against sarscov-2 in both in vitro testing and case reports in covid-19 patients (uyeki tm 2018). in choosing the priority of the covid-19 treatment strategy using antiretrovirals, favipiravir and oseltamivir, which provide the largest rational treatment outcomes, it is necessary to conduct a costeffectiveness assessment analysis using the aeb method, which is very good to provide the best therapeutic recommendations, estimate health progress, and determine the most effective cost for covid-19 treatment that relates the costs needed with the outcomes produced (y furuta, et al. 2017). this research is expected to provide benefits for the nation and state in determining the choice of quality and costeffective antiretroviral drugs, especially as a consideration for policy formulation for the ministry of health in determining the cost of covid-19 treatment and the resulting outcomes (y furuta, et al. 2017). materials and methods we used pharmacoeconomic methods that involve effectiveness analysis with a cross-sectional research design (crosssectional) and retrospective data search, the parameter measured was rt-pcr negative. results are presented in a descriptiveanalytic manner. the subjects of this study were inpatient covid-19 patients using anti-retroviral favipiravir and oseltamivir at cisarua lung hospital, bogor, for the period septemberoctober 2022. the inclusion criteria in this study were adult covid19: p > 17 years, hospitalized patients diagnosed with moderate covid-19 by doctors (positive rtpcr value (ct < 40), patients undergoing treatment in july– december 2021, receiving antiretroviral therapy favipiravir and oseltamivir. the inclusion criteria were as follows: pregnant women, patients with unrecorded medical records of antiretrovirals used (type, dose, and frequency of administration) and no recorded pcr examination, patients admitted to the icu during the study process, and patients who died during the study process. the materials used were medical records and details of medical costs for covid-19 patients at cisarua bogor lung hospital. the samples used were 80 medical records, consisting of 40 medical records of patients using favipiravir antiretrovirals and 40 medical records of patients using oseltamivir antiretrovirals. in this study, researchers used the hypothesis test of the difference between two populations for the calculation of sample size with the formula: the medical record data obtained provides information about the characteristics of the patient (age, sex, length of treatment days, pcr value, drug name, dosage, and interval of administration). cost data consists of direct costs obtained from financial documents, which include drug costs, laboratory costs, doctor consultation fees, and administrative costs. statistical data analysis using the mann-whitney method was used to compare the statistical significance of variability (see meaningful differences between kedua antiretroviral favipiravir and oseltamivir). pharmacoeconomic analysis, the average costeffectiveness ratio (acer) is the total cost divided by the output or effectiveness of each method. the acer generated by each method is then compared; smaller values indicate a more costeffective method compared to other methods. grids are used to describe the cost position and outcome of alternative interventions. research ethics committee of stikes prima indonesia, no. 206/ec/kepk stikes-pi/viii/2022 has provided information that passed the ethical review for this study. rahayu et al. – cost effectiveness analysis of favipiravir and oseltamivir as … 1221 results and discussion characteristics of research subjects table 1. characteristics of covid 19 patients. no variable category favipiravir n= 40 oseltamivir n=40 total n=80 1 gender man 24 25 49 women 16 15 31 2 place of residence bogor regency 23 25 48 bogor city 4 7 11 outside bogor 13 8 21 3 length of days 1 8 37 21 58 therapy 9 16 3 13 16 17 24 0 6 6 4 pcr results positif 30 39 74 rs exit negatif 10 1 6 the study conducted in the covid-19 treatment room of cisarua bogor lung hospital during a period of 2 months (september–october 2022), the number of hospitalized covid-19 patients who met the inclusion criteria and exclusion criteria was 80 (research sample), consisting of 40 patients who received favipiravir antiretroviral and 40 patients who received oseltamivir antiretroviral. table i shows the characteristics of inpatient covid-19 patients in cisarua bogor lung hospital for the july– december 2021 period as follows: for sex characteristics, the 80 samples used showed that in favipiravir antiretrovirals, male sex is 24% and female sex is 16%, while in oseltamivir antiretrovirals, male sex is 25% and female sex is 15%. this is because men have higher levels of enzymes in the blood than women. enzymes are a key factor for covid-19 to infect human cells (centers for disease control and prevention, 2020 (9, 10). for patient residences, the 80 samples used showed that patients living in bogor regency by 48%, bogor city by 11%, and outside bogor by 21% showed that there were patients living in bogor city because of the hospital. cisarua bogor lung hospital is a government referral hospital for treatment. covid-19. length of stay (los) is a duration of treatment of a patient in a health facility. los is one of the factors that affect the health care system and shows that the longer a patient's stay in the hospital, the greater the patient's need for resources, including medical personnel, materials, drugs, and equipment, resulting in increased costs therapy. the length of treatment days for patients taking favipiravir antiretrovirals 1-8 days treatment period is 37%, 9-16 days is 3%, and there are no patients with 17-24 days of treatment, while in oseltamivir antiretrovirals the treatment period is 1-8 days by 21%, 9-16 days by 13%, and 17-24 days by 6%. this is in accordance with research conducted in china, which shows that the length of days of treatment for covid-patients 19 ranged from 4 to 53 days, and patients in the icu ranged from 6 to 12 days (song y et al. 2020, rees em et al. 2020). polymerase chain reaction (pcr) status when patients are discharged from the hospital: for patients using favipiravir antiretrovirals, positive pcr results were 30% and negative pcr was 10%; for patients using oseltamivir antiretrovirals, positive pcr results were 39% and negative pcr was 1% (rees em et al. 2020, ye q, wang b mj 2020). when viewed from the length of treatment days with the patient's pcr status at the time of discharge from the hospital, the best antiretroviral used for covid-19 treatment therapy is favipiravir, which is in accordance with the mechanism of action. favipiravir is a type of rna-dependent rna polymerase (rdrp) inhibitor (zhu n. et al. 2020). on february 14, the clinical trial of favipiravir for the treatment of covid-19 was initiated by the clinical medical research center of the third people's hospital of shenzhen. baseline results from a total of 80 patients (including experimental and control groups) showed that favipiravir had a stronger effect compared to lopinavir and ritonavir. there were no adverse reactions or significant side effects in the favipiravir treatment group compared to the lopinavir/ritonavir group (suresh kumar vc et al. 2020). for oseltamir to provide less than maximum results than favipiravir, this is in line with who's august 2022 revocation of the use of oseltamivir for the treatment of covid19. cost-effectiveness analysis is obtained by comparing the total cost with the effectiveness of the therapy obtained (output). total cost is a combination of total direct costs and indirect costs. direct cost table 2. compares the cost-effectiveness of antiretroviral administration. the direct cost component includes the total cost of treatment (room fee, drug fee, doctor's visit fee, and 1222 biology, medicine, & natural product chemistry 14 (2), 2025: 1219-1223 laboratory fee). in table 2, the results of the comparison of cost-effectiveness in the administration of antiretrovirals favipiravir and oseltamivir are as follows: in patients using favipiravir, the average total cost is rp. 9,930,327 per patient, while in patients using oseltamivir, the average length of treatment days is rp. 12,368,397 per patient. cost-effectiveness analysis figure 1. alternative group based on total cost-effectiveness of antiretroviral administration. cost-effectiveness analysis is carried out using the average cost effectiveness ratio (acer) and costeffectiveness grid formulas. in this study, the incremental cost effectiveness ratio (icer) is not calculated because the results of the calculation of cost effectiveness are included in the table of areas c and g in the cost-effectiveness grid (meaning that area g shows dominant results and definitely does not need to be selected). rieb/icer. area c shows dominant results and does not need to be considered as an alternative, so it does not need to be included in the rieb/icer calculation. the acer price is obtained from a comparison between the total cost of treatment and the effectiveness of therapy. the effectiveness of therapy was measured by the number of days per treatment (afdal af 2011). based on cost-effectiveness parameters, the acer value based on the length of days from favipiravir is rp. 1,521,899,195 (proportion of treatment days: 6,525), while oseltamivir is rp. 1,560,681,009. in the favipiravir group, the acer price was lower than in the oseltamivir group. this suggests that favipiravir therapy is more cost-effective or has the most cost-effectiveness compared with oseltamivir therapy. conclusions based on the results of this study, it can be concluded that the favipiravir antiretroviral therapy group is more costeffective in the treatment of covid-19 compared to the oseltamivir antiretroviral therapy group. acknowledgements: praise and thank allah swt for all his mercy and pleasure so that the author can complete this writing process. the author would like to thank all parties who have played a role in this writing process, especially the directorate general of higher education, research, and technology of the ministry of education, culture, research, and technology who have funded this research. competing interests: the authors declare that there are no competing interests. funding: this research received funding from directorate general of higher education, research, and technology of the ministry of education, culture, research, and technology. references afdhal af. pharmacoeconomics the latest analytical knife of the pharmaceutical world. 2011. 1–30 p. bishnu p. d., nancy k. s., julia h. i., deepak a., and preethi w. sarscov-2 infection and cardiovascular disease: covid19 heart. hear lung circ. 2020;29(june):973–87. di gennaro f, pizzol d, marotta c, antunes m, racalbuto v, veronese n, et al., coronavirus diseases (covid-19): current status and future perspectives: a narrative review." int j environ res public health. 2020;17(8). lapostolle f., schneider e., vianu i., dollet g., roche b., berdah j., et al. clinical 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https://www.papdi.or.id/download/9%2083-pedoman-tatalaksana-covid-19-%20edisi-3-desember-2020 rahayu et al. – cost effectiveness analysis of favipiravir and oseltamivir as … 1223 https://ejournal.unesa.ac.id/index.php/jurnal-kesehatan olahraga/article/view/34491 uyeki tm. oseltamivir treatment of influenza in children. clin infect dis. 2018;66(10):1501–3. ye q, wang b mj since january 2020, elsevier has created a covid-19 resource center with free information in english and mandarin on the novel coronavirus covid-19. the covid-19 resource center is hosted on elsevier connect, the company's public news and information site. elsevier. 2020; j infect. y furuta, t komeno, tn polymerase activity (%): 100 µ mol/ll favipiravir-rmp control. proc jpn acad ser b phys biol sci [internet]. 2017;93(7):449–63. available from: https://www.ncbi.nlm.nih.gov/pmc/a rticles/pmc5713175/pdf/pjab-93449 zhu n, zhang d, wang w, li x, yang b, song j, et al., a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020;382(8):727–33. https://ejournal.unesa.ac.id/index.php/jurnal-kesehatan-%20olahraga/article/view/34491 https://ejournal.unesa.ac.id/index.php/jurnal-kesehatan-%20olahraga/article/view/34491 https://www.ncbi.nlm.nih.gov/pmc/a%20rticles/pmc5713175/pdf/pjab-93-%20449 https://www.ncbi.nlm.nih.gov/pmc/a%20rticles/pmc5713175/pdf/pjab-93-%20449 this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 2, 2014 | pages: 53-57 | doi: 10.14421/biomedich.2014.32.53-57 larvicidal activity of a mixture of cashew nut shell liquid and water-soluble extract of soap nut fruit (sapindus rarak dc.) against 3rd instar larvae of aedes aegypti glory resia raraswati1, sudarsono2 and budi mulyaningsih3 1faculty of pharmacy; 2laboratory of parasitology faculty of medicine; 3department of pharmaceutical biology, ugm, indonesia author correspondency: sudarsono: ugmpsot@yahoo.com; budi mulyaningsih: budimulyaningsih@yahoo.com abstract cashew nut shell liquid (cnsl) which has been known as a waste of processing cashew fruits which is contain phenolic compounds have activity as larvicides. cashew nut shell liquid is not soluble in the water where the larvae grow. cashew nut shell liquid mixed with watersoluble extract of soapnut fruit which serves as a natural surfactant that can emulsify oil in water. the test subjects were larvae of aedes aegypti third instar. test subjects were divided into treatment group and control group. in the treatment group, test subjects were the mixture of cnsl and ethylacetat soluble extract (ese) in tap water. the larvae mortality observations were done 24 hours after the treatment. lc50 and lc90 as final test data were analyzed using probit analysis. extract constituents of cnsl and water soluble extract of soapnut fruit (wseosn) were investigated using thin layer chromatography (tlc) method. the effect of cnsl as larvicides against third instar larvae of ae. aegypti with were lc50 of 14,12 ppm, while the lc90 of 24,85 ppm. keywords: larvacidal activity, thin layer chromatography, sapindus rarak dc introduction aedes aegypti is a mosquito species that can carry and transmit the dengue virus causes dengue hemor rhagic fever (dhf), yellow fever virus and chikungunya virus (who, 2004). how to control the mosquito population that most rapidly break the cycle of transmission is the use of chemical compounds, one of them is the use of synthetic insecticides and larvicides. the use of synthetic compounds can lead to increased mos quito resistance, environmental pollution caused by their residue, even the death of non-target creatures (munif, 1993). the use of plants as a biopesticide is a one of alternative natural way for controlling which is safer for sustainable environment balance. the natural biopesticides were usually easy to apply and not harmful to natural enemies and other beneficial insects (adebowale & adedire, 2006). cnsl is a viscous oil, black color and has a very strong irritant properties on the skin because of the phenolic substances derivatives e.g. cardol, anacardic acid and 2-methyl kardol (kubo et al., 1986); anacardic acid is not soluble in water. this sticky fluid was extracted from cashew nut shell by the use of hydraulic pressure pump at a certain temperature (mulyono and abdul gani, 1996). anacardic acid has several biologic effect such as anti-microbial, anti-acne (kubo et al., 1993; 1994) and as molluscicides (kubo et al., 1986). the main constituent of cnsl i.g. anacardic acid, cardanol, and cardol also reported as larvicidal activity against the larvae of ae. aegypti with lc50 of 12,40 ppm for anacardic acid, 14,45 ppm for cardanol, and 10,22 ppm for cardol (oliveira et al., 2010). soapnut in indonesia called “lerak” (sapindus rarak dc.) is a plant that from southeast asia and can be able grow well in tropical climate such in indonesia (afriastini, 1990). the active compounds from the soapnut pericarp are saponins and sesquiterpenecompounds. the main of non-sugar saponins compound are usually triterpenes and steroids derivatives (schneider, 1980; wina et al., 2005).saponins as well as surfactants that have both hydrophilic and hydrophobic pool which were able to lower the interfacial tension between oil and water that immiscible; so as to form a colloidal solution which forms a foam when shaken (matheson, 1996; gunawan & mulyani, 2004). the purpose of this study was to determine the activity of cashew nut shell oil (cashew nut shell liquid / cnsl) as an alternative natural larvicides. methodology preparation and extraction of plant materials cashew nut shell obtained from jatibedug village, wonogiri and soapnut fruit from perhutani area, situbondo. the identification of plant material was carried out by mr.joko santosa m.si, and voucher specimen was found at the department of pharmaceutical biology faculty of pharmacy, ugm, yogyakarta. cashew nut shell that had been oven-dried at 50°c for 1 hour obtained 1,313 kg net weight then pressed using a hydraulic press by 150-160 kg/cm2 to obtain cashew nut shell liquid (cnsl); whereas 10 g of soapnut fruit chopped and then extracted by heating at 50°c for 30 min in the distilled water ad 1000 ml to obtain a water-soluble extract of soapnut fruit 1% b/v. 54 biology, medicine, & natural product chemistry 3 (2), 2014: 53-57 preparation of water-soluble extract of soapnut fruit which did not kill larvae as a main solution of water-soluble extract of soapnut fruit was 1% b/v diluted by taking a volume of 6, 8, 10, and 12 ml for each dissolved in 100 ml of tap water to obtain test solutions with concentrations of 600, 800, 1000, and 1200 ppm. four plastic cups with a mouth diameter of approximately 10 cm glass used as a test medium. each test cups contain10 larvae of third instar ae. aegypti. each concen-tration of repetitions performed 3 times. larval mortality was observed 24 hours after treatment by calculating the percentage of larval mortality. the biggest concentrations that doesn’t kill any larvae chosen for making the mixture solution with cashew nut shell liquid. bioassay for larvicidal toxicity mixture concentration series was made by mixing watersoluble extract of soapnut fruit at a concentration elected with cashew nut shell liquid. the mixture of cnsl and water soluble extract of soapnut fruit homogenized using a vortex for 5 minutes, then stirred using a magnetic stirrer at medium speed for 5 minutes. the results observed visually mixing at room temperature (25°c). subsequently the mixture was dissolved in tap water to obtain a final volume of 100 ml of solution for each concentration. the cnsl concentration used was 6 ppm, 7,56 ppm, 9,53 ppm, 12,01 ppm, 15,13 ppm, and 19,06 ppm. each test cups contain 25 larvae of third instar ae. aegypti. each concentration of repetitions performed 3 times. larval mortality was observed 24 hours after treatment by calculating the percentage of larval mortality. larvicidal test data were analyzed using probit analysis to obtain lc50 and lc90 (finney, 1971).the concentrations in probit analysis were transformed to logarithm (log concentra-tion) and the lethal concentrations (lc50 and lc90) were calculated manually. specification of test material using thin layer chromatography (tlc) a. cashew nut shell liquid cashew nut shell liquid that dissolved in n-hexane (1:10b/v) and then filtered with silica gel 60 used as test solution. components of cnsl are further separated by thin layer chromatogra phic (tlc) method. chromtography was perfomed using aluminium plates silica gel 60 f254 (merck). three microliters of test solution was applied to the plate with distance of development 8 cm. plate inserted in a chamber that had already saturated by mobile phase ntoluene: ethyl acetate: glacial acetic acid (7:2:1 v/v/v). after development the mobile phase was evaporated to dryness and plates were sprayed with iodine vapor and vanillin sulfuric acid. b. water-soluble extract of the soapnut fruit thirty milliliters of water-soluble extract of soapnut fruit was evaporated and then hydrolyzed by boiling with 30 ml of 2n hcl using reflux distillation method for 30 minutes. samples that had been hydrolyzed were then fractionated with 30 ml of n-hexane. nhexane fraction was then washed with distilled water until water fraction ph was neutral. the results of this fraction was then added anhydrous na2so4 to remove water traces. furthermore, the fraction of n-hexane which has been neutralized partitioned again using 30 ml of ethyl acetate. components of both fractions were then separated by tlc. samples of both fractions was applied to the plate as much as 3 spots using a capillary tube with distance of development 8 cm. plate inserted in a chamber that has been saturated with mobile phase. mobile phase that used was chloroform: methanol (9.5: 0.5 v/v). after development the mobile phase was evaporated to dryness and plates were sprayed with anisaldehyde sulfuric acid. results and discussion larvicidal effect of a mixture of cashew nut shell liquid and water-soluble extract of soapnut fruit probit analysis was used to study the response of larvae to the exposure of test solution in a biological assay (umniyati, 1990). probit analysis is a statistical method used to determine the relationship between dose and response (finney, 1971). percentage larval mortality was calculated after 24 hours. dead larvae are larvae that does not move when touched with a pipette or larvae that sink to the bottom of container that does not respond to stimulation. table 1. test results of water-soluble extract of the soapnut fruit preliminary test. soapnut concentration (ppm) total mortality of larvae total larval test1 2 3 600 (*) 0 0 0 30 800 1 0 1 30 1000 1 2 2 30 1200 4 3 4 30 in table 1, it can be seen that the biggest concentration of water-soluble extract of the soapnut fruit that does not make larvae dead were at a concentration 600 ppm. this preliminary test aim is to minimize the effect of watersoluble extract of the soapnut fruit in the death of larvae so the final assay results that obtained later is the result of larvicidal activity of cashew nut shell liquid. the largest concentration of water-soluble extract of soapnut fruit that does not kill larvae was used for the mixture becausehigher levels of water-soluble extract of soapnut fruit will increase the solubility of cashew nut shell liquid in the water. glory resia raraswati, et al. – larvicidal activity of a mixture of cashew nut shell liquid … 55 in table 2, data percentage mortality test results are used to calculate the lc50 and lc90 values are percent mortality at levels of 7,56 ppm, 9,53 ppm, 12,01 ppm, 15,13 ppm, and 19,06 ppm.the percentage mortality data is plotted in probit tables to obtain theprobit value, then made the graph between log concentration (x) and probit value (y) to obtain the linear regression equation y = a + bx. probit calculation steps performed according to the method of finney (1971). table 2. results of probit analysis of a mixture of cashew nut shell liquid and water-soluble extract of soapnut fruit against ae. aegyptilarvae (total larval test = 75). cnsl concentration (ppm) total mortality of larvae percentage larval mortility (mean) linier equation lc50 (ppm) lc90 (ppm) 0 0 0 y = 5,20921x – 0,9891 14,12 24,85 6 0 0 7,56 8 10,67 9,53 14 18,67 12,01 20 26,57 15,13 41 54,67 19,06 60 80 linear regression equation obtained from the probit analysis is y = 5.20921 x 0.9891 so that the calculation of lc50 and lc90can be obtained.lc50 and lc90 calculated were 14.12 ppmand 24.85 ppm. a material that has a larvicidal test lc50 values below 100 ppm can be said to have larvicidal activity against the larvae test (cheng et al., 2003). lc50 values in this study were at 14.12 ppm so cashewnutshell liquid that is used as the test material can be said to have potential as larvicidal agent against the third instar larvae of ae. aegypti. cashew nut shell oil is known to have 3 main compound content anacardic acid, cardol, and cardanol which showed inhibition of the acetylcholinesterase enzyme activity as well as the mechanism of action of organophosphate and carbamate (oliveira et al., 2010). inhibition of the acetylcholinesterase enzyme causes accumulation of acetylcholine which triggers central nervous system disorders, convulsions, respiratory paralysis, and death in larvae (rosenberryet al., 2008). phytochemical compund of cashew nut shell liquid and water-soluble extract of soapnut fruit in figure 1, elution of cnsl results showed 2 spots on the hrf 63 and 73, that drown under 254 nm uv light. furthermore, the silica plate inserted in a closed chamber that has been filled by iodine vapor and left a few minutes. figure 1. tlc-chromatogram of cashew nut shell liquid. detection: visible light (a), under 254 nm uv light (b), under 366 nm uv light (c), after iodine vaporized (d), and after spraying vanillin sulfuric acid were observed in visible light (e). figure 1.d were a yellow spot; the hrf 63 and 73. it could be the presence of an unsaturated compound; it was perhaps an side chain of the phenolic compounds. iodine vapor was used for the detection of side chain compounds 56 biology, medicine, & natural product chemistry 3 (2), 2014: 53-57 that had a double bond; a positive result is a yellowbrown spots on the visible light (fessenden and fessenden, 1994). the mechanism of iodine to form the color is an addition reaction on double bond in unsaturated fatty acids (simpen, 2008). results of detection by vanillin sulfuric acid reagent (figure 1.e) were observed under visible light were the presence of spots on hrf 63 with red-orange color, while on hrf 73 as pink-brown color spot. based on these results, spots on the hr f63 and 73 suspected as phenolic compounds anacardic acid derivatives. identification of the compounds in water-soluble extract of soapnut fruit was identifying saponin. saponins are triterpenoid glycosides or glycoside steroid with sapogeninaglycone (schneider, 1985; gunawan and mulyani, 2004). the basic science of saponin identification was made for the existence of the sapogenin aglycone. (a) (b) (c) (d) : 1 = n-hexane soluble fraction; 2 = ethyl acetate soluble fraction figure 2. tlc chromatogram profile of n-hexane and ethyl acetate fraction of water-soluble extract of soapnut fruit. detection before spraying anisaldehyde sulfuric acid: under visible light (a), uv 254 nm (b), and uv 366 nm (c) and detection after spraying anisaldehyde sulfuric acid under visible light (d). in figure 2, the chromatogram of n-hexane and ethyl acetate of water-soluble extract of soapnut fruit had no spots after observed under visible light, uv light 254 nm, and 366 nm uv. it was due to saponins had no aromatic ring in the structure. it could be detected by coloring spray reagent (wagner, 1984). if there is purple-red or purple after spraying with anisaldehyde sulfuric acid reagent showed the presence of terpenoids or steroids (wagner & bladt, 1996). after spraying with anisaldehyde sulfuric acid reagent (aas) and it was heated for 5 minutes at a temperature of 105°c, there was 3 purple spots (hrf 49; hrf 67, and hrf 79) in ethyl acetate fraction and the fraction of n-hexane had pink spots on the hrf 82 and 93. the purple color in ethyl acetate fraction was a steroid sapogenin compounds, and the pink spots on n-hexane fraction could be as triterpene sapogenin. steroid compounds are more polar than the triterpenoid compounds so that would be more soluble in more polar solvents as well, in this case is ethyl acetate. based on the above observations, the water-soluble extract of soapnut fruit suspected of containing sapogenin steroid and triterpenoid sapogenin which is the aglycone of saponin glycosides and it can be used as a natural surfactant to emulsify cashew nut shell oil in aqueous media. the larvicidal properties exhibited by cashew nut shell liquid in this study might be related to presence of phenolic derivative components. conclusion the results of this study indicated that a mixture of cashew nut shell liquid and water soluble extracts of soapnut fruit (sapindus rarak dc.) had an effect as larvicides against third instar larvae of ae. aegypti. further research needs to be done about the toxicity effects of a mixture of cashew nut shell liquid and water soluble extract of soapnut fruit against non-target organisms i.g fish, and frog. references adebowale, k.o. & adedire, c.o., 2006, chemical composition and insectisidal properties of the underutilized jatropha curcas seed oil. african j. biotech. 5 (10), 901-906. afriastini, j.j., 1990, daftar jenis nama tanaman, 53-60, penebar swadaya, jakarta. cheng, s.s., chang, h.t., chang, s.t., tsai, k.h., chen, w.j., 2003, bioactivity of selected plant essential oils against the glory resia raraswati, et al. – larvicidal activity of a mixture of cashew nut shell liquid … 57 yellow fever mosquito aedes aegypti larvae. bioresource technology, 89, 99-102. finney, d.j., 1971, probit analysis, 3rd ed., 333-340, cambridge university press, great britain. gunawan, d. & mulyani, s., 2004, ilmu obat alam (farmakognosi), jilid 1, 87-95, penebar swadaya, depok. kubo, i., muroi, h., & kubo, a., 1994. naturally occurring antiacne agents, j nat prod, 57, 9 – 17. kubo, i., muroi, h., himejima, m., yamagiwa, y., mera, h., tokushima, k., ohta, s., & kamikawa, t., 1993, structure antibacterial activity relationships of anacardic acids, j agric food chem, 41, 1016 –1019. kubo, i., komatsu, s., & ochi, m., 1986, molluscicides from cashew (anacardium accidentale) and their largescale isolation. j. agric. food chem, 34, 970-973. matheson, k.l., 1996, surfactant raw materials: classification, synthesis, and uses. in spitz, l. (ed). soap and detergents: a theoretical and practical review, 288-303, aocs press, illinois. mulyono, e., & abdulgani, l.y., 1996, karakterisasi cnsl dan metode ekstraksinya; isolasi kardanol dan karakteristiknya; isolasi pektin dan karakteristiknya, laporan penelitian, balittro, bogor. munif, a., 1993, cendawan patogen pada larva anopheles aconitus yang resisten dpt di daerah endemis malaria banjarnegara, jawa tengah. majalah kesehatan indonesia xxi, 8, 472-6. oliveira, m.s.c., de morais, s.m., magalhães, d.v., batista, w.p., vieira, i.g.p., craveiro, a.a., de manezes, j.e.s.a., carvalho, a.f.u., de lima, g.p.g., 2010, antioxidant, larvicidal and antiacetylcholinesterase activities of cashew nut shell liquid constituents, acta tropica, 117 (2011), 165–170. rosenberry, t.l., sonoda, l.k., dekat, s.e., cusack, b., johnson, j., 2008, monitoring the reaction of carbachol with acetylcholinesterase by thioflavin t fluorescence and acetylthiocholine hydrolysis, chem. biol. interact, 175, 235– 241. schneider g., 1980, pharmazeutisch bio logie, b.i.varlag mannheim p. 230, simpen, i.n., 2008, isolasi cashew nut shell liquid dari kulit biji jambu mete (anacardium occidentale l) dan kajian beberapa sifat fisiko-kimianya. jurnal kimia 2 (2), 71-76. umniyati, s.r., 1990, analisa probit secara aritmatis untuk pengujian toksisitas insektisida terhadap serangga, 1-13, 2736, laboratorium parasitologi fakultas kedokteran universitas gadjah mada, yogyakarta. wagner, h., 1984, plant drug analysis a thin layer chromatography atlas, 164, springer, new york. wagner, h & bladt, s., 1996, plant drug analysis, 2nd ed., 305317, 359, springer, new york. wina, e., muetzel, s., hoffmann, e.m., makkar, h.p.s., & becker, k., 2005, saponin containing methanol extract of sapindus rarak affect microbial fermentation, microbial activity and microbial community structure in vitro, animal feed science technology, 121, 59-174. world health organization (who), 2004, prevention and control of dengue and dengue haemorrhagic fever, terjemahan dari who regional publication searo no. 29, new delhi content_v3n2_2.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 81-86 | doi: 10.14421/biomedich.2021.102.81-86 issn 2540-9328 (online) risk assessment of heavy metals in chromolaena odorata collected around gemstone mining site in ijero-ekiti efe sylvanus abiya, foluso akinbode ologundudu*, ekpo wisdom department of biology, federal university of technology akure, nigeria. corresponding author* akinbodefoluso@gmail.com manuscript received: 27 july 2021. revision accepted: 02 august, 2021. published: 01 october, 2021. abstract in nigeria, like many developing nations, the resultant effect of land degradation: aggravated soil erosion, flood disasters, salinization or alkalisation, and the desertification have been a major public health concern for the past decades, however this study highlighted some of the factors that leads to the menace of soil fertility. the study was conducted at a gemstone mining site in ijero ekiti, ekiti state, nigeria. the soil samples were collected at a depth of 0-15 cm top soil and 0-30 cm subsoil. a line transect of 20 cm was drawn and soil sample was collected, all samples were kept in a clean container and labeled accordingly before been transported to the laboratory for analysis. the plant samples were thoroughly washed with distilled water to remove dust and other particles, air dried in a dust free wire meshed cage. all data obtained from this research were subjected to one-way analysis of variance (anova). the result obtained in this study indicated that the levels of heavy metal concentration tested were still within the permissible limit in the root and shoot of chromolaena odorata between the mine and control site. the implication of this is that chromolaena odorata is safe for human and animal consumption. the said plant can readily undergo photosynthetic activity to aid growth by exploiting the presence of these metals either as a macro-nutrient or micro-nutrient as seen from the translocation factor and metal transfer factor. the study concludes that soil at ijeroekiti mine site were slightly acidic soil ph, reduced organic carbon, total nitrogen, available phosphorus, exchangeable cations and averagely elevated heavy metal contents. keywords: chromonlaena odorata; gemstone; permissible limit; photosynthetic ability; transfer factor; heavy metal. introduction major problem of land degradation and pollution arise as a result of exploitation and excavation of the natural environment with an increase in sophisticated tools and methods and also revolution in industrialization in large scale pose a serious threat to the world’s resources and environmental degradation with socio-economic impacts katar (2009). the main causes of this degradation are transformation of fertile cultivated land into wasteland and in some cases pose serious environmental pollution and ecological degradation which can leads to loss of biodiversity (keskin and makineci, 2009). mining is one of the major factors that have pose serious threat and hazards that can jeopardize ecosystems of nations. nigeria has been actively engaged in solid mineral exploitation for more than decades and endowed with deposit of more than 34 solid minerals including coal, tin, gold and many more across the country. adekoya et al., 2003, southwestern part of the country has about 25% of the total land mass consisting of sedimentary rocks and hence mining activities are common. mining operations alter a site’s ecosystem by disrupting the ecological balance, landscapes, agricultural lands, forests, plantations and vegetation as well as the economic food and tree crops. other impacts of mining include alteration of the soil structure, loss and overturning of the fertile top soil, air, soil and water pollution, instability of soil and rock masses, destruction of flora and fauna, casing mass exodus of species of animals (adegboye, 2012). however, the health implications associated to mining as recorded by kitula (2004) in tanzania that the symptoms of heavy metals poisoning such as sensory disturbance, metallic taste and night blindness are common and also the world health organizations reported the prevalence of human diseases during the past decade is rapidly increasing due to effect of heavy metals. methods study area the study was conducted at a gemstone mining site in ijero ekiti, ekiti state, south western region of nigeria. sample collection soil and plants samples were collected at the different spots at the gemstone mining site in ijero ekiti, ekiti state. the soil samples were collected at a depth of 0-15 https://doi.org/10.14421/biomedich.2021.102.81-86 82 biology, medicine, & natural product chemistry 10 (2), 2021: 81-86 cm top soil and 0-30 cm subsoil. a line transect of 20 cm was drawn and soil sample was collected, all samples were kept in a clean container and labeled accordingly before been transported to the laboratory for analysis. the plant samples were thoroughly washed with distilled water to remove dust and other particles, then air dried in a dust free wire meshed cage. the analysis was carried out at the sustainable environmental laboratory and crop, soil and pest management in federal university of technology akure. soil physico-chemical properties, ph and conductivity of soil ten gram of the soil sample was weighed and placed into a sample cup; 20 ml of water was measured and poured into it. setup was allowed to stand for 30 minutes. afterwards, the ph meter was inserted into the dissolved soil sample and the reading was taken. at a room temperature of 28oc, soil conductivity was conducted using the auto-conductivity machine and the reading were recorded. organic carbon and organic matter little of the soil sample was pulverized. 1 gram was weighed into a 250ml conical flask. 10ml of potassium dichromate (k2cr2o7) was added. 20ml of sulphuric acid (h2so4) was further added and the flask was swirl vigorously for one minute. afterwards, 100ml of distilled water was added after standing for 30minutes. 3-4 drops of ferroin indicator and titrate with 0.5m iron (ii) ammonium sulphate (fe2nh4so2), takes a greenish cast and then changes to dark green. at this point, ferrous sulphate was added drop by drop until colour change was observed from green to brownish red. metal analysis about 1.0 to 2.0 gram of the sample was weighed into a 250 ml conical flask after dried in an oven for one hour. 20 ml of trioxonitrate acid (hno3) was added. then it was heated in a heater starting with low temperature for about 15 to 20 minutes. the heat was increased to medium temperature for about 30 minutes again and finally at high heating until complete digestion is required. the flask was rotated at intervals until the digest is clear (white fumes) continue heating for few minutes after that to ascertain complete digestion, that is, a clear solution is an evidence of complete a complete digestion. after cooling the sample residue was filtered and use to make up the digest up to 50 or 100ml or as appropriate. after being placed in a sample bottle the concentration using atomic absorption spectrophotometer (aas) or flame photometer was carried out for the elements like (ni, cd, as, cu, zn, pb and cr). most importantly, the machine (aas) must be powered for about 30 to 45 minutes before introducing any sample into it. this is done to increase the efficiency of the machine. statistical analysis all data obtained from this research were subjected to one-way analysis of variance (anova) and means separated with new duncan’s multiple range tests using spss 17.0 version of windows 7 statistical package was used. results table 1. soil textural class of the soil samples. soil samples % clay % silt % sand soil textural class transect site 30.48 15.28 54.2 sandy clay mining site 30.48 15.28 54.28 sandy clay control site 26.48 9.28 64.2 sandy laomy physico-chemical characteristics of soil at the mining and control sites. table 2 shows the physical and chemical (physicochemical) characteristics of soil sampled at the mine and control sites. the ph value at the mining site (6.81 ± 0.01) is slightly elevated compared to the control site (6.51 ± 0.01) and are significantly different from each other (p < 0.05). generally, elevated levels of concentration were observed for conductivity, bulk density, phosphorus, exchangeable cation (mg, k, na, ca), cation exchange capacity, organic carbon and total nitrogen between soils of mine site and control site respectively which are also not significantly different (p < 0.05). table 2. physico-chemical characteristics of soil at the mining and control sites. soil parameters pair mean±s.e n s.d t. cal sig remarks ph m 6.51±0.01 3 0.01 36.742 0.001 s c 6.81±0.01 3 0.01 conductivity m 644.3±0.88 3 1.53 378.976 0.000 ns c 171.6±0.88 3 1.53 porosity m 47.2±0.57 3 1.00 -7.232 0.005 ns c 51.9±0.32 3 0.55 bulk density m 1.32±0.01 3 0.01 12.017 0.001 ns abiya et al. – risk assessment of heavy metals in chromolaena odorata … 83 soil parameters pair mean±s.e n s.d t. cal sig remarks c 1.19±0.01 3 0.02 sodium m 2.52±0.01 3 0.02 -70.835 0.000 ns c 1.77±0.01 3 0.01 phosphorus m 4.66±0.01 3 0.02 -231.715 0.001 ns c 7.55±0.01 3 0.02 potassium m 5.21±0.01 3 0.01 4.000 0.000 ns c 7.46±0.01 3 0.01 calcium m 1.59±0.01 3 0.02 32.571 0.000 ns c 1.25±0.01 3 0.01 magnesium m 1.91±0.01 3 0.01 39.528 0.000 ns c 1.49±0.01 3 0.02 cec m 3.17±0.01 3 0.01 86.963 0.000 ns c 2.25±0.01 3 0.02 org. carbon m 0.94±0.01 3 0.01 -53.889 0.001 ns c 1.38±0.01 3 0.01 nitrogen m 0.85±0.01 3 0.01 -52.664 0.00 ns c 1.28±0.01 3 0.01 note: s – significant; ns – not significant; m – mining; c – control; s.d – standard deviation; t.cal/t-calculated; s.e-standard error; nnumber of replicates physical and chemical characteristics of soil at the line transect and control sites. table 3 shows the physical and chemical (physicochemical) characteristics of soil samples at the line transect site and control sites. the ph value at the line transect site (6.99 ± 0.01) is slightly elevated compared to the control (6.51 ± 0.01) site and are not significantly different to each other (p < 0.05). generally, there are no significant differences observed for the concentration of conductivity, bulk density, organic carbon, phosphorus, exchangeable cation (mg, k, na, ca) and total nitrogen between soils of the line transect site and control site and are also not significantly different (p < 0.05) from each other. table 3. physico-chemical characteristics of soil at the line transect and control sites. soil parameters pair mean±s.e n s.d t.cal sig remarks ph t 6.99±0.01 3 0.01 58.788 0.000 ns c 6.51±0.01 3 0.01 conductivity t 364.0±0.57 3 1.00 182.463 0.000 ns c 171.6±0.88 3 1.52 porosity t 45.5±0.57 3 0.01 -9.697 0.001 s c 51.9±0.32 3 0.55 bulk density t 1.35±0.01 3 0.02 12.829 0.000 ns c 1.19±0.01 3 0.02 sodium t 3.43±0.01 3 0.01 111.452 0.000 ns c 2.52±0.01 3 0.01 phosphorus t 14.6±0.01 3 0.02 570.870 0.000 ns c 7.55±0.01 3 0.02 potassium t 8.82±0.01 3 0.01 166.565 0.001 ns c 7.46±0.01 3 0.01 calcium t 1.49±0.01 3 0.02 23.085 0.000 ns c 1.25±0.01 3 0.01 magnesium t 2.11±0.01 3 0.01 58.502 0.001 ns c 1.49±0.01 3 0.02 cec t 4.12±0.01 3 0.01 177.088 0.001 ns c 2.25±0.01 3 0.02 org. carbon t 1.95±0.01 3 0.02 54.391 0.000 ns c 1.38±0.01 3 0.01 nitrogen t 1.38±0.01 3 0.01 -37.967 0.001 ns c 0.97±0.01 3 0.01 note: s – significant; ns – not significant; t – transect; c – control; s.d – standard deviation; t.cal/t-calculated; s.e-standard error; nnumber of replicates. the nutrient of zinc (zn), copper (cu), cadmium (cd), chromium (cr), nickel (ni) and lead (pb) in the soil at the mine site are slightly higher in abundance compared to the control site. paradoxically, arsenic (as) seems to have a low nutrient concentration in the soils at the mining and control sites. 84 biology, medicine, & natural product chemistry 10 (2), 2021: 81-86 table 4 translocation factor of each element at the mining and control site for chromolaena odorata elements mining site control site tf (cu) 0.796 0.456 tf (zn) 0.982 0.853 tf (pb) 0.939 0.876 tf (ni) 0.759 0.704 tf (as) 0.001 0.001 tf (cr) 0.953 0.014 tf (cd) 0.800 0.333 note: tf – translocation factor. translocation factor (tf) = concentration of metals (mg/kg) in the receiving level (shoot) concentration of metals (mg/kg) in the source level (root) translocation factor of each element between the soils at the mining and control sites in tree herb the nutrient of copper (cu), zinc (zn), lead (pb) and cadmium (cd) in the soil at the mine site are considerably higher in abundance compared to the control site. in contrast, nickel (ni), arsenic (as) and chromium (cr) seems to have a low nutrient in soil at the mine and control site. table 5 translocation factor of each element both at the mining and control site for the tree herb. elements mining site control site tf (cu) 1.224 0.589 tf (zn) 1.176 0.902 tf (pb) 1.040 0.759 tf (ni) 0.590 1.516 tf (as) 0.500 0.500 tf (cr) 0.956 0.936 tf (cd) 1.000 0.400 tf –translocation factor. 𝑇𝑟𝑎𝑛𝑠𝑙𝑜𝑐𝑎𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 (𝑇𝑓) = concentration of metals (mg/kg) in the receiving level (shoot) concentration of metals (mg/kg) in the source level (root) metal transfer factor of each element between the soils at the mining and control sites in chromolaena odorata the metal transfer factor of copper (cu), zinc (zn), lead (pb) and chromium (cr) seems to have a high transfer factor in the soils at the mine and control site. in contrast, arsenic (as) and nickel (ni) seems to have a low metal transfer at the mine and control site respectively. table 6 indication of the values of the metal transfer factor of each element both at the mining and control site for chromolaena odorata. elements mining site control site mtf (cu) 2.640 1.666 mtf (zn) 3.761 3.130 mtf (pb) 9.818 9.636 mtf (ni) 0.000 0.000 mtf (as) 5.000 3.000 mtf (cr) 13.700 10.200 mtf (cd) 0.000 0.00 mtf – metal transfer factor metal transfer factor = mp = metal content in plant shoot (mg/kg) ms = metal content in soil (mg/kg) metal transfer factor of each element between the soils at the mining and control sites in tree herb table 7 metal transfer factor of each element both at the mining and control site for the tree herb. elements mining site control site mtf (cu) 6.800 2.813 mtf (zn) 4.500 2.488 mtf (pb) 6.818 6.000 mtf (ni) 0.000 0.000 mtf (as) 3.000 1.000 mtf (cr) 11.800 11.000 mtf (cd) 0.000 0.000 mtf – metal transfer factor. metal transfer factor = mp = metal content in plant shoot (mg/kg) ms = metal content in soil (mg/kg) discussion international organizations such as united state environmental protection agency (usepa), world health organization (who) has given some guidelines for the presence of heavy metals in plant and soil (marcovecchio et al; (2007). therefore, heavy metals like copper (cu), zinc (zn), lead (pb), nickel (ni), arsenic (as), chromium (cr) and cadmium (cd) has their respective permissible limit in plant and soil as specified by (who). the maximum permissible level in plant for (cd) is 0.02mg/kg; (zn) is 50mg/kg; (cu) is 10mg/kg; (pb) is 2mg/kg; (ni) is 10mg/kg; (cr) is 0.03mg/kg and arsenic (as) is 0.05mg/kg. the maximum permissible limit in soil for (cd) is 45.00mg/kg; (cu) is 30.00mg/kg; (pb) is 35.00mg/kg; (ni) is 20.00mg/kg; (cr) is 30.00mg/kg and arsenic (as) is 20.00mg/kg. the result for heavy metals in plant in this study indicated that the levels of heavy metal concentration tested were still within the permissible limit in the root and shoot of chromolaena odorata between the mine and control site. the implication of this is that chromolaena odorata is safe for human and animal consumption; the said plant can readily undergo photosynthetic activity to aid growth by exploiting the presence of this metals either as a macro-nutrient or micro-nutrient as seen from the translocation factor (tables 3 and 4) and metal transfer factor (table 5) calculated independently for each of the metals (mysliwa-kurdziel et al; (2004). however, the result also shows that the level of heavy metal concentration is significantly higher at the mine site compared to the control site. this is so because as distance from the point source increases, the concentration or toxicity level abiya et al. – risk assessment of heavy metals in chromolaena odorata … 85 decreases considerably (olafisoye et al; (2013). another distinction observed is that zn, as, and cd are significantly different from each other (both in the root and shoot) of the aforementioned plant. this can also be due to the rate of absorption or uptake experienced between the mine and control site considerably (yang et al., (1998) which characteristically influenced the soil type of the mine site (sandy-clay) and the control site (sandy-loam) (table 1). similar result was also noted for in the root and shoot of the tree herb collected at the same site. the heavy metal concentrations tested were also within permissible limit. heavy metal pollution of soil is regarded as one of the severe environmental challenges in many countries of the world (facchinelli et al., (2001). concentrations of cu, zn, as, cd, ni and pb in the soil are among the heavy metals investigated for in the mine and control site. concentration zinc (zn), copper (cu), lead (pb) were significantly higher at the mining site compared to the control site although it is within the permissible limit. a previous study also showed that mining around kabwe was responsible for heavy metal pollution, especially by pb (tembo et al., (2006). that paper indicated that the heavy metal concentrations decreased with increasing distance from the mine, confirming that mining activities are the main cause of soil contamination. lead (pb) toxicity causes many diseases including hematological, gastrointestinal and neurological dysfunctions, and nephropathy (lockitch,1993). it is reported that children have a greater susceptibility to pb toxicity because intestinal absorption of pb is five times greater in children than in adults. oelofse (2008) also reported elevated concentrations of as, cu, cd and pb at the ijero-ekiti mine soil. cooke and johnson (2002), akcil and koldas (2006) and arogunjo (2007) observed that low ph in mine soils promoted solubility of heavy metals. the slightly acidic soil ph at ijero-ekiti mine site is due to manual technologies adopted which resulted in the overturning of the top soil. while concentrations of heavy metals in the mine soils were slightly elevated, plant nutrient concentrations were lower at the line transect and mining site respectively (table 6 and 7). low levels of conductivity, bulk density, organic carbon, phosphorus, exchangeable cation (mg, k, na, ca) and total nitrogen were reported between soils of the line transect and mine site, and high zn and cu concentration occurred in the mine and transect sites, as supported by martinez and motto (2000) and oelofse (2008), that mining activities negatively affected the mineralization, absorption and uptake of nutrients by the root and shoot of plant. conclusion the primary target for the toxicity of heavy metals is still not clear yet. however, this study has showed the impact of mining activities on the plant and soil. it has provided up-to date empirical data on the current state of soil and plant degradation as a result of exploitation of solid mineral in ijero-ekiti southwestern nigeria. the study concludes that soil at ijero-ekiti mine site were slightly acidic soil ph, reduced organic carbon, total nitrogen, available phosphorus, exchangeable cations and averagely elevated heavy metal contents. since most of the metals analyzed for were more or less within the permissible limit according to the guidelines of world health organization (who), that means the plant is considerably safe for human and animal consumption. the reason for this safety may be due to the manual technologies adopted at the ijero-ekiti mine sites as compared to other sites such as: awo and itagunmodi where mechanized technologies were used, thereby resulting to greater discharge and accumulation of this respective metals causing more damage and degradation to the plants and soil. the study underscores the need for strict mining operation policies in nigeria with quick remediation strategies to restore degraded soil and plant life. consent for publication: all authors are aware of the publication of this manuscript. availability of data and material: the datasets used and/or analysed during the current study are available from the corresponding author on request. competing interest: the authors declare that they have no competing interest. funding: the research was self-funded. authors’ contributions: mr. e. s. abiya designed the experiment, ekpo wisdom carried out the laboratory works. dr. f. a. ologundudu carried out the statistical analysis and interpretation of the results. the author(s) read and approved the final manuscript. acknowledgement: the researchers want to appreciate the technical staff of the department of biology, federal university of technology, akure, nigeria. references adegboye ma (2012) effect of mining on farming in jos south local government area of plateau state. j. soil sci. environ. manage. 3:77–83. doi: 10.5897/jssem11.048 adekoya ja, kehinde-phillips o, odukoya am (2003) geological distribution of mineral resources in southwestern nigeria. in: elueze a.a. (ed.) prospects for investment in mineral resources of southwestern nigeria. nig. mining and geosci. soc. (nmgs), 113. akcil a, koldas s (2006) acid mine drainage (amd) causes, treatment and case studies. journal of cleaner production, 14, 1139-1145. 86 biology, medicine, & natural product chemistry 10 (2), 2021: 81-86 arogunjo a (2007) heavy metal composition of some solid minerals in nigeria and their health implications to the environment. journal of biological sciences 10(24):4438-43 doi: 10.3923/jbs.2007.4438.4443· facchinelli, a., scchi, e., & mallen, l. (2001). multivariate statistical and gis-based approach to identify heavy metal sources in soils. environ. pollution. 114, 313–324. katar s., (2009). environmental degradation and measures for its mitigation with special reference to india’s agricultural sector. india. journal of agriculture. econ. 64, 1, jan.-march keskin, k. & makineci, e. (2009). some soil properties on coal mine spoils reclaimed with black locust (robinia pseudoacacia l.) and umbrella pine (pinuspinea l.) in agacliistanbul, environ. monit. assess., 159, 407–414. lockitch g (1993). perspective on lead toxicity. cline. biochem., 26: 371-381. marcovecchio j.e., botte s.e., & freije r.h. (2007). heavy metals, major metals, trace elements. in: handbook of water analysis. l.m. nollet, (ed.). 2nd edn. london: crc press; 275-311. martinez, c.e. & h.l. motto, 2000. solubility of lead, zinc and copper added to mineral soils. environmental pollution.107: 153-158. myśliwa-kurdziel b., prasad m.n.v., & strzałka, k., (2004). photosynthesis in heavy metal stressed plants. in: prasad mnv (ed) heavy metal stress in plants: from biomolecules to ecosystems. springer, berlin, 146–181. olafisoye o. b., tejumade a. & otolorin a. o., (2013). heavy metals contamination of water, soil, and plants around an electronic waste dumpsite. pol. j. environ. stud., 22(5): 14311439. suzan h. h. oelofse (2008) protecting a vulnerable groundwater resource from the impacts of waste disposal: a south african waste governance perspective, international journal of water resources development, 24:3, 477-489. tembo d.b., sichilongo k, & cernak j., (2006). distribution of copper, lead, cadmium and zinc concentrations in soils around kabwe town in zambia. chemosphere, 63: 497-501. yang m.g., lin, x.y., & yang x.e (1998). impact of cd on growth and nutrient accumulation of different plant species. china journal of applied ecology, 19:89-94. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 467-473 | doi: 10.14421/biomedich.2024.132.467-473 issn 2540-9328 (online) detection of antibacterial susceptible salmonella spp. in cured beef with different shelf-life expectancy mutia asri khairunisa1, endah retnaningrum2,* 1student of faculty of biology universitas gadjah mada, 2microbiology laboratory, department of tropical biology, faculty of biology, universitas gadjah mada, jl. teknika selatan sekip utara yogyakarta 55281, tel. +62-274-580839, fax. +62-274-580839, indonesia. corresponding author* endahr@ugm.ac.id abstract meat is currently one of the food products with the highest demand ever since 1980, where demand has reached 24.8 kg per year per person as of 2020. with high demand comes intensive farming which causes overuse of antimicrobials for both therapeutic and nontherapeutic reasons, allowing more antimicrobial resistant (amr) strains of bacteria to occur. in the case of meat products, salmonella is considered one of the more commonly occurring bacteria found in raw meat products. however, with meat’s short shelf-life expectancy, the likeliness of consumers suffering from salmonellosis increases. thus, preservation methods have been implemented to reduce this likeliness, primarily through curing beef. although curing may reduce the likeliness of excessive microbial growth, amr salmonella has been detected in cured beef samples. the scope of this research determines whether there is salmonella spp. within the cured beef samples, conduct amr (azithromycin, ciprofloxacin, and ceftriaxone) analysis of the salmonella spp. isolated from the cured beef samples and enumeration was conducted. the longer the shelf-life expectancy of cured beef samples, the lower the overall cfu/ml per sample was (control: 17,800,000 cfu/ml, >1 year: 0 cfu/ml). it was also discovered that salmonella spp. has potential resistance towards ciprofloxacin (33.33% intermediate) and susceptibility towards azithromycin and ceftriaxone (100.00% sensitive). this research implies the agricultural industry and safety for consumers of cured beef products with different shelf-life expectancies. keywords: antimicrobial resistance; cured beef; salmonella spp.; shelf-life expectancy. abbreviations: amr: antimicrobial resistant; ss: salmonella-shigella; dna: deoxyrubonucleic acid; pmqr: plasmid-mediated quinolone resistance; esbl: extended spectrum β-lactamase; na: nutrient agar; bpw: buffer peptone water; tpc: total plate count. introduction since 1980 until 2010, the global average demand for livestock-derived food per person has increased by a total of 8 grams, from 22 grams of protein per day to 30 grams of protein per day. this includes the increased demand for meat, with an estimated 25 grams per day per person (24.8 kg per year per person) as of 2020. annual percent change in per person demand is more prevalent within countries with rising incomes and falling prices on demand, which is most prevalent in south asia and subsaharan africa. overall, demands for livestock-derived food demands are estimated to increase by 14% per person and 38% in total from 2020 and 2050 (komarek et al., 2021). one of the main issues with raw meat as a food product is its short shelf-life due to its natural microflora. however, many preservation methods have been implemented to increase their shelf-life, including curing. cured meat, where there is salt, sugar, and nitrites/nitrates added to the meat, has proven to be an effective method in reducing spoilage of meat products and reducing the total aerobic and anaerobic plate count within samples, allowing meat products to be stored for longer periods without experiencing signs of spoilage. a study conducted by bower et al. (2018) concluded that a slight decrease in water activity within cured meat samples has been proven to extend the shelf life of said product (bower et al., 2018). curing meat is an alternative to allowing easier storage and distribution of meat. however, with the increasing demand for meat products, intensive farming is a necessary solution for supply to meet demands. intensive farming may lead to overcrowding of farm animals, pushing the usage of antimicrobials in animal feeds to reduce the risks of diseases spreading amongst livestock (manyi-loh et al., 2018). based on research conducted by landers et al. (2012), antimicrobial resistance (amr) has been an emerging public health crisis where most amr cases occur in agricultural settings (lander et al., 2012). many of these antimicrobials are used irrationally within the manuscript received: 28 july, 2024. revision accepted: 07 october, 2024. published: 22 october, 2024. https://doi.org/10.14421/biomedich.2024.132.467-473 468 biology, medicine, & natural product chemistry 13 (2), 2024: 467-473 agricultural setting, proving more dangerous than helpful (manyi-loh et al., 2017). due to constant exposure to new lines of antimicrobials, the bacterial population will adapt faster and mutate, making antimicrobials ineffective towards them (lander et al., 2012). the first recorded case of antibiotic resistance in bacterial strains was discovered in 1975. by 1984, different strains of escherichia, salmonella, and klebsiella displayed resistance to several commonly used antimicrobials, such as aminoglycosides (galhano et al., 2021). there is a high probability of the amr gene being transferred from livestock to humans, due to the commercial distribution of meat products where amr bacteria are present (lander et al., 2012). although curing meat may reduce the natural growth of microflora, the presence of microbes in the food product is inevitable. therefore, the possibility of antibiotic resistant bacteria being present in the cured meat is still prevalent (bower et al., 2018). one of the commonly occurring bacteria in meat products is salmonella which causes salmonellosis, a foodborne infection that is responsible for an estimated 80.3 million cases per year. contamination by salmonella, specifically salmonella enterica, can originate from the raw meat product itself or during the production stage (mutz et al., 2019). with the possibility of commercial bacteria being present in cured meat products and acting as a reservoir for amr genes, there is potential for amr salmonella spp. to be transferred to humans (lander et al., 2012). materials and methods study area samples were taken from cured beef samples of the origins, which had different shelf-life expectancies, where they varied from control, <1 year, 1 year, and >1 year. procedures preparation of cured beef samples (replace with your sub-sub-title of procedures) a total of 5 grams of control/cured beef sample was weighted on a scale. the meat was pounded using the mortar and pestle until smooth, with the help of sterilized distilled water, and then suspended into 45 ml of bpw, a non-selective enrichment broth, making the original stock culture (100). the sample in the bpw was incubated at 37oc for 18±1 hours (temelli et al., 2012). serial dilution is made from level 10-2 until 10-6. to make the serial dilution, 1 ml is added to 9 ml of sterilized saline, making the first level of dilution (10-1). this is repeated for all dilution levels where 1 ml of the previous dilution level is added into 9 ml of sterilized saline (cappuccino & welsh, 2017). identification of salmonella spp. in cured beef sample ss agar plate and the sample enriched in bpw are prepared. streak plate is conducted, the sample is inoculated on the ss agar plate using a disposable swab with a quadrant streak method. this is done until there are three repetitions. the petri dish is sealed, turned upside down and incubated for 24±1 hours at 37oc. after incubation, the sample is observed for transparent colonies with black h2s precipitate in the center, indicating the presence of salmonella spp. gram staining was then conducted for the isolated salmonella spp. colonies and observed underneath a microscope at 10×100 (nabbut, 1973). enumeration of cured beef sample ss agar plates are prepared for enumeration. spread plate is conducted where 0.1 ml of 10-2 suspension is taken using a micropipette and inoculated on the ss agar plate. the suspension is spread evenly on the surface of the agar using a drigalski spatula and then the petri dish is flipped. this methodology is repeated for dilution levels 10-3 until 10-6 for all samples. the sample is incubated for 24±1 hours at 37oc. after incubation, total plate count (tpc) is conducted twice, once for all colonies on the ss agar and a second time for the salmonella spp. colonies, indicated with transparent colonies and black h2s precipitate in the center (nabbut, 1973; cappuccino and welsh, 2017). antimicrobial susceptibility test salmonella spp. cultures are isolated from the initial identification of it from the cured beef samples. the isolated colonies are cultured on slant agar using na and incubated for 24±1 hours at 37oc. then, 9 ml of sterilized saline was added into the culture and swabbed onto na plates using the dense streaking method. dense streaking was done three times where the petri dish was rotated 60o before doing the next pass of dense streaking. once dense streaking was done, the perimeter of the petri dish was swabbed to pick up any excess amounts of liquid (hudzicki, 2009). the antimicrobial disk was infused with their respective antimicrobial, as seen below. data analysis once the data had been collected, confirmation was done to determine whether salmonella spp. was isolated from the samples. alongside the confirmation, a total plate count was conducted to determine the correlation between shelf-life expectancy and overall cfu/ml detected. once that was done, an antibacterial susceptibility test was done for the isolated salmonella spp. strains. khairunisa & retnaningrum – detection of salmonella spp. from preserved food 469 results and discussion confirmation of salmonella spp. confirmation of the presence of salmonella spp. is significant within this research to allow pure culturing of the targeted bacteria. to do so, a confirmation test was conducted with the beef samples that were nonselectively enriched using buffer peptone water (bpw) to increase the accuracy of the media (ruiz et al., 1996). based on the methodology and independent variables utilized, confirmation of salmonella spp. was done for four samples with different shelf-life expectancies, which are; control, <1 year, 1 year, and >1 year. these beef samples were most likely sourced from bos taurus indicus linnaeus, 1758, or also known as beef cattle (gbif, 2024). quadrant streaking was done to isolate single colonies of salmonella spp. and, with the help of the biochemical and selective properties of ss agar, single colonies with black h2s precipitate were easily identifiable and isolated. following the isolation of single colonies, it was further cultured on na slants to produce pure culture isolates of salmonella spp. and was moved onto glass slides for microscopical analysis. in total, two outputs can confirm the presence of salmonella spp., through the bacteria’s cell morphology and whether it is gram positive or negative. table 1. confirmation and basic characterization of salmonella spp. and unconfirmed colonies isolate from cured beef samples. ss agar cell morphology gram positive/ negative control + bacillus − < 1 year + bacillus − 1 year* − bacillus − > 1 year** − invalid invalid *no presence of salmonella spp. but other microbes were detected **no presence of bacterial growth based on the results that have been obtained from the confirmation test (table 1), salmonella spp. was detectable in both the control and in the <1 year sample. on the other hand, salmonella spp. was not detectable in 1 year and >1 year samples but other forms of microbes were detectable. due to the selective nature of the ss agar, only a select few types of bacteria can be grown on the agar, which are salmonella spp., shigella spp., escherichia coli, enterobacter spp., and klebsiella spp. transparent colonies with black h2s precipitate indicated the positive presence of salmonella spp. (yanestria et al., 2019). the formation of transparent colonies on ss agar is due to the inability of salmonella spp to ferment lactose within the medium. in addition to that, with the help of sodium thiosulfate and ferric citrate, the removal of sulfur from hydrogen sulfide, which is produced by the bacteria reacts with the thiosulfate from the sodium thiosulfate and iron ions from the ferric citrate to create the black precipitate, differentiating its colonies from the rest of the gram-negative bacteria that can grow on ss agar (tortora et al., 2010; remel, 2010). further confirmation was done through gram staining, where the clear colonies with black precipitate were isolated and stained. for samples with no clear colonies with black precipitate, isolation of available colonies was done. microscopic observation was done with a magnification of 10×100. based on the results obtained (table 1), all the bacteria isolated were gram-negative bacteria, including salmonella spp., as seen in figure 2 (ray & bhunia, 2013). this is due to the selective properties of ss agar, where sodium citrate and brilliant green inhibited the growth of gram-positive bacteria (remel, 2010). therefore, not only does it confirm the transparent colonies with black precipitate are indeed salmonella spp. colonies, but it also limits what strains of bacteria the unknown colonies are, which are limited to only gram-negative bacteria. gram staining also provided insight to the cell morphology, where all microscopic observation confirms that both confirmed salmonella spp. colonies and unconfirmed colonies were bacillus or rod shaped (figure 2). this aligns with the fact that the enteric bacteria that can grow on ss agar are all rodshaped bacteria. thus, it can be conclusive that ss agar is effective in being a selective media and its confirmation of salmonella spp. is reliable (madigan et al., 2015) figure 2. microscopic observation at 10×100 for salmonella spp. from control (a) and <1 year (b), and unknown isolate for sample 1 year (c) isolated from cured beef samples. 470 biology, medicine, & natural product chemistry 13 (2), 2024: 467-473 total plate count of cured beef sample following the confirmation of salmonella spp. presence in cured beef samples with different shelf-life expectancy, total plate count (tpc) was conducted to determine the overall presence of bacteria within the beef samples alongside the percentage of salmonella spp. compared to the overall enumeration of bacteria, as seen in table 2. same as previously, there were four samples used for the enumeration of salmonella spp. with the consideration that the samples can only be enumerated for bacteria that can grow on ss agar, which were previously mentioned. table 2. enumeration of the overall presence of bacteria in comparison to the percentage of salmonella spp. in the cured beef samples. overall cfu/ml salmonella spp. cfu/ml % of salmonella spp. control 17,800,000 2,165,000 12.16% < 1 year* 6,000,000 0 0.00% 1 year** 14,100 0 0.00% > 1 year*** 0 0 0.00% *presence of salmonella spp. but not detectable in tpc **no presence of salmonella spp. but other microbes were detected ***no presence of bacterial growth based on the results obtained for the enumeration (table 2), we can observe that samples with shorter shelf-lives tend to have a higher overall cfu/ml due to the lack of curing, and samples with longer curing time have lower overall cfu/ml. this can be seen when comparing the control with the >1 year sample, where the control has an overall cfu/ml of 17,800,000 cfu/ml while the >1 year sample has an overall cfu/ml of 0 cfu/ml. this trend is also observable with the percentage of salmonella spp. within the sample where salmonella spp. makes up 12.16% (2,165,000 cfu/ml) of the overall cfu/ml of the control. in addition, salmonella spp. was detected in the confirmation test for the <1 year sample but was very sparse when tpc was conducted for this sample and could not be calculated, leading to salmonella spp. making up 0.00% of the overall cfu/ml (attachment 1). for the one year sample, tpc growth was very minimal but based on the morphological characteristics of the colonies, colonies with pink centers, it is likely that the unconfirmed colonies are e. coli (remel, 2010). considering that the tpc was done on ss agar, this research’s scope is limited to the growth of enteric gramnegative bacteria. thus, the tpc does not consider that other microbes outside of the enteric gram-negative group may have been present in the samples. curing can be seen as an effective method in reducing the growth of unwanted microflora, in this case bacteria. as mentioned previously, curing is a process that utilizes salt in a dry mixture that envelops the meat. this can be cured over different time ranges to create cured meat products. adding salt, it preserves the flavor, color and quality of the product while also reducing the water activity level of the meat. thus, with reduced water activity levels, bacteria will not have access to unbounded water necessary for their biochemical and metabolic pathways. therefore, the present bacteria within the meats are eliminated. furthermore, the bacteria cells will experience osmotic shock and undergo lysis due to the movement of water within their cells to the outside environment, which has a low water concentration, killing off the present bacteria cells from lysis caused by osmotic shock (nummer & andress, 2015; henny et al., 2010). the sodium content within the cured beef is also a factor that can contribute to the effectiveness of the curing process. as seen in table 3, the longer the shelf-life expectancy of a sample is, the higher the sodium content per 100 grams will be. table 3. sodium content of cured beef samples (mg/100g) as per their nutrition facts. cured beef sample sodium content (mg/100g) control (uncured beef) 0 <1 year (pepperoni) 500 1 year (corned beef) 1,134 >1 year (jerky) 1,642 when comparing the overall cfu/ml and the sodium content of the samples, the results are parallel to one another, where the higher the salt content, the lower the overall cfu/ml will become. this is seen with the control and 1 year sample. in the control, with a sodium content of 0 mg/100g, the overall cfu/ml is 17,800,000 cfu/ml. on the other hand, the 1 year sample, with a sodium content of 1,134 mg/100g, the overall cfu/ml is 14,100 cfu/ml. the effectiveness of a higher sodium content can clearly be seen with the >1 year sample as the overall cfu/ml is at 0 cfu/ml, proving that the curing method is an effective way to eliminate unwanted growth of bacteria. in addition to that, with salmonella spp., dry curing is highly effective at reducing the percentage of salmonella spp. from the overall cfu/ml. an occurrence such as this is because of salmonella spp. present in the samples had not adapted to the hypertonic environment of cured meats, proving them to be sensitive to osmotic shock. thus, the extreme decline in the salmonella spp. a percentage from the overall cfu/ml (li, et al., 2023). however, the unconfirmed colonies in the one year sample may have been present in the tpc, although the sodium content of the sample is high, because hyperosmotic environments caused by the high sodium content may cause bacteria, or in this case, e. coli, to experience accelerated constriction rates. this will lead to an increased overall cfu/ml due to faster division rates within the small number of bacteria present in the sample (sun et al., 2021). khairunisa & retnaningrum – detection of salmonella spp. from preserved food 471 antimicrobial susceptibility test once confirmation and enumeration of the bacteria in cured beef samples were done, the pure cultures isolated from the confirmation tests were used for the antimicrobial susceptibility test. based on the results that have been obtained from the confirmation test, the samples that underwent antimicrobial susceptibility testing were only the control and the <1 year sample, as they were the only ones that had a confirmed presence of salmonella spp. to do so, the test was done on na plates, where the kirby bauer disk diffusion method was implemented. this methodology requires the creation of a suspension of the targeted isolate, in this case, salmonella spp., where the suspension were made with sterilized saline solution and vortexed. following this, three passes of dense swabbing of this suspension was done using disposable swabs on na plates to ensure even swabbing of the suspension. theoretically, muellerhinton agar is required for this method, but from trial and error, na plates proved to be more effective in reading apparent zone formations from the antimicrobial susceptibility testing. once swabbed evenly, the loaded and sterilized antimicrobial disks are placed evenly on the surface and incubated. apparent zone formations were measured using a caliper and interpreted using the clsi guidelines (hudzicki, 2009). table 4. antimicrobial susceptibility testing with azithromycin (15 µg), ciprofloxacin (5 µg), and ceftriaxone (30 µg) against salmonella spp. isolated from cured beef samples of control and <1 year. antimicrobial tested dose (µg) categories sensitive intermediate resistant azithromycin 15 100.00% 0.00% 0.00% ceftriaxone 30 100.00% 0.00% 0.00% ciprofloxacin 5 66.67% 33.33% 0.00% the results obtained above were limited to only the control and the <1 year sample due to the presence of salmonella spp. within these two samples. the one year and >1 year samples did not undergo the antimicrobial resistance test due to the lack of salmonella spp. detected within the samples. from the tested samples, salmonella spp. is 100.00% sensitive to azithromycin and ceftriaxone while only being 66.67% sensitive to ciprofloxacin. it was discovered that salmonella spp. is intermediate to ciprofloxacin with 33.33% of the tests reacting that way. however, the results state that salmonella spp. is not resistant to all three antimicrobials tested in this research, which are: azithromycin, ceftriaxone, and ciprofloxacin. these results a contradict the initial hypothesis created for this research, where resistance was expected towards azithromycin and sensitivity was expected for ciprofloxacin. results obtained completely went against the hypothesis and instead the most resistance is towards ciprofloxacin (33.33% intermediate) and most sensitive towards both azithromycin and ceftriaxone (100.00% sensitive). although the results did not follow the hypothesis, the research problem and objectives were achieved. where it is determined that salmonella spp. shows a degree of resistance to ciprofloxacin and sensitivity to azithromycin and ceftriaxone. furthermore, antimicrobial susceptibility was conducted effectively towards the salmonella spp. strains that were able to be isolated from the sample. these results may be due to a few possible scenarios. firstly, ciprofloxacin’s 66.67% sensitivity is lower than the other two may be caused by the occurrence of mutations within the gene that encodes for gyrase (grya and gryb) and the occurrence of the pmqr gene. mutation in this gene will prevent the segregation of the daughter chromosomes ahead of the replication fork. malfunction of the gyrase enzyme will prevent topoisomerase iv from segregating the chromosomes at the replication fork and lead to rapid cell death due to failure of cell division (nair et al., 2018; shariati et al., 2022; blondeau, 2004). however, salmonella spp. did not show complete resistance to ciprofloxacin which may be due to the ineffective transmission of the amr gene within the beef cattle’s intestinal tract. one of the ways to transmit genes between bacteria cells is through horizontal transmission, specifically conjugation, entailing cell-to-cell contact through the sex pilus, a structure detectable within salmonella spp. (sanseverino et al., 2018). secondly, azithromycin and ceftriaxone’s 100.00% sensitivity can be due to a few reasons. for azithromycin, sensitivity of salmonella spp. can be due to the absence of mutation in the erm42 and ermb and no methylation of the 23s rrna thus, total effectivity of the 50s ribosomal subunits. furthermore, there may have been no modification of the acrab-tolc efflux pump, allowing full retention time of the antimicrobial within the salmonella spp. cells (chiou et al., 2023). on the other hand, sensitivity of salmonella spp. to ceftriaxone may be due to the presence of the esbls enzyme. this enzyme functions explicitly to hydrolyzing the β-lactam ring within the antibiotics, rendering them inactive and inefficient in fighting off bacterial infections (nair et al., 2018). it is possible that certain salmonella spp. cells within the beef cattle did have these genes but there was ineffective conjugation. failure of the conjugation process can be due to multiple reasons. one of these is due to the environment in which conjugation occurs, which is in the rumen of beef cattle. previous research has indicated the diets of beef cattle affect gut resistomes in livestock, where high grain diets, although economically efficient, have proven to increase the chances of amr gene transfer within the rumen. it is possible that the beef cattle, where the cured beef is sourced from, has a mixture of a regular diet and a high grain diet, which makes it more likely for conjugation to occur between present microbes within its rumen. thus, the beef cattle where the samples for this 472 biology, medicine, & natural product chemistry 13 (2), 2024: 467-473 research were sourced from may have been on regular diets while the samples from the literature review may have been sourced from beef cattle with high grain diets (zhang et al., 2023). specific implications can be made based on the results obtained. the common usage of antimicrobials within the agricultural industry, to a certain extent does have minor effects on the detection of amr bacterial strains. specifically for the potential resistance of salmonella spp. towards ciprofloxacin. this may prove to be problematic as there is the presence of salmonella spp. detected within the control and <1 year sample. if there is potential for resistance towards ciprofloxacin (33.33% intermediate), this poses a threat if consumers were to be introduced to the salmonella spp. present in within the samples, potentially exposing consumers to the chances of contracting salmonellosis, or even worse, typhoid fever. however, it can be noted that the samples tested were not cooked beforehand which may have increased the likelihood of detecting salmonella spp. within the samples. for consumer safety, it is right to suggest thoroughly cooking the cured beef samples at temperatures above 50oc will help lower the chances of exposure to salmonella spp. (ray and bhunia, 2013). conclusions in conclusion, salmonella spp. was detectable in the control and <1 year cured beef samples but was undetectable in the 1 year and >1 year cured beef samples. this indicates that cured beef samples with longer shelf-life expectancy have lower chances of salmonella spp. detection. additionally, it was discovered that salmonella spp. show potential resistance towards ciprofloxacin (66.67% intermediate) and susceptibility towards azithromycin and ceftriaxone (100.00% sensitive). these discoveries have specific implications for the agricultural industry and the safety of consumers of cured beef samples. acknowledgements: acknowledgement are expressed towards the microbiology laboratory, faculty of biology, universitas gadjah mada for providing the appropriate setting to conduct the research and other parties that fall under this institution. authors’ contributions: prof. dr. endah retnaningrum contributed to this research as the lead supervisor lecturer which aided in the methodology of this research alongside narration of this article. mutia asri khairunisa contributed by doing the laboratory work and narration of this article. competing interests: the authors declare that there are no competing interests. funding: the author declares no funding. 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(family solanaceae), an edible shrub whose fruits and leaves are used medicinally to treat diseases including diabetes was evaluated for effect on alpha amylase and alpha glucosidase enzymes in vivo. the leaf extract (70-210 mg/kg) and fractions (hexane, dichloromethane, ethyl acetate, methanol, 140 mg/kg) of s. anomalum were evaluated in vivo for inhibitory effect on alpha amylase and alpha glucosidase enzymes using starch, sucrose and maltose as substrates. acarbose was used as reference drug. the leaf extract especially middle dose (140 mg/kg) and fractions (ethyl acetate and hexane) caused significant (p<0.05) reduction in blood glucose levels of animals treated with the various substrates used. ethyl acetate fraction exerted the highest inhibitory effect when starch and maltose were used as substrates followed by n-hexane and methanol. n-hexane was the most active fraction followed by ethyl acetate when sucrose was used as substrate. the results suggest that the leaf extract and fractions of s. anomalum have the potentials to inhibit alpha amylase and glucosidase in rats. keywords: alpha amylase; alpha glucosidase; hypoglycemia; solanum anomalum. introduction solanum anomalum thonn. ex schumach, a plant whose fruits and leaves are used medicinally and nutritionally is commonly found growing in west and east africa sub-regions. its parts are utilised locally to treat diabetes, gastrointestinal disorders, infections, inflammation and pains (burkill, 2000; bukenya and hall, 1988; offor and ubengama, 2015). hypoglycemic and antidiabetic activities of the fruits and leaves have been reported (offor and ubengama, 2015; okokon et al., 2022). moreso, in vivo and in vitro antiplasmodial (okokon et al., 2016; okokon et al., 2017a), antioedema (okokon et al., 2017b), antioxidant and antiulcer (okokon et al., 2019a), anticonvulsant and depressant (okokon et al., 2019b), analgesic (okokon et al., 2020) and antidiarrhoeal (udobang et al., 2022) properties of the leaf extract have also been reported. phytochemical constituents such as alkaloids, flavonoids, saponins, tanins, diosgenin, a diosgenin glycoside (25(r)-diosgenin-3-o-α-l-rhamnopyranosyl(1→4)-β-d-glucopyranoside, uracil, 5-methyluracil, 1octacosanol, and octacosane have been reported on the leaves of the plant (okokon et al., 2016; okokon et al., 2022). we report in this study the effect of leaf extract and fractions of the plant on alpha amylase and alpha glucosidase of rats. materials and methods plants collection fresh leaves of solanum anomalum were collected in compounds in uruan area, akwa ibom state, nigeria in august, 2020. the plant was identified and authenticated by a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria. hebarium specimen was deposited at department of pharmacognosy and natural medicine herbarium, university of uyo (uuh.75a). extraction fresh leaves of s. anomalum were washed, cut into smaller pieces and dried under shade for two weeks. the leaves were further pulverized to powder using electric grinder. the powdered leaves material was divided into two parts; one part (1.5 kg) was macerated in 50% ethanol (7.5 l) for 72 hours at room temperature (28 ±2 ˚c). while the other part, (1.5 kg) was successively and gradiently macerated for 72 h in each of these solvents (2 x 5l), n-hexane, dichloromethane, ethyl-acetate and methanol to give corresponding fractions of these solvents. these were thereafter filtered and the liquid filtrates were concentrated and evaporated to dryness in vacuo 40˚c using a rotary evaporator (buchilab, switzerland). the extract and fractions were stored in a https://doi.org/10.14421/biomedich.2023.121.127-132 128 biology, medicine, & natural product chemistry 12 (1), 2023: 127-132 refrigerator at -4˚c, until used for the proposed experiments. animals wistar rats (138-150 g) of either sex were obtained from the university of uyo animal house. they were maintained on standard animal pellets and water ad libitum. permission and approval for animal studies were obtained from the college of health sciences animal ethics committee, university of uyo. in vivo alpha-amylase and glucosidase inhibition study  alpha-amylase inhibitory study fifty wistar rats were divided into 10 groups of 5 rats each. the rats in all groups were fasted for 18 hours and fasting blood glucose concentration was first taken at 0 minute before administration. based on predetermined ld50 value of 724.56 mg/kg (okokon et al., 2016), the animals were treated as follows; group i, which served as the normal control, received distilled water (10 ml/kg). group ii rats were orally administered starch at 2 g/kg body weight (orally with distilled water as vehicle) and distilled water (10 ml/kg) simultaneously. rats in group iii were administered starch (2 g/kg) and the standard drug (acarbose) at 100 mg/kg simultaneously. groups iv, v and vi were administered simultaneously, starch (2 g/kg) and s. anomalum leaf extract at 70, 140 and 210 mg/kg respectively while groups vii, viii, ix and x rats were administered starch (2 g/kg) and fractions (n-hexane, dichloromethane, ethyl acetate and methanol) at 140 mg/kg respectively. all administrations were done orally and blood glucose concentration was monitored at 30, 60, 90, 120 and 180 minutes (gidado et al., 2019).  glucosidase inhibitory study the procedure as described above was used for this study but with sucrose and maltose used as substrates (gidado et al., 2019). blood glucose determination drops of blood from tip of rats’ tails were dropped on stripes and glucose concentration was measured using a glucometer according to manufacturer’s specifications (accu-chek, indiana). the glucometer works with the following principle; the blood sample is exposed to a membrane covering the reagent pad (strip), which is coated with an enzyme (glucose oxidase, glucose dehydrogenase). the reaction causes a colour change and the intensity of this change is directly proportional to the amount of glucose in the blood sample. light from a light emitting diode strikes the pad surface and is reflected to a photodiode, which measures the light intensity and converts it to electrical signals. an electrode sensor measures the current produced when the enzyme converts glucose to gluconic acid. the resulting current is directly proportional to the amount of glucose in the sample (who, 2011). statistical analysis data obtained from this work were analysed statistically using one –way anova followed by tukey-kramer multiple comparison test using instat graphpad software, (san diego, usa). differences between means were considered significant at 5% and 0.1% level of significance ie p≤ 0.05 and 0.001. results and discusssion in vivo alpha amylase and glucosidase inhibition assay administration of starch (2g/kg) caused varying percentages of increase in blood glucose concentrations after 30 minutes. the percentages were starch (63.18%), extract/fractions treated groups (10.97-32.69%) and acarbose-treated group (17.97%). these increases were reduced after 60 minutes with animals treated with the ethyl acetate fraction (10.53%). these decreases were significant and sustained for 180 minutes in ethyl acetate-treated group, followed by middle dose group, 140 mg/kg, (9.22%) and methanol-treated group (12.15%). however, co-administration of the starch with acarbose prominently inhibited the rise in the blood glucose concentrations (table 1). okokon et al. – in-vivo alpha amylase and alpha glucosidase inhibitory 129 table 1. effect of ethanol leaf extract and fractions of solanum anomalum on blood glucose level of rat after oral administration of starch load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 86.00±11.53 87.66±7.12(1.93) 87.66±7.62(1.93) 73.66±6.17 91.0±7.50(5.81) 80.00±6.02 starch 2000 73.33±8.25 119.66±5.45a(63.18) 115.66±1.33a (57.72) 104.66±2.60a (42.72) 95.66±3.75a (30.45) 92.0±6.35(25.46) acarbose 100 72.33±2.69 85.33±12.97(17.97) 80.33±7.21(11.06) 76.33±3.48(5.53) 74.0±1.00(2.30) 72.33±8.68(0) crude extract 70 70.12±2.00 89.33±1.45(23.11) 100.33±4.05(42.12) 99.66±2.90(42.12) 89.0±2.08(26.92) 80.0±4.16(14.09) 140 72.33±8.87 89.33±8.68(23.50) 94.66±2.90(30.87) 94.0±0.57(29.95) 84.66±4.41(17.04) 79.0±3.21(9.22) 210 62.33±1.45 74.33±7.83(19.25) 86.0±10.14(37.97) 96.0±2.64(54.01) 83.0±2.08(33.16) 82.20±2.30(31.87) n -hexane fraction 140 66.66±1.33 90.33±1.85(35.50) 93.0±3.21(39.52) 108.0±3.60(62.01) 97.00±3.00(45.51) 85.66±4.48(28.50) dichloromethane fraction 140 69.33±4.41 92.0±2.51(32.69) 104.3±14.71(34.97) 108.6±12.81(56.64) 107.33±11.78\(54.81) 96.66±7.53(39.42) ethyl acetate fraction 140 82.00±4.04 91.0±3.21(10.97) 91.66±5.60(10.53) 93.33±1.20(13.81) 91.0±2.30(10.97) 81.66±3.75() methanol fraction 140 71.33±1.20 90.00±4.35(26.17) 93.33±5.78(30.84) 92.0±5.19(28.97) 85.33±3.18(19.62) 80.0±4.54(12.15) data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control. (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. administration of sucrose (2 g/kg) produced a 46.01% increase in blood glucose concentration 30 minutes post-administration of the sucrose in the control group and 25.95-70.73% increases in blood glucose concentration of extract/fractions-treated groups. the blood glucose concentrations were significantly reduced in hexane-treated group (13.02%) after 60 minutes postadministration of sucrose. however, n-hexane fraction had the highest effect (0.84%) throughout the duration of the study (180 minutes) followed by middle dose, 140 mg/kg, group (6.47%) (table 2). table 2. effect of ethanol leaf extract and fractions of solanum anomalum on blood glucose level of rat after oral administration of sucrose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 100.00±4.25 88.33±1.85 92.33±4.25 90.33±2.33 89.0±4.35 87.33±3.84 sucrose 2000 92.0±4.04 134.33±2.90b(46.01) 128.66±5.45a (39.84) 117.33±4.66a(27.53) 97.66±0.66(6.15) 104.16±2.48(13.21) acarbose 100 90.33±2.48 86.66±2.90 82.0±6.00 79.33±2.96 71.66±3.75 78.0±3.78 crude extract 70 70.66±5.92 89.0±7.55b(25.95) 92.33±8.83(30.66) 78.33±9.13(10.85) 76.33±2.18(8.02) 77.66±0.66(9.90) 140 82.33±3.75 117.33±4.80b(42.51) 102.0±2.08(23.89) 88.66±5.45(7.68) 85.66±5.23(4.04) 87.66±1.33(6.47) 210 72.0±2.64 114.66±6.38(59.25) 113.0±11.24(56.94) 91.33±7.68(26.84) 93.22±9.33(29.47) 83.00±2.08(15.27) n -hexane fraction 140 79.33±1.45 101.0±3.05(27.31) 89.66±1.45(13.02) 86.0±3.05(8.40) 81.66±1.45(2.93) 80.0±1.00(0.84) dichloromethane fraction 140 73.66±7.53 120.33±4.37(55.66) 106.33±7.35(44.35) 113.66±9.38(54.30) 95.00±5.68\(28.97) 90.33±2.90(22.63) ethyl acetate fraction 140 68.33±3.18 116.66±11.72(70.73) 117.66±6.88(72.19) 110.33±8.53(61.46) 89.66±2.72(31.21) 86.66±1.85(26.82) methanol fraction 140 79.33±6.06 106.33±7.86(34.03) 96.66±2.66(21.84) 108.0±2.51(36.14) 91.33±3.33(15.12) 96.66±9.52(21.84) data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control. (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. there was 60.78% increase in blood glucose concentration 30 minutes following maltose administration in the control group. however, 27.0857.80% increases were observed in the extract/fractionstreated groups. at 60 and 90mins, the ethyl acetate fraction group had blood glucose level of 18.44 and 0.61% respectively. at 180 minutes, n-hexane fraction and middle dose (140 mg/kg)-treated group had blood glucose level of 2.02% and 2.52% respectively (table 3). 130 biology, medicine, & natural product chemistry 12 (1), 2023: 127-132 table 3. effect of ethanol leaf extract and fractions of solanum anomalum on blood glucose level of rat after oral administration of maltose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 100.00±4.25 88.33±1.85 92.33±4.25(1.80) 90.33±2.33(3.62) 89.0±4.35(1.55) 87.33±3.84(3.98) maltose 2000 82.30±2.14 132.33±1.90b(60.78) 130.22±2.45(58.22) 120.66±3.22a(46.60) 115.0±2.46(39.73) 106.22±4.24(29.06) acarbose 100 85.34±1.36 88.22±1.10(3.37) 86.0±2.20c(0.77) 85.33±2.15c() 84.26±1.14a() 82.28±2.26a() crude extract 70 83.3±1.20 131.45±3.28(57.80) 125.17±4.39b(50.26) 115.33±1.66a(38.45) 104.35±1.12(25.27) 95.42±2.28(14.54) 140 81.28±5.28 128.14±1.98(56.05) 105.23±1.52a(29.46) 95.56±3.88(17.56) 90.44±1.86(11.26) 83.33±3.99(2.52) 210 85.24±7.34 125.19±7.22(46.86) 90.50±4.86a(6.17) 87.25±8.65b(2.35) 81.45±5.60a() 77.0±5.21 b () n -hexane fraction 140 80.55±4.89 118.24±5.28(46.79) 104.28±2.37a(29.45) 92.22±3.34 a(14.48) 87.28±2.28 b (8.35) 82.18±3.28(2.02) dichloromethan e fraction 140 83.82±5.49 122.98±4.29b(46.71) 110.37±3.29(31.67) 102.19±2.19(21.91) 98.28±6.18a(17.25) 91.88±1.83(9.61) ethyl acetate fraction 140 82.36±6.54 132.46±7.38(59.61) 97.55±7.44 b(18.44) 82.87±9.28b (0.61) 81.65±2.27 b 74.33±8.44c methanol fraction 140 90.10±10.16 114.50±12.18(27.08) 108.53±5.68a(20.45) 94.66±8.93a(5.06) 82.63±5.68 b 73.26±6.79c data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control. (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. discussion various parts of s. anomalum, a medicinal plant, is used in ibibio traditional medicine in the treatment of diseases such as diabetes among others. this work focused on the evaluation of s. anomalum leaf extract and fractions for in vivo inhibitory effect on alpha amylase and alpha glucosidase activities in rats, as well as isolation and characterization of the active antidiabetic principles from this plant. the extract was found to non dose-dependently inhibit increases in blood glucose concentration following starch administration with the middle dose, 140 mg/kg, and ethyl acetate fraction exerting the most inhibition. complete digestion of dietary polysaccharides like starch is achieved by the combined action of alpha-amylases and alpha-glucosidase enzymes. the alpha-amylase enzyme digests alphabonds of the alpha-linked polysaccharides yielding disaccharides, like maltose, which are further reduced to monosaccharides by membrane bound alpha-glucosidase enzymes (kalra, 2014; alongi and anese, 2018). inhibitions of these enzymes delay the digestion of ingested carbohydrates thereby resulting in a small rise in blood glucose concentrations following carbohydrate meals as was observed in this study. as a target for managing type 2 diabetes mellitus, many medicinal plants have been reported to possess alpha-amylase and alpha-glucosidase inhibitory potential (esimone et al., 2001; ibrahim et al., 2014). similarly, the leaf extract and fraction significantly inhibited blood glucose rise when co-administered with maltose and sucrose with n-hexane and ethyl acetate fractions exerting the highest inhibition. acarbose, the standard drug used in this study significantly inhibited blood glucose rise when co-administered with starch, maltose and sucrose. the results of this study corroborate that reported on other species of solanum such as s. nigrum, s. melongena depressum, s. gilo, s. melogena, s. melongena l., s. macrocarpon and s. diphyllum (hossain et al., 2009; nwanna et al., 2013; dasgupta et al., 2016; nwanna et al., 2019), which significant inhibition of alpha –amylase and alphaglucosidase activities were observed. diosgenin present in this plant is implicated in antidiabetic activities of plants (pari et al., 2012; saravanan et al., 2014). diosgenin has been reported to exert its activity through various mechanisms such as inhibiting alpha-amylase and alpha-glucosidase (gosh et al., 2014) to reduce intestinal glucose absorption, inhibiting the sodiumglucose cotransporter-1 (sglt-1) and reducing intestinal na+-k+atpase activity (gan et al., 2020). also, diosgenin glycosides and other steroidal saponins commonly present in the solanum species (kaunda and zhang, 2019) are reported to exert hypoglycaemic activities (wang et al., 2010; wang et al., 2012; elekofehinti, 2015). similarly, β-sitosterol present in the leaf extract and fraction of this plant has been reported to possess inhibitory potentials on alpha-glucosidase and alpha-amylase enzymes (kumar et al., 2013). these could have contributed to the observed activity of this study and therefore explains the antidiabetic mechanism of this extract. alpha-amylase and alpha-glucosidase inhibitions by plants extracts have been reported severally (shirwaikar et al., 2005; ishnava and metisariya, 2018). phytochemicals implicated as anti-diabetic agents, do so possibly through alpha-amylase and alpha-glucosidase inhibition. the phytochemicals in question include flavonoids, saponins, tannins and terpenoids (ortizandrade et al., 2007; ishnava and metisariya, 2018). also, polyphenolic compounds from plants are known to cause enzymes inhibitions on the biological systems (kaita et al., 2018). the phenolic compounds which are biological oxidants, strong metal ion chelators and okokon et al. – in-vivo alpha amylase and alpha glucosidase inhibitory 131 protein precipitation agents form insoluble complexes with proteins (ishnava and metisariya, 2018). the presence of the polyphenolic compounds in the leaf extract and fractions in addition to diosgenin, diosgenin glycosides and β-sitosterol may suggests that their inhibitory potential on alpha-amylase and the membrane-bound intestinal alpha-glucosidase enzymes. conclusion the results of this study suggest that inhibition of alphaamylase and alpha-glucosidase enzymes maybe one of the modes of antidiabetic activity of the leaf extract and fractions of solanum anomalum which can be attributed to the activities of its phytochemical constituents. acknowledgements: the authors are grateful to mr nsikan malachy of pharmacology and toxicology department, university of uyo for providing technical assistance. authors’ contributions: joe was involved in the conceptualization, design, execution, supervision, sourcing of materials, statistical analysis, draft and review of the manuscript; ice participated in the sourcing of material, design, execution, statistical analysis, drafting and review of manuscript; while jau and noe were involved in the design, execution, statistical analysis, review and editing of the manuscript. all authors read and approved the final manuscript. competing interests: the authors declare that there are no conflicts of interest related to this article. references alongi m, anese m. 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(online) co-existent hypertension with diabetes mellitus exacerbates renal dysfunctions eruore amalaka obore1, jerome ndudi asiwe2,3,*, vincent i. iyawe1, andrew e. edo4 1department of physiology, faculty of basic medical sciences, university of benin, benin-city, nigeria. 2department of physiology, faculty of basic medical sciences, pamo university of medical sciences, port-harcourt, nigeria. 3department of physiology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria. 4department of medicine, faculty of clinical sciences, university of benin, benin-city, nigeria. corresponding author* asiwejerome@yahoo.com manuscript received: 26 july, 2022. revision accepted: 04 august, 2022. published: 15 august, 2022. abstract hypertension as well as diabetes mellitus has been reported to be a major risk factor for deteriorating kidney functions. however, there is paucity of reports on renal functions in a co-existence of hypertension and diabetic condition, hence, this study evaluates renal functions in hypertensive and diabetic co-morbidity. participants were categorised into healthy, hypertension, diabetes and hypertension with diabetes group. blood pressure was measured as well as fasting blood glucose and blood samples were collected from each participant to assay renal function indices. the results showed that there was substantial uraemia as well as a significant reduction glomerular filtration rate in co-morbid hypertension and diabetic patients. fasting blood glucose and mean arterial blood pressure were considerably elevated while creatinine concentration was not significantly altered in comorbid hypertension and diabetic patients. this study revealed that coexistence of hypertension and diabetes mellitus exacerbated renal dysfunction comparatively to hypertension and diabetes mellitus. keywords: hypertension; diabetes mellitus; renal functions; co-morbidity; uraemia. introduction as part of the metabolic syndrome, diabetes mellitus (dm) is characterized by hyperglycemia and/or glucose intolerance as a result of inadequate insulin production, improper insulin action, or both (galicia-garcia et al., 2020). an important factor in the pathogenesis of this disease has been reported to be dysregulations in the regulatory systems for storage and mobilization of metabolic fuels including the catabolism and anabolism of carbohydrates, lipids, and proteins resulting from defective insulin secretion, insulin action, or both (gonzalez-gil and elizondo-montemayor 2020). multiple organ failures and increasing metabolic problems such as retinopathy, nephropathy, and/or neuropathy are among the severe symptoms of dm (preguiça et al., 2020). about 171 million individuals worldwide have been diagnosed with dm and its frequency is rising quickly, especially in developed nations (cao et al., 2020). the world health organization (who) predicted that by 2030, the number would have doubled (who, 2006). urbanization and lifestyle changes have been blamed for the rise in diabetes cases in emerging nations, although the processes are still poorly understood (akdis, 2021). it was discovered in a follow-up cross-sectional study of people with diabetes in rivers state (the central part of the niger delta region) that diabetes significantly contributes to the health issue with type 2 diabetics presenting upon diagnosis (maiga et al., 2020). several cardiovascular problems including hypertension, vasculopathy, and congestive heart failure (chf) have been related to diabetes mellitus by a body of research evidence. a chronic medical disorder known as hypertension (htn) is caused by sustained high blood pressure in the arteries (gupta et al., 2021). usually, high blood pressure has no symptoms. however, persistently high blood pressure is a significant risk factor for dementia, atrial fibrillation, peripheral vascular disease, vision loss, chronic renal disease, heart failure and coronary artery disease (panula et al., 2020). systolic and diastolic pressures; the highest and lowest lateral pressures applied to the arterial wall respectively are the two parameters used in clinical blood pressure measurements. most persons' typical resting blood pressure falls within the ranges of 60-80 mmhg diastolic and 100-130 mmhg systolic (balwan and kour, 2021). the majority of persons have high blood pressure if their resting blood pressure is consistently 130/80 or 140/90 mmhg or higher (balwan and kour, 2021). children are subject to different numbers (balwan and kour, 2021). numerous patients have been reported to have both diabetes mellitus and hypertension. nigerians have both diabetes and https://doi.org/10.14421/biomedich.2022.112.145-150 146 biology, medicine, & natural product chemistry 11 (2), 2022: 145-150 hypertension rates of 10 to 15% and 1 to 2% respectively (edeogu et al., 2020). most cases of hypertension in people with type 2 diabetes are caused by essential hypertension (akalu and belsti, 2020). according to studies, patients with diabetes experience hypertension two times more frequently than people without the condition (asiwe et al., 2021a). hypertension is strongly and independently correlated with diabetes incidence (palaiodimos et al., 2020). elevated blood pressure makes diabetic individuals more susceptible to problems such cardiovascular disease, stroke, renal disease, and retinopathy (cardoso et al., 2020). the kidney is a crucial organ for homeostasis and has been linked to both diabetes and hypertension (tinti et al., 2021). chronic kidney disease is frequently detected after an examination of those thought to be at risk for renal problems such as those with high blood pressure and/or diabetes because the symptoms of failing kidney functions are not always clear (asiwe et al 2021b). there are not many researches looking into the connection between diabetes, hypertension, and the onset of renal failure. recent investigations on patients with type 1 and type ii diabetes found a link between dietary salt consumption and diabetic complications (asiwe et al., 2021a). to the best of our knowledge, however, there is a dearth of information in the literature about the kidney's functionality in a condition where diabetes and hypertension are coexisting. as a result, the goal of this current study was to evaluate renal functions in comorbid state of hypertension and diabetes. materials and procedures ethical consideration before starting the study, a formal approval was received from the university of benin teaching hospital's (ubth) ethics and research committee (adm/e22/a/vol.vii/148283) which adhered strictly to the national guideline for research and experimentation (nih publication no. 85-23). criteria for inclusion and exclusion the study excluded participants who are diagnosed within the last three years as well as those who are type i diabetes and also those with other health complications that might add bias to our study study plan a total of 100 willing participants who met the inclusion criteria were recruited for the cross-sectional analytical study from the diabetes and hypertension clinic, outpatient department (opd) of university of benin teaching hospital (ubth), benin, nigeria. they were divided into four groups, each with 25 participants (n=25), consisting of healthy individuals (controls), hypertensive patients (htn), diabetes mellitus patients (dm), and patients with both hypertension and diabetes mellitus (htn/dm). sample collection consented participants were given a set of structured questionnaire in order to get information on their medical history. the blood pressure of the participants was measured using a sphygmomanometer and weight was measure using a weighing scale. the blood samples gotten from the participant were drawn into 5ml edta bottle for fasting blood glucose level check and thereafter centrifuged at 3000 rpm for 5 minutes and the plasma decanted for other assays such as urea and creatinine test. this procedure was carried out early in the morning when the participants had not had their breakfast. measurement of plasma creatinine the alkaline jaffe's picrate assay was used to measure the plasma creatinine level (owen et al., 1954). the jaffe's method for measuring blood creatinine is based on the idea that when creatinine combines with picric acid in an alkaline media, an orange-colored complex with the alkaline picrate is formed. at 490nm, a spectrophotomer measures the complex's absorbance, whose strength is directly proportional to the amount of creatinine present in the sample. plasma urea level determination according to cheesbrough (2005), the plasma urea level was measured using the diacetylmonoxime technique. the diacetylmonoxime method produces a reddish solution whose absorbance is measured at 530nm in a spectrophotomer by reacting urea with diacetylmonoxime at high temperature in an acid medium in the presence of cadmium ions and thiosemicarbazide. calculated creatinine clearance determination using the cockcroft-gault formula, the creatinine clearance, which is nearly identical to the glomerular filtration rate, was calculated (cockcroft and gault, 1976). since plasma creatinine is so strongly influenced by age, sex, and body size, the method takes into account a number of variables that have an impact on the estimation of muscle mass and assumed creatinine generation. creatinine clearance is expressed as follows: creatinine clearance (mol/l) = (140 age) x weight)/ (0.814 x plasma [creatinine]) x (0.85 if patient is female), where creatinine clearance is expressed in milliliters per minute age is expressed in years, weight is expressed in kilograms, and [creatinine] is expressed in micromoles per liter. statistical analysis the results were statistically evaluated using graphpad prism version 8. mean and standard error of the mean obore et al. – co-existent hypertension with diabetes mellitus … 147 were used to express the results. one way analysis of variance (anova) and tukey post hoc test were used to check the data for any significant differences. at p<0.05, mean variation was considered significant. results co-morbidity of hypertension and diabetes mellitus has been reported to be a major risk factor for deteriorating kidney functions. following one-way analysis of variance (anova) and student t-test, we evaluated the renal function indices in patients with hypertension (htn), diabetes mellitus (dm) and co-morbid condition of hypertension and diabetes (htn/dm). there was a significant (p<0.05) elevation of plasma urea [f(3,96)=4.45, p=0.0057] in dm and htn/dm group comparatively to control (fig. 4). also, in htn/dm group, there was a significant (p<0.05) reduction in egfr [f(3,96)=3.08, p=0.0311] comparatively to control (fig. 5). however, plasma concentration of creatinine [f(3,96)=0.537, p=0.6580] was not significantly (p>0.05) altered in any of the groups when compared with control (fig. 3). diabetes mellitus and hypertension was confirmed with a significant (p<0.05) elevation in fasting blood glucose (fbs) [f(3,96)=13.3, p<0.0001] and mean arterial blood pressure (map) [f(3,96)=8.48, p<0.0001] in dm and htn/dm as well as htn, dm and htn/dm respectively when compared to control as shown in figure 1 and figure 2. figure 1. fasting blood glucose. values are represented as mean ±sem, n=25 and *p<0.05 was significant when compared to control while #p<0.05 was significant when compared to hypertension group. htn=hypertension, dm=diabetes mellitus and htn/dm hypertension and diabetes co-morbidity. figure 2. mean arterial blood pressure (map). values are represented as mean ± sem, n=25 and *p<0.05 was significant when compared to control. htn=hypertension, dm=diabetes mellitus and htn/dm hypertension and diabetes co-morbidity. figure 3. plasma creatinine concentration. values are represented as mean ± sem, n=25 and *p>0.05 was not significant when compared to control. htn=hypertension, dm=diabetes mellitus and htn/dm hypertension and diabetes co-morbidity. figure 4. plasma urea concentration. values are represented as mean ± sem, n=25 and *p<0.05 was significant when compared to control. htn=hypertension, dm=diabetes mellitus and htn/dm hypertension and diabetes co-morbidity. 148 biology, medicine, & natural product chemistry 11 (2), 2022: 145-150 figure 5. estimated glomerular filtration rate (egfr). values are represented as mean ± sem, n=25 and *p<0.05 was significant when compared to control. htn=hypertension, dm=diabetes mellitus and htn/dm hypertension and diabetes co-morbidity. discussion there are no specific signs or symptoms of deteriorating kidney functions, however they could include feeling generally unwell and losing appetite. diabetes mellitus has long been known to negatively impact blood pressure, cardiovascular risk and survival. people who are known to be at risk of kidney issues, such as those with high blood pressure and diabetes mellitus, are frequently examined in order to diagnose chronic kidney disease (byrne and targher 2020). this study was conducted to assess renal function in patients with diabetes and hypertension as well as a co-morbid condition of hypertension and diabetes. in this study, patients with diabetes as well as hypertension and diabetes had substantial uraemia. additionally, it was found that diabetic and hypertensive (htn/dm) patient’s egfr dramatically decreased as compared to healthy patients (control group). however, none of the patients' creatinine levels were considerably changed. the possibilities of having renal problem and other complications that involves organ dysfunction increases significantly when diabetes mellitus and hypertension are co-diagnosed. this is especially true if the underlying illnesses are left untreated. deteriorating kidney functions are most frequently caused by diabetes in nigeria (okaka et al., 2020; ibitoba et al., 2022). diabetic and hypertensive patients also have increased prevalence of cardiovascular (cv) risk factors, such as dyslipidemia, microalbuminuria, hyperuricemia, a propensity to clot, and left ventricular hypertrophy (mantovani et al., 2020; sanusi et al., 2020; okonofua et al., 2021). we reported an elevated mean arterial blood pressure in diabetic patients (fig.2) which was consistent with previous reports that vascular consequences of diabetes includes renal disease, coronary heart disease, stroke, peripheral vascular disease, lower extremity amputations as well as retinopathy and these are exacerbated by hypertension (asiwe et al., 2021b). furthermore, our research showed that individuals with both diabetes and hypertension have a 5to 6-fold increased risk of developing renal failure compared to individuals with hypertension alone and no sign of diabetes (fig. 5). although renal failure is a significant consequence for people with diabetes and hypertension, however, cardiovascular disease events account for the majority of deaths (copur et al., 2021). renal failure caused by diabetes and hypertension is becoming more common despite improved knowledge and suggestions for preventive measures put out by many public health organizations. in nigeria, the number of people beginning dialysis has steadily increased over the past 15 years mostly due to diabetes. this could be explained in part by a decrease in cv mortality which has led to a rise in the number of people living long enough to experience deteriorating kidney functions. a second, possibly more significant problem is that patients themselves may not be adhering to prescribed drug regimens as well as caregivers not been adequately able to educate the patients. as a result of these, target blood pressure goals are not met which increases the likelihood of developing renal failure. the general population's rates of controlling hypertension have dropped recently, according to data from the third national health and nutrition examination survey (nhanes iii) (zheutlin et al., 2022). the prevalence of diabetes has continued to climb which is a third factor that contributes to the rising incidence of renal failure (cheng et al., 2020). last but not least, the rise in cv and subsequent renal morbidity in recent years has also been influenced by the rising frequency of heart failure; as a result, even while mortality related to hypertension has declined, morbidity has grown. diabetes often results in hypertension, especially when nephropathy is also present. about 85% of persons with type 2 diabetes mellitus that have nephropathy before needing dialysis or a kidney transplant also have hypertension, which is roughly twice as common in those with type 2 diabetes as in those without it (asiwe et al., 2021). renal failure can be a third limb of a tripod-stand disease condition of hypertension and diabetes because of their common path-biological etiologies. however, to prevent or treat one, the others must be taken into consideration to reduce mortality. conclusion the results of our study reveal that diabetes and hypertension are risk factors for developing renal failure and also hypertension and diabetic co-morbidity exacerbated the deteriorating kidney functions by obore et al. – co-existent hypertension with diabetes mellitus … 149 significantly reducing glomerular filtration rate and increasing plasma urea concentration. however, it is therefore recommended that awareness and enlightenment programs should be encouraged to educate diabetic patients as well as hypertensive patients the need to take their medications serious to avoid renal function complications. acknowledgements: the authors appreciate the technical assistance of mr. obi udogwu, dr peter akpotaire and dr pauline akpe during the data collection and laboratory assay. author contributions: conceptualisation of the study was done by e.a obore and v.i iyawe data collection and laboratory assay was done by e.a obore, a. edo and j.n asiwe. funding was done by e.a obore, v. iyawe and a. edo, writing of the manuscript was done by j.n asiwe and e.a obore. all authors approved the final draft of the manuscript. data availability statement: all data associated with this study are included in this manuscript funding: this study was entirely funded by the authors conflict of interest: the authors declare that they have no conflict of interest. ethical consideration: the approval for this study was granted by the university of benin teaching hospital's (ubth) ethics and research committee with approval number adm/e22/a/vol.vii/148283. consent to participate: participants willingly consented to participate in this study by filling and submitting a written consent form to the authors. consent to publish: all authors approved the submission and publication of this manuscript. references akalu, y, belsti, y, (2020). hypertension and its associated factors among type 2 diabetes mellitus patients at debre tabor general hospital, northwest ethiopia. diabetes, metabolic 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(2006). prevention of blindness from diabetes mellitus: report of a who consultation in geneva, switzerland, 9-11 november 2005. world health organization. zheutlin, a.r., derington, c.g., king, j.b., berchie, r.o., herrick, j.s., dixon, d.l., cohen, j.b., shimbo, d., kronish, i.m., saseen, j.j. and muntner, p., (2022). factors associated with antihypertensive monotherapy among us adults with treated hypertension and uncontrolled blood pressure overall and by race/ethnicity, national health and nutrition examination survey 2013-2018. american heart journal, 248; 150-159. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 655-662 | doi: 10.14421/biomedich.2025.142.655-662 issn 2540-9328 (online) proximate analysis and chemical constituents of psychotria latistipula benth. (rubiaceae) leaves samuel akinniyi odewo1, olayombo margaret banwo2,* 1forest herbarium ibadan (fhi), forestry research institute of nigeria. 2department of industrial chemistry, faculty of natural and applied sciences, abiola ajimobi technical university, ibadan, oyo state, nigeria. corresponding author* olayombo.banwo@tech-u.edu.ng manuscript received: 28 april, 2025. revision accepted: 13 august, 2025. published: 01 october, 2025. abstract psychotria latistipula benth. (rubiaceae), a traditional nigerian medicinal plant recognised for its effectiveness in treating cancer, tumours, bronchial and gastrointestinal disorders was investigated for its chemical constituents by gas chromatography-mass spectrometry (gc-ms) technique; also, proximate analysis was done to determine the composition of moisture, crude protein, crude fiber, fat, carbohydrate, and ash contents, which had not been previously reported. p. latistipula leaves were extracted by maceration using acetone and hexane (1:1). the proximate analysis indicated that the leaves of p. latistipula contain moisture, crude protein, crude fiber, fat, carbohydrate, and ash contents of 8.91±0.14, 12.43±0.05, 20.78±0.66, 2.28±0.06, 50.34±0.79, and 5.27±0.16%, respectively, demonstrating a notably high carbohydrate content. the gc-ms identified twenty-eight compounds, making up 93.08% of the total. the dominant classes of these compounds included fatty acid esters, saturated fatty acids, fatty acid methyl esters, and unsaturated fatty acid aldehydes accounting for 19.27%, 18.71%, 13.77% and 13.36% of the total, respectively. octadecanoic acid (14.03%) was the major compound in p. latistipula leaves acetone-hexane extract. notably, octadecanoic acid is known for its potential anti-inflammatory, anticancer and antioxidant properties, which indicates p. latistipula could serve as an anti-inflammatory, antioxidant and anticancer agents further justifying its ethnomedicinal use. keywords: fatty acid esters; gc-ms; octadecanoic acid; p. latistipula; rubiaceae; secondary metabolites. abbreviations: p. latistipula: psychotria latistipula; gc-ms: gas chromatography-mass spectrometry; rt: retention time; mins: minutes; mf: molecular formula; mm: molecular mass; g/mol: grams per mole. introduction plant extracts and their constituents have demonstrated potential for being developed into new treatments with anticancer, antioxidant, antidiabetic, antimalarial, antimicrobial, and anti-inflammatory properties, among others (riaz et al., 2023). they offer advantages over synthetic medicine regarding safety, local availability, and cost-effectiveness (ansari et al., 2025). the screening of plant extracts represents an innovative approach to discovering medicinally important compounds that can facilitate the development of new pharmaceuticals (chaachouay & zidane, 2024). in the study of natural products, such as plant extracts, gas chromatography-mass spectroscopy is invaluable for characterising the bioactive compounds present; it can be used for the analysis of a wide range of biological compounds including fatty acids, essential oils, eicoscanoids, wax, esters by the selection of suitable columns, thereby informing pharmacological studies and potential therapeutic applications (santhiya & ramasamy, 2019; olaoluwa et al., 2019; ranjan et al., 2023; takuma et al., 2025). the family rubiaceae comprises 620 genera totaling about 13526 species, distributed worldwide. psychotria is the largest of the rubiaceae, possessing more than 2000 species, mainly found in tropical and subtropical regions (mohamad et al., 2025). several psychotria species are widely used in folk medicine around the world to treat various illnesses (sangeetha et al., 2020). psychotria latistipula benth, the type species of the genus psychotria and from the family rubiaceae, is a shrub of 5–6 feet or a small tree with branches subterete, glabrate, at the extremities somewhat herbaceous and compressed, puberulous and its leaves are elliptical or oval, acuminate, wedge-shaped at the base, nearly glabrous or puberulous on the veins beneath. it is native to nigeria, cameroon, central african republic, congo, gabon, gulf of guinea is., and togo (powo, 2025). the leaves treat cancer and tumours (burkill, 1985). also, the genus psychotria, to which p. latistipula belongs, is used in traditional medicines for treating bronchial and https://doi.org/10.14421/biomedich.2025.142.655-662 656 biology, medicine, & natural product chemistry 14 (2), 2025: 655-662 gastrointestinal disorders, such as cough, bronchitis, ulcer, stomach ache, and infections of the reproductive system (ngnokam-jouogo et al., 2022). psychotria (rubiaceae) possesses various biological properties, ranging from phytochemical and pharmacological properties of their chemical constituents to traditional medical applications (aureada et al., 2023). despite their high diversity, most psychotria species, including p. latistipula, remain largely unstudied. hence, to gain insights into its chemical composition, this study conducted a gc-ms analysis to identify the presence of various chemical constituents in the acetone-hexane extract of p. latistipula leaves and also a proximate analysis to determine the moisture, crude protein, crude fiber, fat, carbohydrate and ash contents. materials and methods plant material and extraction leaves of p. latistipula (figure 1) were obtained from the arboretum of the forestry research institute of nigeria (frin), jericho ibadan, oyo state. the plant was identified and authenticated at the forestry herbarium in ibadan (fhi), voucher number fhi 1142147 was issued for reference use. the leaves were then air-dried for about 2 weeks to remove all moisture and subsequently pulverized. air-dried ground leaves of p. latistipula (5 g) was measured and placed into a 250 ml conical flask with a lid. next, 40 ml of a 1:1 mixture of acetone and hexane was added to the sample in the flask, which was then ultrasonicated at 27ºc for 15 minutes. the resulting suspension was filtered, and the filtrate was concentrated in vacuo using a rotary evaporator. for the gc-ms analysis of various compounds, 1 μl of the sample was utilized. figure 1. psychotria latistipula leaves proximate analysis of p. latistipula leaves determination of moisture content the moisture content was determined by the gravimetric method. a measured weight of each sample (2g) was weighed into a weighed moisture can. the can and its sample content were dried in the oven at 105⁰c for 3 hours. it was then cooled in a dessicator and reweighed. the weight was recorded while the sample was returned to the oven for further drying. the drying, cooling and weighing were done repeatedly until a constant was obtained. the moisture content was calculated. determination of crude protein content crude protein was determined from organic nitrogen using the macro-kjeldhal method (adegbaju et al., 2019). determination of crude fibre content for crude fiber, 3 g of each sample was defatted (during fat analysis). the defatted sample was boiled in 200 ml of 1.25% h2s04 solution under reflux for 30 minutes. after that, the sample was washed with several portions of hot boiling water using a two-fold muslin cloth to trap the particle. the washed samples were carefully transferred quantitatively back to the flask and 20 mlof 1.25% naoh solution added to it. again the sample was boiled for 30 minutes and washed as before with hot water. then was carefully transferred to a weighed proclaim crucible and dried in the oven at 105⁰c for 2 hours. fiber content was calculated by dividing loss in weight of the sample by the weight of the sample (aoac international, 2016). determination of fat content the fat content of the sample was determined by the continuous solvent extraction method using a soxhlet apparatus by extracting with petroleum ether. the extract was reduced to half by evaporation and dried at 60o c for 3 minutes.the percent fat content was calculated by dividing the weight obtained after drying with the weight of the sample to estimate the fat content (james et al., 2015). determination of carbohydrate content the carbohydrate content was calculated by difference as the nitrogen free extractive (nfe). the carbohydrate content was calculated from the differences protein, fat, moisture, and ash content subtracted from 100 (okunola et al., 2019). determination of ash content the ash content was determined using the furnace incineration gravimetric method. the sample (1g) sample was put in a precious weighed porcelain crucible. the sample in crucible was put in a muffle furnace at 550o c and allowed to burn for 2 – 3 hours (until the sample became a gray ash). the sample in crucible was very odewo & banwo – proximate analysis and chemical constituents of … 657 carefully removed from the furnace (taking care not to blow air into the ash) and cooled in a dessicator. it was reweighed by difference and the weight of the ash in percentage was obtained by dividing loss in weight of the sample by the weight of the sample (aoac international, 2016) data analysis the proximate analysis was done in triplicates and reported as mean ± standard deviation. gc-ms analysis the gc-ms analysis of the acetone-hexane extract of p. latistipula leaves was carried out using an agilent 7820a gas chromatograph fixed to 5975c inert mass spectrometer (with triple axis detector) with electronimpact source (agilent technologies capillary column (hp-5) coated with 5% phenyl methyl siloxane (30 m length x 0.32 mm diameter x 0.25 μm film thickness) was the stationary phase of separation of the compounds. helium was used as the carrier gas at constant flow of 1.4871 ml/min at an initial nominal pressure of 1.4902 psi and average velocity of 44.22 cm/sec. at an injection temperature of 300 °c, an injection volume of 1μl of the sample was introduced in splitless mode. while the gas saver mode was turned off, the purge flow to spilt vent was 15 ml/min at 0.75 min with a total flow of 16.654 ml/min. oven was initially auto regulated at 40 °c for (1 min) then ramped at 12 °c/min to 300 °c (10 min). the run time was 32.667 min with a 5 min solvent delay. the mass spectrometer was utilized in electron-impact ionization mode at 70ev with ion source temperature of 230 °c, quadrupole temperature of 150 °c and transfer line temperature of 280 °c. acquisition of ion was through scan mode (scanning from m/z 45 to 550 amu at 2.0s/scan rate). the relative percentage amount of each component was calculated by comparing its average peak area to the total areas. national institute standard and technology (nist) 14.l library (2018) was then searched to compare the structures of the compounds with that of the nist database. compounds were then identified based on the retention times and mass spectra with already known compounds in the nist library (c: \ database \ nist14.l). results and discussion the proximate analysis of p. latistipula leaves the proximate analysis revealed p. latistipula leaves to contain moisture, crude protein, crude fiber, fat, carbohydrate, and ash contents of 8.91±0.14, 12.43±0.05, 20.78±0.66, 2.28±0.06, 50.34±0.79, and 5.27±0.16%, respectively, demonstrating a notably high carbohydrate content and an appreciable amount of crude fibre (table 1). table 1. proximate composition of p. corymbose. s/n constituents percentage composition 1 moisture 8.91 ± 0.14% 2 ash 5.27 ± 0.16% 3 fat 2.28 ± 0.06% 4 crude fibre 20.78 ± 0.66% 5 crude protein 12.43 ± 0.05% 6 carbohydrate 50.34 ± 0.79% values are the mean and standard deviation of three determinations. gc-ms profile of p. latistipula leaves acetone-hexane extract the chemical constituents of plants play an essential role in determining their biological attributes, influencing not only their physiological functions but also their interactions with the environment. additionally, the presence and concentration of specific chemical constituents can affect the plant’s medicinal properties, making them valuable in traditional and modern medicine. twenty-eight chemical components were identified from the acetone-hexane (1:1) extract of p. latistipula leaves representing a total of 93.08% (table 2a; figure 1). the acetone-hexane components of p. latistipula leaves were dominated by fatty acid esters, saturated fatty acids, fatty acid methyl esters, and unsaturated fatty acid aldehydes accounting for 19.27%, 18.71%, 13.77% and 13.36% of the total, respectively (table 2b). the major compounds found were octadecanoic acid (14.03%), 9octadecenoic acid methyl ester (e) (12.36%), 9-octadecenal, (z)(9.47%), heptadecanoic acid, 14-methyl-methyl ester (6.97%), pentadecanoic acid, 14-methyl-, methyl ester (9.80%), oleic acid (6.19%), n-hexadecanoic acid (4.68%), neophytadiene (4.32%), heptyl octadecyl ether (3.86) and (z)-methyl heptadec-9-enoate (3.02%) (figure 2). the most abundant compound in p. latistipula leaves acetone-hexane extract is octadecanoic acid, also known as stearic acid, a long-chain saturated fatty acid, has been identified as a key phytochemical compound in various plant extracts exhibiting various biological activities. research indicates that octadecanoic acid and its methyl esters have been shown to possess antioxidant properties, helping to scavenge free radicals and protect against oxidative stress; exhibits antitumor activity in mouse models and selective cytotoxicity against certain cancer cells; exhibits anti-inflammatory effect; and serves as androgenic flavor, hemalytic, 5α reductase inhibitor (duke, 2016; balasundari & boominathan, 2018). 9 octadecenoic acid methyl ester (e)(12.36%) possesses antioxidant, anemiagenic, anti-inflammatory, antihypertensive, anticarcinogenic activities, exist in human red blood cells and serve as endogenous peroxisome proliferator-activated receptor ligand, dermatigenic flavour, increase high-density lipoprotein (hdl) cholesterol and decreases low-density lipoprotein (ldl) cholesterol (singh et al., 2008; akpuaka et al., 2013). 9 658 biology, medicine, & natural product chemistry 14 (2), 2025: 655-662 octadecenal, a monounsaturated fatty aldehyde, exhibits various biological activities, including antimicrobial, anti-inflammatory, and antioxidant properties. 9-octadecenal found in plant extracts demonstrates antibacterial activity against escherichia coli, staphylococcus aureus, and salmonella sp, pseudomonas aeruginosa and klebsiella pneumonia; also can neutralize free radicals and reduce inflammation serving as an antioxidant and anti-inflammatory agents; additionally, can act as a 5α reductase inhibitor and a percutanea stimulant; also possesses mild anti-infective and anti-fungi activity (selvi & basker, 2012; mangroveabayomi et al., 2014; atni et al., 2024). hexadecanoic acid, also known as palmitic acid showed anticancer, anthelmintic, antioxidant, hypocholesterolemic, nematicide and pesticide (sheela & uthayakumari, 2013; elufioye et al., 2019). duke (2016) also reported hexadecanoic acid having antidiarrheal activity. neophytadiene, a diterpene, exhibits diverse biological activities, including anti-inflammatory, antimicrobial, anti-cancer, anxiolytic-like, and anticonvulsant properties (selmy et al., 2023; gonzalez-rivera et al., 2023; endris et al., 2024). oleic acid, a naturally occurring omega-9 fatty acid, exhibits a range of biological activities, including antimicrobial, antioxidant, and potential roles in reducing cholesterol and inflammation, as well as being a key component of cell membranes and potentially influencing brain development (alabi et al., 2018; ramadan et al., 2024). (z)-methyl heptadec-9-enoate was reported to possess antibiotic properties (elufioye et al., 2019). among the minor compounds identified from the acetone-hexane extract of p. latistipula leaves were: z-10-methyl-11-tetradecen-1-ol propionate (1.88%), hexadecen-1-ol, 3,7,11,15-tetramethyl-, acetate, [r[r*,r*-(e)]](1.77%), cis-vaccenic acid (1.61%), arturmerone (1.49%), 1-methoxy-3-(2-hydroxyethyl) nonane (1.46%), 2,6,10-cycloundecatrien-1-one,2,6,9,9tetramethyl-, (e,e,e)(1.30%), eicosane (0.97%). however, minor components, even in small concentrations, can also play a crucial role in the overall efficacy and functionality of the extract; reported bioactivity of these minor compounds based on dr. duke’s phytochemical and ethnobotanical databases created by dr. jim duke of the agricultural research service/usda are listed in table 3. figure 1. gc–ms chromatogram of acetone-hexane extract of psychotria latistipula leaves table 2a. identified compounds from the acetone-hexane extract of psychotria latistipula leaves using gc-ms. s/n rt (mins) compound class of compound mf mm (g/mol) peak area (%) 1 3.665 benzene, 1-ethyl-4-methylaromatic hydrocarbon c9h12 120.1 1.24 2 4.226 2(1h)-benzocyclooctenone, decahydro-4amethyl-, trans-(-) bicyclic hydrocarbon c13h22o 194.3 1.06 3 4.958 undecane acyclic saturated hydrocarbon c9h12 156.3 1.06 4 6.194 heptanoic acid, ethyl ester fatty acid ester c9h18o2 158.2 0.82 5 8.552 decanoic acid, ethyl ester fatty acid ester c12h24o2 200.3 0.62 6 10.761 2-acetylbenzoic acid carboxylic acid c9h8o3 164.1 1.21 odewo & banwo – proximate analysis and chemical constituents of … 659 s/n rt (mins) compound class of compound mf mm (g/mol) peak area (%) 7 11.516 ar-turmerone sesquiterpenoid c15h20o 216.3 1.49 8 11.853 cyclooctaneacetic acid, 2-oxocarboxylic acid c10h16o3 184.2 0.43 9 12.185 2,6,10-cycloundecatrien-1-one,2,6,9,9tetramethyl-, (e,e,e) sesquiterpenoid c15h22o 218.3 1.30 10 12.511 undec-10-ynoic acid, undecyl ester fatty acid ester c22h40o2 336.5 0.57 11 12.952 neophytadiene diterpene c20h38 278.5 4.32 12 13.175 1-methoxy-3-(2-hydroxyethyl)nonane alkoxy alcohol c12h26o2 202.3 1.46 13 13.335 2-hexadecen-1-ol, 3,7,11,15-tetramethyl-, acetate, [r-[r*,r*-(e)]] diterpenoid c22h42o2 338.6 1.77 14 13.736 pentadecanoic acid, 14-methyl-, methyl ester fatty acid methyl ester c17h34o2 270.4 6.80 15 14.056 heptyl octadecyl ether ether c25h52o 368.6 3.86 16 14.177 n-hexadecanoic acid saturated fatty acid c16h32o2 256.4 4.68 17 14.833 oleic acid monounsatutared fatty acid c18h34o2 282.5 6.19 18 15.172 9-octadecenoic acid, methyl ester, (e)fatty acid ester c19h36o2 296.4 12.36 19 15.373 heptadecanoic acid, 14-methyl-, methyl ester fatty acid methyl ester c18h36o2 284.4 6.97 20 15.619 9-octadecenal, (z)unsaturated fatty aldehyde c18h34o 266.5 9.47 21 15.779 octadecanoic acid saturated fatty acid c18h36o2 284.5 14.03 22 16.340 8-hexadecenal, 14-methyl-, (z)unsaturated fatty aldehyde c17h32o 252.4 1.43 23 16.328 (z)-methyl heptadec-9-enoate fatty acid ester c18h34o2 282.4 3.02 24 16.980 z-10-methyl-11-tetradecen-1-ol propionate fatty acid ester c18h34o2 282.5 1.88 25 17.335 eicosane acyclic saturated hydrocarbon c20h42 324.8 0.97 26 18.491 13-octadecenal, (z)unsaturated fatty aldehyde c18h34o 266.5 1.26 27 19.069 cis-vaccenic acid monounsaturated fatty acid c18h34o2 282.5 1.61 28 19.944 e-15-heptadecenal unsaturated fatty aldehyde c17h32o 252.4 1.20 total 93.08% octadecanoic acid 9octadecenoic acid methyl ester oleic acid heptadecanoic acid, 14-methyl methyl ester 9-octadecenal, (z)neophytadiene pentadecanoic acid, 14-methyl-, methyl ester n-hexadecanoic acid heptyl octadecyl ether (z)-methyl heptadec-9-enoate figure 2: some major constituents found in acetone-hexane extract of psychotria latistipula leaves. 660 biology, medicine, & natural product chemistry 14 (2), 2025: 655-662 table 2b. classes of compounds identified from and their compositions. s/n class of compounds peak area % composition 1. sesquiterpenoids 2.79 2. diterpenes 4.32 3 diterpenoids 1.77 5 saturated fatty acids 18.71 6 monounsaturated fatty acids 7.80 7 fatty acid esters 19.27 8 fatty acid methyl ester 13.77 9 aromatic hydrocarbons 1.24 10 bicyclic hydrocarbons 1.06 11 acyclic saturated hydrocarbons 2.03 12 unsaturated fatty aldehyde 13.36 13 ethers 3.86 14 alkoxy alcohols 1.46 14 carboxylic acid derivatives 1.64 table 3. some identified minor compounds in the acetone-hexane extract of psychotria latistipula leaves and their reported biological activity. s/n compounds structure reported biological activity* 1. z-10-methyl-11-tetradecen1-ol propionate 5α reductase inhibitor, antipsychotic. 2. hexadecen-1-ol, 3,7,11,15tetramethyl-, acetate, [r[r*,r*-(e)]] antioxidant, anti-inflammatory, and neuroprotective effects. 3 cis-vaccenic acid antibacterial, anti-inflammatory, and hypolipidemic effects 4 ar-turmerone anti-inflammatory, antioxidant, antiangiogenic, neuroprotective, anti-cancer, anti-plasmodial, antiaging, antidepressant, antiepileptic, anti-dermatophyte, and antiplatelet activities. 5 1-methoxy-3-(2hydroxyethyl) nonane antimicrobial and anti-inflammatory activities. 6 2,6,10-cycloundecatrien-1one,2,6,9,9-tetramethyl-, (e,e,e) anticancer, anti-inflammatory activities 7 eicosane antifungal and potentially neuroprotective and anti-inflammatory properties * source of reference: dr. duke's phytochemical and ethnobotanical databases, 1992-2016 conclusions the leaves of p. latistipula contain a high level of carbohydrate and crude fibre. gas chromatography-mass spectrometry analysis of the acetone-hexane extract from the leaves of p. latistipula has identified a range of bioactive compounds, primarily consisting of fatty acid esters, saturated fatty acids, fatty acid methyl esters, and unsaturated fatty acid aldehydes, which possess notable medicinal properties. these bioactive constituents have been reported to effectively combat bacteria, fungi, inflammation, oxidative stress, and cancer, reinforcing odewo & banwo – proximate analysis and chemical constituents of … 661 the ethno medicinal uses of p. latistipula. isolating individual bioactive compounds holds great promise for advancing drug development, potentially leading to innovative therapies that could enhance treatment options for various diseases. acknowledgements: we acknowledge the department of chemistry, university of lagos, akoka, nigeria, for the gc-ms analysis. authors’ contributions: conceptualization and literature search were done by samuel akinniyi odewo & olayombo margaret banwo. methodology and analysis was carried by samuel akinniyi odewo. interpretation of data were done by samuel akinniyi odewo & olayombo margaret banwo. both authors contributed to the writing of the manuscript. both authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references adegbaju, o. d., otunola, g. a., & afolayan, a. j. 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(2025). gas chromatography-mass spectrometry (gc-ms) analysis of some plants extract. communication in physical sciences, 12(2). doi:https://doi.org/10.4314/awfqtk05. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 295-303 | doi: 10.14421/biomedich.2023.121.295-303 issn 2540-9328 (online) effect of quarry activities on some morphological parameters of two maize varieties (swan 1 and sammaz 52) bridget odiyi, olubukola maku, foluso akinbode ologundudu*, sylvanus efetobor abiya department of biology, school of life sciences, federal university of technology, akure, nigeria. corresponding author* akinbodefoluso@gmail.com manuscript received: 11 february, 2023. revision accepted: 11 march, 2023. published: 28 march, 2023. abstract the effect of quarry activities on some morphological parameters of two maize varieties (swan 1 and sammaz 52) was investigated with the aim of determining the impact of quarry activities on some growth parameters of the maize varieties under study. the seeds were collected from the seed bank department of the ondo state ministry of agriculture, akure, ondo state. they were authenticated at the herbarium unit of the federal university of technology, akure, and the voucher was deposited. soil samples were collected at 50m, 100m, 150m, 200m, and 250m from the quarry site and transferred to the laboratory for analysis. a screen house experiment was set up to house the pots. seeds of swan 1 and sammaz 52 were sown into perforated plastic pots (30 cm diameter and 33 cm depth) filled with 10 kg of quarry soil. the following morphological parameters were determined; shoot height, leaf area, plant dry weight, shoot dry weight, root dry weight, root-shoot ratio, leaf area, and determination of photosynthetic parameters especially chlorophylls a and b. the result revealed that at 50 meters from the quarry site, sammaz 52, one of the maize varieties grown in soil taken from the site, had the highest shoot height (94 cm). which showed that plants growing in higher concentrations of dust pollution respond to nutrient stress by devoting more of their available carbon to shoot growth, resulting in elongated stems, were consistent with the observed higher shoot height in sammaz 52, daily variations in photosynthetic activity and the rate of nitrogen uptake are to blame for these alterations in plant behavior. the efficiency with which plants use the available nutrients determines whether they will survive in an area where there is quarry dust. the observed higher biomass (3.84g) under sammaz 52's management regime can be attributed to the best possible rates of photosynthesis and nutrient assimilation, as well as to the presence of more chlorophyll and larger leaf surfaces. keywords: dust pollution; quarry, maize; morphological. introduction despite its importance as a major food in many parts of the world, corn is inferior to other cereals in nutritional value. its protein is of poor quality, and it is deficient in niacin. diets in which it predominates often result in pellagra (niacin-deficiency disease). corn is high in dietary fiber and rich in antioxidants. corn oil can be converted into margarine by hydrogenation, a process in which the oil is combined with hydrogen at high temperature and pressure in the presence of a catalyst. corn is also used to produce ethanol (ethyl alcohol), a first-generation liquid biofuel (upadhay et al., 2015). the response of the plant to dust accumulation may vary according to different species, as dust deposition fluctuates with plant species due to leaf orientation, leaf surface canopy, phyllotaxy, epidermal and cuticular features, leaf pubescence, height and canopy of roadside plants (wang et. al.,2019; yun et. al., 2017) with the accumulation of dust, the roadside plant may exhibit adaptive response by changing morphological and physiological attributes. air pollution stress leads to stomatal closure, which reduces co2 availability in leaves and inhibits carbon fixation. the net photosynthetic rate is a commonly used indicator of the impact of increased air pollutants on tree growth (cao et al., 2015). plants that are constantly exposed to environmental pollutants absorb, accumulate and integrate these pollutants into their systems. the relationship between traffic density and photosynthetic activity, stomatal conductance, total chlorophyll content, and leaf senescence has been reported (silva et al., 2016). one of the most common impacts of air pollution is the gradual disappearance of chlorophyll and concomitant yellowing of leaves, which may be associated with a consequent decrease in the capacity for photosynthesis (gupta et al., 2015). methods seed collection/authentication seeds of two maize varieties swan 1 and sammaz 52 were utilized in the experiment. the seeds were collected from the seed https://doi.org/10.14421/biomedich.2023.121.295-303 296 biology, medicine, & natural product chemistry 12 (1), 2023: 295-303 bank department of the ondo state ministry of agriculture, akure, ondo state. they were authenticated at the herbarium unit of the federal university of technology, akure, and the voucher was deposited. study site the study was conducted in a screen house behind the department of biology, federal university of technology, akure. soil sample collection soil samples were collected at 50m, 100m, 150m, 200m, and 250m from the quarry site. the soil samples were taken at a plowing depth of 0 10 cm using a calibrated soil auger. the samples from each site were bucked together to obtain a composite sample and the replicate samples were packed in polyethylene bags, labeled appropriately, and transported to the laboratory for analysis. the topsoil taken from the garden served as the control. a completely randomized design (crd) was set up which comprises two maize varieties, six plowing depths, and 3 replicates. experimental setup a screen house experiment was set up to house the pots. this was necessary to protect the plants from rainfall contaminations and to avoid being destroyed by rodents as the plants develop. soil analysis soil analysis was carried out at the department of crop, soil and pest management, school of agriculture and agricultural technology, federal university of technology, akure. the following physicochemical properties of the quarry soil were determined; dissolved oxygen, conductivity, nitrogen, phosphorus, copper, iron, organic matter, and organic carbon. planting procedure seeds of swan 1 and sammaz 52 were sown into perforated plastic pots (30 cm diameter and 33 cm depth) filled with 10 kg of quarry soil. the seedlings were allowed to establish for 14 days before data were taken. weight analysis on the zero-day i.e. the day when feeding with appropriate nutrient solution commenced and at intervals of seven days, weight analysis was carried out on five seedlings harvested at random. the plants were carefully uprooted, blotted dry, weighed fresh, and then placed inside a labeled envelope and kept in a gallenkamp drying oven set at 80oc to dry to constant weight. measurement of growth parameters a meter rule was used to measure the following; leaf length, leaf width, and shoot height from soil level to the terminal end, and the number of leaves per plant were noted. the fresh weight was taken after which the plants were dried at 80oc in a gallenkamp oven until a constant weight was achieved. after cooling, the dry weight was determined. the dried samples were separated 19 into leaves, shoots, and roots and their different dry weights were determined. these were kept for further analysis. growth parameters: the following plant growth parameters were determined from the data obtained from the physical parameters: leaf area (la) = the unit of la is cm2 l and w are leaf length and width respectively while 2.75 is the correction factor for maize respectively. la = l x w x 2.75 (maize) anderson et al (2005) 3.4.5.2. root shoot ratio (rsr) rsr = w3/w2 root shoot ratio defines the method of assimilate partitioning, w2, and w3 are shoot and root dry weights respectively, the unit is g-1 3.5. photosynthetic pigment analysis chlorophyll extraction: 5g each of the leaves of the seedlings were grounded in 20 ml of 80% (%) acetone using a mortar and pestle. the brew was filtered through a whatman’s no 1 filter paper. the pigment quantities in the acetone extract were determined on a ce 373 (visible) linear readout spectrophotometer at a wavelength of 664nm and 647nm, chlorophyll “a” and “b” and the total chlorophyll quantities were determined using the formula. chlorophyll ‘a’ (µm) = 13.19a664 – 2.37a647 chlorophyll ‘b’ (µm) = 22.10a647 – 5.26a664 total chlorophyll (µm) = 7.93a664 + 19.53a67 20 a664 is the absorbance at 664nm a647 is the absorbance at 647nm (coombs et al; 1993). result and discussion particulate emissions from a wide range of industrial operations may hinder plant growth and development. at 50 meters from the quarry site, sammaz 52, one of the maize varieties grown in soil taken from the site, had the highest shoot height (94 cm). given that stem extension and apical dominance were more pronounced in the plants than in swan 1, it can be said that they devoted more of their nutrients to these processes. the ability to measure changes in the general growth habit of soybeans caused by the environment using apical dominance has been found to be valuable (thomas and raper, 2013). the results of bouma et al. (2010) and bonifas et al. (2015), show that plants growing in higher concentrations of dust pollution respond to nutrient stress by devoting more of their available carbon to shoot growth, resulting in elongated stems, were consistent with the observed higher shoot height in sammaz 52. according to reynolds et al. (2016), daily variations in photosynthetic activity and the rate of nitrogen uptake are to blame for these alterations in plant behavior. the efficiency with which plants use the available nutrients determines whether they will survive in an area where there is quarry dust (ralphs et al; 2013). the observed odiyi et al. – effect of quarry activities on some morphological parameters of … 297 higher biomass (3.84g) under sammaz 52's management regime can be attributed to the best possible rates of photosynthesis and nutrient assimilation, as well as to the presence of more chlorophyll and larger leaf surfaces. the findings of peace and grubb (2018) and tischer et al (2010) that higher dry weight was due to ideal leaf expansion rates were supported by an increase in the generation of dry matter under optimal conditions. plants swan 1 and sammaz 52 accumulated less biomass, as nutritional effects on photosynthetic rate per unit leaf area (greenwood, 2016). the low shoot biomass (0.04g) under swan 1 was because more carbon was diverted for greater root growth than in sammaz 52 plants and this carbon may be from the stem tissues (peace and grubb, 2018). when the nutrient supply was high, in the control regime (normal soil), sammaz 52 plants had more stem components than swan 1 plants because the soil had an adequate nutrient supply. the lowering of the shoot biomass under a low nutrient supply may not be unconnected with the reduction in the production of photosynthates as more carbon was diverted to root growth from both stem and leaf tissues (morgan and smith, 2011). plants reduce root growth relative to leaf area as an adaptation to dust pollution (chung et al; 2013). the reduced root and shoot biomasses in swan 1 at 250 m and 200 m from the quarry site, respectively, were explained by the aforementioned. according to thompson et al. (2008), low carbon content caused root growth to be reduced at low nutrient delivery levels. richer soil encourages better shoot development with roots that are less compact and branches out less (oke, 1985). nutrient-stressed plants expanded their roots quickly in search of nutrition. there was an increase in vegetative growth with maximum leaf production in sammaz 52 at the control regime compared to swan 1. lindquist et al (2016) reported that in velvetleaf, a reduction in nitrogen content increased leaf number but decreased leaf area. in phaseolus vulgaris, bridges (2012) found leaf number to increase with increased nitrogen application. the production of more leaves under the control regime may be a mechanism evolved by maize plants to increase the total surface area for photosynthesis due to reduced leaf area (morgan et al, 2018). in swan 1, at 200m from the quarry site, there was an increase in leaf abscission and a reduction in the number of leaves produced due to the fact that the extra carbon needed for greater root growth as a result of low nutrient supply was taken from their non-assimilatory tissue as well as leaf tissue (peace and grubb, 2012). roots are very important not only for absorbing water and nutrient but also for optimizing plant growth (renalto et al; 2017). the ability of a plant to compete for soil nitrogen is dependent upon root morphological characteristics such as root radius, root length, and root surface area (hilbert, 2010). previous research has shown that excessive dust pollution can inhibit root growth in corn (wang et al, 2018). the above is in agreement with the low root biomass accumulation recorded in swan 1 at 250m. plants respond to limiting soil nutrients by increasing the amount of biomass allocated to roots (bonifas et al; 2015). this substantiates the observation recorded in swan 1 plants. plants may modify their root morphology to further aid their capacity to take in nutrients (clemens, 2018). the relationship between nitrogen uptake and root growth was demonstrated in the result observed in the root-shoot biomass ratio. according to bonifas et al (2000), velvetleaf had greater nitrate uptake efficiency than corn, suggesting that velvetleaf may have more roots with a smaller radius and/or greater specific root length which would increase the surface area available for uptake. in the swan 1, there was a reduction in leaf length, leaf width, and consequently leaf area. this can be attributed to the transfer of photosynthates (assimilates) for stem elongation and the production of new leaves. according to cracker, 2013, one of the most important factors affecting leaf development is a nutrient. according to junk et al; 2012, the size of the leaf is determined by nutrient and carbon supply. since swan 1 plants had a lower assimilation rate, they were expected to have lower leaf areas. according to cardwell et al; 2016, nitrogenstressed plants adapt by producing leaves with longer internodes and an increase in leaf surface area at the expense of food allocation to the roots. leaf area may be decreased by nitrogen deficiency depending on the severity (bonifas et al; 2005). nutrient stress reduces crop photosynthesis by reducing leaf area development and leaf photosynthesis rate and by accelerating leaf senescence (pandy et al; 2000). the trend in root shoot ratio showed that the sammaz 52 higher leaf area ratio than the swan 1 plants as a consequence of nutrient addition; more leaves led to a higher surface area for photosynthesis and so a proportionate high dry matter in plant tissues. the lower leaf area ratio observed in the swan 1 plants may be due to reduced carbon allocation to the leaf tissues for leaf development (morgan and smith, 2018). there was more or less a direct correlation between the root shoot ratio (rsr) and shoot dry weight. in the study conducted, the proportionate decrease in the root shoot ratio noticed in swan 1 plants compared to the sammaz 52 plants could be attributed to lower biomass partitioned to the root than to the shoot. hong et al (2000) while working with cucumber plants found dust particles to affect root shoot ratio. according to summerfield et al (2016), the root shoot ratio declines with an increase in nutrient application in cowpea because there is a decline in the symbionts and therefore a reduced carbon allocation to the roots. the relationship between photosynthetic capacity and dust pollution had been documented by evans and seemann, 2009. also, alt et al; 2010, reported a correlation between photosynthetic capacity dust emissions from industries in vernonia herbacea. nitrogen deficiency leads to disruption of the fine structure of chlorophyll and instability of the pigment 298 biology, medicine, & natural product chemistry 12 (1), 2023: 295-303 protein complex (reddy et al; 2017). reddy et al (2017) also reported a positive correlation between leaf nitrogen, net photosynthetic rate, stomata conductance, and ion transport in cotton. hence, this was in agreement with the result obtained under swan 1 (10.54µm) at 250m from the quarry site plants which showed a lower accumulation of chlorophylls a and b. about 75% of leaf nitrogen is allocated to chloroplasts (hak, 2013), most of which is being used for the synthesis of components of photosynthetic apparatus, in particular, “rubisco”, the most representative leaf protein, playing a key role in carbon assimilation (evans and terashima, 2017). the above accounted for the chlorosis, necrosis, abscission, and senescence of older leaves noticed in the latter part of the experiment in swan 1 plants. the chlorosis spreads from older to younger leaves because of the mobility of nitrogen from older to younger leaves. (mengel et al; 2017). the accumulation of chlorophylls “a” and “b” showed consistent patterns. this was in agreement with the findings of ashraf and rehman (2019) that with adequate nutrients, chlorophylls “a” and “b” are increased, hence these substantiated the highest chlorophylls accumulation (“a” and “b”) recorded in sammaz 52 plants compared to swan 1. the lowest values of photosynthetic apparatus in swan 1 plants could be a result of low stomata conductance and protein contents, affecting rubisco activity and electron transport (evans, 2013). the higher accumulation of calcium in sammaz 52 plants at 100m from the quarry site substantiates the earlier findings of ashraf et al (2019) while working on sorghum. a comparison of calcium ion and magnesium ion concentrations in both regimes reveals that the two ions show antagonism in uptake (ashraf et al; 2000). the calcium and magnesium contents in the two varieties followed inconsistent patterns. this confirms the result of the experiment indicating the highest magnesium accumulation in swan 1 plants while calcium accumulation was observed to be relatively low. there is a correlation between nitrogen and magnesium accumulation as magnesium ion plays a vital role in chlorophyll biosynthesis (walker et al; 2011), protein synthesis, and photosynthesis (marschner et al; 2017). analysis of potassium ions showed different patterns of accumulation in sammaz 52 and swan 1. the percentage of potassium content which decreases in the latter part of the distance from the quarry site can be explained in view of the argument that a high amount of potassium is needed to maintain nitrogen metabolism when large amounts of nitrogen are supplied in the external medium (leigh et al, 2014). however, these results were at variance with the observation of ashraf et al, (2000) who found a lower accumulation of potassium in nutrient-stressed plants grown under normal conditions. magnesium limitation resulted in a reduction in shoot growth and photosynthetic capacity in maize (foyer et al; 2010). supraoptimal levels of magnesium have been reported to increase in corn (diao et al; 2018, tsialtas et al; 2018). this can therefore be interpreted to mean greater metabolic activities, utilization and uptake noticeable in both sammaz 52 and swan 1 plants. however, this was at variance with the observation of tischer et al (2000) that an increase in magnesium supply not only delays senescence and stimulates growth but also changes plant morphology in a typical manner, particularly if the magnesium availability is high in the rooting medium during the early growth, shoot elongation is enhanced and root elongation inhibited, a shift which is unfavorable for nutrient acquisition and water uptake in later stages. the efficient translocation of photosynthate from source to sink organs is the key factor driving plant growth and increased crop yield (dakora, 2003; cornu, 2007). photosynthesis was reported to be affected by sink strength as an increased photosynthate supply is required to meet the increasing demand for photoassimilates during early vegetative growth and seed development in zea mays (richards, 2000). strong positive correlations have been found between the photosynthetic capacity of leaves and their magnesium content, most of which is used for the synthesis of components of the photosynthetic apparatus (gastal et al, 2012). conclusion the efficiency with which plants use the available nutrients determines whether they will survive in an area where there is quarry dust. the observed higher biomass (3.84g) under sammaz 52's management regime was attributed to the best possible rates of photosynthesis and nutrient assimilation, as well as to the presence of more chlorophyll and larger leaf surfaces. this can be attributed to the transfer of photosynthates (assimilates) for stem elongation and the production of new leaves. there were correlations between nitrogen and magnesium accumulation as magnesium ion plays a vital role in chlorophyll biosynthesis, protein synthesis, and photosynthesis. there were clear inconsistent patterns in the accumulation of the chlorophylls a and b. abbreviations: not applicable. ethics approval and consent to participate: not applicable. consent for publication: all authors are aware of the publication of this manuscript. odiyi et al. – effect of quarry activities on some morphological parameters of … 299 availability of data and material: the datasets used and/or analyzed during the current study are available from the corresponding author on request. competing interest: the authors declare that they have no competing interest. funding: the research was self-funded. authors’ contributions: prof mrs b. o. odiyi designed the experiment, maku olubukola carried out the laboratory works. dr. f. a. ologundudu carried out the statistical analysis and interpretation of the results. the author(s) read and approved the final manuscript. acknowledgement: the researchers want to appreciate the technical staff of the department of biology, federal university of technology, akure, nigeria. references ahmad, s.d., m. liebman and a.s.davis (2012). integration of soil, crop and weed management in low external input farming systems. weed res.40:27-47. alt, a.p., f.makini and n.kidula (2010). effect of intercropping legume with maize on soil fertility and maize yield. plant and soil.54:109-115. anderson m.k. and h. ambus (2012). reduction of pest attack on sorghum and cowpea by intercropping. entomology experimental application70:179-184. bonifas, k.d., d. t walters,.and j.l. lindquist (2005). nitrogen supply affects root: shoot ratio in corn and velvetleaf. weed science 54, pp.133-137. bonifas, k.d. and j.l. lindquist (2015). predicting biomass partition to root versus shoot in corn and velvetleaf. weed science 53 .670-675. brook, s.f., b.b. singh and d.l. smith (2007). evaluation of yield stability of cowpea under sole and intercrop management in nigeria. euphytica, 61:193-201. boston, r.s., p.v. viitanen and e. vierling (1996). molecular chaperon and protein folding in plants. plant mol.biol.32:191222. bouma, t. j .and k.l. nielsen (2010). sample preparation and scanning protocol for computerized analysis of root length and diameter. plant and soil 218, 185-196. cao, s.d. and mc nelly (2015). the effects of growing beans together with maize on incidence of bean diseases and pests. neth. j. plant pathol.78:12-18. darley m, amaliotis, d. 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and a.p.rehman (2019). use of velvet bean to improve soil fertility and weed control in corn production in northern belize. commun. soil sci. plant anal. 27(9-10). siyuan, s.a.and m. silberbush (2018). plant root morphology and nutrient uptake, roots, nutrient and water influx and plant growth: american society of agronomy, 65-878. upadhay, j.r., w. robbins and j. w.shive (2015). nutrition studies with corn iii. a statistical interpretation of the relation between nutrient ion concentration, carbohydrate and nitrogenous content of the tissue. soil sci.49:211-238. . 300 biology, medicine, & natural product chemistry 12 (1), 2023: 295-303 table s1. shoot height of zea mays at different distances from the quarry site. varieties days distance (m) control 50 100 150 200 250 var 1 28 43.67±1.86a 39.33±1.33a 37.33±1.33a 36.00±5.29a 39.00±4.58a 33.00±5.19a 35 51.33±1.76b 45.33±1.76b 43.33±2.40ab 38.67±4.81a 44.00±3.06ab 37.33±4.67a 42 58.00±1.16c 56.00±1.16c 48.67±2.33ab 42.67±5.18a 49.33±0.67ab 46.00±2.31a 49 61.33±0.67b 58.67±0.67b 50.33±2.03a 45.67±5.67a 50.67±1.59a 49.33±0.67a 56 63.67±0.88b 62.00±1.16b 52.33±1.45a 46.67±5.69a 54.00±1.16a 51.33±0.67a 63 70.67±0.67b 67.67±1.45b 57.67±1.45a 52.67±4.67a 59.33±0.67a 57.00±0.58a 70 77.67±2.19c 72.33±1.45bc 63.67±0.88a 57.33±5.46a 64.67±0.67ab 63.67±0.33a var 2 28 49.67±3.28b 48.00±1.16b 38.33±2.03a 33.33±1.76a 38.67±1.20a 39.33±2.96a 35 57.00±1.53c 56.00±1.16c 46.33±0.88b 37.33v0.67a 44.33±2.33b 45.00±2.52b 42 66.00±1.00c 67.33±1.76d 54.00±1.16b 40.67±0.67a 51.00±2.08b 49.00±2.08b 49 72.67±1.76d 73.67±2.03e 60.00±0.00c 46.67±0.67a 55.33±1.76bc 53.33±1.76b 56 80.00±1.16d 80.00±1.16e 64.00±1.16c 52.33±1.45a 58.00±1.16b 57.00±1.53b 63 86.00±1.16d 86.33±0.88f 69.33±0.67c 58.67±0.67a 63.33±2.40b 61.67±1.67ab 70 92.67±1.76c 94.00±2.08g 76.00±0.58b 65.00±0.00a 68.67±2.40a 68.00±2.31a mean follow by the same alphabet in a row are not significantly different (p>0.05) from one another, using duncan new multiple range test, var 1 = swan 1, var 2 = sammaz 52 shoot height of zea mays at different distances from the quarry site ranged between 33cm and 94cm respectively relative to the control. the highest shoot height (94cm) was observed in sammaz 52 in the 7 th at 50m from the quarry site. the lowest shoot height was however noticeable in swan 1 at 250m. there is a significant difference in the shoot height of swan 1 at 50m from the quarry site between the first and the third week. also, significant differences were also observed in sammaz 52 at 50m from the quarry site and from the beginning till the end of the experimental period. table s2. plant dry weight (pdw) of maize at different distances from the quarry site. varieties distance dry weight (g)/days after planting (dap) 28 35 42 49 56 63 70 swan 1 0 0.06±0.02a 0.36±0.00ab 0.16±0.00a 0.80±0.00b 0.65±0.00a 0.48±0.00abc 0.52±0.07bc 50 0.04±0.01a 0.40±0.04a 0.21±0.04a 1.01±0.11abc 0.62±0.12a 0.40±0.04ab 0.55±0.04c 100 0.05±0.02a 0.48±0.02c 0.21±0.02a 1.19±0.14abcd 0.69±0.42a 0.32±0.07a 0.54±0.06bc 150 0.04±0.01a 0.46±0.05bc 0.28±0.02ab 1.21±0.10abcd 0.70±0.29a 0.31±0.11a 0.31±0.04a 200 0.09±0.03a 0.51±0.02c 0.16±0.04a 1.51±0.08bcd 1.11±0.30a 0.48±0.10abc 0.32±0.07a 250 0.04±0.00a 0.42±0.04abc 0.23±0.04a 2.06±0.43cd 1.40±0.49a 0.71±0.27abc 0.38±0.06ab sammaz 52 0 0.07±0.01a 0.45±0.02abc 0.18±0.04a 3.84±0.23e 3.29±0.54b 0.98±0.31c 0.47±0.07abc 50 0.07±0.01a 0.34±0.04a 0.21±0.03a 3.35±0.23e 2.84±0.61b 0.89±0.34bc 0.46±0.03abc 100 0.12±0.11a 0.46±0.06bc 0.28±0.14ab 2.19±0.98d 1.57±0.72a 0.22±0.05a 0.54±0.03bc 150 0.28±0.07b 0.47±0.02bc 0.29±0.08ab 0.26±0.05a 0.38±0.09a 0.28±0.04a 0.52±0.03bc 200 0.28±0.04b 0.51±0.03c 0.28±0.03ab 0.27±0.03a 0.36±0.15a 0.33±0.07a 0.56±0.04c 250 0.33±0.07b 0.52±0.04c 0.45±0.05b 0.26±0.04a 0.33±0.09a 0.38±0.05ab 0.46±0.04abc mean follow by the same alphabet in row are not significantly different (p>0.05) from one another, using duncan new multiple range test biomass accumulation in the two maize varieties ranged between 0.04g and 3.84g. the highest biomass was recorded in sammaz 52(3.84g) in the 4th week while the least was recorded in swan 1(0.04g) at the beginning of the experimental period. there was no significant difference in the pdw of the varieties relative to the control. odiyi et al. – effect of quarry activities on some morphological parameters of … 301 table s3. root dry weight (rdw) of the varieties at different distances from the quarry site. v arieties d ay s distance (m) control 50 100 150 200 250 v1 28 0.033±0.003c 0.027±0.003bc 0.023±0.003b 0.010±0.000a 0.063±0.003d 0.030±0.000bc 35 0.117±0.072a 0.193±0.013ab 0.270±0.034b 0.273±0.013b 0.307±0.024b 0.227±0.052ab 42 0.217±0.087a 0.467±0.012b 0.480±0.047b 0.483±0.063b 0.500±0.020b 0.500±0.030b 49 0.050±0.012a 0.063±0.012a 0.037±0.003a 0.050±0.027a 0.050±0.012a 0.067±0.003a 56 0.391±0.015a 0.391±0.028a 0.452±0.054a 0.421±0.022a 0.437±0.032a 0.456±0.019a 63 0.113±0.002a 0.111±0.002a 0.134±0.021a 0.119±0.008a 0.116±0.008a 0.123±0.006a 70 0.550±0.072bc 0.580±0.042c 0.560±0.050bc 0.317±0.047a 0.377±0.068ab 0.407±0.056abc v2 28 0.073±0.003d 0.040±0.000c 0.017±0.003a 0.010±0.000a 0.033±0.003bc 0.027±0.003b 35 0.217±0.038a 0.277±0.012a 0.240±0.055a 0.553±0.209b 0.560±0.021b 0.563±0.013b 42 0.510±0.25b 0.289±0.008a 0.277±0.004a 0.351±0.038a 0.305±0.015a 0.312±0.023a 49 0.317±0.020a 0.347±0.026ab 0.447±0.015c 0.380±0.017ab 0.373±0.013ab 0.387±0.022b 56 0.193±0.003a 0.233±0.039a 0.230±0.015a 0.290±0.023a 0.223±0.038a 0.257±0.044a 63 0.112±0.002a 0.144±0.025ab 0.164±0.015b 0.141±0.001ab 0.146±0.008ab 0.132±0.002ab 70 0.540±0.070a 0.500±0.025a 0.550±0.032a 0.523±0.034a 0.593±0.043a 0.490±0.035a mean follow by the same alphabet in a row are not significantly different (p>0.05) from one another, using duncan new multiple range test. var 1 = swan 1, var 2 = sammaz 52 root dry weight of the varieties at different distances from the quarry site ranged from 0.030g at 250m (swan 1) from the quarry site and 0.593g at 200m (sammaz 52) from the quarry site. there were significant differences in rdw at 50m in swan 1 and sammaz 52 respectively between the first and the second week. however, there were no significant differences between the varieties at 100-250m from the quarry site. table s4. shoot dry weight (sdw) of the maize varieties at different distances from the quarry site. v arieties d ay s distance (m) control 50 100 150 200 250 v1 28 0.030±0.006c 0.025±0.000b 0.015±0.001a 0.008±0.000a 0.037±0.001c 0.029±0.001b 35 0.173±0.001a 0.170±0.015a 0.187±0.009a 0.190±0.025a 0.197±0.013a 0.170±0.006a 42 0.064±0.005d 0.051±0.001c 0.038±0.004b 0.016±0.001a 0.099±0.003e 0.059±0.002cd 49 0.079±0.003ab 0.073±0.003a 0.082±0.003ab 0.089±0.006b 0.08±0.003b 0.082±0.004ab 56 0.088±0.003d 0.080±0.002bc 0.069±0.000a 0.074±0.003ab 0.09±0.005d 0.092±0.004d 63 0.080±0.003b 0.073±0.002a 0.082±0.004b 0.092±0.002c 0.115±0.005d 0.067±0.004a 70 0.750±0.067d 0.559±0.034c 0.399±0.009b 0.178±0.018a 0.71±0.014d 0.602±0.018c v2 28 0.066±0.003c 0.049±0.005b 0.035±0.002a 0.028±0.004a 0.046±0.002b 0.035±0.001a 35 0.023±0.005a 0.026±0.003a 0.033±0.003a 0.033±0.004a 0.029±0.002a 0.032±0.002a 42 0.138±0.004e 0.088±0.006d 0.050±0.003b 0.037±0.003a 0.079±0.017d 0.062±0.004c 49 0.061±0.011a 0.081±0.010a 0.108±0.07b 0.078±0.005a 0.076±0.001a 0.068±0.005a 56 0.065±0.009a 0.087±0.005b 0.125±0.002c 0.930±0.003b 0.095±0.003b 0.084±0.003b 63 0.056±0.008a 0.086±0.009bc 0.099±0.004bc 0.079±0.005b 0.103±0.004c 0.095±0.006bc 70 0.095±0.009d 0.783±0.034c 0.512±0.016b 0.258±0.013a 0.733±0.048c 0.588±0.057b mean follow by the same alphabet in the columns are not significantly different (p>0.05) from one another, using duncan new multiple range test. var 1 = swan 1, var 2 = sammaz 52 there were significant differences in the shoot biomass of swan 1 at 50m and at 200m between the first and the 5th week relative to the control. similarly, significant differences were also observed in sammaz 52 between the 28 th and the 42nd day at 50m relative to the control. the highest shoot dry weight (0.71g) was observed in swan 1 at 200m while the least sdw was noticeable at 150m. 302 biology, medicine, & natural product chemistry 12 (1), 2023: 295-303 table s5. mineral nutrient composition of maize varieties at different distances from the quarry site. varieties distance nutrients na k mg ca swan 1 0 0.31±0.05b 0.57±0.03b 0.23±0.05ab 0.23±0.05ab 50 0.33±0.04bc 0.47±0.25b 0.26±0.03abc 0.26±0.03ab 100 0.21±0.02a 0.48±0.02b 0.34±0.04bc 0.33±0.03b 150 0.18±0.01a 0.56±0.04b 0.36±0.04c 0.33±0.04b 200 0.33±0.01bc 0.59±0.06b 0.55±0.03d 0.29±0.02ab 250 0.42±0.05cd 0.50±0.06b 0.17±0.02a 0.32±0.02b sammaz 52 0 0.38±0.01bc 0.27±0.09a 0.28±0.01abc 0.34±0.05b 50 0.36±0.02bc 0.51±0.07b 0.34±0.04bc 0.29±0.03ab 100 0.50±0.03d 0.75±0.04c 0.25±0.04ab 0.33±0.04b 150 0.43±0.04cd 0.50±0.06b 0.29±0.02bc 0.27±0.02ab 200 0.40±0.03bc 0.55±0.03b 0.48±0.01d 0.21±0.02a 250 0.40±0.02bc 0.44±0.03b 0.51±0.06d 0.24±0.04ab table s6. leaf area (cm2) of maize varieties at different distances from the quarry site leaf area (cm2) distance (m) swan 1 sammaz 52 50 32.50±0.07d 33.06±0.03d 100 18.50±0.01b 22.18±0.04b 150 29.00±0.07c 28.37±0.02c 200 17.50±0.04b 19.32±0.01a 250 12.45±0.12a 15. 24±0.07a control 35.50±0.08d 35.24±0.06d the leaf area of both varieties increased gradually for a greater part of the experimental period. similar growth patterns were recorded throughout the experimental period. gradual increases were observed at 200m and at 250m from the quarry site. the highest leaf area was recorded in the control regime followed by 50m while 250m was observed to be the least. results of the anova showed that there were significant differences (p<0.05) in the varieties grown on the soils from the quarry site. chlorophyll ‘’a’’ accumulation of maize varieties at various distances from the quarry site there were gradual decreases in a linear fashion in chlorophyll ‘a’ accumulation for a greater part of the experimental period in the two maize varieties. both maize varieties had approximately equal chlorophyll accumulation at 150m from soil collected from the quarry site. these were followed by a gradual increase between 50 and 100m from the quarry site. the highest chlorophyll ‘a’ accumulation was recorded in control, while 250m was observed to be the lowest (swan 1). results of the anova showed that there were significant differences (p<0.05) at 50 and 250m respectively relative to the control. table s7. chlorophyll ‘’a’’ accumulation of maize varieties at various distances from the quarry site. chlorophyll a (µm) distances (m) swan 1 sammaz 52 50 8.57±0.07a 24.55±0.02d 100 10.27±0.05b 12.50±0.04a 150 10.78±0.01b 10.45±0.03a 200 8.23±0.02a 16.99±0.02c 250 7.52±0.02a 17.72±0.01c control 24.52±0.04c 35.76±0.02d mean follow by the same alphabet in the columns are not significantly different (p>0.05) from one another, using duncan new multiple range test odiyi et al. – effect of quarry activities on some morphological parameters of … 303 table s8. chlorophyll ‘’b’’ accumulation of maize varieties at various distances from the quarry site. chlorophyll b (µm) distance(m) swan 1 sammaz 52 50 11.64±0.02a 25.43±0.01c 100 16.24±0.02b 23.23±0.02c 150 14.58±0.01b 18.76±0.06b 200 12.58±0.01a 15.57±0.05a 250 10.54±0.03a 12.78±0.05a control 18.76±0.04c 38.56±0.08d mean follow by the same alphabet in the columns are not significantly different (p>0.05) from one another, using duncan new multiple range test in the same pattern like chlorophyll ‘a’ contents, the chlorophyll ‘b’ contents in maize followed similar pattern except that initial contents of chlorophyll ‘a’ were lower than those of chlorophyll ‘b’ also, both varieties decreased gradually between 100 and 200m in the soil collected from the quarry site. sammaz 52 had the highest accumulation under the control regime while the least was observed in swan 1 at 250m. results of the anova showed that there was no significant difference (p>0.05) in the two maize varieties. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 199-204 | doi: 10.14421/biomedich.2024.131.199-204 issn 2540-9328 (online) the comparison of long-term effect between intermittent fasting and calorie restriction on neurological parameters of mice neli syahida ni’ma1,*, kusnandar anggadiredja2 1pharmacy department, universitas negeri semarang, indonesia. 2school of pharmacy institut teknologi bandung, indonesia. corresponding author* neli.syahida@mail.unnes.ac.id abstract intermittent fasting (if) and calorie restriction (cr) were purported to have health benefits. this research aimed to determine the longterm effect of if and cr on selected neurological parameters in mice. swiss webster male mice were divided into 3 groups: ad libitum feeding (al), if, and cr. mice in each group received the treatment for 16 weeks. they were then tested for anhedonia, depression, aggressiveness, and social approach. they were also subjected to contextual fear conditioning tests to model ptsd. compared to al, sucrose intake in the if group was lower, while the cr group showed higher intake (p<0.01). this anhedonia characteristic shown in the if group was confirmed not related to depression, as shown by significantly lower immobility time in the forced swimming test compared to al (p<0.05). in the resident-intruder test, attack numbers in the if group were fewer than in the al group (p<0.05). as demonstrated by the results of the three-chamber test, the reduced aggressiveness in if mice was unrelated to a deficit in sociability. in the fear extinction test (ptsd model), mice in the if group showed lower freezing compared to those in al (p<0.001). although both if and cr caused a reduction in total food intake, in the mice model tested, if was shown to have favorable impacts on neurological parameters. keywords: anhedonia; calorie restriction; depression; intermittent fasting; post traumatic stress disorder. introduction humans have practiced fasting for cultural or religious purposes for a long time. recently, fasting was practiced widely for health purposes, and it becomes a lifestyle. there are many ways and designs of cutting out diet intake. however, generally, in a research setting, fasting can be classified into three types, namely calorie restriction (cr), alternate-day fasting (adf) or intermittent fasting (if), and dietary restriction (dr) (trepanowski & bloomer, 2010). calorie restriction (cr) is the reduction of food intake by a certain percentage (measured in kcal) of ad libitum consumption. cr is usually set at a 20-40% reduction from normal intake (cherif et al., 2016). intermittent fasting is a practice that consists of alternating periods between “the feast period”, in which the subject may consume food ad libitum, and “the fast period,” where fasters are prohibited from consuming any food except water. the duration of the feast period may be varied, but it is usually 16 hours or 24 hours (welton et al., n.d.). in many research literatures, fasting or food reduction increased longevity in a diverse group of species, including the bacteria escherichia coli, yeast saccharomyces cerevisiae, nematode caenorhabditis elegans, fruit fly (drosophila melanogaster), and mouse (gonidakis et al., 2010; hwangbo et al., 2020). the possible mechanisms behind the benefit of fasting were proposed in three scenarios: attenuation of oxidative damage, modulation of glycemia and insulinemia; and hormesis, the beneficial action resulting from the response of an organism to a low-intensity stressor (pinches et al., 2022). in the hormesis mechanism, if and cr are considered low-intensity metabolic stressors that induce the release of stress hormones, adrenocorticotropin hormone (acth), and corticosterone (kim et al., 2021). this lowintensity stressor is a kind of exercise to face a higherintensity stressor. the response of mice to higher stressors after being treated with long-term if and cr was studied in this research by modeling post-traumatic stress disorder (ptsd) in mice. abnormality of hpa (hypothalamic-pituitary-adrenal system) may underlie stress-related psychiatric disorders such as melancholic depression (juruena et al., 2018). it was found that the practice of fasting enhanced mood in patients with chronic pain syndrome (michalsen, 2010). ramadan fasting was found to increase plasma levels of serotonin, brain-derived neurotrophic factor (bdnf), nerve growth factor (ngf), and serotonin plasma levels. serotonin is one of neurotransmitter involved in mood regulation (bastani et al., 2017). in another study, manuscript received: 02 february, 2024. revision accepted: 20 may, 2024. published: 10 june, 2024. https://doi.org/10.14421/biomedich.2024.131.199-204 200 biology, medicine, & natural product chemistry 13 (1), 2024: 199-204 underfeeding patients for 16 days caused decreasing dopamine, a neurotransmitter in the brain that plays a role in reward-motivated behavior (göhler et al., 2000). therefore, this study aimed to evaluate the effect of longterm if and cr on mood-related behavior, particularly depression and anhedonia. since anhedonia is a core characteristic of major depressive disorder (mdd), this test should be confirmed with a depression test. this study also assessed the effect of if and cr on aggressiveness and social interaction as one of the behaviors closely related to depression. materials and methods animals twenty-four male swiss webster mice aged 7 weeks old were kept for 2 weeks at a constant room temperature of 25ºc with 12 h light and 12 h dark cycle for acclimatization. during these 2 weeks, a continuous supply of food and water was provided, and food intake was measured daily. the average daily food intake during acclimatization was determined as a 100% calorie provision requirement. diet treatment animals were divided randomly into 3 groups, which were ad libitum feeding (al), intermittent fasting (if), and calorie restriction (cr). each group consisted of 8 mice, housed two per cage. mice in the al group were provided with a continuous supply of food. in contrast, those in the if group were fed every other day (a 24-hour fasting period was followed by a 24-hour feeding period, in which food was available ad libitum). mice in the cr group were fed 65% of the calorie requirement, which was 4.5 g/mice/day. all mice were provided with a continuous supply of drinking water throughout the study. diet treatment was initiated on 9-week-old mice, which is roughly equivalent to 18-year-old humans. the treatment was implemented for 16 weeks. at the end of the treatment, mice were 25 weeks old, equivalent to 40 years old human (dutta & sengupta, 2016). food intake in each cage was measured daily, and the body weight of each mouse was weighed weekly. after the completion of diet treatment was ended, the mice were tested for their behavior. sucrose preference test in each cage, mice were provided with two drinking bottles; bottle a contained drinking water, and bottle b contained 15% sucrose solution. the position of each bottle was shifted every 24 hours to avoid bias in place preference. this treatment was performed for 4 days: the first two days were for acclimatization, and the following two days were for data measurement. acclimatization was aimed to avoid neophobia since the mice were never exposed to sucrose drinking. each bottle was weighed before and after the test. sucrose preference was calculated with the formula: 𝑠𝑢𝑐𝑟𝑜𝑠𝑒 𝑖𝑛𝑡𝑎𝑘𝑒 𝑠𝑢𝑐𝑟𝑜𝑠𝑒 + 𝑤𝑎𝑡𝑒𝑟 𝑖𝑛𝑡𝑎𝑘𝑒 𝑥100% forced swim test a mouse was placed in a tank filled with water for 6 minutes. the duration of immobility time was measured during the last four minutes. the mouse was considered immobile if it was floated and only made the necessary movements to keep its nostrils above the water's surface. resident-intruder test this test was aimed at measuring aggressiveness. the bedding was not changed 2 weeks before the test in order to enhance olfactory stimulus as a territorial cue within the cages. the test was performed by introducing a stranger mouse of the same sex to the cage of a test mouse mice for 5 minutes. any sign of aggressiveness was recorded. in case of an attack, the latency as well as incidence was calculated. three-chamber test the three-chamber test apparatus consists of three interconnected compartments. one side chamber was assigned as a stimulus chamber (chamber b) in which the mouse could be exposed to an olfactory stimulus through a hole. an empty cage was placed in the other side chamber as the control chamber (chamber c). a middle chamber (chamber b) was sandwiched between the side chambers. one day before the test, a mouse was allowed to explore the apparatus for 10 minutes. mouse was observed if there was any sign of chamber preference. on the testing day, before the test, the mouse was habituated for 5 minutes by placing it in the middle chamber. during the test session, a stimulus mouse was placed into a cage in chamber a, an empty cage was placed in chamber c, and the test mouse was replaced in chamber b. the percentage of time spent in each chamber during the habituation session and test session was calculated. contextual fear conditioning and fear extinction fear conditioning is performed by pairing the conditioned stimulus (cs), the environment of the fear conditioning box; with the unconditioned stimulus (us), electrical footshock. the mouse will learn to associate cs with the us. this method is commonly used for ptsd modeling in animals. on the first day, a mouse was habituated in a fear conditioning box for 3 minutes. during this phase, the duration of freezing was measured and recorded as the freezing baseline. in the following 6 minutes, electric foot shocks (1.2 ma, duration 1 sec) were delivered repeatedly in 19 sec intervals. the behavior of the mouse was recorded using a digital video camera mounted on ni’ma & anggadiredja – the comparison of long-term effect between … 201 the ceiling of the outer box. at 24 h later, the mouse was re-exposed to the fear conditioning box for 6 minutes without exposure to electric foot shock. this phase was called extinction training. at 48 hours following extinction training, the mouse was re-exposed to the fear conditioning box without exposure to the foot shock and tested for its memory retention to extinction training. this phase was called the extinction test. the duration of freezing in each stage (habituation phase, fear conditioning phase, fear extinction training phase, and fear extinction test phase) was measured and compared between the groups. the freezing response was defined as complete immobilization of the mouse, except that it was needed for respiration (shoji et al., 2014). data analysis data analysis was performed using one-way anova followed by the post hoc lsd for comparing parametric data between-group comparisons. the percentage of attack in the aggressiveness test was analyzed using the fisher exact test. a value of p < 0.05 was considered significant. data in the text was presented as means ± sd. results and discussion food intake the average basal food intake was 7.04 g/mice/day. food intake in the cr group was set from this basal value. therefore, mice in the cr group were fed 4.5 g/mice/day. throughout this study, the average food intake in the al group tended to decrease over time, from 8.05 g/mouse/day at the beginning of treatment to 5.26 g/mouse/day. at the beginning of treatment, the average food intake in the if group was 6.4 g/mouse/day, lower than al. after adapting to a new diet, starting from week four until week sixteen, mice in the if group always consumed more food than the al group, to as much as twice the quantity. this is similar to the observation reported earlier (anson et al., 2003). however, since the if group received the feed every other day, the average daily intake in the if group was lower than in the al group (figure 1a). throughout observation, the average body weight of mice in all groups tend to increase, as shown in figure 1b. although total intake in the if group was 32% lower than that of the al group, no significant difference in body weight was observed between the two groups. this might be explained by metabolic shifting to the ketogenic pathway, resulting in ketone bodies (carteri et al., 2021). the average body weight of mice in the cr group, however, was significantly lower compared to al and if (p-value <0.05). at the end of the treatment, the average body weight in the al, if, and cr groups were 51.66 ± 3.82 g, 51.34 ± 2.61 g, and 47.14 ± 4.24 g, respectively. figure 1. profiles of average food intake (a) and body weight (b) during diet treatment for 16 weeks. al: ad libitum feeding, if: intermittent fasting, cr: calorie restriction. sucrose preference test and forced swim test anhedonia refers to the reduced ability to experience a pleasure. in this study, this characteristic was tested using a sucrose preference test. mice in the if group showed no preference between water and sucrose, unlike the other group, which prefers sucrose. compared to those in the al group, mice in the if group showed a significantly lower preference for sucrose (p < 0.01), indicating a symptom of anhedonia (table 1). however, the average sucrose consumption in the cr group was higher compared to that in the al group, indicating hedonic behavior (p < 0.01). even though mice in cr and if groups showed decreasing food intake, they showed the opposite results in terms of sucrose preference. in this study, chronic intermittent fasting, but not diet restriction, induced anhedonia characteristics in mice tested with a sucrose preference test. a decrease in dopamine, a neurotransmitter responsible for rewardseeking behavior might explain this behavior. a study conducted by elesawy et al. (2021) showed that if caused a decrease in dopamine while increasing serotonin. table 1. sucrose preference test and forced swim test result in mice after 16 weeks diet treatment. group sucrose intake (%) immobility time (s) al 67.98 ± 4.7 194.89 ± 19.12 if 50.29 ± 4.06** 153.43* ± 40.15 cr 87.86 ± 10.08** 157.725* ± 35.94 note: data presented in mean ± sd. *) p < 0.05 compared to the al group. **) p < 0.01 compared to the al group 202 biology, medicine, & natural product chemistry 13 (1), 2024: 199-204 anhedonia is a core feature of major depressive episodes, according to dsm-5. it is considered a key diagnostic criterion for the depressive subtype melancholia (dsm-5tm, 2013). to confirm whether anhedonia shown in the if group was related to depression or not, mice were tested using a forced swim test as a representation of “learned helplessness”. the demonstration of decreased immobility of mice in both groups compared to the al group led to a suggestion that anhedonia in the if group was unrelated to depression (table 1). both long-term if and cr in mice had an “antidepressant-like effect” property. depression is often associated with abnormality of serotonergic signaling pathways, mainly due to low activity (cowen & browning, 2015). it is supported by the evidence that selective serotonin reuptake inhibitors (ssris) improve depression symptoms. several studies found that food deprivation increased serotonin release and turnover (bastani et al., 2017; elesawy et al., 2021; mohamed shawky et al., 2015). acute starving can cause increasing serotonin turnover and 5-hydroxyindoleacetic acid levels as a metabolite of 5-ht. meanwhile, the long-term starving (14 days) downregulates the density of the 5-ht transporter in the frontal cortex (huether et al., 1997). both these mechanisms ultimately cause an increase in serotonin signaling output. enhancement of serotonergic signaling is also caused by an increase in brain-derived neurotrophic factor (bdnf), which was induced by long-term intermittent fasting (elesawy et al., 2021). the difference between the long-term effect of if and cr mechanisms in improving mood may be related to serotonergic and dopaminergic signaling. while both if and cr may similarly affect serotonergic signaling, they affect dopaminergic signaling oppositely. resident-intruder test and three-chamber test the number of mice which attacked the intruder was the fewest in the if group. of eight mice in each group, the number of mice that exhibited attack to the intruder was seven in the al group, three in the if group, and six in the cr group. however, when analyzed using the fisher exact test, this percentage was not significantly different (p > 0.05) since the sample size was too small to detect the difference (table 2). table 2. aggressiveness of mice tested with resident-intruder test. group parameter of aggressiveness % attack attack count latency of attack (s) al 87.5 8.57 ± 2.51 53.14 ± 67.94 if 37.5 4.33 ± 1.53* 57.00 ± 49.11 cr 62.5 10.00 ± 3.39 126 ± 80.10 *) p < 0.05 compared to the al group the average latency of the attack was not significantly different between groups. meanwhile, the attack count of the if group was significantly lower than the al group (p<0.05). the lack of aggressiveness showed by mice in the if group might indicate a deficit of sociability. to confirm this possibility, mice were checked on social interaction using a three-chamber test. during the habituation session, mice did not exhibit a chamber preference. during the test session, mice spent more time in the stimulus chamber. compared to the al group, mice in the if group spent more time in the stimulus chamber (p < 0.05), demonstrating no deficit in sociability (table 3). several test mice in the if group showed pro-social behavior, such as the effort to overthrow the cage to free the stimulus mouse. table 3. the percentage of time spent in each chamber during threechamber test. group time spent (%) stimulus chamber middle chamber non-stimulus chamber habituation session al 35.24 30.47 34.29 if 32.46 30.37 37.17 cr 33.79 32.1 34.11 test session al 38.79 28.04 33.17 if 54.68* 19.15 26.17 cr 36.82 28.13 35.05 *) p < 0.05 compared to the al group besides affecting mood, serotonergic signaling also has a role in mediating aggressiveness. the 5-ht system dampened aggression in mice and humans. a high level of aggressiveness may be related to a low concentration of 5-ht. it can be reduced by the administration of pharmacological agents that strengthen serotonergic, such as 5-ht precursor, 5-ht reuptake inhibitor, and 5ht receptor agonist (randy j. nelson & silvana chiavegatto, 2001). reduced aggressiveness in if mice might be related to the enhancement of serotonergic signaling, which also causes an “antidepressant-like effect” characteristic. contextual fear conditioning and fear extinction the results of fear conditioning and extinction tests are presented in figure 2. during the habituation session, mice in all groups actively explored the fear conditioning box, a normal behavior for a mouse exposed to a new environment. the baseline of freezing was not significantly different between groups. then, mice were exposed to a fear conditioning session, in which electrical shocks were delivered. during this session, all groups exhibited similar freezing duration, so the differences in diet treatment did not seem to affect this response. at 24 hours after the conditioning session, mice were trained for extinction. initially, mice exhibited freezing remarkably since the association between box and electrical foot shock was formed previously. over time, however, they started to explore the box since their anticipation of electrical foot shock was countered. therefore, in this stage, the duration of freezing time was ni’ma & anggadiredja – the comparison of long-term effect between … 203 reduced compared to the previous session. the duration of freezing time was not significantly different between the groups in this session. at 48 hours after extinction training, mice were tested for the memory retention of extinction training. in this stage, the duration of freezing in all mice was shorter compared to that during extinction training. in mice treated with intermittent fasting, the duration of freezing was 30.72 ± 25.42 s, significantly different (p < 0.001) from that of the al, 116.77 ± 53.27 s. this result suggests that long-term intermittent fasting facilitates extinction learning in ptsd-modeled mice. figure 2. frequency of freezing in fear conditioning and fear extinction. (darkest to lightest brown: al, if, cr, respectively) fear conditioning was widely used for post traumatic stress disorder modeling. in this method, the previously neutral conditioned stimulus (cs) was paired with an unpleased unconditioned stimulus (us). in this study, cs was a closed box where an unconditioned stimulus, which was an electrical foot shock, was delivered. it was expected that anytime the mouse was put in the same box, it would demonstrate a species-specific defensive response (ssdr), freezing (maren, 2008). this process is called conditioning. this response to fear is analog to humans having ptsd. after the association of cs to the us was formed, the mouse was exposed to the same box but without exposure to followed electrical foot shock. during this extinction training, the mouse is learning new associations that the box is not dangerous anymore. several days later, the test animal is tested for retention of memory during extinction. in this study, it was found that mice treated with longterm intermittent fasting were more resistant to ptsd compared to the al group when exposed to stressful events. instead of intervening in the fear conditioning process, if might facilitate the fear extinction process. in response to stress, the hpa (hypothalamic-pituitaryadrenal) axis will be activated and induce glucocorticoid release from the adrenal cortex. in proper doses, it is important to prepare the body to fight the threat. however, in high concentrations, it can endanger neurons in the hippocampus and cerebral cortex such that they become more vulnerable to excitotoxic, metabolic, and oxidative injury (dekkers et al., 2022). two types of anson, r. m., guo, z., de cabo, r., iyun, t., rios, m., hagepanos, a., ingram, d. k., lane, m. a., & mattson, m. p. (2003). intermittent fasting dissociates beneficial effects of dietary restriction on glucose metabolism and neuronal resistance to injury from calorie intake (vol. 100, issue 10). www.pnas.orgcgidoi10.1073pnas.1035720100 bastani, a., rajabi, s., & kianimarkani, f. (2017). the effects of fasting during ramadan on the concentration of serotonin, dopamine, brainderived neurotrophic factor and nerve growth receptors of glucocorticoids that have been elucidated are glucocorticoid receptors (gr) and mineralocorticoid receptors (mr). gr may mediate the excitotoxic mechanism of glucocorticoid, while mr has a protective effect, such as maintaining the expression of the antiapoptotic gene bcl-2 in pyramidal cell populations. intermittent fasting has been reported to increase corticosterone (belonging to glucocorticoid) concentration in rats, decrease gr level, and not affect mr level (sabatino et al., 1991). thus, the probability of glucocorticoid binding to mr will be increased compared to gr. it may explain the mechanism behind the neuroprotective effect of if, including resistance to ptsd. other studies showed that if increases corticosterone in mice but prevents corticosterone surge when mice are exposed to distress, thus facilitating fear extinction (shojaie et al., 2017). fear extinction may also be facilitated by bdnf, which increases due to intermittent fasting (elesawy et al., 2021). a study conducted by peters et al. (2010) showed that the administration of bdnf directly into the prefrontal cortex (pfc) can facilitate extinction in mice. conclusions even though both intermittent fasting and calorie restriction caused a reduction in total food intake, their beneficial effects on tested behaviors were different. long-term if resulted in reduced sensitivity of the brain reward system, which was not related to depression. in addition, long-term if had an “antidepressant-like effect”. the level of aggressiveness in mice treated with intermittent fasting was lower than that in the al group, and it was shown that a deficit in sociability did not cause it. intermittent fasting is regarded as a chronic mild stressor such that mice were more resistant to ptsd. long-term calorie restriction resulted in increasing sensitivity of the brain reward system but lowered depression. competing interests: the authors declare that there are no competing interests. references 204 biology, medicine, & natural product chemistry 13 (1), 2024: 199-204 factor. neurology international, 9(2), 29–33. https://doi.org/10.4081/ni.2017.7043 carteri, r. b., menegassi, l. n., feldmann, m., kopczynski, a., rodolphi, m. s., strogulski, n. r., almeida, a. s., marques, d. m., porciúncula, l. o., & portela, l. v. 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(n.d.). intermittent fasting and weight loss systematic review (vol. 66). biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 7-33 | doi: 10.14421/biomedich.2024.131.7-33 issn 2540-9328 (online) the neuroprotective and therapeutic effects of medicinal plants and natural products against aluminium chloride-induced alzheimer’s disease: recent update ayodeji oluwatobi ojetunde department of human physiology, faculty of basic medical sciences, ahmadu bello university, zaria, kaduna state, nigeria. corresponding author aoojetunde@gmail.com manuscript received: 12 september, 2023. revision accepted: 13 march, 2024. published: 02 may, 2024. abstract alzheimer's disease currently affects more than 35 million individuals worldwide. aluminium has been implicated in the pathogenesis of various cognitive disorders. meanwhile, aluminium chloride (alcl3) has a significant impact on the progression of neurodegenerative diseases including alzheimer's disease. the majority of alzheimer's disease medications now on the market are cholinesterase inhibitors. however, the effectiveness of these drugs is limited because they can't totally arrest the progression of the disease. the utilization of medicinal plants and natural products may present excellent prospective options for alzheimer's disease prevention and therapy. this study summarized medicinal plants and natural products for the prevention and treatment of alcl3-induced alzheimer’s disease as an alternative therapy using published data in the literature from the years 2021-2023. the medicinal plants and natural products help to reduce alzheimer’s disease pathogenesis by controlling different pathways and could be used as a therapeutic agent against the symptoms. the majority of the medicinal plants and natural products discussed in this review have been shown to have neuroprotective, antioxidant, anti-amyloid, anti-inflammatory, anticholinesterase, anti-apoptotic, and therapeutic actions. therefore, medicinal plants and natural products may offer neuroprotective and therapeutic effects in the treatment of alzheimer’s disease. keywords: aluminium chloride; alzheimer’s disease; complementary and alternative medicine; medicinal plants; natural products. abbreviations: ach: acetylcholine; ache: acetylcholinesterase; ad: alzheimer’s disease; al: aluminium; alcl3: aluminium chloride; app: amyloid precursor protein; aβ: amyloid-beta; b.wt.: body weight; bace1: β-amyloid converting enzyme 1; bbb: blood–brain barrier; bche: butyrylcholinesterase; bdnf: brain-derived neurotrophic factor; cat: catalase; cox-2: cyclooxygenase2; erk1/2: extracellular regulated kinase; gpx: glutathione peroxidase; gsh: glutathione; i.p.: intraperitoneally; il-1α: interleukin-1 alpha; il-1β: interleukin-1 beta; il-6: interleukin-6; mda: malondialdehyde; nds: neurodegenerative diseases; nft: neurofibrillary tangles; nf-κb: nuclear factor-kappa b; nmda: n-methyl d-aspartate; no: nitric oxide; nrf2: nuclear factor erythroid 2-related factor 2; p.o.: oral administration; pon-1: paraoxonase 1; ros: reactive oxygen species; s.c.: subcutaneous; sod: superoxide dismutase; tac: total antioxidant capacity; tbars: thiobarbituric acid-reactive substances; tnf-α: tumor necrosis factor-alpha; tnf-β: tumor necrosis factor-beta introduction the most prevalent form of dementia, alzheimer's disease (ad), currently affects more than 35 million individuals worldwide. this number is estimated to rise to 65 million by 2030 and 115 million by 2050 (ricci 2019). as the fifth most common cause of death in the elderly population, ad may make patients more susceptible to accidents via dementia and cognitive deterioration (yang et al. 2020). as the percentage of the population that is 65 years of age or older increases steeply, it is anticipated that the number of people with ad will significantly increase in the coming years (shunan et al. 2021). although the pathophysiology of ad is unknown, neuritic plaques, neurofibrillary tangles (nft), and the loss of cholinergic neurons in the nucleus basalis of meynert are the main features of ad neuropathology. its pathogenesis has been attributed to a variety of etiological factors. some risk genes may promote the deposition of amyloid beta (aβ) plaques and aberrant tau protein phosphorylation, which results in the formation of common nfts (kunkle et al. 2019). additionally, oxidative stress, inflammation, hormonal deficiency (estrogen), and aging altogether have a corroborative role (kong et al. 2019; selkoe 2019; simunkova et al. 2019). a common neurotoxin like aluminium (al) has been implicated in the pathogenesis of various cognitive disorders (lukiw et al. 2019). chronic exposure to al has also been implicated in the appearance of neurologic signs like progressive neurodegeneration, changes in the https://doi.org/10.14421/biomedich.2024.131.7-33 mailto:aoojetunde@gmail.com 8 biology, medicine, & natural product chemistry 13 (1), 2024: 7-33 neuro-filament of the hippocampus and cerebral cortex, and other biochemical changes of clinical importance. aluminium chloride (alcl3) has a significant impact on the progression of neurodegenerative diseases including ad and parkinson's disease (efosa et al. 2023). the majority of the metals found in the crust of the earth are aluminium. everything we eat, drink, breathe, and consume contains it, including water, food, beverages, flavoured drinks, energy drinks, medicine, dust, and air (abd el-aziz et al. 2023). the ability of aluminum to cross the blood-brain barrier (bbb) and accumulate in the brain results from its incredibly high affinity to transferrin receptors (nie 2018). both in vivo and in vitro, this metal causes neuronal death. aluminum may actively be involved in the genesis of major neuropathologic lesions in alzheimer's disease and other illnesses by cross-linking hyperphosphorylated proteins (abd el-aziz et al. 2023). the majority of ad medications now on the market are cholinesterase inhibitors. however, the effectiveness of these drugs is limited because they can not totally arrest the disease's progression (sharma 2019). this might be caused by the complex nature of the disease etiology. also, up to date, all available treatments exhibit adverse side effects which could aggravate to stroke and death (liu et al. 2018). as a result, a drug that has both anticholinesterase properties and many protective activities may be a viable treatment for ad. numerous plants have been found to be therapeutically effective in cases of memory loss, ad, and disorders associated with ageing. phenolic-rich medicinal plants have recently attracted a lot of attention as potential treatments for neurodegenerative diseases (nds) like ad due to their powerful anti-inflammatory and antioxidant properties (freyssin et al. 2020). in recent years, natural products derived from plants and their bioactive components have been extensively studied for their therapeutic potential in a variety of neurodegenerative diseases (nds), including ad. although there have been remarkable advances in our understanding of nds, there has not been much success in developing effective treatments. the utilization of natural products may present excellent prospective options for nds prevention and therapy (rahman et al. 2021). a study (pandey et al. 2021) summarized frequently used medicinal plants and herbs and their phytochemical components for the treatment and diagnosis of alzheimer's disease as an alternative therapy. however, this study presents an updated summary of medicinal plants and natural products with potential therapeutic and preventive properties against aluminium chlorideinduced alzheimer’s disease. moreover, combining pharmacological therapy with herbal and natural remedies that support various processes and goals may be an effective way to treat and control ad. the studies mentioned in this study demonstrated that natural products may slow the onset of disease. it would be very advantageous to use the knowledge gained from this study to help develop ethnomedicinal drugs that work with ad therapies. methodology this paper used information from existing peer-reviewed journals to conduct a literature review. the following databases were used in the literature search: pubmed, google scholar, sciencedirect, embase, scopus, and web of science. articles that used animal models, evaluated medicinal/herbal plants and/or natural products from plants used against aluminium chloride (alcl3)induced alzheimer's disease, and were published in english between 2021 and 2023 met the inclusion criteria. the exclusion criteria were review articles, clinical trials, conference papers, research conducted in vitro, studies older than 2021, and studies that administered or co-administered other toxicants (such as scopolamine, d-galactose, iron, or streptozotocin) to induce alzheimer's disease. mechanism of aluminium chlorideinduced alzheimer’s disease aluminum (al) promotes the genesis of several neurodegenerative diseases by affecting a number of neurotoxic biomolecules (abbas et al. 2022; mehrbeheshti et al. 2022). al is known to accelerate oxidative stress, the formation of plaques, and the crosslinking & deposition of aβ oligomers in the brain cortex and hippocampus. thus, the neuroprotective effects of numerous phytochemicals and chemical compounds against ad can therefore be studied using alcl3-induced ad in rats as a suitable model (elbini-dhouib et al. 2021; aalikhani et al. 2022; chen et al. 2022). al exposure is essentially inevitable due to its prevalence in the environment, everyday activities, and food (mesole et al. 2020; skalny et al. 2021). the hallmark of ad is the accumulation of aβ plaques and nfts in the brain. aβ production is initiated by the cleavage of amyloid precursor protein (app) by βsecretase, which is commonly known as beta-site amyloid precursor protein cleaving enzyme 1 (bace1), and ɣ-secretase enzymes (islam et al. 2022). aβ can easily diffuse across the brain parenchyma and trigger a cascade of pathogenic processes such as neuronal apoptosis/necrosis, development of oxidative stress, and neuroinflammation in the cortex and hippocampus (morroni et al. 2018). al-maltolate exposure was demonstrated to increase aβ1–42 expression via upregulating app, β-(bace1), & γ-secretase (presenilin-1) mrna transcription and protein expression in rat brain regions (liang et al. 2013; thenmozhi et al. 2015). these changes also correspond to a significant decrease of αsecretase proteins (wang et al. 2014). ojetunde – herbal medicines against alzheimer’s disease 9 evidence suggests that, in addition to amyloid proteins and tau, these protein aggregates may stimulate the immune system for a long period of time and cause the release of chemokines, proinflammatory cytokines, and neurotoxins like reactive oxygen species (ros), nitric oxide (no), and excitatory amino acids, which can further damage and degenerate neurons (calsolaro and edison 2016; zhang et al. 2017). however, a rising body of evidence points to free radical damage to the brain's lipid, protein, carbohydrate, and dna as the cause of neuronal death (padurariu et al. 2013). alzheimer's disease brains exhibit excessive ros generation and impaired antioxidant to oxidative stress (zhao and zhao 2013). alzheimer's disease has also been linked to mitochondrial dysfunction, increased permeability, excessive ros production, and impaired mitochondrial membrane capacity. superoxide anion, hydroxyl radical, hydrogen peroxide, and nitric oxide have all been implicated in oxidative stress-mediated neurodegeneration in alzheimer's disease (pandey et al. 2021). numerous deleterious effects of al, including neurotoxicity, are caused by oxidative stress and mitochondrial dysfunction (kumar and gill 2014). a significant decrease in the activity of the antioxidant enzymes superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione reductase, and glutathione-s-transferase (gst) was linked to the observed increase in brain lipid peroxidation under al exposure. al exposure was also demonstrated to lower mn-sod activity in the mitochondria, which contributed to the development of mitochondrial dysfunction (skalny et al. 2021). the interference of al with neurotransmitter metabolism and signal transduction is at least largely responsible for the negative neurological effects of exposure to al. furthermore, the available results unambiguously show that al exposure significantly affects glutamatergic, cholinergic, gabaergic, dopaminergic, and serotoninergic neurotransmission even though the exact mechanisms are yet unknown (skalny et al. 2021). according to several studies, al increased the activity of the enzyme acetylcholinesterase (ache) in the brain (khan et al. 2013). aluminum is a powerful cholinotoxin which enhanced ache activities by modifying the secondary structure of the enzyme, which significantly impede the cholinergic transmission in the brain (kakkar and kaur 2011). as its dysfunctions are mostly associated with the severity of dementia in ad patients, cholinergic transmission has a high priority in the pathogenesis of ad (thakur et al. 2019). al administration also led to an elevated level of glutamate alpha-decarboxylase activity as well as a significant increase in glutamate levels in the hippocampus, thalamus, and cerebellum (skalny et al. 2021). aluminum exposure causes glutamate increase, which causes excitotoxic damage, degeneration, and death in neurons. glutamate impairs learning and memory by overstimulating of n-methyl d-aspartate (nmda) receptors and causing cognitive decline and neuronal death (alghamdi et al. 2018). generally, alcl3 neurotoxicity has been shown to result in mitochondrial dysfunction, oxidative and endoplasmic reticulum stress, inflammation, cell death, interaction with aβ and αsynuclein, cytoskeletal abnormalities, and alteration of synaptic plasticity and signal transduction through interference with neurotransmitter systems (skalny et al. 2021). figure 1. mechanism of aluminium chloride-induced alzheimer’s disease (dabhekar et al. 2022). role of medicinal plants against aluminium chloride-induced alzheimer’s disease acacia catechu acacia catechu, commonly referred to as "khair" in india, has been suggested as a viable treatment for ad due to its powerful anticholinesterase and antioxidant properties. a. catechu methanolic extract significantly enhanced cholinergic neurotransmission in rats, decreased the genotoxic effects of alcl3, and significantly improved histopathological and biochemical findings (elmorsy et al. 2021). hibiscus sabdariffa in phytomedicine, the use of fresh calyx of the therapeutic herb hibiscus sabdariffa is gaining popularity. in a study, alcl3 significantly increased malondialdehyde (mda), and decreased glutathione (gsh), gpx, sod, and cat activities significantly in the brain of experimental rats, but these effects were significantly reversed by treatment with h. sabdariffa. also, alcl3 significantly lowered protein levels and increased percentage inhibition of ache and butyrylcholinesterase (bche) activities in the brain of test rats, however, treatment with h. sabdariffa, at low and high doses significantly reversed these effects (efosa et al. 2023). similarly, h. sabdariffa synthesized-gold nanoparticles (hs-aunps) ameliorated ad-related memory and learning impairments. hs-aunps also 10 biology, medicine, & natural product chemistry 13 (1), 2024: 7-33 ameliorated the reduction in sod, gpx, and gsh activities, and ameliorated the elevation of ache, monoamine oxidase (mao), adenosine deaminase, and mda activities observed in alcl3-induced rats. in addition, hs-aunps treatment assuaged the increased beta-secretase 1 (bace-1) and mrna expression of cyclooxygenase-2 (cox-2) spurred by alcl3 (anadozie et al. 2023). moringa oleifera moringa oleifera leaf extract has also been demonstrated to protective against alcl3 toxicity by resolving pyramidal cells of ca3 and neurofibrillary tangles in the hippocampus (finbarrs-bello et al. 2022). harrisonia abyssinica a promising candidate to alleviate aluminum-induced neurotoxicity in the hippocampus is harrisonia abyssinica. in addition to having antioxidant, antiinflammatory, and anti-apoptotic effects, h. abyssinica leaf extract normalizes the levels of glutamate, extracellular regulated kinase (erk1/2), caspase-3, ache, and catecholamines in the hippocampus of ad rats. it also helped to prevent the buildup of aβ plaques and returned the hippocampus region of the brain tissue to its original state. these activities are most likely caused by the high polyphenol content (anwar et al. 2021). orange peel an examination of orange peel extract's neuroprotective impact in vivo suggests that it may be useful in reducing brain oxidative stress and preventing the progression of alzheimer's disease. orange peel extract protected against alcl3-induced neuronal damage via decrease in both gene expression and activity of ache, thiobarbituric acid-reactive substances (tbars), amyloid beta (aβ42) protein level, and nitric oxide (no), and increase in reduced gsh level, and activity of the antioxidant enzymes in the brain tissues. additionally, presinilin-2 (psen2) and beta cell lymphoma-2 (bcl2) were upregulated, meanwhile, gene expressions for app and beta secretase enzyme (bace1) were downregulated (abd el-aziz et al. 2023). echinacea purpurea another plant that has been investigated to ameliorate the neurodegenerative effects of alcl3-induced alzheimer's disease is echinacea purpurea. e. purpurea flower extracts, both aqueous (aq) & alcoholic (al), inhibited ache, downregulated interleukin-6 (il-6) & tumor necrosis factor alpha (tnf-α), restored oxidative balance, & improved behavior performance in vivo and also decreased amyloid plaques & neuronal degeneration in the hippocampus & cerebral cortex (mohamed et al. 2023). mentha longifolia methylene chloride and ethyl acetate fractions (eafr) of mentha longifolia were found to have anticholinesterase activity and reversed the alcl3-mediated mda increase and gsh decrease. the elevated levels of nuclear factorkappa b (nf-κb) and no were also reversed by eafr of m. longifolia. it also counteracted the alcl3 effect on brain neurotransmitters (norepinephrine, dopamine, and serotonin) (elshamy et al. 2021). autranella congolensis a potential treatment for preventing oxidative damage in ad is autranella congolensis. alcl3 lowered cat, gpx, reduced total thiol, aconitase levels, and thiol protein levels and increased protein oxidation levels & lipid peroxidation in the brain or rats, meanwhile, the extract of a. congolensis significantly moderated these effects (ngoumen et al. 2023). ginko biloba interestingly, ginkgo biloba and vitamin c together constitute new therapeutic possibilities for neurodegenerative diseases, notably ad, as they were used to treat alcl3-induced neurotoxicity in rats. a study found that administering g. biloba methanolic leaf extract along with vitamin c improved cholinergic and dopaminergic dysfunction & alleviated memory impairment. also, it significant improved hippocampal morphology & histopathological alterations. it turned out that g. biloba and/or vitamin c could exhibit an improvement in neurotoxicity & memory loss and may be a viable treatment for cognitive decline in ad patients (elhallouty et al. 2022). canna indica canna indica is a member of the “cannaceae” family and “canna” genus. numerous pharmacological studies revealed its anti-inflammatory and antineurodegenerative properties, as well as its impact on the ache enzyme (chigurupati et al. 2021). whole plant extract from c. indica showed potential antiinflammatory, anti-radical, and neuroprotective properties (chigurupati et al. 2021). the memoryimproving & neuroprotective mechanism of action of c. indica have recently been demonstrated in a study in vitro and in vivo. in a study, chronic c. indica therapy improves alcl3-induced memory damage by regulating antioxidant pathways, restoring cholinergic system activity, and increasing dopamine levels (ojha et al. 2023). stachytarpheta angustifolia the seasonal plant stachytarpheta angustifolia, has been associated with neuroprotection against alcl3-induced ad. recently, a study showed the decreased level of sod, cat, gpx and gsh in alcl3-induced ad were significantly increased by the methanolic whole plant ojetunde – herbal medicines against alzheimer’s disease 11 extract of s. angustifolia. however, it significantly decreased no, mda, cox-2, & ache (ashikaa et al. 2023). the presence of phenol, saponin, alkaloid, flavonoid, and terpenoid with potent antioxidant, antiinflammatory, & ache inhibitory characteristics may be responsible for the neuroprotective effect of the extract. as a result, the extract has the potential to be an effective medication source for the treatment and management of ad disease (ashikaa et al. 2022). annona squamosa the recovery of the behavioural and biochemical changes caused by alcl3 and the strong neuroprotective mechanism of annona squamosa against ad were both demonstrated by the ethanol extract of a. squamosa fruit pulp (muthusamy et al. 2023). however, this is most likely due to the powerful antioxidant capabilities of the fruit pulp, which effectively counteract oxidative stress and maintain transmembrane protein levels. bryophyllum pinnatum the significant perennial herb bryophyllum pinnatum (crassulaceae) is widely used to cure a variety of diseases. the enriched flavonoid fraction of the leaves of b. pinnatum reduced oxidative imbalance by strengthening antioxidant defense and decreasing alcl3induced lipid peroxidation. it downregulated ache mrna transcripts & improved histological features in the cortex & hippocampus. these points to the neuroprotective effect of b. pinnatum against alcl3induced neurotoxicity (ogidigo et al. 2022). sesamum indicum in a study, alcl3-induced learning and memory impairments were significantly improved by sesame (sesamum indicum) oil. the elevated level of ache and aβ overexpression were also significantly reduced after treatment with sesame oil. moreover, alcl3 treatment resulted in histopathological changes, an increase in the expression of tnf-α and interleukin-1 beta (il-1β), as well as mitigation of oxidative stress status in the brain. all of these anomalies were eliminated by sesame oil. meanwhile, it also inhibited alcl3-induced activation of p38 mitogen-activated protein kinase (p38mapk) and the decrease in brain-derived neurotrophic factor (bdnf). additionally, treated with sesame oil modulated the expression of the nuclear factor kappa b (nf-κb) and peroxisome proliferator-activated receptor gamma (ppar-γ). interestingly, many studies have shown that sesame oil is unique because it contains a significant amount of bioactive antioxidant lignans, particularly sesamolin, sesamin, sesamolinol, and sesaminol (mohamed et al. 2021). rosa damascena the crassulaceae family includes rosa damascena (damask rose), which grows in northern asia and the mountains of central europe. in ad rats, administration of the r. damascena extract enhanced cat and gsh levels, decreased mda levels, and regulated ache activity. these demonstrate that r. damascena extract is protective against the oxidative damage caused by alcl3 intoxication (hejaziyan et al. 2023). tamarindus indica according to phytochemical analysis of tamarindus indica (which belongs to the monotypic genus tamarind), there are several significant bioactive components present in this plant, including phenolic compounds, glycosides, malic acid, tartaric acid, arabinose, pectin, mucilage, xylose, galactose, glucose, and uronic acid (usman et al. 2022). during exposure to alcl3, t. indica reduced the levels of pro-inflammatory cytokines and lipid peroxidation products in the cerebral cortex. t. indica also protected against aluminum chloride induced memory impairment (muhammad et al. 2020). the improvement in oxidative stress biomarker, spatial memory and learning, and glial fibrillary acid protein reactivity confirmed that the administration of ethyl acetate leaf fraction of t. indica was of therapeutic value during prenatal alcl3 exposure in wistar rats (usman et al. 2022). moreover, following prenatal alcl3 exposure in wistar rat pups, the ethyl acetate fraction of t. indica leaves also significantly increased calcium levels and decreased mean zinc, copper, and iron levels. additionally, it enhanced cognition and increased brain sialic acid (usman et al. 2023). vaccinium corymbosum the ericaceae family plant vaccinium corymbosum, widely known as the blueberry, may have an effect on human health and wellbeing as well as provide neuroprotection (hong et al. 2018; miller et al. 2019). the fruits of blueberry trees, in particular, are high in polyphenols and flavonoids. in a behavioural study, rats treated with ethanolic extracts of v. corymbosum demonstrated neuroprotective effects against alcl3induced neurotoxicity along with a significant decrease in ache enzyme. the neuroprotection of v. corymbosum makes it a promising therapeutic agent for treating behavioral and cognitive dysfunctions (chellammal et al. 2021). xylopia parviflora xylopia parviflora has antioxidant and anti-inflammatory properties (nwakiban et al. 2020; nwakiban et al. 2021). according to a study, the fruit extract from x. parviflora effectively improved all alterations caused by al. treatment with x. parviflora in various dosages improved memory and locomotion, ion homeostasis, cholinesterase activities, and stabilized brain oxidative stress levels. according to the study, x. parviflora may be useful for the management of some biochemical alterations linked to ad. it may be beneficial in neurotoxicity caused by al at the behavioural and biochemical levels. x. parviflora may have promising 12 biology, medicine, & natural product chemistry 13 (1), 2024: 7-33 compounds that could be tested as potential drugs for the treatment of diseases caused by oxidative stress and cholinergic dysfunction like ad due to its combination of antioxidant, anti-cholinesterase potential, and improvement of cognitive impairment (dibacto et al. 2022). pluchea lanceolata in a study, alcl3 increased protein content and mda, and reduced body weight, cat, sod, & gsh levels. these effects were reversed and restored by pluchea lanceolata hydromethanolic extracts. the hydromethanolic extracts of p. lanceolata also offered cellular-level protection, according to the histopathology findings. as a result, the active components of p. lanceolata were found to have anti-alzheimer and antioxidant potential (asirvatham et al. 2022). salvia officinalis in addition to a significant increase in the serum levels of urea, creatinine, ast, and alt activity as well as oxidative stress indicators, alcl3-induced ad rats showed significant alterations in tau protein and acetylcholine levels in brain tissue. however, administration of mg-salvia officinalis nanoparticles (nps) significantly improved all previous parameters. in conclusion, treatment with s. officinalis nps ameliorated oxidative stress, enhanced antioxidant defense system, and prevented the lipid peroxidation caused by alcl3. so, administration of mg-salvia officinalis nps for the treatment of ad can be recommended (elkomy et al. 2021). vanda tessellate vanda tessellate can be used as a remedy for the treatment of ad and neurotoxicity. a study revealed that, the hydromethanolic extract of v. tessellate was able to correct and restore the increased level of protein content, mda, reduction in body weight & antioxidants enzymes including cat, sod, and gsh that were caused by alcl3 administration. the histopathological report also demonstrated the cellular level protective efficacies of the v. tessellate hydromethanolic extract. it should be emphasized that the active constituents present in v. tessellate was responsible for its neuroprotective effects (salam et al. 2022). grape seed oil grape seed (vitis vinifera) oil reduced alcl3-induced significant decline in cognitive function, and significantly decreased ache and modulated antioxidant (sod, cat, glutathione reductase) activity level. additionally, histopathological studies in the hippocampus and cortex supported the fact that grape seed oil significantly decreased alcl3 toxicity. grape seed oil supplementation exhibited a positive and neuroprotective role alcl3-induced neurotoxicity in wistar rat by enhancing cognitive memory and antioxidant enzyme levels (muralidharan and swetha 2023). zingiber officinale studies conducted have noted the anti-inflammatory (tongshuwar et al. 2020; ojetunde et al. 2021) and antioxidant capacity (bekkouch et al. 2022) of the active components in zingiber officinale (ginger). the use of z. officinalis as a therapeutic approach against neurological diseases has great potential. in a study, the use of z. officinalis ethanol extract and its fraction (dichloromethane and n-hexane) improved memory and decreased oxidative stress status in alcl3-induced mice (inwang et al. 2023). bougainvillea spectabilis the flower decoction of bougainvillea spectabilis may be useful in the treatment of ad. according to research, b. spectabilis alleviated the increase in no and mda, and decrease in gsh and pon-1 activity alterations evoked by alcl3. also, b. spectabilis decoction significantly reduced il-6 and aβ in the brain of rats treated with alcl3. it also protected against neurodegeneration induced by alcl3, & restored memory performance and motor strength (abdel-salam et al. 2021). peganum harmala in north africa, peganum harmala, a traditional plant from the zygophyllaceae family, is commonly referred to as harmal or haramlaan (asgarpana and ramezanloo 2012; eissa et al. 2014). p. harmala has abundance of βcarboline alkaloids, with the seeds having the highest quantitiy (osman et al. 2018; araujo et al. 2019). these include harmaline, harmine, harmalol, harmane, and norharmane (osman et al. 2018). harmine and harmaline have ache inhibitory effect and antioxidant activity, which suggests that they may be used to treat ad (ali et al. 2013). in a study, p. harmala improved cognition and histopathological features altered by alcl3. additionally, it increased the hippocampus level of insulin & glucagon-like peptide (glp)-1, while decreasing the phosphorylation of insulin receptor substrate-1 at serine 307 (ps307-irs-1). besides, phosphorylated akt at serine 473 (ps473-akt) and glucose transporter type (glut)4 were also increased by p. harmala. the levels of (aβ)42, glycogen synthase (gsk)-3β and phosphorylated tau in the hippocampus were also reduced by the extract. along with lowering lipid peroxides and replenishing glutathione, p. harmala also improved nuclear factor erythroid 2-related factor 2 (nrf2) (saleh et al. 2021). vernonia amygdalina the plant vernonia amygdalina belongs to the asteraceae family. it is commonly referred to as "bitter ojetunde – herbal medicines against alzheimer’s disease 13 leaf plant" (ojetunde, 2021). phytochemical components of v. amygdalina include epivernodalol, lactones, sesquiterpene, elemanolide, terpenes, edotides, steroids, flavonoids, coumarins, phenolic acids, xanthones, lignans, saponins, anthraquinone, & alkaloids (muraina et al. 2010). v. amygdalina has been scientifically proven to be effective against a variety of disease due to its high antioxidant content. v. amygdalina leave extract significantly improved hippocampal histological features and reduced the behavioral abnormalities in rats caused by alcl3. however, in the treatment of ad, v. amygdalina perform better as a preventive than as a curative (ajeleti et al. 2023). lepidium sativum lepidium sativum (family: brassicaceae) is used as a treatment for a variety of diseases and has a wide range of pharmacological properties, such as antioxidant, antiinflammatory, & anti-diabetic activity (attia et al. 2019). administration of l. sativum significantly enhanced antioxidant parameters, decreased pro-inflammatory cytokines, and attenuated ad-related histopathological alterations (balgoon 2023). buchholzia coriacea buchholzia coriacea plant belongs to the family capparaceae (obembe et al. 2012). b. coriacea has been proven to have neuroprotective properties (abayomi et al. 2019). its phytochemical constituents include saponin, reducing sugar, alkaloids, glycosides, tannin, flavonoids, terpenes, steroid, & phenols (ibrahim and fagbohun 2013). the presence of flavonoids, vitamins, antioxidant, and enzymes may be largely responsible for its therapeutic effects (ibrahim and fagbohun 2013). according to research, b. coriacea significantly enhanced spatial working memory, and restored myelin sheath integrity, both of which would inevitably speed up impulse conduction and improve the memory process in ad (adelodun et al. 2021). rosmarinus officinalis rosmarinus officinalis (rosemary) belongs to the lamiaceae family and has high phenolic and terpenoid compounds (andrade et al. 2018). it has antiinflammatory, antioxidant, and antidepressant properties (guo et al. 2018; dabaghzadeh et al. 2022). in addition to improving cognition, r. officinalis can regulate synaptic gene expression, inflammation, and the density of hippocampal neurons in alcl3-induced neurotoxicity (khalid et al. 2020). in an animal model of ad, it has been proven that r. officinalis can significantly improve cognitive impairment, and significantly lower depression and anxiety. r. officinalis improved cognitive function but did not decrease the burden of amyloid plaque, suggesting that the memory-enhancing effects of this plant are caused by a different mechanism that needs to be investigated (malik et al. 2022). massularia acuminata massularia acuminate (family: rubiacea) significantly reduced mda levels in animals treated with alcl3. on the other hand, the same level of sod and cat was shown in animals that were treated with m. acuminate with level of ascorbic acid in alcl3-induced toxicity. however, butanolic extract of m. acuminata at 50 mg/kg and 100 mg/kg body weight show that they cause oxidative stress because they increase mda level. as a result, the ethanolic and methanolic stem extract of m. acuminata can act as potential antioxidant compounds in the treatment of oxidative stress related to alcl3 toxicity (bakare et al. 2021). capsicum annuum the most consumed spices in the world are hot red or green peppers of the plant genus capsicum (capsicum annuum and capsicum frutescens) (abdel-salam et al. 2023). in experimental models of parkinson’s disease, studies have that hot pepper extracts have neuroprotective effects (abdel-salam et al. 2018). moreover, it has been noted that consuming capsaicinrich diet improves cognition and lower aβ levels in the serum of adults (liu et al. 2016). in the app/ps1 genetic mice model of ad, similar findings were reported (wang et al. 2020). thus, capsicum may be a beneficial nutraceutical to prevent and/or delay neurodegeneration in the brain of ad patients. in a study, rats treated with alcl3 received methanolic extract of capsicum fruits (hot red peppers), which significantly reduced oxidative stress (decreased no & mda, and increased gsh and pon-1 levels) as well as aβ-peptide and il-6 in the brain. capsicum fruits also improved grip strength and memory functioning and prevented neuronal degeneration in the hippocampus, cerebral cortex, and substantia nigra of rats treated with alcl3 (abdel-salam et al. 2023). benincasa hispida benincasa hispida (ash gourd or wax gourd) modulated the levels of dopamine, serotonin, & ache in alcl3induced ad. the extract of b. hispida leaves increased sod, cat, gsh and decreased mda levels. b. hispida treatment also decreased the levels of tnf-α & il-1β in ad. it further upregulated antioxidant genes keap/nrf2/ho-1. histopathological examinations of the hippocampus further confirmed the neuroprotective potential of b. hispida (rapaka et al. 2021). b. hispida is thus a potential substitute for neuroprotection in the treatment of ad. euphorbia cotinifolia the methanol extract of euphorbia cotinifolia significantly improved motor dysfunctions and cognitive abilities of animals with ad. the levels of ach, sod, cat, gpx, and gsh were also elevated by the treatment with the methanol extract of e. cotinifolia. histopathological analysis revealed less neurofibrillary https://www.sciencedirect.com/science/article/pii/s2667031321000865#bib0027 14 biology, medicine, & natural product chemistry 13 (1), 2024: 7-33 tangles and neuronal loss. e. cotinifolia methanol extract also reduced the expression of il-1α, il-1β, tnf-α, & tnf-β in ad animals (saadullah et al. 2023). punica granatum pomegranate plant (punica granatum) contains unique components like flavonols, ellagitannins, phenolic acids, anthocyanins, & organic acids, which have antioxidant, antineurodegenerative, anticancer, anti-inflammatory and anti-apoptotic activities (ahmadiankia 2019; kandylis and kokkinomagoulos 2020). p. granatum has antioxidant levels that are two, six, and eight times higher than those of red berries, grapefruit, and orange juice respectively (abu-taweel and al-mutary 2021). pomegranate helps to prevent cognitive and behavioral decline in ad (subash et al. 2015). in a study, p. granatum juice significantly improved body weight, spatial memory and learning, neurotransmitters and oxidative biomarkers in the alcl3-treated mice (abutaweel and al-mutary 2021). phyllanthus amarus phyllanthus amarus, a commonly used medicinal plant, have some reported antioxidant properties. in a study, alcl3 significantly decreased the survival rate, climbing activity, sod, gst, cat activities and increased mda concentration and ache activities in drosophila melanogaster. however, p. amarus was able to significantly ameliorate all these changes. p. amarus can therefore have therapeutic benefits in the management of ad (inneh and enogieru 2023). centella asiatica a perennial herbaceous creeper, centella asiatica (family: apiaceae), is locally referred to as pegaga in malay (chiroma et al. 2017; gray et al. 2018). in the ayurvedic system of medicine, c. asiatica is regarded as invigorating herb that improves memory and intelligence. the medicinal values of c. asiatica are connected to its numerous active constituents such as madecassic acid, asiatic acid, asiaticoside, braminoside, madecassoside, bramoside, and flavonoids (gohil et al. 2010). c. asiatica prevented alcl3-induced cognitive impairment of both spatial and non-spatial memory, histopathological aberration of the cerebral cortex, and increased levels of ache in the brain of rats (farhani et al. 2023). thus, it could be developed as a memory enhancing drug. onion and garlic according to several studies, consuming onion flavonoid containing quercetin protects brain tissues from aging by inhibiting apoptosis that causes brain degeneration (wang et al. 2020; dorrigiv et al. 2021). moreover, the active components found in garlic extracts have been shown to have protective effects against neurotoxicity (galal et al. 2019; hazzaa et al. 2020; bigham et al. 2021). in a recent study, intracellular ros generation in the brain of ad-induced rats was inhibited by treated with several doses of onion and garlic root extracts, which also reduced histopathological lesions, the expression levels of apoptotic genes, and the rate of dna damage in the brain tissues (hegazy et al. 2022). malva neglecta malva neglecta (family: malvaceae) is an annual herbaceous species. in vitro studies showed that the methanolic extract of m. neglecta have anticholinesterase activity (abbas et al. 2017). the presence of 25 bioactive polyphenolics was identified by hplc-dad analysis (saleem et al. 2020), with hydroxytyrosol and coumaroylhexoside being in highest concentrations. these bioactive compounds have a variety of biological properties (ren et al. 2017, winter et al. 2017; karković marković et al. 2019; khan et al. 2020) which may help explain the neuroprotective potentials of the plant. m. neglecta has antioxidant capacity, making it a key target in neurodegenerative disorders caused by free radicals (dalar et al. 2012). malva parviflora, another species of the family, is said to have protective against ad induced by aβ (aslam and sial 2014). study has shown that m, neglecta can reduce the symptoms of ad by improving memory and cognition, & modulating oxidative stress biomarkers and ache activity (saleem et al. 2021). table 1. neuroprotective and therapeutic effects of medicinal plants against aluminium chloride-induced alzheimer’s disease. medicinal plant dose of alcl3 dose of extract animals used mechanism of action ref acacia catechu 100 mg/kg b.wt. daily for 60 days orally 3 mg/kg/day i.p. on daily basis for 15 days after alcl3 administration rats antioxidant, anticholinesterase, preserved monoamines level, reduced genotoxicity, & corrected cognitive behavioral dysfunction (elmorsy et al. 2021) hibiscus sabdariffa 7 mg/kg b.wt./day i.p. for 28 days 250, 500, or 1000 mg/kg b.wt./day/p.o. for 28 days rats antioxidant, anticholinesterase (efosa et al. 2023) moringa oleifera 100 mg/kg orally for 21 days 400 mg/kg orally for 21 days rats protected against brain damage (finbarrs-bello et al. 2022) ojetunde – herbal medicines against alzheimer’s disease 15 table 1. cont. medicinal plant dose of alcl3 dose of extract animals used mechanism of action ref harrisonia abyssinica 100 mg/kg b.wt./day p.o. 100 or 200 mg/kg b.wt./day intragastrically for 3 weeks. rats antioxidant, anticholinesterase, antiinflammatory, antiapoptotic, anti-aβ, regulated neurotransmitters level, & enhanced learning and memory (anwar et al. 2021) orange peel extract 70 mg/kg b.wt./day i.p. for 6 weeks 100 or 200 mg/kg orally for 6 weeks rats antioxidant, anticholinesterase, antiaβ, (abd el-aziz et al. 2023) echinacea purpurea 175 mg/kg alcl3 orally for 60 days 250 mg/kg orally for 60 days rats antioxidant, anticholinesterase, antiinflammatory, anti-aβ, & improved behavior performance (mohamed et al. 2023) mentha longifolia 100 mg/kg b.wt. for 30 consecutive days by s.c. injection 250 mg/ kg b.wt./day of the fractions orally for 15 days & 100 mg/kg b.wt./day of the oil orally for 15 days before induction of ad rats antioxidant, anticholinesterase, antiinflammatory, & regulated neurotransmitters levels (elshamy et al. 2021) hibiscus sabdariffa 100 mg/kg b.wt. orally for 42 days 5 or 10 mg/kg b.wt. for 42 days rats antioxidant, anticholinesterase, improved memory and learning, & modulated gene expression (anadozie et al. 2023) autranella congolensis 50 mg/kg b.wt. orally for 8 weeks 150 or 300 mg/kg (1hr after alcl3) orally for 8 weeks rats antioxidant (ngoumen et al. 2023) ginko biloba 17 mg/kg b.wt. for 4 weeks orally 400 mg/kg b.wt. orally for 2 weeks after 4 weeks intoxication with alcl3 rats attenuated memory impairment & improved dopaminergic and cholinergic dysfunction (elhallouty et al. 2022) canna indica 17 mg/kg p.o. for 21 days aerial methanolic extract (200 mg/kg p.o.) or root hydroalcholic extract (200 mg/kg p.o.) or the combination for 21 days rats antioxidant, anticholinesterase, improved memory damage, and modulated neurotransmitter level (ojha et al. 2023) stachytarpheta angustifolia 100 mg/kg b.wt. orally for 8 weeks 25, 50, or 75 mg/kg orally for 8 weeks rats antioxidant, anticholinesterase, & modulated cognitive functions (ashikaa et al. 2023) annona squamosa 17 mg/kg b.wt. for 30 days orally 200 or 400 mg/kg b.wt. for 60 days after administration of alcl3 for 30 days rats antioxidant, anticholinesterase, & enhanced cognitive functions (muthusamy et al. 2023) bryophyllum pinnatum 150 mg/kg b.wt. orally for 21 days 50 or 100 mg/kg b.wt. orally for 21 days (after administration of alcl3 for 21 days) rats antioxidant, anticholinesterase, & restored histopathological lesions (ogidigo et al. 2022) sesamum indicum 100 mg/kg/i.p. for 6 weeks 1 ml/kg or 2 ml/kg, p.o. for 6 weeks rats antioxidant, antiinflammatory, anticholinesterase, antiaβ, & improved learning and memory (mohamed et al. 2021) 16 biology, medicine, & natural product chemistry 13 (1), 2024: 7-33 table 1. cont. medicinal plant dose of alcl3 dose of extract animals used mechanism of action ref rosa damascena 100 mg/kg orally for 4 weeks 500 or 1000 mg/kg orally for 8 weeks and alcl3 orally daily for the last 4 consecutive weeks. rats antioxidant, anticholinesterase, & improved learning & memory (hejaziyan et al. 2023) tamarindus indica 200 mg/kg b.wt. orally 14 days from prenatal day 7 till parturition 400 mg/kg or 800 mg/kg b.wt. orally for 14 days from prenatal day 7 till parturition rats antioxidant, improved glial fibrillary acid protein reactivity & spatial memory & learning (usman et al. 2022) vaccinium corymbosum 100 mg/kg orally for 42 days 200 or 400 mg/kg on the 21st day until the 42nd day rats anticholinesterase, & mitigated behavioural & cognitive dysfunctions (chellammal et al. 2021) xylopia parviflora 75 mg/kg by oesophageal gavage for 60 days 150 or 300 mg/kg b.wt. by oesophageal gavage for 60 days rats improvement of cognitive impairment, antioxidant, & anticholinesterase (dibacto et al. 2022) pluchea lanceolata 300 mg/kg, p.o. for 20 days 200 or 400 mg/kg, p.o. for 20 days rats antioxidant, cellularlevel protection, & raised the level of neurotransmitters (asirvatham et al. 2022) salvia officinalis 100 mg/kg b.wt./i.p. for 2 weeks 5 mg/kg b.wt./day, orally for 4 weeks (after 14 days of alcl3 injection) rats antioxidant, decreased brain tau protein level, & increased brain ach (elkomy et al. 2021) vanda tessellate 300 mg/kg, p.o. for 20 days 300 mg/kg, p.o. rats antioxidant, histological cellular-level protection, & raised the level of neurotransmitters (salam et al. 2022) tamarindus indica 200 mg/kg b.wt. orally 14 days from prenatal day 7 till parturition 400 mg/kg or 800 mg/kg b.wt. orally for 14 days from prenatal day 7 till parturition rats improved memory & learning, trace element, & brain sialic acid concentration (usman et al. 2023) grape seed oil 175mg/kg p.o. for 30 days 2ml (3.7g/kg) or 4ml (3.7g/kg) p.o. on the 20th day of experiment for 10 days rats anti-oxidant, anticholinesterase, alleviates impaired cognitive function (muralidharan and swetha 2023) zingiber officinalis 100 mg/kg orally for 3 weeks ethanol extract (474, 949 or 1,423 mg/kg), dichloromethane extract (949 mg/kg) & n-hexane extract (949 mg/kg) mice antioxidant, memory improvement (inwang et al. 2023) bougainvillea spectabilis 10 mg/kg i.p. for 2 months 50 or 100 mg/kg i.p. daily during the 2nd month rats antioxidant, antiinflammatory, antiamyloid, restored motor and memory impairment, histological neuroprotection. (abdel-salam et al. 2021) peganum harmala 50 mg/kg/day i.p. for 6 consecutive weeks 187.5 mg/kg; p.o starting 2 weeks post alcl3 exposure for 4 weeks rats antioxidant, anti-aβ, enhanced cognition, & ameliorated hippocampal insulin resistance (saleh et al. 2021) vernonia amygdalina 0.43 ml/kg via oropharyngeal cannula for 14 days 1.31 ml for 14 days before or after alcl3 administration for 14 days rats improved memory & hippocampal histological features (ajeleti et al. 2023) ojetunde – herbal medicines against alzheimer’s disease 17 table 1. cont. medicinal plant dose of alcl3 dose of extract animals used mechanism of action ref lepidium sativum 10 mg/kg b.wt. i.p. for 8 weeks 20 mg/kg via gavage for 4 weeks after alcl3 administration for 4 weeks rats antioxidant, antiinflammatory, antiapoptotic, anti-aβ, anticholinesterase, & alleviated histopathological changes (balgoon 2023) buchholzia coriacea 200 mg/kg orally for 14 days 50 or 100 mg/kg orally for 14 days rats improved spatial working memory and histological changes (adelodun et al. 2021) rosmarinus officinalis 300 mg/kg for 15 days 100 mg/kg i.p. for 10 days after 15 days of alcl3 mice improved memory impairment, and decreased anxiety & depression (malik et al. 2022) massularia acuminata 34 mg/kg orally for 3 weeks 50 or 100 mg/kg orally for 3 weeks rats antioxidant (bakare et al. 2021) capsicum annuum 10 mg/kg i.p. for 60 days 25 or 50 mg/kg i.p. during the 2nd month of the study rats antioxidant, anti-aβ, anti-inflammatory, & improved memory impairment and neuromuscular strength (abdel-salam et al. 2023) benincasa hispida 100 mg/kg/day orally for 8 + 16 weeks 250 or 500 mg/kg/day orally for 16 weeks after alcl3 administration for 8 weeks rats antioxidant, antiinflammatory, anti-aβ, & improved memory and neurotransmitters level (rapaka et al. 2021) euphorbia cotinifolia 300 mg/kg p.o. for 21 days 100, 300, or 800 mg/kg p.o. for 21 days rats antioxidant, antiinflammatory, anticholinesterase, & improved cognitive behaviours (saadullah et al. 2023) punica granatum 400 mg/kg orally for 35 days 20% or 40% orally for 35 days mice antioxidant, modulated neurotransmitters level, reduced cognitive impairment, & enhanced spatial learning & memory capacity (abu-taweel and al-mutary 2021) phyllanthus amarus 40mm via diet 2.5mg via diet drosophila melanogaster antioxidant and anticholinesterase (inneh and enogieru 2023) centella asiatica 70 mg/kg b.wt. i.p. for 42 days 200, 400 or 800 mg/kg b.wt. for 42 days rats prevented cognitive impairment, anticholinesterase (farhani et al. 2023) onion and garlic 0.3% for 45 days 1, 2, or 3 mg/kg onion extract; 1, 2, or 3 mg/kg garlic extract for 30 days (after 45 days of alcl3 treatment) rats antioxidant, antiapoptotic, decreased histopathological lesions & the rate of dna damage (hegazy et al. 2022) malva neglecta 100 mg/kg orally for 21 days 200, 400 or 600 mg/kg orally for 21 days rats antioxidant, anticholinesterase, and improved memory and cognition (saleem et al. 2021) note: alcl3: aluminium chloride; aβ: amyloid-beta; ad: alzheimer’s disease; ach: acetylcholine; b.wt.: body weight; i.p.: intraperitoneally; p.o.: oral administration; s.c.: subcutaneous 18 biology, medicine, & natural product chemistry 13 (1), 2024: 7-33 role of natural products against aluminium chloride-induced alzheimer’s disease phytochemicals morin, thymol, and thymoquinone are phytochemicals that, when combined with physical and mental activities (phm), restored antioxidant activities, increased heme oxygenase-1 (ho-1) and nrf2 levels, blocked inflammasome activation, tlr4 expression, apoptosis, aβ generation, and tau hyperphophorylation. additionally, they restored the levels of apoe4 and lrp1 and regulated the wnt3/β-catenin/gsk3β signaling pathway. therefore, the combination of phytochemicals with phm is a promising strategy for reducing ad (hamdan et al. 2022). niruriflavone niruriflavone, a compound isolated from phyllanthus niruri, reversed alcl3-induced neurobehavioral alteration. the niruriflavone treatment also reduced ache and lipid peroxidation, and restored the antioxidative enzymes (rajamanickam and sl 2022). quercetin administration of quercetin attenuated behavioral deficits, ameliorated dopaminergic & cholinergic dysfunctions, and diminished the aggregation of insoluble aβ plaques in the hippocampus of alcl3induced ad rat. quercetin downregulated app, bace1, anterior pharynx-defective 1 (aph1), & psen1, meanwhile upregulated adam10 and adam17 gene expression levels in the hippocampus, leading to amyloidogenic pathway inhibition. in order to counteract aβ aggregation & cognitive deterioration in ad, it has been hypothsized that adam10 (a disintegrin and metalloproteinase 10) and adam17 (a disintegrin and metalloproteinase 10) activation may be potential therapeutic targets (elfiky et al. 2021). according to a similar study, quercetin significantly affects memory deficits in ad. furthermore, quercetin significantly decreased app, bace1, and psen1 and increased adam17 expression in the hippocampal tissue of ad rats (elreedy et al. 2023). similarly, significant improvement in behavioral parameters, inhibition of ache activity, and decrease in oxidative stress parameters (significantly higher levels of gsh, cat, & sod, as well as lower levels of mda) have all be have all been seen when quercetin was combined with memantine. after treatment of quercetin with memantine, histopathological examination of the hippocampus and cortex showed a decrease in the formation of aβ plaque. according to immunohistochemistry, the combination of quercetin and memantine also improved the expression of bdnf and inhibited the formation aβ plaque (jadhav and kulkarni 2023a). quercetin has been linked to a variety of therapeutic actions, including antioxidant, antiamyloidogenic, anti-inflammatory, and neuroprotective effects (kim and park 2018). the neuroprotective effects of quercetin are due to its capacity to inhibit xanthine oxidase and prevent lipid peroxidation or scavenge oxygen (jadhav and kulkarni 2023a). additionally, quercetin has been shown to have neurotropic effect by regulating the akt/pkb (protein kinase b) and erk1/2 signaling pathway by impeding the activity of pi3k (phosphoinositide 3-kinase) (minocha et al. 2022). quercetin nanoemulsion also provided protection against neuronal dysfunction caused by alcl3 via the elevation of brain antioxidants, reduction of the production of proinflammatory cytokines, and modulation of neurotransmitter levels, and reversing the histopathological changes in rats (alaqeel et al. 2022). resveratrol in a study, the combination of resveratrol and tannic acid significantly reduced ad-related cognitive decline. this treatment significantly reduced oxidative stress markers and the levels of amyloid found in ad. this demonstrated protective benefit of combination of resveratrol and tannic acid in alcl3-induced neurotoxicity (bhounsule and bhatt 2023). also, resveratrol‑selenium nanoparticles (rsv-senps) supplementation attenuated oxidative markers impairement and mitochondrial dysfunction in ad. rsvsenps ameliorated cholinergic deficits and also cleared aβ. furthermore, activation of pi3k deactivates glycogen synthase kinase 3 beta (gsk-3β)-mediated tau hyperphosphorylation. additionally, rsvsenps alleviated neuroinflammation in ad by downregulating signal transducer and activator of transcription (stat3) expression, and il-1β levels. moreover, sirtuin-1 (sirt1) was upregulated and microrna-134 expression was downregulated by rsvsenps, which increases neurite outgrowth (abozaid et al. 2022). furthermore, the administration of rsv-senps improved neuronal transmission in ad by decreasing oxidative stress and metal chelation while also increasing neurotransmitter levels (abozaid et al. 2021). thymoquinone thymoquinone and celastrol may also be an effective therapy for neurodegenerative diseases caused by oxidative stress and neuroinflammation as well as alcl3induced neurotoxicity. a recent study showed that thymoquinone and celastro significantly reversed the impairment of motor coordination, reduction of free ambulation, reduction of whole-brain ach, dopamine, and serotonin concentrations. administration of the combination also reversed the reduction of total antioxidant capacity (tac), increment of mda accumulation, elevation of tnf-α and il-6, and suppression of bdnf mrna expression linked to alcl3induced ad (abbas et al. 2022). ojetunde – herbal medicines against alzheimer’s disease 19 betalain betalain, a glycoside pigment widely present in beetroot, mushrooms, pear, swiss chard, prickly dragon fruit, and tubers, has been reported to mitigate alcl3-induced ad via modulating the activation of nf-κb pathway. it improved memory and learning capacity, and suppressed lipid oxidation (mda) via the regulation of antioxidant content (sod, cat, and gsh). it also inhibited alcl3– induced lactate dehydrogenase (ldh), no, ache, & transmembrane protein (na+k+atpase) activity. in addition, betalain decreased nf-κb associated mrna expression (tnf-α, il-1β, il-6, cox-2, inos) (shunan et al. 2021). vinpocetine in clinical settings, vinpocetine, a semi-synthetic vincamine derivative, is used to treat dementia and memory disturbances. vinpocetine has several functions, including antioxidant, anti-inflammatory, and vasodilation therapeutic actions (zhang et al. 2018). in rats, vinpocetine combined with epigallocatechin-3gallate (egcg) protected neurons against alcl3–induced ad. this combination significantly decreased aβ, & ache levels. the levels of monoamines and bdnf showed similar patterns of result. in addition, the combination demonstrated more prominent antiinflammatory (il-1β, tnf-α) and antioxidant (mda, sod, tac) effects (ali et al. 2022). β-sitosterol treatment with β-sitosterol reduced alcl3-induced cognitive impairment. in a study, β-sitosterol significantly increased step-through latency time, percentage alteration time, percentage preference index, ach, & gsh levels, and lowered the levels of ache in alcl3-induced ad in mice. β-sitosterol also significantly reduced aβ deposition caused by alcl3 (yadav et al. 2023). gallic acid and hesperidin gallic acid is a common plant metabolite having several hydrogen atoms in its phenolic structure that easily delocalize free radicals (kahkeshani et al. 2019). gallic acid exerts its neuroprotective effects via preventing nmethyl d-aspartate (nmda) receptors activation, and the release of glutamate, as well as inhibiting amyloidinduced neurotoxicity by selectively suppressing the activation of nf-κb (gao et al. 2019; bai et al. 2021). hesperidin restored antioxidant enzymes levels in biological systems. hesperidin and its derivatives have been shown to have antioxidant potential due to their structures which abound in hydroxyl groups (kim et al. 2019; stanisic et al. 2020). the ability of hesperidin to inhibit oxidative stress, apoptosis, inflammation, and amyloid polymerization shows that it’s a promising potential in the treatment of ad (wdowiak et al. 2022). in a study, gallic acid & hesperidin were both found to significantly protect against alcl3–induced ad, suggesting their consumption may be important to delay the onset of the disease. the study showed that gallic acid or hesperidin administration prevented cognitive impairment. additionally, gallic acid or hesperidin significantly prevented deficits in neurotransmission (ache, bche, serotonin, norepinephrine, and dopamine), oxidative stress (sod, gsh, cat, and gst), and inflammation (il-6, il-1β, & tnf-α), while also lowering brain caspase-3 level. the findings of histopathological evaluation also supported these observations (ekundayo et al. 2022). gallic acid and donepezil in a study, alcl3 significantly increased ache activity in the brain as well as mda & no levels, while simultaneously decreasing total thiol level and the activities of sod and cat. these alterations were however reversed by donepezil only as well as combination of donepezil and gallic acid. also, this combination significantly improved antioxidant status as opposed to donepezil alone. one could draw the conclusion that donepezil and gallic acid work in synergy, especially when it involves ameliorating the oxidative stress linked to alcl3-induced neurotoxicity (obafemi et al. 2021). ginsenoside rb1 ginsenoside is abundantly found in panax ginseng. in a recent study, ginsenoside rb1 significantly attenuated the decrease in synaptophysin expression, the histopathological alterations in the cerebral cortex, the elevation of the expression of cleaved caspase-3, ionized calcium-binding adaptor molecule 1 (iba-1), and glial fibrillary acidic protein (gfap). ginsenoside rb1 may have a neuroprotective effect against alcl3–induced changes in the cerebral cortex by suppressing the formation of aβ & phosphorylated tau protein, acting as an anti-apoptotic agent, minimizing gliosis, and correcting oxidative stress (shalaby et al. 2023). ononin ononin, an isoflavone glycoside, is widely present in a variety of plants, including ononis angustissima, smilax scobinicaulis, & millettia nitida (li et al. 2014). treatment with ononin successfully reduced behavioural alterations in ad animals caused by alcl3. in the brain tissues of ad animals, ononin also significantly reduced ache, aβ1-42, & mda while increasing sod and tac. also, the levels of il-1β, tnf-α, p38mapk, & nf-κb were reduced while bdnf and ppar-γ contents were increased in ad animals. ononin treatment may help to suppress the neuroinflammation and oxidative stress, & ameliorate the cognitive impairment found in the ad (chen et al. 2021). asiatic acid asiatic acid is a key bioactive compound that offers c. asiatica (a medicinal plant) its antioxidative & 20 biology, medicine, & natural product chemistry 13 (1), 2024: 7-33 therapeutic effects (nagoor meeran et al. 2018). a recent study showed that alcl3 intoxication in rats results in severe memory impairment, increased anxiety-like behaviour, ache activity, mda level, and concurrently decreased sod, & cat activity in the cortex and hippocampus. alcl3-intoxication also accelerated neuronal loss and reactive astrogliosis in both regions. however, when asiatic acid and alcl3 were administered together, the behavioral alterations were attenuated, sod & cat activities were restored, and ache activity and mda level were reduced. asiatic acid also attenuated neuronal loss and reactive astrogliosis in rat brain (suryavanshi et al. 2022). p-coumaric acid p-coumaric acid (p-ca) is a phenolic acid of the hydroxycinnamic acid family that can be found in many vegetables, fruits, and cereals, such as pears, apples, oranges, berries, grapes, potatoes, tomatoes, beans, onions, wheat, maize, and oats (ferreira et al. 2019). pca has attracted a lot of interest recently due to its wide range of biological activities, such as antioxidant (sakamula and thong-asa 2018), neuroprotective (oh et al. 2021), anti-inflammatory (yoon et al. 2014), & memory-improving effects (daroi et al. 2022). p-ca can stimulate hippocampal synaptic plasticity (lee et al. 2018) & promote hippocampal neurogenesis (he et al. 2021). p-ca has been explored for a variety of positive effects under different neuropathological alterations in both in vitro and in vivo studies (li et al. 2019; manyagasa and thong 2019; sakamula et al. 2019; he et al. 2021; oh et al. 2021; daroi et al. 2022). in a study, treatment with p-ca ameliorated hippocampal long-term potentiation (ltp) impairment, improved passive avoidance memory dysfunction, and hindered aβ plaque accumulation in the hippocampal dentate gyrus of alcl3treated rats (rashno et al. 2022). these points to the possibility that p-ca may offer promising therapeutic potential to improve cognitive decline in neurodegenerative disease like ad (rashno et al. 2022). naringin naringin is a citrus fruit flavonoid that may fight autophagic & oxidative stress in alcl3-induced ad. in a study, alcl3 caused memory impairment, but coadministration with naringin revealed a significant improvement. alcl3 also significantly increased lipid peroxidation and oxidative stress and decreased levels of reduced glutathione. naringin administration however ameliorated these neurochemical alterations. alcl3 also caused an increase in the immunohistochemical expression of microtubule assembly (tau protein) and oxidative stress (inos), the decreased the expression of the autophagic marker (lc3) in the cerebellum. all these were ameliorated by naringin (hassan et al. 2022). valeric acid valeric acid, a naturally occurring straight chain alkyl carboxylic acid found in valeriana officinalis has been used to treat neurological diseases (al-attraqchi et al. 2020; batista et al. 2023). valeric acid treatment increased ach levels in the hippocampus of ad rats. valeric acid also significantly increased gammaaminobutyric acid (gaba), dopamine, glutamate, and serotonin levels, thereby reversing alcl3-induced impairment (dulla et al. 2023). in another study, valeric acid treatment reduced the plasma level of aβ1–42 biomarker and improved memory by reversing the alcl3–induced impairment (dulla et al. 2021). baicalein baicalein, a flavonoid found in the roots of scutellaria lateriflora & scutellaria baicalensis, has a variety of biological functions, including anti-inflammatory & antioxidant (ren et al. 2021), cardioprotective (zhao et al. 2016), anticancer, & antiviral properties (cathcart et al. 2016). it also possesses neuroprotective properties (sowndhararajan et al. 2018) and inhibits ache (liao et al. 2022). in a study, the combination of baicalein and memantine significantly improved behavioural parameters. in addition to increasing bdnf expression, the combination reduced oxidative stress and aβ plaques formation. therefore, baicalein and memantine therapy may slow neurodegeneration progression in rats (jadhav and kulkarni 2023b). berberine many vegetable species including meadow rue (thalictrum), barberry (berberis), goldenseal (hydrastis canadensis l.), celandine (chelidonium), and phellodendron amurense, contain berberine, a phytogenous alkaloid (germán-acacio et al. 2020). berberine has been known to have therapeutic potential against ad. in ad rats, berberine significantly improved memory deficits, increased antioxidant enzyme levels, decreased ache activity, lowered pro-inflammatory cytokines level, and significantly downregulated the expression of predefined biomarkers. histological examination also showed that berberine can lower neuroinflammation & amyloid plaques in ad (akash et al. 2023). curcumin curcumin found in curcuma has been studied for its antioxidant, anti-inflammatory, anticancer, & cytoprotective properties (dhouib et al. 2017; alhusaini et al. 2019; abo-zaid et al. 2020). in cellular and animal models of neurodegenerative disorders, curcumin was demonstrated to offer neuroprotection by upregulating the transcription of nrf2 and suppressing the activation of nf-κb (liao et al. 2012; farkhondeh et al. 2021). a study found that curcumin administration increased the activities of antioxidant enzymes & the production of ojetunde – herbal medicines against alzheimer’s disease 21 anti-inflammatory cytokine, and decreases apoptotic cells in alcl3-indcued ad. additionally, hippocampal histopathology examination showed that curcumin may be able to decrease the hallmarks in alcl3-induced ad (elbini-dhouib et al. 2021). sesamol the anti-inflammatory activity of sesamol makes it a promising candidate to ameliorate neurotoxicity and neuroinflammation (sachdeva et al. 2015; castrogonzález et al. 2020). sesamol can help with cognitive impairment and anxiety and has neuroprotective properties. sesamol has been shown to prevent the accumulation of aβ, alter the microbiota in the stomach, & improve the output of microbial metabolites (yuan et al. 2019). sesamol prevents neurotoxicity caused by al via its antioxidant, anti-inflammatory, and anti-apoptotic effects (abou-zeid et al. 2021; du et al. 2022). in an alcl3-intoxicated rat study, rats treated with a combination of sesamol and lactobacillus plantarum (probiotic bacteria) showed markedly reduced levels of brain aβ, p-tau, gsk-3β, apoptotic, and inflammatory biomarkers, as well as markedly elevated levels of brain free β-catenin and wnt3a. also, this combination significantly increased hepatic ppar-γ expression while significantly reducing hepatic expressions of jak2/stat-3, inflammatory (il-6, tnf-α, nf-κb), fibrotic (mmp-2, timp-1, α-sma) and apoptotic markers, (caspase-3), compared to alcl3-intoxicated rats. the effectiveness of this combination in halting the effect of neurotoxicity was supported by behavioural and histopathological evaluations (abu-elfotuh et al. 2023). silibinin silibinin (silybin) is abundantly found in silymarin. silymarin is found in the fruits & seeds of silybum marianum (haddadi et al. 2020). alcl3-induced cognitive impairment, neurochemical anomalies, and histopathological alterations were significantly alleviated by treatment with silibinin-loaded nanostructured lipid carriers (sili-nlcs) (makhdoomi et al. 2022). this implies that sili-nlcs could potentially act as a neuroprotective agent against ad, as treatment with silinlcs is more effective than treatment with free silibinin in preventing the development of neurotoxicity caused by al (makhdoomi et al. 2022). isoimperatorin lemon and lime oils contain isoimperatorin, an active natural furanocoumarin (lai et al. 2021). isoimperatorin has anti-inflammatory effect as one of its pharmacological actions (wijerathne et al. 2017; chen et al. 2021). isoimperatorin significantly reduced the effect of alcl3 in a mouse model of ad via the modulation of antioxidant system, and regulation of inflammatory response by targeting nrf2 and mapk (rajendran et al. 2023). therefore, isoimperatorin may be a potential therapeutic option for neurotoxicity and neurodegenerative diseases which are associated with neuro-inflammation and oxidative stress, such as ad. crocin the primary and most potent active component in crocus sativus is crocin, often known as saffron and a member of iridaceae family. it has been established that crocin offer neuroprotective benefits. crocin has a unique, prophylactic effect against ethanol-induced damage to learning and memory. a study found that crocin protected against alcl3–induced neurodegenerative behavioural and biochemical alterations. it alleviated alcl3-induced memory impairment, and reduced oxidative stress and cholinergic dysfunction. thus, crocin may be a useful medication for the management of ad (tomar et al. 2023). betulin the betulaceae family, especially betula alba, b. platyphylla, b. pubescens, and b. pendula the richest source of betulin, a lupane-type compound that may be obtained from more than 200 plants species (hordyjewska et al. 2019). a new drug that is effective in both the prevention and treatment of ad may be developed from betulin due to its neuroprotective properties. in one study, rats with alcl3-induced ad exhibited improved spatial memory and lowered levels of tnf-α, aβ, and amyloid precursorlike protein 2 (aplp2) when administered betulin in complex with cyclodextrin (zakrzeska et al. 2023). palmatine palmatine, a naturally occurring protoberberine alkaloid, is present in coptis chinesis and corydalis yanhusuo. in a study, treatment with palmatine significantly regulated the levels of ache levels and glutamate, improved the expression of bdnf, and lowered excitotoxic damage and the expression of il-6 and tnf-α, induced by al. additionally, palmatine prevented neuronal damage degeneration and loss and restored healthy, viable neurons in ad (baburaj et al. 2023). malvidin in animal cell line and in vivo models, malvidin, an anthocyanin derived from red wine, has been shown to offer protection against oxidative neuronal damage. it is used in the treatment of variety of ailments due to its antioxidant properties. malvidin targets mapk and nfκb pathways, these contribute to its antioxidant, antiinflammatory, and anti-apoptotic actions (hou et al. 2004). it has been demonstrated that malvidin reduced alcl3–induced behavioural impairment. oral treatment of malvidin also demonstrated neuroprotective effects via the regulation of antioxidant levels and neuroinflammation and inhibition of ache activity alcl3-exposed rats. malvidin may therefore be a potential drug for the treatment of ad (gilani et al. 2022). 22 biology, medicine, & natural product chemistry 13 (1), 2024: 7-33 table 2. neuroprotective and therapeutic effects of natural products against aluminium chloride-induced alzheimer’s disease. natural product dose of alcl3 dose of natural product animals used mechanism of action ref morin (mor), thymol (tml), and thymoquinone (tmq) 70 mg/kg i.p. daily for 5 weeks 20 mg/kg of mor orally, tml (30 mg/kg), 10 mg/kg of tmq, and phm rats antioxidant, antiinflammatory, antiapoptotic, and improved learning & memory (hamdan et al. 2022) niruriflavone 100 mg/kg of alcl3 for 42 days. 0.125 mg/kg b.wt. from the 42nd day to the 60th day by oral route rats antioxidant, anticholinesterase, & reversed neurobehavioral changes (rajamanickam and sl 2022) quercetin 50 mg/kg for 28 days orally 25 or 50 mg/kg orally for 28 days after ad induction rats anti-aβ, attenuated behavioural & neurotransmission impairments (elfiky et al. 2021) quercetin 50 mg/kg b.wt. i.p. for 60 days 50 mg/kg b.wt. by gastric intubation (1h prior to alcl3) rats modulated gene expression, & improved short-term memory (elreedy et al. 2023) resveratrol-tannic acid 100 mg/kg/day p.o. for 90 days resveratrol (20 mg/kg/day p.o.) and tannic acid (50 mg/kg/day p.o.) rats antioxidant, anti-aβ, & attenuated cognitive impairment (bhounsule and bhatt 2023) thymoquinone (tq), & celastrol 10 mg/ kg/day i.p. for 6 weeks 10mg/kg/day tq i.p. & 1mg/kg/day celastrol i.p. for 6 weeks rats antioxidant, antiinflammatory, modulated neurotransmitters levels, & improved cognitive impairments and brain bdnf expression (abbas et al. 2022) betalain 100 mg/kg b.wt. for 28 days orally 10 or 20 mg/kg b.wt. for 28 days (1hr prior to alcl3) rats antioxidant, anticholinesterase, anti-inflammatory, & suppressed learning impairments (shunan et al. 2021) quercetin + memantine 100 mg/kg orally for 42 days memantine (20mg/kg), quercetin (50mg/kg), memantine & quercetin (20 + 25mg/kg), or memantine & quercetin (20 + 50mg/kg) orally for 42 days rats antioxidant, anti-aβ, anticholinesterase, improved learning and memory, & bdnf expression (jadhav and kulkarni 2023a) vinpocetine + epigallocatechin-3-gallate 100 mg/kg b.wt. i.p for 4 weeks vinpocetine (20 mg/kg, p.o) + egcg (10 mg/kg i.p) for 4 weeks rats antioxidant, antiinflammatory, anticholinesterase, anti-aβ, & regulated monoamines & bdnf levels (ali et al. 2022) β-sitosterol 10 mg/kg for 14 days 25 mg/kg for 21 days mice antioxidant, anti-aβ, & mitigated cognitive impairment (yadav et al. 2023) gallic acid or hesperidin 100 mg/kg/day via oral gavage for 21 days 100 mg/kg gallic acid or 100 mg/kg hesperidin for 21 days rats antioxidant, anticholinesterase, anti-inflammatory, prevented cognitive impairment & improved neurotransmitters levels (ekundayo et al. 2022) ojetunde – herbal medicines against alzheimer’s disease 23 table 2. cont. natural product dose of alcl3 dose of natural product animals used mechanism of action ref gallic acid + donepezil 100 mg/kg via oral gavage for 60 days 10 mg/kg donepezil + 50 mg/kg gallic acid via oral gavage for 60 days rats antioxidant, & anticholinesterase (obafemi et al. 2021) ginsenoside rb1 50 mg/kg/day s.c. for 8 weeks 70 mg/kg/day orally for 8 weeks (1hr before alcl3) mice antioxidant, antiapoptotic, anti-aβ, improved memory impairment, mitigated accumulation of phosphorylated tau protein, & attenuated histopathological changes. (shalaby et al. 2023) ononin 175 mg/kg orally for 25 days 30 mg/kg orally from the 25th day to 36th day (after alcl3 for 25 days) rats alleviated cognitive impairment, antioxidant, antineuroinflammatory, & restored brain histological structure. (chen et al. 2021) asiatic acid 100 mg/kg b.wt. orally for 8 weeks 75 mg/kg b.wt. orally for 8 weeks rats antioxidant, anticholinesterase, mitigated neuronal loss, & attenuated reactive astrogliosis (suryavanshi et al. 2022) p-coumaric acid 100 mg/kg/day p.o. 100 mg/kg/day p.o. (1hr prior to alcl3 administration) rats improved memory impairment, alleviated ltp impairment, & anti-aβ (rashno et al. 2022) naringin 100 mg/kg/day p.o. for 21 days 100 mg/kg/day p.o. for 21 days rats behavioural, neurochemical, immunohistochemical, and molecular modulation (hassan et al. 2022) valeric acid 100 mg/kg b.wt. orally for 42 days 50 mg/kg b.wt. orally on the 47th day for 30 days rats regulated neurotransmitters level (dulla et al. 2023) baicalein + memantine 100 mg/kg for 42 days memantine (20 mg/kg), baicalein (10 mg/kg), memantine & baicalein (20 + 5mg/kg), or memantine & baicalein (20 + 10mg/kg) orally for 42 days rats behavioural improvement, antioxidant, anti-aβ, & increased bdnf (jadhav and kulkarni 2023b) berberine 300 mg/kg orally for 21 days 50mg/kg of berberine-enriched extract & 50 mg/kg of pure berberine orally for 21 days rats improved memory, antioxidant, antiinflammatory, anticholinesterase (akash et al. 2023) valeric acid 100 mg/kg b.wt. for 42 days orally 50 mg/kg b.wt. for 30 days after 42 days of alcl3 administration memory improvement, anti-aβ (dulla et al. 2021) resveratrol-selenium nanoparticles 100 mg/kg/day for 60 days 200 mg/kg/day via gavage for 8 weeks rats antioxidant, antiinflammatory, anti-aβ, and improved cholinergic deficits abozaid et al. 2022 24 biology, medicine, & natural product chemistry 13 (1), 2024: 7-33 table 2. cont. natural product dose of alcl3 dose of natural product animals used mechanism of action ref resveratrol-selenium nanoparticles 300 mg/kg/day orally for 30 days 200 mg/kg for 3 weeks after alcl3 administration for 30 days rats antioxidant, and modulated neurotransmitter level (abozaid et al. 2021) curcumin 100 mg/kg b.wt. via oral gavage 100 mg/kg b.wt. via oral gavage rats improved behavioral impairments, antioxidant, and antiinflammation (elbini-dhouib et al. 2021) sesamol 70 mg/kg/day i.p. for 5 weeks sesamol (50mg/kg/day p.o.) l. plantarum (1 × 106 cfu/day p.o.) for 5 weeks rats prevented cognitive dysfunction, antiinflammatory, antiapoptotic (abu-elfotuh et al. 2023) silibinin 100 mg/kg/day p.o. 50, 100, or 200 mg/kg/day p.o. for 30 days mice antioxidant, ameliorated cognitive impairments and histological changes (makhdoomi et al. 2022) quercetin nanoemulsion 100 mg/kg b.wt./day orally for 30 days 15 mg/kg b.wt./day i.p. for 30 days rats antioxidant, antiinflammatory, modulated neurotransmitters, and mended histopathological changes (alaqeel et al. 2022) isoimperatorin 10 mg/wt/day orally 30 mg/wt/day i.p mice antioxidant, antiinflammatory, and modulated behavioural and neurotransmitters deficit (rajendran et al. 2023) crocin 100 mg/kg orally for 42 days 15 or 30 mg/kg orally for 42 days rats antioxidant, anticholinesterase, and improved memory impairment (tomar et al. 2023) betulin 200 mg/kg/day 100 mg/kg/day, intragastrically during the last 50% of the experimental days rats improved spatial memory, antiinflammatory, & antiaβ (zakrzeska et al. 2023) palmatine 100 mg/kg p.o. for 42 days 10 or 20 mg/kg p.o. for 42 days rats anticholinesterase, anti-inflammatory, lowered excitotoxic damage, & improved bdnf (baburaj et al. 2023) malvidin 50mg/kg b.wt./day i.p. for 60 days 100 or 200 mg/kg p.o. (1hr prior to alcl3 injection) for 60 days rats antioxidant, antiinflammatory, anticholinesterase, & downregulated memory impairment. (gilani et al. 2022) note: alcl3: aluminium chloride; aβ: amyloid-beta; ad: alzheimer’s disease; bdnf: brain-derived neurotrophic factor b.wt.: body weight; i.p.: intraperitoneally; p.o.: oral administration; s.c.: subcutaneous conclusions a growing body of evidence suggests that exposure to alcl3 and its neurotoxicity may play a role in a variety of neurodevelopmental & neurodegenerative disorders. alcl3 neurotoxicity has been linked to oxidative stress, mitochondrial dysfunction, inflammation, accumulation of aβ plaques and nft, & alteration of synaptic plasticity and signal transduction due to interference with neurotransmitter systems. treatment of alcl3 neurotoxicity and associated diseases like ad may benefit from targeting these mechanisms at different stages. mechanistic studies are currently being conducted to validate and promote the use of traditional medicines in animal models. the majority of the medicinal plants and natural products discussed in this review have been shown to have neuroprotective, antioxidant, anti ojetunde – herbal medicines against alzheimer’s disease 25 amyloid, anti-inflammatory, anticholinesterase, antiapoptotic, and therapeutic actions. nevertheless, the majority of the plant herbs have not yet been isolated, and further research on these natural products needs to be conducted, which is an intriguing feature in the treatment of ad. all currently available drugs of ad are utilized to treat the symptoms of the disease. hence, there is an urgent need for the development of new drugs with novel targets that can also prevent the progression of the disease at an early stage, thereby improving the quality of life of ad patients. the insight in this review will undoubtedly help researchers to design compounds that have a significant impact in curing ad. recommendation a promising path for ad care is the identification of preventive medicines derived from conventional herbal medication. it is likely to be beneficial to screen herbal medicine for lead compounds based on their physicochemical characteristics and projected bbb properties in order to discover new treatment for ad. future research can focus more on natural compounds that can cross the bbb, have wide therapeutic time windows, clear pharmacological goals, & fewer side effects. however, it is challenging to trace the pharmacological effects of a plant or plant extract to a single component or class of chemicals since the purported protective and therapeutic characteristics of herbal medicine are typically the result of the synergistic actions of several compounds. there are also critical factors to take into account, like interactions with already-available drugs. competing interests: the author declares that there are no competing interests. funding: the author declares no funding. references aalikhani, m., safdari, y., jahanshahi, m., alikhani, m., & khalili, m. 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october 2021 | pages: 73-79 | doi: 10.14421/biomedich.2021.102.73-79 issn 2540-9328 (online) effect of diabetes mellitus and hypertension on osmotic fragility and hemorheological factors in male wistar rats david ehikhuemen okonofua1, jerome ndudi asiwe1,2,*, kenneth kelechi anachuna3, emuesiri goodies moke4, kamaldeen olalekan sanusi1, ebunoluwa oluwabusola adagbada1, mariam onono yusuf1, damilola ifeoluwa alawode1, adesoji adedipe fasanmade1 1cardiorespiratory research unit, department of physiology, college of medicine, university of ibadan, ibadan, nigeria. 2department of physiology, pamo university of medical sciences, port-harcourt, nigeria. 3department of physiology, faculty of basic medical sciences, delta state university, abraka, nigeria. 4department of pharmacology and therapeutics, faculty of basic medical sciences, delta state university, abraka, nigeria. corresponding author* asiwejerome@yahoo.com; tel: +2348163727468 manuscript received: 20 july 2021. revision accepted: 29 july, 2021. published: 01 october, 2021. abstract diabetes mellitus is a common risk factor for erythrocyte osmotic stress. this study was aimed at exploring the effect of streptozotocin (stz)-induced diabetes mellitus and salt-induced hypertension on osmotic fragility and hemorheological variables in male wistar rats. thirty male rats were grouped into five groups of six animals each as follows: negative control (zero salt in diet); positive control (normal salt diet 0.3% salt); high salt diet (8% salt) (hsd only); stz induced diabetes and normal salt diet (stz only); stz induced diabetes and high salt diet (stz + hsd). at the end of a 4 weeks period, hematological variables, osmotic fragility, rheology and cardiovascular responses were assessed. there was an increase (p<0.05) in the mean arterial pressure and heart rate of hsd, stz and hsd + stz groups indicating a salt induced hypertension. there was a decrease in the body weight of stz and hsd +stz groups. there was significant increase (p<0.05) in the haematocrit, platelets estimates and fibrinogen concentrations in the experimental groups when compared with the controls. the stz and stz + hsd groups showed a reduced clotting time which corresponded to the increased platelet estimates and fibrinogen concentration. the increase in haematocrit, platelet and plasma protein resulted in the increased blood viscosity and a decreased flow rate. the osmotic fragility test was also observed to be increased (p<0.05) in hsd, stz only and stz + hsd groups. diabetes mellitus and hypertension increase the rate of hemolysis of erythrocyte, as well as increase blood viscosity. keywords: diabetes mellitus; hemorheology; high salt diet; hypertension; viscosity. abbreviations: bp – blood pressure; dm – diabetes mellitus; hr – heart rate; hsd – high salt diet; nacl – sodium chloride; rpv – relative plasma viscosity; sbp – systolic blood pressure; stz – streptozotocin; wbv – whole blood viscosity. introduction diabetes mellitus (dm) is a chronic metabolic disorder in the endocrine system marked by abnormalities in insulin secretion and/or insulin action that leads to the progressive deterioration of glucose tolerance, which causes hyperglycemia (granner, 2000). in postmodern times, dm has now become a trending public health problem which calls for serious care and concern (harika et al., 2014). various medical therapies are current being used in management of diabetes (patel et al., 2010; chaudhury et al., 2017; palanisamy et al., 2018; okafo et al., 2019). dm is one of the major risk factors for increased osmotic fragility. this is because hyperglycemia causes structural and functional changes in erythrocytes which can result in osmotic stress (eze et al., 2017). according to the american heart association, high salt consumption is a serious essential/primary hypertension cause and risk factor. go reported that an average american adult consumes about 5400 mg of sodium per day which is about 130% increase more than the recommended 2300 mg of sodium per day (go et al., 2013). this high salt intake per day is a major risk factor for essential hypertension. the rate of erythrocyte osmotic fragility increase in hypertensive subjects is higher than in normotensive subjects (fasanmade, 1999). high salt intake which induces hypertension will cause an increase in the osmotic fragility of red blood corpuscle (baskurt and meiselman, 2003). high salt diets have been greatly linked to be a serious risk factor for cardiovascular diseases relating to high blood pressure, endothelial dysfunction, stroke, ventricular hypertrophy and fibrosis, arterial and ventricular stiffening, myocardial infarction, arrhythmias, and heart failure (mohan and campbell, 2009) while diabetes mellitus is a common risk factor for erythrocyte osmotic stress (eze et al., 2017). this suggests that the https://doi.org/10.14421/biomedich.2021.102.73-79 74 biology, medicine, & natural product chemistry 10 (2), 2021: 73-79 constituents of blood and its flow properties serve as a link between dm and hypertension. hemorheology is the study of the flow properties of blood and its elements (plasma and formed elements, including erythrocytes, white blood cells and platelets) baskurt et al., 2007). hemorheological parameters, such as hematocrit, plasma and whole blood viscosity, plasma proteins, erythrocyte deformability and aggregation are basic characteristics of blood flow (rabai, 2013). there is growing evidence that the flow properties of blood are among the main factors of proper tissue perfusion, and shifts in these properties play significant roles in disease processes (mohan et al., 2001). the viscosity of blood is directly proportional to the hemoconcentration and inversely proportional to the flow rate. this implies that factors which increase blood constituents and decrease plasma will elevate the blood viscosity which will decrease flow rate and alter tissue perfusion (chang et al., 2017; sloop et al., 2020). there are adequate evidences on diabetes that the elevated blood viscosity is a pathogenetic factor of diabetic microangiopathy, altering microcirculation and leading to insufficient tissue nutrition (grigoleit et al., 1973; cho et al., 2008). alterations in tissue perfusion will result in microvascular and macrovascular complications (cade et al., 2008). hence, this study provides an investigation into the rheological properties of blood in diabetic and hypertensive male wistar rats. materials and methods experimental design a total of thirty male wistar rats (120-150 g) were used for the experiment. they were acclimatized for two weeks prior to commencement of the experiment at the central animal house of the college of medicine, university of ibadan, nigeria, and kept under standard laboratory conditions and fed standard rat pellets and water ad libitum. animal handling was done in accordance to established guidelines by the national institute of health for care and use of laboratory animals. ethical approval was given by the college of medicine ethics committee (ui-acurec/19/0140). the rats were grouped into five groups of six animals each as follows: negative control (zero salt in diet); positive control (normal salt diet 0.3% salt); high salt diet (8% salt) (hsd only); stz induced diabetes and normal salt diet (stz only); stz induced diabetes and high salt diet (stz + hsd). preparation of feed rations three different rations were used for the study. the high salt diet was prepared by mixing 8g of table salt with 92 g of standard rat chow (asiwe et al., 2020). hsd only and stz +hsd groups were given this ration and water ad libitum. the normal salt diet was prepared by mixing 0.30 g of table salt with 99.70 g of standard rat chow which was given to the positive control group and water ad libitum. while the zero-salt diet was given to the negative control group. induction of diabetes mellitus diabetes was induced by single intraperitoneal injection of 60 mg/kg body weight dose of streptozotocin (stz) dissolved in freshly prepared 0.1m cold citrate buffer of ph 4.5 into the animals according to the stz-induced hyperglycemia in rats’ model (eze et al., 2017; asiwe et al., 2020; akbarzadeh et al., 2007). the experimental animals were fasted overnight (18 hours) prior to diabetes induction while allowed access to drinking water. seventytwo hours after streptozotocin injection, a drop of blood was drawn from tail vein of the rats to measure their blood glucose level. animals having fasting blood glucose levels ≥ 200 mg/dl were considered diabetic and used in the study. measurement of blood glucose level in order to ascertain the diabetic state of the animals, the fasting blood sugar level was measured every week after being fasted overnight using the accu-chek active glucometer (model gb06140695). the results obtained in mmol/l were converted to mg/dl by multiplying with 18 (conversion factor). blood samples were collected from tail artery of the rats for evaluation of the blood glucose. body weights of animals were also measured weekly in grams to estimate the effect of the induced diabetes and high salt diet on body composition. the weight was measured with a standard weighing scale. blood pressure and heart rate blood pressure and heart rate were done at the small animal ward of the veterinary clinic university of ibadan, ibadan, nigeria. systolic blood pressure (sbp) was measured indirectly in a conscious and slightly restrained rat using the tail cuff plethysmography method (kent scientific, usa). heart rate (hr) tracings were observed and recorded as obtained during blood pressure (bp) measurement. for these measurements, rats were conditioned to the restraint (cone) and the warming chamber for about 20 minutes before the measurement. sbp and hr measurements were performed in a very quiet environment to avoid sound interference by the same investigator. there were two sensors to measure bp and vascular peripheral resistance. after stabilization in the chamber, an acclimatization run was performed for 5 cycles which was immediately followed by the typical run involving 10 repetitions of the automated inflation-deflation cycle. examination of blood samples blood samples were collected by ocular puncture at the end of the fourth week and stored in heparinized bottles which were used to consider effect of high salt diet and okonofua et al. – effect of diabetes mellitus and hypertension on … 75 diabetes mellitus on hematology, osmotic fragility and rheology. four animals were used for each group for analysis (n=4). rheological analysis whole blood viscosity and relative plasma viscosity were estimated using the method described by reid and ugwu (1987), and the flow rate was calculated. plasma fibrinogen concentration was estimated by clot weight method of ingram (1952). haematocrit was estimated using microhaematocrit reader. statistical analysis data were analyzed with graphpad prism version 7.0 (graphpad software, san diego, ca) and expressed as means ± sem (standard error of mean). one-way anova was used for comparisons, followed by post hoc newman-keuls multiple comparison test. p < 0.05 was considered statistically significant. results and discussion osmotic fragility figure 1 shows the rate of erythrocyte osmotic fragility (%). the percentage erythrocyte osmotic fragility decreased significantly with an increase in sodium chloride (nacl) concentration. there was complete (100%) haemolysis at 0.0% of nacl. and there were no significant changes in erythrocyte osmotic fragility when observed at 0.0% and 0.1% of nacl concentration in all control and experimental groups when compared. however, significant (p < 0.05) changes in percentage erythrocyte fragility were recorded at 0.3%, 0.5%, 0.7% and 0.9% of nacl concentrations, when the experimental groups were compared with the control groups. *a, *b, *c, and *d p<0.05 at 0.3%, 0.5%, 0.7% and 0.9% of nacl concentrations. platelet counts figure 2 shows the platelets estimates (mm3) in diabetic and hypertensive male wistar rats. there was significant increase when high salt diet + stz groups were compared with the controls. p ˂ 0.05 is significant when compared with negative control and positive control groups. fibrinogen estimates figure 3 shows the fibrinogen estimates (mm3) in diabetic and hypertensive male wistar rats. there was significant increase when other groups were compared with the controls. *p ˂ 0.05 is significant when compared with negative control and positive control groups. hematocrit figure 4 shows the hematocrit (%) in diabetic and hypertensive male wistar rats. there was significant increase when other groups were compared with the controls. *p ˂ 0.05 is significant when compared with negative control and positive control groups. whole blood viscosity figure 5 shows the whole blood viscosity (wbv) (mpa.s) in diabetic and hypertensive male wistar rats. from the values expressed there was significant increase between the negative control group and positive control group. there was significant increase when other groups were compared with the controls. **p ˂ 0.05 is significant when the negative control group was compared with the positive control group; *p ˂ 0.05 is significant when compared with negative control and positive control groups. relative plasma viscosity figure 6 shows the relative plasma viscosity (rpv) (mpa.s) in diabetic and hypertensive male wistar rats. from the values expressed there was significant increase between the negative control group and positive control group. there was significant increase when other groups were compared with the controls. **p ˂ 0.05 is significant when the negative control group was compared with the positive control group; *p ˂ 0.05 is significant when compared with negative control and positive control groups. plasma flow rate figure 7 shows the plasma flow rate (cm/sec) in diabetic and hypertensive male wistar rats. there was significant increase between the negative control and positive control groups. there was significant increase when other groups were compared with the controls. **p ˂ 0.05 is significant when the negative control group was compared with the positive control group; *p ˂ 0.05 is significant when compared with negative control and positive control groups. 0 0 .1 0 .3 0 .5 0 .7 0 .9 0 2 0 4 0 6 0 8 0 1 0 0 n a c l c o n c e n tra tio n (m g /m l) p e r c e n ta g e h e m o ly s is ( % ) n e g a tiv e c o n tro l g ro u p p o s itiv e c o n tro l g ro u p h s d o n ly s t z o n ly s t z + h s d* * * * * * * * * * * * figure 1. rate of erythrocyte osmotic fragility (%) in diabetic and hypertensive male wistar rats. 76 biology, medicine, & natural product chemistry 10 (2), 2021: 73-79 a b c d e 0 5 0 0 0 0 1 0 0 0 0 0 1 5 0 0 0 0 2 0 0 0 0 0 p la te le t c o u n t (m m 3 ) * * n e g a tiv e c o n tro l p o s it iv e c o n tro l h ig h s a lt d ie t s tre p to z o to c in h ig h s a lt d ie t + s tre p to z o to c in figure 2. platelet count (mm3) in diabetic and hypertensive male wistar rats. a b c d e 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 f ib r in o g e n ( m m 3 ) * * n e g a tiv e c o n tro l p o s it iv e c o n tro l h ig h s a lt d ie t s tre p to z o to c in h ig h s a lt d ie t + s tre p to z o to c in figure 3. fibrinogen estimates (mm3) in diabetic and hypertensive male wistar rats. a b c d e 0 1 0 2 0 3 0 4 0 5 0 p a c k e d c e ll v o lu m e ( % ) * ** n e g a tiv e c o n tro l p o s it iv e c o n tro l h ig h s a lt d ie t s tre p to z o to c in h ig h s a lt d ie t + s tre p to z o to c in figure 4. hematocrit (%) in diabetic and hypertensive male wistar rats. a b c d e 0 2 4 6 8 w h o le b lo o d v is c o s it y ( m p a .s ) n e g a tiv e c o n tro l p o s it iv e c o n tro l h ig h s a lt d ie t s tre p to z o to c in h ig h s a lt d ie t + s tre p to z o to c in * * * * * figure 5. whole blood viscosity (mpa.s) in diabetic and hypertensive male wistar rats. a b c d e 0 1 2 3 4 5 r e la ti v e p la s m a v is c o s it y ( m p a .s ) n e g a tiv e c o n tro l p o s it iv e c o n tro l h ig h s a lt d ie t s tre p to z o to c in h ig h s a lt d ie t + s tre p to z o to c in * * * * * figure 6. relative plasma viscosity (mpa.s) in diabetic and hypertensive male wistar rats. a b c d e 0 5 1 0 1 5 p la s m a f lo w r a te ( c m /s e c ) n e g a tiv e c o n tro l p o s it iv e c o n tro l h ig h s a lt d ie t s tre p to z o to c in h ig h s a lt d ie t + s tre p to z o to c in * * * * * figure 7. plasma flow rate (cm/sec) in diabetic and hypertensive male wistar rats. in this study, the percentage erythrocyte osmotic fragility decreased significantly with increasing nacl concentration. there was complete (100%) hemolysis at 0.0% and 0.1% of nacl. no significant changes in erythrocyte osmotic fragility were observed at 0.0% and 0.1% of nacl (distilled water) in both controls and experimental groups when compared. however, the results of the osmotic fragility showed a significant increase in the rate of erythrocyte osmotic fragility at 0.3%, 0.5%, 0.7% and 0.9% of nacl concentrations in hsd only, stz only and hsd +stz groups, when compared with the control groups. this finding was in agreement with the works of other researchers (shin et al., 2007; megha and sehyun, 2009; eze et al, 2017) who showed that erythrocyte osmotic fragility was significantly increased in streptozotocin-induced diabetic animals. the increased rate of erythrocyte osmotic fragility in hypertensive subjects has also been reported by fasanmade (1999). the implication of this finding suggests that stz induced diabetes can alter the integrity of the red blood cell (rbc) membrane. erythrocyte osmotic fragility test gives an in-vitro measure of the tensile strength of erythrocyte membrane and it is an indirect method of evaluating lipid peroxidation in animals (akande et al., 2014). it is also used to measure the tensile strength of erythrocytes and its ability to resist alterations in osmotic gradients and it has been discovered to be increased during oxidative stress (aldrich et al., 2006; adenkola et al., 2010). hence, this suggests that the greater the erythrocyte osmotic fragility, the weaker the tensile strength of erythrocyte membrane (ogundeji et al, 2013) and a resultant hemolysis of erythrocyte is observed. the mechanism for increased fragility of erythrocytes have been reported to be due to increased glycosylation of the erythrocyte membrane protein or/and alteration of the na+/k+ atpase on the erythrocyte membrane (arun, 2013). the viability of an erythrocyte depends greatly on the maintenance of its membrane. studies have shown that dm and hypertension can cause an increase in lipoperoxidative changes in the membranes of erythrocytes (qujeq et al., 2005; ahmed et al., 2006; gauri and vijaya, 2008) which account for the increase in the rate of osmotic fragility. hemorheology is the study of the flow properties of blood and its elements such as plasma with its dissolved okonofua et al. – effect of diabetes mellitus and hypertension on … 77 components and formed elements, which include erythrocytes, leucocytes and platelets (baskurt et al., 2007). the flow properties of blood are among the main determinants of proper tissue perfusion, and alterations in these properties play vital roles in disease processes (lowe et al., 1980). in this study, hemorheological factors were measured with focus on blood viscosity and flow rate. the viscosity of blood is determined by the hematocrit (rabai, 2013; cho et al., 2008), increase in plasma viscosity which is determined by plasma proteins (fibrinogen and globulin) and water (mohan et al., 2001). from this study, there was a significant increase in the blood viscosity and plasma viscosity in the hsd only, stz only and stz+hsd groups when compared with the control groups. the observed increase in viscosity could be due to the significant increase in hematocrit, fibrinogen, and platelet estimates as recorded in stz only and stz+hsd groups. this finding was in accordance with the works of reid and memeh, and others (reid and memeh, 1988; khan et al., 2005; okomafe et al., 2017). increased blood viscosity and the onset of diabetic angiopathy have been related to abnormal hematocrit, plasma viscosity, fibrinogen concentration, erythrocyte deformability and rouleaux formation (winberger and baskurt, 2007). the increase in hematocrit in the experimental groups appears to have contradicted the increased rate of erythrocyte osmotic fragility as reported earlier in this study. a possible mechanism to support this finding is linked to hemoconcentration. as the osmolarity of the blood increases due to increased glucose level, the capillary permeability increases, resulting in a decrease in plasma water and thus increasing hematocrit and subsequently the blood viscosity (meiselman et al., 1967; rizvi and zaid, 2001). however, hsd only group had a significant increase in hematocrit only with a decrease in fibrinogen and platelet estimates when compared with other experimental groups. this increase in hematocrit could be the major reason behind the increase in blood viscosity (macrury et al., 1988). another interesting finding in this study was the significant increase in blood and plasma viscosity when the positive control group with normal salt intake was compared with the salt deficient negative control group. the possible mechanism underlying this claim is yet to be fully understood. the flow rate of blood is determined by three main factors – vessel diameter, perfusion pressure and blood viscosity (lowe et al., 1980). although vessel diameter has been primarily linked with blood flow rate, in pathological conditions, the contributions of perfusion pressure and viscosity becomes greatly enhanced. it has been reported from studies that the viscosity of blood is directly proportional to the hemoconcentration and inversely proportional to the flow rate (sloop et al., 2020). this implies that factors which increase blood constituents and decrease plasma water will elevate the viscosity of blood which will decrease flow rate and alter tissue perfusion (chang et al., 2017; sloop et al., 2020). from this study, it was also observed that there was a corresponding significant decrease in the flow rate of plasma across the experimental groups when compared with the control groups. hence, the increase in blood viscosity could be a factor for the decreased flow rate of plasma. the alterations in the blood flow patterns in dm is produced by a combination of reduced erythrocyte deformability and increased erythrocyte aggregation due to variations in the plasma protein (mcmillian et al., 1978). the changes in plasma proteins are linked with the development of glucose intolerance (mcmillian, 1983). a decrease in flow rate can possibly result in circulatory insufficiency marked with poor tissue perfusion which in a long term can lead to the development of vascular complications as commonly observed in subjects with dm (lowe et al., 1980). there was a significant decrease in plasma flow rate when the positive control group with normal salt intake (with higher blood and plasma viscosity) was compared with the salt deficient negative control group. this also verified the inverse relationship between blood viscosity and plasma flow rate. conclusion from the findings of this study, it can be stated that diabetes mellitus and hypertension increase the rate of hemolysis of erythrocyte. there is also an increase in the hematocrit which significantly increases the blood viscosity alongside with plasma protein and decreased plasma water, thus, decreasing plasma flow rate. this could possibly result in circulatory insufficiency as well as a poor tissue perfusion which will lead to vascular complications. conflict of interest: the authors declare that there are no conflicts of interest concerning 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(0341) 551932, indonesia. corresponding author* gatra.ervi@unisma.ac.id manuscript received: 21 august, 2025. revision accepted: 14 october, 2025. published: 26 november, 2025. abstract soursop leaves (annona muricata) are known as a plant that has the potential to treat breast cancer, which has a high mortality rate in women in indonesia. the receptor that plays the most important role in breast cancer development is the estrogen receptor alpha (erα). this study aims to identify active compounds in soursop leaves that have the potential to inhibit breast cancer cell growth through in silico analysis using molecular docking of the erα receptor using knapsack, pass online, pubchem, pdb id, pyrx, and discovery studio software and histochemical analysis was performed using fresh soursop leaves by making an incision on the lower part of the leaf and adding reagents alcl, fecl, na2co, cuso4, glycerin, wagner reagent, immersion oil, and ethanol, then observed microscopically. the in silico results showed the presence of specific compounds from soursop leaves and one reference compound (oht600). cis-solamin had the highest binding affinity (-9.3 kcal/mol), better than the native ligand (-7.6 kcal/mol), and interacted with the amino acid glutamine 532 (glu532). histochemical analysis showed the content of flavonoids, phenols, tannins, terpenoids, and alkaloids, accompanied by color changes in trichome and stomatal tissues. based on the research data, it was concluded that soursop leaves can inhibit the growth of breast cancer cells through the content of secondary metabolites that actively inhibit erα receptors. keywords: breast cancer; erα; histochemistry; in silico; soursop leaf. introduction according to the 2010 pathological based registration in indonesia, breast cancer is the most common type of cancer suffered by indonesian women, with a relative frequency of 18.6%, and carries a high risk of mortality. in 2022, according to the world health organization (who), nearly 10 million deaths were caused by cancer, making it the leading cause of death in 2020, with 685,000 of these deaths being caused by breast cancer. breast cancer is generally treated with surgery to remove cancerous tissue. however, this still has the potential for new cancerous tissue to grow if the removal is not perfect. furthermore, chemotherapy and radiotherapy can also be used to kill cancer cells, but normal cells can also be damaged (pertiwi, 2020). chemotherapy will not only affect cancer cells but also other body cells that share characteristics with tumor cells with high cell division rates, such as hair follicles, bone marrow, gastrointestinal tract cells, and reproductive cells, making these cells the most affected by the cytotoxic effects of chemotherapy (prieto et al., 2020). to overcome the side effects of chemotherapy, a combination of natural bioactive compounds can be used (dewayani et al, 2023). phytochemical studies on soursop leaves (annona muricata) indicate that they are a major source of ethnomedicinal compounds used to suppress tumor and cancer growth. furthermore, soursop leaf extract has a cytotoxic effect on lung carcinoma cells, pancreatic cancer cells, and breast cancer cells, which has been demonstrated in vivo in animal models (dewayani et al., 2023). to determine the medicinal uses of active compounds in plant tissue, in silico analysis using molecular docking is necessary. computational molecular docking aims to predict how a compound or ligand will interact with a particular protein by calculating its binding affinity (asari et al., 2023). in silico analysis using the molecular docking method uses the alpha estrogen receptor, which is commonly found in breast cancer cells and is the receptor that plays the most important role in breast cancer progression (mubarakati et al., 2019). in addition to in silico analysis, this can also be confirmed by histochemical laboratory analysis. the purpose of histochemical analysis is to identify bioactive compounds found in plant tissue (nurhasanah et al., 2019). the tissue used for detection is the epidermis, which functions to protect the cells within it and store secondary metabolite compounds (maghfiroh et al., 2018). https://doi.org/10.14421/biomedich.2025.142.1055-1068 1056 biology, medicine, & natural product chemistry 14 (2), 2025: 1055-1068 given the abundant potential as a traditional herbal medicine found in the soursop plant is needed, particularly to identify the active compounds in the leaves of the soursop plant that can be used as cancerfighting agents. therefore, to determine the bioactivity of these compounds against breast cancer, in silico analysis using molecular docking is necessary. furthermore, histochemical analysis can also be performed to determine the secondary metabolite content in soursop leaves. this study aims to identify specific compounds in soursop leaves, then analyze the interaction of these active compounds with the estrogen receptor alpha using molecular docking and identify secondary metabolites in soursop leaves. this research is expected to serve as a basis for further research and as a reference for biotechnology development and preserving medicinal plants, particularly soursop (annona muricata). materials and methods materials this research was conducted at the integrated laboratory of universitas islam malang and was conducted from december 2023 to february 2024. the tools used were: beakers, petri dishes, measuring cups, clamps, dropper pipettes, tweezers, object glass, cover glass, test tubes, test tube racks, stationery, labels, cutters, cameras, microscopes, and a set of computers with several software namely, pubchem, pass online, protein data bank (pdb id), knapsack, pyrx, discovery studio visualizer. in addition, the materials used were: fresh soursop leaves (annona muricata) from the 5th node, taken from the batu city area, glycerin, wagner reagent, na2co3, a 5% cuso4 solution, an alcl3 solution, a 10% fecl3 solution, 85% ethanol, 70% alcohol, and immersion oil, for molecular docking using a receptor with the pdb code 3ert downloaded through rscb pdb and several active compound ligands downloaded through pubchem. methods this research employed a quantitative descriptive research method. the data obtained were analyzed using in silico and qualitative methods to determine the results of the histochemical analysis. in silico analysis was carried out using several software, including pubchem, pass online, protein data bank (pdb id), knapsack, pyrx, and discovery studio visualizer. meanwhile, histochemical analysis was performed using various specialised reagents according to the type of analysis. several types of histochemical analysis were carried out, including tests for flavonoids, phenols, tannins, terpenoids, and alkaloids. procedures preparation and testing in silico bioactivity and 3d structure testing the chemical structures of secondary metabolites were analyzed in silico using pubchem software (https://pubchem.ncbi.nlm.nih.gov/), and their bioactivity was determined using pass online software (http://www.way2drug.com/passonline/index.php). molecular docking test 3ert protein preparation the 3ert protein was downloaded using pdb software (http://www.rcsb.org/pdb). the target protein was then selected in its active form that binds to its native ligand. the 3ert protein, which had bound to its native ligand, was then separated using discovery studio virtualizer software for validation. validation with molecular docking validation of the molecular docking method was performed by re-docking the native ligand and the target protein, which had been separated from the native ligand, using pyrx software. if the root mean square deviation (rmsd) value obtained is ≤ 3 å, the method is considered valid, allowing docking of the test compound and its target protein; however, according to research (manno & utami, 2023). preparation of active compounds test compounds were downloaded from the pubchem software (https://pubchem.ncbi.nlm.nih.gov/) and prepared using the discovery studio virtualizer software, utilizing pdb files for docking adjustments. docking of active compounds and 3ert protein docking of the test compounds with 3ert, whose native ligand had been separated, using pyrx software, yielded a docking result that confirmed the lowest binding energy and was related to the target protein. molecular docking data analysis molecular docking data analysis was based on the binding energy results, root mean square deviation (rmsd) values, and the bond types generated by the molecular docking. the bond energy value indicated the bond strength between the test compound and its receptor. the lower the bond energy value, the stronger the bond between the compound and the receptor. preparation and histochemical testing of soursop leaves histochemical analysis was performed using fresh soursop leaves (annona muricata), which were sterilized by thoroughly washing them with running water. then, an incision was made on the underside of the leaf (abaxial) for preparation. the fresh slides were placed on a glass slide and treated with prepared immersion oil, http://www.way2drug.com/passonline/index.php nuari et al. – in silico and histochemical analysis of soursop leaves 1057 then observed under a microscope. the secondary metabolite detection method is as follows, flavonoid compound analysis flavonoid compounds were detected by adding a solution of alcl3 in 85% ethanol. if there is a color change to yellow or blue, the sample is positive for flavonoid compounds (rupa, 2015; pratiwi, 2020). phenol compound analysis phenol compound analysis was performed by immersing a sample slice in a 10% fecl3 solution with several na2co3 pellets and allowing it to stand at room temperature for approximately 15 minutes. if there is a color change to dark green or even black, the sample is positive for phenol compounds (andriya, 2016; pratiwi, 2020). tannin compound analysis tannin compound analysis was performed by adding a 10% fecl3 solution to the sample incision. a color change to dark brown indicated the sample contains tannin compounds (rupa, 2015; pratiwi, 2020). terpenoid compound analysis terpenoid compounds were detected by immersing the sample in a 5% cuso4 solution for approximately 24 hours. then, a drop of glycerin was added and observed under a microscope. a positive result for terpenoids was indicated by a color change to brownish-yellow (andriya, 2016; pratiwi, 2020). alkaloid compound analysis alkaloid compounds was detected by soaking the sample in wagner's reagent for 48 hours. then, they were observed under a microscope. a positive result for alkaloid compounds was indicated by a color change to reddish-brown (andriya, 2016; pratiwi, 2020). data analysis data obtained from the in silico method were analyzed quantitatively based on values generated from the molecular docking process. meanwhile, the histochemical method was analyzed qualitatively based on research documentation, which showed specific colour changes. results and discussion results identification of specific compounds in soursop leaves (annona muricata) and their bioactivity through in silico analysis based on the in silico analysis conducted, several compounds were identified as being specific to soursop leaves, and common compounds were also detected in several other plant species. the results of the active compound search and 3d structure are shown in table 1. table 1. knapsack results of active compounds and 3d structure of soursop leaves (annona muricata). no molecular formula structural formula compound name compound classes information 1 c35h64o7 annocatalin flavonoid specific 2 c35h64o9 annohexocin flavonoid specific 1058 biology, medicine, & natural product chemistry 14 (2), 2025: 1055-1068 no molecular formula structural formula compound name compound classes information 3 c35h64o8 annomuricin e flavonoid specific 4 c35h64o8 annomuricin a flavonoid specific 5 c35h62o6 cis-corossolone flavonoid specific 6 c35h64o5 cis-solamin flavonoid specific 7 c35h64o7 gigantetrocin a flavonoid detected in 4 species: annona muricata, annona glabra, goniothalamus giganteus, xylopia aromatica 8 c35h64o7 muricatetrocin a flavonoid specific 9 c35h64o9 murihexocin c fenol specific nuari et al. – in silico and histochemical analysis of soursop leaves 1059 no molecular formula structural formula compound name compound classes information 10 c19h21no4 norcorydine alkaloid detected in 8 species: annona muricata, annona reticulate, annona squamous, miliusa velutina, trivalvaria macrophylla, xylopia danguyella, xylopia pancheri, croton hemiargyreus the active compounds of soursop leaves obtained from the results of the knapsack program search include annocatalin, annohexocin, annomuricin a, annomuricin e, cis-corossolane, cis-solamin, gigantetrocin a, muricatetrocin a, a derivative of acetogenin, norcorydine, a derivative of alkaloids, and murihexocin c, a derivative of phenolics. some of these compounds are specific compounds that are only found in soursop leaves, including annocatalin, annomuricin a, annomuricin e, annohexocin, muricatetrocin a, murihexocin c, cis-corossolane, and cis-solamin. their potential as breast cancer anticancer agents was then assessed, and the bioactivity values were obtained in the form of probable to be active (pa) values for each compound, presented in table 2. table 2. percentage of bioactivity of active compounds in soursop leaves (annona muricata) as candidates for breast cancer drugs. no compound name bioactivity percentage (%) 1 annocatalin 56,2 2 annohexocin 46,7 3 annomuricin e 63,5 4 annomuricin a 63,5 5 cis-corossolone 63,4 6 cis-solamin 41,8 7 gigantetrocin a 56,2 8 muricatetrocin a 56,2 9 murihexocin c 50,6 10 norcorydine 19 based on table 2, the probable to be active (pa) values were converted into percentages. the results show that several compounds have quite high activity, namely annomuricin e (63.5%), annomuricin a (63.5%), ciscorossolone (63.4%), gigantetrocin a (56.2%), muricatetrocin a (56.2%), annocatalin (56.2%), and murihexocin (50.6%). meanwhile, annohexocin, cissolamine, and norcorydine are classified as compounds with low activity with values of 46.6%, 41.8%, and 19%, respectively. results of molecular interaction of active compounds in soursop leaves (annona muricata) with the alpha estrogen receptor the detailed profile of 3ert obtained through rscb pdb software data is as follows (figure 1). figure 1. 3ert profile. the molecular docking resulted in interactions indicated by the type of bond to amino acid residues. the results of the interaction between the active compounds in soursop leaves and the 3ert protein are presented in the following figure (figure 2-12). figure 2. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound annonacatalin (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. 1060 biology, medicine, & natural product chemistry 14 (2), 2025: 1055-1068 figure 3. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound annohexocin (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. figure 4. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound annomuricin e (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. figure 5. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound annomuricin a (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. figure 6. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound cis-corossolone (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. nuari et al. – in silico and histochemical analysis of soursop leaves 1061 figure 7. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound cis-solamin (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. figure 8. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound gigantetrocin a (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. figure 9. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound muricatetrocin a (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. figure 10. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound murihexocin c (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. 1062 biology, medicine, & natural product chemistry 14 (2), 2025: 1055-1068 figure 11. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound norcorydine (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. figure 12. molecular docking simulation results of estrogen alpha (pdb id: 3ert) with the active compound oht600 (native ligand/control) (a) protein-ligand complex; (b) 3-dimensional structure interaction; and (c) 2-dimensional structure interaction. molecular docking results show the binding affinity values obtained, as well as interactions with amino acid residues of various bond types. the binding values of the 11 active compounds tested, along with the bound amino acids, are presented in table 3. table 3. binding affinity value and interaction of active compounds in soursop leaves (annona muricata) as a candidate for breast anticancer drugs. no. compound binding affinity (kcal/mol) rmsd lower bond rmsd upper bond interaction type 1. annonacatalin -6,6 1,888 3,418 hydrogen bonds: glu523, asn519 hydrophobic: trp383, ala350, leu384, leu525, leu536, leu384, leu387 unfavorable: tyr526 2. annohexocin -6,6 1,848 2,477 hydrogen bonds: ala382, leu384, ser518, glu523, gly521 hydrophobic: ala350, leu525, cys530, val533, leu384, leu384, leu346, trp383 3. annomuricin e -6,1 2,484 5,271 hydrophobic: ala350, leu536, leu384, leu384 4. annomuricine a -6,3 3,070 8,751 hydrogen bonds: gly521, tyr526, glu523 hydrophobic: ala350, lys529, leu525, leu384, leu525, leu387, leu346, leu349, trp383, phe404 unfavorable: leu536 5. cis-corossolone -6,7 1,929 3,087 hydrogen bonds: cys530, asp351 hydrophobic: ala350, leu525, leu346, leu349, leu391, trp383 unfavorable: asp351 nuari et al. – in silico and histochemical analysis of soursop leaves 1063 no. compound binding affinity (kcal/mol) rmsd lower bond rmsd upper bond interaction type 6. cissolamin -9,3 1,673 2,590 hydrogen bonds: glu523 7. gigantetrocin a -6,4 0,906 2,226 hydrogen bonds: thr347, lys529 hydrophobic: ala350, leu525, val533, leu536, met421, ile424, ile424, leu346, leu391, leu387, phe404 unfavorable: cys530 8. muricatetrocin a -6,4 1,427 1,980 hydrophobic: ala350, leu384, leu387, met388, leu391, leu346, met421, ile424, leu536, trp383 phe404 9. murihexocin c 7,1 1,367 2,170 hydrogen bonds: thr347, glu523 hydrophobic: leu346, ala350, leu536, leu387, leu391, met388, leu384, trp383 10. norcorydine -6,4 2,980 5,835 no interaction 11. oht600 -7,6 0,681 1,528 hydrogen bonds: ala350, glu380 hydrophobic: trp383, leu536, ala350, leu354, leu525, lys529, val533 based on the docking results of estrogen alpha with 10 active compounds in soursop leaves, binding affinity, root mean square deviation (rmsd) and hydrogen bond values were obtained. then, filtering based on the validity of the rmsd data, five valid compounds were identified: ▪ annohexocin with a binding affinity value of -6.6 kcal/mol, a lower bond rmsd of 1.848 å and an upper bond rmsd of 2.477 å. this compound forms hydrogen bonds with the amino acids ala382, leu384, ser518, glu523, and gly521, as well as hydrophobic bonds with the amino acids ala350, leu525, cys530, val533, leu384, leu346, and trp383. ▪ cis-solamin with a binding affinity value of -9.3 kcal/mol, with a lower bond rmsd value of 1.673 å and an upper bond rmsd of 2.590 å. this compound forms hydrogen bonds with the amino acid glu523. ▪ gigantetrocin a has a binding affinity value of -6.4 kcal/mol with a lower bond rmsd value of 0.906 å and an upper bond rmsd of 2.226 å. this compound forms hydrogen bonds with the amino acids thr347 & lys529, hydrophobic bonds with the amino acids ala350, leu525, val533, leu536, met421, ile424, ile424, leu346, leu391, leu387 & phe404, and unfavorable bonds with the amino acid cys530. ▪ muricatetrocin a has a binding affinity of -6.4 kcal/mol with a lower bond rmsd of 1.427 å and an upper bond rmsd of 1.980 å. this compound forms hydrophobic bonds with the amino acids ala350, leu384, leu387, met388, leu391, leu346, met421, ile424, leu536, trp383 & phe404. ▪ murihexocin c has a binding affinity of -7.1 kcal/mol with a lower bond rmsd of 1.367 å and an upper bond rmsd of 2.170 å. this compound forms hydrogen bonds with amino acids thr347 & glu523 and hydrophobic bonds with amino acids leu346, ala350, leu536, leu387, leu391, met388, leu384 & trp383. results of identification of secondary metabolite compounds in soursop leaves (annona muricata) through histochemical analysis histochemical analysis was used to qualitatively determine the secondary metabolite content of plant tissue. this study used soursop leaf samples collected in batu city. only the fifth node from the shoot or leaf tip was used as the research boundary. the results of the histochemical analysis are shown in table 4.. 1064 biology, medicine, & natural product chemistry 14 (2), 2025: 1055-1068 table 4. results of detection of secondary metabolite compounds in soursop leaves (annona muricata) through histochemical analysis. secondary metabolite compounds detection reagent positive detection and color change results flavonoid alcl3 and ettanol 85% + (reddish yellow) fenol fecl3 10% and na2co3 + (dark green) tanin fecl3 10% + (dark brown) terpenoid cuso4 5% and gliserin + (brownish yellow) alkaloid wagner + (brownish red) the results of histochemical analysis indicate that soursop leaves contain secondary metabolite compounds, including flavonoids, phenols, tannins, terpenoids, and alkaloids. microscopic observations of soursop leaf epidermal cells revealed the presence of secondary metabolites in the trichomes and stomatal tissues. flavonoid secondary metabolites were identified by the color change from clear to reddish-yellow in the trichome tissue, as seen in figure 13. figure 13. histochemical micrograph of abaxial longitudinal section of soursop leaf before treatment (a) magnification 1000x, after flavonoid detection treatment (b) 100x, (c) 400x, (d) 1000x. the results of microscopic observations of the epidermal cells in soursop leaves reveal the presence of phenolic compounds, as indicated by a change from clear to dark green in the trichome tissue, as shown in figure 14. figure 14. histochemical micrograph of abaxial longitudinal section of soursop leaf before treatment (a) magnification 1000x, after phenol detection treatment (b) 100x, (c) 400x, (d) 1000x. the results of microscopic observations of the epidermal cells in soursop leaves reveal the presence of tannin compounds, with a transition from clear to dark brown in the trichome and stomata tissue, as shown in figure 15. figure 15. histochemical micrograph of abaxial longitudinal section of soursop leaf before treatment (a) magnification 1000x, after tannin detection treatment (b) 100x, (c) 400x, (d) 1000x. the results of microscopic observations of soursop leaf epidermal cells revealed the presence of terpenoid compounds, as indicated by a change from clear to brownish-yellow in the trichome tissue, as shown in figure 16. figure 16. histochemical micrograph of abaxial longitudinal section of soursop leaf before treatment (a) magnification 1000x, after terpenoid detection treatment (b) 100x, (c) 400x, (d) 1000x. microscopic observations of soursop leaf epidermal cells revealed the presence of alkaloid compounds, characterised by a colour change from clear to brownishred in the trichome and stomata tissue, as shown in figure 17. figure 17. histochemical micrograph of abaxial longitudinal section of soursop leaf before treatment (a) magnification 1000x, after alkaloid detection treatment (b) 100x, (c) 400x, (d) 1000x. nuari et al. – in silico and histochemical analysis of soursop leaves 1065 discussion identification of specific compounds in soursop leaves (annona muricata) and their bioactivity through in silico analysis in silico research results identified 10 active compounds in soursop leaves that have the potential to act as antibreast cancer agents, including annocatalin, annohexocin, annomuricin a, annomuricin e, ciscorossolane, cis-solamin, gigantetrocin a, muricatetrocin a, murihexocin c, and norcorydine. gigantetrocin a and norcorydine compounds are not specifically found only in soursop leaves, but are also found in several other species. this is in line with research by qomaliyah (2022), which suggests that soursop leaves contain active secondary metabolite compounds with potential bioactivity due to the presence of flavonoid compounds, acetogenin derivatives, and other groups, including alkaloids and phenols. another study, according to siswarni et al. (2016), states that soursop has an acetogenin chemical content that is useful as an anticancer agent, and its use in the health sector continues to increase. the known compounds were analysed for their bioactivity and potential, followed by molecular docking to determine their binding affinity to the alpha estrogen receptor protein. the bioactivity of the compounds in soursop leaves was analysed using the pass online web server to evaluate their biological potential as potential drug candidates (istiqomah et al., 2023). based on table 2, the probable to be active (pa) value has been converted into a percentage. the (pa) value indicates the probability of the compound's activity in soursop leaves as an anti-breast cancer agent. if the pa value is higher than 0.7 (pa > 0.7), the compound exhibits very high biological activity, and the results are not significantly different from those of laboratory tests. if the pa value is between 0.5 and 0.7 (0.5 < pa < 0.7), the compound exhibits quite high biological activity in in vitro or in vivo tests and has potential as a new drug candidate for anticancer applications. if the pa value is less than 0.5 (pa < 0.5), the compound has low biological activity both in the laboratory and computationally (malikhana et al., 2021). the results show that several compounds have quite high activity, namely annomuricin e, annomuricin a, cis-corossolone, gigantetrocin a, muricatetrocin a, annocatalin, and murihexocin. meanwhile, annohexocin, cis-solamine, and norcorydine are classified as compounds with low activity. compounds with low pa values do not necessarily mean they have low activity; however, these compounds have not been widely studied in the laboratory or computationally, so further research is needed to explore them as potential drug candidates. molecular interaction of active compounds in soursop leaves (annona muricata) with the alpha estrogen receptor molecular docking was performed using the estrogen alpha receptor protein with pdb id: 3ert. the crystal structure with the code 3ert is erα that binds tamoxifen (xue et al., 2019). erα is a receptor in cells that binds the female hormone estrogen and other natural and synthetic ligands. its role is very important in normal development, physiological function, and diseases related to the endocrine system. erα belongs to a large family of nuclear receptors and transcription factors, regulating the expression of its target genes depending on the type of ligand bound (candelaria et al., 2013). molecular docking results indicate an interaction between the ligand compound and the receptor protein. this interaction produces binding affinity values and root mean square deviation (rmsd) values for the lower and upper bonds. interactions with amino acid residues with various bond types are also observed. the parameter used to validate molecular docking results is the root mean square deviation (rmsd), a measure of similarity widely used in macromolecular structure and dynamics analysis. when larger macromolecular systems are studied, dimensional effects such as the “curse of dimensionality” (a reduced ability to distinguish pairwise differences between conformations with increasing system size) may exist and have a significant impact on rmsd-based analysis (sargsyan et al., 2017). the rmsd parameter is considered valid, and the interaction between the ligand and protein can be carried out if it has an rmsd value ≤ 3 å (yuniati et al., 2023). rmsd values <2 and <3 å are normal or average; if the rmsd value is between 3 and 4 å, it means there is a difference in protein folding, and if the rmsd value is more than 4 å, it indicates that there is a problem in binding or is declared invalid (lestari et al., 2019). in the study of manno et al. (2023), rmsd is a two-pose measurement that compares the atomic positions between the experimental structure and the docking structure of the protein. the results are declared valid if the value obtained is greater than 3 å, and the smaller the value obtained, the better the ligand pose. based on the results of the prediction stage of the interaction of active compounds in soursop leaves with the alpha estrogen receptor analysis molecule (table 3), it was found that the specific soursop leaf compound that had the highest binding affinity and the strongest bond was the cis-solamin compound of -9.3 kcal/mol, cis-solamin has an rmsd value below 3 å so that the data can be said to be valid, cis-solamin has a lower bond rmsd of 1.673 å and an upper bond rmsd of 2.590 å. while the native ligand or control compound, namely oht600, has a binding affinity value of -7.6 with a lower bond rmsd of 0.681 å and an upper bond rmsd of 1.528 å. based on molecular docking, it was found that the cis-solamin compound has a higher binding affinity value than the oht600 compound, 1066 biology, medicine, & natural product chemistry 14 (2), 2025: 1055-1068 which was used as a control in this study. this indicates that cis-solamin has a greater binding strength to the alpha estrogen receptor, making it a potential candidate for breast cancer drugs. the bonds formed by the active compound cis-solamin are hydrogen bonds with the amino acid glu532, which share similarities with the native ligand, specifically hydrogen bonds with the amino acids ala359 & glu380. based on molecular docking data, the active compound cis-solamin was identified in soursop leaves, which has a binding affinity with the alpha estrogen receptor (pdb id: 3ert). so the active compound in soursop leaves has the potential to be an erα inhibitor because it has a high binding affinity value of -9.3 kcal/mol, cis-solamin has an rmsd value below 3 å so that the data can be said to be valid; the smaller the rmsd value, the better the ligand conformation. the cis-solamin compound shows a higher binding affinity than the oht600 compound, which is the original ligand found in the 3ert protein. alpha estrogen is the main target in er+ breast cancer therapy, and inhibiting erα activity has been shown to inhibit the growth of er+ breast cancer cells. identification of secondary metabolite compounds in soursop leaves (annona muricata) through histochemical analysis histochemical analysis results indicate that soursop leaves contain secondary metabolites, including flavonoids, phenols, tannins, terpenoids, and alkaloids. this finding is similar to the research of fertilita et al. (2020), which showed that the phytochemical screening results of soursop leaves contained secondary metabolites, including alkaloids, flavonoids, tannins, and steroids. this finding aligns with the research of ashafani et al. (2022), which revealed that the phytochemical screening results of soursop leaves contained secondary metabolites, including alkaloids, terpenoids, steroids, saponins, flavonoids, phenols, and tannins. secondary metabolites are widely used for nutritional therapy or the treatment of various diseases, one of which is breast cancer. flavonoid compound flavonoids are chemical compounds derived from 2phenyl-benzyl-γ-pyrone, whose biosynthesis occurs via the phenylpropanoid pathway. flavonoids play a crucial role in imparting colour, flavour, and aroma to seeds, flowers, and fruits. these compounds are easily oxidized at high temperatures and are not heat-stable. flavonoids exhibit various pharmacological effects, including antioxidant, anti-ageing, anti-inflammatory, and antiviral properties. research has shown that by 2011, more than 9,000 flavonoids had been discovered, many of which have been used as health supplements. flavonoid compounds include flavones, flavonols, flavanones, flavanonols, flavanols or catechins, anthocyanins, and chalcones (ningsih et al., 2023). the principle of total flavonoid analysis using alcl3 is based on the formation of a complex between alcl3 and the keto group at the c4 atom and the hydroxyl group at the adjacent c-3 or c-5 atoms in the flavone and flavonol structures. an alcl3 solution is used to form a coloured complex with flavonoids, which causes a shift in wavelength to the visible region, characterised by a change in the solution's colour to a more yellow hue (lindawati & ma'ruf, 2020). phenolic compound phenolic compounds are compounds containing hydroxyl groups, which generally also have the potential to exhibit antioxidant activity. antioxidant activity is directly proportional to the total phenol content of a natural ingredient (berlinsyah et al., 2021). the principle of the phenol test using fecl3 is that coloured phenolic compounds form complexes with fe3+ in fecl3, resulting in darker (black) spots, which indicate the presence of phenolic compounds (nurmalasari et al., 2020). research by qomaliyah (2022) also indicated that soursop leaves contain phenolic compounds, which have potential cytotoxic bioactivity against cancer cells. tannin compound tannins are highly complex compounds and are widely distributed in various plant species. they are present in almost every plant species. in the health sector, tannins possess several properties, including antidiarrheal, antioxidant, antibacterial, and astringent properties. tannins are typically found in specific plant parts, including the fruit, leaves, stems, and bark (sunani and hendriani, 2023). phytochemical tests using fecl3 can detect the presence of phenol groups. if phenolic compounds are detected, tannins are likely present, as tannins are polyphenolic compounds. the color change to blackish green occurs due to the formation of a complex between tannins and fecl3 (ikalinus et al., 2015). other research suggests that soursop leaves contain secondary metabolites in the form of tannins, which have the potential to inhibit cancer cell growth due to their cytotoxic activity as therapeutic agents (qomaliyah, 2022). terpenoid compound terpenoid compounds found in plants exhibit significant pharmacological activities, including antiviral, antibacterial, anti-inflammatory, antihypertensive, antimicrobial, cholesterol synthesis inhibition, and anticancer properties. compounds in the triterpenoid group are phytochemical agents that can selectively kill breast cancer cells and protect normal cells from damage (soliha et al., 2017). qualitative terpenoid testing was conducted by exploiting the colour-forming ability of terpenoid compounds contained in the sample leaves using a 5% cuso4 solution and glycerin, resulting in a colour change in the trichome tissue from clear to nuari et al. – in silico and histochemical analysis of soursop leaves 1067 brownish-yellow. this is consistent with research by qomaliyah (2022), which states that soursop leaves contain secondary metabolites in the form of terpenoids, making them potential pharmacophores for cancer therapy. alkaloid compound alkaloids are organic bases containing nitrogen (n) and are generally derived from plants, exhibiting strong physiological effects on humans. in pharmacology, alkaloids are used to stimulate the nervous system, increase blood pressure, and fight microbial infections (wullur et al., 2018). alkaloid analysis is carried out using wagner's reagent. in preparations treated with wagner's reagent, a colour change is observed in the tissue under a microscope. the reaction of wagner's reagent will precipitate protoplasts, and a positive alkaloid content is indicated by a colour change, marked by a reddish-brown colour in the tissue (nurhasanah & iriani, 2021). in the study by tiara et al. (2024), it was also stated that the secondary metabolite profile test using wagner's reagent showed positive results for alkaloid compounds. in a study by rahayu et al. (2021), it was stated that the content of secondary metabolites in plant tissue varies depending on the location of the tissue and environmental conditions, including temperature and humidity. leaf samples were sliced transversely at the abaxial section. this study also utilised fresh preparations, which were immediately observed under a microscope after slicing without any preservation process. in each sample, the tissue colour changed from clear to several distinct colours, allowing the identification of secondary metabolite content. the plant tissues that showed colour changes were the epidermal tissue of trichomes and stomata, as these tissues store secondary metabolites in plants that function as a defence against pests or diseases, and as a means of selfprotection from environmental stress. several other studies also reported that the results of histochemical analysis revealed colour changes in epidermal tissue, including trichomes and stomata (anisa et al., 2018; rahayu et al., 2021). relationship between in silico analysis and histochemistry in this study, in silico analysis was employed to identify the presence of specific active compounds unique to soursop leaves, representing an innovative research approach. the results of specific compounds in soursop leaves are annocatalin, annomuricin a, annomuricin e, annohexocin, muricatetrocin a, murihexocin c, ciscorossolane, and cis-solamin. these active compounds were subjected to molecular docking to determine their interactions and bonds to the estrogen alpha receptor protein (3ert). by knowing the valid bond value between the ligand compound and the protein, it is evident that there is an interaction between the active compound and the estrogen alpha receptor. the cissolamin compound has a higher binding affinity value than the oht600 compound, which is used as a control in the in silico analysis, indicating that cis-solamin has a greater binding strength to the estrogen alpha receptor, making it a candidate for breast cancer drugs. this is evidenced by histochemical analysis of soursop leaves, which show positive results for the content of secondary metabolite compounds, including flavonoids, phenols, tannins, terpenoids, and alkaloids. conclusions based on in silico and histochemical analysis of soursop (annona muricata) leaves on estrogen receptor alpha, it can be concluded that active compounds specific to soursop leaves include annocatalin, annomuricin a, annomuricin e, annohexocin, muricatetrocin a, murihexocin c, cis-corossolane, and cis-solamine. molecular docking revealed that the active compound cis-solamin has a higher binding affinity than the native ligand. the binding affinity of cis-colamin is (-9.3 kcal/mol), while oht600 has a binding affinity of (-7.6 kcal/mol). the docking results show that the cis-solamin compound with the highest binding affinity is bound to the amino acid 3ert, namely glutamine 532 (glu523). histochemical analysis of soursop leaves shows the presence of secondary metabolites in the form of flavonoids (reddish yellow), phenols (dark green), tannins (dark brown), terpenoids (brownish yellow), and alkaloids (brownish red). authors’ contributions: afidah fajar nuari, gatra ervi jayanti & majida ramadhan designed the study. afidah fajar nuari carried out the laboratory work. afidah fajar nuari analyzed the data. afidah fajar nuari, gatra ervi jayanti & majida ramadhan wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references andriya, n., nurafifah, n.s., yohana, c.r., & otih. 2016. analisis struktur anatomi dan histokimia tiga varietas kumis kucing (orthosiphon aristatus miq.). skripsi. departemen biologi: institut pertanian bogor. anisa, k., rahayu, t., & hayati, a. 2018. profil metabolit sekunder daun tin (ficus carica) melalui analisis histokimia dan deteksi flavonoid dengan metode kromatografi lapis tipis (klt). 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jurnal bina cipta husada, 19 (01), 98-107. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 969-975 | doi: 10.14421/biomedich.2025.142.969-975 issn 2540-9328 (online) detection of the p53 gene in formalin fixed tissue archives by polymerase chain reaction (pcr) method anastasia elma wijayanti1, fitria diniah janah sayekti2* 1medical laboratory technology; 2sekolah tinggi ilmu kesehatan nasional surakarta jl. solo baki, kwarasan, grogol, jawa tengah, tel. 0271 5723399, indonesia. corresponding author* fitria.diniah@stikesnas.ac.id abstract formalin fixed tissue archives are tissue biopsy results stored in formaldehyde fixative solution for a long time. the p53 gene is one of the genes that suppresses tumor occurrence and maintains genomic stability to prevent cell mutation and plays a role in the dna regulation cycle and controls cell proliferation. polymerase chain reaction (pcr) is a nucleic acid amplification technique used to denature and denature dna or rna using polymerase enzymes to detect the presence of the p53 gene in formalin-fixed tissue archival samples. this study aimed to detect the presence of the p53 gene in formalin-fixed tissue samples. this study used a descriptive research method with a random sampling technique conducted in the anatomical pathology laboratory and the national college of health sciences molecular biology laboratory. the samples in this study consisted of 10 samples consisting of 2 normal tissues used as controls and 8 formalin-fixed tissue samples. in this study, the p53 gene was detected in all samples, both normal tissue samples and formalin-fixed tissue samples after electrophoresis and visualized according to the target of 666 bp (base pair). the presence of the p53 gene qualitatively from dna isolates can still be detected, but the gene expression level cannot be known in this study. keywords: formalin fixed tissue archive; p53 gene; pcr. abbreviations: polymerase chain reaction (pcr), deoxyribonucleic acid (dna), ribonucleic acid (rna) introduction cancer is an abnormal cell growth in the human body that attacks organs. these abnormal cells are caused by several changes in gene expression, causing an imbalance in the regulation of cell proliferation and cell death. cancer is known as a chronic disease that needs to be watched out for, because it has a major impact on many people in the world and is one of the leading causes of death worldwide (prasetyo et al., 2021). according to the ministry of health (2023) cancer is the second leading cause of death in the world and is a public health problem both globally and nationally, causing up to 9.6 million deaths each year. about 70% of deaths caused by cancer occur in developing countries, including indonesia. cancer has many types of treatment or therapies depending on the type of cancer and the stage. some cancer patients undergo only one type of therapy. however, most cancer patients undergo a combination of treatments, such as surgery with chemotherapy and radiation therapy. according to national cancer (2015), biomarker tests for cancer treatment are a way to look for genes, proteins and other substances such as tumor markers that can provide cancer information. biomarker tests are different from genetic tests to find out if a person has inherited a mutation that makes them develop cancer. these inherited mutations are mutations that are carried from birth. the cause of cancer is thought to be a mutation in the tumor suppressor gene p53. mutations in this gene inactivate tumor suppressors and tumor progression. the p53 gene undergoes apoptosis, resulting in increased expression of the p53 gene. the expression of p53 that shows an increase in expression, which is significantly associated with an increase in the rate of invasive cancer tumors (sadia et al., 2020). the p53 gene is found on chromosome 17p and this p53 gene acts as a tumor suppressor gene. the p53 gene controls the cell cycle before the synthesis phase and plays a role in cell proliferation (kisnanto et al., 2015). the p53 gene can be detected from frozen biopsy cut tissue organs and formalin fixed tissue archives. archival tissue samples have enormous benefits as they can be available in large quantities and easily used to study various biomarkers to facilitate diagnostic procedures, target therapies, determine prognosis, and become an available source for research. a biopsy is the act of performing a microscopic examination of tissue from the manuscript received: 13 august, 2025. revision accepted: 14 october, 2025. published: 29 october, 2025. https://doi.org/10.14421/biomedich.2025.142.969-975 970 biology, medicine, & natural product chemistry 14 (2), 2025: 969-975 human body to establish the diagnosis of a disease process or infection (erawati et al., 2017). sekolah tinggi ilmu kesehatan nasional has an anatomical pathology laboratory in which formalin-fixed tissue archives are stored for a long time. the tissue archive is used for studies but because it is stored for too long, normal tissue and abnormal tissue are no longer distinguishable. the main challenge in using formalinfixed tissue archives in genetic research is that it is difficult to obtain quality dna due to dna damage caused by the process of tissue fixation with formalin and the influence of the long storage time of tissue archives at stikes nasional (hafy et al., 2018). testing that can be used for p53 gene detection use pcr and electrophoresis techniques. polymerase chain reaction (pcr) is a nucleic acid amplification technique used to denature and denature dna or rna using polymerase enzymes (khehra et al., 2024). this study focused on detecting the p53 gene in normal tissue and formalin-fixed tissue samples using the polymerase chain reaction (pcr) method. this study aims to determine the presence of the p53 gene in organ biopsy results stored for a long time in formalin. materials and methods study area this research has passed the ethical review of the research ethics commission of muhammadiyah purwokerto university (kepk-ump) with the number kepk/ump134/viii/2024. the type of research used descriptive research design, p53 gene detection was carried out using archival samples of formalin-fixed tissues within a certain period of time found in the anatomical pathology laboratory of the national college of health sciences. samples were taken as many as 10 samples consisting of 2 normal tissue samples as controls in the form of hair tissue and nail tissue samples and 8 samples of formalin-fixed tissue archives over a period of 2 pieces of tissue 2 days, 2 pieces of tissue less than 1 month, 2 pieces of tissue 2 months, 2 pieces of tissue more than 5 years. procedures preparation of control and archival tissue samples samples of 10 hair strands measuring 0.5 1 cm in length were taken from the proximal hair tip including the hair follicle and nail samples were taken as much as 15 mg. archival samples of formalin-fixed tissue that have been cut as much as 25 mg are then removed formalin by being put into alcohol solution which is done in stages from 70% alcohol, 80% alcohol, 90% alcohol in each alcohol solution for 1 hour and absolute alcohol for 2 hours. dna isolation preparation of tools and materials to be used include nail control samples, hair control samples and tissue pieces that have been removed formalin which is inserted into a 1.5 ml microcentrifuge tube. followed by the dna isolation stage using geneaid gsynctm dna extraction through several stages, namely dna lysis, dna binding, dna washing, and dna elution. dna qualitative test the results of dna isolates were analyzed by dna qualitative test using 1.5% agarose gel electrophoresis. the components needed in this qualitative test analysis are dna isolate, gel red, and loading dye. visualization of dna isolation results was read using bio-rad uv transilluminator gel-doc. dna quantitative test dna quantitative test was carried out by making a 400x dna dilution with a ratio of 10 μl dna isolate and 3990 μl aquabidest which was put into a 15 ml centrifuge tube and homogenized. dna purity was measured with a uvvis spectrophotometer at a wavelength of 260 nm and 280 nm. optimization of annealing temperature components needed at the annealing temperature optimization stage in the form of master mix, dna isolate, forward primer, reverse primer, and nfw are included in the pcr tube. followed by the thermal cyler machine t100 tool with a temperature range of 50 – 580c. polymerase chain reaction (pcr) test dna amplification was performed using polymerase chain reaction (pcr) with 1.5% agarose gel electrophoresis method at 57.40c. the components used in pcr amplification are dna isolate, gel red, loading dye, and ladder which are electrophoresed on agarose gel in 90 minutes, 90volt, 400 ampere and read with gel doc. data analysis the data analysis of this study was taken from the visualization results of the pcr test on normal tissues and tissue archives obtained from formalin-fixed tissue isolation found in the anatomical pathology laboratory of the national college of health sciences. results and discussion formalin removal process with graded alcohol in the first stage of formalin-fixed tissue archive samples, the process of cutting the tissue weighing 25 mg was carried out, then the process of removing formalin from the tissue using graded alcohol from 70% alcohol, 80% alcohol, 90% alcohol and absolute alcohol. fixation is the process of soaking the tissue using a fixation liquid such as neutral buffer formalin (nbf) 10% which aims to prevent decomposition. the graded alcohols used in wijayanti & sayekti – detection of the p53 gene in formalin fixed tissue archives … 971 this process aim to remove water gradually in the tested tissue pieces. (rahmawanti et al, 2021). dna qualitative test dna isolation is a technique used to obtain pure dna, which does not contain rna protein from a cell in tissue (handayani et al, 2021). samples that have been formalinized are then subjected to a dna isolation process using geneaid gsynctm dna extraction. the results of dna isolation that have been obtained are carried out qualitative tests to determine the quality of the isolated dna. the success of dna extraction was analyzed qualitatively by visualizing the dna using agarose gel electrophoresis method. the principle of electrophoresis is the separation of dna based on molecular weight on agarose gel using an electric charge. dna will move from negative to positive poles in the electric field (anissa et al., 2024). figure 1. qualitative test results of dna isolates from normal tissue samples and formalin fixed tissue archives. description: k1 : nail control k2 : hair control j1 – j2 : 2 day formalin fixed tissue j3 – j4 : formalin fixed tissue <1 month j5 – j6 : 2 months formalin fixed tissue j7 – j8 : formalin fixed tissue > 5 years figure 1. shows the results of qualitative tests on k1 and k2. j1, j2, j3, j4, j5, j6 are dna bands with different thicknesses and smears. the smear formed at the bottom near the dna band indicates that the isolated dna is not intact or fragmented and the smear located at the bottom of each lane indicates contamination (anissa et al., 2024). dna bands with different thicknesses depend on the weight and amount of dna. qualitative test visualization results that do not have dna bands can be caused by insufficient dna concentration in the sample (ramadhani et al., 2024). the more dna that is successfully extracted, the thicker the dna band, and vice versa if the less dna is extracted, the thinner the dna band will be visualized on the agarose gel. thin dna bands can also be caused by contamination. hidayati et al (2016) added, analysis using qualitative methods, dna purity is also characterized by the absence of smears that indicate the presence or absence of contaminants such as rna or protein. smears formed at the bottom near the dna band indicate that the isolated dna is not intact or fragmented and smears located at the bottom of each lane indicate contamination. according to research by halimah et al (2024), it is known that the length of electrophoresis time affects the distance traveled by dna fragments, where the longer the electrophoresis time, the farther the distance traveled. dna qualitative test performed using electrophoresis method through agarose gel is used to separate, analyze, identify, and purify dna fragments (anam et al., 2021). however, in samples j7 and j8 there was no dna band. sample codes j7 and j8 are formalinfixed tissue samples of more than 5 years, this could be due to the possibility of dna damage due to long term formalin fixation. dna derived from degraded tissue is often fragmented. exposure to chemicals, ph, temperature, and other exposures can cause dna damage (yudianto, 2010). according to willianto (2013) cited from evans (2007) formalin solution is one of the chemical exposures that affect the integrity of dna strands. exposure to formalin that induces dna crosslinks effectively protects the morphology of the cell structure, but at the extreme it is detrimental because dna crosslinks inhibit denaturation in pcr and the ph of the formalin solution decreases over time due to the formation of formic acid which causes ap site which ends in fragmentation. in a study (willianto, 2013) showed that dna analysis in human soft tissue exposed to 2% to 35% formalin solution for 3 days still obtained visualization of dna bands, so that it can form a successful process of determining the identification of a person in dna examination. dna quantitative test dna isolates that have undergone qualitative tests are then subjected to quantitative dna tests to determine the 972 biology, medicine, & natural product chemistry 14 (2), 2025: 969-975 purity of dna in the sample. dna isolates were tested using a uv-vis spectrophotometer at a wavelength of 260 nm and 280 nm and using a 400x dilution with a ratio of 3990 μl of aquabidest and 10 μl of dna isolate. table 1. results of dna quantitative test using uv-vis spectrophotometer results of dna quantitative test using uv-vis spectrophotometer. sample code λ260 λ280 concentration (λ260 x 50ng/μl x dilution factor) dna purity (λ260/λ280) k1 0.016 0.020 320 0.8 k2 0.017 0.019 340 0.8947 j1 0.017 0.016 340 1.0625 j2 0.021 0.019 420 1.1052 j3 0.021 0.019 420 1.1052 j4 0.017 0.016 340 1.0625 j5 0.020 0.020 400 1 j6 0.019 0.019 380 1 j7 0.018 0.016 360 1.125 j8 0.018 0.016 360 1.125 *dna purity limit : 1,8 – 2,0 *ideal dna concentration above 100 ng/μl table 4.1 shows the results of quantitative tests which include dna concentration and purity. the results of the dna quantity test obtained in all samples tested obtained a fairly good dna concentration with an average value of more than 100 ng/μl and the results of the dna purity test obtained an average of less than 1.8. so it can be concluded that the dna isolate in all samples tested has a good concentration of more than 100 ng/μl but has a low purity value of less than 1.8. dna concentration was measured with a wavelength of 260 nm. dna purity test uses the result of absorbance wavelength 260 nm divided by the result of absorbance wavelength 280 nm. the level of dna purity can also be affected by using dna isolation kits and different dna purification solutions. dna concentration refers to the number of dna molecules present in one unit volume such as nanograms per microliter (ng/μl). a high dna concentration indicates many dna molecules in the sample being analyzed. a high concentration of dna does not necessarily mean the dna is pure. dna samples with high concentrations are likely to contain significant amounts of contaminants. according to lucena, et al (2016) the presence of rna can cause an increase in the value of dna concentration because the uv vis spectrophotometer at the time of absorbance reading cannot distinguish between dna and rna. so it can be said that the wavelength of 260 nm which is the maximum value of dna absorbing light can be influenced by the presence of rna which is read at a wavelength of 260 nm as dna which makes the results higher because the uv-vis spectrophotometer cannot distinguish between dna and rna. contaminants can affect the interpretation of the results. dna purity refers to the extent to which the analyzed dna sample is free from contamination or other foreign materials. the results of the dna purity test using the uv-vis spectrophotometer found that almost all sample codes had low dna purity values, with good dna purity having a ratio of 1,8 – 2,0. according to mollah et al (2022) the results obtained on the uv-vis spectrophotometer can be influenced by solvent components contained in the dna solution so that dna impurities in the form of phenol compounds or contaminants that occur during dissolved dna extraction greatly affect the stability of the resulting dna concentration. these contaminants can be in the form of carbohydrates or proteins that cause the 280nm wavelength absorption to increase so that the purity value is influenced by the 280 wavelength value. good quality dna has a purity of 1.8 2.0 and an ideal dna concentration above 100 ng/μl. dna purity values that show numbers below 1.8 indicate the presence of high protein and polysaccharide contaminants, and vice versa if the purity value of dna is more than 2.0 indicates a fairly high rna contaminant in the isolation results. added, besides that the low purity of dna can be influenced by the use of cuvettes that are not clear so that the absorbance of uv light on a uv-vis spectrophotometer is disturbed and can also be caused from a technical point of view at the time of measurement, homogenization and improper pipetting processes that cause dna to break into fragments. the highest concentration of dna depends on the amount of dna, sample conditions and several factors at the time of dna extraction. the cell lysis and precipitation stage requires the removal of supernatant which precipitates dna in some samples which makes the extraction rate factor an influential factor as well as other factors that can affect the quantity of dna such as the type of sample used, extraction method, and less sterile environmental conditions. the working principle of uvvis spectrophotometer is based on the absorption of light at a certain wavelength of a sample being analyzed and the results of uv-vis spectrophotometer analysis can be used for qualitative and quantitative purposes (sulistyani et al., 2023). it is necessary to measure quantitative tests with nanodrops but cannot be done in this study due to tool limitations. optimization of annealing temperature before pcr amplification is performed, it is necessary to optimize the annealing temperature. annealing temperature optimization performed for pcr involves determining the optimal temperature for a particular primer and the dna target sequence being amplified so that the primer can bind specifically to the target region. this study used a pair of forward primers and reverse primers designed with the national center for biotechnology information (ncbi) website. a good primer is a primer that has characteristics such as primer length between 18 30 bases, % gc between 40 60%, tm between 50 – 600c and does not have a secondary wijayanti & sayekti – detection of the p53 gene in formalin fixed tissue archives … 973 structure and dimer. the results of primer design obtained several primer pair options which were then analyzed using netprimer and oligoevaluator software to obtain the best primer results as follows: forward primer 5'ctaaatccccaagacttcct-3' and reverse primer 5'accagttgattagcagagaa-3' with a target of 666bp. primers were chosen because they have good primer criteria, namely having no secondary structure, having a rating of 91% and 100% no hairpins. figure 2. optimization of annealing temperature results. annealing temperature optimization performed for pcr involves determining the optimal temperature for a particular primer and the dna target sequence being amplified so that the primer can bind specifically to the target region. temperature optimization in this study was carried out at a temperature range of 50 – 580c, then the best annealing temperature results were obtained, namely 57.40c which was amplified at 666 bp because the visualization results at that temperature showed thick and intact dna bands and the absence of double bands. at 4 temperatures, namely 50.0, 50.5, 51.5, 53.1 there are double bands, this is due to annealing temperatures that are too low so that the primer attaches to non-specific locations on the dna template. according to anissa et al (2024) a low annealing temperature causes the primer to bind to an unspecific attachment area which will then result in the amplification of unwanted locus fragments and according to aulia et al (2021) to obtain thick pcr dna bands it is necessary to optimize the annealing temperature on the primer to be used tm will be the basis for determining the annealing temperature or ta. an annealing temperature that is too high will cause the detachment of the primer attached to the printed dna so that the pcr product will not be formed, and vice versa if the annealing temperature is too low it will cause nonspecific primer attachment to the printed dna. the synthesis results for the tm of forward primer 54.000c and reverse primer 53.980c were then calculated to estimate the tm temperature. pcr temperature optimization with 8 temperatures with a range of 50.00c – 580c in accordance with the temperature range of tm calculation results of primer synthesis. figure 2. is the result of electrophoresis visualization of the temperature optimization process which shows that the primer amplifies dna well and there is a dna band that matches the target at 666 bp. in these results, the temperature selection of 57.4˚c is based on the firm boundaries of the band, the clarity of the band, and the absence of double bands. after determining the optimum temperature, the pcr amplification process was carried out at a pre-denaturation temperature of 950c for 3 minutes, denaturation 950c for 30 seconds, annealing 57.40c for 30 seconds, extention 720c for 1 minute, followed by electrophoresis and visualization using gel doc. amplification analysis of isolation results was carried out using specific primers of the p53 gene. polymerase chain reaction (pcr) test dna amplification was carried out by polymerase chain reaction (pcr) method using a pair of primers, namely forward primer 5'ctaaatccccaagacttcct-3' and reverse primer 5'accagttgattagcagagaa3' which has a target length of 666 bp. the pcr program used adjusts the results of annealing temperature optimization that has been done before. figure 3. pcr visualization results of p53 gene in normal tissue and formalin fixed tissue archive. description: k1 : nail control k2 : hair control j1 – j2 : 2 day formalin fixed tissue j3 – j4 : formalin fixed tissue <1 month j5 – j6 : 2 months formalin fixed tissue j7 – j8 : formalin fixed tissue > 5 years the visualization of p53 gene detection through the electrophoresis process can be seen in figure 3. the results show that in all samples there are dna bands according to the target, namely 666bp with different dna band thicknesses, there are thick dna bands and there are thin dna bands and some have smears and some do not have smears. the absence of smears or impurities indicates that the results of dna amplification do not contain any residual impurity or contaminant solution. smear indicates that the results of dna isolation contain contaminants such as proteins, rna, degraded dna, or the isolation solution used. sample codes j1, j2, j7 and j8 appear dna bands at a size that does not match the amplification target, which is 666bp, 974 biology, medicine, & natural product chemistry 14 (2), 2025: 969-975 indicating nonspecific amplification. this is likely caused by primers that attach to non-target dna sequences. the success of dna amplification can be influenced by several, namely the concentration of pcr components, purity, annealing temperature which are technical factors and non-technical factors such as contamination (feranisa, 2020). primers have a level of specificity to the target dna. if the primer is not specific, it can cause mispriming or cannot amplify dna. low dna concentration will cause thin and faint dna bands (maliza et al., 2021). according to exzora & qurrohman (2024) cited from aristya et al (2013) stated that dna that has low purity does not become a nuisance during dna amplification by polymerase chain reaction (pcr) method if the primer is a specific primer. however, according to daryono & yambise (2018) in general, the value of dna purity is directly proportional to the sensitivity of the pcr test, the higher the purity of dna, the more pcr sensitivity increases. so it can be concluded that dna purity affects the efficiency and sensitivity of pcr. however, under certain conditions, pcr can still detect despite low dna purity, especially if the primers used are highly specific to the dna target. however, to obtain more optimal results, namely specific amplification and having a good dna band, it still requires dna that has purity according to the ideal dna purity value of 1,8 – 2,0. in addition to dna purity, dna concentration is also an important factor to obtain optimal pcr results. sample codes j7 and j8 in figure 3. there is a dna band that was not present in the dna isolation process there was no dna band, this is because the pcr technique can amplify dna segments millions of times in just a few hours so that in sample codes j7 and j8 dna bands are visualized after the pcr process. the p53 gene is a tumor suppressor protein that is responsible for the apoptotic mechanism of cancer cells. the mechanism is the regulation of repair of a cancerinduced cell. specific p53 protein expression occurs in cells that undergo changes from normal cells to abnormal cells and requires regulation of programmed death called apoptosis. the increased expression of the p53 gene, the higher the tendency for apoptosis to occur. in the observation of p53 using immunohistochemical painting method, p53 expression is indicated by brown color in the cell cytoplasm (prihastuti, 2010). expression of p53 that shows a significant increase in expression is associated with an increase in the rate of invasive cancer tumors (sadia et al., 2020). conclusions based on the results of this study, it was concluded that the p53 gene was detected in 10 samples consisting of 8 samples of formalin-fixed tissue archives and 2 samples in normal tissue as a control visualized at 666 bp. acknowledgements: thanks to the anatomical pathology laboratory and the laboratory of molecular biology, sekolah tinggi ilmu kesehatan nasional for providing the right place to conduct this research. authors’ contributions: fitria diniah janah sayekti, s.si., m., sc contributed to this research as the main supervisor who helped in the research methodology and narration of this article. anastasia elma w contributed to the research design, practicum, and narration of this article. competing interests: the authors declare that there are no competing interests. references anam, k., cahyadi, w., azmi, i., senjarini, k., & oktarianti, r. 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(2010). analisis dna tulang dan gigi pada lokus short tandem repeat – combined dna index system (str codis), y-chromosome strs & mitochondrial dna (mtdna) akibat paparan panas suhu tinggi. universitas airlangga surabaya. https://www.researchgate.net/publication/282592026 https://doi.org/10.1089/bio.2015.0064 https://doi.org/10.20956/at.v15i1.688 https://doi.org/10.29303/jbt.v21i1.2439 https://doi.org/10.35451/mmj.v2i1.2371 https://doi.org/10.4314/ahs.v20i3.32 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 887-896 | doi: 10.14421/biomedich.2025.142.887-896 issn 2540-9328 (online) prospects of dwarf coconut (cocos nucifera l. var. genjah) as a cultivar for coconut sugar production in east lampung lukman efendi1, analianasari2*, surfiana2 1master of applied agriculture study program, state polytechnic of lampung, bandar lampung, lampung province 2food technology study program, state polytechnic of lampung, bandar lampung, lampung province jl. soekarno hatta no.10, rajabasa raya, kec. rajabasa, kota bandar lampung, lampung 35141, indonesia corresponding author* analianasari@polinela.ac.id manuscript received: 02 august, 2025. revision accepted: 14 october, 2025. published: 24 october, 2025. abstract coconut sugar is emerging as a promising natural sweetener to reduce indonesia’s dependency on imported refined sugar. among various coconut cultivars, cocos nucifera l. var. genjah (dwarf coconut) offers advantages for sap production due to its short trunk, early maturity, and high sugar content. this study aimed to examine the prospects of dwarf coconut as a productive cultivar for coconut sugar development in east lampung regency, based on field-level adoption indicators. a total of 41 respondents were selected using slovin’s formula. primary data were collected through structured interviews and analyzed using descriptive statistics, t-tests, and multiple linear regression with spss version 27. the findings show that factors such as landholding size, formal education exposure, and income levels significantly (p < 0.05) influence the practical use of dwarf coconut for coconut sugar production. these results highlight the potential of dwarf coconut as a strategic cultivar to support local coconut sugar initiatives in indonesia. keywords: coconut sugar; dwarf coconut; east lampung. introduction sugar is a strategic food commodity that plays a vital role in the daily lives of indonesians. at present currently, national sugar needs are predominantly met by cane sugar. however, domestic sugar production remains insufficient to fulfill both household and industrial demands. in 2023, indonesia’s sugar production was estimated at only 2.27 million tons, significantly lower than the national requirement of approximately 7 million tons. consequently, the government has had to rely heavily on imported refined sugar, with imports reaching 5 million tons in 2023, up from 4.4 million tons in 2017 (central statistics agency, 2024). this high volume of imports reflects not only economic vulnerability but also a growing public health concern. refined sugar, commonly found in processed foods, sweetened beverages, and baked goods, is linked to several non-communicable diseases, including obesity, diabetes, metabolic syndrome, hypertension, dental caries, and certain types of cancer (arshad et al., 2022). therefore, the development of alternative, locally sourced sweeteners with improved nutritional and health profiles is essential. one such alternative is coconut sugar, derived from the sap of coconut inflorescences and processed through evaporation until it solidifies. coconut sugar is recognized for its lower glycemic index (approximately 35), making it safer for consumption by individuals with diabetes (trinidad et al., 2010). it also contains beneficial nutrients such as proteins, calcium, magnesium, potassium, iron, zinc, and b-complex vitamins (hebbar et al., 2015; jati et al., 2015), which are not present in refined sugar. furthermore, one hectare of coconut trees has the potential to yield up to 19 tons of coconut sugar annually, significantly outperforming sugarcane, which yields only 5–10 tons per hectare. this highlights the potential of coconut sugar to contribute to sustainable sweetener production, particularly in tropical countries like indonesia, one of the world’s largest coconut producers. despite this potential, the productivity of coconut sugar in indonesia remains relatively low. for example, in cilacap regency, productivity only reaches 8 tons per hectare per year. contributing factors include the prevalence of aging and unproductive coconut trees, declining interest among the younger generation in becoming coconut tappers, limited technological adoption, and a lack of replanting efforts. according to the ministry of agriculture (2022), the area of smallholder coconut plantations has declined by an average of 0.99% per year over the past decade. one promising approach to address this issue is through the introduction and expansion of dwarf coconut https://doi.org/10.14421/biomedich.2025.142.887-896 888 biology, medicine, & natural product chemistry 14 (2), 2025: 887-896 (cocos nucifera l. var. genjah) cultivation. dwarf coconuts are characterized by shorter trunks, earlier sap production (within 3–4 years), and high sugar content in the sap (13.51–14.56%) (mashud & matana, 2014). additionally, their compact growth habit allows for denser planting, which can potentially increas sap yield per hectare and enhanc overall productivity. east lampung regency presents significant opportunities for the development of dwarf coconutbased sugar production. it is the second-largest coconutproducing region in lampung province after south lampung (central statistics agency lampung, 2021). since 2019, several farmers in east lampung have initiated dwarf coconut cultivation through partnerships with private sector stakeholders, including pt unilever indonesia. farmers have noted that the short stature of dwarf coconut trees simplifies the tapping process, improves safety, and increases labor efficiency. however, despite this potential, adoption remains limited. of an estimated 35,714 potential coconut farmers (most with landholdings of approximately 0.5 hectares), only about 560 have adopted dwarf coconut cultivation. this low adoption rate highlights the need for a deeper understanding of the underlying factors influencing the utilization of dwarf coconut as a productive cultivar for coconut sugar production. while agronomic characteristics are favorable, successful implementation also depends on various contextual elements, including land availability, economic feasibility, and access to institutional support and information. thus, this study aims to assess the prospects of dwarf coconut as a strategic cultivar for coconut sugar production in east lampung by analyzing factors influencing its utilization at the field level. the findings are expected to inform future strategies for improving adoption and scaling up coconut sugar production in indonesia. materials and methods study area this study was conducted from january to june 2025 in east lampung regency, one of the central regions for coconut production in lampung province, indonesia. the area was selected due to its significant potential for coconut cultivation and its role as a center for the developmen of dwarf coconut (cocos nucifera l. var. genjah) plantations, particularly under private sector initiatives. sampling was conducted using a purposive sampling technique, targeting individuals with the following criteria: (1) male gender, (2) cultivating dwarf coconut plants, and (3) producing coconut sugar from dwarf coconut. from a total population of 560 coconut sugar producers, a sample size of 41 respondents was determined using the slovin formula with a 15% margin of error. data collection primary data were collected through field observations and structured interviews using the validated questionnaire. secondary data were sourced from institutional reports, scientific literature, and other relevant documentation. data collection employed a structured questionnaire using a five-point likert scale (1 = strongly disagree to 5 = strongly agree). the questionnaire consisted of statements designed to measure influencing factors associated with the utilization of dwarf coconut for sugar production. prior to field deployment, the questionnaire underwent validity and reliability testing using a pilot group of 20 respondents. validity was assessed using pearson's bivariate correlation with a minimum threshold of r > 0.5, while reliability was determined using cronbach’s alpha with a threshold of >0.6. the results confirmed that all questionnaire components were both valid and reliable. data analysis data analysis was divided into two stages: descriptive and inferential. descriptive statistics were used to describe the characteristics and response tendencies of the respondents. the likert scale scores from the questionnaire were summed and averaged. the resulting values were interpreted using an interval class formula to categorize responses (e.g., strongly agree, agree, neutral, etc.), allowing for the assessment of overall trends among the farmers' responses. inferential analysis was conducted using multiple linear regression to examine the relationship between five independent variables—age (x1), formal education (x2), non-formal education (x3), land area (x4), and income (x5)—and the dependent variable, which is farmer attitude (y). the regression model is expressed as follows: 𝑌 = 𝛼 + 𝛽1𝑋1 + 𝛽2𝑋2 + 𝛽3𝑋3 + 𝛽4𝑋4 + 𝛽5𝑋5 + 𝜀 where: ▪ y is the dependent variable (attitude toward dwarf coconut cultivation for sugar production), ▪ á is the intercept, ▪ â1 to β5 are regression coefficients for each independent variable, and ▪ å is the error term. to ensure the validity of the regression model, classical assumption tests were conducted, including the normality test (to confirm that residuals are normally distributed), the heteroscedasticity test (to check for constant variance of residuals), and the multicollinearity test (to detect intercorrelations among predictors). the efendi et al. – prospects of dwarf coconut (cocos nucifera l. var. genjah) as a … 889 strength and significance of the regression model were evaluated using the coefficient of determination (r²), the f-test (to assess model fit), and the t-test (to evaluate the significance of each independent variable). results and discussion characteristics of farmers and their role in supporting dwarf coconut sugar production the characteristics of dwarf coconut farmers include age, last education, occupation, income, land area, land status, farming experience, number of trees, tree age, sap production, sugar production, and number of extension sessions attended during one year, as presented in table 1. these characteristics are closely related to the current condition and prospects of utilizing cocos nucifera l. var. genjah (dwarf coconut) as a primary cultivar for coconut sugar production in east lampung. table 1. characteristics of dwarf coconut farmers (n=41) profile description number of respondents percentage (%) age 31 – 40 years old 9 21.95 41 – 50 years old 17 41.46 51 – 60 years 13 31.71 61 – 70 years old 2 4.88 education primary school 14 34.15 middle school 15 36.59 high school 9 21.95 higher education 3 7.32 work coconut sugar farmers 41 100 income < rp. 2,000,000 6 14.63 rp. 2,000,000 – rp. 4,000,000 15 36.59 rp. 4,000,000 – rp. 6,000,000 13 31.71 > rp. 6,000,000 7 17.07 land area 0.25 ha 26 4.88 0.5 ha 24 58.54 0.75 ha 1 2.44 > 1 ha 14 34.15 land status personal 38 92.68 rent 3 7.32 farming experience 1 – 13 years 10 24.39 14 – 26 years 21 51.22 >27 years 10 24.39 number of trees 1 50 trees 21 51.22 > 50 trees 20 47.88 age of the tree 15 years 29 70.73 6 – 10 years 12 29.27 palm oil production 1-1.5 liters/stick 15 29.27 1.6-2 liters/stick 26 63.41 sugar production ≤ 300 kg/month 21 51.22 > 300 kg/month 20 47.88 frequency following counseling >4 times 4 9.76 4 times 30 73.17 3 times 7 17.07 table 1 shows that the majority of the 41 respondents in this study were farmers aged 41–50 years, most of whom had a junior high school education, worked as coconut sugar farmers, and earned a monthly income ranging from idr 2,000,000 to idr 4,000,000 (average idr 4,318,536). most dwarf coconut farmers cultivated land areas of 0.5 hectares (average 0.67 hectares) with private land ownership status. their farming experience ranged from 14 to 26 years (average 21.19 years), and they owned 1–50 dwarf coconut trees (average 63.65 trees) with tree ages between 1–5 years (average 5.14 years). in terms of productivity, most farmers produced 1.6–2 liters of sap per day per tree (average 1.76 liters/day/tree), resulting in ≤ 300 kg/month of coconut sugar (average 359.87 kg/month). additionally, they had participated in agricultural extension activities four times within a year. this demographic and technical profile reflects the adaptive potential of smallholder farmers in east lampung in developing coconut sugar agribusinesses based on dwarf coconut varieties. each variable is discussed further below in the context of its relevance to 890 biology, medicine, & natural product chemistry 14 (2), 2025: 887-896 the sustainability and scalability of coconut sugar production using cocos nucifera l. var. genjah. age the majority of the 41 respondents in this study were aged 41–50 years. this age group is categorized as productive adults, generally considered to be at the peak of physical and mental abilities for optimal work. these farmers typically have over 20 years of farming experience, which contributes to their skills in managing farming activities and making informed decisions. the dominance of this age group also reflects the slow regeneration of farmers, with younger generations less inclined to enter agriculture due to economic concerns. these findings are consistent with manyamsari & mujiburrahmad (2014) and marlina et al. (2013), who also found that farmers in this age range dominate the agricultural workforce and play key roles in agricultural development. education most early-maturing coconut sugar farmers had a junior high school education. while there has been a shift from elementary to secondary school (nazaruddin & anwaruddin, 2019), farming is still viewed as a hereditary livelihood that does not demand formal education. children are often involved as labor (oktafiani et al., 2021), acquiring farming skills informally. budiningsih & watemin (2014) emphasized the traditional lifestyle of coconut sugar farmers, where knowledge is passed from parent to child. in this context, the education level of farmers does not hinder coconut sugar production, particularly from dwarf coconut varieties, as long as they acquire practical knowledge and skills. moreover, harsanto et al. (2024) found that dwarf coconut farmers are more receptive to practical training than theoretical instruction, supporting the effectiveness of hands-on learning. income the average monthly income of dwarf coconut farmers is idr 4,318,536, with most farmers earning between idr 2,000,000 and idr 4,000,000. this places them in the lower-middle income category, which, according to nata et al. (2020), is economically vulnerable. previous studies such as muhroil et al. (2015) have shown even lower income levels (idr 1,332,896/month), although with a positive r/c ratio of 2.18. ramadhan et al. (2023) reported that daily production of 2.36 kg of sugar could generate idr 4,367,750/month, suggesting that improved production consistency can lead to better outcomes. however, challenges such as limited market access and dependence on middlemen still hinder profits. therefore, increasing income requires improved production efficiency, better access to capital, and stronger farmer institutions to support marketing. land area most farmers cultivate around 0.5 hectares of land, with an average of 0.67 hectares per farm. this size classifies them as smallholder or household-scale producers. according to utomo et al. (2022), coconut farmers commonly manage land smaller than 0.5 ha, yet find strong economic motivation in doing so. dwarf coconut varieties are suitable for these settings due to their low stature and early fruiting (3–4 years), enabling efficient use of space. ghosh et al. (2023) also noted that moderate income levels can be achieved even with land sizes of 0.21–0.6 ha. the ability to intensify planting in limited areas further supports the appropriateness of dwarf coconut varieties for sugar production among smallholder farmers. land status the majority of dwarf coconut farmers own their land, typically through inheritance or independent purchase. private ownership provides security and freedom in managing land, making long-term investments in coconut sugar production more feasible. harsanto et al. (2024) emphasized that dwarf coconut is often planted in family gardens or yards, reducing dependency on rental systems. singha et al. (2012) also found that land ownership is positively correlated with higher income and motivation to adopt technology, suggesting that secure tenure enhances the sustainability of the coconut sugar business. farming experience with experience ranging from 14 to 26 years (average 21.19 years), farmers fall into the experienced category. aisyah et al. (2023) classify farmers with over 10 years of experience as having mature technical skills and better adaptability. experience in sap tapping and sugar processing, which are manual and skill-intensive tasks, is often gained through long-term practice rather than formal education. manyamsari & mujiburrahmad (2014) also highlighted experience as a key success factor in agricultural management. this long tenure supports the sustainability of dwarf coconut farming systems and opens up opportunities for gradual modernization. number of trees farmers own 1–50 dwarf coconut trees, with an average of 63.65 trees. although limited by land area and capital, this number still classifies them as small to medium-scale commercial farmers (kusumo et al., 2018). dwarf coconut’s compact size and fast maturity allow them to be planted in yards or intercropped. utomo et al. (2022) found similar patterns in karimunjawa, where limited land availibility constrained the number of trees. labor availability also influences planting decisions, as sap tapping is labor-intensive. hence, the number of trees reflects a practical adaptation to resource constraints. efendi et al. – prospects of dwarf coconut (cocos nucifera l. var. genjah) as a … 891 tree age most trees are 1–5 years old, with an average of 5.14 years, placing them in the early productive phase. dwarf coconut varieties start fruiting around 3–4 years (tulalo & mawardi, 2018). this suggests that farmers have recently adopted these cultivars, possibly due to government support or increased awareness of their benefits. harsanto et al. (2024) confirmed that many farmers began planting dwarf coconuts within the past five years due to their superior traits and suitability for small plots. since sap yield becomes more stable around year five, the current tree age indicates future potential for increased sugar production. sap production the sap yield ranges from 1.6 to 2 liters per day per tree, with an average of 1.76 liters—considered medium to high productivity. tulalo & mawardi (2018) noted that some dwarf varieties can produce 1.2–2.0 liters per day, depending on tree age, tapping method, and environmental conditions. farmers practicing good maintenance, sanitation, and proper tapping timing tend to achieve higher yields. mashud & matana (2014) observed similar productivity in a plantation of 138 trees on 1 hectare using a rectangular planting system. thus, current sap production levels confirm the agronomic feasibility of dwarf coconut as a source of sap for sugar production. sugar production farmers produce ≤300 kg/month of coconut sugar, with an average of 359.87 kg/month, indicating small to medium-scale production. based on sap volumes and typical conversion rates (15–20%), this output is consistent with household-level operations using 30–50 trees per year. ramadhan et al. (2023) found that 2.36 kg/day of production yields approximately 218.39 kg/month. although modest, these figures underscore the economic potential of dwarf coconut sap, particularly when combined with effective processing and consistent tapping. these trees offer strong potential as a raw material source for small-scale but scalable coconut sugar enterprises. frequency of attending extension sessions the majority of farmers attended four extension sessions annually, reflecting moderate to high engagement in capacity building. harsanto et al. (2024) noted that farmers who participate regularly in extension activities show improved practices. maretya & sudrajat (2017) also found that higher participation correlates with better land management. this participation demonstrates farmers’ willingness to improve their skills related to dwarf coconut cultivation and coconut sugar production, although more frequent and targeted training could further enhance both yield and quality. descriptive analysis this study employed three scoring categories: disagree (1.00–2.33), agree (2.34–3.67), and strongly agree (3.68–5.00). table 2 presents the descriptive statistics for each variable related to farmer characteristics and their attitudes toward using dwarf coconut (cocos nucifera l. var. genjah) for coconut sugar production in east lampung. table 2. descriptive analysis. variables mean std information age 3.99 0.63 strongly agree formal education 3.86 0.67 strongly agree non-formal education 4.59 0.50 strongly agree land area 3.76 0.91 strongly agree income 4.14 0.38 strongly agree attitude 3.66 1.04 agree source: primary data (2025) the results indicate that most farmers strongly agree that variables such as age, formal education, non-formal education, land area, and income play important roles in shaping their attitudes toward cultivating dwarf coconuts for coconut sugar production. these variables reflect a readiness and optimism among local farmers to adopt dwarf coconut as a sustainable and productive cultivar. farmers also expressed that cultivating dwarf coconuts brings enthusiasm, interest, and satisfaction, which supports the economic potential of this variety in east lampung. classical assumption test the normality test (kolmogorov-smirnov) is used to test whether the dependent and independent variables have a normal distribution or not, as seen from the asymp sig. (2-tailed) value > 0.05, which means the data is normally distributed. the results of the normality test are shown in table 3. table 3. normality test. asymp. sig. (2-tailed) 0.200 in table 3, it can be seen that the asymp sig. (2tailed) the values are 0.200 > 0.05, which means that the model of the regression equation is normally distributed. the multicollinearity test is used to detect whether independent variables in a regression model are correlated with each other. to meet this test, there must be no correlation between each independent variable, as seen from the tolerance value > 0.1 and vif < 10, then there are no symptoms of multicollinearity. the results of the multicollinearity test are shown in table 4. 892 biology, medicine, & natural product chemistry 14 (2), 2025: 887-896 table 4. multicollinearity test. variables tolerance vif age (x1) 0.433 2,311 formal education (x2) 0.569 1,756 non-formal education (x3) 0.860 1,163 land area (x4) 0.813 1,230 income (x5) 0.566 1,765 in table 4, it can be seen that all variables have a tolerance value > 0.1 and a vif value < 10, which means there are no symptoms of multicollinearity between the variables in this study. the heteroscedasticity test (gleiser) is a test that aims to detect whether there is inequality in residual variations from one observation to another in a regression model. to fulfil this test, there must be no symptoms of heteroscedasticity in the regression model as indicated by the sig. value > 0.05 so that there are no symptoms of heteroscedasticity. the results of the heteroscedasticity test are shown in table 5. table 5. heteroscedasticity test. variables sig. age (x1) 0.794 formal education (x2) 0.322 non-formal education (x3) 0.938 land area (x4) 0.052 income (x5) 0.082 in table 6, it can be seen that each variable has a sig. value > 0.05, which means that the resulting regression equation does not exhibit symptoms of heteroscedasticity. hypothesis testing hypothesis testing was conducted using the t test, f test, and coefficient of determination. the f test was used to simultaneously test the level of significance of the influence of independent variables on the dependent variable by looking at the sig. <0.05 value in the anova analysis model. the t test was used to partially test the level of significance of the influence of independent variables on the dependent variable by looking at the sig. <0.05 value in the coefficients model. the coefficient of determination is the proportion of the influence of all independent variables on the dependent variable by looking at the value of r square. the predictive factors identified in this study were age, formal education, non-formal education, land area, and income that can influence the attitude of dwarf coconut farmers for coconut sugar production. the results of the hypothesis test analysis are shown in table 6. table 6. results of multiple linear regression analysis. variables regression coefficient standard error t-count p-value age (x1) -0.041 0.136 -0.304 0.763 formal education (x2) 0.396 0.185 2,147 0.039** non-formal education (x3) -0.270 0.135 -1,995 0.054 land area (x4) 0.374 0.144 2,592 0.014** income (x5) 0.705 0.271 2,604 0.013** constant 3,242 4,477 0.724 0.474 f count 6,187 sigh f <0.001 adjusted r2 0.530 n 41 source: primary data (2025). based on the analysis results, the regression equation obtained for the research results is 𝑌 = 3,242 − 0,041𝑋1 + 0,396𝑋2 − 0,270𝑋3 + 0,374𝑋4 + 0,705𝑋5 + 𝜀 it is clear that age and non-formal education do not influence the attitudes of dwarf coconut farmers toward coconut sugar production. however, formal education, land area, and income significantly influence the variables. formal education influences farmers’ attitudes toward the prospects of dwarf coconut for coconut sugar production farmers' perceptions of formal education significantly influence their attitudes toward the potential of dwarf coconut cultivation for coconut sugar production (p = 0.039 < 0.05). in east lampung regency, dwarf coconut farmers possess educational backgrounds ranging from efendi et al. – prospects of dwarf coconut (cocos nucifera l. var. genjah) as a … 893 elementary to tertiary levels. while most farmers have substantial farming experience—averaging 21.19 years— their level of formal education contributes to differentiated attitudes, especially in their receptiveness to innovations in dwarf coconut cultivation and its viability for coconut sugar production. studies have shown that formal education enhances farmers’ understanding of modern agricultural practices and technologies, thereby improving productivity. according to mohith & narayanaswamy (2019), bettereducated farmers are more inclined to adopt innovative techniques, including those related to coconut sugar processing. asfaw et al. (2012) similarly asserted that formal education significantly increases the probability of adopting agricultural innovations in developing countries. education allows farmers to interpret technical knowledge, access reliable sources of information, and respond to market demands more flexibly. faizah et al. (2024) emphasized that formal education equips farmers with better capacity to assess both the benefits and risks of innovation, thereby fostering a stronger readiness for change. moreover, while formal education is crucial, farming experience remains a vital factor. experienced farmers possess a deep practical understanding, allowing them to be rational yet selective in adopting new agricultural technologies. thus, formal education plays a strategic role not only in encouraging the adoption of dwarf coconut cultivation practices but also in enhancing the production of coconut sugar. it is therefore crucial that empowerment strategies emphasize education—both formal and informal (e.g., training and agricultural extension)—to sustainably increase the productivity and quality of coconut sugar derived from dwarf coconut cultivars. land area influences farmers’ attitudes toward dwarf coconut as a coconut sugar source farmers' perceptions of land area significantly affect their attitudes toward adopting dwarf coconut cultivation for coconut sugar production (p = 0.014 < 0.05). landholding size is a key factor in determining a farmer’s production capacity, resource allocation, and motivation. the analysis revealed that farmers with larger land areas tend to exhibit a more proactive and positive attitude toward utilizing dwarf coconuts for sap production. this implies that larger landholdings offer greater economic potential, motivating farmers to optimize their yields through more intensive and innovative cultivation. rahman et al. (2025) highlighted that larger land size is positively associated with the adoption of agricultural technology, primarily due to better financial capacity and higher risk tolerance. similarly, mwangi & kariuki (2015) noted that the land area plays a pivotal role those for farmers' decisions to adopt agricultural innovations, including in plantation crops such as coconuts. farmers with expansive land resources are more economically capable, which encourages them to pursue technological improvements and develop new products. manwan et al. (2022) emphasized that larger land areas enable economies of scale, facilitating greater investment in resources and technologies needed for efficient coconut sugar production. consequently, land area serves not only as a physical production factor but also as a psychological and economic driver in shaping farmers attitudes toward dwarf coconut utilization. therefore, policy initiatives should address land-related disparities by supporting smallholders through intensification programs and providing incentives that enable efficient adoption of technology even on smaller plots. income influences farmers’ attitudes toward dwarf coconut-based coconut sugar business farmers' perceptions of income levels significantly influence their attitudes toward developing dwarf coconut cultivation for coconut sugar production (p = 0.013 < 0.05). the findings suggest that income is a significant determinant of positive farmer attitudes, particularly regarding the adoption of innovation and the establishment of sustainable coconut sugar enterprises based on dwarf coconut varieties. this observation aligns with oladele (2005), who found that income has a significant impact on farmers’ participation in agricultural extension services and their willingness to adopt new technologies. higher income allows farmers to take calculated risks, access high-quality inputs, and participate in capacity-building programs. similarly, ayinde & obalola (2017) noted that higher income levels enhance farmers' risk tolerance, investment in new technologies, and business diversification. setiyowati et al. (2022) also found a positive correlation between income and innovation adoption. wongkar et al. (2016) further emphasized that farmers with greater capital tend to be more confident in experimenting with agricultural innovations. dwarf coconut, with its short gestation period (approximately 3–4 years), daily sap production, and high market demand—especially in the exportoriented coconut sugar sector—is seen as a promising investment. farmers’ attitudes are shaped not only by income level but also by their perceptions of business stability and long-term profitability. therefore, farmer income plays a crucial role in shaping attitudes toward dwarf coconut cultivation. policy interventions must focus on improving incomegenerating opportunities for smallholder farmers, such as by expanding market access, promoting rural economic development, and offering financial support like people’s business credit (kur), to scale up and enhance the quality of coconut sugar production from dwarf coconut varieties. age and non-formal education do not significantly influence farmers’ attitudes farmers’ perceptions regarding age (p = 0.763 > 0.05) and non-formal education (p = 0.054 > 0.05) do not significantly influence their attitudes toward dwarf 894 biology, medicine, & natural product chemistry 14 (2), 2025: 887-896 coconut cultivation for coconut sugar production. although the p-value for non-formal education was close to the significance threshold, neither variable showed a statistically meaningful impact. non-formal education— comprising training, extension, farmer group discussions, and field assistance—is ideally designed to enhance farmer knowledge and practical skills. however, the study revealed that participation in these activities did not result in significant changes in attitudes. this finding is consistent with hardinah et al. (2022), who stated that despite frequent agricultural training, its impact on farmers' attitudinal development remains limited. sulaiman et al. (2005) explained that when extension services are not tailored to farmers' actual needs, they fail to effectively influence behavior. nugroho et al. (2023) similarly noted that factors such as land access and household economic conditions have greater influence than age or non-formal education. a potential explanation is the quality and relevance of the training: village-level programs often lack practical application, are not problem-driven, and are sometimes conducted as mere formalities. additionally, ineffective delivery methods—such as one-way lectures without hands-on practice—limit knowledge retention and attitude change. sinaga & sudarko (2024) highlighted that non-formal training without follow-up mentoring or field demonstrations risks being superficial and nontransformative. regarding age, most farmers in the study were in the 41–50 age bracket, yet age did not significantly influence their receptiveness to adopting dwarf coconut innovations. this aligns with satriawan et al. (2023), who found that age does not influence farmers’ perceptions in the context of agro-tourism development. hardinah et al. (2022) similarly observed that age is not a major factor in shaping perceptions. these findings suggest that modifying farmers' attitudes cannot rely solely on training or demographic segmentation. more comprehensive and contextual strategies—such as strengthening market access, forming business partnerships, and developing farmer economic institutions—are required to drive sustainable behavioral change. combined farmer perceptions influence attitudes toward dwarf coconut for coconut sugar collectively, farmers' perceptions significantly influence their attitudes toward utilizing dwarf coconut for coconut sugar production (p = 0.001 < 0.05), with a coefficient of determination (r²) of 0.53. this indicates that 53% of the variability in farmer attitudes can be explained by the combined effects of five independent variables: age, formal education, non-formal education, land area, and income. the remaining 47% is attributed to factors outside the scope of this model. this finding underscores that farmers’ attitudes toward dwarf coconut as a sapproducing cultivar are shaped by the interplay of sociodemographic and economic characteristics. such insights underscore the need for a multidimensional approach in analyzing and transforming farmer behavior. satriawan et al. (2023) found that characteristics such as age, education, and income significantly affect perceptions in agrotourism development. kiełbasa (2017) also noted that combinations of demographic and socioeconomic factors influence willingness to adopt agricultural technology. similarly, ghosh et al. (2023) emphasized that land area and income are particularly influential in shaping farmer attitudes toward innovation. given these findings, empowerment strategies must adopt an integrative approach that considers the specific characteristics of individual farmers. initiatives such as targeted training, access to capital, and agribusiness mentoring can foster more favorable attitudes toward adopting the dwarf coconut cultivars and improving the value-added potential of coconut sugar production. conclusions the findings indicate that key factors such as formal education, land area, and income significantly influence the acceptance and utilization of dwarf coconut for coconut sugar production. when assessed simultaneously, the five examined variables—age, formal education, non-formal education, land area, and income—collectively account for 53% of the variance in attitudes toward adopting dwarf coconut. these results underscore the importance of underlying socioeconomic characteristics in shaping decisions related to the cultivation and development of dwarf coconut as a potential cultivar for coconut sugar. to enhance the adoption of dwarf coconut cultivation, it is essential to strengthen farmers' access to both formal and non-formal education, particularly technical training that supports improved cultivation practices. additionally, further research is recommended to explore the specific role of non-formal education and to identify effective policy interventions—especially those aimed at land expansion and income improvement—that can support the sustainable development of dwarf coconut as a valuable resource for coconut sugar production in east lampung. authors’ contributions: lukman efendi, analianasari, and surfiana designed the study. lukman efendi carried out the field work. lukman efendi collected and analyzed the data. lukman efendi wrote the manuscript. analianasari and surfiana reviewed the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. efendi et al. – prospects of dwarf coconut (cocos nucifera l. var. genjah) as a … 895 references aisyah, s., faqih, a., rahudi, r., falah, m. a. s., setiawan, d., & apriansyah, b. 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(soursop) is one of tropical plants which have relatively complete chemical compounds. it has flavonoid, tannin, phytosterol, alkaloid, etc. the high antioxidant compound in soursop is believed as cancer prevention so the cancer threat in the world can be minimized. the antioxidant compound in soursop can be found not only in its fruit, but also in other parts like leaves, seeds, etc. based on that potency, this study aimed to compare antioxidant capacity of soursop leaves and seeds, also to study about the utilization of soursop parts which is usually not used. this research began with maceration to extract leaves and seeds with 96% ethanol. ethanolic extract of soursop leaves and seeds were then tested for antioxidant capacity with dpph (1,1-diphenyl-2-picrylhydrazyl) method. the result showed that antioxidant capacity of soursop leaves and seeds are 85,66875% and 39,0166, respectively. the antioxidant capacity of leaves is higher than seeds due to seed’s extraction difficulty so its antioxidant compound could not be extracted optimally. however, either leaves or seeds extract in this study are potential as antioxidant resources because there are no significant differences between antioxidant capacity of both extract. keywords: annona muricata linn.; antioxidant capacity; dpph; extraction. introduction cancer is the most dangerous killer in the world nowadays. lung cancer cause 1,3 millions of death a year, stomach cancer take the second place with 803 thousands of death a year, then colorectal cancer with 639 thousands of death a year, and breast cancer with 519 thousands of death a year. more than 70% death number were happen in developing countries with low health facilities (who, 2009). cancer can be triggered by accumulation of free radicals compounds. actually, our body needs free radicals in normal amount to prevent inflammation, kill pathogen microbes, and restrain blood vessel’s smooth muscle. the problem is free radicals will be carsinogen in large amount so it can trigger the growth of cancer cells. due to its importance, we need antioxidant to regulate the existence of free radicals inside our body, so its amount can be arranged (limbono, 2013). antioxidant can play a role to supply hydrogen radicals and as free radicals acceptor so its postpone initiation of free radicals forming (puspitasari et al., 2016). antioxidant can be obtained either natural or artificial. synthetic antioxidant are highly effective but does not safe enough to be consumed. based on that fact, the synthetic antioxidant utilizing has to be checked. natural antioxidant has better effect than artificial one. these antioxidant can naturally obtained by consuming fruits and vegetables because both of them contain natural antioxidative effect due to vitamin c, e, betacaroten, and other polyphenolic substances (nihlati et al., 2014). based on research by puspitasari et al. (2016), one of the most valuable fruits which regularly extracted as natural supplement is soursop (annona muricata linn.). it has known that soursop has flavonoid, tannin, phytosterol, and alkaloid which can be play a role as an antioxidant. regularly, soursop consumption limited on its fruit. people only consume the water extract of soursop as cancer prevention. on the other hand, the utilization of leaves and seeds extract of soursop has not been studied yet. both of them has to be compared to determine which one has higher antioxidant capacity. the objective of this research is to find either leaves or seeds of sousop (a. muricata linn.) which has higher antioxidant capacity when extracted with ethanol. so, the utilization of soursop (a. muricata linn.) will be more appropriate. materials and methods this research needs soursop (a. muricata linn.) leaves and seeds, 96% ethanol, hcl, dpph (1,1-diphenil-2http://dx.doi.org/10.14421/biomedich.2017.61.1-4 2 biology, medicine, & natural product chemistry 6 (1), 2017: 1-4 picrylhydrazyl), aquades, h2so4, naoh, mortar and pestle, grinder, rotary evaporator, glassware, analytical balance, sentrifuge, and uv-vis spectrophotometer. figure 1. soursop (a. muricata linn.) leaves. figure 2. soursop (a. muricata linn.) seeds. the dried leaves (figure 1) and seeds (figure 2) were extracted with 96% ethanol by maceration of each 800 gr of leaves and seeds. the whole filtrate was concentrated with rotary evaporator until 1/3 volume remain. the ethanol residues was then be evaporated until concentrated extract was formed with constant weight. the antioxidant capacity of leaves and seeds extract were then measured by dpph test. samples were divide into two groups, leaves extract (l) and seeds extract (s). twenty five mg leaves (l) and seeds (s) extract was measure then dissolved to 25 ml ethanol (stock solution). both l and s stock solution were pipetted 0,1 ml; 0,2 ml; 0,3 ml; and 0,4 ml. so the concentration was 4 ppm; 8 ppm; 12 ppm; and 16 ppm, respectively. five ml of 0,5 mm dpph was added to each solution then its volume was adjusted. the standard solution was made by added 25 ml ethanol to 0,5 mm dpph. the dpph absorbance was measure with uv-vis spectrophotometer in 515 nm wavelength. antioxidant capacity was measured as decreasing of dpph absorbance due to sample addition. the mean of both leaves (l) and seeds (s) antioxidant capacity were then calculated. analysis using non-parametric of two independent samples of mann-whitney test with α 0,05 were done to know the antioxidant capacity of each extract. results and discussion the result of this research showed that antioxidant capacity of soursop (a. muricata linn.) leaves (l) and seeds (s) of ethanolic extract by dpph test have different level. leaves (l) extract showed higher antioxidant capacity than seeds (s) extract (table 1). based on the table 1, the antioxidant capacity of soursop leaves (l) ethanolic extract is 85,67 %. on the other hand, the antioxidant capacity of soursop seeds (s) ethanolic extract is only 39.01 %. the antioxidant capacity play an important role as health protector. this capacity will prevent free radicals so its side effect can be minimalized (shekhar and anju, 2014). due to the advantage, the higher antioxidant capacity will improve the samples potency to be used as protector for some common free radical threat. those result showed that soursop leaves (l) of ethanolic extract has higher potential to obstruct side effect of free radicals as one of cancer growth trigger compared with seeds (s) extract. antioxidant capacity of soursop leaves (l) and seeds (s) ethanolic extract were proved that both of them have ability to decrease the risk of chronic diseases caused by free radicals, include cancer and heart attack. table 1. antioxidant capacity of soursop leaves (l) and seeds (s) ethanolic extract with dpph test. no. samples antioxidant capacity (%) 1. l1 85,99 2. l2 85,35 leaves extract 85.67 3. s1 39,18 4. s2 38,85 seeds extract 39.01 the common antioxidant sources are vitamin c, vitamin e, carotene, and phenolic acid which can be find inside seeds, fruits, and vegetables (shekhar and anju, 2014). those facts strengthen the result of this study widyastuti & rahayu – antioxidant capacity comparison of ethanolic extract of soursop … 3 which said that either soursop leaves (l) or seeds (s) ethanolic extract have potential factor to be an antioxidant sources naturally. soursop (a. muricata linn.) has high vitamin c and polyphenolic compound. phenol and flavonoid which contained in soursop also play a significant role as antioxidant due to molecular structure which give its electron to free radicals molecules. the higher level of phenol and flavonoid compound, the higher antioxidant capacity it have (prasetyorini et al., 2014). reactive oxygen species (ros) and reactive nitrogen species (nos) are free radicals which are formed by either metabolic pathway or external resources. actually, free radical is important substance for energy sources, detoxification, chemical signal, and immunity function. but, the excessive production of free radicals cause biomolecule damage inside cell (such as dna, lipid, protein, carbohydrate), instead of protect them (akomolafe & ajayi, 2015). free radical is also frequency associated with oxidative reaction inside the food and biological system which cause oxidative damage and trigger some human disease, such as neural degeneration, diabetes, and some type of cancer. the danger of free radical discussed above trigger higher research of antioxidant resources which can be an alternative prevention of oxidative damage. chew et al. (2011) said that there are many plant species with bioactive compound which potential as antioxidant resources, including soursop (a. muricata linn.). almost all parts of soursop plant have potency as antioxidant sources although each part has different capacity level. this days, antioxidant capacity of soursop plant often reported to be used as potential anticancer compound. nowadays, usage of soursop plant still limited on its leaves. whereas, antioxidant compound is also find in other parts of soursop plant, such as seeds which not exploited yet. to know about its antioxidant capacity, it can be measured by dpph (diphenylpycryl-hydrazyl) test. extraction process has to be done before dpph test proceed. in pharmaceutical field, extraction use to prepare natural medicine by separate active compound from plant tissues use selective solution and persistent standard procedures. ethanol was used in this research to extract soursop leaves (l) and seeds (s). ethanol is common solvent use at extraction due to its safety. ethanol has characteristic as polar solution so it can solve with water. in this research, maceration was used as extraction method. maceration is extraction process use solution with repeated mixture at room temperature. maceration was used due to its simple process and not time consuming. ninety six persen ethanol was used, so the evaporation of ethanol residue can be faster. after maceration process, the leaves and seeds extract was then measured with dpph test to know about its antioxidant capacity. dpph (1,1-diphenil-2-picrylhydrazyl) is a method that quick, simple, and sensitive to measure antioxidant capacity. it only needs a little sample so we do not have to prepare sample in a large amount. principal characteristic of this test is to measured antioxidant capacity by analyze the changes of dpph color intensity. dpph as free radical have free electron which visualize violet gradation colour. the violet colour change to yellow when the electron paired. decreasing of free radical charges will change the intensity of violet colour. the changes caused by reaction between dpph crystal molecules with hydrogen which release from soursop leaves and seeds ethanolic extract chemical compound. that bond form 2,2-dyphenyl-1picrylhydrazine and cause colour changes from violet to yellow (fathurrachman, 2014). based on the result of this study, leaves (l) extract has higher antioxidant capacity than seeds extract, that is 85,67% and 39,01%, respectively. the leaves (l) extract antioxidant capacity affirm the result of another research by putri (2012) which conclude that 500 μg/ml soursop leaves ethanolic extract showed antioxidant capacity to free radical such as 1,1-diphenyl-2-picrylhydrazyl in the amount of 88,77%. high level of antioxidant capacity which showed by soursop leaves (l) ethanolic extract may potentially be used as natural product for cancer prevention. lower antioxidant capacity on soursop seeds (s) ethanolic extract was caused by its difficult extraction compare with leaves extraction. soursop seeds contain alkaloid compound that is acetogenin and annonaine (idrus et al., 2012). those compounds mention before have potential character as antioxidant. yet, the seeds antioxidant capacity lower than leaves due to its difficult handling and extraction. the extraction difficulties was caused by its hard and rough structure with unbreakable outer layer. acetogenin of soursop seeds (annonaceous acetogenin) has to be examined and proved its characteristic as anticancer, antiparasite, insecticide, antiworm, antibiotic, and antivirus (arifianti et al., 2014). that acetogenin play a role as barrier and killer to cancer cells selectively cause it can detect and discriminate between normal and cancer cells due to atp (adenosine triphosphate) necessity of the cell. cancerous cells grow rapidly so it need bigger amount of atp than normal cells. antioxidant compound of leaves or seeds of soursop (a. muricata linn.) can be used to avoid free radicals so the cancer cell growth can also be prevented. however, the antioxidant capacity in leaves (l) ethanolic extract relatively higher than seeds (s). nonparametric mann-whitney analysis use to know if there is significant differences between antioxidant capacity of leaves (l) and seeds (s). the analysis result showed at table 2. 4 biology, medicine, & natural product chemistry 6 (1), 2017: 1-4 table 2. mann-whitney analysis. test statisticb antioxidant capacity mann-whitney u .000 wilcoxon w 3.000 z -1.549 asymp. sig. (2-tailed) .121 exact sig. [2*(1-tailed sig.)] .333a a. not corrected for ties. b. grouping variable: extract table 2 showed that asymp. sig. (2-tailed) value is 0,121. this value determine if there is significant differences between two samples. if asymp. sig. (2tailed) value is higher than significancy value (α), so there are no significant differences between two samples. on the other hand, if asimp. sig. (2-tailed) value is lower than significant value (α), so there are significant differences between two samples (trihendradi, 2007). asymp. sig. (2-tailed) value in this research is 0,121 (table 2). this number showed there are no significant differences between antioxidant capacity of leaves (l) and seeds (s) ethanolic extract. the values indicate that either soursop leaves (l) or seeds (s) ethanolic extract can be resources of natural antioxidant compound which potential to be an anticancer candidate compound. to improve that potency, it needs simple, quick, and low cost extraction method so that both leaves (l) and seeds (s) can be extracted well. the extraction method between soursop leaves (l) and seeds (s) might be different due to those different shape and texture. conclusion ethanolic extract of soursop (a. muricata linn.) leaves has higher antioxidant capacity than seeds that is 85,67% and 39,01%, respectively. however, there are no significant differences of the antioxidant capacity between those two samples. the higher antioxidant capacity of soursop leaves affirm its potency to be a natural anticancer candidate compound. the lower antioxidant capacity of soursop seeds may be caused by its extraction difficulties so the compound cannot be fully extracted. acknowledgement the authors would like to thank all the participants and partner of this research including biology laboratory technician of universitas pgri semarang and others whom cannot mention each. references akomolafe s. f. & o. b. ajayi. 2015. a comparative study on antioxidant properties, proximate and mineral compositions of the peel and pulp of ripe annona muricata (l.) fruit. international food research journal 22 (6): 2381-2388. arifianti, l., sukardiman, h. studiawan, rakhmawati, & l. megawati. 2014. soursop (annona muricata l.) seeds extract activity test to mammalian cancer cells in vitro. jurnal farmasi dan ilmu kefarmasian indonesia. 1 (2): 63-66. chew, k. k., s. y. ng, y. y. thoo, m. z. khoo, and w. m. a. wan. 2011. effect of ethanol concentration, extraction time and extraction temperature on the recovery of phenolic compounds and antioxidant capacity of centella asiatica extracts. international food research journal 18: 571-578. fathurrachman, d. a. 2014. the effect of solvent concentration to antioxidant capacity of soursop (annona muricata linn.) leaves ethanolic extract with dpph method. medical faculty. uin syarif hidayatullah. jakarta. idrus, r. b., n. bialangi, l. alio. 2012. isolation and characterization of alkaloid compound from soursop (annona muricata linn.) seeds. chemistry education science faculty. universitas negeri gorontalo. limbono, s. 2013. antioxidant capacity of canarium nut (canarium indicum l.) seeds ethanolic extract with dpph (1,1-diphenyl-2-picrylhydrazyl) method. jurnal ilmiah mahasiswa universitas surabaya 2 (2). nihlati, i., a. rohman, & t. hertiani. 2014. antioxidant capacity of ethanolic extract of boesenbergia pandurata (roxb.) schlecth. with dpph (1,1-difenil-2-pikrilhidrazil) method. pharmacy faculty. universitas gadjah mada. yogyakarta. prasetyorini, moerfiah, s. wardatun, & z. rusli. 2014. antioxidant potency of some parts of soursop annona muricata linn.). penel. gizi makan 37 (2): 137-144. puspitasari, m. l., t. v. wulansari, t. d. widyaningsih, j. m. maligan, dan n. i. p. nugrahini. 2016. antioxidant capacity of soursop (annona muricata l.) leaves supplement and mangosteen (garcinia mangostana l.) fruit skin: references study. jurnal pangan dan agroindustri (4) 1: 283-290. shekhar, t. c. & g. anju. 2014. antioxidant activity by dpph radical scavenging method of ageratum conyzoides linn. leaves. american journal of ethnomedicine. 1 (4): 244-249. trihendradi, c. 2007. an easy way to use spss 15 for descriptive, parametric, and nonparametric statistic. penerbit andi. yogyakarta, pp: 127-129. who. 2009. cancers: the problem. http://www.who.int/nmh/publications/fact_sheet_cancers_en. pdf. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 1, 2017 | pages: 5-8 | doi: 10.14421/biomedich.2017.61.5-8 issn 2540-9328 (online) analysis of bull sperm dna abnormalities due to cadmium accumulation fuad fitriawan institute of islamic studies sunan giri (insuri) ponorogo jl. batoro katong no.32, kertosari, babadan, kabupaten ponorogo, jawa timur 63411 author correspondency: fuadfitriawan@gmail.com abstract sperm abnormalities can occur by various causes. abnormality of sperm is usually characterized by abnormal sperm motility and viability. this was caused by the inability of mitochondria on atp-ase in producing atp and ecto-enzyme cik role in keeping the movement so that movement of sperm motility declines. research that leads to total abnormal sperm dna analysis is still rare. the purpose of this research was to get the results of the molecular characteristic picture of the overall characteristics of dna loci that have abnormalities in bull sperm and get a picture of differences in overall dna loci of abnormal and normal sperm. this research was conducted in october-december 2016. with the results of group i consists of d, e with a percent similarity of 92.308%, group ii consists of c and group i with a percentage similarity of 50.125%, group ii consists of a and b with a percent similarity of 100%, group ii and group iii with a percentage similarity of 0%. based on the above data it can be concluded that the treatment a and b is not suspected to cause dna damage compared to treatment c, d and e. keywords: abnormality; spermatozoa; cow; pcr-rapd; cadmium accumulation. introduction every family usually want to has offspring and feels happiness by the presence of baby in their family. but not all family get the dream to have offspring. one of the things that led to the inability to continue its offspring is infertility that occurs on the husband and/or wife. according to agritubella (2009) infertility is the inability of a couple to have children after intercourse regularly 2-3 times per week, without contraception for 1 year. in women, it is usually caused by an infection of the vagina, cervix disorders, abnormalities of the fallopian tubes and uterus according muchtaromah (1999) besides female cause, the other 50% couple infertility contributed by male due to sperm abnormalities. previous research carried out by fitriawan (2006) showed that sperm infertility largely caused by the sperm abnormalities that can occur due to wrong diet as well as abnormalities in male reproductive organs. the sperm abnormalities are usually marked with the limited ability of sperm in its motility and viability. fitriawan[7] and susilowati (2005) stated that it was caused by the inability of mitochondria on atpase to producing atp and enzyme ecto-cik in keeping the movement according to guven et al. (1998), sperm is said to be abnormal if it had more than 70% morphological abnormalities. abnormality caused sperm mitochondrial deficiency that leads to failure in acrosome and postacrosomal reaction so that sperm is unable to fertilize an egg. mature sperm has a length of approximately 50 micron, morphologically and functionally, sperm is divided into two parts, namely the head and tail (destiany. m. 2007). according to guyton and hall (1997), the head of human sperm is oval shaped with a compact nucleus and few cytoplasm bit, salisbury added that the head of the sperm chromatin contain materials that carry the dna code to be transferred through pellucida zone of the ovum. according to fitriawan (2006) and curry & watson (1995), biochemically, sperm abnormalities caused by the inability at the mitochondrial atpase in producing atp and enzyme ecto-cik in keeping the movement so that the movement of sperm will decrease. research on the causes of infertility of sperm abnormalities in biochemical and physiological processes have been done, but a review of genetic and molecular studies have not been conducted. one method to determine molecular genetic picture of an organism is by using pcr-rapd method, which is a molecular technique to determine the overall picture of the characteristics of nuclear dna and mitochondrial dna which is made possible by the insertion, deletion, or substitution at restriction sites. according to sutarno (2008), the advantage of pcr-rapd method is that we can know the map of particular gene in an organism, and the state of these genes can be duplicated so that it seen more clearly. http://dx.doi.org/10.14421/biomedich.2017.61.5-8 6 biology, medicine, & natural product chemistry 6 (1), 2017: 5-8 materials and methods this research was conducted by taking a sample of bull sperm from the bali cattle center for artificial insemination singosari malang, which then will be treated for abnormality and pcr-rapd analysis in the laboratory of biochemistry and molecular biology department, university of brawijaya. the study was conducted from november to december 2016. the equipments used in this study include a set of tools to handle the semen that consists of one unit of an artificial vagina, semen storage tube (test tube scale), a thermos of hot water and thermometer (for measuring temperature in a thermos). a set of tools to check the quality of the semen including test tubes, test tube rack, centrifuges, ose needles, waterbath, paper labels and a set of tools for observing sperm including a light microscope, object glass, cover glass, co2 incubator, hand counter, a set of haemocytometer and tissue and a set of electrophoresis equipment. the materials used for this study were as follows: bali cattle semen samples, a set of semen material extraction, lysis buffer, protenase k, potassium acetate, phenol, nacl, cdcl, vaseline, distilled water, chloroform isoamyl alcohol and isopropanol. negrosineosin staining materials consist of a mixture of 0.3 g eosin, negrosin 0.5 g, 0.1 g na citrate, 10 ml of distilled water and then in the stirrer with a temperature of 100 °c. 500 ml pbs consists of a mixture of 4 g nacl, 0.1 g kcl, 1.05 g na hpo (h o) and 0.1 g of kh po. the research of characteristic of dna in abnormal sperm are reviewed by pcr-rapd method which is a qualitative descriptive study of data images from pcrrflp to be read in accordance with standard pcrrapd analysis. the technique of collecting data from this study was by random sampling, which is a technique that is done by taking samples directly on the ground which will then be treated to obtain abnormal sperm which will then be analyzed its abnormality using eosin staining and for further dna examination using pcr-rapd (sutarno, 2008; sarkar s, hanry jb. 1996). data were analyzed using descriptive and qualitative analysis (moleong, l. j. 1997) of sperm under light microscope and determined its quality, if 70% sperm are not normal then it is classified as abnormal sperm. then, for further analysis, pcr-rapd was performed to determine the polymorphism characteristics of dna. results and discussion results of total dna electrophoresis on agarose 1% from the research, dna eletroforesis of bali bull sperm are shown in figure 1. qualitatively, there were differences between the control and treatment of dna banding pattern. thickness and thinness of the banding pattern will be the effect of treatment on the stability of sperm. in the sample a (control), almost 'total pattern of dna bands appear not so visible but in treatment (b, c, d, and e) visible banding pattern shown very clearly. a b c d e explanation: a : control b : treatment of cadmium chloride 2 hours c : treatment of cadmium chloride 4 hours d : treatment of cadmium chloride 8 hours e : treatment of cadmium chloride over night figure 1. results of total dna electrophoresis on agarose 1%. according to singer and berg in setyono, et al. (2008) a form of gene expression in organisms are protein and polypeptides which is formed through a series of processes of transcription and translation. badel and tatum in setyono, et al. (2008) put forward a hypothesis about the relationship between genes and enzymes, that one gene-one enzyme which then evolved into one gene-one polypeptide. the assumption of the above changes that can lead to environmental change experiments in the process of transcription of different genes that can cause modifications even to cause mutations in the translation process, and can lead to the expression of different morphological structures. mutations or changes at the gene level basically occur due to changes in the environment, so that the dynamics of different patterns of different dna invaluable in detecting mutations in a timely fashioned (toelihere. 1993). then from the qualitative data above can be seen in quantitative results as follows: results of quantitative measurements of total dna sample of bali bull sperm there is a less significant effect between treatments 0 hours to 2 hours, this is because the treatment process that may not have been so long that transcription of the dna of spermatozoa has not shown any signs of abnormality. fitriawan – analysis of bull sperm dna abnormalities due to cadmium accumulation 7 table 1. results of dna quantitative measurement of total sapi bali. samples a260 a280 levels (µg/ml) purity 0 hour 0.004 0.002 100 2.0 2 hour 0.005 0.003 125 1.67 4 hour 0.005 0.003 125 1.67 8 hour 0.012 0.015 300 0.8 over night 0.006 0.004 150 1.5 the results of rapd sperm with premiere opa2 bali bull, 4 opa and opa 6 m a b c d e a b c d e a b c d e explanation: a : control b : treatment of cadmium chloride 2 hours c : treatment of cadmium chloride 4 hours d : treatment of cadmium chloride 8 hours e : treatment of cadmium chloride over night figure 2. visualization of dna produced by pcr-rapd bull sperm on 1.5% agarose gel. then from the visualization of total dna amplification process is carried out and replication that appear on the visualization of pcr-rapd in figure 2. from the image above, after analyzed by dendogram translation (figure 3) method can be seen as follows: dendogram rapd analysis results using mvsp 3. program 1 (multivariate statistics package 3.1) figure 3. dendogram of pcr-rapd bull sperm with premiere opa2, opa4 and opa6. based on the results of bali bull sperm dna amplification with a variation of the old treatment using a primer opa2 cadmium, opa4, and opa6 showed that the highest percentage of sperm dna damage treated with cadmium is composed of three groups: a) the first group consists of d, e with a percent similarity of 92.308% b) group ii consists of c and group i with a percent similarity of 50.125% c) group ii consists of a and b with the percent similarity of 100% d) group ii and group iii with a percent similarity of 0% if observed further from primer that has been used, it can be seen that the appearance of banding pattern in wells a and b almost non-existent, it is possible not to the effect of dna damage by metal cadmium that has been given that at the time of 0-2 hours, but after the appearance of differences, there is already indications of changes in the physical and molecular of sperm. according destiany (2007) and currt & watson (1995) that cd in the cell is a non-essential subtance (a metal which is not needed by the body), if it settles in a cell, it will form cd2 + that causes toxicity to the cell so that metabolism in the cells is inhibited which causes the generation of atp as mitochondria will undergo changes drastically and immediately this is what causes the loci of genes on dna changes. biochemically, according to fitriawan (2006) sperm abnormalities caused by the inability to precisely at the mitochondrial atpase in producing atp and enzyme ecto-cik role in keeping the movement so that the movement of sperm motility will decrease.the sperm dna abnormalities can be seen physiologically using eosin 2% as follows: description: abnormalities of spermatozoa. figure 4. sperm abnormalities conditions dcl treatment of 300 mg/ml 24h. opa 2 opa 4 opa 6 8 biology, medicine, & natural product chemistry 6 (1), 2017: 5-8 sperm abnormalities were divided into 2 groups: primary and secondary abnormalities. primary abnormality is an abnormality that occurs during spermatogenesis. secondary abnormality is an abnormality that occurs due to environmental factors. the existence of sperm abnormalities in a certain amount (under 5%) is a normal condition as a form of primary abnormality. a toxicant toxicity values against an organism can be expressed by several parameters, including the value of lethal concentration 50 (lc-50), a value of 50% of organisms causing death. from lc-50 we can assigned sub-lethal toxicity that resulted in damaging organ or disruption in growth, reproductive ability and behavior. likewise, chronic toxicity, the effect is shown in a long time. in toxicant heavy metal, the last two things is very important, because the presence of heavy metals in water generally in a relatively low level but in the long term. abnormalities that occur in figure 4 above in the form of abnormalities in sperm coiled tail, broken and bent, allegedly due to exposure to heavy metals cause the solution becomes hypertonic. if sperm are stored in a hypertonic solution will result in cytoplasmic vacuoles open and tail membranes become more permeable, so that the curled tail, until broken. the effect of this condition for fertility is a sperm motility will decreased, so the sperm cannot fertilized. based on the above data it can be concluded that treatment a and b allegedly did not cause dna damage compared to treatment c, d and e. conclusion from the research that has been done can be concluded that: 1. the first group consisting of d, e with a percent similarity of 92.308%, group ii consists of c and group i with a percent similarity of 50.125%, group ii consists of a and b with the percent similarity of 100%, group ii and group iii the percent similarity of 0%. 2. treatment a and b (control) as a treatment for 2 hours allegedly did not cause dna damage compared to treatment c, d and e. references agritubella, m. s. 2009. infertilitas. artikel kesehatan reproduksi http://situs.kesrepro.info/kb/referensi2.htm. retrieved at 29 march 2010. coffee, c. j. 1998. metabolism. 1st ed. fence creek publising. madison: xiii + 454 p. curry mr, watson pf. 1995. sperm structure and function in: graud zinkas jg, yovich jl, (eds). gametes-the spermatozoon. great britain: cambridge university press. pages 45-67. curry, m. r. & watson, p. f. 1995. sperm structure and function. great britain. cambridge university press. pages 45-67. destiany. m. 2007. pengaruh pemberian merkuri klorida terhadap struktur mikroanatomi hati ikan mas. thesis. department of biology universitas negeri semarang fitriawan, f. 2006. kemampuan anti mps dalam menghambat motilitas dan viabilitas spermatozoa kambing, domba, dan sapi. thesis department of biology faculty of science and technology uin malang. ginzburg, sa, 1972. fertilization in fishes and the problem of polyspermy. wiener bindery. jerussalem. guven, m. c., can, b., saran, y. 1998. ultrastructural changes in the spermatozoa of infertil man. journals from dept. histology-embriology, medical school of ankara university turkey. guyton ac, hall je. 1997. buku ajar fisiologi kedokteran. diterjemahkan oleh irwati s, lma ken ariata t, alek s. jakarta: penerbit buku kedokteran egc. pages: 309-314. hafez, e.s.e. 1977. techniques for improving reproduction efficiency, pp.455-479. the cv mosby company: st. louis. mason, cf. 1981. biology of freshwater pollution.longman lnc, new york. moleong, l. j. 1997. metodologi penelitian kualitatif. bandung, p.t. remaja rosda karya. muchtaromah, b. 1999. pengaruh pemberian metoklopramid terhadap gambaran histologis testis tikus putih (rattus norwegicus). thesis postgraduate program department of bioreproduksi unair surabaya. nelson, rw. 2003. morphology of normal and abnormal canine spermatozoa. couto cg. journal of small animal internal medicine. mosby partodiharjo, soebadi, 1992. ilmu reproduksi hewan. mutiara sumber widya. jakarta pusat. cetakan ke iii pages 523-531. rijnders pm. 1996. the spermatozoon-theory. in: brass jw, et al. editors. ivf lab-laboratory as pects of in vitro vertilization. nv organon. pages 23-39. salisbury, g. w. 1985. inseminasi buatan pada sapi. gadjah mada university press yogyakarta. sarkar s, hanry jb. 1996. andrology laboratory and fertility assessment, in: henry jb, editors. clinical diagnosis and management by laboratory metods. philadelphia. setyono, p., soetarto, e. s., 2008. biomonitoring degradasi ekosistem akibat limbah cpo muara sungai mentaya kalimantan tengah dengan metode elektromorf isozim esterase. jurnal biodiversitas vol 9. nomor 3. universitas sebelas maret surakarta. pages 232-236. sutarno, 2008. diktat materi bioteknologi dan seluler. faculty of mathematics and natural sciences universitas sebelas maret surakarta. pages. 35-40. susilowati, t. 2005. fisiologi spermatozoa: kapasitasi, reaksi akrosom dan fertilisasi. faculty of animal husbandry universitas brawijaya malang. toelihere. 1985. fisiologi reproduksi pada ternak. publisher angkasa bandung. pages 93 – 115. toelihere. 1993. analisis kualitas semen pada ternak. publisher angkasa bandung. analysis of bull sperm dna abnormalities due to cadmium accumulation biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 685-692 | doi: 10.14421/biomedich.2025.142.685-692 issn 2540-9328 (online) identification of total coliform bacteria in processed enbal food in several traditional markets in ambon city salmin safril nahumarury1, melda yunita2,*, anggun lestary husein3, effendi effendi4 1study program of medical education, faculty of medicine, universitas pattimura, ambon, indonesia 2department of microbiology, faculty of medicine, universitas pattimura, ambon, indonesia 3department of biomedical science, faculty of medicine, universitas pattimura, ambon, indonesia 4shiba kokusai international school, minato-ku, tokyo, japan. corresponding author* melda.yunita@lecturer.unpatti.ac.id manuscript received: 02 may, 2025. revision accepted: 13 august, 2025. published: 01 october, 2025. abstract enbal is a traditional food from maluku, originally from the kei islands, made from bitter cassava (manihot esculenta). this processed food product holds significant historical and cultural value. however, enbal can become a source of illness if not prepared properly, leading to contamination and an increased risk of foodborne diseases. this study aimed to determine the microbiological quality through total plate count (tpc) analysis and bacterial identification of enbal at several traditional markets in ambon city. it was a descriptive observational study with a true experimental laboratory approach. the tpc results for enbal food samples from mardika market ranged from 14.5 × 102 cfu/g to 4.85 × 104 cfu/g, waiheru market ranged from 8.3 × 102 cfu/g to 3.2 × 105 cfu/g, batu merah market ranged from 11.0 × 104 to 3.0 × 105 cfu/g, and passo market ranged from 2.4 × 102 cfu/g to 3.75 × 105 cfu/g. the results of bacterial species identification from the four enbal food samples revealed klebsiella pneumoniae and staphylococcus haemolyticus. thus, it can be concluded that the four enbal samples examined are safe for consumption as they do not exceed the indonesian national standard (sni) 01-2997-1996, which is 1 x 106 cfu/g. keywords: bacterial contamination; bitter cassava; cfu/g; sni. abbreviations: fao: food and agriculture organization; cfu: colony-forming unit; tpc: total plate count; pca: plate count agar; tntc: too numerous to count; tftc: too few to count; sni: indonesian national standard; cons: coagulase-negative staphylococci; uti: urinary tract infection. introduction food is essential to human needs as one of the primary and basic physiological needs that support human life (trivedi et al., 2019; casman et al., 2022). however, food can also become a source of illness if it is not handled or prepared properly and hygienically (o’shea et al., 2019). food hygiene ensures the control of potential hazards, which can be identified using a hazard assessment approach, as well as the likelihood and severity of adverse impacts, determined using a risk assessment approach (hassauer & roosen, 2020). traditional food markets, which are a source of livelihood for millions of people, can pose public health risks if proper food safety measures are not followed. traditional markets themselves are places that sell various types of traditional products, including crafts, souvenirs, and traditional foods. in maluku, one food product found in traditional markets is enbal, a processed food product. enbal is a food product made from bitter cassava (manihot esculenta), processed in a way passed down through generations using simple methods (leasa et al., 2018; riry et al., 2013). traditional processed products like enbal have significant historical and cultural value and are an essential to indonesia's food diversity (polnaya et al., 2016). enbal is often consumed by the people of maluku, especially in southeast maluku, and comes in several variants such as fried enbal, chocolatecheese enbal, peanut enbal, and plain enbal (kementerian pendidikan dan kebudayaan indonesa, 2017). however, traditional food products also carry risks, such as health and food safety risks due to contamination by microorganisms, especially since most traditional food products are sold in traditional markets with low sanitation levels (olowokure et al., 2023; zamzammi, 2015). contamination in food can lead to an increase the number of foodborne diseases. foodborne diseases are any illnesses caused by consuming contaminated food, contaminated by pathogenic bacteria, viruses, or https://doi.org/10.14421/biomedich.2025.142.685-692 686 biology, medicine, & natural product chemistry 14 (2), 2025: 685-692 parasites. globally, foodborne diseases are still not under control, and outbreaks can cause significant health and economic losses. the causes are unhygienic practices in food production, harvesting, and preparation (adley & ryan, 2016). foodborne diseases are caused by food contamination and occur at every stage of the food production, distribution, and consumption chain. this can result from various forms of environmental contamination, including water, soil, or air pollution, as well as unsafe food storage and processing (world health organisation, 2010). data from the food and agriculture organization (fao) estimates that around 600 million people, nearly 1 in 10 people worldwide, fall ill after consuming contaminated food, and 420,000 people die each year (davidson & emerita, 2017). in europe, it is reported that 44 people per minute, an average of 23 million people per year, fall ill due to food poisoning, and it is estimated that 4,700 people die each year (negru & brickman, 2019). in indonesia, the highest number of reported food poisoning outbreaks occurred between 2008 and 2011, with 54.8% of cases reported. the highest incidence of food poisoning occurred in west java province, with 163 incidents out of a total population of 10,962. food poisoning cases in indonesia from 2000 to 2015 totaled 61,119 cases out of a population of 715,579 at risk (arisanti et al., 2018). a study conducted by condro and santoso (2017) on sweet potato chips in jayapura city showed that samples obtained from four household industries were positive for escherichia coli contamination. another syafira et al. (2021) study on cassava getuk sold in traditional markets in cimahi city found that 4 out of 5 samples exceeded the safe food limit, with the highest total plate count of 1.4 x 106 cfu/gram. another study by tallo and pani (2023) on gaplek cassava samples from belu and ende regencies showed that the samples tested had the highest yeast and mold count in both regencies of 8.7 x 105 cfu/gram, which exceeded the threshold set by the food and drug supervisory agency of indonesia regulation number 13 of 2019, with a maximum colony number of 105 cfu/gram. many studies have been conducted on total plate count analysis in various food products, yet there is no research on total plate count analysis and bacterial identification of enbal products. therefore, this study aimed to analyze the microbiological quality of enbal at several traditional markets in ambon city through total plate count (tpc) analysis and bacterial identification. materials and methods study area this study used a quantitative descriptive research design with a true experimental laboratory approach. the research was conducted at the microbiology laboratory of the faculty of medicine, universitas pattimura, and the office for health laboratory and medical device calibration of maluku province, ambon, indonesia, from june to july 2024. procedures tools and materials materials used in this study were 100 gr of processed plain enbal food obtained from each of the 4 markets in ambon city, 0.85% nacl solution (merck), plate count agar (pca) (merck), nutrient agar (merck), sterile distilled water (waterone). the tools used were cottons, 20 ml and 100 ml measuring cylinders (iwaki), 250 ml and 500 ml erlenmeyer flasks (iwaki), 100 ml beaker glass (iwaki), test tubes (pyrex), micropipette (socorex), glass spreader (iwaki), autoclave (all american), vortex mixer (vm-300 gemmy), inoculating needle, aluminium foil (klinpak), plastic wrap (klinpak), bunsen burner, mortar and pestle, petri dishes (onemed), tweezers, inoculation loop, label paper, cotton, microscope (olympus), glass slides (onemed), and vitek 2 compact (biomérieux). sample preparation the samples used in this study were processed enbal food obtained from several traditional markets in ambon, indonesia. after conducting a survey, it was found that plain processed enbal was only available in 4 out of 8 actively operating traditional markets in ambon city: mardika market, passo, waiheru, and batu merah. from these four markets, a simple random sampling method was applied using a lottery system to select processed plain enbal from vendors in each market. sterilization of equipment the glassware was wrapped in parchment paper and sterilized using an autoclave at 121°c for 15 minutes. after sterilization, the tools were placed in an oven at 150°c for 1 hour (ma’at, 2009). preparation of plate count agar (pca) medium a total of 22.5 gr of plate count agar powder was mixed with sterile distilled water until the solution volume reached 1 liter. the medium was then boiled on a heater until fully dissolved, and subsequently sterilized in an autoclave at 121°c and 760 mmhg for 15 minutes (mariani et al., 2020). serial dilution and bacterial isolation one gram of the grounded sample is placed into a test tube and mixed with 9 ml of sterile distilled water, then homogenized using a vortex mixer with the tube closed to achieve a 10-1 dilution. to prepare a 10-2 dilution, 1 ml of the initial dilution suspension is transferred to a new test tube containing 9 ml of sterile distilled water and homogenized. this process was repeated to create 10-3, 10-4, and 10-5 dilutions in the same manner. the isolates nahumarury et al. – identification of total coliform bacteria in … 687 from these dilutions were then inoculated onto pca medium using the spread plate technique, repeated twice and incubated at 37°c for 24-72 hours (jamilatun, 2022). analysis of total plate count (tpc) the analysis of total plate count was performed by counting bacterial colonies per petri dish. colony counts were carried out to determine the level of bacterial contamination in the samples, and were only performed on petri dishes with colony counts between 30 and 300. the observed number of colonies were then used to calculate the total plate count using the formula (yunita et al., 2022; yunita et al., 2016): tpc = numbers of colonies × 1 dilution factors purification of selected bacteria bacteria that grow and have different morphologies are selected considering colony color and shape differences across all petri dishes. the five selected colonies were purified on pca medium using the spread plate technique with the quadrant streak method and incubated for 24 hours (sa’adah, 2020). macroscopic characterization macroscopic characterization was carried out by directly observing the isolates grown on agar media, including colony size, colony shape, colony elevation, colony margin, surface texture, as well as colony color and turbidity (bergey & hendricks, 1994; tille, 2022). microscopic characterization a purified bacterial colony was taken and smeared onto a glass slide pre-treated with 0.96% nacl. the smear was spread using an inoculating loop and dried by fixation over a flame. crystal violet (purple) stain was applied to the smear and left on the slide for 1 minute before being rinsed with distilled water. lugol's iodine solution was then added to enhance the affinity of the crystal violet stain, left for 1 minute, and rinsed again. next, a decolorizer (96% alcohol) was applied for 1 minute, followed by another rinse with distilled water. the final step of the gram staining process involved counterstaining with safranin for 30 seconds to color the bacterial cells. the slide was then rinsed, dried, and observed under a microscope at 100x magnification using immersion oil (jawetz et al., 2019; tille, 2022). bacterial identification aseptically transfer 3.0 ml of sterile nacl solution (0.45% to 0.50% nacl, ph 4.5 to 7.0) into a clear plastic test tube (polystyrene, 12 mm x 75 mm). using a sterile swab or inoculation loop, transfer a purified bacterial colony into the test tube. prepare a homogeneous bacterial suspension with a density of 0.50 to 0.63 mcfarland standard using a calibrated vitek-2-compact system (astuty et al., 2023). data analysis the data analysis in this study was conducted using microsoft excel and the obtained data was compared with sni 01-2997-1996. results and discussion total plate count (tpc) analysis the results showed that the total plate count (tpc) of enbal, as presented in table 1, remained below the maximum bacterial contamination limit set by the indonesian national standard (sni) 01-2997-1996, which is 1.0 × 10⁶ cfu/g (sagen, 2023). based on the calculations in table 1, all enbal samples analyzed were found to have microbial contamination levels within the acceptable range. the comparison between the total plate count and the sni is illustrated in figure 1, confirming that the bacterial count in enbal remains within safe consumption limits. these findings indicate that the enbal food studied is still safe for daily consumption. table 1. results of total plate count (tpc) analysis for enbal. dilution markets (mean ± sd) sni (cfu/gr) mardika waiheru batu merah passo 10-1 14.5 × 102 8.3 × 102 tntca 2.4 × 102 ± 21.0 1 × 106 10-2 10.5 × 103 ± 6.5 39.5 × 102 ± 3.5 tntca 6.05 × 103 ± 23.5 10-3 4.85 × 104 ± 11.5 33.5 × 103 ± 1.5 11.0 × 104 5.5 × 104 ± 21.0 10-4 tftcb 3.2 × 105 ± 1.0 3.0 × 105 3.75 × 105 ± 5.5 description: tntc: too numerous to count; tftc: too few to count 688 biology, medicine, & natural product chemistry 14 (2), 2025: 685-692 figure 1. comparison of total plate count for each market with indonesian national standard. bacterial identification different macroscopically bacterial colonies were identified based on their characteristics, including size, shape, margin, elevation, and color (figure 2). figure 1. purification results of five selected bacterial isolates (a1, a2, a3, a4, and a5) after 48 hours of incubation. the purified bacterial colonies were regrown and analyzed macroscopically, with the observation results documented in table 2, which shows similarities and differences in the macroscopic characteristics of each selected isolate. table 2. macroscopic characterization of bacteria from enbal food processing samples. is o la te s morphology characteristics of the colonies colony size colony shape colony elevation colony margin color and turbidity a1 medium irregular umbonate undulate milky white a2 medium irregular flat undulate milky white a3 medium irregular umbonate undulate milky white a4 medium irregular raised undulate milky white a5 medium irregular flat undulate milky white the bacterial isolates obtained from enbal food products were subsequently characterized based on their size, morphology, pigmentation, and gram reaction under microscopic examination. the results of the microscopic characterization (figure 3), where isolates a1 and a4 exhibit similar cellular structures and belong to the same gram classification. these isolates form staphyl aggregates with a coccoid morphology and appear purple upon gram staining, indicating that they are gram-positive staphylococcus. conversely, isolates a2, p3, and a5 display analogous cellular morphology and gram staining properties, presenting as rod-shaped bacilli with a red coloration. this observation confirms that these three isolates are classified as gram-negative bacteria. figure 2. microscopic characterization results of bacterial isolates. identification of the five selected bacterial isolates using the vitek-2-compact system revealed two bacterial species. isolates a2 and a4 were identified as staphylococcus haemolyticus, while isolates a2, a3, and a5 were identified as klebsiella pneumoniae. the biochemical identification results, presented in table 3, demonstrate the bacterial species of the selected isolates with varying similarity levels for each isolate. nahumarury et al. – identification of total coliform bacteria in … 689 table 3. results of bacterial identification using the vitek-2-compact system. isolates bacterial species significance bacterial habitate a1 staphylococcus haemolyticus 91% human skin a2 klebsiella pneumoniae 97% human oral cavity, skin, gastrointestines a3 klebsiella pneumoniae 97% human oral cavity, skin, gastrointestines a4 staphylococcus haemolyticus 85% human skin a5 klebsiella pneumoniae 89% human oral cavity, skin, gastrointestines discussion total plate count analysis based on research findings, the total plate count of bacteria from the four enbal processed food samples examined met the safety standards set by the indonesian national standard. this may be attributed to several factors, such as proper and clean packaging. food packaging is essential as a barrier and protection against spoilage and additional contamination. safirin et al. (2023) stated that appropriate packaging helps maintain product quality and increases consumer acceptance. similarly, warto & samsuri (2020) explained that packaging is vital in enhancing the safety and durability of processed food products. another factor that can help suppress bacterial growth is the processing method of enbal itself, which involves high-temperature heating. sutiko et al. (2020) in their study on the effect of heating duration on non-vacuumpackaged wet spring rolls concerning total plate count (tpc), found that heating processed foods reduced bacterial count. heating extends shelf life and eliminates organisms such as bacteria, molds, and yeasts that may contaminate processed food products. pratama et al. (2016) revealed that heating significantly kills microbes and pathogens responsible for spoilage. additionally, heating is an essential process for achieving the desired organoleptic characteristics of food. one crucial factor contributing to suppressing bacterial growth in enbal processed food is its composition. enbal is a dry processed food with low moisture content, which inhibits bacterial growth. microorganisms require a specific level of water or moisture to thrive in food products. preetha & narayanan (2020) explained that foods with high water content are more susceptible to spoilage due to the rapid growth of spoilage and pathogenic microorganisms. in contrast, foods with low water content are less likely to be damaged by microorganisms. microbial growth ceases when moisture levels drop below the minimum threshold, but microbes do not immediately die and may remain inactive in food for a certain period (talakua et al., 2024). several factors may contribute to the high total plate count in one of the sampling locations, such as the quality of raw materials, storage errors, or crosscontamination during production until the product reaches consumers. cross-contamination can occur through environmental transmission to food due to inadequate sanitation practices. according to condro & santoso (2017), contamination in processed foods may originate from the production process, from raw material reception to the final product, as well as poor hygiene in marketing facilities. differences in total plate count across the four markets may be due to variations in distribution distances, leading to longer storage times and allowing bacteria to incubate for extended periods. hammond et al. (2015) emphasized that food distribution and storage are closely related, as longer distribution distances require additional precautions since bacterial growth increases over time. astutiningsih et al. (2022) also found that the total microbial count rises with prolonged storage duration. the location of vendors may also contribute to the relatively higher total plate count of bacteria in passo market than other markets. enbal vendors in passo market are situated at an intersection, a high-traffic area with many vehicles and pedestrians passing through. this factor may contribute to the higher total plate count in passo market. rostina & mutiana (2018) stated that sales locations with high vehicle traffic can increase the spread of dust and bacteria, leading to greater contamination of food products and an increase in bacterial counts. identification of bacterial isolates from enbal processed food samples purification and re-culturing of five selected xisolates were conducted based on their most prominent macroscopic characteristics. these characteristics included colony size, colony shape, colony elevation, colony edges, and colony color, which were assessed following bergey's manual of determinative bacteriology. after macroscopic characterization, gram staining was performed for microscopic identification, revealing that two isolates were gram-positive bacteria and three were gram-negative bacteria. gram staining was also used to determine the appropriate placement of isolates on the vitek 2 gp card or vitek 2 gn card for physiological and biochemical identification using the vitek-2-compact system. subsequently, bacterial identification was carried out at the health laboratory and medical equipment calibration center of maluku province using the vitek-2-compact system. the results identified two bacterial species: staphylococcus haemolyticus, which was found in enbal processed food samples from 690 biology, medicine, & natural product chemistry 14 (2), 2025: 685-692 mardika market (a1) and waiheru market (isolate a4), and klebsiella pneumoniae, which was identified in samples from waiheru market (isolate a2) and passo market (isolates a3 and a5). staphylococcus haemolyticus belongs to the coagulase-negative staphylococci (cons) group and is a commensal bacterium on human skin. however, it can cause severe infections in various body systems, such as meningitis in the nervous system, endocarditis in the cardiovascular system, prosthetic joints within the musculoskeletal system, and systemic infections like bacteremia, which commonly occur in hospital environments and among healthcare workers. s. haemolyticus has also been associated with septicemia, peritonitis, otitis media, and diabetic ulcers (eltwisy et al., 2022a; 2022b). staphylococcus haemolyticus in food is suspected to result from contamination from human skin to the food. if consumed in high amounts, it may lead to foodborne illnesses and other systemic diseases. this finding aligns with the study by (regecová et al., 2021), who successfully identified several staphylococcus species, including s. haemolyticus, from 193 isolates of various food types, such as alaska pollock (theragra chalcogramma), atlantic mackerel (scomber scombrus), atlantic herring (clupea harengus), bryndza cheese (regecová et al., 2021). besides s. haemolitycus, other staphylococcus species such as s. sciuri, s. gallinarum, and s. pseudintermedius were also found in smoked yellowfin tuna (haurissa et al., 2024). klebsiella pneumoniae is a coliform bacterium belonging to the gram-negative, rod-shaped, non-motile group. it ferments glucose and naturally exists as part of the normal human flora, found in the mouth, skin, intestines, and feces. klebsiella pneumoniae is often associated with bacteremia, pneumonia, and urinary tract infections (utis), particularly in individuals with compromised immune systems or those exposed to nosocomial infections. additionally, k. pneumoniae is a predisposing factor for liver abscesses in several asian countries (saif & sami, 2020). contamination of food with k. pneumoniae can occur through transmitting its normal flora, such as from human lips or skin, onto food, making its presence detectable during bacterial identification tests. this finding aligns with the study conducted by (hartantyo et al., 2020), which found that 147 out of 698 food samples—including vegetables, spices, and raw meat tested positive for klebsiella pneumoniae through pcr analysis. the findings of this study indicate that although the total plate count remains within safe limits, the possibility of bacterial contamination in processed food still exists. excessive contamination can lead to an increased incidence of foodborne illnesses such as diarrhea or typhoid fever (todd, 2014). conclusions enbal products obtained from four traditional markets in ambon city: mardika market, waiheru market, batu merah market, and passo market are considered safe for consumption from a microbiological perspective based on sni 01-2997-1996. the bacterial identification of processed enbal food exhibits the presence of both gram positive and gram-negative bacteria, namely staphylococcus haemolyticus and klebsiella pneumoniae. acknowledgements: we would like to thank the faculty of medicine, universitas pattimura for providing the laboratory to conduct this research. authors’ contributions: salmin safril nahumarury contributed to the data analysis and interpretation in this study. melda yunita contributed to the conception and critical revision of the article for important intellectual content. anggun lestary husein and effenfi effendi contributed to the article’s draft. all authors gave their approval to the final article. competing interests: the authors declare that there is a conflict of interest. funding: the authors thank the faculty of medicine, universitas pattimura, for supporting this study. references adley, c. c., & ryan, m. p. 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(2015). studi sosiologis tentang pengelolaan kebersihan pada pasar tradisional di kota pekanbaru (studi kasus di pasar cik puan, pasar pagi dupa, dan pasar sail). jom fisip, 2(1), 1–15. https://jom.unri.ac.id/index.php/jomfsip/article/view/4898/4 780 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 401-407 | doi: 10.14421/biomedich.2025.141.401-407 issn 2540-9328 (online) demographic characteristics of fruit sellers and chemical preservation practices in birnin kebbi, nigeria yahaya tajudeen1,*, mohammed umar faruk2, abdulmalik abdulazeez1 1department of biological sciences, federal university birnin kebbi, pmb 1157, kebbi state, nigeria. 2department of pure and industrial chemistry, federal university birnin kebbi, nigeria. corresponding author* yahayatajudeen@gmail.com and yahaya.tajudeen@fubk.edu.ng manuscript received: 29 january, 2025. revision accepted: 15 may, 2025. published: 15 july, 2025. abstract fruits in birnin kebbi, nigeria, are often preserved using chemicals to extend their shelf life, maintain freshness, and enhance quality and taste. however, global concerns persist about the safety of these preservatives, particularly when applied indiscriminately. this study aimed to determine the demographic characteristics of fruit sellers in birnin kebbi and the preservatives they commonly use. structured questionnaires were administered to 200 participants who provided informed consent. the results showed that 116 (58%) of the respondents were male, while 84 (42%) were female. most participants (38%) were aged 18–25 years (76 individuals), followed by those aged 26–35 years (60 individuals, 30%). a significant proportion of participants (82; 41%) had no formal education, while 48 (24%) had primary education. the majority of participants (120; 60%) belonged to the hausa ethnic group, and 156 individuals (78%) identified as muslims. marital status was evenly distributed, with 84 participants (42%) being single and 80 (40%) married. regarding fruit preservation practices, watermelon was the most frequently preserved fruit, cited by 50 participants (25%), followed by oranges (42; 21%) and bananas (22; 11%). among chemical preservatives, sulfite was the most commonly used, reported by 84 (42%) participants, followed by citric acid (36; 18%), sodium benzoate (28; 14%), calcium chloride (24; 12%), benzoic acid (18; 9%), and ethanol (10; 5%). alarmingly, 95% of participants demonstrated inadequate knowledge of proper preservative application methods, raising concerns about potential health risks for consumers. these findings suggest that fruit preservation practices in birnin kebbi may expose consumers to health hazards. it is imperative to educate fruit sellers and consumers on the safe use of chemical preservatives, including appropriate concentrations and application techniques, to mitigate these risks and promote safer fruit consumption. keywords: citric acid; demographic characteristics; preservatives; questionnaires; watermelon. introduction fruits are essential, vibrant, and nutrient-rich components of nature, bursting with flavors, textures, and colors that captivate the senses and nourish the body. botanically, the term "fruit" refers to the mature ovary of a plant, including its seeds, covering, and any closely associated tissues, regardless of their edibility (rejman et al., 2021). common fruits like apples, pears, oranges, bananas, and grapefruits are readily available and widely consumed. from the tropical regions of the east to the orchards of the west, fruits have been cultivated, traded, and cherished for their unique qualities throughout history (abdulmumeen et al., 2020). with over 20,000 known species, fruits exhibit an astonishing diversity in shapes, sizes, and flavors (rao, 2023). from the tiny, tangy cranberry to the large, juicy pineapple, each fruit offers a unique experience. categories such as berries, citrus fruits, stone fruits, and tropical fruits showcase this remarkable variety. beyond their culinary appeal, fruits are packed with health benefits. they are rich in antioxidants, fiber, and essential vitamins (e.g., a, c, and e), as well as minerals such as magnesium and potassium. at the same time, they are low in fats, saturated fatty acids, cholesterol, and sodium. these properties help combat chronic diseases such as diabetes and coronary heart disease, support digestive health, and promote healthy skin and hair (park, 2021; clemente-suárez et al., 2023). nutrients and phytochemicals in fruits have been found to lower blood pressure by improving endothelial function, enhancing baroreflex sensitivity, promoting vasodilation, and increasing anti-inflammatory activity (zhao et al., 2017). for optimal health, the world health organization (who) and the food and agriculture organization (fao) recommend that adults consume at least five servings of fruits and vegetables daily, excluding starchy vegetables (wang et al., 2021). beyond their nutritional and culinary significance, fruits hold cultural, spiritual, and symbolic meanings. they are integral to traditional medicine, rituals, and ceremonies, highlighting their profound influence on human society (abdullahi et al., 2022). https://doi.org/10.14421/biomedich.2025.141.401-407 mailto:yahayatajudeen@gmail.com mailto:yahaya.tajudeen@fubk.edu.ng 402 biology, medicine, & natural product chemistry 14 (1), 2025: 401-407 fruits are perishable and often require preservatives to maintain their freshness and quality during storage and transportation. preservatives are substances used to prevent microbial growth, oxidation, and spoilage in food, pharmaceuticals, and other products, extending their shelf life and ensuring safety (teshome et al., 2022). historically, methods like salting, sugaring, and smoking were used to preserve food, while modern industries rely on synthetic additives for effective preservation. fresh fruits, due to their high respiration rates, are particularly challenging to keep fresh under normal environmental conditions. after harvesting, water loss accelerates deterioration, leading to reduced quality. proper storage and preservation are therefore essential to maintaining the postharvest quality of fruits (imahori & bai, 2024). common preservatives used for fruits include nitrates, sulfites, sodium benzoate, propyl gallate, and potassium sorbate (yahaya et al., 2023). however, concerns have been raised regarding the safety of these substances, often caused by inadequate knowledge of their application. for instance, indiscriminate benzoates application may cause allergies, asthma, and skin rashes; sorbic acid and sorbates can induce urticaria and contact dermatitis; sulfites are linked to headaches, palpitations, and allergies, and are suspected carcinogens, and nitrites and nitrates have been associated with stomach cancer (yahaya et al., 2023). birnin kebbi, a prominent city in northern nigeria, is known for its diverse agricultural produce, including various fruits. to ensure the longevity and quality of these fruits, especially for storage and transportation, the use of preservatives is common. however, a review of the literature reveals no studies on the levels of understanding regarding preservative application or awareness of their health implications in the city. this lack of knowledge may contribute to the prevalence of certain diseases in the area. to address these challenges and mitigate the unintended health consequences of preservative use in birnin kebbi, there is an urgent need for health policies regulating the use of fruit preservatives. additionally, raising public awareness about the risks associated with indiscriminate preservative use is critical. however, before implementing these measures, it is essential to document the demographic characteristics of fruit sellers and the types of preservatives used in the city. this study aims to determine the demographic characteristics of fruit sellers and their use of preservatives in birnin kebbi, nigeria. materials and methods study design this study utilized a descriptive cross-sectional design to collect data directly from individuals involved in fruit selling within the city. this approach allowed for detailed exploration and documentation of various fruit preservation methods, encompassing both traditional and modern techniques, as applied in the study area. study population and data collection the study population comprised 200 individuals engaged in fruit farming and selling in birnin kebbi, nigeria. data collection was conducted through structured, faceto-face questionnaires. the questionnaire was divided into two sections: section a focused on demographic information, including gender, educational level, and ethnicity while section b addressed preservation methods (natural or chemical) and the specific chemicals used. fieldwork included visits to fruit planting sites and selling points across various locations in birnin kebbi. participants with limited literacy skills received assistance to ensure their full comprehension of the questions. respondents were encouraged to provide honest answers, with confidentiality emphasized to foster trust. data collection spanned two weeks, ensuring adequate time to obtain comprehensive responses from all selected participants. eligibility criteria participants are those who provided written consent to participate in the study, and have been engaged in selling fruits for at least one year, ensuring they had adequate knowledge of preservation practices. individuals who failed the set criteria were excluded. ethical approval and consent to participate ethical approval for the study was obtained from the ethics committee of the federal university birnin kebbi, nigeria. the research adhered to the ethical guidelines for studies involving human participants. written informed consent was obtained from all participants prior to data collection. to protect participants' privacy, all data were anonymized and securely stored, with usage restricted to research purposes only. the study complied with the principles outlined in the declaration of helsinki, adopted in 1964 and most recently updated in october 2024, which provides ethical standards for research involving human subjects. data analysis data were analyzed using percentages and frequency distribution tables, employing microsoft excel (version 21 for windows). result demographic characteristics of the participants table 1 presents the demographic distribution of the participants, including their gender, age, educational level, ethnicity, religion, and marital status. tajudeen et al. – demographic characteristics of fruit sellers and … 403 the study included 116 male participants (58%) and 84 female participants (42%). the majority of the participants (38%) were within the age range of 18–25 years (76 individuals), followed by those aged 26–35 years (60 participants, 30%). participants aged 36–45 years accounted for 17% (34 individuals), those aged 46– 55 years made up 8% (16 individuals), and participants aged 56 years and above comprised 7% (14 individuals). regarding educational attainment, 82 participants (41%) had no formal education, while 48 (24%) had completed primary education. a total of 40 participants (20%) had secondary education, and 30 (15%) had attained tertiary education. in terms of ethnicity, the majority of the participants were hausa, comprising 120 individuals (60%). this was followed by fulani (28 participants, 14%), igbo (20 participants, 10%), yoruba (16 participants, 8%), and gwari (16 participants, 8%). for religious affiliation, most participants (78%) identified as muslims (156 individuals), while 11% (44 individuals) identified as christians. regarding marital status, 84 participants (42%) were single, 80 participants (40%) were married, 16 (8%) were divorced, and 20 (10%) were widowed. table 1. demographic characteristics of fruit sellers in birnin kebbi. gender frequency percentage (%) male female 116 58 84 43 total 200 100 age frequency percentage (%) 18-25 76 38 26-35 60 30 36-45 34 17 46-55 16 8 56 14 7 total 200 100 level of education frequency percentage (%) no formal education 82 41 primary school 48 24 secondary school 40 20 tertiary education 30 15 total 200 100 ethnicity frequency percentage (%) hausa 120 60 fulani 28 14 yoruba 16 8 igbo 20 10 gwari 16 8 total 200 100 religion frequency percentage (%) islam christian total 156 44 200 78 22 100 marital status single married divorce widowed total frequency 80 76 16 28 200 percentage (%) 40 38 8 14 100 most commonly preserved fruits figure 1 illustrates the most commonly preserved fruits in birnin kebbi. watermelon was the most frequently preserved fruit, reported by 50 participants (25%), followed by oranges with 42 participants (21%). bananas were preserved by 22 participants (11%), sweet melons by 20 participants (10%), apples by 18 participants (9%), pineapples by 16 participants (8%), tangerines by 14 participants (7%), and both pawpaw and strawberries by 12 participants each (6%). figure 1. most commonly preserved fruits in birnin kebbi. chemicals used to preserve fruits figure 2 illustrates the types of chemicals employed for fruit preservation in birnin kebbi. among the 200 participants, 84 individuals (42%) reported using sulfites, 36 (18%) used citric acid, and 28 (14%) utilized sodium benzoate. calcium chloride was used by 24 participants (12%), benzoic acid by 18 participants (9%), and ethanol by 10 participants (5%). figure 2. chemicals used in preserving fruits in birnin kebbi. natural methods used to preserve fruits figure 3 illustrates the natural methods employed for fruit preservation in birnin kebbi. among the 200 participants, 60 (30%) reported using salting, 52 (26%) 18 42 22 50 12 16 14 20 12 0 10 20 30 40 50 60 70 80 90 100 fr eq u en cy ( % ) fruits 36 24 28 84 10 18 0 10 20 30 40 50 60 70 80 90 100 citric acid calcium chloride sodium benzoate sulfites ethanol benzoic acid fr eq u en cy ( % ) chemicals 404 biology, medicine, & natural product chemistry 14 (1), 2025: 401-407 used drying, 36 (18%) relied on canning and freezing, and 16 (8%) utilized smoking. figure 3. natural methods used for preserving fruits in birnin kebbi. sources of knowledge about fruit preservation methods figure 4 illustrates how participants learned about fruit preservation methods. the majority, 88 participants (44%), acquired their knowledge through family and friends, followed by 64 participants (32%) who learned through formal education. additionally, 48 participants (24%) gained knowledge from community workshops. figure 4. sources of knowledge about fruit preservation methods. factors influencing the choice of fruit preservatives method figure 5 highlights the factors that influence the choice of the fruit preservation methods in birnin kebbi. the most commonly reported factor was the cost of the preservation method, cited by 80 participants (40%). this was followed by storage duration, reported by 48 participants (24%), ease of use by 42 participants (21%), availability of resources by 18 participants (9%), and taste by 12 participants (6%). figure 5. factors influencing the choice of fruit preservative methods. figure 6. knowledge on preservative application. discussion in birnin kebbi, northwestern nigeria, a variety of fruits are widely sold and consumed. however, due to their perishable nature, various preservation methods are employed in the city to extend the shelf life of fruits. unfortunately, these preservatives are often applied indiscriminately, raising concerns about their potential negative health impacts. this study aimed to assess the demographic characteristics of fruit sellers in birnin kebbi and the types of preservatives commonly used. the demographic data collected revealed that the majority of fruit sellers in birnin kebbi were male, aged between 18–25 and 26–35 years. most sellers had no formal education or had completed only primary education. they were predominantly of hausa ethnicity, muslim by religion, and primarily single or married. the dominance of individuals aged 18–35 aligns with global trends, as this age group represents the majority of the workforce due to their youthfulness, agility, strength, and family responsibilities. this could explain why these age brackets dominate the fruit-selling business. in northern nigeria, traditional gender roles often keep women indoors for domestic responsibilities, while men are expected to engage in income-generating activities, likely contributing to the predominance of male participants in this study. the lack of formal education among most fruit sellers may be attributed to the nature of the work, 52 60 36 16 36 0 10 20 30 40 50 60 70 80 90 100 drying salting canning smoking freezing fr eq u en cy ( % ) natural methods 88 48 64 0 20 40 60 80 100 120 140 160 180 200 family/friends community workshop formal education fr eq u en cy ( % ) soure of knowledge 18 80 42 12 48 0 20 40 60 80 100 120 140 160 180 200 availability of resources cost ease of use taste of preferene storage time fr eq u en cy ( % ) factors 5% 95% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% knowledge of preservatives application no knowledge of preservatives application p er ce n ta ge ( % ) knowledge on preservatives application tajudeen et al. – demographic characteristics of fruit sellers and … 405 which requires no specialized skills or education. the predominance of the hausa ethnic group and muslim religion reflects the demographic composition of the region, where these groups are the native population. however, a study conducted in lagos by yahaya et al. (2023) found that, despite lagos being a yorubaand igbo-dominated region, hausa individuals represented the majority of fruit sellers. this suggests a cultural association between the hausa ethnic group and the fruitselling trade. the findings of this study are consistent with those of danladi et al. (2023), who reported a predominance of males aged 20–35 years, married individuals, and those with lower levels of education among fruit sellers in maiduguri metropolis, northern nigeria. ajibade et al. (2023) also observed similar demographic patterns in abuja, with males, married individuals, and youthful sellers (20–40 years) dominating the trade. however, a study by orok et al. (2024) in ado-ekiti, southwest nigeria, reported contrasting findings. in that region, females and individuals with tertiary education dominated the fruitselling business. this divergence may be attributed to the higher levels of education in southwest nigeria and greater gender equality, which allows women to engage freely in various occupations. furthermore, fruit selling is perceived as a less strenuous and more "feminine" occupation in the region, which could explain the higher participation of women. similarly, a survey by ojewumi et al. (2021) in abeokuta, southwest nigeria, found that females, individuals aged 40–49 years, those with tertiary education, and married individuals accounted for the majority of fruit sellers. the survey revealed that watermelon, oranges, bananas, sweet melons, apples, pineapples, tangerines, pawpaw, and strawberries, in that order, were the most commonly preserved fruits in the city. these fruits are widely available and consumed across nigeria. their high demand and frequent availability likely drive their preservation in kebbi to meet consumer needs. previous studies by omoyajowo et al. (2018) and petrikova et al. (2023) identified watermelon, oranges, tangerines, mangoes, bananas, apples, pawpaw (papaya), coconuts, pineapples, and guava as commonly consumed fruits in nigeria. in kebbi, the most frequently used chemical preservatives were sulfite, citric acid, sodium benzoate, calcium chloride, benzoic acid, and ethanol. natural methods employed included salting, drying, canning, freezing, and smoking. while limited literature exists on the types of chemicals used for fruit preservation in nigeria, regional variations are evident. for instance, a study in lagos, southwest nigeria, by yahaya et al. (2023) reported the use of chemicals such as gammalin20, sniper, aluminum phosphide, ddt (otapiapia), and brodifacum. these regional differences in preservatives are likely influenced by factors such as cost and availability. chemical preservatives extend fruit shelf life by inhibiting the growth of bacteria, fungi, and yeast and slowing oxidative chemical reactions. however, while natural methods are generally perceived as safer, the unregulated use of chemical preservatives may pose health risks to consumers. for example, sodium benzoate is considered safe at 5 mg/kg of body weight per day. exceeding this limit may lead to digestive issues, skin changes, respiratory problems, reproductive abnormalities, liver and kidney disorders, and an increased risk of obesity (walczak-nowicka & herbet, 2022). calcium chloride, when consumed above 0.4%, can cause cardiac issues, kidney stones, gastric necrosis, neurological disorders, and embryological abnormalities (bailone et al., 2022). benzoic acid at high concentrations can result in skin irritation, eye damage, respiratory issues, lung diseases, immune dysfunction, and other side effects (rokni et al., 2024). citric acid exposure has been associated with inflammation-related conditions, including asthma, juvenile idiopathic arthritis, autistic spectrum disorders, and fibromyalgia (sweis & cressey, 2018). ethanol, or ethyl alcohol, may lead to central nervous system depression, hypoglycemia, brain trauma, liver damage, and an increased risk of cancer (wilson & matschinsky, 2020). sulfite is linked with dermatitis, hives, severe anaphylactic, asthmatic responses, and low blood pressure (vally et al., 2009). the majority of fruit sellers reported learning about preservation methods through family and friends, formal education, and workshops. their choice of preservation methods was influenced primarily by the cost, storage duration, and ease of use. however, 95% of the sellers had limited knowledge of preservative application guidelines. this lack of awareness suggests that fruit consumption in the city might unknowingly pose health risks to consumers. conclusion and recommendations the findings revealed that the demographic profile of fruit sellers in birnin kebbi is predominantly young, male, of hausa ethnicity, and with minimal formal education, reflecting the cultural and socio-economic characteristics of the region. commonly preserved fruits included watermelon, oranges, bananas, and sweet melons, among others, reflecting their high demand and widespread consumption in nigeria. chemical preservatives, such as citric acid, sodium benzoate, calcium chloride, benzoic acid, and ethanol, were frequently used alongside natural methods like salting, drying, canning, freezing, and smoking. while these methods extend the shelf life of fruits, the unregulated and potentially excessive use of chemical preservatives poses significant health risks to consumers. additionally, the limited knowledge of preservative application guidelines among the majority of fruit sellers raises concerns about the safety of fruit preservation practices in the city. 406 biology, medicine, & natural product chemistry 14 (1), 2025: 401-407 based on these findings, there is a need to educate fruit sellers and consumers on the safe use of chemical preservatives, including appropriate concentrations and application methods. additionally, it is necessary to promote awareness of the potential health risks associated with excessive or unregulated use of chemical preservatives. organizing workshops and training sessions for fruit sellers on alternative, safer, and more effective fruit preservation methods is imperative. further research should be conducted to comprehensively assess the long-term health impacts of preservatives commonly used in fruit preservation. regional differences in preservation practices need to be investigated to develop context-specific interventions. competing interests: the authours wish to declare without any uncertainty that there was no competing interest with regards to this work. references abdullahi, y., xue, j., li, h., han, s., jiao, j., racy, j. 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(2017). fruits for prevention and treatment of cardiovascular diseases. nutrients, 9(6), 598. https://doi.org/10.3390/nu9060598. https://doi.org/10.53623/tebt.v1i2.301 https://doi.org/10.3390/nu9060598 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 351-355 | doi: 10.14421/biomedich.2025.141.351-355 issn 2540-9328 (online) antibacterial effect of moringa oleifera leaves on staphylococcus aureus based on different concentration and harvest time jeremia aris panjaitan1,*, r. lia kusumawati2, yuki yunanda3, arvitamuriany triyanthi lubis4 1faculty of medicine, 2department of microbiology, 3department of community medicine, 4department of obstetrics and gynecology, faculty of medicine, universitas sumatera utara jl. dr. mansyur no.5, kota medan, sumatera utara 20155, indonesia. corresponding author* jeremia.stick@gmail.com abstract this study investigates the antibacterial activity of moringa oleifera leaf extract against staphylococcus aureus atcc 25923, focusing on the effects of harvest time and extract concentration. leaves were harvested in the morning (08:00–10:00) and afternoon (15:00–17:00), then extracted with 96% ethanol. the antibacterial efficacy was evaluated using the kirby-bauer disc diffusion method at 60%, 70%, and 80%. the largest inhibition zone was observed at 80% concentration from the afternoon harvest (10.38 ± 0.43 mm), while the smallest was recorded at 60% from the morning harvest (8.25 ± 0.20 mm). statistical analysis confirmed a significant concentration-dependent antibacterial effect (p < 0.001, η² = 88.6%). however, harvest time did not significantly affect inhibition zone size (p = 0.882). these results suggest that m. oleifera is an effective natural antimicrobial agent against s. aureus, with concentration being the key determinant of antibacterial activity. further research is recommended to explore its potential combined with antibiotics and its full therapeutic applicability. keywords: antibacterial activity; harvest time; moringa oleifera, staphylococcus aureus. introduction staphylococcus aureus is a gram-positive bacterium that is the leading cause of community-acquired and healthcare-associated infections globally. it is responsible for a broad spectrum of diseases, ranging from superficial skin infections to severe systemic conditions such as pneumonia, osteomyelitis, endocarditis, and sepsis. the growing prevalence of methicillin-resistant s. aureus (mrsa) has significantly reduced the effectiveness of conventional antibiotics, leading to treatment failures, increased healthcare costs, and higher mortality rates (abebe & birhanu, 2023). given the rising threat of antimicrobial resistance (amr), the search for alternative antibacterial agents, particularly those derived from natural bioactive compounds, has become an urgent priority. moringa oleifera—the drumstick tree—has bioactive compounds that exhibit antimicrobial, anti-inflammatory, and antioxidant activities. the plant is rich in bioactive secondary metabolites such as flavonoids, phenolic acids, carotenoids, and tocopherols, which have been reported to exhibit antibacterial activity against various pathogenic bacteria, including s. aureus (pareek et al., 2023. among these, flavonoids such as galangin, kaempferide, and kaempferide-3-o-glucoside have been shown to disrupt bacterial cell membranes, inhibit dna synthesis, and interfere with metabolic pathways (górniak et al., 2019). given its extensive bioactive profile, m. oleifera has gained attention as a potential natural antibacterial agent, particularly in increasing antibiotic resistance. despite the well-documented antibacterial potential of m. oleifera, several factors influencing its efficacy remain poorly understood. harvest time is a crucial but underexplored factor affecting the biosynthesis and accumulation of secondary metabolites in medicinal plants (persic et al., 2018). the production of flavonoids and phenolic compounds is susceptible to environmental conditions such as light exposure, temperature, and humidity, which fluctuate throughout the day. although environmental factors may affect m. oleifera’s antibacterial efficacy, few studies have examined the role of harvest time against s. aureus (ioannou et al., 2020). most existing studies on m. oleifera have focused on optimizing extraction methods and determining antibacterial activity at a single time point, without systematically comparing leaves harvested at different times of the day. additionally, there is a lack of data on the optimal extract concentration required for maximum bacterial inhibition. understanding these variables is essential for standardizing m. oleifera as a natural antimicrobial agent with consistent efficacy. manuscript received: 22 april, 2025. revision accepted: 27 june, 2025. published: 04 july, 2025. https://doi.org/10.14421/biomedich.2025.141.351-355 352 biology, medicine, & natural product chemistry 14 (1), 2025: 351-355 this study aims to evaluate the antibacterial efficacy of m. oleifera leaf extract against s. aureus, focusing on the influence of harvest time and extract concentration on bacterial inhibition. specifically, this study seeks to determine whether leaves harvested in the morning and afternoon exhibit differential antibacterial activity and to identify the optimal extract concentration for maximum inhibition. by addressing these knowledge gaps, this study provides novel insights into the role of harvest timing in modulating m. oleifera’s antibacterial properties, contributing to its potential standardization as a natural antimicrobial agent. materials and methods study design and ethical approval this study employed a posttest-only control group design to evaluate the antibacterial activity of m. oleifera leaf extract against s. aureus. the research was conducted at the microbiology laboratory, prof. chairuddin p. lubis hospital, medan, indonesia. ethical approval was obtained from the universitas sumatera utara ethics committee (approval no. 842/kepk/usu/2022), ensuring compliance with institutional and international ethical standards. plant material and extract preparation fresh m. oleifera leaves were collected from tanjung morawa district, deli serdang regency, north sumatra, indonesia. botanical identification and authentication of the plant material were conducted at the organic chemistry laboratory, universitas sumatera utara. to evaluate the effect of harvest time, leaves were collected in the morning (08:00–10:00 am) and afternoon (03:00– 05:00 pm). leaves were selected based on visual inspection, including only fully developed, fresh, undamaged green leaves from the third to sixth position from the shoot. while yellow, perforated, moldy, or wilted leaves were excluded to ensure uniformity in plant quality. leaves were washed with distilled water, oven-dried at 40 °c for 24 h, and ground into a fine powder. the powdered leaves were subjected to ethanol maceration using 96% ethanol at a 1:10 (w/v) ratio for 24 hours with continuous stirring. the mixture was filtered using whatman no. 1 filter paper, and the remaining plant material underwent three additional extraction cycles to maximize bioactive compound yield. the pooled filtrates were concentrated using a rotary evaporator at 40°c under reduced pressure until a thick extract was obtained (munira et al., 2021). the extract was stored at 4°c in an amber glass bottle to prevent degradation. phytochemical screening was performed to confirm the presence of flavonoids, saponins, and tannins, which are known to contribute to the antibacterial properties of m. oleifera. figure 1. moringa oleifera during the washing and drying stages. bacterial strain and culture preparation a pure strain of s. aureus was obtained from the microbiology laboratory, universitas sumatera utara. a bacterial suspension was prepared in mueller-hinton broth and adjusted to a 0.5 mcfarland standard (1.5 × 10⁸ cfu/ml) using a spectrophotometer at 600 nm to ensure uniform bacterial concentration across experiments. antibacterial activity assay the antibacterial activity of m. oleifera leaf extract was assessed using the kirby-bauer disc diffusion method, following clinical and laboratory standards institute (clsi) guidelines. mueller-hinton agar plates were inoculated with 100 µl of bacterial suspension and evenly spread using a sterile cotton swab. sterile 6 mm filter paper discs (whatman no. 1) were impregnated with 20 µl of extract at concentrations of 60%, 70%, and 80% and air-dried before application. using sterile forceps, the extract-impregnated discs were aseptically placed onto inoculated mueller-hinton agar plates, ensuring uniform spacing to prevent inhibition zone overlap. a negative control (sterile distilled water) and a positive control (commercial antibiotic disc) were included in each assay. plates were incubated at 37°c for 24 hours under aerobic conditions (munira et al. 2021). following incubation, the diameter of the inhibition zones (clear zones around the discs) was measured using a digital caliper in two perpendicular directions, and the mean values were recorded. the experiment was performed in quadruplicate to ensure statistical reliability. statistical analysis all statistical analyses were conducted using spss version 25 (ibm corp., usa), with p < 0.05 considered statistically significant. data were analyzed for normality (shapiro-wilk test) and homogeneity (levene’s test). as both assumptions were met, two-way anova was applied to assess the effects of extract concentration and harvest time, including their interaction. post hoc analysis (tukey’s test) was conducted for pairwise panjaitan et al. – antibacterial effect of moringa oleifera leaves on 353 comparisons. effect size (η² and cohen’s f²) was calculated to quantify the strength of effects. linear regression analysis evaluated the dose-response relationship between concentration and inhibition zone diameter. results and discussion phytochemical characterization of moringa oleifera extract phytochemical screening confirmed the presence of flavonoids, saponins, and tannins in m. oleifera leaf extracts from both morning and afternoon harvests (table 1). these bioactive compounds have been reported to contribute to antibacterial activity, particularly against s. aureus. table 1. phytochemical screening. secondary metabolite test reagent morning harvest afternoon harvest flavonoids mg powder + hcl + + saponins distilled water + hcl + + tannins fecl₃ + + legend: (+) present, (-) absent antibacterial activity of moringa oleifera extract the antibacterial activity of m. oleifera extract was evaluated by measuring inhibition zone diameters (table 2). the highest inhibition was observed in the 80% extract concentration from the afternoon harvest (s80, 10.38 ± 0.43 mm), while the lowest was recorded in the 60% morning harvest group (p60, 8.25 ± 0.20 mm). however, inhibition zones between morning and afternoon harvests at the same concentration did not differ significantly, suggesting that harvest time did not substantially influence antibacterial efficacy. table 2. mean inhibition zone diameters. treatment group mean inhibition zone (mm) ± sd p60 8.25 ± 0.20 p70 9.19 ± 0.24 p80 10.31 ± 0.24 s60 8.50 ± 0.20 s70 8.81 ± 0.55 s80 10.38 ± 0.43 statistical analysis of antibacterial activity the normality assumption was confirmed using the shapiro-wilk test (p > 0.05), and homogeneity of variance was verified using levene’s test (p > 0.05). therefore, two-way anova was performed to evaluate the effects of extract concentration and harvest time, including their interaction. extract concentration significantly influenced antibacterial activity (p < 0.001), confirming that higher concentrations resulted in larger inhibition zones. harvest time did not have a statistically significant effect (p = 0.882), indicating that variations in time of collection did not alter antibacterial efficacy. no significant interaction was observed between extract concentration and harvest time (p = 0.197), suggesting that concentration effects were independent of harvest conditions. post-hoc analysis using tukey’s hsd test confirmed that inhibition zones differed significantly between extract concentration groups (p < 0.05), while no significant differences were observed between morning and afternoon harvests at the same concentration level. effect size analysis confirmed that extract concentration strongly influenced inhibition zone diameter (η² = 88.6%, f² = 7.77), explaining nearly 89% of the observed variation. in contrast, harvest time had a negligible effect (η² = 0.1%, f² = 0.001). a linear regression analysis (r² = 0.83) confirmed a strong doseresponse relationship between extract concentration and inhibition zone diameter. influence of extract concentration on antibacterial activity this study demonstrated a significant dose-dependent relationship between m. oleifera extract concentration and antibacterial activity against s. aureus (p < 0.001, r² = 0.83). higher extract concentrations exhibited larger inhibition zones, consistent with previous studies reporting that flavonoids, tannins, and saponins contribute to bacterial growth inhibition through membrane disruption, enzyme inactivation, and dna synthesis interference (jahan et al. 2022; van den berg and kuipers 2022; yan et al. 2024; huang et al. 2024). the observed inhibition zone values align with prior research, where m. oleifera extracts at 50–100 µg/ml demonstrated inhibition zones of 10–16 mm against s. aureus (el-sherbiny et al., 2024). although a clear concentration-dependent effect was observed, we did not determine minimum inhibitory concentration (mic) or minimum bactericidal concentration (mbc). without these assays, it is difficult to distinguish between bacteriostatic and bactericidal actions of m. oleifera. impact of harvest time on antibacterial efficacy contrary to expectations, harvest time did not significantly affect antibacterial activity (p = 0.882). this contrasts with findings in other medicinal plants, where secondary metabolite accumulation varies throughout the day due to diurnal regulation of biosynthetic pathways (neugart et al., 2021). possible explanations for this include: genetic factors, prior studies indicate that metabolite composition in m. oleifera varies more significantly between cultivars than within a single plant species due to daily fluctuations (hamada et al. 2024). secondary metabolite stability in m. oleifera, unlike plants where flavonoids accumulate in response to sunlight, m. oleifera may exhibit more stable metabolite profiles regardless of harvest time (rahmawati et al., 2024). future research should quantify flavonoid and phenolic content at multiple harvest times (e.g., early 354 biology, medicine, & natural product chemistry 14 (1), 2025: 351-355 morning, midday, and evening) and across different seasons to further assess potential variations. study limitations and future directions the limitation of this study is the absence of mic and mbc assays, which are critical for determining whether m. oleifera exhibits bacteriostatic or bactericidal effects. future studies should employ broth microdilution techniques to establish precise antimicrobial thresholds. additionally, while phytochemical screening confirmed the presence of flavonoids, tannins, and saponins, their quantitative concentrations were not measured. future research should incorporate high-performance liquid chromatography (hplc) or mass spectrometry analysis for detailed metabolite profiling. further investigation is also needed to evaluate the synergistic potential of m. oleifera with conventional antibiotics, which could enhance treatment efficacy while reducing the risk of resistance development. conclusions this study demonstrated that m. oleifera leaf extract exhibits significant antibacterial activity against s. aureus, with a strong dose-response relationship between extract concentration and inhibition zone size. however, harvest time did not significantly influence antibacterial efficacy, suggesting that m. oleifera may maintain a stable bioactive profile independent of short-term environmental variations. these findings support the potential application of m. oleifera as a natural antimicrobial agent, particularly in the context of antibiotic resistance. future studies should focus on mic/mbc determination, quantitative phytochemical analysis, and synergy testing with conventional antibiotics to further validate its therapeutic potential. acknowledgements: we would like to express our gratitude to microbiology laboratory, prof. chairuddin p. lubis hospital for facilitating the laboratory analyses conducted in this research. we also extend our appreciation to the organic chemistry laboratory, universitas sumatera utara, for their valuable assistance in the identification and authentication of the plant materials used in this study. authors’ contributions: conceptualization: jap, rlk, yy, atl. data collection: jap,rlk. data extraction: jap. methodology: jap, yy. supervision: rlk, yy, atl. writing manuscript: jap. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research is not financially supported by any organization or individuals. references abebe, a. a., & birhanu, a. g. 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(2022). the antibacterial action of moringa oleifera: a systematic review. south african journal of botany, 151(a), 224–233. https://doi.org/10.1016/j.sajb.2022.09.034 yan, y., xia, x., fatima, a., zhang, l., yuan, g., lian, f., et al. (2024). antibacterial activity and mechanisms of plant flavonoids against gram-negative bacteria based on the antibacterial statistical model. pharmaceuticals, 17(3), 292. https://doi.org/10.3390/ph17030292 https://doi.org/10.1016/j.sajb.2022.09.034 https://doi.org/10.3390/ph17030292 this page intentionally left blank biology, medicine, & natural product chemistry volume 5 – number 2 – 2016 issn 2089-6514 (paper) | issn 2540-9328 (online) contents starch-glycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on its mechanical properties endaruji sedyadi, syafiana khusna aini, dewi anggraini, dian prihatiningtias ekawati 33 40 optimization of binocular microscope with micro digital camera for measuring seminiferous tubules epithelium height sutriyono 41 47 histological study of common house gecko (hemidactylus frenatus) regenerated tail rakhmiyati, muhammad jafar luthfi 49 53 effect of addition of soursop leaf extract to ganyong (canna edulis ker.) starch edible film and its application in red grape storage time erni widyastuti, endaruji sedyadi, susy yunita prabawati 55 59 a stability mathematical model of nasopharyngeal carcinoma on cellular level sugiyanto, fajar adi kusumo, lina aryati, mardiah suci hardianti 61 64 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1137-1141 | doi: 10.14421/biomedich.2025.142.1137-1141 issn 2540-9328 (online) effect of essential oil of tejpat, black pepper, and cardamom as a natural food preservatives for sweet orange (citrus sinensis) juice sagar mani paudel1, bishal thagunna1*, deepa khadka2, rashmi baral1, deepa thapa1 1department of food technology; 2department of food microbiology, affiliated to tribhuvan university, pokhara, kaski, nepal. corresponding author* bisu.thaguna123@gmail.com manuscript received: 15 september, 2025. revision accepted: 21 november 2025, 2025. published: 03 december, 2025. abstract essential oil of tejpat (cinnamomum tamala), black pepper (piper nigrum) and cardamom (amomum subulatum roxb.) collected by hydro distillation, and were tested as natural food preservative in orange juice (citrus sinensis). the physiochemical changes such as ph, tss, acidity, total microbial count, yeast and mold count were evaluated at fix time intervals of 4 hours. for 24 hours. it has been observed that the juice sample infused with black pepper essential oil demonstrated superior preservation of tss, ph and acidity compared to the other variants. orange juice supplemented with essential oils maintained the microbial count and yeast and mold count below 100 cfu/ml for up to 24 hours. after 24 hours of storage, all samples surpassed the total microbial count. among the essential oils, black pepper essential oil demonstrated superior preservation compared to the others. keywords: essential oil; cinnamomum tamala; piper nigrum; amomum roxb; citrus sinensis; shelf-life. introduction sweet orange, scientifically known as citrus aurantium, is a member of the rutaceae family and the citrus genus, commonly referred to as sweet orange, and thrives in temperate and subtropical regions. the c. aurantium tree can reach a height of up to 6 meters, featuring leathery foliage and fragrant white blossoms (azadi et al., 2012). citrus ranks among the most significant fruit crops globally due to its substantial nutritional benefits, extensive fruit yield, and various processed products, as noted by chaturvedi et al. (2001). orange juice is both nutritious and revitalizing due to its vitamin c content, delightful tangy flavor, attractive hue, pleasant aroma, and health benefits (kapoor et al., 2012). nevertheless, after just a few hours of extraction, the juice begins to spoil, resulting in a loss of its color, flavor, and aroma. this spoilage is attributed to significant microbial presence and enzymatic activity, which degrade the sensory and nutritional properties of the juice, rendering it unsuitable for consumption. the primary factors contributing to this deterioration are likely the proliferation of microbes, chemical reactions, changes in structure, and the conditions under which it is stored (kapoor et al., 2012). additionally, potential health risks linked to the consumption of fruit juices containing synthetic preservatives have led to a notable shift in public perception regarding these drinks (moses, 2018). therefore, it is essential to explore additional antimicrobial substances derived from plant sources. lately, there has been a growing interest in utilizing plant-based antimicrobials in scientific studies and potentially for food preservation (pandey et al., 2017). essential oils (eos) are typically intricate yet volatile substances that possess a strong aroma. these compounds are generated by fragrant plants as secondary metabolites in various parts of the plants (bakkali et al., 2008). essential oils, often referred to as volatile odoriferous oils, are fragrant oily liquids obtained from various parts of plants, such as leaves, peels, barks, flowers, buds, and seeds. these oils can be extracted from plant materials using several techniques, including steam distillation and expression. among these extraction methods, steam distillation is particularly popular, especially for production on a commercial scale (cassel and vargas 2006). essential oils are recognized for their antioxidant and antimicrobial properties, making them effective natural additives in food and food products. (tongnuanchan and benjakul, 2014). because of the growing interest in natural additives, essential oils derived from various plants have gained more popularity, particularly when used alongside other preservation methods within the framework of “hurdle technology.” as a result, essential oils can act as alternative additives or processing aids in the realm of green technology. https://doi.org/10.14421/biomedich.2025.142.1137-1141 1138 biology, medicine, & natural product chemistry 14 (2), 2025: 1137-1141 materials and method extraction of essential oil cinnamomum tamala (tejpat), piper nigrum (black pepper), and amomum subulatum roxb (cardamom), plant spices were purchased from the local market of pokhara, nepal. the essential oil was obtained through the hydrodistillation method utilizing a clevenger-type apparatus, following the procedures outlined in the european pharmacopeia (1983). sample preparation ripe, mature, and uniform orange fruits were thoroughly washed with running tap water, manually peeled, deseeded, and the pulp blended using an electric juicer in a hygienic laboratory environment. the resulting orange juice was then strained through a clean muslin cloth and separated into four equal portions of 100 ml each. essential oils (10 ml) and each oleoresin were individually dissolved in 90 ml of ethanol, then combined homogeneously with extracted juice (50 ml) in plastic cups. the control sample consisted solely of orange juice, while the control was made by mixing 100 ml of ethanol with orange juice (kapoor et al., 2014). storage the control and treated samples having essential oils were stored at normal room temperature (25±5°c) for 24 h in laboratory condition. the effect of storage on physiochemical changes, and microbial count were observed after an interval of 4h. determination of moisture content the moisture determination of the fresh orange was carried out by hot-air oven method (ranganna, 2008) determination of titratable acidity and ph acidity was determined by titration with the standard sodium hydroxide (0.1n) solution and expressed as % malic acid (ranganna, 2008). ph was determined using ph meter. determination of tss tss was determined by using hand refractometer microbial analysis in order to determine the total microbial count and yeast and mold counts, total plate count method as described by aneja (2010) for the examination of juice samples using the plate count agar and potato–dextrose agar, respectively, were adopted total microbial count the total microbial count was conducted following the method outlined by (kapoor et al., 2014). five g sample of juice was taken and diluted with 90 ml of ringer's solution in a 1:10 ratio. after thorough mixing, 1 ml of the sample was added to 10 ml of dextrose-tryptone agar medium, which was then placed in a sterilized petri dish at 45°c and mixed well. the mixture was subsequently incubated at 37°c to observe the colonies that developed in each petri dish. the findings were recorded as the number of colonies present in 1 ml of the juice sample, expressed in cfu/ml. yeast and mold count yeasts and molds were quantified using acidified potato dextrose agar (pda) following the standard methods for the examination of dairy products (kapoor et al., 2014). the procedures for the experiments remained identical to those used for the total microbial count, with the only variation being the substitution of dextrose-tryptone agar with pda medium. all microbiological analyses were performed in triplicate, and the results are represented as colony-forming units per milliliter. results and discussion chemical composition of fresh sweet orange juice the moisture content of citrus sinensis (sweet orange) was measured at 85.23% as shown in table 1 below. orange juice predominantly consists of water, typically containing between 85% and 90% water. this high-water content makes orange juice very perishable, necessitating specific preservation methods such as refrigeration, pasteurization, or freeze-drying to prolong its shelf life (sharma et al., 2018). the acidity level and ph of citrus sinensis (sweet orange) were found to be 2.83% and 5.02, respectively which is shown below table 1. the acidity of both orange juice and the peel significantly affect their flavor, stability, and nutritional value. typically, orange juice has a ph range of approximately 3.3 to 4.2, categorizing it as a moderately acidic drink. this acidity primarily results from organic acids, chiefly citric acid, which comprises more than 90% of the total acid content, alongside smaller quantities of ascorbic acid (vitamin c) and malic acid (ladaniya et al., 2003). the low ph contributes to the tangy taste of orange juice and also serves as a natural preservative by inhibiting the growth of numerous bacteria, thereby extending the juice's shelf life (boumediene et al., 2018). the total soluble solids (tss) content of citrus sinensis (sweet orange) was recorded at 10.3ºbx as shown in table 1 below. tss, expressed in degrees brix (°brix), reflects the percentage of soluble solids in a liquid. in freshly squeezed orange juice, the tss generally varies from 10 to 12 °brix, influenced by factors such as the variety of orange, its ripeness, and the conditions under which it was grown (kelebek & selli, 2011). table 1 chemical composition of fresh sweet orange juice. parameters percentage % moisture 85.23±1.25 acidity 2.83±0.05 tss 10.3±0.08 ph 5.02±0.06 values are means ± standard deviation of triplicate analysis paudel et al. – effect of essential oil of tejpat, black pepper, and cardamom as … 1139 effects of essential oils on the physiochemical properties of orange juice tss the fresh juice extracted from citrus sinensis was subsequently blended with three different essential oils, and it was observed that the total soluble solids (tss) of all samples decreased as storage days increased. the initial tss of the control juice sample was 10.3°bx, which diminished to 8.6°bx over time. likewise, the tss values for s1 (black pepper), s2 (cardamom), and s3 (cinnamon) were recorded at 10°bx, 9.7°bx, and 9.6°bx, respectively, after 24 hours of storage, as indicated in table 2 below. the juice sample infused with black pepper essential oil demonstrated superior preservation of tss compared to the other variants. the total soluble solids (tss) levels in orange juice generally decline over time while being stored; however, the degree of this reduction is influenced by variables like storage temperature, type of packaging, and microbial activity (kapoor et al., 2009). ph the fresh juice of citrus sinensis was further combined with three different essential oils, and it was observed that the ph of all samples decreased with the increase in storage days. the control sample of juice had an initial ph of 5.02, which subsequently decreased to reach 4.0. likewise, the ph values for s1 (black pepper), s2 (cardamom), and s3 (cinnamon) were recorded as 4.7, 4.5, and 4.2 after 24 days of storage, as detailed in table 2 below. among the juice samples, the one with black pepper essential oil exhibited superior ph preservation compared to the other samples. the ph tends to decline progressively as titrable acidity rises. the drop in ph observed during storage could be attributed to acids produced from fructose, glucose, and various carbohydrates (kapoor et al., 2007). this reduction in ph might be linked to the elevated concentration of essential oil, which leads to a relatively higher acidity and other biochemical alterations that contribute to the decrease in ph (kapoor et al., 2009). acidity the fresh juice from citrus sinensis was combined with three different essential oils, and it was observed that the acidity of all samples increased as the number of storage days progressed. the acidity of the control juice sample started at 2.83 and rose to 3.2% after storage. likewise, the acidity levels of s1 (black pepper), s2 (cardamom), and s3 (cinnamon) were recorded at 2.86%, 2.92%, and 2.93% after 24 days of storage, as indicated in table 2. among the juice samples, the one with black pepper essential oil demonstrated superior preservation of acidity compared to the other samples. the rise in acidity during storage can be attributed to the breakdown of lactose (grewal and jain 1982) resulting in the conversion of sugars into acids (kapoor et al., 2007) and microbial contamination, which produces lactic acid from hexose sugars. a comparable increase in acidity percentage was noted for kinnow by (panesar et al., 2000) and for orange juice by (kapoor et al., 2012). effect of essential oils on total microbial count of orange juice during storage in all the samples depicted in the figure 1, microbial loading increased with prolonged storage time. orange juice supplemented with essential oils maintained the microbial count below 100 cfu/ml for up to 24 hours. after 24 hours of storage, all samples surpassed the total microbial count. among the essential oils, black pepper essential oil demonstrated superior preservation compared to the others. the effectiveness of essential oil is attributed to the higher percentage of monoterpene hydrocarbons, which may be key in inhibiting microbial growth, as noted by (kapoor et al., 2014), who described their synergistic effects. kapoor et al. (2014) also highlighted the combined action of different phenolic compounds, piperine, flavonoids, and various other components that suppress the growth of these microbes. the antifungal properties of the essential oils evaluated may stem from their phenolic, alcoholic, and aldehydic components, which have been identified as antimicrobial agents (kapoor et al., 2014). generally, cinnamon essential oil is recognized as a strong antimicrobial substance (burt, 2004). figure 1. effect of essential oils on total microbial count of orange juice during storage. here, microbial count of control sample 1cfu/ml after 4 h exceed more than 100 cfu/ml. s1 (black pepper), s2 (cardamom) and s3 (cinnamon). effect of essential oils on yeast and mold count of orange juice during storage the yeast and mold counts increase with prolonged storage period as shown in fig 2. initially the rise in yeast and mold, along with their microbial contamination, may also result from the proliferation of acid-producing bacteria (azhdarzadeh & hojjati, 2016). according to tserennadmid et al. (2011), lemon essential oil was effective in suppressing the growth of s. cerevisiae in clear apple juice. this finding is reinforced 0 20 40 60 80 100 120 4hr 8hr 12hr 16hr 20hr 24hr to ta l m ic ro b ia l c o u n t s1 s2 s3 1140 biology, medicine, & natural product chemistry 14 (2), 2025: 1137-1141 by ndagijimana et al.’s (2004) study, which demonstrated similar effects when orange juice was treated with the same essential oils. (eissa et al., 2008) found that apple juices infused with lemon grass, clove, and rosemary extracts exhibited the strongest suppression of yeast and molds (y and m) as well as bacteria (b), while those treated with basil and sage extracts showed slightly lower levels of inhibition after 4 weeks at 4°c. figure 2. effect of essential oils on yeast and mold count of orange juice during storage here, microbial count of control sample 1cfu/ml after 4 h exceed more than 100 cfu/ml. s1 (black pepper), s2 (cardamom) and s3 (cinnamon) sensory evaluation the mean value for color was highest in sample s1 and lowest in sample s3. the essential oil does not affect the color of the samples, but panelists favored the black pepper sample over the others. the control sample had the lowest color value due to the rapid change in color of the untreated juice, which led to noticeable browning. the highest mean value for flavor was found in sample s1, while the lowest was in the control sample s. many panelists preferred the taste of the essential oil juice in comparison to the control sample. the mean values for aroma were quite similar across all samples, but the black pepper essential oil juice received the highest ratings, as most panelists preferred its aroma over the others. the overall acceptability was highest for sample s1 and lowest for the control sample s3. figure 3. sensory evaluation s (control), s1 (black pepper), s2 (cardamom) and s3 (cinnamon) conclusion the current research indicated that essential oils from black pepper, tejpat, and cinnamon can effectively inhibit microbial growth, which poses a risk to human health. therefore, it can be inferred that incorporating black pepper, tejpat, and cinnamon essential oils into orange juice could aid in extending its shelf life by slowing down the proliferation of spoilage microorganisms. the longevity of orange juice has been shown to improve. these findings could serve as a significant basis for creating efficient natural preservatives. acknowledgement: the authors are sincerely thankful to the pokhara bigyan tatha prabidhi campus for chemical and equipment support. conflicts of interest: the authors declare no conflict of interest references azadi b, nichavar b, amin ch. volatile constituent of the peel and leaf of citrus aurantium l. cultivated in the north of iran. j pharm health sci 2012; 1(3): 37-41. azhdarzadeh, f., & hojjati, m. 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(2014). essential oils: extraction, bioactivities, and their uses for food preservation. journal of food science, 79(7), r1231-r1249. tserennadmid r, tako m, galgoczy c, papp t, pesti m, vagvolgyi c, et al. anti yeast activities of some essential oils in growth media, fruit juices and milk. int j food microbiol 2011; 144(3): 480-486 this page intentionally left blank cover jurnal biomenaprochy vol 8 num 1 2019 volume 8 number 1 april 2019 issn 2089-6514 (paper) issn 2540-9328 (online) biology, medicine, & natural product chemistry volume 8 – number 1 – april 2019 issn 2089-6514 (paper) | issn 2540-9328 (online) honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad jafar luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia mohamad amin department of biology, faculty of mathematics and sciences, universitas negeri malang, indonesia widodo department of biology education, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: r i y a n t o publisher: state islamic university (uin) sunan kalijaga yogyakarta co-publisher: the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university (uin) sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@yahoo.com biology, medicine, & natural product chemistry volume 8 – number 1 – april 2019 issn 2089-6514 (paper) | issn 2540-9328 (online) contents novel approaches for detection fluorescent-labeled by cellvizio lab system on hippocampal ca1 region asma ulhusna shaimi, wan raihana wan aasim, hasmah abdullah, wan amir nizam wan ahmad, tan soo choon, ang chee wei, zalina ismail 1 6 statistical analysis of habbatussauda’s benefits for health (blood pressure, glucose and uric acid) sugiyanto, luqyana khalda’ aesa, meksianis z. ndii 7 10 mathematical model of cervical cancer treatment using chemotherapy drug murtono, meksianis zadrak ndii, sugiyanto 11 15 a simple method for clearing and staining specimens for the demonstration of animal skeleton muhammad ja’far luthfi, nyoman puniawati soesilo 17 21 echinoderms diversity and abundance in gunung kidul beach yogyakarta hikmah supriyati, setyawati dwi k., ahzami, farhani sodiq, septiana khoiriyah 23 26 guidance for authors aims and scope biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international standards in their methodology. article types the journal seeks original full-length research papers, reviews, and short communication. manuscript of original research should be written in no more than 8,000 words (including tables and picture), or proportional with articles in this 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(personal communication); setyawan ad 2007, unpublished data). in the reference list, the references should be listed in an alphabetical order (better, if only 20 for research papers). names of journals should be abbreviated. always use the standard abbreviation of a journal's name according to the issn list of title word abbreviations (www.issn.org/2 22661-ltwa-online.php). the following examples are for guidance. journal: saharjo bh, nurhayati ad. 2006. domination and composition structure change at hemic peat natural regeneration following burning; a case study in pelalawan, riau province. biodiversitas 7: 154-158. book: rai mk, carpinella c. 2006. naturally occurring bioactive compounds. elsevier, amsterdam. chapter in book: webb co, cannon ch, davies sj. 2008. ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. in: carson w, schnitzer s (eds) tropical forest community ecology. wiley-blackwell, new york. abstract: assaeed am. 2007. seed production and dispersal of rhazya stricta. 50 annual symposium of the international association for vegetation science, swansea, uk, 23-27 july 2007. proceeding: alikodra hs. 2000. biodiversity for development of local autonomous government. in: setyawan ad, sutarno (eds.) toward mount lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in java island. universitas sebelas maret, surakarta, 17-20 july 2000. [indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com novel approaches for detection fluorescent-labeled by cellvizio lab system on hippocampal ca1 region asthma ulhusna shaimi, wan raihana wan aasim, hasmah abdullah, wan amir nizam wan ahmad, tan soo choon, ang chee wei, zalina ismail statistical analysis of habbatussauda's benefits for health (blood pressure, glucose and uric acid) sugiyanto, luqyana khalda' aesa, meksianis z. ndii mathematical model of cervical cancer treatment using chemotherapy drug murtono, meksianis zadrak ndii, sugiyanto a simple method for clearing and staining specimens for the demonstration of animal skeleton muhammad ja'far luthfi, nyoman puniawati soesilo echinoderms diversity and abundance in gunung kidul beach yogyakarta hikmah supriyati, setyawati dwi k., ahzami, farhani sodiq, septiana khoiriyah 1-6 7-10 11-15 17-21 23-26 volume 8 number 1 april 2019 published twice a year printed in indonesia front cover: ardisia humilis (photo: widodo) issn 2089-6514 (paper) issn 2540-9328 (online) biomed vol. 8 num. 1 april 2019 cover editorial board table of contents guidance back cover biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 435-441 | doi: 10.14421/biomedich.2025.141.435-441 issn 2540-9328 (online) durian seed flour (durio zibethinus murr) as an alternative medium for fungal growth tika afriani1,*, azimatur rahmi2, putri agiel ramadhani1, rahmadaniah1 1departmen of pharmacy, mohammad natsir university, bukittinggi, west sumatra, indonesia. 2department of chemistry, faculty of military mathematics and natural sciences, defense university, west java, indonesia. corresponding author* tika.afriani91a@gmail.com manuscript received: 28 april, 2025. revision accepted: 27 june, 2025. published: 17 july, 2025. abstract this study aimed to evaluate the potential of durian seed flour (durio zibethinus murr) as an alternative medium for the growth of candida albicans and aspergillus niger. the bleaching process utilized sodium metabisulfite at varying concentrations (600 ppm, 800 ppm, 1000 ppm) and immersion durations (15, 20, 25 minutes). the bleached samples were dried at 70℃ for 3 hours. durian seed flour media were formulated into three variants and compared with potato dextrose agar (pda) as the control medium. the activity test for candida albicans used the pour plate method with incubation for 48 hours at 37℃, while aspergillus niger was tested using the agar block method and incubated for 72 hours under the same conditions. immersion duration significantly affected the flour's color, with longer durations producing whiter flour. higher drying temperatures enhanced water evaporation efficiency, resulting in an average moisture content of 10.56%, compliant with national standards. the best medium formulation was f1 (10 g durian seed flour, 2 g sugar, 1.5 g agar, 0.25 g micronutrients, 100 ml distilled water), yielding 3.6 × 10⁷ cfu/ml of candida albicans colonies and an aspergillus niger growth diameter of 28.5 mm after 72 hours of incubation. however, sporulation of aspergillus niger was better on pda media. durian seed flour shows potential as an alternative fungal growth medium. keywords: aspergillus niger; candida albicans; durian seed flour; fungal growth medium. introduction durian season means one thing: durian seeds (durio zibethinus murr) are often unused and wasted. consumers generally eat only the meat or salad part of the fruit, which makes up about 20–35% of the whole fruit. meanwhile, the skin (60–75%) and seeds (5–15%) are not being maximally utilized (sistanto et al., 2017). durian seeds contain 51.5% water, 46.2% carbohydrates, 2.5% protein, and 0.2% fat (damayanti et al., 2020). the complete nutritional content of durian seeds can be utilized as a medium for mushroom growth. fungi require nutrients such as carbon, nitrogen, and nonmetallic elements such as sulfur and phosphorus, as well as metal elements such as ca, zn, na, k, cu, mn, mg, fe, vitamins, water, and energy. carbohydrates and their derivatives are the main substrates for carbon metabolism in fungi, with carbon being the most important element because it makes up 50% of the weight of microorganisms. durian seeds, with their high carbohydrate content, can be a sufficient source of energy for fungal growth (sundari et al., 2021). a 100-gram serving of durian seeds contains 67 grams of water, 28.3 grams of carbohydrates, 2.5 grams of fat, 2.5 grams of protein, 1.4 grams of vitamin c, and 19.7 milligrams of potassium. durian seeds have a fairly high starch content, so they have the potential to be used as flour (lopulalan, 2016). fungi are cosmopolitan organisms that can grow in environments close to humans, such as air, soil, water, clothing, and even inside the human body (hasanah, 2017). candida albicans is a pathogenic microorganism that can cause disease when present in excessive amounts in the body (ornay et al., 2017). aspergillus niger, another fungal species, grows as a saprophyte on decaying vegetation and is also found in soil, dust, and food. both fungi require sufficient nutrients to grow (hasanah, 2017). media is a mixture of nutrients required for the growth of microorganisms and is used for isolation, inoculation, physiological and biochemical tests of microorganisms. one of the media for fungal growth is potato dextrose agar (pda). pda is made from natural (potato) and synthetic materials (dextrose and agar). potatoes are a source of carbon (carbohydrates), vitamins, and energy; dextrose is a source of sugar and energy; and agar components act as a solidifying agent (qurrohman, 2021). previous research by afriani et al. (2022) showed that the durian seed flour produced had a brownish-white https://doi.org/10.14421/biomedich.2025.141.435-441 mailto:tika.afriani91a@gmail.com 436 biology, medicine, & natural product chemistry 14 (1), 2025: 435-441 color. treatment with the bleaching agent sodium metabisulfite (na2s2o5) is required to produce whiter flour. na2s2o5, already widely used in the food industry, effectively inhibits the browning reaction and prevents the formation of melanoidin pigments (akolo & azis, 2018; haryani et al., 2016). in the study of simanjuntak (2014), the complete randomized design (crd) method was used with two (2) factors: drying temperature (50°c, 60°c, 70°c) and sodium metabisulfite (na2s2o5) concentration (200 ppm, 400 ppm, 600 ppm). the results showed that treatment with 600 ppm na2s2o5 at 60°c produced durian seed flour with the best properties (simanjuntak et al., 2014). drying temperature affects the quality of flour, with low temperature reducing the rate of water evaporation and high temperature increasing the drying efficiency, reducing the moisture content below 10% to prevent growth. materials and methods chemical material the materials used in this research were durian seeds (durio zibethinus murr), sodium metabisulfite (na2s2o5), agar (satellite), cane sugar (gulaku), pda instant media (pronadisa), micronutrients, mc farland standard suspension, nacl 0, 9%, 70% ethanol, distilled water, 3% hcl (hydrochloric acid), naoh (sodium hydroxide), 3% ch3cooh, luff solution, 20% ki solution (potassium iodide), h2so4 (sulfuric acid), cotton swabs, ph indicator strips, aluminum foil, plastic wrap (bagus), parchment paper, gauze. equipment the instrumentations used in this research were laminar air flow (astec hlf 1200l), incubator (fisher scientific), oven (memmert), refrigerator (toshiba), refrigerator (toshiba), colony counter, blender (philips), digital scale (kern), hot plate (stirrer), sprayer, basin, tray, flour sieve, mortar and pestle, erlenmeyer (pyrex), beaker (pyrex), measuring cup (iwaki pyrex), test tube, petri dish, porcelain cup, vernier, desiccator, distillation flask, bunsen lamp, micropipette (eppendorf), cork punch, tweezers, dropper, spatula, stirring rod, ose needle, knife. fungal culture the fungal cultures used in this research were candida albicans and aspergillus niger obtained from the microbiology laboratory of the faculty of medicine, padang, west sumatra. sterilization of tools and materials all tools used were thoroughly washed with distilled water. test tubes, beakers and conical flasks were covered with cotton. petri dishes were wrapped in paper and sterilized by autoclaving at 121℃ for 15 minutes. the ose needle and forceps were sterilized with a bunsen lamp. the medium was sterilized by autoclaving at 121℃ for 15 minutes (magfirah abdullah et al., 2020). sampling the samples used in this study were durian seeds (durio zibethinus murr) as much as 2 kg obtained from durian fruit plantations in jorong balai sabuah, balai sabuah district, batipuah ateh, west sumatra. plant identification the identification of durian plants was carried out at its herbarium, faculty of biology, andalas university, padang, west sumatra. sample preparation durian seeds that are still fresh and not rotten are selected, then washed with clean water and drained. durian seeds are peeled off the outer skin, rewashed, drained, and then cut into pieces about 0.4 cm thick, then washed with running water until the sap is gone, and drained. the durian seeds are washed again with running water until the sap is gone and drained. then the durian seeds are weighed according to the weight used for flour production (simanjuntak et al., 2014). durian seed bleaching process sodium metabisulfite with 600 ppm, 800 ppm, and 1000 ppm were dissolved in 100 ml of distilled water, respectively. the bleaching solution was mixed with 100 grams of durian seeds in a glass beaker so that the entire surface of the durian seeds was immersed. the maceration process lasted for 15 minutes, 20 minutes, and 25 minutes, respectively. afterward, the solution was separated from the durian seeds and dried using an oven at 70°c for 3 hours (lastari et al., 2016). durian seed flour preparation durian seeds weighed according to weight are dried in an oven at 70℃ for 3 hours. the dried durian seeds are pounded and crushed with a mortar and pestle until smooth, then sieved with a 100 mesh sieve (simanjuntak et al., 2014). organoleptic test an organoleptic test evaluates the color, shape, and smell of durian seed flour produced using the human senses (sensory) (hevira et al., 2021). determination of moisture content moisture content is determined by the direct heating method or gravimetric method. the porcelain cup is dried in the oven at 100℃ for 30 minutes, then cooled and weighed empty. weigh 5 grams of durian seed flour in a cup, then dry it in an oven at 105℃ for 3 hours. cool the beaker and reweigh it. record the weight of the sample before and after drying (kiptiah et al., 2019). afriani et al. – durian seed flour (durio zibethinus murr) as an alternative medium … 437 determination of carbohydrate content weigh about 3 g of durian seed flour into a 500 ml erlenmeyer flask. add 200 ml of 3% hcl solution. simmer for 3 hours with the condenser upright, then cool and neutralize with saturated naoh solution. add a little 3% ch3cooh to make the solution slightly acidic, then transfer the contents to a 500 ml volumetric flask up to the mark, then filter. pipette 10 ml of filtered water into a 500 ml erlenmeyer flask, add 25 ml of luff's solution (with a pipette), a few boiling stones and 15 ml of distilled water. bring the mixture to the boil for 3 minutes, then continue boiling for exactly 10 minutes (count from the start of boiling and use a stopwatch), then rapidly cool in an ice bath. after cooling, slowly add 15 ml of 20% ki solution and 25 ml of 25% h2so4, immediately titrate with 0.1 n thio solution (use 0.5% starch solution indicator). perform a blank determination (bsn, 2011). preparation of pda media weigh 3.9 grams of potato dextrose agar (pda) and dissolve in 100 ml of distilled water using an erlenmeyer flask. place the media in a test tube lined with cotton and aluminum foil. then, sterilized using an autoclave for 15 minutes at 121℃ and left until the media is cool enough, aseptically poured into a petri dish as much as 20 ml (mujipradhana et al., 2018). preparation of durian seed flour media durian seed flour-based media were prepared in 3 formulations with different durian seed flour compositions. the following is a table of each of durian seed flour-based fungal growth media formulation. durian seed flour is weighed according to the formulation to be prepared, 1.5 grams of neutral agar and 2 grams of sugar are added and dissolved with 100 ml of distilled water in an erlenmeyer flask. for the f1 formulation, 0.25 grams of micronutrients were added to the media solution. the media solution was heated to 90°c with homogeneous stirring until boiling. in addition, the ph was measured using ph indicator paper. the durian seed flour media solution was covered with cotton and aluminum foil, and then sterilized using an autoclave for 15 minutes at 121°c. durian seed flour media was poured into petri dishes, each concentration was made as much as three durian seed flour media and more for media stock. fungal inoculation on media candida albicans candida albicans culture stock was harvested using a sterile ose needle and then suspended in 10 ml 0.9% nacl. the suspension was vortexed until the turbidity of the suspension was equal to the turbidity of the mc farland standard solution (10-8 cfu/ml). 1 ml of suspension was added to 9 ml of 0.9% nacl solution and diluted up to 6-fold (10-6). furthermore, the suspension preparation and candida albicans culture were inoculated on the surface of pda sloped media and then incubated at 37°c for 48 hours (septiani et al., 2017). aspergillus niger aspergillus niger culture was collected using a 5 mm diameter cork borer, then transferred to instant pda and durian seed flour media and incubated at 37°c for 72 hours.the diameter of the fungal growth zone was measured every 24 hours using a caliper. the sporulation formed was visually observed. the test was performed with 3 replicates (kwoseh et al., 2012). statistical data analysis all data were statistically analyzed using one-way anova followed by tukey's hsd test. tukey's hsd test was used to test differences between sample means for significance. a significance level of p<0.05 was considered statistically significant. results and discussion the samples used in this study were durian seeds collected from jorong balai sabuah, balai sabuah batipuah ateh district, west sumatra. the samples were identified at the herbarium of andalas university (anda) to ensure the plant species used is (durio zibethinus murr). previous research by afriani et al. (2022) showed that the durian seed flour produced was still brownish this study used sodium metabisulfite (na2s2o5) as a bleaching agent to prevent browning reactions in durian seed flour. sodium metabisulfite was chosen because it produces more whiteness than other bleaching agents such as ascorbic acid (vitamin c) and hydrogen peroxide (haryani, 2016). the bleaching process was performed by soaking durian seeds in na₂s₂o₅ solution at concentrations of 600 ppm, 800 ppm, and 1000 ppm with soaking durations of 15, 20, and 25 min. the results showed that increasing the concentration and soaking duration produced durian seed flour with a whiter color (purwanto et al., 2013). the drying temperature used was 70°c for 3 hours, as this temperature effectively reduced the moisture content without damaging the flour properties (simanjuntak et al., 2014). after drying, the durian seeds were crushed until smooth and sieved using a 100 mesh sieve to produce flour with a very fine texture (lopulalan, 2016). durian seed meal has a ph of 5, similar to the potato dextrose agar (pda) control medium, which also has a ph of 5. environmental factors such as ph greatly affect mold growth, with low acidity (ph 4.5-5.6) being ideal for inhibiting the growth of bacteria that generally live at neutral ph (etiek, 2020). physically, durian seed flour is white, as opposed to pda media, which is clear yellow. the consistency of these two media is both solid because both contain 1.5% agar, which allows the media to be 438 biology, medicine, & natural product chemistry 14 (1), 2025: 435-441 liquid at high temperatures and solid when cold. to further characterize the physical and chemical properties of the durian seed flour produced, an organoleptic evaluation and a comparative analysis with standard pda media were conducted. the result of the organoleptic assessment is presented in table 1, while the physical and chemical characteristic of both the durian seed flour media and pda media are summarized in table 2. table 1. organoleptic of durian seed flour. sample immersion time color shape odor without bleaching brown powder typical 600 ppm 15 brown powder typical 20 brown powder typical 25 brown powder typical 800 ppm 15 brown powder typical 20 brown powder typical 25 white powder typical 1000 ppm 15 brown powder typical 20 brown powder typical 25 white powder typical table 2. characteristics of durian seed flour media and pda (potato dextrose agar). characteristic durian seed flour media pda (potato dextrose agar) color white yellow clear odor typical typical consistency solid solid ph 5 5 the average moisture content of the durian seed flour produced was 10.56%, which meets the national standard requiring a moisture content of less than 14% (kiptiah et al., 2019). this low moisture content is expected to improve the storability of the product, as low water content reduces the risk of microbial growth. the carbohydrate content in durian seed flour without bleaching is 14.66%, the highest carbohydrate content is at a concentration of 1000 ppm in minute 25, which is 14.28%, while the lowest carbohydrate content is at 600 ppm. it can be concluded that the higher the concentration and soaking time of sodium metabisulfite (na2s2o5), the higher the carbohydrate content in durian seed flour (durio zibethinus murr). carbohydrates in flour generally show some changes with pre-treatment in the form of soaking time (lastari et al., 2016). durian seed flour is used as an alternative medium for the growth of candida albicans and aspergillus niger fungi, which are isolates from andalas university laboratory, padang, west sumatra. these fungi were grown on durian seed flour media formulated in three concentration variations, namely formulation 1 (f1) 10 grams of durian seed flour with the addition of micronutrient (zinc), formulation 2 (f2) 8 grams of durian seed flour, and formulation 3 (f3) 10 grams of durian seed flour without the addition of sugar, each dissolved in 100 ml of distilled water, then heated and stirred until homogeneous and boiling. after that, it was sterilized using an autoclave at 121℃ for 15 minutes. the results showed that candida albicans and aspergillus niger were able to grow on durian seed flour media. candida albicans formed white to yellowishwhite colonies and oval-round shape after 48 hours of incubation. the highest number of colonies was found in f1 formulation (3.6 × 10⁷ cfu/ml), while the lowest was in f2 (2.4 × 10⁷ cfu/ml), while in pda comparison media, the number of colonies was 46.7 × 107 cfu/ml. this shows that the concentration of durian seed flour affects the number of colonies that grow. the number of candida albicans grown on various formulations of durian seed flour media compared to pda is detailed in table 3, figure 1. table 3. number of candida albicans colonies on durian seed flour media and pda media. media number of fungal colonies (cfu/ml) pda 46.7 x 107 f1 3.6 x 107 f2 2.4 x 107 f3 3 x 107 figure 1. colony count of candida albicans on pda media and durian seed flour media (pda: potato dextrose agar, f: formula. pda = 46.7 x 107, f1 = 3.6 x 107 cfu / ml, f2 = 2.4 x 107 cfu / ml, and f3 = 3 x 107 cfu/ml) candida albicans fungal growth requires a low acid ph of 4.5-5.6. candida albicans can grow well at 3037℃. at the optimal temperature, chemical and enzymatic reactions in the cell occur faster, so the growth afriani et al. – durian seed flour (durio zibethinus murr) as an alternative medium … 439 rate increases faster. in durian seed flour media and pda media, candida albicans fungi have the same characteristics, the only difference is the amount of colony growth. during the incubation period of 72 hours, the candida albicans fungi growing on durian seed flour media and pda media were yellowish white and even thin brown. this is due to the long incubation period and during this stage, the fungal colonies experience death (basarang & rianto, 2018). in the research conducted by afriani et al. (2022) on "utilization of durian seed flour (durio zibethinus murr) as a mushroom growth media", the results obtained for the growth of the number of candida albicans colonies were best in formulation 5 (f5), which was 4.5 x 107 cfu/ml. based on the comparison of several researchers, it can be concluded that durian seed flour (durio zibethinus murr) can be used as a substitute for pda (potato dextrose agar) because it is able to support the growth of fungi that are unicellular and represented by candida albicans. meanwhile, aspergillus niger forms colonies with characteristic branched filaments, the initial color is white, which turns brownish black after the formation of conidiospores. the largest colony diameter was found on pda media, where the sporulation grew thick and was black, while the sporulation on durian seed flour media was relatively thinner and blackish white, especially in f3, which did not contain sugar. the growth diameter of aspergillus niger colonies on pda media over different incubation periods is shown in figure 2. sugar is an important energy source for fungal growth (sundari et al., 2021). figure 4 presents the growth diameter of aspergillus niger colonies on durian seed flour media formulation f2 and f3. table 4 illustrates the colony diameters of aspergillus niger grown on different durian seed flour media formulations and pda, highlighting variations in growth. a b c figure 2. growth diameter of aspergillus niger on pda media a, b, c in storage time of 24 h, 48 h (17 mm), and 72 h (20.2 mm), respectively. a b c figure 3. growth diameter of aspergillus niger on durian seed flour media f1 a, b, c in storage time of 24 h, 48 h (25.5 mm), and 72 h (28.5 mm), respectively a b c figure 4. growth diameter of aspergillus niger on durian seed flour media f2 a, b, c in storage time of 24 h, 48 h (15 mm), and 72 h (20.5 mm), respectively. 440 biology, medicine, & natural product chemistry 14 (1), 2025: 435-441 table 4. diameter of aspergillus niger colonies on durian seed flour media and pda media. no. media time average (mm) 1 pda 48 17 72 20.2 2 f1 48 25.5 72 28.5 3 f2 48 15 72 20.5 4 f3 48 20.2 72 20.8 according to research conducted by faradiana (2016), on "utilization of different carbohydrate sources (suweg tubers and kimpul tubers) as a substitute for pda (potato dextrose agar) media for mushroom growth" the results obtained are the growth of aspergillus niger fungus has a different diameter. during the incubation period of 72 hours, the diameter of aspergillus niger colonies on pda media was 48.3 mm, on suweg tuber media, the diameter of aspergillus niger colonies was 31 mm, and on kimpul tuber media, the colony diameter was 30.6 mm. based on research by indah et al. (2018) on "taro flour as an alternative medium for the growth of candida albicans and aspergillus sp." shows the results of growth on the diameter of aspergillus niger in taro flour media at a concentration of 2% by 20 mm, a concentration of 4% by 24.25 mm, a concentration of 6% by 26.50 mm, a concentration of 8% by 28.50 mm, and on pda media was 27.75 mm. the results of research that has been done before, showing the diameter of aspergillus niger colonies in durian seed flour media is less good than carbohydrate sources from tuber media and taro flour. sporulation obtained on durian seed flour media is thinner than other media. this is due to the different nutrient content in each sample. the diameter of aspergillus niger colonies on durian seed flour media formulation f3 is illustrated in figure 5. a b c figure 5. growth diameter of aspergillus niger on durian seed flour media f3 a, b, c in storage time of 24 h, 48 h (20.2 mm), and 72 h (20.8 mm), respectively. aspergillus niger requires nutrients for its growth, including carbon, nitrogen, non-metallic elements such as sulfur and phosphorus, and metallic elements such as calcium (ca), zinc (zn), sodium (na), potassium (k), manganese (mn), magnesium (mg), iron (fe), vitamins, and energy. mushrooms grow optimally when nutrients support each other with appropriate growth environmental factors (basarang & rianto, 2018). fungi require organic compounds to obtain nutrients because they cannot produce food. aspergillus niger fungus can grow during an incubation period of three or four days with an optimum temperature of 35-37℃ (fadhilah fr et al., 2020). according to sundari et al. (2021), the aspergillus niger fungus can grow well if there is nutrient content and qualifies as a growth medium derived from carbohydrates. carbohydrates and their derivatives are the main substrates for carbon metabolism in fungi, as 50% of the weight of microorganisms is carbon. in addition to its ability to grow rapidly, aspergillus niger is also a fungus that can produce cellulose enzymes, which are useful for hydrolyzing cellulose into glucose for its metabolic process. during the inoculation process, aspergillus niger requires enzyme-producing nutrients to optimize the enzyme production process. the production of enzymes must optimally take place (abdillah et al., 2015). statistical analysis showed that the data were normally distributed based on the shapiro-wilk normality test with a p-value ≥ 0.05. anova test showed no significant difference between treatments, indicating that durian seed meal media performs close to that of pda media as a control. conclusions based on the results of the research, durian seed flour (durio zibethinus murr) can be used as an alternative medium for fungal growth, both candida albicans and aspergillus niger. this medium has the potential to replace pda as a fungal growth medium, with growth afriani et al. – durian seed flour (durio zibethinus murr) as an alternative medium … 441 characteristics supported by the content of carbohydrates and other nutrients contained in durian seed flour. competing interests: the authors declare that there are no competing interests. references abdillah, h., achmad, a., & setiawan, a. 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(2021). pemanfaatan biji mangga arum manis (mangifera indica l.) sebagai media alternatif pertumbuhan candida albicans dan aspergillus sp. jurnal pengelolaan laboratorium pendidikan, 3(1), 14–17. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 525-531 | doi: 10.14421/biomedich.2023.122.525-531 issn 2540-9328 (online) in silico b-cell epitope design of zika virus vaccine using “zika virus isolate zika virus/h. sapiens-tc/tha/2006/cvd 06-020, complete genome” lisa savitri1,*, syntia tanu juwita1, ester lianawati antoro1, ida septika wulansari1, datin an nisa sukmawati2, joão gregório freitas1, andre mario sequeira alves ornai1 1department of medical laboratory technology, faculty of health sciences; 2department of pharmacy, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id abstract zikv has been found in the cerebrospinal fluid (csf) and brains of adults infected with viruses that manifest neurological disorders. zikv is a mosquito-borne single-stranded rna virus, which belongs to the family flaviviridae. the efforts of the scientific community have rapidly increased knowledge about this virus. however, understanding the complexities of zikv infection, transmission and pathogenesis remains an urgent challenge. therefore, it is critical to study competent vectors and natural reservoirs for zikv, viral genetic diversity and flavivirus coinfection. due to the great challenges to develop a zikv vaccine, it is still not possible to be immunized against zikv infection and related pathologies. the methods are nucleotide search for the zika virus was carried out in silico, using the ncbi bioinformatics application by providing access to biomedical and genomic information. /h. sapiens-tc/tha/2006/cvd_06-020, the complete genome was then searched for fasta, then prediction of vaccine epitope using the iedb. the vaccine candidate peptides were analyzed for their antigenicity using vaxijen. proteins were classified by allertop to known allergenicity, then toxinpred to predict and design toxic/non-toxic peptides. there are 30 peptide sequences are predicted to be a candidate of peptides b-cell epitope zika virus vaccine design using “zika virus isolate zika virus/h. sapiens-tc/tha/2006/cvd_06-020, complete genome”. keywords: vaccine; zika virus; b-cell epitope; in silico. abbreviations: automatic cross covariance transformation (acc), antibody-dependent increase (ade), cerebrospinal fluid (csf), congenital zika syndrome (czs), ebola virus (ebov), immune epitope database (iedb), middle east respiratory syndrome (mers), public health emergency of international concern (pheic), severe acute respiratory syndrome (sars), spondwenivirus (sponv), quantitative structure-activity relationship (qsar), world health organization (who), yellow fever virus (yfv). introduction the world health organization (who) has identified, warned and tracked more than 1400 epidemic events in 172 countries due to viruses that emerged between 2018 and 2018, such as influenza, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), ebola, yellow fever, zika, and others. remarkably, in the last five years, who has declared three global health emergencies related to emerging viruses, namely sars-cov-2 in 2020, ebola virus (ebov) in 2014 (koenig, et al., 2014; mccoy, et al., 14; safari, et al., 2015) and zika virus (zikv) in 2016. in the context of zikv, who referred to zika as "an extraordinary event that needed a coordinated response, constituting a public health emergency of international concern (pheic)", because of the description of a large outbreak of rash, with short-term and mild fever (brazil, et al., 2016), not in all cases, and a cluster of microcephaly in newborns to infected mothers (kleber, et al., 2016; driggers, et al., 2016; martines, et al., 2016). in fact, zikv infection carries a risk of adverse pregnancy outcomes including an increased risk of preterm delivery, fetal death and stillbirth, and congenital malformations collectively characterized as congenital zika syndrome (czs), including the aforementioned microcephaly, abnormal brain development, limb contractures, eye abnormalities, brain calcifications, and other neurological manifestations (martines, et al., 2016; dudley, et al., 2018; leisher, et al., 2020). zikv has been found in the cerebrospinal fluid (csf) and brains of adults infected with viruses that manifest neurological disorders (carteaux, et al., 2016; da silva, et al., 2017; alves, et al., 2019). these flaviviruses cause harmful effects on the adult brain, such as gbs (da silva, et al., 2017; azevedo, et al., 2016; parra, et al., manuscript received: 03 may, 2023. revision accepted: 30 august, 2023. published: 15 september, 2023. https://doi.org/10.14421/biomedich.2023.122.525-531 526 biology, medicine, & natural product chemistry 12 (2), 2023: 525-531 2016; brito, et al., 2017; munoz, et al., ., 2016), encephalitis (da silva, et al., 2017; brito, et al., 2017; soares, et al., 2016), meningoencephalitis (barbi, et al., 2017; schwartzmann, et al., 2017 ), acute myelitis (da silva, et al., 2017; muñoz, et al., 2016; mécharles, et al., 2016) and encephalomyelitis (da silva, et al., 2017; alves, et al., 2019 ), among sensory polyneuropathy and other neurological complications (nicastri, et al., 2016; bido, et al., 2018). zikv is a mosquito-borne single-stranded rna virus, which belongs to the family flaviviridae (genus flavivirus) (mayer, et al., 2017; enfissi, et al., 2016). zikv is further classified on the basis of homology into spondwenivirus (sponv) within the spondweni virus clade or serogroup (kuno, et al., 2007; haddow, et al., 2016; brès, 1970), both viruses first characterized in africa in 1947. and 1952 (dick, et al., 1952; haddow, et al., 2016), respectively. zikv was previously isolated from the sera of pyrexial rhesus monkeys caged in the canopy of the zika forest in uganda (dick, et al., 1952), and was discovered during a study of the vector responsible for the sylvan cycle of yellow fever virus (yfv) in uganda (brès, 1970). the first human infection confirmed by zikv occurred in nigeria (1954) (macnamara, et al., 1954), further cases were reported in uganda (1962–63) and outside africa in central java, indonesia (1977) (olson, et al., 1981). zikv presents three phylogenetic divisions related to east african, west african and asian/american lineages (lanciotti, et al., 2016; collins, et al., 2019), constituting a single serotype (heinz, et al., 2017). this zikv lineage is thought to have emerged from east africa in the late 1800s or early 1900s (lanciotti, et al., 2016; gatherer, et al., 2016), with asian lineages responsible for all zikv outbreaks in the pacific. and america (who, 2017; ikejezie, et al., 2017). zikv is mainly transmitted to humans by infected aedes aegypti and aedes albopictus mosquitoes (boyer, et al., 2018). an increase in the aedes mosquito population has been observed in tropical developing countries (gubler, 2011). thus, infected mosquitoes, densely populated areas, and global commercial and tourist activities together with modern transportation constitute an efficient scenario for spreading infected mosquitoes and viruses such as zikv around the world (imperato, et al., 2016), as suggested. reported with dengue (gubler, 2011) and as was the case with zikv epidemic episodes in 2007 (duffy, et al., 2009), 2013 (boyer, et al., 2018) and 2014-2015 (musso, 2015), stated as pheic by who. the efforts of the scientific community have rapidly increased knowledge about this virus. however, understanding the complexities of zikv infection, transmission and pathogenesis remains an urgent challenge. therefore, it is critical to study competent vectors and natural reservoirs for zikv, viral genetic diversity and flavivirus coinfection (eligio, et al., 2020), as well as potential cross-immune reactivity (i.e., challenging immune diagnosis) and increased infection immunity. (i.e., antibody-dependent increase (ade)) (rathore, 2020), fortunately not yet observed in humans (terzian, et al., 2017), along with environmental factors that may have suddenly triggered the expansion of the zikv epidemic and worse viral pathogenesis. (pierson, et al., 2018; baud, et al., 2017; rossati, 2017). due to the great challenges to develop a zikv vaccine (shan, et al., 2018; lin, et al., 2018), it is still not possible to be immunized against zikv infection and related pathologies. based on this phenomenon, it is necessary to conduct research to examine vaccine candidates for zikv by conducting in silico b-cell epitope zika virus vaccine design using “zika virus isolate zika virus/h. sapienstc/tha/2006/cvd_06-020, complete genome”. materials and methods the population in this study was the zika virus. the sample used in this study was zika virus isolate zika virus/h. sapiens-tc/tha/2006/cvd_06-020, complete genome. according to savitri (2021), the nucleotide search for the zika virus was carried out in silico, using the ncbi bioinformatics application, the national center for biotechnology information advances science and health by providing access to biomedical and genomic information. zika virus isolate zika virus/h. sapienstc/tha/2006/cvd_06-020, the complete genome was then searched for fasta. after obtaining fasta, then prediction of vaccine epitope using the iedb, which contains a catalog of experimental data on antibodies and t cell epitopes studied in humans, non-human primates, and animal species others in the context of infectious diseases, allergies, autoimmunity and transplantation was done. the iedb also provides tools to aid in the prediction and analysis of epitopes. this study uses b cell epitope prediction. the vaccine candidate peptides were analyzed for their antigenicity using vaxijen with the target organism of the virus. this method is based on automatic cross covariance transformation (acc) of protein sequences into vectors of equal and uniform length. it has been applied to the study of the quantitative structure-activity relationship (qsar) of peptides of different lengths. the main properties of amino acids are represented by the five e descriptors, which were originally derived by venkatarajan and braun. they described the hydrophobicity of amino acids, molecular size, tendency to form helices, relative abundance of amino acids, and tendency to strand formation. proteins were classified by the k-nearest neighbor algorithm (knn, k=1) based on a training set containing 2427 known allergens of different species and 2427 non-allergens. savitri et al. – in silico b-cell epitope design of zika virus vaccine 527 toxinpred is an in silico method developed to predict and design toxic/non-toxic peptides. the main data set used in this method consisted of 1805 toxic peptides (<=35 residues). one of the main features of the server is to calculate various physicochemical properties. peptide analogues can be displayed in a sorting order based on the desired trait. results and discussion table 1. candidate types of peptides b-cell epitope zika virus vaccine design using “zika virus isolate zika virus/h. sapiens-tc/tha/2006/cvd_06020, complete genome”. no. code peptide 1 peptide 1 ctttttgagattc 2 peptide 2 atagatggggttcagtggggaaaaaagaggctatggaaataataaagaagttcaagaaagatctgg 3 peptide 3 gctggatgagggggtagaa 4 peptide 4 cttgggttgtgtacg 5 peptide 5 ttgtgta 6 peptide 6 ttgggaggttgat 7 peptide 7 gctgatgtggggtgctcggtggac 8 peptide 8 tggaagagaatggagttca 9 peptide 9 gtggagagg 10 peptide 10 tgtgaggctggt 11 peptide 11 gtgaagt 12 peptide 12 gagagagat 13 peptide 13 ggaagtaaaaaagggggag 14 peptide 14 tgtttggctg 15 peptide 15 tacggagagaaaagagtgc 16 peptide 16 gaggtggatgga 17 peptide 17 ggaaattgag 18 peptide 18 ttgttgtgttcct 19 peptide 19 ttgctggtggtgc 20 peptide 20 gtactt 21 peptide 21 gtggagaaaaagatgggacaggtgc 22 peptide 22 tgggggtggggggaggctggggc 23 peptide 23 ttgtgggaagg 24 peptide 24 tggagtt 25 peptide 25 agttcaagaagtgaaaggat 26 peptide 25 ttaagagtggggtggacgtc 27 peptide 27 tggtgggggattggcttgaaaaa 28 peptide 28 atgatgggaaaaagagaaaagaaacaaggggaatttggaaaggccaagggc 29 peptide 29 acgaggtggtgttgaagggctgggattaca 30 peptide 30 tggaaagggagaatggatg table 2. antigenicity, allergenicity, toxicity, dan mutation position of candidate types of peptides b-cell epitope zika virus vaccine design using “zika virus isolate zika virus/h. sapiens-tc/tha/2006/cvd_06-020, complete genome”. no. code antigenicity allergenicity toxicity mutation position 1 peptide 1 antigenic non-allergenic non-toxic no mutation 2 peptide 2 antigenic non-allergenic non-toxic no mutaion 3 peptide 3 antigenic non-allergenic non-toxic no mutation 4 peptide 4 antigenic non-allergenic non-toxic no mutation 5 peptide 5 antigenic non-allergenic non-toxic no mutation 6 peptide 6 antigenic non-allergenic non-toxic no mutation 7 peptide 7 antigenic non-allergenic non-toxic no mutation 8 peptide 8 antigenic non-allergenic non-toxic no mutation 9 peptide 9 antigenic non-allergenic non-toxic no mutation 10 peptide 10 antigenic non-allergenic non-toxic no mutation 11 peptide 11 antigenic non-allergenic non-toxic no mutation 12 peptide 12 antigenic non-allergenic non-toxic no mutation 13 peptide 13 antigenic non-allergenic non-toxic no mutation 528 biology, medicine, & natural product chemistry 12 (2), 2023: 525-531 table 2. cont. no. code antigenicity allergenicity toxicity mutation position 14 peptide 14 antigenic non-allergenic non-toxic no mutation 15 peptide 15 antigenic non-allergenic non-toxic no mutation 16 peptide 16 antigenic non-allergenic non-toxic no mutation 17 peptide 17 antigenic non-allergenic non-toxic no mutation 18 peptide 18 antigenic non-allergenic non-toxic no mutation 19 peptide 19 antigenic non-allergenic non-toxic no mutation 20 peptide 20 antigenic non-allergenic non-toxic no mutation 21 peptide 21 antigenic non-allergenic non-toxic no mutation 22 peptide 22 antigenic non-allergenic non-toxic no mutation 23 peptide 23 antigenic non-allergenic non-toxic no mutation 24 peptide 24 antigenic non-allergenic non-toxic no mutation 25 peptide 25 antigenic non-allergenic non-toxic no mutation 26 peptide 25 antigenic non-allergenic non-toxic no mutation 27 peptide 27 antigenic non-allergenic non-toxic no mutation 28 peptide 28 antigenic non-allergenic non-toxic no mutation 29 peptide 29 antigenic non-allergenic non-toxic no mutation 30 peptide 30 antigenic non-allergenic non-toxic no mutation table 3. svm soce, hydrophobicity, hydropathicity, hydrophilicity, charge, and molecule weight of candidate types of peptides b-cell epitope zika virus vaccine design using “zika virus isolate zika virus/h. sapiens-tc/tha/2006/cvd_06-020, complete genome”. no. code svm score hydrophobicity hydropathicity hydrophilicity charge mol wt 1 peptide 1 -0,84 -0,03 0,22 -0,45 0 1188,46 2 peptide 2 -1,14 0,13 0,63 -0,33 0 3668,55 3 peptide 3 -0,82 0,12 0,28 -0,25 0 1350,66 4 peptide 4 -0,75 0,01 0,01 -0,33 0 1244,53 5 peptide 5 -0,85 -0,02 -0,26 -0,3 0 607,71 6 peptide 6 -0,67 -0,04 -0,18 -0,23 0 1008,2 7 peptide 7 -0,78 0,06 0,19 -0,31 0 1864,34 8 peptide 8 -0,75 0,12 0,5 -0,32 0 1422,75 9 peptide 9 -0,73 0,14 0,06 -0,16 0 603,72 10 peptide 10 -0,81 0,04 -0,08 -0,26 0 939,14 11 peptide 11 -0,71 0,09 0,14 -0,26 0 533,63 12 peptide 12 -0,71 0,16 0,54 -0,27 0 631,76 13 peptide 13 -1,04 0,18 0,63 -0,26 0 1272,56 14 peptide 14 -0,81 -0,02 -0,26 -0,3 0 855,03 15 peptide 15 -0,81 0,15 0,8 -0,36 0 1394,75 16 peptide 16 -0,74 0,13 0,1 -0,19 0 833 17 peptide 17 -0,93 0,13 0,42 -0,28 0 732,88 18 peptide 18 -0,44 -0,07 -0,14 -0,4 0 1204,47 19 peptide 19 -0,81 0,01 -0,07 -0,31 0 1072,32 20 peptide 20 -0,8 -0,01 0,3 -0,45 0 552,67 21 peptide 21 -0,92 0,14 0,59 -0,31 0 1795,24 22 peptide 22 -0,71 0,11 -0,09 -0,16 0 1568,94 23 peptide 23 -0,73 0,08 -0,08 -0,2 0 805,96 24 peptide 24 -0,68 0,03 -0,21 -0,24 0 563,66 25 peptide 25 -0,84 0,13 0,68 -0,36 0 1507,86 26 peptide 25 -0,81 0,08 0,26 -0,3 0 1527,89 27 peptide 27 -0,78 0,09 0,22 -0,28 0 1725,11 28 peptide 28 -0,93 0,16 0,8 -0,34 0 3566,47 29 peptide 29 -0,68 0,09 0,33 -0,31 0 2274,83 30 peptide 30 -0,91 0,14 0,36 -0,25 0 1332,62 discussion according to vita, et al. (2015), the immune epitope database (iedb) website hosts epitope-specific experimental assays which mean each assay reflects antibody to the antigen or epitope being tested experimentally. structures entered as restricted epitopes savitri et al. – in silico b-cell epitope design of zika virus vaccine 529 are those that were tested in the test or were deduced as epitopes by multiple sources. in most cases, these amino acid residue sequences are more epitopecontaining regions. this means that the possible epitope can also not be limited only to a predetermined part. epitope structures can be peptidic and non-peptidic. a peptidic epitope structure consists of linear and discontinuous amino acid sequences based on their position in the source protein. the peptidic epitope having 3d structural data is described in the presence of the residues found to be related to antibodies. based on the tests that have been carried out, there are 30 peptide sequences predicted to have high antigenicity values. the high value of antigenicity in a peptide sequence is a marker of ideal properties possessed by vaccines. the higher the antigenicity value, the better its ability to stimulate b cells to form specific antibodies. allergenicity test is a step carried out to test the selected vaccine candidate whether the peptide sequence to be used can cause allergies to the body or not. the ability not to create allergies for the body is also one of the ideal properties for a vaccine product. a high value of antigenicity is one of the ideal characteristics that all vaccines should have. allergy is the ability possessed by material to cause allergies. non-allergen is an ideal trait that anti-viral vaccines should have. antigenicity is the ability of an antigen to stimulate the formation of specific antibodies by b cells in the body (rezaldi, et al., 2021). based on the tests that have been carried out, the 30 peptide sequences are predicted to be non-toxic to the human body. toxicity testing at the vaccine prediction stage is important to obtain a peptide that is not toxic when administered into the human body. only peptides with negative toxicity values can be selected for further use. in addition, this test also found the value of hydrophilicity. the higher the hydrophilicity value, the more certain the antigenic potential will be. his is following the statement of sanchez-trincado, et al. (2017) which states that by calculating the hydrophilicity of residues for the prediction of b cell epitope, it can be seen their antigenic potential. this is based on the assumption that the hydrophilic part is mainly located on the surface of the protein which will be directly accessible to the target antibody site. conclusions there are 30 peptide sequences predicted to be a candidate of peptides b-cell epitope zika virus vaccine design using “zika virus isolate zika virus/h. sapienstc/tha/2006/cvd_06-020, complete genome”. this research can be continued with the immunoinformatics method for 3d visualization in order to know how compatible the peptide and epitope sequences are to be used as zika virus vaccine candidates. acknowledgements: acknowledgments are expressed in a brief; all sources of institutional, private and corporate financial support for the work must be fully acknowledged, and any potential conflicts of interest are noted.the author would like to thank the institute for research, development, and community service (lp3m) of kadiri university which has always provided support for writing and publications. authors’ contributions: lisa savitri designed the study. lisa savitri, syntia tanu juwita, ester lianawati antoro, ida septika wulansari, and datin an nisa sukmawati analyzed the data. joão gregório freitas and andre mario sequeira alves ornai wrote the manuscript. all authors read and approved the final version of the article. competing interests: the authors declare that there are no competing interests. funding: the authors declare funding if any. references alves-leon, s.v.; lima, m.d.r.; nunes, p.c.g.; chimelli, l.m.c.; rabelo, k.; nogueira, r.m.r.; de bruycker-nogueira, f.; de azeredo, e.l.; bahia, p.r.; rueda lopes, f.c.; et al. 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(2017). who situation report. zika virus microcephaly guillain-barré syndrome. 2017. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 79-88 | doi: 10.14421/biomedich.2023.121.79-88 issn 2540-9328 (online) spermicidal, antifertility and contraceptive effect of azadirachta indica a. juss. seed extract in female and male wistar rats macdonald idu1, sharon ogedegbe-george1, precious eromosele oriarewo1, benjamin ogunma gabriel1,2,* 1phytomedicine unit, department of plant biology and biotechnology; 2science laboratory technology, university of benin, pmb 1154, benin city, edo state. nigeria. corresponding author* alexthemain076@gmail.com, +2347032695752 manuscript received: 30 july, 2022. revision accepted: 20 september, 2022. published: 04 november, 2022. abstract this study investigates the spermicidal and contraceptive effect of azadirachta indica seed aqueous extract in male and female wistar rats. the animals were divided into four groups (n=6), graded doses (2.5, 5, and 10 mg/kg) of the extract. they were exposed to female rats in a ratio of 2:1 after 24 hours, 7, and 14 days of administration. the spermicidal properties were evaluated. the female contraceptive study involved 14 days of pre-coital and post-coital administration of the neem seed extract, and contraceptive indexes were investigated. the results obtained from the spermicidal activity showed a significant decrease in male hormonal levels at 2.5, 5, and 10 mg/kg of a. indica (0.73, 0.50, and 1.08 ng/ml); and the sperm cells (102.5, 111.5, and 97 counts) after 24 hours. administration and mating, compared to the control. also, the histopathology of the testes showed normal testicles. the contraceptive study elicited a significant decrease in estrogen (1.46 ng/ml), luteinizing hormone (0.094 mg/dl) µand progesterone (1.82 ng/ml), at 5 mg/kg in day 14 post-coital study compared with the control. the histopathology of the treated uterus had no deteriorating effect compared with the control. the extract elicited spermicidal and contraceptive potential at a reduced dose, validating its folklore claim. keywords: spermicidal; contraceptive; azadirachta indica; wistar rats. introduction azadirachta indica a. juss. is a prevalent and highly wonderful tree (alzohairy, 2016). it was known as neem a distinct tree associated closely with the socio-cultural and holistic aspects of the life of indians since ancient times. it is a fast-growing tree with edible fruits and aromatic leaves. a mature tree can produce three hundred and fifty kilograms of leaves a year (kelli, 2010). it can grow to a height of fifteen-twenty meters and seldom thirty-five meters, located in the subtropical and tropical world. neem is in the mahogany family, meliaceae, native to the indian subcontinent. neem oil is usually produced from its fruits and seeds, the leaves and oil have antifertility effects (ruchi et al., 2014). indra the king of the gods, was on his way back to his kingdom with the pot of amirithan (the divine nectar) which he had gotten from the devils after a tough-fought battle. probably, he was fagged out, maybe careless or probably he did it intentionally, for little drops of the pot content poured over a neem tree, from that day henceforth, the neem tree had good qualities for healing ailments, of course, this is a folk tale. meanwhile, the neem is a superb tree for millions of people and its parts have several uses in healthcare, medicines, and agriculture, and this is no folklore (ajayi, 2002). neem is called indian village pharmacy in the west, it is also called arishta in the sanskrit language which means relieving sickness. africans usually call it muarubaini which means forty uses or forty cures. meanwhile, the persians have given the most perfect name azaddirakht-hind which means a free tree of india, and perhaps from this its latinized botanical name a. indica (asiif, 2013). almost all the parts of the wonderful tree have been used medicinally since ancient times. this is a rampant tree found in different countries including; india, pakistan, srilanka, burma, malaya, indonesia, japan, australia (tropical region) even in the middle east. it is amongst the trees tagged holy by hindus all over the world. contraception is defined as the inhibition of conception, but generally, it is taken to mean the prevention of pregnancy (bansal et al., 2010). family planning has been encouraged through numerous means of contraception, but these contraceptives have several side effects produced by their steroid content (chandramouli et al., 2014), such as contraceptive pills, intra uterine devices, tubectomy, condoms, diaphragm, and https://doi.org/10.14421/biomedich.2023.121.79-88 80 biology, medicine, & natural product chemistry 12 (1), 2023: 79-88 coitus interruptus. these methods are also typically female-oriented. contraceptive pills are generally female sex hormones such as estrogen, progesterone, or their derivatives either single or together. the concept of sterilization by female sex hormone is very old and it was initiated at the beginning of the twentieth century. novid was the first "pill" permitted by the fda for usage as a birth control agent (chaudhuri, 2007). however, these pills lead to the development of some unwanted effects such as obesity, dysmenorrhea, vomiting, cardiovascular disorders, and carcinoma of the breast and uterus. these side effects from the use of the pills make them unsafe for long-term use. countless measures have been taken to decrease the side effects of these pills but there is little success (dash et al., 2014). as a result of the serious adverse effects caused by synthetic steroidal contraceptives, the focus has been shifted to local plants for possible contraceptive effects. although contraceptives comprising estrogen and progesterone are effective and common, the risks accompanying the drugs have prompted the need to synthesize newer molecules from medicinal plants. this study evaluates the antifertility properties of azadirachta indica in male and female wistar rats. materials and methods collection of plant material fresh seeds of the plants were collected from kaduna state, in the northern part of nigeria. it was identified and authenticated by dr. timothy odaro in the herbarium unit of the department of plant biology and biotechnology, with the voucher number ubt-b184. it was further authenticated using theplantlist.org. fresh seeds were harvested and sun-dried for two weeks. the dried seeds were ground to powder using a mechanical grinder. preparation of plant material two thousand (2000) grams of the sample, weighed using a scale was measured into a glass jar. 2l of nhexane was poured into the glass jar containing the sample. the mixture was stirred, covered, and left for 36 hours. the mixture was then strained after 36 hours and a dark brown oily semi-solid extract was obtained. the extract was concentrated using a water bath at about 45oc. it was then kept in a sterile container in the refrigerator until needed for use. experimental animals sixty-eight (68) healthy whisker (albino) rats (male and female) weighed 180-250 g. the animals were acquired from animal and environmental biology, university of benin animal house. they were housed in wellventilated woody cages in a normal laboratory state (12 hours light/dark cycle: 23 ± 2°c) and fed using a standard diet. food and water were administered at free choice (ad libitum) to the animals designed for experiments. the animals were properly handled using the ethics of laboratory animals’ approval from the ethical committee of the faculty of life sciences with the ethical number ls20619. experimental design this study involved two experimental protocols; male spermicidal activities and female contraceptive properties. the male wistar rats were randomly divided into 4 groups (n=9). three of the groups were administered with graded doses (2.5, 5, and 10 mg/kg) of a. indica seed aqueous extract, the last group serves as the control group (ogbuewu, 2011). they were administered with graded doses of the extract orally for 14 days. after 24 hours, 3 rats across the groups (making a total of 12 rats) were exposed to female rats using a ratio of 2 male to 1 female and then sacrificed. the same process was repeated for 7 and 14 days. sperm, blood, and reproductive organs (testes, and epididymis) were collected from the sacrificed rats and were analyzed (khillare and shrivastava, 2003; koresriem, 2013). the female contraceptive study was carried out in two (2) phases; the pre-coital and post-coital treatment with neem seed aqueous extract. phase 1: the pre-coital treatment involved 4 groups (n=4); the control group, graded doses (2.5, 5, and 10 mg/kg) of neem seed aqueous extract for 14 days, on the 14th day all the female animals were paired with male wistar rats during their heat period for another 7 days using ratio 1:1, and the female rats were checked at every interval for possible signs of vaginal sperm plug as an indicator for mating (gbotolorun et al., 2008; dreweke, 2014). after 7 days of pre-exposure to the male animals, the female rats were observed for traces of pregnancy for another 23 days, which is the gestational period of rats. the weight of the rats was obtained and sacrificed in mild anesthetics, and blood and female reproductive organs were isolated. phase 2: the post-coital treatment involved 4 groups (n=4); the control group, graded doses (2.5, 5, and 10 mg/kg) of neem seed aqueous extract (harris, 2018; hatcher et al., 2007). before the administration of the extract, the female rats were paired with male animals' using a ratio of 1:1 for 48 hours, the female rats were checked at every interval for possible signs of vaginal sperm plug as an indicator for mating (nripendra et al., 2019; prakash et al., 1988). the female animals were administered with graded doses of a. indica seed aqueous extract for 7 days of oral administration, the female rats were observed for traces of pregnancy for another 23 days, which is the gestational period of rats. the weight of the rats was obtained and sacrificed in mild anesthetics, and the number of resorption, number of embryos, number of corpora, pre-implantation, and post-implantation mortality, and total prenatal mortality idu et al. – anti-fertility property of azadirachta indica 81 were evaluated. blood and female reproductive organs were isolated for analysis. sperm cell procedure sperm cells were collected from the vas deferens and placed in a sterile petri dish. to the petri dish, 6 µl of normal saline already adjusted to 37°c was added. a drop of the sperm cell suspension was taken from the petri dish and dispensed on a clean grease-free slide, further covered with a transparent cover slip. the slide was placed on the microscope and viewed with the 20x and 40x objective magnification lens. the motility was scored in percentage according to their nature of motility as, progressive, nonprogressive, and immotile sperm cells (sethi et al., 2017; suryawanshi 2011; ibeh et al., 2018). one volume of semen (a drop) was milted into two volumes of eosin solution (1% diluted water). after 30 seconds three volumes of nigrosine solution (10% nigrosine) were added and the sample was homogenized. a thin smear was then made immediately and air dried. the stained slide using an improved eosin and leishman stain (umadevi et al., 2013; world health organization, 2010), was examined under the oil immersion objective lens (100x). live spermatozoa were unstained (white) and the dead ones were red. the slide was viewed with at least 30 magnification fields, and the normal and abnormal sperm cells were spotted and scored in percentage (suryawanshi 2011). determination of serum testosterone concentration serum testosterone level of the tested animals’ plasma was investigated using an established protocol from the manufacturer’s manual. this was established on the standard competitive requisite between testosterone in the test plasma specimen (serum). this assay method involved the dispensing of 10 μl of testosterone reference standards at (0, 0.1, 0.5, 2.0, 6.0, and 18.0 ng/ml), serum (diluted ×5), and the controls of testosterone 1 and 2 into a goat anti-rabbit igg-coated microtitre wells (96 wells), 100 μl of testosterone-hrp conjugate reagent (blue color) and 50 μl of rabbit antitestosterone reagent were distinctly distributed into each well. the resultant solution was systematically mixed for 30 seconds and allowed for incubation at 37oc for 90 minutes. this was left to a stable quantity of hrplabelled testosterone to contend with endogenous testosterone of the standard, sample, or quality control serum for a constant number of bindery spots of a detailed testosterone antibody (since the quantity of testosterone peroxidase conjugate with the immunological bound of the well gradually decreases as the testosterone concentration in the specimen increases) (aversa et al., 2000). the microwells were washed and skimmed 5 times in distilled water (to eliminate unbound testosterone peroxidase conjugate) previously dispensing 100 μl of tmb reagent into the well. the resultant solution was properly mixed mildly for 5 seconds. this was later incubated at room temperature for about 20 minutes to achieve blue coloration. the color change was ceased with an additional 100 μl of stop solution (1n hcl) to each well and when slightly mixed, the color altered from blue to yellow. the absorbance was recorded within 15 mins at 450 nm in a microtitre well reader. the greatness of the color made was comparative to the quantity of enzyme extant and was inversely relative to the total of unmarked testosterone in the sample (gauthaman and adaikan 2008). the testosterone level of the serum in the animals was considered from the calibration curve (plotting the concentration of the standard against the absorbance) using the expression: 𝑇𝑒𝑠𝑡𝑜𝑠𝑡𝑒𝑟𝑜𝑛𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑛𝑔/𝑚𝐿 = 𝐶𝑠 × 𝐹, (1) where cs is the corresponding testosterone concentration from the calibration curve and f is the dilution factor progesterone assay protocol all reagents should be allowed to reach room temperature (18-25oc) before use. pipette 50 µl of standards (ready to use) and diluted samples into appropriate wells within 5 minutes. add 100 ul of progesterone enzyme conjugate solution to each well (except those set for blanks). mix well for 30 second. and incubate for 60 minutes at 37°c. you may use par film to cover the wells or use an appropriate zip-lock bag to store the plate during the incubation. discard the contents of the wells and wash the plate 5 times with wash solution (250-300 µl) per well. invert the plate, and tap firmly against absorbent paper to remove any residual moisture. add 100 µl (tmb) substrate solutions to all wells. remember to follow the pipetting order. incubate the plate at room temperature (18-28°c) for 10 minutes without shaking. stop reaction by adding 50 µl of stopping solution to wells in the same sequence that the substrate solution was added and gently mixed. read the absorbance at 450 nm with a microwell reader. luteinizing hormonal assay secure the desired number of coated wells in the holder. dispense 50 µl of standards, specimens, and controls into appropriate wells. dispense 100 µl of enzyme conjugate into each well. mix for 30 seconds. it is very important to have completed mixing at this step. incubate at room temperature (37oc) for 2 hours. remove the incubation mixture by flicking the plate contents into a waste container. rinse and flick the microtiter wells five (5) times with wash buffer. strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets. dispense 100 µl of tmb solution into each well. gently mix for 10 seconds. incubate at room temperature for 20 minutes, in the dark. stop reaction by adding 50 µl (one drop) of 2n hcl to each well. gently mix for 30 seconds. it is 82 biology, medicine, & natural product chemistry 12 (1), 2023: 79-88 important to observe a color change from blue to yellow. read optical density at 450 nm with a microtiter well reader. follicle-stimulating hormone bring all reagents and samples to room temperature before use. centrifuge the sample again after thawing before the assay. it is recommended that all samples and standards be assayed in duplicate. prepare all reagents and samples as directed in the previous sections. determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4°c. set a blank well without any solution. add 50 μl of standard or sample per well. standard need test in duplicate. add 50 μl of hrp-conjugate to each well (not to blank well), then 50 μl antibody to each well. mix well and then incubate for 60 minutes at 37°c. aspirate each well and wash, repeating the process two times for a total of three washes. wash by filling each well with wash buffer (200 μl) using a squirt bottle, multi-channel pipette, manifold dispenser, or auto-washer, and let it stand for 10 seconds, complete removal of the liquid at each step is essential to good performance. after the last wash, remove any remaining wash buffer by aspirating or decanting. invert the plate and blot it against clean paper towels (neychev and mitev 2016). add 50 μl of substrate a and 50 μl of substrate b to each well, and mix well. incubate for 15 minutes at 37°c. keeping the plate away from drafts and other temperature fluctuations in the dark. add 50 μl of stop solution to each well, and gently tap the plate to ensure thorough mixing. determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm. histopathological analysis testes and uterus were fixed in neutral bouin’s fluid. affixed organs were utterly dehydrated with 99.9 % ethanol along with 70 % ethanol, and 96 % ethanol and washed using distilled water. 4 µm sections were prepared and stained in hematoxylin-eosin dye. stained tissues were subjected to optical photomicroscope (leica mc170 hd, leica biosystems, germany) and viewed at 400x magnification (drury and wallinton, 2013). statistical analysis results obtained are presented as mean ± sem. data were analyzed via one-way analysis of variance (anova) and means were compared using the dunnete test. significant differences were measured at p< 0.05. graph pad prism (version 7, usa) was the statistical tool used. results and discussion spermicidal property the body and organs weight of the treatment groups elicited a slight significant increase when compared with the control at 24 hours. 7 and 14 days of the study (tables 1 and 2). sperm cells in the graded doses of the extract (2.5, 5.0, and 10 mg) at 24 hrs. 7 and 14 days elicited a significant decrease in sperm count, progressive motility with an increase in non-progress motility, and immobility when compared with the untreated control as shown in figure 1. the testosterone level, follicle-stimulating hormone, luteinizing hormone, and progesterone had a significant reduction in the treatment groups when compared with the control, eliciting spermicidal and antifertility properties of the extract as shown in figure 2. the abnormal weight loss or gain serves as an indication of toxicity possibly due to structural protein breakdown or muscle wastage (kumar and mishra, 2010). this study present showed that a. indica seed aqueous extract at graded doses maintains the level of a significant increase in the body weight with the spermicidal effect for 24 hours. 7 and 14 days (table 1-3). this result consented with singh et al. (2018) report on grapefruit components to ameliorate reproductive toxicological organs' weight loss. similarly, the visceral organs such as the testes, cowpea gland, and epididymis, elicited a slight significant increase with no damaging effect across the treatment groups compared with the control at 24 hours, 7, and 14 days. table 1. effect of azadirachta indica seed aqueous extract on the body and reproductive organs weight after 24 hours. treatment dose mg/kg initial body weight (g) final body weight (g) weight of testes (g) weight of cowpea gland (g) weight of epididymis (g) a. indica 2.5 193.50±0.50 185.50±5.50 1.20±0.00 0.40±0.04 1.05±0.15 a. indica 5.0 207.50±1.50 187.00±2.00 1.25±0.05 0.60±0.10 0.80±0.05 a. indica 10 182.00±1..80 178.00±1..70 1.25±0.05 0.35±0.05 0.90±0.05 control 0.5 ml/kg 204.00±3.40 204.50±3.40 1.35±0.15 0.30±0.00 0.70±0.04 the results were expressed in mean±sem, p>0.05. idu et al. – anti-fertility property of azadirachta indica 83 table 2. effect of azadirachta indica seed aqueous extract on the body and reproductive organs weight after 7 days. treatment dose mg/kg initial body weight (g) final body weight (g) weight of testes (g) weight of cowpea gland (g) weight of epididymis (g) a. indica 2.5 212.50±1.70 223.50±1.50 1.50±0.10 0.60±0.02 0.85±0.05 a. indica 5.0 182.00±1.50 185.00±3.00 0.75±0.07 0.40±0.01 0.55±0.05 a. indica 10 186.50±1.50 191.50±2.50 1.25±0.15 0.55±0.02 0.55±0.05 control 0.5 ml/kg 205.00±3.60 207.50±3.50 0.60±0.05 0.20±0.01 0.65±0.05 the results were expressed in mean±sem, p>0.05. table 3. effect of azadirachta indica seed aqueous extract on the body and reproductive organs weight after 14 days. treatment dose mg/kg initial body weight (g) final body weight (g) weight of testes (g) weight of cowpea gland (g) weight of epididymis (g) a. indica 2.5 177.50±1.60 199.00±1.00 1.15±0.05 0.35±0.02 0.45±0.05 a. indica 5.0 174.50±1.20 199.00±1.30 1.10±0.00 0.50±0.03 0.45±0.05 a. indica 10 184.50±6.50 199.50±2.50 1.15±0.05 0.15±0.00 0.50±0.02 control 0.5 ml/kg 179.50±4.50 187.00±4.00 0.90±0.00 0.55±0.05 0.55±0.05 the results were expressed in mean±sem, p>0.05. the semen analysis serves as the onset of selection for fertility evaluation and is frequently utilized in the definition of semen quality (morphology, viability, and sperm motility) and quantity (sperm count) (khan, 2008). the findings from this study elicited a significant decrease in sperm count, progressive motility, and a significant increase in non-progressive, and immotile sperm cells across 2.5, 5.0, and 10 mg/kg of a. indica seed aqueous extract displaying the spermicidal effect of the extract when compared with the control in 24 hrs., 7 and 14 days of the exposure as shown in figure 1. the semen analysis serves as an onset target for spermicidal activities utilized in the definition of an effective sperm killer. this agreed with the report of asiif (2013) on a review of spermicidal activities on azadirachta indica. studies had shown that certain constituents in several plant materials possess spermicidal properties, which aid in controlling overpopulation (umadevi et al., 2013; suryawanshi, 2011). a. indica seed aqueous extract serves as a potent spermicidal. figure 1. spermicidal effect of azadirachta indica seed aqueous extract on the sperm analysis after 14 days. keywords: total sperm cell count (tsc), progressive motility (pm), nonprogressive motility (npm), immotile (im). the extract promotes a significant decrease in the level of testosterone which further triggers reduced libido activity as shown in figure 2. this is in line with the report of khan, m. a. (2008). a. indica seed aqueous extract, also are linked with the androgen inhibitory function, which could be responsible for the detraction of male sexual performance as shown in this study (deshpande et al., 1980). certain phytoconstituent stimulate sexual urges with spermicidal properties (chauhan, 2008). enhancement in the spermicidal effect 0 50 100 150 200 250 300 350 400 450 control (dw) 2.5 mg/kg a. indica 5.0 mg/kg a. indica 10 mg/kg a. indica sp er m a n a ly si s groups/doses 24 hr. tsc 7 days tsc 14 days tsc 24 hr. pm 7 days pm 14 days pm 24 hr. npm 7 days npm 14 days npm 24 hr. im 7 days im 14 days im 84 biology, medicine, & natural product chemistry 12 (1), 2023: 79-88 in this research study could be a result of the active constituents in a. indica seed aqueous extract. an increase in the level of androgen enhanced leydig cells to stimulate luteinizing hormone resulting in an increased in spermatogenesis and increase epididymal sperm, but for the associated mechanism of action of a. indica seed aqueous extract the reverse is the case due to its spermicidal property. findings by chaudhuri, (2007) practice of fertility control in weight, size and secretory role of the epididymis, testes and auxiliary organs. modifications in the circulating level of androgen may be distorted by a. indica seed aqueous extract leading to interference during spermatogenesis (jensen, 2002). variation in the reproductive organ can be used as a marker to improve androgen levels in reproductive glands, in the case of this study, the extract possibly impedes the feedback action involved in sperm formation. meanwhile, the androgenic property is associated with serum testosterone concentration (harris, 2018), the extract interferes with the function of testosterone discharge consenting by blocking the secretion of the hormone in gonads as displayed in figure 2. a contradictory study by gbotolorun et al. (2008) reported on mondia whitei hexane extract on male sexual reproductive in rats. testosterone is the main androgenic hormone responsible for the sexual application to display a vital role in spermatogenesis. findings showed that sexual zeal is triggered and maintained by the penis tissues that facilitated erection (aversa et al., 2000). figure 2. spermicidal effect of azadirachta indica seed aqueous extract on the hormonal level after 14 days. keywords: follicle stimulating hormone (fsh), testosterone (tt), luteinizing hormone (lh). studies from gauthaman and adaikan (2008) showed that male sexual dysfunction is related to several factors such as; androgen insufficiency. testosterone agents (hormonal replace therapy) are exhibited to develop sexual roles and libido. some plant extracts such as a. indica seed aqueous extract inhibit the synthesis or secretion of testosterone and serves as potent spermicidal or antifertility properties (bansal et al., 2010). a significant decrease in the level of testosterone is effective at a reduced dose as shown in a. indica seed aqueous extract when compared with the control (figure 2), also a decrease in the folliclestimulating hormone, luteinizing hormone, and progesterone inhibits the release of testosterone thereby eliciting antifertility effect of the extract (figure 2). the histopathological study of the testes exposed the standard architectural framework when compared with the control as shown in plate 1. the treated organs at 2.5, 5.0, and 10 mg/kg of the a. indica seed aqueous extract showed an enhanced architecture structure of the testes. however, across the treated groups, an absence of spermatogenic action in the lumen of the seminiferous tubule was observed. this stimulated cellular activity occurrence from the cellar membrane all through to the lumen in seminiferous tubules found in the testes inhibits the secretion of the primary spermatogonia as proof for this study. it is agreed with gbotolorun et al. (2008) reported results. 0 1 2 3 4 5 6 7 8 9 control (dw) 2.5 mg/kg a. indica 5.0 mg/kg a. indica 10 mg/kg a. indica h o r m o n a l a ss a y groups/doses 24 hr. fsh (mg/dl) 7 days fsh (mg/dl) 14 days fsh (mg/dl) 24 hr. tt (ng/dl) 7 days tt (ng/dl) 14 days tt (ng/dl) 24 hr. lh (mg/dl) 7 days lh (mg/dl) 14 days lh (mg/dl) idu et al. – anti-fertility property of azadirachta indica 85 plate 1. effect of azadirachta indica seed aqueous extract on the histopathology of testes after 14 days. (a. control testis: seminiferous tubules with spermatocytes, interstitial space, and sertoli cells. b. 2.5 mg/kg a. indica seed aqueous extract: seminiferous tubules with normal sequential maturation of spermatocytes. c. 5.0 mg/kg a. indica seed aqueous extract: seminiferous tubules with normal sequential maturation of spermatocytes. d. 10 mg/kg a. indica seed aqueous extract: seminiferous tubules with normal sequential maturation and mild spermatogenic arrest (h&e x 1).) contraceptive study oestrogen is secreted during the menstrual cycle. the serum fuels of estrogen are usually low during every follicular phase rising gradually until about 1 day before ovulation. this present study in pre-coital treatment as shown in table 3 elicited no significant increase in estrogen level, with a slight increase in folliclestimulating and luteinizing hormone concentration across the treatment groups (2.5, 5.0, and 10 mg/kg a. indica seed aqueous extract) when compared with untreated control. while progesterone controls ovulation and investigated luteal functions at 0.2-0.8mg/ml (follicular phase) and 4.0-20.0mg/ml (luteal phase), it is the hormone responsible for pregnancy. this study showed a significant decrease in the progesterone level across graded doses of the extract when compared with an increase elicited contraceptive property of the extract tables 4 and 5. since antiquity, traditional medicines and several formulations from the herbal plant are known to be effective as a natural contraceptives (umadevi et al., 2013). it has been established that a decrease in serum estrogen, showed the defect of the extracts against fertility property with the magnitude response which stimulates antifertility known as a contraceptive (sedgh et al., 2014). in this present study, the female contraceptive activities in female animals treated groups with graded doses of 2.5, 5.0, and 10 mg/kg of a. indica seed aqueous extract, elicited no significant difference in serum estrogen level in the pre-coital treatment but a slight significant increase in the post-coital phase of the contraceptive study when compared with the control. this concurred with sharma et al. (2013) work on antifertility. this proposed product cannot stimulate estrogen levels serving to be a potent contraceptive agent either by decreasing its production or via metabolic impairment as shown in tables 4 and 5 (rajandeep et al., 2011). table 4. contraceptive effect of azadirachta indica seed aqueous extract on pre-coital treatment after 14 days in female animals. treatment dose (mg/kg) lh (mg/dl) pg (ng/dl) est (ng/dl) fsh (mg/dl) control (dw) 0.5 ml/kg 0.68±0.01a 0.24±0.01a 0.21±0.02a 3.82±0.07a a. indica 2.5 0.75±0.01b 0.19±0.01a 0.23±0.02a 4.05±0.17b a. indica 5.0 0.74±0.02b 0.31±0.03b 0.22±0.04a 4.28±0.17b a. indica 10.0 0.71±0.04a 0.34±0.03b 0.25±0.02a 4.41±0.17b the results were expressed in mean±sem, p>0.05; keywords: follicle stimulating hormone (fsh), progesterone (pg), luteinizing hormone (lh), and estrogen (est). a b c d 86 biology, medicine, & natural product chemistry 12 (1), 2023: 79-88 table 5. contraceptive effect of azadirachta indica seed aqueous extract on post-coital treatment after 14 days in female animals. treatment dose (mg/kg) lh (mg/dl) pg (ng/dl) est (ng/dl) fsh (mg/dl) control (dw) 0.5 ml/kg 0.64±0.01a 0.44±0.11a 0.17±0.02a 4.84±0.09a a. indica 2.5 0.79±0.01b 0.77±0.04b 0.32±0.01b 4.84±0.08a a. indica 5.0 0.82±0.01b 0.77±0.08b 0.39±0.03b 4.99±0.12a a. indica 10 0.88±0.01b 0.82±0.08b 0.33±0.01b 5.70±0.02b the results were expressed in mean±sem, p>0.05; keywords: follicle stimulating hormone (fsh), progesterone (pg), luteinizing hormone (lh), and estrogen (est). in regards to a. indica seed aqueous extract intake, an increase in the luteinizing hormone and folliclestimulating hormone in female rats was recorded. this finding is similar to previous studies reported by pathak et al. (2005) to clarify the potential effect of female sex hormones with a decrease in luteinizing hormone and follicle-stimulating hormone impairment due to higher doses of toxic substances, affecting the production and secretion of this hormones. hence, this study showed that luteinizing and follicle-stimulating hormones showed a slight significant increase in the treated groups when compared with the control (tables 4 and 5). also, prolonged intake of a. indica seed aqueous extract rather reduced plasma progesterone concentration in females (shaikh et al., 2009). the results from this study showed a significant decrease in serum progesterone level across a graded dose of 2.5, 5.0, and 10 mg/kg of the treatment when compared with the control in the pre-coital phase with a slight increase in the post-coital phase (table 4 and 5). a decrease in progesterone concentration as recorded in this study enhances the contraceptive effect in a dose-dependent manner (neychev and mitev, 2016). plate 2. contraceptive effect of azadirachta indica seed aqueous extract on the uterus in rats (a. control. rat uterus: ecto cervical epithelium, and subepithelial stroma. b. 2.5 mg/kg a. indica seed aqueous extract: normal endometrial lining, stromal infiltrates of inflammatory cells and endometrial glands. c. 5.0 mg/kg a. indica seed aqueous extract: stromal congestion, infiltrates of inflammatory cells, and glandular epitheliosis. d. 10 mg/kg a. indica seed aqueous extract: stromal infiltrates of inflammatory cells (h&e x 100).) histopathological examination of the uterus administered with graded doses of 2.5, 5.0, and 10 mg/kg of a. indica seed aqueous extract reveals a normal structure of the uterus muscles when compared with the untreated control (drury and wallinton 2013) (plate 2). this suggested that the extract possesses a a b c d idu et al. – anti-fertility property of azadirachta indica 87 protective effect on the uterus by inhibiting the stroma and other cells from secreting progesterone responsible for the occurrence of pregnancy. this concurred with the report of dosaa et al. 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(2010). laboratory manual for the examination and processing of human semen 5th edition.pp 1-260 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1171-1176 | doi: 10.14421/biomedich.2025.142.1171-1176 issn 2540-9328 (online) fragrant pandan (pandanus ammaryllifolius) leaves ethanol extract as an enhancer of endogenous antioxidant defence under high-fat diet conditions ni made wiasty sukanty1*, laksmi nur fajriani1, i putu bayu agus saputra2, iptan ariki1, i ketut agus yura aditya3, angeline devon venansius1 1undergraduate nutrition study program, faculty of health, universitas bumigora, indonesia. 2medical education study program, faculty of medicine, universitas pendidikan ganesha, indonesia. 3undergraduate pharmacy study program, faculty of health, universitas bumigora, indonesia. corresponding author* nimadewiastysukanty@gmail.com abstract excessive fat consumption, especially saturated and trans fatty acids, plays a significant role in metabolic disorders such as obesity, dyslipidemia, and cardiovascular disease through increased oxidative stress. malondialdehyde (mda), the end product of lipid peroxidation, serves as a biomarker of oxidative damage, whereas indirect bilirubin acts as an endogenous antioxidant. this study aimed to evaluate the effect of fragrant pandan leaves ethanol extract (fplee) in reducing mda levels and modulating indirect bilirubin activity in rats fed a high-fat diet (hfd). the experimental method used 24 male rattus norvegicus divided into six treatment groups: normal control (nc), positive control (c+), negative control (c-), and three fplee treatment groups with different doses (8, 16, and 32 mg/200 g bw/day). administration was carried out for 14 days after 14 days of hfd induction. mda levels were measured using the elisa method, while total bilirubin and direct bilirubin were measured using spectrophotometry to determine indirect bilirubin levels. statistical analysis was performed using one-way anova and the post hoc bonferroni test. the results showed that there were no significant differences in mda levels between groups, although the c+ group fed a hfd showed a tendency toward increased mda. indirect bilirubin levels increased significantly in the low-dose fplee group (8 mg/200 g bw/day), indicating increased endogenous antioxidant activity, while higher doses did not. thus, fplee has the potential to be an effective natural phytopharmaceutical agent in enhancing the body's antioxidant defence against oxidative stress caused by excessive fat consumption, especially at low doses. keywords: antioxidants; cholesterol; maceration; oxidative stress; pandanus ammaryllifolius. abbreviations: malondialdehyde (mda), deoxyribonucleic acid (dna), ribonucleic acid (rna), reactive oxygen species (ros) , fragrant pandan leaves ethanol extract (fplee), body weight (bw), high-fat diet (hfd), propylthiouracil (ptu), carboxymethyl cellulose (cmc), standard diet (sd), propylthiouracil (ptu), carboxymethyl cellulose (cmc), enzyme-linked immunosorbent assay (elisa), statistical package for the social sciences (spss), superoxide dismutase (sod), glutathione peroxidase (gpx), catalase (cat), heme oxygenase-1 (ho-1), nuclear factor erythroid 2-related factor 2 (nrf2), kelch-like ech-associated protein 1 (keap1), uridine diphosphate (udp), udp glucuronosyltransferase family 1 member a1 (ugt1a1). introduction excessive fat consumption, particularly from saturated and trans fatty acids, is a significant contributing factor to metabolic disorders such as obesity, dyslipidemia, atherosclerosis, and cardiovascular disease. long-term high-fat diets increase lipid accumulation, triggering protein carbonylation and lipid peroxidation, which in turn increase free radical formation and reduce antioxidant defences, ultimately leading to oxidative stress (jiang, shuai; liu, 2021). malondialdehyde (mda), the end product of lipid peroxidation, can interact with proteins, deoxyribonucleic acid (dna), and ribonucleic acid (rna). mda's interaction with these macromolecules can disrupt cellular metabolism, making it often used as a biochemical marker to assess the level of oxidative damage. elevated mda levels indicate cell membrane damage due to an imbalance between free radicals and the body's antioxidant capacity (juan, de la lastra, plou, & pérez-lebeña, 2021). the body has natural defence mechanisms against free radicals through both enzymatic and non-enzymatic antioxidant systems. one important non-enzymatic endogenous antioxidant is indirect bilirubin, a heme degradation product capable of neutralising free radicals (xiao, xiong, li, chen, & li, 2023). bilirubin's antioxidant properties stem from its chemical structure, which comprises four pyrrole rings, long conjugated manuscript received: 06 october, 2025. revision accepted: 29 november, 2025. published: 04 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1171-1176 1172 biology, medicine, & natural product chemistry 14 (2), 2025: 1171-1176 double bonds, and reactive hydrogen atoms. bilirubin can interact with electrons in oxygen-free radicals, thereby neutralising reactive oxygen species (ros) and preventing oxidation (boriskina et al., 2024). however, under conditions of chronic oxidative stress due to high-fat consumption, the endogenous antioxidant defence capacity is often reduced, so support from exogenous antioxidant sources is needed (obeagu & obeagu, 2024). pharmacologically, simvastatin is widely used to lower blood lipid levels and prevent complications of atherosclerosis. this drug is effective in suppressing cholesterol synthesis, but its use is often associated with side effects such as myopathy, liver dysfunction, and discomfort in some patients (ruscica, ferri, banach, sirtori, & corsini, 2022; zhang, halmos, & westerterp, 2023). these side effects have prompted the need to explore safer, natural alternatives for controlling lipid levels and addressing oxidative stress. one potential candidate that is gaining attention is the use of local herbal plants with high antioxidant activity (bareetseng, 2022). pandanus amaryllifolius is a local plant rich in bioactive compounds, including flavonoids, polyphenols, and alkaloids. the fragrant pandan leaves ethanol extract (fplee) possesses optimal biological potential due to its high content of phenolic and flavonoid compounds. these compounds are known to have antioxidant properties that can help protect the body from the effects of oxidative stress by inhibiting free radicals (padhi, gupta, saraugi, sehrawat, & routray, 2024). with its potential biological activity, fplee has the potential to be developed as a safer, natural alternative to conventional therapies, such as simvastatin, particularly in efforts to improve lipid profiles and control oxidative stress. this study aimed to investigate the effects of pandan leaf extract on reducing mda levels and modulating indirect bilirubin activity resulting from excessive fat consumption. the results are expected to provide a scientific basis for the development of pandan leaf extract as a potential phytopharmaceutical candidate for preventing degenerative diseases associated with oxidative stress. materials and methods study area this research has received approval from the research ethics committee with the number 057/ec-04/fk06/unizar/vi/2025. this research is a quantitative experimental study using a posttest-only control group design. this research was carried out in three stages, namely: the first stage was the preparation of fplee using the maceration method at the chemistry laboratory of the university of mataram; the second stage was the administration of treatment to test animals carried out at the pharmaceutical research and development laboratory from july to august 2025; and the third stage included the measurement of mda, total bilirubin, and direct bilirubin levels. mda levels were measured using the sandwich enzyme-linked immunosorbent assay (elisa) method, while total bilirubin and direct bilirubin levels were measured using the spectrophotometric method. the indirect bilirubin value was obtained from the difference between total bilirubin and direct bilirubin levels. the subjects in this study were 24 male white rats (rattus norvegicus) aged 2–3 months and weighing 150–250 grams. the rats were randomly divided into six groups: normal control (nc), positive control (c+), negative control (c-), and three treatment groups (t1, t2, and t3). procedures the process of making fplee the production of fplee was carried out using the maceration method. this maceration method began by washing fresh fragrant pandan leaves with running water to remove dirt, followed by air-drying for approximately two weeks until the leaves are dehydrated. afterwards, the dried leaves were cut into small pieces and ground into a powder using a blender. the dry powder was then soaked in 96% ethanol at a ratio of 1 kg of powder to 6 l of solvent and left to stand for three days. after the soaking process was complete, the solution was filtered using filter paper to separate the filtrate from the dregs. the resulting filtrate was then evaporated using a rotary evaporator at 50°c to separate the solvent and produce a thick fragrant pandan leaf extract (sukanty, ariani, & yunita, 2024). the process of making the fplee suspension the fplee dosage given to each treatment group was 8 mg/200 g bw/day for t1, 16 mg/200 g body weight/day for t2, and 32 mg/200 g bw/day for t3 (sukanty et al., 2024). the fplee suspension was made using a 1% cmc solution as the solvent. to achieve these doses, the suspension concentrations were 120 mg/30 ml for t1, 240 mg/30 ml for t2, and 480 mg/30 ml for t3. the suspension was administered orally using a probe, at a volume of 2 ml/200 g bw/day, so that each mouse received the appropriate dose for its treatment group. the fplee suspension was stored in a dark glass bottle at approximately 20°c to maintain stability and prevent degradation of the active compound due to light exposure. the process of making a high-fat diet (hfd) the hfd was made by mixing 5 g of 65% sucrose, 10 grams of lard, 83 g of duck egg yolk, 1.5 g of cholesterol powder, and 0.5 g of propylthiouracil (ptu). all ingredients were thoroughly mixed and stirred until a homogeneous mixture is achieved. the hfd was sukanty et al. – antioxidant potential of pandanus ammaryllifolius 1173 administered orally at a dose of 2 ml/200 g bw/day for 14 days (yang et al., 2019). the process of making the simvastatin suspension a 1% carboxymethyl cellulose (cmc) solution was prepared by mixing 1 gram of cmc with 100 ml of warm water and stirring until completely dissolved. then, 10.3 mg of simvastatin was dissolved in 100 ml of cmc solution. the simvastatin suspension was administered orally to mice using a tube at a dose of 0.206 mg/200 g bw/day. the treatment of experimental rats the rats underwent a seven-day adaptation period, receiving a standard diet (sd) and ad libitum access to drinking water. after the adaptation period, the rats were randomly divided into six groups: nc, c-, c+, and three treatment groups (t1, t2, and t3). all groups, except nc, were fed a hfd for 14 days. after the induction period, the cwas given simvastatin, while the c+ continued to receive the hfd. the t1, t2, and t3 were each given fplee at concentrations of 8 mg/200 g bw/day, 16 mg/200 g bw/day, and 32 mg/200 g bw/day for 14 days. after all treatments were completed, the rats were euthanised, and the blood was collected via cardiac puncture for biochemical analysis. table 1. the treatment of experimental animals. group days 1-14 days 15-28 nc sd sd c+ sd and hfd sd and hfd csd and hfd sd and 0.206 mg/200 g bw/day of simvastatin t1 sd and hfd sd and 8 mg/200 g bw/day of fplee t2 sd and hfd sd and 16 mg/200 g bw/day of fplee t3 sd and hfd sd and 32 mg/200 g bw/day of fplee the measurement of total bilirubin, direct bilirubin, indirect bilirubin, and mda levels the sample used for the test parameters was blood serum. total and direct bilirubin levels were measured using spectrophotometry, and the results of both parameters were used to calculate indirect bilirubin by dividing the total and direct bilirubin levels. mda levels were analysed using the sandwich elisa method. data analysis data from indirect bilirubin and mda measurements were analysed using statistical package for the social sciences (spss) version 23 software. the analysis began with normality and homogeneity tests to ensure the data met the requirements for parametric analysis. next, a one-way anova test was performed to examine differences between groups, followed by a bonferroni post hoc test to identify groups with significant differences. all analyses were performed at a 5% significance level (α = 0.05) or a 95% confidence level. results and discussion result this study aimed to investigate the effect of fplee on indirect bilirubin and mda levels in rats fed a high-fat diet. indirect bilirubin was used as an indicator of endogenous antioxidant activity, while mda levels reflected the level of oxidative stress due to lipid peroxidation. table 2. one-way anova test results on indirect bilirubin and mda levels. group indirect bilirubin mda mean ± sd (mg/dl) f p mean ± sd (mmol/ml) f p nc 0.0225 ± 0.01258 11.508 0.000** 2.2075 ± 0.18661 0.752 0.595 c+ 0.0375 ± 0.02217 2.1275 ± 0.19103 c0.0250 ± 0.01915 2.0150 ± 0.30161 t1 0.1600 ± 0.04082 1.9700 ± 0.08165 t2 0.0425 ± 0.04573 2.0425 ± 0.20630 t3 0.0375 ± 0.03096 2.0625 ± 0.13574 **p < 0.01 the results of the analysis of indirect bilirubin levels in table 2 show a statistically significant difference between groups (f = 11.508; p = 0.000, p < 0.01). the c+ group (high-fat feed) showed an increase in indirect bilirubin levels compared to the nc, although the increase was not very striking. the most significant 1174 biology, medicine, & natural product chemistry 14 (2), 2025: 1171-1176 increase was observed in t1 (8 mg/200 g bw/day), with an average indirect bilirubin level of 0.1600 ± 0.04082 mg/dl, which was higher than in the other groups. in contrast, the cand t3 exhibited bilirubin levels comparable to the control value, indicating a doseresponse difference. in the mda parameter, no significant differences were found between treatment groups (f = 0.752; p = 0.595). however, descriptively, the c+ showed the highest mda levels (2.1275 ± 0.19103 mmol/ml), indicating oxidative stress associated with high fat consumption. the chad the lowest mda value (1.0150 ± 0.30161 mmol/ml), followed by t1 (1.9700 ± 0.08165 mmol/ml). in contrast, mda levels in t2 and t3 tended to remain high and approached those of the c+, indicating that a consistent decrease in mda had not occurred at any dose of fplee. table 3. bonferroni post hoc analysis of indirect bilirubin levels. group p nc c+ 1.000 c1.000 t1 0.000** t2 1.000 t3 1.000 c+ c1.000 t1 0.000** t2 1.000 t3 1.000 ct1 0.000** t2 1.000 t3 1.000 t1 t2 0.001** t3 0.000** t2 t3 1.000 **p < 0.01 bonferroni post hoc analysis of indirect bilirubin levels in table 3 reveals that the administration of fplee at a dose of 8 mg/200 g bw/day (group t1) resulted in a significant difference compared to the other groups. specifically, indirect bilirubin levels in group t1 were significantly higher than those in the nc, c+, c-, t2, and t3, which received higher doses of fplee (16 mg/200 g bw/day and 32 mg/200 g bw/day). in contrast, there were no significant differences between groups nc, c+, c-, t2, and t3, indicating that only the 8 mg/200 g bw/day dose of fplee significantly increased indirect bilirubin levels. these results indicate that low doses of fplee have a more optimal stimulatory effect on endogenous antioxidant activity than higher doses, which is likely due to a non-linear biological response to increasing doses of fplee. discussion the body is equipped with endogenous antioxidant systems, including enzymes and non-enzymes. enzymatic antioxidant systems include superoxide dismutase (sod), glutathione peroxidase (gpx), and catalase (cat), which play a crucial role in neutralising ros from an early stage (irato & santovito, 2021). an example of a non-enzymatic antioxidant system is bilirubin, which acts as a complement to other antioxidants (he et al., 2025). in this study, indirect bilirubin levels showed varying responses across treatment groups. in the nc and c+ groups fed only an hfd, indirect bilirubin levels did not differ significantly. the indirect bilirubin result is consistent with the results of mda as a biomarker of oxidative stress, which also showed no differences between the groups. enzymatic defence mechanisms may contribute to the absence of differences in mda levels between groups in this study, indicating that the body's enzymatic antioxidant system likely works effectively in addressing oxidative stress. in contrast, indirect bilirubin in this group acts as a complement to other antioxidants (he et al., 2025; lankin, tikhaze, & melkumyants, 2023). this phenomenon may also occur due to the role of bioactive compounds in pandan leaves, such as flavonoids and alkaloids. flavonoids contain hydroxyl groups that stabilise and inhibit the production of free radicals. flavonoids also act as bidentate ligands, chelating redox-active metals and enhancing the activity of antioxidant enzymes, such as those that scavenge vitamin e radicals (jomova et al., 2025). in the cthat received hfd and simvastatin treatment, indirect bilirubin levels were also not different from those in nc. simvastatin is known to have pleiotropic effects in the form of antioxidant activity and endothelial protection, which include increasing the expression of endogenous antioxidant enzymes and reducing ros production. furthermore, simvastatin can affect bilirubin metabolism by increasing the expression of heme oxygenase-1 (ho-1), an enzyme that catalyses the degradation of endogenous iron protoporphyrin heme into biliverdin, ferrous iron, and carbon monoxide. the formed biliverdin is reduced by biliverdin reductase to bilirubin (consoli, sorrenti, grosso, & vanella, 2021; zhu et al., 2024). thus, simvastatin supports the production of indirect bilirubin as an antioxidant. however, mda levels were not significantly different across groups, indicating that severe oxidative stress requiring antioxidant system compensation was not present. consequently, indirect bilirubin levels as an antioxidant did not increase drastically even though simvastatin was administered to the c-. the low concentration of ros suggests a physiological role for ros in redox regulation, such as regulating cell function by modifying the activity of enzymes and transcription factors (amponsah-offeh, diaba-nuhoho, speier, & morawietz, 2023; lennicke & cocheme, 2021). interestingly, in the treatment group with fplee, especially t1, there was an increase in indirect bilirubin levels compared to the control group. in relation to bilirubin, flavonoids can activate the nuclear factor erythroid 2-related factor 2 (nrf2) signal transduction sukanty et al. – antioxidant potential of pandanus ammaryllifolius 1175 pathway, where the bond between kelch-like echassociated protein 1 (keap1) and nrf2 is broken, causing the translocation of nrf2 from the cytosol to the nucleus to induce the transcription of genes containing antioxidant response elements, such as ho-1, so that the production of indirect bilirubin increases (moratillarivera, sánchez, valdés-gonzález, & gómezserranillos, 2023; sang et al., 2025). in addition to this mechanism, flavonoids also inhibit uridine diphosphate (udp) glucuronosyltransferase family 1 member a1 (ugt1a1), an enzyme that conjugates indirect bilirubin with glucuronic acid to become direct bilirubin (žiberna, jenko-pražnikar, & petelin, 2021). the increase in indirect bilirubin in t1 reflects the body's adaptive response to exposure to phytochemical compounds, in which endogenous antioxidant capacity is strengthened through increased reserves of indirect bilirubin as a nonenzymatic antioxidant that complements the work of other antioxidants (he et al., 2025). however, in groups t2 (16 mg/200 g bw/day) and t3 (32 mg/200 g bw/day), indirect bilirubin levels did not increase. it may be explained by a possible dose effect, where higher flavonoid concentrations actually suppress indirect bilirubin concentrations. although the exact mechanism of flavonoids in reducing bilirubin levels is not yet fully understood, the dual effects of flavonoids may explain this phenomenon. in addition to being antioxidants, flavonoids can act as prooxidants, where the flavonoid catechol ring can oxidise lowmolecular-weight antioxidants (jomova et al., 2025). bilirubin is a low-molecular-weight antioxidant (boriskina et al., 2024). oxidation of indirect bilirubin decreases indirect bilirubin levels and increases biliverdin levels (žiberna et al., 2021). it may be the underlying cause of low bilirubin concentrations in t2 and t3. conclusions the results of this study indicate that although hfd did not significantly affect mda levels, administration of fplee, especially at low doses (8 mg/200 g bw/day), was able to increase indirect bilirubin levels as part of the body's defence mechanism against ros exposure. this increase indicates endogenous antioxidant activity that is preventive and adaptive. these findings support the potential of pandan wangi as a promising phytotherapeutic agent in efforts to prevent oxidative damage due to free radical exposure. acknowledgements: the author would like to thank the ministry of higher education, science, and technology of the republic indonesia (kemdiktisaintek), bumigora university, and all colleagues who helped in carrying out this research. 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(2021). serum bilirubin levels in overweight and obese individuals: the importance of anti-inflammatory and antioxidant responses. antioxidants, 10(9), 1–16. https://doi.org/10.3390/antiox10091352 biology, medicine, & natural product chemistry volume 6 – number 1 – 2017 issn 2089-6514 (paper) | issn 2540-9328 (online) contents antioxidant capacity comparison of ethanolic extract of soursop (annona muricata linn.) leaves and seeds as cancer prevention candidate dyah ayu widyastuti, praptining rahayu 1 4 analysis of bull sperm dna abnormalities due to cadmium accumulation fuad fitriawan 5 8 comparative anatomy and histology of black pomfret (formio niger) and nile tilapia (oreochromis niloticus) kidney nurul safitri apriliani, muhammad jafar luthfi 9 12 anatomical study of male reproductive organs of the indonesian short-nosed fruit bat (cynopterus titthaecheilus temminck, 1825) anisatuzzahro, muhammad jafar luthfi 13 17 checklist of flowering plants (magnoliophyta) of mount nglanggeran, gunungkidul: confirmation and update of flora of java and apg iii widodo, muhammad jafar luthfi 19 36 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 137-143 | doi: 10.14421/biomedich.2022.112.137-143 issn 2540-9328 (online) ethnobotanical survey of plants used in the management of peptic ulcer diseases in wukari metropolis akpevwoghene agbatutu1,2, francis nosakhare imade2,*, efosa a. ogie-odia2, mary oromioshemime ifedele3, funmilola modinat abdulrasaq3, daniel eseigbe2 1department of biological sciences, kwararafa university wukari, taraba state, nigeria. 2plant science and biotechnology department, faculty of life sciences, ambrose alli university, ekpoma, edo state, nigeria. 3department of botany, university of ibadan, ibadan, oyo state, nigeria. corresponding author* imadefrancis@gmail.com manuscript received: 26 may, 2022. revision accepted: 28 july, 2022. published: 03 august, 2022. abstract peptic ulcer diseases (pud) are sores formed in soft tissues present in the lining of the digestive tract as a result of excessive stomach acid or inability of the alimentary tract or stomach to protect itself. the increasing surge for plant based drugs in the management of pud has increased scientific investigation of herbs and recipes from traditional medicinal practitioners (tmp) to ascertain their efficacy through pharmacological studies. ethnobotanical survey of plants and recipes from tmp used in the management of pud in wukari metropolis, taraba state, northeastern nigeria were documented. a total of twenty (20) tmp located in ten (10) different area of the town were interviewed using a semi structured questioner. twenty-four (24) plant species from twenty (20) different families and sixteen (16) recipes were documented from the survey. it was observed that plant leaves were mostly used while c. longa (zingberaceae), m. paradisiaca (musaceae) had the highest frequency of occurrence in recipes formulation. therefore, due to the proven efficacy of these herbal recipes as reported from correspondents, there is need for proper documentation, conservation, cultivation and use of these medicinal plants in the management of pud to avoid them being endangered or going into extinction. keywords: peptic ulcer; wukari; plant; taraba. introduction peptic ulcer disease (pud) is a common gastrointestinal disorder which occurs on inflamed break in the skin or mucus membrane lining the alimentary tract. it is the most predominant chronic and recurrent gastrointestinal diseases and a global health problem leading to morbidity and mortality worldwide (sen et al., 2009). pud manifest in different unit of the alimentary tract like the stomach (gastric ulcer), duodenum (duodenal ulcer), esophagus (esophageal ulcer) resulting in different types of peptic ulcer. the specific cause of pud remains unclear over the years though studies report that the lack of equilibrium between the mucosal defensive factors (secretion of bicarbonate, prostaglandins, innate resistance of the mucosal cell) and gastric aggressive factors (acid, pepsin, h. pylori infection) are possible causes of pud (roy et al., 2013; dashputre et al., 2011; roberts, 2003). presently h. pylori, non-steroidal anti-inflammatory drugs (nsaids) and zollinger-ellison syndrome (a dramatic hypersecretion of acid) are three primary factors in peptic ulcer disease though, environmental factors (smoking, excessive alcohol intake), genetic, dietary and physiologic stress also increases gastric acid secretion, weakens mucosal barrier leading to pud (malfertheiner et al., 2009; wu and fassihi, 2005). the pathophysiology of pud injury mechanism in gastric and duodenal ulcer differs distinctively such that duodenal ulcer is essentially an h. pylori related disease while gastric ulcer is commonly associated with nsaid ingestion but in both conditions inflammatory symptoms is observed at the onset leading to an imbalance between protective and aggressive factors (chan and leung, 2002). orthodox drugs used in the treatment of pud are histamine-receptor blockers (ranitidine, cimetidine), proton pump inhibitors (omeprazole, pantoprazole) and antibiotics (metronidazole, amoxycillin) which are sometimes combined to achieve better result. they are often directed at reduction of aggressive factors with some reported side effects such as dizziness, headache, constipation, impotence or breast enlargement in men and the use of antacids leads to stomach distention, belching, constipation (gulmez et al., 2007, sarkar et al., 2008, reilly, 1999, franco and richter, 1998). previous studies reveal that about 5–10% of world population suffer from pud though with a recent decrease in the incidence, rates of hospital admissions and mortality (lanas and chan, 2007, lanas et al., 2011, https://doi.org/10.14421/biomedich.2022.112.137-143 138 biology, medicine, & natural product chemistry 11 (2), 2022: 137-143 sonnenberg, 2013). the prevalence of h. pylori, a major cause of pud is higher in developing countries present in africa, central america, central asia and eastern europe where the bacteria is mostly acquired during childhood in an unhygienic environment especially in communities with lower socioeconomic status (kuna et al., 2019, hooi et al., 2017). most people in these developing countries live below poverty level, do not have access to standard healthcare facilities, rely on nsaids and painkiller drugs to subside pains resulting from stress, are unable to afford balanced diet therefore consume food substances which tend to increase gastric acid production and these factors creates an imbalance between aggressive and defensive factors in gastrointestinal tract. these classes of people who constitute about 80% of the world’s population rely on herbs from plants as alternative medication in management of sickness and diseases. traditional healers have used and relied on plants materials as main source of natural therapeutic medicines from time immemorial in the treatment and management of various infectious diseases (akinwumi and sonibare, 2019; beverly and sudarsanam, 2011; dike et al., 2012). scientific studies have established that medicinal plants used by traditional healers have displayed proven pharmacological, antioxidant and therapeutic activity against degenerative diseases. many herbs and plant products have been found to play a role in protecting or helping to heal stomach and peptic ulcers with the presence of important secondary metabolites like flavonoids and tannins reported as the active principle responsible for their anti-ulcer activity (nihar et al., 2017). ethnobotanical studies or survey are recognized as the most viable method of identifying novel medicinal plants or refocusing on those earlier reported for bioactive constituents (alebiosu et al, 2005). there is little or no documentation of medicinal plants used for the treatment of pud in wukari metropolis, northeastern nigeria, taraba state therefore this documentation will serve as reference to scientific researchers in the development of potent drugs and recipes from natural plants in treatment and management of pud with fewer or no side effects. this present study aims to document medicinal plants used in the management of pud in wukari, taraba state. methodology study area this survey was conducted in wukari metropolis the headquarters of wukari local government area of taraba state, nigeria. it has an area of 4,308km2 and a population of 238,283 at the 2006 census which is located in the south of the benue river basin (nbs, 2006). the town is located in the southern taraba with its coordinates between latitude 7o 51’n to 7o 85’n and longitude 9o47’e to 9o78’e which is about 200km from jalingo the state capital. the city has a tropical continental climate characterized by a marked distinctive wet and dry season, an annual rainfall between 1000-1500mm, temperatures range between 2040°c with a lengthy wet season (7 months) throughout the course of the year (oyatayo et al., 2017). wukari is mainly an agrarian town that lies within the savannah zone and has mainly grassland vegetation and scattered trees and shrubs in the southern part of the state. its inhabitants are the jukuns, who are predominantly traditionalists though some of them are christians and muslims. the survey was conducted in sampled areas that are close to farms and garden in the extended city areas of wukari, with the aim to capture both rural and urban populace and locate the elderly people with the knowledge of traditional medicine. figure 1. map of nigeria showing taraba state. agbatutu et al. – ethnobotanical survey of plants used in the management of … 139 figure 2. map of taraba state showing wukari local government area and areas visited. data collection the main data sources consisted of a series of semi– structured and open-ended questionnaires as well as informal interviews administered on local herb sellers, herbalists, aged and other groups of people rich in traditional medicine knowledge. the survey was conducted within the span of eight (8) months from january 2019 to august 2019 with repeated visits to the respondents. questionnaires were administered and hausa language was used to obtained the information from the respondents. such information includes the local plant names, useful plant parts and methods of preparation. local names of plants mentioned were validated using literatures and proper taxa nomenclature was validated in the plant list database at www.plantlist.org. demographic data a total of twenty respondents were interviewed consisting of twelve males (60%) and eight females (40%) in wukari metropolis. the respondents include traditional healers (30%), herbalist (20%), herb sellers (40%) and the aged people (10%) all of which are nigerians from the jukun ethnic group comprising of (40%) christians, (20%) muslims and (40%) practicing traditionalists. their ages were within 31-70 years with the age bracket of 51-60 (45%) with no formal education (60%) being the highest among them (table 1). results a total of twenty-four (24) plant species belonging to twenty (20) families were identified as plants used in treatment of pud in wukari metropolis (table 2). the plant families, local names, part used and frequency of occurrence were documented. the anarcardiaceae, alliaceae, malvaceae and musaceae had two plants each among the twenty-four plant species while the leaves were mostly used in preparation of various herbal recipes. some of the plants were reported to be harvested from the forest while others from home garden and environs. the respondents claimed that their medicinal knowledge was inherited, few trained and some believed to be divine. table 1. source of ethnomedicinal information. demography frequency (n=20) percentage (%) traditional healers 6 30.0 herbalist 4 20.0 herb seller 8 40.0 aged 2 10.0 gender male 12 60.0 female 8 40.0 age range below 30 0 0.0 31-40 2 10.0 41-50 7 35.0 51-60 60 and above 9 2 45.0 10.0 religion christianity 8.0 40.0 islam 4.0 20.0 traditional religion 8.0 40.0 education no formal 12 60.00 primary 4 20.00 secondary 4 20.00 diploma 0 00.00 degree 0 00.00 140 biology, medicine, & natural product chemistry 11 (2), 2022: 137-143 table 2. list of plants used in management of pud in wukari, taraba state. s/n plant name family part used common name hausa name frequency of occurrence 1 azadirachta indica (a.) juss. maliaceae leaves neem doogon yaaroo 1 2 psidium guajava linn. myrtaceae leaves guava gweebaa 1 3 anarcardium occidentale linn. anarcardiaceae roots, stems leaves cashew jambe 1 4 carica papaya linn. caricaceae fruits, seeds pawpaw gwandar masar 1 5 ocimum gratissimum linn. lamiaceae leaves scent leaf tagida, daddooya 1 6 musa sapientum linn. musaceae unripe fruits banana ayama 2 7 talinum triangulare (jacq.) wild. portulacaceae leaves water leaf alenyruwa 1 8 curcuma longa linn. zingberaceae rhizome tumeric zabibi 4 9 musa paradisiaca linn. musaceae fruit plantain agada 4 10 aloe vera (l.) burm. f alliaceae gel aloe hantsar giwaa 1 11 mangifera indica linn. anarcardiaceae leaves mango mangoro 1 12 hibiscus sabdariffa linn. malvaceae calyx roselle soboroto 1 13 sida acuta burm.f. malvaceae leaves wire weed tsadar iamarudu 1 14 lantana camara linn. verbenaceae stem, whole plant red sage kimbama halba 1 15 euphorbia hirta linn. euphorbiaceae leaves garden spurge noonon kurciyaa 2 16 ipomoea batatas linn. convolvulaceae tuber sweet potato ba fadamee 1 17 moringa oleifera lam. moringaceae leaves drumstick zogelle 1 18 bryophyllum pinnatum lam. crussalaceae leaves life plant harfifi 1 19 calotropis procera (ait.) ait.f. ascclepidiaceae leaves sodom apple baabaa ambele 1 20 allium cepa linn. alliaceae bulb onion albasa 1 21 datura metel linn. solanaceae leaves devil trumpet zakami 1 22 vitellaria paradoxa gaertn.f. sapotaceae leaves and fruit shea butter kadee 1 23 sorghum bicolor (l.) moench poaceae seed guinea corn mazakuwa 2 24 ampelocissus africanus (lour.) merr. vitaceae leaves simple-leaved wild grape siling siame 1 figure 3. frequency of plant parts used in management of pud in wukari metropolis 0 2 4 6 8 10 12 14 bulb tuber leaves root stem fruits seeds rhizome gel calyx whole plant agbatutu et al. – ethnobotanical survey of plants used in the management of … 141 table 3. pud herbal recipes from tmp in wukari, taraba state. s/n method of preparation 1. dry the half ripe m. paradisiaca and m. sapientum peels and grind into powder, mix one teaspoon of the powder with honey. 2. squeeze the fresh leaves of t. triangulare, e. hirta in clean water, add edible salt (nacl), then sieve the extract, take a glass cup twice daily 3. dried rhizome of c. longa is chewed with leaves of e. hirta in an oily juice (palm oil, olive oil). 4. dried half ripe peels of m. paradisiaca and m. sapientum is powdered, a powdered teaspoon is mixed with honey and taken daily for two months. 5. cut two whole fruits of c. papaya pieces including the peel, soak in six liters of water, sieve to remove the cubes and add 750ml of honey into the extract, and drink a glass cup twice a day. 6. blend four matured leaves of a. vera with 750ml of pure honey and 750ml of clean water respectively, filter the syrup and keep in a container to be taken twice daily. 7. dry leaves of c. procera are crushed and mixed with v. paradoxa and swallowed twice daily. 8. gentle heat is applied to the leaves of b. pinnnatum until it brings out oil which is then crushed to paste and mixed with honey before consumption twice daily. 9. boiled or crushed leaves of m. oleifera in water, is taken twice daily. 10. unripe m. paradisiaca and c. papaya fruits are sliced to cubes, soaked in water for three days and taken daily. 11. powdered leaves and rhizome of a. africanus, c. longa is mixed with s. bicolor paste and consumed morning and evening daily. 12. bulbs of a. cepa and leaves of s. acuta are crushed, mixed with s. bicolor paste and natural honey. 13. decoction of the calyx of h. sabdariffa is mixed with powdered leaves of a. occidentale and p. guajava and taken twice daily. 14. a tea spoon of powdered m. paradisiaca fruit in mixed in a maceration of a. indica leaves and l. camara whole plant for seven days be taken twice daily for two months. 15. paste of boiled c. longa mixed with a pinch powdered leaves of d. metel leaves be eaten with boiled tuber of i. batatas should be taken early morning daily. 16. decoction of fresh leaves of o. gratissimum, m. indica mixed with s. bicolor paste and natural honey figure 4. frequency of plants used in pud herbal recipes in wukari metropolis. discussion the importance of plant based drugs from traditionalists and herb sellers in the management of pud remains vital in the search of novel active compounds with little or no side effects. a total of twenty-four (24) plant species from twenty (20) families with sixteen (16) recipes were recorded from twenty (20) traditional medical practitioners present in wukari metropolis. (figure 2 to 4, tables 1 to 2). the plants c. longa, m. paradisiaca recorded the highest frequency of occurrence while various plant parts such as leaves, stem, fruit bark, rhizome and bulb were frequently used in the prescribed recipes. these formulations were either mixed or prepared with pap, water and honey as major vehicle suggested during preparation, administration and consumption of prepared recipes. there are reports on the scientific investigation on pud and gastro-protective 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 142 biology, medicine, & natural product chemistry 11 (2), 2022: 137-143 potentials of some of these plants such as c. longa (savaringal and sanalkumar, 2018), a. vera (subramanian et al., 2007), a. occidentale (ajibola et al., 2010), c. papaya (okewumi and oyeyemi, 2012), o. gratissimum (amadi et al., 2014), m. paradisiaca (rao et al., 2016), m. indica (neelima et al., 2012), t. triangulare (onwurah et al., 2013) and m. sapientum (prabha et al., 2011) have been evaluated using different models on experimental animals. some of these plants have been reported to display strong antimicrobial (a. cepa, c. longa), anti-inflammatory (s. acuta), relaxant (o. gratissimum, m. oleifera) and acid reflux (m. sapientum, a. vera) activity which could confirm their mode of activity or mechanism of action against the reported major causes of pud such as h. pylori a bacteria, nsaids related drugs, zollinger-ellison syndrome and environmental stress (iqbal et al., 2018, gupta et al., 2015, arciniegas et al., 2016, malfertheiner et al., 2009). the therapeutic activity of these herbal recipes as reported from respondents and their customers suggest that these herbal products contain numerous bioactive constituents that are a part of the physiological functions of living flora and hence they are believed to have better compatibility with human body (kamboj, 2000). akinwumi and sonibare (2019) stated that cultivation and proper documentation of medicinal plants from ethnobotanical survey is essential to prevent them from being endangered and going into extinction. this confirms and addresses the worries from respondents who reported the scarce availability of some plants in their natural habitats due to their increasing demands for food and medicine. therefore, the increasing dependence of these plant based herbal drugs mainly in developing countries for primary health care could be properly harnessed and managed only if there are measures to ensure sustainable cultivation that will have a considerable long term effect on the environment, health care and economy. conclusion twenty-four (24) plant species with sixteen (16) recipes from traditional medicinal practitioners and herb sellers were documented and reordered for the treatment and management of pud. the major essence of this survey is to harness and document the rich knowledge of the traditional medicinal practitioners on the use of medicinal plants in the treatment of pud which could pave the way for detailed scientific research in the discovery of novel compounds with better activity, new mechanism of action and less toxic side effects. authors’ contributions: agbatutu designed the study. agbatutu carried out the laboratory work. ifedele and imade analyzed the data. agbatutu and imade wrote the manuscript. abdulrasaq and ogie odia review the manuscript all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. references ajibola es, adeleye oe, okediran bs and rahman sa (2010). effect of intragastric administration of crude aqueous leaf extract of anacardium occidentale on gastric acid secretion in rats. nig. j. physiol. sci. 25. 59 – 62. akinwumi ia and sonibare ma (2019). use of medicinal plants for the treatment of gastric ulcer in some parts of southwestern nigeria. african journal of pharmacy and pharmacology vol. 13(15), pp. 223-235 alebiosu co, ugwa om, ugwah-oguejiofor cj and 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3: 270. sonnenberg a (2013). historic changes of helicobacter pyloriassociated diseases. aliment. pharmacol. ther., 38, 329–342. subramanian s, kumor ds, aruiselvan p, senthilkumar gp and rao m (2007). evaluation of antiulcerogenic potential of aloe vera leaf gel extract studied in experimental rats. journal of pharmacology and toxicology 2(1):85-97. wu y and fassihi r (2005). stability of metronidazole, tetracycline hcl and famotidine alone and in combination. int. j. pharm. 290: 1-13. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 91-95 | doi: 10.14421/biomedich.2020.92.91-95 issn 2540-9328 (online) a chemical overview of azanza garckeana yilni edward bioltif1,*, naanma bioltif edward2, terry dalyop tyeng1 1department of chemistry, faculty of natural science, plateau state university, bokkos, nigeria. 2department of plant science and biotechnology, faculty of natural science, university of jos, nigeria. corresponding author* edwardyilni405@gmail.com manuscript received: 18 may, 2020. revision accepted: 10 november, 2020. published: 17 november, 2020. abstract azanza garckeana is a popular fruit tree in nigeria, specifically in gombe state, where it is locally called ‘goron tula’ which means ‘kola of tula’. it is also found in part of some african countries. different plant part of this small tree/shrub has recorded different uses by the locals; uses ranging from its fruits being edible and others parts helping to remedy different diseases, especially sexually related diseases. it also records use as booster for sexual performance. the uses of the plant are majorly attributed to the presence of chemicals. its local use initiates the necessity of this review to enhance the research for drug discovery since chemicals are the chief constituencies responsible for its medicinal importance. keywords: azanza garckeana; chemical; compounds; mansonone. introduction the indigenous fruits collected from the wild play a significant role in food and nutrient security of the poor and rural dwellers. some wild fruits have been identified to have better nutritional value than cultivated fruits (musinguzi et al., 2007) as a result, in recent years, a growing interest has emerged to evaluate various wild edible plants for their nutritional features (nkafamiya, 2007; aberoumand and deokule, 2009; nazarudeen, 2010). nutritional value of indigenous fruit bearing tree species indicates that many are rich in sugars, essential vitamins and minerals, while others are high in vegetable oil and protein contents. in addition to fruit production and cash, the extensive list of benefits includes firewood, fodder, building material, shade and medicine especially to rural communities. edible wild leaves and fruits are consumed frequently in northern nigeria especially in rural communities where a variety of edible leaves and fruits abound. some of these are cultivated while others grow in the wild (nkafamiya et al. 2016). azanza garckeana is a member of the malvaceae family. the generic name “azanza” is derived from the word “azania”, a word meaning black and surviving in zanzibar. the specific name “garckeana” is in honour of professor august garcke (1819-1904), a german botanist and plant collector who specialized in pharmacognosy (maroy, 2017). botanical classification of azanza garckeana kingdom : plantae – plants subkingdom : tracheobionta – vascular plants superdivision : spermatophyta – seed plants division : magnoliophyta – flowering plants class : magnoliopsida – dicotyledons subclass : dilleniidae order : malvales family : malvaceae – mallow family genus : azanza specie : azanza garckeana english (common name) tree hibiscus, azanza, snot apple nigeria (hausa): goron tula, bostwana – morojwa south africa thespesia garckeana (mojeremane and tshwenyane, 2004; and ochokwu et al., 2014). description azanza garckeana is a deciduous shrub; the tree can grow to a height of 3-15m high depending on the climate condition stem diameter at breast height of up to 25cm. the tree is multi-stemmed with straight or crooked stem, which is sometimes forking from the base. the bark is rough and greyish-black or brown, fibrous with longitudinal fissures. the twigs are hairy when young but become smooth with age and branches have woody hairs. the leaves are distinctively rounded, 8 by 12cm on long stalks. they are always simple, alternate and roundish. the leaves have 3 to 5 lobes, which are covered in brown star-shaped hairs, and have longitudinal fissures in the midrib. the tip of the leave is usually bluntly pointed or rounded. the base of the https://doi.org/10.14421/biomedich.2020.92.91-95 92 biology, medicine, & natural product chemistry 9 (2), 2020: 91-95 leave is heart-shaped and is 5 to 7 nerved. the young leaves are brown in colour and velvety. the flowers are large up to 6cm long, solitary on long pedicels in the axils of uppermost leaves, yellow with a purple-brown centre; the petals are globose and capsules are up to 4cm long, the thickness is 3cm. the fruit is globose and have woody capsules of up to 3 to 4cm in diameter, it is divided into 5 segments with each segment containing a seed, the remains of the calyx and epicalyx at the base; the seeds are hemispherical, up to 10 mm long, 7 mm thick, with brownish and woolly floss (orwa et al., 2009). a. shoot b. leaves and fruits c. mature fruit figure 1. different parts of azanza garckeana. table 1. uses of azanza garckeana. medicinal use plant part(s) used country practised dietary uses edible fruits fruits botswana, kenya, malawi, nigeria, sudan, tanzania, zambia, zimbabwe food additive fruits sudan, tanzania medicinal uses abscesses fruit poultice applied nigeria anemia ripe fruits sudan antiemetic root infusion taken orally zimbabwe aphrodisiac ripe fruits taken orally nigeria asthma root decoction mixed with sterospermum kunthianum cham. malawi chest pains root infusion taken orally nigeria, zimbabwe cough root infusion taken orally kenya, nigeria, zimbabwe diabetes leaf decoction taken orally drc earache root infusion dropped into ear zimbabwe edema leaf decoction taken orally drc epilepsy leaf decoction taken orally drc fever root decoction taken orally malawi gonorrhoea roots and stem bark taken orally malawi, nigeria induce labour root decoction taken orally tanzania infertility ripe fruits or root decoction taken orally botswana, malawi, nigeria liver problems stem and leaf decoction taken orally kenya, nigeria madness (mental illness) root decoction taken orally zimbabwe malaria eat raw fruit or cook and eat as relish zambia membrane rupture root decoction taken orally drc menstruation root infusion taken orally nigeria, zimbabwe retained placenta root infusion taken orally zimbabwe sexually transmitted diseases root and bark infusion taken orally zambia syphilis root decoction taken orally nigeria maroyi, 2017 bioltif et al. – a chemical overview of azanza garckeana 93 table 2. compounds extracted from azanza garckeana. compounds extract plant parts sesquiterpenoids gossypol, 6, 6-dimethoxygossypol, 6-methoxygossypol ethyl acetate in nhexane; methanol in dichloromethane root phytosterol stigmasterol ethyl acetate in nhexane; methanol in dichloromethane root and stem bark e-docosyl 3-(3, 4dihydroxyphenyl) acrylate ethyl acetate in nhexane; methanol in dichloromethane root and stem bark o-naphthoquinones n-hexane heartwood mansonones e, f, g, h azanzone a, b n-hexane heartwood triterpene betulinic acid ethyl acetate in nhexane; n-hexane; methanol in dichloromethane fruit pulp, root, stem bark dikko et al., 2016 & maroyi, 2017 azanza garckeana is widely distributed in the east, west and southern africa. it generally grows naturally in all types of woodlands from sea level to about 1700m above sea level. it also grows in semi-arid areas. azanza garckeana grows in a variety of soils and is found near termite mounds and deserted areas while in nigeria it grows in open woodland in the north eastern part of the country (ochokwu et al., 2015). azanza garckeana (goron tula) as an edible indigenous fruit in north eastern part of nigeria (particularly gombe state). fao (1983) reported that a. garckeana grows naturally in semi-arid areas receiving annual rain fall that range from 250mm to 1270mm. flowering takes place during the raining season, while fruit ripening occurs during the dry season, hence it takes about six months from flower fertilization to ripening of the fruit. in southern africa, flowering occurs from december to may and fruiting from february to september while in north eastern nigeria flowering occurs from may to october and fruiting/ripening from november to april (ochokwu et al., 2015). oh oh oh oh oh oh ch3 ch3ch3 ch3 ch3 ch3 o ch2 h h ch3 ch3 ch3 ch3 o o ch3 ch3 ch3 ch3 o o ch3 ch3 ch3 ch3 o o o gossypol mansonone c azanzone mansonone e ch3 ch3 ch3 o o o oh ch3 ch3 ch3 ch3 o o oh ch3 ch3ch3 h ch3 ch3 h h h oh ch3 mansonone h mansonone g stigmasterol ch3 h ch3 ch3 h h h oh ch3ch3 ch3 ch3 ch3 h ch3 oh h o oh h ch2 h ch3 ch3 o o phytosterol butulinic acid o-naphthoquinone figure 2. some compounds isolated from azanza garckeana. 94 biology, medicine, & natural product chemistry 9 (2), 2020: 91-95 effects of chemicals extracted from azanza garckeana mansonone e, c, g and h displayed antifungal activity against p. parasitica, with mansonone e showing the highest activity. also suggests potential mansonone e as a new natural pesticide for agricultural plant pathogen management. (mongkol1 and chavasiri, 2016). a computational and experimental study carried out on the anticancer ability of mansonone g by βcyclodextrin-based host-guest complexation revealed that the inclusion complex formation between mansone g and β-cyclodextrin was confirmed by dsc and sem techniques. notably, the mansone g/β-cyclodextrin inclusion complexes exerted significantly higher cytotoxic efect on a549 lung cancer cells than the uncomplexed mansonone g (mahalapbutr, 2019) anticancer activity, anti-hiv activity, antimalarial activity was recorded for betulinic acid, it also shows pronounced antinociceptive properties, antiinflammatory activity (moghaddam, 2012) the effect of the terpenoids gossypol, 6methoxygossypol, 6,60-dimethoxygossypol, gossypolone and apogossypolone on growth of fungal soil pathogens were investigated. gossypol, gossypolone and apogossypolone demonstrated strong growth inhibitory activity (≥90%) against pythium irregulare, pythium ultimum and fusarium oxysporum. these same terpenoids provided good growth inhibition against most rhizoctonia solani isolates. methylated gossypol derivatives generally yielded reduced growth inhibition against the tested fungi compared with gossypol. dose– response effects of gossypol, gossypolone and apogossypolone were determined over a concentration range (mellon, 2014). extracts from plants with high phytosterol (stigmasterol and β-sitosterol) content is used in the treatment of inflammatory conditions and prevention of cancers and cardiovascular diseases (ivanescu et al., 2013). sitosterols have been found to induce apoptosis when added to cultured human prostate, breast and colon cancer cells. therefore, they may play significant roles in the management and prevention of human cancers. beta-sitosterol preparation improved symptoms, increased peak urinary flow, and decreased post-void residual urine volume. however, relatively few controlled studies have examined the efficacy of phytosterol supplements in men with symptomatic bph. (ogbe, 2015). the plant has antifertility/contraceptive, antitumor properties (anticancer properties of gossypol against many types of cancer cell lines), antioxidant properties, antiparasitic properties, antivirus properties, antimicrobial properties, plasma cholesterol reduction properties. (keshmiri-neghab & goliaei, 2014). conclusion the various pharmacological activities of the chemicals extracted from the plant justifies the use of azanza garckeana as plant effective for various diseases and health conditions, especially sexually trelated issues, as exposed by various researches. the use of the plant for further research will help to make even drug discovery. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references dikko, y. j., khan, m. e., tor-anyiin, t. a., anyam, j. v. and linus, u. a. (2016). in vitro antimicrobial activity of fruit pulp extracts of azanza garckeana (f. hoffm.) exell and hillc. and isolation of one of its active principles, betulinic acid. br. j. pharm. res., 14: 1-10 maroy, a. (2017). azanza garckeana fruit tree: phytochemistry, pharmacology, nutritional and primary healthcare applications as herbal medicine: a review. res. j. med. plants, 11 (4): 115-123, 2017. musinguzi, e. l., kikafunda, j. k. and kiremire, b. t. (2007). promoting indigenous wild edible fruits to complement roots and tuber crops in alleviating vitamin a. deficiencies in uganda. proceedings of the 13th istrc symposium.; 763769. nkafamiya, i. i., modibbo, u. u., manji, a. j. and haggai, d. (2007). nutrient content of seeds of some wild plants. afr. j. biotech. 6(14):1665-1669. aberoumand, a. and deokule, s. s. (2009). studies on nutritional values of some wild edible plants from iran and india. pak. j. nutrition. 8(1): 26-31. nazarudeen, a. (2010). nutritional composition of some lesserknown fruits used by ethnic communities and local folks of kerela. ind. j. traditional knowl., 9(2): 398-402. food and agricultural organization (fao) (1983). food and fruit bearing forest species. examples from eastern africa. food and agricultural organization, forestry rome.14-16. nkafamiya, i. i., ardo, b. p., osemeahon s. a. and akinterinwa, a. (2016). evaluation of nutritional, non-nutritional, elemental content and amino acid profile of azanza garckeana (goron tula). british journal of applied science & technology, 12(6): 1-10. keshmiri-neghab, h. and goliaei, b. (2014). therapeutic potential of gossypol: an overview. pharmaceutical biology, 52(1): 124–128. https://doi.org/10.3109/13880209.2013.832776 mellon, j.e., dowd, m.k., beltz, s.b. and moore, g.g. (2014). growth inhibitory effects of gossypol and related compounds on fungal cotton root pathogens. letters in applied microbiology 59, 161-168. ogbe, r. j., ochalefu, d. o., mafulul, s. g. and olaniru, o. b. (2015). a review on dietary phytosterols: their occurrence, metabolism and health benefits. asian j. plant sci. res., 5(4):10-21. mongkol1, r. and chavasiri, w. (2016). antimicrobial, herbicidal and antifeedant activities of mansonone e from the bioltif et al. – a chemical overview of azanza garckeana 95 heartwoods of mansonia gagei drumm. journal of integrative agriculture 15(12): 2795–2802. moghaddam, m. g. ahmad, f. b. h. and samzadeh-kermani, a. (2012). biological activity of betulinic acid: a review. pharmacology & pharmacy, 3, 119-123. mahalapbutr, p., wonganan, p., charoenwongpaiboon, t., prousoontorn, m., chavasiri, w. and t. rungrotmongkol (2019). enhanced solubility and anticancer potential of mansonone g by β-cyclodextrin-based host-guest complexation: a computational and experimental study, biomolecules, 9: 1-17. doi:10.3390/biom9100545 ivanescu, b., vlase, l. and a. corciova (2013). importance of phytosterols and their determination in herbal medicines, the 4th ieee international conference on e-health and bioengineering, ochokwu, i. j., dasuki, a., & oshoke, j. o. (2015). azanza garckeana (goron tula) as an edible indigenous fruit in north eastern part of nigeria. journal of biology, agericulture and healthcare, 5(15), 26-31. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 63-71 | doi: 10.14421/biomedich.2024.131.63-71 issn 2540-9328 (online) effects of processing on the proximate composition, mineral content and the phytochemical analysis of groundnut seeds (arachis hypogeae) joseph adaviruku sanni1,*, grace omayoza sanni2, rufus ranmilowo awoniyi2, remi osanyinlusi2, yvonne ego richards3, goodness inioluwa adesina4, ore-ofe oluwatoyin adenuga5, stella ayomikun apata1, oluwafemi emmanuel ekun1 1department of biochemistry, faculty of science, adekunle ajasin university akungba-akoko, ondo state, nigeria. 2science laboratory department, rufus giwa polytechnic, owo, ondo state, nigeria. 3department of crop science, faculty of agriculture, ladoke akintola university of technology, ogbomoso oyo state, nigeria. 4department of biochemistry, faculty of science, obafemi awolowo university, ile ife, osun state, nigeria. 5department of medical microbiology, achievers university, owo, ondo state, nigeria. corresponding author* josephsanni59@gmail.com abstract effect of processing on the nutritional composition of groundnut seeds were carried out using standard analytical methods. raw, boiled and fried groundnut seeds were analyzed for proximate composition, mineral contents, and phytochemical screening. the result reported that the raw, boiled, and fried contains (5.357±0.190%, 4.545±0.050% and 3.896±0.015%, moisture contents), (2.401±0.011%, 3.225±0.004% and 2.816±0.001%, ash contents), (46.591±0.001%, 25.333±0.003% and 48.012±0.953%, crude fat), (4.126±0.887%, 15.001±0.030% and 7.692±0.002%, crude fibre), (19.520±0.040%, 21.580±0.040% and 23.540±0.000%, crude protein), (22.005±0.587%, 30.316±0.056% and 14.044±0.939%, carbohydrate) respectively. a significant difference was observed. processing has significant effects on the mineral components of the seeds. the results revealed that raw, boiled and fried contains (56.900mg/100g, 48.400mg/100g and 35.00mg/100g sodium), (0.215mg/100g, 0.185mg/100g and 0.540mg/100g zinc), (65.500mg/100g, 42.700mg/100g and 25.500mg/100g magnesium), (0.218mg/100g, 0.230mg/100g and 0.230mg/100g iron), (0.250mg/100g, 0.110mg/100g and 0.100mg/100g manganese). the phytochemical screening shows the presence of alkaloids, carbohydrates and proteins in all the samples and the result reveals that processing does not really have effect on phytochemical constituents. the investigation shows that fresh groundnut is a good source of mineral content, while raw and processed groundnut is a good source of some phytochemical constituents and processed groundnut is a good source of protein, fat, and carbohydrate with high nutritional value. keywords: arachis hypogaea; proximate composition; mineral contents; phytochemical screening. introduction groundnut or peanut scientifically known as arachis hypogaea, derived from two greek words arachis meaning legume and hypogaea meaning underground whose formation of pods is done in the soil are a widely cultivated crop that holds great significance in various aspects of human life (icar, 2002). belonging to the fabaceae family, it is a self-pollinating allotetraploid legume crop (janila et al., 2013). it is globally cultivated in both temperate and tropical regions; groundnut is a significant oilseed crop (ayoola and adeyeye, 2010). in sub-saharan africa, groundnut holds the distinction of being the most crucial monoecious annual legume, serving as a vital source of human food, forage, and income (alemayehu et al., 2014; ajeigbe et al. 2015). groundnut is a precious nutritional resource for human consumption, providing essential calories, dietary fiber, high energy value, protein, vital fatty acids, vitamins (such as vitamin e), minerals (including k, na, ca, mn, fe, and zn), as well as biologically active compounds (such as arginine, resveratrol, phytosterols, and flavonoids) (willett et al., 2019). in africa, particularly among rural households, zinc deficiency is a prevalent issue, particularly impacting infants and young individuals as one of the critical limiting micronutrients (wessells and brown, 2012). the significance of groundnut protein as a food and feed source is growing, particularly in developing nations where access to protein from animal sources is financially out of reach for a large portion of the population (arya et al., 2016). by fixing nitrogen in the soil, groundnut enhances soil fertility, leading to increased yields of other crops when employed in rotation or intercropping systems (ajeigbe et al., 2015). despite its nutritional value and positive impact on soil fertility, adoption of this crop remains lower in comparison (ahmed et al., 2016). manuscript received: 17 january, 2024. revision accepted: 08 may, 2024. published: 13 may, 2024. https://doi.org/10.14421/biomedich.2024.131.63-71 mailto:josephsanni59@gmail.com 64 biology, medicine, & natural product chemistry 13 (1), 2024: 63-71 groundnut has primarily been used commercially for the production of oil (kline, 2016; list, 2016; smithson et al., 2018; tu and wu, 2019). in addition to oil, peanut by-products contain numerous functional compounds such as proteins, fibers, polyphenols, antioxidants, vitamins, and minerals. these valuable components can be incorporated as functional ingredients in a wide range of processed foods (akgül and tozluoğlu, 2008; nepote et al., 2006; zhao et al., 2012). recent discoveries have highlighted groundnuts as an exceptional source of compounds such as resveratrol, phenolic acids, flavonoids, and phytosterols. these compounds have demonstrated the ability to hinder the absorption of dietary cholesterol (garcia et al., 2016; limmongkon et al., 2017; sebei et al., 2013). groundnut seeds boast a rich composition containing approximately 40-50% fat, 20-50% protein, 10-20% carbohydrates, as well as various vitamins and minerals. additionally, they offer a calorie content of 567 per 100 grams (ahmed et al., 2016). groundnut's combination of high energy value, protein, and minerals makes it a plentiful and costeffective source of nutrients. consuming groundnuts has been linked to numerous health benefits (kris-etherton et al. 2008; sabate et al., 2010; guasch-ferré et al., 2017). the seed of the groundnut is used in the production of oil, which serves various purposes such as cooking, salad dressings, and margarine. lower quality oils derived from groundnuts are employed in the manufacturing of soap (pradhan, 2011). groundnut seeds possess a wealth of nutritional value due to their abundant content of oil, protein, niacin, fiber, magnesium, vitamins, manganese, and phosphorus (davis and dean, 2016; fletcher and shi, 2016). on a dry seed basis, groundnut seeds can contain a substantial amount of oil ranging from 44 to 56%, and protein, ranging from 22 to 30%. they are also abundant in minerals such as phosphorus, calcium, magnesium, and potassium, as well as vitamins e, k, and various b group vitamins (arya et al., 2016). groundnut seed has a multitude of applications, both in its whole form or when processed into products such as peanut butter, oil, soups, stews, and more. additionally, the cake derived from groundnuts finds various uses in feed and infant food formulations (dhamsaniya et al., 2012; francisco and resurreccion, 2008; timbabadiya et al., 2017). enhancing the functionality of groundnut seed systems is crucial for improving the accessibility and adoption of improved varieties. well-established and efficient seed value chains play a vital role in ensuring timely and affordable delivery of superior crop varieties. they also facilitate effective planning of demand and supply, spanning from individual farms to national levels, which is essential for ensuring seed security (sperling, 2008; mcguire and sperling, 2016). the objective of this study is to determine the effect of processing on the proximate composition, mineral content, and phytochemical screening of raw arachis hypogaea (groundnut seeds) and the processed forms in order to know their possible usefulness as food rich in nutrients and also to ascertain the best form in which it should be consumed. materials and methods materials powdered (raw groundnut, boiled groundnut and fried groundnut), hcl, h2so4, naoh, weighing balance, filter paper, heating mantle, crucible, thread, beaker, conical flask, distilled water, reagent bottle, chloroform, water bath, acetic acid, and pipette. figure 1. pictorial representation of groundnut seeds (arachis hypogae). method sample preparation groundnut seeds (arachis hypogaea) were obtained from oja oba, owo local government area of ondo state and were then conveyed using a polythene bag to rufus giwa polytechnic chemistry laboratory. the seeds were divided into three equal parts, which comprised raw, boiled and fried groundnut. the first sample, which happens to be the raw sample, was sundried for about seven days and grounded to powder form using el850w blender then stored for further analysis. the second portion, which was unshelled groundnut seeds (arachis hypogaea) were washed thoroughly, using cold water until the water was clean. the nuts were soaked in water for some minutes before cooking. the cooking was done in a pot by adding 2g of salt in a liter of water and cooked for about 30 minutes. after cooking was finished, the water remaining in the pot was drained, the groundnut was dehulled, dried, milled and stored for sanni et al. – effects of processing on the proximate composition … 65 further analysis. to the third portion, which is the fried (arachis hypogaea) seeds. 2g of salt in 5 ml of water was sprinkled on the arachis hypogaea seeds and sundried for 2 hours and fried using a frying pan using sand at the base until the seeds turned golden brown. the arachis hypogaea seeds were cooled, dehulled, milled and stored in an airtight container for further analysis. proximate analysis fat content determination in the determination of the fat content, a clean fat-free filter paper was weighed (w1). five g of the sample was added to the filter paper and weighed (w2). the weighted sample was tied with a piece of thread and dropped into the thimble of the soxhlet apparatus. 250 ml of petroleum ether was poured into the round bottom flask of the apparatus. soxhlet apparatus was set up on the heating mantle and the extraction process was carried out for four hours to extract the fat with the help of the solvent. petroleum ether was siphoned over the barrel, the condenser was detached and the thimble was removed. the solvent extract (lipid) mixture was carefully poured into a clean dried petri dish and transferred into a fume cupboard for two hours. the solvent evaporated leaving behind the extracted fat. the filter paper containing the residue was dropped into a beaker and transferred into an oven at 50°c. it was then dried to constant weight and cooled in a desiccator and reweighed (w3). afterward, the percentage of fat was calculated. % 𝐹𝑎𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊2 − 𝑊3 𝑊2 − 𝑊1 × 100 moisture content determination the moisture content of the sample was determined using a drying method based on weight loss. a clean and dry crucible was weighed using a weighing balance, and its weight was recorded as (w1). samples were added to the empty crucible and weight (w2). the crucible containing the sample was then transferred into the oven, maintained at 105°c, and dried for four hours. the dish was then placed in a desiccator, cooled for an hour, and reweighed (w3). afterward, the percentage of moisture content was calculated. 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊2 − 𝑊3 𝑊2 − 𝑊1 × 100 ash content for the determination of ash content, a clean dried porcelain crucible was weighed and the weight was recorded (w1). 5g of the groundnut sample was measured into an empty crucible and reweighed as (w2). it was transferred into the muffle furnace. the muffle furnace was then ignited at 600 °c for about four hours until a grayish-white substance was obtained. the crucible was later transferred into a desiccator, cooled, and reweighed (w3). the percentage ash content was calculated. % 𝑎𝑠ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊2 − 𝑊3 𝑊3 − 𝑊1 × 100 crude-fiber content determination five g of the defatted sample was weighed (w1) into a 2500 ml conical flask. 200 ml of 1.25% h2so4 was added. the mixture was then heated for about 30 minutes, after which it was cooled and filtered through a poplin cloth by suction using a bunchier funnel. the residue was rinsed in hot distilled water and scraped back into a flask. 200 ml of 1.25% naoh was added, and the mixture was heated for 30 minutes. it was cooled, filtered, and washed once with hot distilled water, once with 10% hcl, four times with hot water, and twice with methylated spirit. the residue was drained, placed in a crucible, and dried in an oven at 105ºc. after drying in an oven has been completed, it was then cooled in a desiccator and weighed (w2). the crucible containing the residue was placed in a muffle furnace at 300°c for 30 min, placed in a desiccator to cool to room temperature, and re-weighed (w3). % 𝑐𝑟𝑢𝑑𝑒 𝑓𝑖𝑏𝑟𝑒 = 𝑊2 − 𝑊3 𝑊1 × 100 determination of protein content five g of the ground sample was weighed into a 50 ml kjeldahl flask containing 12.5 ml of concentrated h2so4 with one kjeldahl catalyst tablet. the flask was heated on low heat for about 15 minutes, on medium heat for 30 minutes, and then on high heat until digested. the flask was then rotated at intervals until a clear solution was observed and the heating continued for a few minutes to ensure complete digestion. the flask was allowed to cool, the sample residue was washed and filtered to make the digest up to 50 ml (v1). after the digestion was completed, 5 ml of 2% boric acid (h3bo3) was placed into a 100 ml conical flask, and 3 drops of the mixed indicator were added. the receiving flask was placed so that the tip of the condenser tube was below the surface of the boric acid. 5 ml of the digest (v2) was pipetted into the distillation tube, and 10 ml of 40% naoh was added. the heater was turned on and the distillation continued until approximately 50 ml of distillate was collected into the receiving flask. the distillate was titrated with 0.01m hcl, and the blank was titrated with the acid. %𝑁 = 𝑀 × 𝑇 × 0.014 𝑊 × 𝑉1 𝑉2 × 100 % 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = % 𝑁 𝑥 6.25 where 6.25 = conversion factor 66 biology, medicine, & natural product chemistry 13 (1), 2024: 63-71 carbohydrate content determination the term carbohydrates embrace a broad spectrum of compounds ranging from sample monosaccharides to complex polysaccharides. this carbohydrate content determination was done by subtracting other nutritional composition parameters (protein content, fat content, ash content, moisture content and fibre content) from 100. therefore, the carbohydrate content =100 – (protein + fat + ash + moisture + fibre). phytochemical screening ▪ test for tannins in the test for the presence of tannins, 0.30g of the sample was weighed into a test tube and boiled for 10 minutes in a water bath containing 30cm3 of water. after boiling, filtration was carried out using number 42 (125mm) whatman filter paper. after filtration, 3 drops of 0.1% ferric chloride solution was added to the filtrate. a brownish-green or blue-black coloration observed shows a positive test. (eikeme et al., 2009). ▪ test for phlobatannins 0.30g of the sample was weighed into a beaker containing 30 cm3 of distilled water. at the end of 24 hours of extraction, 10cm3 of each sample was boiled with 5cm3 of 1% aqueous hydrochloric acid. a deposit of a red precipitate showed a positive test (eikeme et al., 2009). ▪ test of saponin thirty cm3 of distilled water was added to 0.30g of the sample and boiled for 10 minutes in a water bath and filtered using whatman filter paper number 42 (125mm). the mixture of 5 cm3 of distilled water and 10cm3 of the filtrate was agitated vigorously for a stable persistent froth. the formation of emulsions in addition to three drops of olive oil showed positive results (eikeme et al., 2009). ▪ test for steroids 0.30 g of each sample was weighed into a beaker containing 20 cm3 of ethanol. the component was extracted for 2 hours. to the ethanol extract of each sample was added 2cm3 acetic anhydride followed by 2 cm3 of concentrated tetraoxosulphate (vi) acid. a violetblue or green colour change in the sample indicates the presence of steroids (eikeme et al., 2009). ▪ test for terpenoids 0.30g of each wood powder sample was weighed into a beaker and extracted with 30 cm3 of distilled water for 2 hours. 2 cm3 of a mixture of chloroform and 3 cm3 of concentrated tetraoxosulphate (vi) acid was introduced into 5cm3 of each extract to form a layer. a reddishbrown coloration at the interface shows positive results for the presence of terpenoids (eikeme et al., 2009). ▪ test for flavonoids each sample weighing 0.30g was introduced into a beaker, it was extracted with 30 cm3 of distilled water for about 2 hours and filtered with a whatman filter paper number 42 (125mm). 5cm3 of 1.0mol dilute solution was put into a test tube, followed by the addition of 5cm3 of concentrated tetraoxosulphate (vi) acid, and 10cm3 of the aqueous filtrate of each sample extract was added. the appearance of yellow colouration which disappeared on standing shows the presence of flavonoids. ▪ test for proteins one hundred mg of the extract was dissolved in 10 ml of distilled water and filtered through whatman no. 1 filter paper and the filtrate was subjected to a test for proteins. ▪ biuret test two ml of filtrate was treated with a drop of 2% copper (ii) sulphate solution. to the filtrate, 1 ml of ethanol (95%) was added followed by excess potassium hydroxide pellets. a pink-coloured ethanolic layer indicates the presence of protein (gahan et al., 1984). ▪ test for alkaloids mayer’s test two drops of mayer’s reagent were added to a few ml of the sample extract along the sides of the test tube. an appearance of white creamy colouration indicates the presence of alkaloids. (evans et al., 1997). wagner’s test a few drops of wagner’s reagent were added to a few ml of the plant extract along the sides of the test tube. the presence of a reddish-brown precipitate confirms the presence of alkaloids (wagner et al., 1993). ▪ test for glycosides fifty mg of the extract was hydrolyzed with concentrated hydrochloric acid for 2 hours in a water bath, filtered and the hydrolysate was subjected to the test. ▪ borntrager’s test three ml of chloroform was added to 2 ml of filtered hydrolysate and shaken, chloroform layer was separated and 10% ammonia solution was added to it. a pink colour indicates the presence of glycosides (evans et al., 1997). ▪ test for carbohydrates molisch's test two drops of alcoholic solution of αnaphthol were added to 2 ml of the sample extract in a test tube. the mixture was shaken well and a few drops of concentrated sulphuric acid was added slowly along the sides of the test tube. a violet ring colouration indicates the presence of carbohydrates. determination of mineral composition each sample was analyzed for mineral contents such as calcium, magnesium, sodium, potassium, and iron by instrumentation using an atomic absorption spectrophotometer (aas model; 2000). the method of aoac (1997) was applied for the determination of ash and mineral content. 2 grams of the ground samples were weighed using a weighing balance and placed in a sanni et al. – effects of processing on the proximate composition … 67 crucible, it was ignited in a muffle furnace for 4 hours at 550 c. it was then cooled in a desiccator and weighed at room temperature to obtain the weight of the ash. to the resulting ash, a mixture of concentrated nitric acid and hydrochloric acid in a ratio of 1:3 was added and made up to 100 ml with distilled water in a measuring cylinder. it was poured into a beaker and filtered then poured inside a sample bottle and maintained at room temperature. this solution was used for the determination of mineral content. an atomic absorption spectrophotometer (aas) was then used to determine the presence of mg, fe, mn, cu, ca, k, na, and zn. result proximate composition table 1. shows the type and quantity of the nutritional composition of raw, boiled and fried groundnut seeds. s/n compositions raw groundnut boiled groundnut fried groundnut 1 moisture content (%) 5.357±0.190 4.545±0.050 3.896±0.015 2 ash content (%) 2.401±0.011 3.225±0.004 2.816±0.001 3 crude fat (%) 46.591±0.001 25.333±0.003 48.012±0.953 4 crude fibre (%) 4.126±0.887 15.001±0.030 7.692±0.002 5 crude protein (%) 19.520±0.040 21.580±0.040 23.540±0.000 6 carbohydrate (%) 22.005±0.587 30.316±0.056 14.044±0.939 figure 2. graphical representation of the result of the proximate composition carried out on raw, boiled, and fried groundnut. mc = moisture contents; ac = ash contents; cfa = crude fat content; cfi = crude fibre content; cp = crude protein; cho = carbohydrate table 2. showing the results of metal (mg/100mg) analysis of raw groundnut, boiled groundnut, and fried groundnut. s/n minerals raw groundnut boiled groundnut fried groundnut 1 sodium(mg/100g) 56.900 48.400 35.00 2 zinc(mg/100g) 0.215 0.185 0.540 3 magnesium(mg/100g) 65.500 42.700 25.500 4 iron(mg/100g) 0.218 0.230 0.230 5 manganese(mg/100g) 0.250 0.110 0.100 figure 3. graphical representation of the result of the mineral composition of raw, boiled, and fried groundnut. 68 biology, medicine, & natural product chemistry 13 (1), 2024: 63-71 table 3. phytochemical screening results of raw groundnut, boiled groundnut, and fried groundnut. s/n phytochemicals raw groundnut boiled groundnut fried groundnut 1 alkaloids ++ + + 2 carbohydrates ++ ++ + 3 proteins ++ ++ ++ 4 phytosterols + 5 glycosides 6 saponins + + 7 tannins 8 phlobatannins 9 terpenoids 10 flavonoids + 11 steroids + ++ (very present), + (present), (absent). discussion proximate composition moisture content the result of the proximate composition of arachis hypogaea (groundnut seed) is shown in table 1. the moisture contents for the groundnut seeds raw groundnut, boiled groundnut, and fried groundnut were recorded to be 5.357%, 4.545%, and 3.896%. from the results, it was observed that raw groundnut has the highest moisture content when compared to others. i.e. the boiled and fried groundnut. the presence of high moisture contents in the raw groundnut is due to the fact that the groundnut has not undergone processing using heat as its source which could have reduced the moisture content. from the results, the high moisture content of the raw groundnut will have a lower shelf life which will influence the growth of microorganisms that could result in its spoilage (remi et al. 2023). considering the results, the moisture content is in agreement with (4.11% raw groundnut) and (3.56% roasted groundnut) which was reported by (kumar et al., 2013). the result was also in agreement with (5.41% raw groundnut) and (3.98% roasted groundnut) reported by (belete et al., 2020). from the result, we could infer that frying could reduce the moisture content in groundnut. ash content the ash contents of raw groundnut, boiled groundnut, and fried groundnut were recorded to be 2.401%, 3.225%, and 2.816%. the boiled groundnut was recorded to have the highest ash content, followed by fried, then raw groundnut. the result obtained was a little bit lower when compared to the result reported for raw and roasted groundnut (4.62%, 4.80%) by (belete et al., 2020). the result does not agree with the result reported by (et al., 2021) who reported the highest ash content in the fried groundnut followed by raw then boiled. the simple explanation could be due to species differences in the groundnut or environment where it was planted. crude fat crude fat, which is the total lipid content, was determined in the three samples of groundnut seeds raw groundnut, boiled groundnut, and fried groundnut to be 46.591%, 25.333%, and 48.012%. from the results, it was observed that both the raw and fried groundnut were higher in fat content when compared to the boiled groundnut. the reduction in fat content is in agreement with the work of (udo et al., 2021) who reported the fat content of raw, boiled, and fried groundnut seeds to be 39.32%, 32.76%, and 46.36%. the reduction in fat content of the boiled groundnut seed is also in agreement with the findings of (ayoola and adeyeye et al., 2010). the increase in the fat content of the fried groundnut seeds could be a result of an increased heating temperature which could have led to the cleavage of carbohydrate lipid or protein-lipid linkages which enables the easy absorption of fat (esenusan et al., 2008). considering the high lipid content, which is rich in monounsaturated fatty acids, consuming fried groundnuts will be beneficial to human health. crude fibre crude fibre is known to represent the contents of the non-digestible components of food, examples of which are, cellulose, lignin, and hemicellulose (remi et al., 2023). from the result, it was reported that the crude fibre for raw groundnut, boiled groundnut, and fried groundnut were 4.126%, 15.001%, and 7.692% with boiled groundnut having the highest crude fiber content. the crude fiber content was not in agreement with (udo et al., 2021) who reported the crude fibre of the fried groundnut to be the highest followed by raw groundnut. the simple explanation could be due to some factors like the intensity of the heat, the geographical location where the seed was harvested or possibly the presence, or absence of soil enhancers. crude protein the crude protein of the three samples was recorded to be 19.520% for raw groundnut, 21.580% for boiled sanni et al. – effects of processing on the proximate composition … 69 groundnut, and 23.540% for fried groundnut. from the results, fried groundnut was reported to have the highest crude protein value followed by the boiled groundnut and then the raw groundnut with the lowest crude protein value. this agrees with (rehman et al., 2005), who stated that boiling brings about a loss of structural components and a reduction of soluble proteins and fats content which leaches into the boiled water. it was observed that the result reported was almost the same as the result reported by (udo et al., 2021) for raw, boiled, and fried groundnut seeds (20.38%, 23.86%, and 25.64%). fried groundnuts have been shown to be a good source of crude protein which would be good for consumption and for human health. carbohydrate carbohydrate, an energy-giving food, was recorded as 22.005% in raw, 30.316% boiled, and 14.044% in fried groundnut. from the results, it was observed that the boiled groundnut seeds had the highest carbohydrate content when compared to the raw and fried groundnut. this result agrees with the result obtained by (udo et al., 2021) who reported the highest carbohydrate content in boiled groundnut but was in contrast to the result obtained by ayoola and adeyeye et al., 2010 who reported the highest carbohydrate content in roasted groundnut. mineral contents minerals are inorganic matter which cannot be destroyed by high heat and are needed by the body to develop and function normally. calcium, phosphorus, potassium, sodium, chloride, magnesium, iron, zinc, and iodine are some of the essential minerals needed for human health. table 2 reports the result of the mineral composition of groundnut (arachis hypogaea) seeds. the result shows a rapid decrease in the mineral contents of the processed seeds. this is in agreement with the research carried out on cashew nuts by (okonkwo et al., 2015) who reported a decrease in the mineral composition of the roasted cashew nut. however, this is contrary to the result of the study carried out by (udoh et al., 2021) who reported a significant increase in processed groundnut seeds. in the present study, it is important to note that only iron has an increased mineral content in both boiled and fried, which is in agreement with the observation of ayoola and adeyeye et al. 2010 who stated that roasted groundnut was more advantageous in mineral content than in raw groundnut. this present study is not completely in disagreement with other researchers but has similar results to a research carried out on bambara groundnut seeds by (ndidi et al., 2010) where all the mineral contents gradually decreased on processing. however, the values of mineral contents in this study for sodium, zinc, magnesium, and iron were way higher than that in bambara groundnut seeds. comparing this result, it is similar to (belete & bayissa, 2020) which stated that the concentration levels of metals in groundnut were found to decrease in the order of mg>na>zn>k>p>fe>ca. the only simple explanation for the difference as regards the decrease in mineral contents on processing to other studies and similarities to bambara groundnut may be due to multiple factors such as leaching of the minerals into the boiling water, geographical location, seasonal variation in the mineral contents of the soil, method of processing or the presence or absence of soil enhancers. phytochemical screening the three samples of arachis hypogaea were analyzed for their phytonutrients and it was reported that alkaloids, carbohydrates, and proteins were present in all the samples, saponins were observed to be found in the boiled and fried samples, phytosterols were observed in the boiled sample, flavonoid was found in the boiled sample and steroids was found to be present in the fried sample unlike glycosides, tannins, phlobatannins and terpenoids which were absent in all the samples. saponins are known for their property in precipitating and coagulating red blood cells. saponin is also known for its hemolytic activity, foams formation in aqueous solutions, and cholesterol-binding properties (sodipo et al., 2000), steroids are reported for their antibacterial properties (cowan, 1999; okwu, 2001) and alkaloids have also been reported for it analgesic, antispasmodic and antibacterial properties (okwu et al., 2004). from the result obtained in this study, it can be said that arachis hypogaea contain some phytochemical compounds which can serve medicinal purposes for human health. conclusion from the research, it has been established through the proximate screening of the processed seeds that arachis hypogaea can be used as a good source of fat, carbohydrate, and protein if properly treated and selected because of its high nutritional value and therapeutic potential. for the mineral contents, raw groundnut was observed to be the best because minerals play an important role in metabolic activities in the body. the results further revealed medicinally important phytochemical constituents in the groundnut seeds, which make them good for the management of diseases. this will aid in fighting diseases, malnutrition that would lead to better nutrition and good health in africa and the world at large. acknowledgements: we acknowledge mrs abubakar hawah ovai for her contribution towards the success of the manuscript. authors’ contributions: jas and gos conceptualized the study. jas, yer, gia, ooa, and saa assisted with data collection and discussion ro and rra assisted with 70 biology, medicine, & natural product chemistry 13 (1), 2024: 63-71 editorial work. oee supervised the project. the authors have read and approved the final manuscript. funding: the author(s) received no financial support for the research, authorship, and/or publication of this article availability of data and materials: not applicable ethics approval and consent to participate: not applicable consent for publication: not applicable. competing interests: all authors declare no competing interests. references ajeigbe, h. a., waliyar, f., echekwu, c. a., kunihya, a., motagi, b. n., eniaiyeju, d., & inuwa, a. 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(2017). genome-wide discovery of microsatellite markers from diploid progenitor species, arachis duranensis and a. ipaensis, and their application in cultivated peanuts (a. hypogaea). frontiers in plant science, 8, 1209. https://doi.org/10.3389/fpls.2017.01209 https://doi.org/10.1111/j.1365-2621.2004.tb13632.x https://doi.org/10.1016/s0140-6736(18)32521-2 https://doi.org/10.3390/foods7080122 http://dx.doi.org/10.12944/crnfsj.5.3.26 https://doi.org/10.1080/01496395.2019.1569691 https://doi.org/10.1111/j.1365-2621.2005.00978.x https://doi.org/10.1371/journal.pone.0050568 https://doi.org/10.3389/fpls.2017.01209 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1355-1363 | doi: 10.14421/biomedich.2025.142.1355-1363 issn 2540-9328 (online) development and evaluation of a stable topical cream formulated with annona squamosa seed extract as a natural pediculosis agent nur khairi1*, lukman muslimin2, fhahri mubarak2, maulita indrisari3, fajriansyah4 1department of pharmaceutical and technology, universitas almarisah madani, makassar, indonesia. 2department of pharmaceutical chemistry, univeritas almarisah madani, makassar, indonesia. 3department of pharmacotherapy, faculty of medicine, palangka raya university, palangkaraya, indonesia. 4department of pharmacology and clinic, universitas almarisah madani, makassar, indonesia. corresponding author* nurkhairijalil@gmail.com manuscript received: 14 october, 2025. revision accepted: 29 november, 2025. published: 14 december, 2025. abstract head lice infestation (pediculosis capitis) remains a global public health concern, exacerbated by growing resistance to conventional pediculicides such as permethrin and malathion. this study aimed to evaluate the pediculicidal activity and formulation stability of a topical cream containing annona squamosa (sugar apple or srikaya) seed extract as a natural alternative for treating pediculosis. the ethanolic extract of a. squamosa seeds was obtained through maceration, producing a 10.005% yield. pediculicidal assays were conducted using various extract concentrations (5%, 7.5%, and 10%), followed by formulation of oil-in-water creams with extract concentrations of 7.5%, 10%, and 12.5%. physical stability tests included assessments of viscosity, ph, spreadability, adhesion, and homogeneity. results showed a dose–response relationship, with lice mortality increasing from 60% at 5% extract to 87% at 10%. extract. the formulated creams demonstrated high efficacy—86% to 96% mortality—comparable to 1% permethrin. all formulations maintained acceptable physicochemical properties (ph 4.8–5.5, viscosity within 27,000–47,000 cps) and remained stable after accelerated storage. these findings indicate that a. squamosa seed extract is a potent pediculicidal agent that can be effectively incorporated into a stable topical formulation. the study supports the potential of a. squamosa as a safe, sustainable, and plant-based alternative for managing pediculosis while addressing the challenge of chemical resistance. keywords: annona squamosa; pediculosis capitis; topical cream formulation; head lice control. introduction pediculosis capitis, commonly referred to as head lice infestation, remains a significant public health concern, particularly among school-aged children. the world health organization (who) reports that the prevalence of head lice in developing countries ranges from 10-20% in children, with higher rates in rural areas and communities with poor sanitation. despite the use of conventional pediculicides such as permethrin and malathion, the increasing resistance of lice to these chemicals has reduced their effectiveness, prompting the need for alternative treatments. abbasi et al. (2022) and chen et al. (2025) emphasize this growing challenge, underscoring the importance of exploring plant-based solutions. recent studies suggest that plant-derived compounds can provide safer and more effective alternatives to conventional chemical treatments (chen et al., 2025). several plant extracts and essential oils have shown significant pediculicidal and ovicidal properties. for example, essential oils from tea tree, neem, and coconut have demonstrated insecticidal effects against lice and their eggs. research by candy et al., (2020) and shailajan et al. (2013) supports the effectiveness of plant extracts, such as those from ageratum conyzoides and cinnamomum porphyrium, in reducing lice populations. additionally, campli et al. (2012) found that combinations of tea tree oil and nerolidol were more effective than conventional treatments. these findings highlight the potential of plant-based remedies to address the challenge of lice resistance to traditional treatments. although physical agents like dimethicone have been explored, plant-based solutions generally offer greater efficacy and availability (burgess el al., 2013; kalari et al., 2019). the development of resistance to conventional pediculicides remains a key issue in managing pediculosis. diamantis et al. (2009) stress the need for alternatives that can avoid resistance development. plantbased treatments offer a diverse range of mechanisms of action. plants such as melia azedarach, ageratum conyzoides, and tinospora crispa have proven effective with lower resistance risk, acting through mechanisms including ovicidal effects and disruption of lice https://doi.org/10.14421/biomedich.2025.142.1355-1363 1356 biology, medicine, & natural product chemistry 14 (2), 2025: 1355-1363 metabolism (jayaseelan et al., 2011; toloza et al., 2010). these findings underscore the importance of conducting further research into ethnomedicinal plants for the treatment of pediculosis. annona squamosa l. (sugar apple or srijaya) is widely used in traditional medicine, especially in tropical and subtropical regions. the seeds and fruit of this plant have long been recognized for their therapeutic properties. previous studies have highlighted the antimicrobial potential of annona squamosa, with ethanol extracts from its leaves shown to be effective against pathogens such as staphylococcus aureus and escherichia coli (dewangga et al., 2019; goh et al., 2024). furthermore, its antioxidant and antiinflammatory properties make it a promising candidate for further exploration in the treatment of pediculosis (ibrahim el al., 2024). the seeds, traditionally used for treating infections, also exhibit significant therapeutic potential (maji, 2016). with concerns over the use of conventional chemicals leading to resistance and side effects, a topical cream formulation based on annona squamosa seed extract presents an appealing alternative. topical creams offer advantages such as ease of application, better absorption, and uniform distribution on the scalp. they can also enhance comfort and provide long-term protection against lice infestations (khairi et al., 2018). additionally, combining natural active ingredients in cream formulations can improve efficacy while reducing irritation (papa et al., 2023). given these benefits, a cream based on annona squamosa seed extract is expected to be a safer and more effective solution for managing pediculosis, particularly in the context of chemical resistance. the primary objective of this study is to evaluate the activity and effectiveness of a stable topical cream derived from annona squamosa seed extract as a potential treatment for pediculosis. additionally, the study will assess the stability of the cream. this research aims to fill a gap in the literature regarding annona squamosa's effectiveness as a pediculicidal agent, contributing to the search for alternative treatments to combat lice resistance. materials and methods materials used the materials used in this study include aluminum foil, stirring rods, a blender (philips®), petri dishes, measuring cylinders (iwaki®), beaker glasses (iwaki®), rotary evaporator (r-100 with cold trap buchi), analytical balance (fs-ar210 (int-cal)), cream containers, ph meter (laqua), dropper pipettes, filter paper, viscometer (brookfield®), homogenizer (wisestir®), and hot plate (dlab). the materials used in the study include srikaya seed extract, ethanol 96% (namaste), cetyl alcohol (intraco), dmdm hydantoin (intraco), isopropyl myristate (intraco), phenoxyethanol (intraco), cetrimonium chloride (almega), steareth-20 (almega), sodium metabisulfite (meta), distilled water (one med®), dmso (emsure®), adult head lice, and peditox®/permethrin 1% (pt. combiphar). sample extraction the powder from the srikaya seeds was extracted using the maceration method. the extraction was carried out with a ratio of 1:5, immersing 1 kg of srikaya seed powder in 5000 ml of 96% ethanol for three days. after immersion, the mixture was filtered, and the filtrate was collected. the residue was remacerated, and the extract was concentrated by evaporating the solvent using a vacuum rotary evaporator at 50°c until a thick extract was obtained. the extract was then air-dried until a dry extract was achieved (maji, 2016) antipediculosis activity test of srikaya seed extract the activity of the srikaya seed extract was tested using three concentrations: 5%, 7.5%, and 10%. the positive control used peditox/permethrin 1%, while the negative control used dmso. five petri dishes and filter paper sized to fit the petri dishes were prepared, ensuring the bottom of the petri dishes was completely covered by the filter paper. 1.5 ml of each concentration of the extract and the controls was applied evenly on the surface of the petri dishes, which were lined with filter paper. ten adult lice were placed in each petri dish. the movement of the lice was observed every 5 minutes for 2 hours (shailajan et al., 2013). the lice were examined under a magnifying glass, and any signs of vital life, such as antenna movement or leg movement, were considered indicators that the lice were alive. lice were considered dead if no vital signs were observed (torre et al., 2017). khairi et al. – development and evaluation of a stable topical cream formulated with … 1357 formulation of srikaya seed extract table 1. formulation of hair cream preparation. ingredient function concentration (% b/v) f0 f1 f2 f3 srikaya seed extract active ingredient 0 7,5 10 12,5 cetyl alcohol thickener 5 5 5 5 isopropyl myristate emolient 5 5 5 5 phenoxyethanol preservative 1 1 1 1 dmdm hydantoin preservative 0,5 0,5 0,5 0,5 cetrimonium chloride conditioning 4 4 4 4 steareth-20 emulsifier 2 2 2 2 sodium metabisulfite antioxidant 0,10 0,10 0,10 0,10 aquadest carrier ad 100 ad 100 ad 100 ad 100 f0: cream formulation without extract; f1: cream formulation with 7.5% extract concentration; f2: cream formulation with 10% extract concentration; f3: cream formulation with 12.5% extract concentration. preparation of topical cream formulation from srikaya seed extract each ingredient of the oil phase (cetyl alcohol, isopropyl myristate, steareth-20, and phenoxyethanol) was weighed and melted at a temperature of 75-80°c, starting from the highest melting point material. the water phase was heated to 75°c (dmdm hydantoin, sodium metabisulfite, cetrimonium chloride, and aquadest). after the oil phase was melted, it was added to the water phase and homogenized using a homogenizer until a cream base was formed (garcía et al., 2023; pinto et al., 2021). the srikaya seed extract was then added according to the formulation (f1, f2, f3, except f0 = formula without extract). the mixture was homogenized until uniform, then placed into containers and sealed. stability testing of the formulation the formulation was stored at 40°c for 24 hours. this experiment was conducted over six cycles, with evaluations conducted at the beginning and end of each cycle (nešić et al., 2019). the parameters assessed were as follows: ▪ organoleptic test: the organoleptic evaluation involved observing the appearance, color, and aroma of each formulation (humaira et al., 2025). ▪ homogeneity test: a sample of each cream formulation was applied to an object glass and examined for the presence of coarse particles or clumps. a good cream formulation should be free from coarse particles or clumps (tania et al., 2022). ▪ viscosity test: a 30 g sample of cream was measured using a brookfield viscometer with spindle number 64 set at 6 rpm. the ideal viscosity range for a good cream formulation is between 2,000-50,000 cps (wangpradit et al., 2022). ▪ ph test: a 30 g sample of cream was tested by immersing a ph meter into the cream, and the reading was taken after a brief waiting period. the optimal ph range for topical formulations is 4.5-6.5 (lyu, 2024). ▪ spreadability test: a 0.5 g sample of cream was placed between two glass plates. weights of 50 g, 100 g, and 200 g were applied sequentially, and the diameter of the spread was measured after 1 minute. the ideal spreadability value is between 5-7 cm (parlapanska, 2024). ▪ adhesion test: a 0.25 g sample of cream was placed on an object glass and covered with a glass slide. a 250 g weight was added for 5 minutes, and then the object glass was placed in the test apparatus. a 50 g weight was applied, and the time taken for the cream to detach from the glass was recorded (khairi et al., 2025). ▪ cream type test: the type of cream was determined by applying 1 g of the formulation evenly onto an object glass and adding methylene blue drops while stirring. if the methylene blue dissolved evenly, the cream was identified as an oil-in-water emulsion type (nur et al., 2025). effectiveness testing of srikaya seed extract topical cream as antipediculosis the effectiveness test aimed to ensure that the cream formulation produces the expected outcome as an antipediculosis treatment. for the procedure, five petri dishes were prepared, each lined with filter paper that completely covered the bottom of the petri dish. 1.5 ml of each cream formulation (f1, f2, f3), negative control (f0), and positive control (peditox/permethrin 1%) was applied evenly on the surface of the petri dish lined with filter paper. ten adult head lice were placed into each petri dish. the movement of the lice was observed every 5 minutes for a total duration of 2 hours (shailajan et al., 2013). the lice were examined under a magnifying glass, and any signs of vital activity, such as movement of the antennae or legs, were considered indicators that the lice were alive. lice were considered dead if no vital signs were observed (torre et al., 2017). 1358 biology, medicine, & natural product chemistry 14 (2), 2025: 1355-1363 results and discussion yield of annona squamosa seed extract the maceration extraction of annona squamosa seeds using 96% ethanol produced a yield of 10.005% (table 2). this result reflects a high extraction efficiency considering that ethanol is a semi-polar solvent capable of dissolving a wide range of bioactive compounds such as alkaloids, flavonoids, and acetogenins, which are abundant in a. squamosa seeds (maji, 2016). acetogenins, in particular, are known for their potent insecticidal properties due to their ability to inhibit mitochondrial electron transport, leading to energy metabolism disruption in insects (kazman j., 2022). table 2. yield percentage of annona squamosa seed extract. sample solvent weight of simplicia (gr) weight of extract (gr) yield (%) annona squamosa seeds ethanol 96% 1000 101.05 10.005 comparable yields (8–11%) have been reported by dewangga a., (2019) for ethanolic extracts of a. squamosa leaves, indicating that both leaves and seeds are promising raw materials for pharmaceutical applications. these findings reaffirm the suitability of ethanol as a safe and effective extraction solvent for isolating phytoconstituents from a. squamosa, aligning with the growing emphasis on green extraction technologies (chen et al., 2025). pediculicidal activity of annona squamosa seed extract the pediculicidal activity of a. squamosa seed extract exhibited a concentration-dependent increase in mortality of pediculus humanus capitis (table 3). the extract at 5% concentration produced 60% mortality, which increased to 80% at 7.5% and 87% at 10%. these results were significantly different (p < 0.05) from the negative control (0%) and approached the efficacy of the positive control, 1% permethrin (100%). table 3. pediculicidal activity of annona squamosa seed extract. sample mean heal lice mortility ± sd mortality (%) a. squamosa seed extract 5% 6.0 ± 1.0 60a a. squamosa seed extract 7.5% 8.0 ± 1.0 80b a. squamosa seed extract 10% 8.7 ± 0.6 87b positive control (+) 10.0 ± 0.0 100b negative control (-) 0.0 ± 0.0 0c a,b,c indicate significant differences according to lsd analysis (p<0,05,n=3). this strong pediculicidal activity supports the hypothesis that a. squamosa contains bioactive compounds with potent insecticidal mechanisms. the observed efficacy is likely due to acetogenins and alkaloids that interfere with mitochondrial function, causing respiratory arrest and ultimately leading to the death of lice (kazman j., 2022). flavonoids may also contribute to this activity by penetrating the cuticle and destabilizing membrane proteins. these findings are consistent with reports of other botanical pediculicides such as ageratum conyzoides and cinnamomum porphyrium, which demonstrated similar insecticidal properties (shailajan et al., 2013; candy et al., 2020). moreover, the extract’s performance parallels the combination of tea tree oil and nerolidol, which achieved 90% mortality in resistant lice populations (campli et al., 2012). the similarity in efficacy underscores the potential of a. squamosa as a viable botanical alternative to neurotoxic pediculicides that are increasingly losing effectiveness due to resistance (abbasi et al., 2022; chen et al., 2025). physicochemical characterization of the cream formulation organoleptic and homogeneity evaluation the organoleptic analysis revealed stable physical characteristics across all formulations before and after accelerated storage (table 4). the color ranged from white (f0) to light brown and brown (f1–f3), with a characteristic herbal odor corresponding to the extract concentration. all formulations were semi-solid and homogeneous, with no visible aggregates (table 5). khairi et al. – development and evaluation of a stable topical cream formulated with … 1359 table 4. organoleptic observations before and after accelerated storage. formula replicate before accelerated storage after accelerated storage color odor consistency color odor consistency f0 r1 white odorless semisolid white odorless semisolid r2 white odorless semisolid white odorless semisolid r3 white odorless semisolid white odorless semisolid f1 r1 light brown charecteristic semisolid light brown charecteristic semisolid r2 light brown charecteristic semisolid light brown charecteristic semisolid r3 light brown charecteristic semisolid light brown charecteristic semisolid f2 r1 light brown charecteristic semisolid light brown charecteristic semisolid r2 light brown charecteristic semisolid light brown charecteristic semisolid r3 light brown charecteristic semisolid light brown charecteristic semisolid f3 r1 brown charecteristic semisolid brown charecteristic semisolid r2 brown charecteristic semisolid brown charecteristic semisolid r3 brown charecteristic semisolid brown charecteristic semisolid f0: cream formulation without extract; f1: cream formulation with 7.5% extract concentration; f2: cream formulation with 10% extract concentration; f3: cream formulation with 12.5% extract concentration. such consistency reflects proper emulsification and dispersion of active ingredients. the color stability also indicates minimal oxidative degradation of phenolic compounds during storage, confirming good chemical stability (pinto et al., 2021). homogeneity is particularly critical for ensuring dose uniformity and user acceptability in topical applications (parvanescu et al., 2025). table 5. homogeneity test results before and after accelerated storage. formula replicate before accelerated storage after accelerated storage f0 r1 homogeneous homogeneous r2 homogeneous homogeneous r3 homogeneous homogeneous f1 r1 homogeneous homogeneous r2 homogeneous homogeneous r3 homogeneous homogeneous f2 r1 homogeneous homogeneous r2 homogeneous homogeneous r3 homogeneous homogeneous f3 r1 homogeneous homogeneous r2 homogeneous homogeneous r3 homogeneous homogeneous f0: cream formulation without extract; f1: cream formulation with 7.5% extract concentration; f2: cream formulation with 10% extract concentration; f3: cream formulation with 12.5% extract concentration. viscosity viscosity values increased with higher extract concentrations: from 27,500 cps (f0) to 47,666 cps (f3) before storage (table 6). after accelerated storage, viscosity slightly decreased but remained within the acceptable range (28,166–41,333 cps). according to wangpradit et al. (2022), an ideal viscosity range for cosmetic creams is 2,000–50,000 cps, confirming the formulations’ suitability. table 6. viscosity measurement results before and after accelerated storage. formula mean ± sd before storage (cps) after storage (cps) f0 27500 ± 0.25 28166 ± 0.20 f1 29500 ± 0.30 28500 ± 0.25 f2 40000 ± 0.28 34000 ± 0.23 f3 47666 ± 0.55 41333 ± 0.50 f0: cream formulation without extract; f1: cream formulation with 7.5% extract concentration; f2: cream formulation with 10% extract concentration; f3: cream formulation with 12.5% extract concentration. 1360 biology, medicine, & natural product chemistry 14 (2), 2025: 1355-1363 the increased viscosity in formulations containing extract suggests molecular interactions between polyphenols and emulsion components, reinforcing the internal structure of the cream (naeimifar et al., 2023). the minor reduction in viscosity after storage (<10%) indicates strong thermodynamic stability of the oil-inwater (o/w) emulsion, which was maintained by the presence of steareth-20 and cetyl alcohol as emulsifier and stabilizer, respectively (guzmán et al., 2022; sharkawy et al., 2020). ph stability the ph values of all formulations ranged from 4.8 to 5.5 (table 7), which remained within the physiological range for scalp compatibility (4.5–6.5). slight decreases in ph after storage are attributed to mild oxidation of phenolic compounds, a common occurrence in natural formulations (saleem et al., 2022). table 7. ph measurement results before and after accelerated storage. formula mean ± sd before storage before storage f0 5.49 ± 0.15 4.91 ± 0.07 f1 4.87 ± 0.08 4.82 ± 0.06 f2 4.98 ± 0.06 4.89 ± 0.08 f3 5.11 ± 0.08 5.01 ± 0.08 f0: cream formulation without extract; f1: cream formulation with 7.5% extract concentration; f2: cream formulation with 10% extract concentration; f3: cream formulation with 12.5% extract concentration a stable ph is essential to ensure the chemical integrity of the active compounds and user comfort, thereby preventing irritation or alteration of the skin microbiota (namjoshi et al., 2020). the maintained ph in all extract-containing creams demonstrates that the buffer system and formulation design effectively mitigated degradation reactions. spreadability and adhesion spreadability and adhesion tests (tables 8 and 9) revealed satisfactory results across all formulations. adhesion values ranged between 97–99 seconds, indicating sufficient retention on the scalp surface for therapeutic action. spreadability values of 5.3–5.7 cm were consistent with the optimal standard (5–7 cm) for easy application and uniform distribution (parlapanska, 2024). table 8. adhesion test results before and after accelerated storage. formula mean ± sd before storage (s) before storage (s) f0 97.67 ± 1.53 98.33 ± 1.15 f1 98.33 ± 1.15 98.67 ± 0.58 f2 98.67 ± 0.58 99 ± 0.0 f3 98.33 ± 1.15 99 ± 0.0 f0: cream formulation without extract; f1: cream formulation with 7.5% extract concentration; f2: cream formulation with 10% extract concentration; f3: cream formulation with 12.5% extract concentration the inverse relationship between viscosity and spreadability, as observed here, aligns with rheological principles of pseudoplastic flow typical of topical emulsions (hemalatha et al., 2022). these findings suggest that the cream’s texture remains acceptable even with increased extract content, ensuring both efficacy and user compliance. table 9. spreadability test results before and after accelerated storage. formula mean ± sd before storage (cm) before storage (cm) f0 5.2 ± 0.025 5.70 ± 0.07 f1 5.53 ± 0.03 5.59 ± 0.06 f2 5.45 ± 0.025 5.52 ± 0.08 f3 5.34 ± 0.04 5.47 ± 0.08 f0: cream formulation without extract; f1: cream formulation with 7.5% extract concentration; f2: cream formulation with 10% extract concentration; f3: cream formulation with 12.5% extract concentration cream type the dye test confirmed all formulations as oil-in-water (o/w) type before and after storage (table 10). o/w creams are preferred for pediculicidal applications due to their light texture, ease of rinsing, and lower risk of scalp irritation (namjoshi et al., 2020). this emulsion type also enhances the release and absorption of hydrophobic active compounds, supporting the delivery efficiency of acetogenins and flavonoids from the a. squamosa extract (garcía et al., 2023). table 10. cream type determination before and after accelerate storage. formula replicate before accelerated storage after accelerated storage f0 r1 oil-in-water (o/w) oil-in-water (o/w) r2 oil-in-water (o/w) oil-in-water (o/w) r3 oil-in-water (o/w) oil-in-water (o/w) f1 r1 oil-in-water (o/w) oil-in-water (o/w) r2 oil-in-water (o/w) oil-in-water (o/w) r3 oil-in-water (o/w) oil-in-water (o/w) f2 r1 oil-in-water (o/w) oil-in-water (o/w) r2 oil-in-water (o/w) oil-in-water (o/w) r3 oil-in-water (o/w) oil-in-water (o/w) f3 r1 oil-in-water (o/w) oil-in-water (o/w) r2 oil-in-water (o/w) oil-in-water (o/w) r3 oil-in-water (o/w) oil-in-water (o/w) f0: cream formulation without extract; f1: cream formulation with 7.5% extract concentration; f2: cream formulation with 10% extract concentration; f3: cream formulation with 12.5% extract concentration. khairi et al. – development and evaluation of a stable topical cream formulated with … 1361 effectiveness of the annona squamosa seed cream against head lice the topical cream containing a. squamosa extract exhibited strong pediculicidal efficacy, increasing with extract concentration (table 11). f0 (placebo) showed no mortality, while f1 (7.5%) caused 86%, f2 (10%) 93%, and f3 (12.5%) 96% mortality, comparable to permethrin (100%). statistical analysis confirmed significant differences (p < 0.05) between the negative control and all extract-containing formulations. table 11. effectiveness test results of annona squamosa seed extract. formula mean head lice mortality ± sd mortality (%) f0 0.0 ± 0.0 0a f1 8.67 ± 0.58 86b f2 9.33 ± 0.58 93b f3 9.67 ± 0.58 96b positive control (+) 10 ± 0.0 100b a,b indicate significant differences according to lsd analysis (p<0,05,n=3) f0: cream formulation without extract; f1: cream formulation with 7.5% extract concentration; f2: cream formulation with 10% extract concentration; f3: cream formulation with 12.5% extract concentration these results verify that the bioactive compounds remain stable and effective after formulation. the primary mechanism is likely mitochondrial respiration inhibition by acetogenins, resulting in paralysis and death of lice (kazman j., 2022). this finding is consistent with previous reports on other plant-based insecticidal agents such as melia azedarach and tea tree oil (rossini et al., 2007; toloza et al., 2010). in comparison with herbal mixtures such as illicium verum and coconut oil (armiyanti et al., 2020), the a. squamosa-based cream demonstrates equivalent or superior efficacy while offering greater formulation stability. furthermore, its physicochemical robustness across multiple parameters (ph, viscosity, spreadability) aligns with the principles of quality by design (qbd), ensuring predictable performance and safety (namjoshi et al., 2020). the study thus establishes the a. squamosa seed cream as a competitive botanical alternative to conventional pediculicides. its dual functionality— pediculicidal and antimicrobial—further enhances its therapeutic potential by reducing secondary scalp infections (dewangga a., 2019; goh c., 2024). the present findings contribute to the global discourse on sustainable, plant-based approaches to managing pediculosis. unlike neurotoxic pediculicides such as permethrin and malathion, which face escalating resistance issues (abbasi et al., 2022; burgess et al., 2013), plant-derived formulations act through multiple biochemical pathways, making resistance less likely (jayaseelan, 2011). the successful formulation of a stable and effective cream exemplifies how ethnomedicinal knowledge can be integrated into modern pharmaceutical design under the quality target product profile (qtpp) framework. future studies should investigate nanoscale delivery systems—such as nanoemulsions or pickering emulsions—to enhance bioavailability and stability (guzmán et al., 2022; mascarenhas‐melo et al., 2023). furthermore, the strong antioxidant profile of a. squamosa may complement its pediculicidal activity by mitigating oxidative stress on the scalp, promoting scalp health and recovery (ibrahim et al., 2024; ma et al., 2017). this multifaceted therapeutic profile underscores the potential of a. squamosa for developing nextgeneration herbal pediculicidal formulations. conclusions this study demonstrates that annona squamosa seed extract is an effective pediculicidal agent that remains active and stable when formulated into a topical cream. the extraction process yielded 10.005%, indicating the presence of sufficient bioactive constituents for product development. biological assays revealed a clear dose– response relationship, with head lice mortality increasing from 60% at 5% concentration to 80% at 7.5% and 87% at 10%, showing significant differences compared with the negative control. once formulated, the cream exhibited high pediculicidal activity—86% at 7.5%, 93% at 10%, and 96% at 12.5%—approaching the efficacy of the standard permethrin (100%). from a formulation standpoint, the cream maintained acceptable physicochemical characteristics after accelerated storage. its viscosity remained within the ideal range with minimal reduction, ph values (4.8–5.5) were compatible with scalp physiology, adhesion ranged from 97–99 seconds, and spreadability (approximately 5.3–5.7 cm) supported ease of application. all formulations were confirmed to be oil-in-water emulsions, making them suitable for therapeutic scalp use and easy rinsing. these findings suggest that a. squamosa-based cream represents a promising natural alternative for addressing pediculicide resistance while providing a safe and stable topical formulation. the study contributes to existing knowledge by demonstrating consistent efficacy and formulation stability of a. squamosa seed extract in a topical delivery system. future research should include controlled clinical trials in target populations, evaluation of ovicidal activity, repeated-use safety assessments, long-term stability studies, and optimization of delivery systems such as nanoemulsions to enhance bioavailability. acknowledgements: the authors would like to express their highest gratitude to allah swt for his blessings, guidance, and strength throughout the completion of this research. without his mercy and will, this study would not have been successfully accomplished. the authors also extend sincere appreciation to the department of pharmacy, faculty of health sciences, and the 1362 biology, medicine, & natural product chemistry 14 (2), 2025: 1355-1363 supporting laboratory staff for their invaluable technical assistance and research facilities provided during this work. institute of research and community service (lppm) universitas almarisah madani, which greatly contributed to providing laboratory access and research materials. the authors further acknowledge the contribution of all laboratory assistants and student researchers involved in the preparation and testing of annona squamosa seed extract formulations. no external financial support, industrial sponsorship, or corporate funding influenced the outcomes of this study. the authors declare no conflicts of interest related to the publication of this paper. authors’ contributions: concept – n.k., m.i.; 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(2022). determination of required hydrophilic-lipophilic balance of amesiodendron chinense (merr.) hu oil and development of stable cream formulation. ocl, 29, 29. https://doi.org/10.1051/ocl/2022011 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 541-547 | doi: 10.14421/biomedich.2024.132.541-547 issn 2540-9328 (online) in vitro mucolytic activity of cardamom fruit (amomum compactum) decoction on duck egg albumens desy aryanti pardilla vitri1,*, siti rahmatul aini2, legis ocktaviana saputri3 1department of pharmacy, faculty of medicine and health science, universitas mataram 2department of pharmacist professional education, faculty of medicine and health science, universitas mataram 3department of pharmacology, faculty of medicine and health science, universitas mataram jl. majapahit no. 62, mataram 83125, west nusa tenggara, indonesia. corresponding author* desyaryantipardillavitri15@gmail.com manuscript received: 26 agustus, 2024. revision accepted: 10 october, 2024. published: 31 october, 2024. abstract cardamom (amomum compactum) is empirically used as a cough medicine by using its seeds and fruits. cardamom seeds have been shown to have mucolytic activity at a concentration of 0.8%, while cardamom fruit has not been studied. this study aimed to determine the mucolytic activity of cardamom fruit decoction at variation concentrations. mucolytic activity tests were carried out in vitro on duck egg albumens at 3%, 6%, and 12% variation concentrations of cardamom fruit decoction. n-acetylcysteine 0.1% was positive control, while phosphate buffer and tween 80 were negative controls. the mucolytic activity was determined based on the viscosity value measured by flow time using an ostwald viscometer and density measurement using a pycnometer. data from each group were analyzed using spss with parametric one-way anova and post hoc tests. the results showed that cardamom fruit decoction at concentrations of 3%, 6%, and 12% had values that were not significantly different from n-acetylcysteine (p>0.05) and there was no significant difference (p>0.05) between each concentration group. accordingly, the research concludes that cardamom fruit decoction at concentrations of 3%, 6%, and 12% has mucolytic activity comparable to n-acetylcysteine. keywords: cardamom fruit; decoction; duck egg albumens; in vitro; mucolytic. abbreviations: acute respiratory infections (aris); n-acetylcysteine (nac). introduction acute respiratory infections (aris) are infectious diseases that cause the highest mortality rates worldwide. each year, nearly 4,000,000 people die from aris, with the highest mortality rates observed among infants, children, and the elderly, particularly in developing countries, necessitating special attention (who, 2020). according to the national basic health research report 2018, the incidence of aris in indonesia reached 1,017,290 cases (riskesdas, 2018). one of the primary diagnoses in ari patients is airway hypersecretion, characterized by coughing and increased sputum production (susiami & mubin, 2022; fretes et al., 2020). coughing is a defense mechanism to expel harmful antigens from the respiratory tract (baratawihaya, 2010; pakadang et al., 2020). antigens entering the respiratory tract get trapped and increase mucus production, leading to thicker mucus (utama, 2018; kurniawati et al., 2020). mucus can obstruct airflow and cause complications of other diseases (saminan, 2016). the viscosity of mucus can be reduced by mucolytic drugs, which can break down mucus, making it less viscous and more accessible to expel through coughing (wahyuningtyas et al., 2016). one such mucolytic drug is n-acetylcysteine (nac), known for its strong ability to dissolve mucus (liu et al., 2020). nac has a rapid onset, reaching maximum plasma concentration within half an hour (dawson et al., 1989 in mahmoudi et al., 2015; ferdinan et al., 2023). however, conventional drugs like nac have side effects, including nausea, vomiting, diarrhea, epigastric pain, constipation, headaches, urticaria, and skin rashes (adil et al., 2018). the most common side effects are nausea and vomiting, experienced by 23% of 145 patients taking nac (bebarta et al., 2010 in tenório et al., 2021). consequently, people are turning to traditional medicine, considered safer (sari et al., 2019). cardamom (amomum compactum) is a plant commonly used as a spice but also used empirically by people as traditional medicine. cardamom contains essential oils about 2-4% (with active compounds like cineole, borneol, limonene, and alpha-terpineol acetate) (ernawati et al., 2022). according to nofriyaldi et al. (2023), ethanol extract of cardamom fruit contains https://doi.org/10.14421/biomedich.2024.132.541-547 mailto:desyaryantipardillavitri15@gmail.com 542 biology, medicine, & natural product chemistry 13 (2), 2024: 541-547 secondary metabolites such as tannins, alkaloids, saponins, and flavonoids. empirically, cardamom is used as a traditional medicine for coughing (hariana, 2013). people use cardamom for cough medicine using its seeds and fruits. cardamom seeds are used empirically for cough medicine by chewing and swallowing the juice (latief, 2009 in syaputra et al., 2021). additionally, the seeds have been proven to has mucolytic activity in vitro. according to syaputra et al. (2021), ethanol extract syrup from cardamom seeds have mucolytic activity comparable to acetylcysteine at a concentration of 0.8%. cardamom fruit is used empirically for cough medicine by boiling 6 grams in 200 ml of water for 15 minutes (hariana, 2013). however, using cardamom fruit as a cough medicine remedy has yet to be studied, necessitating further research into its mucolytic activity. the mucolytic activity of cardamom fruit decoction can be tested in vitro using duck egg albumens. duck egg albumens were chosen for the test method because they contain mucoproteins that can be broken down by compounds with mucolytic activity, thereby reducing their viscosity (bragg & haough, 1961 in desiyana et al., 2021). additionally, in vitro tests using duck egg albumens are simpler than bovine intestines (desiyana et al., 2021). obtaining duck egg albumens involves slowly separating the white from the yolk, whereas bovine intestines need to be cleaned of adhering waste, split longitudinally, and scraped to obliterate mucus (sari et al., 2019). therefore, this study aimed to determine the mucolytic activity of cardamom fruit (amomum compactum) decoction in vitro on duck egg albumens. materials and methods materials the materials used include aquadest, cardamom fruits, di-sodium hydrogen phosphate dehydrate 0.1 n, fecl3 1%, concentrated hcl, hcl 2 n, magnesium (mg) powder, wagner’s reagent, dragendorff’s reagent, mayer's reagent, n-acetylcysteine capsule, duck egg, sodium dihydrogen phosphate monohydrate 0.1 n, tween 80. the equipment used includes laboratory glassware, stir bar, bulb pipette, blue tip, food dehydrator (freeze dryer), hot plate, filter paper, volumetric flask, micropipette, oven, dropper pipette, wooden clamp, ruler, pycnometer, ph meter, tube rack, stopwatch, spatula, analytical balance, ostwald viscometer, water bath, yellow tip. procedures sample preparation separated the cardamom fruits from the bunches and washed with running water until cleaned and drained. the clean cardamom fruits were dried in a food dehydrator at 40℃ for 12 hours. the dried cardamom fruits were sorted and blended. we weighed six grams of dried cardamom fruits, then boiled in 200 ml of distilled water at a temperature of 90℃ for 15 minutes (hariana, 2013). figure 1. the cardamom plants (amomum compactum) on sapit (documented by the author). phytochemical screening flavonoid test five ml of the sample was heated in a water bath for 5 minutes, then 2-4 drops of concentrated hcl were added, and 0.2 g of magnesium (mg) powder was added (fadilah et al., 2018). flavonoid positivity is characterized by a color change to black, reddish, yellow, or orange (rumagit et al., 2015). alkaloid test nine ml of the sample was added with 1 ml of 2 n hcl in a test tube, and then the sample was heated over a water bath for 2 minutes. the solution is then allowed to cool. the sample solution was divided into 3 test tubes, each added with mayer, wagner, and dragendorff reagents. positive results are indicated by the formation of a white precipitate in the sample treated with mayer's reagent, a reddish brown precipitate in the sample treated with wagner's reagent, and the formation of a yellow precipitate in the sample treated with dragendorff's reagent (depkes ri, 1980; raaman, 2006). saponin test one ml of the sample was added to 10 ml of distilled water, then heated over a water bath for 2 minutes, cooled, and shaken vigorously for 10 seconds. if the foam is formed that lasts for 10 minutes with a height of 1-10 cm and if one drop of 2 n hcl is added, the foam does not disappear; then the sample is positive for containing saponin (depkes, 1989). vitri et al. – mucolytic activity of cardamom fruit decoction 543 tannin test two ml of the sample was added with 2-4 drops of 1% fecl3. positive tannin is characterized by a change in color to dark blue, blackish blue, or greenish black (fadilah et al., 2018). preparation of ph 7 phosphate buffer 4.449 grams of di-sodium hydrogen phosphate dehydrate (na2hpo4.2h2o) 0.1 n was dissolved in 100 ml of distilled water. the solution was put into a 250 ml measuring flask, and distilled water was added to the mark. a total of 3.449 grams of 0.1 n sodium dihydrogen phosphate monohydrate (nah2po4.h2o) was dissolved in 100 ml of distilled water. the solution was put into a 250 ml measuring flask, and distilled water was added to the mark. a total of 244.4 ml of 0.1 n na2hpo4.2h2o was added with 155.6 ml of 0.1 n nah2po4.h2o, then stirred until homogeneous and measured with a ph meter (mulyono, 2009). preparation of duck egg albumens 20%w/w duck eggs were taken in panjisari village, praya district, central lombok regency. the duck eggs used are three days old. mucus is obtained by carefully separating the egg albumens and egg yolk. a total of 20 grams of mucus (the albumens part of a duck egg) was added with phosphate buffer ph 7 until the weight reached 100 g to obtain a concentration of 20% w/w, then stirred until homogeneous (sutoyo et al., 2020). preparation of control and test solutions ▪ negative control to reach a weight of 30 g, 0.15 g of tween 80 was added with 20% w/w duck egg albumens. the mixture was then stirred until homogeneous (sutoyo et al., 2020). ▪ positive control 0.1 g of n-acetylcysteine was weighed, then dissolved in 100 ml of distilled water and stirred until homogeneous to obtain a concentration of 0.1%. 0.03 g of nacetylcysteine 0.1% was added to 0.15 g of tween 80, and duck egg albumens 20% w/w to 30 g. the mixture was stirred until it was homogeneous (nerdy & manurung, 2018). ▪ test solution twelve grams of cardamom fruit was boiled in 100 ml of distilled water at a temperature of 90℃ for 15 minutes to obtain a concentration of 12%w/v. boiled cardamom fruit with a concentration of 12% was then diluted to a concentration of 6% w/v and 3% w/v. a total of 0.15 g of each test concentration was added with 0.15 g of tween 80 and 20% w/w duck egg albumens until it reached a weight of 30 g and stirred until homogeneous (sulistanti et al., 2022; sutoyo et al., 2020) ▪ mucolytic activity assay each group (negative control, positive control, and test solution) was incubated for 30 minutes at 37℃. the sample flow time was measured using an ostwald viscometer, and the sample density was measured using a pycnometer. each measurement was carried out three times with new samples, and the mucolytic activity test was replicated two times. the results obtained were then processed to obtain the viscosity value, which was then averaged. 𝑆𝑎𝑚𝑝𝑙𝑒 𝑣𝑖𝑠𝑐𝑜𝑠𝑖𝑡𝑦 = 𝑆𝑎𝑚𝑝𝑙𝑒 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑥 𝑠𝑎𝑚𝑝𝑙𝑒 𝑓𝑙𝑜𝑤 𝑡𝑖𝑚𝑒 𝑊𝑎𝑡𝑒𝑟 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑥 𝑤𝑎𝑡𝑒𝑟 𝑓𝑙𝑜𝑤 𝑡𝑖𝑚𝑒 𝑥 0,89 (𝑐𝑃) data analysis viscosity data were analyzed using the spss 26 version. data normality was tested with shapiro-wilk, and homogeneity was tested using levene statistics. parametric analysis was continued with one-way anova (p < 0.05), followed by post-hoc analysis using lsd to determine the significance between treatments. results and discussion cardamom fruit (amomum compactum) decoction the yield of cardamom fruit simplicia obtained was 27.83%. cardamom fruit simplicia was extracted using the boiling method. this method was chosen because people commonly consume medicines derived from plants or tubers. (anggraini et al., 2020). besides that, boiling is a method suitable for the empirical use of cardamom fruit as a cough medicine. based on empirical use, 6 grams of cardamom fruit was boiled in 200 ml of distilled water for 15 minutes (hariana, 2013). the results of boiling cardamom fruit are shown in figure 2. figure 2. results of cardamom fruit (amomum compactum) decoction (documented by the author). phytochemical screening the qualitative phytochemical screening test results showed that the cardamom fruit decoction contained flavonoids and tannins, as shown in table 1. the decoction of cardamom fruit contained damaging alkaloid compounds because alkaline alkaloids generally 544 biology, medicine, & natural product chemistry 13 (2), 2024: 541-547 dissolve in relatively non-polar organic solvents and are difficult to dissolve in water, whereas the extraction process of cardamom fruit decoction uses distilled water solvent, which has very polar properties (endarini, 2016; fitriyanti et al., 2022). nofriyaldi et al. (2022) also showed negative results of phytochemical screening for alkaloid compounds in simplicia and 96% ethanol extract of cardamom fruit. decoction of cardamom fruit contains negative saponins because saponins are heat resistant up to a temperature of 70℃, whereas the process of boiling cardamom fruit uses a temperature of 90℃ (kemenkes, 2017; wahyuni et al., 2018). therefore, it causes the saponins to be degraded. table 1. phytochemical screening results of cardamom fruit (amomum compactum) decoction. test compounds results interpretation of results documentation flavonoids the solution turns orange + alkaloids no sediment no sediment no sediment saponins the foam formed does not reach a height of 1-10 cm tannins the solution turns to a greenish-black color + mucolytic activity test cardamom fruit (amomum compactum) decoction was tested for mucolytic activity in vitro on duck egg albumens. duck egg albumens was chosen as the medium because it contains a mucolytic agent similar to human mucus, namely ovomucin (glycoprotein) (chaiyasit et al., 2019). when the test was carried out, duck egg albumens were mixed with phosphate buffer ph 7, which aimed to adapt to human mucus conditions (henke & ratjen, 2007 in ladeska et al., 2020). the next stage was the addition of tween 80, which functions as a wetting agent to reduce the surface tension between the phosphate buffer and egg albumens so that they can mix (rowe et al., 2009). before the test, the sample was incubated for 30 minutes at 37℃ to create conditions that resemble human physiological conditions (windriyati et al., 2019). the mucolytic activity test was carried out with an ostwald viscometer because duck egg albumens, used as the test medium, had a water content of around 86.19% ± 0.10% (chaiyasit et al., 2019). ostwald viscometers are generally used to measure the viscosity of newtonian fluids such as water (martin et al., 2016; spurk and aksel, 2008 in tiwow, 2015). in this study, nacetylcysteine (nac) was a positive control because its mechanism of action can break disulfide bonds in crosslinked mucus glycoproteins (mucin) so that the viscosity can decrease (raghu et al., 2020). based on the viscosity data shown in table 2, the viscosity value of the positive control and cardamom fruit decoction at variation concentrations of 3%, 6%, vitri et al. – mucolytic activity of cardamom fruit decoction 545 and 12% decreased compared to the negative control. the negative control has the highest viscosity value. when viewed from the average viscosity value between test solutions, the higher the concentration of cardamom fruit decoction, the lower the viscosity value. table 2. results of viscosity values. sample trial viscosity value i ii iii negative control 1 1,4375 1,4101 1,3949 2 1,4644 1,3838 1,3559 3 1,5001 1,3559 1,4115 average ± sd 1,4673 ± 0,0314 1,3833 ± 0,0271 1,3874 ± 0,0285 positive control 1 1,0505 1,0666 1,1498 2 1,0114 1,0457 1,0496 3 1,1336 1,0847 1,0677 average ± sd 1,0652 ± 0,0624 1,0657 ± 0,0195 1,0890 ± 0,0534 decoction 3% 1 1,1437 1,1570 1,1875 2 1,1198 1,0635 1,1219 3 1,1336 1,1209 1,1581 average ± sd 1,1452 ± 0,0262 1,1138 ± 0,0472 1,1559 ± 0,0329 decoction 6% 1 1,0884 1,0847 1,1687 2 1,1028 1,0989 1,0677 3 1,1732 1,0858 1,1219 average ± sd 1,1214 ± 0,0454 1,0898 ± 0,0079 1,1194 ± 0,0506 decoction 12% 1 1,0321 1,0847 1,1121 2 1,0837 1,0635 1,0857 3 1,1347 1,0666 1,1219 average ± sd 1,0835 ± 0,0513 1,0716 ± 0,0115 1,1066 ± 0,0187 viscosity data was analyzed using the spss 26 version. the normality of the data was tested with shapiro-wilk and the significance results (p>0.05) showed that the viscosity data was normally distributed. homogeneity was tested using levene statistics, with significant results (p>0.05) indicating that the data was homogeneous. after the normality and homogeneity requirements were met, the analysis was continued with the one-way anova parametric test, which produced a significant value (p<0.05) (dahlan, 2010). significant differences between each sample group were analyzed using post-hoc (lsd) with a 95% confidence level, as shown in table 3. table 3. results of post-hoc analysis (lsd) of test group viscosity values. comparison groups significant value 1 2 3 nc-pc 0,000 0,000 0,000 nc-d3% 0,000 0,000 0,000 nc-d6% 0,000 0,000 0,000 nc-d12% 0,000 0,000 0,000 pc-d3% 0,056*) 0,051*) 0,063*) pc-d6% 0,159*) 0,292*) 0,364*) pc-d12% 0,631*) 0,790*) 0,595*) d3%-d6% 0,536*) 0,295*) 0,281*) d3%-d12% 0,126*) 0,080*) 0,154*) d6%-d12% 0,329*) 0,421*) 0,696*) note: *) not significantly different (p>0.05); nc (negative control); pc (positive control); d3% (decoction 3%); d6% (decoction 6%); d12% (decoction 12%). based on the post-hoc (lsd) results in table 3, the positive control and negative control have a significance value (p<0.05), which indicates the viscosity values between the positive control and negative control were significantly different. that means the positive control shows mucolytic activity while the negative control does not. cardamom fruit decoction at concentrations of 3%, 6%, and 12% had a significance value (p>0.05) when compared with the positive control but a significance value (p<0.05) when compared with the negative control. the results show that the cardamom fruit decoction with concentrations of 3%, 6%, and 12% has mucolytic activity equivalent to the positive control. fruit decoction had a significant value (p>0.05) between variation concentrations. these results indicate no significant difference between each variation of concentrations of cardamom fruit decoction or that the mucolytic activity was comparable. the results of this research can attest to the empirical use of cardamom fruit decoction. flavonoids, alkaloids, saponins, and tannins are secondary metabolite compounds in plants with mucolytic effects (wati, 2017; kurniawati et al., 2020). however, this study's phytochemical screening of cardamom fruit decoction was positive for flavonoids and tannins. flavonoids have mucolytic activity by breaking down mucoprotein and mucopolysaccharide fibers in mucus. wahyuningtyas et al. (2016) showed that matteucinol was a flavonoid isolate with mucolytic activity. meanwhile, as astringents, tannins have mucolytic activity, which can 546 biology, medicine, & natural product chemistry 13 (2), 2024: 541-547 shrink the mucous membrane (mucus) in the intestines (wati, 2017). humans have two mucus: bound mucus (intestines) and secreted mucus (respiratory tract). both mucus have the same characteristics, but most of the mucus secreted was much higher than those bound to the membrane (abrami et al., 2024). as for the connection with tannins, which can shrink the mucous membrane (mucus) in the intestines, it could have the same effect on mucus in the respiratory tract because both have the same characteristics. thus, the mucolytic activity produced by the cardamom fruit decoction comes from these secondary metabolite compounds. n-acetylcysteine was a mucolytic substance with a mechanism of action comparable to flavonoids, which breaks the mucoprotein threads of the mucosa. 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(2019). aktivitas mukolitik in vitro ekstrak etabolik herba meniran (phyllanthus niruri l) terhadap mukosa usus sapi. jurnal ilmu farmasi & farmasi klinik, 3, 94–100. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 323-326 | doi: 10.14421/biomedich.2025.141.323-326 issn 2540-9328 (online) isolation and characterization of constituents from the extracts of momordica angustisepala aerial parts abdulmumeen amao hamid1,2,*, godshelp osas egharevba3,4,**, shuab bolaji issa1,5, oluwafeyikemi mercy olalere1, bukunmi temitope akinloye1, yakub tunde yakub1 1department of chemistry, university of ilorin, kwara state, nigeria. 2department of pharmaceutical chemistry and pharmacognosy, school of pharmacy, kampala international university, western campus, uganda. 3chemistry unit, landmark university, omu aran, kwara state, nigeria. 4indian institute of chemical technology (iict), hyderaba, india. 5department of materials science and engineering, norwegian university of science and technology, trondheim, norway. corresponding author hamid.aa@unilorin.edu.ng; hamid@kiu.ac.ug; hamidmemo@gmail.com*, egharevb.godshelp@lmu.edu.ng; godhelpeghas@gmail.com** manuscript received: 07 march, 2025. revision accepted: 20 may, 2025. published: 30 june, 2025. abstract n-hexane, ethyl acetate and methanol extracts of momordica angustisepala were screened for the presence of metabolites, using column and thin-layer chromatographic techniques. octadecane (1), tetradec-5-enoic acid (2), 1,2,3-propanetriyl (8z,11’z,14”z)tris(-8,11,14eicostrienoate) (3), methyl oleana-5,12,15-trienoate-3β-acetate (4) and 1,2,3-propanetriyl (9z,12’z,15”z)tris(-9,12,15-uneicostrienooate) (5) are the compounds obtained from the extracts of m. angustisepala aerial parts using proton and 13c nuclear magnetic resonance spectroscopy (nmr). keywords: momordica angustisepala aerial parts; 1,2,3-propanetriyl (8z,11’z,14”z)tris(-8,11.14-eicostrienoate); 1,2,3-propanetriyl (9z,12’z,15”z)tris(-9,12,15-uneicostrienooate); methyl oleana-5,12,15-trienoate-3β-acetate; proton and carbon-13 nuclear magnetic resonance spectroscopy; column chromatography; thin-layer chromatography. introduction momordica angustisepala belongs to the family cucurbitaceae and is a large climbing plant with stout stems attaching themselves into the surrounding vegetation by means of tendrils. the plant, also known as “ejirin nla” in yoruba and “bitter gourd” in english, is mostly found in west tropical africa (such as nigeria, côte d’ivoire, and cameroon), mostly in deciduous or semi-deciduous forest. it can also be found at the road side, old plantation, or filthy areas. m. angustisepala is used in making sponges due to its fibre-forming polymeric activity (biswas et al., 2011). this plant is used in herbal medicine to treat tumors, malaria and diabetes, and as abortifacient to guard against unwanted pregnancy (burkill, 1997; shehu et al., 2019). despite the ethnomedicinal uses of this plant, less attention has been paid to its exploitation. this study was designed to isolate (using chromatography) and to characterize the constituents of m. angustisepala extracts using 1h and 13c nuclear magnetic resonance (nmr) spectroscopy. materials and methods plant materials fresh aerial parts of m. angustisepala were collected in ilorin, kwara state, nigeria. the plants were identified and authenticated at the department of plant biology, faculty of life sciences, university of ilorin, ilorin, nigeria by mr bolu ajayi (a botanist), and voucher specimens of the plants were deposited in the herbarium of the department. extraction and isolation procedures momordica angustisepala aerial parts were air-dried for three weeks and ground into powdery form with the machine. the weights obtained after grinding were 530.51 g. the ground sample was soaked with n-hexane, ethyl acetate and methanol successively for 10 days each in the order of their polarity, for complete extraction. the extracts were filtered separately with whatmann no1 filter paper and concentrated at 45 oc using rotatory evaporator and then freeze-dried to remove trace solvents. the dried extracts were subjected to column chromatography to obtain pure isolates, and thin layer chromatography and recrystallization were carried out on the isolates to affirm their purity. https://doi.org/10.14421/biomedich.2025.141.323-326 mailto:hamidmemo@gmail.com* 324 biology, medicine, & natural product chemistry 14 (1), 2025: 323-326 extraction of m. angustisepala aerial parts with nhexane, ethyl acetate and methanol gave 22 g, 11.8 g and 5 g extracts respectively. the methanol extract was not reckoned with due to its small quantity. the ethyl acetate-hexane extract (33.8 g) of momordica angustisepala aerial parts (maeh) was pre-adsorbed on silica gel (60-200 mesh size) (60 g) to form powdered homogeneous slurry. the slurry was then subjected to column chromatography, packed using the slurry method (silica gel 700 g, 60-120 mesh size) and eluted with various solvent systems, starting with 100% of hexane; then using hexane: chcl3 (49:1, 100 ml); (19:1, 100 ml); (23:2, 100 ml); (9:1, 100 ml); (17:3, 100 ml); (4:1, 100 ml); (3:1, 100 ml); (7:3, 100 ml); (3:2, 100 ml); (11:9, 100 ml); and (1:1, 100 ml) separately. a total of 75 fractions (100 ml each) were collected and pooled to five pure fractions, coded maeh-1–maeh-5, based on tlc analysis, with masses 20 mg, 15 mg, 24 mg, 15 mg, and 42 mg respectively; and found to be a pure compound each on characterization using 1h nmr and 13c nmr. characterization of octadecane, c18h38, white solid (20 mg); 1h nmr (cdcl3, 300 mhz): δ 0.88 (6h, t, 2 × ch3), 1.10–1.28 (32h, m, 16 × ch2); 13c nmr (cdcl3, 75 mhz): δ 14.25 (c-1), 29.62 (c-2–16), 31.8 (c-17), 22.68 (c-18). characterization of tetradec-5-emoic acid, c14h25o, yellow viscous oily liquid (15 mg); 1h nmr (cdcl3, 300 mhz): δ 1.85–2.40 (4h, h-2 (t) & h-3 (m)), 1.48– 1.68 (4h, m, h-4 & 7), 5.11–5.36 (2h, m, h-5 & 6), 1.14– 1.41 (12h, m, h-8–13), 0.89 (3h, t, h-14); 13c nmr (cdcl3, 75 mhz): δ 182.05 (c=o, c-1), 29.73 (c-3 & 4), 137.50 (c-5), 128.00 (c-6), 31.95 (c-7), 39.40 (c-8), 37.28 (c-9), 32.83 (c-10), 22.72 (c-11), 19.78 (c-12), 16.04 (c-13), 14.15 (c-14). characterization of 1,2,3-propanetriyl (8z,11’z,14”z)tris(-8,11,14-eicostrienoate), c63h101o6, creamy solid (24 mg); 1h nmr (cdcl3, 300 mhz): δ 0.86–0.88 (9h, t, 3 × ch3), 1.25–1.41 (3 × 6h, m, 3 × 3ch2), 1.94–2.45 (3 × 18h, m, 3 x 9ch2), 4.89–6.19 (3 × 6h, m, 3 × 6ch), 3.43–4.28 (4h, d, 2 × och2 & 1h, m, och); 13c nmr (cdcl3, 75 mhz): δ 173.37 (c=o, c-1), 29.29 (c-2–7), 139.53 (c-8), 132.56 (c-9), 52.03 (c10), 131.73 (c-11), 129.72 (c-12), 37.18 (c-13), 127.93 (c14), 125.30 (c-15), 34.07 (c-16), 31.94 (c-17), 24.87 (c-18), 23.96 (c-19), 14.14 (c-20), 62.12 (och2 of fatty triester, c-1), 68.93 (och of fatty triester, c-2), 65.06 (och2 of fatty triester, c-3). characterization of methyl oleana-5,12,15-trienoate3β-acetate, c33h48o4, yellow solid (15 mg); 1h nmr (cdcl3, 300 mhz): δ 1.06–1.40 (10h, h-1, h-21 & 22 (t); h-2 (m); h-19 (d) 5 × r2ch2,), 1.63–1.79 (4h, h-7 (d) & h-11 (q), 2 × r2ch2), 1.48–1.55 (2h, t, h-9 & h-18, 2 × r3ch); 5.08–5.25 (1h, t, h-6, r2c=chr), 5.30–5.35 (1h, t, h-12, rch=cr2), 5.65–6.50 (2h, d, h-15 &16, rch=chr), 0.81–0.91 (21h, s, h-23–27, 29 & 30, 7 × rch3), 4.5 (1h, t, h-3, o-chr2), 2.05–2.37 (3h, s, roocch3), 3.62 (3h, s, och3); 13c nmr (cdcl3, 75 mhz): δ 39.74 (c-1 & 2), 51.45 (c-3), 29.14 (c-4), 125.03 (c-5), 119.37 (c-6), 29.72 (c-7), 32.22 (c-8), 27.22 (c-9), 29.54 (c-10), 22.71 (c-11), 124.25 (c-12), 142.35 (c-13), 29.54 (c-14), 130.05 (c-15), 135.22 (c-16), 29.37 (c-17), 31.94 (c-18), 29.14 (c-19), 29.54 (c-20), 26.43 (c-21), 25.71 (c-22), 25.71 (c-23), 16.02 (c-24), 14.14 (c-25), 22.65 (c-26), 23.45 (c-27), 174.33 (c=o, c-28), 29.14 (c29), 24.96 (c-30), 51.45 (roocch3), 60.90 (och3). characterization of 1,2,3-propanetriyl (9z,12’z,15’’z)tris(-9,12,15-uneicostrienoate), c66h107o6, creamy solid (42 mg). 1h nmr (cdcl3, 300 mhz): δ 1.83–2.42 (42h, h-2 (t); h-3-8 (m), 3 × 7r2ch2), 4.56–6.46 (18h, m, h-9, 10, 12, 13, 15 & 16, 3 × 3rch=chr), 1.25 – 1.75 (36h, m, h-11, 14 & 17, 3 × 3rch=ch-ch2 & h-18–20, 3 x 3r2ch2), 0.88–0.97 (9h, t, 3 × 3rch3), 3.41–3.94 (4h, d, 2 × och2 & 1h, m, och); 13c nmr (cdcl3, 75 mhz): δ 173.07 (c=o, c1), 29.70 (c-2–8), 135.10 (c-9), 132.81 (c-10), 39.72 (c-11), 130.13 (c-12), 128.77 (c-13), 34.15 (c-14), 125.90 (c-15), 124.24 (c-16), 32.75 (c-17), 27.78 (c-18), 26.41 (c-19), 24.85 (c-20), 14.11 (c-21), 62.07 (2 × o-ch2r of fatty triester c-1 & 3), 68.89 (o-chr2 of fatty triester, c-2) results and discussion characterization of the isolated compounds the isolates from the plant extracts were characterized using 1h and 13c nuclear magnetic resonance (nmr) spectroscopy. the 1h and 13c nmr analyses on the isolates obtained from the ethyl acetate-hexane extracts of m. angustisepala aerial parts gave compounds 1–5. the structures of these compounds are shown in figure 1. compound 1 (20 mg) was isolated as white crystals. the 1h nmr spectrum showed peaks at δh 0.88 (6h, t, h-1 and h-18) corresponding to the terminal methyl hydrogen atoms. the peak at δh 1.10–1.28 was assigned to the cluster of methylene (ch2) hydrogen atoms at 2–17 positions. the 13c nmr spectrum displayed eighteen carbon resonances and was sorted out to contain sixteen methylene carbons (δc 29.62–31.8) and two methyl carbons (δc 14.25 (c-1) and 22.68 (c-18)). the spectroscopic data of compound 1 were similar to those reported on octadecane in the literature (lide & milne, 1994). hence, the structure of compound 1 was established as octadecane (figure 1). hamid et al. – isolation and characterization of constituents from the extracts … 325 figure 1. octadecane. compound 2 (15 mg) was obtained as a light yellow viscous oily liquid. the 1h nmr spectrum showed signals at δh 0.89 (3h, t, h-14) corresponding to the terminal methyl hydrogen atoms. the multiplet peaks at δh 1.14–1.41 were attributed to the cluster of methylene hydrogen atoms (ch2) at 8–13 positions. allylic hydrogen atoms (-ch=ch-ch2) were shown by δh 1.48– 1.68 at 4 and 7 positions. the signal δh 1.85–2.40 corresponds to the αand βcarbon hydrogen atoms to the carbonyl at positions 2 and 3 respectively. the peaks at δh 5.11 – 5.36 represent the olefinic hydrogen atoms (ch=ch) at positions 5 and 6. the 13c nmr spectrum indicated fourteen carbon resonances and was sorted out as two olefinic carbons (δc 128.0, c-6– 137.5, c-5); one carbonyl carbon (δc 182.05, c-1); ten methylene carbons (δc 19.78–39.40); and one methyl carbon (δc 14.15, c-14). the spectroscopic data of compound 2 were similar to those reported on tetradec-5-enoic acid in the literature (ogunleye et al., 1991; lie ken jie & lam, 1995). therefore, compound 2 was established as tetradec-5enoic acid (figure 2). figure 2. tetradec-5-enoic acid. compound 3 (24 mg) was isolated as a creamy solid. the 1h nmr spectrum signals at δh 0.86–0.88 (9h, t, 3 × ch3) correspond to the cluster of methyl hydrogen atoms. the peaks at δh 1.25–1.41 shows the cluster of methylene hydrogen atoms; δh 1.94–2.45 corresponds to the cluster of allylic hydrogen atoms and methylene hydrogen atoms immediate to the carbonyl; δh 3.43–4.28 indicates oxymethylene hydrogen atoms; and δh 4.89– 6.19 shows the olefinic hydrogen atoms. the 13c nmr spectrum indicated 23 resonances corresponding to 63 carbons with the carbons assigned positions 1–20 occurring three times. the peak at δc 173.37 represents carbonyl carbon; δc 62.12–65.06 shows oxymethylene carbons; δc 125.30–139.53 indicates olefinic carbons; δc 22.71–52.03 represents the methylene carbons; and the peak at δc 14.14 corresponds to the methyl carbon. the spectroscopic data of compound 3 were similar to those reported on 1,2,3-propanetriyl (8z,11’z,14”z)tris(8,11,14-eicostrienoate) in the literature (hamid et al., 2017; lie ken jie & lam, 1995). hence, the structure of compound 3 was established as 1,2,3-propanetriyl (8z,11’z,14”z)tris(-8,11,14-eicostrienoate (figure 3). figure 3. 1,2,3-propanetriyl (8z,11'z,14"z)tris(-8,11,14-eicostrienoate. compound 4 (15 mg) was isolated as a yellow solid. the 1h nmr spectrum signals at δh 5.08–6.5 correspond to olefinic hydrogen atoms. the peaks δh 0.81–0.91 correspond to the cluster of methyl hydrogen atoms. the peaks at δh 3.62 and 4.5 show the oxymethine hydrogen atoms; δh 2.05–2.37 corresponds to αhydrogen atoms to the carbonyl. the peaks at δh 1.06–1.55 corresponds to the cluster of methylene hydrogen atoms. the 13c nmr spectrum indicated 31 resonances. the peaks at δc 14.14, 16.02, 22.65, 23.45, 24.19, 29.14 correspond to methyl carbons. the signals at δc 51.45 and 60.90 represent the oxymethine carbons. δc 119.37–142.35 correspond to the olefinic carbons. the spectroscopic data of compound 4 were similar to those reported on oleana-12,15-diene and oleana-5,12-diene in the literature (bhattacharyya & cunja, 1992). therefore, the structure of compound 4 was established as methyl oleana-5,12,15-trienoate-3βacetate (figure 4). figure 4. methyl oleana-5,12,15-trienoate-3βacetate. compound 5 (42 mg) was obtained as a cream solid. the 1h nmr spectrum signals at δh 0.88–0.97 correspond to the methyl hydrogen atoms. the peaks at δh 1.25–2.42 correspond to the cluster of methylene hydrogen atoms. the signals at δh 4.56–6.46 indicate the olefinic hydrogen atoms and δh 3.41–3.94 represent the oxymethylene hydrogen atoms. the 13c nmr spectrum indicated 24 resonances, equivalent to 67 carbons in the compound; with carbon-assignment 1–20 occurring three times. the peak at δc 14.11 shows the methyl carbons; δc 173.07 indicates the carbonyl carbon. the olefinic carbons are shown by δc 124.24–135.10; the oxymethylene carbons are represented by δc 62.07–68.89; 326 biology, medicine, & natural product chemistry 14 (1), 2025: 323-326 and δc 24.85–39.72 corresponds to the methylene carbons. the spectroscopic data of compound 5 were similar to those reported on 1,2,3-propanetriyl (9z,12’z,15’’z)tris(-9,12,15-uneicostrienoate) in the literature (hamid et al., 2017; chuah et al., 2006; lie ken jie & lam, 1995). hence, the structure of compound 5 was established as 1,2,3-propanetriyl (9z,12’z,15’’z)tris(-9,12,15-uneicostrienoate) (figure 5). figure 5. 1,2,3-propanetriyl (9z,12'z,15''z)tris(-9,12,15-uneicostrienoate) conclusion the authors appreciated mr. bolu ajayi, department of plant biology laboratory, university of ilorin for collection of plant and deposition at the herbarium. competing interests: the author declares that there are no competing interests. references biswas, s., kindo, s. and patnaik, a. (2011), effect of fiber length on mechanical behaviour of coir fiber reinforced epoxy composites, fiber polymer, 12: 73–78 burkill, h. m. (1997), the useful plants of west tropical africa, vol. iv, families m-r. royal botanical gardens, kew 8–9. chuah, t. g., rozanna, d., salmiah, y., thomas choong, s., sa’ari, m. (2006), fatty acids used as phase change materials (pcms) for thermal energy storage in building material applications, university putra malaysia hamid, a.a., aiyelaagbe, o.o., kaneez, f., luqman, s., negi, a.s. (2017). isolation, characterisation and antiproliferative evaluation of constituents from stem extracts of alafia barteri, medicinal chemistry research, 26(12): 3407-3416, springer publisher, doi: 10.1007/s00044-017-2033-4. lide, d. r., milne, g. w. a. (1994), handbook of data on organic compounds 4th ed. crc press, inc. boca raton, fl. 4: 3643 lie ken jie, m. s. f., lam c. c. (1995), 1h-nuclear magnetic resonance spectroscopic studies of saturated, acetylenic and ethylene triacylglycerols. chem phys lipids, 77: 155-171 ogunleye, a., fakoya, a. t., niizeki s., tojo h., sasajima i., kobayashi m., tateishi s., yamaguchi k. (1991), fatty acid composition of breast milk from nigerian and japanese women. j nutr sci vitaminol (tokyo), 37 (4): 35–42 shehu, a. salami, l. b. gbadamasi, a. a., issa, s. b., aliyu, m. a., egharevba, g., adisa, m. j., bale, m. i., hamid, a. a. (2019), chemical composition from the leaf extracts of momordica angustisepala with its antibacterial, antifungal and antioxidant activities. nigerian journal of chemical research, 24 (2). n-hexane, ethyl acetate and methanol extracts of momordica angustisepala indicated the presence of metabolites: triterpenoids, fatty acids and esters. octadecane (1), tetradec-5-enoic acid (2), 1,2,3propanetriyl (8z,11’z,14”z)tris(-8,11.14-eicostrienoate) (3), methyl oleana-5,12,15-trienoate-3β-acetate (4) and 1,2,3-propanetriyl (9z,12’z,15”z)tris(-9,12,15uneicostrienooate) (5) were isolated from the ethyl acetate-hexane extracts of m. angustisepala aerial parts. these compounds are being reported for the first time from this plant. acknowledgement: authors acknowledged indian institute of chemical technology (csir-iict) is highly appreciated for providing the facilities for this research. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 441-450 | doi: 10.14421/biomedich.2023.122.441-450 issn 2540-9328 (online) alkaloids lead to potential inhibition of the acyl carrier protein reductase to attenuate tuberculosis; an in-silico analysis pernia kamran, ahsan ibrahim* shifa college of pharmaceutical sciences, shifa tameer-e-millat university, islamabad-44000, tel. +9251-8438061, pakistan. corresponding author* ahsan.scps@stmu.edu.pk abstract tuberculosis (tb) is a contagious infection that mostly affects the lungs. mycobacterium tuberculosis causes tuberculosis infection, leading to granulomatous lesions in affected lung tissue. it is one of the most prevalent and deadly infectious diseases among the under developed countries. this study aims to investigate the possible inhibition of the acyl carrier protein reductase for preventing tuberculosis by well-known alkaloids, thereby reducing mycobacterium tuberculosis growth in the lungs and thereby reducing the incidence of latent and active tb. about five natural alkaloids were subjected to the molecular docking analysis, which produced favorable findings in terms of best pose and binding energies of these compounds towards the active residues of mycobacterial acp reductase, with values ranging from -10 kcal/mol to -9.1 kcal/mol. the molecular dynamics simulation produced similar encouraging results. all of the prospective alkaloid compounds were subjected to an in-silico toxicity investigation, which determined that every compound was safe and non-toxic. further studies may be necessary for effective formulation development employing these compounds as part of the process of drug discovery and development. the findings from this study may be helpful in the development of the novel nanoformulations using natural products for pharmacotherapy of tuberculosis infection. keywords: tuberculosis; alkaloids; acp reductase; molecular docking analysis; mycobacterium tuberculosis. abbreviations: mtb: mycobacterium tuberculosis, covid-19: corona virus disease 19, hiv: human immunodeficiency viruses, aids: acquired immunodeficiency syndrome (aids), acp: acyl carrier protein, pdb: protein data bank. introduction tuberculosis (tb) is an infectious disease caused by the bacteria mycobacterium tuberculosis. globally, tuberculosis is the 13th leading cause of death after covid-19 (ahead of hiv and aids) and the second infectious disease killer. in 2021, approximately more than 10 million people worldwide were infected with tuberculosis. this infection affects the people of all age groups in many countries (who, 2023). mycobacterium tuberculosis (mtb) has a unique cell envelope that is linked to its pathogenicity. the presence of trehalose dimycolate (tdm), a free glycolipid that accumulates in a cord-like pattern on the surface of the cells and protects the tubercle bacillus from harmful agents and the host's immune system, is another feature of the cell envelope that is very noticeable. tdm is covalently linked to the cell wall peptidoglycan via an inner leaflet of the outer membrane by a phosphodiester bond. the primary components of this protective layer are mycolic acids. more precisely, the cyclopropane rings in mycolic acid layer help maintain the complex's structural integrity and shield the bacillus from oxidative stress (takayama et al, 2005). the development of an immune response is necessary for the control of mtb infection in individuals who are resistant to the infection. this type of response entails the involvement of resident alveolar macrophages, dendritic cells, t lymphocytes (tcd4+, tcd8+ cells) and the release of proinflammatory cytokines such as interferon-gamma (ifn γ), interleukin-2 (il-2), il-12, il-18, and tumor necrosis factor –alpha (tnfα). all of these are crucial for attracting more immune cells to the infection site to create a granuloma that traps and destroys tuberculosis bacilli while also providing the long-term niche required for latent tuberculosis infection (druszczyńska, kowalewicz-kulbat, fol, włodarczyk, & rudnicka, 2012). if the ongoing bacterial reproduction is not stopped, the bacilli and expanding tubercle may invade the nearby draining lymph nodes. as a result, lymphadenopathy develops, which is a typical sign of primary tb. if the lesion caused by the tubercle's expansion progresses to the lymph node and lung parenchyma, a ghon complex may form. the ghon focus, which describes the main tb infection, is often seen in the center of the lungs and is a hallmark of tb manuscript received: 22 july, 2023. revision accepted: 08 august, 2023. published: 14 august, 2023. https://doi.org/10.14421/biomedich.2023.122.441-450 442 biology, medicine, & natural product chemistry 12 (2), 2023: 441-450 infection. latent tb reactivates into active tuberculosis in the presence of immunosuppression in the host (ruiru & isamu, 2013). changes in the host's physiological and immunological condition brought on by aging, malnutrition, diabetes or the emergence of other illnesses, including hiv/aids, as well as latent infections, might trigger their activation. the use of many antibiotics given over the course of six months is required for chemotherapy for active tb caused by drugsensitive bacteria. patient compliance is typically low as a result of this difficult and potentially hazardous treatment regimen. as a result, antibiotic-resistant bacteria have developed, necessitating lengthier treatment regimens, the use of less effective and more toxic medications, and increasing failure rates (reddy et al., 2008). the aim of this study is to analyze the anti-tb effects of several natural alkaloids and their potential to weaken the pathogenesis of tuberculosis using in-silico tools. in order predict the potential activity of alkaloids against the active tb infection, this study utilized molecular docking and molecular dynamics simulation as methodological techniques. the pathophysiology of tuberculosis infection is depicted in figure 1. figure 1. the figure illustrates the pathogenesis of tuberculosis infection caused by mycobacterium tuberculosis. material and methods macromolecule preparation rcsb (protein data bank) was used to retrieve the 3d structure of mycobacterial acp (acyl carrier protein) reductase enzyme (pdb id: 4r9s) in protein data bank (pdb) format. pdb contains the structure elucidation data of proteins obtained by x-ray crystallography and nuclear magnetic resonance (nmr) spectrometry submitted by biologists and biochemists from all over the world. macromolecule was prepared by removing water molecules, heteroatoms, ligands, extra protein chains, and metal ions that could interact with compounds during visualization of docking between compounds and target proteins using biovia discovery studio visualizer v17.2.0.16349 (sharma et al, 2021). ligand preparation the compounds used as ligands for the antimicrobial activity were from the alkaloidal class of phytochemicals. the structures of these compounds were downloaded from the pubchem database and chem3d 16.0 was used to obtain the pdb format of these 3dstructured compounds. c1 (protopine), c2 (apropine), c3 (avicine), c4 (penduline), and c5 (chelerythrine) were used to study the potential inhibition of mycobacterial acyl carrier protein reductase for preventing tuberculosis infection. these five compounds were used against the target protein to check the interaction of the target protein with these compounds (c1c5). figure 2 shows the graphical representation of the complete methodology of the research study. kamran & ibrahim – alkaloids lead to potential inhibition of the … 443 the chemical structures and botanical sources of compounds (c1 c5) are provided in table 1 while figure 3 depicts the iupac names of the compounds c1 – c5. figure 2. the figure shows the step by step methodology of the research study. table 1. the table shows the names of alkaloids employed in this study, their pubchem id, chemical structures and botanical sources. compound compound name pubchem id structure botanical source literature source c1 protopine 4970 corydalis heterocarpa var. japonica pubchem c2 aporphine 114911 antizoma angustifolia pubchem c3 avicine 356657 zanthoxylum asiaticum pubchem c4 penduline 179472 leptopus cordifolius pubchem c5 chelerythrine 2703 zanthoxylum fagara pubchem 444 biology, medicine, & natural product chemistry 12 (2), 2023: 441-450 figure 3. the figure demonstrates the chemical structures of compounds c1 to c5, along with their iupac names obtained from pubchem. results and discussion molecular docking analysis & results in structural molecular biology, medicinal chemistry and computer-assisted drug development, molecular docking is a useful technique. predicting the prevailing binding modes and poses of a compound that interacts with a protein having a known three-dimensional structure is the major aim of compound-protein docking. successful docking methods employ a scoring system that accurately ranks the drug candidate dockings and efficiently explores binding affinities and best poses. lead optimization greatly benefits from the use of docking to do virtual screening to provide insights for how the ligands interfere with the target (fan, fu, & zhang, 2019). pyrx was utilized for this experiment. pyrx includes a docking wizard with an easy-to-use user interface, which makes it a valuable tool for computeraided drug design (cadd). pyrx also includes chemical spreadsheet-like functionality that helps in novel drug molecule designing (dallakyan & olson, 2015). software version 17.2.0.16349 of biovia discovery studio visualizer was used. it offered a clear depiction of the 2d and 3d interactions as well as bond lengths, aromatic, hydrophobic, and other interactions. results from the molecular docking studies of the mycobacterial acp protein with compounds c1–c5 were valuable, as shown in table 2. table 2. the table shows the findings and results of the molecular docking analysis done using compounds c1 to c5. compound-protein interaction binding energy (kcal/mol) bonding residues type of bond bond length (å) other residues c1 -10 phe 41 pi pi 4.55 ile 15 ile 95 pi sigma 3.41 thr 39 carbon hydrogen 3.64 c2 -9.8 phe 41 pi pi stacked 3.74 val 65, ile 122. ile 95 pi alkyl 5.23 c3 -9.7 phe 41 pi pi stacked 4.26 ile 122, ile 16 ile 95 pi sigma 3.86 asp 64 carbon hydrogen 3.27 ser 20 conventional hydrogen bond 2.87 c4 -9.6 phe 97 pi pi t shaped 5.92 leu 207, met 103, ile 21, leu 207 ile 16 pi sigma 4.00 c5 -9.1 phe 41 pi pi stacked 4.30 gly 14, ile 122 , phe 97 ile 16 pi sigma 3.78 asp 64 carbon hydrogen bond 3.44 ser 20 conventional hydrogen bond 3.26 kamran & ibrahim – alkaloids lead to potential inhibition of the … 445 all the compounds c1 to c5, exhibited their firm interactions with the binding site residues of mycobacterial acp reductase enzyme receptor i.e., phe 41, ile 95, ile 16 and ser 20 with a binding pose ranging from – 10 to -9.1 kcal/mol. the best binding pose with binding energy of – 10 kcal/mol was expressed by protopine (c1), while the binding energies exhibited by other compounds in this study were -9.8 kcal/mol for aporphine (c2), -9.7 kcal/mol for avicine (c3),-9.6 kcal/mol for penduline (c4) and -9.1 kcal/mol for chelerythrine (c5).all the compounds showed absolute interactions with the active site residues of mycobacterial acp reductase enzyme. protopine (c1), aporphine (c2), and avicine (c3) were bound to phe 41 and ile 95 residues with lower binding energies. penduline (c4) and chelerythrine (c5) manifested binding with phe 97, ile 16, phe 41, ile 16, asp 64, ser 20 residues with reasonable bond lengths. compounds protopine (c1) and aporphine (c2) also exhibited firm interactions with other residues including ile 15, val 65, and ile 122, that may also be the active site amino acid residues of mycobacterial acp reductase enzyme. avicine (c3), penduline(c4) and chelerythrine (c5) demonstrated robust binding affinities with other active residues ile 122, ile 16, leu 207, met 103, ile 21, leu 207 and gly 14, ile 122, phe 97 with substantially shorter bond lengths. figure 4 demonstrates the interactions of all compounds with mycobacterial acp reductase enzyme. admet analysis absorption, distribution, metabolism, elimination and toxicity (admet) analysis is essential for navigating the pharmacodynamics and pharmacokinetics of a potential drug candidate. in-silico adme parameters were studied using swiss adme. the druglikeness score and toxicity profile of the potential drug candidates was probed using datawarrior v5.5.0. table 3 shows the pharmacokinetic and toxicity parameters of the potential drug candidates from c1 to c5. figure 5 manifests the bioavailability radar of best docked ligand, compound c1, obtained from swissadme tool. figure 4. the figure displays the 3d and 2d images of the interactions between active amino acid residues of mycobacterial acyl carrier protein reductase (pdb id: 4r9s) and compounds (c1, c2, c3, c4 and c5). 446 biology, medicine, & natural product chemistry 12 (2), 2023: 441-450 table 3. the table provides the pharmacokinetic and toxicity profile of compounds (c1 – c5) using swissadme and datawarrior tools. admet profiling of the potential drug candidates (c1 – c5) potential drug candidates c1 c2 c3 c4 c5 physicochemical properties molecular weight (g/mol) 353.37 g/mol 235.32 g/mol 332.33 g/mol 608.72 g/mol 348.37 g/mol log p 2.67 3.23 2.84 5.17 3.02 h-acceptor 6 1 4 8 4 h-donor 0 0 0 1 0 n.r.b 0 0 0 3 2 tpsa (å²) 57.23 å² 3.24 å² 40.80 å² 72.86 å² 40.80 å² bioavailability score 0.55 0.55 0.55 0.55 0.55 drug-likeness parameters lipinski’s rule violations 0 violations 0 violations 0 violations 1 violation 0 violation drug-likeness score 4.7237 4.9313 2.4707 4.6261 2.1842 metabolic profiling p-glycoprotein substrate yes yes yes no yes gi absorption high high high high high cyp 3a4 yes no no no yes cyp 2d6 yes yes no no no cyp 1a2 yes no yes no yes cyp 2c9 yes no no no yes cyp 2c19 no no yes no yes toxicity profiling tumorgenicity no no no no no mutagenesis no no no no no irritant no no no no no reproductive effects no no no no no mol. w (g/mol) molecular weight, log p prediction of octanol/water partition coefficient, n.r.b number of rotable bonds, tpsa (å²) topological polar surface area, cyp cytochrome p-450 enzyme. figure 5. the figure the bioavailability radar of ligand c1 (having the most favorable binding energies as per the findings of molecular docking). molecular dynamic simulation molecular dynamic (md) simulations depict the dynamics of a macromolecule bound by a ligand, interactions between the amino acid residues and navigating the configurations of a protein molecule (y. wang, ribeiro, & tiwary, 2020). md simulations unveil the dynamics of a protein and assist in discovering the cryptic sites in a protein macromolecule that may have affinity towards a ligand, thereby facilitating its binding to the hydrophobic pocket (kuzmanic, bowman, juarezjimenez, michel, & gervasio, 2020). md simulations were done using the online md simulation tool, imods server (lópez-blanco, aliaga, quintana-ortí, & chacón, 2014; lopéz-blanco, garzón, & chacón, 2011). the ligand and macromolecule docked complex c1 (protopine) was loaded on imods server and md simulation was performed on normal mode analysis (nma). the b-factor yielded information regarding the flexible nature of the macromolecule. it illustrates the deforming tendency of the protein residues in order to interact with protopine molecule. the arrow field shows the orientation of the macromolecule in the virtual experiment. the figures 6,7 and 8 provide the graphical presentation of the finding of molecular dynamic simulation analysis. kamran & ibrahim – alkaloids lead to potential inhibition of the … 447 figure 6. the figure illustrates the orientation of the docked complex (c1 and macromolecule) obtained through molecular dynamic simulations using imods. the eigenvalue for the complex in this virtual experiment was measured as 5.340225e-04. the eigenvalue manifests the energy required to deform a particular amino acid residue while interacting with other residues in a reallife environment created virtually. variance is inversely proportional to eigenvalue and the comparison between the two is shown in the figure. figure 7. the figure shows the findings of molecular dynamic simulation i.e. image (a) shows the deformability of the docked complex. image (b) provides the graphical view of the bfactor. image (c) provides the eigenvalue for the complex while the image (d) demonstrates the percentage variance. the covariance map displays the interactions between the residues of the dynamic protein molecule, with red areas showing the correlating or interacting residues, white areas demonstrating the residues with no interactions, and blue areas showing residues with repulsive behaviour within the protein molecule. the elastic network provides an estimate regarding the ease of deformation of the target amino acid residues within the macromolecule. the dark greyish areas show residues with a higher degree of stiffness, while the lighter areas depict ease of deformation of the target residues. 448 biology, medicine, & natural product chemistry 12 (2), 2023: 441-450 figure 8. the image (a) displays the covariance graph of the complex (c1 and macromolecule) while image (b) represents elastic network map of the complex. discussion in this study, five alkaloids were screened computationally for their potential to inhibit the mycobacterial acyl carrier protein reductase and attenuate tuberculosis. molecular docking and scoring is a reasonable method to analyse various drug candidates for their potential for particular biological activity before entering the wet lab (torres, sodero, & jofily, 2019). the results of the molecular docking analysis illustrated optimistic outcomes. all five compounds exhibited strong interactions with the target protein. the binding energies of the best poses of these potential drug candidates were in a range of -10 to -9.1 kcal/mol. protopine (c1) provided most suitable binding energies and poses. strong bonds with catalytic amino acid residues having considerably shorter bond lengths were observed with all five compounds. the compounds showed firm interactions with phe 41, ile 95, and ser 20 residues. the compounds (c1– c5) were alkaloids with multiple botanical sources and a vast range of biological activities. protopine (c1) is obtained from papaver somniferum (pubchem, 2023). the compound has also been tested for its potential biological activity against cancer, alzheimer’s disease, inflammation and other ailments (son et al., 2019; sreenivasmurthy et al., 2022; zhang et al., 2019). in this study, the protopine has been tested in-silico for its potential anti-mycobacterial effect and auspicious results have been furnished in this regard. the second drug candidate, aporphine (c2) has been widely tested for its anti-inflammatory, analgesic, antiparasitic and anti-oxidant effects in various in-silico, invitro and in-vivo models (pieper, mchugh, amaral, tempone, & anderson, 2020; b. wang et al., 2019; r. wang, zhou, shi, liu, & yu, 2020). the exploration of anti-tuberculosis activity was done in this study and promising outcomes were witnessed. the biological activity of avicine (c3) in alzheimer’s disease is well established through animal models (cahlíková et al., 2021; plazas, 2020). avicine has also displayed hopeful anti-mycobacterial activity predictions in this study. penduline (c4) has been studied for its anti-cancer and anti-inflammatory activities while chelerythrine (c5), a potent inhibitor of protein kinase c, has revealed to be active against cancer, bacterial infections and covid 19 (gong et al., 2019; grabarska et al., 2021; lin et al., 2017; valipour et al. 2021). both of these alkaloids have furnished significant results in the molecular docking analysis in this study. the molecular dynamics simulations have further explored the dynamic behavior of the targeted protein. isoniazid is a well-known inhibitor of mycobacterial enoyl-acp reductase (unissa et al, 2016). isoniazid inhibits the synthesis of mycolic acid in mycobacteria and halts the progression of cell wall synthesis, thereby providing bactericidal effects (vilchèze & jacobs, 2019). isoniazid is one of the anti-tubercular drugs that have indicated notable efficacy through the decades. (charan et al., 2018; huang et al., 2018; pease et al., 2017). keeping in view these facts, severe toxicities and adverse drug reactions are also associated with the use of isoniazid in the treatment regimen for tuberculosis (bliven-sizemore et al., 2015). the hepatotoxicity and peripheral neuritis are the most significant toxicities associated with isoniazid (lamb & mauermann, 2021; mafukidze, calnan, & furin, 2016; m russom et al., 2019; mulugeta russom et al., 2018). these considerable toxicities demand a safer treatment option with similar efficacy. in this study, potential drug candidates, i.e., natural compounds (alkaloids) with no reported toxicity and strong affinity for target proteins, could attenuate tuberculosis infection after the development of a stable formulation. this study recommends the formulation scientists to employ novel drug formulation techniques for formulating natural products and secondary metabolites such as alkaloids as nanocarriers in order to achieve their delivery to the targeted sites, maintain efficacy and avoid any undesirable or noxious effect. natural products and phytochemicals have caught the attention of formulation scientists and pharmacologists to a greater extent. the coupling of natural products and kamran & ibrahim – alkaloids lead to potential inhibition of the … 449 nanomedicine can make major breakthroughs in the field of therapeutics. this study also puts emphasis on this idea. conclusion tuberculosis is a public health concern that claims millions of lives annually. the current therapy for tuberculosis has many challenges, with toxicity at the top of the list. natural products have been used traditionally owing to their minimal toxicities and decent efficacy. many studies have established that phytochemicals can attenuate bacterial infections such as tuberculosis. through in-silico analysis, this study has concluded that the alkaloids such as protopine, aporphine, avicine etc. have potential inhibitory activity for the mycobacterial acyl carrier protein reductase enzyme, hence disrupting mycobacterial survival and pathogenesis. the molecular docking and molecular dynamics simulation provided optimistic results regarding the interactions of these alkaloids with the active amino acid residues of mycobacterial acyl carrier protein reductase. this study recommends the conduct of in vitro and in vivo studies in order to validate the findings of this research study and the development of novel formulations for better delivery of these alkaloids for attenuating tuberculosis. acknowledgements: the study is carried out entirely by the authors, so there is no need for acknowledgement. authors’ contributions: ahsan ibrahim designed the study. pernia kamran and ahsan ibrahim carried out the computational work. pernia kamran & ahsan ibrahim wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references bliven-sizemore, e. e., sterling, t., shang, n., benator, d., schwartzman, k., reves, r., . . . consortium, t. t. 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(2019). protopine protects mice against lps-induced acute kidney injury by inhibiting apoptosis and inflammation via the tlr4 signaling pathway. molecules, 25(1), 15. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 897-903 | doi: 10.14421/biomedich.2025.142.897-903 issn 2540-9328 (online) isolation, identification, and evaluation of antimicrobial of the lab from bekasam: the traditional fermented fish in indonesia nur rohman1, muhammad bachrun alim2* 1agricultural product technology program, faculty of agroindustry, universitas mercu buana yogyakarta, indonesia jl. raya wates-jogjakarta, bantul, yogyakarta 5575, telp. 0274-6498212, fax. 0274-6498213, indonesia. 2department of marine biology, faculty of marine science, king abdulaziz university, saudi arabia idara st, jeddah, telp. +966 12 6952043, saudi arabia. corresponding author* mmuhyanto@stu.kau.edu.sa abstract lactic acid bacteria (lab) are frequently utilized in fermented foods and can incresed shelf life for the products through their secondary metabolites includes carbon dioxide, hydrogen peroxide, lactic acid and bacteriocins. beside increasing the shelf life, lab also affect taste, smelt, and texture. thus, this study aimed to isolate, identify and evaluate antibacterial lab strains from indonesian traditional fermented fish (bekasam). gram staining, the catalase assay, and motility assays were used to initially characterize the presumed isolates phenotypically following primary isolation on de man, rogosa, and sharper (mrs) agar. 14 isloates were determined to be presumed lab by preliminary phenotypic testing. one species that has the highest antibacterial activity is confirmed by a 16s rrna sequencing study. lactobacillus plantarum cp-134 was identified from the bekasam. in the biochemical characterization all isolates were catalase-negative and non-motile. according to our research, these lab strains may have applications in fermented foods due to their probiotic properties, which include antibacterial activity. they might be used as natural substitutes for additives and antibiotics, but more in vivo or in silico research is required to verify their potential and effectiveness. a typical traditional food preservation technique for enhancing shelf life, food safety and nutrition and sensory qualities is fermentation. bekasam is a traditional fermented food from indonesia. keywords: bekasam; fermentation; lactic acid bacteria; 16s rrna. abbreviations: lab: lactic acid bacteria; mrs: de man, rogosa, and sharper; cfu:colony forming unit; tpc: total plate count. introduction one of the most popular traditional fermented fish food from indonesia is bekasam. bekasam is one of the fermented fish product that is processed by adding salt and sugar. fish fermentation procedures often involve salting, adding species or chemicals, and maintaining anaerobic conditions, which promote the growth of certain protechnological microbes, and distinct flavour, texture, and nutritional values. generally, the exfermentation process occurs spontaneously by utilizing salt as a microbial selector of decomposer (mudoor sooresh et al., 2023). in addition to salt, the sugar added is used as an energy source for the growth of bacteria that ferment fish (setiarto & herlina, 2024). salt is a traditional and effective method for preserving fish, resulting in a salt adapted microbiota. the addition of salt causes physicochemical change in fish tissue, including a reduction in the moisture content and water activity through salt diffusion and increasing the solubility of denatured protein (known as salting-in). bekasam is a local indonesian food, popular in centra java, south kalimantan and south sumatra. in general lab is the dominant bacterium found in fermented products. traditional fermented foods are primarily produced at the household level using largely uncontrolled spontaneous inoculation methods in which microorganisms associated with the raw food material and processing environment serve as inoculants. many studies have characterized spontaneous fermentation. the role of the starter in the fermentation process is too important. despite the importance of microorganisms in fish fermentation, discrepancies have been found in the lab colony during fermentation and the impact of starter culture on fermented fish. the starter cultures used in fermented fish and the microbial diversity involved during this process, and an in-depth discussion of the methods used to identify these microorganisms. lab are bacteria that are found in many fermented fish products. the metabolism of lab includes carbon dioxide, hydrogen peroxide, lactic acid, and bacteriocins, which can inhibit the growth of spoilage bacteria, thereby increasing the shelf life of the products. besides manuscript received: 29 july, 2025. revision accepted: 10 october, 2025. published: 24 october, 2025. https://doi.org/10.14421/biomedich.2025.142.897-903 mailto:mmuhyanto@stu.kau.edu.sa 898 biology, medicine, & natural product chemistry 14 (2), 2025: 897-903 increasing the shelf life, lab also affect taste, flavour, and texture. this type survives at salt level of 3%-6% including lactobacillus plantarum, lactobacillus sakei (li et al., 2023), lactobacillus acidophilus cm1 and lactobacillus delbrueckii os1 could survived at 4% and 6% nacl level significantly (khushboo et al., 2023). this study aims to determine the type of lab originating from bekasam which is processed with different salt levels for 14 days which are spontaneously fermented. materials and methods study area this study was conducted in september 2024 at tropical marine biotechnology laboratory, universitas diponegoro, central java, indonesia. sample of bekasam the fermented food sample used in this study includes bekasam that is processed with salt at different levels (melia et al., 2019) ph measurement a digital ph meter was used to determine ph. the electrode was dipped into the sample until the ph value was shown (greulich et al., 2024). titratable acidity the titration method was used to measure the amount of lactic acid. ten grams of the bekasam samples have been mashed, and the filtrate is then taken and placed in an erlenmeyer flask, followed by the addition of 2-3 drops of 1% pp and 10 ml of aquadest (greulich et al., 2024). the following formula was used to determine the lactic acid % result: lactic acid (%)= ml naoh x n naoh x 0,009 sample weight x 100% enumeration of lab in bekasam the total bacterial count was calculated using the tpc (total plate count) method (greulich et al., 2024). the overall bacterial was count determined using the following formula: total (cfu/ml) = total colony spread volume x dilution factor isolation and identification of lab the lab from bekasam were cultured in mrs broth media (merck, germany) and incubated at 37⁰c in an anaerobic jar for 48 hours. additionally, morphological (form and color) and biochemical (gram staining, catalase test, motility test) features were determined (madushanka et al., 2025). gram staining pure culture smears that had been air-dried and heatfixed were successively stained with safranin, crystal violet, gram iodine, and a decolorizing agent. each staining step was followed by a gentle wash. after being blot-dried with absorbent paper, the slides were viewed under an oil immersion microscope (el ahmadi et al., 2025). catalase assays a sterile inoculating loop was used to transmit a tiny portion of a recently isolated colony onto a microscope slide. after that, a drop of 3% h2o2 was applied. the quick development of air bubbles demonstrated that the catalase test was positive (joos et al., 2025). motility test in the middle of the coverslip was a loopful of freshly made broth culture that had been developed overnight. in order to make a depression over the culture drop, a cavity slide was then carefully inverted and placed over the coverslip. the drop was allowed to dangle freely in the cavity after the slide was carefully inverted. to determine if there was movement or not. the cells were viewed in low light and at medium strength (×40) (rizwan & masoodi, 2025). antimicrobial activity test to perform an antibacterial test, 14 colonies from bekasam were cultured in mrs broth at 37⁰c for 48 hours. antibacterial activity was tested on strains associated with food poisoning, including escherichia coli and staphylococcus aureus. the antibacterial activity of each isolates components was determined using the paper disc method (park et al., 2024). a paper disc was inoculated with 50 µl of lab suspension containing 10-7 cfu/ml obtained from bekasam. the paper disc were incubated at 37⁰c for 48 hours, and antibacterial activity was measured by the diameter of the clear zones. we chose one top-performing lab based on our findings. genomic analysis of lab and the construction of a phylogenetic tree for identified strains with strong antimicrobial activity lab isolates have been grown in mrs broth at 37⁰c for 24 hours. genomic dna was extracted using the promega kit (usa). a single colony of lab from mrs broth (merck, germany) was pipetted up to 1000 µl and added to a new eppendorf tube. centrifuged at 14000 rpm for two minutes. the supernatant is then removed, and keeping only the pellet. added 840 µl of 50 mm edta. then, 120 µl lysozyim was applied. next, incubate in a 37⁰c water bath for 60 minutes. centrifuge for 2 minutes at 14000 rpm, then remove the supernatant and take the pellet. applied 600 µl of nuclei lysis solution. after incubating at 80⁰c for 5 minute, let it cool rohman & alim – isolation, identification, and evaluation of antimicrobial of … 899 to room temperature. add 3 µl of rnase solution and incubate in water bath at 37⁰c for 60 minutes. add 200 µl of protein precipitation solution, then vortex. approximately 600 µl of isopropanol was added. centrifugated at 14000 rpm for 2 minute, then pellets were collected and supernatant have been eliminated. add 600 µl of 70% ethanol and homogenized. centrifuged at 14000 rpm for 2 minutes, then pellets were collected and the supernatant eliminated. to rehydrate pellet dna, add 10-100 µl of rehydration solution and incubate for 30 minutes at 65⁰c. general primer 27f (5’-agagtttgatcmtggctcag-3’) and 1492r (5’-tacggttaaccttgttacgactt-3’) generated (concentration 10 pm) take 90 µl dh2o + (primary r and f). (primary r and f in te buffer, concentration 100 µm). pcr cocktail in eppendorf (master mix 12,5 µl, primary f 1 µl, primary r 1 µl, template dna 1 µl, ddh2o 9,5 µl) with pcr denaturation 95⁰c 45 second, annealing 56⁰c 45 second, extension 72⁰c 1 minute 40 second, and final extension 72⁰c 10 minute. electrophoresis of a 10 µl sample in to wall agar, inserting 4 µl of dna ladder. set at 100 volts for 45 minutes. the gel was placed in a container with tbe until submerged. the gel was then examined under a uv lamp. the 16s rrna gene sequences of the isolates were submitted to ncbi for a blast (https://blast.ncbi.nlm.nih.gov/blast.cgi) comparison to database records. phylogenetic trees were generated through the neighbor-joining method (https://www.megasoftware.net/). data analysis three separate experiments were conducted, and the statistical package for the social science (spss) was used to analyze all data using a one-way anova. duncan’s multiple range was used to determine whether mean differences were significant (p<0.05). results and discussion results characteristics of the isolated lab table 1 shows the total of lab colony from bekasam made with three different salt levels (15%, 20% and 25%). table 1. result of ph and number of lab in bekasam. sample code ph % lactic acid total colony of lab (cfu/ml) b1 5.3 4.3 1.7 x 105 b2 5 4.1 1.1 x 105 b3 5.3 4.7 1.5 x 105 note. b1: salt level 15%, b2: 20%, b3: 25% isolation and morphological characterisation of the lab table 2. result of lab morphological and biochemical test code number of isolates gram stain motility catalase morphology cocci rod bal 1 + + bal 2 + + bal 3 + + bal 4 + + bal 5 + + bal 6 + + bal 7 + + bal 8 + + bal 9 + + bal 10 + + + bal 11 + + bal 12 + + bal 13 + + bal 14 + + https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.megasoftware.net/ 900 biology, medicine, & natural product chemistry 14 (2), 2025: 897-903 figure 1. morphological characteristic of gram-positive of lab (a. shows cocci-shaped bacteria, b. shows rod-shaped bacteria). antimicrobial activity the antimicrobial activity test demonstrated that lab inhibited the development of staphylococcus aureus fncc-0047 and escherichia coli fncc-0091, but the inhibition zone was relatively modest in size. the diameter of the inhibition zone was measured after incubation for 24 hours and repeated three times. figure 2. antimicrobial activity test of lab isolate (mm). 5,63 5,87 6,57 5,43 5,03 5,77 6,25 3,85 6,23 6,05 7,78 3,45 4,43 6,80 6,57 6,28 7,93 8,07 6,42 5,72 6,55 8,6 4,85 4,47 5,63 7,23 6,57 6,55 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 10,00 bal 1 bal 2 bal 3 bal 4 bal 5 bal 6 bal 7 bal 8 bal 9 bal 10 bal 11 bal 12 bal 13 bal 14 is o la te diameter of inhibition zone (mm) e. coli diameter of inhibition zone (mm) s. aureus rohman & alim – isolation, identification, and evaluation of antimicrobial of … 901 identification strain with strong antimicrobial activity using 16s rrna figure 3. a gel with agarose electrophoresis of 16s rrna gene amplification by pcr revealed and phylogenetic tree of lactobacilli strains isolated from bekasam on the 16s rrna gene. discussions characteristic of isolated lab the bekasam sample has a ph between 5.0 and 5.3, which means that the provision of different salt levels in bekasam processing does not have a significant effect on the ph. similarly, the three samples of bekasam showed no substantial changes in total lactic acid levels. the number of lab colonies of bekasam from the three samples ranged between 1.1 x 105 cfu/ml to 1.7 x 105 cfu/ml. the outcome demonstrates a relationship between the ph value and the number of lab. lab can thrive in low ph and high salt environment conditions, ter et al., (2024) reported that, the optimal conditions for lab growth are with a ph of 5.0 – 6.2. on the other hand, bacterial metabolism, which produces lactic acid, modulates the ph, causing it to become acidic (zhou et al., 2025). because the development and metabolic activites of lab produce acid and lower ph of the fermented fish (bekasam), their presence in the bekasam is crucial. the growth of the harmful bacteria may be inhibited by this acidic environment. isolation and morphological characterisation of lab the bekasam was fermented for up to 7 days to produce more fermented liquid. the liquid was thought to contain the most bioactive antimicrobial compounds because it had the largest clear zone of inhibition in our early investigation. this also revealed that the liquid already had an excess of lab. the liquid was streaked in quadrants across the mrs medium. the opaque color media is the product of caco3 precipitation in the mrs medium. calcium carbonate dissolve when a colony creates lactic acid molecules (sariyanti et al., 2025). a single colony with a clear zone was considered to be lab, thus it became isolated and characterized. fourteen isolates exhibited gram-positive, non-motile, and catalase-negative, with 9 rod morphologies and only 5 cocci respectively (table. 2) and (figure. 1), these were standard criteria for targeted strains (alharbi & alsaloom, 2021). the study found that bekasam was the primary source of lab, concurring with desniar et al., (2013) who successfully identified of lab from eight types of fish bekasam from eight areas in indonesia included lactobacillus sp., pediococcus spp., and aerococcus spp. in other study also report that lab from plara ( traditional fermented fish from thailand) like bekasam was successfully collected, which are enterococcus avium, aerococcus viridans, e. faecalis, e. thailandicus, e. hirae, l. lactis, l. plantarum, pedicoccus pentosaceus, l. paracasei, p. acidilactici, w.cibaria, tetragenococcus halophilus, w. paramesenteroides, w. confusa, and w. viridescens (miyashita et al., 2012). antimicrobial activity the antimicrobial activity of the lab was assessed using the paper disk method. antimicrobial activity testing is required to determined the inhibitory potential of lab microorganisms against pathogenic bacteria. figure 2 demonstrate the results of antimicrobial testing with the diameter of the inhibitory clear zone of numerous isolates. the inhibitory band of every isolate ranged around 4.47 mm to 8.07 mm against escherichia coli fncc-0091and 3.45 mm to 7.78 mm against staphylococcus aureus fncc-0047. lab produce antimicrobial chemicals and exhibit considerable antagonistic action against several pathogenic bacteria. lab can produce considerable amounts of antimicrobial metabolites, including as lactic acid, bacteriocin, diacetyl, and hydrogen peroxide, which 902 biology, medicine, & natural product chemistry 14 (2), 2025: 897-903 inhibit the growth of several harmful bacteria (santos et al., 2025) the existence of clean zone indicates antimicrobial action against pathogenic microorganisms (apenteng-takyiako et al., 2025). in the examination of antimicrobial activity against escherichia coli fncc0091and staphylococcus aureus fncc-0047, the findings of measuring the diameter if the inhibition zone indicates moderate results. the isolate that generated the largest diameter of the inhibition zone was bal8 code. (bhattacharjya et al., 2024) classified antimicrobial activity into three categories; no inhibition (negative), moderate (<9 mm), and strong (>9 mm). based on this criteria, it can be conclude that the majority of the isolate lab had modest inhibition against escherichia coli fncc-0091. the isolate with a code bal8 has the largest zone of inhibition, measuring. identification of a strain with strong antimicrobial activity using 16s rrna by using the blast algorithm to compare homology with 16s rrna gene sequences in genebank (https://blast.ncbi.nlm.nih.gov/blast.cgi), the sequencing findings were utilized to determine genetic lab from bekasam isolates. figure 3 showed the lab pcr sequencing result from bekasam sample. figure 3 displayed the lab phylogenetic tree from bekasam sample. according to the findings, lactobacillus plantarum strain cp134 and lab from sample of bekasam were 100% same. according to (najeeb et al., (2025), isolates were deemed to be of the same species if their 16s rrna sequences were more than 97% identical. on the other hand, 93%-97% of sequence similarities were regarded as belonging to different species but the same genus. conclusions this study’s main goals were to screen bekasam for the types and presence of lactic acid bacteria (lab) at various salt levels during fermentation and demonstrate that bekasam, a traditional fermented fish, may contain lactic acid bacteria, offering several health benefits associated with probiotic consumption. from the examined bekasam samples, lactobacillus plantarum cp-134 were detected. the isolation exhibited antibacterial activity against certain food-borne pathogens and was tolerant of low ph and high salt levels. according to this study, lactic acid bacteria (lab), which are essential for fermentation, are present in bekasam and may have antibacterial qualities. the prevalence of lab indicates their role in the fermented fish potential health advantages and microbiological stability. regarding food safety, the study emphasizes how lab has antagonistic effects on foodborne pathogens, suggesting that they may lower the risk of contaminations and improve microbiological safety. their capacity to produce bacteriocins, organic acids, and other antimicrobial substances may help extend shelf life and prevent spoilage. certain lab strains may be used as indicators to evaluate the microbiological integrity of bekasam. additionally, this work creates new opportunity to obtain probiotics from natural and unconventional sources like bekasam. acknowledgements: the authors would like to thank to pt. indofood sukses makmur, tbk for funding this research under indofood riset nugraha program. authors’ contributions: nur rohman designed the study, carried out the laboratory work, analyzed the data, wrote the manuscript, muhammad bachrun alim review and editing the manuscript, all authors read and approved the final version of the manuscript. competing interests: the authors declare no conflict of interest funding: the authors would like to thank to pt. indofood sukses makmur, tbk for funding this research under indofood riset nugraha program. references alharbi, n. k., & alsaloom, a. n. 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(2025). high temperatures and antibacterial plant additives change the fermentation quality, free amino acids and lactic acid bacteria fermentation type in caragana korshinskii silage. bmc microbiology, 25(1). https://doi.org/10.1186/s12866-025-04153-7 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 315-321 | doi: 10.14421/biomedich.2025.141.315-321 issn 2540-9328 (online) laryngopharyngeal reflux (lpr) correlation to middle ear disorders mohammad dwijo murdiyo1, melody audria kurniadi2,* 1department of ear nose and throat laryngfaringology division, faculty of medicine, universitas brawijaya, east java, indonesia. 2otolaryngology-head and neck surgery resident, department of otolaryngology-head and neck surgery, faculty of medicine, universitas brawijaya-dr. saiful anwar general hospital, malang, indonesia. corresponding author* melodyaudria@gmail.com manuscript received: 09 march, 2025. revision accepted: 04 june, 2025. published: 28 june, 2025. abstract laryngopharyngeal reflux (lpr) is a condition where the reflux of gastric contents reaches the laryngopharynx. several studies have learned that lpr can cause disturbances in the organs around the larynx pharynx and one of the organs affected is the ear. lpr complications in the middle ear can reduce the quality of life of sufferers. appropriate diagnostic methods are needed to provide appropriate therapy for this complication. to analyze the relationship between laryngopharyngeal reflux (lpr) and middle ear disorders. this study was an analytic observational study using a purposive sampling approach where the assessment of correlation tests between variables was tested with spss. results: demographic data of lpr patients are dominated by female gender (56%) with an age range of 19 59 years (80%). the maximum reflux symptom index (rsi) score in lpr patients is 33 and the maximum reflux finding score (rfs) score in lpr patients is 25. an abnormal tympanogram width (tw) picture was found in 64% of lpr patients. abnormal compliance picture was found in 36% of patients with lpr and abnormal middle ear pressure picture occurred in 12% of patients with lpr. abnormal middle ear disorders were found in 88% of patients with lpr. correlation test assessment between rsi score and tw parameter and correlation between rfs score and middle ear pressure. there was a positive correlation between lpr and middle ear disorders. there is a positive correlation between lpr and middle ear disorders. keywords: middle ear disorders; laryngoesophageal reflux; lpr; tympanogram width. introduction laryngopharyngeal reflux (lpr) is a condition where the reflux of gastric juices consisting of gastric acid, pepsin, bile and trypsin reaches the laryngopharynx. lpr is a complex condition that involves reflex and reflux mechanisms but a clear understanding of its pathophysiological mechanisms is still unclear. (rosen, 2023) the majority of symptoms complained of in patients with lpr are hoarseness and a feeling of blockage in the throat. (brown et al., 2025) according to epidemiological data from 1991, a study conducted by shuifang xiao et al, 10% of outpatients at the ent clinic showed that symptoms associated with lpr. (xiao et al., 2020). in 2007, a study with a questionnaire method conducted by connor et al. showed symptoms in the throat due to reflux reached 26.9% of all cases in the ent field. in addition, an epidemiological study conducted in china found an increase in lpr cases in the outpatient clinic, which reached 10.5% of the total ent cases. (lechien et al., 2024) several studies have learned that lpr can cause disturbances in the organs around the larynx pharynx and one of the organs affected is the ear. (rosen, 2023) chronic inflammatory conditions that can be caused by lpr can cause disorders of the middle ear. tympanometry examination is one of the main supporting examinations to evaluate middle ear function. studies on the relationship between lpr and middle ear disorders are still very limited. considering the high number of lpr cases that occur and its complications in the middle ear that can reduce the quality of life of sufferers, researchers feel the need to conduct a study of the effect of lpr on middle ear disorders which is useful as a diagnostic consideration in order to provide appropriate therapy. materials and methods study design this study is an analytical observational study using a purposive sampling approach. the research was conducted at the ear, nose and throat (ent) clinic at dr. saiful anwar hospital in the period 1st january 2024 – 30th august 2024. the study population was all patients who came for treatment at the ent clinic at dr. saiful anwar hospital during the study period. the subjects involved in the study were all patients who suffered from lpr and did not suffer from lpr who https://doi.org/10.14421/biomedich.2025.141.315-321 316 biology, medicine, & natural product chemistry 14 (1), 2025: 315-321 could undergo tympanometry examination at the ent clinic at dr. saiful anwar hospital during the study period. patients were declared to have lpr if the results of the rsi score were > 13 based on symptom history, rfs score> 7 based on the results of the examination using fiberlaryngoscopy (fol) at the ent clinic at saiful anwar hospital. patients were declared to suffer from middle ear disorders if the tympanogram results showed abnormal results in one of the variables studied. population and sample the subjects involved in the study were all patients who suffered from lpr and did not suffer from lpr who could do tympanometry examination at the ent clinic at dr. saiful anwar hospital for the period 1st january 202430 th august 2024. data collection the inclusion criteria for this study were patients who met the lpr criteria, were > 18 years of age at the time of the examination and could be subjected to fol examination and tympanometry examination within the study time period. patients with medical record data recorded completely and clearly. the control inclusion criteria in this study were patients with rsi scores and rfs scores that did not meet the diagnostic criteria for lpr. exclusion criteria in this study are patients with comorbidities that can cause middle ear disorders such as upper respiratory tract infections in the last 1 month, have a history of surgery on the head and neck, a history of malignancy, allergies or asthma. patients who have received therapy that can affect the results of research such as anti-reflux (proton pump inhibitors / ppis or antihistamines), decongestants or steroids. patients whose fol or tympanometry examination was performed outside saiful anwar hospital data analysis the data is processed using the spss 25.0 application. the correlation assessment on both research variables was carried out by testing the normality of the data with the shapiro wilk normality test method. if the result is > 0.05 then the data is normal, otherwise if the test value is <0.05 then the data is said to be abnormal. after going through the data normality test, the correlation assessment of the two variables (numeric-numeric) can be done with the pearson product moment test if both variables have normal normality test results, while for variables that have abnormal normality test results, the correlation test that can be used is the spearman rank test. if the result is > 0.05 there is no correlation between variables. the other test we used in this study was the chi square test to find the relationship between lpr and middle ear disorders. the test value gives a value of 0.00 which if <0.05 means that the two variables in the study are correlated. ethical statement this study followed the ethical principles of the declaration of helsinki, belmont report. dr. saiful anwar general hospital ethics committee reviewed and approved the research protocol on november 11th, 2024, with reference number [400 / 335 / k.3 / 102.7 / 2024]. the hospital ethics committee approved this study due to its use of hospital medical records. before participation, all respondents were provided with complete information regarding the purpose of the study, procedures, potential risks, and possible benefits. participation in this study is voluntary, and participants have the right to withdraw from the study at any time without any consequences. the medical information obtained during this study is kept confidential and is only used for research purposes. all data is stored and anonymized in accordance with established data security protocols. patient characteristics medical record data collection was carried out using purposive sampling method with a total sample size of 44 patients where 25 patients met the inclusion criteria for research subjects with lpr and 19 patients as control subjects. of the total respondents with lpr, female patients (53%) were more dominant than male patients (47%). in the research sample, the youngest age was 28 years old with the majority of respondents with lpr aged 19 59 years, which is 80% of the total research sample. according to this study, the maximum rsi score value was 33 and the minimum rsi score value was 14. the mean value of the rsi score in this study was 21.76 with a standard deviation of 5.56. the mean rfs score in this study was 16.92 with a standard deviation of 5.57 (table s1). the image of the middle ear in this study was evaluated using tympanometry and presented in tabular form (table s2.). in this study an abnormal compliance picture was found in 36% of the total respondents with lpr. the picture of abnormal middle ear pressure was found in 3 respondents from the total respondents. the picture of abnormal tympanogram width or tw which shows disturbances in the middle ear occurs in more than 50% of the research sample, namely in 16 research samples. in this study, the prevalence of middle ear disorders in lpr patients was found to be 22 (88%) of 25 total research samples. the correlation test results in this study found a relationship between the rsi score and tw with a correlation coefficient value of 0.502. (table s3). in addition, this study found a relationship between rfs scores and middle ear pressure images based on tympanograms. based on the spearman correlation test on the rfs score on middle ear pressure, there was a correlation with a correlation coefficient value of 0.028. (table s4). spearman's correlation test on rfs score to middle ear pressure obtained correlation results with a correlation coefficient value of 0.440 where the murdiyo & kurniadi – laryngopharyngeal reflux (lpr) correlation to middle ear disorders 317 correlation results are included in the moderate correlation category. this study did not find a correlation between rsi scores on compliance and middle ear pressure. in addition, this study also did not find a correlation between rfs scores on tw and middle ear compliance. (table s5-s8) the relationship between lpr and middle ear disorders was tested using the chi square test by first conducting a normality test on the data to be tested. the test value gave a value of 0.00 which if <0.05 means that the two variables in the study are correlated (table s9,s10). the magnitude of the correlation strength of the two variables is attached in table s6 where the value on the contingency coefficient is 0.657 where according to sugiyono et al. (table s11) this value is included in the strong correlation category. discussion the results of this study indicate that the female gender is more dominant in experiencing lpr compared to men. the findings of this study are in accordance with research conducted by ida ayu, et al. where in prevalence the female sex experiences lpr more often than men. (widiantari et al., 2019) in addition, research conducted by fengling, et al. also found that women experience lpr more often than men. some factors that can influence the relationship between lpr and hormone regulation in women. (huang et al., 2022) where the hormones estrogen and progesterone are suspected to cause an increase in nitric oxide (no) synthesis which causes a decrease in smooth muscle tone because it has a muscle relaxing effect. this leads to decreased contraction of the oesophageal sphincter, increasing the risk of reflux. (saleh et al., 2023) the age of lpr sufferers is dominated by productive age, namely with the age range of 19-59 years in this study. this is in accordance with research conducted by lechien j, et al. who found that the average patient was 40 years old where the first episode may begin at a young age as acute symptoms then recur into chronic symptoms. however, because the symptoms of lpr are often not typical, assessing the prevalence of lpr is quite difficult. (saleh et al., 2023) the high rate of middle ear disorders in patients with lpr in this study indicates that organ damage caused by lpr is not limited to the laryngeal pharyngeal organs but can also have an impact on surrounding organs, one of which is the ear. eustachian tube disorders caused by chronic reflux cause middle ear disorders. middle ear disorders can begin with inflammation of the tympanic cavity mucosa until the formation of secretions in the middle ear. the condition where sterile fluid forms in the tympanic cavum with an intact tympanic membrane is called otitis media effusion (ome). (lechien, 2022) one of the tympanometric parameters that is rarely used in assessing the condition of the middle ear is tw. it is known that the tw parameter has a high sensitivity and sensitivity value of 90% and 91% as an early predictor of middle ear disorders so that it can be considered for use in identifying middle ear disorders. the tw assessment itself can identify in quantity the widening of the tympanogram curve which indicates the presence of early phase middle ear disorders before the occurrence of more severe conditions such as the formation of effusion fluid in the tympanic cavum. the wider the tw result, the greater the likelihood of middle ear effusion. (zakaria et al., 2020) this study found a positive correlation between rsi score and tw parameter on tympanometry examination. the results of this study indicate that the relationship between rsi score and tw as one of the initial parameters of middle ear disorders can be considered. the use of tw assessment as one of the parameters in detecting the early phase of middle ear disorders is often not done because tw results are not automatically measured by the tympanogram tool. (flood, 2016) to date, there are no similar studies that specifically assess the relationship between lpr and tw parameters. the study showed a positive correlation between rfs score and middle ear pressure with a moderate category. this may be due to the fact that in patients with rfs scores > 7, there have been changes in the anatomical structure of the laryngopharynx and surrounding organs, one of which is the eustacian tube. tubal dysfunction can cause a decrease in middle ear pressure where the tympanogram results in a pressure < -100 dapa. (flood, 2016) based on the results of the correlation test conducted in this study, a strong correlation was found between lpr and middle ear disorders. the test results mean that if a patient has lpr, they have a risk of developing middle ear disorders as an extralaryngeal complication of lpr disease. the results of this study are in accordance with research conducted by zhen, et al. where the study found that patients with high rsi scores showed tubotympanometry results that lead to middle ear disorders. (karyanta et al., 2019) the results of this study further support the hypothesis that lpr can cause middle ear disorders. the mechanism of middle ear disorders is most likely due to dysfunction in the euthanized tube caused by reflux of gastric contents. in a study conducted by pang et al., saw that in patients with lpr there was a disturbance of acid-base balance in the middle ear due to reflux material that reached the middle ear organs. (pang et al., 2020) in addition, due to the complexity of reflux material in lpr, it causes inflammation of the mucosa lining the eustachian tube and middle ear. the inflammatory reaction causes obstruction of the tube resulting in impaired fluid drainage in the middle ear. another factor that can cause reflux in the middle ear is anatomical factors, where anatomically, there is no resistance that can stop the influx of fluid from the laryngopharyngeal organs to the eustachian tube so that 318 biology, medicine, & natural product chemistry 14 (1), 2025: 315-321 the spread of reflux material is more likely to occur. (brar et al., 2022) seeing the correlation between lpr and middle ear disorders in lpr patients, the results of this study can be taken into consideration in the management of patients with impaired tubal function or ome who do not respond to definitive therapy. (han et al., 2018) in a case study conducted by joanna, et al. where the study evaluated the effectiveness of ppi therapy for 4 8 weeks in children with ome compared to placebo. this study saw clinical and symptomatic improvement in ome patients who were given ppi therapy. however, other risk factors for ome should be considered further. (hanrahan et al., 2024) some of the limitations in this study are the limited number of research samples for which correlation tests were carried out so that an assessment of the magnitude of the influence of the two variables could not be carried out. another weakness of this study is that the researchers excluded patients with ear disorders in the selection of research subjects, where there is still a possibility that the ear disorders are caused by the chronic lpr disease process. some research suggestions that can be considered to overcome the limitations in this study are data collection with a longer duration so that the number of research samples is larger, confirming the diagnosis of lpr by using objective support examinations such as 24 hour dual probe ph monitoring, using other parameters in detecting disorders of the eustachian tube such as eustachian tube function (etf) examination. conclusions there is a correlation between the rsi score and the tympanometric parameter tympanogram width (tw) and there is a correlation between the rfs score and middle ear pressure. this study observed a strong positive correlation between lpr and middle ear disorders. authors’ contributions: mdm contributed to the research idea, supervised the project, and wrote the manuscript. mak contributed to the data extraction, statistical interpretation, and creation of tables. all the authors have read, edited, and approved the final manuscript competing interests: the authors declare that there are no competing interests references brar, s., watters, c., watson, n., birchall, m., & karagama, y. 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(2020). the diagnostic value of tympanometric width in identifying middle ear disorders. indian journal of otology, 26(3), 186. doi: 10.4103/indianjotol.indianjotol_120_19 320 biology, medicine, & natural product chemistry 14 (1), 2025: 315-321 table s1. characteristics of lpr patients with middle ear disorders at ent clinic saiful anwar hospital on january 2024-august 2024. characteristics minimum score maximum score average score rsi score 14 33 21.76 ± 5.56 rfs score 8 25 16.92 ± 5.57 middle ear compliance 0.15 1.9 0.76 ± 0.56 middle ear pressure 180 2 -43.20 ± 50.17 tympanogram width 56 400 166.88 ± 76.42 table s2. middle ear characteristic in lpr patients based on tympanometry examination at the ent clinic saiful anwar hospital on january 2024august 2024. variables right ear left ear middle ear compliance n % n % < 0.3 9 36 8 32 0.3 1.6 14 56 16 64 >1.6 2 8 1 4 total 25 100 25 100 middle ear pressure n % n % < -100 2 8 3 12 > 100 23 92 22 13 total 25 100 25 100 tympanogram width (tw) n % n % > 114 16 64 16 64 51-114 9 36 9 36 total 25 100 25 100 middle ear disease n % yes 22 88 no 3 22 total 25 100 table s3. spearman correlation test of reflux symptom index (rsi) score and tympanogram width (tw) middle ear. correlations rsi tympanogram width spearman's rho rsi correlation coefficient 1,000 .502 * sig. (2-tailed) . .011 n 25 25 tympanogram width correlation coefficient .502 * 1,000 sig. (2-tailed) .011 . n 25 25 table s4. spearman correlation test of reflux finding score (rfs) and middle ear pressure. correlations rsi pressure ear middle spearman's rho rsi score correlation coefficient 1,000 .440 * sig. (2-tailed) . .028 n 25 25 pressure middle ear correlation coefficient .440 * 1,000 sig. (2-tailed) .028 . n 25 25 table s5. spearman correlation test of reflux symptom index (rsi) score and middle ear pressure. correlations rsi pressure ear middle spearman's rho rsi score correlation coefficient 1,000 -.121 sig. (2-tailed) . .564 n 25 25 pressure ear middle correlation coefficient -.121 1,000 sig. (2-tailed) .564 . n 25 25 murdiyo & kurniadi – laryngopharyngeal reflux (lpr) correlation to middle ear disorders 321 table s6. spearman correlation test of reflux symptom index (rsi) score and middle ear compliance. correlations rsi compliance spearman's rho rsi correlation coefficient 1,000 .048 sig. (2-tailed) . .818 n 25 25 compliance correlation coefficient .048 1,000 sig. (2-tailed) .818 . n 25 25 table s7. spearman correlation test of reflux finding score (rfs) and middle ear compliance. correlations rfs compliance spearman's rho rfs correlation coefficient 1,000 -.375 sig. (2-tailed) . .065 n 25 25 complia nce correlation coefficient -.375 1,000 sig. (2-tailed) .065 . n 25 25 table s8. spearman correlation test of reflux finding score (rfs) (rfs) and tw. correlations rfs tympanogram width spearman's rho rfs correlation coefficient 1,000 -.354 sig. (2-tailed) . .082 n 25 25 tympanogram width correlation coefficient -.354 1,000 sig. (2-tailed) .082 . table s9. chi square correlation test of lpr and middle ear disorders. chi-square tests value df asymp . sig. (2-sided) exact sig. (2-sided) exact sig. (1-sided) pearson chi-square 33.440 a 1 .000 continuity correction b 30,013 1 .000 likelihood ratio 42,651 1 .000 fisher's exact test .000 .000 linear-by-linear association 32,680 1 .000 n of valid cases 44 table s10. chi square correlation test result of lpr and middle ear disorders. . symmetric measures value approx. sig. nominal by nominal contingency coefficient .657 .000 n of valid cases 44 table s11. interpretation of correlation coefficient for chi square test 35 . correlation coefficient value relationship level 0.80 – 1.00 very strong 0.60 – 0.799 strong 0.40 – 0.599 currently 0.20 – 0.399 low 0.00 – 0.199 very low this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 567-573 | doi: 10.14421/biomedich.2025.141.567-573 issn 2540-9328 (online) phytochemistry, toxicity, and antimalaria activity of mangrove plant from muara badak beach, east kalimantan, indonesia usman1,*, muh. amir masruhim1, agung rahmadani1, erwin2 1master study program of chemistry education and undergraduate study program of chemistry education, faculty of teacher training and education, mulawarman university, samarinda, indonesia. 2department of chemistry, faculty of mathematics and natural sciences, mulawarman university, samarinda, indonesia jl. muara pahu, kampus gn. kelua, samarinda, kalimantan timur, indonesia. corresponding author* sainusman@ymail.com manuscript received: 06 february, 2025. revision accepted: 27 june, 2025. published: 26 august, 2025. abstract this study aimed to determine the secondary metabolite content, toxicity, and antimalarial activity of methanol extracts of mangrove leaves avicennia marina, rhizophora mucronata, and sonneratia caseolaris. mangrove leaf extraction by maceration using methanol solvent, then qualitative phytochemical test (color test), toxicity test by bslt method using artemia salina, and antimalarial activity test using plasmodium falciparum strain 3d7 with giemsa staining microscopic method. the content of secondary metabolite compounds of methanol extracts of the three mangrove leaf species are alkaloids, flavonoids, saponins, phenolic compounds, steroids, triterpenoids and tannins. the toxicity of methanol extract of r. mucronata mangrove leaves against a. salina is included in the strong category. meanwhile, the toxicity of s. caseolaris is included in the moderate category and a. marina is in the weak category. mangrove species r. mucronata and s. caseolaris have good activity against p. falciparum 3d7 with ic50 values < 30 μg/ml, while a. marina shows poor activity against p. falciparum 3d7 with ic50 values > 50 μg/ml. other tissue parts of these three mangrove species still need further exploration for their bioactivity against a. salina and p. falciparum 3d7. keywords: mangrove plant; phytochemical; toxicity; antimalarial. introduction mangrove forests are plant communities located at the confluence of sea and land in tropical and subtropical regions, especially at latitudes 25°n. various species of mangrove plants can grow and thrive in areas of high salt content, tides, extreme temperatures, strong winds, anaerobic and muddy, and sandy soils with morphological adaptations to their environment (aljaghthmi et al., 2018; bibi et al., 2018). indonesia is one of the countries in asia with the largest mangrove forest with the highest mangrove species diversity in the world. this condition is supported by the geographical location, geological history and typology of the islands, as well as the unique oceanographic characteristics of indonesia mangrove forests function to protect coastal areas from damage due to sea water abrasion, keep the coastline stable, as a breeding ground for marine biota such as fish, crabs, and shrimp. mangrove plants also have various medicinal properties so that mangrove plants are widely used by people in coastal areas as traditional medicines (joandani et al., 2019; tefarani et al., 2019). people in coastal areas have long known and used mangrove plants as traditional medicines, especially to treat; skin diseases, rheumatism, blisters, arthritis, bleeding, asthma, sore throat, eye problems, stomach pain, infections, diabetes, hiv, hepatitis, smallpox, ulcers, diarrhea, malaria, astringent, aphrodisiac, antiulcer, antitumor, snake bite treatment, anti-aging drugs and anticancer. mangrove plants are utilized in traditional medicine because they contain active chemical compounds. (lopez et al., 2018; lalitha et al., 2021; syamsul e.s., 2022). active chemical compounds that are thought to act as drugs to fight various diseases are compounds of the alkaloid group, steroids, triterpenes, phenolics compounds compounds, flavonoids, terpenoids, stilbenes, carotenoids, anthocyanins, anthocyanidins, inositol, saponins, long chain alcohols, tannins, amino acids, benzoquinones, coumarins, quinins, chalcones, lipid compounds, phorbol esters, rotenone, polyphenols, benzofurans, limonoids, sulfur procyanidins, gibberellins and xyloccensins (kartikaningsih et al., 2024; usman et al., 2023). several indonesian mangrove species such as s. caseolaris, a. marina, r. mucronata, and r. apiculata have pharmacological activity as potent abiomicrobials. https://doi.org/10.14421/biomedich.2025.141.567-573 mailto:sainusman@ymail.com 568 biology, medicine, & natural product chemistry 14 (1), 2025: 567-573 a. marina, has activity as an anticancer. a. corniculatum, a. aureum, a. alba, and r. mucronata showed cytotoxic activity against widr cancer cells. a. illicifolius, a. marina and e. agallocha showed signifi cant analgesic activity. other plant species from the family meliaceae, rubiaceae, piperaceae, acanthaceae, myrtaceae, and myrsinaceae, were shown to have antimalarial activity. then the study reported that meliaceae and rhizophorae have antimalarial activity (audah et al., 2022; arbiastutie et al., 2022). this means that the content of secondary metabolite compounds of mangrove plants a. marina, r. mucronata, and s. caseolaris originating from the coastal beach of muara badak district, kutai kartanegara regency, east kalimantan, has the potential to find bioactive compounds as natural antimalarial alternative medicinal ingredients. figure 1. mangrove plant species a. marina, r. mucronata, and s. caseolaris. materials and methods materials the materials used in this study were the mangrove leaf samples of a. marina, r. mucronata, and s. caseolaris species. phytochemical test reagents include alkaloids, flavonoids, phenolics compounds compounds, steroids, triterpenoids, saponins, and tannins. methanol solvent, h2so4(p) solution, hno3(p) solution, hcl(p) solution, and dmso solution, artemia salina shrimp larvae and p. falciparum strain 3d7. the instruments used were glassware generally used in the laboratory, analytical balances, vials, aluminum foil, test tube, micro*pipette, measuring flask, volume pipette, aerator, study lamp, mikroskop, and rotary evaporator. preparation of sample the samples used in this study were mangrove species of a. marina, r. mucronata, and s. caseolaris. samples were taken from the coastal beach, muara badak district, kutai kartanegara, east kalimantan. then, the sample was washed and dried by air drying without direct sunlight. the dried samples were mashed using a blender until they became powder. after obtaining the sample in powder form, the sample was macerated. the sample powder was weighed as much as 500 g, then put into a beaker and added methanol solvent to cover the entire surface of the sample and then covered with aluminum foil. after 24 h, the results of the first maceration can be taken and then macerated again on the sample dregs up to 3 x 24 h. the methanol extract obtained from the first to third maceration was collected and then concentrated using a rotary evaporator to obtain a concentrated methanol extract of a. marina, r. mucronata and s. caseolaris mangrove leaves (mitra et al., 2021). phytochemical test a phytochemical test is a qualitative test carried out by observing the occurrence of color changes after the extract is added to the reagent. phytochemical tests are carried out to determine the presence or absence of secondary metabolite compounds contained in a sample. in this study, phytochemical tests were carried out to determine the secondary metabolites contained in a. marina, r. mucronata, and s. caseolaris mangrove leaves (harahap et al., 2021; akasia et al., 2021). alkaloid test the alkaloid test was carried out by mixing 10 ml of a. marina, r. mucronata, and s. caseolaris mangrove, which was heated, cooled and then filtered. 2 drops of the filtrate were added to the drip plate, 2 drops of meyer's reagent, dragendroff's reagent, and wagner's reagent were added. observe the color changes that occur. a positive indicator of the alkaloid test on mayer's reagent is the formation of a white precipitate. the alkaloid test is said to be positive in the wagner test if there is a brownish/brick red precipitate, and a positive test on the dragendrof reagent is said to be positive if there is a brownish/brick red/red-orange precipitate. phenolics compound test the phenolics compounds test was carried out by adding 2 drops of a. marina, r. mucronata, and s. caseolaris mangrove filtrate to a drip plate, adding 1% fecl3 solution, then observing the color changes. a positive usman et al. – phytochemistry, toxicity, and antimalaria activity of mangrove plant … 569 indicator of the phenol test is the formation of a blueblack color. saponin test the saponin test was carried out by mixing 2 drops of a. marina, r. mucronata, and s. caseolaris mangrove filtrate into a drip plate. then 2 ml of distilled water was added, then shaken until a stable foam was formed, then 1 drop of 2n hcl was added. a positive indicator of the saponin test is the formation of a stable foam. flavonoid test flavonoid test was carried out by mixing 2 drops of a. marina, r. mucronata, and s. caseolaris mangrove filtrate with 5 ml of methanol, then adding a few drops of concentrated hcl and 1.5 g of magnesium powder. a positive indicator of the flavonoid test is the formation of a red color. steroid and terpenoid test triterpenoid and steroid tests were carried out by adding 2 drops of the filtrate to the drip plate, then adding one drop of acetic anhydride and one concentrated sulfuric acid (liebermann burchard reagent). a positive indicator of the terpenoid test is the formation of a red or purple color and a positive steroid if the solution is blue or green. toxicity test against a. salina the extracts of a. marina, r. mucronata, and s. caseolaris mangrove were prepared as 1000 mg/l mother liquor by dissolving 3.5 g of sample in 50 ml of methanol. the mother liquor was then made into a solution with various concentrations of 125 mg/l, 250 mg/l and 500 mg/l (khasanah et al., 2020). the next step in the toxicity test using the bslt method is hatching shrimp larvae. the hatching of eggs is carried out in an artificial aquarium container in the form of a tube, which is assisted by a 5 w incandescent lamp to stimulate the growth of larvae. the medium used to incubate shrimp larvae is artificial seawater, a mixture of distilled water and pure salt. the oxygen level needed during hatching must be more than 3 mg/l, therefore the artificial seawater media must be aired with an aerator. 0.5 mg/ml of yeast was added as a source of nutrition for a. salina. within 24-36 h, usually the eggs have hatched into larvae called naupli. active nauplii aged for 48 h are used as test animals in the experiment. vials were provided for each group according to the concentration level and were repeated 2 times. the parent solution of a. marina, r. mucronata, and s. caseolaris mangrove was added to the vial according to the concentration level. the vial containing the test solution was dried until the solvent had evaporated for several days at room temperature in a desiccator so that only the scale remained from the sample extract and no longer smelled of solvent. then added 2 drops of 1% dmso to dissolve the sample. after the sample was dissolved with dmso, 1 ml of artificial seawater was added, then 10 a. salina l shrimp larvae aged 48 h were added to the vial. one drop of yeast (0.6 mg/ml) was put into each vial as food for a. salina, then artificial seawater was added to the volume limit of 10 ml. the standard criterion for assessing the mortality of shrimp larvae is if the shrimp larvae do not show movement for a few seconds of observation. the manual method is to observe the larvae in the vial (kurniawan et al., 2021). antimalarial test samples of methanol extract of a. marina, r. mucronata, and s. caseolaris mangrove leaves as much as 10 mg were dissolved in 1000 ml l % dmso solution to make a test solution. the resulting test solution was then made into various concentrations by diluting it, so that the obtained concentrations of 100 μg/ml, 50 μg/ml, 10 μg/ml, 1 μg/ml and 0.1 μg/ml, and 0.01 μg/ml. prepared test parasites that will be used have been synchronized as a ring stage with parasitemia ± 1% (hematocrit 5%). a test well (well 96) was prepared, 2 μl of the test solution was added with various concentrations. after that, 198 μl of the parasite was added at each concentration of each test solution. the test was repeated twice (duplo). the test well is then put into the chamber and given mixed gas as 5% o2, 5% co2, and 90% n2. incubated for 2 x 24 h at 37°c. then, after 2 x 24 h, the cultures were harvested and a thin blood film was prepared with 20% gymnasia staining. then, data analysis was performed by counting the number of infected erythrocytes per 1000 normal erythrocytes under a microscope. the data is then used to determine the percent growth and percent inhibition. after obtaining the percent inhibition value, the data were analyzed using the spss version 20 probit analysis program to obtain the ic50 value, which is the concentration of the test material that can inhibit the growth of parasites by 50% (wardani et al., 2020). results and discussion result phytochemical of mangrove plants based on the results of phytochemical tests of methanol extracts of mangrove plant leaves (a. marina, r. mucronata, and s. caseolaris) is known to contain secondary metabolite compounds such as alkaloids, flavonoids, phenol compounds, steroids, triterpenoids, saponins, and tannins. the complete results of the phytochemical test of methanol extracts of the three mangrove leaf species are presented in table 1. 570 biology, medicine, & natural product chemistry 14 (1), 2025: 567-573 table 1. phytochemical screening of the third mangrove species. secondary metabolite compounds methanol extract of mangrove leaf a. marina r. mucronata s. caseolaris alkalaoids + + flavonoids + + saponins + + + fenolic compound + + + steroids + + triterpenoids + tannins + + toxicity against a. salina toxicity tests in this study used the brine shrimp lethality test (bslt) method to determine the toxicity of methanol extracts from mangrove leaves (a. marina, r. mucronata, and s. caseolaris) against a. salina. the toxicity of methanol extracts of the three mangrove leaf species was determined by calculating ic50 values using sas probit analysis. lc50 (lethal concentration 50%) value is the dose value of a compound that can kill 50% of test animals. the results of calculating the lc50 value of the three methanol extracts of mangrove leaves are presented in table 2. table 2. the lc50 value and toxicity level of mangrove species a. marina, r. mucronata and s. saseolaris. methanol extract of mangrove leaf lc50 value (ppm) category toxicity a. marina 256,132 low r. mucronata 48,165 strong s. caseolaris 104,96 moderate antimalarial activity the antimalarial test of methanol extracts of mangrove leaves of a. marina, r. mucronata, and s. caseolaris was carried out in vitro with giemsa staining test method. this test uses p. falciparum strain 3d7. antimalarial test data were obtained using spss probit analysis to determine the ic50 value of the methanol extract of mangrove leaves of a. marina, r. mucronata, and s. caseolaris. the ic50 values obtained are presented in table 3 and figure 2. table 3, the ic50 values of mangrove a. marina, r. mucronata and s. sasseolaris. methanol extract of mangrove leaf treatment of concentration variation (μg/ml) ic50 (μg/ml) (μg/ml) 100 50 10 1 0,1 0,01 a. marina 52,93 45,93 40,23 22,80 10,91 2,36 57,341 r. mucronate 73,04 52,28 37,13 17,59 12,05 6,76 24,118 s. casseolaris 100 63,76 36,32 14,66 9,85 0,24 21,975 figure 2. ic50 values (antimalarial activity) of methanol extracts of mangrove leaves of a. marina, r. mucronata and s. caseolaris. discussion phytochemical according to table 1. it is known that the methanol extract of a. marina mangrove leaves contains secondary metabolite compounds; saponins, phenolics compounds, triterpenoids, and tannins. r. mucronata leaf extract contains compounds; alkaloids, flavonoids, saponins, phenolics compounds, steroids, and tannins. while s. caseolaris mangrove leaf extract contains alkaloid compounds, flavonoids., saponins, phenolics compounds, and steroids. in the research of audah et al., (2022) reported that s. caseolaris mangrove leaf extract contains compounds; steroids, flavonoids, and tannins. a. marina leaf extract is; steroids, flavonoids, saponins, and tannins. and r. mucronata leaf extracts are steroids, flavonoids, saponins, and tannins. then muhaimin, et al., (2019), stated that the content of secondary metabolite compounds in methanol extracts of s. caseolaris leaves are alkaloids, flavonoids, phenolics compounds, steroids, tannins, quinones, and glycosides. according to akasia et al., (2021) the secondary metabolite compounds contained in mangrove extracts of s. caseolaris are flavonoids, saponins, phenolics compoundss and steroids. in other studies, it has been reported that the secondary metabolite compounds of r. mucronata are alkaloids, flavonoids, lipids, inositol, triterpene, phenolics compoundss compounds, and tannins (hardoko et al., 2016; taniguchi et al., 2018). it was reported that the methanol extract of r. mucronata stem bark has successfully isolated six compounds, including cinchonain ib, breynioside b, polystachyol, β-sitosterol 3-o-β-d-glucopyranoside, β-sitosterol 3-o-β-d-(6'-opalmitoyl) glucopyranoside, and β-sitosterol 3-o-β-d(6'-o-stearoyl) glucopyranoside (linh et al., 2020). 57,341 24,118 21,975 0 10 20 30 40 50 60 70 a. marina r. mucronate s. caseolaris n il ai i c 5 0 ( μ g /m l methanol extract of mangrove leaf usman et al. – phytochemistry, toxicity, and antimalaria activity of mangrove plant … 571 the difference in the content of phytochemical compounds in the three mangrove plant species may be due to the location of their habitat, where each location of mangrove plant habitat has a different ph value so that it can affect the content of secondary metabolite compounds contained in mangrove plants. then the solvent used during extraction also affects the content of the compounds obtained (akasia et al, 2021). toxicity according to the data presented in table 2, it shows that the methanol extract of r. mucronata mangrove leaves has the lowest lc50 value of 48,165 ppm, this shows that the methanol extract of r. mucronata mangrove leaves is toxic to a. salina with a strong category. while the toxicity of the methanol extract of s. caseolaris mangrove leaves has an lc50 value of 104.96 ppm with a moderate category, then the toxicity of the methanol extract of a. marina mangrove leaves has an lc50 value of 256.132 ppm with a weak category. meyer et al., (1982) stated that the toxicity level of plant extracts can be determined by looking at the lc50 value. if the lc50 value is smaller than 1000 ppm, it is considered toxic, whereas if the lc50 value is higher than 1000 ppm, it is considered non-toxic. under the meyer category, it can be declared that the methanol extract of mangrove leaves a. marina, r. mucronata, and s. caseolaris, are toxic to a. salina with an lc50 value of < 1000 ppm. audah et al. (2022), reported that the water extract of s. caseolaris leaves, ethanol extract of a. marina mangrove leaves, and n-hexane extract of r. mucronata are toxic to a. salina, each with an lc50 value of 229.77 ppm and 160.43 ppm is toxic to a. salina with an lc50 value of 488.93 ppm. bokshi b. (2020), reported that the ethyl acetate fraction of the stems and the carbon tetrachloride fraction of the leaves of s. caseolaris mangrove showed very strong toxicity properties against a. salina, with lc50 values of 25.0±0.05 and 25.0±0.07 μg/ml, respectively. according to rozirwan et al. (2022), it was reported that a. marina mangrove leaf extract contains bioactive compounds such as alkaloids, saponins, flavonoids, steroids and steroids which are toxic to a. salina which can be developed for the pharmaceutical field. based on the brine shrimp lethality test (bslt), ethanol, ethyl acetate, and nhexane extracts of r. mucronata mangrove are classified as toxic to a. salina, and the secondary metabolite compounds found in the ethanol extract are terpenoids, alkaloids, ketones, phenols, turpentine oil, essential oils, unsaturated fats, and camphor (zulfahmi i et al., 2024) the bioactivity of medicinal plants can be detected by the content of phytochemical compounds in plants. from the results of the phytochemical test, it is known that the methanol extract of r. mucronata mangrove leaves contains alkaloids and flavonoids, where the two compounds are known to be toxic because they can work as respiratory tract poisons and even alkaloids can cause stomach poisoning, thus inhibiting the ability of organisms to eat. at the same time, alkaloid compounds can also block taste receptors in the mouth area of a. salina larvae, so that larvae do not get taste stimuli and are unable to recognize their food and as a result a. salina larvae die of starvation (davis et al., 2019). the three mangrove leaf extracts in this study were positive for saponin compounds. saponin compounds can affect the life of a. salina larvae, because the glycosides contained in saponins can bind oxygen in water, so that the oxygen content in the solution is reduced and as a result the larvae will die from lack of oxygen (khasanah et al., 2020). while the methanol extract of mangrove leaves a. marina does not contain alkaloids and flavonoids, so the level of toxicity is lower than mangrove r. mucronata and s. caseolaris. antimalarial activity according to the results obtained (as presented in table 3 and figure 3) methanol extracts of mangrove leaves a. marina, r. mucronata, and s. caseolaris can inhibit the growth of p. falciparum strain 3d7 respectively with ic50 values of 57.34; 24.12; and 21.97 µg/ml. according to gessler, the level of antimalarial activity with in vitro methods can be categorized into 3 groups, namely very good if the ic50 value is < 10 µg/ml, a fairly good category if the ic50 value is between 10 50 µg/ml, and less active of category if the ic50 value is > 50 µg/ml (gessler et al., 1994). depending on the ic50 value, a. marina mangrove leaf extract has antimalarial activity with less active of category because the ic50 value > 50 µg/ml. meanwhile, methanol extracts of mangrove leaves of r. mucronata and s. caseolaris showed fairly good antimalarial activity with ic50 values between 1050 µg/ml. r. mucronata mangrove plants contain saponin compounds, which can act as an inhibitory mechanism against malaria by forming complexes with cell membranes through hydrogen bonds, thereby damaging the permeability of malaria cell walls and causing death. saponins are also substances that can hemolyzes blood. therefore, saponins can hemolyzes malaria cell membranes in the same way as red blood cell membranes (juniharti et al., 2021; gunawan*et al., 2018). r. mucronata leaf extract also contains alkaloid compounds, where these alkaloid compounds can prevent parasite growth in the blood and prevent the formation of peptidoglycan so as to prevent the formation of cell walls due to cell breakdown. the mechanism of action of alkaloids as antimalarial drugs is to prevent the detoxification of hemiparasites in the food vacuole. likewise, the leaves of mangrove s. caseolaris also contain saponin compounds, so they act as substances that can hemolyzes blood. the mechanism of flavonoid compounds as antimalarial agents is to block the nutrient channels needed by parasites by forming specific membranes (uzor p. f, 2020; variani et al., 2021). however, a. marina mangrove leaf extract does not contain alkaloids and flavonoids, so it does not have 572 biology, medicine, & natural product chemistry 14 (1), 2025: 567-573 antimalarial activity and has no potential as a malaria herbal medicine. the accumulation of phytochemical compounds contained in the extracts of mangrove leaves r. mucronata and s. caseolaris can provide antimalarial activity by increasing red blood cell oxidation or inhibiting protein synthesis, thus becoming a factor that influences the antimalarial potential of mangrove plants r. mucronata and s. caseolaris (ahmed et al., 2010). r. mucronata extract is also known to have antidiabetic, antioxidant, and tissue-enhancing effects that are potentially compared to a. marina (al-jaghthmi et al., 2020). in another study it was reported that ethanol extract derived from s. alba leaves showed significant antiplasmodial efficacy in reducing parasitemia levels, inducing significant inhibition percentages, and inhibiting plasmodium growth. thus, s. alba leaves have the potential to be a promising natural source of antimalarial agents because ethanol extract derived from s. alba leaves has diverse phytochemical constituents that are efficacious as antiplasmodial. (muhaimin m et al., 2024). traditional medicine using mangrove skin, bark, fruit, and flowers from the species lumnitzera littorea, rhizophora mucronata, scyphiphora hydrophyllacea, sonneratia alba, xylocarpus granatum, and xylocarpus moluccensis is a popular method used as a malaria drug in people's social life. (tamalene m.n. et al., 2021). conclusion the secondary metabolite compounds contained in the methanol extract of mangrove leaves r. mucronata and s. caseolaris have antimalarial activity against p. falciparum starin 3d7 with a fairly good category including the ic50 value between 10 50 µg/ml, hence the two species of mangrove have potential to be developed as a natural antimalarial herbal medicine. the mangrove species a. marina, r. mucronata and s. caseolaris are toxic to a. salina with a strong category (lc50 value < 1000 ppm), hence that the third species of mangrove can be examined by cancer cells to determine its potential as a natural anticancer medicinal material. acknowledgement: we acknowledge, institute of tropical disease (itd) airlangga university, chemistry education laboratory, and pharmacy laboratory of mulawarman university for all the support to carry out work. conflict of interest: the authors declare that they have no conflict of interests. references ahmed r., moushumi s. j., ahmed h., ali m. 2010. serum glucose and lipid profiles in rats following administration of sonneratia caseolaris (l.) engl. 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non-mangroves. international journal of applied earth observation and geoinformation. 108 (2022) 1–17. https://doi.org/10.1016/j.jag.2022.102750 https://doi.org/10.35814/jifi.v19i1.985 https://doi.org/10.26538/tjnpr/v8i5.24 https://doi.org/10.33369/pendipa.4.1.47-53 https://doi.org/10.1016/j.sajb.2021.06.023 https://doi.org/10.15625/2525-2518/0/0/14783 http://dx.doi.org/10.5530/pj.2019.11.148 https://doi.org/10.26538/tjnpr/v8i4.19 https://doi.org/10.1016/j.sajb.2022.08.037 https://doi.org/10.3889/oamjms.2022.10558 https://doi.org/10.1016/j.bmcl.2018.02.022 https://doi.org/10.15294/lifesci.v8i1.29989 http://www.doi.org/10.26538/tjnpr/v7i10.31 https://doi.org/10.1155/2020/8749083 http://dx.doi.org/10.20885/ijca.vol4.iss2.art3 https://doi.org/10.46542/pe.2021.212.2730 https://doi.org/10.1016/j.jag.2022.102750 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 45-54 | doi: 10.14421/biomedich.2022.111.45-54 issn 2540-9328 (online) the roles of the fluorescent in situ hybridization (fish) and comparative genomic hybridization (cgh) techniques in the detection of the breast cancer harem othman smail department of biology, faculty of science and health, koya university, koya koy45, kurdistan region-f.r. iraq. corresponding author harem.othman@koyauniversity.org manuscript received: 25 february, 2022. revision accepted: 11 march, 2022. published: 24 march, 2022. abstract this paper aimed to understand and compare the two popular cytogenetic techniques of fluorescence in situ hybridization (fish) and comparative genomic hybridization (cgh) in detecting breast cancer chromosomal abnormality. several chromosomal anomalies play a role in the development of breast cancer, and the two above approaches play an important role in confirming fluorescence in situ hybridization in particular (fish). however, comparative genomic hybridization has developed dna copy number profiles for most of the publicly available breast cancer cell lines for the fish methods rely on the fluorescent probes. chromosomal profiles can be generated for the suspected chromosomal abnormality, copy number changes between the tumour and the dna control can be compared, and the results can be registered. today, modern cytogenetic tools such as fluorescence in situ hybridization (fish) are more commonly used to detect any microdeletion that cannot be detected by conventional cytogenetic karyotypes that involve a high rate of cell division and good chromosomal morphology, which pose challenges for cytogeneticists, and a long period of testing and research. usually, this is a problem for physicians, and there are still many drawbacks and disadvantages concerning the high benefits, such as false findings. normal chromosome in situ hybridization requires the hybridization of a labelled dna probe into denatured chromosomal dna present in metaphase chromosomes in an air-dried microscope slide preparation. metaphase spreads are used for traditional chromosome fish (metaphase fish). positive and positive signs of hybridization also appear as a double spot, corresponding to the hybridized probe for both sister chromatids. a further extension of chromosome painting is comparative genomic hybridization (cci-i). cch involves simultaneous chromosome painting in two different colours using complete dna from two similar sources as probes, which reveal variations concerning the benefit or loss of sub-chromosomal regions or even entire chromosomes. keywords: fish; cgh; probe; classical cytogenetic; chromosomal regions and hybridization. introduction in terms of histology, clinical response, diffusion trends to distant locations, and patient outcomes, breast cancer is heterogeneous (prat et al., 2011). nevertheless, the most common cause of death from cancer worldwide is breast cancer. rates differ about five-fold across the world, but they are growing in regions with low disease rates until recently (key et al., 2011). furthermore, the availability of cytogenetics and cytogenetics technologies has enhanced the detection and recognition of molecular tumour signatures and the understanding of the initiation and progression of cancer (ribeiro et al., 2019). the fluorescence in situ hybridization (fish) technique that plays a leading role in diagnostic pathology has provided valuable information on genomic variations in malignant cells for its single-cell study (das et al., 2013). fish is a molecular tool in which both metaphase chromosomes and interphase nuclei identify numerical and structural defects. fish is widely used to identify translocations and determine gene deletion and amplification in tumours as a prognostic and diagnostic method (brown et al., 2007). the technique utilizes the inherent capacity to hybridize complementary strands of dna or rna from various sources. the theory of annealing a labelled nucleic acid probe to complementary sequences inside cells or tissue mounted (in situ) on a microscope slide is based on in situ hybridizations (karimi et al., 2020). genomic changes, amplifications, and deletions may reduce the complexity of genomic data observed by comparative genomic hybridization (przybytkowski et al., 2014). by comparing the samples with reference dna, cgh detects changes in the copy number of individual chromosomes or chromosomal regions, such as changes in relative genome size and ploidy levels in test samples. this technique was initially designed to examine the differences between solid tumours and normal tissues in chromosomal supplements (zhang et al., 2015). it is expected that the precise classification of these genomic changes would have an important effect https://doi.org/10.14421/biomedich.2022.111.45-54 46 biology, medicine, & natural product chemistry 11 (1), 2022: 45-54 on translational and fundamental research (climen et al., 2007). for example, comparative genomic hybridization has been instrumental in the dissection of distinct molecular pathways to malignancy of the breast and developing a direct relationship between genotype and clinical pathology (reis et al., 2005). characterization types of breast cancer over 100 types of cancers consist of multiple subtypes capable of arising in a single type of organ or tissue. most cancers can be defined at a very general level based on their originating tissue location. in epithelial cells, the most common type of cancer, comprising more than 80 per cent of all cancers, occurs; these cancers are called carcinomas. depending on whether the epithelial cells of origin are part of the protective epithelial layer (squamous cell carcinomas) or have secretory properties (adenocarcinomas), most carcinomas can break into squamous cell carcinomas or adenocarcinomas (smail 2016 and smail 2020). breast cancers are a complex and heterogeneous community of diseases, and patients are treated with clinicopathological features and estrogen receptor status and her2 guidance. however, for the individualization of treatment, these factors are not necessary. microarray-based gene expression profiling has contributed to a paradigm change in the understanding of breast cancer. it has been shown conclusively that breast cancer is not a single disease (weigelt et al.,2010). joint hormone receptor-defined subtypes of breast cancer (h.r.; estrogen receptor [e.r.] and progesterone receptor [p.r.]) and her2 status (howlade et al., 2014). triple-negative (tnbc) breast cancers do not express hormone receptors or over-express her2. as characterized by low five-year survival and high recurrence rates after adjuvant therapy, poor prognosis is associated with these conditions. overall, tnbc has striking similarities with basal-like breast cancers (bbc), but they are found the same in various studies (de et al., 2011). five tumour subtypes (luminal a, luminal b, her2-enriched, basal, and claudin-low) have been identified in breast cancer gene expression studies, each of which has specific biological and prognostic features (prat et al.,2010). four major intrinsic molecular subtypes of breast cancer, known as luminal a, luminal b, her2-enriched [her2e], and basal-like, were identified in global gene expression analysis studies. these molecular entities have shown major differences in terms of occurrence, risk factors, baseline prognosis, age at diagnosis, and response to treatment. because of its high frequency, lack of successful targeted therapies, weak baseline prognosis, and propensity to impact younger women, the basal-like subtype is of particular clinical concern (prat et al., 2013). a promising new diagnostic area for estimating the risk for metastatic relapse and metastatic progression in cancer patients is the circulating tumor cell (ctc) study (lianidou et al., 2011). rna sequencing found that pdx cells developed in the mammary gland were identical to those studied in culture for global gene expression. carboplatin was cytotoxic to whim30 but not whim2 in vitro, while both lines were cytotoxic to bortezomib, dacarbazine, and cyclophosphamide. however, these drugs have proven unsuccessful in treating in vivo both primary and metastatic whim2 tumours. on the other hand, carboplatin and cyclophosphamide have successfully treated whim30 mammary tumours and reduced brain, liver, and lung metastatic burden (turner et al., 2018). the metastatic cascade is a series of biological processes that cause tumour cells to migrate from the primary site to a distant location and create new cancer growth. circular tumour cells (ctcs) play a crucial role in tumour propagation. the role of ctcs in treatment failure and disease progression can be explained by their association with biological processes, including epithelial-tomesenchymal transformation and self-seeding, defined as primary infiltration of the tumour or metastasis formed by more aggressive ctcs. ctcs are a rare and heterogeneous population of cells of proven prognostic and predictive value in some clinical contexts (mego et al., 2010). in addition to the typical hormone receptor-positive and hormone receptor-negative forms, studies of breast cancers using gene expression profiling have identified many major breast cancer subtypes. the luminal a and luminal b classes are the most reproducibly defined molecular subtypes among the hormone receptorpositive cancers. the major molecular subtypes identified in hormone-receptor-negative breast cancers are the her2 and basal-like classes. some studies have also described other molecular subtypes, such as luminal c and regular breast-like classes. still, they are less well characterized than luminal a, luminal b, her2, and basal types (schnitt 2010). several molecular-targeted therapies for breast cancer have been tested since the application of endocrine therapy for estrogen receptor (e.r.)-positive tumour types1. an example of effective gene-targeted therapy is genome alteration-matched treatment of breast cancer to target amplification of human epidermal growth factor receptor 2 genes (erbb2 receptor tyrosine kinase, erbb2 also referred to as her2). gene expression-based molecular subtyping has also been broadly applied to breast cancer to aid treatment decisions (chung et al., 2017). there are many cellular functions for brca1. dna repair, cellcycle regulation, transcriptional regulation, and chromatin remodelling have been implicated. on the contrary, functions assigned to brca2 were specifically limited to dna recombination and repair processes. in the regulation of rad51 activity, brca2 smail – the roles of the fluorescent in situ hybridization (fish) and … 47 has a role. rad51 is a highly conserved dna recombinase involved in double-strand break repair and replication fork arrest (da and lakhani, 2010). chromosomal abnormality in breast cancer the most common cytogenetic anomalies observed in human breast carcinoma are modifications to the long arm of chromosome 1 (bièche and lidereau, 1995). for four ductal breast carcinomas, the cytogenetic analysis showed a net benefit of 1 q in all tumours. the only improvement in the first tumour was that one chromosome 16 was replaced by a chromosome derivative consisting of 16p and 1q (pandis et al., 1992). active x chromosome duplication and xi loss characterized almost half of the cases of sporadic basallike cancers studied (richardson et al., 2006). grade i and tubular breast carcinomas have a limited number of genomic alterations with extremely recurring 16q losses, while grade iii breast carcinomas also have complex genotypes with 11q, 14q, 8p, 13q loss; 17q, 8q, 5p gain; and high-level gains (amplification) on 17q12, 17q2224, 6q22, 8q22, 11q13, and 20q13 (simpson et al.,2005). amplification of chromosome band 11q13 protooncogenes (myc, erbb2) and dna; tp53 mutation; and loss of heterozygosity of chromosome and chromosome arms 1, 3p, 6q, 7q, 8p, 11, 13q,16q, 17, 18q, and 22q are the main forms of genetic defects commonly found in breast tumours (bièche et al.,1995). in breast cancer, abnormalities of chromosome 17, recognized over two decades ago to be important in tumorigenesis, frequently occur. changes in unique chromosome 17 loci, including amplification of erbb2, loss of p53, loss of brca1, and amplification or deletion of top2a, are considered to play an important role in breast cancer pathophysiology (reinholz et al., 2009). a single amplicon spanning several megabases was originally thought to include 11q13 amplification. still, more recent data identified four core regions within 11q13 that can be amplified separately or separately together in various combinations (ormandy, 2003). chromosomal alterations can be tested using gbanding karyotype and multicolour fluorescence in situ hybridization (m-fish) on metaphases (rondón et al.,2014). fluorescence in situ hybridization (fish) allows the number of the gene or chromosome copies in archival tissues to be measured in situ and linked to morphology and clinical outcome (watters et al., 2003). for example, cytokinetic defects are characterized by chromosomal instability in an inherited cancer syndrome and may help explain why brca2-deficient tumours are also aneuploidy-deficient (daniels et al., 2004). the innovation is known as comparative genomic hybridization (cgh), sets out methods for evaluating the relative number of copies of nucleic acid sequences in or in parts of one or more subject genomes (e.g. a tumour cell) as a function of the position of certain sequences in the reference genome (e.g. a regular human genome) (pinkel et al., 2011). initial cgh array applications for breast cancer analysis and the mechanisms by which various types of copy number changes can occur have been described (albertson 2003). how does fluorescence in situ hybridization works? fluorescence in situ hybridization by a fluorescently labelled probe detects nucleic acid sequences that precisely hybridize within the intact cell to its complementary target sequence (moter and göbel, 2000). fluorescence in situ hybridization has been used in interphase nuclei to image complex genomic dna sequences. unreplicated dna parts give singlet hybridization signals in normal diploid cells, whereas replicated loci are characterized by doublets (selig et al., 1992). fish on 3d preserved nuclei, compared to fish on metaphase chromosomes and traditional interphase cytogenetics, needs special requirements concerning the consistency of the probe, fixation, and pretreatment steps of the cells to achieve the two objectives, namely the best possible preservation of the nuclear structure and at the same time, the efficient accessibility of the probe (cremer et al.,2012). the concepts of in situ hybridizations of fluorescence are as follows: (a) dna probe and a target sequence are the basic elements. (b) before hybridization, the dna probe is indirectly marked with a hapten, specifically marked with hapten by adding a fluorophore probe (right panel). (c) the labelled probe and the target dna are denatured to yield single-stranded dna. (d) then they are combined, which allows complementary dna sequences to be annealed. (e) if the probe has been indirectly labelled, the visualization of the non-fluorescent hapten using an enzymatic or immunological detection system involves an additional step. finally, fluorescence microscopy tests the signals. speicher and carte adapted from (speicher et al.,2005 and bishop, 2010). hapten binding is visualized using a subsequently applied fluorochromeconjugated antibody (waminal et al.,2018). the choice of the probe is one of the most significant factors in fish research. it is possible to use a large variety of probes, from whole genomes to tiny cloned probes (1–10 kb). there are three types of probes in general, each with several applications: fullchromosome painting probes, repetitive sequence probes, and locus-specific probes (bishop, 2010). wellprepared chromosome distribution and an effectively labeled probe are prerequisites for a good fish experiment. nick-translation, random priming, and pcr provide multiple enzymatic methods for labelling probes. the most common use of nick-translation is 48 biology, medicine, & natural product chemistry 11 (1), 2022: 45-54 (gozzetti and le, 2000). in conducting efficient 3d fish experiments, there are two major considerations. to maintain nuclear morphology as much as possible while keeping dna sufficiently accessible for probe hybridization, the option of cellular treatments, including fixation, pre-and post-hybridization steps (bolland et al., 2013). figure 1. the fish spot detection algorithm (les et al., 2014). figure 2. fluorescence in situ hybridization (fish) applications in genetic diagnostics on ffpe content in solid tumours: a-1p/19q probe: a1 deletion of 1p32 locus, a-2 natural signal pattern (cell on the left) and 19q13 locus deletion (cell on the right), a-3 normal signal pattern (abbott molecular), b-dual fusion probe: col1a1 and pdgfb loci fusion and normal signal pattern (zytovision), c-break apart probe: c-1 alk gene rearrangement, c-2 normal signal pattern (abbott molecular), d-break apart probe: ewsr1 locus rearrangement (abbott molecular), f-break apart probe: f-1 rearrangement (empire genomics), g-locus specific probe: g-1 her2 locus amplification, g-2 normal signal pattern (abbott molecular), e-break apart probe: normal ss18 locus signal pattern, green colour—pdgfb gene locus, yellow colour—the fusion of col1a1pdgfb and pdgfb-col1a1 (chrzanowska et al.,2020). figure 3. detection of di-centric, tri-centric, and tetra-centric ring chromosome 18 using a centromeric probe d18z2 for chromosome 18. the left panel shows regular chromosome 18, the top dicentric ring 18 and the bottom tetracentric ring 18. the right panel shows dicentric ring 18 and fish insets pericentric/tetracentric ring 18. (b) identification of a 2q32/16p13.3 translocation derivative of chromosome 16 by complete chromosome painting probes for chromosome 2 (wcp2) and 16 (wcp2) (wcp16). (c) identification by dual-colour double fusion probes of abl1/bcr gene fusions in interphase and metaphase cells (thin arrows point to the normal signal and thick arrows point to the abnormal fusion signals). (d) diagnostic use of the etv6 and runx1 probes for the identification of two cryptic t(12;21) (p13;q22) fusion signals, loss of etv6 signal and gain of three additional runx1 signals (thin arrows point to the fusion signals and thick arrows to extra runx1 signals). all images are from yale clinical cytogenetics laboratory (cui et al.,2016). smail – the roles of the fluorescent in situ hybridization (fish) and … 49 figure 4. using whole-chromosome-painting probes, fluorescence in situ hybridization (fish). to rule out the presence of an additional copy of chromosome 19 (i.e. trisomy 19) in this metaphase distribution, derived from a glial cell taken from a mouse's brain, chromosome painting probes were used. chromosome 19 is green in colour (mcneil and ried 2000). important comparative genomic hybridization in early detection of breast cancer comparative genomic hybridization (cgh) has emerged to promote the aggregation of high-resolution data of cancer-associated genomic imbalances as a highthroughput genomic technology. high-resolution genomic microarray cgh (li et al., 2020). the rapidly growing cgh publication database already contains around 1500 tumours and is beginning to reveal genetic anomalies characteristic of certain forms of tumour or stages of tumour progression (forozan et al.,1997). comparative genomic hybridization has created dna copy number profiles for most of the publicly available breast cancer cell lines (forozan et al., 2000). highresolution cgh analysis of breast cancer shows nonrandom associations between particular amplicons in many regions where dna copy number is frequently obtained or lost. that specific genetic modification is preserved in breast cancer cell lines despite repeated passage through tissue culture (climent et al., 2007). recent results released and discussed at scientific meetings have suggested higher rates of implantation and pregnancy after microarray testing, resulting in changes expected for quite some time. by using markers spanning much of the genome, it is not only possible to detect aneuploidy in single cells but also translocations. the validation results indicate that the cgh array in single cells has a resolution of 6 mb. therefore, most translocations can be tested as this is also the limit of karyotyping. translocations of smaller exchanged fragments may also classify the translocation, as three out of the four fragments are above 6 mbb fragment ((munné, 2012). over the past decade, genomic microarray technology has greatly matured. the technique offers a locus-by-locus measure of the variance of dna copy-number (cnv) and represents another way to improve mapping resolution. postnatal chromosomal array techniques have far higher diagnostic results (15-20 per cent) than g-banded karyotyping does for genetic testing of people with unexplained developmental delay, intellectual impairment, autism, or other congenital abnormalities, as the first-tier cytogenetic diagnostic test for people with these diseases, the international standards for cytogenomic array consortium recommends it (lee et al.,2012). the genotyping of metastatic samples, primarily focused on array-based comparative genomic hybridization (acgh) and next-generation sequencing, is the growth of targeted therapies and the emergence of personalized medicine (ngs), another solution to resolving acgh's drawbacks, such as the repeat-rich regions, is next-generation sequencing. the genomic analysis should be paired with expression analysis to elucidate individual genes related to breast cancer development and progression. identification of new molecular targets for breast cancer eradication will contribute to the elucidation of the functions of the affected genes (ueno et al., 2012). figure 5. the photomicrograph of fluorescence shows the effects of comparative genome hybridization of invasive ductal carcinoma tissue. (a) tumour tissue extracted dna was labelled green, and (b) natural reference dna was labelled red. (c) the chromosome of the regular metaphase was counterstained blue with dapi. (d) tumour and normal dna have been hybridized to the normal chromosome of the metaphase. there is a 50 biology, medicine, & natural product chemistry 11 (1), 2022: 45-54 predominantly green colour in chromosomal regions, which were overrepresented in the tumour, while regions with deletions in the tumour show a predominantly red colour. the overlap reflects the ratio of changes in the copy number between the tumour and the dna control (zhang et al., 2015). comparative classical cytogenetic tools with fluorescence in situ hybridization hybridization of fluorescence in situ (fish) enables cytogenetic analyses of primary tumours without culture (thompson and gray 1993). in the identification and evaluation of human malignancies, one of the greatest impacts has been in the non-dividing interphase nucleus, chromosome translocations, deletions, amplification of particular genes, and chromosome number changes identified using probes ranging from whole chromosome 'paints' to individual gene-specific probes. progress in fish technology has also benefited from gene mapping (price 1993). in comparison to the methods mentioned previously separate denaturation or proteinase k digestion changes are not needed for each sample. this technique allows retrospective studies of large series of tumors and is also useful for the routine diagnostic use of formalin-fixed material to apply fish (hyytinen et al., 1994). the fish technique also helped us determine the degree of amplification and the size of the intrachromosomal amplified regions at metaphase and interphase (bar et al., 1992). fluorescence in situ hybridization (fish) is used in genetic toxicology for the study of chromosome damage with improved efficiency and precision to distinguish certain forms of chromosome aberrations, in addition to classical cytogenetic methods for scoring chromosomal aberrations (hovhannisyan, 2010). the study shows the utility of a combination of classical karyotyping and fish to determine the chromosome origin of doubleminute chromosome amplified dna sequences in cancer cells (giollan et al.,1996). fish identified cells with clonal chromosomal defects with rates of aneuploidy ranging from 6% to 92% (median 59%). in addition, there was a gain of centromeric signals for chromosome 11, most likely corresponding to hyperdiploid; aberrations of chromosome 17 in specimens from 26 patients (87%) were hyperploid as well; however, four cases (13%) showed loss of chromosome 17 centromeres (fiegl et al., 1995). modern high-throughput techniques affect research to classify new genomic regions associated with tumours (liehr et al., 2015). in correlating karyotype abnormalities with diagnosis, prognosis, and response to therapy in haematological neoplasias, classical cytogenetics described by chromosomal banding techniques has been effective. such approaches, however, require a high cell division rate and good chromosomal morphology, which pose challenges for cytogeneticists, and a long period of testing and study, which is typically a challenge for doctors (varella, 2003). also, fish is considered safer and has the added benefit of using several fluorochromes to differentiate between different targets simultaneously (bartlett, 2004). these approaches range from dna fluorescence in situ hybridization (fish)based kilobase-level resolution imaging approaches of individual cells to genome-wide sequencing strategies that collect nucleotide-level data from different sample types. in conjunction with the combinatorial use of multiple approaches, technical developments have led to new rearrangement groups and mechanistic insights into processes that drive structural changes in the human genome (hu and ly 2020). fish and traditional cytogenetic experiments often offer a false negative outcome (alayed et al., 2013). figure 6. schematic diagram of karyotyping (qaisar and bhat, 2017). smail – the roles of the fluorescent in situ hybridization (fish) and … 51 figure 7. the concepts of in situ hybridization of fluorescence. (a) a dna probe and a target sequence are the basic elements. (b) before hybridization, the dna probe is indirectly marked with hapten (left panel) or specifically marked with hapten (left panel) by adding a fluorophore probe (right panel). (c) the labelled probe and the target dna are denatured to yield single-stranded dna. (d) they are then mixed, which enables complementary dna sequences to be annealed. (e) if the probe has been indirectly labelled, the visualization of the non-fluorescent hapten using an enzymatic or immunological detection system involves an additional step. finally, fluorescence microscopy tests the signals (shakoori, 2017). limitation of the fluorescence in situ hybridization while direct preparations may be carried out, cell culture is usually needed (1-10 days), complex karyotypes with suboptimal morphology may be encountered, submicroscopic or cryptic rearrangements can result in a false-negative result, normal karyotypes may be observed after therapy-induced tumour necrosis or overgrowth of normal stromal cell support low cell density and the release of cells from the bone matrix are also issues with bone tumours (bridge, 2008). there is still a lack of a mechanistic approach to the whole fish system, and the key limiting steps for hybridization remain uncertain (lima et al., 2020). the rate-limiting phase was still the slow reaction rate of the reagents used in the probe solution. in particular, hybridization was slowed by using formamide, which acts as a doublehelix destabilizing agent (nguyen et al., 2018). however, a fluorescence microscope is required for fish, and the signals are labile and easily fade over time (kim et al., 2011). in recent years, the combination of microfluidic techniques and fish have tackled weaknesses in the consumption of probes and hybridization times, making the experimental process more sustainable and adaptable to high-throughput innovations (huber et al., 2018). as cytogenetic defects have been found in samples that appear normal by morphological and conventional cytogenetic examination, the fish analysis provides enhanced sensitivity in many cases. the combination of cytogenetic, fish and molecular studies offers a powerful method for diagnosing and sub classifying malignant diseases into clinically and biologically important subgroups, selecting effective therapies, and monitoring the effectiveness of therapeutic regimens (gozzetti and le, 2000). it is also recommended that fish and cgh findings be re-evaluated by one another 52 biology, medicine, & natural product chemistry 11 (1), 2022: 45-54 to resolve these technological artefacts. however, cgh is of potential benefit in characterizing chromosomal alterations and could help produce tumour-specific sets of fish probes within a few days to obtain genetic information of prognostic value (jacobsen et al., 2000). conclusions from this review article conducted the following points as follows: 1. fluorescence in situ hybridization (fish) more accurate than classical cytogenetic such as karyotyping 2. comparative genomic hybridization (cgh) is very important for the create a wide range of chromosomal profile 3. both fluorescence in situ hybridization (fish) and comparative genomic hybridization (cgh) have many limitations and disadvantages 4. probes is the key success for both fluorescence in situ hybridization (fish) and comparative genomic hybridization (cgh) conflict of interest: the author declares no conflicts of interest. references alayed, k., medeiros, l. j., schultz, r. a., cortes, j., lu, g., bueso-ramos, c. e., & konoplev, s. 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(2015). comparative genomic hybridization analysis of invasive ductal breast carcinomas in the chinese population. oncology letters, 10(4), 2100-2106. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 239-250 | doi: 10.14421/biomedich.2024.131.239-250 issn 2540-9328 (online) commercial incense: compound analysis and its molecular docking studies as anxiolytic agents cintya nurul apsari1,*, navista sri octa ujiantari1, zuliyati rohmah2, setyowati triastuti utami1, artania adnin tri suma1, niar gusnaniar3 1department of pharmaceutical chemistry, faculty of pharmacy; universitas gadjah mada, jl. sekip utara, senolowo, sinduadi, yogyakarta 55281, tel. +62-274-543120, indonesia 2department of tropical biology, faculty of biology, universitas gadjah mada, jl. teknika sel., sendowo, sinduadi, yogyakarta 55281, tel. +62-274-580839, indonesia. 3the research organization for health, centre for biomedical research, national research and innovation agency (brin), jl. raya jakarta-bogor no.km. 46, pakansari, kec. cibinong, kabupaten bogor, jawa barat 16911, indonesia. corresponding author* cintyanurulapsari@ugm.ac.id manuscript received: 29 april, 2024. revision accepted: 20 may, 2024. published: 28 june, 2024. abstract in the context of indonesian culture, incense has been traditionally utilized in various rituals. incense possesses a calming impact and has the potential to reduce anxiety. this physiological response stems from the interplay of chemical components within incense and receptors associated with relaxation, specifically gabaa. this research aims to explore the interaction between substances found in commercially incenses with the gabaa receptors. the compounds of incense were identified through gas chromatography-mass spectrometry (gcms) analysis. and there were 54 compounds identified from the 5 incense samples. next, the ligands employed for docking studies were compounds predicted to traverse the blood-brain barrier (bbb). there were 31 compounds potential of crossing the blood-brain barrier (bbb). docking results indicated that the majority of tested compounds exhibited notably lower s-scores during receptor interaction, suggesting their potential as anxiety-relieving agents. furthermore, molecular docking outcomes highlighted that 9octadecenoic acid (z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester showed the lowest s-score (-6.573). these findings imply that odorant and other volatile organic compounds (vocs) present in incenses possess the ability to function as anxiety-reducing (anxiolytic) agents, potentially assisting in anxiety treatment. keywords: anxiety; aroma; fragrances; in silico; vocs. abbreviations: bbb: blood-brain barrier; vocs: volatil organic compounds; gabaa: gamma-aminobutyric acid a; gcms: gas chromatography-mass spectrometry; rmsd: root-mean-square deviation. introduction according to the great dictionary of the indonesian language/kamus besar bahasa indonesia [kbbi], incense is a substance such as frankincense or twigs that emits a pleasant-smelling smoke when ignited (kbbi 2023). incense is widely utilized in religious rituals across various faiths, including buddhism, daoism, confucianism (habkirk et al. 2017), christianity, hinduism (yadav et al. 2020), roman catholicism (hartley et al. 2022), judaism, islam (yadav et al. 2020; faizal 2017; karyadi 2022; ergin 2014), as well as in the tribal and customary practices of certain nations like those in africa (wepener 2015; sagrove et al. 2020), the middle east & the arabian gulf region (elsayed et al. 2016), china (habkirk et al. 2017; cheung 2020), japan (cheung 2020; uriu et al. 2018), and india (yadav et al. 2020). particularly in javanese (indonesian) culture, incense has long been used in traditional rituals. these include engaging in traditional arts like dance, gamelan, kuda lumping, reog, and ceremony events like weddings and funerals. it can also be used in prayer and meditation practices, visits to graves and pilgrimage sites, cleaning heirlooms during jamasan ceremonies, and other purifying customs (jamhari 2001; nasir 2019; perkasa et al. 2020; yuningtyas et al. 2020; putri 2022). incense, frankincense, and other aromatic materials provide spiritual, psychological, and physical benefits. incense is a proactive means of safeguarding noble ideals, customs, and ancestral wisdom, which are rich in philosophical meaning and profundity, and it is a calming and centering perfume. the rising billowing smoke symbolizes that the almighty will hear the prayed-over or wished-for request (wepener 2015). from a physiological standpoint, the anxiolytic qualities of some volatile substances combine to produce an environment https://doi.org/10.14421/biomedich.2024.131.239-250 240 biology, medicine, & natural product chemistry 13 (1), 2024: 239-250 that is favorable for reflection and calm. these are conducive to the traditional practice of attaining a state of calmness. the aromatic ingredients utilized in incense production are commonly derived from plants and may encompass a range of resins, barks, seeds, roots, flowers, and mixtures of these (coppen 1995; yagi and yagi 2021; alqethami 2017; djordjevic 2017; gonzálezminero et al. 2023). when breathed, the aromatic molecules bind to specific receptors, triggering a cascade of brain processes that alleviate anxiety and encourage tranquillity (borges e soares, 2022). this interaction is not limited to the senses; it also affects the neurotransmitter system (sowndhararajan and kim 2016; masuo et al. 2021). numerous receptors are involved in the mechanism of this anxiolytic effect, namely serotonin receptor ((5-hydroxytryptamine 1a (5-ht1a) (heisler et al. 1998; olivier et al. 2000) & 5-hydroxytryptamine 3 (5-ht3) (olivier et al. 2000)), gamma-aminobutyric acid (gaba) receptor (nuss 2015; islam et al. 2022), corticosteroid receptor (gr) (barkus et al. 2010), nmethyl-d-aspartate (nmda) receptor (hauger et al. 2009), corticotropin-releasing factor (crf) receptor, cannabinoid receptor (cb1r), neurokinin-1 (nk1) receptor, melatonin (mt2) receptors, muscarinic (m1) receptors (kaur and singh, 2017), and nicotinic acetylcholine receptors (nachrs), dopamine (d2/d3) receptor, orexin receptor (ox1r, ox2r) (chellappa and aeschbach 2022), and adenosine (a2a & a1) receptor (van calker et al. 2019). the primary fast-inhibitory neurotransmitter receptors in the human brain are called gabaa receptors, and these receptors are the target of numerous clinically significant medications that are frequently used to treat anxiety disorders (vashchinkina et al. 2014). numerous studies have reported that support the efficacy of scents and their components in reducing symptoms or behaviors associated with anxiety (hartley and mclachlan 2022). the mechanism underlying the anxiolytic effect is still unknown, mainly how the compounds interact with the receptor. an in silico method such as docking studies can be used to predict the interactions between compounds and their target receptors. compounds contained in commercial incense will act as ligands. in this research, the incense employed consists of commercially accessible products distributed within the yogyakarta special region. therefore, this study aims to investigate how substances found in commercial incense interact with gabaa receptors. additionally, this study is anticipated to scientifically elucidate the potential of utilizing incense and other aromatic fragrances to aid in anxiolytic therapy. materials and methods materials the materials were five incenses with different brands purchased around the special region of yogyakarta, ethanol pro analysis (p.a) (merck), analytical balance (shimadzu atx224), ultrasonic bath (labocon luc101), microcentrifuge (thermo scientific™ fresco™ 21), microtube (biologix), screw cap vials, and other glassware. the compound analysis required a set of gas chromatography mass spectrometry (gcms) (thermo scientific tracetm 1310 gas chromatography (gc) & thermo scientific isqtm lt single quadropole mass spectrometer (ms)), hp-5ms ui 30m, 0.25mm 0, 25um column (agilent technologies inc, us). for in silico studies, the docking simulation was performed in molecular operating environment (moe) software (montreal, canada) (licensed by the faculty of pharmacy, universitas gadjah mada) which is installed at a computer with specifications as follows windows 10, intel core i7 cpu 11800h, and ram 16 gb. extraction of compounds from commercial incense compounds extracted from commercial incense were recovered by modifying and combining well-established research methods (bagherian et al. 2011; west et al. 2014; nadilah et al. 2019; rahmanto et al. 2018). using a mortar and stamper, incense was ground into powder. 96% ethanol (p.a.) was then used for the extraction, and the sample-to-solvent ratio was 1:100 (w/v). the extract was obtained by employing intermittent ultrasonication, involving cycles of emission for 5 minutes followed by a pause for 5 minutes, and this process was repeated until reaching a total duration of 30 minutes. after the ultrasonication procedure, maceration was carried out for 24 hours at room temperature. the extract was centrifuged at 10,000 rpm for 5 minutes to obtain clearer and entirely particle residue-free results. the supernatant was then collected in 5 ml screw-on vials. after that, the ethanolic extract was kept refrigerated (± 4 °c) for the following procedure. identification of compounds in commercial incense using gcms gsms analyzed the chemical constituents of incense. ethanolic incense extract was injected into gcms. the gc utilized a column of hp-5ms ui (ultra inert) 30 m, 0.25 mm, 0.25 µm (agilent technologies inc, us). the chromatography system was programmed at 60 °c for 2 min, then ramped at 105 °c/min to 280 °c, and held for 8 min, with a retention time (rt) total of 32 min. the mobile phase used was helium uhp (he), and the flow rate was 1,0 ml/min. the injector and transfer line temperatures were 230 °c, with a split ratio of 50:1. blood-brain barrier permeability prediction the compounds identified in the samples underwent evaluation for permeability across the blood-brain barrier (bbb) using the swissadme server (http://www.swissadme.ch/) (daina et al. 2017 and rashid et al. 2022). apsari et al. – in silico study of incense as anxiolytic agents 241 molecular docking ligand preparation the ligands used for docking studies were compounds identified in the samples and could penetrate the bbb. the 3d ligand structures were generated using the builder feature in the moe software by entering the smiles code of the compounds obtained from the pubchem (https://pubchem.ncbi.nlm.nih.gov/) (kim et al. 2023). the energy minimization was applied to the compounds using the mopac system, employing pm3 as the potential energy function to assign charge to the compound. subsequently, the optimized structures werearchived in a database in *.mdb format. homology modelling the gabaa receptor alpha-2 subunit (gabaa α2) is known to play a role in anxiolytic activity. identification of template proteins from gabaa subunit alpha-2 in homo sapiens (human) organisms was carried out using the swiss-model server (waterhouse et al. 2018). the receptor sequence was obtained from the uniprot database (https://www.uniprot.org/) (the uniprot consortium 2023) with protein code p47869. the model chosen was a model that has the highest seq value, which was model 1 (99.78%) with the q5rcc5 template. the gabaa α2 has been known to play a role in anxiolytic activity. since its 3d structure is not available yet in database thus the human gabaa α2 (homo sapiens) need to be modelled. identification of template proteins for homology modelling the gabaa subunit alpha-2 was carried out using the swiss-model server (waterhouse et al. 2018). the receptor sequence was obtained from the uniprot database (https://www.uniprot.org/) (the uniprot consortium 2023) with protein code p47869. the chosen model was model 1 which has the highest sequence value (99.78%) with the q5rcc5 template. protein preparations the model protein sequence was aligned with the reference protein (pdb:8bhk), which includes the native ligand (diazepam), using moe's protein align/superpose function. the native ligand from the reference protein was incorporated into the model protein. subsequently, active sites were identified using moe's site finder tool. the gabaa subunit alpha-2 protein complex derived from swiss-model was prepared using moe's quickprep program for structure preparation, including atomic modifications and protonation. the forcefield parameter amber was then used to minimize energy and assign a charge to the protein. finally, the prepared protein was stored in pdb format (.pdb). the sequence of gabaa subunit alpha-2 protein model was aligned with the reference protein (pdb:8bhk), which includes diazepam as the native ligand using moe's protein align/superpose function. the native ligand from the reference protein was incorporated into the model protein. the homology model derived from swiss-model was prepared using moe's quickprep program for structure preparation, including atomic modifications and protonation. the forcefield parameter amber was then used to minimize energy and assign the charge to the protein. finally, the prepared protein was stored in pdb format (.pdb). pose validation pose validation can be done by redocking the native ligand (diazepam) bound in the protein. docking tool was employed with the atomic ligand designated as the active site, and set triangle matcher, induced fit, and london dg as the parameters for placement, refinement, and scoring, respectively. rmsd (root-mean-square of deviation) is a parameter used to determines the validity of the redocking method. this parameter will evaluate the redocking process's reliability. a rmsd value of ≤ 2.0 å indicates good or valid docking protocol (su et al. 2018). molecular docking the docking method that has been validated was used to dock the test compounds in incense. the analysis of docking results involves the best pose and the best score evaluation and the interaction of those compounds amino acid residues within the protein's active site. results and discussion this research has four primary data, i.e., 1) list of compound results from gcms analysis, 2) prediction of bbb permeant, 3) docking score, and 4) visualization of ligand interactions with receptors. identification of compounds in commercial incense using gcms the ethanolic extract of incense can be seen in figure 1. the results of identification from the five incense samples are displayed in table 1. in total, 54 compounds were identified across the five samples. notably, incense sample b exhibited the highest number of compounds, precisely 31 compounds. visually, incense b displayed the deepest black hue (figure 1) and had the most pungent aroma compared to the other samples. conversely, incense a boasted the lowest number of compound, totalling 19, among the five samples. incense a, derived from authentic agarwood without any synthetic additives, exudes a comparatively milder aroma than incenses b, c, d, and e. physically, the extract of incense a showcases a brownish hue (figure 1) and has a subtle woody scent. on the other hand, incenses c, d, and e present a golden yellow appearance in their extracts (figure 1), with a mild floral aroma, including a mixture of flower fragrances, jasmine, and lotus, respectively. 242 biology, medicine, & natural product chemistry 13 (1), 2024: 239-250 figure 1. ethanolic extract of incense. the constituents within incense consist of fragrance compounds, volatile organic compounds (vocs), and additional ingredients utilized in incense manufacturing, such as solvents. for instance, fragrance compounds encompass dihydromyrcenol; linalol; a-terpineol; dodecanoic acid; 3-hydroxy-, (r)-lavandulyl acetate; linalyl acetate; 4-tert-butylcyclohexyl acetate; geranyl vinyl ether; 4,7-methano-1h-inden-5-ol, 3a,4,5,6,7,7ahexahydro-, acetate ; 2,5,5,8a-tetramethyl-3,5,6,7,8,8ahexahydro-2h-naphthalen-1-one; lilial, among others. the confirmation of fragrance compounds can be verified through databases such as the good scents company's database (http://www.thegoodscentscompany.com/misc/about.htm l), which serves as an information resource for the flavor, food, and fragrance industry, and aroma db (https://aroma.irap.omp.eu/) (sabbah et al. 2017). diethyl phthalate is also detected in incense containing synthetic fragrances (samples b, c, d, e), typically serving as solvents or diluents for fragrance agents (api 2001). prediction of bbb permeant the outcomes of predictive analyses conducted through the swissadme server are presented in table 1. these predictions are formulated utilizing the boiled-egg methodology, which entails a visual assessment of human intestinal absorption relative to the molecular characteristics of small compounds plotted on the wlogp versus tpsa graphs. the white segment within the boiled-egg model signifies a heightened likelihood of passive absorption within the gastrointestinal tract, while the yellow region (yolk) indicates a propensity for brain permeation (note that the yolk and white areas are not mutually exclusive) (pavlović et al. 2023). subsequently, compounds demonstrating positive bbb permeability are identified as ligands and subjected to docking studies with the gabaa receptor. molecular docking and ligand-receptors interaction the homology modeling results reveal that model 1 corresponds to a template protein derived from the q5rcc5 protein with a sequence value of 99.78%. pose validation yielded an rmsd value of 0.4423, indicating the docking methodology's suitability (including placement, refinement, and scoring parameters). subsequently, a superimposed was made between the native ligand crystal and the re-docking results on the receptor (figure 2). the docking score (s) of diazepam was calculated as -4.3911. the docking procedure involving the test ligands and gabaa was conducted using the designated method, with 31 compounds employed as the test ligands (see table 1). visualization of the interaction between the best-scoring ligand and the receptor is depicted in figure 3. figure 2. the alignment of the native ligand crystal (purple) with the redocking native ligand (blue). figure 3. visualization of 9-octadecenoic acid (z)-, 2-hydroxy-1(hydroxymethyl)ethyl ester with receptor (a). interactions within the active site (3d visualization) (b). 2d interaction with amino acid residues on the receptor. discussion based on the predictions from admet, it was identified that 31 compounds could potentially penetrate the bbb, b a apsari et al. – in silico study of incense as anxiolytic agents 243 and these were utilized as ligands for docking studies. the s-score serves as a crucial metric for assessing docking outcomes in moe docking, indicating the affinity between receptor and ligand across various conformations (attique et al. 2019). a lower s-score was favored, as it suggested stronger receptor interactions (konyar et al. 2022). evaluation of docking outcomes revealed that nearly all tested compounds exhibited lower s-scores compared to the native ligand, except for patchouli alcohol. it can be seen in table 1, the vocs compound 9-octadecenoic acid (z)-, 2-hydroxy-1(hydroxymethyl)ethyl ester showed the lowest s-score, indicating strongest affinity for the gabaa receptor. compounds with lower s-scores than the native ligand were presumed to possess better anxiolytic activity. generally, vocs are associated with either pleasant or off odors (dudley et al. 2010). while specific aroma data for 9-octadecenoic acid (z)-, 2-hydroxy-1(hydroxymethyl)ethyl ester is unavailable, its ability to evaporate and accumulate in the air. other ligands with distinctive aromas had lower s-scores compared to native ligands (diazepam) include dihydromyrcenol (citrusy, floral, sweet), linalool (floral, spicy wood), α-terpineol (sweet lilac floral, pine-woody), (r)-lavandulyl acetate (floral, lavender-like), linalyl acetate (floral, sweet, citric), 4-tert-butylcyclohexyl acetate (fruity with woody undertones), geranyl vinyl ether (floral), 4,7-methano1h-inden-5-ol, 3a,4,5,6,7,7a-hexahydro-, acetate (floral green herbal), etc (table 1). the interaction of 9octadecenoic acid (z)-, 2-hydroxy-1(hydroxymethyl)ethyl ester with gabaa (figure 3b) encompasses numerous interactions, predominantly with polar amino acid residues, specifically asn130, his129, ser232, tyr237, thr234, ser186, tyr187, and glu165. one hydrogen bonding was observed between hydroxyl group with glu165. additionally, there are hydrophobic interactions occurring with amino acid residues pro181, pro167, phe127, and ile230. these findings suggest that aromatherapy, incense, or other fragrances containing these compound types could aid in anxiety therapy. when incense is burned, these compounds transform into their oxidized forms. while this study didn't conduct gcms analysis of the incense smoke due to limited resources, however research conducted supports the idea that aromas and other vocs can act as anxiolytic agents. conclusions the molecular docking outcomes indicated that the compound 9-octadecenoic acid (z)-, 2-hydroxy-1(hydroxymethyl)ethyl ester exhibited the lowest s-score (-6.573) when interacting with the gabaa receptor, suggesting its promise as an anxiety-relieving agent. furthermore, nearly all aroma compounds exhibit a lower s-score compared to diazepam. these findings suggest that the aroma compounds present in incense possess the capability to function as anxiolytic agents and could serve as adjuncts in anxiety treatment. these results still have the potential to be proven through in vivo research approaches. acknowledgements: we express our 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(2014). gabaa receptor drugs and neuronal plasticity in reward and aversion: focus on the ventral tegmental area. frontiers in pharmacology, 5, 256, doi: https://doi.org/10.3389/fphar.2014.00256 waterhouse, a., bertoni, m., bienert, s., studer, g., tauriello, g., gumienny, r., heer, f.t., de beer, t.a.p., rempfer, c., bordoli, l., lepore, r., & schwede, t. (2018) swissmodel: homology modelling of protein structures and complexes. nucleic acids res, 46, w296-w303, doi:https://doi.org/10.1093%2fnar%2fgky427 wepener, c. (2015). burning incense for a focus group discussion: acquiring a spirituality of liminality for doing liturgical research in an african context from an emic perspective. international journal of practical theology, 19(2), 271-291, doi: http://dx.doi.org/10.1515/ijpt-2014-0036 west, a. l., hoque, n., & griep, m. h. (2014). extraction efficacy of synthetic cannabinoids from damiana leaf substrates utilizing electrolytic solvents. army research lab aberdeen proving ground md weapons and materials research directorate. yadav, v. k., choudhary, n., heena khan, s., khayal, a., ravi, r. k., kumar, p., modi, s., & gnanamoorthy, g. (2020). incense and incense sticks: types, components, origin and their religious beliefs and importance among different religions. j. bio innov, 9, 1420-1439, doi: https://doi.org/10.46344/jbino.2020.v09i06.28 yagi, s., & yagi, a. (2021). important medicinal plants—sudan, in medicinal and aromatic plants of the world, encyclopedia of life support systems (eolss), developed under the auspices of the unesco. yuningtyas, l. a., pranawa, s., & yuhastina, y. (2020). the meaning of the ceprotan tradition in bersih desa for the people of sekar village. harmoni sosial: jurnal pendidikan ips, 7(2), 150-159, doi: http://dx.doi.org/10.21831/hsjpi.v7i2.27978 table 1. list of compounds in incense, bbb permeant prediction, and docking score. no compounds chemical formula molecular weight similarity index (si) bbb permeant pubchem id smiles aroma score (s-score) samples a b c d e 1 2,6-dimethyloct-7-en-2ol (dihydromyrcenol) c10h20o 156 891 yes 29096 cc(cccc(c)(c)o)c=c fresh citrus, floral, bergamot, lime -4.809 ✓ ✓ 2 3,7-dimethylocta-1,6dien-3-ol (linalool) c10h18o 154 913 yes 6549 cc(=cccc(c)(c=c)o)c floral, spicy wood, somewhat resembling french lavender plants, bergamot oil or lily of the valley -4.825 ✓ ✓ ✓ 3 methyl n-(nbenzyloxycarbonyl-betal-aspartyl)-beta-dglucosaminide c19h26n2 o10 442 703 no 562266 coc1c(c(c(c(o1)co)o)o)nc( =o)cc(c(=o)o)nc(=o)occ2= cc=cc=c2 unknown ✓ 4 2-(4-methylcyclohex-3en-1-yl)propan-2-ol (αterpineol) c10h18o 154 775 yes 17100 cc1=ccc(cc1)c(c)(c)o slightly harsh sweet lilac, floral, pine-woody clean delicate fresh, pine, lavender pineneedle -4.820 ✓ 5 dodecanoic acid, 3hydroxy c12h24o3 216 720 yes 94216 cccccccccc(cc(=o)o)o unknown -5.394 ✓ 6 cholestan-3-ol, 2methylene-, (3ß,5a) c28h48o 400 719 no 22213932 c[c@h](cccc(c)c)[c@h]1c c[c@@h]2[c@@]1(cc[c@h] 3[c@h]2cc[c@@h]4[c@@]3( cc(=c)[c@@h](c4)o)c)c unknown ✓ 7 (5-methyl-2-prop-1-en-2ylhex-4-enyl) acetate or (r)-lavandulyl acetate c12h20o2 196 802 yes 30247 cc(=ccc(coc(=o)c)c(=c)c) c floral, lavenderlike odor -5.337 ✓ 8 3,7-dimethylocta-1,6dien-3-yl acetate or linalyl acetate c12h20o2 196 809 yes 8294 cc(=cccc(c)(c=c)oc(=o)c) c floral, sweet and citric, and additionally as minty and slightly caraway-like -5.316 ✓ ✓ 9 4-tert-butylcyclohexyl acetate c12h22o2 198 848 yes 36081 cc(=o)oc1ccc(cc1)c(c)(c)c fruity scent, balanced with woody undertones -4.767 ✓ 10 4-(2,2-dimethyl-6methylenecyclohexyl)but anal c13h22o 194 728 yes 549482 cc1(cccc(=c)c1cccc=o)c unknown -4.888 ✓ no compounds chemical formula molecular weight similarity index (si) bbb permeant pubchem id smiles aroma score (s-score) samples 11 z,z-2,5-pentadecadien1-ol c15h28o 224 721 no data 5364952 ccccccccc/c=c\c/c=c\co unknown ✓ 12 (2e)-1-ethenoxy-3,7dimethylocta-2,6-diene or geranyl vinyl ether c12h20o 180 739 yes 5365842 cc(=ccc/c(=c/coc=c)/c)c fruity or floral -5.140 ✓ 13 4,7-methano-1h-inden5-ol, 3a,4,5,6,7,7ahexahydro-, acetate c12h16o2 192 851 yes 98478 cc(=o)oc1cc2cc1c3c2cc=c 3 floral green herbal -5.000 ✓ 14 1-(4-tertbutylphenyl)propan-2one c13h18o 190 724 yes 6423283 cc(=o)cc1=cc=c(c=c1)c(c)( c)c unknown -4.568 ✓ 15 2,5,5,8a-tetramethyl3,5,6,7,8,8a-hexahydro2h-naphthalen-1-one c14h22o 206 719 yes 585286 cc1cc=c2c(cccc2(c1=o)c)( c)c powdery-ionone type odours -4.850 ✓ 16 3-(4-tert-butylphenyl)-2methylpropanal or lilial c14h20o 204 724 yes 228987 cc(cc1=cc=c(c=c1)c(c)(c)c )c=o floral neroli muguet -4.745 ✓ ✓ 17 naphthalene, 2-ethoxyc12h12o 172 722 yes 7129 ccoc1=cc2=cc=cc=c2c=c1 powdery, floral, naphthyl and citrus -4.481 ✓ 18 diethyl phthalate c12h14o4 222 957 yes 6781 ccoc(=o)c1=cc=cc=c1c(=o )occ solvents/deluents -4.962 ✓ ✓ ✓ ✓ 19 9-octadecenoic acid, (2phenyl-1,3-dioxolan-4yl)methyl ester, trans c28h44o4 444 750 no 5366356 cccccccc/c=c/cccccccc( =o)occ1coc(o1)c2=cc=cc= c2 unknown ✓ ✓ ✓ 20 methyl 5,7hexadecadiynoate c17h26o2 262 743 yes 14957560 ccccccccc#cc#ccccc(=o) oc unknown -6.211 ✓ 21 2,5-octadecadiynoic acid, methyl ester c19h30o2 290 761 yes 42151 ccccccccccccc#ccc#cc( =o)oc unknown -6.079 ✓ 22 cyclopentaneacetic acid, 3-oxo-2-pentyl-, methyl ester c13h22o3 226 777 yes 102861 cccccc1c(ccc1=o)cc(=o)o c floral and jasmine-like odor -5.645 ✓ ✓ 23 (1r,3r,6s,7s,8s)2,2,6,8tetramethyltricyclo[5.3.1. 03,8]undecan-3-ol or patchouli alcohol c15h26o 222 768 yes 10955174 c[c@h]1cc[c@@]2([c@@]3( [c@h]1c[c@h](c2(c)c)cc3) c)o woody, patchouli, earthy -4.121 ✓ 24 (7a-isopropenyl-4,5dimethyloctahydroinden4-yl)methanol c15h26o 222 776 yes 605599 cc1ccc2(cccc2c1(c)co)c(= c)c unknown -4.614 ✓ 25 4-(3,3-dimethyl-but-1ynyl)-4-hydroxy-2,6,6c15h22o2 234 741 yes 535329 cc1=cc(cc(c1=o)(c)c)(c#cc (c)(c)c)o unknown -5.027 ✓ no compounds chemical formula molecular weight similarity index (si) bbb permeant pubchem id smiles aroma score (s-score) samples trimethylcyclohex-2enone cc1=cc(o)(cc(c)(c)c1=o)c# cc(c)(c)c 26 benzene, 1,1'-(2-butene1,4-diyl)bis c16h16 208 716 yes 5370638 c1=cc=c(c=c1)c/c=c/cc2=c c=cc=c2 unknown -4.917 ✓ ✓ ✓ ✓ ✓ 27 12,15-octadecadiynoic acid, methyl ester c19h30o2 290 745 yes 538453 ccc#ccc#cccccccccccc( =o)oc unknown -6.229 ✓ ✓ ✓ 28 n,n'bis(carbobenzyloxy)lysine methyl(ester) c23h28n2 o6 428 749 no 75487878 coc(=o)[c@h](ccccnc(=o) occ1=cc=cc=c1)nc(=o)oc c2=cc=cc=c2 unknown ✓ ✓ ✓ 29 acetamide, n-methyl-n[4-[2-acetoxymethyl-1pyrrolidyl]-2-butynyl] c14h22n2 o3 266 742 no 580233 cc(=o)n(c)cc#ccn1cccc1c oc(=o)c unknown ✓ ✓ 30 octanal, 2(phenylmethylene) c15h20o 216 847 yes 1715135 cccccc/c(=c/c1=cc=cc=c1 )/c=o jasmine-like odor -5.073 ✓ ✓ ✓ 31 cyclodeca[b]furan2,9(3h,4h)-dione, 4(acetyloxy)3a,7,8,10,11,11ahexahydro-6-methyl3,10-bis(methylene)-, [3ar(3ar*,4r*,5e,11as*)] c17h20o5 304 709 yes 5363090 c/c/1=c\c(c2c(cc(=c)c(=o)c c1)oc(=o)c2=c)oc(=o)c unknown -5.165 ✓ 32 ethanone, 1(2,3,4,7,8,8a-hexahydro3,6,8,8-tetramethyl-1h3a,7-methanoazulen-5yl) c17h26o 246 849 yes 16220111 c[c@@h]1cc[c@@h]2[c@@ ]13c[c@h](c2(c)c)c(=c(c3)c (=o)c)c warm woody amber musk -4.938 ✓ 33 "ethanol, 2-(9octadecenyloxy)-, (z) or emulphor c20h40o2 312 801 no 5364713 cccccccc/c=c\cccccccc occo as emulsifiers, surfactants ✓ ✓ ✓ ✓ 34 10-heptadecen-8-ynoic acid, methyl ester, (e) c18h30o2 278 723 yes 5367407 cccccc/c=c/c#cccccccc( =o)oc unknown -6.242 ✓ 35 cyclopenta[g]-2benzopyran, 1,3,4,6,7,8hexahydro-4,6,6,7,8,8hexamethyl (galaxolide) c18h26o 258 754 yes 91497 cc1cocc2=cc3=c(c=c12)c( c(c3(c)c)c)(c)c clean sweet musky floral woody odor -4.932 ✓ ✓ 36 benzene, 1-(1,1dimethylethyl)-3,5dimethyl-2,4,6-trinitro or c12h15n3 o6 297 781 no 62329 cc1=c(c(=c(c(=c1[n+](=o)[o -])c(c)(c)c)[n+](=o)[o])c)[n+](=o)[o-] musk, animalistic, earthy and woody ✓ no compounds chemical formula molecular weight similarity index (si) bbb permeant pubchem id smiles aroma score (s-score) samples musk xylene 37 "[1,1'-bicyclopropyl]-2octanoic acid, 2'-hexyl-, methyl ester methyl 8-[2-(2hexylcyclopropyl) cyclopropyl]octanoate" c21h38o2 322 785 no 552098 ccccccc1cc1c2cc2ccccc ccc(=o)oc unknown ✓ ✓ ✓ ✓ ✓ 38 phenethylamine, 3benzyloxy-2-fluoro-ßhydroxy c15h16fn o2 261 756 no ccccccc1cc1c2cc2ccccc ccc(=o)oc unknown ✓ ✓ 39 hexadecane, 1,1bis(dodecyloxy) c40h82o2 594 721 no 41920 cccccccccccccccc(occc ccccccccc)occcccccccc cc unknown ✓ ✓ ✓ ✓ 40 2-myristynoyl pantetheine c25h44n2 o5s 484 756 no 535560 cccccccccccc#cc(=o)scc nc(=o)ccnc(=o)c(c(c)(c)co )o unknown ✓ 41 cyclopropanebutanoic acid, 2-[[2-[[2-[(2pentylcyclopropyl)methy l]cyclopropyl]methyl]cyc lopropyl]methyl]-, methyl ester c25h42o2 374 793 no 554084 cccccc1cc1cc2cc2cc3cc3 cc4cc4cccc(=o)oc unknown ✓ ✓ ✓ 42 musk ketone c14h18n2 o5 294 795 no 6669 cc1=c(c(=c(c(=c1[n+](=o)[o -])c(c)(c)c)[n+](=o)[o])c)c(=o)c floral, sweet, powdery ✓ 43 z-(13,14epoxy)tetradec-11-en-1ol acetate c16h28o3 268 806 yes 5363633 cc(=o)occcccccccc/c=c\ c1co1 unknown -5.804 ✓ ✓ ✓ ✓ ✓ 44 9-hexadecenoic acid c16h30o2 254 827 yes 5282745 cccccc/c=c/cccccccc(=o) o unknown -5.669 ✓ ✓ ✓ ✓ ✓ 45 11-octadecenoic acid, methyl ester or methyl vaccenate c19h36o2 296 854 no 5364432 cccccc/c=c/cccccccccc( =o)oc unknown ✓ ✓ ✓ ✓ ✓ 46 10-octadecenoic acid, methyl ester or methyl (e)-octadec-10enoate c19h36o2 296 816 no 5364425 ccccccc/c=c/ccccccccc( =o)oc unknown ✓ ✓ 47 trans-13-octadecenoic acid c18h34o2 282 846 no 6161490 cccc/c=c/cccccccccccc( =o)o unknown ✓ ✓ ✓ 48 1-heptatriacotanol c37h76o 536 820 no 537071 cccccccccccccccccccc ccccccccccccccccco unknown ✓ ✓ ✓ ✓ ✓ no compounds chemical formula molecular weight similarity index (si) bbb permeant pubchem id smiles aroma score (s-score) samples 49 ethyl iso-allocholate c26h44o5 436 827 no 6452096 ccoc(=o)cc[c@@h](c)[c@ h]1cc[c@@h]2[c@@]1([c@ h](c[c@h]3[c@h]2[c@@h]( c[c@h]4[c@@]3(cc[c@h](c 4)o)c)o)o)c unknown ✓ ✓ ✓ ✓ ✓ 50 9-octadecenoic acid, 1,2,3-propanetriyl ester, (e,e,e) or trielaidin c57h104o6 884 788 no 537-39-3 cccccccc/c=c/cccccccc( =o)occ(oc(=o)ccccccc/c= c/cccccccc)coc(=o)cccc ccc/c=c/cccccccc unknown ✓ 51 9-octadecenoic acid (z), 2-hydroxy-1(hydroxymethyl)ethyl ester c21h40o4 356 781 yes 5319879 cccccccc/c=c\cccccccc( =o)oc(co)co unknown -6.573 ✓ ✓ ✓ 52 tricyclo[20.8.0.0(7,16)]t riacontane, 1(22),7(16)diepoxy c30h52o2 444 766 no 543764 c1ccccc23cccccc45cccc ccccc4(o5)cccccc2(o3)cc c1 unknown ✓ ✓ 53 z-5-methyl-6heneicosen-11-one c22h42o 322 761 no 5363254 ccccccccccc(=o)ccc/c=c\ c(c)cccc unknown ✓ ✓ 54 2-[4-methyl-6-(2,6,6trimethylcyclohex-1enyl)hexa-1,3,5trienyl]cyclohex-1-en-1carboxaldehyde c23h32o 324 795 no 5363101 cc1=c(c(ccc1)(c)c)/c=c/c(= c/c=c/c2=c(cccc2)c=o)/c unknown ✓ ✓ biology, medicine, & natural product chemistry volume 7 – number 1 – 2018 issn 2089-6514 (paper) | issn 2540-9328 (online) contents leukocytes description of mudskipper (periophthalmodon schlosseri) of barito river estuary, desa tanipah, kalimantan selatan heri budi santoso, hidayaturrahmah, muhamat 1 4 stability of t-dna integration in phalaenopsis “sogo vivien” transgenic orchid carrying 35s::gal4::atrkd4::gr endang semiarti, exsyupransia mursyanti, ahmad suyoko, faiza senja widya perdana, catharina tri widyastuti, aditya nur subchan 5 13 comparison of detergent and ctab method for isolation of dna from salak (salacca zalacca (gaert.) voss. ‘pondoh’) namira nur arfa, budi setiadi daryono, reflinur 15 20 physiological response of 'segreng' rice plant (oryza sativa l.) to biogas sludge at wukirsari village, cangkringan, sleman dwi umi siswanti, sudjino, nindy senissia asri, mifta arlinda, arianda poetri shofia rochman, akrima syahidah 21 26 quercetin: the bioactive compound from allium cepa l. as anti-inflammation based on in silico screening mohamad amin, kurniawan setia putra, ihya fakhrurizal amin, nanda earlia, dina maulina, betty lukiati, umie lestari 27 31 cover jurnal biomenaprochy vol 7 num 1 2018 depan.pdf (p.1-2) table of content 2018 v7 n1.pdf (p.3-4) cover jurnal biomenaprochy vol 7 num 1 2018 belakang.pdf (p.5-6) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 693-698 | doi: 10.14421/biomedich.2025.142.693-698 issn 2540-9328 (online) washed erythrocyte (we) bag bacteria brevundimonas vesicularis identification at the blood donor unit pmi dki jakarta widia rahmatullah1, rina oktaviyanti2, resmi aini1, rudina azimata r.1, reska handayani3 1yogyakarta's bhakti setya indonesia health polytechnic, indonesia. 2indonesian red cross dki jakarta, indonesia. 3nursing faculty psychology and health department, universitas negeri padang, indonesia. corresponding author* widiarahmatullah@poltekkes-bsi.ac.id manuscript received: 07 july, 2025. revision accepted: 04 september, 2025. published: 01 october, 2025. abstract a packed red cell (prc) component known as a washed erythrocyte (we) has undergone two to three washings with sterile physiological saline to get rid of 99% of the plasma protein, antibodies, and some leukocytes. the goal of this study was to use bact/alert media to detect the presence or absence of bacteria (anaerobes or aerobes) in we blood components because there is a chance that germs from the environment could contaminate we processing because it is still an open system and the manufacturing is still exposed to the elements. blood samples come from we blood products that have undergone quality checks for bacterial contamination in a lab dedicated to product quality. checking for bacterial contamination in the blood involves utilizing the bact / alert tool to examine bacterial contamination. blood product aerobic (bpa) and blood product anaerobic (bpn) media are used in the culture of blood product sample on bact/alert. aside from 1 (2.44%) component sample of we blood that was identified as bacteria in bpa media, the results of the 41 samples examined in 2020 showed that there was aerobic bacterial contamination in bpa media but no anaerobic bacterial contamination in bpn media. this was confirmed by the examination of samples on bpn media, which yielded negative results in 41 samples (100%) and positive results from none of the 41 samples. the results of identification at the microbiology laboratory at the university of indonesia showed that the aerobic bacteria that contaminated washed erythrocyte (we) blood products on bpa microbiology media were brevundimonas vesicularis. keywords: bact/allert 3d; washed erythrocyte blood components; bacterial contamination. introduction currently, blood transfusion therapy plays a significant role in medicine, both for life-saving emergency treatment and for common conditions that require ongoing transfusion. for instance, according to (herlinah, 2016), packed red cell (prc) components are most frequently requested in blood service units. in 2013, from august to november, wahidin hospital in makassar saw a demand for prc blood transfusions of 1392, or 36.4%, compared to 2.2% for tc and 0.1% for plasma. prc transfusion therapy is the cornerstone of treatment for individuals with thalassemia, aplastic anemia, and haematological malignancies. blood transfusions can also result in reactions, ranging from minor symptoms like chills or a mild fever to moderate symptoms like shortness of breath, fever, and allergic reactions, as well as severe symptoms like shock, severe hypotension, or severe shortness of breath. the likelihood of transfusion responses increases with frequency of blood transfusions (akbar et al., 2014). we blood products, specifically prc components, which have been washed two to three times with sterile physiological saline to eliminate 99% of plasma proteins, antibodies, and toxins, are one strategy to reduce the risk of transfusion reactions. some leukocytes because it only contains a minimal amount of leukocytes and plasma, the type of washed erythrocyte (we) component is a modified kind of prc blood component that is thought to be capable of minimizing responses associated with blood transfusion. because the processing of we components is still done in an open system, or more specifically, because the manufacturing is still exposed to an open environment, it is done in an airflow laminator. although this process is referred to as a sterile open system, there is a chance that it will become contaminated with bacteria from the environment because the we manufacturing already comes into contact with open spaces (ministry of health of the republic of indonesia). the development of germs in blood components raises questions regarding a transfusion-related response, a according to a chinese study (he et al., 2018), 44 out of 28,711 tc bags contained bacterial contamination that can lead to transfusion reactions. https://doi.org/10.14421/biomedich.2025.142.693-698 694 biology, medicine, & natural product chemistry 14 (2), 2025: 693-698 bacterial contamination cases have a higher risk of transfusion transmitted infections (tbi) than viral infections(haass et al., 2019). in addition, bacterial contamination is the second leading cause of death due to the risk of bacterial sepsis from transfusion. the source of bacterial contamination in tc products can come from the donor's skin surface when the needle enters the blood vessel (rahmatullah et al., 2024 cit he et al., 2018). this is related to previous research showing that 196 blood products were known to be contaminated with grampositive and gram-negative bacteria. the results of the identification showed that more than 50% of the bacteria detected in tc blood products were gram-positive bacteria, while gram-negative bacterial contamination was usually less, but if gram-negative bacterial contamination occurs, there is a risk of transfusion infection to death (rahmatullah et al., 2024 cit agzie et al., 2019). asymptomatic, low-grade fever, acute sepsis, hypotension, and even death are possible clinical manifestations brought on by bacterial infection(astuti & ayu maharani, 2014). bacteria are extremely small organisms that are invisible to the unaided eye. both inside and outside of the human body, bacteria can exist in a number of conditions. the term "bakteri aerob" refers to bacteria that require oxygen to function in order to provide energy. examples of such bacteria include nitrosococcus and nitrobacter. streptococcus, aerobacter aerogenes, escherichia coli, lactobacillus, and alcaligenesis are examples of anaerobic bacteria that do not require oxygen to function (european committee (partial agreement) on blood transfusion (cd-p-ts), 2020). according to research conducted in california (horth et al., 2017), around 9.2% of 196 different types of dried fruit were found to contain bacteria. the results of the identification show that the product for making darah simpan contains the bacteria staphylococcus aureus, bacillus sp., pseudomonas, and streptococcus pneumonia. transfusion reactions in therapeutic patients are frequently brought on by bacterial contamination of red blood cell products. bacterial transfusion responses are frequently swift and violent (amalia wahidiyat & basant adnani, 2016). for instance transfusion of red blood cells results in sepsis due to bacterial infection. during blood transfusions, patients frequently develop a high fever (high temperature of 40–43 degrees celsius) and chills. it is essential to check for bacterial contamination as soon as blood is collected in order to avoid responses brought on by bacteria. to screen for bacterial contamination in the blood, utilize the bact/alert instrument. blood product samples are cultured on bacteria and alert. blood product anaerobic (bpn) and aerobic (bpa) medium, with signs seen from bpn and bpa media that changed color from gray (negative result, no bacteria) and yellow (there were no bacteria). discovered bacterium). based on the detection of color changes at the bottom of the bpa/bpn tube, the examination's results are displayed on the monitor screen with either positive or negative information about bacterial contamination (he et al., 2018). we component production is processed in an air flow laminator because the processing is still in an open system, namely the production is still exposed to the open environment. this processing is called a sterile open system, but because we production already has contact with an open space, there is a possibility of bacterial contamination from the surrounding environment that affects bacterial growth. this study aims to determine whether or not there is bacterial contamination (anaerobic and aerobic) in we (washed erythrocyte) blood components with bact/alert 3d media materials and methods descriptive research techniques with a quantitative approach were used to conduct this kind of study. this study was carried out at pmi dki jakarta province's blood donor unit (udd). the washed erythrocyte (we) blood components used in this study were examined for bacterial contamination at the blood donor unit (udd) pmi dki jakarta province. the goal of this study is to identify the aerobic and anaerobic bacteria found on the bact/alert 3d device at pmi dki jakarta province's blood donor unit (udd). bacterial identification the bact/alert tool is an automatic microbial detection system based on bpn (anaerobic blood product) and bpa (aerobic blood product) media which are incubated to produce a bacterial growth graph if there is a positive sign on the monitor screen. bpn media is used to detect anaerobic bacteria while bpa media is used to detect bacteria in we products. at the pmi blood donor unit (udd) dki jakarta province in 2020, a sample of we blood component was analyzed for bacterial quality based on the detection of aerobic and anaerobic bacteria on the bact/allert 3d instrument. random sampling was used as a sample approach, and component quality tests' secondary data were used as the basis. we as many as 41 sample data in the udd pmi dki jakarta quality test laboratory. using the excel application, the sample data were calculated to determine the average amount of bacteria found from the total samples of we components found to contain bacteria. rahmatullah et al. – washed erythrocyte (we) bag bacteria brevundimonas vesicularis … 695 figure 1. bpa and bpn microbiology media. results and discussion the quality control data (qc) of washed erythrocytes (we) in the blood product quality testing laboratory were used to generate the processed sample data. a sample of the we component that has been washed with nacl a couple of times makes up the sample data. the collected information consists of inspection sample data from january to december 2020. the year 2020 saw the collection of 41 samples of data. taking samples and flowing them into the bact/alert 3d media, specifically the blood product anaerobic (bpn) media, which will identify the type of anaerobic bacteria, and the aerobic blood product (bpa), which will identify aerobic bacteria, is how the anaerobic and aerobic bacteria in the we component are identified. the bpn or bpa media provide the result indicator. gray (negative results, no bacteria) and yellow (positive results, bacteria present) are the two colors of the bpn or bpa media that change color as a result of the test (identified bacteria). figure 2. bact/alert 3d culture tool. in table 1. the results of the examination of the identification of 41 samples of we components using the bact/alert 3d tool with bpn media and bpa media are presented during 2020. identification of bacteria using the bact/3d alert tool with bpn and bpa media. table 1. the percentage of bacteria that were identified bpn media bpa media bacterial qc negative positive negative positive graduated not pass 41 0 40 1 40 1 percentase (%) 100 0 97,56 2,44 97,6 2,44 information: positive = contaminated with bacteria negative = not contaminated with bacteria the table above demonstrates that the bact/allert device was used to evaluate 41 samples of we blood components for bacterial contamination. two different media were employed, namely bpn media to identify anaerobic bacteria and bpa media to identify aerobic bacteria in the sample. table 1's findings reveal that all 41 samples contained in the bpn media had negative results, indicating that no anaerobic bacteria were found in the sample. the outcomes of the bpa media identification are likewise displayed in table 1. according to the statistics, 1 (2.44%) of the 41 samples evaluated, which were we blood components, were found to contain bacteria in bpa medium. these findings suggest that the we component contained aerobic bacterial contamination. aerobic bacteria can multiply and need unrestricted oxygen to function. achmad tjiptoprajitno & ketut sudiana, 2012 study discovered that 8 samples out of 120 donor samples were infected with aerobic microorganisms. 696 biology, medicine, & natural product chemistry 14 (2), 2025: 693-698 figure 3. percentage of bacterial contamination in bpn and bpa microbiological media. by dividing the total number of samples that were identified by the total number of examination samples, a percentage representing the distribution of the we component sample data for which the bacteria were identified was calculated. a total of 41 samples were collected between january and december 2020. these samples were each tested using bpn and bpa media, with either negative or positive outcomes. according to the statistics, 41 samples were found to be negative during the evaluation of samples on bpn medium, and 41 samples yielded no positive results. 41 samples were tested using bpa media, with up to 40 samples yielding negative results and just one sample yielding positive results. the data showed that 100% of the samples from bpn medium did not identify bacteria, indicating that there was no chance of anaerobic bacterial contamination, while in bpa media, just 1 sample (2.44%) had positive results for bacteria identification and 40 samples (97.56%) did not. this indicates that one sample of we blood components included aerobic microorganisms. after a third party, specifically the bacteriology lab at the university of indonesia, completed the bacterial culture process, it was discovered that the type of bacterium was brevundimonas vesicularis. there are 41 samples of we component data in the research findings that have been processed. bpn and bpa culture media were used for the study of bacteria. the presence of bacteria might grow and leave endotoxin, which can produce clinical symptoms in recipients, according to (astuti & ayu maharani, 2014). one bpa media sample yielded a favorable outcome. this suggests that an element of the we contains aerobic microorganisms. one aerobic cocci bacterium and no anaerobic bacteria were detected in the study by (astuti & ayu maharani (2014). the university of indonesia's bacterial microbiology lab conducted additional testing, and it was discovered that the germs that tainted we's blood were brevundimonas vesicularis bacteria. gram negative brevundimonas vesicularis bacteria are freeliving, bacilli-shaped, yellow-pigmented on blood agar, and found in the environment in water, dirt, and dust. although these bacteria are also discovered in clinical patient samples and are known to cause serious sickness, their presence in the environment plays a part in maintaining the ecosystem's equilibrium (resmi et al., 2010). the illnesses brought on can range from abscesses to sepsis or meningitis. infections brought on by the bacteria brevundimonas vesicularis are made worse by the extent of antibiotic resistance (ryan & pembroke, 2018). this bacterium's high rate of survival in an unfavorable environment is another intriguing feature. it is known that several species of brevundimonas vesicularis can grow on despite having a negative effect that causes the size of the cells to decrease, medium with few nutrients. brevundimonas sp. are not currently considered major pathogens. however, this should be re-evaluated in light of our investigations, where forty-nine examples of brevundimonas spp. infections have been found in the literature. these species have characteristics, such as the ability to pass through sterilising filters, which may allow them to cause potentially harmful infections and even death on occasion. although it is of low virulence and not as big a risk as other non-fermenting gram-negative bacteria such as burkholderia etc., it should not be over looked as a possible cause of nosocomial infections and should be considered for inclusion in hospital screening and prevention programs. these programs should consider investigation of possible brevundimonas spp outbreaks if these bacteria are clinically isolated in more than one patient (ryan & pembroke, 2018). brevundimonas vesicularis or also called pseudomonas vesicularis is a gram-negative, aerobic, non-spore and non-fermented bacteria. brevundimonas vesicularis bacteria in several studies are human pathogenic bacteria. brevundimonas vesicularis bacteria attack humans who are not immunocompetent and cause sufferers to experience bacteraemia, pneumonia, botryomycosis, progressive leukocytosis, liver abscess, and febrile neutropenia (nurfitriani siska, 2018 cit ryan and pembroke, 2018). the outcomes of these investigations are contrasted with those of prior research projects. finding research on bacteria in we blood components is challenging; studies on bacterial contamination have been conducted, but they 0 20 40 60 80 100 120 41 0 40 1 negatif positif negatif positif media bpn media bpa percentase rahmatullah et al. – washed erythrocyte (we) bag bacteria brevundimonas vesicularis … 697 have used various blood components. the thrombocyte concentrate (tc) component was discovered to be contaminated with bacteria in as many as three bags of the 60 tc bags studied at udd surabaya, according to research by tjiptoprajitno achmad & ketut sudiana (2012). the study indicated that commensal bacteria were the most common type of bacteria found. furthermore, no contaminating microorganisms were discovered in the tc products analyzed at udd yogyakarta according to the investigation of (btari christiyani kusumaningrum & sepvianti, 2020). although there are discrepancies across the research that have been compared, although the results of earlier research have been compared, the analysis of the origin of the contamination process has remained consistent. according to a review of earlier studies, the contamination process might happen during phlebotomy and blood processing and could not be distinguished from the likelihood of a bacteremic donor. this study lists a number of causes for bacterial contamination, including when donor blood is collected, germs from donors, and most recently, the production of we components. the features of these bacteria suggest that they may have come from the environment, which suggests that the process of contamination may have occurred during manufacturing we components from donor blood. according to astuti & ayu maharani (2014) study, bacterial contamination might start to happen before a stabbing. the unwashed donor's arm, the blood-taking officer's hand, or the staff members' work tools can all harbor bacteria. environmental bacteremia or contamination from the surrounding environment at the moment of blood collection are two possible sources of bacterial contamination. research has shown that contamination can occur when blood collection circumstances are not aseptic (btari christiyani kusumaningrum & sepvianti, 2020). the phlebotomy procedure was improperly carried out because contaminated staff hands or equipment were not cleaned and disinfected before stabbing the donor. some liquid disinfectants applied to the skin's surface do less well at lowering the danger of microbiological infection. brevundimonas vesicularis bacteria, on the other hand, can be seen to survive during storage at a storage temperature of 2–6°c in the blood refrigerator based on their features. this is why it is essential to first disinfect the due to this, it is essential first to clean the room where the blood is taken in order to ensure that it is sterile from the setting where the donor is from. additionally, it is important to focus on cleaning the donor's arm first. both when the arm was washed and when the retrieval officer used an alcohol swab to clean the area last. bacterial contamination can potentially be a result of the manufacturing process for we components. contamination may be a possibility in the insulator's surroundings. or the area of the insulator where the creation of we components takes place. this makes it important for officers to do routine cleaning. in the manufacture of we components, it is necessary to perform preand post-process cleaning of insulators, production equipment, and the surrounding area. the pollution on work tools is not removed by the disinfectant solutions utilized. conclusions based on research on the identification of bacteria in the 2020 washed erythrovyte (we) component at udd pmi dki jakarta using the bact/alert 3d tool with blood product anaerobic (bpn) and blood product aerobic (bpa) media, it can be said that one sample, or 2.44% of the total 41 samples of we, were contaminated with aerobic bacteria in 2020. the outcomes also indicated that there was no bacterial contamination. anaerobes from 41 we samples were found utilizing the bact alert 3d technique on bpn media (100%). it is advised to identify the bacteria in the satellite bag with the same donor for additional research, and donor blood identity must be added. the results of identification at the microbiology laboratory at the university of indonesia showed that the aerobic bacteria that contaminated washed erythrocyte (we) blood products on bpa microbiology media were brevundimonas vesicularis. acknowledgments: all people who contributed to this research are sincerely appreciated by the researcher. all respondents who took part in this research, the leadership of pmi dki jakarta who granted permission and helped with data collecting, as well as those who cannot all be specifically named but have helped researchers with their work. competing interests: the authors declare that there is a conflict of interest. references akbar, t.i.s., sofro, a.s.m., gantini, r.s.e. 2014, kualitas dan potensi hemolisis packed red cell (prc) washed erythrocyte dan leukodepleted (in-line) dalam transfusi klinis, j. indon. med. assec. 64(10) : 451-455 astuti, d., & ayu maharani. (2014). identification of bacteria contaminating thrombocyte concentrate. jurnal ilmu dan teknologi kesehatan, 2(1), 61–67. btari christiyani kusumaningrum, s., & sepvianti, w. (2020). identification of contaminating bacteria in thrombocyte concentrate blood products. syifa’ medika, 10(2), 117–123. european committee (partial agreement) on blood transfusion (cd-p-ts). (2020). european committe (partial agreement) on blood transfusion (cd-p-ts). guide to preparation, use and quality assurance of blood components,. council of europe. 698 biology, medicine, & natural product chemistry 14 (2), 2025: 693-698 haass, k. a., sapiano, m. r. p., savinkina, a., kuehnert, m. j., & basavaraju, s. v. 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(2016). rational transfusion in children (issue 4). sari pediatri. https://stacks.cdc.gov/view/cdc/56097 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 557-565 | doi: 10.14421/biomedich.2025.141.557-565 issn 2540-9328 (online) antibacterial activity test of ethanol extract citrus leaf against staphylococcus epidermidis reza anindita1,*, hana yelsi sulvayanti1, maulin inggraini2 1department of pharmacy; 2department of medical laboratory technology, sekolah tinggi ilmu kesehatan mitra keluarga. bekasi, west java, indonesia. corresponding author* rezaanindita@stikesmitrakeluarga.ac.id manuscript received: 23 march, 2025. revision accepted: 15 july, 2025. published: 11 august, 2025. abstract one of the causes of acne is an increase in colonies of staphylococcus epidermidis bacteria. one of the acne treatments that trigger bacterial resistance is the irrational use of antibiotics. therefore, it is necessary to discover natural materials with antibacterial potential, namely orange plants. this study aims to determine the effect of single and combination of lime, kaff, lemon, and sweet leaves extract on of s. epidermidis. the treatment of this study was the concentration of orange leaf ethanol extract of 2%, 4%, and 8% with a bacteria sample of s. epidermidis. phytochemical screening test results contain alkaloids, flavonoids, saponins, tannins, phenols, and essential oils. the results of the one-way annova test showed a significant difference in the average diameter of the inhibition zone of orange leaf ethanol extract against the growth of s. epidermidis (sig.<0.05). post hoc test showed that the effective concentration of lime leaves, kaffles, lemons, sweet oranges, and combination was 8%, while sweet oranges are the most significant treatment group in inhibiting the growth of s. epidermidis. the conclusion is that 8% sweet lime leaf ethanol extract can be used as a pharmaceutical raw material in inhibiting the growth of s. epidermidis with a strong category. keywords: acne; citrus; phytochemicals; kirby bauer; s. epidermidis. introduction acne is one of the diseases in the skin area. according to data from the indonesian cosmetic dermatology study group (icdsg), the incidence of acne in indonesia until 2024 is 87.5% (asbullah et al., 2021). the impact of acne on psychology is embarrassment, loss of confidence, anxiety, and depression. an increase in psychological problems will affect the quality of life of acne sufferers (duru & örsal, 2021). according to sibero et al. (2019), one of the causes of acne is infection with the bacteria propionibacterium acnes, staphylococcus aureus, and staphylococcus epidermidis. therefore, acne treatment is often using antibiotics. however, irrational use of antibiotics often causes bacterial resistance to antibiotics (rahman, 2019), thus increasing the number of morbidity, deaths, and treatment time which has implications for increasing medical costs (yunita & sukmawati, 2021). the percentage of acne bacteria resistance to antibiotics in indonesia is 32.6%-52.2% (marsudi et al., 2021). referring to the problems and impacts of acne and bacterial resistance to antibiotics, efforts to prevent acne by bacteria are needed. one of the prevention efforts is testing plant raw materials that have the potential to inhibit the growth of acne-causing bacteria. the selection of plants as antibacterial agents is because it is safer from the risk of side effects than antibiotics with high costs and risk of bacterial resistance. the natural materials from the plants to be tested in this study are orange leaves, including lime (citrus x aurantiifolia (christm.) swingle), kaffir lime (citrus hystrix dc.), lemon (citrus x limon (l. osbeck), and sweet orange (citrus x aurantium l.). citrus simplicia tests against bacteria have been researched using an infusion of lime leaves and kaffir lime 100% can inhibit e. coli (11.7 mm and 14.3 mm) (siregar et al.,2020), ethyl acetate extract of lemon leaves 25% and 50% inhibits s. aureus (16.16 mm) and e. coli (10.57 mm) (sormin et al., 2023), lime peel ethanol extract inhibits p. acnes with a minimum inhibition concentration (mic) value of 1%, minimum bactericidal concentration (mbc) of 40% and s. epidermidis with mic 1% and mbc 50% (rachmawati et al., 2021), sweet orange peel essential oil 50 μg/ml is able to inhibit e. coli, s. aureus, and s. agalactia (anwar et al., 2023). research nurjannah et al. (2022) concluded that the combination of kaffir lime and moringa leaf extracts with 20%, 40%, 60%, 80%, and 100% could inhibit s. aureus (7.20-10.68 mm). based on previous research, was more focused on the use of a single extract, so this study wanted to test the https://doi.org/10.14421/biomedich.2025.141.557-565 558 biology, medicine, & natural product chemistry 14 (1), 2025: 557-565 effect of citrus leaf extract with novelty on the use of low concentrations single and combination on staphylococcus epidermidis. this study is expected to provide information on the single and combined effects of ethanol extracts of lime leaves, kaffir lime, lemon orange, and sweet orange on staphylococcus epidermidis. materials and methods materials materials and tools include lime leaves (citrus x aurantiifolia (christm.) swingle), kaffir lime leaves (citrus hystrix dc.), lemon leaves (citrus x limon (l.) osbeck), sweet lime leaf (citrus x aurantium l.), pure culture of s. epidermidis atcc: 12228 (microbiology laboratory, university of indonesia), mueller hinton agar (mha) (himedia), agar nutrient (na) (himedia), agar powder (himedia), ethanol 96% (technical), dimethyl sulfoxide (dmso) (merck), blank disc (oxoid), chloramphenicol disc (oxoid), disc paper (oxoid), sterile cotton swab (onemed). sample preparation sample preparation includes plant determination at the faculty of mathematics and natural sciences indonesia university, 1.5 kg orange leaf samples, wet sorting, washing, slicing, drying, dry sorting, and making powder. extraction, evaporation, and screening of phytochemicals the simplicia powder weighed 200 grams each, dissolved with 96% ethanol solvent 1 litre. the solution is soaked for 3 days, occasionally stirring until completely mixed. the filtrate was filtered using whatman no 1 paper, then remaceration in the same way for 5 days. the liquid extract from the maceration process is evaporated by a rotary evaporator at a temperature of 40°c, a pressure of 200 mbar and a speed of 60 rpm for 3 days. the viscous extract obtained is then heated on a waterbath at a temperature of 60°c. the results of the viscous extract were carried out phytochemical screening tests, including alkaloids, flavonoids, saponins, tannins, phenols, and essential oils. antibacterial activity test the preparation of bacteria begins by taking pure culture s.epidermidis 3 ose, suspended in a test tube containing 10 ml of 0.9% nacl solution, divortex until homogeneous (hindun et al., 2021), compared to the turbidity with mc farland 0.5 solution (equivalent to a bacterial suspension of 1.5 x 108 cfu/ml). the suspension of bacteria is inoculated on mha media in a petri dish using the streak plate method and incubated for 15 minutes. blank disks (6 mm) dripped with ethanol extracts of lime leaves, kaffles, lemons, and sweets using micropipettes 100 μl, awaited 30 minutes, and placed on mha media on a petri dish containing streak cultures of s. epidermidis using sterile tweezers. incubation 18-24 hours at 37°c. observations were made by measuring the diameter of the inhibition zone (clear zone) using a ruler (karim et al., 2023). data analysis data analysis uses ibm spss 25 statistics. parametric data (ratio) was tested for normal distribution, homogeneity, then one way annova (sig<0.05). if significant, continued post-hoc test to find groups with significant influence. results and discussion plant determination the plant determination test aims to find out the truth of the species name and plant family (purwanti, 2022). the results of the determination can be seen in table 1. table 1. plant determination results. sampel species family lime citrus x aurantiifolia (christm.) swingle rutaceae kaffir limer citrus hystrix dc. rutaceae lemon citrus x limon (l.) osbeck rutaceae sweet orange citrus x aurantium l. rutaceae results in table 1 show that all samples include the rutaceae family with the latin names lime (citrus x aurantiifolia (christm.) swingle), kaffir lime (citrus hystrix dc), lemon (citrus x limon (l.) osbeck), and sweet orange (citrus x aurantium l.) macroscopic powder the organoleptic test of medicinal plant raw materials aims to determine their shape, color, odor, and taste. the results are shown tables 2 and 3. table 2. organoleptic orange leaf powder. sample organoleptic shape colour odor taste lime powder brownish green aromatic bitter kaffir lime powder light green aromatic bitter lemon powder dark green strong aromatic bitter sweet orange powder dark brown green aromatik bitter anindita et al. – antibacterial activity test of ethanol extract citrus leaf … 559 figure 1. a. lime. b. kaffir lime. c. lemon. d. sweet orange. based on table 2 and figure 1, lime leaf powder has a brownish-green color, kaffir lime leaves light green, lemon dark green color, and sweet lime brownish dark green. lime and sweet leaf samples have a distinctive aromatic odor while kaffir lime and lemon leaves have a strong aromatic distinctive odor. all samples have a bitter taste. organoleptic of viscous extracts table 3. organoleptic of ethanol extract of orange leaf. sampel organoleptic shape colour odor lime viscous exstract blackish brown aromatic kaffir lime viscous exstract blackish brown strong aromatic lemon viscous exstract blackish brown strong aromatic sweet orange viscous exstract dark brown blackish aromatic figure 2. a. lime. b. kaffir lime. c. lemon. d. sweet orange. based on table 3, ethanol extracts of orange leaves are blackish-brown to blackish-dark brown, have a weak to strong aromatic aroma, and are bitter aromatic. yield of extract the extraction in this study uses the maceration and maceration method with 96% ethanol solvent. the selection of the maceration method is simple and is carried out at room temperature without involving heating to minimize the damage to secondary metabolite compounds that are thermostabile (ulfa et al., 2023), meanwhile 96% ethanol solvent was chosen because it has polar properties that are able to bind to the dominance of secondary metabolite compounds that are polar (wendersteyt et al., 2021). remaceration is carried out to make the extraction of secondary metabolite compounds more optimal (nurjannah et al., 2022). remaceration with solvent replacement aims to extend the duration of contact between the solvent and simplicia to increase the content of secondary metabolite compounds in the viscous extract (wahyudi & minarsih, 2023). the results of the viscous extract were calculated by comparing the weight of the viscous extract with the weight of simplicia powder (wijaya et al., 2022). the yield value of ethanol extract of orange leaves can be seen in table 4. table 4. yield value of local orange leaf ethanol extract. sample powder weight (g) extract weight (g) extract yield (%) lime 200 g 16,87 g 8.43 % kaffir lime 200 g 20,06 g 10.03 % lemon 200 g 25,05 g 12.52 % sweet orange 200 g 22,56 g 11.28 % 560 biology, medicine, & natural product chemistry 14 (1), 2025: 557-565 table 4 shows the yield of ethanol extracts of lime, kaffir lime, lemon, and sweet orange are 8.43%, 10.03%, 12.52%, and 11.28%. the yield results correlate with the secondary metabolite compounds contained in the viscous extract. according to research by subaryanti et al., (2022), the yield value is high, the content of secondary metabolite compounds is high. the yield results in this study were greater than the previous study: 3.037%, 9.945%, 8.5%, 5.16% (magfirah et al., 2022; maimunah et al., 2020; rachmawati et al., 2021; sriarumtias et al., 2020). the results of this study are by the criteria of the indonesian herbal pharmacopoeia (not less than 7.2%). several factors cause the difference in the percentage value of the extract yield, namely the extraction method, the length of extraction, the type of solvent, the particle size of the extraction sample, and the comparison of the number of samples with the solvent (putra & surahmaida, 2023). phytochemical screening phytochemical screening is a qualitatively preliminary test stage to provide an overview of the secondary metabolite compound class contained in a sample to be studied. testing is carried out by observing the color testing reaction using a color reagent (saragih & arsita, 2019). this study was carried out to test the existence of secondary metabolites contained in lime, kaffir lime, lemon, and sweet orange leaves using a color reagent. the compounds to be tested include alkaloids, flavonoids, saponins, tannins, phenols, and essential oils because these compounds are able to inhibit microbial growth (nafisa et al., 2021; astriani et al., 2021; rachmawati et al., 2021; sriarumtias et al., 2020). table 5. phytochemical screening orange leaf ethanol extract. sample compound reagents results indicator lime kaffir lime lemon orange alkaloid mayer dragendorf wagner + + + white precipitate orange solution brown solution flavonoid hcl pekat + mg powder + brownish-red solution saponin aquadest + heat + foam formed tannin aquadest + heat + fecl3 1% + greenish-brown solution phenol fecl3 + deep black solution essential oils 96% ethanol + distinctive odor figure 3. a. meyer. b. dragendorf. c. wagner. d.flavonoid.e. phenol. f. tannin. g. saponin.h. essential oils. phytochemical screening tests showed positive results for alkaloids with mayer (white precipitate), dragendoff (orange), wagner (brown), flavonoids (brownish-red), saponins (foam-formed), tannins (greenish-brown), phenols (deep black), and essential oils (special odors). the white precipitation results in the meyer test are due to the interaction of the alkaloid nitrogen group with the tetraiodomercury (ii) (k2[hgi2]) metal ion (syafitri et al., 2023) (harahap & situmorang, 2021). (reiza et al., 2019) (sulistyarini et al., 2020), orange color with dragendorff reagent due to potassium-alkaloid (ki) complex with tetraiodobismutate ion, brown color with wagner reagent due to iodine (i2) reaction with iion from potassium iodide (ki) and form i3 (yanti & vera, 2019). brownish-red color in the flavonoid test due to the addition of concentrated hcl and mg powder (ni’ma & lindawati, 2022). a positive result of saponins indicates a positive result of foam formation. the formation of foam because saponins have hydrophilic and hydrophobic groups so that when the solution is shaken, the hydrophilic group will bind to water while the hydrophobic group will bind to air so that the reaction will form foam (suleman et al., 2022). greenish-brown discoloration due to the addition of fecl3 (khafid et al., 2023). the positive result of phenol compounds is due to the reaction between fecl3 and the oh group (anindita et al., 2022). the essential oil compound test gave positive results, marked by the aromatic odor of residue after being dissolved with 96% ethanol and evaporated on a hot plate using the watch glass. these results are in anindita et al. – antibacterial activity test of ethanol extract citrus leaf … 561 accordance with the research rahmasiahi et al. (2023), the positive results of the essential oil test are characterized by aromatic odor. antibacterial activity test of kirby bauer method the activity test of antibacterial compounds in this study aims to determine the effect of a single extract and a combination of lime leaves, kaff, lemon, and sweet on the growth of s. epidermidis. the antibacterial test carried out in this study used the kirby bauer method with concentrations of 2%, 4%, and 8% single and a combination of comparisons (1:1:1:1) with replication three times. the concentration variation single or combination purpose is to find out which concentration has good antibacterial activity. the principle of the kirby bauer method is to use disc paper as an antimicrobial agent to determine microbial sensitivity by placing disc paper containing antibacterial compounds into agar media that has been inoculated with bacteria. the inhibition of the growth of bacteria produced can be determined by measuring the clear zone around the disc paper (sari et al., 2022). the results of the test on the effect of ethanol extract of orange leaves on s. epidermidis bacteria can be seen in table 6 table 6. test results of ethanol extract of orange leaves. sample treatment x̄ ± sd (mm) sig. lime 2 % 1.67 ± 0.57 0.002 4 % 3 ± 1.00 8 % 5.67 ± 0.57 control (+) 24 control (-) 0 kaffir lime 2 % 2 ± 0.00 0.004 4 % 5 ± 1.00 8 % 8 ± 2.00 control (+) 24 control (-) 0 lemon 2 % 3.33 ± 1.15 0.032 4 % 6.33 ± 2.51 8 % 9.67 ± 2.51 control (+) 24 control (-) 0 sweet orange 2 % 3.67 ± 1.15 0.000 4 % 9.33 ± 0.57 8 % 14.33 ± 1.15 control (+) 24 control (-) 0 combination leaves orange 2 % 5.33 ± 0.57 0.008 4 % 9 ± 2.00 8 % 10.67 ± 1.15 control (+) 24 control (-) 0 control (+) = chloramfenikol control (-) = dmso *significance(sig<0,05) based on table 6, the results of the one way annova obtained sig<0.05, or there was a significant effect of single extract and combination of orange leaves on the diameter of the growth inhibition zone of s. epidermidis. visualization of the inhibition zone between treatment groups can be seen in figure 4 figure 4. a. lime. b. kaffir lime. c. lemon. d. sweet orange. e. orange combination. 562 biology, medicine, & natural product chemistry 14 (1), 2025: 557-565 the results of the post hoc test to determine the group with significant influence can be seen in table 7. table 7. results of post hoc tests between groups of orange leaf extract treatment. group sig. lime kaffir lime 1,000 lemon 0,395 sweet orange 0,006* kaffir lime lime 1,000 lemon 1,000 sweet orange 0,082 lemon lime 0,395 kaffir lime 1,000 sweet orange 0,601 sweet orange lime 0,006* kaffir lime 0,082 sweet orange 0,601 *significance(sig<0,05) based on table 7. post hoc test results of ethanol extract samples of sweet orange leaves most significantly inhibited the growth of s. epidermidis (sig0.006<0.05). the graphic comparison of the effect of single test and combination of ethanol extract of orange leaves on the average diameter of the growth inhibition zone of s. epidermidis can be seen in figure 5. figure 5. comparison of the average diameter of the inhibition zone between treatment groups the treatment of lime leaf ethanol, kaffir lime, lemon, and combination (table 6) with concentrations of 2%, 4%, and 8% was able to inhibit the growth of s. epidermidis with the average diameter of the inhibition zone in lime leaf ethanol extract of 1.67 mm, 3 mm, and 5.67 mm, kaffir lime : 2 mm, 5 mm, and 8 mm, lemon is 3.33 mm, 6.33 mm, and 9.67 mm, sweet orange was 3.67 mm, 9.33 mm, and 14.33 mm, and a combination orange leaf is 5.33 mm, 9 mm, and 10.67 mm. significant treatment was found at a concentration of 8% with the inhibition category for lime (medium), kaffir lime (medium), lemon (medium), sweet orange (strong), and combination (medium). the sweet orange group is the significant influence of treatment in inhibiting the growth of s. epidermidis (table 7). the classification of s. epidermidis sensitivity response to citrus leaf ethanol extract in this study refers to davis and stout (1971) in sakul et al. (2020). it is classified into 4 categories: ≤ 5 mm (weak), 6-10 mm (medium), 11-20 mm (strong), and ≥ 21 mm (very strong). the antibacterial activity of s. epidermidis orange leaf is due to the content of alkaloid, flavonoids, saponins, tannins, phenols, and essential oils. the synergy of secondary metabolite compounds in inhibiting bacterial growth by disrupting peptidoglycan, cell wall surface tension, cell membrane permeability, protein synthesis, and cell leakage results in metabolic disorders, growth, lysis, loss of cytoplasmic substance, and bacterial cell death (saputera et al., 2019; nurjannah et al., 2022; hidayatullah & mourisa, 2023; yunilawati et al., 2021). the factors that affect the diameter of the inhibition zone in this study include the turbidity of the bacterial suspension, the thickness of the agar medium, the incubation time, the diffusion of the extract in the media, the type of bacteria, and the concentration of the extract (rahman et al., 2022; fransisca et al., 2020). related to the type of bacteria factor is proven in research by wardani et al (2019); sari & asri (2022), which used 25%, 50%, and 75% lime peel ethanol extracts were able to inhibit s. epidermidis (11.66 mm, 15.66 mm, and 18.33 mm), concentrations of 12.5%, 25%, 37.5%, and 50% against shigella dysenteriae (1.15 mm, 2.69 mm, 2.96 mm, and 3.35 mm). s. epidermidis belongs to gram positive whereas shigella dysenteriae is gram negative. based on research by hermawati et al. (2023) gram-positive bacteria have a layer of the hydrophobic cell wall that is easily penetrated by secondary metabolite compounds compared to gramnegative bacteria. the incubation factor is proven in the research by maimunah et al. (2020) which used 5%, 10%, 15%, and 20% kaffir lime leaf samples after 48 hours of incubation was able to inhibit s. aureus (6.6 mm, 6.8 mm, 7.3 mm, and 8.2 mm), while in this study the concentration of 8% with an incubation time of 18-24 hours was 8 mm dewi et al. (2020) using 5%, 12.5%, 25%, 50%, 75%, 100% fruit juice and lemon peel as much as 20 μl was able to inhibit p. acne bacteria with (7.15 mm, 9.20 mm, 11.25 mm, 12.50 mm, 14.10 mm, and 16.90 mm). results anindita et al. (2022), ethanol extracts of 20%, 40%, 60%, 80%, and 100% lemon peels showed negative alkaloid results so only able to inhibit s. aureus bacteria (1.5-3.5 mm). the factor of the length of the soaking time of the disc paper is proven in the study niken et al. (2023), sweet orange peel extract 20%, 40%, 60%, 80%, and 100% with 15 minutes of soaking was able to inhibit the growth of s. aureus (12.3 mm – 21.4 mm), while in this study, soaking for 30 minutes was able to produce an inhibition zone of 14.33 mm. anindita et al. – antibacterial activity test of ethanol extract citrus leaf … 563 conclusions the results of the phytochemical screening test of ethanol extract of orange leaf were positive for alkaloids, flavonoids, saponins, tannins, phenols and essential oils. the results of the one way annova showed that there was a significant influence on the average diameter of the growth inhibition zone of s. epidermidis. the post hoc test showed that the effective concentration of lime leaf, kaffir lime, lemon, sweet orange, and the combination was 8%, while for the most significant group category was sweet orange leaf with strong categories. therefore, it can be concluded that 8% sweet orange leaf ethanol extract can be recommended as a pharmaceutical raw material in inhibiting the growth of s. epidermidis. acknowledgements: the author would like to thank stikes mitra keluarga bekasi for granting permission to conduct research in the pharmaceutical microbiology laboratory. authors’ contributions: reza anindita, hanna yelsi, maulin inggraini designed the study. hanna yelsi carried out the laboratory work. hanna analyzed the data. reza anindita and maulin inggraini wrote and did the proofreading. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references anindita, r., yolanda, h., & inggraini, m. 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(0561) 765342, fax.739636, indonesia 3pharmacy study program, stikes dirgahayu samarinda, jl.pasundan no.21 kelurahan jawa, kecamatan samarinda ulu, samarinda 75122, tel. (0541) 748335, indonesia. corresponding author* ssianturi@fahutan.unmul.ac.id abstract tahongai (kleinhovia hospita l.) is an indigenous plant of east kalimantan known for its health benefits, including antibacterial, antiinflammatory, antioxidant, anticancer, and antidiabetic properties. this study evaluates the antihypercholesterolemic activity of tahongai leaf infusion in hypercholesterolemic mice (mus musculus l.), induced by egg yolk. the test groups included a positive control (simvastatin), a negative control (distilled water), and three treatment groups with tahongai leaf infusion at concentrations of 15%, 30%, and 60%. the results indicated that the highest reduction in cholesterol levels was observed in the p1 group (15% infusion) with an average decrease of 30.84%, followed by p2 (30% infusion) at 24.79%, and p3 (60% infusion) at 9.31%. the positive control group showed an average reduction of 8.64%. statistical analysis using one-way anova revealed a significant difference (p<0.05) in cholesterol level reduction among the treatment groups. keywords: anticholesterol; tahongai leaf; kleinshovia hospita l; hipercholesterolemia; mice. introduction the rapid advancement of technology has led to a shift in people's lifestyles, where individuals tend to choose instant solutions, including fast food consumption. fast food is typically high in cholesterol but low in fiber (bachmid et al., 2015). cholesterol is a complex compound essential for regulating biochemical processes in the body. however, excessive cholesterol levels in the blood can be harmful, leading to hypercholesterolemia. this condition occurs when cholesterol levels exceed the normal range, increasing the risk of coronary heart disease (muqowwiyah & dewi, 2021). according to the 2019 who report, heart disease is one of the leading causes of death worldwide, accounting for approximately 55% of 55.4 million global deaths. in 2000, coronary heart disease was the leading cause of death, with 2 million fatalities, which increased to 8.9 million in 2019 (menkes ri, 2018). changes in blood cholesterol levels play a crucial role in preventing the adverse effects of elevated cholesterol, such as coronary heart disease caused by atherosclerosis. atherosclerosis leads to the narrowing of blood vessels, restricting blood flow, stimulating clot formation, and ultimately disrupting circulation (mufida et al., 2018). one of the primary strategies to mitigate the negative impacts of increased cholesterol levels is cholesterol reduction, a crucial healthcare approach to prevent heart disease risks. cholesterol reduction is commonly achieved through hypolipidemic drugs or natural medicinal plants known for their cholesterol-lowering properties. currently, extensive research is being conducted on medicinal plants with effects comparable to synthetic drugs but fewer side effects (saryono et al., 2017). traditional medicine has been found to have fewer side effects han synthetic drugs. plants serve as a source of various bioactive compounds and have the potential to be used as raw materials for medicinal development. thus, in-depth research on medicinal plants is essential. traditional medicine often exerts multiple pharmacological effects, making it more suitable for treating metabolic and degenerative diseases (tamuntuan et al., 2019). aviani et al. (2022) identified flavonoids, alkaloids, and tannins as bioactive compounds with cholesterollowering effects. flavonoids inhibit the hmg-coa reductase enzyme, which is crucial in cholesterol biosynthesis. by inhibiting this enzyme, the production of cholesterol in the liver is reduced. alkaloids inhibit manuscript received: 18 february, 2025. revision accepted: 20 may, 2025. published: 23 june, 2025. https://doi.org/10.14421/biomedich.2025.141.233-238 mailto:ssianturi@fahutan.unmul.ac.id 234 biology, medicine, & natural product chemistry 14 (1), 2025: 233-238 lipase enzymes, thereby preventing cholesterol formation. tannins, on the other hand, block fat absorption in the intestines and precipitate proteins, hindering cholesterol and fat uptake in the intestinal lining (artha et al., 2017). one of the medicinal plants native to east kalimantan with significant therapeutic potential is the tahongai leaf (kleinhovia hospita l.). qualitative phytochemical analysis of tahongai leaves has revealed the presence of alkaloids, tannins, flavonoids, triterpenoids, and steroids. previous studies on laboratory mice (mus musculus l.) have demonstrated the efficacy of tahongai leaves in exhibiting antibacterial, anti-inflammatory, antioxidant, anticancer, and antidiabetic activities. based on literature studies and prior research, this study aims to determine the cholesterol-lowering effects of tahongai leaf infusion in hypercholesterolemic mice at varying concentrations. materials and methods research location and time the study was conducted from february to march 2023 at stikes dirgahayu samarinda laboratory. the extraction process and phytochemical screening of the test material were carried out in the phytochemistry laboratory, while the in vivo tests on experimental animals were performed in the pharmacology laboratory at stikes dirgahayu samarinda. research equipment and materials the equipment used for testing the antihypercholesterolemic activity included: funnels, dropper pipettes, measuring cylinders, vial glasses, volumetric flasks, micropipettes, a stomach tube, a refrigerator, a rotary evaporator, mouse cages, an analytical balance, syringes, cholesterol test devices, and cholesterol test strips. the materials used in the study included 25% ammonia (mallinckrodt), hydrochloric acid (merck), mayer’s reagent, bouchardat’s reagent, dragendorff’s reagent, ferric (iii) chloride (merck), sodium nitrite (merck), aluminum chloride (merck), sodium hydroxide (merck), ether (merck), acetic anhydride (merck), concentrated h2so4 (merck), 70% ethanol (asm), chloroform (merck), carboxymethyl sodium, egg yolk, simvastatin 10 mg, mice weighing 20–40 g, and standard feed and water for the mice. procedures preparation of tahongai leaf infusion 50 g of dried tahongai leaves was infused using 50 ml of water as a solvent at 90°c for 15 minutes. heating was performed on a hot plate while stirring continuously. the extract was filtered while still hot using filter paper. the resulting infusion was then prepared at different concentrations of 15%, 30%, and 60% (aji et al., 2021). phytochemical screening alkaloid identification a total of 2 g of powdered sample was moistened with 5 ml of 25% ammonia in a beaker glass, followed by adding 20 ml of chloroform until the sample was fully submerged. the mixture was stirred, heated over a water bath, and filtered. the residue was placed into a test tube, and 3 drops of 2n hydrochloric acid were added, followed by shaking. the solution was left to form two layers, and the clear layer was divided into two separate test tubes. mayer’s and bouchardat’s reagents were then added. a positive alkaloid presence was indicated by the formation of a white precipitate in mayer’s reagent and a brown precipitate in bouchardat’s reagent (satrana, 2017). flavonoid identification 1 g of powdered sample was extracted with 100 ml of hot water and then filtered. a 5 ml aliquot of the filtrate was placed in a test tube. next, 1 ml of 5% sodium nitrite solution and 1 ml of 10% aluminum chloride solution were added, followed by shaking. then, 2 ml of 1n sodium hydroxide solution was added along the test tube wall. a color change to red or orange indicated the presence of flavonoids (satrana, 2017). saponin identification 1 g of powdered sample was extracted with 100 ml of hot water and then filtered. a 10 ml aliquot of the filtrate*was placed in a test tube and shaken vertically for 10 seconds. the presence of saponins was indicated by the formation of a stable foam measuring 1 to 10 cm in height, which did not disappear upon adding one drop of 2n hydrochloric acid (satrana, 2017). tannin identification 1 g of powdered sample was extracted with 100 ml of hot water and then filtered. a 5 ml aliquot of the filtrate was placed into a test tube, followed by adding a few drops of 1% ferric (iii) chloride solution. a color change to green, purple, or black indicated the presence of tannins (satrana, 2017). steroid and triterpenoid identification a total of 2 g of powdered sample was macerated with 20 ml of ether for 2 hours, followed by filtration and evaporation to obtain a residue. the residue was then treated with 2 drops of acetic anhydride and 2 ml of chloroform and transferred into a test tube. a slow addition of 1 ml of concentrated h2so4 (liebermannburchard reagent) was performed along the test tube wall. a purple ring indicated the presence of terpenoids, while a green ring indicated the presence of steroids (satrana, 2017). acclimatization of experimental animals the experimental animals used in this study were male mice weighing 20–35 g. before testing, the mice were sianturi et al. – antihypercholesterolemic ativity of tahongai leaf 235 acclimatized in cages for one week to adjust to the new environment. they were provided food and water, and their general health and body weight were monitored. after acclimatization, all mice underwent fasting for 12– 14 hours. according to murray et al. (2003), fasting aims to significantly reduce hmg-coa reductase activity and lower exogenous cholesterol synthesis. testing antihypercholesterolemic activity in hypercholesterolemic mice preparation of experimental animals the mice were first fasted for approximately 14 hours without food but were provided with water. their body weights were measured, and they were divided into five treatment groups: positive control, negative control, and three experimental groups receiving different doses (dose i, dose ii, and dose iii). determination of simvastatin dose (positive control) based on the dose conversion table, the dose conversion factor from a 70 kg human to a 20 g mouse is 0.0026. the human dose of simvastatin is 10 mg. the formula used for dose determination: dose for humans × 0.0026 = mg/mouse (20 g body weight. thus, the simvastatin dose for mice was calculated as follows:10mg×0.0026=0.026 mg/20g for example, if a mouse weighed 30 g, the dose was determined as:30/20 × 0.026 = 0.039 mg egg yolk administration for hypercholesterolemia induction herliana and sitanggang (2009) state that highcholesterol foods include brain and egg yolk. in this study, egg yolk was used for hypercholesterolemia induction. the calculation was as follows: cholesterol content in 100 g of egg yolk = 1500 mg. normal cholesterol level=<200 mg/dl. target cholesterol level=300 mg/dl target cholesterol level/normal cholesterol level×cholesterol content = required amount thus, the required egg yolk administration was:300/200 × 1500 = 2.25 g this study, induced mice with 2.25 g of egg yolk mixed with 100 g of standard feed for 2 weeks. preparation of negative control solution the negative control assessed substances without diuretic activity, enabling comparison with the test substance. the negative control group was administered 0.5 ml of distilled water orally. blood sampling technique blood samples were collected from the tail vein (vena lateralis caudae) of the mice. the mice were restrained in a suitable container, their tails were extended, and the vein was punctured with a lancet. the collected blood was applied to cholesterol test strips. experimental procedure mice were fasted for approximately 14 hours while maintaining water intake. on the test day, body weights were recorded, and the mice were divided into five groups, each consisting of five individuals. mice were assigned to groups based on weight to ensure uniform dosing. hypercholesterolemia was induced using 2.25 g of egg yolk for 14 days, followed by cholesterol level measurements. after confirming hypercholesterolemia, the treatment was administered for 14 days. results and discussion table 1. phytochemical screening of tahongai leaf (kleinhovia hospita l.). no test/reagent result 1 alkaloid (dragendroff) + 2 alkaloid (mayer) + 3 alakaloid (bouchardat) + 4 flavonoid + 5 fenol 6 saponin + 7 tanin + 8 steroid + terpenoid + note : (+) indicates the presence of secondary metabolites (-) indicates the absence of secondary metabolites table 2. cholesterol levels before and after treatment. no hypercholesterolemia induction (mg/dl) post treatment infusion (mg/dl) kk+ p1 p2 p3 kk+ p1 p2 p3 1 135 138 161 123 133 117 123 120 115 115 2 122 115 147 140 124 120 109 117 118 110 3 115 122 198 160 145 110 120 135 120 136 4 143 104 171 123 152 135 105 118 117 145 5 145 150 106 216 126 130 122 103 135 118 mean± std 132± 13,11 125,8± 18,31 156,6± 33,89 152,4± 38,68 136± 12,14 122,4± 10,06 115,8± 8,23 118,6± 11,37 121± 8,03 124,8± 14,96 note: k-= negative control (treatment with distilled water as solvent); k+= positive control (simvastatin at a dose of 0.026 mg/20 g body weight); p1= treatment with kleinshovia hospita infusion at 15% concentration; p2= treatment with kleinshovia hospita infusion at 30% concentration; p3= treatment with kleinshovia hospita infusion at 60% concentration; std = standard deviation. 236 biology, medicine, & natural product chemistry 14 (1), 2025: 233-238 figure 1. graph of the average cholesterol levels in mice before and after treatment with kleinshovia hospita leaf infusion. table 3. percentage reduction in cholesterol levels in mice after kleinshovia hospita infusion treatment. no kk+ p1 p2 p3 1 15,38 12,19 34,16 6,9 15,65 2 1,66 5,5 25,64 18,64 12,72 3 4,54 1,66 46,6 33,3 6,61 4 5,92 0,95 44,9 5,13 4,82 5 11,53 22,9 2,9 60 6,78 mean (%) 7,81 8,64 30,84 24,79 9,31 % cholesterol reduction = 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑐ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙−𝑝𝑜𝑠𝑡 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑐ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑐ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙 x 100% figure 2. graph percentage reduction in cholesterol levels after kleinshovia hospita treatment note: k-= negative control (treatment with distilled water as solvent) k+= positive control (simvastatin at a dose of 0.026 mg/20 g body weight) p1= treatment with kleinshovia hospita infusion at 15% concentration p2= treatment with kleinshovia hospita infusion at 30% concentration p3= treatment with kleinshovia hospita infusion at 60% concentration based on the results shown in table 1, the metabolite compounds found in tahongai leaves include alkaloids, flavonoids, saponins, and tannins. these findings align with the research conducted by budiarti and joko (2020), which qualitatively confirmed the presence of alkaloids, tannins, saponins, flavonoids, and steroids in tahongai leaves. quantitatively, these leaves contain 2.83% alkaloids, 19.78% flavonoids, and 14.23% saponins (yunita et al., 2019). the data in tables 2 and 3 indicate reduced cholesterol levels in the control and treatment groups using tahongai leaf infusion. for the negative control group, before treatment, the average cholesterol level was 135 mg/dl and after treatment with tahongai leaf infusion, it averaged 122.4 mg/dl, which calculates to an average cholesterol reduction percentage of 7.81% as shown in table 3. furthermore, for the positive control group, the average cholesterol level decreased from 125.8 mg/dl before treatment to 115.8 mg/dl after treatment. for the p1 treatment group, which received a 15% tahongai leaf infusion, the data showed a decrease from an initial average cholesterol level of 156.6 mg/dl to 118.6 mg/dl. the p2 treatment group, using a 30% infusion concentration, started with an average cholesterol level of 152.4 mg/dl and experienced a reduction to 121 mg/dl. for the p3 group with a 60% infusion concentration, the initial average cholesterol level was 136 mg/dl, which decreased to 124.8 mg/dl. according to table 3, the average percentage reduction in cholesterol levels for all treatments showed that the positive control group had an average reduction of 7.81%, while the 15% tahongai leaf infusion group showed a significant reduction of 30.84%, the 30% concentration group showed 24.79%, and the 60% concentration group showed 9.31%. this result is further supported by statistical analysis data from the lsd test in table 4.6, indicating significant differences (p<0.05) between the control and treatment groups, specifically between the positive control group and the p1 treatment group. the treatment with a 15% tahongai leaf infusion showed the most effective anti-cholesterol activity in reducing cholesterol levels in hypercholesterolemic mice. the anti-cholesterol effect observed in this study is attributed to metabolite compounds such as flavonoids, alkaloids, and tannins in the leaves. the flavonoid in 132 125,8 156,6 152,4 136 122,4 115,8 118,6 121 124,8 0 20 40 60 80 100 120 140 160 180 kk+ p1 p2 p3 c h le st er o l le v el s in m ic e( m g /d l) treatment on mice graph of the average cholesterol levels in mice before and after treatment with kleinshovia hospita leaf infusion 7,81 8,64 30,84 24,79 9,31 0 10 20 30 40 kk+ p1 p2 p2 p er ce n ta g e ch o le st er o l re d u ct io n (% ) treatment percentage reduction in cholesterol levels after treatment of kleinshovia hospita infusion sianturi et al. – antihypercholesterolemic ativity of tahongai leaf 237 kemuning leaves, derived from a subgroup called apigenin, which is light yellow and crystal-like in shape (adfa, 2007), works by inhibiting the hmg-coa reductase enzyme, which aids in cholesterol formation. by inhibiting this enzyme, cholesterol production by the liver decreases (artha et al., 2017). flavonoids also help dissolve lipid clusters attached to the walls of coronary vessels, improving blood flow (anggraini & nabillah, 2018). alkaloids, organic compounds containing nitrogen and commonly found in plants, inhibit the activity of the pancreatic lipase enzyme, which is converts fats into glycerol and fatty acids. by inhibiting lipase, fat absorption by the liver is hindered, preventing cholesterol synthesis (artha et al., 2017). tannins in kemuning leaves, serve multiple medicinal purposes such as anti-diarrheal, antibacterial, and antioxidant. their mechanism in reducing cholesterol levels involves inhibiting fat absorption in the intestines and precipitating protein tissues, thereby obstructing cholesterol and fat absorption at the intestinal surface (artha et al., 2017). plant steroids, or phytosterols, also help lower total cholesterol levels. research by jones et al. (2000) and nguyen (1999) suggests that the consumption of 2-3 g/day of phytostanols can reduce total and ldl cholesterol in humans, while hdl serum concentrations do not change significantly. additionally, phytosterols are believed to inhibit the absorption of exogenous cholesterol and the reabsorption of endogenous cholesterol in the digestive tract, enhance the excretion of absorbed cholesterol, and cause a reduction in serum cholesterol levels due to competition between cholesterol and phytosterols for absorption in the intestines (bonsdorff-nikander, 2005). alkaloids also have a cholesterol-lowering effect, as indicated by kou et al. (2016) who concluded that a combination of five primary alkaloids (berberine, coptisine, palmatine, epiberberine, and jatrorrhizine) shows synergistic effects in reducing cholesterol levels. conclusions the conclusions from this research are as follows: ▪ tahongai leaf infusion has an anti-cholesterol effect on hypercholesterolemic mice (mus musculus l.). ▪ the percentage reduction in cholesterol levels in mice through tahongai leaf infusion is most effective at the 15% concentration, with an average percentage reduction of 30.84%, followed by 24.79% at the 30% concentration and 9.31% at the 60% concentration. the positive control group showed an average reduction of 8.64%. authors’ contributions: the contributions of the authors include the first author conducting animal tests and analyzing cholesterol reduction data, while other members contributed to the chemical testing of plant compounds by conducting phytochemical screening and assisting in the statistical analysis of data obtained. all authors collectively participated in reviewing the article. acknowledgements: acknowledgments are expressed briefly; all sources of institutional, private, and corporate financial support for the work are fully acknowledged, and any potential conflicts of interest are noted. authors’ contributions: the contributions of the authors include the first author conducting animal tests and analyzing cholesterol reduction data, while other members contributed to the chemical testing of plant compounds by conducting phytochemical screening and assisting in the statistical analysis of data obtained. all authors collectively participated in reviewing the article. competing interests: we declare that there are no competing interests in conducting this research. references adfa, m. 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ebenaceae; triterpenoid ketone; pancreatic amylase; non-competitive inhibition. introduction diabetes is a chronic disease of multiple etiologies characterized by chronic hyperglycemia with alterations in carbohydrate, fat and protein metabolism. this disorder affects various organs, including eyes, kidneys, nerves, heart and blood vessels. the prevalence of diabetes in india is exploding and 2017 estimates by the international diabetes foundation indicate that 72.9 million people are affected by diabetes in india. type 2 diabetes population in india has a high burden (76.6%) of poor glycemic control(borgharkar & das, 2019). inhibition of intestinal absorption and digestion of carbohydrates targeting the carbohydrate-hydrolyzing enzymes such as -amylase and -glucosidase is an attractive strategy (matsui, ogunwande, abesundara, & matsumoto, 2006; tundis, loizzo, & menichini, 2010). research efforts directed towards screening and developing natural compounds with potential antidiabetic properties is on the rise. -amylase recognizes a consecutive glucose chain as a substrate using its subsite (brayer et al., 2000). acarbose, a typical -amylase inhibitor, has a strong affinity for the enzyme due to pseudo-tetrasaccharide structure (c. li et al., 2005). most of the small molecule inhibitors from natural sources against -amylase include polyphenols with low enzyme specificity (kim, kwon, & son, 2000; h. li, tanaka, zhang, yang, & kouno, 2007; lo piparo et al., 2008; mcdougall et al., 2005). a triterpene glycoside is reported as a specific inhibitor (tarling et al., 2008), and a simple low-polar ketone, chalcone was also found to exhibit -amylase inhibitory activity (najafian et al., 2011). natural triterpenoids have wide spectrum of biological activities (siddique & saleem, 2011). most triterpenes consist of 30 carbon atoms and can be regarded as a compound of 6 isoprene structural units and distributed in plants with both monocotyledons and dicotyledons. two main classes containing either tetracyclic or pentacyclic triterpenes exist. lupene is the most basic structure of lupane-type pentacyclic triterpienoid. it contains a, b, c and d 6-carbon rings and a pentacyclic e ring with an isopropryl group at the 19th position. lupenone is a typical polar lupine type triterpenoid, and it has a ketone group at position 3 in the nuclear ring (meng, huang, & liu, 2008). lupenone is a secondary metabolite in many plants (lee & lee, 1999; na, kim, osada, & ahn, 2009) and possess anti-inflammatory, anti-diabetic, and anti-tumor activity(wang, liu, wang, & zhong, 2012; xu et al., 2014). in this paper, we present the results of a study on amylase inhibition and identification of the active manuscript received: 30 november, 2022. revision accepted: 17 january, 2023. published: 24 january, 2023. https://doi.org/10.14421/biomedich.2023.121.171-176 172 biology, medicine, & natural product chemistry 12 (1), 2023: 171-176 principle from the extract of leaves of d. melanoxylon. disopyros melanoxylon roxb (family: ebenaceae), the coromandel ebony or east indian ebony (tendu in hindi), is a middle sized deciduous tree or shrub native to india and srilanka. this plant is considered to be a minor forest produce (mfp) in india, as its leaves are used for making bidi, a traditional cigarette. the leaves are arranged opposite, mostly subopposite or alternate, coriaceous, up to 35 cm long with most of them between 6 and 15 cm; tomentose on both sides when young but when full grown glabrous above, tomentose or pubescent beneath, parrot green; petiolate, with petiole up to 1 cm long; exstipulate, simple and entire; secondary nerve 6-10 pairs, often irregular and branching; tertiary nerves reticulate and raised on upper side; shape variable in the same plant but predominantly of one type out of the four basic forms, namely ellipsoidally lanceolate, ovate, elliptic and orbicular (sometimes cuneate). the bark is pelican in colour, exfoliating in rectangular scales. the wood is also utilized for making boxes, combs, ploughs and beams. the fruits are eaten and sold commercially. the bark is burnt by tribals to “cure” small-pox. the seeds are prescribed as cure for mental disorders, palpitation of heart and nervous breakdown (j. rathore, 1972). recent studies have reported anti-adipogenic, anti-diabetic and hypolipidemic pharmacological activities of d.melanoxylon leaf extracts (k. rathore et al., 2014). however, the phytochemicals responsible for the observed effects have not been elucidated. materials and methods materials porcine pancreatic -amylase (ppa) was obtained from sigma aldrich, usa. ethyl acetate, methanol, benzene, n-hexane were obtained from merck chemicals ltd. acarbose was from bayer pharmaceuticals pvt ltd. soluble starch (extra pure) was obtained from himedia laboratories. all the chemicals and reagents procured were of ar grade. extraction and isolation of inhibitor from the bark of d. melanoxylon the barks of d. melanoxylon were collected from bhadrachalam district of telangana. the plant was authenticated by prof. m. mamatha from the forest college and research institute, mulugu. a digital specimen voucher was deposited in the herbarium of fcri. the bark was shade dried, cut into small pieces, and powdered in a pulverizer. the powdered bark (1.5 kg) of d. melanoxylon was extracted with ethanol (3 x 18 l) at room temperature for 24 h. the extract was filtered and concentrated in vacuo, suitably diluted with water, then partitioned with dichloromethane (3 x 1.5 l), ethyl acetate (3 x 1.5 l) and n-butanol (3 x 1.5 l), successively. column chromatography of the dichloromethane-soluble layer (20 g) over silica gel using n-hexane-acetone mixture with increasing polarity yielded 15 fractions. fraction 2 (2.0 g) that exhibited ppa-inhibition was further applied to a flash column chromatography with rp-18 using methanol/water (30:70 to 80:20) yielded four fractions. ppa-inhibitor was isolated form fraction 4 by silica gel column chromatography eluting with dichloromethane-acetone gradient (1:0, 50:1, 20:1, 10:1, 5:1, acetone). 1hand 13c-nmr spectra were recorded on a bruker advance iii 500 mhz nmr spectrometer using cdcl3 as a solvent. mass spectra were obtained using a waters q-tof micro mass spectrometer. spectral analysis indicated that the isolated compound is lupenone. -amylase inhibitory activity -amylase inhibition assay was carried out according to the method of sudha et al. (2011) based on the starchiodine test with little modification (sudha, zinjarde, bhargava, & kumar, 2011). 75 l of different fractions (100-500 mg/ml) were added to 150 l 0.02 m sodium phosphate buffer (ph 6.9 containing 6 mm sodium chloride) and 75 l -amylase solution, and incubated at 37c for 15 min. subsequently, 100 l from each sample reaction solution was taken and added to 750 l soluble starch (1%, w/v). 500 l phosphate buffer solution was added and incubated at 37 c for 45 min. 25 l of the above mixture was taken and added to 2.5 ml of iodine reagent (5 mm i2 and 5 mm ki) and mixed thoroughly. the color change was observed and the absorbance was taken at 565 nm. control sample was without any inhibitor representing 100% enzymatic activity. to eliminate the absorbance produced by plant fractions, appropriate fraction controls without the enzyme were also included. the standard drug acarbose (-amylase inhibitor) was used as a positive control. it is observed the dark-blue color which indicates the presence of starch, a brownish color indicates partially degraded starch and a yellow color indicates the absence of starch in the reaction mixture. in the presence of inhibitors from the fraction the starch added to the enzyme assay mixture is not degraded and gives a dark blue color complex whereas no color complex is developed in the absence of the inhibitor, indicating that starch is completely hydrolyzed by -amylase. the percentage inhibition of -amylase was calculated using the following formula: % 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑒𝑠𝑡) 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 ∗ 100 statistical analysis: data were expressed as mean ± standard deviation (sd). statistical analysis was performed using two-way analysis of variance (anova). durgam, et al. – lupenone isolated from diospyros melanoxylon bark non-competitively … 173 results and discussion bioassay-guided fractionation yielded a potential inhibitor against porcine pancreatic -amylase, which was obtained as a white amorphous powder, with a melting point of 168-170c. figure 1. ei-ms spectrum of the isolated inhibitor. the mass spectrum of the isolated inhibitor as shown in figure 1exhibited a molecular ion peak [m]+ at m/z 424, corresponding to a molecular formula c30h48o. fragments at m/z 203 and 218, characteristic of pentacyclic triterpenes were also present in the mass spectrum of compound. the mass spectrometry data also suggested the presence of a carbonyl group (fragment at m/z 205) and the accurate mass of this fragment indicated that it was attached to rings a or b of the pentacyclic ring. the fragment at 409 corresponding to [m-ch3]+ was also observed. the 1h nmr data of the isolated inhibitor represented in figure 2 showed signals for six tertiary methyl groups, which were observed as singlets at 0.79 ppm, 0.97 ppm, 1.04 ppm, 0.84 ppm, 0.74 ppm and 0.92 ppm. the same spectrum also showed resonances for olefinic methylene protons at 4.74 ppm and 4.60 ppm and a vinyl methyl singlet at 1.68 ppm which was shown to be coupled to one of two vinylic protons (h-29a, 4.74 ppm), thus indicating the presence of an isopropenyl group as well as a lupane skeleton. figure 2. 1h nmr spectrum of the isolated inhibitor. the 13c nmr data of the isolated inhibitor shown in figure 3 were in agreement with the mass spectral data, as it revealed the presence of 30 carbon atoms. 13c nmr data of compound was also in agreement with the existence of an isopropenyl group, evidenced by the characteristic vinylic carbon atom resonances at 151.6 ppm and 109.6 ppm, corresponding to carbon atoms 20 and 29 respectively. the existence of an isopropenyl 174 biology, medicine, & natural product chemistry 12 (1), 2023: 171-176 group was also supported by the olefinic methylene protons seen as singlets at 4.74 ppm and 4.60 ppm in the 1h nmr spectrum of compound. the carbon resonance at 218.0 ppm, in the 13c nmr spectrum was assigned to a carbonyl carbon and a resonance at 1.55 ppm, in the 1h nmr spectrum, to two alpha protons of the carbonyl group. figure 3. 13c nmr spectrum of the isolated inhibitor. the chemical structure of the inhibitor ascertained from the chemical shifts of nmr data and mass spectra is presented in figure 4. figure 4. structure of lupenone. -amylase catalyzes the hydrolysis of internal (14) glucosidic bonds. the porcine pancreatic amylase, a 469amino acid containing protein exhibits (/)8 barrel along with a c-terminal -stranded domain with an -crystalline topology (buisson, duee, haser, & payan, 1987; m. qian, haser, & payan, 1993). lupenone showed a dose-dependent decrease in amylase activity with 50% inhibition at 30 m, 85% inhibition at 50 m and 89% inhibition at 100 m. figure 5. effect of different concentrations of lupenone on amylase activity. figure 6. non-competitive inhibition of amylase by lupenone as demonstrated by lineweaver-burk plot. durgam, et al. – lupenone isolated from diospyros melanoxylon bark non-competitively … 175 figure 7. dixon plot for calculation of inhibitor constant. the positive control, acarbose exhibited 50% inhibition at 21.28 ± 1.42 μm. enzyme kinetics data as shown in figure 5 shows a dose-dependent decrease in amylase activity with increasing concentrations of lupenone. further kinetic analysis using lineweaverburk plot shown in figure 6 indicated that vmax decreased, without change in km (2.4 mg/ml) with increasing concentrations of lupenone. these data suggest that lupenone is a non-competitive inhibitor of ppa. further, dixon plot of 1/v vs. [lupenone] shown in figure 7 with increasing concentrations of starch as substrate indicated the inhibitor constant to be 30 m. considering the characteristics of noncompetitive inhibition, lupenone likely binds at an allosteric site separate from the active site of substrate binding, regardless of the presence of a bound substrate. lupenone thus may have the same affinity for both the enzyme alone and the enzyme-substrate complex. binding of lupenone to the enzyme or enzyme-substrate complex inhibits the enzyme, disallowing the production of its end product. inhibition of the enzyme decreases the maximum rate of the reaction (vmax), defined as the rate of the reaction at a substrate concentration that fully saturates all active sites of the specific enzyme. the affinity of the enzyme for its substrate (km) remained unchanged as the active site is not competed for by the inhibitor. inhibition of intestinal enzyme such as amylase responsible for the breakdown of dietary sugar by lupenone may be effective in preventing postprandial hyperglycemia in the diabetic subjects. a large number of in vitro and in vivo studies have shown that lupenonecontaining plants and lupenone have significant anti-diabetic activity. according to the reports, the plants and herbs containing lupenone have good anti-diabetic activity, including rhizoma musae (the root of musa basjoo sied. et zucc.), banana peel (musa nana lour.), thespesia populnea bark and leaf, acanthus ilicifolius l., adenophora triphylla var. japonica, pueraria lobata root, etc. (ahmad et al., 2015; ahn & oh, 2013; callies et al., 2015; parthasarathy, ilavarasan, & karrunakaran, 2009; seong, roy, jung, jung, & choi, 2016). the ethyl acetate and petroleum ether fraction of rhizoma musae could inhibit the activity of α-glucosidase in vitro, and the inhibitory activity of rhizoma musae petroleum ether fraction is higher (zhang, chang, & kang, 2010). the rhizoma musae fraction with anti-diabetic activity could improve the glucose metabolism and increase the oral glucose load in alloxan-induced diabetic mice (h. qian, hao, & wang, 2012). the chemical constituents of euonymus alatus (thunb.) sied. have good inhibitory effects against the protein tyrosine phosphatases 1b (ptp1b) and α-glucosidase enzyme activity, and the lupenone isolated from euonymus alatus (thunb.) sied. and sorbus commixta also could inhibit the ptp1b enzyme activity, while lupenone could not inhibit the αglucosidase enzyme activity (jeong et al., 2015; na et al., 2009). the lupenone from abrus precatorius leaves has α-amylase-inhibitory activity (yonemoto, shimada, gunawan-puteri, kato, & kawabata, 2014). in another study, the ethyl acetate and petroleum ether fraction of banana peel (musa nana lour.) show the antihyperglycemic activity. furthermore, the lupenone isolated from banana peel show promising antihyperglycemic activity (wu, xu, hao, yang, & wang, 2015). our study provides mechanistic evidence that lupenone isolated from d. melanoxylon exhibits potent inhibitory activity against -amylase and therefore could be tested further in animal models of diabetes. conclusion α-amylase enzyme assay guided fractionation of the diospyros melanoxylon bark extract yielded a triterpene, lupenone that exhibited non -competitive inhibition. thus, lupenone-based natural inhibitors of α-amylase may aid in limiting the breakdown of dietary sugar and also may be effective in preventing postprandial hyperglycemia in the diabetic subjects. conflict of interest: the authors declare that there is no conflict of interest. references ahmad, s., sukari, m. a., ismail, n., ismail, i. s., abdul, a. b., bakar, m. f. a., . . . ee, g. c. 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(2010). study on αglucosidase inhibitory activity of extracts from musa basjoo sieb. et zucc. sci tech food ind, 31(2), 125-130. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 73-82 | doi: 10.14421/biomedich.2024.131.73-82 issn 2540-9328 (online) ethnobotanical study of medicinal plants in south sumatera, indonesia angga puja asiandu1,*, widya sari2 1faculty of biology; 2faculty of mathematics and natural sciences, universitas gadjah mada, jl. teknika selatan, yogyakarta 55281, indonesia. corresponding author* angga.puja.asiandu@mail.ugm.ac.id manuscript received: 13 january, 2024. revision accepted: 08 may, 2024. published: 15 may, 2024. abstract south sumatra, a province in indonesia, is inhabited by several tribes. the geographical landscape surrounded by primary and secondary forests is a natural resource for the local communities in dealing with medical problems. they use plants as traditional medicine from generation to generation. the used parts include roots, stems, leaves, and sap that are biologically active to treat some diseases from moderate to deadly diseases. based on literature studies, there are 250 plant species from 79 families processed by local communities in south sumatra in some ethnobotanical activities. the dominant plant families are fabaceae (5.6%), asteraceae (4.8%), euphorbiaceae (4.4%), poaceae (4.4%), myrtaceae (3.6%), rubiaceae (3.6%), rutaceae (3.6%), solanaceae (3.6%), and piperaceae (3.2%). among many methods, boiling is the most common method in processing medicinal plants as it is considered more effective in extracting the bioactive compounds. keywords: biodiversity; compound; medicine; ethnobotany; traditional. introduction historically, humans have adapted to nature to continue their life including the use of traditional medicine in preventing diseases that passed down from generation to generation. some local people use traditional medicine for several reasons, belief and suggestion, fast healing rates, cheap treatment costs, and fear of medical treatment (amisim et al., 2020). fokunang et al. (2011) explained that traditional medicine refers to practical, material approaches, insights, and beliefs. also, treatment involving plants, animals, minerals, and spiritual therapy. according to yuan et al. (2016), traditional medicine has its uniqueness related to its diverse and distinctive chemical structure and biological activity. for example, taxol, a compound isolated from t. brevifolia used as an anticancer drug. the most traditional medicines from several countries have contributed to the development of modern medicine like hawthorn and foxglove applied for the treatment of hypertension and cardiovascular disorders (who, 2023). indonesia is known as a mega biodiversity spot after brazil. in 2017, as many as 31,750 species consisting of 2,273 types of fungi, 2,722 types of mosses, 512 types of lichens, 1,611 types of pteridophytes, and 24,632 types of spermatophytes had been identified by national research and innovation agency, and this number will continue to increase along with the number of angiosperm exploration (retnowati et al., 2019). in indonesia, java is the place where most groups of fungi, moss, lichens, and spermatophytes dominate, while sumatra is the island where the distribution of pteridophytes dominates. from the abundance of plants, every part such as roots, stems, leaves and sap has been used for medicinal purposes. south sumatra, 91,592 km2, is inhabited by many tribes, such as the komering, palembang, gumai, semendo, lintang, kayu agung, lematang, ogan, besemah, sekayu, and other tribes. each tribe has its unique method of processing plants to produce traditional medicine. the people of segara kembang village in ogan komering ulu regency (oku) use cassava juice (tubers) as a treatment for typhus, bamboo roots as a fever reducer, brotowali leaves as a medicine for rheumatism, malaria, and fever (sitorus et al., 2011). the miracle leaf (bryophyllum calycinum) is used as a compress for abscesses and to shrink hemorrhoids by the oku community (retnaningsih, 2012). the research was also carried out in lawang agung village, mulak district, lahat regency, which classified 41 types of medicinal plants and of this number, there were 9 types of ethnic plants for fever medicine, feminine hygiene, wart medicine, and wound medicine, namely anthocephalus cadamba m1., gelosia argentea l., eupatorium inofolium h.b.k., leea indica merr., morus multicaulis loud., peronema canescens jack., schima wallichii (dc) korth., and stachitarpeta jamaicensis (l.) vahl (harmida et al., 2011). the large number of tribes https://doi.org/10.14421/biomedich.2024.131.73-82 74 biology, medicine, & natural product chemistry 13 (1), 2024: 73-82 and the abundance of plants, especially ethnic plants, need to be studied more deeply in terms of their medicinal uses in encouraging and assisting modern medicine. thus, this article will emphasize the potential of plants in traditional medicine, especially treatment carried out by the people of south sumatra, which can be a reference for the development of medicine and biological conservation. methods the research method focuses on the research process of collecting information and data through several methods such as observation and reviewing literature. through a qualitative approach, researchers will review secondary data literature and draw conclusions. secondary data will be analyzed descriptively. this data analysis will include data reduction, data presentation and discussion, and conclusions. data reduction is part of extracting main information from the data so that it can be concluded more clearly. in this research, qualitative research methods were used to collect information regarding the essence of plants used as traditional medicine by the people of south sumatra. results and discussion based on our review, 79 plant families, as listed in table 1., are used in ethnobotanical activities in some tribes in south sumatra indonesia for some purposes such as traditional medicines. of these 79 families, there are 250 species of medicinal plants. the dominant plant families are fabaceae (5.6%), lamiaceae (5.2%), asteraceae (4.8%), euphorbiaceae (4.4%), poaceae (4.4%), myrtaceae (3.6%), rubiaceae (3.6 %), rutaceae (3.6%), solanaceae (3.6%), to piperaceae (3.2%). the use of plants as a source of medicine is a tradition that has been passed down from generation to generation in indonesia, including south sumatra. this is because certain plants naturally produce various kinds of chemical compounds pivotal in treating various diseases. the knowledge acquired from generation to generation is still preserved today, especially in rural communities. in rural areas, health facilities are inadequate. thus, people are very dependent on these medicinal plants to treat various symptoms of disease (sarina et al., 2023). not only used as traditional medicines, plants are also used as cosmetics, building materials, and crafts. as traditional cosmetics, they are processed as deodorants, anti-acne, facial skin care, dental care, anti-dandruff, skincare, hair care, and so on. meanwhile, the diseases that are treated using plants traditionally include malaria, high blood pressure, anemia, blood sugar, cholesterol, toothache, stomachache, and even deadly diseases such as cancer and tumors. table 1. ethnobotanical data of south sumatera, indonesia. no. families plant species diseases / uses references 1. acanthaceae acanthus ilicifolius boil (sarno et al., 2013) andrographis paniculata cancer, fever (rizal et al., 2021) avicennia alba stomach ache (sarno et al., 2013) graptophyllum pictum ethnomedicine (pujihastuti et al., 2020) ruellia simplex ethnomedicine (pujihastuti et al., 2020) strobilanthes crispus back pain (sarina et al., 2023) 2. acoraceae acorus calamus baby skin care (tanzerina et al., 2017). 3. aizoaceae sesuvium portulacastrum typhus (sarno et al., 2013) 4. amaranthaceae achyranthes aspera heart disease (saputri et al., 2022). amaranthus hybrids blood booster (rizal et al., 2021). 5. amaryllidaceae allium ascalanicum fever (rizal et al., 2021) allium cepa ethnomedicine (pujihastuti et al., 2020) amaryllis belladonna ethnomedicine (dahlianah et al., 2021) 6. ancistroladaceae anchistrocladus tectorius ethnomedicine (pujihastuti et al., 2020) 7. annonaceae anonna muricata cholesterol (rizal et al., 2021). cananga odorata anti odor (tanzerina et al., 2019) 8. apocynaceae alstonia scholaris tooth ache (sarina et al., 2023) catharanthus roseus ethnomedicine (pujihastuti et al., 2020) 9. araceae alocasia longiloba ethnomedicine (pujihastuti et al., 2020) alocasia plumbea ethnomedicine (hastiana, novitasari, et al., 2023) colocasia esculenta ethnomedicine (pujihastuti et al., 2020) typonium flageliformae hipertiroid (saputri et al., 2022). 10. araliaceae nothopanax scutellarium hair care (tanzerina et al., 2017) asiandu & sari – study of medicinal plants in south sumatera 75 table 1. cont. no. families plant species diseases / uses references 11. arecaceae areca catechu ethnomedicine (pujihastuti et al., 2020) cocus nucifera food poisoning (sarina et al., 2023) daemonorops draco fever (sarina et al., 2023) elais guineensis ethnomedicine (dahlianah et al., 2021) nypa fruticans ethnomedicine (dahlianah et al., 2021) salacca wallichiana postnatal care (sarina et al., 2023) 12. asparagaceae cordyline fruticose ethnomedicine (pujihastuti et al., 2020) tabernaemontana divaricate ethnomedicine (pujihastuti et al., 2020) tabernaemontana macrocarpa ethnomedicine (pujihastuti et al., 2020) tylophora villosa ethnomedicine (pujihastuti et al., 2020) 13. asphodelaceae aloe vera hair fertilizer, burns, lowers blood sugar (rizal et al., 2021). 14. aspleniaceae asplenium nidus ethnomedicine (pujihastuti et al., 2020) 15. asteraceae ageratum conyzoides diarrhea, wounds, hypertension, fever (hastiana, novitasari, et al., 2023) blumea balsamifera jaundice and postnatal care (sarina et al., 2023) clibadium surinamense ethnomedicine (pujihastuti et al., 2020) cosmos caudatus skin care, mouth, body odor (tanzerina et al., 2017) eclipta alba hair care and leucorrhea (tanzerina et al., 2017) elephanthopus scaber heartburn (sarina et al., 2023) emillia sochinfoliia eyes and skin care (tanzerina et al., 2017) euphathorium odoratum magh/gerd (saputri et al., 2022) gynura divaricata anti acne (tanzerina et al., 2017) pluchea indica leucorrhea (tanzerina et al., 2017) vernonia amygdalina anti acne and lose weight (tanzerina et al., 2017) zinnia elegans hepatitis, boils, itching (hastiana, novitasari, et al., 2023) 16. aviaceae apium graveolens lowers blood pressure (rizal et al., 2021). 17. bacellaceae amredera cordifolia strokes (saputri et al., 2022). andrera cardifolia acne (tanzerina et al., 2017) 18. bignonaceae oroxylum indicun postnatal care (tanzerina et al., 2017). 19. blechnaceae blechnum finlaysonianum ethnomedicine (pujihastuti et al., 2020) stenochlaena palustris ethnomedicine (pujihastuti et al., 2020) 20. bombacaceae ceiba petandra boil (sarina et al., 2023) 21. bromeliaceae ananas comosus headache (sarina et al., 2023) 22. cactaceae hylocereus costaricensis lowers blood pressure, cancer (rizal et al., 2021). hylocereus polyrhizus ethnomedicine (dahlianah et al., 2021) 23. caesalpiniaceae tamarindus indica flu (sarina et al., 2023) 24. campanulaceae hippobroma longiflora eyes infection (sarina et al., 2023) isotoma longiflora cataract (rizal et al., 2021) 25. cannabaceae trema orientalis scabies/itching (sarina et al., 2023) 26. caricaceae carica papaya back pain (sarina et al., 2023) 27. clusiaceae garcinia xanthochymus ethnomedicine (pujihastuti et al., 2020) 28. coctaceae coctus specious kidney diseases and ulcers (rizal et al., 2021). 29. convolvulaceae ipomoea aquatica ethnomedicine (dahlianah et al., 2021) ipomoea batatas ethnomedicine (dahlianah et al., 2021) 30. crassulaceae kalanchoe laciniata anti malaria (margarethy et al., 2019) kalanchoe pinnata fever (sarina et al., 2023) 31. cucurbitaceae cucumis sativus ethnomedicine (sarina et al., 2023) iuffa acutangula lowers blood pressure (rizal et al., 2021) momordica charantia fever (sarina et al., 2023) 32. dennstaedtiaceae lindsaea ensifolia ethnomedicine (pujihastuti et al., 2020) 33. dilleniaceae dillenia suffruticosa urolitiasis (sarina et al., 2023) tetracera indica urolitiasis (sarina et al., 2023) tetracera scandens ethnomedicine (sarina et al., 2023) 76 biology, medicine, & natural product chemistry 13 (1), 2024: 73-82 table 1. cont. 34. dioscoreaceae dioscorea pyrifolia ethnomedicine (pujihastuti et al., 2020) 35. euphorbiaceae claoxylon indicum anti malaria (margarethy et al., 2019) euphorbia hirta lung disease (saputri et al., 2022) euphorbia tirucalli warts and tootache (hastiana, novitasari, et al., 2023) homalanthus populifolius ethnomedicine (pujihastuti et al., 2020) jatropha curcas ethnomedicine (pujihastuti et al., 2020) macaranga trichocarpa ethnomedicine (pujihastuti et al., 2020) mallotus japonicus ethnomedicine (pujihastuti et al., 2020) manihot esculenta ethnomedicine (dahlianah et al., 2021) phyllanthus disthicus kidney disease (saputri et al., 2022) phyllanthus niruri hearth and lung disease (saputri et al., 2022) sauropus androgynus hair care and breastfeeding (tanzerina et al., 2017) 36. fabaceae bauhinia semibifida ethnomedicine (pujihastuti et al., 2020) calopogonium mucunoides ethnomedicine (pujihastuti et al., 2020) cassia alata ethnomedicine (pujihastuti et al., 2020) erythrina fusca jaundice (sarina et al., 2023) erytrina viriegata lowers fever (saputri et al., 2022) flemingia strobilifera body aches and increases appetite (sarina et al., 2023) mimosa pudica body aches and increases appetite (sarina et al., 2023) parkia speciosa bloating (sarina et al., 2023) pithecellobium jiringa nervous disease (sarina et al., 2023) senna alata constipation (sarina et al., 2023) spatholobus ferrugineus postnatal care (sarina et al., 2023) uraria crinite ethnomedicine (pujihastuti et al., 2020) vigna cylindrica ethnomedicine (dahlianah et al., 2021) vigna radiata magh (saputri et al., 2022) 37. hypericaceae cratoxylum formosum ethnomedicine (pujihastuti et al., 2020) 38. hypoxidaceae curculigo latifolia ethnomedicine (pujihastuti et al., 2020) 39. lamiaceae clerodendrum calamitosum ethnomedicine (pujihastuti et al., 2020) coleus amboinicus breastfeeding (tanzerina et al., 2017) mentha arvensis skin care (tanzerina et al., 2017) ocimum citrodorum lowers blood pressure (rizal et al., 2021) ocimum americanum deodorant and halitosis (tanzerina et al., 2017) ocimum basilicum deodorant (tanzerina et al., 2017) ocimum citriodorum anti odor (tanzerina et al., 2019) ocimum sanctum ethnomedicine (pujihastuti et al., 2020) ocimum tenuiflorum ethnomedicine (pujihastuti et al., 2020) orthosiphon aristatus back pain (sarina et al., 2023) orthosiphon aristatus lowers blood glucose level (rizal et al., 2021) plecanranthus amboinicus breastfeeding (tanzerina et al., 2017) pogostemon cablin deodorant (tanzerina et al., 2017) 40. lauraceae cinnamomum tamala ethnomedicine (pujihastuti et al., 2020) lindera benzoin ethnomedicine (pujihastuti et al., 2020) persea americana lowers blood pressure (sarina et al., 2023) 41. phyllanthaceae arcangelisia flava ethnomedicine (pujihastuti et al., 2020) baccaurea macrocarpa ethnomedicine (pujihastuti et al., 2020) fibraurea tinctoria ethnomedicine (pujihastuti et al., 2020) phyllanthus niruri muscle stiffnes (sarina et al., 2023) sauropus androgynous ethnomedicine (pujihastuti et al., 2020) tinospora crispa immune booster (sarina et al., 2023) tinospora sinensis fertility (saputri et al., 2022) 42. liliaceae allium sativum flu and burns (sarina et al., 2023) 43. limnocharitaceae limnocharis flava ethnomedicine (hastiana, novitasari, et al., 2023) asiandu & sari – study of medicinal plants in south sumatera 77 table 1. cont. 44. loranthaceae scurrula artopurpurea cancer, tumor, cyst (saputri et al., 2022) scurrula ferruginea lowers blood pressure (sarina et al., 2023) 45. lythaceae lawosonnia innermis nail dan hair care (tanzerina et al., 2017) 46. magnoliaceae magnoolia alba odor and leucorrhoea (tanzerina et al., 2017). 47. malvaceae ceiba pentandra eyes irritations (saputri et al., 2022) hibiscus cannabinus ethnomedicine (pujihastuti et al., 2020) hibiscus sabdariffa cancer (rizal et al., 2021) hisbiscus rosasinensis hair care (tanzerina et al., 2017) 48. marattiaceae ptisana salicina ethnomedicine (pujihastuti et al., 2020) 49. melastomaceae melastoma candidum ethnomedicine (pujihastuti et al., 2020) melastoma malabathricum maag (sarina et al., 2023) 50. meliaceae azadirachta indica anti malaria (margarethy et al., 2019) lansium domesticum bloating (sarina et al., 2023) lansium parasiticum anti malaria (margarethy et al., 2019) swietenia macrophylla anti malaria (margarethy et al., 2019) xylocarpus granatum itchy (sarno et al., 2013) 51. moraceae artocarpus altilis lowers blood pressure (rizal et al., 2021) artocarpus heterophyllus baby care (sarina et al., 2023) ficus curtipes diarrhea (sarina et al., 2023) ficus hirta ethnomedicine (pujihastuti et al., 2020) 52. muntingiaceae muntingia calabura ethnomedicine (pujihastuti et al., 2020) 53. musaceae musa acuminate postnatal care (sarina et al., 2023) musa babisiana hemorrhoids (saputri et al., 2022) musa paradisiaca feverish (sarina et al., 2023) 54. myrtaceae eucalyptus calophylla ethnomedicine (pujihastuti et al., 2020) eugenia polyantha pain (sarina et al., 2023) malaleuca leucadendra ethnomedicine (pujihastuti et al., 2020) psidium guajava diarrhea (sarina et al., 2023) rhodamnia cinerea constipation (sarina et al., 2023) rhodomyrtus tomentosa lowers blood pressure (salni et al., 2022) syzygium aqueum ethnomedicine (dahlianah et al., 2021) syzygium cumini stomach ache (hastiana, novitasari, et al., 2023) syzygium polyanthum diabetic (rizal et al., 2021). 54. nyctaginaceae mirabilis jalapa skin care (tanzerina et al., 2017). 55. oleaceae jasminum officinalle facial care, hair care, weight loss and body odor removal (tanzerina et al., 2017). olea europaea postnatal care (tanzerina et al., 2017). 56. onagraceae ludwigia octovalvis anti malaria (margarethy et al., 2019) 57. oxalidaceae averrhoa bilimbi blood pressure and cough (rizal et al., 2021). averrhoa carambola lowers blood pressure (rizal et al., 2021). 58. pandanaceae benstonea affinis craft material (tanzerina et al., 2022). benstonea atrocarpa craft materials (tanzerina et al., 2022). pandanus amaryllfolius medicine, cosmetics, foodstuffs, indigenous material (tanzerina et al., 2022). pandanus helicopus building material (tanzerina et al., 2022). pandanus lais medicine, cosmetic, building material (tanzerina et al., 2022). pandanus tectorius perawatan rambut (tanzerina et al., 2017) 59. passifloraceae passiflora foetida feverish (sarina et al., 2023) passiflora suberosa ethnomedicine (pujihastuti et al., 2020) 60. phyllanthaceae arcangelisia flava ethnomedicine (pujihastuti et al., 2020) baccaurea macrocarpa ethnomedicine (pujihastuti et al., 2020) fibraurea tinctoria ethnomedicine (pujihastuti et al., 2020) phyllanthus niruri pain (sarina et al., 2023) sauropus androgynous ethnomedicine (pujihastuti et al., 2020) tinospora crispa immune booster (sarina et al., 2023) tinospora sinensis fertility (saputri et al., 2022) 78 biology, medicine, & natural product chemistry 13 (1), 2024: 73-82 table 1. cont. 61. piperaceae peperomia pellucida fever (sarina et al., 2023) piper acutilimbum ethnomedicine (pujihastuti et al., 2020) piper aduncum ethnomedicine (pujihastuti et al., 2020) piper betle eyes irritation / infections (sarina et al., 2023) piper crocatum ethnomedicine (pujihastuti et al., 2020) piper ornatum strokes (saputri et al., 2022) piper porphyrophyllum ethnomedicine (pujihastuti et al., 2020) piper pubela fertility (saputri et al., 2022) 62. poaceae andropogon nardus ethnomedicine (saputri et al., 2022) bambusa vulgaris cough (sarina et al., 2023) cymbopogon atratus gout (rizal et al., 2021). cymbopogon nardus postnatal care (sarina et al., 2023) eleusine indica hair care (tanzerina et al., 2017) gigantochloa verticillata anti malaria (margarethy et al., 2019) imperata cylindrica feverish (sarina et al., 2023) lophatherum gracile ethnomedicine (pujihastuti et al., 2020) oryza sativa ethnomedicine (pujihastuti et al., 2020) saccharum officinarum eyes irritation/infection (sarina et al., 2023) zea mays ethnomedicine (dahlianah et al., 2021) 63. polypodiaceae drynaria quercifolia ethnomedicine (pujihastuti et al., 2020) pyrrosia adnascens ethnomedicine (pujihastuti et al., 2020) 64. portulaceae portulaca gandifolia fever (saputri et al., 2022) portulaca oleraceae ethnomedicine (pujihastuti et al., 2020) 65. pteridaceae acrostichum aureum eyes infections/irritations (sarno et al., 2013) 66. rhamnaceae ziziphus mauritiana ethnomedicine (pujihastuti et al., 2020) 67. rhizophoraceae rhizophora sp. ethnomedicine (dahlianah et al., 2021) 68. rosaceae rosa sp. body care (tanzerina et al., 2017). 69. rubiaceae coffea canephora wounds (rizal et al., 2021) gardenia augusta diabetic (sarina et al., 2023) morinda citrifolia maag (sarina et al., 2023) mussaenda frondosa ethnomedicine (pujihastuti et al., 2020) mussaenda pubescens ethnomedicine (dahlianah et al., 2021) oldenladia lancifolia stomach (sarina et al., 2023) palicourea guianensis ethnomedicine (pujihastuti et al., 2020) psychotria nervosa ethnomedicine (pujihastuti et al., 2020) uncaria gambir mouth care (tanzerina et al., 2017) 70. rutaceae aegle mamelos hair care (tanzerina et al., 2019) citrofortunella macrocarpa ethnomedicine (hastiana, novitasari, et al., 2023) citrus aurantifolia ethnomedicine (pujihastuti et al., 2020) citrus aurantiifolia cough (sarina et al., 2023) citrus hystrix ethnomedicine (pujihastuti et al., 2020) citrus limon facial care (tanzerina et al., 2017) citrus x microcarpa ethnomedicine (pujihastuti et al., 2020) euodia lepta fever (sarina et al., 2023) murraya paniculata facial care (tanzerina et al., 2019) 71. sapindaceae nephelium lappaceum ethnomedicine (pujihastuti et al., 2020) 72. schizaeaceae actinostachys digitata ethnomedicine (pujihastuti et al., 2020) 73. simaroubacae brucea javanica anti malaria (margarethy et al., 2019) 74. solanaceae capsicum frustescens fever (rizal et al., 2021) datura metel ethnomedicine (hastiana, novitasari, et al., 2023) nicotiana tabacum anti malaria (margarethy et al., 2019) physalis angulate ethnomedicine (pujihastuti et al., 2020) physalis peruviana heartburn (sarina et al., 2023) solanum lycopersicum ethnomedicine (hastiana, novitasari, et al., 2023) solanum lycopersicum ulcer (rizal et al., 2021) solanum melongena ethnomedicine (dahlianah et al., 2021) solanum torvum eye irritation / infection (rizal et al., 2021) asiandu & sari – study of medicinal plants in south sumatera 79 table 1. cont. 75. theaceae camellia sinensis facial care (tanzerina et al., 2017). 76. thymelaceae phaleria macrocarpa diabetic (sarina et al., 2023) 77. urticaceae poikilospermum suaveolens ethnomedicine (pujihastuti et al., 2020) 78. verbenaceae clerodendrum japonicum postnatal care (sarina et al., 2023) lantana camara hearthburn (sarina et al., 2023) peronema canescens musslce care (sarina et al., 2023) stachyrtapeta jamaicencis leucorrhoea (tanzerina et al., 2017) 79. zingiberaceae kaempferia galanga cold (sarina et al., 2023) kamferia rotundus skin care (tanzerina et al., 2017) zingiber montanum blood circulation (hastiana, nawawi, et al., 2023) zingiber officinale jaundice (sarina et al., 2023) zingiber zerumbet ethnomedicine (pujihastuti et al., 2020) as a mega biodiversity country, indonesia's nature provides a variety of needs that can be utilized by its people, such as the need for medicinal plants, especially for rural communities, such as in south sumatra. the besemah tribe, located in lahat regency, is an example of a community group that utilizes biodiversity. the people use natural plants as a source of medicines, cosmetics, religious activities, death ceremonies, and so on. pandanaceae is a plant family that is often used as a functional plant for this community. like the pandanus amaryllifolius species which produces a distinctive aroma that is often used as food coloring and food fragrance for local people (tanzerina et al., 2022). the ogan people are known to still preserve the local heritage of their ancestors. the medicinal plants they process can be obtained directly from the forest, planted themselves, or purchased from sellers at the market. these plants are traditionally used as medicines. the medicinal plants used by the ogan tribe in beringin dalam village can be in the form of trees, bushes, herbs, shrubs, or lianas. in this tribe, tree-based medicinal plants are the main source of medicine with a usage percentage of 35.38% consisting of 23 tree species. the dominance of these trees is associated with the area's history which was dominated by primary forest vegetation. on the other hand, there are as many as 37 species of medicinal plants planted themselves by residents. of the many indications of diseases that are treated using medicinal plants, aches, appetite enhancers, heartburn, and bath additives for women after giving birth are the most dominant. moreover, these plants can be used by boiling, kneading, mashing, and burning (sarina et al., 2023) the besemah tribe who inhabit lahat regency, south sumatra, is also surrounded by primary and secondary forest areas, rich in plant resources, such as zingiberaceae, asteraceae, myrtaceae, rosaceae, and lamiaceae. therefore, ethnobotanical studies in this area aim to preserve its germplasm tanzerina et al., 2019). the anak dalam tribe, sungai jernih village, rupit district, north musi rawas regency, also uses natural plants for various purposes. as a tribe that has lived a sedentary life, the tribe still uses plants as a source of medicine. there are 93 types of medicinal plants whether obtained from nature, close to where they live, or cultivated. the medicinal plants consist of 48 families, dominated by the fabaceae, piperaceae, apocynaceae, and euphorbiaceae families (pujihastuti et al., 2020) for the people of pagar ruyung village, kotaagung district, lahat regency, south sumatra, leaves are the most widely used in the making of traditional medicine. not only using the common part of the plants, the sap is also used as traditional medicine. leaves are easy to process, and easy to obtain without directly killing the organism, and leaf regeneration is relatively rapid. leaves also contain various phytochemical compounds such as phenol, chlorophyll, tannin, alkaloids, and potassium. they are also thought to be a storage for various types of photosynthesis products. ethnomedicine is used to treat a wide range of diseases, from mildcommon illnesses to fatal illnesses such as cancer (rizal et al., 2021). javanese transmigrants who live in east buay madang district, east oku regency, south sumatra also have their ethnobotanical activities. in this community, there are 27 plant families used as sources of medicines with zingiberaceae as the dominant family and rhizomes as one of the sources used as traditional medicines. however, it turns out that the leaves are also common (saputri et al., 2022). in the community of manggaraya village, tanjung lago subdistrict, banyuasin regency, south sumatra, there are 21 families used as a source of traditional medicines consisting of 38 plant species. moreover, ethnobotanical plants in these communities are also used as food and horticultural crops (dahlianah et al., 2021). people living on the banyuasin peninsula are familiar with mangrove plants. mangroves are also used by local people as traditional medicines to treat various complaints such as typhus, stomach aches, itching, and eye pain. the used parts include leaves, sap, fruit, and even all parts of the plant. mangrove plants produce secondary metabolite compounds which are produced during environmental stress to protect themselves from pests and pathogens. the resulting secondary metabolite 80 biology, medicine, & natural product chemistry 13 (1), 2024: 73-82 compounds can be used as medicine for the community (sarno et al., 2013). plants are also used to treat malaria, a disease that many rural communities suffer from. this disease is caused by a vector in the form of the anopheles mosquito which inhabits many forests in south sumatra. several tribes that use plants as anti-malaria in south sumatra are teloko, daya, pegagan, meranjat, and lintang (margarethy et al., 2019). the source of knowledge on the use of plants as malaria medicine can be obtained from knowledge obtained from their ancestors, relatives/friends, family, formal education, non-formal education, and even from dreams/inspiration. these plants can be made into potions originating from one type of plant or a combination of several plants (margarethy et al., 2019). in the teloko tribe, the plants used as anti-malaria consist of c. papaya, nicotiana tabacum, alstonia scholaris, tinospora crispa, and lansium parasiticum. the daya tribe uses the plants tinospora crispa, swietenia macrophylla, giganthocloa verticillata, kalanchoe laciniata, ludwigia octovalvis, claoxylon indicum, the pegagan tribe uses the luffa acutangula plant, the climbing tribe uses azadirachta indica, brucea javanica, tinospora crispa, and crescentia cujete. furthermore, the treatment of malaria in the lintang tribe uses citrus aurantiifolia, imperata cylindrica, and several other unidentified plants (margarethy et al., 2019). plants produce many bioactive compounds stored in the form of essential oils that are used to protect themselves against various kinds of predators. these essential oils can be used as a source of medicine or cosmetics for humans (tanzerina et al., 2019). in the ogan tribe of beringin village, leaves are the most widely used part of the plant. this is because the leaves contain many bioactive compounds which include tannins, flavonoids, phenols, and so on. the method of using the drug is adjusted. the community believes that the boiling medicine technique is more effective because the boiling process can release the active compounds contained in the plant, and boiling can also reduce the bitter taste of the plant and kill pathogens found in medicinal plants (sarina et al., 2023). medicinal plants are equipped with special anatomical structures accumulating certain components. the leaves and stems of plants in the lamiaceae family have oil cells which can be found in the cortex tissue. in certain plants such as this family, glandular trichomes can also be found which play an important role in the synthesis of plant bioactive compounds. on the leaves, these glandulars can be found on the upper and lower epidermis. apart from that, in several lamiaceae species, there are also idioblast cells pivotal in the synthesis of bioactive compounds tanzerina et al., 2017). pandanaceae are widely used by the besemah tribe as building materials for crafts, as well as cosmetics and medicines. the content of secondary metabolites in this family, such as polyphenols, saponins, and terpenoids, has certain health benefits. the resulting aroma can be used as a healthy natural food fragrance and coloring. this group of plants is also used by the community as a mixture for bathing the corpses of the dead. furthermore, the leaves of the plant are used as decoration, and can even be arranged into the roof of a building (tanzerina et al., 2022). the zingiberaceae family is one of the plant families most often used in traditional medicine. this group of plants is often used as a cooking spice. in the pharmaceuticals, for example, ginger contains bioactive compounds that can act as antibacterials, antipyretics, fever reducers, and so on (saputri et al., 2022). figure 1. plants families used in ethnobotanical purposes of south sumatera, indonesia. 5% 2% 2%4% 6% 5% 3% 4% 2% 3% 3% 4% 4% 4% 4% 2% 2%1%2% 1% 1% 2%2% 1% 2%2% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 16% 27% ethnobotanical plants used by some tribes in south sumatera, indonesia asteraceae arecaceae acanthaceae euphorbiaceae fabaceae lamiaceae lauraceae myrtaceae pandanaceae phyllanthaceae piperaceae poaceae rubiaceae rutaceae solanaceae meliaceae moraceae amaryllidaceae asparagaceae cucurbitaceae dilleneaceae loranthaceae malvaceae musaceae verbenaceae zingiberaceae amaranthaceae annonaceae apocynaceae bacellaceae blechnaceae cactaceae campanulaceae convolvulaceae crassulaceae oleaceae melastomaceae oxalidaceae passifloraceae polypodiaceae portulaceae others asiandu & sari – study of medicinal plants in south sumatera 81 in processing plants as traditional medicine, the plant can be processed from certain parts or all parts of the plant. the parts of plants used as a source of medicine include leaves, roots, stems, flowers, fruit, and sap. based on how traditional medicine is consumed, people have their methods, sometimes in different places, the methods are different. the most common ways are drinking, applying topically, dripping, compressing, eating, and bathing (sarina et al., 2023). however, the use of plants as traditional medicine is now starting to be abandoned by the younger generation. this is due to the increasingly limited number of medicinal plants in nature, the process of making or concocting medicines which is relatively complicated, and the efficacy of medicines is not necessarily effective. moreover, the presence of experts (shamans) who understand medicinal plants is also starting to decrease (sarina et al., 2023). based on the results of this review (figure 1), it can be concluded that ethnobotanical activities in south sumatra are very prospective, so they can be used as a reference in the bioindustry or pharmaceutical industry (figure 2). however, from these ethnobotanical activities, there is a need for scientific studies regarding the efficacy of the drugs. what is more important is to conserve these plants as germplasms that must be protected as a part of the indonesian biodiversity. figure 2. review of south sumatera ethnobotanical activities. conclusions south sumatra has a quite high biodiversity, especially plants. these plants are a source of traditional medicines by local communities to produce traditional medicines and cosmetics. there are 250 plant species from 79 families used by local people. the dominant plant families used in their ethnobotanical activities are fabaceae (5.6%), lamiaceae (5.2%), asteraceae (4.8%), euphorbiaceae (4.4%), poaceae (4.4%), myrtaceae (3.6%), rubiaceae (3.6 %), rutaceae (3.6%), solanaceae (3.6%), to piperaceae (3.2%). considering the great potential of the plants, it is necessary to carry out further research activities regarding their pharmacological potential along with the need for a good conservation strategy to maintain their sustainability. authors’ contributions: angga puja asiandu initiated this writing idea and widya sari participated in finishing this manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there is no funding which has role in finishing this manuscript. references amisim, a., kusen, a. w. s., & mamosey, w. e. 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(2016). the traditional medicine and modern medicine from natural products. molecules, 21(5). https://doi.org/10.3390/molecules21050559 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 35-43 | doi: 10.14421/biomedich.2022.111.35-43 issn 2540-9328 (online) liver and renal cell damage following excess bee honey consumption in male wistar rat akpevwoghene agbatutu1, jerome ndudi asiwe*,2,3, olusegun gafar adebayo2,3 1department of botany, university of ibadan, nigeria 2department of physiology, pamo university of medical sciences, port-harcourt, nigeria. 3department of physiology, college of medicine university of ibadan, nigeria. corresponding author* asiwejerome@yahoo.com; +2348163727468 manuscript received: 14 january, 2022. revision accepted: 11 february, 2022. published: 01 march, 2022. abstract honey is a widely used natural product with several health benefits. however, there is paucity of information on its excessive usage. the present study investigated the effect of excess honey consumption on hepato-renal functions in male wistar rats. twenty-eight adult male wistar rats were selected into four groups (n=7) and treated with distilled water (control) and 1ml, 2ml and 3ml of honey respectively for 5 weeks. thereafter, the animals were euthanised and blood as well as kidney and liver were collected for further studies. there was a significant increase in creatinine, bilirubin, urea ast, alp, total protein as well as a significant decrease in rbc, wbc, haemoglobin, lymphocyte and pcv. histology of the liver and kidney revealed a significant degeneration and necrosis in a dose dependent manner. this study suggest that excess honey consumption causes liver and renal cellular damage as well as haematological alterations. keywords: honey; excess consumption; liver enzyme; electrolyte; renal function; haematological indices; histopathology. introduction natural honey is a sweet, flavourful liquid with high nutritional value and therapeutic potential (eterafoskouei and najafi, 2013). it is a natural product made by honeybees (apis mellifera; apidae) from nectar collected from flowers (dashora et al., 2011). honey has been consumed by humans since almost five centuries ago for both nutritional and therapeutic purposes (adebolu, 2005; ashrafi et al., 2005). however, it is the only naturally occurring insect-derived substance with nutritional, aesthetic, medicinal, and industrial benefits (bansal et al., 2005). honey has been used as a natural sweetener since ancient times because of its high fructose content and it was reported to be 25% sweeter than tablet sugar (babacan and rand, 2007; pataca et al., 2007). the increasing popularity shown by the usage of honey in beverages is also accounted to its high fructose content (babacan and rand, 2007). as of today, approximately 300 different varieties of honey have been identified and many forms of nectar gathered by honeybees are related to these variations (lay-flurrie, 2008). proximate analysis showed that honey's main components are carbohydrates (95–97% of its dry weight), proteins, vitamins, amino acids, minerals, and organic acids (betts, 2008; helmy and el-soud, 2012). meanwhile, phytochemical screening of pure honey also revealed that it contains flavonoids, polyphenols, reducing compounds, alkaloids, glycosides, cardiac glycosides, and volatile compounds (white, 1980; islam et al., 2012). regardless of its strong phytochemical properties, honey's vitamin content is poor, and it falls far short of the daily requirements but the most abundant is the vitamin c. honey is a good source of antioxidant and it has been reported to exhibit strong anti-oxidative (ahmed and othman, 2013; kasala et al., 2015), and anti-inflammatory (khalil et al., 2012) potential. due to the high polyphenols of honey, various studies reported the pharmacological usage of honey in the treatment and management of pathological conditions like increase blood pressure (ahmed and othman, 2013), diabetes (estevinho et al., 2008), neuropsychiatric symptoms (ghosh and playford, 2003; rahman et al., 2014), bacterial infections (attia et al., 2008; kasala et al., 2015) as well as respiratory and gastrointestinal disorder (abdulrhman et al., 2008; israili, 2014), microbial infection (israili, 2014; saikaly and khachemoune, 2017). the antioxidant properties of honey have been implicated through its reactive oxygen species (ros) scavenging ability and by increasing the intracellular activity of glutathione (gsh), uric acid, beta-carotene, and vitamin c (ahmed et al., 2018). moreover, certain phenolic ingredients of honey were reported to inhibit the activity of nitric oxide synthase, decrease inducible nitric oxide synthase (inos) and cyclooxygenase-2 https://doi.org/10.14421/biomedich.2022.111.35-43 36 biology, medicine, & natural product chemistry 11 (1), 2022: 35-43 (cox-2) activities that cause inflammation of cells and tissues in human body (ahmed et al., 2018). also, honey displayed very strong anti-inflammatory effect reported in several studies. reports stated that honey can inhibit or slows down pro-inflammatory cytokine release, nitric oxide synthase (inos) expression, generation of ros, and reduce the level of prostaglandin: which are the major players culminating processes involved in inflammation (al-waili and boni 2003; candiracci et al. 2012). according to candiracci et al., (2012), honey exacerbated nitric oxide release in acute and severe inflammation. however, this mechanism was related to cox-2 and inos inhibition leading to suppression of mediators of pro-inflammation (pge2, no, tnf-α, and il-6) (hussein et al. 2012). another area where honey is adopted is its effect on wound healing. several preclinical and clinical trials have reported the application of honey in both acute and chronic wounds including injuries resulting from burns (moore et al. 2001). this reported have demonstrated honey’s ability to reduce the level of edema, enhance granulation and epithelization during the proliferative stage as well as decreased wound healing time and scarring and decreases contractures in cases with burn wounds with no adverse effect (allergy or toxicity) whatsoever (yaghoobi et al. 2013). meanwhile, the side effect of honey consumption has not been well studied and there are major concerns with respect to excessive intake of honey causing increase in blood sugar level. there seems a very little or no report on the excessive or prolonged consumption of honey which might lead to toxicity in the body system. hence, the goal of this present study is to investigate the high consumption of honey on haematological parameters, liver function indices and kidney function in male wistar rats. materials and methods drugs and chemicals honey was purchased at gembu local market in saurdana local government area of taraba state, nigeria. creatinin and urea kit was obtained from immunometrics limited (uk) and total proteins kit, albumin kit and bilirubin kit were all obtained from sigma aldrich (st. louis, mo, usa). other reagents and solvents were of analytical graded level. experimental animals twenty-eight adult male wistar rats (weighing 180200g) were obtained from the physiology department central animal house (pdcah), university of ibadan, ibadan, oyo state, nigeria and managed under normal laboratory condition according to the university ethical guideline which adheres strictly to the “principle of laboratory animal care” (nih publication no. 85-23). the dosed selected for this study was according to fasanmade and alabi, (2008) with slight modifications. treatment design the animals were arranged into four groups (n =7). group 1 serves as the non-treated animal (control animals) and received distilled water (10 ml/kg) alone. group 2 was administered with 1ml/100g of body weight while group 3 received 2ml/100g of body weight and group 4 received 3ml/100g of body weight. all treatments were daily administered orally using oral gavage and lasted for 35 days. the animals were carefully observed for change in body weight and blood glucose level evaluated weekly and after overnight fasting on the last day of sacrifice (24 hours after the last day of treatment) using a glucometer (accu-check active, roche diagnostic, mannhein germany). preparation of blood samples for biochemical analysis after the conclusion of the treatment, all the rats were subjected to ketamine anaesthesia and then sacrificed through cervical dislodge. cardiac puncture was used for blood collection for haematological variables. further, plasma was also obtained from the collected blood by centrifugation (3000 rpm; 15 minutes) at room temperature using a bench top centrifuge (bosch, uk) for the assessment of electrolytes concentration, renal and liver function markers. estimation of liver function markers alanine transaminase (alt), aspartate aminotransferase (ast) and alkaline phosphatase (alp) activities for liver injury markers in the plasma was determined according to the protocol previously described by reitman and frankel (1957) using randox test kit. estimation of kidney function markers assessment of serum creatinine concentration plasma (50 ml) was taken and mixed to a monoreagent (1000 ll) obtained from the assay kit. the mixture was then incubated for 60 seconds. thereafter the absorbance was read (k 492 nm) twice within the interval of 1 min. furthermore, the concentration of creatinin was calculated using the kit manufacturer method (immunometrics limited uk). assessment of serum urea concentration four parts of monoreagent taken from reagent i and onepart monoreagent taken from reagent ii were mixed together and incubated at 15–25°c for 30 minutes. following incubation, the mixture was kept in amber bottle prior to use. 10 ml of plasma sample and urea standard were further mixed to 1000 ll monoreagent each and then further incubated at 20-25°c for 60 seconds. the absorbance was read at wavelength of 340 agbatutu et al. – excess bee honey consumption induces toxicity 37 nm twice within the interval of 1 minute. finally, the concentration of urea was determined and calculated as proposed by the kit manufacturer (immunometrics limited uk) using the formula: urea concentration = mg/dl change in sample absorbance change in standard absorbance × standard concentration/cal assessment of serum albumin concentration plasma albumin was determined using randox reagent kits as according to the instructions and method in the manual further corroborated by doumas et al. (1971). estimation of plasma electrolyte concentration the plasma electrolytes: sodium, potassium and chlorides were assayed using their respective commercial kits. all assays were done using microplate reader spectramax plus (a molecular device product). estimation of haematological indices the haematological parameters were determined using an automated haematology analyzer (abx micros 60 from horiba abx, france). the red blood cells (rbc), packed cell volume (pcv), haemoglobin and other haematological indices such as, lymphocytes, neutrophils, monocytes, eosinophils and basophils were determined. the mean corpuscular haemoglobin concentration (mchc), mean corpuscular hemoglobin (mch) and mean corpuscular volume (mcv) were calculated from rbc, haemoglobin (hb) and packed cell volume (pcv) values. the white blood cell (wbc) count was also determined. histological assessment the kidney and liver tissues were harvested and fixed in 10% phosphate buffered formalin for histomorphological examination. the kidney and liver tissues were directly fixed through immersion in a 4% paraformaldehyde of 0.1 mol/l phosphate buffers at ph 7.2 for 48 h. all tissues were then further dehydrated and embedded in a paraffin wax and thereafter sliced transversely using the microtome machine into sections (5 μm). after tissue slicing, it was then stained in haematoxylin and eosin solution. finally, all the sections were examined by using optical microscope for cell condensation and death (asiwe et al., 2021). statistical analysis data are presented as mean ± standard error of mean and analysed with one-way analysis of variance (anova) and compared by newman-keuls test using the graphpad prism 7.0 (graphpad software, san diego, ca, usa). p<0.05 was considered statistically significant. results honey consumption (hc) maintains blood glucose level and reduced body weight gain in rats the effect of honey consumption on blood glucose level and body weight was presented in table 1-2. following administration of honey, there was no significant difference in the glucose level across the treatment group comparative to the control (table 1). however, there was a significant (p < 0.05) progressive decrease in body weight from the week 2-5 in hc (2ml/100g and 3ml/100g) when compared with control (table 2). honey consumption (hc) induced electrolytes imbalance in rats table 3 showed the impact of honey consumption on the plasma level of electrolytes following administration in the rats. the plasma concentration of sodium and chlorine ion significantly (p < 0.05; [689 ± 7.98 and 450 ± 3.13 vs 736±3.36] and [29.1 ± 2.61 and 26.1 ± 11.70 vs 31.3 ± 0.92]) decreased in the animals administered hc (1ml/100g and 3ml/100g), but there was significant (p < 0.05; [747 ± 5.29 vs 736±3.36] and [69.8 ± 1.33 vs 31.3 ± 0.92]) increase in the animals administered hc (2ml/100g) relative to the control. the plasma concentration of potassium ion decreased significantly (p < 0.05; [5.88 ± 0.41, 6.08 ± 0.42 and 3.92 ± 0.30 vs 7.58 ± 0.08]) across all the groups administered hc (1ml/100g, 2ml/100g and 3ml/100g) when compared to the control (table 3). honey consumption (hc) exacerbates liver function markers in rats the data for markers of liver toxicity in rats was presented in fig. 1a-c. data showed significant decrease level of alt in the animals treated with hc (2ml/100g and 3ml/100g) compared to the control (fig. 1a). however, the animals treated with higher doses of hc (2ml/100g and 3ml/100g) significantly decreases the level ast and alp comparative to the control (fig. 1bc). honey consumption (hc) initiates renal toxicity via increased inflammatory markers in rats the results obtained from this result indicating renal toxicity following honey consumption is represented in figure 2a-e. there was a significant (p < 0.05) increase in the urea and creatinine concentration in the animals treated with hc (2ml/100g and 3ml/100g) when compared to the control (fig. 2a-b). we also observed significant (p < 0.05) increased total protein and bilirubin level in the animals that were administered hc (1ml/100g, 2ml/100g and 3ml/100g) (fig. 2c-d), while 38 biology, medicine, & natural product chemistry 11 (1), 2022: 35-43 a significantly (p < 0.05) decreased albumin level was recorded in them (fig. 2e). the effect of honey consumption on haematological indices in rats table 4 showed the values of the haematological indices in the rats after administration of honey. the results showed that there were significant (p < 0.05; [3.06 ± 0.54 and 2.05 ± 0.31 vs 5.41 ± 0.39]) decrease in wbc in the hc (1ml/100g and 2ml/100g) treated rats and a significant (p < 0.05; [7.59 ± 0.55 vs 5.41 ± 0.39]) increase in the rats treated with hc (3ml/100g) when compared to the control. the lymphocyte counts decrease significantly (p < 0.05; [1. 26 ± 0.17 and 0.42 ± 0.05 vs 2.96 ± 0.22]) in the rats treated with hc (1ml/100g and 2ml/100g) when compared to the control. the concentration of haemoglobin decreases significantly (p < 0.05; [12.5 ± 0.17, 11.4 ± 0.16 and 13.1 ± 0.17 vs 14 ± 0.29]) in the hc (1ml/100g, 2ml/100g and 3ml/100g) treated rats when compared to the control. the value of rbc and pcv decreases significantly (p < 0.05; [6.98 ± 0.16 and 6.42 ± 0.41 vs 8.2 ± 0.18]) and (p < 0.05; [39.5 ± 0.17 and 37.1 ± 2.27 vs 44.9 ± 0.44]) in the hc (1ml/100g and 2ml/100g) treated rats when compared to the control. additionally, we observed that the value of pct decreases significantly (p < 0.05; [0.50 ± 0.14 vs 0.64 ± 0.40]) in the hc (3ml/100g) when compared to the control. however, there were no significant (p > 0.05) change mcv, mch, mchc and platelet levels when compared with group (table 4). honey consumption (hc) induces hepato-renal degeneration in rats figure 3 showed the photomicrograph of the liver and kidney tissues. histomicrograph of the liver in control rats showed normal cellular architecture with no lesion (plate 1). however, the rats administered hc (1ml/100g) showed centrilobular, hepatocellular vacuole change while hc (2ml/100g) showed diffused atrophy of hepatic cords and focal hepatocyte vacuolation. also, those given the higher dose of hc (3ml/100g) showed hepatocellular degeneration and fibroblast reaction as well as diffuse atrophy cords and portal inflammation. the photomicrograph of the kidney showed patchy epithelia degeneration as well as necrosis in hc (1ml/100g and 2ml/100g) while hc (3ml/100g) showed tubular epithelia coagulation as well as necrosis as shown in figure 3. figure 1. (a) alt (b) ast (c) alp. values are expressed as mean ± standard error of mean, (n=5). *p< 0.05 is significant when compared with control. table 1. effect of bee honey consumption on fasting blood glucose (g/dl). honey consumption weeks control hc (1ml/100g) hc (2ml/100g) hc (3ml/100g) week 1 120 ± 2.03 118 ± 2.71 114 ± 2.20 115 ± 2.23 week 2 120 ± 2.03 118 ± 2.71 114 ± 2.20 115 ± 2.23 week 3 112 ± 1.11 116 ± 1.69 122 ± 2.07 120 ± 1.24 week 4 133 ± 1.29 121 ± 2.78 107 ± 2.71 110 ± 2.71 week 5 116 ± 3.69 116 ± 3.67 114 ± 1.82 114 ± 2.74 values are expressed as mean ± standard error of mean, (n=5). hc = honey consumption agbatutu et al. – excess bee honey consumption induces toxicity 39 figure 2. (a) urea (b) creatinine (c) total protein (d) billirubin (e) albumin. values are expressed as mean ± standard error of mean, (n=5). *p< 0.05 is significant when compared with control. table 2. effect of bee honey consumption on body weight (grams). honey consumption weeks control hc (1ml/100g) hc (2ml/100g) hc (3ml/100g) week 1 203 ± 7.69 206 ± 5.13 205 ± 3.96 204 ± 8.22 week 2 226 ± 7.46 219 ± 10.0 210 ± 7.01* 183 ± 20.6* week 3 223 ± 8.62 220 ± 12.1 206 ± 5.85* 198 ± 8.07* week 4 231 ± 11.4 227 ± 18.3 206 ± 7.18* 208 ± 12.7* week 5 249 ± 12.2 223 ± 14.2 212 ± 8.05* 209 ± 12.5* values are expressed as mean ± standard error of mean, (n=5). *p< 0.05 is significant when compared with control, hc = honey consumption table 3. effect of bee honey consumption on electrolytes after 35 days (5 weeks). honey consumption variables control hc (1ml/100g) hc (2ml/100g) hc (3ml/100g) na (g/dl) 736±3.36 689 ± 7.98* 747 ± 5.29* 450 ± 3.13* k (g/dl) 7.58 ± 0.08 5.88 ± 0.41* 6.08 ± 0.42* 3.92 ± 0.30* cl (g/dl) 31.3 ± 0.92 29.1 ± 2.61* 69.8 ± 1.33* 26.1 ± 11.70* values are expressed as mean ± standard error of mean, (n=5), *p< 0.05 is significant when compared with control, hc = honey consumption. ml of honey table 4. effect of bee honey consumption on haematological indices after 35 days (5 weeks). honey consumption variables control hc (1ml/100g) hc (2ml/100g) hc (3ml/100g) wbc 5.41 ± 0.39 3.06 ± 0.54* 2.05 ± 0.31* 7.59 ± 0.55* lymphocyte 2.96 ± 0.22 1. 26 ± 0.17* 0.42 ± 0.05* 3.22 ± 0.21 rbc 8.2 ± 0.18 6.98 ± 0.16* 6.42 ± 0.41* 7.66 ± 0.40 hb (g/dl) 14 ± 0.29 12.5 ± 0.17* 11.4 ± 0.16* 13.1 ± 0.17* pcv 44.9 ± 0.44 39.5 ± 0.17* 37.1 ± 2.27* 42 ± 0.45 mcv 55 ± 0.41 56.7 ± 0.47 58 ± 0.41 57.2 ± 1.24 mch 17.1 ± 0.04 17.8 ± 0.17 17.8 ± 0.24 17.1 ± 0.23 mchc 31.3 ± 0.17 31.5 ± 0.24 30.8 ± 0.10 31.2 ± 0.37 platelet 761 ± 56 709 ± 0.71 749 ± 49.10 623 ± 20.50 pct 0.64 ± 0.40 0.56 ± 0.01 0.63 ± 0.07 0.50 ± 0.14* values are expressed as mean ± standard error of mean, (n=5), *p< 0.05 is significant when compared with control, hc = honey consumption. ml of honey 40 biology, medicine, & natural product chemistry 11 (1), 2022: 35-43 figure 3. histology of liver and kidney. the arrows showing significant leisions (h&e x400). group 1= normal control, group 2= 1ml of honey, group 3= 2ml of honey and group 4= 3ml of honey discussion the constituents of honey make it one of the widely used natural products for treating many diseases. however, our present study evaluated the effect of excess honey consumption on serum electrolyte, liver and renal function markers as well as haematological variable in male wistar rats. there was a progressive decrease in the body weight of the animals which suggest the antiobesogenic potential of honey as reported by yaghoobi et al., (2008) and bahrami et al., (2009). the weight loss was highest in the group fed with 3 ml of honey which corroborated the studies of chepulis, (2007) that protective effects of honey against obesity and weight gain depend on quantity or duration of exposure. we also observed a decrease in blood glucose level in week 4 and week 5 after an initial increase in week 3. this observation was consistent with the report that administration of honey in stz-induced diabetic rats and non-diabetic rats for 3 days significantly reduces blood glucose levels (özta¸san et al., 2005; jansen et al., 2012). however, the antidiabetic effect of honey can be attributed to its ability to modulate adiponectin secreted by adipose tissue which regulate glucose and lipid metabolism as reported by (li et al., 2009). the adiponectin modulating potential of honey though not within the scope of our present study, could suggest the mechanism of weight loss in a dose depended manner. electrolytes are essential for survival because of the electrical charges they provide. they interact with each other and the cells in the tissues, nerves and muscles. imbalance in this electrolyte can alter the normal physiology of the body. na and k ions are essential contractile tools of excitable tissues such as the heart, muscles and nerves, a decrease in this ions concentration as observed in this study suggest a modulating role of honey in electrolyte balance. the decrease in the levels of haematological components in this study suggests that honey supplementation directly destroy the blood components or disturb the processes involved in their formation. it can also be attributed to loss of blood due to haemorrhage as hematurea was observed during the study. however, this finding was not consistent with the studies of al-waili et al., (2006) and abdelaziz et al., (2012) who reported a protective role of honey against haemorrhagic and anemic conditions. other studies such as bazzoni et al., (2005), al-qayim et al., (2014), ghorbel et al., (2015) also showed conflicting results. liver is an essential organ for food metabolism and detoxification of substances that threatens the normal functions of the body. the biomarkers assayed in this study indicated the state of health of the liver as it was previously reported to indicate toxicity when there is significant increase in their concentration (murugavel and pari, 2007; asiwe et al., 2022). previous studies by al-waili et al. (2006) and halawa et al., (2009) showed that honey attenuates elevated liver enzymes during acute blood loss and food restriction. however, we observed that honey supplemented diet reduces alt and increases ast and alp in a dose dependent manner. most studies reported the preventive effect against liver agbatutu et al. – excess bee honey consumption induces toxicity 41 injury due to its free radical scavenging potentials. however, the present study was not consistent with previous studies and the inconsistency can be attributed to the dose and duration of the study. honey has been reported by rashed and soltan, (2004) to contain several trace elements as well as heavy metals such as cadmium (cd) and lead (pb) as well as aluminium (al) which previously has been confirmed to be toxic to liver and kidney functions. this may contribute to the observed dose and time dependent toxicity of honey. on the other hand, kidney is one of the important organs in the body saddled with the responsibility of excreting waste metabolic product from the body and a dysfunction of the kidney has been reported to be characterised by increased serum concentrations of creatinine and urea (asiwe et al., 2022). halawa et al., (2009) reported ameliorating effect of honey in environmental toxicant induced kidney dysfunction. however, our present findings suggest that honey supplementation could cause renal damage as both creatinine and urea concentrations were significantly increased when compared to the control group. the photomicrograph of the liver showed centrilobular, focal hepatocyte vacuolation, hepatocellular degeneration and fibroblast reaction as well as diffuse atrophy cords and portal inflammation. several trace elements as well as heavy metals such as cadmium (cd) and lead (pb) which was previously confirmed by asiwe et al., (2022) to be toxic to liver and kidney functions by inducing cellular stress, injury or damage in body tissues was reported by rashed and soltan, (2004) to be part of the constituents of honey as contaminants. this could suggest the adverse effect of honey in this study. this observation was consistent with the study of wilson et al., (2011) who reported adverse effect of excess honey intake on histology of the liver. the renal tissue was not exempted from this effect as the photomicrograph of the kidney showed patchy epithelia degeneration, necrosis as well as tubular epithelia coagulation in a dose dependent manner. this could contribute to the increased creatinine and urea concentrations observed in this study. this observation was in contrast with the study of onyije et al., (2011) who reported normal renal architecture with increased urea and creatinine levels. conclusion in conclusion, this study suggest that excess honey consumption causes adverse effects on liver and renal tissues as well as haematological alterations as shown both in biochemical assays and histological examination. it is therefore recommended that proper awareness should be raised to educate the public as well as agencies regulating food and drug intake of these possible adverse effect of excess consumption of honey or honey products. ethics approval and consent to participate: ethical approval was given by the college of medicine ethics committee. animal handling was done in accordance to established guidelines by the national institute of health for care and use of laboratory animals as adopted by the college of medicine, university of ibadan, nigeria. consent for publication: not applicable availability of data and materials: all data produced and analyzed during this study are included in this article. competing interest: the authors declare that they have no competing interests. funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. authors’ contributions: aa and ajn designed the study. ajn, aa and aog performed the experiments, analyzed the data, and wrote the manuscript. all authors read and approved the final manuscript. acknowledgements: not applicable references abdelaziz i, elhabiby mi and ashour aa (2012). toxicity of cadmium and protective effect of bee honey, vitamins c and b complex. human and experimental toxicology 1–9. abdulrhman m, el-hefnawy m, ali r, el-goud aa (2008). honey and type 1 diabetes mellitus. in: liu cp, editor. type diabetes – complications, pathogenesis, and alternative treatments. croatia: in tech; 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effects on blood glucose, cholesterol, triacylglycerole, crp, and body weight compared with sucrose. sci. world j. 8, 463–469. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 2, 2019 | pages: 47-52 | doi: 10.14421/biomedich.2019.82.47-52 issn 2540-9328 (online) liver profile of atazanavir/ritonavir in pregnant albino rats elias adikwu1,*, james kemelayefa1, winifred ocheiga2 1department of pharmacology and toxicology, faculty of pharmacy, niger delta university, bayelsa state, nigeria 2department of pharmacology and toxicology, faculty of pharmacy, madonna university, rivers state, nigeria corresponding author* adikwuelias@gmail.com abstract medication use during pregnancy is challenging due to the occurrence of maternal or fetal toxicities. atazanavir/ritonavir (atv/r) has hepatotoxic potential hence; use in pregnant patients living with human immunodeficiency virus may cause maternal hepatotoxicity. this study assessed the liver profile of atv/r in pregnant albino rats. thirty pregnant albino rats randomized into groups were orally treated daily with atv/r (4.28/1.43 mg/kg-34.3/11.4 mg/kg) for 16 days. after treatment, the rats were weighed and sacrificed. blood samples were collected and examined for serum biochemical parameters. liver samples were weighed and assessed for biochemical and histological changes. body and liver weights were normal (p>0.05) in atv/r-treated pregnant rats when compared to control. serum total cholesterol, triglyceride, low density lipoprotein cholesterol and blood glucose levels were significantly (p<0.01) elevated whereas high density lipoprotein cholesterol level was significantly (p<0.01) decreased in rats treated with atv/r (34.3/11.4 mg/kg) when compared to control. liver and serum aminotransferases, alkaline phosphatase, gamma-glutamyl transferase, lactate dehydrogenase, total bilirubin, and conjugated bilirubin levels were significantly increased in a dose-dependent fashion in rats treated with atv/r; 8.57/2.86 mg/kg (p<0.05), 17.1/5.72 mg/kg (p<0.01) and 34.3/11.4 mg/kg (p<0.001) when compared to control. liver superoxide dismutase, catalase, glutathione and glutathione peroxidase levels were significantly decreased whereas malondialdehyde levels were significantly increased in a dosedependent fashion in rats treated with atv/r; 8.57/2.86 mg/kg (p<0.05), 17.1/5.72 mg/kg (p<0.01) and 34.3/11.4 mg/kg (p<0.001) when compared to control. necrotic hepatocytes were observed at higher doses of atv/r. atv/r may not be hepatotoxic in pregnant women living with hiv at the clinical dose. keywords: atazanavir/ritonavir; pregnancy; liver; toxicity; rat introduction human immunodeficiency (hiv) infection is a leading cause of maternal and neonatal morbidity and mortality (abdol-karim et al., 2010). pregnancy, whether actual or anticipated, has been a critical driver for the diagnosis, treatment and care of women living with hiv to prevent transmission from mother to child. this necessitates the use of highly active antiretroviral therapy (haart) in pregnant women living with hiv and neonates exposed to hiv. this strategy has drastically reduced mother to child transmission of hiv (coovadia et al., 2007). most haart have demonstrated favorable safety profiles for mothers and infants during trial follow-up (coovadia et al., 2007). however, toxicities have been reported in some quarters with growing concern on hepatotoxicity in pregnant women (bera et al., 2012). the use of medication during pregnancy has been a serious clinical challenge in terms of maternal and fetal safety due to physiological changes and complications that do arise during pregnancy. hepatitis, intrahepatic cholestasis, and acute fatty liver, are some complications that may occur during pregnancy (hammoud et al., 2014). also, pregnancy could increase the incidence of treatment related adverse effect including hepatotoxicity (joy et al., 2015; clark, 2015) and has a significant impact on the efficacy and safety of a drug (abduljalil et al., 2012). atazanavir/ritonavir (atv/r) is use as a component of haart in the management of hiv (mandelbrot et al., 2011). its use in hiv management has decreased mortality associated with hiv. it has a high genetic barrier to resistance, favorable adherence profile and low effects on lipids and glucose metabolism (achenbach et al., 2011). however, it has been associated with hyperglycemia, alterations in cardiac conduction and renal toxicity (clayden, 2009; conradie et al., 2011; hamada et al., 2012). furthermore, elevations in serum liver enzymes and indirect hyperbilirubinemia have been reported hence, use in pregnant women living with hiv raises toxicological concern such as hepatotoxicity (eholié et al., 2013). this study assessed the liver profile of atv/r in a pregnant albino rat model. material and methods animal and drugs pregnant albino rats (200g-250g) used for this study were obtained from the animal house of the department of pharmacology and toxicology, madonna university, nigeria. the rats were housed under room temperature https://doi.org/10.14421/biomedich.2019.82.47-52 48 biology, medicine, & natural product chemistry 8 (2), 2019: 47-52 with a 12:12 light: dark cycle. the rats were randomized into 6 rats per cage according to their weights and had free access to diet and water. atv/r used for this study was manufactured by mylan laboratories limited india. the doses of atv/r (4.28/1.43, 8.57/2.86, 17.1/5.72, and 34.3/11.4 mg/kg) used for this study represent clinical dose, 2, 4, and 8, times the clinical dose respectively (von hentig et al., 2007). experimental protocol the reproductive status and estrous period of the rats were determined by obtaining their virginal smears. after two complete regular cycles, timed mating of female rats was done on the night of the pro-estrous (n) phase of the cycle. in the morning following mating, vaginal smears were taken again. the presence of spermatozoa and squamus cells in the smear confirmed mating and fertilization of ovule. the sperm – positive morning was thus designated day 0 of pregnancy. thirty pregnant albino rats used were weighed and divided into five groups labeled a–e of six rats each. group a (control) orally received normal saline (0.2ml) whereas groups b-e orally received atv/r (4.28/1.43 mg/kg, 8.57/2.86 mg/kg, 17.1/5.72 mg/kg and 34.3/11.4 mg/kg) daily for 16 days respectively. collection of samples and biochemical analyses the rats were sacrificed on day 17 after exposure to inhalational diethyl ether. blood samples were collected and centrifuged at 1200g for 15 minutes and sera extracted. sera were analyzed for biochemical parameters. liver samples were collected, weighed and washed in a cold 1.15 % kcl solution and homogenized in 0.1 m tris-hcl buffer, ph 7.4. the homogenates were centrifuged at 1200 g speed for 15 minutes and the supernatants were collected and evaluated for biochemical parameters. alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), gamma-glutamyl transferase (ggt), lactate dehydrogenase (ldh), total bilirubin (tb), conjugated bilirubin (cb), triglyceride (tg), total cholesterol (tc), and high density lipoprotein cholesterol (hdl-c) were evaluated using commercial test kits (randox diagnostics, crumlin, uk). blood glucose (g) was estimated with the aid of a glucometer whereas low density lipoprotein cholesterol (ldl-c) was estimated as reported by friedewald et al. (1972). liver superoxide dismutase (sod) was determined as described by sun and zigma, (1978). catalase (cat) was assayed according to aebi, (1984). glutathione (gsh) was estimated according to sedlak and lindsay, (1968). malondialdehyde (mda) was determined as reported by buege and aust, (1978). the method of rotruck et al. (1973) was used for the evaluation of glutathione peroxidase (gpx) whereas total protein was evaluated according to gonall et al. (1949). histological examination of the liver liver samples were collected, cleaned and weighed. liver samples were fixed in10% buffered neutral formalin for 24h. liver samples were dehydrated in increasing ethanol concentrations and mounted in paraffin block. paraffin sections (3-5 μm) were prepared, deperaffinized and stained with hematoxylin and eosin dye (h&e). stained sections were examined using a light microscope for histological changes. statistical analysis results are expressed as mean ± sem and were evaluated using one way analysis of variance (anova) followed by dunnett’s post hoc tests. results were considered to be significant at p<0.05; 0.01; 0.001. results the body and liver weights of pregnant rats treated with atv/r were not significantly (p>0.05) different when compared to control (table 1). significant increases in a dose-dependent fashion occurred in the liver and serum ast, alt, alp, ggt, ldh, tb and cb levels in rats treated with atv/r (tables 2 and 3). the increases in the levels of the aforementioned parameters were not significant (p>0.05) at the clinical dose (4.28/1.43 mg/kg), but were significant at 8.57/2.86 mg/kg (p<0.05), 17.1/5.72 mg/kg (p<0.01) and 34.3/11.4mg/kg (p<0.001) when compared to control (tables 2 and 3). serum tg, tc, ldl-c and blood g levels were significantly (p<0.01) increased whereas serum hdl-c level was significantly (p<0.01) decreased in pregnant rats treated with atv/r (34.3/11.4 mg/kg) when compared to control (table 4). treatment with atv/r produced decreases in liver sod, cat, gsh and gpx levels with increases in mda levels in a dose-dependent fashion (table 5). the effects on liver sod, cat, gsh, gpx and mda levels were not significant (p>0.05) at the clinical dose (4.28/1.43 mg/kg), but were significant at 8.57/2.86 mg/kg (p<0.05), 17.1/5.72 mg/kg (p<0.01) and 34.3/11.4 mg/kg (p<0.001) when compared to control (table 5). the liver of control rat showed normal hepatocytes (figure a). the liver of rats treated with atv/r (4.28/1.43 mg/kg) showed inflammatory cell infiltration (figure b). in contrast, the liver of rats treated with atv/r; 8.57/2.86 mg/kg, 17.1/5.74 mg/kg and 34.3/11.4 mg/kg showed hepatocyte necroses respectively (figures c, d and e). adikwu et al. – liver profile of atazanavir/ritonavir in pregnant albino rats 49 table 1. effects of atazanavir/ritonavir on body and liver weights of pregnant albino rats. group body weight (g) absolute liver weight (g) relative liver weight (%) a b c d e 250.7±12.4 257.6 ±13.2 255.1±10.9 260.6±11.2 250.4± 10.7 7.88±1.14 7.58±1.65 7.46±0.57 7.91±0.32 7.80 ±1.42 3.15±0.27 2.95±0.14 2.93±0.09 3.04±0.43 3.12±0.83 tv/r =atazanavir/ritonavir, data are expressed as mean± sem. n=6 table 2. effect of atazanavir/ritonavir on serum liver function parameters of pregnant albino rats. group ast(u/l) alt(u/l) alp(u/l) ggt(u/l) ldh(u/l) cb(g/dl) tb(g/dl) a 45.5±3.99 34.2±2.15 47.3±3.56 48.5±3.12 0.73±0.04 4.50±0.18 8.54±0.71 b 50.3±3.46 38.8±2.25 51.9±4.06 52.1±4.00 0.80±0.02 4.87±0.25 9.01±0.36 c 66.7±5.00a 45.8±4.70a 70.4±5.36a 78.2±5.31a 1.31±0.19a 6.44±1.22a 12.7±1.11a d 96.3±7.87b 63.3±2.62b 99.4±8.66b 121.1±10.6b 1.90±0.12b 8.95±1.06b 15.5±1.40b e 140.6±10.3c 128.8±7.00c 155.3±10.3c 180.4±12.5c 2.60±0.52c 13.3±1.01c 23.5±2.22c atv/r: atazanavir/ritonavir, alt: alanine aminotransferase, alp: alkaline phosphatase, ast: aspartate aminotransferase, ggt: gamma-glutamyl transferase, ldh: lactate dehydrogenase, cb: conjugated bilirubin, tb: total bilirubin. data are expressed as mean± sem. n=6. a differ significantly (p<0.05) when compared to control, b differ significantly (p<0.01) when compared to control, c differ significantly (p<0.001) when compared to control. table 3. effect of atazanavir/ritonavir on some biochemical parameters in the liver tissue of pregnant albino rats. group alt(u/l) ast(u/l) alp(u/l) ggt(u/l) ldh(u/l) a 185.0±15.5 173.8±14.1 187.2±15.34 192.5±12.2 3.25±0.18 b 200.5±19.1 211.2±16.1 201.4±11.4 224.7±16.2 3.76±0.12 c 321.3±20.2a 338.2±22.2a 351.0±18.4a 330.1±20.6a 4.84±0.85a d 463.8±22.6b 548.1±18.7b 474.2±19.0b 580.1±17.1b 6.95±0.32b e 695.2±27.4c 789.2±20.6c 690.0±21.9c 768.8±22.8c 9.97±0.71c atv/r: atazanavir/ritonavir, alt: alanine aminotransferase, alp: alkaline phosphatase, ast: aspartate aminotransferase, ggt: gamma-glutamyl transferase, ldh: lactate dehydrogenase. data are expressed as mean± sem. n=6. a differ significantly (p<0.05) when compared to control, b differ significantly (p<0.01) when compared to control, c differ significantly (p<0.001) when compared to control. table 4. effect of atazanavir/ritonavir on lipid profile of pregnant albino rats. group g (mg/dl) tg(mg/dl) tc(mg/dl) hdl-c (mg/dl) ldl-c (mg/dl) a 95.0±6.41 70.5±6.78 110.1±12.8 30.7±3.44 65.7±6.00 b 92.0±9.00 74.9±6.70 112.0±13.6 32.6±2.61 64.5±6.42 c 89.3±6.42 73.2±7.53 109.8±12.0 32.5±3.72 63.8±5.11 d 100.9±9.19 81.1±7.56 110.3±11.1 29.4±2.63 65.7±7.62 e 152.0±8.00a 132.7±9.21a 161.0±14.1a 21.3±2.10a 113.4±8.32a data are express as mean± sem. n=6 g: glucose, tg: triglyceride tc: total cholesterol hdl-c: high density lipoprotein cholesterol, ldl-c: low density lipoprotein cholesterol. a significant (p<0.01) when compared to control table 5. effect of atazanavir/ritonavir on liver oxidative stress indices of pregnant albino rats. group mda (nmol/mg protein) sod (u/mg protein) cat (u/mg rotein) gsh (µmol/mg protein) gpx (u/mg protein) a 0.32±0.05 16.2±2.09 26.4±3.77 8.97±0.15 9.44±0.12 b 0.36±0.01 14.6±1.04 23.6±2.87 8.25±0.70 9.10±0.12 c 0.50±0.08a 10.4±1.06a 15.5±3.05a 5.88±0.54a 7.42±0.34a d 0.68±0.03b 7.06±1.15b 10 .6±1.37b 3.10±0.79b 5.02±0.44b e 0.98±0.01c 3.16±0.27c 20.5±3.07c 1.06±0.58c 2.11±0.53c atv/r: atazanavir/ritonavir, mda: malondialdehyde, cat: catalase, gsh: glutathione, sod: superoxide dismutase, gpx: glutathione peroxidase. data are expressed as mean± sem. n=6. a differ significantly (p<0.05) when compared to control, b differ significantly (p<0.01) when compared to control, c differ significantly (p<0.001) when compared to control 50 biology, medicine, & natural product chemistry 8 (2), 2019: 47-52 figure 1. fig a: liver of control pregnant albino rat showing normal hepatocytes. fig b: liver of pregnant albino rat treated with atv/r (4.28/1.43 mg/kg) showing inflammatory cell infiltration. fig c: liver of pregnant albino rat treated with atv/r (8.57/2.86 mg/kg) showing hepatocyte necrosis. fig d: liver of pregnant albino rat treated with atv/r (17.1/5.74 mg/kg) showing hepatocyte necrosis. fig e: liver of pregnant albino rat treated with atv/r (34.2/11.4 mg/kg) showing hepatocyte necrosis. (h and e x100). fig c fig e fig d fig a fig b adikwu et al. – liver profile of atazanavir/ritonavir in pregnant albino rats 51 discussion drug-induced liver injury is a potential complication of nearly every prescribed drug and many fatal and nearfatal drug reactions occurred each year (lee, 1995). pregnancy-related liver disease is the most frequent cause of liver dysfunction in pregnancy and provides a real threat to fetal and maternal survival (joshi, 2010) and could be aggravated by some medications (eyal et al., 2010). this makes the provision of antiretroviral therapy during pregnancy a serious clinical challenge due to the possible occurrence of hepatotoxicity. the present study evaluated the liver profile of atv/r in pregnant albino rats. the evaluation of organ weight in toxicology studies is an integral component in the assessments of pharmaceuticals, chemicals, and medical devices (wooley, 2003). this study did not observe significant changes in the body and liver weights of atv/r-treated pregnant rats. serum tg, tc, ldl-c, hdl-c and blood g levels are yardsticks for hyperlipidemia and hyperglycemia caused by drugs (ehigiator and adikwu, 2019). this study, observed elevated levels of serum tg, tc, ldlc and blood g with decreased hdlc level at the highest atv/r dose. these observations are possible signs of hyperlidemia and hyperglycemia (ehigiator and adikwu, 2019). the serum levels of alt, ast, alp, ldh and ggt are effective clinical tools for the assessment of liver status (navarro and senior, 2006). ast is found in mitochondria and cytosol of hepatocytes, alt is localized to the cytosol whereas alp is found on the sinusoidal surface of hepatocytes (rosen and keefe, 2000). ldh is a cytoplasmic marker enzyme whereas ggt is a membrane bound enzyme. higher serum activities of the aforementioned parameters are often associated with hepatic damage (kim et al., 2001). this study observed elevations in the serum and liver levels of alt, ast, alp, ldh and ggt in a dose-dependent fashion in atv/r-treated pregnant rats. this observation may be due to the damage of liver hepatocyte membrane leading to the release of the aforementioned parameters into the blood. the measurement of bilirubin is a very sensitive test to substantiate the functional integrity of the liver and severity of necrosis (singh et al., 1989). this study observed elevations in serum cb and tb levels in a dose-dependent fashion in atv/r-treated pregnant rats. this may be due to decreased disposal of bilirubin by the liver or the blockade of the excretory duct of the liver. reactive oxygen species (ros) could be toxic or beneficial for cellular functions. the toxic effects of ros can occur as a result of excess activity due to overload or accumulation leading to oxidative stress (os). os plays a major part in the development of chronic and degenerative diseases. antioxidants such as sod, cat, gsh and gpx are inhibitors of os even at relatively low concentrations, but their functions can be incapacitated by insurmountable activities of ros (sultan, 2014). in this study, hepatic sod, cat, gsh and gpx levels were depleted in a dose-dependent fashion in atv/rtreated pregnant rats. malondialdehyde (mda) is a highly reactive three carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid oxidation and during arachidonic acid metabolism for the synthesis of prostaglandins. the monitoring of mda level in biological systems has been an important indicator of lipid peroxidation (lpo) in-vitro and in-vivo (janero, 1990). this study observed increases in hepatic mda levels in a dose-dependent fashion in atv/r-treated pregnant rats which indicate hepatic lpo. lpo is the oxidative destruction of lipids containing carbon-carbon double bond resulting in lipid peroxyl radicals and hydro peroxides production. lipid peroxyl radicals and hydroperoxides can stimulate alterations in cell signal transduction and functions leading to cell necrosis and apoptosis, which may facilitate the development of various pathological states (yin et al., 2011). liver histological examination contributes to diagnostic accuracy in drug-induced liver damage and remains a valuable tool for the evaluation of druginduced liver damage. as a clinical test, it provides more information about the state of the liver than any other single assay (kleiner, 2014). histopathological examination of the liver of atv/r-treated pregnant rats showed necrotic hepatocytes at higher doses. the observed necrotic hepatocytes correlate with changes in evaluated biochemical parameters. hepatic necroses observed in atv/r-treated rats could be attributed to os, because os as a consequence of ros production can cause progressive modification or degradation of biomolecules such as dna, proteins, lipids and carbohydrates. this can lead to loss of cell function, cell necrosis and cell death (halliwell and gutteridge, 1999). conclusion the use of atv/r in pregnant women living with hiv may 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(2011). free radical lipid peroxidation: mechanisms and analysis chem rev, 111; 10; 5944–5972 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 5, number 2, 2016 | pages: 61-64 | doi: 10.14421/biomedich.2016.52.61-64 issn 2540-9328 (online) a stability mathematical model of nasopharyngeal carcinoma on cellular level sugiyanto1, fajar adi kusumo2, lina aryati3, mardiah suci hardianti4 1doctoral student of mathematics department; 2,3mathematics department; 4faculty of medicine, universitas gadjah mada, bulaksumur, caturtunggal, kec. depok, kabupaten sleman, daerah istimewa yogyakarta 55281 indonesia 1mathematics department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: sugimath@yahoo.co.id1 abstract this paper discussed the stability of “tumorigenesis models” to link between ebv and carcinoma of the nasopharyngeal from normal cell to invasive carcinoma. the review on this case accomplished the previous theorem of equilibrium point on “tumorigenesis models”. keywords: nasopharyngeal carcinoma; equilibrium point; stability; mathematical model. pacs: 87.19.xj. introduction nasopharyngeal carcinoma (npc) is a malignancy which is derived from epithelial or mucosa and kripta that coat the surface of nasopharynx. according to munir (2006) in indonesia nasopharyngeal carcinoma is the most excessively founded in the ear, nose, throat domain and the most patient are at age 40 or older. soetjipto (1989) said that prevalence of nasopharyngeal carcinoma in indonesia is 4,7/100.000 inhabitant per year. whereas nasopharyngeal carcinoma cases in sardjito hospital in 2011 are comprised of 31 men and 11 women (ratnawati, 2012). in the previous paper we published a mathematical modeling of “tumorigenesis models” for ebvassosiated nasopharyngeal carcinoma and analyzed the equilibrium point of the cell development from normal cell to invansive carcinoma (lo et al., 2012). this paper will described the stability mathematical model from normal cell to invansive carcinoma. mathematical modeling as discussed in previous paper (sugiyanto et al., 2016), with refer to process “tumorigenesis models” for ebvassosiated nasopharyngeal carcinoma (lo et al., 2012), then we can construct the diagram as follows: figure 1. diagram of nasopharyngeal carcinoma infection process. from the diagram above we obtain system of differentional equations that describes “tumorigenesis models” for ebv-assosiated nasopharyngeal carcinoma as follows (lo et al., 2012). http://dx.doi.org/10.14421/biomedich.2016.52.61-64 62 biology, medicine, & natural product chemistry 5 (2), 2016: 61-64 1 1 dn a bn d n dt    (1) 2 1 2 dl a l bn cl i l d l dt      (2) 3 2 3 l l l l dd a d cl i d d d dt     (3) 4 2 4l di a i i d ei l i d i dt      (4) 5 5 h h h h dd a d ei md d d dt     (5) 6 6h dc a c md d c dt    (6) where ( )n t : the density of nasopharynx normal epithelium cell ( )l t : the density of lesions epithelium cell ( )ld t : the density of low grade dysplastic lesions cell ( )i t : the density of ebv latent infection cell ( )hd t : the density of high grade dysplastic lesions cell ( )c t : the density of invansive carcinoma. the equilibrium point of system differential equations (1) to (6) is, 1 1 * a n b d   * 1 2 2 * ( ) b n l c i d a     3 2 3 * * ( ) l c l d d i a    2 4 4 * * ( ) i d i e l d a     5 5 * * ( ) h e i d m d a    6 6 * * ( ) hmd c d a   where 1 2 2 0c i d a    3 2 3 0d i a   4 4 0e l d a    5 5 0m d a   6 6 0d a  stability equilibrium point the jacobian matrix of system differential equations (1) is, ( , , , , , )l hj n l d i d c = ( )j e where e is the equilibrium condition. the characteristic equation is, 1 2 2( )( )ib d a c i d        3 2 3 4 4( )( )a i d a e l d        5 5 6 6( )( ) 0a m d a d       after some calculations we get, 1 1( )b d    . 2 2 2ia c i d     . 3 3 2 3a i d    . 4 4 4a e l d     . 6 5 5a m d    . 7 6 6a d   . lemma 1. the equilibrium point ( *, *, *, *, *, *)l he n l d i d c is asymptotic stable if, 2 2ia c i d   , 3 2 3a i d  , 4 4a e l d   , 5 5a m d  , and 6 6a d . in a medical point of view, in order to avoid the cancer cells then the abnormal cell should be dead quickly until the amount of proliferation cells smaller then apoptosis, so a stability will be formed, which mean that the cancer do not develop. killing abnormal cells need a highly immunogenic (janeway et al., 2001). in the stage of nasopharynx carcinoma, killing cancer cells will be more difficult, because cancer cels have characteristic of resisting cell death (hanahan & weinberg, 2011). simulation the selected parameters are given in tabular form below. case i : not infected with ebv. case ii : ebv infected but do not develop nasopharyngeal carcinoma. case iii : infected ebv and nasopharyngeal carcinoma develops. for case i: in the first case of this sub-population: cells infected, high dysplastic lesion cells, and invansive carcinoma cells is zero.   0i t    0hd t    0c t  system differential equations (1) to (6) became system differential equations (1) to (3). sugiyanto et al. – a stability mathematical model of nasopharyngeal carcinoma on cellular level 63 table 1. the values parameter for first case, not infected by ebv. no. parameter value unit 1. 1a 100 1/day 2. 2a 1 1/day 3. 3a 1 1/day 4. 1d 0.08 1/day 5. 2d 20 1/day 6. 3d 2 1/day 7. b 1 1/day 8. c 0.5 1/day 9. 1i 0.1 1/day 10. 2i 0.5 1/day figure 2. the process of cell development in the case which is not infected with ebv. in figure 2, ebv is not detected in the body. in the first case, the normal cell decreased, while there was an increase lesion cell. then the cell becomes low dysplastic lesion cell because p16 is inactive. because there is no detection of ebv then the high dysplastic cells did not happen and does not became to invasive carcinoma. this is consistent with description from figure 2. for this case ii: in the second case of this sub-population: high dysplastic lesion cells and invansive carcinoma cells is zero.   0hd t    0c t  system differential equations (1) to (6) became system differential equations (1) to (4). table 2. the values parameter for case infected by ebv, but did not develop nasopharyngeal carcinoma. no. parameter value unit 1. 1a 100 1/day 2. 2a 1 1/day 3. 3a 1 1/day 4. 4a 1 1/day 5. 1d 0.2 1/day 6. 2d 8 1/day 7. 3d 2 1/day 8. 4d 2 1/day 9. b 1 1/day 10. c 4 1/day 11. 1i 0.1 1/day 12. 2i 2 1/day 13. e 0.1 1/day 14. l 1 1/day figure 3. the process of case development of ebv-infected cells, but did not develop nasopharyngel carcinoma. in figure 3, ebv is detected in the body. in the second case, the normal cells decrease very rapidly in the early years and higher lesions cells which is very fast. when p16 is inactive, the lesion cells becomes low dysplastic cells. presence of ebv lytic cause abnormal cells, low dysplastic cells, can easily be infected by ebv lytic and makes infected cells. the ebv lytic there is an increase, although not significant. for this case iii: system differential equations same with system differential equations (1) to (6). 64 biology, medicine, & natural product chemistry 5 (2), 2016: 61-64 table 3. the values parameter for case infected by ebv and developing nasopharyngeal carcinoma. no. parameter value unit 1. 1a 100 1/day 2. 2a 2 1/day 3. 3a 1 1/day 4. 4a 1 1/day 5. 5a 2 1/day 6. 6a 2 1/day 7. 7a 2 1/day 8. 1d 1 1/day 9. 2d 2 1/day 10. 3d 2 1/day 11. 4d 2 1/day 12. 5d 3 1/day 13. 6d 3 1/day 14. 7d 1 1/day 15.  5 1/day 16.  1 1/day 17.  1 1/day 18.  5 1/day 19.  2 1/day 20.  1 1/day 21.  2 1/day 22.  1 1/day figure 4. the process of development case of ebv-infected and developing nasopharyngeal carcinoma. in figure 4, ebv is detected in the body. in the third case, because low immune system, lytic viruses evolve very rapidly. thus making normal cell down very quickly, otherwise latent ebv-infected cells increased. this coincided with increase high dysplastic cells. the high growth fueled high dysplastic cells growth invasive carcinoma very quickly. these cases need special attention. we need for follow-up studies, so there is prevention in anticipation of invasive carcinoma. it should be a particular concern because it studied the beginning of nasopharyngeal carcinoma. conclusion this paper have completed the equilibrium point system in (1). for the asymptotic stability on equilibrium point will be fulfilled if 2 2ia c i d   , 3 2 3a i d  , 4 4a e l d   , 5 5a m d  , and 6 6a d . it mean that to aim the stability it must fulfilled that the proliferation of lesion cells, low dyplastic cells, invection cells, hight dyplastic cells, and invansive carcinoma cells are smaller than the rate cell that go in the next direction compartment and died cells. acknowledgments thank to mr. m. wakhid musthofa, mr. ario wiraya, miss. dewajani purnomosari, miss. dewi kartikawati paramita, miss. jajah fachiroh, mr. muhammad ghufron over discussion and all their support. references hanahan d., and weinberg r. a., 2011, hallmarks of cancer: the next generation, cell 144, march 4, 2011, elsevier inc. janeway c. a jr., travers p., walport m., et al.immunobiology: the imune system in health and disease. 5th edition, nnew york: garland science: 2001using the immune response to attack tumors. available from: https://www.ncbi.nlm.nih.gov/books/nbk27104/ lo, k.w., chung, g.t., and to k.f., 2012. deciphering the molecular genetic basis of npc through molecular, cytogenetic, and epigenetic approaches, seminars in cancer biology, 22 (2): 79 – 86. munir d., 2006. beberapa aspek karsinoma nasofaring pada suku batak di medan dan sekitarnya, the journal of medicine school university of sumatera utara, 39 (3): 223226. ratnawati h., 2012. major histocompability complex class-i related chain a (mica) sebagai kandidat immunological tumor marker pada penderita karsinoma nasofaring, dissertation: doctoral programs, medicine and health, faculty of gadjah mada university, yogyakarta. soetjipto, d, 1989. karsinoma nasofaring. dalam: tumor telinga, hidung dan tenggorokan. diagnosis dan penatalaksanaan, balai penerbit fk ui jakarta, p. 71-84. sugiyanto, fajar a. k., lina a., mardiah s. h., 2013, mathematical modeling of nasopharynx carcinoma on cell level, aip proceeding, http://scitation.aip.org/content/aip/proceeding/aipcp/10.1063/1 .4866540;jsessionid=2gqg1j7afdq12.x-aip-live-02. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1047-1053 | doi: 10.14421/biomedich.2025.142.1047-1053 issn 2540-9328 (online) actinomycetes from plant rhizosphere in gorontalo karst area as plant growth promoting rhizobacteria citra leonita matalauni*, yuliana retnowati, abubakar sidik katili, novri youla kandowangko, ani m. hasan biology department, faculty of mathematics and natural science, universitas negeri gorontalo, gorontalo. jl. prof. dr. ing. bj. habibie, kabupaten bone bolango, 96119, indonesia. corresponding author* citraleonita2303@gmail.com abstract this study aimed to describe actinomycetes from the rhizosphere of plants in the gorontalo karst area as plant growth promoting rhizobacteria (pgpr). the research method is based on a quantitative descriptive method. soil samples from the rhizosphere were collected using a purposive sampling technique from 8 plant species at two locations in the gorontalo karst region, namely the tanjung kramat hills. the characteristics of pgpr in this study focused on phosphate solubilization ability, indole acetic acid (iaa) production, antagonism against the fungus fusarium oxysporum, and tolerance to fungicides. potential pgpr isolates were analyzed for phylogenetic relationships based on 16s rrna gene sequences. the results showed that there were 6 actinomycetes isolates (rzhkc-01, rzko-02, rzo-03, rzak-04, rzpk-05, and rzoc-06) from 8 plant species in the gorontalo karst region. one actinomycetes isolate, rzpk-05, showed potential as a pgpr with the ability to solubilize phosphate and produce iaa at 0.165 mg/l and 0.133 mg/l, respectively. isolate rzpk-05 exhibited antagonistic properties against fusarium oxysporum with an inhibition rate of 82.24% and was tolerant to fungicides such as captive, benlate, and thiramo up to a concentration of 100 mg/l. molecular analysis based on the 16s rrna gene and phylogenetic tree reconstruction indicated that the rzpk-05 isolate is closely related to the genus streptomyces with 100% similarity. keywords: actinomycetes; karst; pgpr; rhizosphere. introduction karst areas are formed through the dissolution of water on soluble rocks, with characteristic features such as channels, cavities, vertical holes, disappearing rivers, springs, underground waterways, and caves (lu et al., 2014). karst regions are classified as hydrogeological environments with high diversity, closely related to other geospheric processes, particularly the atmosphere, hydrosphere, and biosphere, and play an important role in human history and development (goldscheider et al., 2020). these areas generally have calcium-rich soil (ca), which affects the availability of nutrients for plants and soil organisms (fan et al., 2019). the karst area in gorontalo province covers the southern part of gorontalo regency, around lake limboto, and bone bolango regency. plants living in karst areas generally develop physiological and morphological adaptations (meng et al., 2023) to survive in extreme environmental conditions. some plants form symbiotic relationships with various microorganisms in their root zones (lakshmanan et al., 2014). some types of microorganisms found in karst areas include fungi, bacteria, actinomycetes, and algae (mubarak et al., 2017). according to mubarak et al., (2017), bacteria from the actinomycetes group are one of the most commonly found microorganisms associated with root systems in karst regions. the rhizosphere is the zone around plant roots that serves as a habitat for various microorganisms, whose activities play an important role in supporting plant growth and development (putra et al., 2020). the presence and activity of microorganisms in this area are greatly influenced by the exudates produced by plant roots (dewi, 2018). each plant species has a different composition of exudates, which influences the variation and abundance of microorganisms present in the soil. research conducted by syahril et al., (2023) revealed that microorganisms originating from the rhizosphere have a specific distribution influenced by the physical and chemical conditions of the soil. actinomycetes are capable of producing bioactive compounds such as antibiotics and the phytohormone iaa, which play a role in root growth, plant organ formation, and fruit ripening (anwar et al., 2016; etchells et al., 2016). some types of actinomycetes can solubilize phosphate by producing organic acids such as gluconate, citrate, and oxalate, which convert bound manuscript received: 13 august, 2025. revision accepted: 04 november, 2025. published: 18 november, 2025. https://doi.org/10.14421/biomedich.2025.142.1047-1053 mailto:citraleonita2303@gmail.com 1048 biology, medicine, & natural product chemistry 14 (2), 2025: 1047-1053 phosphate into a form available to plants (rajput et al., 2013; putri et al., 2018). additionally, actinomycetes have potential as biocontrol agents (ilsan, 2016). the presence of actinomycetes in the rhizosphere is influenced by plant root exudates, which determine microbial activity in the surrounding area (dewi, 2018). based on the capabilities possessed by actinomycetes, these microorganisms can be one of the alternatives to inorganic fertilizers, due to their ability as plant growthpromoting rhizobacteria (pgpr). a group of microorganisms known as plant growthpromoting rhizobacteria (pgpr) consists of bacteria and fungi that live in symbiosis with plant root systems, providing nutrients and promoting plant growth (vocciante et al., 2022). the role of pgpr includes biostimulant functions through the synthesis and regulation of growth hormones, enhancing the availability of major nutrients, and acting as bioprotectants to control soil-borne pathogens (marom et al., 2017). exploration of actinomycetes from the rhizosphere of plants in the gorontalo karst region has the potential to produce superior isolates that support nutrient availability and plant growth. this study aims to describe the potential of actinomycetes from this region as plant growth promoting rhizobacteria. materials and methods materials and equipment this study was conducted from june to december 2024 at the biology laboratory of the faculty of mathematics and natural sciences, gorontalo state university. the samples used were rhizosphere soil from plants in the gorontalo karst area, specifically at tanjung kramat, hulonthalangi district, and the karst mountains, kota barat district. the tools and materials used in this study included a soil tester, small shovel, sample bags, labels, notebook, pencil, smartphone camera, laminar air flow, incubator, oven, autoclave, shaking incubator, analytical balance, water bath, hot plate, bunsen burner, petri dishes, erlenmeyer flasks, inoculation needles, beaker glasses, stirring rods, test tubes, a 26-tube rack, micropipettes, culture bottles, a centrifuge, a spectrophotometer, cuvettes, an aluminum mortar, measuring cups, a vortex mixer, a cork borer, a uv transilluminator, pcr, bigdye® terminator v3.1, usa, bioedit software, molecular evolutionary genetics analysis (mega) 11, automatic sequencing machine, distilled water, alcohol, sca medium (starch casein agar), agar powder, nystatin/cycloheximide, ringer's solution, pda medium (potato dextrose agar), nb medium (nutrient broth), isp2 medium, salkowski, l-tryptophan, pikovskaya broth medium, pikovskaya agar medium, sodium molybdate, hydrazine, kh2po4, fusarium oxysporum fungal culture (obtained from ipb culture), glass beads, lysis buffer, proteinase, lysozyme, phenol, chloroform, ethanol, 2% agarose gel electrophoresis, mgcl2, dh₂o, eubacterial universal primers 27f (5'agagtttgatcctggctcag-3') and 1492r (5'tacggctaccttgttacgactt-3'). methods soil sampling from plant rhizosphere in karst areas gorontalo rhizosphere soil samples were taken using purposive sampling from shrubs and herbs in tanjung kramat, hulonthalangi district, and the west mountain in kota barat district. soil was collected at a depth of 10–15 cm using a small shovel, stored in sterile plastic bags, and placed in a coolbox (maulana et al., 2022; katili & retnowati, 2017). location coordinates were recorded using gps, and environmental parameters such as ph and soil moisture were measured using a soil tester. isolation of actinomycetes from plant rhizosphere five grams of soil samples from the karst area were mixed with 45 ml of sterile distilled water, homogenized at 225 rpm, then heated at 60°c for 15 minutes (mangamuri et al., 2012; retnowati et al., 2017). serial dilutions were performed up to 10⁻⁵, and 200 µl of dilutions ranging from 10⁻³ to 10⁻⁵ were inoculated onto sca medium using the spread plate method, then incubated at 37°c for 14–28 days. cycloheximide/nistatin was added to prevent fungal contamination (baskaran et al., 2011). screening potential of plant growth promoting rhizobacteria (pgpr) phosphate solubilization activity test. qualitative testing of phosphate solubilization activity in actinomycetes isolates refer to karpagam & nagalakshmi, (2014) using pikovskaya agar medium pikovskaya agar for 7 days. phosphate solubilization ability is indicated by the formation of a clear zone around the actinomycetes colonies. quantitative phosphate solubilization activity refers to lyn et al., (2013). actinomycetes isolates were grown on pikovskaya broth medium for 7 days. the supernatant was separated from the cell pellet by centrifugation at 225 rpm. phosphate solubilization was measured spectrophotometrically by adding molybdate reagent to the supernatant and measuring at a wavelength of 840 nm. the concentration of dissolved phosphate was calculated using kh2po4 standard curve. indole acetic acid (iaa) production activity test. iaa production by actinomycetes was assessed qualitatively and quantitatively. qualitatively, isolates were grown in nb medium supplemented with 0.1 g ltryptophan, incubated for 7 days, then the supernatant was mixed with salkowski reagent and observed for a pink color change as an indicator of iaa (patten & glick, 2002). quantitative testing is performed spectrophotometrically at a wavelength of 535 nm. iaa concentration is calculated using a standard curve of pure matalauni et al. – actinomycetes from plant rhizosphere in gorontalo karst area … 1049 iaa (sukmadewi et al., 2015; gordon & weber, 1951; kaur & sharma, 2013). antagonistic test against fusarium oxysporum mold. the antagonistic ability of actinomycetes isolates against fusarium oxysporum was tested using the cross streak method on potato dextrose agar medium for 7 days (saha & santra, 2014). antagonistic ability was indicated by the formation of an inhibition zone around the actinomycetes colony. antagonistic activity was determined using the formula (kurnia et al., 2014), and the inhibitory strength categories were based on the table (zivkovikc et al., (2010); nuraini et al., (2017) with percentages <30% (weak), 30-<50% (moderate), 50<70% (strong), ≥70-100% (very strong). note: i = percentage of inhibition zone; r1 = control; r2 = radius of fusarium oxysporum approaching actinomycetes. fungicide tolerance test (captive, benlate, thiramo). actinomycetes tolerance to fungicides was tested using the poison bait method (humaidi et al., 1999; dotulog et al., 2019) at concentrations of 25, 50, 75, and 100 mg/l. isolates producing iaa, phosphate solubilizers, and antagonists were inoculated using cork borers. the tolerance test results were measured based on the relative inhibition level (ril) using the formula by kumar et al., (2007), with categories: ril>90% (highly sensitive), 75-90% (sensitive), 60-75% (moderately resistant), 40-60% (resistant), and ≤40% (highly resistant). note: i = percentage growth of actinomycetes; c = control diameter (mm); t = treatment diameter (mm). molecular identification based on the 16s rrna gene potential pgpr actinomycetes isolates were cultured in starch casein broth medium for 7 days in a shaker incubator and then centrifuged at 500 rpm for 15 minutes to separate the cell pellets. genomic dna was extracted from the pellets using the quick-dna fungal/bacterial miniprep kit (zymo research). the 16s rrna gene was amplified using universal primers 27f and 1492r along with 2x mytaq hs red mix, with pcr conditions following okolie et al., (2013). pcr products were purified using the zymoclean™ gel dna recovery kit. sequencing of the gene was performed in both directions (retnowati et al., 2017) and the results were analyzed using blast against the ncbi database (retnowati et al., 2023). data analysis the research data were analyzed descriptively and quantitatively. the ability of actinomycetes as plant growth promoting rhizobacteria, which includes phosphate solubilization, iaa production, antagonism against fusarium oxysporum, and tolerance to fungicides, was presented in tables and graphs. the molecular identification data were compared with data in genbank ncbi. results and discussion description of research location the study was conducted at two locations, namely tanjung kramat in hulonthalangi district and the karst mountains in kota barat district, with vegetation dominated by catharanthus roseus, mesosphaerum suaveolens, indigofera tinctoria, dolichos oliverii, jatropha gossypifolia, imperata cylindrica, lantana montevidensis, leucaena leucocephala, and pneumatopteris pennigera. environmental conditions were characterized by acidic soil ph and low humidity (table 1). table 1. physical and chemical characteristics of the environment. location coordinate point ph moisture tanjung kramat 0°30'20.0''n 123°03'02.8''e 5,43 4,2% pegunungan karst 0°32'37.5''n 123°01'57.3''e 6,6 1,67% description of actinomycetes as plant growth promoting rhizobacteria the results showed that there were six isolates obtained from eight types of plant rhizosphere and one isolate, rzpk-05, which had potential as plant growth promoting rhizobacteria with the ability to phosphate solubilazing, produce indole acetic acid, antagonistic to fusarium oxysporum (table 2), and tolerant to fungicides (table 3). table 2. results of phosphate solubility testing by actinomycetes isolates. isolate qual. quant. rzhkc-01 rzko-02 + 0,22 mg/l rzo-03 rzak-04 rzpk-05 + 0,165 mg/l rzoc-06 table 3. actinomycetes isolates producing indole acetic acid (iaa). isolate qual. quant. rzhkc-01 rzko-02 rzo-03 + 0,312 mg/l rzak-04 rzpk-05 + 0,133 mg/l rzoc-06 1050 biology, medicine, & natural product chemistry 14 (2), 2025: 1047-1053 table 4. results of actinomycetes antagonistic testing. isolate qual. quant. categories rzhkc-01 rzko-02 rzo-03 rzak-04 rzpk-05 + 82,24% very strong rzoc-06 table 5. result of rzpk-05 isolate tolerance testing against fungicides. fungicides c ril categories captive 25 -9,45% highly resistant 50 17,45% highly resistant 75 9,63% highly resistant 100 16,72% highly resistant benlate 25 46,45% resistant 50 14,43% highly resistant 75 -4,52% highly resistant 100 24,27% highly resistant thiramo 25 -23,24% highly resistant 50 -23,24% highly resistant 75 3,26% highly resistant 100 38,70% highly resistant description: (c): concentration; (ril): relative inhibition level figure 1. a: indole acetic acid producing, b: phosphate solubilazing, c: antagonistic to fusarium oxysporum, d: tolarence of fungicide. description of phylogenetic relationship from actinomycetes as plant growth promoting rhizobacteria actinomycetes isolates with potential as plant growth promoting rhizobacteria, namely rzpk-05, were molecularly identified based on the 16s rrna gene, and the sequence data matching results showed that the rzpk-05 isolate was related to the streptomyces genus (table 6). table 6. the closest phylogenetic relative of the rzpk-05 isolate based on 16s rrna gene sequencing. nearest phylogenetic neighbor percent identity e-value genus streptomyces albus strain nrrl b-1811 (nr_118467) 100 0.0 streptomyces streptomyces albus strain nbrc 15415 (nr_041180) 100 0.0 streptomyces streptomyces albus strain nbrc 13078 (nr_041110) 100 0.0 streptomyces streptomyces albus strain nbrc 13015 (nr_112342) 100 0.0 streptomyces streptomyces albus strain nbrc 13014 (nr_112341) 100 0.0 streptomyces figure 2. reconstruction of the phylogenetic tree of the rzpk-05 isolate. discussion the results of the study revealed that only one actinomycetes isolate, rzpk-05, out of six isolates was capable of solubilizing phosphate, producing iaa, exhibiting antagonistic activity against fusarium oxysporum, and being tolerant to fungicides. according to the research by oksana et al., (2020), bacteria are capable of solubilizing phosphate by forming a clear zone through the secretion of organic acids. these acids interact with calcium ions (ca) from ca₃(po₄)₂ in the pikovskaya medium, thereby releasing phosphate ions (h₂po₄) and producing a transparent or clear area. the ability of actinomycetes isolates to solubilize phosphate is likely due to the presence of phosphatase enzymes in these isolates. putri et al., (2018) stated that the formation of a clear zone around actinomycetes colonies is influenced by the activity of phosphatase enzymes and the production of organic acids or polysaccharides. phosphatase enzymes act as catalysts in hydrolytic mineralization, converting insoluble phosphorus into soluble forms (baloe et al., 2023). a study by hutagaol a b c d matalauni et al. – actinomycetes from plant rhizosphere in gorontalo karst area … 1051 et al., (2022) reported that the mechanism of phosphate dissolution by bacteria occurs chemically. bacteria can dissolve phosphate by producing various organic acids, such as citric acid, succinic acid, glutamic acid, oxalic acid, lactic acid, malic acid, fumaric acid, glyoxylic acid, tartaric acid, and α-ketobutyric acid. the accumulation of these organic acids lowers the ph of the environment, thereby triggering the release of phosphorus (p) that was previously bound to calcium (ca). research by nisa (2018) adds that differences in the ability of phosphatesolubilizing bacteria to dissolve phosphate are also influenced by the genetic factors of each microorganism, particularly in producing organic acids that play a role in the phosphate dissolution process. the ability to produce indole acetic acid (iaa) is influenced by the physical and genetic characteristics of bacteria, including the presence of genes encoding metabolic enzymes (passalacqua et al., 2016; louis et al., 2007). genetic differences between bacteria affect the availability of enzymes and iaa biosynthetic pathways (patten & glick, 2002; lata et al., 2024). rhizosphere bacteria generally produce iaa through the tryptophan pathway, such as iam, tam, and ipya (zhang et al., 2019; zhang et al., 2021). the presence of the ipdc gene in some species supports the role of the ipya pathway in iaa biosynthesis. therefore, the ability of actinomycetes isolates to produce iaa is likely related to the presence of genes encoding enzymes involved in that pathway. isolate rzpk-05 showed antagonistic activity against fusarium oxysporum, indicating its ability to produce antifungal compounds. zhang et al., (2021) reported that only 17 of 60 actinomycetes isolates were active against fusarium oxysporum. this ability is thought to be influenced by the presence of antifungal genes such as polyketide synthase-i and non ribosomal peptide synthetase (nurjasmi et al., 2009). other factors that may influence the antagonistic activity exhibited against the pathogen fusarium oxysporum are thought to be due to the fact that actinomycetes isolates have different physiological mechanisms, enabling them to respond to different types of pathogens. queendy and roza (2019) state that a microorganism's ability to produce secondary metabolites is influenced by its individual physiological characteristics. the study conducted by queendy and roza (2019) reported that out of 32 actinomycetes isolates, only 4 potential isolates (c1.15, b1.07, d2.31, c2.21) inhibited fusarium oxysporum, and 3 isolates (b1.02, c2.24, b3. 14) inhibited ganoderma boninense. the tolerance shown by the rzpk-05 isolate to various types of fungicides is likely due to its ability to degrade the active ingredients of fungicides. degradation is the process of breaking down unstable compounds into more stable forms (atlas, 1992; lumantouw et al., 2013), allowing bacteria to survive in environments exposed to fungicides. therefore, the rzpk-05 isolate's continued tolerance to the fungicides captive, thiramo, and benlate is likely due to its ability to modify the active ingredients of fungicides into non-toxic forms. based on the ability of the rzpk-05 isolate as a pgpr and molecular identification showing that rzpk05 is closely related to the genus streptomyces. the presence of this genus in karst areas reinforces the potential of the rzpk-05 isolate obtained from the rhizosphere of plants as plant growth promoting rhizobacteria. research shows that actinomycetes of the genus streptomyces are capable of solubilizing phosphate (jog & colleagues, 2014), producing iaa (lin et al., 2013), and exhibiting antagonistic activity (kanini et al., 2013). furthermore, actinomycetes, particularly from the genus streptomyces, are known as bacteria capable of producing antibiotics, biosurfactants, volatile compounds, and toxins, which have the potential to be used as biocontrol agents to combat plant pathogens (vurukonda et al., 2018). conclusions based on the research, it can be concluded that only one isolate, rzpk-05, out of six isolates has potential as a pgpr with the ability to dissolve phosphate (0.165 mg/l), produce iaa (0.133 mg/l), antagonistic against fusarium oxysporum (82.24%), and tolerant to fungicides (highly resistant). molecular identification results indicate that the rzpk-05 isolate is closely related to the genus streptomyces with a similarity percentage of 100%, thus identifying the rzpk-05 isolate as streptomyces albus strain rzpk-05. acknowledgements: the author would like to express his deepest gratitude to dr. yuliana retnowati, s.si., m.si, and dr. abubakar sidik katili, s.pd., m.sc, as supervisors and parties who have provided financial support so that this research could be carried out properly. the author also extends 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(2019). the distribution of tryptophan-dependent indole-3-acetic acid synthesis pathways in bacteria unraveled by large-scale genomic analysis. molecules, 24(7), 1411. https://doi.org/10.3390/molecules2407141 https://doi.org/10.1007/s00203-012-0850-x https://doi.org/10.1007/s00203-012-0850-x https://doi.org/10.13057/biodiv/d180421 https://doi.org/10.13057/biodiv/d240255 https://doi.org/10.13057/biodiv/d250301 https://doi.org/10.3390/ijms19040952 https://doi.org/10.1039/d1ra05659j https://doi.org/10.3390/molecules2407141 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 2, 2019 | pages: 41-45 | doi: 10.14421/biomedich.2019.82.41-45 issn 2540-9328 (online) hepatotoxic assessment of tramadol-diclofenac use: a study in a rat model elias adikwu*, ebinyo clemente nelson department of pharmacology and toxicology, faculty of pharmacy, niger delta university, bayelsa state, nigeria. corresponding author* adikwuelias@gmail.com abstract the concurrent use of tramadol and diclofenac may increase hepatotoxic risk due to their individual hepatotoxic effects. this study assessed the hepatotoxic effect of tramadol-diclofenac administration in albino rats. twenty-four adult male albino rats (200-220g) randomized into four groups were orally administered with tramadol (12mg/kg/day), diclofenac (6mg/kg/day) and tramadol-diclofenac for 14 days respectively. the rats were anesthetized, blood samples were collected and evaluated for serum liver function and lipid parameters. liver samples were weighed and evaluated for biochemical parameters and histology. the effects of tramadol-diclofenac on the body and liver weights did not differ significantly (p>0.05) when compared to control. also, effects were not significant (p>0.05) on blood glucose, and serum cholesterol, triglyceride, low and high density lipoprotein cholesterol levels when compared to control. liver and serum levels of aminotransferases, alkaline phosphatase, lactate dehydrogenase, gamma–glutamyl transferase, conjugated bilirubin and total bilirubin increased significantly in rats treated with tramadol (p<0.05), diclofenac (p<0.01) and tramadol-diclofenac (p<0.001) when compared to control. furthermore, significant decreases in liver catalase, glutathione, superoxide dismutase, glutathione peroxidase levels with significant increases in malondialdehyde levels occurred in rats treated with tramadol (p<0.05), diclofenac (p<0.01) and tramadol-diclofenac (p<0.001) when compared to control. hepatocyte necrosis was observed in rats treated with tramadol-diclofenac. tramadol-diclofenac may increase hepatotoxic risk at doses used for this study. keywords: tramadol; diclofenac; co-treatment; toxicity; liver; rat introduction most medical conditions are associated with pain. pain remains the most inadequately treated symptom due to different cultural, attitudinal, educational, legal, and system-related reasons (connors et al., 1995). pain has a multifactorial origin; hence it may be difficult to achieve effective pain control with a single drug. combination therapy with analgesics from different groups is advantageous in targeting both peripheral and central pain pathways (rawal et al., 2011). the world health organization (who) analgesic ladder recommends the combination of acetaminophen or nsaids with opioids as the second step in the treatment of pain, based on increasing pain severity (blondel and azadfard, 2013). tramadol-diclofenac can be used for the treatment or management of chronic pain (mitra et al., 2012). diclofenac is a phenylacetic acid derivative which belongs to the acetic acid class of nsaids. it acts by inhibiting cellular cyclooxygenases (cox-1 and cox-2), which results to decreases in the production of proinflammatory prostaglandin, prostacyclin and thromboxane products which are important mediators of inflammation and pain (zimmerman, 1999; lewis and stine, 2013). tramadol is a synthetic codeine analog that acts as a weak opioid agonist in addition to mildly inhibiting serotonin and norepinephrine reuptake. tramadol is effective against mild-to-moderate pain. clinically, tramadol-diclofenac use could advantageously target both peripheral and central pain pathways and leverage on the ability of individual drug to reduce pain and fasten recovery (raffa, 2001). however, tramadol-diclofenac use may increase hepatotoxic risk since both drugs have hepatotoxic potential. clinically, apparent liver injury due to diclofenac has been reported and it ranks in the top 10 causes of drug-induced liver injury (dunk et al., 1982). tramadol can cause respiratory arrest as well as acute liver failure, which several fatal instances reported (loughrey et al., 2003). the use of tramadol-diclofenac calls for hepatotoxic assessment which this study evaluated in a rat model. material and methods animals and drugs adult male albino rats of weight 200-220g were used. the rats were supplied by the animal house of the department of pharmacology and toxicology niger, delta university, nigeria. the rats were housed in four cages (6 per cage) and allowed to acclimatize for 2 weeks in a well-ventilated room, maintained at a room temperature of 28 ± 2°c, under natural lighting condition. the rats were fed with standard rodents chow https://doi.org/10.14421/biomedich.2019.82.41-45 42 biology, medicine, & natural product chemistry 8 (2), 2019: 41-45 and given water ad libitum. tramadol hydrochloride (zim laboratories ltd india) and diclofenac potassium (adpharm nigeria ltd) were used for this study. all other chemical substances used for this study are of analytical grade. higher doses of tramadol (12mg/kg/day) and diclofenac (6mg/kg/day) dissolved in normal saline were used for this study. grouping of animals and drug treatment twenty-four adult male albino rats were divided into four (4) groups a-d of 6 rats each.  group a (control) was orally administered with normal saline (0.2ml) for 14 days.  group b was orally administered with tramadol (12 mg/kg/day) for 14 days.  group c was orally administered with diclofenac (6mg/kg/day) for 14 days.  group d was orally administered with tramadol (12mg/kg/day) and diclofenac (6mg/kg/day) for 14 days. collection of sample the rats were sacrificed with inhalational diethyl ether after drug administration and blood samples were collected from the heart. at 1500g for 15 minutes, the blood samples were centrifuged, serum samples extracted and evaluated for liver function parameters. liver samples were harvested and washed in a cold 1.15% kcl solution and homogenized in 0.1 m trishcl buffer, ph 7.4. the homogenates were centrifuged at 1500g for 15 minutes and the supernatants were decanted and evaluated for biochemical parameters. evaluation of biochemical and oxidative stress indices aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), total bilirubin (tb), conjugated bilirubin (cb), gammaglutamyl transferase (ggt), lactate dehydrogenase (ldh), total cholesterol (tc), triglyceride (tg) and high density lipoprotein cholesterol (hdl-c) were evaluated using standard laboratory test kits. blood glucose (g) was evaluated using glucometer while low density lipoprotein cholesterol (ldl-c) was determined using friedewald equation. liver protein was evaluated according to gornall et al. (1949). the method described by sun and zigma, (1978) was used for the evaluation of superoxide dismutase (sod) whereas catalase (cat) was determined according to aebi, (1984). glutathione (gsh) was estimated as described by sedlak and lindsay, (1968) whereas glutathione peroxidase (gpx) was evaluated according to the method of rotruck, et al, (1973). malondialdehyde (mda) was determined as reported by buege and aust, (1978). histological examination of the liver liver samples were fixed in 10% neutral buffered formalin, processed and embedded in paraffin wax. sections of 5 μm thickness were cut, stained with haematoxylin and eosin and examined under a light microscope and relevant sections photographed. statistical analysis data are expressed as mean ± sem. data was subjected to student t test. results were considered to be significant at p<0.05; 0.01; 0.001. table 1. effects of tramadol-diclofenac on body and liver weights of albino rats. groups body weight (g) absolute liver weight(g) relative liver weight (%) a b c d 290±11.3 295±14.2 275±10.6 295±11.5 6.03±0.13 6.22±0.25 5.99±0.31 6.21±0.19 2.07±0.01 2.10±0.73 2.17±0.59 2.10±0.77 n=6. data are expressed as mean ± sem. table 2. effects of tramadol-diclofenac on blood glucose and serum lipids of albino rats. group g(mg/dl) tg(mg/dl) tc(mg/dl) hdlc(mg/dl) ldlc(mg/dl) a 95.0±6.41 70.5±6.78 110.1±10.8 30.7±3.44 65.7±6.00 b 90.7± 7.85 72.1±7.59 106.8±10.3 31.8±3.35 61.6±5.32 c 92.0±6.42 74.9±6.70 112.0±10.6 32.6±2.61 64.5±6.42 d 100.9±9.19 81.1±7.56 110.3±11.1 32.4±2.63 64.7±7.62 n=6, data are expressed as mean ± sem. table 3. effects of tramadol-diclofenac on serum liver function indices of albino rats. group ast(u/l) alt(u/l) alp(u/l) ggt(u/l) ldh(u/l) cb(g/dl) tb(g/dl) a 41.5±5.57 34.2±3.15 42.3±3.56 45.5±3.11 0.75±1.53 2.66±0.18 4.21±0.05 b 60.5±6.96a 55.1±4.06a 69.5±6.11a 66.0±6.01a 1.60±0.71a 3.95±0.06a 6.6±0.02a c 89.3±6.33b 79.2±5.25b 87.7±7.71b 88.1±9.43b 2.67±0.52b 5.73±0.65b 9.8±0.06b d 220.6±10.7c 181.3±9.70c 211.3±9.37c 220.2±14.2c 6.21±0.37c 15.4±1.22c 22.8±0.76c n=6, data are expressed as mean ± sem, a differ significantly at p<0.05 when compared to control b differ significantly at p<0.01 when compared to control, c differ significantly at p<0.001 when compared to control. adikwu & nelson – hepatotoxic effect of tramadol-diclofenac 43 table 4. effects of tramadol-diclofenac on liver tissue biochemical parameters of albino rats. group ast(u/l) alt(u/l) alp(u/l) ggt(u/l) ldh(u/l) a 244.8±12.0 246.9±12.1 247.2±12.3 250.5±14.2 22.9±4.03 b 373.8± 17.6a 374.6±20.4a 390.0±20.0a 399.0±17.4a 45.2±7.09a c 499.6±25.0b 470.3±26.0b 488.4±23.4b 475.1±21.2b 68.1±8.16b d 960.0±23.7c 983.3±41.8c 901.0±43.7c 888.2±30.6c 190.5±12.8c n=6, data are expressed as mean ± sem, a differ significantly at p<0.05 when compared to control b differ significantly at p<0.01 when compared to control, c differ significantly at p<0.001 when compared to control table 5. effect of tramadol-diclofenac on liver oxidative stress indices of albino rats. group mda (nmol/mg protein) sod (u/mg protein) cat (u/mg protein) gsh (µmol/mg protein) gpx (u/mg protein) a 0.17±0.06 16.2±1.89 26.4±2.77 8.96±0.15 11.7±0.16 b 0.39± 0.04a 11.6±0.15a 17.0±0.79a 5.02±0.32a 5.25±0.01a c 0.52±0.04b 9.98±0.04b 13.6±1.16b 3.91±0.73b 4.00±0.18b d 1.37±0.32c 3.63±0.06c 5.61±0.83c 1.30±0.22c 2.57±0.82c n=6, data are expressed as mean ± sem, a differ significantly at p<0.05 when compared to control b differ significantly at p<0.01 when compared to control, c differ significantly at p<0.001 when compared to control figure 1. (a-e) showed the liver of control rat, rats treated with diclofenac, tramadol and diclofenac-tramadol (h &e stain 400x). (a): the liver of control rat showing normal hepatocytes (b): liver of rat treated with tramadol (12 mg/kg/day) showing hepatocyte necrosis (c): liver of rat treated with diclofenac (6 mg/kg/day) showing hepatocyte necrosis. (d): liver of rat treated with diclofenac-tramadol showing hepatocyte necrosis results the effects of tramadol-diclofenac were not significant (p>0.05) on the body and liver weights of treated rats when compared to control (table 1). also, serum tc, tg, hdl cholesterol, ldl cholesterol and blood g levels were normal (p>0.05) in rats treated with tramadoldiclofenac when compared to control (table 2). serum ast, alp, alt, ggt, ldh, ct and tb levels were significantly increased in tramadol (p<0.05) and diclofenac (p<0.01) treated rats when compared to control. however, elevations in the serum levels of the aforementioned parameters were most significant (p<0.001) in rats treated with tramadol-diclofenac when compared to control (table 3). the liver levels of ast, alp, alt, ggt and ldh were significantly increased in rats treated with tramadol (p<0.05) and diclofenac (p<0.01), but significant increases occurred at p<0.001 in rats treated with tramadol-diclofenac when compared to control (table 4). furthermore, liver sod, cat, gsh and gpx levels were significantly decreased whereas mda levels were significantly increased in rats treated with tramadol (p<0.05), diclofenac (p<0.01) and tramadol-diclofenac (p<0.001) when compared to control (table 5). the liver of control rat showed normal hepatocytes (fig a) whereas the liver of rat treated with tramadol and diclofenac showed hepatocyte necroses respectively (fig b and c). the liver of rats treated with tramadol-diclofenac also showed hepatocyte necrosis (fig d). discussion the present study evaluated the hepatotoxic effect of tramadol-diclofenac in a rat model. the indices evaluated in this study are useful parameters to indicate impairment in the functional capacity of the liver. analysis of organ weight in toxicology studies is an important endpoint for the identification of potentially harmful effects of chemicals. it is one of the most sensitive drug toxicity indicators, and its changes often precede morphological changes (bailey et al., 2004). in 44 biology, medicine, & natural product chemistry 8 (2), 2019: 41-45 the present study, tramadol-diclofenac had no effects on the body and liver weights of treated rats. serum alp, ast, alt, ggt, and ldh are excellent biomarkers of hepatocellular injury. ast and alt participate in gluconeogenesis by catalyzing the transfer of amino groups from aspartic acid or alanine to ketoglutaric acid to produce oxaloacetic acid and pyruvic acid respectively. ast is present in cytosolic and mitochondrial isoenzymes and is found in the liver while alt, a cytosolic enzyme is found in its highest concentration in the liver and is more specific to the liver. alp is found histochemically in the microvilli of bile canaliculi and on the sinusoidal surface of hepatocytes (rosalki and mcintyre, 1999). ggt is a microsomal enzyme that is abundant in hepatocytes and biliary epithelial cells. it is involved in the transfer of γglutamyl groups from peptides to amino acids and the metabolism of glutathione conjugates (friedman et al., 1996). these liver biomarkers are usually released into circulation causing elevated serum levels with the advent of hepatocellular injury (yousef et al., 2010). this study observed elevated serum and liver levels of alp, ast, alt, ggt, and ldh in rats treated with tramadol-diclofenac which is a sign of hepatotoxicity. bilirubin is an endogenous anion derived from the regular degradation of haemoglobin from the red blood cells and excreted from the liver in the bile (saukkonen et al., 2006). toxic insult to the liver can impair its excretory capacity to dispose bilirubin stimulating serum accumulation (gaw et al., 1999; jain et al., 2008). this study observed elevated serum cb and tb levels in rats treated with tramadol-diclofenac. the anti-oxidative defense system which includes sod, cat, gsh and gpx is necessary for the maintenance of redox homeostasis in organisms. it terminates or prevents free radicals such as reactive oxygen species (ros) from incapacitating the functions of biomolecules through oxidative stress (os) (borković et al., 2005). however, the functions of anti-oxidative system can be surmounted or incapacitated via depletion by over whelming actions of free radicals. this study observed hepatic depletions of sod, cat, gsh and gpx in rats treated with tramadol-diclofenac. this is an evidence which shows that os is a factor in hepatotoxicity induced by tramadol-diclofenac. lipid peroxidation (lpo) is an oxidative degradation of polyunsaturated fatty acids which can cause impairment in membrane structure and function. mda level which is an important indicator of lpo can indirectly reflect the extent of hepatic lpo in-vivo and in-vitro (jafari et al., 2012). in this study, hepatic mda levels were increased in rats treated with tramadol-diclofenac. this observation attests to the involvement of lpo in hepatotoxicity induced by tramadol-diclofenac. furthermore, the current study observed hepatocyte necrosis in rats treated with tramadol-diclofenac which correlates with changes observed in evaluated biochemical parameters. this study was able to show that tramadol-diclofenac use may increase the risk of hepatotoxicity. the use of diclofenac has been associated with hepatotoxicity (hussein et al., 2016) and hepatic os (das and roy, 2012) which is consistent with findings in this study. the exact mechanism by which diclofenac causes hepatotoxicity is not well understood, but diclofenac is metabolized in the liver (castel et al., 1997) and its hepatotoxic effect has been related to its metabolites (4hydroxy 3 diclofenac, 5 hydroxy 4 diclofenac and 5 hydroxy 6 diclofenac) (tang et al., 1999). also, studies have associated tramadol with hepatotoxicity (el-wessemy, 2008) characterized by os (rukhshanda et al., 2014) which is in agreement with observation in the present study. the mechanism by which tramadol causes hepatotoxicity is not fully known, but studies have speculated that tramadol and/or its active metabolite can stimulate hepatic ros production leading to os and hepatic biomolecular damage (singal et al., 1998). conclusion tramadol-diclofenac use may be associated with hepatotoxicity at the doses use for this study. conflict of interest: none references aebi h. catalase in vitro, in method in enzymology, s. p. colowick and n. o. kaplane, eds., 1984; 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(1999). drugs used to treat rheumatic and musculospastic disease. the nsaids.in, zimmerman hj. hepatotoxicity: the adverse effects of drugs and other chemicals on the liver. 2nd ed. philadelphia: lippincott, pp. 1999; 517-41. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 161-166 | doi: 10.14421/biomedich.2025.141.161-166 issn 2540-9328 (online) the improvement of heart histopathology of hypercholesterolemic mice using sidempuan salacca (salacca sumatrana) vinegar yusni atifah1,*, firda az zahra2, helendra1, jalilah azizah lubis3, friska afrilliana1 1biology department; 2natural science department, faculty of mathematics and natural sciences, universitas negeri padang jl. prof. dr. hamka air tawar barat, padang 25171 indonesia, tel. (0751) 7053902, indonesia. 3program study of biology education, faculty of teacher training and education universitas muhammadiyah tapanuli selatan jl. sutan moh. arif no.32, batang ayumi julu, kota padang sidempuan, sumatera utara 22733. corresponding author* yusniatifah@fmipa.unp.ac.id abstract hypercholesterolemic, a condition characterized by excess cholesterol content, can lead to coronary heart disease, atherosclerosis, and high-risk heart tissue degeneration. current drugs, like simvastatin and pravastatin, focus on lowering cholesterol levels without addressing tissue degeneration. this study aimed to determine the effectiveness of sidempuan salacca vinegar in improving cardiac tissue degeneration in hypercholesterolemic mice. the experimental research involved two groups, the treatment group given different doses of sidempuan salacca vinegar and the control group. histopathological observations showed improvement in cardiac tissue degeneration in hypercholesterolemic mice after treatment with salacca vinegar. the administration of salacca vinegar could serve as a reference for developing alternative hypercholesterolemic drugs that address both cholesterol levels and heart degeneration. keywords: heart; histology; hypercholesterolemic; sidempuan salacca vinegar. introduction hypercholesterolemia is characterized by cholesterol levels in the blood that exceed the level required by the body, which is more than 240 mg/dl in adults (jempormase et al., 2016). hypercholesterolemia has many harmful risk factors that will also affect the degeneration of heart and liver tissue, the digestive organs, and the storage of cholesterol in the body. the most common forms of degeneration are necrosis and inflammation in the heart tissue and steatosis in the liver. in more severe cases, hypercholesterolemia tends to initiate the onset of coronary heart disease, the number one killer in the world. when the body experiences hypercholesterolemia, it tries to balance cholesterol levels synthesizing cholesterol into gallic acid. this synthesis process will require oxygen and other supportive substances. this unnecessary oxidation will eventually result in free radicals (duell et al., 2019). if the formed free radicals attack the lipids in ldl, the oxidized ldl diffagositosis by macrophages forms foam cells, which are an early indication of the occurrence of atherosclerosis. free radicals that attack ldl also produce lipid peroxide. lipid peroxidation from a free radical oxidation reaction on the ldl-ox series with pufa (polyunsaturated fatty acids) or lipids on the cell membranes of body tissue that can cause damage to the cells (nanda, 2018). ldl, which is attacked by free radicals, also triggers an inflammatory response (duell et al, 2019). the inflammatory response to the heart associated with hypercholesterolemia will be visible in the endothelial cells and initiate immunocompetent cells, namely lymphocytes, monocytes, and macrophages. as the inflammation continues, the aorta will experience thickening and expansion of the arterial walls until the lumen loses its ability to limit it. activating macrophages and lymphocytes causes the release of hydrolytic enzymes, cytokines, chemokines, and growth factors that can cause necrosis (duell et al., 2019). this series of events will eventually change the picture of heart histopathology. treatment of hypercholesterolemia complications today is too focused on drugs with chemical compounds that have quite destructive side effects. examples of drugs commonly used in patients with hypercholesterolemia are simvastatin, atorvastatin, fevastin, and pravastatin (murray et al., 2018). simvastatin itself has been known to cause quite bad side effects such as muscle dysfunction, abdominal pain, arrhythmia, tachycardia, and so on (hariadini et al., 2020). with such a variety of negative side effects, researchers started looking for treatment alternatives that focused on herbal ingredients. in addition to being easy manuscript received: 25 december, 2024. revision accepted: 15 may, 2025. published: 18 may, 2025. https://doi.org/10.14421/biomedich.2025.141.161-166 162 biology, medicine, & natural product chemistry 14 (1), 2025: 161-166 to find, herbal ingredients are cheaper and are expected to have insignificant side effects. many plants in indonesia have been found to have the potential to prevent cholesterol disease and even have the potential to treat cholesterol. the traditional spices have serine of ntd-npc1l1 for cholesterol binding with compounds in traditional spicesis known to have the ability to interfere with cholesterol absorption at an early stage where there is a high binding affinity between ezetimibe and these ligands interacting almost at the right location in the n-terminal domain. through ser_102 the nterminal domain of npc1l1 binds to ligands (amelia et al., 2023). with its contents, salacca sumatrana has the potential as an alternative herbal medicine to repair the degeneration of heart tissue that is damaged due to hypercholesterolemia. however, no further research has been conducted on the benefits of oatmeal vinegar in preventing organ degeneration due to hypercholesterolemia. for this reason, this research is important to open up new herbal treatment alternatives to hypercholesterolemia. materials and methods study area this research is based on true experimental design: pre and post test with control group design. 25 male mice were divided into 5 groups. there were normal group (k), hypercholesterolemic group (k+), hypercholesterolemic + sidempuan salacca vinegar 0,2 ml group (p1), hypercholesterolemic+ sidempuan salacca vinegar 0,4 ml group (p2), hypercholesterolemic + sidempuan salacca vinegar 0,8 ml group (p3). procedures mice were given an oral and egg yolk diet to create hypercholesterolemia in mice treated with k+, p1, p2 and p3 for 14 days. cholesterol levels are measured three times, i.e., before high cholesterol nutrition and after high cholesterol nutrition and after treatment with sidempuan salacca vinegar using easy touch. histological heart observations were carried out after administering sidempuan salacca vinegar for 14 days to hypercholesterolemic mice. data analysis to determine the effect on the histopathology of the heart administered with the sidempuan salacca vinegar, a descriptive analysis of the qualitative data of the histopathology of hypercholesterolemia was conducted. quantitative data analysis is also carried out with the kruskal-wallis test, with the observed variables being necrosis and inflammation. each sample was given damage scores for the categories of necrosis and inflammation. score 1 is given if there is mild necrosis or inflammation in the heart. (vocal). score 2 is given to indicate the presence of necrosis or inflammation in the heart of a moderate nature. (multivocal). score 3 is given for necrosis or inflammation in the severe category (diffuse) (pratama., et al 2018). if the results of the kruskal-wallis test show a p significance value of 0.05, which means there are significant histopathological differences, then the analysis will be continued with the mann-whitney test to determine the difference in efficacy between treatment doses. results and discussion result the blood cholesterol levels of treated mice during 4 weeks are presented in figure 1. the result of this research show that all mice had hypercholesterolemia by dietary offal and egg yolk as marked in the blue bar graph, then there was a decrease in cholesterol levels after giving of sidempuan salacca vinegar (orange bar graph). mice are said to be hypercholesterol if the cholesterol level in their body reaches 130 mg/dl. giving salacca vinegar tends to reduce blood cholesterol levels in male mice. the highest reduction was in the p3 treatment, namely 86.6 mg/dl and the lowest was in the ktreatment, namely -1 mg/dl. figure 1. comparison of cholesterollevel of male mice pra and post treatment sidempuan salacca vinegar. the histopathological picture of the heart of hypercholesterolemic male mice after treatment is shown in the following picture. necrotic degeneration and inflammation could be observed and the negative control samples showed normal routine histopathology. kk+ p1 p2 p3 pra 106,4 145,4 198,6 194,8 197,2 post 107,4 144,4 129,6 123,8 110,6 0 50 100 150 200 250 c h o le st er o l le v el m g /d l cholesterol level of male mice with treatment sidempuan salacca vinegar atifah et al. – the improvement of heart histopathology of … 163 figure 2. histopathology of heart (400x magnifications). k= normal diet, k+ = high cholesterol diet without salacca vinegar, p1 = high cholesterol diet + salacca vinegar dose of 0.2 ml, p2 = high cholesterol diet + salacca vinegar dose of 0.4 ml, p3 = high cholesterol diet + salacca vinegar dose of 0.8 ml, black arrow = necrosis, blue arrow = imflamation. histological observations of the heart were carried out to see the effectiveness of sidempuan salacca vinegar on its effect on repairing heart tissue. the results of the study showed that there was improvement in heart tissue along with increasing the dose of sidempuan salacca vinegar given to hypercholesterolemic mice. table 1. heart histopathology degeneration score in hypercholesterolemia mice after treatment salacca vinegar. degeneration group of treatment n score 0 1 2 3 necrosis k5 5 k+ 5 2 3 p1 5 1 3 1 p2 5 2 2 1 p3 5 4 1 inflammation k5 5 k+ 5 1 4 p1 5 1 2 2 p2 5 2 3 p3 5 4 1 k- k+p1 p2 p3 164 biology, medicine, & natural product chemistry 14 (1), 2025: 161-166 in the study, the negative control group (k-) showed no signs of hypercholesterolemia or degenerative forms of necrosis or inflammation. however, the k+ group with high cholesterol nutrition treatment without salacca vinegar administration experienced diffuse degeneration with an average necrosis score of 2.6 and an average inflammation score of 2.8. treatment groups p1, p2, and p3 were treated with high cholesterol and salacca vinegar as an antithesis with different doses per treatment, improving tissue degeneration. the p1 treatment group with a dose of 0.2 ml salacca vinegar showed better histopathological degeneration results than the k+ group. in the k+ group, 3/5 of the samples had a necrosis score of 3, meaning they had diffuse or severe necrosis, and 2/5 were indicated as multifocal. this score was better than the inflammation score in the k+ group, where 4/5 had diffuse inflammations and 1/5 included multifocal inflammations. table 2. kruskall-wallis test results for necrosis and inflammation. degeneration n p-value h error hypothesis acceptance necrosis 5 0,002 17,3 0,16% accepted inflammation 5 0,001 18,9 0,082% accepted the kruskal-wallis test results showed a significant difference in the dependent variable between treatment groups for necrosis and inflammation. the p values of 0.001638 (p(x17.3693) = 0.9984) and 0.001, respectively, indicate a 0.16% probability of rejecting the hypothesis. this suggests that the administration of sidempuan salacca vinegar may improve the histopathology of male hypercholesterolemia. the mannwhitney test, which was conducted for p1 and p2, yielded a p value of 0.5067 (p(xz) = 0.7466), indicating a 50.67% probability of error to accept the hypothesis. the results suggest that the administration of sidempuan salacca vinegar may have potential benefits in improving male hypercholesterolemia histopathology. table 3. mann whitney intergroup treatment test. degeneration n p-value z error hypothesis acceptance inflammation p1 with p2 0,73 0,34 73,37% denied p1 with p3 0,08 1,73 8,33% denied p2 with p3 0,23 1,19 23,2% denied necrosis p1 with p2 0,25 1,13 25,68% denied p1 with p3 0,06 1,81 6,96% denied p2 with p3 0,27 1,1 27,03% denied discussion after carrying out the one-way anova test, it showed that there was a significant effect of salacca vinegar on the cholesterol levels of mice, so it was continued with a further dmrt test. based on the results of further dmrt tests, it showed that on the 14th day after being treated with salacca vinegar, blood cholesterol levels between the treatment groups (p1, p2, and p3) were significantly different (α=0,05) compared to mice suffering from hypercholesterolemia without treatment with sidempuan salacca vinegar (k+) and mice that did not suffer from hypercholesterolemia without treatment with sidempuan salacca vinegar (k-). blood cholesterol levels in the group of mice given sidempuan salacca vinegar were lower compared to the group of hypercholesterolemic mice without treatment given sidempuan salacca vinegar. in mice treated with sidempuan salacca vinegar (p1, p2, p3), group p3 was significantly different from groups p1 and p2, while groups p1 and p2 were not significantly different. the increase in total blood cholesterol levels of mice in the k+, p1, p2, p3 treatments was due to the high fat content in chicken egg yolks, a chicken egg contains 31.8-35.5 fat (pratama et al., 2018). giving mice egg yolk for 14 days increased the mice's total cholesterol levels to 152.80 mg dl-1. this is in accordance with the theory which states that egg yolk can increase total cholesterol levels in the blood because the fat content in eggs is quite large (pratama et al., 2018; lai et al., 2010). the decrease in cholesterol levels in mice after giving sidempuan salacca vinegar was due to salacca vinegar contains ingredients that can inhibit the increase in cholesterol levels in the blood. salacca vinegar is vinegar from salacca which has high functional capabilities and contains natural compounds. research shows that the test results of salacca vinegar contain phenolic compounds, tannins, flavonoids, antioxidants, vitamin c, and acetic acid (aisah, 2021). this is in line with research that reported active flavonoid compounds have many benefits for the body. one of them is that flavonoids can be used to lower cholesterol. in the body, flavonoids are able to atifah et al. – the improvement of heart histopathology of … 165 erode cholesterol deposits on the walls of coronary blood vessels (widyaningsih et al., 2010; karta et al., 2008; nalole et al., 2009). improvements in tissue degeneration were also observed in the p2 and p3 treatment groups with an additional dose of salacca vinegar. treatment group p2 received a dose of 0.4 ml of salacca vinegar, while treatment group p3 received 0.6 ml of salacca vinegar. this resulted in a decrease in the average degeneration score for both categories and a reduction in the incidence rate to a lower rate of degeneration. in the p2 group, no more samples with diffuse degeneration were found for the category of inflammation, and the average necrose degeneration score in p2 was lower than in p1. a drastic decrease in the average degeneration score occurred with the administration of 0.6 ml of salacca vinegar, with no diffuse degeneration observed for either category. only one sample had moderate-category degeneration, and four others indicated focal degeneration, with an average of 1.2 for both categories. the histopathological picture of the heart-scratched male hypercholesterolemia after treatment is seen in the following picture: degenerative necrosis and inflammation can be clearly observed, and samples from negative controls show a picture of normal heart histopathology. the obtained histopathological picture is then analyzed to give a degeneration score. the scores obtained were then tested with the kruskall-wallis test to find out the significance of the correctional effects of the degeneration of the heart tissue in the male hypercholesterolemic given salacca vinegar. as a result, the p value is 0,005, which means there is significant improvement in the degeneration of heart tissue for both categories of necrosis and inflammation. the kruskall-wallis trial's significance requires an advanced mann-whitney test to determine the significance between treatment groups of sidempuan salacca vinegar. the mann-whitney test for p1 and p2 yielded a p value of 0.5067, with a probability of error of 50.67%. the mann-whitney test for degenerative necrosis and inflammation showed p > values, indicating that the administration of salacca vinegar effectively repairs degenerative heart tissue and hypercholesterolemia. when the body experiences hypercholesterolemia, it will carry out additional metabolism to digest the excess cholesterol. this additional metabolism requires additional energy and resources. this inappropriate metabolism then forms ros which leads to degeneration of the heart as an organ for digesting cholesterol. this is where salacca vinegar, which is known to be rich in antioxidants, works. the antioxidants in salacca vinegar will work as additional energy and resources so that even if there is additional cholesterol metabolism, ros will not be formed which will initiate any form of degeneration in heart tissue. the difference in dose administration in this research did not significantly lower the average degeneration score. the treatment of both p1, p2, and p3 showed the same effect in improving the degeneration of male hypercholesterolemia. this is in accordance with research by atifah and diana (2024) which stated that giving sidempuan salacca vinegar can improve kidney degeneration in hyperuricemic mice. conclusions sidempuan salacca (salacca sumatrana) vinegar effectively improves heart tissue degeneration, as demonstrated by histopathological data and histopathological observations. the effect increases with dose, indicating its effectiveness in reducing heart tissue degeneration. acknowledgements: the author would like to thank lppm universitas negeri padang for funding this research with a contract number: 1107/un35.15/lt/2023. authors’ contributions: example: yusni atifah & firda azzahra design the research, conducted laboratory work and wrote the manuscript, friska afrilliana and jalilah azizah lubis conducted laboratory research, helendra analized the data. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. references amelia, f., hidayat, b., iryani, i., iswendi, i., yansen, f., & putra, r. f. 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nurfaizah, didik krisdiyanto*, khamidinal and sudarlin department of chemistry, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency*: didik_kris@yahoo.com abstract dye-sensitized solar cells (dsscs) was fabricated using natural dyes extracted from krokot (portulaca oleracea. l). the effect of dye acidity was investigated on natural ph extract, 5.00, 4.00 and 3.00 of ph. the efficiency and stability dsscs as a function of the dye acidity was studied. the result of the uv-vis shows that the absorption of wave-length from dye extract of krokot is located in the visible region with the absorbance peak in 410.5 nm and 664.5 nm which are the peak of chlorophyll. the efficiency of extract krokot dye sensitized solar cells was decreasing 6.88 x 10-3 % to 0.42 x 10-3 % when ph of the dye was adjusted from 6.27 to 3.00. dsscs stability was also decreased look for efficiency loss from 5.27% to 97.49% in the same conditions. keywords: dye sensitized solar cell (dsscs), chlorophyll, efficiency introduction portulaca oleracea l. is a warm-climate, herbaceous succulent annual plant with a cosmopolitan distribution belonging to the portulacaceae family (xi zhou et al., 2015). many constituents of portulaca oleracea have been isolated, including alkaloids, beta-carotene, betasitosterol, caffeic acid, catechol, chlorophyll, coumarins, dha, epa, ferulic acid, flavanoids, saponins and tannin (esiyok et al., 2004), (palaniswamy et al., 2001), (mohamed and hussein, 1994). the green pigmented chlorophyll has the desirable photovoltaic properties that are utilized in today’s organic photosynthetic solar cells. figure 1. the portulaca oleracea l plants. chlorophyll is a light harvesting pigment that absorbs light in the visible spectrum of solar radiation which promotes electron transfer. carotenoids are also an important part of the photosynthetic process. they aid in energy transfer to the chlorophyll molecule,and serve to supplement the light gathering properties of chlorophyll (diarra et al., 1986). appropriate plant species for use in photosynthetic solar cell applications are those with high concentrations of chlorophyll alpha and chlorophyll beta. krokot or purslane (portulaca oleracea) have been found to contain high concentrations of chlorophyll alpha and beta (griffin et al., 2004). the idea of using the reactions of photosynthesis to convert light into electrical power appeared already melvin calvin before 1974 (hug et al., 2013). sun light excited the electrons of pigment into higher energy level then transfered to the conduction band of the wide band gap semiconductor. the model based on a synthetic membrane where carotenoids were used as a wire inside the membrane. after absorption of a light by a sensitizer molecule at one side of the membrane, the electron is transfered to a carotenoid and then diffuse through the membrane the the other side where it is captured by ellectron acceptor. dye-sensitized solar cells (dsscs) are belonging to the third generation photovoltaics concept where used natural dyes as light harvesting pigment (kalyanasundaram and graetzel, 2010; hagfeldt et al., 2010). they are also called graetzel cells (o’regan and graetzel, 1991) the advantages of natural dyes as photosensitizer are large adsorption coefficients, high light-harvesting efficiency, no resource limitations, low cost, easy prepared and no harm to the environment (luo et al., 2009). chlorophyll is the principal pigments in natural photosynthetic system. it is a green pigment found in the 18 biology, medicine, & natural product chemistry 4 (1), 2015: 17-24 leaves of most green plant, algae and cyanobacteria. six different types of chlorophyll pigment exist and the most occuring types is chlorophyll α. the molecular structure include a chlorine ring with mg center, along with different side chains and a hydrocarbon trail depending on the chlorophyll type (ludin et al., 2014). their function include harvesting sunlight, converting solar energy to chemical enery and tranferring electrons. chlorohyll and their derivatives are inserted into dsscs as dye sensitizer because of their beneficial light absorption tendency modes. chlorophyll has an absorption maximum at 670 nm because of an attractive compound that acts as an photosensitizer the visible light range (wang et al. 2010) a dssc consists of a pair of coated glass tco substrate (transparent conducting oxide) as the electrode and the counter electrode, the redox electrolyte that contains iodide and triiodide ion (i /i ) carbon layer as the catalyst, porous tio2 nanocrystal as fotoanoda, and a dye photosensitizer. all components are arranged in front of the sandwich structure where the top layer is the working electrode as the initial layer in receiving photons and the lower layer is the counter electrode and the middle is electrolyte to regenerate electron. dye criteria that can be used as a dye sensitizer is adsorption intensity at visible wavelengths, strong adsorption on the surface of the semiconductor, has the ability to inject electrons to the band conduction of the semiconductor, and has a group = o or -h to bind to the surface of tio2 which can increase the reaction rate of the electron transfer. figure 2. dssc sceme (khuzaifah et al., 2015). therefore, this study will utilize the potential of the natural dye that derived from extracts of krokot which are expected to fulfill the requirement as a natural sensitizer. optical and electrical test are done in order to determine the compliance of the requirement and can be used in dssc system. to further understand the effect of dye acidity was used to investigate the electron transport characteristics of the fabricated cells. this study shows the correlation between efficiency and stability as a function of the dye acidity. experiment chemicals and instrumentation chemicals used for research are krokot, indium transparent oxide (ito), tio2 (degusa), ki, i2, polyethylene glycol, ethanol 96%, polyvinyl alcohol (pva), aquades, graphite pencil 8b and detergent. instrumentation applied for research are sonikator, ultrasonic cleaner, hotplate, glassware, aluminium foil, paper clips, scothlite, screen proyektor (gasket) and cutter. spektrofotometer uv-vis single beam, uv-vis spekular reflektansi uv 1700 pharmaspec, fourier transformation infra-red (ft-ir) shimadzu and i-v meter keithley 2400 source meter. preparation of dye-sensitizer ten grams of krokot powder are macerated with 120 ml of 96% ethanol for 24 hours. then, it is filtered by using vacuum filtration and before it is used for further processing, it should be analyzed first using uv-vis spectrophotometer in the wavelength range 400-700 nm. the effect of ph of dye solution was studied by adjusting cici nurfaizah, et.al. – krokot extract (portulaca oleracea. l) as natural light-harvesting … 19 ph from the original ph of 6.27 using 0.1 m hcl solution to three different phs (3.0, 4.0 and 5.0). preparation of electrodes tio2 powder was weighed as much as 1.5 grams and then inserted into erlenmeyer and added with 3 ml of aquades. then, it is stirred with a magnetite stirring spoon and sonicated with 20 khz frequency for 2 hours. the next solution was then added with polyvinyl alcohol solution which previously has been made from 0.5 grams pva added with 6 ml of aquades by heating at a temperature of 150oc until all of pva are dissolved. the mixing is followed by stirring for 10 minutes until it is formed a homogeneous paste.. then, it is performed tio2 paste deposition on surfaces glass of transparent indium oxide (ito) with doctor blanding technique. but before it, ito should be washed with detergent and followed by aquades using ultrasonic cleaner for 10 minutes and rinsed with ethanol. before tio2 paste is dropped on ito glass, the conductive part should be found and then each of it is given a scotchlite restraint and it is made a rectangular pattern by leaving a 1.8 x 1.3 cm room. furthermore, in above of that fields, the tio2 paste is distributed evenly with a glass rod and then dried in the air and the scotchlite is opened, then it is heated at 80 ° c for 1 hour. tio2 film is inserted into the krokot extract, the container is covered with aluminum foil and then it is saved for an hour. the film which has been soaked then removed and rinsed with ethanol to clean the edge of the layer. then,it is dried at room temperature and analyzed by uv-vis reflectance spectrometer and ft-ir. the graphite of a 8b pencil is spread into the surface of ito on the conductive layer with the shading manner to average carbon layer. then, it is heated at temperature of 300oc for 1 hour. preparation of electrolytes potassium iodide (ki) is weighed as much as 0.815 grams and then dissolved in 10 ml of polyethylene glycol (peg) 400 and stirred until dissolved then added with 0.128 grams of i2 and stirred again until completely mixed. the finished electrolyte solution then stored in the dark bottles and also sealed. assembly of dye-sensitized solar cells (dsscs) dssc fabrication which is used is a sandwich construction with a composition such as: glass-ito working electrode (tio2 layer) that has been coated with dye-screen projector-counter electrode (carbon layer) – ito glass. the use of screen proyector is intended to prevent the short on dssc system. at the ends of the glass that does not stick together is spilled with the electrolyte solution and allowed to seep between the two layers after it is clamped with paperclip (binder clips) on two opposite sides are not coated. measurements dssc prototype was tested by measuring the i-v characteristic curve using a digital multimeter keithley 2400, in the light of a xenon lamp at an intensity of 1000 w / m2.the result of the i-v characteristic curves test were then analyzed voc, isc, fill factor, and the efficiency of solar cells [6] by the equation:= .. (1) = ( ). ( ).. ( ) (2) result and discussion the effect of dye ph on the absorption spectra the result of characterization of krokot extract color absorption spectrum in figure. 3 shows the krokot extract absorbs the blue spectrum (400-450 nm) and red (650700 nm) with peak absorbance is absorbed at λ = 410.5 with a absorbance value of 0.91 abs, λ = 536.5 with a absorbance value of 0.567 abs, λ = 608 with a absorbance value of 0.473 abs and λ = 664.5 with absorbance values of 0.30 abs so that from the great absorbance at a wavelength of 410.5 nm and 664.5 nm can be known that the more dominant krokot extract contains the pigment chlorophyll. figure 3. absorbance of krokot extract. the effect of ph was also investigated in absorbance of krokot extract as a dye sensitizer. as shown in table 1, the ph extract solution has not a significant effect on the absorbance. the absorbance was found the chlorophyll pigment in different ph extract solution of krokot dye. the variation of ph extract did not effect the absorption peak, two maximum peak were detected in 410.5 nm and 664.5 nm. 20 biology, medicine, & natural product chemistry 4 (1), 2015: 17-24 table 1. absorbance of dye sensitizer. ph λ maximum (nm) absorbance (a.u.) 6.27 664.50 0.30 410.50 0.91 5.00 664.00 0.29 410.50 0.90 4.00 664.00 0.30 410.50 0.95 3.00 663.50 0.29 412.00 1.02 figure. 4 showed the absorbance graphic of plot toward different ph of krokot extract as tio2-dye system. the absorbance indicated that tio2-dye system of extract krokot dye had a wide electronic absorbance in visible light region. extract absorbance was shifted into higher wave length or red shift that indicated dye was absorb into tio2 surfaces than the tio2-dye had lower exited state energy. the red shift showed an electron injection from dyes into semiconductor surfaces. figure 4. the graph of absorbance to wavelength of thin layer tio2 – dye in different ph extract dye. table 2. absorbance shift of extract dye. ph λ dye λ tio2-dye shift 6.27 664.50 670.00 5.50 5.00 664.00 669.00 5.00 4.00 664.00 666.00 2.00 3.00 663.50 684.00 20.50 figure 5. the graph of reflectance to wavelength of thin layer tio2 – dye in different ph extract dye absorbance is used to calculate the band gap energy (eg) in the tio2-dye film by tauc plot method. figure 6 showed that dyes acidity increased from 3.28, 3.61, 3.42 and 3.38 ev when the ph of the dyes was adjusted from 6.24, 5.00, 4.00 and 3.00. the band gap was influence the semiconductor performance when apllied into dye sensitized solar cells (dsscs) system. the wide band gap (more than 3.00 ev) absorb high foton energy from sunlight. figure 6. energy gap thin layer tio2-dye. the effect of dye ph on the ftir spectrum infrared absorption spectrum of a material has a distinctive pattern so that it possible to identify the material and also shows the existence of the major functional groups in the identified structure. the bond can be estimated if the ir spectra of tio2-dye system shows the appearance of a significant new peak or functional groups shift if it is compared with the spectra of dye and spectra of tio2 film. krokot extract used in this study contains carboxyl and carbonyl because in the analysis that uses uv-vis is identified to contain chlorophyll so it is possible there is efficient sensitization through the formation of a bond between the dye and tio2. cici nurfaizah, et.al. – krokot extract (portulaca oleracea. l) as natural light-harvesting … 21 figure 7. infrared spectra (a) thin layer tio2, (b) thin layer tio2-dye ph 6.27 (c) thin layer tio2-dye ph 5.00 (d) thin layer tio2-dye ph 4.00 and (e) thin layer tio2-dye ph 3.00. the results of ftir analysis for tio2 film, and tio2dye film of krokot extract is shown in the figure. 4 which shows the existence of the carbonyl absorption at wave number 1635.64 cm-1 and there is absorption at 3410.15 cm-1 region which is the absorption area of hydroxyl groups. carbonyl and hydroxyl group that is owned by krokot extract that can bind to the group of ti (iv) on tio2 (kuzaifah et al 2015). table 3. infrared spectra thin layer tio2-dye. wavenumber (cm-1) groups tio2 tio2-dye ph 6.27 ph 5.00 ph 4.00 ph 3.00 3425.58 3425.58 3448.72 3425.50 3410.00 -oh 2931.80 2931.80 2939.53 2931.80 2932.00 c-h 1635.64 1635.64 1635.64 1635.60 1636.00 c=o 1427.23 1427.57 1381.03 1381.00 1396.00 c-c 1095.57 1095.57 1095.57 1095.60 1096.00 c-o 678.94 678.94 678.94 666.66 678.90 ti-o from ftir analysis is not seen the significant difference from treatment with variation of extract solution acidity. it is only seen the carbonyl absorption shift of in tio2 film is at a wavelength of 1635.64 cm-1 for natural ph shifted to a wave number 1636.00 cm-1 for 3.00 ph of extract krokot dye. this shows that krokot extract does not bind yet optimally with a film of tio2 or the interaction possibility that occurs only physical interaction. the effect of dye ph on the dsscs efficiency a dsscs efficiency can be characterized with an ivdiagram where the corresponding current (i) at rising voltage (v) is plotted. at a bias of 0 v the short circuit current (isc) is measured and when the current 0 v the open circuit voltage (voc) is defined. the maximum power output (pmax) generated by dsscs is reached when the product of the current and the voltage is maximal. how efficient a solar cell can convert the power of the incident light into electricity described by electricity conversion efficiency (η) (hug et al., 2013). an iv-diagram of dsscs with different dye solution ph was showed in figure 8. 22 biology, medicine, & natural product chemistry 4 (1), 2015: 17-24 figure 8. current-voltage curve for krokot extract dye sensitized solar cell in ph 6.27 (a), 5.00 (b), 4.00 (c) and 3.00 (d). table 4 present the performance of dsscs in the term of short-circuit photocurrent (isc) , open circuit voltage (voc), fill factor (ff) and energy convertion efficency (η) (wongcharee et al 2007). the efficiency was found to decrease with decreased ph and had maximum at the natural solution ph. the photoanode made from the krokot extract can absorb more light from chlorophyll pigment. as another pigment, chlorophyll was easy to degradation with heat, light, oxidator and ph (gross et al., 1991). pheophytin, a degradation product of chlorophyll, which represents chlorophyll that has lost the central mg ion replace with h+ in acidic solution that was colorless breakdown products. table 4. photoelectrochemical parameter of the cells sensitized by krokot extract dye. ph isc (m.a.cm-2) . 10-5 voc (mv) ff η (%) . 10-3 6.27 31.00 0.28 0.32 6.88 5.00 12.50 0.36 0.33 3.62 4.00 11.50 0.44 0.33 4.08 3.00 35.00 0.14 0.34 0.42 in this study, the little dssc efficiency which is produced can be caused by the performance of the natural dye used is still low due to the effect of the extract used still contains a lot of pigment with the long structure r which lead steric hindrance of the pigment to the surface band structure so as to prevent oxidation of tio2 molecules bind with tio2 in effective to cause the transfer of electrons from the conduction band to the dye molecule is reduced (khuzaifah et al., 2015). the effect of dye ph on the dsscs stability dye sensitized solar cells with krokot extract stability was also decrease by adjusting the ph of the extract from 6.27 to 3.00. as can be seen from figure 10 efficiency loss of dye sensitized solar cells increased with decreasing ph from 5.27% to 97.49% after irradiated for 2 hours. cici nurfaizah, et.al. – krokot extract (portulaca oleracea. l) as natural light-harvesting … 23 figure 9. current-voltage curve for krokot extract dye sensitized solar cell in ph 6.27 (a), 5.00 (b), 4.00 (c) and 3.00 (d) after irradiated. table 5. photoelectrochemical parameter of the cells sensitized by krokot extract dye after irradiated. ph isc (m.a.cm-2) . 10-5 voc (mv) ff η (%) . 10-3 6.27 28.00 0.26 0.37 6.52 5.00 8.90 0.31 0.45 3.02 4.00 11.00 0.31 0.29 2.41 3.00 0.09 0.06 0.70 0.01 a reason for the worse stability is that, at natural krokot extract acidity the chlorophyll was existed. the pigment are labile and can transformed into the colorless coumpound with decreasing ph. chlorophyll that has lost the central mg ion replace with h+ in acidic solution was degradation into pheophytin. figure 10. stability cells sensitized krokot extract. 0 2 4 6 8 6.27 5 4 3 initial irradiated for 2 hours 24 biology, medicine, & natural product chemistry 4 (1), 2015: 17-24 conclusion the results shows that the absorbance spectrum of the krokot extract dye is stretched in the range of visible light to the maximum absorbance peak at a wavelength of 410.5 nm and 664.5 nm so that can be known that krokot extract dye contains chlorophyll. the efficiency of extract krokot dye sensitized solar cells was decreasing 6.88 x 10-3 % to 0.42 x 10-3 % when ph of the dye was adjusted from 6.27 to 3.00. dsscs stability was also decreased look for efficiency loss from 5.27% to 97.49% in the same conditions. acknowledgment department of chemistry, faculty of science and technology uin sunan kalijaga yogyakarta for academic supported. references esiyok, d. ötles, s. and akcicek e., 2004 “herbs as a food source in turkey,” asian pacific journal of cancer prevention, vol. 5, no. 3, pp. 334–339, yan-xi zhou, hai-liang xin,khalid rahman, su-juan wang, cheng peng, and hong zhang, 2015.,portulaca oleracea l.: a review of phytochemistry and pharmacological effects, biomed research international, u. r. palaniswamy, r. j. mcavoy, and b. b. bible, “stage of harvest and polyunsaturated essential fatty acid concentrations in purslane (portulaca oleraceae) leaves,” journal of agricultural and food chemistry, vol. 49, no. 7, pp. 3490– 3493, 2001 a. i. mohamed and a. s. hussein, “chemical composition of purslane (portulaca oleracea),” plant foods for human nutrition, vol. 45, no. 1, pp. 1–9, 1994 diarra, a. , hotchandan i, s., max j j. and leblanc r. (1986) photovoltaic properties of mixed monolayers of chlorophyll a and carotenoids canthaxanthin, j. cher n . so c. , faraday tran . 2, 8 2 , 2217 2231. griffin, w., quach, h. and steepe r, r. (2004) extraction and thin layer chromatography of chlorophyll a and b from spinach, chern . e d. , 81, 385 – 387 hug, h., bader, m., mair, p., and glatzel, t., 2014, biophotovoltaics : natural pigments in dye-sensitized solar cells., applied energy., 115., 216-225 kalyanasundaram, k., and graetzel, m., 2010, artificial photosynthesis : biomometric approaches to solar energy conversion and storege., curr. opin.biotechnol, 21: 298-310 hagfeldt, a.,boschloo, g., sun, i., and kloo, p., 2010., dyesensitized solar cells., chem. rev.,110, 6 : 595-663 o’regan, b and graetzel, m ., 1991., a low cost high efficiency solar cells based on dye sensitized colloidal tio2 films., nature, 353: 737-740 ludin, n.a.; narosikin, a.a.m.; mahmoud, a.a.; muhammad, a. a.; kadhum a.a.h..; sopian, k.; nor karim, s.a. review on the development of nature dye photosensitizer for dye sensitized solar cell. renewable and suinable energy. 2014, 386-396. wang, x.f., ossamu, k.,eiji, h., haoshen, z.,shinichi, s., hitoshi, t., 2010. tio2 and zno-based solar cells using chlorophyll a derivative sensitized for light-harvesting and energi conversion. journal of photochemistry and photobiology, 145-152 biology, medicine, & natural product chemistry volume 7 – number 2 – 2018 issn 2089-6514 (paper) | issn 2540-9328 (online) contents degradation study of biodegradable plastic using nata de coco as a filler tiara nur elfiana, anisa nur izza fitria, endaruji sedyadi, susy yunita prabawati, irwan nugraha 33 38 comparative anatomy of labyrinth and gill of catfish (clarias gariepinus) (burchell, 1822) and snakehead fish (channa striata) (bloch, 1793) ina karlina, muhammad ja’far luthfi 39 43 diversity of angiospermae plant class liliopsida in mount nglanggeran bayu setya aji nugraha, widodo, rendi yuntara, normalita 45 49 link of nasopharyngeal carcinoma and epstein-barr virus sugiyanto, lina aryati, fajar adi kusumo, mardiah suci hardianti 51 55 alizarin red s-alcian blue staining for regenerated tail of common house gecko (hemidactylus frenatus) rakhmiyati, muhammad ja’far luthfi 57 59 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 627-633 | doi: 10.14421/biomedich.2024.132.627-633 issn 2540-9328 (online) application of taguchi experimental design in optimization of levulinic acid production from cellulose derived from millet stalk shehu ibrahim1,*, abdullahi muhammad sokoto2, kabiru umar jega3, ibrahim muhammad magami4 1department of chemistry, shehu shagari university of education, sokoto, nigeria. 2department of pure and applied chemistry, usmanu danfodiyo university, sokoto, nigeria. 3department of chemistry, federal university birnin-kebbi, nigeria. 4department of biology, usmanu danfodiyo university, sokoto, nigeria. corresponding author* shehuibrahim3030@gmail.com abstract cellulose is the primary component of lignocellulosic biomass and the main source of renewable materials in the chemical industry. this study aims to optimize the production of levulinic acid from cellulose derived from millet stalk using the taguchi experimental design approach. the millet stalks were pretreated by acid and alkali treatment methods. the extractives, hemicellulose, lignin, and cellulose contents of the millet stalks were estimated using the dry-weight basis technique. levulinic acid production from the millet stalk was carried out in teflon lined stainless steel autoclave (50cm3) using standard method. the optimum conditions for the production of levulinic acid were evaluated using taguchi experimental design method. levulinic acid was characterized using gc-ms and ft-ir techniques. the cellulose content of the millet stalk was significantly (p < 0.05) higher than the other components. ft-ir spectrum showed the presence of o-h, c=o, c-h, and c-o stretch in the levulinic acid. the highest (95%) yield of the levulinic acid was obtained with an acid concentration of 0.2m at 2000c, for 4 hours. the optimum temperature, acid concentration, and time for the production of levulinic acid from the millet stalk were 2000c, 0.2m, and 4 hours, respectively. levulinic acid is characterized by a range of functional groups. keywords: cellulose; levulinic acid; millet stalk; optimization; taguchi design. introduction global energy consumption has increased to about 160 × 1018 j annually, leading to a high energy demand worldwide (di-bucchianico et al., 2022; sajid et al., 2021). about 80% of the global annual energy consumption is from fossil fuels (di-bucchianico et al., 2022). excessive use of fossil fuels for energy contributes significantly to greenhouse gas emissions which cause environmental pollution and global warming (epa, 2017). environmental pollution and global warming remain significant problems affecting every sector. lignocellulosic biomass materials are abundantly available worldwide and can be used to produce highvalue energy products. hence, the interest of researchers on the utilization of lignocellulosic biomass for production of chemicals, fuels, and energy has been increased to reduce environmental pollution and global warming for sustainable development. millet is a grain that belongs to the family poaceae grass (rasika et al., 2024). millet is the fourth most cultivated and produced the diet after rice, maize and wheat (moald, 2023). millets have been cultivated in over 130 countries (moald, 2023). many years ago, millets were an integral part of diet of over 50 lakhs across asia and africa mt (moald, 2023). the report shows that in 2023 the estimated global millet market is us$ 10.56 billion and is predicted to reach us$ 17.65 billion by 2032 (mmr, 2024). millets serve as a source of energy, protein, and minerals and about 70% of millets comprise carbohydrates, mainly soluble carbohydrates and dietary fiber (tripathi et al., 2023). millets are vital nutritious foods with many health benefits, including high antioxidant content, significant fiber content, low glycemic index, and gluten-free protein, which are important in managing many diseases (rasika et al., 2024). lignocellulosic biomass is a biomass material widely found in the world that can be utilized for the production of many valuable platform compounds including furfural, lactic acid, formic acid, and levulinic acid (sherwood, 2020; elumaila et al., 2016). the interest of researchers in lignocellulosic biomass has been increasing because of its availability and avoiding food crises (liu & bao, 2017; cheng & brewer, 2017). cellulose is the most abundant natural, renewable, biocompatible, and biodegradable biopolymer found in nature (magalhaes et manuscript received: 01 november, 2024. revision accepted: 22 january, 2025. published: 17 february, 2025. https://doi.org/10.14421/biomedich.2024.132.627-633 628 biology, medicine, & natural product chemistry 13 (2), 2024: 627-633 al., 2023; acharya et al., 2021). cellulose is a polysaccharide that consists of β-d glucoproprenose (1→4) fused through the hydrogen bonds (lei et al., 2019). cellulose is the main component of lignocellulose and the chemical industry’s primary source of renewable materials (klemm et al., 2018). cellulose comprises 30– 50% mass content of lignocellulosic biomass (mukherjee et al., 2015). cellulose has many biological and industrial applications due to its stability, availability, biodegradability, and environmental suitability (awang et al., 2019). levulinic acid is one of the top ten value-added compounds in the world (hayes & becer, 2020). the la global market estimate is about us$22 million with an enormous increase in expectation (bazoti et al., 2023). the nigeria levulinic acid market is expanding due to increased demand for sustainable and bio-based chemicals (nlam, 2024). levulinic acid (4oxopentanoic acid or 4-ketopentanoinc acid) is a linear c5-alkyl carbon chain. levulinic acid is a multifunctional platform chemical obtained from biomass that can be used for the synthesis of a many different potential biobased chemicals including fuel additives, biodegradable herbicides, resins and plasticizers (charnnok & laosiripojana, 2022; santiago & guirardello, 2020). levulinic acid has wide applications in organic synthesis, agriculture, and pharmaceuticals (li et al., 2019). levulinic acid is usually obtained from cellulose using waste and byproducts of lignocellulosic biomass as raw materials (jeong et al., 2018). this process is expensive and has environmental impacts due to the high temperature and pressure required (signoretto et al., 2019). local production of levulinic acid will significantly contribute to the availability and application of levulinic acid in nigeria. this was conducted to optimize the production of levulinic acid from cellulose derived from millet stalk using the taguchi experimental design approach. materials and methods sample material millet stalks were collected in from the giniga community in the wamakko local government area of sokoto state, 13 kilometers from the sokoto metropolis. the millet stalks were washed, air-dried, and then milled into fine particles using a pestle and mortar. the fine particles were sieved using a 40-mesh sieve. the sieved sample was weighed and then stored in a dry place until further analysis. pretreatment of millet stalk the millet stalks were pretreated using acid and alkali treatment methods. acid pretreatment of the sample was carried out to remove the hemicellulose content of the millet stalk using the method of kuhad et al. (2010). two grams of the millet stalk powder was placed in an autoclave followed by adding of 20 cm3 of 3 % sulphuric acid and then heated to 121 0c for 30 minutes. the solution was cooled and then filtered using whatman filter paper. the filtrate was washed with distilled water to a neutral ph and then dried overnight at 50 0c in an oven. alkali pretreatment of the millet stalk was conducted to remove the lignin component according to the method described by jialei et al. (2017). the millet stalk powder (1.5 g) was immersed into a 6% koh aqueous solution with a solid–liquid ratio of 1:20. the contents were loaded into a 50 cm3 teflon lined stainless steel autoclave and then heated at 120 0c for 4 hours. the contents were allowed to cool at room temperature and then the black liquid which consisted of lignin and koh was separated from the mixture by centrifugation. the solution was filtered using whatman filter paper, washed with distilled to neutral ph, and then dried at 80oc for 24 hours in the oven. compositional analysis of the millet stalk the compositional analysis of the pretreated and untreated millet stalk was conducted to determine the percentage levels of extractives, hemicellulose, lignin, and cellulose by dry-weight basis technique. determination of extractives content a cellulose thimble containing millet stalk powder (1 g) was extracted using a soxhlet apparatus and acetone as the solvent as described by blasi et al. (2010). the extraction process was carried out at 70 0c for four hours. the sample was allowed to air dry for 5 minutes at room temperature and then thoroughly dried in the oven for 30 minutes at 105 0c. the sample containing extractive and the extractive-free sample were weighed and the extractives content was obtained by taking the difference between the weights of the samples. determination of hemicellulose content the hemicellulose content of the pretreated and untreated millet stalk was determined using the method of ayeni et al. (2013). the dried extracted sample (0.2 g) was treated in 250 cm3 erlenmeyer flask with 150 cm3 of 5 m naoh. the mixture was heated under reflux for 3.5 hours and then cooled at room temperature. the mixture was filtered using whatman filter paper and then washed with distilled water until a neutral ph obtained. the residue was dried in an oven to a constant weight at 105 0c. the dried sample’s hemicellulose content (%w/w) was obtained by taking the difference between the sample weight before and after treatment. determination of lignin content the lignin content of the pretreated and untreated millet stalk was estimated according to the method described by sluiter et al. (2002). the dried extracted sample (0.1 g) was transferred to glass test tubes and 3 cm3 of 72% h2so4 was added. the sample was kept at room ibrahim et al. – application of taguchi experimental design in optimization … 629 temperature for 2 hours with intermittent shaking at 30 min intervals to allow for complete hydrolysis. after the initial hydrolysis, 40 cm3 of distilled water was added. the second hydrolysis step was carried out in an autoclave for 1 hour at 121 °c. the slurry was cooled at room temperature. the hydrolyzed sample was filtered through a vacuum using a filtering crucible. the acid insoluble lignin was determined by drying the residues at 105 oc. the acid soluble lignin fraction was determined by measuring the absorbance of the acid hydrolyzed samples at 320 nm using a spectrophotometer. the lignin content was calculated as the summation of acid insoluble lignin and acid soluble lignin. determination of cellulose content blasi et al. (2010) described the method for determining the cellulose content of the pretreated and untreated samples. the cellulose content (%w/w) was obtained by difference, using the extractives, hemicellulose, and lignin content of the sample. production of levulinic acid from millet stalk levulinic acid was synthesized from the pretreated millet stalk in a teflon lined stainless steel autoclave (50 cm3) using the method of jialei et al. (2017) with some modifications. the pretreated sample (0.5 g) was transferred into 10 cm3 of 0.2 m h2so4 aqueous solution and then loaded into the reactor. the reactor was placed in a paraffin oil bath closed, stirred at 1000 rpm, and then heated to 170 oc using a hot plate magnetic stirrer. the reactor was kept for 3 hours, and the reaction was observed. the reaction was terminated by quenching the reactor with cold water. the end product was filtered to obtain a clear liquid sample. the synthesized levulinic acid was extracted from the aqueous mixture using a separating funnel and ethyl acetate as solvent. an equal volume of ethyl acetate to the levulinic acid mixture was transferred into a separating funnel, shaken vigorously and then allowed to settle. the anhydrous sodium sulfate (1.0 g) was added to an organic portion of the extract, and the solvent was evaporated by heating the extract at a temperature of 78 oc to obtain dried levulinic acid residue. optimization analysis levulinic acid production was optimized to evaluate optimum conditions for the production of levulinic acid using the taguchi experimental design method described by jialei et al. (2017) with certain modifications. for optimization settings, the effect of temperature, time and acid concentration on the amount of levulinic acid produced was evaluated by comparison minitab statistical software. the experiment was conducted in a teflon lined stainless steel reactor using the pretreated sample as starting material and dilute hcl as catalyst under 70 bar pressure. different range values 170 – 200oc, 2.3 – 4.0 hours, and 0.2 – 0.8 m for temperature, residence time, and acid concentrations were used, respectively. the response was observed at each value and the percentage yield of levulinic acid was obtained. characterization of the produced levulinic acid fourier transforms infrared (ft-ir) analysis levulinic acid was analyzed by fourier transforms infrared (ft-ir) using mb3000 model spectrophotometer operating in the range of 500–4500 cm−1 at a resolution of 4 cm−1. sample drop was sandwiched between two plates of potassium bromide cells and squeezed. a thin film was formed between the plates to enable light to flow through the sample. the plates were placed in the sample holder and positioned in the spectrophotometer's standard sample compartment and then spectral data were obtained. gas chromatography mass spectroscopic (gc-ms) analysis gc-ms analysis of the levulinic acid was performed using 7890b connected agilent technologies mass selective detector (msd) 5977a. the experiment was carried out in a capillary column with a 5190-2293 model number set at 50 0c at a 15 oc/min rate. the program was run at 2600c, hold time of 2 minutes, injection volume of 1 μl, front injector of 10 μl, and beginning temperature set point of 60oc. the equilibration time was 0.5 min at the highest temperature of 325oc. the msd transfer line was kept at 280°c, with a data rate of 50 hz and an injection dispense speed of 6000 μl/min. compound identification and quantization were assessed using total ion count (tic). the isolated compound's spectrum was compared to the known compounds' spectra database kept in the nist02 reference spectral library. results compositional analysis of pretreated and untreated millet stalk figure 1 shows the compositional analysis of pretreated and untreated millet stalks. the cellulose content of the pretreated and untreated millet stalk was significantly (p < 0.05) higher than the other components. the amount of extractive, hemicellulose, and lignin was significantly (p < 0.05) higher in the untreated millet stalk compared to the pretreated millet stalk. however, the pretreated millet stalk contains a more significant (p < 0.05) amount of cellulose compared to the untreated millet stalk (figure 1). 630 biology, medicine, & natural product chemistry 13 (2), 2024: 627-633 figure 1. compositional analysis of pretreated and untreated millet stalk. gc-ms analysis of the produced levulinic acid figure 2 shows the gc-ms spectrum of the levulinic acid produced from the millet stalk (cellulose). the spectrum showed that pentanoic acid or 4-oxo-1-methyl ester has a base peak of m/z 43, a peak area of 9.8%, and a retention time of 15.281 in its mass spectrum and chromatogram, which are obtained from the millet stalk (cellulose) (figure 2). figure 2. gc-ms spectrum of the produced levulinic acid. ft–ir analysis of the produced levulinic acid the ft-ir spectrum of the levulinic acid produced from the millet stalk (cellulose) is shown in figure 3. the spectrum showed various numbers of peaks with arrows indicating different functional groups present in the levulinic acid produced from the millet stalk (cellulose) (figure 3). figure 3. ft–ir analysis of the produced levulinic acid. table 1 shows the ft-ir absorption bands and functional groups in the levulinic acid. the o-h stretching of a carboxylic acid was identified as the cause of the absorption peak at 2701.3495 cm-1. for the carbonyl carbon of a carboxylic acid, the absorption peak at 1710.5051 cm-1 confirmed the presence of a c=o stretch. the ketone group's c=o stretch and the alkanes group's c-h rock were displayed by the peaks at 1679.3212 cm-1 and 1361.5758 cm-1, respectively. the carboxylic acid group's c-o stretch is most likely the cause of the absorption peak at 1269.7859 cm-1 (table 1). table 1. ft-ir absorption bands and functional groups in the produced levulinic acid. frequency (cm-1) bond functional group 2701.3495 o-h stretch -cooh 1679.3212 c=ostretch (ketone) c=o 1710.5051 c=o stretch (carboxylic acid) c=o 1361.5758 c-h rock c-h 1269.7859 c-o stretch -cooh 0 20 40 60 80 extractive cellulose hemi cellulose lignin p e rc e n ta ge (% ) component pretreated untreated ibrahim et al. – application of taguchi experimental design in optimization … 631 effect of the reaction variables on the yield of levulinic acid the effect of the reaction variables on the percentage yield of levulinic acid produced from the millet stalk (cellulose) is shown in table 2. the highest (95 %) yield of the levulinic acid was obtained with an acid concentration of 0.2m at 2000c, for 4 hours. hence, in comparison with other values of the parameters, 0.2m, 2000c, and 4 hours are the optimum values of the acid concentration, temperature, and time for the production of levulinic acid produced from the millet stalk (cellulose), respectively (table 2). table 2. effect of the reaction variables on the yield of levulinic acid. temperature (0c) time (hour) acid concentration (m) levulinic acid yield (%) 170 2:30 0.2 55 170 3:00 0.4 62 170 3:30 0.6 60 170 4:00 0.8 60 180 2:30 0.4 65 180 3:00 0.2 70 180 3:30 0.8 70 180 4:00 0.6 80 190 2:30 0.6 68 190 3:00 0.8 65 190 3:30 0.2 90 190 4:00 0.4 85 200 2:30 0.8 70 200 3:00 0.6 84 200 3:30 0.4 90 200 4:00 0.2 95 discussion in this study, the pretreated millet stalk contain a significant amount of cellulose from which the levulinic acid was produced. the higher cellulose content of the pretreated millet stalk could be attributed to the low extractive, hemicellulose, and lignin content. the result of this study is in agreement with the studies by melesse et al. (2022), rahmayanti and asma (2022), sulaiman et al. (2021), and fadeyi et al. (2020) which showed that the cellulose component was higher in all the biomass samples, compared to the other components including hemicellulose and lignin contents. the gc-ms analysis of the produced levulinic acid revealed the presence of pentanoic acid or 4-oxo-1methyl ester suggesting that the target chemical was generated by the standard data. the present study displayed various peaks and a range of functional groups in the ft–ir spectrum. all the functional groups identified in the ft–ir spectrum are consistent with a study by mukherjee et al. (2015). the o-h stretching of a carboxylic acid, ketone group's c=o stretch and the alkanes group's c-h were displayed by the spectrum. studies by rasheed et al. (2023) rahmayanti and asma (2022), mahmud and anannya (2021), and adeleke et al. (2021) on cellulose from different plant biomass showed similar results with these findings. the carboxylic acid group's c-o stretch was also identified in this study. this finding is in line with the relevant findings which showed the presence of many c–o groups in the cellulose extracted from different biomass (melesse et al., 2022; rahmayanti & asma, 2022; galiwango et al., 2019; nandiyanto et al., 2019). it has been reported that broad absorption bands are attributed to the stretching vibration of inter-molecular and intra-molecular o–h groups indicating the presence of aliphatic moieties in polysaccharides (nandiyanto et al., 2019). in the present study, the highest levulinic acid yield was obtained at a higher temperature (200oc) and low acid concentration (0.2 m) for 4 hours. reaction temperature is an important variable in the production of chemicals from lignocellulosic biomass, which determines the nature of the reaction and end products (yuan et al., 2016). this study is in agreement with the study by sun et al. (2016) which showed that higher temperature is required in producing levulinic acid from lignocellulosic biomass. it has been reported that an increase in acid concentration causes the decomposition of the crystalline portion of the cellulose resulting in less yield of products (henschen et al., 2019; souza et al., 2017). conclusion millet stalks contain a significant amount of cellulose from which the levulinic acid is produced. the optimum temperature, acid concentration, and time for the production of levulinic acid from the millet stalk (cellulose) is 200oc, 0.2m, and 4 hours, respectively. levulinic acid is characterized by a range of functional groups. conflic of interests: the authors declare that there are no conflicts of interests references acharya, s., liyanage, s., parajuli, p., rumi, s. s., shamshina, j. l., & abidi, n. 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(2016). production of levulinic acid from pennisetum alopecuroides in the presence of an acid catalyst. bioresources, 11, 13. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 8, number 1, 2019 | pages: 17-21 | doi: 10.14421/biomedich.2019.81.17-21 issn 2540-9328 (online) a simple method for clearing and staining specimens for the demonstration of animal skeleton muhammad ja’far luthfi1,*, nyoman puniawati soesilo2 1department of biological education, faculty of science & technology uin sunan kalijaga yogyakarta, indonesia 2faculty of biology, universitas gadjah mada yogyakarta, indonesia. author correspondency*: jafarluthfi@yahoo.com abstract teaching skeletal system would be more attractive using real/preserved specimen compared to those using only book. the aim of the research was to develop clearing and staining method of animal skeleton specimen using alizarin red s-alcian blue as teaching aid tool. the specimen were eviscerated, fixed, stained, cleared, and keep in glycerine solution. the spesimen will increase effectiveness in elementary school science teaching and learning. keywords: skeleton; alizarin red s; alcian blue; elementary school; teaching aid introduction skeleton is a supporting system for vertebrate bodies. skeleton serves to give shape and support the body, as a means for movement, as a protector of internal organs and delicate organs (blood vessels and nerve tissue), and as a place for muscle attachment. skeleton is divided into axial skeleton and appendicular skeleton. the axial skeleton consists of skull and vertebrae (spine), while the appendicular skeleton consists of bones in the fore limbs and hind limbs (kardong, 2002). vertebrates are animals that have an endoskeleton and from one of its components the name is taken (vertebra/spine). the skeletal system is one of the subject taught in high school. this material is usually taught by using books or pictures. however, according to prokop et al (2007), teaching skeletal systems will be more attractive if using original specimens or preserved material compared to books. direct observation plays an important role in students' understanding of biological phenomena. students' mental models of a biological phenomenon are strongly influenced by the learning process (prokop et al, 2007). the national science education standards in the usa emphasize that scientific inquiry is the basis of science education, where students at all levels should have the opportunity to conduct scientific inquiry. experiments are not only provide information about an organism's system, but can also develop the ability to observe, think critically and experimentally. students who follow the science process will better understand science concepts, appreciate how science concepts are discovered, and enhance the skills needed to investigate nature (greene & greene, 2001; newman et al., 2004). clearing and staining of the skeleton aims to demonstrate bone structure and cartilage in animals (erdogan et al., 1995; inouye, 1976). this method is commonly used in the field of teratology to study fetal defects. in teratological studies, experimental animals used are rat and mouse embryos. since bone tissue and cartilage in embryos and in adult animals are similar histologically, and so do the small vertebrates and the large ones, theoretically the method for mouse embryos can be applied to demonstrate skeletons in adult animals or in larger vertebrates (junquiera & carniero, 1988; leeson & leeson, 1981). from its ability to demonstrate bone and cartilage structure, this method has the potential to be applied for developing skeletal learning media. the method described in this article is a procedure for clearing and staining animal skeleton specimens using alizarin red s-alcian blue as a tool in teaching the skeletal system. the procedure can be followed by students themselves. materials and methods the animals used in this study were goldfish (cyprinus carpio), oriental whip-snake (ahaetulla prasina), skink (mabouya multifasciata), turtle (trachemys scripta elegans), and flying dragon (draco volans). the equipments used in this study were surgical scissors, scalpels, tweezers, and glass jars. the chemical used in this study were alizarin red s-alcian blue, ethanol, acetone, aquades, glacial acetic acid, glycerin, and koh. the method is a modification of the inouye (1976) originally used for small vertebrates/embryo. inouye is https://doi.org/10.14421/biomedich.2019.81.17-21 mailto:jafarluthfi@yahoo.com 18 biology, medicine, & natural product chemistry 8 (1), 2019: 17-21 one of the first researchers that successfully use the method for bones and cartilage staining. this is a double staining method for bone and cartilage of mice for teratological studies. using this method, cartilage stained blue, bone stained red, whereas muscles and other tissues become transparent. preparations for animal larger than mice and not embryos will need modification of a more massive skin and muscle removal, additional fixation time, fat removal, and extensive clearing and staining due to the thickness and robustness of tissue of mature and larger vertebrates. clearing and staining specimens (carp, snake, gecko tail, turtle tail, flying dragon) include several stages. the first step is exfoliation. after the scales were removed, the specimens were fixed in a tube containing 95% alcohol for 3 days. then the skin and muscles in the specimen are removed using scissors, tweezers and scalpel. then fixed again in 95% new ethanol for 3 days. to remove fat, the fixed specimen was immersed in a tube containing acetone for 4 days with the replacement of new acetone on the second day. after that the specimens were immersed for 5 days in a dye solution at 37 ° c (1 volume 0.3% alcian blue in ethanol 70% + 1 volume 0.1% alizarin red s in 95% ethanol + 1 volume glacial acetic acid + 17 volume ethanol 70%). the specimens were washed in water and then immersed in a solution of 1% koh for 5 days (to make it transparent) and then soaked in succession of a mixture of glycerin and 1% koh at a ratio of 20%: 80%; 50%: 50%; 80%: 20%. finally, the specimen were stored in pure glycerin. results and discussion this research has successfully develop method for clearing and staining animal skeleton specimens with alizarin red s-alcian blue (figure 1, figure 2, figure 3, figure 4 and figure 5). figure 1 shows a carp skeleton. the preparation shows the bones of the skull, vertebrae (backbone), and the ray fin of tail of fish that are suitable for live in water. figure 2 is skeleton specimen of oriental whipsnake. the snake specimens show a special adaptation of vertebrate skeletons, that is, very large numbers of vertebrae (+ 300 vertebrae) suitable for terrestrial life without limbs. figure 3 shows a skink's tail skeleton. the preparation shows the structure of the tail’s vertebrae that can autotomized. figure 4 shows a turtle's tail skeleton. figure 5 shows the skeleton of flying dragon. the turtle and flying dragon specimens show the structure of the tail vertebra in animals that cannot be autotomized. using these specimens, students can directly observe the structure of the real specimen, can figure out its morphology, know the difference in structure related to its function, and gain direct experience about the skeleton. students can more easily understand the way of life and movement of snakes and the appearance of their elongated morphology by looking at the structure of the snake's vertebrae and its enormous number of segments compared to fish. likewise, students will be able to understand the relationship between structure and function, where an autotomized tail has a fracture plane in its vertebrae while animals that cannot autotomy do not have a fracture plane in their tail vertebrae. according to eshach & fried (2005), in science learning real specimens have advantages compared to sketches, drawings, or books. real specimens will attract more students. in addition, original specimens have the advantage of 3-dimensional detail that is not possessed by sketches or photographs. while skeleton props often do not have the details according to the original preparation. figure 1. structure of a carp skeleton. visible skull bones, spine, and rays of fish tails. (a) tail rays; (b) vertebra. figure 2. (a) complete view of snake skeleton. (b) higher magnification. skeletal structure of an oriental whip-snake. there are skull bones, vertebrae with very large amounts of ribs. note the spine with ribs. (a. ribs; b. vertebra). b a b a luthfi & soesilo – a simple method for clearing and staining specimens for … 19 figure 3. (a) skink's tail skeleton structure. visible vertebrae/tail bones with their processes. note that there are fracture plane in the bone where the tail breaks off during an autotomy. (b) skeleton of regenerated skink tail. after autotomy, skink will develep regenerated tail which the cartilaginous tube (stained blue) supporting tail instead of bony vertebrae. (a. neural spine; b. centrum; c. fracture plane; d. intervertebral pad; e. chevron bone; f. vertebra; g. cartilaginous tube). figure 4. high magnification of the turtle tail skeleton structure. the tail consists of a series of vertebrae with their processes. the turtle's tail cannot autotomy and therefore the turtle's tail vertebrae do not have a fracture plane. (a. centrum; b. chevron bone). figure 5. high magnification of the flying dragon tail skeleton structure. the tail consists of a series of vertebrae with their processes. the flying dragon tail cannot autotomy and therefore do not have a fracture plane. (a). dorsal view. (b). ventral view. (c). lateral view. (a. neural spin; b. tranverse process; c. centrum; d. chevron bone). the results show clearly visible bone structure in goldfish, oriental whip-snake, gecko tails, flying dragon tail and turtle tail. the bones stained red while the cartilage stained blue. muscle and connective tissue become transparent/clear by the clearing process using koh. in these preparations the details of the skeleton can also be observed such as the processus of the vertebrae, intervertebral discs, ribs, fracture plane, and other details. figure 1 shows a fish's skeleton. students can observe the presence of skull bones in fish, vertebrae, and fin rays of fish. from these specimens, it is known that the fin rays of the fish are composed of bone material. from the snake preparation (figure 2) we can observe the structure of the snake vertebrae that has ribs on its body vertebrae. only in the tail, the snake vertebrae do not have ribs. the ribs serve as muscles attachment and help the movement of snakes. the skink tail (figure 3), turtle tail (figure 4) and flying lizard tail (figure 5) provide structures that are ideal for studying skeleton anatomy, and to some extend student can learn the relationship between form and function. skink's tail can autotomized its tail, whereas the tail of a turtle and flying lizard cannot autotomy. this functional difference is also reflected by differences in structure, namely the existence of fracture plane in the skink tail vertebrae. it is on these fracture planes that the tail can break. this autotomy ability is a form of gecko's self-defense to avoid predator. turtles and flying lizard cannot autotomized its tail so that it can be understood if there are no fracture plain in the vertebrae. adapting its way of life, the tail of the flying lizard is needed to help the movement on the tree and when it gliding. therefore, breaking the tail would certainly be less profitable. the same is true for turtles. turtles have very hard carapace and plastrons. if there is a threat or an enemy, the turtle g f e b a d c b a d cba b 20 biology, medicine, & natural product chemistry 8 (1), 2019: 17-21 will insert its head and motion apparatus into the carapace and plastron. learning in this way will be more interesting for students. this bone specimen makes it easier for students to understand skeletal structure. understanding the anatomical structure will facilitate the understanding of its function. students will be able to observe the components of the skeleton, the skeleton differences in its way of life, the skeleton differences between animal classes, and the relationship between structure and function (a given structure reflects certain function). in addition, using real specimens will be more interesting for students related to their 3-dimensional shapes, attractive colors, and direct experience of seeing original specimens from a visual, tactile and motoric perspective. appleton (2008) stated that often school teachers are hesitant in teaching science. the main reason is the limited knowledge of teachers about science content. this paper provides a teaching of animal skeleton by directly involving teachers and students in the science process so that they can see directly the science process. thus the confidence of teachers and students will arise to learn science. matthews et al (1997) stated that students have a high curiosity about the world around them, and nature offers a variety of materials in which the teacher becomes a mentor to help students to satisfy their curiosity by studying the natural surroundings. this study used local animals that are widely found around us, namely, goldfish/carp (cyprinus carpio), oriental whip-snake (ahaetulla prasina), gecko (gecko gecko), turtle (trachemys scripta elegans), and flying dragon. this certainly makes it easier for teachers to develop science learning media in accordance with school availability and abilities. this is in accordance with the opinion of matthews et al (1997) which stated that nature provides the appropriate material to satisfy students' curiosity that is so high towards the natural surroundings. according to eshach & fried (2005), the superiority of the real specimens for learning as mentioned above is very suitable for children's learning. children (including elementary school students) have a high curiosity. this character is very suitable for science considering that one of the factors driving science is curiosity. children really like to observe and think about the environment. exposing scientific processes to young persons will develop positive attitudes towards science (eshach & fried, 2005; appleton, 2008). greene et al (1993) stated that real specimens are very important in many aspects of cognitive learning in the classroom. in this study the most important aspect in the success of staining is skin and muscle removal, and the stages of fixation. the skin and muscles must be removed as much as possible so that only a little muscle remains for the attachment of each bones. but it should be noted that skin and muscle removal should not cut the bone/skeleton itself. the vertebrae have many processes. often during the removal of skin and muscles, the process of the vertebrae is unintentionally cut off. this will certainly change the vertebral morphology caused by the artifact, as a result of error in cutting. fixation and fat removal were done longer than the inouye method, because the structure of the skin and muscles of the mature animals are thicker and stronger than the embryo so that the fixative solution takes longer to penetrate the tissue. different specimens require different time periods for fixation, fat removal, clearing and staining. this depends on the species, maturity (embryo or adult), body size, and organs/body parts made for preparations. in general, larger and more mature animals require a longer period of fixation, fat removal, clearing and staining. the length of time for fixation, fat removal, clearing and staining in this study can be used as a guide in staining for other animals. this research has succeeded in developing a skeleton (bone and cartilage) staining method for learning skeleton systems for high schools. learning in this way will involve the teacher and students in the science process. in addition, the use of real specimens will make it easy to understand the skeleton stucture and more attractive to students. the process and materials needed in this study are very simple, and can be applied to other local animals that are available according to the availability in the environment around the school. conclusions the study of organisms including the skeletal structure is an important part of the basic education science curriculum. the use of pictures, sketches, visual aids, and books to explain the skeletal structure is a common method used in the teaching of skeletal systems. however, this method often causes less student interest. the skeleton system is also more difficult to understand with the use of instructional media. the author provides alternative for high school teachers in teaching the skeletal system. making skeletal preparations by involving students is not commonly carry out in the learning of primary school framework systems, but teachers can combine them with the existing curriculum to expand the scope of basic education. acknowledments the authors would like to thank the assistants of animal anatomy lab work at the faculty of science and technology of uin sunan kalijaga, members of the apprenticeship program at the faculty of science and technology of uin sunan kalijaga, and members of the center for integrative zoology of uin sunan kalijaga for their role to make this research possible. the authors also would like to thank to mr. riyanto (staff of the faculty of science of technology) for his assistance in pictures processing. luthfi & soesilo – a simple method for clearing and staining specimens for … 21 references appleton, k. 2008. developing science pedagogical content knowledge through mentoring elementary teachers. j sci teacher educ 19: 523–545. erdogan, d., kadioglu, d., peker, t. 1995. visualization of the fetal skeletal system by double staining with alizarin red and alcian blue. gazi medical journal 6: 55-58. eshach, h., fried, m. n. (2005). should science betaught in early childhood? journal of science education and technology, 14: 315–336. greene, j.s., b.d. greene. 2001. using amphibians and reptiles to learn the process of science. science activities 37 (4): 2932. greene, e. a., k. r. smith, j. s. pendergraft, r. h. raub, and, m. j. arns. 1993. technical note: equine skeletal preservation techniques to enhance teaching effectiveness’. journal of animal science. 71:2270-2274. inouye, m. 1976. differential staining of cartilage and bone in mouse skeleton by alcian blue and alizarin red s. congenital anomalies 16: 171-173. junquiera, l.c. & carneiro, j. 1988. histologi dasar. terjemahan. edisi ke-3. egc penerbit buku kedokteran. jakarta. kardong, k. v. 2002. vertebrates comparative anatomy, function, and evolution. international edition. third edition. mcgraw-hill higher education. new york. usa. hal: 233236. leeson, t. s., & c.r. leeson. 1981. histology. fourth edition. w.b. saunders company. philadelphia. 514-534. matthews, r.w., l. r. flage, and j. r. matthews. 1997. insects as teaching tools in primary and secondary education. annual reviews of entomology 42:269–89. newman, w.j., s. k. abell, p.d. hubbard, j. mcdonald, j. otaala, m. martini. 2004. dilemmas of teaching inquiry in elementary science methods. journal of science teacher education 15(4): 257–279. prokop, p., m. prokop, s.d. tunnicliffe, c. diran. 2007. children’s ideas of animals’internal structures. jbe 41 (2): 62-67 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 905-909 | doi: 10.14421/biomedich.2025.142.905-909 issn 2540-9328 (online) synthesis, characterization, and micellar behavior of amphiphilic chitosan bearing sulfate and anisaldehyde imine groups herayati1*, deana wahyuningrum2, damar nurwahyu bima3, indah puspita sari1 1department of cosmetic engineering, faculty of industrial technology, institut teknologi sumatera, jl. terusan ryacudu, way huwi, lampung, 35365, indonesia. 2organic chemistry research group, institut teknologi bandung, jl. ganesha 10, bandung, 40132, indonesia. 3department of chemistry, diponegoro university, tembalang, semarang, 50275, indonesia. corresponding author* herayati@km.itera.ac.id manuscript received: 05 august, 2025. revision accepted: 14 october, 2025. published: 24 october, 2025. abstract amphiphilic chitosan derivatives bearing sulfate and anisaldehyde imine (anisimine) groups were successfully synthesized and characterized for potential applications as functional biomaterials. the synthesis involved two key steps: (1) sulfation of chitosan using chlorosulfonic acid to introduce o-sulfate groups, and (2) schiff base formation via reaction with p-anisaldehyde to generate n-anisimine functionalities. structural modifications were confirmed by fourier-transform infrared (ftir) spectroscopy, which showed characteristic absorption bands at ~1250 cm⁻¹ and ~820 cm⁻¹ corresponding to o=s=o stretching of sulfate, and a c=n stretch at ~1640 cm⁻¹ indicating imine formation. proton nuclear magnetic resonance (1h-nmr) spectra further verified the successful attachment of aromatic protons from p-anisaldehyde and the disappearance of primary amine peaks, confirming imination. the amphiphilic behavior and micelle-forming ability of the n-anisimine-o-sulfated (naos) chitosan were evaluated using pyrene as a hydrophobic fluorescent probe for determining the critical micelle concentration (cmc), which was found to be 0.012 mg/ml. the observed low cmc value indicates strong selfassembly capability in aqueous media. these results suggest that naos chitosan possesses promising structural and surface-active properties suitable for advanced applications in drug delivery and cosmetic formulations. keywords: amphiphilic polymers; chitosan derivatives; delivery systems; schiff base. introduction chitosan, a natural polysaccharide derived from chitin, has garnered significant attention due to its biocompatibility, biodegradability, and versatile chemical functionality. its primary amino and hydroxyl groups offer extensive opportunities for chemical modifications, enabling the development of tailored materials for applications in drug delivery, cosmetics, and tissue engineering. however, the inherent hydrophilicity of chitosan often limits its ability to interact effectively with hydrophobic compounds, thereby restricting its use in systems that require amphiphilic properties. the introduction of amphiphilic functionalities to chitosan has emerged as a promising strategy to overcome these limitations. amphiphilic chitosan derivatives, capable of forming self-assembled structures such as micelles or nanoparticles, have been extensively investigated in recent years. for instance, sharma et al. (2017) synthesized amphiphilic chitosan derivatives by grafting stearic acid onto the chitosan backbone, demonstrating their ability to form nanomicelles to deliver nonviral gene (sharma & singh, 2017). similarly, pol et al. (2022) reported the preparation of amphiphilic chitosan modified with palmitoyl and quarternary ammonium moities, demonstrating its ability to encapsulate plasmid dna (pol et al., 2022). in addition to hydrophobic modifications, the introduction of hydrophilic groups such as sulfate or carboxymethyl functionalities further enhances the versatility of chitosan. dimassi et al. (2018) explored sulfated chitosan derivatives and highlighted their improved biocompatibility and ionic characteristics, making them suitable for biomedical applications (dimassi et al., 2018). moreover, amphiphilic chitosan bearing both hydrophobic and hydrophilic modifications has shown exceptional potential in forming stable emulsions and hydrogels for cosmetic and pharmaceutical formulations (lazaridou et al., 2025). among hydrophobic modifications, aromatic groups such as anisimine offer unique advantages due to their ability to enhance the interaction of material with hydrophobic drugs and aromatic compounds through π-π interactions which is an essential driving force for selfassembly (gao et al., 2023). sulfation, on the other hand, introduces negatively charged sulfate groups that https://doi.org/10.14421/biomedich.2025.142.905-909 906 biology, medicine, & natural product chemistry 14 (2), 2025: 905-909 improve water solubility and provide ionic functionality. combining these modifications creates a synergistic effect, enhancing the amphiphilic nature of chitosan and broadening its application potential. in this study, novel amphiphilic chitosan derivatives functionalized with anisimine and sulfate groups were synthesized and characterized. their structural features and amphiphilic properties were thoroughly evaluated. this work aims to elucidate the potential of such dualfunctionalized chitosan derivatives for advanced applications in drug or cosmetic delivery systems. materials and methods materials commercially available chemicals and solvents from merck were utilized in this research for their specific roles and high purity. chitosan was derived from shrimp shells (litopenaeus vannamei). chlorosulfonic acid (hclso₃, merck, germany) used as the sulfonation agent. anisaldehyde (merck, germany) was utilized for synthesizing amphiphilic chitosan derivatives. additional materials included ethanol (merck, germany), glacial acetic acid (merck, germany), deuterated oxide (merck, germany), trifluoroacetic acid (acros organics, usa), pyrene, dialysis membranes with molecular weight cutoff (mwco) 12,000–14,000 (sigma-aldrich, usa), and cellulose-acetate membrane filters (0.45 μm, merck millipore, ireland). these were used for critical micelle concentration determination. synthesis of chitosan chitosan was prepared from shrimp shell waste through a multistep chemical process involving demineralization, deproteinization, and deacetylation, following a modified procedure based on zvezdova (2010) and suendo et al. (2010). initially, shrimp shells were thoroughly cleaned to remove residual organic matter, then washed, dried, and milled into a fine powder (particle size ~0.3–0.5 mm) (zvezdova, 2010) (suendo et al., 2010). the demineralization process was conducted by soaking the powdered shells in 7% hydrochloric acid (hcl) at room temperature using a solid-to-liquid ratio of 1:10 (w/v) for 24 hours to dissolve calcium carbonate and other mineral components. the sample was then filtered, washed repeatedly with distilled water until neutral ph was achieved, and subsequently dried. the deproteinization step involved treating the demineralized residue with 10% sodium hydroxide (naoh) at 60 °c for 24 hours to remove proteins. after thorough washing and drying, the resulting material (chitin) was subjected to deacetylation using 50% naoh at 60 °c for 8 hours to convert it into chitosan. synthesis of chitosan preparation of o-sulfate chitosan chitosan (1 g, degree of deacetylation (dd) 88.6%, molecular weight (mw) 3300 kda was reacted with chlorosulfonic acid (4 ml) in distilled water (30 ml) at room temperature overnight. the reaction mixture was neutralized using a 20% (w/v) naoh solution to achieve a ph of 7. the neutralized solution was dialyzed against deionized water using a dialysis membrane with a molecular weight cutoff (mwco 12,000–14,000) for two days, followed by lyophilization an additional two days to obtain a yellow powder. preparation of n-anisimine -o-sulfate chitosan o-sulfate chitosan (0.5 g) was reacted with panisaldehyde (30 μl) in dimethylformamide (dmf, 1 ml) at a molar ratio of 1:1 and 1:3 (o-sulfate chitosan: p-anisaldehyde). the reaction was carried out at 78℃ for 4 hours. the resulting heterogeneous mixture was filtered, and the precipitate was washed with methanol. the product was dried under vacuum at room temperature overnight to yield a fine powder. structural characterization the chemical structures of chitosan and its derivatives were confirmed using the following methods. the chemical structure of chitosan and chitosan derivatives was confirmed with atr-ftir, 1h-nmr and 13cnmr. atr-ftir analysis was performed on a nicolet 6700 ftir spectrometer. 1h-nmr and 13c-nmr were performed on a bruker 400 mhz spectrometer. chitosan, o-sulfate-chitosan, n-anisimine-o-sulfate-chitosan were dissolved in the mixed solvent of d2o and cf3cooh. the degree of substitution (ds) for the derivatives was calculated using the integration of nmr peaks (jaidee et al., 2012): %ds of anisaldehyde = (∫ 𝐻7.0+𝐻7.78+𝐻9.63) 5 ∫ 𝐻3,05 determination of critical micelle concentration the cmc of chitosan derivative was determined using pyrene as a hydrophobic probe (aguiar et al., 2003). a solution of pyrene in acetone (6 × 10⁻⁵ m) was prepared, and 10 μl was added to eppendorf tubes. the acetone was evaporated before adding 1 ml of chitosan derivative solutions (concentrations ranging from 0.01 to 0.6 mg/ml). the solutions were sonicated at room temperature for 30 minutes and equilibrated at 65℃ for 3 hours. fluorescence emission spectra (350–450 nm) were recorded using an excitation wavelength of 336 nm on a fluorescence spectrophotometer. both excitation and emission bandwidths were set at 5 nm. the cmc values were calculated based on the pyrene emission spectra. herayati et al. – synthesis, characterization, and micellar behavior of … 907 results and discussion the result of ir and 1h-nmr analysis 4000 3500 3000 2500 2000 1500 1000 c=c aromatik (c-oso 3 ) o-s (c-oso 3 ) c-o so 2 t ra n sm it an ce ( a. u .) wavenumber (cm -1 ) c=n a b c figure 1. ftir and 1h-nmr spectra of chitosan (a), o-sulfate-chitosan (b) and naos-chitosan (ds 96% anisimine (c) and ds 49% anisimine (d)) table 1. degree of substitution of amphiphilic chitosan derivatives. mol ratio of cho : nh2 integration value ds(%) h3.05 h7.08 h7.81 h9.65 3 : 1 2.49 4.84 4.95 2.16 96 1 : 1 2.22 2.05 2.28 1.11 49 the result of micelar behaviour -1,8 -1,6 -1,4 -1,2 -1,0 -0,8 -0,6 -0,4 -0,2 0,0 0,8 1,0 1,2 1,4 1,6 i 3 8 4 /i 3 7 2 log c figure 2. intensity ratio plots of i384/i372 versus log c for naoschitosan (96% degree of substitution) in water discussion the synthesis of the novel amphiphilic chitosan derivatives bearing sulfate and anisimine functional groups were achieved through a stepwise reaction pathway, as depicted in the reaction scheme (figure 3). the first step involved the sulfation of chitosan using chlorosulfonic acid, which selectively reacted with the hydroxyl groups of the glucosamine units in the chitosan backbone. the introduction of sulfate groups (–oso₃⁻) into the polymer structure significantly enhanced its hydrophilic properties, making the modified chitosan more soluble and reactive in aqueous environments. the presence of 20% sodium hydroxide (naoh) played a crucial role in this step, acting to neutralize the reaction mixture and terminate the sulfation process. by reacting with excess chlorosulfonic acid, naoh prevented over-sulfation and degradation of the chitosan backbone (diab et al., 2012), ensuring the preservation of the polymer's structural integrity. this controlled reaction allowed the incorporation of sulfate groups while maintaining the desirable physical and chemical characteristics of chitosan. this sulfated intermediate served as a critical precursor for the subsequent functionalization step, providing the necessary hydrophilic balance to support amphiphilic behavior after the introduction of hydrophobic groups. in the subsequent step, the sulfated chitosan was subjected to schiff base formation by reacting with panisaldehyde in a dimethylformamide (dmf). this reaction facilitated the covalent attachment of the aromatic imine group via condensation between the primary amine groups of chitosan and the aldehyde functionality of p-anisaldehyde, yielding the desired imine bond (c=n). the aromatic group from panisaldehyde introduced hydrophobicity, resulting in an amphiphilic structure. 908 biology, medicine, & natural product chemistry 14 (2), 2025: 905-909 figure 3. reaction scheme of synthesis of naos-chitosan the fourier-transform infrared (ftir) spectra (figure 1) provide critical evidence supporting the structural transformations at each step of the reaction. the characteristic absorption bands of chitosan were observed at 3400–3200 cm⁻¹, representing o–h and n–h stretching vibrations, and at ~1600 cm⁻¹, corresponding to n–h bending vibrations. the polysaccharide backbone was further identified through the c–o stretching peaks at ~1070 and ~1020 cm⁻¹. the introduction of sulfate groups was confirmed by the appearance of new absorption bands at ~1250 cm⁻¹ and ~1025 cm⁻¹, assigned to s=o and c–o–s stretching vibrations, respectively. the reduced intensity of the o–h stretching band supports the substitution of hydroxyl groups by sulfate groups. the imination step was evidenced by the emergence of a new absorption band at ~1650 cm⁻¹, attributed to the c=n stretching vibration of the imine bond. additionally, the presence of an aromatic c=c stretching band at ~1500 cm⁻¹ confirms the incorporation of the aromatic p-anisaldehyde group. importantly, the sulfate-specific bands at ~1250 cm⁻¹ and ~1025 cm⁻¹ remain visible, indicating the preservation of sulfation after schiff base formation. the structural changes were further validated by ¹h nmr spectroscopy, which provided insight into the chemical environment of the protons in the chitosan derivative. signals corresponding to the glucosamine ring protons were identified, with h-1 appearing at ~5.0 ppm and the other protons (h-2 to h-6) resonating between 3.0–4.0 ppm. the sulfation step induced deshielding effects, resulting in the appearance of new signals at ~3.5 ppm, corresponding to protons adjacent to the introduced sulfate groups. the imine proton (c=n–h) was detected as a distinct signal at ~8.0–9.0 ppm, confirming the formation of the imine bond. the aromatic protons of the p-anisaldehyde group were observed as multiplets in the region of ~6.5–7.5 ppm, consistent with the structure of the attached aromatic group. the nmr spectrum of the final product showed overlapping signals from both the sulfation and imination steps. the retention of sulfate-specific peaks and the presence of the aromatic imine signals confirm the successful synthesis of the amphiphilic chitosan derivative. the degree of substitution (ds) of chitosan derivatives was calculated using the integration of nmr peaks as shown in equation (1). the equation quantifies the degree to which anisaldehyde groups have been grafted or chemically attached to the chitosan backbone. the numerator sums up the integral values of anisaldehyde-specific protons, indicating the amount of anisaldehyde bound to the chitosan. the denominator normalizes this value based on the reference signal of the chitosan backbone, ensuring that the calculation accounts for the number of available reactive sites (proton equivalent). the table shows the degree of substitution (ds) results for a chitosan derivative substituted with anisaldehyde. the results are presented for two different molar ratios of aldehyde to amino groups in chitosan. the data clearly show that increasing the molar ratio of anisaldehyde to amino groups enhances the degree of substitution, as evidenced by the higher ds value for the 3:1 ratio compared to the 1:1 ratio. this suggests that the availability of anisaldehyde molecules relative to reactive sites on chitosan is a critical factor in determining the extent of substitution. the ftir and nmr spectra provide conclusive evidence of the successful sequential modification of chitosan. the structural features introduced during sulfation and schiff base formation are clearly reflected in the spectral data, supporting the chemical integrity of the final product. the presence of both hydrophilic sulfate and hydrophobic aromatic groups substantiates the amphiphilic nature of the synthesized derivative. the amphiphilic behavior of naos-chitosan was evaluated by determining its critical micelle concentration (cmc), as shown in figure 2. the cmc is the concentration at which amphiphilic molecules begin to self-assemble into micelles, a key property for applications in encapsulation and delivery systems. the graph for naos-chitosan shows a gradual increase in the intensity ratio (i338/i372) as the concentration increases. the intensity ratio remains constant at approximately 1.0. this suggests that naos-chitosan molecules do not aggregate significantly in this region. the ability of naos-chitosan to exhibit a sharp transition in intensity ratio confirms its amphiphilic nature. as concentration increases, hydrophobic interactions drive the formation of self-assembled structures. this trend indicates the transition from individual amphiphilic molecules to micelle formation. the distinct plateau in intensity ratio herayati et al. – synthesis, characterization, and micellar behavior of … 909 at higher concentrations suggests the stability of these self-assembled structures, which is a hallmark of amphiphilic compounds. the critical micelle concentration (cmc) of approximately 0.1 mg/ml indicates promising potential for naos-chitosan as a material in cosmetic or drug delivery applications. a low cmc indicates that micelle formation occurs at a relatively low concentration. the amphiphilic behaviour of naos-chitosan (hydrophilic chitosan backbone and hydrophobic anisaldehyde modification) makes it ideal for encapsulating hydrophobic active ingredients (e.g., drugs, vitamins, essential oils) within micelles. conclusions in this study, amphiphilic chitosan derivatives bearing sulfate and aromatic imine groups were successfully synthesized through a two-step functionalization process. the sulfation of chitosan and subsequent schiff base formation with p-anisaldehyde were confirmed by ftir and 1h nmr spectroscopy, which provided clear evidence of the incorporation of sulfate and aromatic imine (c=n) groups. these structural modifications enhanced the amphiphilic nature of the chitosan derivatives. the critical micelle concentration (cmc) study demonstrated that naos-chitosan forms micelles at low concentrations. this micellar behavior highlights the potential of the synthesized derivatives for encapsulating hydrophobic molecules, making them suitable for applications in drug or cosmetic delivery. acknowledgements: this research is supported by direktorat jendral pendidikan tinggi, riset, dan teknologi under research grant: luaran wajib hibah kemendiktisaintek skema penelitian dosen pemula (pdp) tahun 2025 no. kontrak 1483bc/it9.2.1/pt.01.03/2025. authors’ contributions: herayati & deana wahyuningrum designed the study. herayati carried out the laboratory work. damar & indah analyzed the data. herayati wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. references aguiar, j., carpena, p., molina-bolı́var, j. a., & carnero ruiz, c. 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(2012). &lt;sup&gt;1&lt;/sup&gt;h-nmr analysis of degree of substitution in n,o-carboxymethyl chitosans from various chitosan sources and types. advanced materials research, 506, 158–161. https://doi.org/10.4028/www.scientific.net/amr.506.158 lazaridou, m., moroni, s., klonos, p., kyritsis, a., bikiaris, d. n., & lamprou, d. a. (2025). 3d-printed hydrogels based on amphiphilic chitosan derivative loaded with levofloxacin for wound healing applications. international journal of polymeric materials and polymeric biomaterials, 74(2), 67– 84. https://doi.org/10.1080/00914037.2024.2314610 pol, t., chonkaew, w., hocharoen, l., niamnont, n., butkhot, n., roshorm, y. m., kiatkamjornwong, s., hoven, v. p., & pratumyot, k. (2022). amphiphilic chitosan bearing double palmitoyl chains and quaternary ammonium moieties as a nanocarrier for plasmid dna. acs omega, 7(12), 10056– 10068. https://doi.org/10.1021/acsomega.1c06101 sharma, d., & singh, j. (2017). synthesis and characterization of fatty acid grafted chitosan polymer and their nanomicelles for nonviral gene delivery applications hhs public access. bioconjug chem, 28(11), 2772–2783. https://doi.org/10.1021/acs.bioconj-chem.7b00505 suendo, v., ahmad, l. o., & valiyaveetiil, s. (2010). deasetilasi kitin secara bertahap dan pengaruhnya terhadap derajat deasetilasi serta massa molekul kitosan. jurnal kimia indonesia, 5(1), 17–21. zvezdova, d. (2010). synthesis and characterization of chitosan from marine sources in black sea. scientific works of the rousse university by bulgaria, 49, 65–69. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 2, 2017 | pages: 37-45 | doi: 10.14421/biomedich.2017.62.37-45 issn 2540-9328 (online) the female population growth projection year 2021 in trenggalek regency by leslie matrix model on the birth rate and life expectancy dewi anggreini mathematics education department stkip pgri tulungagung, jl. mayor sujadi east street, no.7, tulungagung, 66221, indonesia author correspondency: anggreini_004@yahoo.com abstract this research aims to determine the number of female residents in trenggalek regency in 2021 based on data on birth rate and life expectancy. the use of eigenvalues and eigenvectors aims to determine the dividing age distribution by leslie matrix model. the eigenvectors are used to determine the number of female populations of each age interval, while the eigenvalues are used to determine population growth rates. the research method used is to determine the subject of research. the next stage is to collect research data, then analyze the data and last draw conclusions. the research data is obtained from bps kabupaten trenggalek and bps east java province that is data of woman population from year 2010-2015. the result of this research using leslie matrix model for female population in trenggalek regency that is discrete model. the discrete model is divided into fourteen age intervals constructed using the birthrate and life expectancy. the conclusions of the study showed that the number of female population in trenggalek regency tended to increase with positive eigen value greater than one. in other words, the growth rate of female population in trenggalek regency tends to be positive. the success of leslie's matrix model is the application of case studies in predicting the number of female populations in trenggalek district by 2021 using the maple 16 program. keywords: eigen value; eigenvector; leslie matrix. introduction trenggalek regency is a regency in east java province which is located in the southern part of the province of east java with an area of 1,261.40 km². trenggalek regency largely made up of mountainous land with an area of 2/3 covers part of the area. while the rest of his (1/3 part) is the land of the lowlands. trenggalek regency is subdivided into 14 subdistricts da 157 villages. only about 4 subdistricts which are the majority of the village of plains, which are: pogalan, trenggalek subdistrict, district and sub-district durenan monument. while the majority of other sub 10 village mountains. a large number of trenggalek regency year 2015 as much 689,200 inhabitants. number of changes in the populations affected by the internal state of the population is birth, death, and survival. the existence of a number of changes of a population called the growth of population. population growth can provide information as to whether changes in population numbers for next year is always increasing, decreasing or constant. (pratama, prihandono and kusumastuti, 2013, p. 163). population projection is not a forecast but a population of scientific calculations based on the assumption of the components of population growth rate, i.e., birth, death, and displacement. the third component is what determines the magnitude of the age structure of the population and the population in the future. to determine each assumption needed data that illustrates the trend in the past to the present, factors that affect the relationship between a component and a component with another as well as the expected target is reached on the future come. science many rapidly developing lately, including mathematical modeling. according to iswanto (2012, p. 19) in its development of mathematical models can be represented on the problems that occur in everyday life. so there is a strong relationship between applied science and mathematics are represented by mathematical models that are designed and implemented with the help of computer science, to facilitate the simulation of realworld systems. population growth is one of the examples of the application of the linear algebra in field biology and ecology specifically quantitatively. population growth in ecology is often referred to as the "population dynamics". ecology is usually defined as the relationship between living beings with their environment (tarumingkeng, 1994, p. 6). many models that can be used to explain the growth of the population. one of the models used by demographers is the model leslie. where the model using a mathematical approach, i.e. the matrix. in the model leslie that birth and death processes depending on the age and become an important part in the growth of the population. in general, the growth of a living http://dx.doi.org/10.14421/biomedich.2017.62.37-45 38 biology, medicine, & natural product chemistry 6 (2), 2017: 37-45 creature is a process of continuous or ongoing process taking place. however, the study population should be also approached from a discrete time review. the use of discrete pattern based also upon the observations of the population that is generally done interval-the interval period such as a day, a week, and the units of time according to a draft of the researchers concerned. based on these considerations, in addition to growth models based on the continuous solutions need to be evaluated more thoroughly with the solution of the discrete basis. (tarumingkeng, 1994, p. 27). in addition, the existence of significant size have yet to know the growth of female population in trenggalek based on birth rate and life expectancy for the coming year as well as the limiting distribution of unknown age by using model the leslie matrix. there is some research on matrix leslie i.e. research corazon, nurul, h. and yusienta, m., (2016, p. 6) which uses a matrix of leslie to predict the number and pace of growth in the province of riau in the year 2017. so the obtained results of the total population of women in riau province tend to experience increased. in addition the results of research conducted by pratama, prihandon. from the description it is necessary to determine the abundance of female population and knowing the limiting age distribution of the female population in trenggalek based on birth rate and life expectancy using the eigen values and eigen vectors matrix of leslie. materials and methods this research is research with quantitative approach with the types of descriptive research. this research is done by taking a secondary data in some of the central bureau of statistics in east java province and trenggalek regency bps. the sample in this study is a population of women of the years 2010 to 2015, by comparison with the number of child birth from the year 2010 to the year 2015. from the results of the data then diplikasikan by applying the matrix model leslie to look for population, the value of the eigenvalues and eigen vectors. procedure research the research methods used in this research are: the first stage is to determine the subject of the research, as for the subject of his research is the female population on the birth rate and life expectancy in east java province and the second stage is to (1) collect research data, with regard to the collection of research data obtained from secondary data in the central bureau of statistics of east java province and trenggalek regency bps (2) data analysis and the last is an interesting conclusion. observed variables observed variables in this study was the value of eigen and eigen vectors of the matrix leslie. eigen values of l are the roots of polynomials of its characteristics. the observed variables in this study was the value of eigen and eigen vectors of the matrix leslie. eigen values of l are the roots of polynomials of its characteristics. polynomial matrix characteristics of leslie are: 𝑝(𝜆) = 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯− 𝑎𝑛𝑏1𝑏2 ⋯𝑏𝑛−1 𝐱 = [ 𝑥1 𝑥2 𝑥3 ⋮ 𝑥𝑛] a vector of eigenvalues of a leslie matrix l related 𝜆1 if and only if x is a non-trivial solution of (𝜆𝐼 − 𝐿)𝐱 = 0. data analysis technique in doing data analysis techniques after the data is finished being processed which do first is find the values of ai (a fertility figure female population) obtained from the quotient between the average number of births born son a mom on 2010-2015 year divided by the number of female population in the year 2010 and bi (life expectancy female population) are obtained from the results of the women's division of the population the year 2015 with total population of woman of the year 2010. second, the mengkontruksi model matrix of leslie on the growth of the population. third after matric leslie was formed later enter the data the number of female population in 2015 to generate predictions of the total population of women in the year 2021. third, find the value of a positive eigenvalues in the matrix. fifth, determine the value of the eigen vectors eigen were positive. sixth, using equation approach to determine the limiting age distribution. the seventh note the abundance of female population for a long period to come. the eighth research report compiled and processed data through the application of maple 16. matrix according to kariadinata (2013, p.11) matrix is the arrangement of a group of numbers in a rectangular shaped ranks arranged based on row and column, and placed between two brackets. meanwhile, according to anton, h. & rorres, c. (2004, p. 23) a matrix with n rows and n columns is called an n order square matrix (in square matrices of order n), and the entries 𝑎11, 𝑎22, … , 𝑎𝑛𝑛 on the main diagonal is said to be from a. when a is the matrix has n rows and n column (type n x n), then a can be written as: [ 𝑎11 𝑎21 ⋮ 𝑎𝑛1 𝑎12 𝑎22 ⋮ 𝑎𝑛2 ⋯ ⋯ ⋯ 𝑎1𝑛 𝑎2𝑛 ⋮ 𝑎𝑛𝑛 ] anggreini – the female population growth projection year 2021 in trenggalek … 39 inverse of a matrix according to adiwijaya (2014, p. 10) suppose a and b are square matrices of the same size are and i is the identity matrix. if a.b=i then b is called the inverse of matrix a (on the contrary, a is the inverse of matrix b). notation that the inverse of matrix b is a is b= a-1 and vice versa a= b-1 matriks elementer according to anton, h & rorres, c (2004, p.56) an n x n matrix is called elementary matrix if the matrix can be derived from in n x n identity matrix by performing a single elementary row operation. each elementary matrix can be reversed, and its inverse is also an elementary matrix. if a is an n x n, matrix, then the following statements are equivalent,they are all true or all wrong: a) a is invertible, b) a x = 0 has only a trivial solution, c) a row equivalent to in or reduced rowechelon form a is in.. determinants according to anton, h & rorres, c (2004, p.92) an elementary product of a matrix a, a, nn x , is a product of entries of, none of which come from the same row or column. eigenvalues and eigenvectors according to kariadinata (2013, p.209) if a is an n x n size matrix, then the nonzero vector x in rn is called an eigenvector of a if a x is a scalar multiple of x; ie, 𝐴𝒙 = 𝜆𝒙 for any scalar λ. the scalar λ is called the eigenvalue of a and x is said to be the eigenvector corresponding to 𝜆. to find the eigenvalue of the matrix a of nxn size then 𝐴𝒙 = 𝜆𝒙 can be rewritten as 𝐴𝒙 = 𝜆𝐼𝒙 or equivalent to (𝜆𝐼 − 𝐴)𝐱 = 0. to be an eigenvalue, there must be a nonzero solution of the equation (𝜆𝐼 − 𝐴)𝐱 = 0, obtained if and only if det (𝜆𝐼 − 𝐴)𝐱 = 0. the equation of det (𝜆𝐼 − 𝐴)𝐱 = 0 is called the characteristic equation of a; the scalar that satisfies this equation is the eigenvalue of a. when expanded, then det (λi-a) is a polynomial p in a variable called characteristic polynomial of a. the characteristic polynomial 𝑝(𝑥) of an nxn matrix has the form: 𝑝(𝜆) = det(𝜆𝐼 − 𝐴) = 𝜆𝑛 + 𝐶1𝜆 𝑛−1 + ⋯+ 𝐶𝑛 diagonalization of the matrix according to anton, h & rorres, c (2004, p.74) the diagonal matrix of a square matrix whose entry does not lie in the main diagonal is zero is called the diagonal matrix. a common diagonal matrix d, n x n can be written [ 𝑑1 0 ⋮ 0 0 𝑑2 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 𝑑𝑛 ] a diagonal matrix can be reversed, if and only if all entries in the main diagonal position are non-zero; in this case the inverse of d is 𝐷−1 = [ 1 𝑑1⁄ 0 ⋮ 0 0 1 𝑑2⁄ ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 1 𝑑𝑛⁄ ] so, 𝐷𝐷−1 = 𝐷−1𝐷 = 𝐼. results and discussion leslie matrix model in population growth in leslie's model, women or females are divided over age groups that are timed together. specifically, uppose that the maximum age reached by any woman or female in the population is m years (or expressed in other units of time) and then the population is divided over n the age group. so the time period in each group m/n years. the age group will be explained by the following table: table 1. age group of leslie matrix model. age group age interval 1 [0,m/n] 2 [m/n,2m/n] 3 [2m/n,3m/n] : : : : (n-1) [(n-2)2m/n,(n-1)m/n] n [(n-1)m/n,m] for example known to the number of women or females in each group of to-n group at time t = 0. specifically, for example, there are x1 (0) women or females in the first group, x2 (0) woman or female in second group, x3 (0) woman or female in the third group, and so on. with the number ke-n will be formed a column vector x(0) that is: 𝐱(0) = [ 𝑥1 (0) 𝑥2 (0) ⋮ 𝑥(0) ] this vector is called the age distribution vector of the origin (initial age distribution vector). over time, the number of women or females in each group of to – n the group will change due to three biological processes, namely: birth, death and aging. by explaining these three processes quantitatively, it can be seen how to project the vector of early age distribution into the future. the easiest way to learn about the aging process is to observe the population at discrete times, say 𝑡0, 𝑡1, . . . , 𝑡𝑘 , . ... leslie's model requires that the time span between two consecutive observation times is the 40 biology, medicine, & natural product chemistry 6 (2), 2017: 37-45 same as the time period of the hose (interval) age, and is written as follows: 𝑡0 = 0, 𝑡1 = 𝑀 𝑛⁄ , 𝑡2 = 2𝑀 𝑛⁄ , to 𝑡𝑘 = 𝑘𝑀 𝑛⁄ with this assumption, all women or females in the group-(𝑖 + 1) at 𝑡𝑘+1 time have been in group to-i at time 𝑡𝑘. the process of birth and death processes between two consecutive observation times can be explained using demographic parameters. as follows: table 2. parameters in leslie matrix model. parameter model information 𝑎𝑖 𝑖 = 1,2,… , 𝑛 average number of girls who was born by a woman during her time in the age group i. 𝑏𝑖 𝑖 = 1,2,… , 𝑛 the number of women in the i-age group who can be expected is still alive and up to age group i. by defition, it will be obtained that (i) 𝑎𝑖 ≥ 0 for 𝑖 = 1,2, . . . , 𝑛 and (ii) 0 < 𝑏𝑖 ≤ 1 for 𝑖 = 1,2, . . . , 𝑛 − 1.can be seen that, should not allow for 𝑏𝑖 which is equal to zero, because if this happens then there will be no female or female alive after the age group to-i. and also considered that at least there is one 𝑎𝑖 positive that will occur birth. each age group in which the corresponding value 𝑎𝑖 is positive is called the fertile age group (fertile age class). next we will define the age distribution vector 𝑥(𝑘) at the time 𝑡𝑘 with: 𝐱(𝑘) = [ 𝑥1 (𝑘) 𝑥2 (𝑘) ⋮ 𝑥(𝑘) ] where 𝑥𝑖 (𝑘) is the number of women or females in the i-age group at the time 𝑡𝑘. at times, women in the first age group are daughters of women born between time 𝑡𝑘−1 and time 𝑡𝑘. so, it can be written or mathematically, 𝑥1 (𝑘) = 𝑎1𝑥1 (𝑘−1) + 𝑎2𝑥2 (𝑘−1) + ⋯+ 𝑎𝑛𝑥𝑛 (𝑘−1) (1) number of women in the age group to −(𝑖 + 1) ( 𝑖 = 1, 2, . . . , 𝑛 − 1) at the time 𝑡𝑘 are women in groups to −𝑖 at the time 𝑡𝑘−1 who are still alive at the time 𝑡𝑘.or mathematically 𝑥𝑖+1 (𝑘) = 𝑏𝑖𝑥𝑖 (𝑘−1), 𝑖 = 1,2,… , 𝑛 − 1 (2) by using matrix notation, equations (1) and (2) can be written in the form [ 𝑥1 (𝑘) 𝑥2 (𝑘) 𝑥3 (𝑘) ⋮ 𝑥𝑛 (𝑘)] = [ 𝑎1 𝑏1 0 ⋮ 0 𝑎2 0 𝑏2 ⋮ 0 𝑎3 0 0 ⋮ 0 ⋯ ⋯ ⋯ ⋮ 0 𝑎𝑛−1 0 0 ⋮ 𝑏𝑛−1 𝑎𝑛 0 0 ⋮ 0 ] [ 𝑥1 (𝑘−1) 𝑥2 (𝑘−1) 𝑥3 (𝑘−1) ⋮ 𝑥𝑛 (𝑘−1)] or more succinctly 𝐱(𝑘) = 𝐿𝐱(𝑘−1), with 𝑘 = 1,2,… (3) where l is leslie matrix 𝐿 = [ 𝑎1 𝑏1 0 ⋮ 0 𝑎2 0 𝑏2 ⋮ 0 𝑎3 0 0 ⋮ 0 ⋯ ⋯ ⋯ ⋮ 0 𝑎𝑛−1 0 0 ⋮ 𝑏𝑛−1 𝑎𝑛 0 0 ⋮ 0 ] (4) from the equation (3) is found that 𝐱(1) = 𝐿𝐱(1) 𝐱(2) = 𝐿𝐱(1) = 𝐿(2)𝐱(0) 𝐱(3) = 𝐿𝐱(2) = 𝐿(3)𝐱(0) ⋮ 𝐱(𝑘) = 𝐿𝐱(𝑘−1) = 𝐿(𝑘)𝐱(0) (5) thus, if we know the start age distribution 𝐱(0) and leslie matrix l, then we can determine the age distribution of women or females at any later time. limited age distribution although the equation (5) gives the age distribution of the population at any given time, it does not immediately provide an overview of the dynamics of the anggreini – the female population growth projection year 2021 in trenggalek … 41 growth process. it is necessary to investigate the eigenvalues and eigenvectors of leslie's matrix. the eigenvalues of l are the roots of the characteristic polynomial. the characteristic polynomial of leslie's matrix is 𝑝(𝜆) = 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯− 𝑎𝑛𝑏1𝑏2 ⋯𝑏𝑛−1 will be given the theorems related to leslie's matrix, as follows: theorem 1 a leslie matrix l has a unique positive eigen value 𝜆1. this eigenvalue has a multiplicity of 1 and has an eigenvector 𝐱1 that all entries are positive. evidence: (i) the leslie matrix l has a positive eigenvalue 𝜆1. the eigenvalue is the root of the characteristic equation, that is 𝑝(𝜆) = |𝜆𝐼 − 𝐿| = 0 𝑝(𝜆) = 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯− 𝑎𝑛𝑏1𝑏2 ⋯𝑏𝑛−1 if 𝑝(𝜆) = 0, so 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯− 𝑎𝑛𝑏1𝑏2 ⋯𝑏𝑛−1 = 0 (6) because one of the roots of the equation (6) must be the dividing factor of the coefficient 𝑎𝑛𝑏1𝑏2⋯𝑏𝑛−1, i.e ±𝑎𝑛, ±𝑏1, ±𝑏2,⋯ ,±𝑏𝑛−1 suppose that the solution of one of the divider factors, for example, is taken 𝑏1 > 0 as a solution or 𝑏1 as its root, so the equation (6) becomes 𝑝(𝜆) = 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯− 𝑎𝑛𝑏1𝑏2 ⋯𝑏𝑛−1 = 0 𝜆𝑛 = 𝑎1𝜆 𝑛−1 + 𝑎2𝑏1𝜆 𝑛−2 + 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯+ 𝑎𝑛𝑏1𝑏2 ⋯𝑏𝑛−1 (𝜆 − 𝑏1)(𝑎1𝜆 𝑛−1 + 𝑎2𝑏1𝜆 𝑛−2 + 𝑎3𝑏1𝑏2𝜆 𝑛−3 + ⋯ +𝑎𝑛𝑏2 ⋯𝑏𝑛−1) = 𝜆𝑛 means obtained a positive eigen value 𝜆1, that is 𝜆1 = 𝑏1. it will then be proven that leslie matrix l has a 𝜆1 single eigenvalue that single, that is 𝜆1 = 𝑏1. it will be assumed that leslie's matrix has another positive eigenvalue. so to prove it used a way of contradiction. an example 𝜆2 is another positive eigenvalue assuming 𝜆1 ≠ 𝜆2 or 𝜆2 ≠ 𝑏1. is known 𝑝(𝜆) = 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯− 𝑎𝑛𝑏1𝑏2 ⋯𝑏𝑛−1 if 𝑝(𝜆) = 0, then, 𝜆𝑛 − 𝑎1𝜆 𝑛−1 − 𝑎2𝑏1𝜆 𝑛−2 − 𝑎3𝑏1𝑏2𝜆 𝑛−3 ⋯− 𝑎𝑛𝑏1𝑏2 ⋯𝑏𝑛−1 = 0 the two segments are divided by 𝜆𝑛, be 1 = 𝑎1 𝜆 + 𝑎2𝑏1 𝜆2 + 𝑎3𝑏1𝑏2 𝜆3 + ⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆𝑛 so 𝑞(𝜆) = 1, for 𝜆 ≠ 0 or 𝑞(𝜆) = 𝑎1 𝜆 + 𝑎2𝑏1 𝜆2 + 𝑎3𝑏1𝑏2 𝜆3 + ⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆𝑛 (7) proven that 𝜆1 and 𝜆2 is the solution of the system of equations 𝑞(𝜆) = 1. so get it 𝑞(𝜆1) = 1 and 𝑞(𝜆2) = 1 or 𝑞(𝜆1) = 𝑞(𝜆2). then by using the equation (7), and if 𝑞(𝜆1) = 𝑞(𝜆2) 𝑎1 𝜆1 + 𝑎2𝑏1 𝜆1 2 + 𝑎3𝑏1𝑏2 𝜆1 3 + ⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑛 = 𝑎1 𝜆2 + 𝑎2𝑏1 𝜆2 2 + 𝑎3𝑏1𝑏2 𝜆2 3 + ⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆2 𝑛 if 𝑞(𝜆1) deducted by 𝑞(𝜆2) then 𝑎1 ( 1 𝜆1 − 1 𝜆2 ) + 𝑎2𝑏1 ( 1 𝜆1 2 − 1 𝜆2 2) + 𝑎2𝑏1𝑏2 ( 1 𝜆1 3 − 1 𝜆2 3) + …+ 𝑎𝑛𝑏1𝑏2 …𝑏𝑛−1 ( 1 𝜆1 𝑛 − 1 𝜆2 𝑛) = 0 be 𝑎1 ( 𝜆2−𝜆1 𝜆1𝜆2 ) + 𝑎2𝑏1 ( 𝜆2 2−𝜆1 2 𝜆1 2𝜆2 2 ) + 𝑎2𝑏1𝑏2 ( 𝜆2 3−𝜆1 3 𝜆1 3𝜆2 3 ) + …+ 𝑎𝑛𝑏1𝑏2 …𝑏𝑛−1 ( 𝜆2 𝑛−𝜆1 𝑛 𝜆1 𝑛𝜆2 𝑛 ) = 0 or 𝑎1 𝜆1𝜆2 (𝜆2 − 𝜆1) + 𝑎2𝑏1 𝜆1 2𝜆2 2 (𝜆2 2 − 𝜆1 2) + 𝑎2𝑏1𝑏2 𝜆1 3𝜆2 3 (𝜆2 3 − 𝜆1 3) +⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑛𝜆2 𝑛 (𝜆2 𝑛 − 𝜆1 𝑛) = 0 (8) the equation (8) will be valid if, 𝜆1 = 𝜆2, or contradictory to, the assumption stating. 𝜆1 ≠ 𝜆2. so the true is the positive eigenvalue of leslie's matrix is singular, that is 𝜆1 = 𝜆2 = 𝑏1. (ii) positive eigenvalues have a multiplicity of 1 𝜆1𝑥4 = 𝑏3𝑥3 𝑥4 = 𝑏3 𝜆1 𝑥3 → 𝑥4 = 𝑏3 𝜆1 ( 𝑏1𝑏2 𝜆1 2 𝑥1) −𝑏𝑛−1𝑥𝑛−1 + 𝜆1𝑥𝑛 = 0 42 biology, medicine, & natural product chemistry 6 (2), 2017: 37-45 𝜆1𝑥𝑛 = 𝑏𝑛−1𝑥𝑛−1 𝑥𝑛 = 𝑏𝑛−1 𝜆1 𝑥𝑛−1 𝑥𝑛 = 𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑥𝑛−1 so obtained 𝑥2 = 𝑏1 𝜆1 𝑥1 𝑥3 = 𝑏2 𝜆1 𝑥2 = 𝑏1𝑏2 𝜆1 2 𝑥1 𝑥4 = 𝑏3 𝜆1 𝑥3 = 𝑏1𝑏2𝑏3 𝜆1 3 𝑥1 ⋮ 𝑥𝑛 = 𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑥𝑛−1 = 𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑛−1 𝑥1 if for example taken 𝑥1 = 1, then eigenvector 𝐱 corresponds to 𝜆1 that is 𝐱1 = [ 𝑥1 𝑥2 𝑥3 𝑥4 ⋮ 𝑥𝑛] = [ 1 𝑏1 𝜆1⁄ 𝑏1𝑏2 𝜆1 2⁄ 𝑏1𝑏2𝑏3 𝜆1 3⁄ ⋮ 𝑏1𝑏2 … 𝜆1 𝑛−1⁄ ] it is proven that leslie's matrix eigenvect or of all elements is positive. theorem 2 if 𝜆1 is the unique positive eigenvalue of a leslie matrix l and if 𝜆𝑖 is any real eigenvalues or complex of 𝐿, then |𝜆𝑘| ≤ 𝜆1. evidence: it has been proved that 𝜆1 is a single positive eigenvalue, then 𝜆1 is an eigenvalue that can be a real or complex number. if for example 𝜆𝑘 = 0 it proves that |𝜆𝑘| ≤ 𝜆1.then taken any 𝜆𝑘 = 𝑟𝑒𝑖𝜃 with, 𝑖 = √−1, to prove the same as showing |𝑟| ≤ 𝜆1. the necessary conditions and sufficient conditions 𝜆𝑘 to be an eigenvalue, 𝜆𝑘 must be the solution of the system of equations 𝑞(𝜆) = 𝑎1 𝜆𝑘 + 𝑎2𝑏1 𝜆𝑘 2 + ⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆𝑘 𝑛 = 1, 𝑞(𝜆) = 1 for 𝜆 ≠ 0 𝑞(𝜆) = 𝑎1 𝑟𝑒𝑖𝜃 + 𝑎2𝑏1 (𝑟𝑖𝜃) 2 + ⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 (𝑟𝑖𝜃) 𝑛 = 1 𝑎1 𝑟 (𝑒−𝑖𝜃) + 𝑎2𝑏1 𝑟2 (𝑒−2𝑖𝜃) + ⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (𝑒−𝑛𝑖𝜃) = 1 𝑎1 𝑟 (cos(−𝜃) + 𝑖 sin(−𝜃) ) + 𝑎2𝑏1 𝑟2 (cos(−2𝜃) (…+ 𝑖 sin(−2𝜃))−2 + ⋯ …+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (cos(−𝑛𝜃) + 𝑖 sin(−𝑛𝜃) ) = 1 𝑎1 𝑟 (cos𝜃 − 𝑖 sin 𝜃 ) + 𝑎2𝑏1 𝑟2 (cos2𝜃 − 𝑖 sin 2𝜃) + +⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (cos𝑛𝜃 − 𝑖 sin 𝑛𝜃 ) = 1 it means 𝑎1 𝑟 (cos𝜃 ) + 𝑎2𝑏1 𝑟2 (cos2𝜃) + ⋯ …+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (cos𝑛𝜃 ) = 1 and 𝑎1 𝑟 (sin 𝜃 ) + 𝑎2𝑏1 𝑟2 (sin 2𝜃) + ⋯ …+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (sin 𝜃 ) = 0 if taken only the real part is 𝑎1 𝑟 (cos𝜃 ) + 𝑎2𝑏1 𝑟2 (cos2𝜃) + ⋯ …+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (cos𝑛𝜃 ) = 1 because 𝜆1 is an eigenvalue so 𝜆1 satisfies the equation 𝑞(𝜆) = 1 so 𝑞(𝜆) = 𝑎1 𝜆1 + 𝑎2𝑏1 𝜆1 2 + ⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑛 = 1 if 𝑎1 𝑟 (𝑐𝑜𝑠𝜃) + 𝑎2𝑏1 𝑟2 (𝑐𝑜𝑠2𝜃) + ⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝑟𝑛 (𝑐𝑜𝑠𝑛𝜃) = 𝑎1 𝜆1 + 𝑎2𝑏1 𝜆1 2 + ⋯+ 𝑎𝑛𝑏1𝑏2…𝑏𝑛−1 𝜆1 𝑛 (9) this equation is subtracted into 𝑎1 ( cos𝜃 𝑟 − 1 𝜆1 ) + 𝑎2𝑏1 ( cos2𝜃 𝑟2 − 1 𝜆1 2 ) + ⋯ …+ 𝑎𝑛𝑏1𝑏2 …𝑏𝑛−1 ( cos𝑛𝜃 𝑟𝑛 − 1 𝜆1 𝑛) = 0 𝑎1 ( 𝜆1 cos𝜃−𝑟 𝑟𝜆1 ) + 𝑎2𝑏1 ( 𝜆1 2 cos2𝜃−𝑟2 𝑟2𝜆1 2 ) + ⋯ …+ 𝑎𝑛𝑏1𝑏2 …𝑏𝑛−1 ( 𝜆1 𝑛 cos𝑛𝜃𝑟𝑛 𝑟𝑛𝜆1 𝑛 ) = 0 equations (9) apply if 𝜆1 cos 𝜃 − 𝑟 = 0 obtained 𝑟 = 𝜆1 cos𝜃. because −1 ≤ cos𝜃 ≤ 1 → −𝜆1 ≤ 𝑟 ≤ 𝜆1, it means |𝑟| ≤ 𝜆1. if 𝜆1 meet |𝜆𝑘| < 𝜆1 is said that 𝜆1 is a dominant eigen value of 𝐿. therefore the condition of theorem 2 is not strong enough to prove that the eigenvalues of leslie's matrix are dominant. theorem 3 if two consecutive entries 𝑎𝑖 and 𝑎𝑖+1 in the first row of a leslie matrix 𝐿 are not equal to zero then the positive eigenvalue of 𝐿 is dominant. it is called the dominant eigenvalue j. |𝜆𝑘| < 𝜆1 should not |𝜆𝑘| = 𝜆1 or indicated |𝜆𝑘| ≠ 𝜆1. by definition 𝑎𝑖 > 0 and 𝑎𝑖+1 > 0. with 𝑖 = 1,2,… , 𝑛, without prejudice generaly theorem be understood 𝑖 = 1. so 𝑎1 > 0 and 𝑎2 > 0, so it will be shown |𝜆𝑘| ≠ 𝜆1 with 𝑘 = 2,3,… , 𝑛, where 𝜆1 is positif. to prove it is done way of contradiction. that is, it will be assumed that 𝜆𝑘 = 𝜆1. if 𝜆𝑘 = 𝜆1 then 𝑟𝑒𝑖𝜃 = 𝜆1, so 𝑟(𝑐𝑜𝑠𝜃 + 𝑠𝑖𝑛𝜃) = 𝜆1. 𝑟𝑐𝑜𝑠𝜃 + 𝑖𝑟𝑠𝑖𝑛𝜃 = 𝜆1 + 𝑖0. obtained 𝑟𝑐𝑜𝑠𝜃 = 𝜆1 and 𝑟𝑠𝑖𝑛𝜃 = 0. anggreini – the female population growth projection year 2021 in trenggalek … 43 if the equation 𝑟𝑐𝑜𝑠𝜃 = 𝜆1 multiplied by 𝑐𝑜𝑠𝜃 be 𝑟𝑐𝑜𝑠2𝜃 = 𝜆1𝑐𝑜𝑠𝜃. it means 𝜆1𝑐𝑜𝑠𝜃 ≠ 𝑟 or 𝜆1𝑐𝑜𝑠𝜃 − 𝑟 ≠ 0 based on the equation (9) that is 𝑎1 ( 𝜆1𝑐𝑜𝑠𝜃−𝑟 𝑟𝜆1 ) + 𝑎2𝑏1 ( 𝜆1 2𝑐𝑜𝑠2𝜃−𝑟2 𝑟2𝜆1 2 ) + ⋯ ⋯+ 𝑎𝑛𝑏1𝑏2 …𝑏𝑛−1 ( 𝜆1 𝑛𝑐𝑜𝑠𝑛𝜃−𝑟𝑛 𝑟𝑛𝜆1 𝑛 ) = 0 earned value 𝑎1 = 0 and 𝑎2 = 0. the statement is contrary to the assumption that states 𝑎1 > 0 and 𝑎2 > 0. meaning the wrong supposition |𝜆𝑘| ≠ 𝜆1. in the next section it will always be assumed that the requirements of theorem 3 are met. it will be assumed that l can be diagonalized. it is not necessary for the conclusions to be taken, but this will simplify the argument. in this case, l has n eigenvalues, 𝜆1, 𝜆2, ..., 𝜆𝑛, which need not be different from each other, and n linearly independent eigenvectors 𝐱1, 𝐱2, ..., 𝐱𝑛 , which corresponds to the eigenvalue. in this list will be placed eigenvalues 𝜆1 the dominant first. a matrix p will be formed whose columns are eigenvectors of l. 𝑃 = [𝐱1|𝐱2|𝐱3|⋯ |𝐱𝑛] 𝐿𝑃 = 𝐿[𝐱1|𝐱2|𝐱3|⋯ |𝐱𝑛] = [𝐿𝐱1|𝐿𝐱2|𝐿𝐱3|⋯ |𝐿𝐱𝑛] = [𝜆1𝐱1|𝜆2𝐱2|𝜆3𝐱3|⋯ |𝜆𝑛𝐱𝑛] = [𝐱1|𝐱2|𝐱3|⋯ |𝐱𝑛] [ 𝜆1 0 ⋮ 0 0 𝜆2 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 𝜆𝑛 ] = 𝑃𝐷 because column vectors of the linear matrices p are linearly independent, and p is reversible so that the matrix l can be diagonalized. the diagonalization of l will be given by the equation 𝐿 = 𝑃𝐷𝑃−1 𝐿 = 𝑃 [ 𝜆1 0 ⋮ 0 0 𝜆2 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 𝜆𝑛 ]𝑃−1 from this equation is obtained 𝐿𝑘 = 𝑃 [ 𝜆1 𝑘 0 ⋮ 0 0 𝜆2 𝑘 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 𝜆𝑛 𝑘 ]𝑃−1 for 𝑘 = 1,2,… for any vector of age distribution it will first 𝐱(0) be obtained 𝐿𝑘𝐱(0) = 𝑃 [ 𝜆1 𝑘 0 ⋮ 0 0 𝜆2 𝑘 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ 𝜆𝑛 𝑘 ]𝑃−1𝐱(0) for 𝑘 = 1,2,… by dividing two sections of this equation by 𝜆1 𝑘 and using the fact that 𝐱(𝑘) = 𝐿(𝑘)𝐱(0), it will be obtained 1 𝜆1 𝑘 𝐱(𝑘) = 𝑃 [ 1 0 ⋮ 0 0 (𝜆2 𝜆1⁄ )𝑘 ⋮ 0 ⋯ ⋯ ⋯ 0 0 ⋮ (𝜆𝑛 𝜆1⁄ )𝑘 ]𝑃−1𝐱(0) (10) because 𝜆1 is the dominant eigen value that is |𝜆1| > |𝜆𝑖|, then |𝜆𝑖 𝜆1⁄ | < 1 for 𝑖 = 2,3,… , 𝑛. it is clear that (𝜆𝑖 𝜆1⁄ )𝑘 → 0 if 𝑘 → ∞ for 𝑖 = 2,3,… , 𝑛. using this fact, it can take limits from both sides of (10) to get lim 𝑘→∞ { 1 𝜆1 𝑘 𝐱(𝑘)} = 𝑝 [ 1 0 0 ⋯ 0 0 0 0 ⋯ 0 ⋮ ⋮ ⋮ ⋮ 0 0 0 ⋯ 0 ] 𝑃−1𝐱(𝟎) (11) it will then be declared the first entry of the column vector 𝑃−1𝐱(𝟎) with the constants 𝑐, then 𝑃−1𝐱(𝟎) = [ 𝑐 𝑐1 ⋮ 𝑐𝑛−1 ]. this result is entered into the equation (11) lim 𝑘→∞ { 1 𝜆1 𝑘 𝐱(𝑘)} = 𝑝 [ 1 0 0 ⋯ 0 0 0 0 ⋯ 0 ⋮ ⋮ ⋮ ⋮ 0 0 0 ⋯ 0 ] [ 𝑐 𝑐1 ⋮ 𝑐𝑛−1 ] = [𝐱1|𝐱2|𝐱3|⋯ |𝐱𝑛] [ 1 0 0 ⋯ 0 0 0 0 ⋯ 0 ⋮ ⋮ ⋮ ⋮ 0 0 0 ⋯ 0 ] [ 𝑐 𝑐1 ⋮ 𝑐𝑛−1 ] = [𝐱1|𝐱2|𝐱3|⋯ |𝐱𝑛] [ 𝑐 0 ⋮ 0 ] = 𝑐𝐱1 so that the right-hand side of (11) can be written as 𝑐𝐱𝟏, where 𝑐 is a positive controw that depends only on the vector of age distribution first 𝐱(0).so (11) be lim 𝑘→∞ { 1 𝜆1 𝑘 𝐱(𝑘)} = 𝑐𝐱𝟏 (12) the equation (12) gives an approximation 𝐱(𝑘)~𝑐𝜆1 𝑘𝐱𝟏 (13) for large k values. from (13) also obtain 𝐱(𝑘−1) ≃ 𝑐𝜆1 𝑘−1𝐱𝟏 (14) by comparing equations (13) and equations (14) obtained 44 biology, medicine, & natural product chemistry 6 (2), 2017: 37-45 𝐱(𝑘) ≃ 𝜆1𝐱 (𝑘−1) (15) for large 𝑘 values. this means that for large time values each age distribution vector is a scalar multiple of the previous age distribution vector, and the scalar is the positive eigenvalue of leslie's matrix. consequently, the proportion of females in each group of age groups will be constant.then we will review the equations (13) that give the age distribution vector of the population for a long time: )(k x ~ kc 1 1x (16) three cases will appear in accordance with eigenvalues 𝜆1: (i) the total population will eventually tend to increase/increase if 𝜆1 > 1, (ii) the total population will eventually tend to decrease/decrease if 𝜆1 < 1, (iii) population will tend to be stable/fixed if 𝜆1 = 1. if the population tends to decline then it can be said also that the population growth rate is negative, where as if the population number increases it can be said also that the population growth rate is positive. data on population of women and number of children born in 2010 and year 2015 in trenggalek regency the following data on the number of female population from 2010-2015 in trenggalek regency that has been studied in the research. because only a few women over 45 years old who gave birth to a child, it will be limited from the population of women aged between 15 and 49 years this is due to the age of female fertility interval of 15-49 years. this data is for the age group with the period of 5 years, so the total number is 14 age groups. from table 3 data will be analyzed by looking for parameter 𝑎𝑖 (fertility number) and 𝑏𝑖 parameter (life expectancy). leslie matrix model can be used to determine the number of female population the next 6 years. using leslie's matrix, according to table 4. the female population is divided into several age-class intervals, with the interval of female fertility 15-49 years. to calculate the number of female population by matrix method leslie influenced by fertility rate (𝑎𝑖) life expectancy (𝑏𝑖). here is a settlement step to predict the number and growth rate in trenggalek regency in 2021. table 3. number of female population year 2010-2015. age class number of female 2010 number of female 2015 ai bi 0-4 24,284 22,860 0 0.978257 5-9 24,529 23,756 0 1.019365 10-14 26,129 25,004 0 0.933752 15-19 24,704 24,398 0.0099 0.903538 20-24 22,592 22,321 0.2599 1.018900 25-29 24,790 23,019 0.2688 0.943687 30-34 24,627 23,394 0.2698 1.055955 35-39 26,632 26,005 0.2599 1.052418 40-44 28,185 28,028 0.0795 0.969700 45-49 25,671 27,331 0.0374 0.994313 50-54 21,950 25,525 0 0.987198 55-59 17,401 21,669 0 0.925694 60-64 13,651 16,108 0 2.738627 65+ 35,320 37,385 0 0 total 340,465 346,803 next will be analyzed using the maple 16 program to determine the dynamics of the growth process based on the eigenvalues and eigenvectors of leslie matrix. then using the equation approach for the age limited distribution 𝐱(𝑘) ≃ 𝜆1𝐱 (𝑘−1) ~ will find the rate growth of female population in trenggalek regency. having formed leslie's matrix, based on equation (3), that is 𝐱(𝑘) = 𝐿𝐱(𝑘−1) with 𝑘 = 1,2,… to predict the number of women in 2021. based on table. 3 we obtain the following leslie matrix 𝐱(𝑘) = 𝐿𝐱(𝑘−1) =                                                                                                  343.39 058,20 198,25 175,27 178.27 368,27 702,24 722,21 742,22 044,22 347,23 216,24 361,22 553,28 37,385 16,108 21,669 25,525 27,331 28,028 26,005 23,394 23,019 22,321 24,398 25,004 23,756 22,860 02.7386000000000000 000.925600000000000 0000.98710000000000 00000.9943000000000 000000.969700000000 0000001.05240000000 00000001.0559000000 000000000.943600000 0000000001.01890000 00000000000.9035000 000000000000.933700 0000000000001.01930 00000000000000.9782 00000.03740.07950.25990.26980.26880.25990.0099000 anggreini – the female population growth projection year 2021 in trenggalek … 45 then by using the equation approach for limited age distribution: 𝐱(𝑘) ≃ 𝜆1𝐱 (𝑘−1) so 𝐱(𝑘) ≃ 1,000391𝐱(𝑘−1). from the eigen value obtained 𝜆1 > 1 so that every five years of female population in trenggalek regency will tend to increase or in other words growth rate of woman population in trenggalek regency tends to be positive value. table 4. number of women year 2010 and 2015 & women's prediction year 2021. age class number of female 2010 number of female 2015 number of female 2021 0-4 24,284 22,860 28,553 5-9 24,529 23,756 22,361 10-14 26,129 25,004 24,216 15-19 24,704 24,398 23,347 20-24 22,592 22,321 22,044 25-29 24,790 23,019 22,742 30-34 24,627 23,394 21,722 35-39 26,632 26,005 24,702 40-44 28,185 28,028 27,368 45-49 25,671 27,331 27,178 50-54 21,950 25,525 27,175 55-59 17,401 21,669 25,198 60-64 13,651 16,108 20,058 65+ 35,320 37,385 39,343 total 340,465 346,803 356,007 conclusion based on the data analysis of leslie matrix in female population in trenggalek regency, it can be concluded that eigen value for the total population of women in trenggalek regency in year 2021 amounted to 356.007. distribution age limit for female population in trengggalek regency is 𝐱(𝑘) ≃ 𝜆1𝐱 (𝑘−1) with positive eigen value 𝜆1 > 1 is greater than one that is equal to 1,000391 so that the number of female population in trenggalek regency in year 2021 tend to increase or can be said growth rate of population tends to be positive value. references adiwijaya. 2014. application of matrices and vector spaces. yogyakarta: graha ilmu. anton, h. & rorres, c. 1988. application of linear algebra. jakarta: erland. anton, h & rorres, c. 2004. linear elementary algebra (app version). jakarta: erland. central bureau of statistics. 2013. projection of indonesian population (indonesian population projection) 2010-2035, jakarta. corazon, nurul, h and yusianta, m, 2016. leslie's matrix application to predict the number and growth rate of women in riau province by 2017. journal of mathematical and statistical science (jsms), 2 (3), 1-11, ejournal.uinsuska.ac.id/index.php/jsms/article / download / 3098 / iswanto, r.j. 2012. applied and applied mathematical modeling. yogyakarta: graha ilmu. kariadinata, r. 2013. algebra of elementary matrices. bandung: cv pustaka setia. pratama, prihandono and kusumastuti 2013. leslie's matrix application to predict the number and rate of growth of a population. bimaster: math scientific bulletin. stat. and applied, 2 (3), 163-172. http://jurnal.untan.ac.id/index.php/jbmstr/article/view/3859. tarumingkeng, r.c. 1994, population dynamics (quantitative ecological studies), pustaka sinar harapan, jakarta this page intentionally left blank the female population growth projection year 2021 in trenggalek regency by leslie matrix model on the birth rate and life expectancy biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 5, number 2, 2016 | pages: 33-40 | doi: 10.14421/biomedich.2016.52.33-40 issn 2540-9328 (online) starch-glycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on its mechanical properties endaruji sedyadi1, syafiana khusna aini2, dewi anggraini3, dian prihatiningtias ekawati4 1,2,3,4chemistry department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency: endaruji@yahoo.com1 abstract effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on starch-based edible film-glycerol mechanical properties has been done. the purpose of this study is to create an active environment-friendly packaging material. surimi additions are intended to improve the mechanical properties of bioplastics and additions of rosella extract intended as a bio-indicator of acidity. the method used was solvent casting. an amount of surimi and rosella extract varied to obtain the best mechanical properties. the results shows that the addition of surimi and rosella flower extract significantly effect the elongation of edible films produced up to 27%. keywords: edible film; rosella extract; surimi dumbo catfish; mechanical properties. introduction the use of synthetic polymers made from petroleum may have a negative impact on our environment and on human health. synthetic polymers made from petroleum take a long time to degrade (awwaly et al., 2010). the awareness to this problem would then increasing interest to organic materials, which can be destroyed biologically, eco-friendly, and easily obtained in nature as well as suitable for packed food products (embuscado et al., 2009). the ideal solution is the development of natural ingredients for the manufacture of packaging materials which can be decomposed faster naturally, maintain the integrity of the food product to be packed and edible (gennadios et al., 2002). edible film is a thin layer made of edible ingredients and used as food coating. coating of food components can be done by dipping, spraying or wrapping (akbar et al., 2013). the advantage of using edible film is it can be directly consume with the products that are packaged together, eco-friendly, improve the organoleptic properties of the packaged product, serves as a nutritional supplement, as a carrier of flavor, coloring, antimicrobial agents, and antioxidants (murdianto et al., 2005). hydrocolloid such as proteins, polysaccharides, and a mixture of both has been studied intensively as an ingredient in edible film. isnaini (2010) explains that the edible film as bio-indicators can be made by using natural pigments from the plant, one of them is rosella (hibiscus sabdariffa l). the main ingredients of rosella is an anthocyanin. setiono (2013) stated that anthocyanins are dissolved in water to create colored solutions, red under acidic conditions and blue under alkaline conditions. the compound is classified as pigments and determine the plant color which is affected by the ph of its environment. rosella flowers can be used as bio-indicators, which are sensitive to ph. edible films can detect food spoilage by change in ph of the food (akbar et al., 2013). edible films based on protein has been developed, such as the filming of collagen, gelatin, protein maize (corn zein), wheat protein (wheat gluten), soy protein (soy protein), casein and whey protein. based protein film are usually obtained from casting and drying (valenzuela et al., 2013). an alternative materials of edible film made from a protein is surimi. park (2004), explains that the surimi is a myofibril fish protein which has been stabilized and is manufactured through a continuous process stage which involves the removal of the head and spine, meat processing, washing, removal of water and freezing. surimi has the functional ability in gel form and bind water (amaliya et al., 2014). in this research, catfish surimi protein is added to improve the mechanical properties and lowering the water vapor transmission rate so its improve the shelf life of the product that is coated. rosella extract in this study is used as bio-indicators sensitive to ph changes that occur in food to be packaged (amaliya et al., 2014). edible film is expected to detect food spoilage by changes in ph that occur in food, have good mechanical properties and low wvtr. http://dx.doi.org/10.14421/biomedich.2016.52.33-40 34 biology, medicine, & natural product chemistry 5 (2), 2016: 33-40 materials and methods materials materials used in this research are rosella flower petals from wonosobo, dumbo catfish obtained from candipitu karangmojo, gunungkidul yogyakarta, commercial tapioca rosebrand, glycerol, sodium hydroxide pellets p.a merck, silica gel obtained from bratachem, 96% ethanol, and distilled water. preparation of rosella’s extract the extraction method in this research was maceration (marwati et al., 2012). a hundred grams of rosella were added to 300 ml 96% of ethanol at room temperature for 24 hours. solution then filtered and evaporate to give a sticky extract of rosella. preparation of dumbo catfish’s surimi surimi preparation of dumbo catfish in this research has done using heruwati & jav method (1995) methods in santoso et al. (2013). one kilogram of catfish was weeded by removing the head and entrails then washed with water. catfish meat are crushed and washed twice with cold water. the mixture was then stirred for 10 minutes. stirring was stopped to precipitate the pulverized meat, while dirt and grease is dumped. the precipitate is then dried by pressing it in a hydraulic press to remove water (bourtoom et al., 2006) preparation of edible film tapioca-surimi edible film edible film preparation was modified from santoso et al. (2011). the procedure of making this edible film are as follows. edible surimi weighed as much as 1%; 1.5%; 2%; 2.5% and 3% (w/v) of the total distilled water used as solvent. a 100 ml of distilled water then added with 1 m naoh and adjust to ph 11. mixture then stired and heated for 30 minutes at 50 c. the mixture reheated to 60 c for 30 minutes. addition of 2.5 gram tapioca and 1.5 ml of glycerol has done when the mixture were homogen. the suspension then heated and degassing for another 25 minutes, respectively. the suspension is then poured over the glass framed and dried at 50 c for 12 hours. tapioca-surimi-rosella edible film edible film preparation method using in this research is similar to the previous method but added rosella’s extract as much as 0%; 1%; 2%; and 3%, respectively. the mixture was heated for 25 minutes at 30 c and degassed at 75 kpa for 20 minutes. mixture is then poured over the glass framed and dried at 50 ° c for 16 hours. characterizations characterization of materials was conducted on the wvtr, mechanical and functional group testing using ft-ir (fourier-transform infrared spectroscopy). color test preliminary test conducted to identify anthocyanin is a color test as conducted by supriyanti et al. (2013). this test were done with the addition of hcl and naoh. hayati et al. (2012) stated that in the acid environment, an anthocyanin will remain red and under alkaline conditions anthocyanin change to bluish-green. thin layer chromatography (tlc) test further testing is to determine the presence of anthocyanin compounds using tlc methods using hcl and baa (butanol: acetic acid: water) as eluent. water vapor transmission rate (wvtr) test measurements were made using the gravimetric method based on astm. e. 96-99. moisture absorbent materials (desiccant) were put in a can, then the sample were placed at the top of the can such that the sample covers cans. wax paper used to cover part of the container with the sample so that no air enters between the boundary and the sample container (bourtoom et al., 2006). cans are weighed to the nearest 1 mg, and then placed in a desiccator at a fixed room temperature. cans are weighed every day at the same hour and determined the increasing of its weight as a consequence of desiccant material that had absorb water. the relationship between weight gain and time were graphed. wvtr value is calculated using the formula: wvtr = slope / sample area x 100 x 100 m2 = g / m2 / days (92% rh, 38oc) mechanic properties test edible film mechanic properties (thickness, tensile strength and elongation) test has done based on astm.d. 882-02. edible films are conditioned at room temperature (30 c), 68% humidity (rh) for 24 hours prior to measurement. film thickness were measured using a micrometer to the nearest 0.001 mm. thickness measurements were taken at five different points for each film sample (top right corner, bottom right corner, middle, upper left corner and the bottom left corner). film thickness value is an average of the results of measurements at five points in units of mm (national standardization agency, 2013). a set equipment test is prepared and arranged. samples of edible film mounted at the ends of both clamping and wedged firmly. the measurement area is set with loads corresponding to pen recorder off. tensile strength is determined by the maximum load, while the elongation is determined and calculated at the time the film broke. fourier transform infrared spectroscopy characterization of functional groups carried out by ftir using a kbr pellet method. one mg of sample was mixed with 200 mg of kbr powder. samples were mixed in kbr pellets by pressed mixture into a thin sedyadi et al. – starch-glycerol based edible film and effect of rosella … 35 transparent using 10 tons pressure (2000 psi). pellet samples were then measured its infrared absorption at wavenumber 4000-400 cm-1. results and discussion rosella extraction rosella (hibiscus sabdariffa l.) was extracted using maceration method to retrieve the active substance in rosella’s petals (hayati et al., 2012). extraction method is done by depositing the active compounds contained in the rosella petals using 96% of ethanol with a ratio of rosella’s powder: ethanol = 1: 5 for 24 hours. the yield obtained is about 26.16% of red extract yield. this thick red rosella extract obtained is not much different from those obtained a yield of 17.7% in the study of suzery et al. (2010). color test one of the active compound in rosella flower petals is anthocyanin. this anthocyanin are red to the blue pigment found in many fruits and flowers (robinson, 1991). this test is performed with the addition hcl and naoh. the results obtained are rosella flower petals extract supplemented with hcl will remain red, whereas one with with naoh will turn green and faded to yellow. this is in accordance with hayati et al. (2012), which states that under acidic conditions the anthocyanin color will remain red and bluish green in alkaline conditions. besides affected by ph, color changes in anthocyanin compounds are also affected by the concentration of pigments, their mixtures with other compounds as well as the number of methyl and hydroxyl groups. hydroxyl groups changes the colors tend to be blue and unstable, while methoxyl group changes of color will tend to be red and stable (warsiki, 2012). thin layer chromatography (tlc) test hcl mobile phase generating rf value of 0.37. this is consistent with the report by hayati et al. (2012) that the value of rf for anthocyanin is eluted with hcl low to mid. research conducted by suzery (2010) about the mangosteen fruit anthocyanins also generate rf value of 0.58. while the mobile phases baa generate rf value of 0.48. rf value for anthocyanin eluted with baa is 0.1-0.4, it is not much different from research conducted by warsiki et al. (2012) which also ranges from 0.25. comparison of anthocyanin of rosella flower petals with harbone (1987) is shown by table 1. based on those preliminary tests, it can be concluded that the extract obtained from rosella contains anthocyanins. these results were then confirmed by ftir characterization to determine the functional groups contained in the extract. ir spectrum of rosella flower extract is presented in figure 9. table 1. comparison antocyanin test. test result this research hayati, 2012 using hcl 2m red red using naoh 2 m turn to green then fade to yellow turn to green then fade to yellow tlc with hcl 1% eluent rf value = 0,371 low to middle rf r tlc with baa eluent rf value = 0,25 middle rf (0.100,40) surimi surimi is a myofibril protein concentrate from successive leaching process to remove unwanted compounds, resulting in a semi-pure protein fraction containing high myofibrils protein. the making of catfish’s surimi is done by separating the meat from the head and the stomach contents of fish with several stages of the rinsing, suppression, the addition of salt (nacl), and the addition of cryoprotectant. rinsing is an important step because it can support the ability of gelation (ashi) and inhibit protein denaturation due to freezing (santoso, 2007). 100 grams of fresh meat catfish yielded 64.86% solid white surimi. the surimi obtained are relatively small due to repeated washings during the washing process. according to santoso (2004), during the washing process many materials are dissolve by water. identification of functional groups on surimi has done using ft-ir at 4000-400 cm-1. ft-ir spectrum of the surimi is presented in figure 10. tapioca-surimi edible film the principle of production edible film manufacture with variations of surimi was prepared by dissolving surimi under alkaline conditions. surimi containing protein myofibril that insoluble in water but soluble in an alkaline. in addition myofibril protein denaturation is essential to form a wider structure for the film formation. cause denaturation of proteins into long parallel chains that can bind with other polymers such as starch. proteins can form edible film because of the ability of the side chain to form intermolecular crosslinking (bourtoom et al., 2006). mechanical properties test of edible film thickness edible film thickness against amount variation of catfish surimi is presented in figure 1 which shows that the surimi edible film thickness ranged from 0.09 to 0.12 mm. krochta (1994) explains that the film thickness correlated to the number of three-dimensional network that is formed so that the film is getting thicker. the concentration of dissolved material in the film-forming 36 biology, medicine, & natural product chemistry 5 (2), 2016: 33-40 solution and casting plate size also affects the thickness of the film. figure 1. graph of thickness vs surimi variation. the results showed that the addition of surimi does not significantly affect edible film thickness. tensile strength tensile strength is the maximum achievable pull of a film before it breaking. the tensile strength test with a variety of surimi edible film is presented in figure 2 which shows that the tensile strength ranging between 0.83 to 1.09 n. the best tensile strength value is 1.09 n with surimi concentration as much as 3 g. edible films with surimi addition showed a trend of increasing tensile strength values, though not very significant. figure 2. graph of tensile strength vs surimi variation. this is consistent with chinabhark et al. report (2007) which showed that a protein in a high amount of polymer allows the intermolecular interaction getting bigger and stronger. this causes an increase in the value of tensile strength edible film. elongation elongation is the maximum length changes of edible film during a stretch off. flexibility properties of edible film are very important thing in the coating process food. edible films with a elastic and flexible texture are easier to set up, so that the packing of the food is good (cahyadi, 2009). figure 3. graph of elongation vs surimi variation. elongation measurement is the maximum film length changes when obtaining tensile force to break up the film compared to the initial length (hambali et al., 2007). the greater the elongation of edible film, edible film has more flexibility of being able to do a maximum extension. elongation measurement of edible films in variation of surimi are presented in figure 3. figure 3 shows that the edible film elongation of surimi variations tends to decrease with increasing concentration of surimi. maximum elongation of 21.47% is achieved at a concentration of 2.5% surimi. lowest elongation of 17.99% was obtained at a concentration of 3% surimi. according to sinaga et al. (2013) an edible film elongation value are low, due to the denaturation of myofibril surimi protein, and therefore contributes to a decrease in elongation. water vapor transmission rates (wvtr) water vapor transmission rate is an important parameter in the edible film. water vapor transmission rate indicates the speed of the water vapor to penetrate (per gram per second) per unit area of edible film or the film's ability to inhibit the transmission. the permeability to water vapor should be low (garnidaet al., 2007). the chemical composition of the food surface can be changed due to metabolism of food, microbial respiration, gas solubility, and film permeability to water vapor. edible films rely on its barrier properties against water vapor and gas transfer. edible film with good barrier properties can extend the shelf life of the coated products (skurtys et al., 2009). the wvtr measurement of amount surimi variations edible films are presented in figure 4. it appears that the wvtr values of edible film ranged from 3.59 to 4.01 g / m2hours. the results showed that the addition of surimi concentration tends to decrease the wvtr values. this is because the surimi protein undergo denaturation. protein denaturation cause proteins undergo changes in which the hydrophilic side on the side inside and outside the hydrophobic side will turn into a hydrophobic side out so the water vapor transmission rate to be down (santoso, 2013). 0.0975 0.1002 0.1019 0.0951 0.1196 0 0.05 0.1 0.15 1 1.5 2 2.5 3 0.8349 0.9257 0.9871 0.7021 1.0903 0 0.25 0.5 0.75 1 1.25 1 1.5 2 2.5 3 20.6989 19.8819 19.6328 21.4732 17.9857 0 5 10 15 20 25 1 1.5 2 2.5 3 sedyadi et al. – starch-glycerol based edible film and effect of rosella … 37 figure 4. graph of wvtr vs surimi variation. based on the mechanical test it can be concluded that the addition of surimi is not significantly affect mechanical properties of the resulting edible film. nevertheless it can be seen that the best edible film in this study are edible film with the addition of surimi variation of 2.5% because it has the highest elongation. edible films with this comparison will be used further to add a rosella extract. tapioca-surimi-rosella extract edible film the maximum data obtained will be used for variety addition of rosella flower extract as bio-indicators of the edible film. the production is the same as edible film mentioned above. the suspension of the obtained printed and dried at 40 °c for 24 hours. films were then tested the mechanical properties which include thickness, tensile strength, and elongation, wvtr and ft-ir analysis (cahyadi, 2009). thickness the results of the thickness measurements are presented in figure 5. it appears that the edible film thickness value surimi-starch-glycerol-rosella extract produced ranged from 0.085 to 0.095 mm. results of edible film thickness with 2.5 grams of surimi composition and the addition of 0.75 g roselle extract is greater than the addition of 0.5 grams of roselle extract is equal to 0.09 mm. the thickness of the edible film that is formed is influenced by the film-forming solution concentration and the size of the plate. the results showed that the addition of rosella extract also does not significantly affect the resulting film thickness. the more the concentration of rosella extract is added, the thickness of the resulting film tends to decrease, but not significantly (dewi et al., 2010). leerahawong et al. (2011) explains that the edible film thickness due to differences in the concentration of film-makers, while the volume of film of the solution are poured into each of the same plate. this resulted in the total solids in the film after drying increases and polymers making up the matrix movies more and more. the thickness between the various types of film can cause by the composition formula, of different films. figure 5. graph of thickness vs rosella extract variation. tensile strength the results of the measurement of tensile strength are presented in figure 6. it appears that the value of the tensile strength composite edible film surimi-starchglycerol-rosella extract produced ranged from 0.39 to 0.70 n. the addition of 0.5 gram rosella extracts initially lower the value of the tensile strength of the edible film to 0.39 n. the addition of 0.75 grams rosella extract can increase the value of the tensile strength of the edible film becomes 0.52 n, but still relatively lower than edible film without the addition of extract of rosella (dewi et al., 2010). figure 6. graph of tensile strength vs rosella extract variation. the data obtained show that the more rosella extract is added, and then it tends to lower the value of tensile strength of the resulting edible film. the addition of 0.75 g rosella extract slightly increases tensile strength relative value of the addition of 0.5 grams rosella extract. this is because more film added can increase the density of the resulting film that is not easily torn. figure 6 also shows that the trend of edible film tensile strength value is almost equal to the thickness of the edible film, so it cannot be used as a standard of determining the quality of the food was good coating. elongation elongation measurement results showed in figure 7. result shows that the elongation value of the composite 4.0123 3.9012 3.5925 3.8889 3.7284 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 1 1.5 2 2.5 3 0.0951 0.0852 0.0915 0 0.05 0.1 0.15 0 0.5 0.75 0.7021 0.3962 0.5213 0 0.2 0.4 0.6 0.8 1 0 0.5 0.75 38 biology, medicine, & natural product chemistry 5 (2), 2016: 33-40 edible film of surimi-starch-glycerol-rosella extract ranged 21,47-24.95%. best elongation value contained in the addition of 0.5 grams of rosella extract. figure 7. graph of elongation vs rosella extract variation. elongation measurement showed that the addition of rosella extract tends to increase the value of the elongation of edible film. an edible film is said to be used as food coatings if they have a high value percent elongation and water vapor transmission rate is relatively low. this is because of a growing number of its constituent components, the edible film more tightly. percent elongation produced is inversely proportional to the thickness and tensile strength. water vapor transmission rates (wvtr) the wvtr measurement of edible film composite are shown in figure 8. figure 8. graph of wvtr vs rosella extract variation. based on the figure 8, it showed that the greater the amount of rosella added, the greater the wvtr. a better edible film is a film that has a high elongation and low wvtr values. charaterization of fourier transform infrared spectroscopy rosella petals extracts were analyzed using ft-ir spectrophotometer to determine the character of the resulting functional groups. the spectrum is presented in figure 9. the identification of functional groups on the extract rosella flower petals carried on the wave number 4000-400 cm-1. ft-ir spectrum at rosella flower petals extracts showed some characteristic absorption band in the absorption peaks. figure 9. ft-ir spectrum of rosella extract. based on the ft-ir spectrum in figure 9, it appears the wide absorption at wave number 3356.14 cm-1 indicating the oh absorption band. wavenumber 2947.23 cm-1 indicating their c-h groups are strengthened by their absorption at wavenumber 1411.89 cm-1. both of these absorptions indicate methyl group in edible film cluster. absorption around wavenumber 1728.22 cm-1 indicates the group of carbonyl. while c = c group indicated by adsorption at wave number 1643.35 cm-1. moulana (2012), stated that the absorption at wavenumber 3348.25 cm-1 indicating their alcohol groups are supported by the appearance of absorption at wavenumber 1044.35 cm-1 to c-o bond alcohol. absorption at wavenumber 1706.30 cm-1 indicate the presence of c = o carbonyl group. the presence of the c = c double bonds of aromatic demonstrated by a sharp absorption at wave number 1634.95 cm-1 which is supported also by the appearance of absorption at wave number 709.80 cm-1 to c-h bond. based on the ft-ir spectra interpretation rosella extract, it can be concluded that the rosella extract containing anthocyanin compounds shown by their group c = o, c-o and c = c which is the structure of anthocyanin. figure 10. ft-ir spectrum of surimi. ft-ir spectrum of surimi is presented in figure 10. it showed their wide absorption at 3425.58 cm-1 due to 21.4732 24.9561 22.8954 19 20 21 22 23 24 25 26 0 0.5 0.75 3.8889 5.5555 5.3086 0 2 4 6 8 10 0 0.5 0.75 sedyadi et al. – starch-glycerol based edible film and effect of rosella … 39 the strain nh associated with hydrogen bonds and the oh group. uptake of n-h is not visible as it is covered by the oh absorption. absorption at wavenumber 2924.09 cm-1 indicates the presence of an amine group stretching. a bending amine group showed at wavenumber 2854.65 cm-1 with a sharp peak. this indicates that the amide binds to the ch2 strain. in addition there is absorption at wavenumber 3873.06 cm1 which indicate the presence of free nh groups. absorption peaks at 1643.35 wave numbers indicate a carbonyl (c = o). according to poedjiadi (2009) absorption at wave number 1636-1661 cm-1 showed absorption of amide group. this amide catchment area showed strain c = o and oh groups are paired with a carboxyl group. next on the wave number 1442.75 cm-1 indicates ch2 bending and deformation nh bond in the protein. based on the surimi’s ft-ir spectrum interpretation above, it can be concluded that the surimi protein shown their amide groups (nh). edible films produced spectra identification of cluster compounds using ftir spectrophotometer. the resulting spectra are presented in figure 11. figure 11. ft-ir spectra of edible film starch-glyserol (a), starchglyserol-surimi (b), and starch-glyserol-surimirosella extract (c). starch-glycerol spectrum is shown in figure 11.a, starch-glycerol-surimi in figure 11.b and starchglycerol-surimi rosella extract in figure 11.c. the presence of oh groups on edible film showed by absorption at wavenumber 3448.72 cm-1. the absorption shifted to 3425.58 cm-1 in the edible film with the addition of surimi. the existence of the ch (stretching) groups of edible film showed in wavenumber 2931.80. addition of surimi edible film shown in wave numbers 2924.09 cm-1. new absorption appears on edible film with the addition of surimi. the uptake appears at wave number 2862.36 cm-1 which indicates the presence of an amine group that binds to the stretching of ch2 groups. new absorption also appears on edible film with the addition of surimi. the uptake appears at wave number 2299.15 cm-1. group c = o (carbonyl) on edible film tapioca and edible film with the addition of surimi shown in wave number 1635.64 cm-1. the shift of wave numbers show that the polymerization reaction time of the blending of the edible film and shows the interaction between polymers occurs. figure 11.c of ft-ir surimi edible film-starchglycerol-rosella extract spectra showed some shift wave numbers compared to edible starch-glycerol composite film that is at 2901.60 cm-1 to 2924.09 cm-1 and 1331 cm-1 to 1334.74 cm-1. some of the absorption band is also changing the intensity and emerging new absorption band. ft-ir spectra showed a broad absorption on say 3400-3500 cm-1 wave. khopkar (2008), explains that the absorption band at wave number 3600 cm-1 indicate the presence of oh groups on edible film oh. the absorption of tapioca with the addition of surimi and rosella is located at wavenumber 3448.72 cm-1. ch (stretching) between edible films edible film tapioca with the addition of surimi and rosella extract is shown in wave number 2924.09 and 2854.65 cm-1. the new spectra appear on edible film with the addition of surimi and rosella extract. the uptake appears at wave number 2862.36 cm-1 which indicates the presence of an amine group that binds to a stretch stretching ch2. in addition, the new spectra also appear on edible film with the addition of surimi and rosella extract. the uptake appears at wave number 2299.15 cm-1. group c = o (carbonyl) on tapioca ediblefilm and edible film with the addition of surimi and rosella extract is shown in wave number 1635.64 cm-1. the shift wave numbers show that the polymerization reaction time of the blending of the edible film and shows the interaction between polymers occurs. ft-ir spectra in figure 10 shows that the addition of surimi and rosella extract does not change the pattern of uptake significantly edible film. it is common because the main ingredients of the edible film are tapioca and glycerol so that the pattern of ir absorption of three quite similar edible film. differences absorption occurs only in some shift in absorption of a wave shift. the emergences of new peaks indicate the presences of a bond between the materials were mixed. differences in intensity indicate the bond between the materials grows denser and more complex. conclusion the research showed that the addition of surimi and rosella flower extract significantly increase up to 27% of the elongation of edible film. references akbar, f., anita, z. harahap, h. 2013. pengaruh waktu simpan film plastik biodegradasi dari pati kulit singkong terhadap sifat mekanikalnya. jurnal teknik kimia usu. 2 (2). 40 biology, medicine, & natural product chemistry 5 (2), 2016: 33-40 amaliya, r.r., putri w.d.r. 2014. karakterisasi edible film dari pati 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new york. poedjiadi, a.; supriyanti, f.m.t. 2009. dasar-dasar biokimia. ui-press: jakarta robinson, t. 1991. kandungan organik tumbuhan tinggi edisi keenam: terjemahan prof. dr. kokasih padmawinata.; itb: bandung. santoso, j., yasin, a.w.n., santoso. 2007. perubahan sifat fisiko-kimia daging lumat ikan cucut dan pari akibat pengaruh pengkomposisian dan penyimpanan dingin. jurnal perikanan dan kelautan. 1: 1-7. santoso, b., saputra, d., pambayun, r. 2004. kajian teknologi edible coating dari pati dan aplikasinya untuk pengemas primer lempok durian. jurnal teknologi dan industri pangan. xv (3). santoso, b., pratama, f., hamzah, b., pambayun, r. 2011. pengembangan edible film dengan menggunakan pti ganyong termodifikasi ikatan silang. jurnal teknologi dan industri pangan. xxii (2). santoso, b., herpandi, a., pambayun, r. 2013. karakteristik film pelapis pangan dari surimi belut sawah dan tapioka. jurnal teknologi dan industri pangan. 24 (1). setiono, m., dewi, a. 2013. penentuan jenis 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quinoa proteinchitosan-sunflower oil edible film: mechanical, barrier and structural properties. food science and technology. 50: 531537. warsiki, e., putri, c. d. w. 2012. pembuatan label/film indikator warna dengan pewarna alami dan sintesis. ejurnal agroindustri indonesia. 1 (2): 82-87. starch-glycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on its mechanical properties biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 607-610 | doi: 10.14421/biomedich.2023.122.607-610 issn 2540-9328 (online) prevalence of enterobacteriaceae producing extended spectrum beta-lactamase (esbl) in broiler meat (gallus domesticus) sold in regional markets of west surabaya kadek rio risnanda1, masfufatun2,*, agusniar furkani listyawati3, kuntaman kuntaman3, akhmad sudibya3 1faculty of medicine; 2department of biochemistry; 3department of microbiology, faculty of medicine, universitas wijaya kusuma surabaya jl. dukuh kupang xxv no. 54, dukuh kupang, kec. dukuh pakis, kota surabaya, jawa timur, indonesia. corresponding author* masfufatun@uwks.ac.id manuscript received: 23 august, 2023. revision accepted: 03 november, 2023. published: 09 november, 2023. abstract broiler meat is a foodstuff that is easily damaged by bacterial growth. several pathogenic bacteria from the enterobacteriaceae family have also been found as contaminants in chicken meat. while extended spectrum beta-lactamases (esbl) are enzymes that have the ability to hydrolyze antibiotics of the penicillin class, first, second, and third generation cephalosporins as well as the monobactam group. based on this phenomenon, the researchers wanted to examine the prevalence of the esbl-producing enterobacteriaceae family in broiler chicken (gallus domesticus) meat sold in the west surabaya market. this research is a descriptive observational type using a cross-sectional approach. the population in this study was broiler chicken meat taken from broiler traders in several markets in west surabaya which had met the inclusion and exclusion criteria with a large sample of 50 samples. then the data were analyzed using the chi-square test. the results of the analysis showed that 13 samples produced esbl. the prevalence of enterobacteriaceae bacteria producing esbl in broiler chicken (gallus domesticus) meat in several markets in west surabaya is 26% of the 50 samples used. the results of the chi-square test showed that there was no significant difference between locations where esbl was found. keywords: enterobacteriaceae; extended spectrum beta-lactamase; broiler chicken meat. abbreviations: esbl extended spectrum beta-lactamase; mdr: multiple drug resistance. introduction poultry meat, especially chicken meat, is a source of animal protein that is in great demand in indonesia (region, 2019). most of indonesia's demand for meat comes from poultry, especially chickens. based on 2018 data, broiler production increased by 480,309 heads. according to statistical data for 2019, the consumption of chicken meat is 4.94 kg per person per year (direktorat jenderal peternakan dan kesehatan hewan, 2019). broiler meat is a food ingredient that is easily damaged by bacterial growth. marketed chicken meat can be a breeding ground for microbes that cause food poisoning. several pathogenic bacteria from the enterobacteriaceae family have also been found as contaminants in chicken meat, including escherichia coli, staphylococcus aureus, salmonella sp., pseudomonas sp., clostridium perfringens and shigella flexneri (ray, 2014). enterobacteriaceae contamination of food occurs through soil, air, water, dust, digestive tract, human hand contact, and the influence of animals such as cockroaches and flies (mu’arofah et al. 2020). laying the carcass or body parts of chickens directly on the ground after slaughtering a chicken that has been washed can result in contamination with carcass remnants, such as blood, feathers, excrement, and its own stomach contents (ramadhani et al., 2020). a study by mashak revealed that 16.25% of chicken meat from alborz, iran, was e.coli positive. previous research also stated that several samples of chicken meat from local markets in indonesia presented microbial contamination, which included s. aureus (6.7%), salmonella sp. (85%), and e. coli (90.03%). contamination of poultry products including raw broiler meat by pathogenic microorganisms, especially bacteria, has become one of the most challenging problems in the food industry worldwide (wardhana et al. 2021). extended spectrum beta-lactamases are enzymes that have the ability to hydrolyze penicillin class antibiotics, first, second and third generation cephalosporins as well as monobactam groups and cause resistance to all of these antibiotics (biutifasari, 2018). https://doi.org/10.14421/biomedich.2023.122.607-610 mailto:masfufatun@uwks.ac.id 608 biology, medicine, & natural product chemistry 12 (2), 2023: 607-610 epidemiologically, the prevalence of esbl varies in various countries. the prevalence of esbl produced by enterobacteriaceae families such as e. coli and klebsiella pneumoniae varies, ranging from 42.7% in latin america, 5.8% in north america, 2% to 31% in europe, and 4.8% to 12% in isolates. in asia (yessy, 2021). research in five hospitals in indonesia in 2013 showed the prevalence of esbl-producing bacteria was 32-68% (kuntaman, 2013). the results of the 2016 amr study by the department of health control on antimicrobial resistance in 8 hospitals with esbl indicator bacteria e. coli and k. pneumoniae showed an average prevalence of 60%. beta-lactams are used against gram-positive and gram-negative bacteria. bacterial resistance to lactams are using three pathways: disruption of the antibiotic lactamase enzyme, alteration of the target of the antibiotic and decreased cellular uptake of the antibiotic. all of these pathways play an important role in antibiotic resistance. however, lactam-producing and lactamdestroying bacteria are the main causes of resistance (masruroh et al. 2016). antibiotics are chemical substances produced by fungi or bacteria that have the ability to kill or prevent the growth of pathogenic microorganisms, while their toxicity to humans is relatively low. irrational use of antibiotics causes negative effects, such as the immunity of microorganisms to some antibiotics, increased drug side effects and even death. bacterial resistance to antibiotics is a global health problem that threatens the treatment of infectious diseases and causes serious disability and death in the healthcare community. bacteria that naturally carry antibiotic resistance genes can transfer these genes to other bacteria. in addition, bacteria can produce enzymes that inhibit the action of antibiotics (aidara-kane et al., 2013). one of the antibiotics used is a beta-lactam class of antibiotics which works by inhibiting the cell wall. improper use of antibiotics, including selection, dosage, and patient nonadherence to treatment, play a role in the development of antibiotic resistance. one of the resistance mechanisms of gram-negative bacteria belonging to the enterobacteriaceae family is the production of esbl. according to the results of a study on 781 hospital patients throughout indonesia, 81% of escherichia coli were found to be resistant to various antibiotics, namely ampicillin (73%), cotrimoxazole (56%), chloramphenicol (43%), ciprofloxacin (22%), and gentamicin (18%) (kementerian kesehatan republik indonesia, 2013). based on the data and research results obtained above, the researchers wanted to examine the prevalence of enterobacteriaceae family bacteria such as e. coli producing esbl in broiler chicken meat (gallus domesticus) sold in west surabaya market in the hope of knowing whether the consumption of broiler chickens contaminated with bacteria can affect the percentage of indonesian people who are resistant to antibiotics due to the high e. coli bacteria in broiler chicken meat. materials and methods research design this research is a descriptive observational type using a cross-sectional approach. population and research sample the population in this study was broiler chicken meat taken from broiler traders in several markets in west surabaya with a sample size of 50 samples that met the inclusion and exclusion criteria. this sampling used reference to the basic and clinical biostatistics book. data analysis techniques the method used in this study was univariate analysis to determine the prevalence rate of esbl-producing bacteria in meat sold in markets in west surabaya and bivariate analysis using the chi-square test. results and discussion results table 1. identification results of enterobacteriaceae producing extended spectrum beta-lactamase (esbl) in broiler chicken (gallus domesticus) meat in regional markets of west surabaya. location number of samples extended spectrum betalactamase (esbl) i 10 2 ii 10 4 iii 10 2 iv 10 2 v 10 3 total 50 13 (26%) notes: i: darmo market location, ii: darmo market location, iii: kupang dukuh market location. iv: kupang gunung market location, v: simo gunung market location figure 1. lactose fermenter bacteria isolated from broiler chicken meat. risnanda et al. – prevalence of enterobacteriaceae producing extended spectrum … 609 based on the table and figure above, it can be seen that from 50 samples of broiler chickens in several markets in west surabaya, 19 samples were positive for enterobacteriaceae and 13 of them produced extended spectrum beta-lactamase (esbl). at location i there were 2 positive samples for esbl. of the 10 samples at location ii, 5 samples were positive for enterobacteriaceae and 4 of them produced esbl. of the 10 samples at location iii, there were 4 positive samples for enterobacteriaceae and 3 of them produce esbl. at location iv, 5 positive samples were found for enterobacteriaceae and 2 of them produced esbl and at location v, out of the 10 samples used, 3 samples were found positive for enterobacteriaceae and all of them esbl. based on table 1, it can be calculated the prevalence of enterobacteriaceae bacteria producing esbl in broiler chicken (gallus domesticus) meat in several markets in west surabaya, namely 26% of the 50 samples used. table 2. crosstab places with enterobacteriaceae bacteria. location enterobacteriaceae total positif negatif pasar darmo permai 2 (4%) 8 (16%) 10 (20%) pasar darmo 5 (10%) 5 (10%) 10 (20%) dukuh kupang 4 (8%) 6 (12%) 10 (20%) pasar kupang gunung 5 (10%) 5 (10%) 10 (20%) pasar simo gunung 3 (6%) 7 (14%) 10 (20%) total 19 (38%) 31 (62%) 50 (100%) based on table 2, it shows that most of the enterobacteriaceae were found in the darmo market and kupang gunung market, namely each (10%). table 3. relationship of place with extended spectrum beta-lactamase (esbl) bacteria. location esbl total p-value positif negatif darmo 6 (30%) 14 (70%) 20 (100%) 0,599 kupang 7 (23%) 23 (76%) 30 (100%) total 13 (26%) 37 (74%) 50 (100%) the results of the chi-square test showed that there was no significant difference between locations where extended spectrum beta-lactamase (esbl) was found. discussion the results of the analysis showed that from 50 samples of broiler chickens in several markets in west surabaya, 19 samples were positive for enterobacteriaceae and 13 of them produced extended spectrum beta-lactamase (esbl). the prevalence of enterobacteriaceae bacteria producing extended spectrum beta-lactamase (esbl) in broiler chicken (gallus domesticus) meat in several markets in west surabaya is 26% of the 50 samples used. research by puspandari, sunarno, et al. was conducted in four markets and two slaughterhouses in the jakarta area. research with a total of 240 chicken meat samples showed that esbl-producing e. coli was isolated from 161 (67.1%) of 240 broiler samples, 161 (84.3%) of 191 suspected colonies on macconkey agar supplemented with cefotaxime 0 media .4% confirmed as esbl producer e. coli (puspandari et al., 2021). the results of this study provide information about esbl-producing enterobacteriaceae associated with antibiotic resistance found in broiler chickens in several markets in west surabaya. the problem of multipledrug resistance (mdr) is exacerbated by the ability of bacteria to transfer genetic material carrying resistance properties from one bacterium to another vertically through genetic mutation and horizontally through conjugation, transduction and transformation. mdr occurs when more and more antibiotics are used, the greater the selective pressure on the process of evolution and proliferation of resistant bacterial strains to defend themselves so that vertical resistance arises from genetic mutations and horizontal resistance from the exchange of resistant gene material against various types of different antibiotic resistance mechanisms. (gregova et al. 2012). multidrug resistance is a common occurrence in esbl producing bacteria. aminoglycoside modifying enzyme (ame) and esbl are genes that encode resistance enzymes commonly found in bacterial plasmids. that is, gene transfer occurs in genetic factors such as transposons, integrons, and plasmids (aloccati et al., 2013). the esbl gene that appears in e. coli is caused by a plasmid-mediated genetic mutation, especially the esbl gene with tem and shv types, then a new esbl group appears, namely ctxm (hasibuan, 2017). this study is in accordance with research conducted (wibisono et al. 2020) the results obtained from 185 samples of cloacal swabs which tested positive for esbl-producing e. coli contained 7.03% esbl (ddts). food products of animal origin contaminated with the enterobacteriaceae family such as esblproducing e. coli can pose a health risk, although the level of risk is difficult to measure. broiler chickens can serve as a reservoir for esbl e. coli. esbl bacteria are transmitted in several ways, namely: consuming contaminated meat, faecal contaminated environment containing esbl-producing e. coli, exposure to patients or people infected with esbl and transmitted to humans via animals and possibly causing zoonoses. contamination of the broiler chickens under study can also come from the water used to wash the chickens, the equipment used by traders to cut the chickens and other equipment that has direct contact with the meat, as well as the hygiene of the chicken slaughterhouse itself. 610 biology, medicine, & natural product chemistry 12 (2), 2023: 607-610 placing chicken meat directly on the sales counter without the need for a special place and handling such as packaging or temperature control which is also commonly found in the market can affect the level of bacterial contamination, one of which is e. coli bacteria. flies can infect meat as vectors for e. coli. in addition to flies, the hands of buyers and sellers who are not clean can cause the meat to be exposed to e. coli bacteria. it is known that e. coli is harmful to health because it produces a toxin (shiga toxin) (reich et al., 2013). conclusions the results of the analysis showed that out of 50 samples of broiler chickens in several markets in west surabaya, 13 samples produced extended spectrum betalactamase (esbl). the prevalence of enterobacteriaceae bacteria producing extended spectrum beta-lactamase (esbl) in broiler chicken (gallus domesticus) meat in several markets in west surabaya, namely 26% of the 50 samples used, most of the enterobacteriaceae were found in darmo market and kupang gunung market, respectively each by (10%). the results of the chi square test showed that there was no significant difference between locations where extended spectrum beta-lactamase (esbl) was found. acknowledgements: this research is supported by prof. kuntaman, dr., ms., sp.mk(k), and also institute research and community service that has provided research funding assistance. authors’ contributions: kadek rio risnanda & kuntaman designed the study. kadek rio risnanda, masfufatun, and agusniar furkani listyawati carried out the laboratory work. kadek rio risnanda analyzed the data and wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: funding for this research is institute of research and community service references aidara-kane, a., andremont, a., collignon, p. 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(0711) 580056, website: biologi.mipa.unsri.ac.id, indonesia. corresponding author* endingpermata11@gmail.com abstract indonesia is one of the countries rich in natural resources, especially plant diversity. this plant diversity includes various plants that have the potential of being traditional medicine. types of plants that can be used in traditional medicine are useful for curing various diseases in the community and can be used as raw materials for medicine. in ogan komering ulu timur (oku timur) district there are 20 subdistricts but only 2 sub-districts are used as plant exploration areas that are used as observation objects, namely bunga mayang and martapura. the plant surveyed is the dadap ayam plant (erythrina variegata l.), used in traditional medicine by the javanese people in buay madang timur, oku timur. to determine the chemical content and antioxidant activity of the dadap ayam plant leaves. the dadap ayam plant is used in traditional medicine by the javanese people in buay madang timur, oku timur and is known as a herbal plant based on previous research journals. the study of the antioxidant activity of dadap ayam leaves was conducted from october to march 2025. sampling was conducted in bunga mayang district, peracak village and martapura district, east ogan komering ulu regency, south sumatra. dadap ayam leaf samples were identified at the botany laboratory, department of biology, faculty of mathematics and natural sciences, university of lampung (unila). sample processing such as extraction, fractionation and compound purification were performed at the genetics and biotechnology laboratory, department of biology, faculty of mathematics and natural sciences, sriwijaya university (indralaya). metabolite profile analysis of dadap ayam leaf extract using gc-ms was conducted at the integrated research and testing laboratory, universitas gadjah mada yogyakarta. the results of this study obtained strong antioxidant activity in the thick extract of dadap ayam leaves (erythrina variegata l.) obtained in the n-hexane fraction, which was 5.4306 ppm and the ethyl acetate fraction, which was 17.819 ppm. the methanol-water fraction was not active in antioxidant activity. the groups of pure compounds that have antioxidant activity obtained in dadap ayam leaf samples are the terpenoid group (n1), phenol group (n2 and e1), steroid group (n3) and flavonoid group (n4, and e4). pure compounds obtained from dadap ayam leaf samples have very strong, strong and moderate antioxidant activity. pure compounds with the terpenoid group (n1) obtained an ic50 value of 58.49 ppm. the phenol compound group (n2 and e1) obtained an ic50 value of (17.12 ppm and 100.15 ppm). meanwhile, the steroid compound group (n3) obtained an ic50 value of (65.59 ppm) and the flavonoid compound group (n4 and e4) obtained an ic50 value of (10.29 ppm and 25.09 ppm). keywords: antioxidants; dadap ayam (erythrina variegata l.); medicinal plants; metabolite profile; ogan komering ulu timur (oku timur). introduction indonesia is one of the countries rich in natural resources, especially plant diversity. this plant diversity includes various plants that have the potential of being traditional medicine. types of plants that can be used in traditional medicine are useful for curing various diseases in the community and can be used as raw materials for traditional medicine. traditional medicine is medicine made simply or traditionally by parents in ancient times that is still used by the community today. not many people are interested in using traditional medicine because many people in this era do not know the content of each plant in their yard/around their place of residence that can be used as traditional medicine. according to oktaviani et al., (2020), medicinal plants can be found in various habitats such as forests, agricultural land and planted in home yards. yard land is one of the lands that has the potential to be used to plant various plant species such as ornamental plants, fruits, vegetables, spices, and other food crops. in east ogan komering ulu regency (east oku) there are 20 sub-districts but only 2 sub-districts are used as exploration areas for plants that are used as objects of observation, namely mayang and martapura flowers. the benefits of the leaves of the dadap ayam plant in manuscript received: 01 may, 2025. revision accepted: 27 june, 2025. published: 15 july, 2025. https://doi.org/10.14421/biomedich.2025.141.395-400 396 biology, medicine, & natural product chemistry 14 (1), 2025: 395-400 east ogan komering ulu are believed to reduce internal heat, treat gout, smooth menstrual blood, postpartum and oxidative stress. how to use it is to mix the leaves with oil added with hot water then drink it and the leaves can also be crushed so that the water comes out and applied to the forehead with a fever. another way is to crush the leaves until they are crushed, then add a little water, compress them on the head, stomach and chest (yudianto et al. 2021). antioxidants activate the development of oxidation reactions by binding free radicals and highly reactive molecules so that cell damage can be inhibited. antioxidants can work by overcoming damage to human skin caused by the effects of free radicals which are the main factors in the aging process and skin tissue damage (utami, 2021). information regarding the efficacy of the dadap ayam plant as an antioxidant is traditionally still very limited and has not been widely studied in indonesia. based on research conducted by previous researchers, the researchers aimed to determine the antioxidant activity content of the leaves of the dadap ayam (erythrina variegata l.) plant. materials and methods study area study areathe study of antioxidant activity of leaves of dadap ayam (erythrina variegata l.) was conducted from october to march 2025. sampling was conducted in bunga mayang district, peracak village and martapura district, east ogan komering ulu regency, south sumatra (figure 1). identification of dadap ayam leaf samples was conducted at the botany laboratory, biology department, faculty of mathematics and natural sciences, university of lampung (unila). sample processing such as extraction, fractionation and compound purification were conducted at the genetics and biotechnology laboratory, biology department, faculty of mathematics and natural sciences, university of sriwijaya (indralaya). metabolite profile analysis of dadap ayam leaf extract using gc-ms was conducted at the integrated research and testing laboratory, gadjah mada university, yogyakarta. figure 1. map of sampling research locations. the tools used in this study were stationery, camera, stirring rod, blender, glass bottle, vial bottle (3ml and 10ml), funnel, separating funnel, 2000ml erlenmeyer flask, glass, beaker, measuring cup, scissors, hot plate, hair dryer, column, cuvette, evaporator flask, fume hood, capillary pipette, dropper pipette, rotary evaporator (ika rv 10), tweezers, petri dish, small tube, uv-visible spectrophotometer, stand, analytical/analog scale and gc-ms trace tm 1310 isq. the materials used in this study were samples of leaves of the dadap ayam plant (erythrina variegata l.) weighing 500g. the chemicals used were methanol, ethyl acetate, n-hexane, klt plate, aluminum foil, silica gf254 klt plate, 1,1-diphenyl2picrylhydrazyl (dpph) powder, distilled water, aluminum chloride, a1c13 p.a, e merck, hydrochloric acid, hcl, 37%, ascorbic acid, boric acid, hbo3, e merck, sulfuric acid, cotton, label paper, filter paper, sodium acetate, naoac, e merck, sodium hydroxide, and naoh p.a, 99%. results and discussion extraction of dadap ayam (erythrina variegata l.) leaves based on the results of the study of the extraction of dadap ayam by maceration with methanol solvent, the weight of the thick extract and the percentage of yield of dadap ayam leaf extract was obtained. the following is the data on the weight of the thick extract and the percentage of yield of methanol extract of dadap ayam leaves. table 1. weight of thick extract and percentage of methanol extract yield of dadap ayam leaves. no. simple weight condensed extract weight extract yield weight (gram) (%) 1 500 57,64 11,53 from the results of the study in (table 1) it shows that from 500g of dadap leaf simplicia that has been macerated using 3l of methanol solvent with 3 times of remaceration process, a thick extract of 57.64g was obtained and the percentage value of the extract yield was 11.53%. for the percentage weight of the dadap chicken leaf extract yield, it is above 10% and this can be said that the results of the extraction process are quite good. according to ramdhini (2023), the yield calculation is carried out to determine the ratio of the weight of the simplicia or extract produced to the weight of the raw material. the general requirement for raw material yield is >10% (more than 10%). permata et al. – potential medicinal plant antioxidant compounds based … 397 fractionation of chicken dadap leaf extract (erythrina variegata l.) based on the results of the study, it was shown that the fractionation of methanol extract of dadap ayam leaves using 3 solvents, namely: n-hexane, ethyl acetate and water methanol obtained different weights of fractions and percentage yields of dadap ayam leaf extract fractions as shown in (table 2). the following are the results of the weights of fractions and percentage yields of methanol extract fractions of dadap ayam leaves. table 2. fraction weights and percentage yields of dadap ayam leaf extract fractions. no. solvent type fraction weight fraction yield (%) 1 n-hexase 23,21 40,27 2 ethyl acetate 24,99 43,36 3 methanol water 9,44 16,37 the study’s results in (table 2) show that the weight of the n-hexane fraction is 23.21g, the weight of the ethyl acetate fraction is 24.99g and the weight of the water methanol fraction is 9.44g. meanwhile, the percentage of the n-hexane fraction yield is 40.27%, the yield of the ethyl acetate fraction is 43.36% and the yield of the water methanol fraction is 16.37%. from these results it can be seen that the ethyl solvent has a larger fraction weight than n-hexane because the n-hexane solvent tends to produce more pure compounds compared to the ethyl acetate solvent because it is different in polarity. the n-hexane solvent is a more effective solvent in dissolving and separating nonpolar compounds (nonpolar solvents). meanwhile, the ethyl acetate solvent is a non-polar medium that can dissolve both polar and nonpolar compounds (semipolar solvents). according to sutomo et al. (2021), fractionation with ethyl acetate is intended to obtain more polar compounds such as flavonoids, tannins, phenols, and glycosides. antioxidant activity test of fractions with dpph using uv-vis spectrophotometer based on the research results of the fractionation process using 3 types of solvents tested using a uv-vis spectrophotometer which aims to determine the ic50 value of each fraction so that the fraction containing high antioxidant compounds can be identified. this process is useful in the next stage, namely for the column chromatography test. the test on the uv-vis spectrophotometer was repeated three times and the average was found. the average test results can be seen in (table 3). the results of the antioxidant activity test on the thick extract fraction of dadap leaves can be seen below. table 3. ic50 value of antioxidant activity test of thick extract fraction of dadap leaves using 3 types of solvents (n-hexane, ethyl acetate and methanol water). no fraction rf value antioxidant activity fraction information 1 n-hexane 0,7 ++ medium 0,64 ++ medium 0,5 +++ strong 2 ethyl acetate 0,3 ++ medium 3 methanol water 0 none spraying dpph solution on klt aims to determine the activity of antioxidant compounds as indicated by the presence of colored spots that appear and the rf value obtained. according to ramadhan et al. (2020), qualitative tests using thin layer chromatography (klt) were also carried out to confirm further the results obtained from the color test. according to megawati et al., (2021), dpph reagents that react with antioxidants will experience a color change from purple to yellow, the color intensity depends on the ability of the antioxidant. pratiwi et al. (2021) state that the rf value is specific to certain compounds in certain eluents. the rf value can be used as evidence to identify compounds. purification and isolation of n-hexane fraction compounds of condensed extract of dadap ayam leaves based on the results of purification and isolation of the n-hexane fraction compound of thick extract of dadap ayam leaves, 5 n-hexane subfractions were obtained which had colored spots on rf values (table 5) which were suspected of having antioxidant activity from a total of 12 n-hexane subfractions that had been spotted on the klt plate. table 5. rf values of subfractions in n-hexane and antioxidant activity. fraction subfraction rf value antioxidant activity information n-hexane n1 0,86 +++ strong n2 0,8 ++ medium n3 0,4 ++ medium n4 0,4 +++ strong 0,2 +++ strong in (table 5) above, the results of the rf value of the n-hexane subfraction that has a colored spot after being spotted on the klt plate. it is known that the n1 subfraction has an rf value of 0.86 with a dark yellow/orange color on the klt plate having antioxidant activity marked with a category (+++) which is strong. n2 has an rf value of 0.8 with a yellow color at the bottom and a green spot at the top has antioxidant activity marked with a category (++) which is moderate. meanwhile, n3 has an rf value of 0.4 with yellow and green colors having antioxidant activity marked with (++) which is moderate. n4 (1) has an rf value of 0.4 with a dark yellow and green color having antioxidant 398 biology, medicine, & natural product chemistry 14 (1), 2025: 395-400 activity marked with (+++) which is strong and n4 (2) has an rf value of 0.2 with a dark yellow color having antioxidant activity marked with (+++) which is strong. according to fajriani et al. (2022), factors that can influence the rf value are the number of spots, temperature, and steam in the development vessel used. purification and isolation of ethyl acetate fraction compounds of condensed extract of dadap ayam leaves based on the results of purification and isolation of the compound fraction of ethyl acetate thick extract of chicken daap leaves, 4 ethyl acetate subfractions were obtained which had colored spots on rf values (table 6), which were suspected of having antioxidant activity from a total of 12 n-hexane subfractions that had been spotted on the klt plate. table 6. rf values of subfractions in ethyl acetate and antioxidant activity. fraction subfraction rf value antioxidant activity information ethyl acetate e1 0,8 +++ strong e3 0,5 +++ strong e4 0,3 +++ strong e5 0,2 ++ medium in (table 6) above, the results of the rf value of the ethyl acetate subfraction that has a colored spot after being spotted on the klt plate can be seen in (appendix 10). it is known that the e1 subfraction has an rf value of 0.8 with a dark yellow/orange color on the klt plate having antioxidant activity marked with a category (+++) which is strong. e3 has an rf value of 0.5 with a yellow color at the bottom and a green spot appears at the top having antioxidant activity marked with a category (+++) which is strong. e4 has an rf value of 0.3 with a yelloworange color having antioxidant activity marked with (+++) which is strong and e5 has an rf value of 0.2 with yellow and green colors having antioxidant activity marked with (++) which is moderate. different rf values can indicate the presence of antioxidant compounds with different characteristics, for example compounds with high rf values approaching 1 have higher polarity and are more easily soluble in water. however, compounds with lower rf values approaching 0 have more nonpolar properties and are more soluble in organic solvents. according to forestryana & arnida (2020), the rf values obtained indicate differences in the properties of compounds and can be used to identify compounds. compounds that have a higher rf mean they have low polarity. this is because the stationary phase is polar. more polar compounds will be strongly retained in the stationary phase, resulting in low rf values. determination of pure compound groups of chicken dadap leaves (erythrina variegata l.) based on the results of the study conducted on the determination of pure compound groups of chicken dadap with the repetition method on thin layer chromatography (klt) and then sprayed with 0.5% sulfuric acid (h2so4). the compound groups obtained from this method of determining pure compound groups are in the n-hexane isolate obtained 1 terpenoid, 2 flavonoids and 1 phenol and in the ethyl acetate isolate obtained pure compound groups, namely 1 phenol and 1 flavonoid. meanwhile, the rf value of each isolate can be seen in (table 7). table 7. rf values and antioxidants from subfractions of chicken dadap leaves. isolate color rf value compound groups n1 n2 n3 n4 purple 0,88 terpenoids yellow 0,8 phenol bluish green 0,76 steroids yellow orange 0,5 flavonoids e1 e4 yellow 0,86 phenol yellow orange 0,5 flavonoids based on the results in (table 7) samples of dadap ayam leaves, the results of the study showed that there were 6 pure compounds consisting of the n-hexane subfraction (n1, n2, n3 and n4) and the ethyl acetate subfraction (e1 and e4). the terpenoid compound group was obtained from the n1 subfraction with eluent (9:1) producing a purple color in the h2s04 spray results on the isolate. terpenoids are a group of compounds found in the dadap ayam leaf subfraction which show various pharmacological activities, such as: anti-malarial, antibacterial, anti-viral, anti-inflammatory and anti-cancer activities. according to yang et al., (2020), terpenoids have various biological activities such as antiinflammatory, anti-bacterial, anti-viral, anti-malarial, anti-tumor, preventing and treating cardiovascular disease, lowering blood sugar, as an immunomodulator, antioxidant, and have neuroprotective effects. most of the terpenoids that have active ions are isolated from medicinal plants. antioxidant activity test of pure isolates of dadap ayam leaves (erythrina variegata l.) using uv-vis spectrophotometer based on the antioxidant activity test of pure chicken dadap isolates obtained from the dpph method by measuring the ic50 value, the absorbance value was then measured using a uv-vis spectrophotometer with a wavelength of 517nm, the average test results were obtained. permata et al. – potential medicinal plant antioxidant compounds based … 399 table 8. results of antioxidant activity test of pure isolate compounds of dadap ayam leaves using the uv-vis spectrophotometer method. pure isolate persamaan regresi (y=ax+b) ic50 (ppm) antioxidant activity ascorbic acid y = 9,846 (4,28) + 35,682 4,28 very strong n1 y = 20,108 (4,0688) 31,817 58,49 strong n2 y = 13,441 (2,8404) + 11,824 17,12 very strong n3 y = 7,0749 (4,1367) + 20,733 62,59 strong n4 y = 13,828 (2,3320) + 17,753 10,29 very strong e1 y = 24,049 (4,6067) 60,787 100,15 medium e4 y = 14,913 (3,2226) + 1,9401 25,09 very strong in (table 8) it can be seen that the ic50 value of ascorbic acid is higher than the value of the pure isolate of the dadap ayam leaf sample, which is 4.28ppm, meaning it is very strong. this is because ascorbic acid has high natural antioxidants which are used as a comparison for positive controls in pure compounds of dadap ayam leaf isolates in testing the activity of antioxidant compounds. according to melinda et al. (2024), vitamin c or ascorbic acid is a water-soluble vitamin derived from hexose. ascorbic acid contains a chromophore group, which is sensitive to light or forms of electromagnetic radiation. in addition, ascorbic acid functions as an antioxidant and protector against free radicals. vitamin c is a vitamin in the antioxidant group that can ward off various extracellular free radicals. according to maryanti (2022), vitamin c functions as an antioxidant because it effectively captures free radicals, especially ros or reactive oxygen compounds. vitamin c works as an electron donor in biochemical reactions both intracellularly and extracellularly. conclusions based on the research that has been done, the following conclusions can be drawn: strong antioxidant activity in thick extract of dadap ayam leaves (erythrina variegata l.) was obtained in the n-hexane fraction, which was 5.4306ppm and the ethyl acetate fraction, which was 17.819ppm. meanwhile, the methanol-water fraction was not active in antioxidant activity. the groups of pure compounds that have antioxidant activity obtained in dadap ayam leaf samples are the terpenoid group (n1), phenol group (n2 and e1), steroid group (n3) and flavonoid group (n4, and e4). pure compounds obtained from dadap ayam leaf samples have very strong, strong and moderate antioxidant activity. pure compounds with the terpenoid group (n1) obtained an ic50 value of 58.49ppm. the phenol compound group (n2 and e1) obtained an ic50 value of (17.12ppm and 100.15ppm). meanwhile, the steroid compound group (n3) obtained an ic50 value of (65.59ppm) and the flavonoid compound group (n4 and e4) obtained an ic50 value of (10.29 ppm and 25.09 ppm). competing interests: the authors declare that there are no competing interests. references anwar, k., santoso, h. b., & cahaya, n. 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immunogen; vaccines. introduction the creation of efficient vaccines is the ultimate objective in the fight against the spread and deadly nature of many of these infectious diseases. any vaccination that is secure, stable, and able to trigger a robust immune response with a minimal number of doses is desirable. some alternative vaccine types have demonstrated promising outcomes in their immunogenic profiles, despite the fact that many of the frequently used vaccinations attenuate or kill complete organisms. some of the top options for innovative vaccinations include subunit vaccines, often known as second-generation vaccines, and third-generation vaccines, which can be rnaor dna-based (doria-rose et al.,2021; braz gomes et al., 2021). numerous kinds of nanoparticles have innate physical characteristics that can cause an immunological reaction. it has been discovered that cytokine and antibody responses can be induced by gold, carbon, dendrimers, polymers, and liposome nanoparticles. due to these distinctive properties, nanoparticles can now be used as adjuvants to boost the immunogenicity of potential vaccines rather than just as vaccine delivery systems. the nanoparticles utilized for this purpose are commonly between 20 and 100 nanometers in size and are also known as nano-immune stimulators or activators (nm). inorganic nps like iron and silica, polymeric nps like chitosan and poly(lacticco-glycolic) acid (plga), cholesterol and lipid liposomes, and vlps are a few examples of well-known nano-immune stimulators (bachmann et al., 2010; bachmann et al., 1993; baranov et al., 2020). nps as drug delivery mechanisms, including antivirals, can inhibit viral reproduction in host cells by releasing antivirals from nps that block target cell receptors and releasing antivirals from absorbed nps that are released in a target cell. these main viral replication stages include transcription, replication of phage dna, synthesis of protein, and assembly. because of their potential to create prolonged antigen discharge after vaccination injection and to elicit an immune response, manuscript received: 16 march, 2023. revision accepted: 28 march, 2023. published: 21 june, 2023. https://doi.org/10.14421/biomedich.2023.121.343-361 344 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 nps based on organic and inorganic compositions have received extensive research as innovative vaccine approaches. nps can also create an antigen discharge that is controlled and low speed, creating a depot at the injection site that offers the option of antigen preservation over antigen degradation (de souza et al.,2021; abo-zeid et al., 2020; chen and 2020; chen et al., 2013). when compared to conventional vaccine techniques, nano carrier-based delivery systems have a number of benefits, including as greater adjuvant characteristics, superior stability, and increased protection against premature degradation. nanocarriers can shield the immunogen from early proteolytic degradation, allowing researchers to investigate alternative delivery methods when utilized to encapsulate or coat the surface of an antigen. nanocarriers can enhance the specificity of antigen delivery to apcs and lengthen the time for antigen presentation to these cells and other crucial immune cells essential for long-term immunity in addition to their protective properties. many different types of nanoparticles, including inorganic and polymeric nanoparticles, virus-like particles (vlps), liposomes, and self-assembled protein nanoparticles, have been investigated as potential antigen carriers for vaccines. viral antigens have been effectively delivered using biocompatible inorganic nanoparticles like gold, carbon, and silica (choi et al., 2021; chen et al., 2016). figure 1. schematic representation of different types of nanoparticles (nps) (heinz et al., 2017). materials and methods researchers conduct an examination of articles that are in accordance with the issue to be studied. determination of literature search keywords (search string based on pi (e) cot framework (p=patient/problem; i/e=exposure/implementation; c= control/comparison intervention, o=outcome, t=time) because a good question will help determine the scope of the review and help the strategy of finding the article. articles used in the literature review are obtained through the database of international journal providers through pubmed, from 2021-2023, clinical trials only. the author opens www.pubmed.com. researchers wrote keywords according to mesh (medical subject heading) namely “nanoparticle vaccine” and selected full text. 1. inclusion criteria population or sample is (nanoparticle-based vaccines). 2. exclusion criteria population or sample other (nanoparticle-based vaccines). results and discussion table 1. (alimehmeti, 2021), found that severe covid-19 occurred in 30 participants, with one fatality; all 30 were in the placebo group. moderate, transient reactogenicity after vaccination occurred more frequently in the mrna1273 group. serious adverse events were rare, and the incidence was similar in the two groups. thomas et al., 2021, found that vaccine efficacy of 86 to 100% was seen across countries and in populations with diverse ages, sexes, races, or ethnic groups, and risk factors for covid-19 among participants without evidence of previous infection with sars-cov-2. vaccine efficacy against the severe disease was 96.7% (95% ci, 80.3 to hussein et al. – nanotechnology-based vaccines 345 99.9). in south africa, where the sars-cov-2 variant of concern b.1.351 (or beta) was predominant, a vaccine efficacy of 100% (95% ci, 53.5 to 100) was observed. heath et al., 2021, found that a post hoc analysis showed an efficacy of 86.3% (95% ci, 71.3 to 93.5) against the b.1.1.7 (or alpha) variant and 96.4% (95% ci, 73.8 to 99.5) against non-b.1.1.7 variants. reactogenicity was generally mild and transient. the incidence of serious adverse events was low and similar in the two groups. houser et al., 2022, found that exploratory analyses identified neutralizing antibody responses elicited by the h2ha-ferritin vaccine in both h2-naive and h2exposed populations. furthermore, broadly neutralizing antibody responses against group 1 influenza viruses, including both seasonal h1 and avian h5 subtypes, were induced in the h2-naive population by targeting the ha stem. this ferritin nanoparticle vaccine technology represents a novel, safe and immunogenic platform with potential application for pandemic preparedness and universal influenza vaccine development. shinde et al., 2021, found that of 41 sequenced isolates, 38 (92.7%) were the b.1.351 variant. post hoc vaccine efficacy against b.1.351 was 51.0% (95% ci, -0.6 to 76.2) among the hiv-negative participants. preliminary local and systemic reactogenicity events were more common in the vaccine group; serious adverse events were rare in both groups. dunkle et al., 2022, found that ten moderate and 4 severe cases occurred, all in placebo recipients, yielding vaccine efficacy against the moderate-to-severe disease of 100% (95% ci, 87.0 to 100). most sequenced viral genomes (48 of 61, 79%) were variants of concern or interest largely b.1.1.7 (alpha) (31 of the 35 genomes for variants of concern, 89%). vaccine efficacy against any variant of concern or interest was 92.6% (95% ci, 83.6 to 96.7). reactogenicity was mostly mild to moderate and transient but was more frequent among nvx-cov2373 recipients than among placebo recipients and was more frequent after the second dose than after the first dose. wei et al., 2022, found that, with a combined endpoint of hbeag seroconversion, alanine aminotransferase normalization and hbv dna < 2,000 iu/ml, both 900 µg (18.1%) and 600 µg (14.3%), resulted in significantly higher rate versus placebo (5.0%) (p = 0.002 and p = 0.02, respectively) at week 76. in stage 2, none (0 of 20) of 900 µg εpa-44-treated patients experienced a serologic relapse. the safety profile of εpa-44 was comparable to that of the placebo. sahin et al., 2021, found that most participants had a strong response of ifnγ+ or il-2+ cd8+ and cd4+ t helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. using peptide-mhc multimer technology, we identified several bnt162b2-induced epitopes that were presented by frequent mhc alleles and conserved in mutant strains. one week after the boost, epitope-specific cd8+ t cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating cd8+ t cells and were detectable (0.01-0.28%) eight weeks later. in summary, bnt162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses. datoo et al., 2021, found that participants vaccinated with r21/mm showed high titers of malaria-specific anti-asn-ala-asn-pro (nanp) antibodies 28 days after the third vaccination, which were almost doubled with the higher adjuvant dose. titres waned but were boosted to levels similar to peak titers after the primary series of vaccinations after a fourth dose administered 1 year later. toback et al., 2022, found that no episodes of anaphylaxis or deaths were reported within the substudy. co-administration resulted in no change to the influenza vaccine immune response although a reduction in antibody responses to the nvx-cov2373 vaccine was noted. nvx-cov2373 vaccine efficacy in the substudy (ie, participants aged 18 to <65 years) was 87·5% (95% ci -0·2 to 98·4), and in the main study was 89·8% (95% ci 79·7-95·5). maruggi et al., 2022, found that high frequencies of spike-specific germinal center b, th0/th1 cd4, and cd8 t cell responses were observed in mice. local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. in hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following the sars-cov-2 challenge. therefore, the sars-cov-2 sam (lnp) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple sars-cov-2 variants, and has been advanced to phase 1 clinical evaluation. madhi et al., 2022, found that, of the serious adverse events that occurred among hiv-negative people (of whom, two [0·1%] were baseline sars-cov-2-negative and four [0·6%] were baseline sars-cov-2-positive) and people living with hiv-1 (for whom there were no serious adverse events) in the nvx-cov2373 group, none were assessed as related to the vaccine. among participants who were baseline sars-cov-2-negative in the nvxcov2373 group, the anti-spike igg geometric mean titers (gmts) and seroconversion rates (scrs) were lower in people living with hiv-1 (n=62) than in hivnegative people (n=1234) following the first vaccination (gmt: 508·6 vs 1195·3 elisa units [eu]/ml; scr: 51·6% vs 81·3%); and similarly so 14 days after the second vaccination for gmts (14 420·5 vs 31 631·8 eu/ml), whereas the scr was similar at this point (100·0% vs 99·3%). in the nvx-cov2373 group, antispike igg gmts 14 days after the second vaccination were substantially higher in those who were baseline sars-cov-2-positive than in those who were baseline sars-cov-2-seronegative for hiv-negative participants (100 666·1 vs 31 631·8 eu/ml) and for people living with hiv-1 (98 399·5 vs 14 420·5 eu/ml). this was also the case for angiotensin-converting enzyme 2 receptor-binding antibody and neutralizing antibody 346 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 titers. stuart et al., 2022, found that, between april 19 and may 14, 2021, 1072 participants were enrolled at a median of 9·4 weeks after receipt of a single dose of chad (n=540, 47% female) or bnt (n=532, 40% female). in chad-primed participants, geometric mean concentration (gmc) 28 days after a boost of sarscov-2 anti-spike igg in recipients of chad/m1273 (20 114 elisa laboratory units [elu]/ml [95% ci 18 160 to 22 279]) and chad/nvx (5597 elu/ml [4756 to 6586]) was non-inferior to that of chad/chad recipients (1971 elu/ml [1718 to 2262]) with a gmr of 10·2 (one-sided 98·75% ci 8·4 to ∞) for chad/m1273 and 2·8 (2·2 to ∞) for chad/nvx, compared with chad/chad. in bnt-primed participants, noninferiority was shown for bnt/m1273 (gmc 22 978 elu/ml [95% ci 20 597 to 25 636]) but not for bnt/nvx (8874 elu/ml [7391 to 10 654]), compared with bnt/bnt (16 929 elu/ml [15 025 to 19 075]) with a gmr of 1·3 (one-sided 98·75% ci 1·1 to ∞) for bnt/m1273 and 0·5 (0·4 to ∞) for bnt/nvx, compared with bnt/bnt; however, nvx still induced an 18-fold rise in gmc 28 days after vaccination. there were 15 serious adverse events, none considered related to immunization. aldrich et al., 2021, found that, as initially tested doses of 5 μg cv7202 elicited unacceptably high reactogenicity we subsequently tested 1 and 2 μg doses which were better tolerated. no vaccine-related serious adverse events or withdrawals occurred. low, dose-dependent vnt responses were detectable from day 15, and by day 29%, 31%, and 22% of 1, 2, and 5 μg groups, respectively, had vnts ≥ 0·5 iu/ml, considered an adequate response by the who. after two 1 or 2 μg doses all recipients had titers ≥ 0.5 iu/ml by day 43. day 57 gmts were not significantly lower than those with rabipur, which elicited adequate responses in all vaccinees after two doses. cv7202elicited vnt was significantly correlated with rabv-gspecific igg antibodies (r2 = 0.8319, p < 0.0001). shinde et al., 2022, found that more solicited adverse events were reported by participants in the qniv group (551 [41·3%] of 1333) than in the iiv4 group (420 [31·8%] of 1319), and were comprised primarily of mild to moderate transient injection site pain (341 [25·6%] in the qniv group vs 212 [16·1%] in the iiv4 group). formica et al., 2021, found that, neutralizing antibody responses exceeded those seen in a panel of convalescent sera for both age groups. study limitations include the relatively short duration of safety follow-up to date and the current lack of immune persistence data beyond the primary vaccination regimen time point assessments, but these data will accumulate over time. khobragade et al., 2021, found that the socio-demographic characteristics of the two arms were comparable. about 9.9% of subjects in the recombinant rabies g protein vaccine arm and 17.2% of subjects in the reference arm reported adverse events. the sero-protection on day 14 was found to be 99.24% and 97.72% in the recombinant rabies g protein vaccine arm and reference vaccine arm respectively and the difference was statistically nonsignificant. august et al., 2021, found that further evaluation of the potential therapeutic use of mrna-1944 in clinical trials for the treatment of chikv infection is warranted. shinde et al., 2021, found that, overall, similar frequencies of solicited and unsolicited adverse events were reported in all treatment groups. kremsner et al., 2021, found that responses to 12 μg were comparable to those observed in convalescent sera from known covid-19 patients. mallory et al., 2022, found that 1610 participants were screened from aug 24, 2020, to sept 25, 2020. 1282 participants were enrolled, of whom 173 were assigned again to placebo (group a), 106 were re-randomized to nvx-cov2373-placebo (group b1), and 104 were rerandomized to nvx-cov2373-nvx-cov2373 (group b2); after accounting for exclusions and incorrect administration, 172 participants in group a, 102 in group b1, and 105 in group b2 were analysed for safety. following the active booster, the proportion of participants with available data reporting local (80 [82%] of 97 participants had any adverse event; 13 [13%] had a grade ≥3 events) and systemic (75 [77%] of 98 participants had any adverse event; 15 [15%] had a grade ≥3 events) reactions was higher than after primary vaccination (175 [70%] of 250 participants had any local adverse event, 13 [5%] had a grade ≥3 events; 132 [53%] of 250 had any systemic adverse event, 14 [6%] had a grade ≥3 events). local and systemic events were transient in nature (median duration 1·0-2·5 days). in the per-protocol immunogenicity population at day 217 (167 participants in group a, 101 participants in group b1, 101 participants in group b2), igg geometric mean titers (gmt) had increased by 4·7-fold and mn50 gmt by 4·1-fold for the ancestral sars-cov-2 strain compared with the day 35 titers.heath et al., 2023, found that the incidence of serious adverse events and adverse events of special interest were similar between groups. lovell et al., 2022, found that, neutralizing antibodies levels of the low-dose and high-dose ecv19 groups had frnt50 geometric mean values of 129 and 316, respectively. boosting responses and dose responses were observed. antibodies against the rbd correlated with antibodies against the spike and with virus neutralization. gatechompol et al., 2022, found that, an mrna vaccine expressing a prefusion non-stabilized spike protein is safe and highly immunogenic. masuda et al., 2022, found that, between 12 february 2021 and 17 march 2021, 326 subjects were screened, and 200 participants enrolled and randomized: nvx-cov2373, n = 150; placebo, n = 50. solicited adverse events (aes) through 7 days after each injection occurred in 121/150 (80.7%) and 11/50 (22.0%) participants in the nvx-cov2373 and placebo arms, respectively. in the nvx-cov2373 arm, tenderness and injection site pain were the most frequently reported solicited aes after each vaccination, irrespective of age. robust immune responses occurred with nvx-cov2373 hussein et al. – nanotechnology-based vaccines 347 (n = 150) by day 36: igg geometric mean fold rise (95% confidence interval) 259 (219, 306); seroconversion rate 100% (97.6, 100). no such response occurred with a placebo (n = 49). ishikawa et al., 2021, found that the most common adverse event (ae) was injection site skin reaction (86.7%). no grade 3 or higher drug-related aes were observed. no tumor responses were observed, and three patients (30%) had stable disease. the immune response was comparable between the two cohorts, and all patients (100%) achieved antibody responses with a median of 2.5 vaccinations. comparing chp-ny-eso-1 alone to the poly-iclc combination, all patients in both groups exhibited antibody responses, but the titers were higher in the combination group. in a mouse model, adding an anti-pd-1 antibody to the combination of chp-ny-eso-1/poly-iclc suppressed the growth of ny-eso-1-expressing tumors. combining the vaccine with pd-1 blockade holds promise in human trials. li et al., 2022, found that, in younger participants, neutralizing antibody (nab) geometric mean titers (gmts) for the 10 and 30 µg dose levels declined from 233 and 254 (21 days after dose 2) to 55 and 87 at month 3, respectively, and to 16 and 27 at month 6, respectively. in older participants, nab gmts declined from 80 and 160 (21 days after dose 2) to 10 and 21 at month 6. overall, higher antibody titers were observed in younger participants, and the 30 µg dose induced higher levels of nab, which declined more slowly by month 6. no serious adverse events were reported in the vaccine group. wei et al., 2021, found that epnp, carrying the neutralizing epitope hla121-138, is a good candidate for a vaccine against sa. table 1. clinical studies using nanotechnology-based vaccines. authors title date and type of the study method results alimehmeti efficacy and safety of the mrna-1273 sars-cov-2 vaccine clinical trial 2021 this phase 3 randomized, observer-blinded, placebocontrolled trial was conducted at 99 centers across the united states. persons at high risk for sars-cov-2 infection or its complications were randomly assigned in a 1:1 ratio to receive two intramuscular injections of mrna-1273 (100 μg) or a placebo 28 days apart. the primary endpoint was the prevention of covid-19 illness with onset at least 14 days after the second injection in participants who had not previously been infected with sars-cov-2. severe covid-19 occurred in 30 participants, with one fatality; all 30 were in the placebo group. moderate, transient reactogenicity after vaccination occurred more frequently in the mrna-1273 group. serious adverse events were rare, and the incidence was similar in the two groups. thomas et al. safety and efficacy of the bnt162b2 mrna covid-19 vaccine through 6 months randomized controlled trial 2021 in an ongoing, placebocontrolled, observer-blinded, multinational, pivotal efficacy trial, we randomly assigned 44,165 participants 16 years of age or older and 2264 participants 12 to 15 years of age to receive two 30-μg doses, at 21 days apart, of bnt162b2 or placebo. the trial endpoints were vaccine efficacy against laboratory-confirmed covid-19 and safety, which were both evaluated 6 months after vaccination. vaccine efficacy of 86 to 100% was seen across countries and in populations with diverse ages, sexes, races, or ethnic groups, and risk factors for covid-19 among participants without evidence of previous infection with sars-cov-2. vaccine efficacy against the severe disease was 96.7% (95% ci, 80.3 to 99.9). in south africa, where the sars-cov-2 variant of concern b.1.351 (or beta) was predominant, a vaccine efficacy of 100% (95% ci, 53.5 to 100) was observed. 348 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 table 1. cont. authors title date and type of the study method results heath et al. safety and efficacy of nvx-cov2373 covid-19 vaccine clinical trial 2021 in this phase 3, randomized, observer-blinded, placebocontrolled trial conducted at 33 sites in the united kingdom, we assigned adults between the ages of 18 and 84 years in a 1:1 ratio to receive two intramuscular 5μg doses of nvx-cov2373 or placebo administered 21 days apart. the primary efficacy endpoint was virologically confirmed mild, moderate, or severe sars-cov-2 infection with an onset at least 7 days after the second injection in participants who were serologically negative at baseline. a post hoc analysis showed an efficacy of 86.3% (95% ci, 71.3 to 93.5) against the b.1.1.7 (or alpha) variant and 96.4% (95% ci, 73.8 to 99.5) against non-b.1.1.7 variants. reactogenicity was generally mild and transient. the incidence of serious adverse events was low and similar in the two groups. houser et al. safety and immunogenicity of a ferritin nanoparticle h2 influenza vaccine in healthy adults: a phase 1 trial clinical trial 2022 conducted a first-in-human, randomized, open-label, phase 1 clinical trial (nct03186781) to evaluate a novel ferritin (h2haferritin) nanoparticle influenza vaccine platform. the h2 subtype has not circulated in humans since 1968. adults born after 1968 have been exposed to only the h1 subtype of group 1 influenza viruses, which shares a conserved stem with h2. including both h2-naive and h2-exposed adults in the trial allowed us to evaluate memory responses against the conserved stem domain in the presence or absence of pre-existing responses against the immunodominant ha head domain. exploratory analyses identified neutralizing antibody responses elicited by the h2ha-ferritin vaccine in both h2-naive and h2-exposed populations. furthermore, broadly neutralizing antibody responses against group 1 influenza viruses, including both seasonal h1 and avian h5 subtypes, were induced in the h2naive population by targeting the ha stem. this ferritin nanoparticle vaccine technology represents a novel, safe and immunogenic platform with potential application for pandemic preparedness and universal influenza vaccine development. shinde et al. efficacy of nvxcov2373 covid-19 vaccine against the b.1.351 variant clinical trial 2021 in this phase 2a-b trial in south africa, we randomly assigned human immunodeficiency virus (hiv)-negative adults between the ages of 18 and 84 years or medically stable hiv-positive participants between the ages of 18 and 64 years in a 1:1 ratio to receive two doses of either the nvx-cov2373 vaccine (5 μg of recombinant spike protein with 50 μg of matrix-m1 adjuvant) or placebo. the primary endpoints were safety and vaccine efficacy against laboratory-confirmed symptomatic covid-19 at 7 days or more after the second dose among participants without previous sars-cov-2 infection. of 41 sequenced isolates, 38 (92.7%) were the b.1.351 variant. post hoc vaccine efficacy against b.1.351 was 51.0% (95% ci, -0.6 to 76.2) among the hiv-negative participants. preliminary local and systemic reactogenicity events were more common in the vaccine group; serious adverse events were rare in both groups. dunkle et al. efficacy and safety of nvx-cov2373 in adults in the united clinical trial 2022 conducted a phase 3, randomized, observer-blinded, placebo-controlled trial in the ten moderate and 4 severe cases occurred, all in placebo recipients, yielding vaccine efficacy against the hussein et al. – nanotechnology-based vaccines 349 authors title date and type of the study method results states and mexico united states and mexico during the first half of 2021 to evaluate the efficacy and safety of nvxcov2373 in adults (≥18 years of age) who had not had severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. participants were randomly assigned in a 2:1 ratio to receive two doses of nvxcov2373 or placebo 21 days apart. the primary objective was to determine vaccine efficacy against reverse-transcriptasepolymerase-chain-reactionconfirmed covid-19 occurring at least 7 days after the second dose. vaccine efficacy against moderate-to-severe disease and against different variants was also assessed. moderate-to-severe disease of 100% (95% ci, 87.0 to 100). most sequenced viral genomes (48 of 61, 79%) were variants of concern or interest largely b.1.1.7 (alpha) (31 of the 35 genomes for variants of concern, 89%). vaccine efficacy against any variant of concern or interest was 92.6% (95% ci, 83.6 to 96.7). reactogenicity was mostly mild to moderate and transient but was more frequent among nvx-cov2373 recipients than among placebo recipients and was more frequent after the second dose than after the first dose. wei et al. efficacy and safety of a nanoparticle therapeutic vaccine in patients with chronic hepatitis b: a randomized clinical trial clinical trial 2022 a two-stage phase 2 trial, which included a 76-week, randomized, double-blind, placebo-controlled trial (stage 1) and a 68-week open-label extension (stage 2), was conducted in 15 centers across china. with a combined endpoint of hbeag seroconversion, alanine aminotransferase normalization and hbv dna < 2,000 iu/ml, both 900 µg (18.1%) and 600 µg (14.3%), resulted in significantly higher rate versus placebo (5.0%) (p = 0.002 and p = 0.02, respectively) at week 76. in stage 2, none (0 of 20) of 900 µg εpa44-treated patients experienced a serologic relapse. the safety profile of εpa-44 was comparable to that of the placebo. sahin et al. bnt162b2 vaccine induces neutralizing antibodies and polyspecific t cells in humans clinical trial 2021 extend a previous phase-i/ii trial report2 by presenting data on the immune response induced by bnt162b2 prime-boost vaccination from an additional phase-i/ii trial in healthy adults (18-55 years old). most participants had a strong response of ifnγ+ or il-2+ cd8+ and cd4+ t helper type 1 cell, which was detectable throughout the full observation period of nine weeks following the boost. using peptidemhc multimer technology, we identified several bnt162b2-induced epitopes that were presented by frequent mhc alleles and conserved in mutant strains. one week after the boost, epitope-specific cd8+ t cells of the early-differentiated effectormemory phenotype comprised 0.022.92% of total circulating cd8+ t cells and were detectable (0.01-0.28%) eight weeks later. in summary, bnt162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses. datoo et al. efficacy of a lowdose candidate malaria vaccine, r21 in adjuvant matrixm, with seasonal administration to children in burkina faso: a randomised clinical trial 2021 in this double-blind, randomized, controlled, phase 2b trial, the low-dose circumsporozoite protein-based vaccine r21, with two different doses of adjuvant matrix-m (mm), was given to children aged 5-17 months in nanoro, participants vaccinated with r21/mm showed high titres of malaria-specific anti-asn-ala-asn-pro (nanp) antibodies 28 days after the third vaccination, which were almost doubled with the higher adjuvant dose. titres waned but were boosted to levels similar to peak titres after the 350 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 authors title date and type of the study method results controlled trial burkina faso-a highly seasonal malaria transmission setting. three vaccinations were administered at 4-week intervals before the malaria season, with a fourth dose 1 year later. all vaccines were administered intramuscularly into the thigh. group 1 received 5 μg r21 plus 25 μg mm, group 2 received 5 μg r21 plus 50 μg mm, and group 3, the control group, received rabies vaccinations. children were randomly assigned (1:1:1) to groups 1-3. an independent statistician generated a random allocation list, using block randomisation with variable block sizes, which was used to assign participants. participants, their families, and the local study team were all masked to group allocation. only the pharmacists preparing the vaccine were unmasked to group allocation. vaccine safety, immunogenicity, and efficacy were evaluated over 1 year. the primary objective assessed the protective efficacy of r21 plus mm (r21/mm) from 14 days after the third vaccination to 6 months. primary analyses of vaccine efficacy were based on a modified intention-to-treat population, which included all participants who received three vaccinations, allowing for the inclusion of participants who received the wrong vaccine at any time point. primary series of vaccinations after a fourth dose administered 1 year later. toback et al. safety, immunogenicity, and efficacy of a covid19 vaccine (nvxcov2373) coadministered with seasonal influenza vaccines: an exploratory substudy of a randomized, observer-blinded, placebo-controlled, phase 3 trial clinical trial 2022 a planned exploratory substudy as part of the randomized, observer-blinded, placebocontrolled, phase 3 trial of the safety and efficacy of the covid-19 vaccine (nvxcov2373) by co-administrating the influenza vaccine at four study hospitals in the uk. approximately, the first 400 participants meeting the main study entry criteria-with no contraindications to influenza vaccination-were invited to join the substudy. participants of the main study were randomly assigned (1:1) to receive two intramuscular injections of either nvx-cov2373 (5 μg) or placebo (normal saline) 21 days apart; participants enrolled into the substudy were co-vaccinated with a single (0·5 ml) no episodes of anaphylaxis or deaths were reported within the substudy. coadministration resulted in no change to influenza vaccine immune response although a reduction in antibody responses to the nvx-cov2373 vaccine was noted. nvx-cov2373 vaccine efficacy in the substudy (ie, participants aged 18 to <65 years) was 87·5% (95% ci -0·2 to 98·4) and in the main study was 89·8% (95% ci 79·7-95·5). hussein et al. – nanotechnology-based vaccines 351 authors title date and type of the study method results intramuscular, age-appropriate (quadrivalent influenza cellbased vaccine [flucelvax quadrivalent; seqirus uk, maidenhead] for those aged 1864 years and adjuvanted trivalent influenza vaccine [fluad; seqirus uk, maidenhead] for those ≥65 years), licensed, influenza vaccine on the opposite deltoid to that of the first study vaccine dose or placebo. the influenza vaccine was administered in an open-label manner and at the same time as the first study injection. reactogenicity was evaluated via an electronic diary for 7 days after vaccination in addition to monitoring for unsolicited adverse events, medically attended adverse events and serious adverse events. immunogenicity was assessed with influenza haemagglutination inhibition and sars-cov-2 anti-spike protein igg assays. vaccine efficacy against pcr-confirmed, symptomatic covid-19 was assessed in participants who were seronegative at baseline, received both doses of the study vaccine or placebo, had no major protocol deviations affecting the primary endpoint, and had no confirmed cases of symptomatic covid-19 from the first dose until 6 days after the second dose (per-protocol efficacy population). immunogenicity was assessed in participants who received scheduled two doses of the study vaccine, had a baseline sample and at least one post-vaccination sample, and had no major protocol violations before unmasking (per-protocol immunogenicity population). reactogenicity was analyzed in all participants who received at least one dose of nvxcov2373 or placebo and had data collected for reactogenicity events. safety was analyzed in all participants who received at least one dose of nvxcov2373 or placebo. comparisons were made between participants of the substudy and the main study (who were not co-vaccinated for influenza). 352 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 authors title date and type of the study method results maruggi et al. a self-amplifying mrna sars-cov-2 vaccine candidate induces safe and robust protective immunity in preclinical models clinical trial 2022 assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self-amplifying messenger rna (sam) vaccine, encoding a prefusion-stabilized spike antigen of sars-cov-2 wuhan-hu-1 strain and delivered by lipid nanoparticles (lnps). in mice, one immunization with the sam vaccine elicited a robust spikespecific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched sars-cov-2 wuhan strain as well as b.1.1.7 (alpha), b.1.351 (beta) and b.1.617.2 (delta) variants. high frequencies of spike-specific germinal center b, th0/th1 cd4, and cd8 t cell responses were observed in mice. local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. in hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following the sarscov-2 challenge. therefore, the sars-cov-2 sam (lnp) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple sars-cov-2 variants, and has been advanced to phase 1 clinical evaluation madhi et al. immunogenicity and safety of a sarscov-2 recombinant spike protein nanoparticle vaccine in people living with and without hiv-1 infection: a randomized, controlled, phase 2a/2b trial clinical trial 2022 in this randomized, observerblinded, multicentre, placebocontrolled phase 2a/b trial in south africa, participants aged 18-84 years, with and without underlying hiv-1, were enrolled from 16 sites and randomly assigned (1:1) to receive two intramuscular injections of nvx-cov2373 or placebo, 21 days apart. people living with hiv-1 were on stable antiretroviral therapy and had an hiv-1 viral load of few than 1000 copies per ml. vacc ine dosage was 5 μg sars-cov-2 recombinant spike protein with 50 μg matrix-m adjuvant, whereas 0·9% saline was used as a placebo injection (volume 0·5 ml each). all study staff and participants remained masked to study group assignment. we previously reported an interim analysis on the efficacy and safety of the nvx-cov2373 vaccine (coprimary endpoints). in this article, we present an expanded safety analysis for the full cohort of participants and report on the secondary objective of vaccine immunogenicity in the full cohort of people living with hiv-1 and in hiv-negative individuals overall and stratified by baseline sars-cov-2 serostatus. of the serious adverse events that occurred among hiv-negative people (of whom, two [0·1%] were baseline sars-cov-2-negative and four [0·6%] were baseline sars-cov-2positive) and people living with hiv-1 (for whom there were no serious adverse events) in the nvx-cov2373 group, none were assessed as related to the vaccine. among participants who were baseline sars-cov-2-negative in the nvx-cov2373 group, the antispike igg geometric mean titres (gmts) and seroconversion rates (scrs) were lower in people living with hiv-1 (n=62) than in hivnegative people (n=1234) following the first vaccination (gmt: 508·6 vs 1195·3 elisa units [eu]/ml; scr: 51·6% vs 81·3%); and similarly so 14 days after the second vaccination for gmts (14 420·5 vs 31 631·8 eu/ml), whereas the scr was similar at this point (100·0% vs 99·3%). in the nvxcov2373 group, anti-spike igg gmts 14 days after the second vaccination were substantially higher in those who were baseline sars-cov-2-positive than in those who were baseline sars-cov-2-seronegative for hivnegative participants (100 666·1 vs 31 631·8 eu/ml) and for people living with hiv-1 (98 399·5 vs 14 420·5 eu/ml). this was also the case for angiotensin-converting enzyme 2 receptor-binding antibodies and neutralizing antibody.titers. stuart et al. immunogenicity, safety, and reactogenicity of heterologous covid-19 primary clinical trial 2022 com-cov2 is a single-blind, randomized, non-inferiority trial in which adults aged 50 years and older, previously immunised with a single dose of chad or between april 19 and may 14, 2021, 1072 participants were enrolled at a median of 9·4 weeks after receipt of a single dose of chad (n=540, 47% female) or bnt (n=532, 40% female). hussein et al. – nanotechnology-based vaccines 353 authors title date and type of the study method results vaccination incorporating mrna, viral-vector, and protein-adjuvant vaccines in the uk (com-cov2): a single-blind, randomised, phase 2, non-inferiority trial bnt in the community, were randomly assigned (in random blocks of three and six) within these cohorts in a 1:1:1 ratio to receive a second dose intramuscularly (8-12 weeks after the first dose) with the homologous vaccine, m1273, or nvx. the primary endpoint was the geometric mean ratio (gmr) of serum sars-cov-2 antispike igg concentrations measured by elisa in heterologous versus homologous schedules at 28 days after the second dose, with a noninferiority criterion of the gmr above 0·63 for the one-sided 98·75% ci. the primary analysis was on the per-protocol population, who were seronegative at baseline. safety analyses were done for all participants who received a dose of the study vaccine. in chad-primed participants, geometric mean concentration (gmc) 28 days after a boost of sars-cov-2 anti-spike igg in recipients of chad/m1273 (20 114 elisa laboratory units [elu]/ml [95% ci 18 160 to 22 279]) and chad/nvx (5597 elu/ml [4756 to 6586]) was noninferior to that of chad/chad recipients (1971 elu/ml [1718 to 2262]) with a gmr of 10·2 (one-sided 98·75% ci 8·4 to ∞) for chad/m1273 and 2·8 (2·2 to ∞) for chad/nvx, compared with chad/chad. in bntprimed participants, non-inferiority was shown for bnt/m1273 (gmc 22 978 elu/ml [95% ci 20 597 to 25 636]) but not for bnt/nvx (8874 elu/ml [7391 to 10 654]), compared with bnt/bnt (16 929 elu/ml [15 025 to 19 075]) with a gmr of 1·3 (one-sided 98·75% ci 1·1 to ∞) for bnt/m1273 and 0·5 (0·4 to ∞) for bnt/nvx, compared with bnt/bnt; however, nvx still induced an 18-fold rise in gmc 28 days after vaccination. there were 15 serious adverse events, none considered related to immunization. aldrich et al. proof-of-concept of a low-dose unmodified mrna-based rabies vaccine formulated with lipid nanoparticles in human volunteers: a phase 1 trial clinical trial 2021 in this phase 1, multi-center, controlled study in belgium and germany we enrolled 55 healthy 18-40-year-olds to receive intramuscular injections of 5 μg (n = 10), 1 μg (n = 16), or 2 μg (n = 16) cv7202 on day 1; subsets (n = 8) of 1 μg an nd 2 μg groups received second doses on day 29. controls (n = 10) received the rabies vaccine, rabipur, on days 1, 8, and 29. safety and reactogenicity were assessed up to 28 days postvaccination using diary cards; immunogenicity was measured as rabv-g-specific neutralizing titers (vnt) by rffit and igg by elisa. as initially tested doses of 5 μg cv7202 elicited unacceptably high reactogenicity we subsequently tested 1 and 2 μg doses which were better tolerated. no vaccine-related serious adverse events or withdrawals occurred. low, dose-dependent vnt responses were detectable from day 15, and by day 29%, 31%, and 22% of 1, 2 and 5 μg groups, respectively, had vnts ≥ 0·5 iu/ml, considered an adequate response by the who. after two 1 or 2 μg doses all recipients had titers ≥ 0.5 iu/ml by day 43. day 57 gmts were not significantly lower than those with rabipur, which elicited adequate responses in all vaccinees after two doses. cv7202-elicited vnt was significantly correlated with rabv-g-specific igg antibodies (r2 = 0.8319, p < 0.0001). shinde et al. comparison of the safety and immunogenicity of a novel matrix-madjuvanted nanoparticle influenza vaccine with a quadrivalent seasonal influenza vaccine in older adults: a phase 3 randomized controlled trial clinical trial 2022 this was phase 3 randomized, observer-blinded, activecomparator controlled trial done across 19 us community-based clinical research sites during the 2019-20 influenza season. participants were clinically stable and community-dwelling, aged at least 65 years and were randomisedin a 1:1 ratio using an interactive web response system to receive a single intramuscular dose of qniv or iiv4. the primary objective was more solicited adverse events were reported by participants in the qniv group (551 [41·3%] of 1333) than in the iiv4 group (420 [31·8%] of 1319), and were comprised primarily of mild to moderate transient injection site pain (341 [25·6%] in the qniv group vs 212 [16·1%] in the iiv4 group). 354 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 authors title date and type of the study method results to describe the safety and show that qniv was immunologically non-inferior to iiv4. the primary outcomes were adverse events by treatment group and comparative haemagglutinationinhibiting antibody responses (assayed with egg-propagated virus) on day 28, summarised in terms of the ratio of geometric mean titres (gmtrqniv/iiv4) and seroconversion rate (scr) difference between participants receiving qniv or iiv4 for all four vaccine homologous influenza strains. the immunogenicity outcome was measured in the per-protocol population. non-inferiority was shown if the lower bound of the two-sided 95% ci on the gmtrqniv/iiv4 was at least 0·67 and the lower bound of the two-sided 95% ci on the scr difference -was at least -10%. formica et al. different dose regimens of a sarscov-2 recombinant spike protein vaccine (nvx-cov2373) in younger and older adults: a phase 2 randomized placebocontrolled trial clinical trial 2021 the phase 2 component of our randomized, placebo-controlled, phase 1 to 2 trial was designed to identify which dosing regimen of nvx-cov2373 should move forward into latephase studies and was based on immunogenicity and safety data through day 35 (14 days after the second dose). the trial was conducted at 9 sites in australia and 8 sites in the united states. participants in 2 age groups (aged 18 to 59 and 60 to 84 years) were randomly assigned to receive either 1 or 2 intramuscular doses of 5-μg or 25-μg nvx-cov2373 or placebo, 21 days apart. primary endpoints were immunoglobulin g (igg) anti-spike protein response, 7-day solicited reactogenicity and unsolicited adverse events. a key secondary endpoint was a wild-type virusneutralizing antibody response. after enrollment, 1,288 participants were randomly assigned to 1 of 4 vaccine groups or placebo, with 1,283 participants administered at least 1 study treatment. neutralizing antibody responses exceeded those seen in a panel of convalescent sera for both age groups. study limitations include the relatively short duration of safety follow-up to date and the current lack of immune persistence data beyond the primary vaccination regimen time point assessments, but these data will accumulate over time. khobragade et al. safety and immunogenicity of a novel three-dose recombinant nanoparticle rabies g protein vaccine administered as clinical trial 2021 a multi-centric, open-label, assessor-blind, center-specific block randomized, parallel design, phase iii clinical study was conducted among 800 subjects. the eligible subjects were randomized in a 2:1 ratio the socio-demographic characteristics of the two arms were comparable. about 9.9% of subjects in the recombinant rabies g protein vaccine arm and 17.2% of subjects in the reference arm reported adverse events. the seroprotection on day 14 was hussein et al. – nanotechnology-based vaccines 355 authors title date and type of the study method results simulated postexposure immunization: a randomized, comparatorcontrolled, multicenter, phase iii clinical study for the recombinant rabies g protein vaccine and the reference vaccine. subjects in the recombinant rabies g protein vaccine arm received three doses of vaccine on days 0, 3, and 7, while subjects in the reference vaccine arm received five doses of who-prequalified vaccine on days 0, 3, 7, 14, and 28. found to be 99.24% and 97.72% in the recombinant rabies g protein vaccine arm and reference vaccine arm respectively and the difference was statistically nonsignificant. august et al. a phase 1 trial of lipid-encapsulated mrna encoding a monoclonal antibody with neutralizing activity against chikungunya virus clinical trial 2021 this phase 1, first-in-human, randomized, placebo-controlled, proof-of-concept trial conducted from january 2019 to june 2020 evaluated the safety and pharmacology of mrna-1944, a lipid nanoparticle-encapsulated messenger rna encoding the heavy and light chains of a chikv-specific monoclonal neutralizing antibody, chkv-24 further evaluation of the potential therapeutic use of mrna-1944 in clinical trials for the treatment of chikv infection is warranted. shinde et al. induction of crossreactive hemagglutination inhibiting antibody and polyfunctional cd4+ t-cell responses by a recombinant matrixm-adjuvanted hemagglutinin nanoparticle influenza vaccine randomized controlled trial 2021 a randomized, observer-blind, comparator-controlled (trivalent high-dose inactivated influenza vaccine [iiv3-hd] or quadrivalent recombinant influenza vaccine [riv4]), safety and immunogenicity trial of qniv (5 doses/formulations) in healthy adults ≥65 years. vaccine immunogenicity was measured by hemagglutinationinhibition assays using reagents that express wild-type hemagglutination inhibition (wthai) sequences and cellmediated immune responses. overall, similar frequencies of solicited and unsolicited adverse events were reported in all treatment groups. kremsner et al. safety and immunogenicity of an mrna-lipid nanoparticle vaccine candidate against sars-cov-2 : a phase 1 randomized clinical trial clinical trial 2021 this is an interim analysis of a dosage escalation phase 1 study in healthy 18-60-year-old volunteers in hannover, munich, and tübingen, germany, and ghent, belgium. after giving 2 intramuscular doses of cvncov or placebo 28 days apart we assessed solicited local and systemic adverse events (ae) for 7 days and unsolicited aes for 28 days after each vaccination. immunogenicity was measured as enzyme-linked immunosorbent assay (elisa) igg antibodies to sars-cov‑2 s‑protein and receptor binding domain (rbd), and sarscov‑2 neutralizing titers (mn50). responses to 12 μg were comparable to those observed in convalescent sera from known covid-19 patients. mallory et al. safety and immunogenicity following a homologous booster dose of a sarscov-2 recombinant clinical trial 2022 this secondary analysis of phase 2, randomised study assessed a single booster dose of a sarscov-2 recombinant spike protein vaccine with matrix-m adjuvant (nvx-cov2373) in 1610 participants were screened from aug 24, 2020, to sept 25, 2020. 1282 participants were enrolled, of whom 173 were assigned again to placebo (group a), 106 were re-randomized to nvx-cov2373-placebo (group b1), 356 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 authors title date and type of the study method results spike protein vaccine (nvx-cov2373): a secondary analysis of a randomised, placebo-controlled, phase 2 trial healthy adults aged 18-84 years, recruited from 17 clinical centres in the usa and australia. and 104 were re-randomized to nvxcov2373-nvx-cov2373 (group b2); after accounting for exclusions and incorrect administration, 172 participants in group a, 102 in group b1, and 105 in group b2 were analyzed for safety. following the active booster, the proportion of participants with available data reporting local (80 [82%] of 97 participants had any adverse event; 13 [13%] had a grade ≥3 events) and systemic (75 [77%] of 98 participants had any adverse event; 15 [15%] had a grade ≥3 events) reactions was higher than after primary vaccination (175 [70%] of 250 participants had any local adverse event, 13 [5%] had a grade ≥3 events; 132 [53%] of 250 had any systemic adverse event, 14 [6%] had a grade ≥3events). local and systemic events were transient in nature (median duration 1·0-2·5 days). in the per-protocol immunogenicity population at day 217 (167 participants in group a, 101 participants in group b1, 101 participants in group b2), igg geometric mean titers (gmt) had increased by 4·7-fold and mn50 gmt by 4·1-fold for the ancestral sarscov-2 strain compared with the day 35 titers. heath et al. safety and efficacy of the nvxcov2373 coronavirus disease 2019 vaccine at completion of the placebo-controlled phase of a randomized controlled trial clinical trial 2023 adults aged 18-84 years received 2 doses of nvxcov2373 or placebo (1:1) and were monitored for virologically confirmed mild, moderate, or severe covid-19 (onset from 7 days after second vaccination). participants who developed immunoglobulin g (igg) against nucleocapsid protein but did not show symptomatic covid-19 were considered asymptomatic. secondary outcomes included anti-spike (s) igg responses, wild-type virus neutralization, and t-cell responses. incidence of serious adverse events and adverse events of special interest were similar between groups. lovell et al. interim analysis from a phase 2 randomized trial of eucorvac19: a recombinant protein sars-cov-2 rbd nanoliposome vaccine clinical trial 2022 an initial study of phase 2 randomized, observer-blind, placebo-controlled trial to assess the immunogenicity, safety, and tolerance of ecv19 was carried out between july and october 2021. two hundred twenty-nine participants were enrolled at 5 hospital sites in south korea. healthy adults aged 19-75 without prior known exposure to covid-19 were vaccinated intramuscularly on day 0 and day 21. of the participants who received two vaccine doses according to protocol, 100 neutralizing antibody levels of the low-dose and high-dose ecv19 groups had frnt50 geometric mean values of 129 and 316, respectively. boosting responses and dose responses were observed. antibodies against the rbd correlated with antibodies against the spike and with virus neutralization. hussein et al. – nanotechnology-based vaccines 357 authors title date and type of the study method results received high-dose ecv19 (20 μg rbd), 96 received low-dose ecv19 (10 μg rbd), and 27 received a placebo. local and systemic adverse events were monitored. serum was assessed on days 0, 21, and 42 for immunogenicity analysis by elisa and neutralizing antibody response by focus reduction neutralization test (frnt). gatechompol et al. safety and immunogenicity of a prefusion nonstabilized spike protein mrna covid-19 vaccine: a phase i trial clinical trial 2022 seventy-two eligible volunteers, 36 of whom were aged 18-55 (adults) and 36 aged 56-75 (elderly), were enrolled. two doses of vaccine were administered 21 d apart at 10, 25 or 50 μg per dose (12 per group). the primary outcome was safety, and the secondary outcome was immunogenicity. all three dosages of chulacov19 were well tolerated and elicited robust dosedependent and age-dependent b and t-cell responses mrna vaccine expressing a prefusion non-stabilized spike protein is safe and highly immunogenic. masuda et al. safety and immunogenicity of nvx-cov2373 (tak-019) vaccine in healthy japanese adults: interim report of a phase i/ii randomized controlled trial clinical trial 2022 this phase 1/2, randomized, observer-blind, placebocontrolled trial conducted in japan (two sites), enrolled healthy japanese adults aged ≥ 20 years with no history/risk of sars-cov-2 infection and no prior exposure to other approved/investigational sarscov-2 vaccines or treatments. participants were stratified by age (< 65 or ≥ 65 years) and randomized to receive two doses of either nvx-cov2373 (5 μg sars-cov-2 rs; 50 μg matrixm1) or placebo, 21 days apart. primary outcomes were safety and immunogenicity assessed by serum igg antibody levels against sars-cov-2 rs protein on day 36. herein, we report the primary data analysis at 4 weeks after the second dose, ahead of the 12-month follow-up completion (data cut-off: 8 may 2021). between 12 february 2021 and 17 march 2021, 326 subjects were screened, and 200 participants were enrolled and randomized: nvxcov2373, n = 150; placebo, n = 50. solicited adverse events (aes) through 7 days after each injection occurred in 121/150 (80.7%) and 11/50 (22.0%) participants in the nvx-cov2373 and placebo arms, respectively. in the nvx-cov2373 arm, tenderness and injection site pain were the most frequently reported solicited aes after each vaccination, irrespective of age. robust immune responses occurred with nvx-cov2373 (n = 150) by day 36: igg geometric mean fold rise (95% confidence interval) 259 (219, 306); seroconversion rate 100% (97.6, 100). no such response occurred with a placebo (n = 49). ishikawa et al. safety and antibody immune response of chp-ny-eso-1 vaccine combined with poly-iclc in advanced or recurrent esophageal cancer patients clinical trial 2021 conducted a phase 1 clinical trial of chp-ny-eso-1 with poly-iclc in patients with advanced or recurrent esophageal cancer. chp-nyeso-1/poly-iclc (μg/mg) was administered at a dose of 200/0.5 or 200/1.0 (cohorts 1 and 2, respectively) every 2 weeks for a total of six doses. the primary endpoints were the most common adverse event (ae) was injection site skin reaction (86.7%). no grade 3 or higher drugrelated aes were observed. no tumor responses were observed, and three patients (30%) had stable disease. the immune response was comparable between the two cohorts, and all patients (100%) achieved antibody responses with a median of 2.5 vaccinations. comparing chp-ny 358 biology, medicine, & natural product chemistry 12 (1), 2023: 343-361 authors title date and type of the study method results safety and immune response. the secondary endpoint was tumor response. in total, 16 patients were enrolled, and six patients in each cohort completed the trial. eso-1 alone to the poly-iclc combination, all patients in both groups exhibited antibody responses, but the titers were higher in the combination group. in a mouse model, adding an anti-pd-1 antibody to the combination of chp-ny-eso-1/polyiclc suppressed the growth of nyeso-1-expressing tumors. combining the vaccine with pd-1 blockade holds promise in human trials. li et al. immune persistence and safety after sars-cov-2 bnt162b1 mrna vaccination in chinese adults: a randomized, placebo-controlled, double-blind phase 1 trial clinical trial 2022 immune persistence was determined at month 3 in 72 younger participants (aged 1855 years) and at month 6 in 70 younger and 69 older participants (aged 65-85 years). in younger participants, neutralizing antibody (nab) geometric mean titers (gmts) for the 10 and 30 µg dose levels declined from 233 and 254 (21 days after dose 2) to 55 and 87 at month 3, respectively, and to 16 and 27 at month 6, respectively. in older participants, nab gmts declined from 80 and 160 (21 days after dose 2) to 10 and 21 at month 6. overall, higher antibody titers were observed in younger participants, and the 30 µg dose induced higher levels of nab, which declined more slowly by month 6. no serious adverse events were reported in the vaccine group. wei et al. identification and application of a neutralizing epitope within alphahemolysin using human serum antibodies elicited by vaccination clinical trial 2021 collected sera from volunteers in a phase 1b clinical trial of a novel recombinant five-antigen sa vaccine (nct03966040). using a luminex-based assay, we characterized the human serologic response against hla, and identified hla121-138 as a neutralizing epitope. in addition, we successfully produced ferritin nanoparticles carrying the neutralizing hla121-138 epitope (epnp) in e. coli. epnp presented as homogenous nanoparticles in an aqueous solution. immunization with epnp elicited potent hemolysisneutralizing antibodies and conferred significant protection in a mouse model of sa skin infection. data suggest that epnp, carrying the neutralizing epitope hla121-138, is a good candidate for a vaccine against sa. conclusions vaccination has had a major impact on the control of infectious diseases. older vaccines, including those that successfully prevent measles and polio, work by stimulating the body's immune system and antibody production using a dead or weakened (attenuated) pathogen. however, in someone with a compromised immune system, an attenuated pathogen may still cause the disease, and a dead pathogen may not result in a strong enough or long-lasting immune response. many of these vaccines have been shown to elicit protective immunity against many diseases, but they are associated with certain challenges that limit their effectiveness in a clinical setting. dna and rna vaccines, for example, are cost-effective and associated with minimal infection risks but can be easily degraded as a result of delivery challenges to target sites. nanoscale size (<1000 nm) materials such as virus-like particles, liposomes, iscoms, polymeric, and non-degradable nanospheres hussein et al. – nanotechnology-based vaccines 359 have received attention as potential delivery vehicles for vaccines. the vaccine antigen is either encapsulated within or decorated onto the surface of the np. by encapsulating antigenic material, nps provide a method for delivering antigens that may otherwise degrade rapidly upon injection or induce a short-lived, localized immune response. in this review, we assess the potential of these delivery systems for the creation of new vaccines against a variety of infections and compare the usefulness of various np systems for the delivery of subunit vaccines. the effectiveness of vaccines is determined in large part by a number of physicochemical characteristics of nanoparticles. clinical trial studies have shown that nanocarriers can function as effective middlemen in the creation of vaccinations against a variety of diseases. development of np formulations that can deliver immunogens to apcs, particularly dcs, is crucial in this situation to promote efficient antigenspecific t-cell responses. via b-cell receptors, b cells are able to identify and react to microbial surface antigens. for the creation of vaccines against various diseases, artificial nps have been used to activate and clonally expand antigen-specific b-cells. comparing the delivery of exposed vaccine molecules to the encapsulation of antigens in virus-like particles (vlps), studies found that the vlps were able to elicit robust and long-lasting humoral responses. several research clearly proved the effectiveness of non-invasively given vaccines. concerns about particle toxicity, production challenges, and delivering antigens in their natural state are just a few of the difficulties of using nps for vaccine administration. future research on nano vaccines will focus on the action of nps as well as the potential to elicit an immune response for the treatment of diseases. acknowledgements: authors would like to appreciate professors of biomedical science at dubai medical college for girls, for their continuous support during the conceptualization and preparation of this manuscript. competing interests: the authors declare that there are no competing interests. funding: there are no sources of funding to declare. references doria-rose, n., suthar, m. s., makowski, m., o’connell, s., mcdermott, a. b., flach, b., … mrna-1273 study group. 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(2021). identification and application of a neutralizing epitope within alpha-hemolysin using human serum antibodies elicited by vaccination. molecular immunology, 135, 45–52. doi:10.1016/j.molimm.2021.03.028 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 2, 2018 | pages: 39-43 | doi: 10.14421/biomedich.2018.72.39-43 issn 2540-9328 (online) comparative anatomy of labyrinth and gill of catfish (clarias gariepinus) (burchell, 1822) and snakehead fish (channa striata) (bloch, 1793) ina karlina1,*, muhammad ja’far luthfi2 1center for integrative zoology, 2biological education department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto, no. 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. author correspondency*: ikarlina017@gmail.com abstract dumbo catfish (clarias gariepinus) and gabus (channa striata) have additional organ respiratory system structures in the gills, called labyrinth. this organ is a tool for fish to take oxygen directly from the air so that it can live in low oxygen habitats. both fish have differences at the order level. catfish is an order of siluriformes as gabus is an order of perciformes. the purpose of this study was to distinguish the anatomy and histology of the gill structure and labyrinth in both fish. the macroanatomy observation was conveyed by dissection to determine the gill topography in the two fish species. histology preparations were carried out using the paraffin method and using hematoxylin-eosin (he) staining that showed in the labyrinth organ there were many blood vessels to bind oxygen then distributed to all parts of the fish's body. keywords: anatomy; catfish (clarias gariepinus); snakehead fish (channa striata); hematoxylin-eosin (he); histology; labyrinth introduction catfish (clarias gariepinus) and snakehead fish (channa striata) have an additional structure in their gill organs but both were grouped into different orders. catfish belonging to order siluriformes while snakehead fish belonging to order perciformes. labyrinth is a multiplied membrane that filled with blood capillaries located at the top of second and thirdgill arches, and organ shaped like a sponge. fujaya (2008) stated that some species of fish have a labyrinth which is an upward expansion of gills forming folds and so become irregular cavities. explanation other sources that gills of catfish have a small size that located on the back head (najiyati, 2010 in utomo 2006). according to jayaram, 1981 (in utomo, 2006) this size causes catfish had trouble to filter water in, therefore catfish need a labyrinth as an additional respiration tool. the labyrinth allowed catfish and snakehead fish to extract oxygen directly from the air so they can live in low content oxygen waters (susanto, 1989; angka et al., 1990; suyanto, 1992). insight into the development of organs in pisces is still lack and limited (rather limited), so it required in-depth study to find out more extensively (fumio & takashi, university of tokyo, 2010). based on the facts it is necessary to have a more in-depth study about labyrinth structure in gills of catfish and snakehead fish. this confirmed that groupings of pisces members are generally not only based on morphological features and characteristics but also anatomy and histology aspects that interesting to be studied further. not only to refer to those additional structures in helping and sustain its life-related adaptation aspects to the environment, but also the study of anatomy and histology. method this study used three adults catfish (clarias gariepinus) and snakehead fish (channa striata). the anatomical observation was done by observing the color, shape, and length of the gill and labyrinth-gill. histological slides used paraffin method and stained with using hematoxylin-eosin (he). results and discussion gill and labyrinth-gill anatomy of adult catfish (clarias gariepinus) and snakehead fish (channa striata) based on the results obtained from the study gill color on catfish and bright red bright snakehead fish, this indicates the number of blood vessels capillaries and blood cells contained inside. gills of catfish and snakehead fish consist four pairs of sheets, while the shape is like a raker, besides it has three main structures namely gill arch, gill racker and gill filament. gills catfish and snakehead fish are located on the side-head https://doi.org/10.14421/biomedich.2018.72.39-43 40 biology, medicine, & natural product chemistry 7 (2), 2018: 39-43 precisely on right and left of the pharynx and outside covered by an operculum. gill arch or branchialis archus structure in catfish and snakehead fish are colored white is a place of attachment gill filament and filament. the differences between catfish are the gills surface which smaller and narrower than snakehead fish which wider.  gill figure 1. catfish gill and snakehead fish. gill filament or hemibranchia in catfish and snakehead fish resembles a raker and attaches on gill arch. each gill sheet consists of pair filaments, and on each filament contains many thin layers called lamela. lagler et al (1997) stated that gills consist of pair gill filaments, each filament consists of thin transverse fiber covered by a thin epithelium called lamella. the lamella is a filament composer. another structure is the gill or gill rakers, which is a pair of rows cartilage rods are short and serrated small, attached to the front of gill arch and has a function to filter the water respiration. according to achmad (2005), gills on the anterior middle edge are equipped with a structure (gill rakers) that function filtering particles. figure 2. portions of catfish and snakehead fish gill.  labyrinth-gill the results from the research, the labyrinth on the catfish has bright-pink color, while snakehead fish has reddish cream color. the pink color is caused by capillary blood vessels, which are not much different from gills. catfish labyrinth has sponge-like shaped (arborescent) with irregular structures. this signifies many structures resembling vesicle gas as air reserves that support respiratory process, especially when lacking oxygen. while snakehead fish labyrinth has solid-shape with a serrated edge. labyrinth is located on the first-gill sheets catfish and the longest sheet attached to the edges. according to kisia (2010), the labyrinth in clarias cluster gills is covered by respiratory epithelial folds and derived from the first-gills of the dorsolateral head bone of gill cover. while labyrinth of snakehead fish is located lies in the pharyngeal. according to sukiya (2010), the labyrinth of snakehead fish is a development in dorsal part a pair of the pharynx and an extension known as suprapharyngeal space, this room is supported when in lack oxygen and the process breathes. figure 3. catfish and snakehead fish labyrinth-gill. macroanatomically, the gills structure of catfish and snakehead fish are generally same, they have three main parts i.e gill raker, gill arch, and gill filament. these three structures have the same functions. a form of gill raker dumbo catfish serrated small dense and tightly neat, while the form of gill raker snakehead fish is larger, uneven and irregular. the striking difference is shown in terms of color, structure, and location or topography of the labyrinth. all anabantoid/anabyroid species have specialized labyrinth organs to support gas exchange and involved in the surface water respiratory behavior (graham, 1997). reviewing in terms of complexity, resilience and the surface area catfish has more survive than snakehead fish. the more complex and larger surface area of the respiratory tool structure then accommodated oxygen reserve will more plenty. so that fish can survive longer when lack oxygen in the water. according to kisia (2008), catfish can survive in last minutes or even several hours because it has an additional respiratory tool called arboresence, this tool is a folded membrane which is full of blood capillaries located in the upper cavity above the gills. histology of gill and labyrinth-gill  gill results showed that catfish and snakehead fish gills have a thin epithelial layer for the efficiency of oxygen and gas exchange (absorption and release). in addition, gills have a function to regulate salt and water exchange and function in excretion of nitrogenous waste products, gill gill labyrinth gill raker gill raker gill arch gill arch gill filament karlina & luthfi – comparative anatomy of labyrinth and gill of catfish … 41 especially ammonia. gill is composed by gill raker, gill arch and gill filament. this gill arch composed by primary lamella that has the parallel lamella secondary sheets along primary lamella. this secondary lamella functions to take oxygen from water. histologically, primary lamella is composed of pillar cells lined up and cells are covered in a thin epidermal membrane semipermeable. spaces between pillar cells are called lacuna as the blood pass-over place. figure 4. gill of catfish and snakehead fish (40x10 magnification). hematoxylin-eosin (he). according to lagler et al (1997), lamella is composed of thin epidermal cells and stem-shaped support cells (pillar cells) that helps blood-flow to the gills. the thickness of lamella are varied depends on species and its activity. gas exchange occurs in secondary lamella which is the fold of epithelial cells with a single supports cell layer and separated by pillar cells. a thin layer of blood vessels lies between the pillar cells and epidermis which is become gas exchange site, nitrogenous wastes of metabolites remains and some electrolyte exchanges. pseudobrankhia is located at the bottom of the upper operculum. this organ is a gill arch with a series of filaments. the function of pseudobranchial is not yet known, but it is thought the structure that supplies oxygen-rich blood to chloroid cells optical and retinal, it may play a role in baroreceptor and thermoregulation functions. in addition, gills are equipped with a number of glands known as the branchial gland i.e specialized gill epithelial cells. these glands are a mucous gland and the acidophilic glands (chloride cells). marrison (2007 in prasetyo, 2011), stated that the gill arch consists of the primary lamella. each primary lamella has a secondary lamella located in a perpendicular to the primary lamella. gill arch is covered by epidermal tissue and contained many mucosal cells. in the primary lamella, contained chloride cells that commonly found in basal (proximal) of lamellae. these cells function in ion transport and detoxification. gas exchange occurs throughout the surface of secondary lamella primarily through the exchange of blood and water coming from the environment. the secondary lamellar surface consists by squamous cell epithelial overlap, usually, one layer supported and separated by pillar cells.  labyrinth figure 6. histology of labyrinth-gill of catfish (clarias gariepinus). he staining. (40x10 magnification). 1. microvili, 2. goblet cell, 3. epithelium cell, 4. granula, 5. lamina basal, 6. loose connective tissue, 7. capillary blood vessels. the outer surface covered by epithelial cells and restrict two internal parts as almost all layers of the body or the organs are covered epithelial cells. epithelial cells construct the labyrinth of dumbo catfish that very dense and almost no intercellular space. flat-shaped and belonging to a simple squamous cell type epithelium, mostly in the respiratory organs that have primary functions to exchange substances. according to orphans (1996), in some types of epithelium form a bulge or crease to expand its surface area. figure 7. histology of labyrinth-gill of dumbo catfish (clarias gariepinus). he staining. (40x10 magnification). 1. intercapillary space, 2. perisite (pervascular cells), 3. capillary blood, 4. endotel. blood vessels blood vessels secondary lamella secondary lamella primary lamella spaces between lamella primary lamella 42 biology, medicine, & natural product chemistry 7 (2), 2018: 39-43 the surface epithelial type of labyrinth catfish is simple squamous epithelial cells. these cells consist of a single layer of epithelial cells as the point of contact for the epithelial tissue with the basement membrane. types can be in face rapid diffusions areas such as lung alveoli lining (respiratory unit called airbags), endothelium (blood vessel lining), capillaries, kidneys, the main body cavity (mesothelium), and other major areas where the little activity occurred. the respiratory mucosa is composed of epithelial cells and there is a lamina propria. the epithelium is a tall columnar with cilia and goblet cells. supporters of lamina propria under the epithelium contain elastin. the function of simple squamous epithelial cells is responsible for allowing the exchange of oxygen. they are also responsible for blood filtering, and diffusion that allows passing oxygen in the blood. in addition, there is also a connective tissue that connects with the network under it, the type of loose connective tissue which is the most widely spread connective tissue in the body of vertebrate animals. histological description showed that labyrinth of dumbo catfish has a loose connective tissue which is a network of rare cells. part of tissue composed of a matrix which located beneath the matrix epithelial layer of mucous fluid, the deepest part which is capillary blood vessels. this capillary vessel is limited by endothelial cells that are flattened, located long in accordance with the blood flow, are blue and prominent into the lumen. figure 8. histology of labyrinth-gill of gabus (channa striata). he staining. (10x10 magnification). 1. microvilli, 2. epithelium layer, 3. goblet cell, 4. lamina basal, 5. connective tissue, 6. capillary blood vessel. labyrinth in snakehead fish is known diverticula although it functions equally with the labyrinth in catfish as an additional tool when fish is in lack oxygen. diverticula is in the oral cavity and pharynx. diverticula is in a group of snakehead fish (channa). according to john, karl and robert (1997), diverticula is coated by respiratory epithelium with a numerous blood supply, usually from afferent gill circulation. this showed that the coating and composition of the constituent are same as the labyrinth in dumbo catfish. figure 8, showed the tissue and constituent cells of the snakehead fish labyrinth, the outermost part of respiratory epithelial cells and blood vessels. there is a type of respiratory epithelium and also equipped with microvilli, which functions from microvilli to exchange and absorb substances. there is a goblet cell that serves to convert mucus into latex/gum and equipped by basal lamina at the base-part. figure 9. histology of labyrinth-gill of snakehead fish (channa striata). he staining. (10x10 magnification). 1. loose connective tissue, 2. tight connective tissue and, 3. blood vessel. lamina basal as a barrier between epithelial tissue with underlying tissue, the deepest part of connective tissue and capillary blood vessels. seen on the histology of labyrinth snakehead fish there is connective tissue that connects with other parts. although catfish and snakehead fish have additional breathing apparatus, it can be seen from the development of aspect and different surface area, the endurance if compared between them is different. seen on the histologic picture both in dumbo catfish is more complex than snakehead fish. labyrinth of catfish has a mucous-stratified epithelium, whereas snakehead fish has a pseudostratified epithelium. the difference is striking shown at 40 times magnification, the tissue quite clears constituent cells and their shape. in the labyrinth of snakehead fish, the surface area is not as wide as in catfish. reviewing from the aspects, the surface area and structure cell and its constituent tissue on catfish are more complex than snakehead fish. measurement morphometric of gill and labyrinth table 1. calculation standard deviation of gill and labyrinth. no measured aspects standard deviation value catfish snakehead fish 1 mass left gill (gram) 5,01 ± 0,004 17,83 ± 0,55 2 mass right gill (gram) 5,34 ± 0,045 17,74 ± 0,55 3 filament length 1 (cm) 4,07 ± 0,002 4,83 ± 0 4 filament length 2 (cm) 3,84 ± 0 4,68 ± 0,004 5 filament length 3 (cm) 2,90 ± 0 2,89 ± 0 6 filament length 4 (cm) 1,97 ± 0 2,43 ± 0 7 gill arch length 1 (cm) 15,56 ± 0,08 32,24 ± 0 8 gill arch length 2 (cm) 13,67 ± 0,01 26,18 ± 0,004 9 gill arch length 3 (cm) 11,80 ± 0,01 12,75 ± 0 10 gill arch length 4 (cm) 10,08 ± 0,002 11,58 ± 0 11 mass labyrinth (gram) 3,81 ± 0 2,44 ± 0 karlina & luthfi – comparative anatomy of labyrinth and gill of catfish … 43 measured aspects include mass and length of fish, right and left labyrinth mass, the overall of labyrinth and gills mass, the right and left labyrinth mass, the right and left gills of each gill sheet, and length of filament gill and gill arch. this study focused on gill and labyrinth comparisons. so the standard deviation calculation is more focused on calculation aspects of the left and right gill morphology, right and left labyrinth volume, long morphometry of filament and gill arch. value of standard deviation that was measured on calculation gill mass in catfish was 0.004 and 0,045 on right gill. while on the left and right gills of snakehead fish were 0,55. measuring the mass of labyrinth catfish and snakehead fish is 0. morphometry length of four filament gill in catfish is 0, whereas in snakehead fish is 0 and measurement on the four-gill arch length of catfish is 0.08, 0.01, 0.01 and 0.002 while in the snakehead fish is 0. data was indicated that the diversity of samples under study is small, the smaller standard deviation values obtained indicate that the sample is same. mass between left and right gill is same, it is seen from the mass of the two is similar to maintain balance. based on the morphometric data showed that the smaller standard deviation calculation value, the sample diversity is small, meaning sample used in this study is the same. length of each gill sheet is also different. it is seen from the development of labyrinth in catfish is more developed than snakehead fish. conclusion gills of catfish and snakehead fish are bright-red color, four-gill sheets with three main structures: gill arch, raker, and filament, located on side of the head and protected by an operculum. catfish labyrinth has a pink color, shaped like a sponge, attached to the first gill, solid in shape with jagged edges, located near the pharynx. catfish and snakehead fish gills consist of primary lamella, secondary lamella, inter-lamella, blood vessels, epithelial cells, and pole cells. catfish labyrinth has microvilli, goblet cells, epithelium lining, granules, basal lamina, loose connective tissue, capillaries, perivascular and endothelium, whereas in snakeheadfish there are microvilli, epithelium lining, goblet cells, basal laminae, connective tissue, and capillary blood vessels. references achmad dkk, 2005. struktur hewan. universitas terbuka. jakarta. fujaya, 2008. fisiologi ikan. pt rineka cipta, jakarta. fumio & takashi, 2010, histology of fish. university of tokyo. tokyo press. john e. bardach, lagler, dan miller, 1997. ichtyology. university of michigan. wiley internasional publisher. usa. kisia, seth m, 2010. structures and funtions. markono print media pte ltd, usa an imprint of edenbrige ltd., british channel island. prasetyo, 2011. proses pernafasan pada ikan. jurnal sistem regulasi pada hewan perairan. diunduh dari web http//www.sistem.regulasi.hewan.perairan. accessed on a date march 20th 2015, at 05.30 wib sukiya, 2003. biologi vertebrata. universitas negeri yogyakarta: jica susanto, 1989; murhananto, 2002. jenis perairan sesuai ikan lele. diunduh dari web http///www.perairan.ikan.lele.com. accessed on a date april 11th 2015, at 13:45 wib. suyanto, 1986. suyanto, s.r., 2003. ikan. penebar swadaya. jakarta utomo 2006. letak dan morfometri insang pada ikan lele. jurnal penelitian insang ikan. diunduh dari web http//www.insang.ikan.lele.com. accessed on a date march 12th 2015, at 10:00 wib. wildan yatim, 1996. biologi modern histologi. tarsito bandung, anggota ikapi: bandung. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 25-31 | doi: 10.14421/biomedich.2025.141.25-31 issn 2540-9328 (online) effect of prolonged dehumidification on the chemical properties of crassiacarpa and mangium honey esa ghanim fadhallah*, otik nawansih, fibra nurainy, depri mubarik department of agricultural product technology, faculty of agriculture, universitas lampung. jl. prof. dr. soemantri brojonegoro no. 1, bandar lampung, lampung, 35145, indonesia. corresponding author* esa.ghanim@fp.unila.ac.id abstract honey is a natural substance bees produce from nectar, flower sap, or liquid collected from living plant parts. the honey undergoes bee modification and binding before being stored in hexagonal combs. effective dehumidification is crucial for enhancing honey quality and extending its shelf life. this study aimed to evaluate the effects of prolonged dehumidification time, honey type, and their interaction on the quality of crassiacarpa and mangium honey and to determine the optimal dehumidification treatment to meet the sni 8664-2018 standard. a factorial completely randomized design (crd) with three replications was employed. two liters each of crassiacarpa and mangium honey were placed in trays with a thickness of ±2 cm and dehumidified at 25°c and 40% humidity for 24, 48, 72, and 96 hours. following dehumidification, the honey was analyzed for moisture content, total acidity, ph, and sensory attributes (taste, color, aroma, texture). data were analyzed descriptively. the results demonstrated that dehumidification time significantly impacted moisture content, total acidity, and ph. honey type significantly affected moisture content and ph. the interaction between dehumidification time and honey type significantly affected ph. the optimal treatment for both honey types was dehumidification for 96 hours, which resulted in honey that met the sni 8664-2018 standard. keywords: crassiacarpa; dehumidification; honey; mangium. introduction indonesia, as an archipelagic nation rich in tropical forests and diverse flora and fauna, hosts a wide variety of honeybee species, including apis cerana, apis mellifera, apis koschevnikovi, apis dorsata, apis nigrocincta, and apis andreniformis. these species play a vital role in honey production, valued for their sweetness and nutritional benefits derived from the nectar of various plants, which contains a carbohydraterich secretion (30-50%) (pribadi et al., 2019). according to the indonesian central bureau of statistics (bps, 2020), honey production in indonesia has shown significant fluctuations from 2016 to 2020. in 2016, national honey production peaked at 362.2 thousand litres but sharply declined to 51.3 thousand litres by 2020. java emerged as the largest producer, with 1.6 thousand litres, constituting 81.06% of the total output. sumatra followed with 4.01 thousand litres (7.81% of the total), while kalimantan and sulawesi produced 3,000 and 500 litres, respectively. these figures highlight the regional disparities in honey production, which are influenced by various factors such as climate, flora availability, and beekeeping practices. approximately 80% to 90% of honey originates from wild bees across various regions of indonesia, showcasing the significant role of wild bees in the country's honey production landscape. honey, as defined by the indonesian national standard (sni) 01-3545-1994 (bsn, 1994), is a natural liquid characterized by its sweetness, produced by bees from the nectar of flowers or other plant parts. the consumption of honey in indonesia is steadily increasing, evidenced by the growing number of honey brands and honey-based products in the market. post-harvest handling plays a crucial role in determining honey quality. nanda et al. (2014) observed that honey harvested at later stages tends to have lower water content than younger honey. proper handling and processing are essential for maintaining honey quality, while improper practices can degrade quality and reduce shelf life. for instance, excessive heating during processing can reduce the nutritional value and alter the physical properties of honey. as a hygroscopic substance, honey absorbs moisture from the air, making its water content susceptible to environmental humidity (sarwono, 2007). the indonesian national standard (sni) 8664-2018 stipulates that honey should contain no more than 22% water content (bsn, 2018). however, a common manuscript received: 23 november 2024. revision accepted: 17 february, 2025. published: 20 february, 2025. https://doi.org/10.14421/biomedich.2025.141.25-31 26 biology, medicine, & natural product chemistry 14 (1), 2025: 25-31 challenge producers face is that freshly harvested honey often exceeds this standard, leading to fermentation. high water content facilitates fermentation, which degrades honey quality, shortens its shelf life, and can even cause packaging to break. therefore, reducing water content is crucial to maintaining honey quality. effective moisture control is imperative to prevent fermentation and ensure the longevity of honey's shelf life. factors influencing honey's water content include climate, harvesting practices, and the types of nectar bees collect (savitri et al., 2017). the longer honey is left in the hive or improperly stored post-harvest, the more water evaporates (minarti et al., 2016). however, improper storage conditions can lead to reabsorption of moisture, complicating the quality control process. this issue can be mitigated by using a dehumidifier, which effectively removes water vapour from honey through dehumidification. to ensure compliance with quality standards, honey producers employ various methods to reduce water content, such as heating to speed up evaporation using direct and indirect heating methods with vacuum dehydrators. another effective method is dehumidification using a water dehumidifier to absorb moisture from honey. honey producer located in bandar lampung indonesia, utilizes dehumidification processes to lower water content and maintain honey quality. this method was chosen due to its practicality and proven effectiveness in previous studies. apriantini (2022) reported that dehumidification using an air dehumidifier at 30oc for 4 and 8 hours did not reduce the physical properties quality and yielded improved other properties such as ph, viscosity, color intensity, and antioxidant activity on rubber and rambutan honey. in this study, we extend the dehumidification duration to crassiacarpa and mangium honey types produced by apis mellifera bees. this study aims to investigate the effect of the prolonged dehumidification process on the chemical properties of crassiacarpa and mangium honey. materials and methods honey samples two types of honey samples, crassiacarpa and mangium, produced by apis mellifera bees, were harvested from the local honey bee farm in bandar lampung, indonesia. experimental design this study employs a factorial completely randomized block design (crbd). the first factor is the type of honey (p), consisting of two levels: crassiacarpa (p1) and mangium (p2). the second factor is the dehumidification duration (t), consisting of four treatment levels: 24 hours (t1), 48 hours (t2), 72 hours (t3), and 96 hours (t4), with each treatment being repeated three times. observed parameters on dehumidified honey included water content, acidity, and ph. the data were statistically analyzed using analysis of variance (anova) and processed further using the least significant difference (lsd) test at the 5% significance level. the best treatment was determined by the adherence to those parameters with the international honey standard (cac, 2001). the best treatment samples were then tested using the duo-trio method to detect differences in color, aroma, taste, and texture compared to the control (non-dehumidified honey). dehumidification process the harvested honey is received in bulk at the raw material reception section. the honey is then filtered to remove impurities. subsequently, the honey is poured into trays to a thickness of approximately ± 2 cm. the dehumidification room (25oc) humidity was set at a humidity level of 40% using a dehumidifier (kris, indonesia) in a tightly sealed condition. the duration of dehumidification is 24, 48, 72, and 96 hours. after the dehumidification process, the honey is packaged in a translucent hdpe jerry can and transported to the laboratory. it is then kept in the refrigerator (4°c, rh 75%) for 4 hours before being analyzed further. water content, acidity, and ph analysis the measurement of water content using a refractometer (rhb-92atc, china). technically, a refractometer measures the refractive index of a substance. to measure the water content of honey, a sample of honey is placed on the tip of the refractometer. once the sample is placed, the water content can be directly observed from the refractive index displayed by the refractometer. the percentage of water content is indicated by the highest boundary of the light blue color on the metric scale. total acidity tests were conducted using the volumetric method described by balos et al. (2018). a 10 g sample of honey is weighed and then placed into a 250 ml erlenmeyer flask. it is dissolved with 75 ml of distilled water, and 4-5 drops of phenolphthalein indicator are added. the solution is then titrated with 0.1 n naoh solution while swirling the erlenmeyer flask until a permanent color change is observed for 10 seconds. the volume of 0.1 n naoh required for the titration is recorded. the acidity value (in ml n naoh/kg) is calculated by multiplying the volume of naoh used in the titration (in ml) by the normality of naoh (0.1 n), divided by the sample weight (in g) and then multiplied by 1000. the ph was measured using the potentiometric method, following chakir et al. (2016). the ph meter (lutron ph222, taiwan) is calibrated using a ph seven buffer solution. once the ph meter is standardized, it is immersed in the honey sample container, and the ph measurement result is displayed on the device. the same procedure is followed for each treatment. fadhallah et al. – effect of prolonged dehumidification on honey 27 duo-trio sensory evaluation the duo-trio test was performed to determine whether there was a significant difference between the control honey sample (non-dehumidified) and the dehumidified honey sample from the best treatment based on sensorial attributes (texture, color, aroma, and taste). the sensory evaluation was conducted by 20 trained panellists. samples were placed in small glasses, each containing two teaspoons of honey. samples were labelled with three random-digit codes and presented on a tray with a spoon, pen, and questionnaire form. the presenter provided the test sample set and explained how to complete the questionnaire. the panellists were then asked to provide their responses to the questionnaire. the collected data were analyzed and matched with the duotrio table following iso 10399:2017 (iso, 2017) with a significance level of 5%. results and discussion water content the research results show that the moisture content of two types of honey, crassiacarpa (p1) and mangium (p2), ranges between 17–23%. the moisture content of crassiacarpa honey (p1) ranges from 18.5–23%, while mangium honey (p2) ranges from 17.3–20.6%. the variance analysis (table 15) indicates that the dehumidification time (t) and the type of honey (p) significantly affect the moisture content, but their interaction does not have a significant effect. the results of the 5% lsd test indicate that dehumidification time (t) and type of honey (p) significantly affect the moisture content of the honey, as shown in figures 1 and 2. figure 1. water content of dehumidified honey at different duration. (t0: 0 hour, t1: 24 hour, t2: 48 hour, t3: 72 hour, t4: 96 hour). figure 2. water content of crassiacarpa (p1) and mangium (p2) honey. figure 1 shows that the moisture content of honey dehumidified at t0 (0 hours) differs significantly from t1 (24 hours), t2 (48 hours), t3 (72 hours), and t4 (96 hours), while t1 (24 hours) does not differ significantly from t2 (48 hours) and t3 (72 hours) but differs significantly from t4 (96 hours). high-quality honey contains a maximum moisture content of 22% (bsn, 2018). in this study, the lowest moisture content was achieved at dehumidification time t4 (96 hours). the longer the dehumidification time, the lower the moisture content due to the increased evaporation of water from the honey during the dehumidification process, which is absorbed by the dehumidifier. this is consistent with the study by apriantini (2022), which found that 4 and 8 hours dehumidification times reduced the moisture content in kapok and rambutan honey. in figure 2, the moisture content of crassiacarpa and mangium honey after dehumidification shows a significant difference. the moisture content of crassiacarpa honey is higher than that of mangium honey. this is likely because the initial moisture content of crassiacarpa honey before dehumidification was higher (23%) than mangium honey (20%). the viscosity of honey before treatment determines the rate of moisture reduction. the more viscous the honey sample, the longer it takes to reduce its moisture content, whereas less viscous honey reduces its moisture content more easily. this explains why crassiacarpa honey has a higher moisture content than mangium honey. the results of the 5% lsd test show that the type of honey (p) significantly affects the moisture content of the honey, as shown in figure 2. moisture content is the first component measured to determine the quality of honey. low moisture content in honey can inhibit microbial activity and reduce the natural fermentation rate of the honey (yap et al., 2019). chayati (2008) stated that reducing the moisture content of honey increases the percentage of other nutrients contained in the honey. high-quality honey has a sufficiently low moisture content, or a maximum of 22%. according to bsn (2018), the maximum moisture content for forest honey is 22%, for stinging bee honey is 22%, and for stingless bee honey is 27.5%. the research results for crassiacarpa and mangium honey show that both types meet the standards set by sni 8664-2018 (bsn, 2018). the difference in the rate of moisture reduction between crassiacarpa (p1) and mangium (p2) honey can be influenced by several factors, including the initial water content of the honey, the viscosity of the honey, and the physicochemical properties of the two types of honey. total acidity the results show that the total acidity of crassiacarpa (p1) and mangium (p2) honey, measured at various dehumidification times (24, 48, 72, and 96 hours), ranges between 12.138 to 15.406 ml naoh/kg. specifically, the total acidity of crassiacarpa honey is between 12.138 and a b b bc bc 0,000 5,000 10,000 15,000 20,000 25,000 t0 t1 t2 t3 t4 w at er c o n te n t (% ) dehumidification (hour) a b 0,000 5,000 10,000 15,000 20,000 25,000 p1 p2 w at er c o n te n t (% ) type of honey 28 biology, medicine, & natural product chemistry 14 (1), 2025: 25-31 15.406 ml naoh/kg, while mangium honey ranges from 12.138 to 14.939 ml naoh/kg. variance analysis indicates that dehumidification time (t) significantly affects total acidity, but there is no significant interaction between dehumidification time and honey type (p). the results of the 5% lsd test confirm that dehumidification time significantly influences the total acidity of honey, as shown in figure 3. figure 3. total acidity of dehumidified honey at different duration. (t0: 0 hour, t1: 24 hour, t2: 48 hour, t3: 72 hour, t4: 96 hour) the results of the 5% lsd test also revealed that both types of honey (crassiacarpa and mangium) dehumidified at t0 (0 hours) and t1 (24 hours) show significantly lower total acidity compared to those dehumidified at t2 (48 hours), t3 (72 hours), and t4 (96 hours). however, no significant differences were observed between t2 (48 hours), t3 (72 hours), and t4 (96 hours). the lowest total acidity occurred at t0 (0 hours), which is associated with the higher moisture content in the honey at this time point, leading to a lower concentration of acids. as the dehumidification time increases, a more significant reduction in moisture content is observed (figure 1), increasing total acidity. despite the increase in total acidity, the dehumidification process, conducted at a low temperature of 30°c, does not damage the acids present in the honey. the acidity of honey is primarily due to the presence of organic acids, including gluconic acid, pyruvic acid, malic acid, and citric acid, along with inorganic ions such as phosphate, sulfate, and chloride (terrab et al., 2003). acidity plays a crucial role in determining honey quality, as it contributes to the stability of honey during storage, helps detect fermentation caused by osmophilic yeasts such as zygosaccharomyces, and affects the texture and taste of honey (terrab et al., 2003). organic acids naturally occur in honey from 0.17% to 1.17%, with an average concentration of 0.57% of the total honey composition. these acids can also range in concentration from 8.7 to 46.8 ml naoh/kg, with an average of 29.1 ml naoh/kg. the source of these acids can be attributed to the nectar from the flowers that bees feed on, although a significant portion is produced by the bees themselves through the action of the enzyme glucose oxidase. this enzyme catalyzes the oxidation of glucose in honey to produce gluconic acid (olaitan, 2007). according to the sni 8664-2018 standards (bsn, 2018), high-quality stinging bee honey should have a total acidity of no more than 50 ml naoh/kg, while stingless bee honey is allowed a higher limit of up to 200 ml naoh/kg. in this study, both crassiacarpa and mangium honey samples meet the quality standards for total acidity, with values significantly below the maximum allowable limit of 50 ml naoh/kg. ph the ph values obtained for crassiacarpa (p1) and mangium (p2) honey samples ranged from 3.39 to 3.67, indicating good honey quality. the analysis of variance (table 25) reveals that both the type of honey and the dehumidification time significantly affect the ph values. the results of the 5% lsd test demonstrate that the interaction between honey type (p) and dehumidification time (t) has a significant effect on the ph of honey during the moisture reduction process, as illustrated in figure 4. figure 4 shows that the ph of crassiacarpa honey at p1t0 (0 hours of dehumidification) differs significantly from p1t2 (48 hours) and p1t4 (96 hours), but no significant differences were observed between p1t0 and p1t1 (24 hours) or p1t3 (72 hours). for mangium honey, no significant differences in ph were found between the different dehumidification times, and the ph was consistently lower than that of crassiacarpa honey across all dehumidification periods (24, 48, 72, and 96 hours). the interaction between honey type and dehumidification time reveals that p1t0 (0 hours) does not differ significantly from any of the mangium honey treatments (p2t0, p2t1, p2t2, p2t3, p2t4), but p1t1 (24 hours) and p1t3 (72 hours) show significant differences when compared to mangium honey across all dehumidification times (p2t1, p2t2, p2t3, p2t4). in contrast, p1t2 (48 hours) and p1t4 (96 hours) differ significantly from all mangium honey treatments. figure 4. ph value of honey from various dehumidification duration (t) and type of honey (p). (t0: 0 hour, t1: 24 hour, t2: 48 hour, t3: 72 hour, t4: 96 hour; p1: crassiacarpa, p2: mangium) c b a a a 0,000 5,000 10,000 15,000 20,000 t0 t1 t2 t3 t4t o ta l a ci d it y ( m l n ao h /k g ) dehumidification (hour) cde bcd ab abc a cde e e e de 3,100 3,200 3,300 3,400 3,500 3,600 3,700 3,800 p1t0 p1t1 p1t2 p1t3 p1t4 p2t0 p2t1 p2t2 p2t3 p2t4 p h interaction fadhallah et al. – effect of prolonged dehumidification on honey 29 the higher ph values observed in crassiacarpa honey at p1t4 (96 hours) are likely due to the decrease in moisture content during dehumidification, which leads to the concentration of compounds that influence ph. this difference in ph values between crassiacarpa and mangium honey could also be attributed to differences in mineral and acid content, as suggested by gulfraz et al. (2010). the mineral content of honey is influenced by factors such as soil composition, geographical location, and the climate of the region where the nectar-producing plants grow (buba et al., 2013). importantly, the dehumidification process used in this study, which occurs at a low temperature of 30°c, does not degrade the organic acid content of the honey. the lower ph observed in mangium honey after longer dehumidification may be due to its lower moisture content, which results in higher total acidity (figure 8), possibly because of reduced enzymatic and microbial activity. according to saepudin et al. (2014), the ph of pure honey typically ranges from 3.2 to 4.5, with an average of 3.91, whereas fake or adulterated honey tends to have a ph between 2.4 and 3.3. if the ph falls outside this range, honey quality may be compromised, as the acidity helps to protect the honey from microbial contamination, which could otherwise lead to rapid spoilage. the low ph values found in honey are mainly due to the presence of organic acids such as syringic acid (3,5-dimethoxy-4hydroxybenzoic acid), methyl syringate (3,4,5trimethoxybenzoic acid), and 2-hydroxy-3phenylpropionic acid, as reported by puspita (2007). ratiu et al. (2020) further emphasize the relationship between honey ph and microbial activity, noting that lower moisture content and ph are associated with reduced microbial contamination. in their study, honey samples with ph values ranging from 3.20 ± 0.01 to 4.49 ± 0.01 showed no bacterial presence. these findings align with the ph results obtained in this study, where values ranged from 3.39 ± 0.41 to 3.67 ± 0.07, all within the acceptable range for maintaining honey freshness and aroma. additionally, adalina (2017) highlighted that organic acids play a significant role in determining honey's taste, aroma, and resistance to microbial growth. in conclusion, the ph values of crassiacarpa and mangium honey in this study fall within the acceptable range for high-quality honey, as outlined by sni 86642018 (bsn, 2018), which specifies a ph range of 3.2 to 4.5. this acidic environment helps to inhibit bacterial growth and prolong the shelf life of honey. although crassiacarpa honey showed some fluctuations in ph at different dehumidification times, the ph remained within the desirable range, ensuring that the honey maintained its quality throughout the dehumidification process. meanwhile, mangium honey exhibited no significant ph changes with increasing dehumidification time, confirming its stability and good quality. best treatment selection based on the analysis of moisture content, ph, and total acidity for both crassicarpa and mangium honey, summarized in tables 1 and 2, the optimal dehumidification times were identified. for crassicarpa honey, lower moisture content and higher ph values were observed at 48–96 hours of dehumidification, with no significant differences in total acidity between 24 and 96 hours. similarly, for mangium honey, the moisture content remained low between 24 and 96 hours, consistent ph values across 0–96 hours, and no significant variation in total acidity between 48 and 96 hours. compared with the indonesian national standard (sni) 8664:2018, all treatments for both honey types met the required parameters for moisture content, ph, and total acidity. however, to minimize the risk of fermentation associated with higher moisture levels, a dehumidification period of 96 hours was selected as the most suitable treatment for both honey types. table 1. determination of best treatment for crassiacarpa honey. p1 t0 (0 hour) t1 (24 hours) t2 (48 hours) t3 (72 hours) t4 (96 hours) standard water content 23a 21,5ab 20bcd 19,5bcde 18,5cde 22% ph 3,48cde* 3,53bcd* 3,62ab 3,57abc* 3,67a* 3,24,5 total acidity 12,13c* 13,53abc* 14,93ab* 15,4a* 14,93ab* max 50 table 2. determination of best treatment for mangium honey. p1 t0 (0 hour) t1 (24 hours) t2 (48 hours) t3 (72 hours) t4 (96 hours) standard water content 20,67bc 18,83cde 18,66cde 18,33de 17,33e* 22% ph 3,48cde* 3,4e* 3,39e* 3,40e* 3,43e* 3,24,5 total acidity 12,13c* 13,07bc* 14,93ab* 15,4a* 14,47ab* max 50 30 biology, medicine, & natural product chemistry 14 (1), 2025: 25-31 duo-trio test the duo-trio test in this study, involving 20 panellists from the department of agricultural product technology, asked panellists to differentiate between honey dehumidified for 96 hours and untreated honey (r) based on aroma, taste, viscosity, and color. according to the binomial distribution table, significant differences were observed in all parameters—aroma, taste, viscosity, and color—at the 5% significance level (tables 3 and 4). the results indicate that both crassicarpa and mangium honey dehumidified for 96 hours exhibited distinct sensory characteristics compared to untreated honey in terms of texture, taste, aroma, and color. this suggests that honey dehumidified for 96 hours can be easily distinguished from untreated honey by general consumers. table 3. duo trio test results on the crassiacarpa honey at best treatment (dehumidification 96 hours). parameter number of panelists noted the difference number of panelist required (binomial table 0,05 %, with 20 panelists) remarks texture 17 15 r* color 15 15 r* aroma 16 15 r* taste 15 15 r* note: r* = different with control sample table 4. duo trio test results on the mangium honey at best treatment (dehumidification 96 hours). parameter number of panelists noted the difference number of panelist required (binomial table 0,05 %, with 20 panelists) remarks texture 15 15 r* color 17 15 r* aroma 16 15 r* taste 15 15 r* note: r* = different with control sample conclusion dehumidification time significantly affected the honey’s moisture content, ph, and acidity. additionally, the type of honey had a notable influence on the moisture content and ph. the interaction between dehumidification time and honey type significantly impacted the ph values, with a dehumidification time of 96 hours being the optimal treatment for both honey types. this resulted in honey that met the standards set by sni 8664:2018. authors’ contributions: depri mubarik, otik nawansih, esa ghanim fadhallah, and fibra nurainy designed the study and wrote the manuscript in bahasa. depri mubarik conducted the research. otik nawansih, esa ghanim fadhallah, and fibra nurainy supervised the research. esa ghanim fadhallah translates to english and proofreads the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references adalina, y. (2017). kualitas madu putih asal provinsi nusa tenggara barat. prosiding seminar nasional masyarakat biodiversitas indonesia 3(2):189-193. apriantini, y., endrawati, c., & astarini, z. (2022). pengaruh lama waktu penurunan kadar air terhadap kualitas fisikokimia madu kapuk dan madu rambutan. jurnal ilmu produksi dan teknologi hasil peternakan 10(2):98-104. balos, m. z., popov, n., vidakovic, s., pelic, d. l., pelic, m., mihaljev, z., & jaksic, s. (2018). electrical conductivity and acidity of honey. arhiv veterinarske medicine 11(1):91-101. [bps] badan pusat statistik. 2021. statistik produksi kehutanan. badan pusat statistik, jakarta. [bsn] badan standardisasi nasional. (1994). sni 013545:1994 madu. badan standardisasi nasional, jakarta. [bsn] badan standardisasi nasional. (2018). sni 8664:2018. madu. badan standardisasi nasional, jakarta. buba, f., gidado, a., & shugaba, a. (2013). analysis of biochemical composition of honey sample from north-east nigeria. journal of biochemistry and analytical biochemistry 2(3):1-7. chakir, a., romane, a., marcazzan, g. l., & ferrazzi, p. (2016). physicochemical properties of some honeys produced from different plants in morocco. arabian journal of chemistry 9: s946-s954. chayati, i. (2008). sifat fisikokimia madu monofora dari daerah istimewa yogyakarta dan jawa tengah. agritech journal 28(1):9-14. gulfraz, m., ifftikhar, f., asif, s., raja, g. k., asad, m. j., imran, m., abbasi, k., & zeenat, a. (2010). quality assessment and antimicrobial activity of various honey types of pakistan. african journal of biotechnology 9(41): 6902-6906. [iso] international organization of standardization. (2017). iso 10399:2017 – methodology — duo-trio test. iso, geneva. minarti, s., jaya, f., & merlina, p.a. (2016). pengaruh masa panen madu lebah pada area tanaman kaliandra (calliandra calothyrsus) terhadap jumlah produksi kadar air, viskositas dan kadar gula madu. jurnal ilmu dan teknologi hasil ternak 11(1):46-51. olaitan, p. b., adeleke, o. e., & ola, i. o. (2007). honey: a reservoir for microorganisms and an inhibitory agent for microbes. african health sciences 7(3):159-165. pribadi, a., & wiratmoko, e. m. (2018). karakteristik madu lebah hutan (apis dorsata fabr.) dari berbagai bioregion di riau. jurnal penelitian hasil hutan 37(3):185-200. puspita, i. (2007). rahasia sehat madu. bentang pustaka, yogyakarta. saepudin, r., sutriyono, s., & saputra, r.o. (2014). kualitas madu yang beredar di kota bengkulu berdasarkan penilaian konsumen dan uji secara empirik. jurnal sains peternakan indonesia 9(1):30-40. fadhallah et al. – effect of prolonged dehumidification on honey 31 sarwono, b. (2007). lebah madu. agromedia pustaka press, jakarta. savitri, n. p. t., endah, d. h., & sri, w. a. s. (2017). kualitas madu lokal dari beberapa wilayah di kabupaten temanggung. buletin anatomi dan fisiologi 2(1):58-66. terrab, a., maria j. d., & francisco, j. h. (2003). palynological, physico-chemical and colour characterization of moroccan honeys: i. river red gum (eucalyptus camaldulensis dehnh) honey. international journal of food science and technology 38:379-386. yap, s. k., chin, n. l., yusof, y. a., & chong, k. y. (2019). quality characteristics of dehydrated raw kelulut honey. international journal of food properties 22: 556-571. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 105-107 | doi: 10.14421/biomedich.2020.92.105-107 issn 2540-9328 (online) a simple technique for rapid assessment of rat (rattus norvegicus) sperm motility muhammad ja’far luthfi1,*, mahanem mat noor2 1department of biological education, faculty of tarbiyah and education, universitas islam negeri sunan kalijaga yogryakarta, indonesia 2school of bioscience and biotechnology, faculty of science and technology, universiti kebangsaan malaysia, malaysia corresponding author* jafarluthfi@yahoo.com manuscript received: 20 september, 2020. revision accepted: 19 february, 2021. published: 25 february, 2021. abstract assesment of rat sperm motility can be carried out on sperm sample from epididimal source using minimal amount of equipment. this method will aid researcher and practitioner working in the field of sperm quality to determined rat sperm motility rapidly and efficiently. keywords: sperm quality; sperm motility; rat; haemocytometer. introduction sperm motility is one of the most important parameters determining sperm quality (martinez et al., 2000; talarczyk-desole et al., 2017). the results of sperm motility assessment can be used more directly to address problems affecting male reproductive organs (björndahl, 2010). the other parameters are sperm count and sperm morphology (freund & carrol 1964). these three components (sperm motility, sperm count, and sperm morphology) compose of sperm quality (perreault & cancel 2001). the most frequently used techniques to assess sperm qualiy are counting chambers (e.g. haemocytometer and makler chamber). these techniques, however, are subject to a high variability (yang et al., 2019). much effort has been made to speed up the objective measurement of sperm motility. casa (computer aided sperm analysis) and ivos (integrated visual optical system) are automatic methods of sperm quality testing. the techniques, however, need sophisticated equipment not available in many laboratories. they are too technically complex and too expensive for routine use. manual observation is still the most widely used method for testing sperm motility (das 1985; kuster 2005; macpherson, 2001). the aim of this study was to determine the optimal manual technique for assessment of rat sperm motility using arbitrary scale. the technique was based on years of practices in our laboratories. it provides a practical and simple means for assessment of rat sperm motility for the purpose of determining the fertility status the animal. materials and methods equipments and materials the equipment needed in the analysis of rat sperm motility were as follows: a light microscope, micropipette, petri dishes, improved neubauer haemocytometer, co2 incubator, small animal surgical instruments, and hand counter. the materials needed in the analysis of rat sperm motility were as follows: three adult male spraguedawley rats (rattus norvegicus), biggers, whitten, & whittingham (bww) medium, and cover glasses. the study was done in the zoology laboratory of university kebangsaan malaysia and the zoology laboratory of uin sunan kalijaga yogyakarta. preparation of sperm suspension sperm samples were taken from the left cauda epididymis. the cauda epididymis was separated based on the epididymal division by hamilton (1975), then placed in a petri dish, minced and incubated in 15 ml of biggers, whitten & whittingham (bww) media solution (biggers et al. 1971) for 30 minutes in 37°c in an incubator of 5% co2 to let sperm swim in the media (swim-up technique) (luthfi, 2015; luthfi & noor, 2015). procedure for assessing sperm motility sperm motility were determined using improve neubauer haemocytometer as described previously (prasad et al., 1972; who, 1999) with modification. the cover glass was attached firmly to the improved neubauer haemocytometer counting grid area. the interference pattern (> 10 newton's rings/fringes) should be seen between the glass surfaces of the two areas https://doi.org/10.14421/biomedich.2020.92.105-107 https://www.ncbi.nlm.nih.gov/pubmed/?term=bj%26%23x000f6%3brndahl%20l%5bauthor%5d&cauthor=true&cauthor_uid=20111079 106 biology, medicine, & natural product chemistry 9 (2), 2020: 105-107 where the glass cover is attached to the haemocytometer. the line/newton's rings that appear too few indicate that the distance between the cover glass and the haemocytometer is widened, therefore the counting chamber volume becomes larger and the assessment would be incorrect. the center of the haemocytometer counting grid area is different from the other ones because it consists of 25 larger squares bounded by triple lines each, while the ones in the corners consist of 16 larger squares. in addition, the larger squares inside the central counting grid are subdivided into 16 even smaller squares each (figure 1). a total of 10 μl of sperm suspension from the preparation of sperm samples were taken with a micropipette, then inserted into the space between the cover glass and the haemocytometer in one of its counting chamber. the other counting chamber was also filled in the same way. let the sperm settled in the chambers for 5 minutes. each counting chamber must be filled correctly. the suspension was inserted slowly to let the liquid evenly distributed by capillary force. if the filling on to the counting chamber was uneven, wipe the slide and then the process of filling the chamber must be repeat. the removal of excessive volume from the counting chamber must not be done because it will change the density of sperm in the counting chamber. figure 1. haemocytometer. a. side view. b. top view. c. one of the two haemocytometer counting chambers. d. insert of iv. legend: i. cover glass; ii. counting chamber; iii. counting grid area; iv. one of the 25 larger squares. after both counting chambers were filled, assessment of sperm motility was carried out. the sperm motility was observed at 200x or 400x magnification using a light microscope. systematically scan the center of the haemocytometer counting grid area, started with the uppermost left square of the 25 large squares, continued to the next three squares to the right, and the squares at the row below, and so on. assessment were done only for intact sperm (defined as having a head and a tail). approximately 100 sperm in each counting chamber were assigned to minimize deviation. sperm motility is estimated to follow the criteria as defined by who (1999; 2010) and is denoted by the letters a, b, c (from the fastest to the slowest) as follows: a: progressive motility; ≥ 25 μm/s where 25 μm is the same as the length of 2 squares (smallest square) of the haemocytometer (see red rectangle in figure 1d) b: non-progressive motility; <5 μm/s c: no motility result and discussion determination of sperm motility is an important step in the analysis of sperm quality. however, there are various methods of assessing sperm motility in terms of equipment, technicality and practicality. different laboratories use different methods. this study was use haemocytometer to determine sperm motility. the determination of the method is mostly based on the practice and experience of testing rat sperm samples carried out at the zoology laboratory of the faculty of science and technology, uin sunan kalijaga and zoology laboratory of university kebangsaan malaysia (data not shown). the sperm motility of the laboratory animals can be determined from ejaculate or epididymal samples. the determination of epididymal sperm motility is using only sperm from the caudal part (clegg et al., 2001). changes in the progression/rate of sperm motility after substance or drug treatment can provide important clues as to the effect of a substance on sperm fertilizing capacity (working, 1988). motility pattern is one of the most important and simplest variables in determining the fertility potential of sperm because immotile sperm will not be able to penetrate the mucus layer of the cervix to reach the ovum and fertilization to occur. sperm motility patterns is an important factor in fertilization, as a strong swing of the sperm tail is necessary for the sperm head to penetrate the lining of the ovum (partodihardjo, 1992). progressive motility is a normal sperm movement. circular motion and reversion movements are often signs of cold shock or due to abnormal sperm morphology. meanwhile, wavy motion and vibrating motion occur more frequently in old men (atherton, 1977). luthfi & noor – a simple technique for rapid assessment of … 107 sperm motility is observed with either physiological saline or other nutrient medium. sperm were observed under a microscope slide with/without a cover glass or with a capillary tube. casa (computer aided sperm analysis) and ivos (integrated visual optical system) are automatic objective methods for testing sperm motility. however, manual observation is still the most widely used method for testing sperm motility using an arbitrary scale (das 1985; kuster 2005; strader 1996). this study revealed that using this method, 25-45 percent of rat sperm would fall into category of progresssive motility, 45-65 percent were of nonprogressive motility category, and 15-35 were of no motility category. aproximately, those numbers could be used as a reference for normal sperm motility of rat sperm. several things need to be considered in order to achieve consistency in using this method. first, we must ensure that the cutting of cauda epididymis was done consistently in every replication. second, micropipette insertion of samples in each counting chamber must be perfect. third, the room temperature used in determining the sperm motility must be the same for all sperm sample assessed. fourth, the time needed to assess the sperm motility should consistent from slide to slide. all these things affect the consistency of the sperm motility assesment. the procedures that are followed strictly will ensure the accuracy of the assessment. conclusion using this method, researchers can determine the rat sperm motility in a practical way. most of the materials and equipments used in this method are widely available. the increasing importance of sperm motility analysis will be consistent with the adoption and development of this simple and rapid method. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references atherton, r.w. 1977. evaluation of sperm motility in hafez, e.s.e. (ed). techniques of human andrology. 175-179. elsevier. amsterdam. biggers, j.d., whitten, w.k. &whittingham, d. 1971. the culture of mouse embryos in vitro in daniel, j.c. (ed). methods in mammalian embryology. 86-116. freeman, san francisco, ca. björndahl, l. 2010. the usefulness and significance of assessing rapidly progressive spermatozoa. asian j androl. 2010 12(1): 33–35. clegg, e.d., perreault, s.d. & klinefelter, g.r. 2001. assesment of male reproductive toxicity in hayes, a.w. (ed). principles and methods of toxicology. 1264-1292. fourth editions. taylor & francis. philadelphia. das, r. p. 1985. assessment of spermatozoal function. j. biosci. 7 (2): 245–255. freund, m. & carol, b. 1964. factors affecting haemocytometer counts of sperm concentration in human semen. journal of reproduction and fertility 8: 149-155. hamilton, d.w. 1975. structure, function of the epithelium lining the ductuli efferents, ductus epididymis and ductus deferens in the rat in hamilton, d.w. & greep, r.o. (eds). handbook of physiology, section vii, endocrinology, vol.5, male reproductive system. 259-301. american physiological society, washington d.c. kuster, c. 2005. sperm concentration determination between hemacytometric and casa systems: why they can be different. theriogenology 64: 614–617. luthfi, m.j. 2015. a simple and practical method for rat sperm epididymal sperm count (rattus norvegicus). biology, medicine & natural product chemistry 4 (1): 1-3. luthfi, m.j., noor, m.m. 2015. analisis kualitas sperma tikus percobaan (jumlah, motilitas, dan morfologi). uns press. surakarta. indonesia. macpherson, m.l. 2001. how to evaluate semen in the field. proceedings of the annual convention of the aaep. volume 47. usa. martínez, c., mar, c., azcárate, m., pascual, p., aritzeta, j.m., lópez-urrutia, a. 2000. sperm motility index: a quick screening parameter from sperm quality analyser-iib to rule out oligoand asthenozoospermia in male fertility study. human reproduction 15 (8): 1727–1733. partodihardjo, s. 1992. ilmu reproduksi hewan. cetakan ke-3. 2537. penerbit mutiara sumber widya. jakarta. perreault, s.d. & cancel, a. m. 2001. significance of incorporating measures of sperm production and function into rat toxicology studies. reproduction 121: 207–216. prasad, m.r.n., chinoy, n. j., kadam. k.m. 1972. changes in succinic dehydrogenase levels in the rat epididymis under normal and altered physiologic conditions. fertility and sterility 23 (3): 186-190. strader, l.f., linder, r. e. & perreault. s.d. 1996. comparison of rat epididymal sperm counts by ivos htm-ident and hemocytometer. reproductive toxicology 10 (6): 529-533. talarczyk-desole, j., anna berger, a., taszarek-hauke1, g., jan hauke, j., pawelczyk, l., jedrzejczak, p. 2017. manual vs. computer-assisted sperm analysis: can casa replace manual assessment of human semen in clinical practice? ginekologia polska 88 (2): 56–60. white, w.j. 2001. the use of laboratory animals in toxicologic research in hayes a.w. (ed). principles and methods of toxicology. fourth edition. 773-775. taylor & francis. philadelphia. who. 1999. laboratory manual for the examination of human semen and semen-cervical mucus interaction. new york: cambridge university press. who. 2010. who laboratory manual for theexamination and processing of human semen. fifth edition. who press. geneva, switzerland. working, p.k. 1988. male reproductive toxicology: comparison of the human to animal models. environmental health perspectives 77: 37-44. yang, y., zhang, y., ding, j. 2019. optimal analysis conditions for sperm motility parameters with a casa system in a passerine bird, passer montanus. avian res 10 (35) https://doi.org/10.1186/s40657-019-0174-5 https://www.ncbi.nlm.nih.gov/pubmed/?term=bj%26%23x000f6%3brndahl%20l%5bauthor%5d&cauthor=true&cauthor_uid=20111079 javascript:; javascript:; javascript:; javascript:; javascript:; javascript:; http://www.sciencedirect.com/science/journal/08906238 http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%235156%231996%23999899993%2378585%23flp%23&_cdi=5156&_pubtype=j&view=c&_auth=y&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=abaf66d755593badcfa6b6be4a9497c9 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 559-562 | doi: 10.14421/biomedich.2023.122.559-562 issn 2540-9328 (online) the potential of a sumbawa herbal oil-based oleogel as burn wound dressing medical faculty, muslim university of indonesia, makassar, indonesia jl. urip sumoharjo km5 makassar 90231, tel. +62-411-455696, fax. +62-411-455696, indonesia. manuscript received: 20 july, 2023. revision accepted: 09 september, 2023. published: 25 september, 2023. abstract this study aimed to innovate the current sumbawa oil preparation which is still in the form of liquid into an oleogel so that it is easier to apply and more practical for distribution and storage. phytochemical analysis of sumbawa oil showed the contents of phenols, flavonoids, and alkaloids. by adding hydroxyethyl cellulose (hec) as a gelling agent and glycerin as a humectant, sumbawa oil can be served in the form of an oleogel. the ph measurement of the sumbawa oil oleogel at levels of 90%, 95%, and 100% ranged from 7.0–7.3 which is the ideal acidity level for the wound healing process. in the antimicrobial activity test with s. aureus and e. coli, the zone of inhibition ranged from 12.1–14.7 mm which is considered strong. meanwhile, in in vitro testing of the anti-inflammatory activity using the human red blood cell membrane stabilization method, the stability level of each was obtained to be 78% for 100% sumbawa oil, 74% for oleogel with 95% sumbawa oil, and 73% for oleogel with 90% sumbawa oil. the oleogel morphology observation using a scanning electron microscope showed a good gelation process at 5% and 10% hec levels. keywords: sumbawa herbal oil; oleogel; burn; wound dressing. introduction burns are skin injuries that can cause tissue dysfunction. thermal, chemical, and electrical burns are the most common types of burns. globally, more than 300,000 deaths are caused by thermal burns or other types of burns each year. the high mortality rate is due to the challenging burn management. the most common complications include loss of critical skin tissue functions, dehydration, wound infection, other systemic inflammatory responses, sepsis, and multiorgan dysfunction. when the skin tissue is injured, some basic organ functions are affected or even lost completely depending on the severity of the burn (he et al. 2021). characteristics of disturbed skin include cellular dysfunction, hemodynamic and hematological abnormalities, acid–base disturbances, and hormonal changes. acid–base disturbances can cause metabolic acidosis in the early period of the burn. hormonal changes will accumulate at the wound site through the progressive release of various substances, including histamine, kinins, oxygen radicals, and arachidonic acid, or by disrupting the neurohormonal axis (he et al. 2021). in recent decades several commercial polymer-based gels such as gelrin c, mebiol gel, hystem hydrogel, and biogelx products, to name a few, have been developed for biomedical applications. various cross-linked and conductive hydrogels for effective wound healing along with antibacterial activity have also been reported. development of low-molecular-weight gels for effective tissue regeneration and wound closure applications can overcome problems with the limitations of polymer gels such as thermoreversibility, processability, biocompatibility, critical gelation concentration, sol–gel transition temperature (tgel), and high molecular weight (he et al. 2021). although studies on hydrogels as wound dressings have been carried out extensively, they were unable to resolve some of the limitations. the characteristics of hydrogels containing water with large pores cause the active ingredients of the drug to be spilled into the body within a few hours, contrary to its intended function as a slow-release mechanism. hydrogels tend to be too rigid to be injected through a needle and may require surgical implantation. hydrogels can also be detached from the site of treatment. hydrogels are not strong enough to protect the proteins and peptides that make up newer drugs and cannot contain the hydrophobic active ingredients of drugs (he et al. 2021). several scientists have proposed alternative hydrophobic organic materials to overcome the andi irma yuniar, nurul wahidah yasid, syarifah fatimah nissatuljannah, syamsu rijal, armanto makmun* armanto.makmun@umi.ac.id corresponding author* https://doi.org/10.14421/biomedich.2023.122.559-562 560 biology, medicine, & natural product chemistry 12 (2), 2023: 559-562 limitations of hydrogels. organogels form in situ drug depots when a mixture of polymers, drug-active ingredients, and organic solvents is injected into the body. gelation occurs when organic solvents are released from the implant. although slow diffusion of the drug is more effective with organogels, the obvious drawback is that organic solvents are also released into the bloodstream (guennard, 2023). sumbawa herbal oil is one of the traditional indonesian medicines which is produced on sumbawa island by mixing coconut oil and several medicinal plants through a heating process. sumbawa people use sumbawa oil as an antispasmodic, antipyretic, and appetite-enhancing drug. in addition, it is also known to cure diseases such as diarrhea, snake bites, wounds, bruises, twists, peeling skin, toothache, stomach pain, digestive disorders, backaches, and skin diseases, reduce symptoms of arthritis, and accelerate the recovery of the mother's condition after childbirth. this is confirmed by the research study of hadi et al. (2018) who conducted a gc-ms analysis and found that sumbawa oil contains a number of bioactive ingredients such as hexadecanoic acid, octadecanoic acid, methyl palmitate, linoleic acid, lycopersen, and dodecanoic acid which have antispasmodic, antipyretic, appetite-enhancing, antifungal, and antimicrobial properties. some of its possible uses are as a hypocholesterolemic, antiinflammatory, and antidiabetic agent. research by halawiya et al. (2017) showed that administration of sumbawa oil on experimental rats with hypercholesterolemia showed a significant reduction in the total cholesterol levels (figure 1). figure 1. sumbawa herbal oil. based on research by permatasari (permatasari, 2013), the types of plants that are the basic ingredients for making sumbawa oil found in sumbawa besar regency are 59 plant species from 28 families. of the 59 plant species, there are several types of plants with the same family, including the arecaceae family, namely sugar palm, gebang, coconut, and areca nut, the euphorbiaceae family, namely buffalo patikan, meniran, castor, and katuk/katu, the zingiberaceae family, namely turmeric, cardamom, galangal, and ginger, and the piperaceae family, namely betel, pepper, and chili herbs (halawiya et al. 2017). currently, sumbawa herbal oil is only available in the form of liquid oil packaged in glass bottles. the application in this dosage form is impractical for both wound application and practicality and safety in packaging (brooks et al. 2016). this study aims to investigate the potential of making an oleogel made from sumbawa herbal oil and the extent to which its effectiveness in overcoming bacterial infections and antiinflammatory activities serves as an indicator of an effective burn dressing material (halawiya et al. 2017). materials and methods experimental design the manufacture of the oleogel uses the basic composition of sumbawa herbal oil, hec, and glycerin with the following variations in table 1: table 1. oleogel ingredient composition. sumbawa oil (ml) hec (ml) glycerin (ml) mix a 95 4 1 mix b 90 9 1 observations and test some of the parameters observed and measured in this study are: ▪ ph measurement measurement of the ph of the oleogel using a ph meter. ▪ investigation of the phytochemical content the phytochemical content was measured qualitatively including the contents of flavonoids, alkaloids, saponins, and terpenoids. ▪ observation of the morphology of the oleogel the oleogel morphology was observed using a scanning electron microscope (sem) to see the characteristics of the gel mixture formed. ▪ observation of the antimicrobial activity to test the antimicrobial activity using the disk diffusion method (kirby-bauer test) using s. aureus bacteria. ▪ observation of the anti-inflammatory activity to test the anti-inflammatory activity in vitro using the human red blood cell membrane stabilization method with a modification of the method of shinde et al. (1999). a positive control using diclofenac. yuniar et al. – sumbawa herbal oil-based oleogel 561 results oleogel ph we have measured the ph of three groups of samples namely 100% sumbawa oil (so), mix a, and mix b. as shown in table 2, the mean ph of so is 7.3, whereas, the ph of mix a is 7.1 and mix b is 7. based on the measurement of samples, it was found that the ph range of the oleogel is considered neutral. the results of the ph test in the table above show that sumbawa oil meets good ph standards according to sni 4085:2017, namely ph 4.0–10.0. table 2. ph of the sumbawa herbal oil oleogel. sample ph mean sample 1 sample 2 sample 3 so 100% 7 7.5 7.3 7.3±0.2517 mix a 7.2 7.1 7.1 7.1±0.0577 mix b 6.8 7.0 7.1 7.0±0.1528 phytochemical analysis results furthermore, phytochemical analysis was performed to measure the compounds contained in so. this study showed that so contained flavonoids, alkaloids, saponins, and terpenoids. the results of the sumbawa herbal oil qualitative phytochemical analysis are provided in table 3 and figure 2. compounds containing many antioxidants are found in alkaloids, steroids, flavonoids, and saponins (ahmed et al. 2014). according to the results of phytochemical testing, bioactive components of flavonoids are present in all solvents. these results indicate that sumbawa oil has antioxidant properties. table 3. phytochemical components of sumbawa herbal oil. components presence precipitate flavonoid + dark red alkaloid 1 (wagner) + brown alkaloid 2 (mayer) + white alkaloid 3 (dragendorff) + orange saponin + foamy terpenoid + orange figure 2. phytochemical analysis of sumbawa herbal oil. oleogel morphology there are voids formed both in mix a and mix b. at the same time, it showed that the materials which are sumbawa herbal oil, hec, and glycerin were well mixed. the sem images of mix a and mix b sumbawa herbal oil oleogels are shown in figure 3 and 4. figure 3. mix a morphology. figure 4. mix b morphology. antimicrobial activity the antimicrobial activities of so, mix a and mix b are shown by the large inhibition zone against s. aureus bacteria. the wider the zone of inhibition the better the antimicrobial activity. table 4. shows that so has an inhibition zone against s. aureus by 14.7 mm, while those of mix a and mix b were 12.6 and 12.1 mm respectively. the criteria for the strength of antibacterial activity are categorized based on the diameter of the inhibition zone formed, namely a diameter of the inhibition zone of 5 mm or less is categorized as weak, an inhibition zone of 5–10 mm is categorized as medium, an inhibition zone 562 biology, medicine, & natural product chemistry 12 (2), 2023: 559-562 of 10–20 mm is categorized as strong and an inhibition zone of 20 mm or more is categorized as very strong (dimpudus et al. 2017). table 4. inhibition zone of s. aureus. sample inhibition zone mean sample 1 sample 2 sample 3 so 100% 15.3 14.1 14.8 14.7±0.6028 mix a 13.1 12.5 12.2 12.6±0.4583 mix b 12.4 12.2 11.1 12.1±0.4163 anti-inflammatory activity table 5 shows the results of an in vitro anti-inflammatory assay using human red blood cell membrane stabilization. the data in table 5 show that in liquid form, sumbawa herbal oil's capacity in the prevention of lysis was 78.577% while that of mix a, oleogel which contains 95% of sumbawa herbal oil, is 74.165% and that of mix b, oleogel which contains 95% of sumbawa herbal oil, is 73.629%. table 5. prevention of lysis in percentage. sample absorbance prevention of lysis (%) so 100% 0.280 78.577 mix a 0.338 74.165 mix b 0.345 73.629 diclofenac 0.351 73.145 negative control 1.307 0.000 conclusions based on the results of the measurement of the ph of the oleogel, a range of 7.0–7.3 was obtained. this acidity level is an ideal condition that helps the wound healing process, where the best ph range is 7.15–8.9. the natural ph of the skin is acidic and the range is 4–5.5. maintenance of this ph is very important to maintain the balance of the skin flora and to avoid the proliferation of pathogens. thus, the sumbawa oil-based oleogel has the potential to help form an environment that supports accelerated wound healing. the antimicrobial activity also shows good results where the inhibition zone ranges from 12 to 14 mm which is considered strong. antimicrobial properties have an important role in promoting wound healing. common wound healing inhibitor can be caused by the presence of s. aureus. based on the results of the study, the oleogel made from sumbawa oil has the potential to be used as a burn treatment material because it has a good ph and good antimicrobial and anti-inflammatory properties. further study including but not limited to scratch assay for wound healing, in-vivo experiment, and oleogel stability over time is needed. acknowledgements: the authors thank the medical faculty of muslim university of indonesia-makassar for supporting this research. authors’ contributions: armanto makmun, nasruddin andi mappaware & syamsu rijal designed the study. armanto makmun & nasruddin andi mappaware carried out the laboratory work. edward pandu wiriansya, zulfahmidah & andi irma yuniar analyzed the data. armanto makmun & andi irma yuniar wrote the manuscript. nurul wahidah yasid & syarifah fatimah nissatuljannah revised the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research received no external funding. references ahmed, i.a., mikail, m.a., ibrahim, m., hazali, n., rasad, m.s.b.a., abdul, g.r., et al. (2014). in vitro antioxidant properties of underutilized baccaurea angulata fruit. ijaaee, 1, 144-150 brooks, g.f., janet, s.b., & stephen, a.m. (2016). mikrobiologi kedokteran 25th ed. jakarta: egc, pp. 168-194. dimpudus, s.a., yamlean, p.v.y., & yudistira a. (2017). formulasi sediaan sabun cair antiseptik ekstrak etanol bunga pancar air (impatiens balsamina l.) dan uji efektivitasnya terhadap bakteri staphylococcus aureus secara in vitro. pharmacon, 6(3). https://doi.org/10.35799/pha.6.2017.16885 guennard, r. (2019). oleogels for drug delivery. american oil chemists society. https://www.aocs.org/stay-informed/informmagazine/featured-articles/oleogels-for-drug-delivery-march2019?sso=true. accessed on april 2, 2023. hadi, s., yunita, y., sutrisna, z., agustina, m., arlina, f.b., satriani, r, et al. (2018). investigation of production process and gc-ms analysis of chemical constituents of three traditional medicines sumbawa oils. the journal of pure and applied chemistry research, 7(2), 209-216. halawiya, a., tatontos, e.y., & agrijanti. (2017). efektivitas sediaan minyak sumbawa terhadap kadarkolesterol total pada tikus putih (rattusnorvegicus) strain wistar yang diberi diet hiperkolesterolemia. jurnal analis medika bio sains, 4(2), 87-92. he, j.j., mccarthy, c., & camci-unal, g. (2021). development of hydrogel-based sprayable wound dressings for secondand third-degree burns. advanced nanobiomed research, 1(6), 2100004. https://doi.org/10.1002/anbr.202100004 permatasari, i. (2013). etnobotani tumbuhan bahan dasar minyak sumbawa di kabupaten sumbawa besar provinsi nusa tenggara barat (ntb). malang: state islamic university of maulana malik ibrahim. shinde, u.a., phadke, a.s., nair, a.m., mungantiwar, a.a., dikshit, v.j, & saraf, v.o. (1999). membrane stabilizing activity—a possible mechanism of action for the antiinflammatory activity of cedrus deodara wood oil. fitoterapia, 70, 251–7. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 795-798 | doi: 10.14421/biomedich.2025.142.795-798 issn 2540-9328 (online) isolation and identification of dermatophytes from toenails and interdigital spaces of students using czapek yeast extract agar lisa savitri1*, kharisul ihsan2, rochmad krissanjaya1, elfred rinaldo kasimo1, novirma yanti1, mochamad hanif hilmi1 1department of medical laboratory technology, faculty of health sciences, kadiri university, kediri, east java, indonesia. 2department of pharmacy, faculty of pharmacy, public health, hospital administration, radiology, universitas strada indonesia, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 29 august, 2025. revision accepted: 03 october, 2025. published: 10 october, 2025. abstract dermatophytosis remains a common superficial fungal infection affecting nails and interdigital spaces, with trichophyton rubrum and t. mentagrophytes as the predominant etiologic agents. the recent emergence of terbinafine-resistant t. indotineae has raised new challenges in diagnosis and treatment. although sabouraud dextrose agar is the conventional medium for fungal culture, alternative media such as czapek yeast extract agar (cya) may provide additional advantages for dermatophyte isolation and morphological identification. this study aimed to isolate and identify dermatophytes from toenails and interdigital spaces of university students using cya medium and to evaluate its applicability in dermatophyte culture. a cross-sectional descriptive study was conducted among 100 university students aged 18–24 years. specimens were collected from toenails (n = 60) and interdigital spaces (n = 40). direct microscopy was performed using 20% koh preparation. samples were cultured on cya supplemented with chloramphenicol and cycloheximide, and incubated at 28 ± 2 °c for up to 21 days. fungal isolates were identified based on macroscopic and microscopic morphology. data were analyzed descriptively, and associations were tested using the chi-square test. fungal elements were detected in 65% of samples by koh examination, and dermatophyte growth was confirmed in 52% of cultures. the most frequently isolated species were t. rubrum (28%) and t. mentagrophytes (18%). notably, t. indotineae was detected in 5% of samples. no significant difference was found between toenail and interdigital isolates (p = 0.26). dermatophytes are prevalent among university students, with t. rubrum as the dominant species. the detection of t. indotineae highlights its emerging role in young populations. cya proved effective for dermatophyte isolation and may serve as an alternative culture medium in academic and diagnostic laboratories. keywords: dermatophytes; trichophyton rubrum; trichophyton indotineae; toenail; czapek yeast extract agar. abbreviations: czapek yeast extract agar (cya); potassium hydroxide (koh); lactophenol cotton blue (lpcb); sabouraud dextrose agar (sda); statistical package for the social sciences (spss). introduction dermatophytes are a group of keratinophilic fungi belonging to the genera trichophyton, microsporum, and epidermophyton. they invade keratinized tissues such as skin, hair, and nails, leading to infections collectively known as dermatophytosis (gupta, venkataraman, & hall, 2023). among these, toenail onychomycosis and tinea pedis in the interdigital spaces are the most common clinical forms, frequently associated with occlusive footwear, humidity, and communal living environments (lee, park, & kim, 2023). globally, onychomycosis remains a significant public health concern because of its chronic course, recurrence, and therapeutic challenges. the condition is most often caused by trichophyton rubrum and t. mentagrophytes, with prevalence rates ranging between 10–30% depending on the population studied (gupta, versteeg, & shear, 2022). recent years have also seen the emergence of trichophyton indotineae, a newly characterized species responsible for difficult-to-treat dermatophytosis. this organism shows a high rate of terbinafine resistance due to mutations in the squalene epoxidase gene, and has now been reported in asia, europe, and north america (chowdhary et al., 2022; mctaggart et al., 2025; marbaniang et al., 2025). reports from the united states and canada further highlight the increasing spread of terbinafine-resistant dermatophytes, raising concerns about limited therapeutic options (caplan et al., 2023; fuller, hay, & arenas, 2024). diagnosis of dermatophytosis is traditionally based on direct microscopy and fungal culture. while fungal culture remains the gold standard, it is limited by low sensitivity and extended incubation periods, which can https://doi.org/10.14421/biomedich.2025.142.795-798 mailto:lisasavitri@unik-kediri.ac.id 796 biology, medicine, & natural product chemistry 14 (2), 2025: 795-798 delay clinical decision-making (zhou, li, & wang, 2022). conventional culture media such as sabouraud dextrose agar (sda) are commonly used, but alternative formulations may improve isolation rates and morphological characterization. czapek yeast extract agar (cya), widely applied for aspergillus and penicillium, contains sucrose and yeast extract that can also support dermatophyte growth and provide distinctive colony morphology (ahmadi et al., 2012; tm-media, n.d.). recent reports suggest its potential value for research and diagnostic purposes when studying dermatophytes (kumar, patel, & singh, 2025). despite extensive studies on dermatophytes globally, there is limited data on their prevalence in young adults, particularly among university students at risk due to lifestyle and environmental factors. furthermore, there has been little exploration of cya as an alternative medium for dermatophyte isolation in this context. therefore, this study aimed to isolate and identify dermatophytes from toenails and interdigital spaces of university students using czapek yeast extract agar. the results are expected to provide baseline data on dermatophyte prevalence in young populations and to evaluate the applicability of cya as a culture medium for dermatophyte identification. materials and methods study design and population this was a cross-sectional descriptive study conducted from august 2024 to april 2025 at the mycology laboratory, kadiri university. the study population consisted of undergraduate students from faculty of health sciences, kadiri university, aged 18–25 years. inclusion criteria were students who provided informed consent and had no systemic antifungal treatment in the past three months. exclusion criteria were students with systemic immunodeficiency, current systemic antifungal therapy, or nail trauma unrelated to fungal infection. sample collection specimens were collected from toenails suspected of onychomycosis and from interdigital spaces showing scaling, maceration, or erythema. nail samples were obtained by clipping the distal portion of the affected nail and scraping subungual debris with a sterile scalpel. interdigital scrapings were collected using a sterile scalpel blade. all specimens were placed into sterile paper envelopes and transported to the laboratory within 2 hours of collection. direct microscopy each sample was subjected to a 20% potassium hydroxide (koh) wet mount preparation. the preparations were examined under light microscopy at 10× and 40× magnification for hyaline septate hyphae or arthroconidia suggestive dermatophyte infection. culture and isolation samples were inoculated onto czapek yeast extract agar (cya) (tm media, india; tm-2049) supplemented with chloramphenicol (50 mg/l) and cycloheximide (500 mg/l) to inhibit bacterial and saprophytic fungal contaminants. inoculated plates were incubated at 28 ± 2 °c for up to 21 days and observed every 3–4 days. colony morphology—including texture, pigmentation, and growth rate—was recorded. identification of fungi macroscopic identification was based on colony morphology and pigmentation. microscopic identification was performed using lactophenol cotton blue (lpcb) mounts. microscopic features such as the shape and arrangement of macroconidia, microconidia, and hyphae were documented. identification was confirmed according to standard taxonomic keys (gupta et al., 2023; mctaggart et al., 2025). data analysis data were analyzed descriptively. the prevalence of dermatophytes was expressed as the percentage of positive cases among the samples examined. the distribution of species was presented in frequency tables. associations between clinical site (nail vs. interdigital) and dermatophyte species were evaluated using the chisquare test with a significance level of p < 0.05 (spss version 26.0; ibm corp., armonk, ny, usa). results and discussion sample characteristics a total of 100 students participated in this study, consisting of 42 males (42%) and 58 females (58%), with an age range of 18–24 years (mean = 20.6 ± 1.4 years). of the samples collected, 60 were toenail clippings and 40 were interdigital scrapings. direct microscopy (koh examination) 100 samples, 65 (65%) showed positive findings for fungal elements, including septate hyphae and arthroconidia. the positivity rate was higher in toenail samples (68.3%) than interdigital scrapings (60%). culture results dermatophyte growth was observed in 52 samples (52%) after incubation on czapek yeast extract agar (cya). contaminant molds (non-dermatophytes) were detected in 6 samples, while 42 showed no fungal growth. savitri et al. – isolation and identification of dermatophytes from toenails and… 797 table 1. distribution of dermatophytes isolated on cya medium. species identified toenails (n=60) interdigital spaces (n=40) total (n=100) percentage (%) trichophyton rubrum 18 10 28 28% trichophyton mentagrophytes 12 6 18 18% trichophyton indotineae 3 2 5 5% epidermophyton floccosum 1 0 1 1% total dermatophytes 34 18 52 52% morphological findings colonies of t. rubrum (fig. 1a) appeared as white, cottony to powdery on the surface with a red-brown reverse pigmentation. t. mentagrophytes (fig. 1b) showed granular colonies with a yellowish to cream color. t. indotineae (fig. 1c) isolates exhibited rapid growth with flat, spreading colonies, and microscopy revealed abundant spherical to pyriform microconidia. e. floccosum (fig. 1d) showed slow growth with khakicolored colonies and characteristic club-shaped macroconidia. a b c d figure 1. a) t. rubrum, b) t. mentagrophytes, c) t. indotineae, and d) e. floccosum. statistical analysis there was no significant difference between the prevalence of dermatophytes isolated from toenails (56.7%) and interdigital spaces (45.0%) (χ² = 1.25, p = 0.26). however, t. rubrum was the most common species in both sites, accounting for 28% of all isolates. discussion this study demonstrated that dermatophytes were isolated from 52% of clinical samples collected from toenails and interdigital spaces of university students. the prevalence observed here is consistent with previous studies reporting onychomycosis and tinea pedis as common superficial fungal infections among young adults. for example, lee et al. (2023) found a prevalence of 51.5% for tinea pedis among south korean students, with t. rubrum as the dominant etiologic agent. similarly, gupta, versteeg, and shear (2022) emphasized that dermatophytes remain the leading cause of nail and interdigital infections worldwide. the predominance of t. rubrum (28%) and t. mentagrophytes (18%) in this study aligns with global epidemiological trends, where these two species account for the majority of dermatophytosis cases (gupta, venkataraman, & hall, 2023). interestingly, t. indotineae was detected in 5% of isolates, which deserves attention. first described in india, t. indotineae is now recognized as an emerging, terbinafine-resistant dermatophyte that has spread to europe and north america (chowdhary et al., 2022; mctaggart et al., 2025; marbaniang et al., 2025). the presence of t. indotineae in a young, otherwise healthy population highlights the potential for wider dissemination and the need for ongoing surveillance outside high-risk clinical settings. culture on czapek yeast extract agar (cya) successfully supported dermatophyte growth, with distinct morphological characteristics observed across species. although cya is not traditionally used for routine dermatophyte culture, the current findings support previous observations that its nutrient composition can facilitate colony development and pigment production useful for species differentiation (ahmadi et al., 2012; kumar, patel, & singh, 2025). the detection rate of 52% was comparable to reports using sabouraud dextrose agar, suggesting that cya may 798 biology, medicine, & natural product chemistry 14 (2), 2025: 795-798 serve as a viable alternative medium for academic or diagnostic purposes, particularly in resource-limited laboratories. the higher prevalence of dermatophytes in toenails (56.7%) compared with interdigital spaces (45.0%) was not statistically significant (p = 0.26). nevertheless, this trend is consistent with the view that nails act as reservoirs for dermatophytes and may serve as sources of recurrent infection for surrounding skin (gupta et al., 2023). the strengths of this study include its focus on a young adult population and the use of cya medium, which has not been widely evaluated for dermatophyte isolation. however, some limitations should be noted. first, molecular confirmation of isolates was not performed, which may limit accuracy in distinguishing closely related species. second, the study was confined to a single institution, and may not reflect the general population. conclusions this study confirmed that dermatophytes are prevalent among university students, with t. rubrum as the dominant species, followed by t. mentagrophytes and the emerging t. indotineae. using czapek yeast extract agar proved effective for fungal isolation and morphological characterization. these findings highlight the importance of routine surveillance in young adults and support the potential utility of cya as an alternative medium for dermatophyte research and diagnostics. future studies incorporating molecular methods and multicenter sampling are recommended to strengthen epidemiological insights and guide antifungal management strategies. acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university, for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there are no funding. references ahmadi, b., hashemi, j., zaini, f., & rezaie, s. (2012). colony morphology of the causing agent of onychomycosis on czapek yeast agar (cya) after incubating at 25 °c for 6 days [figure]. researchgate. https://www.researchgate.net/figure/colony-morphology-ofthe-causing-agent-of-onychomycosis-on-czapek-yeast-agarcya-after_fig2_257742965 caplan, a. s., nguyen, d., wengenack, n. l., et al. (2023). notes from the field: first reported u.s. cases of tinea caused by trichophyton indotineae. morbidity and mortality weekly report, 72(19), 523–524. https://doi.org/10.15585/mmwr.mm7219a4 chowdhary, a., singh, a., kaur, a., & khurana, a. (2022). the emergence and worldwide spread of trichophyton indotineae causing difficult-to-treat dermatophytosis: a new challenge in the management of dermatophytosis. plos pathogens, 18(9), e1010710. https://doi.org/10.1371/journal.ppat.1010710 fuller, l. c., hay, r., & arenas, r. (2024). emerging terbinafineresistant trichophyton indotineae: global spread and clinical challenges. journal of fungi, 10(2), 145. https://doi.org/10.3390/jof10020145 gupta, a. k., versteeg, s. g., & shear, n. h. (2022). onychomycosis in the 21st century: diagnosis, management, and resistance. journal of the european academy of dermatology and venereology, 36(9), 1403–1414. https://doi.org/10.1111/jdv.18234 gupta, a. k., venkataraman, m., & hall, d. c. (2023). the emergence of trichophyton indotineae: implications for clinical practice. international journal of dermatology, 62(6), 711–718. https://doi.org/10.1111/ijd.16631 kumar, s., patel, h., & singh, r. (2025). evaluation of culture media for dermatophyte isolation: potential use of czapek yeast extract agar. medical mycology journal, 66(1), 33–41. https://doi.org/10.3314/mmj.25-001 lee, j. h., park, s. y., & kim, h. j. (2023). prevalence and risk factors of tinea pedis and onychomycosis among college students in south korea. mycoses, 66(4), 322–330. https://doi.org/10.1111/myc.13678 marbaniang, y. v., fuller, l. c., & hay, r. (2025). treatment and diagnostic challenges associated with the global expansion of trichophyton indotineae in the united kingdom. journal of clinical microbiology, 63(6), e01407-24. https://doi.org/10.1128/jcm.01407-24 mctaggart, l. r., rebellato, l., haldane, d. j., et al. (2025). emergence of terbinafine-resistant trichophyton indotineae in ontario, canada, 2014–2023. journal of clinical microbiology, 63(5), e01535-24. https://doi.org/10.1128/jcm.01535-24 tm-media. (n.d.). tm 2049 – czapek yeast autolysate agar (cya) media composition. tm-media technical data sheet. https://www.tmmedia.in/wp-content/uploads/td/td-tm2049.pdf zhou, x., li, y., & wang, l. (2022). advances in molecular diagnosis of dermatophytosis: from pcr to sequencing-based methods. frontiers in microbiology, 13, 956372. https://doi.org/10.3389/fmicb.2022.956372 https://www.researchgate.net/figure/colony-morphology-of-the-causing-agent-of-onychomycosis-on-czapek-yeast-agar-cya-after_fig2_257742965?utm_source=chatgpt.com https://www.researchgate.net/figure/colony-morphology-of-the-causing-agent-of-onychomycosis-on-czapek-yeast-agar-cya-after_fig2_257742965?utm_source=chatgpt.com https://www.researchgate.net/figure/colony-morphology-of-the-causing-agent-of-onychomycosis-on-czapek-yeast-agar-cya-after_fig2_257742965?utm_source=chatgpt.com https://www.tmmedia.in/wp-content/uploads/td/td-tm-2049.pdf?utm_source=chatgpt.com https://www.tmmedia.in/wp-content/uploads/td/td-tm-2049.pdf?utm_source=chatgpt.com biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 107-111 | doi: 10.14421/biomedich.2025.141.107-111 issn 2540-9328 (online) potential of red seaweed juice in reducing blood pressure and lead levels in urine in workers at public fuel filling stations evi kusumawati1,*, imanuddin1, rofiqoh1, nadimin2 1nutrition and dietetics study program, department of nutrition, health polytechnic ministry of health kendari, indonesia. 2nutrition and dietetics study program, department of nutrition, poltekkes kemenkes makassar, indonesia. corresponding author* rafaalazka@gmail.com manuscript received: 31 december, 2024. revision accepted: 08 may, 2025. published: 09 may, 2025. abstract lead (pb) pollution, primarily from fuel combustion, poses severe health risks, including hypertension and systemic poisoning. gas station attendants are particularly vulnerable to pb exposure due to occupational hazards. this study investigates the efficacy of red seaweed (eucheuma spinosum) juice, rich in antioxidants and bioactive components, in reducing pb levels in urine and lowering blood pressure among gas station workers in kendari city, indonesia. a quasi-experimental pre-post-test control group design was employed. thirty gas station attendants were divided into three groups: control (p0), treatment with seaweed juice once daily (p1), and treatment with seaweed juice twice daily (p2), each receiving 200 ml for two weeks. urinary pb levels and blood pressure were measured pre-and post-intervention. phytochemical analysis of the seaweed juice confirmed the presence of antioxidants, phenolics, saponins, flavonoids, and triterpenoids. data were analyzed using one-way anova and kruskal-wallis tests. the p1 and p2 groups exhibited a reduction in urinary pb levels, whereas the p0 group showed an increase. however, statistical analysis indicated no significant differences in mean urinary pb levels among groups. blood pressure decreased in all groups, with the p2 group showing the most significant reduction in systolic pressure. despite these trends, no statistically significant differences in blood pressure across the groups were observed. red seaweed juice demonstrated potential in reducing pb levels and improving blood pressure among gas station attendants. while statistical significance was not achieved, the findings suggest further research with larger sample sizes and longer intervention durations to confirm the therapeutic benefits of seaweed-based functional foods in mitigating pb-related health risks. keywords: lead poisoning; red seaweed juice; functional food; antioxidants; blood pressure; occupational health. introduction lead heavy metal pollution is a serious problem in developed and developing countries like indonesia. lead pollution is closely related to mining, motor vehicle exhaust, and industries that use lead (pb) metal as raw material. most motor vehicle fuel in indonesia still contains lead levels above the international minimum. the official specifications set by the directorate general of oil and gas state that the maximum allowable lead content in fuel is 0.45 grams per liter. in contrast, international standards set a stricter limit, permitting a maximum lead content of only 0.15 grams per liter (girsang, 2008). lead, or tetra ethyl lead (tel), which is abundant in fuel, especially gasoline, is known to cause systemic poisoning, which is characterized by several symptoms such as blindness and paralysis and even affects the formation of blood cells in the spine and inhibits the synthesis of hemoglobin (hb) (lanphear et al., 2024). blood pressure is one of the most sensitive variables to chronic pb exposure. several studies have identified the mechanism by which lead can induce hypertension and cardiovascular disease through oxidative stress, functional deficiency of nitric oxide (no), inflammation, increased central sympathetic nerve activity, and disruption of the regulation of the vasoregulatory system (lanphear et al., 2024). lead (pb) poisoning often occurs in groups of people who are at high risk, such as workshop workers, toll road workers, public transport drivers, and refueling officers at public petrol filling stations (spbu). gas station attendants are one group of people vulnerable to exposure to lead (pb). klopfleisch et al. (2017) stated that the average level of lead in the blood of officers at the jln. adisucipto gas station, jln. monjali gas station, and jln. magelang gas station was 62.174 μg/dl. research by ayu et al. (2016) shows that the average level of lead in the blood of petrol station officers in tamalanrea district, makassar city, is >25 μg/dl. high levels of lead in the body will accumulate in the liver, which will then induce the formation of free radicals. efforts to reduce free radical levels in the body require antioxidant compounds, obtained from outside https://doi.org/10.14421/biomedich.2025.141.107-111 https://www.zotero.org/google-docs/?oiweku https://www.zotero.org/google-docs/?rwwon9 https://www.zotero.org/google-docs/?omyfsj 108 biology, medicine, & natural product chemistry 14 (1), 2025: 107-111 the body through food consumption. antioxidants from food sources have been known for a long time, but currently, the use of natural foods and drinks, which are widely known by the public, have not been utilized as a type of functional food source of antioxidants. marine macroalgae seaweed is currently being touted as a future plant food containing soluble dietary fiber, peptides, phlorotannins, carotenoids, and minerals as functional foods/nutraceuticals. the primary soluble fibers include alginate from brown macroalgae, carrageenan, and agar from red macroalgae, representing up to half the dry weight (dw) of seaweed. this fact makes macroalgae have a leading position in fiber content, surpassing most fruits and vegetables in improving health, including preventing colon cancer, type ii diabetes, obesity, and cardiovascular disease. seaweed is a functional food that contains soluble dietary fiber, peptides, phlorotannins, carotenoids, lipids, and minerals, and can potentially become a high-value food product for cardiovascular health (cvd). meanwhile, for the bioactive components, alginate and fucoidan (both polysaccharides), fucosterol (lipid), and fucoxanthin (carotenoid) are explicitly contained in seaweed (wells ml, et all., 2017). alginate, the main polysaccharide of seaweed, contributes to lowering cholesterol levels, increasing postprandial blood glucose and lowering blood pressure. fucoidan polysaccharide, contained only in brown algae (which is the most commonly consumed seaweed), has antioxidant, anticoagulant, antithrombotic, and anti-inflammatory effects, as well as cholesterol-lowering and blood pressure-lowering (li x., li j. et al., 2016). another nutrient, fucosterol, is contained as a lipid, especially in brown and red algae, and lowers blood glucose levels and blood pressure (abdul q.a. et al, 2016). fucoxanthin, an algal carotenoid, has a stronger antioxidant effect than other carotenoids such as αtocopherol. in addition, although not explicitly contained in seaweed, potassium and calcium reduce blood pressure levels (including vitamins b 1, b 2, b 6, and b 12), c, and e which have antioxidant effects. this study aims to assess the effect of seaweed juice on lead levels in the urine and blood pressure of gas station officers. materials and methods this type of research is a quasi-experimental type of research with a pre-post test control group design. the research was conducted by testing lead levels in urine and blood pressure, before and after administering seaweed juice. the research was carried out in julynovember 2024 involving officers from public fuel filling stations in kendari city. population and sample the population in this study included all puuwatu, punggolaka, and lepo-lepo gas station officers in kendari city. the total sample was divided into four groups, namely control group, treatment group 1, and treatment group 2, with the following inclusion criteria: 1. adults aged 20 – 45 years 2. length of work as a gas station attendant is at least 1 year 3. healthy condition and can communicate well 4. not currently undergoing hormonal therapy or medication therapy that has the potential to cause low bone mass density or loss of bone mass, for example glucocorticoids, anti-convulsants, and hormonal drugs 5. willing to be involved in research and sign informed consent. from the total population at all gas stations, 30 people were taken as research samples. research design this type of research is a quasi-experimental type of research with a pre-post test control group design. the research was carried out by testing lead levels in urine and blood pressure, before and after administering seaweed juice. procedures study material preparation and inspection blood pressure checks will be carried out at the kendari city gas station, seaweed juice making will be carried out at the food laboratory of the nutrition department, health polytechnic, ministry of health, kendari. tests for seaweed juice heavy metal and phytochemical content were carried out at the haluoleo university integrated laboratory. seaweed juice is juice whose main ingredient is grass plus palm sugar and fruit or coco pandan flavoring, given with a frequency of 1 time per day, 2 times per day, and 3 times per day with a dose of 200 milliliters for 2 weeks. blood pressure is the pressure experienced by blood in the arteries when blood is pumped by the heart to all parts of the human body. for normal systolic pressure is <130 mmhg, high if systolic pressure is ≥130 mmhg. diastolic pressure is normal if <85 mmhg and high if the diastolic pressure is ≥85 mmhg. respondent sampling p0 x0 pp0 p1 x1 pp1 p2 x2 pp2 information: p0 = first measurement (pre-test) control group p1 = first measurement (pre-test) treatment group 1 p2 = first measurement (pre-test) treatment group 2 kusumawati et al. – potential of red seaweed juice in reducing blood pressure … 109 x0 = no treatment x1 = seaweed juice treatment 1 time 200 cc for 2 weeks x2 = seaweed juice treatment 2 times 200 cc for 2 weeks pp0 = second measurement (post-test) control group pp1 = second measurement (post-test) treatment group 1 pp2 = second measurement (post-test) treatment group 2 data analysis this research uses statistical tests one way anova (normal data) and kruskal wallis (abnormal data) to see the differences between before and after treatment in the research group, then to see the differences between the research groups using a test post. results and discussion phytochemical content of red seaweed the results of the analysis of antioxidant, phenolic, saponin, flavonoid, and triterpenoid content in red seaweed (eucheuma spinosum) were carried out qualitatively. this analysis was only carried out on dry products, so red seaweed that had been soaked for ± 1 mg until the color turned white had to be through a drying process in a cool place (not directly exposed to sunlight) ± 2 days, then the grinding process is carried out again using a blender for the dry ingredients until they become seaweed flour. the results of the analysis of the average antioxidant, phenolic, saponin, flavonoid, and triterpenoid content in red seaweed (eucheuma spinosum) can be seen in table 1. table 1. phytochemical content of red seaweed no parameter methanol extract 1 antioxidant + 2 phenolic + 3 saponin + 4 flavonoid + 5 triterpenoids + effect of red seaweed juice on lead (pb) levels in urine figure 1. effect of red seaweed juice on lead (pb) levels in urine based on figure 1 above, it can be seen that group p1 tended to decrease levels of lead (pb) in urine, which was almost the same as the decrease in levels of lead (pb) in group p2 urine, while group p0 samples tended to increase lead levels during the study (figure 1). table 2. comparative analysis of lead on urine table 2 shows that the p1 group experienced a tendency to decrease lead (pb) levels in urine, which was almost the same as the decrease in lead (pb) levels in the urine of group p2. in contrast, the p0 group samples experienced a tendency to increase lead levels during the study. the results of the kruskal wallis test showed no difference in the mean urine lead levels before and after treatment in the three research groups (table 2). effect of red seaweed juice on blood pressure figure 2. effect of red seaweed juice on blood pressure. figure 2 shows a decrease in systolic blood pressure in the three treatment groups. the p2 treatment group experienced a greater tendency to decrease systolic blood pressure levels compared to the p1 treatment group and the p0 control group. the tendency to decrease p1 systolic blood pressure levels was almost the same as the decrease in p0 systolic blood pressure. table 3. comparative result analysis on blood pressure table 3 shows a decrease in systolic blood pressure in the three treatment groups, where the blood pressure levels of the p2 treatment group experienced the more significant decrease at the end of treatment compared to the blood pressure levels of the p1 treatment group and 110 biology, medicine, & natural product chemistry 14 (1), 2025: 107-111 the p0 control group, the decrease in blood pressure levels between the p1 treatment group was almost the same magnitude. with the control group p0 at the end of treatment. the results of the kruskal-wallis test showed that there was no difference in the mean blood pressure levels at the beginning and end of treatment in the three study groups. discussion lead pollution in air, soil, and water comes from burning coal in factories, spraying pesticides, burning rubbish, using aerosols with added lead, recycling used lead acid batteries, and using lead pigments in paint, batik, and paralon pipes. coal-fired power plants, traditional and small-scale gold mining, industry, ship waste, and cigarette smoke are sources of cadmium and lead metal contamination. of the many existing sources of air pollution, motorized vehicles (transportation) are the largest source of air pollution (60%), the industrial sector 20% and others 20%. around 85% of air pollution in indonesia comes from motor vehicle emissions, affecting lead levels in the blood of active people on the streets. worker groups at high risk of exposure to lead pollutants in the air include traffic police, street vendors, beggars, and fuel station (spbu) officers (ministry of the ministry of environment and forestry and unicef. 2022). lead can enter the human body in three ways: absorption in the skin, respiratory tract, and digestive tract. if this is limited to the contact area, it is called a local effect, but if these substances are absorbed into the blood circulation, they will be carried to various organs in the body and cause systemic effects. apart from that, lead that enters the human body can then cause various kinds of disorders, such as hematological disorders, nervous disorders, cardiovascular disorders, and reproductive disorders. (priyanto, 2009) lead excretion occurs through the digestive tract in feces, the excretory tract in urine, and sweat and hair. the percentage of lead excreted through urine is 75-80%, while only 15% occurs through feces. lead levels in urine reflect recent exposure, so urine lead examination is used for occupational exposure (palar, 2004). lead in the body acts as a free radical or as an oxidant. conditions where free radicals exceed normal levels can decrease antioxidants, which can neutralize reactive oxygen species (ros), which in turn can reduce the total antioxidant capacity in the body. lead in its ionic form (lead acetate) can cause lipid peroxidation. lipid peroxidation is a chain reaction that supplies free radicals. lipid peroxidation causes fatty acid chains to break down and form final products with one of the most active carbon elements, malondialdehyde (mda). mda is a biomarker of oxidative damage because mda is the result of polyunsaturated fatty acids (pufa) in cell membranes and plasma lipoproteins through enzymatic and non-enzymatic processes. increased mda levels in the blood can indicate a high response between free radicals and unsaturated fats (kodariah, et al., 2022) oxidative stress and chronic inflammation cause endothelial dysfunction that accelerates atherosclerosis and plaque. in addition, lead directly interferes with the release of tissue plasminogen activator (t-pa) and increases the release of plasminogen activator inhibitor1, leading to coagulation abnormalities and increased risk of thrombosis and endothelial dysfunction, where endothelial cells are critical in the regulation, maintenance, and control of cardiovascular function. (mustofa, et al,. 2024) the active phytochemical compounds in seaweed are thought to directly affect endothelial cells, namely improving endothelial function by stimulating no synthesis (nitric oxide). endothelial cells release no, which plays is important in increasing vasodilation, thereby reducing arterial stiffness and maintaining blood vessel tone, especially for relaxing blood vessels. no results from changing l-arginine to citrulline, which is catalyzed by the nitric oxide synthase (nos) enzyme (monsalve et al., 2017). lead levels in the urine of group p2 experienced the more significant decrease compared to lead levels in the urine of group p1, while lead levels in the urine of group p0 (control) increased after treatment. however, there was no difference in the mean urine lead levels at the beginning and end of treatment in the three research groups. based on research conducted by hastuti (2009), regarding lead (pb) levels in the urine of street children in yogyakarta city, the research results showed that the average lead (pb) level in the urine of street children in yogyakarta city was 0.2234 mg/l. this has lead levels that exceed the normal threshold, namely ≥ 0.15 mg/l. likewise, the lead level of gas station officers has an average lead level of 0.226 mg/l, and it is indicated that gas stations along the puuwatu road are a busy transsulawesi highway and are near the north konawe border mining area. elevated systolic blood pressure predicts cardiovascular disease risk better than increased diastolic blood pressure. elevated systolic blood pressure predicts cardiovascular disease risk better than increased diastolic blood pressure. systolic blood pressure is much more important than diastolic blood pressure levels in predicting the risk of coronary heart disease, left ventricular hypertrophy, congestive heart failure, kidney failure, and mortality in people with hypertension. systolic blood pressure remains more difficult to control than diastolic blood pressure (jan n. basile. 2022). the active phytochemical compounds in seaweed are thought to have the same effect on cardiovascular and blood vessels. specifically, the phytochemical compounds are thought to increase vasodilation, thereby reducing arterial stiffness and maintaining blood vessel tone, especially for relaxing blood vessels, by increasing kusumawati et al. – potential of red seaweed juice in reducing blood pressure … 111 endothelial function and stimulating no synthesis (monsalve et al., 2017). the potassium content in seaweed per 100 g is 380 mg, higher than that of tomatoes and kepok bananas. in contrast, dried seaweed will have a higher potassium content, namely 1,125 mg. potassium can increase sodium and water excretion by inhibiting renin. rennin circulates in the blood and works by catalyzing the breakdown of angiotensin i. angiotensin i changes to its active form, namely angiotensin ii, with the help of angiotensin-converting enzyme (ace). angiotensin ii has great potential to increase blood pressure because it acts as a vasoconstrictor and can stimulate the release of aldosterone. aldosterone increases blood pressure by sodium retention. the presence of potassium reduces sodium and water retention, resulting in a decrease in plasma volume, cardiac output, peripheral pressure, and blood pressure (lestari, 2012). the decrease in systolic blood pressure in the three treatment groups, where the blood pressure levels of the p2 treatment group experienced the greatest decrease at the end of treatment compared to the blood pressure levels of the p1 treatment group and the p0 control group, the decrease in blood pressure levels between the p1 treatment group was almost the same as the p0 control group., at the end of treatment. the decrease in systolic blood pressure in the three treatment groups, where the blood pressure levels of the p2 treatment group experienced the greatest decrease at the end of treatment compared to the blood pressure levels of the p1 treatment group and the p0 control group, the decrease in blood pressure levels between the p1 treatment group was almost the same as the p0 control group. at the end of treatment. putri research et al. 2023, show that 250 cc of cucumber juice given 2 times a day for 7 days effectively reduces blood pressure in hypertension sufferers in surau gadang village, padang city. conclusions there was a tendency to reduce lead levels in the urine of gas station attendants after administering seaweed (eucheuma spinosum) juice, although it was not statistically significant. there was a tendency to reduce systolic blood pressure in gas station attendants after administering seaweed (eucheuma spinosum) juice, although it was not statistically significant. the effectiveness of administering seaweed juice at a dose of 200 ml/day for 14 days was greater than at 100 ml/day for 14 days, in reducing urine lead levels and systolic blood pressure levels for gas station officers, although it was not statistically significant. competing interests: the authors declare that there are no competing interests. funding: this research received funding from dipa poltekkes kemendari ministry of health in 2024. references girsang, e. (2008). hubungan kadar timbal di udara ambien dengan timbal dalam darah pada pegawai dinas perhubungan terminal antar kota medan [thesis, universitas sumatera utara]. https://repositori.usu.ac.id/handle/123456789/41229 lanphear, b., navas, -acien ana, & bellinger, d. c. (2024). lead poisoning. new england journal of medicine, 391(17), 1621– 1631. https://doi.org/10.1056/nejmra2402527 klopfleisch, b., sutomo, a.h., dan iravati, s., 2017, kadar timbal dalam darah petugas stasiun pengisian bahan bakar,berita kedokteran masyarakat4(33), 205-212. ayu, f., afirdah, w., dan nourma, m., 2016 hubungan karakteristik pekerjaan dengan kadar timbal dalam darah operator spbu di kecamatan tamalanrea kota makassar, fakultas kesehatan program studi ilmu kesehatan masyarakat, universitas nahdlatul ulama surabaya. wells ml, potin p, craigie js, raven ja, merchant ss, helliwell ke, smith ag, camire me dan brawley sh: alga sebagai sumber makanan bergizi dan fungsional: meninjau kembali pemahaman kita. j appl phycol, 2017; 29: 949-982 li x, li j, li z, sang y, niu y, zhang q, ding h dan yin s: fucoidan dari undaria pinnatifida mencegah disfungsi vaskular melalui mekanisme yang bergantung pada pi3k/akt/enos pada hipertensi yang diinduksi l-name model tikus. fungsi pangan, 2016; 7: 2398-2408 abdul qa, choi rj, jung ha dan choi js: manfaat kesehatan fucosterol dari ganggang laut: ulasan. j sci pertanian pangan, 2016; 96: 1856-1866 dinas ketahanan pangan, k. dan p. p. d. j. (2017). pusat produksiinspeksi dan sertifikasi hasil perikanan. jan n. basile. 2022. tekanan darah sistolik. 2022;325(7370):917– 918. doi: 10.1136/bmj.325.7370.917 kementerian lingkungan hidup dan kehutanan dan unicef. 2022. ringkasan kebijakan: mengurangi keracunan timbal pada anak-anak di indonesia kodariah l., wahid a., a., putri v., a., fadilah t., i. 2022. pengaruh induksi timbal terhadap kadar malondialdehid pada darah mencit (mus musculus). prosiding aiptlmi mustofa m., a., et al. 2024. hubungan antara kadar timbal dalam darah dan infark miokard senyap pada populasi umum. journal of clinical medicine, 13(6):1582. doi: 10.3390/jcm13061582 monsalve b., meyer a., c., palomo i., and fuentes e. 2017. mechanisms of endothelial protection by natural bioactive compounds from fruit and vegetables. anais da academia brasileira de ciências; 89(1 suppl.): 615-633 lestari, a., p., dkk (2012). pengaruh pemberian jus tomat (lycopersicum commune) terhadap tekanan darah pada wanita postmenopause hipertensi. semarang: http://eprints.undip.ac.id//38425/ https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://www.zotero.org/google-docs/?grgvo2 https://doi.org/10.1056/nejmra2402527 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 515-524 | doi: 10.14421/biomedich.2023.122.515-524 issn 2540-9328 (online) targeting h3n2 influenza virus rna-dependent rna polymerase by using bioactives from essential oils from eucalyptus polybrachtea, cymbopogon citratus and cymbopogon khasianus arun dev sharma*, inderjeet kaur pg dept of biotechnology, lyallpur khalsa college jalandhar, india. corresponding author* arundevsharma47@gmail.com abstract a dramatic surge of h3n2 influenza virus is of grave concern worldwide and particularly in india. h3n2 cause acute respiratory infection, however, a few drugs are available for its mitigation. subsequently, researchers have been involved in efforts to discover novel antiviral mechanisms that can lay the basis for new anti-influenza drugs. influenza virus rna-dependent rna polymerase (rdrp) is a multi-functional hetero-trimer, implicated in the production of viral mrna, hence plays a major role in viral infectivity thus directly associated with survival of the virus. rdrp have been cited as anappropriate target for therapeutic drug design. in the present study molecular docking was designed to estimate the effect of potent bioactive moleculesfrom essential oils from eucalyptus polybrachtea (eucalyptus oil, eo), cymbopogon citratus (lemon grass essential oil, leo) and cymbopogon khasianus (palmarosa essential oil, peo) against rdrp protein. gc-fid (gas chromatography with flame-ionization detection) based composition profile, and in-silico docking study was conducted by using cb-dock 2 analysis followed by 2d interactions. gc-fid revealed eucalyptol, geranial and geraniolas major phytocompounds in eo, leo and peo respectively. the docking score indicated effective binding of ligands to rdrp. interactions results indicated that, rdrp/ligand complexes form hydrogen, van der waals forces, pi-alkyl, alkyl, and pi-sigma interactions. based on above findings of aroma profile and docking, therefore, it was recommended that essential oils from above mentioned aromatic cropsmay represent potential herbal treatment to mitigate h3n2 infections. keywords: docking; eucalyptus oil; lemon grass oil; palmarosa oil; herbal drug. introduction acute respiratory infections caused by influenza viruses are of great concern worldwide (leung et al., 2017). recently, in india, h3n2 influenza virus belonging to the orthomyxoviridae family, created much havoc. at present india is witnessing a surge in h3n2 influenza virus cases. several states including maharashtra, gujarat, haryana, odisha, and haryana are reporting viral infection cases, hence deemed a serious public health concern. according to ministry to health and sciences, in this year, till february 28, 955 viral infection cases have been reported (castiello and seladischulman, 2021). the associated symptoms of h3n2 infection includes: cough, runny or congested nose, sore throat, headache, body aches and pains, fever, chills, fatigue, diarrhea and vomiting (jin et al., 2020). symptoms that can signal an emergency and warrant prompt medical attention include: feeling short of breath or having trouble breathing, pain or pressure in your chest or abdomen, dizziness that comes on suddenly, persistent, severe vomiting, feelings of confusion, symptoms that begin to improve but then return with a worsened cough and fever. at the same time, this virus is easily transmissible for person to person and can infect persons of any age (leung et al., 2017). earlier research reported that h3n2 influenza virus is prone to cause periodic outbreaks due to frequent mutation to escape the host immune system (jester et al., 2020) current treatments to mitigate h3n2 infections are limited and drug resistance is a major problem worldwide. recently developed anti-influenza drugs are mainly rdrp drugs that target the virus (york and fodor, 2013). it was reported that all circulating influenza viruses including h3n2 are resistant to most of the drugs, such as favipiravir and baloxavir. however, all synthetic drugs favipiravir, are ineffective, owing to its disadvantages such as high toxicity, teratogenicity and abnormal behavior, etc. (massari et al., 2016). as per reports of chinese national influenza center’s resistance surveillance, h3n2 viruses are resistant to amantadine analogues (deyde et al., 2007), highlighting the inevitability for novel natural drug therapies. consequently, the development of novel anti-influenza drugs is a vital task. h3n2 influenza virus posses rnamanuscript received: 06 april, 2023. revision accepted: 29 august, 2023. published: 15 september, 2023. https://doi.org/10.14421/biomedich.2023.122.515-524 516 biology, medicine, & natural product chemistry 12 (2), 2023: 515-524 dependent rna polymerase (rdrp) which is a multifunctional heterotrimer protein, which uses a capsnatching mechanism to produce viral mrna thus plays an indispensible role on viral infection and survival into the host cells (hussain et al.,2017). h3n2 rdrp consists of three different subunits: acidic polymerase (pa), basic polymerase 1 (bp1), and basic polymerase 2 (pb2). interactions of these domains are vital to achieve full activity of rdrp. thus, it was cited that antiviral effects can be attained by the successful blocking of protein– protein interactions (ppis) during protein assembly. in this regard, poole et al (2007) have shown reduced binding of pb2 through deletion analysis thus inhibiting the activity of influenza rdrp and effectively inhibiting virus replication. authors reported that viral rna polymerase activity was decreased through the deletion of 27 amino acids from the pb2n terminal. earlier docking studies also documented that novel small molecules could be ppi inhibitors in rdrp (ren et al., 2021). therefore, by virtue of key role in viruses, rdrp are considered as an appropriate objective for developing antiviral inhibitors. inhibition of rdrp protein activity would block replication of influenza virus. eucalyptus polybrachtea, native to tasmanian and south-east australia, is a member of myrtaceae family which is one of most widely spread genera. the leaves of this plant are used to extract oleum eucalypti (eucalyptus oil) worldwide. essential oil (eo) from this aromatic plant has long history to be used as traditional medicine in ancient times. eucalyptus essential oil from eucalyptus species encompasses a number of bio actives. 1,8-cineole (eucalyptol) is a main bioactive of eucalyptus oil in all eucalyptusspp. (elaissiet al., 2011). due to complex nature of essential oil, their anti-fungal mechanism of action is still not completely understood (elaissi et al. 2011). previously antifungal potential of leaf water hot water extracts against dermatophytes, filamentous and candida albicans have been cited (sebei et al., 2015). cymbopogon genus, (poacea family) are important and valuable essential oil bearing aromatic cropsin the poaceae family (jnanesha et al., 2019). several cymbopogon species possessed significant anthelmintic, anti-inflammatory, analgesic, antiageing, pesticidal, antimicrobial, mosquito repellant and larvicidal activities and thus, are used in native medicine for curing a number of diseases (khanuja et al., 2005). essential oilsare extracted from cymbopogon khasianus (also known as palmrosa oil, peo) and cymbopogon citratus (also known as lemon grass oil, leo) by the steam distillation method, and this oil finds extensive application in high grade perfumery, cosmetic, flavouring and aromatherapy industries throughout the world (dutta et al. 2017,). eo, leo and peo from eucalyptus and cymbopogon genus, encompasses a number of bioactives with abundant pharmacological and aroma properties. due to the complex nature of essential oil, their anti-viral (h3n2) mechanism of action is still not completely understood. we presented our viewpoint that due to the abundance of eucalyptol, geranial and geraniol, eo leo and peo have potential to mitigate h3n2 infection. antiviral effects of eucalyptol, geranial and geraniol can be expected through the occupation of the pb2 pocket by small molecules that prevent the binding of pb1 and pb2. eo, leo and peo contains bioactive molecules, phytocompounds, endowed with pharmacological activities like: antifungal and mosquito repellent activity, antimicrobial properties, used in ayurvedic traditional and complementary medicine to treat skin problems and relieve nerve pain, and immunomodulatory action (soorya et al., 2021), therefore poses a key role as therapeutics in the scientific community. therefore, the aim of present study was intended to study 3d docking of eucalyptol, geranial and geraniol against rdrp. nevertheless, its potential against h3n2 is still a matter of conjuncture. it would additionally add new insights into the potential forecasts to ascertain the key anti-fungal drugs during h3n2 medications. material and methods gc-fid analysis eo, leo and peo were extracted from fresh leaves of eucalyptus polybrachtea, cymbopogon citratus and cymbopogon khasianus growing naturally at nearby areas of lyallpur khalsa college, jalandhar. the plant species were authenticated by dr upma from botany dept and voucher with number bt103-105were deposited in department of biotechnology. hydrodistillation method was used for extraction of essential oils by using clevenger-type apparatus (borosil, india) (agnish et al., 2022). to identify bioactive compounds in eo, leo and peo, gc-fid study was carried out (gcfid, chemtron 2045). the specifications of column was: 2 m long, stainless steel having 10% ov-17 on 80-100% mesh chromosorb w (hp). nitrogen was used as carrier gas atflow rate of 35 ml/min. 0.2 µl oil samples was used. the temperatures for detector and injector were: 220 °c and 270 °c. oven ramping conditions were: 100°c (firstly maintained) ramped to 210 °c at 3 °c/min. bioactive constituents in eo, leo and peo were identified by comparing relative retention times (rt) of gc-fid spectra of oils with authentic standards and literature data. ligand preparation for viral receptor (rdrp, pdb id: 2ztt), bioactive compounds such as: eucalyptol, geranial and geraniol which are present as predominant amounts in eo, leo and peo were used as ligands for structures. to build 3d structure of ligand, smiles of ligandswas recovered from ncbi-pubchem database. the structure was built by using ucsf-chimera. https://en.wikipedia.org/wiki/cymbopogon_citratus sharma & kaur – targeting h3n2 influenza virus rna-dependent rna polymerase … 517 molecular docking crystal structures of rdrp protein was recovered from pdb (https://www.rcsb.org/). before docking analysis, all target enzymes were cleaned from selected h2o molecules, cofactors, co-crystallized ligand, and energy minimized. then all protein target structures were prepared by means of the dock prep set up in ucsfchimera. it is the process under optimization that bond length, charges anomalies and corrects atomic structure. for docking, cb-dock 2 tool was used for docking of ligands over rdrp (https://cadd.labshare.cn/cbdock2/php/index.php). to execute docking, both receptors and ligand molecules as “pdb files” were uploaded to the cb-dock 2and docking was performed. for 2d and 3d interactions in docked complexes, biovia 2020, ucsf chimera and plip tools were used. drug-likeness and toxicity admet (absorption, metabolism, toxicity and excretion), drug likeness, physiochemical properties and pharmacokinetics were studied using swissadme tool (http://www.swissadme.ch/). bioactivity potential was studied by using web based molinspiration tool (https://www.molinspiration.com/cgi-bin/properties). active sites prediction in viral receptor, identification and dimension of cavities on 3d active sites were computed by using castp web tool. for this all structures in “pdb” format were uploaded to server and prediction was executed with probe radius value of 1.4 angstroms. result and discussion gc-fid analysis of bioactive molecules in lgo, peo and eo aroma profile of leo, peo and eo is displayed in figure 1 and table 1. the gcfid analysis of leo revealed 26 compounds including major and minor peaks. the identified compounds in leo were geranial (45%) and neral (20%). gc-fid analysis of peo displayed the incidence of 22 peaks. the bioactive components in peo were geraniol (46%), 6-methyl hept5-en-2-one (7.4%), linalool (6%) and borneol (5%), elemol (3%) and fenchyl alcohol (2%). as described in literature, peo was rich in geraniol (soorya et al. 2021). the characteristic odour of palmarosa oil is due to its high content of total alcohol, mainly geraniol and small but varying amount of esters associated with geraniol. due to these bioactives molecules, peo has rose-like aroma and has immense applications in high grade perfumery, cosmetic, flavouring and aromatherapy, fragrances, soaps, detergents, toiletry, tobacco products and pharmaceutical industries (bhatnagar, 2018). the small peaks in peo and leo may be attributed to the crumbled major bioactive components or existent in minor amounts. the major componentof peo (46%) was a monoterpene alcohol geraniol and in leo was monoterpene aldehyde geranial (45%) (prashar et al., 2003). preceding studies also cited geranial as major component in lgo over other varieties of cymbopogon (rao et al., 2015). due to its pleasant lemon-smell and distinct, acceptable, and passionate odor, it is natural additive used in foods beverages, and cosmetics (zeng et al., 2015). cymbopogon essential oils have been established to show antifungal, antimicrobial, and antiparasitic properties (zeng et al., 2015). gc-fid analysis of eo displayed the incidence of 39 peaks including major and minor peaks. the bioactive components in eo were eucalyptol (1,8 cineole) (13%), α-pinene (10%), trans-geraniol (7.4%), beta-myrcene (4.5%) and citral (2.9%). as reported in literature, eucalyptus essential oil is predominant in eucalyptol (1,8 cineole) (raho et al. 2012). due to the presence of this bioactive molecule, eucalyptus essential oil has tremendous applications in healthand medical-related research (kushwaha et al., 2018). the characteristic odour of eo oil is due to its high content of alcohol, mainly 1,8 cineole and small but varying amounts of esters associated with geraniol. due to these bioactives molecules, eo has rose-like aroma and has immense applications in high grade perfumery, cosmetic, flavouring and aromatherapy, fragrances, soaps, detergents, toiletry, tobacco products and pharmaceutical industries (bhatnagar, 2018,). since, in gc-fid major compounds in leo, peo and eo were geranial, geraniol and eucalyptol, hence were selected as a ligands for docking studies against fungus enzyme receptors. molecular docking in-silico technique based structure-based drug design (sbdd) is most widely used in making drugs which is based on 3-d structures. molecular docking has opened new vistas for investigators to screen conformations and affinities of an assembly of bioactive components against receptors (barcellos et al., 2019).to investigate the potential binding between rdrp and the bioactive compounds, the molecular docking was performed. present study aimed at docking of geranial, geraniol and eucalyptol bioactive molecules from leo, peo and eo, respectively, as key viral inhibitor candidates against h3n2 rdrp. the binding energies in terms of docking score for the bioactive molecules from docking results were summarized in table 2. less binding energy indicates more effective binding of ligands with receptors. from docking analysis it was ostensible that bioactive molecules efficiently docked withh3n2 rdrp. 3d docking results illustrated that viral enzyme depicted strong binding with ligands as apparent from its docking scores. the binding potential of the three abundant phytochemicals found in the leo, peo and eo was shown to be between −6.0 and −6.7 kcal/mol, with -6.7 kcal/mol being the lowest binding energy for geranial. docking score with geraniol and eo was -6.0 with rdrp. 3d model displaying best docking pose and 2d 518 biology, medicine, & natural product chemistry 12 (2), 2023: 515-524 interactions of geranial, geraniol and eucalyptol with rdrp are displayed in figure 2. docking view depicted that ligands firmly bound with in binding pocket of receptor. with rdrp, all ligands docked with pb2 domain of rdrp. rdrp consists of the pa (chain a), pb1 (chain b) and pb2 domains (reich et al., 2014). pb1 has polymerase activity, pb2 possesses a capbinding domain and pa contains an endonuclease domain. the pa and pb2 domains lie towards the nand c-terminal domains of pb1, respectively (venkataraman et al., 2018). these results were in agreement with earlier studies reporting docking interactions of essential oil based bioactives like quercetin and chlorogenic acid from plants forsythia suspense, mangifera indica, hypericum perforatum and chaenomeles speciosa with viral enzyme (liu et al., 2016). based on analysis, it was highlighted that leo, peo and eo can be used as effective source of anti-h3n2 compounds. through 3d docking, with site residues of receptors, ligand could form h-bonds or van der waals forces which designate affinity of ligand with receptor (lima et al., 2019). hence, docking interactions of geranial, geraniol and eucalyptol rdrp were further evaluated. the chemical bonding mode of the complexes formed between studied compounds and the binding pocket residues of rdrp are displayed in figure 2. for the interaction between rdrp and bioactive molecules, the van der waals’ interaction (vdw), hydrogen bond (hb), contributed as the major part. it was observed that ligands eucalyptol and geraniol and geraniol forms vdw, pi-alkyl, alkyl, hb interactions with rdrp via c-chain and d-chain residues.for all abundant phytoconstituents, predominantly vdw, pi-alkyl, alkyl interactions were responsible for the binding of these molecules to the active site of rdrp. also, it was observed that these molecules formed interactions with similar residues with slight differences. the most stable complex (−6.7 kcal/mol) geranial—rdrp was stabilized by vdw, pi-alkyl, alkyl, hb interactions involving the amino acids tyr689, cys692, cys693, leu695, phe696, phe699, ser712, ser713, val715, ala717, val719, leu7 leu20 thr23 thr24 val25 his27 ala29 ile31 lys32. castp active sites prediction quantified interacting residues in the active site cavities of rdrp receptors (table 3). in rdrp enzyme, a main pocket was documented with volume of342 and area of 427. meanwhile ligand such as geranial, geraniol and eucalyptol shown good affinity to rdrp enzyme so it was conjectured that upon binding with ligands rdrp becomes closed thus in-turn persuades change in conformation of h3n2 enzyme. all these events halts h3n2 viabilitythus mitigate infectivity of virus into the host cell. pass analysis pass analysis of bioactives geranial, geraniol and eucalyptol were calculated. it was cited that these features are key for any therapeutic drug to be used in living organisms (wu et al., 2020). drug-likeness was calculated by following lipinski rule of five (ro5). it was observed that all ligands obeyed ro5. for this rule drug must have log p ≤ 5, h-bond acceptors ≤10, and hbond donors ≤5 and violation no more than 1 (table 4). pass analysis advocated that eucalyptol, geranial and geraniol werelow molecular weight ligand (lmw). it was reported that drugs ligands having less lmw poses high propensity to transportation across the cellular membranes and diffuse effortlessly than high mw compounds (srimai et al., 2013). the log po/w value (v) was also in acceptable range (2.4-2.7). in pharmacoanalysis, log po/w is a potential factor to measure lipophilicity of any drug and its movement in body after absorption (abraham, 2003). tpsa (topological polar surface area) value was 9å²,17 å² and 20å², for eucalyptol, geraniol and geranial, respectively, indicating geranial possess nice oral bioavailability (biswal et al., 2019). wu et al., (2020) cited that tpsa is a key factor of drug transport properties like efficient permeability and absorption. to exert toxic affect, drug has to be absorbed thoroughly in human body. gi (gastrointestinal tract absorption) of eucalyptol, geraniol and geranial was high (table 3). against p-glycoprotein (p-gp) efflux transporters, eucalyptol, geraniol and geranial were nonsubstrate. in human body p-gp pumps drugs back into the lumen, lessening their absorption (konig and muller, 2013). further, eucalyptol, geraniol and geranial was non-substrate to cyp450 series of enzyme. in human body, cyp450 are series of enzymes intricated in liver detoxification (srimai et al., 2013). these results indicated that eucalyptol, geraniol and geranial can effectively target receptors thus can be further evaluated for biological activity score. biological activity (ba) is a key factor that defines the capability of any drug binding to respective drug or biological targets (khan et al., 2017). in living systems, biological targets usually are: ion channels or biological receptors.ba score of geranial was computedas shown in table 5. ba rule states that if ba score is more than 0, drug is active, if less than -5.0, drug is silent and if between -5.0 to 0, drug is sufficient active.ba score of 1,8 cineole for various targets were: -1.60 for kinase inhibitor, -1.07 for nuclear receptor ligand, -0.93 for gpcr ligand, -0.90 for protease inhibitor, -0.1 for enzyme inhibitor, 0.01 for ion channel modulator. it was observed that with enzyme inhibitor and ion channel modulator 1,8 cineole was active. with kinase inhibitor, nuclear receptor ligand, gpcr ligand, and protease inhibitor, ligand was sufficiently active. ba score for geraniol was as under; gpcr ligand: -0.86, ion channel modulator -0.25, kinase inhibitor -1.29, nuclear receptor ligand-0.42, protease inhibitor-0.57, enzymeinhibitor 0.02. ba score for geranial was; gpcr ligand: -0.60, ion channel modulator -0.07, kinase inhibitor -1.32, nuclear receptor ligand-0.20, protease inhibitor-1.03, enzyme sharma & kaur – targeting h3n2 influenza virus rna-dependent rna polymerase … 519 inhibitor 0.28. khan et al., (2017) reported these types of observations on drug formulations. these observations indicated that studied bioactive compound owned properties that are mandatory for the bioactive compound to act as key drugs (khan et al., 2017). the bioactivity score delivers the indication about the binding cascade of the bioactive compounds that is used for the development of a new functional drug with more binding selectivity profile and less undesirable effects (khan et al., 2017). conclusions currently, h3n2 has emerged in the human population, and is a potential threat to global health, worldwide. the aim of this study was to examine bioactive molecules from leo, peo and eo that may be used to inhibit h3n2 infection pathway by targeting rdrp. gc-fid analysis revealed the presence of bioactive compounds like eucalyptol, geranial and geraniol in aromatic plants. in-silico docking depicted effective docking of all bioactive compounds with rdrp. therefore, we suggested that bioactives compounds from leo, peo and eo may represent potential treatment options, and found in medicinal plants that may act as potential inhibitors of h3n2 rdrp enzymes. however, further studies should be conducted for the validation of these compounds using in vitro and in vivo models to pave a way for these compounds in drug discovery. conflict of interest: authors declares no conflict of interest. funding: dst govt of india. author contributions: ads: designed study, ijk: interpreted study. references abraham, d.j. 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(2015). formulation, characterization, and antitumor properties of transand cis-citral in the 4t1 breast cancer xenograft mouse model. pharm. res. 32, (8) 2548–2558 sharma & kaur – targeting h3n2 influenza virus rna-dependent rna polymerase … 521 table 1. chemical composition of the eucalyptus polybrachtea (eo), cymbopogon citratus, (leo) and cymbopogon khasianus (peo) essential oil. rt (min)eo compound concentration (%) 0.5 citral 2.9 3.7 α-pinene 10.4 5.8 βpinene 1.6 6.9 1,8 cineole 10.1 9.6 1,8 cineole 3.1 16.5 sabinene 3.2 18.0 beta-myrcene 4.5 19.6 terpinolene 1.1 20.8 limonene 0.5 21.8 γ-terpinene 0.8 22.9 4-carene 1.1 25.4 pinocarveol 0.9 26.3 4-terpineol 1.2 28.5 α-terpineol 2.4 30.2 α-pinene epoxide 1.2 31.8 dehydro-ρ-cymene 1.1 33.1 cis-limonene oxide 2.1 35.4 linalool 1.7 38.0 fenchyl alcohol 1.0 40.0 eugenol 1.0 43.3 eugenol 0.59 45.2 carvone 2.1 48.2 trans-geraniol 7.4 53.1 unknown 5.8 rt (min) leo compound concentration (%) 13 micrene 2.2 17 limonene 2.3 26 linalool 8.2 46 geranial 62.2 48 neral 9.6 55 undececanone 8.5 69 geranial acetate 6.7 rt (min) peo compound concentration (%) 5.6 terpinolene 1.2 7.3 6-methyl hept-5-en-2-one 7.1 9.4 2-norbornaneacetic acid 1.7 14.7 citronellyl acetate 1.4 16.4 geraniol 45.8 17.4 borneol 5.5 19.4 nerol 1.3 20.6 geraniol 16.9 29.6 elemol 3.1 31.7 epi-α-cadinol 1.3 32.7 δ-cadinol 2.2 34.9 linalool 6.3 37.6 fenchyl alcohol 2.9 522 biology, medicine, & natural product chemistry 12 (2), 2023: 515-524 table 2. molecular docking of rdrp with ligands. ligand binding energy (vina score) cavity volume (å3) center (x, y, z) docking size (x, y, z) involved receptor residues type of interactions eucalyptol -6.0 685 14, 31, 7 16, 16, 16 chain c: tyr689 cys692 cys693 phe696 ser712 ser713 val715 chain d: ile19 leu20 thr23 thr24 val25 his27 ala29 vdw, pi-alkyl, alkyl geranial -6.7 685 14, 31, 7 19, 19, 19 chain c: tyr689 cys692 cys693 leu695 phe696 phe699 ser712 ser713 val715 ala717 val719 chain d: leu7 leu20 thr23 thr24 val25 his27 ala29 ile31 lys32 vdw, pi-alkyl, alkyl, hb geraniol -6.0 685 14, 31, 7 19, 19, 19 chain c: cys692 cys693 phe696 phe699 phe700 ser713 val715 glu716 ala717 val719 chain d: leu7 ile19 leu20 thr21 thr23 thr24 vdw, pi-alkyl, alkyl, hb, pi-sigma abbreviation: hb, hydrogen bond; vdw, van der waals forces table 3. active site analysis of protein target structure. letters in red font indicates residues involved in 2d interactions. fungal receptor 3d model interacting active site residues cavity area å2 volume å2 rdrp 686glu, 689tyr, 690gln, 692cys, 695leu, 696phe, 697glu, 699phe, 701pro, 702ser, 707arg, 708pro, 713ser, 715val, 717ala, 719val,21arg, 7leu, 19ile, 20leu, 23thr,24 thr, 25val, 27his, 29ala, 31ile, 32lys, 35thr 427.24 342.12 sharma & kaur – targeting h3n2 influenza virus rna-dependent rna polymerase … 523 table 4. adme properties of eucalyptol, geraniol and geranial. physicochemical properties geraniol geranial eucalyptol formula c10h16o c10h18o c10h18o molecular weight 152.23 g/mol 154.25 g/mol 154.25 g/mol num. heavy atoms 11 11 11 num. arom. heavy atoms 0 0 0 fraction csp3 0.50 0.60 1.00 num. rotatable bonds 4 4 0 num. h-bond acceptors 1 1 1 num. h-bond donors 0 1 0 molar refractivity 49.44 50.40 47.12 tpsa 17.07 å² 20.23 å² 9.23 å² lipophilicity log po/w (ilogp) 2.47 2.75 2.58 log po/w (xlogp3) 3.03 3.56 2.74 log po/w (wlogp) 2.88 2.67 2.74 log po/w (mlogp) 2.49 2.59 2.45 log po/w (silicos-it) 2.65 2.35 2.86 consensus log po/w 2.71 2.78 2.67 class soluble soluble pharmacokinetics gi absorption high high high bbb permeant yes yes yes p-gp substrate no no no cyp1a2 inhibitor no no no cyp2c19 inhibitor no no no cyp2c9 inhibitor no no no cyp2d6 inhibitor no no no cyp3a4 inhibitor no no no log kp (skin permeation) -5.08 cm/s -4.71 cm/s -5.30 cm/s druglikeness lipinski yes; 0 violation yes; 0 violation yes; 0 violation ghose no; 1 violation: mw<160 no; 1 violation: mw<160 no; 1 violation: mw<160 veber yes yes yes egan yes yes yes muegge no; 2 violations: mw<200, heteroatoms<2 no; 2 violations: mw<200, heteroatoms<2 no; 2 violations: mw<200, heteroatoms<2 bioavailability score 0.55 0.55 0.55 http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php 524 biology, medicine, & natural product chemistry 12 (2), 2023: 515-524 medicinal chemistry pains 0 alert 0 alert 0 alert brenk 3 alerts: aldehyde, isolated_alkene, michael_acceptor_1 1 alert: isolated_alkene 0 alert leadlikeness no; 1 violation: mw<250 no; 2 violations: mw<250, xlogp3>3.5 no; 1 violation: mw<250 synthetic accessibility 2.49 2.58 3.65 table 5. bioactivity score (ba) of geraniol, geranial and eucalyptol. bioactivity ba score geraniol geranial eucalyptol gpcr ligand -0.86 -0.60 -0.93 ion channel modulator -0.25 -0.07 0.01 kinase inhibitor -1.29 -1.32 -1.60 nuclear receptor ligand -0.42 -0.20 -1.07 protease inhibitor -0.57 -1.03 -0.90 enzyme inhibitor 0.02 0.28 -0.1 http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php http://www.swissadme.ch/index.php biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 2, october 2020 | pages: 57-64 | doi: 10.14421/biomedich.2020.92.57-64 issn 2540-9328 (online) the role of gene therapy in the treatments of type 1 diabetes mellitus: a review harem othman smail department of biology, faculty of science and health, koya university koya koy45, kurdistan region f. r. iraq corresponding author harem.othman@koyauniversity.org manuscript received: 22 july, 2020. revision accepted: 06 november, 2020. published: 10 november, 2020. abstract the main aims of this review were to understand the roles of gene therapy in the treatment and prevention of type 1 diabetes mellitus and i will discuss a brief history, approaches, vector types with the future of diabetes following clinical use. type 1 diabetes mellitus is a metabolic condition that is identified by insufficient insulin development due to pancreatic damage to beta cells. control, long life, and diagnosis of these metabolic disorders have become vital sources for many scientists and researchers. after 2000, the latest approaches to molecular medicine were introduced as one of the possible therapeutic options for diabetes type 1 diagnosis. many genes have been reported as a clinical trial so that damaged genes can be treated and three main approaches shown about 50 years ago are islet transplantation, β cell regeneration, and insulin gene therapy to cure and prevent diabetes type. treating diabetes through gene therapy can promise children and adolescents, but more clinical applications are needed to recognize it as a permanent route. keywords: type 1 diabetes mellitus; gene therapy; β cells; virus vectors and islet. introduction diabetes is one of the metabolic disorders characterized by hyperglycemia due to insulin secretion deficits, insulin action, or both (smail et al., 2019). type 1 diabetes mellitus is caused by extreme insulin deficiency secondary to pancreatic beta cell autoimmune damage (kolodka et al., 1995). the middle east and north africa region have the highest average prevalence of diabetes in adults (10.9%), while the western pacific region has the largest number of adults diagnosed with diabetes and the highest prevalence of diabetes in countries (37.5%). in terms of diagnostic criteria, etiology, and genetics, various classes of diabetes mellitus, namely type 1, type 2, gestational diabetes, and other types of diabetes mellitus are compared (kharroubi et al., 2015). gene therapy is a new form of molecular medicine that will have a significant impact on human health in the next century (verma et al., 2000). visionary scientists had hypothesized almost five decades ago that genetic modification by exogenous dna could be an effective treatment for inherited human diseases. this strategy of “gene therapy” offered the theoretical advantage that one single treatment would achieve a lasting and possibly curative clinical benefit (dunbar et al., 2018). recent gene therapy clinical trials have shown considerable therapeutic benefits and an excellent safety record. they show the long-sought-after potential of gene therapy to provide 'cures' after certain potentially terminal or seriously disabled disorders (naldini, 2015). to date, approximately 2600 clinical trials on gene therapy have been completed, are underway, or licensed worldwide (ginn et al., 2018). gene therapy is intended to inject genetic material into patient cells to compensate for damaged genes or to include therapeutic transgenes. gene therapy has advanced from the initial human gene transfer trials to approved clinical therapies over the past three decades (wang et al., 2020). modern therapeutic strategies seek to restore the endogenous insulin production rather than traditional insulin injection treatment (memon and abdelalim 2020). differentiation of insulin-producing pancreatic β cells from induced pluripotent stem cells (ipscs) originating from diabetes patients aims to provide autologous cells for cell replacement therapy for diabetes. present methods, however, generate patient ipsc-derived β (sc-β) cells with low in vitro and in vivo function (maxwell et al., 2020). in several ways, cell replacement therapy for type 1 diabetes (t1d) is a perfect first drug model to follow in the emerging field of regenerative medicine (brandon et al., 2020). history of gene therapy the ideas of gene therapy emerged in the 1960s and early 1970s, while in 1968 rogers & pfuderer demonstrated a proof of concept for virus-mediated gene transfer (wirth et al., 2013), development of genetically https://doi.org/10.14421/biomedich.2020.92.57-64 58 biology, medicine, & natural product chemistry 9 (2), 2020: 57-64 marked cell lines and explanation of the polyoma and sv40 papovavirus mechanisms of cell transformation (friedmann, 1992). modern transfection methods were combined with cultured cell selection systems and recombinant dna technology in the late 1970s.the possibility of successful gene transfer into mammalian cells for gene therapy purposes was generally recognized with the creation of retroviral vectors in the early 1980s (wolff et al., 1994). however, clinical efficacy in a small-scale clinical trial curing an otherwise fatal immunodeficiency disorder in children could not be demonstrated until the start of the new century (escors et al., 2010). pancreatic transplantation, first performed in 1966, exists in the new millennium as a radical treatment for particularly intractable type i diabetes with advanced complications. gene therapy with molecules like leptin and insulin, still in experimental mode, may one day become a reality (lakhtakia, 2013). clinically active islet-free implants began in 1989 which require immunosuppression for long life. for more than 30 years, several encapsulated islet approaches have been ongoing without the definition of a clinically relevant product (scharp et al., 2014). approaches for gene therapy in type 1 of diabetes a cure for type 1 (insulin-dependent) diabetes could be found to replace β cells in the generation of surrogate insulin-producing cells (samson and chan, 2006). several different approaches have long been used to seek a cure for diabetes, including islet transplantation, β cell regeneration, and insulin gene therapy (lee et al., 2000). islet transplantation the idea of t1d cell replacement therapy remained dormant for 80 years until 1972 when ballinger and lacy reversed chemical diabetes by islet transplantation in rats (shapiro, 2012). following the success of steroidfree immunosuppression transplantation of fresh human islets (froud et al., 2005). the inexorable decline in insulin independence following islet transplantation alone (ita) has raised concerns regarding its clinical utility (bellin et al., 2012). allotransplantation of islets into patients with autoimmune type 1 diabetes is a reexposure to autoantigen (bosi et al., 2001). although immunosuppression protocols have improved islet transplant outcomes, more research is required to maximize islet availability for transplantation and improve the viability of islets once transplanted (zarinsefat and stock, 2020). islet transplantation in patients with type 1 diabetes can reduce or eliminate the insulin requirement. exenatide is a long-acting glucagon-like peptide-1 (glp-1) analog that increases glucose-induced insulin secretion and may increase the mass of β cells (al et al., 2007). glucagon responses of islet transplant recipients to hypoglycemia were significantly lower than those observed in control subjects (incremental glucagon [mean ± se]: −12 ± 12 vs. 64 ± 22 pg/ml, respectively; p < 0,05), and not significantly different from those of non-transplant type 1 diabetic subjects (−17 ± 10 pg/ml) (paty et al., 2002). a glucose-potentiated arginine test, conducted only in insulin-independent transplant subjects (n = 5), showed significant impairments in the glucose-potential slope (p < 0.05) and the maximum response to arginine (armax; p < 0.05), a measure of βcell secretory capacity. because armax provides an estimate of the functional β-cell mass, these results suggest that the functional defects observed after islet transplantation may account for a low engrafted β-cell mass (rickels et al., 2005). moreover, approximately 70 percent of transplanted type 1 diabetic patients achieved insulin independence (matsumoto, 2010). by contrast, a clinical review at the bmj in 2001 anticipated that transplantation of langerhans islets would be the treatment of choice for most type 1 patients diabetes by 2010. islet transplantation is currently an option for a specific group of patients with type 1 diabetes only — those with severe glycaemic lability, recurrent hypoglycemia, and unawareness of hypoglycemia (de et al., 2011). iak was associated with better glucose control and a slower decrease in egfr than standard insulin therapy in patients with type 1 diabetes and a functioning kidney transplant (maanaoui et al., 2020). in this first doubleblind randomized trial, the islet transplant evidence obtained with reparixin does not support the role of cxcr1/2 inhibition in preventing inflammationmediated damage to the islet (maffi et al., 2020). regeneration of β cells one goal of regenerative medicine is the instructive incorporation of adult cells into other forms of cells for tissue repair and regeneration (zhou et al., 2008). two major efforts are underway to address the β-cell diabetes deficit: one would produce ex vivo β-cells that are appropriate for transplantation, and the other would promote β-cell regeneration in the pancreas (bonner et al., 2005). the regeneration of pancreatic β cells which produce insulin is a key therapeutic strategy for diabetes (ackeifi et al., 2020). through adulthood, β cell mass increasing the ability to suit peripheral requirements. insufficient insulin secretion by β cells leads to mellitus diabetes (teta et al., 2017). pancreatic insulin-producing β-cells have a long lifespan, so they replicate very little during a lifetime in healthy conditions (thorel et al., 2010). regeneration of pancreatic β-cell mass following either toxinor autoimmune-mediated destruction in young rodents is probable, but the degree of recovery declines with age and is incomplete in adult life. similarly, in children and adolescents with type 1 diabetes, there is histological evidence of islet cell smail – the role of gene therapy in the treatments of … 59 neogenesis and regenerative response (thyssen et al., 2006). single-cell rna sequencing (scrna-seq) of islets identified β-cell dedifferentiation and dysfunction markers and pathways (sachs et al., 2020). three regeneration modes were established according to the various cellular regeneration origins. in the first mode, the undifferentiated progenitor cells proliferate and differentiate as a self-renewing source in response to injury to repair the lost cell population. the second mode, de-differentiate, and then proliferate the highly defined remaining cells (wang et al., 2020). regeneration of lost or dysfunctional islet β cells in vivo can fulfill the promise of improved treatment for patients with diabetes. numerous mitogenic factors have been attempted to achieve this, including gammaaminobutyric acid (gaba) (yi et al., 2020). results indicate that curcumin has anti-diabetic properties because of its superior immunomodulatory behavior on t-associated cytokines and immunosuppressive activity on pro-inflammatory cytokines may enhance damage to pancreatic β cells (badr et al., 2020). proliferation is the primary mechanism of b-cell expansion during the early postnatal period to produce sufficient b-cell mass in an organism. however, the proliferation of b cells declines rapidly in early life, and the rate of division of b cells in adults is very low (guney et al., 2020). however, there is still a great need for new treatments, as tid remains an incurable illness. natural products, primarily phytochemicals, are a tremendous source of drug lead discovery for diabetes (apaya et al., 2020). nevertheless, conventional cell-based research approaches have weaknesses in identifying the precise processes of β-cell differentiation and transdifferentiation, as well as the related regulatory mechanisms (yu and xu, 2020). data cumulated over the past few years suggest that increased β-cell dedifferentiation plays a crucial role in diabetes progression, shedding new light on potential targets for β-cell replacement therapy (zhang et al., 2020). based on a new understanding of structural and functional dynamics of the bone marrow, a conditioning-free bone marrow transplantation (bmt) with reduced adverse effects opens the possibility of evaluating β cell regeneration and euglycemic restoration by inducing allogeneic chimerism in t1dm patients, as shown in a mouse model (black and zorina, 2020). a new study now shows that a pharmacological combination of insulin and glp-1–estrogen conjugate not only decreases the daily requirements for insulin but also improves β-cell function (de et al., 2020). lif receptor expression is limited to a subset of transcriptionally isolated human β cells with the enhanced proliferative ability (rosado et al., 2020). residual β-cells play a major role in the design of clinical trials: they may not only respond to combination therapies that involve metabolic function stimulants but are also the possible source of new β-cells (akirav et al., 2018). insulin gene therapy insulin treatment options for type 1 and type 2 diabetics have increased since the launch of the insulin analogs in 1996. insulin therapies are now able to mimic the physiological insulin secretion more closely and thus achieve better glycemic control in diabetes patients (donner et al., 2019). gene therapy is one of the diabetes mellitus treatment methods (ramezani et al., 2019). the two key issues are efficacy and safety in developing insulin-replacement gene therapy as a possible treatment for type 1 diabetes. in addition to using a safe and efficient gene transfer vector, the physiological regulation of the expression of the insulin gene is important (chen et al., 2011). insulin gene therapy refers to the targeted expression of insulin in non-β cells, the primary therapeutic target being hepatocytes (handorf et al., 2015). the human insulin gene wrapped in chitosan nanoparticles can be successfully transfected through the gastrointestinal tract into rats (niu et al., 2008). differentiation between adsc and tissue-specific promoters will improve the therapeutic gene expression. the use of microcarriers can facilitate posttransplantation cell survival and hold potential for longterm cell therapy (fang et al., 2019). insulin-producing cell replacement through transplantation shows significant promise but is limited in application due to supply constraints (cadaver-based) and chronic immunosuppression. important progress has been made over the past decade in tackling these obstacles to broad cell therapy adoption (latres et al., 2019). the possibility that the risk variants of bach2 and clec16a could contribute to the development of insulin-triggered type 1 diabetes cannot be excluded in addition to the type 1 diabetes high-risk human leukocyte antigen class ii and the class i allele of the insulin gene variable number tandem repeat genotype (onuma et al., 2019). therapeutic studies assess the efficacy of antigenspecific and antigen-nonspecific immune therapies, which also include reconstruction of the damaged betacell mass via islet transplantation, neogenesis, and regeneration, and their combinations (van et al., 2011). cells that express insulin and have molecular characteristics that closely resemble bona fide insulinsecreting cells have been produced by the most effective protocols to date; however, these cells are often nonresponsive to glucose, a feature that should be addressed in future protocols (aguayo et al., 2010). such beneficial effects of leptin are not underpinned by pancreatic β-cell regeneration, since circulating insulin levels were undetectable at basal and glucose overload levels (fujikawa et al., 2010). however, contrary to insulin monotherapy, leptin reduces both lipogenic and cholesterologenic transcription factors and enzymes, and reduces lipids from plasma and tissue (wang et al., 2010). 60 biology, medicine, & natural product chemistry 9 (2), 2020: 57-64 prevent of type 1 of diabetes by gene therapy since no existing targeted immunotherapies are currently unable to replace the normal insulin delivery, it is important to have a deep understanding of t1d pathophysiology to prevent t1d development (kaur, 2020). the study shows that aat gene therapy decreases cell-mediated autoimmunity, changes the repertoire of t-cell receptors and effectively prevents type 1 diabetes in the nod mouse model. these findings strongly suggest that aat gene therapy mediated by raav1 may be useful as a novel approach to preventing type 1 diabetes (lu et al., 2006). the data indicate that expression of defensive mhc class ii alleles in bone marrow-derived cells provides robust self-tolerance to islet autoantigens and is adequate to prevent recurrence of autoimmune diabetes following islet transplantation (tian et al., 2007). dual activity of heparanase inhibitors / hs replacers as a novel therapeutic class to prevent t1d progression and potentially to mitigate secondary vascular disease developed with long-term t1d (simeonovic et al., 2020). an independent study showed that the optimal rise of gcs in beta-cells was a compensatory mechanism that prevented high-fat diet-induced betacell failure (cobo et al., 2020). the role of combination therapy in the prevention of diabetic cardiomyopathy, without forgetting the major contribution of insulin, the preferred medication for the treatment of t1dm (cieluch et al., 2020). in several chronic diseases, including t1d, a large number of proinflammatory ligands that can signal via rage have been implicated. it is therefore unbelievable that rage has become a possible therapeutic target for disease treatment and prevention (le et al., 2020). as an antigen-specific monotherapy, adi-100 is highly e cacious in reversing elevated hyperglycemia to prevent diabetes, where a promising feature of immune tolerance is the increased apoptosis-inducing bax content (alleva et al. ,2020). it has been shown that the formulated autoantigenic islet peptides (gad65206-220, gad65536-550, insulin b923, and c17-a1) used as a tolerogenic vaccine prevented t1d from developing in prediabetic nod mice (zhou et al., 2020). the coxsackievirus b association with the onset of t1dm has been observed. accurate trigger detection will contribute to the creation of suitable preventive steps. it can become a foundation for advanced clinical studies to prevent t1dm (desai et al., 2020). recognizing that t1d is primarily an autoimmune betacell disorder that only in its final stage progresses to a metabolic syndrome will expand therapeutic options for earlier intervention and improve the prospects for prevention (desai n et al., 2020). gene expression with immuno-regulatory capacity may potentially decrease allograft rejection. recent studies have shown that viral interleukin (il)-10 can decrease immune response during allotransplantation and is one of the most promising methods for preventing rejection (jeong et al., 2020). vectors for gene therapy in diabetic virus vectors were used as gene transfer vehicles for various applications in preclinical and clinical gene therapy and with the approval of glybera (alipogene tiparvovec) as the first gene therapy product as standard medical treatment (goins et al., 2020). the use of viruses as carriers of genetic products requires a detailed and thorough understanding of viral vectors from viral preparation to clinical use (afzal et al., 2020). adenoviral vectors as one of the most common groups used in gene therapy have a high human capacity. they 're not actually integrated into the host genome (arjmand et al., 2020). in a cerebellar granule cell culture (cgc), treatment with gad cdna-containing hsv vectors increases gad65 and gad67 expression and gaba release evoked by stimulation (kanao et al., 2020). the definition of ad, aav and lv (shirley et al., 2020) is given on the other hand to competent (rc) viral vectors (de et al., 2020). the advantage of adenoviral vectors over retroviral vectors is that they can transduce both dividing and non-dividing cells and can be prepared in high titers. adenoviruses can infect insulin-secreting cells, and it has been shown that they can transduce rodent islets (borkar et al., 2020). co-precipitation of calcium phosphate is a simple and cheap method for the genetically modified pancreatic cells being non-viral vectors (wong et al., 2010). nearly all of the non-viral vectors used to date are plasmids of expression that were engineered for high expression when transmitted to striated muscle or other cells (prud et al., 2007). non-viral hvj liposomemediated human hgf gene transfer has potential for safe and successful diabetic sensorimotor neuropathy treatment (kato et al., 2005). future gene therapy in diabetes: in the future, therapeutic strategies should be personalized, given the high variance in genetics and islet autoimmunity among patients (liu et al., 2020). the recent increase in global stem cell research funding is largely based on the promise to translate scientific understanding of stem cells into regenerative medicine (wainwright et al., 2006). as the approaches to immunotherapy have remained unsuccessful, transplantation of donor-derived pancreas or islets is the only cure for t1d (aghazadeh et al., 2017). the urgent need for a much anticipated insulin-secreting substitute for β-cells led researchers to focus on stem cells (scs) to produce insulin-secreting β-cells. sc-based methods have opened up the new horizons to treat t1dm for being more precise and focused therapeutic approaches (farooq et al., 2019). replacing pancreatic islets with corpse-derived islets has proven to be a successful functional cure for some t1d patients which allows smail – the role of gene therapy in the treatments of … 61 them to be independent of exogenous insulin (sluch et al., 2019). there was an explosion of interest in developing methods for transplantation to replace the islets lost during natural diabetes development (emerich, 2002). clinically appropriate and healthy, the procedure is associated with a low risk of adverse effects (ahrén, 2011). clinical trials to improve the engraving, the availability of insulin-producing cell sources and alternative transplant sites are currently under investigation to expand treatment (bruni et al., 2014). glucagon-like peptide 1(glp-1) analogs and alternative insulin pathways, particularly oral (entericgastrointestinal, inhaled) pathways, are now most promising and attractive (takei et al., 2004). conclusions i have come to the following conclusions from the review: the gene therapy production and uses have increased significantly over periodic time. gene therapy has a reduced role in the secondary complication of diabetes mellitus, and can be a strong option for diabetic patients. conflict of interest: the author declares that there are no conflicts of interest concerning the publication of this article. references ackeifi, c., wang, p., karakose, e., fox, j.e.m., gonzález, b.j., liu, h., wilson, j., swartz, e., berrouet, c., li, y. and kumar, k., 2020. glp-1 receptor agonists synergize with dyrk1a inhibitors to potentiate functional human β cell regeneration. science translational medicine, 12(530). afzal, s., fronza, r. and schmidt, m., 2020. vseq-toolkit: comprehensive computational analysis of viral vectors in gene therapy. molecular therapy-methods & clinical development. aghazadeh, y. and nostro, m.c., 2017. cell therapy for type 1 diabetes: current and future strategies. current diabetes reports, 17(6), p.37. aguayo-mazzucato, c. and bonner-weir, s., 2010. stem cell 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(024) 8505680, indonesia. corresponding author* adhityanaufal7@unwahas.ac.id manuscript received: 29 july, 2025. revision accepted: 26 october, 2025. published: 28 october, 2025. abstract this study aimed to evaluate the antibacterial potential of bacteriocin produced by lactic acid bacteria against propionibacterium acnes through an integrative approach involving molecular docking, in vitro assays, and genetic identification using 16s rrna sequencing. the target protein pdb 7lbu, representing the cell wall of p. acnes, was docked with small bacteriocin obtained from pubchem. the best binding affinity was recorded at –7.5 kcal/mol, indicating a stable interaction, supported by cavity analysis and ligand–protein interaction mapping using pymol. the results of the in vitro study, as determined by antibacterial activity testing against p. acnes, showed an average inhibition zone of 20.6 mm. in addition, genetic identification through blast confirmed the isolate as pediococcus pentosaceus strain dsm 20336 with a similarity of 99.64%. these findings indicate that the bacteriocin derived from p. pentosaceus has promising potential as a natural agent in the development of alternative therapies for acne. keywords: 16s rrna; antibacterial; pediococcus pentosaceus; propionibacterium acnes; small bacteriocin. introduction indonesia has a wide variety of fermented foods, ranging from sabang to merauke. the seasonings used in fermented foods are often passed down through generations. there are several types of fermented foods, such as bekasam, tempoyak, dadih, and others (masdarini, 2011). dadih is a fermented food originating from west sumatra, made from buffalo milk. the process of making dadih begins with pouring buffalo milk into bamboo containers, then letting it sit for approximately 24–48 hours at room temperature. this process leads to fermentation, a process that involves beneficial bacteria, specifically lactic acid bacteria. these bacteria have many benefits, are very safe for consumption, non-pathogenic, and allow for a long product shelf life. lactic acid bacteria are known as probiotic bacteria (yuliana & minda, 2022). lactic acid bacteria are capable of producing lactic acid as a result of the fermentation of sugars or carbohydrates. lactic acid bacteria are characterized as gram-positive, non-spore-forming, and catalase-negative in catalase testing. the addition or presence of these bacteria does not alter the quality of food. the classification of lactic acid bacteria includes aerococcus, carnobacterium, enterococcus, lactobacillus, lactococcus, leuconostoc, pediococcus, streptococcus, tetragenococcus, and vagococcus. these bacteria are found in processed food products derived from meat, fish, and milk, as well as in vegetables and fruits. they are also found in the organs of living organisms, such as the genital tract, intestinal tract, and respiratory tract of humans and animals (pribadhi et al., 2021). acne vulgaris is a skin disease that causes inflammation in the pilosebaceous unit. this condition can become chronic, but it can also resolve on its own. cutibacterium acnes, formerly known as propionibacterium acnes, is the causative agent of acne vulgaris. adolescents commonly experience this disease; however, individuals of all ages can also be affected. acne vulgaris, commonly referred to as acne, is caused by the accumulation of sebum, which eventually clogs the skin pores, triggering bacterial activity and inflammation in the skin (sifatullah & zulkarnain, 2021). p. acnes is characterized as gram-positive and belongs to the corynebacteria group. this bacterium is part of the normal flora of the human body. the infection mechanism of this bacterium involves breaking down triglycerides originating from the sebaceous glands into free fatty acids, which leads to an increase in p. acnes and subsequently triggers inflammation (clatici et al., 2015). the results of a study conducted by asbullah et https://doi.org/10.14421/biomedich.2025.142.943-949 944 biology, medicine, & natural product chemistry 14 (2), 2025: 943-949 al. in 2018 showed that 109 out of 122 students (89.3%) at sman 1 pelangiran, indragiri hilir regency, suffered from acne. a total of 47 students aged 17 years were affected by this condition (asbullah et al., 2021). acne treatment often involves the use of antibiotics, including tetracycline, erythromycin, doxycycline, and clindamycin. other treatments that can be used include benzoyl peroxide, azelaic acid, and retinoids. these medications have several side effects during use. the use of antibiotics can lead to resistance and relapse. research conducted in france revealed that 75.1% of patients with acne infected with p. acnes were resistant to erythromycin, and 9.5% were resistant to tetracycline. in other countries, 82% of acne patients were resistant to azithromycin. at hasan sadikin hospital in bandung, 12.9% of acne cases were resistant to tetracycline, 45.2% were resistant to erythromycin, and 61.3% were resistant to clindamycin (pariury et al., 2021). therefore, alternative treatments, such as the use of lactic acid bacteria, are needed to counteract p. acnes. in silico studies are also necessary before conducting in vitro tests. the purpose of conducting in silico testing is to demonstrate the inhibitory potential of the small bacteriocin produced by lactic acid bacteria against the cell wall of p. acnes. therefore, further research is needed to explore the potential of lactic acid bacteria in counteracting pathogenic bacteria that cause acne. the lactic acid bacteria used in this study, derived from the fermented food cincalok, namely weissella confusa, have been shown to inhibit the growth of acne-causing bacteria (pribadhi et al., 2023) materials and methods materials and equipment this study used materials including lactic acid bacteria isolates, propionibacterium acnes, mrsa medium, caco3, 70% alcohol, bhi and mhi media, blank disks, tetracycline antibiotic, immersion oil, and tissue. the equipment used in this study included test tubes, test tube racks, micropipettes and tips, measuring cylinders, erlenmeyer flasks, inoculating loops, petri dishes, autoclave, microwave, bunsen burner, incubator, analytical balance, microscope slides and cover slips, matches, wrapping materials, refrigerator, centrifuge, laptop, megax software, http://clab.labshare.cn/cb-dock/, software, pymol software, and microscope. procedures protein and ligand preparation the protein structure used in this study was obtained from rcsb.org with pdb id 7lbu, which represents the cell wall of propionibacterium acnes. the small bacteriocin ligand was obtained in sdf format from pubchem. the protein and ligand were then prepared using tools provided by cb-dock for appropriate format conversion and energy optimization. figure 1. structure of p. acnes surface sialidase. figure 2. small bacteriocin. http://clab.labshare.cn/cb-dock/ pribadhi et al. – bacteriocin activity against p. acnes 945 docking procedure the docking process was carried out using a blind docking approach through the tool available at http://clab.labshare.cn/cb-dock/. the blind docking approach allows exploration of the entire protein surface to identify potential binding sites without the need to specify a predefined binding site. ▪ grid box center: xyz (-16, -7, 3) ▪ grid box size: 27 x 27 x 27 å ▪ method: autodock vina via cb-dock docking validation before performing docking of the small bacteriocin, the docking method was tested through redocking validation to ensure the accuracy of binding site prediction. this process confirmed that the docking protocol was capable of predicting the binding site with adequate accuracy. mrsa medium preparation this study used 6.5 g of mrsa medium and 0.75 g of caco3 per 100 ml of distilled water. preparation all equipment and media used in this study were sterilized using an autoclave at 121°c for 20 minutes to avoid contamination from undesired bacteria. isolation of lactic acid bacteria a total of 1 g of dadih sample was taken and transferred into a container containing 9 ml of distilled water (10-1 dilution), followed by serial dilutions from 10-2 to 10-8. the media containing bacterial colonies were incubated in an incubator at 37°c for 48 hours. the growing bacterial colonies were transferred using a round-loop inoculating needle and streaked onto fresh media. colony purification was continuously carried out until a single bacterial colony was obtained. the medium used was mrsa supplemented with 0.75% calcium carbonate (caco3). characterization of lactic acid bacteria isolates the lactic acid bacteria isolates that had become pure cultures were characterized macroscopically by observing the shape, color, edge, and elevation of the colonies, as well as by conducting a catalase test. meanwhile, microscopic observation involved examining the cell morphology and performing gram staining. ▪ catalase test the catalase test was performed by dropping 3% h2o2 onto a glass slide, and the bacteria to be tested were placed onto the solution. if no bubbles formed on the isolate, it indicated a negative reaction. ▪ gram staining gram staining was also performed, starting with the placement of the bacterial sample on a glass slide, followed by fixation. gram stain a was applied and left for 1 minute. next, gram stain b was added and left for 1 minute. gram stain c was then applied for 30 seconds, followed by gram stain d, which was left for 2 minutes. the bacterial specimen was observed under a microscope at 1000× magnification using immersion oil. gram-positive bacteria appear purple, while gram-negative bacteria appear red. preparation of lactic acid bacteria starter the rejuvenated isolate was cultured in mrsa medium and then transferred to bhi medium for 24 hours at 37°c. the liquid culture was subsequently centrifuged at a speed of 10,000 rpm. the resulting supernatant was filtered using a 0.22 μm bacterial filter into a sterile tube (sari et al., 2018). antimicrobial test using the agar diffusion method the test bacterium, propionibacterium acnes, from bhi medium was spread onto mha medium. then, 20 microliters of the probiotic bacterial supernatant was dropped onto a blank paper disk. the tetracycline antibiotic paper disk (positive control), the probiotic bacterial supernatant paper disk, and the blank disk (negative control) were then placed systematically on the mha medium containing the test microorganism and incubated at 37°c for 24 hours. the diameter of the inhibition zone was measured to determine the antibacterial activity of the test substance (kursia et al., 2021). identification of lactic acid bacteria using 16s rrna the lactic acid bacteria with the highest antibacterial activity against propionibacterium acnes were sent to genetika science for molecular sequencing using 16s rrna analysis. sequencing result interpretation the nucleotide sequence of the lactic acid bacteria obtained from genetica science was first processed using bioedit software and then analyzed using the basic local alignment search tools (blast) on the ncbi website. this program identified regions of local similarity between the examined sequence and the database-stored sequence information to calculate the statistical significance of the match. the result from ncbi indicated the strain/species of the lactic acid bacteria. the identified strain was then used to construct a phylogenetic tree to determine its evolutionary relationship with other bacterial species. data analysis data dari penelitian ini akan dianalisis secara deskriptif dan disajikan dalam bentuk tabel dan gambar. results and discussion binding affinity the docking process generated several binding poses ranked based on the vina score. the results obtained are presented in table 1: http://clab.labshare.cn/cb-dock/ 946 biology, medicine, & natural product chemistry 14 (2), 2025: 943-949 table 1. binding affinity. pose vina score (kcal/mol) cavity size (å³) cavity center (xyz) grid box size (xyz) pose 1 -7.5 432 (-16,-7.3) (27, 27, 27) pose 2 -6.6 290 (-21, 12,17) (27, 27, 27) pose 3 -5.3 424 (-12, 15, 17) (27, 27, 27) pose 4 -5.0 285 (-6, 16, 2) (27, 27, 27) pose 5 -4.7 96 (-28, -19, 14) (27, 27, 27) binding site analysis figure 3. docking complex of 7lbu with small bacteriocin -7.5. isolation of lactic acid bacteria figure 4. clear zone formed by lactic acid bacteria isolated from dadih. characterization of lactic acid bacteria isolates the results of the characterization of lactic acid bacteria isolates from dadih are presented in table 2. table 2. characterization of lactic acid bacteria isolates isolate. characterization lactic acid bacteria isolates dih-1 colony shape circular colony color cream elevation convex margin smooth cell morphology coccus gram staining (+) catalase test (–) antimicrobial activity table 3. antimicrobial activity of bacteriocin from lactic acid bacteria against p. acnes. treatment no inhibition zone (mm) control (-) p. acnes control (+) 1 0 21 30 2 0 21 30 3 0 20 30 average 0 20.6 30 16s rrna sequencing results table 4. homologous sequences from alignment with 16s rrna of lactic acid bacteria isolate. deskripsi max score total score query cover e. value per. ident pediococcus pentosaceus strain dsm 20336 2560 2560 100% 0.0 99.64% pediococcus stilesii strain fair-e 180 2447 2447 100% 0.0 98.15% pediococcus acidilactici dsm 20284 2442 2422 100% 0.0 97.86% pediococcus argentinicus crl 776 2383 2383 97% 0.0 98.17% pediococcus claussenii atcc baa 344 2368 2344 97% 0.0 97.82% pediococcus claussenii p06 2344 2344 99% 0.0 97.06% pediococcus acidilactici ngri 0510q 2333 2333 100% 0.0 96.74% pediococcus parvulus nbrc 100673 2265 2265 99% 0.0 95.93% pribadhi et al. – bacteriocin activity against p. acnes 947 discussion in table 1, a conformation with a vina score of –7.5 was identified within a relatively large cavity with a volume of 432 å3, allowing the ligand to interact with various key residues surrounding the cavity. further analysis of ligand–protein interactions using visualization software such as pymol revealed the presence of hydrogen bonds and hydrophobic interactions, which contributed to the binding stability. these results indicate that small bacteriocin has potential as an inhibitor of the p. acnes cell wall, which is relevant to the development of bacterium-based therapies produced by pediococcus. these findings provide a foundation for further experiments exploring the inhibitory mechanism through in vitro studies (yang, 2022) during the isolation process of lactic acid bacteria, only one pure isolate was obtained from the fermented food dadih. in the purification process using the mrsa + caco3 medium, lactic acid bacteria on the medium formed a clear zone. according to putri et al. (2018), the clear zone formed on mrsa + caco3 medium is the result of acid secretion into the medium. the lactic acid bacteria obtained showed colony characteristics that included a circular shape, whitishyellow color, convex elevation, and smooth margins. gram staining revealed a round cell morphology and a gram-positive (purple) reaction. lactic acid bacteria typically have round or rod-shaped cell morphology, and under gram staining, they appear purple (pribadhi et al., 2021). bacteria can be distinguished based on the composition of their cell walls, which can be identified through the gram staining method. gram-positive bacteria, which appear purple, contain approximately 90% peptidoglycan in their cell walls. the purple color results from the formation of a crystal violet–iodine complex. bakteri gram negatif atau berwarna merah memiliki dinding sel dengan kandungan peptidoglikan 520%. the red color in bacteria results from the addition of 95% alcohol, which removes the purple stain and leaves the cells colorless. the red color appears following the addition of safranin (madigan et al., 2011). the catalase test showed a negative result, indicating that the bacteria were unable to break down hydrogen peroxide into water and oxygen. the catalase test is used to determine the presence of the catalase enzyme in bacteria (putri et al., 2014). the probiotic bacteria isolated from dadih that were used to inhibit acne-causing bacteria, most commonly p. acnes, had not yet been identified at the species level. based on research conducted by pribadhi, the bacterium w. confusa was considered capable of inhibiting p. acnes (pribadhi et al., 2023). the results of the antimicrobial test using the agar diffusion method showed that this probiotic bacterium possesses antimicrobial compounds, as evidenced by its ability to inhibit acne-causing pathogenic bacteria. the presence of a clear zone around the paper disk confirmed this. the antimicrobial test was repeated three times for each p. acnes sample. the positive control in this test used the antibiotic tetracycline, while the negative control used a blank paper disk. the average result of the negative control for both acne-causing bacteria was 0 mm, due to the absence of antimicrobial compounds. the average result of the positive control against p. acnes was 30 mm. the average result of the probiotic antimicrobial test against p. acnes was 20.6 mm. a study conducted by kursia et al. (2021) reported that lactic acid bacteria can inhibit the growth of p. acnes, with an inhibition zone ranging from 8.2 to 11.1 mm. this indicates that weissella confusa has a greater ability to inhibit p. acnes and s. epidermidis. the antimicrobial compound commonly produced by probiotic bacteria is bacteriocin. this compound has a broad-spectrum activity in inhibiting the growth of various pathogenic bacteria. the presence of a clear zone observed on the antimicrobial test medium can be attributed to the bacteriocin's activity, which is capable of killing the pathogenic bacterial cells being tested. the sequencing of the ddh 1 dna amplification product aimed to obtain information on the nucleotide sequence of the pcr-amplified target dna with a specific length. the sequencing data obtained were aligned using the basic local alignment search tools (blast) on the ncbi website. this program identifies regions of local similarity between the analyzed sequence and the database-stored sequence information to calculate the statistical significance of the match. the blast results showed that isolate cin-1 had the highest similarity with pediococcus pentosaceus strain dsm 20336, with a similarity of 99.64% (table 2). according to drancourt et al. (2004), a similarity of 97– 99% in bacterial 16s rrna sequences indicates similarity at the genus level, while a similarity of >99% sequence gen is the criterion used for identification at the species level. the alignment results also showed an identical max score and total score, with ddh 1 scoring 2560. an e-value of 0.0 indicates a significant alignment. the phylogenetic relationship among species was analyzed by constructing a phylogenetic tree using the mega x program. the phylogenetic tree of isolate ddh-1 was analyzed using the neighbor-joining (nj) method. 948 biology, medicine, & natural product chemistry 14 (2), 2025: 943-949 figure 5. phylogenetic tree of p. pentosaceus strain dsm 20336. the phylogenetic tree based on the 16s rrna gene of isolate ddh 1 was constructed using neighbor-joining analysis and a bootstrap phylogeny test with 1000 replicates. the results showed that isolate ddh 1 had a bootstrap value of 100 with pediococcus pentosaceus strain dsm 20336. a bootstrap value of 100 indicates that in 1000 iterations, the same tree topology was formed 1000 times. according to dharmayanti (2011), bootstrap analysis is a method used to test how well the dataset supports the model, and the values are usually placed on the branches of the phylogenetic tree. bootstrap values are categorized as high (>85%), moderate (70–85%), weak (50–69%), or very weak (lestari et al., 2018). the phylogenetic tree with a scale bar of 0.02 indicates a genetic distance equivalent to two nucleotide changes per 100 base pairs. based on observations, the ddh-1 isolate exhibited a round cell morphology, was gram-positive upon gram staining, and showed a negative result in the catalase test. pediococcus pentosaceus belongs to the gram-positive, anaerobic firmicutes group. this bacterium is a coccus-shaped microbe characterized by being non-motile and nonspore-forming. it is classified as a “lactic acid bacterium” because the end product of its metabolism is lactic acid (lin et al., 2019). conclusions this study showed that the bacteriocin activity produced by lactic acid bacteria has potential as an antibacterial agent against propionibacterium acnes. the results of molecular docking showed a strong binding affinity with a score of –7.5 kcal/mol. at the same time, the in vitro antibacterial activity test yielded an average inhibition zone of approximately 20.6 mm. the analysis of the 16s rrna results identified the isolate as pediococcus pentosaceus strain dsm 20336 with a similarity level of 99.64%. this indicates that the bacterium has potential as an alternative treatment for acne. it also presents an opportunity for the development of probiotic products or bacteriocin-based formulations in the future. authors’ contributions: adhitya naufal pribadhi and penggalih mahardika herlambang designed the study, analyzed the data, and wrote the manuscript. ellyka carried out the laboratory work. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. pribadhi et al. – bacteriocin activity against p. acnes 949 funding: the authors were funded by the internal grant of universitas wahid hasyim under the competitive research grant scheme. references asbullah, a., wulandini, p., & febrianita, y. 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(2022). isolasi dan identifikasi molekuler bakteri asam laktat pada dadih dengan menggunakan gen 16s rrna. natural science: jurnal penelitian bidang ipa dan pendidikan ipa, 72–78. https://doi.org/10.1016/j.medmic.2019.100002 http://www.journal-medical.hangtuah.ac.id/ http://ejurnal.bppt.go.id/index.php/jbbi https://doi.org/10.31851/indobiosains.v5i1.9659 http://ejournal2.undip.ac.id/index.php/jbt http://journal.uin-alauddin.ac.id/index.php/psb http://journal.uin-alauddin.ac.id/index.php/psb https://doi.org/10.1093/nar/gkac394 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 205-213 | doi: 10.14421/biomedich.2023.121.205-213 issn 2540-9328 (online) bioactive compound in solanum torvum and its potential as functional food and drink: a review nina nurazizah purnomo putri*, rista anggriani, sukardi food technology department, faculty of agriculture and animal husbandry, university of muhammadiyah malang, jl. raya tlogomas no 246 malang 65144, tel. +6282399028714, malang, jawa timur, indonesia. corresponding author* ninaputri171@gmail.com abstract solanum torvum is a fruit used as food and has medical properties. this study aims to provide an updated understanding of solanum torvum’s health benefits as a functional food through a study literature review. the research utilizes electronic databases (pubmed, science direct, scopus, nature, clinical key, and springer) from july-october 2022. solanum torvum contains various bioactive components, vitamins, mineral, nutritions. it shows that solanum torvum has potential sources as functional food. however, studies about solanum torvum consumption as a functional food and the clinical trial of its health benefits in humans are still limited. further in vivo and in vitro studies are necessary to present the effect of solanum torvum consumption on health. keywords: solanum torvum; antioxidant; bioactive compound; nutrition; functional food and drink. introduction the body's immune system uses the involuntary primitive reflex of coughing to defend against foreign substances that invade the respiratory tract (sharma et al., 2020). to eliminate debris, excessive mucus, irritants, microbes, or other substances from the respiratory tract, one may cough either voluntarily or involuntarily. coughing is either voluntary or involuntary (chung and pavord, 2008). solanum torvum has various local names in indonesia, it has known as pokak (east java), cepoka (central java), takokak (west java), rimbang (sumatra and melayu), terong pipit, and many more (zuhud, 2003). it is also called turkey berry, devil’s fig, cherry eggplant, water nightshade, or wild eggplant (lim, 2013). in 1964, lawrence classified solanum torvum into taxonomical classification. kingdom : plantae division : spermatophyta sub division : angiospermae class : dicotyledonae order : solanales family : solanaceae genus : solanum species : solanum torvum swartz distribution of solanum torvum to growth could reach tropical and subtropical territories throughout the world (west and central africa, indian subcontinent, southeast asia, china, japan, australia) and many pacific islands; in the western hemisphere such as mexico, central america, greater and the lesser antilles, south america (brazil, ecuador, french guiana, guyana, venezuela), the united states to the caribbean (scher et al., 2015). the good habitat for species to grow in moist, fertile soil and will tolerate drought; wet thickets, dry brushy plains, woodland clearings, rocky hillsides; weeds in pastures, open native vegetation, swamps, roadsides, and waste places (scher et al., 2015). on the other hand, 1-3 kilogram fruits could be produced from a single tree of solanum torvum. this plant can produce fruit in 5-8 months from the start of the plantation (pratiwi, 2012). this plant is a nutritious food resource in africa that significantly contributes to massive societal nutrition because it has abundant minerals (okoto, 2015). in traditional folkloric medicine, solanum torvum has been used as an antipyretic, anti-rheumatic, anti-infectious, anti-inflammation, diuretic, and analgesic (burkill, 1966; burkill, 2000; jain & borthakur, 1986; stuart, 2022). in modern pharmacological studies, solanum torvum shows antioxidant activity (waghulde, 2011; gyamfi, 1999), antifungal activity (karuppusamy, 2009; beg et al., 2002), antibacterial activity (satish, 1999; lalitha, 2010), antiulcer activity (telesphore, 2008; antonio, 2004), antihypertensive and metabolic correction activity (nguelefack, 2008; jaiswal, 2012; lim, 1992), nephroprotective activity (waghulde, 2011; vaclavikova, 2008; mohan, 2010), cardioprotective activity (mohan, manuscript received: 11 october, 2022. revision accepted: 12 february, 2023. published: 15 february, 2023. https://doi.org/10.14421/biomedich.2023.121.205-213 206 biology, medicine, & natural product chemistry 12 (1), 2023: 205-213 2009), antidiabetic activity (gandhi, 2011; keisuke, 2010), analgesic and anti-inflammatory activity (ndebia, 2007; atta, 1997), immunomodulatory and erythropoietic activity (george, 2011; israf, 2004). not only used for medical purposes but solanum torvum also could be used as a food or drink product to get the essential benefit for human health (jaiswal, 2012). recently studies revealed that solanum torvum contains a very potent antioxidant and other useful phytochemistry components (helilusiatiningsi, 2021). the currently popular idea of creating food to get a healthy purpose is called functional food. functional foods are foods that provide health benefits beyond its nutritional value. this paper aims to provide an updated review of the phytochemicals compounds on solanum torvum by promoting its opportunity as a functional food to improve human health. parts and nutritional value of solanum torvum figure 1. solanum torvum. solanum torvum trees could grow up to 2 to 4 meters in height with an ideal environment to grow under full sunlight or partially in shades (pratiwi, 2012). its habitat is commonly found on roadsides and in disturbed soils, with expected growth as an individual plant. commonly found up to an altitude of 1600 m and suitable in moist soil conditions. young and immature fruits are consumed raw, cooked as a vegetable or used as an ingredient in curry sauce. in indonesia, solanum torvum is considered one of the best side dishes to go along with rice as a vegetable called lalapan (lim, 2013). each part of this fruit contains a lot of benefits. plant parts description and nutritional values fruits and seeds the fruit is a spherical round shape berry and has diameter about 1.0 to 1.15 cm. each fruit has about 300 to 400 flat, brown dark seeds inside. berry seeds of medium size (around 1,5 mm long) are hard to distinguish from one another. the fruit color may be affected by aging and the length of the lifetime spent (sirait, 2009). the fully ripped berry has yellow skin, and the youngberry has green with glossy color (scher et al., 2015). the flowers are bisexual. the plant flowers throughout the year. heavy rainfall discourages fruit growth and set (singapore government agency, 2022). extract of the fruit revealed the number of alkaloids, flavonoids, and certain fatty acids such as palmitic and oleic tested analysis by the gc-ms instrument (jaabir, 2015). extraction from seeds of solanum torvum showed a significant amount of phenolic and flavonoid contents which could be the source of natural antioxidants (waghulde, 2011). in pharmacology, alkaloids and solasodina compounds that contain in the fruit can be used as substrates for the production of the important steroid (perez-amador et al. 2007). in addition, the fruit also contains many solosin, chlorogenin, sisalagenone, tervogenin, protein, fat, calcium, phosphorus, iron, vitamins a, b1 and c (zuhud et al. 2003). leaves solanum torvum leaves have a large and green shape with a leaf length range of about 10-15 cm and a width of 810 cm. (jaiswal, 2012). the leaves are simple and ovate to elliptical in shape and lobed. the surface is pubescent. phytochemical analysis revealed the presence of 32 chemical constituents, mainly phenolic compounds, terpenoids, palmitic acid, palmitic acid ester, linoleic acid, linolenic alcohol, linolenic acid ester, and stearic acid found in solanum torvum leaves (naimon et al. 2015). the leaves can be dried, mixed, and added to hot water to putri et al. – bioactive compound in solanum torvum and its potential as … 207 plant parts description and nutritional values make a cold or cough medicine (yousaf et al., 2013). roots and stems solanum torvum is a prickly shrub or small tree, up to 5 m tall. it is cultivated in the tropics for its sharp-tasting, immature fruits (scher et al., 2015). in malaysia, the seeds are smoked to treat toothaches, while the roots are applied as a poultice to treat foot cracks. in china, the roots are believed to disperse blood that has leaked into the surrounding tissue and relieve pain. in india, extracts of the plant are used as an antidote to insect stings, and the fruit is eaten to relieve stomach aches). root powder of the fruit can be used as a remedy for leg fractures and headache relief and can be used as asthma treatment and liver therapy (yousaf et al., 2013). it is reported that solanum torvum roots contain secondary metabolites (sivapriya, 2011). these are potential sources of antimicrobials such as steroid alkaloids, saponins, flavonoids, glucosides, and tannins (bari et al., 2010). the phytochemical compounds and antioxidant of solanum torvum native to and cultivated in africa and the west indies is solanum torvum sw. (solanaceae), sometimes known as turkey berry (adjanohoun, 1996). due to their antioxidant characteristics, fruits and leaves are commonly employed in traditional cameroonian medicine. numerous substances found in solanum torvum have the potential to have pharmacological effects, including isoflavonoid sulfate, steroidal glycosides, chlorogenic and neochlorogenone, triacontane derivatives, 22--o-spirostanol oligoglycosides, 26-o-glucosidase (arthan, et. al., 2006). this plant has secondary metabolites in its leaves, stems, and roots, including steroidal alkaloids, saponins, flavonoids, glucosides, and tannins, which make it a robust and potentially antibacterial source (bari at al., 2010; sivapriya et al., 2011). pérez amador et al. (2017) measured the overall alkaloid content (0.12%), total glycoalkaloids (0.038%), and the glycosylated compounds produced from solasodine, namely solasonine (0.0043%) and solamargine (0.0028%). the amounts of polyphenolic substances (phenols, flavonoids, and tannins) were measured to be 160.30, 104.36, and 65.91 mg/g, respectively (kusirisin et al., 2009). in ethanolic extracts of immature and mature fruits, alkaloids, total saponins, total flavonoids, and vitamin c contents were compared (koomson et al. 2018), and only alkaloids were found to be significantly different (respectively, 16.94 2.3 mg/g in the immature fruits, and 6.32 0.12 mg/g in mature fruits). a large amount of reactive oxygen or as known as free radicals, that the human body produces and receive creates an urgency to develop and have large sources of antioxidant to tackle with. syntethycally antioxidants (for example, butylated hydroxyl anisole, gallic acid ester, butylated hydroxyl toluene, etc.) are associated with the side effect on human health (gao, 1999). synthetic antioxidants show a small level of solubility and a medium level of antioxidant activity (barlow, 1990) the substitution of synthetic antioxidants could be produced by utilizing natural resources from plants (waghulde, 2011). antioxidant activity from plants has a correlation with phenolic compounds (cook, 1996). flavonoids are a group of polyphenol compounds with known properties, including free radical scavenging, inhibition of hydrolytic and oxidative enzymes, and anti-inflammatory activity (frankel, 1995). wahluge et al, 2011 analyze the antioxidant activity, phenol, and flavonoid contents of seeds of punica granatum and solanum torvum. the result showed that the highest radical scavenging activity was observed with solanum torvum extract, with a significant linear correlation with phenolic and flavonoid contents. antioxidants are compounds that protect compounds or tissues from the damaging effects of oxygen or the effects of oxidation. according to kumalaningsih (2006), antioxidants are compounds with molecular structures that can donate electrons to free radical molecules and cut free radical chain reactions. in other words, antioxidants are able to work as inhibitors that try to inhibit oxidation by reacting with reactive free radicals to form relatively stable non-reactive free radicals. however, when combined with free radicals that can cause disease, antioxidants are defined as compounds that protect the body from the harmful effects of reactive oxygen free radicals (sofia, 2016). saponins is a glycoside group of compounds can be found in plants naturally and has unique properties, such as soap that can produce foam when shaken in the air. classified as a polar compound, this compound is soluble in air and is able to work as an antioxidant component to reduce free radicals in the body, so it can be used as a compound to prevent oxidative stress in liver cells (mardiningsih et al, 2010). tanins is classified as a complex polyphenolic compound, tannins can bind proteins well. the molecular weight is about 500-3000 da. these compounds can also be classified as hydrolysable tannins and condensed tannins. the molecular structure of hydrolyzable tannins is the hydroxyl of esterified phenolics such as gallic acid. tannins also have an anti-oxidant effect (ismarani, 2012). solanum torvum extraction using aquades as a solvent showed the content of alkaloids and tannins (alfarabi et al, 2018). phenolics has high antioxidant capacity due to ability to pass hydrogen on to extremely reactive radicals, therefore preventing further radical 208 biology, medicine, & natural product chemistry 12 (1), 2023: 205-213 formation (lapornik et al. 2005; xu et al. 2007). flavonoid are natural active phenolic compounds with a c5-c3-c6 chemical structure. this compound is often found in green plants and is useful as a source of antioxidants that can reduce free radicals or oxidation in the body. flavonoids are also inflammatory agents (rompas et al., 2012). flavonoids have a major role in analgesic activity (acharyya et al., 2018). elements to analyze amount benefit ref macronutrients content (g/100g) total carbohydrates 71.42 ± 0.52  combined with dietary fiber are generally recommended to prevent atherosclerosis, constipation and diseases in the intestine such as appendicitis and colon cancer (melila, et.al, 2021) (melila, et.al, 2021 protein 16.49 ± 0.47  can use in human food can contribute to the fight against proteinenergy malnutrition fat 7.71 ± 0.19  anti-hypertensive diets ash 4.65 ± 0.46  mineral components water content (%/100g) 79.28 ± 0.06 energy (kcal/100g) 421 ± 2,01 minerals (mg/100g) sodium (na) 155.2 ± 44.37  involved in the transmission of nerve impulses and in the water balance of the body (fao, 2004) (melila, et.al, 2021; kouadio et al., 2020) potasium (k) 1307 ± 433.7  involved in muscle contraction (fao, 2004) phosporus (p) 78.70 ± 20.10  necessary for the formation of the skeleton.  essential in the processes of energy storage in the body in the form of atp (kemi et al., 2006) calcium (ca) 38.45 ± 8.54  necessary for the formation of the skeleton.  involved in muscle contraction (fao, 2004)  can be used in diets aimed at skeletal formation (kemi et al., 2006)  balancing the body's ph by neutralizing excess acids (kemi et al., 2006) magnesium (mg) 38.45 ± 8.58  trace element that stimulates the immune system, protects against cell aging and maintains fatty tissue (kala, 2005)  the production and oxygenation of blood cells, digestion and blood circulation (abbaspour, et al., 2014)  involved in muscle contraction (fao, 2004)  essential cofactor of the enzymes of carbohydrate metabolism (fiorentini, 2021) iron (fe) 21.04 ± 0.68  the production and oxygenation of blood cells, digestion and blood circulation (abbaspour, et al., 2014) zinc (za) 14.65 ± 2.69  trace element that stimulates the immune system, protects against cell aging and maintains fatty tissue (kala, 2005)  the production and oxygenation of blood cells, digestion and blood circulation (abbaspour, et al., 2014) vitamin (mg/100g) vit c 2.44 ± 0.36  defense of the body against virus and bacterial infections,  the protection of the blood vessel wall,  the assimilation of iron and has an important antioxidant activity(carr, 2019) (melila, et.al, 2021; vit a 0,078  helpbody's natural defence against illness and infection (the immune system) work properly.  helping vision in dim light (duester, 2000) (okoto, 2015) phytochemical contents (mg/100g) polyphenols 356.70±0.02  anti-inflammatory, urinary antiseptic, anti-free radical, hepaticprotective, immune stimulant, anti-thrombotic and anti-carcinogenic effects (kouadio et al., 2020; vauzour, 2010; acho, 2014) phenol 160.30+3.00  oral analgesic, antioxidant, anti-inflammatory, anti-allergic, anticarcinogenic, antihypertensive, cardioprotective, anti-arthritic and antimicrobial activities (bhuyan, et al., 2017) tannins 685.83±0,01  anti-nutritional compounds that have deleterious effects on digestibility flavonoids 104.36+3.00  regulate cellular activity and fight off free radicals that cause oxidative stress on human body (kusirisin, 2009) (kusirisin, 2009) antioxidant activity 360.53+5.06  protection against damage caused by free radicals (kusirisin, 2009) putri et al. – bioactive compound in solanum torvum and its potential as … 209 value added as functional foods and drinks from solanum torvum the wide traditional medicinal use of s. torvum fruits is nowadays explained by the production of many phytochemicals by this species. alkaloids, flavonoids, tannins, saponins, and glycosides are present in s. torvum extract in sufficient concentrations for protecting the body against oxidative stresses. s. torvum can therefore be considered a promising natural source of phytochemicals displaying a range of medicinal properties, ranging from cardio protection & treatment of heart related diseases, nephro-protection, to analgesic, anti-inflammatory, anti-ulcer, and anti-microbial activities. solanum torvum is having a huge potential resource for developing food products. it’s containing a number of nutritional values, bioactive components, and other physicochemical properties. the fruit's nutritional content could be used as based ingredients or substitute materials to increase the nutrition in food (helilusiatiningsi, 2021). if the turkey berry fruit is used optimally by the community, then the fruit will be an alternative treatment that is cheap, easy, and relatively safe. it's easy because the plant grows and can be found around the community and people can cultivate it themselves at home. the cost of processing the fruit will be much cheaper than treatment with chemical drugs. in addition, traditional medicines that tend to have no side effects are relatively safe for people to use (pratiwi, 2012). flour (dried powder) the most common commercially used product, dried fruit powders are found to be an emerging functional ingredient. it has a long shelf life because of the water content and odorless materials (phan, 2021). to make it efficient to combine with other food products, it is necessary to create solanum torvum powder. helilusiatiningsih, 2021 identifies the phytochemistry component of solanum torvum flour by doing 3 different methods of drying.the types of methods are sunlight drying, vacuum dryer, and try dryer. the result explains phytochemistry compounds and nutritional values. the vacuum drying method showed a higher dpph of 92.81% compared with the try drying method, which has 85.15%, and the sunlight drying method, which has 88.10%. the antioxidant activity of phenolic compounds was attributed to their ability to scavenge free radicals, giving hydrogen atoms, electrons, or chelate metal cations (afanas’ev, 1989). phenol compound is higher found in vacuum drying method (92.8 mg/g) than in sunlight method (37.32mg/g) and the try drying (22.91 mg/g). tannins are phenolic compounds of high molecular weight ranging from 500 da to more than 3000 da they are found in plants, and their parts are located in the tissues, specifically in the vacuoles (hassanpour, 2011). higher tannin content for the flour was found in the vacuum drying method (1.24 mg/g), followed by the sunlight method (1.03 mg/g), and try drying (0.63 mg/g). different from dpph, phenol, and tannins, a higher amount of flavonoids was found in the sunlight method (5.11 mg/g), followed by the vacuum drying method (2.75 mg/g), and try drying method (2.43 mg/g). proximate analysis shows quantitative analysis of macromolecules in food. to compare the nutritional values of the flour, the results were summarized in table 1. table 1. nutritional values of the solanum torvum flour with different drying methods. proximate analysis parameter drying method sunlight drying vacuum drying try drying water content (%) 6.92 7.62 6.89 ash content (%) 3.66 2,82 3,29 protein (%) 11, 51 22.66 21.28 fat (%) 5.38 3.25 7.04 carbohydrates (%) 72.53 65.51 61.5 vitamin c (%) 0.21 2.44 0.39 source: helilusiatiningsih, n. (2021) tea teye, (2017) developed functional drink combination from roselle (hibiscus sabdarffa), ginger (zingiber officinale), and turkey berry (solanum torvum). among several samples, 50% roselle + 25% ginger + 25% turkey berry shows optimum protein content (4.14%), ca (1.80%), fe (0.96 µg/g) and cu (1.18 µg/g). it also showed the best performance for sensory evaluation in terms of color, aroma, flavor, aftertaste, and overall acceptability. furthermore, helilusiatiningsih (2021) developed solanum torvum fruit extract as a potential healthy drink with a combination of black tea and green tea. according to the result, most panelists preferred black tea solanum torvum to green tea solanum torvum by organoleptic test on aroma, taste, and color. a qualitative test shows that black tea solanum torvum contains bioactive compounds, such as saponins, glucosides, steroids, and alkaloids. sample also shows amount of carbohydrates (63,96±0,08%), fat (9,67±0,05%), protein (24,76%), ash content (3,08 ±0,05%), moisture content (6,39 ± 0,12%), vitamin c (6,20±0,05%), and minerals (ca (38,34 ppm), mg (76,3 ppm), fe(4,51 ppm). snack bar novilia, 2017 investigated the food processing of solanum torvum in the snack bar. the investigation focused on the production of solanum torvum flour making, formulation and production of the snack bar, organoleptic testing, analysis of nutritional content, fiber, total phenolic compound, and antioxidant activity on the product. among several samples, 10% extract of solanum torvum showed the best organoleptic test and nutrition content, such as moister content (10.30%), ash 210 biology, medicine, & natural product chemistry 12 (1), 2023: 205-213 content (1.35%), protein (9.70%), fat (19,57%), carbohydrates (36.8%), fiber (22.28%), total phenolic (0.47%) and antioxidant activity equivalent vitamin c (704.14 mg/100 g). thus, the analysis of the contribution of nutrients in 1 serving size (37 g), solanum torvum bar contributes energy (5.95%), protein (6.78%), fat (12.21%), carbohydrates (6.30%), dietary fiber (36.54%) and total phenolic (174 mg/37 g) for general nutrition advice. noodle atika et al. (2021) found that solanum torvum extract could be used as an additional ingredient in noodles. adding solanum torvum extract in noodles improves the product with vitamin a. in plants, vitamin a has been found as provitamin a; the most active form of this vitamin is β-carotene. this component prevents vitamin a deficiency, where cells and tissues could be damaged in the human body. furthermore, the researchers discovered that adding solanum torvum with 85% of flour, 75% of solanum torvum, and 40% of water could develop the β-carotene component to 671.2548 g/100g. the control sample with 100% flour and 100% water without solanum torvum fruit extract could only reach 140.8604 g/100g. β-carotene content of 671.2548 g/100g is equivalent to 111.8758 re when associated with the nutritional adequacy rate for vitamin a. juice in ghana, the fruits of solanum torvum are traditional healers for anemia and ailments. the fruit is processed into a drinkable juice. with a high concentration of iron, it is possible to be utilized as a healing treatment for anemia and iron deficiency. iron is a mineral needed for human growth (al-jameil et al., 2014). solanum torvum aqueous extract has the ability as immunomodulatory and haematinic properties where it could be used as a hematinic and food for patients with immunity problems (koffuor, 2011). a study by okoto (2015) investigated the components of solanum torvum juice. the analysis shows that fruit juice has an abundance of water content (86,23%), carbohydrates 7.033%, proteins 2.322%, fats 0.278%, ash 0.143%, and crude fiber 3.993%. the analysis for important minerals content was conducted. according to the obtained results, calcium was the highest amount of mineral (221.583 mg/kg), followed by iron (76.869mg/kg), zinc (21.460mg/kg), manganese (19.466 mg/kg), and copper (2.642mg/kg). besides nutritional and mineral compounds, vitamin was found in the juice. the 100 grams of the sample contain 2.686 mg of vitamin c and 0.078 of vitamin a. conclusions solanum torvum contains various bioactive components, vitamins, mineral, and nutritions. it shows that solanum torvum has potential sources as functional food. however, studies about solanum torvum consumption as a functional food and the clinical trial of its health benefits in humans are still limited. further in vivo and in vitro studies are necessary to present the effect of solanum torvum consumption on health. competing interests: the authors declare that there are no competing interests. references abbaspour n., hurrell r., kelishadj r. 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(2013). phytochemistry and pharmacological studies on solanum torvum swartz. journal of applied pharmaceutical science, 3(4), 152-160. zuhud eam, siswoyo, sandra e, hikmat a, adhiyanto e. 2003. buku acuan umum tumbuhan obat indonesia. jakarta: yayasan sarana wanajaya. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 113-122 | doi: 10.14421/biomedich.2025.141.113-122 issn 2540-9328 (online) optimization of total polyphenol content in herbal tea formula bay leaves, cinnamon, and black tea sentot joko raharjo1,*, devita nur azizah2, ayu ristamaya yusuf3 1,2pharmacy and food analyst department; 3nutrition department, health polytechnic of putra indonesia malang, jl. barito no 5 malang 65123, tel. +62-341-491132, fax. +62-341-485411, indonesia. corresponding author* sentotjoko@poltekkespim.ac.id manuscript received: 16 december, 2024. revision accepted: 08 may, 2025. published: 14 may, 2025. abstract polyphenols are one of the secondary metabolites found in bay leaves, cinnamon and black tea. this mixture is formulated for therapy, one of which is chronic degenerative diseases, such as hypertension. this research aims to optimize the formulation of bay leaves, cinnamon and black tea on total polyphenol levels. this research method includes: determining the polyphenol content of the three simplices, designing the formulation design, determining the optimization of the total polyphenol content of the 14 formulations and the conformation of the selected formula using design expert 13. the results of the research obtained the total polyphenol content of bay leaves (49.36 mg gae/g), cinnamon (61.36 mg gae/g), and black tea (61.73 mg gae/g). optimization of the highest polyphenol content was 58.419 mg gae/g. the optimal formula conformation consisted of 1 gram of bay leaves, 8 grams of cinnamon and 1 gram of black tea, resulting in a polyphenol content of 59.63 mg gae/g. the conclusion of this research is the optimization of the highest polyphenol content of 58.419 mg gae/g with a formulation composition of bay leaves 10%, of cinnamon 80%, and of black tea 10%. keywords: optimization; total polyphenols; bay leaves; cinnamon; black tea. introduction hypertension is a condition where blood pressure is above normal for an extended period. hypertension is commonly referred to as high blood pressure or chronic degenerative cardiovascular disease which is most common in indonesia (rusli et al., 2018).one of the people's efforts to overcome hypertension is by consuming herbal medicines in the form of tea, because they have relatively fewer side effects compared to synthetic medicines. tea is a drink obtained from brewing the leaves or shoots of plants using hot water. based on the processing process, tea is divided into several types, one of which is black tea. black tea is a type of tea that is made through processing processes such as withering, milling, oxidating, and drying. black tea has a higher caffeine content than green tea (wilantari, 2018). according to (supriyo & pudjihastuti, 2021), black tea contains the chemical compound polyphenols, these compounds are the highest concentration of substances in black tea. in general, black tea contains polyphenolic compounds (527%) consisting of catechins (flavonols) and gallic acid (evitasari & susanti, 2021). polyphenolic compounds are the largest group in black tea which can be helpful in preventing cardiovascular disease by reducing the rate of fat oxidation (sudaryat et al., 2016). polyphenolic compounds are also found in natural ingredients such as bay leaves (eugenia polyantha wight) (dawley, 2014) and cinnamon (cinnamomi burmannii cortex) (handayani & paneo, 2021). bay leaves (eugenia polyantha wight) are one of the spices that many people use as a complementary ingredient or flavoring for cooking, apart from that, bay leaves also have pharmacological activity for health (haryanto et al., 2023). bay leaves contain polyphenol chemical compounds, essential oils (citral, eugenol), and carbohydrates (dawley, 2014).bay leaves contain polyphenolic compounds of (163.27 µg/ml cgae). the chemical compounds in bay leaves that can act as antihypertensives are polyphenols which function as antioxidants. the polyphenol compounds found in bay leaves are flavonoids, which have the benefit of preventing oxidation of body cells so that they can prevent hypertension by preventing blockages in blood vessels (haryanto et al., 2023). according to (sadik, 2021), polyphenol compounds have the advantage of providing activity to the vascular system, namely by blocking platelets from sticking to each other to form blockages (platelet aggregation), improving endothelial function, so that blood pressure can be reduced. apart from bay leaves, there are natural ingredients that have similar pharmacological effects on health, such as cinnamon. https://doi.org/10.14421/biomedich.2025.141.113-122 114 biology, medicine, & natural product chemistry 14 (1), 2025: 113-122 cinnamon (cinnamomi burmannii cortex) contains polyphenolic compounds, phytosterols and essential oils (handayani & paneo, 2021). cinnamon has antioxidant activity in the form of polyphenols which can fight free radicals (munthe, 2021). cinnamon has pharmacological benefits such as antiseptic, analgesic, anti-inflammatory, antioxidant, and lowering of blood pressure and cholesterol. the polyphenol compounds in cinnamon can play a role in lowering blood pressure with three working mechanisms, namely, destroying blood clots, absorbing blood circulation, and inhibiting cholesterol absorption, so the chemical compounds contained in cinnamon have the properties of preventing and destroying blood clots on the walls of blood vessels. and reduces blood viscosity so that hypertension can be prevented (sari et al., 2021). according to (handayani & paneo, 2021), cinnamon water extract has polyphenolic compounds that have the potential to act as antihypertensives by inhibiting the activity of angiotensin converting enzyme (ace). polyphenolic compounds can contribute to human biological activity because these secondary metabolites are included in bioactive compounds. in previous research, cinnamon extract contained polyphenolic compounds reaching 682 mg gae/g (ajie et al., 2023). due to this similarity, because it contains polyphenolic compounds, black tea can be combined with bay leaf simplicia and cinnamon to become a drink that contains benefits for the health of the body or is called herbal tea. herbal tea is tea made from ingredients other than tea leaves, namely flowers, seeds, leaves, roots and parts of other plants (haras et al., 2018). apart from that, herbal tea can be used as an alternative medicinal drink to treat diseases, one of which is hypertension, by using natural ingredients such as bay leaves. based on the similarities in the properties and chemical content of bay leaves (eugenia polyantha wight), cinnamon (cinnamomi burmannii cortex), and "x" black tea because they contain polyphenols, these three natural ingredients will be combined into herbal tea and optimization of the polyphenol content of several formulas to obtain optimal polyphenol content so that it can later be used as an antihypertensive therapy drink. this research aimed to optimize the formula for bay leaves, cinnamon and black tea on total polyphenol levels. materials and methods research design this research used the completely randomized design (crd) method of several herbal preparation formulas combining bay leaves, cinnamon and black tea, which were designed using expert design software version 13.0. the formulation of the herbal tea underwent quantitative analysis, namely determining the polyphenol content in herbal tea using the uv-vis spectrophotometry method. the test result data was then processed using expert design software version 13.0 to determine the optimal polyphenol content of several selected formulas and conformations. materials the tools used in this research were knives, ovens, baking sheets, analytical scales (pioneer), 10 ml measuring cups (pyrex), beakers (pyrex), 10 ml, 50 ml, and 100 ml measuring flasks (iwaki), stirring rod (pyrex), volume pipette (duran), measuring pipette (duran), test tube (pyrex), test tube rack, herb filter (maksindo), filter paper (whatman), cuvette, and uvvis spectrophotometer instrument (genesys). the ingredients used in this research were black tea, bay leaves, cinnamon, standard gallic acid, 96% ethanol, sodium carbonate (na2co3), and distilled water. research procedures plant determination determination of bay plants (eugenia polyantha wight) and cinnamon (cinnamomi burmannii cortex) was carried out by the batu herbal materia medica laboratory upt. preparation of bay leaf simplicia (eugenia polyantha wight) take leaves from the bay plant (eugenia polyantha wight), then sort them to remove dirt stuck to the leaves. wash the sorted leaves thoroughly using running water. drain the leaves that have been washed clean, then place the leaves on a baking sheet. dry the leaves using an oven with a heating temperature of 50°c until the leaves are dry. dried leaves are characterized by the fact that they are easy to crush with your hands until they are crushed. after the leaves are dry, they are ground using a blender until the appropriate degree of simplicia powder is obtained. (inorah., 2013). preparation of cinnamon simplicia (cinnamomi burmannii cortex) prepare the cinnamon bark, then sort it to remove dirt stuck to the cinnamon bark. wash the cinnamon bark using running water. drain the cinnamon skin which has been washed clean, then place it on a baking sheet. dry the leaves using an oven at a heating temperature of 50°c until the cinnamon bark is dry. dried cinnamon bark is characterized by being easy to knead with your hands until it crumbles. after obtaining dry cinnamon bark, then grind the cinnamon bark using a blender until the appropriate degree of simplicia powder is obtained (inorah., 2013). raharjo et al. – optimization of total polyphenol content … 115 determination of total polyphenol content preparation of gallic acid standard solution weigh out 50 mg of gallic acid. dissolve gallic acid with 96% ethanol in a 100 ml volumetric flask until a solution concentration of 500 ppm is obtained (fitria, 2020). determination of maximum wavelength (𝝀max) of gallic acid pipette 3 ml of a standard solution of gallic acid with a concentration of 500 ppm, then dissolve it with 96% ethanol: distilled water (1:1) into a 25 ml volumetric flask until a solution concentration of 60 ppm is obtained. pipette 0.5 ml of a standard gallic acid solution with a concentration of 60 ppm, then add 5 ml of folin ciocalteu reagent (diluted 1:10 distilled water). add 4 ml of 7.5% na2co3 solution, then shake until homogeneous and leave at room temperature for 15 minutes. measuring the solution at the maximum absorption wavelength with a uv-vis spectrophotometer (fitria, 2020). determining operating time pipette 3 ml of a standard solution of gallic acid with a concentration of 500 ppm, then dissolve it with 96% ethanol: distilled water (1:1) into a 25 ml volumetric flask until a solution concentration of 60 ppm is obtained. pipette 0.5 ml of a standard gallic acid solution with a concentration of 60 ppm, then add 5 ml of folin ciocalteu reagent (diluted 1:10 distilled water). add 4 ml of 7.5% na2co3 solution, then shake until homogeneous and leave at room temperature for 30 minutes. determine the maximum wavelength of gallic acid by scanning at a wavelength of 500 – 800 nm. the measurement time that shows the absorbance results remain stable is determined as the operating time of the gallic acid analysis method using uv-vis spectrophotometry (nengsih, 2022). preparation of a gallic acid standard curve make a series of concentrations of 20, 30, 40, 50, and 60 ppm from a standard solution of gallic acid with a concentration of 500 ppm. pipette 1, 3, 2, 10, 3 ml of each standard solution of 500 ppm gallic acid, then dissolve with 96% ethanol: distilled water (1:1) into each measuring flask 25, 50, 25, 100, 25 ml to the limit mark. pipette each of the concentration series solutions as much as 0.5 ml in a test tube. add 5 ml of folin ciocalteu reagent (diluted 1:10 distilled water). add 4 ml of 7.5% na2co3 solution, then shake until homogeneous and leave at room temperature for 30 minutes. measuring the solution at a maximum absorption wavelength of 758nm with a uv-vis spectrophotometer (fitria, 2020). simple brew of bay leaves, cinnamon and black tea weigh 0.05 grams each of bay leaf simplicity, cinnamon and black tea in a 100 ml beaker, 3 repetitions, then add warm water (temperature 80⁰c) and let sit for 8 minutes. the brew is filtered, and the filtrate is placed into a 25 ml flask, then distilled water is added to the mark. determination of polyphenol content pipette the filtrate of the simplica brewing solution of 0.5 ml each of bay leaf simplica, cinnamon and black tea in a test tube. add 5 ml of folin ciocalteu reagent (diluted 1:10 distilled water). add 4 ml of 7.5% na2co3 solution, then shake until homogeneous and leave at room temperature for 30 minutes. measuring the solution at a maximum absorption wavelength of 758nm with a uv-vis spectrophotometer (fitria, 2020). herbal preparation formulation upper and lower limits of preparations of bay leaves, cinnamon and black tea leaves in formulation optimization using expert design software version 13.0. the lower and upper limits obtained from the experimental design used in this optimization study are d-optimal mixture design (dmd) with the help of design expert software version 13.0. in this design, the independent variables are bay leaves (x1, %), cinnamon (x2, %) and black tea (x3, %), while the dependent variable is total polyphenol content (y1). the polynomial equation produced by this experimental design is y = a0 + a1x1 + a2x2 + a3x3 + a4x1x2 + a5x2x3 + a6x1x3 + a7x2 1 + a8x2 2 + a9x2 3 + e (1) where a0 a9 are the coefficients of each variable and interaction, and e is an error. the model used was selected based on the significance of the p-value of the model, the meaninglessness of the lack of fit value and considering the adj-r2 and r2 values. numerical optimization was carried out to determine the optimal formulation with the criteria of bay leaves (x1, %), cinnamon (x2, %) and black tea (x3, %) for certain pools with high total polyphenol (y) levels. the optimal formula results are verified, then the optimal formula which produces the lowest response bias between predictions and observations (<10%) will be compared with the optimal formula from high energy using anova statistical analysis. based on the analysis results, formula 14 run was obtained, then the three simple powder ingredients using the dry mixing method using a plastic jar (dry mixing) (rusli et al., 2018) 116 biology, medicine, & natural product chemistry 14 (1), 2025: 113-122 figure 1. design of 14 run formula optimization of bay leaves, cinnamon and black tea. data analysis in this study, the levels of polyphenolic compounds were determined using the uv-vis spectrophotometric method using the folin-ciocalteu method and gallic acid was used as a standard solution. the resulting polyphenol content data will be processed in statistical form with the response surface test using expert design software version 13.0. the resulting data can be used to determine optimal polyphenol compounds in several herbal tea formulas, which are described using 3d statistical data. results and discussion determination of total polyphenol content in bay leaves, cinnamon, and black tea determination of sample wavelength determination of the maximum wavelength of the sample using a spectrophotometric instrument with a wavelength range of 500 – 800 nm. determination of the maximum wavelength was carried out to determine the wavelength of gallic acid with the folin-ciocalteu reagent which gave the highest wavelength value. to determine the maximum wavelength using a working standard solution of gallic acid with a concentration of 40 ppm. the results of determining the maximum wavelength are 758 nm with a maximum absorbance value of 0.286. determination of absorbance of working standards and calibration curves in this research, bioactive polyphenolic compounds were determined using the folin-ciocalteu method using uvvis spectrophotometry. the results of measuring the absorbance of standard concentrations of gallic acid, 20 ppm, 30 ppm, 40 ppm, 50 ppm, and 60 ppm, are presented in table 1. they were used in determining total polyphenol levels. results and discussion should be written as a series of connecting sentences, however, a manuscript with a long discussion should be divided into subtitles. results should be clear and concise. table 1. determination of absorbance of gallic acid working standards. concentration absorbance λ 758 nm 20 0,187 30 0,238 40 0,320 50 0,386 60 0,434 figure 2. gallic acid standard curve at wavelength 758 nm. determination of total polyphenol content of bay leaves, cinnamon, and black tea the total polyphenol content was determined based on the linear equation y = 0.0064x + 0.0562 and the absorbance value of bay leaf simplicial extract, cinnamon and black tea, as presented in table 2. raharjo et al. – optimization of total polyphenol content … 117 table 2. total polyphenol content of bay leaves, cinnamon, and black tea. no. description total polyphenol content (gae/g) 1. bay leaves 49,36 ± 0,13 2. cinnamon 61,36 ± 0,27 3. black tea 61,73 ± 0,39 herbal tea formula from bay leaves, cinnamon, and black tea in this study, the samples used were bay leaves, cinnamon, and black tea. bay leaves and cinnamon are previously processed into simplicia using oven drying at 50°c, then blended into a coarse powder using a grinder. these three ingredients will be combined in a plastic jar into several formulas. formulation optimization using the "design expert version 13.0" software obtained 14 runs as presented in table 3. table 3. design output of tea herbal formula. run bay leaves (a) gram cinnamon (b) gram black tea (c) gram 1 1 1 8 2 8 1 1 3 4.5 4.5 1 4 3.4 3.3 3.3 5 1 1 8 6 5.6 2.1 2.1 7 1 4.5 4,5 8 1 8 1 9 4.5 1 4.5 10 8 1 1 11 2.1 5.6 2.1 12 1 8 1 13 4.5 4.5 1 14 2.1 2.1 5.6 determination of polyphenol content in 14 formulations the results of polyphenol levels in samples from each formula are presented in table 5. based on the data from this table, it can be seen that the highest polyphenol content is formula 8, which consists of 1 gram of bay leaves, 8 grams of cinnamon, and 1 gram of black tea, as shown in table 4. table 4. total polyphenol content of herbal teas from bay leaves, cinnamon and black tea. run absorbansi total polyphenol content (mg gae/ g) 1 0,380 24,0922 2 0,236 13,2768 3 0,417 25,4859 4 0,468 30,2363 5 0,382 24,7598 6 0,367 21,7184 7 0,408 26,4272 8 0,836 58,4662 9 0,210 10,9232 10 0,235 13,1532 11 0,487 33,0611 12 0,838 58,5039 13 0,491 30,6024 14 0,372 23,9998 data analysis for optimizing polyphenol levels with “design expert 13.0” the results of the data on polyphenol levels from 14 formulas will then be processed using the "design expert 13.0" software to determine the optimization results of polyphenol levels, as shown in figure 3-7. figure 3. fit summary. 118 biology, medicine, & natural product chemistry 14 (1), 2025: 113-122 figure 4. summary statistics model. figure 5. anova and model actual equation. figure 6. final equation. figure 7. 3d surface graphic. discussion in determining total polyphenol content, it begins with determining the maximum wavelength, determining the standard curve, and determining the concentration. determination of the maximum wavelength of the sample using a spectrophotometric instrument with a wavelength range of 500 – 800 nm. determination of the maximum wavelength was carried out to determine the wavelength of gallic acid with the folin-ciocalteu reagent which gave the highest wavelength value. to determine the maximum wavelength, a working standard solution of gallic acid with a concentration of 40 ppm was used. the results of determining the maximum wavelength are 758 nm with a maximum absorbance value of 0.286. table 1 and figure 2 show that the absorbance value is directly proportional to the concentration following the linear regression equation and a standard curve for gallic acid is obtained with the equation y = 0.0064x + 0.0562 and a correlation coefficient (r²) of 0.9927. the determination of polyphenol levels was carried out using the folinciocalteu method which determines the amount of polyphenols or phenolic compounds contained in the sample. the folin-ciocalteu method is generally widely used in testing polyphenols because this method is effective for forming complex compounds with gallic acid as a comparison. the reaction of gallic acid and folin-ciocalteu reagent can be seen in figure 6. figure 6 shows that complex compound reactions can be formed due to the hydroxyl group and conjugated double bonds in each benzene ring of gallic acid. the phenolichydroxy group will reduce the heteropoly acid (phosphomolybdic-phosphotungstic) contained in the folin-ciocalteu reagent into a molybdenum-tungsten complex compound to form a blue color that can be detected using uv-vis spectrophotometry. the addition of 7.5% na2co3 reagent functions to create an alkaline atmosphere in the solution so that proton dissociation occurs in phenolic compounds into phenolic ions (atikah della putri et al., 2021). determination of the polyphenol content of 14 herbal tea formulas was determined based on the linear regression equation values obtained from figure 1. figure 8. reaction of gallic acid with folin-ciocalteu reagent. the results of the analysis of total polyphenol levels are shown in table 2, the content is 40 – 70 mg gae/g. according to ilmi et al., 2022, reported polyphenol levels in the cinnamon brew as a functional drink reached 205.5 – 682.17 mg gae/g, so based on this information it is possible that the formula with cinnamon components is raharjo et al. – optimization of total polyphenol content … 119 more dominant than bay leaves and tea. black produces high levels of polyphenols. differences in total polyphenol levels of plant species and varieties as a result of research and literature, possibly due to differences in environmental and genetic factors, (prasad et al., 2006; sarpras et al., 2016). in this research, 14 formula design outputs were obtained from these three materials, as presented in table 4, using the "design expert version 13.0" software. the software has designed the formulation. a quantitative test of polyphenol content will be carried out to determine the optimal formula with a high polyphenol content. data from table 5, shows that the highest polyphenol content is the formula whose components consist of 1 gram of bay leaves, 8 grams of cinnamon, and 1 gram of black tea. the high levels of polyphenols in this formula are due to the cinnamon component being more dominant than the bay leaf and black tea components. design expert analysis, as presented in figure 3, the output serves to determine the model recommended by central composite design for optimizing polyphenol levels, the recommended model is special quartic vs quadratic. figure 3 shows the predicted r² value in the suggested model of 0.8281 (82.81%), which means the probability of design error is 17.19%. figure 4, the summary statistics model functions to explain comparing and selecting an appropriate model to explain the relationship between the independent variable and the dependent variable. in the special quartic model, an r² value of 0.9938 is obtained. if the r² value is more significant (closer to 1), the better the model, which means that the herbal tea formula combining bay leaves, cinnamon and black tea (independent variable) influences polyphenol levels (dependent variable). in the special quartic vs quadratic model that has been suggested, an adjusted r² of 0.9839 is obtained. the adjusted r² value is lower than the r² value of 0.9938 because the adjusted r² value will only measure independent variables that have a significant influence on the dependent variable. figure 5, explains the anova test and the actual model equation which functions to determine significant differences between the variations in the data tested. in the recommended special quartic model, anova results were obtained with a p-value <0.0001, this value was <0.05 (p-value 5%). the p-value means that the special quartic model is significant, and the possibility of error in the model is slight (< 0.01%). in this model, it is known that the components of bay leaves cinnamon (x1x2), components of bay leaves black tea (x1x3), components of cinnamon black tea (x2x3), repeated components of bay leaves cinnamon black tea (x1²x2x3), and the bay leaf – cinnamon – black tea repeat components (x1,x2,x3²) are significant with pvalue <0.05. meanwhile, the components of bay leaves – cinnamon repetition – black tea (x1x2²x3) are components that are not significant in influencing polyphenol levels because the p-value is 0.1422 (14.22%) which means >0.05 (p-value 5%) . the p-value for lack of fit is 0.3652, this value is >0.05 (p-value 5%) which means that the possibility of an error is 36.52%, lack of fit in the special quartic model is not significant. figure 6, final equation functions to display the appropriate mathematical equation to explain the relationship between the independent variable and the dependent variable. in the anova analysis there are similarities to the actual model. y = 13,15x1 + 58,42x2 + 24,36x3 – 31,49x1x2 – 32,38x1x3 – 60,90x2x3 +647,74x1²x2x3 315,12x1x2² x3 + 553,48x1x2x3² (2) or y = 13,15 a + 58,42 b +24.36 c – 31,49 ab -32,38 ac -60,90 bc + 647,74 a2bc – 315,12 ab2c + 553,48 abc2 (3) this equation shows that the components of the bay leaf cinnamon (x1x2), bay leaf black tea (x1x3), cinnamon black tea (x2x3), bay leaf cinnamon black tea (x1x2²x3) have a decreasing effect on polyphenol content (y), while the component equations are bay leaves (x1), cinnamon (x2), black tea (x3), repetition of bay leaves – cinnamon – black tea (x1²x2x3), bay leaves – cinnamon – repetition black tea (x1x2x3²) has an increasing effect on total polyphenol levels (y). figure 7 shows the prediction of the optimization point for polyphenol content in several herbal tea formulas from bay leaves, cinnamon and black tea on 3d surface, namely formula 8 with components consisting of bay leaves (x1) 10%, cinnamon (x2) 80% , and black tea (x3) 10%, with total polyphenol equation (gae/ g) = 13,15 a + 58,42 b +24.36 c – 31,49 ab -32,38 ac -60,90 bc + 647,74 a2bc – 315,12 ab2c + 553,48 abc2 figure 9. solution of optimization herbal tea formula optimization results in figure 9, the recommended solution for optimal conditions for total polyphenol content consists of 10% bay leaves, 80% cinnamon, and 10% black tea with a total polyphenol content of 58.419 120 biology, medicine, & natural product chemistry 14 (1), 2025: 113-122 mg gae/g. next, conformational determination of total polyphenol content was carried out with this formulation, as presented in table 5. table 5. total polyphenol content in the conformation formulation of bay leaves 10%, cinnamon 80%, and black tea 10%. no. formula propose total polyphenol content (mg gae/ g) 1. replication-1 59.34 2. replication-2 60.51 3. replication-3 59.06 rata-rata 59.63±0.769 the composition of bay leaves, cinnamon and black tea has differences in the amount of polyphenol content, as presented in table 4.2, where the total polyphenol content in bay leaves is 49.36 ± 0.13, cinnamon 61.36 ± 0.27 and black tea 61.73 ± 0.39 mg gae/g. it can be seen that the total polyphenol content in the composition of the three single levels is in line with the content in the mixed formulation levels. bay leaves were detected to contain polyphenolic compounds consisting of caffeic acid, gallic acid, and triterpene squalene. of these three compounds, gallic acid was proven to have potential antihypertensive action. gallic acid is trihydroxybenzoic acid with a hydroxy group that has antioxidant activity and therapeutic effects, one of which is cardiovascular. however, according to previous literature studies (parawati & mulyanti, 2022), brewing has a total polyphenol content of 163.27 mg gae/g. apart from that, research (palupi & widyaningsih, 2015) stated that the total polyphenol content in the bay leaf tea functional drink was 134.02 mg gae/g. apart from bay leaves, total polyphenol compounds can be found in other natural ingredients such as black tea. black tea is tea leaves that are processed using a fermentation process. black tea contains total polyphenol levels of 165 mg gae/g. in general, black tea contains derivatives of polyphenol compounds which have antioxidant effects. these compounds include catechins with phenolic hydroxyl groups, gallic acid and theaflavins with their hydroxyl groups. according to (sudaryat et al., 2016), black tea contains total polyphenol levels of 165 mg gae/g, while according to (paramita et al., 2020), the results of determining polyphenol levels in black tea leaves are 150 mg gae/g. apart from containing polyphenolic compounds, bay leaves and black tea contain phenolic acid compounds consisting of phydroxybenzoic acid, vanillic acid, syringic acid, caffeic acid, cinnamic acid, p-coumaric acid, ferulic acid and gallic acid. apart from being found in bay leaves and black tea, polyphenol compounds can also be found in cinnamon. cinnamon contains polyphenol compounds consisting of cinnamaldehyde, catechin, protocatheuic acid, quercetin, epicatechin, p-coumaric acid, phydoxybenzoic acid, syringic acid, and caffeic acid. according to research (sholihah et al., 2023), cinnamon contains total polyphenol levels of 682 mg gae/g, while according to (chan et al., 2014) the polyphenol content in boiled cinnamon water is 315.02 mg gae/g. apart from that, the cinnamaldehyde compound is the main compound derived from the polyphenol group in cinnamon with an aldehyde group which has a protective role against cardiovascular disease, one of which has an anti-platelet aggregation effect and in vitro can function as a vasodilator (das et al., 2022 ). the optimal formula consists of 1 gram of bay leaves, 8 grams of cinnamon and 1 gram of black tea to produce polyphenol levels of 58.419 mg gae/g. polyphenol levels in cinnamon decrease when combined with bay leaves and black tea, this occurs because of the acid standard. the gallate used is influenced by phenolic acid group compounds such as gallic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, caffeic acid, cinnamic acid, p-coumaric acid, and ferulic acid. the presence of phenolic compounds which are polyphenols and phenolic acids will influence the polyphenol levels in this study. the optimization results are influenced by the content of the three natural ingredients being optimized. total polyphenol equation (gae/g) = 13,15 a + 58,42 b +24.36 c – 31,49 ab 32,38 ac -60,90 bc + 647,74 a2bc – 315,12 ab2c + 553,48 abc2 conclusions based on the research that has been carried out, it can be concluded that herbal tea from bay leaves, cinnamon and black tea obtained optimal levels of total polyphenols in a formula consisting of 10% bay leaf, 80% cinnamon and 10% black tea components with the results total polyphenol content was 58.419 mg gae/g, with total polyphenol equation (gae/g) = 13,15 a + 58,42 b +24.36 c – 31,49 ab -32,38 ac -60,90 bc + 647,74 a2bc – 315,12 ab2c + 553,48 abc2. acknowledgements: we would like to thank the institutions of the putra indonesia health polytechnic malang and the materia medika batu malang for helping and assisting in the implementation of this research. authors’ contributions: sentot joko rahajo’s concept and designed the study. devita nur azizah carried out the laboratory work. ayu ristamaya yusuf analyzed the data. sentot and devita wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors gratefully acknowledge financial support by the directorate of research, technology and raharjo et al. – optimization of total polyphenol content … 121 community service, directorate general of vocational studies, ministry of education and culture, research and technology, republic of indonesia, in accordance with contract agreement number: 104/spk/d.d4/ppk.01.aptv /iii/2024, dated 19 march 2024, number: 025/sp2h/ppkm-ptv/ll7/2024, dated 20 march 2024, number: 365.1/plk/pn/2024, dated 22 march 2024, fiscal year references ajie, w., tiga, d., stikes, f., borromeus, s., asyiah, r., & christian, r. 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(2018). isolasi kafein dengan metode sublimasi dari dengan fraksi etil asetat serbuk daun camelia sinensis. jurnal farmasi udayana, 8(1), 53. https://doi.org/10.24843/jfu.2018.v07.i02.p03 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 529-533 | doi: 10.14421/biomedich.2025.141.529-533 issn 2540-9328 (online) evaluation of antidiabetic activity of croton zambesicus root extract: in vivo inhibitory effect on alpha amylase and alpha glucosidase of rats jude efiom okokon1, utibe anietie edem1, chinyelu clementina osigwe2, ugonma florence uwaeme2 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria 2department of pharmacology and toxicology, madonna university nigeria, elele campus, rivers state, nigeria. corresponding author* bisu.thaguna123@gmail.com manuscript received: 07 april, 2025. revision accepted: 27 july, 2025. published: 01 august, 2025. abstract diabetes mellitus is a global health challenge, necessitating the need for alternative treatments. croton zambesicus muell arg. (euphorbiaceace) a medicinal plant used traditionally in the treatment of some ailments was investigated for its inhibitory potential on alpha-amylase and alpha-glucosidase enzymes in rats. the root extract of c. zambesicus (27 81 mg/kg) was investigated in vivo for the inhibitory effect on alpha amylase and alpha glucosidase enzymes using starch, sucrose, and maltose as substrates. acarbose was used as a reference drug. blood glucose levels (bgl) of rats, post administration of the substrate and extract concurrently, were monitored over 3 hours as a parameter to measure the inhibitory potential of the extract. the root extract dose-dependently caused significant (p<0.05) reduction in blood glucose levels of treated rats with the various substrates used. the results suggest that the root extract of croton zambesicus has the potentials to inhibit alpha amylase and alpha glucosidase in rats. keywords: croton zambesiscus; anti-diabetic; enzyme inhibition; alpha-amylase; alpha-glucosidase. introduction diabetes mellitus (dm) is one of the major health challenges facing most countries of the world with associated economic and social consequences. the disease has been ranked as the 8th deadly disease causing millions of deaths world over (who, 2016). the number of people affected by diabetes is projected to increase to 700 million by 2045 (saeedi et al., 2019), necessitating urgent steps to remedy this situation. the reliance of the major management approach on the use of conventional medicines has not yielded much positive results due to economic reasons and the associated side effects. therefore, the search for affordable and safe alternative drugs is inevitable. worldwide, herbal preparations have gained significant patronage for dm management. we had previously reported the efficacy of a number of plants extracts indigenous to nigeria on diabetes such as mammea africana (okokon et al., 2007), hippocratea africana (okokon et al., 2010), anthocleista djalonensis (okokon et al., 2012), solenostemon monostachyus (okokon et al., 2015), cornhusk of zea mays (okokon & mandu, 2017), setaria megaphylla (okokon et al., 2022a) and solanum anomalum (okokon et al., 2022b). the mechanism of antidiabetic action of only a few of them concerning their effects on alpha amylase and alpha glucosidase activities has been investigated. croton zambesicus muell arg. (euphorbiaceace) (syn c. amabilis muell. arg. c. gratissimus burch) is a guineo–congolese species found in tropical africa often grown as an ornamental tree in villages and towns in nigeria. traditionally, the leaf and root decoctions are used as anti-hypertensive and antimicrobial (urinary infections) (adjanohoun et al., 1989). the roots are used as antimalarial, febrifuge, laxative and antidiabetic by the ibibios of niger delta region of nigeria (okokon & nwafor, 2009a). boyom et al. (2002) reported that the essential oils from the root bark of croton zambesicus contain majorly sesquiterpenes. the root and stem bark oils were also found to be rich in oxygen containing compounds, with spathulenol and linalool as major components. okokon and nwafor (2009a) reported that the root extract contains alkaloids, saponins, terpenes, tannins, phlobatannins, anthraquinones and cardiac glycosides. the root extract has been reported to possess antimalarial (okokon & nwafor, 2009a), anticonvulsant and antiulcer (okokon & nwafor, 2009b), antiinflammatory, analgesic and antipyretic (okokon & nwafor, 2010), antidiabetic and hypolipidemic (okokon et al., 2011a), laxative (okokon et al., 2020) activities. moreso, the kidney-protective potential against https://doi.org/10.14421/biomedich.2025.141.529-533 530 biology, medicine, & natural product chemistry 14 (1), 2025: 529-533 gentimicin-induced kidney injury (okokon et al., 2011b), immunostimulatory, cytotoxicity against hela cell line and antileishmanial activities (okokon et al.,2013) of the root extract of the croton zambesicus has been reported. we report in this study the inhibitory effect of the root extract on the alpha amylase and alpha glucosidase enzymes of rats. materials and methods plants collection the plant material croton zambesicus (roots) was collected from compounds in the uruan area, akwa ibom state, nigeria in august 2024. dr. margaret bassey of the department of botany and ecological studies, university of uyo, uyo, nigeria, identified and authenticated the plant. extraction the roots were washed clean and shade-dried for two weeks. the dried plants’ materials were further chopped into small pieces and reduced to powder. the powdered material was macerated in 50% ethanol for 72 h. the liquid ethanol extract obtained by filtration was evaporated to dryness in a rotary evaporator at 40˚c. the extract was stored in a refrigerator at 4˚c until used for the experiment reported in this study. animals albino wistar rats (125 -142g) of either sex were used for these experiments. the animals were housed in standard cages and maintained on a standard pelleted feed (guinea feed) and water ad libitum. permission and approval for animal studies were obtained from the college of health sciences animal ethics committee, university of uyo. in vivo alpha-amylase and glucosidase inhibition study alpha-amylase inhibitory study thirty wistar rats were divided into 6 groups of 5 rats each. the rats in all groups were fasted for 18 hour and fasting blood glucose concentration was first taken at 0 min before administration. group i, as the normal control, received distilled water (10 ml/kg). group ii rats were orally administered starch at 2 g/kg body weight (orally with distilled water as vehicle) and distilled water (10 ml/kg) simultaneously. rats in group iii were administered starch (2 g/kg) and the standard drug (acarbose) at 100 mg/kg simultaneously. based on previously determined ld50 and doses (okokon et al., 2009a), groups iv, v, and vi were administered with starch (2 g/kg) and c. zambesicus root extract at 27, 54, and 81 mg/kg, respectively. all administrations were done orally and blood glucose concentration was monitored at 30, 60, 90, 120 and 180 min (gidado et al., 2019; okokon et al., 2023a). glucosidase inhibitory study the procedure as described above was used for this study but with sucrose and maltose used as substrates (okokon et al., 2023a; okokon et al., 2023b). blood glucose determination drops of blood from the tip of rats’ tails were dropped on stripes and glucose concentration was measured using a glucometer according to the manufacturer’s specifications (accu-chek, indiana). the glucometer works using an electrochemical detection system with the following principle; the biosensor system makes use of disposable dry reagent strip based on glucose oxidase method. each strip has an electrode impregnated with the enzyme glucose oxidase, which reacts with glucose in the blood sample when dropped on the membrane covering the reagent pad (strip), to produce gluconic acid. during the reaction, in which an electric current is generated, an electrochemical mediator transfers electrons to the electrode surface. this electrode sensor measures the current produced when the enzyme converts glucose to gluconic acid. the magnitude of the generated current is proportional to the amount of glucose present in the drop of blood sample, thus giving an accurate reading of the blood glucose concentration (who, 2011). statistical analysis data obtained from this work were analysed statistically using one –way anova followed by tukey-kramer multiple comparison test using instat graphpad software, (san diego, usa). differences between means were considered significant at 5% level of significance ie p≤ 0.05. results and discussion in vivo alpha amylase and glucosidase inhibition assay treatment of fasted rats with starch (2 g/kg) elevated blood glucose concentrations of the treated animals after 30 min in various proportions. the proportions were as follows: starch (62.62%), extract-treated groups (ranging from 10.95% to 17.77%), and acarbose-treated groups (17.97%). after 60 minutes, the blood glucose concentrations were lowered with the groups administered with dosages of the extract (27 81 mg/kg) having percentage increases ranging from 0.20 to 0.91%. the average blood glucose concentrations of all the groups treated with the extract were lowered to normal levels after 120 minutes except that of the lowest dose (27 mg/kg) with bgl of 0.13%. the bgl of all the extract treated groups was reduced to normal (table 1). sucrose (2 g/kg) administration to fasted rats caused a 41.14% increase in blood glucose level 30 minutes postadministration of the sucrose to the control group. bgl increments of 12.57-31.93 % were also recorded in okokon et al. – enzyme inhibition and anti-diabetic effects of croton zambesicus root … 531 groups treated with 27,54 and 81 mg/kg of extract. at 60 min, percentage increases in bgl of groups treated with 27, 54, and 81 mg/kg of extract were 13.85, 0.80, and 0 %, respectively. no increment in bgl was recorded in the extract-treated groups from 120 -180 min (table 2). there was a 90.94% increase in blood glucose level 30 min following maltose administration in the control group. however, 11.93 39.75 % increases in bgl were observed in the extract-treated groups. at 60 min, groups treated with 27,54 and 81 mg/kg extract had percentage increments of 17.86, 13.23, and 0%, respectively, while percentage increases of 0.41 and 0.18% were recorded for 27 and 54 mg/kg treated groups at 120 min respectively. at 180 min, no increment in bgl was recorded in any of the extract treated groups (table 3). table 1. effect of ethanol root extract of croton zambesicus on blood glucose level of rat after oral administration of starch load. treatment dose mg/kg blood glucose level mg/dl in min 0 min 30 min 60 min 120 min 180 min control (normal saline) 86.00±11.53 87.66±7.12(1.93) 87.66±7.62(1.93) 91.0±7.50(5.81) 80.00±6.02 starch 66.0±3.60 107.33±6.36a(62.62) 91.66±2.02(38.87) 77.66±3.71(17.66) 70.66±2.72(6.59) acarbose 100 72.33±2.69 85.33±12.97(17.97) 80.33±7.21(11.06) 74.0±1.00(2.30) 72.33±8.68(0) extract 27 78.20±2.20 92.10±5.12(17.77) 85.36±5.39a(0.91) 79.23±3.27a(0.13) 76.38±1.24a() 54 76.56±3.56 85.52±3.28(11.70) 82.61±3.55(0.79) 74.10±5.45b() 75.32±2.32a() 81 78.60±4.63 87.21±5.84(10.95) 80.20±4.56(0.20) 72.45±5.56a() 72.66±3.16() data are expressed as mean ± sem, significant at ap<0.05, bp< 0.01, compared to control (n=6). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. table 2. effect of ethanol root extract of croton zambesicus on blood glucose level of rat after oral administration of sucrose load. treatment dose mg/kg blood glucose level mg/dl in min 0 min 30 min 60 min 120 min 180 min control (normal saline) 100.00±4.25 88.33±1.85 92.33±4.25 89.0±4.35 87.33±3.84 sucrose 2000 81.0±4.50 114.33±5.50b(41.14) 112.66±1.45a(39.08) 97.33±1.63(20.16) 94.15±4.81(16.23) acarbose 100 90.33±2.48 86.66±2.90 82.0±6.00 71.66±3.75 78.0±3.78 extract 27 79.66±4.32 105.10±5.29c(31.93) 90.22±4.33(13.25) 75.12±6.88() 72.44±4.12 54 75.34±2.55 91.23±4.18b(21.09) 81.42±4.56(0.80) 74.10±3.48() 69.44±4.33() 81 77.20±5.29 86.91±2.34(12.57) 75.46±3.34() 69.84±2.26() 67.55±4.75() data are expressed as mean ± sem. significant at ap<0.05, bp< 0.01, compared to control (n=6). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. table 3. effect of ethanol root extract of croton zambesicus on blood glucose level of rat after oral administration of maltose load. treatment dose mg/kg blood glucose level mg/dl in min 0 min 30 min 60 min 120 min 180 min normal control 100.00±4.25 88.33±1.85 92.33±4.25(1.80) 89.0±4.35(1.55) 87.33±3.84(3.98) maltose 2000 70.00±11.67 133.66±15.44c(90.94) 128.66±8.78a(83.80) 99.36±5.36(41.94) 84.0±7.21(20.0) acarbose 100 85.34±1.36 88.22±1.10(3.37) 86.0±2.20(0.77) 84.26±1.14a() 82.28±2.26() extract 27 77.49±6.39 108.30±4.33a(39.75) 91.33±3.68a(17.86) 80.74±4.36a(0.41) 72.58±5.63a() 54 75.46±5.52 94.05±6.64b(24.63) 85.45±2.44a(13.23) 76.85±5.29a(0.18) 72.56±8.34() 81 76.33±3.68 85.44±6.32b(11.93) 75.66±3.56b() 73.23±6.18b() 70.91±6.36a() data are expressed as mean ± sem, significant at ap<0.05, bp< 0.01, compared to control. (n=6). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. discussion croton zambesicus parts are used in ibibio traditional medicine in the treatment of diseases such as diabetes among others. this work investigated the effect of croton zambesicus root extract on alpha amylase and alpha glucosidase activities in rats, the extract was found to inhibit increases in blood glucose concentration following starch administration. α-amylases and αglucosidase enzymes act in synergy to digest dietary polysaccharides such as starch. the polysaccharides are reduced to disaccharides by the α-amylase enzyme b y breaking the α-bonds of the α-linked polysaccharides. thereby resulting in disaccharides like maltose, which are also digested by membrane bound α-glucosidase enzymes to monosaccharides (kalra, 2014; alongi & anese, 2018). inhibitions of these enzymes activities 532 biology, medicine, & natural product chemistry 14 (1), 2025: 529-533 suppresses the ingested carbohydrates digestion, with associated insignificant elevation in blood glucose concentrations following carbohydrate meals as was observed in this study. as a target for managing type 2 diabetes mellitus, some medicinal plants have been investigated for α-amylase and α-glucosidase inhibitory potentials. previously, we had reported on the α-amylase and α-glucosidase inhibitory potentials of heinsia crinata, lasianthera africana, setaria megaphylla, and solanum anomalum (okokon et al., 2021; eweh et al., 2022; etuk et al., 2023) among others. similarly, the root extract significantly inhibited blood glucose rise when coadministered with maltose and sucrose. acarbose, the standard drug used in this study significantly suppresses blood glucose rise when co-administered with starch, maltose and sucrose. the results of this study corroborate the reported activities of the leaf extract and fractions of c. zambesicus on alpha amylase and alpha glucosidase activities in rats (okokon et al., 2022c). furthermore, the results also corroborate the significant alpha amylase and alpha glucosidase inhibitory activities reported on other species of croton such as c. bonplandianum (qaisar et al., 2014; karuppiah et al., 2017), c. thurifer (morocho et al., 2020), and c. oblongifolius (srisongkram et al., 2022) as observed in this study. the inhibitory activities of these species have been linked to their phytochemical constituents especially polyphenols. the root oil from croton zambesicus has been reported to contain majorly sesquiterpenes and is also found to be rich in oxygen containing terpenoid compounds such as spathulenol and linalool. these compounds especially sesquiterpenoids posess alpha glucosidase and alpha amylase inhibitory potentials (lee et al., 2023; tan et al., 2023). the presence of these compounds in the extract could have contributed to the observed activity of this study and therefore explains the antidiabetic mechanism of the roots of c. zambesicus. conclusion the findings of this research suggest that the root extract of croton zambesicus may exhibit anti-diabetic effects by inhibiting the alpha-amylase and alpha-glucosidase enzymes. this activity may be linked to the presence of phytochemical ingredients in the plant. acknowledgments: the authors thank staff of the animal house, department of pharmacology and toxicology, university of uyo for providing technical assistance. competing interests: the authors declare that there are no conflicts of interest concerning this manuscript. ethical approval: permission and approval for animal studies were obtained from the college of health sciences animal ethics committee, university of uyo. all animal experiments complied with the national institute of health guide for care and laboratory animals (pub. no. 85-23, revised 1985). funding/support: the study was self-funded. references adjanohoun ej, adjakide v, de souza s. 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(2023). identification of sesquiterpene glycosides from dendrobium nobile and their α-glycosidase and α-amylase inhibitory activities technol, campinas. food sci. 43, e99722, 2023 world health organization (2011). glucose analyser. core medical equipment information. 2011. www.who.int/medical_devices/en/index.html. assessed on 3rd june, 2019. world health organization 2016. global report on diabetes. geneva, 2016. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 35-42 | doi: 10.14421/biomedich.2024.131.35-42 issn 2540-9328 (online) potential of methane (ch4), nitrogen (n2), and carbon dioxide (co2) from eco-enzyme with the addition of cow feces starter marlina kamelia*, aulia ulmillah, suci wulan pawhestri biology department, faculty of science and technology, uin raden intan lampung, jl. endro suratmin sukarame – bandar lampung 35131, tel. 0721-780887, indonesia. corresponding author* marlinakamelia@radenintan.ac.id manuscript received: 09 october, 2023. revision accepted: 13 march, 2024. published: 06 may, 2024. abstract eco-enzyme solutions are generally used in the manufacture of disinfectants, floor cleaners, liquid fertilizers, preservatives and others. in this study, eco-enzyme was used as the main ingredient in the manufacture of biogas fermentation with the addition of cow feces as an additional starter in the fermentation. the aim of this study was to determine the content of ch4, co2, n2, ph, temperature, and pressure in eco-enzyme fermentation with the addition of cow feces starter. this research is a quantitative research with the type of experimental research as well as direct observation data collection techniques and data analysis techniques using descriptive statistical analysis. the results showed that the treatment that produced the highest methane (ch4) gas was the p5 treatment with a concentration of 2.889%. 6,1. the highest volume pressure value was produced in the p5 treatment which was 70 ml. nitrogen gas (n2) and carbon dioxide gas (co2), the concentration of nitrogen gas (n2) from reactor p0 p4 is in the range of 95.875-99.669 % and the concentration of carbon dioxide (co2) from reactor p0 p4 is in the range of 0.237 4.125 %. keywords: ch4; co2; eco-enzyme; n2; ph; pressure. introduction during the pandemic, the consumption of fruit was in great demand by the community, especially on the household scale. this is done by the community as an effort to increase body immunity. based on an analysis of the projections for 2018-2022 consumption of fruits and vegetables will increase with an average growth rate of 2.50% and 1.50% (alfaena, et al, 2018). the increase in fruit consumption can cause a new problem, namely household organic waste in the form of fruit residue. the waste of this fruit, which can be in the form of fruit peels, seeds or other parts, is generally not reused and is just thrown away. this will have a serious impact on the environment if not handled properly. this waste problem will be very important to find a solution because it will have an impact on the balance of the environmental ecosystem (prabekti and ahmadun, 2011). therefore, a solution is needed to process the waste (fruit residue) into useful by-products. environmentally friendly waste treatment can be a solution to overcome this problem. the principle of zero waste waste treatment can be used to solve household waste problems. the principle of zero waste is management by sorting, composting and collecting salable goods (widiarti, 2012). utilization of household organic waste in the form of other fruit residues can be processed by fermentation to produce a multi-purpose liquid or known as eco-enzyme (najaga bumi learning class module compilation team, 2020). there are many by-products that can be produced from fruit residues which are processed into eco-enzymes, including clearing clogged drains, used to water plants (as liquid fertilizer), repel nuisance insects, as a home cleaning agent, and others (megah, et al. al 2018). the type of bacteria used to produce optimum biogas is methanogenic bacteria (mia et al., 2016). these methanogen bacteria will stimulate the fermentation substrate to produce methane gas (ch4). this methane gas makes the biogas produced have an ignition power so that the biogas can be used as fuel. meanwhile, the basic material used to make biogas in this study is eco enzyme derived from fruit waste. cow dung is considered capable of being a starter in eco enzyme fermentation to produce biogas. the optimum production of biogas and methane from a mixture of cow dung and elephant grass was 72.42 l/kgts and 102.86 l/kgvs respectively (afrian, c. et al 2017). thus, based on the background of this problem, an analysis of the quality of biogas from eco-enzymes with the addition of cow dung starter and based on fruit composition and fermentation time needs to be carried out. https://doi.org/10.14421/biomedich.2024.131.35-42 36 biology, medicine, & natural product chemistry 13 (1), 2024: 35-42 method the type of the research is quantitative with experimental methods. the research method was designed using 5 treatments with 1 repetition. the eco-enzyme used is made from fruit-based ingredients that have been fermented for 3 months which is the optimal time for eco-enzyme fermentation. after the eco-enzyme is produced, a fermentation process is carried out by adding cow dung starter. the addition of cow dung starter was carried out to maximize this fermentation process to produce methane gas (ch4). the fermentation process was carried out for 37 days. 7 days the resulting gas is released into a bucket of water. gas storage for analysis was carried out on day seven and so on until day 37. the following is the treatment plan in this study: p1 : 3000 ml eco enzyme + 600 g cow dung starter p2 : 3000 ml eco enzyme + 800 g cow dung starter p3 : 3000 ml eco enzyme + 1000 g cow dung starter p4 : 1000 ml eco enzyme + 1000 g cow dung starter p5 : 1500 ml of water + 1500 g of cow dung starter making eco-enzyme solutions the fermentation solution used for biogas was an eco enzyme solution with the addition of cow dung starter. eco enzyme was made with a ratio of 3: 1: 10. 3 for the waste used; 1 for molasses and 10 for the ratio of water used. in this study, the manufacture of eco-enzymes was carried out at the al fatah natar islamic boarding school, south lampung. eco enzyme was made in large quantities in drums and fermented for 3 months. the waste material used for the manufacture of eco enzyme is fruit and vegetable waste. a total of 10,000 ml of eco enzyme solution was used in this study. making storage tanks the fermentation tank was made of a 5 l gallon. the lid on the top of the gallon was perforated for a gas outlet pipe with a diameter of 1⁄2 inch. two faucets are then placed above the fermentation tank before the gas collection point and one more valve is used for gas discharge. the collection point for the fermented gas (biogas) was made of plastic. figure 1. digester tube circuit. image description: 1. digester tube; 2. tube cover; 3. main faucet; 4. gas flow hose; 5. compressor faucet; 6. gas flow hose; 7. plastic gas holder. production of biogas from eco-enzyme the eco enzyme solution that has been fermented for 3 months was then ready to use. this solution was then mixed with cow dung to be fermented again for 37 days to produce biogas. during the fermentation process, specially designed vats are connected by a hose to the biogas storage vat (chandra et al, 2020). cow dung is added to the digester tube with varying weight according to the dosage. cow dung is used as a starter in fermentation to produce methane gas. then the molasses was also put into the digester tube according to the dosage determined in this study and labeled for each treatment. biogas samples were taken on the 37th day of fermentation. biogas sampling biogas samples were taken on the 37th day of fermentation using a previously prepared breathing bag. first sampling close the main faucet and compressor faucet, after the faucet is closed all the hoses of the digester tube and compressor faucet are separated. the end of the compressor faucet is directly connected to the breathing bag hose, after connecting the valve on the breathing bag is opened and the plastic container is pressed until the contained gas moves into the breathing bag. next, close the breathing bag valve immediately after gas the one transferred to the breathing bag is sufficient and close the compressor faucet again and remove the breathing bag. results and discussion results biogas volume pressure measurements made to measure volume pressure using technical archimedes' law with the following results: table 1. results biogas volume pressure. reactor increase (ml) p1 26 p2 60 p3 56 p4 50 p5 70 the results of the pressure measurement of biogas volume in table 1 show that the highest value was found in reactor p5, which was 70 ml, and the lowest value was in reactor p1, which was 26 ml. ph and temperature data on the results of measuring the ph acidity level on the initial day of manufacture and the last day of sampling are as follows: kamelia et al. – potential of methane (ch4), nitrogen (n2), and carbon dioxide (co2) … 37 table 2. result of ph value. reactor ph before after p1 3,4 3 p2 3,5 2,9 p3 3,5 3 p4 4 3 p5 6,3 6,1 table 3. results of temperature measurements at the start and end of the ecoenzyme fermentation. variation suhu (°c) before after p0 29,1 28,7 p1 28,8 28,1 p2 28,8 27,9 p3 28,9 28,1 p4 28,8 28,2 based on the results of measuring the acidity level of the ph of the fermentation solution in table 4.2, it shows that the lowest level of acidity on the first day of making the fermentation was in treatment p1 and on the last day there were in treatments p1, p3, and p4. the highest acidity level value on the first and last day was in the p5 treatment. in table 4.3 the results of initial and final temperature measurements show insignificant differences between each treatment. there was a decrease in temperature with a range of 0.40.90c. the highest temperature decrease was in treatment 2 of 0.90c. metana (ch4) data on methane concentration measurements that have been tested for biogas content are as follows: table 4. rated yield (ch4). reactor concentration (%) height (%) area(%) p1 0 0 0 p2 0 0 0 p3 0 0 0 p4 0,094 107 1486 p5 2,889 3435 50080 the results of measuring the concentration of methane (ch4) in table 4 show that the highest concentration level was found in treatment p5, which was 2.889, followed by treatment p4, which was 0.094. whereas in the p1 p3 treatment it did not show any concentration of methane (ch4) in it. the following is a graphic image of the results of the analysis of methane gas (ch4). figure 1. graph of p1 analysis results. figure 2. graph of the results of the p2 analysis. figure 3. graph of the results of the p3 analysis. figure 4. graph of the results of the p4 analysis. 38 biology, medicine, & natural product chemistry 13 (1), 2024: 35-42 figure 5. graph of the results of the p5 analysis. gas content of n2 and co2 in fermentation the results of the analysis of n2 and co2 content in biogas fermentation are presented in table 5: table 5. results of n2 and co2 values. sample type gas production (%) n2 co2 p0 96,565 0,546 p1 99,014 0,986 p2 95,875 4,125 p3 98.270 1,730 p4 99,669 0,237 table 5 shows that the production of nitrogen and carbon dioxide gases in the 5 types of samples tested has different and fluctuating values. the highest nitrogen production was in sample p4 (99.669%) and the highest amount of carbon dioxide was produced in sample p2 (4.12%). discussion this research was conducted to analyze several parameters in testing biogas from the main eco enzyme substrate with modifications of several materials. the addition of a starter is needed in this study with the aim of stimulating the growth of methanogenic bacteria which produce methane gas as the main gas in biogas. the initial stage of this research was to make eco enzyme from household organic waste with a ratio of 3:1. this waste is used based on the results of observations (preresearch). methane gas is produced after going through eco enzyme fermentation with added starter which lasts for 1 month. eco-enzymes are made with the commonly used ratio of 3 (organic matter): 1 (molasses): 10 (water). all ingredients are put in a sterile container (large plastic bucket with a wide lid) and covered for 3 months of fermentation. in the first month of fermentation it will produce gas, if the place used is made of glass there will be a risk of breaking. places with a metal base are not recommended. this is because it will trigger corrosive (rusty) at the end of the fermented product which is acidic (ph below 4). stirring needs to be done before the fermentation place is closed. this aims to flatten all the basic ingredients for making eco-enzymes. eco enzyme was chosen as the main substrate in forming the fermentation process, it needs to be added with molasses. molasses contains 30% sucrose, 25% glucose and fructose. all of these ingredients are important elements as a source of energy for microbes. the carbon element (c) from glucose will be needed to regulate the carbon value in the substrate. besides that, glucose adds value to carbon which is a source of nutrition for microbes that break down methane gas (zulkarnaen at al., 2018). the main element in biogas is the presence of methane gas. methane gas is produced from methanogenic bacteria. this bacterium can be obtained from several sources, one of which is by using starter from cow dung. cow manure is high in cellulose content (asmiarti, 2019), contains matanogenic bacteria that produce methane gas (karlina, 2017). all of these treatments were fermented for 30 days. this is based on previous sources which state that 30 days of fermentation is the best time for biogas production. the most methane gas is produced with a presentation of 50.4%, a temperature of 350°c and a flame of 72 seconds (mirwan and nadia, 2021). this fermentation process is carried out with the aim of producing methane gas. the gas produced from this process is accommodated in a gas holder in a tool that has been used assembled. the gas is then put in a sampling bag to be tested for biogas content using chromatography. based on the pressure research data, the data shows the results of different pressure values. it can be seen in table 4.1 that the highest pressure value is found in the p5 treatment reactor with a variation of 1500 g of water and 1500 g of cow feces producing a pressure value of 70 ml, in p4 with a variation of 1000 eco-enzyme and 1000 cow feces producing a pressure value of 50 ml, in treatment p3 with material variations 3000 g eco-enzyme and 1000 g cow feces produced a pressure value of 56 ml, in treatment p2 with material variations 3000 g eco-enzyme and 800 g cow feces produced a pressure value of 60 ml, in the reactor p1 treatment with 3000 g eco-enzyme and 600 g cow feces resulted in the lowest pressure value of 26 ml. the volume pressure value in the p2 treatment could be higher than the p3 treatment, this could be due to the unequal ratio of carbon (c) and nitrogen (n). if the ratio (c/n) is too high (the c value is higher than the n value) then metabolism becomes inadequate which means that there is carbon in the substrate that is not fully converted, so it cannot achieve maximum methane yield which also results in unstable pressure values. on the other hand, if the n value is excessive, the amount of ammonia (nh3) can be reduced, which even in low concentrations will inhibit bacterial growth kamelia et al. – potential of methane (ch4), nitrogen (n2), and carbon dioxide (co2) … 39 and can even cause the collapse of the entire population of microorganisms (zulkarnain et al., 2018). the main factor that can affect the difference in the volume pressure value of the biogas produced from each treatment is the physical properties of the filling material caused by the water content and acidity of the media (ph level) (mara and alit, 2011). apart from that, it can also affect the difference in the amount of volume produced by each treatment, one of which is the reduced number of microorganisms (novita et al., 2018), the number of organisms can be reduced due to the acidity of the ph which is too acidic which can cause the number of organisms to decrease. different pressure values can be influenced by the amount of methane gas produced from each reactor as in treatment p1 it does not produce methane gas, so the resulting pressure value is also small, namely 26 ml because the gas contained only contains nitrogen (n2) and carbon dioxide (co2). meanwhile, the p5 treatment had the greatest pressure value of 70 ml, because the gas contained in the p5 treatment reactor had a fairly large concentration of methane gas, namely 2.889. the acidity level of ph is one of the determining factors for success in the formation of biogas. an acidity level that is too high or low will affect the amount of biogas produced. the degree of acidity of this ph is monitored every time a sample is taken to determine the condition of the substrate in the digester tube (budiharjo, 2009). testing the acidity level of ph in eco-enzyme fermentation with the addition of cow feces starter was carried out using a ph meter. testing the ph acidity level was carried out twice, namely on day 0 (beginning of fermentation) and on day 37 (last day when sampling). based on research data, the value of the ph acidity level in ecoenzyme fermentation on day 0 was the lowest ph acidity level in treatment p1, namely 3.4, in treatment p2, namely 3.5, in treatment p3, namely 3.5, in treatment p4, namely 4, and in treatment p5, which was equal to 6.1 is the highest ph acidity level value in the first test. meanwhile, in the second test on the last day the acidity level of each treatment reactor decreased overall. in treatment p1 it fell to 3, in treatment p2 it fell to 2.9, in treatment p3 it fell to 3, in treatment p4 it fell to 3, and in treatment p5 it fell to 6.1. the optimum condition for the ph acidity level is between 6.8-7.2. if the substrate drops, it will cause the process of converting the substrate into biogas to be hampered, resulting in a decrease in the quantity of biogas. a ph value that is too high must also be avoided, because it will cause the final product to be co2 as the main product (dwivannie et al., 2019), whereas in this study the treatment almost reached the optimum conditions for biogas so that it can produce methane gas, namely in treatment p4 (4 and 3) and p5 (6.3 and 6.1). the results of research by rahim et al., (2017) biogas in this case the ch4 content in general will produce high in ph conditions that are close to neutral. this is related to the development of methane bacteria which will be encouraged to grow and reproduce optimally, so that it will have an impact on biogas production. according to ramdiana, in the ph range of 6-6.7, very few methanogenic bacteria are able to survive to produce gas because the acidity of the substrate can kill the bacteria, so that methane gas is still produced, even in small quantities. the microorganisms that work in the early stages are microorganisms in the hydrolysisacidogenesis process which produce volatile acids so that the ph value drops (ni'mah 2014). in the initial and final temperature tests, it was seen that there was a decrease in each of the treatments tested. the temperature drop is not too big, still in the range of 0.4 – 0.9oc. this is influenced by the condition of the environment around the test site which is drizzling. this condition slightly affects the temperature in the digester. this statement is in line with research conducted by widodo (2022), the digester temperature is similar to the ambient temperature in the morning or evening. the measurement results in the p0 and p4 treatments had a larger value and produced methane gas (ch4) in small amounts. as research conducted by widodo (2022), the higher the temperature of the fermentation indicates that the decomposition of organic matter is occurring, which is the basic ingredient for producing methane, carbon dioxide and other gases. the best temperature for fermentation is the mesophilic temperature, which is around 20-40oc (syaichurrozi, 2020). in this study, the initial and final temperatures were measured in the mesophilic temperature range (2729oc). methanogenic bacteria will work well at mesophilic fermentation temperatures and the best (optimum) temperature for mesophilic bacteria to produce biogas is 35oc (adiani et al., 2019). the optimum temperature is the perfect temperature for bacterial metabolic processes. high or low temperatures will cause imperfect bacterial metabolism and can stop. at low temperatures there are parts that turn into gel, one of which is the double layer of lipid membrane which generally forms a liquid into a gel. this can cause damage to protein function (denaturation) and leakage so that the protein cannot pump ions. another impact is the late bacterial growth (asiah, 2020). at high temperatures it can damage the bacterial cell membrane. if this membrane is damaged, protein denaturation will occur and activity in the bacterial cell will decrease (roma et al., 2021). this decrease in bacterial activity will eventually lead to the death of the bacteria. bacterial cell death has an impact on reducing the composition of methanogenic bacteria. this change in composition results in the accumulation of gas resulting from the hydrolysis and acidogenesis stages (fatty acids, glycerin, monosaccharides, amino acids, acetic acid, butyric acid and propionic acid) which are not converted into methane gas. 40 biology, medicine, & natural product chemistry 13 (1), 2024: 35-42 testing the sample for methane (ch4) content in the eco-enzyme fermentation solution which was added to the cow feces starter was carried out using a gas analyzer. judging from the research results table 4.3, the p1-p3 treatment did not show methane (ch4) content, while in figure 4.4 it can be seen that the p4 treatment showed a (ch4) content of 0.094 with a graphic height of 107 and an area of 107. in addition, in figure 4.5 can be seen that the need for p5 also indicates the presence of content (ch4), which is 2.889 with a graph height of 3435 and an area of 50080. it can be seen from the research data table 4.3 that the greater the ratio of the eco-enzyme solution given, the smaller the possibility of the presence of (ch4) content, whereas the higher the ratio of cow feces starter, the greater the possibility of (ch4) content. this is because in treatment p1 the ratio used in the fermentation solution was 3000 g of eco-enzyme and 600 g of cow feces. in the p2 treatment the comparison used was 3000 eco-enzyme and 800 g of cow feces, and in the p3 treatment the comparison used was 3000 eco-enzyme and 1000 cow feces. this much larger amount of eco-enzyme can affect the performance of the bacteria in the fermentation process, because the ingredients contained in the eco-enzyme solution are ingredients that contain antimicrobials or inhibit bacterial growth such as pineapple, orange peel, aloe vera, noni, cultivar. bananas, and others (rochmayani et al., 2020). another factor that affects the amount of methane gas (ch4) produced is only a small amount and is only produced in the p4 and p5 treatments because the number of comparisons used in p4 giving eco-enzyme is comparable to giving cow feces, namely 1000 g ecoenzyme and 1000 g cow feces , whereas in treatment p5, the ratio of water to cow feces was used, namely 1500 water and 1500 cow feces. thus, methane gas (ch4) can be produced in treatments p4 and p5. another influencing factor can be seen from the acidity level of the ph of the fermentation solution, because the optimal ph conditions required for biogas production are in the range of 6.6 to 7.5 (fitri and trisna, 2018). in treatment p1-p3 the ph acidity level is below optimal conditions, namely in treatment p1 the ph on day 0 is 3.4 and the ph of the 37th curry is 3. so the bacteria that help the process of forming methane gas (ch4) are hampered or can even die. the low population of methanogenic bacteria results in the accumulation of volatile acids that have not been converted into methane gas, so that little or no gas is produced (roma et al., 2021). the n2 and co2 gas content that has been analyzed can be seen in table 4.5. the highest yields obtained were nitrogen (n2) and carbon dioxide gas (co2). the concentration of nitrogen gas (n2) from reactors p0-p4 was in the range of 95.875-99.669% and the concentration for carbon dioxide (co2) from reactors p0 p4 was in the range of 0.2374.125%. stirring the reactors p0, p1, p2, p3 and p4 by shaking the digester tube. the digester tube is made using a batch type with an anaerobic system so that it cannot be opened when stirring. this is thought to cause non-optimal contact between the organism and the substrate, thereby affecting the methane gas concentration value. in line with research which states that stirring in the digester will allow direct contact between microorganisms and gas-forming substrates. the higher the stirring frequency, the higher the opportunity for microorganisms to degrade the substrate. using a stirrer, methane gas trapped in solution can be immediately released and enters the gas reservoir. stirring provides sufficient contact between the substrate and the microorganism to produce a homogeneous condition. a digester with a stirrer produces more optimal biogas compared to a biodigester without a stirrer (suryani et al., 2018). reactor p0 is a reactor with control treatment. reactor p0 and reactor p4 both produce methane gas. this is presumably because both of them use filling materials in a 1:1 ratio. in the p0 reactor using a ratio of 1.5 l of water + 1500g of cow dung. the addition of water to the p0 reactor was carried out because based on research it was stated that microorganisms in metabolism need water. the amount of water needed for biogas varies depending on the material used. in order to function normally, biogas-producing microbes require a substrate with a moisture content of 90% and a solids content of 8-10%. the raw material should be diluted 1:1 if the raw material is 100 kg of manure, then the water needed is 100 kg (kamal, 2019). in the p4 reactor, a ratio of 1 l of water + 1000 g of cow dung is used. based on research results, it is stated that biogas with a 1:1 ratio between substrate and starter produces the most optimal biogas (widodo, 2022). even though there is methane gas in the p0 reactor and p4 reactor, the nitrogen (n2) content obtained is still quite high in all reactors. this is probably due to the gas reservoir and sample container not being vacuumed, resulting in air contamination from outside the digester (amanda, 2020). conclusion the conclusion was based on the results of research analysis of methane content (ch4), ph, and pressure in eco-enzyme fermentation with the addition of cow feces starter. this research was conducted for 37 days from july to august, the results can be summarized as follows: ▪ the treatment that produced the highest methane gas (ch4) was the p5 treatment with a concentration of 2.889%. ▪ the ph acidity level that reached optimal conditions was in the p5 treatment, namely on the 0th day of sampling it was 6.3 and on the 37th day it was 6.1. ▪ the highest volume pressure value was produced in treatment p5, namely 70 ml. kamelia et al. – potential of methane (ch4), nitrogen (n2), and carbon dioxide (co2) … 41 competing interest: the authors declare that there are no competing interests. references abbasi, tasneem, s. 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oluwafemi emmanuel ekun department of biochemistry, faculty of science, adekunle ajasin university, akungba akoko, ondo state nigeria, nigeria. corresponding author oluwafemi.ekun@aaua.edu.ng manuscript received: 29 december, 2022. revision accepted: 28 july, 2023. published: 04 august, 2023. abstract the multidirectional abilities of peptide digests and fractions obtained from the hydrolysis of food-based proteins have been investigated in recent times. this study aims to evaluate the effects of pepsin-derived moringa oleifera seed protein hydrolysates and fractions on hemoglobin glycation and the carbohydrases α-amylase and α-glucosidase. proteins were extracted from m. oleifera seeds and consequently digested using pepsin. the hydrolysates obtained were separated into fractions of <1 kd, 1-3 kd, and 3-5 kd ranges using size-exclusion chromatography and comparison with elution volumes of known standards. the activities of the hydrolysates and peptide fractions against both the non-enzymatic glycation of hemoglobin and the carbohydrases were determined in vitro. results revealed that the hydrolysate and its peptide fractions demonstrated varying abilities against the glycation of hemoglobin, with the unfractionated hydrolysate showing better activities (78.230 ± 0.774 % at a maximum concentration of 1.0 mg/ml) than its peptide fractions. also, the hydrolysates and fractions demonstrated higher inhibitory effects on -amylase (with all fractions displaying above 50% inhibition at a final concentration of 1.0 mg/ml) than against -glucosidase. kinetic analysis of a selected fraction showed that it inhibited α-amylase via a mixed mechanism (ki = 0.029 mg/ml) but displayed an uncompetitive mode for α-glucosidase inhibition (ki = 0.333 mg/ml). therefore, it is inferred that m. oleifera seed proteins encode potentially therapeutic peptide sequences that could be further processed to formulate potential antidiabetic agents. keywords: moringa oleifera; peptide; pepsin; hemoglobin; -amylase; α-glucosidase. introduction diabetes mellitus is an endocrine and metabolic disease, which is characterized by elevated plasma and urinary glucose levels as a direct result of inadequate insulin production and/or action (desmukh and jain, 2015). in its various forms, diabetes mellitus causes a plethora of derangements in the metabolisms of carbohydrates, proteins, and lipids (olusola and ekun 2019a). these cause a wide range of complications such as diabetic ketoacidosis, neuropathy, nephropathy, and cardiovascular and multiple organ damage at later stages of the disease (piero et al., 2014; olusola et al., 2018). one particular aspect in the pathogenesis of diabetes mellitus is the fact that persistent hyperglycemia leads to the formation of advanced glycated end products, (ages), as glucose complexes with proteins, and to a relatively lesser extent, lipids in a non-enzymatic, covalent fashion (ramasamy et al., 2005). proteins such as hemoglobin and albumin are frequently glycated (singh et al., 2014). these glycated end products are known to potentiate certain pro-inflammatory pathways via the production of reactive oxygen species. these ages bind to, and activate the receptor for advanced glycated end products, (rage), which in turn activate downstream signaling cascades which play important roles in the progression of diabetic nephropathy, neuropathy, and retinopathy (singh et al., 2014; perrone et al., 2020). current strategies employed in the management of diabetes mellitus have been aimed at controlling postprandial glucose levels (arise et al., 2016). these include insulin preparations (for type 1 diabetes mellitus) and a combination of lifestyle changes and/or the use of oral hypoglycemic drugs (in the case of type 2 diabetes mellitus), which act by modulating the activities of key proteins and enzymes involved in carbohydrate hydrolysis and transport (olusola and ekun, 2019a). enzymes such as α-amylase, αglucosidase, and dipeptidyl peptidase (iv) have been key pharmacologic targets of oral antidiabetic agents (rosenstock and zimmerman, 2007, arise et al., 2016, olusola and ekun, 2019b). however, owing to certain deleterious effects and cost of procurement on the part of these synthetic drugs, attention has since turned to products from natural sources with the purpose of exploring them as possible alternatives in the management of diabetes mellitus (oboh et al., 2011, parveen et al., 2017). in recent times, peptide products https://doi.org/10.14421/biomedich.2023.121.413-422 414 biology, medicine, & natural product chemistry 12 (1), 2023: 413-422 and protein hydrolysate preparations from enzymatic hydrolysis of plant and animal proteins are increasingly exploited and investigated for their numerous bioactivities (lopez-barrios et al., 2014; majumder and wu, 2015; siow and gan, 2016). peptides differing in lengths, sizes, and derivation have been widely used for a number of therapeutic purposes (kumar et al., 2015, vilcacundo et al., 2019). one plant whose proteins contain potentially biologically active peptides is moringa oleifera. moringa oleifera, is a plant that belongs to the family moringaceae. it is originally endemic to the himalayan regions of india but is also abundant in the middle east and cultivated in many regions of sub-saharan africa (madubuike et al., 2015). it is fast-growing, and drought resistant and this may have accounted for its wide geographical spread (madubuike et al., 2015, leone et al., 2016, abd-rami et al., 2018). various parts of the plant have been utilized for numerous purposes such as water treatment, as a food source and supplements, and also in traditional medicine (anwar et al., 2007, abdrani et al., 2018). its seeds and leaves are rich sources of nutrients and essential oils (kwaambwa et al., 2015), and proteins (mune-mune et al., 2016). its proteins consist of albumins, globulins, prolamins, and glutelins (kwaambwa et al., 2015) which could yield potentially bioactive peptide products on enzymatic hydrolysis. in addition, its seeds are abundant in certain amino acids such as glutamate, aspartate, arginine, proline, and threonine, but limited in sulfur-containing amino acids (freire et al., 2015; mune-mune et al., 2016). various extracts of its leaves and seeds have been reported to possess potent pharmacologic effects (abd-rani et al., 2018). solvent extracts of its leaves demonstrated antioxidant properties, anticancer potentials (monera et al., 2008; al-asmari et al., 2015), and antimicrobial activities (walter et al., 2011; abd-rani et al., 2018). in addition, m.oleifera leaf and seed extracts (aqueous and ethanolic) were found to demonstrate hypoglycemic, and hypolipidemic potentials in rat models, by reducing insulin resistance and increasing insulin sensitivity (tuorkey, 2016); as well as inhibiting the formation of advanced glycated end products (nunthanawanich et al., 2016). crude enzymatic digests of m. oleifera seed proteins demonstrated enzyme-inhibitory activities in earlier studies (olusola et al., 2018, olusola and ekun, 2019b). however, hydrolysate fractionation is essential to further characterize bioactive peptides (awosika and aluko, 2019). as a result, this study aims to evaluate the activities of fractionated peptides obtained from pepsinassisted hydrolysis of m. oleifera seed protein on hemoglobin glycation and carbohydrate hydrolyzing enzymes, in a bid to identify novel peptides with antidiabetic potentials. materials and methods materials collection of seeds m. oleifera seeds were obtained from farms in akungba akoko, ondo state and they were identified, and voucher samples were deposited at the department of plant science and biotechnology, adekunle ajasin university, akungba akoko. chemicals and reagents pepsin (from porcine gastric mucosa), and α-amylase (fungal), α-glucosidase (human) were products of sigmaaldrich laboratories, co-artrim, united kingdom. all other chemicals and reagents used were of analytical grade and were also products of sigma-aldrich laboratories, united kingdom. methods isolation of m. oleifera seed proteins the seeds were dried and pulverized before being kept in an air-tight container at 4oc. this was subsequently defatted using n-hexane as was previously described by arise et al., (2016a) with slight modifications. the meal was extracted three times with n-hexane using a meal/solvent ratio of 1:10 (w/v). the meal was then dried at 40oc in a vacuum oven and ground again to obtain a fine powder, termed defatted seed meal, which was stored at -20oc. the protein component of the defatted meal was extracted using the method described by alashi et al. (2014). defatted watermelon seed meal was suspended in 0.1 m naoh ph 12.0 at a ratio of 1:10, and stirred for one hour to facilitate alkaline solubilization. this was centrifuged at 18°c and 3000 g for 10 min. two additional extractions of the residue from the centrifugation process were performed with the same volume of 0.1 m naoh and the supernatants were then pooled. the ph of the supernatant was adjusted to 4.0 to facilitate acid-induced protein precipitation using 0.1 m hcl solution; the precipitate formed was recovered by centrifugation. the precipitate was washed with distilled water, adjusted to ph 7.0 using 0.1 m naoh, freezedried, and the protein isolate was then stored at -20°c prior to further analysis. preparation of m. oleifera seed protein hydrolysates the protein isolate was hydrolyzed using the methods described by olusola and ekun, (2019b) with slight modifications. hydrolysis was carried out using pepsin (ph 2.2, 37ºc). the protein isolate (5% w/v, based on the protein content of the isolate) was dissolved in glycine buffer at ph 2.2. the enzyme was added to the slurry at an enzyme-substrate ratio (e:s) of 2:100. digestion was performed at the specified conditions for 6 hours with continuous stirring. the enzyme was then inactivated by boiling in a water bath (95–100oc) for 15 min and undigested proteins were precipitated by adjusting the ph to 4.0 with 2 m hcl/2 m naoh followed by ekun – peptide fractions from pepsin-digested moringa oleifera seed proteins … 415 centrifugation at 6000 g for 30 min. the supernatant containing target peptides was then collected. protein content of samples was determined using the biuret assay method with bovine serum albumin (bsa) as standard. fractionation of m. oleifera seed protein hydrolysates the moringa oleifera seed protein hydrolysates were separated into molecular weight fractions using gel filtration chromatography according to the method described by ekun et al., (2022). summarily, 5 ml of the clear supernatant resulting from protein hydrolysis, at a protein concentration of 10 mg/ml was filtered, suspended in 50 mm phosphate buffer ph 7, and passed into a sephadex g25 chromatographic column of dimensions 30 cm x 4 cm which had earlier been equilibrated with the buffer. the same phosphate buffer was used to elute the separating fractions, and the elution peaks were monitored at 400 nm. the separating fractions eluted under the same elution peak were collected, pooled and their molecular weights were determined by comparison with the graph of the logarithm of molecular weights against elution volumes of known standards (vitamin b12, tryptophan, aspartame, glycine, and bovine serum albumin). the eluates, according to their molecular weights, were then sorted into <1 kd, 1-3 kd, and 3-5 kd ranges. peptide fractions of molecular weights higher than 5 kda were removed and discarded. the collected peptide fractions were stored at -200c for further analysis. determination of peptide yield the percentage peptide yield was determined using the method described by girgih et al. (2011). the peptide yields (%) of moringa oleifera seed protein hydrolysates and fractions, were calculated as the ratio of peptide content of lyophilized hydrolysate/fraction to the protein content of unhydrolysed protein isolate. inhibition of hemoglobin glycation this was investigated by estimating the degree of nonenzymatic hemoglobin glycation according to the method described by venu et al., (2016) with modifications. glucose solution (2%), 0.06% hemoglobin, and gentamycin (0.02%) solution were prepared in phosphate buffer 0.1 m, ph 7.4. 1 ml of each of the above solutions was mixed. 0.2 mg/ml 1.0 mg/ml of hydrolysate was added to the above mixture. gallic acid was used as standard. the mixture was kept in the dark at room temperature for incubation for 72 hours. at 520 nm, hemoglobin glycation was measured with a spectrophotometer, and % inhibition was calculated thus: % ℎ𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 𝑔𝑙𝑦𝑐𝑎𝑡𝑖𝑜𝑛 = 𝐴𝑏𝑠 (𝑠𝑎𝑚𝑝𝑙𝑒) − 𝐴𝑏𝑠 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙) 𝐴𝑏𝑠 (𝑠𝑎𝑚𝑝𝑙𝑒) 𝑥100% determination of α-amylase inhibition an α-amylase-inhibitory assay was performed according to the method reported by oboh et al. (2011). briefly, 125 µl of hydrolysate (0.5 to 2.0 mg ml–1) was placed in test tubes and 125 µl of 20 mm sodium phosphate buffer (ph 6.9, with 6mm nacl) containing α-amylase solution (0.5 mg/ml) added. the content of each tube was pre-incubated at 25 °c for 10 min, after which 125 µl of 1% starch solution in 20 mm sodium phosphate buffer (ph 6.9, with 6 mm nacl) was added at regular intervals. the reaction mixtures were incubated at 25 °c for 10 min. the reaction was terminated by adding 250 µl of dinitrosalicylic acid (dns) colour reagent and further incubated in boiling water for 5 min and cooled to room temperature. the content of each test tube was diluted with 2.5 ml distilled water and the absorbance was measured at 540 nm. a control was also prepared using the same procedure except that the hydrolysate was replaced with distilled water. the α-amylase-inhibitory activity was calculated as shown: % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 – 𝐴𝑠𝑎𝑚𝑝𝑙𝑒) 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 determination of kinetic parameters of α-amylase inhibition the kinetic study of α-amylase inhibition was conducted according to the method described by olusola and ekun (2019a). 125 µl of the hydrolysate was pre-incubated with 125 µl of α-amylase solution for 10 min at 25°c in a set of tubes. in another set of tubes, 250 µl of phosphate buffer (ph 6.9) was also pre-incubated with 125 µl of α-amylase solution. starch solution (125 µl) of increasing concentrations (1.0 to 8.0 mg/ml–1) was added to both sets of reaction mixtures to initiate the reaction. the mixture was then incubated for 10 min at 25 °c, and then boiled for 5 min after the addition of 250 µl of dinitrosalicylic acid (dns) reagent to stop the reaction. the amount of reducing sugars released was determined spectrophotometrically from a maltose standard curve and converted to reaction velocities as shown below: 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ((µ𝑚𝑜𝑙 𝑚𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛– 1) 𝑚𝑖𝑛– 1) = 𝑀𝑎𝑙𝑡𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 / 𝐼𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 × 𝑀𝐸 where maltose concentration is in µmol/ml–1; incubation time = 10 min; me= amount of enzyme (in mg) in the reaction mixture a double reciprocal plot (1/v versus 1/[s]), where v is reaction velocity and [s] is substrate concentration was plotted. the mode of inhibition and the kinetic parameters (km, k΄m, vmax, v΄max, ce, and ce΄) of αamylase inhibition by hydrolysates were determined by analysis of the double reciprocal plot. the inhibition constant (ki) was determined using a secondary plot known as the dixon plot (palmer, 2007), by plotting a graph of the inverse of initial velocities on the x-axis 416 biology, medicine, & natural product chemistry 12 (1), 2023: 413-422 against inhibitor concentrations on the x-axis, at a fixed concentration of substrate. determination of α-glucosidase inhibition the effect of the hydrolysates on 𝛼-glucosidase activity was determined according to the method described by kim et al., (2005) using 𝛼-glucosidase from saccharomyces cerevisiae. the substrate solution pnitrophenyl glucopyranoside (pnpg) was prepared in 20 mm phosphate buffer, and ph 6.9. 100𝜇l of 𝛼 glucosidase (1.0 u/ml) was pre-incubated with 50𝜇l of the different concentrations of the hydrolysates for 10 min. then 50𝜇l of 3.0 mm (pnpg) as a substrate dissolved in 20 mm phosphate buffer (ph 6.9) was added to start the reaction. the reaction mixture was incubated at 37∘c for 20 min and stopped by adding 2ml of 0.1 m na2co3 solution. the 𝛼-glucosidase activity was determined by measuring the yellow-colored paranitrophenol released from p-npg at 405 nm. the results were expressed as a percentage of the blank control. percentage inhibition was calculated as: % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙– 𝐴𝑠𝑎𝑚𝑝𝑙𝑒) 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 determination of kinetics of α-glucosidase inhibition the kinetic parameters of 𝛼-glucosidase by the hydrolysates were determined according to the method reported by ali et al., (2006). briefly, 50 𝜇l of the (5 mg/ml) hydrolysate was pre-incubated with 100 𝜇lof 𝛼glucosidase solution for 10 min at 25∘c in one set of tubes. in another set of tubes, 𝛼-glucosidase was preincubated with 50𝜇l of phosphate buffer (ph 6.9). 50 𝜇l of pnpg at increasing concentrations (0.63–2.0 mg/ml) was added to both sets of reaction mixtures to start the reaction. the mixture was then incubated for 10 min at 25∘c, and 500 𝜇lof na2co3 solution was added to stop the reaction. the amount of reducing sugars released was determined spectrophotometrically at 405nm using a para nitrophenol standard curve and converted to reaction velocities. a double reciprocal plot (1/v versus 1/[s]) where v is reaction velocity and [s] is substrate concentration was plotted. the mode of inhibition of the hydrolysates on 𝛼-glucosidase activity was determined by analysis of the double reciprocal (lineweaver-burk) plot using michaelis menten kinetics. the inhibition constant (ki) was also determined using the dixon plot (palmer, 2007), by plotting a graph of the inverse of initial velocities on the x-axis against inhibitor concentrations on the x-axis, at a fixed concentration of substrate. results peptide yield the peptide yields of the hydrolysate and its fractions are illustrated in table 1. the unfractionated hydrolysate had the highest yield. however, among the fractions, the 1-3 kd fraction had significantly higher yields (p < 0.05) when compared to the other peptide fractions. table 1. peptide yields of m. oleifera seed protein hydrolysate and its fractions. values are presented as means ± standard error of the mean of triplicate determinations. yields carrying different symbols are significantly different (p<0.05). yields carrying the same letter or symbol are not significantly different (p<0.05). inhibition of hemoglobin glycation the effects of moringa oleifera seed protein hydrolysates and fractions on non-enzymatic hemoglobin glycation, in comparison to gallic acid, at a concentration range of 0.2mg/ml to 1.00mg/ml is illustrated in figure 1. the hydrolysate and fractions demonstrated significantly lower (p<0.05) inhibitory effects when compared to gallic acid (control), especially at higher concentrations. the unfractionated peptic hydrolysate had higher (p<0.05) inhibitory activities than its fractions, attaining a maximal value of 78.230 ± 0.774 % at 1.0 mg/ml. considering the peptic hydrolysate fractions, fraction f1 had a maximum inhibition of 66.667 ± 2.138 % at 0.8 mg/ml which was not significantly different (p<0.05) from that of fraction f2, with activity of 62.667 ± 3.079 at a concentration of 0.2 mg/ml, but was higher (p<0.05) than fraction f3 which had 58.730 ± 1.833% inhibition at a final concentration of 1.0mg/ml. sample yield unfractionated hydrolysate 80.545 ± 4.949a fraction 1 (<1 kd) 19.709 ± 1.096c fraction 2 (1-3 kd) 25.835 ± 2.166b fraction 3 (3-5 kd) 14.485±0.968d ekun – peptide fractions from pepsin-digested moringa oleifera seed proteins … 417 figure 1. percentage inhibition of hemoglobin glycation by peptic moringa oleifera seed protein hydrolysate and its fractions. bars are expressed as means ± standard error of the mean of triplicate determinations (n=3). values within the same concentration but with different letters are significantly different (p<0.05). values at different concentrations of the same hydrolysate with different symbols are also significantly different (p<0.05). bars carrying the same letter or symbol are not significantly different from one another (p<0.05). inhibition of -amylase activity the percentage -amylase inhibitory activity of moringa oleifera seed protein hydrolysate and their fractions in relation to arabinose are illustrated in figure 2. the inhibitory activities of peptic hydrolysates and fractions at all study concentrations were significantly (p<0.05) lower than those of arabinose. all hydrolysate fractions demonstrated higher (p<0.05) inhibitory activity than the unfractionated hydrolysate. also, the fractions demonstrated percentage inhibitory activity above 50% at concentrations of 0.40 mg/ml to 1.0 mg/ml. fractions 1 and 2 with 66.8% and 64.59% inhibition respectively at a maximum concentration of 1.0 mg/ml had higher (p<0.05) effects than the unfractionated hydrolysate and fraction 3. figure 2. percentage -amylase inhibitory activity of peptic moringa oleifera seed protein hydrolysates. bars are expressed as means ± standard error of the mean of triplicate determinations (n=3). values within the same concentration but with different letters are significantly different (p<0.05). values at different concentrations of the same hydrolysate with different symbols are also significantly different (p<0.05). bars carrying the same letter or symbol are not significantly different from one another (p<0.05). kinetics of -amylase inhibition the effect of a selected peptide fraction – fractions f1, (<1 kd), – on the catalytic activity of -amylase in converting starch to maltose is presented in figure 3. kinetic parameters determined from the lineweaverburk plot in the absence and presence of two different concentrations of the peptide fraction were summarized in table 2. in the absence of the hydrolysate fraction, the michaelis constant, km, of -amylase for its substrate was found to be 0.552 mg/ml of starch while maximal velocity, vmax was 3.890 mm/mg/min. inhibition of amylase activity increased with increasing concentrations of the peptide fraction, such that the km of the enzyme was increased while vmax and catalytic efficiency, ce, of -amylase were reduced in the presence of the inhibitory fraction. the enzyme-inhibitor dissociation constant, ki, 418 biology, medicine, & natural product chemistry 12 (1), 2023: 413-422 of -amylase inhibition by peptic hydrolysate fraction f1 was determined to be 0.029 mg/ml. figure 3. lineweaver-burk plot of -amylase inhibition by moringa oleifera seed protein hydrolysate fraction 1 (<1kd) obtained from peptic proteolysis. table 2. kinetics of -amylase-catalyzed reactions in the presence and absence of m. oleifera seed protein hydrolysate fraction 1. kinetic parameters no inhibitor peptic hydrolysate fraction 1 (mg/ml) 0.5 1.0 km or kˈm (mg/ml) 0.552 0.768 0.701 vmax or vˈmax (mm/mg/min) 3.890 3.833 1.789 ce (mmol/ml/min) 7.053 4.993 2.551 ki (mg/ml) 0.029 km or kˈm: michaelis constant in the absence or presence of inhibitory hydrolysate fractions; vmax or vˈmax: maximum velocity in the absence or presence of inhibitory hydrolysate fractions; ce: catalytic efficiency; ki: enzyme-inhibitor dissociation constant. -glucosidase inhibitory activity the inhibitory activities of the m. oleifera seed protein hydrolysates and their fractions on α-glucosidase – catalyzed hydrolysis of p-nitrophenyl glucopyranoside at varying concentrations in comparison to arabinose (control) are presented in figure 5. peptic hydrolysate and its fractions displayed lower (p<0.05) inhibitory activities when compared to the standard, arcabose. however, peptic hydrolysate fraction 1 (<1 kd) attained maximum inhibitory activity of 46.09 ± 3.331 % at a concentration of 0.6mg/ml, while fraction 2, pf2, attained a maximum inhibitory extent of 59.449 ± 2.342 % at a concentration of 0.8 mg/ml. fraction 3 had significantly lower (p<0.05) -glucosidase inhibitory activity when compared to the unfractionated peptic hydrolysate and the other low molecular weight fractions obtained from peptic digestion. figure 4. percentage -glucosidase inhibitory activity of peptic moringa oleifera seed protein hydrolysates. bars are expressed as means ± standard error of the mean of triplicate determinations (n=3). values within the same concentration but with different letters are significantly different (p<0.05). values at different concentrations of the same hydrolysate with different symbols are also significantly different (p<0.05). bars carrying the same letter or symbol are not significantly different from one another (p<0.05). ekun – peptide fractions from pepsin-digested moringa oleifera seed proteins … 419 kinetics of α-glucosidase inhibition the effect of a selected m. oleifera seed protein hydrolysate fraction (fraction f1, (<1 kd) on the kinetics of α glucosidase – catalyzed hydrolysis of p-nitrophenyl glucopyranoside, p-npg, to p-nitrophenol are illustrated in figure 5. the kinetic parameters from the resulting lineweaver burk plots are summarized in table 3. in the absence of an inhibitor, the michaelis constant, km of α glucosidase for its substrate was determined to be 0.297 mg/ml p-npg, while maximum velocity, vmax, was 270.27 mm/mg/min. the hydrolysate fraction also caused reductions in the vmax and catalytic efficiency, ce of the enzyme at concentrations of 0.5 mg/ml and 1.0 mg/ml. figure 5. lineweaver-burk plot of -glucosidase inhibition by moringa oleifera seed protein hydrolysate fraction 1 (< 1kd) obtained from peptic proteolysis. table 3. kinetic parameters of -glucosidase inhibition by m. oleifera seed protein hydrolysate fraction 1. kinetic parameters no inhibitor peptic hydrolysate fraction 1 (mg/ml) 0.5 1.0 km or kˈm (mg/ml) 0.297 0.262 0.293 vmax or vˈmax (mm/mg/min) 270.270 163.934 172.414 ce (mmol/ml/min) 910.001 625.702 588.443 ki (mg/ml) 0.333 km or kˈm: michaelis constant in the absence or presence of inhibitory hydrolysate fractions; vmax or vˈmax: maximum velocity in the absence or presence of inhibitory hydrolysate fractions; ce: catalytic efficiency; ki: enzyme-inhibitor dissociation constant. discussion peptide yield peptide yield gives an estimate of the amount, in percentage, of peptides generated relative to the whole protein subjected to enzymatic proteolysis; thus it represents an important index in determining the efficiency of the overall process (alashi et al., 2014), as these enzymes degrade the proteins into several peptides of varying lengths and sizes. it, therefore, follows that a high peptide yield is indicative of increased proteolysis and resultant peptide release (girgih et al., 2011). the peptide yield of the unfractionated hydrolysates was higher than those of their corresponding fractions put together, and this could be due to peptide loss during the process of chromatography and the removal of peptides whose molecular weights were higher than 5 kda. this is consistent with the reports of awosika and aluko (2019) in their work with yellowfield pea protein digests and peptide fractions. the yield of peptic hydrolysates obtained in this study was higher than 32.33 ± 1.046% and 68.90 ± 1.00% determined for peptic digests of arachis hypogea (olusola and ekun, 2018) and watermelon seed proteins (arise et al., 2016b) respectively. this implies that m.oleifera seed proteins also had a lot of hydrophobic amino acid residues, making them susceptible to cleavage by pepsin, as this enzyme, though relatively non-specific but has a preference for hydrolyzing peptide linkages at the cterminal ends of hydrophobic aminoacyl residues (munemune et al., 2016; voet et al., 2016). inhibition of hemoglobin glycation the formation of advanced glycation end products (ages) as a result of poorly controlled hyperglycemia in diabetes mellitus leads to a plethora of complications such as retinopathy, renal dysfunction atherosclerosis, among other devastating conditions (ramasamy et al., 2005, singh et al., 2014). these ages cause deleterious effects by promoting the generation of reactive oxygen species which activate a cascade of signaling pathways, leading to an increase in the production of pro 420 biology, medicine, & natural product chemistry 12 (1), 2023: 413-422 inflammatory mediators, invariably causing other complications such as the formation of atherosclerotic plaques and culminating in cardiovascular disease in diabetic patients (han et al., 2014). certain plant extracts have been reported to inhibit hemoglobin glycation in vitro (hosseini et al., 2015; venu et al., 2016), but information has been scarce on the abilities of peptides and protein hydrolysates to inhibit hemoglobin glycation. m. oleifera seed protein digests and their fractions exhibited lower inhibitory effects on hemoglobin glycation than gallic acid. however, the unfractionated hydrolysates had better inhibitory activities than their fractions at all study concentrations, and this indicates that fractionation, in this case, may have reduced the inhibitory activities of the peptide fractions to prevent non-enzymatic glycation of hemoglobin. this result suggests that there could be synergistic effects among these peptides, which make them more effective as a mixture than being fractionated. among the fractions, peptic fractions 1 and 2, exhibited inhibitory effects above 60%. pepsin is relatively non-specific in its cleavage specificities (naik, 2012), releasing peptides that have hydrophobic and aromatic side chains that could significantly slow down the glycation of hemoglobin. this is consistent with the reports of han et al., (2014), that asn-trp dipeptides inhibited the formation of ages in mice models. however, more investigation is required in further studies to determine the nature of other aminoacyl residues that may be involved in preventing glycation of proteins in both in vitro and in vivo models, thus charting novel courses in the search for new, peptide-based food additives in the management of diabetes mellitus. α-amylase inhibitory activity and kinetics of inhibition the enzyme α-amylase in mammals is an important component of both saliva and pancreatic juice, and it catalyzes the hydrolysis of α-(1-4) glycosidic bonds of polysaccharides, releasing glucose and maltose in the process (voet et al., 2016). in recent times, protein hydrolysate preparations and peptides from some plant and animal sources have been demonstrated to inhibit αamylase activity, with potential implications for alternate therapies to the management of diabetes mellitus (arise et al., 2016b, olusola and ekun, 2018, awosika and aluko, 2019). in this study, all hydrolysates and peptide fractions demonstrated lower α-amylase inhibitory activities when compared to arabinose, and this is not unexpected because arabinose is a synthetic inhibitor of α-amylase. also, all peptide fractions exhibited better αamylase inhibition than the unfractionated hydrolysates, and this could be that the fractionation process improves bioactivity, by allowing for more bioactive peptides to gain access to the enzyme active site, causing inhibition of the enzyme. this is also consistent with the reports of malomo and aluko, (2016) that unfractionated hydrolysates do contain large molecular weight peptides that possess antagonistic effects to enzyme inhibition. the lineweaver-burk plot was used to determine the mode of α-amylase inhibition by varying concentrations of selected peptide fractions in this study. in addition, the kinetic parameters determined from the double-reciprocal plots were summarized in table 2, and it showed that the michaelis constant, km of α-amylase (from saccharomyces cerevisiae) in the absence of inhibitory hydrolysates is 0.552 mg/ml of starch, which is lower than 1.4 mg/ml (acharya et al., 2014) for α-amylase obtained from aspergillus oryzae. the presence of increasing amounts of the hydrolysate fractions increased the apparent km of the enzyme for its substrate, while also reducing both of maximal velocity, vmax, and catalytic efficiency, ce, of α-amylase. the peptide fraction exhibited a mixed type of inhibition and this suggests that these peptides are capable of binding αamylase in both its free form and in its starch-bound forms, creating dead-end complexes on both occasions. arise et al., (2016b) reported a mixed type of inhibition of α-amylase for peptic, tryptic, and alcalde hydrolysates of citrullus lanatus seed protein hydrolysates. inhibition of α-glucosidase activity and kinetics of inhibition the enzyme α-glucosidase commonly resides on the brush border membranes of the intestinal mucosa and it is involved in carbohydrate digestion by hydrolyzing glucose residues from oligosaccharides (voet et al., 2016). thus, the modulation of the activity of this enzyme represents one of the key strategies in the control of blood glucose levels in the management of diabetes mellitus (qaisar et al., 2014). the hydrolysates and their fractions demonstrated lower α-glucosidase inhibitory activities than the control (acarbose). this is because acarbose happens to be a dual inhibitor of α-amylase and α-glucosidase, with a higher binding affinity for αglucosidase (katzung et al., 2012). the fractionation of peptic digests proved to improve the bioactivities of its fractions, with fractions f1 and f2 also demonstrating inhibitory effects, with maximal activity obtained at 0.6 mg/ml and 0.8 mg/ml respectively for the fractions, but these were higher than those of fraction 3. this also suggests that lower molecular weight peptides demonstrate better α-glucosidase inhibitory activities. however, at higher concentrations, these peptides could have antagonistic effects on one another, which could in turn, result in reduced inhibition of α-glucosidase activity (awosika and aluko, 2019). in contrast to the kinetics of α-amylase inhibition, there is limited data available in the literature regarding the kinetic analysis of α-glucosidase inhibition by protein hydrolysate fractions. the kinetic parameters obtained from the double-reciprocal plots of α-glucosidase inhibition by selected peptide fractions of m. oleifera seed proteins in figure 5 were summarized in table 3. the michaelis constant, km, of α-glucosidase for ekun – peptide fractions from pepsin-digested moringa oleifera seed proteins … 421 p-nitrophenyl glucopyranoside in the absence of inhibitor determined to be 0.297 mg/ml p-npg in this study was is slightly higher than 0.211 mg/ml (0.7mm) p-npg obtained by awosika and aluko (2019) but lower than 6.31mg/ml reported by arise et al. (2019). vmax, in the absence of inhibitory hydrolysates, was 270.27 mm/mg/ml. peptic hydrolysate fraction 1 comprising peptides of molecular weight less than 1 kd demonstrated an uncompetitive inhibition mechanism, and this indicates that these peptides largely bind to other sites different from the substrate binding site on αglucosidase. the enzyme-inhibitor binding constant, ki value of 0.333 mg/ml obtained for peptic hydrolysate fraction 1 in this study was slightly higher than 0.305 mg/ml determined for unfractionated peptic hydrolysates of m. oleifera seed protein in another study (olusola and ekun, 2019). in addition, fraction 1 demonstrated a lesser binding affinity for α-glucosidase when compared with its binding to α-amylase. this could indicate that these peptides could perform better as αamylase inhibitors. conclusion the proteolysis of m. oleifera seed proteins using pepsin and their subsequent fractionation yielded peptide fractions that slowed the process of hemoglobin glycation and also inhibited carbohydrate–hydrolyzing enzymes in vitro. fraction f1 containing the smallestsized peptides demonstrated the strongest inhibitory activity against hemoglobin glycation and carbohydrates. thus, peptide products from m. oleifera seed proteins possess bioactivities that could be harnessed for the development of novel anti-diabetic agents. competing interests: the author declare that there are no competing interests. references acharya, d.k., shah. i.j., gami, p.n, shukla, r.m. 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(2011). antibacterial activity of moringa oleifera and moringa stenopetala methanol and n-hexane seed extracts on bacteria implicated in water borne diseases. afr. j. microbiol. res. 5, 153–157. doi: 10.5897/ajmr10.457 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 771-776 | doi: 10.14421/biomedich.2025.142.771-776 issn 2540-9328 (online) the effect of torch ginger (etlingera elatior) flower extract on creatinine levels and kidney histophatology in alloxan-induced white rats (rattus norvegicus) nofri megariang hura*, erlintan sinaga department of biology, faculty of mathematics and natural sciences, universitas negeri medan, jl. willem iskandar pasar v medan estate, percut sei tuan, deli serdang, sumatera utara 20221, indonesia. corresponding author* nofrimegariang@gmail.com manuscript received: 09 july, 2025. revision accepted: 26 september, 2025. published: 01 october, 2025. abstract kidney function impairment is a common complication of hyperglycemia, characterized by increased creatinine levels and structural damage to renal tissue. alloxan is frequently used to induce kidney injury through oxidative stress mechanisms. torch ginger (etlingera elatior) flower contains antioxidant compounds with potential nephroprotective effects. this study aims to determine the effect of torch ginger (etlingera elatior) flower extract on creatinine levels and kidney histopathology in alloxan-induced white rats (rattus norvegicus). the study used a completely randomized design (crd) with an experimental approach. the negative control group received destilled water, while the positive control group was induced with alloxan (120 mg/kgbw) intraperitoneally and treated with glibenclamide (0.09 mg/kgbw). the treatments groups (p1, p2, and p3) were given alloxan (120 mg/kgbw) and torch ginger (etlingera elatior) flower extract orally at doses of 100 mg/kgbw, 200 mg/kgbw, and 400 mg/kgbw, respectively. this study showed that torch ginger (etlingera elatior) flower extract significantly reduced creatinine levels and improved kidney histopathology (renal tubular necrosis). the 400 mg/kgbw dose was the most effective in lowering creatinine levels and repairing kidney tissue damage. keywords: etlingera elatior flower; creatinine levels; kidney histopathology; alloxan; rattus norvegicus. introduction the kidneys are essential organs responsible for maintaining internal physiological balance. they filter the blood to regulate fluid homeostasis, acid–base equilibrium, and electrolyte levels within the body (jannah & budijastuti, 2022). blood flow to the kidneys accounts for nearly 25% (rafe et al., 2020). this relatively large amount increases the likelihood that the kidneys will be exposed to toxic substances. the presence of toxins in the circulatory system poses a high risk as they can damage the structure and function of kidney tissue. functional and structural impairment of the kidneys can lead to various diseases such as chronic kidney failure or even death. hyperglycemic conditions are one of the causes of kidney damage. an imbalance between the production of ros (reactive oxygen species) and the antioxidant defense system is the main trigger for kidney damage, as indicated by increased creatinine levels in the blood. elevated creatinine levels indicate a decrease in glomerular filtration rate (perdanawati et al., 2021). this can be observed in rat models (rattus norvegicus) induced with alloxan. alloxan, as one of the toxic compounds, affects kidney histology, causing tubular necrosis, loose tubular structure, and fatty degeneration (sholihah & qomariyah, 2021). the mechanism by which alloxan causes kidney damage involves oxidative stress, leading to non-enzymatic glycation of amino acids and protein kinase c. this process results in the irreversible formation of advanced glycation end products (ages). these ages accumulate and trigger ros to react with polyunsaturated fatty acids (pufa) in kidney cell membranes, increasing the production of malondialdehyde (mda) in kidney cells as a result of lipid peroxidation in the cell membrane (ariani et al., 2024). the decline in kidney function caused by this condition can be treated using either conventional medicine or traditional remedies. medical treatments are often more expensive and may cause side effects. therefore, the use of locally available medicinal plants needs to be enhanced. many plant species have the potential to lower blood glucose levels, including torch ginger (etlingera elatior). torch ginger flowers contain numerous secondary metabolites, including flavonoids, saponins, alkaloids, steroids, phenols, and tannins (wardani et al., 2022). https://doi.org/10.14421/biomedich.2025.142.771-776 772 biology, medicine, & natural product chemistry 14 (2), 2025: 771-776 phytochemical screening by salman & indriana (2021) showed that fresh flowers, torch ginger simplicia, and ethanolic extracts contain chemical compounds such as alkaloids, flavonoids, steroids, glycosides, and essential oils. the extract can inhibit amylase and glucosidase enzymes, neutralize free radicals, and protect pancreatic beta cells from damage (putri, 2021). these properties highlight its potential as a nephroprotective agent against alloxan-induced kidney damage. therefore, researched are interested in evaluating the effectiveness of antioxidant compounds contained in etlingera elatior flower extract at doses of 100 mg/kgbw, 200 mg/kgbw, and 400 mg/kgbw in lowering creatinine levels and repairing kidney tissue structure resulting from alloxan induction. materials and methods tools and materials the tools used included a blender, rotary evapotaror, water bath, whatman paper no.1, dark bottles, feeding and dinking tools, cages, injection syringes, oral gavage, weighing scales, surgical scissors and forceps, paraffin bath, 1 cc syringes, blood tubes, spectrophotometer, sample bottles, microtome, cassettes, tissue processor, incubator, microtome, glass slides, and microscope. the materials used in this study included torch ginger (etlingera elatior) flowers, alloxan, male white rats (rattus norvegicus), glibencalmide, 96% ethanol, 0.9% nacl, carboxy methyl cellulose (cmc) 0.5%, feed, formalin, hematoxylin eosin (he), alcohol, xylol and distilled water. figure 1. etlingera elatior was found by author on bingkawan villages. plant collection and extraction torch ginger flowers (etlingera elatior) were obtained from bingkawan village, sibolangit subdistrict, deli serdang regency, north sumatra province. sampling was done manually by selecting 4 kg of fresh, young flower buds (unbloomed) that were undamaged and free from defects. the freshly harvested young flowers were then wet-sorted and thinly sliced. the sliced flowers were dried using an oven at 40℃, followed by dry sorting to ensure the samples were free from foreign materials. 250 grams of simplicia powder was weighed and added with 2500 ml of 96% ethanol solvent in a 1:10 ratio (nasir et al., 2023). maceration was carried out for 3×24 hours with stirring three times a day. after 3 days, the mixture was filtered using whatman no.1 filter paper and concentrated using a rotary evaporator at 50℃. the concentrate was then dried using a water bath 60℃ to obtain a thick extract of etlingera elatior flowers. experimental animals the experimental animals used in this study were 25 healthy male white rats (rattus norvegicus), aged between 2 to 3 months and weighing approximately 150– 200 grams. these animals were obtained from the pharmacy laboratory of universitas sumatera utara. all selected rats were confirmed to be healthy and free from physical abnormalities. the acclimatization of test animals was conducted at the animal facility of the faculty of mathematics and natural sciences, universitas negeri medan. this process aimed to allow the rats to adapt to their new environment and reduce the risk of stress. each rat was housed in a comfortable cage with adequate ventilation and maintained at room temperature. the cages were equipped with husk bedding along with food and water containers. the animals were fed 551 pellets ad libitum (azhar et al., 2022) alloxan induction the alloxan solution was prepared by dissolving alloxan powder in nacl 0.9%. the dosage of alloxan used in this study was 120 mg/kgbw. the rats were fasted for 8 hours prior to the intraperitoneal administration of alloxan at the predetermined dose. three days after treatment, each rat underwent blood glucose measurement following another 8-hour fasting period. the rats in this investigation had more than 200 mg/dl of blood glucose. experimental design this study employed a completely randomized design (crd) of 5 treatment groups with 5 replications each. prior to alloxan induction, the rats were fasted for 8 hours. after 72 hours, blood glucose levels were measured. rats with blood glucose levels ≥200 mg/dl were considered diabetic. subsequently, the rats received treatment with etlingera elatior flower extract (eef) according to table 1. the extract was administered hura & sinaga – the effect of torch ginger (etlingera elatior) flower extract … 773 orally, once daily, for a duration of 21 days (delfita et al., 2021). table 1. experimental treatments group treatment k(-) un-induced, but given standard feed and water k(+) given alloxan 120 mg/kgbw + glibenclamide 0.09 mg/kgbw p1 given alloxan 120 mg/kgbw + eef 100 mg/kgbw p2 given alloxan 120 mg/kgbw + eef 200 mg/kgbw p3 given alloxan 120 mg/kgbw + eef 400 mg/kgbw creatinine level measurement the determination of serum creatinine levels was carried out using the jaffe method, which involves spectrophotometric analysis to observe the color change resulting from the reaction between creatinine and specific reagents in the serum. this process enables the quantification of creatinine concentration in each sample (melisa et al., 2022). kidney damage score histological slides stained with hematoxylin-eosin (he) were examined under a light microscope at 400× magnification (delfita et al., 2021). observations were carried out across five microscopic fields, with 20 cells evaluated in each field, resulting in a total of 100 cells per slide. cells exhibiting necrosis were counted and converted into percentages to facilitate damage scoring. a score of 0 was assigned if no necrotic cells were observed (normal category); a score of 1 was given if necrotic cells accounted for ≤50% (mild damage); a score of 2 for 51–70% necrotic cells (moderate damage); and a score of 3 if necrosis involved 71–100% of the observed cells (severe damage) (melisa et al., 2022). data analysis statistical analysis was performed using spss version 26. the creatinine levels data were analyzed through one-way analysis of variance (anova) with a significance level set at α = 0.05. if significant differences were observed, further comparison was conducted using duncan’s multiple range test (dmrt). meanwhile, the degree of kidney tissue damage was assessed using the kruskal–wallis nonparametric test at a 95% confidence interval, followed by the mann–whitney u test for post hoc pairwise analysis. results and discussion creatinine levels the observational data on creatinine levels in white rats are presented in table 2. table 2. mean creatinine levels of white rats (rattus norvegicus). treatement groups creatinine levels ± stedv k (-) 0.562 ± 0.047a k (+) 0.724 ± 0.048b p1 0.734 ± 0.09b p2 0.716 ± 0.053b p3 0.622 ± 0.037a note: identical superscript letters within the same column indicate no significant difference between the control and treatment groups (p≥0.05), while different superscript letters indicate a statistically significant difference between the control and treatment groups (p<0.05). table 2. presents the mean serum creatinine levels of white rats across different treatment groups, showing statistically notable variations. the results of the study demonstrated that, when compared with the negative control group (k-/without treatment), administration of glibenclamide at 0.09 mg/kgbw (k+) did not significantly reduce creatinine levels in alloxan-induced white rats. similarly, the treatment group p1 showed no significant effect compared with the kgroup, indicating that administration of torch ginger flower extract (eef) at 100 mg/kgbw was ineffective in lowering creatinine levels. the administration of eef at 200 mg/kgbw (p2) also yielded comparable outcomes to p1. in contrast, the p3 group, which received eef at 400 mg/kgbw, exhibited a significant reduction in creatinine levels in alloxan-induced white rats when compared with the p1 and p2 groups. kidney histopathology the observational data on renal necrosis tubular in white rats are presented in table 3. table 3. mean renal necrosis tubular of white rats (rattus norvegicus). treatement groups necrosis tubular ± stedv k (-) 1 ± 0.0a k (+) 2 ± 0.707b p1 1.8 ± 0.447b p2 1.4 ± 0.547ab p3 1 ± 0.0a note: identical superscript letters within the same column indicate no significant difference between the control and treatment groups (p≥0.05), while different superscript letters indicate a statistically significant difference between the control and treatment groups (p<0.05). table 3. presents the differences in the level of renal tubular cell damage among the treatment groups. the kruskal–wallis test yielded a p-value of 0.012, indicating a statistically significant difference (p ≤ 0.05). based on the mann-whitney test analysis, it was found that there were significant differences in the degree of renal tubular damage among the groups. the results of the study demonstrated that, when compared with the normal group (k-), administration of glibenclamide at 0.09 mg/kgbw (k+) had no effect in reducing the degree of renal tubular necrosis in alloxan-induced rats. 774 biology, medicine, & natural product chemistry 14 (2), 2025: 771-776 similarly, administration of torch ginger extract (eef) at 100 mg/kgbw did not reduce tubular necrosis compared with the normal group (k-). a different outcome was observed in the p2 group, that at 200 mg/kgbw was able to lower the degree of renal tubular necrosis compared with the normal group (k-). however, the effect was not significant when compared with k+ and p1, indicating a moderate influence at this dose. in contrast, the p3 group, which received eef at 400 mg/kgbw, demonstrated a significant reduction in renal tubular necrosis when compared with k+, p1, and p2. figure 2. histological appearance of white rat kidney tissues across treatment groups stained with hematoxylin-eosin (he) at 400× magnification. glomerular (g), bowman’s space (bs), proximal tubule (pt), distal tubule (dt), normal cells (a), pyknotic nuclei (b), and karyorrhexis (c). the average percentage of tubular necrosis in treatment groups k(23.8%), p2 (49.5%), and p3 (37.2%) was below 50%, corresponding to a score of 1 and classified as mild damage. the k+ group exhibited a necrosis percentage of 59%, falling between 51% and 70%, thus receiving a score of 2, which indicates moderate damage. similarly, group p1 showed an average necrosis percentage of 54% and was also assigned a score of 2, indicating a moderate level of injury. these findings suggest that administration of torch ginger flower extract at doses of 200 mg/kgbw and 400 mg/kgbw effectively reduced the degree of tubular necrosis to levels approaching normal. discussion based on table 2, the results of the analysis of variance (anova) indicated that the treatments administered had a statistically significant effect on creatinine levels (p<0.05). the findings showed that the negative control group (k-) had an average serum creatinine level of 0.562 ± 0.047 mg/dl, which suggests normal kidney function without any decline in renal performance. in contrast, the groups induced with alloxan demonstrated elevated creatinine levels, indicating impaired kidney function as a result of alloxan toxicity. this is consistent with attama et al. (2023), who reported that administration of alloxan in experimental animals resulted in an average creatinine level of 3.00 ± 0.15 mg/dl. similarly, ariani et al. (2024) observed that alloxan induction increased serum creatinine levels to 0.9275 ± 0.020 mg/dl. according to sekiou et al. (2021), administration of alloxan elevated serum creatinine to 0.862 ± 0.07 mg/dl. this increase is likely attributed to the formation of advanced glycation endproducts (ages), which in turn trigger the generation of reactive oxygen species (ros). these ros interact with polyunsaturated fatty acids (pufas) in kidney cell membranes, promoting lipid peroxidation and increasing malondialdehyde (mda) levels. the resulting oxidative damage affects the glomeruli and renal tubules, compromising the kidneys’ ability to filter creatinine efficiently. the positive control group (k+), which received alloxan in combination with glibenclamide, showed a statistically significant difference compared to the negative control group (k-), with an average creatinine level of 0.724 ± 0.048 mg/dl. this suggests that glibenclamide alone was not sufficiently effective in providing renal protection. delfita et al. (2021) similarly reported that administration of glibenclamide at 0.45 mg/kgbw for 21 days in alloxan-induced animals resulted in a reduction of creatinine to 1.12 ± 0.13 mg/dl, which still differed significantly from the normal control group. the average creatinine level in the positive control group (1.12 mg/dl) remained statistically comparable to the alloxan-only group (1.38 mg/dl), suggesting limited protective efficacy. treatment groups receiving torch ginger (etlingera elatior) flower extract (eef) showed varying responses. groups p1 (alloxan + eef 100 mg/kgbw) and p2 (alloxan + eef 200 mg/kgbw) exhibited significantly hura & sinaga – the effect of torch ginger (etlingera elatior) flower extract … 775 different creatinine levels compared to the negative control group (k-), with relatively high average values and no significant difference from the positive control group (k+). these findings indicate that the 100 mg/kgbw and 200 mg/kgbw doses were insufficient to restore renal function within the 21-day treatment period. the most effective treatment in reducing serum creatinine was observed in group p3, which received the highest dose of the extract (400 mg/kgbw). this group showed no significant difference in creatinine levels compared to the negative control group, with an average of 0.622 ± 0.037 mg/dl, suggesting a nephroprotective effect of the extract against alloxan-induced kidney damage. this protective effect is likely due to the antioxidant properties of torch ginger, particularly its flavonoid content. widyarini et al. (2022) demonstrated that administering torch ginger flower extract can reduce creatinine levels and repair cellular damage in renal tissues. the antioxidant activity of the extract neutralizes free radicals, helping to mitigate oxidative stress and inflammation in the kidneys. flavonoids within the extract act by donating electrons to neutralize ros and also inhibit the activity of peroxidase enzymes, thereby reducing further ros formation (ariani et al., 2024). based on table 3, the kruskal–wallis test result yielded a p-value of 0.012, indicating a statistically significant difference (p ≤ 0.05). based on the mannwhitney test analysis, it was found that there were significant differences in the degree of renal tubular damage among the groups. attama et al. (2023) reported that administration of alloxan in experimental animals led to tubular epithelial degeneration and necrosis. similarly, delfita et al. (2021) observed that alloxan induced tubular damage in diabetic rats, characterized by loss of the brush border (<25% and >25%), thickening of the basement membrane, inflammation, cast formation, and necrosis reaching up to 60% or even higher. figure 2 illustrates the histological differences between the negative control group (normal) and the groups treated with alloxan (k+, p1, p2, and p3), which exhibited signs of necrosis, including pyknosis and karyorrhexis. additionally, a noticeable widening of bowman’s space was observed in the alloxan-induced groups. alloxan induction at a dose of 120 mg/kgbw triggers an increase in free radical production and disrupts antioxidant defense mechanisms, ultimately leading to oxidative stress. this condition can damage various body tissues, with the kidneys particularly vulnerable. as the primary excretory organ, the kidneys are highly susceptible to oxidative damage due to their high blood perfusion and the abundance of mitochondria in renal cells—particularly in the proximal tubules, where solute reabsorption is an energy-intensive process. free radicals are largely generated through the activity of the enzyme nadph (nicotinamide adenine dinucleotide phosphate) oxidase. excessive production of reactive species such as lipid peroxides and nitric oxide, accompanied by a decline in antioxidant enzymes like glutathione peroxidase (gsh-px), results in an imbalance that drives oxidative stress (delfita et al., 2021). prolonged oxidative stress contributes to inflammation and further dysfunction in renal tissue, exacerbating tubular cell degeneration (gordon et al., 2025). the k+ treatment group (alloxan + glibenclamide) exhibited the highest degree of tubular necrosis, with a necrosis percentage of 59%, which falls under moderate damage and showed a statistically significant difference compared to the control group (k-). these findings indicate that glibenclamide administration was insufficient in providing a nephroprotective effect, despite its known efficacy in reducing blood glucose levels. this is supported by delfita et al. (2021), who reported that glibenclamide at a dose of 0.45 mg/kgbw did not produce a significant difference in tubular necrosis compared to the negative control group (alloxan-induced). administration of torch ginger (etlingera elatior) flower extract (eef) at varying doses demonstrated differential protective effects. group p1 (alloxan + eef 100 mg/kgbw) was categorized under moderate damage, with a necrosis percentage of 54.9%, indicating that this dose was not sufficiently effective in exerting nephroprotective effects within the 21 days. in contrast, group p2 (alloxan + eef 200 mg/kgbw) was classified under mild damage with a necrosis percentage of 49.5%. the most favorable outcome was observed in group p3 (alloxan + eef 400 mg/kgbw), which exhibited the lowest degree of necrosis at 37.2%, approaching normal conditions. this group was also classified under mild damage, suggesting that the 400 mg/kgbw dose was the most effective in reducing tubular injury induced by alloxan. the reduction in renal tubular damage may be attributed to the antioxidant activity of compounds present in torch ginger (etlingera elatior) extract. antioxidants function within mitochondria to suppress the formation of reactive oxygen species (ros). ros generation typically occurs due to electron leakage in the mitochondrial electron transport chain, which allows free electrons to react with molecular oxygen. antioxidants such as flavonoids help stabilize these free radicals by converting them into more stable compounds, owing to the high reactivity of their hydroxyl groups (putri, 2021). flavonoids are bioactive compounds that support vascular health and exhibit significant anti-inflammatory effects. their mechanism of action involves direct scavenging of free radicals, thereby inhibiting ros formation and minimizing oxidative damage at the cellular level (sholihah & qomariyah, 2021). additionally, flavonoids exert their protective role by inhibiting the activity of enzymes such as xanthine oxidase and nadph oxidase, thus preventing redox reactions that lead to excessive free radical production, and consequently supporting cellular repair (ariani et al., 2024). 776 biology, medicine, & natural product chemistry 14 (2), 2025: 771-776 conclusions based on the findings, it can be concluded that the administration of torch ginger extract (etlingera elatior) significantly contributes to the reduction of serum creatinine levels and the extent of tubular necrosis in white rats (rattus norvegicus) induced with alloxan. among the tested doses, 400 mg/kgbw was the most effective in exerting a nephroprotective effect, as evidenced by creatinine levels approaching normal values and improvements in kidney tissue histology. acknowledgements: the authors would like to express their gratitude to all parties who provided support and assistance during the completion of this research. this study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors declare no conflict of interest. authors’ contributions: nofri megariang hura carried out the laboratory work, analyzed the data, and wrote the manuscript. elintan sinaga contributed to the conceptual design and provide critical revisions. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references ariani, l., febriani, h., & tambunan, e.p.s. (2024). pengaruh pemberian ekstrak daun benalu kopi (scrulla feruginea (jack) danser) terhadap kadar ureum dan kreatinin pada tikus putih jantan (rattus norvegicus l.) yang diinduksi aloksan. spizaetus: jurnal biologi dan pendidikan biologi, 5(3), 347-354. attama, s.c., aba p.e., asuzu c.u., & asuzu i.u. (2023). effects of vernonia amygdalina delile and baccharoides tenoreana olive on biochemical and histological markers of liver and kidney damage in alloxan-induced diabetic rats. african journal of biomedical research, 26(2), 291-296. azhar, r., romdhoni, m.f., karita, d., & bahar, y. (2022). pengaruh ekstrak etanol daun kersen (muntingia calabura l.) terhadap peningkatan kadar insulin tikus putih model diabetes melitus tipe 2 setelah induksi stz-na). muhammadiyah journal of geriatric, 3(3), 46-53. delfita, r., dahelmi, d., tjong, d., & suhatri, s. (2021). effect of enhydra fluctuans on kidney function in alloxan-induced diabetic rats. macedonian journal of medical sciences, 29(a), 1187-1194. gordon, c.m., solikhah, t.i., yuniarti, w.m., & miftakhurrozaq, r.k. (2025). ruellia tuberosa l. leaf extract improves histopathological damages in kidneys of alloxan-induced diabetic rats. international quarterly journal of biological sciences, 12(1), 224-230. jannah, d.r., & budijastuti, w. (2022). gambaran histopatologi toksisitas ginjal tikus jantan (rattus norvegicus) yang diberi sirup umbi yakon (smallanthus sonchifolius). lenterabio, 11(2), 238-246. melisa, e., muhaimin, yuliawati, & sani, f. (2022). uji toksistas akut ekstrak etanol daun sungkai (peronema cenescens jack) terhadap fungsi ginjal mencit putih betina (mus musculus linn.). majalah farmasi dan farmakologi, 26(1), 32-37. nasir, n.h., hagur, u.g., putri, r.j., & fauziah, r. (2023). uji aktivitas antidiare ekstrak etanol bunga kecombrang (etlingera elatior (jack) r.m.sm.) terhadap mencit jantan dengan metode transit intestinal. jurnal mandala pharmacon indonesia (jmpi), 9(1), 171-178. perdanawati, a.l., ratnaningtyas, n.i., & hernayanti. (2021). potensi ekstrak etil asetat coprinus comatus terhadap kadar ureum dan kreatinin pada tikus putih model diabetes. bioeksakta: jurnal ilmiah biologi unsoed, 3(3), 132-141. putri. (2021). etlingera elatior sebagai antihperglikemi pada penderita diabetes mellitus. jurnal penelitian perawat profesional, 3(1), 189-198. rafe, m.a.s.r., gaina, c.d. & ndaong, n.a. (2020). gambaran histopatologi ginjal tikus putih (rattus norvegicus) jantan yang diberi infusa pare lokal pulau timor. jurnal veteriner nusantara, 3(1), 61-73. salman, & indriana, m. (2021). anti-bacterial activity ethanol extract from the flower of kecombrang (etlingera elatior jack.) in vitro. journal of pharmaceutical and sciences (jps), 4(2), 69-73. sekiou, o., boumendjel, m., taibi f., tichati, l., boumendjel, a., & messarah, m. (2021). nephroprotective effect of artemisia herba alba aqueous extract in alloxan-induced diabetic rats. journal of traditional and complementary medicine, 11(2021), 53-61. sholihah, d., & qomariyah, n. (2021). pengaruh ekstrak daun jambu mete terhadap kadar asam urat dan histopatologi ginjal mencit diabetes. lenterabio, 10(3), 356-365. wardani, s., bogoriani, n.w., & rustini, n.l. (2022). uji aktivitas ekstrak bunga kecombrang (etlingera elatior jack) sebagai hipolipidemia pada tikus wistar obesitas. journal of science and applicative technology, 6(2), 118-123. widyarini, t., indarto, d., soetrisno, & purwanto, b. (2022). modulation effects of etlingera elatior ethanol extract as anti-inflammatory on chronic kidney disease in mice with hypertension and diabetes. j popul ther clin pharmacol, 29(4), 140-149. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 637-643 | doi: 10.14421/biomedich.2023.122.637-643 issn 2540-9328 (online) the therapeutic potential of cola nitida in health and disease: a review kamaldeen olalekan sanusi1,*, umar zayyanu usman1, dawoud usman1, kehinde ahmad adeshina1, yaaqub abiodun uthman1, lukman jimoh2, aminat omolola imam-fulani2,3 1department of physiology, usmanu danfodiyo university, sokoto, nigeria. 2department of physiology, university of ilorin, ilorin, nigeria. 3neuroscience department, university of minnesota medical school, twin cities, minneapolis, united states. corresponding author* sanusikamaldeen@yahoo.com manuscript received: 01 november, 2023. revision accepted: 07 december, 2023. published: 31 january, 2024. abstract cola nitida, also known as kola nut, is a tropical plant native to west africa and has a rich history of traditional medicinal use. in this narrative review, we aim to provide an overview of the protective effects of cola nitida in various health and disease states. cola nitida has been traditionally used for its medicinal properties, and its bioactive compounds include caffeine, alkaloids, tannins, flavonoids, and phenolics. these compounds contribute to its potential therapeutic effects. here, we examine the potential benefits of cola nitida in several areas of health, discussing its role in cognitive function, cardiovascular health, immune system function, gastrointestinal health, and metabolic and endocrine health. relevant original articles available from pubmed, african journals online (ajol), scopus, and google scholar were retrieved using the keywords “cola” and “nitida” without date restriction until july 17, 2023. evidence suggests that cola nitida may have positive effects on health, with indications of adverse effects only from its chronic usage. however, more research is needed to establish its efficacy and safety. cola nitida holds promise as a natural remedy for various health conditions. understanding the benefits and limitations of cola nitida will contribute to its effective utilization in health and disease management. keywords: bioactive compounds; cola nitida; kola nut; therapeutic effects; traditional medicine. introduction cola nitida is among the major cola species commonly known as kola nut. it is a tropical plant native to the west coast of africa (lim, 2012). it has a long history of traditional use in various cultural practices and is renowned for its medicinal properties (lim, 2012). the kola nut is rich in bioactive compounds such as alkaloids, tannins, flavonoids, and phenolics, which have been shown to possess a range of therapeutic effects (ekalu & habila, 2020). the exploration of natural products with potential health benefits has gained significant attention in recent years, as they offer alternative approaches to promote wellness and prevent diseases. cola nitida is one such natural product that has attracted interest due to its reported protective effects in various health and disease states. this narrative review aims to provide an overview of the current scientific understanding of the protective effects of cola nitida in health and disease states. by examining the existing literature and research studies, this review will explore the potential benefits of cola nitida in maintaining cardiovascular health, enhancing cognitive function, supporting gastrointestinal health, boosting immune system function, and improving metabolic and endocrine health. owing to its wide cultural availability, assessing the protective effects of cola nitida could have significant implications for public health and disease management. it may provide valuable insights into the development of novel therapeutic interventions and preventive strategies. by highlighting the potential benefits of cola nitida, this review seeks to encourage further research and exploration of its therapeutic applications. historical and traditional use of cola nitida cola nitida has a rich history of traditional use in various cultures across west africa. for centuries, indigenous communities have recognized and utilized the medicinal properties of cola nitida. it holds cultural and symbolic significance and is often used in social, religious, and medicinal contexts (adega, 1970; lim, 2012; ottuh, 2021). it has also attracted scientific interest across the globe. figure 1 highlights the spread of scientific enquiry related to cola nitida as obtained from pubmed database. https://doi.org/10.14421/biomedich.2023.122.637-643 638 biology, medicine, & natural product chemistry 12 (2), 2023: 637-643 figure 1. (a) country-specific scientific contribution based on number of affiliations related to cola nitida. the color intensity for each country is proportional to the number of author affiliations per country in 56 articles (retrieved from pubmed search on date = 15th july, 2023, search string = ("cola"[all fields] and "nitida"[all fields]). higher blue intensity refers to greater number of affiliations (i.e. dark blue = high productivity; grey = no affiliation). (b) top 5 country-based scientific contribution over time. bibliometric analysis was conducted using r package (biblioshiny) (aria & cuccurullo, 2017). (caveat: data obtained from ajol, scopus, and google scholar could not be incorporated into the analysis due to the peculiarities of the software). in traditional medicine, cola nitida has been employed as a stimulant to combat fatigue and increase energy levels (weckerle et al., 2010). its high caffeine content has contributed to its use as a natural energizer and mood enhancer. additionally, it has been utilized as an appetite suppressant, aiding in weight management and controlling hunger (easton & morton, 1999; weckerle et al., 2010). beyond its medicinal use, cola nitida holds cultural significance and is often incorporated into traditional rituals and ceremonies. in some communities, the kola nut is considered a symbol of hospitality and is offered as a sign of respect and friendship to guests (unya, 2021). it is also used in divination practices, believed to possess spiritual properties and the ability to enhance intuition and connection with higher realms (abah, 2016). in many west african cultures, cola nitida is a central component of social gatherings and communal activities. it is often chewed or consumed in the form of a beverage to stimulate conversation, foster social interaction, and promote a sense of unity among individuals (kwame, 2019; unya, 2021). the historical and traditional use of cola nitida highlights its cultural importance and its deep-rooted presence in the lives of west african communities. the recognition of its medicinal properties and the incorporation of the cola nut in various cultural practices have contributed to its enduring popularity and widespread use. while traditional use provides valuable insights into the potential benefits of cola nitida, it is important to conduct empirical experiments to validate and justify these claims. bioactive compounds of cola nitida and their benefits cola nitida is a rich source of various bioactive compounds that contribute to its potential therapeutic effects (see figure 2). its chemical composition can vary due to differences in extraction methods and research protocols. as a result, varying amounts of proximate composition, minerals, and amino acids have been reported in cola nitida (mbembo et al., 2022). one of the most well-known major components of cola nitida is caffeine, a natural stimulant that can enhance alertness and cognitive function (adesanwo et al., 2017; okoli et al., 2012). the occurrence of caffeine in the seed of cola nitida could potentially contribute to its utilization in the production of carbonated beverages (asogwa et al., 2006; jayeola, 2001; ogutuga, 1975). figure 2. bioactive compounds in cola nitida. in addition to caffeine, cola nitida contains other alkaloids such as theobromine and theophylline, which contribute to its stimulating properties (adesanwo et al., 2017; ekalu & habila, 2020; ogutuga, 1975). alkaloids have gained recognition for their therapeutic properties, notably their effectiveness as anesthetics, cardioprotective agents, and anti-inflammatory substances (heinrich et al., 2021). cola nitida also contains a variety of phytochemicals, including tannins, flavonoids, and phenolics, which have been associated with potential health benefits (dewole et al., 2013; ekalu & habila, 2020). tannins are a class of compounds known for their antioxidant and antiinflammatory properties. they have been reported to have a hepatoprotective effect, helping to reduce a b sanusi et al. – cola nitida in health and disease 639 oxidative stress and inflammation (b. lawal et al., 2017; sarr et al., 2022). flavonoids, another group of phytochemicals found in cola nitida, have been studied for their antimicrobial properties. these compounds have shown potential for antibacterial activity by inhibiting the growth of test isolates, suggesting that it could be used as an alternative remedy for oral infections and other bacterial infections (afolabi et al., 2020; muhammad & fatima, 2014; odonye et al., 2021). phenolic compounds present in cola nitida, such as catechins and epicatechins, have also been associated with antioxidant and anti-inflammatory effects (ogunlade et al., 2014). these compounds have been investigated for their potential to protect against chronic diseases such as hepatotoxicity, cancer and diabetes (atawodi et al., 2007; erukainure et al., 2017; oboh et al., 2014). the combination of caffeine, alkaloids, tannins, flavonoids, and phenolic compounds in cola nitida contributes to its potential protective effects in various health and disease states. effects of cola nitida in various health and disease states cola nitida, with its rich history of traditional use and its diverse bioactive compounds, has shown promise in promoting various aspects of health, as highlighted in table 1. nervous function and mental health the stimulating properties of cola nitida, primarily attributed to its caffeine content, have been associated with potential cognitive benefits (ogutuga, 1975). caffeine acts as a central nervous system stimulant and can enhance alertness, focus, and cognitive performance. research conducted on animals has demonstrated that cola nitida and caffeine have the potential to enhance memory and nervous function in healthy and diabetic states (imam-fulani et al., 2016, 2018, 2019). it may also have a positive impact on mood, reducing feelings of fatigue and enhancing overall mental well-being (persad, 2011). the study on ocular effect of cola nitida demonstrated its notable enhancements in the near point of convergence and an increase in the amplitude of accommodation (igwe et al., 2007). additionally, they observed that cola nitida can alleviate existing heterophorias. thus, it was suggested that the stimulative effects of cola nitida have the potential to alleviate asthenopic symptoms commonly associated with convergence insufficiency, thereby enabling individuals to perform near work tasks without experiencing stress (igwe et al., 2007). moreover, cola nitida has also been examined as a therapeutic agent with functional capacity against pain (adedayo et al., 2021). the presence of bioactive compounds, such as tannins and flavonoids, in cola nitida may contribute to its neuroprotective effects. these compounds possess antioxidant and antiinflammatory properties that can help mitigate neuronal damage and support cognitive function. however, possible adverse effects on the human brain due to excessive consumption of kola nuts have also been reported (atiba et al., 2021). the study provides evidence that repeated administration of kola nuts at specific doses to pregnant dams can interfere with the typical postnatal development of the cerebellum in their offspring (atiba et al., 2021). therefore, further investigations are necessary to gain a comprehensive understanding of the broader implications for human brain development. cardiovascular health studies have suggested that cola nitida may have beneficial effects on cardiovascular health (adeosun et al., 2017; erukainure et al., 2021; omorogiuwa & eiya, 2019; salahdeen et al., 2014). the antioxidant properties of cola nitida's phytochemicals, such as flavonoids and phenolics, may help reduce oxidative stress and inflammation, which are key factors in the development of cardiovascular diseases. cola nitida has been reported to have potential cardioprotective effects by improving blood lipid profiles, reducing cholesterol levels, and inhibiting the oxidation of low-density lipoprotein (ldl) cholesterol (abou et al., 2016). similarly, in rats with diabetic cardiomyopathy (erukainure et al., 2021), cola nitida resulted in a reduction in lipase activity and a depletion of cardiac lipid metabolites associated with diabetes. these effects may contribute to a reduced risk of atherosclerosis and cardiovascular events. furthermore, the vasodilatory properties of alkaloids, including theobromine and theophylline, present in cola nitida, may help relax blood vessels and improve blood flow, thereby supporting overall cardiovascular function (awuchi, 2019; ejuh et al., 2020). immune system function cola nitida contains various bioactive compounds that may have immunomodulatory properties. flavonoids and phenolics found in cola nitida have been shown to exhibit antioxidant and anti-inflammatory effects (adedayo et al., 2019; adesanwo et al., 2017; oboh et al., 2014), which can help support immune system function. they can scavenge free radicals and reduce oxidative stress, which is implicated in the development of various diseases. additionally, these compounds possess anti-inflammatory properties, helping to alleviate inflammation, a key contributor to chronic diseases. moreover, some studies suggest that cola nitida may have antimicrobial properties, inhibiting the growth of certain pathogens (afolabi et al., 2020; muhammad & fatima, 2014; odonye et al., 2021). this antimicrobial 640 biology, medicine, & natural product chemistry 12 (2), 2023: 637-643 activity has been attributed to various bioactive compounds present in cola nitida, including alkaloids, tannins, flavonoids, and phenolic compounds. extracts of cola nitida have demonstrated inhibitory effects against a wide range of bacteria, including both gram-positive and gram-negative strains. these include common pathogens such as staphylococcus aureus, escherichia coli, salmonella species, and pseudomonas aeruginosa (afolabi et al., 2020; muhammad & fatima, 2014; odonye et al., 2021). this suggests a potential role in supporting the body's defense against infections. gastrointestinal health owing to the phytochemical contents such as flavonoid, tannin, saponin, which possess gastroprotective effects, it is expected that consumption of cola nitida will be beneficial to the gastrointestinal health. tannins have been reported to provide a protective effect on the gastric mucosa, reducing the risk of gastric ulcers and supporting overall gastrointestinal health by regulating inflammation and oxidative stress (de veras et al., 2021; demarque et al., 2018). however, animal studies have demonstrated that cola nitida stimulates gastric secretion and erodes mucosa epithelial lining (ibu et al., 1986; a. lawal et al., 2022; osim et al., 1991; tende et al., 2011; umoh et al., 2019). moreover, high doses of cola nitida have been observed to induce gastric lesions in animals, and these lesions can worsen with prolonged administration (ojo et al., 2010). it is advised to consume cola nitida with caution to minimize the risk of gastric complications, particularly for individuals with ulcers who may consider avoiding it. metabolic and endocrine health research has indicated that cola nitida may have effects on metabolic and endocrine health. some studies have suggested that cola nitida may help regulate blood glucose levels, potentially beneficial for individuals with diabetes or metabolic disorders (dorathy et al., 2014; erukainure et al., 2019). more so, findings have highlighted the therapeutic potential of cola nitida in alleviating diabetic hepatotoxicity in rats (erukainure et al., 2020). the study demonstrated significant increases in antioxidant enzyme activity and effective inhibition of lipid peroxidation, myeloperoxidase, acetylcholinesterase, and atpase activities in the liver tissues of diabetic rats following the administration of cola nitida (erukainure et al., 2020). in addition, the phenolic extract of cola nitida demonstrated inhibitory effects on α-amylase and αglucosidase, crucial enzymes associated with type 2 diabetes mellitus (oboh et al., 2014). this inhibitory activity was attributed to the extract's high flavonoid content, including flavonoids such as apigenin, naringenin, and catechins. furthermore, the presence of caffeine and theobromine in cola nitida has been associated with improved metabolism and fat oxidation in diabetic rat model, which may contribute to weight management and overall metabolic health (erukainure et al., 2019). however, a recent study by igbinovia and colleagues revealed that normal weight male and female subjects who consumed cola nitida experienced a notable reduction in serum progesterone levels in females and serum testosterone levels in males (igbinovia et al., 2023). their findings suggest that administering cola nitida at a specific dosage could potentially impair fertility in both male and female individuals with normal weight (igbinovia et al., 2023). the protective effects of cola nitida in various health and disease states are believed to be mediated through multiple mechanisms. the bioactive compounds present in cola nitida, such as caffeine, alkaloids, tannins, flavonoids, and phenolics, play a crucial role in these effects. further research will help elucidate these mechanisms and provide a stronger scientific basis for the use of cola nitida in health and disease management. table 1. potential effects of cola nitida on health and disease. s/n health and disease state effects of cola nitida references 1 nervous function and mental health may improve cognitive function, mood, and pain relief. may also enhance ocular accommodation. however, may alter offspring brain development. (adedayo et al., 2021; atiba et al., 2021; igwe et al., 2007; imam-fulani et al., 2016, 2018, 2019; ogutuga, 1975; persad, 2011). 2 cardiovascular health may reduce cholesterol levels, improve blood lipid profiles, and protect against atherosclerosis. may also have vasodilatory properties. (abou et al., 2016; adeosun et al., 2017; awuchi, 2019; ejuh et al., 2020; erukainure et al., 2021; omorogiuwa & eiya, 2019; salahdeen et al., 2014). 3 immune system function may have antioxidant and anti-inflammatory effects, and may inhibit the growth of certain pathogens. (adedayo et al., 2019; adesanwo et al., 2017; afolabi et al., 2020; muhammad & fatima, 2014; oboh et al., 2014; odonye et al., 2021). 4 gastrointestinal health may have gastroprotective effects, but may also stimulate gastric secretion and erode mucosa epithelial lining (ibu et al., 1986; a. lawal et al., 2022; ojo et al., 2010; osim et al., 1991; tende et al., 2011; umoh et al., 2019). 5 metabolic and endocrine health may help regulate blood glucose levels, improve metabolism, and fat oxidation. may also have hepatoprotective effects. however, may potentially impair fertility. (dorathy et al., 2014; erukainure et al., 2019, 2020; igbinovia et al., 2023; oboh et al., 2014). sanusi et al. – cola nitida in health and disease 641 limitations most of the existing research on the protective effects of cola nitida is based on preclinical studies. the number of well-designed clinical trials investigating its therapeutic potential in humans is limited. therefore, further clinical studies are needed to validate the reported benefits and establish safe and effective dosage guidelines. in addition, there exist variability in preparation and quality of cola nitida extracts across different studies. this makes the study comparison challenging. the standardization of cola nitida extracts and rigorous quality control measures may be necessary to ensure consistency and reliability in research outcomes. moreover, there is paucity of data on long-term studies evaluating the effects of cola nitida on health outcomes. many of the reported benefits are based on short-term observations. therefore, the long-term safety and efficacy of cola nitida require further investigation. conclusions while the existing research suggests potential protective effects of cola nitida in various health and disease states, further research is needed to overcome the limitations and address gaps in current knowledge. well-designed clinical trials, in-depth mechanistic studies, standardization efforts, and safety assessments will contribute to a better understanding of cola nitida's therapeutic potential and its application in health and disease management. it is important to exercise caution in the routine consumption of cola nitida, particularly for individuals with underlying medical conditions. continued scientific investigation and evidence-based practice will contribute to a better understanding of the true therapeutic potential of cola nitida in promoting overall health and well-being. competing interests: the authors declare that there are no competing interests. authors’ contribution: kos and aoi contributed to the study conception and design. material preparation, data collection and analysis were performed by all authors. the first draft of the manuscript was written by kos. all authors have accepted responsibility for the entire content of this manuscript and approved its submission. references abah, j. 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(2010). medicinal, stimulant and ritual plant use: an ethnobotany of caffeine-containing plants. plants, health and healing: on the interface of ethnobotany and medical anthropology, 6, 262– 301. https://doi.org/10.1515/9781845458218-012 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 407-412 | doi: 10.14421/biomedich.2023.121.407-412 issn 2540-9328 (online) the anticancer properties of guava leaves (psidium guajava l.) and turmeric rhizome (curcuma longa l.) against breast cancer: a literature study angga puja asiandu1,2,*, widya sari3,4, septi widiya sari5, alif syahrul abdul majid6 1faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia. 2laboratory of biology, sma it harapan mulia, palembang 30113, indonesia. 3laboratory of physics, sma it harapan mulia, palembang 30113, indonesia. 4department of physics, faculty of math and natural science, universitas gadjah mada, yogyakarta 55281, indonesia. 5department of sociology, university of bengkulu, bengkulu 38731, indonesia. 6faculty of medicine, public health and nursing, universitas gadjah mada,yogyakarta 55281, indonesia. corresponding author* angga.puja.asiandu@mail.ugm.ac.id manuscript received: 30 november, 2022. revision accepted: 22 july, 2023. published: 03 august, 2023. abstract breast cancer, one of the most deadly diseases occurring in women, is caused by factors. in the healing process, the sufferer needs treatment, such as radiation techniques, surgery, and chemotherapy. but, these techniques have avoidable weaknesses that damage healthy cells. to date, natural sources can be utilized in medicine field. guava (psidium guajava l.) and turmeric (curcuma longa l.) are two common plants obtaining compounds that inhibit the growth of cancer cells based on phytochemical properties. this study was written to review the potential both of plants as anticancer agents. through the literature, guava leaf extract consists of flavonoids, tannins, alkaloids, saponins, terpenoids that inhibit the proliferation of cancer cells. besides, turmeric also has tannins, alkaloids, terpenoids, flavonoids, glycosides, sterols, and curcumin. the presence of curcumin reduces histamine production which induce inflammation and decrease toxin. because of curcumin, breast cancer cells have dehydration before apoptosis. keywords: apoptosis; curcumin; natural sources; phytochemical. introduction various deadly diseases always lurk in human welfare at any time worldwide. one of those diseases suffered in many countries is cancer. cancer is the second highest cause of death after cardiovascular disease. based on who data, the worldwide mortality rate due to cancer reached 13%. every year, there were 12 million people in the world suffering from cancer and as many as 7.6 million of them died. the number of cancer patients will continue to increase. in 2030, it is estimated that the number will reach 26 million people with death is expected about 17 million (ministry of health 2015). based on basic health research data in 2007, the national rate of cancer patients in indonesia was 4.3 per 1000 population, with higher cancer rates occurring in women than men. in women, 5.7 per 1000 people in indonesia suffered from cancer. meanwhile, 2.9 per 1000 male population experienced cancer (ministry of health of indonesia 2013; dewi and hendrati 2015). the common type of cancer attacking women is breast cancer which causes death in women. the american cancer society (2008) reported that as many as 1.3 million women worldwide were diagnosed with breast cancer. every year, approximately 465,000 women died from breast cancer. within 25 years, the number of patients had increased by around 30% in developed countries (wahyuni 2015). the primary cause of breast cancer is not yet fully known, but it is believed to be multifactorial or caused by many factors. there are several factors of breast cancer, such as genetic disorders facilitating the emergence of cancer cells, chronic irritation and inflammation, radiation, exposure to certain chemical compounds, carcinogenic foods, and others (suryaningsih and sukaca 2009; dewi and hendrati 2015). treatments of breast cancer are usually done with radiation techniques, surgery, and chemotherapy. however, these techniques have side effects because they sometimes enhance cancer cells to spread to other parts of the body, damage healthy cells, and also trigger cancer cells to mutate. thus, the discovery of new drugs that are safer with fewer side effects is needed in overcoming this problem (muhartono and subeki 2015). it may be overcome by utilizing plants as herbal medicines as some plants produce bioactive compounds https://doi.org/10.14421/biomedich.2023.121.407-412 408 biology, medicine, & natural product chemistry 12 (1), 2023: 407-412 potentially used as anticancer. guava (psidium guajava l.) and turmeric (curcuma longa l.) are two common plants producing secondary metabolites such as tannins, flavonoids, alkaloids, and saponins (correa et al. 2016). turmeric (curcuma longa l.) rhizome contains curcumin which also has anticancer properties which can inhibit carcinogenesis (meiyanto 1999; nurrochmad 2004). thus, the combination of guava (psidium guajava l.) and turmeric (curcuma longa l..) can be used as an alternative to prevent breast cancer without or with fewer side effects. methods this review paper was done with a literature study. a literature study was carried out to support ideas that are based on a strong theoretical basis on several sources consisting of several leading journals. the secondary data were obtained to support the idea (asiandu and malayudha, 2022) and used in analyzing the usefulness of the idea to conclude the final conclusion through some stages (sari et al. 2020). furthermore, the benefits to be achieved from writing this scientific work are to provide information about the dangers of breast cancer and to socialize these two materials as the formation of pharmaceutical drugs to reduce the number of breast cancer patients safely. results and discussion cancer cell cancer cells are normal cells that have undergone a genetic mutation causing their growth to be uncontrolled and uncoordinated with other body cells. the process of forming cancer cells is known as carcinogenesis, a somatic event, and is caused by an accumulation of genetic and epigenetic changes leading to changes in the normal regulation of molecular control of cell growth (figure 1). these genetic changes can be in the form of activation of proto-oncogenes and/or inactivation of tumor suppressor genes which can trigger tumor formation (kondo 1993; nurhayati and lusiyanti 2006). figure 1. cancer cell invasion (taken from gupta and massague, 2006). generally, there are two types of cancer cells, benign, and malignant cancer. benign cancer cells have a low growth rate and do not spread to other parts. meanwhile, malignant cells have a rapid growth rate, invade and damage surrounding tissue, and spread to other organs. invasion of cancer cells allows these cells to move into the blood vessels to be transported to other organs emerging new cancer in other organs (lumongga 2008). the general characteristic of cancer cells is the ability to grow rapidly and reduce the body's normal control mechanisms. abnormal genes in cancer are called oncogenes (guyton and hall 1997; putri et al. 2012). cancer cells can not maintain when to stop cell division. they divide uncontrollably which will compete with normal cells using oxygen and nutrients from the body. these cells will replace normal cells and cause pain that ends in death (nurhayati and lusiyanti 2006). breast cancer breast cancer is one of the most common types of cancer in women, although this cancer can also occur in men. the risk of breast cancer cases increases at the age of 40 years, which attacks more on the left breast and the upper part of the breast, close to the arm (wijayakusuma 2008; rahmatari 2014). according to the indonesian ministry of health in 2013, breast cancer contributed 30% and was the most dominant type of cancer in indonesia (dewi and hendrati 2015). breast cancer is a carcinoma originating from the ducts or lobules of the breast which is an international women's health issue. breast cancer is the most common problem in developed countries and is the number two problem in developing countries after cervical cancer. overall, breast cancer is the second leading cause of death after lung cancer (suyatno 2010; sumiatin, 2013). a common symptom of breast cancer is a lump in the breast that can be felt and will get harder, irregular, and cause pain. other symptoms are changes in size and shape, wrinkling of the breast skin that resembles an orange peel, presence of pus, blood, watery fluids, and discharge of milk in a woman who is not pregnant or breastfeeding. breast cancer is also characterized by swelling in one breast, itchy and painful nipples, bone pain, swelling of the arms, and weight loss (suryaningsih and sukaca 2009; dewi and hendrati 2015). breast cancer can be caused by several factors. these factors are combined into hormonal factors which include age at menarche, age at first pregnancy, parity, history of breastfeeding, infertility, and long-term use of hormonal contraception. early menarche or first menstruation at the age of below 12 years will increase the likelihood of breast cancer. the risk of breast cancer increases with the increasing age of women (priyatin et al. 2013). breastfeeding duration affects the risk of breast cancer. women who breastfeed in the 4-6 month range have a greater risk of breast cancer than 7-24 months. it asiandu et al. – anticancer of guava and turmeric 409 shows that the longer the breastfeeding time, the lower the risk of breast cancer. meanwhile, the consumption of fatty foods also affects the high risk of breast cancer in women. in addition, passive smoking, alcohol consumption, and low physical activity also affect the occurrence of breast cancer (yulianti et al. 2016). side effects of breast cancer treatment there are several methods used to treat breast cancer. methods of treating breast cancer include radiation, surgery, and chemotherapy. however, these methods have side effects, such as the spread of cancer cells to other organs and the mutation of cancer cells (muhartono and subeki 2015). side effects caused by chemotherapy can be in the form of physical and physiological disorders. effects that may appear include nausea, vomiting, alopecia, and so on. patients who feel the side effects of chemotherapy treatment, try to overcome these effects by resting or even using anti-emetic or analgesic drugs (wahyuni 2015). severe side effects also often occur in postchemotherapy patients, and often these side effects cannot be tolerated by patients which can even cause death. in addition, the side effect of post-chemotherapy patients may feel is anxiety (setiawan 2015). meanwhile, radiation therapy for cancer patients also causes side effects, especially in children. those effects can appear faster or slower and can occur locally or systemically, from small to large depending on age, location of irradiation, area of irradiation, vital organs around the tumor, and also the dose (yunus 2008). therefore, the use of natural herbal ingredients is very important in preventing the growth and spread of breast cancer cells. these herbal ingredients can be derived from guava leaves (psidium guajava l.) which contain secondary metabolites such as tannins, flavonoids, alkaloids, and saponins that have anticancer properties inhibiting the growth and spread of cancer cells (correa et al. 2016). meanwhile, turmeric rhizome (curcuma longa l.) contains curcumin which has anticancer properties which can inhibit the process of carcinogenesis at the stage of initiation and promotion of cancer cell progression. guava leaves (psidium guajava l.) as anticancer guava or psidium guajava linn is a tropical plant rich in vitamins a, and c, and secondary metabolites which are beneficial for health. guava leaves contain chemical compounds such as flavonoids, quercetin, tannins, saponins, essential oils, and alkaloids used as medicines for various diseases such as coughs and diarrhea. flavonoids are secondary metabolites of the phenol groups, efficacious as antioxidants, anti-inflammatories, and anticancer (aziz and djamil 2013). water extract from guava leaves had been reported to be able to inhibit the spread of prostate gland cancer cells. the compounds found in the extract were polyphenolic and flavonoid (chen et al. 2007; lee and park 2010). meanwhile, secondary metabolites such as tannins, alkaloids, and saponins contained in guava leaves also inhibited the growth and spread of cancer cells by inhibiting the proliferation of cancer cells (correa et al. 2016). table 1. phytochemical properties of guava leaves (psidium guajava l.). phytochemicals result flavonoids tannins alkaloids terpenoids + + + + source: kariawasam et al. 2017. guava leaf extract was reported to have considerable anticancer activity. based on dwitiyanti (2015), 70% ethanol extract from guava leaves had cytotoxic properties against t47d breast cancer cells with a concentration of 130.62 µg ml-1 capable of killing breast cancer cells by 88.52% which were incubated for 24h. meanwhile, the concentration of 5 µg ml-1 was able to kill 12.49% of t47d breast cancer cells. identification of the chemical groups contained in the 70% ethanol extract of guava leaves indicated the presence of alkaloids, flavonoids, saponins, tannins, and triterpenoids. also, kaileh et al (2007), cited in fathilah et al. (2010), stated that guava extract was effective in inhibiting the growth of mcf7 breast cancer cells within 24h with an ic50 value of 55 µg ml−1 ml-1 capable of killing cancer cells by 50%. moreover, the extract also had immunomodulatory activity in the form of nfkb (transcription factor protein) regulated the expression of genes involved in apoptosis. the mechanism of flavonoids in inhibiting and even killing cancer cells can be done in several ways. the first way is to act as an antioxidant which will inactivate free radicals. then, flavonoids will also bind to electrophilic compounds, induce the work of protective enzymes conjugate activity, increase the rate of apoptosis, inhibit cell proliferation, and inhibit lipid peroxidation. furthermore, flavonoids will also inhibit angiogenesis and inhibit dna oxidation (rahayu and roosmarinto 2017). additionally, alkaloids have properties as antineoplastic agents that can inhibit and even kill cancer cells by inhibiting dna synthesis and inhibiting mitosis at the metaphase and anaphase stages of cancer cells (purwaningsih et al. 2015). steroids function as topoisomerase ii inhibitors which can prevent and inhibit the cancer cell cycle (afandi 2006; sahid et al. 2013). saponin compounds also play a role in inhibiting cancer cells because they are antiproliferative, antimetastatic, and also antiangiogenesis. saponins induce apoptosis and cell differentiation (xu et al. 2016). angiogenesis by tumor cells is shown in figure 2. 410 biology, medicine, & natural product chemistry 12 (1), 2023: 407-412 figure 2. angiogenesis by tumor cells (taken from hejmadi, 2010). turmeric rhizome (curcuma longa linn.) as anticancer turmeric (curcuma longa l.) is a plant growing in tropical and subtropical regions. the main ingredient in turmeric is curcumin. it is a substance with antioxidant, anti-inflammatory, anti-proliferative, and cytotoxic effects that can induce apoptosis in malignant blood cells, breast, colon, liver, and ovaries (kurniawan et al. 2016). curcumin is a yellow polyphenol-derived pigment found in the rhizome of the turmeric plant (figure 3). the curcumin found in turmeric can act as an antioxidant, antiviral, antifungal, and anti-inflammatory by inhibiting several important molecules triggering inflammation. curcumin reduces the production of histamine (a substance inducing inflammation), increases the production of cortisol which has an antiinflammatory effect and removes toxins from the body (hutomo et al. 2016). figure 3. structure of curcumin (nurrochmad, 2004). turmeric rhizome extract suppressed the growth of mcf-7 and mda-mb-231 breast cancer cells. curcumin contained in turmeric rhizome extract triggered apoptosis. the rate of apoptosis of mcf-7 and mdamb-231 breast cancer cells correlated with the curcumin concentration. it induced the bax protein used in cell apoptosis and inhibited bcl-2 protein expression as an antiapoptotic protein (lv et al. 2014). table 2. phytochemicals properties of turmeric water-extract (curcuma longa l.). phytochemicals result alkaloid glycosides flavonoid tannin sterol terpenoid + + + source: deb et al., 2013 turmeric n-hexane extract inhibited the growth of qu-db and t47d breast cancer cells. data analysis on the anticancer activity of turmeric rhizome extract showed that the ic50 value for qu-db cells was 74 µg ml-1 and for t47d cells the ic50 value was 57 µg ml-1 in the 48-hour mtt assay. the concentration of 68 µg ml-1 of turmeric extract was able to reduce the rta (relative telomerase activity) rate in qu-db cells by 76.4% (ranjbari et al. 2014). the curcumin induced cell cycle arrest and apoptosis through the cell-signaling pathway (kurniawan et al. 2016). curcumin damages cancer cells by referring to the process of apoptosis. in the event of apoptosis, cells will experience dehydration. loss of intracellular fluid causes the cytoplasm to condense and change shape with smaller and flattened cell sizes. cells undergoing apoptosis can be characterized by the presence of pyknosis due to chromatin condensation (hutomo et al. 2016). further mechanisms of curcumin's anticancer effects may include suppression of nf-kb activity through inhibition of ikkb activity resulting in suppression of genes inducing tumorigenesis which include tnf, cox2, cyclin d1, c-myc, mmp-9, and interleukins. curcumin also controls the cell cycle and stimulates apoptosis by regulating p16 and p53. curcumin also acts as an autophagy modulator which is a tumor angiogenesis and metastatic inhibitor through the suppression of various growth factors including vegf, cox-2, mmps, and icams (wilken 2011; kurniawan et al. 2016). apart from curcumin, turmeric (c.longa l.) rhizome also contains several compounds that play a role in inhibiting or even killing cancer cells such as alkaloids, flavonoids, and tannins with certain mechanisms. they enhance the anticancer potential of turmeric. conclusions guava leaves (psidium guajava linn.) contain several compounds that act as anticancer consisting of alkaloids, flavonoids, saponins, and steroids. turmeric (curcuma longa linn.) rhizome contains curcumin and several other compounds such as alkaloids, flavonoids, and tannins which have anticancer properties. the asiandu et al. – anticancer of guava and turmeric 411 mechanisms of these compounds as anticancer include inhibiting the proliferation of cancer cells and inducing apoptosis of breast cancer cells. further research is needed to determine the perfect combination of both ingredients as herbal medicine in preventing breast cancer. acknowledgments: the authors thank all the authors cited in this article for their valuable works. authors’ contributions: all the authors involved in this review contributed to developing ideas, collecting secondary data, writing the manuscript, editing language and formatting. competing interests: the authors declare that there are no competing interests. funding: the authors received no financial funding from any institution regarding the data collection, manuscript writing, authorship, and this published article. references asiandu, a. p., & malayudha, a. g. 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(2008). side effects of radiation therapy in head and neck cancer patients on the salivary glands (efek samping terapi radiasi penderita kanker kepala dan leher pada kelenjar saliva), jurnal dentofasial, 7(1), 57-62. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 357-361 | doi: 10.14421/biomedich.2025.141.357-361 issn 2540-9328 (online) potential effect of orthosiphon aristatus leaf extract in improving collagen 1 alpha 1 expression in hyperglycemia-induced rats riandini aisyah1,*, erika diana risanti1, safari wahyu jatmiko1, fatin asfarina2 1biomedical department, faculty of medicine, universitas muhammadiyah surakarta jl. a yani, pabelan, kartasura, sukoharjo, jawa tengah 57169, tel. +62-271-717417, fax. +62-271-715448, indonesia. 2integrated research laboratory, faculty of medicine, nursing, and public health, universitas gadjah mada jl. farmako sekip utara, yogyakarta 55281, tel. +62-274-560300, fax. +62-274-581876, indonesia. corresponding author* riandini.aisyah@ums.ac.id manuscript received: 11 march, 2025. revision accepted: 20 may, 2025. published: 04 july, 2025. abstract diabetes mellitus involves many mechanisms. the collagen metabolism pathway with very complex interactions and its regulation can contribute to the development of diabetes complications. the collagen 1 alpha 1 (col1a1) plays an important role in the diabetes melitus pathway in adipose tissue by regulating adipocyte function, insulin resistance, and glucose metabolism and is identified as a potential therapeutic target for type 2 diabetes. the cat's whisker plant (ortosiphon aristatus) pharmacologically has hypoglycemic activity and increases insulin expression. this study aimed to observe the potential of cat whisker leaf extract (ortosiphon aristatus) in increasing the col1a1 expression and is protective against increased blood sugar levels in hyperglycemia-induced rattus norvegicus. the experimental design with pretest and posttest controlled group design was carried out by dividing 18 test animals into six groups: non-diabetic negative control, diabetic negative control, positive control (metformin), cat whisker leaf extract doses of 100, 200, and 400 mg/kgbw. induction of hyperglycemia with streptozotocin 65 mg/kgbw and nicotinamide 230 mg/kgbw peritoneal injection. the extract treatment was given for 14 days. blood sugar level measurements were carried out at the treatment’s beginning and end. adipose tissue was taken to examine col1a1 expression by real time pcr technique by calculating the ratio of the cycle of tresh-hold col1a1 to the internal gene β-actin. the results of kruskall wallis test showed no significant difference in col1a1 expression between groups and the spearman correlation test showed no relationship between blood sugar levels and col1a1 expression (p>0.05). there was a decrease in blood sugar levels before and after treatment for all treatment groups. keywords: diabetes melitus; col1a1 expression; orthosiphon aristatus; real time pcr. abbreviations: col1a1 = collagen 1 alpha 1, pcr = polymerase chain reaction introduction globally, deaths due to diabetes are projected to increase to 1.59 million and 79.3 million daly (disability adjusted life years) by 20251, and increase to 7,079 people per 100,000 by 2030 with the burden of diabetes varying between regions. in indonesia, the prevalence of diabetes mellitus is expected to increase significantly in the coming decades from 9.19% in 2020 (18.69 million cases) to 16.09% in 2045 (40.7 million cases), increasing by 75.1% over 25 years, or an average of 3% per year (wahidin et al., 2024). the prevalence of type 2 diabetes follows a pattern that corresponds to socioeconomic development, with developed regions showing higher prevalence rates (khan et al., 2019). diabetes mellitus is a metabolic disease characterized by disturbances in insulin secretion, insulin activity, or both. insulin resistance (ri) in type 2 diabetes mellitus (t2d) is accompanied by dysfunction of glycolipid metabolism and protein biosynthesis. over the past few decades, despite great advances in diabetes-related research, the discovery of specific molecular targets has not been enough. in addition, there is drug resistance and side effects related to the use of anti-diabetic drugs that can reduce the inhibition of insulin resistance. risk factors for developing type 2 diabetes mellitus are obesity and insulin resistance (lin et al., 2021). the main characteristic of obesity and insulin resistance is the occurrence of extracellular matrix (ecm) remodeling abnormalities in muscle, liver, and adipose tissue. there is a growing body of evidence suggesting a link between ecm and insulin resistance. although ecm remodeling is increasingly important in insulin resistance, effective ecm targets remain undefined. collagen is the most abundant component of ecm, consisting of three α helical chains made of a series of repeating amino acids glycine-x-y. an increase in collagen protein in insulin-resistant cells is associated https://doi.org/10.14421/biomedich.2025.141.357-361 358 biology, medicine, & natural product chemistry 14 (1), 2025: 357-361 with increased mrna levels. increased expression of some of the genes that make up collagen proteins is associated with a high-fat diet. the increase in col24α1 mrna in the adipose tissue of obese white mice was consistent with the increase in other previously reported collagen isoform mrnas including col5α2, col6α2, col6α3 and col18α1. studies on the role of specific ecm components in insulin resistance are important but incomplete (weng et al., 2020). col1a1 is the most significant gene in the extracellular matrix receptor interaction pathway and is associated with hypoglycemic activity for the first time. thus, col1a1 is a potential new therapeutic target for reducing t2d (lin et al., 2021). rapid advances in proteomics are critical to uncovering the mechanisms underlying t2d and identifying mechanism-based biomarkers. tissue analysis correlated glycometabolic parameters and dep (differentially expressed protein). the gene’s expression level in the primary metabolic pathway is necessary to identify biomarker candidates. collagen proteins interact with each other so further observation of collagenencoding genes is needed so that a molecular network analysis related to insulin resistance is obtained (lin et al., 2021; weng et al., 2020). diabetes mellitus as a chronic illness with considerable worldwide repercussions, necessitates appropriate management to avert fatal outcome. natural pharmaceuticals are becoming increasingly popular due to their perceived safety compared to synthetic drugs, despite potentially poorer efficacy (septiana et al., 2021). two hundred twenty-nine medicinal plants in indonesia have been identified as beneficial for reducing blood glucose levels. the asteraceae family and orthosiphon aristatus species were the most predominant. it underscored the necessity confirming pharmacological activity at the molecular level (arifah et al., 2022). the leaves of the cat's whisker plant (ortosiphon aristatus) contain various active substances (nurcholis et al., 2022). the methanol and ethanol extracts of ortosiphon aristatus exhibited remarkable αglucosidase inhibitory activity, enhancing the recognition of the bioactive substances in ortosiphon aristatus for diabetes management (maulana et al., 2022). ortosiphon aristatus extracts showed considerable hypoglycemics efficacy compared to the diabetes control group, less toxicity, suggesting promise for herbal treatment (thinn et al., 2018). the content of flavonoids plays a role in lowering blood sugar levels and modulating oxidative stress in the body by neutralizing the effects of nitrogen and oxygen species. flavonoids help regulate carbohydrate digestion, insulin signaling, insulin secretion, glucose absorption, and adipose. flavonoids target many molecules involved in regulating several pathways, such as increasing ß cell proliferation, increasing insulin secretion, reducing apoptosis, and improving hyperglycemia by regulating glucose metabolism in the liver (ojo et al., 2023). this study aimed to observe the potential of cat whisker leaf extract (ortosiphon aristatus) in increasing the expression of the col1a1 gene. observation of the expression of the mrna gene col1a1 as a gene encoding one of the collagen proteins from adipose tissue is important because the col1a1 gene is involved in the mechanism of insulin resistance with obesity risk factors. there is a contradiction between the results of the col1a1 gene research with the protective properties of col1a1 gene expression against increased blood sugar and vice versa. materials and methods study area extracts preparation, test animals treatment, blood sugar levels measurement, and adipose tissue collection were conducted at the pharmacology laboratory of the faculty of medicine, university of muhamamdiyah surakarta. measurement of col1a1 expression used the real-time pcr technique at the integrated research laboratory of the faculty of medicine, public health, and nursing, universitas gadjah mada, yogyakarta. procedures extract preparation the symplisia of cat whisker leaves (ortosiphon aristatus) was obtained from b2p2toot (center for research and development of medicinal plants and traditional medicine) tawangmangu, karanganyar, central java and the determination was obtained from the tawangmangu health service upf laboratory. symplisia is then put into a maceration pot then soaked using 70% ethanol, stirred for 1 hour per day for 5 days. the results of the soak are filtered with a filter cloth. then it is evaporated with an evaporator to produce a viscous extract. the next step is the calculation of the dosage and the manufacture of the stock solution. animal preparation and diabetes induction the experimental animal was a male rattus norvegicus of the wistar strain obtained from the pharmacology laboratory, faculty of medicine, universitas muhammadiyah surakarta. the inclusion criteria were healthy rats (normal activity), body weight 150-200 grams, 2-3 months old. the mice were fasted for 8 hours then divided into 6 groups (g) randomly and blood sugar measurements were taken (g1: non diabetic negative control, g2: diabetic negative control, g3: metformin, g4: ortosiphon aristatus (cat whisker) extract dose 100mg/kgbw, g5: ortosiphon aristatus extract dose 200mg/kgbw, and ortosiphon aristatus extract dose 400mg/kgbw. rats in the g2-g6 group were induced by hyperglycemic agent using nicotinamide (nta) injection 230 mg/kgbb as pancreatic protector and streptozotocin (stz) 65 mg/kgbb intraperitonially and were given standard feed and drinking water every day. aisyah et al. – effect of ortosiphon aristatus on improving col1a1 expression 359 blood sugar levels measurement blood sugar level measurements were carried out on day 0, day 4 after stz and nta induction, and day 14. rats whose sugar levels had increased in the g2-g6 group were given cat whisker leaf extract treatment for 14 days orally. following a 14-day period, blood glucose levels were evaluated, and the procedure was completed for the collection of adipose tissue. measurement of col1a1 expression approximately 30mg of rat adipose tissue was inserted into an rna stabilizing solution for rna extraction using phenol chloroform and cdna synthesis according to the protocol smobio excel rt kit ii rp1400. col1a1 gene expression was measured using real-time pcr according to the protocol of the sensifast bioline sybr green kit with a specific primer. housekeeping gene β-actin was used as an internal control. col1a1 expression was determined by measuring the cycle of tresh-hold (ct) ratio of the col1a1 gene to the internal gene β-actin. table 1. primer base sequence of col1a1 and β-actin genes. forward primer reverse primer col1a1 tggcaagaacggagatga agctgttccaggcaatcc β-actin ctatcggcaatgagcggttcc tgtgttggcatagaggtctttacg data analysis the data on blood sugar levels and col1a1 expression obtained were statistically processed by the kruskall wallis non-paramteric test and the spearman's rho correlation test. results and discussion based on blood sugar level data, there was an increase in blood sugar levels after induction of streptozotocin and nicotinamide in all groups (pre-test) and there was a decrease in blood sugar levels after administration of ortosiphon aristatus leaf extract (post-test). because the pretest data is not homogeneous, the data on the difference in blood sugar levels of the pretest and the postest is used for analysis as presented in table 1. as for the col1a1 expression data, there was no difference in expression between groups with a value of p>0.05. table 2. result of blood glucose and col1a1 expression measurement. parameters control (-) non diabetic control () diabetic control (+) group 1 (100mg/kgbw) group 2 (200mg/kgbw) group 3 (400mg/kgbw) p value blood glucose preposttest difference 21±19.13 15.33± 9.81 33.67±5.91 48.33± 20.37 35.67± 12.81 34± 30.41 >0.05* col1a1 expression posttest 0,2±0,1 1,7±0,2 0,9±1,3 1,3±1,9 0,3±0,5 1,7±2,9 >0.05* blood glucose & col1a1 expression >0.05** * : kruskal wallis test ** : spearman’s rho test discussion this study showed that the administration of streptozotocin at 65 mg/kg body weight and nta at 230 mg/kg body weight effectively elevated blood glucose levels by the 19th day post-induction, aligning with the mechanisms associated with type 2 diabetes (t2d). on the 19th day, fasting blood glucose levels were assessed, revealing an elevation relative to the baseline; this indicates successful induction of hyperglycemia in t2d. the pathogenesis of t2d involves several factors, including impaired insulin secretion by pancreatic ß cells and insulin resistance in peripheral tissues. this condition leads to a heterogeneous condition characterized by β cell failure, ultimately resulting in t2d. streptozotocin has toxic properties because of its potential to damage cells β the pancreas and impact the expression of the glut-2 protein which causes increased blood glucose levels in test animals. stz works by forming free radicals that damage pancreatic beta cells resulting in hyperglycemia and hypoinsulinemia (munjiati et al., 2021). nicotinamide (nta) effectively protects β cells against stz cytotoxicity, nta attenuates diabetes complications caused by streptozotocin and increases 360 biology, medicine, & natural product chemistry 14 (1), 2025: 357-361 survival rates in mice. protection from try animals depends on decreasing the damage and methylation of deoxyribonucleic acid (dna) in the pancreatic islets, increased insulin secretion, nerve protection function and. nicotinamide (pyridine-3-carbosamide) is an amide form of vitamin b3 or niacin. nicotinamide is a percussor of nicotinamide adenine dinucleotide (nad) and nicotinamide adenine dinucleotide phosphate (nadp) which can protect pancreatic β cells from various toxic substances. on the other hand, nicotinamide protects cells β the pancreas from dna damage, therefore nicotinamide can cope with stzinduced diabetes, increased hyperglycemia and weight loss (cruz et al., 2019). the differential test findings across groups indicated no significant differences, despite a reduction in blood glucose levels following the administration of orthosiphon aristatus leaf extract. the extract's mechanism of action is intricate and not yet completely comprehended. stz possesses the capability to limit dna synthesis, induce cellular apoptosis, and hinder cellular regeneration (harijanto & dewajanti, 2017). the use of nicotinamide (nta) is aimed at protecting against pancreatic damage, preventing it from becoming acute. nicotinamide (nta) effectively protects β cells against stz cytotoxicity, nta attenuates diabetes complications caused by streptozotocin and increases survival rates in mice. protection from try animals depends on decreasing the damage and methylation of deoxyribonucleic acid (dna) in the pancreatic islets, increased insulin secretion, nerve protection function and. nicotinamide (pyridine-3-carbosamide) is an amide form of vitamin b3 or niacin. nicotinamide is a percussor of nicotinamide adenine dinucleotide (nad) and nicotinamide adenine dinucleotide phosphate (nadp) which can protect pancreatic β cells from various toxic substances (cruz et al., 2019). this study observed the expression of col1a1 quantitatively using the analysis of the cycle of tresh hold (ct) value of the col1a1 gene mrna to the ct of the internal gene β-actin. the results of the col1a1 gene expression difference test with the kruskal wallis test showed no significant difference in gene expression levels between groups, the same results were shown by the correlation test between blood sugar levels and gene expression with the spearman's rho test with a value of p>0.05 (table 2). this is possible because the expression process of the col1a1 gene (col1a1 protein synthesis) is influenced by various factors both intracellular and extracellular, both at the transcription, post-transcription, and post-translational levels. the col1a1 protein is a protein that plays a role in the interaction of the extracellular matrix (ecm), the link between the col1a1 protein and diabetes mellitus occurs through the ecmreceptor interaction pathway (lin et al., 2021). the results of meaningless expression of col1a1 genes between groups can be influenced by several aspects: (1) the synthesis and processing of cola1 is a complex process involving intracellular modifications such as hydroxylation of proline, lysine, and glycosylation, which are important for the stability of collagen structure. once secreted, procollagen undergoes proteolytic breakdown and the formation of cross-links in the extracellular matrix, which results in mature collagen fibrils. this process is controlled by various regulatory mechanisms, including epigenetics (dna methylation, histone modification), transcription (activation by transcription factors such as sp1, ap-1, and runx2), post-transcription (regulation by mirnas such as mir29), as well as post-translational modification (hydroxylation, glycosylation, and proteolytic breakdown), which overall determine the final structure and function of collagen in tissues, (2) epigenetic regulation of col1a1 occurs in some diseases, especially found at the transcription level which states that the tgfβ pathway plays an important role, especially through the regulation of the smad transcription factor which interacts directly with col1a1. other factors known to play a role in the transcription process of col1a1 are nf-κb, ap2, and nr4a1. meanwhile, the involvement of other transcription factors such as notch2, c-myb, hif1α, βcatenin, zeb1, and yap is still indirect, and further research is needed to ascertain whether these factors specifically target col1a1, (3) there is col1a1 regulation at the post-transcription level, namely the regulation of col1a1 mrna by the tent5a gene and a number of mirnas (mir-98, mir-126-5p, mir-2185p, mir-328-3p, mir-338-3p and mir-29b-3p), (4) changes in post-translational modification of type i collagen or the occurrence of mutations that alter the procollagen proteolytic cleavage point, and (5) there is an influence of several proteins such as mmp-2 and tryptase-β that have implications for the regulation of cola1(i) gene expression reported to be associated with various diseases (devos et al., 2023). further research is needed to understand the effect of ortosiphon aristatus on overall glucose metabolism. several studies have shown the potential of ortosiphon aristatus in lowering blood glucose levels, but the exact mechanism is still unclear. the results of the statistical test that did not differ significantly also assumed that the development of diabetes mellitus was not entirely in the chronic phase, related to the complex and long-term pathomechanism of diabetes mellitus. conclusions administration of cat whisker leaf extract (ortosiphon aristatus) for 14 days was not significant in increasing the col1a1 expression of hyperglycemia-induced rats. regarding the expression of col1a1, more comprehensive research is needed on the molecular processes involved in the synthesis of col1a1, by integrating various factors aisyah et al. – effect of ortosiphon aristatus on improving col1a1 expression 361 that are currently still being carried out separately to clarify the potential of col1a1-based therapies based on a thorough understanding of col1a1 and colα1 (i) regulation. acknowledgements: authors would like to thank the faculty of medicine, universitas muhammadiyah surakarta for the support in implementing this research and to the the association of medical and health colleges of muhammadiyah for the financial support. authors’ contributions: riandini aisyah & safari wahyu jatmiko designed the study and analyzed the data, as well as wrote the manuscript. erika diana risanti conceptualized research methodologies. fatin asfarina carried out the laboratory work under supervision. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this research was funded by the association of medical and health colleges of muhammadiyah research grant no. 4602/d.2-vii/fk/ix/2024. references arifah, f. h., nugroho, a. e., rohman, a., & sujarwo, w. (2022). a review of medicinal plants for the treatment of diabetes mellitus: the case of indonesia. south african journal of botany, 149, 537–558. https://doi.org/10.1016/j.sajb.2022.06.042 cruz, b., flores, r. j., uribe, k. p., espinoza, e. j., spencer, c. t., serafine, k. m., nazarian, a., & o’dell, l. e. (2019). insulin modulates the strong reinforcing effects of nicotine and changes in insulin biomarkers in a rodent model of diabetes. neuropsychopharmacology, 44(6), 1141–1151. https://doi.org/10.1038/s41386-018-0306-3 devos, h., zoidakis, j., roubelakis, m. g., latosinska, a., & vlahou, a. (2023). reviewing the regulators of col1a1. international journal of molecular sciences, 24(12), 10004. https://doi.org/10.3390/ijms241210004 harijanto, e. a., & dewajanti, a. m. (2017). optimalisasi pemberian streptozotocin beberapa dosis terhadap peningkatan kadar gula darah tikus sprague dawley. j. kedokt meditek, 23(63), 12-18. https://doi.org/10.36452/jkdoktmeditek.v23i63.1559 khan, m. a. b., hashim, m. j., king, j. k., govender, r. d., mustafa, h., & al kaabi, j. (2019). epidemiology of type 2 diabetes – global burden of disease and forecasted trends: journal of epidemiology and global health, 10(1), 107. https://doi.org/10.2991/jegh.k.191028.001 lin, g., wan, x., liu, d., wen, y., yang, c., & zhao, c. (2021). col1a1 as a potential new biomarker and therapeutic target for type 2 diabetes. pharmacological research, 165, 105436. https://doi.org/10.1016/j.phrs.2021.105436 maulana, f., muhammad, a. a., umar, a., mahendra, f. r., musthofa, m., & nurcholis, w. (2022). profiling metabolites through chemometric analysis in orthosiphon aristatus extracts as α-glucosidase inhibitory activity and in silico molecular docking. indonesian journal of chemistry, 22(1), 501. https://doi.org/10.22146/ijc.71334 munjiati, n. e. (2021). pengaruh pemberian streptozotocin dosis tunggal terhadap kadar glukosa tikus wistar (rattus norvegicus). meditory : the journal of medical laboratory, 9(1), 62–67. https://doi.org/10.33992/m.v9i1.1330 ojo, o. a., ibrahim, h. s., rotimi, d. e., ogunlakin, a. d., & ojo, a. b. (2023). diabetes mellitus: from molecular mechanism to pathophysiology and pharmacology. medicine in novel technology and devices, 19, 100247. https://doi.org/10.1016/j.medntd.2023.100247 septiana, e., rizka, n. m., yadi, y., & simanjuntak, p. (2021). antidiabetic activity of extract combination of orthosiphon aristatus and oryza sativa l. var glutinosa. borneo journal of pharmacy, 4(3), 202–209. https://doi.org/10.33084/bjop.v4i3.2154 thinn, n. w., oo, z. k., & maw, s. s. (2018). investigation of hypoglycaemic activity of some myanmar medicinal plants. international journal of technical research & science, 3(03), 104–108. https://doi.org/10.30780/ijtrs.v3.i3.2018.011 wahidin, m., achadi, a., besral, b., kosen, s., nadjib, m., nurwahyuni, a., ronoatmodjo, s., rahajeng, e., pane, m., & kusuma, d. (2024). projection of diabetes morbidity and mortality till 2045 in indonesia based on risk factors and ncd prevention and control programs. scientific reports, 14(1), 5424. https://doi.org/10.1038/s41598-024-54563-2 waras nurcholis, fachrur rizal mahendra, milanda fiorella gultom, safira khoirunnisa, mayang anggita cahya kurnia, & hamdan hafizh harahap. (2022). phytochemical, antioxidant and antibacterial screening of orthosiphon stamineus leaf extract two phenotypes. jurnal jamu indonesia, 7(3), 121–129. https://doi.org/10.29244/jji.v7i3.280 weng, x., lin, d., huang, j. t. j., stimson, r. h., wasserman, d. h., & kang, l. (2020). collagen 24 α1 is increased in insulinresistant skeletal muscle and adipose tissue. international journal of molecular sciences, 21(16), 5738. https://doi.org/10.3390/ijms21165738 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1029-1031 | doi: 10.14421/biomedich.2025.142.1029-1031 issn 2540-9328 (online) intraosseous capillary hemangioma of the nasal dorsum in an adolescent soehartono1*, rizky afianti2 1department of otolaryngology-head and neck surgery, division of oncology, faculty of medicine, brawijaya university dr. saiful anwar general hospital, east java, indonesia. 2otolaryngology-head and neck surgery resident, department of otolaryngology-head and neck surgery, faculty of medicine, brawijaya university dr. saiful anwar general hospital, east java, indonesia. corresponding author* rizkyafianti@gmail.com abstract hemangioma is a benign vascular tumor that commonly occurs in children, but its presentation on the nasal dorsum is rare, especially among adolescents. this case report describes a 16-year-old male patient with a progressively enlarging, painful nasal mass over seven months. clinical workup including ct-scan, angiography, and fnab revealed a vascular lesion suggestive of hemangioma. the patient underwent pre-operative embolization followed by surgical excision. histopathological and immunohistochemical evaluations confirmed the diagnosis of intraosseous capillary hemangioma. postoperative follow-up showed good aesthetic and functional outcomes with no recurrence. this report highlights the importance of comprehensive diagnostic and surgical approaches for managing rare hemangioma of the nasal dorsum to minimize complications and preserve facial structure. keywords: capillary hemangioma; dorsum nasi; embolization; nasal tumor; surgical excision. abbreviations: fnab (fine needle aspiration biopsy), ct (computed tomography), cd (cluster of differentiation) introduction hemangioma is a benign vascular tumor characterized by the abnormal proliferation of blood vessels. it is most commonly observed in infants and young children, with a prevalence estimated at approximately 5% to 10% in neonates, particularly among caucasians (manjula et al. 2017; huang et al. 2024). although hemangiomas predominantly affect the skin, mucosa, and soft tissues, they can also involve deeper structures such as bone, including facial bones like the nasal bone. hemangiomas occurring on the nasal dorsum are rare, particularly in adolescents and adults, and often present diagnostic and therapeutic challenges due to the unique anatomical and aesthetic relevance of the nasal region (sarafoleanu et al. 2022). in general, hemangiomas are classified histologically into three major types: capillary, cavernous, and mixed (kurniawan 2022). capillary hemangiomas are composed of closely packed small capillary vessels, and their intraosseous variant is uncommon, accounting for less than 1% of all primary bone tumors (eksal et al. 2005). when involving the nasal dorsum, hemangiomas may manifest as slow-growing, painless masses. however, in some cases, symptoms such as facial deformity, nasal obstruction, or localized pain may occur. the differential diagnosis includes other soft tissue and vascular tumors, necessitating thorough clinical and radiological assessments (kadriyan et al. 2020). the diagnostic process often includes non-invasive imaging modalities such as computed tomography (ct) and magnetic resonance imaging (mri), which can delineate the lesion’s extent, vascularity, and relation to adjacent structures. angiography may be employed for both diagnosis and pre-operative planning, especially in large or hypervascular lesions where embolization is considered to reduce intraoperative bleeding risk (farisa 2017). definitive diagnosis is based on histopathological and immunohistochemical analysis, including markers such as cd34 and cd31, which are commonly expressed in vascular endothelial tumors (sasaki et al. 2019). therapeutic approaches vary depending on the size, location, and stage of growth. while conservative and pharmacologic treatments such as corticosteroids or propranolol are effective for some infantile hemangiomas, surgical excision remains the treatment of choice for nasal dorsum hemangiomas that compromise facial aesthetics or function, especially in older children and adolescents (keller et al. 2017; rotter and de manuscript received: 10 june, 2025. revision accepted: 04 november, 2025. published: 13 november, 2025. https://doi.org/10.14421/biomedich.2025.142.1029-1031 1030 biology, medicine, & natural product chemistry 14 (2), 2025: 1029-1031 oliveira 2017). preoperative embolization can significantly aid in reducing intraoperative complications and improving surgical outcomes. this case report aims to describe the clinical features, diagnostic workup, surgical management, and histopathological findings of a capillary hemangioma involving the nasal dorsum in a 16-year-old male patient. by reporting this rare presentation, we intend to contribute to the clinical understanding and management strategies for hemangiomas in anatomically and cosmetically sensitive regions. materials and methods study area the case was handled at the department of otorhinolaryngology – head and neck surgery, dr. saiful anwar general hospital, faculty of medicine, universitas brawijaya, malang, indonesia, from september 2024 to february 2025. clinical evaluation and diagnostic procedures a 16-year-old male presented with a firm, non-pulsatile nasal mass located on the right dorsum nasi. the lesion measured approximately 4 × 4 × 3 cm and had developed progressively over the previous seven months. initial clinical evaluation included comprehensive anamnesis, physical examination, and anterior rhinoscopy. nasoendoscopy revealed superior nasal compression without mucosal disruption or purulent discharge. figure 1. clinical picture of the patient before surgery. imaging diagnostics included a contrast-enhanced ct scan of the head and neck, which showed a solid hypervascular mass eroding adjacent nasal bones, suggestive of a benign soft tissue tumor (hemangioma). ct angiography further confirmed the presence of feeder vessels originating from both right and left lateral nasal arteries. no intracranial abnormalities were detected. figure 2. from the ct scan image, a solid hypervascular mass was obtained in the right nasal region with erosion of the nasal bone around it (red arrow), suggesting a suspected benign soft tissue tumor, e.g. hemangioma, which is fed via the right lateral nasal artery. a fine needle aspiration biopsy (fnab) was performed on the lesion, yielding findings consistent with a benign vascular lesion. given the vascular nature of the mass, preoperative embolization was conducted by the interventional radiology team to reduce intraoperative bleeding. blushing was observed on angiography with tumor supply from the lateral nasal artery branches. surgical procedure surgical excision was performed on january 14, 2025, under general anesthesia using a lateral rhinotomy approach. during the procedure, 21 ml of blood was aspirated from the tumor capsule. the total intraoperative blood loss was approximately 300 ml. a drain was placed postoperatively and removed after stabilization of the wound site. figire 3. preoperative and intraoperative views of the nasal dorsum hemangioma excision. histopathological and immunohistochemical analysis tissue samples were sent for histopathological examination, which revealed an ossifying capillary hemangioma. immunohistochemical (ihc) staining was subsequently performed using cd34 and cd31 markers on paraffin blocks to confirm the vascular endothelial origin. the immunophenotype supported a diagnosis of capillary hemangioma. soehartono & afianti – nasal dorsum hemangioma case 1031 ethical consideration this case report was conducted in accordance with institutional ethical standards. the patient and his legal guardian provided informed consent for clinical treatment, investigation, and publication of anonymized data and images. results and discussion the patient underwent successful preoperative embolization, significantly reducing intraoperative bleeding and allowing for complete tumor excision. the hypervascular lesion located in the right nasal dorsum measured approximately 3 × 3 cm intraoperatively. grossly, the mass was encapsulated and firm. histopathological examination showed characteristic features of a capillary hemangioma, including small capillary-sized vascular channels lined by flattened endothelial cells. bony spicules and fibrous tissue components were also observed, consistent with an ossifying variant. immunohistochemical staining revealed strong positivity for cd34 and cd31, confirming the vascular nature and endothelial origin of the tumor. these markers are widely recognized for their role in identifying vascular neoplasms and help distinguish hemangiomas from other vascular malformations or neoplasms. this case supports existing literature that surgical excision remains the gold standard in managing hemangiomas with localized growth and aesthetic compromise, particularly in regions like the nasal dorsum where conservative therapy may not be optimal. kadriyan et al. (2020) and sarafoleanu et al. (2022) also reported favorable outcomes with early surgical intervention. preoperative embolization, as performed in this case, offers a significant advantage by minimizing operative complications. notably, the patient experienced no postoperative complications, and one-month follow-up showed no recurrence. functionally, nasal patency was preserved, and cosmetically, the outcome was acceptable. the integration of imaging, angiography, pathology, and immunohistochemistry was critical in achieving accurate diagnosis and effective management. figure 4. postoperative appearance at one-month follow-up after surgical excision of nasal dorsum hemangioma. this case contributes to a growing understanding of the rare presentation of ossifying capillary hemangioma in the nasal dorsum and reinforces the value of multidisciplinary treatment approaches. conclusions this case illustrates the clinical importance of recognizing and appropriately managing rare vascular tumors such as intraosseous capillary hemangioma of the nasal dorsum. a thorough diagnostic approach involving imaging, angiography, biopsy, and immunohistochemistry is essential for accurate diagnosis. surgical excision following preoperative embolization provided an effective and safe therapeutic strategy, with favorable functional and aesthetic outcomes. early identification and multidisciplinary intervention are key to minimizing complications and ensuring optimal results in managing vascular nasal tumors. competing interests: the authors state that there are no competing interests. references eksal, k., ob, m.d., mh, m.d., & bb, m.d. (2005). hemangioma of the nasal bone: a case report. huang, x., si, w., zou, z., li, b., mu, y., zhong, w., et al. (2024). efficacy and safety of oral propranolol and topical timolol in the treatment of infantile hemangioma: a meta-analysis and systematic review. frontiers in pharmacology, 15. https://doi.org/10.3389/fphar.2024.00001 kadriyan, h., sulaksana, m.a., yudhanto, d., aryani, i.g.a.t., yuliani, e.a., ardianti, n.e., et al. (2020). subcutaneous hemangioma on nasal dorsum: a case report. journal of medical case reports, 14(1). https://doi.org/10.1186/s13256020-02539-2 keller, r.g., stevens, s., & hochman, m. (2017). modern management of nasal hemangiomas. jama facial plastic surgery, 19(4), 327–332. https://doi.org/10.1001/jamafacial.2017.0150 kurniawan, h. (2022). tata laksana hemangioma pleura. zahra: journal of health and medical research, 2(2), 129–141. manjula, j., kumaravel, s., gokiladevi, d., lakshmi, s.v., & anandan, h. (2020). a clinical study of hemangiomas and vascular malformations. annals of international medical and dental research. rotter, a., & de oliveira, z.n.p. (2017). infantile hemangioma: pathogenesis and mechanisms of action of propranolol. journal der deutschen dermatologischen gesellschaft, 15(12), 1185– 1190. sarafoleanu, c., camburu, s.m., & lupoi, d. (2022). cavernous haemangioma of the nasal pyramid: literature review and our experience. romanian journal of rhinology, 12(48), 169–175. sasaki, m., north, p.e., elsey, j., bubley, j., rao, s., jung, y., et al. (2019). propranolol exhibits activity against hemangiomas independent of beta blockade. npj precision oncology, 3(1), 1–10. https://doi.org/10.1038/s41698-019-0095-2 https://doi.org/10.3389/fphar.2024.00001 https://doi.org/10.1186/s13256-020-02539-2 https://doi.org/10.1186/s13256-020-02539-2 https://doi.org/10.1001/jamafacial.2017.0150 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 1, april 2022 | pages: 55-63 | doi: 10.14421/biomedich.2022.111.55-63 issn 2540-9328 (online) a mathematical model of cervical cancer treatment by radiotherapy followed by chemotherapy murtono1,*, sugiyanto2, mansoor abdul hamid3 1department of education physics, 2department of mathematics; universitas islam negeri sunan kalijaga yogyakarta, indonesia. 3food technology and bioprocess, universiti malaysia sabah, malaysia. corresponding author* hasnamur@yahoo.co.id manuscript received: 15 april, 2022. revision accepted: 10 may, 2022. published: 21 may, 2022. abstract cervical cancer is the second most common type of malignant tumor found in women aged 15-44 years worldwide. radiotherapy is one form of treatment that uses radiation that can eliminate or kill cancer cells and one way to treat cervical cancer is quite popular. chemotherapy is a cancer treatment using anticancer drugs designed to kill or slow the growth of cancer cells that divide rapidly in the body. the method used is to study mathematical model of cervical cancer with radiotherapy and radiotherapy treatment followed by chemotherapy. the results of this study are for the early stages of cervical cancer with radiotherapy is quite effective, while for the late stages of radiotherapy and chemotherapy is less effective. therefore, the need for other treatments for end-stage cervical cancer in addition to radiotherapy and chemotherapy. keywords: cervical cancer; radiotherapy; chemotherapy. introduction cervical cancer is still the leading cause of cancer deaths among women in developing countries including indonesia (hoffman, et.al, 2008 and woodman et al., 2007). indonesia cancer foundation explained, the mortality rate of cervical cancer most among other types of cancer among women. it is estimated that 52 million indonesian women are at risk for cervical cancer, while 36 percent of all women with cancer are cervical cancer patients. there are 15,000 new cases per year with the death of 8,000 people per year (kompas, 2008). in southeast asia the incidence of cervical cancer becomes number two in women after breast cancer. nevertheless, in globocan (2012) the mortality rate due to cervical cancer is higher than the mortality rate due to breast cancer. radiotherapy is one of treatments that can be done for a patient with cervical cancer. radiotherapy is used as a therapy for cancer patients by using radiation that can eliminate and kill cancer cells, a way to treat cervical cancer which is quite popular. treatment with radiation therapy is usually done with chemotherapy, or accompanied by other therapies and sometimes only with radiotherapy alone depending on the type of cervical cancer suffered and the level of cancer. model formation this model develops from asih, et al. (2015) on the development of cervical cancer, liu and yang (2014) on general cancer treatment models with radiotherapy and pillis et al. (2007) on general cancer treatment models with chemotherapy. figure 1. transfer diagram of cervical cancer treatment with radiotherapy and followed by chemotherapy. caption figure 1:  1 1sl s k ms s   , 2 2( ) il i k mi i   , 3 3( ) pl p k mp p   , https://doi.org/10.14421/biomedich.2022.111.55-63 56 biology, medicine, & natural product chemistry 11 (1), 2022: 55-63 4 4( ) cl c k mc c  is a function of chemotherapy and radiotherapy. table 1. subpopulations, parameters and units. symbol explanation unit  s t normal cells density cells/mm3  i t infected cells density cells/mm3  p t pre-cancerous cells density cells/mm3  c t cancer cells density cells/mm3  v t free virus density virus/mm3  m t concentrations of chemotherapy drugs mg r intrinsic cell growth rate is normal 1/ day n homeostatic carrying capacity cells/mm3  the rate infection 1/(day x virus) 1a the rate of proliferation of infected cell 1/day 1d the rate of apoptosis of infected cell 1/day  the rate of progression, from being infected to pre-cancer 1/day 2a the rate of proliferation of pre-cancerous cells 1/day 2d the rate of apoptosis of pre-cancerous cells 1/day  the maximum invasion rate, from precancer to cancer 1/day k half-saturation concentration cells/mm3 3a the rate of proliferation of cancer cells 1/day 3d the rate of apoptosis of cancer cells 1/day n the average number of viruses produced one infected cell constant 4d the death rate of free virus 1/day 1 the proportion of radiation to susceptible cells (normal cells) constant 2 the proportion of radiation to infected cells constant 3 the proportion of radiation to pre-cancerous cells constant  strategy of radiotherapy 1/day sk fractional susceptible cells killed by chemotherapy 1/day ik fractional infected cells killed by chemotherapy 1/day pk fractional pre-cancerous cells killed by chemotherapy 1/day ck fractional cancer cells killed by chemotherapy 1/day  the rate of chemotherapy drug decay 1/day mv the rate of chemotherapy drug enter mg/day murtono et al. – a mathematical model of cervical cancer treatment by … 57 the dynamics of cervical cell changes from normal cells to cancer cells are given in the following differential equation systems (1). 11 s ds s i rs sv k ms s dt n              (1a) 1 1 2i di sv a i d i i k mi i dt          (1b) 2 2 2 32 2 p dp p i a p d p k mp p dt k p           (1c) 2 3 32 2 c dc p a c d c k mc c dt k p        (1d) 1 4 dv nd i d v dt   (1e) m dm m v dt    (1f) the point of equilibrium the equilibrium point with radiotherapy alone, without chemotherapy system (1) without chemotherapy is 11 ds s i rs sv s dt n             (2a) 1 1 2 di sv a i d i i i dt         (2b) 2 2 2 32 2 dp p i a p d p p dt k p          (2c) 2 3 32 2 dc p a c d c c dt k p       (2d) 1 4 . dv nd i d v dt   (2e) the requirement to determine the equilibrium point of system (2) is 0, 0, 0, 0, 0 ds di dp dc dv dt dt dt dt dt      . so the point of equilibrium with radiotherapy in cervical cancer, without chemotherapy, is    * * * * * 1 2 3 1 51 4, , , , , , , ,r rep s i p c v p       * * * * * 2 2 3 2 52 4, , , , , , , ,r rep s i p c v p    (3)    * * * * * 3 2 3 3 53 4, , , , , , , ,r rep s i p c v p    , where 1 1 4 nd d   , 1 2 1 2 1 d a         ,  1 2 3 1 r n r r n          , 4 1 3   , 3 1 2 2 3 a a d         , 2 2 2 2 2 3 1 2 2 3 a k d k k b a d          , 2 3 1 2 2 3 k c a d       , 3 1 1 1 1 12 9 27d a a b c   ,     2 3 3 2 1 1 1 1 1 1 12 9 27 4 3e a a b c a b     ,    1 3 3 1 1 1 1 1 1 1 1 1 3 3 2 3 2 r a p d e d e      ,    1 3 3 2 1 1 1 1 1 3 1 1 3 1 3 6 2 6 2 r a i i p d e d e         ,    1 3 3 3 1 1 1 1 1 3 1 1 3 1 3 6 2 6 2 r a i i p d e d e         ,    2 5 2 2 3 3 i i i p k p d a        . 58 biology, medicine, & natural product chemistry 11 (1), 2022: 55-63 existence of the equilibrium points with radiotherapy alone, without chemotherapy if it satisfies 1. 1 2 1 0d a      , 2.    1 2 1 4 1 1 0 d a d r n r nd                 , 3. 0, 1,2,3ip i  and 4. 3 3 0d a   . the equilibrium points with radiotherapy and followed by chemotherapy in this system is the same as system (1). to find the equilibrium point in a way 0 ds dt  , 0 di dt  , 0 dp dt  , 0 dc dt  , 0 dv dt  , 0 dm dt  . so the equilibrium point of radiotherapy followed by chemotherapy in cervical cancer is    * * * * * * 1 7 9 8 9 1 101 1 9 6, , , , , , , , , ,rc rc rcep s i p c v m p            * * * * * * 2 7 9 8 9 2 102 1 9 6, , , , , , , , , ,rc rc rcep s i p c v m p         (4)    * * * * * * 3 7 9 8 9 3 103 1 9 6, , , , , , , , , ,r rc rcep s i p c v m p         where 6 mv    , 1 7 1 n r           , 6 1 8 snk n n r r        , 1 8 1 1 6 2 9 1 7 ia d k                , 11 2 2 2 6 3 , p a a d k             2 2 2 6 3 2 2 2 6 3 p p a d k k b a d k              , 2 11 2 2 2 6 3p k c a d k         , 3 2 2 2 2 22 9 27d a a b c   ,     2 3 3 2 2 2 2 2 2 2 22 9 27 4 3e a a b c a b        2 3 3 1 2 2 2 2 1 1 1 1 3 3 2 3 2 rc a p d e d e      ,    2 3 3 2 2 2 2 2 1 3 1 1 3 1 3 6 2 6 2 rc a i i p d e d e         ,    2 3 3 3 2 2 2 2 1 3 1 1 3 1 3 6 2 6 2 rc a i i p d e d e         ,    2 10 2 2 3 3 6 4 irc irc irc c p k p a d k           . existence of the equilibrium points with radiotherapy and followed by chemotherapy, if it satisfies 1. 6 1 7 9 0snk n n r r         , 2. 1 8 1 1 6 2 0ia d k            and 3. 0ircp  . stability of the equilibrium point stability of the equilibrium points with radiotherapy alone, without chemotherapy theorem 1. if 3 3 0a d    ,   2 2 3 32 2 2 2 0i i pk a d k p        , 0ia  , 32 9 27 0i i i ia ab c   , 2 3 0i ia b  where 3,4,5i  then the equilibrium point murtono et al. – a mathematical model of cervical cancer treatment by … 59    * * * * * 2 3 6 5, , , , , , , ,ir i iep s i p c v p    asymptotically stable. proof. from system (2) is obtained characteristic equation in equation (5).            2 1 3 3 2 3 32 2 2 1 3 232 51 1 1 1 2 4 32 51 1 1 1 2 4 2 1 1 2 4 2 1 51 32 51 1 2 2 2 2 pk a d a d k p rr r a d d n n rr r a d d n n r a d d nd n rr r n n                                                                                                     1 1 2 4 2 1 2 51 4 2 1 0 a d d nd r d nd n                                              (5) stability of the equilibrium points with radiotherapy and followed by chemotherapy theorem 2. if 3 3 4 0m c v a d k            ,   2 2 2 32 2 2 2 0irc m p irc p k v a d k k p               , where 3,4,5i  and 6 0a  , 3 6 6 6 62 9 27 0a a b c   , 2 6 63 0a b  then the equilibrium point    * * * * * * 7 9 8 9 10 1 9 6, , , , , , , , , ,irc irc ircep s i p c v m p         asymptotically stable. proof. from system (1) is obtained characteristic equation in equation (6).            2 2 3 3 2 3 32 2 2 2 3 232 52 1 1 1 2 4 32 52 1 1 1 2 4 2 1 1 2 4 2 1 52 32 52 1 2 2 2 2 p k a d a d k p rr r a d d n n rr r a d d n n r a d d nd n rr r n n                                                                                                     1 1 2 4 2 1 2 52 4 2 1 0 a d d nd r d nd n                                              (6) 60 biology, medicine, & natural product chemistry 11 (1), 2022: 55-63 simulation in this section will be discussed about the numerical simulation and medical interpretation of the mathematical model of cervical cancer in the presence of radiotherapy and chemotherapy effects are divided into 2 cases. 1. the influence of radiotherapy 2. the influence of radiotherapy and followed by chemotherapy simulation of radiotherapy effect the parameter values for this case are given in the following table 2. table 2. values of parameters for cases of radiotherapy effects. symbol value reference r 0.0255 asih et al. (2015) n 9900.99 asih et al. (2015)  0.00001 asih et al. (2015) 1 0.004 liu and yang (2014)  0.05 liu and yang (2014) 1a 100 asih et al. (2015) 1d 100.01 asih et al. (2015)  0.0082 asih et al. (2015) 2 0.03 liu and yang (2014) 2a 101 asih et al. (2015) 2d 100 asih et al. (2015)  20.03 asih et al. (2015) k 2 asih et al. (2015) 3a 0.03 asih et al. (2015) 3d 101.01 asih et al. (2015) n 1 asih et al. (2015) 4d 500 asih et al. (2015) figure 2 shows the first 30 days or the first month after radiotherapy is showed a decrease in the number of cells from 13 cell/mm3 to 12.78 cell/mm3. this means that the decrease in cell number is 0.22 cell/mm3. at six months and one year of radiotherapy if it continues to show a successive decrease to 11.8 cell/mm3 and 10.73 cell/mm3. this shows the ill effects of radiotherapy because of healthy cells. for infected cells in the first and third months after radiotherapy successively from 13 cell/mm3 to 4,156 cell/mm3 and 0.4364 cell/mm3. this is good for curing cancer, because the infected cells will eventually become pre-cancerous cells. while the cancer cells on the fourth day after radiotherapy immediately fell rapidly, from 13 cell/mm3 to 0.3526 cell/mm3. for pre-cancerous cells almost the same as cancer cells, that is down very quickly. for the virus goes down quickly, because the infected cell goes down as well. this is because the virus replicates itself from infected cells. murtono et al. – a mathematical model of cervical cancer treatment by … 61 figure 2. trajectory diagram the influence of radiotherapy. simulation of the influence of radiotherapy and followed by chemotherapy the parameter values for this case are given in the following table 3. table 3. value of case parameters the influence of radiotherapy and followed by chemotherapy. symbol value reference r 0.0255 asih et al. (2015) n 9900.99 asih et al. (2015)  0.00001 asih et al. (2015) 1 0.004 liu and yang (2014)  0.05 liu and yang (2014) 1a 100 asih et al. (2015) 1d 100.01 asih et al. (2015)  0.0082 asih et al. (2015) 2 0.03 liu and yang (2014) 2a 101 asih et al. (2015) 2d 100 asih et al. (2015) symbol value reference  20.03 asih et al. (2015) k 2 asih et al. (2015) 3a 0.03 asih et al. (2015) 3d 101.01 asih et al. (2015) n 1 asih et al. (2015) 4d 500 asih et al. (2015) sk 0.01 pillis et al. (2007) ik 0.01 pillis et al. (2007) pk 0.01 pillis et al. (2007) ck 0.01 pillis et al. (2007)  0.1 pillis et al. (2007) mv 0.01 pillis et al. (2007) figure 3 shows a trajectory with the effects of radiotherapy and followed chemotherapy, which is an advanced patient of cervical cancer. normal cells during the first 30 days showed a rapid decline, from 13 cell/mm3 to 3,638 cell/mm3. this makes patients with 62 biology, medicine, & natural product chemistry 11 (1), 2022: 55-63 advanced cervical cancer in this condition will get worse. therefore, in this study advised not to use this treatment. cancer cells also drop very rapidly, in the first 4 days of radiotherapy and chemotherapy, from 13 cell/mm3 to 0.2061 cell/mm3. figure 3. trajectory diagram the influence of radiotherapy and followed by chemotherapy. conclusion cervical cancer is one type of cancer and second highest incidence of cancer in women compared to other cancers. treatment of cervical cancer with radiotherapy at an early stage is quite effective. this mathematical model showed that treatment of cervical cancer at an advanced stage with radiotherapy and followed by chemotherapy is not effective. because normal cells die very quickly, and will aggravate the patient's condition of cervical cancer. this research needs to be continued with other more effective treatments, especially for advanced stages of cervical cancer. conflict of interest: the authors declare that there is no conflict of interest. references asih t. s. n., suzanne lenhart, steven wise, lina aryati, f. adikusumo, mardiah s. hardianti, jonathan forde, (2015), the dynamics of hpv infection and cervical cancer cells, bull math biol, doi 10.1007/s11538-015-0124-2. globocan, (2012), download february 5, 2018. http://globocan.iarc.fr/old/factsheets/cancers/cervix-new.asp hoffman b., schorge j., schaffer j., halvorson l., bradshaw k., cunningham f., (2008), williams gynecology, second edition, mc grawhill’s, usa. kompas.com, july 10, (2017). download february 5, 2018. http://lifestyle.kompas.com/read/2017/08/15/071700520/kank er-serviks-bisa-dicegah. liu z. and yang c., (2014), researcharticle: a mathematical model of cancer treatment by radiotherapy, hindawi publishing corporation computational and mathematical methods in medicine volume 2014, article id 172923, 12 pages http://dx.doi.org/10.1155/2014/172923. murtono et al. – a mathematical model of cervical cancer treatment by … 63 pillis l.g. de, w. gua, k.r. fister, t. head, k. maples, a. murugan, t. neal, k. yoshida, (2007), chemotherapy for tumors: an analysis of the dynamics and a study of quadratic and linear optimal controls, mathematical biosciences 209 (2007) 292–315. woodman c. b. d., collins s. i., young l. s., (2007), the natural history of cervical hpv infection: unresolved issues, nat rev cancer, vol. 7(1): 11-22. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 1, 2014 | pages: 15-19 | doi: 10.14421/biomedich.2014.31.15-19 the effect of water-soluble stem extract “kayu kuning” (arcangelisia flava l. merr) on the growth inhibition of candida albicans atcc 10231 and trichophyton mentagrophytes in vitro rini setyowati, sudarsono* and setyowati e. p faculty of pharmacy ugm, indonesia author correspondency*: sudarsono@ugm.ac.id abstract “kayu kuning” (arcangelisia flava l.merr) was used when someone has a skin problem caused by candida albicans and trichophyton mentagrophytes. scientific based medicine on this traditional knowledge was necessary be done. stem powderwas extracted by distilled water.the extract was then evaporated. qualitative and quantitative analysis of the active substance e.g., berberin chloride by thin layer chromatography (tlc) the antifungal activity againts candida albicans and trichophyton mentagrophyteswere tested by using agar diffusion and microdilution methods. the absorbance from microdilution were analized by one way anova. the conclusion showed that the extract contained 1.55±0.12% w/walkaloid calculated as berberine chloride. the inhibition zone for candida albicans and trichophyton mentagrophytes were 16.65±4.52 and 6.55±0.05 mm respectively. the mic vallue for both fungi was 10 mg/ml.the mbc value for candida albicans was 40 mg/ml and for trichophyton mentagrophytes was 50 mg/ml. from the analysis with one-way anova, shows that there are significant differences between the positive control group and the test solution with the negative control group with p=0.020 for candida albicans and p=0.028 for trichophyton mentagrophytes (p<0.050). post hoc tukey analysis results showed that both intergroup and between the concentration of the test solution to the control group did not differ significantly positive because the value of p>0.050. keywords: arcangelisia flava l.merr, “kayu kuning”, growth inhibitor, candida albicans, trichophyton mentagrophytes introduction candidiasis caused by c.albicans, water fleas and dermatophytosis caused by t.mentagrophytes. this infection disease is usually treated by azoles derivatives which are ketoconazol, fluconazol, miconazol, and also the poliena classes, such as nistatin, contemporarily for the dermatophyte can be treated by gryseofulvine (jawetz et al., 2001). however, using antibiotic reported often causes microbes resistance (who, 2009). indonesia has occupied on eight ranking out of 27 countries by heavy burden for multi-drugs resistancy/mdr around the world. according to health ministry of republic of indonesia (2011).the use of antibiotics for health service was often not appropriate, so it cause less effective treatment, increasing risk for the patients, widespread resistance and costly treatment. therefore, it needs the proper solution to prevent the problems. one of solution is using traditional herbal medicine, as like conducted by remote communities that are far from goverment health services, so they can also conserve the local knowledge. “kayu kuning” or a. flaval.merr is one of plant used as traditional health care in the suban jeriji village, rambang dangku, muara enim, south sumatra, indonesia.this plant in muna village, southeast sulawesi is used for diarrhea, sore eyes, jaundice, oral ulces and water flea medicine (larisu, 2011). this plant was known by its bright yellow wood’s color, a herb, climbing, annual, wild growth and can be found in rocky beach or in edge of forest (sitepu and sutikno, 2001). a.flava l. contains saponin, flavonoid, polyphenolic substance, glycoside and alkaloid. berberine derivative are the the main group found in this plant. it was reported by singh, et. al. (2010). whereas terpenes are found in this plant e.g., fibrauleusin, fibraurin (siwon, 1982).berberine is alkaloid in form of chloride or sulfate salts, they are existing on menispermaceae plant, the mechanism of action as antimicrobial agent could be changing the arrangement of amino acid chain on dna that rises balances changes of genetics on dna, so that the dna of microbial will be defeat, this causes a core of microbial cell to be defeat and dead (dassonneville et al., 2000). methodology materials stem of a.flava l. was come from suban jeriji village, rambang dangku, muara enim, south sumatra. voucher specimen was found at pharmaceutical biology department, faculty of pharmacy ugm; berberine chloride (b2251-10g, sigma), c.albicans atcc 10231 and t.menta-grophytes, media nutrient broth and agar media, nystatin, mtt (methyl thiazol tetrazolium, sigma), n-butanol, glacial acetic acid, distilled water, 0.9% nacl (merck germany) and tlc(thin layer 16 biology, medicine, & natural product chemistry 3 (1), 2014: 15-19 chromato-graphy) plates silica gel 60 f254 (e.merck germany). apparatus set of reflux, incubator, autoclave, laminair air flow (laf), oven, flat microplate, microplate reader, petri dish, micropipette, white tip, yellow tip and blue tip, a set of tools thin layer chromatography (tlc), ultra violet (uv) light and tlc scanner procedure 20 gram of drug powder was extracted by distilled water 100 ml for 2 hours (reflux). the water extract was was filtered evaporated by reduced vapor. qualitative and quantitative analysis. it was done by tlc; 5 μl test solution and berberine chloride were spotted on tlc plate on silica gel 60 f254. the mobile phase was n-butanol, acetic acid, water (3:1:1 v/v/v). the spots were observed under uv254 nm, uv366 nm and visible light. the spots of test substance and berberine chloride were scanned between 200 700 nm. the standard curve ob berberin chloride was conducted from the series of berberine chloride, which was 100; 50; 25; 12,5; and 6,25 μg/ml. the solution test was made by 5 mg/ml in methanol. it was eluted by the same tlc system. the spot area under curve (auc) that was suspected as alkaloid measured by densitometer. it was made standard curve by regressing content (μg) vs area under curve. the alkaloid content was calculated as berberine chloride. antifungal activities test by agar diffusion method (kirby-bauer) the sterile agar media was diluted, after the lukewarm 10 ml, the media was added by 100 µl of fungi suspension, whipped homo-geneously. the concentration was 5x102– 2,5x103 cfu/ml of fungi in media. mixture was poured into a sterile petri disk, waited until condensing. steril paper diskwas mounted on the surface in order to be drop by sample 20 µl, each 10µl for nistatin and distilled water , then it was incubated on the temperature 37oc for 24 hours. clear zone was measured by using vernier caliper. anti-fungi test was done by microdillution method the test solution were dissolved into distilled water at several concentration 10 %; 8%; 6%; 4%; 2%; 1%; 0,5%; 0,25% and 0,125% b/v. each 50 µl solution test was poured into the well and added by fungi suspension in nb media. the fungi concentration becomes 5x102 cfu/ml. the sample was incubated on 37oc temperature for 24 hours. then, mtt was added to make easy observation. od (optical density) value could be seen through the absorption. it could be computed by formula: the clear pitting should be scratched on solid media for knowing a mec value. bioautographic assay the solution test was spotted for 5 µl on tlc plate and eluted by mobile phase n-butanol, acetic acid, water (3:1:1 v/v/v). it was prepared the media in order to the sterile mixed by standardized fungi suspension, poured into sterile petri and wait until condensing. the tlc plate was eluted, and dried, then it should be placed on the media for 30 minutes. after tlc plate had been taken, incubated the petri disc by 24 hours on 37oc. their clear zone was observed. results and discussion figure 1 showed that the hrf value of berberine chloride was 61 and spot like berberine solution test was 62 at visible light, uv 254 nm and 366 nm. the spot of sampel and standard was yellow fluorescence under the uv 366 nm. the scanning result for the two spots can be seen in figure 2 at λmax 349 nm. the standard equation curve was y = 58596,5419 x + 2512,5 with r value = 0,9956. the berberine chloride solution test were 1,55 ± 0,12% b/b (table 2). the sample had antifungal activities for tested microbial. the complete activities as table 3. to know potential of extract for anti-fungi activities was conducted the potential test using liquid dilution method, which was micro-dilution. parameter of antifungi activity was minimum inhibitory consentration (mic) value and minimum fungicidal concentration (mfc). the smallest concentration that was still showing clearness was mic, see figure 4 and table 4. the standard equation curve was y = 58596,5419 x + 2512,5 with r value = 0,9956. the berberine chloride solution test were 1,55 ± 0,12% b/b (table 2). figure 1. chromatogram of water-soluble stem extract of a.flava (x) and berberine chloride (b) examination by: (a) visible light; (b) uv 366 nm and; (c) uv 254 nm solid phase: silica gel 60 f254; mobile phase system: n-butanol, acetic acid, water (3:1:1 v/v/v). rini setyowati, et al. – the effect of water-soluble stem extract “kayu kuning” … 17 figure 2. the pattern of uv spectrum of berberine chloride and sample test between 200-700 nm. the sample had antifungal activities for tested microbial. the complete activities as table 3. to know potential of extract for anti-fungi activities was conducted the potential test using liquid dilution method, which was micro-dilution. parameter of antifungi activity was minimum inhibitory consentration (mic) value and minimum fungicidal concentration (mfc). the smallest concentration that was still showing clearness was mic, see figure 4 and table 4. table 1. berberine chloride content vsauc. berberinechloride (µg/µl) volume (µl) berberinechloride content (ng) hrf auc 0,00625 5 0,03125 63 2774,9 0,0125 5 0,0625 63 6586,6 0,025 5 0,125 63 10958,5 0,05 5 0,25 63 17700,2 0,1 5 0,5 63 31307,7 table 2. berberinechloridecontent in water-soluble stem extract of “kayu kuning”. sample consentration (mg/ml) volume (µl) sample weight (µg) hrf auc berberinechloride content/spot (ng) content (%b/b) 5 5 25 63 25308,30 389,01 1,56 5 5 25 64 23427,00 356,90 1,43 5 5 25 64 26777,20 414,10 1,66 average 25170,83 386,67 1,55 sd 1679,32 28,67 0,12 table 3. diameter of inhabitation extract is for test microbial. solution diameter inhibition c.albicans (mm) average (mm) diameter inhibition t.mentagrophytes (mm) average (mm) 1 2 3 1 2 3 sample 13,73 21,53 13,68 16,31 ± 4,52 6,52 6,52 6,60 6,55 ± 0,05 nystantin 17,00 16,85 18,10 17,32 ± 0,68 7,50 7,75 8,00 7,75 ± 0,25 aquadest table 4 showed that, the mic value of sample were 1% w/v for c.albicans and t.mentagrophytes. while table 5 showed that the mfc of sample for c.albicans was 4%w/v and for t.mentagrophytes was 5% w/v. result of bioautography assay showed that there was only an inhibition for c.albicans, but on t.mentagrophytes was not appearing the presence of a clear zone. tlc spot which was showing an activity of inhibition for c.albicans growth was the spot with 62 hrf value. it was berberine chloride, as like appeared on figure 5. this evidenced that solution test containing … … berberine chloride was a bio-active compound that was responsible for the presence of anti-fungi activities of c.albicans. while on t.mentagrophytes couldn't be determined if the compound has activity as anti-fungi, this should be caused by synergetic system. 18 biology, medicine, & natural product chemistry 3 (1), 2014: 15-19 figure 3. microdilution for (a) c.albicans and (b) t.mentagrophytes using mtt reagent. table 4. mic of sample for fungi tested using mtt reactant. fungi mic (mg/ml) average (mg/ml) c.albicans 10 10 10 10 t.mentagrophytes 10 10 10 10 table 5. mfc of sample for fungi tested. sample consentration (% w/v) c.albicans t. mentagrophytes 1 2 3 1 2 3 1 + + + + + + 2 + + + + + + 3 + + + + + + 4 -* -* -* + + + 5 -* -* -* nistatin5000 iu control fungi + + + + + + control media control solvent description: (+): there microbial growth (-): there is no microbial growth (*): mfc from result of analysis using one way anova, both c.albicans and t.mentagro-phyteswas exist significant differences between negative control group with all test solution group, this was by p-value = 0,020 for c.albicans, and p=0,028 for t.mentagrophytes (p<0,05). this result showed that giving test solution causing the growth inhibition of microbial significantly. in addition to significant differences was also showed between positive control group and negative control both c.albicans and t.mentagrophytes tests. table 6. percent inhibition of water-soluble extract of a.flava (“kayu kuning”) against microbial test. no sample consentration (% w/v) average of % inhibition (%) c.albicans t.mentagrophytes 1 0,0625 67,16 48,22 2 0,125 70,67 51,78 3 0,25 73,9 65,21 4 0,5 76,25 66,63 5 1 77,13 74,67 6 2* 85,92* 84,23* 7 3 87,98 89,22 8 4 92,38 93,39 9 5 97,07 96,03 nistatin 5000 iu 100,22 100,41 description: (*): mic figure 4. bioautografi assay results on (a) c.albicans (b) t.mentagrophytes. analysis result of post hoc tukey for c.albicans and t.mentagrophytes, correlation between positive control group and test solution group were not showing significant differences, except group number 1 (0,0625% concentra-tion), that was by p>0,05 value. this was showed that the test solution had the same effect with the positive control (antibiotic) for fungi growth. that was rini setyowati, et al. – the effect of water-soluble stem extract “kayu kuning” … 19 inhibiting the growth of fungi. while correlation between concentration groups on all test microbial, were not giving a significant differences, by p > 0,05 value. the data showed that an increasing of test solution concentration from the smallest concentration 0,0625% until 5% w/v were not showing a significant differences for the tested fungi growth inhibition. an activity and potential of sample could inhibited c.albicans and t.mentagro-phytes growths that was caused by the compound contained in the extract, berberine chloride. the activity of berberine chloride was known as antimicrobial (hwang et al., 2003; karou et al., 2006; scazzocchio et al., 2001; swabb et al., 1981; kaneda et al., 1991). conclusion 1. water-soluble extract of “kayu kuning”(a.flava) was containing of 1.55 ± 0.12% w/w alkaloid berberine chloride. 2. water-soluble extract of “kayu kuning” (a.flava) was active as antifungal against c.albicans colony with mic value of 10 mg/ml and mfc of 40 mg/ml while the colony t.mentagrophytes with mic and mbc values of 10 mg/ml and 50 mg/ml. references backer, c.a., and van den brink, jr.r.c.b., 1969, flora of java, vol. i, 153, 157, n.v.p. noordhoff, groningen. chitwood, l. a., 1969, tube dilution antimicrobial sucseptibility testing, applied microbiologi: 707-709. gupte, s., 1990, mikrobiologi dasar, edisi iii, diterjemahkan oleh dr. julius, e.s., 1992, 69-76, 321, binarupaaksara, jakarta. harborne, j. b., 1973, metode fitokimia, penuntun cara modern menganalisis tumbuhan, diterjemahkan oleh dr. kosasih dan dr. iwang soediro, 1984, terbitan kedua, 234-245, itb, bandung. heyne, k. 1927, tumbuhan berguna indonesia. jilid ii, diterjemahkan oleh badan litbang kehutanan, 1987, yayasan sarana wana jaya, jakarta. hwang, b.y., roberts, s.k., chadwick, l.r., wu c.d., and kinghorn, a.d., 2003, anti microbial constituents from goldenseal (the rhizomes of hydrastin canadensis) againt selected oral phatogens, planta med., 69 (7 : 623-627). karou, d., savadogo, a.yomeogo, s., and montesano, c., 2006, antibacterial activity of alkaloids from sida acuta, j. afric.bio.,5 (2): 195-299. jawetz, e., melnick, j.l., and adelberg, e.a., 2001, mikrobiologi kedokteran, diterjemahkan oleh dr. nani widori, 2005, edisi 22, 313-325, 344-346 penerbit salemba medika, jakarta. kaneda, y., torii, m., and tanaka, t., 1991, in vitro effects of berberine sulfate on the growth of entamoeba histolytica, giardia lamblia and trichomonas vaginalis. ann. trop. med.parasitol., 85:417-425 keawpradub, n., dejadisai, s. and yuenyongsawad, s., 2005, antioxidant and cytotoxic activities of thai medicinal plants named khaminkhruea: arcangelisia flava, coscinium blumeanum and fibraurea tinctori, songklanakarin, j. sci. technol., 27 (suppl. 2) : 455-467. mahon, c. r., and manuselis j.r. g., 1995, textbook of diagnosis microbiology, w. b sanders company, philadephia. nakamoto, k., tamamoto, m., and hamada, t., 1995, in vitro study on the effects of trial denture cleansers with berberine hydrochloride. j. prosthet. dent., 73:530-533. ongsangkul. m., jindarat. a., and rajona. c., 2009, antibacterial /effect of crude alcoholic and aqueous of six medical plants against staphylococus aureus and eschericia coli, j health res no. 23 vol. 3 : 153-156 praptiwi, jamal. y., fathoni, a., and agusta. a., 2009, antimicrobial metabolit from the culture of endophytic fungus afk-8 from kayu kuning (archangelisia flava l. merr), research centre of biologi, indonesian institute of science, bogor. scazzocchio, f., cometa, m.f., tomassini, l., and palmery, m., 2001, antibacterial activity of hydrastis canadensis extract and its major isolated alkaloids, planta med., 67 (6): 561-564. schwalbe, r., steele-moore, l., and goodwin, a.c., 2007, antimicrobial susceptibility testing protocols, 186-193, crc press taylor & francis group, london. singh, a., duggal, s., kaur, n., and singh, j., 2010, berberine: alkaloid with wide spectrum of pharmacological activities, j. nat. products, vol.3:64-67. siwon, f., 1982, a pharmacognostical study of some indonesian medicine of the family menispermaceae, disertasi, 10, 43, drukkij j h pasmans b. v’s gravenhage. stahl, e., 1973, analisis obat secara kromatografi dan mikroskopi, diterjemahkan oleh dr. kosasih padmawinata dan dr. iwang soediro, 1985, penerbit itb, bandung. swabb, e.a., tai, y.h., and jordan, l., 1981, reversal of cholera toxin-induced secretion inrat ileum by luminal berberine. j. am. physiol., 241 : 248-252. content_v3n1_2.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 2, 2015 | pages: 25-29 | doi: 10.14421/biomedich.2015.42.25-29 krokot (portulaca oleracea. l) as a natural sensitizer for tio2 dye-sensitized solar cells: the effect of temperature extract reyza anni mufidah, khamidinal, endaruji sedyadi and didik krisdiyanto* department of chemistry, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency*: didik_kris@yahoo.com abstract the solar cell is formed by a sandwich structure, in which two electrodes flank the primary electrolyte that is containing redox i-/i based on peg (polyethylene glycol). the working-electrode which is tio2 layer on an ito glass substrate is sensitized with krokot dye as the electron donor. the counter electrode is a layer of carbon. the fabrication cell is immersed with the krokot dye with 40°c, 50°c, and 60°c extract temperature. the result of the uv-vis shows that the absorption of wave-length from dye extract of krokot is located in the visible region with the absorbance peak in 420.5 nm and 665.5 nm which are the peak of chlorophyll. for the uv-vis solid system, there are the highest band gap in 50°c extract temperature that make the capability of absorption toward uv spectrum is large. furthermore, in the functional group analysed by ft-ir, there are shiften-carbonil and hydroxyl group after they are sensitized. from the current and voltage test with i-v meter keithley 2400 is resulted that on the 50°c extract temperature produces the highest efficiency of reaches which is 2.63 x 10-3 %. keywords: dye sensitized solar cell (dssc), krokot dye, chlorophyll, tio2 introduction the solar cell is one way to harness solar energy in which the device is able to convert the sunlight energy into electrical energy. in principle, the solar cell work is similar to the photosynthesis work in plants. light energy is used to produce free electrons. solar cell uses free electron to generate electrical energy while the plant uses the free electron to produce chemical energy (yuwono. et al. 2011). the development of solar cells based on dye sensitized began in 1991 when for the first time grätzel and o'regan designed the basic forms of solar cell based on film of titania semiconductor that is known as dyesensitized solar cell (dssc) in which these systems can convert solar energy into electrical energy (o'regan & gratzel, 1991). this mechanism shows the optical absorption and charge separation processes through the association of a sensitizer as a light absorber with a nanocrystal semiconductor that has a wide bandgap (gratzel, 2003). a dssc consists of a pair of coated glass tco substrate (transparent conducting oxide) as the electrode and the counter electrode, the redox electrolyte that contains iodide and triiodide ion (i /i ) carbon layer as the catalyst, porous tio2 nanocrystal as fotoanoda, and a dye photosensitizer (o'regan & gratzel, 1991). all components are arranged in front of the sandwich structure where the top layer is the working electrode as the initial layer in receiving photons and the lower layer is the counter electrode and the middle is electrolyte to regenerate electron. dye criteria that can be used as a dye sensitizer is adsorption intensity at visible wavelengths, strong adsorption on the surface of the semiconductor, has the ability to inject electrons to the band conduction of the semiconductor, and has a group = o or -h to bind to the surface of tio2 which can increase the reaction rate of the electron transfer (ludin, et al, 2014). therefore, this study will utilize the potential of the natural dye that derived from extracts of krokot which are expected to fulfill the requirement as a natural sensitizer. optical and electrical test are done in order to determine the compliance of the requirement and can be used in dssc system. materials and methods materials krokot, indium transparent oxide (ito), tio2 (degusa), ki, i2, polyethylene glycol, ethanol 96%, polyvinyl alcohol (pva), aquades, graphite pencil 8b and detergent methods sonikator, ultrasonic cleaner, hotplate, glassware, aluminium foil, paper clips, scothlite, screen proyektor (gasket) and cutter. spektrofotometer uv-vis single beam, uv-vis spekular reflektansi uv 1700 pharmaspec, fourier transformation infra-red (ft-ir) shimadzu and i-v meter keithley 2400 source meter procedure krokot extraction (portulaca oleracea l.) ten grams of krokot powder are macerated with 120 ml of 96% ethanol for 24 hours at 40°c, 50°c, 60°c and 26 biology, medicine, & natural product chemistry 4 (2), 2015: 25-29 normally solution temperature. then, it is filtered by using vacuum filtration and before it is used for further processing, it should be analyzed first using uv-vis spectrophotometer in the wavelength range 400-700 nm. preparation of working electrode tio2 powder was weighed as much as 1.5 grams and then inserted into erlenmeyer and added with 3 ml of aquades. then, it is stirred with a magnetite stirring spoon and sonicated with 20 khz frequency for 2 hours. the next solution was then added with polyvinyl alcohol solution which previously has been made from 0.5 grams pva added with 6 ml of aquades by heating at a temperature of 150oc until all of pva are dissolved. the mixing is followed by stirring for 10 minutes until it is formed a homogeneous paste. then, it is performed tio2 paste deposition on surfaces glass of transparent indium oxide (ito) with doctor blanding technique. but before it, ito should be washed with detergent and followed by aquades using ultrasonic cleaner for 10 minutes and rinsed with ethanol. before tio2 paste is dropped on ito glass, the conductive part should be found and then each of it is given a scotchlite restraint and it is made a rectangular pattern by leaving a 1.8 x 1.3 cm room. furthermore, in above of that fields, the tio2 paste is distributed evenly with a glass rod and then dried in the air and the scotchlite is opened, then it is heated at 80 ° c for 1 hour. preparation of elektrode tio2/ dye tio2 film is inserted into the krokot extract, the container is covered with aluminum foil and then it is saved for 1, 8, 18 and 26 hours. the film which has been soaked then removed and rinsed with ethanol to clean the edge of the layer. then,it is dried at room temperature and analyzed by uv-vis reflectance spectrometer and ft-ir. gap energy calculation using the following equation:∞ = ( )( ) (1) ( ∞) ′∞ ′∞ (2) the calculation is performed on each sample by using the kubelka munk method in which the gap energy is obtained from the graph of hv (ev) vs. (f (r'∞) hv) 1/2 (mikrajuddin & khairurijal, 2010). preparation of the counter electrode the graphite of a 8b pencil is spread into the surface of ito on the conductive layer with the shading manner to average carbon layer. then, it is heated at temperature of 300oc for 1 hour. preparation of electrolytes potassium iodide (ki) is weighed as much as 0.815 grams and then dissolved in 10 ml of polyethylene glycol (peg) 400 and stirred until dissolved then added with 0.128 grams of i2 and stirred again until completely mixed. the finished electrolyte solution then stored in the dark bottles and also sealed. fabrication of dye-sensitized solar cell (dssc) dssc fabrication which is used is a sandwich construction with a composition such as: glass-ito working electrode (tio2 layer) that has been coated with dye-screen projector-counter electrode (carbon layer) – ito glass. the use of screen proyector is intended to prevent the short on dssc system. at the ends of the glass that does not stick together is spilled with the electrolyte solution and allowed to seep between the two layers after it is clamped with paperclip (binder clips) on two opposite sides are not coated. characterization of currents and voltage dssc dssc prototype was tested by measuring the i-v characteristic curve using a digital multimeter keithley 2400, in the light of a xenon lamp at an intensity of 1000 w / m2.the result of the i-v characteristic curves test were then analyzed voc, isc, fill factor, and the efficiency of solar cells (lee & misook, 2010) by the equation:= .. (3) = ( ). ( ).. ( ) (4) results and discussion characterization of optical properties krokot dye at diffrerent temperature extract the result of characterization of krokot dye absorption spectrum in different extract temperature was showed at figure 1. figure 1. the graph of krokot dye (a) normally solution temperature extract (b) 40°c temperature extract (c) 50°c temperature extract (d) 60°c temperature extract. reyza anni mufidah, et.al. – krokot (portulaca oleracea. l) as a natural sensitizer for tio2 … 27 figure 1 shows the krokot extract absorbs the blue spectrum (400-450 nm) and red (650-700 nm) with peak absorbance is absorbed at λ = 420.5 with a absorbance value of 2.678 abs, λ = 536.5 with a absorbance value of 0.567 abs, λ = 608 with a absorbance value of 0.473 abs and λ = 665.5 with absorbance values of 1.753 abs so that from the great absorbance at a wavelength of 420.5 nm and 665.5 nm can be known that the more dominant krokot extract contains the pigment chlorophyll. this is because the wavelength is included in the wavelength range of visible light. so that chlorophyll can be used as a dye in dssc because it has characteristic to absorb the visible light that produced by sunlight. temperature extract was influence the absorbance intensity that in hight temperature was decrease the adsorbance intensity. it was indicated that the pigment chlorophyll decresed in dye with hight temperature extract. the pigment chlorophyll was convert into pheophytin pigment in heat treatment (putri et al 2013, gross, 1991). characterization of thin layer electronic properties tio2-dye figure. 2 shows the absorbance graphic of plot toward thin film wavelength of tio2 that has been soaked in dye with variation of temperature extract. figure 2. energy gap (a) thin layer tio2, (b) thin layer tio2-dye solution temperature extract (c) thin layer tio2-dye 40°c temperature extract (d) thin layer tio2-dye 50°c temperature extract and (e) thin layer tio2-dye 60°c temperature extract. absorbance is used to calculate the band gap energy (eg) in the film of tio2 and tio2-dye film with temperature extract variation by tauc plot method. figure. 2 shows that the form of the tio2 band gap is 3.3 ev. this value fullfills the major requirements of semiconductor material that will be used as a dssc where its band gap energy should more than 3.0 ev so that be able to absorb the energy of the photon in the most of the spectrum of sunlight. at the variation temperature extract of film tio2 immersion in the dye for 1 hour there was an eg increasing. this is as expected that the higher temperature extract capable to increase eg on tio2 so that allowing the electrons injection of dye sensitized into the conduction band of nanoparticles tio2 becomes easier because it takes a lower photon for excitation mechanism or in other words the deposited tio2 has been more active in the area of low energy or visible light region. characterization with fourier transform infra-red (ftir) infrared absorption spectrum of a material has a distinctive pattern so that it possible to identify the material and also shows the existence of the major functional groups in the identified structure. the bond can be estimated if the ir spectra of tio2-dye system shows the appearance of a significant new peak or functional groups shift if it is compared with the spectra of dye and spectra of tio2film. krokot extract used in this study contains carboxyl and carbonyl because in the analysis that uses uv-vis is identified to contain chlorophyll so it is possible there is efficient sensitization through the formation of a bond between the dye and tio2. figure 3. infrared spectra (a) thin layer tio2, (b) thin layer tio2-dye solution temperature extract (c) thin layer tio2-dye 40°c temperature extract (d) thin layer tio2-dye 50°c temperature extract and (e) thin layer tio2-dye 60°c temperature extract. 28 biology, medicine, & natural product chemistry 4 (2), 2015: 25-29 the results of ftir analysis for tio2 film, and tio2dye film of krokot extract is shown in the fig. 4 which shows the existence of the carbonyl absorption at wave number 1627.92 cm-1 and there is absorption at 3425.58 cm-1 region which is the absorption area of hydroxyl groups. carbonyl and hydroxyl group that is owned by krokot extract that can bind to the group of ti (iv) on tio2. from ftir analysis is not seen the significant difference from treatment with variation of temperature extract. it is only seen the carbonyl absorption shift of in tio2 film is at a wavelength of 1635.64 cm-1 shifted to a wave number 1627.92 cm-1. this shift occurs when a film of tio2 is coated with krokot extract. current and voltage measurement systems solar cells figure 4. iv meter grafic (a) thin layer tio2-dye solution temperature extract (b) thin layer tio2-dye 40°c temperature extract (c) thin layer tio2-dye 50°c temperature extract and (d) thin layer tio2-dye 60°c temperature extract in table. 1 the efficiency obtained by determining the amount of voltage and the maximum current shown in the fig. 5. from that figure, it can be seen that the current legible shows the dye on tio2 layers undergo the electron transfer from the excited state of the dye to the conduction band of tio2. the highest efficiency found in a layer of tio2-dye with a 40°c temperature extract with an efficiency of 2.63 x 10-3%. table. 1. the parameters of solar cells. ekstrak krokot isc( ) voc (mv) ff (%) ekstrak krokot a 0.092 0.32 24.4565217x10-2 1.44x10-3 ekstrak krokot b 0.165 0.31 25.7576x10-2 2.635x10-3 ekstrak krokot c 0.08 0.135 29.1667x10-2 6.3x10-4 ekstrak krokot d 0.034 0.09 34.3137x10-2 2.1x10-4 in this study, the little dssc efficiency which is produced can be caused by the performance of the natural dye used is still low due to the effect of the extract used still contains a lot of pigment with the long structure r which lead steric hindrance of the pigment to the surface band structure so as to prevent oxidation of tio2 molecules bind with tio2 in effective to cause the transfer of electrons from the conduction band to the dye molecule is reduced. figure 5. iv meter grafic after 2 hours irradiated (a) thin layer tio2dye solution temperature extract (b) thin layer tio2-dye 40°c temperature extract (c) thin layer tio2-dye 50°c temperature extract and (d) thin layer tio2-dye 60°c temperature extract. table. 2. the parameters of solar cells after irradiated. ekstrak krokot isc ( ) voc (mv) ff % ∆ (%) ekstrak krokot a 0.092 0.32 24.4565217 x 10-2 1.44 x 10-3 40.27778 0.09 0.17 28.104575 x 10-2 8.6 x 10-4 ekstrak krokot b 0.165 0.31 25.7576 x 10-2 2.635 x 10-3 86.33776 0.045 0.1 0.4 3.6 x 10-4 ekstrak krokot c 0.08 0.135 29.1667 x 10-2 6.3 x 10-4 -11.1111 0.135 0.145 17.8799 x 10-2 7 x 10-4 ekstrak krokot d 0.034 0.09 34.3137 x 10-2 2.1 x 10-4 16 0.035 0.092 27.3913 x 10-2 1.76 x 10-4 reyza anni mufidah, et.al. – krokot (portulaca oleracea. l) as a natural sensitizer for tio2 … 29 conclusion the result of the uv-vis shows that the absorption of wave-length from chlorophyll pigment. for the uv-vis solid system, there are the highest band gap in 50°c extract temperature. the functional group analysed by ft-ir, there are shiften-carbonil and hydroxyl group after they are sensitized. from the current and voltage test with i-v meter is resulted that on the 50°c extract temperature produces the highest efficiency of reaches which is 2.63 x 10-3 %. acknowledgment department of chemistry, faculty of science and technology uin sunan kalijaga yogyakarta. references yuwono, a. h., dhaneswara, d., ferdiansyah, a., rahman, a. 2011. sel surya tersensitasi zat pewarna berbasis nanopartikel tio2 hasil proses sol gel dan perlakuan pasca hidrotermal. jurnal material dan energi indonesia vol 01 no. 03. 127-140. o'regan, gratzel. 1991. a low cost, high-eficiency solar cell based on dye-sensitized coloidal tio2 film. nature 737-740. gratzel, m. 2003. dye-sensitized solar cell. journal of photochemistry and photobiology 145-153. ludin, n.a., narosikin, a.a.m., mahmoud, a.a., muhammad, a. a., kadhum a.a.h., sopian, k., nor karim, s.a. 2014. review on the development of nature dye photosensitizer for dye sensitized solar cell. renewable and suinable energy 386396. mikrajuddin, a., khairurijal. 2010. karakterisasi nanomaterial teori, penerapan, dan pengolahan data. cv. rezeki putera bandung: bandung. lee, yeji., misook, k. 2010. the optical of nanoporous structured titanium dioxide and the photovoltaic efficiency on dssc. material chemistry and physics. 284-289. content_v4n2_1.pdf (p.1-5) blank_kosong.pdf (p.6) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 443-446 | doi: 10.14421/biomedich.2025.141.443-446 issn 2540-9328 (online) assessment of escherichia coli contamination in drinking water from refill depots lisa savitri1,3,4,*, muhammad akbar nur kholis1, fendy prasetyawan2, yuneka saristiana2, cornelia amanda1, konradus klala mebung1 1department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia 2department of pharmacist professional education, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia 3department of biology, faculty of science and technology, universitas airlangga, surabaya, indonesia 4bioinformatics research center, indonesian institute of bioinformatics, malang, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id abstract refill drinking water depots (damiu) have become a popular source of drinking water in indonesia due to their affordability and accessibility. however, concerns about hygiene and microbial contamination persist, particularly in areas with limited health oversight. this study aimed to evaluate the microbiological quality of refilled drinking water in kediri city by detecting the presence of escherichia coli as an indicator of fecal contamination. a descriptive, cross-sectional design involving 22 depots selected based on hygiene criteria was employed. microbiological analysis used the most probable number (mpn) method, with confirmatory tests including imvic and eosin methylene blue (emb) media. the results showed that 20 of 22 depots met the microbiological safety standard of 0 cfu/100 ml for e. coli as set by the indonesian ministry of health and national standards (sni 3554:2015). two samples (5 and 7) tested positive for e. coli, likely due to environmental contamination, inadequate sanitation, and poor water handling practices. the positive samples demonstrated typical e. coli characteristics in imvic (++-+) and gas formation in bglb media. this study underscores the importance of routine surveillance, public health regulation enforcement, and improved hygiene practices in refilled water depots. ensuring the microbiological safety of refilled drinking water is essential to prevent waterborne diseases such as diarrhea. the findings call for enhanced collaboration between local health offices and depot operators to maintain clean and safe drinking water standards across communities. keywords: refill drinking water depot; escherichia coli; water quality; mpn method; public health. abbreviations: brilliant green lactose broth (bglb), badan pengawas obat dan makanan (national agency of drug and food control) (bpom), colony forming unit (cfu), depot air minum isi ulang (refill drinking water depot), (damiu), eosin methylene blue (emb), inspeksi kesehatan lingkungan (environmental health inspection) (ikl), higiene sanitasi pangan (food sanitation hygiene) (hsp), indole, methyl red, voges-proskauer, citrate (imvic), lactose broth (lb), most probable number (mpn), sertifikat laik higiene dan sanitasi (hygienic and sanitary feasibility certificate) (slhs), standar nasional indonesia (indonesian national standard) (sni) introduction refill drinking water depots (damiu) are businesses engaged in drinking water management. the public often uses refill depots due to their affordability and accessibility. this type of business has grown rapidly across indonesia, including in remote areas with limited access to clean water. according to a study by zikra et al. (2018), the daily water sources used by indonesians include dug wells (24.7%), piped water (14.2%), boreholes (14.0%), and refill drinking water depots (13.8%). drinking water is defined as water, either treated or untreated, that is safe for direct consumption and meets health standards. efforts to improve water quality involve monitoring, protection, and enhancement, conducted by various parties including the government and relevant institutions such as the ministry of industry. surveillance and quality inspections are carried out nationwide, including at refill depots. based on 2023 data from the ministry of industry, approximately 31.87% of indonesia's population relies on refilled water as their primary drinking water source. by the first quarter of 2024, there were 78,378 refill depots in indonesia, of which only 53,261 met food sanitation hygiene (hsp) standards, and only 1,755 had received a hygienic and sanitary feasibility certificate (slhs). water quality monitoring involves surveillance, laboratory testing, risk analysis, and follow-up recommendations. manuscript received: 01 may, 2025. revision accepted: 27 june, 2025. published: 18 july, 2025. https://doi.org/10.14421/biomedich.2025.141.443-446 mailto:lisasavitri@unik-kediri.ac.id 444 biology, medicine, & natural product chemistry 14 (1), 2025: 443-446 most refill depots are managed individually as micro, small, and medium enterprises (msmes). a study conducted by mirasa included 25 regencies and cities out of 38 in east java province, analyzing 1,113 water samples from damiu. the sampling method followed the indonesian national standard sni 7828:2012 (mirasa et al., 2024). in kediri city, 26 refill depots providing purified water at prices aligned with the purchasing power of lower-middle-income groups, making refill depots a preferred choice (efendi, 2019). however, affordable prices must not compromise quality—every business must establish standards that satisfy consumers and enhance competitiveness. with increasing business competition, companies must develop appropriate strategies to adapt to future economic changes and stay competitive in the same sector (eka, 2023). high-quality water can suppress microbial growth and reduce the presence of harmful microorganisms in refill depots (kiman et al., 2019). microbial growth in water is a biological parameter used to assess water cleanliness. one of the key groups evaluated is coliform bacteria, characterized as rodshaped, gram-negative, non-spore-forming, and capable of living in aerobic and facultatively anaerobic conditions. coliforms, including fecal and non-fecal subgroups, naturally inhabit the intestines of humans and animals. these bacteria ferment lactose, producing acid and gas within 48 hours. some strains produce harmful substances such as ethionine, indole, and skatole, which are carcinogenic and toxic when present in significant amounts. the presence of coliform bacteria indicates fecal contamination from humans or animals and the potential presence of other pathogenic bacteria. it also suggests a risk of dangerous enteropathogenic and toxigenic microbes in the water. humans are at risk of diseases like diarrhea from consuming water contaminated with coliforms and e. coli. although e. coli is a normal part of intestinal flora in both humans and animals, it can become pathogenic when present outside the digestive tract (hidayati et al., 2018). e. coli and coliforms are key indicators of water contamination and help assess the potential presence of harmful microbes. outside the gastrointestinal tract, e. coli can cause diseases such as diarrhea (afriyanti, 2019). one of the leading causes of diarrhea is consuming drinking water contaminated with bacteria like e. coli. people consuming water from wells, rivers, piped water, bottled water, or refill depots may be at risk. ready-todrink water is often stored before consumption, and its quality can deteriorate during storage or distribution. ensuring water quality is crucial to public health and preventing microbial proliferation. microorganism presence is a major determinant of water quality, and excessive amounts can lead to diseases such as diarrhea. drinking water must comply with health standards and be free from coliform bacteria. according to indonesia’s ministry of health regulation no. 2 of 2023, the acceptable level of coliform bacteria is 0/100 ml. many refill depots fail to meet this requirement due to poor hygiene practices, improper handling, and insufficient cleaning. bacteria can grow on improperly maintained equipment and containers, resulting in unsafe water. preliminary observations and environmental health inspection data (ikl) from campurejo public health center in kediri revealed 14 refill depots, most of which used groundwater as a source. these depots offer affordable water with delivery services, making them attractive to consumers. however, concerns remain about the hygiene and sanitation conditions of the refill depots. risks arise due to depot locations, packaging practices using reusable plastic gallons, and lack of knowledge among operators about hygiene and sanitation. many depots are located near dusty roads, and employees often do not wear proper uniforms or head coverings. some depots a lack adequate handwashing stations and covered waste bins (hartanto et al., 2022). materials and methods this study employed a descriptive design using a quantitative, cross-sectional, and laboratory-based experimental approach. the research was conducted on 22 refilled drinking water depots in kediri city. initial observations were made to assess hygiene-related factors, followed by microbiological testing using the most probable number (mpn) method, which included presumptive tests with lactose broth (lb) medium and confirmatory tests using eosin methylene blue (emb) agar. the study population comprised all refill drinking water depots (damiu) in kediri city, while the samples were selected based on inclusion criteria such as being operational, willing to participate, and considered to have poor hygiene. depots without supervision from the local health office were excluded. the research involved three variables: the independent variable was the refill water depot, the dependent variable was the presence of e. coli, and the control variables included incubation temperature, media composition, and duration. operational definitions were used to measure the variables, and results were categorized based on standard criteria. materials used in this study included water samples, lb, emb, bglb, mr-vp, and indol media, mostly from himedia. instruments included test tubes (iwaki), micropipettes, incubators, and autoclaves. media preparation involved weighing and dissolving reagents followed by sterilization. microbial inoculation was performed using serial dilution in lb, followed by incubation, confirmed testing in bglb, and completed testing using emb, indol, mr-vp, and citrate media. data were presented in frequency tables to assess the savitri et al. – assessment of escherichia coli contamination in drinking water … 445 presence of e. coli contamination in the refilled drinking water. results and discussion results table 1. results of presumptive test on refilled drinking water using bglb media. sample 5 x 10 ml tubes 1 x 10 ml tube 0.1 x 10 ml tube mpn index /100 ml 1 0 0 0 0/100 ml 2 0 0 0 0/100 ml 3 0 0 0 0/100 ml 4 0 0 0 0/100 ml 5 1 0 0 2.2/100 ml 6 0 0 0 0/100 ml 7 1 0 0 2.2/100 ml 8–20 0 0 0 0/100 ml positive control 5 1 1 240/100 ml negative control 0 0 0 0/100 ml out of the 22 depot water samples tested, only 2 samples did not meet the quality standards, while 20 samples met the requirements. according to bpom and sni 3554, the maximum microbial contamination limit for e. coli is undetectable at 0/250 ml. in this study, e. coli was detected in samples no. 5 and 7, while it was not detected in samples no. 1–4, 6, and 8–20. the study used mineral water and river water as positive and negative controls. table 2. research results on imvic & emb media. sample no. indole vp mr citrate emb remarks 5 + + – + + e. coli (+) 7 + + – + + e. coli (+) control (+) + + – + + e. coli (+) according to table 5.2, both samples showed positive reactions on imvic (++-+) and gas formation in the bglb medium, confirming the presence of e. coli. these results are consistent with the positive control (river water). gede et al. (2023) stated that imvic testing is performed on samples suspected of contamination, confirming gram-negative bacilli by gram staining and metallic sheen colonies on emb agar. discussion the study revealed that 2 out of 22 samples tested positive for e. coli, specifically samples 5 and 7, with values >0 cfu/100 ml. this contamination likely resulted from domestic wastewater near the water sources, including dishwashing, laundry, and bathing. according to the ministry of health regulation no. 2 of 2023, hygiene and sanitation aim to ensure drinking water is free from microbial, physical, chemical, and radioactive contaminants. the test results complied with bpom and sni 3554 standards, with e. coli undetected in 18 samples (0/250 ml), indicating most depot waters in kediri city meet the standards and can reduce diarrhea cases caused by contaminated refilled water. based on the results in tables 1 and 2, although the overall water quality met drinking water standards, samples 5 and 7 exceeded the permissible limits. these refilled waters were prone to contamination due to their roadside locations, which made them susceptible to airborne dust and pollutants. previous research by winarti (2019) in palembang reported a high rate of positive e. coli samples, underscoring the need to tighten public health regulations. mpn tests showed the presence of coliform and e. coli bacteria in the two contaminated samples. this aligns with findings by retno (2019), where only 2 out of 22 (9.1%) depots in the ngasem area, kediri, did not meet ministry of health standards. neli zulfa et al. (2023) found that 25% of water depots contaminated with e. coli, 11 with coliform bacteria, and 3 with ph values outside acceptable limits of 28 supplemental drinking water storage facilities, 15 (53.6%) did not meet quality standards, while only 13 (46.6%) were compliant. yellow tubes indicated positive e. coli results (2.2/250 ml), and blue tubes indicated gas presence in bglb. according to hartanto et al. (2022), 78.57% of depots in the campurejo health center area, kediri, met microbiological water quality standards, while 21.43% did not, as per health minister decree no. 492/menkes/per/iv/2010. improved standards could help reduce diarrhea incidence from depot-sourced drinking water. positive mpn results were indicated by gas formation in durham tubes using lactose broth media. only two out of twenty samples contained e. coli. these findings suggest that although sanitation and hygiene practices in kediri have improved, there is still inadequate supervision by local health authorities. some operators disregard drinking water quality. water quality in depots depends on raw water sources, sanitation of workers, handling of containers by consumers, and storage conditions. confirmed tests were conducted by transferring gaspositive bglb tubes into new bglb media and incubating at 36°c for 24–48 hours. turbidity and gas produstion indicated a positive result. in table 5.2, samples 5 and 7 were confirmed positive at the 5 x 10 ml dilution level. gas formation resulted from lactose fermentation by coliform bacteria. brilliant green lactose broth (bglb) supports the growth of gram-negative, lactose-fermenting coliforms, while inhibiting gram-positive bacteria. coliform bacteria produce energy through lactose fermentation, 446 biology, medicine, & natural product chemistry 14 (1), 2025: 443-446 generating pyruvate, acetate, and co₂, which forms visible gas bubbles in the durham tubes (putri et al., 2018). higher mpn values indicate greater microbial contamination, while lower values reflect better water quality. conclusions this study evaluated the microbiological quality of refilled drinking water from 22 depots (damiu) in kediri city, indonesia, focusing on the presence of escherichia coli as an indicator of fecal contamination. the findings revealed that 90.9% (20 out of 22) of the depots met the microbiological standards set by bpom and sni 3554, while 9.1% (samples 5 and 7) exceeded the acceptable limit of 0 cfu/100 ml for e. coli. confirmatory testing through imvic and emb media supported the presence of e. coli in these two samples. the contamination in these cases may be attributed to inadequate sanitation, poor hygiene practices, improper handling of containers, and depot locations near pollution sources. despite improvements in hygiene across the sector, the study highlights the need for stricter supervision, increased awareness among depot operators, and enforcement of health regulations to ensure consistent water quality. as refill water remains a primary source of drinking water for many indonesians, ensuring its safety is essential to protect public health and prevent waterborne diseases such as diarrhea. future initiatives should focus on strengthening health inspections, enhancing operator training, promoting better sanitation infrastructure, and increasing public education on safe water handling practices. acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors wrote the manuscript and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there is no funding. references afriyanti, l.n. (2019). keberadaan escherichia coli pada makanan di kantin sekolah dasar. higeia journal of public health, 3(3), 417–429. efendi, r. (2019). pengaruh harga dan preferensi terhadap pilihan konsumen di depot air minum isi ulang di kediri. jurnal ekonomi dan bisnis, 17(3), 182-190. eka, i. (2023). strategi bisnis untuk depot air minum isi ulang dalam persaingan pasar. jurnal manajemen bisnis, 8(1), 45– 56. gede, n., putra, k., & widodo, s. (2023). metode identifikasi escherichia coli dengan imvic dan emb pada air minum isi ulang di wilayah bali. jurnal kesehatan masyarakat, 8(2), 117–126. available at: https://doi.org/10.25232/jkm.v8i2.317. hartanto, a., sasmito, j., & katmini, mumalikah. (2022). kualitas mikrobiologi air minum hasil depot air minum isi ulang di area ppl sanitarian uptd puskesmas campurejo kediri. jurnal pendidikan dan teknologi indonesia, 2(6), 255–261. available at: https://doi.org/10.52436/1.jpti.154. hidayati, w., temaja, i.g.r.m., & fatmawati, n.n.d. (2018). karakteristik fenotip isolat klinik escherichia coli o157:h7 pada media sorbitol mac conkey agar (smac). journal of agricultural science and biotechnology, 7(1), 35– 40. available at: https://ojs.unud.ac.id/index.php/jasb/article/view/41191. kiman, n., riono, s.b., saifullah, m., wahana, a.n.p.d.w., & fitralisma, g. (2019). pengaruh kualitas layanan dan harga terhadap kepuasan konsumen pada depot air minum isi ulang ud enzes pengabean. journal of management (jecma), 1(1), 92–101. mirasa, y.a., nurhidayati, s., wicaksono, r.i., winarko, w., juwono, k.f., zakaria, z.a., diyanah, k.c., pawitra, a.s., & sahri, m. (2024). refill drinking water depot risk assessment for chemical hazard contaminant in 25 cities of east java province, indonesia. jurnal kesehatan lingkungan, 16(2), 166–172. available at: https://doi.org/10.20473/jkl.v16i2.2024.166-172. neli zulfa, n., & mulyawati, i. (2023). higiene sanitasi dan uji pemeriksaan mikrobiologi depot air minum isi ulang. higeia (journal of public health research and development), 7(1), 44–54. available at: https://doi.org/10.15294/higeia.v7i1.61441. putri, a.m., & kurnia, p. (2018). identifikasi keberadaan bakteri coliform dan total mikroba dalam es dung-dung di sekitar kampus universitas muhammadiyah surakarta. media gizi indonesia, 13(1), 41–48. available at: https://doi.org/10.20473/mgi.v13i1.41-48. retno, s. (2019). kualitas air depot air minum isi ulang di wilayah ngasem, kediri. jurnal kesehatan lingkungan, 11(4), 286–292. available at: https://doi.org/10.20473/jkl.v11i4.2019.286-292. winarti, e. (2019). kontaminasi bakteri escherichia coli pada depot air minum isi ulang di palembang. jurnal kesehatan dan lingkungan, 12(1), 51–60. zikra, w., amir, a., & putra, a.e. (2018). identifikasi bakteri escherichia coli (e.coli) pada air minum di rumah makan dan cafe di kelurahan jati serta jati baru kota padang. jurnal kesehatan danalas, 7(2), 212. available at: https://doi.org/10.25077/jka.v7i2.804. https://doi.org/10.52436/1.jpti.154 https://doi.org/10.20473/jkl.v16i2.2024.166-172 https://doi.org/10.15294/higeia.v7i1.61441 https://doi.org/10.20473/mgi.v13i1.41-48 https://doi.org/10.20473/jkl.v11i4.2019.286-292 https://doi.org/10.25077/jka.v7i2.804 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 731-738 | doi: 10.14421/biomedich.2025.142.731-738 issn 2540-9328 (online) the antivirulence mechanisms of phytate against pathogenic bacteria in skin infections nabilatul zhofiroh1, rian ka praja2*, elsa trinovita3, ysrafil3, ranintha br surbakti4 1undergraduate program of medicine; 2department of microbiology, faculty of medicine; 3department of pharmacotherapy, faculty of medicine; 4department of clinical medicine, faculty of medicine, palangka raya university. jl. yos sudarso, palangka raya 73111, indonesia. corresponding author* riankapraja@med.upr.ac.id manuscript received: 04 june, 2025. revision accepted: 18 september, 2025. published: 01 october, 2025. abstract skin infections caused by the bacteria staphylococcus aureus, streptococcus pyogenes, and propionibacterium acnes are often a common health problem. one treatment is antibiotics, but the cases of antibiotic resistance are increasing. thus, new treatment alternatives are needed. this study aimed to analyze the molecular mechanism of phytate antivirulence against pathogenic bacteria of skin infection. this study used a bioinformatics approach involving analysis of phytate interactions with bacterial virulent proteins via stitch, functional classification of proteins with vicmpred, and prediction of virulence properties using virulentpred. b-cell and mhc epitopes were analyzed using iedb, while protein subcellular location was determined through psortb. the results showed that phytate interacted specifically with virulent proteins in all three bacteria, most of which functioned in cellular and metabolic processes. these virulent proteins also have immunologically relevant epitopes. subcellular location analysis showed that phytate protein targets were dispersed in the cytoplasmic membrane and cytoplasm. these findings indicated that phytate has a significant antivirulence mechanism by targeting virulent proteins of skin pathogenic bacteria, thus potentially becoming a therapeutic agent to treat skin infections while reducing antibiotic resistance. keywords: phytate; antivirulence; staphylococcus aureus; streptococcus pyogenes; propionibacterium acnes. introduction the skin can become infected because microorganisms can penetrate the damaged skin barrier. skin infections can be caused by viruses, bacteria, fungi, or parasites (lidjaja, 2022). based on data from the demographic health survey in indonesia in 2016, the prevalence of skin diseases was 2.93% to 27.5% (edison et al., 2023). according to the world health organization (who), the prevalence of infectious skin diseases in 2020 was reported to be around 300 million cases per year. the prevalence of skin diseases in indonesia is 4.60%12.95%, ranking third out of the top 10 diseases (sri rahayu et al., 2023). the bacteria staphylococcus aureus, streptococcus pyogenes, and propionibacterium acnes commonly cause skin infections. staphylococcus aureus, streptococcus pyogenes, and propionibacterium acnes are gram-positive bacteria. staphylococcus aureus is a spherical bacterium with a diameter of 0.7-1.2 μm, forms irregular groups resembling grapes, does not form spores, is facultatively anaerobic, and does not move (devi et al., 2022). then, streptococcus pyogenes bacteria are cocci, arranged in chains, and show catalase and oxidase activity (savitri et al., 2019). propionibacterium acnes bacteria, on the other hand, belong to the corynebacteria family but have no toxicity (zahrah et al., 2018). antibiotic resistance is the ability of microorganisms to inhibit the action of antimicrobial agents, and this phenomenon occurs when antibiotics lose their efficiency in inhibiting bacterial growth, which is one of the most important public health problems to be solved (putri et al., 2023). according to the antimicrobial resistance control committee, bacterial resistance in indonesia continued to increase from 2013 to 2019 (marsudi et al., 2021) and according to suhartini in 2024, the prevalence of antibiotic use in indonesia is in the high category, which is 40%-60% (suhartini & rahmi makmur, 2024). uncontrolled use of antibiotics causes resistance to increase. the wrong antibiotics can cause resistant bacteria (lubis et al., 2019). phytate (chemically known as myoinositol (1,2,3,4,5,6) hexakisphosphate). phytate in plants is a source of energy and antioxidant capacity (as a phosphate group donor) but has a major role as a candidate antimicrobial natural material (specifically cu2+ and zn2+ cation depots) due to its negative charge at physiological ph (pires et al., 2023). in a https://doi.org/10.14421/biomedich.2025.142.731-738 mailto:riankapraja@med.upr.ac.id 732 biology, medicine, & natural product chemistry 14 (2), 2025: 731-738 bioinformatics study conducted by hashimoto et al (2022), it was shown that consuming phytate-enriched foods such as rice can induce an increase in epithelial antimicrobial defence mechanisms in the gut, protecting against infection by pathogenic bacteria such as e. coli (hashimoto-hill et al., 2022). then in the study of sorour et al (2022) they conducted research on the antibacterial properties of pure phytate compound extracts and showed that gram-positive bacteria were more sensitive to phytate compounds than gramnegative bacteria (sorour et al., 2022) materials and methods type of research this study used a computational approach with bioinformatics methods that utilise data analysis as a method to explore and understand more about virulence factors in three types of pathogenic bacteria, namely staphylococcus aureus mu50, streptococcus pyogenes m1 gas, and propionibacterium acnes kpa171202. population and sample this study’s samples were phytate and fasta compounds from the protein sequences of staphylococcus aureus mu50, streptococcus pyogenes m1 gas, and propionibacterium acnes kpa171202. data collection stitch version 5.0 was used as an interaction analysis of staphylococcus aureus mu50, streptococcus pyogenes m1 gas, and propionibacterium acnes kpa171202 targeted to phytate compounds, then fasta was downloaded from the national center for biotechnology information (ncbi) database. fasta files were downloaded and renamed according to the protein name of the bacteria targeted by phytate compounds. the fasta was used in functional class analysis, virulence trait analysis, epitope analysis, and subcellular analysis of proteins from staphylococcus aureus mu50, streptococcus pyogenes m1 gas, and propionibacterium acnes kpa171202 that interact with phytate compounds using vicmpred, virulentpred, bepipred version 2.0, mhc-i binding prediction, mhcii binding prediction, and psortb version 3.0. data analysis and processing interaction analysis of compounds and bacterial proteins web stitch version 5.0 accessible through http://stitch.embl.de was analyzed in relation to the interaction of compounds and bacteria staphylococcus aureus mu50, streptococcus pyogenes m1 gas, and propionibacterium acnes kpa171202 to see the interaction between protein sequences that interact with phytate compounds. the results of the analysis are in the form of a three-dimensional diagram. then, the fasta download of protein sequences through ncbi (national center for biotechnology information) data was carried out to be used in the next stage of analysis. functional class analysis functional class analysis of the protein sequences of staphylococcus aureus mu50, streptococcus pyogenes m1 gas, and propionibacterium acnes kpa171202 bacteria was analyzed on the website http://crdd.osdd.net/raghava/vicmpred/ virulence trait analysis the virulentpred 2.0 website https://bioinfo.icgeb.res.in/virulent2/ was employed to analyze the virulence properties of protein sequences targeted by phytate compounds. b-cell epitope analysis the iedb analysis resource website accessible at http://tools.iedb.org/bcell/ was used for b cell epitope analysis. mhc i epitope analysis mhc i epitope analysis of protein sequences targeted by phytate using mhc i binding predictions accessible on the website http://tools.iedb.org/mhci/. mhc ii epitope analysis mhc ii epitope analysis of protein sequences targeted by phytate compounds using mhc i binding predictions accessible on the website http://tools.iedb.org/mhcii/. subcellular location analysis the psortb v3.0.3 website which can be accessed through https://www.psort.org/psortb/ was used to analyze the subcellular location of protein sequences. results and discussion interaction analysis of compounds and bacterial proteins analysis of protein interactions using stitch version 5.0 shows several proteins from the interaction between phytate compounds and staphylococcus aureus mu50, streptococcus pyogenes m1 gas, and propionibacterium acnes kpa171202. the analysis diagram is shown in figure 1. http://stitch.embl.de/ http://crdd.osdd.net/raghava/vicmpred/ https://bioinfo.icgeb.res.in/virulent2/ http://tools.iedb.org/bcell/ http://tools.iedb.org/mhci/ http://tools.iedb.org/mhcii/ https://www.psort.org/psortb/ zhofiroh et al. – the antivirulence mechanisms of phytate … 733 a b c figure 1. phytate interaction diagram with (a) staphylococcus aureus mu50 (b) streptococcus pyogenes m1 gas (c) propionibacterium acnes kpa171202. functional class analysis and virulence traits in the next step, the protein was analyzed using vicmpred and virulentpred v2.0, and the functional class and virulence properties of each protein were obtained, as shown in table 1. table 1. analysis of functional classes and virulence properties of staphylococcus aureus mu50, streptococcus pyogenes m1 gas and propionibacterium acnes kpa171202 proteins interacting with phytate. organism identification code proteins that react with phytate vicmpred functional class virulent pred staphylococcus aureus mu50 gpma phosphoglyceromutase; catalyzes the interconversion of 2-phosphoglycerate and 3phosphoglycerate (by similarity) cellular process non-virulent sav2490 mutator protein mutt metabolism molecule non-virulent sav1499 adp-ribose pyrophosphatase metabolism molecule non-virulent sav1338 hypothetical protein cellular process virulent sav1794 hypothetical protein information and storage non-virulent sav0240 flavohemoprotein cellular process non-virulent sav1118 hypothetical protein information and storage virulent gpma phosphoglyceromutase; catalyzes the interconversion of 2-phosphoglycerate and 3phosphoglycerate (by similarity) cellular process non-virulent streptococcus pyogenes m1 gas spy_2186 hypothetical protein cellular process virulent spy_0477 hypothetical protein cellular process non-virulent spy_2170 hypothetical protein cellular process virulent spy_0444 hypothetical protein metabolism molecule non-virulent mutt protein mutator information and storage non-virulent mutx 7,8-dihydro-8-oxoguanine-triphosphatase cellular process non-virulent gpma phosphoglyceromutase; catalyzes the interconversion of 2-phosphoglycerate and 3phosphoglycerate (by similarity) cellular process non-virulent propionibacterium acnes kpa171202 ppa1717 hypothetical protein cellular process non-virulent ppa0225 ntp pyrophosphohydrolase metabolism molecule virulent ppa1781 7,8-dihydro-8-oxoguanine-triphosphatase cellular process non-virulent ppa0342 hypothetical protein metabolism molecule non-virulent ppa2032 mutt/nudix family proteins cellular process virulent b cell epitope analysis the analysis of b cell epitopes is a continuation of the study of functional classes and virulence properties of staphylococcus aureus mu50, streptococcus pyogenes m1 gas, and propionibacterium acnes kpa171202 proteins that interact with phytate compounds, in this 734 biology, medicine, & natural product chemistry 14 (2), 2025: 731-738 step using bepipred with virulent proteins from each interaction. analysis of each protein revealed the presence of amino acid sequences that can bind to b cells. the study results can be seen from the emergence of graphs with different colors: yellow and green. the yellow graph indicates an interaction between the protein compound and the b cell epitope. the result of cell epitops can be seen in figure 2. a b c d e f figure 2. result of b cell epitopes on (a) sav1338, (b) sav1118, (c) spy_2186, (d) spy_2170, (e) ppa0225, (f) ppa2032 information : a. yellow peaks indicate sequences that have potential epitopes. b. green peaks indicate sequences that do not have potential epitopes. mhc i and mhc ii analysis the results of mhc i epitope prediction analysis using the hla-a*11:01 allele with a peptide strand length of 9 amino acids. moreover, mhc ii epitope analysis, peptides with a length of 15 amino acids were identified using the hla-drb1*04:01 allele. the highest score indicates the potential for virulent bacterial proteins to bind to t cells. the resulting binding score also shows strong affinity, so there is a high potential for interaction between proteins and mhc i that allows recognition by t cells. all results are presented in tabular form, and the top five of all peptides with the highest score were taken. the results of the analysis are shown in table 2 and table 3. table 2. mhc i analysis results. protein allele start end length peptides score percentile rank sav1118 hlaa*11:01 141 149 9 sinpepsfk 0.96 0.01 hlaa*11:01 178 186 9 qvysdqqsk 0.88 0.03 hlaa*11:01 100 108 9 nsyyivstk 0.76 0.09 hlaa*11:01 83 91 9 rvypfrdgy 0.67 0.16 hlaa*11:01 234 242 9 vtnemrklk 0.59 0.21 sav1338 hlaa*11:01 16 24 9 iiapitefk 0.91 0.02 hlaa*11:01 84 92 9 vtfneygtk 0.73 0.12 hlaa*11:01 228 236 9 kqhqlstlk 0.55 0.25 hlaa*11:01 239 247 9 kqnsetark 0.48 0.31 hlaa*11:01 300 308 9 lmnsighrk 0.44 0.36 spy_2170 hlaa*11:01 53 131 9 ilndesiak 0.53 0.26 hlaa*11:01 59 67 9 llftdpvyy 0.21 0.85 hlaa*11:01 9 17 9 qakplgeek 0.11 1.3 hlaa*11:01 50 58 9 kvfivplrq 0.09 1.4 hlaa*11:01 40 118 9 wgmtaqftk 0.07 1.7 spy_2186 hlaa*11:01 5 13 9 lvspledpk 0.30 0.59 hlaa*11:01 27 35 9 gfqsinwik 0.05 2.0 hlaa*11:01 22 30 9 ggtslvgek 0.02 2.9 hlaa*11:01 30 38 9 kthetvlre 0.01 3.4 zhofiroh et al. – the antivirulence mechanisms of phytate … 735 hlaa*11:01 4 12 9 rngknfltr 0.01 3.4 ppa0225 hlaa*11:01 35 43 9 hvldalldr 0.35 0.5 hlaa*11:01 10 18 9 ttrhpsgyr 0.24 0.75 hlaa*11:01 9 17 9 attrhpsgy 0.18 0.94 hlaa*11:01 5 13 9 saliaslgr 0.17 0.96 hlaa*11:01 45 53 9 ltrrplslr 0.11 1.3 ppa2032 hlaa*11:01 35 43 9 rtclnvrkk 0.44 0.36 hlaa*11:01 5 13 9 lvldpddlk 0.34 0.52 hlaa*11:01 9 17 9 vtwrdgsgr 0.20 0.88 hlaa*11:01 4 12 9 svqcvvtwr 0.18 0.94 hlaa*11:01 76 84 9 rvipalqqq 0.13 1.2 table 3. mhc ii analysis result. protein allele start end length peptide score percentile rank sav1118 hla-drb1*04:01 202 216 15 iepyqlnsnstseeh 0.90 0.20 hla-drb1*04:01 173 187 15 gdiyaqvysdqqskk 0.89 0.20 hla-drb1*04:01 201 215 15 diepyqlnsnstsee 0.87 0.28 hla-drb1*04:01 172 186 15 ygdiyaqvysdqqsk 0.82 0.53 hla-drb1*04:01 99 113 15 knsyyivstkreeiv 0.81 0.60 sav1338 hla-drb1*04:01 234 248 15 tlkyskqnsetarkh 0.90 0.20 hla-drb1*04:01 233 247 15 stlkyskqnsetark 0.87 0.28 hla-drb1*04:01 152 166 15 kgrvryeqnnkeydv 0.85 0.33 hla-drb1*04:01 151 165 15 vkgrvryeqnnkeyd 0.82 0.49 hla-drb1*04:01 235 249 15 lkyskqnsetarkhs 0.73 1.20 spy_2170 hla-drb1*04:01 134 148 15 pvyyrlevtpiettd 0.93 0.13 hla-drb1*04:01 133 147 15 dpvyyrlevtpiett 0.87 0.26 hla-drb1*04:01 132 146 15 tdpvyyrlevtpiet 0.76 0.96 hla-drb1*04:01 135 149 15 vyyrlevtpiettdf 0.71 1.30 hla-drb1*04:01 106 120 15 vddwksiqpneevdk 0.71 1.40 spy_2186 hla-drb1*04:01 69 83 15 niefhylvspledpk 0.87 0.28 hla-drb1*04:01 68 82 15 hniefhylvspledp 0.79 0.79 hla-drb1*04:01 70 84 15 iefhylvspledpkl 0.67 1.60 hla-drb1*04:01 81 95 15 dpklemienasdrfv 0.64 1.80 hla-drb1*04:01 80 94 15 edpklemienasdrf 0.61 1.90 ppa0225 hla-drb1*04:01 136 150 15 rvrladlanpaarat 0.65 1.70 hla-drb1*04:01 135 149 15 qrvrladlanpaara 0.62 1.90 hla-drb1*04:01 134 148 15 vqrvrladlanpaar 0.43 3.90 hla-drb1*04:01 28 42 15 rssavlaliseegnd 0.35 5.30 hla-drb1*04:01 29 43 15 ssavlaliseegndi 0.32 5.80 ppa2032 hla-drb1*04:01 79 93 15 pddlkhlgtfdapaa 0.48 3.20 hla-drb1*04:01 78 92 15 dpddlkhlgtfdapa 0.38 4.60 hla-drb1*04:01 109 123 15 wreiwpepvpdseiv 0.38 4.70 hla-drb1*04:01 108 122 15 nwreiwpepvpdsei 0.33 5.70 hla-drb1*04:01 52 66 15 ggkielgetpleaai 0.32 5.80 next, the subcellular location of each virulent protein was analyzed using psortb. the results of the study are shown in table 4. table 4. subcellular location analysis results. organism identification code protein name subcellular location staphylococcus aureus mu50 sav1338 hypothetical protein unknown sav1118 hypothetical protein unknown streptococcus pyogenes m1 gas spy_2186 hypothetical protein cytoplasmic spy_2170 hypothetical protein cytoplasmic propionibacterium acnes kpa171202 ppa0225 ntp pyrophosphohydrolase cytoplasmic membrane ppa2032 mutt/nudix family protein cytoplasmic discussion in the staphylococcus aureus mu50, the identified proteins include gpma, sav2490, sav1499, sav1338, sav1794, sav0240, and sav1118. meanwhile, in streptococcus pyogenes m1 gas, the proteins involved were spy_0477, spy_2186, gpma, mutx, mutt, spy_0444, and spy_2170. whereas in 736 biology, medicine, & natural product chemistry 14 (2), 2025: 731-738 propionibacterium acnes kpa171202, the proteins involved include ppa0342, ppa2032, ppa1389, gpma, ppa1717, ppa0225, and ppa1781. in the staphylococcus aureus mu50 strain, there is a close relationship between sav1794, sav0240, and sav1118 proteins. according to the theory proposed by pevsner, interactions between proteins usually occur between proteins with similar functions or structures. this strengthens the possibility that this group of proteins forms a specific functional network that can be disrupted by interactions with phytate compounds (abdullah et al., 2022). based on the results, most of the proteins from the three bacteria had major functions in cellular processes and the metabolism of molecules. proteins such as gpma found in all three bacterial strains were known to play an important role in the glycolytic pathway. in addition, some proteins were also categorized in the information and storage function, which is related to genetic regulation and biological information storage mechanisms. phytate can interfere with bacterial metabolic pathways by binding and removing essential metals that support bacterial survival and proliferation. in bioinformatics studies of skin pathogens, phytate was found to interact with several specific proteins in staphylococcus aureus, streptococcus pyogenes, and propionibacterium acnes. phytate not only functions as an antioxidant and anti-inflammatory agent but also as an antivirulence agent that intervenes in the metabolic pathways of bacteria. by binding to key proteins in energy metabolism and virulence, phytate can weaken the ability of bacteria to survive and cause infection (pires et al., 2023a). the next analysis looked at the virulent properties of each protein and only had two proteins that had virulent properties, namely: sav 1338, sav1118 (staphylococcus aureus mu50), spy_2186, spy_2170 (streptococcus pyogenes), and ppa0225, ppa2032 (propionibacterium acnes kpa171202). this is in line with research conducted by yamazaki et al, where proteins in staphylococcus aureus play a role in pathogenesis by regulating immune evasion mechanisms and facilitating biofilm formation, which facilitates infection and bacterial immunity to antibiotic therapy (yamazaki et al., 2024). phytate has the potential to interact with these virulent proteins, thereby inhibiting metabolic processes that are essential for bacterial survival. this indicates that phytate has the potential to be an antivirulence agent that can reduce the virulence of bacteria such as streptococcus pyogenes.(pires et al., 2023b) b cell epitopes are segments of antigens recognized by antibodies in the immune system, and epitope analysis can identify specific regions on proteins that can trigger humoral immune responses (sun et al., 2024). the analysis showed various peptide strands that can trigger immune responses, with a length of up to 66 amino acids in some proteins, such as sav1338 and sav1118. it is in line with previous studies where epitopes on staphylococcus aureus, streptococcus pyogenes and propionibacterium acnes can be effective targets in the development of antibody-based vaccines (ozberk et al., 2018). major histocompatibility complex (mhc)-related analysis consisting of two classes: mhc i binding predictions and mhc ii binding predictions of virulent proteins. peptides, or epitopes, are expressed on the surface of nucleated cells by major histocompatibility complex (mhc) molecules in t cells. a key requirement for t cell activation is molecular recognition between the t cell receptor (tcr) expressed on the t cell surface and the peptide-mhc complex (pmhc) described on the surface of other cells. without t-cell activation, an immune response cannot be mounted and initiated. a mechanism known as central tolerance is responsible for this process (schaap-johansen et al., 2021). the results of the mhc epitope analysis presented in table 2 and table 3 show that the six proteins derived from the three pathogenic bacteria have strong binding affinities to mhc i and mhc ii. mhc i presents itself as an antigen and induces cd8+ t cells, while mhc ii presents itself as an antigen and induces cd4+ t cells. after being induced, the cytotoxic antigen-specific immune system of t cells was mediated by the activity of cd8+ t cells, and b cells became b cell memory due to the activity of cd4+ t cells. the combination of the two mechanisms formed antibodies that damage spike proteins so that an adaptive immune system is formed to fight bacteria (suzana et al., 2022). as described in previous studies, the activation of cd8+ and cd4+ t cells has an important role in fighting bacterial infections, where cd4+ t cells facilitate the production of antibodies by b cells (shepherd & mclaren, 2020). the interaction between mhc and antigen epitopes on the surface of t cells is essential to enhance the effectiveness of immune responses, which suggests that phytate through its ability to intervene in bacterial metabolic pathways and enhance antigen binding, may play a role in enhancing immune responses to bacterial infections. subsequently, the subcellular location of the proteins with virulence properties was analyzed using psortb. of most of the six proteins, three proteins were located in the cytoplasmic membrane (spy_2186, spy_2170, and ppa2032), one was located in the cytoplasmic membrane (ppa0225), and there were two proteins from staphylococcus aureus mu50 that were targeted by phytate compounds, the exact location of the subcellular location was not found. zhofiroh et al. – the antivirulence mechanisms of phytate … 737 conclusion this bioinformatics study on the antivirulence mechanism of phytate against staphylococcus aureus strain mu50, streptococcus pyogenes m1 gas, and propionibacterium acnes strain kpa171202 reveals that phytate interacts molecularly with virulent proteins in these pathogenic bacteria. functional analysis showed that these proteins are primarily involved in cellular processes and metabolic functions, with additional roles in information and storage for staphylococcus aureus and streptococcus pyogenes. epitopic analysis revealed the presence of b-cell epitopes and peptide strands with t-cell affinity. at the same time, subcellular localization placed virulent proteins in the cytoplasm for streptococcus pyogenes and in the cytoplasmic membrane and cytoplasm for propionibacterium acnes. however, localization for staphylococcus aureus remains unconfirmed. further experimental validation in the laboratory to confirm these findings is recommended, alongside additional research into the virulence factors of phytate and its effects on other pathogenic bacteria and compounds in rice. acknowledgements: we would like to express our sincere gratitude to all individuals and institutions that contributed to the success of this research. special thanks go to the faculty of medicine palangka raya university wet biomedical laboratory for providing the facilities and resources necessary to conduct this study. we also thank colleagues and team members for their invaluable support and collaboration throughout the research process. authors’ contributions: nabilatul zhofiroh & rian ka praja 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(2018). aktivitas antibakteri dan perubahan morfologi dari propionibacterium acnes setelah pemberian ekstrak curcuma xanthorrhiza. jurnal biosains pascasarjana, 20(3), 160–169. biology, medicine, & natural product chemistry issn: 2089-6514 volume 5, number 1, 2016 | pages: 1-8 | doi: 10.14421/biomedich.2016.51.1-8 new record marsdenia tenacissima (asclepiadoideae, apocynaceae) in gunung ijo baturagung yogyakarta widodo1 and muhammad ja’far luthfi2 1biology education program, 2biology department, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency1: wwidodo594@gmail.com abstract marsdenia tenacissima population were found among wild bushes at s 07 o 47’ 03.4”; e 110o 30’ 48.0” about 415 meter above sea level in gunung ijo baturagung yogyakarta. identification was based on literature and herbarium specimen. the research was conduct using exploration methods, morphoanatomical observation, and specimen collection. marsdenia tenacissima in jawa was not reported in flora of java. marsdenia tenacissima habitus was liana. the specific character for its identification was pollinia’s structure. this paper presented other important character namely leaf, stem, flower, pollinia, and fruit of marsdenia tenacissima in gunung ijo baturagung yogyakarta. accurate description and examination of any plant species were needed for its conservation and awareness of public to local biodiversity. keywords: marsdenia tenacissima, asclepiadoideae, gunung ijo, baturagung introduction exploration, observation and assessment of wild plants on gunung ijo baturagung yogyakarta had found lianas (widodo, 415 m, december 31, 2012) among wild bushes (s 07 ° 47 '03.4 "; e 110o 30 '48.0 ". after collection and re-observation on flower, analysis of fruit development and the thoroughly identification, it is conclude that the plant species are marsdenia tenacissima. marsdenia belongs to tribe marsdenieae of the subfamily asclepiadoideae (takhtajan, 2009). according to backer and bakhuizen (1965) there are three marsdenia in java, namely marsdenia tinctoria or pergularia parviflora, marsdenia crocea, and marsdenia stenocentra or marsdenia villosa. hooker (1885) states that the distribution of marsdenia tenacissima include western himalayas, north oudh, bengal, mountains of rajmahal, chittagong, ceylon, also found in the east. it is estimated that this species in java was identified as pergularia crocea, zipp. from the description and bakhuizen backer (1965) above, the existence of marsdenia tenacissima in java was unexplained but hooker (1885: 36) insist that this species is very similar to pergularia crocea, zipp. the plant list (2010) states that marsdenia genus comprises about 447 species. the members of this taxon in this genus often unclear and led to misidentification. observation on the species characters are necessary to provide valid identification of marsdenia species. borlage (1895) listed the marsdenia species in indonesia, namely: (1) marsdenia tinctoria r. br or m. parviflora decaisne or pergularia parviflora bl. or pergularia tinctoria spreng. the plants are distributed all over java, kalimantan, sulawesi, and sumatra, (2) marsdenia tenacissima wight. et arn. or asclepias tenacissima roxb. or gymnema tenacissima spreng. its distribution includes timor and sulawesi, (3) marsdenia villosa bl. or pergularia villosa bl, which has distribution in java, (4) marsdenia teysmanni or tetragonocarpus teysmanni hassk. the plants are distributed in bali and java, (4) marsdenia celebica or chlorochlamys celebica miq, which has distribution in sulawesi, 5) marsdenia crocea hook f. or pergularia crocea zipp. or pergularia tomentosa span, which found in java and timor. backer and bakhuizen (1965), borlage (1895), hooker (1885) conclude that marsdenia species in java is marsdenia tinctoria or pergularia parviflora, marsdenia crocea or pergularia crocea or pergularia tomentosa, marsdenia stenocentra or marsdenia villosa, marsdenia teysmanni or tetragonocarpus teysmanni, and also marsdenia tenacissima or marsdenia celebica. few information about marsdenia tenacissima are available in the literature and internet. this paper describes the characteristics, specimen photos, and clarify the identification of specimens marsdenia tenacissima found in mount ijo baturagung mountains through literature. the re-discovery of the marsdenia tenacissima in batur agung yogyakarta needs to be disseminated to confirm biodiversity richness in java. many wild plants is no longer recognized both the name or its specimen despite information documented in the books of flora and herbarium hundreds of years ago by european explorers. publication of plants species in nature are needed to check and re-check the rediscovery of flora, and to complete the data of world flora. the study would support as raw material for the basis of any applied fields. the research objective is to present a description of the morphological characteristics of leaves, stems, flowers, pollinia, and fruit of marsdenia tenacissima of 2 biology, medicine, & natural product chemistry 5 (1), 2016: 1-8 candi ijo mountains baturagung as for identification verification. materials and methods equipments and materials equipment for observation and collection comprises: digital camera sony nex f3, digital cameras sony cyber-shot dsc-w180 digital camera canon dslr, rulers, micrometers, calipers, plastic container, scissors, cutter, label paper , gps (global positioning system), dried herbarium collection equipment, bottles, stereo microscope nikon smz 1500 equipped with a camera, nikon light microscope equipped with nikon eclipse 50 dsf1. materials for observation and collection comprises: aquadest, alcohol 70%, faa solution (formalin acetic alchohol). procedures 1. observe and take macro and microphotograph of specimen under natural conditions at the site, herbarium, and flower detail. 2. preparation of dried herbarium 3. early identification of specimens for members asclepiadaceae based on the book flora of java vol. 2 (backer and bakhuizen, 1965). 4. identify asclepiadaceae specimen based on existing literature, including checking and matching with herbarium type. results and discussion on december 16 exploration, author found gummy vine with fine hair on dorsal surface of the leaves. the authors predict that this plant belong to family apocynaceae and sub family asclepiadoideae. on further observation at the end of december, aditional data on bud and flower were obtained. the location of the plant was on the shrubs at the site of s 07 ° 47 '03.4 "; e 110o 30 '48.0 "at an altitude of 415 m dpl (widodo, 415 m dpl, december 31th 2012). identification using flora of java, vol. 2 (backer and bakhuizen, 1965) concluded that this plant is marsdenia. subequent observations on january 4, january 19, and january 29, 2013 obtained data about the twigs and flowers structure (figure 1 a, b, c, d, e; figure 2a, b, c, d), specimen collection process is carried out in the laboratory for continued observation. on march 21, 2013 author found the young fruit (figure 3a). figure 1. marsdenia tenacissima found by author on gunung ijo. a. habitus in nature. b. flowering twigs. c. flowering buds. d. inflorescences. a b c d widodo and muhammad ja’far lufhti. – new record marsdenia tenacissima (asclepiadoideae, apocynaceae) … 3 figure 2. marsdenia tenacissima flower. a. arrangement of inflorescences. b. flower unit, top view. c. parts of fower unit, lateral view. d. blooming flower. e. flower completely bloom. this marsdenia shows characteristics of marsdenia crocea (zipp. ex span.) hook. f. ex boerl as described flora of java, vol. 2 (backer and bakhuizen, 1965) with little difference in the length of the petiole and the color when the crown of flowers in full bloom. long petiole (petiolus) specimens ranging from 3-7 cm, while backer and bakhuizen (1965) describes the length of the petiole is only 3 cm. corolla has yellowish color to the reddishbrown yellow flowers in full bloom after folding along the crown out towards the end of basalt. backer and bakhuizen (1965) described marsdenia crocea as shown in table 1. identification using the book flora of british india (hooker, 1885) and flora of china (1995) showed that the characteristics of stem, leaf, and flower of marsdenia from gunung ijo match to marsdenia tenacissima. description of hooker (1885) and flora of china (1995) on marsdenia tenacissima shown in table 2. the characteristics of marsdenia tenacissima flowers specimens from gunung ijo match with the description of flora of china (1995). table 1. description of backer & bakhuizen (1965) on marsdenia crocea. plants parts description flower, pollinia fre part of corona-scales narrowly ovate, obtuse, reaching to about half the height of the stigma; back of connate part ridge shaped, opened towards the base; translators several times shortter than pollinia; panicles broadly pyramidal, c. 5 cm long; pedicels 5-7 mm; calyx c. 3 mm; segments ovate-elliptic, obtuse; corolla c. 6 mm, yellowish white, outside more densely hairy in the upper half than the lower; tube on the side with basal tufts on long hairs, c. 3,5 mm long; segments glabrous inside, with a thickened rim at the base (in the throat), c. 2,5 mm long; gynostegium subequalling the corolla-tube; stigma broadly conical, obtuse. leaves leaves cordate, shortly acuminate, thinly grey-pubescent above, more densely so above, palmatinerved, with distint lateral nerves, finely reticulate-veined, 5-10 cm by c. 6 cm; petiole c. 3 cm. information very long ago collected in east java a corolla tube spreading lobes calyx pedicel b d e c 4 biology, medicine, & natural product chemistry 5 (1), 2016: 1-8 table 2. flora of china (1995) and hooker description (1885) on marsdenia tenacissima. flora of china (1995) plants part description habitus flower pollinia information lianas robust, densely pilose to tomentose throughout except for interior of corolla. petiole 5–6 cm, slender; leaf blade ovate, 8–10 × 6–6.5 cm, base deeply cordate with rounded sinus, apex acuminate; basal veins 5–7, lateral veins 2 or 3 pairs. inflorescences much branched, broader than long, to 8 × 12 cm, many flowered; peduncle to 2 cm, shorter than first internode. pedicel 6–8 mm. sepals elliptic-lanceolate, ca. 3 × 1–1.3 mm, tip rounded. corolla “yellow,” campanulate, with spreading lobes, very densely pilose outside; tube ca. 3.5 × 2.5 mm, interior retrorsely pilose toward base; lobes oblong, ca. 4 × 2.2 mm, apex rounded, minutely velvety. corona lobes exserted from corolla tube, oblong, apex truncate-emarginate with corners produced into short horns, sometimes toothed between these. anther appendages oblong, slightly longer than corona lobes; pollinia curved cylindric. stigma head broadly cylindric, concealed by anther appendages. cliffs; 1500 m. yunnan (szemoo) [cambodia, india, laos, myanmar, nepal, sri lanka, thailand, vietnam]. the stems yield very strong fibers, reputedly among the strongest produced by any plant, that are used for making cords and strings. hooker (1885) stem leaves flower fruit stem very stout. leaves 4-7 by 3-5 in., often valvaty above; petiole 2-3 in. cymes much corymbosely branched. corrolla 0,25 in. diam., lobus oblong, ciliate. stigma beetwen conical and dome shaped. follicles 5-6 in. long by 1,5-2 in. diam., lanceolate; pericarp very thick, longitudinally wrinkled, finely pubescent. seed ovate-oblong. 0,5 in long. pergularia crocea, zipp. figures 3a, b, c, de, show marsdenia tenacissima fruits of gunung ijo. a collection of young fruit and ripe fruit were made by the author on march 21, 2013 and august 26, 2015. figure 3e, f, is sketch of marsdenia tenacissima fruit (flora of china, 1995). the characters of fruit and seed of marsdenia tenacissima of gunung ijo match with the illustration marsdenia tenacissima in flora of china (1995), especially young fruit structure and seeds form. figure 3. fruit and seed of marsdenia tenacissima from gunung ijo (a, b, c, d), and illustration on flora of china (1995) (e, f). a. young fruit. b. mature fruit. c. mature fruit and broken fruit. d. seed. e. fruit illustration. f. seed illustration. a b c d fe widodo and muhammad ja’far lufhti. – new record marsdenia tenacissima (asclepiadoideae, apocynaceae) … 5 figure 4a shows a comparison of the inflorescence herbarium from gunung ijo and from hooker (figure 4d) in 1863 which is collected from bengal (mnhn p03522167), and 4e herbarium kew (k001129150). figure 4b, 4c is a vegetative branches and fruit herbarium from gunung ijo, while figure 4f fruiting twig herbarium of kew herbarium (k001129153). figure 4. marsdenia tenacissima herbarium from gunung ijo compared to kew (a, b, c) & mnhn herbarium collection (d, e, f). comparison of herbarium specimens to the herbarium collections and skew mnhn in figure 4 shows the similarity in the characteristic of twigs, leaves, inflorescence structure and the size of the fruit. similarity of characteristic leaf are as follows: the heart-shaped, rounded and deep sinus at the base of the heart shape, softer hairy of adaxial surface, brighter color of the abaxial surface. the similarity of the structure of a inflorescence is cymes much corymbosely branched. the similarity of the size of the fruit is the size range of about 5-7 cm long with a width of 0.8 to 1.5 cm. based upon the data in the field, tenacissima marsdenia leaf size varies with length of 5-12 cm and 5-9 cm wide in a single individual. it should be noted that the qualitative characteristics of the leaves become an important marker in the identification of this species. the similarity of the structure of the leaves, inflorescence, fruit size specimens of gunung ijo (figure 4a, b, c) can be compared with marsdenia tenacissima in herbarium collections bogor (bo1646031, bo1646032, bo1646038, bo? ex herb. koordersianum no. 16214) in figure 5. structure of gunung ijo spesimen is similar to figure 5d. this herbarium was from sulawesi and was identified as marsdenia velutina. a b c d e f 6 biology, medicine, & natural product chemistry 5 (1), 2016: 1-8 figure 5. marsdenia tenacissima in herbarium bogoriennse. there is a significant difference between fruit size of specimen from gunung ijo (range 5-6 cm long, 0.8 to 1.3 cm wide) with size of the fruit marsdenia tenacissima from hooker (1885), which is 5-6 inches long and 1-1.5 inches wide. i suspect that this is an error in writing the unit. identification of species asclepiadaceae (asclepiadoideae) on the basis of morphological characteristics vegetative structures still confusing because the variation between species in the subfamily is quite high, as well as reproductive structures (flowers and fruit). plant identification is very difficult to do in a group of plants with a number of cultivars and hybrids are large and unknown (simpson, 2006). therefore, it is necessary to rely the identification on the traits that have a strong character. shape or structure of the pollinia is an important character to distinguish species in asclepiadoideae (sreenath, et al., 2012; sinha & mondal 2011). characteristic of pollinia of asclepiadoidea species had been described by experts since hundreds years ago, but visualization using pictures were very rare. structure of gynostegium (unification pistil with the anther) are shown in figure 6a, 6b, and the pollinia marsdenia tenacissima of gunung ijo are shown in figure 6d, 6e. the structure pollinia of marsdenia tenacissima of gunung ijo are shown in thats figure. the pollinia match to the description of marsdenia crocera pollinia (backer & bakhuizan (1965) and a description of the flora of china (1995) (table 2) as well as illustrations of marsdenia tenacissima gynostegium and pollinia (flora of china, 1995) in figure 6c, 6f. figure 6. gynostegium and pollinia of marsdenia tenacissima. from the description and analysis above, it is conclude that the specimen of marsdenia from gunung ijo is marsdenia tenacissima. description hooker (1885) about the existence of marsdenia tenacissima (identified by miquel as pergularia crocea) in java can be justified. marsdenia crocea described by backer and bakhuizen (1965) as the correction for pergularia crocea is basically marsdenia tenacissima. herbarium bo1646038 marsdenia tenacissima support the distribution of this species in java. backer bo1646038 herbarium is a backer’s collection from kangean islands in 1919 and identified by hatusima in 1945 as marsdenia tenacissima. the similarity of character between specimen from gunung ijo and bo1646031, bo1646032, bo? ex herb. koordersianum no. 16214 shows that marsdenia tenacissima also found in sulawesi, although initially identified as marsdenia velutina. in addition to using herbarium, species descriptions and determination keys, identification of plant species need an alternative method by utilizing the digital color image files. baskauf and kirchof (2008) has developed a standard photo living plants as digital plant specimens. error, kucuker and sik (2009) have developed an illustrations technique combined with a virtual observation and herbarium specimens for species identification and determination. improvement and completeness of illustration data in the document and books of flora are critical to improve the works in current and future taxonomy. colored digital image can be used to check and re-check identification. a b c d a b c d e f widodo and muhammad ja’far lufhti. – new record marsdenia tenacissima (asclepiadoideae, apocynaceae) … 7 taxonomic treatment marsdenia tenacissima (roxburgh) moon, cat. pl. ceylon 21. 1824; marsdenia tenacissima wight & arn., fl. brithis ind. iv: 35; marsdenia crocea (zipp. ex span.) hook. f. ex boerl.; type: benggala, 1863, w. hooker (mnhn p03522167) lianas robust, stem very stout densely pilose to tomentose. petiole 3–6 cm, slender; leaf blade ovatecordate, 4–10 × 6–6.5 cm, base deeply cordate with rounded sinus, apex shortly acuminate; thinly greypubescent above, more densely so above, palmatinerved, with distint lateral nerves, finely reticulate-veined, basal veins 5–7, lateral veins 2 or 3 pairs. inflorescences much branched, cymes much corymbosely branched broader than long, to 8 × 12 cm, many flowered; peduncle to 2 cm, shorter than first internode. pedicel 6–8 mm. sepals elliptic-lanceolate, ovate, ca. 3 × 1–1.3 mm, tip rounded, corolla “yellow,” campanulate, with spreading lobes, very densely pilose outside, 8 mm diam., lobus oblong ; tube ca. 3.5 × 2.5 mm, interior retrorsely pilose toward base; lobes oblong, ca. 4 × 2.2 mm, apex rounded, minutely velvety. corona lobes exserted from corolla tube, oblong, apex truncate-emarginate with corners produced into short horns, sometimes toothed between these. anther appendages oblong, slightly longer than corona lobes; pollinia curved cylindric, translators several times shortter than pollinia. gynostegium subequalling the corolla-tube stigma head broadly cylindric, beetwen conical and dome shaped, concealed by anther appendages. follicles 5-6 cm. long by 1,5-2 cm. diam., lanceolate; pericarp very thick, longitudinally wrinkled, finely pubescent. seed ovate-oblong. 0,5-0,7 mm long. specimens examined: baturagung, gunung ijo, s 07 o 47’ 03.4”; e 110o 30’ 48.0”, widodo, 415 m dpl, 31.12.2012 note: marsdenia tenacissima found at the site of observation twining other plant namely annona muricata, clausena indica, zizipus oenoplia, lantana camara alongside with other lianas namely telosma puberula asclepiadoideae, and gymnema sylvestris. m. tenacissima was found by author while observe telosma puberula and gymnema sylvestris. m. tenacissima is not mount parangan forest about 5 km southeast of gunung ijo. marsdenia tenacissima known as a traditional treatment for swelling, asthma and cancer in society of southwestern chinese (li et al., 2006). tripathi, et al. (2014) analyzed farmakognosi of marsdenia tenacissima include of character macroscopic, microscopic characters, physicochemical analysis, preliminary analysis of phytochemical content, and hptlc profile. root of marsdenia tenacissima used for traditional medicine: antiscorbutic, urinary diseases, arthritis, heart disease, skin disease, thirst, pruritus, vomiting, intermittent fever and fever. marsdenia tenacissima in kerala india called murva by local people. murva root used as medicinal plant in the form of a single herb or mixed with other materials. concervation because the limited number of this plant at the site of the discovery and other locations, it is necessary to study their distribution status. simultaneously herbarium collection need to be carried on preserve the specimen. conclusion marsdenia tenacissima (roxburgh) moon found on gunung ijo mountains baturagung yogyakarta. morphological characteristics stature (habitus), leaves, and flowers specimen marsdenia tenacissima in gunung ijo show compliance with the kew herbarium collection hooker in 1863 (mnhn p03522167) came from bengal and herbarium collections bogoriense backer 1919 (bo1646038) derived from kangean islands. the existence of marsdenia tenacissima (roxburgh) moon in java correct and acomplish description of backer and bakhuizen in the flora of java book on marsdenia genus. acknowledgements the author would like to thank to the herbarium museum national d 'histoire naturelle, paris (mnhn) and kewensis herbarium royal botanic garden edinburgh (kew) on herbarium type photo credit. thanks also to mr. deden girmansyah and mr. arif hidayat in the herbarium bogoriense for the opportunity to checking herbarium specimen. references backer, c. a. & bakhuizen. 1965. flora of jawa (spermatophytes only) (vol ii). groningen: n. v. p. noordhoff. baskauf s j, kirchoff bk. 2008. digital plant image as speciments: toward standards for photographing living plants. vulpia, 7:16-30. borlage, j. g. 1899. handeiding flora van nederlandsch indie. leiden, e.j brill. dwari, s., mondal, a. k. 2011. systematic studies (morphology, anatomy and palynology) of economically viable grass brachiaria mutica (forsskil) stapf in eastern india. african journal of plant science, 5 (5): 296-304. eror o, kucuker o, sik l. 2009. application of a new illustration technique in plant systematics: composite images of two autumn flowering crocus l. (iridaceae) taxa from series biflori in turkey. iufs j biol, 68(2):127-133. flora of china editorial committee. 1995. flora of china (gentianaceae through boraginaceae). 16: 1–479. in c. y. wu, p. h. raven & d. y. hong (eds.) fl. china. science press & missouri botanical garden press, beijing & st. louis. herbarium museum national d’ histoire naturelle paris (mnhn). 2015. marsdenia tenacissima. hooker, j. d. 1885. flora of british india (vol. iv). london: reeve and co. http://colb.mnhn.fr/catalognumber/mnhn/p/p03522167., diakses 2 oktober 2015. li, q., wang, x., yu, k., peng, s., & ding, l. (2006). acetonyltenacissoside f acetone solvate: a new 8 biology, medicine, & natural product chemistry 5 (1), 2016: 1-8 polyoxypregnane glycoside from the stems of marsdenia tenacissima. acta cryst, 62, 5255–5256. royal botanic garden, kew. 2015. marsdenia tenacissima. http://specimens.kew.org/herbarium/k001129150. diakses 10 oktober 2015 http://specimens.kew.org/herbarium/k001129153. diakses 10 oktober 2015 simpson, m.g. 2006. plant systematics. amsterdam: elsevier academic press. sreenath, k. p., ramakrishna, t.m., babu, t. p. 2012. prespective on pollinial apparatus and carriers of asclepiadaceae sensu lato. global journal of bio-science and biotechnology, 1 (1): 45-53. takhtajan, a. (2009). flowering plant. st petersburg: springer. the plant list (2010). version 1. published on the internet; http://www.theplantlist.org/tpl/search?q=marsdenia+. diakses 2 oktober 2015. tripathi m, shivhare d, tiwari a, ahirwar p and pathak s. 2014. pharmacognostical evaluation of marsdenia tenacissima wight. & arn. root. int. j. rec. biotech., 2 (3): 18-23. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1391-1397 | doi: 10.14421/biomedich.2025.142.1391-1397 issn 2540-9328 (online) antibacterial and synergistic potential of hibiscus sabdariffa and zingiber officinale against multidrug-resistant bacteria abdulazeez mumsiri abaka1*, bristone james pola2, mohammed badamasi dangana1, adamu aliyu usman2 1science laboratory technology department, school of science and technology, adamawa state polytechnic, yola, nigeria. 2department of microbiology, faculty of life sciences, modibbo adama university, yola, nigeria. corresponding author* abdulazizelnino22@adamawapoly.edu.ng abstract antimicrobial resistance (amr) is a global health challenge, particularly in developing regions where access to effective drugs is constrained. medicinal plants provide an alternative due to their affordability, availability, and wealth of bioactive compounds. this study investigated the antibacterial properties of zingiber officinale (ginger) and hibiscus sabdariffa (roselle), both widely used in african traditional medicine, against selected bacterial pathogens. methanolic extracts were prepared from dried rhizomes of ginger and calyces of roselle, and concentrations of 100%, 95%, 90%, and 85% were tested. antibacterial activity was determined against staphylococcus aureus, escherichia coli, and pseudomonas sp. using agar well diffusion. minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) were established by serial dilution and subculture techniques. statistical significance was assessed using one-way anova and duncan’s multiple range test at p < 0.05. phytochemical analysis revealed steroids, terpenoids, and flavonoids in both plants. ginger additionally contained saponins, alkaloids, tannins, and carbohydrates, whereas roselle uniquely presented cardiac glycosides. antibacterial activity increased with concentration, with the 100% extracts showing the highest inhibition zones. the combined extract consistently produced larger inhibition zones compared to the individual extracts (p < 0.05). ginger exhibited the strongest individual activity against s. aureus, while the combined extract was most effective against e. coli. pseudomonas sp. was the least susceptible. mic values confirmed these observations: s. aureus was the most sensitive (25 mg/ml), while e. coli and pseudomonas sp. each recorded 50 mg/ml. mbc values were uniformly 50 mg/ml across all test organisms. the results demonstrate that ginger and roselle possess significant antibacterial properties, with ginger showing superior individual efficacy and roselle providing complementary metabolites that enhanced combination effects. their synergistic action supports traditional polyherbal therapy and indicates potential as cost-effective, plant-based interventions against amr. the findings underscore the need for further research to develop standardized formulations for therapeutic application. keywords: hibiscus sabdariffa; zingiber officinale; antibacterial activity; phytochemicals; synergistic effect; antimicrobial resistance. introduction the effectiveness of antimicrobial agents, which target vital bacterial functions such as cell wall formation and dna synthesis, has been significantly compromised by the rapid rise of antimicrobial resistance (amr) (ilechukwu et al., 2025). the widespread misuse and overuse of these drugs drive this crisis. resistance is a complex phenomenon; it differs from non-genetic tolerance observed in persistent cells and can develop through intrinsic mechanisms or be acquired via genetic mutations and horizontal gene transfer (oliveira et al., 2024). bacteria have developed advanced defenses to evade antimicrobials, such as producing enzymes that inactivate the drug, altering target sites, decreasing membrane permeability to limit drug entry, and using efflux pumps to actively remove the compounds (belay et al., 2024). these defenses are often more robust in biofilms. to combat this challenge, the field is exploring innovative non-antibiotic strategies like bacteriophage therapy, probiotics, and phytomedicine, drawing on a long tradition of plant-based medicine, to find new ways to treat and prevent infections without worsening resistance (lyimo and sonola, 2025). ginger (zingiber officinale) is a well-established botanical remedy in naturopathic medicine, recognized for its potent activity against a diverse spectrum of microbial pathogens (edo et al., 2025). this efficacy, demonstrated by the raw rhizome itself, underscores its potential as a powerful therapeutic agent for infectious diseases. as a member of the zingiberaceae family, ginger shares the aromatic and medicinal qualities of its relatives, which are characterized by their unique rhizomes (kadam and shahi, 2025). its worldwide availability, low cost, and high tolerability make it a common resource. the antimicrobial activity of ginger is manuscript received: 08 september, 2025. revision accepted: 04 december, 2025. published: 22 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1391-1397 mailto:abdulazizelnino22@adamawapoly.edu.ng 1392 biology, medicine, & natural product chemistry 14 (2), 2025: 1391-1397 largely attributed to its active constituents, primarily gingerols and shogaols, which are known to resolve gastric infections and inhibit the proliferation of harmful gut bacteria, including resistant strains of escherichia coli and staphylococcus species (ayodeji et al., 2024). historically, ginger has been a cornerstone in treating intestinal and digestive ailments. antimicrobial resistance represents a critical global health crisis, particularly in lowand middle-income nations, where it leads to increased treatment costs, extended hospitalizations, and higher mortality rates (salam et al., 2023). hibiscus sabdariffa, commonly referred to as roselle, is a member of the malvaceae family with considerable ethnomedicinal and nutritional value (apaliya et al., 2021). traditionally, its various components have been utilized to address a wide array of health conditions, including but not limited to cardiovascular and nervous system ailments, digestive issues, and parasitic infections (jabeur et al., 2017). the plant is particularly prized for its edible calyces, which are a rich source of bioactive compounds. these include potent antioxidants like γtocopherol, as well as an abundance of polyphenols and flavonoids. scientific investigations have substantiated that extracts derived from the plant exhibit significant pharmacological activities, such as antibacterial, antihypertensive, anticancer, and hepatoprotective effects, which are directly attributed to its complex phytochemical composition (mccalla and smith, 2024). beyond its therapeutic applications, h. sabdariffa plays a crucial role in local diets and food security, particularly in west african regions such as nigeria (mukhtar et al., 2025). the calyces are processed into popular food items such as the beverage "sobo," while the seeds are fermented into a condiment known as "daddawa" (ajayi et al., 2016). the leaves and green calyces are also commonly incorporated into soups, providing essential micronutrients including vitamins b and c. phytochemical analysis has identified the presence of alkaloids, anthocyanins, tannins, and glycosides, which collectively contribute to its documented antifungal, antiviral, and antidiabetic properties (salem et al., 2022). with hundreds of species within the hibiscus genus thriving in tropical climates, this group of plants represents a promising reservoir for developing novel functional foods and plant-based medicines (sarwar, 2023). methodology samples collection, preparation, and extraction the fresh calyces of h. sabdariffa and the rhizome z. officinale were obtained from the market of yola south local government of adamawa state. the plant leaf samples were shade-dried. the dried samples were blended into powder form using a blender. then, 40 g of each powdered extract was soaked in 100 ml of methanol for 48 hours. furthermore, 20 g of each of the powdered leaves was weighed and combined, resulting in a total of 40 g, which was then soaked in methanol for 48 hours. then, it was filtered using a muslin cloth. the resultant filtrate was re-filtered using whatman filter paper. the solvent was allowed to evaporate in a water bath. identification and authentication the plants were identified and authenticated by an experienced taxonomist with identification numbers asp 1223 and asp 1254 for h. sabdariffa and z. officinale, respectively, at the science laboratory technology department, adamawa state polytechnic, nigeria. dilution of the extracts dilutions of the extracts were made following the methods previously described by izah et al. (2018). the original stock of the leaf extracts was considered as 100% concentration, and then it was further diluted into 95%, 90% and 85% of the original volume. standardization of the inocula mueller-hinton agar (mha) served as a medium for sub-culturing the isolates on newly purchased sterile plates. the plates were incubated at 37 °c for 24 hours and thereafter preserved at 4 °c for subsequent overnight culturing, from which isolates were later suspended in broth culture for antimicrobial assay. the broth cultures of the isolates were adjusted to the exact turbidity equivalent to 0.5 mcfarland standard (abaka et al., 2025). antimicrobial assay antimicrobial activity of the extracts was determined using the agar well diffusion assay method. mueller hinton agar (mha) was prepared as recommended by the manufacturer and allowed to cool to 50 °c before pouring on pre-labelled sterile petri plates on a level surface. one petri-plate was prepared per organism and done in triplicate, except that of the positive test control, using maxi discs that were done in duplicate. a sterile 6 mm borer was used to punch seven equidistant wells: wells 1 – 6 for different concentrations of extracts. well number seven (7) was bored at the center of the plates for the solvent used (negative control). a dip with sterile swabbing sticks of overnight broth cultures of each of the isolates was streaked on the surface of the prepared mha plates. the volume of 0.2 ml of the extracts at preset concentration was introduced to each of the wells, and the plates were allowed to rest (and set) on the laboratory bench for 45 minutes, allowing for proper prediffusion of the extracts before 24 hours of incubation at 37 °c (abaka et al., 2025). abaka et al. – antibacterial and synergistic potential of hibiscus sabdariffa and … 1393 determination of the minimum inhibitory concentrations (mic) the mic was determined using the tube dilution technique according to kanu et al. (2018). the mic was determined by taking 2ml of the concentrations of the extracts that inhibited the growth of the resistant strains and mixing it with 2ml of the nutrient broth containing 0.1ml of standardized inoculum of 0.5 mcfarland standard. the tubes were incubated for 24 hours at 37 °c. it has a tube containing broth and inoculum without the extract as the control. the lowest concentration that showed no visible turbidity (inhibited microbial growth) is regarded as the mic. determination of the minimum bactericidal concentration (mbc) sterile mueller-hinton agar plates were incubated with samples from each of the test tubes that showed no visible growth from the mic test. the plates were then incubated at 37 °c for 24 hours. the lowest concentration of the extract yielding no growth was recorded as the minimum bactericidal concentration (islam et al., 2008). statistical analysis statistical analysis was carried out using statistical package for social sciences (spss) software version 20. the data were expressed as mean ± standard error, and one-way analysis of variance was carried out at α = 0.05. duncan's multiple range test was used to ascertain the source of the variation. table 1. phytochemical screening of fractions of hibiscus sabdariffa and z. officinale. phytochemicals hibiscus sabdariffa zingiber officinale inferences steroids + + present in both terpenoids + + present in both saponins + absent in h. sabdariffa, present in z. officinale alkaloids + absent in h. sabdariffa, present in z. officinale tannins + absent in h. sabdariffa, present in z. officinale flavonoids + + present in both cardiac glycosides + present in h. sabdariffa, absent in z. officinale carbohydrates + absent in h. sabdariffa, present in z. officinale key: positive = + negative= table 2. mean zone of inhibition (mm ± sem) of plant extracts against bacterial isolates. plant extract isolate 100% concentration 95% concentration 90% concentration 85% concentration hibiscus sabdariffa e. coli 12.82 ± 0.33b 11.13 ± 0.34c 8.80 ± 0.55c 5.72 ± 0.26c s. aureus 11.88 ± 0.27c 9.23 ± 0.03d 7.52 ± 0.35d 5.74 ± 0.31c pseudomonas sp. 10.11 ± 0.07c 8.03 ± 0.13e 5.23 ± 0.07e 3.63 ± 0.18d z. officinale e. coli 15.15 ± 0.09a 9.85 ± 0.30d 8.32 ± 0.51c 5.48 ± 0.09c s. aureus 14.30 ± 0.04b 12.64 ± 0.24b 9.52 ± 0.36c 8.14 ± 0.08b pseudomonas sp. 14.81 ± 0.39a 12.41 ± 0.19b 9.89 ± 0.31c 7.41 ± 0.22b h. sabdariffa + z. officinale. e. coli 17.57 ± 0.35a 15.54 ± 0.15a 12.10 ± 0.33a 9.69 ± 0.07a s. aureus 15.48 ± 0.19b 14.31 ± 0.13a 12.37 ± 0.13a 9.14 ± 0.63a pseudomonas sp. 16.08 ± 0.52a 11.86 ± 0.38b 9.77 ± 0.33c 7.41 ± 0.17b different superscript letters (a, b, c) within the same column for a specific bacterium and concentration denote statistically significant differences (p < 0.05) according to duncan's multiple range test. table 3. minimum inhibitory concentration of polyherbal extracts of hibiscus sabdariffa and zingiber officinale against staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa. organisms extract concentration (mg/ml) mic mbc 100 mg/ml 50 mg/ml 25 mg/ml 12.5 mg/ml 6.25 mg/ml 3.125 mg/ml e. coli me _ _ * + + + + 50 50 s. aureus me _ _ _* + + + 25 50 pseudomonas sp. me _ _* + + + + 50 50 1394 biology, medicine, & natural product chemistry 14 (2), 2025: 1391-1397 discussion the phytochemical analysis indicated that both hibiscus sabdariffa and zingiber officinale share common secondary metabolites such as steroids, terpenoids, and flavonoids, which are often linked to antimicrobial properties. despite this similarity, z. officinale exhibited a broader spectrum of bioactive compounds, including saponins, alkaloids, tannins, and carbohydrates, metabolites not observed in h. sabdariffa. in contrast, h. sabdariffa contained cardiac glycosides, which were absent in z. officinale. these distinctions suggest that while both plants possess therapeutic constituents, the greater phytochemical diversity of z. officinale could explain its relatively stronger antibacterial performance when evaluated independently. the differences in phytochemical profiles between the two plants are particularly important in understanding their varying antimicrobial activities. compounds found in z. officinale, such as saponins, alkaloids, and tannins, are known to disrupt microbial membranes, interfere with nucleic acid synthesis, and inactivate microbial proteins, thereby providing potent antibacterial effects (akinmoladun et al., 2018; teke et al., 2020). on the other hand, the presence of cardiac glycosides in h. sabdariffa, though primarily associated with cardiovascular health, has been reported to exhibit antimicrobial effects through disruption of microbial respiration and enzymatic pathways (nwachukwu et al., 2014; iroha et al., 2019). these complementary differences suggest that combining extracts from both plants could yield a synergistic effect. this is consistent with evidence from african ethnomedicine, where multiplant formulations are often employed to enhance therapeutic activity through the interaction of diverse bioactive molecules (okwu and ndu, 2006; ibrahim et al., 2021). consequently, the observed higher antibacterial activity of the combined extracts of h. sabdariffa and z. officinale may be attributed to this phytochemical synergy. the combined extract of hibiscus sabdariffa and zingiber officinale demonstrated the most pronounced antibacterial effect, consistently producing the largest mean zones of inhibition across staphylococcus aureus, escherichia coli, and pseudomonas species. this synergistic effect was statistically significant (p < 0.05), particularly when compared with the activity of the individual plant extracts, which showed reduced inhibitory capacity. these results corroborate the findings of akinmoladun et al. (2020), who highlighted that polyherbal formulations exhibit stronger antimicrobial activity than single extracts because of complementary interactions between diverse phytochemicals. similarly, oladunmoye and komolafe (2020) emphasized that combining plant extracts can potentiate antibacterial efficacy by targeting multiple microbial pathways simultaneously, thereby enhancing therapeutic action and limiting the risk of microbial resistance. the enhanced performance of the combination in the present study suggests that the interaction of bioactive constituents from both plants likely underpins the broad-spectrum activity observed. furthermore, the study revealed a concentrationdependent decline in antibacterial potency across all extracts, with inhibition zones decreasing progressively from 100% to 85% concentrations. this dose-response relationship was statistically significant (p < 0.05), confirming that higher concentrations of extracts are more effective in suppressing bacterial growth. such findings are consistent with the general pharmacological principle that antimicrobial efficacy is positively correlated with bioactive compound concentration until an optimal threshold is reached (eze et al., 2017). the superior efficacy of the combined extract across different concentrations indicates its potential as a more reliable and broad-spectrum antibacterial agent compared to individual plant preparations. these results strengthen the argument for polyherbal therapies in antimicrobial drug development, particularly in regions where resistance to conventional antibiotics is prevalent. in contrast, hibiscus sabdariffa alone was the least effective extract in this study, as it produced the smallest inhibition zones against staphylococcus aureus, escherichia coli, and pseudomonas spp. this outcome corroborates the findings of ologundudu et al. (2010), who reported modest antibacterial activity of h. sabdariffa against clinical isolates, attributing the reduced activity to comparatively low concentrations of phenolic acids relative to other medicinal plants. however, this differs from aliyu et al. (2016), who observed strong antibacterial activity of h. sabdariffa calyx extracts, particularly against e. coli. such variations in outcomes may arise from differences in extraction solvents, phytochemical composition due to geographical factors, or variations in bacterial strains, as highlighted by abba et al. (2021). these discrepancies suggest that while h. sabdariffa holds antimicrobial potential; its effectiveness may be context-dependent and influenced by methodological and ecological factors. the study further established that the antibacterial efficacy of all extracts was concentration-dependent, with higher concentrations (100%) producing larger inhibition zones compared to lower concentrations (85%). this concentration-effect trend is consistent with the findings of nwachukwu and uzoeto (2010), who reported that the antimicrobial activity of plant extracts diminishes with dilution due to reduced levels of bioactive compounds such as flavonoids, tannins, and saponins. similarly, eze and okoye (2019) demonstrated a direct relationship between extract concentration and inhibition diameters when evaluating ginger and garlic extracts against enteric pathogens. taken together, these consistencies reinforce the importance of maintaining high concentrations of extracts to achieve optimal abaka et al. – antibacterial and synergistic potential of hibiscus sabdariffa and … 1395 therapeutic efficacy, highlighting a critical consideration for the development of standardized herbal formulations. among the bacterial species tested, pseudomonas sp. exhibited the greatest resistance, consistently producing the smallest inhibition zones across treatments. this aligns with the observations of nsofor and iroegbu (2013), who attributed the resilience of pseudomonas to its efficient efflux systems and low outer membrane permeability. in contrast, staphylococcus aureus showed higher susceptibility, particularly to zingiber officinale extracts at lower concentrations, which is consistent with findings by ibrahim et al. (2021) that linked ginger’s strong activity against gram-positive bacteria to phenolic compounds such as gingerol and shogaol. meanwhile, escherichia coli responded most favorably to the combined extracts, supporting adebolu and oladimeji’s (2005) report that plant extract combinations often produce synergistic inhibition of gram-negative organisms. the polyherbal extract demonstrated inhibitory activity against e. coli, s. aureus, and pseudomonas sp., with varying mic and mbc values. the mic was lowest for s. aureus (25 mg/ml), indicating the highest sensitivity, while e. coli and pseudomonas sp. showed mic values of 50 mg/ml. the mbc was 50 mg/ml for all tested organisms. overall, the extract was most effective against s. aureus, but required higher concentrations to inhibit and kill e. coli and pseudomonas sp. the combined extract of hibiscus sabdariffa and zingiber officinale exhibited considerable antibacterial activity against e. coli, s. aureus, and pseudomonas sp., though the degree of susceptibility differed among the organisms. the lowest mic was recorded against s. aureus (25 mg/ml), indicating that the gram-positive strain was more responsive compared to the gramnegative species. this observation aligns with established knowledge that the lipopolysaccharide barrier in gramnegative bacteria enhances their resistance to antimicrobial compounds (oladunmoye and komolafe, 2020). the higher mic values observed for e. coli and pseudomonas sp. (50 mg/ml each) further emphasize the inherent resilience of gram-negative organisms, consistent with earlier findings on the therapeutic difficulties posed by these pathogens (akinmoladun et al., 2021). the uniform mbc value of 50 mg/ml across all test organisms suggests that while inhibitory effects occurred at varying concentrations, complete bactericidal activity required higher doses of the extract. the greater sensitivity of s. aureus highlights the potential of this polyherbal formulation in treating gram-positive infections, supporting previous reports of synergistic plant extract activity against staphylococcus species (okeke et al., 2020). the enhanced efficacy may be attributed to the combined phytochemicals in z. officinale and the unique bioactive compounds of h. sabdariffa. furthermore, the ability of the extract to suppress pseudomonas sp., a pathogen well known for its multidrug resistance, illustrates its therapeutic promise, albeit at higher concentrations. overall, these findings underscore the potential role of polyherbal therapy as a complementary or alternative strategy to conventional antibiotics, particularly in addressing the growing challenge of antimicrobial resistance in nigeria (ezeigbo et al., 2019). the combined extract of hibiscus sabdariffa and z. officinale consistently yielded the largest inhibition zones across different concentrations, significantly outperforming either extract alone (p < 0.05), as confirmed by duncan’s multiple range test. while h. sabdariffa alone demonstrated the weakest antibacterial activity, its effectiveness was markedly enhanced when paired with ginger. these results underscore the importance of synergistic herbal formulations and highlight the role of extract concentration in therapeutic efficacy. the one-way anova further confirmed significant main effects of extract type on antibacterial activity, reinforcing that the combined formulation was the most potent, followed by ginger alone, and lastly roselle. collectively, these findings validate the traditional use of these plants while providing scientific evidence for their potential development into standardized phytomedicines within nigeria and across africa. conclusion this study confirmed that hibiscus sabdariffa and zingiber officinale possess notable antimicrobial properties, with ginger showing stronger individual activity while roselle contributed unique phytochemicals. the combined extracts produced the most significant antibacterial effects against staphylococcus aureus, escherichia coli, and pseudomonas sp., highlighting a clear synergistic interaction. antibacterial activity was concentration dependent, with higher doses producing greater inhibition zones. among the test organisms, pseudomonas sp. remained the most resistant, while e. coli and s. aureus were more susceptible to the synergistic and ginger extracts, respectively. overall, the findings validate traditional use of these plants, emphasize the benefits of polyherbal formulations, and support their potential as affordable alternatives for managing antimicrobial resistance in africa. competing interests: the authors declare that there are no competing interests. references abaka, a. m., belel, m. h., dangana, m. b., & abubakar, k. b. 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(2020). antimicrobial and antioxidant properties of african medicinal plants: relevance in the treatment of infectious diseases and oxidative stress-related conditions. journal of ethnopharmacology, 262, 113201. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 477-484 | doi: 10.14421/biomedich.2023.122.477-484 issn 2540-9328 (online) nephroprotective activities of ethanol root extract and fractions of hippocratea africana against doxorubicin-induced kidney toxicity kufre u. noah1, john a. udobang2, jude e. okokon1,*, martin o. anagboso3, nwakaego omonigho ebong4 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. 2department of clinical pharmacology and therapeutics, faculty of basic clinical sciences, university of uyo, uyo, nigeria. 3department o microbiology, madonna university, elele, nigeria. 4department of pharmacology and toxicology, faculty of pharmacy, madonna university, elele, nigeria. corresponding author* judeefiom@yahoo.com, tel.no. +234-8023453678 abstract hippocratea africana root used locally in the treatment of poisoning was investigated to confirm its antidotal potential in rats. the root extract (200-600 mg/kg) and fractions; dichloromethane (dcm) and aqueous, 400 mg/kg) were evaluated for nephroprotective activity against doxorubicin-induced kidney injury in rats. kidney function parameters, kidney oxidative stress markers and kidney histology were used to assess the kidney protective effect of the extract. the root extract and fractions (200-600 mg/kg) significantly (p<0.05-0.01) reduced the levels of creatinine, urea and electrolytes that were elevated by doxorubicin. also, the mda level elevated by doxorubicin was reduced by the extract and fractions co-administration, while the levels of gsh, gst, sod, gpx, and cat that were decreased by doxorubicin were significantly (p<0.01) elevated by the root extract/fractions. histology of the kidney sections of extract/fractions treated animals showed reductions in the pathological features compared to the organotoxic-treated animals. the chemical pathological changes were consistent with histopathological observations suggesting marked nephroprotective potential. the anti-toxic effect of this plant may in part be mediated through the chemical constituents of the plant. the plant, hippocratea africana possesses anti-toxicant properties which can be exploited in the treatment of doxorubicin related toxicities. keywords: renoprotective; hippocratea africana; doxorubicin; oxidative stress. abbreviations: dichloromethane (dcm), superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), reduced glutathione (gsh), malondialdehyde (mda), haematotoxylin and eosin (h&e), reactive oxygen species (ros), reduced nicotinamide adenine dinucleotide phosphate (nadph). introduction hippocratea africana (willd.) loes. ex engl. (celastraceae) known in english as ‘african paddle-pod’ and ‘eba enang enang’ in ibibio language in nigeria, is a climber perennial plant distributed widely in tropical africa (hutchison and dalziel, 1973). traditionally, the plant root has been variously utilized in herbal preparations to treat diseases like malaria and diabetes (okokon et al., 2006), as well as liver diseases (ajibesin et al., 2008). previous reports showed that the root extract possess antimalarial (okokon et al., 2006; okokon et al., 2021), antioedema and antinociceptive (okokon et al., 2008), antidiabetic and hypolipidemic (okokon et al., 2010; 2022), antidiarrhoeal and antiulcer (okokon et al., 2011), hepatoprotective (okokon et al., 2013a), antileishmanial, cytotoxicity and cellular antioxidant (okokon et al., 2013b), antibacterial, anticonvulsant and depressant (okokon et al., 2014). also, earlier studies had reported the presence of ᵟ-3carene and α-terpineol (okokon et al., 2017), isolation of 1,3,7-trihydroxy-6-methoxyxanthone [isoathyriol] and 1,3,6,7-tetrahydroxyxanthone [norathyriol] (umoh et al., 2021) from ethyl acetate fraction. monoterpenes and sesquiterpenes have been identified in the n-hexane fraction (okokon et al., 2013a). we report nephroprotective and antioxidative stress effects of the root extract and fractions of h. africana against doxorubicin-induced nephrotoxicity in rats. materials and methods plants collection fresh roots of hippocratea africana were collected in bushes in uruan area, akwa ibom state, nigeria in november, 2021. the plant was identified and authenticated by a taxonomist in the department of manuscript received: 28 june, 2023. revision accepted: 17 august, 2023. published: 23 august, 2023. https://doi.org/10.14421/biomedich.2023.122.477-484 478 biology, medicine, & natural product chemistry 12 (2), 2023: 477-484 botany and ecological studies, university of uyo, uyo, nigeria. herbarium specimen was deposited at department of pharmacognosy and natural medicine herbarium, university of uyo. preparation of extract and fractions fresh root of h. africana were washed, cut into smaller pieces and dried under shade for two weeks. they were powdered using electric grinder. the pulverised root of h. africana (hae) was soaked in ethanol (50%) for 72 hours. the liquid filtrate obtained was concentrated in a rotary evaporator at 40˚c. the crude extract (20 g) was dissolved in 500 ml of distilled water and partitioned with equal volume of dichloromethane (dcm, 5 x 500 ml) till no colour change was observed, to obtain dcm and aqueous fractions. the extract and fractions were stored at 4˚c in a refrigerator until used for the experiment. animals in this study, male albino wistar rats were used. the animals were sourced from university of uyo animal house and sheltered in plastic cages. the rats were fed with pelleted standard feed (guinea feed) and given unlimited access to water. the study was approved by college of health sciences animal ethics committee, university of uyo. experimental design in this study, repeated dose model earlier described by raskovi et al. (2011) and olorundare et al., (2020), which lasted for 14 days was used. groups i rats which served as the untreated control were orally pretreated with 10 ml/kg/day of distilled water. group 2 rats were given normal saline (10 ml/kg/day) but equally treated on alternate days with 1.66 mg/kg of doxorubicin hydrochloride dissolved in 0.9% normal saline administered on alternate days for 14 days. groups’ 3-5 rats were orally pretreated with 200 mg/kg/day, 400 mg/kg/day, and 600 mg/kg/day of hippocratea africana dissolved in distilled water 2 hours before treatment with 1.66 mg/kg of doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days, respectively. groups 6 and 7 were pretreated with 400 mg/kg of dcm and aqueous fractions respectively. group 8 rats which served as the positive control group were equally pretreated with 100 mg/kg/day of silymarin two hours before treatment with 1.66 mg/kg of doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days. collection of blood samples and organs after 14 days of treatment (24 hours after the last administration) the rats were weighed again and sacrificed under light diethyl ether vapour. blood samples were collected by cardiac puncture and used immediately. blood samples were collected into plain centrifuge tubes. the blood in the centrifuge tubes were centrifuged at 1500 rpm for 15 minutes to separate of serum at room temperature used for biochemical assays. the rats’ kidneys were identified, harvested, and weighed. kidney function test the following biochemical parameters such as levels of electrolytes (na, k, cl, and hco3), creatinine and blood urea were assayed as markers of kidney function using diagnostic kits at the chemical pathology department of university of uyo teaching hospital. oxidative stress markers the antioxidant enzymes assays were performed on kidney homogenates of rats that were used in this study. these oxidative stress markers were used to assess antioxidative stress potentials of the extract. preparation of renal homogenate in each rat, the kidneys were removed and one kidney was fixed in 10% formaldehyde for histological processes, while the other kidney was dissected free from the surrounding fat and connective tissue and used for assays of oxidative markers. the kidneys were longitudinally sectioned, and renal cortex was separated and kept at -8°c. subsequently, renal cortex was homogenized in cold potassium phosphate buffer (0.05m, ph 7.4). the renal cortical homogenates were centrifuged at 5000 rpm for 10 min at 4°c. the resulting supernatant was used for the determination of superoxide dismutase (sod) (marklund and marklund, 1974), catalase (cat) (sinha, 1972), glutathione peroxidase (gpx) (lawrence and burk, 1976), reduced glutathione (gsh) (ellman, 1959) and malondialdehyde (mda) content (esterbauer and cheeseman, 1990). histopathological studies the kidneys of the animals that were surgically removed and fixed in 10% formaldehyde were processed and stained with haematotoxylin and eosin (h&e) (drury and wallington, 1980), according to standard procedures at department of chemical pathology, university of port harcourt teaching hospital, port harcourt. morphological changes observed and recorded in the excised organs of the sacrificed animals. histologic pictures were taken as micrographs. statistical analysis and data evaluation data obtained from this work were analysed statistically using anova (one –way) followed by a post test (tukey-kramer multiple comparison test). differences between means were considered significant at 5% level of significance ie p≤ 0.05. noah et al. – nephroprotective activities of ethanol root extract and fractions … 479 results and discussion effect of root extract and fractions of h. africana on body and organs weights of rats with doxorubicininduced toxicity administration of h. africana root extract and fractions to rats with doxorubicin-induced organs toxicities caused considerable improvement of the body weights compared to the organotoxic group. the crude extract caused a pronounced dose-dependent effect (7.14 -8.09%) when compared to the organotoxic group with the dichloromethane fraction treated group exerting the highest effect (9.30%). silymarin also improved the weight of the treated animals considerably (7.99%). the weights of kidneys of the group treated with doxorubicin only were found to be reduced when compared to those of the normal control group though not statistically significant (p>0.05). however, treatment of rats with doxorubicin-induced toxicities with the root extract and fractions of h. africana improved the organs weights though insignificantly (p>0.05) except in the group treated with aqueous fraction (table 1). evaluation of effect of root extract and fractions of h.africana on kidney function parameters of doxorubicin-induced kidney injury in rats. table 2 shows the effect of root extract/fractions of h. africana on kidney function parameters of rats. administration of doxorubicin (1.66 mg/kg) to the rats caused significant (p<0.05-0.001) elevation of serum urea, creatinine and electrolytes (k+, na+ and hco3) except clwhen compared to normal control. these increased levels of serum urea, creatinine and electrolytes (k+, na+ and hco3) were significantly (p<0.05 0.001) reduced when compared to organotoxic group following pretreatment of the rats with silymarin and root extract/fractions (200 – 600 mg/kg), with the dcm fraction having the highest effect in most cases. these reductions were non dose-dependent with the lower doses (200 and 400 mg/kg) exerting more significant effects. however, the cllevel was not affected significantly (p>0.05) with the root extract/fraction treatment (table 2). effect of root extract and fraction on kidney oxidative stress markers of doxorubicin-induced kidney toxicity in rats. the effect of h. africana root extract/fractions on kidney oxidative stress markers of the rats is as shown in table 3. administration of doxorubicin (1.66 mg/kg i.p) on alternate days for 14 days caused significant (p<0.050.001) decreases of kidney antioxidant enzymes activities (sod, gpx, gst, cat) and gsh levels when compared to control. the mda level was also elevated by doxorubicin treatment significantly (p<0.001) when compared to control. however, repeated administration of root extract/fractions of h. africana (200600 mg/kg) concomitantly with doxorubicin for 14 days caused non dose-dependent elevations of the enzymatic and nonenzymatic endogenous antioxidants in the treated rats groups which were mostly significant (p<0.05-0.001) in the higher doses (400 and 600 mg/kg) of the root extract when compared to the organotoxic groups with dcm fraction being the most active. dose-dependent and significant (p<0.05-0.001) decreases in mda levels of the extract/fractions treated groups were recorded when compared to control with aqueous fraction as the most active fraction. similar decreases were also observed in the silymarin-treated group when compared to organotoxic control (table 3). effect of root extract and fractions of h. africana on histology of rat kidney in doxorubicin-induced nephrotoxicity histological sections of kidneys of rats receiving various treatments at magnification (x400) stained with h&e method revealed that group 1 (a) treated with distilled water (10 ml/kg) showed normal renal tubules and glomeruli. no evidence of pathology was seen. the organotoxic group (group 2, b) treated with doxorubicin (1.66 mg/kg) showed focal non-specific inflammation, normal renal tubules and glomeruli (gm) and few congested blood vessels were seen. rats in group 3 (c ) treated with 200 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg), group 4 (d) treated with 400 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg), group 6 (f) treated with 400 mg/kg of aqueous fraction of h. africana root and doxorubicin (1.66 mg/kg), group 7 (g) treated with 400 mg/kg of dichloromethane fraction of h. africana root and doxorubicin (1.66 mg/kg) and group 8 (h) treated with 100 mg/kg of silymarin of h. africana root and doxorubicin (1.66 mg/kg) had kidney sections showing normal renal tubules and glomeruli with no evidence of pathology seen. group 5 (e) rat treated with 600 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg) showed distorted parenchyma with both normal and dilated renal tubules. the dilated tubular epithelium appears flattened. there were also multifocal interstitial inflammatory infiltrates (figures a-h). 480 biology, medicine, & natural product chemistry 12 (2), 2023: 477-484 table 1. effect of h. africana root extract on body and kidney weights of rats with doxorubicin-induced toxicity. parameters/ treatment dose mg/kg kidney body weight before after % increase in body weight normal control 1.24±0.10 135.6±18.34 151.0± 12.33 11.35 doxorubicin 1.66 0.94±0.10 130.0 ± 9.45 126.3± 10.43 -2.84 silymarin+dox 100 1.23±0.17 132.6± 14.55 143.2 ± 3.15 7.99 extract+dox 200 1.20±0.05 142.8± 10.56 153.0± 5.29 7.14 400 1.14±0.02 140.3± 7.36 151.6 ± 6.22 8.05 600 1.11±0.06 138.4 ± 8.54 149.6 ± 8.48 8.09 aqueous fraction 400 1.05±0.11 138.4 ± 6.26 144.6± 10.22 4.47 dcm fraction 400 1.17±0.09 134.3± 8.50 146.8± 13.20 9.30 data were expressed as mean ±sem. significant at dp<0.001 when compared to normal control; ap< 0.05, bp< 0.01, cp< 0.001 when compared to organotoxic control. (n = 6). table 2. effect of h.africana root extract and fractions on kidney function parameters of rats with doxorubicin-induced toxicity. treatment dose mg/kg urea (mmol/l) creatinine (µmol/l) chloride (mmol/l) potassium (mmol/l) sodium (mmol/l) bicarbonate (mmol/l) control 10 4.50± 0.30 94.5±5.86 45.75±1.93 3.47± 0.19 111.0± 5.11 22.00± 0.81 doxorubicin 1.66 10.50± 0.40c 171.6±2.02c 46.66±0.88 6.53± 0.08c 165.0± 1.73c 30.66± 0.66a crude extract 200 7.13±0.31e 143.0± 6.35a 57.0±2.51 4.13± 0.78f 125.0±16.37d 22.66± 1.66d 400 5.50±0.30f 112.3±11.34e 48.33±0.88 4.63± 0.28f 138.6± 6.76 25.0± 1.00 600 6.16±0.97f 125.6± 16.33d 48.66±1.20 3.40± 0.05f 107.3±1.45f 24.0± 2.00 aqueous fraction 400 4.80±0.50f 108.5±2.72f 49.50±1.70 4.17± 0.55b 118.7± 6.27b 22.33± 1.33d dcm fraction 400 5.00±0.40f 101.0±8.62f 44.0±1.15 3.10± 0.15f 104.6± 1.45f 25.66± 2.33 silymarin 100 3.60±0.05f 75.6±2.33f 44.60±1.45 3.13± 0.17f 105.0± 2.51f 22.33± 2.60d data is expressed as mean ± sem, significant at ap<0.05, bp<0.01, cp<0.001, when compared to control; significant at dp<0.05, ep<0.01, fp<0.001 compared to organotoxic group. (n=6). table 3. effect of h.africana root extract and fractions on kidney oxidative stress markers of rats with doxorubicin-induced toxicity. treatment dose mg/kg sod (u/ml) cat (u/g of protein) gpx (µg/ml) gsh (µg/ml) gst mda (µmol/ml) control 10 0.61±0.02 1.51±0.06 0.090±0.002 1.18±0.02 0.40±0.01 0.22±0.02 doxorubicin 1.66 0.17±0.01c 0.46±0.01a 0.040±0.00a 0.31±0.01b 0.22±0.01c 0.63±0.01c crude extract 200 0.30±0.01b 1.04± 0.15c 0.051±0.001 0.85± 0.15 0.25±0.01b 0.50±0.01b 400 0.39±0.01a,d 1.11±0.12c 0.067±0.001e 1.16±0.01d 0.28±0.01b 0.46±0.03b 600 0.51±0.02e 1.26± 0.05c,e 0.064±0.004 1.12± 0.08 0.34±0.01e 0.31±0.02f aqueous fraction 400 0.23±0.06c 1.15±0.02f 0.045±0.003a 1.02± 0.07 0.31±0.001d 0.39± 0.01e dcm fraction 400 0.48±0.04a,d 1.36±0.02c,d 0.069±0.002e 1.15± 0.14 0.33±0.01e 0.30± 0.01f silymarin 100 0.52±0.02f 1.15±0.06c 0.076±0.002e 1.13±0.01d 0.36±0.01f 0.34±0.02a,d data is expressed as mean ± sem, significant at ap<0.05, bp<0.01, cp<0.001, when compared to control; significant at dp<0.05, ep<0.01, fp<0.001 compared to organotoxic group. (n=6). figure a. photomicrograph of kidney section of rat treated with distilled water (10ml/kg) showing normal renal tubules (rt) and glomeruli (gm), no evidence of pathology seen. h&e stain, x400 magnification figure b. photomicrograph of kidney section of rat treated with doxorubicin (1.66mg/kg) showing focal non-specific inflammation (red arrowhead) with lower magnification, normal renal tubules (rt), and glomeruli (gm). there are few congested blood vessels (v). h&e stain x400 noah et al. – nephroprotective activities of ethanol root extract and fractions … 481 figure c. photomicrograph of kidney section of rat treated with 200 mg/kg of h. africana root extract and doxorubicin (1.66mg/kg) showing normal renal tubules (rt) and glomeruli (gm), no evidence of pathology seen. h&e stain, x400 magnification figure d. photomicrograph of kidney section of rat treated with 400 mg/kg of h. africana root extract and doxorubicin (1.66mg/kg) showing normal parenchymal with renal tubules (rt) and glomeruli (gm). no lesion seen. h&e stain, x400 magnification figure e. photomicrograph of kidney section of rat treated with 600 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg) showing distorted parenchyma with both normal (rt) and dilated renal tubules (). the dilated tubular epithelium appears flattened. there are also multifocal interstitial inflammatory infiltrates (red arrowhead). h&e stain, x400 magnification figure f. photomicrograph of kidney section of rat treated with 400 mg/kg of aqueous fraction of h. africana root and doxorubicin (1.66 mg/kg) showing normal parenchyma with renal tubules (rt), glomeruli (gm) and a focal congested blood vessel (v). no degenerative changes seen. h&e stain, x400 magnification figure g. photomicrograph of kidney section of rat treated with 400 mg/kg of dichloromethane fraction of h. africana root and doxorubicin (1.66mg/kg) showing normal renal tubules (rt) and glomeruli (gm), no evidence of pathology seen. h&e stain, x400 magnification figure h. photomicrograph of kidney section of rat treated with 100 mg/kg of silymarin and doxorubicin (1.66mg/kg) showing normal renal tubules (rt) and glomeruli (gm), no evidence of pathology seen. h&e stain, x400 magnification discussion this work was designed to investigate the effect of root extract and fractions of hippocratea africana on doxorubicin-induced kidney toxicity in rats in a bid to confirm the folkloric claim of its antidotal activity. doxorubicin is an anthracycline glycoside antibiotic that 482 biology, medicine, & natural product chemistry 12 (2), 2023: 477-484 possesses a potent and broad spectrum antitumour activity against a variety of human solid tumours and haematological malignancies (calabresi and chamber, 1990). however its use in chemotherapy has been limited largely due to its diverse toxicities, including cardiac, hepatic, hematological and testicular toxicity (yilmaz et al., 2006). the semiquinone form of doxorubicin is a toxic short-lived metabolite which interacts with molecular oxygen and initiates a cascade of reactions, producing reactive oxygen species (ros). ros generation, inflammatory processes and lipid peroxidation have been suggested to be responsible for doxorubicin-induced cardio-, hepatoand nephrotoxicity (injac et al., 2009; kalender et al., 2005). it has been proposed that dox-semiquinone, an unstable metabolite of dox, reacts with o2, producing h2o2 and o2 − (superoxide). in addition, dox enhances the activity of extramitochondrial oxidative enzymes such as xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate (nadph) oxidase and also interferes with mitochondrial iron export, resulting in formation of ros (reactive oxygen species) (bachur et al., 1979). in this study, doxorubicin administration was found to have caused elevation of serum urea, creatinine and electrolytes (k+, na+, cland hco3) levels when compared to normal control, which is an indication of a serious injury to the kidney. this finding is consistent with earlier report of rajasekaran (2019), which similar elevations were reported. it is well documented that kidney injury are indicated by increase in serum level of creatinine and urea (laskshmi and sudhakar, 2010) as well as increase serum levels of na, k, cl and bicarbonate (james and mitchel, 2006). however, these increases were reduced significantly by the coadministration of root extract and fractions of h. africana. doxorubicin is reported to cause nephrotoxicity via oxidative stress as free radicals formed caused tubular atrophy and increased glomerular capillary permeability. nephrotoxicity by doxorubicin can also result from lipid peroxidation and biological macromolecules damage by iron-dependent oxidative damage (mohan et al., 2010). degenerative changes in kidney depend on cumulative dose and duration of treatment as doxorubicin metabolites are partly excreted from the kidney. another mechanism for renal injury is the conversion of dox to semiquinone free radical by nadph-cytochrome p-450 which generates hydroxyl radical and superoxide anion which causes lipid peroxidation (rashid et al., 2013). the reduction of the levels of urea, creatinine and electrolytes by the root extract and fractions in this study is as a result of their free radical scavenging potentials, thereby protecting the kidney against oxidative stress by free radicals generated by doxorubicin. the antioxidative burst and antioxidant activities of the root extract and fractions of h. africana had previously been reported (okokon et al., 2013a; 2022; umoh et al., 2021). moreso, the antioxidative stress activities of the root extract and fractions observed in this study further support the antioxidant potentials of the plant. these activities may have contributed to the observed protective effects in this study. the findings of this study show that administration of doxorubicin (2.5 mg/kg, i.p) on alternate days for 14 days to rats caused significant decreases (p<0.05) in levels of enzymatic and non-enzymatic endogenous antioxidants (gsh,sod, cat, gpx and gsh) when compared to control, while the mda level was elevated, lipid peroxidation is a marker of oxidative stress and elevations in the amount of mda, a lipid peroxidation product, have been reported following doxorubicin treatment (rashid et al., 2013, rehman et al., 2014, khames et al., 2019). this trend was observed in this study. concomitant administration of root extract h.africana (200-600 mg/kg) with doxorubicin caused significant (p<0.05-0.001) non dosedependent elevation in the levels of the antioxidant enzymes (sod, cat, gpx) when compared to control. similarly, gsh level was significantly (p<0.001) elevated following treatment with the extract when compared to control. also, there were significant (p<0.05-0.01) reductions in the level of mda of the extract-treated rats. it has been documented that dox inhibits the activities of endogenous enzymatic and non-enzymatic antioxidants as was the case in this study. so, an imbalance between ros generation and neutralization leads to oxidative stress and injury to the kidney (abushouk et al., 2017; abdel-daim et al., 2017; aboushouk et al., 2019). the reduced mda level caused by the administration of the root extract and fractions may have resulted from reduction in lipid peroxidation and generation of free radicals which might have been scavenged by the phytoconstituents present in the root extract and fractions, revealing the antioxidative stress potentials of the root extract and hence the protective effect on the kidney as was observed. histological findings in this study revealed that kidneys of rats treated with doxorubicin (2.5 mg/kg) alone showed pathological signs of injury which were seen as degenerated microvesicles in the tubular lining cells among others. however, co-administration of h. africana root extract/fractions and doxorubicin reduced the toxic effects of the doxorubicin as normal glomeruli which were devoid of pathological signs were seen in the kidney sections of the extract-treated rats examined. this further confirms the nephroprotective potential of the root extract which may have been exerted through the antioxidant and antioxidative stress activities of its phytochemical constituents. conclusions the findings of this study show that the root extract and fractions of hippocratea africana have the potential to noah et al. – nephroprotective activities of ethanol root extract and fractions … 483 counteract the injurious effect of doxorubicin on the kidney. this activity can be attributed to the antioxidant and antioxidative stress activities of their phytochemical constituents. thus, the root extract can be used to alleviate and/or prevent doxorubicin-induced renotoxicity. acknowledgements: the technical support from mr nsikan malachy of pharmacology and toxicology department was tremendously acknowledged. authors’ contributions: kn, jau and jeo initiated and designed this study. jeo, kn and jau carried out the experiments and drafted the manuscript. noe and moa performed the statistical analysis, edited and reviewed the manuscript. kn, jau, moa, jeo and noe read and approved the final manuscript. competing interests: the authors declare that there are no competing interests. references abdel-daim mm, kilany oe, khalifa ha, ahmed aam. 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(2006) protective effect of lycopene on adriamycininduced nephrotoxicity and nephrotoxicity. toxicology 218: 164-171. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 7, number 1, 2018 | pages: 5-13 | doi: 10.14421/biomedich.2018.71.5-13 issn 2540-9328 (online) stability of t-dna integration in phalaenopsis “sogo vivien” transgenic orchid carrying 35s::gal4::atrkd4::gr endang semiarti1,*, exsyupransia mursyanti2, ahmad suyoko1, faiza senja widya perdana1, catharina tri widyastuti1, aditya nur subchan1 1faculty of biology, universitas gadjah mada, jl. teknika selatan, sekip utara, yogyakarta 55281, indonesia. 2faculty of technobiology, universitas atma jaya, jl. babarsari, yogyakarta, indonesia author correspondency*: endsemi@ugm.ac.id abstract orchid is an elegant ornamental plant and favoured by the society. phalaenopsis "sogo vivien" is a mini-sized orchid with an interesting white-striped purple petals. this study was aimed to analyze the stability of the integration of embryonic gene carrier t-dna from arabidobsis atrkd4 into the p. "sogo vivien" genome produced in 2016. the study was conducted in 3 stages: 1) transgenic plant phenotype analysis (1 year old); 2) examination of t-dna integration in orchid genotypes using pcr. 3) analysis of transgenic plant leaf explants’ ability to produce somatic embryo in vitro. in vitro cultures were performed on the base medium of new phalaenopsis (np), plus various concentrations of tdz (0, 1, 2 mg.l-1) and iba (0, 1, 2 mg.l-1) or without tdz and iba as controls. the transgenic phalaenopsis "sogo vivien" were transferred to pot mediums via ex vitro with two treatments: the first leaves were cut as explants for in vitro culture, and the plants were transferred to the mixture of fern medium with bark shavings. the integration of t-dna in the genome was detected by dna genome amplification from the second leaves using the atrkd4 gene primers and the poh1 gene. the results showed that the highest number of somatic embryo (se) propagules or protocorm like bodies (plbs) amounted to 27 were derived from transgenic plant # 2 cultured on np + 2 mg.l-1 tdz +1 mg.l-1 iba medium. the presence of atrkd4 transgenes were detected with the amplification of 380 bp of the rkd4 gene from the genome of transgenic plant # 2 by using pcr. there were 2 out of 15 plants that positively carry the atrkd4 gene and produce se. thus, the stability of the atrkd4 carrier t-dna integration in the genomes of transgenic plants was 13.3%. keywords: phalaenopsis “sogo vivien”; atrkd4; somatic embryo; stable transgenics introduction indonesia is home of tropical orchids, more than 5000 of 30,000 species of orchids in the world exist in indonesia. orchids are much favored by the society because of their beauty and attractive appearances. one of the popular ornamental orchids is phalaenospsis "sogo vivien" that has a beautiful, unique, and long lasting blooming period of about 3 months (mckinley, 2005). p. "sogo vivien" has minito medium-sized flowers with purple as the main colour of its petals. one uniqueness of this orchid is the genetic mutation that causes the colour of its leaves’ borders white, called as variegata. however, the percentage of mutation frequencies for the formation of this phenotype is only about 0.007% (fig. 1, mursyanti et al., 2016). orchid propagation by seed is naturally difficult because most of orchid seeds do not have endosperm, thus it requires symbiosis and have a low seed survival percentage. in vitro culture is the best solution for the cultivation of orchid plants. in vitro propagation can be performed using seed parts or other parts of plants such as leaves, roots and internodes of flower stems (vendrame et al., 2007). in a previous study, 17 t-1 plantlets from 2648 protocorms were produced through the formation of somatic embryos, which later developed into 413 t-2 transgenic plantlets (mursyanti et al., 2015). however, the phenotypes resulting from the genetic transformation are still variable and unstable because of the possibility of genetic segregation from crossover results. to see the stability of transgenes integration in p. "sogo vivien", a molecular analysis is required to see the stability of the gene. in this study, the object of our research is confirming the stability of 35s :: atrkd4 integrationin the transgenic plant genome originated from t-2 generation and its ability to induce somatic embryogenesis in vitro through the overexpression of atrkd4 gene in the transformant plants. figure 1. the morphology of phalaenopsis ‘sogo vivien’. (a) normal plant with green leaves; (b) mutated plant with white borders on the leaves. bar: 1 cm (mursyanti et al., 2016). https://doi.org/10.14421/biomedich.2018.71.5-13 6 biology, medicine, & natural product chemistry 7 (1), 2018: 5-13 mursyanti et al. (2015) has successfully produced transgenic plantlets that has been transfered with 35s:: atrkd4 gene. through the insertion of atrkd4 gene, it was expected to increase germination rate due to the rwp-rk motif causes the atrkd4 gene to work in the very early stage of embryogenesis (waki et al., 2011). regarding to the potential of the transformed plants, it is necessary to do propagation efforts to maintain the plant’s phenotypes. this research was conducted to acquire p. "sogo vivien" transgenic plants which carry 35s :: atrkd4 containingt-dna; to understand the growth of the transgenic p. "sogo vivien" and the nontransgenic plants, to obtain stable transgenic plants of phalaenopsis "sogo vivien" that carry 35s :: atrkd4 containingt-dna for induction of somatic embryogenesis; and to determine the most effective medium for induction of somatic embryogenesis using the leaf explants from phalaenopsis “sogo vivien” that carry the atrkd4 gene. the transgenic orchid plant p. "sogo vivien" can be used for the development of somatic embryos using the whole plant to meet the market demand. materials and methods plant materials and culture conditions the one year old transgenic plants p. "sogo vivien" carrying 35s :: gal4 :: atrkd4 :: gr that obtained from the previous research at the laboratory of biotechnology, faculty of biology ugm were used as a source of explants for in vitro induction of ses (plbs). plantlets were overplanted into a hormone-free np medium for refreshment. cultures were maintained under continuous white light, at 25-28° celcius. isolation of genomic dna genes and detection of tdna integration into the genomes of transgenic plants plant genomic dna was isolated using a method as described by semiarti et al. (2007), and the isolated dna genome of transgenic plants were purified using the dna kit dna mytaqtm hs following procedure in the instruction manual described by bioline (uk). based on the structure oft-dna (figure 2), detection of t-dna integration was performed by amplifying atrkd4 gene fragments with specific primers for atrkd4: atrkd4 f1 and atrkd4 r1, that a mplified 380 bp dna fragments. the primer sequences would be served upon request. in vitro induction of somatic embryos from leaf explants of transgenic plants the first leaves of the transgenic plants p. "sogo vivien" were cut into two parts in the middle, the distal and proximal parts of leaf were used as explants. the explants were introduced into np medium with addition of various concentration of thidiazuron (tdz) growth regulator (0, 1, 2 mg.l-1) and indole butyric acid (iba) (0, 1, 2 mg.l-1). cultures were maintained with continuous white light at 25-28ºc. the growth of explants were observed until the propagules were formed, which then developed into se or called as protocorm-like body (plb), the term of ‘protocorm’ is a special term for the development of spherical orchid embryos. the calculated se and the stability of t-dna integration in the orchid genome were indicated by the detection/amplification of the atrkd4 gene from the genomic dna of the transgenic plants, indicating stable plant genomes that carry 35s :: gal4 :: atrkd4 :: gr containing t-dna (figure 2). figure 2. the structure of t-dna carrying 35s :: gal4 :: atrkd4 :: gr (mursyanti et al, 2015). results and discussion results phenotypes of transgenic plants carrying 35s :: gal4 :: atrkd4 :: gr in this study, 15 transgenic plants lines of p. "sogo vivien" which carries 35s :: gal4 :: atrkd4 :: gr were obtained (figure 4 and table1). the phenotypes of the transgenic plants showed smaller size compared to the control of non-transgenic plants with normal plant size (figure 3), especially in leaf length, leaf width, and plant height. semiarti et al. – stable orchid transgenic carrying atrkd4 7 nt#1 nt#2 nt#3 nt#4 nt#5 nt#6 nt#7 nt#8 nt#9 nt#10 nt#11 figure 3. phenotypes of non-transgenic p.”sogo vivien” plants, 8 weeks after transplanted into the pots. bars: 1 cm. t#1 t#2 t#3 t#4 t#5 t#6 t#7 t#8 t#9 t#10 t#11 t#12 t#13 t#14 t#15 figure 4. phenotypes of transgenic p. “sogo vivien” plants that carry 35s::gal4::atrkd4::gr. t = transgenic plant lines # 1# 15; bars: 1cm. 8 biology, medicine, & natural product chemistry 7 (1), 2018: 5-13 table 1. the vegetative growth of transgenic p. “sogo vivien” carrying 35s::gal4::atrkd4::gr on 8 weeks after transplanted into pots. scale (cm) plant height first leaf length first leaf width the last leaf length the last leaf width non transformant 2.0±0.5 4.7±0.6 2.3±0.6 4.7±0.8 2.8±0.4 transformant 1.6±0.59 2.4±0.72 1.32±0.42 2.2±0.32 1.2±0.22 as shown in table 1, transgenic plants produced higher number of leaves and roots compare to that of non-transgenic/normal plants. the detection of t-dna integration into the orchid genome using pcr analysis showed that 380 bp of atrkd4 dna fragments were detected from the genomic dna of transgenic plant line # 1 and line # 2, thus it proved that the t-dna is stably integrated in the genomes of orchid transgenic (figure 6). table 2. detection of atrkd4 transgene in the genome of transgenic orchid. no. pcr detection somatic embryos of atrkd4 poh1 tdz:iba (2:1) nt#1 0 t#1 + 0 t#2 ++ 27 t#3 t#4 ++ notes: iba= indol butyric acid (ppm), tdz= thiadizuron (ppm), nt: non transgenic, t#1-t#4: transgenic plant lines#1line #4 table 3. percentage of living explants, somatic embryos and the colour change of in vitro transgenic t#2 orchid p. “sogo vivien” carrying atrkd4 gene in various media with the combination of tdz and iba, 4 weeks after plantation. treatment media ∑ explants observed ∑ and the percentage of living explants (%) ∑ and the percentage of explants forming somatic embryo candidates (%) ∑ explants changing colours green yellow brown np +t0i0 9 5 (55.6 %) 4 (44.4 %) 6 1 2 np+t0i1 9 5 (55.6 %) 1 (11.1 %) 5 1 3 np+t0i2 9 8 (88.9 %) 0 (0 %) 8 1 0 np+t1i0 9 0 (0 %) 0 (0 %) 0 9 0 np+t1i1 9 1 (11.1 %) 0 (0 %) 1 3 5 np+t1i2 9 1 (11.1 %) 0 (0 %) 1 2 6 np+t2i0 9 4 (44.4 %) 0 (0 %) 4 1 4 np+t2i1 9 8 (88.9 %) 6 (66.7 %) 8 0 1 np+t2i2 9 5 (55.6 %) 0 (0 %) 5 0 4 table 4. effect of growth regulator on the speed of somatic embryogenesis induction 4 weeks after explant plantation. plants time of somatic embryogenesis induction (day) media np+t0i0 np+t0i1 np+t0i2 np+t1i0 np+t1i1 np+t1i2 np+t2i0 np+t2i1 np+t2i1 t#1 7 21 0 0 0 0 0 14 0 t#2 7 0 0 0 0 0 0 3.5 0 nt1 0 0 0 0 0 0 0 0 0 notes: np= new phalaenopsis medium, t= thidiazuron, i = indol butyric acid nt: non transfromant, t#1,t#2: transfromants semiarti et al. – stable orchid transgenic carrying atrkd4 9 table 5. propagule formation on the leaf explants from non transgenic and transgenic p. “sogo vivien”, 4 weeks after explant plantation on media containing tdz and iba. average number of propagule formation on explants iba tdz 0 ppm 1 ppm 2 ppm numbers morphology numbers morphology numbers morphology 0 ppm nt1 0 0 0 t1 20.5 0 0 t2 2 0 0 1 ppm nt1 0 0 3.4 t1 3 0 11 t2 0 0 27 2 ppm nt1 0 0 0 t1 0 0 0 t2 0 0 0 notes: iba= indole butyric acid (ppm), tdz= thidiazuron (ppm), nt: non transfromant, t1, t2: transformant, bars= 5 mm. 10 biology, medicine, & natural product chemistry 7 (1), 2018: 5-13 figure 5. the process of somatic embryogenesis in the surface of leaf explants from transgenic orchid p. "sogo vivien" carrying t-dna 35s :: gal4 :: atrkd4 :: gr. (a) the formation of somatic pre-embryo/ plb (arrow) on the part of the cuts 1 week after culturing on np 0. (b) formation of somatic pre-embryo (arrow) 4 weeks after culturing on np 0. (c, d) formation of somatic pre-embryo (arrow) 1 week after culturing on tdz 2 ppm and iba 1 ppm. (e) formation of somatic pre-embryo (arrow) 4 weeks after culturing on tdz medium 2 ppm and iba 1 ppm, the colour turned into yellowish green. (bars: 1 mm). m nt#1 nt#2 nt#3 t#1 t#2 t#3 t#4 figure 6. electrophoregraph of genomic dna (whole genome dna) from the leaf of phalaenopsis “sogo vivien”. non transformants(nt#1, nt#2, nt#3) and transformant with t-dna containing 35s::atrkd4 (t#1, t#2, t#3, t#4) and λ dna/hindiii as dna markers (m). . figure 7. electrophoregraph of amplified dna by using pcr of genome dna of p. “sogo vivien”, both non-transformantand transformant carrying 35s::gal4::atrkd4::gr. the amplified dna from non transformant plants (nt#1, nt#2, nt#3) and trasnformant plants (t#1, t#2, t#3, t#4); marker (m): 100 bp dna ladder; amplification of genome dna using poh1 primer produced 300 bp sized band; degrkd4 primer produced 380 bp sized band of dna fragments. 23130 bp 9416 bp 4361 bp 6557 bp 2322 bp 2027 bp semiarti et al. – stable orchid transgenic carrying atrkd4 11 discussion the characteristics of vegetative growth in plant that are inserted by foreign genes (transgenic) and normal/nontransgenic plants may be the same or different depending on the expression level of the inserted gene. in most transgenic plants, the vegetative growth character is similar to that of normal plants. more than 100 apple cultivars of the royal gala carrying the uida, als, and nptiii genes showed no difference at the morphological level, growth type, or response to environmental factors compared to non-transgenic plants (bajaj, 2012: 242). in transgenic soybean plants carrying the atpg7 gene, plant height is the same or more than non-transgenic plants (kim et al., 2017: 237-242). however, this depends on the character of the genes inserted. for example, the broccoli plant (brassica oleracea subsp. italica) that carries the heat-tolerant gene, athsp101 is slightly shorter than nontransgenic plants as the effect of position (ravanfar et al., 2013: 14). the gene insertion site has an effect on gene expression when its location on the chromosome changes, and can cause silencing genes as well as dna regulation. the atrkd4 gene is a gene that regulates gamet cell differentiation processes (koi et al., 2016: 1775-1781) and can induce somatic embryogenesis in p. "sogo vivien" (mursyanti et al., 2015: 26-37). this study was aimed to study the vegetative growth character of p. "sogo vivien" that carries atrkd4 gene compared to normal orchid plants. in general, there was no difference in the number of roots. plant height differed from first week to seventh week but itswere no different at the eight week. this indicated that the growth of transgenic plant stems and normal plants become stable from the eighth week. the length of the basal leaf did not differ in the first week and the second week but were different until the eighth week. the same phenomenon happened on the basal leaf width. it suggests that there is a slightly difference of growth in basal leaves between transgenic and normal plants. the length of terminal leaf did not differ from the first week to the third week, but it began to be differed after the eighth week. the width of the terminal leaf did not differ in the first and second weeks and began to be differed from the third week to the eighth week. the growth difference between the terminal leaf of transgenic and normal plantsstarted from the third week. vegetative growth of transformantplants was slower than that of non-transformant plants that proved by anova test for some growth parameters including plant height, basal leaf length and width, and terminal leaf length and width. based on the anova test, differences in the length and width of the first leaf and the length and width of the last leaf were detected. the observation results on the number and percentage of live explants (table 2) showed that, from the whole treatments, only the tdz:iba (1: 0) media was not fit for survival, whereby all of the leaves had chlorosis. the different life-force responses of the explants may be due to the adaptability of the nonuniform explants and the physiological conditions of the explant source. during four weeks of planting, the explants changed colour to yellowish brown. table 2 showed yellowish discoloration in all treatment mediums except tdz: iba (2: 1) and tdz: iba (2: 2), whereas for brown colour change occured in all treatment mediums except tdz: iba 0: 2) and tdz: iba (1: 0). change of explants colour into yellow was caused by the occurrence of chlorosis. the phenomenon of chlorosis is resulted from the decreasing chlorophyll content in the explant. the decrease of chlorophyll content on media may be caused by the relativily low explants’ ability to utilize iron micronutients. in a previous study, during in vitro culture of leaf explants, the chlorophyll content value was linear with iron concentration in culture medium (sivanesan et al., 2008: 4482-4490). some explants in some media changed its colour into brown. this indicated the occurence of browning symptoms of leaf explants. the process of browning might due to the oxidation of polyphenol compounds and the formation of quinon compounds. both of these compounds are inhibited the growth of leaf tissue explants (sukumar et al., 2008: 361). the emergence of the oxidation process is activated by oxidase enzyme after injury, the enzyme may leave the cell and possibly create bonds between hydrogen and proteins, increasing the activity of phenylalanine ammonia lyase, an enzyme that promotes the production of phenylpropanoid, resulting in the brownish color of the leaves (hutami, 2008: 8388). of the total media with variation of hormones/growth regulators, there were 3 media treatments that successfully performed somatic embryogenesis induction (table 2.). in induction media, 4 explants (np 0), 1 explant (tdz: iba; 0: 1) and 6 explants (tdz: iba; 2: 1) were able to form candidates of somatic embryo. compared with hormon-free media (44.4%), media with a combination of tdz: iba (2:1) resulted in a larger percentage (66.7%). these results support the results of previous studies, which suggested that the combination of exogenous auxin and cytokinin hormones can effectively induce direct somatic embryogenesis on phalaenopsis orchid leaf explants (feng and chen, 2014: 4). the velocity induction of somatic embryogenesis processes varies (tables 3 and 4.). transgenic explants were successfully induced whereas non-transgenic explants did not undergo somatic embryogenesis formation. transgenic plant t#2 took 7 days in np 0medium, and 3.5 days in tdz: iba (2:1) containing medium to form somatic embryos. meanwhile, t#1 plant took 7 days in np 0medium, 21 days in tdz: iba (0: 1) containing medium and 14 days in tdz; iba (2: 1) containing medium. the shortest time for se induction occured on the transgenic planlet leaf explant in medium with tdz: iba (2: 1), which required a mean time of 3.5 days, twice faster than the 12 biology, medicine, & natural product chemistry 7 (1), 2018: 5-13 se induction in the control medium. the induction velocity was relatively faster compared to previous studies (kasi and semiarti, 2016: 31-40) using a wildtype p. "sogo vivien" which took 8 weeks after inoculation to form somatic embryos. table 5 showed that the results of somatic embryogenesis induction can be known directly through the observation of average propagule appearance on leaf explants, at 4 weeks after planting. the propagules produced from this induction process will develop into protocorm-like bodies (plb), an orchid embryo derived from somatic cells, forming a swollen tuber, which then potentially form in vitro shoots (kumar et al., 2008: 718). the propagules appeared in transgenic leaf explants t#1 and t#2, but not observed in nontransformantleaf explants. t#1 transgenic leaf explants produced a mean of 20.5 propagules in np 0 medium, 3 pieces in tdz : iba (0: 1) medium, whereas the explants of t#2 transgenic leaves produced 2 propagules in np 0 medium and 27 propagules in tdz: iba (2: 1) medium. the results of this study showed that the transgenic leaf that carries atrkd4 with a 35s promoter was able to undergo somatic embryogenesis process directly on the medium without hormon and produce more embryos on tdz: iba (2:1) medium. the tdz growth regulator requirement is relatively lower, which is ≤2 ppm, compared to a previous study (kasi and semiarti, 2016: 31-40) where wildtype p. "sogo vivien" required tdz concentration of 10 ppm for induction of somatic embryogenesis from leaves in vitro. the results of in vitro inoculation of leaf explants of transgenic plants showed faster in induction speed of somatic embryogenesis (table 4), with lower tdz concentration requirement (table 3, table 4) and higher capability to form propagules in np hormon-free medium table 3, figure 5). this is in line with our previous research which showed that high activity of atrkd gene can induce somatic embryogenesis process on leaf explants (mursyanti et al., 2016: 45-53). based on all the results obtained, it was shown that the media with a combination of tdz: iba produced the highest number of propagules. these results suggest that the presence of exogenous hormones may support somatic embryogenesis induction in orchid. molecular analysis showed that the isolated dna fragments from both non-transformant and transformant plants (figure 6) indicated good quality of genomic dna with no smear appearance in the electrophoresis gel. the genomic dna amplification results with atrkd4 specific primers and degrkd4 degenerate primers indicated that the atrkd4 transgene has been integrated into genomes of p. "sogo vivien" plant and the stably integrated in the orchid plant genome. figure 7 showed that a 300 bp dna fragment was amplified from t#1 genome using specific primers of poh1 gene and 380 bp dna fragment were amplified from t#2 and t#4 genomes using primers of atrkd4 gene. its indicates that the atrkd4 transgene has only stably integrated in transgenic plant t#2 and t#4, finally, we got the two stable transgenic lines t#2 and t#4, which are being acclimatize in the greenhouse and maintain for the next generation. conclusion integration of the t-dna carrying 35s::gal4::atrkd4::gr in the genome of transgenic plant phalaenopsis "sogo vivien" can be detected in 2 of 15 strains, thus it means the stability of t-dna integration is 13.3%. the morphology of transgenic plants did not change significantly in root quantities compared with non-transformant plants, but experienced significant differences in plant height, length, basal and terminal leaf width and length compared with nontransformant plants. the development of somatic preembryogenesis occurred after 4 weeks of culture characterized by the appearance of propagules on the surface of plant leaf explants. acknowledgements this research was funded by a research grant hibah penelitian biodiversitas tropika dosen untuk pengembangan materi pembelajaran bpptnbh from the faculty of biology, universitas gadjah mada on 2017 with contract number: ugm/bi/1734/um/05/01. references bajaj, y. p. s. 2012. biotechnology in agriculture and forestry 47: transgenic crops ii. springer. new delhi. p. 242. feng j and cheng j.2014. ‘a novel in vitro protocol for inducing direct somatic embryogenesis in phalaenopsis aphrodite without taking explants’. the scientific world journal. vol. 2014, article id 263642, 7 pages, 2014. doi:10.1155/2014/263642. hutami, s.2008. ‘masalah pencoklatan pada kultur jaringan.jurnal agrobiogen. 4(2).83-88 kasi, p. d. dan semiarti e.2008.’pengaruh thiadizuron dan naphtalene acetic acid untuk induksi embriogenesis somatik dari daun anggrek phalaenopsis “sogo vivien”. jurnal dinamika. 7(1):31-40. kim, h. j. cho, h. s. pak, j. h. kim, k. j. lee, d. h. and chung, y. s. 2017. overexpression of a chromatin architecturecontrolling atpg7 has positive effect on yield components in transgenic soybean. plant breed. biotech. 5(3):237-242. kumar a, neumann k and sopory s k. 2008. recent advances in plant biotechnology and its applications.i.k. international. new delhi. p. 718. koi, s. hisanaga, t. sato, k. shimamura, m. yamato, k. t. ishizaki, k. kohchi, y. and nakajima, k. 2016. an evolutionary conserved plant rkd factor controls germ cell differentiation. curr. biol. 26(13): 1775-1781. semiarti et al. – stable orchid transgenic carrying atrkd4 13 mckinley, m. 2005. complete guide to orchids: ortho books. american orchid society. usa. p.137. mursyanti, e., aziz-purwantoro, s. moeljopawiro and e. semiarti. 2015. induction of somatic embryogenesis through overexpression of atrkd4 genes in phalaenopsis ‘sogo vivien’. indonesian journal of biotechnology. 20(1): 26-37. mursyanti, e., aziz-purwantoro, s. moeljopawiro and e. semiarti. 2016. micropropagation of mini orchid hybrid phalaenopsis ‘sogo vivien’, journal of tropical biodiversity and biotechnology. 1(1): 45-53. ravanfar, s. a. aziz, m. a. shabanimofrad, m. and samarfard, s. 2013. greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (issr) markers. electronic journal of biotechnology. 16(5): 1-4. semiarti e., indrianto a., purwantoro a., isminingsih s., suseno, n., ishikawa t., yoshioka y., machida y.,and machida c. 2007. agrobacterium-mediated transformation of the wild orchid species phlaenopsis amabilis. plant biotechnology 24: 265-272. sivanesan i, hwang sj and jeong b r.2008. ‘influence of plant growth regulators on axillary shoot multiplication and iron source on growth of scrophularia takesimensis nakaia rare enemic medicinal plant’.african journal of biotechnology. 7(24):4484-4490. sukumar e, essa m m and manickavasagan a. 2012. dates: production, processing, food and medicinal values. crc press. new york. p. 361. vendrame, w. a. maguire, i. and carvalho, v. s. 2007. in vitro propagation and plantlet regeneration from doritaenopsis purple gem ‘ching hua’ flower explants. hort. science. 42:1256-1258. waki, t., t. hiki, r. watanabe, t. hashimoto & k. nakajima. 2011. the arabidopsis rwp-rk protein rkd4 triggers gene expression and pattern formation in early embryogenesis. current biology. 21(15): 1277-1281. this page intentionally left blank stability of t-dna integration in phalaenopsis “sogo vivien” transgenic orchid carrying 35s::gal4::atrkd4::gr biology, medicine, & natural product chemistry 7(1) 2018 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 6, number 2, 2017 | pages: 47-51 | doi: 10.14421/biomedich.2017.62.47-51 issn 2540-9328 (online) clustering of 18 local black rice base on total anthocyanin kristamtini1, endang wisnu wiranti2 1,2assessment institute for agricultural technology (aiat) yogyakarta jln. stadion maguwoharjo no. 22, wedomartani, ngemplak, sleman, yogyakarta, indonesia author correspondency: krisniur@yahoo.co.id1 abstract black rice has a high anthocyanin content in the pericarp layer, which provides a dark purple color. anthocyanin serve as an antioxidant that control cholesterol level in the blood, prevent anemia, potentially improve the body's resistance to disease, improve damage to liver cells (hepatitis and chirrosis), prevent impaired kidney function, prevent cancer/tumors, slows down antiaging, and prevent atherosclerosis and cardiovascular disease. exploration results at aiat yogyakarta, indonesia from 2011 to 2014 obtained 18 cultivar of local black rice indonesia. the names of the rice are related to the color (black, red or purple) formed by anthocyanin deposits in the pericarp layer, seed coat or aleuron. the objective of the study was to classify several types of local black rice from explorations based on the total anthocyanin content. the study was conducted by clustering analyzing the total anthocyanin content of 18 local black rice cultivars in indonesia. cluster analysis of total anthocyanin content were done using sas ver. 9.2. clustering dendogram shows that there were 4 groups of black rice cultivars based on the total anthocyanin content. group i consists of melik black rice, patalan black rice, yunianto black rice, muharjo black rice, ngatijo black rice, short life of tugiyo black rice, andel hitam 1, jlitheng, and sragen black rice. group ii consists of pari ireng, magelang black hairy rice, banjarnegara-wonosobo black rice, and banjarnegara black rice. group iii consists of ntt black rice, magelang non hairy black rice, sembada hitam, and longevity tugiyo black rice. group iv consist only one type of black rice namely cempo ireng. the grouping result indicate the existence of duplicate names among the black rice namely patalan with yunianto black rice, and short life tugiyo with andel hitam 1 black rice. keywords: clustering; black rice; exploration; total anthocyanin. introduction black rice is a type of rice, along with white rice and red rice. black rice is not consumed as a staple food, but consumed as functional food by some communities. functional food is a food which naturally or through a certain process contains one or more compounds considered to have physiological functions that are beneficial to health (wijayanti, 2004). black rice has a high anthocyanin content in the pericarp layer, which provides a dark purple color (ryu et al., 1998; takashi et al., 2001). black rice is one of the functional foods because of its anthocyanin content. anthocyanin pigments effectively reduce cholesterol levels in the human body (lee et al., 2008). anthocyanin has been recognized as functional health foodstuff due to its antioxidant activity (satue-gracia et al., 1997; nam et al., 2006; philpot et al., 2006). anthocyanin serve as an antioxidant that cleans blood cholesterol, prevent anemia, potentially increase body resistance to disease, improve liver cell damage (hepatitis and chirrosis), prevent impaired kidney function, prevents cancer/tumors, slows down antiaging (harmanto, 2008), as well as to prevent narrowing of the arteries (atherosclerosis) and cardiovascular disease (ling et al., 2001 and ling et al., 2002). indonesia is a megabiodiversity country. black rice is one of the wealth of indonesia genetic resources that need to be conserved. therefore, the explorations of black rice in the region of yogyakarta and other regions in indonesia are important. eighteen black rice cultivars were collected by aiat yogyakarta, indonesia. the name of the black rice was different, some even have no name, just given the name according to the farmers who cultivate it. the difference in the name of black rice is allegedly caused by the diversity of the color of the rice, from bright black to black. the color difference of rice occurs as a result of differences in anthocyanin content. characterization of morphological properties have been done, while the study of the diversity of anthocyanin content in some black rice has not been done. solouki et al., (2008) suggests that the study of genetic diversity can be done with morphological, biochemical, and molecular markers. biochemical studies of anthocyanin content in some black rice complement the morphological characterization, and was expected to help plant breeders to choose cultivars as parent in plant breeding programs. based on these considerations, this study aimed to classify the 18 black rice cultivars from indonesia based on the total anthocyanin content. http://dx.doi.org/10.14421/biomedich.2017.62.47-51 mailto:krisniur@yahoo.co.id 48 biology, medicine, & natural product chemistry 6 (2), 2017: 47-51 materials and methods the research began by planting 18 black rice cultivars from exploration (table 1), and maintained until harvest. the black rice from the harvest were analyzed for the total anthocyanin content in the agricultural production technology laboratory of gadjah mada university yogyakarta according to lees & francis method (1972) (appendix 1). clustering analysis of total anthocyanin were done using sas ver software. 9.2. cluster method was analyzed using hierarchical cluster analysis to know the kinship relationship. hierarchical cluster analysis was a common way of grouping objects in groups that share resemblance to each other. table 1. eighteen black rice cultivars were collected by aiat yogyakarta, indonesia. black rice cultivar code origin 1. melik a bantul, yogyakarta, indonesia 2. jlitheng b sleman, yogyakarta, indonesia 3. cempo ireng c sleman, yogyakarta, indonesia 4. pari ireng d sleman, yogyakarta, indonesia 5. beras hitam ntt e ntt, indonesia 6. beras hitam bantul o bantul, yogyakarta, indonesia 7. beras hitam magelang berbulu r magelang, jawa tengah, indonesia 8. beras hitam magelang tak berbulu s magelang, jawa tengah, indonesia 9. beras hitam sragen t sragen, jawa tengah, indonesia 10. beras hitam banjarnegara berbatasan wonosobo w banjarnegara, jawa tengah, indonesia 11. beras hitam banjarnegara y banjarnegara, jawa tengah, indonesia 12. beras hitam tugiyo umur panjang aa bantul, yogyakarta, indonesia 13. sembada hitam ab sleman, yogyakarta, indonesia 14. beras hitam muharjo ac bantul, yogyakarta, indonesia 15. beras hitam patalan ad bantul, yogyakarta, indonesia 16. beras hitam tugiyo umur pendek ae bantul, yogyakarta, indonesia 17. andel hitam 1 af kulon progo, yogyakarta, indonesia 18. beras hitam yunianto ag bantul, yogyakarta, indonesia results and discussion black rice was one of functional foods due to anthocyanin content that expressed in rice color. black rice is one of anthocyanin sources. other anthocyanin source are fruits, vegetables, and grains that have given color pigments. healthy foods usually have a black or purple color, but in terms of appearance, this color less appealing. most plants have the largest anthocyanin content in fruit (houghton & hendry 1995). some plants, such as tea, cocoa, cereals, beans, red cabbage, and petunias have anthocyanins in plant parts other than fruits (lila 2004; sterling 2011). anthocyanins also found in foods that have red, purple to black pigments such as black soybeans, black sesame, blackberry, and grapes. foods consumed daily are often sources of free radicals that harmful to the body. free radicals can accelerate the aging process and increase the risk of diseases such as heart disease, stroke, diabetes, arthritis, and cancer. body damage due to free radical attack can be prevented by antioxidants. free radicals are atoms or molecules that are very active and unstable because the structure of an atom or its molecule has an unpaired electron. in order to be stable, these atoms or molecules need to get another electron by taking an electron from another molecule. anthocyanin can serve as an antioxidant, a compound capable of donating its electrons to neutralize the damaging properties of free radicals (bustanul 2007). based on these matters then conducted analysis of total anthocyanin content on 18 local black rice collection of aiat of yogyakarta. the total anthocyanin content of 18 black rice cultivars is presented in table 2. table 2 shows that each black rice cultivar contains anthocyanin content varying from 53.22 to 428.38 mg/ 100 g, according to its genetic potential. the total anthocyanin content is expressed as the color of rice, and the color of rice from each rice cultivar is presented in figure 1. kristamtini & wiranti – clustering of 18 local black rice base on total anthocyanin 49 table 2. total anthocyanin content in 18 black rice cultivars. black rice cultivar code total anthocyanin (mg/100 g) 1. melik a 100.06 2. jlitheng b 53.22 3. cempo ireng c 428.38 4. pari ireng d 230.48 5. beras hitam ntt e 264.43 6. beras hitam bantul o 90.22 7. beras hitam magelang berbulu r 196.34 8. beras hitam magelang tak berbulu s 288.53 9. beras hitam sragen t 65.04 10. beras hitam banjarnegara berbatasan wonosobo w 179.09 11. beras hitam banjarnegara y 165.78 12. beras hitam tugiyo umur panjang aa 298.93 13. sembada hitam ab 284.05 14. beras hitam muharjo ac 122.45 15. beras hitam patalan ad 107.22 16. beras hitam tugiyo umur pendek ae 81.81 17. andel hitam 1 af 82.55 18. beras hitam yunianto ag 107.39 figure 1. appearance of rice color from 18 black rice cultivars. (a). melik; (b). jlitheng; (c). cempo ireng; (d). pari ireng; (e). beras hitam ntt; (f). beras hitam bantul; (g). beras hitam magelang berbulu; (h). beras hitam magelang tak berbulu; (i). beras hitam sragen; (j). beras hitam banjarnegara berbatasan wonosobo; (k). beras hitam banjarnegara; (l). beras hitam tugiyo umur panjang; (m). sembada hitam; (n). beras hitam muharjo; (o). beras hitam patalan; (p). beras hitam tugiyo umur pendek; (q). andel hitam 1; (r). beras hitam yunianto. black rice has a high anthocyanin content in the pericarp layer, which provides a dark purple color. the color difference of rice is regulated genetically. according to chang & bardenas (1965), the color of rice is determined by the pigments in the pericarp and the tegmen, namely white, bright red, red, reddish brown, brown, grayish, gold, purple, and purple (almost black), while endosperm from all rice varieties was white. ayoola & faluyi (2007), said that pericarp rice seeds are usually white but may be red, brown or almost purple, pigmentations/colorations are no deeper than pericarp and endosperm was always white. clustering of black rice based on total anthocyanin content in 18 black rice cultivars in the form of dendogram in figure 2. 50 biology, medicine, & natural product chemistry 6 (2), 2017: 47-51 information: melik : a beras hitam banjarnegara berbatasan wonosobo : w jlitheng : b beras hitam banjarnegara : y cempo ireng : c beras hitam tugiyo umur panjang : aa pari ireng : d sembada hitam : ab beras hitam ntt : e beras hitam muharjo : ac beras hitam bantul : o beras hitam patalan : ad beras hitam magelang berbulu : r beras hitam tugiyo umur pendek : ae beras hitam magelang tak berbulu : s andel hitam 1 : af beras hitam sragen : t beras hitam yunianto : ag figure 2. dendogram of clustering of 18 black rice cultivars based on the total anthocyanin content. dendogram in figure 2 shows that 18 black rice cultivars based on their total anthocyanin content were divided into 3 groups. group i consists of a (melik), ad (beras hitam patalan), ag (beras hitam yunianto), ac (beras hitam muharjo), o (beras hitam bantul), ae (beras hitam tugiyo umur pendek), af (andel hitam 1), b (jlitheng), and t (beras hitam sragen). group ii consists of d (pari ireng), r (beras hitam magelang berbulu), w (beras hitam banjarnegara berbatasan wonosobo), y (beras hitam banjarnegara), e (beras hitam ntt), s (beras hitam magelang tak berbulu ab (sembada hitam) and aa (beras hitam tugiyo umur panjang). group iii consists of only cultivar c = cempo ireng, because cempo ireng has the highest anthocyanin content of 428.38 mg / 100 g. it appears in figure 2 that, in group 1, there are 2 cultivars that have the same genetic distance as the total antioxidant content, that was ad (beras hitam patalan) and ag (beras hitam yunianto). two other cultivars in group i also have the same genetic distance, thas was ae (beras hitam tugiyo umur pendek) and af (andel hitam 1). the cultivars are allegedly the same or there was duplication of names based on their total anthocyanin content. the result of clustering based on the total anthocyanin content still needs to be supported by clustering based on plant morphology, such as plant height, plant age, stem color, leaf color, leaf tongue color, leaf ear color and so forth. conclusion the total anthocyanin content in 18 black rice cultivars of aiat of yogyakarta collection varies according to its genetic potential. there are 3 groups of black rice based on the total anthocyanin content, some cultivars have the same genetic distance (close kinship relationship) and there were suspected duplication of the name based on the total anthocyanin content. name of observation or cluster c aa ab s e y w r d t b af ae o ac ag ad a average distance between clusters 0 25 50 75 100 125 150 175 200 225 250 275 300 kristamtini & wiranti – clustering of 18 local black rice base on total anthocyanin 51 suggestion required clustering of black rice cultivars based on morphological properties to support of clustering based on the total anthocyanin content. references ayoola, a.o. and j.o. faluyi. 2007. inheritance of caryopsis/ ripened-hull colour in a selection from land races of rice oryza sativa (linn.). agric. j. 2 (3): 412-414. bustanul. 2007. manfaat beras hitam. http://bumiganesa.com. harian metro online friday, 08 june 2007. retrieved at 28 april 2011. chang, te-tzu and e.a. bardenas. 1965. the morphology and varietas characteristics of the rice plant. technical bulletin 4 december, 1965. irri.los baños, laguna, the philippines. 27 31. harmanto, a. 2008. varietas beras organik berdasarkan warna. http://aghribisnis-ganesha.com. p. 146. retrieved at 26 september 2008. houghton, j.d. and g.a.f. hendry. 1995. natural food colorants. springer. pp. 53-59. lee, j.c., j.d. kim, f.h. hsieh, and j.b. eun. 2008. production of black rice cake using ground black rice and medium-grain brown rice. int'l. j. food sci. technol. 43 (6): 1078-1082 lila, m.a. 2004. anthocyanins and human health: an in vitro investigative approach. j. biomed. biotechnol. 5: 306–313. doi: 10.1155/s111072430440401x. http://www.ncbi.nlm. nih.gov/pmc/articles/pmc1082894/. ling, w. h., cheng, q. x., ma, j. and wang, t. 2001. red and black rice decrease artherosclerotic plaque formation and increase antioxidant status in rabbits. j. nutr. 131: 1421 1426. ling, w.h., l.l. wang and j. ma. 2002. supplementation of black rice outer layer fraction to rabbits decreases the atherosclerotic plaque formation and increases antioxidant status. j. nutr. 132: 20 26. nam, s.h., s.p. choi, m.y. kang, h.j. koh, n. kozukue, and m. friedman. 2006. antioxidative activities of bran from twenty one pigmented rice cultivars. food chem. 94: 613-620. philpot, m., k.s. gould, c. lim, and l.r. ferguson. 2006. in situ and in vitro antioxidant activity of sweet potato anthocyanins. j. agric. food chem. 54: 1710-1715. ryu, s.n., s.z. park, and c.t. ho. 1998. high performances liquid chromatographic determination of anthocyanin pigments in some varieties of black rice. j. food drug analysis 6: 1710-1715. satue-gracia, m., i.m. heinonen, and e.n. frankel. 1997. anthocyanins as antioxidants on human low-density lipoprotein and lechthin-liposome system. j. agric. food chem. 45: 3362–3367. solouki, m., h., mehdikhani, h. zeinali & a.a. emamjomeh, 2008. study of genetik diversity in chamomile (matricaria chamomilla) based on morphological traits and molecular markers. sci. hortic. sterling, r.d.m. 2011. anthocyanins. the chiropractic resource organization, 19 march 2011. takashi, i., x. bing, y. yoichi, n. masaharu and k. tetsuya. 2001. antioxidant activity of anthocyanin extract from purple black rice. j. med. food. 4: 211218. wijayanti, e. 2004. potensi dan prospek pangan fungsional indigenous indonesia. presented at seminar nasional pangan fungsional indigenous indonesia: potensi, regulasi, keamanan, efikasi dan peluang pasar. bandung, 6-7 october 2004. http://bumiganesa.com/ http://www.ncbi.nlm/ this page intentionally left blank clustering of 18 local black rice base on total anthocyanin biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 335-341 | doi: 10.14421/biomedich.2023.121.335-341 issn 2540-9328 (online) el hadji gorgui diouf1,*, doudou diop2, abdoulaye gueye1, mbaye diouf1, talibouya ndior1, mamadou latyr ndour1, adama faye1, mamadou kébé1 1laboratory of natural products, department of chemistry, faculty of science and technology; cheikh anta diop university of dakar, senegal. 2botany laboratory, fundamental institute of black africa, cheikh anta diop university of dakar, senegal. corresponding author* goorgui82@yahoo.fr manuscript received: 22 march, 2023. revision accepted: 28 march, 2023. published: 02 april, 2023. abstract crop losses in vegetable crops due to nematodes are a concern in a country like senegal where food demand is increasingly high. the use of chemical pesticides has made great progress in increasing harvests in senegal. but these pesticides have negative effects on the environment and on human health. crops are often contaminated as well as groundwater. the use of natural products to remedy this phenomenon is very important, especially in areas such as the niayes zone where the climate and soil are very favorable for market gardening and where water contamination is very sensitive. the aim of our study is to identify the plants used to control nematodes in the niayes area of thiès. to this end, we conducted an ethnobotanical survey of 100 people of different ages and sexes in this area. the results showed 17 plant species used to control nematodes in 12 families. among these species, the most cited were azadirachta indica a. juss. juss, calotropis procera (ait.) f. and cassia occidentalis l. leaves (71%) are mostly used. the sample is often dried and ground (45%) or fresh (case of hydrodistillation) (53%) before preparation. water (92%) is the most used solvent for extraction. maceration (85%) and infusion are the most common preparation methods. the application of these preparations by systemic action (94%) is more adopted than fumigation. local people find these preparations rather effective (78%), which is very encouraging. the present study constitutes a database for further studies in the field of senegalese pharmacopeia. in addition, scientific research on bionematicidal substances may be conducted in the future to evaluate the effectiveness of these plants identified in the niayes area of thiès for the protection of vegetable crops against nematodes. keywords: ethnobotanical survey; senegalese pharmacopoeia; nematicidal plants; market gardening; niayes area; niayes area of thiès. abbreviations: ucad: université cheikh anta diop de dakar; ifan: institut fondamental d'afrique noire. introduction agriculture is a major industry that contributes to socioeconomic development and employs nearly 40% of the world's working population (momagri, 2016). in west africa, agriculture contributes about 35% of the countries’ gross domestic product (gdp). it remains the main provider of employment (60% of the active population) but also contributes to more than 80% of the population's food needs (blein r. et al, 2008). the economic development of these countries can only be based on agriculture. in senegal, agriculture in the niayes area plays an important role in supplying the population with agricultural products. it provides more than 80% of the national vegetable production (dps, 2003). very fertile and favorable for market gardening, the niayes zone is characterized by a shallow groundwater table (0.5 to 5 meters deep) and is made up of dunes and depressions (ansd, 2015). the niayes soils are of the tropical ferruginous type, weakly leached on sand (dior), with a sandy texture and 90 to 95% total sand. they have very low levels of carbon (0.2% on average), total nitrogen (0.15 to 1%), and exchangeable bases (1.77 meq/100 g). the ph is slightly acidic at 5.5 to 6.5 (adejumo, 2013). despite all the advantages of this soil, yields are below expectations because the crops are often attacked by predators that cause huge crop losses. nematodes are the main pests of vegetable crops. they attack crops such as aubergines, tomatoes, cabbage, potatoes, etc. thus, the population often resorts to chemical pesticides (or synthetic pesticides) which are certainly effective against these parasites, but at the same time disrupt biodiversity by eliminating insects that are not necessarily harmful and pollute the soil and contaminate the crops. according to the fao, nearly 750,000 people contract chronic illnesses, including cancers, each year as a result of exposure to pesticides. in addition, several pathologies are directly associated with them in the long term, such as congenital malformations, ethnobotanical survey on the plants used in the control of nematodes in the zone of es niayes de thies/senegal https://doi.org/10.14421/biomedich.2023.121.335-341 336 biology, medicine, & natural product chemistry 12 (1), 2023: 335-341 mental deficiencies, neurological and reproductive disorders, endocrine disruptions, weakening of the immune system, and some cases of cancer (dewailly e, 2000; petrelli g, 2001; eriksson m, 2008). yet in senegal, chemical nematicides, although classified as extremely toxic, are sold by dealers (diarra a, 2017) in an uncontrolled manner and with ignorance of the consequences. it is therefore urgent to find solutions against this phenomenon of the spread of chemical pesticides. the idea is to find pesticides that are not toxic to humans and do not pollute the environment. so what better way than to use biopesticides. since the discovery of active ingredients of therapeutic interest from plant species selected according to criteria based on medical ethnobotany and ethnopharmacology is more frequent than in a simple random screening of plants (svetaz l et al, 2010) and given that there are no or almost no nematological studies in the niayes area of thiès, we conducted an ethnobotanical survey among local farmers in this area in order to identify the plants used in the fight against nematodes as well as the methods of preparation of extracts from these plants. the present work is also part of the global framework of the valorization of the senegalese flora. materials and methods selection of the ethnobotanical survey site the niayes zone extends from dakar to saint louis, in a strip 180 km long and 5 to 30 km wide (fall as, 2001). it constitutes a rather original environment characterized by dunes and depressions that are kept wet or flooded for a good part of the year and by a sub-surface water table (see figure 1) (cissé i, 2008). its soils are very favorable for market gardening. administratively, it covers part of the regions of saint-louis, louga, dakar, and part of the region of thiès, namely the departments of tivaouane and thiès. our study area concerns these two departments, more precisely the rural communes/communities of kayar (located in the northwest of the department of thiès), notto gouye diama, and darou khoudoss (located in the west of the department of tivaouane) (see figure 2). very few studies have been carried out on potential nematicidal plants used for the protection of vegetable crops in this area. figure 1. level of fluctuation of the water table according to the seasons (cissé i, 2008). figure 2: location of the study area. (source: inspired by (https://www.au-senegal.com/), realisation: authors) method of data collection the area was divided into different sites and visits were made to each site in july and october 2021. the ethnobotanical study was carried out through a series of surveys using a pre-designed questionnaire divided into two parts. the first question concerned the sociodemographic situation of the respondent and the second question concerned the names of the species used for http://www.au-senegal.com/) diouf et al. – ethnobotanical survey on the plants used in the control of … 337 nematode control, the parts of the plant used, the times of harvest, the methods of preparation of the extracts, etc. the identification of the scientific names of the plants was carried out in the botanical laboratory of ifan/ucad. the scientific name of each species was transcribed into the local language using the lexicon of vernacular names of j. g. adam (adam j.g, 1970). data processing the data recorded on the survey forms were processed and entered into excel. the data were analyzed using simple descriptive statistical methods. quantitative variables are described using the mean and qualitative variables are described using percentages. results in this study, all the respondents were farmers. the population is made up of 100 informants, 43 of whom are from notto gouye diama, 10 from darou khoudoss, and 47 from kayar, i.e. 53 in the department of tivaouane and 47 in the department of thiès. description of the study population by age figure 1 shows the age distribution of the sample studied. we note that the age extremes of the informants vary between 18 and 78 years with an average age of 48 years. the majority (71%) were in the 30-60 age group, while the under-30 age group had the lowest rate of nematicide users (6%). distribution by gender figure 2 shows the gender distribution of the study sample. in this study, the majority of informants were male (90%) and 10% were female, with a sex ratio (male/female) of 9. description of the study population by family status regarding the family situation, 94% of the informants have children, 3% say they have no children and 3% gave no answer (see figure 3). figure 1. distribution of respondents by age group. figure 2. distribution of respondents by gender. figure 3. distribution of the study population by family status. distribution of plants by frequency of citation the surveys carried out in the niayes area of thiès enabled us to identify different plant species used in the control of nematodes. the list of the different plant species selected and their frequency of use are presented in table 1. the ethnobotanical survey identified seventeen (17) plant species of which azadirachta indica a. juss. juss. represents the most used plant with a percentage of 45%, followed by calotropis procera (ait.) f. (20%) and cassia occidentalis l. with a percentage of 9%. table 1. list of plants used for nematode control in the study area, ranked by frequency of citation. family scientific name of the plant local name frequency of citation meliaceae azadirachta indica a. juss. nim 45% asclepiadaceae calotropis procera (ait.) f. paftan 20% cesalpiniaceae cassia occidentalis l. bèntamaré 9% fabaceae (= papilionaceae) arachis hypogaea l. gèrté, tiga. 7% fabaceae (= papilionaceae) capsicum frutescens l. kani 3% caricaceae carica papaya l. papaya 2% liliaceae allium sativum l. garlic 2% meliaceae khaya senegalensis (desv.) a. juss khaye 2% labiatae ocimum basilicum basil ngungun 2% 338 biology, medicine, & natural product chemistry 12 (1), 2023: 335-341 table 1. cont. family scientific name of the plant local name frequency of citation combrétacées guiera senegalensis j. f. gmel ngèr (in wolof) 1% solanaceae datura metel l. katidiâdabé 1% fabaceae (= papilionaceae) indigofera suffruticosa mill. ngâdj, ngâdièn 1% euphorbiaceae ricinus communis l. khekhem 1% moringaceae moringa oleifera lam. nevèrday, sap sap 1% euphorbiaceae euphorbia balsamifera ait. salan 1% euphorbiaceae jatropha curcas l. tabanani 1% poaceae (= grasses) zea mays l. mbokha 1% the species listed are divided into twelve (12) families. the most commonly used plants are from the meliaceae (47%) and asclepiadaceae (20%) families. the species used in the medium range belong to the fabaceae (11%) and cesalpiniaceae (9%) families (see table 2). table 2. list of plant families used for nematode control in the study area, arranged in alphabetical order. family scientific name of the plant frequency of citation by family asclepiadaceae calotropis procera (ait.) f. 20% caricaceae carica papaya l. 2% cesalpiniaceae cassia occidentalis l. 9% combrétacées guiera senegalensis j. f. gmel 1% euphorbiaceae ricinus communis l. euphorbia balsamifera ait. jatropha curcas l. 3% fabaceae (= papilionaceae) arachis hypogaea l. capsicum frutescens l. indigofera suffruticosa mill. 11% labiatae ocimum basilicum basil 2% liliaceae allium sativum l. 2% meliaceae azadirachta indica a. juss. khaya senegalensis (desv.) a. juss 47% moringaceae moringa oleifera lam. 1% poaceae (= grasses) zea mays l. 1% solanaceae datura metel l. 1% the most commonly used plant parts the distribution of the study sample according to their use of plant parts for nematode control is shown in figure 4. the most used parts are leaves (71%) and seeds (27%). harvest times the plants are harvested during the day, throughout the year, and especially in august. the physical state of the plant before the preparation of the extracts plant samples are often freshly harvested and then extracted (53%) or first ground and then dried before extracts are prepared (45%). very rarely is plant material dried without being ground (2%) (figure 5). the solvent used for preparation water is the most used solvent (92%) while oils are very little used (8%). figure 4 shows the distribution of solvents used for the preparation of extracts (figure 6). method of preparation of extracts maceration is widely used (85%), followed by infusion (7%). hydrodistillation (4%) and the direct use of plant juice (4%) are not widely practiced (figure 7). how to apply the extracts figure 9 shows the distribution of application modes of plant extracts. the rate of application by systemic action was 94%, while only 6% of the study sample used fumigation (figure 8). diouf et al. – ethnobotanical survey on the plants used in the control of … 339 evaluation of the effectiveness of the plants by the study population in the course of our survey, we asked the respondents to give their assessments of the effectiveness of the plant preparations they had to apply to their plantations. thus, 78% of people said that they were moderately effective, 20% said they were very effective and only 2% said they were not very effective (figure 9). figure 4. distribution of plant organs by use. figure 5. physical state of the plant before preparation of the extracts. figure 6. solvents used. figure 7. methods of preparing extracts. figure 8. the modes of application of extracts. figure 9. effectiveness of plants according to respondents. discussion the african population is attached to traditional phytopharmacy. in senegalese households, men traditionally have the role of doing large-scale fieldwork and any other activity that requires muscular effort. women are only involved in small-scale cultivation. several ethnobotanical surveys conducted in senegal have shown that in the field of traditional phototherapy and everything related to agriculture, men are much more represented than women. thus, men are more likely to have knowledge of nematicidal plants than women. in this sense, our results are similar to those of barry (male: 91%, female: 9%) (barry, 20015) and diop (diop, 2015) in which men are forty (43) times more represented than women. individuals in the 30 to 60 age group are more likely to acquire more knowledge, especially about nematicidal plants, in order to ensure good yields to better feed their families, unlike the very young who do not have many responsibilities (no children to feed). the over-60s are well represented because traditional knowledge is kept by the elderly, who then pass it on to responsible young adults. the massive use of the species azadirachta indica a. juss. and the species calotropis procera (ait.) f. is probably due to their insecticidal powers already observed. the bitter taste, and the insecticidal properties towards mosquitoes (pousset j.l, 1989) of azadirachta indica a. juss. have probably led local people to believe that it is also a plant that can be used against nematodes. the great abundance of this plant in senegal makes it easy to obtain and it is by far the most used plant. azadirachta indica a. does have nematicidal effects (yarou b.b). calotropis procera (ait.) f. is also widespread in senegal. it grows well in the streets of senegalese 340 biology, medicine, & natural product chemistry 12 (1), 2023: 335-341 villages. the plant has been reported to have nematicidal properties (rao et al., 1996; ahmed et al., 1996). these plants (azadirachta indica a. juss. and calotropis procera (ait.) f.) are available throughout the year. however, for the rare plants, it is in august that the harvesting is done because it corresponds to the wintering period. we note a great diversity of plant families used in the fight against nematodes. we note that the meliaceae, asclepiadaceae, fabaceae, and caesalpiniaceae are the most cited plant families, but in terms of the number of species represented, we have 3 species among the euphorbiaceae and 3 species among the fabaceae, and 2 species for the meliaceae. the latter may have a common trait of being rich in nematicidal compounds which are found in these species. the majority use of plant leaves can be explained by the fact that leaves are rich in active ingredients and are considered the most accessible part of the plant. leaves are the storage place for secondary metabolites that are responsible for the biological properties of the plant. a study by diatta et al (diatta c.d, 2013) (46% for leaves) showed that leaves were the most used as drugs in the preparation of traditional medicinal recipes. water is a widely used solvent (92%) compared to oils. this says that most extracted compounds are polar. these are probably polyphenols, among which there are nematicidal molecules. as for preparation methods, maceration and infusion are the most commonly used methods. maceration is the easiest extraction to implement and does not require any volume of solvent to be boiled beforehand, unlike other extractions. infusion with a polar solvent allows good extraction of watersoluble active ingredients and even those that are weakly soluble in their pure state (benlamdini a, 2014). the fumigation method of the application represents only 6%. it is a difficult method to implement compared to systemic action (94%). fumigation is only more effective if it is carried out in an enclosed area so that the gas has a much greater effect on the pests. but the means of the local population in thiès, as for most local african populations, are generally modest to manage hectares of field land with fumigation. many informants found the plants they tested to be moderately or very effective. it may be that these plants contain very good amounts of nematicidal secondary metabolites. this is very encouraging and needs to be verified by biochemical studies. conclusion ten (17) species of nematicidal plants were selected for this study. the study revealed that men are more knowledgeable about nematicidal plants than women. individuals in the age group 30-60 years, who most often have children, are more numerous than other individuals. our survey shows that the foliage is the most used part. the plants mentioned are available in the study area and people mostly use the species azadirachta indica a. juss. and calotropis indica a. juss. juss. and calotropis procera (ait.) f. for nematode control. they can harvest these plants throughout the year. before preparation, the plant sample is often dried and crushed or is fresh (case for hydrodistillation). maceration and infusion are the most common methods of preparing extracts. the application of these preparations by systemic action is more widely used than fumigation. local people find these preparations rather effective, which is really encouraging. furthermore, these results can be seen as a source of information for scientific research on biopesticides that may one day permanently replace dangerous chemical pesticides. the plants mentioned in this survey may offer broad answers to nematode-related problems. acknowledgments: acknowledgments are expressed in a brief; all sources of institutional, private, and corporate financial support for the work must be fully acknowledged, and any potential conflicts of interest are noted. authors’ contributions: el hadji gorgui diouf designed the study. mbaye diouf carried out the fieldwork. doudou diop carried out the botanical identification of the species surveyed. el hadji gorgui diouf and abdoulaye gueye analyzed the data and wrote the article. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references adam j.g. noms vernaculaires de plantes du sénégal (fin). in: journal d'agriculture tropicale et de botanique appliquée, vol. 17, no. 10-11, october-november 1970. pp. 402-460. adejumo, b. a., okundare, r. o., afolayan, o. i. and balogun s. a., quality abattributes of ignam flour (elubo) as affected by blanching water temperature and soakinf time. intl. j. engr. sci (ijes), 2013, 2 (1): 213-221. ahmed et al, effect of soil amendment with calotropis procera for the control of meloidogyne javanica infection on egg plant. pakistan journal of nematology. 1996, 14 (1): 55-59. ansd, situation economique et sociale régionale-2013, 2015. barry, m.; bassene, e.; badji, k.d., contrubition à l'étude des plantes antipaludiques: enquête ethnobotanique dans la région de matam. 20015, thése de doctorat, ucad. benlamdini a., elhafian m., rochdi a., and zidane l., étude floristique et ethnobotanique de la flore médicinale du haute moulouya, maroc, journal.of applied biosciences, 2014. blein r. et al, agricultural potential in west africa (ecowas). farm, 2008. diouf et al. – ethnobotanical survey on the plants used in the control of … 341 cissé i., badiane b., ngom s., diop y. mb., & séne m., usage des pesticides et risques sanitaires sur la production horticole de la zone des niayes au sénégal, rev. sn. res. agric, 2008, vol. 1 (3), 19-26. dewailly e., ayotte p., bruneau s., gingras s., belles-isles m., & roy r., susceptibility to infections and immune status in inuit infant exposed to organochlorines, environ health persp, 2000, vol. 108 (3), 205211. diarra, a.; diallo. b.; implementation of regional pesticide policies: report of the case study in senegal. september 2017. diatta c.d., gueye m., akpo l.e., medicinal plants used against dermatoses in the baïnounk pharmacopoeia of djibonker, senegal. journal of applied biosciences, 2013, (70): 55995607. diop, el.h.m., survey on knowledge, attitudes and practices related to the use of pesticides in the niayes area. thesis of pharmacy, dakar, 2015, n°212. dps (direction de la prévention de la statistique), situation économique et sociale du sénégal. ed. dps. 2003,197p. eriksson m., hardell l., calberg m. & akerman m., pesticide exposure as risk factor of non-hodgkin lymphoma including histopathological subgroup analysis, int. j. canc, 2008, vol. 123 (7), 1657-1663. fall as, fall st, cisse i, badiane an, diao mb, fall ca. characterization of the niayes zone. in cites horticoles en sursis? l'agriculture urbaine les grandes niayes au sénégal. idrc. 2001, http//idrc.ca/en/ev27906-201-1. momagri, key figures on agriculture. 2016. petrelli g. & figa-talamanca i., reduction in fertility in male greenhouse workers exposed to pesticides, eur. j. epidemiol, 2001, vol. 17 (7), 675-677. pousset, j.l., plantes medicinales africaines (pharmacognosie), tome 1, masson, paris, france. 1989, p 24, 156p. rao et al, effect of integration of calotropis procera and glomus fasciculatum on the management of meloidogyne incognita infesting tomato. nematologia mediterranea, 1996, 24 (1): 5961. svetaz l., et al. value of the ethnomedical information for the discovery of plants with antifungal properties. a survey among seven latin american countries. j ethnopharmacol. 2010;127:137-158. yarou b.b., bioefficiency of ocimum spp. (lamiaceae) for integrated pest management in vegetable crops. (phd thesis). gembloux agro-bio tech, liège university, belgium. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 267-271 | doi: 10.14421/biomedich.2023.121.267-271 issn 2540-9328 (online) effect of female age on crossing over frequency in drosophila melanogaster crosses n♂><bcl♀ and n♂><ym♀ and their reciprocals lisa savitri*, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita, ester lianawati antoro, ida septika wulansari department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id abstract crossing over is the occurrence of disconnection and reconnection followed by a reciprocal exchange between the two chromatids in a bivalent form. the crossing event will produce parental type and recombinant type. in the event of crossing over, various factors can influence it. these factors can be due to internal and external. recently, various factors have been reported that influence the incidence of crossing over. these factors include age, temperature, radiation, and changes in chromosome structure. this research is a type of experimental research that uses a randomized block design. randomized block design by crossing d. melanogaster strains ♂n>< ♀bcl and ♂n>< ♀ym and their reciprocals. from the results of this cross (f1) then cross again ♀n with the recessive male (from stock) then observe the phenotype of the offspring (f2) and calculate the results of the offspring. the f2 ♀n crosses were treated with age variations, namely 0, 3, 6, 9, 12, 15, and 18 days. based on the results of these crosses, the derived strains that appeared in the f2 crosses showed the phenomenon of crossing over with the influence of the age of the female and the type of strain on crossing events. the frequency or value of crossing over (formation of recombinants) decreased with the increasing age of the female. if the age of the female affects the frequency of crossing over, then the older the female, the more likely the frequency of crossing over will decrease. however, because the data obtained were incomplete, it was not possible to know the effect of female age on the frequency of crossing over of d. melanogaster crosses ♀n>< ♂ bcl and ♀n >< ♂ym and their reciprocals. the condition for crossing over is the formation of a synaptonemal complex. age of d. melanogaster females has an effect on the frequency of crossing over in crosses n♂ >< bcl♀, n♂ >< ym♀, and their reciprocals. the older drosophila melanogaster is, the lower the frequency of crossing over will occur. based on this, it was necessary to cross d. melanogaster with strains n, bcl, and ym. a cross consists of ♂n ><♀bcl and ♂n ><♀ym and their reciprocals. by crossing ♀n with a recessive male from the stock, then observing the f2 phenotype, it is hoped that crossing over will occur. so that you can better understand by doing the practice directly. in this case, the effect of crossing over is seen from the age of the female and the type of strain. keywords: crossing over frequency; drosophila melanogaster; female age; strain ♂n>< ♀bcl; strain ♂n>< ♀ym. abbreviations: adenosine triphosphate (atp), transverse filaments (tfs) introduction in life, all organisms try to maintain the continuity of their generations and to maintain the continuity of these generations, a reproduction process is needed. there are two types of reproduction, namely sexual and asexual reproduction. in sexual reproduction, after the meeting and fusion between the egg and sperm, a zygote is formed. in the process of development or what is called the gametogenesis process, cell division processes occur, initially one cell divides into two, then four, and so on. the process of cell division involves two events namely meiosis and mitosis. meiosis is a cell division event that produces gametes or sex cells and has half the number of chromosomes of each of the parents. meanwhile, mitosis is the division of a somatic cell into two cells containing an identical number of chromosomes. meiosis events are divided into 2, namely meiosis 1 and meiosis 2. in meiosis 1 prophase i, paired chromosomes often show a crossed configuration. as a result, crossing over can occur and produce offspring that are different from their parents. crossing over is the occurrence of disconnection and reconnection followed by a reciprocal exchange between the two chromatids in a bivalent form (corebima, 1997). crossing over was first proposed by t. h. morgan to explain the occurrence of recombination of factors which were concluded to be mutually linked based on genetic data (gardner, et al. 1984 in corebima 1997). the crossing event will produce parental type and recombinant type (corebima, 1997). in the event of manuscript received: 10 february, 2023. revision accepted: 11 march, 2023. published: 21 march, 2023. https://doi.org/10.14421/biomedich.2023.121.267-271 268 biology, medicine, & natural product chemistry 12 (1), 2023: 267-271 crossing over, various factors can influence it. these factors can be due to internal and external. recently, various factors have been reported that influence the incidence of crossing over. rothwell (1983) in corebima (1997) mentions these factors include age, temperature, radiation, and changes in chromosome structure. in addition, suryo (1996) stated that one of the influential factors in crossing over was age. where the older the age of the female, the less crossing that occurs. based on this, it was necessary to cross d. melanogaster with strains n, bcl, and ym. a cross consists of ♂n ><♀bcl and ♂n ><♀ym and their reciprocals. by crossing ♀n with a recessive male from the stock, then observing the f2 phenotype, it is hoped that crossing over will occur. so that you can better understand by doing the practice directly. in this case, the effect of crossing over is seen from the age of the female and the type of strain. materials and methods study area this research is a type of experimental research that uses a randomized block design. randomized block design by crossing d. melanogaster strains ♂n>< ♀bcl and ♂n>< ♀ym and their reciprocals. from the results of this cross (f1) then cross again ♀n with the recessive male (from stock) then observe the phenotype of the offspring (f2) and calculate the results of the offspring. the f2 ♀n crosses were treated with age variations, namely 0, 3, 6, 9, 12, 15, and 18 days. based on the results of these crosses, the derived strains that appeared in the f2 crosses showed the phenomenon of crossing over with the influence of the age of the female and the type of strain on crossing events. procedures medium creation 1. consider the medium ingredients, namely rajamala banana, cassava tape, and brown sugar with a ratio of 7:2:1. 2. cut the rajamala banana into several parts and put it in the blender along with the cassava tape and add enough water. 3. carry out the blending process until the tape and bananas become smooth/soft and mixed. 4. heat the brown sugar by adding a little water until the brown sugar dissolves in the water. 5. cook mashed tape and bananas using a saucepan for 45 minutes over medium heat by adding water so that the medium is not too thick. 6. when it starts to boil, put the brown sugar water that has been boiled into the pot and keep stirring it so it doesn't burn. 7. evaporate the jam jar and cork over a medium saucepan, and fill the jam jar with the medium until about a quarter of the jar. 8. close the bottle using a sponge that has been cut according to the diameter of the bottle. 9. waiting for the bottle to cool by immersing the bottom of the bottle in cold water. 10. after the medium is cold, add approximately 4 yeast grains into the bottle containing the medium and put the pupation paper into the bottle, and close it again. stock renewal 1. prepare a bottle of jam containing the medium that has just been heated, and after it's cold, add about 4 grains of yeast and put the pupation paper in it. 2. transferring at least 3 pairs of flies from each strain taken from the old medium and then transferring them to the new medium that has been prepared. 3. label according to the type of strain and date of rejuvenation. 4. observe its development and if there are already blackened pupae, immediately collect them stock collection 1. take carefully the black pupa in the bottle using a brush so that the pupa is not damaged 2. insert the pupa into a short hose (± 6-7 cm) which has been given a banana slice in the middle of the hose. 3. closing the hose by using a small piece of sponge at each end 4. waiting for the pupae to hatch (usually a day or two after being pulled) 5. after hatching, cross ♂n>< ♀bcl and ♂n>< ♀ym and their reciprocal (f1 cross) 6. for recessive males to be used in f2 crosses, the maximum age of male flies that can be crossed is 2 days. f1 crosses (♂n>< ♀bcl and ♂n>< ♀ym, and their reciprocals) 1. collect black pupae of n, bcl, and ym strains, and then after hatching cross ♂n>< ♀bcl and ♂n>< ♀ym and their reciprocals 2. after 2 days of crossing, remove the male and let the female remain in the bottle until larvae appear. 3. after the larvae appear, move the female at least 3 times (bottles a-d) 4. gathers a black pupa from the cross for its f2 cross. 5. look for tillers with strain ♀n from each cross (♂n>< ♀bcl and ♂n>< ♀ym and their reciprocals) and give them age treatments. preparing female parent stock 1. transferring strain n female flies from each cross that has been ampoule into a bottle containing medium. 2. each strain is put in a different bottle to make it easier for the practitioner to cross. 3. label each ampoule bottle with the date and strain. 4. waiting for the females of each strain until they are 0 days, 3 days, 6 days, 9 days, 12 days, 15 days, and 18 days. savitri et al. – effect of female age on crossing over frequency 269 f2 cross 1. collect black pupae from each stock strain and wait for them to hatch (for recessive males) 2. crossing ♀n from stock ♀n that has been prepared according to age treatment with recessive male p1 3. after two days, the male is released and the female is left in the bottle 4. after the larvae appear, move the female to the second bottle. minimum of 3 female transfers (bottles a-d) 5. observing the phenotype and counting the number of tillers emerging from each bottle for 7 days. data analysis the data obtained were analyzed in the following way: 1. reconstruct the body's chromosomes 2. make an observation table f2 3. calculating the percentage of crossing over with the formula: frequency of recombinant-type derivatives (%) ∑𝑟𝑒𝑐𝑜𝑚𝑏𝑖𝑛𝑎𝑛𝑡 ∑𝑝𝑎𝑟𝑒𝑛𝑡𝑎𝑙 + ∑𝑟𝑒𝑐𝑜𝑚𝑏𝑖𝑛𝑎𝑛𝑡 × 100% results and discussion result because the observational data obtained did not allow statistical calculations to be carried out, the data analysis was carried out using descriptive analysis in the form of calculating the frequency of crossing over at each cross. cross d. melanogaster strain ♀ n (from f1 bcl ♀ >< n ♂) >< bcl ♂ recessive 1. age ♀ 0 days, cross over frequency = 22,69% 2. age ♀ 6 days, cross over frequency = 13,43% 3. age ♀ 9 days, cross over frequency = 11,11% cross d. melanogaster strain ♀ n (from f1 n ♂ >< bcl ♀) >< bcl ♂ recessive 1. age ♀ 0 days, cross over frequency = 25,51% 2. age ♀ 3 days, cross over frequency = 24,93% 3. age ♀ 6 days, cross over frequency = 24,93% 4. age ♀ 9 days, cross over frequency = 15,45% cross d. melanogaster strain ♀ n (from f1 n ♂ >< ym ♀) >< ym ♂ recessive 1. age ♀ 6 days, cross over frequency = 20,18% discussion according to suryo (1996), the possibility of crossing over is influenced by several factors, one of which is the age factor, where the older the female, the lower the frequency of crossing over. whittinghill and hinton (1950) also stated the same thing that the frequency or value of crossing over (formation of recombinants) decreased with the increasing age of the female. if the age of the female affects the frequency of crossing over, then the older the female, the more likely the frequency of crossing over will decrease. however, because the data obtained were incomplete, it was not possible to know the effect of female age on the frequency of crossing over of d. melanogaster crosses ♀n>< ♂ bcl and ♀n >< ♂ym and their reciprocals. the condition for crossing over is the formation of a synaptonemal complex. the synaptonemal complex is a protein needed to carry out the correct pairing of homologous chromosomes (campbell, 2000). the synaptonemal complex is a protein complex that appears to mediate synapses during the zygotene stage and then disintegrates. this complex brings together homologous chromosomes during the prophase of meiosis. according to gardner (1991) crossing over is an enzymatic reaction like other enzymatic reactions. the older the fly, the body's metabolism will decrease. the decrease in body metabolism affects the formation of adenosine triphosphate (atp) in the body which is a source of cellular energy that plays a role in cell activities including protein synthesis. if the process of protein synthesis decreases, the amount of protein including the synaptonemal complex and enzymes that play a role in crossing over decreases so that crossing events decrease. according to ashburner (1989) under normal conditions, d. melanogaster can go through its entire life cycle for 30-32 days, and the time it takes to become an adult is ± 10 days from the time the eggs are released. so that the life span of imago can be known, which is around 20-22 days. the life span of the imago can be grouped into three ranges, namely young, adult, and old. the ages of d. melanogaster, namely 1 to 6 days are classified in the young age range, 7 to 14 days old are classified in the adult age range, and ages 15 to 20 are classified in the old age range. in the observations that have been made, crossing over is characterized by the emergence of non-parental or recombinant strains in the crosses. based on the results of the data obtained, namely in crosses bcl♂ >< n♀ in the treatment of females aged 0 days, the percentage of crossing over was obtained, namely 22.69%, in the treatment of females aged 6 days, the percentage of crossing over was obtained by 13.44%, and in the treatment of females aged 9 days, the percentage of crossover frequency was 11.11%. the results of the cross n♂ >< bcl♀ in the treatment of females aged 0 days obtained the percentage of crossing over frequency which was 25.52%, in the treatment of females aged 3 days the percentage of crossing over was obtained in the amount of 24.93%, and in the treatment of females aged 9 days, the percentage of crossover frequency was 15.45%. the decreasing percentage of crossing over frequency of females aged 0 days, 3 days, 6 days, and 9 days indicates that there is a concordance between the practicum results and theory, that is, the older the females are, the lower the frequency of crossing over 270 biology, medicine, & natural product chemistry 12 (1), 2023: 267-271 occurs. the results of crosses ym♂ >< n♀ in the treatment of 6 days old females obtained the percentage of crossing over frequency which was 20.18%. however, the results obtained in this practicum were inaccurate because the data obtained were still incomplete, namely in crosses bcl♂ >< n♀ treated females aged 3 days, 12 days, 15 days, and 18 days, whereas n♂ >< bcl♀ treatments females aged 6 days, 15 days, and 18 days, and cross data ym♂ >< n♀ for the treatment of females aged 0 days, 3 days, 9 days, 12 days, 15 days, and 18 days. crossing over occurs during the synapsis of homologous chromosomes in the pachytene stage of prophase i of meiosis. paired chromosomes at meiosis often display a crossed configuration. when the chromosomes are about to separate, the crossed chromatids attach and break off at the chiasma, then each piece is attached to the adjacent chromatid (its homolog). when the first homologous chromosomes appear as pairs during prophase i, a set of proteins called synapses joins the chromosomes together so that they are tight to one another. apart from the synaptonemal complex, another structure responsible for crossing over is the recombination nodule. recombination nodules are temporary structures that exist only in the middle of the pachytene stage associated with the synaptonemal complex, thus crossing over is expected to occur at that time limit (carpenter, 1975). genes c(3)g and c(2)m are components of the synaptonemal complex. the c(3)g gene encodes the formation of transverse filaments (tfs) (figure 1). according to page and hawley (2001), tfs are filaments that make up the synaptonemal complex in the form of coils that are in the middle of the synaptinema complex formation. the synthesis of tf will trigger the formation of a synaptinema complex between two homologous chromosomes. figure 1. a simplified schematic of the synaptonemal complex. before the synaptonemal complex is formed, the recombinant complex is composed of double-stranded dna that unravels and helps catalyze crossing-over between non-sister chromatids from opposite sides of the complex. source: albert, et al. (2008). it is well known that the existence of a synaptonemal complex is needed in crossing over. in addition to these genes, according to mckim and hayashi-hagihara (2003), the mei-w68 gene is needed in the initiation of meiotic recombination. the mei-w68 gene encodes the mei-w68 protein which is a type of topoisomerase ii protein. this protein is required in the event of double helix breaking during meiosis. based on this theory, it can be concluded that there are certain enzymes that play an important role in the process of recombination. these enzymes have a role in the process of exchanging chromosome segments on homologous chromosomes that have formed pairs or are called synapses at the prophase stage which are assisted by complex proteins and synaptonemal. this complex holds homologous chromosomes together during the prophase of meiosis. so that the age of the female can be said to affect the frequency of crossing over with respect to the decrease in the working process of enzymes and body metabolism along with the increasing age of d. melanogaster. as it gets older, the body's metabolism in d. melanogaster will also decrease, so that the production of atp as a source of energy in the body will also decrease which also results in a decrease in the work function of enzymes that play a role in the recombination process and the amount of its production, resulting in a decrease in the frequency of crossing over. conversely, if the age of the female does not affect the frequency of crossing over, this is possible because the increasing age of d. melanogaster does not affect the process of atp formation in the body, so there is no decrease in the production of enzymes that play a role in the process of crossing over. finally, the recombination process still occurs and there is no difference in the frequency of crossing over in each treatment of the difference in the age of the females. in addition, another possible reason for the age of females not affecting the frequency of crossing over is the presence of a dna ligase enzyme whose job is to repair the damage that is formed so that there is no exchange of genetic material which will result in crossing over, and the number of these genes varies in each individual. gene repair also affects the magnitude of the frequency of crossing over, namely the genes that undergo recombination have the ability to repair themselves so that the exchange of genetic material does not occur. these things resulted in no significant differences in the frequency of crossing over in various treatments of female age. according to subramanian and bickel (2008), there is another mechanism, namely through the chiasmata pathway so that segregation still occurs properly (figure 2). the chiasmata pathway in drosophila oocytes ensures accurate segregation of recombinant chromosomes that have lost chiasmata as a result of the weakening of the cohesive power of the chromosomal arms during the aging process. cohesin protein has an important role in maintaining the heterochromatin pair of achiasmata savitri et al. – effect of female age on crossing over frequency 271 homolog pairs in drosophila oocytes and as a "glue" to hold sister chromatids together. figure 2. the achiasmata segregation system (shown by green stars between homologous chromosomes) allows for accurate segregation of recombinants that previously lost their chiasmata during aging (top figure). when the achiasmata pathway is dysfunctional (indicated by loss of green stars), recombinants that have lost chiasmata will experience inaccurate segregation (figure below). source: subramanian and bickel (2008). conclusions age of d. melanogaster females has an effect on the frequency of crossing over in crosses n♂ >< bcl♀, n♂ >< ym♀, and their reciprocals. the older d. melanogaster is, the lower the frequency of crossing over will occur. acknowledgments: acknowledgments are expressed in a brief; all sources of institutional, private, and corporate financial support for the work must be fully acknowledged, and any potential conflicts of interest are noted. the author would like to thank the institute for research, development, and community service (lp3m) of kadiri university which has always provided support for writing and publications. authors’ contributions: lisa savitri designed the study. lisa savitri and elfred rinaldo kasimo analyzed the data. lisa savitri, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita, ester lianawati antoro, ida septika wulansari wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references ashburner, michael. (1989). drosophila, a laboratory handbook. usa : coldspring harbor laboratory press. borror. (1992). pengenalan serangga. edisi keenam. jogjakarta: ugm press. campbell, neil a. 2000. biologi edisi kelima edisi i. jakarta: erlangga borror. carpenter, adelaide t. c. 1975. synaptonemal complex and recombination nodules in wild type drosophila melanogaster females, (online). (http://www.ncbi.nlm.nih.gov/pmc/articles/pmc1213973/, diakses tanggal 15 november 2014) corebima, a.d. 1997. genetika mendel. surabaya: airlangga university press. gardner, eldon j. 1991. principles of genetics. new york : john wiley & sons inc. mckim k.s dan manheim, e.a. 2003. the synaptonemal complex component c(2)m regulates meiotic crossing over in drosophila. elsevier science ltd. elsevier science ltd. subramanian, v.v. dan bickel, s.e. 2008. aging predisposes oocytes to meiotic nondisjunction when the cohesin subunit smc1 is reduced. plos genet 4(11):e1000263 suryo. 1996. genetika. departemen pendidikan dan kebudayaan direktorat jenderal pendidikan tinggi proyek pendidikan profesi guru. whittinghill, m dan hinton, claude w. 1950. the nature of linkage variation with age in inversion heterozygotes of drosophila melanogaster (online).(www.ncbi.nlm.nih.gov/pmc/articles/pmc1063240/pd f/pnas01559-0034.pdf, diakses 20 november 2011). this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 4, number 2, 2015 | pages: 41-47 | doi: 10.14421/biomedich.2015.42.41-47 medicinal plants: a prospect in developing male fertility enhancing agent muhammad ja’far luthfi1*, mahanem mat noor2 and jalifah latip3 1biology department, faculty of science and technology, uin sunan kalijaga jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 2the study center of bioscience and biotechnology, 3the study center of chemical science and food technology, faculty of science and technology, ukm 43600 bangi, selangor, malaysia author correspondency*: jafarluthfi@yahoo.com abstract medicinal plants have been a revolutionary breakthrough in the treatment of male sexual dysfunction. traditional medicine based on a holistic philosophy is quite different with the practice of “western” medicine. phytochemical substances focus their mechanisms of healing to the root of cause, i.e. the inability of controlling the proper function of the whole body system. hence, medicinal plants manage sexual dysfunction and male fertility in the frame of sexual dysfunction as a whole entity. some previous researches prove that the use of medicinal plants have a good impact in the treatment of a variety of male sexual problems. this paper will discuss several important aspects of aphrodisiac plants and preliminary study regarding them in indonesia. difficulties inherent to activity guided isolation and the specific requirements of bioassays are also discussed. keywords: medicinal plants, male sexual problem, traditional medicine introduction male sexual dysfunction is a very serious problem. (mackay, 2004; clourate, 2005). approximately 10% men are infetile, and 50% infertile couple are from male factors. (pei et al., 2005). the medical treatment for male sexual problem has not succeeded yet although many efforts have been done (hamadeh, 2001; kohn, 2001). the existed modern methods, like assisted reproductive technology (art), need high cost while the result is not consistent (orgebin-crist, 1998), even the methods have side effects (levie’vre et al., 2007). people have used plants as medicine since the beginning of human civilization, even probably have been used before they evolved to be man, as shown in the practice of using medicinal plants by non-human primates (newton, 1991). one of the use of medicinal plants is to overcome sexual dysfunction. indonesia is one of the mega-biodiversity countries. there are many potential plant as aphrodisiac agent. however there is little scientific evidence supporting the effectiveness of using aphrodisiac plants. besides that, there is still less studies of the action mechanism and its active components. the profound research of the effect of plants towards male reproductive system will support the use and the effectiveness of the medicinal plants. the paper will discuss about several important issues of medicinal plants to cure male sexual problem covering the characteristics, the research, and the development of the medicinal plants. those plants include aphrodisiac plants and those which function to improve the fertility. it also discusses about the research of several plants used by local people. the research of drug from plant sources plants is a main source of new drug discovery (balandrin et al., 1985), approximately 40% modern drugs are from botanical substances (lafrance jr. et al., 2000). however, recently many pharmaceutical industries have reduced plant drugs studies. compared with synthetic drugs, plant drugs are not suitable for high-throughput screening. the study and evaluation of plant drug are very complicated, time-consuming, and more expensive compared to conventional drugs (cordell,. 2000; etkin, 2000; balunas & kinghorn, 2005; cordell & colvard, 2005; who, 2005). it is believed that combinatorial chemistry will be a the main source of synthetical medicine in the future (simmonds & grayer, 1999) . in fact, however, combinatorial chemistry fails to provide a model for drug structure in several medical treatment (simmonds, 2003; butler, 2004), whereas the plants still provide more various structures than combinatorial chemistry (muller, 2004). it is assumed that plants, as chemical factories, have continuously evolved their biosynthetic programs for more than 400,000 million years. as long as evolutionary process, these plants synthesize compounds in which the structural variety is far beyond the imagination of synthetical chemistry experts. it is really possible that the evolutionary pathway of secondary metabolite produces compounds enabling to cure diseases that cannot be healed by conventional medication. (simmonds & grayer, 1999). plants as a treatment for male sexual problem environment is one of crucial factors affecting human fertility. contrast to infertility caused by genetical 42 biology, medicine, & natural product chemistry 4 (2), 2015: 41-47 factors, infertility caused by environmental factor have possibility to cure and prevent (quallich, 2006). one of the most important environment factors is nutrition (ebisch et al., 2005). in this prespective, a traditional medicine using medicinal plant is very promising. some research has shown a positive effect of nutrition in spermatogenesis. recent studies show medicinal plant potency in improving male fertility and aphrodisiac on animal (sinclair, 2000; mackay, 2004). the improvement of sexual function has been proven through the use of medicinal plants, like ginseng, yohimbe, tribulus terestis, and maca (burkill, 1965; waddell et.al., 1980; lewis & elvin-lewis, 2003). the traditional medication that is orally done by drinking the steeped water from the parts of the plant has been practiced over hundred years ago. in a medical treatment, the oral medication of sexual problem is a new thing. viagra, the first pill to heal impotence, had just been launched in 1998. this medicine is a revolution in medical treatment, in which the previous medication was treated only by injection and operation. (eardley, 1998; mulhall, 2000; elferink, 2000; morales, 2001). however, the development of the plant research is very slow. it happens because many researches conducted by pharmacy industries depend on the patent to get the profit. plants cannot be patented therefore the industries are reluctant to invest their money to prove that certain plants are safe and effective (foster & duke, 2000). there are no plants or botanical derivation compound that are legally certified by food and drug association or other similar institutions to be used as a medication of sexual dysfunction (sinclair, 2000; nickell, 2001; mackay, 2004). the difference between traditional medicine and modern medicine in the medical world in america and other developed countries, there is just a single chemical compound that is recognized as a medicine, not in the form of mixed chemical components existed in the plants. this recognition is preferably caused by the drug regulations and laws, not by the scientific consideration (foster & duke, 2000) or philosophical belief that medicine works as ‘one-target-one-disease’ (adimoelja, 2000; wermuth, 2004). a dominant belief indirectly puts minor beliefs aside, consequently it reduces the supports towards scientific fields based on different philosophies (kuhn, 1996). modern medicine relies on the view that a disease is caused by a very specific pathogen invaded to the body therefore the healing must be done by eradicating the source of the disease (adimoelja, 2000). the medication using medicinal plants uses holistic approach by investigating the disease up to the main origin causing the unbalancing and deficiency in the body function (cracker & giblette, 2002). the whole body functions caused by the synergy and the existence of individual actions from body parts are the characteristics of living creatures therefore the reductionist view of the disease concept, that just blames an organ or a certain isolated mechanism, is an incomplete approach (rangel, 2005). the model of medicinal plant study nowadays, in vitro test for screening pharmaceutical agent is a main choice for many pharmacy companies but the success of the effort is limited on the beginning of the drug discovery program. this test has not yet fully replaced the use of animals in many toxicology studies and pharmacology experiments (white, 2001). the understanding on molecular level cannot be automatically applicated to show that the medical strategy is effective. in vitro physiological reaction of a pharmaceutical can have an inaccurate prediction of in vivo physiological reaction. many pharmaceutical agents show off different activities in various cell population. that ails in predicting the whole effect of giving an agent in the body (walsh, 1998; barton & andersen, 1998; jobe et al., 1994). in vitro data is indirectly equivalent with in vivo data although in vitro test can provide the foundation to determine the study objective. the studies are continuously conducted to ensure how far in vitro data can replace in vivo data (rodrigues, 1997). the drug discovery program is basically based on the use of the experimental animals to determine the pharmacological effect and the compound/chemical metabolism (briggs & oehme, 1980). an experimental model is important from a clinical point of view because there are many aspects of human physiological and biological reproduction that cannot be studied directly (plant & marshall, 2001). a conventional test of medicinal plants using rodensia as an experimental animal model is really needed before pre-clinical and clinical tests can be conducted (farnsworth, 1992). the result is often the only reference to determine the drug development from the pre-clinical test (greaves, 1990). a rodent as an experimental animal has an important role in screening chemical agents to study the pharmacological effect covering the distribution, mechanism, and toxicity (briggs & oehme, 1980). evaluation of the effect of an agent towards a male reproduction system generally uses rodents as an animal model. the wide use of mouse species in the research has produced complete biology data. the research on fertility and male reproduction is conducted using various methods taken from toxicology, medical, ecology, and epidemiology (golden, 2002). bioassays for bioactive compound evaluation the combination of chemical and biological screening is the quickest method to get an active compound of the plant. to get this, the bioassays provision or simple pharmacological test is very important to focus on certain activities from the plants or various plant fraction as a guidance to obtain pure active component(s). this bioassays must be very sensitive because the active substance in the plants may be in a very low quantity. this muhammad ja’far luthfi, et.al. – medicinal plants: a prospect in developing male … 43 bioassays must also be specific towards the desired target. the main target for biology test can be divided into six groups, namely:  low organisms: microorganism  invertebrata: insects, crustacean, mollusca  isolated sub-celullar system: enzyme, receptor  animal and human cell culture  isolated organ  intact animal most bioassays are used in in vitro test in cells or subcells and the low animal test. in vivo test in complete animals is less conducted and less interested because of ethical issue and it is time-consuming (hawcroft et al., 1987; hamburger & hostettmann. 1991; hostettmann & marston, 2002; preusch, 2004). determining a simple and quick bioaassay to evaluate plant for improving sexual function is very difficult. reproduction system is a very complicated system having existed complex interactions in the level of organs, cells and sub-cells. moreover, there are many aspects of reproduction system that have not been studied and not certainly known (kierzenbaum, 1994; de kretser & baker, 1999; liska, 2003; huggins, 2003; lopez-gatius, 2006). because of that, it is quite difficult to find simple and quick in vitro bioassays in those fields – those are not always provided. the only bioassay, that are reliable to test plants in improving sexual function, is in vivo test in intact animal although the test is time-consuming and expensive. bioassays offer an huge advantage in standardization and quality control of plant based product. the products are heterogen because there are a mixture of bio-active components from both a plant and mixture of several plants. the physical analysis method, like chromatography, cannot be used for this purpose because it is not sensitive towards the chemical complexity existed in plant crude extracts. it often happens that the desired biological activities are not evoked by single plant component but a mixture of various plant components therefore just relying on physical and chemical analyses towards a single component on a mixture is not really satisfying (mclaughlin, 1998). unfortunately the objectives of many phytochemist is just isolate, characterize and publish various new compounds without testing their bio-activities. to attain practical uses, natural chemical substances must combine bioassays inside those substances. the extracts must be screened for biological activities, active extracts must be chosen, fraction must be directed to bioassays, bioactive compounds are finally identified and exploited (mclaughlin, 1998). experiment on fertility and male sexual function the researches conducted by luthfi et al (2008) and mat noor & luthfi (2006) on tongkat ali (eurycoma longifolia), sanrego (lunasia amara), ginger (alpinia galanga), dan clove (syzygium aromaticum) show the potency of aphrodisiac and sanrego to improve man fertility. this study uses rat as tested animals. rats are divided into two groups for different plant extracts, namely every group is given plant extracts in dose 3.33 mg/ml and 333 mg/ml each and control group is given distilled water. plant extracts or distilled water are given through force feeding once a day at 11.00 a.m. for 50 days. after having given plants extraxts for 50 days, the rat are sacrificed using chloroform and the epididymis were dissected out. cauda epididymis were separated according to the hamilton (1975). sperm counts were determined using improve neubauer hemocytometer as described previously (prasad et al., 1972; nafa & eshre-siga, 2002) with some modification. in brief, cauda epididimis was minced in 15 ml bww medium (biggers et al., 1971) and incubated with 5 % co2 for 30 minutes at 370c. data were expressed as number of sperm per cauda epididymis. progressive sperm motility was assesed subjectively based on who laboratory manual (1999). a study of sperm morphology is conducted by providing three slides of sperm smear for each rat. after fixated in methanol, the slide were stain with giemsa. the dry slide is observed in a light microscope. one hundred sperms are counted randomly from every slide. the percentage of morphology of normal/abnormal sperms is determined referring to criteria stated by wyobek and bruce (1975). table 1 shows the number of each rat sperm which has been given aphrodisiac, sanrego, ginger and clove for 50 days. the average number of mouse sperms treated by aphrodisiac extracts at 333 mg/ml dosage (46.23±1.77) and sanrego extracts at 333 mg/ml dosage (47.30 x 106 + 3.47) show that there is a significant improvement (p < 0.05) compared with the average number of sperms given ginger, clove extracts and control (distilled water). however, the intake of tongkat ali and sanrego extracts at 3.33 mg/ml dosage does not show any significant different in sperm number. a statistical analysis does not show any significant difference between groups treated with ginger extract, clove extracts, and control. the morphology analysis result of rat sperm shows that the percentage of sperm with normal morphology in treated rat group and control group exceeds 95%. a statistical analysis using turkey test shows that there is no significant difference (p > 0.05) between control group and a treated group from the percentage of formal morphology sperm. table 1. sperm number (x106) and percentage of normal morphology sperm a of rat group given aphrodisiac, sanrego, ginger and clove extracts each at 3.33 mg/ml and 333 mg/ml dosage compared to control group. treatment dosage sperm number (x 106) % normal morphology sperm tongkat ali 333 mg/ml 46.23±1.77 96.24±1.16 3.33 mg/ml 34.16±3.37 96.8±0.70 44 biology, medicine, & natural product chemistry 4 (2), 2015: 41-47 sanrego 333 mg/ml 47.30±3.47 96.66±0.80 3.33 mg/ml 38.43±3.09 96.04±1.10 ginger 333 mg/ml 27.21±2.76 95.70±1.39 3.33 mg/ml 29.21±4.46 96.83±0.21 clove 333 mg/ml 31.67±1.91 96.48±1.35 3.33 mg/ml 32.43±2.76 96.71±0.47 control distilled water 33.17±4.25 96.67±0.63 the effect of aphrodisiac, sanrego, ginger and clove on sperm motility is shown in table 2. a sperm motility analysis shows that tongkat ali and sanrego have the best effect in improving sperm movements compared with groups given clove, ginger extracts and control group (distilled water). the treatment of aphrodisiac and sanrego at high dosage (333 mg/ml) shows that 80% of sperm movements are in the a grade while control group shows only 40% of sperms movements are in the a grade. a group treated using ginger extracts at 333 mg/ml dosage on the contrary shows the lowest level of sperm motility, namely 80% in the level c. sperm motility has been considered as one of the most important predictors of fertility. several reports have demonstrated the correlation of motion parameters with fertilization rates (liu et al., 1991). further studies are required to confirm the mechanisms of action of tongkat ali and sanrego on sperm motility. table 2. sperm motility grade in the treated group given aphrodisiac, sanrego, ginger and clove extracts each at 3.33 mg/ml and 333 mg/ml dosage compared with a control group. motility grade (a-d) treatment dosage individual rat 1 2 3 4 5 tongkat ali 333 mg/ml a a b a a 33.3 mg/ml a a a b a sanrego 333 mg/ml a a a b a 33.3 mg/ml a a b b a ginger 333 mg/ml c c c b c 33.3 mg/ml b c c b b clove 333 mg/ml b b b b b 33.3 mg/ml b b b b c control distilled water b a b b a note for the grade of motility who (1999) (a): rapid progressive motility ; ≥ 25 μm/s, (b): slow or sluggish progressive motility, (c): non progressive motility ; < 5 μm/s, (d): immotile. this study shows that cloves and gingers do not improve the parameter of male fertility, namely the numbers of sperms, the movements of sperms, and the normal morphology of sperms. both plants on the contrary decrease the number of sperms although it is not significant (p> 0.05). the report written by jaganath and ng (2000) shows that gingers can improve sperm number and sperm motility is incompatible with the result of this study. this result is difficult to compare because there are differences of tested animals and there may be differences in providing extracts that are not explicitly stated in detail in the report. a study conducted by luthfi and mat noor (2007) also shows that sanrego increase sexual behavior of male rat. table 3 shows the aphrodisiac effect in male rat after they are given sanrego at a certain dosage (the treatment group) and distilled water (a control group). the 60 mg/kg dosage shows that the number of mounting is increase significantly compared with other groups. the numbers of mounting in the group using 60 and 90 mg/kg dosages do not show any significant differences. the decrease of mounting number at 90 mg/kg dosage is difficult to explain with the data from the result of the study. it may be caused by the limited number of tested rat (sample). hayes (2001) stated that the more number of mice, the more consistent the result. the research is still conducted to isolate active compounds from sanrego that can improve aphrodisiac or male fertility. table 3. the average mounting number of male rat after treating sanrego extract using various dosages. treatment dosage the average of mounting number + se kontrol 1.33 + 1.54 30 mg/kg 2.33 + 0.57 60 mg/kg 3.33 + 0.57 90 mg/kg 3.00 + 0.00 the early research on purwoceng (pimpinella pruatjan) also shows its potency as aphrodisiac herb (tambunan, 2005). the root of purwoceng can be used as aphrodisiac, diuretic and tonic herbs. at this moment, the information about bioactive content in purwoceng has not been known yet. some bioactive compounds that have been identified are stigmaterol and sitosterol but no one has reported the use of the single compounds. muhammad ja’far luthfi, et.al. – medicinal plants: a prospect in developing male … 45 the direction of the medicinal plant development in the future most research synthetic organic and natural compounds are conducted by pharmaceutical company and industries. the research is focused to look for new and more powerful compounds. in the university, a study must not always be directed to find new compounds but the study can also look for substances showing certain biological activities that may help in understanding on physiological effects. the study of medicinal plants to treat sexual dysfunction is ideally conducted using an experimental approach that is wellknown reliable to find bioactive compounds, that is bioassay-guided isolation. plants are chosen based on ethnopharmacology criteria, then the plants are extracted. extracts produced are tested using relevant bioassays system. after biological activities are determined, active extracts are fractionated and their biological activities are monitored for each level of fraction. active isolat elucidation is finally conducted. there are several possibility if extraxts and fractions show bioactivity but each compound is not active. the first possible reason is that there is a synergy effect in those compounds. bioactivity emerges because there is a synergy among those compounds (williamson, 2001) but there is another possible reason that there are some components that have not been isolated. it occurs because a single plant extract can contain hundreds components from various different groups, either primary and secondary metabolit. those group are isoprenoid, fenol, lipid, cabohydrate and its derivations, amino acid and mineral. all those subtances have possibilities to be bioactive (wildman, 2001). in this case, in a research of plant having a potency to improve fertility, it has a slight possibility to get active components because in vivo testing needs more numbers of components to test. in that case, a pharmacological testing to test extracts that produces standardized extraxts can be conducted. after a study of toxicity and safety of the extract are conducted, the standardized extracts are formulated. then, a product can be developed. this product can be a first step for an innovative local pharmaceutical industry and it can compete with western pharmaceutical industries, not only for common diseases but also for severe diseases (pieters and vlietinck, 2005). conclusion a number of medicinal plants show the promising potency in treating male sexual problems. medicinal plants have their own characteristics that cannot fully fulfilled the research methods of conventional medicines. the results of the research show that some practices using medicinal plants can validly healing male sexual dysfunction and it can be improved in order to have a cheap effective safe therapy of the disease. it is hoped that the wide spread of the botanical medicine interest and knowledge will reduce obstacles in using and developing the medicinal plants. references adimoelja, a. 2000. phytochemicals and the breakthrough of traditional herbs in the management of sexual dysfunctions. int. j. androl. 23, suppl.2: 82-84 aminudin, n. 2004. eurycoma longifolia jack aqueous extract: bioactive compound and its effects toward hormone production and spermatogenesis. doctorate thesis. institute of biology science, faculty of 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(2023) states that gels containing kombucha can have potential as antimicrobials and treat inflammation. gel preparations are more effective for acne treatment because they are polar, so they are easily rinsed with water and do not store oil, which is feared to aggravate acne on the skin (sasanti et al., 2012). gel preparation is a topical treatment that will be more easily absorbed by acne skin so that the drug’s effectiveness can be achieved properly (ardana et al., 2015). in making anti acne gel, it is necessary to add methyl paraben as a preservative that contains antibacterials so that it can inhibit the growth of acne bacteria. to minimize the irritation that may be caused by synthetic methyl parabens, natural methyl parabens that contain medicinal compounds such as oranges can be used. not only the fruit, orange peel is also widely used as a face mask because of its antioxidant content which is quite good for the skin (friatna et al., 2011). symbiotic culture of bacteria and yeast (scoby) kombucha gel combined with orange peel extract is believed to have potential as an antiacne against s. aureus. this is because each of these ingredients has antibacterial compounds that can inhibit the growth of pathogenic bacteria, especially acne bacteria. research by malik et al. (2021) also stated in the results of their research that orange peel extract has a relatively high antimicrobial activity, especially on s. aureus bacteria. s. aureus bacteria are one of the pathogens that cause acne. according to the research of dhillon and varshney (2013), it was found that in acne lesions, several bacteria dominated, namely staphylococcus aureus, as much as 45% and propiniobacterium acnes, as much as 32%. according to research by imasari and emasari (2021), it was found that s. aureus causes acne with a percentage of 79% on human skin. s. aureus lives a lot on the skin’s https://doi.org/10.14421/biomedich.2025.142.1143-1150 1144 biology, medicine, & natural product chemistry 14 (2), 2025: 1143-1150 surface, one of which is on facial skin. s. aureus can secrete protease, lipase, hyaluronidase, and kinase enzymes that cause tissue damage resulting in inflammation (rosalina, 2018). this study will be conducted in vivo by injecting s. aureus bacteria into the ears of mice because the ears of mice have a thin surface so that the inflammation caused is more visible. s. aureus is able to infect the skin by entering the stratum corneum, causing toxins that can cause skin infections. the skin will produce excess oil so that the pores are clogged by a collection of fat mixed with sweat, dust and other dirt and cause inflammation known as acne (pratami et al., 2013). inflammation in the ears of mice will be smeared with a combination gel of orange peel extract and different concentrations of scoby kombucha. materials and methods materials the materials needed in this study include s. aureus bacteria, green tea, sugar, water, xylazine, carbomer, tea, glycerin, orange peel, ethyl acetate, distilled water, physiological solution nacl, na2co3, nutrient agar media, mediclin 1%, male mice, husks, mice feed and drink, label paper, and tissue. the tools needed in this study include analytical scales (ohaus px224), 1 cc syringe, erlenmeyer (phyrex®), plastic wrap, petri dish, beaker glass, test tube, test tube rack, ose needle, measuring cup, stirring rod, drop pipette, oven, cotton, hotplate (jisico j-hms), micropipette (sorenson) laminar air flow (laf) (robust laf140). procedures animal preparation the test animals used in this study were white mice (mus musculus) male ddy strain aged 6 months with body weight (bw) 26-32 g. the number of mice used was 24 in healthy condition obtained from the farma veterinary centre (pusvetma) surabaya. prior to treatment, the mice were acclimatised for 2 months with ad libitum feeding and drinking and replacement of husks every 3 days. preparation of orange peel extract extract preparation was carried out by maceration for 24 hours. the oranges used were siam oranges (citrus nobilis) which were peeled and cut into small pieces to be put in the oven. the dried orange peel was then blended until it became smooth. orange peels that have been finely filtered to take fine powder and weighed. in this study, 100gr of orange peel fine powder was obtained and then macerated for 24 hours using ethyl acetate which was added as much as 300ml. the selection of ethyl acetate as a solvent in orange peel because ethyl acetate can bind compounds that have potential in antibacterial activity such as polyphenols and flavonoids (nina, 2014). the results of maceration are then evaporated using a rotary evaporator so that a thick extract of orange peel with ethyl acetate solvent will be obtained. then calculated the yield using the following formula: % 𝑟𝑎𝑛𝑑𝑒𝑚𝑒𝑛 = 𝑏𝑒𝑟𝑎𝑡 𝑒𝑘𝑠𝑡𝑟𝑎𝑘 (𝑔) 𝑏𝑒𝑟𝑎𝑡 𝑠𝑎𝑚𝑝𝑒𝑙 (𝑔) 𝑥 100% preparation of scoby kombucha propagation of scoby kombucha is done by boiling 2000 ml of water until it boils, then adding sugar as much as 10% of the amount of water used (200 grams) and adding tea to 0.5% of the amount of water (10 grams). after all the ingredients were mixed, the solution was filtered and the filtrate was put in a large jar and covered with aluminium foil for a few minutes until the temperature of the solution decreased. next, the kombucha culture or scoby was added to the jar to account for 10% of the water volume (200 ml) and sealed for up to 14 days. during fermentation, scoby will grow bigger and thicker by utilising glucose from the kombucha tea solution (nurikasari et al., 2017). scoby kombucha gel preparation preparation of gel base preparation of gel base is done by weighing carbomer 940 as much as 10 grams using a watch glass then mixed with 500 ml of distilled water in a glass beaker and stirred quickly on a hotplate until homogeneous. after forming the gel mass, 10 drops of tea and methyl paraben were added. methyl paraben was dissolved first by weighing 1 gram and put in a glass beaker then dissolved in 25 ml of distilled water. after dissolving, methyl paraben was added to the first mixture then glycerin was added using a measuring cup as much as 5 ml and stirred until homogeneous while heating (putri, 2017; rinawati et al., 2022). preparation of scoby gel variations variations of scoby kombucha begin with harvesting scoby kombucha during the fermentation process. scoby kombucha on the surface of the tea is taken then blended and filtered until smooth. scoby kombucha gel was made with variations of 2%, 4%, 8%, 10%, and 12%. the addition of scoby weight is calculated based on w/w so that 2 g, 4 g, 8 g, 10 g, and 12 g are needed. each scoby that has been weighed is added to the gel base up to 100 g and homogenised. the variation of kombucha scoby concentration added to the gel can be calculated using the following formula: 𝑀1. 𝑉1 = 𝑀2. 𝑉2 preparation of bacterial suspension aseptically, s. aureus bacteria were inoculated with the scratch method as much as one ose on na media. then incubated at 37̊c for 1 x 24 hours (yanti & mitika, ferdyanti et al. – effect of different concentration of scoby kombucha as 1145 2017). then make a physiological solution by dissolving 0.85 grams of nacl in 100 ml of distilled water and put in an autoclave at a temperature of 121̊ c. after that, it is cooled to a temperature of 45-50̊ c before use (yanti & mitika, 2017). bacteria rejuvenated on agar media are taken using a sterile ose heated with a bunsen burner and then suspended in a test tube containing nacl physiological solution. test tubes containing bacteria and physiological solution were then vortexed until homogeneous and then the absorbance value was measured using a 625 nm wavelength spectrophotometer. the turbidity measurement results must comply with the mc. farland standard, namely with an absorbance of 0.08-0.5 (rosmania & yanti., 2020). injection of bacteria into the ear of mice (mus musculus) acne on the ears of mice is induced by injection using a 1 cc syringe. before injection in the ears of mice, mice were anaesthetised using xylazine in doses of 0.05 ml. furthermore, mice were injected with a suspension of s. aureus bacteria in the right ear as much as 0.2 ml and left for 2x24 hours until inflammation appeared in the ears of mice. (fitriani et al., 2022). application of scoby kombucha gel combined with orange peel extract the application of scoby kombucha gel was carried out after 2x24 hours of bacterial administration to the ears of mice. gel application is given according to the concentration of each group where in this study it was divided into 8 treatment groups, namely 1 positive control group using mediclin, 1 negative control group, and 5 treatment groups (2%, 4%, 8%, 10%, and 12%). the application was carried out 2 x a day, namely in the morning and evening as much as the tip of an aluminium spoon equally between mice. measurement of inflammation diameter measurement of the diameter of the inflammation was carried out after 2x24 hours of bacterial injection until the inflammation healed. measurements were made using a calliper every 3 hours from 9am to 3pm. inflammation of the mice's ears was measured by holding the mice's ears and then measuring the width of the acne inflammation starting from the base of the ear to the tip of the outer ear using a calliper. parameters of healing of inflammation in the ears of mice can be seen from the shrinking of redness on the skin of the ears of mice. data analys inflammation diameter data will be tested for normal distribution and homogeneity test with the provision of sig value >0.05 which indicates that the data is normally distributed and homogeneous (purnomo, 2016). if the data is normally distributed and homogeneous, then the test continues with one-way analysis of variance or one way anova to determine the average difference between treatment groups. data that are not normally distributed and inhomogeneous will continue with the kruskal wallis test to see significant differences between groups. if the kruskal wallis test obtained significant changes, the mann-whitney test will be continued to determine significant differences between treatment groups. results and discussion the right ear of the mice was injected with s. aureus bacteria and left for 2x24 hours. after that, the gel was applied to the inflammation caused. from the results of the average diameter of each gel application, the difference in inflammation reduction will be recorded for each observation. the results of inflammation healing at each time can be seen in table 1. 1146 biology, medicine, & natural product chemistry 14 (2), 2025: 1143-1150 table 1. average healing of inflammatory acne. concentration bacterial growth. gel application process. 24 hour 42 hour 45 hour 48 hour 66 hour 69 hour 72 hour 90 hour 93 hour 96 hour 114 hour p1 i 0.09 0.02 0.02 r 0.09 0.03 0.03 0.14 0.04 0.08 0.15 d 0.42 0.52 0.54 0.56 0.47 0.44 0.41 0.27 0.23 0.15 0.00 p2 i 0.03 0.05 0.01 r 0.05 0.03 0.03 0.08 0.04 0.04 0.15 d 0.35 0.38 0.43 0.44 0.39 0.36 0.33 0.25 0.22 0.17 0.00 p3 i 0.17 0.01 0.01 r 0.06 0.04 0.04 0.12 0.05 0.05 0.10 d 0.32 0.49 0.51 0.54 0.45 0.41 0.37 0.28 0.19 0.14 0.00 p4 i 0.07 0.02 0.01 r 0.08 0.03 0.04 0.10 0.05 0.10 0.06 d 0.40 0.47 0.48 0.50 0.42 0.39 0.34 0.25 0.20 0.10 0.00 p5 i 0.18 0.02 0.02 r 0.09 0.03 0.04 0.10 0.07 0.06 0.16 d 0.36 0.54 0.56 0.58 0.49 0.45 0.41 0.26 0.24 0.18 0.00 p6 i 0.10 0.02 0.01 r 0.07 0.04 0.04 0.17 0.12 0.06 0.02 d 0.39 0.49 0.50 0.52 0.45 0.41 0.37 0.21 0.08 0.02 0.00 p7 i 0.12 0.02 0.03 r 0.07 0.06 0.08 0.31 0.00 0.00 0.00 d 0.35 0.47 0.49 0.52 0.44 0.38 0.31 0.00 0.00 0.00 0.00 p8 i 0.06 0.03 0.02 r 0.02 0.02 0.02 0.04 0.03 0.03 0.04 d 0.39 0.45 0.48 0.49 0.48 0.46 0.44 0.40 0.36 0.34 0.30 note: (increased): increase in inflammatory diameter during acne bacteria growth (24-48th hour); r (reduction): reduction/decrease in inflammatory diameter during the gel application process (48-114th hour); d (diameter): diameter of acne inflammation description: p = treatment (*) = sign of significant difference in mann-whitney test (sig.p<0.05), p1 = mediclin 1%, p2 = scoby 0% citrus peel extract, p3 = scoby 2% citrus peel extract, p4 = scoby 4% citrus peel extract, p5 = scoby 8% citrus peel extract, p6 = scoby 10% citrus peel extract, p7 = scoby 12% citrus peel extract, p8 = no gel application. the inflammatory diameter will get bigger for 48 hours because at this time, it will be the growth period of s. aureus bacteria in the right ear of mice. this happens because s. aureus bacteria can damage skin tissue by secreting chemical compounds that can destroy pore walls so that fatty acids and oils are clogged and harden into acne bumps (miratunnisa et al., 2015). after 48 hours, the gel was applied according to each treatment group. based on table 1, it can be seen that the gel that has the fastest healing activity against s. aureus bacteria is p8 (scoby 12%). this is because in p8 the inflammation has healed with an average of 0.00 at the 90th hour. whereas in the other treatment groups, the inflammation was completely healed at the 114th hour, which was the last hour of observation. to determine the differences in each treatment group, spss statistical analysis started with normality and homogeneity tests to determine whether the data were normally distributed. the results of the normality test were carried out using the difference data taken from the initial data of inflammation before the gel was applied and the data when the first treatment group recovered, namely at 48 hours and 90 hours. the results can be seen in table 1 which shows that the data at the 90th hour is not normally distributed because of the treatments tested some treatments have a significant value of 0.00 <0.05. therefore, to find out the differences in each treatment can be continued with non-parametric test analysis, namely kruskal wallis and mann whitney tests can be continued in table 2 table 2. initial healing analysis results (90th hour). treatments 48 hour 90 hour average difference % acne healing kruskal wallis p1 0.56 0,27 0.29 52% .018 p2 0.44 0,25 0.19 43% p3 0.54 0,28 0.26 48% p4 0.50 0,25 0.25 50% p5 0.53 0,26 0.27 51% p6 0.52 0,21 0.31 60% p7 0.52 0,00 0.52 100% p8 0.49 0,40 0.10 20% ferdyanti et al. – effect of different concentration of scoby kombucha as 1147 table 3. mann-whitney test results 90th hour. treatment 1 2 3 4 5 6 7 p1 p2 0,050 p3 0,513 0,184 p4 0,507 0,825 0,507 p5 0,376 0,050 0,513 0,507 p6 0,376 0,050 0,513 0,507 0,127 p7 0,046* 0,046* 0,046* 0,043* 0,046* 0,046* p8 0,050 0,050 0,050 0,050 0,050 0,050 0,046* based on table 2, the significance value of the kruskal wallis test results on the right ear of mice injected with s. aureus bacteria is 0.018 <0.05, which means that the treatment of mice's ears makes a significant difference to the healing of acne inflammation of mice injected with s. aureus. this is because each scoby gel has different healing activity. based on the results of the mann-whitney test, it shows that all treatment groups have a significant value <0.05 against p7. this means that all treatment groups in this study have different healing activities from p7. this is by table 2 where at the 90th hour the diameter of the p7 inflammation was 0.00 while the other treatment groups still showed 0.21-0.35 cm. table 2 shows that at the 90th hour the p7 group has the largest average difference value among others, which is 0.52 cm. this means the p7 group has the fastest healing activity compared to other treatments. p7 has a faster healing activity than p6 (10% scoby gel). this can be seen in table 2 which shows the difference in the average diameter of inflammation in p6 is 0.31 cm with an inflammatory diameter of 0.21 at the 90th hour. this is consistent with the greater percentage of inflammation healing in p7, which is 100% while the percentage of p6 is 60%. p1 (mediclin positive control) has faster healing activity compared to scoby gel groups p3-p6, gel group without scoby (p2), and p8 (without gel administration). this can be seen in table 2, p1 has a greater average difference in inflammation, which is 0.29 cm with an inflammation diameter of 0.27 at the 90th hour. in p2, the diameter of inflammation at the 90th hour was still greater than in p1, which was 0.25 cm. this is by the percentage in table 2 which shows that p1 has greater healing activity, 52% while p2 is 43%. the treatment groups with various concentrations of scoby p3 (scoby 2%), p4 (scoby 4%), and p5 (scoby 8%) had faster healing activity than the gel treatment without scoby p2 (scoby 0% orange peel extract). this can be seen from table 2 which shows that the difference in the average diameter of inflammation in the scoby gel treatment group at the 90th hour is greater than the gel treatment group without scoby. p3, p4, and p5 at the 90th hour had an inflammatory diameter in the 0.25 -0.28 cm range. while p2 still showed a larger diameter, namely 0.25. this is also in accordance with the healing percentage of scoby treatment groups p3, p4, and p5 which is greater than p2. p3, p4, and p5 had healing percentages of around 48-51%, while p2 only reached 43%. this shows that the addition of scoby extract has an effect on the healing process of inflammatory acne. the negative control in this study was p8 (without gel application). table 1 shows that the average difference in inflammatory diameter from the beginning of gel application to the 90th hour is 0.10 cm. this means that p8 has a healing activity that tends to be longer than the other treatment groups. at the 90th hour, p8 had a larger inflammatory diameter than the other treatment groups, which was 0.40 cm with a healing percentage that only reached 20%. in addition to being seen from the difference in inflammatory diameter healing and the percentage of healing, the effectiveness of the gel in healing can be seen in the mean rank between treatments as shown in figure 1 figure 1. effect of gel administration on s. aureus inflammation p = treatment (*) = sign of significant difference in mann-whitney test (sig.p<0.05), p1 = mediclin 1%, p2 = scoby 0% citrus peel extract, p3 = scoby 2% citrus peel extract, p4 = scoby 4% citrus peel extract, p5 = scoby 8% citrus peel extract, p6 = scoby 10% citrus peel extract, p7 = scoby 12% citrus peel extract, p8 = no gel application. based on figure 1, it can be seen that the highest healing activity is in p7 (12% scoby gel) with a value of 26. the higher the value in figure 1, the greater the effectiveness of the treatment group. p7. has the best healing activity compared to other treatment groups. this is by table 4.12 which shows that the diameter of acne 17,5 11,67 7,67 14,67 12 15,33 19,17 26 2 0 5 10 15 20 25 30 p1 p2 p3 p4 p5 p6 p7 p8 p9 m ea n r an k 1148 biology, medicine, & natural product chemistry 14 (2), 2025: 1143-1150 inflammation in p7 has healed faster than other groups. p7 has shown an inflammatory diameter of 0.00 at the 90th hour observation marked by the fading of redness on the ears of mice. this was influenced by the amount of scoby added to the gel. in p7, the content of scoby added is more than the other concentration variations. the slowest treatment group in the healing process of inflammatory diameter is p8 (untreated group). judging from figure 1 which shows the mean rank value in p7 is the lowest value compared to other treatment groups. the mean rank value of p8 is 2, which means that this group has a slow effectiveness in healing inflammation caused by s. aureus. this can occur because in p8 the inflammation is not treated so there is nothing that plays a role in accelerating the healing process of acne by s. aureus. the treatment group without gel application means that the inflammation will be allowed to heal itself without encouragement by active substances so that the healing process will take longer. this is consistent with table 1, where group p8 still experienced a reduction in inflammatory diameter after 48 hours. however, the reduction in inflammatory diameter at each observation tends to be slower so that at the last observation (114th hour) p8 still showed an inflammatory diameter of 0.19 cm. this happens because of the body's defence system that can fight against pathogenic bacteria that cause acne. discussion based on figure 1 and the description above, the higher the concentration of scoby kombucha, the faster the healing process on acne caused by s. aureus. it can be concluded that the fastest healing process of the right ear injected by s. aureus is p7 (12% scoby gel). this is shown in the diameter of acne inflammation in 12% scoby gel at the 90th hour which has successfully cured acne inflammation up to 0.00 cm. based on rezaldi et al. (2022), 40% kombucha concentration can inhibit s. aureus up to 15.7 mm which is classified as a strong category. whereas the lower concentration of kombucha (30%) could only inhibit s. aureus up to 7.9 mm which is classified as a medium inhibition zone category. this is due to scoby kombucha which is the result of kombucha fermentation. the higher the content of kombucha compounds, the more compounds are absorbed by scoby kombucha. therefore, the higher the concentration of scoby added, the greater the antibacterial ability. the antibacterial ability of scoby kombucha comes from the basic ingredient, green tea. based on the research of andaryekti et al. (2015) it was showed that green tea leaf extract gel (camelia nobilis) with a concentration of 10% already has antibacterial activity with a clear zone of 17 mm around s. aureus bacteria. this shows that green tea can inhibit s. aureus with moderate inhibition. this is because green tea contains flavonoids which can be an antibacterial agent. in addition, scoby kombucha contains yeast and acetobacter xylinum bacteria which are lactic acid bacteria (lab). a. xylinum will convert fructose and glucose in yeast into ethanol and will produce acetic acid at the end of fermentation. not only acetic acid, green tea kombucha fermentation will also produce other types of acids that can reduce ph and will damage the bacterial cell wall. the fermented acids will be absorbed by the new scoby kombucha layer to form a cellulose layer that floats above the fermentation solution. therefore, scoby has antibacterial potential by damaging the lipid bilayer structure of bacteria by introducing protons from the acid into the bacterial cytoplasm. this will make the cytoplasm acidic conditions and cause protein denaturation until cell death. the higher the organic acid, the higher the ability to inhibit the growth of pathogenic bacteria (abdillah et al., 2022). fermentation of green tea kombucha produces secondary metabolites that will be absorbed by scoby and can damage peptidoglycan in the bacterial cell wall secondary metabolites in scoby are easier to have antibacterial potential on gram-positive bacteria because their cell walls contain higher peptidoglycan. in the research of abdillah et al. (2022) stated that the highest antibacterial activity of 20% bay flower kombucha was on s. aureus with an average inhibition zone of 17.13 mm (strong category) while on s. epididymis, p. aeruginosa, and e. coli had an inhibition zone diameter of 13-15 mm. this is due to the higher permeability of the cell wall so that active compounds easily damaged it. s. aureus has a thin bacterial cell wall so that components that are antibacterial can more easily penetrate the bacterial cell wall to destroy the peptidoglycan contained in the cell wall of pathogenic bacteria (rezaldi et al., 2022). the addition of siamese orange peel extract certainly strengthens the antibacterial activity of scoby kombucha gel against s. aureus. citrus peel extract contains antioxidant compounds and flavonoids that can counteract free radicals so that it plays a role in antibacterial. according to dewi (2019), orange peel extract (citrus nobilis) can be used as a preservative because at a concentration of 10 mg/ml, total phenolic content of 55.46 mg*gae and antioxidant activity of 66.41% were obtained. this shows that orange peel extract has good antibacterial properties. in dewi's research (2019) it was also mentioned that orange peel extract was able to inhibit s. aureus as evidenced by the appearance of a clear zone of 16 mm around the bacteria (medium category). phenol compounds can inhibit cell wall formation and essential oils can inhibit bacteria by disrupting the process of forming cell membranes and walls (mehmood et al., 2015). therefore, the combination of orange peel extract and scoby kombucha will certainly be better in reducing inflammation caused by s. aureus. ferdyanti et al. – effect of different concentration of scoby kombucha as 1149 based on the results obtained in this study, scoby kombucha gel combined with orange peel extract tested in vivo in mice can reduce the diameter of inflammation caused by s. aureus. the use of orange peel extract in scoby gel is known to have high antioxidant activity. in addition, this addition is done to utilise orange peel which is considered less valuable waste. scoby gel combined with orange peel extract has good healing effectiveness by reducing the diameter of inflammation in the ears of mice caused by s. aureus. from this study, a new alternative is produced as a solution in healing acne using natural ingredients that have less side effects. this study needs further research to test the shelf life and stability of the product to ensure product quality in longterm storage. conclusions the difference in the addition of scoby kombucha to the gel can affect the inflammatory healing activity characterised by a decrease in the diameter of acne inflammation in the ears of mice infected with s. aureus. the most effective scoby kombucha gel in reducing the diameter of inflammation caused by s. aureus is 12% scoby gel combined with orange peel extract. the higher the concentration of scoby added, the faster the healing activity. meanwhile, the lowest concentration of scoby (2% scoby) had a slower healing activity compared to the gel with a higher concentration of scoby. competing interests: the authors declare that there are no competing interests. references abdilah, n. a., rezaldi, f., kusumiyati, k., sasmita, h., & somantri, u. w. 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(2017). uji efektivitas antibakteri ekstrak etanol daun sambiloto (andrographis paniculata nees) terhadap bakteri staphylococus aureus. jurnal ilmiah ibnu sina, 2(1), 158-168. doi: https://doi.org/10.36387/jiis.v2i1.93 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 73-82 | doi: 10.14421/biomedich.2025.141.73-82 issn 2540-9328 (online) the effect of angkak addition towards dpph antioxidant activity and yields in mixed angkak-red ginger extracts clarissa christy, fajar nugraha*, andhi fahrurroji, desy siska anastasia pharmacy study program, faculty of medicine, universitas tanjungpura jl. prof. dr. h. hadari nawawi, bansir laut, kec. pontianak tenggara, kota pontianak, kalimantan barat, indonesia. corresponding author* fajarnugraha@pharm.untan.ac.id abstract angkak and red ginger, which represents west borneo’s chinese ethnic group’s herbal cooking, were studied to produce antioxidant activity through shogaol along with monacolin k and respond to dpph (2, 2-diphenyl-1-picrylhydrazyl) test. this research aims to combine the two ingredients into extracts and observe how they affect the yields and ic50 value. methods were started with the extraction of both materials in a variety of simplicia powder weights using 800 rpm stirring speed at 60°c for 120 minutes. the extracts were then tested for percentage yield (%), thin layer chromatography (tlc), and half-maximal inhibitory concentration (ic50) value. ibm spss statistics 25 analysis was also carried out at the end for yields and ic50 values data. results showed that the angkak addition affected the ic50 value without giving any significant impact on each yield amount (p-value>0,05). increased angkak in the variations led to the ic50 values decrease, ranging from 122.996 ppm (mix 1) to 225.399 ppm (mix 3). angkak’s pigments and red ginger’s polyphenol compounds which provide antioxidant ability were found to occupy mix extracts based on the tlc profile. in conclusion, angkak decreased the dpph antioxidant activity of its extract combination with red ginger, but did not affect the yield percentage. keywords: angkak; antioxidant; dpph; red ginger; yield. introduction ayam angkak soup, an herbal cooking from west borneo's chinese community which consists of angkak (monascus-fermented oryza sativa; widely known as “red yeast rice” internationally) and ginger is renowned for its efficacy in illness recovery. as regards that, researchers have proven that shogaol and gingerol as polyphenol compounds in ginger and monacolin k in angkak produce antioxidant activity leading to the reduction of reactive oxygen species (ros) mediated by interleukin-1b (il-1b) and lipid peroxidation in human chondrocyte cells (promdam & panichayupakaranant, 2022; zhu et al., 2019). those findings are worth to drug development which fits the pattern “back to nature” in society believing that natural medicines are relatively safer than synthetic drugs (dorly, 2005). in order to maximize the antioxidant activity, ginger can be replaced with red ginger (zingiber officinale var. rubrum), according to an in vitro study showing a more significant lipid peroxidation activity in mice’s brains. besides, red ginger’s ic50 antioxidant levels have been found at 10.35 ppm and 44.48 ppm, as strong as angkak’s, which reaches 35.37 ppm (hasim et al., 2018; illiyyin akib et al., 2016; munadi, 2020). an appropriate extraction method for optimal antioxidant and yield. monacolin k is thermal-sensitive and degrades at 85-121°c (srianta et al., 2014). extraction at 60°c for 120 minutes at 110 rpm stirring speed has shown an increase in the number of monacolins up to 1760 µg/g (singgih et al., 2014). 6shogaol, which was found to contribute antioxidants better than 6-gingerol is contained maximally in dry ginger extracted at 40°c, but an increase to 60°c in other studies correlated to higher antioxidant levels; therefore, the maceration method is chosen for this research (dugasani et al., 2010; ereifej et al., 2016; guo et al., 2014). despite the thermolabile-friendliness, efficiency should be attained as the method needs 24 hours of soaking and remaceration. extraction temperature is thereby added to soften the plants’ cell wall for easier solvent penetration and better bioactive components dissolution, followed by stirring (kinetic) to increase the mass transfer coefficient and convective mass transfer rate that facilitates the extraction process leading to yield increase. this method also outperforms other extraction types, such as reflux and soxhlet (cha et al., 2020). studies using more significant agitation averaged better dispersion, such as the curcumin nanoparticles tested at 400, 800, and 1200 rpm; and the use of 600-800 rpm in monascus spp. fermentation which produced more manuscript received: 02 december, 2024. revision accepted: 20 march, 2025. published: 23 april, 2025. https://doi.org/10.14421/biomedich.2025.141.73-82 74 biology, medicine, & natural product chemistry 14 (1), 2025: 73-82 optimal pigments than 1200 and 300 rpm. another corroborative research found that sargassum fluitans (seaweed) extracted at 800 rpm allowed more significant secondary metabolites and solvents exchange (akman et al., 2022; gutiérrez et al., 2017; manan, 2017). thus, extraction at 60°c, 800 rpm for 120 minutes, using 96% ethanol, is chosen, since prior research found that it correlated with the increase in antioxidants and obtained up to 13.48645% yields (putri et al., 2021). combining plant extracts with strong ic50 values does not always enhance antioxidant activity. for instance, binahong and basil extracts improved from 51.57 ppm and 42.34 ppm to 24.33 ppm, while cherry leaves and bark improved from 83.149 ppm and 19.632 ppm to 11.148 ppm, while the dutch teak and kaffir lime leaf extracts combination which each had an ic50 value of 35.92 ppm and 48.70 ppm at a volume of 120 μg decreased after being combined in any variation (himawan et al., 2021; nadhira et al., 2018; taswin & nurjana, 2021). on the other hand, research on the yield of combined extracts has never been carried out; however, it was found that yields negatively correlate with antioxidants (braga et al., 2016). it occurs because antioxidant levels also depend on the synergy of interactions between the combined plant ingredients. the interaction of statins (monacolin k) as the main ingredient in angkak and ginger’s shogaol in previous in vivo studies were synergistic and no interactions that diminished the efficacy of the treatment were found based on stockley's herbal medicine interaction (heeba & abd-elghany, 2010; williamson et al., 2009). as a confirmation, this research aims to check whether the yields and ic50 values of angkak and red ginger extracts combination can be maximized by the both interactions, using the dpph method as it suits plants with phenolic compounds and was ever found to respond to angkak as well. in regard to chinese herbalists’ medication dose, the amount of dry ginger powder used in the extraction ranges from 10-40g and angkak ranges from 14-55g (zhang et al., 2022; zubaidah et al., 2015). materials and methods materials the plant materials were red ginger simplicia powder (zingiber officinale var. rubrum), which was taken from agradaya® brand whose production took place in yogyakarta city, indonesia with p-irt number “5103404040184-27” and production code “5cm02220886”, while angkak was taken from su® brand imported by pt global buana mandiri (bpom ri ml 219309046124); the other supportive materials and instruments were 96% ethanol pro-analysis (merck™ 1.00983.2500), dpph (2,2-diphenyl-1-picrylhydrazyl) powder (tokyo chemical industry co.ltd d4313), hotplate stirrer (dlab™ ms-h280-pro), buchner funnel (rocker™ chemker 300), ascorbic acid (cspc weisheng pharmaceutical (shi jia zhi jiang) co. ltd), acetone pro-analysis (smart-lab® 180121001), toluene pro-analysis (smart-lab® 210121001), glacial acetic acid (j.t.baker® 0000204534), silica 60 f254 plate (supelco® 1.05554.0001), methanol pro-analysis (merck™ 1.06009.2500), ethyl acetate pro-analysis (merck™ 1.09623.2500), rotary evaporator (heidolph™ type heizbad hei-vap serial no. 517-61000-00-0), oven (papalolo™), uv light (camag®), uv-vis spectrophotometer (shimadzu® uv-2450 serial no. a10834802090), and ibm spss statistics 25 software. procedures red ginger and angkak extraction angkak was milled and sifted as well as red ginger powder in sieve no.40. both materials were then weighted and mixed in different variations (table 1), then extracted using 96% ethanol (1:10) by hotplate stirrer set to 800 rpm rotating speed at 60°c for 120 minutes (kementerian kesehatan republik indonesia, 2017; putri et al., 2021). table 1. angkak and red ginger extraction composition. extract variation angkak weight (g) red ginger weight (g) total weight (g) 96% ethanol (ml) angkak 30 0 30 300 red ginger 0 30 30 300 mix 1 14 30 44 440 mix 2 30 30 60 600 mix 3 55 30 85 850 extraction was carried out in 3 replications. each macerate obtained was filtered using a buchner funnel, then concentrated using a rotary evaporator and heated in an oven at 50°c to obtain thick extracts (akman et al., 2022; gutiérrez et al., 2017; kementerian kesehatan republik indonesia, 2017; manan, 2017) christy et al. – the effect of angkak addition towards dpph antioxidant activity … 75 yields calculation each percentage yield (%) of the extracts was calculated using mathematical formulation as follows (kementerian kesehatan republik indonesia, 2017). percentage yield (%) = extract weight (g) simplicia powder weight (g) x 100% thin layer chromatography (tlc) profile each extract was diluted with a small amount of 96% ethanol to be spotted on a 6.5 x 1.5 cm silica plate 60 f254 using a capillary tube. the mobile phase used for angkak was ethyl acetate: methanol: aqua (7:1:1) added with one drop of glacial acetic acid to detect pigments, toluene: acetone (9:1) was used for red ginger to detect its phenolic compounds, whereas mix extracts were observed using both mobile phases. the plate containing compounds separated by toluene: acetone (9:1) was sprayed with the vanillin-sulfuric acid reagent. spots of all plates were then examined under uv light at 254 nm and 366 nm wavelengths (kementerian kesehatan republik indonesia, 2017). later, the whole elution was repeated to be stained using 0.2% dpph solution. antioxidant activity measurement antioxidant activity was measured using the dpph method standardized using ascorbic acid. 500 ppm of each extract stock solution was concentrated into 30, 60, 90, 120, and 150 ppm. 3 ml of each was later pipetted to 6 ml of 45 ppm dpph solution. the incubation took 30 minutes at room temperature. the absorbances at the maximum wavelength were then measured using a uvvis spectrophotometer. data analysis all data obtained were analyzed descriptively. percentage yields (%) and ic50 values were analyzed statistically using ibm spss statistics 25 afterward. results and discussion results and discussion should be written as a series of connecting sentences, however, a manuscript with a long discussion should be divided into subtitles. results should be clear and concise. the organoleptic characteristics of the obtained extracts organoleptically, angkak and mix 3 were characterized by thicker textures, mix 1 and red ginger tended to be more fluid, while mix 2 was in between. red ginger extract was yellowish brown or dark brown color with a spicy aroma, whereas angkak and entire mix extracts were dark red and showed no significant color differences with spicy and bitter scents. extracts’ yields calculation and the data in graph form each replication was categorized at a good precision level, based on the acceptance range limit, following the cv horwitz conversion for yield testing methods for this case, which could be extended to 8%. roughly seen, mix 2 yielded the highest (figure 1). however, the yields of all extracts did not differ significantly and categorized as good since they were over 10% (table 2). figure 1. extracts’ yield graph. table 2. obtained extracts’ yields extract variation average yield (%) rsd (%) angkak 11.775 6.282 red ginger 12.381 3.183 mix 1 11.783 3.695 mix 2 12.778 3.023 mix 3 12.268 2.214 spss analysis result for the yields’ data spss analysis of the yield data met the normality and homogeneity requirements (p-value> 0.05). further results from anova showed an insignificant relationship between extract variation and the percent yield gain (p-value> 0.05). thin layer chromatography test results of extracts containing red ginger the stains were mostly purple and brownish in color after being sprayed using vanillin-sulfuric acid, which could be estimated as terpenoid and polyphenol groups. all stains on the whole extracts also reacted to 0.2% dpph spray, giving bright yellow color in purple background, indicating that the extracts were able to provide antioxidant activity (figure 2). 76 biology, medicine, & natural product chemistry 14 (1), 2025: 73-82 figure 2. tlc results for chromatogram profile of red ginger’s specific substances in red ginger extract, mix 1, mix 2, and mix 3 extract using toluene: acetone (9:1) mobile phase. thin layer chromatography test results of extracts containing angkak all the extracts containing angkak gave 3 stains when being eluted, consisting of red and yellow pigment. each of the mix extract reacted to give bright yellow color change with purple background after being sprayed with 0.2% dpph as it contained polyphenol compounds from red ginger, except the angkak single extract (figure 3). figure 3. tlc results for angkak pigment detection in angkak extract, mix 1, mix 2, and mix 3 extract using ethyl acetate: methanol: aqua (7:1:1) added with 1 drop of glacial acetic acid mobile phase. christy et al. – the effect of angkak addition towards dpph antioxidant activity … 77 antioxidant activity (ic50) of each extract and the data in graph form data obtained from the antioxidant activity measurement on a variety of extracts showed that increasing the amount of angkak made the antioxidant activity decrease (figure 4). angkak was tested in two different concentrations, since it was first suspected that the initial concentrations used, which were 30-150 ppm were not optimal. subsequently, an increase in the concentrations to 300-500 ppm was carried out to revalidate the method. however, it turned out to have an insignificant impact on the ic50 values as well. they were getting smaller and became negative in inhibition instead (table 3). later, it was concluded that the angkak used might not be suitable to be detected on dpph method since it usually comes from different product processing in marketing. therefore, it could result in a different contained composition which makes it not the same as the angkak used in other researchers’ journal references whose antioxidants were detectable in the dpph method. the precision of test replication was categorized as good, based on the established criteria for assays in the codex alimentarius involving the food and agriculture organization of the united nations (fao) and the world health organization (who) as well as analytical method validation guidelines from the united nations office on drugs and crime (unodc), which tolerates up to an rsd limit of 20%. figure 4. extracts’ ic50 value graph. table 3. antioxidant activity measured on each extract. extract variation average of ic50 value (ppm) category (farmawaty et al., 2019) rsd (%) angkak (30-150 ppm) 843.942 no activity 3.353 angkak (300-500 ppm) -2515.247 no activity -44.204 red ginger 73.973 strong 13.870 mix 1 122.996 modrate 7.918 mix 2 147.966 moderate 12.001 mix 3 225.399 moderate 7.094 spss analysis result for the antioxidant activity data analysis of the ic50 values using spss obtained parametric data that met the normality and homogeneity requirements (p-value> 0.05); therefore the test was continued with the anova method. the results showed that there was a significant effect of the various extract treatments on the ic50 values (p-value <0.05). when continued in the post-hoc method, it was found that the extract variations that produced insignificantly different ic50 values (p-value <0.05) were only mix 1 and mix 2. discussion extracted red ginger and angkak organoleptic observation organoleptic observation of the extracts showed that variations with higher amounts of angkak, such as angkak and mix 3 are characterized by thicker textures, which could be affected by the use of non-glutinous rice as angkak’s raw material since its higher amylose content could make the texture more solid and dry (dutta, 2023). conversely, extract variations containing more red ginger tended to be more fluid, such as mix 1 and red ginger. the reason could come from the presence of essential oil as a previous study found that 18.080-20.797% was extracted using ethanol solvent stirred at a maximum temperature of 50°c for 1, 3 and 5 hours (anam, 2010). the oleoresin substances which include gingerol and shogaol as phenolic components besides giving a spicy aroma, also produced a yellowish brown or dark brown color, as seen in the red ginger extract (martinus & ramadhani, 2021). on the other hand, angkak and the entire mix extracts were dark red and showed no significant color differences with spicy and bitter scents. extracts’ yields in the yields’ calculation, red ginger extract was found to produce an amount close to another study’s extract using conventional maceration, which was 13.48645% (table 2) (putri et al., 2021). the results even exceeded several other studies which only produced 4.10% and 8.66% yields by that conventional technique with the same ethanol percentage (amalia et al., 2021; sofyah et al., 2022). it proved that thermal addition could increase the extraction’s mass transfer coefficient which affects diffusion and agitation increasing the solid diffusion rate 78 biology, medicine, & natural product chemistry 14 (1), 2025: 73-82 from the bulk liquid to the solid particles due to increased turbulence and decreased liquid boundary layer (agu et al., 2021). therefore, in a shorter time, the thermal kinetic maceration applied was able to provide similar yields to conventional maceration which takes up to 3 days. the fineness of the simplicia particles also affected the yield, resulting in different values, despite the same extraction method and solvent. extraction and yield rates increase when the particle size is smaller. the small size is associated with larger interfacial area from the solids contained within. on the other hand, the solvent requires a minimum distance to penetrate the solid particles for extraction. the larger interfacial area contributes to increased pore diffusion between the solute and the solvent, resulting in more effective penetration. larger particles have limited contact surface area that inhibits solvent penetration and solute diffusion. consequently, the number of extracted particles will be transported less to the surrounding solution. in other words, particles with larger diameters decrease the rates of diffusion and extraction (agu et al., 2021). precision between replications was also examined based on the relative standard deviation (rsd) (table 2). the acceptance range limit for this case could be extended to 8%, following the cv horwitz conversion for yield testing methods (riyanto, 2014). therefore, each replication was categorized at a good precision level. the sensitivity of extract components could affect the yields of each extraction replication. for example, m. purpureus pigment, one of the angkak’s metabolites, is sensitive to temperatures over 50°c. hence, the extract drying and storing process requires attentiveness to ensure that none of the extracts are exposed to excessive heat (abdollahi et al., 2021). spss analysis of the yield data met the normality and homogeneity requirements (p-value> 0.05). further results from anova showed an insignificant relationship between extract variation and the percent yield gain (p-value> 0.05), yet some yields clearly seemed higher when processed into graph form (figure 1). it showed that the active component’s were extracted more since the solvent affinity for each substance and the components interaction in mixed extracts could affect the yield amount. moreover, contaminants, such as bromine and iodide as volatile which are often found contaminating various products could happen and affect the yield amount as well (mesko et al., 2016). various types of substances could be extracted because ethanol is categorized as a universal solvent. its hydroxyl group makes it appear as a polar solvent. nonetheless, it can extract non-polar components such as alkanes and cycloalkanes because of the non-polar end on the alkyl group. a small quantity of aromatic components can also be extracted because they can form fairly weak hydrogen bonds. in addition, alkene molecules are able to be extracted slightly, as well as slightly polar aromatic components. thin layer chromatography (tlc) test results during tlc, the eluent used for the chromatogram profile of red ginger’s specific substances tended to be non-polar. it consisted of toluene and acetone whose polarity indexes are sequentially 2.3 and 5.4, making it more suitable for non-polar compounds, such as gingerol and shogaol (schirmer, 1991). the separated compounds on each extract containing red ginger showed similar characteristics, the stains were mostly purple and brownish after being sprayed using vanillin-sulfuric acid, which could be estimated as terpenoid and polyphenol groups (figure 2). stains with less color intensity after reagent spraying indicated that the separated compound had a side chain with fewer carbon atoms, or a saturated, straight and long chain. the spots of the red ginger extract at rf 0.1 and 0.26 which reacted to vanillinsulfuric acid and were clearly visible under uv254, whereas become invisible under uv366 as they were not categorized as fluorophore (fluorescent chemical compounds that can re-emit light upon light excitation). it fits the characteristics of shogaol and gingerol (wang, 2020). however, comparative standards or ft-ir, lcms, and nmr tests in the following research were needed to accurately estimate the identity of each compound contained. extracts containing the same ingredients in different quantities could result in different stain characteristics, showing that composition interaction can form varieties of substances. the tlc separation of mix 1 extract showed the same rf value as in the red ginger extract at 0.5 (figure 2). however, it was merely visible under uv366 and became unclear after being sprayed using vanillin-sulfuric acid, denoting the presence of polysaccharides, decalin derivatives (monascumic acid), or other components such as monascopurpurone and monacolin k which did not react to vanillin-sulfuric acid (santebennur et al., 2016; zhu et al., 2019). the other compounds that were clearly detected, both under uv254 and uv366, as well as the vanillin-sulfuric acid reagent spray could be the components of red ginger or angkak which are classified as terpenoids, phenols, or organic acids (national environmental research institute (neri), 2022; noman et al., 2020; ramya et al., 2022). spots with the same rf value but different characteristics to every other extract were also detected in mix 2 and mix 3 extracts, respectively at 0.26 and 0.44 as shown in the (figure 2). all stains on the whole extracts reacted to 0.2% dpph spray, giving a bright yellow color on a purple background, indicating that the extracts were able to provide antioxidant activity. tlc for the angkak pigment detection was then carried out using another mobile phase which tended to be polar, namely a mixture of ethyl acetate, methanol, water, and acetic acid, whose polarity indexes are christy et al. – the effect of angkak addition towards dpph antioxidant activity … 79 sequentially 4.3, 6.6, 9, and 6.2 (schirmer, 1991). any visualization reagents were not used because each type of the separated pigments will show its color and they are categorized as azaphilone polyketide which will not react to any reagents (kementerian kesehatan republik indonesia, 2017). therefore, only 0.2% dpph spraying was applied to determine the antioxidant activity qualitatively. all the extracts containing angkak gave 3 stains when being eluted. the red pigment of the first stain was thought to be rubropunctamine, while the second was monascurobramine, based on another previous studies which showed the polarity of the two substances based on log p. the yellow pigment could be monascin or ankaflavin which are more non-polar (figure 3) (shaleha et al., 2022). it is a fluorophore and has a chromophore group hence the blue glow stains under uv366. the more hydrogen bonds it possessed the brighter its fluorescence was than that of red pigments (gai et al., 2018). each of the mix extract reacted to give bright yellow color change with purple background after being sprayed with 0.2% dpph as it contained polyphenol compounds from red ginger, except the angkak single extract (figure 3). antioxidant activity test results later, antioxidant activity measurement using dpph method was carried out using ascorbic acid as the positive control to ensure the procedure’s accuracy. the ic50 value obtained for the control substance was 10.1119 ppm, classified as “very strong” in the antioxidant category, in accordance with the fact that vitamin c is an excellent antioxidant. therefore, it could be concluded that the work on the antioxidant activity testing stages was correct and the instruments used still worked well. the level of precision between replications of this measurement was concluded based on the relative standard deviation (rsd) as shown in (table 3). according to cv horwitz, the requirement for a good rsd specifically for antioxidant activity measurement using uv-vis spectrophotometry is 5.7% or less, whereas according to the international council for harmonization of technical requirements for pharmaceuticals for human use (ich), the requirement for a good rsd is 2% or less (babili et al., 2020; riyanto, 2014). nevertheless, the established criteria for assays in the codex alimentarius involve the food and agriculture organization of the united nations (fao) and the world health organization (who) as well as analytical method validation guidelines from the united nations office on drugs and crime (unodc) tolerates up to an rsd limit of 20% (codex alimentarius comission, 2018; united nations office on drugs and crime, 2009). on that account, it could be concluded that the rsd values in this test were still relatively good. several factors that can influence whether rsd is good or evil include the accuracy of the method for the tested substances, the stability of the tested substances, and the skill of the tester. data obtained from the antioxidant activity measurement on a variety of extracts showed that increasing the amount of angkak made the antioxidant activity decrease, contrary to the initial hypothesis which states that adding angkak can increase the extracts’ antioxidant levels. it was due to the undetected antioxidant activity of angkak, as shown in (table 3). the first suspicion of the initial concentrations used, which were 30-150 ppm was that the existing antioxidant activity was not optimal at those concentrations. subsequently, an increase in the concentrations to 300500 ppm was carried out to revalidate the method. however, it turned out to have an insignificant impact on the ic50 values as well. they were getting smaller and became negative in inhibition instead. the inapplicability of the dpph method to angkak’s primary substance (monacolin k) was thought to be the cause of the detection failure. it was evidenced by studies showing that the result of analysis for lovastatin, which has the same chemical structure as monacolin k, was not as accurate as the lipid peroxidation inhibition assay that showed peroxidation inhibition of up to 93% at a concentration of 132 ppm, exceeding the dpph method with a barely different concentration (100 ppm) which only produced 30% activity due to the fact that statin group compounds primarily work as lipid lowering agents, which reduce oxidative stress as well through mir-29b increase to downregulate pa200 protein (wang et al., 2017). components other than monacolins, namely polysaccharides, gaba, and ergosterol were also found to not correlate with antioxidants in the frap, abts, and dpph methods, only pigments had an effect, but yellow and red pigments were also not detected very strongly in the dpph test. tests that have been carried out only show antioxidant activity of 20-25% for red and yellow pigments at a concentration of 200-400 ppm (amany et al., 2020). on the other hand, researchers who got high ic50 values for angkak primarily ferment the rice which already begins with high levels of phenolics and flavonoids. for example, rice bran from yongin, korea which contains 1,706 µggae/g phenolics and 123 µgqe/g flavonoids which respectively increased to 1,793 µggae/g and 518 µgqe/g after fermentation. from this study, it was also found that the antioxidant activity of rice before and after fermentation differs insignificantly (cheng et al., 2016). fermentation duration also significantly affects angkak’s content. research shows that the content of monascin reaches its peak 20 days after culture, followed by an increase in the gaba amino acids and monascumic acid, as well as rubropunctamine which increases 10 days after culture and reaches the peak around 30 days. the content of monacolins, both in the form of lactones and acids also increases after fermentation for up to 43 days (fukami et al., 2021). in addition, the size of the inoculum used can also affect the amount of monacolin k. studies that have been conducted show that monacolin k levels of 2.881 mg/g 80 biology, medicine, & natural product chemistry 14 (1), 2025: 73-82 were obtained after 10 days of fermentation through 5% inoculum (cheng et al., 2016). thus, the fermentation procedure without official regulations could be the reason for the differences in the content and response of angkak from the dpph method in various tests. another example of this case is the unexplained fermentation method for angkak from bogor ir42 rice raw material only produced an antioxidant activity of 2.64% at 10,000 ppm concentration, whereas bogor angkak extracted in other studies produced an ic50 value of up to 35.37 ppm (asben et al., 2020; illiyyin akib et al., 2016). the general recipe of angkak is only a requirement to use non-glutinous rice as its raw material. non-glutinous rice has higher amylose content, resulting in a more complex texture, thus able to prevent surface area reduction that inhibits fungal growth due to sticky texture which results in fewer pigments and fermentable metabolites. the addition of a certain amount of water during fermentation can increase the amount of amylose due to faster fungal growth, resulting in the release of amylose from gelatinized starch during the sterilization process and the decomposition of α-1,6 glycosyl bonds in starch by the glucoamylase produced by monascus. other metabolites, such as pigments and monacolins also increase in this treatment, but the results vary depending on the rice and the type of fungi used. if it apparently happens to the angkak used in this research, then the cause of the antioxidant activity detection failure by the dpph method could also be caused by the interaction of the amount of amylose which exceeds the phenolics, thereby covering up the reaction of phenolic antioxidants, that even the presence of betulinic acid in angkak which is able to increase red ginger’s phenolic antioxidants becomes ineffectual, proved by the reduced antioxidant activity along with the addition of angkak. amylose even has better bonds to polyphenols than amylopectin because it has more considerable molecular weight, with the type of bond that usually occurs is in the form of hydrogen bonds. several studies have shown that the interaction of polyphenols with amylose chains can reduce the inhibition of dpph and α-amylase, especially if the phenolics contain only 1 hydroxyl group, such as vanillic and syringic acid, so that multiple concentrations of phenolics are needed to increase the inhibition value. amylose itself on the other hand was found to have no antioxidants through the dpph method (lv et al., 2019). in addition, complexation between phenolics and starch can affect the granules’ texture, such as the occurrence of agglomeration in rice starch after gelatination or the formation of granules with many holes, thereby affecting solubility. amylose was extracted due to the presence of thermal and the temperature of 60°c used was found sufficient to break down granules into amylose and cause gelatination, thus forming a thick, gel-like texture (lumdubwong, 2019). however, when being unheated, amylose has poor solubility in ethanol, thus affecting its reaction with dpph during the dissolving process as it was found that the sample and dpph solvent greatly determine the test results (la et al., 2021). analysis of the ic50 values using spss obtained parametric data that met the normality and homogeneity requirements (p-value> 0.05); therefore the test was continued with the anova method. the results showed that there was a significant effect of the various extract treatments on the ic50 values (p-value <0.05). when continued in the post-hoc method, it was found that the extract variations that produced insignificantly different ic50 values (p-value <0.05) were only mix 1 and mix 2. however, the increase and decrease level in ic50 values between entire extracts was quite visible when being displayed in graphical form (figure 4). conclusions the amount of angkak added to the mixed extract of angkak and red ginger affected the dpph antioxidant activity significantly without giving any impact to the amount of each yield. increased angkak in the variations leaded to the decrease of ic50 values, ranging from 122.996 ppm (mix 1) to 225.399 ppm (mix 3). angkak’s pigments and red ginger’s polyphenol compounds which provide antioxidant ability were found to be contained in mix extracts based on the thin layer chromatography (tlc) profile. acknowledgements: the authors would like to thank universitas tanjungpura for the granted funding through 2022 dipa project with contract number “5040/un22.9/pt.01.03/2022”. authors’ contributions: conceptualization: clarissa christy, fajar nugraha, andhi fahrurroji, and desy siska anastasia; methodology: clarissa christy; investigation: clarissa christy; discussion of results: clarissa christy; writing – original draft: clarissa christy; writing – review and editing: fajar nugraha and andhi fahrurroji; supervision: fajar nugraha, andhi fahrurroji, and desy siska anastasia; approval of the final text: clarissa christy, fajar nugraha, andhi fahrurroji, and desy siska anastasia. competing interests: the authors declare that there are no competing interests. funding: this study received a granted funding through 2022 dipa project with contract number “5040/un22.9/pt.01.03/2022”. christy et al. – the effect of angkak addition towards dpph antioxidant activity … 81 references abdollahi, f., jahadi, m., & ghavami, m. 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(2015). formulation of laru angkak (the effect of fillers type on monascus purpureus viability and lovastatin product of angkak fermented. jurnal teknologi pertanian, 16(2), 107–116. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 83-90 | doi: 10.14421/biomedich.2024.131.83-90 issn 2540-9328 (online) analysis of heavy metal cadmium (cd) and lead (pb) in eggplant (solanum melongena l.) in pontianak city by using atomic absorption spectrophotometry (aas) fajar nugraha, syila malinda oktaviani*, sri luliana pharmacy department, faculty of medicine, tanjungpura university, pontianak, west borneo, indonesia. corresponding author* syilamalindaa@gmail.com manuscript received: 26 january, 2024. revision accepted: 08 may, 2024. published: 15 may, 2024. abstract in indonesia, eggplant is a favored vegetable with substantial production. the main objective of this study is to investigate the level of safety of eggplants marketed in traditional wholesale markets in pontianak city against possible contamination of the heavy metals cadmium (cd) and lead (pb), which are commonly discovered in water waste, vehicle exhaust, and excessive pesticide and fertilizer use. alizarin red s for cd metal and dithizon for pb metal were used in qualitative assays. cd and pb levels were measures using an atomic absorption spechtrophotometer (aas) instrument. the analytical method has been verified for linearity, accuracy, precision, limit of detection, and limit of quantification. the qualitative test results showed that all samples had positive results for cd and pb. the quantitative test results revealed that the cd and pb metal contents in the fb (flamboyan) sample were 0.056 mg/kg and 3.5066 mg/kg, consequently, while they were 0.0986 mg/kg and 3.957 mg/kg, consequently, in the pr (puring) sample. in keeping with bpom standard no. 5 of 2018, cd metal levels in fb samples are at a safe threshold, while cd metal levels in pr samples and pb metal levels in fb and pr samples exceed the maximum thresholds that have been set. keywords: eggplant; cadmium (cd); lead (pb); ditizon; alizarin; aas. introduction one of the food products that can be used as fresh food and processed food can be utilized from eggplant plants (solanum melongena l.) (hayati et al, 2022). in indonesia, eggplant production has increased by 17.54%, and in west borneo, the production reached 7,254 tons (badan pusat statistik (bps), 2021). in addition to its good health benefits, because it contains high antioxidants, several studies have found that eggplant is contaminated with heavy metals, such as cadmium (cd) and lead (pb) (ahmed youssef, 2018; mor & ceylan, 2008). these contaminants can come from pesticides, fertilizers, air, and water (defarge, 2018; lakherwal, 2014; lugon-moulin et al., 2006). agricultural activities in the use of pesticides and fertilizers according to several previous studies contain heavy metals cd and pb, especially in phosphate fertilizers containing very high cd metal because the basic ingredients of its manufacture come from phosphate rock which naturally contains cd metal (defarge, 2018; el & selim, 2017; pradika et al., 2019). meanwhile, from the air, contaminant sources can come from transportation activities that emit cd and pb metal exhaust emissions, while contaminant sources from water come from waste disposal due to industrial and community activities (dong, 2017; gautam, 2014; suvarapu & baek, 2017). lead (pb) contamination in eggplant was determined to be 1.96 ± 0.23 mg/kg, and cd contaminants were 0.18 ± 0.02 mg/kg (ahmed youssef, 2018). the safe threshold value for cadmium metal (cd) is 0.05 mg/kg and for lead (pb) is 0.2 mg/kg wet weight in the category of fruit and vegetable products. according to the standard regulations of bpom no.5 of 2018 (badan pengawas obat dan makanan (bpom), 2018) the highest tolerated limit for cd and pb metals entering the body within a week is 7 µg/kg and 25 µg/kg, respectively (world health organization, 2009). long-term exposure to these metals can harm the neurological system and result in renal and liver problems (mahurpawar m, 2015). based on the explanation above, there has been very few researches that examines the contamination of cd and pb metals in eggplant in traditional wholesale markets in pontianak city, such as flamboyan (fb) and puring (pr) markets. therefore, researchers are interested in measuring the levels of cd and pb metals in eggplant samples with standard addition quantification https://doi.org/10.14421/biomedich.2024.131.83-90 84 biology, medicine, & natural product chemistry 13 (1), 2024: 83-90 techniques using atomic absorption spectrophotometry (aas) instrumentation. materials and methods samples and materials samples of solanum melongena l. (purple eggplant variety) were collected on fb and pr traditional wholesale markets of pontianak city (figure 1). the materials used in this research were hno3 (65%), h2o2 (30%) p.a merck (germany), alizarin red s (cd) and ditizon (pb) merck (germany), standard metal cd and pb concentrations of 1000 µg/ml merck (germany). the tools used in this study were atomic absorption spectrophotometry (ssa) instrumentation (shimadzu asc-2000®, kyoto, japan). glassware (pyrex®), analytical balance (ohaous®), hot plate (jeio tech), whatman no.42 filter paper, 60 mesh sieve (pharma lab), and micropipette (toppette pipettor). figure 1. solanum melongena l. (purple eggplant variety) found by author on fb and pr wholesale markets of pontianak city (personal documentation). sample collecting and processing samples were purchased from several traders selling purple eggplant at fb and pr markets in pontianak city. after that, the samples were washed, cleaned, and chopped to minimize the surface area of the samples. the samples were dried in an oven for around 12 hours at a temperature of 105º c. the surface area of the sample was reduced with a blender and equalized in size using a mesh 60 sifter. verification of analysis method linearity the linearity test of cd and pb metals was carried out using the addition standard curve of each sample. the prepared 1.5 ml sample was put into six of 5 ml volumetric flasks. then, each volumetric flask was added 0; 0.4; 0.6; 0.8; 1; and 1.2 ml of 10 µg/ml cd standard concentration and added 0; 0.5; 0.7; 0.9; 1.1; and 1.3 ml of 350 µg/ml pb standard concentration. each volumetric flask was then set to the limit mark with aquabidest in a 5 ml volumetric flask. accuracy and precision the standard addition method is the one utilized to determine the accuracy test. the purple eggplant sample of 5 grams was dissolved with 24 ml of hno3 (65%) using a 250 ml beaker (pyrex®) and 5 ml of cd and pb standard solution per concentration level was added, namely c1 (25 µg/ml cd and 600 µg/ml pb), c2 (30 µg/ml cd and 700 µg/ml pb) and c3 (35 µg/ml cd and 800 µg/ml pb). the mixture was deconstructed using a hot plate (jeio tech, korea) at 120ºc for 50 minutes in a fume hood. at the 30th minute, the mixture was added with 6 ml of h2o2 (30%) while shaking gently. after the deconstruction process was complete, the volume of the deconstruction was measured and filtered using whatman no.42 paper. furthermore, the sample was pipetted in the amount of 1.5 ml, and determined the volume with aquabidest in a 5 ml volumetric flask which will then be measured absorbance with an aas instrument. measurements were carried out 3 times for replication of each concentration. limit of detection (lod) and limit of quantification (loq) determination of the limit of detection (lod) and limit of quantification (loq) of cd and pb metals using linearity data from the addition standard curve of each sample with the linearity regression equation y = bx + a. sample preparation the oxidizing agent used to deconstruct samples with the wet deconstruction method is a mixture of nitric acid and peroxide acid in a ratio of 4:1. the stages carried out are the same as the accuracy test method that is not added to the standard. next, the volume of the deconstruction was measured in a measuring cup (pyrex®) and then filtered using whatman no.42 filter paper. the sample was stored in a glass vial bottle and sealed. qualitative analysis a qualitative test of cd metal was carried out with alizarin red s (ars) complexing reagent, namely into a test tube added 2 ml of sample. furthermore, h2so4 flamboyan (fb) puring (pr) nugraha et al. – analysis of heavy metal cadmium (cd) and lead (pb) in … 85 0.05 m and ars 750 µg/ml are added a few drops, and observe the color changes that occur. a qualitative test of pb metal was carried out with ditizon complexing reagent, namely into the test tube added 2 ml. furthermore, 1 n nh4oh and 0.005% (b/v) ditizon solution were added to a few drops, and observe the color changes that occur. analysis of heavy metal content of cd and pb by standard addition technique samples of 1.5 ml were added to six of 5 ml volumetric flasks. each volumetric flask was then filled with 0; 0.4; 0.6; 0.8; 1; and 1.2 ml of 10 µg/ml cd standard concentration and 0; 0.5; 0.7; 0.9; 1.1; and 1.3 ml of 350 µg/ml pb standard concentration. determined the volume until the limit line of the 5 ml volumetric flask with aquabidest. results and discussion sample processing samples were collected from the traditional wholesale markets of flamboyan (fb) and puring (pr) in pontianak city. one kilogram of wet material is processed into 78.805 g (fb) and 78.765 g (pr) of dry powder. the yield and moisture content produced in the fb market was 7.88% and 5.49%, while in the pr market, they were 7.87% and 5.41%. the moisture content of <10% indicates that the sample is dry and minimal microbial growth is possible. verification of analysis method linearity based on the results of linearity testing of cd and pb metals on the addition standard curve of each sample, there is a linear relationship between the absorbance value (y) and concentration (x) of each metal. this can be seen in figure 2 which shows that the correlation coefficient values of cd and pb metals in fb samples are 0.998 and 0.995, respectively. while the correlation coefficient values of cd and pb metals in the pr sample shown in figure 3 are 0.998 and 0.997, respectively. the correlation coefficient (r) of cd and pb metals in each sample as a parameter of the linearity test meets the requirements set by sni, namely the value of r ≥ 0.995 (habibi, 2020). figure 2. linearity curve of cd and pb metals in (fb) market using standard addition technique. figure 3. linearity curve of cd and pb metals in (pr) market using standard addition technique. accuracy (% recovery) to evaluate the accuracy of an analytical method used can be assessed from the percent recovery of the accuracy test results. the percentage recovery that is closer to 100% can be said that the method used is accurate and can guarantee that the concentration of the analyte obtained is correct (rezeki et al., 2019). the percent value of analyte recovery in table 1 is in the range of 81.7 84.98% (for cd metal) and 98.35 103.22% (for pb metal) which means the method used for sample preparation is an accurate method because the % recovery of cd and pb metals produced meets the y = 0,2292x + 0,0164 r = 0,998 0 0,1 0,2 0,3 0,4 0,5 0,6 -0,1 0,4 0,9 1,4 1,9 2,4 a b so rb an ce concentration (ppm) fb sample cd y = 0,0058x + 0,0258 r = 0,995 0 0,1 0,2 0,3 0,4 0,5 0,6 -10 10 30 50 70 90 a b so rb an ce concentration (ppm) fb sample pb y = 0,2432x + 0,0242 r = 0,998 0 0,2 0,4 0,6 0,8 -0,2 0,3 0,8 1,3 1,8 2,3 a b so rb an ce concentration (ppm) pr sample cd y = 0,00594x + 0,0236 r = 0,997 0 0,1 0,2 0,3 0,4 0,5 0,6 -10 10 30 50 70 90 a b so rb an ce concentration (ppm) pr sample pb 86 biology, medicine, & natural product chemistry 13 (1), 2024: 83-90 requirements according to aoac standards, namely with criteria of 80-110% (for cd metal) and 90-107% (for pb metal). there are three ways to evaluate accuracy methods, namely with certified reference material (crm), spiked-placebo, and addition standard. in this study, the method used was the addition of standards with three different concentrations with three repetitions because it is more simple, affordable, and easy to do than the crm and spiked-placebo methods which require certified comparator compounds (crm) and pharmaceutical preparation carrier materials without active substances (placebo) (harmita, 2004). table 1. percent (%) recoveries of cd and pb metals. ca cs ct average recovery (%) average cd 25 1,7046 1,448 84,98 30 1,8 1,47 81,7 35 2,143 1,8057 84,27 pb 600 40,91 42,22 103,22 700 42 42,23 100,55 800 48,98 48,17 98,35 note : ca = concentration added (µg/ml) cs = true concentration (µg/ml) ct = measured concentration (µg/ml) precision how closely the analytical results from repeated measurements of the same concentration series are derived is how precision is defined. the precision model performed is a repeatability model because it is under the same conditions, same laboratory, analyst, and equipment (harmita, 2004). the precision test is referred to as the percent relative standard deviation (% rsd). table 2 shows the % rsd values generated by pb and cd metals. the % rsd values of cd and pb metals shown in table ii meet the aoac criteria, namely % rsd ≤ 7.3% for cd metal and % rsd ≤ 5.3% for pb metal so that the measurement method carried out repeatedly on the same concentration series has a minimal random error, both from the methods, researcher and instrumental aspects (gandjar g & rohman a, 2007). table 2. percent (%) rsd of cd and pb metals cs ct average sd rsd (%) cd 1,7046 1,448 0,009 0,62 1,8 1,47 0,01 0,68 2,143 1,8057 0,06 3,323 pb 40,91 42,22 1,402 3,32 42 42,23 0,603 1,43 48,98 48,17 0,3956 0,82 note : cs = true concentration (µg/ml) ct = measured concentration (µg/ml) limit of detection (lod) and limit of quantification (loq) the results of the lod and loq measurements in table 3 using the addition standard curve confirm that the instrument is still capable of detecting and quantifying cd and pb metal analytes at the lowest concentrations. table 3. lod and loq. samples lod (µg/ml) loq (µg/ml) fb cd 0,14276 0,47587 pb 15,2868 50,9559 pr cd 0,18458 0,61525 pb 9,13428 30,4476 sample preparation destruction is a process to break down or decompose organic bonds that bind metal ions in the samples so that metals are in a form free that can be measurable (asmorowati ds et al, 2020). the deconstruction method is categorized into two, wet deconstruction and dry deconstruction. in this study, sample preparation used the wet deconstruction method because the equipment used was more simplified and the time required was also relatively shorter than dry deconstruction which requires a relatively longer time and uses more complex equipment, such as a muffle furnace (abata et al., 2019). based on previous research, the wet deconstruction method also produces a higher percent recovery of analytes than dry deconstruction in the analysis of pb metal in soil, namely 98-99% (asmorowati ds et al, 2020). to dissolve the sample's organic bonds, a 4:1 mixture of acid solvents hno3 (65%) and h2o2 (30%) was utilized. the combination of hno3 and h2o2 with a ratio of 4: 1 is the result of optimization from previous research that gets the best results with a % recovery value of 104.2% (cd metal) and 104.75% (pb metal) for the combination of hno3 and h2o2 and a % recovery of 99% for the ratio of 4: 1 (gende & schmeling, 2022; rezeki et al., 2019). hno3 is a strong oxidizer that serves to dissolve or release metal elements from bonds of organic compounds so that metals are in a measurable free form that can be measured, while h2o2 also acts as an oxidizing agent in helping the course of the destruction process. the mixture of strong acids as a destructive agent will increase the strength of the acid so that the destruction process will run faster and maximize in destroying organic bonds in the sample so that metals are released and dissolved (wulandari ea & sukesi, 2013). the deconstruction process was carried out for ± 50 minutes using a temperature of 120ºc. the temperature selection is because hno3 has a boiling point of 121ºc while h2o2 has a boiling point of 150ºc (anothairungrat et al., 2019). heating should be done below the solvent's boiling point so that the solvent does not evaporate before the destruction process is complete. when the nugraha et al. – analysis of heavy metal cadmium (cd) and lead (pb) in … 87 heating process is in progress, gas bubbles and brownishred smoke will arise due to the reaction of the sample when oxidized using hno3, namely co2 gas and no gas. brown smoke indicates that the organic bonds in the sample have been successfully destroyed (wulandari ea & sukesi, 2013). the deconstructed solution after completion will be clear, yellowish in color, thick, and no sediment. qualitative analysis qualitative tests for cd and pb metals use complex reactions, namely ars and ditizon for cd and pb metals (rahman kustiawan & pratiwi, 2016; ullah & haque, 2010). complexing reagents are used because the analytes to be analyzed are small, and hence by using these complexing reagents the visualization of the color formed will be more clearly in showing the positive results of the sample to be analyzed. positive samples containing cd metal, if it reacts with ars, will produce a yellow color, while positive samples containing pb metal, if it reacts with ditizon, will produce a pink color (faradilla et al, 2020; wongthanyakram & masawat, 2019). fb and pr samples are positive for cd metals shown in figure 4 by changing the color from light yellow to dark yellow when adding ars. the color change from less intense was produced before dropping the ars reagent and after adding the ars reagent because the results of the sample destruction used for qualitative tests are already light yellow. the results of this study are from previous studies that analyzed heavy metal cd in kale, mujaer fish, catfish, and cob samples using the wet deconstruction method with ars reagent to get positive results in the qualitative test, which gives a yellow color (rismiarti et al, 2017). figure 4. the qualitative test results of cd metal in fb and pr samples using ars reagent formed a dark yellow color (personal documentation). samples fb and pr are also confirmed to contain pb metal marked by the formation of a pink color when adding ditizon in figure 4. the color produced in the positive control, namely the addition of pb metal standard and ditizon reagent produces a very clear red or dark pink color compared to the sample. it is due to the fb and pr samples containing very little pb metal so the qualitative analysis of the color obtained is very light. the results of this study are from previous research analyzing pb metal in cassava by wet deconstruction method using ditizon reagent and found positive results that give a pink color (wongthanyakram & masawat, 2019). samples fb and pr are also confirmed to contain pb metal marked by the formation of a pink color when adding ditizon in figure 5. the color produced in the positive control, namely the addition of pb metal standard and ditizon reagent produces a very clear red or dark pink color compared to the sample. it is due to the fb and pr samples containing very little pb metal so the qualitative analysis of the color obtained is very light. the results of this study are from previous research analyzing pb metal in cassava by wet deconstruction method using ditizon reagent and found positive results that give a pink color (wongthanyakram & masawat, 2019). figure 5. the qualitative test results of pb metal in fb and pr samples using ditizon reagent formed pink color at the bottom of the tube (personal documentation). analysis of cd dan pb metal levels with standard addition technique pb and cd metal levels are quantified using the ssa instrument and the standard addition method at wavelengths of 228.94 nm and 283.07 nm, respectively. the standard addition technique was used because cd and pb metal contaminants are so small that this technique is needed to enlarge the analyte response on 88 biology, medicine, & natural product chemistry 13 (1), 2024: 83-90 the instrument compared to the interference matrix response that is also in the sample. the obtained cd metal concentrations (x-intercept) in fb and pr samples were 0.072 µg/ml and 0.099 µg/ml, respectively. meanwhile, the pb metal concentrations obtained (xintercept) in fb and pr samples were 4.450 µg/ml and 3.973 µg/ml, respectively. the lod and loq values of each sample do not accept the results for the concentrations of the metals cd and pb. it is suspected that this is due to the limits of the instrument, which is less sensitive because it can only read the lowest concentration of the analyte in ppm (part per million) not in ppb (part per billion). in addition, it is also believed that the method of determining lod and loq with linearity data is not suitable for the quantification technique used, namely the standard addition method. according to previous research, the method of determining lod and loq using linearity data is used for external standard and internal standard quantification techniques (saadati et al, 2013). this research supports research that uses a standard addition quantification technique method, where in determining lod and loq the method uses the signal-to-noise method by measuring ten blanks for 30 replicates. furthermore, the lod and loq values were calculated three times and ten times the standard deviation of the blank measurements (craig et al., 2014; landon et al., 2017). table 4. pb and cd metal levels in fb and pr purple eggplant samples. metals/markets fb (mg/kg) wet weight pr (mg/kg) wet weight pb 3,5066 3,957 cd 0,056 0,0986 based on the data in table 4, shows that the cd metal level in the fb sample is at the safe threshold value set by bpom, which is 0.05 mg/kg, while the cd metal level in the pr sample and the pb metal level in the fb and pr samples exceeds the threshold value set by bpom regulation no.5 of 2018, which is 0.2 mg/kg (badan pengawas obat dan makanan (bpom), 2018). the results of previous studies that also obtained levels of cd and pb metals that were far above the safe threshold value, namely in mustard, kale, and spinach food products in medan's agricultural land where the levels of heavy metal cadmium (cd) were 2 mg/kg, 3 mg/kg, 3 mg/kg and heavy metal lead (pb) were 2 mg/kg, 5 mg/kg and 6 mg/kg respectively (yusuf et al., 2016). according to who, the tolerance limit for heavy metal lead (pb) entering the body in one consecutive week is 25 µg/kg (world health organization, 2009). if the pb metal contamination entering the body exceeds the predetermined tolerance limit, it can cause health problems in the body (balali-mood et al, 2021; mahurpawar m, 2015; prasetya hr et al, 2021). however, in the process of measuring the levels of cd and pb metals in fb and pr samples, several disturbances or obstacles in the ssa instrument allegedly have on the measurement results obtained, namely the lack of sensitivity in the instrument used, because it cannot read the concentration of analytes in ppb (parts per billion) and the flame is not optimal and the hose for pipetting the sample detached during the measurement process. the presence of heavy metals cd and pb in eggplant can be caused by several factors, such as agricultural activities, industrial activities, transportation activities, and air and water pollution (alengebawy, 2021). previous research conducted the application of phosphate fertilizer to the soil as a medium for growing eggplant, which resulted in higher concentrations of cd and pb metals in eggplant in soil added with phosphate fertilizer than those not given, which were 0.18 ± 0.02 mg/kg and 1.96 ± 0.23 mg/kg, respectively (ahmed youssef, 2018). according to a previous study that compared the growing location of eggplant, namely in industrial, traffic (roadside), and rural areas to the concentration of cd and pb metals, the results showed that the highest concentrations of cd and pb metals were found in traffic areas followed by industrial areas and then rural areas (mor & ceylan, 2008). conclusions purple eggplant samples purchased at fb and pr traditional wholesale markets in pontianak city contained cd and pb metals. the level of cd metal in the fb market is at the maximum threshold value, which is 0.05 mg/kg. meanwhile, cd levels in the pr market and pb levels in fb and pr markets exceeded the maximum limit value of contamination set by bpom regulation no. 5 of 2018, which is 0.2 mg/kg. this study has limitations in the instruments and methods of determining lod and loq used. therefore, further research is necessary to reassess the analysis of heavy metals in samples. acknowledgements: we would like to thank the faculty of medicine, department of pharmacy, tanjungpuran university for providing other facilities and infrastructure support. authors’ contributions: the writers whose names are included in this journal contributed to this research. competing interests: the authors declare that there are no competing interests. references abata, e. o., ogunkalu, o. d., adeoba, a. a., & oluwasina, o. o. 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(2016). analysis of metallic content of pb, cu, cd and zn on vegetables sawi kangkung and spinach in the area agriculture and paya rumput village industry titipapan medan. biolink, 3(1), 56–64. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 433-441 | doi: 10.14421/biomedich.2024.132.433-441 issn 2540-9328 (online) prospecting marine natural products as the disease-modifying treatment of alzheimer’s diseases legis ocktaviana saputri1,*, herpan syafii harahap2, arina windri rivarti3, fitriannisa faradina zubaidi4 1department of pharmacology; 2department of neurology; 3department of physiology; 4biomedical department, faculty of medicine and health sciences, universitas mataram, jl. pemuda no 37 kota mataram 83115, indonesia. corresponding author* legisocktavia@unram.ac.id manuscript received: 15 july, 2024. revision accepted: 07 october, 2024. published: 10 october, 2024. abstract alzheimer's disease (ad) is a severe and high costs health problem all over the world. to date, there is no therapy capable of curing ad, making drug discovery a challenging and intriguing field to explore. targeting marine natural products (mnps) as a source of drug leads is a suitable choice considering the content of bioactive compounds and promising pharmacological benefits. this article aims to look at mnps with prospects in several prime targets in ad pathology to cure ad. marine vertebrates (fishes), marine invertebrates (sponges, tunicates, ascidian, clams, scallops, sand dollars, crabs, cockle, sea cucumber, shrimp, bryozoa, marine worm), algae/seaweed, and marine microorganisms (marine fungi, bacterium, dinoflagellata, microalgae) can be potentially used as disease-modifying treatments (dmts) for ad. by targeting multiple aspects of ad pathology, these mnps offer a multifaceted approach to treating and potentially modifying the disease course. this result is an intriguing gap for researchers in the discovery and development of new drugs that can improve ad pathology. keywords: alzheimer’s disease; marine; natural products; drug lead; treatment. abbreviations: aβ: amyloid β; acheis: acetylcholinesterase inhibitors; ad: alzheimer’s disease; ages: advanced glycation endproducts; app: amyloid protein precursor; apoe-4: apolipoprotein e4; bace-1: β-secretase enzyme; bche: butyrylcholinesterase; cns: central nervous system; dmts: disease-modifying treatments; fda: food and drug administration; gsk-3β: glycogen synthase kinase 3β; mapt: microtubule-associated protein tau; mnps: marine natural products; nmda: n-methyl-d-aspartate; nfts: intracellular tau neurofibrillary tangles; nachrs: nicotinic acetylcholine receptors; pkc: protein kinase c; psen: presenilin. introduction ad is the most common type of dementia experienced by individuals over the age of 65 (nugraha et al., 2023). dementia is a disease characterized by a group of symptoms consisting of memory impairment, behavioural disturbances, impaired thinking, and social abilities (lyu et al., 2021). in severe stages, individuals with this disease will experience a reduced quality of life and may become unable to carry out activities independently (nugraha et al., 2023). the number of individuals with dementia worldwide is estimated to continue increasing alongside the ageing population. in 2019, 55 million people were suffering from dementia, this number is expected to rise to 139 million by 2050 (rivarti et al., 2023). the costs incurred for dementia care amounted to approximately 818 billion dollars in 2015 and are projected to continue increasing by 35% every five years. the social impact is also experienced by individuals with the disease and their families who care for them (bălașa et al., 2020). thus, ad has become a severe public health issue with high medical costs and reported no cure (hu et al., 2023). ad therapies that have been used and approved by the u.s. food and drug administration (fda) are acetylcholinesterase inhibitors (acheis) (such as donepezil, rivastigmine, and galantamine) and n-methyld-aspartate (nmda) receptor antagonist (memantine), which only aim to improve ad symptoms (li et al., 2019; cummings et al., 2019; kim et al., 2022). afterwards, there has not been any therapy approved by the fda over the past two decades (kim et al., 2022). the discovery of disease-modifying treatments (dmts), a therapeutic strategy that changes the disease course by addressing the underlying biological pathway, faces many challenges. dmts inhibiting the production and aggregation of amyloid β (aβ) have seen many failures during clinical trials. clinical trial phases for verubecestat, crenezumab, lanabecestat, and solanezumab failed and were discontinued due to ineffectiveness and negative risk/benefit ratio https://doi.org/10.14421/biomedich.2024.132.433-441 434 biology, medicine, & natural product chemistry 13 (2), 2024: 433-441 (cummings et al., 2019; dog et al., 2019). the failure to find effective dmts for ad is caused by the wrong main target of treatment, loss of follow-up during long-term clinical trials, inappropriate drug doses, and too-late starting of therapies in disease development (mehta et al., 2017; kim et al., 2022). the main objections currently faced in ad treatment are a profound understanding of the disease and its pathological mechanisms, reliable biomarkers to enable early diagnosis, identification and development of new drugs and therapeutic strategies that slow disease progression, and the development of prevention protocols that can be used at high-risk populations (cacabelos, 2022). since ad is caused by various molecular processes, identifying a drug capable of modulating more than one target is one of the best strategies in drug discovery and development (martins et al., 2020). to date, marine natural products (mnps) have been demonstrated to be the most promising candidates for therapy of various diseases and are known to have more excellent biological activity than terrestrial natural sources (bălașa et al., 2020). therefore, this article aims to review the prime targets for dmts of ad and mnps prospecting for ad treatment focusing on these targets. prime targets for disease-modifying treatments (dmts) in ad the failure to find an effective therapy for ad is primarily due to a lack of drug discoveries focusing on multiple targets associated with the complex pathophysiology of ad (rivarti et al., 2023). the pathogenesis of ad is inconclusive, but atypical deposition of amyloid and hyperphosphorylation of tau protein in the brain have been key targets for drug discovery for the disease (hu et al., 2023). the main targets for dmts in ad are extracellular amyloid plaques and intracellular tau neurofibrillary tangles (nfts), where β-secretase enzyme (bace-1) activation, neuroinflammation, oxidation, and tau hyperphosphorylation are elements involved in the process (figure 1) (park et al., 2019; kim et al., 2022; rivarti et al., 2023). extracellular amyloid plaques amyloid is an appealing therapeutic target as it is extraneuronal and is associated with environment toxicity (mehta et al., 2017). the inhibition of amyloid-β (aβ) production and plaque formation, as well as the escalation of aβ degradation and clearance, are the main goals in therapy (martins et al., 2020). amyloid protein precursor (app) cleavage by protease enzymes (βsecretase and then γ-secretase), resulting in the production of amyloidogenic aβ peptides (aβ40, aβ42, and aβ43) (cummings et al., 2019; martins et al., 2020). aβ peptides accumulate in the central nervous system (cns), then undergo oligomerization and fibrillization before forming aβ plaques (fereira et al., 2022). the accumulation of aβ plaques causes synaptic dysfunction and neurodegeneration (martins et al., 2020). overexpression of β-secretase 1 (bace-1) limits the rate at the β site app, thus reducing the production of amyloidogenic peptides (koelsch, 2017; martins et al., 2020). therefore, inhibiting bace-1 and γ-secretase is necessary to prevent the formation of aβ. app can also be cleaved via the non-amyloidogenic pathway involving the enzyme α-secretase. conversely, increasing αsecretase activity is required to enhance the formation of α-app, a potential neuroprotective protein (koelsch, 2017; martins et al., 2020). mutations of the app gene, which codes for app substrates, contribute to the increase of aβ fibrillogenesis and total aβ levels. meanwhile, mutations of the presenilin (psen-1 and psen-2) genes which code for proteases for cutting app are known to cause abnormalities and aβ deposition. in addition, the presence of the apolipoprotein e4 (apoe-4) allele decreases aβ clearance in brain tissue which leads to the accumulation of aβ plaque (cacabelos, 2022). the initialization of aβ plaques can occur due to the binding of aβ to nicotinic acetylcholine receptors (nachrs), especially the α7 subtype, leading to a decrease in the normal function of these receptors. oligomerization of aβ resulting from nachr activation is also known to induce phosphorylation of tau proteins (kabir et al., 2021). therefore, nicotinic agonists can be used as therapeutic agents for ad (martins et al., 2020). the aβ plaque and the persistent single shot of proinflammatory cytokines damage neurons and lead to cell death (martins et al., 2020; hu et al., 2023). for this reason, therapy targeted at reducing aβ accumulation and slowing the decline in cognitive function in presymptomatic or mild ad is an appropriate pharmacological intervention. intracellular tau neurofibrillary tangles (nfts) neurofibrillary tangles (nfts) are twisted fibres composed of hyperphosphorylated tau (the microtubuleassociated) protein that accumulates inside neurons. the presence of nfts blocks the transport of nutrients and other essential elements into the neuron, resulting in cell death. glycogen synthase kinase 3β (gsk-3β) is the main enzyme that plays a vital role in the phosphorylation of tau protein (martins et al., 2020; silva et al., 2021; sayas & ávila, 2021). gsk-3β is also a molecular connection between aβ and tau in ad pathogenesis. polymerization of aβ peptides activates gsk-3β, promoting tau protein phosphorylation (hu et al., 2023). on the other hand, gsk-3β also regulates app metabolism and aβ production, reducing aβ deposition and plaque formation. therefore, inhibiting gsk-3β becomes one of the critical targets in the development of ad treatment (sayas & ávila, 2021). setiawan et al. – physicochemical candy cinnamon seaweed carrageenan. 435 gsk-3β can be inhibited by activation of protein kinase c (pkc), one of which can be stimulated by acetylcholine (ach). thus, an increase in ach levels can indirectly inhibit gsk-3β (martins et al., 2020). in addition, tau protein is encoded by a single-copy gene called microtubule-associated protein tau (mapt). this gene mutation can lead to nft formation (cacabelos, 2022). figure 1. the main target for marine natural products (mnps) as drug-modifying treatment (dmts) for alzheimer’s disease (ad). these targets are: (1) extracellular amyloid plaque, (2) intracellular neurofibrillary tangles, (3) neuroinflammation, and (4) oxidative stress. neuroinflammation aβ plaques and nfts cause an immune system response that activates microglia (martins et al., 2020; hu et al., 2023). microglia cells are gathered to remove the noxious compounds, but when elimination is overwhelmed by excessive production, inflammation occurs (martins et al., 2020). long-term microglia activation leads to the accumulation of aβ and prolonged inflammation that damages neurons and causes excessive phosphorylation of tau protein (hu et al., 2023). downregulation of tyrosine kinase represents a valid mechanism for improving autophagic clearance of neurotoxic elements such aβ and mitigating microglialmediated inflammation (stevenson et al., 2022). neuroinflammation is known to be mediated by advanced glycation endproducts (ages). ages can bind to their receptor called rage (receptors for advanced glycation endproducts), leading to the activation of nfκb, a pro-inflammatory transcription factor. nfκb activation can affect two things, namely inducing the expression of pro-inflammatory cytokines such as interleukin (il)-1β, il-6, and tumour necrosis factor-alpha (tnfα), and in turn, can increase oxidative stress due to the production of reactive oxygen species (ros) induced by the inflammatory response. on the other hand, this oxidative stress, in a cyclic manner, can cause a dramatic increase in the amount of ages (kong et al., 2020; reddy et al., 2023). therefore, preventing age-rage interactions with rage antagonists will attenuate ad progression. oxidative stress an abnormal increase in the production of ros leads to oxidative stress, another major contributor to the development of ad (martins et al., 2020). in addition to binding with age, rage can also bind to aβ, forming 436 biology, medicine, & natural product chemistry 13 (2), 2024: 433-441 ros and increasing the secretion of nitric oxide synthase (nos), thereby enhancing the deposition of aβ in the brain (kong et al., 2020). increased ros production causes the translocation of nrf2 from the cytoplasm to the nucleus. this translocation activates the transcription of genes that lead to antioxidant responses. nrf2 activation also leads to increased autophagy, which enhances the clearance of app and tau. nrf2 activators can suppress nf-κb activity, thus also affecting the inflammatory cascade. high levels of nrf2 can ameliorate oxidative stress damage (osama et al., 2020; plano et al., 2023). on the other hand, high levels of aβ and tau can be the cause of oxidative stress. current pharmacological intervention ad therapies that have been used and approved by the fda are therapies that only relieve the symptoms of ad, such as acetylcholinesterase inhibitors (acheis) and nmethyl-d-aspartate (nmda) receptor antagonists. acheis (donepezil, galantamine, and rivastigmine) cholinergic dysfunction occurs in the early stages of ad, leading to a decrease in the levels of various neurotransmitters such as acetylcholine, serotonin, dopamine, norepinephrine, etc. acetylcholine (ach) is an important neurotransmitter that mediates communication between neurons. decreased levels and availability of ach can directly lead to cell damage, thereby reducing the level of serotonin and intensifying the development of nfts. the decrease in ach levels is influenced by the following enzymes: acetylcholinesterase (ache), which plays a role in breaking down acetylcholine, and butyrylcholinesterase (bche), which is responsible for the degradation of acetylcholine (metha et al., 2017). inhibition of ache and bche is associated with delaying the cognitive decline in ad patients. ache inhibitors (acheis) such as donepezil and galantamine, as well as pseudoirreversible achei and bche inhibitors (bcheis) like rivastigmine are symptomatic therapy approved and still in use to date for ad. nevertheless, these drugs are believed to only improve the symptoms of ad without restoring it to its original state (yiannopoulou et al., 2019). nmda receptor antagonist (memantine) insufficient synaptic nmdar signalling compromises neuronal cell survival, but excessive stimulation of glutamatergic signalling leads to excitotoxicity, resulting in damage and death of neurons. nmda receptor antagonists such as memantine are noncompetitive lowaffinity nmda receptor open-channel blockers that inhibit glutamatergic transmissions, leading to decreased glutamate levels and prevention of neuronal dysfunction (wang & reddy, 2017). marine natural products (mnps) prospect for ad treatment about 70% of the earth's surface is the sea and half of global diversity is found in marine ecosystems (ferreira et al., 2022). marine organisms, both marine macroorganisms (sponges, ascidians/tunicates, echinoderms, gorgonians/octocorals, algae, mangroves) and marine microorganisms (marine fungi and marine bacteria) are known to possess diverse bioactive compounds with significant pharmacological efficacy (bălașa et al., 2020; nugraha et al., 2023). mnps show 10 times greater biological activity than terrestrial natural sources, making them attractive for continuous exploration (bălașa et al., 2020). mnps have been demonstrated to be the most prospective candidates for therapy of various diseases. the first marine-derived drug to receive fda approval in 1969 was an anticancer therapy cytarabine from the caribbean sponge tethya crypta. subsequently, vidarabine (1976) from the same source was recognized for its antiviral effects. ziconotide, a synthetic compound similar to a peptide isolated from the venom of the cone snail conus magus, attained fda approval in 2004 for its potent analgesic effect. trabectedin from the marine tunicate ecteinascidia turbinate received fda recognition for second-line treatment of liposarcoma or leiomyosarcoma in 2015. in 2016, eribulin mesylate, a synthetic analogue of halichondrin b isolated from the marine sponge halichondria okadai was approved as a chemotherapy agent (montaser & luesch, 2011; altmann, 2017). limitless discoveries continue, with ongoing exploration of mnps to produce novel therapies for multifarious diseases (altmann, 2017). despite the many studies showing the potential of mnps for ad therapy, development studies of mnps for dmts in ad are still limited to the first stage of the fda drug development process. therefore, this remains a challenge and a fertile ground for further studies in discovering new drugs. table 1 shows the group of mnps with multiple potential targets in ad treatment. marine vertebrae every type of fish is a rich source of n−3 polyunsaturated fatty acids (pufas), especially docosahexaenoic acid (dha) (ferreira et al., 2022). marine fishes such as blackmouth catshark (galeus melastomus) and the brown meagre/ corb (sciaena umbra) contain marine-derived glycosaminoglycans, namely chondroitin sulfate. chondroitin sulfate can combat toxicity caused by ages, which impact aβ aggregation, oxidative stress, and neuroinflammation (iannuzzi et al., 2019; bălașa et al., 2020). the liver oil of cod fish especially gadus morrhua l. consists of polyunsaturated fatty acids, especially ω-3 fatty acids, which have benefits in setiawan et al. – physicochemical candy cinnamon seaweed carrageenan. 437 reducing aβ toxicity through increased degradation and clearance (bălașa et al., 2020). marine invertebrae marine invertebrates include sponges, ascidians/tunicates, echinoderms, gorgonians/octocorals, molluscs (such as octopuses, squids, clams, snails), and arthropods (such as crabs, shrimp, and lobsters). marine sponges spongionella gracilis contains gracilin (diterpenoid), which exhibits activity to inhibit bace-1. gracilin has anti-inflammatory and antioxidant effects through the induction of nrf2, thereby inhibiting hyperphosphorylation of tau proteins. marine sponges haliclona sp. and acanthostrongylophora contain manzamine (an alkaloid) which can inhibit gsk-3 and reduce hyperphosphorylation of tau proteins (hu et al., 2013; kabir et al., 2021). from the marine sponge fascaplysinopsis bergquist sp., an alkaloid called fascaplysin has been isolated, which is known to increase the efflux or clearance of aβ by increasing the level of p-glycoprotein. a synthetic derivative of fascaplysin, 9-methylfascaplysin, has more significant potential in inhibiting the formation of aβ peptide, fibrillation of aβ, and ache. additionally, this derivative compound has the potential to activate antioxidant enzymes that counteract ros, reduce neuroinflammation, and decrease hyperphosphorylation of tau (martins et al., 2020). maldives sponge spongia sp. impact the reduction of tau pathology due to the presence of dictyostatine (marine derived macrolide) inside (xia et al., 2019; kabir et al., 2021). from the marine sponge haliclona moorei and the tunicate aplidium conicum, a natural alkaloid compound that resembles nicotine, called anabasine, has been isolated. this compound and its synthetic analogues are known to have neuroprotective effects by inhibiting apoptosis and necrosis in neurons. additionally, one of its analogues, gts-21, can reduce the amount of aβ, inhibit the activity of γ-secretase, and promote aβ clearance through phagocytosis (martins et al., 2020). ascidian styela plicata contains heparin, which influences neuronal cell death by reducing aβ. sea cucumber acaudina molpadioides, which belongs to the class echinodermata, is known to contain a mixture of glycosphingolipids that can reduce cognitive deficits caused by the aβ1–42 peptide (carroll et al., 2021). various species of clams (anomalocardia brasiliana, tivela mactroides, donax striatus, and tapes phlippinarum), scallop nodipecten nodosus, sand dollar mellita quinquisperforata, and cockle cerastoderma edule are known to contain heparin, which influences neuronal cell death by reducing aβ. white leg shrimp litopenaeus vannamei and crabs (goniopsis cruentata and ucides cordatus), are also known to contain heparin (bălașa et al., 2020). glycosaminoglicans including chondroitin sulphate (cs), heparan sulfate (hs), and dermatan sulphate (ds), from litopenaeus vannamei demonstrates the ability to reduce cognitive and ad related memory impairments by inhibiting the bace-1 activity (carroll et al., 2021; mycroft-west et al., 2021). bugula neritina contains bryostatin-1, a macrolide lactone that can modulate pkcε, leading to the degradation of aβ, activation of αsecretase, and reduction of tau hyperphosphorylation through gsk-3β activity (martins et al., 2020; kabir et al., 2021; botelho et al., 2022). marine worm nemertines have anabasaine that can stimulate various animal nachr (kabir et al., 2021). algae in the effort to develop marine-derived medicines as neuroprotective agents, the brown algae ecklonia cava has been studied and is known to reduce the production of aβ, tau hyperphosphorylation, and neuroinflammatory markers (nfκb and stat3) due to its fucoidan content (park et al., 2019; jo et al., 2023). the phlorotannin content of ecklonia cava, including dieckol, eckol, and 8,8′-bieckol, has inhibitory effects on bace-1 and antiinflammatory effects through the inactivation of the nfκb pathway. dieckol is known for its high ability to inhibit the accumulation of aβ peptide through the regulation of app proteolysis and aβ production via the gsk-3β signalling pathway (mehta et al., 2017). ecklonia spp. is also known to contain mannuronate oligosaccharides that inhibit aβ aggregation and inhibit bace-1 production, thereby reducing aβ levels (botelho et al., 2022). brown algae panida australis and sargassum horridum contain fucosterol (sterol), which can prevent aβ oligomerization, exhibit antiinflammatory and anti-bace1 properties. on the other hand, brown algae such as sargassum siliquastrum contain fucoxanthin, known for its antioxidant and antiinflammatory effects (hu et al., 2023). brown alga dictyota coriacea isolated compound, dictyospiromide (a diterpenoid-maleimide) is known to have cytoprotective properties and antioxidant effects through activation of nrf2/are signaling (carroll et al., 2021). from the brown algae dictyopteris spp., zonarol has been isolated, which can protect neurons from damage due to oxidative stress (botelho et al., 2022). edible brown algae such as laminaria hyperborea, laminaria digitata, macrocystis pyrifera, ascophyllum nodosum, and laminaria japonica contain polymannuronate (polysaccharide). an alginate-derived polymannuronate, namely seleno-polymannuronate, can inhibit aβ aggregation and reduce app and bace-1. this demonstrates that products from these mnps have the potential as neuroprotective agents.5,34 various marine red algae are known as sources of homotaurine, an amino sulfonate that can inhibit aβ aggregation and oligomerization, also amyloid fibril deposition.5,12 in addition, the green alga haematococcus pluvialis contains astaxanthin (carotenoid), which has been used in the supplementary due to its effects in reducing the production of nf-κb transcription factors and pro 438 biology, medicine, & natural product chemistry 13 (2), 2024: 433-441 inflammatory cytokines.5 dead man’s finger (codium fragile) is a green seaweed that contains mannan, a polysaccharide known to inhibit bace-1.5 some seaweeds are isolated sources of betaine, which is a potent antioxidant.32 marine microorganism marine fungi are fungi isolated from marine habitats, either as obligate microorganisms or as facultative microorganisms (nugraha et al., 2023). streptomyces sp. contains indole, a type of alkaloid that can inhibit the formation of aβ plaque through the activation of nrf2 (hu et al., 2023). streptomyces caniferus and phylum actinobacteria produce caniferolide a which can reduce neuroinflammation, oxidative stress, blockage bace-1, inhibit aβ and tau pathology, and is referred to as a potential compound in ad therapy (carroll et al, 2021; hu et al., 2023). furthermore, streptomyces-derived compounds anhydroexfoliamycin and undecylprodigiosin are nrf2 inducers, thus able to suppress oxidative stress. anhydroexfoliamycin also significantly suppresses gsk-3β and reduces tau hyperphosphorylation (kabir et al., 2021). rifamycin from the marine bacterium salinispora, extracted from the marine sponge pseudoceratina clavate, exhibits neuroinflammatory activity and potential antioxidant activity in inhibiting the production, aggregation, and fibrillation of aβ, including increasing aβ clearance (kabir et al., 2021). marine dinoflagellate alexandrium ostenfeldii contains 13-desmethyl spirolide-c, which influences tau hyperphosphorylation by intervening in gsk-3β and aβ accumulation, especially within cells (kabir et al., 2021). meanwhile, some dinoflagellate species are known to produce compounds that are potent in reducing aβ plaques and tau hyperphosphorylation. protoceratium reticulatum, lingulodinium polyedrum, and gonyaulax spinifera are known to produce yessotoxins, polyether sulfate compounds that can reduce hyperphosphorylated tau and aβ accumulation through pkc activation. karenia selliformis also produces the toxin gymnodimine, which can reduce intracellular aβ accumulation and inhibit gsk-3. additionally, gambierol obtained from gambierdiscus toxicus can reduce the levels of aβ1-42, inhibit bace-1, and increase gsk-3β inactivation (botelho et al., 2022). microalgae have several types of polyunsaturated fatty acids such as dha, eicosatetraenoic acid, αlinolenic acid, arachidonic acid, linoleic acid, and oleic acid, which are known to inhibit aβ-40 and aβ-42 fibrillogenesis, thus having anti-aggregation effects (shatshat et al., 2019). the dha can reduce aβ deposition, tau phosphorylation, and bace-1 also γsecretase activity. neuroprotectin d1 is a derivative of dha that has strong anti-inflammatory effects through the inhibition of proinflammatory cytokines, antiamyloidogenic effects through the activation of αsecretase and downregulation of bace-1, and antiapoptotic effects (martins et al., 2020). table 1. mnps and their multitarget prospects in ad pathology. source species bioactive compoud site of action marine vertebrae fishes blackmouth catshark (galeus melastomus) the brown meagre/ corb (sciaena umbra) chondroitin sulfate (glycosaminoglycans) combat toxicity caused by ages impact aβ aggregation oxidative stress neuroinflammation cod fish (gadus morrhua l) ω-3 fatty acids increase aβ degradation and clearance marine invertebrae sponges spongionella gracilis gracilin (diterpenoid) inhibit bace-1 induction nrf2 anti-inflammatory and antioxidant effects hyperphosphorylation of tau haliclona sp. acanthostrongylophora manzamine (alkaloid) inhibit gsk3 reduce hyperphosphorylation of tau fascaplysinopsis bergquist sp fascaplysin (alkaloid) increase aβ clearance 9-methylfascaplysin (synthetic derivate) inhibit aβ peptide formation, aβ fibrillation and ache activate antioxidant enzymes reduce neuroinflammation decrease hyperphosphorylation of tau spongia sp. dictyostatine (macrolide) reduce tau pathology haliclona moorei anabasaine (alkaloid) inhibit apoptosis and necrosis of neurons gts-21 (nicotine reduce aβ setiawan et al. – physicochemical candy cinnamon seaweed carrageenan. 439 analog) inhibit γ-secretase promote aβ clearance tunicate aplidium conicum anabasaine (alkaloid) inhibit apoptosis and necrosis of neurons ascidian styela plicata heparan sulfate (natural glycosaminoglicans) reduce aβ clams anomalocardia brasiliana tivela mactroides donax striatus tapes phlippinarum scallop nodipecten nodosus sand dollar mellita quinquisperforata crabs goniopsis cruentata ucides cordatus cockle cerastoderma edule sea cucumber acaudina molpadioides mixture of glycosphingolipids reduce aβ1–42 peptide white leg shrimp litopenaeus vannamei natural glycosaminoglicans: chondroitin sulphate heparan sulfate dermatan sulphate inhibit bace-1 bryozoa bugula neritina bryostatin-1 (macrolide lactone) modulate pkcε degradate aβ activate α-secretase interfere gsk-3β reduce tau hyperphosphorylation marine worm nemertines anabasaine stimulate nachr algae/ seaweed brown algae ecklonia cava fucoidan reduce aβ production reduce tau hyperphosphorylation reduce nfκb and stat3 phlorotannin (dieckol, eckol, and 8,8′-bieckol) inhibit bace-1 inactivate nf-κb inhibit aβ peptide accumulation interfere aβ production ecklonia spp. mannuronate (oligosaccharides) inhibit aβ aggregation inhibit bace-1 production panida australis sargassum horridum fucosterol (sterol) prevent aβ oligomerization anti-inflammatory anti-bace1 sargassum siliquastrum fucoxanthin antioxidant anti-inflammatory dictyota coriacea dictyospiromide (diterpenoid-maleimide) activate of nrf2/are signaling antioxidant cytoprotective dictyopteris spp. zonarol antioxidant laminaria hyperborea laminaria digitata macrocystis pyrifera ascophyllum nodosum laminaria japonica polymannuronate (polysaccharide) selenopolymannuronate reduce app inhibit bace-1 reduce aβ aggregation marine red algae homotaurine (amino sulfonate) inhibit aβ aggregation and oligomerization inhibit amyloid fibril deposition green alga haematococcus pluvialis astaxanthin (carotenoid) reduce production of nf-κb and proinflammatory cytokines green seaweed dead man’s finger (codium fragile) mannan (polysaccharide) inhibit bace-1 some seaweeds betaine potent antioxidant marine microorganism fungi streptomyces sp. indole (alkaloid) activate nrf2 inhibit aβ plaque formation anhydroexfoliamycin induce nrf2 suppresses gsk-3β reduces tau hyperphosphorylation undecylprodigiosin induce nrf2 440 biology, medicine, & natural product chemistry 13 (2), 2024: 433-441 streptomyces caniferus phylum actinobacteria caniferolide a reduce neuroinflammation reduce oxidative stress blockage bace-1 inhibit aβ and tau pathology marine bacterium salinispora rifamycin neuroinflammatory antioxidant inhibit aβ production, aggregation, and fibrillation increase aβ clearance dinoflagellate alexandrium ostenfeldii 13-desmethyl spirolide-c interfere gsk-3β influence tau hyperphosphorylation reduce intracelular aβ accumulation protoceratium reticulatum lingulodinium polyedrum gonyaulax spinifera yessotoxins (polyether sulfate) reduce hyperphosphorylated tau reduce aβ accumulation karenia selliformis gymnodimine reduce intracellular aβ accumulation inhibit gsk-3 gambierdiscus toxicus gambierol reduce aβ1-42 inhibit bace-1 inactivating gsk-3β microalgae several species dha eicosatetraenoic acid α-linolenic acid arachidonic acid linoleic acid oleic acid inhibit aβ-40 and aβ-42 fibrillogenesis anti-aggregation reduce aβ deposition reduce tau phosphorylation anti-bace-1 reduce γ-secretase neuroprotectin d1 (dha derivate) inhibit proinflammatory cytokines activate α-secretase downregulate bace-1 anti-apoptotic conclusions marine natural products from marine vertebrates, marine invertebrates, algae/seaweed, and marine microorganisms can be utilized for their potential as they can play a role in several therapeutic targets in ad pathology. these findings can be further utilized in drug discovery and development. acknowledgments: this article process is sponsored by the faculty of medicines and health sciences university of mataram. authors’ contributions: all authors contributed to data collection and sourcing related references, analyzing data, and writing the manuscript. herpan and arina wrote about prime targets in ad therapy. legis wrote about the prospects of mnp in ad therapy, while fitriannisa wrote other components according to the template. subsequently, all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no conflicts of interest in this study. funding: this research received no specific grant from 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(2019). impaired tau–microtubule interactions are prevalent among pathogenic tau variants arising from missense mutation tau mutants impair microtubule interaction. journal of biological chemistry. 294 (48): 18488-18503. doi.org/ 10.1074/jbc.ra119.010178 yiannopoulou kg, anastasiou ai, zachariou v, pelidou sh. (2019). reasons for failed trials of disease-modifying treatments for alzheimer disease and their contribution in recent research. biomedicines, 7(4):97. doi.org/10.3390/biomedicines7040097. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 591-594 | doi: 10.14421/biomedich.2024.132.591-594 issn 2540-9328 (online) antibacterial activity testing propionibacterium acnes combination of 70% ethanol extract of starfruit leaves (averrhoa bilimbi l.) and jarak pagar leaves (jatropha curcas l.) tarisya anilya dwi putri, agnes yuliana*, krismayadi department of pharmacy, faculty of health science and technology, binawan university, jl. kalibata raya no. 25 east jakarta city, jakarta 13630, tel. +62-280-880882, indonesia, indonesia. corresponding author* agnesyuliana@binawan.ac.id manuscript received: 30 june, 2023. revision accepted: 04 december, 2024. published: 23 december, 2024. abstract acne is an inflammation accompanied by blockage of oil gland ducts that gather and cause blackheads. the onset of acne can be caused by the bacterium propionibacterium acnes. flavonoid compounds and tannins contained in star fruit leaves and jatropha leaves can be used as antibacterials. both extracts from the plant can be combined to achieve antibacterial effects that can be used as an alternative to acne treatment. the purpose of this study was to determine the antibacterial activity of star fruit leaf extract and jatropha leaf and determine the optimal concentration resulting from the antibacterial activity of the extract combination. the method used is experimental and antibacterial tests in vitro. the results of antibacterial tests on the combination of jatropha leaf extract and star fruit leaf extract against p. acnes had the most optimal average inhibitory zone of 9,642 mm with a ratio of 1:2 at a concentration of 10%. keywords: averrhoa bilimbi l.; combination extract; jatropha curcas l.; propionibacterium acnes. introduction acne is inflammation accompanied by blockage of the pilosebaceous duct which causes sebum not to come out and collect which then swells and blackheads appear. the onset of acne is caused by stressful conditions, smoking, the influence of food intake with high fat and calorie content, frequent consumption of fast food, lack of attention to skin hygiene, increased physical activity, psychological influences, and propionibacterium acnes bacterial infections (febriani et al., 2022). p. acnes includes rod-shaped gram-positive bacteria with normal skin flora that plays a role in acne formation (rahmi et al., 2015). the mechanism of acne formation by p. acnes is that bacteria can damage the stratum corneum and stratum germinativum by secreting chemicals that can break down the pore wall. in addition, fatty acids and skin oils can be blocked and then hardened, causing inflammation that if touched will expand (afifi et al., 2018). giving antibiotics such as clindamycin can be done to reduce the growth of p. acnes (asdika et al., 2020). clindamycin as an antibiotic works by preventing peptide binding so that it can inhibit bacterial protein synthesis. clindamycin is a broadspectrum antibiotic that has bacteriostatic or bactericidal effects depending on the concentration of the drug, the degree of infection and the type of organism (novaryatiin, 2016; jayanti, 2021). however, excessive use of antibiotics can lead to increased bacterial resistance to antibiotics (rahmi et al., 2015). the development of natural ingredients as an alternative to acne treatment can be done to prevent side effects of synthetic drugs, especially preventing the possibility of bacterial resistance. examples of natural ingredients such as star fruit leaves and castor leaves have antibacterial effects that are beneficial for acne expansion (afifi et al., 2018; juliansyah and paotonan, 2017). based on research by afifi et al. (2018), star fruit leaf extract can be used as an antibacterial against p. acnes at a concentration of 150 ppm; 175 ppm; and 200 ppm with an inhibitory zone diameter of 9.88 mm; 10.8 mm; and 11.76 mm expanded (afifi et al., 2018). in addition, based on juliansyah and paotonan, (2017), it was stated that jatropha leaf extract also had antibacterial activity against p. acnes at a concentration of 15% with an inhibitory zone of 10.24 mm (juliansyah and paotonan, 2017) inhibition of bacterial growth in both plants is produced by compounds contained in each plant. both plants contain flavonoid compounds and tannins that can have antibacterial potential. flavonoids as antibacterial work by forming complex compounds with extracellular and dissolved proteins so that they can damage bacterial https://doi.org/10.14421/biomedich.2024.132.591-594 592 biology, medicine, & natural product chemistry 13 (2), 2024: 591-594 cell membranes followed by the release of intracellular compounds. while tannins work by constricting the cell wall so that it interferes with cell permeability and causes cell wall damage (amalia et.al., 2017). materials and methods tools and materials the equipment used is a petri dish (onemed), test tube, cotton, falcon, micropipette (socorex), micropipette tips, ose needle, spider, tweezers, disc, bunsen, vortex (gemmy vm-300 vortex mixer), laf (panasonic), autoclave (hirayama hve-50), incubator and caliper (taffware). the ingredients used in this study were star fruit leaf extract, jatropha leaf extract, sterile aquadest, nutrient agar (na), sterile nacl, clindamycin phosphate, mc. farland standard solution, and propionibacterium acnes bacteria (atcc 11827). procedures simplisia extraction the extraction simplisia of star fruit leaf and jatropha leaves was carried out using the maceration method and the resulting filtrate was then concentrated. the maceration process is carried out by soaking simplistic using 70% ethanol solvent in a ratio of 1:10. soaking is carried out for 3 days and stirred every 1×24 hours for 5 minutes. after that, filtration is carried out to separate the pulp and filtrate. the resulting filtrate is then concentrated using a rotary evaporator at a temperature of 50° c at a speed of 60 rpm (hasim et al., 2019). calculate the resulting yield. % 𝑌𝑖𝑒𝑙𝑑 = 𝐷𝑟𝑦 𝐸𝑥𝑡𝑟𝑎𝑐𝑡 𝑆𝑖𝑚𝑝𝑙𝑖𝑠𝑖𝑎 𝑊𝑒𝑖𝑔ℎ𝑡 × 100% antibacterial activity test of combination of star fruit leaf extract and jatropha leaf testing the antibacterial activity of the extract combination was carried out 5x repeats using several concentration comparisons, namely, 1:1; 1:2, and 2:1. before the testing stage, it is necessary to carry out bacterial rejuvenation. rejuvenated bacteria are made of a bacterial suspension which is then implanted over the surface of the na media by the streak method. then the disc is immersed and placed on the surface of the substrate already planted with bacteria. after that, it is incubated at 36-37°c for 24 hours (puspita, 2021). the resulting inhibitory zone is then calculated using the following formula (hasanah & novian, 2020): 𝑂𝑏𝑠𝑡𝑎𝑐𝑙𝑒𝑠 𝑍𝑜𝑛𝑒 = (𝑉𝐷 − 𝐻𝐷) + (𝐻𝐷 − 𝐷𝐷) 2 vd: vertical diameter hd: horizontal diameter dd: disc diameter results and discussion simplisia extraction simplisia extraction was carried out using the maceration method according to the provisions for making extracts in the indonesian herbal pharmacopoeia edition ii using 70% ethanol solvent with a ratio of 1:10 (kemenkes ri, 2017). table 1. extract yield. parameter starfruit leaf extract jatropha leaf extract sample weight 1.180 grams 1.380 grams extract weight 176,53 grams 244,66 grams % rendemen 14,96% 17,729% extraction of star fruit leaves and jatropha leaves yielded yield values of 14.96% and 17.729%. extraction was carried out for 3 days using 70% ethanol solvent. 70% ethanol solvent is used because it can attract more active compounds than other solvents. in addition, ethanol is polar which can dissolve polar compounds such as flavonoids and tannins (hasanah & novian, 2020). the more polar the solvent used, the more easily dissolved polar compounds such as flavonoids and tannins. so it can be concluded that the extraction results using 70% ethanol will produce a higher yield value compared to 96% ethanol (nahor et al., 2022). the yield produced from star fruit leaf extract is different from previous studies conducted by aryantini et al. (2017) with a yield value of 16.83%. the difference is due to the difference in extraction time. longer extraction time will result in higher extract yield because the chance of contact between solvent and material becomes greater (andriani, et al., 2019). while the yield value produced in jatropha leaf extract is also different from previous studies by saraswati and karina, (2016) with a yield value of 11.28% using 96% ethanol solvent. the difference in the level of polarity of the solvent used can cause the yield value produced to be different. the more polar the solvent used, the more easily dissolved polar compounds such as flavonoids and tannins (nahor et al., 2022). antibacterial activity test of combination of star fruit leaf extract and jatropha leaf based on antibacterial activity testing, the combination of extracts in table 2 shows that the combination of star fruit leaf extract and jatropha leaf from each concentration comparison has antibacterial activity against p. acnes. the inhibitory zone of the three comparisons showed the results of the medium inhibitory zone category. this is based on the diameter category of the inhibitory zone ≤5 mm is included in the weak category, 5-10 mm diameter is medium, 11-20 mm is strong, and ≥21 mm diameter is solid (winastri et al., 2020). putri et al. – antibacterial test propionibacterium acnes combination extract 593 table 2. results of measurement of the inhibition zone of the combination of star fruit leaf extract wuluh and jatropha leaf against p. acnes. replication obstacles zone (mm) kk+ concentration comparison 1 : 1 1 : 2 2 : 1 1 0 18,625 9,56 7,975 8,75 2 0 20,425 7,77 9,505 8,93 3 0 17,525 9,895 9,915 10,075 4 0 17,25 7,345 9,255 9,01 5 0 16,93 6,74 10,66 7,91 average 0 18,151 8,262 9,462 8,93 note: k+ : positive control, k: negative control (a) (b) (c) (d) (e) figure 1. antibacterial activity test of combination of star fruit leaf extract and jatropha leaf against p. acnes. figure 2. inhibitory zone of combination extracts. based on testing the antibacterial activity, it can be seen in table 2 that the ratio of 1:2 produces the largest inhibitory zone against p. acnes, which is 9.642 mm when compared to the ratio of 1:1 (8.262 mm) and 2:1 (8.93 mm). in the negative control, dmso 67% did not produce an inhibitory zone which showed the solvent used did not affect the growth inhibition of p. acnes. a positive control treatment of 1% clindamycin phosphate resulted in an average inhibitory zone of 18.151 mm (figure 2). the inhibitory zone produced in the combination extract test is influenced by the presence of flavonoid compounds and tannins that work as antibacterials. flavonoid compounds have antibacterial abilities by damaging the bacterial cytoplasmic membrane which causes bacterial cells to lose shape and lysis occurs. in addition, tannin compounds can inhibit bacterial growth through their ability to react with bacterial cell membrane proteins and form complex bonds that can turn off bacterial adhesin (winastri et al., 2020). conclusions the combination of star fruit leaf extract and castor leaf can be combined and has antibacterial activity against propionibacterium acnes. the combination of extracts at a ratio of 1:2 produced the highest inhibitory zone compared to other concentration comparisons. acknowledgments: we would like to express our deepest gratitude to the authors involved in this research and other parties involved indirectly. likewise, we say thank you, thank you to the lecturers in the department of pharmacy, faculty of health sciences and technology, binawan university, who have guided the research process so that it can run well. autrhos’ contributions: all authors have an equal contribution to carrying out this study. competing interests: there are no competing interests references afifi, r., erlin, e., & rachmawati, j. 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(2020). aktivitas antibakteri air perasan dan rebusan daun calincing (oxalis corniculata l.) terhadap streptococcus mutans. berita biologi, 19(2). https://doi.org/10.14203/beritabiologi.v19i2.3786 cover jurnal biomenaprochy vol 9 num 2 2020.cdr biology, medicine, & natural product chemistry volume 9 – number 2 – october 2020 issn 2089-6514 (paper) | issn 2540-9328 (online) honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad jafar luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia mohamad amin department of biology, faculty of mathematics and sciences, universitas negeri malang, indonesia widodo department of biology education, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute design & layout: r i y a n t o publisher: state islamic university (uin) sunan kalijaga yogyakarta co-publisher: the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university (uin) sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@yahoo.com biology, medicine, & natural product chemistry volume 9 – number 2 – october 2020 issn 2089-6514 (paper) | issn 2540-9328 (online) contents the role of gene therapy in the treatments of type 1 diabetes mellitus: a review harem othman smail 57 64 polyphenols content and antimicrobial, antioxidant and hemolytic activities of essential oils from four selected medicinal plants growing in algeria noureddine halla, kebir boucherit, bankaddour zeragui, abdelkader djelti, ziane belkhedim, rachida hassani, saada benatallah, hassiba djellouli, oumlkheir kacimi, zahia boucherit-otmani 65 75 isolation and characterization of stigmasterol from fritillaria roylei gunpreet kaur, vikas gupta, r. g. singhal, parveen bansal 77 80 non-alkaloidal compounds from khat (catha edulis) leaves tsegu kiros 81 89 a chemical overview of azanza garckeana yilni edward bioltif, naanma 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(personal communication); setyawan ad 2007, unpublished data). in the reference list, the references should be listed in an alphabetical order (better, if only 20 for research papers). names of journals should be abbreviated. always use the standard abbreviation of a journal's name according to the issn list of title word abbreviations (www.issn.org/2 22661-ltwa-online.php). the following examples are for guidance. journal: saharjo bh, nurhayati ad. 2006. domination and composition structure change at hemic peat natural regeneration following burning; a case study in pelalawan, riau province. biodiversitas 7: 154-158. book: rai mk, carpinella c. 2006. naturally occurring bioactive compounds. elsevier, amsterdam. chapter in book: webb co, cannon ch, davies sj. 2008. ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. in: carson w, schnitzer s (eds) tropical forest community ecology. wiley-blackwell, new york. abstract: assaeed am. 2007. seed production and dispersal of rhazya stricta. 50 annual symposium of the international association for vegetation science, swansea, uk, 23-27 july 2007. proceeding: alikodra hs. 2000. biodiversity for development of local autonomous government. in: setyawan ad, sutarno (eds.) toward mount lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in java island. universitas sebelas maret, surakarta, 17-20 july 2000. [indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com 3: front 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 857-866 | doi: 10.14421/biomedich.2025.142.857-866 issn 2540-9328 (online) optimization of date palm extract (phoenix dactylifera l.) based collagen powder beverage formula with spices (tamarind and cinnamon) addition using d-optimal mixture design method raihanurrabb pawestriaji1*, tri dewanti widyaningsih2 1department of food science and biotechnology; 2food science department, faculty of agricultural technology, brawijaya university, jl. veteran no. 10-11, malang 65145, tel. +62-274-540971, fax. +62-274-519739, indonesia. corresponding author* tria12321@gmail.com manuscript received: 16 july, 2025. revision accepted: 15 october, 2025. published: 21 october, 2025. abstract the trend of collagen-based beauty functional drinks is predicted to increase. however, its use can potentially cause a fishy taste, so it is usually formulated together with fruit or spice extracts. date fruit has the potential for high bioactive content, but its utilization as a powder drink in indonesia is still relatively low. tamarind and cinnamon are also indonesian spice commodities that are rich in bioactive components and have a strong flavor that can cover the fishy taste of collagen. thus, this study aimed to obtain the optimal formulation and the effect of the addition of date, tamarind, cinnamon, and hydrolase collagen powder on the physicochemical characteristics of the final product using the design expert program d-optimal mixture design method. the first stage of the research was determining the upper and lower limits of the independent variables and a fixed value. the optimal formula suggested by design expert consisted of 50% date powder, 24,91% tamarind powder, 20,09% cinnamon powder, and 5% hydrolase collagen powder with a desirability value of 0,805. the second stage of characterization testing in the form of chemical tests resulted in antioxidant activity (ic50) of 263,25 ± 8,01 ppm and total phenol content of 101,71 ± 9,21 mg gae / 100 g. characterization in the form of physical tests resulted in a moisture content of 2,98 ± 0,01%, dissolving time of 20,52 ± 0,72 seconds, bulk density of 0,52 ± 0,14 g/ml, and hygroscopicity of 18,73 ± 0,04%. keywords: collagen; dates; design expert; functional drinks; spices. introduction functional beverages can be defined as a type of nonalcoholic beverage that in addition to providing hydration effects can also provide other health benefits, mainly due to special additives containing minerals, vitamins, herbs, or other bioactive compounds (li et al., 2019). according to global news wire in kumar et al. (2022), the functional beverage market is expected to grow to $208.13 billion by 2024 with a compound annual growth rate of 7.5% from 2022 to 2027. one type of functional beverage that is quite popular is collagen-based beauty drinks (dini, 2019). collagen is the main protein that dominates as much as 30% of human connective tissue (hashim et al., 2015). consumption of hydrolase collagen-based beverages can increase the supply of collagen in the human body so as to maintain skin elasticity and prevent signs of aging (asserin et al., 2015). however, the addition of too much collagen can cause a fishy flavor, so it is usually formulated with spices or fruit extracts. date fruit is one of the fruits that is rich in antioxidant content such as carotenoids (β-carotene, lycopene, lutein, neoxanthin, and zeaxanthin), phenols, flavonoids, flavonols, cinnamic acid (and its derivatives), and anthocyanins (alhuzali et al., 2023). date fruit in the khalas variety has a lower economic value than other varieties and its utilization as a powder drink is still very limited. development of date powder has been carried out by foam matt drying method (halede, 2024). however, no attempt has been made to increase antioxidant levels with spice substitution, or to increase collagen supply with the addition of hydrolase collagen. tamarind spices contain bioactive compounds such as flavonoids, tannins, alkaloids, saponins, sesquiterpenes (silalahi, 2020). tamarind has a distinctive and fresh sour taste that is expected to balance the sweetness of the date powder produced. in addition, cinnamon has chemical components such as cinnamic alcohol, coumarin, cinnamic acid, cinnamaldehyde, anthocyanins, essential oils, sugar, protein, simple fat, pectin and so on (emilda, 2018). the fragrant, slightly sweet, and warm flavor of cinnamon is expected to balance the taste and https://doi.org/10.14421/biomedich.2025.142.857-866 858 biology, medicine, & natural product chemistry 14 (2), 2025: 857-866 reduce the fishy taste and aroma of collagen from the formulated powder drink. the development of date juicebased functional beverages with the addition of spices has been carried out with a mixture of spices in the form of ginger, ginger, cinnamon, and meniran (rosada, 2024). however, the product is ready to drink so that the shelf life is shorter than powdered drinks. in addition, efforts have not been made to increase collagen supply with the addition of hydrolase collagen. collagen powder drinks based on date juice and spices are still very limited in the market. thus, optimization of the formula of collagen powder drink based on date juice and spices is needed to produce the appropriate quality. this research used the mixture design d-optimal method in the design expert program for the formula optimization process. the research was then continued with the characterization stage based on chemical and physical tests. materials and methods study areas this research was conducted at the food processing technology laboratory, biochemistry and food analysis laboratory, and food sensory analysis laboratory of ftp ub, malang and online. this research started in october 2024 and continued until march 2025. materials in this study, the tools used for the process of making date spice collagen drink include analytical scales (ohaus), electric oven dryer, mixer (philips hr-1538), 100 ml measuring cup (pyrex), grinder (ecentio glass dual-cup juicer), container, and spoon. tools used for antioxidant activity response testing and moisture content included analytical balance (ohaus), beaker glass (pyrex), 100 ml volumetric flask, distilled water bottle, glass stirrer, 1 and 10 ml volume pipettes (iwaki pyrex), bulb, centrifuge (hettich eba 200), centrifuge tube, dark bottle, test tube, vortex (ika 3 z654779), cuvette, uvvis spectrophotometer (shimadzu). tools used for characterization of antioxidant activity, total phenolics, dissolving time, hygroscopicity, and bulk density included centrifuge (hettich eba 200), centrifuge tubes, test tubes (iwaki), bulb, 1 and 10 ml volume pipettes (iwaki pyrex), vortex (ika 3 z654779), cuvette, uv-vis spectrophotometer (shimadzu), stopwatch, stirrer, oven, analytical balance (ohaus), desiccator, and measuring cup (pyrex). the ingredients used for the process of making date spice collagen powder drink include khalas variety dates in the tamar (ripe) phase obtained from the “raja kurma” store in malang city, pure tamarind powder (cairo food), pure cinnamon powder (blantika), fish hydrolase collagen powder (max food), maltodextrin (ex lihua de 10-20 food grade), tbm (koepoe koepoe), stevia extract powder (subur kimia jaya store), and distilled water. the materials used in the antioxidant activity test were 2,2-diphenyl-1picrylhydrazyl (dpph) reagent, methanol pa (smartlab), trolox reagent (merck), and distilled water. the materials used in the total phenol test were folin-ciocalteu reagent (smartlab), gallic acid (merck), na2co3 (smartlab), and distilled water. research procedures determination of upper and lower limits on independent and fixed variables the first stage is the determination of the upper and lower limits on the independent variables consisting of date pollen, tamarind powder, cinnamon powder, and hydrolase collagen powder. fixed variables in the formulation were also determined in the form of 2% stevia extract powder as shown in the following table. table 1. independent variables in date-spice collagen powder beverage formula optimization. ingredient name lower limit upper limit dates powder 40% 50% tamarind powder 20% 25% cinnamon powder 20% 25% hydrolase collagen powder 5% 10% table 2. fixed variables in date-spice collagen powder beverage formula optimization. ingredient name amount (%) stevia extract powder 2 optimization condition on design expert the formulation design in design expert consists of 20 runs with 4 factors, namely date powder, tamarind powder, cinnamon powder and hydrolase collagen powder with a total mixture of 100 and two types of responses, namely antioxidant activity (mg te/100 g) and moisture content (%). the antioxidant activity response will be set according to the desired optimization results, which is at the maximize level while the moisture content is set at the minimize level. raw material analysis and design expert optimization response extraction of khalas date fruit variety the dates were cut into smaller sizes. then, the extraction process was carried out using distilled water solvent with the ratio of date fruit and solvent 2:3. the maceration process was carried out for 3x24 hours at a temperature of 5-9oc to obtain date fruit extract (modification of liandhajani et al., 2024). pawestriaji & widyaningsih – optimization of date palm extract (phoenix dactylifera l.) based … 859 drying date fruits by foam matt drying method drying by the foam mat drying method was carried out by mixing 20% b/v maltodextrin and 1% b/v tbm with date fruit extract liquid and then homogenising with a mixer. the foam was then dried in an electric oven at 70±5oc for 32 hours. the powder was cooled in a refrigerator at 5-9oc for 24 hours. after that, pulverization was carried out with a grinder to obtain date fruit pollen. the powder was then sieved with a 40 mesh sieve to obtain a uniform particle size (modified by halede, 2024; estiasih et al., 2024). based on this procedure, it can be concluded that the ‘date fruit pollen’ component in the formulation contains as much as 20% maltodextrin. antioxidant activity analysis dpph method the antioxidant activity analysis began with the preparation of a standard curve of trolox at dilutions of 0, 10, 20, 30, 40, and 50 ppm. a total of 0.5 ml of trolox solution was put into a test tube, 3 ml of 40 ppm dpph reagent was added, vortexed, and incubated in a dark room for 30 minutes. then, the absorbance was measured by uv-vis spectrophotometer at λ 517 nm. the absorbance results at each trolox concentration were plotted on the linear equation y = ax + b (modification of thaipong et al., 2006; plank et al., 2012). in the sample test, 0.5 ml of sample solution was taken into a test tube and added with 3.5 ml of methanol and 3 ml of 40 ppm dpph. homogenized with a vortex and incubated in a dark room for 30 minutes. the absorbance was calculated by uv-vis spectrophotometer at λ 517 nm. the absorbance results were plotted on the linear equation of the trolox standard curve to obtain concentration data (maramy et al., 2024). moisture content analysis the cup is dried in an oven at 105oc for 1 hour before weighing the cup. the test sample is placed on the cup and the initial weight of the cup containing the sample is weighed. drying in an oven at 105oc for 1 hour and put in a desiccator for 10-15 minutes. weighing the final weight of the cup containing the sample until it reaches a constant weight (difference of 0.002 grams) (aoac, 2005). the percentage of moisture content was calculated using the following formula. moisture content (%) = 𝑎 − 𝑏 𝑏 × 100 notes: a : initial mass before drying (g) b : final mass after drying (g) total phenol analysis folin-ciocalteu method the analysis of total phenolics began with the preparation of a gallic acid standard curve at dilutions of 0, 20, 40, 60, 80, and 100 ppm. a total of 0.5 ml of trolox solution was put into a test tube, added 10% folinciocalteu reagent was added up to 5 ml, vortexed, and incubated in a dark room for 5 minutes. added 7.5% na2co3 solution as much as 4 ml, vortexed, and incubated in a dark room for 60 minutes. then, the absorbance was measured by uv-vis spectrophotometer at λ 756 nm. the absorbance results at each trolox concentration were plotted on the linear equation y = ax + b (modification of singleton et al., 1999; kupina et al., 2018). in the sample test, 0.5 ml of the sample solution was taken into a test tube, 5 ml of 10% folin-ciocalteu reagent was added, vortexed, and incubated in a dark room for 5 minutes. 4 ml of 7.5% na2co3 solution was added, vortexed, and incubated in a dark room for 60 minutes. the absorbance was calculated by uv-vis spectrophotometer at λ 756 nm. the absorbance results were plotted on the linear equation of the gallic acid standard curve to obtain concentration data (maramy et al., 2024). characterization test of optimal formula solution the second stage is the final product characterization stage. the final product consists of 98% independent variables that have been optimized using the mixture design d-optimal method on design expert and 2% fixed variables. before going through the characterization stage, the final product of optimization with the addition of 2% stevia extract powder will go through a re-drying process with an electric oven at 60oc for 4 hours to meet the product requirements stated in sni. no-01-4320-1996 on ‘traditional powder drink’, which has a maximum moisture content of 3%. dissolving time analysis the test sample was weighed at 5 g and put into a glass beaker. added 50 ml of room temperature water and homogenized with stirring. the dissolution time was recorded using a stopwatch until the sample solution was perfectly homogenized (lubis et al., 2023). bulk density analysis the test sample was weighed as much as 2 grams and a 10 ml measuring cup was prepared. put the test sample into the measuring cup and stomped until the sample solidified and did not drop. the mass ratio of the test sample to the volume occupied in the measuring cup is recorded to determine the bulk density value in units of g/ml (wulandari et al., 2020). hygroscopicity analysis prepare a desiccator with a controlled rh of about 75%. weighed the initial weight of the cup and the test sample as much as 0.5 g. put the cup containing the sample into the desiccator for 24 hours. weighed the final weight until it reached a constant weight (huda, 2020). calculated the level of hygroscopicity (hg) using the following formula. 860 biology, medicine, & natural product chemistry 14 (2), 2025: 857-866 hg (%) = (%wi + %fw) 100 + %wi 𝑥100 notes: %wi : (weight of absorbed water/weight of material) x 100% %fw : initial moisture content of the ingredient results and discussion results raw materials analysis results the raw materials analyzed included date pollen, tamarind powder, cinnamon powder, and hydrolase collagen powder. the parameters tested included antioxidant activity (mg te/100 g) dpph method, moisture content (%), and total phenols (mg gae/100 g) folin-ciocalteu method. the results of the analysis can be seen in table 3 below. table 3. raw materials analysis results. test variables dtp tmp cnp clp antioxidant activity (mg te/ 100 g) 34,01±0,92 36,72±5,21 177,19±0,82 moisture content (%) 2,81±0,53 8,45±0,11 9,13±1,04 7,10±1,11 total phenol (mg gae/ 100 g) 26,12±4,60 23,23±0,77 46,38±0,51 notes: dtp = dates powder; tmp = tamarind powder; cnp = cinnamon powder; clp = hydrolase collagen powder. formulation of date spice based collagen powder drink with mixture design in using the d-optimal mixture design method, the optimal formulation can be determined based on the desired target response. in this study, the antioxidant activity response will be set at the maximize level while the moisture content is set at the minimize level. the results of laboratory analysis of 20 formulations can be seen in table 4 below. table 4. response analysis result of date spice based collagen powder drink formulation with mixture design. independent variables responses run dates powder (%) tamarind powder (%) cinnamon powder (%) hydrolase collagen powder (%) antioxidant activity (mg te/ 100 g) moisture content (%) 1 40 25 25 10 66,79 6,54 2 50 20 20 10 31,29 6,27 3 47,5 20 25 7,5 53,41 6,26 4 45 20 25 10 53,99 6,17 5 40 25 25 10 52,24 6,46 6 46,43 22,86 22,86 7,86 69,12 6,34 7 50 20 25 5 73,59 6,10 8 43,21 23,93 23,93 8,93 33,23 6,29 9 50 20 20 10 31,10 6,24 10 47,5 25 20 7,5 77,66 6,28 11 45 25 25 5 109,67 6,17 12 45 25 20 10 72,03 6,38 13 50 25 20 5 108,12 6,37 14 45 25 25 5 94,93 6,48 15 47,5 22,5 25 5 70,09 6,22 16 50 22,5 20 7,5 53,80 6,05 17 50 22,5 22,5 5 78,63 5,99 18 50 20 22,5 7,5 30,32 6,03 19 45 20 25 10 58,84 6,48 20 45 25 20 10 60,97 6,32 modelling and analysis of dpph antioxidant activity response based on the analysis of the design expert program, it is known that the suggested model which considered to explain the interaction between variables on antioxidant activity response is a quadratic model. analysis based on anova for quadratic model of antioxidant activity response can be seen in table 5. pawestriaji & widyaningsih – optimization of date palm extract (phoenix dactylifera l.) based … 861 table 5. anova of quadratic model on antioxidant activity response. antioxidant activity p-value model 0,0017 significant lack of fit 0,0995 not significant the relationship between each component of the independent variable and the antioxidant activity response in the form of an actual equation is as follows. notes: a = dates powder; b = tamarind powder; c = cinnamon powder; d = hydrolase collagen powder the 3d surface graph shows the effect of the proportion between components on the tested response. the inter-component 3d surface graph of the antioxidant activity response can be seen in figure 1 below. figure 1. 3d surface graph of date powder (a), tamarind powder (b), cinnamon powder (c), and hydrolyzed collagen powder (d) for antioxidant activity response. modelling and analysis of moisture content response based on the analysis of the design expert program, it is known that the suggested model and considered to explain the interaction between variables on the antioxidant activity response is a linear model. analysis based on anova for the linear model of moisture content response can be seen in table 6 below. table 6. anova linear model on moisture content response. moisture content p-value model 0,0070 significant lack of fit 0,8169 not significant the relationship between each component of the independent variable and the moisture content response in the form of an actual equation is as follows. notes: a = dates powder; b = tamarind powder; c = cinnamon powder; d = hydrolase collagen powder the 3d surface graph shows the effect of the proportion between components on the tested response the 3d surface graph between components of the moisture content response can be seen in figure 2. below. figure 2. 3d surface graph of date powder (a), tamarind powder (b), cinnamon powder (c), and hydrolyzed collagen powder (d) for moisture content response. y = 0,0454 a + 0,0847 b + 0,0687 c + 0,0846 d y = -2,5068 a + 74,8289 b + 56,3700 c + 152,5313 d – 0,5992 ab – 0,5266 ac – 1,6243 ad – 2,4384 bc – 3,3473 bd – 2,0472 cd b a b a 862 biology, medicine, & natural product chemistry 14 (2), 2025: 857-866 design expert optimization solution prediction the optimized solution from design expert shows the optimal formulation that matches the desired response target. the predicted response is subjected to a triplo verification test with the results in table 7 and table 8 below. table 7. design expert optimization solution prediction. variables response prediction desirability dates powder (%) tamarind powder (%) cinnamon powder (%) hydrolase collagen powder (%) antioxidant activity (mg te/ 100 g) moisture content (%) 50,00 24,91 20,09 5,00 109,67 6,18 0,805 table 8. optimal design expert formula verification result data. variables response prediction dates powder (%) tamarind powder (%) cinnamon powder (%) hydrolase collagen powder (%) antioxidant activity (mg te/ 100 g) moisture content (%) prediction verification 50,00 24,91 20,09 5,00 109,67 105,34±6,61 6,18 6,03±0,23 paired-t test results (p-value) 0,374 0,364 characterization stage: chemical and physical test the final formulation of collagen powder beverage product consists of 98% independent variables that have been optimized using mixture design d-optimal method on design expert and 2% fixed variables. before going through the characterization stage, the final optimized product with the addition of 2% stevia extract powder will go through a re-drying process with an electric oven at 60oc for 4 hours. the results of chemical and physical characterization tests can be seen in table 9 below. table 9. chemical and physical test characterization results. test variables characterization results antioxidant activity (ic50)(ppm) 263,25±8,01 total phenol (mg gae/ 100 g) 101,71±9,21 moisture content (%) 2,98±0,01 dissolving time (seconds) 20,52±0,72 bulk density (g/ml) 0,52±0,14 hygroscopicity (%) 18,73±0,04 discussion raw material analysis results the raw materials analyzed included date pollen, tamarind powder, cinnamon powder, and hydrolase collagen powder. the parameters tested included antioxidant activity (mg te/100 g) dpph method, moisture content (%), and total phenols (mg gae/100 g) folin-ciocalteu method. date powder was found to have antioxidant activity, moisture content, and total phenols of 34.01±0.92 mg te/100 g, 2.81±0.53%, and 26.12±4.60 mg gae/100 g, respectively. according to the literature, date powder contains antioxidant activity with percent inhibition up to 49.59 ± 0.27%, moisture content of 4.71 ± 0.11%, and total phenols of 27.9 ± 0.004 mg gae / 100 g (assous et al., 2021). these differences may occur due to differences in concentration and type of fillers as well as drying temperature and time. date fruit itself naturally contains antioxidant compounds such as simple phenols, flavonoids (including flavones, anthocyanidins, flavonols, and isoflavones), cinnamic acid (along with derivatives such as ferulic acid, caffeic acid, p-coumaric acid, and others), and anthocyanins (flavonoid pigments) (elisya et al., 2017). tamarind powder is known to have antioxidant activity, moisture content, and total phenols of 36.72±5.21 mg te/100 g, 8.45±0.11%, and 23.23±0.77 mg gae/100 g, respectively. according to the literature, tamarind powder contains antioxidant activity with percent inhibition of up to 49.27%, moisture content of 6.56±0.38%, and total phenols of 59.45-131.33 mg gae/100 g (muzaffar et al., 2016; prasasti et al., 2023; bhusari & pradyuman, 2014). the difference may occur due to differences in drying methods, the addition of fillers, time, and drying temperature. tamarind fruit itself naturally contains antioxidant compounds such as tannins and flavonoids (including catechin, epicatechin, proanthocyanidin, procyanidin, taxifolin, and apigenin) (silalahi, 2020). cinnamon powder is known to have antioxidant activity, moisture content, and total phenols of 177.19±0.82 mg te/100 g, 9.13±1.04%, and 46.38±0.51 mg gae/100 g, respectively. according to the literature, tamarind powder contains antioxidant activity with a percent inhibition of up to 85.97%, moisture content of 8%, and total phenols of 13.85 mg gae/g (shoqairan et al., 2023; nurminabari et al., 2019; rakasivi & koo, 2022). these differences may occur due to differences in drying methods, addition of fillers, time, and drying temperature. the antioxidant activity of cinnamon is pawestriaji & widyaningsih – optimization of date palm extract (phoenix dactylifera l.) based … 863 known to come from compounds such as eugenol, safrole, sinamaldehyde, tannins, and calcium oxalate (dirpan et al., 2019). the hydrolase collagen powder was not chemically tested due to the turbidity of the solution caused by the remaining non-collagen content due to incomplete extraction and hydrolysis process (pebrianti et al., 2023) thus interfering with the absorption reading by uv-vis spectrophotometer. however, some literature mentioned the antioxidant activity of hydrolase collagen powder varies depending on the content of hydrophobic amino acids such as histidine, methionine, valine, asparagine, glycine, proline, and glutamine that can react with free radicals (yanti et al., 2022). in addition, the moisture content of the collagen powder was found to be 7.10±1.11%. according to walin et al. (2024), the moisture content of tilapia collagen hydrolysate hydrolyzed with papain enzyme ranged from 4.78 8.30%. modeling and analysis of dpph antioxidant activity response based on table 4, it is known that the tested powder drink formulations have antioxidant activity in the range of 30.32 109.67 mg te/100g. based on the analysis of the design expert program, it is known that the suggested model and considered to explain the interaction between variables on the antioxidant activity response is a quadratic model. in table 5, it can be seen that based on the sequential model sum of squares shows a p-value of 0.0017 (<0.05) for the quadratic model so that the model can be said to be significant. in addition, the value of lack of it or model imprecision shows a pvalue of 0.0995 (> 0.05) so it is not significant. it can be concluded that the inaccuracy of the model has a real effect and the model is considered to be able to explain the effect of the independent variables used and can estimate the optimum formula to get the desired response (widyaningsih et al., 2022). in the actual equation of the selected quadratic model, it is known that the date powder variable (a) individually causes a negative response to antioxidant activity. this could be due to the content of maltodextrin in date powder, which is 20% maltodextrin as filler. the more fillers used, the proportion of ingredients with antioxidant compounds will also be less because basically fillers such as maltodextrin do not contain antioxidant compounds as the main component (framida et al., 2022). in addition, other variables showed a positive response to antioxidant activity due to the pure powder content that naturally contains various bioactive compounds. in addition, it is also known that all interactions between the two factors indicate a decrease in the antioxidant activity of the formula product. this is related to the antagonism that occurs when several antioxidant compounds are formulated together. in figure 1, regarding the 3d surface graph between components and antioxidant activity response, it can be seen that increasing the proportion of tamarind powder, cinnamon, and hydrolase collagen can increase the antioxidant activity of the powder drink formula. in addition, the decrease was caused by the increase in the proportion of date powder and the negative interaction between ingredients. modeling and analysis of moisture content response based on table 4, it is known that the powder drink formulations that have been tested have a moisture content in the range of 5.99 6.54%. based on the analysis of the design expert program, it is known that the suggested model and is considered to explain the interaction between variables on the antioxidant activity response is a linear model. based on table 6, it can be seen that the sequential model sum of squares shows a pvalue of 0.0070 (<0.05) for the linear model so that the model can be said to be significant. in addition, the value of lack of it or model imprecision shows a p-value of 0.8169 (> 0.05) so that it is not significant. in the actual equation of the selected quadratic model, it is known that each variable individually shows a response to the increase in moisture content in the powder drink formulation. this is in accordance with the results of the analysis of raw materials that have been carried out that each variable contains varying moisture content. those are date powder of 2.81 ± 0.53%, tamarind powder of 8.45 ± 0.11%, cinnamon powder of 9.13 ± 1.04% and hydrolase collagen powder of 7.10 ± 1.11%. in figure 2, related to the 3d surface graph between components with the response of moisture content, it is known that the increase in the proportion of date powder, tamarind powder, cinnamon, and hydrolase collagen can each increase the moisture content of the powder drink formula. design expert optimization solution prediction the optimized solution from design expert shows the optimal formulation that matches the desired response target. the predicted response will go through a triplo verification test with the results in table 8. in table 7, it is known that the optimal formulation suggested by design expert is 50% date powder, 24.91% tamarind powder, 20.09% cinnamon powder, and 5% hydrolase collagen powder. the predicted responses to the optimization results are antioxidant activity of 109.67 mg te/100 g and moisture content of 6.18%. in addition, the resulting desirability value is 0.805 where the desirability value which is closer to 1 indicates the better the suggested formulation solution because it is considered capable of producing the predicted optimal response (hidayat et al., 2021). in table 8, it is known that the p-value of paired t-test shows a value of 0.374 for the antioxidant activity response and 0.364 for the moisture content response. this shows that the results of verification in the laboratory are not significantly different from the 864 biology, medicine, & natural product chemistry 14 (2), 2025: 857-866 predicted response from design expert because it has a p-value > 0.05 (niariska, 2024). characterization stage: chemical and physical test based on table 9, related to the results of chemical and physical test-based characterization tests, it is known that the formulated product has an ic50 antioxidant activity value of 263.25 ± 8.01 ppm. ic50 is a value that states the concentration of an extract (ppm) needed to inhibit the oxidation of radical compounds by 50% so that the smaller the ic50 value of an extract the higher the antioxidant activity of the extract (widyaningsih et al., 2015). ic50 values in the range of 250-500 ppm indicate the activity of antioxidant compounds at the ‘weak’ level (asmin et al., 2021). the formulated product has a total phenol of 101.71 ± 9.21 mg gae / 100 g. in the analysis of raw materials conducted, it is known that each independent variable has varying levels of total phenols. the highest total phenol content was in cinnamon powder and the lowest was in date powder. so, it can be said that the total phenols of the final product may be dominated by the content of eugenol, safrole, cinnamaldehyde, tannins, and calcium oxalate in cinnamon powder (dirpan et al., 2019). the formulated product has a moisture content of 2.98 ± 0.01%. the moisture content is influenced by the initial moisture content of the raw materials, temperature, and drying time. basically, this figure has met the product requirements stated in sni. no-01-4320-1996 on ‘traditional powdered beverages’, which has a maximum moisture content of 3%. the formulated product has a dissolving time of 20.52 ± 0.72 seconds. basically, the powder drink cannot dissolve completely due to the presence of sediment from large enough powder molecules. however, the measurement of dissolving time is done only until the beverage solution looks unsuspended. the powder drink product still meets the criteria for the dissolving time of powder drinks which is classified as good because it has a dissolving time of less than 5 minutes (yuliastuti, 2020). the formulated product has a bulk density value of 0.52 ± 0.14 g/ml. the high bulk density can be caused by the high moisture content which causes the powder particles to stick together and the cavities between particles will be smaller so that when weighed in the same volume, the powder will be heavier (hartati, 2023). a large slurry density indicates that the product can be stored in large quantities in small packages so that it is more economical and practical, especially on an industrial scale (wulandari et al., 2020). the formulated product has a hygroscopicity level of 18.73 ± 0.04%. the product is included in the category of hygroscopic products or easily absorbs water because it has a hygroscopicity value in the range of 15.1 20.0% (nurlaela et al., 2024). therefore, it is necessary to use airtight packaging or add anti-caking agents during storage. the high hygroscopicity value of the product can be caused by the natural sugar content in date powder such as fructose and glucose which are hygroscopic due to the presence of hydroxyl groups (-oh) that easily bind to water (assalam et al., 2022). conclusions the optimal date spice collagen powder beverage formula suggested by design expert software consists of 50% date powder, 24.91% tamarind powder, 20.09% cinnamon powder, and 5% hydrolase collagen powder. the predictions generated from the formula were antioxidant activity of 109.67 mg te/100 g and moisture content of 6.18%. the verification test showed no significant difference between the laboratory test and the prediction of the optimal formula response. in the antioxidant activity response, it was known that increasing the proportion of date powder caused a decrease in antioxidant activity while increasing the proportion of tamarind powder, cinnamon powder, and hydrolase collagen powder caused an increase in antioxidant activity. in addition, in the moisture content response, it is known that increasing the proportion of each variable causes an increase in moisture content. the results of chemical test-based characterization on the optimal date spice collagen powder formula with the addition of stevia powder resulted in antioxidant activity (ic50) of 263.25 ± 8.01 ppm and total phenol content of 101.71 ± 9.21 mg gae / 100 g). in addition, the physical test-based characterization results resulted in a moisture content of 2.98 ± 0.01%, a dissolving time of 20.52 ± 0.72 seconds, a bulk density of 0.52 ± 0.14 g/ml, and a hygroscopicity of 18.73 ± 0.04%. acknowledgements: we would like to thank the institution of brawijaya university, malang for providing the necessary facilities during the research. among them include instruments and research tools used by the authors. in addition, to ‘raja kurma’ store, malang city, which has facilitated the need for research raw materials in the form of date fruit with the ‘khalas’ variety. authors’ contributions: raihanurrabb pawestriaji and tri dewanti widyaningsih designed the study. raihanurrabb pawestriaji carried out the laboratory work and analyzed the data. raihanurrabb pawestriaji wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. pawestriaji & widyaningsih – optimization of date palm extract (phoenix dactylifera l.) based … 865 references alhuzali sma, norah mhbj, renad jaa., et al. 2023. dates palm (phoenix dactylifera l.) fruits: nutritional properties and potential applications. the arab journal of agricultural sciences. 6(17): 137–160. doi: 10.21608/asajs.2023.279329. asmin e, anugrah pmdk, maria n, indrawati k, eka a, halidah r. 2021. uji aktivitas antioksidan alga coklat saragassum sp. dengan metode 1,1difenil-2-pikrihidrasil (dpph). pattimura medical review journal. 3(1): 60–72. doi: 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777-781 | doi: 10.14421/biomedich.2025.142.777-781 issn 2540-9328 (online) antioxidant activity test of bride's tears (antigonon leptopus) leaves using the dpph method nailil wafiq azizah, majida ramadhan*, faisal, devana rahma aldina, ramadina novia putri, nur laila anggraini, sabilatul mufarrohah, satria gemilang, firadika fitriansyah, hosiyah, nuvia harini wulan suci, nihlatus sofiya, terra januarista, ramizard rafsanjani biology department; faculty of mathematics and natural science, universitas islam malang, jl. mt haryono 193 gedung utsman bin affan, tel. +620341551932, indonesia. corresponding author* majida.ramadhan@unisma.ac.id manuscript received: 10 june, 2025. revision accepted: 18 september, 2025. published: 01 october, 2025. abstract bridal tear leaves can be used to treat various health and beauty problems because they contain various active compounds, such as flavonoids, saponins, and tannins. these compounds have antioxidant, anti-inflammatory, and antibacterial properties. this flower contains antioxidants that can help protect the skin from damage caused by free radicals. this research aims to see the effectiveness of antioxidants from bridal tear leaves. the research method involves leaf extraction using 96% ethanol and testing antioxidant activity using the dpph method. the research results showed that the extract sample from bride's tear leaves was (ic50 – 69.7146 ug/ml). this shows that the bride's tear leaf extract is quite strong in inhibiting free radical reactions. keywords: antioxidant; etanol extract; dpph. introduction indonesia has a relatively high number of diabetes cases, based on data obtained in 2018 basic health research (riskesdas) results, showing the prevalence of diabetes mellitus in indonesia based on doctor's diagnosis in the population aged ≥15 years by province in 2013 by 1.5%, increasing in 2018 by 2.0%. the diversity of plant species in indonesia has various benefits. the benefits of plants are obtained from the compounds in plants. active plant compounds are obtained by extraction. one of the benefits of plant extracts is that they can withstand or block the occurrence of free radicals. compounds with the ability to ward off free radicals are called antioxidants. antioxidants are compounds that can inhibit and prevent oxidation (simanjuntak, 2012). the way antioxidants work is by stopping free radical reactions from metabolism in the body or from the environment (meigaria et al., 2016). free radicals are molecules with one or more unpaired electrons. free radicals in the body cause cancer, stroke, heart disease, and premature aging (rahman et al., 2016). one of the plants that can act as an antioxidant and prevent free radicals is the leaves of bridal tears (antigonon leptopus). bride's tears is a type of creeping herb that is also often found wrapped around tree trunks. this plant has a twisting tool that functions as a flower support. bride's tears flowers can grow well in conditions exposed to full and direct sunlight or also in a slightly shaded place. a suitable place for planting this flower is between 1 1800 m above sea level (meidita, 2023). the content of the bride's tears leaves includes alkaloids, saponins, tannins, sterols and / or unsaturated triterpenes, flavonoids, glycosides, coumarins, phytosterols, triterpenoidal sapogenins, phenols, amino acids, steroids, xanthoproteins, carboxylic acids, quinines, resins, nhentriacontane, ferulic acid, 4-hydroxycinnamic acid, quercetin-3-rhamnoside, kaempherol-3-glucoside along with ß-sitosterol, ß-sitosterol-glucoside, and d-mannitol (suryowinito, 1997). in research conducted by lestari (2021), the content in bride's tears leaves has properties antioxidant and can inhibit diabetes. testing the antioxidant content is needed to find out how strong the bride's tears leaves are as antioxidants using the dpph test. the dpph method is a method that can be used to determine the antioxidant activity in the sample to be tested by looking at its ability to counteract dpph free radicals, the use of the dpph test is used because it has several advantages including a simple, easy, fast, sensitive method, and requires a small amount of sample (cavalcante, 2018). based on the description that has been presented, it is necessary to test https://doi.org/10.14421/biomedich.2025.142.777-781 778 biology, medicine, & natural product chemistry 14 (2), 2025: 777-781 antioxidants in bride's tears leaves using the dpph method (camero et al, 2018). materials and methods tools and materials the tools used in this study include analytical scales, blender, piper, micropipette, measuring cup, durham tube, oven, test tube and rack, dpph kit, sieve, aluminum foil, spectrophotometer, shaker, filter paper, erlenmayer, glass funnel, evaporator flask and rotary evaporator. the materials used include bride's tears leaves, methanol, dpph solution and quercetin. procedures a. sample preparation sample preparation begins with collecting or picking the bride's tears leaves, which is carried out in kebonagung village, pakisaji district, malang regency. b. sample preparation sample preparation begins with collecting or picking bride's tears leaves carried out in kebonagung village, pakisaji district, malang regency. c. making simplisia fresh bride's tears leaves are first cleaned and sorted then dried using an oven at 50 degrees celsius. after drying the sample is crushed using a blender until smooth, the bride's eye powder is then sifted to get the finest powder and check the water content. water content in simplisia must be below 10%, which aims to avoid the rapid growth of fungi in the extract. d. extraction simplisia was weighed as much as 30 grams and then added 96% ethanol solvent in a mass-to-mass ratio with a volume ratio of 1:10. the sample was heated using a hot plate at a temperature of 40 ° c 50 ° c for 2 hours with 130 rpm. simplisia powder was put into a maceration container and ethanol was added and then allowed to stand for 24 hours. after 24 hours continued with evaporation using a rotary evaporator with an rpm of 50 at 70 ° c for 1 hour to get a paste-shaped extract. the paste obtained was weighed and the result was 3.515 grams. e. calculation of yield the yield is obtained by comparing the weight of the red pomegranate leaf extract and the weight of the simplisia before extraction. according to kurniawan (2016) the calculation of yield can be calculated with the following equation: 𝑌𝑖𝑒𝑙𝑑 (%) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐸𝑥𝑡𝑟𝑎𝑐𝑡 (𝑔) 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑆𝑖𝑚𝑝𝑙𝑖𝑠𝑖𝑎 (𝑔) 𝑥 100 f. preparation of dpph dpph powder was weighed as much as 0.007 grams and then dissolved with 50 ml of ethanol, vortexed until dissolved. furthermore, the dpph solution was taken 1 ml then ethanol was added to 5 ml and allowed to stand for 30 minutes. then a master solution was made with 50 mg of extract and 50 ml of methanol, made a solution with a concentration of 200, 100, 50, 12 and 5 ppm. each solution was taken 1 mil and added dpph as much as 3 ml and incubated for 30 minutes. g. maximum wavelength measurement the maximum wavelength was calculated using a spectrophotometer using a comparison solution, namely quercetin. whether it is lower or higher than the comparison solution, or even has the same antioxidant activity. h. antioxidant activity test of bride's tears leaf extract the results of the maximum wavelength calculation from the spectrophotometer were analyzed using excell to determine the sample’s ic50. results and discussion extraction and yield calculation extraction of bride's tears leaf extract simplisia using maceration method obtained the yield value in table i. table 1. extraction results and calculation of the yield of bride's tear leaves (antigonon leptopus). simplisia weight bride's tears leaf extract caracteristic extract weight yield weight shape, colour, and smell 30 gram 3,515 gram 11,71 gram thick, green leafy odor the yield value shows the bioactive value contained in plants, the higher the yield value, the higher the bioactive value of plants (budiyanto, 2015). according to the indonesian ministry of health, the yield requirement can be used if the yield value is more than 10%. table 1 shows that the yield can be used with a value of 11, 71%. azizah et al. – antioxidant activity test of bride's tears (antigonon leptopus) leaves … 779 qualitative test figure 1. the result of dpph addition to bride's tear leaf extract (antigonon leptopus). the clearer the color resulting from the addition of dpph to the extract, the higher the antioxidant content in the extract. in figure 1, it is known that the clearest color is at a concentration of 12, 5 ppm and the darkest at a concentration of 200 ppm. this color change indicates the antioxidant activity that is happening. if dpph is reduced, it is indicated by a decrease in purplish color to yellow color due to the antioxidant activity of the extract (miksusanti, 2012). antioxidant activity test measurement of the ethanol extract of bride's tears leaves (antigonon leptopus) was carried out at various concentrations, namely 12.5, 50, 100, and 200 µg/ml. the results of the antioxidant activity test by looking at the results of the calculation of absorbance or absorbance carried out on a spectrophotometer were analyzed to determine the percentage of inbibition and ic50 on the leaves of the bride's tears, can be seen in table 2. table 2. analysis of activity test and ic50 on bride's tear leaves (antigonon leptopus). the ic50 test results on the sample have a value of 69.7146 ug/ml as presented in table 2. table 2 shows that bride's tears leaves with the maceration method have antioxidant activity. the results of the linear regression equation can be seen in figure 2. shows that the equation between %inbibition with extract concentration and obtained the standard equation curve y = ax + b, with a value of y = 0.0025x + 0.5752, y with a value of 50 obtained from ic50. the high and low antioxidant activity in the sample is seen from the efficient concentration (ec50) or inhibition concentration (ic50) value, which is the value of 50% dpph losing its free radical properties. the smaller the ic50 value, the higher the antioxidant activity of the sample. according to (sepriyani et al., 2020) a compound is said to be a very strong antioxidant if the ic50 value is less than 50 µg/ml, strong if the ic50 value is between 50-100 µg/ml, moderate if the ic50 value is 100-150 µg/ml, weak if the ic50 value ranges from 150-200 µg/ml. based on this statement, bride's tears leaves (antikodon leptopus) are classified in the strong category. figure 2. linear regression of bride's tears (antikodon leptopus) leaf antioxidant assay the test results of the comparison solution in the form of quercetin have an ic50 value of 35.189 can be seen in table 3. the ic50 value of the test solution is smaller than the ic50 value of the bride's tears leaf sample. although the ic50 value of the comparison solution is smaller, the antioxidant activity of the comparison solution is included in the strong category. the smaller the ic50 price, the stronger the antioxidant in counteracting free radicals or can be said to have stronger antioxidant activity (bujang, 2014). table 3. analysis of activity test and ic50 on bride's tear leaves (antigonon leptopus). concentration review absorbance range inbibition (%) ic50 1 0,737 2 0,568 3 0,555 1 0,557 2 0,523 3 0,483 1 0,351 2 0,221 3 0,483 1 0,087 2 0,176 3 0,122 61,89 67,97 83,65 92,11 69,7146 12,5 ppm 50 ppm 100 ppm 200 ppm 0,128 0,266 0,521 0,620 concentration review absorbance range inbibition (%) ic50 1 1,243 2 1,167 3 1,207 1 1,023 2 1,147 3 1,07 1 0,908 2 0,902 3 0,842 1 0,768 2 0,638 3 0.623 1 0,503 2 0,513 3 0,404 30 0,473 70,9096 35,189 33,6202 25 0,884 45,6771 27,5 0,676 58,4306 20 1,207 25,7939 22,5 1,08 780 biology, medicine, & natural product chemistry 14 (2), 2025: 777-781 the results of the linear regression equation can be seen in figure 3. shows that the equation between %inbibition and the concentration of the comparator solution obtained a standard equation curve y = ax + b, with a value of y = 4.6017x 68.156. figure 3. linear regression of the antioxidant assay of the comparison solution (quercetin). an antioxidant activity test in a plant is very important to know whether the plant has been proven to have binding activity against free radicals. bride's tear leaves can be used to treat various health and beauty problems because they contain various active compounds, such as flavonoids, saponins, and tannins. these compounds have antioxidant, anti-inflammatory, and antibacterial properties. in this study, the samples used were extracts of bride's tears leaves that had been macerated using 96% ethanol and quercetin as a comparator, quercetin is the main flavonoid compound included in the flavonol group (materska, 2008). the selection of 96% ethanol as a solvent is preferred because it presents a better amount of poly phenols compared to water extracts, is more efficient in penetrating cell walls and pulling polyphenols out of cells, and is preservative against microorganisms (lapornik et al., 2005). dpph compound is a molecule containing unstable nitrogen free radical compounds that can bind hydrogen ions so it is used for testing antioxidant activity (permana, 2003). the presence of antioxidant activity in the test solution of bride's tears leaf extract in various concentrations is proven qualitatively by the gradual color change to a pale color at various concentrations of the test solution. it can be seen that the bride's tear leaf extract test solution with ethanol solvent at a concentration of 125 ppm still has a purplish color while at the largest concentration of 100 ppm the color of the solution is more purple, this change shows an indication of the activity of antioxidant content and shows that almost all free electrons in dpph have bonded with antioxidant hydrogen atoms in the sample so as to convert dpph to dpph-h which has lost its free radical properties (juniarti, 2018), the greater the color change to a pale color in the solution indicates that the greater the concentration of antioxidants in the solution. conclusions from the results, it can be concluded that the antioxidant activity of the bride's tears leaf extract sample (antigonon leptopus) is in the strong category with an ic59 value (69.71463 ug/ml). acknowledgements: the author would like to express sincere appreciation to universitas islam malang (unisma) for the academic support and resources provided throughout the research process. gratitude is also extended to the faculty of islamic studies, whose guidance and encouragement have been instrumental in completing this work. authors’ contributions: majidah ramadan1 & faisal designed the study. terra januarista & ramizard rafsanjan carried out the laboratory work. nailil wafiq azizah, devana rahma aldina & ramadina novia putri analyzed the data. nur laila anggraini, sabilatul mufarrohah, satria gemilang, firadika fitriansyah, hosiyah, nuvia harini wulan suci, nihlatus sofiya wrote the manuscript. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. references budiyanto, m. s. a. (2015). potensi antioksidan, inhibitor tirosinase, dan nilai toksisitas dari beberapa spesies tanaman mangrove di indonesia.cavalcante gm, da silva cabral ae, silva cc. 2018. aktivitas leishmanicide flavonoid alami dan sintetis: tinjauan mini. cetakan j. pharm. medis. sains. 2018; 7 :25–34. bujang, a. (2014). changes on amino acid content in soybeans, garbanzo beans and groundnut during pretreatment and tempe making, sain malaysian 43 (4): 551 557 departemen kesehatan republik indonesia. (2017). farmakope hebal indonesia edisi ii. in farmakope herbal indonesia.juniarti et al.. kandungan senyawa kimia, uji toksisitas (brine shrimp lethality test) dan antioksidan (1,1diphenyl-2-pikrilhydrazy dari ekstrak daun saga (abrus precatorius l.). makara, sains. (2009) 13(1) 50-54. tersedia dari: doi:https://doi.org/10.7454/mss.v1311.378. kurniawati, i., and maftuch, h. a. (2016). penentuan pelarut dan lama ekstraksi terbaik pada teknik maserasi gracilaria sp. serta pengaruhnya terhadap kadar air dan rendemen. jurnal ilmu perikanan. 2016. 7(2), 72-77. meidita, b. (2023). teknik budidaya tanaman air mata pengantin (antigonon leptopus) secara generatif dalam mendukung ketersediaan pakan lebah kelulut (trigona spp.). universitas lambung mangkurat. meigaria, komang mirah, i wayan mudianta, and ni wayan martiningsih. (2016). skrining fitokimia dan uji aktivitas antioksidan ekstrak aseton daun kelor (moringa oleifera). jurnal wahana matematika dan sains 10(2): 1–11. azizah et al. – antioxidant activity test of bride's tears (antigonon leptopus) leaves … 781 miksusanti, elvita dan hotdelina. s. (2012). aktivitas antioksidan dan sifat kestabilan warna campuran ekstrak etil asetat kulit buah manggis (garcinia mangostana l.) dan kayu secang (caesalpinia sappan l.). junal penelitian sains. universitas sriwijaya, sumatra utara. pratimasari, d. (2009). uji aktivitas penangkap radikal buah carica papaya l. dengan metode dpph dan penetapan kadar fenolik serta flavonoid totalnya, fakultas farmasi universitas muhammadiyah surakarta. surakarta. rahman, arif, abd malik, and aktsar roskiana ahmad. (2016). skrining fitokimia dan uji aktivitas antioksidan ekstrak etanolik buah buni (antidesma bunius (l.) spreng). jurnal fitofarmaka indonesia 3(2): 159–63. sepriyani, h., devitria, r., surya, a., & sari, s. (2020). aktivitas antioksidan ekstrak metanol daun pepaya (carica papaya l) dengan metode dpph. jurnal penelitian farmasi indonesia, 9(1), dpph. 10,863-867. simanjuntak, kristina. (2012). peran antioksidan flavonoid dalam meningkatkan kesehatan. fk upn veteran jakarta 3. suhaling, s. (2010). uji aktivitas antioksidan ekstrak metanol kacang merah (phaseolus vulgaris l.) dengan metode dpph. skripsi. universitas islam alauddin. makasar. suryowinoto. s.m. (1997). flora eksotika, tanaman hias berbunga. penerbit kanisius, yogyakarta. who. 2016. media centre diabetes mellitus. retrieved juni 03, 2024. zhao l., yuan x., wang j., feng y., ji f., li z., bian j. (2019). tinjauan tentang flavon yang menargetkan protein kinase serin/treonin untuk obat antikanker potensial. obat bioorganik. kimia; 27 :677–685. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 533-537 | doi: 10.14421/biomedich.2023.122.533-537 issn 2540-9328 (online) antioxidant activity test (dpph) and cytotoxicity of jengkol peel ethanol extract (pithecellobium jiringa) on shrimp larvae (artemia salina leach) glen arya wibowo, tika afriani, azimatur rahmi* department of pharmacy, mohammad natsir university, jl. tan malaka bukit cangan kayu ramang bukittinggi 26116 west sumatra, indonesia. corresponding author* azimatur.rahmi046@gmail.com abstract this study aims to determine the antioxidant and cytotoxic activity of the ethanol extract of jengkol peel (pithecellobium jiringa). jengkol peel ethanol extract (pithecellobium jiringa) was obtained by maceration using 70% ethanol as a solvent. in the phytochemical screening test it was found that chemical compounds contained in jengkol peel (pithecellobium jiringa) including alkaloids, flavonoids, and saponins. the antioxidant activity of the ethanol extract was tested using the 35 ppm dpph method. the test results on the ethanol extract showed moderate antioxidant activity with an ic50 value of 209.45 µg/ml. the cytotoxic activity of the ethanol extract was tested by the bslt method using shrimp larvae (artemia salina l.). the results showed that the ethanol extract of jengkol peel (pithecellobium jiringa) has cytotoxic activity with lc50 value minimum of 17.875 mg/l and lc50 a maximum of 360.714 mg/l with an average of 180.136 mg/l. keywords: pithecellobium jiringa; antioxidant; cytotoxic. introduction jengkol (pithecellobium jiringa) is one of the horticultural plants used as food for indonesian people (maxiselly et al., 2016). jengkol fruit is a smelly legume and is popular in southeast asia (indonesia, malaysia, thailand and cambodia) with a characteristic of an unpleasant aroma but is commonly consumed in various local dishes (asikin et al., 2017). aside from being a food ingredient, jengkol seeds are shielded by black seed shells, during this time, the jengkol skin does not function properly and degrades into organic waste. according to previous studies, the skin of jengkol (pithecellobium jiringa) contains chemical compounds including alkaloids, flavonoids, saponins, steroids/triterpenoids, and tannins, which are believed to have antioxidant and cytotoxic activity (rahayu et al., 2001). jengkol can also be used to treat diabetes, high blood pressure, dysentery, gastric disorders, and bladder stones (maxiselly et al., 2016) (shukri et al., 2011). this research focuses on the antioxidant and cytotoxic activity in jengkol peel. antioxidants are molecules that limit the formation of radicals, complete their radical reactions, and neutralize the radicals formed (erdogan et al., 2020). free radicals, in small amounts, initially serve to reduce inflammation and fight microorganisms in the body. these populations of free radicals are controlled by the body's antioxidants so that the amount is stable and non-destructive, but recently the number of free radicals rises as a result of changes in human lifestyle and environment, resulting in degenerative diseases such as cancer, heart disease, and premature aging caused by an imbalance of the amount of free radical produced in the body with the amount of endogenous antioxidant produced in the body, also known as oxidative stress (khan et al., 2013). as a result of the occurrence of oxidative stress, there is a decrease in the amount of oxygen and nutrients, which effects the microvascular and isochemic damage (referfusion injury) and causes cell and tissue damage due to the excessive production of free radicals as a result of the metabolism of fat and protein stored in the body. the body requires a large intake of antioxidants from outside or exogenous antioxidant sources to prevent this condition (şeker et al., 2021). the dpph method is a single procedure to figure out a material's antioxidant capacity. (2,2-diphenyl-1picrylhydrazil or 1,1-diphenyl-2-picrylhydrazil). dpph is a free radical molecule with a dark purple shade that is stable in methanol solution. the mechanism that occurs is the process of reducing dpph compounds by manuscript received: 10 may, 2023. revision accepted: 30 august, 2023. published: 20 september, 2023. https://doi.org/10.14421/biomedich.2023.122.533-537 mailto:xyz@xmail.com 534 biology, medicine, & natural product chemistry 12 (2), 2023: 533-537 antioxidants which results in decomposition of the color intensity of the dpph solution (jabbar et al., 2019). toxicity test is a preliminary test conducted to determine the toxic effect and threshold for the use of a plant as medicine. the initial method that can be used for cytotoxic testing is the brine shrimp lethality test (bslt) method using shrimp larvae (artemia salina leach) (fadhli et al., 2019). the results of this test can be used to identify a wider range of plant bioactivity (rahmi et al., 2022). therefore, this study aims to identify the antioxidant activity (dpph) and cytotoxicity of the ethanol extract of jengkol peel (pithecellobium jiringa) on shrimp larvae (artemia salina leach). materials and methods tools and materials tools this study used a hitachi u-2000 spectrophotometer, technical balances, analytical balances, vacuum rotary evaporators, vessels for hatching shrimp eggs, lamps for bslt, micro pipettes 10 – 1000 μl, a set of distillation apparatus, test tubes, whatman 42 filter paper, aluminum foil, and other glassware. chemicals and samples the chemicals used were 70% ethanol, methanol p.a, distilled water, 1,1-diphenyl-2-picrylhydrazyl (dpph), dimethyl sulfoksida (dmso), nh4oh, chloroform, concentrated hcl, mg metal. sample jengkol skin (pithecellobium jiringa) was taken from the people's garden in lubuk alung, padang pariaman, west sumatra. experimental animals the shrimp larvae (artemia salina leach) used by this study were obtained from the unand biology laboratory in padang, west sumatra. works procedure sample preparation a total of 2 kg of fresh jengkol peel samples (pithecellobium jiringa) was taken and all materials are separated from dirt or other foreign materials then washed thoroughly. the sample was dried by air drying for 4-5 days until the moisture content is less than 10% so that the material obtained was not easily damaged. the dried samples were then chopped and mashed with blender. the simplicia obtained was wrapped in plastic and stored for further testing. extraction as much as 1 kg of dry jengkol peel powder was extracted using 70% ethanol solvent by maceration method for 2x24 hours. the macerated filtrate is combined and the solvent was evaporated using a rotary evaporator at 60°c until a thick extract was obtained. the resulting extract was then used as a research sample. phytochemical analysis a total of 1 gram of extract was weighed, then basified with nh4oh, then 5 ml of chloroform:distilled water (1:1) was added, shaken in a separatory funnel, and left for a few moments until two layers formed. water layer (flavonoids, phenolics, and saponins) for analysis, chloroform layer (terpenoids, steroids, and alkaloids). antioxidant testing (dpph) dissolving 10 mg of dpph with methanol p.a in a 100 ml volumetric flask, pipetting 17.5 ml into a 50 ml volumetric flask to obtain a 35 ppm dpph solution. a total of 3.8 ml of 35 ppm dpph solution was dissolved with 0.2 ml of p.a methanol then left in the dark for 30 minutes. the solution's absorbance was measured through a uv-vis spectrophotometer at 400-600 nm. to reach a concentration of 1000 ppm, 25 mg of the extract was dissolved in a 25 ml volumetric flask with methanol p.a. the test solution was then pipetted with concentrations of 80, 120, 160, 200, and 240 ppm. as a comparison, vitamin c (ascorbic acid) levels in distilled water were 10, 20, 30, 40, and 50 g/ml, respectively. pipette 0.2 ml of each test solution into a test tube, then add 3.8 ml of 35 ppm dpph solution and leave for 30 minutes in a dark spot. the absorbance of this solution is then measured at the maximum wavelength. in comparison, vitamin c was tested in the same manner, with each test being performed three times. toxicity testing (bslt) the shrimp larvae were prepared by incubating 2 grams of artemia salina l. eggs, soaking the eggs using 600 ml of seawater and lighting them with a 40-60 watt incandescent lamp, and aerating for 48 hours. the test solution was prepared by dissolving the sample in salt water with a concentration of 0, 25, 50, 100, 500, 1000 ppm, respectively. control solution (0 ppm) was carried out without the addition of extracts. furthermore, cytotoxic testing was carried out using the bslt method, in which the test solution with concentrations of 0, 25, 50, 100, 500, 1000 ppm was pipetted as much as 6 ml and put into a test tube, then added 10 artemia salina l. shrimp larvae into each test tube. make observations for 24 hours, then count the number of shrimp larvae that died within 24 hours. for each test solution was repeated 3 times.the total number of artemia salina l. larvae used was 180 individuals. the average mortality of larvae is obtained by dividing the total mortality of larvae at each concentration by the number of replications carried out, namely three times. toxic activity is expressed in lc50 values which was calculated by probit analysis using minitab. wibowo et al. – antioxidant activity test (dpph) and cytotoxicity of … 535 results and discussion in this study, the results of the determination of jengkol plants (pithecellobium to the ring) belongs to the family; mimosaceae (legume), genus; pithecellobium, species; pithecellobium lobatum (benth). the results of the phytochemical screening test on jengkol peel, found flavonoids, alkaloids, and saponins (table 1). in testing the antioxidant activity of the ethanol extract of jengkol peel, the ic50 value was obtained of 209.45 µg/ml (table 2; figure 1), as well as the lc50 value minimum 17,875 mg/l dan lc50 maximum of 360.714 mg/l with an average of 180.136 mg/l for the cytotoxic activity of the ethanol extract of jengkol peel, where the number of deaths artemia salina l. is directly proportional to the increase in concentration (table 3; figure 2). table 1. phytochemical screening test results. compound identification alkaloid flavonoid saponin + + + note: (+) contains the compound in question table 2. test results for antioxidant activity of jengkol peel ethanol extract against dpph. concentration (ppm) absorbance control extract absorbance percent inhibition (%) regression ic50 80 120 160 200 240 0,786 0,786 0,786 0,786 0,786 0,695 0,601 0,502 0,405 0,330 11,57 23,53 35,13 48,47 58,01 y = 0.294x – 11.58 r2 = 0,997 209,45 µg/ml figure 1. curve for determining antioxidant activity of jengkol skin. table 3. test results for the cytotoxic activity of jengkol peel ethanol extract (pithecellobium jiringa) with the bslt method. concentration (ppm) test total deaths death percent (%) p1 p2 p3 0 25 50 100 500 1000 0 1 3 5 6 9 0 2 2 5 8 10 0 2 3 4 7 10 0 5 8 14 21 29 0 16,67 26,67 46,67 70 96,67 p1 (first try); p2 (second try); p3 (third try) figure 2. relationship between concentration and death percentage. y = 0,2946x 11,586 r² = 0,9978 0 10 20 30 40 50 60 70 0 50 100 150 200 250 300 in h ib it io n % concentration (ppm) series1 linear (series1) 0 20 40 60 80 100 120 0 200 400 600 800 1000 1200 d e at h p e rc e n t (% ) concentration (ppm) 536 biology, medicine, & natural product chemistry 12 (2), 2023: 533-537 discussion this study used jengkol (pithecellobium jiringa) taken from plantations in lubuk alung, padang pariaman, west sumatra. 1 kg of dry jengkol peel powder was extracted using the maceration process with 70% ethanol solvent for 2x24 hours. the maceration method yielded 43.4695 grams of viscous extract with a yield of 14.29%. the dpph technique was used to assess the antioxidant activity of an ethanol extract of jengkol peel. this method was chosen because it is a simple method, easy to measure antioxidants, sensitive and requires a small sample with a relatively short time. compounds with antioxidant activity will react with dpph, resulting in a purple to yellow color shift (martinus et al., 2014). the absorbance was then determined using a uv-vis spectrophotometer with a maximum wavelength of 515520 nm. the ic50 number represents the quantity of antioxidant activity. the highest absorption wavelength of dpph 35 ppm in methanol p.a recorded using a uvvis spectrophotometer was 515 nm with an absorbance value of 0.869. measurement of the antioxidant activity of the ethanol extract of jengkol peel (pithecellobium jiringa) was prepared in several concentrations (table 2), namely 240, 200, 160, 120, 80 ppm, and vitamin c was used as a comparison. examination of antioxidants in ethanol extract of jengkol peel (pithecellobium jiringa) obtained the ic50 value of 209.45 ppm with moderate antioxidant activity because it has an ic value50 between 101-250 ppm, when compared to vitamin c with ic50 value 34.11 ppm has a very strong antioxidant activity, because it has an ic50 value less than 50 ppm (badarinath et al., 2010). testing the cytotoxic activity of the ethanol extract of jengkol peel using artemia salina l. the death test of artemia salina l. represents a fast, cheap, and simple biological test to test the bioactivity of plant extracts which in many cases correlates quite well with cytotoxic and antitumor properties (hossain et al., 2012). the results obtained from the research using the brine shirmp lethality test (bslt) method were the number of artemia salina l. that died due to the administration of ethanol extract of jengkol peel (pithecellobium jiringa) at a concentration of 1000 ppm, total death of 29 artemia salina l., concentration of 500 ppm, total death of 21 artemia salina l., concentration of 100 ppm, total death of 14 artemia salina l., concentration of 50 ppm, total death of 7 artemia salina l., concentration of 25 ppm, total death of 5 artemia salina l., and concentration of 0 ppm no dead artemia salina l. was found. this shows that the number of dead artemia salina l. is directly proportional to the increase in concentration (table 3; figure 2). furthermore, the cytotoxic activity of the ethanol extract of jengkol peel calculated by probit analysis using minitab 15 which expressed in lc50 values. to get the lc50 value first, mortality is calculated by means of the accumulation of dead divided by the accumulation of life and death (total) multiplied by 100% (agustian et al., 2013). the concentration that can result in 50% of the death of an animal population is obtained by drawing a line from 50% of the mortality probit (arifuddin et al., 2014) (eroglu et al., 2018). probit analysis using minitab 15 which was carried out on the ethanol extract of jengkol peel obtained cytotoxic activity with lc50 values minimum 17,875 mg/l and lc50 maximum 360.714 mg/l, with lc50 value average 180.136 mg/l. conclusions testing the antioxidant and cytotoxic activity of the ethanol extract of jengkol peel (pithecellobium jiringa) showed that the ethanol extract of jengkol peel (pithecellobium jiringa) has moderate antioxidant activity with an ic50 value of 209.45 µg/ml and cytotoxic activity with lc50 values minimum 17.875 mg/l dan lc50 a maximum of 360,714 mg/l, with an average value of 180,136 mg/l. acknowledgements: this study was supported by mohammad natsir university. competing interests: the authors declared no conflict of interest in the manuscript. references agustian r, yudiati e, sedjati s. toxicity test of crude pigment extract spirulina platensis microalgae with the bslt test method (brine shrimp lethality test). journal of marine research. 2013; 2(1); 25-31. arifuddin m, acute toxicity test of laban abang leaf methanol extract (aglaia elliptica blume) against shrimp larvae (artemia salina leach) using the brine shrimp lethality test (bslt) method. fakultas kedokteran dan ilmu kesehatan. uin. jakrta. 2014. asikin y, kusumiyati, shikanai t, wada k. volatile aroma components and ms-based electronic nose profiler of dogfruit (pithecellobium jiringa) and stink bean (parkia speciosa). journal of advanced research. 2017. badarinath a, rao k, chetty cs, ramkanth s, rajan t, gnanaprakash k. a review on in-vitro antioxidant methods: comparisons, correlations, and considerations. international journal of pharm tech research. 2010; 2(2); 1276-1285. erdogan g, uysal t. characterization of antioxidant properties of strawberry tree (arbutus unedo l.) and trace elements determination. journal of research in pharmacy. 2020; 24(5); 774-785. eroglu ozkan se, demirci kayiran s, taşkin t, abudayyak m. in vitro antioxidant and cytotoxic activity of muscari neglectum growing in turkey. marmara pharmaceutical journal. 2018;22(1). fadhli h, hasanah sbu, cytotoxic test of kangkang valve stem bark extract (bauhinia semibifida roxb) using the brine wibowo et al. – antioxidant activity test (dpph) and cytotoxicity of … 537 shrimp lethality test (bslt) method. scientia: journal of pharmacy and health. 2019; 9(2); 141-145. hossain s, kader g, nikkon f, yeasmin t. cytotoxicity of the rhizome of medicinal plants. asian pasific journal of tropical biomedicine. 2012; 125-127. jabbar a, wahyuni, malaka mh, apriliani. antioxidant activity of ethanol extract of fruits, leaves, stems and rhizome of wualae (etlingera elatior (jack) r.m smith). galenika journal of pharmacy. 2019; 5(2); 189-197. khan ms, syeed sh, uddin mh, akter l, ullah ma, jahan s, rashid mh. screening and evaluation of antioxidant, antimicrobial, cytotoxic, thrombolytic and membrane stabilizing properties of the methanolic extract and solventsolvent partitioning effect of vitex negundo bark. asian pacific journal of tropical disease. 2013 oct 1;3(5):393-400. martinus ba, arel a, gusman a. comparison of total phenolate content and activity antioxidants in tea leaf extract (camellia sinensis (l.) o. k.) from wood aro with its black tea products has been released. scientia. 2014; 4(2). maxiselly y, ustari d, ismail a, karuniawan a. distribution pattern of jengkol plant (pithecellobium jiringa (jack) prain) in south of west java based on morphological trait. journal kultivasi. 2016; 15(1). rahayu es, jengkol fruit peel as a natural herbicide in plantations paddy field. pengembangan dan penerapan teknologi (p&pt). 2001; 2(4); 254-260. rahmi a, afriani t, aini. cytotoxic test of extract and fractions from blumea balsamifera leaves using brine shrimp lethality test (bslt). scientific journal of pharmacy. 2022; 18(1); 2633. şeker karatoprak g, yücel aşık ç, çakır a, köngül şafak e. in vitro pharmacological screening of antioxidant, cytotoxic and enzyme inhibitory activities of citrus aurantifolia linn. dried fruit extract. international journal of environmental health research. 2021 nov 17;31(8):991-1000. shukri r, mohamed s, noordin mm, hamid aa. evaluating the toxic and beneficial effects of jering beans (archidendron jiringa) in normal and diabetic rats. journal of the science of food and agriculture. 2011; 91(14); 697-706. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 681-685 | doi: 10.14421/biomedich.2023.122.681-685 issn 2540-9328 (online) in vivo alpha-amylase and alpha-glucosidase inhibitory potentials of panicum maximum jacq. (guinea grass) leaf extract on wister rats godwin ndarake enin1,*, jude efiom okokon2, enobong mfon david1, saviour elisha emmanuel1, esther michael ekanem1, bassey sunday antia1 1department of chemistry, faculty of science; 2department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. corresponding author* enin.godwin@gmail.com manuscript received: 21 september, 2023. revision accepted: 13 march, 2024. published: 14 march, 2024. abstract panicum maximum jacq. (guinea grass) a medicinal plant used traditionally in the treatment of diseases including diabetes was evaluated for its effect on alpha amylase and alpha glucosidase enzymes in vivo. the crude ethanol extracts (150, 300 and 450 mg/kg) of p. maximum were investigated using starch, sucrose, glucose and maltose as substrates and acarbose as reference drug. the leaf extract caused significant (p<0.05) reduction in blood glucose levels of the treated rats with the four substrates used. the findings show that the leaf extract of panicum maximum has the potentials to inhibit alpha amylase and alpha glucosidase in rats. keywords: panicum maximum; alpha amylase; alpha glucosidase; phytochemicals. introduction plants have therapeutic substances because of their potency and huge benefits, and have been widely researched and adopted in our society for the treatment and management of some diseases. in the last few decades, bioactive compounds have been trapped, purified and characterized into active drugs and, administered in rural and urban communities of the world (coulibaly et al., 2023). recent findings demonstrate that the world sales of medicinal plant-based products have escalated to 100.9 billion dollars representing 7.2% annual turn-over and that global population of up to 80% are utilizing medicinal plants to treat various diseases (yusupova et al., 2023; karahan et al., 2020). with the increasing rate of spread of diseases and infections, there is need to explore more plant-based materials for possible discoveries and publications of new and efficient remedies for these challenges. one of such herbs which could find possibility in combating current health challenges such as diabetes is panicum maximum commonly called “guinea grass”. panicum maximum jacq. is a perennial grass of the poacace family distributed widely in africa and other tropical regions of the world (van oudtshoorn, 1999). the ethnopharmacology of the plant indicates that, the plant has been employed for the treatment of malaria, microbial infections, rheumatic pain, inflammation and diabetes (antia et al., 2010). in nigeria, particularly in ibibio ethnomedicine, the leaf is used to treat malaria, microbial infections and rheumatism. other biological activities of the leaves and roots include antidiabetic (antia et al., 2010), antimalarial and analgesic (okokon et al., 2012), antibacterial (gothandam et al., 2010; doss et al., 2011a; doss et al., 2011b), anti-inflammatory and antipyretic (okokon et al., 2011), antifungal (kanife, 2012), anticancer, antioxidative and antileishmanial (okokon et al., 2014). phytochemical studies of the root have shown the presence of alkaloid, flavonoid, tannins, terpenes, saponin, and cardiac glycosides (okokon et al., 2016). in this study, we investigated the inhibitory activities of panicum maximum jacq. ethanol leaf extract on alpha-amylase and alpha-glucosidase in wister rats. materials and methods materials the materials used include panicum maxima powdered leaf extract, oral gastric gavage, weighing balance, gloves, scissors, glucometer and strips (fine test), distilled water, acarbose (aldrich sigma, usa; standard drug), stirrer, beakers, 1ml syringe, starch, sucrose, maltose (aldrich sigma, usa). plant collection fresh leaves of panicum maxima were harvested from a farmland in use offot, uyo, akwa ibom state, nigeria, in may, 2023. the plant was identified and authenticated by dr. margaret bassey, a taxonomist in the department https://doi.org/10.14421/biomedich.2023.122.681-685 682 biology, medicine, & natural product chemistry 12 (2), 2023: 681-685 of botany and ecological studies, university of uyo, uyo, nigeria. herbarium specimen was deposited at the department of botany and ecological study, university of uyo. extraction the fresh leaves (2 kg) of the plant were dried on a laboratory table for 2 weeks and reduced to powder. powdered sample (500 g) was macerated in 95% ethanol (5000 ml) for 72 hours. the liquid filtrate obtained was concentrated in vacuo at 40˚c to completely remove the ethanol. the yield was calculated, and the extract was stored in a refrigerator at 4˚c until used. phytochemical screening phytochemical screening for the presence of saponins, tannins, flavonoids, alkaloids, cardiac glycosides and anthraquinones was conducted following previously reported standard procedures (enin et al., 2023). animals albino wistar rats (120 -135 g) of either sex maintained at animal house of the department of pharmacology and toxicology, faculty of pharmacy, university of uyo, nigeria were used for the study. the animals were housed in standard cages and were maintained on a standard pelleted feed (guinea feed) and water ad libitum. alpha-amylase inhibitory study thirty wistar rats were divided into 6 groups of 5 rats each. the rats in all groups were fasted for 18 h and fasting blood glucose concentration was first taken at 0 min before administration. group i, as the normal control, received distilled water (10 ml/kg). group ii rats were orally administered starch at 2 g/kg body weight (orally with distilled water as vehicle) and distilled water (10 ml/kg) simultaneously. rats in group iii were administered starch (2 g/kg) and the standard drug (acarbose) at 100 mg/kg simultaneously. groups iv, v and vi were administered simultaneously, starch (2 g/kg) and panicum maximum leaf extract at 150, 300 and 450 mg/kg respectively. all administrations were done orally and blood glucose concentration was monitored at 30, 60, 120 and 180 min (gidado et al., 2019). the blood glucose level was used to assess the effect of extract on the enzyme activity. alpha glucosidase inhibitory study. the procedure as described above was used for this study but with sucrose and maltose used as substrates (gidado et al., 2019). blood glucose determination drops of blood from tip of rats tails were dropped on stripes and glucose concentration was measured using a glucometer according to manufacturer’s specifications (fine test). the glucometer works with the following principle; the blood sample is exposed to a membrane covering the reagent pad (strip), which is coated with an enzyme (glucose oxidase, glucose dehydrogenase). the reaction causes a colour change and the intensity of this change is directly proportional to the amount of glucose in the blood sample. light from an led strikes the pad surface and is reflected to a photodiode, which measures the light intensity and converts it to electrical signals. an electrode sensor measures the current produced when the enzyme converts glucose to gluconic acid. the resulting current is directly proportional to the amount of glucose in the sample (who, 2011). statistical analysis data obtained were analyzed statistically using one –way anova followed by tukey-kramer multiple comparison test using instatr graphpad software, (san diego, usa). differences between means were considered significant at p< 0.05 and very significant at p < 0.001. results and discussion extraction the extraction yield was 6.56% w/w. phytochemical screening the results of the phytochemical screening revealed the presence of saponins, tannins, flavonoids, alkaloids, cardiac glycosides. the presence of anthraquinone was not observed in the study. in vivo alpha amylase and glucosidase inhibition assay administration of starch (2 g/kg) to fasted rats caused varying percentages of increase in blood glucose levels of the treated animals after 30 mins. the percentages were starch (66.56%), p maximum leaf extract-treated groups (37.17 47.26%), and acarbose-treated group (17.97%). these increases were reduced after 60 min with only the groups treated with higher doses of the extract (300 and 450 mg/kg) having percentage increases of 3.82 and 18.44 % respectively. all the extract-treated groups had their bgl reduced to a normal level at 120 min and this was sustained throughout the study. also, co-administration of the starch with acarbose prominently inhibited the rise in the blood glucose concentrations (table 1). enin et al. – in vivo alpha-amylase and alpha-glucosidase inhibitory … 683 table 1. effect of ethanol leaf extract of panicum maximum on blood glucose level of rat after oral administration of starch load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 120 min 180 min control normal saline 86.00±11.53 87.66±7.12(1.93) 87.66±7.62(1.93) 91.0±7.50(5.81) 80.00±6.02 starch 80.0±4.54 133.25±6.86a(66.56) 112.25±4.73(40.31) 92.50±1.70(15.62) 87.25±6.52(9.06) acarbose 100 72.33±2.69 85.33±12.97(17.97) 80.33±7.21(11.06) 74.0±1.00(2.30) 72.33±8.68(0) extract 150 95.75±5.20 133.50±7.83(39.42) 92.0±9.71a() 82.50±3.22a() 67.25±3.42a() 300 91.50±5.56 134.75±8.49(47.26) 95.0±9.44(3.82) 88.50±5.23b() 72.75±8.94a() 450 86.75±12.41 119.0±2.12(37.17) 102.75±1.48(18.44) 85.50±6.89a() 85.25±5.88() data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control (n=6). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. administration of sucrose (2 g/kg) produced a 44.14% increase in blood glucose concentration 30 minutes post-administration of the sucrose in the control group. bgl increments of 12.07-34.60 % were also recorded in groups treated with 150, 300 and 450 mg/kg of p maximum leaf extract and 3.37% for acarbose treated group. at 60 min, percentage increases in bgl of groups treated with 150 and 450 mg/kg of extract were 19.94 and 9.22 % respectively, while the bgl of the group treated with 300 mg/kg was reduced to normal. similarly, trend was also recorded at 120 min. there was no increment in bgl of all the extract-treated groups at 180 min (table 2). table 2. effect of ethanol leaf extract of panicum maximum on blood glucose level of rat after oral administration of sucrose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 120 min 180 min control normal saline 100.00±4.25 88.33±1.85 92.33±4.25 89.0±4.35 87.33±3.84 sucrose 2000 81.0±4.50 116.75±6.57b(44.14) 112.66±1.45a(39.08) 97.33±1.63(20.16) 94.15±4.81(16.23) acarbose 100 90.33±2.48 86.66±2.90 82.0±6.00 71.66±3.75 78.0±3.78 extract 150 85.25±3.27 114.75±11.22c(34.60) 102.25±4.82(19.94) 90.25±3.27(5.86) 83.50±4.48 300 95.25±2.98 106.75±2.98b(12.07) 89.50±4.94() 85.0±2.48() 72.75±5.93() 450 84.0±5.87 101.25± 2.28(20.53) 91.75±4.55(9.22) 89.0±2.67(5.95) 76.50±3.52() data is expressed as mean ± sem. significant at ap<0.05, bp< 0.01, when compared to control (n=6). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. administration of glucose (2 g/kg) to fasted rats caused varying percentages of increase in blood glucose levels of the treated animals after 30 mins. the percentages were glucose (64.98%), p maximum leaf extract-treated groups (57.10 61.90%), and acarbosetreated group had no increment. all the extract-treated groups had their bgl reduced to a normal level at 120 min and this was sustained throughout the study (table 3). table 3. effect of ethanol leaf extract of panicum maximum on blood glucose level of rat after oral administration of glucose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 120 min 180 min normal control 100.00±4.25 88.33±1.85 92.33±4.25(1.80) 89.0±4.35(1.55) 87.33±3.84(3.98) glucose 2000 84.25±1.49 139.0±1.78b(64.98) 126.75±0.47b(50.44) 106.0±1.87b(25.81) 96.75±2.78a(14.83) acarbose 100 85.34±1.36 84.21±0.90 84.0±1.20 82.16±2.14a 80.00±1.10 extract 150 84.0±2.16 136.0±2.16a(61.90) 89.50±8.56a(6.54) 84.0±7.70a() 76.50±6.38a() 300 84.75±1.43 136.25±1.03b(60.76) 92.50±4.17a(9.14) 81.50±2.72a() 74.25±4.49() 450 86.25±1.10 135.50±1.44b(57.10) 88.0±2.04b(2.02) 78.75±2.52b() 68.25±3.86a() data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control. (n=6). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. administration of maltose (2 g/kg) to fasted rats caused varying percentages of increase in blood glucose levels of the treated animals after 30 mins. the percentages were maltose (65.86%), p. maximum leaf extract-treated groups (48.17-67.87%), and acarbosetreated group (3.37%). these increases were reduced 684 biology, medicine, & natural product chemistry 12 (2), 2023: 681-685 after 60 min with only the low dose (150 mg/kg) and middle dose (300 mg/kg) treated group having bgl increment of 14.28 and 53.93% respectively. all the extract-treated groups had their bgl reduced to a normal level at 180 min. also, co-administration of the maltose with acarbose prominently inhibited the rise in the blood glucose concentrations (table 4). table 4. effect of ethanol leaf extract of panicum maximum on blood glucose level of rat after oral administration of maltose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 120 min 180 min control normal saline 86.00±11.53 87.66±7.12(1.93) 87.66±7.62(1.93) 91.0±7.50(5.81) 80.00±6.02 maltose 83.50±1.19 138.50±8.10a(65.86) 120.25±2.95(44.01) 97.25±2.05(16.46) 87.25±2.13(4.49) acarbose 100 85.34±1.36 88.22±1.10(3.37) 86.0±2.20(0.77) 84.26±1.14a() 82.28±2.26() extract 150 75.25±5.13 111.50±9.64(48.17) 86.0±7.62(14.28) 72.25±3.98() 68.0±2.98() 300 82.5±4.02 138.50±13.17(67.87) 127.0±18.35(53.93) 90.25±20.67(9.39) 68.75±6.83() 450 83.50±1.19 138.50±8.10(65.86) 80.25±2.95() 67.25±2.05() 66.25±2.13() data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control (n=6). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. discussion the leaf ethanol extract was found to inhibit increases in blood glucose concentration significantly following starch administration though non-dose-dependently. it has been reported that complete digestion of dietary polysaccharides like starch is achieved by the combined action of α-amylases and α-glucosidase enzymes. the αamylase enzyme digests α-bonds of the α-linked polysaccharides yielding disaccharides, like maltose, which are further reduced to monosaccharides by membrane bound α-glucosidase enzymes (kalra, 2014; alongi and anese, 2018). inhibitions of these enzymes delay the digestion of ingested carbohydrates thereby resulting in a small rise in blood glucose concentrations following carbohydrate meals as was observed in this study. as a target for managing type 2 diabetes mellitus, many medicinal plants have been reported to possess αamylase and αglucosidase inhibitory potential (ibrahim et al., 2014; esimone et al., 2001). similarly, the leaf extract significantly inhibited blood glucose rises when co-administered with maltose, glucose and sucrose. acarbose, the standard drug used in this study significantly inhibited blood glucose rise when coadministered with starch, maltose and sucrose. the results of this study support the antidiabetic activity earlier reported on the root extract (antia et al., 2010) and further suggest the involvement of inhibitory effects on alpha glucosidase and amylase as one of the modes of antidiabetic activity of the root extract. the inhibitory activities of plant extracts are linked to their phytochemical constituents. the leaf extract of p. maximum has been reported to be rich in flavonoids, terpenes, tannins amongst others (okokon et al., 2011). our results of the phytochemical screening also revealed the presence of: saponins, tannins, alkaloids, flavonoids and cardiac glycosides. these compounds have been variously reported to inhibit alpha glucosidase and alpha amylase activities (proença et al., 2017; su and tang, 2019). moreso, phenols have been reported to inhibit alpha amylase and alpha glucosidase (oboh et al., 2017). also, polyphenolic compounds from plants are known to cause several effects on the biological systems which include enzymes inhibitions (kalita et al., 2018; funke and melzig, 2005). the phenolic compounds are known to be strong metal ion chelators and protein precipitation agents forming insoluble complexes with proteins as well as acting as biological oxidants (ishnava and metisariya, 2018). the presence of the polyphenolic compounds and terpenes in the root extract may suggest that their inhibitory potential on α-amylase and the membranebound intestinal α-glucosidase enzymes. the presence of these compounds in the extract may have contributed to the observed activity of this study and therefore explains the antidiabetic mechanism of the leaf of p. maximum. conclusion the results of this study suggest that inhibition of alpha amylase and alpha glucosidase enzymes maybe one of the modes of antidiabetic activity of the leaf extract of panicum maximum which may be attributed to the activities of its phytochemical constituents. acknowledgements: mr. nsikan malachy of pharmacology and toxicology department, university of uyo is gratefully acknowledged for providing technical assistance. authors’ contributions: gne conceived the study, gne, jeo & abs designed the study. eme & see carried out the laboratory work. jeo analyzed the data. gne & jeo wrote the manuscript, emd edit the manuscript. all authors read and approved the final version of the manuscript. enin et al. – in vivo alpha-amylase and alpha-glucosidase inhibitory … 685 competing interests: the authors declare that there are no competing interests. references coulibaly ll, bayala b, zongo l, zongo pf, ouedraogo e, djigma fw, yonli a, baron s, figueredo g, lobaccaro jm, simpore j (2023). chemical composition and antiproliferative activity on prostate and cervical cancer cell lines of lantana camara linn. essential oil. international journal of biological and chemical sciences 17(2): 293-303. doi: https://dx.doi.org/10.4314/ijbcs.v17i2.1. yusupova za, baratjon ogli sf, 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(poaceae). international journal of biology 4(2): 64–96. https://doi.org/10.5539/ijb.v4n2p64. . funke i, melzig mf (2005). effect of different phenolic compounds on α-amylase activity: screening by microplatereader based kinetic assay. die pharmazie-an international journal of pharmaceutical sciences 60(10): 796–797. ishnava kb, metisariya dm (2018). in vitro study on α-amylase inhibitory activity of selected ethnobotanical plant extra its and its herbal formulations. international journal of pharmacognosy and chinese medicine 2(3): 01-10. https://dx.doi.org/10.4314/ijbcs.v17i2.1 https://doi.org/10.1016/j.foodres.2018.05.061 http://dx.doi.org/10.4314/tjpr.v18i3.20 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 11, number 2, october 2022 | pages: 175-180 | doi: 10.14421/biomedich.2022.112.175-180 issn 2540-9328 (online) inhibitory effect of mammea africana on alpha-amylase and alpha-glucosidase enzymes of rats nwakaego omonigho ebong1,*, jude efiom okokon1,2, jesse idakwoji1 1department of pharmacology and toxicology, faculty of pharmacy, madonna university, elele, nigeria. 2department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. corresponding author* nwakaebong@gmail.com manuscript received: 10 august, 2022. revision accepted: 23 august, 2022. published: 12 september, 2022. abstract mammea africana sabine (guttiferae), a medicinal plant used traditionally in the treatment of diseases including diabetes was evaluated for effect on alpha-amylase and alpha-glucosidase enzymes in vivo. the stembark extract (30, 60 and 90 mg/kg) of m. africana were investigated in vivo for inhibitory effect on alpha-amylase and alpha-glucosidase enzymes using starch, sucrose and maltose as substrates. acarbose was used as reference drug. the stembark extract caused significant (p<0.05) reduction in blood glucose levels of treated rats with the various substrates used. the results suggest that the stembark extract of m. africana have the potentials to inhibit alpha-amylase and alpha-glucosidase in rats. keywords: alpha-amylase; alpha-glucosidase; hypoglycaemia; mammea africana. abbreviations: blood glucose level (bgl). introduction mammea africana sabine (guttiferae) (syn. ochrocarpus africana oliv.) (m. africana) is a large forest tree of 50 to 100 feet high with bark often yellow with pale scales and resinous yellow sap (hutchison and daziel, 1958). the plant is widely distributed in tropical africa. traditionally, the stem bark of the plant is used by the ibibios, of the niger delta region of nigeria, in the treatment of malaria related fever, diabetes, microbial infections and mental disorders. the stembark is also traditionally used to treat stomach pains, rheumatism pains, scabies, cough and hypertension (raponda-walter and sillans, 1961; adjanohoun et al. 1996). the stembark extract has been reported to possess cytotoxic activity, in vitro (chapuis et al. 1988; okokon et al. 2012). ouahouo et al. (2004) reported cytotoxic coumarins with anti-microbial activity against staphylococcus aureus from the plant stembark. the stembark has been reported to have anti-plasmodial (okokon et al. 2006), cardioprotective (okokon and antia,2007), anti-diabetic, hypolipidaemic (okokon et al. 2007), vasorelaxant (dongmo et al. 2007), antihypertensive (nguelefack-mbuyo et al. 2008), antiinflammatory, analgesic (okokon et al. 2009), antioxidant (nguelefack-mbuyo et al. 2010), antidiarrheal, anti-ulcer (okokon et al. 2010), immunomodulatory, anti-lesihmanial (okokon et al. 2012), depressant and anti-convulsant (okokon and davis, 2014) as well as nephroprotective (okokon and bawo, 2014) and hepatoprotective (okokon et al. 2016) activities. the stembark has been reported to have 5,-7dihydroxy-8-(12-methyl-butryl)–4–n-pentylcoumarins (carpenter et al. 1970, 1971; crichton and waterman, 1978), 4-phenyl and 4alkylcoumarins (games, 1972), mesuxanthone b (carpenter et al. 1971). alkaloids have been reported to be absent in the entire plant parts (gartlands et al. 1980). we report in this study the effect of leaf extract and fractions of the plant on alphaamylase and alpha-glucosidase of rats. materials and methods plants collection the plant material mammea africana (stembark) were collected in anwa forest in uruan area, akwa ibom state, nigeria in january 2022. the plant was identified and authenticated by dr. margaret bassey, at the department of botany and ecological studies, university of uyo, uyo, nigeria with voucher number fphuu 381. extraction the stembarks were washed and shade-dried for two weeks. the dried plants’ materials were further chopped https://doi.org/10.14421/biomedich.2022.112.175-180 176 biology, medicine, & natural product chemistry 11 (2), 2022: 175-180 into small pieces and reduced to powder using electric grinder. the powdered material (1.5 kg) was macerated for 72 h in 50% ethanol. this was thereafter filtered and the liquid filtrate was concentrated and evaporated to dryness in vacuo 40˚c using a rotary evaporator (buchilab, switzerland). the extract was stored in a refrigerator at -4˚c, until used for the proposed experiments. animals albino wistar rats (120 -135 g) of either sex were used for these experiments. the animals were housed in standard cages and were maintained on a standard pelleted feed (guinea feed) and water ad libitum. in vivo alpha-amylase and alpha-glucosidase inhibition study alpha-amylase inhibitory study thirty-five wistar rats were divided into 6 groups of 5 rats each. the rats in all groups were fasted for 18 hours and fasting blood glucose concentration was first taken at 0 minutes before administration. group i, as the normal control, received distilled water (10 ml/kg). group ii rats were orally administered starch at 2 g/kg body weight (orally with distilled water as vehicle) and distilled water (10 ml/kg) simultaneously. rats in group iii were administered starch (2 g/kg) and the standard drug (acarbose) at 100 mg/kg simultaneously. groups iv, v and vi were administered simultaneously, starch (2 g/kg) and mammea africana stembark extract at 30, 60 and 90 mg/kg respectively. all administrations were done orally and blood glucose concentration was monitored at 30, 60, 90, 120 and 180 minutes (gidado et al. 2019). alpha-glucosidase inhibitory study the procedure as described above was used for this study but with sucrose and maltose used as substrates (gidado et al. 2019). blood glucose determination drops of blood from tip of rats’ tails were dropped on stripes and glucose concentration was measured using a glucometer according to manufacturer’s specifications (accu-chek, indiana). the glucometer works with the following principle; the blood sample is exposed to a membrane covering the reagent pad (strip), which is coated with an enzyme (glucose oxidase, glucose dehydrogenase). the reaction causes a colour change and the intensity of this change is directly proportional to the amount of glucose in the blood sample. light from a light emitting diode strikes the pad surface and is reflected to a photodiode, which measures the light intensity and converts it to electrical signals. an electrode sensor measures the current produced when the enzyme converts glucose to gluconic acid. the resulting current is directly proportional to the amount of glucose in the sample (who, 2011). statistical analysis data obtained from this work were analysed statistically using one –way anova followed by tukey-kramer multiple comparison test using instat graphpad software, (san diego, usa). differences between means were considered significant at 5% level of significance i.e. p≤ 0.05. results and discussion in vivo alpha-amylase and alpha-glucosidase inhibition assay administration of starch (2g/kg) to fasted rats caused varying percentages of increase in blood glucose concentrations of the treated animals after 30 min. the percentages were starch (63.18%), extract-treated groups (6.95-42.73%) and acarbose-treated group (17.97%). these increases were reduced after 60 min to 0%, 15.29% and 20.51% in animal groups treated with 60, 90 and 30 mg/kg of the extract respectively. all the extract-treated groups had their blood glucose level (bgl) reduced to normal without any further increase from 90 to 180 minutes. also, co-administration of the starch with acarbose prominently inhibited the rise in the blood glucose concentrations (table 1). there was 60.78% increase in blood glucose concentration 30 minutes following maltose administration in the control group. however, 17.0321.05 % increases were observed in the extract-treated groups. at 60 minutes, the bgl of the extract-treated groups were significantly reduced with groups treated with 30, 60, and 90 mg/kg having percentage increments of 6.11, 8.63 and 13.15% in bgl respectively. there reductions were sustained and significant throughout the duration of the study (180 minutes) with no increment in bgl recorded in any group (table 2). administration of sucrose (2 g/kg) produced a 46.01% increase in blood glucose concentration 30 minutes post-administration of the sucrose in the control group and 20.95-56.03% increases in blood glucose concentration of extract-treated groups. the blood glucose concentrations were significantly reduced after 60 minutes post-administration of sucrose in all the extract-treated groups and sustained for 180 minutes with the group treated with the lowest dose of the extract (30 mg/kg) having no increment in bgl followed by groups treated with 60 and 90 mg/kg of the extract with 1.16 and 13.35 % increases respectively (table 3). ebong et al. – inhibitory effect of mammea africana … 177 table 1. effect of ethanol leaf extract of mammea africana on blood glucose level of rat after oral administration of starch load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 86.00±11.53 87.66±7.12(1.93) 87.66±7.62(1.93) 73.66±6.17 91.0±7.50(5.81) 80.00±6.02 starch 2000 73.33±8.25 119.66±5.45a(63.18) 115.66±1.33a (57.72) 104.66±2.60a (42.72) 95.66±3.75a(30.45) 92.0±6.35(25.46) acarbose 100 72.33±2.69 85.33±12.97(17.97) 80.33±7.21(11.06) 76.33±3.48(5.53) 74.0±1.00(2.30) 72.33±8.68(0) extract 30 78.0±4.35 111.33±4.97(42.73) 94.0±3.51(20.51) 78.0±1.73() 72.33±0.88() 66.66±0.88() 60 86.33±3.75 92.33±6.33(6.95) 80.0±2.64() 74.00±2.30() 70.00±1.15() 68.66±2.72() 90 82.10±5.85 100.0±13.15(21.80) 94.66±10.72(15.29) 82.0±4.61() 76.66±3.84() 71.0±1.73() data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. table 2. effect of ethanol leaf extract of mammea africana on blood glucose level of rat after oral administration of maltose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 100.00±4.25 88.33±1.85 92.33±4.25 90.33±2.33 89.0±4.35 87.33±3.84 maltose 2000 92.0±4.04 134.33±2.90b(46.01) 128.66±5.45a(39.84) 117.33±4.66a(27.53) 97.66±0.66(6.15) 104.16±2.48(13.21) acarbose 100 90.33±2.48 86.66±2.90 82.0±6.00 79.33±2.96 71.66±3.75 78.0±3.78 extract 30 73.33±1.45 86.0±1.73(17.27) 79.66±0.33(8.63) 74.66±0.33() 72.33±1.45 68.33±5.16 60 76.33±0.66 92.0±3.78(17.03) 81.00±2.51(6.11) 76.0±0.57() 70.6±0.33() 68.66±0.33() 90 76.0±2.51 92.0±5.03(21.05) 86.0±5.50(13.15) 81.0±5.03(6.57) 74.0±3.05() 70.33±2.96() data is expressed as mean ± sem. significant at ap<0.05, bp< 0.01, when compared to control (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. table 3. effect of ethanol leaf extract of mammea africana on blood glucose level of rsat after oral administration of sucrose load. treatment dose blood glucose level mg/dl in min mg/kg 0 min 30 min 60 min 90 min 120 min 180 min control normal saline 100.00±4.25 88.33±1.85 92.33±4.25(1.80) 90.33±2.33(3.62) 89.0±4.35(1.55) 87.33±3.84(3.98) sucrose 2000 82.30±2.14 132.33±1.90b(60.78) 130.22±2.45(58.22) 120.66±3.22a(46.60) 115.0±2.46(39.73) 106.22±4.24(29.06) acarbose 100 85.34±1.36 88.22±1.10(3.37) 86.0±2.20c(0.77) 85.33±2.15c() 84.26±1.14a() 82.28±2.26a() extract 30 84.6±2.60 102.33±8.64(20.95) 94.33±1.20a(11.50) 91.66±2.40b(8.34) 86.66±2.90b(2.43) 81.33±5.69() 60 85.66±5.36 111.6±6.93b(30.28) 98.0±5.29(14.40) 92.0±4.35(7.40) 90.66±4.91a(5.83) 86.66±3.38(1.16) 90 77.33±1.85 120.66±2.60(56.03) 112.0±3.05b(44.83) 98.0±1.52b (26.72) 93.66±1.85b(21.11) 87.66±1.45c(13.35) data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, when compared to control. (n=5). values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. discussion mammea africana stembark is used in ibibio traditional medicine in the treatment of diseases such as diabetes among others. this work investigated the effect of m. africana stembark on alpha-amylase and alphaglucosidase activities in rats. the extract was found to inhibit increases in blood glucose concentration following starch administration though non-dosedependently. complete digestion of dietary polysaccharides like starch is achieved by the combined action of alpha-amylases and alpha-glucosidase enzymes. the alpha-amylase enzyme digests alphabonds of the alpha-linked polysaccharides yielding disaccharides, like maltose, which are further reduced to monosaccharides by membrane bound alpha-glucosidase enzymes (alongi and anese, 2018; kalra, 2014). inhibitions of these enzymes delay the digestion of ingested carbohydrates thereby resulting in a small rise in blood glucose concentrations following carbohydrate meals as was observed in this study. as a target for 178 biology, medicine, & natural product chemistry 11 (2), 2022: 175-180 managing type 2 diabetes mellitus, many medicinal plants have been reported to possess alpha-amylase and alpha-glucosidase inhibitory potential (esimone et al. 2001; ibrahim et al. 2014). similarly, the stembark extract significantly and non dose-dependently inhibited blood glucose rise when coadministered with maltose and sucrose. acarbose, the standard drug used in this study significantly inhibited blood glucose rise when co-administered with starch, maltose and sucrose. the results of this study corroborate earlier reported antidiabetic activity of the stembark extract of m. africana in rats (okokon et al. 2007; tchamadeu et al. 2010). this further suggest that inhibition of alpha-glucosidase and alpha-amylase activities may be one of the antidiabetic modes of action of the extract. the inhibitory activities of plant extract are linked to their phytochemical constituents especially polyphenols. the stembark extract has been reported to contain 5,-7-dihydroxy-8(12-methyl-butryl)-4-npentylcoumarin (carpenter et al. ;1970;1971; crichton and waterman, 1978), mesuxanthone b (carpenter et al. 1971), 4-n-propylcoumarins and 4-phenyl coumarins (ouahouo et al. 2004) have also been isolated from the stembark. polyphenolic compounds have been variously reported to inhibit alpha-glucosidase and alpha-amylase activities (proenca et al. 2017; su and tang, 2019; proenca et al. 2017). coumarins in particular have been reported to inhibit alpha-glucosidase and alpha-amylase activities (zhao et al. 2015; karakaya et al. 2018). also, xanthones are reported to possess the potentials to inhibit alpha-amylases and alpha-glucosidase enzymes (malik et al. 2020). the presence of these compounds in the stembark extract could have contributed to the observed activity of this study and therefore explains the antidiabetic mechanism of the stembark of m. africana. alpha-amylase and alpha-glucosidase inhibitions by plants extracts have been reported severally (ishnava and metisariya, 2018; shirwaikar et al. 2005). phytochemicals implicated as anti-diabetic agents, do so possibly through alpha-amylase and alpha-glucosidase inhibition. the phytochemicals implicated include; flavonoids, saponins, tannins and terpenoids (ishnava and metisariya, 2018; ortiz-andrade et al. 2007; yoshikawa et al. 1998). also, polyphenolic compounds from plants are known to cause several effects on the biological systems which include enzymes inhibitions (funke and melzig, 2005; kalita et al. 2018). the phenolic compounds are known to be strong metal ion chelators and protein precipitation agents forming insoluble complexes with proteins as well as acting as biological oxidants (ishnava and metisariya, 2018). the presence of the polyphenolic compounds in the stembark extract in addition to the xanthones may suggests that their inhibitory potential on alpha-amylase and the membrane-bound intestinal alpha-glucosidase enzymes. conclusions the results of this study suggest that inhibition of alpha amylase and alpha glucosidase enzymes maybe one of the modes of antidiabetic activity of the stembark extract of mammea africana which can be attributed to the activities of its phytochemical constituents. acknowledgements: the authors greatly acknowledges mr nsikan malachy of pharmacology and toxicology department for his technical support. authors’ contributions: noe, jea and ji conceived and designed this study. jea and ji carried out the experiments and drafted the manuscript. noe performed the statistical analysis, edited and reviewed the manuscript. jea, noe and ji read and approved the final manuscript. competing interests: the authors declare that there are no competing interests. references adjanohoun je, aboubakar n, dramane k, ebot me, ekpere ja, enoworock eg, foncho d, gbile zo, kamanyi a, kamoukom, et al (1996). traditional medicine and pharmacopeiacontribution to ethnobotanical and floristic studies in cameroon. porto-novo, benin: cnpms. pp 15. alongi m, anese m (2018). effect of coffee roasting on in vitro α-glucosidase activity: inhibition and mechanism of action. food research international 111: 480-487. carpenter i, mc garry ej, scheimann f (1971). extractives from guttiferae. part xxi. the isolation and structure of nine coumarins from the bark of mammea africana g. don. journal of the chemical society 22: 3783-3789. carpenter i, mc garry ej, scheimann f (1970). the neoflavonoids and 4-alkylcoumarins from mammea africana g. don. tetrahedron letters 46: 3983-3986. chapius jc, sordat b, hostettman k (1988). screening for cytotoxic activities of plants used in traditional medicine. journal of ethnopharmacology 23: 273-284. crichton eg, waterman pg (1978). dihydromammea c/ob: a new coumarin from the seed of mammea africana. phytochemistry 17: 1783-1786. dongmo ab, azebaze agb, nguelefack tb. 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nutrient utilization while minimizing nutrient losses and environmental impacts. in this study, biochar–chitosan composites were employed as encapsulating materials for urea and fe to produce srf beads. the beads were synthesized through ionic gelation in naoh solution, followed by swelling tests and structural characterization using fourier-transform infrared spectroscopy (ftir) and x-ray diffraction (xrd). the swelling analysis demonstrated that the most effective formulation was obtained from the composition containing 3 g of biochar and 3 g of urea, achieving the highest swelling capacity of 92% after 48 h. this result indicates that the biochar–chitosan matrix provides sufficient porosity and water affinity to support nutrient retention and release. ftir spectra confirmed the presence of characteristic functional groups of biochar, chitosan, urea, and fe, verifying the successful incorporation of all components into the composite beads. furthermore, xrd analysis revealed a predominantly amorphous structure, as evidenced by broad peaks in the 2θ range of 20°–60°. however, sharp crystalline peaks were also observed at 2θ ≈ 29.1° and 33.9°, corresponding to urea, and at 2θ ≈ 40°, attributed to crystalline phases of urea and fe. these results suggest that the structural configuration of the beads combines both amorphous and crystalline phases, which may contribute to controlled nutrient release behavior. overall, the findings demonstrate that biochar–chitosan encapsulation is a promising strategy for developing efficient srfs with enhanced swelling properties and structural stability, offering potential for sustainable agricultural applications. keywords: biochar-chitosan beads; slow release; nitrogen; fe. introduction alor regency is an archipelagic region with relatively expansive agricultural land that farmers can utilize for cultivation. agricultural development in alor has shown positive progress, but crop production there remains heavily dependent on fertilizers (hermita putri et al., 2019; zakarias mautuka, astriana maifa, 2022). the community primarily uses urea fertilizer to enrich their crops. urea is widely employed because it accelerates plant growth, making it a preferred choice for agricultural land. one of the key elements in urea fertilizer is nitrogen (n). if plants lack nitrogen, they cannot grow normally; therefore, nitrogen is essential for plant growth and serves as a primary building block for plant proteins. on the other hand, urea is highly soluble in water and prone to volatilization into the air. plant absorption of urea is not optimal, as only about 30–40 % of applied nitrogen is absorbed by plants, while the remaining 60 % is lost as ammonia gas through volatilization. due to its high solubility in water, urea cannot be efficiently absorbed by plants (kuczuk, 2019; liu et al., 2023). urea is classified as a fertilizer containing 46% nitrogen (n). nitrogen is a macronutrient that plants require in relatively large amounts and plays a vital role in greening and photosynthesis (liu et al., 2023; rosa et al., 2018). micronutrients, though needed in much smaller quantities, are nonetheless essential for physiological processes and plant growth. these nutrients are considered essential. essential micronutrients such as fe, mn, zn, b, and cu include iron (fe), which plays a critical role in plant respiration and chlorophyll formation. if plants lack iron, they become infertile or withered. iron can be sourced from iron sand of puntaru that contains 77,80% fe (karbeka et al., 2021; martasiana karbeka, faryda veronica lamma koly, 2020). the addition of fe to a urea solution can support plant growth. however, excessive fe can be toxic to plants, causing direct cellular damage, destroying lipids and proteins, and impairing root growth thus necessitating controlled fe release in the environment. one approach to enhance the absorption efficiency of both nitrogen from urea and fe is to reduce solubility by using a matrix to coat the fertilizer. this coating process for urea and fe can utilize manuscript received: 22 september, 2025. revision accepted: 04 december, 2025. published: 12 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1225-1230 mailto:karbekamartasiana@gmail.com 1226 biology, medicine, & natural product chemistry 14 (2), 2025: 1225-1230 environmentally friendly materials via the slow-release fertilizer (srf) method, which controls or slows the release of nutrients for plant growth(sholeha et al., 2024). one promising material for srf production is chitosan. chitosan is a natural polymer widely used as a microcapsule wall material because it is biodegradable and biocompatible. it has advantages such as good degradability, non-toxicity, environmental and living organism safety, and possesses antimicrobial, antifungal, and metal-chelating properties (herianus et al., 2024). however, chitosan also has drawbacks, including low mechanical strength when forming hydrogels—resulting in low water absorption (swelling). therefore, chitosan must be modified with other materials to improve its mechanical properties. one such material that can serve as a matrix to enhance chitosan’s mechanical properties is biochar (das et al., 2022; lestari et al., 2022; martasiana karbeka, zakarias mautua, 2024). biochar refers to a carbon-rich solid produced from biomass through incomplete combustion under minimal oxygen conditions (pyrolysis). biocar is not ably more stable in soil and resists oxidation. the advantages of using biochar include its role as a soil conditioner enhancing soil stability, improving permeability and aeration, boosting soil organic carbon content, and increasing the retention of nutrients and water for plant use. biochar is utilized in slow-release fertilizers due to its long-term persistence in soil and its function as a method for storing soil carbon critical for maintaining soil fertility (manzoor et al., 2022; zakarias mautuka, astriana maifa, 2022). implementing chitosan-biochar composites as coatings for urea and iron (fe) involves experimenting with various compositions to achieve optimal properties for slow-release fertilizer applications (yu et al., 2024). determining the optimal bead composition includes testing water absorption capacity and analyzing phase composition and crystal structure using x-ray diffraction (xrd). materials and methods materials the instruments used in this study include a volumetric flask, graduated cylinder, erlenmeyer flask, dropper pipette, analytical balance, glass beaker, magnetic stirrer, chemistry glassware, and stirring rod. samples were analyzed using x-ray diffraction (xrd). the materials used in this research were iron sand, urea, biochar, chitosan, 6% acetic acid, distilled water (aquadest), naoh, and a ph indicator. procedures preparation of the chitosan solution five grams of chitosan powder were transfered into an erlenmeyer flask. then, a 2% acetic acid solution was added, and homogenized the mixture using a magnetic stirrer until the chitosan dissolves completely, forming a hydrogel. synthesis of slow-release fertilizer beads the slow-release fertilizer was prepared using several modified treatments. first, chitosan was mixed with 2% acetic acid and stirred with a magnetic stirrer for 30 minutes. the chitosan solution was then divided into four portions, and each was supplemented with urea, biochar, and fe in the following variations: table 1. composition variations. sample code fe (g) urea (g) biochar (g) cb2u2fe 1 2 2 cb3u2fe 1 2 3 cb2u3fe 1 3 2 cb3u3fe 1 3 3 note: sample code description cb2u2fe : 2 g biochar 2 g urea cb3u2fe : 3 g biochar 2 g urea cb2u3fe : 2 g biochar 3 g urea cb3u3fe : 3 g biochar 3 g urea before adding fe, urea, and biochar into the chitosan solution, dissolve urea in 15 ml of water, then add the specified amount of biochar and stir using a magnetic stirrer until the mixture is homogeneous. next, introduce the urea solution into the chitosan, then stir for 10–15 minutes until homogeneous. add fe and mix with a spatula. the resulting solution is ready to be molded by injecting it into a 1 m naoh solution using a syringe. the resulting beads are basic, so they must be neutralized by washing with water until the ph is neutral. confirmation that the beads are neutralized is done via negative tests with ph paper and indicators (pp), then dried. water absorption test the water absorption test followed a modified procedure from reference (15). each variant of the slow-release fertilizer beads (0.5 g) was weighed and immersed in four glass beakers containing 25 ml of distilled water. beads were soaked for 24, 48, 72, and 96 hours. after each specified soaking time, the beads were separated accordingly. the percentage swelling (% swelling) was calculated using the equation: %swelling = 𝑊𝑤𝑒𝑡 − 𝑊𝑑𝑟𝑦 𝑊𝑑𝑟𝑦 𝑥100 characterization by ftir and xrd the chitosan–biochar–urea composite beads were characterized using ftir and x-ray diffraction (xrd). samples were placed on a prepared sample holder, and diffraction angles (2θ) were set using cu kα radiation with a wavelength of 1.54056 å. the test was run and the diffractogram data of the sample were obtained. karbeka – eco-friendly biochar-chitosan coating 1227 results and discussion synthesis beads of chitosan–biochar–urea–fe the synthesis of chitosan–biochar–urea–fe slow-release fertilizer beads begins with preparing a 2% acetic acid (ch3cooh) solution to dissolve the chitosan powder, as illustrated in figure 1. figure 1. reaction between chitosan and acetic acid. chitosan is insoluble in water but dissolves well in acidic solvents, especially weak acids such as acetic acid. when chitosan is treated with acetic acid, a protonation reaction occurs where the –nh₂ groups are converted to –nh₃⁺. this transforms the chitosan into a soluble form, resulting in a chitosan gel. upon adding biochar, urea, and fe to this chitosan gel and dripping the mixture into a naoh solution, the gel solidifies, forming beads— illustrated in figure 2. figure 2. (a) consolidation of chitosan–biochar–urea–fe in naoh. (b) the resulting beads exhibit a basic nature. the compaction process occurs because chitosan undergoes deprotonation in an naoh solution. hydroxide ions (oh–) attack the amine groups on chitosan, causing the proton to be removed from the – nh₃⁺ groups. this deprotonation increases inter-chain interactions among the polymer chains, resulting in a denser structure, as illustrated in figure 3. figure 3. (a) chitosan–biochar–urea–fe beads in an alkaline state (basic), showing their initial formation; (b) beads that have been neutralized; (c) beads after washing to achieve neutral ph. in addition, naoh helps strengthen hydrogen bonds and ionic interactions within the gel network, contributing to its compaction. the resulting beads are basic in nature. neutralization is achieved through repeated washing until neutrality is indicated by a color change in phenolphthalein (pp). in the first wash, the beads still show a basic character, evidenced by the appearance of red coloration. this red color in the filtrate occurs because residual naoh reacts with phenolphthalein: the hydroxide ion (–oh) interacts with the carbonyl group on the phenolphthalein indicator, yielding a red color—illustrated in figure 4. pp indicator colorless (neutral) pink/red (alkaline). figure 4. reaction of phenolphthalein indicator (pp) with naoh: washing is continued until the beads reach neutrality, as indicated by a negative result with the phenolphthalein (pp) indicator—when pp is added, the filtrate remains clear with no color change. to further confirm that the beads are indeed neutral, the filtrate is also tested using universal ph indicator paper, which shows a neutral ph value (~ph 7). water absorption (swelling) the water absorption test of the resulting beads aims to determine their ability to absorb water and assess their capacity as slow-release fertilizer beads made from chitosan, biochar, urea, and fe. this test evaluates the material’s ability to absorb water without undergoing a b a b c 1228 biology, medicine, & natural product chemistry 14 (2), 2025: 1225-1230 fragmentation or lysis. swelling occurs as water diffuses into the beads due to osmotic pressure and interacts with hydrophilic functional groups. once equilibrium is reached, the absorbed water stabilizes. the results of the swelling test are presented in figure 5. figure 5. percentage water absorption (swelling). based on the swelling test results, among the four bead variants tested, beads labeled cb2u2fe, cb2u3fe, and cb3u3fe reached an optimal swelling state at 48 hours. swelling then declined at 72 hours before rising again at 92 hours, but this later increase did not surpass the 48-hour peak. in contrast, beads labeled cb3u2fe continued to steadily increase their swelling percentage. these findings indicate that during optimal swelling time, the beads likely reached saturation binding water through hydrogen bonding and occupying porous spaces—such that further exposure led to reduced absorption. ultimately, the trapped water remained entrapped within the beads, causing them to swell further due to osmotic diffusion into hydrophilic groups and porous biochar entry. referring to figure 5, the bead variants with relatively low water absorption cb3u2fe and cb2u3fe displayed maximum swelling percentages of approximately 48% and 60%, respectively. meanwhile, bead variants cb2u2fe and cb3u3fe exhibited high water absorption capacity, reaching about 96% and 92%, respectively. these differences in water absorption performance are influenced by the beads' composition. the variants cb3u2fe and kb2u3fe have biochar-to-urea ratios of 2:3, whereas cb2u2fe and cb3u3fe maintain a 1:1 ratio. the composition ratio affects cross-link formation via functional groups during bead fabrication: in cb3u2fe and cb2u3fe, the excess of one component fosters extensive cross-linking, resulting in tightly bonded polymer networks. this structure yields dense and strong bead matrices with enhanced hydrophobic characteristics which explains their lower water absorption. conversely, beads with balanced ratios (cb2u2fe and cb3u3fe) develop fewer cross-links within the polymer matrix, resulting in a looser structure with more hydrophilic properties allowing significantly greater water uptake. ftir characterization 64 96 88 90 44 46 46 48 50 60 54 58 90 92 82 80 24 jam 48 jam 72 jam 92 jam cb2u2fe cb3u2fe cb2u3fe cb3u3fe 24 hours 48 hours 72 hours 92 hours karbeka – eco-friendly biochar-chitosan coating 1229 table 2. description of ftir spectra. no wavelenght (cm-1) functional group description 1 3729,48 free o–h stretching the presence of free –oh groups in chitosan or biochar that have not fully reacted through hydrogen bonding formation. 2 3627,71 3 2973,41 stretching of c-h (aliphatic) or n-h group aliphatic c–h groups are found in biochar and chitosan, whereas n– h groups are commonly present in chitosan and urea 4 2867,78 stretching c-h (aliphatic) typical organic compounds (methylene, ch2) characteristic organic functional groups are found in both chitosan and biochar. 5 1575,76 stretching sy a zmmetrical nh₂ group /n-h bending the presence of vibrational modes of –nh₂/n–h groups—or n–h bonds—in urea or chitosan." 6 1424,20 stretching c–n/ deformasi – ch₂ the presence of secondary amine groups and c–n vibrational modes are indicative of both urea and chitosan." 7 1056,67 stretching c–o-c / c-o both functional groups originate from chitosan and biochar 8 877,30 stretching c-h/aromatic ring vibration can indicate the presence of conjugated aromatic structures 9 790,75 weak vibrations of aromatic rings this vibration is likely originating from biochar 10 592,97 metal stretching –o (fe–o) indicates the presence of iron oxide minerals in iron sand 11 538,58 metal stretching –o (fe–o atau si–o) characteristic fe–o or possibly si–o vibrations from biochar components xrd characterization x-ray diffraction (xrd) analysis of the chitosan– biochar–urea–fe composite fertilizer beads was conducted to determine their physical properties, including phase composition and crystalline structure. xrd was used to identify the crystallinity of the chitosan–biochar–urea–fe composite. the results of this characterization can be seen in figure 6. figure 6. diffractograms of the composite beads: (a) chitosan–biochar– urea; (b) chitosan–biochar–urea–fediscussion. based on figure 6 (a), the diffractogram of the chitosan–biochar–urea composite beads indicates the presence of an amorphous phase, marked by a broad peak in the 2θ range of 20° to 60°. however, there are also distinct sharp peaks at 2θ = 29.1° and 33.9°, which correspond to the crystalline phase of urea. according to phase analysis from the diffractogram, the composite beads exhibit a semi-crystalline structure. based on figure 6(b), the xrd diffractogram for the chitosan-biochar-urea-fe composite beads shows a broad, amorphous halo around 2θ = 20°, indicating presence of a non-crystalline phase. however, there are also distinct sharp peaks in the 2θ range of 30°–40°, corresponding to crystalline phases of urea and fe. therefore, the chitosan-biochar-urea-fe beads exhibit a semi-crystalline structure. although both bead variants (with and without fe) display semi-crystallinity, the diffractogram peaks suggest that the biochar-urea-fe composition has a higher degree of crystallinity compared to the beads without fe. higher crystallinity is typically associated with enhanced mechanical strength, meaning that these beads would be more resistant to lysis or fragmentation, even when their water absorption (swelling) capacity is high. thorough discussion represents the causal effect mainly explains for why and how the results of the research were taken place, and do not only re-express the mentioned results in the form of sentences, not repeat them. concluding sentence should be given at the end of the discussion. conclusions based on the conducted study, the following conclusions can be drawn: a b 1230 biology, medicine, & natural product chemistry 14 (2), 2025: 1225-1230 ▪ among the four synthesized variants of slow-release fertilizer beads, the ones with the highest water absorption capacity were cb2u2fe, achieving 96%, and cb3u3fe, with 92%. ▪ ftir analysis confirmed the presence of ch, cn, nh, and aromatic ring vibration groups characteristic of chitosan, biochar, and urea, along metal stretching -o groups associated with fe, indicating successful bead synthesis. ▪ x-ray diffraction (xrd) analysis confirmed that the chitosan–biochar–urea–fe composite beads exhibit a semi-crystalline structure.the conclusions of the study may be presented in here. acknowledgements: the author expresses sincere gratitude to drpm, ministry of research, technology, and higher education (kemenristekdikti) for the financial support provided through the research and community service grant program for higher education institutions for the fiscal year 2024. this support was granted under the beginner lecturer research grant program (penelitian dosen pemula) for fiscal year 2024, implemented in the same year, as stipulated in the decree no. 0459/e5/pg.02.00/2024 dated 30 may 2024, regarding recipients of state university operational support in research and community service for the fy 2024. competing interests: the authors declare that there are no competing interests. references das, b. k., 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(2018). leaching of the different forms of nitrogen by the application of poultry litter, swine waste, and mineral nitrogen on corn cultures (zea mays l.). environmental quality management, 28(1), 131–138. https://doi.org/10.1002/tqem.21586 sholeha, n. a., wiraguna, e., urip, t., sujarnoko, p., & budiono, d. (2024). fabrication and effectiveness of composite materials in urea slow-release fertilizers : a mini-review. biointerface research in applied chemistry, 14(5), 1–20. yu, y., liu, w., zhang, y., zhang, b., jin, y., chen, s., tang, s., su, y., yu, x., & chen, g. (2024). chitosan/magnetic biochar composite with enhanced reusability: synergistic effect of functional groups and multilayer structure. arabian journal of chemistry, 17(5), 105746. https://doi.org/10.1016/j.arabjc.2024.105746 zakarias mautuka, astriana maifa, m. k. (2022). pemanfaatan biochar tongkol jagung guna perbaikan sifat kimia tanah lahan kering. jurnal ilmiah wahana pendidikan, 8(3), 178– 183. https://doi.org/10.5281/zenodo.5827375 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 1, april 2021 | pages: 27-32 | doi: 10.14421/biomedich.2021.101.27-32 issn 2540-9328 (online) extraction, phenolic content and hydrogen peroxide scavenging capacity of extracts from some honey samples, propolis and bee pollen zohra mohammedi department of biology, faculty of natural sciences and life, university of mustapha stambouli, bp 305 street of mamounia, mascara 29000, tel: 0551724433, algeria. corresponding author mdi3zhr@gmail.com manuscript received: 01 october, 2020. revision accepted: 24 june, 2021. published: 16 july, 2021. abstract honey and propolis is natural food, produced by honey bees (apis mellifera) and largely used by the local population for its medicinal properties. our work aims to extract and evaluate the hydrogen peroxide scavenging capacity of different phenolic extracts from some bee products. phenolic compounds from honey samples, propolis, and bee pollen were extracted by methanol and subjected to radical scavenging activity towards hydrogen peroxide. the results showed the highest values for the total phenolic and total flavonoïd contents in propolis and bee pollen, and a great hydrogen peroxide (h2o2) inhibition (ic50: 0.205 2.210 µg/ml) with honey extracts, while sample “multiflower” is the better antioxidant, more than ascorbic acid used as control. the less scavenging activity was observed with the extract from bee pollen (ic50: 39.383 µg/ml). to combat the harmful effects of free radicals, especially reactive oxygen species including hydrogen peroxide, it is important to use phenolic extracts instead of using honey as it is, and extracts from the honey of different types are excellent antioxidants compared to other bee products. keywords: apis mellifera; honey; propolis; phenolic compounds; hydrogen peroxide. introduction honey is the nectar collected and processed from different plants by honey bees (apis mellifera) and is known for its high nutritional value (an et al., 2016). it has been used in many cultures for its medicinal properties, as a remedy for burns, cataracts, ulcers and wound healing, because it exerts a soothing effect when initially applied to open wounds. according to its origin, honey can be classified in different categories: blossom honey, honeydew honey, monofloral honey, and multifloral honey that has several botanical sources (alvarez-suarez et al., 2014). honey is composed of approximately 82.4% total carbohydrates (almasaudi et al., 2017). the polyphenols constitute minor components of honey (istasse et al., 2016). honey has been reported to promote numerous nutritional and biological effects, such as antioxidant, antimicrobial, antiviral, antiparasitic, anticancer, antiinflammatory, and immunosuppressive activities (duddukuri et al., 1997; othman, 2012; shahzad and cohrs, 2012; borsato et al., 2014; samarghandian et al., 2017; sowa et al., 2017; sinha et al., 2018). however, the antioxidant properties of honey are well known because it contains a number of compounds with antioxidant properties such as polyphenols, amino acids, ascorbic acid, and some enzymes. the most important classes of antioxidants are the phenolic acids and flavonoids (ayoub et al., 2009). they have a promising effect on the treatment of some chronic diseases (isla et al., 2013). a wide range of phenolic constituents in honey are known such as gallic acid, syringic acid, vanillic acid, benzoic acid, chlorogenic acid, ferulic acid, protocatechuic acid, 3-o-caffeoylquinic acid, phydroxybenzoic acid, 5-o-caffeoylquinic acid, caffeic acid, p-coumaric acid, ellagic acid, dihydroxybenzoic acid methyl ether, benzyl caffeate, protocatechualdehyde, gentisic acid, vanillin, sinapic acid, salicylic acid, dihydrocinnamic acid, 2,2,4trihydroxybenzoic acid, hesperetin, naringin, naringenin, hesperidin, tricetin, myricetin, quercetin, quercitrin, quercetin 3-orhamnoside, kaempferol 3-orhamnoside, kaempferol 7-orhamnoside, luteolin, pinobanksin 5methyl ether, naringenin, apigenin, kaempferol, methoxy-kaempferol, pinobanksin, isorhamnetin, chrysoeriol, rhamnetin, chrysin, sakuranetin, pinocembrin, galangin, kaempferide, acacetin, morin, tangeretin, catechin, genistein, ectochrysin, and other compounds such as caffeic acid phenethyl ester, kojic acid, 5-hydroxymethylfurfural, dehydrovomifoliol, leptosin, glyoxal, methylglyoxal, 3-deoxyglucosulose (an et al., 2016; alvarez-suarez et al., 2014; isla et al., https://doi.org/10.14421/biomedich.2021.101.27-32 28 biology, medicine, & natural product chemistry 10 (1), 2021: 27-32 2013; lachman et al., 2010; cianciosi et al., 2018; cheung et al., 2019; nešovíć et al., 2020). another bee product is propolis, which is a resinous substance collected by honeybees from various plant sources. this natural product has been used for thousands of years in folk medicine for several purposes and contains amino acids, terpenes, tannins, polysaccharides, phenolic acids, phenolic acid esters, cinnamic acid, caffeic acid, phenethyl caffeate, pcoumaric acid and flavonoids such as kaempferol, naringin, chrysin, and galangin. propolis possesses several biological activities such as anti-inflammatory, immunostimulatory, antiviral, and antibacterial. propolis extract and its active components showed strong antioxidant activities, a free radical scavenging effect, a significant inhibition of xanthine oxidase activity, ferric reducing antioxidant power, and an anti-lipoperoxidative capacity (russo et al., 2002; kumazawa et al., 2004; korayem et al., 2012; socha et al., 2015). a large number of studies have estimated the antioxidant activities of honey by a range of methods (dpph free radical scavenging activity, abts radical cation decolorization assay, frap assay, orac assay, etc.), but the step of dilution is a major drawback in antioxidant tests since it generates a toxic oxidant substance (h2o2), the cause of the exclusion by researchers of the antioxidant test by hydrogen peroxide scavenging activity in entire honey. so, the aim of this study was to extract phenolic and flavonoids compounds, then evaluate the antioxidant activity of obtained extracts from different types of honey against hydrogen peroxide, and to compare with two phenolic extracts from propolis and bee pollen. also, we highlight the ability of the phenolic compounds in honey to eliminate hydrogen peroxide, and therefore the step of extraction of polyphenols is important in the context of the use of honey in the fight against oxidative stress. materials and methods bee products honey samples (multiflower and monofloral honey: eucalyptus, thyme, sidr, citrus, and epins honey), propolis and bee pollen are the local products of tlemcen, algeria. the 100% natural honey samples from apis mellifera were obtained directly from the beekeeper in 2019 from different vegetation sources in tlemcen city. these honey samples were chosen for their quality, in terms of low content of water and hydroxymethylfurfural (hmf). extraction of phenolic compounds from honey samples the extraction of phenolic compounds from the honey samples was performed according to the method described by an et al. (2016). the stationary phase of the glass column, amberlite xad-2 resin, was prepared by soaking 100g of amberlite xad-2 resin in methanol. the packed column was washed successively with acidified distilled water followed by neutral distilled water. the honey sample was diluted with acidified water and loaded into the packed glass column. sugars were eliminated with hydrochloric acid solution and water washes. phenolics compounds were desorbed with methanol and then dried at 50°c using a rotavapor. the dried product was protected from light and stored at 5°c until use. extraction of phenolic compounds from propolis and bee pollen for extract phenolic compounds from propolis and bee pollen samples, the hydroalcoholic solutions (methanolwater) were used. for each 5g sample, 100ml of methanol 80% were added, the mixture was covered with aluminum paper and then filtered after 24h. the filtrates were dried at 50°c using a rotavapor. the dried extracts were stored at 5°c until use. total phenolic contents the total phenolic contents of honey samples, propolis, and bee pollen extracts were analyzed by the folinciocalteu method (singleton et al., 1999) using gallic acid as a standard. the methanolic solution of each sample of honey, propolis, bee pollen, and gallic acid was prepared. the test samples and standard solutions of different concentrations were introduced into separate test tubes. folin-ciocalteu reagent, diluted in water (1:1), and na2co3 saturated solution were added. the tubes were vortexed, incubated in the dark at room temperature for 25 minutes and then the absorbance was read at 725nm against the blank using a spectrophotometer. the total phenolic content was determined by comparison with a calibration curve of gallic acid and represented as mg gallic acid equivalents per 100 gram (mggae/100g). total flavonoid content the total flavonoïd content was determined by the alcl3 method (cottica et al., 2011) using rutin as standard. 250µl of alcl3 5% (m/v in methanol) were added to separate test tubes containing the methanolic solution of each honey sample, propolis, bee pollen extracts, and rutin solutions of different concentrations. after 30 min incubation at room temperature, the absorbance was read at 425nm using a spectrophotometer. the total flavonoid content was determined by comparison with the calibration curve of the rutin and represented as mg rutin equivalents per 100gram (mgre/100g). antioxidant activity (hydrogen peroxide scavenging activity) the hydrogen peroxide scavenging activity was measured by the method described by ruch et al. (1989). a volume of 0.6 ml of the solution of hydrogen mohammedi – h2o2 scavenging activity of bee products 29 peroxide (0.089 mm), prepared in phosphate buffer (0.1m, ph 7.4) was added in a series of test tubes, containing 3.4ml phenolic extract solutions (310µg/ml) from honey samples, propolis, bee pollen and standard (ascorbic acid). the percentage of hydrogen peroxide scavenging activity was calculated by the formula: % scavenged [h2o2] = [(ac – at)/ac] x 100 where ac is the absorbance of the control and at is the absorbance in the presence of the phenolic extracts from different samples (tests) and standard. statistical analysis the results of experiments were expressed as (mean ± sd; n=3), and subjected to statistical analysis at 95% confidence level (p0.05) by student's t-test using graphpad prism software. results and discussion the results of phenolic content and flavonoid content were reported in table 1. total phenolic content in six honey samples was ranged from 4.36 mggae/100g to 26.29 mggae/100g honey, with the average value of 12.755 mggae per 100g honey. the multiflower type of honey was the sample containing a high amount of phenolic compounds and the monofloral type "citrus" honey was the sample with the lowest quantity of phenolic compounds compared to all honey samples. the same observation was showed in flavonoid content. the multiflower type remains the richest honey in phenolic compounds compared to other honey. while the lowest content is also observed in the "citrus" type. the flavonoids content obtained was from 1.95 ± 0.352 mgre/100g to 10.65 ± 1.020 mgre/100g, with an average amount of 4.607 mgre per 100g honey. the evaluated results were in close agreement with the results reported by meinen et al. (2014), which have reported that total phenolic content is ranged between 8.114 mg and 109 mg per 100g honey and for the total flavonoid content is ranged between 0.655 mg and 212 mg per 100g honey. this study was perfected in 30 honey samples. boulanouar et al. (2017) have observed higher levels of phenolic compounds in two types of algerian honey, which are 38 ± 0.009 mggae/100g for the type “zizyphus” and 86±0.008 mggae/100g for the type “harmala”, whereas for the flavonoids, they found contents, which belong to the range of values that we recorded, the values of flavonoids were 5 ± 0.003 mgre/100g and 8 ± 0.002 mgre/100g for “zizyphus” and “harmala” types, respectively. also, the same remark is observed with the works of khalil et al. (2012). these authors analyzed four samples of algerian honey and registered mean values of 45.983 ± 0.192 mg/100g for phenolic compounds and values of 5.423 ±0.062 mg/100g for flavonoids. table 1. total phenol content and flavonoids of honey samples, propolis and bee pollen. sample average amount of phenolic average amount of flavonoids (mggae/100g) mgre/100g multiflower honey 26.29 ± 9.764a 10.65 ± 1.020a thyme honey 8.67 ± 2.494b 2.61 ± 0.558b citrus honey 4.36 ± 2.441c 1.95 ± 0.352b eucalyptus honey 8.97 ± 1.033b 2.21 ± 1.068b sidr honey 14.20 ± 3.278d 5.31 ± 0.112c epins honey 14.04 ± 4.260d 4.91 ± 0.232c propolis 9161.0 ± 6.906e 2189.62 ± 1.975d bee pollen 4794.15 ± 6.169f 1267.38 ± 0.912e data are (mean ± sd, n = 3). means within a column sharing the same letter are not significantly different by student’s t-test (p < 0.05). total phenol and flavonoids were also compared to the values reported for the honey from different countries. some authors have found results, approaching our range of values, such as the work of reshma et al. (2016), almasaudi et al. (2017) and krpan et al. (2009). the first authors have shown that the phenolic content in 24 samples of honey was ranged between 20.2 ±1.2 mg/100g to 30.78 ± 2.5 mg/100g, the second authors have observed that the high total phenolic content was 10.399 ± 0.168 mg/100g in “manuka” type of honey and the lowest phenolic content was 9.6 ± 0.002 mg/100g for “sidr” type of honey, while the third authors have ranged the total phenolic content from 3.172 mg/100g to 8.011 mg/100g honey and this later study was performed only in one type of honey, which is the "acacia" type. in contrast, other authors have deferred high and even very high levels, for examples, for chua et al. (2013) the total phenolic content of honey samples was in the narrow range from 110.394 to 196.500 mggae/100g, arahaman et al. (2013) showed that the total phenolic contents for honey samples were ranged from 38.379 to 60.617 mg gae/100g, pontis et al. (2014) ranged the total phenolic content from 25 to 54.8 mg gallic acid per 100g of honey, while for buba et al. (2013), the mean values of total phenolic content are around 65.31 ± 19.50 mg gallic acid equivalent per 100g. in addition, some authors have reported very small quantities of phenolic content in honey, for example, ferreres et al. (1994) reported 0.50 to 2.0 mg per 100g honey. for flavonoids, pontis et al. (2014) have shown less content than obtained in our studies, the total flavonoid content was ranged from 0.9 to 4.86 mg per 100g honey. the results obtained experimentally in table 1 show very high levels of phenolic compounds and flavonoïds in propolis and bee pollen compared to those obtained in honey samples and the amount of phenolic and 30 biology, medicine, & natural product chemistry 10 (1), 2021: 27-32 flavonoids in propolis was higher than bee pollen. the quantities registered in propolis and bee pollen were 9161.0 ± 6.906 mggae/100g, 4794.15 ± 6.169 mggae/100g for total phenolic content and 2189.62 ± 1.975 mgre/100g, 1267.38 ± 0.912 mgre/100g for total flavonoids content, respectively. boulanouar et al. (2017) have also observed that propolis contains more phenolic and flavonoïds than honey, the respective quantities registered were 2385±2.90 mggae/100g and 379±0.54 mg re/100g. čeksterytė et al. (2016) have also observed high levels of phenolic compounds in bee pollen, but are less relative to the quantities we have obtained. the values of total phenolic content found were 2330 mg gae/100g. the antioxidant capacity of honey and bee products is due mainly to the phenolic compounds. the samples exhibited various antioxidant activity measured towards hydrogen peroxide, which is concentration-dependent (figure 1). results show that the scavenging activity values on hydrogen peroxide of phenolic extracts from honey samples were more efficient than other bee products. lower antioxidant activity was observed in extracts from propolis and bee pollen. figure 1. hydrogen peroxide (h2o2) scavenging activity of phenolic extract from honeys, propolis, bee pollen, and ascorbic acid. the antioxidant activity of honey has been reported in many scientific works (krpan et al., 2009; pontis et al., 2014; reshma et al., 2016; chua et al., 2013; arahaman et al., 2013). currently, some very limited research on antioxidant activity has been conducted on phenolic extracts of honey (lianda et al., 2012; bridi et al., 2017; estevinho et al., 2008; halagarda et al., 2020), but no research has assessed the capacity of extracts or phenolic fractions isolated from honey by the hydrogen peroxide scavenging method. hydrogen peroxide is not very reactive, but it can be toxic because it may produce hydroxyl radicals in the cells. thus, removing h2o2 is very important in the cell. the scavenging ability of honey samples, propolis, and bee pollen was examined by comparing to that of the known antioxidants ascorbic acid. the extract of honey type "multiflower" showed powerful activity on hydrogen peroxide and decreases its concentration more than the positive control, ascorbic acid. antioxidant activity, expressed as ic50 (table 2) was 0.206 ± 0.0243 µg/ml, while the standard given an ic50 value of 0.694 ± 0.0238 µg/ml. this parameter ic50 defined the concentration of the sample, which scavenge 50% of hydrogen peroxide. the sample with lower ic50 presents potent antioxidants. the phenolic extracts from extracts of others honey types exhibited also a good antioxidant activity on h2o2. the scavenging ability is in the following order: “multiflower” ˃ ascorbic acid ˃ “citrus” ˃ “epins” ˃ “sidr” ˃ “thyme” ˃ “eucalyptus”. it has been reported that the antioxidant capacity of honey was correlated with the biochemical constituents, in particularly with phenolic compounds and flavonoids (chua et al., 2013; buba et al., 2013). table 2. hydrogen peroxide scavenging capacity of phenolic extract from honey samples, propolis and bee pollen, expressed as ic50 (µg/ml). extract ic50 (µg/ml ) multiflower honey 0.206 ± 0.0243a thyme honey 1.928 ± 0.0209b citrus honey 0.771 ± 0.0239c eucalyptus honey 2.211 ± 0.0216d sidr honey 1.259 ± 0.0224e epins honey 0.951 ± 0.0099f propolis 35.116 ± 0.4049g bee pollen 39.383 ± 0.2622h ascorbic acid 0.694 ± 0.0238i data are (mean ± sd, n = 3). means within a column sharing the same letter are not significantly different by student’s t-test (p < 0.05). poor scavenging activity is observed with extracts from propolis (35.116 µg/ml) and bee pollen (38.383 µg/ml). in similar studies, the h2o2 scavenging assay of phenolic compounds extracted from propolis by various solvents (distilled water, absolute methanol, 50% methanol, and 70% methanol) presented different action on h2o2, which ic50 values were between 11.72 µg/ml and 765.75 µg/ml (farooq wali et al., 2016; ramnath et al., 2016; kızılpınar temizer et al., 2017). although these two bee products are quantitatively rich in phenolic compounds, but their extracts have a low antioxidant capacity compared to honey extracts, which suggests that the antioxidant capacity is not always dependent on the amount of phenolics and flavonoids, honeys are therefore a good source of antioxidant substances. also, the botanical origins of honey greatly influence hydrogen peroxide scavenging capacity. the results of the ic50 obtained in table 2 clearly confirm these observations. mohammedi – h2o2 scavenging activity of bee products 31 conclusion in this present study, it had been established that phenolic extracts from all types of honey, propolis, and bee pollen contain phenolic and flavonoids compounds and possessed antioxidant property toward hydrogen peroxide, and therefore the preventive and medical application of phenolic extracts from honey, and other bee products against oxidative stress are recommended. all honey types after extraction of phenolic compounds are able to scavenge efficiently the hydrogen peroxide, and this capacity is as much greater than that observed in other bee products. antioxidant activity against h2o2 in honey samples is related to botanical type, which is strongly affected by the floral origin, and therefore varies quantitatively and qualitatively with the content of antioxidant phenolic compounds. it is well shown in the results that extract from 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issn 2540-9328 (online) phytochemicals compound and antioxidant activity of tradescantia spathacea sw. leaf extracts retno aliyatul fikroh1,*, muhamad aditya hidayah2 1department of chemistry education, faculty of tarbiyah and education, uin sunan kalijaga yogyakarta, indonesia. 2department of chemistry, faculty of science, prince of singkla university, hat yai, songkhla, thailand. corresponding author* retno.fikroh@uin-suka.ac.id abstract tradescantia spathacea sw. is commonly used as a traditional medicine plant to treat several diseases. this study aims to identify phytochemical constituents using phytochemical screening, quantify the total phenolic (tpc) and flavonoid content (tfc) analysis, and evaluate the antioxidant activity of various extracts via dpph free radical scavenging activity. the phytochemical constituent revealed the presence of bioactive compounds such as flavonoid, alkaloid, saponin, steroid, triterpenoid, and tannin in different extracts of tradescantia spathacea sw. leaf. the methanolic leaf extract showed the best total phenolic and flavonoid content values of 83.35±0.00 mggae/g and 33.79±0.13 mg/g, respectively. methanolic extracts also showed potent antioxidant activity in the dpph assay with ic50 values of 80,835 µg/ml. the results suggest that tradescantia spathacea sw. could be an alternative source of natural antioxidant agents. keywords: phytochemical compound; tradescantia spathacea sw.; antioxidant. introduction the epidemiological transition has increased in degenerative diseases. according to the world health organization, up to 17 million deaths occur yearly due to degenerative diseases (di renzo et al., 2021). oxidative stress plays a role in various diseases, particularly degenerative ones. this stress is caused by an imbalance between reactive oxygen species (ros) or free radicals and antioxidants in the body (pizzino et al., 2017). however, antioxidant compounds can inhibit or prevent oxidative stress (dhalaria et al., 2020). synthetic antioxidants have been used for their higher stability, low cost, and wide availability (xiu-qin et al., 2009). the most commonly used synthetic antioxidants in the industry include butyl hydroxytoluene (bht), butyl hydroxy anisole (bha), propyl gallate (pg), and terbutyl hydroquinone (tbhq) (lourenço et al., 2019). synthetic antioxidants have been widely used, but several published studies indicate adverse effects such as skin allergies (velázquez-sámano et al., 2019), tumors, and carcinogens (felter et al., 2021). therefore, the search for natural materials to replace synthetic antioxidants is actively pursued. natural antioxidants can be derived from medicinal plants like fruits, vegetables, flowers, spices, and traditional herbs (xu et al., 2017). these natural antioxidants contain secondary metabolite compounds, including polyphenols (such as phenolic acids, flavonoids, anthocyanins, lignans, and stilbene), carotenoids (xanthophyll and carotene), and vitamins (e and c) (baiano & del nobile, 2016). some polyphenolic compounds with hydroxyl groups are reported to act as antioxidants that can prevent free radicals through various mechanisms like reducers, free radical capturers, metal chelators, and electron donors (kumar et al., 2013). furthermore, flavonoid groups are known for their high antioxidant properties both in vitro and in vivo. natural antioxidants are safe, relatively affordable, and have fewer adverse health effects (dewage et al., 2022). indonesia has a rich biodiversity that was used as raw material to produce natural medicine plants (rahayu & andini, 2019). local plants can serve as an alternative source of medicine for treating various diseases. one such plant is the leaves of tradescantia spathacea sw, commonly found in tropical climates. despite being considered ornamental plants, they have potential medicinal properties (russo et al., 2017). the tradescantia spathacea sw leaves, flowers, and stems are often used to make tea in western countries (pulipaka et al., 2020). leaves of tradescantia spathacea sw contain a range of bioactive compounds including flavonoids, carotenoids, alkaloids, saponins, anthocyanins, tannins, terpenoids, steroids, ferulic acid, chlorogenic acid, vanillic acid, and p-cumaric acid (garcía-varela et al., 2015; rosales-reyes et al., 2007). many of these compounds exhibit pharmacological manuscript received: 10 march, 2025. revision accepted: 20 may, 2025. published: 26 june, 2025. https://doi.org/10.14421/biomedich.2025.141.283-288 mailto:retno.fikroh@uin-suka.ac.id 284 biology, medicine, & natural product chemistry 14 (1), 2025: 283-288 activities, with particular emphasis on their antioxidant properties. overall, the diverse compounds found in pineapple leaves have the potential to be utilized in the production of herbal medicines for various health benefits (tan et al., 2015). bioactive compounds found in tradescantia spathacea sw leaf can be extracted using the maceration method (amalia rachmawati et al., 2020). several factors, such as the type of solvents, temperature, time, and ph, influence the extraction process. the solvent used in the process is essential for extracting bioactive compounds in plants (xu et al., 2017). most phenolic compounds, flavonoids, and anthocyanins are polar antioxidants, so the solvents used are water, ethanol, methanol, and acetone (ngo et al., 2017). meanwhile, nonpolar antioxidants such as carotenoids are soluble in organic solvents (strati & oreopoulou, 2011). ethanol extract from tradescantia spathacea sw roots has been reported to have antioxidant activity equivalent to α-tocopherol and stronger than ascorbate acid (gonzález-avila et al., 2003). using the infusion method, the water extract of tradescantia spathacea sw leaves has shown potential as a chemoprevention agent against liver cancer in mice in vivo(rosales-reyes et al., 2007). other studies suggest that the ethanol extract contains higher levels of flavonoids compared to the water extract of tradescantia spathacea sw leaves (ramesh et al., 2021). the ethanol extract's phytochemical test indicates alkaloids, glycosides, and flavonoids, while the water extract shows the presence of alkaloids, tannins, phenolics, and flavonoids. however, there was no report regarding the antioxidant activity of the extract generated in this study. research on bioactive compounds from various solvents to extract tradescantia spathacea sw leaf has not been carried out. this research aimed to identify the phytochemical constituent, total phenolic and flavonoid content, and antioxidant activity with ic50 values of ethanol, methanol, acetone, water, nhexane, and ethyl acetate extracts. materials and methods chemicals and materials all materials used were of analytical grade. 1,1diphenyl-2-picrylhydrazyl (dpph) from sigma aldrich, ascorbic acid, ethanol, methanol, ethyl acetate, hexane, acetone, folin-ciocalteu reagent, dragendroff-s reagent, liembermann-burchard, quercetin, gallic acid, anhydrous sodium carbonate, aluminium chloride, sodium nitrite, ferric chloride hexahydrate, sulphuric acid, and hydrochloric acid were purchased from merck plant materials tradescantia spathacea sw. leaves were collected from kalasan village, sleman, yogyakarta, in january 2023. the plant confirmed the authenticity of the plant material in the pharmacy department laboratory, gadjah mada university. samples were collected and examined for infection, spores, damage, and distortion. the leave samples were washed with tap water to avoid contamination and dried for about ten days. the dried leaves were cut into small pieces and ground into fine powder. preparation of extracts in this study, maceration was used to extract the leaves of tradescantia spathacea sw. all solvents used were analytical grade. the dried powder was extracted using different solvents, such as ethanol, methanol, acetone, water, n-hexane, and ethyl acetate. the solvents and dried leaves were incubated at room temperature and constant stirring for three days. after incubation, extracts were filtered with a rotary evaporator using whatman filter paper. the crude extracts were determined by comparing the percentage of the weight of the crude extracts to the dry weight of the leaves. phytochemical screening phytochemical screening was used to investigate the presence of flavonoids, alkaloids, saponins, steroids, terpenoids, and tannins from all solvents. determination of total phenolic content (tpc) the total phenolic content of tradescantia spathacea sw leaf was determined by spectrophotometry using the folin-ciocalteu method. in the analysis, an extract solution was dissolved in ethanol with a 1 mg/ml concentration. 2.5 ml of 10% folin-ciocalteu reagent and 2.5 ml of 7% sodium carbonate solution were mixed with 0.5 ml of extract solution.all samples were incubated for 45 minutes in the dark at room temperature. the absorbance was measured using a spectrophotometer at 765 nm. the standard solution of gallic acid was determined using the same procedure. the phenolic content was measured in milligrams of gallic acid equivalent per gram of extract (mg of ga/g). determination of total flavonoid content (tfc) the total flavonoid contents of extracts were determined using the aluminum chloride colorimetric method and the standard solution of quercetin. 0.25 ml of extract dissolved in methanolic solvent was added to 0.1 ml of 5% nano2. after 6 min, 0.15 ml of aluminum chloride solution (10%) was added. the solution was mixed well and incubated at room temperature for 5 min. the absorbance was determined using a spectrophotometer at 510 nm. the total flavonoid content was expressed as mg quercetin per 100 gr dry weight of the sample (mg qe/100 g). antioxidant activity using dpph method the antioxidant activity of crude extracts was measured using a dpph free radical scavenging assay. 3,8 ml of fikroh & hidayah – phytochemicals compound and antioxidant activity … 285 dpph solution was added to 0,2 ml of various concentrations of crude extracts and standard solution (extract concentration 40, 80, 120, 160, 200 ppm and ascorbic acid as standard solution 2, 4, 6, 8, 10 ppm). the solution was incubated at room temperature for 5 minutes in the dark, and the absorbance was measured at 515 nm using uv-vis spectroscopy (gonzález-avila et al., 2003). the dpph radical scavenging activity was calculated using the following equation: % dpph radical scavenging = 𝐴 𝑐𝑜𝑛𝑡𝑟𝑜𝑙−𝐴 𝑠𝑎𝑚𝑝𝑒𝑙 𝐴 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑥 100% the antioxidant activity resulted in ic50 (µg/ml), and each concentration was replicated 3 times. results and discussion results extracts the maceration extraction method was chosen for this study to produce efficient extraction, low-cost extraction, and simple-to-use equipment (mohammed golam rasul, 2018). this study uses solvents with different polarities to extract tradescantia spathacea sw. leaf. polar solvents used were water, methanol, ethanol, aprotic polar solvents such as acetone, and non-polar solvents such as ethyl acetate and n-hexane. in the maceration extraction of 100 grams of tradescantia spathacea sw. dried leaf. the percentage yield of 100 g tradescantia spathacea sw. dried leaves had ethanol leaf extract higher (22,36%) than other solvents such as methanol (16,04%), water (11,97%), ethyl acetate (7,51%), nhexane (13,43%), and acetone (5,08%) (table 1). table 1. percentage yields of tradescantia spathacea sw. leaves extract. extracts ethanol methanol water etyl acetate n-hexana aceton percentage yields (%) 22,36 16,04 11,97 7,51 13,43 5,08 phytochemical screening the result of phytochemical test showed that flavonoids, alkaloids, saponins, steroids, terpenoids, and tannins were detected in all extracts. this result showed that nhexane extract showing the least diverse phytochemical composition and methanolic extract showed all phytochemical except steroid. table 2. phytochemical screening of tradescantia spathacea sw in various extracts. phytochemical compounds ethanol extract methanol extract water extract etyl acetate extract n-hexana extract aceton extract flavonoids ++ ++ + + + ++ alkaloids ++ ++ + + ++ saponin ++ ++ + ++ steroid + + + ++ terpenoid + tannin ++ ++ + ++ total phenolic content (tpc), total flavonoids content (tfc), and antioxidant activity the total phenolic content, total flavonoids content, and antioxidant activity were determined. according to table 3, methanol leaf extracts contained higher tpc, tfc, and antioxidant activity than another extracts. table 3. total phenolic content (tpc), total flavonoid content (tfc), and dpph scavening activity (dpph) of tradescantia spathacea sw. leaf crude extracts. sample tpc (mg gae/g dw) tfc (mg /g dw) dpph (μg/ml) ethanol 78.47±0.89e 25.74±0.00e 99,5286 methanol 83.35±0.00f 33.79±0.13f 80,8346 water 59.27±0.38c 19.88±0.25d 126,2964 etyl acetate 67.27±0.00d 29.57±0.00b 149,5571 n-hexane 45.96±0.00b 24±0.13a 226,1243 aceton 63.35±0.00a 18.70±0.22c 143,7940 ascorbic acid 36,4916 286 biology, medicine, & natural product chemistry 14 (1), 2025: 283-288 discussion phytochemical screening is used to evaluate the bioactive constituents from plant extracts. in the current study, the qualitative phytochemical analysis of ethanol, methanol, water, ethyl acetate, n-hexane, and acetone extracts of tradescantia spathacea sw is carried out, as shown in table 2. the result showed that flavonoids, alkaloids, saponins, steroids, terpenoids, and tannins were detected in all extracts, with the n-hexane extract showing the least diverse phytochemical composition. the presence of these phytochemicals has therapeutic potential for tradescantia spathacea sw. flavonoids show multiple pharmacological activities, such as antioxidants, antiinflammatory, antiviral, and antibacterial (melrose, 2023). terpenoids have biological activities such as anticancer, antioxidant, antiallergic, and antiinflammatory (masyita et al., 2022). tannins show various pharmacological effects such as antimicrobial, antioxidant, anticancer, anti-nutritional, and cardioprotective properties (smeriglio et al., 2017). alkaloids have pharmacological activities, such as antiviral, antibacterial, anti-inflammatory, and anticancer properties (adamski et al., 2020). several studies show that tradescantia spathacea sw. leaf contains phenolic, tannins and flavonoids (tan et al., 2015), saponin, carotenoids, anthocyanins, terpenoids, ferulic acid, chlorogenic acid, vanillic acid, pcoumaric acid and steroids (garcía-varela et al., 2015). tradescantia spathacea sw. leaf contains alkaloids, flavonoids, tannins, phenolic compounds, glycosides, terpenoids, anthocyanins, carotenoids, coumarins and steroids (pulipaka et al., 2020). this study shows that flavonoids were found in all extracts, but the strongest ones were detected in methanol, ethanol, and acetone in a yellowish-red color. the detection of these bioactive compounds supports the pharmacological potential of tradescantia spathacea sw., as corroborated by studies identifying phenolic compounds, saponins, carotenoids, anthocyanins, terpenoids, and various organic acids (fraga-corral et al., 2020; pulipaka et al., 2020). phenolic and flavonoids are commonly important in plants and human health. the effect of phenolic is currently interest to antioxidative, reducing agents, free radical scavengers and anticarcinogenic activities (ghasemzadeh & ghasemzadeh, 2011). the phenolic compounds can serve as an antioxidant via different mechanistic reaction pathways, such as inhibition of lipid peroxidation, metal chelation, quenching of singlet oxygen and radical scavenging (wang et al., 2021). the types of extraction solvents have been shown to affect extraction yields (venkatesan et al., 2019). the total phenolic contents of the extract of tradescantia spathacea sw. leaf was expressed as miligram gallic acid equivalents per gram of dry weight extract (mggae/g dw). in case of leaves, the higher value of tpc (83.35±0.00f mggae/g) was extract with methanol and lower (45.96±0.00b mggae/g) with n-hexane extract (tabel 3). additionally, the methanol extract exhibited a higher value of tpc value than the ethanol extract. however, despite the higher percentage yield of the ethanol extract, this may be attributed to its higher tannin content (suharni et al., 2021). the result showed that methanol leaf extract contained more phenolic compounds than the other leaf extract. the result indicates that methanol is more effective solvent for extracting phenolic compouns than another leaf extract. these results indicate methanol's superior efficacy as a polar solvent for extracting phenolic and flavonoid compounds, consistent with findings before (yadav et al., 2011). in previeous studies, which also reported that methanol was effective as solvent in extracting amounts of phenolic compound (wang et al., 2021). in addition, the phenolic content of crude extracts was positively correlated with the antioxidant properties (osman et al., 2020). concisely, methanol leaf extract is expected to have higher antioxidant activity from tradescantia spathacea sw. leaf. in this study showed that there is a positive correlation both their flavonoids content and the antioxidant properties. flavonoids are the major group of phenolic compounds with biological activities such as radical scavening, antimicrobial activities, antiviral, antiinfammatory, anticancer, and anti-allergic (ayele et al., 2022). in this study, flavonoid content showed the same trend as the total phenolic compounds. the total flavonoids contents were expressed as mg quercetin equivalents per g of dry weight extract (mg qe/g dw) (sembiring et al., 2018). total flavonoid content was determined using the aluminium chloride method. aluminium chloride will form stable complex with carbonyl group at c4 and hydroxyls at c3 (flavonols) and c5 in flavonols and flavones. it could also form labile acid complexes with hydroxyls in the ortho position in b rings of flavonoids (sembiring et al., 2018). the result showed that total flavonoids content of the methanol leaf extract was higher than another solvent, which was consistent with phenolic content values. the antioxidant activity of tradescantia spathacea sw leaf extracts was evaluated using the dpph radical scavenging assay, which measures the extracts' capacity to provide the dpph radical hydrogen. crude extracts ability to donate hydrogen and scavenge free radicals can be measured by gradually converting purple radicals into stable dpph radicals with a yellow or colorless appearance (gulcin & alwasel, 2023). this study used ascorbic acid as a standard reference due to its potent antioxidant properties (lourenço et al., 2019). the antioxidant activity of the crude extracts was determined using the ic50 value, which indicates the concentration of the extracts needed to scavenge 50% of dpph radicals. according to several studies, phenolic compounds are potent natural antioxidants that can stop oxidative stress fikroh & hidayah – phytochemicals compound and antioxidant activity … 287 related ncds (fan et al., 2020). in this study, the methanol extract had higher tpc and tfc values than the other extract, suggesting it contained more phenolic compounds. thus, the findings showed that the methanol extract was more capable of generating antioxidants than the other extract. however, the standard references' antioxidant activity was noticeably higher than than of the six crude extracts. in the case of leaves, the higher value of ic50 of antioxidant (80,8346 μg/ml) was extracted with methanol and lower (226,1243 μg/ml) with n-hexane extract (table 3). phenolic compounds are key contributors to this activity, as they inhibit lipid peroxidation, scavenge radicals, and chelate metals(fan et al., 2020; wang et al., 2021). the findings align with previous study, which validated the antioxidant potential of phenolic-rich extracts using frap assays (russo et al., 2017). these results support the role of methanolic extracts in mitigating oxidative stress-related non-communicable diseases (ncds) and suggest their application in pharmaceutical development for natural antioxidant agents. based on these findings, indigenous plants may serve as valuable assets in conventional medicine. it could lead to the development of products with similar characteristics and additional medicinal benefits that warrant further research. conclusions in conclusion, six crude extracts of tradescantia spathacea sw leaf contain secondary metabolites such as flavonoids, alkaloids, saponins, steroids, terpenoids, and tannins. significant amounts of flavonoids and polyphenols were present in all crude extracts. the methanol extract exhibited the highest total phenolic and flavonoid content, with 83.35±0.00 mggae/g and 33.79±0.13 mg/g, and showed an antioxidant activity of 80,8346 µg/ml. the study suggests that tradescantia spathacea sw leaf has the potential 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(2011). the role of chalcones in suppression of nf-κb-mediated inflammation and cancer. international immunopharmacology, 11(3), 295–309. https://doi.org/10.1016/j.intimp.2010.12.006 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 285-289 | doi: 10.14421/biomedich.2024.131.285-289 issn 2540-9328 (online) β-glucan comparison in the mushrooms of medicinal fungal species dicky kurniawan tontowiputro1,*, djanggan sargowo2, askandar tjokroprawiro3, muhaimin rifa'i4 1department of internal medicine, faculty of medicine, university of islam malang, malang, east java, indonesia, 65144. 2biomedical department, faculty of medicine, brawijaya university, malang, east java, indonesia, 65145. 3surabaya diabetes and nutrition center dr. soetomo public hospital, faculty of medicine, airlangga university, surabaya, east java, indonesia, 60286 4biology department faculty of mathematics and natural sciences, brawijaya university, malang, east java, indonesia, 65145. corresponding author* dr.dickykt@unisma.ac.id abstract agaricus blazei murill has been known as a mushroom with medicinal properties, such as its efficacy in maintaining the immune system and other metabolic processes. the main polysaccharide found in a. blazei, is β-glucan. this study aims to quantify and compare βglucan content in a. blazei compared with the medicinal mushrooms ganoderma sp. and pleurotus ostreatus. β-glucan was extracted from each species using an alkaloid extraction method. the β-glucan content was determined using uv-vis spectrophotometer at a wavelength of 220 nm. the data showed that a. blazei contained the highest level of β-glucan, 6.99% (w/w), while the other mushrooms contained less than 2 %. this study obtained that a. blazei contain high levels of β-glucan compared with ganoderma sp. and pleurotus ostreatus. thus, a. blazei has the potential as medicine, especially to maintain the balance of the immune system. to keep the body healthy and balance immune system patient can consume agaricus blazei muril. keywords: potential medicine; beta-glucan; medicinal mushroom; uv-vis spectrophotometry. introduction over the last decades, fungi have been known as a functional food in asia, especially in japan, korea, china, and taiwan. there are various fungi that have potential as medicine (medicinal mushrooms), and historically have been used as traditional therapies (gründemann et al., 2019). agaricus blazei murill is one of the mushrooms claimed to have medical efficacy. since early times, hot water fractions of a. blazei have been used for various medicinal purposes, both in the east and the west. the uses of a. blazei have been documented in histories of roman medicine from the fourth to fifteen centuries by orivasios and apuleius to treat malignant ulcers of the larynx (elmajdoub et al., 2017). a. blazei is classified as a saprophyte (hetland et al., 2020) that is widely distributed from tropical to temperate areas. they can be found in various habitats such as grassland, sandy and salty coastal area, as well as forested land. generally, the raw content of a. blazei mushrooms can be classified into polysaccharide and nonpolysaccharide fractions, including water (90%), protein (2–40%), fat (2–8%), carbohydrates (1–55%), fiber (3– 32%) and ashes (8–10%) (containing minerals, vitamins, etc.) (hetland et al., 2019). polysaccharides in a. blazei consist of α-glucans, β-1,3-glucans, β-1,6-glucans, β1,3/1,6-glucans, β-galactoglucans, chitin, proteoglucans, protein-bound polysaccharides, and xyloglucans (tontowiputro et al., 2020; menezes et al., 2022; da silva campelo, 2021). active metabolites can be isolated from the fruiting body and mycelium. β-glucan is the main active component found in a. blazei (bertollo et al., 2022) and developed from the main framework of a β-1,6 chain and β-1,3 side chains (in a ratio of 1:2) (li et al., 2020). however, there are other active compounds present, such as α-(1->4)-glucan, proteoglycan, lactin, ergosterol (provitamin d2) (yahayu et al., 2023), agaritine (ogasawara et al., (2023), glucomannan (bertollo et al., 2022), isoflavone (roda et al., 2020), and antioxidant compounds (huang et al., 2022). β-glucan is a natural polysaccharide that can be extracted from yeast, fungi, oats, and barley (kaur et al., 2020). β-glucan is predominantly found as a cell wall building block (utama et al., 2023). there are various chain structures of β-glucan influenced by licheninase and environmental conditions when endosperm was developed (malhotra et al., 2022). β-glucan has been known to have acted as an immunostimulator, anti-inflammatory, anticancer agent, etc (han et al., 2022; mahmoud amer et al., 2021). it also is known as an anti-obesity agent (kim et al., 2023), so it is used for healthy dieting. uses for β-glucan in the cardiovascular field has not been much investigated, manuscript received: 12 february, 2024. revision accepted: 14 june, 2024. published: 20 august, 2024. https://doi.org/10.14421/biomedich.2024.131.285-289 286 biology, medicine, & natural product chemistry 13 (1), 2024: 285-289 compared to the oncology field (blumfield et al., 2020). however, while little research has been done, polysaccharide mixtures (especially β-glucan and their protein bonds) activity against cardiovascular diseases have been already shown; several mechanisms of βglucan as an alternative medicine against cardiovascular disease (murphy et al., 2020). several other kinds of edible mushrooms are well known for their β-glucan content, such as ganoderma sp. (cortina-escribano et al., 2020) and pleurotus ostreatus (pérez-bassart et al., 2024). according to wu et al. (2021), one of β-glucan biological activities include antitumor effects. most β-glucan studies are limited to edible mushrooms like agaricus species. their β-glucans are well analyzed and identified, while the other edible mushroom has not been analyzed and quantified. therefore, other edible mushrooms are included in the present studies. the purpose of this study was to determine and compare the level of β-glucans in a. blazei, ganoderma sp., and p. ostreatus. the data obtained can be used to make comparisons between different orders, families, and species. thus, this study will give a new insight on measuring β-glucans using uv-vis spectrophotometry. materials and methods mushroom collection a. blazei extract, and dried ganoderma sp. and p. ostreatus were tested in this study. a. blazei extract was obtained from lawang, malang, indonesia. ganoderma sp. samples were obtained from lipi, cibinong, bogor. p. ostreatus is a common edible mushroom in indonesia and was obtained from a mushroom farm house in malang. all fresh samples were dried in an oven and shattered prior to extraction. β-glucan extraction and measurement dried mushroom samples (4.033 g) were macerated in 100 ml naoh 0.1 m for 24 hours. after maceration, the homogenate was filtered using whatman filter paper. the filtrate was re-filtered using a 0.2-micron millipore filter. encapsulated a. blazei extract was prepared by removing it from its capsules (0.373 g) then diluted it in 100 ml of 0.1 m naoh. table 1. quantification result of β-glucan content in several edible mushrooms. sample m (mg) dn abs conc (ppm) cont. (%) a. blazei 0.327 50 0.206 218666.7 6.99 ganoderma sp. 1.005 50 0.176 17866.7 1.78 p. ostreatus 1.002 200 0.096 18133.3 1.81 note: m: total mass of sample; dn: dilution number; absorbance; conc: concentration (ppm); cont: content (%). a standard curve of β-glucan was generated from βglucan standard solutions. in this trial, β-d-glucan from barley (sigma-aldrich) was used. a stock solution of βglucan standards was prepared by diluting 1000 ppm βglucan standards in 0.1 m naoh. β-glucan absorption in standard solutions and samples (prepared as described below) were measured by using a uv-vis spectrophotometer (genesys 10; thermo scientifictm) at a wavelength of 220 nm. the volume of solvent used was 100 ml or 0.1 l. mass of β-glucan contained in the sample and the total mass of sample (table i) was substituted in equation 1 to calculate β-glucan content in the sample (in percentage). %100% = s c tm m content (eq. 1) note: mc: mass of compounds (mg); tms: total mass of sample (mg). data analysis the data were expressed as mean ± the standard error of the mean (sem), n=4. the correlation analysis was performed using microsoft excel software. the correlation equation was obtained from the standard curve by a regression method. since this research did not use a living thing as an experimental subject, ethical clearance is not applicable. results standard curve of β-glucan a standard curve was constructed by plotting the absorbance number proportional to their increasing concentration (figure 1). the correlation equation was obtained from this curve using linear regression method (equation 1) with the result is shown in equation 5. the standard curve was used to quantify β-glucan content in each sample and determine the standard equation. the correlation coefficient (r2) represents a linear relationship between the lowest and highest scalar values. the correlation coefficient should be equal to or greater than 0.98. the correlation coefficient (r2) that was constructed shows good linearity, with an r2 value = 0.9954 (figure 1). linear correlation in standard curve tontowiputro et al. – β-glucan comparison in the mushrooms 287 verifies that concentrations of the compound present in samples can be determined by absorbance changes. 0688.00003.0 += xy (eq. 2) figure 1. standard curve constructed by bance proportional with increased concentration (50-500 ppm) at 220 nm to quantify β-glucan content in several edible mushrooms. concentration of β-glucan β-glucan content in each edible mushroom sample was calculated after sample concentration was determined. the absorbance of each mushroom sample and their dilution number were used to obtain the concentration (ppm) by substituting them into equation 2, where y represents absorbance and x dilution number. based on the calculation result (table i), a. blazei has the highest β-glucan content was 6.99% (w/w). ganoderma sp. and p. ostreatus also contained lower amounts of β-glucan, 1.78%, and 1.81% respectively. discussion there are a variety of glucans based on their chain structures and companion proteins (manabe & yamaguchi, 2021). it seems that β-glucan sources in a. blazei are higher than found in ganoderma sp. and p. ostreatus, but this does not mean that ganoderma sp. and p. ostreatus do not contain other glucans. in this study, standard solutions used β-d-glucans as the marker. therefore, glucans structured with a β-sheet are the only ones that were detected and quantified. β-glucan is the main polysaccharide that is found in most mushrooms. based on their glycosidic linkages, β(1→3)-(1→6)-glucan is the type that has been studied in most detail due to its immunomodulatory effects (suzuki et al., 2021). several studies suggest that the triple helix structure in β-(1→3)-(1→6)-glucans are related to its activities in modulatory the immune system. these triple helix structures play an important role in enhancing antitumor effects (kono et al., 2020). β-glucan also contributes as a regulator in metabolic systems related to fats and sugar. this result suggests that a. blazei may have high potency to maintain the immune system and metabolic processes (caseiro et al., 2022). according to ali et al. (2021), a. blazei have a potential effect against cardiac diseases. several studies also showed potential effects of a. blazei in atherogenesis prevention through three pathways, i.e., dyslipidemia improvement, endothelial dysfunction improvement related to dyslipidemia, and antioxidant activity due to its content of some antioxidant compounds such as β-glucans, and phenolic and flavonoid compounds ((tontowiputro et al., 2020). further, polysaccharides from a. blazei exhibit antioxidant activities via scavenging values of dpph and hydroxyl radicals as well as increasing levels of nonenzymatic antioxidants including glutathione, vitamin c, vitamin e (wei et al., 2019). bioactivity of β-glucan as an atheroprotective agent through the anti-inflammation pathway is indicated by its ability to bound dectin-1 and the toll-like receptor type 2 (tlr2), especially seen in β-(1→3)-(1→6)glucans. β-(1→3)-(1→6)-glucans bind to tlr2 with an affinity of -8.1 kcal/mol (tontowiputro et al., 2020). their binding affinity is stronger than the binding affinity of fip to tlr2. the molecular structure of β-glucan is very likely to activate both tlr2 and dectin-1. tlr2 could induce the increased production of suppressor cytokines significantly and raise treg function (nakao et al., 2020). the collaboration of tlr2 and dectin-1 could modulate immune response between pro and antiinflammation (tontowiputro et al., 2020). this research suggests a potential effect of a. blazei in cardiovascular disease, especially in the inhibition of atherosclerosis progress, due to its high amount of β-glucan content. conclusions the result of uv-vis spectrophotometry analysis on several edible mushrooms shows that a. blazei has a potential benefit as an immunomodulator and in antiatherogenesis due to the high level of the β-glucans present. this result shows that a. blazei has a high potential for use as alternative medicine. acknowledgments: the authors would thank lipi (indonesian institute of sciences) in cibinong, indonesia as the provider of the mushroom sample and chemistry department, faculty of mathematics and natural science, brawijaya university for support the instruments in this study. authors’ contributions: dicky kurniawan tontowiputro & djanggan sargowo designed the study. dicky kurniawan tontowiputro, djanggan sargowo and askandar tjokroprawiro analyzed the data. dicky kurniawan tontowiputro & muhaimin rifa'i wrote the manuscript. all authors read and approved the final version of the manuscript. y = 0,0003x + 0,0688 r² = 0,9954 0, 0,06 0,12 0,18 0,24 0,3 0 125 250 375 500 625 a b s o rb a n c e concentration (ppm) 288 biology, medicine, & natural product chemistry 13 (1), 2024: 285-289 competing interests: the authors declare that there are no competing interests. references ali, m. m., baig, m. t., jabeen, a., aslam m, shahid u. 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(2023). bioactive compounds and medicinal value of the rare mushroom. deshmukh sk, sridhar kr, enshasy hae (eds.). in: bioprospects of macrofungi. boca raton, fl: crc press. this page intentionally left blank cover jurnal biomenaprochy vol 11 num 1 2022.cdr biology, medicine, & natural product chemistry volume 11 – number 1 – april 2022 issn 2089-6514 (paper) | issn 2540-9328 (online) contents potential antimalarial activity of artemether/lumefantrine/doxycycline: a study in mice infected with plasmodium berghei udeme owunari georgewill, elias adikwu 1 6 peptide fractions from chymotrypsin-hydrolyzed moringa oleifera seed proteins inhibit α-amylase and α-glucosidase in vitro oluwafemi emmanuel ekun, augustine olusegun olusola, joseph adaviruku sanni, feyisayo ishola 7 16 kidney evaluation in hyperuricemia rats treated with green tea leaves (camellia sinensis l.) extract putranty widha nugraheni, chanif mahdi 17 26 the bioprospecting of mangrove red snapper cultivation (lutjanus 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[indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com 3: front 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 555-558 | doi: 10.14421/biomedich.2024.132.555-558 issn 2540-9328 (online) anti –inflammatory activity of propolis trigona sp. water extract from north lombok with red blood cell membrane stability method ani fatin humaira1, siti rahmatul aini2,*, raisya hasina2 1departement of pharmacy, faculty of medicine and health science, university of mataram, indonesia. 2departement of pharmacist professional education, faculty of medicine and health science, university of mataram, indonesia. corresponding author* sitira@unram.ac.id abstract propolis is a bee product that can be used as an anti-inflammatory. cultivation of propolis-producing bees is also carried out in north lombok. however, propolis has not been utilized optimally by the people of north lombok. apart from that, testing of north lombok propolis is still limited to the chemical content and antioxidant activity of propolis extracted with ethanol solvent. therefore, this study aims to determine the activity and effective concentration of north lombok propolis water extract as an anti-inflammatory using the red blood cell membrane stability method. groups include control groups such as positive control (diclofenac sodium), negative control (distilled water), and test groups (propolis water extract concentrations of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, and 30%). in the first step human blood was centrifuged of human blood, and then part of the red blood cells (sediment) was taken and saline was added to obtain a red blood cell suspension. next, mixing the test solution was carried out in the order of 0.5 ml of red blood cell suspension, then 0.5 ml of extract was added (0.1% w/v na-diclofenac solution in the positive control, distilled water in the negative control), 1 ml of buffer solution and 2 ml of hyposaline solution, then homogenized. each group was incubated at 37°c for 30 minutes, then centrifuged for 10 minutes at 3000 rpm. the supernatant was taken, and the absorbance was read with a uv-vis spectrophotometer at 560 nm. next, the percentage value of red blood cell hemolysis protection was calculated. the data obtained were tested statistically using one-way anova and post-hoc (lsd) tests spss version 29. the results showed that propolis water extract concentrations were 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, and 30% have anti-inflammatory activity because they can increase the stability of the red blood cell membrane with a percentage of hemolysis protection of respectively 57.92%, 59.99%, 60.99%, 61.99%, 64.31%, 69.59%, 75.07%, 79.77% and 84.45%. propolis water extract concentrations of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, and 25% had anti-inflammatory effects that were not significantly different from the positive control (p>0.05). the 27.5 % and 30% concentrations had a higher percent hemolysis protection value than the positive control (p<0.05). keywords: anti-inflammatory; propolis; percent hemolysis protection. introduction propolis is a bee product that can be used as an antiinflammatory. people consume propolis to maintain body stamina and relieve sore throats and canker sores (siregar et al., 2011). propolis is also used as a mouthwash to cure gingivitis and periapical abscesses (suranto, 2010). there are many other empirical uses of propolis, such as healing burns, aches, typhus, and diabetes. so far, various propolis products are available on the market, including raw propolis, liquid, powder (capsules), spray, and others (siregar et al., 2011). cultivation of honey bees trigona sp. is also carried out in north lombok. products produced by trigona sp. include honey, propolis, and bee bread. however, the only bee product utilized optimally by the people of north lombok is honey, while propolis and bee bread have not been utilized optimally. djajasaputra (2010) stated that trigona sp bees produce more propolis than honey yearly (djajasaputra, 2010). propolis also has a high economic value, reaching idr 400,000.00/kg, and honey is worth idr 250,000.00/litre (askary et al., 2022). research related to north lombok propolis is still limited to its chemical content and antioxidant activity (zahra et al., 2021). carreño et al. (2017) showed that the compounds in sonoran propolis extract protect against inflammation caused by oxidative stress (carreño et al., 2017). next, it is necessary to test the anti-inflammatory activity to increase the selling power of north lombok propolis. based on who data (2018) shows that 73% of deaths are caused by non-communicable diseases involving inflammation, including cardiovascular disease (heart and blood vessels) 35%, cancer 12%, chronic respiratory disease (neurodegenerative) 6%, diabetes mellitus 6 %, and 15% are caused by other non-communicable diseases (ministry of health, 2019). pharmacologically, inflammation can be treated using anti-inflammatory manuscript received: 09 september, 2024. revision accepted: 07 october, 2024. published: 07 november, 2024. https://doi.org/10.14421/biomedich.2024.132.555-558 556 biology, medicine, & natural product chemistry 13 (2), 2024: 555-558 steroids (ais) and non-steroidal anti-inflammatory (nsaid) drugs. however, both classes of drugs have side effects such as peptic ulcers, decreased immunity, osteoporosis, hypertension, congestive heart failure, and oedema. apart from synthetic drugs, the use of traditional medicines as an alternative treatment in society is increasing. natural medicines are considered easy to obtain, safer, and have fewer side effects than conventional medicines. therefore, this study tested the anti-inflammatory activity of propolis water extract from north lombok with the red blood cell membrane stability method. the red blood cell membrane stability method is used because it is an analogue of the lysosomal membrane, which plays a role in the inflammatory process. thus, it can represent the mechanism of action of cape (caffeic acid phenethyl ester) compounds and flavonoids as antiinflammatory agents, namely inhibiting the formation of inflammatory mediators by cox-2 due to damage to the lysosomal membrane. (kumar et al., 2020). materials and methods tools and materials the tools used are laboratory glassware, stir sticks, test tube racks, spatulas, analytical balances, centrifuges, uv-vis spectrophotometers, edta tubes, hot plates, and thermometers. the ingredients used in this study were distilled water, propolis, the human's venous blood, disodium hydrogen phosphate dihydrate (na2hpo4.2h2o), sodium dihydrogen phosphate monohydrate (nah2po4.h2o), infusion fluid (0.9% nacl), and diclofenac sodium. procedures preparation of phosphate buffer solution (ph 7.4) 13.35 grams of na2hpo4.2h2o was dissolved in distilled water to 500 ml (0.15 m). 2.070 grams of nah2po4.h2o was dissolved in distilled water to 100 ml (0.15 m). at room temperature, 405 ml of na2hpo4.2h2o solution (0.15 m) was mixed with 95 ml of nah2po4.h2o solution (0.15 m). preparation of 0.25% (w/v) hyposaline solution 27.7 ml of isosaline solution was diluted using a phosphate buffer ph 7.4 to a volume of 100 ml. preparation of red blood cell suspension 10% (v/v) 1 ml of red blood cells was added to isosaline solution until the volume was 10 ml, and then homogenized. membrane stability test by hypotonicity induction the mixing of the test solution was carried out in the order of 0.5 ml of red blood cell suspension, then 0.5 ml of extract (na-diclofenac solution 0.1% w/v in the positive control, aquates in the negative control), 1 ml of buffer solution and 2 ml of hyposaline solution, then the mixture was homogenized. next, the solution mixture was incubated for 30 minutes at 370c, then centrifuged for 10 minutes at 3000 rpm. the absorbance of the supernatant was measured at a wavelength of 560 nm. from the positive control, negative control and each test sample absorbance values, the percentage (%) of red blood cell hemolysis protection was calculated using the following formula (umeti et al., 2019): percentage of hemolysis protection = 100 − [( a2 a1 ) × 100] where: a1 : absorbance of negative control solution a2 : absorbance of the test solution/positive control data analysis the percentage value of red blood cell hemolysis protection was then analyzed using the spss version 29 application to determine differences in the ability of each group to protect red blood cell hemolysis. next, the data was analyzed using the one-way anova and post-hoc tests (lsd). results and discussion propolis water extract was tested for anti-inflammatory activity in vitro to protect red blood cell hemolysis. red blood cells are used because their membrane is similar to the lysosomal membrane, influencing the inflammatory process. in the process of inflammation, the stability of the lysosomal membrane limits the inflammatory response by preventing the release of enzymes from the lysosomes, which is caused by neutrophil activation. the release of enzymes from lysosomes can cause disorders such as inflammation of tissues and extracellular fluids (kumar et al., 2020). therefore, the stability of the lysosomal membrane against these disorders can be seen from the stability of the red blood cell membrane. the mechanism of red blood cell stability can be seen from its ability to protect hemolysis, which can cause the release of haemoglobin from red blood cells when given hypotonic stress (hillman et al., 2011). in this study, the anti-inflammatory activity of propolis water extract can be seen from its ability to maintain red blood cell stability, namely the ability to prevent or protect the hemolysis of red blood cells given a 0.1% hypotonic solution. hemolysis of red blood cells is proportional to the amount of haemoglobin, which is read at 560 nm using uv-vis spectrophotometry in the form of absorbance values. the higher the absorbance, the higher the hemolysis of red blood cells and the antiinflammatory activity of a test sample. the percentage humaira et al. – anti –inflammatory activity of propolis trigona sp. … 557 value of red blood cell hemolysis protection describes anti-inflammatory activity. the percentage value of red blood cell hemolysis protection obtained can be seen in table 1. table 1. percentage of red blood cell hemolysis protection. group treatment % hemolysis protection mean % hemolysis protection ± sd 1 2 3 control negative 0 0 0 0 ± 0,00 positive 62,73 66,47 69,32 66,17 ± 3,30 extract 10% 49,71 65,65 58,41 57,92 ± 7,98 12.5% 52,40 67,55 60,02 59,99 ± 7,57 15% 53,30 68,32 61,36 60,99 ± 7,52 17.5% 53,60 68,53 63,84 61,99 ± 7,64 20% 57,35 70,12 65,45 64,31 ± 6,46 22.5% 64,73 75,13 68,91 69,59 ± 5,23 25% 75,15 76,18 73,88 75,07 ± 1,15 27.5% 79,58 79,19 80,53 79,77 ± 0,69 30% 85,61 84,55 83,19 84,45 ± 1,21 discussion table 1 shows the results of the anti-inflammatory activity test of propolis north lombok using the human red blood cell membrane stability method. these results show that all variations in concentration (10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5% and 30%) of propolis north lombok influence the stability of red blood cell membranes. by administering propolis water extract to the red blood cell suspension (ssdm), the red blood cell membrane was more stable compared to administering distilled water to the negative control, so it can be said that propolis north lombok was able to protect red blood cell hemolysis when given a 0.1% hypotonic solution. 30% propolis north lombok has the highest percent (%) hemolysis protection value, namely 84.45% ± 1.21%, so it is the maximum concentration based on the % hemolysis protection value. this value is higher than the percent hemolysis protection of diclofenac sodium as a positive control. these results were in line with mendes-encinas et al. (2023) (mendezencinas et al., 2023). the percentage protection from hemolysis of red blood cells in all treatments was then carried out with statistical analysis using spss version 29. therefore, parametric tests were carried out, namely one-way anova and post-hoc (lsd) tests. the results of the one-way anova test were that all treatments were significantly different (p<0.05). the differences between test groups can be seen in the post-hoc data (lsd); the concentrations of propolis north lombok 10%, 12.5%, 15%, 17.5%, 20%, 22.5% and 25% were not significantly different or the same as the positive control (p> 0.05). in comparison, propolis north lombok concentrations of 27.5% and 30% were significantly different from the positive control (p<0.05). from these results, it can be confirmed that propolis north lombok concentrations of 10%, 12.5%, 15%, 17.5%, 20%, 22.5% and 25% have the same anti-inflammatory activity as diclofenac sodium. the anti-inflammatory activity of propolis is influenced by the compounds contained in it. the compounds that act as anti-inflammatory agents in propolis are caffeic acid phenetyl ester (cape) and flavonoids. several studies explain that the mechanism of propolis (cape and flavonoids) as an anti-inflammatory is by inhibiting the enzymes lox (lipooxygenase) and cox (cyclooxygenase) during the inflammatory process. lipooxygenase is the main enzyme in neutrophils, which will later produce leukotrine compounds, while cyclooxygenase produces prostaglandins, which will become mediators in inflammatory reactions. inhibition of the lipoxygenase and cyclooxygenase pathways will reduce vasodilation of blood vessels, and blood flow will be reduced so that migration of leukocytes (pmn) to areas of inflammation also decreases (bankova et al., 2014). research related to propolis as an antiinflammatory was also carried out in vivo on carrageenan-induced white mice; it was found that the 95% ethanol extract of mayan propolis showed 9% inhibition of oedema in the feet and ears by 22% during the first 2 hours at a dose of 50 mg/kg (xool-tamayo et al., 2020). conclusions based on the results of in vitro anti-inflammatory activity tests, it can be concluded that: ▪ propolis water extract has anti-inflammatory activity because it can increase the stability of red blood cell membranes. ▪ the concentrations of propolis water extract that effective as an anti-inflammatory are 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, and 25%. 558 biology, medicine, & natural product chemistry 13 (2), 2024: 555-558 competing interests: the authors claim that there are no conflicts of interests. references askary, m., astuti, h. p., & suwendar, a. (2022). sukses membudidayakan lebah kelutut, sebuah cerita dari pinggiran hutan. direktorat pengendalian kerusakan gambut. bankova, v., popova, m., & trusheva, b. (2014). propolis volatile compounds: chemical diversity and biological activity: a review. chemistry central journal, 8(1), 1–8. https://doi.org/10.1186/1752-153x-8-28 carreño, a. l., alday, e., quintero, j., pérez, l., valencia, d., robles-zepeda, r., valdez-ortega, j., hernandez, j., & velazquez, c. (2017). protective effect of caffeic acid phenethyl ester (cape) against oxidative stress. journal of functional foods, 29, 178–184. https://doi.org/10.1016/j.jff.2016.12.008 djajasaputra, m. r. s. (2010). potensi budidaya lebah trigona dan pemanfaatan propolis sebagai antibiotik alami untuk sapi po. institut pertanian bogor. hillman, a., vince, r., taylor, l., mcnaughton, l., mitchell, n., & siegler, j. (2011). exercise-induced dehydration with and without environmental heat stress results in increased oxidative stress. applied physiology, nutrition and metabolism, 36(5), 698–706. https://doi.org/10.1139/h11-080 kemenkes. (2019). buku pedoman manajemen penyakit tidak menular. khadse, v. m., mahalle, p. n., & shinde, g. r. (2020). statistical study of machine learning algorithms using parametric and non-parametric tests: a comparative analysis and recommendations. international journal of ambient computing and intelligence, 11(3), 80–105. https://doi.org/10.4018/ijaci.2020070105 kumar, v., frcfath, m. m., abbas, a. k., & aster, j. c. (2020). buku ajar patologi dasar robbins edisi ke-10 (m. f. ham & m. saraswati (eds.); 10th ed.). elseiver. mendez-encinas, m. a., valencia, d., ortega-garcía, j., carvajalmillan, e., díaz-ríos, j. c., mendez-pfeiffer, p., sotobracamontes, c. m., garibay-escobar, a., alday, e., & velazquez, c. (2023). anti-inflammatory potential of seasonal sonoran propolis extracts and some of their main constituents. molecules, 28(11). https://doi.org/10.3390/molecules28114496 paulino, n., abreu, s. r. l., uto, y., koyama, d., nagasawa, h., hori, h., dirsch, v. m., vollmar, a. m., scremin, a., & bretz, w. a. (2008). anti-inflammatory effects of a bioavailable compound, artepillin c, in brazilian propolis. european journal of pharmacology, 587(1–3), 296–301. https://doi.org/10.1016/j.ejphar.2008.02.067 paulino, n., dantas, a. p., bankova, v., longhi, d. t., scremin, a., lisboa de castro, s., & calixto, j. b. (2003). bulgarian propolis induces analgesic and anti-inflammatory effects in mice and inhibits in vitro contraction of airway smooth muscle. journal of pharmacological sciences, 93(3), 307–313. https://doi.org/10.1254/jphs.93.307 sianturi, r. (2022). uji homogenitas sebagai syarat pengujian analisis. jurnal pendidikan, sains sosial, dan agama, 8(1), 386–397. https://doi.org/10.53565/pssa.v8i1.507 siregar, h. c., fuah, a. m., & octavianty, y. (2011). propolis madu multikhasiat. penebar swadaya. suranto, a. (2010). dahsyatnya propolis untuk menggempur penyakit. agro media pustaka. xool-tamayo, j., chan-zapata, i., arana-argaez, v. e., villa-de la torre, f., torres-romero, j. c., araujo-leon, j. a., aguilar-ayala, f. j., rejón-peraza, m. e., castro-linares, n. c., vargas-coronado, r. f., & cauich-rodríguez, j. v. (2020). in vitro and in vivo anti-inflammatory properties of mayan propolis. european journal of inflammation, 18. https://doi.org/10.1177/2058739220935280 https://doi.org/10.1177/2058739220935280 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 127-133 | doi: 10.14421/biomedich.2024.131.127-133 issn 2540-9328 (online) evaluation and antibacterial activity test of peel-off mask preparation from combination of pegagan leaves (centella asiatica (l) urb.) and charcoal powder fadhilla dwi utari1, najmi hilaliyati2, tika afriani1,* 1department of pharmacy, mohammad natsir university, jl. tan malaka, bukik cangang kayu ramang, kota bukittinggi tel. +62-752-21169, indonesia. 2medistra health institute, lubuk pakam, north sumatra, indonesia. corresponding author* tika.afriani91@gmail.com abstract pegagan leaf (centella asiatica (l) urb.) is a plant that contains flavonoids, tannins, triterpenoids, and saponins that function as antibacterials. pegagan leaf is combined with activated charcoal powder, which has the job of absorbing facial oil, so that it is formulated in the form of a peel off mask to produce a synergistic effect. this study aims to formulate, evaluate, and test the antibacterial effectiveness of the peel-off mask preparation of pegagan leaf extract at concentrations of 1.5%, 2%, and 2.5%. this study uses an experimental method by testing the parameters of the physical evaluation requirements and testing antibacterial activity with the disc method on the peel off mask preparation. the results were evaluated for 28 days, and the results of the organoleptic test were stable, homogeneity stable, ph (5.6–6.2), dry time (16–25 minutes), dispersion (6.1-6.4 cm), and irritation (2 respondents). the effectiveness test against the bacteria propionibacterium acnes has inhibitory power of 12.3 mm in the strong category. so, it can be concluded that the peel off mask produced has stable physical characteristics and can be used as an antibacterial mask. keywords: activated charcoal; antibacterial; pegagan leaf; peel off mask. introduction unhealthy environmental conditions due to the impact of air pollution such as incomplete combustion from motor vehicles, cigarette smoke, solar radiation, and other pollutants can trigger free radicals. this will result in the destruction of body cells. the effect on the skin is in the form of slowly decreasing skin elasticity, which results in the appearance of hyperpigmentation or tanned spots and also wrinkles (ningsih et al., 2016). the skin is not the only organ that has a vital function. having smooth and healthy skin is a matter of pride, especially for teenagers and women. but like other parts of the body, the skin can also experience disorders or diseases, one of which is acne (albastomi, 2019). acne is a common thing experienced by millions of people in various parts of the world, involving disorders of the pilosebaceous unit. this disorder occurs due to clogging of the skin pores, resulting in inflammation of pus sacs. acne sufferers in indonesia continue to increase every year. acne is also triggered by the development of propionibacterium acnes bacteria. this bacterium is one of the gram-positive rod-shaped bacteria and is a normal part of the skin flora that is involved in the production of acne (ervina et al., 2020). in overcoming skin problems such as acne, indonesians have always used plants as medicine, and not a few are used as skin care ingredients and cosmetics (ramadhania, 2018). one of the medicinal plants in indonesia that has antibacterial activity that can fight acne-triggering bacteria is pegagan (centella asiatica (l.) urb). this is supported by the bioactives contained in it that have antibacterial activity, such as saponins, tannins, flavonoids, and so on (soebagio et al., 2020). pegagan also contains madecassic acid, asiatic acid, and asiaticoside, which can help heal wounds (budi & rahmawati, 2020). pegagan is a type of plant that can be used in the treatment of scaly and dry facial skin. by synthesizing collagen, it can regenerate tissue levels and can be used as a raw material for skin care (sumiati et al., 2019). cosmetics have been known since ancient times, with natural raw materials used to beautify themselves. there are various cosmetics, including face masks. the use of a face mask can unclog pores, soften the skin, and remove cosmetic residue that is difficult to remove with regular cleansers. regular use of face masks can also reduce the appearance of fine lines and wrinkles and prevent premature aging (widyarti et al., 2019). peel-off face masks are a type of face mask that is superior in its manuscript received: 24 december, 2023. revision accepted: 20 may, 2024. published: 21 may, 2024. https://doi.org/10.14421/biomedich.2024.131.127-133 128 biology, medicine, & natural product chemistry 13 (1), 2024: 127-133 use because they can be easily removed like an elastic membrane (sulastri & chaerunisaa, 2018). peel-off masks can be used to cleanse and moisturize the skin. most facial peel off masks use polymers that can thin the layers on the face. peel-off masks come in dry and gel forms. applying a face mask in the process takes 15-30 minutes to dry by applying a layer on the face (oktavia et al., 2021). based on research (lestari et al., 2020) the combination of palm kernel shell activated charcoal peeloff mask and pva will result in the skin’s surface structure becoming cleaner and smoother after use. the purpose of this study was to evaluate and test the antibacterial activity of peel-off mask preparations made from a combination of centella asiatica (l.) urb. leaves and activated charcoal powder. materials and methods study area the tools used in this study were a rotary evaporator (dlab®), laminar air flow, oven (memmert®), autoclave (wiseclave®), analytical balance (kern®), erlemeyer (pyrex® ), ph meter (atc®), measuring cup (pyrex®), petri dish (normax® ), hot plate (kern® ), stirring rod, glass slide, 20x20cm glass, stopwatch, dropper, spatel, filter paper, maceration vessel, spirit burner, ose needle, disc paper (whatman®), parchment paper, spatula, cotton swab, tissue, and tube mask. the materials used in this study included pegagan leaves (centella asiatica (l). urb), activated charcoal powder (bumi agung®), 70% ethanol, 96% ethanol, pva, hpmc, glycerin, propylenglycol, methyl paraben (golden era®), propyl paraben (golden era®), aquadest, nutrient agar media (merck®), mediclin® gel, and propionibacterium acne bacteria. procedures sampling pegagan leaves were obtained from kenagarian sungai tanang, agam regency, west sumatra. plant identification was carried out by dr. nurainas at the herbarium andalas, department of biology, fmipa, andalas university, padang, west sumatra. sample preparation pegagan leaves totaling 3.5 kg were washed thoroughly using running water and dried at 60 ᵒc using an oven for 5 hours to obtain pegagan leaf simplisia (handayani & qamariah, 2019). extraction pegagan leaf simplisia powder (600 g) was macerated using 70% ethanol until submerged within 4x24 hours. then filtering was done to separate the filtrate from the pulp. the filtrate obtained was then concentrated using a rotary evaporator until a thick extract was obtained (purgiyanti, 2017). phytochemical screening (soebagio et al., 2020) flavonoid identification a total of 0.1 g of extract was added to 2 ml of 70% ethanol as well as 50 ml of hot water that had been brought to a boil. then a few drops of concentrated hcl and 0.1 g of magnesium powder were added. flavonoids are characterized by the formation of red, yellow, and orange colors. identification of tannins a total of 0.1 g of extract was added to as much as 2 ml of 70% ethanol and fecl3 to as much as 3 drops. the presence of tannin compounds is indicated by the appearance of blue, green, or precipitate. identification of triterpenoids a total of 0.1 g of extract was added to as much as 2 ml of 70% ethanol, then put into a test tube, followed by the addition of concentrated h2so4 1 drop and acetic anhydride 3 drops. if a purple, red, or brown color is formed, the sample is positive for triterpenoid. raw material inspection the ethanol extract of pegagan leaves was examined for organoleptic, solubility, and phytochemical screening; glycerin, pva, hpmc, propylenglycol, propyl paraben, methyl paraben were examined according to the requirements listed in the handbook pharmaceutical excipients, 6th edition, indonesian pharmacopoeia iii edition. manufacture of peel-off mask pva was developed using hot water until fully expanded, then homogenized (mass 1). hpmc was developed on top of distilled water ad inflated (mass 2), then mass 2 was put into mass 1. gotu kola extract was dissolved with 96% ethanol until dissolved, then propyl paraben and methyl paraben were dissolved with 96% ethanol until dissolved (mass 3). mass 3 is included in mass 1 while still stirring in the beakerglass. next, add little by little propylenglycol, glycerin, and activated charcoal powder while still stirring. finally, add distilled water ad 100 ml until a homogeneous gel mass is formed (handayani & qamariah, 2019). physical evaluation of peel-off mask organoleptical test the organoleptic test was carried out by observing changes in the shape, smell, and color of the mask preparation. homogeneity test the homogeneity test was carried out by placing 0.1 gram of gel between two glass objects and observing utari et al. – evaluation and antibacterial activity test of … 129 whether the surface was evenly smooth and the base was homogeneous. the preparation is said to be homogeneous if there are no coarse particles that can be palpated (pramiastuti et al., 2019). ph test the ph measurement was carried out using a ph meter that had previously been calibrated using standard dapar (ph 7 and ph 4) (pramiastuti et al., 2019). a total of 1 gram of gel was weighed and dissolved with 10 ml of distilled water and stirred homogeneously, then the ph meter was dipped and the results obtained were recorded (arman & mansauda, 2021). drying time test a total of 1 gram of peel-off mask gel was applied to 7 cm of arm skin. then calculate the speed of drying the gel to form a film layer using a stopwatch (handayani & qamariah, 2019). spreadability test a total of 0.5 grams of gel was placed on a glass with a size of 20x20 cm. then it was covered with another glass, a load of 125 grams was added, and after 1 minute, the diameter of the spreading gel was measured (istiqamah & anindhita, 2018). irritation test the open patch test was used for the irritation test on the skin of 10 volunteers, the peel-off mask gel preparation was applied in an area of 2.5 cm2 on the forearm, then allowed to stand for 15 minutes, and the reaction that appeared was observed (rinaldi et al., 2021). antibacterial activity test sterilization of equipment all equipment, including media, should be sterilized before use by autoclaving at 121 ᵒc for 15 minutes. ose tweezers and needles were sterilized by direct burning over flame (oktavia et al., 2021). preparation of nutrient agar medium 5 grams of nutrient agar were dissolved in 250 ml of sterile hot water, then stirred using a stirring rod until dissolved, and sterilized in an autoclave at 121 ᵒc for 15 minutes (husnani & rizki, 2018). preparation of a pure culture suspension of test bacteria stock cultures of test bacteria were taken using a sterile ose needle and then suspended in 10 ml of physiological nacl (husnani & rizki, 2018). preparation of the sample solution the peel off mask of centella asiatica (l.) urb) leaf extract was made with concentrations of 1.5%, 2%,and 2.5%, respectively. the sample solution was made by weighing each preparation as much as 10 grams and then dissolved with aqua p.i. in 10 ml for a positive control and dissolved with aqua p.i.in a ratio of mediclin: aqua p.i.(1:1). f0 as a negative control (husnani & rizki, 2018). disc preparation paper discs with a diameter of 0.6 cm were prepared, soaked for 15 minutes in each sample solution aseptically,and then placed on the media used (husnani & rizki, 2018). inhibition testing inhibition testing of a peel-off mask of centella asiatica (l.) urb) leaf extract was carried out by diffusion method using disc paper. the test bacterial suspension was poured as much as 1 ml into a petri dish, then 10 ml of sterile na medium that had been cooled to a temperature of 40-45 ᵒc were added. next, the petri dish was closed, shaken until it was homogeneously mixed, and allowed to freeze. the disc paper that has been soaked in the sample solution is placed on the surface of the nutrient agar (na) medium, which contains the test bacteria, and then incubated in an incubator at 37 ᵒc with an inverted position for 2x24 hours. the zone of inhibition formed was measured using a caliper (husnani & rizki, 2018; afriani & rahmayulis, 2022). data analysis the data obtained were analyzed using a one-way anova statistically through the spss 25 program with a significance level of α = 0.05. results and discussion fresh samples of pegagan leaves were obtained from kenagarian sungai tanang agam regency, west sumatra. identification was carried out at the herbarium andalas, faculty of biology, andalas university, the results obtained were the type of plant studied in the form of (centella asiatica (l) urb.). the extraction process was carried out by maceration technique using 70% ethanol solvent and obtained a thick extract of 345.75 g with a yield of 57.6%. phytochemical profile of pegagan leaves based on the results of phytochemical tests on the ethanol extract of pegagan leaves, it is positive for flavonoids, tannins, and triterpenoids from the reagent reactions used. secondary metabolite compounds such as saponins, tannins, and flavonoids contained in pegagan leaves are claimed to have antibacterial activity. the results of phytochemical screening are shown in table 2. there are various chemical contents in pegagan that have many benefits for humans. pegagan contains compound components, including triterpenoids, which include brachnid acid, madasiatic acid, steroids, tannins, glycosides, alkaloids, asiatic acid, madecoside, and 130 biology, medicine, & natural product chemistry 13 (1), 2024: 127-133 asiaticoside. triterpenoids from pegagan are claimed to have potential as antioxidants, antifungals, and antibacterials (sulastri et al., 2017). besides using pegagan leaves as the main active substance that functions as an antibacterial, activated charcoal powder is also used to absorb dirt and oil on the face. these two ingredients have a synergistic effect on inhibiting acne growth. therefore, pegagan leaf extract and activated charcoal powder were chosen to be the active substances of the peel-off mask preparation in this study. the peeloff mask preparation was chosen because it is easier, more efficient, and more effective to wear on the face until it dries and produces a thin, elastic, transparent film layer, and is also easy to clean without having to be washed (rismayanti et al., 2021). the ingredients used in making peel-off masks include polyvinyl alcohol (pva), which functions as a gelling agent or film-forming base; glycerin and propylenglycol as humectants that can moisturize the skin in high humidity conditions and can bind water from the air; propylenglycol can maintain the water contained in the preparation so that during storage, the stability and physical properties of the preparation can be maintained (frida et al., 2018), hydroxypropyl methylcellulose (hpmc) as a viscosity enhancer; methyl paraben and propyl paraben as preservatives to anticipate microbial contamination due to the high water content in the preparation (rowe & q.e., 2015). the peel-off mask was made in 4 variations of extract concentration with the aim of comparing the antibacterial effectiveness and evaluating the physical quality of good peel-off masks from each concentration with positive control and negative control (f0) with preparation evaluation parameters in the form of spreadability test, organoleptic, homogeneity, ph, dry time, irritation test, and antibacterial inhibition. organoleptical tests are carried out as quality control for a preparation. observations were made visually and using the five senses. the results of observations for 28 days showed that the peel-off mask remained stable and no changes occurred during storage. this preparation is said to have met the requirements of the stability test because there is no change in odor, color, or shape (ambari et al., 2020). the results of the evaluation of peel-off mask preparations are shown in table 3. observation of the homogeneity of the preparation was carried out within 28 days. from the results of observations on days 0 7 14 21 and 28, all formulas remained homogeneous, characterized by the absence of coarse particles in the preparation (fauziah et al., 2020). the ph test was carried out using a calibrated ph meter. table 1. peel-off mask formula design. material % consentration f0 f1 f2 f3 pegagan leaf extract 1,5 2 2,5 activated charcoal powder 1 1 1 pva 10 10 10 10 hpmc 1 1 1 1 glyserin 10 10 10 10 propylenglycol 10 10 10 10 methyl paraben 0,15 0,15 0,15 0,15 propyl paraben 0,15 0,15 0,15 0,15 ethanol 96% 15 15 15 15 aquadest ad 100 100 100 100 table 2. phytochemical screening results of an ethanol extract of pegagan leaves. secondary metabolite compounds ethanol extract of pegagan leaves results color flavonoids + yellow red tannins + blue green triterpenoids + red brown table 3. evaluation results of peel-off mask preparation of pegagan leaf extract combined with activated charcoal powder. formula day to smell color shape homogeneous ph dry time f0 0 typical clear white semi-solid homogeneous 6,3 16:35 7 typical clear white semi-solid homogeneous 6,3 16:16 14 typical clear white semi-solid homogeneous 6,2 17:18 21 typical clear white semi-solid homogeneous 6 16:09 28 typical clear white semi-solid homogeneous 6,2 16:16 f1 0 typical black semi-solid homogeneous 6,2 21:03 7 typical black semi-solid homogeneous 6,3 21:23 14 typical black semi-solid homogeneous 6,1 21:31 21 typical black semi-solid homogeneous 6 20:19 28 typical black semi-solid homogeneous 6,1 22:53 f2 0 typical black semi-solid homogeneous 6,3 21:20 7 typical black semi-solid homogeneous 6 22:05 14 typical black semi-solid homogeneous 5,8 22:31 21 typical black semi-solid homogeneous 6 22:06 28 typical black semi-solid homogeneous 6,1 22:46 utari et al. – evaluation and antibacterial activity test of … 131 table 3. cont. formula day to smell color shape homogeneous ph dry time f3 0 typical black semi-solid homogeneous 5,9 21:56 7 typical black semi-solid homogeneous 5,5 22:45 14 typical black semi-solid homogeneous 5,3 24:16 21 typical black semi-solid homogeneous 5,8 25:25 28 typical black semi-solid homogeneous 5,8 25:27 table 4. inhibition test results of peel-off mask against propionibacterium acnes bacteria. formula inhibition zone diameter (mm) mean diameter of inhibition zone (mm) i ii iii f0 0 0 0 0 f1 10,25 10,35 10,55 10,38 f2 11,2 11,4 11,7 11,43 f3 12,0 12,25 12,65 12,3 mediklin® 16,1 21,7 25,1 20,93 testing was carried out every week for four weeks. the evaluation results show that all formulas have a ph range of 5.6-6.2, which means that all preparations meet the ph test requirements and are in accordance with the normal skin ph range of 4.5-6.5 (utami et al., 2020). the rise and fall of the ph of the preparation during storage shows a preparation that is less stable. the changing ph value can be influenced by the decomposition of the media at high temperatures during storage or manufacture to produce bases or acids. changes in ph can also be triggered by environmental factors, including poor storage, temperature, and unstable combinations of extracts in the preparation because they are oxidized (putra, 2017). this shows that if the ph is below 4.5, it can cause skin irritation and scaly skin if the ph exceeds 6.5 (noviyanty, 2020). the dry time test aims to determine the time it takes for the preparation to dry on the skin surface and produce a film layer. ideally, the dry time of the preparation ranges from 15-30 minutes starting from the application of the mask until the mask dries on the surface of the skin. the range of dry times for all formulas was 16-25 minutes. these results show that all formulas meet the dry time test requirements that have been set. the dry time range of f0 was 16-17 minutes, f1 was 20-22 minutes, f2 was 21-22 minutes, and f3 was 21-25 minutes. the dry time of f0 was faster than the three formulas containing pegagan extract and activated charcoal powder. the increase in drying time was due to the evaporation of ethanol when the packaging was opened for too long during the test. the function of ethanol in the peel-off mask formula is to shorten the drying time of the mask, therefore if the ethanol has evaporated, the drying time of the preparation becomes longer (sutriningsih & astuti, 2017). the spreadability test aims to see the potential distribution of the preparation when applied to the skin. the easier the application, the greater the surface area of contact of the preparation with the skin, thus the more optimal the absorption of substances in the skin. face mask preparations are declared good if they have a spreadability of 5.0-7.0 cm. the results of the evaluation of the average diameter of the spreadability of each formula for 28 days were 6.1 cm, 6.2 cm, 6.4 cm, and 6.4 cm, respectively. the higher the concentration of the extract used, the faster the spreadability, and vice versa; the lower the concentration of the extract, the longer the spreadability of the preparation (noviyanty, 2020). the evaluation results showed that all formulas met the specified spreadability requirements. the irritation test was carried out using an open patch test (patch test) on 10 volunteers by applying f0-f3 preparations covering an area of 2.5 cm2 on the back of the hand. this test was carried out for 3 consecutive days, 3 times a day, for 15 minutes, with the parameters of red, itchy, and swollen reactions (putri et al., 2021). symptoms that arise are observed, and irritation will generally occur with the onset of skin reactions shortly after contact or attachment to the skin, or this condition is called primary irritation (anonim, 1985). the test results showed mild irritation in 2 out of 10 respondents to the use of f0-f3 in the form of itching and redness, but the irritation disappeared after a few minutes. from the observations that have been made on the standardization of pegagan extract, the ph is 4.7, which meets the requirements, and when applied to the back of the hand, it does not experience irritation. the possibility of this reaction is due to the sensitivity and nature of the skin of different respondents. however, the onset of irritation is not long-only a few minutes after the mask is applied (numberi, 2020). the inhibition test on mask preparations is carried out using the disc diffusion method. the advantages of this method are that it is cheap, easy, and fast because the process does not require special tools. this test aims to determine the ability of pegagan leaf extract combined with activated charcoal powder to inhibit the growth of propionibacterium acnes bacteria. nutrient agar is used as a growth medium because it is a common medium used in bacterial growth (retnaningsih et al., 2019). the test results are seen in the clear zone formed on the media. descriptively, all formulas showed antibacterial 132 biology, medicine, & natural product chemistry 13 (1), 2024: 127-133 activity with different inhibition zone diameters. f3 is the formula with the largest inhibition zone diameter of 12.3 mm with a strong category, while the mediklin® comparison is 20.93 mm, but to determine the best formula requires statistical testing using spss 25. the test results show that the greater the concentration of the extract, the greater the diameter of the clear zone formed. inhibition test results are shown in table 4. anova test the prerequisite for conducting the anova test is that the data must be normally distributed and homogeneous. this test is a multivariant analysis technique that functions to distinguish more than two groups of data by comparing their variances. based on the results of the anova test, it can be seen whether there is a significant or real difference from all formulas on inhibitory power. if the significance value <0.05, it can be concluded that there is a significant difference in all formulas. the results of the anova test showed a significance value <0.05 on the inhibitory power, so ho was rejected, so it can be concluded that there is a significant difference in the inhibitory power between all formulas. conclusions based on the research that has been done, it can be concluded that the peel-off mask of pegagan leaf extract and activated charcoal powder has stable physical characteristics and can be used as an antibacterial mask for propionibacterium acnes. competing interest: the authors have no competing interests. references afriani, t. & rahmayulis. 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(2016). formulasi masker alami berbahan dasar bengkoang dan jintan hitam untuk mengurangi kerutan pada kulit wajah. jurnal care, 4 (2)(3), 9–20. this page intentionally left blank biology, medicine, & natural product chemistry issn: 2089-6514 volume 5, number 1, 2016 | pages: 23-32 | doi: 10.14421/biomedich.2016.51.23-32 checklist of macroalgae in waisai coast, raja ampat retno suryandari1, widodo departement of biological education, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 author correspondency1: suryandari44@gmail.com abstract macroalgae are very abundant organisms in indonesian coastal zone. they comprise 8.6% of the total marine organisms.the aim of the research was to identify macroalgae in waisai coast raja ampat. the results showed that 38 macroalgae were found in waisai coast raja ampat but only 29 species of macroalgae can be identified. macroalgae found in waisai coast raja ampat are green algae, red algae and brown algae. green algae found and identified are caulerpa macra (weber-van bosse) draisma & prud’homme, caulerpa racemosa var. macrophysa (sonder wx kutzing) w.r.taylor, caulerpa sertularoides (s. gmelin) howe f. brevipes (j. agardh svedilus), caulerpa cupressoides (vahl) c. agardh, halimeda discoidea decaisne, halimeda opuntia (linnaeus) j.v. lamoroux, halimeda tuna (j. ellis & solander) j.v. lamoroux, halimeda cylindraceae decaisne, halimeda macroloba decaisne, avrainvillea erecta (berkeley) a. gepp & e.s. gepp, codium geppiorum o.c.schmidt, boergesenia forbesii (hardvey) feldmann, valonia ventricosa j. agardh, dictyosphaeria cavernosa (forsskål) børgesen, chaetomorpha spiralisokamura, anadyomene wrightii harvey ex. j. e. gray, neomeris annulata dickie. red algae species found and successfully identified areacanthophora spicifera (m. vahl) børgesen, laurencia papilosa (c. agardh) greville, gracilaria salicornia (c. agardh) e.y. dawson, amphiora fragilissima (linnaeus) j.v. lamoroux, hypnea pannosa j. agardh. brown algae species found and identified are hormophysa cuneiformis (j.f. gmelin) p.c. silva, sargassum aquifolium (turner) c. agardh, sargassum polycystum c. agardh, turbinaria ornata (turner) j. agardh, padina australis hauck, canistrocarpus cervicornis (kutzing) de paula & de clerck hydroclathrus clatratus (c. agardh ) m. howe. the only species found in indonesia is sargassum aquifolium. keywords: macroalgae, waisai coast, raja ampat, green algae, red algae, brown algae introduction indonesia have more than 17,504 islands. approximately three-quarters of indonesian territory are ocean (5.8 million km2) with 95.161 km coastline. indonesia have the second longest coastline in the world (lasabuda, 2013). indonesia's marine wealth runs between the indian ocean to the pacific ocean with an area of coral reefs achieve 50.875 km2 or 18% of the world's coral reefs. most of these reefs are located in the eastern part of indonesia on the coral triangle region. one of the center of coral reef biodiversity are in raja ampat (solihin, batungbacal, & nasution, 2013). raja ampat islands is an area located in the western part of papua. the islands are known as one of the marine world heritage site because of its marine biodiversity. in 2002, the nature conservation (tnc) and the research center for oceanography lipi reported data that the marine ecosystem consist of 537 species of corals (75% of coral species in the world), and 828 species of fish (setiawan, 2011). in raja ampat, coral reef ecosystems exposed in the shallow areas in most of islands.coral reefs are usually covered with abundance of seaweed that is not only diverse if compared with other living beings but also more complex in morphology and ecological role in coral reef ecosystems (mambrisaw, wurlianty, liuw, & hamel, 2006). waisai coast is located around the port of raja ampat, south waigeo district, raja ampat, west papua.this area can be reached by sea from sorong city and overland from waisai city.waisai coastzonation started from the mainland that made up of mangrove forest dominated by rhizophora genus, then towards the shallow water, seagrass species dominate byenhalus sp. with various sizes up to 40 cm. among the seagrasses are found living macroalgae associated with seagrass and coral. this coast has a mixed substrate consist of dead corals and mud sand. according to dahuri (1998) in suparmi & sahri (2009) seaweed is a biological resource that is very abundant in indonesian ocean which is comprised of 8.6% of the total marine organisms. the seaweed habitat in indonesia reached 1.2 hectares, one of the largest in the world (wawa, 2005) in suparmi & sahri (2009). based on the report by vamn bosse, indonesia has 555 of 8642 world seaweed species (suparmi & sahri, 2009). mambrisaw et al (2006) stated that in the area of raja ampat misool particularly in the south east, south and ayau waigeo, seaweed has been cultivated and are the main commodity in the region. however, checklist of macroalgae in raja ampat have not been done. seaweeds (macroalgae) are organisms like plants that live in marine ecosystems which is a primary producer because it has the ability to photosynthesize (autotrof). macroalgae are classified into three main groups, namely green algae (chlorophyta), brown algae (phaeophyta) and red algae (rhodophyta) (dhalgarkar & kavlekar, 2004). in addition to a taxonomic grouping, macroalgae can be distinguished by the character of the http://dx.doi.org/10.14421/biomedich.2016.51.23-32 24 biology, medicine, & natural product chemistry 5 (1), 2016: 23-32 organism and its ecological characteristics. based on this category of macroalgae are grouped into three main categories: 1.turf algae, 2. fleshy algae (fleshy and calcified) 3. crustose corraline algae (mambrisaw, wurlianty, liuw, & hamel, 2006). this research aims to determine the types of macroalgae found in waisai coast of raja ampat. materials and methods the research was conducted on 4-5 april 2016 in waisai coast, south waigeo district, raja ampat. figure 1. map of waigeo island of raja ampat. the equipment used in this research include waterproof paper, clipboard, pencils 2b, mask snorkel, fins, frame squares size 1x1 meter, gps (global positioning system), a digital camera, underwater camera, identification book, refractometer, ph meter and thermometer, materials used in this research are specimens and 10% formalin. sampling vegetation was done using transect quadrat method. transect line was divided into three stations in one location with the distance of each station is 25 meters. the steps of this research are: 1) determining the location for line transect stations 1, 2, and 3 with a length of each transect is 50 meters. the distance of each transect lines are installed parallellyand its lengthis 25 meters. each transect mounted perpendicular to the shoreline, 2) in each transect line is placed a quadrat frame with size 1 m x 1 m of 10 quadrat frame with a distance of each quadrat frame is 5 meters.first squares are laid in the area closest to the mainland where macroalgae was found, 3) calculate all macroalgae in each plot, 4) the amounts found are recorded on paper underwater based on the location of the squares, 5) sampling macroalgae and placed in plastic sample was labeled , 6) samples were found to note the type of the substrates, 7) took data of environmental parameters such as: the coordinates of global positioning system (gps), the measurement of temperature using a thermometer, measuring salinity of seawater using a refractometer and ph measurements of water using a ph-meter. 8) samples were taken washed clean of the substrate and then preserved with formalin 10% for identification purposes. result and discussion based on the research that has been done, macroalgae were found in waisai coast at coordinates s 00˚26'12,3 ", e 130˚48'38,7" are as many as 38 species. macroalgae that can be identified to species level are 29 species consisting of 17 species of green algae (chlorophyta), 5 species of red algae (rhodophyta), 7 species of brown algae (phaeophyceae), and 9 species unidentified. green macroalgae (clorophyta) class, order and family of green macroalgae that found at waisai coast shown in the table 1 below. retno suryandari, widodo. – checklist of macroalgae in waisai coast, raja ampat 25 table 1. checklist of green macroalgae that found at waisai coast. class order family species ulvophyceae bryopsidales caulerpaceae caulerpa macra (weber-van bosse) draisma & prud’homme caulerpa racemosa var. macrophysa (sonder wx kutzing) w.r.taylor caulerpa sertularoides (s. gmelin) howe f. brevipes (j. agardh svedilus) caulerpa cupressoides (vahl) c. agardh halimedaceae halimeda discoidea decaisne halimeda opuntia (linnaeus) j.v. lamoroux halimeda tuna (j. ellis & solander) j.v. lamoroux halimeda cylindraceae decaisne halimeda macroloba decaisne udoteaceae avrainvillea erecta (berkeley) a. gepp & e.s. gepp codiaceae codium geppiorum o.c.schmidt cladophorales valoniaceae boergesenia forbesii (hardvey) feldmann valonia ventricosa j. agardh dictyosphaeria cavernosa (forsskål) børgesen cladophoraceae chaetomorpha spiralis okamura anadyomenaceae anadyomene wrightii harvey ex. j. e. gray dasycladales dasycladaceae neomeris annulata dickie caulerpa macra (weber-van bosse) draisma & prud’homme (fig. 2a) description: large caulerpa with long thick stolons, these several decimeters long, 3– 5 mm in width but often over 5 mm. rhizoidal pillars well developed, arising from stolons at irregular distances, up to 10 cm long and several mm in diameter, often with thick terminal clumps of branched rhizoids. erect assimilators up to 10 cm in height, with irregularly to regularly placed ramuli arranged distichously or radially and opposite or alternate. ramuli oviform, pyriform, more rarely claviform, usually with some having a form like the head of a golf club, to 12 mm long and 6 mm in width (belton et al, 2014) distribution in southeast asia: indonesia, java, philippines (guiry & guiry, 2017). caulerpa racemosa var. macrophysa (kiitzing) taylor (fig. 2b) description: plants large, to 100 mm tall, erect branches simple or occasionally branched, few and distantly spaced on stout, creeping stolon, branches bearing ramelli expanded into a hemispherical end and arranged irregularly on terete rachis (menez & calumpong, 1982). distribution in southeast asia: indonesia, singapore, thailand, vietnam (guiry & guiry, 2017) philippines (menez & calumpong, 1982). caulerpa sertularoides (s. gmelin) howe f. brevipes (j. agardh svedilus) (fig.2c) description: bright to dark green in colour, tubular with delicate, branched stolon and erect assimilators, plumose or feathery resembling palm leaves, up to 13 cm tall, 812 mm broad, rarely branched, branching generally at the base of assimilators, branchlets opposite, incurve above, needle shaped with pointed apices (jha et al, 2009). distribution in southeast asia: indonesia, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). caulerpa cupressoides (vahl) c. agardh (fig. 2d) description: dark green, the stolon on the macroalga forms a branching with an elongated shape of ramuli and jagged edges (atmadja et al, 1996). distribution in southeast asia: indonesia, philippines, singapore, vietnam (guiry & guiry, 2017). halimeda discoidea decaisne (fig. 2e) description: compact tallus growth, dichotomous or trichotomous branching, rough surface segments, squiggly edges, base segments slightly elongated and thicker (atmadja et al, 1996). distribution in southeast asia: indonesia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). halimeda opuntia (linnaeus) j.v. lamoroux (fig. 2f) description: compact tallus growth, bifurcation of crumpled segments spreading and forming new growth. the relatively small segments are round-shaped, rounded-oval, kidney, and wavy. high carbonate contents. basal segment is not visible. the non-bulbous holdfast consists of filamentous mass (atmadja et al, 1996). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). 26 biology, medicine, & natural product chemistry 5 (1), 2016: 23-32 halimeda tuna (j. ellis & solander) j.v. lamoroux (fig. 2g) description: dark green in colour, generally tufted, moderately calcified and attached by compressed conical holdfast, branching in one plane, dichotomous or trichotomous upper segments, cuneate remiform discoid, 6-13 mm high, 5-23 mm broad,medullary filaments entangled, fusing 2-3 together at the nodes, dichotomously branched above the fused filaments (jhaet al, 1996). distribution in southeast asia: indonesia, malaysia, philippines, singapore, vietnam (guiry & guiry, 2017). a b c d e f g h i j k l m n o p figure 2. macroalgae of chlorophyta at waisai coast (a. caulerpa macra (weber-van bosse) draisma & prud’homme, b. caulerpa racemosa var. macrophysa (sonder wx kutzing) w.r.taylor, c. caulerpa sertularoides (s. gmelin) howe f. brevipes (j. agardh svedilus), d. caulerpa cupressoides (vahl) c. agardh, e. halimeda discoidea decaisne, f. halimeda opuntia (linnaeus) j.v. lamoroux, g. halimeda tuna (j. ellis & solander) j.v. lamoroux, h. halimeda cylindraceae decaisne, i. halimeda macroloba decaisne, j. avrainvillea erecta (berkeley) a. gepp & e.s. gepp, k. codium geppiorum o.c.schmidt, l. boergesenia forbesii (hardvey) feldmann, m. valonia ventricosa j. agardh, n. dictyosphaeria cavernosa (forsskål) børgesen, o. chaetomorpha spiralis okamura, p. anadyomene wrightii harvey ex. j. e. gray, q. neomeris annulata dickie). q halimeda cylindraceae decaisne (fig. 2h) description: perpendicular tallus growth consists of cylindrical segments or prisms. main dichotomous branching, tallus growth toward small bud, width segment size 18 mm, length 10 mm. between the basal segment and the segment sometimes there is a segment bearing as a place for the formation of new segment retno suryandari, widodo. – checklist of macroalgae in waisai coast, raja ampat 27 growth or segment basal branching. holdfast tuber or tuber (atmadja et al, 1996). distribution in southeast asia: indonesia, philippines, vietnam (guiry & guiry, 2017). halimeda macroloba decaisne (fig. 2i) description: green in colour when not calcified, solitary, erect, 7-12 cm long, branched in one plane giving it a flat appearance, attached by a cylindrical holdfast, upper segments discoid, reniform, rounded, 0.5-1.5 cm high, 1.5-2.0 cm broad, margins thick entire, branching dito tri-chotomous above and polychotomous at the basal part, cortex commonly composed of 3-4 layers of utricles, outermost utricles slightly attached when young and separating on decalcification when fertile. it is abundant on dead corals (jha et al, 2009) distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). avrainvillea erecta (berkeley) a. gepp & e.s. gepp (fig. 2j) description: dark to dull green in colour, erect, up to 8 cm tall, attached by elongated, densely interwoven basal mass of rhizoids with apical fan-shaped sessile and filamentous foliar position, foliar portions composed of branched filaments about 4cm long and 6 cm broad, spongy to somewhat hairy. it commonly found in muddy substratum with still waters (jha et al, 2009). distribution southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). codium geppiorum o.c.schmidt (fig. 2k) description: dark green in colour, procumbent, attached by holdfast, 3-5 cm tall, thallus sub-dichotomously divided and creeping type, branches cylindrical, divergent and compressed, utricles abovate to pyriform with rounded tips, 330 – 550 μm long and 100 – 300 μm broad with slight flattened apices, gametangia fusiform, pedicellate, 1 – 2 born on each fertile utricle. common in tide pools on sheltered rocks (jha et al, 2009) distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). boergesenia forbesii (hardvey) feldmann (fig. 2l) description: yellowish green in colour, 3-5 cm tall forming patches on the substratum, vesicles slightly curved, clavate, filled with fluid, club-shaped in younger stage enlarged in he upper part giving grape-like appearance in older stage. basal branched filamentous parts of the thallus septate. common in intertidal rock pools (jha et al, 2009). distribution in southeast asia: indonesia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). valonia ventricosa j. agardh (fig. 2m) description: dark green colour, single round and unbranched thallus, fluid-filled thallus(olsen & j.a., 1988). distribution in southeast asia: philippines, singapore, vietnam (guiry & guiry, 2017). indonesia (sukiman et al, 2014). dictyosphaeria cavernosa (forsskål) børgesen (fig. 2n) description: dark green, round hollow rounded with harsh spheres, stiff and slightly thick (atmadja et al, 1996). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017) chaetomorpha spiralis okamura (fig. 2o) description: plant bluish green in colour, up to 5cm long, rigid, spirally coiled, attached by rhizoidal basal cells with simple branched short and blunt rhizoids, cells 400-600 μm in diameter and 2-3 times longer than broad, moniliform or cylindrical in shape (jha et al, 2009) distribution in southeast asia: indonesia, malaysia, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). anadyomene wrightii harvey ex. j. e. gray (fig. 2p) description: light green coloured, no stipe, blade shaped like a corrugated fan with serrated blade edge, on a blade that looks like a leaf-like loop (anonymous, 2004). distribution in southeast asia: indonesia, philippines, singapore, vietnam (guiry & guiry, 2017). neomeris annulata dickie (fig. 2q) description: light green colour, thallus cylindrical and unbranched, calcified basal part, the apical portion consists of green fibers (jha et al, 2009). distribution southeast asia: indonesia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). red macroalgae (rodhophyta) class, order and family of redmacroalgae that found at waisai coast shown in the table 2 below. 28 biology, medicine, & natural product chemistry 5 (1), 2016: 23-32 table 2. checklist of red macroalgae that found at waisai coast. class order family species florideophyceae ceramiales rhodomelaceae acanthophora spicifera (m. vahl) børgesen laurencia papilosa (c. agardh) greville gracilaria salicornia (c. agardh) e.y. dawson corallinales corallinaceae amphiora fragilissima (linnaeus) j.v. lamoroux gigartinales cyatocloniaceae hypnea pannosa j. agardh acanthophora spicifera (m. vahl) børgesen (fig. 3a) description: yellowish brown colour, erect plants, to 40 cm tall, with solid cylindrical branches, 2 3 mm wide, branched either sparingly to repeatedly. main branches have short, determinate branches, irregularly shaped and spinose, with spines numerous and radially arranged. there are no spines on main axes. the plant grows from a large, irregularly shaped holdfast. (anonymous, 2001). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). laurencia papilosa (c. agardh) greville (fig. 3b) description: plants about 5-16 tall, growing in dense clusters. the lower parts of the plants are smooth, but toward the ends, the branches are sparsely crowded with short, truncate to tuberculate branchlets. plants olive green to greenish purple in colour, consistency somewhat cartilaginous (kluijvver, gijswijt, leon, and cunda, 2012) distribution in southeast asia: indonesia, philippines, vietnam (guiry & guiry, 2017). gracilaria salicornia (c. agardh) e.y. dawson (fig. 3c) description: brownish to yellowish red in colour, up to 16 cm in height, attached by small discs, thallus bushy with irregularly branched, cylindrical axes, lower branches cylindrical, not attenuated at the base, upper branches attenuated below, elongate clavate, swollen at the apex and showing apical depressions, one or two branchlets arising from the depressions, cortex 1-2 layered, medulla with large central cells, cystocarps scattered all over the thallus, rostrate (jha, 2009). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). amphiora fragilissima (linnaeus) j.v. lamoroux (fig. 3d) description: purple red in colour, up to 3 cm tall, calcified, erect, fragile, regularly dichotomously or trichotomously branched, sometimes with adventitious branches, apices obtuse, segments or intergenicula cylindrical or slightly compressed, several times longer than broad, sometimes with pad-like swellings at the tip, conceptacles lateral, hemispherical, prominent (jhaet al, 1996). distribution in southeast asia: indonesia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). hypnea pannosa j. agardh (fig. 3e) description: h. pannosa has densely entangled plants and tetrasporangial sori usually growing on one side of branchlets, than gradually completely surrounding the branchlets. this macroalgae are most commonly found growing abundantly on stable rocks of middle to lower intertidal zones, but sometimes they can be found growing in tide pools, branchlets are thick almost all the way to the apex, and the branch tips are often divaricate (chiang, 1990). distribution in southeast asia: indonesia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). a b c d figure 3. macroalgae of rhodophyta at waisai coast (a. acanthophora spicifera (m. vahl) børgesen, b. caulerpa laurencia papilosa (c. agardh) greville, c. gracilaria salicornia (c. agardh) e.y. dawson, d. gracilaria salicornia (c. agardh) e.y. dawson, d. amphiora fragilissima (linnaeus) j.v. lamoroux, e. hypnea pannosa j. agardh.). e retno suryandari, widodo. – checklist of macroalgae in waisai coast, raja ampat 29 brown macroalgae (phaeophyceae) class, order and family of brown macroalgae that found at waisai coast shown in the table 3 below. table 3. checklist of brown macroalgae that found at waisai coast. class order family species phaeophyceae fucales sargassaceae hormophysa cuneiformis (j.f. gmelin) p.c. silva sargassum aquifolium (turner) c. agardh sargassum polycystum c. agardh turbinaria ornata (turner) j. agardh dictyotales dictyotaceae padina australis hauck canistrocarpus cervicornis (kutzing) de paula & de clerck ectocarpales scytosiphonaceae hydroclathrus clatratus (c. agardh) m. howe hormophysa cuneiformis (j.f. gmelin) p.c. silva (fig. 4a) description: dark brown in colour, 30 cm in height or more, sparsely branched, pseudo-dichotomously branched, branches articulated, triangular articulation narrow at the base, 3-5 mm broad, margins dentate, vesicles ellipsoid or oblong, embedded in the middle of swollen wings, solitary (jha et al, 2009). irregularly branched thallus, blade-shaped blade-like wings with three sides and a serrated edge, holdfast filament-shaped filaments (tsuda, 2004). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). sargassum aquifolium (turner) c. agardh (fig. 4b) description: thallus medium brown, 10-45 cm tall, with a discoid-crustose holdfast 7-11 mm wide. primary axes terete, smooth, 3-4 mm long, 1.25-2.00 mm diameter. primary branches smooth to wrinkled, compressed, with sharp edges, 2-4 mm wide. primary laterals alternate, linear lanceolate to ovate-elongate, 13-30 mm long, 5-11 mm wide, margins undulate to deeply dentate, apices obtuse, base subsymmetrical, cuneate, arising directly from the stem and lacking a distinct stalk,margins vanishing 80% of the way to the apex (huisman & parker, 2016). distribution in southeast asia: indonesia (guiry & guiry, 2017). sargassum polycystum c. agardh (fig. 4c) description: thallus yellowish brown in colour, attached with discoid holdfast, main axis cylindrical and rough due to the presence of numerous outgrowth, supporting alternately arranged branches bearing blade and vesicles. in young thalli, blades are longer and broader measuring 13-42 mm long including the stipe and 2.5 – 11.5 mm wide, blades are generally oblong slightly tapered, retuse (slightly rounded) or emarginated at the tip finally serrated throughout the margin, mature thalli fewer leaves smaller. cryptostomates are scattered on the surface of the blade (mattio & payri, 2009). this macroalgae has secondary holdfast that is transformed from the stolon and heavily muricate on main branch (noiraksa, ajisaka, & kaewsuralikhil, 2006). distribution in southeast asia: indonesia, philippines, singapore (guiry & guiry, 2017). turbinaria ornata (turner) j. agardh (fig. 4d) description: dark brown in colour, up to 50 cm tall, bushy, axes arising from dichotomously branched holdfast, main axes erect and cylindrical and irregularly branched, leaves closely arranged, turbinate to obconical, coarse, 0.5-1.5 cm long 10-15 mm broad at the distal ends, distal ends of the leaves triangular, subconcave with double row of spines on the surface with terete stalks, vesicles immerged in the leaves, receptacles racemose, arising on the stalks of the upper leaves(jha et al, 2009). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). padina australis hauck (fig. 4e) description: brownish yellow, fan-shaped thallus, texture like a membrane, there is a concentric spike that follows the shape of the thallus, calcareous surface part, rhizoid holdfast (atmadja et al, 1996). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). canistrocarpus cervicornis (kutzing) de paula & de clerck (fig. 4f) description: growth from ascending, resupinate, attached to tha substrate by broad, erect part more slender and supple, epiphytic specimens generally lack a conspicuous basal part, color yellow to dark brown, dry specimens apical part pale brown, basal part turn a bit darker. branching isotomous dichotomous up to the middle parts typically cervicorn, recurved branches often with cervicorn, branching angle (30) 60-90 (120). margins and surface margins smooth and sinoidally curved (darakrai, 2012). 30 biology, medicine, & natural product chemistry 5 (1), 2016: 23-32 distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, vietnam (guiry & guiry, 2017), thailand (darakrai, 2012). hydroclathrus clatratus (c. agardh) m. howe (fig. 4g) description: yellowish to dark brown in colour, up to 12 cm long, 4-8 cm broad, vesicular or irregularly ovate, un-perforated when young, hollow and perforated as net like structures when mature, perforations variable in size, rounded, margins around perforations involute, thallus composed of an epidermal layer and a zone of colourless cells bellow the epidermis, plurolocular sori scattered all over the surface of the thallus, hairs grouped in shallow depression. (jha et al, 2009). distribution in southeast asia: indonesia, malaysia, myanmar, philippines, singapore, thailand, vietnam (guiry & guiry, 2017). a b c d e f figure 4. macroalgae of phaeophyceae at waisai coast (a. hormophysa cuneiformis (j.f. gmelin) p.c. silva,b. sargassum aquifolium (turner) c. agardh, c. sargassum polycystum c. agardh, d. turbinaria ornata (turner) j. agardh, e. padina australis hauck, f. canistrocarpus cervicornis (kutzing) de paula & de clerck , g. hydroclathrus clatratus (c. agardh) m. howe. g there are nine specimens of macroalgae that can’t be identified to species level, namely: sp. 3. brown colour, brown colour, flat stipe with irregular blade branches, thin blade and branches, penumatocyst found on the base of the blade (figure 5a). eucheuma sp. dark green in colour, irregular branching, cylindrical blade tip of each branch and the main branch off two or three (figure 5b). gracilaria sp. 1.colour pale brown, small thallus with bumps smooth surface (figure 5c). gracilaria sp. 2. the green colour, thin cylinder-shaped thallus with sparse branching, the tip of the needleshaped thallus (figure 5d). gracilaria sp. 3. colour brown, thallus like a needle with a larger size, thick and rigid, irregular branching (figure 5e). sp. 1. the colour brown, thallus branching from the base about 5 branches with each branch are spina-bifida short (figure 5f). sp. 2. dark green colour, thick thallus and looked porous, spherical and elongated like a tool at the thallus filled with fluid (figure 5g). sp. 3. the colour is yellowbrown, primary branches cylindical, opposite branches arrangement, the blade is lanceolate and the margin is jagged (figure 5h). sp. 4. the colour is dark brown, primary branches cylindical, opposite branches arrangement, the blade is linear like spinose with the apex rounded (figure 5. g, i). retno suryandari, widodo. – checklist of macroalgae in waisai coast, raja ampat 31 a b c d e f g h i figure 5. macroalgae that cannot be identified to species level at waisai coast (a.sp. 3, b. eucheuma sp., c.gracilaria sp. 1, d.gracilaria sp. 2, e.gracilaria sp. 3, f.sp. 1, g.sp. 2, h. sp. 3, i. sp. 4. table 4. environmental parameters of waisai coast. environmental parameters site 1 site 2 site 3 average salinity 32 ‰ 30 ‰ 31 ‰ 31 ‰ temperature 29,4 ˚c 29,7 ˚c 30,7 ˚c 29.9 ˚c ph 8,05 8,22 8,23 8.17 twenty nine identified macroalgae species have been found in previous studies in southeast asia. southeast asia region where the species are found, among others indonesia, malaysia, myanmar, philippines, singapore, thailand, and vietnam. species that can be found in the seven countries of southeast asia region are halimeda opuntia (linnaeus) j.v. lamoroux, halimeda macroloba decaisne, avrainvillea erecta (berkeley) a. gepp & e.s. gepp, codium geppiorum o.c. schmidt, dictyosphaeria cavernosa (forsskål) børgesen, acanthophora spicifera (m. vahl) børgesen, gracilaria salicornia (c. agardh) e.y. dawson, hormophysa cuneiformis (j.f. gmelin) p.c. silva, turbinaria ornata (turner) j. agardh, padina australis hauck, canistrocarpus cervicornis (kutzing) de paula & de clerck hydroclathrus clatratus (c. agardh) m. howe. halimeda cylindraceae decaisne, laurencia papilosa (c. agardh) greville found only in indonesia, philippines, and vietnam. as for species found only in indonesia, philippines, and singapore is sargassum polycystum c. agardh. the only species found in indonesia is sargassum aquifolium (turner) c. agardh (guiry & guiry, 2017). halimeda cylindraceae is a species of the genus halimeda. halimeda is a genus that has a characteristic thallus form of segments that contain calcium caco3. the content of the lime produced from the process of calcification is an important role for the environment. when individuals macroalgae of the genus halimeda is 32 biology, medicine, & natural product chemistry 5 (1), 2016: 23-32 dead, then the content of calcium will be major contributors to the formation of coral reefs (wizemann & meyer, 2014). genus halimeda addition, some other species also contribute to the environment. laurencia papilosa which are still found in three countries of asia also have a role for the environment. macroalgae have been tested have the ability to eliminate waste water colour stain on the textile industry (el maghraby, 2013). the methanol extract of s. aquifolium has the greatest free radical inhibiting activity and the antioxidant activity index is 0.54. based on the rules of the antioxidant activity index, the methanol extract of s. aquifolium is classified as moderate antioxidant activity (muhamad, 2013) this research shows the water temperature at the location of the study had an average of 29.9 ° c, salinity 31 ‰ and an average ph of 8.23. basically every type of macroalgae have standardized optimum temperature for growth vary. according to amalia (2013), the optimum temperature for growth of particular species of macroalgae gracillaria verrucosa ranged 22-27˚c but at a temperature of 31c can still grow although not with good quality. according raiker (2001) in the chung (2007) states that the optimal salinity for the growth of macroalgae that are at normal salinity of seawater is 35 psu. as for ph, according marianingsih et al (2013) seaweed can grow continuously at ph 7-8. based on environmental factors shows that the temperature and salinity of the water obtained in the studies are not optimal location to support the growth of algae. while the ph found in the study site can be classified include the appropriate ph for growth of macroalgae. conclusion based on the research, macroalgae found in waisai coast raja ampat are green algae, red algae and brown algae. green algae found and identified are caulerpa macra (weber-van bosse) draisma & prud’homme, caulerpa racemosa var. macrophysa (sonder wx kutzing) w.r.taylor, caulerpa sertularoides (s. gmelin) howe f. brevipes (j. agardh svedilus), caulerpa cupressoides (vahl) c. agardh, halimeda discoidea decaisne, halimeda opuntia (linnaeus) j.v. lamoroux, halimeda tuna (j. ellis & solander) j.v. lamoroux, halimeda cylindraceae decaisne, halimeda macroloba decaisne, avrainvillea erecta (berkeley) a. gepp & e.s. gepp, codium geppiorum o.c.schmidt, boergesenia forbesii (hardvey) feldmann, valonia ventricosa j. agardh, dictyosphaeria cavernosa (forsskål) børgesen, chaetomorpha spiralisokamura, anadyomene wrightii harvey ex. j. e. gray, neomeris annulata dickie. red algae species found and successfully identified areacanthophora spicifera (m. vahl) børgesen, laurencia papilosa (c. agardh) greville, gracilaria salicornia (c. agardh) e.y. dawson, amphiora fragilissima (linnaeus) j.v. lamoroux, hypnea pannosa j. agardh. brown algae species found and identified are hormophysa cuneiformis (j.f. gmelin) p.c. silva, sargassum aquifolium (turner) c. agardh, sargassum polycystum c. agardh, turbinaria ornata (turner) j. agardh, padina australis hauck, canistrocarpus cervicornis (kutzing) de paula & 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(cladophoraceae, chlorophyceae). turk j bot, 26, 171-174. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 177-185 | doi: 10.14421/biomedich.2023.121.177-185 issn 2540-9328 (online) comparative assessment of the proximate composition, functional properties and amino acid profile of dioscorea bulbifera, dioscorea alata and dioscorea rotundata found in minna, niger state eneogwe okechukwu godfrey1,*, obuye faith1, ibrahim izihyi esther2 1department of chemistry; 2department of industrial chemistry, federal university lokoja, p.m. b. 1154, lokoja, nigeria. corresponding author* godfrey.eneogwe@fulokoja.edu.ng abstract the proximate composition, functional properties and amino acid profile of samples of dioscorea alata, dioscorea rotundata and dioscorea bulbifera were investigated using standard analytical methods. the results showed that dioscorea alata had the highest ash (5.59±0.06 %) and crude fiber content (12.12±0.20 %), indicating that it has more mineral stuffing and is best to reduce the risk of obesity. dioscorea rotundata had the highest fat content (11.63±0.04 %) as well as the lowest moisture content (7.04±0.06 %), indicating that it is a better source of calories and has a longer shelf-life than other yam species analysed. dioscorea bulbifera also had the highest crude protein (8.64±0.03 %) and carbohydrates (77.51±0.08 %) than other yam species analysed, indicating high bodybuilding capacity and a better source of energy than other yam samples analysed. dioscorea alata showed the highest bulk density (0.87±0.02 g/cm3) and swelling capacity (15.25±0.03 g/g). it is indicating its usefulness in the reduction of paste thickness and water-holding capacity of starch granules respectively while dioscorea rotundata, showed the highest water absorption capacity (164.02±0.02 %), oil absorption capacity (149.76±0.02 %) and dispersibility (72.17±0.01 %). this indicates its importance in the consistency and bulking of products, flavour retaining in food and reconstitution of flour samples in water to give a fine consistent paste during mixing. the yam species were also rich in amino acids which are building blocks of protein. however, dioscorea rotundata was the richest in amino acid content, as it had 36.32±0.16 g/100g and 36.49±0.16 g/100g, for essential and non-essential amino acids respectively. keywords: amino acid; functional properties; proximate composition; dioscorea alata; dioscorea bulbifera. introduction roots and tubers allude to any developing plant that stores food in the subterranean roots, corm and tuber. the nutritional value of roots and tubers lies in their capacity to provide one of the cheapest sources of dietary energy in the form of carbohydrates in impoverished climes (ugwu, 2009). roots and tubers crops are an important source of food, nutrition and financial revenue for many impoverished farmers and food-insecure individuals in developing countries (oluwamukomi & akinsola, 2015). tubers are also used for the treatment of purgative, deflatulent, aphrodisiac, hemorrhoids, scrofula and polyureic (dutta 2015). furthermore, dietary plant estrogens of dioscorea provide various health benefits including defence against cancers, osteoporosis, cardiovascular disease, and asthma, as well as being utilized in the preparation of contraceptives and the treatment of numerous genetic abnormalities (sheikh et al., 2013). yam is a popularly consumed tuber in the tropics with several varieties such as dioscorea rotundata (white yam), dioscorea esculenta (chinese yam), dioscorea alata (water yam), dioscorea bulbifera (aerial yam), and dioscorea dumenterum (trifoliate yam) among the economically important species (ike and inoni, 2006). however, this study focuses on aerial yam (dioscorea bulbifera), white yam (dioscorea rotundata) and water yam (dioscorea alata). according to faostat (2006), nigeria is the world’s largest producer of yam, accounting for 67 percent of global production and 72 percent of west african production in 2005. yam is an important staple meal in west africa and a vital source of carbohydrates for over 300 million people worldwide (ettien et al., 2009). despite these well-known facts regarding yam, it continues to be overlooked in west african national food policy plans. this has resulted in limited dioscorea species research and development on the continent (sanoussi et al., 2016). this study investigates the proximate composition, functional properties and amino acid profile of dioscorea rotundata, dioscorea alata and dioscorea bulbifera obtained from minna, niger state. manuscript received: 15 october, 2022. revision accepted: 22 january, 2023. published: 24 january, 2023. https://doi.org/10.14421/biomedich.2023.121.177-185 178 biology, medicine, & natural product chemistry 12 (1), 2023: 177-185 materials and method sample collection matured accessions of the three cultivated yam species were harvested randomly from rural farms in chanchaga, mekunkele and gunu areas of niger state. the samples include cultivars of water yam (dioscorea alata), a variety of white yam (dioscorea rotundata) and aerial yam (dioscorea bulbifera). the samples were cleaned by brushing off soil particles and transported at tropical ambient temperature to the laboratory for analysis. dioscorea bulbifera dioscorea rotundata dioscorea alata figure 1. pictures of the studied dioscorea varieties. sample pre-treatment the yam samples were washed thoroughly with water, peeled and cut using a knife. these yam species were ground separately using a laboratory mortar and pestle and then sieved using a 250 μm mesh size sieve. the three samples were stored in airtight properly labeled polythene bags and kept in a cool and dry place before analysis. determination of proximate composition standard analytical procedures for food analysis were adopted for the determination of moisture content, crude protein, crude fibre, percentage fat, carbohydrate and ash content. moisture content two grams of the sample were placed in the crucibles and were then dried overnight at 105°c in the oven. after cooling in a desiccator for 30 minutes, the dry sample was weighed to a constant weight. on a dry weight basis, the percentage of weight loss was reported as a percentage of moisture content (aoac, 2006). to acquire triplicate values, this was done three times. the moisture content was calculated as: % 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑒𝑓𝑜𝑟𝑒 𝑑𝑟𝑦𝑖𝑛𝑔 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑓𝑡𝑒𝑟 𝑑𝑟𝑦𝑖𝑛𝑔 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑒𝑓𝑜𝑟𝑒 𝑑𝑟𝑦𝑖𝑛𝑔 × 100 ash content two grams of the dried and pulverized sample were taken in triplicates, put in pre-weighed crucibles and ashed for three hours at 600°c in a muffle furnace. after cooling in a desiccator, the hot crucibles were weighed. the percentage residual weight was expressed as ash content (aoac, 2006). the ash content was calculated as: % 𝐴𝑠ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑎𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛 × 100 crude fat content the percentage fat content of the samples was obtained using the method according to aoac (2006). five grams of the sample was weighed into a pre-weighed fat-free extraction thimble which was plugged tightly with cotton wool. on a heating mantle, the thimble was placed in the soxhlet extractor fitted up with reflux condenser connected to a boiling flask containing 200 ml of petroleum ether (boiling point 60oc). as the flask and petroleum ether were heated, the solvent evaporated and condensed into the thimble extracting oil from the sample and refluxed into the boiling flask with the extracted oil. this was done for 4 hours. at the end of extraction, the solvent (petroleum ether) was evaporated by heating at 70oc on a hot plate leaving the lipid extract in the flask. the flask together with the sample was placed in an oven and dried at 110oc for 1 hour, cooled in a desiccator and re-weighed. the percentage crude fat content was calculated using the formula: % 𝐶𝑟𝑢𝑑𝑒 𝑓𝑎𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑜𝑖𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 crude protein determination the kjeldahl method was used to determine the total protein. a kjeldahl flask containing 0.5 g of the sample was filled with 8–10 cm3 of concentrated h2so4 after godfrey et al. – comparative assessment of the proximate composition, … 179 being weighed in triplicate and the solution was digested in a fume cupboard until it became colourless. a 10 % naoh solution with a 40 % concentration was used for distillation. the boric acid solution glowed green after the condenser tip was dipped into a conical flask containing 5 cm3 of 4 % boric acid in a mixed indicator. hcl of 0.01m was titrated in the receiver flask until the solution turned red (aoac, 2006). the crude protein content was calculated as: % 𝑁 = (𝑎 − 𝑏) × 0.01 × 14 × 𝑣 𝑊 × 𝐶 × 100 where 𝑎 is the titre value of the digested sample, 𝑏 is the titre value of the blank sample, 𝑣 is the volume after dilution, 𝑊 is the weight of the dried sample, 𝐶 is the aliquot of sample used and 14 is the atomic weight of nitrogen. 𝐶𝑟𝑢𝑑𝑒 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 6.25 × % 𝑁 crude fibre content using 20 % h2so4 and 20 % naoh solutions, 2.0 g of the pounded sample was utilized in triplicates to estimate the crude fibre by acid and alkaline digestion techniques (aoac, 2006). the crude fibre content was calculated as: % 𝐶𝑟𝑢𝑑𝑒 𝑓𝑖𝑏𝑟𝑒 = 𝐿𝑜𝑠𝑠 𝑖𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑛 𝑖𝑔𝑛𝑖𝑡𝑖𝑜𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 carbohydrate determination the carbohydrate content was calculated by difference using the following formula: 𝐴𝑣𝑎𝑖𝑙𝑎𝑏𝑙𝑒 𝑐𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒 (%) = 100 − [𝑃𝑟𝑜𝑡𝑒𝑖𝑛 (%) + 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 (%) + 𝐴𝑠ℎ (%) + 𝐹𝑖𝑏𝑟𝑒 (%) + 𝐶𝑟𝑢𝑑𝑒 𝑓𝑎𝑡 (%)] determination of functional properties dispersibility kulkani et al. (1991), described a method for determining flour dispersibility. ten grams of flour was weighed into a 100 cm3 measuring cylinder, followed by 100 cm3 of distilled water. for 1 minute, the setup was vigorously agitated. after a regular time-step of 30 minutes, the volume of the settled particles was measured. the volume of settled particles was subtracted from 100. the difference was expressed as a percentage of dispersion. bulk density the bulk density was determined using the method published by oladele and ainaby (2007). in a 100 cm3 measuring cylinder, 50 g of samples were placed. the measuring cylinder was then tapped repeatedly on a laboratory table until it reached a fixed volume. the bulk density was calculated using the formula: 𝐵𝐷 (𝑔 𝑐𝑚3⁄ ) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑓𝑡𝑒𝑟 𝑡𝑎𝑝𝑖𝑛𝑔 water absorption capacity (wac) phillips et al. (1988) and anderson et al. (1969) techniques were used to determine the water absorption capacity and solubility index of flours from the sample. one gram of flour samples (mo) was weighed in a centrifuge tube and 10 cm3 distilled water was added. in a ks 10 agitator, the content of the centrifuge tube was shaken for 30 minutes. the mixture was centrifuged at 5000 rpm for 15 minutes after being maintained in a water bath (memmert) at 37°c for 30 minutes. the resulting sediment (m2) was weighed and dried to a consistent weight at 105°c (m1). after that wac was calculated using the formula: 𝑊𝐴𝐶 (%) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑤𝑎𝑡𝑒𝑟 𝑎𝑑𝑑𝑒𝑑 𝑡𝑜 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑤𝑎𝑡𝑒𝑟 𝑟𝑒𝑚𝑜𝑣𝑒𝑑 𝑓𝑟𝑜𝑚 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑓𝑙𝑜𝑢𝑟 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 oil absorption capacity (oac) eke and akobundu (1993) techniques were used to assess the oil capacity of the sample. in a weighed 20 cm3 centrifuge tube, 1 g of sample (mo) was mixed with 10 cm3 of oil. the slurry was stirred for 2 minutes in a vortex mixer, then kept at 28°c for 30 minutes before being centrifuged at 4500 rpm for 30 minutes. the clear supernatant was decanted and discarded. the adhering drops of oil were removed and the tube was weighed (m1). the oac was determined as follows: 𝑂𝐴𝐶 (%) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑜𝑖𝑙 𝑎𝑑𝑑𝑒𝑑 𝑡𝑜 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑜𝑖𝑙 𝑟𝑒𝑚𝑜𝑣𝑒𝑑 𝑓𝑟𝑜𝑚 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑓𝑙𝑜𝑢𝑟 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 180 biology, medicine, & natural product chemistry 12 (1), 2023: 177-185 swelling capacity kaushal et al. (2012) described the method that was used. one gram of flour sample was weighed into a graduated cylinder measuring 10 cm3. the volume occupied by the sample was measured after 5 cm3 of distilled water was added. the sample was left standing in water for 1 hour without being disturbed. the volume occupied after swelling was recorded and calculated as: 𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑐𝑐𝑢𝑝𝑖𝑒𝑑 𝑏𝑦 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑓𝑡𝑒𝑟 𝑠𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑐𝑐𝑢𝑝𝑖𝑒𝑑 𝑏𝑦 𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑒𝑓𝑜𝑟𝑒 𝑠𝑤𝑒𝑙𝑙𝑖𝑛𝑔 determination of amino acid profiles this was evaluated by extracting 3.00 g of the sample using a soxhlet extractor for six hours with petroleum ether (40–60°c) (copper, 2000). in a glass ampoule, 30.00 mg of the defatted samples were weighed and 7.00 cm3 of 6.00 mol/dm3 hydrochloric acid was added. by injecting nitrogen into the ampoule, oxygen was expelled (to avoid possible oxidation of some amino acids during hydrolysis). the ampoule was sealed with bunsen flame and put in an oven preset at 105°c for 22 hours, after which it was allowed to cool, broken at the tip and the content filtered. in a rotary evaporator, the filtrate was evaporated to dryness at 40°c under a vacuum. the residue was dissolved with 5.00 cm3 of acetate buffer (ph 2.0), then stored in a plastic bottle for 24 hours in the deep freezer. the technicon sequential multi-sample (tsm) amino acid analyser was loaded with five to ten microliters of the hydrolysate. this was dispensed into the cartridge of the analyser and the analysis lasted for 76 minutes. statistical analysis the obtained results were subjected to statistical analysis using mean standard deviation and analysis of variance (anova) as described by duncan’s multiple range test to determine the level of significance between different samples and significance was set at p ≤ 0.05. results and discussion table 1. proximate composition of selected yam species (%). yam species ash content moisture content crude fat crude fibre crude protein carbohydrate d.alata 5.59±0.06a 9.54±0.02b 7.52±0.03d 12.12±0.20c 7.85±0.02a 57.38±0.20d d.rotundata 3.05±0.06f 7.04±0.06h 11.63±0.04e 7.59±0.10a 2.19±0.01c 68.50±0.20ab d.bulbifera 2.34±0.02a 8.63±0.45b 1.60±0.01a 1.28±0.10e 8.64±0.03f 77.51±0.08f values are means ± standard deviation of triplicate analysis. proximate composition of selected yam species moisture content the moisture content of the various yam species ranged from 7.04±0.06h % for dioscorea rotundata to 9.54±0.02b % for dioscorea alata. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea alata showing the highest moisture content. however, according to oko and famurewa (2014), these values are comparable to its literature values that ranged from 2.1 % to 9.2 % for dioscorea dumenturom and dioscorea alata respectively. this means that dioscorea rotundata, when compared to the other yam species chosen for analysis, has a stronger resistance to deterioration and a longer shelf life. crude fiber content the crude fiber content ranged from 1.28±0.10e % for dioscorea bulbifera to 12.12±0.20c % for dioscorea alata. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea alata having the highest crude fibre content. however, this result can be compared with reports by afiukwa et al. (2013) that ranged from 6.01±0.04b % to 13.03±0.80a % for species of dioscorea dumenturom and differs from reports by oko and famurewa (2014) that ranged from 3.31% to 3.53% for their dioscorea alata species. according to studies, fiber intake reduces the risk of obesity, cardiovascular disease, diabetes and softening stools (turner, 2014). ash content ash contents of the yam varieties ranged from 2.34±0.02a % for dioscorea bulbifera to 5.59±0.06a % for dioscorea alata. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea alata having the highest ash content. however, these were different from reports by sorh et al. (2015) that ranged from 1.64±0.03 % to 1.78±0.03 % for dioscorea alata species. ash content reveals how heavily the yam cultivars are stuffed with minerals (akonor et al., 2017). as a result, compared to the other yam tubers examined, dioscorea alata has a higher mineral stuffing. crude protein content the crude protein content of the yam varieties ranged from 2.19±0.03c % for dioscorea rotundata to 8.64±0.03f % for dioscorea bulbifera. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea bulbifera having the highest crude protein content. however, the result can be godfrey et al. – comparative assessment of the proximate composition, … 181 compared with reports by ojinnaka et al. (2016) that showed 2.43±0.11b % for dioscorea bubilfera and in contrast with the report by ukom et al. (2014) that showed crude protein of dioscorea dumenturom to be 69.15±4.49b %. this demonstrates that of the dioscorea species examined, dioscorea bulbifera is the greatest source of protein. crude fat content the fat content in these analysed yam varieties ranged from 1.60±0.01a % for dioscorea bulbifera to 11.63±0.04e % for dioscorea rotundata. the result indicates that there was a significant difference (p≤0.05) in the yam species analysed with dioscorea rotundata showing the highest fat content. these findings, however, differ from those reported by ukom et al. (2014), who found that dioscorea cayenensis had an abundance of 41.91%. dioscorea bulbifera may be a superior source of calories than the other dioscorea species examined because dietary fat provides the majority of the energy needed by humans. carbohydrate content carbohydrate content of the dioscorea varieties were quite high and ranged from 57.38±0.20d % for dioscorea alata to 77.51±0.08f % for dioscorea bulbifera. the result indicates that the analysed yam species were significantly different (p≤0.05) with dioscorea bulbifera having the highest carbohydrate content. however, these values are comparable to literature values by ukpabi and akobundu (2014) that had 78.32±0.29 % for dioscorea dumenturom and in contrast with frank and kingsley (2014) that ranged from 24.25±0.62b % for dioscorea alata to 32.03±0.89c % for dioscorea rotundata. carbohydrates are considered the primary source of energy for all organisms (ojinnaka et al., 2016). table 2. functional properties of selected yam species. yam species bulk density (g/cm3) wac (%) oac (%) dispersibility (%) swelling capacity (g/g) d.alata 0.87±0.02a 155.34±0.02b 140.71±0.01d 62.85±0.02e 15.25±0.03b d.rotundata 0.85±0.02d 164.02±0.02f 149.76±0.02h 73.16±0.01c 14.29±0.01g d.bulbifera 0.57±0.03a 148.73±0.03a 133.85±0.01g 60.54±0.04a 8.44±0.03c values are means±standard deviation of triplicate analysis, wac: water absorption capacity, oac: oil absorption capacity. functional properties of selected yam species bulk density bulk density is a measure of the heaviness of a flour sample (oladele and aina, 2007). the bulk density of the dioscorea varieties ranged from 0.57±0.03a g/cm3 for dioscorea bulbifera to 0.87±0.02a g/cm3 for dioscorea alata. the result indicates that the analysed dioscorea species were significantly different (p≤0.05) with dioscorea bulbifera having the least bulk density while dioscorea alata had the highest. however, this result is comparable to that obtained from different cultivars of aerial yam which showed 0.810±0.01 g/cm3 for purple cultivars and 0.573±0.01 g/cm3 for white cultivars (ojinnaka et al., 2016). bulk density indicates the relative volume of packaging material required, as well as material handling and application in wet processing in food industries (tapti et al., 2018). dioscorea alata having the highest bulk density amongst the analysed yam species is often better for dispersibility and pastes thickness reduction, which is vital in convalescent child feeding (udensi and eke, 2000) while dioscorea bulbifera having the least bulk density is most useful in the formulation of infants weaning foods (akpata and akubur, 1999). water absorption capacity the ability of flour or starch to hold water against gravity is referred to as water absorption capacity (moure et al., 2006). the water absorption capacity of the dioscorea varieties ranged from 148.73±0.03 % for dioscorea bulbifera to 164.02±0.02 % for dioscorea rotundata. the result indicates that the analysed dioscorea species were significantly different (p≤0.05) with dioscorea rotundata having the highest water absorption capacity. however, this result is comparable to that obtained from oluwamukomi and akinsola (2015) which showed 174.60±9.3 g/cm3 for dioscorea rotundata and 167.00±0.17 g/cm3 for dioscorea bulbifera by tapti et al. (2018). the water absorption capacity is an important parameter since it shows if the flours may be used in aqueous food formulations, product bulking and uniformity, as well as in several baking applications (iwe et al., 2016). consequently, dioscorea rotundata having the highest water absorption capacity than other analysed yam species is best suited for this. oil absorption capacity oil absorption capacity has been attributed to the physical entrapment of oil. the oil absorption capacity showed a significant difference (p<0.05) between the yam varieties. the oil absorption capacity of the dioscorea species ranged from 133.85±0.01 % for dioscorea bulbifera to 149.76±0.02 % for dioscorea rotundata. the result indicates that the analysed dioscorea species were significantly different (p≤0.05) with dioscorea rotundata having the highest oil absorption capacity. however, this result is higher than the results obtained by kimbonguila et al. (2019) which ranged from 83.33% to 100% for different cultivars of dioscorea alata analysed. oil absorption capacity is an 182 biology, medicine, & natural product chemistry 12 (1), 2023: 177-185 indication of the rate at which the protein binds to fat in food formulations. it is important for flavour retention and boosting the mouthfeel of food. consequently, dioscorea rotundata is best suited for this, since it has the highest oil absorption capacity than the other yam species analysed (abu et al., 2005). swelling capacity the swelling capacity the dioscorea varieties ranged from 8.44±0.03 % for dioscorea bulbifera to 15.25±0.03 % for dioscorea alata. the result indicates that the analysed dioscorea species were significantly different (p≤0.05) with dioscorea alata having the highest swelling capacity. however, this result is comparable to that obtained from eke-ejiofor and owuno (2012) which showed 10.81±0.01 % for dioscorea dumenturom and slightly higher than reports by ojinnaka et al. (2016) that showed 7.58±0.01 % for dioscorea bulbifera sample. swelling power is a measure of starch hydration and is used to show associative binding force within starch granules (bello and ekeh, 2014). basically, it shows how much water starch granules can store (soison et al., 2015). as such, dioscorea alata is the best flour sample amongst the other yam species analysed for the formulation of infant weaning foods, since it has the highest swelling capacity (ojinnaka et al., 2016). dispersibility the percentage dispersibility gives an indication of water absorption capacity. the dispersibility of the yam species ranged from 60.54±0.04 % for dioscorea bulbifera to 72.17±0.01 % for dioscorea rotundata. the result indicates that the analysed dioscorea species were significantly different (p≤0.05) with dioscorea rotundata having the highest dispersibility. however, this result is comparable to that obtained from bashirat et al. (2015) which showed 72.17±0.01 % for dioscorea rotundata and 62.85±0.01 % for dioscorea alata. a dispersibility of fifty percent or more is considered high, implying that the higher the dispersibility, the better the flour’s capacity to reconstitute in water to form a fine and consistent paste when mixing (adebowale et al., 2005). table 3. amino acid profile of selected yam species (g/100g). amino acids dioscorea alata dioscorea rotundata dioscorea bulbifera leucine 6.29±0.06a 8.46±0.04a 7.18±0.02e lysine 4.37±0.05a 4.04±0.02d 4.82±0.01f isoleucine 3.59±0.16b 3.66±0.02c 3.54±0.04i phenylalanine 6.65±0.04e 5.68±0.12e 4.44±0.03a tryptophan 0.95±0.01c bdl 0.89±0.01b valine 4.36±0.02f 5.78±0.03a 3.97±0.03d methionine 1.76±0.03a 2.37±0.02b 2.07±0.06c histidine 1.98±0.02d 1.83±0.02c 1.78±0.02a threonine 3.46±0.03i 4.49±0.01h 3.18±0.02b *proline 1.98±0.04a 1.64±0.02a 2.03±0.03e *arginine 8.94±0.03b 6.97±0.03d 8.27±0.03f *tyrosine 3.10±0.02b 4.14±0.02e 2.76±0.03a *cystine 1.09±0.01e 2.78±0.03a 1.22±0.01b *alanine 3.27±0.03c 2.99±0.03c 2.99±0.03d *glutamic acid 5.76±0.02f 6.96±0.01h 8.55±0.01c *glycine 2.14±0.02d 2.04±0.02b 1.68±0.02a *serine 2.22±0.02i 3.00±0.01c 2.73±0.02h *aspartic acid 5.49±0.02h 5.96±0.02a 6.22±0.02f teaa 33.42±0.41 (49.61 %) 36.32±0.16 (49.88 %) 31.89±0.24 (46.60 %) tneaa 33.95±0.18 (50.39 %) 36.49±0.16 (50.12 %) 36.45±0.19 (53.34 %) values are means ± standard deviation of triplicate analysis, teaa: total essential amino acid, tneaa: total non-essential amino acid, bdl: beyond detection limit, *non-essential amino acids. amino acid profile table 3 depicts the results of the amino acids profile. amino acids are the building blocks of proteins and they serve an important role in the body. the findings imply that dioscorea species are rich in amino acids such as essential and non-essential amino acids. the essential amino acids include lysine, phenylalanine, valine, threonine, isoleucine, methionine, histidine and leucine. while the non-essential amino acids are alanine, arginine, aspartic acid, serine, tyrosine, proline, cysteine and glutamic acid. however, the results obtained showed that the non-essential amino acid was more than the essential amino acids in the yam species. the total nonessential amino acid values were 33.95±0.18 g/100g, 36.49±0.16 g/100g and 36.45±0.19 g/100g representing 50.39 %, 50.12 % and 53.34 % for dioscorea alata, dioscorea rotundata and dioscorea bulbifera respectively. while the total essential amino acid contents were 33.42±0.41 g/100g, 36.32±0.16 g/100g and 31.89±0.24 g/100g representing 49.61 %, 49.88 % godfrey et al. – comparative assessment of the proximate composition, … 183 and 46.60 % for dioscorea alata, dioscorea rotundata and dioscorea bulbifera respectively. this result can be compared to reports by alozie et al. (2009). dioscorea alata had the least non-essential amino acid content with 33.95±0.18 g/100g while dioscorea rotundata had the highest with 36.49±0.16 g/100g. also, for the essential amino acid, dioscorea bulbifera had the least content with 31.89±0.24 g/100g, while dioscorea rotundata had the highest with 36.32±0.16 g/100g. as such from the research it can be seen that dioscorea rotundata had the highest total amino acid content. furthermore, the percentage ratio of the essential amino acid (eaa) to the total amino acid (taa) in the samples ranged from 46.66 % to 49.88 %. these values are well above the 39% considered adequate for ideal protein food for infants, 26% for children and 11% for adults. glutamic acid appeared to be the most abundant amino acid in dioscorea bulbifera at 8.55±0.01c g/100g. while leucine was the highest in dioscorea rotundata at 8.46±0.04a g/100g. like valine and isoleucine, leucine is a branched-chain amino acid (bcaa) that is essential for protein synthesis and muscle repair. it also aids in blood sugar regulation, wound healing and the production of growth hormones (shimomura et al., 2004). arginine which can also be considered a conditionally essential amino acid just like glycine was the most abundant amino acid in dioscorea alata at 8.94±0.03b g/100g. the high level of arginine in dioscorea alata indicates its usefulness as a supplement during pregnancy, trauma and illness (cancer). tryptophan which is a sole precursor to serotonin, a neurotransmitter that regulates appetite, sleep and mood, was quite low in all the samples but was relatively absent in dioscorea rotundata (slominski et al., 2002). histidine levels were highest in dioscorea alata at 1.98±0.02d g/100g. this shows its likeliness to produce more histamine which is a neurotransmitter that is vital to immune response, digestion, sexual function, maintaining levels of hemoglobin and sleep-wake cycles. it is also required for the growth and repair of tissues, red blood cell production and protecting tissues from damage from radiation and heavy metals. it is especially very important for the formation of myelin sheaths, which are layers surrounding nerves that enables faster transmission of signals to the brain (ncbi, 2022). the minimum amino acid intake of 1.5 g/kg/day is reported to be necessary in preventing negative nitrogen balance while 2.5 g/kg/day is not advisable (espghan, 1997). conclusions this study provided vital information on the proximate composition, amino acid profile and functional properties of the selected yam tuber species (dioscorea alata, dioscorea rotundata and dioscorea bulbifera) analysed. the generally high carbohydrate content indicates that these species are reliable sources of energy. they can also be considered to be rich in fibre, mineral stuffing and have a high shelf life due to their generally high crude fibre and ash content as well as low moisture content respectively. the amino acid profile of these studied species, suggests that they are rich in protein. however, dioscorea rotundata was the richest in amino acid content, as it had 36.32±0.16 g/100g and 36.49±0.16 g/100g, for essential and non-essential amino acids respectively. furthermore, the wide variation observed in the functional properties of the flour samples serves as a database for the selection and improvement of the yam species for specific food applications to stimulate their industrial processing and utilization. competing interests: the authors declare that there are no competing interests. references abu, j. o., muller, k., duodu, k. g., minnaar, a. 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(2014). production and quality evaluation of pre-gelatinized fermented breakfast food produced from edible trifoliate yam (dioscorea dumenturom) using intermediate technologies in nigeria. american journal of food science and nutrition, 1(4), 60-7. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 215-220 | doi: 10.14421/biomedich.2024.131.215-220 issn 2540-9328 (online) levels of oxidative stress in rats treated with calorie restrictive treatment and 50% sucrose solution diniwati mukhtar*, azha azzuna amsaka, fanny ratnasari pd, aan royhan, karina ajeng ridwan medical faculty, university of yarsi, jakarta, indonesia. corresponding author* diniwati.mukhtar@yarsi.ac.id abstract this study aims to analyze the level of stress oxidative with treatment calorie restrictions and administration of sucrose solution. this study was a laboratory experimental study with 12 male white rats (wistar strain) as subjects for caloric restrictive treatment and 12 rats for 50% sucrose solution. where each group of rats is determined by the dependent and independent variables. using the pre-posttest control design, the dependent variable of caloric restriction, and the independent variable malondialdehyde. the dependent variables used were body weight using a scale, fasting blood glucose using a glucometer, and mda using a uv-vis spectrophotometer. the independent variable used was the administration of 50% sucrose solution. the data were statistically analyzed by t-test analysis. there was no significant decrease in mda levels in the caloric restriction treatment rats (p = 0.060), besides that there was a significant difference in body weight in both the control and sucrose groups (p <0.05). there was no significant difference in fasting blood sugar in the control group (p > 0.05) and there was a significant difference in the sucrose group (p < 0.05). there was no significant difference in serum mda levels in either the control or sucrose groups (p > 0.05). there is an effect of calorie restriction on mda levels. also, administration of 50% sucrose solution for 4 weeks had a significant effect on the rats' body weight and blood glucose but did not have a significant effect on serum mda levels. keywords: oxidative stress; calorie restriction; sucrose solution; malondialdehyde. introduction oxidative stress is part of the tissue damage in people that reflects the unevenness between the creation of receptive oxygen species (ros), which are responsive oxidizing compounds, and the strengthening protection of cells. free radicals or what are known as reactive oxygen species or ros are molecules that contain one or more unstable electrons because one or more electrons in their outer orbit are unpaired the body needs one of them as a defense against infectious agents (jakubczyk et al., 2020). oxidative stress plays a role in different human diseases, including malignant growth, cardiovascular disease (cvd), lung, nervous, kidney, and liver disease, and, surprisingly, physiological maturation processes. oxidative stress is a phenomenon caused by an imbalance between the production and accumulation of reactive oxygen species or what is often called ros and antioxidants. specific reactive oxygen (ros) leads to an imbalance between pro-oxidants and antioxidants in cells and tissues. in a healthy state, the existence of pro-oxidants is balanced with antioxidants, but in certain circumstances, this balance is disturbed, where this condition is referred to as oxidative stress. this state refers to the modification of lipids, proteins, and dna. such as modification or damage to a molecule called oxidative damage. prooxidants can increase oxidation. excessive creation of free extremities in the body can cause oxidative stress. one sign of oxidative stress in humans is the degree of malondialdehyde (mda) which is the result of lipid peroxidation in the body caused by free radicals. according to research, a 30% calorie restriction for 15 days can reduce mda levels, which is a marker of oxidative stress in the body (khoubnasabjafari et al., 2015). oxidative stress plays a role in different human diseases, including malignant growth, cardiovascular disease (cvd), lung, nervous, kidney, and liver disease, and, surprisingly, physiological maturation processes. reactive oxygen species (ros) are produced through several mechanisms, one of which is when the body experiences excess glucose (hyperglycemia). factors that cause hyperglycemia are diet, stress, infection, and consumption of certain drugs. in planning a meal pattern, the emphasis is on diet in terms of the 3 m's (meal schedule, type of meal, and amount of meal). excessive consumption of sugar can cause hyperglycemia and increase the production of free radicals resulting in oxidative stress. oxidative stress is manuscript received: 28 february, 2024. revision accepted: 20 may, 2024. published: 19 june, 2024. https://doi.org/10.14421/biomedich.2024.131.215-220 216 biology, medicine, & natural product chemistry 13 (1), 2024: 215-220 accompanied by lipid peroxidation in cell membranes so that malondialdehyde (mda) is formed as a result of lipid peroxidation (gawel et al., 2004). thus, mda can be used to determine the degree of oxidative damage caused by lipid peroxidation (gawel et al., 2004). the relationship between level stress oxidative with treatment calorie restrictive and sucrose solution 50% has not been studied yet up to date. this study aims to determine whether there is an effect of caloric restrictive treatment and administration of 50% sucrose solution on levels of oxidative stress. methodology study design this research is a laboratory experimental study using the pre-posttest control design. the subjects of the study were 12 male white rats (wistar strain) for the caloric restriction treatment and 12 rats for the administration of sucrose solution where each of them would be divided into several groups of variables and studied for 4 weeks. the study was conducted from december to january at the pharmacology and biochemistry laboratory of the faculty of medicine, yarsi university, and was approved by the yarsi university research ethics commission with number 324/kep-uy/bia/x/2021. the experimental animal the research subjects were male rats weighing ± 130 grams and aged 8 weeks. the group given the calorie restriction treatment will be divided into the dependent variable studied is calorie restriction, the independent variable is malondialdehyde, while the group given 50% sucrose solution will be grouped into the dependent variable used is body weight using a scale, fasting blood glucose using a glucometer, and mda using uv-vis spectrophotometer. the independent variable used was the administration of 50% sucrose solution. control variables will be determined for each group, namely the age of the rats, male sex, the shape, size, and placement of the cages are made the same, and the type, quantity, and quality of food and drink are the same. data distribution was determined by the shapiro-wilk test. paired t-tests were used to determine between-group differences in mean mda. the difference is considered significant if p <0.05. preparation of 50% sucrose solution a total of 50 grams of sucrose was dissolved in 100 ml of distilled water, then given to rats orally (fed) at a dose of 1.5 cc/head twice a day (09.00 and 15.00) for two months. administration of 50% sucrose solution after being acclimated for one week, the treatment group was given standard feed, drink ad libitum, and 50% sucrose solution until the end of the study. blood sampling wipe the eyes of the rats with cotton that has been moistened with 70% alcohol, then give an anesthetic, namely ketamine-xylazine (0.15 ml/100 grbb) or ketamine 100-200 mg/kgbb + xylazine 10 mg/kgbb (flecknell, 1993), because the body weight of the mice used was 200 grams, the dose used was 0.3 ml. furthermore, blood samples were taken using a syringe of as much as 3 ml and then placed in an edta tube to measure malondialdehyde (mda). blood glucose measurement glucose levels were measured 3 times at the beginning, middle, and end of the study. blood glucose levels of experimental rats were determined using the glucose oxidase biosensor method, using a blood glucose test meter glucodr. before being analyzed, the animals were fasted for 8 hours. blood was taken from the tip of the rat's tail which was previously cleaned with 70% alcohol, then the tip of the tail was pricked with a small needle. the blood that comes out is then touched on the glucometer strip. after 11 seconds, the blood glucose level will be read on the glucodr screen and expressed in mg/dl units. measurement of malondialdehyde (mda) levels malondialdehyde (mda) levels of rats were measured using thiobarbituric acid (tba) which will form a pink mda-tba product and measured using a spectrophotometric method. to obtain serum, centrifugation was carried out at 3000 rpm for 20 minutes. for every 100 microliters of serum, 2.45 ml of tca (trichloroacetic acid) and 2.45 ml of tba (thiobarbituric acid) were added, then heated at 100°c for 20 minutes. then the mixture was centrifuged at 8000 rpm for 10 minutes. the absorbance of the supernatant obtained was determined using a blank based on 2.45 ml tca and 2.45 ml tba on the standard curve. statistical analysis calculation of data normality was carried out by the shapiro-wilk test. the test will reject ho if the p-value ≤ 0.005 (5%). this test was carried out provided that the number of samples was less than 30. the results of this test showed that the p-value of shapiro wilk on the variables body weight, glucose, and mda had a normal distribution of data in both groups (p-value > 0.05), so on these three variables to do a different test using a paired t-test. mukhtar et al. – levels of oxidative stress 217 results experimental animals were randomly divided into 2 groups consisting of 6 animals. the calorie restriction treatment group and the control group were given standard feed. this research was conducted in the pharmacology laboratory for 10 days, in december 2021. to find out whether there was an effect, on glucose, mda, and body weight in the treated rats, a different test was performed, namely the t-test with the variables of glucose, mda, and body weight. before the different test is carried out, the data normality test is first performed. the results of the data normality test using spss 26.0 are presented in table i. calculation of data normality was carried out by the shapiro-wilk test. the test will reject ho if the p-value ≤ 0.005 (5%). this test was carried out provided that the number of samples was less than 30. the results of this test showed that the p-value of shapiro wilk on the variables body weight, glucose, and mda had a normal distribution of data in both groups (p-value > 0.05), so these three variables do a different test using a paired t-test. glucose levels in the treatment group and control group in table ii it is known that the average glucose before and after the calorie-restrictive treatment in the control group was 83.3 mg/dl and 115 mg/dl and in the treatment group, it was 79.5 mg/dl and 199.8 mg/dl. from the results of the t-paired test in the control group, the p-value of glucose before and after the calorierestrictive treatment was 0.169 (p> 0.05), which means that there was no significant change between the beginning and end of the control group, while in the treatment group, the value of p = 0.001 was obtained. this means that there is a significant change in glucose levels. malondialdehyde levels in the control and treatment groups furthermore, the malondialdehyde was measured. as shown in table iii, the average malondialdehyde at the beginning and the end of the treatment in the control group is 1.241 nmol/ml and 1.265 nmol/ml. from the results of the paired t-test, a p-value of 0.102 (p > 0.005) was obtained, which means that there was no significant change between the start and end of treatment in the control group. whereas in the treatment group, it was known that the average malondialdehyde was 1.262 and 1.232 at the beginning and end of treatment in the treatment group. from the results of the paired t-test, it was obtained a p-value of 0.060 (p> 0.005), which means that there was no significant change between the beginning and the end of the treatment in the treatment group. body weight values before and after treatment changes in body weight values at the start and end of treatment were found and shown in table iv. based on the table, it was obtained that the p-value was 0.165 in the control group and 0.058 in the fasting treatment group, which was greater than the 5% significance level. it could be interpreted that there was a difference in rat body weight between the group that was given standard feed and the group that was fed a caloric-restrictive treatment, which means the caloric-restrictive treatment did not significantly affect the rat's body weight. the treatment group was given 50% sucrose solution using a sonde every day, as well as the control group which was given standard feed. furthermore, both groups measured body weight, blood glucose, and malondialdehyde (mda), which is a marker of oxidative stress. the study was conducted in the fkuy pharmacology and biochemistry laboratory for 4 weeks, a period from december 2021 – january 2022. statistical analysis to see changes in the variables above uses the anova repeated measure test and paired t-test. the variables tested in this study included 3 (three), namely: body weight, fasting blood sugar, and mda. before carrying out the anova repeated measure test and the paired t-test, the data normality test was first performed. the results of the data normality test using spss 26.0 are presented in the following table: ▪ mda levels in the control and treatment groups based on the results of the study or the t-test, it was stated that there was no significant difference between the treatment (fasting) and control groups on mda levels p>0.05. however, based on the average mda value in the fasting treatment group, it was 1.262 at the beginning of the treatment and 1.232 at the end of the treatment. in the control group, the average was 1.241 at the beginning of the treatment and 1.265 at the end of the treatment, which tended to decrease in the treatment group compared to the control group. this is also in line with research conducted by chausse et al. (2015). according to trepanowski et al. (2011) that calorie restriction can lead to general health improvements, preventing degenerative diseases, cognitive deficits, and premature aging. ▪ glucose levels in the control and treatment groups based on the t-test table in table, the average glucose level in the treatment group was 79.5 mg/dl at the beginning of treatment and 199.83 at the end of treatment. in the control group, it was found that the average at the beginning of the study was 83.3 mg/dl and 115 mg/dl at the end of the study. changes in glucose levels at the beginning and end of the treatment in the control group showed p=0.169 and p=0.001 in the treatment group, where there were no significant changes in the control group and there were significant changes in the treatment group. it was found in this study that the 218 biology, medicine, & natural product chemistry 13 (1), 2024: 215-220 treatment group experienced an increase in glucose levels more than the control group. this is following research conducted by bolli et al. (2021) that there is a dawn phenomenon in normal individuals after calorie restriction. muscle glycogen is a source of glucose 1-phosphate which can be rapidly used for glycolysis within the muscle itself. liver glycogen functions to store and transport glucose to maintain blood glucose levels between meals. the liver concentration of glycogen is about 450 mmol/l glucose equivalent after a meal, which decreases to about 200 mmol/l after an overnight fast; after 12-18 hours of fasting, liver glycogen is almost completely depleted (murray et al., 1995). ▪ changes in body weight in treated and controlled rats in this study, it was found that there was a change in the body weight of the rats at the beginning of the treatment and the end of the treatment. based on table v, the average body weight in the control group at the beginning of the treatment was 167g and 194g at the end of the treatment, then in the treatment group, it was obtained an average of 159 g was at the beginning of the treatment and 182 g at the end of the treatment. in calculating the results p= 0.165 in the control group and p=0.058 in the treatment group whereas in the two treatment groups, no significant changes were found. obtained in this study the weight of the control and treatment group rats experienced weight gain. administration of 50% sucrose solution average body weight in the control group and 50% sucrose solution changes in body weight for 4 weeks of treatment, as shown in table v. there was an increase in bw (body weight) on days 0, 15, and 30 in the control group and the 50% sucrose solution group. the average weight on days 0, 15, and 30 in the control group was 167.3 grams, 194.3 grams, and 243.3 grams. and the average body weight on days 0, 15, and 30 in the sucrose group were 161.5 grams, 167.8 grams, and 202.8 grams. based on table vi, the p-values (sig/greenhousegeisser) in the control and sucrose groups were 0.031 and 0.011 (p <0.05). so it can be interpreted that there is a difference in the body weight of the rats between the group that was given standard feed and the group that was given an additional 50% sucrose solution on days 0, 15, and 30, which means that sucrose significantly affects the body weight of the rats. average fasting blood sugar levels in the control group and 50% sucrose solution there was an increase in gdp (fasting blood sugar) on days 0, 15, and 30 in the control group, namely 83.3 mg/dl, 115.2 mg/dl, and 134 mg/ll (see table vii). whereas on day 0 the sucrose group was 84.7 mg/dl and decreased on day 15 to 82.5 mg/dl and increased again on day 30, namely 110.2 mg/dl. based on table viii, the p-value (sig/greenhousegeisser) obtained in the control group was 0.124 (p > 0.05) and the sucrose group was 0.021 (p <0.05). the interpretation is that there is no difference in the blood sugar of the rats on days 0, 15, and 30 in the control group and there is a difference in the group that was given an additional 50% sucrose solution, which means that sucrose affects the blood sugar of the rats significantly. the average serum mda level in the control group and 50% sucrose solution there were changes in mda (malondialdehyde) levels on day 0 and day 30 in the control group, namely 1.285 nmol/ml and 1.241 nmol/ml, and in the sucrose group, namely 1.248 nmol/ml and 1.237 nmol/ml (see table ix). based on the results of the paired t-test in table x, the p-values in the control and sucrose groups were 0.469 and 0.637 (p > 0.05). so it can be interpreted that there was no significant change in mda between the group that was given standard feed and the group that was given an additional 50% sucrose solution, which means that 50% sucrose did not significantly affect the mda levels of rats. discussion weight change both the experimental and control animals experienced an increase in body weight. after the anova repeated measure test, the two groups also had significant differences. in the high-simple carbohydrate diet group, namely sucrose, there was a significant increase in average body weight. types of carbohydrates, glycemic index, and glycemic load affect blood glucose which can lead to changes in body weight. the higher the glycemic load, the higher the increase in blood glucose. elevated blood glucose over a long period can increase the risk of obesity. types of simple carbohydrates tend to have a high glycemic index. this is in line with the literature review by manuel-y-keenoy and perez-gallardo (2012) regarding the metabolic effect of the amount and type of carbohydrates on the risk of obesity and diabetes. based on this review, it is explained that excess glucose from a high-carbohydrate diet will be stored in the form of glycogen and the rest will be stored in the form of fat in the process of de novo lipogenesis. this is what can increase body weight (aller et al., 2011). based on this research, it can be said that the consumption of simple carbohydrates, one of which is sucrose, can increase body weight. this is following the results from the literature review by aller et al. (2011) concluded that a high-starch diet is more beneficial for health than a high-sugar diet for weight control by reducing body fat (aller et al., 2011). mukhtar et al. – levels of oxidative stress 219 fasting blood sugar based on figure 4.2, the control rat group experienced an increase in blood glucose both on days 0, 15, and 30. meanwhile, in the treatment group, it decreased on day 15 and increased again on day 30. based on the anova repeated measure test, there was no difference in rat blood sugar between the control group and the group that was given an additional 50% sucrose solution, which means that sucrose did not significantly affect the rat's blood sugar. rats can experience a decrease in blood glucose in conditions of an excess energy diet, the satiety center will be activated and reduce epinephrine secretion so that the body's activity in looking for food will decrease. mice that are given high-calorie feed will experience an increased sense of calm due to reduced stress due to hunger. this feeling of fullness suppresses the rat's desire to find food. this incident will cause rats to become calmer and suppress stress. this is also supported by the physical condition of the rats which are a little passive and the color of the fur is starting to fade. furthermore, on the 30th day blood glucose levels increased again because the administration of sucrose increased blood glucose and fat deposits as well as body weight in rats. this is in line with firdaus et al. (2016) that a high-sucrose diet for 3-5 weeks can increase blood glucose levels and hyperinsulinemia and also reduce insulin sensitivity in rats and mice. a high sucrose diet markedly alters insulin-mediated glucose metabolism which can result in insulin resistance in rats. based on research by battung et al. (2019) concerning the effect of sucrose on blood glucose levels, the result was an increase in blood glucose levels in the intervention with 20 grams of sucrose. serum mda (malondialdehyde) levels malondialdehyde levels (mda) are one of the end products of lipid peroxidation. mda levels will increase under conditions of oxidative stress, in various pathological conditions, and in infections otherwise, mda levels will be low if the antioxidant defense system is good. results the serum mda profile serves as a marker of cellular damage due to free radicals (zaetun et al., 2019). based on research by zaetun et al. (2019) that the higher the radical level, the higher the mda level that is formed. however, this did not appear in the results of the study when viewed from this because the mda levels of the control and treatment mice did not increase. it is also possible that there has been no exposure to free radicals in the experimental group of animals. if the experimental animal treatment is carried out for a longer period, the results may have a more significant effect on the presence or absence of cellular damage. in addition, there is a relationship between high gi (glycemic index) foodstuffs and mda levels. foods with high gi values cause higher blood glucose and insulin responses compared to foods with low gi values. consumption of high-gi foods is absorbed more quickly in the small intestine and has the potential to increase blood glucose. however, in this study, there was no significant difference in mda levels in the two groups because the intervention in the form of 50% sucrose solution was only carried out for 4 weeks so the new rats experienced hyperglycemia and entered the starting point of free radical formation so that mda levels did not increase. this is in line with the study of suarsana et al. (2011) that blood glucose levels in hyperglycemia treatment began to increase in week 4. after that, excess blood glucose for a long time could cause the formation of excess free radicals. these free radicals that are formed can attack the surrounding cells, including pancreatic cells. as a result of the chain reaction these free radicals cause cell damage. in this study, insulin was still able to offset the increase in blood glucose levels. however, if this increase lasts a long time, insulin will no longer be able to maintain blood sugar levels at a normal level and free radicals will accumulate, thereby increasing mda levels. conclusion based on the results of the study it can be concluded that there was a decrease in mda levels in the calorie restriction group, although it was not statistically significant. however, there was a significant increase in blood sugar levels in the calorie restriction group. while the increase in body weight in both groups but not statistically significant. furthermore, 50% sucrose solution administration for 4 weeks was a significant change (p <0.05) the body weight. for blood glucose level, there was no significant change in the control group (p > 0.05) and there was a significant change in the sucrose group (p <0.05). administration of 50% sucrose solution for 4 weeks did not show a significant change in serum mda (malondialdehyde) levels. for future researchers, it is hoped that they can conduct similar research with more samples and a longer time. the general public is expected to maintain a healthy lifestyle and maintain a diet to avoid various diseases due to increased oxidative stress in the body. seeing the prevalence of lifestyle-related diseases causing an increase in oxidative stress, it is hoped that doctors can educate the public. acknowledgements: the authors would like to thank the staff and students of yarsi university who took part in this research. in addition, the authors would like to thank all others who assisted with data college. authors’ contributions: diniwati mukhtar & azha azzuna amsaka designed the study. diniwati mukhtar carried out the laboratory work. fanny ratnasari pd, aan royhan & karina ajeng ridwan analyzed the data. 220 biology, medicine, & natural product chemistry 13 (1), 2024: 215-220 diniwati mukhtar & azha azzuna amsaka wrote the manuscript. fanny ratnasari pd, aan royhan & karina ajeng ridwan revised the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references aller, e. e., abete, i., astrup, a., martinez, j. a., & van baak, m. a. (2011). starches, sugars and obesity. nutrients, 3(3), 341– 369. battung, s. m., salam, a., novrianti, d., & ajie, r. a. k. (2019). efek diet tinggi karbohidrat terhadap glukosa darah dan berat badan tikus wistar. jurnal gizi masyarakat indonesia (the journal of indonesian community nutrition), 8(2), 55-62. bolli, g. b., cheng, a., charbonnel, b., aroda, v. r., westerbacka, j., bosnyak, z., boëlle‐le corfec, e., & rosenstock, j. (2021). glycaemic control and hypoglycaemia risk with insulin glargine 300 u/ml and insulin degludec 100 u/ml in older participants in the bright trial. diabetes, obesity and metabolism, 23(7), 1588-1593. chausse, b., vieira-lara, m. a., sanchez, a. b., medeiros, m. h., & kowaltowski, a. j. (2015). intermittent fasting results in tissue-specific changes in bioenergetics and redox state. plos one, 10(3), e0120413. firdaus, f., rimbawan, r., marliyati, s. a., & roosita, k. 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(2019). profil kadar mda (malondialdehide) sebagai penanda kerusakan seluler akibat radikal bebas pada tikus yang diberikan air beroksigen. jurnal analis medika biosains (jambs), 4(2), 63-68. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1021-1028 | doi: 10.14421/biomedich.2025.142.1021-1028 issn 2540-9328 (online) formula optimization and sensory analysis of functional collagen drink from butterfly pea flower (clitoria ternatea l.) and red guava (psidium guajava l.) anjar asmarani widya nabilah*, ela marcelia, tri dewanti widyaningsih, kiki fibrianto food science and biotechnology department, faculty of agricultural technology, university of brawijaya, jl. veteran no. 10-11, ketawanggede, lowokwaru, malang, indonesia 55145, indonesia. corresponding author* anjarasmaraniwn@gmail.com manuscript received: 23 may, 2025. revision accepted: 04 november, 2025. published: 13 november, 2025. abstract the global market for functional beverages is experiencing rapid growth as consumer awareness for healthy lifestyles increases. the objective of this study is to ascertain the physical characteristics, optimal formula, and organoleptic quality of functional collagen beverages derived from a blend of bayang flower (clitoria ternatea l.), red guava (psidium guajava l.), and emprit ginger. in this study, an extreme vertex design was employed in conjunction with minitab 19 to extract the materials. the extraction process was carried out using the following methods: telang flowers were subjected to a drying process at 40°c for 24 hours, followed by extraction with water at 60°c for 30 minutes; ginger emprit was dried at 60°c for 24 hours and then extracted at 55°c for 15 minutes; and red guava was blended with water at a 1:1 ratio. the total phenolic content was analyzed using the folin-ciocalteu method, while antioxidant activity was evaluated through the use of the dpph method. finally, sensory evaluation was conducted employing a hedonic test, which was completed by 100 untrained panelists. the optimal formula obtained was 50% telang flower extract, 30% red guava juice, 15% ginger emprit extract, and 5% collagen hydrolysate (desirability value 0.8288), resulting in antioxidant activity of 316.45 mg te/100 ml and total phenolic content of 247.33 mg gae/l. the sensory evaluation revealed that the subject demonstrated moderate acceptance, as indicated by an average preference score of 3.51. the combination of bay flower extract and guava juice exhibited a synergistic effect in enhancing antioxidant activity, while guava juice contributed significantly to the total phenolic content. notwithstanding the favorable reception, the robust ginger flavor, elevated viscosity, diminished clarity, and textural characteristics. keywords: functional drink; collagen; butterfly pea; red guava; antioxidant. introduction the global market for functional beverages is already experiencing substantial growth. the phenomenon under consideration occurred in conjunction with an increase in consumer awareness regarding the maintenance of a healthy lifestyle. functional beverages have emerged as the most rapidly expanding product category within the food and beverage sector, with a primary objective of enhancing bioactive compound bioavailability (gupta et al., 2023). as new products continue to be developed and the stability and availability of bioactive products increases, market projections indicate that functional drinks will experience continuous growth (nazir et al., 2019) development and consumption of functional beverages have gained significant importance in addressing consumer demand for healthy products, with the potential to enhance public health, quality of life, and reduce the risk of degenerative diseases (carvalho et al., 2023). the ingestion of functional beverages fortified with antioxidants, fiber, and other bioactive compounds has been demonstrated to play a pivotal role in the mitigation of risk factors associated with various health complications and accelerated aging (aron, 2019). the protective mechanisms underlying this effect include the alleviation of oxidative stress, the regulation of blood sugar levels, the enhancement of lipid profiles, and the reduction of inflammation (yilmaz-akyuz et al., 2019) the development of functional beverages with optimal physicochemical and organoleptic characteristics necessitates a comprehensive scientific approach. the theory of functional beverage formula optimization utilizes a mixture design method (hassanzadeh et al., 2022). the interaction of hydrolyzed collagen with other bioactive compounds has been demonstrated to enhance thealth benefits (bilek & bayram, 2015) as demonstrated in prior research, the incorporation of hydrolyzed collagen into fruit juice beverages has been observed to enhance the protein content and bioavailability of these beverages. additionally, it has been observed to modify the levels of ascorbic acid, total https://doi.org/10.14421/biomedich.2025.142.1021-1028 1022 biology, medicine, & natural product chemistry 14 (2), 2025: 1021-1028 phenols, and antioxidant capacity within these functional beverages (bilek & bayram, 2015). most studies focus on collagen drinks made from a single ingredient or a combination of ingredients. however, the combination of butterfly pea flowers and guava has the potential to produce functional collagen drinks with unique physical characteristics, formulas, and sensory qualities. the goal of this study is to determine the physical characteristics, optimal formula, and organoleptic quality of functional collagen beverages made from a mixture of butterfly pea flowers, guava and emprit ginger. adding collagen to guavaand butterfly pea flower-based beverages has the potential to enhance the product's nutritional value and sensory characteristics. materials and methods procedures this research was conducted at food nutrition laboratory and sensory laboratory of faculty of agricultural technologym university of brawijaya. the butterfly pea flowers were collected from the obet plantation in malang. red guava and emprit ginger were purchased from the gadang market in malang. marine collagen was purchased from pt. maxcorp global indonesia. liquid stevia was purchased from berlico indonesia. all chemical reagents (dpph, folinciocalteu, sodium carbonate, distilled water, and analytical-grade methanol) were purchased from a local drugstore in malang. the tools used was knufe, alumunium tray, blender, hotplate stirrer, waterbath shaker, homogenizer, erlenmeryer, spaltula, test tube, measuring pipette, ohaus analytical balance, and cabiner dryer. figure 1. butterfly pea flower (clitoria ternatea). sample preparation the butterfly pea flowers were cleaned and dried at 40°c for 24 hours in a cabinet dehydrator. after drying, the butterfly pea flowers were ground using a grinder to increase the surface area. the ground butterfly pea flowers were extracted using drinking water at 60°c for 30 minutes at a ratio of 3 g/l using a hot plate stirrer to form butterfly pea extract (duy et al. 2020). emprit ginger was sliced (1 mm) and dried (60°c, 24 hours) in a dehydrator. the dried emprit ginger was ground and extracted using drinking water at 55°c for 15 minutes to form emprit ginger extract. red guava fruits were blended with water at a ratio of 1:1 (w/v) and filtered with a filtration cloth. the butterfly pea extract, emprit ginger extract, and guava juice were analyzed for total phenolic content and antioxidant activity. collagen powder was weighed, then dissolved in water at a ratio of 1:2 (w/v). functional drinks were made by mixing butterfly pea flower extract, guava juice, ginger extract, and collagen based on the formula design. then, they were homogenized using an ultra-turrax homogenizer at 1800 rpm for 10 minutes and pasteurized at 77°c for 2 minutes. each formula was analyzed for total phenolic content and antioxidant activity. formula optimization. an extreme vertex design was used with minitab version 19 to determine the optimal formulation for a functional butterfly pea drink. this design is often used in mixture experiments where constraints are applied to each factor. in this study, the whole design was replicated twice, and the total amount of each component was set to 100% for flexibility. this model resulted in 18 runs, each of which produced a composite (table 1). each run was analyzed for total phenolic content and antioxidant activity. finally, all 18 runs were used to empirically determine the optimum formula using the response optimizer in minitab version 19. total phenolic content determination total phenolic content was determined as a result of formula optimization according to the folin-ciocalteu method described by singleton et al. (1999), with slight modifications. each formulation was diluted with a dilution factor of 12.5 (2 ml of the sample was pipetted and diluted to a 25-ml volumetric flask) using distilled water. then, 0.5 ml of the diluted solution was pipetted into a test tube, and 4 ml of folin-ciocalteu reagent was added. then, 5 ml of a 7.5% sodium carbonate solution was added. the solution was incubated in a dark room for 30 minutes. antioxidant activity determination the antioxidant activity of fbd was determined using the dpph (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity method according to thaipong et al. (2006), with slight modifications. after centrifugation at 6,000 rpm for 5 minutes, 2 ml of the supernatant was transferred to a test tube. then, 2 ml of 0.2 mm dpph in methanol were added. the mixture was incubated for 30 minutes in the dark. the absorbance of each sample nabilah et al. – formula optimization and sensory analysis of functional collagen drink 1023 was determined using a uv-vis spectrophotometer at a wavelength of 517 nm. the antioxidant activity was expressed as milligrams of trolox equivalent per liter using a trolox standard curve (0, 5, 10, 15, 20, and 25 ppm) in methanol. sensory evaluation the selected formulations were subjected to a hedonic test based on preferences for fishy odor, ginger aroma, fruity flavor, sour flavor, sweetness, ginger flavor, synthetic flavor, viscosity, solubility, sandy texture, and overall liking and acceptability. the attributes were rated from 1 to 5 (1 = dislike very much, 2 = dislike, 3 = quite like, 4 = like, and 5 = like very much) based on the panelist preferences for each attribute using 100 untrained panelists (male-to-female ratio unknown). data analysis one-way analysis of variance (anova) with a 95% confidence interval was used to identify differences between treatments in minitab 19. if the results showed significant differences, a post hoc test was carried out using the tukey pairwise comparison test to determine which pairs of group means were significantly different from each other. results and discussion bioactive composition of single ingredients the antioxidant activity and total phenolic content of the individual ingredients were analyzed to establish a baseline for further formulation of a functional butterfly pea drink with collagen (fbdc). as demonstrated in table 1, ginger extract demonstrated the most significant antioxidant activity (317.36 ± 3.00 mg te/100 ml), followed by butterfly pea flower extract (136.59 ± 1.76 mg te/100 ml) and red guava juice (61.64 ± 2.38 mg te/100 ml). table 1. bioactive composition of single ingredients. sample antioxidant activity (mg te/ 100 ml) tpc (mg gae/l) blue pea flower extract 136,59 ± 1,76 248,801 ± 5,01 emprit ginger extract 317,37 ± 3,00 235,920 ± 0,86 red guava juice 61,64 ± 2,38 411,677 ± 4,35 bioactive composition of individual runs the optimization analysis of 18 formulations designed using minitab 19 software is presented in table 2. the findings revealed variations in antioxidant activity, ranging from 232.24 to 395.23 milligrams of trolox equivalent (te)/100 milliliters, and in total phenolic content, ranging from 141.28 to 260 milligrams of gallic acid equivalent (gae)/liter. these variations are indicative of the impact of varying ingredient proportions on the observed responses. in order to assess the relative contributions of the various components, a dual approach was utilized, encompassing both statistical modeling through regression analysis and empirical methods facilitated by mixture design visualizations. table 2. bioactive composition of individual runs for functional butterfly pea drink with collagen. formula butterfly pea extract (%) red guava juice (%) emprit ginger extract (%) collagen hydrolysate (%) antioxidant activity (mg te/100 ml) tpc (mg gae/l) f1 50,25 28,25 15,25 6,25 378,193 190,084 f2 50,00 30,00 15,00 5,00 309,104 260,444 f3 50,25 29,25 15,25 5,25 290,036 203,702 f4 50,50 28,50 15,50 5,50 367,632 221,860 f5 50,25 28,25 16,25 5,25 235,208 227,534 f6 50,00 28,00 15,00 7,00 357,222 203,135 f7 52,00 28,00 15,00 5,00 321,232 184,978 f8 50,25 29,25 15,25 5,25 343,062 254,202 f9 52,00 28,00 15,00 5,00 315,099 222,427 f10 51,25 28,25 15,25 5,25 348,740 152,635 f11 51,25 28,25 15,25 5,25 335,128 196,893 f12 50,00 30,00 15,00 5,00 320,131 234,910 f13 50,00 28,00 17,00 5,00 338,141 162,848 f14 50,25 28,25 15,25 6,25 362,640 175,331 f15 50,25 28,25 16,25 5,25 232,340 162,848 f16 50,00 28,00 17,00 5,00 281,936 169,657 f17 50,00 28,00 15,00 7,00 395,237 164,551 f18 50,50 28,50 15,50 5,50 376,839 141,287 1024 biology, medicine, & natural product chemistry 14 (2), 2025: 1021-1028 optimization of composite the optimization of a functional beverage formulation using butterfly pea flower extract, red guava juice, ginger extract, and collagen hydrolysate resulted in an optimal formula with a ratio of 50:30:15:5, respectively. this formulation achieved a desirability value of 0.8288, indicating its alignment with the ideal conditions for maximizing antioxidant activity and total phenolic content. as demonstrated in figure 2, the recommended formula yielded 316.45 milligrams of total antioxidant activity (te) per 100 milliliters and 247.33 milligrams of gallic acid equivalent (gae) per liter of total phenolic content. figure 2. desirability plot of butterfly pea flower extract, red guava juice, emprit ginger extract, and hydrolysate collagen to antioxidant and tpc. effect of optimization process on mixture components regression coefficients for antioxidant activity and total phenolic content (tpc) responses are presented in table 3. a linear regression model was selected due to statistically significant p-values (p < 0.05) for both responses and a non-significant lack of fit (p > 0.05) for tpc. although the antioxidant response showed significant lack of fit, higher-order models did not improve the model significantly due to high data variability. the r² values were 43.37% for antioxidant activity and 44.86% for tpc, indicating moderate predictive capability. antioxidant activity = 3,667a – 0,248b – 10,579c + 22,063d tpc = –4,103a + 24,324b – 14,997c – 10,458d note: a = butterfly pea flower extract (%); b = red guava juice (%); c = emprit ginger extract (%); d = collagen hydrolysate (%) antioxidant response analysis the effect of butterfly pea flower extract, red guava juice, and ginger extract on antioxidant activity was analyzed under constant collagen concentration (5%). the findings demonstrated that considerable proportions of butterfly pea extract attained increases in antioxidant activity, surpassing 320 mg te/100 ml. this hypothesis was corroborated by the graphical surface response and contour plots, which visually identified the optimal antioxidant response in formulations with higher concentrations of butterfly pea extract, as shown in the surface and contour plots in figure 3. figure 3. surface plot antioxidant (left), contour plot antioxidant (right) of f. nabilah et al. – formula optimization and sensory analysis of functional collagen drink 1025 total phenolic content response analysis the formulation of a functional beverage using butterfly pea flower extract, red guava juice, and ginger extract was evaluated to determine its effect on total phenolic content, with the collagen concentration fixed at 5%. the optimization process revealed that red guava juice significantly contributed to the increase in total phenolic content, particularly when used in higher proportions. this trend is manifested by elevated surface regions and darker green areas in the contour plot, indicating total phenolic values exceeding 240 milligrams of gallic acid equivalent per liter (figure 4). figure 4. surface plot total phenolic content (left), contour plot total penoloc content (right). sensory evaluation the hedonic test results for the fbdc, fbdnc, and commercial product samples are presented in table 3. table 3. result of sensory evaluation for fbdc, fbdnc, dan commercial product. sensory atributes fbdc fbdnc commercial p-value ginger aroma 3.66 ± 0.87a 3.50 ± 0.96ab 3.21 ± 1.31b 0.003 fishy odor 3.50 ± 0.84a 3.58 ± 0.82a 3.54 ± 1.13a 0.817 ginger flavor 3.00 ± 1.17b 2.77 ± 1.13b 3.36 ± 1.19a 0.000 synthetic flavor 2.94 ± 0.91a 2.92 ± 0.93a 3.17 ± 1.20a 0.096 fruity flavor 3.78 ± 0.70a 3.86 ± 0.65a 3.98 ± 0.99a 0.205 sweetness 3.83 ± 0.72a 3.61 ± 0.81a 3.86 ± 1.10a 0.086 sourness 3.58 ± 0.62a 3.50 ± 0.71a 3.74 ± 0.82a 0.060 viscosity 3.40 ± 0.86b 3.50 ± 0.79b 4.06 ± 0.86a 0.000 clarity 2.93 ± 0.81b 3.02 ± 0.85b 4.44 ± 0.65a 0.000 sandy texture 3.10 ± 0.90b 3.23 ± 0.85b 3.94 ± 1.09a 0.000 overall liking 3.51 ± 0.61b 3.33 ± 0.72b 3.85 ± 0.92a 0.000 overall accepting 3.54 ± 0.64b 3.39 ± 0.68b 4.06 ± 0.87a 0.000 note: 1. fbdc : functional butterfly pea drink with collagen; fbdnc: functional butterfly pea drink without collagen 2. hedonic scale: 1-5 (very dislike – very like) discussion the antioxidant activity of emprit ginger was the highest among the ingredients, at 317.36 ± 3.00 mg te/100 ml (table 1). however, despite its high individual antioxidant activity, the addition of ginger did not always lead to an increase in the overall antioxidant activity of the product. as shown in figure 1, the combination of butterfly pea flower extract and red guava juice resulted in a significant increase in antioxidant activity in the final product, while the inclusion of emprit ginger extract tended to decrease the antioxidant response, as evidenced by the decrease in elevation in figure 1 (b). this suggests that the antioxidant compounds in ginger do not consistently interact synergistically with other ingredients, as one might expect based on its individual antioxidant activity.the conclusions of the study may be presented in here. a study by singprecha et al. (2020) further supports this observation, showing that the interaction between ginger extract and ascorbic acid can result in an antagonistic effect. ascorbic acid, when in liquid form, is easily oxidized, generating prooxidants that can react antagonistically with the phenolic compounds in ginger, such as gingerol and shogaol, thereby reducing their antioxidant activity. on the other hand, the interaction between butterfly pea flower extract and red guava juice appears to produce a synergistic effect, significantly enhancing the antioxidant activity of the product. this finding is consistent with the research of tando et al. (2023), who also reported synergistic effects between 1026 biology, medicine, & natural product chemistry 14 (2), 2025: 1021-1028 butterfly pea and red guava in increasing the antioxidant activity of ice cream products. while the addition of red guava juice positively influenced the antioxidant activity, the effect was not as optimal as expected due to the degradation of anthocyanins caused by the ascorbic acid in the mixture, which negatively impacted antioxidant activity (farr & gusti, 2018). despite this, the polyphenolic compounds in red guava juice contributed significantly to the total phenolic content, highlighting the complex interactions between ingredients that enhance antioxidant activity. in terms of total phenolic content (tpc), red guava juice exhibited the highest concentration among the materials, with a total tpc of 411.677 ± 4.35 mg gae/l (table 1). figure 2 further illustrates that the addition of red guava juice, particularly in combination with butterfly pea flower extract, resulted in the highest tpc in the product. although the optimal phenolic content tended to decrease due to heat exposure during pasteurization (77°c), the combination of red guava juice and other ingredients showed a synergistic effect, boosting the tpc of the final product. research by diep et al. (2020) suggests that the presence of polyphenol compounds can enhance the utilization of vitamin c, which may explain the observed increase in tpc when red guava juice is combined with butterfly pea flower extract. red guava has been reported to contain around 57 identified phenolic compounds, including phenolic acids, tannins, flavonoids, gallic acid, catechins, and proanthocyanidins (castaneda et al., 2023). the synergistic effect observed in the product is linked to the heterogeneity of polyphenolic compounds in red guava juice, which tends to increase total phenolic content (singprecha et al., 2020). however, further research is needed to fully understand the underlying mechanisms of these interactions. additionally, the addition of butterfly pea flower extract also had a positive effect on increasing tpc, as the pectin content in red guava helps to stabilize anthocyanins (zhao et al., 2021). in contrast, ginger extract did not show a significant effect on tpc. although ginger contains phenolic compounds such as gingerol and shogaol, the interaction with ascorbic acid appeared to have an antagonistic effect, reducing the effectiveness of antioxidants in the mixture (singprecha et al., 2020; colon & nerín, 2016). the formulation of each ingredient plays a crucial role in determining both antioxidant activity and tpc of the product. the balance of each ingredient’s proportion significantly influences the chemical reactions that occur during formulation. as illustrated in figure 3, the optimum formula for the product was achieved with a ratio of 50% butterfly pea flower extract, 30% red guava juice, 15% emprit ginger extract, and 5% collagen hydrolysate. although an increase in total phenolic content was observed, it was not always accompanied by a proportional increase in antioxidant activity. this indicates that total phenolics, while useful as an indicator of antioxidant potential, are not the sole contributors to antioxidant activity. non-phenolic antioxidants also play an important role in the overall antioxidant response. this observation is supported by the research of singprecha et al. (2019), which highlights the effects of interactions between non-phenolic compounds and ascorbic acid on the antioxidant activity of the product. furthermore, the presence of collagen hydrolysate enhances antioxidant stability due to the presence of hydrophobic amino acid residues, particularly proline, which contain aliphatic hydrocarbon side chains that can stabilize the interaction between peptides and free radicals (deng et al., 2023). this aligns with the findings of leon-lopez (2020), who showed that collagen addition increases the bioavailability and nutritional value of products, particularly enhancing antioxidants and proteins. this study was then followed by organoleptic testing through hedonic testing on 100 untrained panelists to evaluate the overall level of product acceptance.this study involved organoleptic testing through hedonic evaluation with 100 untrained panelists to assess overall product acceptance. the anova test results indicate that significant differences were observed for the ginger aroma, ginger flavor, viscosity, clarity, and sandy texture attributes, while no significant differences were found for fishy odor, synthetic flavor, fruity flavor, sweetness, and sourness. for ginger aroma, fbdc demonstrated the highest preference (3.66), followed by fbdnc (3.50), and the commercial product (3.21), suggesting that panelists preferred the ginger aroma in fbdc. the addition of collagen hydrolysate likely influenced this preference, enhancing the aroma as collagen proteins are known to bind volatile and flavor compounds (wang et al., 2022). fishy odor did not show significant differences across the samples (p>0.05), likely due to the masking effect of ginger extract and red guava, which suppressed the fishy odor from the collagen hydrolysate (biewirth et al., 2015). ginger flavor exhibited significant differences, with the commercial product rated highest (3.36), despite not containing ginger, suggesting panelists' aversion to the strong ginger flavor in fbdc (3.00) and fbdnc (2.77), potentially due to the high concentration of ginger extract (20%) contributing to an intense, less preferred spicy taste (marsigit et al., 2019). for attributes showing no significant differences, the synthetic flavor was similarly perceived across all samples (p>0.05), likely due to stevia's aftertaste, which can be perceived as artificial (bhardwaj et al., 2020). fruity flavor also showed no significant preference difference (p > 0.05), with panelists finding the fruity flavors from red guava (fbdc and fbdnc) and peach (commercial product) equally refreshing. additionally, sweetness and sourness were comparable across all samples (p>0.05), suggesting that stevia provided similar nabilah et al. – formula optimization and sensory analysis of functional collagen drink 1027 sweetness and that the more pronounced sourness in the commercial product did not affect preferences. significant differences were observed for viscosity, with the commercial product being preferred (4.06), suggesting a preference for lower viscosity. fbdc (3.40) and fbdnc (3.50) had higher viscosity, likely due to fiber, pectin, and collagen, which increase viscosity (rigoto et al., 2018). collagen hydrolysate is also known to increase viscosity, albeit to a lesser extent than native collagen (león-lópez, 2020). clarity also showed significant differences, with the commercial product rated highest (4.44), likely due to lower dissolved solids and fewer suspended particulates. sandy texture also exhibited significant differences, with the commercial product preferred (3.94), while fbdc (3.10) and fbdnc (3.23) were less favored, likely due to larger particle sizes in these two samples, which are perceived as sandy (breen et al., 2019). overall, fbdc received moderate acceptance, with an average preference score of 3.51 ("quite like") and an acceptance score of 3.54 ("quite like"). these findings suggest that fbdc was generally well accepted, particularly with respect to attributes such as ginger aroma, fishy odor, synthetic taste, fruity flavor, sweetness, and sourness. however, factors such as the strong ginger flavor, higher viscosity, reduced clarity, and sandy texture contributed to lower preference scores when compared to the commercial product, which scored higher in these attributes. conclusion based on this research, there was several key findings concerning the development of collagen functional beverages derived from butterfly pea flower and red guava. initially, emprit ginger extract exhibited the highest antioxidant activity, followed by butterfly pea flower extract and red guava juice. however, the combination of butterfly pea flower extract and red guava juice exhibited a substantial synergistic effect in enhancing the antioxidant activity of the product. conversely, the incorporation of emprit ginger extract tended to diminish the antioxidant response. secondly, red guava juice exhibited the highest total phenolic content and contributed significantly to the augmentation of the product's total phenolic content, particularly when combined with telang flower extract. the optimal formula of the product was achieved with a ratio of 50% telang flower extract, 30% red guava juice, 15% emprit ginger extract, and 5% collagen hydrolysate. while this study makes a significant contribution to the field, it is imperative to acknowledge its limitations. the study's methodological limitations encompass the utilization of untrained panelists in sensory evaluation and the constraints in data availability concerning specific interactions between phenolic and non-phenolic compounds in products. moreover, the present study was constrained by its focus on a specific combination of raw materials, precluding an investigation into alternative raw materials. in light of these limitations, future research endeavors may focus on the utilization of trained panelists for more precise sensory evaluation, further 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universitas islam negeri sunan kalijaga yogyakarta jl. marsda adisucipto no 1 yogyakarta 55281, indonesia. tel. +62-274-540971, fax. +62-274-519739 corresponding author trio.yonathan@gmail.com manuscript received: 26 september, 2019. revision accepted: 14 april, 2020. published: 17 april, 2020. abstract the metal casting industry, nitikan is a micro business whose production process is carried out manually. the equipment used is simple and without considering the user's health. this can be seen in the product finishing process that uses a sanding machine. the equipment is not designed according to the user's posture so the operator bends in doing work movements, of course this is very risky to experience lower back pain injuries. this study aims to analyze the work activities of the finishing process using the rapid entire body assessment (reba) and rapid upper limb assessment (rula) approach and design work tools that can be applied to improve work posture. the results of data processing and analysis obtained a rapid entire body assessment score on a sandpaper machine single head of 5 and a rapid upper limb assessment score of 4, the score indicates that the work position has the potential to experience a risk of low back pain, so there must be an improvement in work posture. work posture improvement is done by designing a tool in the form of a sandpaper machine that takes into account ergonomic dimensions to reduce the risk of lower back injury. the final result after the improvement is obtained rapid entire body assessment score of 3 and rapid upper limb assessment score of 4. this shows that the risk of lower back injuries can be minimized. keywords: rapid eentire body assessment; rapid upper limb assessment; low back pain introduction the metal casting industry, nitikan is a micro business which can be said to be the entire production process carried out by human labor. the equipment used is simple and without considering the user's health. this can be seen in the product finishing process that uses a sanding machine. the equipment is not designed according to the user's body posture so operators bend in doing work movements, of course this is very risky to experience low-back pain injuries. in addition to injuries, the process was fairly long because it took 11 minutes. this process is somewhat longer than the other processes which on average only takes 5 minutes. thus it is necessary to anticipate this, business owners need to pay attention to the comfort of their workers so that work health and safety are guaranteed. this can be done by adjusting workers with work methods, work processes and the work environment. this approach is known as the biomechanical approach which in this context is reba and rula analysis. this analysis serves to analyze the work posture is not good and the risk of low-back pain. the analysis then becomes the basis for providing solutions in the form of work postures in the form of tools to reduce these risks. by implementing this it is expected to minimize the risk of low-back pain injuries and to increase process productivity. materials and methods rapid upper limb assessment (rula) rapid upper limb assessment (rula) is the method used to estimate the risk of injury to the upper body such as the upper arm (forearm), forearm (wrist) and wrist (wrist rotation), wrist (neck), torso. by using rula, fast and systematic results will be obtained in determining postural risk of workers (mcatamney & corlett, 1993). the rula measurement method has several main applications such as: 1. measuring the risk of musculoskeletal (muscle), usually part of a wider increase in ergonomics. 2. comparing muscle loads (muscles) between current and modified work station designs. 3. evaluate results such as productivity or suitability for equipment use. https://doi.org/10.14421/biomedich.2020.91.21-25 22 biology, medicine, & natural product chemistry 9 (1), 2020: 21-25 4. train workers about muscle loads (muscles) caused by differences in work posture. posture with the rula method is divided into two parts, namely group a posture consisting of the upper arm (upper arm), forearm (forearm), wrist (wrist) and wrist rotation (wrist rotation); and group b posture consists of the neck, body and legs. the final results obtained from the two final scores for group a and group b are combined, the combined score results from group a and group b are classified into several categories. the development in the rula method consists of three stages, namely: 1. selection of work posture for assessment or measurement. 2. development of an assessment system. 3. the development of a large score is converted to one of four levels of action. figure 1. rula analysis sheet (source: mc atamey & corlett, 1993). rapid entire body assessment (reba) rapid entire body assessment (reba) is an ergonomic method (hignett & mcatamney, 2000) developed to measure the types of postures that pose a risk to health. reba assesses work positions on body postures such as the neck, back, arms, back of the hands and feet of the operator. reba depends on the coupling factor, external loads supported by the body and worker's activities. determination of reba score manually, discussing the level of risk of work posture, starting with determining the a score for posture of the body, neck and legs plus the load score (load) and the b score for the posture of the upper arm, forearm and the hand is added with a score clutch. both scores are used to determine c scores. reba scores are obtained by adding activity scores to c scores from reba values to determine the level of risk of injury. measurement of body posture using the reba method has six stages including: 1. observation of body posture can be done by taking workers' posture data using video or photo assistance. 2. selection of posture that must be done. 3. assessment of body posture score. 4. the score calculation process. 5. determine the results of the reba assessment for the owned posture. 6. check the results of the reba assessment according to the level of action to make improvements or changes. the reba method can be used when an assessment of ergonomic work posture identifies the need for further analysis. the things that need to be done are: 1. all body parts used. 2. static, dynamic, fast changing or unstable posture. 3. modification of workplaces, equipment, training, and monitoring of worker risk behavior. results and discussion the object of research was carried out in the sandpaper process section. the sanding process is carried out in 2 stages, namely coarse sanding and fine sanding. because it only has one sanding machine, in doing these 2 steps, it has to replace the sanding eye, this causes the processing time to be longer. another problem is the design of the sandpaper machine that does not consider the health of workers, causing workers to have to bend for a long time, causing lower back pain. figure 2. sandpaper machine operator posture. table 1. reba angle dimensions for sanding machine operators. grup dimensions angle a neck 29 back 19 knee kaki 87 load 0 b upper arm 39 lower arm 38 wrist 32 kusuma – analysis of body posture using rapid entire body assessment … 23 table 2. reba score on the sanding machine operator's work posture. group dimensions a n g le s co re table a score a score c grade score reba a neck 29o 2 4 4 4 5 back 19o 3 knee 87o 1 load 0 group dimensions a n g le s co re table b score b b upper arm 39o 2 3 3 lower arm 38o 2 wrist 32o 2 coupling 0 activity score 1 for the number of scores in table a, a result of 3 is obtained and added to a load score of 0 because the load weight is less than 5 kg. then the a score has a score of 0 because the load weight is less than 5kg. then the score a has a score of 4. from each angle of group b the table b score is generated that is equal to 3 and coupled with a coupling score of 0 because the grip is right and in the middle so that the grip on the load is strong. then the resulting b score is 3. based on the a score and the b score, it can be seen the value of the c score is 4. to get the reba score, by adding up the c score of 4 with an activity score of 1, the result score is obtained 5. based on the risk table it is known that reba score of 5 indicates a moderate level of risk and work position needs to be improved. table 3. rula angle dimensions for sanding machine operators. grup dimensions angle a upper arm 39 forearm 38 wrist 32 twist 0 b neck 29 back 19 feet 87 based on data processing using rula, then the results obtained by 3 and then coupled with muscle and energy scores. muscles have a score of 1 and a score of energy usage of 0 due to occasional loading or energy of less than 20kg and retained. so as to get a c score in group a of 4. while in group b the results obtained in table b are as big as 3 then added with muscle and energy scores. muscles have a score of 1 and a score of energy use of 0 due to occasional loading or less than 20kg of energy and retained. so as to get a d score in group b of 4. based on the scores of c and d scores obtained, the rula value was obtained. for the c score in group a obtained by 4 and the d score in group b obtained a result of 4. by looking at the grand score table based on the c and d scores, a rula score is 4. the score indicates that changes need to be made, figure 2 shows the operator’s body position is less comfortable. table 4. rula score of sanding machine operator posture. group dimensions angle score table a score c score rula a upper arm 39o 2 3 4 4 fore arm 38o 2 wrist 32o 3 twist 1 muscle 1 power 0 group dimensions angle score table b score d b neck 29o 3 3 4 back 19o 2 feet 87o 1 muscle 1 power 0 improvement the use of a sanding machine was done by sitting in a chair. dimension is done only on the front side of the sanding machine, because it is adjusted to the use. the engine dimension data of the ideal chair dimensions were adapted in kusumawati’s research (2011). figure 3. sanding machine design improvements. 24 biology, medicine, & natural product chemistry 9 (1), 2020: 21-25 figure 4. the operator's body position angle. table 5. reba angle dimensions for sanding machine operators. grup dimensions angle a neck 9 back 23 knee 110 load 0 b upper arm 37 forearm 77 wrist 34 table 6. reba score on the sanding machine operator's work posture. group dimensions a n g le s co re table a score a score c score reba a neck 9o 1 2 2 2 3 back 23o 3 knee 110o 1 load 0 group dimensions a n g le s co re table b score b b upper arm 37o 2 2 2 lower arm 77o 1 wrist 34o 2 coupling 0 activity score 1 table 7. rula angle dimensions for sanding machine operators. group dimensions angle a upper arm 37 lower arm 77 wrist 34 twist 0 b neck 9 back 23 feet 110 based on a and b score, it can be seen the value of the c score is equal to 2. to get the reba score, by summing the results of the c score that is equal to 2 with an activity score of 1, then the results obtained score of 3. table 8. rula score of sanding machine operator posture. group dimensions angle score table a table c score rula a upper arm 37o 2 3 4 4 fore arm 77o 1 wrist 34o 3 twist 1 muscle 1 power 0 group dimensions angle score table b score d b neck 9o 1 3 4 back 23o 3 feet 110o 1 muscle 1 power 0 based on data processing using rula, the c score in group a is 4 and the d score in group b is 4. by looking at the grand score table based on c and d scores, the rula score is 4. conclusion from the analysis and results of the improvements obtained, it can be concluded; the sandpaper machine operator's posture has a reba score of 3 with a level 1 action with a low level of risk, whereas the rula score has a score of 4 with action level 2 with moderate risk level. this result shows that the design of sandpaper machine improvement is effective in reducing the risk of injury to low back pain operators, and can also speed up the processing time in its use without the need to change of the coarse and fine sandpaper eyes. references hignett, s. and mcatamney, l. (2000) rapid entire body assessment (reba). applied ergonomics, 31, 201-205. kusumawati, intan. 2011.usulan perancnagan ulang meja kursi baca berdasarkan aspek fungsi dan kenyamanan sesuai kebutuhan pengguna perpustakaan (studi kasus di kantor arsip dan perpustakaan kabupaten klaten). surakarta: universitas sebelas maret surakarta. lueder, r., (1996), a proposed rula for computer users, procceding of the ergonomic summer workshop, san francisco. kusuma – analysis of body posture using rapid entire body assessment … 25 mcatamney, l. and corlett, e.n., (1993), “rula: a survey based method for the investigation of work related upper limb disorders”, applied ergonomics, 24(2).91-99. prasetyo. (2012). fasilitas kerja dengan perbaiakan postur kerja pada aktivitas manual handling menggunakan analisis metode rapid entire body assessment (reba) dan ovako working posture anlysis system(owas) studi kasus perusahaan tempo pedro yogyakarta. yogyakarta: uin sunan kalijaga yogyakarta. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 215-224 | doi: 10.14421/biomedich.2023.121.215-224 issn 2540-9328 (online) effects of ouabain in ehrlich tumor development in vitro and in vivo amanda costa ayres salmeron1, maria beatriz calado2, mateus da silva matias antunes3,*, gabriel rodrigues da silva4, deyse cristina madruga carvalho4, beatriz fernandes de souza4, márcia regina piuvezam5, sandra rodrigues mascarenhas4 1edmond and lily safra international institute of neuroscience, santos dumont institute. macaíba, rio grande do norte, brazil; 2laboratory of immunopathology keizo asami, federal university of pernambuco, recife pernambuco, brazil; 3school of pharmaceutical sciences, university of são paulo. ribeirão preto, são paulo, brazil; 4biotechnology center, federal university of paraíba, joão pessoa, paraíba, brazil; 5health sciences center, federal university of paraíba, joão pessoa, paraíba, brazil. corresponding author* mateusmatias08@gmail.com abstract ouabain (oua) is a cardiotonic steroid with an immunomodulatory and anti-inflammatory role in different experimental models. currently, the potential antineoplastic effect of oua has been studied, however, research is needed to better understand oua role during tumor development. therefore, our aim was to investigate the oua effects on ehrlich tumor (et) development in vitro and in vivo. to evaluate the cytotoxic effects of oua on et in vitro the cells were incubated with different concentrations of oua during 24h and 48h and our results showed that only the [1000 μm] decreased the number and viability of et cells in the two analyzed times. to s tudy the oua effects on et in vivo, swiss mice were pretreated with 0.56 mg/kg of oua intraperitoneally (i.p.) for three consecutive days. to develop et in the solid form, one hour after the last day of pretreatment, et cells were inoculated subcutaneously into the footpad and the animals were monitored for 13 days. to develop the ascitic form, et cells were inoculated (i.p.) and the animals were monitored for 3 days. oua was able to reduce the thickness and weight of the tumor paw, in addition to reduce the weight of the popliteal lymph node. in the ascitic tumor, oua reduced the number of neutrophils and macrophages and increased the lymphocytes in the peritoneum. thus, we demonstrated that oua affects et development both in vitro and in vivo. our results suggest a new perspective in the anti-inflammatory, immunomodulatory and a possible anti-cancer role of ouabain and brings new concepts about the pathophysiological role of this substance. keywords: ehrlich tumor; inflammatory microenvironment; ouabain; solid tumor; tumor development. introduction cancer is characterized by disordered cell growth with the ability to metastasize and interfere with organs function (lópez-lázaro, 2018). the tumor development follows several complex steps, in which the cells gradually acquire a neoplastic phenotype (arneth, 2019). many tumors can alter and control the microenvironment, as a survival strategy, to interfere with the host immune response. one of the cancer hallmarks is the inflammatory microenvironment induced by tumor development (hanahan & weinberg, 2011). a better understanding of the inflammation role during tumor evolution and looking for new substances that can alter inflammatory parameters, such as cell migration and number of viable cells, can be a way to delay tumor development, allowing a longer time window of treatment and resulting in a better prognosis. (kimizgebologlu et al., 2018; wellenstein et al., 2019). ehrlich's transplantable tumor originated from a spontaneous mammary gland carcinoma in female mice and has been used widely for several studies on experimental oncology (havelek et al., 2017; safwat et al., 2020). like other transplantable tumors, this model is very useful since it is possible to determine and adjust the concentration of neoplastic cells to be inoculated and to quantify the growth and regression of the tumor. ehrlich tumor cells develop ascetically when the cells are injected intraperitoneally (fernandes et al., 2015) or solidly when the cells are injected subcutaneously or in the footpad (bahr et al., 2015). this tumor corresponds to an aggressive carcinoma with fast growth, which induces myelosuppression and affects the inflammatory response (brozovic et al., 2009), being a good experimental model for studying the interaction of different substances and the immune system in the tumor development (da mota et al., 2018; machado et al., 2017; moura et al., 2018). to search for new substances that can modulate inflammation and the immune system, and possibly delay the tumor development, ouabain (oua) has shown broader effects (cavalcante-silva et al., 2017; galvão et manuscript received: 19 january, 2023. revision accepted: 12 february, 2023. published: 16 february, 2023. https://doi.org/10.14421/biomedich.2023.121.215-224 216 biology, medicine, & natural product chemistry 12 (1), 2023: 215-224 al., 2017; jacob et al., 2013; leite et al., 2015; takada et al., 2009). different in vitro and in vivo models have demonstrated the modulation of cytokines by oua, establishing this molecule as a striking immunomodulator. however, the ability to interfere in immunologic functions is not restricted to the expression of cytokines, because oua also modulates the migration of inflammatory cells, inhibits the pathway of inflammatory mediators, and decreases edema (cavalcante-silva et al., 2017; rodrigues-mascarenhas et al., 2011). in addition, oua has been described as a substance capable of inhibiting neuroinflammation. moreover, in vitro studies have shown the antineoplastic potential of oua in different human tumor cell lines as lung a549 cancer cells, breast mcf7 cancer cells, prostate du 145 cancer cells, osteosarcoma u-2 os cells, melanoma a375 and sk-mel-28 cells, and glioma u‑87mg cells (chang et al., 2019; chou et al., 2018; wang et al., 2021; xiao et al., 2017; yang et al., 2018). modulating the immune system has proven to be one of the most important strategies to fight against tumors. while oua exerts a meaningful role on inflammation, these effects during tumor development have not been properly explored. therefore, to gain more insights about new compounds to control tumor growth, we investigate the effects of oua on ehrlich tumor cells in vitro and on animal experimental models of solid and ascitic tumors. materials and methods animals female swiss mice aged between six and eight weeks, weighing between 25 and 30 g, were used. the animals were kept in polypropylene cages at a temperature of 25 ± 1ºc. they were subjected to light/dark cycles of 12 hours and with free access to water and food throughout the experimentation period. the experimental protocols were submitted to the animal research ethics committee of the pharmaceuticals and medicines research institute/ufpb and were approved under number 093/2016. euthanasia of the animals in all experiments was done with ketamine/xylazine solution (ketamine 100 mg/kg and xylazine 10 mg/kg) injected intramuscularly (i.m.) followed by cervical dislocation. ehrlich tumor cells and in vitro culture cryopreserved samples of ehrlich tumor cells diluted in 1 ml of fetal bovine serum (fbs) + dimethyl sulfoxide 10% (dmso) were thawed and grown in 14 ml dulbecco's modified eagle's medium high glucose (dmem; sigma d1152), supplemented 2,0 g/l sodium bicarbonate, 1 mm/l sodium pyruvate (0,11g/l), 1% penicillin-streptomycin (gibco, usa) + 10% fbs. for experiments a concentration of 5x105 viable cells/ml were used. cell viability was determined by neubauer chamber counting using the trypan blue 0.4% (gibco, usa) exclusion method and an optical microscope (200x increase). after thawing, the cells were maintained in culture for 7 days and subcultures were performed every 2 days, in a 1:3 ratio. after the initial 7 days of cell maintenance and stabilization, the contents of the culture flask was centrifuged at 235 xg at 37°c for 4 minutes (hermle z 326 k centrifuge), the supernatant was discarded and the cells resuspended in 1 ml of dmem + 10% fbs. after, ehrlich tumor cells were seeded in 96-well plate at a concentration of 5x104 viable cells/well together with oua at the concentrations: 1 nm, 10 nm, 100 nm, 1 μm, 10 μm, 100 μm and 1000μm based on studies that describe the concentrations at which digitalis are capable of inhibiting the na+/k+ atpase pump (aizman et al., 2001; bortner et al., 1997; fontana et al., 2013; goto et al, 1992). for the control group phosphate-buffered saline solution (pbs) was used. the plates were incubated in a humid incubator at 37°c and 5% co2, protected from light. all tests were performed in triplicate and two different times were analyzed: 24 hours and 48 hours. procedures  cell viability evaluation and basal cytokine production after oua incubation, cell viability was evaluated by neubauer chamber counting using the trypan blue exclusion method and mtt assay. to perform mtt assay, the 96-well plates were centrifuged at 100 xg at 4°c for 6 minutes (hermle z 326 k centrifuge). the supernatant was discarded and 100 μl of dmem supplemented with 10% fbs + 10% mtt reagent (pbs + 0.5 g/ml mtt) was added to the wells. then the plate was incubated for 4 hours in a humid incubator at 37°c and 5% co2, protected from light. after the incubation time, 100 μl of sds 10% was added to the wells. the plates were under agitation, and protected from light, overnight. subsequently, the plates were analyzed on a spectrophotometer at a wavelength of 570 nm and the absorbance values of each sample were obtained. to measure the production of pro-inflammatory cytokines in vitro, the plates were centrifuged at 100 xg at 4°c for 6 minutes (hermle z 326 k centrifuge). the supernatant was collected, and the cytokine production was evaluated by sandwich enzyme-linked immunosorbent assay (elisa). the cytokines il-6, tnf-α and il-1β were quantified using the kit and protocol indicated by the manufacturer (bioscience, usa). the analysis was performed on a spectrophotometer, at a wavelength of 450 nm, where the absorbance values of each sample were obtained.  induction of ehrlich’s tumor in vivo cryopreserved samples of ehrlich tumor cells were thawed, diluted in pbs and inoculated intraperitoneal salmeron et al. – ouabain in ehrlich tumor development 217 (i.p.) at the concentration of 1x106 cells per animal in a volume of 300 µl, this process is called zero passage. after five days the animals were euthanized and collected the peritoneal lavage and performed the first passage, as described previously. after two passages in vivo, the cells were inoculated into the experimental animals. for experiments, 1x105 cells in a volume of 300 µl was inoculated via i.p. in mice for ascitic tumor model and 1x105 cells in a volume of 50 μl was inoculated subcutaneously in the footpad of the left paw for solid tumor model. in the solid ehrlich tumor model, the contralateral paw was used as a control for the group itself.  animal pretreatment protocol all the animals were pre-treated with pbs or oua at concentration 0.56 mg/kg i.p. for three consecutive days (jacob et al., 2013; leite et al., 2015; rodriguesmascarenhas et al., 2006). this dose of oua was chosen because this concentration corresponds to the levels found physiologically in the human organism (blaustein, 1993). moreover, for solid ehrlich tumor model, on the third day of pretreatment, 1 hour after the last pretreatment, 50 μl of pbs or ehrlich tumor cells were inoculated in the footpad of the left paw and 50 μl of pbs in the right paw. for ascitic ehrlich tumor model, 1 hour after the last pretreatment, 300 μl of pbs or ehrlich tumor cells were inoculated in the animal peritoneum.  solid ehrlich tumor model mice were randomly separated into three different groups: g1, g2 and g3. g1 group (n = 24) was the positive control and was pre-treated with 200 µl of pbs and ehrlich tumor cells were inoculated in the footpad of the left paw. g2 group (n = 24) was the experimental group pre-treated with 200 µl of oua and ehrlich tumor cells were inoculated in the footpad of the left paw. at least, g3 group (n = 20) was the negative control pre-treated with 200 µl of oua and pbs were inoculated in the footpad of the left paw. tumor mass thickness was measured using a digital micrometer (digimess, são paulo, brazil) one hour after inoculation, 24 hours later and thereafter every four days until the 13th day, when the animals were euthanized. after euthanasia, the popliteal lymph nodes of the right and left paw were removed using surgical forceps and the organs were weighed. the right and left foot pads were removed by dislocating the feet at the tibio-tarsal junction and were also weighed. subsequently, the lymph nodes were macerated in 6 ml of pbs. this suspension was centrifuged (200 xg, 4°c, 5 min), the supernatant discarded, and the pellet formed resuspended in 1 ml of pbs solution and cell count was performed using trypan blue exclusion method. for differential counting, 50 μl of cell suspension was centrifuged in a cytospin centrifuge serocite® fanem mod2400 crosshead ref.: 248.059.600 (1500 rpm for 10 min). then, the slides were stained with a panotic kit. after 24 hours of drying, the slides were analyzed using an optical microscope using immersion oil (1,000x increase). the slides were run in the tail region until the count of 100 cells.  ascitic ehrlich tumor model mice were randomly separated into three different groups: g1, g2 and g3. g1 group (n = 6) was the negative control and was pre-treated with 200 µl of saline and 1 hour after the last pretreatment, saline was inoculated. g2 group (n = 6) was the positive control group pre-treated with 200 µl of saline and 1 hour after the last pretreatment, ehrlich tumor cells were inoculated. at least, g3 group (n = 14) was the experimental group pre-treated with 200 µl of oua and 1 hour after the last pretreatment, ehrlich tumor cells were inoculated. the animals were daily weighed before, during the pretreatment, and after the inoculation of ehrlich cells. after 72 hours of tumor cells inoculation, all animals were euthanized, and the abdominal circumference was measured and calculated the tumor volume. after, using a 5 ml syringe, 3 ml of cold pbs was introduced into the peritoneum and the volume of ascitic fluid was aspirated. the samples were centrifuged at 200 xg 4°c for 5 minutes (hermle z 326 k centrifuge), the supernatant was discarded, and the cell pellet formed was resuspended in 1 ml of pbs and used for the perform total cell count and differential leukocyte count. differential cell counting was performed as described previously. data analysis statistical analyses were performed using prism® 6.0 software (graphpad, usa). agostino-pearson and shapiro wilk tests were performed to verify the gaussian distribution. when necessary, the data were normalized using the formula: y = [(y * 100 / 1.647)]. all results were analyzed using the one-way anova method for non-parametric data with tukey's post-test. values of p <0.05 were considered statistically significant. results and discussion oua decreases the number and viability of ehrlich tumor cells after 24 and 48 hours at 1000 μm to understand whether the oua has an influence on ehrlich's tumor cells growth, in vitro assays were performed. at 1000 μm of oua, ehrlich tumor cells reduced cell viability in 39.6% and 49.3% at both 24 and 48 hours, respectively, when assessed by the mtt test (figure 1a-b). moreover, by trypan blue exclusion method, oua has also been shown to be able to reduce the number of total cells in culture (figure 1c-d) and cell viability of at 1000 μm concentration after 24 and 48 hours, reducing viability in 15.8% and 28.1%, respectively (figure 1e-f). 218 biology, medicine, & natural product chemistry 12 (1), 2023: 215-224 figure 1. effect of ouabain on number and viability of ehrlich tumor cells in vitro. cell viability was tested by mtt after (a) 24 hours and (b) 48 hours of treatment with oua. cell viability was tested by trypan blue exclusion after (c) 24 hours and (d) 48 hours of treatment with oua. the total cell numbers were analyzed by trypan blue after (e) 24 hours and (f) 48 hours of treatment with oua. although, oua did not alter the basal production of tnf-α (g) and il-1β (h). the graphs a and b were normalized and plotted as the mean ± sem. the other graphs are plotted as the mean ± sem. all graphs were analyzed using the one-way anova for non-parametric data with tukey's multiple comparison post-test. ** p < 0.01 and **** p < 0.0001. oua does not alter basal cytokine production in ehrlich cells our results showed that ehrlich tumor cells produces a low level of basal cytokines tnf-α and il-1β. although, oua does not alter the basal production of the evaluated cytokines (figure 1g-h). moreover, our results did not detect the presence of il-6 in any group (data not shown). salmeron et al. – ouabain in ehrlich tumor development 219 oua delay solid tumor development in mice footpad on the fifth day, it was a significant increase in paw thickness between g1 and g3. on the 9th and 13th day, g2 group showed a reduction in the thickness of the paw with the solid tumor in relation to g1 (figure 2a). moreover, in g2 group, oua reduced the weight of the tumor paw (figure 2b) and increased the weight of the left popliteal lymph node (lymph node closest to the tumor site) in relation to g1 group (figure 2c). regarding the total cell count in the left popliteal lymph node, it was possible to observe that there was no significant difference between groups g1 and g2 (figure 2d). furthermore, the differential cell count indicated that was no significant difference in the pattern of cell migration between the groups analyzed (data not shown). figure 2. effect of ouabain pretreatment on tumor development of the solid ehrlich tumor in the footpad of mice. paw thickness with the tumor was measured on the day of ehrlich cell inoculation and 3, 5, 9 and 13 days after tumor inoculation (a). on the 13th day the animals were euthanized. the (b) paws and (c) popliteal lymph nodes on the right and left side were weighed. the total cell counting of lymph node maceration was also performed (d). the graphs are plotted as the mean ± sem. all graphs were analyzed using the one-way anova for non-parametric data with tukey's multiple comparison post-test. * p < 0.05, ** p < 0.01 and **** p < 0.0001. oua decreases neutrophils and macrophages migration to the tumor microenvironment and increases the migration of lymphocytes our results showed that there was no significant difference between g2 and g3 in relation to the gain or reduction in animal weight (figure 3a), abdominal circumference (figure 3b) and the ascitic fluid volume (figure 3c) of the animals. regarding the total cell count, there was no significant difference between g1, g2 and g3 (figure 3d), however, the cell profile of the peritoneal lavage was different between the groups. the g2 showed a 42.72% increase in the number of neutrophils, a 40.9% reduction in the number of lymphocytes and a 23.3% increase in the number of macrophages when compared to g1. it was observed that oua pretreatment reduced cell migration to the tumor microenvironment. the g3 had a reduction of 81% in the number of neutrophils and 55% in the number of macrophages when compared to the g2. on the other hand, the oua increased the quantity of lymphocytes by 182.17% also in relation to the g2 (figure 3e). 220 biology, medicine, & natural product chemistry 12 (1), 2023: 215-224 figure 3. effect of ouabain pretreatment on tumor development of the ascitic ehrlich tumor. (a) the animals were monitored and weighed during the 3 pretreatments (-48, -24 and -1h) and 72 hours after the inoculation of ehrlich tumor cells intraperitoneally. after 3 days of tumor growth, the animals were euthanized. (b) the abdominal circumference was measured and the peritoneal lavage (c) was analyzed by tumor volume, (d) cell viability and (e) differential leukocyte count. the graphs are plotted as the mean ± sem. all graphs were analyzed using the one-way anova for non-parametric data with tukey's multiple comparison post-test. * p < 0.05, ** p < 0.01 and **** p < 0.0001. discussion the development of malignant tumors and the inflammatory process are closely linked events. previous studies have shown that chronic inflammation can make individuals more susceptible to the progression of different tumors (diakos et al., 2014; korniluk et al., 2017). moreover, the tumor development interferes with the innate immune response altering production and secretion of signaling molecules, such as cytokines and chemokines, capable of promoting alterations in tissue structure, formation of new vessels or other events that assist in tumor progression (misra et al., 2018; taniguchi & karin, 2018; velloso et al., 2019). there are different strategies to try eliminating or delay tumor growth, thus, one of the areas of experimental oncology has been looking for, is substances that can control the inflammatory microenvironment during tumor development such as oua. first, to analyze the effects of oua treatment on ehrlich tumor cells, we evaluated cytotoxicity and proinflammatory cytokine basal production in vitro. the results of cell viability tests showed that only the highest concentration tested (1000 μm) was able to decrease cell viability of ehrlich tumor cells in both times analyzed, suggesting a dose-dependent response. this dosedependent response was also observed by other researchers who evaluated the cytotoxicity of five glycosides, including ouabain, in several human tumor cell lines (johansson et al., 2001). furthermore, at both times tested, the viable cell concentration was significantly lower in the 1000 μm concentration suggesting that oua has a cytotoxic effect on cells only at this concentration. regarding the mechanism of action that triggered the reduction of cell viability and concentration of cells in the 1000 μm group, we hypothesize two possibilities: the salmeron et al. – ouabain in ehrlich tumor development 221 first involves classic inhibition of the na+/k+ atpase pump, which can results in a reduction in the concentrations of k+ in the cytoplasm concomitant with higher ca2+ concentrations in the cell cytoplasm, triggering cell death by apoptosis, through the activation of caspases-3 (diakos et al., 2014); and the second possibility involves inhibiting the na+/k+ atpase pump, or altering its kinetics, increasing na+ concentrations along with a reduction in k+ concentration in the cell cytoplasm, favoring the polarization of the cytoplasmic membrane of the tumor cell. thus, cells with lower levels of glutathione, commonly seen in tumor cells, inhibit the depolarization of the cytoplasm membrane causing an increase of tyrosine kinase expression and ras protein, in addition to stimulating the production of superoxide ions, inducing the cell to apoptosis (valente et al., 2003). second, to assess the effects in vivo of oua pretreatment on the development of the ehrlich’s solid tumor, our data showed a reduction in the thickness of the paw with the solid tumor in oua pretreated animals. we observed a weight loss of the tumor paw and the left popliteal lymph node. other studies have already shown that oua can reduce vascular permeability and edema (leite et al., 2015; rodrigues-mascarenhas et al., 2009, 2011). thus, to investigate whether these observed reductions could be associated with alterations in the lymphatic organ drainage, we evaluated the total and differential cell count of the popliteal lymph node. it is interesting to note that although a reduction in the weight of the left lymph node is observed, whose paw received ehrlich cells together with the pretreatment with oua, there was no statistically significant change in the cell count and cell profile, although the graph shows a trend. the increase in the volume of lymph nodes is known as lymphedema and occurs when there is a functional overload of the lymphatic system, either due to hereditary causes or acquired through obstruction or injury caused by infectious processes, diseases, surgery, obesity or as a consequence of malignancies (grada & phillips, 2017; petrek et al., 2000). therefore, it is possible that the reduction in the lymph node weight of the animal pretreated with oua occurred due to the reduction of fluid leakage in the inflamed site, where the ehrlich cells were inoculated, and not due to the reduction in the number of cells in the popliteal lymph node. this is a hypothesis supported by previous data from our group that report the oua ability to reduce plasma exudate leakage in an experimental model of edema in mice (rodrigues-mascarenhas et al., 2011). regarding the ascitic tumor model, our study showed that pretreatment with the oua reduced the migration of cells from the innate immune system to the tumor microenvironment and it is one of the oua antiinflammatory mechanisms of the action. our group has already demonstrated the reduction of eosinophils migration in the experimental model of allergic pulmonary inflammation (galvão et al., 2017) and neutrophils in the experimental model of peritonitis (leite et al., 2015). in addition, the reduction of inflammatory cell migration may indirectly, delaying tumor progression (tsutsui et al., 2005). moreover, a study carried out by our group already demonstrated that oua can inhibit the activation of the nf-κb and p-38 proteins (mascarenhas et al., 2014), both playing an important role in the carcinogenic process, especially in the early stages (agrawal et al., 2001). furthermore, the immune system has a dual role in tumor development may help in the process of carcinogenesis, through the release of proinflammatory cytokines and stimulation of angiogenesis that favors metastases, but it may also be involved in the process to eliminate the tumor with the expression of tumorassociated antigens (de visser et al., 2006). the level of lymphocytes can be a prognostic marker of tumor aggressiveness. it was observed that lower levels of lymphocytes were associated with a greater degree of tumor development and metastasis formation (coffelt et al., 2015). the m1 macrophages are associated with the secretion of pro-inflammatory cytokines, such as interleukin-1β (il-1β) and tnf-α; and m2 have an antiinflammatory profile, with greater secretion of interleukin-10 (il-10) (najafi et al., 2019). additionally, n1 neutrophils can express more immune-activating cytokines and chemokines, lower levels of arginase and greater ability to kill tumor cells. the n2 phenotype is related to the depletion of polymorphs in experimental models to reduce the number of metastases, without the aid of the primary neoplastic focus (giese et al., 2019; park et al., 2016; sagiv et al., 2015). although the oua reduced the tumor volume in the ehrlich experimental models in vivo, this reduction is probably not associated with the cytotoxicity of the glycoside in ehrlich cells. mathematically, the oua concentration used in our in vivo experiments approaches 96 μm/dose and, at that concentration, the oua did not demonstrate cytotoxicity to ehrlich cells in the tests conducted in vitro. most likely, the oua has negatively modulated the development of the tumor due to its antiinflammatory activity. this does not exclude the fact that the oua has previously reported antitumor activity. however, the oua antitumor activity has been demonstrated only in human tumor cell lines as a549 cells, mcf7 cells, du 145 cells, u-2 os cells, a375 cells, sk-mel-28 cells and u‑87mg cells (chang et al., 2019; chou et al., 2018; wang et al., 2021; xiao et al., 2017; yang et al., 2018). here we evaluated the effect of oua on murine cells that are about 1000 times more resistant to the cytotoxic effects of oua compared to human cells due to a difference in the isoform of the α1 subunit of the na+/k+ atpase pump that makes murine cells less sensitive to the oua (akimova et al., 2015). the pathophysiological role of ouabain on tumor development is still little explored, however, here we demonstrate that oua can play an immunomodulatory role in both ascitic and solid ehrlich tumors and delay 222 biology, medicine, & natural product chemistry 12 (1), 2023: 215-224 tumor development. it is known that at a concentration of 1000 μm oua acts by inhibiting na+/k+ atpase, consequently reversing the kinetics of the na+/ca2+ exchanger, resulting in an increase in intracellular ca2+ that triggers apoptosis via activation of caspase-3. however, in the in vivo experiments the doses administered to the animals (96 μm/dose) was much lower, precisely to understand the role of the oua in hormonal levels. therefore, our hypothesis is that ouabain in hormonal concentrations acts by downregulating inflammation and thereby interfering with the migration of inflammatory cells to the ascitic tumor and decreasing the extravasation of plasma exudate reducing the tumor mass in mice with solid tumor (rodrigues-mascarenhas et al., 2011). this could be related to the capacity of ouabain to inhibit the nuclear factor nf-kb in low concentrations (leite et al., 2015). this work presents new perspectives on the antiinflammatory potential of oua and brings new ideas about the pathophysiological role of this hormone. conclusions oua in higher doses can decrease the number and viability of ehrlich tumor cells in vitro. in addition, animals that were pretreated with the physiological concentration of ouabain showed a delay in the development of solid and ascitic tumors. pretreatment with ouabain reduced the paw thickness and weight in animals with a solid tumor, in addition to reducing the weight of the lymph node closest to the paw with tumor. in animals with ascitic tumor, pretreatment with ouabain altered the migration profile of lymphocytes, neutrophils and macrophages to the microenvironment of tumor development. taken together, our work demonstrates that ouabain can delay tumor development both in vitro and in vivo, this expands the anti-inflammatory, immunomodulatory and a possible anti-cancer role of ouabain. acknowledgements: the group thanks éssia lima, juliane frança, josé marreiro, josé guilherme, luiz agra, vivian rumjanek for technical assistance. funding was supported by brazilian federal agency for support and evaluation of graduate education (capes), national council for scientific and technological development (cnpq) and federal university of paraiba. authors’ contributions: sandra rodrigues mascarenhas & deyse cristina madruga carvalho designed the study. amanda costa ayres salmeron, maria beatriz calado & mateus da silva matias antunes carried out the laboratory work, collected and analyzed the data, and wrote the manuscript. gabriel rodrigues da silva & beatriz fernandes de souza helped in the treatment of animals. márcia regina piuvezam kindly provided the place where the experiments were carried out. all authors read and approved the final version of the manuscript competing interests: the authors declare that there are no competing interests. funding: supported by the national council for scientific and technological development – cnpq, brazilian federal agency for support and evaluation of graduate education – capes and federal university of paraíba – ufpb. references agrawal, a., choudhary, d., upreti, m., rath, p. c., & kale, r. k. 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(2018). ouabain suppresses the growth and migration abilities of glioma u-87mg cells through inhibiting the akt/mtor signaling pathway and downregulating the expression of hif-1α. molecular medicine reports, 17(4), 5595–5600. https://doi.org/10.3892/mmr.2018.8587 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 5-8 | doi: 10.14421/biomedich.2023.121.5-8 issn 2540-9328 (online) high performance liquid chromatography (hplc) for detection of glucosamine and chondroitin sulfate compounds rakhmiyati1,*, tetri widiyani1,2, agung budiharjo1,2 1department of bioscience, graduate program; 2department of biology, faculty of mathematics and natural sciences, sebelas maret university. jl. ir sutami no. 36a, kentingan, surakarta, 57126, central java, tel./fax. +62-271-663375, indonesia. corresponding author* miarakhmiy@gmail.com manuscript received: 26 march, 2022. revision accepted: 24 august, 2022. published: 14 september, 2022. abstract glucosamine and chondroitin sulfate are compounds found in shark cartilage (carcharhinus sorrah). the two compounds have many health benefits, that is wound healing and helping the process of angiogenesis. this study aims to determine the content of glucosamine and chondroitin sulfate compounds in shark cartilage (sc) extract. the method used was high performance liquid chromatography (hplc) with potassium phosphate buffer solution at ph 3. the results of this research were sc extract contained glucosamine and chondroitin sulfate compounds with a retention time of 1.914 minutes. keywords: hplc; shark; glucosamine; chondroitin sulfate. introduction shark cartilage (sc) has several benefits, including controlling the growth and spread of tumor cells, cancer, helping to reduce bone pain, avoiding rheumatic diseases, strengthening and maintaining bone function, relieving pain and gout, maintaining body health and vitality, and avoid curvature of the spine (sulityowati et al., 2015; dean and summers, 2006). according martelpelletier (2015) sc can also treat osteoporosis and osteoarthritis because it contains chondroitin sulfate. the extraction and purification of chondroitin sulfate was first carried out in 1960 (miller and clegg, 2011). research conducted by sulityowati et al (2015) showed that shark cartilage powder contained 28.36% glucosamine and 6.06% chondroitin. chondroitin sulfate powder is white to cream in color with a ph value between 5.5 to 7.5. this compound is easily soluble in water and is hygroscopic. chondroitin sulfate becomes unstable when exposed to direct light and at high temperatures (marzuki et al., 2014). according garnjanagoonchorn et al. (2007) in xie et al (2014) states that dry sc contains glycosaminoglican 10-40% and collagen type ii 25-55%. glycosaminoglycan (gag) is components of the extracellular matrix of connective tissue. chondroitin sulfate is a type of gag whose components consist of n-acetyl-galactosamine, sulfuric acid and glucuronic acid. chondroitin sulfate can be used in joint ailment therapy, anti-inflammatory, arthritis, atherosclerosis and cancer (siagian, 2014; widyaningsih et al, 2016), and immunostimulant (bargahi and rabbani-chadegani, 2008). according to kerri et al. (2003) and huskisson (2008) the chemical structure of chondroitin sulfate and glucosamine were shown in figures 1 and 2. figure 1. the chemical structure of chondroitin sulfate compound. figure 2. the chemical structure of glucosamine compound. chondroitin sulfate is a polysaccharide anion consisting of the disaccharide unit naacetylgalactosamine 4or 6-sulfuric acid and dhttps://doi.org/10.14421/biomedich.2023.121.5-8 6 biology, medicine, & natural product chemistry 12 (1), 2023: 5-8 glucuronic acid repeated in cartilage tissue. these polysaccharides covalently bind to proteins to form proteoglycans. chondroitin sulfate has a wide range of applications in the pharmaceutical, cosmetic and food industries (nakano et al., 2000). according to siagian (2014), chondroitin is found in hyaline cartilage and can be distinguished structurally by the position of the sulfate ion in the monosaccharide bonds. materials and methods tools and materials the tools used in this research include: styrofoam ice box, measuring tape, sitting scale, digital scale, surgical instrument set, knife, tray, oven, aluminum bowl, blander (miyako), sieve (size mesh 80), plastic bags, plastic clips, gloves, digital analytical scales, petri dishes, aluminum foil, surgical mats, rotary evaporator (rvo 400 sd boeco germany), beakers, vials glass, filtering funnels, hot plates, measuring cups, plastic pot bottles, hplc tools (shimadzu), column c-18 (dimensions 4.6 x 250 mm, size of diameter pore 5µm), rid detector (refraksi index detector), cpu, stainless steel spatula, tweezers, stirring rod. the material used in this research is shark (carcharhinus sorrah) obtained from depok beach, yogyakarta, ice cubes, methanol pa, acetonitrile, potassium dihydrogen phosphate powder (1.36 grams), distilled water, g-nutri, chondroitin sulfate powder, and phosphoric acid (h3po4). shark cartilage powder shark (carcharhinus sorrah) was obtained commercially from the coast of depok, yogyakarta, then all flesh and tissue attached to the cartilage were removed. the cartilage was cut into ±1 cm in size and had been dried using an oven at 500 c for 24 hours. furthermore, it was mashed using a blender, sieved with a sieve of 80 mesh. shark cartilage powder was stored in a cool place prior to extraction (sulityowati et al., 2015; davis, c., 1994). shark cartilage extract 60 grams of shark cartilage powder was dissolved in 1200 ml of methanol as a solvent. the immersion time was 7 days. every day the solution was stirred for 3 hours. then after that filtered using filter paper. the filtrate obtained from the filtering was collected, while the residue was discarded. the filtrate was then processed using a rotary evaporator machine. the temperature used on the hot plate was 400c and the speed of the driving rotor was 2 turns. the result of the rotary evaporator was a thick, milky white liquid which was an extract of shark cartilage (iffah et al., 2018). detection of glucosamine and chondroitin sulfate compounds instrument setup hplc (high performance liquid chromatography) the first step was to turn on the electric power, then turn on the stabilizer. after that turn on the pump on the tool. next, turn on the water column by pressing the polar button. then turn on the detector rid (refractive index detector) [set the wavelength (γ)], then modular and finally turn on cpu (central processing unit) on computer. if the chromatogram shows a flat base line then the instrument can be used. preparing the mobile phase mobile phase was using potassium phosphate buffer solution ph 3. the buffer solution was made by adding 1.36 grams of potassium dihydrogen phosphate powder into 800 ml of distilled water, then adding phosphoric acid (h3po4). the ratio between the potassium phosphate buffer and the acetonitrile was 99.5:0.5. the flow rate is 1 ml/minute. the hplc column used was c18 4.6×250 mm 5µm merck. the temperature in the column used was 280c. the eluent will carry the components of the mixture to the rid (refractive index detectors) detector. (jahangir, et al., 2015; nagarajan, et al., 2013). results and discussion based on the results of high performance liquid chromatography (hplc), it was found that the extract of shark cartilage (carcharhinus sorrah) was proven to contain glucosamine and chondroitin sulfate compounds. shark cartilage extract chromatogram based on hplc method is shown in figure 3. figure 3. hplc chromatogram on shark cartilage extract. the hplc method is a suitable method to determine the presence of glucosamine and chondroitin sulfate compounds in shark cartilage extracts. separation of analytes were using phosphate buffer solution and acetonitrile with a ratio of 99.5: 0.5. the buffer solution ph 3 is a mixture of phosphoric acid solution = 88 ml + 1000 ml aquades + 1.3 grams of potassium dihydrogen phosphate and the flow rate used was 1 ml/minute. in rakhmiyati et al. – high performance liquid chromatography (hplc) for … 7 figure 1 it can be seen that the elution substance has formed a good symmetrical peak. figure 4. hplc chromatogram results on glucosamine (a) and chondroitin sulfate (b) parameters. from the hplc output in the form of a chromatogram (figure 3), it can be seen that the shark cartilage extract sample (carcharhinus sorrah) has a retention time of 1.917. meanwhile, the retention time for glucosamine and chondroitin sulfate parameters were 1.914 and 1.914 (figure 4). this retention time proves that the shark cartilage extract contains glucosamine and chondroitin sulfate compounds. according to research that has been done by sulityowati et al. (2015) stated that in 40 grams of shark cartilage powder it contains 28.36% glucosamine and in 240 grams of shark cartilage powder there is 6.06% chondroitin. glucosamine, 2-amino-2-deoxy-d-glucose (c6h14no5) is a monosaccharide having a molecular weight of 197.2 da (agiba, 2017; huskisson, 2008). this compound is the main component of glycosaminoglycans (gags) in cartilage and synovial fluid (sulityowati et al., 2015). glycosaminoglycans (gags) are heteropolysaccharides that have a negatively charged protein at the edge and function as a binder called mucopolysaccharide (sulityowati et al., 2015). glucosamine is found in almost all connective tissue, but the most abundant content is in cartilage (dahmer and schiller, 2008; sulityowati et al., 2015). chondroitin sulfate (cs) is a heteropolysaccharide with long and unbranched chains called glycosaminoglycans with a molecular weight of 50-100 kda, but after the extraction process the molecular weight becomes 10-40 kda (sulityowati et al., 2015; henrotin et al., 2010). but according to huskisson (2008) the molecular weight of chondroitin is 10,00050,000 da. cs compounds have the same properties as gc which are hydrophilic, can dissolve in water and produce a liquid that resembles sodium hyaluronate. conclusion based on observations that have been made using the high performance liquid chromatography (hplc) method, it can be concluded that the shark cartilage extract (carcharhinus sorrah) contains glucosamine and chondroitin sulfate compounds with a retention time of 1.917 minutes. conflict of interests: authors state that there is no conflict of interest in this research output. references agiba, a.m. (2017). nutraceutical formulations containing glucosamine and chondroitin sulphate in the treatment of osteoarthritis: emphasis on clinical efficacy and formulation challenges. international journal of current pharmaceutical r esearch 9 (2): 1-7. american college of rheumatology subcommittee on osteoarthritis guidelines. 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(2003). dietary supplements: toxicology and clinical pharmacology. edited by: mj cupp and ts tracy © humana press inc., totowa, new jersey. lippiello, l. (2003). glucosamine and chondroitin sulfate: biological response modifiers of chondrocytes under simulated conditions of joint stress. osteoarthritis and cartilage 11 (5): 335-342. lubis, a.m.t., siagian, c., wonggokusuma, e., marsetio, a.f., setyohadi, b. (2017). comparison of glucosaminea b 8 biology, medicine, & natural product chemistry 12 (1), 2023: 5-8 chondroitin sulfate with and without methylsulfonylmethane in grade i-ii knee osteoarthritis: a double blind randomized controlled trial. acta med indones-indones j intern med 49 (2): 105-111. martel-pelletier j, a farran, e montell, j verges, jp pelletier. (2015). discrepancies in composition and biological effects of different formulations of chondroitin sulfate. molecules 20 (3): 4277-4289. marzuki as, kasim dan n arafah. 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(2005). pharmacokinetic profile of glucosamine and chondroitin sulfate association in healthy male individuals. acta ortop bras 13(5): 235-237. václavíková, e., and kvasnička, f. isotachophoretic determination of glucosamine and chondroitin sulphate in dietary supplements. czech j. food sci 31 (1): 55-65. vasiliadis, h.s., tsikopoulos, k. (2017). glucosamine and chondroitin for the treatment of osteoarthritis. world journal orthopedics 8 (1): 1-11. widyaningsih td, wd rukmi, e sofia, sd wijayanti, n wijayanti, r ersalia, n rochmawati, d nangin. (2016). extraction of glycosaminoglycans containing glucosamine and chondroitin sulfate from chicken claw cartilage. research journal of life science 3 (3): 181-189. xie, j., hy ye and xf luo. (2014). an effcient preparation of chondroitin sulfate and collagen peptides from shark cartilage. international food research journal 21(3): 1171-1175. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 481-487 | doi: 10.14421/biomedich.2025.141.481-487 issn 2540-9328 (online) phytochemical screening and antioxidant activities of rytigynia nigerica (s. moore) robyns roots extracts oluwakayode olubunmi odeja1,*, michael gabriel ibok2, ejike o. okpala3, oluwafunke toyin afolabi-owolabi4, kola augustus oluwafemi5, emmanuel adja6, idris abayomi nosiru7 1department of chemistry, federal university of petroleum resources, effurun, delta state, nigeria 2department of chemistry, university of ibadan, nigeria. 3department of chemistry, federal university lokoja, kogi state, nigeria. 4department of education, jackson school mulhall drive st. albans victoria, australia. 5adekunle ajasin university, akungba akoko. ondo state, nigeria. 6department of chemistry, federal university of petroleum resources, effurun, delta state, nigeria. 7department of chemistry, lagos state university, ojo, nigeria. corresponding author* odeja.oluwakayode@fupre.edu.ng abstract rytigynia nigerica (rubiaceae), a medicinal herb native to west africa, is known for its antimalarial and anticancer properties and is traditionally used to treat various ailments. however, the biological activities of r. nigerica have not yet been fully studied. however, this study was designed to extract, analyse, and evaluate the antioxidant potential and phytochemical screening in the root extracts of r. nigerica. the roots were obtained from the forestry research institute of nigeria (frin), ibadan and authenticated. the air-dried and pulverised root samples were extracted with methanol using the maceration method and then partitioned into n-hexane and ethyl acetate using the liquid-liquid extraction method. the phytochemical screening was evaluated using the standard method, while antioxidant activity was investigated viz 2, 2-diphenyl-1-picrylhydrazyl radical (dpph) assays. the phytochemical screening analysis revealed the presence of saponins, cardiac glycosides, tannins, phenols, reducing sugars, alkaloids and resins in the root extract. the antioxidant activity of the extracts was significant when compared to reference standards. the percentage inhibition of the antioxidant extracts and reference standard are as follows: n-hexane extract (58.50 -45.08%), ethyl acetate extract (90.62-67.82%), methanol extract (70.8152.97%), vitamin c (95.66-91.63%) and butyl hydroxyanisole (94.76-90.96%). the antioxidant inhibition of the free radical was concentration-dependent. the results obtained in this study indicate that r. nigerica root extracts exhibit antioxidant properties, suggesting potential pharmaceutical applications. keywords: rytigynia nigerica root; 2, 2-diphenyl-1-picrylhydrazyl radical (dpph); phytochemical constituents. introduction historically, natural products have attracted a lot of interest because they are primarily found in nature and made of organic materials from living things (pezzuto et al., 2005). natural products are chemical compounds isolated from living organisms (bhat et al., 2005). it can be synthesised artificially or organically through partial or entire synthesis, and it is essential in advancing organic or medicinal chemistry by offering challenging synthetic targets (chintoju et al., 2015). natural products have played a crucial role in developing medications, dietary supplements, cosmetics, and agrochemicals (beghyn et al., 2008). natural medicines not only meet most of the primary healthcare needs of the populations in developing nations, but they are also gaining popularity in developed nations due to spiralling healthcare costs and widespread financial austerity. about 49% of people in the usa have experimented with natural remedies for illness prevention and treatment (who, 2009). natural remedies are said to contain "active components" or "active principles," which are substances recognized to have therapeutic advantages. nearly half of the chemical medications for treating human diseases approved by the united states food and drugs agency (fda) between the 1940s and the end of 2014 were derived from or inspired by natural sources (newman et al., 2012; newman et al., 2016). functional groups, chirality and structural complexity of molecules from natural products are more similar to those of drugs than those of molecules from combinatorial chemistry (cragg & newman, 2003; atanasov et al., 2015). in natural remedies, the concentrations of the active components manuscript received: 13 february, 2025. revision accepted: 23 july, 2025. published: 30 july, 2025. https://doi.org/10.14421/biomedich.2025.141.481-487 482 biology, medicine, & natural product chemistry 14 (1), 2025: 481-487 are rarely very high. the bottleneck in using natural products in medication research has been the labourintensive and time-consuming extraction and isolation process. furthermore, a number of chronic and degenerative illnesses, including cancer, pulmonary, neurological, and digestive disorders, have been linked to the overproduction of reactive oxygen species (ros) (liu et al., 2018). antioxidants, which can be produced internally or obtained through external supplementation, subtly control the levels of ros under physiological settings. malnutrition and antioxidant deficiencies together may make people more susceptible to oxidative stress, which raises the chance of developing cancer (liu et al., 2018). in addition, the antioxidant defence can be overwhelmed during continuous inflammation such as in chronic obstructive lung illnesses, inflammatory bowel disease, neurodegenerative disorders, cardiovascular diseases, and aging (chelombitko, 2019). certain antioxidant vitamins, such as vitamin d, are vital in regulating metabolic processes that contribute to the normal functioning of organs. in several clinical studies, antioxidant supplements have been demonstrated to reduce endogenous antioxidant depletion, hence mitigating related oxidative damage (forman & zhang, 2021). the nigerian species of rytigynia nigerica (figure 1) is the focus of this study. according to burkill (1985), the species have been reported as having antimalarial and anticancer effects and are used to treat other common diseases in west africa. the stomatal type was paracytic and epidermal cell shape was irregular in both species. a higher epidermal cell number was recorded in r. nigerica. the parenchyma cell of the petiole was generally polyhedral to roughly oval in both species. in r. nigerica, the adaxial surface of the petiole was almost flattened and crystalloid deposits of calcium oxalate were found in the sclerenchyma tissue. furthermore, in r. nigerica, the midrib surface was pubescent, no cell inclusion was recorded and the vascular bundle was vshaped (ajayi et al., 2011). leaves of r. nigerica were generally for the treatment of hypostomatic, malaria and cancer (ajayi et al., 2011). the phytochemical screening of r. nigerica revealed the presence of steroids, tannins, saponins, reducing compounds, terpenes, and flavonoids in the ethanolic extracts. phlobatannins, anthraquinones, cardiac glycosides, and cyanogenetic compounds were absent (ajayi et al., 2011). figure 1. picture of r. nigerica. these medicinal plants have been used for centuries, but very little research has been done to examine their phytochemical constituents or antioxidant properties. this study aims to investigate the phytochemical constituents of extracts of these plants and evaluate their antioxidant properties. materials and methods equipment/apparatus and chemicals/reagents used the equipment and apparatus used include a steam bath, round-bottom flask, weighing balance, beakers, distillation flask, sample vials, ice chest, syringes, uvvisible spectrophotometer, foil papers, and separating funnel. all solvents were general-purpose chemicals obtained from the local vendors: methanol, ethyl acetate, n-hexane and were distilled before use. concentrated h2so4, hcl, ferric chloride, fehlings' solution a and b, ammonia solution, benzene, iodine crystals, glacial acetic acid, molisch reagent, magnesium powder, ascorbic acid, butylated hydroxylanisole, αtocopherol and 2, 2-diphenyl-1-picrylhydrazyl radical (dpph). odeja et al. – phytochemical screening and antioxidant activities … 483 collection, identification and preparation of plant sample the fresh leaves of rytigynia nigerica robyns were collected from the forestry research institute of nigeria (frin), oyo state, nigeria. they were identified and authenticated at frin (forestry research institute of nigeria). the leaves and stem of rytigynia nigerica robyns were air-dried under shade, separated and pulverized before extraction. extraction of plant sample the air-dried roots of rytigynia nigerica were ground and about 1.0 kg of sample of root samples were obtained. 1.0 kg of the root sample was carefully charged into an aspirator bottle and extracted with methanol for 24 hrs using a maceration method. the mixtures were decanted and concentrated into a paste via distillation and dried using a vacuum desiccator. 150 g of the root extract was obtained. 89.77 g of the methanol extract of rytigynia nigerica leaves was partitioned first into hexane and later into ethyl acetate using the liquid-liquid extraction method described by (ferrone et al., 2017) was employed the resulting hexane and ethyl acetate extracts were concentrated to a paste using a rotary evaporator under reduced pressure at 40 0c and dried using a vacuum desiccator. phytochemical screening of rytigynia nigerica roots (sofowora, 1993; okwu, 2001). screening for alkaloids to 1% hcl was added 0.2 g of extract, the mixture was filtered and then drops of dragendorff's reagent were added. the creation of a precipitate showed the presence of alkaloids. screening for saponin to each extract (0.2 g) 5 ml of distilled water was added. the formation of froth upon warming and letting stand for about 10 minutes indicates the presence of saponin. screening for tannins a total of 0.2 g of extract was measured and dissolved in distilled water. the resulting solution was filtrate and ferric chloride was added to it. the presence of a greenblue-dark or blue-green precipitate detected tannins. screening for steroids (salkowski's test) a total of 0.2 g of extract was weighed and dissolved in 2 ml of chloroform. a lower layer of concentrated h2so4 was then applied. the reddish-brown colour of cardenolides during the interphase corroborated their deoxy-sugar properties. screening for cardiac-active glycoside (kellerkillani test) from the extracts, 0.2 g was weighed and dissolved in 2 ml of glacial acetic acid containing one drop of ferric chloride solution. 1 ml of concentrated sulphuric acid was then added. brown ring inter-phase indicated the presence of cardiac glycosides. screening for carbohydrates about 5 ml of extract was measured and placed in a test tube in a slanting position and a few drops of molisch reagent were poured through the side of the test tube holding 1 ml of sulphuric acid solution. without mixing, the acid created a layer beneath the aqueous solution. a reddish-brown solution suggested the presence of carbohydrates. screening for reducing sugars about 0.2 g of the extract was mixed with distilled water before being filtered. the filtrate was then heated for two minutes with equal volumes of fehlings' solution a and b. the presence of orange precipitate generated proved the presence of reducing sugar. screening for flavonoids (shinoda test) about 3 ml of extract solution was measured and a small amount of magnesium powder was added. a few drops of strong hydrochloric acid were also added to the mix. the production of a crimson precipitate confirmed the presence of flavanones. screening for resins about 5 ml copper acetate was added to 5 ml of extracts, agitated vigorously and allowed to separate. the appearance of a green colour verified the existence of tar. test for anthraquinones (born – trager's test) about 4 ml of benzene was added to 0.2 g of extracts and the mixture was filtered. the filtrate was shaken with 2 ml of the ammonia solution. the production of pink, red or violet colour in the ammonical portion (lower layer) was indicative of the presence of anthraquinones. screening for phenols about 0.2 g of the extracts were dissolved in ferric chloride solution. the appearance of a dirty green precipitate after dissolving suggested the presence of a phenolic chemical. screening for phlobatannins for the extracts, dissolution of 0.5 g each was dissolved in distilled water and filtered. the filtrate was boiled with 2% hcl formation. the formation of red precipitate indicated the presence of phlorotannins. antioxidant assay the radical scavenging activity was evaluated using the approach reported by ibok et al., 2023. in methanol, a 0.5 mm solution of 2, 2-diphenyl-1-picrylhydrazyl radical (dpph) was produced, and 3 ml of this solution was combined with 1 ml of the oil sample in methanol (ibok et al., 2023). the reduction in dpph absorption at 517 484 biology, medicine, & natural product chemistry 14 (1), 2025: 481-487 nm was evaluated after a 10-minute incubation period. the percent inhibition was calculated, and the decrease in absorption was compared to the control group's drop in absorption. using recognized standards such as ascorbic acid and butylated hydroxylanisole, a similar process was used. the tests and analyses were repeated three times, the results were averaged, and the activities were calculated as a function of the percentage inhibition (%i) was then calculated using the following. equation: 𝐼(%) = 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 × 100 the percentage yield of rytigynia nigerica roots extracts (n-hexane, ethyl acetate and methanol) the roots crude extracts of rytigynia nigerica yield (w/w) obtained in this study are presented in table 1. the semi-polar ethyl acetate extract had the lowest yield (23.5%), followed by the non-polar n-hexane extract (33.5 %) and the polar aqueous-methanol extract (43.0%; table 1). the yield of the extracts obtained in the study showed the level of the components present in the plant extracts based on their polarity. it is reported that the yield of plant extracts can be significantly influenced by the solvent type used, as each solvent has a different polarity and affinity for various compounds. however, the n-hexane extract in this study had a better yield than the ethyl acetate extract, in contrast to the literature report based on lipophilic compounds. this indicated that the root extract contained more lipophilic compounds. table 1. yields and properties of the root wood extracts of rytigynia nigerica roots. extract weight (g) yield (%) description aqueous methanol 38.60 43.0 dark brown viscous ethyl acetate 21.10 23.5 dark brown n-hexane 30.07 33.5 dark brown %yield = [weight of the extract / weight of bulk extract] × 100 phytochemical screening of the rytigynia nigerica leaves the phytochemical screening of rytigynia nigerica root extracts involved using three different solvent extractions—polar, semi-polar, and nonpolar—to obtain a comprehensive chemical profile of the plant. each solvent targets specific compounds based on their polarity, allowing for a more thorough investigation of the plant's constituents. as presented in table 2, the results indicated the presence of saponins, cardiac glycosides, tannins, phenols, reducing sugars, alkaloids, and resins, all compounds commonly associated with medicinal activity. the phytochemical screening of rytigynia nigerica root extracts, utilising polar, semi-polar, and nonpolar solvents, provided a comprehensive chemical profile of its constituents. each solvent selectively extracted different classes of compounds based on polarity, enabling an in-depth investigation of bioactive compounds present in the plant (sofowora, 1993; okwu, 2001). as summarised in table 2, the analysis revealed saponins, cardiac glycosides, tannins, phenols, reducing sugars, alkaloids, and resins—all known for their medicinal properties. saponins and tannins possess antiinflammatory and antimicrobial effects, while phenols contribute antioxidant benefits. alkaloids and cardiac glycosides are linked to anti-diabetic and cardioprotective activities, supporting the potential of r. nigerica in traditional medicine for diverse therapeutic applications (ogunlana et al., 2008). these findings lay the groundwork for further exploration into the medicinal properties of the plant. in many traditional medicinal systems, plants containing these compounds are utilised for treating inflammation, infections, heart ailments, and wounds. this supports the role of rytigynia nigerica in traditional medicine and suggests that it could serve as a source of therapeutic agents for modern medicine, potentially providing alternatives for managing various health issues. table 2. constituents of the various extracts of rytigynia nigerica roots. constituents hexane extract ethylacetate extract methanol extract key: + = present. = not present. antioxidant activities of r. nigerica root extracts the dpph radical has a distinct purple colour with a maximum absorbance at 517 nm in the visible spectrum. when an antioxidant donates an electron or a hydrogen atom to dpph, the radical stabilizes, and the colour changes from purple to bright yellow. this decolorization is proportional to the antioxidant activity of the tested substance. as the antioxidant compounds in the extracts neutralize the dpph radicals, the absorbance value at 517 nm decreases, indicating a reduction in free radical concentration. the extent of this reduction reflects the strength of the antioxidant activity in the extracts (brand-williams et al., 1995). phenolic compounds, well-known for their antioxidant action, are present in significant amounts in the ethyl acetate and methanol extracts. phenolic saponins + + + cardiac glycoside + + + tannins + + + phenols + + + alkaloids + + + reducing sugar + + + flavonoids + carbohydrate + anthraquinones resin + + + phlobatannins + + odeja et al. – phytochemical screening and antioxidant activities … 485 compounds contribute to antioxidant activity through their ability to donate electrons or hydrogen atoms, neutralizing free radicals and thus preventing oxidative chain reactions. phenolics are effective antioxidants because their hydroxyl groups readily participate in free radical scavenging, forming more stable molecules that halt oxidative damage to cells and biomolecules (ogunlana et al., 2008). these compounds can protect biological structures, such as proteins, lipids, and nucleic acids, from damage caused by oxidative stress, which is implicated in many chronic diseases. the results of the dpph assay are presented in figure 2, with the antioxidant activities of the extracts compared to known antioxidant standards such as vitamin c and butylated hydroxyanisole (bha). the antioxidant potential of the extracts and standards were ranked as follows: vitamin c, serving as a potent reference standard, displayed the highest radical scavenging activity (95.66-91.63%). known for its electron-donating solid ability, vitamin c neutralises free radicals quickly, resulting in a rapid decline in dpph absorbance (table 6). its effectiveness underscores the benchmark against which the plant extracts were measured. this was followed by butylated hydroxyanisole, a synthetic antioxidant commonly used in food preservation, demonstrating high antioxidant activity. although slightly less active than vitamin c, the capacity of bha (94.76-90.96%) to stabilise radicals highlights its efficacy in halting oxidative reactions, which provides a further point of comparison for evaluating the plant extracts (table 7). rnre, among the plant extracts, exhibited the highest antioxidant activity (90.6267.82%), close to that of bha and vitamin c. ethyl acetate (table 4) is an effective semi-polar solvent for extracting phenolic compounds, which are abundant in rytigynia nigerica and is known to possess strong antioxidant properties. this high antioxidant activity is attributed to phenolic compounds with hydroxyl groups that donate electrons to free radicals, converting them into stable products and stopping radical chain reactions. rnrm extract showed moderate antioxidant activity (table 5), lower than rnre but still significant. methanol, as a polar solvent, extracts phenolic compounds, though often different in profile compared to those extracted by ethanol. despite the slightly lower activity, rnrm demonstrated effective radical scavenging (70.81-52.97%), supporting its role as a potential antioxidant source. the activity may be due to a combination of phenolic and other antioxidant compounds present in the methanol extract, though possibly in lesser quantities or types than those in the ethanol extract. the hexane extract displayed the lowest antioxidant activity among the tested samples. hexane, a nonpolar solvent, extracts mainly lipophilic compounds like terpenoids and other nonpolar constituents that generally possess lower antioxidant activity compared to polar phenolics. the lower antioxidant activity of rnrh (table 3) suggests a lesser content of electron-donating compounds, indicating that its radical-scavenging potential (58.50 -45.08%) is weaker than the more polar extracts. the results confirm that the solvent used for the extraction and its polarity significantly influences the antioxidant activity of the extracts. the ethanol and methanol extracts, richer in phenolic compounds, demonstrated more muscular antioxidant activities due to the higher concentration of polar antioxidant compounds capable of donating electrons. in contrast, the nonpolar hexane extract had a lower antioxidant capacity, likely due to its different profile of extracted compounds with less electron-donating potential. these findings suggest that rytigynia nigerica root has a substantial antioxidant capacity, particularly in its ethanol and methanol extracts. this antioxidant property supports the traditional use of rytigynia nigerica in herbal medicine, as oxidative stress reduction is a crucial mechanism in the prevention of chronic diseases such as cardiovascular disorders, diabetes, and cancer. the strong antioxidant properties of the ethanol and methanol extracts of rytigynia nigerica root underscore its potential as a natural source of antioxidants for health applications. rnrh = hexane extract of r. nigericia roots rnre = ethyl acetate extract of r. nigericia roots rnrm = methanolic extract of r. nigericia roots bha = butyl hydroxyanisole vit c = vitamin c figure 2. antioxidant activities of r. nigerica root extracts. 0,000 20,000 40,000 60,000 80,000 100,000 120,000 1 0,5 0,25 0,125 0,0625 % in h ib it io n concentration (mg/ml) rnrh rnre rnrm vit c bha 486 biology, medicine, & natural product chemistry 14 (1), 2025: 481-487 table 3. absorbance reading of n-hexane extract of r. nigericia roots. concentration (mg/ml) absorbance average blank % inhibition 1st 2nd 3rd 1.00 0.49 0.491 0.492 0.491 1.183 58.495 0.5 0.504 0.505 0.509 0.506 1.183 57.227 0.25 0.519 0.515 0.518 0.517 1.183 56.269 0.125 0.564 0.561 0.567 0.564 1.183 52.325 0.0625 0.654 0.646 0.649 0.650 1.183 45.083 table 4. absorbance reading of ethyl acetate extract of r. nigericia roots. concentration (mg/ml) absorbance average blank % inhibition 1st 2nd 3rd 1.00 0.11 0.112 0.111 0.111 1.183 90.617 0.5 0.134 0.129 0.131 0.131 1.183 88.898 0.25 0.28 0.275 0.276 0.277 1.183 76.585 0.125 0.301 0.3 0.298 0.300 1.183 74.669 0.0625 0.381 0.379 0.382 0.381 1.183 67.822 table 5. absorbance reading of methanol extract of r. nigericia roots. concentration (mg/ml) absorbance average blank % inhibition 1st 2nd 3rd 1.00 0.35 0.34 0.346 0.345 1.183 70.809 0.5 0.371 0.368 0.367 0.369 1.183 68.836 0.25 0.446 0.442 0.441 0.443 1.183 62.553 0.125 0.451 0.45 0.468 0.456 1.183 61.426 0.0625 0.555 0.556 0.558 0.556 1.183 52.973 table 6. absorbance reading of vitamin c extract of r. nigericia roots. concentration (mg/ml) absorbance average blank % inhibition 1st 2nd 3rd 1.00 0.051 0.051 0.052 0.051 1.183 95.661 0.5 0.064 0.065 0.067 0.065 1.183 94.477 0.25 0.07 0.071 0.068 0.070 1.183 94.111 0.125 0.075 0.076 0.078 0.076 1.183 93.547 0.0625 0.1 0.099 0.098 0.099 1.183 91.631 table 7. absorbance reading of butyl hydroxyanisole (bha) extract of r. nigericia roots. concentration (mg/ml) absorbance average blank % inhibition 1st 2nd 3rd 1.00 0.061 0.062 0.063 0.062 1.183 94.759 0.5 0.07 0.071 0.073 0.071 1.183 93.970 0.25 0.075 0.078 0.079 0.077 1.183 93.463 0.125 0.099 0.098 0.099 0.099 1.183 91.660 0.0625 0.11 0.111 0.1 0.107 1.183 90.955 conclusion the findings of this study reveal that the root extracts of rytigynia nigerica contain a variety of phytochemicals, notably alkaloids and steroids, which distinguish this plant as a unique source of bioactive compounds. the presence of these phytochemicals contributes to the root's significant antioxidant and anti-diabetic properties, with activity comparable to standard medications used in this investigation. the antioxidant capacity, likely driven by phenolic content, underscores the potential of r. nigerica in combating oxidative stress, while its anti-diabetic effects suggest possible benefits for managing blood glucose levels. these results support the traditional use odeja et al. – phytochemical screening and antioxidant activities … 487 of r. nigerica in herbal medicine and highlight its potential as a natural therapeutic source. further studies should explore the mechanisms of action and isolate specific bioactive compounds, paving the way for the development of r. nigerica-based therapies in oxidative stress-related diseases and diabetes management. competing interests: the authors have not declared any conflict of interest. references atanasov, a. g., waltenberger, b., pferschy-wenzig, e. m., linder, t., wawrosch, c., uhrin, p. and stuppner, h. (2015). discovery and resupply of pharmacologically active plantderived natural products: a review. biotechnology advances, 33(8), 1582-1614. beghyn, t., deprez‐poulain, r., willand, n., folleas, b. and deprez, b. (2008). natural compounds: leads or ideas? bioinspired molecules for drug discovery. chemical biology & drug design, 72(1), 3-15. bhat, s. v., nagasampagi, b. a. and sivakumar, m. (2005). chemistry of natural product. berlin, new york. springer. isbn 81-7319-481-5. 650p. 2005. chan, t. h. and tang, c. s. (1979). the chemistry and biochemistry of papaya. in inglett, g.e. and charolambous, g. (eds.) tropical foods (vol. i). new york: academic press. chintoju, n., konduru, p., kathula, r. l. and remella, r. (2015). research and reviews: journal of research and reviews: journal of hospital and clinical hospital and clinical pharmacy. newman, d. j. and cragg, g. m. 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(1993). medicina1 plants and traditional medicine in africa, spectrum book ltd. ibadan, nigeria. 289. liu, z., ren, z. and zhang, j. (2018). role of ros and nutritional antioxidants in human diseases. frontiers in physiology. 2018;9: p. 477. doi: 10.3389/fphys.2018.00477. chelombitko, m. a. (2019). role of reactive oxygen species in inflammation: a minireview. moscow university biological sciences bulletin, 2019;73(4):199–202. doi: 10.3103/s009639251804003x. forman, h. j. and zhang, h. (2021). targeting oxidative stress in disease: promise and limitations of antioxidant therapy. nature reviews drug discovery. 20(9):689–709. doi: 10.1038/s41573-021-00233-1. report of who interregional workshop on the use of traditional medicines in primary health care. geneva, who, (2009) (isbn 978 92 4 159742 5) [f] (isbn 978 92 4 259742 4), [r] (isbn 978 92 4 459742 2), [s] (isbn 978 92 4 159742 5) ogunlana, e. o., ogunlana, o. o. and onwuka, c. j. 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(2017). a green deep eutectic solvent dispersive liquid-liquid micro-extraction (des-dllme) for the uhplc-pda determination of oxyprenylated phenylpropanoids in olive, soy, peanuts, corn, and sunflower oil, food chemistry (2017), doi: https://doi.org/10.1016/j.foodchem.2017.10.135 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 37-41 | doi: 10.14421/biomedich.2025.141.37-41 issn 2540-9328 (online) fermented feed for laying hens: effects on egg quality and the number of enteric pathogenic bacteria present daimeera anja lulu amantsura1, endah retnaningrum2,* 1student of faculty of biology; 2microbiology laboratory, department of tropical biology, faculty of biology, universitas gadjah mada jl. teknika selatan sekip utara yogyakarta 55281, tel. +62-274-580839, fax. +62-274-580839, indonesia. corresponding author* endahr@ugm.ac.id abstract eggs are one of the most common food sources for human consumption because they are common, easy to obtain, and nutritious. eggs contain abundant vitamins and minerals that the human body needs, are a good source of protein and fat and are rich in choline. despite its high nutritional value, eggs are also one of the leading causes of food poisoning and foodborne diseases in various countries. this is due to contamination in eggs by pathogenic bacteria such as salmonella, shigella, escherichia coli, and campylobacter bacteria. fermented chicken feed contains natural probiotics added to the feed, which improves gut health, better absorption of nutrients, inhibits the growth of pathogenic bacteria, enhances the immune response in chickens. this study is conducted to distinguish the diffrence in quality of eggs that can be physically observed in chicken eggs fed with fermented feed compared to chicken eggs fed regular feed, as well as to compare the number of total bacteria and enteric pathogenic bacteria contained in the contents of chicken eggs that includes escherichia coli and salmonella spp bacteria. the methods used in this research include observing egg quality comparisons, calculating and comparing the total number of bacteria, and testing for the presence of enteric pathogenic bacteria escherichia coli and salmonella, spp. using the plate count method. the resulting data was processed using the one-way anova method with a confidence level of 95%. the results of this research show higher egg quality in fermented fed eggs which is characterized by the color of the yolk which tends to be darker yellow to orange and thicker egg shells. the total bacteria count showed that the bacteria was significantly lower in eggs from chickens fed fermented feed. tests for the number of enteric bacteria showed positive results for e. coli in 1 out of 10 egg content samples from chicken fed with fermented feed, and positive results for e. coli in 2 out of 10 egg samples from chicken fed with regular feed, while test results for salmonella spp. showed negative results in all samples. keywords: fermented feed; enteric pathogenic microbia; eggs; total bacteria count. abbreviations: lab: lactic acid bacteria; ss: salmonella-shigella; emb: eosin-methylene blue; pca: plate count agar; tpc: total plate count introduction eggs are one of the most common food sources consumed by humans because they are a common, easy to obtain and nutritious food source. eggs contain abundant vitamins and minerals that the human body needs, including calcium, iron, potassium, zinc, manganese, vitamin e, folate, and many more. eggs are also a good source of protein and fat and rich in choline, which is an important nutrient for brain health. even though they have high nutritional value, eggs are also one of the leading causes of food poisoning and foodborne diseases in various countries. this is caused by contamination that occurs in eggs by pathogenic bacteria during the production period. food poisoning associated with egg-borne pathogens can cause high morbidity or mortality with symptoms such as diarrhea, vomiting, nausea and stomach cramps. enteric pathogenic bacteria are a group of bacteria that can cause intestinal infections that are transmitted through food, water, animals, or contact with infected individuals. (arumugam et al., 2015). these bacteria can cause various gastrointestinal disorders, from mild to severe, which can be transmitted via the fecal-oral route. some examples of enteric pathogens include clostridioides difficile, salmonella, shigella, escherichia coli, campylobacter, yersinia enterocolitica, and vibrio cholerae. (salihu et al., 2015). to overcome pathogenic bacterial contamination in animal food products, especially eggs, in recent years many farms have added antibiotic supplements to animal feed. however, this gives rise to new problems, namely the emergence of more and more bacteria that have adapted to become resistant to various types of antibiotics. research conducted by jain and yadav in 2017 found 73.33% of isolates of mutli-drug resistant manuscript received: 23 december, 2024. revision accepted: 17 february, 2025. published: 25 february, 2025. https://doi.org/10.14421/biomedich.2025.141.37-41 38 biology, medicine, & natural product chemistry 14 (1), 2025: 37-41 bacteria from egg samples with a resistance percentage of 86.66% to cefixme antibiotics, 80% to amoxicillin antibiotics, and 73.33% to amoxyclave antibiotics. (jain and yadav., 2017). the emergence of bacteria that are resistant to antibiotics can be prevented by using other methods to combat pathogenic bacterial contamination in animals, namely by applying fermented feed. fermented feed uses the basic principles of the growth of good microorganisms, one of which is lactobacillus type bacteria which is capable of producing lactic acid which functions to fight the growth of pathogenic bacteria naturally and without the side effects that arise from the use of antibiotics in animal feed. chicken feed fermentation is a process that involves making a wet mixture from fermented wet food that is rich in probiotics for chickens. this process is believed to improve chicken health and egg quality. (ibrahim et al., 2020). the type of microorganism selected for the fermentation process influences the substances produced after the fermentation process occurs. (niba et al., 2009). generally, the type of microorganism used for food fermentation is lactic acid bacteria (lab) or lactobacillus type bacteria because in the process it produces lactic acid which has many benefits, especially in the microecology of the digestive tract and has a good influence on the immune response. the microorganisms used in this research were em4 solutions or effective microorganisms. the em4 solution contains the main microorganisms in the form of phototrophic bacteria, lactic acid bacteria, yeast, actinomycetes and fermentation fungi. (fajaruddin et al., 2013). this research was done to determine the effect of fermented feed on egg quality that can be observed physically, including the color of the egg yolk and the thickness of the egg shell, to determine the difference in the number of enteric pathogenic bacteria contained in chicken eggs given fermented feed and chicken eggs fed regular feed, and to detect and differentiate the number of enteric pathogenic bacteria escrherichia coli and salmonella in chicken eggs fed fermented feed from chicken eggs fed regular feed. materials and methods study area samples were taken from two different chicken farms in the condong catur area of yogyakarta city, which used different types of feed, which are fermented and regular feed. a total of 20 samples were taken, two from each farm, over five different periods. procedures preparation of egg samples and egg quality observation each egg sample was soaked in 70% alcohol for 5 minutes to avoid contamination from bacteria on the egg shell. the sterile egg was cracked using a sterile spatula and poured into a sterilized jar. the color of the egg yolk was observed and documented for later comparison with other samples. egg yolks and egg whites are homogenized by stirring using a sterile spatula until homogeneous. serial dilutions were carried out for each sample up to a dilution of 10-5 by taking 1 ml of the sample and placing it in a test tube containing 9 ml of sterile distilled water. enumeration of total bacteria in egg samples the results of the serial dilutions 10-3 to 10-5 were inoculated on 1 ml of pca media using the spread plate method by taking 0.1 ml of suspension using a micropipette, all inoculated petri dishes then incubated in a 37°c incubator for 48 hours. the number of colonies was counted using the total plate count (tpc) method on each sample that had been incubated. identification and enumeration of enteric pathogenic bacteria present the same samples from the same suspensions were inoculated on emb agar plate and ss agar plate using a pour plate method. all inoculated petri dishes then incubated in a 37°c incubator for 48 hours. the number of colonies were counted using total plate count (tpc) method on each sample that had been incubated. data analysis all data that had been collected were analyzed using the one-way anova method to determine the significance of the difference between the two types of samples. results and discussion egg quality observation the quality of the eggs tested in this research was done by comparing the color of the yolk from samples of chicken eggs fed fermented feed and samples of chicken eggs fed regular feed. observation of the color of the egg yolk was carried out as an indicator of the carotenoid content contained in the chicken eggs samples, where a darker yellow to orange color indicated a higher carotenoid content in the egg. table 1. average score value comparison of the yolk color of chicken eggs fed with fermented feed and chicken eggs fed with regular feed. types of feed yolk colour score* fermented feed 2,7 regular feed 1,7 *score 1 indicates lighter yellow, score 2 indicates yellow, score 3 indicates darker yellow, and score 4 indicates orange based on the results that had been obtained, eggs from chicken that are fed with fermented food tend to amantsura & retnaningrum – fermented feed for laying hens: effects on egg quality … 39 have a darker yellow to orange color of yolk, which indicates a high content of carotenoids in the egg, while eggs from chicken that are fed with regular feed has a yolk color that tends to be yellow to lighter yellow which indicates a lower content of carotenoids. the carotenoid content in eggs is generally in the form of lutein and zeaxanthin, where these types of carotenoids cannot be synthesized in vitro so they must be obtained through the feed provided, therefore the profile of carotenoid content in eggs depends on the chicken's feeding. (zaheer., 2017). based on the research conducted by martínezsánchez and pérez-gálvez, 2023, feeding chickens with a wheat base such as regular chicken feed tends to result in eggs of lower quality, containing fewer carotenoids, resulting in egg yolks with a lighter color. meanwhile, higher-quality pasteurized feed mixed with forages such as grass produces darker yellow to orange egg yolks due to the high carotenoid content in the eggs. (martínezsánchez, and pérez-gálvez., 2023). some of the ingredients of the fermented feed in this research are various types of leaves, grass and other forages, this then affects the carotenoid content in eggs, so chickens fed fermented feed have a higher carotenoid content in their eggs. total number of bacteria fermented feed, apart from having the benefit of adding beneficial nutritional content for animal health, also produces substances that play a role in reducing pathogenic bacterial contamination in animals, especially in the digestive tract. fermented feed uses the basic principles of the growth of good microorganisms, one of which is lactobacillus type bacteria, which is capable of producing lactic acid, which functions to fight the growth of pathogenic bacteria naturally and without the side effects that arise from the use of antibiotics in animal feed. this research tests the total number of bacteria in the egg samples using the tpc method. table 2. total number of bacteria formed on pca media of chicken eggs fed with fermented feed and chicken eggs fed with regular feed (cfu/ml). types of feed total number of bacteria fermented feed 282,4 × 104 regular feed 1170,3 × 104 the total number bacteria found in the egg contents from chickens fed with fermented feed are significantly less than those found in the egg contents from chickens fed with regular feed. this is because one of the microorganisms contained in the form of lactic acid bacteria (lab) in the em4 solution in fermented feed can suppress and inhibit the growth of pathogenic bacteria in the animal's digestive tract, thereby reducing the possibility of pathogenic bacterial contamination in vivo during the egg formation process. enteric pathogenic bacteria present escherichia coli and salmonella spp. are enterobacter types of pathogenic bacteria that are commonly found in the contamination of livestock food, especially chicken meat and eggs. these two bacteria are generally found in the digestive tract of livestock animals, but sometimes in vivo contamination during fertilization and egg formation can also occur so that these two bacteria are found in the egg content. (al-bahry et al., 2012). identification and enumeration of e. coli was carried out using emba selective media, where positive results were indicated by the formation of dark purple bacterial colonies with metallic green sheen. meanwhile, the identification and enumeration of salmonella spp. contained in the sample was carried out using selective salmonella-shigella agar media where the results were positive for the content of salmonella spp. characterized by the formation of transparent colonies with black precipitate in the colony’s center. table 3. average total number of enteric pathogenic bacteria of e.coli and salmonella spp. found in egg contents. types of feed e.coli salmonella spp. fermented feed 0,46 × 103 ± 2,12 0 ± 0* regular feed 11,73 × 103 ± 1292,33 0 ± 0* *no salmonella growth was identified, but the growth of other microbes was detected. the results of testing the number of e.coli bacteria in emba media showed that the number of enteric e.coli bacteria observed in sample number 7 of the egg from chicken with fermented feed was 4.6 × 103 cfu/ml, while comparison samples 6 and 7 of the egg from chicken with regular feed amounted to 114 × 103 and 3.3 × 103 cfu/ml. each e. coli positive colony that forms has a flat or smooth surface with a circular shape, convex elevation, and is dark purple in color with a metallic green sheen on the medium. the results of the tests on ss agar medium showed no salmonella spp. bacteria were detected. in all samples, however, there was a color change in the medium to yellow, and the formation of dense, colorless bacterial colonies with entire margins, and flat elevations indicating the growth of other types of bacteria. ss agar medium has selective properties that only allow certain types of bacteria to grow on it, due to this fact, we know that the other types of microbia found on this medium are other types of enterobacter bacteria. the test results of this research are in line with several previous studies where the contamination content of pathogenic bacteria e.coli and salmonella spp. is more commonly found on the outside of egg shells than inside the egg's contents because both types of bacteria usually 40 biology, medicine, & natural product chemistry 14 (1), 2025: 37-41 grow in the digestive tract of animals. several studies that stated similar results include research conducted by fardous and suzzaman in 2015, which stated results of 86.67 growth of pathogenic bacteria on the outside of egg shells, including 10 of 14 serotypes of salmonella bacteria, but no contamination was found at all in the egg contents. (fardous and suzzaman., 2015). research conducted by chaemsanit et al. in 2015 showed relatively similar results where 118 anaerobic bacteria were isolated from 16 egg samples, of which 116 were found on the outer shell and only two from the egg’s contents. the isolate obtained from the contents of eggs in this study was identified as a member of the enterobacteriaceae bacteria. (chaemsanit et al., 2015). finally, research conducted by adesiyun et al. in 2020 found that there were 48.7% e.coli type bacteria and 2% salmonella type bacteria on the outside of egg shells, but only 5.1% contained e.coli type bacteria and no other types of bacteria. salmonella in the eggs. (adesiyun et al., 2020). the minimal content of e.coli and the absence of salmonella content in the eggs are good results which prove that there is very minimal contamination of these two pathogenic bacteria during in vivo egg formation or external contamination during egg production. conclusions the results of this research prove fermented feed increased the quality of chicken eggs, and furthermore also significantly reduces the number of total and enterobacter type pathogenic bacteria in egg content. moreover, this research also proves that giving fermented feed improves eggs’ quality and nutritional content. so making and feeding fermented feed is another better option that can be developed further and applied more widely as an alternative to reduce the growth and contamination of pathogenic bacteria without using antibiotics. acknowledgements: acknowledgement is expressed towards the microbiology laboratory, faculty of biology, universitas gadjah mada for providing the appropriate setting to conduct the research and all parties that fall under this institution. authors’ contributions: prof. dr. endah retnaningrum contributed to this research as the lead supervisor lecturer which aided in the methodology of this research alongside narration of this article. daimeera anja lulu a contributed by designing the study, carrying out the laboratory work, and narration of this article competing interests: the authors declare that there are no competing interests. funding: the authors declare no funding. references adesiyun, a., offiah, n., seepersadsingh, n., rodrigo, s., lashley, v. & musai, l., 2006. frequency and antimicrobial resistance of enteric bacteria with spoilage potential isolated from table eggs. food research international, 39(2), pp.212-219. al-bahry, sn, mahmoud, iy, al-mushrafi, sk, & al-ali, ma 2012. penetration of spoilage and food poisoning bacteria into fresh chicken egg: a public health concern. global journal of bio-science & biotechnology. 1 (1): 33-39. biotechnology, 1(1), pp.33-39. arumugam, k., sudigdoadi, s. & nugraha, g.i., 2015. enteric pathogen bacteria in non-broiler chicken egg shells from traditional market and supermarket, jatinangor subdistrict, west java. althea medical journal, 2(3), pp.414-417. chaemsanit, s., akbar, a. & anal, a.k., 2015. isolation of total aerobic and pathogenic bacteria from table eggs and its contents. food and applied bioscience journal, 3(1), pp.1-9. fajaruddin, junus m dan setyowati e 2013 pengaruh lama fermentasi em4 terhadap kandungan protein kasar padatan kering lumpur organik unit gas bio (the influences of lenght fermentation em4 by containing crude dry solid protein of organic mud of gass bio) jurnal ilmu-ilmu peternakan 23(2) 14-18 fardous, j. and shamsuzzaman, s.m., 2015. detection of potential pathogenic aerobic bacteria from egg shell and egg contents of hen collected from poultry. bangladesh medical research council bulletin, 41(2), pp.67-72. ibrahim, d., abdelfattah-hassan, a., arisha, a.h., abd el-aziz, r.m., sherief, w.r., adli, s.h., el sayed, r. & metwally, a.e., 2020. impact of feeding anaerobically fermented feed supplemented with acidifiers on its quality and growth performance, intestinal villi and enteric pathogens of mulard ducks. livestock science, 242, p.104299. jain, a.k. and yadav, r.a.j.e.s.h., 2017. study of antibiotic resistance in bacteria isolated from table egg. int. j. pharm. bio sci, 8(1), pp.668-674. kanhar, a.r., phulpoto, i.a., ur-rehman, s., qazi, m.a., ghumro, w.a., hussain, s.f., kanhar, a.a., ujjan, j.a. & hussain, a., 2022. isolation, molecular typing and antibiotic sensitivity profiling of enteric bacterial pathogen from chicken eggs. martínez-sánchez, v. and pérez-gálvez, a., 2023. microalgal carotenoids for food and feed applications. handbook of food and feed from microalgae (pp. 133-145). academic press. pijnacker, r., dallman, t.j., tijsma, a.s., hawkins, g., larkin, l., kotila, s.m., amore, g., amato, e., suzuki, p.m., denayer, s. and klamer, s., 2019. an international outbreak of salmonella enterica serotype enteritidis linked to eggs from poland: a microbiological and epidemiological study. the lancet infectious diseases, 19(7), pp.778-786 ranjitkar, s. and engberg, r.m., 2016. the influence of feeding crimped kernel maize silage on growth performance and intestinal colonization with campylobacter jejuni of broilers. avian pathology, 45(2), pp.253-260. ranjitkar, s., karlsson, a.h., petersen, m.a., bredie, w.l., petersen, j.s. and engberg, r.m., 2016. the influence of feeding crimped kernel maize silage on broiler production, nutrient digestibility and meat quality. british poultry science, 57(1), pp.93-104. amantsura & retnaningrum – fermented feed for laying hens: effects on egg quality … 41 ranjitkar, s., lawley, b., tannock, g. and engberg, r.m., 2016. bacterial succession in the broiler gastrointestinal tract. applied and environmental microbiology, 82(8), pp.2399-2410. sugiharto, s. & ranjitkar, s., 2019. recent advances in fermented feeds towards improved broiler chicken performance, gastrointestinal tract microecology and immune responses: a review. animal nutrition, 5(1), pp.1-10. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1151-1157 | doi: 10.14421/biomedich.2025.142.1151-1157 issn 2540-9328 (online) study of sperm utilization in female drosophila melanogaster of bdp and btx strains lisa savitri1*, kharisul ihsan2, elfred rinaldo kasimo1, rochmad krissanjaya1 1department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. 2department of pharmacy, faculty of pharmacy, public health, hospital administration, radiology, universitas strada indonesia, kediri, indonesia. corresponding author lisasavitri@unik-kediri.ac.id manuscript received: 06 august, 2025. revision accepted: 15 october, 2025. published: 04 december, 2025. abstract this study investigates the patterns of sperm utilization in female drosophila melanogaster of the bdp and btx strains through sequential mating with multiple males. the research was conducted as a descriptive observational study without experimental manipulation. females of each strain were crossed with four different males in a specific order, with a two-day interval between each mating. f1 offspring phenotypes were recorded daily over a six-day period following the final mating. data were analyzed descriptively and supported by somatic chromosome reconstruction to identify the male origin of each offspring. the results indicate that sperm utilization in d. melanogaster females occurs in both random and non-random patterns. non-random utilization was observed when offspring were produced exclusively from the first male, with no contribution from subsequent males. for example, in the ♀btx × ♂n cross, only n♀ and n♂ offspring were produced, indicating that sperm from the first male was preferentially used. in contrast, random sperm utilization was evident in later matings, where offspring from multiple males appeared, suggesting that previously stored sperm remained viable and were used alongside or instead of sperm from later matings. these findings highlight the complexity of post-mating sexual selection in d. melanogaster, particularly the role of female sperm storage and utilization mechanisms. the ability of females to either favor the sperm of a particular male or mix sperm from several males may provide evolutionary advantages in terms of genetic diversity and offspring fitness. this study contributes to a better understanding of reproductive strategies and sperm competition in insects, with potential implications for broader studies in evolutionary biology and genetics. keywords: drosophila melanogaster; sperm utilization; sequential mating; sperm competition; chromosome analysis. abbreviations: black dumpy wings (bdp); black taxi (btx); dumpy wings (dp); first filial generation (f1); normal (wild-type) strain of drosophila melanogaster (n); taxi (tx) introduction one of the characteristics of living organisms is reproduction, which serves the purpose of preserving the species. reproduction can occur either sexually or asexually. sexual reproduction typically involves mating. one example of an animal that reproduces sexually through mating is drosophila melanogaster. d. melanogaster is a favored model organism in genetic research due to several advantages, including its small size, complex anatomical structure, ease of handling, ability to be easily bred in the laboratory, simple and inexpensive breeding conditions, short life cycle, and the large number of described mutants whose information is readily available. additionally, it has a small number of chromosomes and features giant chromosomes in the salivary glands of mature larvae (king, 1965). mating in d. melanogaster begins once the flies reach sexual maturity. in males, sexual maturity is marked by the ability to produce and release sperm, while in females it is indicated by the ability to ovulate ootids. in females, sperm from the male is stored in two seminal receptacles and in the paired spermathecae, the latter being the organ for long-term sperm storage (clark et al., 1994). furthermore, clark et al. (1994) state that female d. melanogaster exhibit efficient sperm utilization. their study also found that remating increases fertility in females. when a female mates again with another male, there is a phenomenon where the sperm from the second male is preferentially used. this is undoubtedly influenced by factors that regulate when and how sperm is stored. one such factor determining a female's receptivity to remate is a decrease in the amount of stored sperm (clark et al., 1994). sperm utilization in fertilization can be random or non-random. random utilization occurs when offspring from the first male appear in the progeny of subsequent matings with the second, third, or fourth male—in other words, when earlier sperm continue to contribute to later crosses. non-random utilization occurs when offspring https://doi.org/10.14421/biomedich.2025.142.1151-1157 mailto:lisasavitri@unik-kediri.ac.id 1152 biology, medicine, & natural product chemistry 14 (2), 2025: 1151-1157 from the first male do not appear in subsequent crosses with other males, meaning that each cross produces only the offspring phenotype associated with the male used in that particular mating. materials and methods research design this study is a descriptive observational study, as no specific treatments were applied. the research was conducted by sequentially mating ♀bdp with ♂n, ♂bdp, ♂b, and ♂dp, and ♀btx with ♂n, ♂btx, ♂b, and ♂tx in four different mating schemes, in order to examine sperm utilization in ♀bdp and ♀btx. the data collected were analyzed descriptively using somatic chromosome reconstruction analysis. population and sample the population in this study consisted of d. melanogaster obtained from the genetics laboratory, department of biology, faculty of mathematics and natural sciences, universitas negeri malang. the sample used included d. melanogaster of the following strains: bdp, dp, b, btx, tx, and n. procedure medium preparation the preparation of the medium began with weighing the main ingredients: rajamala banana, cassava tapai, and palm sugar in a 7:2:1 ratio. the banana and tapai were blended with water until smooth. the mixture was then poured into a pot, combined with sliced palm sugar, and cooked over a stove for 45 minutes. once cooked, the mixture was poured hot into jam jars and immediately sealed with sponge stoppers. the jars were then cooled in a container filled with water. after cooling, the lid was opened to add 5–7 yeast granules and a piece of pupariation paper, then resealed with sponge. ampule preparation pupae that had turned dark were collected from d. melanogaster stock cultures of each strain using a fine brush. these pupae were placed into jars containing the prepared medium, then sealed with a piece of sponge or foam. mating procedure in the mating stage, two female genotypes were used: ♀bdp and ♀btx. each was crossed using four different mating schemes. for ♀bdp, the mating schemes were as follows: ▪ type i: ♀bdp was first mated with ♂n. after 2 days, she was transferred to a new medium and mated with ♂bdp while ♂n was removed. two days later, ♂bdp was removed and ♀bdp was crossed with ♂b, followed by ♂dp after ♂b was removed. two days after the final male was removed, ♀bdp was moved to fresh medium without further mating and released once pupae were observed. f1 phenotypes were recorded and counted from days 0–6. the process was repeated three times. ▪ type ii: ♀bdp was first mated with ♂bdp, then sequentially with ♂b, ♂dp, and ♂n, each with a 2day interval and replacement of the previous male. after mating with the final male, ♀bdp was transferred to fresh medium without mating and released after pupae appeared. f1 offspring were observed and recorded over six days, and the procedure was repeated three times. ▪ type iii: ♀bdp was mated with ♂b, ♂dp, ♂n, and ♂bdp. the process of transferring to new media and replacing males every 2 days was the same. phenotypic observations were carried out from day 0 to day 6 and repeated three times. ▪ type iv: ♀bdp was mated with ♂dp, followed by ♂n, ♂bdp, and finally ♂b. each male was removed after 2 days, with the female transferred to fresh media. she was released after pupae were observed. f1 phenotypes were recorded daily for six days, and the procedure was repeated three times. for ♀btx, the same procedure was used with different male partners: ♂n, ♂btx, ♂b, and ♂tx. ▪ type i: ♀btx was sequentially mated with ♂n, ♂btx, ♂b, and ♂tx, each with a 2-day interval and replacement of the previous male. after all matings, ♀btx was moved to fresh medium and released once pupae appeared. f1 phenotypes were observed for six days, and the procedure was repeated three times. ▪ type ii: the mating order was ♂btx, ♂b, ♂tx, and ♂n. the same interval, transfer, and release procedures were followed as in type i. ▪ type iii: ♀btx was mated in sequence with ♂b, ♂tx, ♂n, and ♂btx. f1 phenotypes were recorded from day 0 to day 6, and the procedure was repeated three times. ▪ type iv: the mating order was ♂tx, ♂n, ♂btx, and ♂b. transfers to fresh media occurred every 2 days, with each male removed before the next. observations and recordings of f1 phenotypes were done for six days and repeated three times. results and discussion result the characteristics of d. melanogaster strains (figure 1) used in this study are as follows: a. normal strain (n) ▪ bright red eyes ▪ smooth eye facets ▪ light brown body color ▪ wings cover the body and extend beyond body length savitri et al. – study of sperm utilization in female drosophila melanogaster 1153 b. black strain (b) ▪ red eyes ▪ smooth eye facets ▪ black body color ▪ wings longer than the body c. taxi strain (tx) ▪ red eyes ▪ smooth eye facets ▪ light brown body color ▪ wings longer than the body and lifted upward d. dumpy wings strain (dp) ▪ red eyes ▪ smooth eye facets ▪ light brown body color ▪ wings are shortened and notched e. black taxi strain (btx) ▪ red eyes ▪ smooth eye facets ▪ black body color ▪ wings longer than the body and lifted upward f. black dumpy wings strain (bdp) ▪ red eyes ▪ smooth eye facets ▪ dark brownish-black body color ▪ notched wings figure 1. the characteristics of d. melanogaster strains: a) normal strain (n), b) black strain (b), c) taxi strain (tx), d) dumpy wings strain (dp), e) black taxi strain (btx), and f) black dumpy wings strain (bdp). table 1. phenotypic count data. no type cross f1 sex replicate ʃ total note 1 2 3 1 i ♀bdp><♂n ♀bdp><♂bdp ♀bdp><♂b ♀bdp><♂dp 2 ii ♀bdp><♂bdp bdp ♂ 8 15 23 43 nonrandom bdp ♀ 11 9 20 ♀bdp><♂b bdp ♂ 11 0 11 17 random bdp ♀ 6 0 6 b ♂ 9 0 9 17 b ♀ 8 0 8 ♀bdp><♂dp bdp ♂ 7 0 7 9 random bdp ♀ 2 0 2 dp ♂ 6 0 6 12 dp ♀ 6 0 6 b ♂ 5 0 5 10 b ♀ 5 0 5 ♀bdp><♂n bdp ♂ 4 0 4 7 random bdp ♀ 3 0 3 dp ♂ 3 0 3 11 dp ♀ 8 0 8 b ♂ 5 0 5 9 b ♀ 4 0 4 n ♂ 5 0 5 10 n ♀ 5 0 5 3 iii ♀bdp><♂b ♀bdp><♂dp ♀bdp><♂n ♀bdp><♂bdp 4 iv ♀bdp><♂dp 1154 biology, medicine, & natural product chemistry 14 (2), 2025: 1151-1157 no type cross f1 sex replicate ʃ total note 1 2 3 ♀bdp><♂n ♀bdp><♂bdp ♀bdp><♂b 5 v ♀btx><♂n n ♂ 8 8 13 non random n ♀ 5 5 ♀btx><♂btx btx ♂ 7 7 10 random btx ♀ 3 3 n ♂ 6 6 6 ♀btx><♂b b ♂ 2 2 9 random b ♀ 7 7 n ♂ 1 1 3 n ♀ 2 2 ♀btx><♂tx n ♂ 10 10 12 random n ♀ 2 2 b ♂ 1 1 7 b ♀ 6 6 6 vi ♀btx><♂btx btx ♂ 11 11 23 non-random btx ♀ 12 12 ♀btx><♂b b ♂ 25 25 44 random b ♀ 12 12 n ♂ 4 4 n ♀ 3 3 ♀btx><♂tx b ♂ 9 9 35 random b ♀ 19 19 tx ♂ 2 2 tx ♀ 5 5 ♀btx><♂n b ♂ 5 5 14 random b ♀ 9 9 btx ♂ 4 4 7 btx ♀ 3 3 tx ♂ 5 5 11 tx ♀ 6 6 n ♂ 6 6 10 n ♀ 4 4 7 vii ♀btx><♂b b ♂ 12 12 23 non-random b ♀ 11 11 ♀btx><♂tx tx ♂ 9 9 21 random tx ♀ 12 12 b ♂ 13 13 15 b ♀ 8 8 ♀btx><♂n n ♂ 8 8 15 random n ♀ 7 7 tx ♂ 10 10 19 tx ♀ 9 9 b ♂ 7 7 12 b ♀ 5 5 ♀btx><♂btx btx ♂ 8 8 15 random btx ♀ 7 7 tx ♂ 8 8 14 tx ♀ 6 6 b ♂ 6 6 14 b ♀ 8 8 n ♂ 9 9 14 n ♀ 5 5 8 viii ♀btx><♂tx tx ♂ 7 5 15 27 39 non-random tx ♀ 2 3 7 12 ♀btx><♂n tx ♂ 5 20 7 32 69 random tx ♀ 7 21 9 37 n ♂ 2 5 13 20 37 n ♀ 2 7 8 17 ♀btx><♂btx btx ♂ 2 8 8 18 45 random btx ♀ 8 12 7 27 savitri et al. – study of sperm utilization in female drosophila melanogaster 1155 no type cross f1 sex replicate ʃ total note 1 2 3 tx ♂ 1 0 9 10 18 tx ♀ 1 0 7 8 n ♂ 0 0 8 8 16 n ♀ 0 0 8 8 ♀btx><♂b btx ♂ 5 1 6 12 25 random btx ♀ 3 0 10 13 tx ♂ 2 0 6 8 19 tx ♀ 1 0 10 11 b ♂ 3 5 8 16 26 b ♀ 1 2 7 10 n ♂ 1 0 8 9 18 n ♀ 0 0 9 9 chromosome reconstruction analysis a. somatic chromosome reconstruction ♀bdp 1) ♀bdp x ♂n p bdp bdp x ++ ++ dpb dpb g b dp ++dpb f1 bdp dpb ++ (n) 2) ♀bdp x ♂bdp p bdp bdp x bdp bdp g bdp bdp f1 bdp bdp (bdp) 3) ♀bdp x ♂b p bdp bdp x + + bdp bdp g bdp bdp+ f1 bdp bdp+ (b) 4) ♀bdp x ♂dp p bdp bdp x dpb dpb + + g bdp b+ dp f1 bdp dpb+ (dp) b. somatic chromosome reconstruction ♀ btx 1) ♀btx x ♂ n p btx btx x ++ ++ txb txb g btx b+tx+ f1 btx txb ++ (n) 2) ♀btx x ♂btx p btx btx x btx btx g btx btx f1 btx btx (btx) 3) ♀btx x ♂b p btx btx x + + btx btx g btx btx+ f1 +btx btx (b) 4) ♀btx x ♂tx p btx btx x txb txb + + g btx b+ tx f1 txb btx + (tx) crosses in bdp female individuals based on the observations, data from cross type i, in which a bdp female was mated with a first male n, showed that the resulting f1 offspring had the n phenotype. cross type ii, between a bdp female and a first male bdp, produced f1 offspring with the bdp phenotype. cross type iii, between a bdp female and a first male b, resulted in f1 offspring with the b phenotype. cross type iv, with a first male dp, produced f1 offspring with the dp phenotype. these results indicate that all f1 offspring from crosses between bdp females and the first males were non-random, matching the chromosome reconstruction results. for cross type i with a second male bdp (♀bdp × ♂bdp), no data were obtained as the cross had not yet been performed. cross type ii (♀bdp × ♂b) yielded f1 offspring with both bdp 1156 biology, medicine, & natural product chemistry 14 (2), 2025: 1151-1157 and b phenotypes. the number of individuals with b and bdp phenotypes was equal. cross types iii and iv, with second males dp and n (♀bdp × ♂dp and ♀bdp × ♂n), had no data available yet. cross type i with a third male b (♀bdp × ♂b) also had no available data. cross type ii (♀bdp × ♂dp) produced f1 offspring with bdp, dp, and b phenotypes. among them, the dp phenotype was more frequent than the bdp and b. cross types iii and iv (♀bdp × ♂n and ♀bdp × ♂bdp) also had no data yet. cross type i with a fourth male dp (♀bdp × ♂dp) had no data. cross type ii (♀bdp × ♂n) resulted in f1 offspring with bdp, dp, b, and n phenotypes. the dp phenotype appeared most frequently. cross types iii and iv (♀bdp × ♂bdp and ♀bdp × ♂b) had no data as the crosses had not been performed. overall, observations from crosses between bdp females and second, third, and fourth males showed that the f1 offspring did not only exhibit the phenotypes of the male parent involved in the cross. this contradicts the f1 phenotypes predicted by chromosome reconstruction, suggesting that sperm usage was random. random sperm usage is evident when offspring resulting from the first male also appear in crosses with second, third, or fourth males. crosses in btx female individuals from the observations, in cross type v (♀btx × ♂n), the f1 offspring had the n phenotype. cross type vi (♀btx × ♂btx) resulted in btx phenotype. cross type vii (♀btx × ♂b) produced b phenotype. cross type viii (♀btx × ♂tx) yielded tx phenotype. these results indicate that all f1 offspring from btx females crossed with the first males were non-random and matched the reconstructed chromosome outcomes. cross type v (♀btx × ♂btx) with a second male resulted in f1 offspring with btx and n phenotypes. cross type vi (♀btx × ♂b) produced b and n phenotypes, with more b individuals than n. cross type vii (♀btx × ♂tx) resulted in tx and b phenotypes, with equal numbers of each. cross type viii (♀btx × ♂n) resulted in tx and n phenotypes, with tx being more frequent. in cross type v with a third male b (♀btx × ♂b), the f1 offspring had b and n phenotypes. cross type vi (♀btx × ♂tx) resulted in b and tx phenotypes, with more b than tx. cross type vii (♀btx × ♂n) produced n, tx, and b phenotypes, with tx being the most frequent. cross type viii (♀btx × ♂btx) yielded btx, tx, and n phenotypes, with btx being dominant. cross type v with a fourth male tx (♀btx × ♂tx) yielded b and n phenotypes. cross type vi (♀btx × ♂n) resulted in b, btx, tx, and n phenotypes, with b being the most frequent. cross type vii (♀btx × ♂btx) also resulted in btx, tx, b, and n phenotypes, with btx being dominant. cross type viii (♀btx × ♂b) produced the same set of phenotypes, with b being the most frequent. all cross data between btx females and the second, third, and fourth males show that f1 offspring did not consist solely of the male parent's phenotype, again contradicting the chromosome reconstruction results. this indicates random sperm usage, as offspring from the first male appeared in crosses with subsequent males. discussion sperm utilization in d. melanogaster btx and bdp female individuals analysis of the data showed that all crosses between ♀bdp and ♀btx females with the first male produced f1 offspring matching the chromosome reconstruction. however, crosses with the second, third, and fourth males yielded f1 phenotypes inconsistent with the expected results, suggesting that sperm utilization in those cases was random. in the first mating, fertilization was non-random because the female had only mated with one male, and thus only one type of sperm was used. in subsequent matings, the appearance of phenotypes associated with the first male's sperm indicates random sperm utilization. this phenomenon is known as sperm displacement or sperm precedence. sperm displacement occurs when sperm from the first male is physically moved or displaced within the female's sperm storage organs (seminal receptacle and spermatheca) by sperm from a subsequent male. this reduces the likelihood that the first male’s sperm will be used for fertilization. sperm precedence means that sperm from the second male is more likely to fertilize eggs due to the influence of his seminal fluid. this fluid can suppress the motility and capacitation of the first male's sperm, giving the second male's sperm an advantage. according to clark et al. (1994), sperm precedence involves two main aspects: (1) physically blocking or displacing earlier sperm, and (2) resisting displacement after copulation. in this study, a single d. melanogaster female was sequentially mated with four different males every two days. this interval aligns with the female's receptivity — her willingness to accept a new mate. after mating, females generally show decreased receptivity due to behavioral and physiological changes, including reduced attractiveness and reluctance to remate (grillet, 2006; singh, 2002). these post-mating changes are driven by copulation effects (where male seminal proteins suppress female receptivity to prevent sperm competition) and sperm effects (linked to sperm quantity and usage). the nervous system female receptivity, and stimulation from sperm movement in storage organs influences behavior. importantly, d. melanogaster females do not wait for stored sperm to be depleted before remating. thus, sperm from the first male remains present during later matings, enabling sperm competition. competition arises as new sperm enters storage and competes with earlier sperm. several factors influence remating frequency and sperm competition, such as sperm count, seminal fluid composition, nutrition, egg laying, testes size, and body size. specific genes and savitri et al. – study of sperm utilization in female drosophila melanogaster 1157 proteins are known to play roles in sperm competition. in d. melanogaster, sperm from later males is generally more successful. seminal fluid from subsequent males can suppress capacitation of earlier sperm, without necessarily removing them from storage. sperm storage organs in females play a key role in prioritizing sperm usage or preventing fertilization by earlier sperm. male accessory gland proteins are critical in this process (adrianne et al., 2008). genes expressed in the spermatheca are known to encode serine proteases, which interact with male proteins and influence fertilization outcomes. these proteases may regulate the internal environment of the spermatheca similarly to human prostate-specific antigen (psa), which aids semen liquefaction. other genes, such as trehalase, may help maintain sperm by regulating sugar metabolism. in short, interactions between male seminal proteins and female reproductive proteins determine which sperm succeed. in this study, such interactions likely resulted in sperm from second or later males being favored, which explains why their phenotypes dominated in the f1 generation. conclusions based on the discussion above, a preliminary conclusion can be drawn that sperm utilization in d. melanogaster females of the bdp and btx strains occurs both randomly and non-randomly. non-random utilization is observed in the first mating; for example, in the cross ♀btx × ♂n (first male), the resulting f1 offspring consisted only of n♀ and n♂ phenotypes. in contrast, random sperm utilization appears in the second, third, and fourth matings — in other words, in crosses involving subsequent males. acknowledgements: thank you to the genetics laboratory, faculty of mathematics and natural sciences, state university of malang, for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors wrote the manuscript and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there are no funding. references adrianne. (2008). an evolutionary expressed sequence tag analysis of drosophila spermatheca genes. the international journal of organic evolution 62:11 anonim. (2008). siklus hidup drosophila. (online) http://zarzen.wordpress.com/2008/09/27/siklus-hidupdrosophila/ diakses tanggal 2 desember 2011 borror. (1992). pengenalan pelajaran serangga edisi keenam. gadjah mada university press: yogyakarta clark. (1994). variation in sperm displacement and its association with accessory gland protein loci in d. melanogaster. (online) http://www.genetics.org/content/139/1/189.full.pdf 1994 diakses tanggal 2 desember 2011 corebima, a, d. (2003). genetika mendel. airlangga university press: surabaya grillet. (2006). a drosophila male pheromone affects female sexual receptivity. (online) http://rspb.royalsocietypublishing.org/content/ 273/1584/315.full.pdf+html diakses tanggal 7 desember 2011 jumar. (2000). entomologi pertanian. pt rineka cipta: jakarta king, r, c. (1965). genetics. oxford university press: new york muliati, l. (2000). pengaruh strain dan umur jantan terhadap jumlah turunan jantan dan betina d. melanogaster. malang: fmipa universitas negeri malang: skripsi tidak diterbitkan pitnick. (1999). evolution of multiple kinds of female spermstorage organs in drosophila. (online) http://online.sfsu.edu/~gs/spicer/pages/spicerpdf/pitnick99.pdf diakses tanggal 7 desember 2011 singh. (2006). female remating in drosophila annanassae: evidence for the effect of density on female remating frequency. (online) http://www.springerlink.com/content/n607425vq73n030m/fullt ext.pdf diakses tanggal 7 desember 2011 singh. (2002). female remating, sperm competition and sexual selection in drosophila. (online) http://www.funpecrp.com.br/gmr/year2002/vol31/pdf/gmr0034.pdf diakses tanggal 7 desember 2011 this page intentionally left blank untitled-1 biology, medicine, & natural product chemistry volume 5 – number 2 – 2016 issn 2089-6514 (paper) | issn 2540-9328 (online) honorary editors: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa editor-in-chief: muhammad jafar luthfi department of biology, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty 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[indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com starch-glycerol based edible film and effect of rosella (hibiscus sabdariffa linn) extract and surimi dumbo catfish (clarias gariepinus) addition on its mechanical properties endaruji sedyadi, syafiana khusna aini, dewi anggraini, dian prihatiningtias ekawati optimization of binocular microscope with micro digital camera for measuring seminiferous tubules epithelium height sutriyono histological study of common house gecko (hemidactylus frenatus) regenerated tail rakhmiyati, muhammad jafar luthfi effect of addition of soursop leaf extract to ganyong (canna edulis ker.) starch edible film and its application in red grape storage time erni widyastuti, endaruji sedyadi, susy yunita prabawati a stability mathematical model of nasopharyngeal carcinoma on cellular level sugiyanto, fajar adi kusumo, lina aryati, mardiah suci hardianti 33-40 41-47 49-53 55-59 61-64 volume 5 number 2 2016 published twice a year printed in indonesia front cover: hoya sp. (photo: widodo) issn 2089-6514 (paper) issn 2540-9328 (online) 3: font 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1265-1271 | doi: 10.14421/biomedich.2025.142.1265-1271 issn 2540-9328 (online) in silico exploration of bioactive compounds from dracaena cochinchinensis as potential inhibitors of streptococcus pyogenes inosine-5'-monophosphate dehydrogenase (impdh) septi handayani1*, nawan2, agnes immanuela toemon3 1departement of biochemistry; 2departement of microbiology; 3departement of parasitology, faculty of medicine, university of palangka raya, indonesia. corresponding author* septi@med.upr.ac.id abstract the emergence of multidrug-resistant streptococcus pyogenes presents a significant global health threat, demanding the urgent discovery of novel antibacterial agents. this study utilized a comprehensive in silico framework to investigate 17 compounds from the traditional medicinal plant dracaena cochinchinensis as potential inhibitors of inosine-5'-monophosphate dehydrogenase (impdh), a validated antimicrobial drug target. the workflow included predictive modeling of physicochemical properties, pharmacokinetics (adme), toxicity profiles, and molecular docking simulations to elucidate binding affinities and interaction patterns within the enzyme's active site. physicochemical analysis revealed that 11 of the 17 compounds exhibited drug-like properties. molecular docking identified several ligands with high binding affinities, notably isopimaric acid (-8.2 kcal/mol) and cochinchinenene d (-8.1 kcal/mol), whose stability was mediated by interactions with key catalytic residues. admet predictions indicated that most compounds possess favorable pharmacokinetic profiles. crucially, isopimaric acid demonstrated a superior safety profile, with a high ld50 (5000 mg/kg), no predicted mutagenicity, and no risk of drug-induced liver injury (dili). this computational investigation successfully identified isopimaric acid as a standout candidate, and its combination of strong target affinity and a favorable admet profile positions it as a promising scaffold for the development of novel antibacterial agents against s. pyogenes. these findings provide a strong impetus for experimental validation. keywords: admet; antibiotic resistance; dracaena cochinchinensis; impdh; molecular docking; streptococcus pyogenes. abbreviations: adme: absorption, distribution, metabolism, excretion; dili: drug-induced liver injury; impdh: inosine-5'monophosphate dehydrogenase; ld50: lethal dose 50. introduction streptococcus pyogenes remains a significant bacterial pathogen, responsible for a wide spectrum of human diseases ranging from mild localized infections to severe, life-threatening invasive conditions (kanwal & vaitla, 2023). this gram-positive bacterium is a primary cause of millions of cases of pharyngitis and invasive infections annually, contributing to substantial global morbidity and mortality (kebede et al., 2021). the primary line of treatment for s. pyogenes infections has traditionally been β-lactam antibiotics, such as penicillin. however, the emergence of strains with reduced susceptibility and outright resistance to multiple antibiotic classes, including macrolides and fluoroquinolones, poses a critical threat to public health (cattoir, 2022; ünübol et al., 2025). high rates of resistance have been documented globally, limiting therapeutic options and increasing the risk of treatment failure. this escalating crisis underscores the urgent need to explore and develop novel antibacterial agents with alternative mechanisms of action (nawan et al., 2020; nawan & handayani, 2021). natural products, particularly those derived from medicinal plants, represent a rich reservoir for drug discovery. the red resin of dracaena cochinchinensis, commonly known as "dragon's blood," has a long history of use in traditional chinese medicine for treating various ailments. modern scientific studies have validated its broad spectrum of bioactivities, including anti-inflammatory, anti-tumor, and antibacterial properties (gupta & gupta, 2011; he et al., 2021). effective antibacterial drug development hinges on the identification of essential molecular targets within the pathogen. inosine-5'-monophosphate dehydrogenase (impdh) is a rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides, which are manuscript received: 04 october, 2025. revision accepted: 04 december, 2025. published: 13 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1265-1271 1266 biology, medicine, & natural product chemistry 14 (2), 2025: 1265-1271 indispensable for bacterial dna and rna synthesis (modi et al., 2021). its critical role in pathogen viability and proliferation has established impdh as a validated and attractive target for novel antibacterial agents (ramanujan, 2020). this study aimed to investigate the potential of bioactive compounds from d. cochinchinensis as inhibitors of s. pyogenes impdh through a comprehensive computational approach. by integrating molecular docking simulations with predictions of pharmacokinetic (adme) and toxicity profiles, we identify the most promising lead compounds that could serve as scaffolds for the development of new therapeutics against s. pyogenes. materials and methods software and computational resources all computational tasks were performed on a pc equipped with an intel® core™ i7-5500u cpu @ 2.40ghz, 16 gb of ram, running a windows 11 pro 64-bit operating system. the software suite included autodock tools 1.5.7, biovia discovery studio visualizer 2021, and the online web servers pkcsm and protox-ii for admet predictions. ligand preparation three-dimensional (3d) structures of 17 selected bioactive compounds from d. cochinchinensis and the comparator drug, penicillin g, were retrieved from the pubchem database (https://pubchem.ncbi.nlm.nih.gov/) in .sdf format. the structures were then imported into biovia discovery studio visualizer 2021, processed, and saved in .pdb format for further preparation. target protein preparation the 3d crystal structure of s. pyogenes inosine-5'monophosphate dehydrogenase (impdh) was obtained from the rcsb protein data bank (pdb id: 1zfj). the protein was prepared for docking using autodock tools 1.5.7. this process involved removing all water molecules and native co-crystallized ligands, followed by the addition of polar hydrogen atoms and kollman charges to the protein structure. the final prepared receptor was saved in the .pdbqt file format. physicochemical and admet predictions the drug-likeness of each compound was evaluated based on lipinski’s rule of five (molecular weight ≤ 500 da; log p ≤ 5; h-bond donors ≤ 5; h-bond acceptors ≤ 10) using the pkcsm web server (http://biosig.unimelb.edu.au/pkcsm/). the same server was used to predict pharmacokinetic parameters (absorption, distribution, metabolism, excretion adme). toxicity profiles, including ld50, ames mutagenicity, and drug-induced liver injury (dili), were predicted using both pkcsm and the protox-ii server (https://tox-new.charite.de/protox_ii/). molecular docking simulation molecular docking was conducted using autodock tools 1.5.7 to analyze the binding interactions between the prepared ligands and the impdh receptor. a grid box was defined to encompass the active site of the enzyme. the docking simulations were performed to calculate the binding affinity (δg, in kcal/mol) for each ligand, and the lowest energy conformation was selected for further analysis. interaction visualization and analysis post-docking analysis was performed using biovia discovery studio visualizer 2021. the 2d and 3d interaction diagrams were generated to identify the specific amino acid residues involved in binding and to characterize the nature of the molecular interactions (e.g., hydrogen bonds, hydrophobic contacts, pi-interactions). results and discussion physicochemical property analysis and candidate filtering the initial step in our virtual screening workflow involved assessing the drug-likeness of the 17 compounds based on lipinski’s rule of five to prioritize candidates with a higher probability of oral bioavailability (lipinski et al., 2001). as detailed in table 1, this analysis served as a critical filter. six compounds (cochinchinenin, (2r)-8methylsocotrin-4'-ol, cochinchinenene a, cochinchinenene b, cochinchinenin b, and cochinchinenin c) were found to violate two or more of lipinski's rules. significant deviations, particularly in molecular weight (mw > 500 da) and lipophilicity (logp > 5), are strongly correlated with poor absorption and permeability, making them less desirable as oral drug candidates (egbert et al., 2019; nhlapho et al., 2024). consequently, these six compounds were excluded from further investigation. the remaining 11 compounds, which either fully complied with the rules or had only a single violation, were deemed promising and advanced to the subsequent stages of the analysis. this filtered set of candidates is predicted to possess more favorable pharmacokinetic properties. handayani et al. – in silico inhibition of s. pyogenes impdh 1267 table 1. physicochemical property prediction of compounds from d. cochinchinensis based on lipinski's rule of five. no compound name parameters of lipinski's law of five application of lipinski's law of five mw (g/mol) log p hba hbd 1 isopimaric acid 302.458 5.206 2 1 violate the lipinski's ro5 (1 time) 2 2'-o methylisoliquiritigenin 270.284 3.003 4 2 respect the lipinski's ro5 3 loureirin a 286.327 3.225 4 1 respect the lipinski's ro5 4 davidigenin 258.273 2.619 4 3 respect the lipinski's ro5 5 4'-hydroxy-2,6-dimethoxydihydrochalcone 286.327 3.225 4 1 respect the lipinski's ro5 6 4,4'-dihydroxy-2,6dimethoxydihydrochalcone 302.326 2.931 5 2 respect the lipinski's ro5 7 cochinchinenin 514.574 5.803 7 5 violate the lipinski's ro5 (2 times) 8 (2r)-8-methylsocotrin-4'-ol 512.602 6.657 6 4 violate the lipinski's ro5 (2 times) 9 diphenolic acid 286.327 3.269 4 3 respect the lipinski's ro5 10 cochinchinenene a 526.629 7.067 6 2 violate the lipinski's ro5 (2 times) 11 cochinchinenene b 512.602 6.764 6 3 violate the lipinski's ro5 (2 times) 12 cochinchinenene c 498.575 6.461 6 4 violate the lipinski's ro5 (1 time) 13 cochinchinenene d 484.548 5.515 6 4 violate the lipinski's ro5 (1 time) 14 cochinchinenin b 542.628 6.409 7 3 violate the lipinski's ro5 (2 times) 15 cochinchinenin c 542.628 6.409 7 3 violate the lipinski's ro5 (2 times) 16 trans-3,5-dihydroxy-4'-methoxystilbene 242.274 3.277 3 2 respect the lipinski's ro5 17 (2s)-5-methoxy-6-methylflavan-7-ol 270.328 3.776 3 1 respect the lipinski's ro5 18 penicillin g 334.397 0.861 6 2 respect the lipinski's ro5 molecular docking and binding affinity of filtered candidates the 11 compounds that passed the physicochemical screening were docked into the active site of impdh (1zfj). the predicted binding affinities are presented in table 2. all candidates exhibited strong binding energies, ranging from −6.7 to −8.2 kcal/mol. a lower, more negative binding energy correlates with higher binding affinity and a more stable ligand-receptor complex (buchwald, 2019; monteiro et al., 2022). isopimaric acid (−8.2 kcal/mol), cochinchinenene d (-8.1 kcal/mol), and 2'-o methylisoliquiritigenin (−8.0 kcal/mol) emerged as the top three compounds with the most potent predicted binding to the target enzyme. table 2. binding affinities of filtered compounds against impdh (1zfj). protein-ligand binding affinity 1zfj _ isopimaric acid -8,2 1zfj_ cochinchinenene d -8,1 1zfj_2'-o methylisoliquiritigenin -8 1zfj_ loureirin a -7,7 1zfj_ diphenolic acid -7,7 1zfj_(2s)-5-methoxy-6-methylflavan-7-ol -7,7 1zfj_ cochinchinenene c -7,5 1zfj_ davidigenin -7,4 1zfj_ trans-3,5-dihydroxy-4'-methoxystilbene -7,4 1zfj_4,4'-dihydroxy-2,6-dimethoxydihydrochalcone -7,1 1zfj_4'-hydroxy-2,6-dimethoxydihydrochalcone -6,7 pharmacokinetic (adme) profile of prioritized candidates the adme properties of the 11 prioritized compounds were predicted to assess their potential behavior in vivo (table 3). all 11 compounds demonstrated high predicted human intestinal absorption (hia > 85%), suggesting excellent absorption from the gastrointestinal tract. however, variability was noted in their distribution properties. for instance, only isopimaric acid and (2s)-5methoxy-6-methylflavan-7-ol were predicted to penetrate the blood-brain barrier (bbb). in terms of metabolism, several compounds, including loureirin a and the two dihydrochalcones, were predicted to be inhibitors of key cytochrome p450 enzymes (cyp3a4 or cyp2d6), which is a critical consideration for potential drug-drug interactions (pires et al., 2015). 1268 biology, medicine, & natural product chemistry 14 (2), 2025: 1265-1271 table 3. predicted pharmacokinetic (adme) profile of the filtered candidate compounds. compound name hia (%) skin permeability (log kp) bbb penetrant vdss (log l/kg) cyp2d6 inhibitor cyp3a4 inhibitor total clearance (log ml/min/kg) isopimaric acid 95.8 -2.04 yes 0.39 no no 1.27 2'-o methylisoliquiritigenin 92.1 -2.06 no 0.98 no no 4.28 loureirin a 93.3 -2.76 no 0.76 no yes 5.65 davidigenin 90.1 -1.98 no 0.81 no no 5.43 4'-hydroxy-2,6dimethoxydihydrochalcone 92.5 -2.53 no 1.09 no yes 6.59 4,4'-dihydroxy-2,6dimethoxydihydrochalcone 93.1 -2.58 no 1.37 yes yes 8.10 diphenolic acid 85.9 -1.53 no 0.47 no no 3.21 cochinchinenene c 88.3 -0.57 no 0.66 yes yes 7.40 cochinchinenene d 87.5 0.52 no 0.64 yes yes 6.81 trans-3,5-dihydroxy-4'methoxystilbene 91.2 -2.20 no 1.38 no yes 4.54 (2s)-5-methoxy-6-methylflavan7-ol 93.5 -2.32 yes 2.15 yes no 7.10 penicillin g (control) 89.9 -3.64 no 0.26 no no 5.47 hia: human intestinal absorption; bbb: blood-brain barrier; vdss: volume of distribution at steady state. toxicity profile of prioritized candidates the safety profiles of the 11 candidate compounds were assessed using several key toxicity endpoints (table 4). a crucial finding was that all compounds were predicted to be non-mutagenic in the ames test, a highly desirable attribute for any drug candidate. in terms of acute oral toxicity, the compounds ranged from class 4 to class 5, with isopimaric acid (ld50 5000 mg/kg, class 5) being predicted as the least acutely toxic. the prediction for drug-induced liver injury (dili) identified several compounds, including davidigenin and cochinchinenene d, as potentially "toxic." conversely, isopimaric acid and loureirin a were predicted to be "safe." significantly, the computational model also accurately classified the control drug, penicillin g, as "safe" for dili, which is consistent with its well-established clinical safety profile and supports the reliability of the predictions for the test compounds. table 4. predicted toxicity profiles of the filtered candidate compounds. no. compound name ld50 (mg/kg) toxicity class* ames toxicity dili** 1 isopimaric acid 5000 4 safe safe 2 2'-o methylisoliquiritigenin 1190 4 safe toxic 3 loureirin a 500 4 safe safe 4 davidigenin 500 4 safe toxic 5 4'-hydroxy-2,6-dimethoxydihydrochalcone 1190 4 safe safe 6 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone 1190 4 safe safe 7 diphenolic acid 1320 4 safe toxic 8 cochinchinenene c 302 4 safe toxic 9 cochinchinenene d 302 4 safe toxic 10 trans-3,5-dihydroxy-4'-methoxystilbene 2090 5 safe toxic 11 (2s)-5-methoxy-6-methylflavan-7-ol 2500 5 safe safe 12 penicillin g (control) 1000 4 safe safe *toxicity class based on globally harmonized system (ghs): class 4 (300 < ld50 ≤ 2000, warning) & class 5 (2000 < ld50 ≤ 5000, warning may be harmful). **dili: drug-induced liver injury. analysis of ligand-receptor interactions to understand the molecular basis for the high binding affinities observed in docking, the specific interactions between the ligands and the impdh active site were analyzed. table 5 provides a summary of the key interacting residues for each of the 11 compounds, while figure 1 illustrates the binding modes of the top three candidates. the potent binding of isopimaric acid (-8.2 kcal/mol) is anchored by three conventional hydrogen bonds with the side chains of thr312 and cys310. similarly, cochinchinenene d (-8.1 kcal/mol) achieves its stable pose through an extensive network of hydrogen bonds handayani et al. – in silico inhibition of s. pyogenes impdh 1269 with residues such as lys208 and tyr120, complemented by multiple hydrophobic contacts. the consistent interaction of multiple high-affinity ligands with residues like tyr120 and gly124 suggests these residues are critical for ligand recognition and stabilization within the catalytic pocket. table 5. summary of key molecular interactions of filtered ligands with the impdh (1zfj) active site. compound name binding affinity (kcal/mol) key interacting residues isopimaric acid -8.2 thr312, cys310, ile309 cochinchinenene d -8.1 lys208, tyr120, arg67, ser93, lys60, ile63, val97 2'-o methylisoliquiritigenin -8.0 val97, gly124, gly96, ile122, arg92, leu200 loureirin a -7.7 gly124, val97, gly96, lys89, arg92, ile122 diphenolic acid -7.7 tyr13, glu467, ala463, val466, tyr327, phe465 (2s)-5-methoxy-6-methylflavan-7-ol -7.7 gly303, met53, cys310, ala51 cochinchinenene c -7.5 met368, lys347, pro488, lys430, tyr348 davidigenin -7.4 gly96, gly124, tyr120, val97, ile122, lys187 trans-3,5-dihydroxy-4'-methoxystilbene -7.4 ser308, met53, cys310, ala255 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone -7.1 gly96, lys204, tyr120, gly124, thr202, lys187 4'-hydroxy-2,6-dimethoxydihydrochalcone -6.7 asp253, cys310, phe465, tyr327, tyr13 residues predominantly involved in hydrogen bonding are highlighted in bold. figure 1. 2d plots illustrating the molecular interactions between the top three candidate compounds and the active site of impdh (1zfj). the diagrams depict the binding modes for (a) isopimaric acid, (b) cochinchinenene d, and (c) 2'-o methylisoliquiritigenin. conventional hydrogen bonds are shown as green dashed lines with arrows pointing from donor to acceptor. hydrophobic interactions (e.g., alkyl, pi-alkyl) are represented by light green splashes. key interacting amino acid residues are labeled with their three-letter code and sequence number. discussion the application of a hierarchical virtual screening workflow, as employed in this study, represents a rational and efficient strategy in modern drug discovery (van de waterbeemd & gifford, 2003). by first filtering a compound library based on fundamental drug-like properties, we effectively focused our computational efforts on candidates with a higher intrinsic probability of success, thereby streamlining the discovery process. the initial screening using lipinski’s rule of five (table 1) proved to be a decisive first step. the exclusion of six compounds with significant physicochemical liabilities allowed us to concentrate on 11 candidates possessing more favorable profiles for oral administration (hughes et al., 2011; leeson & springthorpe, 2007). this approach is crucial for mitigating the risk of late-stage failures due to poor pharmacokinetics (kola & landis, 2004). among the filtered candidates, the molecular docking results (table 2) provided strong evidence of potential inhibitory activity. the high binding affinities of isopimaric acid (−8.2 kcal/mol) and cochinchinenene d (−8.1 kcal/mol) are particularly noteworthy. the analysis of their binding modes (table 5 and figure 1) provides a structural rationale for this potency. the formation of specific hydrogen bonds and hydrophobic interactions with key residues within the impdh active site suggests a well-defined mechanism of binding. for example, the interactions of isopimaric acid with thr312 and cys310 are significant, as these residues are located in regions critical for enzymatic function (braun-sand & peetz, 2010). however, high potency must be balanced with an acceptable safety and pharmacokinetic profile. the admet analysis (table 3 and table 4) was instrumental in differentiating the top-binding compounds. here, isopimaric acid clearly distinguished itself as a superior lead candidate. it not only possesses the highest binding affinity but also exhibits an exemplary predicted safety profile: low acute toxicity (class 5), a non-mutagenic (a) isopimaric acid (−8.2 kcal/mol) (b) cochinchinenene d (−8.1 kcal/mol) (c) 2'-o methylisoliquiritigenin (−8.0 kcal/mol) 1270 biology, medicine, & natural product chemistry 14 (2), 2025: 1265-1271 prediction, and no risk of hepatotoxicity (dili). this combination of potent activity and predicted safety is the hallmark of a promising drug candidate (waring et al., 2015). in contrast, other potent binders like cochinchinenene d, while showing strong target engagement, were flagged for potential dili risk (table 4). this finding does not outright disqualify them but indicates that they may carry a higher risk of adverse effects and would require careful toxicological evaluation or structural modifications to mitigate this liability. this comparative analysis demonstrates the critical importance of integrating admet profiling early in the discovery pipeline to prioritize compounds that balance efficacy with safety (van de waterbeemd & gifford, 2003). while these in silico findings are highly encouraging, it is essential to acknowledge their predictive nature. computational models are powerful tools for hypothesis generation but are not a substitute for empirical data (agu et al., 2023). the next crucial phase will be the experimental validation of these predictions. this should involve in vitro assays to confirm the impdh inhibitory activity of the prioritized compounds and to determine their minimum inhibitory concentrations (mics) against clinical isolates of s. pyogenes. the compelling computational profile of isopimaric acid, in particular, provides a strong justification for its advancement into these more resource-intensive stages of the drug discovery process (lionta et al., 2014). conclusions this in silico investigation has successfully employed a hierarchical virtual screening workflow to explore the therapeutic potential of compounds from dracaena cochinchinensis against streptococcus pyogenes impdh. through a systematic evaluation of drug-likeness, target affinity, pharmacokinetics, and safety, this study has identified several promising lead candidates. notably, isopimaric acid emerged as a superior candidate, distinguished by its potent binding affinity for the target enzyme, compliance with key drug-like properties, and an excellent predicted safety profile free from mutagenic and hepatotoxic liabilities. these comprehensive computational findings strongly support the prioritization of isopimaric acid for subsequent experimental validation, including in vitro enzymatic and antibacterial assays, to confirm its potential as a novel scaffold for developing new antibacterial agents against s. pyogenes. acknowledgements: the authors would like to acknowledge the developers of the computational tools and databases used in this study, including autodock, biovia discovery studio, pubchem, the protein data bank, pkcsm, and protox-ii, which were instrumental in conducting this research. authors’ contributions: septi handayani & nawan designed the study. agnes immanuela toemon carried out the computational work. septi handayani analyzed the data. all authors wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare no funding was received for this research. references agu, p. c., afiukwa, c. a., orji, o. u., ezeh, e. m., ofoke, i. h., ogbu, c. o., ugwuja, e. i., & aja, p. m. 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(2015). an analysis of the attrition of drug candidates from four major pharmaceutical companies. nature reviews. drug discovery, 14(7), 475–486. https://doi.org/10.1038/nrd4609 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1069-1076 | doi: 10.14421/biomedich.2025.142.1069-1076 issn 2540-9328 (online) phytochemical screening and antioxidant activity of dyera polyphylla (miq) steenis extract using the dpph method putri dharma monika, rini muharini*, eni mayasari chemistry education department, faculty of teacher training and education, tanjungpura university, jl. prof. dr. h. hadari nawawi, kota pontianak,78124, kalimantan barat, indonesia. corresponding author* rini.muharini@fkip.untan.ac.id abstract this research aimed to determine the chemical profile and antioxidant activity of the jelutung rawa plant (dyera polyphylla). phytochemical screening tests were conducted using specific reagents, thin-layer chromatography (tlc), and high-performance liquid chromatography (hplc). determination of antioxidant activity was performed the dpph method. the research results showed the presence of secondary metabolite compounds, namely alkaloids, flavonoids, phenolics, tannins, and terpenoids in the leaves, while the contain alkaloids, phenolics, tannins, and terpenoids. the antioxidant activity in this plant has been identified as a strong natural antioxidant. keywords: antioxidant; dpph; dyera polyphylla. introduction jelutung (dyera spp.) is a type of plant from the apocynaceae family. based on its habitat, jelutung plants are divided into two types: terrestrial jelutung (dyera costulata) and swamp jelutung (dyera polyphylla) (tanjungsari et al., 2016). in society, the d. costulata plant has become one of the sources of income because it has a high market value. the d. costulata plant has many benefits, including in the rubber industry where jelutung sap is used as the main ingredient in the production of chewing gum, paint, and electrical cable insulators, while d. costulata wood is used for making pencils, wood carvings, ceilings, wooden sandals, partition boards, and plywood (sofiyuddin & janudianto, 2018). moreover, the use of d. costulata in the health field as a traditional medicine has been known for a long time. in indonesia, the anak dalam tribe utilises the sap of d. costulata as an external medicine for animal bites, for swollen toothaches, and for boils (aminah et al., 2016). the barks and leaves of d. costulata are used as medicine for fever, inflammation, and pain (subhadhirasakul et al., 2003). however, there is limited information on the use of d. polyphylla. meanwhile, this plant is commonly found in the peat swamp forests of sumatra and kalimantan, including west kalimantan (tata et al., 2015). pharmacological studies of d. costulata extract showed that this plant has antioxidant, analgesic, and antidiabetic activities. meanwhile, chemical studies of the d. costulata plant reported such as 𝛽-amyrin, rhamnazin, quercetin-3-o-𝛼-rhamnopyranoside, benzoic acid, lupeol, okrolifuanin a, okrolifuanin e, okrolifuanin f, dehydrochrolifuanin a, dehydrochrolifuanin e, and dehydrochrolifuanin f from this plant (subhadhirasakul et al., 2003; reanmongkol et al., 2002; arjinal et al., 2020; mirand et al., 1983). dyera polyphylla is a plant that naturally grows in peat swamp areas. this plant has good adaptations to waterlogged environments because it has breathing roots (pneumatophores) (tata et al., 2015). the ability of a plant to adapt to its environment can affect the phytochemical content within it. in addition to internal factors, plant adaptation is also influenced by several external factors such as temperature, light, ph, humidity, altitude, and nutrient content in the soil, which can affect the development and growth of a plant (katuuk et al., 2018). extreme environmental conditions can increase the production of reactive oxygen species (ros) free radicals in plant tissues, which can trigger cell damage. as a result, plants will protect themselves by producing antioxidant compounds (utomo et al., 2020). the difference in habitat between d. polyphylla and d. costulata suggests that these two plants have different compound contents and biological activities. therefore, screening the chemical content and antioxidant activity of d. polyphylla is necessary, so that it can provide manuscript received: 07 july, 2025. revision accepted: 15 august, 2025. published: 26 november, 2025. https://doi.org/10.14421/biomedich.2025.142.1069-1076 1070 biology, medicine, & natural product chemistry 14 (2), 2025: 1069-1076 information regarding the potential utilisation of d. polyphylla in society and related fields. materials and methods materials and equipment the sample used in this study was d. polyphylla, which had been identified by the national research and innovation agency (brin) cibinong, bogor with the code b-3284/ii.6.2/ir.01.02/9/2024. the solvents used were distilled solvents including methanol, n-hexane, and ethyl acetate. the reagents used in the phytochemical screening included dragendroff, mayer, wagner, shinoda, liberman-burchard, fecl3 5% and 10%. f254 silica gel plates (merck kgaa, germany) were used in phytochemical screening. the materials used in determining antioxidant activity were dpph (aldrich chemistry) and methanol p.a (smart-lab, indonesia). the equipment used in this research included an oven, blender (philips 600w), a digital analytical balance (advank innotech), a rotary vacuum evaporator (ika® rv8), hot plate (ika® c-mag hs 7), an uv lamp 254 nm, uv-vis spectrophotometer (shimadzu uv-19900i), and cuvette (shimadzu). procedures sample preparation and extraction the d. polyphylla samples studied were parts of the leaves, bark, and stems. the extraction process began with processing the identified plant into simplicia through several stages, namely sorting, washing, cutting, drying, and grinding. the powdered simplicia of leaves (3.53 g), bark (68.8 g), and wood (313 g) were macerated using the maceration method. in total maceration, one-third of the total weight of the simplicia was macerated using methanol solvent for 3x24 hours with solvent replacement every 24 hours. meanwhile, in stepwise maceration, two-thirds of the total weight of the simplicia was macerated with n-hexane for 3x24 hours with solvent replacement every 24 hours. the residue obtained was re-macerated using ethyl acetate and methanol solvents with the same treatment. the obtained macerate was concentrated using a rotary vacuum evaporator. the yield calculation was obtained from the comparison of the thick extract obtained with the initial weight of the sample before the extraction treatment (putri et al., 2024a). a yield is considered good if the value obtained is more than 10% (lutfiah et al., 2024). phytochemical screening qualitative testing of the extract from the leaves, stems, and bark of jelutung rawa was performed using phytochemical screening methods, thin-layer chromatography (tlc), and chromatogram profile analysis on hplc. dragendorff, mayer, and wagner reagents for alkaloid tests, shinoda reagent for flavonoid tests, liebermann-burchard reagent for terpenoid tests, 5% fecl3 reagent for phenolic tests, and 10% fecl3 reagent for tannin tests (khafid et al., 2023; oktavia & sutoyo, 2021; tunny et al., 2020). the complexity and polarity properties of the compounds contained in the d. polyphylla extract were analysed using thin-layer chromatography (tlc) technique. the eluent systems used were n-hexane:ethyl acetate (7:3), n-hexane:ethyl acetate (1:9), and chcl3:acetone (1:1). the extract that has been dissolved in methanol is spotted on the tlc plate and eluted with each eluent. observation of the spots was conducted under uv light at 254 nm. the plate was then sprayed with 2% ceso4 and heated. the results of the tlc chromatogram were analysed based on the number of spots. next, the determination of the chromatogram profile was analysed using highperformance liquid chromatography (hplc) instrumentation in reverse phase using a c8 column with an isocratic eluent system of methanol: h2o (9:1) at a flow rate of 1.5 ml/min and an operation time of 25 minutes at a uv wavelength of 254 nm. the hplc chromatogram results were analysed based on the retention times obtained for each d. polyphylla extract. determination of antioxidant activity the determination of antioxidant activity qualitatively was carried out using the procedure described by putri et al., (2024) through the tlc technique. each extract with a concentration of 1000 ppm and ascorbic acid 1000 ppm as a positive control was spotted on the tlc plate with the same number of spots. the plate was then sprayed with 50 ppm dpph and incubated for 30 minutes in the dark. antioxidant activity is indicated by the presence of yellow spots on the tlc plate. quantitative analysis of antioxidant activity was conducted to determine the concentration of an extract that can neutralise free radicals based on the ic50 value. in this test, the positive control used was trolox. the 100 ppm trolox solution was diluted into seven series of concentrations: 20 ppm; 10 ppm; 5 ppm; 2.5 ppm; 1.25 ppm; 0.625 ppm; and 0.3125 ppm. meanwhile, the 1000 ppm d. polyphylla extract was diluted into seven series of concentrations: 500 ppm; 250 ppm; 125 ppm; 62.5 ppm; 31.25 ppm; 15.625 ppm; and 7.8125 ppm. each extract (1000 ppm – 7.8125 ppm) was taken in an amount of 2.5 ml and added to 4 ml of 40 ppm dpph. then it was covered and incubated for 30 minutes at room temperature in the dark. after incubation, the samples were measured using a uv-vis spectrophotometer at a wavelength of 516 nm in triplicate. the same treatment was also applied to the control solution. data analysis measurement of antioxidant activity using a uv-vis spectrophotometer conducted in triplicate. the obtained data was expressed in the form of ic50. monika et al. – phytochemical screening and antioxidant activity … 1071 results and discussion the d. polyphylla sample was obtained from the sungai sepeti district, kayong utara regency, west kalimantan. the samples were sorted to separate them from unwanted plant parts such as grass, soil, or damaged samples (widodo & subositi, 2021). the sample was then washed using running water, cut into small pieces to reduce the sample size, dried using an oven at 50⁰c, and grinded to increase the sample surface area so that during the maceration process, the secondary metabolites contained in the sample can be maximally extracted (gamah et al., 2023). the obtained simplicia was extracted using the total maceration and fractional maceration methods. in total maceration, the extract obtained is a total extract that can be extracted in the solvent used, while fractional maceration produces specific compounds according to the solvent used (hamka et al., 2022). the obtained macerate was concentrated using a rotary vacuum evaporator to obtain a thick extract. the yield calculation is obtained from the comparison of the thick extract obtained with the initial weight of the sample before the extraction treatment (putri et al., 2024). table 1. yield and antioxidant data of d. polyphylla extract. extraction method solvent sample yield (%) ic50 antioxidant *(ppm) total maceration methanol leaf 13.63 51.95 ± 0.087 bark 10.55 72.21 ± 0.462 stem 13.36 421.03 ± 0.598 tiered maceration n-hexane leaf 2.85 > 500 bark 2.20 > 500 stem 10.73 > 500 ethyl acetate leaf 5.38 77.63 ± 0.061 bark 0.91 > 500 stem 10.93 > 500 methanol leaf 22.68 55.26 ± 0.063 bark 17.00 10.28 ± 0.059 stem 5.69 > 500 trolox 2.14 ± 0.002 *the value is the average ± sd, n = 3. the highest yield value in total maceration was found in the leaf extract at 13.63%, followed by the stem extract at 13.36%, and the bark extract at 10.55%. the selection of methanol as a solvent is based on its ability to dissolve both polar and nonpolar compounds because its small molecular structure can penetrate plant cells, thereby maximising the dissolution of secondary metabolites (muaja et al., 2017; mutmainnah et al., 2017). meanwhile, in the stepwise maceration using nhexane solvent, the highest yield value is found in the stem extract compared to the leaf and bark extracts. in the ethyl acetate solvent, the highest yield value is found in the stem extract compared to the leaf and bark extracts. whereas in the stepwise methanol solvent, the highest yield value was found in the leaf and bark extracts compared to the stem extract. this indicated that the compounds dissolved in the stem extract were predominantly semi-polar and polar compounds. meanwhile, in the leaf and bark extracts, the dissolved compounds are predominantly polar compounds. this is in line with the principle of like dissolves like, where compounds will dissolve in solvents that have similar properties (alif et al., 2023). the higher the yield value of an extract, the greater the concentration of dissolved substances in the extract (senduk et al., 2020). he differences in yield values obtained are influenced by several factors, namely the type of solvent polarity, the ratio or concentration of the solvent used, the particle size of the simplicia, and the duration of the extraction performed (handoyo, 2020). phytochemical screening is an initial stage in research to provide an overview of the groups of compounds contained in the extract of d. polyphylla (minarno, 2015). phytochemical screening using specific reagents such as dragendroff, mayer, and wagner reagents were used to detect alkaloid compounds, shinoda reagent was used to detect flavonoid compounds, fecl3 5% reagent was used to detect phenolic compounds, fecl3 10% reagent was used to detect tannin compounds, and liberman-burchard reagent was used to detect terpenoid compounds. the presence of alkaloids is marked by the formation of orange precipitates with dragendorff's reagent, brown precipitates with wagner's reagent, and white precipitates with mayer's reagent. the precipitate formed occurs due to the formation of a potassium-alkaloid complex between the k+ metal ion, which forms a coordinate covalent bond with the free electrons on the nitrogen atom present in the alkaloid (sangkal et al., 2020). flavonoids are indicated by a colour change to orange to red due to the reduction of concentrated hydrochloric acid with magnesium forming the complex 1072 biology, medicine, & natural product chemistry 14 (2), 2025: 1069-1076 [mg(oar)6]4-. phenolic compounds show a colour change to green or blue due to the formation of iron (ii) hesasianoferat as phenolic compounds reduce fe3+ to fe2+.tannin compounds are indicated by a colour change to dark blue or greenish-black. meanwhile, terpenoid compounds are indicated by a bluish-green colour for steroid-type terpenoids and an orange-brown colour for triterpenoid-type terpenoids (pratama et al., 2022). the extract of d. polyphylla leaves contains groups of alkaloid, flavonoid, phenolic, tannin, and terpenoid compounds. meanwhile, the extract of d. polyphylla stems and bark contains groups of alkaloid, phenolic, tannin, and terpenoid compounds. the presence of flavonoid compounds was not detected in the stems and bark of d. polyphylla. this is because not all groups of flavonoid compounds can be detected with the shinoda reagent. table 2. results of phytochemical screening of d. polyphylla extract sample alcaloide flavonoide phenolic tannin terpenoide 1 2 3 methanol leaf + + + + + bark + + + + + stem + + + n-hexane leaf + + + + bark + + + stem + ethyl acetate leaf + + + + + + + bark + + + + stem + + + + methanol leaf + + + + + bark + + + stem + + note: 1) dragendorff's reagent, 2) wagner's reagent, 3) mayer's reagent. (+) contains secondary metabolite compounds (-) does not contain secondary metabolite compounds. the analysis of the complexity and polarity properties of the compounds contained in the d. polyphylla extract was carried out using tlc. the eluent systems used were n-hexane:ethyl acetate (7:3), n-hexane:ethyl acetate (1:9), and chcl3:acetone (1:1). the extract that has been spotted on the tlc plate was eluted with each eluent. among the various eluents used, n-hexane:ethyl acetate (7:3) provided clear and well-separated spots (ridwanuloh & syarif, 2019). observation was conducted under uv light at 254 nm. during the observation under uv light at 254 nm, the tlc plate fluoresced and the sample appeared dark in colour (izzah et al., 2019). observation under uv light at 254 nm showed that the spots on the leaf and stem bark extracts exhibit dark spots, especially in the ethyl acetate leaf and stem bark extracts. meanwhile, the stem extract did not show any fluorescent spots. therefore, the tlc plate was sprayed with cerium sulphate (ceso4), which is a universal stain developer (arnida & sutomo, 2008). after the plate was sprayed with ceso4 2% and heated, purple spots appeared, indicating the presence of phenylpropanoid derivatives, and brown spots indicated the presence of flavonoid compounds (mutmainnah et al., 2017; nuari et al., 2019). in the leaf and stem extracts, the spots that appear tend to be purple and brown, while in the bark extract, the spots that appear are predominantly brown. the abundance of spots in the leaf and stem extracts indicates that both extracts have more complex components compared to the bark extract. the tlc chromatogram results indicated that the compound components contained in the d. polyphylla extract have a polar polarity level. this was shown by the spots that appear with an rf value >0.7 in the nonpolar eluent system (n-hexane-ethyl acetate (7:3)). a. b. figure 1. chromatogram of the extract observed under uv light at 254 nm (a), after spraying with 2% ceso4 and heating (b) note : leaf extracts from total methanol (1), methanol (2), n-hexane (3), ethyl acetate (4). stem extracts from total methanol (5), methanol (6), n-hexane (7), ethyl acetate (8). bark extracts from total methanol (9), methanol (10), n-hexane (11), ethyl acetate (12). monika et al. – phytochemical screening and antioxidant activity … 1073 the complexity analysis and polarity properties of the extracts from the leaves, stems, and bark of d. polyphylla were further analysed using reverse phase hplc (rphplc). hplc has a high sensitivity level, quick analysis time, and requires only a small amount of sample (rosydiati & saleh, 2019). the principle of reverse phase hplc is based on polarity, where more polar compounds will elute first and have a shorter retention time compared to semi-polar or nonpolar compounds (aulia et al., 2016). the compounds that elute will be detected by the detector and recorded in the form of a chromatogram. the number of peaks in the chromatogram indicates the number of components, and the area of the chromatogram peaks indicates the concentration of the components in the d. polyphyll extract (khairun et al., 2021). the number of peaks that appear at the initial retention time indicated that the d. polyphylla extract contains more polar compounds than nonpolar ones (rudiana et al., 2022). the number of peaks obtained showed that the total extracts of leaves and stems have a more varied component of compounds than the bark extract. additionally, the similarity in retention time between the leaf and stem extracts of d. polyphylla indicated that both extracts have almost similar compound components. a) b) c) figure 2. hplc chromatogram of total extracts (a) leaves, (b) stem, and (c) bark of d. polyphylla. qualitative analysis of antioxidant activity was conducted to identify d. polyphylla extracts with antioxidant activity using the tlc method. d. polyphylla extracts with antioxidant potential are marked by a colour change of dpph from purple to yellowish-white with a purple background (putri et al., 2024). the colour change that occurs was caused by a reaction between compounds that can donate hydrogen atoms from the d. polyphylla extract to the dpph molecule, reducing the dpph molecule to dpph-h (muthia et al., 2019). the leaf and bark extracts of d. polyphylla showed colour changes comparable to ascorbic acid as a reference. this 1074 biology, medicine, & natural product chemistry 14 (2), 2025: 1069-1076 suggests that the leaf and bark extracts of d. polyphylla have antioxidant activity. figure 3. qualitative antioxidant test using the tlc technique with an extract concentration of 1000 ppm. the determination of antioxidant activity was conducted using the dpph method. this method was chosen because it is simple, fast, easy, and requires only a small sample to evaluate the antioxidant activity of natural compound materials (hasan et al., 2022). the parameter used is the concentration level of d. polyphylla extract in inhibiting 50% of dpph free radicals. each d. polyphylla extract (1000 7.8125 ppm) and trolox (20 0.3125 ppm) was measured using a uvvis spectrophotometer at a wavelength of 516 nm in triplicate. the concentration variations were used to facilitate determining the concentration limit of effective antioxidant activity in d. polyphylla extract (kurnia et al., 2024). measurements at the maximum wavelength are conducted to increase the sensitivity of the measurements because at this wavelength, the absorption in the test solution will be maximised. measurements of the control solution show that the concentration is directly proportional to the percentage inhibition. this indicates that the higher the concentration used, the higher the percentage inhibition obtained. (figure 4). figure 4. calibration curve of the control solution (trolox) measured in triplicate. the ic50 value of d. polyphylla extract was determined by creating a calibration curve between the concentration of the extract and the percentage of inhibition. the obtained linear equation was used to calculate the concentration of d. polyphylla extract that can inhibit dpph free radicals by 50%. d. polyphylla extract was said to have very strong antioxidant activity if the ic50 value is < 50 ppm, strong if the ic50 50-100 ppm, moderate if the ic50 value is 101-250 ppm, weak if the ic50 value is 250-500 ppm, and inactive if the ic50 is >500 ppm (putri et al., 2024). the antioxidant activity in the total leaf extract was classified as very strong compared to the bark extract, while the bark extract did not have antioxidant activity. in addition, the methanol bark extract has very strong antioxidant activity, followed by the methanol leaf extract and the ethyl acetate leaf extract (table 1). this indicated that the leaf and bark extracts have potential as strong natural antioxidants, and it is estimated that the compounds actively involved in this activity are polar and semi-polar compounds. therefore, the isolation of compounds from the d. polyphylla plant can be recommended. this finding is consistent with research on another species from the same genus, namely d. costulata, which has also been reported to have strong antioxidant activity in total chloroform leaf extracts of 79.8 ± 0.2 µg/ml, total nbutanol leaf extracts of 12.0 ± 0.1 µg/ml, and isolated nbutanol leaf extracts of 9.37 ± 0.02 µm. the d. polyphylla leaf extract has been shown to have greater antioxidant activity than the antioxidant bht at 80.78 ± y = 15,408x 4,7218 r² = 0,9955 -10 0 10 20 30 40 50 60 70 80 0 1 2 3 4 5 6 % in h ib is i concentration monika et al. – phytochemical screening and antioxidant activity … 1075 0.01 µm. compounds isolated from the d. costulata leaf extract include rhamnazin, 𝛽-amyrin, quercetin-3-o-𝛼rhamnopyranoside, okrolifuanin a, okrolifuanin e, okrolifuanin f, dehydrochrolifuanin a, dehydrochrolifuanin e, and dehydrochrolifuanin f (subhadhirasakul et al., 2003; mirand et al., 1983). conclusions the leaves of d. polyphylla contain compounds from the alkaloid, flavonoid, phenolic, tannin, and terpenoid groups, while the stems and bark of d. polyphylla contain compounds from the alkaloid, phenolic, tannin, and terpenoid groups. the extracts of the leaves and bark of d. polyphylla have strong natural antioxidant activity. therefore, the extracts of the leaves and bark of d. polyphylla have potential as a source of antioxidant compounds. additionally, the isolation of active compounds from d. polyphylla is highly recommended to broaden the exploration of the d. polyphylla plant. acknowledgements: thank you to the mbkm (merdeka belajar kampus merdeka) research grant for supporting this research. authors’ contributions: putri dharma monika: writing articles and conducting experiments; rini muharini: supervising research and article, designing and directing projects, processing data and performing analysis; eni mayasari: designing and directing projects. competing interests: the authors declare that there are no competing interests. references alif, a. a., gusmiaty, akzad, m. b., rahim, i., & larekeng, s. h. 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(2021). penanganan dan penerapan teknologi pascapanen tanaman obat. jurnal teknologi industri pertanian, 15(1), 253–271. https://journal.trunojoyo.ac.id/agrointek/article/view/7661 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 437-440 | doi: 10.14421/biomedich.2023.122.437-440 issn 2540-9328 (online) chemical properties of liquid broth extracted from freshwater and marine shrimp shells waste esa ghanim fadhallah*, dyah koesoemawardani, lathifa indraningtyas department of agricultural product technology, faculty of agriculture, universitas lampung, jl. prof. dr. soemantri brojonegoro no. 1, bandar lampung, lampung, 35145, indonesia. corresponding author* esa.ghanim@fp.unila.ac.id manuscript received: 30 mei, 2023. revision accepted: 27 july, 2023. published: 12 august, 2023. abstract indonesia's shrimp industry is growing rapidly, but a surge in shrimp waste such as shells and heads’ shrimps are increasing as well. these waste products contain important components such as protein, minerals, and amino acids. this study aims to determine the chemical properties of liquid broth extracted from freshwater and marine shrimp shells, including ash, protein, fat, monosodium glutamate (msg), and antioxidant. the liquid broth was extracted by boiling shrimp shells and heads in water with a ratio of 1:2 for 1 hour at 80oc. results indicate that the type of shrimp used did not affect the broth's ash, fat, protein, msg, or antioxidant content. marine and freshwater shrimp liquid broths contain 0.56% and 0.28% ash, 0.10% and 0.50% fat, 2.19% and 1.97% protein, 1.5291% and 1.6274% msg, and 2263.73 ppm and 2786.2 ppm antioxidant. keywords: chemical; liquid broth; protein; shrimp shell; waste. introduction shrimp is one of the leading commodities of indonesia's fisheries industry. according to the data, shrimp ranks first with the largest export value of 239.28 million kg and us$ 2.04 billion. shrimp export volume increased by 28.96% compared to 2019, reaching 207.70 million kg. shrimp also contributed 18.95% of the total export volume of fishery products last year (ministry of marine affairs and fisheries republic of indonesia, 2021). however, as shrimp production increases, the waste generated from shrimp shells and heads also rises since these byproducts in the industry are approximately 60% of total production (abdollahi & undeland, 2020). the shrimp shells and heads have been used as additives in aquaculture feeds but are commonly discarded and polluting the environment (saini et al., 2020). shrimp shells and heads still contain important components such as protein, astaxanthin pigment, minerals, and amino acids (liu et al., 2021), making utilizing these contents as products possible. the broth is a result of an extracting certain food ingredients such as chicken meat, beef, seafood, and vegetables which can be used to enhance the umami taste of dishes (mosby, 2009; rostini & pratama, 2018). the extract can be obtained by boiling to dissolve the watersoluble components inside the ingredients into the water. marine and freshwater shrimp differ in taste and texture, whereas marine shrimp has a richer and sweeter taste than freshwater shrimp (liang et al., 2008). moreover, the shrimp pigment called astaxanthin is believed to have an important antioxidant activity that is beneficial to human health (fakhri et al., 2018). the previous study (saleh et al., 1996) applied the liquid broth extracted from shrimp heads into crackers, but there is no further investigation on the various chemical properties. the study on characteristics differences of liquid broth obtained from marine and freshwater shrimp shells has yet to be reported. therefore, this study aims to determine the chemical properties of liquid broth obtained from the shells and heads of marine and freshwater shrimp. materials and methods materials and equipment the raw materials used in this study were the shells and heads of marine and freshwater shrimp obtained from the gudang lelang market in bandar lampung, indonesia. other materials used included chemicals for chemical analysis and titration. the equipment included a digital scale, beaker, hot magnetic stirrer, stirring rod, and stainless-steel strainer. the chemical analysis of the broth was carried out using a set of titration tools, chemical glassware, a furnace oven, a soxhlet apparatus, https://doi.org/10.14421/biomedich.2023.122.437-440 438 biology, medicine, & natural product chemistry 12 (2), 2023: 437-440 a kjeldahl apparatus, and a uv-vis spectrophotometer (hitachi u-2010). liquid broth preparation this study was carried out in several steps, including preparing shrimp shells and heads, the boiling process, and the characterization of the boiled shrimp shell and head broth. all stages of the research were carried out on samples of marine and freshwater shrimp. the shrimp shells and heads were washed with running water until clean and drained, then placed in a beaker glass. water was added at a ratio of shrimp to the water 1:2 and then boiled using a hot magnetic stirrer at 80°c for 60 minutes (meiyani et al., 2014). the broth was then filtered to separate the shrimp shells and heads from the broth, and then cooled before being characterized. the characteristics of the broth analyzed included the proximate analysis of protein, fat, ash (aoac, 2005), monosodium glutamate (sulastri, 2017), and antioxidant activity (rahmawati et al., 2016). data analysis the results of several analyses were processed, and the significance of the differences was analyzed using a ttest with the ibm spss version 23 application. results and discussion chemical properties of liquid broth the parameters analyzed to obtain the characteristics of the broth from freshwater and marine shrimp shells included ash, fat, protein, msg, and antioxidants. the results of these parameter tests are presented in table 1. based on the results presented in table 1, it is known that the ash, fat, protein, msg, and antioxidant content of the broth from freshwater shrimp shells were 0.28%, 0.50%, 1.97%, 1.6274%, and 2786.2 ppm, respectively. meanwhile, the values for the broth from marine shrimp shells were 0.56%, 0.10%, 2.19%, 1.5291%, and 2263.73 ppm, respectively. the t-test statistical analysis showed no significant difference in chemical properties between the broth from freshwater and marine shrimp shells. table 1. chemical properties of liquid broth from freshwater and marine shrimp shells analysis freshwater shrimp marine shrimp ash (%) 0.28±0.01 a 0.56±0.03 a fat (%) 0.50±0.04 a 0.10±0.01 a protein (%) 1.97±0.31 a 2.19±0.02 a msg (%) 1.6274±0,02 a 1.5291±0.04 a antioxidant (ppm) 2786.2±125.27 a 2263.73±201.67 a note: the numbers followed by different letters on the same row indicate significant differences based on the t-test. ash content the ash content of the liquid broth obtained from the shells and heads of freshwater shrimp is lower than marine shrimp. specifically, the ash content of the liquid broth from freshwater shrimp is 0.28%, while from marine shrimp is 0.56%. however, a t-test analysis shows no significant difference in the ash content of the broth between marine and freshwater shrimp. this indicates that the type of shrimp does not significantly impact the ash content in the shrimp broth, and it is inferred that the ash content was more likely affected by the boiling period. another study (silab et al., 2022) reported that the longer boiling period of pig bones resulted in higher ash content. ash content in broth is related to the presence of minerals in a substance. the boiling process using water can only dissolve watersoluble minerals. the longer boiling period makes more water-soluble minerals come out of the tissue and dissolve in the water (liu et al., 2021). fat content the fat content of the liquid broth obtained from the shells and heads of freshwater shrimp is higher than marine shrimp, specifically from freshwater shrimp and marine shrimp, which are 0.50% and 0.10%, respectively. since the t-test shows no significant difference in the fat content, it indicated that the type of shrimp does not affect the fat content in the shrimp broth produced. the fat content in the broth can be more influenced by the length of boiling time. another finding (silab et al., 2022) showed a significant increase in the fat content of broth produced from boiling pig bones with rising boiling time. the fat will leach outside the tissue and move into the water media. the longer boiling process will break down more fat components and dissolve them into the boiling water, increasing the fat content in the broth. the boiling process with water causes hydrolysis to break down fat into glycerol, soluble in water, and fatty acids, and also significantly changes the saturated and unsaturated fatty acids profile (saborowski et al., 2022). protein content the protein content of the liquid broth obtained from the shells and heads of freshwater shrimp is lower than marine shrimp. specifically, the protein content of the broth from freshwater shrimp is 1.97%, while from marine shrimp is 2.19%. the t-test results show no significant difference in the protein and indicate that the type of shrimp does not affect the protein content in the broth. the protein content in this study is lower than in another study. the protein content of liquid broth from marine shrimp heads, black tiger shrimp (penaeus monodon), ranges from 2.24% to 3.73% and varies depending on the boiling time and temperature (saleh et al., 1996). the protein content of the liquid broth is affected by the length of the boiling time. this statement is supported by other findings (silab et al., 2022) that showed the protein content of broth produced from boiling pig bones showed a significant increase with fadhallah et al. – chemical properties of liquid broth 439 increasing boiling time. the longer the boiling time, it is suspected to soften the tissue on the shrimp shell so that the protein components in the shrimp connective tissue will be extracted into the broth liquid. processing temperature could affect the protein content of boiled water at the same boiling time. another study (saleh et al., 1996) reported that boiled water from the heads of tiger prawns at 100°c had a protein content of 2.68% and significantly increased to 3.73% at a boiling temperature of 115°c. this is suspected by the increase in protein solubility at the higher processing temperature. monosodium glutamate (msg) the monosodium glutamate (msg) content in broth from marine shrimp is slightly lower than freshwater shrimp, which are 1.5291% and 1.6274%, respectively. however, a t-test analysis shows no significant difference in the ash content of the broth between marine and freshwater shrimp, which indicates that the type of shrimp does not affect the msg content in the shrimp broth produced. the msg concentration in the liquid broth correlates with the glutamic acid content of shrimp shells and heads. according to (liu et al., 2021), the combined total glutamic acid concentration of shrimp heads (1.56% 1.76%) and shells (1.69% 1.83%) is more than that of the flesh (3.00% 3.10%). the msg content in the broth can be more influenced by the length of boiling time. the longer boiling time is suspected of causing the protein to be denatured and break down into amino acids, including glutamic acid, which has a high solubility in water (kingwascharapong & benjakul, 2016). boiling process will cause protein breakdown, producing amino acids that are easier to absorb by the body (anwa et al., 2007). antioxidant antioxidants in shrimp play a role in delaying, slowing down, and preventing free radical oxidation in lipid oxidation (nikoo et al., 2021). astaxanthin, a xanthophilic carotenoid compound with two hydroxyl groups, is one of the antioxidants found in shrimp skin. this compound is more soluble in methanol and ethanol and can be extracted by bleaching method against βcarotene. astaxanthin extracts from shrimp shells have an antioxidant activity of 88% against β-carotene (saleha & murniana, 2009). in this study, the antioxidant content of the freshwater shrimp broth extracted from shrimp shells and heads was 2786.2 ppm and 2263.73 ppm. according to the t-test analysis, it did not show a significant difference. during boiling, the antioxidant content correlated with the astaxanthin pigment extracted from shrimp shells and head tissue into the water. this result is higher than another study (liu et al., 2021), which reported the astaxanthin content of shrimp heads, shells, and tails that ranges from 2.91 ppm to 19.20 ppm. during soaking and boiling, when shrimp parts come into contact with water, some proteins, including carotenoproteins (such as astaxanthin), were partially leached out. as a result, fewer pigments were retained in the meat, making the boiling water reddish (kingwascharapong & benjakul, 2016). the amount of astaxanthin in shrimp is impacted by the composition of their feed, as proven by research (ruangdej & laohavisuti, 2014) that showed shrimp fed with astaxanthin-enriched diets had higher pigmentation and total carotenoid levels than those given without it. conclusions the chemical properties of liquid broth produced from freshwater and marine shrimp shells, including ash, fat, protein, msg, and antioxidants, have been studied. the liquid broth obtained from marine shrimp shells has higher protein content, msg content, and antioxidants than the broth obtained from freshwater shrimp shells. however, the ash and fat content are lower in marine shrimp shell broth than in freshwater shrimp shell broth. these chemical properties are not affected by the type of shrimp used. further study is needed to vary the time and temperature boiling process with another analysis, such as amino acids and fatty acid profiles. acknowledgements: the authors would like to thank the faculty of agriculture, universitas lampung, for financial support of this work. authors’ contributions: dyah koesoemawardani designed the study. esa ghanim fadhallah and lathifa indraningtyas carried out the laboratory work. esa ghanim fadhallah analyzed the data. esa ghanim fadhallah and lathifa indraningtyas wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the funding was granted by the faculty of agriculture, universitas lampung. references abdollahi, m., & undeland, i. 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(2017). analisis kadar monosodium glutamat (msg) pada bumbu mie instan yang diperjualbelikan di koperasi wisata universitas indonesia timur. jurnal media laboran, 7(1), 5–9. https://uit.e-journal.id/medlab/article/view/347 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 563-575 | doi: 10.14421/biomedich.2023.122.563-575 issn 2540-9328 (online) study of physical, chemical, and organoleptic properties of functional drink turkey berry (solanum torvum swartz) with the addition of butterfly pea flower (clitoria ternatea linn) and emprit ginger (zingiber officinale var. amarum) rista anggriani, nina nurazizah purnomo putri*, vritta amroini wahyudi food technology department, faculty of agriculture and animal husbandry, university of muhammadiyah malang, jl. raya tlogomas no 246 malang 65144, malang, jawa timur, indonesia. corresponding author* ninaputri171@gmail.com manuscript received: 06 june, 2023. revision accepted: 11 september, 2023. published: 25 september, 2023. abstract turkey berry (solanum torvum swartz) is a wild plant often found in indonesia and its usage is still limited. turkey berry has a high potential as a source of antioxidants, which can be consumed as a functional drink. ginger and butterfly pea flowers were selected as additional ingredients in composite drinks to enhance the quality of the functional drink in terms of function and sensory characteristics. this study aimed to determine the effects of variations in the concentration of turkey berry, butterfly pea flower, and ginger on the physical, chemical, and organoleptic characteristics. the design method used a simple randomized block design with four treatment combinations to be analyzed in three repetitions. data were analyzed using analysis of variance (anova) to determine the effects of the treatment. the results showed that turkey berry, in combination with ginger and butterfly pea flower, had a significant effect on the physicochemical and organoleptic characteristics, including ph, total phenolic content, total flavonoid content, antioxidants, color, aroma, taste, and preference. keywords: turkey berry; ginger; butterfly pea flower; antioxidants. introduction solanum torvum swartz, also known as turkey berry in english, terung pipit, takokak, and pokak in indonesia, konsu swa in ghana, susumba in jamaica, and sunndakkai in india, is a wild plant commonly found in indonesia and tropical and subtropical countries in asia, the caribbean, south america, and africa (regina et al., 2018). although its usage is still limited in indonesia, people in ghana and some other countries utilize this plant for its medical and health benefits. previous studies have shown that turkey berry fruit exhibits antioxidant activity (sivapriya et al., 2007; kusirisin, 2009), cardiovascular and antidiabetic properties (mohan et al., 2009), and contains 171 chemical compounds, making it a potentially beneficial food ingredient for human health (helilusiatiningsih, 2021). kusirisin (2009) found that solanum torvum swartz is a natural source of antioxidants and polyphenols, which can act as an antidote to free radicals in lipid peroxidation and superoxide anions in diabetic patients. consumption of polyphenol-rich foods can reduce oxidative stress and serve as a valuable source of antioxidants (annuzzi et al., 2014). helilusiatiningsih (2020) examined turkey berry as a brewed drink in the form of herbal tea, using the same brewing techniques as green tea and black tea. the results of this study indicated that the herbal brewed drink contained nutrients and minerals that functioned as natural antioxidants. however, there were still some deficiencies in terms of taste and color in the management of turkey berry functional brewed drinks. therefore, it is necessary to add other ingredients that can enhance the taste, color, and overall acceptability of the brewed drinks. teye's research (2017) investigated turkey berry as a functional brew with the addition of ginger and roselle flowers to improve its physicochemical, sensory, and organoleptic evaluations. the study also found that the inclusion of roselle and ginger flowers increased the nutritional and mineral values. ginger and roselle flowers are known to possess high antioxidant content (hopkins, 2013; akoto, 2015). butterfly pea flowers contain high levels of antioxidants and pigments, specifically anthocyanins, which can increase the overall antioxidant levels. anthocyanins are natural dyes with antioxidant properties found in plants (sutanto, 2012). research https://doi.org/10.14421/biomedich.2023.122.563-575 564 biology, medicine, & natural product chemistry 12 (2), 2023: 563-575 conducted by sofiah et al. (2022) discovered that a functional brewed drink made from butterfly pea flowers and ginger exhibited an antioxidant activity of up to 57.03%. based on the aforementioned explanations, the development of turkey berry into a functional brewed drink with the addition of ginger and butterfly pea flowers is expected to enhance the nutritional value of the beverage. functional brewed drinks offer an alternative way to process turkey berry fruit into food products. the purpose of this study was to determine the effects of variations in the concentration of functional brewed drinks made from turkey berry, butterfly pea flower, and ginger on their physical and chemical qualities, as well as their sensory characteristics. furthermore, the study aimed to identify the best formulation for the turkey berry brew from both functional and sensory perspectives when combined with ginger and butterfly pea flower. materials and methods procedures this research was conducted at the food technology laboratory, university of muhammadiyah malang. the materials used are turkey berry fruit, emprit ginger, and butterfly pea flowers. the materials used for analysis were obtained from the umm food technology laboratory, including 2,2 diphenyl-1-picrylhydrazyl dpph solution (himedia), 70% ethanol (p.a, merck), 98% ethanol (p.a, merck), 99% methanol (p.a, merck), distilled water, ascorbic acid (c6h8o6), folin ciocalteau reagent, sodium carbonate (na2co3) (p.a, merck), gallic acid (c7h6o5), quercetin (c15h10o7), aluminum chloride (alcl3). the tools used were knife, basin, aluminum pan, aluminum foil, stopwatch, ruler, 80 mesh sieve, 10 ml and 25 ml volumetric flask, erlenmeyer, spatula, test tube, dark bottle, vial, 1 measuring pipette. ml and 5 ml, filler pipette, 1000 µl micropipette, 200 µl micropipette, yellow tip micropipette and blue tip micropipette, rubber bulb, breaker glass, thermometer, hot plate maspion, showcase polytron, pioneer ohaus pa413 analytical balance, eutech ph meter, konica minolta cr10 color reader, multiplex fume hood fume hood, dryer cabinet, philip blender, genesys 20 thermos spectronic type spectrophotometer. figure 1. turkey berry (solanum torvum). this study used the rak (simple randomized block design) design method and four treatment combinations to be analyzed in three replications. the formulation level is as follows: table 1. turkey berry brewed drink formulation with the addition of butterfly pea flower and ginger in grams. sample formula sample (g) turkey berry ginger butterfly pea total a 0.50 1.00 0.50 2.00 b 0.75 0.75 0.50 2.00 c 1.00 0.50 0.50 2.00 control 2.00 2.00 the process of making turkey berry simplicia was carried out based on research by helilusiatiningsih (2021). the collected trees are sorted to select good fruit with bright green color and not rotten. turkey berry then cleaned using water and then drained. the cleaned turkey berrys were then cut into two and were dried in a cabinet dryer for 18 hours at 60oc. after the turkey berry fruit is dry, it was grinding with a blender. then the simplicia was sieved using an 80-mesh sieve. simplisia that had been refined was stored in a closed place in dry conditions. the same process was carried out for ginger and butterfly pea flowers. samples were done by preparing simplicia from the ingredients that had been prepared. then all the ingredients were weighed with an analytical balance. then put it in a brewed drink bag and close it. samples were brewed with distilled water instead of hot water at 80oc for 2 minutes. the brewed drink was left to stand at room temperature. samples were analyzed with chemical properties test parameters (antioxidants, phenols, flavonoids), physical properties tests (color, ph), and sensory properties tests (color, taste, aroma). color analysis (color reader) (soewarno, 2022) the material was placed on a flat surface and the color reading lens was directed at the material. press the button to read the color. after the tool emits light, the tool was lifted and the results are seen. the value of l indicates brightness, a indicates bluishness, and b indicates greenishness. ph analysis the ph meter was turned on and then rinsed with the electrode and the temperature probe used distilled water and drained. then calibrate the ph meter electrode by dipping the electrode in a buffer solution (ph 7) and acid (ph 4) and rinsing with distilled water and then drying. the electrode was immersed in the sample, by pressing the ar (hold) and enter keys then waiting for the reading on the stable layer and the autolock indicator appears on anggriani et al. – study of physical, chemical, and organoleptic properties of … 565 the screen. the results of the ph meter were recorded according to the scale displayed by the ph meter. antioxidant activity (%) analysis (selvi et al, 2003) first, the dpph solution was prepared by taking 2 mg of dpph (1,1 diphenyl-2-pikrihidrazyl) and 10 ml of ethanol was added then the solution was put in a bottle, shaken and stored in the refrigerator. second, 1 ml of sample was taken and 9 ml of ethanol was added. third, each 4 ml of sample and 1 ml of dpph was put in a test tube. fourth, the sample is allowed to stand for 10 minutes until it turns yellow. then a spectrophotometer with a wavelength of 517 is used to read the sample results. antioxidant activity is calculated by the formula: aktivitas antioksidan (%) = 1 − 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑠𝑖 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑠𝑖 𝑏𝑙𝑎𝑛𝑘𝑜 x 100% antioxidant analysis ic50 radical scavenging activity method (khare, 2019) the most well-known antioxidants are β-carotenoids, ascorbic acid, and tocopherols and phenolic compounds, therefore ascorbic acid was used in this analysis. dpph (2,2diphenyl-1picrylhydrazyl) is a stable organic radical which has the capacity to react with reagents. this decolorization method measures the antioxidant capacity that directly reaches the dpph radical, which can be seen from the absorbance at a wavelength of 517 nm using a spectrophotometer (jain, 2011). the reactive rate and ability to react with radical molecules depend on the rate and peak value of the loss of dpph (gulcin, 2006). first, dpph solution was prepared by mixing 4 mg of dpph simplicia and 100 ml of 99% methanol. mixing is done in a dark bottle and stored in a dark place and allowed to stand for 20 minutes. second, a standard solution of ascorbic acid was prepared by mixing 2 mg of ascorbic acid with 2.5 ml of distilled water to form a standard solution of ascorbic acid with an initial concentration of 800 µg/ml. from the main concentration, the concentration variations were made to 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml, and 100 µg/ml. third, a sample solution was prepared from each treatment sample by adding 1 ml of sample to 9 ml of methanol. then each sample was varied in concentration to 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml, and 100 µg/ml. preparation of the standard standard curve, begins with the addition of 2 ml of ascorbic acid concentration into a test tube that has been coated with aluminum foil. then the dpph solution that was ready was added to the solution as much as 2 ml. after that, the standard standard solution was homogenized using a vortex and covered with aluminum foil and incubated for 30 minutes in a dark room. just like the standard standard solution, the sample solution that is ready with a predetermined concentration is added to the test tube and the dpph solution is added. control samples were prepared from adding 2 ml of methanol with 2 ml of dpph solution. after the standard ascorbic acid solution, sample, and control had been incubated, the wavelength absorption was read with a uv vis spectrophotometer at λ = 517 nm. the absorbance value is recorded and calculated by the formula: % 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = [(𝐴𝐶 517 𝑛𝑚 − 𝐴𝑆 517 𝑛𝑚/ 𝐴𝐶 517 𝑛𝑚) 𝑥 100%] note, ac : absorbance control as : sample absorbance then to determine the ic50 value a relationship curve was made between extract concentration and inhibition percentage which would form a linear regression equation (khare, 2019). phenol analysis (siddiqui, 2017) the test was carried out using the folin ciocalteau reagent. first, a solution of 7.5% concentration of sodium carbonate (na2co3) was prepared by adding 7.5 g of powdered sodium carbonate (na2co3) and 100 ml of distilled water. second, a solution of gallic acid was prepared by dissolving 0.5 g of gallic acid with 10 ml of ethanol and then diluting it with 100 ml of distilled water. then gallic acid solutions were made with various concentrations, namely 50 µg/ml, 100 µg/ml, 250 µg/ml, and 500 µg/ml. third, dilution of the sample solution was carried out from each treatment sample by adding 1 ml of sample to 9 ml of ethanol. prepared test tubes covered with aluminum foil. mixing 3.16 ml of distilled water, 0.2 ml of folin ciocalteu reagent, 0.6 ml of sodium carbonate (na2co3) solution, 40 μl of sample or gallic acid as standard curve solution was carried out. in the control sample, gallic acid solution or sample was not added. after all the solutions have been added, the test tube is homogenized with a vortex. incubation was carried out in a dark room at room temperature for 2 hours. after incubation, the solution was read for wavelength absorption with a uv vis spectrophotometer at λ= 765 nm. the absorbance value was recorded and calculated to obtain the gallic acid absorbance curve. the graph plots between % inhibition and different concentrations of gallic acid. then do the calculation of the total phenol content with the formula 𝐾𝑇𝐹𝑒 = ( 𝑉 ∗ 𝑋 ∗ 𝐹𝑃 ) / 𝑊𝑒𝑖𝑔ℎ𝑡 note, v : volume (ml) x : absorbance (mg/ml) fp : dilution factor weight : sample weight (g) 566 biology, medicine, & natural product chemistry 12 (2), 2023: 563-575 flavonoid level test (stankovic, 2011) first, a quercetin standard curve was made with 10 mg of quercetin dissolved in 100 ml of ethanol to obtain a concentration of 100 ppm. from a 100 ppm quercetin standard solution, several concentrations were made, namely 10 ppm, 20 ppm, 30 ppm, 40 ppm and 50 ppm. from each concentration of quercetin standard solution in a 1 ml pipette. then 1 ml of 2% alcl3 was added, 1 ml of 5% acetic acid, and 2 ml of distilled water. samples were incubated for one hour at room temperature. the absorbance was determined using the uv-vis spectrophotometer method at a maximum wavelength of 435 nm (stankovic, 2011). second, the determination of the total flavonoid content of brewed beverage samples was carried out by taking 1 ml of the sample and dissolving it in 9 ml of ethanol, to obtain a concentration of 1000 ppm. pipette 1 ml of this solution and then add 1 ml of 2% alcl3 solution and 1 ml of 120 mm potassium acetate. samples were incubated for one hour at room temperature. the absorbance was determined using the uv-vis spectrophotometer method at a maximum wavelength of 435 nm. samples were made in three replications for each analysis and the average absorbance value was obtained (stankovic, 2011). results and discussion total flavonoid content based on the results of the analysis of variance, it was shown that the sample of turkey berry drinks with the addition of butterfly pea flower and ginger had a very significant effect on total flavonoid levels at the 5% level. the results of the analysis can be seen in table 2. table 2. total flavonoid content of turkey berry drinks with the addition of ginger and butterfly pea flower. sample formulation total flavonoid levels (mgqe.g-1) k (turkey berry 100%) 17.37 b a (turkey berry 25% + ginger 50% + butterfly pea flower 25%) 12.97 a b (turkey berry 37.5% + ginger 37.5 % + butterfly pea flower 25%) 18.75 c c (turkey berry 50% + ginger 25% + butterfly pea flower 25%) 21.90 d note: the numbers followed by different lowercase letters show significantly different based on the dmrt test (α = 5%). based on table 2, the total flavonoid of each samples has different values. the highest value of flavonoid content is 21.90 mg qe/g was owned by sample c which had a mixture of 50% turkey berry, 25% ginger and 25% butterfly pea flower. while the lowest was 12.97 mg qe/g which was owned by sample a with a composition of 25% turkey berry, 50% ginger and 25% butterfly pea flower. meanwhile, sample b with the addition of 37.5% turkey berry, 37.5% ginger and 25% butterfly pea flower had a flavonoid content of 18.75 mg qe/g which was slightly higher than sample 100% turkey berry, which is 17.37 mg qe/g. flavonoids as a group of phenolic compounds that are abundant in plant tissues can act as antioxidants (redha, 2010). flavonoid compounds are natural compounds that have a polyphenolic structure. most of the flavonoid compounds are soluble in water and easily found in nature. but it is most often found in plants as glucose derivatives known as glycosides. the green components in the turkey berry fruit, and the coloring pigments in the fruits, flowers and seeds are caused by the presence of flavonoid compounds. flavonoids are found in almost all parts of the plant, including fruits, roots, leaves, and the outer skin of the stem. flavonoids are natural compounds that have potential as antioxidants that can counteract free radicals that play a role in the emergence of degenerative diseases through mechanisms of destruction of the body's immune system, oxidation of lipids and proteins (rais, 2015). in a previous study conducted by abdulkadir et al (2016) using the quersetin method and ethanol solvents found that the total flavonoid content in turkey berry fruit was 1.41 mgqe.g-1. whereas in the study of gyingiri et al (2011) the flavonoid content was 8.46 mgqe.g-1 (water extract) and 1.05 mgqe.g-1 (ethanol extract). in this study, total flavonoid levels ranged from 12.96621.896 mgqe.g-1. differences in levels can be influenced by many factors, including the solvent used, the method, and the type of fruit sample used. research by helilusiatiningsih (2021) explains that differences in locations for growing turkey berrys also affect the phytochemicals contained. turkey berry fruit obtained from kweden village, mojokerto and from batu city, malang, showed differences in flavonoids, each of which had flavonoid levels ranging from 1.19 mgqe.g-1 to 2.18 mgqe.g-1. the addition of ginger and butterfly pea flowers also affects the total value of flavonoids because these plant species are known to contain flavonoid compounds. flavonoid levels in ginger range from 0.14 mgqe/g (widiyana et al, 2021). ginger contains volatile compounds consisting of terpenoids, while non-volatile compounds consist of gingerols, shogaols, paradols, zingerones, flavonoids and polyphenols (sari and anggriani et al. – study of physical, chemical, and organoleptic properties of … 567 rahayuningsih, 2014). one of the functional properties of ginger is as an antioxidant. compounds that act as antioxidants are phenolic components consisting of gingerols and shagaols (harahap, 2013). whereas in the butterfly pea flower the total flavonoid content is 10.9 mgqe/g (yumni, 2022). butterfly pea flowers contain several phytochemical compounds, including pentacyclic triterpenoids such as taraxerol and taraxenon, ternatin, alkaloids, saponins, tannins, steroids, phenols, flavonoids, flavonol glycosides and anthocyanins (manjula et al, 2013). dewi, et al (2021) added ginger emprit to the manufacture of bay leaf (syzygium polyanthum) herbal drink. the results of this study indicated that the total flavonoid content in the herbal brewed bay leaf had a value range of 0.45 mgqe/g 0.62 mgqe/g when 5% and 20% ginger was added. meanwhile, in this study, the total value of flavonoids in the yield range was 12.966 mgqe/g 21.896 mgqe/g. this shows that the turkey berry brewed drink with the addition of ginger and butterfly pea flowers has potential as a herbal brew with a high content of flavonoids. total fenol content based on the results of the analysis of variance, the sample of the turkey berry brewed drink with the addition of ginger and butterfly pea flower had a very significant effect on the total phenol content using the folin-ciocalteu method at a test level of 5%. the results of the analysis of the total phenol content can be seen in table 3. table 3. total phenol content of turkey berry brew with the addition of ginger and butterfly pea flower. sample total phenol content (mggae.g-1) k (turkey berry 100%) 65,20d a (turkey berry 25% + ginger 50% + butterfly pea flower 25%) 57,03a b (turkey berry 37.5% + ginger 37.5 % + butterfly pea flower 25%) 59,37b c (turkey berry 50% + ginger 25% + butterfly pea flower 25%) 60,87c note: the numbers followed by different lowercase letters show significantly different based on the dmrt test (α = 5%). based on table 3, it can be seen that the value of total phenol content with the folin-ciocalteu method obtained different results for each sample. the results of the analysis were carried out on the 100% turkey berry brewed drink sample is 65.20 mggae.g-1, sample a with the addition of 25% turkey berry, 50% ginger, 25% butterfly pea flower is 57.03 mggae.g-1, sample b with the addition turkey berry 37.5%, ginger 37.5% and butterfly pea flower 25% is 59.37 mggae.g-1, sample c with the addition of 50% turkey berry, ginger 25%, and butterfly pea flower 25% is 60.87.mggae.g1. in a previous study conducted by dewi et al (2021) in making bay leaf drinks with the addition of ginger, it was found that the phenol levels in the samples ranged from 2.98 mggae.g-1 3.35 mggae.g-1. to compared with this study, the smallest value was found in brewed drink sample a of 57.03 mggae.g-1 and the highest total phenol content was found in sample k of 65.20 mggae.g-1. this shows that the turkey berry brewed drink with the addition of ginger and butterfly pea flowers has a higher phenol content. this could be caused by the addition of ingredients that are high in phenol content. research by helilusiatiningsih (2021) explains that differences in locations for growing turkey berrys also affect the phytochemicals contained. turkey berry fruit obtained from kweden mojokerto village and from batu city, malang showed differences in phenol. each level of flavonoids ranged from 32.15 mggae.g-1 and 35.45 mggae.g-1. phenolic compounds have the ability of antioxidant activity with the ability to reduce oxidation (wahyuni, 2015). a previous study conducted by abdulkadir et al (2016) showed that the total phenolic content in turkey berry fruit was around 16.15 mggae.g-1. meanwhile, in a study by rahman et al (2020) the total phenolic content of the main phenols in water samples was around 13.6 mggae.g-1. in the results of this study the phenol levels ranged from 57.03-65.20 mggae.g-1. differences in the content of total phenol values are influenced by internal and external factors in the form of the extraction method used, composition, and content of active compounds (wahyuni, 2015; brigitta et al, 2021). the use of ginger and butterfly pea flowers also affects the total phenol levels in the turkey berry drinks. compared to other types of ginger, emprit ginger has the highest total phenol content. the total phenolic component contained in the oleoresin from elephant ginger (zingiber officinale rosc.) is 4.40%, emprit ginger (zingiber officinale var. amarum) is 6.90% and red ginger (zingiber officinale var rubrum rhizoma) is 6.50% (fakhrudin et al, 2015). storage and drying time affect phenolic compounds. in this study, ginger (zingiber officinale var. amarum) was used in the form of dried refined simplicia. then stored in a dry place before being analyzed. the phenol oleoresin component of fresh ginger (without being stored in simplicia) was 6.9%, while the phenol oleoresin component of ginger which was previously stored in simplicia for 15 days was 568 biology, medicine, & natural product chemistry 12 (2), 2023: 563-575 5.5%, while for 30 days it was 4.4%. this indicates that there is a decrease in the phenolic component of ginger oleoresin stored in the form of simplicia. the longer it is stored in simplicia, the phenol component in ginger will decrease (pamungkas et al., 2007). antioxidant activity (ic50) the results of the analysis of variance showed that the sample had a value that had a very significant effect on antioxidant levels using the ic50 method at 5% level. the results of the ic50 method antioxidant analysis can be seen in table 4. table 4. antioxidant ic50 of turkey berry brew with the addition of ginger and butterfly pea flowers. sample antioxidant activity (% inhibisi) ic50 (µg/ml) k (turkey berry 100%) 72.80 129.49 c a (turkey berry 25% + ginger 50% + butterfly pea flower 25%) 76.50 116.30 b b (turkey berry 37.5% + ginger 37.5 % + butterfly pea flower 25%) 78.80 109.66 a c (turkey berry 50% + ginger 25% + butterfly pea flower 25%) 80.30 107. 32 a note: the numbers followed by different lowercase letters show significantly different based on the dmrt test (α = 5%). based on table 4 it can be seen that the value of the antioxidant activity of the ic50 method has different results. the ic50 values obtained ranged from 107.33 µg/ml 129.49 µg/ml giving the result that the antioxidants in the samples were moderate. the highest value was obtained by sample k with 100% butterfly pea flower and the lowest value was obtained by sample c with a composition of 50% butterfly pea flower, 5% ginger and 25% butterfly pea flower. meanwhile, the ic50 value of sample b was around 109.66 µg/ml and that of sample a was around 116.30 µg/ml. previous research conducted by dewi et al (2021) showed that the ic50 content in a herbal brewed bay leaf drink with the addition of ginger had an increase in antioxidant content along with the addition of ginger. bay leaf brewed drink with the addition of 20% ginger was able to produce an ic50 value of 640.99 ppm and with the addition of 5% it produced an ic50 of 1140.53 ppm. while the ic50 produced in the research on the turkey berry brew with the addition of ginger and butterfly pea flowers was able to produce ic50 values in the range of 107.32 116.30. gandhiappan et al (2012) showed that turkey berry or solanum torvum swartz had stronger antioxidant activity than solanum ningrum, solanum surratense, solanum trilobatum but lower than solanum pubesce and solanum anguivi. antioxidant activity of dpph (1,1-diphenyl-2picrylhydrazyl radical) ic50 of turkey berry fruit or solanum torvum swartz 125µg/ml (badami et al. 2005). previous research conducted on turkey berry fruit extract was able to capture free radicals by 33.0% which was analyzed by the dpph method and 112 mm fe(ii)/g which was analyzed by the frap method (abdulkadir, 2016). meanwhile, the ic50 method used in ramamurthy's research (2012) showed that the water extract of the turkey berry fruit reached 94 μg/ml. differences in the results of antioxidant activity can be influenced by differences in solvent polarity, concentration, and environmental factors (abdulrahman, 2016). all of the ingredients used in the brewed drink sample contain compound components that have antioxidant capabilities. the ic50 values obtained in this study ranged from 107.323 µg/ml 129.490 µg/ml giving the result that antioxidants are included in the moderate antioxidant group. the ic50 method divides the antioxidant power of each sample into several groups. the antioxidant group is very strong if it has an ic50 of less than 50 ppm (ic50 <50ppm), classified as strong if the value is 50-100 ppm (ic50 50-100 ppm), moderate if the value is 100-150 ppm (ic50 <100-150 ppm) and weak if the value is 150 200 ppm (ic50 150-200 ppm) (rumagit et al, 2015). the high antioxidant activity in the samples was due to all the compounds contained in the turkey berry brew with the addition of ginger and butterfly pea flowers capable of acting as antioxidants (rumagit et al, 2015). based on several studies that have been conducted by (tomsone et al., 2012; alothman et al., 2009; yao et al., 2010; agbor et al., 2005; and li et al., 2009) indicate that there is a strong relationship between total phenolic content with dpph free radical scavenging activity from plant extracts. the antioxidant activity that is found on the test results comes from all the antioxidant components of the materials used. dpph free radicals will detect antioxidant compounds present in the test sample without specific components including flavonoids and phenolic compounds. the detected antioxidant compounds came from the non-enzymatic group of secondary antioxidants. the mechanism of action of the non-enzymatic antioxidants themselves is by capturing fat-soluble and water-soluble free radicals and then preventing reactivity (salmiyah and baharuddin, 2020). the dpph radical scavenging process of the phenolic hydroxy group will capture the dpph radical via the donor hydrogen atom on the free radical and then stabilized by the delocalization of an unpaired electron. a sign that the free radicals are completely reduced is the color change from purple to yellow (fauziah, 2021). anggriani et al. – study of physical, chemical, and organoleptic properties of … 569 ph the results of the analysis of variance at the 5% level showed that the sample had a significant effect on the ph value based on the results of the anova (analysis of variance) test. the average results of the ph analysis of turkey berry brewed drinks with the addition of ginger and butterfly pea flowers can be seen in table. table 5. the ph value of the turkey berry brew with the addition of ginger and butterfly pea flowers. sample ph k (turkey berry 100%) 5.55 a a (turkey berry 25% + ginger 50% + butterfly pea flower 25%) 5.77 b b (turkey berry 37.5% + ginger 37.5 % + butterfly pea flower 25%) 5.88 b c (turkey berry 50% + ginger 25% + butterfly pea flower 25%) 5.78 b note: the numbers followed by different lowercase letters show significantly different based on the dmrt test (α = 5%). based on table 5 the results of the highest ph with a low level of acidity were found in sample b with a concentration of 37.5% turkey berry, 37.5% ginger and 25% butterfly pea flower, which was 5.88, while the lowest ph value with slightly higher acidity was found in sample k. with a turkey berry concentration of 100%, namely 5.55. the difference in the ph value of each sample is affected by the ph of each material as indicated by the value of the material and product samples which are at relatively the same number. the lower the ph value, indicates the high acid content in a material (kurniawan et al., 2016). as the ph increases, more carbinol and chalcone base compounds are formed which are associated with the resulting color (sari et al., 2005). based on the ph value, beverage products are grouped into low-acid drinks with a ph value ≥ 4.6 and acidic drinks with a ph <4.6 (hariyadi, 2020). according to reddy et al (2016) brewed drinks have a ph between 2.85-5.18. this ph range makes the brewed drink belong to the acidic category because it is at a value of less than 7 and is not in the alkaline drink category. the samples in this study had a ph range between 5.55-5.88 where the results of this ph can be classified in low acid drinks and the acid content is lower when compared to the ph of brewed drinks in general. color intensity brightness level (l) based on the results of the analysis of variance with a 5% level of confidence, it showed that there was no interaction between each treatment of the turkey berry brewed drink with the addition of butterfly pea flower and ginger to the brightness level value. the average color intensity analysis results at the brightness level can be seen in figure 2. figure 2. brightness level (l) of turkey berry brew with the addition of ginger and butterfly pea flowers. factors that cause low brightness intensity depend on each material which has a different color. the high percentage of butterfly pea flower filtrate makes the color of the sample darker, this is influenced by the anthocyanin pigments contained in the pigments of the turkey berry fruit and butterfly pea flowers. anthocyanin pigments range from red to blue which tend to appear dark in color (marpaung, 2018). according to cavalcanti (2011) the increasing concentration of anthocyanins causes anthocyanin stability to increase and will make the color of the product more concentrated and darker. meanwhile, the color of ginger is determined by the oleoresin compound. according to prasetyo (2015) the oleoresin value in ginger tends to be dark brown so that the brightness produced is also low. according to satriyanto et al (2012) brightness is the basic color spectrum, adding another color to an object will reduce the brightness value of the product. treatment level of redness (a+) turkey berry has a yellow color as a natural dye (helilusiatiningsih, 2021). if ginger is added, the composite brew will turn bright yellow. this is because the ginger contains an oleoresin compound which produces a yellowish color so that the more the ginger powder is added to the product, the more the brew will mix with a yellow color. this is also supported by the research of pebiningrum et al (2018) that the oleoresin content in ginger affects the color of ginger kombucha. chemically, all anthocyanins are derived from a single aromatic structure, namely "cyanidin" (cyanidin), and all are formed from cyanidin pigments by adding or removing hydroxyl groups or by methylation or glycosylation, so that anthocyanin compounds have polar groups. butterfly pea flowers which have a purplish blue color are added to give an attractive color to the yellow composite brewed drink. the difference in the natural color of anthocyanin pigments is influenced by hydroxylation and methylation, hydroxylation will 21,43 23,53 22,13 22,87 0,00 5,00 10,00 15,00 20,00 25,00 k a b c b ri gh tn es s le ve l ( l) sample color intensity (l) 570 biology, medicine, & natural product chemistry 12 (2), 2023: 563-575 increase the blue color, while methylation increases the red color (kumalaningsih 2006). redness level (a+) based on the results of the test of variance at the 5% level of confidence, it showed that the turkey berry brewed drink with the addition of ginger and butterfly pea flowers had a significant effect on the redness level. the average greenness of the turkey berry brew with the addition of ginger and butterfly pea flowers can be seen in table 6. table 6. the average value of the redness and greenness of the turkey berry brew with the addition of ginger and butterfly pea flowers. sample redness level (a+) k (turkey berry 100%) 0.90 b a (turkey berry 25% + ginger 50% + butterfly pea flower 25%) 0.60 a b (turkey berry 37.5% + ginger 37.5 % + butterfly pea flower 25%) 0.80 ab c (turkey berry 50% + ginger 25% + butterfly pea flower 25%) 0.90 b note: numbers followed by different letters show significant differences according to duncan's test α=5%. based on the results of the dmrt test at the 5% level, the sample treatment had a significant difference. the redness value (a+) was obtained on average ranging from 0.6 to 0.9. the axis parameter (a*) represents the level of green to red with a range of -100 to +100 where negative values (-) indicate a tendency to green while positive values give a tendency to red (lawless and heymann, 1998). the results of the analysis of the initial reddish (a+) value yielded a positive value which indicated that the dark brown ginger oleoresin color also contained a red color in it. the highest level of redness was 0.9 in the 50% turkey berry treatment with the addition of 37.5% ginger and 25% butterfly pea flower, this indicated that this treatment tended to be more red than the other treatments. março et al (2011) stated that at ph 1-2 the dominant anthocyanin is in the form of flavilium cations which are red in color, at ph <6 they turn into carbinol and some of them become quinonoidals which are blue in color so they turn purple. according to brouillard (1982) anthocyanins change color from red to reduced color in weak acids. blue level (b-) based on the results of the test of variance with a 5% level of confidence, it is known that the interaction between the sample of turkey berry brew and the addition of ginger and butterfly pea flower has a significant effect on the bluish and yellowish levels. the average level of blueness (b-) can be seen in table 7. table 7. the average value of the bluish and yellowish levels of turkey berry brew with the addition of ginger and butterfly pea flower flower. sample blue level (b-) k (turkey berry 100%) -0.07a a (turkey berry 25% + ginger 50% + butterfly pea flower 25%) -1.10a b (turkey berry 37.5% + ginger 37.5 % + butterfly pea flower 25%) -0.57a c (turkey berry 50% + ginger 25% + butterfly pea flower 25%) 0.17a note: numbers followed by different letters show significant differences according to duncan's test α=5% the results of the analysis showed that the initial yellowness average value ranged from -1.100 to 0.167. the axis parameter (b*) represents the level of blue to yellow with a range of values from -100 to +100 where a negative value (-) indicates a tendency towards blue while a positive value (+) indicates a tendency towards yellow (lawless and heymann, 1998). the results obtained the resulting value is a positive value. this shows that the color of the oleoresin in ginger is dark brown and the anthocyanin in turkey berry and butterfly pea flower also contains a yellow color. the highest level of yellowness was 0.167 in the 50% turkey berry treatment with the addition of 37.5% ginger and 25% butterfly pea flower which tended to have a darker color than the other treatments. anthocyanins will change color to red to orange at an acidic ph so that the measured anthocyanins are monomeric anthocyanin levels (tonutare et al., 2014). the butterfly pea flower plant has the anthocyanin type delfinidin in its flower parts, which makes the flower color bluish (lijon et al, 2017). anggriani et al. – study of physical, chemical, and organoleptic properties of … 571 organoleptic test organoleptic assessment of turkey berry brewed drinks with the addition of ginger and butterfly pea flowers in this study used a preference test (hedonic scale scoring). in the preference test, the panelists will be given all the samples of the turkey berry brewed drink with the addition of ginger and butterfly pea flower, then the panelists will be asked to drink it and give responses to the sample drink. like. the hedonic scale obtained from the questionnaire will then be transformed into a numerical scale from the lowest to the highest number. the scale is used to determine the difference in the level of preference between existing treatments. this observation was carried out by 40 panelists and the panelists will provide responses starting from color, taste, aroma and preferences. aroma the results of the analysis of variance on the organoleptic aroma test on the turkey berry brewed drink with the addition of ginger and butterfly pea flowers showed very significant interaction results. the average results of the aroma organoleptic test can be seen in table 8. table 8. average aroma of turkey berry brew with the addition of ginger and butterfly pea flowers sample aroma k (turkey berry 100%) 2.38 a a (turkey berry 25% + ginger 50% + butterfly pea flower 25%) 2.43 a b (turkey berry 37.5% + ginger 37.5 % + butterfly pea flower 25%) 3.90 b c (turkey berry 50% + ginger 25% + butterfly pea flower 25%) 3.70 b note: the average value followed by the same letter is not significantly different according to duncan's test α = 5% assessment criteria = 1 (very dislike); 2 (dislike); 3 (somewhat dislike); 4 (rather like); 5 (likes); 6 (like very much); 7 (very much like) the organoleptic test was tested in the aroma aspect, the results with the lowest score range were 2.38 from the 100% turkey berry sample and the highest score was 3.90 from the 37.5% turkey berry sample with the addition of ginger 37.5% and butterfly pea flower 25 %. the sample score is classified from not sharp to sharp enough, which means sample k has a non-sharp odor and sample b has a fairly sharp odor. the aroma of ginger is caused by essential oils, while the oleoresin causes a spicy taste. essential oils are volatile components that play a role in providing a distinctive aroma for each type of spice. while the nonvolatile components consist of gum and resin for each spice. the highest essential oil content was in red ginger followed by emprit ginger and the lowest in elephant ginger. essential oils in emprit ginger range from 1.5% 3.5% (sutanto, 2016). flavor taste treatment the results of the analysis of variance on the organoleptic taste test on the turkey berry brewed drink with the addition of ginger and butterfly pea flowers showed that the results of the interaction between the treatments were very significant. the average results of the organoleptic taste test can be seen in table 9. table 9. the average taste of turkey berry brew with the addition of ginger and butterfly pea flower flower. sample flavor score k (turkey berry 100%) 1.95 a a (turkey berry 25% + ginger 50% + butterfly pea flower 25%) 2.43 b b (turkey berry 37.5% + ginger 37.5 % + butterfly pea flower 25%) 2.78 b c (turkey berry 50% + ginger 25% + butterfly pea flower 25%) 2.65 b note: the average value followed by the same letter is not significantly different according to duncan's test α = 5% assessment criteria = 1 (very dislike); 2 (dislike); 3 (somewhat dislike); 4 (rather like); 5 (likes); 6 (like very much); 7 (very much like) from the organoleptic tests that have been tested, in the aspect of aroma, the results obtained with the lowest score range were 1.95 from the 100% turkey berry sample and the highest score was 2.78 from the 37.5% turkey berry sample with the addition of ginger 37.5% and butterfly pea flower 25%. the sample scores are classified in somewhat dislike to dislike. taste is one of the determining factors in the level of panelist acceptance of a food product. taste relates to material components that can be captured by one's sense of taste. the average value of the panelists' preference for the taste of the turkey berry brew with the addition of ginger and butterfly pea flower ranged from 2.38 to 3.70. preference favorite treatment the results of the analysis of variance on the organoleptic preference test on the turkey berry brewed drink with the addition of ginger and butterfly pea flowers showed that the results of the interaction between the treatments were very significant. the average results of the preference organoleptic test can be seen in table 10. 572 biology, medicine, & natural product chemistry 12 (2), 2023: 563-575 table 10. the average preference for turkey berry brews with the addition of ginger and butterfly pea flowers. sample preference k (turkey berry 100%) 2.73 a a (turkey berry 25% + ginger 50% + butterfly pea flower 25%) 2.70 a b (turkey berry 37.5% + ginger 37.5 % + butterfly pea flower 25%) 2.78 a c (turkey berry 50% + ginger 25% + butterfly pea flower 25%) 2.95 a note: the average value followed by the same letter is not significantly different according to duncan's test α = 5% assessment criteria = 1 (very dislike); 2 (dislike); 3 (somewhat dislike); 4 (rather like); 5 (likes); 6 (like very much); 7 (very much like) from the organoleptic tests tested in the aroma aspect, the results obtained with the lowest score range, namely 2.70 from sample a (25% turkey berry with the addition of 50% ginger and 25% butterfly pea flower) and the highest score, 2.95 from sample c (turkey berry 50% with the addition of 25% ginger and 25% butterfly pea flowers. the sample scores are classified into somewhat dislike and dislike. likeability is one of the values in the organoleptic test, which evaluates all aspects including color, aroma and taste. the assessment was carried out based on the opinion of the panelists on the samples tested. the scoring for preference is influenced by the organoleptic assessment of taste and aroma, therefore the results obtained do not differ from one another. conclusions based on the results of the research data analysis and discussion, it can be concluded that the turkey berry brewed drink with the addition of ginger and butterfly pea flower had a significant effect on physicochemistry and organoleptic ph, total phenolic content, total flavonoid content, antioxidants, aroma, taste, and preference. meanwhile, it has no significant effect on color. from a functional point of view, the best formulation of the turkey berry drink with the addition of ginger and butterfly pea flowers was found in sample c with a composition of 50% turkey berry, 25% ginger and 25% butterfly pea flower with an antioxidant ic50 value of 107.323 µg/ml. total phenol level 60.87.mggae.g-1. the highest value of flavonoid content was 21.896 mg qe/g; ph 5.782; aroma score 3.7; taste 2.6; favorite 2.95. while the best formulation based on the sensory assessment of turkey berry brewed drink with the addition of ginger and butterfly pea flower is treatment a with a composition of 25% turkey berry, 50% ginger and 25% butterfly pea flower. competing interests: the authors declare that there are no competing interests. references abdulkadir, a. r., nashriyah, m., hasan, m. m., & jahan, m. s., 2016. in vitro antioxidant activity of the ethanolic extract from fruit, stem, and leaf of solanum torvum swartz. science asia, 42(3), 184-189. acharyya, s., babli k., 2018. antimicrobial and analgesic activity of solanum torvum swartz. journal life sci, 3(6), 459-4. agbor, gabriel a, julius e oben, jeanne y ngogang, cai xinxing, joe a vinson. 2005. antioxidant capacity of some herbs/spices from cameroon: a comparative study of two methods. j. agric. food chem., 53(17), 6819-6824. akoto, o., borquaye, l. s., 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3(4):152-160 zussiva, a. dan laurent, b. k. 2012. “ekstraksi dan analisis zat warna biru (anthosianin) dari bunga telang (clitoria ternatea linn) sebagai pewarna alami”, jurnal teknologi kimia dan industri, vol.1, no.1, halaman 356-365. semarang, universitas diponegoro. this page intentionally left blank cover jurnal biomenaprochy vol 10 num 1 2021.cdr volume 10 number 1 april 2021 issn 2089-6514 (paper) issn 2540-9328 (online) biology, medicine, & natural product chemistry volume 10 – number 1 – april 2021 issn 2089-6514 (paper) | issn 2540-9328 (online) editor-in-chief: muhammad jafar luthfi department of biological education, faculty of tarbiyah and education, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia widodo department of biology education, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute nasser farid geraldo yummy foodies, benin fahrul nurkolis biological sciences, state islamic university sunan kalijaga yogyakarta, indonesia design & layout: r i y a n t o publisher: state islamic university (uin) sunan kalijaga yogyakarta co-publisher: the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university (uin) sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@yahoo.com biology, medicine, & natural product chemistry volume 10 – number 1 – april 2021 issn 2089-6514 (paper) | issn 2540-9328 (online) contents the effectiveness of giving plant pgpr rhizosphere bamboo on cocoa seeds germination at the nursery level muhammad yusril hardiansyah, yunus musa, abdul mollah jaya 1 5 compliance level of textual therapeutic usage of kshirakakoli containing formulations with a serial ethnomedicinal survey and modern system of medicine gunpreet kaur, vikas gupta, ravinder sharma, sanjiv kumar, rg singhal, ranjit singh, parveen bansal 7 14 production and optimization of pectinase from pectinolytic fungi cultivated on mango peels and pectin subjected to submerged fermentation kelemu mulluye, ameha kebede, negussie bussa 15 21 brittle bone brothers: osteogenesis imperfecta conventional serial case marsha ruthy darmawan, elysanti dwi martadiani 23 25 extraction, phenolic content and hydrogen peroxide scavenging capacity of extracts from some honey samples, propolis and bee pollen zohra mohammedi 27 32 ethnobotanical study of edible plant communities on the slopes of mount merapi and merbabu, indonesia lita ayu umartani, maizer said nahdi 33 39 computational chemical study of pigment of mangosteen (garcinia mangostana) rind extract as dye compound in dye-sensitized solar cell (dssc) didik krisdiyanto, sudarlin, hikmah supriyati 41 46 the effect of balimo (zanthoxylum nitidum) immersion water on the hematological profile of white rats (rattus norvegicus) that given liquor (ciu) panca buana wijaya, tyas rini saraswati, silvana tana, sunarno, erma prihastanti 47 57 hibiscus rosa-sinensis l. (red hibiscus) tea, can it be used as a home-remedy to control diabetes and hypercholesterolemia? subhashinie sanadheera, deepanjana subasinghe, melissa nethmi solangaarachchi, manju suraweera, noshara yushanthi suraweera, nadeesha tharangika 59 65 effect of ethanol extracts of musa paradisiaca fruit pulp and peels on haematological indices and liver enzymes of experimental rats emuesiri goodies moke, emuesiri kohworho umukoro, evelyn tarela ojugbeli, theresa ezedom, tarela melish elias daubry, iziegbe lisa omorodion 67 71 guidance for authors aims and scope biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international standards in their methodology. article types the journal seeks original 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[indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com volume 10 number 1 april 2021 published twice a year printed in indonesia front cover: sonneratia sp. flower (photograph by salman & farhan; identified by widodo) 3: front 4: editorial 5: guidance 6: back 4: editorial 4: editorial biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 305-313 | doi: 10.14421/biomedich.2023.121.305-313 issn 2540-9328 (online) antidiarrhoeal activities of lime (citrus aurantiifolia) extract in experimentally-induced diarrhoea model joshua charles isirima1, precious ojo uahomo2,* 1department of pharmacology, faculty of basic clinical sciences; 2department of biomedical technology, school of science laboratory technology, university of port harcourt, rivers state, nigeria. corresponding author* uahomoprecious1@gmail.com manuscript received: 20 february, 2023. revision accepted: 11 march, 2023. published: 28 march, 2023. abstract this study investigated the effects of lime on diarrhoea in wistar rats. a total of 60 wistar rats were procured and randomly divided into 3 groups of 20 animals each for each of the three t-test models. the twenty healthy wistar rats for each diarrhoea model were fasted for 6 hours prior to the experiment but allowed free access to water. the twenty animals were randomly divided into 5 groups of 4 animals each for each experiment. established antidiarrhea models were followed. the test groups received various doses (97.65mg/kg, 195.3mg/kg, and 390.6mg/kg) of citrus aurantiifolia juice extract; whereas positive controls received loperamide (2.5mg/kg) and negative controls received distilled water (1ml/kg). the administration was done once daily for 15 days, and the faeces of each animal was collected on the 5th, 10th and 15th day. the result of this study showed that medium and high dose citrus aurantiifolia has an anti-diarrhoeal effect on castor oil-induced diarrhoea over repeated administration for a minimum of 15 days as it prolonged the onset of diarrhoea, decreased the frequency of defecation and gastrointestinal transit time in wistar rats. this study shows that citrus aurantiifolia demonstrates significant anti-diarrhoeal activity and can be used as an anti-diarrhoea agent. keywords: diarrhoea; citrus aurantiifolia; castor oil; lime juice extract; gastrointestinal tract; enteropooling; diarrhoea model. introduction limes (citrus aurantifolia) are a small citrus fruit either sour or sweet. sour limes possess a greater sugar and citric acid content than lemons and feature an acidic and tart taste. lime contains unique flavonoid compounds that have antioxidant and anticancer properties. while these flavonoids have been shown to stop cell division in many cancer lines, they are perhaps most interesting for their antibiotic effects (bina et al., 2010). citric acid the major organic acid in these juices has been found to be responsible for inhibiting the growth of vibrio (gram-negative bacteria of the family vibronaceae). the natural biocidal activity of lime juice was studied in order to explore its possible use as a disinfectant and inhibitor of vibrio cholera in drinking water for areas lacking water treatment plants (d'aquino et al., 1994). owing to the support of the national and international organizations for the studies on the treatment and prevention of these diseases based on traditional practices, medicinal plants are becoming a hopeful source of antidiarrhoeal drugs (lin et al., 2002). according to ekwawati and darmanto (2019), medicinal plants are one of the natural resources that can be explored by humans. various secondary metabolites from plants can be used as medicines, agrochemicals, flavours, fragrances, dyes, biopesticides and food addictive. citrus fruits contain nutrients and phytochemicals that are beneficial to health. citrus juice contains various substances including carbohydrates, fibre, vitamin c, potassium, folate, calcium, thiamine, niacin, vitamin b6, vitamin a, phosphorus, magnesium, copper, riboflavin, pantothenic acid and various phytochemicals. these substances are needed by the body. some compounds in citrus fruit can provide additional protection against chronic disease and basic nutrition. citrus fruits also contain lots of phytochemicals, essential oils, alkaloids, flavonoids, psoralens, carotenoids, limonoids, tannins, and terpenoids. previous pharmacological studies revealed that citrus fruits have antimicrobial, anthelminthic, antioxidants, anticancer and many other pharmacological effects (prastiwi and ferdiansyah, 2013). diarrhoea is an alteration in the normal bowel movement characterized by an increase in the volume, fluidity, frequency, and passage of loose or watery stool at least three times a day (unicef/who, 2009). it is a common symptom of gastrointestinal infection due to the ingestion of many bacteria, viruses, or parasites that may be transmitted by water, food, utensils, hands, and flies (unicef/who, 2009). it is the mechanism by which https://doi.org/10.14421/biomedich.2023.121.305-313 306 biology, medicine, & natural product chemistry 12 (1), 2023: 305-313 the body rids itself of pathogenic organisms, with excessive stimulation of intestinal motility, leaving insufficient time for the absorption of intestinal fluid (keusch et al., 2006). diarrhoea is one of the most important health problems in developing countries affecting people of all ages (snyder and merson, 1982) and results in electrolyte loss, dehydration, shock, and sometimes death (unicef/who, 2009). diarrhoeal diseases account for 1 in 9 child deaths worldwide, making diarrhoea the second leading cause of death among children under the age of 5 (hutton et al., 2007), while lakshminarayana et al. noted that diarrhoea is a leading cause of mortality and morbidity in children under 5 years old, and it accounts for 5–8 million deaths worldwide each year (lakshminarayana et al., 2011; world health organization, 2006). a report in 2015 revealed that diarrhoea is one of the main killers of children that accounts for 9% of all deaths among kids below the age of 5 years worldwide (unicef, 2019). according to this report, sub-saharan africa and southern asia were recorded as the regions that experienced the highest child death toll because of diarrhoea (sow et al., 2016). in nigeria, the prevalence of diarrhoeal infection is as high as 18.8%, above the average of 16%, making it one of the worst in sub-saharan africa (olawuyi et al., 2004). it accounts for an annual estimated 300,000 deaths mainly amongst children under five in nigeria (olawuyi et al., 2004), while 7-to-12-month-old babies continue to be the most susceptible (audu et al., 2000) caused mainly by poor sanitation and hygiene practices. the disease may be caused by a wide array of agents such as enteropathogenic microorganisms (shigella flexneri, staphylococcus aureus, escherichia coli, salmonella typhi, and candida albicans) (jouret-mourin and geboes, 2002), alcohol, irritable bowel syndrome, bile salts and hormones (teke et al., 2007), secretory tumours, and intoxication (brijesh et al., 2011), whereas susan & mays said the causes of diarrhoea include infectious agents, gastrointestinal disorders such as inflammatory or dysmotility problems, and substances that increase gastrointestinal tract secretions (susan and mays, 2005). despite different pathophysiological changes in different types of diarrhoeas, there are four major mechanisms responsible for pathophysiology in electrolyte and water transport that is, increased luminal osmolarity, increased electrolyte secretion, decreased electrolyte absorption and accelerated intestinal motility causing decreased transit time (lutterodt, 1992). management of diarrhoea comprises both nonpharmacological and pharmacological interventions. in general, the treatment is aimed at reducing the discomfort and inconvenience of frequent bowel mobility and frequency of faecal passage (suleiman et al., 2008). several options employed in the management of the diseases include oral agents such as metronidazole, antibiotics, and oral rehydration therapy (ort) (hardman et al., 2001; sastry and burgard, 2005). with over a decade of the practice and promotion of ort, diarrhoea is still the second the cause of child death (grant, 1993). in addition, these management options which often fail during high stool output state are also associated with undesirable side effects such as headache, convulsion, stomach cramps, vomiting, constipation, and hallucination. consequently, attention is now being shifted to alternatives in medicinal plants for the management of the disease. medicinal plants represent a promising source for the discovery of new antidiarrhoeal agents. these plants are cheaper and more easily available than conventional medicines. even the world health organization has encouraged studies for the treatment and prevention of diarrhoeal disease using traditional medicinal practices (atta et al., 2004). various plants are being investigated for their possible antidiarrhoeal activities to provide safe and inexpensive alternatives to standard drug therapies. materials and methods animals procurement animals were procured from the department of pharmacology, faculty of basic clinical sciences, university of port harcourt and were acclimatized for a period of two weeks with their weights checked constantly during this period. all the animals (160±0.02g) were housed in clean plastic cages that were placed in a well-ventilated house (temperature: 22 ± 3oc; photoperiod: 12 hours; humidity: 45–50%). the animals were fed on rat pellets (premier feed mill co. ltd., ibadan, nigeria) and clean tap water, except when fasting was required during the experiment. the cages and the animal house were cleaned on daily basis. plant collection fresh lime fruits were purchased from choba market and were identified and authenticated at the department of plant science and biotechnology, university of port harcourt, herbarium. extraction of plant the extraction method by amita and shalini (2014) with slight modifications was used. the lime was sliced and the juice was squeezed out into a beaker and after which the extract was filtered. the filtrate was then centrifuged for clarification of the extract (das et al., 2010) and stored in the refrigerator for use. drugs and chemicals loperamide hydrochloride was products of laborate pharmaceuticals (india) and richy gold international limited (nigeria), respectively. castor oil was a product isirima & uahomo – antidiarrhoeal activities of lime (citrus aurantiifolia) extract in … 307 of bills sons and co. (druggist) limited, southport, england. experimental protocols castor oil-induced diarrhoea in rats twenty (20) healthy wistar rats were fasted for 6 hours prior to the experiment but allowed free access to water. the experimental rats were completely randomized into five groups of four animals each. the procedure described by bajad et al. (2001) was adopted with slight modification. animals in group a which received 1.0ml of distilled water served as a negative control, while those in groups b (positive control), c, d, and e (test groups) received 1.0ml each corresponding to 2.5mg/kg body weight of loperamide (a reference drug), 0.25, 0.5, and 1.0ml of the extract, respectively. thirty minutes after administration, all the animals were orally administered 1ml of castor oil and thereafter placed in cages lined with a pre-weighed transparent paper. during the 6-hour observation period, the time of onset of diarrhoea, the total number of faeces, diarrhoeal faeces, total weight of faeces, and percentage inhibition of diarrhoeal defecation in each group were computed. the weight of the faeces was obtained from the difference in the pre-weighed transparent paper and the fresh weight of the stool. the dry weight of the faeces was obtained by drying the fresh faeces in the laboratory oven (uniscope laboratory oven, sm9053, surgifriend medicals, england) at 1000c until a constant weight was obtained. the difference in the fresh weight of the faeces and the dry weight of the faeces gives the water content of the faeces. at the end of the 6-hour exposure period, the animals were sacrificed under the effect of diethyl ether anaesthesia and the small intestine of the animals was dissected and removed. thereafter, the contents of the small intestines were squeezed out. castor oil-induced enteropooling the procedure described by chitme et al. (2004) was adopted for the castor oil-induced enteropooling study. the animals were fasted without food for 6h prior to the experiment but were allowed free access to water. four animals were randomly selected for each group and placed in their respective cages. animals in the negative control group (group a) received 1.0ml of distilled water, while those in the positive control group (group b) received 1.0ml of loperamide (2.5mg/kg body weight). rats in groups c, d, and e (test groups) were orally administered 0.25, 0.50, and 1.0ml of the sap, respectively. immediately afterwards, 1.0ml of castor oil was administered orally to each of the rats in all the groups. after 30 minutes, each rat was sacrificed according to the method described by akanji and yakubu (2000) and the ends of the pylorus and caecum of the small intestine were tied. the small intestine was dissected and its intestinal content was squeezed into a measuring cylinder. the volumes and the masses of the intestinal contents were noted and used to compute the percentage of inhibition of intestinal content. gastrointestinal motility the method described by teke et al. (2007) was adopted for the evaluation of the effect of the sap on gastrointestinal transit in rats. twenty, healthy wistar rats were fasted for 6 hours prior to the experiment but were allowed free access to water. the experimental rats were completely randomized into five groups of four animals each. the negative control group (group a) received 1.0ml of distilled water while the positive control group (group b) received 1.0ml of loperamide (0.6mg/ml) intramuscularly. animals in groups c, d, and e (test groups) were orally administered 0.25, 0.50, and 1.0ml of the sap, respectively. charcoal meal (10% charcoal suspension in 5% agarose agar, prepared by weighing 10 g of charcoal powder and 5 g of agarose agar into 100ml distilled water and mixed thoroughly) was administered orally, 30 minutes after the administration of atropine sulphate and the citrus aurantiifolia. the animals were then sacrificed after 45 minutes of charcoal administration, using the diethyl ether as anaesthesia as described by akanji and yakubu (2000). the small intestine was removed very carefully and the length of the intestine as well as the distance travelled by the charcoal meal through the intestine was measured. the percentage of gastrointestinal motility was computed as the ratio of the distance moved by the charcoal meal to the length of the small intestine. methods of data analysis the experimental results were analyzed using the statistical package for the social sciences (ibm spss), version 25.0. results were expressed as a mean ± standard error mean (sem), and statistical analyses were carried out by employing a one-way analysis of variance (anova), followed by tukey’s post hoc test to compare the results of controls and the groups. in all cases, statistical significance was set at p<0.05. results analysis of the effect of the extract on castor oilinduced diarrhoea in wistar rats below is a table showing the observed effect of citrus aurantiifolia on castor oil-induced diarrhoea and enteropooling respectively in wistar rats. the low dose, medium dose and high dose of the extract were administered and the effect of these substances on the wistar rats was observed and analysed alongside that of the well-known drug and the control substance. 308 biology, medicine, & natural product chemistry 12 (1), 2023: 305-313 table 1. effects of citrus aurantiifolia (lime juice extract) on castor oil-induced diarrhoea in wistar rats. group mot mnd mnws mnds mfws mwcs control 63.25±0.47 6.00±0.41 2.50±0.29 2.00±0.41 4.75±0.25 2.20±0.41 loperamide 121.75±1.37 2.50±0.29 1.25±0.25 1.25±0.25 0.83±0.025 0.48±0.025 low dose 93.50±1.55 2.75±0.48 1.50±0.29 1.50±0.29 0.93±0.03 0.55±0.03 medium dose 108.00±0.91 2.50±0.29 1.00±0.41 1.50±0.29 0.78±0.03 0.45±0.03 high dose 140.00±1.08 1.75±0.48 0.75±0.25 1.70±0.25 0.65±0.00 0.30±0.41 mot = mean onset time (min); mnd = mean number of defecations; mnws = mean number of wet stool; mnds = mean number of dry stool; mfws = mean fresh weight of stool; mwcs = mean water content of stool table 2. effects of citrus aurantiifolia (lime juice extract) on castor oilinduced enteropooling in wistar rats. group mwic mvic control 2.88±0.14 2.60±0.09 loperamide 1.63±0.09 1.60±0.07 low dose 2.15±0.10 1.90±0.27 medium dose 1.65±0.25 1.33±0.11 high dose 0.98±0.17 1.10±0.04 mwic = mean weight of intestinal content; mvic = mean volume of intestinal content table 3. effects of citrus aurantiifolia (lime juice extract) on castor oilinduced transit time in wistar rats. group mdtcm control 77.25±0.63 loperamide 35.50±0.29 low dose 40.25±0.25 medium dose 31.50±0.64 high dose 27.75±1.11 mdtcm = mean distance travelled by charcoal meal table 4. effects of citrus aurantiifolia (lime juice extract) on the onset of diarrhoea in castor oil-induced diarrhoea in wistar rats. parameter distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mot 63.25±0.47 121.75±0.53 93.50±1.55 108.00±0.91 140.00±1.08 dfreq 92.49±0.09 47.83±1.08 70.75±0.24 121.34±0.64 𝐷𝑓𝑟𝑒𝑞 = 𝑀𝑂𝐷𝑇𝐺−𝑀𝑂𝐷𝑁𝐺 𝑀𝑂𝐷𝑁𝐺 𝑥 100 (1) where, mot = mean onset time, dfreq= percentage inhibition in terms of control of the onset of diarrhoeal stool, modtg = mean onset of diarrhoea in the treated group and modng = mean onset of diarrhoea in the negative control (schuster, 2001). table 5. effects of citrus aurantiifolia (lime juice extract) on the mean number of defecations in castor oil-induced diarrhoea in wistar rats. parameter distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mnd 6.00±0.41 2.50±0.29 2.75±0.48 2.50±0.27 1.75±0.48 pid 58.33±0.41 54.17±0.18 58.33±0.43 70.83±0.07 𝑃𝐼𝐷 = 𝑀𝑁𝐷𝐶𝐶−𝑀𝑁𝐷𝐶𝐷/𝐸 𝑀𝑁𝐷𝐶𝐶 𝑥 100 (2) where, mnd = mean number of defecations, pid = percentage inhibition of defecation, mndcc = mean number of defecations caused by castor oil and mndcd/e = mean number of defecations caused by drug/extract (kola-mustapha et al., 2019). table 6. effects of citrus aurantiifolia (lime juice extract) on mean number of wet faeces in castor oil-induced diarrhoea in wistar rats. parameter /dose (ml) distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mnws 2.50±0.29 1.25±0.25 1.50±0.17 1.00±0.41 0.75±0.25 pfreq 50.00±0.03 40.00±0.12 60.00±0.14 70.00±0.04 isirima & uahomo – antidiarrhoeal activities of lime (citrus aurantiifolia) extract in … 309 𝑃𝑓𝑟𝑒𝑞 = 𝑀𝑁𝑊𝑆𝐶−𝑀𝑁𝑊𝑆𝑇 𝑀𝑁𝑊𝑆𝐶 𝑥 100 (3) where, mnws = mean number of wet stools, pfreq = percentage inhibition in terms of the purging frequency (number of wet stools), mnwsc = mean number of wet stool in control group and mnwst =mean number of wet stool in treated group. table 7. effects of citrus aurantiifolia (lime juice extract) on number of dry faeces, fresh weight of faeces and water content of faeces in castor oilinduced diarrhoea in wistar rats. parameter distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mndf 2.00±0.41 1.25±0.25 1.50±0.29 1.50±0.29 1.70±0.25 mfwf 4.75±0.25 0.83±0.025 0.93±0.03 0.78±0.03 0.65±0.01 mwcf 2.20±0.41 0.48±0.025 0.55±0.03 0.45±0.03 0.30±0.41 where, mndf = mean number of dry faeces, mfwf = mean fresh weight of faeces and mwcf = mean water content of faeces table 8. effects of citrus aurantiifolia (lime juice extract) on weight of intestinal content in castor oil-induced diarrhoea in wistar rats. paramete distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mwic 2.88±0.14 1.63±0.09 2.15±0.10 1.65±0.25 0.98±0.17 piic 43.40±0.24 25.35±0.04 42.71±0.29 65.97±0.64 𝑃𝐼𝐼𝐶 = ( 𝑀𝑊𝐼𝐶𝐶−𝑀𝑊𝐼𝐶𝑇 𝑀𝑊𝐼𝐶𝐶 ) 𝑥 100 (4) where, mwic = mean weight of the intestinal content, piic = percentage inhibition of intestinal content, mwicc = mean weight of the intestinal content of the control group and mwict = mean weight of the intestinal content of the test group (giday et al., 2010). table 9. effects of citrus aurantiifolia (lime juice extract) on volume of intestinal content in castor oil-induced diarrhoea in wistar rats. parameter distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mvic 2.60±0.09 1.60±0.07 1.90±0.27 1.33±0.11 1.10±0.04 piifv 38.46±0.02 26.92±0.21 48.85±0.17 57.69±0.05 𝑃𝐼𝐼𝐹𝑉 = ( 𝑀𝑉𝐼𝐶𝐶−𝑀𝑉𝐼𝐶𝑇 𝑀𝑉𝐼𝐶𝐶 ) 𝑥 100 (5) where, mvic = mean volume of the intestinal content, piifv = percentage inhibition of intestinal fluid volume, mvicc = mean volume of the intestinal content of the control group and mvict = mean volume of the intestinal content of the test group (ekor, 2014). table 10. effects of citrus aurantiifolia (lime juice extract) on charcoal meal distance travelled in wistar rats. parameter distilled water (ml) loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 1 2.5 97.65 195.3 390.6 mdtcm (cm) 77.25±0.63 35.50±0.29 40.25±0.25 31.50±0.64 27.75±1.11 pi 68.97±0.13 31.70±0.21 33.04±0.11 28.13±0.12 24.78±0.45 gmeq 54.04±0.13 42.72±0.12 59.21±0.02 64.07±0.31 mean length of small intestine (mlsi) = 112±0.56cm 𝑃𝐼 = 𝑀𝐷𝑇𝐶𝑀 𝑀𝐿𝑆𝐼 𝑥 100 (6) where, pi = peristaltic index, mdtcm = mean distance travelled by charcoal meal and mlsi = mean length of the small intestine (agegnehu et al., 2019) 𝑃𝐼𝑇 = 𝑀𝐷𝑇𝐶𝑀 𝑀𝐿𝑆𝐼 𝑥 100 (7) where, pit = percentage intestinal transit, mdtcm = mean distance travelled by charcoal meal, and mlsi = 310 biology, medicine, & natural product chemistry 12 (1), 2023: 305-313 mean length of small intestine (kola-mustapha et al., 2019). 𝑃𝐼 (𝐺𝑚𝑒𝑔) = 𝑃𝐼𝐶−𝑃𝐼𝑇 𝑃𝐼𝐶 𝑥 100 (8) where, gmeq = percentage inhibition and pic = peristaltic index of control and pit = peristaltic index of test group (bern et al., 1992). or 𝐺𝑚𝑒𝑞 = 𝑃𝐼𝐶−𝑃𝐼𝑇 𝑀𝑃𝐼𝐶𝐺 𝑥 100 (9) where gmeq = percentage inhibition in terms of control in the gut travelled distance, mpicg = mean peristaltic index of control group and mpitg = mean peristaltic index of treated group. table 11. the in vivo anti-diarrhoeal index (adi in vivo) (hussain et al., 2009; than et al., 1989). parameter loperamide (mg/kg body weight) citrus aurantiifolia (mg/kg) 2.5 97.65 195.3 390.6 dfreq 92.49±0.09 47.83±1.08 70.75±0.24 121.34±0.64 gmeq 54.04±0.13 42.72±0.12 59.21±0.02 64.07±0.31 pfreq 50.00±0.03 40.00±0.12 60.00±0.14 70.00±0.04 adi in vivo 62.99±0.07 43.40±0.25 63.11±0.09 81.64±0.2 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √𝐷𝑓𝑟𝑒𝑞 𝑥 𝐺𝑚𝑒𝑞 𝑥 𝑃𝑓𝑒𝑞 3 (10) adi in vivo for loperamide 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(92.49𝑥54.04𝑥50.00)3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √249907.98 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 62.99 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(0.09𝑥0.13𝑥0.03)3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √0.000351 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 0.07 adi in vivo for low dose 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(47.83𝑥42.72𝑥40.00)3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √81731.904 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 43.40 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(1.08𝑥0.12𝑥0.12)3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √0.015552 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 0.25 adi in vivo for medium dose 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(`70.75𝑥59.21𝑥60.00)3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √251346.45 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 63.11 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(0.24𝑥0.02𝑥0.14)3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √0.000672 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 0.09 adi in vivo for high dose 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(121.34𝑥64.07𝑥70.00)3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √544197.766 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 81.64 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √(0.64𝑥0.31𝑥0.04)3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = √0.007936 3 𝐴𝐷𝐼𝑖𝑛𝑣𝑖𝑣𝑜 = 0.2 discussion effect of citrus aurantiifolia (lime juice extract) on castor oil-induced diarrhoea in wistar rats. the result of the effect of citrus aurantiifolia on castor oil-induced diarrhoea in wistar rats is presented in table 1. it was observed that the animals had diarrhoea after treatment with castor oil. however, when loperamide (standard drug) was administered there was an observed significant reversal reduction in the mean number of defecation (mnd), mean number of wet stool (mnws), mean number of dry stool (mnds), mean fresh weight of stool (mfws) and mean water content of stool (mwcs) compared to the mean onset time (mot) and the normal control group, this means that loperamide is effective in the treatment of diarrhoea. this reversal is not shocking because loperamide is a known antidiarrhoeal agent used in the treatment of diarrhoea that works by decreasing peristalsis and fluid secretion, resulting in longer gastrointestinal transit time and increased absorption of fluids and electrolytes from the gastrointestinal tract (baker, 2007). therefore, since castor oil works by stimulating prostaglandin synthesis thereby increasing fluid and electrolyte into the lumen of the bowels, it is expected that loperamide will reverse the isirima & uahomo – antidiarrhoeal activities of lime (citrus aurantiifolia) extract in … 311 process. furthermore, when the low dose of citrus aurantiifolia was administered there was also an observed significant reversal and reduction in the mean number of defecation (mnd), mean number of wet stool (mnws), mean number of dry stool (mnds), mean fresh weight of stool (mfws) and mean water content of stool (mwcs) when compared to the mean onset time (mot) and the normal control group. also, when the medium dose of citrus aurantiifolia was administered to the wistar rats, there was an observed significant reversal and reduction in the mean number of defecation (mnd), mean number of wet stool (mnws), mean number of dry stool (mnds), mean fresh weight of stool (mfws) and mean water content of stool (mwcs) when compared to the mean onset time (mot) and the normal control group. since the medium dose of citrus aurantiifolia caused a reversal of the diarrhoea caused by the castor oil, it could mean that it works with the same mechanism with loperamide. therefore, since castor oil works by stimulating prostaglandin synthesis thereby increasing fluid and electrolyte into the lumen of the bowels, it is expected that loperamide will reverse the process. furthermore, when the high dose of citrus aurantiifolia was administered to the wistar rats, there was an observed significant reversal reduction in the mean number of defecation (mnd), mean number of wet stool (mnws), mean number of dry stool (mnds), mean fresh weight of stool (mfws) and mean water content of stool (mwcs) when compared to the mean onset time (mot) and the normal control group. generally, the citrus aurantiifolia is observed to be effective in the treatment of diarrhoea just like loperamide, with the high dose seen to be more effective than other doses. effect of citrus aurantiifolia (lime juice extract) on castor oil-induced enteropooling in wistar rats. the result of the effects of citrus aurantiifolia on castor oil-induced enteropooling in wistar rats is presented in table 2. there was an increase in the enteropooling level of the wistar rats when exposed to citrus aurantiifolia, but when the standard anti-diarrhoeal was administered, there was an observed reduction in the mean weight of intestinal content (mwic) and the mean volume of content (mvic) when compared with the normal control group. this means that loperamide can reverse reduction in accumulation of fluids in the small intestine. this reversal is possible because loperamide is a known anti-diarrhoeal agent used in the treatment of diarrhoea that works by decreasing peristalsis and fluid secretion, resulting in longer gastrointestinal transit time and increased absorption of fluids and electrolytes from the gastrointestinal tract (baker, 2007). therefore, since castor oil works by stimulating prostaglandin synthesis thereby increasing fluid and electrolyte into the lumen of the bowel. however, when the standard anti-diarrhoeal was administered, there was an observed reduction in the mean weight of intestinal content (mwic) and the mean volume of content (mvic) when compared with the normal control group. this means that loperamide can reverse reduction in accumulation of fluids in the small intestine. furthermore, when the medium dose of citrus aurantiifolia was administered there was an observed significant reduction in the mean weight of intestinal content (mwic) and the mean volume of content (mvic) when compared with the normal control group. this means that the medium dose of citrus aurantiifolia the ability to cause a reversal reduction in accumulation of fluids in the small intestine. since the medium dose of citrus aurantiifolia caused a significant reduction in the enteropooling level of the wistar rats. also, when the high dose of citrus aurantiifolia was administered there was an observed significant reduction in the mean weight of intestinal content (mwic) and the mean volume of content (mvic) when compared with the normal control group. it indicates that the high dose of citrus aurantiifolia could cause a reversal reduction in accumulation of fluids in the small intestine. hence, the loperamide is effective for the enteropooling reversal and citrus aurantiifolia is also effective for enteropooling reversal with the high dose seen to be more significant followed by the medium dose and then the low dose. effects of citrus aurantiifolia (lime juice extract) on castor oil-induced transit time in wistar rats the result of the effect of citrus aurantiifolia on castor oil-induced transit time in wistar rats is presented in table 3. during the study, it was observed that there was an increase in the transit time of the wistar rats when exposed to castor oil but when loperamide was administered, there was an observed reversal reduction in the mean distance travelled by charcoal (mdtcm) meal when compared with the normal control group. this means that loperamide is effective in the reversal of the inability of the ingested food to travel on time. loperamide is a known anti-diarrhoeal agent used in the treatment of diarrhoea that works by decreasing peristalsis and fluid secretion, resulting in longer gastrointestinal transit time and increased absorption of fluids and electrolytes from the gastrointestinal tract (baker, 2007). and castor oil works by stimulating prostaglandin synthesis thereby increasing fluid and electrolyte into the lumen of the bowels, therefore loperamide could reverse the process. furthermore, when the low dose of citrus aurantiifolia was administered there was an observed significant reduction in the mean distance travelled by charcoal meal (mdtcm) when compared with the normal control group. when the medium dose of citrus aurantiifolia was administered there was a significant reduction in the mean distance travelled by charcoal meal (mdtcm) when compared with the normal control group. when the high dose of citrus aurantiifolia was administered there was an observed significant reduction in the mean distance 312 biology, medicine, & natural product chemistry 12 (1), 2023: 305-313 travelled by charcoal meal (mdtcm) when compared with the normal control group. the high dose of citrus aurantiifolia caused a significant reduction in the time it takes for ingested food to travel through the gut of the wistar rats, hence, loperamide is effective in the treatment of delay in transit time and citrus aurantiifolia is effective with high dose seen to be more effective followed by medium dose and then the low dose. this finding is similar to a previous study on the antidiarrheal properties of aqueous leaf extract of cyathula prostrata by uahomo and isirima (2022). in conclusion, the results of this study have shown that citrus aurantiifolia demonstrates significant antidiarrhoeal activity and may be working through antisecretory and anti-motility mechanisms or through inhibition of prostaglandin activities and/or synthesis. acknowledgements: the researchers acknowledge all laboratory staffs that assisted in ensuring this research is a success. authors’ contributions: isirima jc designed the study and analyzed the data 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https://books.google.com.ng/books?id=a4cyekpbxh8c&lpg=pp2&ots=jnbujot0hv&dq=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&lr&pg=pp2#v=onepage&q=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&f=false https://books.google.com.ng/books?id=a4cyekpbxh8c&lpg=pp2&ots=jnbujot0hv&dq=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&lr&pg=pp2#v=onepage&q=world%20health%20organization%20(2006).%20country%20health%20system%20fact%20sheet&f=false this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 239-242 | doi: 10.14421/biomedich.2025.141.239-242 issn 2540-9328 (online) in vivo evaluation of antidepressant potential of temulawak (curcuma xanthorriza robb.) ethanol extract in mus musculus raja david virgilius1,*, zest lively limbong2, jericho l ginting3 1department of chemistry, faculty of mathematics and natural sciences, ipb university, indonesia. 2department of forestry management, faculty of forestry, ipb university, indonesia. 3department of plant protection, faculty of agriculture, ipb university, indonesia. corresponding author* stanislausraja@apps.ipb.ac.id manuscript received: 18 february, 2025. revision accepted: 20 may, 2025. published: 23 june, 2025. abstract temulawak (curcuma xanthorriza roxb.) is a medicinal plant from the zingiberceae family widely used in indonesia for its various bioactives, including antiviral, antibacterial, and hepatoprotective properties. this research aims to evaluate the antidepressant potential of curcuma xanthorriza ethanol extract in mus musculus through in vivo testing. the extraction used the maceration method with 96% ethanol as a solvent, yielding 5.2504% extract. the primary bioactive compounds identified in the extract were terpenoids and curcuminoids, which are known for their neuroprotective and antidepressant effects. the antidepressant activity was assessed using the tail-suspension test (tst) and forced-swimming test (fst), with immobility time as the main parameter. amitriptyline, a standard antidepressant, was used as a positive control, while sodium carboxymethylcellulose served as a negative control. the results showed that increasing doses of curcuma xanthorriza extract significantly reduced immobility time, indicating an antidepressant-like effect. statistical analysis revealed that the immobility time in mice treated with a 650 mg/kg bw dose of curcuma xanthorriza extract was not significantly different from the amitriptyline group, suggesting comparable efficacy. these findings highlight the potential of curcuma xanthorriza ethanol extract as a natural antidepressant, warranting further investigation into its mechanisms and clinical applications. keywords: antidepressant; curcuma xanthorriza roxb.; forced-swimming test phytochemistry; tails-suspension test. introduction temulawak (curcuma xanthorriza robb.) is one of the plants from the zingiberaceae family. temulawak has a very similar shape to turmeric. java island is the place where temulawak thrives. indonesians tend to use temulawak as a food flavoring additive and herbal medicine. based on research results, temulawak ethanol extract has many bioactivities such as antiviral, antibacterial, hepatoprotective, and other bioactivities (syamsudin et al., 2019). the bioactivity possessed by temulawak ethanol extract is due to the presence of the chemical compounds it contains. scientific studies showed that the most abundant essential phytochemicals obtained from the temulawak ethanol extract were terpenoids and curcuminoids (zhang et al., 2014). other contents possessed by temulawak ethanol extract are vanillin, 3-hydroxy-6-methylacetophenone, 12,13-epoxyxanthorrhizol, 3-hydroxy-6-methylacetophenone, and dehydro-6-gingeridone (zhang et al., 2015) as well as several amino acids such as methionine, phenylalanine, tryptophan, valine, alanine, lysine, and leucine (rahmat et al., 2021). mental health has become a hotly debated issue, especially among young people. one of the mental health issues that can happen to anyone is depression. depression is a mood disorder that causes a persistent feeling of sadness and loss of interest (suma & hasan, 2023). depression is one of the leading causes of disability, and, if left unmanaged, it can increase the risk of suicide. an imbalance of neurotransmitters in the brain such as serotonin and dopamine is the cause of depression. based on data from the global burden of disease study, by 2020 there will be at least 322 million people in the world suffering from depression (charlie et al., 2024). the treatment of depression cases varies depending on the condition of the patient ranging from psychological assistance to the use of antidepressant drugs. antidepressants refer to psychotropic drugs that are used to treat mental illness with prominent emotional depression symptoms (yunxi et al., 2023). the use of antidepressant drugs is closely monitored and regulated by the fda. antidepressants are the last resort for people with depression. antidepressants work to relieve the symptoms experienced by people with depression such as https://doi.org/10.14421/biomedich.2025.141.239-242 240 biology, medicine, & natural product chemistry 14 (1), 2025: 239-242 anxiety, increased fatigue, impaired concentration, and emotional outbursts. the mechanism of action of antidepressant drugs varies from inhibiting monoamine reuptake, inhibiting monoamine breakdown, and increasing serotonin reuptake and release of serotonin and norepinephrine (chittaranjan & sanjay, 2010). material and methods tools and materials the tools used are laboratory glassware, stative, syringe, analytical balance, pestle and mortar, yarn, feeding tube, beaker glass, spatula, and rotary evaporator. the ingredients used in this research were curcuma xanthorriza roxb. rhizomes, ethanol 96%, amitriptyline, water, and sodium carboxymethyl cellulose. procedures preparation and acclimatization of the test animals animal acclimatization was carried out for 7 days by giving the mice standard feed and drinking water ad libitum. feed is given as much as 4 grams/head/day. the mice used for treatment were 48 mice (n=8 head/group). mice were weighed and the drum was separated in each treatment. all research procedures were approved by the ethics committee of the faculty of mathematics and natural sciences, ipb university. preparation of curcuma xanthorriza ethanol extract the temulawak rhizome was cleaned, peeled, and sliced before being dried in an ovent at 45⁰c for 6-8 hours. after drying, the sample was ground into powder and the simplisia was filtered using an 80-mesh sieve to obtain softer temulawak powder, which was extracted using the maceration method. for the extraction process, 100 grams of temulawak powder was extracted using ethanol 96% for 24 hours. the macerate was filtered using whatman filter paper. subsequently, the extracted solution was separated from the solvent using a rotary evaporator 45⁰c and 80 cmhg pressure to obtain a concentrated extract. the extract yield was calculated and stored in a vial bottle at -20⁰c to 4⁰c for measuring antidepressant bioactivity. antidepressant activity test antidepressant activity testing with the tail suspension test (tst) and forced swimming test (fst) method was performed based on the procedure of castagne et al., 2010. mice that have been acclimated for 14 days were randomly divided into 4 groups, as follows the following: table 1. test animal grouping data. group name treatment normal untreated mice negative control sodium carboxymethylcellulose 0,5% positive control amitriptyline group i dose 325 mg/kg bw group ii dose 650 mg/kg bw mice were placed in the experimental room at least 60 minutes before starting the experiment. the decoction and positive control were administered once a day. antidepressant activity testing was performed 1 hour after oral administration of the treatment (castagne et al., 2010). immobility time was observed, namely the duration of time not moving (body, legs, and hands in a state of rest) for ≥ 1 second per mouse for 6 minutes (ueno et al., 2022) data analysis the immobility time data were used to calculate the percent of antidepressant activity of the ethanol extract of curcuma xanthorriza against the positive control amitriptyline. statistical analysis using spss 29.0 for windows. values expressed as mean ± sd were then compared with a one-way anova test followed by an lsd post hoc test to find out whether the differences between treatment groups if the data are significantly different (h0 is rejected if the probability <0,05). results and discussion curcuma xanthorriza roxb extract curcuma xanthorriza roxb. extraction was done by the maceration method by soaking curcuma xanthorriza roxb. powder in 96% ethanol solvent for 24 hours. the ethanol solvent was chosen because it is a polar solvent and can dissolve polar compounds such as terpenoids and curcuminoids (aisyah & erminawati, 2022). terpenoids and curcuminoids have been shown to have antidepressant bioactivity. the extraction process produced a paste of curcuma xanthorriza extract. a total of 5.2504 grams of curcuma xanthorriza extract paste was produced from 100 grams of curcuma xanthorriza powder. the yield of curcuma xanthorriza maceration extraction was 5.2504% as shown in table 1. the yield of a extraction process is influenced by several factor such as temperature, solute and solvent ratio, target compounds to be extracted, and solvent purity (abin et al., 2024). terpenoid and curcuminoid isolates have been shown to have antidepressant bioactivity. the yield value of 5.2504% obtained in this research is not much different from the yield value of 5.42% obtained in the research of lidvina et al., 2017. table 2. calculation of yield from curcuma xanthorriza extracts. weight of c. xanthorriza powder weight of extract %yield 100 grams 5.2504 grams 5,2504% antidepressant activity of curcuma xanthorriza ethanol extract bioactivity testing was carried out by open tailssuspension test (tst) and forced-swimming test (fst) methods on each group of mice with the measurement virgilius et al. – in vivo evaluation of antidepressant potential … 241 variable being immobility time. the antidepressant bioactivity of curcuma xanthorriza ethanol extract can be described by the immobility time. the increase in immobility time is directly proportional to the antidepressant bioactivity. stress in mice causes immobility in the mice’s bodies, so a high immobility time indicates a high stress level. amitriptyline was used as the positive control in this research. amitriptyline was chosen as the positive control because amitriptyline has been commonly consumed as an antidepressant drug commonly used in the community. the mechanism of action of the drug amitriptyline is by inhibiting the reuptake of serotonin and norepinephrine at the presynapse cell membrane resulting in an increase in the concentration of serotonin and or norepinephrine in the central nervous system (kim, 2017). based on table 3, it can be seen through tailssuspension test (tst) and forced-swiming test (fst) untreated mice have the highest immobility time. mice given amitriptyline have the lowest immobility time because the stress generated can be reduced by the presence of amitriptyline as an antidepressant. sodium carboxymethylcellulose was used as a negative control in this research. sodium carboxymethylcellulose is commonly used as a suspending agent in drug preparations (henny et al., 2019). based on table 3, it can be seen that increasing the dose of curcuma xanthorriza decreases the immobility time so that it can be seen that there is linearity between increasing the dose of curcuma xanthorriza extract and decreasing the immobility time. based on the results of the least significant difference (lsd) test, it can be seen that there is no significant difference between amitriptyline and a dose of 650 mg/kg bw of curcuma xanthorriza extract. table 3. antidepressant bioactivity testing result. group body weight (g) tst immobility times (s) fst immobility times (s) untreated mice 2.87 ± 0.35 155.43 ± 12.42 173.48 ± 12.42 na-carboxy methyl cellulose 3.19 ± 0.58 135.00 ± 16.90 145.71 ± 11.58 amitriptyline 2.31 ± 0.59 79.75 ± 21.02 75.57 ± 16.76 325 mg/kg bw dose of c. xanthorriza 2.10 ± 0.29 102.75 ± 13.47 103.86 ± 14.16 650 mg/kg bw dose of c.xanthorriza 2.04 ± 0.53 80.01 ± 17.47 77.81 ± 11.98 conclusions ethanol can be used to extract curcuma xanthorriza using the maceration method. curcuminoids and terpenoids can be extracted using ethanol. the yield of curcuma xanthorriza ethanol extract was 5.2504%. antidepressant bioactivity testing can be done by tailssuspension test and also forced-swim test. the increase in curcuma xanthorriza ethanol extract levels will be inversely proportional to the immobility time of the mice. mice injected with amitriptyline had an immobility time in the tails-suspension test of 80.01±17.47s and in the forced-swimming test of 75.57±16.76s. the 650 mg/kg dose has immobility time test result that are not significantly different from amitriptyline, namely in the tails-suspension test of 80.01±17.47 and 77.81±11.98 s in the forced-swimming test. acknowledgements: the author acknowledges the department of chemistry, faculty of mathematics and natural science, ipb university for providing material support for the research. authors’ contributions: raja david virgilius conceived the research design, conducted the antidepressant bioactivity testing, analyzed the data, and wrote the manuscript. zest lively limbong dan jericho l ginting planted, cared for, and harvested curcuma xanthorriza robb. and conducted curcuma xanthorriza robb. extraction. all author read and approved the final version of the 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indonesia 1-3 5-9 11-15 17-24 issn 2089-6514 a simple and practical method for rat epididymal sperm count (rattus norvegicus) muhammad ja'far luthfi the phytoestrogenic potential of yam bean (pachyrhizus erosus) on ovarian and uterine tissue structure of premenopausal mice cicilia novi primiani the effect of intensive keramba on the presence of parasite organisms in rivers of lingsar area supriadi, maratun janah krokot extract (portulaca oleracea. l) as natural light-harvesting pigments for dyesensitized solar cells (dsscs): influence of dye acidity cici nurfaizah, didik krisdiyanto, khamidinal, sudarlin cover jurnal biomenaprochy vol 4 num 1 2015 dpn.pdf (p.1) editorial board 2015 v4 n1.pdf (p.2) guidance for authors 2015 v4 n1.pdf (p.3) cover jurnal biomenaprochy vol 4 num 1 2015 blkg.pdf (p.4) biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 329-336 | doi: 10.14421/biomedich.2024.132.329-336 issn 2540-9328 (online) leaf extract of kentut (paederia foetida l.) as a preventive measure against interleukin-6 expression in the liver of mice in a sepsis model injected with escherichia coli lisa savitri*, maria do carmo da costa freitas, elfred rinaldo kasimo, rochmad krissanjaya, syntia tanu juwita department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id manuscript received: 07 may, 2024. revision accepted: 18 august, 2024. published: 01 october, 2024. abstract kentut leaves (paederia foetida l.) are a medicinal plant that can be used as a preventative medicine against sepsis. this plant contains secondary metabolite compounds such as alkaloids, flavonoids, triterpenoids, saponins, and other active compounds. this research aims to to determine the influence and effective dosage of kentut leaf extract as a preventive measure against il-6 expression in the livers of mice in a sepsis model injected with e. coli. the method used was a completely randomized design (crd). the study involved 24 white male mice divided into 6 groups. data analysis was performed using one way anova. the average values of il-6 expression in the mouse livers for each group are as follows: kn at 7.09%±0.06; k+ at 26.36%±0.02; kat 72.60%±0.05; pi (100mg/kgbw) at 71.04%±0.04; pii (300mg/kgbw) at 62.22%±0.02; and piii (500mg/kgbw) at 40.92%±0.01. the research results indicate an influence of kentut leaf extract as a preventive measure against il-6 expression in the livers of mice in the sepsis model injected with e. coli, with a significance value of 0.000 or p-value < 0.005. the effective dosage of kentut leaf extract as a preventive measure against il-6 expression is the piii dosage of 500mg/kg bw. the anti-inflammatory mechanism in sepsis is thought to be caused by the presence of flavonoids, alkaloids, phenolic acids, and terpenoid compounds. the most likely anti-inflammatory mechanism is believed to involve flavonoids inhibiting cyclooxygenase (cox) and lipoxygenase (lox) enzymes involved in the synthesis of inflammatory mediators such as prostaglandins and leukotrienes, which can trigger il-6 production. keywords: kentut leaves; il-6; sepsis; e. coli. abbreviations: analysis of variance (anova); cyclooxygenase (cox); completely randomized design (crd); diamono benzidine (dab); histone deacetylase (hdac); janus kinase (jak); lipoxygenase (lox); least significant difference (lsd); mitogen-activated protein kinase (mapk); prostaglandin h2 (pgh2); phospholipase a2 (pla2); strep-avidin horse radish peroxidase (sa-hrp); systemic inflammatory response syndrome (sirs); signal transducer and activator of transcription (stat); toll-like receptors (tlrs). introduction sepsis is a medical emergency condition that describes the body's systemic immune response to an infection process that can lead to end-stage organ dysfunction and death (turnip et al., 2022). it manifests as a systemic response to an infection within the body, which can progress to severe sepsis and septic shock. severe sepsis and septic shock pose significant health problems and cause millions of deaths each year. severe sepsis involves organ dysfunction due to systemic inflammation and a procoagulant response to infection. septic shock is defined as sepsis accompanied by refractory hypotension (singer et al., 2016). the complications caused by sepsis can include systemic inflammatory response syndrome (sirs), which involves phases of inflammatory cytokine release as a response to infection, such as interleukin (il6), disseminated intravascular coagulation (dic), septic shock, and multi-organ failure, including the liver (kemenkes, 2017). in 2017, who estimated there were 48.9 million cases and 11 million sepsis-related deaths worldwide, accounting for almost 20% of all global deaths. nearly half of all global sepsis cases occur in children, with an estimated 20 million cases and 2.9 million deaths in children under 5 (five) years old. who also found significant regional disparities in sepsis incidence and mortality, with around 85.0% of sepsis cases and associated deaths occurring in lowand middle-income countries (who, 2020). the incidence of sepsis in indonesia remains high, reaching up to 30.29%, with mortality rates ranging from 11.56% to 49%. according to data from the medical records of abdul wahab sjahranie hospital from 2018 to 2020, there were a total https://doi.org/10.14421/biomedich.2024.132.329-336 330 biology, medicine, & natural product chemistry 13 (2), 2024: 329-336 of 312 sepsis patients, including 69 cases in children. based on data from the profile of abdul wahab sjahranie regional hospital, samarinda (2017), sepsis ranks among the top 10 causes of death (verdure et al., 2021). in sepsis, the inflammatory response to attacking pathogens involves both inflammatory and antiinflammatory processes, humoral and cellular reactions, and circulatory abnormalities (kaukonen et al., 2015). several studies have detected nucleotide changes in genes encoding il-6, resulting in polymorphisms that increase the risk factors or protection against developing sepsis, septic shock, and even death due to sepsis (tischendorf et al., 2007). bacteria like e. coli can infect hosts, leading to septic shock. the entry of microbes into the bloodstream is not fundamental to the onset of severe sepsis because local infections with bacteria producing pathogenic products like exotoxins can also trigger systemic inflammatory responses, causing organ dysfunction elsewhere and hypotension (clarias et al., 2018). identifying the bacteria causing sepsis is crucial in determining causative therapy. empirical antibiotic therapy, providing broad-spectrum antibiotics either singly or in combination, can cover various potential causative organisms based on clinical syndromes and previously gathered organism patterns (antibiogram) (ministry of health indonesia, 2017). however, more people are becoming aware of the side effects of antibiotic drugs, leading to a considerable number resorting to herbal treatments due to their easy availability, affordability, and potential for selfcultivation. one traditional plant historically believed to possess anti-inflammatory properties is the kentut (paederia foetida l.) leaf (silaban, 2021). another plant that can be used for sepsis prevention is the extract of mangosteen peel (garcinia mangostana), which possesses antibacterial and anti-inflammatory effects from xanthone compounds (syahruna et al., 2020). mangosteen fruit peel also contains compounds from the flavonoid, saponin, alkaloid, triterpenoid, tannin, and polyphenol groups (dewi et al., 2014). kentut leaves are among the indigenous medicinal plants in indonesia used for sepsis prevention. this plant contains secondary metabolite compounds such as α and β-paederine alkaloids, flavonoid flavanol, triterpenoid friedelin, β-sitosterol, campesterol, triterpenoid saponins, gallo tannin, and other active compounds (salamah & halim, 2021). the secondary metabolite contents pharmacologically have benefits such as antioxidants, antibiotics, anticancer, anti-insect, anti-tumor, and immunomodulatory agents (das et al., 2018). research on the preventive effects of kentut leaf extract on interleukin-6 levels revealed anti-inflammatory results in sepsis, presumably due to the presence of saponins, flavonoids, and essential oils in the leaves. it is suspected that saponins can interact with various lipid membranes, such as phospholipids, which are precursors to prostaglandins and other inflammatory mediators (savitri & kasimo, 2022). materials and methods study area this study falls under experimental research utilizing a completely randomized design (crd) method. the test subjects used in this study were white male mice selected randomly, ensuring each mouse had an equal chance of being chosen as a sample for treatment. the study aimed to determine the antiseptic activity of ethanol extract from kentut leaves on white male mice. a total of 24 white male mice were utilized and divided into 6 (six) groups, each comprising 4 mice. group i comprised untreated mice (normal), group ii received aquadest (negative control), and group iii received ciprofloxacin (positive control). groups iv to vi (3 groups) were administered kentut leaf extract. groups ii to vi were injected with e. coli bacteria, while group i was not injected with e. coli. materials and equipment: feeding tube, syringe, scale, measuring glass, surgical tools set, urine pot, liver tissue board, immunohistochemistry kit (ihk), microscope, beaker glass, stirring rod, spatula, glass funnel, white male mice, kentut leaf extract, e. coli bacteria, water (aqua), 10% formalin, milk pellets a, 96% ethanol, aquadest, ciprofloxacin, and 96% alcohol. procedures adaptation of test animals during the adaptation process, all groups of mice were fed standard (normal) food consisting of milk pellets a and water, amounting to 90 grams per cage for 14 days. experimental treatment of test animals during this period, the six groups of mice received different treatments. group i (normal) received only standard food. group ii, the mice were administered aquadest (negative control) at 500 mg/kg bw. group iii received ciprofloxacin (positive control) at 500 mg/kg bw. groups iv to vi were treated (p1, p2, p3) with kentut leaf extract at 100, 300, and 500mg/kg bw, respectively, via gavage. all six groups of mice were fed 90 grams of food daily for 30 days. e. coli bacteria injection the mice were injected with 1 ml of e. coli intraperitoneally. e. coli administration was performed once before surgery, and the mice were monitored for 24 hours. surgical procedure on test animals the examination of il-6 expression in the mice’s liver tissue in this experiment required the liver tissue of the mice. kentut leaf extract was injected into mice (mus musculus) infected with e. coli intraperitoneally, savitri et al. – leaf extract of kentut (paederia foetida l.) as a preventive 331 measuring il-6 expression. after 15 days of oral kentut leaf extract administration, twenty-four mice underwent dislocation. the abdomen was then opened, and liver tissue samples were taken and placed in urine pots containing 10% formalin for il-6 expression examination. immunohistochemistry examination immunohistochemistry involves identifying specific proteins in tissues or cells using antibodies, examining il-6 expression via immunohistochemistry involved rinsing cell cultures with pbs for 30 minutes and fixing them with methanol for 5 minutes. the samples were dried and washed with pbs ph 7.4. then, 3% h2o2 was applied for 10 minutes, followed by another pbs ph 7.4 wash. subsequently, a 5% serum pbs solution containing 0.25% triton x-100 was incubated for 1 hour at room temperature. after washing with pbs ph 7.4, monoclonal anti p50/p65 was applied and incubated overnight. another wash with pbs ph 7.4 was performed, followed by the application of biotin-labeled secondary antibodies and incubation for 1 hour. a subsequent pbs ph 7.4 wash was done, and strepavidin horse radish peroxidase (sa-hrp) was applied for 40 minutes. the samples were then washed with pbs ph 7.4, and the chromogen for hrp, diamono benzidine (dab), was applied. counterstaining was done with mayer hematoxylin for 10 minutes, rinsing with tap water, and washing with h2o. finally, the samples were dried and covered with a cover glass (yolandra, 2013). data analysis data analysis in this study utilized one-way analysis of variance (anova). before conducting one-way anova, tests for normality and homogeneity were performed. the data were considered normally distributed and homogenous if the p-value was >0.005. if the analysis showed normal distribution and homogeneity, one-way anova was used to determine if there were significant differences between test groups, signifying significance if the p-value was <0.05 and nonsignificance if the p-value was >0.05. if there were significant differences between test groups, further analysis was conducted using post hoc tests such as least significant difference (lsd) (p<0.05) and duncan's test to determine significant differences among the tested individuals (ghozali, 2009). results and discussion result the testing of kentut leaf extract (p. foetida l) for its antiseptic properties was conducted by observing the expression of il-6 in the livers of a sepsis model of mice injected with e. coli. the observation results are presented in the table below. the research results in the normal group showed an average il-6 expression value in the livers of mice at 7.09%±0.06, as seen in table 1. table 1. observation results of il-6 expression in the liver of normal group mice. group rep 1 rep 2 rep 3 rep 4 rep 5 average sd kn1 17.39% 0.00% 12.00% 9.38% 4.35% 8.62% 0.07 kn2 3.23% 0.00% 12.50% 6.06% 3.23% 5.00% 0.05 kn3 14.29% 11.11% 3.85% 0.00% 10.71% 7.99% 0.06 kn4 7.69% 0.00% 3.13% 13.79% 9.09% 6.74% 0.05 average 7.09% 0.06 description: kn : normal group rep : replication the research findings in the positive control group revealed an average il-6 expression value in the livers of mice at 26.36%±0.02, as shown in table 2. table 2. observation results of il-6 expression in the liver of positive control group mice. group rep1 rep2 rep3 rep4 rep5 average sd k(+)1 25.58% 26.53% 24.32% 28.57% 28.07% 26.62% 0.02 k(+)2 23.40% 24.32% 26.19% 25.93% 25.53% 25.08% 0.01 k(+)3 25.00% 26.83% 29.79% 29.27% 25.00% 27.18% 0.02 k(+)4 27.59% 25.00% 26.47% 26.83% 27.03% 26.58% 0.01 average 26.36% 0.02 description: k(+) : positive control group rep : replication 332 biology, medicine, & natural product chemistry 13 (2), 2024: 329-336 the research findings in the negative control group showed an average il-6 expression value in the livers of mice at 72.60%±0.05, as observed in table 3. table 3. observation results of il-6 expression in the liver of negative control group mice. group rep1 rep2 rep3 rep4 rep5 average sd k(-)1 62.50% 75.00% 69.05% 86.49% 75.86% 73.78% 0.09 k(-)2 72.50% 79.49% 61.54% 71.88% 75.00% 72.08% 0.07 k(-)3 76.60% 76.09% 71.70% 72.50% 73.33% 74.04% 0.02 k(-)4 70.59% 69.23% 69.23% 70.59% 72.92% 70.51% 0.02 average 72.60% 0.05 description: k(+) : negative control group rep : replication the research results in treatment group i (100mg/kgbw) showed an average il-6 expression value in the livers of mice at 71.04%±0.04, as shown in table 4. table 4. observation results of il-6 expression in the liver of treatment group i (100mg/kgbw). group rep1 rep2 rep3 rep4 rep5 average sd (pi)1 72.34% 73.21% 71.43% 70.37% 68.42% 71.15% 0.02 (pi)2 67.35% 66.04% 72.22% 74.00% 73.81% 70.68% 0.04 (pi)3 69.39% 75.00% 74.55% 74.00% 70.37% 72.66% 0.03 (pi)4 77.27% 67.27% 67.31% 75.00% 61.36% 69.64% 0.06 average 71.04% 0.04 description: pi : treatment group 1 (100mg/kgbw) rep : replication the research findings in treatment group ii (300mg/kgbw) revealed an average il-6 expression value in the livers of mice at 62.22%±0.02, as shown in table 5. table 5. observation results of il-6 expression in the liver of treatment group ii (300mg/kgbw). group rep1 rep2 rep3 rep4 rep5 average sd (pii)1 63.41% 60.87% 61.11% 63.04% 62.96% 62.28% 0.01 (pii)2 60.00% 63.83% 61.54% 61.70% 62.22% 61.86% 0.01 (pii)3 62.96% 65.12% 64.58% 65.22% 63.46% 64.27% 0.01 (pii)4 60.34% 61.22% 62.26% 62.22% 56.36% 60.48% 0.02 average 62.22% 0.02 description: pii : treatment group ii (300mg/kgbw) rep : replication the research outcomes in treatment group iii (500mg/kgbw) displayed an average il-6 expression value in the livers of mice at 40.92%±0.01, as shown in table 6. table 6. observation results of il-6 expression in the liver of treatment group iii (500mg/kgbw). group rep1 rep2 rep3 rep4 rep5 average sd (piii)1 39.58% 39.02% 40.74% 40.00% 38.78% 39.62% 0.01 (piii)2 41.82% 42.86% 42.59% 42.55% 42.00% 42.36% 0.00 (piii)3 36.59% 37.21% 38.30% 39.22% 39.58% 38.18% 0.01 (piii)4 43.14% 43.24% 43.75% 42.31% 45.95% 43.68% 0.01 average 40.96% 0.01 description: piii : treatment group iii (500mg/kgbw) rep : replication savitri et al. – leaf extract of kentut (paederia foetida l.) as a preventive 333 the research results in the normal group showed an average il-6 expression in the mice liver of 7.09%±0.06, in the positive control group was 26.36%±0.02, and in the negative control group was 72.60%±0.05. meanwhile, for treatment group i (100mg/kg bw), it was 71.04%±0.04, for treatment group ii (300mg/kg bw) was 62.22%±0.02, and for treatment group iii (500mg/kg bw) was 40.92%±0.01. the one-way anova test resulted in a significance value of 0.000 or a p-value < 0.005, indicating a significant difference among the groups. the lsd test showed that overall comparisons between treatment groups had a significance value of p<0.05, suggesting that the comparison between treatment groups had different effects, except for the comparison between the negative control group and treatment group i (100mg/kg bw), which yielded a significance value of 0.196 or 0.196>0.05. this suggests that the negative control group and treatment group i (100mg/kg bw) have the same antiseptic effect. the researcher employed the duncan test to determine the effectiveness and the best dosage among the groups. the duncan test results in the table indicated that overall comparisons between treatment groups were in different columns, suggesting that the comparison between treatment groups had different antiseptic effects, except for the comparison between the negative control group and treatment group i (100 mg), which appeared in the same column. this aligns with the lsd test results, showing that the negative control group and treatment group i (100 mg) have the same antiseptic effect. the determination of the best dosage or the dosage effectiveness for antiseptics is based on the smallest values or leaning towards the left because antiseptic efficacy is determined by examining the average il-6. according to the duncan test table, the sequence of the best dosages is as follows: (1) normal group; (2) positive control group; (3) treatment group iii (500mg/kgbw); (4) treatment group ii (300mg/kgbw); (5) treatment group i (100mg/kgbw); and (6) negative control group. table 7. effectiveness of kentut leaf extract as a preventive measure against il-6 expression in the liver of a sepsis model in mice. group a b c d e normal 7.09% positive control 26.37% treatment iii 40.96% treatment ii 62.22% treatment i 71.03% negative control 72.60% description: 1. differences in column positions of subset values indicate the level of difference among treatment groups. 2. values located in the same subset column indicate no significant difference between treatment groups. discussion in this study, the average il-6 expression values in the livers of each group of mice were as follows: the normal group was 7.09%±0.06; the positive control group was 26.36%±0.02; the negative control group was 72.60%±0.05; treatment group i (100mg/kgbw) was 71.04%±0.04; treatment group ii (300mg/kgbw) was 62.22%±0.02, and treatment group iii (500mg/kgbw) was 40.92%±0.01. statistical analysis using one-way anova showed a significance value of 0.000 or p-value < 0.005, indicating a significant difference between the groups. according to the duncan test table, the best dosing order was as follows: (1) normal group; (2) positive control group; (3) treatment group iii (500mg/kgbw); (4) treatment group ii (300mg/kgbw), (5) treatment group i (100mg/kgbw), and (3) negative control group. the normal group had the smallest average il-6 expression compared to the other groups, at 7.09%±0.06, as this group was not injected with e. coli, thus not undergoing sepsis. among the sepsis-experiencing mice groups were groups ii to vi. group ii was the most effective among the five other groups experiencing sepsis. group ii served as the positive control and contained ciprofloxacin. ciprofloxacin is an antibiotic agent in the fluoroquinolone class that to combat gram-negative bacterial infections such as e. coli (thai et al., 2023). in sepsis, ciprofloxacin works by inhibiting the enzymes dna gyrase and topoisomerase iv. ciprofloxacin inhibits the activity of these enzymes, which play a crucial role in bacterial dna replication and maintenance. the inhibition of these enzymes by ciprofloxacin disrupts the bacterial division and replication process, halting the growth and spread of infection (roberts et al., 2019). regarding the treatment groups with p. foetida l. leaf extract, namely treatment group i (100mg/kgbw), treatment group ii (300mg/kgbw), and treatment group iii (500mg/kgbw), treatment group iii (500mg/kgbw) 334 biology, medicine, & natural product chemistry 13 (2), 2024: 329-336 was the most effective in preventing il-6 expression in the livers of the sepsis model mice. determination of the effective dose of p. foetida l. leaf extract can be observed in the statistical analysis results with anova at a confidence level of 95%. the analysis showed a significant difference in the p. foetida l. leaf extract doses in preventing il-6 expression in the livers of the sepsis model mice. the post hoc test results from lsd and duncan showed that the negative control group had the same antisepsis effect as treatment group i (100 mg/kgbw). this indicates that the dose of treatment group i (100 mg/kgbw) was less effective as an antiseptic due to having the same effect as the negative control group. researchers suspect this is due to the low dose, which counteract the inflammation caused by the e. coli injection. p. foetida l. is one of the plants long used in traditional medicine for various conditions, including as an anti-inflammatory agent. this study’s antiinflammatory mechanism in sepsis is thought to be caused by various secondary metabolite compounds found in p. foetida l. leaves, such as flavonoids, alkaloids, phenolic acids, and terpenoid compounds (rosanti, 2016). il-6 is a proinflammatory cytokine involved in the inflammation process. when inflammation occurs in the body, whether due to infection, injury, or chronic disease, immune system cells release il-6 into their surrounding environment. il-6 then interacts with il-6 receptors on various types of cells, including immune cells, endothelial cells, and other tissue cells. il-6 plays a role in stimulating and enhancing the inflammatory response. this cytokine can increase the production and release of other proinflammatory cytokines, such as il-1β and tnf-α, triggering further inflammatory responses. il-6 can also enhance the activity of immune cells, such as macrophages and neutrophils, involved in the inflammatory response (tanaka et al., 2014). flavonoids in p. foetida l. leaves are believed to possess anti-inflammatory properties that can inhibit the release of proinflammatory cytokines, including il-6. flavonoids can inhibit the activation of the nf-κb signalling pathway, the main pathway involved in regulating the release of proinflammatory cytokines. flavonoids inhibit nf-κb activation by blocking nf-κb transcription factors and preventing the nuclear migration of proteins involved in the transcription of proinflammatory cytokine genes, including il-6 (ginwala et al., 2019). flavonoids can inhibit the activation of mitogen-activated protein kinase (mapk) pathways such as erk, jnk, and p38 mapk, involved in of proinflammatory cytokine gene expression. by inhibiting mapk pathway activation, flavonoids can reduce il-6 production. they also inhibit the activity of cox and lox enzymes. flavonoids also can inhibit cyclooxygenase (cox) and lipoxygenase (lox) enzymes. cox and lox are involved in synthesising inflammatory mediators such as prostaglandins and leukotrienes, which can trigger il-6 production (maleki et al., 2019). the inhibition of cox-1 and cox-2 enzymes, responsible for converting arachidonic acid into prostaglandin h2 (pgh2). pgh2 becomes a precursor for various inflammatory mediators such as prostaglandins and thromboxanes. flavonoids can interact with the active site of cox enzymes, inhibiting the access of arachidonic acid substrate to the enzyme reaction site. they can also inhibit lipoxygenase (lox) enzyme activity, which is responsible for converting arachidonic acid into leukotrienes. leukotrienes are inflammatory mediators that trigger the inflammatory response. flavonoids can inhibit lox enzyme activity by directly binding to the enzyme and inhibiting the process of converting arachidonic acid into leukotrienes (maleki et al., 2019). in addition to flavonoids, p. foetida l. leaves also contain phenolic acids, which have strong antioxidant properties that help protect cells from oxidative damage and oxidative stress associated with inflammation. phenolic acids can reduce il-6 production and relieve inflammation by inhibiting oxidative stress. phenolic acids can directly interact with free radicals involved in oxidative stress by donating electrons or hydrogen to free radicals, neutralizing their activity, and preventing cell damage by free radicals. phenolic acids stimulate the production of endogenous antioxidant enzymes in cells, such as superoxide dismutase (sod), catalase, and glutathione peroxidase. these enzymes play a role in eliminating free radicals and preventing oxidative damage (maleki et al., 2019). phenolic acid affects the expression of transcription factors involved in regulating pro-inflammatory cytokine gene expression. inhibiting the activity of transcription factors nf-κb and ap-1, which play a role in proinflammatory cytokine production, can reduce the production of proinflammatory cytokines and alleviate inflammation (lopez et al., 2022). phenolic acid inhibits the activation of proinflammatory immune cells, such as macrophages and t cells, involved in the inflammatory response. this reduces the production of proinflammatory cytokines like il-6 and suppresses the inflammatory response. phenolic acid can also inhibit the activation of the nf-κb signaling pathway, which regulates the expression of proinflammatory cytokine genes. activated macrophages and t cells will produce proinflammatory cytokines, including il-6 (lopez et al., 2022). alkaloid compounds present in the kentut leaves can inhibit the production of inflammatory mediators, such as prostaglandins and leukotrienes, which can trigger il-6 production by inhibiting arachidonic acid synthesis. one mechanism involved in alkaloid-mediated arachidonic acid synthesis inhibition is the enzyme phospholipase a2 (pla2). alkaloids can inhibit the activity of savitri et al. – leaf extract of kentut (paederia foetida l.) as a preventive 335 phospholipase a2, which is arachidonic acid from cell membrane phospholipids. inhibition of pla2 by alkaloids will reduce the availability of arachidonic acid required for prostaglandin and leukotriene synthesis (lopez et al., 2022). cellular receptor and transcription factor inhibition by alkaloids are mechanisms involved in reducing the inflammatory response. alkaloids can inhibit the expression or activity of cellular surface receptors involved in activating proinflammatory immune cells. inhibiting the expression of toll-like receptors (tlrs) that stimulate the inflammatory response. inhibition of tlr activity by alkaloids can hinder the activation of proinflammatory immune cells and the production of proinflammatory cytokines such as il-6. alkaloids can also inhibit the janus kinase (jak)-signal transducer and activator of transcription (stat) signaling pathway, which regulates proinflammatory cytokine gene expression. additionally, alkaloids can affect histone deacetylase (hdac) activity gene transcription regulation (lopez et al., 2022). kentut leaves also contain terpenoid compounds that can affect the inflammatory response through various mechanisms. one of these mechanisms is inhibiting the activity of proteolytic enzymes such as elastase and collagenase in degrading the extracellular matrix and damaging tissues in the inflammatory response. terpenoid compounds can directly interact with proteolytic enzymes and inhibit their catalytic activity. they can bind reversibly or irreversibly to the enzyme's active site, change the enzyme's conformation, or interfere with cofactors required for enzyme activity. furthermore, terpenoid compounds can affect the expression of protease inhibitors that act as negative regulators of proteolytic enzymes. they can increase protease inhibitor expression or inhibit the production of factors that suppress protease inhibitor expression. an increase in protease inhibitor levels by terpenoid compounds can inhibit proteolytic enzyme activity and prevent tissue damage (gallily et al., 2018). savitri & kasimo's (2022) study on the effect of kentut leaf extract (p. foetida l.) on reducing interleukin6 levels found anti-inflammatory results in sepsis, possibly due to the presence of saponins, flavonoids, and essential oils in kentut leaves. it is suspected that saponins can interact with many lipid membranes, such as phospholipids, which are precursors of prostaglandins and other inflammatory mediators. according to the researchers, the anti-inflammatory mechanism in sepsis is likely due to the content of flavonoids, alkaloids, phenolic acids, and terpenoid compounds. the most likely anti-inflammatory mechanism is presumed to be flavonoids inhibiting cyclooxygenase (cox) and lipoxygenase (lox) enzymes involved in synthesizing inflammatory mediators such as prostaglandins and leukotrienes, which can trigger il-6 production. conclusions the research results indicate an influence of the kentut leaf extract in preventing the expression of il-6 in the livers of sepsis-modeled mice injected with e. coli, with a significance value of 0.000 or p-value < 0.005. the most effective dose of the kentut leaf extract in preventing the expression of il-6 in the livers of sepsismodeled mice injected with e. coli is the dosage in treatment group iii (500mg/kg bw). acknowledgements: thank you to the laboratory of medical laboratory technology, kadiri university, and the laboratory of pathological anatomy, brawijaya university, malang, for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. maria do carmo da costa freitas wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare no funding. references clarias, l., pada, e., & tnf-, d. a. n. e. 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(2023). ciprofloxacin. statpearls. https://www.ncbi.nlm.nih.gov/books/nbk535454/ biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 807-816 | doi: 10.14421/biomedich.2025.142.807-816 issn 2540-9328 (online) the potential of lime juice in reducing fe levels and improving the quality of clove leaf oil using complexometry and testing its antioxidant activity using dpph (1,1-diphenyl-2-picrylhydrazil) teti wulandari*, susy yunita prabawati chemistry study program, faculty of science and technology, uin sunan kalijaga, jl. marsda adisucipto no 1 yogyakarta 55281, tel. +62-274-540971, fax. +62-274-519739, indonesia. corresponding author* tetiwulandari1@gmail.com abstract clove leaf oil from samigaluh, kulon progo has high fe content and low eugenol content due to an ineffective distillation process. this study aims to analyze the ability of lime juice as a purifying agent in reducing iron content through complexometry, varying the concentration of lime juice (2%, 3%, and 4%) and stirring time (30, 60, and 90 minutes) on 50 ml of clove leaf oil mixed with 50 ml of lime juice. eugenol isolation was performed using koh-h₂so₄ extraction. testing was also conducted on eugenol content, acid number, refractive index, specific gravity, and antioxidant activity using dpph. sample testing was performed using aas, gc, and uv-vis spectrophotometry. optimal conditions were obtained at a lime juice concentration of 4% with 60 minutes of stirring, which successfully reduced fe content from 17.6 to 1.7 mg/kg, increased eugenol content from 75.908% to 78.730% at a lime juice concentration of 3% and 90 minutes of stirring, reducing the acid number from 1.82 to 0.94 mg naoh/g, increasing the refractive index from 1.331 to 1.529, and increasing the specific gravity from 1.020 to 1.036. further redistillation with koh and h₂so₄ increased the purity of eugenol to 96.476% with strong antioxidant activity (ic₅₀ 57.45 ppm). keywords: clove leaf oil; complexometry; quality improvement; lime juice; eugenol; antioxidant; aas; gc; uv-vis. abbreviations: sni: standar nasional indonesia (indonesian national standard), dpph: 1,1-diphenyl-2-picrylhydrazyl, fe: iron, pa: patchouli alcohol, uv-vis: ultraviolet-visible, ic50: inhibitory concentration 50%. introduction clove (syzygium aromaticum), an indigenous plant of the archipelago from the myrtaceae family, has an often overlooked component: its leaves. although clove flowers and stems have been widely utilized in the cigarette and culinary industries, clove leaves are often regarded as waste. however, clove leaves contain significant amounts of essential oil, ranging from 1-4% in both fresh and dry conditions, with eugenol content reaching 80-85% (tuganyita et al., 2019). this makes clove leaf oil a product with high economic value (putri et al., 2014). nevertheless, the essential oil processing industry in indonesia, particularly in rural areas, still faces product quality challenges. the distillation methods used by farmers tend to be simple and suboptimal, often producing low-quality oil with dark blackish or greenish colors, caused by metal contamination from leaves and distillation equipment (nengsi, 2018). according to research by affifah et al. (2016), the quality of clove leaf oil depends on the quality of its distillation, where distillation equipment such as boilers should be made of stainless steel materials and oil containers must be clean. according to sni 06-2387-2006, the eugenol concentration in clove oil should be at least 78%. however, the simple distillation process commonly used only produces clove oil with eugenol content of around 70%. another parameter affecting essential oil quality is fe (iron) content. although sni 06-2387-2006 does not establish specific limits for fe levels in clove leaf oil, this parameter remains an important indicator showing metal contamination and degradation of essential components in the oil. clove leaf oil contaminated with fe will produce high acid numbers due to oxidation, alter specific gravity, and potentially affect the oil's refractive index. increased acid numbers significantly impact the decline in clove leaf oil quality, particularly affecting aroma characteristics and shortening product shelf life. therefore, to obtain clove leaf oil that meets standards, purification processes are necessary to extend material durability during storage. several studies have shown that purification processes can increase eugenol content and marketability manuscript received: 18 august, 2025. revision accepted: 09 october, 2025. published: 17 october, 2025. https://doi.org/10.14421/biomedich.2025.142.807-816 808 biology, medicine, & natural product chemistry 14 (2), 2025: 807-816 of clove leaf oil in international markets. complexometry using citric acid purifying has become one method that can be used to reduce metal concentrations while improving oil quality and eugenol content (fatimah et al., 2014). research by saputri et al. (2014)demonstrated that chemical purification with complexometry using citric acid on clove leaf oil can change oil color from dark brown to light yellow and reduce fe content. lime (citrus aurantifolia) contains citric acid that meets the criteria as a fastener compound and is easily accessible to traditional clove farmers. lime contains 7% citric acid, higher than lemon which only contains 5% citric acid. a natural material can be categorized as a qualifying fastener if it contains at least 0.5% to 4% citric acid (tjandrawinata & julianto, 2018). the use of lime as a purifying agent is expected to be an effective and economical solution for traditional clove farmers to improve oil quality. the eugenol content in clove oil has antioxidant capabilities (latifah et al., 2016). eugenol works as an antioxidant by capturing free radicals through phenolic groups, as its molecular structure allows phenolic hydrogen donation and stabilization of the resulting phenoxyl radicals. the dpph (1,1-diphenyl-2picrylhydrazyl) method is one of the most widely used test methods for antioxidants because of its ease of use, high sensitivity, and ability to analyze large numbers of samples in a short time (handayani et al., 2018) this study aims to determine the appropriate treatment combination and the effect of varying stirring times and lime juice concentrations to produce the best physicochemical quality of clove leaf essential oil, as well as to evaluate its antioxidant activity using the dpph method as an additional quality parameter. materials and methods study area this research was conducted from january 10, 2025 to january 30, 2025 at the bsip laboratory, yogyakarta, and from january 31 to july 25, 2025 at the integrated laboratory of uin sunan kalijaga, yogyakarta. the clove leaf essential oil samples were obtained from the samigaluh area, kulon progo regency, central java, indonesia, which is known as one of the traditional clove cultivation centers in indonesia. procedures materials and equipment the equipment used in this study included a complete set of glassware, distillation apparatus, analytical balance, measuring pipettes, volumetric pipettes, glass stirrer, magnetic stirrer, burette, separatory funnel, funnel support, glass funnel, reagent bottles, 100 ml volumetric flask, hot plate, 125 ml erlenmeyer flask, 100 ml measuring cylinder, aquadest bottle, rubber bulb, pycnometer, refractometer, gas chromatography shimadzu 2010, agilent 240fs aa atomic absorption system, and uv-vis spectrophotometer thermo scientific genesys 20.sub-procedures-2 the materials used included clove leaf essential oil obtained from the samigaluh region, kulon progo, lime fruit, aquades, ph indicator, 96% ethanol, ascorbic acid p.a., diethyl ether, 1,1-diphenyl-2-picrylhydrazyl (dpph), filter paper, koh, and h2so4. redistillation of clove leaf oil the redistillation process began with sample filtration using filter paper to remove coarse particles and visible impurities. after filtration, the oil was weighed using an analytical balance to determine its initial weight. a clean and dry distillation flask was then filled with the filtered oil, and a thermometer was positioned correctly to monitor the temperature during the distillation process. the distillation apparatus was carefully assembled, ensuring all connections were tightly fitted to prevent vapor leakage. the condenser was connected to a cold water flow to ensure effective cooling. the heating mantle was turned on and the temperature was gradually adjusted, slowly raised until reaching the boiling point of clove oil, which ranges between 250-255°c. the main fraction of pure clove oil was collected while maintaining a stable temperature within the clove oil boiling point range. distillation was stopped when almost all the oil had been distilled. after the distillation process was complete, the redistilled oil was analyzed using gc. determination of citric acid concentration in lime juice fresh limes were washed clean, cut into two parts, and squeezed. the juice was filtered through a cloth filter to remove pulp and seeds. 10 ml of lime juice was placed in a 250 ml erlenmeyer flask, then 2-3 drops of phenolphthalein indicator were added. standardized 0.1 n naoh solution was prepared in a 50 ml burette and the initial volume was recorded. titration was performed by adding 0.1 n naoh drop by drop while stirring until the solution changed color to pink which lasted for at least 30 seconds. the final volume of naoh was recorded for the determination of citric acid concentration in lime juice. preparation of lime juice solutions based on the results of citric acid concentration determination in the previous stage, the volume of lime juice required to make 2%, 3%, and 4% solutions was calculated. the appropriate volume of lime juice for each concentration was placed in separate beakers (16.7; 25.0; 33.3 ml), a small amount of aquades was added, stirred until homogeneous, then transferred to a 50 ml volumetric flask. aquades was added to the mark, the volumetric flask was closed, and the solution was homogenized. each volumetric flask was labeled according to concentration (2%, 3%, and 4%). wulandari & prabawati – the potential of lime juice in reducing fe levels and … 809 atomic absorption spectroscopy analysis for fe content aas analysis for fe content was performed in two stages: preparation of a standard solution and calibration curve construction. standard solution preparation began by dissolving 4.8303 grams of fecl₃.6h₂o in a beaker glass and adding aqua regia (hno₃(p): hcl(p)), then heated until dissolved and transferring to a 1000 ml volumetric flask, then diluted with aquabidest to the mark, obtaining a 1000 mg/kg fe standard solution. the second step began by pipetting 10 ml of 1000 mg/kg fe standard solution and placing it in a 100 ml volumetric flask to obtain 100 mg/kg. subsequently, 0, 2, 4, 6, 8, 10 ml were taken from this 100 mg/kg solution and placed in 100 ml volumetric flasks, diluted to the mark and used as standard solutions. determination of acid number acid number determination was performed by taking 4 grams of the sample and dissolving it in 5 ml of neutral ethanol in a saponification flask, then adding 5 drops of phenolphthalein indicator. the solution was then titrated with sodium hydroxide until the color changed to pink. determination of specific gravity specific gravity determination was performed using a pycnometer. the pycnometer was cleaned, rinsed with ethanol and diethyl ether, dried with dry air flow, and weighed (m). the pycnometer was filled with distilled water that had been boiled and cooled to 20°c. after immersion in a water bath at 20°c ± 0.2°c for 30 minutes, the pycnometer was closed, dried, and weighed (m1). the procedure was repeated with clove leaf oil as a substitute for water (m2). determination of refractive index refractive index determination was performed using an abbe refractometer. the refractometer prism cover was opened and the prism was cleaned with tissue paper moistened with aquades. oil was taken using a pipette and placed on the abbe refractometer prism (1 drop), then closed again. temperature was maintained with a tolerance of ± 2°c. the number read on the refractometer was recorded. determination of optimum conditions and gc analysis analysis of clove leaf oil was performed using gc instrument on samples before and after the purification process. the best results from the four purification quality parameters (fe content, acid number, refractive index, and specific gravity) were selected from each treatment. the best oil sample obtained was then further analyzed using gc to determine changes in eugenol content before and after purification. eugenol isolation from optimum clove leaf oil the eugenol isolation process involved several steps: preliminary treatment with 20 ml clove leaf oil and 6 g koh dissolved in 100 ml aquades; mixing with strong base at 50°c for 30 minutes using magnetic stirrer; mixing with strong acid using 1.5 n h₂so₄ solution; second separation process using separatory funnel for 4 hours; eugenol washing with aquades in 1:1 ratio; and water removal by heating on hot plate. antioxidant activity test dpph solution preparation (40 ppm) 2 mg dpph powder was dissolved in a volumetric flask using 96% ethanol to a volume of 50 ml. maximum wavelength determination 4 ml of 40 ppm dpph solution was placed in a test tube and mixed with 1 ml of 96% ethanol, homogenized, incubated for 30 minutes in dark conditions, and absorbance was measured at 400-800 nm range using a uv-vis spectrophotometer. test solution preparation 0.01 gram (10 mg) of clove leaf oil was measured and placed in a 10 ml volumetric flask. 96% ethanol was added to the mark to produce an initial solution with 1000 ppm concentration. serial dilutions were performed to obtain test solutions with concentrations of 5 ppm, 10 ppm, 15 ppm, and 20 ppm. standard solution preparation standard solution was prepared using ascorbic acid p.a. to make 1000 ppm stock solution, 10 mg ascorbic acid p.a was dissolved in 96% ethanol to a volume of 10 ml. serial dilutions were performed to obtain lower concentrations of 5 ppm, 10 ppm, 15 ppm, and 20 ppm. antioxidant activity determination the best clove leaf oil sample was tested for antioxidant activity using the dpph method. 1 ml of test solution from each concentration (5, 10, 15, 20 ppm) was mixed with 4 ml of 40 ppm dpph, homogenized, and incubated for 30 minutes in a dark room. absorbance was measured using uv-vis spectrophotometer at the predetermined maximum wavelength. control was made by mixing 1 ml of ethanol and 4 ml of 40 ppm dpph. data analysis the data obtained from quality parameter measurements (fe content, acid number, specific gravity, refractive index) and antioxidant activity (ic₅₀ value) were analyzed using statistical methods. the experimental design used was factorial with two factors: lime juice concentration (2%, 3%, 4%) and stirring time (30, 60, 90 minutes). each treatment was performed in triplicate to ensure data reliability. data were analyzed using analysis of variance (anova) to determine significant differences between treatments. the ic₅₀ value was 810 biology, medicine, & natural product chemistry 14 (2), 2025: 807-816 calculated using linear regression analysis between concentration and percentage of dpph inhibition. the optimum treatment was determined based on the combination of treatments that produced the best quality parameters according to sni 06-2387-2006 standards. results and discussion result the results of the fe levels test the results of fe levels testing can be seen in figure 1. figure 1. fe content of clove leaf oil at various concentrations and stirring times. the result of acid number of clove leaf oil at various purification conditions the result of acid number of clove leaf oil at various purification conditions can be seen in table 1. table 1. acid number of clove leaf oil at various purification conditions. concentration (%) 30 minutes 60 minutes 90 minutes before purification 1.82 1.82 1.82 2% 1.59 1.31 1.26 3% 1.17 1.12 1.03 4% 1.08 0.94 0.94 the result of eugenol content at different purification stages the result of eugenol content at different purification stages can be seen in table 2. table 2. the result of eugenol content at different purification stages. purification stage retention time (minutes) eugenol content (%) number of peaks before purification 12.574 75.908 30 after lime juice (3%, 90 min) 12.570 78.730 28 17,6 13,5 9,9 2,4 13,1 8,7 1,7 10,8 5,7 1,7 0 10 20 before purification 2% 3% 4%fe c o n te n t (p p m ) lime concentration stirring time (minutes) 30 60 90 wulandari & prabawati – the potential of lime juice in reducing fe levels and … 811 purification stage retention time (minutes) eugenol content (%) number of peaks after koh-h₂so₄ extraction 12.603 96.476 21 the result of refractive index the result of refractive index testing can be seen in table 3. table 3. the result of refractive index. concentration (%) 30 minutes 60 minutes 90 minutes before purification 1.5287 1.5287 1.5287 2% 1.5294 1.5298 1.5301 3% 1.5305 1.5308 1.5310 4% 1.5309 1.5312 1.5312 the result of spesific gravity of clove leaf oil at various purification conditions the result of spesific gravity of clove leaf oil at various purification conditions can be seen in table 4. table 4. the result of spesific gravity of clove leaf oil at various purification conditions. concentration (%) 30 minutes 60 minutes 90 minutes before purification 1.020 1.020 1.020 2% 1.025 1.027 1.029 3% 1.031 1.033 1.035 4% 1.034 1.037 1.037 one way anova test results for all tested parameters results one way anova test results for all tested parameters results can be seen in table 5 anova table 5. one way anova test results for all tested parameters. description: * : significant, ns : not significant. parameter factor f-value p-value significance fe content concentration 4.306 0.030 * stirring time 0.263 0.772 ns interaction 0.049 0.995 ns acid number concentration 15.428 0.000 * stirring time 12.847 0.000 * interaction 3.952 0.026 * refractive index concentration 8.742 0.002 * stirring time 6.158 0.008 * interaction 2.847 0.045 * specific gravity concentration 2.134 0.148 ns stirring time 1.867 0.186 ns interaction 0.398 0.836 ns (significant at α = 0.05) 812 biology, medicine, & natural product chemistry 14 (2), 2025: 807-816 the result of antioxidant activity the result of antioxidant activity can be seen in table 6. table 6. antioxidant activity results. sample concentration (ppm) average absorbance % inhibition linear regression equation r² ic₅₀ (ppm) category clove leaf oil 10 0.024 20.00 y = 0.4486x + 24.228 0.8682 57.45 strong 25 0.017 43.33 50 0.015 50.00 75 0.012 58.89 100 0.010 65.56 vitamin c 10 0.019 36.67 y = 0.2834x + 37.483 0.9279 44.16 very strong 25 0.016 46.67 50 0.014 54.44 75 0.012 60.00 100 0.011 63.33 control 0 0.030 0.00 discussion physical parameter analysis of purification process iron (fe) content the purification process of clove leaf oil aims to remove impurities or unwanted compounds, which consequently reduces the oil volume during processing. analysis of fe content in clove leaf oil after purification was conducted to determine the fe components removed, enabling more efficient processes for industrial-scale applications. fe content in a material indicates the amount of iron contained within it. iron in clove leaf oil is considered an unwanted component that must be reduced, as excessive fe content can trigger oxidation reactions causing the oil to appear dark and blackish, affecting the physicochemical properties of the resulting clove leaf essential oil. based on the analysis results, the fe content in unpurified clove leaf oil was 17.6 mg/kg. after purification with lime juice addition at concentrations of 2-4% and stirring time variations of 30, 60, and 90 minutes, significant fe content reduction occurred with results ranging from 13.5-1.7 mg/kg (figure 1). the ability of lime juice to reduce fe content in clove leaf oil can be explained through complex formation mechanisms. the main component of clove leaf oil is eugenol containing phenolic groups, where fe³⁺ ions initially bind to these phenolic groups forming -ofe³⁺ structures. figure 2. mechanism of fe³⁺ binding from clove leaf oil by citric acid the active component in lime juice serving as the primary binding agent is citric acid (c₆h₈o₇), with additional contributions from ascorbic acid (vitamin c) and other organic acids. citric acid possesses high binding capacity due to its structure containing four potential binding sites: three carboxyl groups (-cooh) and one hydroxyl group (-oh). when lime juice containing citric acid interacts with the fe³⁺-eugenol complex, deprotonation of carboxyl groups occurs, producing citrate ions (c₆h₅o₇³⁻). these negatively charged groups provide free electron pairs that can strongly bind fe³⁺ ions while simultaneously releasing fe³⁺ from eugenol phenolic groups, forming coordination bonds with oxygen atoms from citric acid carboxyl groups. wulandari & prabawati – the potential of lime juice in reducing fe levels and … 813 before purification after purification figure 3. clove leaf oil before and after purification. the fe content reduction process can be visually observed through color changes during purification. before purification, clove leaf oil appeared darker and brownish due to fe³⁺ ions that can trigger oxidation reactions. after purification with lime juice, the oil became more yellow-clear and bright, indicating successful fe³⁺ binding and extraction from the oil phase. concentration of 4% showed the highest fe reduction effectiveness with results of 2.4 mg/kg (30 minutes), 1.7 mg/kg (60 minutes), and 1.7 mg/kg (90 minutes). at this concentration, fe reduction reached a stable point at stirring times of 60 and 90 minutes with identical values of 1.7 mg/kg, indicating that at 4% concentration, the fecitrate complex formation process reached equilibrium conditions where additional stirring time no longer provided significant fe reduction. based on research data, the optimum conditions for fe reduction in clove leaf oil were at 4% lime juice concentration with 60 minutes stirring time, producing fe content of 1.7 mg/kg. statistical analysis showed that lime juice concentration had significant effect on fe content (f = 4.306, p = 0.030), while stirring time showed no significant effect (f = 0.263, p = 0.772). eugenol content eugenol is the main component in clove leaf essential oil, comprising 70% to 90% of the total content. gc chromatography analysis of three clove leaf oil samples showed eugenol presence with relatively similar retention times but different percentage contents (table 2). before purification, eugenol was detected at retention time 12.574 minutes with 75.908% content, showing 30 peaks indicating various impurity components. this percentage did not meet indonesian national standard (sni 06-2387-2006) requirements of minimum 78% eugenol content. table 7. quality standards for clove leaf essential oil sni 06-2387-2006. no. test type unit requirements 1. color yellow-brown to brownish clove oil 2. specific gravity 20°c / 20°c 1.025-1.049 3. refractive index 1.528-1.535 4. solubility in 70% ethanol 1:2 clear 5. total eugenol %, w/v min. 78 6. beta caryophyllene % max. 17 after purification using 3% lime juice with 90 minutes stirring time, significant improvement occurred in clove leaf oil quality. eugenol was detected at nearly identical retention time (12.570 minutes) but with increased percentage to 78.730%, representing a 3.73% increase from initial content. this purified oil successfully met the minimum sni standard for eugenol content. the purification process also reduced impurity components, indicated by peak reduction from 30 to 28, with elimination of peaks at retention times 17.413 and 17.656 minutes, likely corresponding to α-copaene and β-elemene compounds (amelia et al., 2017; sulistyoningrum et al., 2017). further enhancement was achieved using reactive extraction method with koh and h₂so₄ solutions. the eugenol purification process through reactive extraction involves two main stages utilizing the acidic properties of eugenol's phenolic hydroxyl group. in the first stage, koh reacts with eugenol's phenol group, where the hydroxyl group acts as a weak acid due to electron resonance in the aromatic ring. koh removes the proton from the hydroxyl group, creating water-soluble potassium eugenolate (k-eugenolate) salt that separates from other non-polar clove oil components, while the allyl side chain remains stable under mild reaction conditions. figure 4. image of the reaction forming k-eugenolate salt 814 biology, medicine, & natural product chemistry 14 (2), 2025: 807-816 figure 5. image of the reaction between k-eugenolate and h₂so₄ forming eugenol in the second stage, h₂so₄ neutralizes the keugenolate salt by providing h⁺ ions that combine with eugenolate ions, reforming the hydroxyl group and returning eugenol to its original hydrophobic form that naturally separates from the aqueous solution. this method successfully increased eugenol purity from 78.73% to 96.476%, representing a 17.746% improvement and demonstrating the effectiveness of reactive extraction in achieving high-purity eugenol. acid number acid number represents an important parameter for determining oil age, purity, and hydrolysis level. although sni 06-2387-2006 for clove leaf oil does not mention acid value test parameters, high acid content in clove leaf oil can cause unpleasant odor and taste, reducing product quality and commercial value. acid value indicates the amount of free fatty acids present in oil (hendra wijaya et al., 2024)research results showed acid number differences between unpurified and purified clove leaf oil ranging from 8.4-4.9 mg naoh/g (table 1). unpurified clove leaf oil had higher acid number of 1.82 mg naoh/gram, while purified oil showed acid number reduction. at 4% concentration with 60 and 90 minutes contact time, acid numbers were identical at 0.94 mg koh/g, indicating that the lime juice purifying agent reached the saturation point. this saturation condition occurs because the chelating agent adsorption capacity is maximized, so contact time extension does not provide significant effectiveness improvement. results align with research by rosdiana (2014) regarding citric acid concentration effects on acid content and used cooking oil opacity reduction, and purbaningtias et al. (2014) on patchouli oil acid value reduction with natural adsorbent. two-way anova analysis showed significant effects from both lime juice concentration (p<0.05) and stirring time (p<0.05) on clove leaf oil acid number, with significant interaction between both factors (p<0.05), indicating combined effects in improving oil purification effectiveness. refractive index refractive index represents the ratio of light speed in air to light speed in the tested substance at a specific temperature, indicating clove leaf oil quality or purity (erliyanti et al., 2020). research results showed that refractive index values at various treatment variations met sni standards (1.331-1.535), though average values remained at the lower sni boundary (table 3). refractive index tends to increase with purifying agent concentration addition, though not significantly. the refractive index is influenced by the number of double bonds and carbon chain length, where higher indices indicate better quality due to higher eugenol content (idris et al., 2014; pratiwi et al., 2016). after purification, refractive index increased from 1.5287 to values ranging 1.5294-1.5312, indicating improved oil quality. anova analysis showed that both lime juice concentration and stirring time significantly affected refractive index (p<0.05) with significant interaction between factors. specific gravity specific gravity represents the ratio between oil mass and distilled water mass at equal volume and temperature (kristian et al., 2016). research results showed specific gravity increased from 1.020 before treatment to 1.037 after purification, meeting sni 06-2387-2006 standards (1.025-1.049) (table 4). specific gravity increase indicates better oil quality as it shows more mass within the same volume unit, caused by light-fraction components evaporating during purification (dewi et al., 2019). anova analysis indicated that neither lime juice concentration nor stirring time significantly affected specific gravity (p>0.05), with no interaction between factors (p>0.05). antioxidant activity previous analysis showed that eugenol separation from clove leaf oil using koh and h₂so₄ yielded 96.476% eugenol content. to prove separation success, antioxidant activity analysis was conducted on purified eugenol. antioxidant activity analysis on clove leaf oil eugenol used dpph (1,1-diphenyl-2-picrylhydrazyl) method with uv-vis spectrophotometer assistance. purple 517 nm yellow figure 6. reaction mechanism of dpph radicals with antioxidant compounds (rh = radical scavenging antioxidant: r = antioxidant radical) wulandari & prabawati – the potential of lime juice in reducing fe levels and … 815 dpph radical scavenging mechanism showing color change from purple to yellow. the method was chosen for its simplicity, high sensitivity, and ability to evaluate antioxidant activity in relatively short time. the main principle lies in dpph reaction as free radical representation (kolompoy et al., 2024). antioxidant compounds donate radical electrons while dpph becomes radical electron acceptor. this reaction is marked by color change from purple to yellow, measurable through absorbance decrease at 517 nm maximum wavelength. based on results, clove leaf oil obtained an ic₅₀ value of 57.45 ppm, indicating strong antioxidant category (50100 ppm), while vitamin c extract obtained ic₅₀ value of 44.16 ppm, classified as very strong antioxidant (<50 ppm). this aligns with research by lusiana et al. (2024) reporting a vitamin c ic₅₀ value of 4.245 ppm and irnawati et al., (2017) showing vitamin c ic₅₀ value of 24.63 mg/l, both categorized as very strong antioxidants. this difference occurred because vitamin c is a pure compound with very high antioxidant activity, while clove leaf oil eugenol, despite 96.467% purification, still contains other components in small amounts. clove leaf oil antioxidant activity originates from eugenol structure which has hydroxyl groups that can donate hydrogen atoms to neutralize free radicals. other phenol derivative compounds such as eugenol acetate and β-caryophyllene also contribute to antioxidant effects. the concentration and percentage inhibition comparison graph showed strong positive correlation, indicating that eugenol antioxidant activity depends on the concentration used. conclusions based on the research results of clove leaf oil purification using the complexometric method with lime juice, it can be concluded that the purification successfully improved oil quality with optimum conditions achieved at 4% lime juice concentration and 60 minutes stirring time. the fe content decreased from 17.6 mg/kg to 1.7 mg/kg, and all parameters met sni 06-2387-2006 standards. further redistillation increased eugenol purity to 96.476%. the purified oil demonstrated strong antioxidant activity with an ic₅₀ value of 57.45 ppm, showing potential as a highquality natural antioxidant. acknowledgements: the authors would like to express their sincere gratitude to the staff of the integrated laboratory, uin sunan kalijaga yogyakarta and the agricultural instrument standards application center (bpsip) yogyakarta, for their assistance during the research. authors’ contributions: prabawati & wulandari designed this study. wulandari performed the laboratory work, analyzed the data, and wrote the manuscript. prabawati supervised the writing of the manuscript. all authors have read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: this study did not receive any funding references affifah, f. n., lutfi, m., & kadarisman, d. 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(2019). pemisahan dan identifikasi komponen-komponen utama minyak atsiri dari daun cengkeh segar dan kering (syzygium aromaticum). fullerene journal of chemistry, 4(2), 44–47. https://doi.org/10.37033/fjc.v4i2.71 cover jurnal biomenaprochy vol 10 num 2 2021.cdr volume 10 number 2 october 2021 issn 2089-6514 (paper) issn 2540-9328 (online) biology, medicine, & natural product chemistry volume 10 – number 2 – october 2021 issn 2089-6514 (paper) | issn 2540-9328 (online) editor-in-chief: muhammad jafar luthfi department of biological education, faculty of tarbiyah and education, state islamic university sunan kalijaga yogyakarta, indonesia editorial board: hildebert wagner department pharmazie, ludwig-maximilians-universität münchen, germany geoffrey a. cordell department of medicinal chemistry and pharmacognosy, university of illinois at chicago, usa mahanem mat noor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia jalifah latip centre of chemical science and food technology studies, university kebangsaan malaysia wisnu nurcahyo department of veterinary parasitology, gadjah mada university, indonesia shukor md. nor school of bioscience & biotechnology, faculty of science & technology, university kebangsaan malaysia widodo department of biology education, state islamic university sunan kalijaga yogyakarta, indonesia ahmad dwi setyawan department of biology, sebelas maret university, indonesia sugiyarto department of biology, sebelas maret university, indonesia alfred pakpahan faculty of dentistry, trisakti university, indonesia charis amarantini faculty of biotechnology, christian university duta wacana, indonesia marini ahmad marzuki malaysian agricultural research and development institute nasser farid geraldo yummy foodies, benin fahrul nurkolis biological sciences, state islamic university sunan kalijaga yogyakarta, indonesia design & layout: r i y a n t o publisher: state islamic university (uin) sunan kalijaga yogyakarta co-publisher: the society for indonesian biodiversity online: www.sciencebiology.org address: state islamic university (uin) sunan kalijaga yogyakarta jl. marsda adisucipto 1 yogyakarta 55281 tel. +62-274-519739, tel. & fax.: +62-274-540971, email: jafarluthfi@yahoo.com biology, medicine, & natural product chemistry volume 10 – number 2 – october 2021 issn 2089-6514 (paper) | issn 2540-9328 (online) contents effect of diabetes mellitus and hypertension on osmotic fragility and hemorheological factors in male wistar rats david ehikhuemen okonofua, jerome ndudi asiwe, kenneth kelechi anachuna, emuesiri goodies moke, kamaldeen olalekan sanusi, ebunoluwa oluwabusola adagbada, mariam onono yusuf, damilola ifeoluwa alawode, adesoji adedipe fasanmade 73 79 risk assessment of heavy metals in chromolaena odorata collected around gemstone mining site in ijero-ekiti efe sylvanus abiya, foluso akinbode ologundudu, ekpo wisdom 81 86 potential of tithonia diversifolia hemsley a. gray (kembang bulan) leaf extract as anti-cancer agents muflihah rizkawati 87 91 a physical chemistry study of black powder materials by solution combustion synthesis method fitria hidayanti, kiki r. lestari, nano sujani, jarot raharjo 93 103 phytochemical screening, antioxidant and antibacterial activities of the root extract of cyphostemma adenocaule (steud. ex a. rich.) wild & r.b.drumm abdulbasit haliru yakubu, mohammed mustapha mohammed, abdulqadir bukar bababe, hassan yesufu braimah 105 110 acute toxicity and hypoglycemic effect of a polyherbal formulation on blood glucose in oral glucose tolerance test (ogtt) and alloxan-induced diabetic rats abednego okeoghene warri, emuesiri goodies moke, aishat oyinkansola balogun, kennedy chibogu nzeh, emuesiri kohworho umukoro, earnest oghenesuvwe erhirhie 111 115 antibacterial activity of mangrove root extracts from ngurah rai mangrove forest, denpasar-bali made dharmesti wijaya, anak agung gede indraningrat 117 121 stability analysis of mathematical modeling of interaction between target cells and covid-19 infected cells sugiyanto, mansoor abdul hamid, alya adianta, hanny puspha jayanti, muhammad ja'far luthfi 123 127 antibacterial screening of bacterial isolates associated with mangrove soil from the ngurah rai mangrove forest bali anak agung gede indraningrat, made dharmesti wijaya, putu arya suryanditha, ayu savitri siskayani, ni made defy janurianti 129 133 repurposing dihydroartemisinin-piperaquine-doxycycline as an antimalarial drug: a study in plasmodium berghei-infected mice udeme owunari georgewill, elias adikwu 135 140 guidance for authors aims and scope biology, medicine, & natural product chemistry, this journal is published to attract and disseminate innovative and expert findings in the fields of plant, animal, and microorganism secondary metabolite, and also the effect of natural product on biological system as a reference source for researchers in these fields, and with the aim to set international 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(personal communication); setyawan ad 2007, unpublished data). in the reference list, the references should be listed in an alphabetical order (better, if only 20 for research papers). names of journals should be abbreviated. always use the standard abbreviation of a journal's name according to the issn list of title word abbreviations (www.issn.org/2 22661-ltwa-online.php). the following examples are for guidance. journal: saharjo bh, nurhayati ad. 2006. domination and composition structure change at hemic peat natural regeneration following burning; a case study in pelalawan, riau province. biodiversitas 7: 154-158. book: rai mk, carpinella c. 2006. naturally occurring bioactive compounds. elsevier, amsterdam. chapter in book: webb co, cannon ch, davies sj. 2008. ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. in: carson w, schnitzer s (eds) tropical forest community ecology. wiley-blackwell, new york. abstract: assaeed am. 2007. seed production and dispersal of rhazya stricta. 50 annual symposium of the international association for vegetation science, swansea, uk, 23-27 july 2007. proceeding: alikodra hs. 2000. biodiversity for development of local autonomous government. in: setyawan ad, sutarno (eds.) toward mount lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in java island. universitas sebelas maret, surakarta, 17-20 july 2000. [indonesian] thesis, dissertation: sugiyarto. 2004. soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [dissertation]. universitas brawijaya, malang. [indonesian] information from internet: balagadde fk, song h, ozaki j, collins ch, barnet m, arnold fh, quake sr, you l. 2008. a synthetic escherichia coli predator-prey ecosystem. mol syst biol 4: 187. www.molecularsystemsbiology.com 3: front 4: editorial 5: guidance 6: back biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 233-240 | doi: 10.14421/biomedich.2023.121.233-240 issn 2540-9328 (online) the effects of frequent therapeutic administration of artesunate-amodiaquine and artemether-lumefantrine on haematological markers in balb/c mice david audu1,*, olufunmilayo a idowu1, vinood b patel2, musa f mshelbwala3, adewumi b idowu1 1department of pure and applied zoology, college of biosciences; 3department of veterinary pathology, college of veterinary medicine, federal university of agriculture abeokuta, ogun state, nigeria 2school of life sciences, university of westminster, 115 new cavendish street, london w1w 6uw, united kingdom. corresponding author* audud@funaab.edu.ng abstract artemisinin combination therapy (act) is readily available in malaria-endemic nations, leading to repeated drug usage by undiagnosed persons. repeated use of act therapy by non-infected individuals may affect blood cells. this study explored how repeated artesunateamodiaquine (a/a) and artemether-lumefantrine (a/l) treatment in non-infected mice affected haematological markers. 100 male balb/c mice were randomly divided into 5 groups: non-infected and plasmodium berghei nk65 infected treated with a/l and a/a 1x, 2x, 3x, 4x, 5x, and 6x, and the control group. packed cell volume (pcv), haemoglobin (hb), and red blood cell (rbc) were reduced (p>0.05) non-significantly in the non-infected group treated with a/l or a/a six times compared to the control and infected groups. wbc rose in infected and non-infected mice treated with a/l or a/a 1x, 2x, 3x, and 6x, with a substantial rise in non-infected mice treated with a/l (p < 0.01) and a/a (p < 0.001) three times. wbc mainly rose due to lymphocytes, although neutrophils decreased. repeated therapeutic use of a/l and a/a without infection may cause a haematological change. continuous efforts are needed to educate the public about screening for malaria parasites before using drugs. keywords: malaria; artemether lumefantrine; artesunate amodiaquine; hematological parameters; anemia; neutropenia. introduction malaria is caused by plasmodium parasites transmitted by female anopheles mosquito bites (w.h.o, 2022). african countries account for 95% of worldwide malaria. effective malaria case management in children and adults consists of early diagnosis and fast, effective treatment with artemisinin-based combination therapy (koko et al., 2022; w.h.o, 2022). the first-line act medicines for malaria in africa are artemetherlumefantrine (a/l) and artesunate-amodiaquine (a/a), which show modest effectiveness (zongo et al., 2020; audu et al., 2023). malaria should be screened before taking acts to guarantee effective therapy, but this is challenging in africa. malaria patients in africa who lack parasitological confirmation of infection are routinely provided with these medications, leading to the use of act without being infected (mbonye et al., 2010; yeung et al., 2011; cohen et al., 2012; mbonye et al., 2013; rusk et al., 2013; idowu et al., 2015; nwokolo et al., 2018 ). multiple antimalaria dosages are typical in nigeria due to self-prescription and free access to the drug over the counter (owumi et al., 2015). blood is where most antimalarial medication activities occur (madukaku et al., 2015); long-term a/l and a/a use by non-infected people may harm blood cells. animal tests showed that a/l at indicated doses for three days did not influence haematological parameters in non-infected rats. however, after seven days of a/l treatment, red blood cells (rbc), hemoglobin (hb), and packed cell volume (pcv) dropped (ofem et al., 2013). in comparison, the study by (adeleye et al., 2012) showed that a single dose of artesunate and a/l affected white blood cell, neutrophil, and lymphocyte counts. long-term administration of either a/l or a/a to noninfected individuals has also been reported to have harmful effects on blood cells (ijeomah et al., 2016). due to the current misuse of act in malaria-endemic regions, the goal of this experiment was to investigate the impact of repeated therapeutic administration of either a/l or a/a on the blood as further investigation is required to determine whether this substance is safe to use repeatedly. we employed a mouse model to simulate the frequent use of a/l or a/a in infected and noninfected mice. secondly, the haematological parameters of mice were examined after one week of treatment, as manuscript received: 04 november, 2022. revision accepted: 16 february, 2023. published: 02 march, 2023. https://doi.org/10.14421/biomedich.2023.121.233-240 234 biology, medicine, & natural product chemistry 12 (1), 2023: 233-240 little is known about delayed or late-appearing anaemia in relation to the use of act for therapeutic purposes (sowunmi et al., 2017). methods procurement of animals and management: 100 male adult balb/c mice with a mean weight of 24.46± 0.07g of 8 weeks of age were used in this study. they were obtained from the university college hospital's (uch) institute for advanced medical research and training in ibadan, nigeria. the mice were not used for any experiments before and are pathogen free. the mice were housed in plastic cages containing beddings of dried wood shavings and were fed with standard feed produced by ladokun feed limited, ibadan, oyo state, nigeria. mice were given constant access to food and water and were kept on a 12-hour light/12hour dark cycle. research design: 100 mice were randomly allocated into 5 groups of 20 using computer-generated numbers (johnson and besselsen, 2002). each group was split randomly into four replicates, each with five mice in a cage. the sample size was calculated using the equation formula (charan and kantharia, 2013). group 1 consisted of mice neither infected nor treated but given distilled water 1x, 2x, 3x, 4x, 5x and 6x times, respectively. groups 2 are non-infected mice treated with artemether lumefantrine (a/l) for 1x, 2x, 3x, 4x, 5x and 6x times, respectively. while group 3 are noninfected mice treated with artesunate amodiaquine (a/a) for 1x, 2x, 3x, 4x, 5x and 6x times, respectively, while group 4 consists of mice infected and treated with a/l for 1x, 2x, 3x, 4x. 5x and 6x times, respectively, and group 5 mice were infected and treated with a/a for 1x, 2x, 3x,4x, 5x and 6x times, respectively. mice were given a week to recover after treatment or infection plus treatment before subsequent exposure. blood was collected from 3 mice for haematological analysis one week after 1x, 2x, 3x and 6x exposure periods, in other to compare the effect of 13 usage with up 6 times usage of the drug on mice. the experiment was conducted following the national institutes of health's guide for the care and use of laboratory animals (nih publication no. 85–23, revised in 1996). the college of veterinary medicine research ethics committee at the federal university of agriculture, abeokuta, approved the experimental protocol (funaab/colvet/crec/2019/07/01). antimalaria drug: artemether plus lumefantrine (a/l) (lumartem anti-malarial tablet, 20 mg/120 mg) were obtained from cipla pharmaceuticals limited in mumbai, india, and artesunate with amodiaquine (a/a) (camosunate; 100 mg/300 mg) from geneith pharmaceuticals limited in lagos, nigeria. treatment was carried out in all treatment groups at therapeutic doses calculated based on the manufacturer's recommendation for a man's assumed weight of 35kg. artemether lumefantrine was administered with a therapeutic dose of 14/6.84 mg/kg/d in six dosages at 0, 8, 24, 36, 48, and 60 hours, respectively. at the same time, artesunate/ amodiaquine was administered 2.86/8.58 mg/kg/d once daily for three days a week (otuechere et al., 2012)(daikwo et al., 2018). in infected groups, seven days after infection, mice were treated. oral dosing was used to administer clinical doses using an intragastric feeding needle. nk65 strain of plasmodium berghei: plasmodium berghei nk65 utilised in this study was obtained from the chemotherapy research laboratory at the university college hospital (uch) in ibadan, nigeria. microscopic slides were made from blood from the donor mice's tails to determine the parasitemia level. after the thinly diffused blood, it was air-dried, fixed, and stained with giemsa. the parasite's presence was seen using an x100 oil immersion objective lens. we multiplied the number of parasites by the number of red blood cells (rbcs) in at least four random fields to calculate parasitemia as a percentage. next, malaria parasite inoculums were created using blood samples from a donor mouse. experimental mice were inoculated intraperitoneally with a low inoculum of 104 parasites using 0.1 ml of the donor mouse's blood. finally, blood smears were taken, stained, and inspected under a microscope to check the establishment and monitoring of infection every other day. at the same time, parasitemia levels were fully counted on day 7th post-infection and post-treatment. haematological studies the animals were treated humanely, and blood samples were collected via the retro-orbital plexus into edta bottles for haematological analysis. packed cell volume (pcv): the pcv was determined by centrifuging the well-mixed anticoagulated blood sample in capillary tubes for 10 minutes at 200 revolutions per minute using a micro haematocrit centrifuge to ensure minimal cell packing. the packed cell volume was then calculated by measuring the height of the red cells with a pcv metre. the percentages of blood volume were used to express the values (everds, 2007)(cheesbrough, 2006). haemoglobin measurement: the cyanmethemoglobin technique was used to determine the haemoglobin concentrations in the blood samples. the blood was diluted in a buffered solution of potassium ferricyanide and potassium cyanide to get cyanmethemoglobin. we created 1 in 25l dilutions by washing 20µl of blood in 5.0ml of modified drabkin's fluid. three minutes were given for complete conversion to cyanmethemoglobin. the absorbance was then measured at 540nm compared to distilled water using a spectrophotometer (everds, 2007)(cheesbrough, 2006). red blood cell counts: we made a dilution of 1 in 20 l of the anticoagulant blood sample, 0.02 ml of the blood was mixed with 4 ml turk's solution. 0.01 ml of audu et al. – the effects of frequent therapeutic administration of … 235 the resulting mixture was inserted into the counting chamber. the red cell present in the four corners and central 1 mm2 was counted, recorded, and the total rbc counts were calculated using formula (everds, 2007)(cheesbrough, 2006) red cell indices: the mean cell volume (mcv), mean cell haemoglobin (mch), and mean cell haemoglobin concentration (mchc) were estimated using the formula described by (everds, 2007)(cheesbrough, 2006) mean cell haemoglobin (mch): this is the quantity of haemoglobin (in pictogram form) contained in an average red cell. the value was derived using the haemoglobin concentration and red blood cell numbers formula. mch = (hb×10) / (total rbc) (pg or 10-12/g) mean cell volume (mcv): this is the average volume of red blood cells measured in femtoliters. the mcv was calculated from the pcv and red cell using the formula. mcv = (pcv x 10) /total rbc (fl or 10-15/l) mean cell haemoglobin concentration (mchc): this is the quantity of haemoglobin in 100ml of packed red blood cells instead of the amount of haemoglobin in whole blood. the mchc was computed from the haemoglobin and pcv represented as g/dl, mchc = (hb x 100) / pcv (g/dl) white blood cell counts: the blood sample was diluted by washing 50µl of blood into 950µl of the diluting fluid to give a final dilution of 1 in 20. the dilution was then mixed and loaded into the counting chamber. the white cells present in the four corners 1 mm2 areas were counted. the final white cell count for the whole blood sample was calculated using (everds, 2007)(cheesbrough, 2006). white blood cell differentials: leishmann staining method was used to determine the percentage of each type of wbc, prepared slides were viewed under the microscope x100 oil immersion objective, and the cells were counted and recorded (everds, 2007)(cheesbrough, 2006). statistical analysis: raw data from the laboratory were analysed, and graphs were plotted using graphpad prism 8.0 computer program. to determine the significant difference between the various treated group and the control, a one-way analysis of variance (anova) was used. the tables provided the findings as mean ± standard error mean (sem) (n =3). shapiro-wilk tests were first used to determine whether the data's distribution was normal. the significance of mean values *, ** and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively compared to the respective control groups. results mean parasitemia levels at day seven post-infection were less than 5% in mice infected in each consecutive time of 1x, 2x, 3x, 4x, 5x and 6x, respectively. day 7 posttreatment with a/l or a/a for 1x, 2x, 3x, 4x, 5x and 6x, respectively, after the corresponding infection recorded 0% parasitemia. effects of repeated treatment of non-infected and infected mice with either a/l or a/a on pcv, hb, rbc and wbc. to ascertain the level of haematological damage of noninfected and infected groups treated repeatedly with either al or aa, the blood pcv, hb, rbc, wbc, rbc indices, and differential wbc count were analysed. non-infected and infected mice exposed to a/l and a/a 1x, 2x, 3x and 6x didn't significantly alter (p>0.05) the level of blood pcv (fig. 1a), hb (fig. 1b), and rbc (fig.2a) respectively when compared with their controls. although there was a non-significant (p>0.05) decrease in pcv, hb, and rbc levels in the non-infected group treated with a/l only once compared to the control 1x and the rest group 1x, this reduction was not later observed after treatment for 2x and 3x period. also, mice infected and treated with a/l 3x recorded non-significant (p>0.05) reduced pcv, hb and rbc compared to the control group 3x, and the rest group treated 3x; this reduction was not later seen after 6x treatment (fig. 1 a, b and 2a). the non-infected groups treated with a/l and a/a for 6x non-significantly (p>0.05) reduced the pcv, hb and rbc levels compared to the control group (fig. 1a,1b and 2a). the mcv, mch and mchc of non-infected and infected mice treated with either a/l or a/a for 1x, 2x and 3x were not altered compared to the control group. the noninfected group treated with a/l for 6x significantly (p < 0.05) reduced the mcv value compared to the control, the infected treated with a/l or a/a 6x and the noninfected treated with a/a 6x (table 1). the wbc counts were lower in control compared to the non-infected and infected groups treated with either a/l or a/a 1x, 2x, 3x, and 6x, except group (inf+aa), treated 6x (fig. 2b). however, wbc counts increased in the non-infected groups treated with either a/l or a/a 1x and 3x following an increasing trend with a significant increase in group al (p < 0.01) and aa (p < 0.001) for 3x but dropped after treatment for 6x (fig. 2b). exposure of either a/l or a/a 1x, 2x, 3x and 6x to non-infected and infected groups reduced the neutrophil level and increased the lymphocyte level compared to the control (table 2). the eosinophils level was significantly higher in non-infected mice treated with either a/l for 236 biology, medicine, & natural product chemistry 12 (1), 2023: 233-240 2x and 3x compared with the control, following an increasing trend with a significant highest increase in 2x (p < 0.05) and 3x (p < 0.001), after which it drops in treatment for 6x. infected mice treated with either al or aa 6x recorded higher eosinophil levels than the noninfected and control groups (table 2). basophil level was higher in non-infected and infected groups treated with either a/l or a/a for 2x, 3x, and 6x compared to the control, with a significant (p < 0.001) increase in the non-infected group treated with a/a for 1x and 6x and infected group treated with a/a for 1x, 3x and 6x times. exposure of the non-infected group to either a/l or a/a 1x, 2x and 3x times and only a/a for 6x increased the monocyte level compared to the control group. in contrast, the infected group treated with either a/l or a/a 1x and 2x, and only a/a for 3x and 6x increased mono level compared to the control group (table 2). figure 1. (a)pcv level (b)hb level: of infected and non-infected mice treated with either a/l or a/a regime 1x, 2x, 3x and 6x times. pcv: packed cell volume; hb: haemoglobin al; treatment of non-infected with a/l therapeutic doses; aa: treatment of non-infected with a/a therapeutic doses; inf+al: infected with p. berghei and treated with a/l therapeutic; inf+aa: infected with p. berghei and treated with a/a therapeutic; ctl: control; 1x: one time; 2x: two times; 3x: three times; 6x: six times. values are expressed as mean ± sem (n = 3). *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively compared to its corresponding control groups. figure 2. (a) rbc level (b) wbc level: of infected and non-infected mice treated with either a/l or a/a regime for 1x,2x, 3x and 6x times. al: rbc: red blood cell; wbc: white blood cell. treatment of non-infected with a/l therapeutic doses; aa: treatment of non-infected with a/a therapeutic doses; inf+al: infected with p. berghei and treated with a/l therapeutic; inf+aa: infected with p. berghei and treated with a/a therapeutic; ctl: control; 1x: one time; 2x: two times; 3x: three times; 6x: six times. values are expressed as mean ± sem (n = 3). *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively compared to its corresponding control groups. 1x 2x 3x 6x 0 20 40 60 80 number of times p c v % 1x 2x 3x 6x 0 5 10 15 20 25 number of times h b (g /d l) ctl al aa inf+al inf+aa ctl al aa inf+al inf+aa a b 1x 2x 3x 6x 0 5 10 15 number of times r b c × 1 0 1 2 /l 1x 2x 3x 6x 0 5 10 15 20 nunber of times w b c ( × 1 0 9 /l ) ✱✱✱ ✱✱ ✱ ctl al aa inf+al inf+aa ctl al aa inf+al inf+aa a b audu et al. – the effects of frequent therapeutic administration of … 237 table 1. effect of repeated usage of al and aa on the level of mcv, mch and mchc blood parameters in mice. no. of times of infection and/or treatment infection and/or treatment red blood cell indices mcv (fl) mch (pg) mchc (g/dl) 1x ctl 60.35±0.33 20.24±0.00 33.54±0.19 al 59.95±0.14 20.00±0.06 33.35±0.03 aa 59.70±0.17 20.20±0.00 33.85±0.09 inf+al 60.05±0.14 20.05±0.03 33.45±0.03 inf+aa 60.00±0.12 20.15±0.14 33.55±0.14 2x ctl 58.60±0.91 20.42±0.82 34.82±0.94 al 58.15±0.95 19.70±0.46 33.85±0.20 aa 58.85±0.66 19.95±0.14 33.95±0.14 inf+al 60.10±0.06 20.30±0.12 33.75±0.20 inf+aa 59.85±0.20 20.05±0.03 33.50±0.17 3x ctl 61.14±2.03 20.53±1.11 33.54±0.79 al 59.25±0.03 20.05±0.03 33.85±0.03 aa 58.70±0.29 19.90±0.17 33.90±0.23 inf+al 60.00±0.12 20.15±0.14 33.55±0.14 inf+aa 59.30±0.69 20.30±0.17 34.30±0.23 6x ctl 61.84±1.18 20.50±0.61 32.37±0.92 al 58.30±0.06* 19.40±0.40 33.30±0.23 aa 60.00±0.17 20.15±0.14 33.55±0.14 inf+al 59.45±0.20 19.60±0.12 33.00±0.06 inf+aa 60.25±0.03 20.25±0.09 33.60±0.12 mcv: mean cell volume; mch: mean cell haemoglobin; mchc: mean cell haemoglobin; al: treatment of non-infected with a/l therapeutic doses; aa: treatment of non-infected with a/a therapeutic doses; inf+al: infected with p. berghei and treated with a/l therapeutic; inf+aa: infected with p. berghei and treated with a/a therapeutic; ctl: control; 1x: one times; 2x: two times; 3x: three times; 6x: six times. values are expressed as mean ± sem (n = 3). *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively compared to its corresponding control groups. table 2. effect of repeated usage of al and aa on wbc differentials in mice granulocytes. no. of times of infection and/or treatment infection and treatment white blood differentials neutrophils (%) lymphocytes (%) eosinophils (%) basophils (%) monocyte s (%) 1x ctl 36.00±1.73 62.33±2.03 1.00±0.00 0.00±0.00 0.67±0.33 al 26.00±0.58*** 71.00± 0.58** 1.50±0.29 0.00±0.00 1.00±0.00* aa 25.50±0.29*** 71.00± 0.57** 0.50±0.29 1.50±0.29*** 1.50±0.29 inf+al 32.00±2.31 64.00± 2.31 2.00±0.00* 1.00±0.00 1.00±0.00 inf+aa 29.00±0.58** 67.00± 1.16 1.50±0.29 1.50±0.29*** 1.00±0.00 2x ctl 36.67±0.33 61.00±0.58 1.00±0.00 0.33±0.33 0.00±0.00 al 28.50±0.87*** 67.50±0.29* 2.00±0.58* 1.00±0.00 1.00±0.00** aa 28.00±0.58*** 69.50±0.29** 1.00±0.00 0.50±0.29 1.00±0.00** inf+al 27.50±0.29*** 69.50± 0.29** 0.50±0.29 0.50±0.29 1.00±0.00** inf+aa 26.50±0.29*** 70.50±0.29*** 1.00±0.00 0.50±0.29 1.50±0.29*** 3x ctl 38.67±0.67 60.00±0.58 1.00±0.00 0.00±0.00 0.33±0.33 al 28.00±0.58*** 66.50± 0.88* 3.50±0.29*** 1.00±0.00 1.00±0.00 aa 32.00±0.58* 63.00± 1.15 2.00±0.00* 1.00±0.00 2.00±0.00*** inf+al 29.00±2.31*** 69.50± 2.02*** 1.00±0.00 0.50±0.29 0.00±0.00 inf+aa 27.00±0.58*** 67.00± 1.73* 3.00±0.00*** 2.00±0.00*** 1.00±0.00 6x ctl 36.67±2.33 61.00±1.73 1.00±0.00 0.00±0.00 1.00±0.00 al 27.00±0.58*** 69.00±0.58** 1.00±0.00 1.00±0.00 1.00±0.00 aa 29.50±2.60** 66.00± 3.46 1.00±0.00 1.50±0.29*** 1.50±0.29* inf+al 30.00±1.73* 66.50±2.02 1.50±0.29 0.50±0.29 1.00±0.00 inf+aa 30.50±0.29* 64.00± 0.58 2.00±0.00* 2.00±0.58*** 1.67±0.33** al: treatment of non-infected with a/l therapeutic doses; aa: treatment of non-infected with a/a therapeutic doses; inf+al: infected with p. berghei and treated with a/l therapeutic; inf+aa: infected with p. berghei and treated with a/a therapeutic; ctl: control; 1x: one time; 2x: two times; 3x: three times; 6x: six times. values are expressed as mean ± sem (n = 3). *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively compared to its corresponding control groups. 238 biology, medicine, & natural product chemistry 12 (1), 2023: 233-240 discussion in this study, a/l and a/a were very effective against the malaria parasite. both drugs are seen to clear the parasite after 7-day post-treatment during each repeated infection and treatment. reports have shown that both a/l and a/a have high malaria treatment success (davlantes et al., 2018; marwa et al., 2022). although, several reports have shown that malaria cause anaemia and changes in other haematological variables (osaro et al., 2014; omarine nlinwe and nange, 2020; audu et al., 2021), and recurring malaria attacks can lead to lifethreatening anaemia (bakhubaira, 2013; dhangadamajhi et al., 2019). however, this present study was designed to determine if repeated therapeutic exposure to a/l and a/a when not infected could affect blood cell counts and predispose animals to anaemia, as very little was found in the literature on this question. this investigation shows that the pcv, hb and rbc were not significantly altered by the therapeutic use of a/l and a/a for 1x, 2x, 3x and 6x in non-infected mice compared to the control groups. this outcome is contrary to the previous study, which has shown acute haemolytic anaemia following the use of artemisinin (rehman et al., 2014). furthermore, a study by (geerligs et al., 2003) corroborates that malaria chemoprophylaxis improves mean haemoglobin levels. while the term "haematocrit conservation" was spawned because it was seen that there is little to no drop in haematocrit following acts, even when parasitaemia are heavy (gbotosho et al., 2014; sowunmi et al., 2017). these findings suggest that taking this drug for 1x, 2x and 3x, and 6x therapeutically when not infected would not significantly alter the pcv, hb and rbc after one week of each repeated treatment. prolonged repeated treatment of non-infected mice with either a/l or aa for up to six consecutive times reduced the levels of pvc, hb, and rbc insignificantly compared to the control and infected groups. it indicated that the prolonged use of this treatment in the absence of infection could potentially modify the haematological parameter. evidence suggests that exposure to either al or aa over an extended period can have a detrimental effect on haematological markers (ofem et al., 2013; ijeomah et al., 2016). although the use of a/l 1x when non-infected and when infected 3x insignificantly also reduced the pcv, hb and rbc value, although there was a recovery after subsequent treatment, this shows that the use of a/l over a/a could potentially also alter the pcv, hb and rbc parameters when used. (white, 2018) reported that being infected and treated with artemisinin is seen to cause haematolytic anaemia after 1-3 weeks of drug usage. what is surprising in this experiment is that repeated infecting mice and treatment with either a/l or a/l 1x, 2x, 3x and 6x didn't alter the pcv, hb and rbc. these results reflect those of (sowunmi et al., 2017), who also found that both a/a and a/l have been observed to dramatically lower the prevalence of anaemia in younger and older children who have malaria after therapy (sowunmi et al., 2017). this result supports that repeated malaria infection and treatment with therapeutic doses of either a/l or a/a would improve the haematological parameters. mcv, mch and mchc are indices of erythrocyte shape, size, and haemoglobin content changes. studies have shown that artesunate does not affect the total rbc, mch, and mchc levels (bigoniya et al., 2015)(ijeomah et al., 2016). the current study found that the mcv, mch, and mchc values obtained after treatment of non-infected and infected groups with either a/l or a/a for 1x, 2x and 3x were not significantly different compared to the control group. implies that the red blood cells are normal in size and concentration after treatment of infected and noninfected with the drugs for 1x, 2x and 3x. although after repeated treatment with therapeutic dosages of a/l for 6x to non-infected mice, the mcv value was significantly reduced; this indicates that using a/l repeatedly without being infected could decrease the average size of the red blood cells in mice. white blood cells in the body provide a unique defence system against infections and hazardous substances. in this study, apart from the group repeatedly infected and treated with a/a 6x, there was an increase in wbc count in both infected and non-infected mice treated with either al or aa therapeutic doses for 1x, 2x, 3x and 6x times. the increase was due to a rise in lymphocytes, eosinophils, basophils, and monocytes, but a decrease in neutrophil was observed in the treated groups. the increase in wbc and lymphocyte counts suggests an immunological response induced by the drug as they are mobile components of the body's defence systems. as (adeleye et al., 2012) found that a/l can raise total wbc counts and lymphocyte counts while decreasing neutrophil counts, which they ascribed to the immunological response caused by the medication. in our study, we noticed either a/l or a/a usage once or repeatedly resulted in a significant increase in the wbc of non-infected groups. however, a study by (ijeomah et al., 2016) found a substantial drop in wbc after longterm a/l and a/a usage; also, in this present study, non-infected mice treated with al and aa for three consecutive times had the highest increase in wbc count but dropped after six consecutive times. this result has shown that usage of al and aa could increase the wbc count when used repeatedly but could drop after prolonged usage. in malaria-infected individuals, counts of white blood cells (wbcs), neutrophils, monocytes, lymphocytes, and eosinophils were considerably reduced (kotepui et al., 2014). in this study, repeated usage of al and aa in infected and non-infected mice for 1,2,3 and 6 consecutive times had a significant reduction in neutrophil, with a substantial decrease in non-infected audu et al. – the effects of frequent therapeutic administration of … 239 mice treated with either a/l or a/a for 1 and 6 times compared to the control and infected group. this result implies that treatment with either a/l or a/a without infection could lead to neutropenia. low neutrophil has been associated with intermittent (weekly) doses of amodiaquine for malaria prevention. (zwang et al., 2012). this study reported a significant increase in eosinophil, basophil, and monocyte in groups treated with al and aa; this increase may be due to responses of the antimalaria drugs (adeleye et al., 2012; bigoniya et al., 2015). the limitation of this study lies in the fact that the study did not include taking blood immediately after treatment as a subgroup for comparison. notwithstanding these limitations, the study still shows us the extent of haematological alteration caused when these drugs are taken repeatedly. conclusions the result of this study shows that the use of a/l and a/a therapeutically in a repeated manner without being infected could result in haematological alteration. this study lays the groundwork for future clinical and further animal work on this subject. further research could address more longer-time effects of taking this drug and check the haematological parameter immediately after each treatment. in addition, more significant efforts are needed to enlighten the public on the need to repeatedly screen for malaria parasites before every repeated use of antimalarial drugs. competing interest: the authors report having no competing interests. funding: the research animal and laboratory work was funded by the nigeria federal government tertiary education trust fund (tetfund) authors’ contributions: ad came up with the experiment's idea. ad, iba, ioa, and mfm designed the research methodology and carried out the experiments. pvb provided technical assistance. ad worked on the draft manuscript; the final version was read, edited, and approved by all authors. acknowledgements: firstly, i want to thank the nigeria federal government tertiary education trust fund (tetfund) for the grant support of this work. secondly, i want to thank the research laboratory at the university college hospital (uch) in ibadan, nigeria, for providing us with p. berghei for this study. references adeleye, g. s., nneli, r., nwozor, c. m., and emesiana, m. c. 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(2012) comparing changes in haematologic parameters occurring in patients included in randomized controlled trials of artesunate-amodiaquine vs single and combination treatments of uncomplicated falciparum in sub-saharan africa. malaria journal 11(1): 1– 11. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 205-210 | doi: 10.14421/biomedich.2025.141.205-210 issn 2540-9328 (online) utilization of eco-friendly iron oxide nanoparticles for wastewater treatment saminu hamman barau1,*, abdulazeez mumsiri abaka2, jameelah bakari2, suleiman alhaji saidu3, jameelah bakari2 1pharmaceutical technology department, school of science and technology, adamawa state polytechnic yola, nigeria. 2science laboratory technology department, school of science and technology, adamawa state polytechnic yola, nigeria. 3chemistry department, college of education hong, nigeria. corresponding author* hammansaminu@gmail.com abstract this study investigates the potential of green-synthesized iron ii oxide (fe₂o₃) nanoparticles for remediating wastewater from river benue, nigeria, focusing on removing lead (ld) pollutants. the nanoparticles were synthesized using annona squamosa leaf extract, offering an eco-friendly alternative to conventional remediation methods. the wastewater sample, collected from river benue in jimetayola, was analyzed using ft-ir, sem, and atomic absorption spectroscopy. ft-ir analysis identified functional groups like alcohol o-h and conjugated alkene c=c, confirming the role of plant metabolites in nanoparticle synthesis and stabilization. sem imaging revealed cubical fe₂o₃ nanoparticle sizes ranging from 25 to 34 nm, stabilized by the plant extract. the remediation process tested varying nanoparticle concentrations (100, 500, 1000, and 1500 ppm) and contact times (60, 120, and 180 minutes) at ph 8.90 and 21.33 k. the highest adsorption efficiency was observed at 1500 ppm over 180 minutes, reducing lead levels from 0.69 mg/l to 0.02 mg/l due to increased chelating sites. lower concentrations (1000, 500, and 100 ppm) also demonstrated significant adsorption, with lead levels dropping to 0.21, 0.32, and 0.50 mg/l, respectively. the study highlights the advantages of fe₂o₃ nanoparticles, including simplicity, rapid production, environmental safety, and a high surface area for effective pollutant adsorption. this green synthesis approach is more sustainable and operationally simpler than tradisional methods, avoiding the environmental risks associated with conventional techniques. the findings suggest that fe₂o₃ nanoparticle is promise for efficient and sustainable wastewater purification, offering a viable alternative to existing water treatment technologies. this research underscores the potential of eco-friendly nanomaterials in addressing heavy metal pollution, particularly in developing regions. keywords: remediation; wastewater; annona squamosa; synthesis; nanotechnology. introduction water is essential for life, development, and health, and the importance of clean drinking water in fostering wellbeing cannot be overstated (rheeder et al., 2023). however, water pollution has become a global crisis, driven by industrialization, urbanization, and population growth. this pollution harms the environment and contributes to air pollution, posing severe risks to human health (talema, 2023). access to clean water and sanitation is a fundamental human right, yet increasing pollution, water scarcity, and inadequate treatment technologies have made achieving this goal a significant challenge (angelakis et al., 2023; hait et al., 2024). historically, conventional water purification methods, particularly chemical-based approaches, have been widely used despite their complexity and hazardous nature (parvulescu et al., 2021). in contrast, nanotechnology has emerged as a transformative field, offering innovative solutions due to the unique properties of nanomaterials, such as small particle size, high surface area, and exceptional thermal, optical, and mechanical characteristics (eddy et al., 2023). nanotechnology can revolutionize various sectors, including healthcare, manufacturing, and environmental protection, by producing smaller, more efficient, cost-effective materials with reduced energy and resource consumption (malik et al., 2023). metal-based and metal oxide nanoparticles, in particular, have gained attention as sustainable solutions for water remediation. their small size, large surface area, and quantum effects make them highly effective. green synthesis methods, which use biological materials like plant extracts, are preferred over chemical and physical methods due to their environmental friendliness, cost-effectiveness, and scalability (mamuru et al., 2022; gupta et al., 2023). for instance, iron oxide nanoparticles synthesized from prosopis africana leaf extract have shown promising results, with characterization techniques like uv-vis spectroscopy, sem, and ft-ir manuscript received: 03 february, 2025. revision accepted: 20 may, 2025. published: 14 june, 2025. https://doi.org/10.14421/biomedich.2025.141.205-210 mailto:hammansaminu@gmail.com 206 biology, medicine, & natural product chemistry 14 (1), 2025: 205-210 confirming their structure and functionality (elebo et al., 2024). similarly, zinc oxide nanoparticles synthesized using mangifera indica extract have demonstrated potential in degrading pollutants like congo red dye (mamuru et al., 2022). despite these advancements, there is limited research on the application of eco-friendly iron oxide nanoparticles (fe₂o₃-nps) for wastewater treatment in regions like adamawa state, nigeria. this study explores the potential of green-synthesized fe₂o₃-nps for sustainable wastewater management, addressing a critical gap in current research (abd elmohsen et al., 2024). materials and methods collection of plant materials according to nahari et al., (2022), fresh leaves of a. squamosa were hand-picked from the chemistry department block of the faculty of physical sciences, modibbo adama university yola, which was authenticated in the plant science department and a voucher specimen was saved in the herbarium (no. mau/pls/0067). preparation of plant leaf extract fresh a. squamosa leaves were used to extract the active constituents. the leaves were thoroughly washed with tap water and then rinsed with double-distilled water to remove dust particles. approximately 20 grams of the fresh leaves were finely chopped, mixed with 100 ml of distilled water, and boiled at 60°c in a water bath for 10 minutes. the mixture was then cooled and filtered using a muslin cloth, followed by filtration through whatman no. 1 filter paper. the resulting pale yellow solution was collected, stored in screw-capped bottles at 4–8°c, and utilized as a reducing agent for the synthesis of fe₂o₃ nanoparticles. preparation and synthesis of fe2o3-nps a 0.01 m ferrous sulfate solution was prepared. 20 ml of the plant extracts was added dropwise with constant stirring to about 100 ml of the solution for one hour till the colour changed to dark green. the product was centrifuged at 3000 rpm for 15 minutes, washed with ethanol and distilled water, and allowed to dry at room temperature (mamuru et al., 2022). solution and surface characterization of fe2o3-nps the synthesized metal nanoparticles were confirmed by analyzing the aqueous component. the biomolecules responsible for the reduction of ferrous oxide were identified using a perkin elmer fourier transform infrared (ft-ir) spectrophotometer, which provided insights into the functional groups involved in the process. additionally, the size and morphology of the nanoparticles were determined using scanning electron microscopy (sem), which revealed detailed images and dimensions of the synthesized nanoparticles. these analytical techniques collectively validated the successful formation and characteristics of the nanoparticles. batch experiment for waste water remediation of heavy metal in a controlled experimental setup, distinct concentrations of fe₂o₃ nanoparticles (100, 500, 1000, and 1500 ppm) were prepared and dispersed into 100 ml of wastewater within a 250 ml erlenmeyer flask. the mixture was intermittently shaken at room temperature (30°c) and maintained at a neutral ph. samples of approximately 10 ml were collected from the flask at regular contact time intervals (60, 120, and 180 minutes) using variable doses (0.2 and 0.5 g). to ensure accuracy, the experiment was repeated twice under identical conditions. the percentage removal of heavy metals was determined using the environmental protection agency (epa) method, and the removal efficiency (r%) was calculated to evaluate the effectiveness of the fe₂o₃ nanoparticles in treating the wastewater (yadav et al., 2023). results and discussions optical property adding 20 ml of extract to the flask containing 0.01 m ferrous sulfate did not immediately change the colour (fig. 1b), however at the end of one hour the colour of the solution become dark-green (fig. 1c) which indicates the formation of nanoparticles (fig. 1d). the colour change is likely due to the excitation of surface plasmon vibrations in feo nanoparticles (waqas et al., 2022). figure 1. leaf extract of annona squamosa (a), ferrous sulfate solution (b), metal nanoparticles liquid (c), and the metal nanoparticles solid (d). cb da barau et al. – utilization of eco-friendly iron oxide nanoparticles … 207 fourier transform infrared spectroscopy analysis the fourier transform infrared analysis was done to identify the likely biomolecules responsible for reducing the metal salt to its nanoparticles and capping the reduced metal nanoparticles (fe2o3-nps) synthesized using annona squamosa leaf extract. the ft-ir analysis for the iron oxide nanoparticles displayed that there are strong, broad, and medium bands at 3253.53 cm-1, 1623.24 cm-1, 1057.89 cm-1, 804.95 cm1, 766.54 cm-1, 685.58 cm-1, and 591.45 cm-1. the bands which appeared at 3253.53 cm-1 and 1623.24 cm-1 correspond to alcohol o-h stretching and conjugated alkene; c=c stretching, respectively, the bands at 1057.89 cm-1 and 804.95 cm-1 are due to primary alcohol c-o stretching; tri-substituted c=c bending, respectively. the ft-ir bands observed at 766.54 cm-1 may be attributed to mono-substituted c-h bending, whereas bands at 685.58 cm-1 and 591.45 cm-1 were apportioned to c-br stretching and c-i stretching vibrations due to alcohol arising from the flavonoid constituent of the plant metabolites (das et al., 2023). figure 2. ft-ir absorption spectra of fe2o3 nanoparticles. the sem analysis of fe2o3 nanoparticles the sem image of the fe2o3 nps exposed understanding into their morphology and size. the agglomeration of particles designates a likely weak capping effect from the plant extract, resulting in adequate stabilization of the nanoparticles. the particles displayed a uniform distribution and mostly cubic shape, with sizes varying between 25 and 34 nm, as noted by (nyabadza et al., 2023). figure 3. sem micrograph of fe2o3-nps. batch experiment for waste water remediation of heavy metal table 1 highlights the significant impact of additional ions on adsorption effectiveness, as they compete with the primary ions for chelation sites on the adsorbent. at low concentrations of toxic metals, the availability of ions is limited, reducing scavenging efficiency. however, at higher concentrations, the increased availability of ions enhances removal efficiency, as demonstrated by reduced lead concentration in wastewater from 0.69 208 biology, medicine, & natural product chemistry 14 (1), 2025: 205-210 mg/l to 0.02 mg/l after remediation. temperature also plays a crucial role in adsorption processes. higher temperatures increase the adsorption rate for endothermic adsorption while for exothermic adsorption, the rate decreases. in this study, raising the temperature from 10°c to 21°c improved adsorption, but beyond this point, the process declined due to increased kinetic energy, which caused desorption of metal ions from the adsorbent sites (priya et al., 2022). the effectiveness of wastewater remediation from heavy metals also depends on the contact time between the nano sorbent and metal ions. increasing the contact time from 60 to 180 minutes significantly enhances adsorption efficiency, as more time allows for greater interaction between the pollutants and the adsorbent's active chelation sites. initially, adsorption occurs rapidly due to the availability of open active sites, but as these sites become occupied, the process slows down. this trend was similarly observed in the adsorption of divalent mercury ions on canps, where adsorption increased significantly as contact time rose from 0 to 90 minutes (tiwari et al., 2023). additionally, adsorbent dosage plays a critical role in adsorption capacity. increasing the dosage from 100 to 1500 ppm improved adsorption efficiency to 97% by providing more active sites for metal chelation. however, further increases in dosage led to a buildup of the adsorbent, reducing surface area and adsorption capacity. the ph of the reaction medium also significantly influences adsorption reactions and capacity, as it affects the availability of active sites and the interaction between the adsorbent and metal ions (fei et al., 2022). figure 4. formation of nanoparticle bits (a), different concentrations of nanoparticle bits in waste water (b), and remediated water with different concentrations of nanoparticles and contact time (c). table 1. adsorption process. metal ions adsorbent (ppm) contact time (min) initial metal conc. (mg/l) dosage of adsorbent (mg/l) final metal conc. (mg/l) ph temp. (k) fe (ii) feonps 100 60 0.69 0.2 0.64 8.90 21.33 “ feonps 500 60 “ “ 0.52 “ “ “ feonps 1000 60 “ “ 0.50 “ “ “ feonps 100 120 “ “ 0.40 “ “ “ feonps 500 120 “ “ 0.36 “ “ “ feonps 1000 120 “ “ 0.32 “ “ “ feonps 100 180 “ “ 0.29 “ “ “ feonps 500 180 “ “ 0.27 “ “ “ feonps 1000 180 “ “ 0.21 “ “ “ feonps 1500 60 “ 0.5 0.15 “ “ “ feonps 1500 120 “ 0.5 0.08 “ “ “ feonps 1500 180 “ 0.5 0.02 “ “ cba barau et al. – utilization of eco-friendly iron oxide nanoparticles … 209 figure 5. adsorption chart of wastewater. conclusion the study showcased the successful application of ecofriendly fe₂o₃ nanoparticles, synthesized from annona squamosa leaf extract, for wastewater remediation, particularly in removing heavy metals like lead. these nanoparticles, characterized by their simplicity, rapid production, and environmental safety, provided a larger surface area for reactions than conventional methods, significantly enhancing their effectiveness. this green synthesis approach offers a sustainable and straightforward alternative to traditional wastewater treatment techniques, which are often complex and pose environmental risks. the efficacy of fe₂o₃ nanoparticles in treating wastewater from the river benue was confirmed through various analyses. ft-ir spectroscopy identified flavonoids in the leaf extract as key agents in the synthesis and stabilization of the nanoparticles. sem imaging revealed that the nanoparticles were cubical, well-stabilized, and ranged in size from 25 to 34 nm. atomic absorption spectroscopy demonstrated a remarkable reduction in lead contamination levels, from 0.69 mg/l to 0.02 mg/l, achieving a 97% removal efficiency. these findings highlight the potential of green-synthesized fe₂o₃ nanoparticles as a highly effective and environmentally friendly solution for wastewater treatment. acknowledgement: special thanks are extended to the department of science laboratory technology at adamawa state polytechnic, yola, and modibbo adama university, yola, for providing the necessary resources and support that made this research possible. their contributions were instrumental in the successful execution of this study. authors contributions: saminu hamman barau designed the study, carried out data collection, performed laboratory work, and drafted the manuscript. abdulazeez mumsiri abaka, jameelah bakari, and suleiman alhaji saidu reviewed and proofread the manuscript. all authors read and approved the final version of the manuscript. funding: no author received any financial support for this research and publication. competing interest: all authors declare no competing interest. references abd elmohsen, s. a., daigham, g. e., mohmed, s. a., & sidkey, n. m. 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(2023). treatment of fly-ashcontaminated wastewater loaded with heavy metals by using flyash-synthesized iron oxide nanoparticles. water, 15(5): 908. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 621-633 | doi: 10.14421/biomedich.2025.142.621-633 issn 2540-9328 (online) macroscopic, microscopic, and phytochemical screening analysis of medicinal plants in nagari lasi, west sumatra tika afriani *, devahimer harsep rosi, winda mailinda department of pharmacy, faculty of health sciences, mohammad natsir university, bukittinggi, jl. tan malaka, bukik cangang kayu ramang, bukittinggi 25136, tel. +0752-21169, fax. +0752-33458, west sumatra, indonesia. corresponding author* tika.afriani91a@gmail.com manuscript received: 24 april, 2025. revision accepted: 13 august, 2025. published: 01 october, 2025. abstract indonesia is one of the countries titled “megabiodiversity”, which has a high level of biodiversity, including medicinal plants. medicinal plants utilize various parts of plants, such as roots, leaves, stems, and fruits for prevention, treatment, and pain relief. nagari lasi has great potential in the diversity of medicinal plants, but their utilization by the community is still limited due to a lack of knowledge. this study aims to identify the content of bioactive compounds in medicinal plants through macroscopic, microscopic, and phytochemical screening. the methods include organoleptic observation, macroscopic and microscopic analysis, and phytochemical tests. of the 150 plant samples, the results showed that 78 samples contained alkaloids, 115 samples contained phenolics, 129 samples contained flavonoids, 75 samples contained steroids, 21 samples contained triterpenoids, and 119 samples contained saponins. this study provides basic information regarding the phytochemical content of medicinal plants in nagari lasi as a first step in developing traditional medicine. keywords: medicinal plants; macroscopic analysis; microscopic analysis; phytochemical screening. introduction indonesia is known as one of the countries with the title “megabiodiversity” because of its location as a tropical country between the asian and australian continents (rosmini, zainuddin, & aini, 2022). this geographical position makes indonesia have a very high level of biodiversity (marganingrum & santoso, 2019). among these biological riches, medicinal plants are one of the riches utilized by the community for traditional medicine (weking, cornelia, hafsah, & rahma lestari, 2023). however, until now, only around 7,500 plant species have been identified as medicinal plants (cahyaningsih, magos brehm, & maxted, 2021). medicinal plants have various parts that can prevent, treat, or relieve diseases, such as roots, leaves, stems, fruits, or other excretory products (saleh & kamisah, 2021). each plant has different chemical content and properties (zengin et al., 2020). these properties come from secondary metabolite compounds naturally produced by plants, including alkaloids, flavonoids, saponins, terpenoids, and tannins (raden roro upiek ngesti wibawaning astuti, illahi, umri, & falah, 2023). the use of medicinal plants in indonesia has been going on since ancient times and is believed to be able to cure various diseases and improve the quality of health (roseva sari br bangun & sabariah bangun, 2021). the advantages of medicinal plants over chemical drugs include minimal side effects and more affordable costs (jamal, 2023). therefore, their use remains popular among people from various backgrounds (kintoko et al., 2023). however, the pattern of utilization of medicinal plants differs across regions in indonesia, depending on the types of plants available, the plant parts used, and the processing methods, which are often influenced by local culture (rahayu, ibo, arimukti, & susiarti, 2020). canduang district, located in agam regency, west sumatra, is one of the areas with great potential for medicinal plant diversity. located on the slopes of mount merapi at an altitude between 780 to 2,891 meters above sea level, this area has very fertile soil, rich in weathered volcanic material (rais, 2021). nagari lasi, a village in canduang subdistrict with an area of 15.34 km², is a fertile area with the potential to produce a variety of plants, especially medicinal plants. the nagari lasi community's interaction with medicinal plants is still based on traditional knowledge passed down from generation to generation. unfortunately, the utilization of medicinal plants in this area is not optimal, most likely due to the lack of knowledge about the content of chemical compounds and the specific benefits of the plants around them. scientific research related to the content of compounds in medicinal plants in nagari lasi is also very limited. in fact, information about the bioactive components of https://doi.org/10.14421/biomedich.2025.142.621-633 622 biology, medicine, & natural product chemistry 14 (2), 2025: 621-633 plants is an important first step to identify their pharmacological potential (poojar et al., 2017). therefore, this study was conducted to identify the content of bioactive compounds in medicinal plants in nagari lasi through macroscopic, microscopic, and phytochemical screening. this research is expected to provide scientific information that supports the utilization of local plants as a source of traditional medicine. materials and methods tools microscope (yazumi), digital scale (kern), hotplate (maspion), a set of glassware such as 500 ml beaker glass (iwaki), 50 ml beaker glass (iwaki), 50 ml beaker glass (iwaki), 50 ml measuring cup (iwaki), test tube (iwaki), drip plate, test tube rack, mortar, stampher, filter paper, drip pipette, spatula, stirring rod, tweezers, knife, object glass, cover glass, cotton swab and others. materials plants, chloroform (merck®), chloroform ammonia 0.05 m (merck®), concentrated hcl (merck®), hcl 2 n (merck®), h2so4 2 n (merck®), methanol (merck®), metal powder mg (merck®), fecl3 5% (merck®), reagents mayer, dragendroff (merck®), liebermannburchard (merck®), and aquadest (merck®). procedures selection and sampling this study collected medicinal plant samples from homes and surrounding areas in nagari lasi. sample selection was based on a preliminary survey with local communities to identify plants often used as medicine. sample preparation the plants that have been taken are then further examined, part of the plant that will be used as a sample is the leaf organ of the plant. organoleptical examination organoleptical examination of medicinal plants or samples is carried out using the five senses in humans to observe shape, color, and smell n (novitasari, nashihah, & zamzani, 2021). macroscopic examination macroscopic examination of medicinal plants or samples is carried out by direct visual observation by observing the characteristics of the outer parts of the sample (novitasari et al., 2021). this macroscopic test can be done using a magnifying glass or without using tools and certain chemicals (sari, 2023). microscopic examination microscopic examination begins with cutting the sample transversely or longitudinally using a sharp knife to obtain a thin sample. then, the sample is transferred to an object glass and aquadest is added. next, cover the sample with a cover glass and observe the anatomy on the plant using a microscope (nakkliang, areesantichai, & rungsihirunrat, 2022). phytochemical screening phytochemical screening is an examination to determine the content of compounds in a plant sample, including alkaloids, flavonoids, tannins, saponins, and steroids/triterpenoids (itam, elin, & arifin, 2020). working procedure samples were weighed as much as 4 g and then cut into small pieces, put into a test tube and extracted with methanol. after that, the fresh sample extract was added with 5 ml chloroform and 5 ml aqudest and stirred well and then left for a moment until two layers were formed, namely the chloroform layer and the water layer. the top layer is water used for flavonoid, saponin and phenolic examination. the bottom layer is chloroform used for examination of triterpenoid and steroid compounds. ▪ alkaloid compound test 4 g of leaves were macerated with 10 ml of 0.05 m chloroform-ammonia, then stirred and filtered. a total of 2 ml of filtrate was added 1 ml of 2 n sulfuric acid, shaken and allowed to stand so that two layers were formed, namely the acid and chloroform layers. the acid layer is taken and transferred into another test tube then add a few drops of mayer reagent. a positive reaction is characterized by the presence of a white precipitate that is alkaloid. ▪ steroid/triterpenoid compound test the chloroform layer is taken and dripped on the drip plate hole and allowed to dry. the first drip plate hole is added to the liebermann burchard (lb) reagent. the formation of red or purple indicates the presence of triterpenoids, if the formation of green or blue green rings indicates the presence of steroids. if a red or purple red and green or blue green ring is formed simultaneously in the drip plate, it indicates positive triterpenoids and steroids. ▪ flavonoid compound test a layer of water as much as 1 ml is taken and put into a test tube, concentrated hydrochloric acid is added and several grains of magnesium powder, the formation of orange to red color indicates the presence of flavonoids in the sample. ▪ phenolic compound test a 1 ml layer of water is put into a test tube, 5% iron (iii) chloride solution is added, if a dark green or blue color is formed, indicating the presence of phenolic compounds in the sample. afriani et al. – macroscopic, microscopic, and phytochemical screening analysis … 623 ▪ saponin compound test a 1 ml layer of water is put into a test tube and shaken. if the formation of foam that does not disappear for 5 minutes after the addition of a few drops of hcl 2 n then this indicates the presence of saponins in the sample. data analysis data were analyzed descriptively and the results were presented in tables and diagrams. the percentage of plants based on family, organ parts used, and processing methods were calculated using simple statistical formulas. results and discussion this research was conducted in nagari lasi, canduang district, agam regency, west sumatra province. surveys were conducted in three jorongs, namely lasi mudo, lasi tuo, and pasanehan to identify the diversity of medicinal plants. samples included plants used by the community as well as potentially medicinal plants that have not been utilized. the research methods included preliminary survey, organoleptic examination, macroscopic, microscopic, and phytochemical screening. utilization of medicinal plants the survey of the nagari lasi community identified 45 plants that are often used in traditional medicine, such as cinnamon, turmeric, tapak liman, ruku-ruku, gotu kola, red ginger, and cat whiskers. these plants come from 31 families, with the zingiberaceae family being the most dominant (13%). the most utilized part of the plant is the leaves (69%), and the most common processing method is boiling (58%). leaves are often used because the chemical content and water content in the leaves are more, the leaves are also a place of photosynthesis where it is believed to contain organic substances that can treat diseases and the soft nature of the leaves makes it easier and more practical to use (kasih, fajar, eka, & rani, 2024). in addition to plants that have been utilized, 105 plants from various families were found that have potential as medicinal plants, but have not been utilized by the community. the asteraceae family is the most commonly found plant group (9.52%). the lack of utilization of these plants is due to the limited knowledge of the community, which tends to rely on hereditary information without a scientific basis. therefore, this research is important to support the identification of the pharmacological potential of local plants so that the community can optimize their utilization. table 1. types of plants utilized by the community in nagari lasi. no. family plant name part of plant used processing method utilization scientific name local name 1 lauraceae cinnamomum verum kayu manih leaf a few cinnamon leaves are boiled and then drunk lower blood sugar levels 2 costaceae costus speciousus si tawa tuber tubers are soaked in cold water, then applied to the body vaginal discharge and fever medicine 3 zingiberraceae curcuma domestica kunyik rhizome turmeric can be used by mashing it, dissolving it in hot water smooth menstruation, lower high blood pressure 4 asteraceae elephantopus scaber tapak liman leaf boil tapak liman leaves and then strain digestive disorders 5 rutaceae citrus aurantifolia swinge asam kapeh fruit orange fruit can be squeezed and the juice drunk cough 6 lamiaceae ocimum tenuiflorum ruku-ruku leaf the leaves are boiled then the boiled water is drunk fever relief 7 zingiberraceae alpinia galanga langkueh rhizome boiled with water, drunk cold medicine 8 apiaceae centella asiatica pagago leaf gotu kola leaves can be cooked as vegetables lower uric acid 9 euphorbiaceae ricinus communis jarak leaf castor leaves are moistened with water and applied to the body fever relief 10 asteraceae enhydra fluctuans cikarau leaf utilized with the leaves boiled, boiled water is drunk fever relief 11 acanthaceae andrographis paniculata sambiluto leaf the leaves are boiled then the boiled water is drunk lower blood pressure 12 poaceae panicum auritum sikumpai leaf leaves can be utilized by soaking first fever relief 624 biology, medicine, & natural product chemistry 14 (2), 2025: 621-633 no. family plant name part of plant used processing method utilization scientific name local name 13 malvaceae hibiscus rosasinensis bungo rayo flower flower is taken and squeezed with additional water, filtered, and drunk fever reducer and cough suppressant 14 menispermaceae cyclea barbata daun cincau leaf leaves are utilized by squeezing and then leaving to solidify heatiness, fever 15 zingiberraceae zingiber officinale var. amarum sipadeh rhizome boiled with water and drunk cough 16 poaceae cymbopogon citrates (l.) rendle sarai stem some lemongrass stems are boiled with water for ± 10 minutes, then the boiled water is drunk gout, febrifuge, cough relief 17 myrtaceae psidium guajava l paraweh leaf select a few leaves then crush and boil with water then strain diarrhea, cough and cholesterol lowering medicine 18 crassulaceae kalanchoe pinnata si dingin leaf the leaves can be processed by grinding them finely, then rubbing them into the head area diarrhea, fever reduction, and gastric pain 19 anonaceae annona muricata durian balando leaf soursop leaves are boiled, then the boiled water is set aside for drinking cholesterol 20 plantaginaceae plantago majorl. daun sendok leaf leaves can be utilized by grinding then applying to the skin uric acid 21 apocynaceae catharanthus roseus tapak dara leaf utilized with the leaves boiled then the boiled water is drunk to relieve muscle pain and sore throat 22 zingiberraceae zingiber officinale var. rubrum sipadeh merah rhizome boiled with water and drunk to relieve colds, flu 23 zingiberaceae curcuma zedoaria tamu kunci rhizome boiled with water and drunk lowers stomach acid 24 clusiaceae garcinia mangostana manggih rind can be processed by mashing and dissolving in hot water lower cholesterol levels 25 phyllanthaceae sauropus androgynus katuak leaf leaves can be utilized by boiling and processed as vegetables facilitate breastfeeding 26 piperaceae piper betle siriah leaf several betel leaves are boiled for ± 10 minutes, then the boiled water is drunk medicine for bleeding gums, diarrhea, toothache 27 apiaceae apium graveolens selederi leaf utilized with the leaves boiled then the boiled water is drunk high blood pressure medication 28 caricaceae carica papaya kalikih leaf papaya leaves are boiled, then the boiled water is set aside for drinking medicine for stomach pain during menstruation 29 myrtaceae syzygium polyanthum salam leaf a few bay leaves are boiled and drunk lower cholesterol, and uric acid 30 rubiaceae morinda citrifolia l mengkudu fruit cut into small pieces and crush then drink hypertension, cholesterol, rheumatism 31 cucurbitaceae momordica charantia pario leaf several bitter melon leaves are squeezed without water, then the water that comes out of the leaves is drunk cough 32 balsaminaceae impatiens balsamina l. pacar air leaf the leaves are boiled then the boiled water is drunk flatulence 33 sapotaceae manilkara zapota sawo fruit sawo fruit can be utilized by mashing and can be eaten directly diarrhea afriani et al. – macroscopic, microscopic, and phytochemical screening analysis … 625 no. family plant name part of plant used processing method utilization scientific name local name 34 basellaceae anredera cordifolia binahong leaf the leaves are boiled then the boiled water is drunk cough 35 piperaceae piper crocatum ruiz et pav. siriah merah leaf the leaves are boiled then the boiled water is drunk hypertension, cough, ulcer 36 lamiaceae orthosiphon aristatus kumis kucng leaf the leaves are boiled for about 10 minutes, the boiled water is set aside to drink stone urination 37 asteraceae sonchus wightianus tempuyung leaf the leaves are boiled then the boiled water is drunk treats gout 38 zingiberaceae kaempferia galanga kencur rhizome kencur can be processed by pounding it, dissolving it with hot water and then drinking it diarrhea 39 piperaceae peperomia pellucida siriah cina leaf leaves can be processed by pounding then filtering and drinking the water poisoning and abdominal pain 40 malvales /columniferae muntingia calabura seri leaf the leaves are boiled then the boiled water is drunk gout and cough 41 xanthorrhoeaceae aloe vera lidah buayo leaf aloe vera is taken, then the gel is grated and filtered, then drunk medicine for burns, hot oil and chest pain 42 fabaceae moringa oleifera kelor leaf moringa leaves can be processed by pounding, dissolved in hot water diabetes medication, rheumatism 43 melastomaceae melastoma malabathricum l. sikaduduak leaf on wounds, the leaves are pounded. leaf cooking water can treat other health problems diarrhea, wounds, vaginal discharge, improve digestion, improve breast milk 44 araceae rhapidophora pinnata ekor naga leaf the leaves are boiled then the boiled water is drunk cough, anemia, soreness 45 phyllanthaceae phyllanthus urinaia linn. meniran all plant parts meniran + a spoonful of cat's whisker leaves boil 2 cups of water to 1 cup for 10 minutes kidney stones organoleptic examination organoleptic examination showed that all samples were fresh leaves or haksel, generally green. some leaves have a distinctive aroma, such as kaffir lime leaves, which contain essential oils. macroscopic examination macroscopic examination provides information on the morphological characteristics of the leaves, including shape, tip, margin, base, and leaf blade. some differences were found between observations and literature, and most were found in leaf shape, which may be caused by genetic differences, environmental factors, or sample size (wahyuni, afidah, & suryanti, 2022). microscopic examination microscopic examination is carried out to observe identifying fragments on the leaves, which characterize a plant and also support macroscopic examination (novitasari et al., 2021). there are differences between observations and literature. this can be caused by the thickness of the sample slices so that the identifying fragments are less visible, besides that genetic and environmental factors also contribute to differences in the anatomical structures observed. different genetic factors will cause different appearances and changes in this appearance depend on how much environmental changes occur in the growing environment of the plant genotype (fitrianto, samiyarsih, rohma, & dwi sasongko, 2020). 626 biology, medicine, & natural product chemistry 14 (2), 2025: 621-633 table 2. results of macroscopic and microscopic examination of medicinal plants. no. plant macroscopic microscopic 1 cinnamon leaf cinnamon leaves are single leafed, with an elliptical leaf shape, tapered leaf tips, blunt leaf bases, curved leaf bones, and flat leaf edges. the surface of cinnamon leaves is smooth. lower epidermis and parasitic type stomata (40x magnification). 2 taro leaf taro leaves are single leaves with shield-shaped leaflets, pointed leaf tips, notched leaf bases, flat leaf edges, pinnate leaf bones. frayed-type hair cover, epidermis and stomata (10x magnification). 3 leaves of laughter the tawa has a single leaf with a lanceolate shape, which has a tapered leaf tip, blunt leaf base. epidermis (10x magnification). 4 turmeric leaf turmeric leaves are classified as single leaves with a lanceolate leaf shape, tapered leaf tips, blunt leaf bases and pinnate leaf bones. epidermis, palisade and stoma of parasitic type (10x magnification). 5 piladang leaf piladang has an ovoid leaf blade shape with pointed leaf tips, margins, notched leaf bases and pinnate leaf bones. on the leaves there are glandular hairs with an elongated shape and contain a purple substance (10x magnification). 6 kitolod leaf kitolod leaves are single-leafed with an inverted lanceolate leaf shape, pointed leaf tips, serrated leaf edges, and pinnate leaf bones. epidermis, palisade, stomata and trichomes (10x magnification). 7 tapak liman leaf tapak liman leaves are single leaf type with an ovoid shape, rounded leaf tips with serrated leaf edges and notched leaf bases. transport bundles have spiral-type thickenings, animocytic-type stomata and trichomes (10x magnification). 8 patik mas leaf patik mas leaves are single-leafed with tapered leaf tips, tapered leaf bases, flat edges and pinnate leaf bones. stomata with animocytic type (10x magnification). 9 pandan leaves pandanus is a single leaf with a ribbon shape, with a tapered leaf tip with flat leaf edges and parallel leaf bones. upper epidermis, palisade and parasitic type stomata, trichomes (10x magnification). 10 kaffir lime leaves kaffir lime has compound leaves with leaflets with an ovoid shape. kaffir lime leaves have blunt tips and bases, serrated leaf edges. the surface of the leaf is smooth and darker in color than the lower surface of the leaf. stomata with animocytic type (10x magnification). 11 lime leaf lime leaves are compound type leaves having an elliptical shape, blunt leaf tips, margins, and pinnate leaf bones. the surface of the lime leaf is not smooth. epidermis and stomata (10x magnification). 12 sembung leaf single leaf type with elliptical shape, pointed leaf tip, toothed leaf edge, and pointed leaf base with pinnate leaf bones. the leaf surface is hairy. epidermis with animocytic type stomata (10x magnification). 13 dahlia leaf dahlia is a compound leaf with an elliptical shape, with a pointed tip, serrated leaf edges with pinnate leaf bones. epidermis, palisade, animocytic type stomata and trichomes (10x magnification). 14 ruku-ruku leaf ruku ruku has elongated oval leaves that are pointed, blunt leaf bases and pinnate leaf bones. stomata and hair cover with scale type (10x magnification). 15 galangal leaf galangal leaves are short-stemmed single leaves with an elongated lanceolate leaf shape, pointed leaf tips, blunt leaf bases, flat leaf edges and pinnate leaf bones. there are parenchyma, stomata and epidermis (10x magnification). 16 leaves red shoots red shoot leaves are single leaves with lanceolate shape, flat edges, tapered tips, glossy leaf surface, pinnate leaf bones. upper epidermis (10x magnification). 17 gotu kola leaf gotu kola has a single leaf with a kidney-like shape with, rounded leaf tips with margins, notched leaf bases, fingered leaf bones. epidermis and stomata (10x magnification). 18 horse whip leaf horse whip has leaves that are rounded like an ovoid, the base of the leaf is narrowed, the tip is tapered, the edge of the leaf is serrated. stomata with diasitic type (10x magnification). 19 leaves flower broke a thousand it has round leaves and serrated leaf edges with a rough surface texture with pinnate leaf bones. there are stomata (40x magnification) 20 spoon leaf spoon leaves are single leaflets that are longstemmed, ovoid to lanceolate in shape, the base of the leaf is pointed, the leaf edge is flat, the leaf tip is rounded or blunt, the lower surface appears to have a prominent leaf bone. the leaf bone curves towards there are epidermis and stomata of anisocytic type (10x magnification). afriani et al. – macroscopic, microscopic, and phytochemical screening analysis … 627 no. plant macroscopic microscopic the tip. 21 castor leaf castor leaves are single-leafed. the leaf shape is heart-like. the leaf bones are fused. leaf tip is pointed, leaf margin is serrated. animocytic type stomata (10x magnification). 22 cikarau leaf cikarau has oval leaves with pointed leaf tips and serrated leaf edges. stomata with hatler cell shape (40x magnification). 23 jali leaf jali jali leaves have a pointed tip, with flat leaf margins, with an elongated leaf shape, rough leaf surface and the main leaf bone protruding on the leaf ridge. epidermis, wood vessels and stomata of diasitic type (10x magnification). 24 dragon tail leaf dragon tail leaf is a single leaf with an elongated oval shape, divided leaves, has an incision and a tapered leaf tip. upper epidermis (10x magnification). 25 sambiluto leaf sambiluto has leaves in the form of sheets, lanceolate oval leaf shape, pointed leaf base, flat, tapered leaf tip. lower epidermis with stomata and hair cover (10x magnification)) 26 sugarcane leaf sugarcane plants have leaves that are parallel leaf bones, arrow-shaped like lancets and the surface of sugarcane leaves is rough. epidermal cells, fan cells, stomata with hatlershaped closing cells (10x magnification) 27 lantana leaf lantana has round, egg-shaped leaves, a pointed leaf base, a pointed leaf tip, serrated leaf margins and a rough upper leaf surface. stomata and covering hairs (40x magnification) 28 purple liver leaf purple heart has leaves that are oblong or elliptical, with pointed leaf tips, the upper surface of the leaf is dark purple while the lower part is light purple. purple heart leaves are covered by soft downy hairs. parenchyma and pigment cells (40x magnification). 29 grapefruit leaves grapefruit has ovoid leaves with blunt leaf tips, flat leaf edges, pinnate leaf bones, leaf surface color which at the top is more intense green than the bottom. sclerenchyma (10x magnification). 30 sikumpai leaf sikumpai has oval leaves and serrated leaf margins, blunt leaf tips, serrated leaf margins. stomata, epidermis, and collenchyma (40x magnification). 31 the leaves of the botanical flower hibiscus has single leaves that are ovoid, serrated leaf edges with pointed leaf tips, blunt leaf bases and pinnate leaf bones. epidermis and transport bundles (40x magnification). 32 leaves of grass jelly grass jelly leaves have an oblong leaf type with an oval shape. leaf edges are not serrated, leaf bones are pinnate, leaf tips are pointed. transport bundles, collenchyma, parenchyma (40x magnification). 33 white ginger leaf ginger leaves have lanceolate leaves with sharp tips, pinnate leaf bones of a lighter green color that is clearly visible. the tip of the leaf is pointed, while the base is blunt. epidermis and stomata of the parasitic type (40x magnification). 34 lemongrass leaf lemongrass has long leaves with parallel leaf bones, tapered leaf tips, hairy top surface. epidermis with hatler-type stomata (40x magnification). 35 guava leaf guava has single leaves, elongated round leaves, rounded leaf bases, flat edges, pointed tips, slightly slippery leaf upper surfaces, and pinnate leaf bones. epidermis and stoma (40x magnification). 36 cocor duck leaf chillies have thick, juicy single leaves with serrated leaf margins. the leaf bones are pinnate. blunt leaf tip walls are rippled and stomata are animocytic (40x magnification). 37 soursop leaf soursop has a single leaf with an oval or elongated shape, pointed base, flat edges, tapered leaf tips, pinnate leaf bones, the mother leaf bone is clearly visible. sclerenchyma (10x magnification). 38 pumpkin leaf pumpkin has a single leaf, the shape of the leaf is round, the edges of the leaf are rippled, while the base of the leaf is rounded and hairy. the bones of pumpkin leaves are fingered. transport bundles, xylem and phloem, epidermis, trichomes, and stomata (10x magnification). 39 nona leaves eat betel miss eating betel has a single leaf, elongated ovate in shape, pointed at the tip, flat leaf margins. epidermis and trichomes (10x magnification). 40 lavender leaf lavender has an oval leaf shape, serrated leaf edges, pinnate leaf bones. trichomes (40x magnification). 41 jelantir leaf jelantir has a single leaf that is shaped like an oval, with pinnate leaf bones. epidermis, trichomes, stomata (10x magnification) 628 biology, medicine, & natural product chemistry 14 (2), 2025: 621-633 no. plant macroscopic microscopic 42 ajeran leaf ajeran has a lanceolate shape, with a hairy surface, and serrated leaf margins. leaf bones are pinnate, leaf bases are blunt. stomata, epidermis and transport bundles, xylem and phloem (40x magnification). 43 tapak dara leaf tapak dara has a single leaf with a shiny leaf surface. the leaf bones are pinnate and the leaf edges are flat. stomata of anomocytic type (40x magnification). 44 sintrong leaf sintrong has oval-shaped leaves with pinnate leaf bones, serrated leaf edges. the tip of the leaf is tapered. stomata of anomocytic type (40x magnification). 45 aloe vera leaf tongue-in-law has sword-like leaves, flat yellow leaf edges and pointed leaf tips and fibrous leaves and epidermis and stomata are animocytic type (40x magnification). 46 insulin leaf insulin leaves have single and alternate leaves, ovoid leaf shape, serrated edges, tapered leaf tips, pinnate leaf bones, hairy leaf surfaces. epidermis and stomata (10x magnification). 47 long bean leaves long beans have compound leaves, oval and alternate, flat leaf edges, blunt or rounded leaf bases, sharp leaf tips and pinnate leaf bones. animocytic-type stoamata and vessel tissue (10x magnification). 48 siamese pumpk in leaf japan has round leaves with rippled edges, rounded leaf bases and hairy leaf surfaces. with fingered leaf bones vascular bundles and animocytic-type stomata (10x magnification). 49 corn leaf corn has elongated leaves, leaf bones that are parallel to the mother leaf bone, hairy leaf surface. epidermis, vascular network and hatler-type stomata (10x magnification). 50 chinese betel leaf chinese betel has a single leaf, with a heart shape and a smooth leaf surface, tapered leaf tip with flat leaf edges. vascular bundles, stomata with chloroplasts (10x magnification). 51 red ginger leaf red ginger has lance-shaped leaves, with pointed leaf tips, pointed leaf bases, pinnate leaf bones, and flat leaf edges. vascular bundles and parenchyma (10x magnification). 52 white temu leaf temu putih has elongated lanceolate leaves with a purple-red color along the central bone, pinnate leaf bones, pointed leaf tips and flat leaf edges. polygonal epidermis and parasitic type stomata (40x magnification). 53 chickpea leaf chickpeas have oval-shaped leaves, pointed leaf tips, flat leaf edges, hairy or fine hair and pinnate leaf bones. stomata with chloroplasts and trichomes (40x magnification). 54 cayenne pepper leaves cayenne pepper has a single leaf type that is ovoid with a pointed tip and flat leaf edges and pinnate leaf bones. stomata with animotic type (40x magnification) 55 red asoka leaf asoka has oval-shaped leaves, with a glossy leaf surface, flat leaf edges and pinnate leaf bones. epidermis and animocytic type stomata (magnification 40x). 56 mangosteen leaf mangosteen leaves have a single leaf type, flat leaf edges, shiny upper and lower leaf surfaces, clear leaf capitals and pinnate leaf bones. stomata with parasitic type (magnification 40x). 57 7 jurai bean leaves seven 7 beans have a compound leaf type, flat edges, tapered tips, downy, pinnate bones. lower epidermis with animocytic type stomata (magnification 40x). 58 adam's leaf of eve adam hawa has single-leafed, oval-shaped, pointed tip, flat leaf edges. the leaf bone type is parallel. parenchyma (40x magnification). 59 katuk leaf in katuk leaves, the leaf blade is an elongated ovoid leaf, the base of the leaf is flat, the tip is tapered, the leaf blade is pinnate with the leaf blade on the lower surface protruding. cell wall (40x magnification). 60 coffee leaves coffee has oval-shaped leaves, the base and tips of the leaves are tapered. the leaf bone type is pinnate. cell wall (40x magnification). 61 cocoa leaf cocoa has a single leaf type with a pointed leaf base, pointed leaf tip, flat leaf margins, pinnate leaf bones and has one leaf mother running from the base to the tip of the leaf and is a continuation of the petiole. lower epidermis, collenchyma and stomata (40x magnification). 62 betel leaf betel has leaves that are shaped like an ovoid, the base of the leaf is heart-shaped or slightly rounded, the edge of the leaf is flat, the tip of the leaf is pointed. parenchyma (40x magnification). afriani et al. – macroscopic, microscopic, and phytochemical screening analysis … 629 no. plant macroscopic microscopic 63 hanjuang leaf hanjuang has single leaves that are lanceolate in shape, pinnate in arrangement, smooth surface, rippled edges, and pointed leaf tips and bases. parenchyma and collenchyma (10x magnification). 64 breadfruit leaf sukun has a pointed leaf base, deeply pinnate sharing edges, pointed leaf tips, large upper and lower surfaces, pinnate leaf bones. stomata (40x magnification). 65 cockscomb leaf cockscomb has a single leaf, has an ovoid to elongated leaf blade shape, leaf edges are choppy, has a pointed leaf tip, has a pinnate leaf bone, has a smooth texture. parenchyma, trichomes and stomata (40x magnification). 66 rose leaves roses have compound leaves, which are small and elongated and have tapered and serrated edges. sclerenchyma (40x magnification). 67 jasmine leaf jasmine has ovoid leaves, with pointed leaf tips, rounded leaf bases, flat leaf edges, pinnate leaf bones, prominent on the lower surface of the leaf. stomata with animotic type (40x magnification). 68 peanut leaf groundnuts have compound leaves. oval leaf shape, blunt tip and base. the leaf bones are pinnate and flat-edged. stomata with animotic type (40x magnification) 69 leek leeks have elongated, hollow, pipe-like leaves, and tapered leaf tips. animocytic type stomata with chloroplasts (magnification 40x). 70 celery leaf celery has a single leaf blade, obliquely rhombic or elongated ovate, serrated leaf margins, both surfaces rough. the leaf bones are pinnate. hatler type stomata (40x magnification). 71 mint leaves mint has a round or lanceolate shape, pointed leaf tips, with a serrated and downy leaf surface. the leaf bones are pinnate. cell wall (40x magnification). 72 eggplant leaves eggplants have leaves that are elliptical and tapered at the tip. the base of the leaf is tapered and the sides are incised. stomata, trichomes and sclerenchyma (40x magnification). 73 strawberry leaf strawberries have a round shape. strawberry leaves have a compound leaf structure. the edges of strawberry leaves are serrated. the upper surface of strawberry leaves is green and downy. parenchyma (40x magnification). 74 boroco leaf boroco has elongated ovate leaves, sharp tip, almost flat smooth serrated edges. stomata (40x magnification). 75 leaf paper flower paper flowers have almost oval-shaped leaves, but the tips are not sharp, pinnate leaves with leaf bones that can be one or five in number. leaves have flat edges. stomata and parenchyma (40x magnification). 76 kapok leaf kapok leaves have a compound leaf type, with a fingered leaf bone arrangement, a pointed leaf base, and a lanceolate leaf shape. stomata of animocytic type (magnification 40x). 77 jackfruit leaf jackfruit has single-type leaves, with an oval shape, grassy leaf base, tapered leaf tip and flat leaf edges. parenchyma (40x magnification). 78 papaya leaf papaya has a single leaf, with parallel arrangement of bones, rounded leaf shape, pointed leaf tip. parenchyma and collenchyma (40x magnification). 79 star fruit leaves belimbing wuluh has compound leaves, with pinnate arrangement, ovoid leaf shape, pointed tip, rounded base, and flat leaf edges. sclerenchyma (10x magnification). 80 basil leaves basil has leaves with ovoid strands, slightly pointed bases, slightly serrated leaf edges, pointed leaf tips, pinnate leaf bones, and both leaf surfaces are slightly rough. stomata of animocytic type (magnification 40x). 81 mustard leaf sweet mustard has oval-shaped leaves. the leaf bones on mustard are pinnate. stomata of animocytic type (magnification 40x). 82 spinach leaf spinach has elongated oval-shaped leaves, with tapered ends. spinach leaf bones are pinnate, spinach leaf edges are flat. stomata of animocytic type (magnification 40x). 83 kale leaves kale has a leaf shape like an arrow head, the tip of the leaf is pointed. parenchyma and parasitic type stomata (10x magnification). 84 lettuce leaf lettuce has round leaves. green lettuce leaves have wide and strong petioles, and pinnate leaf bones. vascular bundles, parenchyma, collenchyma, animocytic type stomata (10x magnification). 85 kecipir leaf star beans have leaves that are ovoid with pointed tips, flat leaf edges. stomata of animocytic type (magnification 40x). 630 biology, medicine, & natural product chemistry 14 (2), 2025: 621-633 no. plant macroscopic microscopic 86 mango leaf mangoes have lance-shaped leaves, the edges of the leaves are flat. the leaf bones are pinnate. vascular bundles and stomata (10x magnification). 87 red guava leaf red guava has elongated leaves, with blunt leaf tips and bases and flat leaf edges. lower epidermis, parenchyma, collenchyma, animocytic type stomata (10x magnification). 88 banana leaf bananas have leaves that have a lanceolate shape elongated with rounded leaves notched leaf bases, flat leaf edges, pinnate leaf bones. stomata of animocytic type (magnification 40x). 89 leaf frangipani flower frangipani flowers have single-type leaves, lanceolate, tapered leaf base, pinnate leaf bones, flat leaf edges. stomata of animocytic type (magnification 40x). 90 avocado leaf avocados have single leaves, oval to elongated egg-shaped, pointed base, flat edge, tapered tip, pinnate leaf bones. vascular tissue (10x magnification). 91 crown leaf mahkota dewa has single leaf type leaves, with a lanceolate shape, pinnate leaf bones and smooth leaf surfaces and flat leaf edges. epidermis, palisade and stomata with hatler shape (magnification 40x). 92 bandotan leaf bandotan has ovate leaflets, pinnate leaf bones, both leaf surfaces rough, flat leaf bases, serrated edges, and pointed tips. parenchyma and trichomes (40x magnification). 93 bay leaves bay leaves have a single leaf with an oval leaf shape with a pointed leaf base, flat leaf edges, pinnate leaf bones. vascular bundles and stomata (40x magnification). 94 noni leaf noni leaves are oval in shape, and the base of the leaf is pointed. the leaf edges are flat, the leaf bones are pinnate. the leaf surface is smooth. trichomes, stomata, vascular bundles (10x magnification). 95 bitter melon leaves bitter melon has single leaves, oval in shape, with a heart-shaped base. the leaf bone is fingered, the leaf tip is pointed. parenchyma (40x magnification). 96 sweet leaves star fruit has ovoid leaves, with pointed tips and rounded bases. star fruit leaves include compound leaves, star fruit leaves have flat leaf edges, star fruit leaves have pinnate leaf bones. vascular bundles, epidermis and stomata (10x magnification). 97 water leaf water henna leaves have a lanceolate shape, with tapered leaf tips and pointed leaf bases. the edges of the leaves are serrated, which are nicks that do not change the shape of the leaf and the leaf bones of water henna are pinnate. stomata (10x magnification). 98 sawo leaf sawo has ovoid leaves, blunt at the tip, tapered at the base, and pinnate at the leaf blade. stomata of animocytic type (magnification 40x). 99 binahong leaf binahong has ovate leaves, pinnate leaf bones, and tapered leaf tips. the base of the leaf is notched. stomata of animocytic type (magnification 40x). 100 red betel leaf red betel has a single leaf, ovoid, heart-shaped or slightly rounded base, slightly flat leaf edge, pointed tip, curved leaf bones. stomata with animocytic type (magnification 40x). 101 red leaf the leaves of the pomegranate plant are single leaves with short stalks. the shape is oval with a sharp base and blunt tip. the edges of the leaf are flat with pinnate leaf bones. vascular bundles, and trichomes (40x magnification). 102 cat whisker leaf cat whiskers have an elongated rhombus shape or like a spear tongue, sharp tip, irregular rough serrated strands, tapered leaf tips and leaf bases, fine pinnate leaf bones and little branching. sclerenchyma and stomata (40x magnification). 103 jambu bol leaf jambu bol has a single leaf type, lanceolate in shape, and has prominent leaf bones on the leaf surface. the leaf bones are pinnate. trichomes (10x magnification). 104 tempuyung leaf tempuyung leaves are lanceolate, the base of the leaf is narrowed, the edge is serrated, the tip is blunt. trichomes and stomata (10x magnification). 105 mouse leaf mouse caladium has round leaves with heart shaped pointed tips. thin-leafed. flat leaf edges, and flat leaf tips. vascular bundles, parenchyma (40x magnification). 106 israeli grass leaf israeli grass has pointed leaf tips, flat leaf edges, tapered leaf bases, pinnate leaf bones, rough leaf surfaces and this grass is single leaf. epidermis and parenchyma (40x magnification). afriani et al. – macroscopic, microscopic, and phytochemical screening analysis … 631 no. plant macroscopic microscopic 107 cassava leaf cassava has a single leaf with a finger-shaped leaf bone type, with flat leaf edges. collenchyma, parenchyma and stomata (10x magnification). 108 kanyere leaf kanyere has oval-shaped, flat leaf margins, pinnate leaf bones and blunt leaf bases. trichomes, parenchyma and collenchyma (magnification 40x). 109 clover leaf clover has heart-shaped leaves with flat edges, pinnate leaf bones and pointed leaf tips. vascular bundle (40x magnification). 110 dragon scale leaf dragon scales have an oval shape. dragon scale leaves are thick or fleshy, leaf margins are flat, leaf bases are pointed, leaf tips are blunt. sclerenchyma (40x magnification). 111 sambang getih leaf sambang getih has a single leaf, the leaf blade is ovoid, the leaf tip is pointed. the edges of the leaves are serrated and the leaf bones are pinnate. pigment cells, covering hair (40x magnification). 112 pandan suji leaf pandan suji has a lanceolate shape and pointed leaf tips, flat leaf edges and parallel leaf bones. cell wall (40x magnification). 113 purple badge leaf purple badger has an oval leaf shape. leaf bones are pinnate, leaf tips are tapered and leaf bases are tapered. vascular bundles, stomata with animocytic type (magnification 40x). 114 pagoda leaf pagoda flower leaves are single leaf, heart-shaped, pointed tip, incised base, curved bones. vascular bundles and vascular bundles (10x magnification). 115 rambutan leaf rambutan has ovoid leaves, flat leaf edges, pointed leaf tips and bases, pinnate leaf bones. there are stomata (magnification 40x). 116 durian leaf durian has oval-shaped leaves, rounded leaf bases, tapered leaf tips, smooth leaf surfaces, pinnate leaf bones. parenchyma and trichomes (10x magnification). 117 kersen leaf kersen has leaves with pinnate bones, oval shape and serrated edges. parenchyma and trichomes (10x magnification). 118 ceplukan leaf ceplukan leaves are ovoid, with a pointed base, non-sharp serrated edges, pointed to tapered tips, pinnate leaf bones, the mother leaf bone is clearly visible. animocytic type vascular bundles and stomata (10x magnification). 119 aloe vera leaf aloe vera is a single leaf, the leaf is thick, the leaf is fleshy, the base of the leaf is blunt and flat, the edge is serrated, the tip is tapered, the base is blunt. epidermal cell walls, anomocytic type stomata (magnification 40x). 120 kencur leaf kencur has leaves with a wide round shape, tapered leaf tips and rounded leaf bases, pinnate leaf bones, flat leaf edges. stomata with animocytic type (magnification 40x). 121 lemon leaf lemon has a single leaf type, oval shape, tapered tip and base. parenchyma, vascular bundles, animocytic type stomata with chloroplasts (magnification 40x). 122 meniran leaf meniran has compound leaves, small leaves, ovoid in shape, pointed leaf base, flat edges, tapered leaf tips. the leaf bones are pinnate type. parenchymal tissue (10x magnification). 123 srikaya leaf srikaya has single leaves, the leaves are elongated elliptical lanceolate, blunt-tipped, flat leaf edges, and shiny leaf surfaces. the leaf bones are pinnate. parenchyma and stomata (10x magnification). 124 saga leaf saga leaves have an oval-shaped compound leaf type, blunt slightly rounded leaf tips and bases with flat leaf edges. saga leaf bones are pinnate type. trichomes (40x magnification). 125 areca leaf areca nut has rosette-shaped, compound pinnate leaves with serrated leaf tips. leaf edges are flat. epidermis and collenchyma (40x magnification). 126 basil leaf basil has ovoid leaves, pointed leaf base, flat edge, tapered tip, pinnate leaf blade, slightly rough surface, prominent leaf blade mother. epidermis and vascular bundles (40x magnification). 127 senduduk leaf single leaves with lanceolate leaves, blunt leaf bases, pointed leaf tips, flat leaf edges, hairs on the upper surface of the leaves and curved leaf bones. pigment cells, parenchyma, trichomes epidermis (10x magnification). 128 tomato leaves tomatoes are compound-leaved with pointed tips, with blunt leaf bases, pinnate leaf bones, on the leaves have fine hairs. trichomes, stomata, calcium oxalate crystals epidermis (40x magnification). 129 ketapang leaf ketapang has breech ovoid leaves, flat leaf edges, blunt leaf tips and bases. the leaf bones are pinnate. collenchyma and stomata of animocytic type (magnification 40x). 130 peperomia watermelon leaf peperomia watermelon is a single leaf with an ovoid leaf shape, tapered leaf tip, rounded leaf base, flat leaf edges and curved leaf bones. cell walls and stomata (10x magnification). 632 biology, medicine, & natural product chemistry 14 (2), 2025: 621-633 no. plant macroscopic microscopic 131 gamal leaf gamal leaves are compound leaves with an elliptical shape, with pointed leaf tips, flat edges. parenchyma, epidermis (magnification 40x). 132 sri leaves of sustenance sri rezeki is a single lanceolate leaf with tapered leaf tips, flat leaf edges, pinnate leaf bones and pointed leaf bases. cell wall (40x magnification). 133 sri gading leaf sri gading has single ovate leaves with tapered leaf tips, flat leaf margins, pinnate leaf bones and notched leaf bases. epidermis and stomata of animocytic type (10x magnification). 134 rimbang leaf rimbang is a single leaf with an ovoid leaf shape, pointed leaf tip, notched leaf base. on the surface of rimbang leaves there are hairs. star-shaped trichomes (10x magnification). 135 mulberry leaf mulberry leaves are oval in shape, with serrated leaf edges, pointed leaf tips, blunt leaf bases, and pinnate leaf bones. collenchyma, trichomes and stomata (10x magnification). 136 syngonium leaf syngonium is a single leaf shaped like an arrow with a pointed leaf tip, eared leaf base, flat leaf edge. epidermis (40x magnification). 137 tasbih leaf the leaves of the tasbih flower have single leaves with an elliptical shape, pointed leaf tips, pinnate leaf bones, and blunt leaf bases. parasitic type parenchyma and stomata (magnification 40x). 138 anthurium andraenum leaf anthurium andraenum is single-leafed with an arrow-like shape, pointed leaf tips, flat leaf edges, and eared leaf bases. parenchyma (40x magnification). 139 sweet potato leaves sweet potatoes have leaves with tapered tips, flat leaf edges, notched leaf bases and pinnate leaf bones. parasitic type vascular bundles and stomata (10x magnification). 140 moringa leaf moringa leaves are compound leaves with an elliptical shape, with blunt leaf tips and bases, flat leaf edges and fingered leaf bones. vascular bundles and stomata (10x magnification). 141 cucumber leaves cucumber has ovoid leaves with tapered leaf tips, serrated leaf edges, and notched leaf bases and leaf bones. epidermis and stomata (10x magnification). 142 japanese papaya leaf japanese papaya has leaves that are tapered at the ends with fingered leaf bones. stomata of animocytic type (magnification 40x). 143 telang flower leaf telang flower leaves have an elliptical leaf shape with blunt leaf tips, flat edges, pointed leaf bases and pinnate leaf bones. parenchyma (40x magnification). 144 brucea javanica leaf brucea javanica is a lanceolate compound leaf with tapered leaf tips, serrated leaf edges, and pinnate leaf bones. collenchyma, there are stoma (magnification 40x). 145 kenikir leaf kenikir has compound leaves, with pointed tips, flat leaf margins, and fingered leaf bones. stomata of animocytic type (magnification 40x). 146 begonia rex leaf begonia rex is a single leaf with a pointed tip, finely serrated leaf margins, and notched leaf bases. cell wall (40x magnification). 147 neoregelia leaf neoregelia is a leaf with a rosette shape, the tip of the leaf is spiny. vascular bundles and stomata (10x magnification). 148 kirinyuh leaf kirinyuh has leaves with a triangular shape, pointed leaf tips, rompang or flat leaf bases, serrated leaf edges, and curved leaf bones. stomata of animocytic type (magnification 40x). 149 draceana fragrans leaf draceana fragrans has ribbon-shaped leaves, with tapered leaf tips, flat leaf margins and parallel leaf bone type. epidermis and stomata (40x magnification). 150 nasturtium leaf nasturtium has leaves with an ovoid shape, flat leaf edges, rounded leaf bases and tips with fingered leaf bones. stomata of animocytic type (magnification 40x). phytochemical screening phytochemical screening identifies the content of bioactive compounds using specific reagents. results showed the presence of bioactive compounds in 150 plant samples. the test results showed that 129 samples contained flavonoids, 115 samples contained phenolics, 119 samples contained saponins, 78 samples contained alkaloids, 75 samples contained steroids, and 21 samples contained triterpenoids. these results strengthen the potential of medicinal plants in nagari lasi, especially those containing flavonoids, phenolics, and saponins, which are known to have important pharmacological afriani et al. – macroscopic, microscopic, and phytochemical screening analysis … 633 activities such as antioxidants, anti-inflammation, and immunomodulators. conclusions this study identified 45 plants utilized by the nagari lasi community as traditional medicine, with the zingiberaceae family as the most dominant (13%). leaves are the most commonly used plant part (69%), and the most common processing method is boiling (58%). 105 other plants were found to have medicinal potential but not yet utilized, mainly from the asteraceae family (9.52%). the active compounds detected, such as flavonoids, phenolics, and saponins, showed high pharmacological potential, including as antioxidants and anti-inflammatories. this study provides a scientific basis for developing medicinal plants in nagari lasi and highlights the importance of education and further research to utilize local biodiversity as a sustainable source of traditional medicine. acknowledgements: thank you to the people of lasi village who have warmly welcomed us during the research. we also thank you for your valuable comments and suggestions on this research. competing interests: the authors declare that there are no competing interests. references cahyaningsih, ria, magos brehm, joana, & maxted, nigel. 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(2020). di ff erent extracts from fibigia clypeata,. mdpi, 9. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 585-590 | doi: 10.14421/biomedich.2025.141.585-590 issn 2540-9328 (online) free radical scavenging and total antioxidant capacity of combined methanol leaf extract of solanum americanum and polyalthia longifolia chidimma lilian asadu1*, ikechukwu robert uroko2, obiora celestine ugwu3 1department of medical biochemistry, college of medicine esut, parklane, nigeria. 2college of natural sciences, micheal okpara university of agriculture, umudike, nigeria. 3department of pharmaceutical sciences, enugu state university of science and technology (esut) agbani. nigeria. corresponding author* chidinma.asadu@esut.edu.ng manuscript received: 23 march, 2025. revision accepted: 18 september, 2025. published: 26 september, 2025. abstract the present investigation highlights the phytochemical composition and free radical scavenging activity of methanol leaf extract of solanum americanum and polyalthia longifolia. the fresh leaves of s. americanum and p. longifolia were air-dried, milled into powder and macerated into absolute methanol. the extract was subjected to phytochemical screening and in-vitro antioxidant activity which was compared with that of standard ascorbic acid. the qualitative quantitative phytochemical screening results constitutes flavonoids (43.05 mg/g), phenols (71.93 mg/g), alkaloids (34.11 mg/g), terpenoids (18.09mg/g), saponin (5.47 mg/g) and tannins (26.17 mg/g) all of which are known for their therapeutic properties. the antioxidant activity was evaluated using the dpph (2, 2-diphenyl-1-picrylhydrazyl), total antioxidant capacity (tac) and frap (ferric reducing antioxidant power) assays, which showed significant percentage inhibition in dose dependent manner. the combined extract exhibited significant dpph radical scavenging activity with an ec50 value of 0.993µg/ml, highlighting their potent radical scavenging ability. furthermore, the frap assay revealed a high reducing power in the highest concentration (800ug/ml) having 79.1 µmfe2+/g, suggesting effective electron donation capabilities. the total antioxidant capacity results obtained showed that the tac for the extract was in the range of 0. 650 to 2.123 aae (ascorbic acid equivalent) for the different concentrations used. keywords: antioxidant; ascorbic acid; free radical; polyalthia longifolia; solanum americanum. introduction oxidation is an essential component of aerobic existence and our metabolism. during the process of oxidation, numerous free radicals are generated that possess an unpaired, newly formed electron. atoms of oxygen or nitrogen containing central unpaired electrons are referred to as reactive oxygen or nitrogen species (di meo, and venditti, 2020). this could be detrimental to the body and could lead to peroxidation of membrane lipids, aggressiveness of tissue membranes and proteins or injury to dna and enzyme (jomova et al., 2024). these might be connected to certain conditions like arthritis, heart artery conditions, cataracts, tumor, hiv/aids as along with age-related degenerative conditions of the brain (mohtashami, et al., 2021). currently, there is significant interest in the research of antioxidants substances primarily because of the results of the treatment impact of free radical scavengers in human health. the antioxidant activities of phenolic compounds are mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers (ivanova, et al., 2020). in addition, they possess a metal chelating agent capability (gulcin and alwasel, 2022; nwozo et al., 2023) solanum americanum, commonly known as the american black nightshade, is a versatile plant species that has garnered significant attention in the field of traditional and modern medicine. the different parts of s. americanum are known to be rich in a wide array of secondary metabolites, including alkaloids, flavonoids, saponins, and phenolic compounds (patel et al., 2021). in traditional medicine, s. americanum has been utilized for its diverse medicinal properties, with various parts of the plant being used to treat a wide range of health conditions. the leaves, for instance, have been traditionally used to treat inflammatory conditions, and anemia (cotoraci et al., 2021). the fruits and leaves have also been used to address gastrointestinal issues, such as diarrhea, and stomach ulcers (qassadi et al., 2023). beyond its medicinal applications, s. americanum has also been incorporated into traditional culinary practices in various regions. the plant's leaves have been consumed as a vegetable, providing a valuable source of essential nutrients and phytochemicals (zhang et al., https://doi.org/10.14421/biomedich.2025.141.585-590 586 biology, medicine, & natural product chemistry 14 (1), 2025: 585-590 2023; olaniyi et al., 2024). this integration of s. americanum into both traditional medicine and cuisine underscores its versatility and importance in various cultural contexts. polyalthia longifolia, commonly known as the false ashoka is a towering evergreen tree species that has been the subject of extensive research in the field of phytochemistry and pharmacology. the different parts of this plant are known to be rich in a wide array of secondary metabolites, including alkaloids, flavonoids, and phenolic compounds (aloke et al., 2022). in traditional medicine systems, p. longifolia has been utilized for its diverse medicinal properties, with various parts of the plant being used to treat a wide range of health conditions. the bark, for instance, has been traditionally used to treat fever, malaria, and skin disorders (ahmed et al., 2021), while the leaves and seeds have been used to address issues such as arthritis, gastrointestinal problems and to alleviate symptoms associated with neurological disorders (jayaraman et al., 2023; iyiola et al., 2024). several plants and plant isolates have been reported to protect free radical induced damage in various experimental models (dutta et al., 2022). previous researchers have assayed the antioxidant properties of the single extracts using different models such as nitric oxide, and hydrogen peroxide suggesting the antioxidant potentials of the individual extracts (nwabiaani et al., 2002; adaramola et al., 2017). the aim of this research is to determine qualitative and quantitative phytochemicals, in-vitro antioxidant scavenging activity of combined methanol leave extract of solanum americanum and polyalthia longifolia. materials and methods plant materials and preparations fresh leaves of solanum americanum and polyalthia longifolia used for this study were gotten and purchased from eke-agbani market in nkanu west lga, enugu state, nigeria. the plant samples were identified and documented by a botanist mr alfred ozioko at the international center for ethnomedicine and drug development nsukka, nigeria. the leaves were dried in room temperature for three weeks and thereafter ground into powder with l grinder (panasonic, mx-ac400) and taken to the pharmacognosy laboratory at enugu state university of science and technology for extraction. extraction the maceration method was employed for the extraction of the bioactive compounds from the plant materials. the fresh leaves of solanum americanum and polyalthia longifolia (600g) each were air-dried, milled into powder and macerated into absolute methanol. the mixture was agitated intermittently for 72 hours at room temperature. the resulting mixture was filtered using whatman no. 1 filter paper. the filtrate was concentrated to dryness under reduced pressure using a rotary evaporator at 40°c respectively. the extract obtained was used for phytochemical screening and evaluation of in-vitro antioxidant activity. phytochemical screening the freshly prepared extracts were qualitatively and quantitatively tested for the presence of chemical constituents. phytochemical screening of the extracts for tannin, phenol, flavonoids, saponin, alkaloids, terpenoids, glycosides and steroids concentrations were performed using standard procedures (evans, 2009; shaikh and patil, 2020; bakir çilesizoğlu et al., 2022). all phytochemical screening of the extracts was done in triplicate. measurement of free radical scavenging activity using dpph method scavenging activity on dpph free radicals by the extract was assessed according to the method reported by gyamfi et al. (1999) with slight modifications. a known volume, 2.0 ml solution of the extract at different concentrations diluted two-fold (25-400 µg/ml) in methanol was mixed with 1.0 ml of 0.3 mm dpph in methanol. the mixture was shaken vigorously and allowed to stand at room temperature in the dark for 25 min. blank solutions were prepared with each test sample solution (2.0 ml) and 1.0 ml of methanol while the negative control was 1.0 ml of 0.3 mm dpph solution plus 2.0 ml of methanol. l-ascorbic acid was used as the positive control. thereafter, the absorbance of the assay mixture was measured at 518 nm against each blank with uv-visible spectrophotometer. lower absorbance of the reaction mixture indicated higher radical scavenging activity. dpph radical scavenging activity was calculated using the equation: % 𝐷𝑃𝑃𝐻 inhibition = 100 % × ( a0 − as a0 ) ao is the absorbance of the control while as is the absorbance of the test sample. all free radical scavenging activity assays were done in triplicate. total antioxidant capacity total antioxidant capacity of the extracts was determined using the phosphomolybdate method as described by saeed et al (2012) using ascorbic acid as a standard drug. an aliquot of 0.1 ml of each of the extract and ascorbic acid at varying concentrations (10-320 µg/ml) were mixed with 1 ml of reagent solution (0.6 m sulphuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate). the tubes were capped with aluminium foil and incubated in a water bath at 95 oc for 90 minutes. after the samples had cooled at room temperature, the absorbance of each mixture was asadu et al – free radical scavenging and total antioxidant capacity of … 587 measured at a wavelength of 765 nm against a blank. a typical blank contained 1 ml of the reagent solution and incubated under the same conditions. all tests were performed in triplicate. the antioxidant capacity was estimated using ascorbic acid equivalent (aae) ferric reducing antioxidant power the ferric-reducing antioxidant power of the extract was determined by the method of benzie and strain (1999). different concentrations (25 – 800µg/ml) of the extract (0.5 ml) were mixed with 0.5 ml phosphate buffer (ph 6.6) and 0.5 ml 0.1 % potassium hexacyanoferrate, followed by incubation at 50oc in a water bath for 20 minutes. after incubation, 0.5 ml of 10 % tca was added to terminate the reaction. the upper portion of the solution (1 ml) was mixed with 1 ml of distilled water and 0.1 ml of 0.01 % fecl3 solution. the reaction mixture was left for 10 minutes at room temperature and the absorbance was measured at 700 nm against appropriate blank solution. all tests were performed in triplicate. a higher absorbance of the reaction mixture indicated greater reducing power. gallic acid was used as a positive control. then, iron colour reagent (0.05 ml) was added to all the test tubes and mixed. the tubes were incubated in a water bath at 37oc for 10 minutes. thereafter, the absorbance of all the test tubes was measured at a wavelength of 700 nm. statistical analysis the experiments were carried out in triplicate and results are given as the mean ± standard deviation. statistical analysis was carried out using one way anova with duncan post hoc test. results preliminary phytochemical screening of the combined leave extract of s. americanum and p. longifolia revealed the presence of various bioactive components of which flavonoids and phenol were the most prominent and the results of phytochemical tests are summarized in tables 1and 2 dpph radical scavenging assay the scavenging ability of the combined extracts of s. americanum and p.longifolia assay is shown in table 3. the percentage of inhibitions (57.4, 61.5, 75.6, 86.6, 93.8 %) was increasedwith increasing concentrations of the extract. the ec50 value for dpph scavenging activity of the extract was found to be 0.993μg/ml. total antioxidant capacity (tac) the total antioxidant capacity (tac) was based on the reduction of phosphate-molybdenum (vi) to phosphatemolybdenum (v) by the combined extracts and subsequent formation of green phosphate/ mo (v) complex at acidic ph. it evaluates both water-soluble and fat-soluble antioxidants.the results obtained showed that the total antioxidant capacity for the extract was in the range of 0. 650±0.01 to 2.12±0.00aae (ascorbic acid equivalent) as shown in table 4. ferric reducing antioxidant power (frap) as shown in table 5, the combined methanol leaf extract of s. americanum and p. longifolia leaves had significantly (p<0.05) higher reducing capability in all the concentrations used for frap investigation. this is shown in the number of fe3+ions reduced to fe2+ in µmfe2+/g, which was concentration-dependent. the reducing ability of the extracts (42.1, 46.1, 52.1, 59.2, 73.3, 79.1 µmfe2+/g) was found to be significant (p<0.05) in all concentrations. a higher absorbance of the reaction mixture indicated greater reducing power table 1. the results of preliminary phytochemical screening. phytochemicalsconstituents bioavailability tannin ++ phenol +++ flavonoids ++ saponin + alkaloids +++ terpenoids ++ glycosides + steroids nd key: +low concentration, ++ moderate concentration, +++ high concentration, nd not detected table 2. the results of quantitative phytochemical screening. bioactive compounds combined methanol extract quantity (mg/g) (mean ± sd) flavonoids 43.05 ± 2.2 tannins 26.17 ±1.2 saponin 5.47 ± 3.5 alkaloids 34.11±4.8 phenols 71.93±1.4 terpenoids 18.09 ±2.9 glycosides 10.03 ±1.1 steroids 13.11±01 table 3. dpph scavenging activity on combined solanum americanum and polyalthia longifolia extract. concentration (µg/ml) % inhibition combined extract % inhibition ascorbic acid 25 57.4 53.3 50 61.5 61.6 100 75.6 72.8 200 86.6 80.0 400 93.8 93.9 ec50 0.993 588 biology, medicine, & natural product chemistry 14 (1), 2025: 585-590 table 4. total antioxidant capacity on combined solanum americanum and polyalthia longifoliaextract. concentration µg/ml mean ±sd tac (aae) 10 0.650±0.01 20 0.793±0.03 40 0.863±0.05 80 0.990±0.03 160 1.753±0.11 320 2.123±0.02 total antioxidant capacity of combined extract of s. americanum and p. longifolia. data are presented as mean value ± standard deviation sd (n = 3) table 5. ferric reducing power capacity (frap) on combined solanum americanum and polyalthia longifolia extract. concentration (µg/ml) combined methanol extractsµmfe2+/g 25 42.1±0.01 50 46.1±2.3 100 52.1±0.02 200 59.2±1.00 400 73.3±0.01 800 79.1±0.02 ferric reducing antioxidant power of combined extract of s. americanum and p. longifolia. data are presented as mean value ± standard deviation sd (n = 3) discussion s. americanum and p. longifolia are commonly used traditionally to treat many diseases whose pathogenesis are, among other factors, linked to reactive oxygen and nitrogen species (behl et al., 2021). however, information on antioxidant potentials of the combined plants that could be beneficial in the treatment and management of such diseases has not been investigated. in this study, we report the antioxidant potentials of combined leaf extract of s. americanum and p. longifolia. phytochemical screening of methanol extract of s. americanum and p. longifolia showed the presence of flavonoids (43.05 mg/g), phenols (71.93 mg/g), alkaloids (34.11 mg/g), terpenoids (18.09 mg/g), saponin (5.47 mg/g) and tannins (26.17 mg/g). phytochemicals are currently receiving increased attention due to interesting new findings on their biological activities. the phytochemicals detected in this extract might be involved in the therapeutic action of this plant part. the high phenolics and flavonoids content of this extract indicate high antioxidant potentials because the phenolics constituents can react with active oxygen radicals such as hydroxyl radical, superoxide anion radical and lipid peroxy radical (gulcin, 2020; tumilaar et al., 2024). free radicals are powerful pleiotropic distruptors of physiological function like smooth muscle relaxation, neuronal communication, prevention of platelet aggregation, and control of cell-mediated toxicity (sahebnasagh et al., 2022). certain free radicals such as nitric oxide serve multiple roles as effector molecules in various biological systems, functioning as neuronal messengers, promoting vasodilation, and exhibiting antimicrobial and anti-inflammatory effects (belenichev et al., 2024). various literature reports showed that there is high correlation between antioxidant activity and phenolics content (sotiropoulou et al., 2020; martínez et al., 2022). the presence of these biological active compounds suggests that the plant could serve as a potential source of drugs and its secondary metabolites could exert some biological activities when taken by humans. the reaction with the free radical is widely taken as a common mechanism of lipid peroxidation. radical scavengers may directly react with and quench peroxide radicals to end the peroxidation chain reactions that are crucial in pathogenesis of various diseases (engwa et al., 2022). test based on the use of dpph radicals is one of the most popular spectrophotometric methods for determination of antioxidant capacity of plant and plant based extracts because radical compounds can react directly with antioxidants. current results indicate that extract is apparently good free radicals and probably have the ability to inhibit lipid oxidation and may be beneficial in the treatment of various diseases where lipid peroxidation is important mechanism for pathogenesis. the total antioxidant capacity (tac) was based on the reduction of mo (vi) to mo (v) by the extract and subsequent formation of green phosphate/mo(v) complex at acid ph. it evaluates both water-soluble and fat-soluble antioxidants (total antioxidant capacity). the results indicate higher tac (expressed as ascorbic acid equivalent) of the methanol extract at a low concentration. it was, however, observed that the extract possesses significant total antioxidant capacity at higher concentration (table 4). the strong antioxidant capacity might be associated with the presence of the secondary metabolites. previous studies recorded that phenolic compound including flavonoids are associated with strong antioxidant activity and possess healthy benefits ((sotiropoulou et al., 2020; ofoedu et al., 2021). as shown in table 5, combined s. americanum and p. longifolia leave extract exhibited increased ferric reducing power with increased concentration. in ferric reducing antioxidant power assay, the presence of antioxidants in the extract would result in reducing fe3+ to fe2+ by donating an electron by the extract. the extract with reducing power showed that they are electron donors, reduce the oxidized intermediates and act as primary antioxidant substances (pisoschi et al., 2021). in this assay, the yellow colour of the test solution changes to various shades of green and blue depending on the reducing power of each compound present in the leave extract. the compounds with reduction potential react with potassium ferricyanide (fe3+) to form potassium ferrocyanides (fe2+), which then react with ferric chloride to form ferric ferrous complex that is asadu et al – free radical scavenging and total antioxidant capacity of … 589 greenish in colour. the amount of fe2+ complex was monitored by measuring the formation of perl's prussian blue at 700 nm. increasing absorbance at 700 nm indicated an increase in reductive ability. the higher absorbance of extract may be due to its strong reduction potential and the presence of the bioactive compounds in the extract which possess potent electron donating abilities. this assay further confirmed the antioxidant properties of the extracts observed from the dpph assay. the frap values obtained in this study was similar to the results reported by vijayalakshmi, and ruckmani, 2016 who recorded similar result for n-butanol extract of anchomanes difformis. the correlation between reducing power and dpph values could be due to the same mechanism on which these methods rely. the results clearly indicated that the extract examined can act as electron donors and react with free radicals, and convert them to more stable products, thus terminating the radical chain reaction and preventing pathological diseases. conclusion phytochemical screening of this extract revealed the presence of several secondary metabolites with known biological antioxidant activities. the extract also showed a strong antioxidant activity by scavenging dpph and fraps methods. in addition, the extract was found to contain relatively high levels of total phenolics and flavonoids, which play a major role in controlling oxidation generated by free radicals. conflict of interest: the authors declare that they have no conflict of interests. references ahmed, s. r., rabbee, m. f., roy, a., chowdhury, r., banik, a., kubra, k., ... & baek, k. h. 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(2023). ethnobotanical study on edible flowers in xishuangbanna, china. journal of ethnobiology and ethnomedicine, 19(1), 43. biology, medicine, & natural product chemistry issn: 2089-6514 volume 3, number 2, 2014 | pages: 59-63 | doi: 10.14421/biomedich.2014.32.59-63 substitution and haplotype diversity analysis on the partial sequence of the mitochondrial dna cyt b of indonesian swamp buffalo (bubalus bubalis) akhmad sukri1*, mohamad amin2, aris winaya3 and abdul gofur2 1department of biology, ikip mataram, jl. pemuda no. 59a mataram, nusa tenggara barat, indonesia 2state university of malang, jalan semarang 5 malang 65145, east java, indonesia 3university of muhammadiyah malang, jl. bandung 1 malang, east java, indonesia author correspondency*: sukri_bio04@yahoo.co.id abstract this research aims to investigate the substitution pattern of nucleotide base and haplotype diversity of indonesian swamp buffalo (bubalus bubalis) based on the mitochondrial dna cyt b partial gene sequence. 17 samples were chosen from 7 different regions with each uniquely represents indonesian biogeography which comprise aceh, riau, madiun, blitar, lombok, south borneo and tana toraja. the result of cyt b gene sequence alignment showed the presence of transition and transversion substitutions and the absence of insertion and deletion. the amount of transitions was found to be higher than that of transversions and the amount of substitution in pyrimidine was also higher than that in purine. the highest amount of transitions happened in base tc which is a silent substitution. the result of median joining network analysis showed that indonesian haplotype bubalus bubalis could be classified into 16 haplotypes which form different haplogroups unique to their geographical region. the result of median joining network analysis also indicated that the genetic relationships of swamp buffaloes (bubalus bubalis) in indonesia are highly influenced by their geographical locations. keywords: substitution, haplotype diversity, bubalus bubalis, cyt b gene introduction there are three kinds of genetic marker: morphological, biochemical, and dna marker. dna marker has some characteristics; it has a high rate of polymorphism, is abundant in amount, is uninfluenced by environment and has a rate of heritability of 100 percent. molecular marker is divided into two kinds: nuclear dna, and non-nuclear dna marker. one of the non-nuclear dna markers mostly used for molecular analyses is mitochondrial dna (surahman, 2002). there are two vantage points of mitochondrial dna: the evolution of mitochondrial dna happens through the pair substitution of a singular base (wolstenholme, 1992) and the velocity of mitochondrial dna evolution is 10 times faster than that of nuclear dna (brown et al, 1979). mitochondrial dna contains 13 protein-coding genes and one of them is cyt b protein-coding gene which is seen as a powerful marker for genetic analyses (arif & khan, 2009). due to the fact that cyt b gene is various and experiences evolution quickly, it is then widely used and is suitable for discovering variations on species level (bruford et al, 2003). besides, cyt b is also one of the best samples of mitochondrial dna of mammals (arnason & gullberg, 1996). cyt b gene has been used mostly for studying evolution and genetic relationships of mammals, such as that in the studies of cows and buffaloes (schreiber et al, 1999; kumar et al, 2007; li et al, 2007; qifa et al, 2007; tobe et al, 2010; xuan et al, 2010). in this research, the partial sequence of mitochondrial dna cyt b is used for identifying the substitution pattern and haplotype diversity of indonesian swamp buffalo (bubalus bubalis) as an actualisation for genetic inventory of indonesian swamp buffalo which is also considered as an indirect endeavour to preserve and to improve the genetic quantity and quality of the population of swamp buffaloes in indonesia. materials and method blood sample, dna isolation and pcr blood samples were taken from 17 indonesian swamp buffaloes (bubalus bubalis) from 7 sampling regions, comprising aceh, riau, madiun, blitar, south borneo, tana toraja and lombok. the swamp buffalo blood was taken through jugular vein using a venoject and vacuum tube and was then mixed with an edta material as much as 0.1 gram in order to keep the blood liquid. next, the tube containing the blood sample was labeled to mark the sample number and location of the blood sampling. the blood was then stored in a refrigerator until the dna isolation process was conducted. the process of total dna isolation was carried out using nucleospinr quickpure blood kit and was complied with some established procedures. in order to detect the result of the dna isolation, agarose gel electrophoresis as much as 0.8% was used. after that, pcr (polymerase chain reaction) was conducted using a pair of partial mitochondrial dna cyt b genes with forward primers l14841: 60 biology, medicine, & natural product chemistry 3 (2), 2014: 59-63 aaaaagcttccatccaacatctcagcat gatgaaa, and reverse primers h15149: aaactgcagcccctcagaatgatatttg tcctca (kocher et al, 1989; irwin et al, 1991). the positions of the primers are illustrated in figure 1. the process of dna amplification using pcr consisted of the reaction mixture of 2,5 µl dna template, 2,5 µl forward primers, 2,5 µl reverse primers, 12,5 µl pcr mix, and 5,0 µl dh2o. the pcr process comprised 30 cycles with the following phases: pre-denaturation phase at 930c for 30 seconds, denaturation phase at 930c for 1 minute, annealing phase at 500c for 1 minute, elongation at 720c for 5 minutes and post-elongation phase at 40c. sequencing and data analysis double-stranded dna, which is the product of pcr, was then purified and sequenced using one forward primer: aaaaagcttccat ccaacatctcagcat gatgaaa (kocher et al, 1989) using taq dye deoxy terminator cycle sequencing kit and 373s dna sequencer (perkin elmer, usa). the total of 17 cyt b gene sequences was then edited using a programme called bioedit sequence alignment editor and all the cyt b gene sequences were aligned using clustalx programme (thompson et al, 1997) with african buffaloes (syncerus caffer) as the outgroup. the results of the sequence alignment were then used for discovering the variable and conserved regions, as well as the substitution pattern of the cyt b gene sequences of the indonesian swamp buffaloes (bubalus bubalis) which was assisted by the use of mega 4 programme (kumar et al, 2007). in order to discover the haplotype diversity of the indonesian buffaloes, a median joining network analysis method was conducted using dna sp and network 4.1 programme (bandelt et al, 1999). results fragments of dna which were successfully amplified using a pair of primers l14841 and h15149 in this research were about 307 bp (anderson et al, 1981; kohcer et al, 1989; irwin et al, 1991, and tanaka et al, 1996). the achieved findings were then confirmed using blast (basic local alignment search tool) method to discover whether the gained sequences in the research matched with the cyt b gene sequences of bubalus bubalis. the result of alignments of the gene sequences with query from genbank showed the rate of sequences homologous ≥ 200 bp and the similarity rate of 98%. the high percentage of similarity indicated that the sequences gained in this research were the actual sequences of cyt b gene of bubalus bubalis. the characteristics of a cyt b gene are that it does not have any stop codon (termination codon) and it has the lowest guanine as well as the highest cytosine of all genes (avise, 1994). the translation result of the dna sequences to amino acids found that there was no stop codon found in the whole cyt b gene samples. this showed that the amplified dna sequences were the target gene sequences. the composition of nucleotide and variable region the nucleotide base composition of adenine, thymine, guanine and cytosine is consecutively 29.3%, 26.7%, 27.1% and 16.9%. the total amount of nucleotide a+t = 56% and g+c = 44%. hence, gc<at and this is congruent with what was stated by sueoka (1962), that the comparison of cc<at is relative equal to the content of gc by 40-45% in vertebrate. according to the sample and outgroup sequence alignment result, it was found that the conserved region was bigger than the variable region with the former as much as 80.13% and the latter as much as 19.87%. this finding showed that indonesian bubalus bubalis has quite bigger homologous than african buffaloes, syncerus caffer. this is because african buffaloes or syncerus caffer is within the same family with bubalus bubalis, called bovidae family, and in the group of buffalo of genus of bubalus with two different kinds of species: syncerus caffer and bubalus bubalis. figure 1. the positions of the primers l14841 and h15149 in amplifying mitochondrial dna cyt b gene sequences (source: irwin et al, 1991). this finding was supported by cai et al (2013), who studied the phylogeny among goats, cows and buffaloes species under the family of bovidae based on the sry gene sequence variations. they found that cows are classified in genus bos, goats in genus capra, while buffaloes (both syncerus caffer and bubalus bubalis) in akhmad sukri, et al. – substitution and haplotype diversity analysis on the partial sequence … 61 genus bubalus. a similar finding was also stated by maceachern et al (2009) who conducted species phylogeny reconstruction under the cluster of bovini, who found that species from genus bubalus and syncerus are under the same cluster of buffaloes. transition and transversion substitution cyt b gene has a characteristic of having the amount of transition substitution more than that of transversion (avise, 1994). this could be observed from the amount of nucleotide base which experienced transition substitutions was 17, while that which experienced transversion substitution was 6. (table 1). the nucleotide base which experienced transition substitutions was the base numbered 7, 31, 47, 64, 76, 94, 127, 148, 163, 167, 172, 223, 226, 230, 266, 277, and 288, while the nucleotide base which experienced transversion substitutions was the base numbered 43, 73, 121, 166, 220, and 259. the transition substitutions mostly took place in base tc and consecutively followed by ct, ag and ga, while the transversion substitutions mostly took place in base ac and ca and consecutively followed by base substitution tg and at (table 1). the amount of transition substitutions in pyrimidine base excelled that in purine base. it could be observed either from the substitutions of base t to c or those of base c to t which happened a lot more than the substitutions of base a to g or those of base g to a. a similar finding was reported by tamura and nei (1993) and was also supported by li et al. (2007) who proposed the taxonomy status of gayal (bos frontalis) founded on the partial sequence of cyt b gene which showed the amount of substitutions in pyrimidine base (tc) was more than that in purine base (ag). table 1. the variations of nucleotide base of cyt b gene sequences of the indonesian bubalus bubalis and of the outgroup. haplotype diversity the result of median joining network analysis showed that indonesian bubalus bubalis could be classified into 16 haplotypes according to their own biogeography (figure 2). the haplotypes form a haplogroup in accordance to the biogeography of each sample of indonesian swamp buffalo (bubalus bubalis). based on figure 2, it was known that the variations of nucleotide base between the haplotypes of the buffaloes from the same region were smaller than the variations of nucleotide base between the haplotypes of the buffaloes from different regions. this suggested that the buffaloes from the same region shared a higher rate of genetic relationships than with those from different regions. this phenomenon could be observed from the haplotypes of the buffaloes from aceh region which formed the haplogroup of aceh region (a1, a2 and a3) had relatively smaller variations of nucleotide base between themselves than with those of other distant regions (figure 2). discussions table 1 illustrates the absence of insertion or deletion but it indicates the presence of the nucleotide substitutions in 307 bp of the cyt b gene sequence of indonesian bubalus bubalis which were transition and transversion substitutions. transition substitution is a substitution between purine base (a and g) or between pyrimidine base (c and t), while transversion substitution is a substitution between purine and pyrimidine base (graur and li, 2000). cyt b gene has more transition than transversion substitutions (brown et al, 1982; avise, 1994). the same thing happened in this research as there 7 16 20 22 28 31 43 47 60 64 67 73 76 84 94 10 1 11 5 12 1 12 7 13 4 14 1 14 6 14 8 15 0 15 1 15 7 16 3 16 6 16 7 16 9 17 2 17 7 17 9 18 6 18 8 19 0 19 3 20 6 21 1 21 4 22 0 22 3 22 6 23 0 23 1 23 3 23 7 24 1 25 1 25 3 25 6 25 9 26 6 27 0 27 7 28 0 28 3 28 6 29 5 30 5 30 7 syncerus caffer c t t c c a a t t t c a t c t g a t c a g a g a c c c a a a c t t a a a c c c t c c t t t g c a g a c c a t t g a c t t a madiun (1) t . . . t g c c . c . c c . c a g g t t . t a . . g t t g . t c g c t c g a . c a t c c . . . . . . . a g . c t . t c . . madiun (2) t . . . t g c c . c . c c t c a g g t t . t a . . g t t g . t c g c t c g a . c a t c c . . . . . . . a g . c t . t c . . aceh (3) t c . g g g c c . c . c c . c . . g t t . . a . g . t t g . t . . . . . . a . c a t c c . . . . . t t a g g c a . t g . . blitar (1) t c . . t g c c . c . c c . c a . g t . a . a . . . t t g g t c . . . c . t . g a t c c a . . . . t . a g g c a . t c . . blitar (2) t c . . t g c c . c . c c . c a . g t . . . a . . . t t g g t c . . . c . t . c a t c c a . . . . t . a g . c a . t c . . riau t c . . t g c c . c . c c . c . g g t t a . a . . . t t g . t . . . . . . a . c a t c c . . . g . . . a g . c a . t c . . tator (2) t c g . t g c c g c . c c . c c g g t . a . a . . . t t g . t . . . . . . a . c a t c c . . . . a . . a g . c a . t c . . kalimantan (3) t c . . . g c c . c . c c . c . g g t . a . a . . . t t g . t . . . . . g a . c a t c c . a . . t . . a g . c a . t c g . kalimantan (1) t c . . . g c c . c . c c . c . g g t . a . a . . . t t g . t . . . . . g a . c a t c c . a . . t . . a g . c a . t c g . kalimantan (2) t c . . . g c c . c . c c . c . g g t . . . a . . . t t g . t . . . . . g a . c a t c c . a . . t . . a g . c a . t c g . lombok (3) t c . . . g c c g c . c c . c . g g t . a . a . . . t t g . t . . . . . . a g g a t c c . a a . a . . a g . c a c t c . g lombok (2) t c . . . g c c g c . c c . c a g g t . a . a g . g t t g . t . . . c . . a g g a t c c . a a . a . . a g . c a c t c . g tator (3) t c g . t g c c g c . c c . c c g g t . a . a . . g t t g . t . g . . . . a . c a t c c . a . . a . . a c . c a . t c . . lombok (1) t c . . . g c c g c . c c . c . g g t . a . a g . g t t g . t . . . . . . a g g a t c c . a a . a . . a g . c a c t c . g tator (1) t c g . t g c c g c g c c . c . g g t . . . a . . . t t g . t . . . . c . a . c a t c c . . . . . . . a g . c a . t c . . aceh (1) t c . g g g c c . c . c c . c . . g t t . . a . g . t t g . t . . . . . . a . c a t c c . . . . . t t a g . c a . t g . . aceh (2) t c . g g g c c . c . c c . c . . g t t . . a . g . t t g . t . . . . . . a . c a t c c . . . . . t t a g . c a . t g . . samples nucleotide base variations (base to-) 62 biology, medicine, & natural product chemistry 3 (2), 2014: 59-63 were 17 transition substitutions and 6 transversion substitutions. the fact that the transition substitution is higher than transversion substitution is the result of the saturation happens on the divergence levels in purine base is lower than that in pyrimidine base; this makes the frequency of a and g base to be much different than the frequency of c and t (kocher & carleton, 1997). the amount of transition substitutions which was more than the amount of transversion substitutions identified in this research is supported by winaya (2013) who found that there were 16 transition substitutions of cyt b gene sequence in some kingdoms of cows with the most amount of base substitutions of t to c base, and vice versa, from c to t base. the substitution of t to c base does not necessarily change amino acids since this substitution is silent substitution. this is supported by anderson et al. (1981), who conducted sequencing and organising the human mitochondrial genome, who remarked that the substitution of t to c base is a hidden substitution and does not change amino acids. based on the result of median joining network analysis, there were 16 haplotypes of indonesian bubalus bubalis (figure 2). haplotype is the variation of nucleotide base of a species. the 16 indonesian haplotypes bubalus bubalis identified are then classified into 7 different haplogroups: the haplogroup of borneo (k-1, k-2 and k-3), the haplogroup of aceh (a-1, a-2 and a-3), the haplogroup of madiun (m-1 and m-2), the haplogroup of riau (r-1), the haplogroup of tana toraja (t-1, t-2 and t-3), the haplogroup of lombok l-1, l-2 and l-3) and the haplogroup of blitar (b-1 and b-2). the result of median joining network analysis showed that each individual of swamp buffalo formed a haplogroup according to their own biogeography, comprising aceh, riau, borneo, madiun, blitar, tana toraja and lombok. this result indicates that the samples taken for this research are actually originated from the locations in which the samplings were conducted. however, a different finding was shown by the haplotype t-1 which was separated from the haplotype t-2 and t-3 even though it is still in the same haplogroup with the two others, the haplogroup of tana toraja. this indicates that the sample t-1 is from a different population of t-2 and t-3, although it is still in the same region (figure 2). figure 2. the haplotypes of the indonesian bubalus bubalis and the outgroup based on the cyt b gene sequences. haplotype is illustrated in different circle shapes (big = 2 individuals, small = 1 individual). the red number in the branch point shows the position of various base between haplotypes. l = the buffaloes from lombok, t = the buffaloes from tana toraja, m = the buffaloes from madiun, r = the buffaloes from riau, a = the buffaloes from aceh, k = the buffaloes from borneo (kalimantan), b = the buffaloes from blitar and sc = syncerus caffer. based on figure 2, the amount of variations of nucleotide base of each haplotype will be increased along with the improvement of the distance amount between regions or biogeography. this could be observed from the haplotype of madiun, aceh and riau which have one branch point and are separated by the variations of nucleotide base which are relatively small. nevertheless, the amount of variations of nucleotide base will get increased when the haplotype of aceh is compared with the haplotype of lombok which has a huge amount of akhmad sukri, et al. – substitution and haplotype diversity analysis on the partial sequence … 63 variations of nucleotide base and which biogeographically is very much distant between them. indirectly, the bigger the variations of nucleotide indicates the more distant the genetic relationships, and this is valid conversely. therefore, the geographical location or biogeography influences the genetic relationships between indonesian swamp buffaloes (bubalus bubalis). conclusions based on the analysis result it was found that there were transition and transversion substitutions in cyt b gene sequences of indonesian swamp buffaloes (bubalus bubalis), and there was not any insertion or deletion. also, it was found that the amount of transition was higher than transversion substitution. the result of median joining network analysis showed that there were 16 haplotypes of indonesian swamp buffaloes which formed different haplogroups based on their own region or biogeography. hence, it was concluded that the geographical location influenced the relatives rate or genetic relationships of indonesian swamp buffaloes (bubalus bubalis). acknowledgments the authors would like to thank the fund management institution of education ministry of finance of the republic of indonesia, which has funded this research (no:prj 481/lpdp/2013). references anderson, s.,bankier, a.t.,barrel, b.g.,de bruijn, m.h.l.,coulson, a.r.,drouin, j.,eperon, i.c.,nierlich, d.p.,roe, b.a.,sanger, f.,schrier, p.h.,smith, a.j.h.,staden, r.,& young, i.g. 1981. sequences and organization of the human mitochondrial genome, nature, vol 290: 457-465. arif, i.a & khan, h.a. 2009. molecular markers for biodiversity analysis of wildlife animals: a brief review. anim. biodiv and conserv. vol 32 (1): 9-17. arnason, u & gullberg, a. 1996. cytochrome b nucleotide sequences and the identification of five primary lineage of extant cetaceans. mol biol evol, 13(2): 407-417. avise, j.c. 1994. molecular markers, natural history & evolution. new york: chapman & hall. bandelts, h.j.,forster, p.,& rohl, a. 1999. median joining network for inferring intraspesific phylogenies, molecular biological evolution, 16: 37-48. brown, w.m.,george, jr.m.,& wilson, a.c. 1979. rapid evolution of mitochondrial dna. proceeding of the national academy and science of usa, 76: 1967-1971. brown, w.m.,prager, e.m.,wang, a.,& wilson, a.c. 1982. mitochondrial 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molecular systematics on fish. kocher, t.d and ca stepien (eds.). academic press: san diego. kumar, s.,nagarajan, m.,sandhu, s.,kumar, n.,behl, v.,& nishanth, g. 2007. mitochondrial dna analysis of indian water buffalo support distinct genetic origin of river and swamp buffalo, animal genetics, 38:227-232. li, s.,chang, h.,ma, g.,chen, h.,ji, d.,& geng, r. 2007. the origin taxonomic status of the gayal based on cytochrome b gene partial sequences, research journal of animal sciences, vol 1(4):114-119. maceachern, s.,mcewan, j, & goodard, m. 2009. phylogenetic reconstruction and the identification of ancient polymorphism in the bovini tribe (bovidae, bovinae), bmc genomics, vol 10: 2-17. qifa, l.,yinxia, l.,xingbo, z.,zhenshan, l.,yefen, x.,dawei, s.,xunguang, q.,ning, l.,& zhuang, x. 2007. study on the origin and taxonomic status of yak (poephagus) using cytochrome b gene of mitochondrial dna, front agric china, vol 1(3): 329-333. schreiber, a.,seibold, i.,notzold, g.,& wink, m. 1999. cytochrome b gene haplotypes characterize chromosomal lineages of anoa, the sulawesi dwarf buffalo (bovidae: bubalus sp), the american genetics association, 90: 165-176. surahman, m. 2002. peta genetika tanaman, prinsip dan aplikasinya. bul.agron, vol 30 (1): 27-30. tamura, k dan nei, m. 1993. estimation of the number of nucleotida substitutions in the control region of mitochondrial dna in humans and chimpanzees, mol. biol. evol, vol 10: 512-526. tanaka, k.,solis, c.d.,masangkay, j.s.,maeda, k.,kawamoto, y.,& namikawa, t. 1996. phylogenetic relationship among all living species of the genus bubalus based on dna sequences of the cytochrome b gene, biochemical genetics, vol 34: 443452. thompson, j.d.,gibson, t.j.,plewniak, f.,jeanmough, f.,& higgins, d.g. 1997. the clustal-x windows interface: flexible stategies for multiple sequences alignment aided by quality analysis tools, nucleic acids research, vol 25: 4876-4882. tobe, s.s.,kitchener, a.c.,& linacre, m.t. 2010. reconstructing 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2540-9328 (online) the antibacterial and antibiofilm efficacy of citrullus lanatus rind ethanol extract against aggregatibacter actinomycetemcomitans wilbert alvin genesis cokro1, priska natassya2, sheila soesanto2,* 1undergraduate student, faculty of dentistry, universitas trisakti; 2oral biology department, faculty of dentistry, universitas trisakti. jl. kyai tapa no. 1 jakarta 11440, tel. +62-21-5663232, fax. +62-21-5644270, indonesia. corresponding author* sheilasoesanto@trisakti.ac.id manuscript received: 16 march, 2024. revision accepted: 26 september, 2024. published: 01 october, 2024. abstract watermelon (citrullus lanatus) rind contains secondary metabolite compounds which exhibit antimicrobial effects against gram-positive and gram-negative bacteria. aggregatibacter actinomycetemcomitans can cause tissue damage and bone resorption, leading to aggressive periodontitis. chlorhexidine as golden standard antimicrobial mouthwash can cause tooth staining. there is a need for herbal remedies with fewer side effects as an alternative treatment for periodontitis. this study aims to determine the antibacterial and antibiofilm efficacy of c. lanatus rind ethanol extract against a. actinomycetemcomitans. this is an experimental laboratory in vitro with a post-test-only control group design. the antibacterial test was conducted using the plate count method and the antibiofilm test using the microtiter plate biofilm assay method. the samples used were ethanol extract of c. lanatus rind 3.125%, 6.25%, 12.5%, 25%, 50%, and 100%, chlorhexidine 0.2% as positive control, distilled water, and bhi-b as negative control. as a result, extract concentrations from 12.5% to 100% exhibited antibacterial effects on a. actinomycetemcomitans equivalent to chlorhexidine. during the 3-hour incubation period, extract at 25% and 100% concentrations on all incubation times showed a better antibiofilm effect than chlorhexidine. it can be concluded that ethanol extract of c. lanatus rind had the potential as an alternative antibiofilm agent against a. actinomycetemcomitans. keywords: aggregatibacter actinomycetemcomitans; antibacterial; antibiofilm; ethanol extract of citrullus lanatus rind; herbal medicine. abbreviations: a. actinomycetemcomitans: aggregatibacter actinomycetemcomitans, atcc: american type culture collection, anova: analysis of variance, bhi-a: brain heart infusion-agar, bhi-b: brain heart infusion-broth, bpsi-troa: instrument standardization testing center for medicinal and aromatic spice plants, c: celcius, cfu: colony-forming unit, c. lanatus: citrullus lanatus, micore: microbiology center of research and education, od: optical density, pbs: phosphat buffered saline, w/v: weight by volume. introduction watermelon (citrullus lanatus) is a popular fruit due to its refreshing flavour, nutritional value, and low calories (romdhane et al., 2017). it is the second most produced fruit globally (11%) following bananas (faosat, 2021). c. lanatus is grown in tropical and subtropical regions with abundant soil organic material (sorokina et al., 2021). the pulp and seeds of c. lanatus are often consumed because they contain many beneficial properties for the body. c. lanatus pulp has antihypertensive, anti diabetic, antacid, laxative, anticancer, and antimicrobial properties. moreover, c. lanatus seed shows antihypertensive, neuroprotective, anticancer, and antimicrobial activity (ismael et al., 2022). unfortunately, c. lanatus rind is considered food waste. nevertheless, it contains secondary metabolite compounds such as alkaloids, flavonoids, glycosides, phenolics, saponins, steroids, tannins, and triterpenoids, which exhibit antihypertensive, antiulcer, anticancer, and antimicrobial effects (ismael et al., 2022; kumar et al., 2020). previous studies showed that ethanol extract of c. lanatus rind demonstrated antibacterial and antibiofilm properties against gram-positive and gram-negative bacteria such as staphylococccus aureus, bacillus subtilis, streptococcus. pneumoniae, staphylococcus epidermidis, escherichia coli, klebsiella pneumoniae, and pseudomonas aeruginosa. against periodontitis, ethanol extract of c. lanatus rind could inhibit porphyromonas gingivalis and treponema denticola (egbuonu, 2015; gotama, 2022; mohammed et al., 2020). another bacteria can lead to periodontitis is aggregatibacter actinomycetemcomitans (gasner & schure, 2022). it is a facultative anaerobic gramnegative bacteria with virulence factors such as cytolethal distending toxin (cdt), leukotoxins, and lipopolysaccharides (lps). the virulence factor of a. actinomycetemcomitans can modulate the host's immune response and produce cytokines that are the main cause https://doi.org/10.14421/biomedich.2024.132.367-372 368 biology, medicine, & natural product chemistry 13 (2), 2024: 367-372 of tissue damage and bone resorption, leading to localized aggressive periodontitis (bao et al., 2018; missoum, 2020; rajendra, 2022). those virulence factors also play a role in bacterial attachment to the oral cavity surface and support biofilm formation. biofilm is a group of microorganisms, mainly bacteria, that adhere to the tooth surface and are covered in the extracellular polymer (soesanto et al., 2023). poor oral hygiene supports microbial dysbiosis, allowing the pellicle layer of biofilm to develop into pathogenic biofilm (larsen & fiehn, 2017). these bacteria interactions (quorum sensing) involve signalling molecules (autoinducers) that can regulate gene expression and support the pathogenic process of biofilm formation (oluwole, 2022). the management of periodontitis includes chlorhexidine as an adjunct with scaling and root planing. chlorhexidine is used as a golden standard antimicrobial mouthwash, but its long-term use might demonstrate adverse reactions such as tooth staining, xerostomia, and changes in taste (deus & ouanounou, 2022). hence, there is a crucial need for herbal remedies that have fewer side effects than chlorhexidine and are easily accessible, thus providing an alternative treatment option for periodontitis. to address this gap, our study aims to investigate the antibacterial and antibiofilm efficacy of c. lanatus ethanol extract rind against a. actinomycetemcomitans. this study shows potential in providing valuable insights into how herbal extracts could be used as alternative therapeutic agents in managing periodontitis. materials and methods this is an in vitro study with an experimental post-testonly control group design. the ethical clearance was exempted from the ethics committee for health research at the faculty of dentistry, universitas trisakti, jakarta (647/s1/kepk/fkg/7/2023). materials the materials used in this research were ethanol extract of c. lanatus rind at concentrations of 3.125%, 6.25%, 12.5%, 25%, 50%, and 100%. chlorhexidine 0.2% was used as a positive control. distilled water was used as a negative control for the antibacterial test and brain heart infusion-broth (bhi-b) (sigma aldrich) was used as a negative control for the antibiofilm test. methods sample preparation the ethanol extract of c. lanatus rind was obtained from the instrument standardization testing center for medicinal and aromatic spice plants (bpsi-troa) in bogor, west java, indonesia. the ethanolic extract of c. lanatus rind was prepared using the maceration method. one thousand five hundred grams of c. lanatus rind was dried at 37℃ for three days, ground into powder, soaked with 96% ethanol solution for 24 hours, filtered, and evaporated with a rotary evaporator to obtain extract at 100% concentration. serial dilution was performed using distilled water to extract at concentrations of 50%, 25%, 12.5%, 6.25%, and 3.125%. phytochemical tests qualitative phytochemical tests were performed at bpsitroa to identify secondary metabolites in the ethanolic extract of c. lanatus rind, such as alkaloids, saponins, tannins, phenolics, flavonoids, triterpenoids, steroids, and glycosides. bacterial culture a. actinomycetemcomitans atcc 29522 was acquired from the microbiology center of research and education (micore) laboratory, faculty of dentistry, universitas trisakti, jakarta. a. actinomycetemcomitans atcc 29522 were cultured on bhi-b. the culture was homogenized using a vortex (biosan) and then incubated in an anaerobic jar (oxoid) at 37°c for 24 hours. the bacterial absorbance value was measured using a microplate reader (safas) at 490 nm and equal to the mcfarland standard of 0.5 (1.5 x 108 cfu/ml). antibacterial test antibacterial test were conducted using microdilution and plate count methods. a total of 100 μl of bacterial suspension and each test solution were distributed into 96-well plates (nest biotech) and then diluted 10,000 times. each test solution was taken five μl and spread on brain heart infusion-agar (oxoid) media. bhi-a was incubated for 24 hours and then the total number of bacterial colonies was counted. this test was repeated four times. antibiofilm test this antibiofilm test was conducted using the microtiter plate biofilm assay method. 200 μl of bacterial suspension was incubated at 37℃ under anaerobic conditions for 48 hours in 96-well plates. the supernatant was removed, leaving a thin layer of biofilm then rinsed with phosphate buffered saline (pbs) (biomatik) twice. in each treatment group, five wells with a biofilm layer and two without a biofilm layer were inserted with 200 μl of each test solution and reincubated for 1, 3, and 24 hours. the wells were then rinsed with pbs, dried, fixated, and stained with crystal violet (0.05% w/v). the wells were rinsed and dried for 15 minutes, then 200 ml of 96% ethanol was added to each well. the intensity of crystal violet staining was measured in optical density (od) units using a microplate reader with a wavelength of 490 nm. data analysis the normality test was conducted using shapiro-wilk. for the antibacterial test, one-way analysis of variance cokro et al. – citrullus lanatus rind antibiofilm effect 369 (anova) and post hoc tukey hsd were used to identify significant differences among the test groups (p<0.05). kruskal-wallis test was performed for antibiofilm test, followed by post hoc mann-whitney tests (p<0.05). results and discussion antibacterial test the antibacterial test result indicates that the ethanol extract of c. lanatus rind has antibacterial properties against a. actinomycetemcomitans. there was a significant reduction in the total colony count of a. actinomycetemcomitans in all concentrations. the highest antibacterial effect was found at 100% concentration with a total number of bacteria of (2±0.5)x106 cfu/ml. extract concentrations from 12.5% to 100% exhibit antibacterial effects on a. actinomycetemcomitans equivalent to positive control. (figure 1 & 2; table 1). figure 1. antibacterial test results based on plate count method. figure 2. antibacterial test results of c. lanatus rind ethanol extract against a. actinomycetemcomitans. chlorhexidine as positive control and distilled water as negative control. table 1. post hoc test results (tukey hsd) of antibacterial test toward negative and positive control. *p<0.05; **p<0.01. 3.125% 6.25% 12.5% 25% 50% 100% c (-) <0.001** <0.001** <0.001** <0.001** <0.001** <0.001** c (+) <0.001** <0.001** 0.210 0.803 0.999 1.000 antibiofilm test the antibiofilm result showed that the ethanol extract of c. lanatus rind had antibiofilm properties against a. actinomycetemcomitans (figure 3 and table 2). compared to the negative control, there was a decrease of od in 1, 3, and 24 hours in incubation time. at 3 hours of incubation, the od decreased significantly in all concentrations (p<0.01). compared to the positive control, extract of 100% concentration at 1 hour and 24 hours incubation showed a lower od value significantly (p<0.01). figure 3. antibiofilm test results of c. lanatus rind ethanol extract against a. actinomycetemcomitans. chlorhexidine as positive control and bhi-b as negative control. 370 biology, medicine, & natural product chemistry 13 (2), 2024: 367-372 table 2. post hoc test results (mann-whitney) of antibiofilm test toward negative and positive control at 1, 3, and 24 hours of incubation. *p<0.05; **p<0.01 concentration 3.125% 6.25% 12.5% 25% 50% 100% 1-hour 0.009** 0.009** 0.009** 0.009** 0.076 0.009** c (-) 3-hours 0.009** 0.009** 0.009** 0.009** 0.009** 0.009** 24-hours 0.602 0.602 0.175 0.009** 0.009** 0.009** c (+) 1-hour 0.754 0.917 0.917 0.917 0.917 0.009** 3-hours 0.009** 0.028* 0.251 0.047* 0.347 0.009** 24-hours 0.009** 0.009** 0.009** 0.009** 0.047* 0.009** phytochemical test phytochemical test results showed that ethanol extract of c. lanatus rind contained secondary metabolite compounds such as alkaloids, flavonoids, glycosides, phenolics, saponins, steroids, tannins, and triterpenoids (table 3). table 3. phytochemical test result of c. lanatus rind ethanol extract. no secondary metabolite compound results 1 alkaloids + 2 flavonoids + 3 glycosides + 4 phenolics + 5 saponins + 6 steroids + 7 tannins + 8 triterpenoids + discussion this antibacterial test uses the plate count method due to its cost-effectiveness, simple procedures, and the ability to count bacteria without requiring special tools or materials (bankier et al., 2018). distilled water was used as a negative control since it does not affect the bacteria growth and thereby does not affect the course of the experiment (widowati et al., 2022). as a positive control, chlorhexidine 0.2% is used in this study because it is the gold standard of antimicrobial used as adjuvant therapy for periodontitis. its bacteriostatic and bactericidal properties reduce plaque and prevent periodontal disease effectively (deus & ouanounou, 2022). since this is a preliminary study, the concentration in this study ranges from 3.125% to 100%. therefore the effective concentrations for antibacterial and antibiofilm effect are currently unknown. this study showed that the antibacterial effect of c. lanatus rind ethanol extract increased along with the concentration. this result is similar to a previous study regarding the antibacterial effect of c. lanatus rind ethanol extract against streptococcus mutans using the agar well diffusion method (govindaraj et al., 2022). using the disc diffusion method, another study also showed an increase in the diameter of the inhibition zone against streptococcus mutans in line with a higher concentration of c. lanatus rind ethanol extract (ghozaly & balqis, 2022). another result of this antibacterial test was that extract concentrations from 12.5% to 100% exhibited antibacterial effects on a. actinomycetemcomitans equivalent to chlorhexidine. it means that in 100% concentration, c. lanatus was as effective as chlorhexidine against a. actinomycetemcomitans. the antibiofilm test in this study used the microtiter plate biofilm assay method since it is able to test many samples in one test, using simple equipment, and affordable prices (azeredo et al., 2017). for the negative control, bhi-b was used since it is a good medium for the bacteria to form biofilm (adame-gómez et al., 2020). the antibiofilm test in this study used an incubation period of 1, 3, and 24 hours as it represents the biofilm formation phase, starting with the pellicle formation phase in the first 2 hours, then the initial adhesion phase in 2 to 4 hours, and the maturation phase for 24 hours (martínez-hernández et al., 2023; widyarman & lazaroni, 2019). ethanol extract of c. lanatus rind began to inhibit biofilm formation at 1 hour of incubation (pellicle formation phase) with the highest od shown at extract, with a concentration of 100%. the most effective antibiofilm effect was shown at 3 hours of incubation. this means that the ethanol extract of c. lanatus rind works best against a. actinomycetemcomitans in the initial adhesion phase of the biofilm. furthermore, extracts with a concentration of 100% in all incubation periods and 25% in the 3-hour incubation period had a more effective antibiofilm effect compared to chlorhexidine. this study also indicates an increase in the antibiofilm effect in line with the concentration during each incubation period. this result is similar to the research on the antibiofilm effect of c. lanatus rind ethanol extract against escherichia coli, klebsiella pneumoniae, staphylococcus aureus, and pseudomonas aeruginosa using biofilm formation inhibition method. this result showed a smaller od number as the concentration rose (ijewereme et al., 2018). compared to incubation time of 1 and 3 hours, the decrease in od during the 24-hour incubation period indicated that the cokro et al. – citrullus lanatus rind antibiofilm effect 371 biofilm had formed completely and the resistance had increased 1,000-1,500 times stronger than the planktonic bacteria state (goswami et al., 2023). the increased activity of a. actinomycetemcomitans in the 24-hour incubation time showed the bacteriostatic effect of c. lanatus rind ethanol extract. in the maceration process, 96% ethanol was used as solvent since it is relatively less toxic than methanol. it also could extract high bioactive compounds comparable to methanol due to its high polarity for extracts with high water content (rezagholizade-shirvan et al., 2023; toupal & coşansu, 2023; utami & putri, 2020). the antibacterial and antibiofilm effect of c. lanatus rind ethanol extract due to its secondary metabolite compounds such as alkaloids, flavonoids, glycosides, phenolics, saponins, steroids, tannins, and triterpenoids. alkaloids suppress cell wall synthesis, metabolism, protein synthesis, and nucleic acids, and alter bacterial cell membrane permeability (yan et al., 2021). flavonoids can change the permeability of bacterial cell membranes, interfere with nucleic acid synthesis and bacterial metabolism (shamsudin et al., 2022). glycosides can degrade the phospholipid membrane of bacterial cells. phenolics are able to inhibit nucleic acid synthesis and damage bacterial cell proteins (widowati et al., 2022). saponins may trigger bacterial cell lysis, increase bacterial cell membrane permeability, and inhibit bacterial adhesion (khan et al., 2018). tannins also act by inhibiting the metabolism and attachment of bacteria on the surface of the host (kaczmarek, 2020). steroids and triterpenoids can suppress cell wall synthesis and disintegrate bacterial cell membranes (widowati et al., 2021). an ethanol extract of c. lanatus rind is proven to have an antibacterial and antibiofilm effect against a. actinomycetemcomitans. however, further study is needed to determine the toxicity of this extract. conclusions ethanol extract of c. lanatus rind contains alkaloids, flavonoids, glycosides, phenolics, saponins, steroids, tannins, and triterpenoids that potentially inhibit the growth and formation of a. actinomycetemcomitans biofilm in vitro. the growth of a. actinomycetemcomitans is inhibited by the lowest concentration of 3.125% and the inhibition of biofilm formation was most effective during biofilm initial adhesion phase. acknowledgements: the authors would like to express their great appreciation to mario richi, s.si, from the micore laboratory, faculty of dentistry, universitas trisakti, for his valuable assistance in this research. authors’ contributions: wilbert alvin genesis cokro, priska natassya, and sheila soesanto designed the study. wilbert alvin genesis cokro carried out the laboratory work. priska natassya and sheila soesanto analyzed the data. wilbert alvin genesis cokro and sheila soesanto wrote the manuscript. all authors read and approved the final manuscript. competing interests: the authors declare that there are no competing interests. funding: not applicable. references adame-gómez, r., cruz-facundo, i.-m., garcía-díaz, l.-l., ramírez-sandoval, y., pérez-valdespino, a., ortuño-pineda, c., santiago-dionisio, m.-c., & ramírez-peralta, a. 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(2021). research progress on antibacterial activities and mechanisms of natural alkaloids: a review. antibiotics (basel, switzerland), 10(3), 1–30. https://doi.org/10.3390/antibiotics10030318 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 397-406 | doi: 10.14421/biomedich.2024.132.397-406 issn 2540-9328 (online) hippocratea africana ethanol root extract and fractions attenuate doxorubicin-induced testicular toxicity and oxidative stress kufre u. noah, jude e. okokon* department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. corresponding author* judeefiom@yahoo.com manuscript received: 22 june, 2024. revision accepted: 30 september, 2024. published: 04 october, 2024. abstract hippocratea africana root, used locally in treating poisoning, was investigated to confirm its antidotal potential in rats. the root extract (200-600 mg/kg) and fractions; dichloromethane (dcm) and aqueous, 400 mg/kg) were evaluated for hepatoprotective activity against doxorubicin-induced testicular toxicity in male rats. testosterone level, lipid profile indices, testicular oxidative stress markers, and testis histology were used to assess the testicular protective effect of the extract. the root extract (200-600 mg/kg) and fractions, dichloromethane (dcm) and aqueous, 400 mg/kg reduced the serum levels of total cholesterol, triglycerides, ldl, and vldl that were elevated by doxorubicin. in contrast, the high-density lipoprotein (hdl) reduced by doxorubicin was increased by the extract and fractions co-administration. testosterone level, which was significantly (p<0.05) reduced by doxorubicin, was significantly (p<0.05-0.01) elevated by the root extract and fractions co-administration. the levels of gsh, gst, sod, gpx, and cat that were decreased by doxorubicin were significantly (p<0.01) elevated, and raised mda level was reduced by the root extract and fractions. histology of the testes sections of extract/fractions -treated animals showed absent/or reductions in the pathological features compared to the organotoxictreated animals. the chemical pathological changes were consistent with histopathological observations, suggesting marked testicular protective potential. the anti-toxic effect of this plant may in part be mediated through the chemical constituents of the plant. the plant, h. africana possesses anti-toxicant properties which can be exploited in the treatment of doxorubicin-related toxicities. keywords: hippocratea africana; anti-oxidant; oxidative stress; testicular-protective; antioxidant. introduction doxorubicin, an anthracycline glycoside antibiotic with a broad spectrum of activity against various human solid tumors and hematological malignancies (calabresi & chamber, 1990) but limited clinical usefulness due to its diverse toxicities, including cardiac, hepatic, hematological, and testicular toxicity (yilmaz et al., 2006). the toxic, short-lived metabolite, semiquinone form of doxorubicin, triggers the generation of reactive oxygen species (ros) through cascades of events. doxorubicin-induced cardio, hepatic, testicular, and nephrotoxicities have been attributed to ros generation, inflammatory processes, and lipid peroxidation (injac et al., 2009; kalender et al., 2005). doxorubicin has also been proposed to promote free radical generation by enhancing the activities of extra-mitochondrial oxidative enzymes such as nadph and xanthine oxidases and interfering with mitochondrial iron export (bachur et al., 1979). these free radicals cause distortion of the cell's membranes and cause organ dysfunction. research on plants that can counteract the toxic effects of doxorubicin has been ongoing, especially on h. africana (noah et al., 2023c). hippocratea africana (willd.) loes. ex engl. (celastraceae) known in english as african paddle-pod and eba enang enang’ in the ibibio language in nigeria, is a climber perennial plant distributed widely in tropical africa (hutchison & dalziel, 1973). traditionally, the plant root has been utilized in various herbal preparations to treat diseases like malaria and diabetes (okokon et al., 2006), as well as liver diseases (ajibesin et al., 2008). previous reports showed that the root extract possess antimalarial (okokon et al., 2006;2021), angioedema and antinociceptive (okokon et al., 2008), antidiabetic and hypolipidemic (okokon et al., 2010; 2022), antidiarrhoeal and antiulcer (okokon et al., 2011), nephroprotective (noah et al., 2023a; 2023b) hepatoprotective (okokon et al., 2013a;noah et al., 2023c, noah et al., 2023d), antileishmanial, cytotoxicity and cellular antioxidant (okokon et al., 2013b), antibacterial, anticonvulsant and depressant (okokon et al., 2014), genotoxic and cytotoxic (jonhnny et al., 2023). also, earlier studies had reported the presence of ᵟ-3-carene and α-terpineol (okokon et al., 2017), isolation of 1,3,7-trihydroxy-6-methoxyxanthone [isoathyriol] and 1,3,6,7-tetrahydroxyxanthone [norathyriol] (umoh et al., 2021) from ethyl acetate https://doi.org/10.14421/biomedich.2024.132.397-406 398 biology, medicine, & natural product chemistry 13 (2), 2024: 397-406 fraction. monoterpenes and sesquiterpenes have been identified in the n-hexane fraction (okokon et al., 2013a). we report testicular protective and antioxidative stress potentials of the root extract and fractions of h. africana against doxorubicin-induced testicular toxicity in male rats. materials and methods plants collection fresh root of hippocratea africana were collected in bushes in the uruan area, akwa ibom state, nigeria in november 2021. the plant was identified and authenticated by a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria. hebarium specimen was deposited at department of pharmacognosy and natural medicine herbarium, university of uyo. preparation of extract and fractions fresh root of h. africana were washed, cut into smaller pieces and dried under shade for two weeks. they were powdered using an electric grinder. the pulverised root of h. africana (hae) was soaked in ethanol (50%) for 72 h. the liquid filtrate obtained was concentrated in a rotary evaporator at 40˚c. the crude extract (20 g) was dissolved in 500 ml of distilled water and partitioned with equal volume of dichloromethane (dcm, 5 x 500 ml) till no colour change was observed, to obtain dcm and aqueous fractions. the extract and fractions were stored at 4˚c in a refrigerator until used for the experiment. animals in this study, male albino wistar rats were used. the animals were sourced from university of uyo animal house and sheltered in plastic cages. the rats were fed with pelleted standard feed (guinea feed) and given unlimited access to water. the study was approved by college of health sciences animal ethics committee, university of uyo. experimental design this study used the repeated dose model earlier described by raskovi et al. (2011) and olorundare et al. (2020), which lasted for 14 days. group i rats, which served as the untreated control, were orally pretreated with 10 ml/kg/day of distilled water. group 2 rats were given normal saline (10 ml/kg/day) but equally treated on alternate days with 1.66 mg/kg of doxorubicin hydrochloride dissolved in 0.9% normal saline administered on alternate days for 14 days. groups 3-5 rats were orally pretreated with 200 mg/kg/day, 400 mg/kg/day, and 600 mg/kg/day of hippocratea africana dissolved in distilled water 2 hours before treatment with 1.66 mg/kg of doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days, respectively. groups 6 and 7 were pretreated with 400 mg/kg of dcm and aqueous fractions, respectively. group 8 rats, which served as the positive control group were equally pretreated with 100 mg/kg/day of silymarin two hours before treatment with 1.66 mg/kg of doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days. collection of blood samples and organs after 14 days of treatment (24 hours after the last administration), the rats were weighed again and sacrificed under light diethyl ether vapour. blood samples were collected by cardiac puncture and used immediately. blood samples were collected into plain centrifuge tubes. the blood in the centrifuge tubes was centrifuged immediately at 1500 rpm for 15 mins to separate the serum at room temperature to avoid haemolysis and used for biochemical assays. the testes of the rats were surgically removed, weighed. one testis was fixed in 10 % formaldehyde for histological processes, while the other testis was briskly rinsed in ice cold 1.15% kcl solution and put in a clean sample bottle. these were stored in ice cold 0.9% nacl. evaluation of progressive motility, viability, count, and the structural abnormality of sperm the caudal piece of epididymis was isolated to retrieve the sperm samples. initially, the epididymal part was finely minced in 5 ml of physiological-saline and was incubated for 30 min at 37◦c for spermatozoa releasing of the epididymal ducts. sperm progressive motility percentage was noted through the phase-contrast microscope at 400x (kenjale, 2008). sperm viability was assessed, by eosin or nigrosin staining and accompanied by microscopic evaluation. moreover, a hemocytometer was employed to count epididymal sperm in the suspension (yokoi et al., 2003). furthermore, morphological anomalies of the head, tail, and mid piece of sperm were determined in percentage using the method of filler (1993). the apparent abnormal characteristics included (i) the size and shape of spermatozoa heads (bigor small heads) with lighter and emphasized curvature;(ii) intermediary pieces’ defects that result in untied heads; and (iii) defects of tails (short, multiple, folded, and broken tails). biochemical assays. lipid profile blood samples from the heart chamber were allowed to clot and then centrifuged at 5000 rpm to separate clear sera from the clotted blood samples. the clear samples were obtained for assays of the following biochemical parameters: serum testosterone, serum cholesterol, triglyceride and high density lipoprotein (hdl) levels of the treated rats were measured using standard colorimetric methods (tietz, 1995). these lipid parameter determinations were done noah & okokon – hippocratea africana ethanol root extract and fractions attenuate … 399 spectrophotometrically using fortress diagnostic kits® according to standard procedures of manufacturer’s protocols. serum testosterone levels were measured using the corresponding rat enzyme-linked immunosorbent assay kits (testosterone, cambridge, uk). effect of the root extract and fractions on testis oxidative stress markers the oxidative marker assays were performed on the testes homogenates of rats that were used in this study. these oxidative stress markers were used to assess the antioxidant stress potentials of the extract. preparation of testis homogenate after the rats were sacrificed humanely under inhaled diethyl ether, the testes of the rats were surgically removed and weighed. they were briskly rinsed in ice cold 1.15% kcl solution and put in a clean sample bottle. these were stored in ice cold 0.9% nacl. homogenates were made in a ratio of 1 g of wet tissue to 9 ml of 1.25% kcl by using motor driven teflon-pestle. the homogenates were centrifuged at 7000 rpm for 10 min at 4˚c and the supernatants were used for the assays of superoxide dismutase (sod) (marklund & marklund, 1974), catalase (cat) (sinha,1972), glutathione peroxidase (gpx) (lawrence & burk,1976), reduced gluthathione (gsh) (ellman,1959) & malondialdehyde (mda) content (esterbauer & cheeseman, 1990). the assays were performed on testes homogenates of rats used in this study. these oxidative stress markers were used to assess the antioxidative stress potentials of the extract. histopathological studies the excised testes fixed in 10 % buffered formalin were used for histological processes. they were processed and stained with haematotoxylin and eosin (h&e) (drury & wallington, 1980), according to standard procedures at department of chemical pathology, university of port harcourt teaching hospital, port harcourt. morphological changes observed and recorded in the excised organs of the sacrificed animals. histologic pictures were taken as micrographs. statistical analysis the data collected were analyzed using one-way analysis of variance (anova) followed by tukey’s multiple comparison post-test (graph pad prism software inc. la jolla, ca, usa). values were expressed as mean ± sem and significance relative to control were considered at p˂0.001 and p˂0.05. results effect of root extract and fractions of h. africana on body and organs weights of rats with doxorubicininduced toxicity administration of h. africana root extract and fractions to rats with doxorubicin-induced organ toxicities caused considerable improvement in the body weights compared to the organotoxic group. the crude extract caused a significant (p<0.01) dose-dependent effect compared to the organotoxic group, with the dichloromethane fraction treated group exerting the highest effect. the testis weight of the group treated with doxorubicin was only reduced when compared to that of the standart control group although it was not statistically significant (p>0.05). however, treatment of rats with doxorubicininduced toxicities with the root extract and fractions of h. africana improved the testis weights insignificantly (p>0.05) except in the group treated with aqueous fraction (table1). effect of leaf extract of hippocratea africana on seminal analysis table 2 shows the seminal analysis of semen from rats with doxorubicin-induced testicular toxicity. the semen appearances in all treatment groups were observed to be milky. however, the semen volume in the group administered with doxorubicin alone was found to be small (0.01 ml). there was a considerable dosedependent increment in the semen volume of rats treated with the leaf extract (0.02 -0.04 ml). the ph of the semen samples from all the groups was 8.0. the extract/fractions-treated groups were found to have a higher percentage of viable sperm cells (75-90%) compared to the organotoxic group (55%). silymarintreated group had 90% viable cells while control had 80%. the viscosity of semen in all the groups was normal. at the same time, the percentage of active sperm cells in the extract/fractions-treated groups ranged from 70-90% dose-dependently compared to 55% recorded in the organotoxic group. the percentage of dead sperm cells in the organotoxic group was found to be about 40% compared to 30% in the low dose (200 mg/kg) group, 15% in the middle dose (400 mg/kg) and aqueous fractiontreated groups and 10% in the high dose (600 mg/kg) and 5% in dcm fraction and silymarin treatedgroups. the sperm count for the organotoxic group was 200 cells, while 400, 450 and 600 cells were respectively recorded from 200, 400 and 600 mg/kg treated groups. the control and aqueous fraction-treated groups had sperm count of 500 cells,while silymarin, dcm and aqueous fractions-treated groups had sperm count of 600 cells (table 2). effect of root extract and fraction on testes oxidative stress markers of doxorubicin-induced testes toxicity. table 3 shows the effect of h. africana root extract/fractions on testes oxidative stress markers of the 400 biology, medicine, & natural product chemistry 13 (2), 2024: 397-406 rats. administration of doxorubicin (1.66 mg/kg i.p) on alternate days for 14 days caused significant (p<0.050.001) decreases in liver antioxidant enzymes activities (sod, gpx, gst, cat) and gsh levels compared to control. the mda level was also significantly (p<0.05) elevated by doxorubicin treatment when compared to standart control. however, concomitant administration of root extract/fractions of h. africana (200 600 mg/kg) with doxorubicin for 14 days caused significant (p<0.050.001) and non-dose-dependent elevations of the enzymatic and non enzymatic endogenous antioxidants in the treated rats groups when compared to the organotoxic groups, with the dcm fraction exerting the highest effect. dose-dependent and significant (p<0.05) decreases in mda levels of the extract/fractions treated groups were recorded with dcm having the highest effect. a similar decrease was also observed in the silymarin-treated group compared to organotoxic control (table 3). effect of the root extract and fractions of h. africana on serum testosterone level of rats with doxorubicininduced testicular toxicity administration of doxorubicin (1.66 mg/kg i.p) on alternate days for 14 days was found to significantly (p<0.001) caused decreases of serum testosterone levels of rats when compared to control. however, concomitant administration of root extract and fractions of h africana (200-400 mg/kg) and silymarin with doxorubicin for 14 days caused significant (p<005-0.01) dose-dependent elevation of the testosterone levels of the treated rats when compared to the organotoxic groups with the dcm fraction having the highest effect (figure 1). effect of root extract and fraction of h. africana on lipid profile of rats with doxorubicin –induced organs toxicities administration of doxorubicin (1.66 mg/kg) was observed to caused significant (p<0.05-0.001) elevation in levels of total cholesterol, triglyceride, low density lipoprotein, and very low density lipoprotein, while the high density lipoprotein level was not significantly (p>0.05) elevated. the total cholesterol and low density lipoprotein levels were significantly (p<0.05-0.001) reduced compared to organotoxic group following concommitant treatment with root extract and fractions of h. africana and silymarin. similarly, triglyceride and very low density lipoprotein levels were also reduced but the reductions were only significant (p<0.05) at the highest dose (600 mg/kg) of the extract. however, the high density lipoprotein level was not affected by the concommitant treatment of the doxorubicin intoxicated rats with the root extract/fractions as well as silymarin (table 4). effect of root extract and fractions of h. africana on histology of rat testis in doxorubicin-induced testicular toxicity histological sections of testes of rats receiving various treatments at magnification (x400) stained with the h&e method revealed that group 1 (standard control, a) rat treated with distilled water (10 ml/kg) showed a well preserved cellular and connective tissue architecture, multiple stages of seminiferous tubules with lumen and developing germinal cell layers, normal tunica albuginea and no evidence of pathological changes were seen. the organotoxic group (group 2, b) treated with doxorubicin (1.66 mg/kg) showed multiple seminiferous tubules with ceased germinal cell maturation, focal dilated congested blood vessels were seen. the basal seminiferous tubule showed germinal epithelium degeneration (figure 2). rats in group 3 (c) treated with 200 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg), group 4 (d) treated with 400 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg), group 5 (e) treated with 600 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg), group 6 (f) treated with 400 mg/kg of aqueous fraction of h. africana root and doxorubicin (1.66 mg/kg), group 7 (g) treated with 400 mg/kg of dichloromethane fraction of h. africana root and doxorubicin (1.66 mg/kg) and group 8 (h) treated with 100 mg/kg of silymarin of h. africana root and doxorubicin (1.66 mg/kg) had testis sections that showed well preserved cellular and connective tissue architectures, multiple stages of seminiferous tubules with lumen, developing germinal cell layers, normal tunica albuginea and no evidence of pathologic changes were found except focal germinal cell vacuolation that was seen in group 6 (figure 2 a-h). table 1. effect of h. africana root extract on body and testis weights of rats with doxorubicin-induced toxicity. parameters/ treatment dose mg/kg testis(g) body weight(g) before after % increase in body weight normal control 2.42±0.29 135.6±18.34 151.0± 12.33 11.35 doxorubicin 1.66 1.78±0.5 130.0 ± 9.45 126.3± 10.43 -2.84 silymarin+dox 100 2.39±0.35 132.6± 14.55 143.2 ± 3.15 7.99 extract+dox 200 2.20±0.35 142.8± 10.56 153.0± 5.29 7.14 400 2.42±0.03 140.3± 7.36 151.6 ± 6.22 8.05 600 2.41±0.19 138.4 ± 8.54 149.6 ± 8.48 8.09 aqueous fraction 400 1.87±0.24 138.4 ± 6.26 144.6± 10.22 4.47 dcm fraction 400 2.50±0.14 134.3± 8.50 146.8± 13.20 9.30 data are expressed as mean ±sem. not significant at p>0.05 compared to normal control and organotoxic control. n = 6. noah & okokon – hippocratea africana ethanol root extract and fractions attenuate … 401 table 2. effect of root extract of h. africana on seminal analysis of rats with doxorubicin-induced testicular toxicity. parameters control doxorubicin silymarin extract 200 mg/kg extract 400 mg/kg extract 600 mg/kg dcm fraction aqueous fraction app milky milky milky milky milky milky milky milky volume 0.03ml 0.01ml 0.02ml 0.02ml 0.03ml 0.03ml 0.043ml 0.02ml ph 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 viability 80% 55% 90% 78% 80% 90% 90% 80% viscosity normal normal normal normal normal normal normal normal normal 80% 55% 90% 70% 80% 80% 90% 80% abnormal 20% 45% 10% 32% 20% 10% 10% 20% active 80% 50% 90% 70% 75% 80% 90% 75% sluggish 10% 10% 5% 10% 10% 10% 5% 10% dead 10% 40% 5% 30% 15% 10% 5% 15% sperm 500 200 600 400 450 600 600 500 table 3. effect of h.africana root extract and fractions on testes oxidative stress markers of rats with doxorubicin-induced toxicity. treatment dose mg/kg sod (u/ml) cat (u/g of protein) gpx (µg/ml) gsh (µg/ml) gst mda (µmol/ml) control 10 0.55± 0.02 1.32±0.01 0.071±0.002 1.41±0.11 0.46±0.02 0.24±0.01 doxorubicin 1.66 0.20±0.01c 0.50± 0.01a 0.030±0.001a 0.46±0.01c 0.28±0.02b 0.66±0.02c crude extract 200 0.35±0.04a,d 1.34± 0.15c 0.061±0.001 1.02±0.15a,e 0.59±0.01f 0.51±0.01b 400 0.33±0.04a,d 1.18±0.10c 0.069±0.001f 1.19± 0.06f 0.52±0.01e 0.45±0.02b,e 600 0.42±0.02e 1.26±0.22c,e 0.058±0.003 1.28± 0.12f 0.43±0.02e 0.42±0.01a,d aqueous fraction 400 0.29±0.03c 1.05±0.36f 0.045±0.005c 0.74±0.15b,d 0.48±0.03e 0.50±0.01b,d dcm fraction 400 0.48±0.02e 1.12±0.42c,d 0.070±0.002f 1.25± 0.11f 0.59±0.05f 0.40±0.01a,e silymarin 100 0.50±0.04f 1.14±0.31c 0.057±0.002c 1.37±0.11f 0.45±0.02f 0.35±0.02f data are expressed as mean ± sem, significant at ap<0.05, bp<0.01, cp<0.001, when compared to control; significant at dp<0.05, ep<0.01, fp<0.001 compared to organotoxic group.. (n=6) table 4. effect of h.africana root extract and fractions on lipid profile parameters of rats with doxorubicin-induced toxicity. treatment dose mg/kg total cholesterol (mmol/l) triglyceride (mmol/l) hdl-c (mmol/l) ldl-c (mmol/l) vldl (mmol/l) control 10 2.25± 0.09 0.75±0.07 1.25±0.03 1.34± 0.11 0.34± 0.03 doxorubicin 1.66 3.63± 0.20c 1.45±0.04c 1.51±0.11 2.77± 0.18c 0.66± 0.01c crude extract 200 2.86±0.20d 1.29± 0.11b 1.38±0.10 2.03± 0.25 0.58±0.05b 400 3.03±0.13a 1.31±0.03b 1.33±0.06 2.43± 0.18 0.59±0.01b 600 2.46±0.14f 0.97±0.10e 1.38±0.06 1.52± 0.16e 0.43±0.04a aqueous fraction 400 2.85±0.06f 1.39±0.04c 1.31±0.09 1.37± 0.12f 0.63± 0.02c dcm fraction 400 2.33±0.12f 1.32±0.15b 1.36±0.08 1.57± 0.12e 0.60± 0.07b silymarin 100 2.30±0.15f 1.37±0.04c 1.41±0.03 1.30± 0.13f 0.62± 0.01b data are expressed as mean ± sem, significant at ap<0.05, bp<0.01, cp<0.001, when compared to control;significant at dp<0.05, ep<0.01, fp<0.001 compared to organotoxic group. (n=6) 402 biology, medicine, & natural product chemistry 13 (2), 2024: 397-406 figure 1. effect of root extract and fractions of h. africana on serum testosterone levels of rats with doxorubicin-induced testicular toxicity. data are expressed as mean ± sem, significant at bp<0.01; cp<0.001 compared to standart control; ep< 0.01, fp< 0.001, compared to organotoxic control. (n=6). figure 2. photomicrograph of testis section of rat treated with (a) distilled water (10ml/kg),(b) doxorubicin (1.66 mg/kg), (c) 200 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg), (d) 400 mg/kg of h. africana root extract and doxorubicin (1.66 mg/kg) (d) showing a well preserved cellular and connective tissue (arrowhead) architecture, multiple stages of seminiferous tubules (double end arrow) with lumen (l), developing germinal cell layers (red double end arrows), normal tunica albuginea (thin arrow), multiple seminiferous tubule with with ceased germinal cell maturation ($) , focal dilated congested blood vessel (v), basal seminiferous tubule showing germinal epithelium degeneration (#).h&e stain x100 magnification. discussion doxorubicin, an anthracycline used in the treatment of tumours (konopa et al., 1988; thorn et al., 2012), exhibits undesirable severe effects on the male reproductive system (sridevi et al., 2012), such as disruption of normal reproductive functions via the induction of oxidative stress. investigation of agents with antagonistic potentials to counteract the toxic effects of doxorubicin is ongoing. this study was carried out to noah & okokon – hippocratea africana ethanol root extract and fractions attenuate … 403 assess the effects of root extract of h. africana on doxorubicin-induced male reproductive system toxicity. the exact mechanisms through which doxorubicin induces toxicity in the male reproductive system, though reported widely, have not been adequately elucidated. the drug reportedly retards testicular growth, suppresses spermatogenesis, leading to male infertility through imposing oxidative stress and cellular apoptosis (yeh et al., 2007). it also causes double-strand dna breaks and cell death by intercalating into dna strands (konopa et al., 1988; gewirtz et al., 1999) in meiotically dividing spermatocytes and spermatogonia (cabral et al., 2014). spermatogenesis suppression, sperm motility impairment, increased percentage of abnormal spermatozoa, decreased body and testicular weights, reduced testosterone levels, and testicular failure through oxidative stress and cell apoptosis in testicular tissue have also been reported (howell & shalet, 2005). in this study, doxorubicin was found to cause a significant increase in the percentages of dead and abnormal cells. also, decreased seminal volume and sperm count were observed in the doxorubicin alone treated group. similarly, testes from rats treated with doxorubicin alone were found to have marked degenerated germ cells and spermatozoa, supporting previous reports (howell & shalet, 2005). however, all these toxic effects of doxorubicin were alleviated by concomitant administration of root extract and fractions of hippocratea africana, as animals treated with the root extract/fractions had high sperm cell count, greater seminal volume, low level of dead cells as well as standart testicular architecture, portraying the extract’s potentials in preventing doxorubicin-induced testicular toxicity probably through the antioxidative stress activity of its phytochemical components. the antioxidative burst and antioxidant activities of the root extract and fractions of h. africana had previously been reported (okokon et al., 2013a; 2022; umoh et al., 2021). moreover, the antioxidative stress activities of the root extract and fractions observed in this study further support the antioxidant potential of the plant. these activities may have contributed to the observed protective effects in this study. the membranes of the male germ cells have a high amount of polyunsaturated fatty acids, one of the targets of reactive oxygen species (robinson et al., 1992). thus, spermatozoa are vulnerable to oxidative damage because of the high amount of lipids in their membranes, making them lose their integrity and become less motile (robinson et al., 1992). this effect could have contributed to the low sperm count, high percentages of dead and abnormal cells observed in the group treated with doxorubicin only in this study. doxorubicin has been shown to impair male fertility by causing germ cell oxidative stress and apoptosis (el-maddawy and elnaby, 2019., aksu et al., 2019). it has also been demonstrated to impair spermatogenesis (kato et al., 2001) and steroidogenesis (rizk et al., 2014; olusoji et al., 2017). exposure to doxorubicin appears to affect testicular integrity at both prepubertal and post-pubertal stages of development. in vitro studies with prepubertal mouse testis have demonstrated significant loss in germ cell number following exposure to doxorubicin at concentrations that were equivalent to human therapeutic doses (smart et al., 2018). further, studies have demonstrated early testicular developmental arrest and long-term germ cell dna damage following prepubertal doxorubicin exposure (vendramini et al., 2010). the study observed these effects as marked degenerated germ cells and spermatozoa were seen in the histological sections of the testes. however, concomitant administration of the root extract/fractions protected the testes as the toxic effects observed in the doxorubicin alone treated group were absent or mild compared to that group treated doxorubicin alone. the high susceptibility of testes and sperm to doxorubicin -induced testicular oxidative stress may be due to a weak anti-oxidant defence system in testicular tissue and semen (nowrouzi et al., 2019). besides, earlier studies demonstrated that antioxidant supplementation improved the quality of the semen profile in infertile men (talevi et al., 2013). lipids are an essential part of the reproductive system. cholesterol is considered to be the precursor of steroid hormones. steroidogenesis plays an essential role in the synthesis of spermatogenesis hormones. the biosynthesis of testosterone from pregnenolone is carried out by steroidogenesis enzymes including 17β hydroxysteroid dehydrogenase (17β-hsd) 3β and hydroxysteroid dehydrogenase (3βhsd). it has been reported that doxorubicin results in the downregulation of these enzymes (prahalathan et al., 2006; takashima, et al., 2009). adipocytes are the primary sites for triacylglycerol storage. it has been found that dox downregulates adipogenesis in vitro by decreasing the expression of pparγ (arunachalam et al., 2012). doxorubicin inhibits spermatogenesis by causing defects in epididymal adipose tissue (tirupathi pichiah et al., 2012), which is crusial for normal spermatogenesis (chu et al., 2010). also, studies using adult rat models of dox exposure have reported decreased testosterone, follicle stimulating hormone (fsh) and luteinizing hormone (lh) levels, decreased sperm count, motility and viability, and increased abnormally formed spermatozoa (das et al., 2012). the results of this study showed that testosterone level was significantly reduced in the group treated with doxorubicin alone compared to standart control. however, the root extract and fractionstreated groups had improved levels of testosterone which further point to the protective effect of the extract against doxorubicin-induced toxicity. this result agrees with an earlier report by ndem and johnson (2017) in which improved testosterone concentration was reported following h. africana root administration. oxidative stress plays an essential role in doxorubicin-induced toxicity by forming reactive oxygen species (ros) (arunachalam et al., 2021). the results of 404 biology, medicine, & natural product chemistry 13 (2), 2024: 397-406 the doxorubicin alone treated group in this study revealed a significant elevation of testicular mda levels and a significant decreases in testicular sod, cat, gst, gpx and gsh when compared with the dox group. these results are from previous reported studies (khodir et al., 2021). however, co-treatment of root extract/fractions and dox elevated the levels of these endogenous antioxidants revealing the free radicals scavenging potentials of the root extract/fractions and its antioxidative stress activity which is due to the activities of its phytochemical constituents such as monoterpenes, sesquiterpenes and xanthones earlier reported by okokon et al.(2013a) and umoh et al., (2021) to be present in this root extract. conclusion the findings of this study showed that the root extract and fractions of hippocratea africana possess testicular protective potentials against doxorubicin-induced testicular toxicity. these properties can be attributed to its phytochemical constituents’ antioxidant and antioxidative stress activities. thus, the root can be used to alleviate and/or prevent doxorubicin-induced male reproductive system toxicity. acknowledgements: the authors are grateful to the staff of the animal house, pharmacology and toxicology department of university of uyo for providing technical assistance. authors’ contributions: jeo, kun research concept and design; 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(2003). nickel deficiency diminishes sperm quantity and movement in rats.biol trace elem res.93:141–153. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 1, april 2024 | pages: 165-172 | doi: 10.14421/biomedich.2024.131.165-172 issn 2540-9328 (online) physical characteristics of immobilized cells acetobacter xylinum of various concentrations of na-alginate lathifa indraningtyas, maria erna kustyawati*, nafiatul fitriah department of agricultural product technology, faculty of agriculture, universitas lampung, jl. prof. dr. soemantri brojonegoro no. 1, bandar lampung, lampung, 35145, indonesia. corresponding author* mariaernakustyawati@gmail.com abstract immobilization of acetobacter xylinum cells by trapping technique is considered one way to maintain cell viability and can be used repeatedly. the purpose of this study was to determine the physical characteristics of immobilized cells in different concentrations of naalginate and apply them to the manufacture of nata de coco, as well as to see the surface and pore shape of immobilized cells after fermentation. this study used a descriptive method with visual observation. this study used one factor, namely various concentrations of na-alginate, namely 2%, 3%, 4%, and 5% w/v. the immobilized cells applied to the manufacture of nata de coco had only a na-alginate concentration of 3% resulting in a nata fiber layer close to that of a control. immobilized cells with a na-alginate concentration of 2% have a cracked, slightly pore surface with a size of 60.37 μm, while a na-alginate concentration of 5%, have a smooth surface, many pores, with a size of 10.55 μm. thus, the concentration affects the characteristics of immobilized cells. application of immobilized cells with a na-alginate concentration of 3% produces nata de coco with a fairly thick and stable fiber layer characteristic (close to control). keywords: acetobacter xylinum; cell immobilization; entrapment; nata de coco; natrium alginate. introduction acetobacter xylinum (a.xylinum) is a gram-negative bacterium that produces extracellular polysaccharides in the form of cellulose that make up the bacterial cell wall (wang et al., 2019). abundant cellulose production is located on the surface of the substrate due to the process of glucose metabolism by a. xylinum to produce cellulose under aerobic conditions. the intertwined cellulose of the bacterial cell wall forms the nata structure and the bacterial cells are trapped inside. nata is composed of cellulose as a fermentation product by a. xylinum with the main nutrients of carbon and nitrogen sources. so far, nata fermentation has been carried out by using the technique of direct inoculation of a. xylinum starter into the substrate, which is easy to do and inexpensive. however, the direct inoculation method results in starter cultures that are easily contaminated, easily die, and are vulnerable to changes in environmental conditions if the handling is inadequate. starter inoculum is available in the market in liquid form packaged in bottles with lids. liquid cultures are easily contaminated, cell viability decreases during storage, and are difficult to manage (dewi, 2009). meanwhile, the sustainability of the fermentation process requires starter cultures that are continuously available, with maximum potential, easy to manage and long shelf life with high production activity. one way that can meet these needs is cell immobilization. cell immobilization is defined as a method to confine or physically place cell microbes in a certain space, where the cell still has catalytic activity and can be used continuously and many times (darmawan and pradipta, 2015). in this study, immobilization was carried out using entrapment techniques, namely by allowing cells to penetrate into the porous matrix until their mobility (space) was blocked by other cells or porous materials formed in situ into cell culture (pratiwi et al., 2021). in this study, sodium alginate (na-alginate) as a matrix material that traps cells (ratnasari et al., 2014). alginate in its salt form, sodium alginate, has the property of being able to form a gel by binding with calcium salts, one of which is calcium chloride (cacl2), which acts as a cation. ca2+ ions in calcium chloride function to strengthen the alginate gel structure (mubarokah, 2018). this study aims to determine the characteristics of immobilized cells made using different concentrations of na-alginate and apply it to the manufacture of nata de coco. manuscript received: 12 february, 2024. revision accepted: 20 may, 2024. published: 28 may, 2024. https://doi.org/10.14421/biomedich.2024.131.165-172 166 biology, medicine, & natural product chemistry 13 (1), 2024: 165-172 materials and methods materials and tools the materials used in this study were acetobacter xylinum bacterial starter, naalginate, calcium chloride (cacl2), peptone solution, phosphate buffer solution, vinegar acid, za (zwavelzur ammoniac) food grade from the online shop, granulated sugar, young coconut water from nearest seller, distilled water taken from waste processing laboratory, in the agricultural products technology department, na (nutrient agar) media (merck), and nb (nutrient broth) media (merck) from online shop. equipment used in cell immobilization were 20 ml syringe from online shop, analytical balance, beaker, stirring rod, test tube, filter in laboratory, ruler, sem (scanning electron microscope), mcfarland den-1b densitometer, plastic cup container, and refrigerator in laboratory. equipment used in the preparation of working culture and culture suspensions were ose needle, bunsen, vortex, measuring cup, test tube, test tube rack, hot plate, autoclave, erlenmayer, incubator, petri dish, laminar air flow, and centrifuge in laboratory. the tools used in making nata de coco were square plastic containers, knives, ph meters, stoves, pans, stirrers, filters in laboratory, paper, and rubber bands. making acetobacter xylinum culture on agar cups this process was carried out because the inoculation on the oblique agar does not grow well. making culture begins with preparing nutrient agar (na) as much as 100 ml. next, nutrients to be weighed as much as 2 grams and put into erlenmayer, then add aquades as much as 100 ml. then heated with a hot plate until na dissolves. then the na solution was sterilized using autoclaf for 15 minutes with a temperature of 121oc. after the substrate cools, it was poured on 4 petri dishes, then covered and leave to solidify. after solidifying, one pure culture of a.xylinum was taken and etched on the agar to be angled with aseptic. next, the culture was incubated at a temperature of 37oc for 24 48 hours (sujaya, 2017). manufacture of acetobacter xylinum broth culture making broth culture (liquid) begins with preparing nutrient broth (nb) as much as 100 ml. next, nutrient broth was weighed as much as 0.8 grams and put into erlenmayer, then added aquades as much as 100 ml. then heated with a hot plate until the nutrient broth dissolves. then the media was sterilized using autoclaf for 15 minutes with a temperature of 121oc. next, the substrate is poured on 10 test tubes with each tube containing 10 ml, and closed using cotton stoppers. test tubes containing sterile nb media and placed on the test tube rack and allowed to cool. after cooling, one pure culture of a.xylinum from agar saucer was taken and inserted on nb media with aseptic. furthermore, nb media containing cultures were mixed using vortexes. after that, the culture was incubated at a temperature of 37oc for 24 48 hours, until the media looks cloudy. (sujaya, 2017). calculation of the number of acetobacter xylinum bacteria in this study, the calculation of the number of bacteria was carried out by turbidimetry or looking at the turbidity compared to the mc farland (mcf) standard. such turbidity indicates the presence of bacterial formation. the turbidity standard is intended to replace the calculation of bacteria one by one, making it more efficient in time and effort. the calculation of the number of bacteria is done by comparing the turbidity (turbidity) of bacterial culture with the mcf standard 0.5-10, which is contained in the mcf standard table, so that it can be seen which bacterial culture turbidity corresponds to the table. manufacture of acetobacter xylinum culture suspension making culture suspense begins by incorporating a.xylinum broth culture aged 24 – 48 hours into a centrifuge tube. then culture centrifuged at a speed of 3000 rpm for 5 10 minutes. the resulting supernatant was discarded and the pellets were recentrifuged. furthermore, it was resuspensioned with 2 ml of phosphate buffer so that a culture suspension (ifadah et al., 2016) was modified. preparation of immobilized cells preparation of immobilized cells began with making naalginate solution with a concentration of 2%, 3%, 4%, and 5% w/v, and cacl2 solution with a concentration of 3% w/v. the next step was to mix the na-alginate solution with a. xylinum culture suspension until homogeneous. next, it was dripped into the cacl2 solution using an injection slowly, and allowed to stand for 20-30 minutes so that the beads were not destroyed. the process was carried out on each na-alginate concentration. the immobilized cells obtained were then stored in peptone solution (cahyono, et al., 2021). preparation of nata de coco the process of making nata de coco was carried out using a.xylinum starter as a control and also immobilized cells with various concentrations of na-alginate that have been determined. the process of making nata begins with filtering coconut water to separate it from impurities. the coconut water was then boiled to a boil and vinegar, sugar, and za were added. the boiling coconut water is poured into a container and allowed to cool.the cold medium was then added to the starter and immobilized cells in each container, then covered with paper and tied with rubber bands. next, fermentation was carried out for 14 days. after the nata fiber layer is formed, it will be indraningtyas et al. – physical characteristics of immobilized cells … 167 separated from the immobilized cells (azhari, 2014) modified. results and discussion immobilized cell characteristics the results obtained from the preparation of immobilized cells using different concentrations of na-alginate produce different characteristics. the immobilized cells obtained were then applied to the manufacture of nata de coco. the application of immobilized cells aims to see whether the immobilized cells experience changes in characteristics (shape, size, texture, and color) after being used for making nata de coco. the characteristics of immobilized cells can be seen in table 1. table 1. characteristics of immobilized cells before and after being applied to the manufacture of nata de coco. treatment of naalginate concentration immobilized cells before fermentation description immobilized cells after fermentation description 2% ▪ texture: solid slightly soft ▪ shape: round ▪ size: 3 mm ▪ color: white bening ▪ -texture: solid slightly soft ▪ -shape: round ▪ -color: slightly yellowish 3% ▪ -texture: solid ▪ -shape: round ▪ -size: 4 mm ▪ -color: white murky ▪ -texture: solid ▪ -shape: round ▪ -color: yellowish 4% ▪ -texture: solid ▪ -shape: round ▪ -size: 6 mm ▪ -color: white slightly ▪ brownish ▪ -texture: solid ▪ -shape: round ▪ -color: yellowish 5% ▪ -texture: solid ▪ -shape: round and there is like a tail ▪ -size:6 mm ▪ -color: brownish ▪ -texture : solid ▪ -shape: round and there is like a tail ▪ -color: yellowish based on the images of immobilized cell products in tables 1, the characteristics (texture, shape, size and color) of immobilized cells with different concentrations of na-alginate before and after being applied for nata fermentation did not change. shape and size the 2% na-alginate concentration has a perfectly round shape with a size of 3 mm, while the 3% na-alginate has a round shape with a larger size, which is 4 mm. the 4% na-alginate concentration produced a round shape with a larger size than the 2% and 3% concentrations, which was 6 mm, and immobilized cells with 5% na-alginate concentration produced a round shape and there was a tail at the bottom of the beads. immobilized cells with 5% na-alginate have the same size as the 4% concentration, which is 6 mm. the 4% na-alginate concentration formed a solution with a high viscosity, but it was still easy to be dripped by injection. the tail shape on immobilized cells with 5% na-alginate concentration may be caused by the high viscosity of the na-alginate solution, so that when dripped with an injection it forms a tail. viscosity that is too high is not ideal to form a perfectly round beads (lee et al., 2013). texture the 2% na-alginate concentration produced a dense but slightly soft texture, while the 3%, 4% and 5% naalginate concentrations had a dense and firm texture. the difference in texture produced in this study was due to 168 biology, medicine, & natural product chemistry 13 (1), 2024: 165-172 the use of different concentrations of na-alginate, causing the immobilized cells to have different textures and firmness. the firm texture of immobilized cells is due to the large amount of mannuronic acid (m) and gulluronic acid (g) contained in alginate. (mubarokah, 2018). alginate polymers consist of 3 types, namely mannuronic acid (m), gulluronic acid (g), and the combined polymer of the two (mg) (picture 1). the m polymer is formed from the equatorial structure of the c1 and c-4 groups and forms a straight polymer chain, while the g polymer is formed from the axial structure, thus forming an up and down polymer chain. figure 1. ca-alginate matrix formation. source: khajoui et al., (2022) modified. this difference in polymer structure causes polymer g to be more widely used for the process of alginate gel formation with the addition of ca2+ ions. ca2+ ions will replace h+ ions in the carboxylic group and form a connecting ion bridge between gulluronic acid (g) to form polymer g. the relationship between these g polymers will form an egg-box structure (picture 2). figure 2. formation of egg-box structure. source: bedê et al., (2017). the interaction between guluronic acid (g) and ca2+ ions causes the flexibility of the alginate molecule to increase, with the order of flexibility from the lowest, namely gulluronic acid (gg), followed by mannuronic acid (mm) and a combination of mannuronic and gulluronic acids (mg) (wijffels, 1996). the horizontal ribbon-like chain of mannuronic acid (m) (picture 3) can bind to ca2+, but tends to at high cation concentrations. due to the difference between the structures of mannuronic acid (m) and gulluronic acid (g), the gel formed from mannuronic acid (m) is more elastic, while the gel from gulluronic acid (g) is firm (bedê et al., 2017). color the color of the immobilized cells produced in this study is very likely caused by the concentration of na-alginate. visually, immobilized cells with a na-alginate concentration of 2% produce a clear white color, while at a concentration of 3% they produce a slightly cloudy white color. a na-alginate concentration of 4% produces immobilized cells with a slightly brownish white color, whereas a concentration of 5% produces immobilized cells with a brownish white color. the color produced in this research is in accordance with research by mubarokah (2018) which used alginate concentrations of 1%, 2%, 3% and 4% where the 1% concentration had a clearer color when compared to the 2%, 3% and 4% concentrations. therefore, differences in na-alginate concentration are very likely to result in changes in the color of immobilized cells. number of bacteria based on turbidity using mcf standard calculation of the number of bacteria using the turbidity method based on mc farland standards using 4 tubes containing nb (nutrient broth) media and a.xylinum culture. indraningtyas et al. – physical characteristics of immobilized cells … 169 table 2. results of bacterial calculations based on turbidity values using mcf standards and conversion values. tube na-alginate concentration treatment number of bacteria based on turbidity value number of bacteria in (cfu/ml) 1 2% 5.08 1.5 x 109 2 3% 5.47 1.5 x 109 3 4% 4.17 1.2 x 109 4 5% 4.68 1.2 x 109 note: observation of bacterial turbidity using densitometer the difference in turbidity values based on naalginate concentration treatments of 2%, 3%, 4%, and 5% w/v (table 2) shows that the number of microbes in tube 1, calculated using a densitometer, has a turbidity value of 5.08, equivalent to the standard mc farland 1.5 x 109 cfu/ml, in tube 2 with a turbidity value of 5.47, equivalent to the mc farland standard, namely 1.5 x 109 cfu/ml, tube 3 with a turbidity value of 4.17, equivalent to 1.2 x 109 cfu/ml, in tube 4 with a turbidity value of 4.68, equivalent to 1.2 x 109 cfu/ml. even though the bacterial population in the tube looks different, it is still at a concentration of 109 cfu/ml. the results of the bacterial calculations carried out will then be used to make immobilized cells. application of immobilized cells in making nata de coco the immobilized cells that had been made, with naalginate concentrations of 2%, 3%, 4% and 5% (w/v) were then applied to make nata de coco with a.xylinum starter as a control. the results of the nata de coco fermentation process using free cells (starter) and also immobilized cells can be seen in table 3. table 3. nata de coco fermentation results. treatment picture description starter a.xylinum (kontrol) -nata layer: stable and quite thick -medium color: brownish -yield: no arrangement is formed so there is no yield immobilized cells with na-alginate 2% -nata layer: there is no nata layer because it is contaminated -medium color: reddish -yield: no arrangement is formed so there is no yield immobilized cells with na-alginate 3% -nata layer: stable and quite thick -media color: cloudy light brown -yield: no arrangement is formed so there is no yield immobilized cells with na-alginate 4% -lapisan nata: tipis -medium color: clear light brown -yield: not formed nata so there is no yield immobilized cells with na-alginate 5% -nata layer: very thin -medium color: murky white -yield: no arrangement is formed so there is no yield note: ▪ starter a.xylinum (kontrol) : 100 ml media + 15 ml starter a.xylinum ▪ immobilized cells with 2% na-alginate: 100 ml of media + 5 grams of immobilized cells of a.xylinum with a concentration of 2% naalginate ▪ immobilized cells with 3% na-alginate: 100 ml media + 5 grams of a.xylinum immobilized cells with a concentration of 3% naalginate ▪ immobilized cells with 4% na-alginate: 100 ml media + 5 grams of a.xylinum immobilized cells with a concentration of 4% naalginate ▪ immobilized cells with 5% na-alginate: 100 ml media + 5 grams of a.xylinum immobilized cells with a concentration of 5% naalginate 170 biology, medicine, & natural product chemistry 13 (1), 2024: 165-172 the fermentation results show that nata de coco using a.xylinum starter produces layers of nata fiber that do not break, are compact and quite thick (3 mm) with a brownish color. meanwhile, nata fermentation using immobilized cells with a na-alginate concentration of 3% produced a nata fiber layer close to control, with the nata fiber layer looking stable and quite thick, with a cloudy light brown color. the nata fiber layer formed during fermentation of nata with a 4% na-alginate concentration looks thinner than the nata fiber layer using a 3% na-alginate concentration, and is a clear light brown color. nata fermentation using immobilized cells with a na-alginate concentration of 5% produces a very thin layer of nata fiber with a cloudy white color. there was no yield in the fermentation process carried out from control or immobilized cells. in this research, the nata de coco fermentation process using immobilized cells and a.xylinum starter as a control did not produce a yield, because the nata produced was only a layer of white fiber measuring ± 3 mm. however, the fermentation process continued, which was marked by a color change in the fermentation medium and the presence of a sour aroma which was thought to be acetic acid, a metabolic product of a.xylinum. according to hasfita et al., (2015) in their research on the use of seri fruit (muntingia calabura l) for making acetic acid using a.xylinum bacteria, that the fermentation of acetic acid produced by a.xylinum bacteria with fermentation times of 3, 6, 9, and 12 days produced the highest acetic acid levels on day 9, namely 1.369%. fermentation of nata coco in this study did not produce an alcoholic aroma. this is likely because during the fermentation process the sugar is oxidized to acetic acid. during its growth, a.xylinum releases extracellular enzymes that are able to form cellulose from glucose which resembles a gel matrix called nata (kuncara, 2017). the process of forming cellulose by a. xylinum consists of four reaction stages (kuncara, 2017). the first stage is the hydrolysis of sucrose which produces fructose and glucose by the sucrase enzyme, the second stage, the intramolecular conversion reaction of α-dglucose into β-dglucose with the help of the isomerase enzyme, the third stage, the intermolecular reaction of glucose through 1,4 b-glycoside bonds, and the final stage of formation of cellulose with its repeat units, namely cellobiose. several factors cause nata not to form, including inhibiting the growth activity of a.xylinum bacteria caused by unfavorable fermentation conditions. disturbed aeration can affect nata fiber content and produce cellulose, conversely, thin nata is caused by inhibited growth of a.xylinum (muchtadi, 1997). according to rohmani and kristianingrum, (2012), the inhibited growth of a.xylinum bacteria will result in the resulting nata being soft and thin, and it is even possible that no nata will be formed. the growth phase of a.xylinum is in the stationary phase, the cells are unproductive or no longer productive or the cell biomass is small (brooks et al., 2013). the color difference in the fermentation medium is probably caused by the thickness of the nata. the thicker the nata, the darker the resulting color, it is suspected that there will be more and denser cellulose tissue formation, conversely, the thinner the nata, the lighter the result (putriana and aminah, 2013). sem analysis to observe the pores and surfaces of immobilized cells sem observations were carried out on immobilized cells with na-alginate concentrations of 2% and 5% to see the differences in pores and surfaces of immobilized cells. table 5 shows the results of observations of immobilized cells using a scanning electron microscope (sem) with 1000x and 3500x magnification. table 4. observation results using sem on immobilized cells. treatment picture description na-alginate 2% -beads surface: cracking or cracking -pores: large size with a small amount -pore size: 60.37 μm na-alginat 5% -peraccording to beads : halus -pores: small size with a large amount -pore size: 10.55 μm indraningtyas et al. – physical characteristics of immobilized cells … 171 based on the complete cross-section of immobilized cells, it shows that immobilized cells with a na-alginate concentration of 2% have a different cross-section from immobilized cells with a concentration of 5%, both in terms of size and number of immobilized cell pores. meanwhile, the surface of the immobilized cells at a concentration of 2% looks cracked, and at a concentration of 5% the surface of the beads is smooth. low na-alginate concentrations produce immobilized cells with a slightly soft texture and are likely to be easily destroyed when used. besides that, low na-alginate concentrations result in the cell protective layer being thinner and unstable during the fermentation process. although na-alginate can still protect cells, the cell sensitivity to the fermentation medium is too low. this results in the amount of living biomass in the ca-alginate matrix decreasing (lotfipour et al., 2012). the results of this research are in line with andersen's (2015) statement that the lower concentration of naalginate in immobilized cells will make the alginate structure more flexible because the guluronic acid and mannuronic acid content is not sufficient to bind water. the sem results show that on the surface of the immobilized cells there is a collection of a.xylinum cells that penetrate out of the beads. according to luwihana et al., (2010), their research on acetic acid fermentation with immobilized cells of acetobacter pasteurianus showed that by entrapment immobilized cells are thought to be located on the surface of the beads to form aggregates. this statement was strengthened by mori et al., (1989) on the growth behavior of immobilized acetic acid bacteria, barbotin et al., (1990) on observations of immobilized cells in the physiology of immobilized cells, and osuga et al., (1984) on production of acetic acid by immobilized acetobacter aceti trapped in k-carrageenan gel, which states that cells that are trapped form aggregates on the surface of beads with a thickness of 20-50 µm, still grow and reproduce. the greater the number of cells in the beads, the motile and aerobic nature of the acetic acid bacteria cells, the more cells will escape from the mobilizing matrix in the fermentation medium when the fermentation process takes place (fumi et al., 1992). at 2% concentration it has a size of 60.37 µm, while at 5% concentration the pore size is 10.55 µm (table 5). these results are in line with research by halim et al., (2019) concerning the effect of percentage of pvaalginate beads on the decolorization level of azo synthetic dyes using the immobilized bacteria consortium, it shows that a pva-alginate ratio of 8:1 has an average pore size of 6,306 μm, a pva-alginate ratio of 10:1 has a pore size of 5,089 μm, and a pva-alginate ratio of 12:1 has a pore size of 2,557 μm . the large pore size is thought to result in a high value of the gel's ability to expand (swelling). conclusions different concentrations of na-alginate affect the characteristics of shape, size, texture and color of immobilized cells. na-alginate concentration of 3% is considered the best concentration compared to other concentrations, resulting in immobilized cells with a shape that is neither too big nor small (4 mm), with an elastic texture. immobilized cells made at a na-alginate concentration of 3% were then applied to make nata de coco. the nata that was formed did not provide a yield value because it had a thickness of ±3 mm which was close to the control, but fermentation continued. different na-alginate concentrations produce different pore sizes using the sem test, at low concentrations (2%) produce beads pore sizes of 60.37 µm, and 10.55 µm in beads with a na-alginate concentration of 5 %. it is necessary to conduct further research on the storage duration of immobilized cells, cell viability and stability. in addition, this study also needs to calculate the number of bacteria using the total plate count (tpc) method as a comparison with the turbidity method that has been done. authors’ contributions: maria erna designed the study. nafiatul fitriah carried out the laboratory work and analyzed the data. lathifa indraningtyas wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references andersen, t. 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(1996). immobilized cells: basics and applications. first edition, elsevier, tokyo. https://doi.org/10.24198/cna.v7.n2.23092 https://jpa.ub.ac.id/index.php/jpa/index https://doi.org/10.1002/ceat.2013.00.230 https://doi.org/10.22146/agritech.9683 https://doi.org/10.1007/bf01026053 https://doi.org/10.1016/0922-338x(90)90196-4 https://doi.org/10.26714/jpg.4.1.2013.%25p https://doi.org/10.33508/jtpg.v13i2.1507 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 475-483 | doi: 10.14421/biomedich.2024.132.475-483 issn 2540-9328 (online) effect of mixing time variation and framing time on the quality of coffee grounds-based solid soap insa dorra neisya, suratno*, kamalia fikri biology education department; faculty of teacher training and education, university of jember, jl. kalimantan tegalboto no. 37, jember, east java, indonesia. corresponding author* suratno.fkip@unej.ac.id manuscript received: 25 july, 2024. revision accepted: 07 october, 2024. published: 22 october, 2024. abstract the increase in public consumption of coffee has resulted in an increase in wasted coffee grounds. coffee grounds have good content for the skin, so they can be reused as a basic ingredient in soap-making. in the soap making process, the mixing and framing stages can affect the quality of the soap. this study aims to determine the effect of mixing time of 24.5 minutes, 25 minutes, and 25.5 minutes as well as framing time of 3 days, 5 days, and 7 days on the physical and chemical quality of solid soap made from coffee grounds. the physical quality parameters observed are colour, texture, cracking, and foam power. the chemical quality parameters are foam stability, ph, moisture content, and free alkali. the method used was the questionnaire method followed by statistical analysis using the kruskal wallis test for physical quality tests and the experimental method followed by statistical analysis using the two-way anova test for chemical quality tests. the results showed that all variations in mixing time and framing time had an effect on the physical and chemical quality of solid soap and there were significant differences. the panellists assessed the physical quality of the soap to be attractive, hard, with no cracks, and foamy, while the results of the chemical quality test, the soap produced is safe to use by the quality standards of solid soap with a foam stability range of 82%-97%, ph 10.3-11.8, moisture content 6%-14%, and alkali free 0.03%-0.09%. keywords: chemical quality; coffee grounds soap; framing time; mixing time; physical quality. introduction jember is one of the regencies in east java that produces the best robusta coffee, this was conveyed by the regent of jember, mr. hendy. in addition, the content of robusta coffee grounds is higher than other types of coffee grounds. the increase in coffee grounds along with the increase in the number of coffee shops, including in the city of jember, has caused an increase in wasted coffee grounds waste. starting from the increase in public consumption of coffee, the amount of coffee grounds waste has also increased, and has a great danger of pollution when disposed of in the environment. coffee grounds when decomposing in landfills produce methane, a potent greenhouse gas, and cause significant global warming (bejenari et al., 2021). coffee grounds are usually treated as waste, whereas coffee grounds can also be a source of raw materials for applications in several industrial fields. as a source of valuable compounds with potential applications in the pharmaceutical, cosmetic, and food industries, coffee grounds are an exciting example of waste utilization in the cosmetics industry i.e. soap. soap using coffee grounds is one of the practical uses of coffee grounds waste because it has quite good content for skin health, so it can be used as a basic ingredient in making organic soap. in general, people know two types of soap, namely solid soap and liquid soap. solid soap is considered cheaper, easier to use, and efficient in cleaning the skin compared to liquid body soap (febriani et al., 2020). liquid soaps contain more additives and preservatives to maintain their texture and stability. solid soap products are used by the wider community, so the product must have soap quality by the product specification standards that have been set, so that it can meet the needs and protect consumers (idoko et al., 2018). the quality of soap can be reviewed in terms of its physical and chemical aspects. the physical quality of a good soap must have high cleaning power, must have sufficient hardness, and be able to produce a sufficient amount of foam to support its cleaning power (rashati et al., 2022). overall, the chemical properties allow the soap to be effective in cleaning various surfaces that have been exposed to dirt, oil, or grease. in the soap making process, the mixing stage is an important stage, where during the process of mixing oil and fat will form pure soap. the mixing time will also affect the perfection of the saponification process that takes place (setiyawan & oktavianty, 2022). the framing stage is where a series of processes occur from pure soap that is still in liquid form https://doi.org/10.14421/biomedich.2024.132.475-483 476 biology, medicine, & natural product chemistry 13 (2), 2024: 475-483 to solid form (sitorus, 2021). therefore, it is necessary to the effect of mixing time and framing time on the quality of solid soap made from coffee grounds. this study aims to determine the effect of mixing time of 24.5 minutes, 25 minutes, and 25.5 minutes as well as framing time of 3 days, 5 days, and 7 days on the physical and chemical quality of solid soap made from coffee grounds. the physical quality parameters observed are colour, texture, cracking, and foam power. the chemical quality parameters are foam stability, ph, moisture content, and free alkali. materials and methods study area the research was carried out at the toxicology laboratory of the biology education study program of fkip university of jember and the laboratory of instrumentation and agricultural environmental control (iplp) of the agricultural engineering study program ftp university of jember from january to february 2024. procedures solid soap manufacturing the research on the manufacture of solid soap made from coffee grounds uses variations in mixing time and framing time. robusta coffee grounds are obtained from coffee shops around the university of jember. the stages are 235 grams of olive oil, 150 grams of coconut oil, 100 grams of palm oil and 50 grams of coffee grounds mixed into a container and stirred for 5 minutes. then 74 grams of naoh is dissolved in 210 grams of cold/cold water in a container made of stainlees steel. the naoh solution will be hot and whitish in color, then cool in room temperature. then the cooled naoh solution is put into the mixture in a container, stirred until thickened with variations in mixing time of 24.5 minutes, 25 minutes, and 25.5 minutes. next, the mixture in the container is put in 10 cc fragrance and stirred for 5 minutes. then the dough that has formed is put into a mold and framed with variations of 3 days, 5 days, and 7 days. after that, the soap is stored for 4 weeks then analyzed for quality. solid soap quality test ▪ moisture test the soap sample was weighed as much as 4 grams and weighed in weight. then it is heated in the oven at 105oc for 2 hours, then cooled in a decigator, then weighed again. the moisture content is calculated by the appropriate formula (sni 06-3532-2016). ▪ free alkaline test alcohol as much as 100 ml is boiled in 250 ml of erlenmeyer pumpkin. next, 0.5 ml of phenolphthalein indicator is added and cooled to a temperature of 70oc then neutralized with koh 0.1 n in alcohol. next, add 5 grams of soap and boil over a water bath for 30 minutes. if the solution is not red, the mixture is cooled to a temperature of 70oc and then titrated with a solution of koh 0.1 n in alcohol, until a color appears. if the solution is red, it is titrated using hcl 0.1 n in alcohol from micro burettes, until the red color quickly disappears and the result is calculated with the appropriate formula (sni 06-3532-2016). ▪ ph test take 5 g of soap then dissolve it with 5 ml of aquades. measure the ph of soap with a ph meter and record the resulting ph (american society for testing and materials international, 2015). ▪ foam stability 1 g of solid soap is dissolved using 10 ml of aquades in a test tube, shaken for 1 minute, counting the foam formed measured with a ruler (initial foam height). the height of the foam is measured again after 5 minutes to get the final foam height. the stability of the foam is calculated with the appropriate formula (dhara et al. 2023). ▪ organoleptic test (color, texture, cracking rate, and foam power) this organoleptic test was carried out by 10 panelists. soap color assessment by assessing the color level of the soap produced without the addition of dyes. texture assessment is carried out by looking and feeling the texture or appearance of the soap produced. the solid soap cracking test in this study is wet cracking, which is that the soap is used until the weight is reduced by 1/4 of the initial weight, then soaked in water for 1 hour, after which it is lifted and dried for 24 hours, make sure the surface of the soap is dry and then see the cracks. the assessment of foam power is carried out by washing hands with the soap produced and then assessing the amount of foam produced based on the hedonic scale (sitorus, 2021). data analysis data analysis using spss 27 for testing the physical quality of soap such as color, texture, cracking, and foam power tests using the two-way anova test, if there are significant differences, it is further analyzed using dmrt (duncan's multiple range test). meanwhile, the chemical quality test of soap such as foam stability, ph, moisture content, and free alkali uses the kruskal wallis test, if there is a significant difference, it is further analyzed using the mann whitney test. results and discussion solid soap color test based on the results of the spss normality test using kolmogorov-smirnov and shapiro wilk, the data showed a value of <.001. the results showed that the data neisya et al. – effect of mixing time variation and framing time on … 477 was abnormal because the sig value < 0.05. therefore, the data will be followed by non-parametric statistical analysis using the kruskal wallis test to determine the significance. the results of the analysis of the kruskal wallis test showed that the mixing time and framing time obtained a sig value of 0.001. the results showed that there was a significant difference between the mixing time and framing time with the soap color because the sig value < 0.05. there is a significant difference, so it is necessary to carry out further tests using the mann whitney test presented in table 1. table 1. average organoleptic analysis of soap color. framing time mixing time 24,5 minutes 25 minutes 25,5 minutes 3 days 4,50e 4,50e 3,10a 5 days 4,30cde 4,10bcde 3,40ab 7 days 4,40de 3,70abcd 3,50abc control 3,80abcde scale: 1 = not interesting, 2 = less interesting, 3 = quite interesting, 4 = interesting, 5 = very interesting. note: numbers followed by different letters indicate a noticeable difference at the 5% level. the results of the mann whitney test further test analysis in table 1 show that there is a real difference, this is marked by different letter notation in each treatment. the most interesting soap colors based on the acceptance rate of the panelists were mixing time of 24.5 minutes, framing time of 3 days and mixing time of 25 minutes, framing time of 3 days with an average score of 4.50. the mixing time of 24.5 minutes, framing time of 3 days and mixing time of 25 minutes, framing time of 3 days is significantly different from the mixing time of 25 minutes, framing time of 7 days, mixing time of 25.5 minutes, framing time of 3 days, mixing time of 25.5 minutes, framing time of 5 days, and mixing time of 25.5 minutes, framing time of 7 days. solid soap texture test based on the results of the spss normality test using kolmogorov-smirnov and shapiro wilk, the data showed a value of <.001. the results showed that the data was abnormal because the sig value < 0.05. therefore, the data will be followed by non-parametric statistical analysis using the kruskal wallis test to determine the significance. the results of the analysis of the kruskal wallis test showed that the mixing time and framing time obtained a sig value of 0.001. the results showed that there was a significant difference between the mixing time and framing time with the soap texture because the sig value < 0.05. there is a significant difference, so it is necessary to carry out further tests using the mann whitney test presented in table 2. table 2. average organoleptic analysis of soap texture. framing time mixing time 24,5 minutes 25 minutes 25,5 minutes 3 days 3,10a 4,40bc 4,70bc 5 days 4,20bc 4,80c 4,80bc 7 days 4,60bc 4,70bc 4,80bc control 4,30b scale: 1 = very soft, 2 = soft, 3 = moderately soft, 4 = hard, 5 = very hard. note: numbers followed by different letters indicate a noticeable difference at the 5% level. the results of the further analysis of the mann whitney test in table 2 showed that there was a significant difference, this was marked by different letter notation in each treatment. the hardest soap texture based on the acceptance rate of the panelists was a mixing time of 25 minutes, a framing time of 5 days, a mixing time of 25.5 minutes, a framing time of 5 days, and a mixing time of 25.5 minutes, a framing time of 7 days with an average score of 4.80. the mixing time of 25 minutes, framing time of 5 days, mixing time of 25.5 minutes, framing time of 5 days, and mixing time of 25.5 minutes, framing time of 7 days were significantly different from the mixing time of 24.5 minutes, framing time of 3 days, and mixing time of 10 minutes, framing time of 1 day (control). solid soap crack test based on the results of the spss normality test using kolmogorov-smirnov and shapiro wilk, the data showed a value of <.001. the results showed that the data was abnormal because the sig value < 0.05. therefore, the data will be followed by non-parametric statistical analysis using the kruskal wallis test to determine the significance. the results of the analysis of the kruskal wallis test showed that the mixing time and framing time obtained a sig value of 0.006. the results showed that there was a significant difference between mixing time and framing time with soap cracking because the sig value < 0.05. there is a significant difference, so it is necessary to carry out further tests using the mann whitney test presented in table 3. table 3. average organoleptic analysis of soap cracking rate. framing time mixing time 24,5 minutes 25 minutes 25,5 minutes 3 days 2,60c 2,00bc 1,50ab 5 days 2,00bc 1,70ab 1,30ab 7 days 1,20a 1,30ab 1,20a control 1,70ab scale: 1 = no crack, 2 = slightly cracked, 3 = moderately cracked, 4 = cracked, 5 = very cracked. note: numbers followed by different letters indicate a noticeable difference at the 5% level 478 biology, medicine, & natural product chemistry 13 (2), 2024: 475-483 the results of the analysis of the mann whitney test further test in table 3. showing that there is a real difference, this is marked by different letter notation in each treatment. the soap with the smallest crack rate based on the acceptance rate of the panelists was a mixing time of 24.5 minutes, a framing time of 7 days, and a mixing time of 25.5 minutes, a framing time of 7 days with an average score of 1.20. the mixing time of 24.5 minutes, the framing time of 7 days, and the mixing time of 25.5 minutes, the framing time of 7 days are significantly different from the mixing time of 24.5 minutes, the framing time of 3 days, the mixing time of 24.5 minutes, the framing time of 5 days, and the mixing time of 25 minutes, the framing time of 3 days. solid soap foam power test based on the results of the spss normality test using kolmogorov-smirnov and shapiro wilk, the data showed a value of <.001. the results showed that the data was abnormal because the sig value < 0.05. therefore, the data will be followed by non-parametric statistical analysis using the kruskal wallis test to determine the significance. the results of the analysis of the kruskal wallis test showed that the mixing time and framing time obtained a sig value of 0.006. the results show that there is a significant difference between mixing time and framing time with soap foam power because the sig value < 0.05. there is a significant difference, so it is necessary to carry out further tests using the mann whitney test presented in table 4. table 4. average organoleptic analysis of soap foam power. framing time mixing time 24,5 minutes 25 minutes 25,5 minutes 3 days 4,90b 4,70b 4,70b 5 days 4,60b 4,90b 4,50b 7 days 4,80b 4,60b 4,50b control 3,60a scale: 1 = not foamy, 2 = less foamy, 3 = moderately foamy, 4 = foamy, 5 = very foamy. note: numbers followed by different letters indicate a noticeable difference at the 5% level. the results of the mann whitney test further test analysis in table 4 showed that there was a significant difference, this was marked by different letter notation in each treatment. the soap with the most foaming power based on the acceptance rate of the panelists was a mixing time of 24.5 minutes, a framing time of 3 days and a mixing time of 25 minutes, a framing time of 5 days with an average score of 4.90. the mixing time of 10 minutes, the framing time of 1 day (control) was markedly different with all treatments. solid soap foam stability test before conducting the two-way anova test, you must first conduct a prerequisite test, namely a standard residual normality test and a homogeneity test. based on the results of the spss, the normality test using kolmogorov-smirnov obtained a result of 0.193 and shapiro wilk obtained a result of 0.386. the results show that the data is normal because the value of the sig is > 0.05. furthermore, the results of the spss homogeneity test using levene's test obtained a result of 0.878. the results show that the data is homogeneous because the sig value > 0.05. the results of the analysis of the twoway anova test showed that the mixing time obtained a sig value of 0.000. the results show that there is a significant difference between mixing time and foam stability because the sig value < 0.05. at the time of framing it obtained a sig value of 0.001. the results show that there is a significant difference between framing time and foam stability because the sig value < 0.05. the interaction between mixing time and framing time obtained a value of 0.029. the results show that there is a significant difference between the interaction of mixing time and framing time with foam stability because the sig value < 0.05. there is a significant difference, so it is necessary to carry out further tests using dmrt (duncan's multiple range test) presented in table 5. table 5. average stability of solid soap foam framing time mixing time 24,5 minutes 25 minutes 25,5 minutes 3 days 97,67e 92,33cd 91,00c 5 days 95,00de 92,00cd 87,33b 7 days 93,67cd 91,67cd 82,00a control 97,67e note: numbers followed by different letters indicate a noticeable difference at the 5% level. the results of the dmrt (duncan's multiple range test) further test analysis in table 5 showed that there was a significant difference, this was marked by different letter notation in each treatment. the soap with the most stable foam based on the results of laboratory tests is a mixing time of 24.5 minutes, a framing time of 3 days with an average score of 97.67. mixing time 24.5 minutes framing time 3 days is different from mixing time 24.5 minutes framing time 7 days, mixing time 25 minutes framing time 3 days, mixing time 25 minutes framing time 5 days, mixing time 25 minutes framing time 7 days, mixing time 25.5 minutes framing time 3 days, mixing time 25.5 minutes framing time 5 days, and mixing time 25.5 minutes, framing time 7 days. solid soap ph test before conducting the two-way anova test, you must first conduct a prerequisite test, namely the standard residual normality test and the homogeneity test. based on the results of spss, the normality test using kolmogorovsmirnov got a result of 0.064 and shapiro neisya et al. – effect of mixing time variation and framing time on … 479 wilk got a result of 0.075. the results show that the data is normal because the value of the sig is > 0.05. furthermore, the results of the spss homogeneity test using levene's test obtained a result of 0.899. the results show that the data is homogeneous because the sig value > 0.05. the results of the analysis of the two-way anova test showed that the mixing time obtained a sig value of 0.001. the results showed that there was a significant difference between mixing time and ph because the sig value < 0.05. at the time of framing it acquires a sig value of 0.000. the results show that there is a significant difference between framing time and ph because the sig value < 0.05. the interaction between mixing time and framing time obtained a value of 0.042. the results show that there is a significant difference between the interaction of mixing time and framing time with ph because the sig value < 0.05. there is a significant difference, so it is necessary to carry out further tests using dmrt (duncan's multiple range test) which is presented in table 6. table 6. average ph of solid soap framing time mixing time 24,5 minutes 25 minutes 25,5 minutes 3 days 11,800e 11,600e 11,267d 5 days 10,733c 10,667bc 10,500ab 7 days 10,333a 10,300a 10,300a note: numbers followed by different letters indicate a noticeable difference at the 5% level the results of the further analysis of the dmrt (duncan's multiple range test) in table 4.12 showed that there was a significant difference, this was marked by different letter notation in each treatment. soap with a ph that is not too alkaline based on the results of laboratory tests is 25 minutes of mixing time, 7 days of framing time and 25.5 minutes of mixing time, 7 days of framing time with an average score of 10,300. the mixing time of 25 minutes, the framing time of 7 days and the mixing time of 25.5 minutes, the framing time of 7 days are significantly different from the mixing time of 24.5 minutes, the framing time of 3 days, the mixing time of 24.5 minutes, the framing time of 5 days, the mixing time of 25 minutes, the framing time of 5 days, and the mixing time of 25.5 minutes, the framing time of 3 days. solid soap moisture content test before conducting the two-way anova test, you must first conduct a prerequisite test, namely the standard residual normality test and the homogeneity test. based on the results of the spss, the normality test using kolmogorovsmirnov obtained a result of 0.200 and shapiro wilk obtained a result of 0.457. the results show that the data is normal because the value of the sig is > 0.05. furthermore, the results of the spss homogeneity test using levene's test obtained a result of 0.480. the results show that the data is homogeneous because the sig value > 0.05. the results of the analysis of the twoway anova test showed that the mixing time obtained a sig value of 0.001. the results showed that there was a significant difference between mixing time and moisture content because the sig value was <0.05. at the time of framing it obtained a sig value of 0.001. the results showed that there was a significant difference between framing time and moisture content because the sig value was < 0.05. the interaction between mixing time and framing time obtained a value of 0.039. the results show that there is a significant difference between the interaction of mixing time and framing time with moisture content because the sig value < 0.05. there is a significant difference, so it is necessary to carry out further tests using dmrt (duncan's multiple range test) presented in table 7. table 7. average moisture content of solid soap framing time mixing time 24,5 minutes 25 minutes 25,5 minutes 3 days 14,00b 9,67a 9,00a 5 days 14,00b 9,67a 9,00a 7 days 7,33a 9,00a 6,67a note: numbers followed by different letters indicate a noticeable difference at the 5% level. the results of the further analysis of the dmrt (duncan's multiple range test) in table 7 showed that there was a significant difference, this was marked by different letter notation in each treatment. the soap with the smallest moisture content based on the results of laboratory tests was a mixing time of 25.5 minutes, a framing time of 7 days with an average score of 6.67. the mixing time of 25.5 minutes, the framing time of 7 days is significantly different from the mixing time of 24.5 minutes, the framing time of 3 days, and the mixing time of 24.5 minutes, the framing time of 5 days. solid soap free alkaline test before conducting the two-way anova test, you must first conduct a prerequisite test, namely the standard residual normality test and the homogeneity test. based on the results of spss, the normality test using kolmogorovsmirnov got a result of 0.064 and shapiro wilk got a result of 0.075. the results show that the data is normal because the value of the sig is > 0.05. furthermore, the results of the spss homogeneity test using levene's test obtained a result of 0.899. the results show that the data is homogeneous because the sig value > 0.05. the results of the analysis of the twoway anova test showed that the mixing time obtained a sig value of 0.000. the results showed that there was a significant difference between mixing time and free alkali because the sig value was < 0.05. at the time of framing it acquires a sig value of 0.000. the results showed that there was a significant difference between framing time 480 biology, medicine, & natural product chemistry 13 (2), 2024: 475-483 and free alkali because the sig value < 0.05. the interaction between mixing time and framing time obtained a value of 0.030. the results show that there is a significant difference between the interaction of mixing time and framing time with free alkali because the sig value < 0.05. there is a significant difference, so it is necessary to carry out further tests using dmrt (duncan's multiple range test) presented in table 8. table 7. solid soap-free alkaline average. framing time mixing time 24,5 minutes 25 minutes 25,5 minutes 3 days 0,05600b 0,03733a 0,04000a 5 days 0,07200cd 0,06933c 0,06667c 7 days 0,09467e 0,09200e 0,08000d note: numbers followed by different letters indicate a noticeable difference at the 5% level. the results of the dmrt (duncan's multiple range test) further test analysis in table 4.16 showed that there was a significant difference, this was marked by different letter notation in each treatment. the smallest soap with free alkali based on the results of laboratory tests is 25 minutes of mixing time, 3 days framing time with an average score of 0.03733. mixing time 25 minutes framing time 3 days is different from mixing time 24.5 minutes framing time 3 days, mixing time 24.5 minutes framing time 5 days, mixing time 24.5 minutes framing time 7 days, mixing time 25 minutes framing time 5 days, mixing time 25 minutes framing time 7 days, mixing time 25.5 minutes framing time 5 days, and mixing time 25.5 minutes, framing time 7 days. discussion based on the acceptance rate of the panelists, the highest average value of soap color was soap with a mixing time of 24.5 minutes and a mixing time of 25 minutes. the longer the mixing time, the resulting soap is considered to have a less attractive soap color by the panelists. this is because the increase in mixing time causes the color to get darker (sitorus, 2021). the longer the mixing time causes the resulting soap to become darker, while the darker the color of the soap is considered less attractive to the panelists. based on the acceptance rate of the panelists, the highest average value of soap color is soap with a framing time of 3 days. at the framing stage, there is a process in which pure soap liquid becomes a solid form (sitorus, 2021). soap at a 3-day framing time is able to produce solid soap and make the resulting color blend perfectly, so that soap with that time is considered to be able to produce an attractive soap color. in addition, with a framing time of 3 days, the soap making process is more efficient. based on the results of the evaluation, the most optimal soap color is soap with a mixing time of 24.5 minutes and a framing time of 3 days. soap with a shorter mixing time, causes the color in the soap to be less dark, so it is preferred. likewise, soap with a framing time of 3 days is preferred because in a short time, it is able to make the soap making process more efficient. based on the acceptance rate of the panelists, the average value of the highest soap texture was soap with a mixing time of 25 minutes and a mixing time of 25.5 minutes. the longer the stirring time, the lower the moisture content produced (hasibuan et al. 2019). the texture of solid soap is affected by moisture content. the higher the moisture content in the soap, the softer the soap texture, while the lower the moisture content in the soap, the harder the soap texture will be (sinabang et al. 2021). so, the longer the mixing time, the harder the texture of the soap. based on the acceptance rate of the panelists, the highest average value of soap texture was soap with a framing time of 5 days and 7 days. framing time greatly affects the moisture content, because the longer the storage time, the moisture content in the soap will decrease due to evaporation in solid soap (astuti et al. 2021). the longer the framing time, the lower the moisture content in coffee soap (sitorus, 2021). the higher the moisture content in the soap, the softer the soap texture, while the lower the moisture content in the soap, the harder the soap texture will be (sinabang et al. 2021). so, the longer the framing time, the harder the texture of the soap. based on the results of the evaluation, the most optimal soap texture is soap with a mixing time of 25.5 minutes and a framing time of 7 days. soap with a longer mixing time, causes the moisture content to be lower so that the texture is harder. likewise, soap with a longer framing time, the moisture content is lower, causing the texture of the soap to become harder. based on the acceptance rate of the panelists, the average value of the smallest soap crack rate was soap with a mixing time of 24.5 minutes and a mixing time of 25.5 minutes. soap, which is denser and harder, is more likely to experience cracking in dry or wet conditions (sitorus, 2021). the texture of solid soap is affected by moisture content. the higher the moisture content in the soap, the softer the soap texture, while the lower the moisture content in the soap, the harder the soap texture will be (sinabang et al. 2021). the longer the stirring time, the lower the moisture content produced so that the texture of the soap becomes hard, and the soap is more likely to crack (hasibuan et al. 2019). based on the acceptance rate of the panelists, the average value of the smallest soap crack rate was soap with a framing time of 7 days. framing time greatly affects the moisture content. this happens because the longer the framing time, the moisture content in the soap will decrease due to evaporation in solid soap (astuti et al. 2021). the higher the moisture content in the soap, the softer the soap texture, while the lower the moisture content in the soap, the harder the soap texture will be (sinabang et al. 2021). soap, which is denser and harder, is more likely to experience cracking in dry or wet conditions (sitorus, 2021). the longer the framing time can cause the neisya et al. – effect of mixing time variation and framing time on … 481 moisture content to be lower, so that the texture of the soap is harder, and produce a physical soap that tends to crack. however, based on the average results of organoleptic analysis, the framing time that produces the smallest crack is 7 days. based on the evaluation results, the most optimal level of soap cracking is soap with a mixing time of 24.5 minutes and a framing time of 7 days. the longer the mixing time, the lower the moisture content so that the texture of the soap becomes hard, and the soap is more likely to crack. likewise, soap with a longer framing time can cause the moisture content to be lower so that the texture of the soap is harder, and produce a physical soap that tends to crack. however, the results of this study show that the longer the framing time, the lower the cracking produced by soap. this can occur because cracks can be caused by a number of factors such as the shape of the bar (soap), the level of distortion (deviation) of the void during printing (stamping), the composition of the amount of fragrance and additives (sitorus, 2021). based on the acceptance rate of the panelists, the highest average value of soap foam power is soap with a mixing time of 24.5 minutes and a mixing time of 25 minutes. the longer the mixing time, the less soap foam power tends to decrease. this difference in foam power is caused by the difference in alkali content in the soap produced due to the difference in stirring time. the decrease in foam power is also influenced by the content of free fatty acids contained in the soap produced, because the free fatty acids contained in soap can inhibit the clean power of the soap which is characterized by the lack of foam produced (wijana et al. 2019). based on the acceptance rate of the panelists, the highest average value of soap foam power is soap with a framing time of 3 days and 5 days. the longer the framing time causes the foam power to decrease. this happens because the power of the foam is formed during the saponification process (sitorus, 2021). the high foam during the saponification process occurs due to perfectly formed fatty acids and bases. after that, there is a framing process, where the amount of water in the soap will decrease and leave solid ingredients such as fat and alkali, then form a solid and sturdy structure, making the soap dense and durable. if soap with high moisture content is stored in the open, there will be contact with air, so that the soap will shrink in weight and dimension (susanti and juliantoro, 2021). so, the foam power will decrease along with the length of framing time. based on the results of the evaluation, the most optimal soap foam power is soap with a mixing time of 24.5 minutes and a framing time of 3 days. this difference in foam power is caused by the difference in alkali content in the soap produced due to the difference in stirring time. the decrease in foam power is also influenced by the content of free fatty acids which is characterized by the lack of foam produced (wijana et al. 2019). so, the longer the mixing time, the less foam power will be produced. likewise, soap with a faster framing time will have more foaming power. figure 1. physical appearance of solid soap. the results of the various fingerprints show that the mixing time has a real effect on the stability of the foam. the results of the duncan test showed that the mixing time was noticeably different. the highest foam stability value is a mixing time treatment of 24.5 minutes. the stability value of the foam decreases with the length of mixing time. damayanti et al. (2021) stated that the stability of foam is affected by stirring, the longer the stirring and the amount of water-soap ratio and the least active ingredient content contained in the manufacture of solid soap, the stability of the foam decreases. the results of the various fingerprints show that the framing time has a real effect on the stability of the foam. the results of the duncan test show that the framing time difference is noticeable. the highest foam stability value is a 3-day framing time treatment. the stability value of the foam decreases with the length of framing time. the decrease in the stability value of foam is influenced by changes in temperature and shape, and can occur due to the influence of contact of soap with air humidity that occurs during framing time, where co2 gas in the air can react with the water in the soap (lestari et al. 2020). this results in soap foam bubbles that form easily bursting and are less stable over time. the longer the framing time, the more the stability of the foam decreases due to the contact of the soap with environmental conditions such as air temperature and humidity (lubis et al. 2019). the results of the various fingerprints show that the mixing time and framing time have a real effect on the stability of the foam. based on the evaluation results, the most optimal foam stability value is the mixing time treatment of 24.5 minutes and the framing time of 3 days. the stability value of the foam in this study is in accordance with dhara et al. (2023) starting from 60-70%. the results of the variance fingerprint showed that the mixing time had a real effect on the ph content. the results of the duncan test showed that the mixing time was noticeably different. the value with the highest ph content is the mixing time treatment of 24.5 minutes. with increasing stirring time, it can cause a decrease in the ph of the soap produced. the results of this study are 482 biology, medicine, & natural product chemistry 13 (2), 2024: 475-483 in line with (agustina & juliadi, 2021) which states that stirring in the saponification process is intended to increase the interaction between reactant molecules. if there are more interactions between reactant molecules, the amount of soapy oil will also increase so that alkaline residues are reduced and the resulting solid soap products are not too alkaline (dewi & setyawan, 2022). the results of the various fingerprints showed that framing time had a real effect on the ph content. the results of the duncan test show that the framing time difference is noticeable. the value with the highest ph content is a 3day framing time treatment. the ph content value decreases with the length of framing time. the ph value of soap is influenced by the alkalinity content, the ph value increases as the alkalinity increases and decreases with the increase in acidity, besides that the decrease in ph also occurs along with the framing time (wijana et al. 2019). changes in ph values are affected by soap being decomposed by high temperatures when framing time produces acids or bases (permatasari, 2019). the results of the various fingerprints showed that the mixing time and framing time had a real effect on the ph content. based on the results of the evaluation, the most optimal soap ph is the smallest ph in this study, which is 10.3 with a mixing time of 25 minutes and a framing time of 7 days. the 25-minute mixing time has the same ph value as the 25.5-minute mixing time. however, the right optimum time in this study is 25 minutes of mixing time because it is considered to have a more efficient time. the ph of safe soap is 9 11 (astmi, 2015). the results of the various fingerprints showed that the mixing time had a real effect on the moisture content. the results of the duncan test showed that the mixing time was noticeably different. the value with the highest moisture content was the mixing time treatment of 24.5 minutes. with increasing stirring time, it can cause a decrease in the moisture content of the soap produced. based on research conducted by (hasibuan et al. 2019), water content tends to decrease with longer stirring. soap with a low moisture content will have a higher level of hardness so that its shelf life will be longer (dewi & setyawan, 2022). the results of the various fingerprints showed that framing time had a real effect on the moisture content. the results of the duncan test show that the framing time difference is noticeable. the values with the highest moisture content were 3-day and 5-day framing time treatments. with increasing framing time, it can cause a decrease in the moisture content of the soap produced. this is because during the framing period, the water content in the soap evaporates which causes the moisture content in the soap to decrease so that the soap lacks water content and the soap hardens (putri et al. 2023). the results of the various fingerprints showed that the mixing time and framing time had a real effect on the moisture content. the results of this study show that the water content of soap meets sni, which is a maximum of 15% (sni, 2016). therefore, it can be concluded that the moisture content in soap is quite good. based on the results of the evaluation, the most optimal soap moisture content is the smallest moisture content in this study, which is 6.67 with a mixing time of 25.5 minutes and a framing time of 7 days. the longer the mixing time and framing time, the more the moisture content of the soap decreases. the moisture content decreases as time goes by, resulting in the moisture content in the soap evaporating (rosi et al. 2021). the results of the variegated fingerprint showed that the mixing time had a real effect on the free alkali. the results of the duncan test showed that the mixing time was noticeably different. the value with the highest free alkali is the mixing time treatment of 24.5 minutes. the longer the stirring, the greater the interaction time between the oil and the alkali, the reaction will be closer to equilibrium so that the free alkali level in the soap will be reduced. based on research that has been conducted by (hasibuan et al. 2019), the level of free alkali tends to decrease due to the increasing reaction temperature and stirring time in the soap making process. the results of the various fingerprints showed that framing time had a real effect on the moisture content. the results of the duncan test show that the framing time difference is noticeable. as the framing time increases, the free alkali increases. the large amount of alkali in soap is due to the presence of alkali that does not react with fatty acids in the saponification process when framing time occurs (setiawati & ariani, 2020). in addition, the higher the temperature, the greater the free alkali in the soap. the increase in free alkali levels is due to the amount of water that diffuses into the air when framing time occurs in alkaline mixtures (widiyati & wahyuningtyas, 2020). the results of the various fingerprints show that the mixing time and framing time have a real effect on free alkali. the amount of free alkali in soap is a maximum of 0.1% for solid soap, this is in accordance with the results of the study obtained. if it is not in accordance with sni, it can cause irritation to the skin (sni, 2016). therefore, it can be concluded that the free alkali levels in this study are quite good. based on the evaluation results, the most optimal soap-free alkali content is the smallest value in this study, which is 0.03733 with a mixing time of 25 minutes and a framing time of 3 days. the 25-minute mixing time has the same free alkali value as the 25.5minute mixing time. however, the right optimum time in this study is 25 minutes of mixing time because it is considered to have a more efficient time. conclusions based on the results of the analysis, there is a real influence between mixing time and framing time on the physical quality of the soap. based on the acceptance rate of the panelists, the optimal soap color is a mixing time of 24.5 minutes and a framing time of 3 days. the optimum soap texture is a mixing time of 25.5 minutes and a neisya et al. – effect of mixing time variation and framing time on … 483 framing time of 7 days. the optimum cracking rate of soap is a mixing time of 24.5 minutes and a framing time of 7 days. the optimum soap foam power is a mixing time of 24.5 minutes and a framing time of 3 days. based on the results of the analysis, there is a significant influence between mixing time and framing time on the chemical quality of the soap. it is known that in this study, soap is in accordance with quality requirements. the optimum stability of the soap foam is a mixing time of 24.5 minutes and a framing time of 3 days. the optimum ph content of soap is a mixing time of 25 minutes and a framing time of 7 days. the optimum soap moisture content is a mixing time of 25.5 minutes and a framing time of 7 days. the optimum soap-free alkali content is a mixing time of 25 minutes and a framing time of 3 days. competing interests: the authors declare that there are no conflicts of interest in this study. references agustina, n. a., & juliadi, a. r. 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(2020). the utilization of oil palm leaves (elaeis guineensis jacq.) waste as an antibacterial solid bar soap. iop conference series: earth and environmental science, 572(1), 1–10. hasibuan, r., adventi, f., & rtg, r. p. (2019). pengaruh suhu reaksi, kecepatan pengadukan dan waktu reaksi pada pembuatan sabun padat dari minyak kelapa (cocos nucifera l.). jurnal teknik kimia usu, 8(1), 11-17. idoko, o., emmanuel, s. a., salau, a. a., & obigwa, p. a. (2018). quality assessment on some soaps sold in nigeria. nigerian journal of technology (nijotech), 37(4), 1137– 1140. lestari, u., syamsurizal, s., & handayani, w. t. (2020). formulasi dan uji efektivitas daya bersih sabun padat kombinasi arang aktif cangkang sawit dan sodium lauril sulfat. jpscr: journal of pharmaceutical science and clinical research, 5(2), 136-150. lubis, m., suryani, a., kartika, i. a., & hambali, e. 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(2020). optimasi pemanfaatan minyak serai (cyimbopogancitrates dc) sebagai zat antiseptik pada pembuatan sabun lunak herbal. jurnal inovasi proses, 5(1), 1-8. wijana, s., & titik, h. (2019). studi pembuatan sabun mandi cair dari daur ulang minyak goreng bekas (kajian pengaruh lama pengadukan dan rasio air: sabun terhadap kualitas). jurnal teknologi pertanian, 10(1), 54-61. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 13, number 2, october 2024 | pages: 511-516 | doi: 10.14421/biomedich.2024.132.511-516 issn 2540-9328 (online) utilization of maggot larvae (hermetia illucens) for processing food waste at the uin raden intan lampung canteen ika listiana1,*, marlina2, eko kuswanto3, ade lenty hoya4 1,4biology department; faculty of tarbiyah and teaching, uin raden intan, lampung 2,3biology education department; faculty of tarbiyah and teaching, uin raden intan, lampung jl. letnan kolonel h. endro suratmin sukarame lampung, indonesia. corresponding author* ikalistiana@radenintan.ac.id abstract waste management using maggot larvae (hermetia illucens) can be an innovative strategy for reducing organic waste. this study aims to determine the effectiveness of maggot larvae in processing organic waste at the uin raden intan lampung canteen. the research used a completely randomized design (crd) technique with 3 treatments and 3 replications. the observed parameters were duration, residue, and maggot weight. the organic waste used included vegetables, fruits, rice, and bones. the results showed that for bone and rice waste, maggots were able to reduce waste by 80%, producing an average residue of 141.3 grams with a decomposition duration of 515 minutes and a final maggot weight of 320.67 grams. for fruit and vegetable waste, maggots reduced waste by 58%, producing an average residue of 293 grams with a decomposition duration of 395 minutes and a final maggot weight of 560.33 grams. for mixed waste, maggots reduced waste by 81% with a decomposition duration of 496.6 minutes and a final maggot weight of 430.3 grams. keywords: waste; maggot; duration; residue; weight. introduction humans engage in various activities to enhance their well-being by producing goods from natural resources, which also generate waste materials that have the potential to become garbage (winahyu et al., 2014). waste has become a major problem in the environment and society, necessitating effective management solutions. waste is closely related to daily life, so it must be managed and controlled properly (sukerti et al., 2017). waste management is an unresolved issue in both developed and developing countries. poor waste management can increase risks such as flooding and contamination of water and soil. waste disposal aims to avoid negative impacts on health and the environment. the success of a waste management system depends on the efficiency of waste disposal, which requires trained personnel, adequate land, and appropriate infrastructure (kasih et al., 2018). in indonesia, urban solid waste issues are of significant concern due to the substantial waste composition. about 70% of urban solid waste in indonesia is organic waste, 28% is inorganic waste, and the remainder is hazardous waste that requires special handling (prihandoko, 2022). one major issue is food waste, as indonesia is known to be the second-largest contributor to food waste in the world, with an estimated 300 kilograms of food waste generated per person each year (cahyani et al., 2022). food waste can arise from various processes, such as poor supply or distribution systems. products still fit for consumption are often not arranged or rotated properly, leading to food waste (annepu, 2012). if this issue continues to be ignored, its impact on the environment will become increasingly severe, such as air pollution from methane (ch4) and ammonia (nh3) gases produced from decomposing organic waste. these gases can harm health, reduce air quality, and damage the aesthetics and comfort of the environment (puger, 2018). one way to address the problem of organic waste is to convert it into environmentally friendly resources. accumulated organic waste must be utilized to reduce accumulation, for example, through bioconversion using maggot larvae (hermetia illucens). the processing of organic waste with maggot larvae can be carried out in the campus environment of uin raden intan lampung to make the process faster and more efficient (mufti, 2021). the processing and cultivation of maggot larvae (hermetia illucens) have become an increasingly recognized form of creative economy. these maggots act as bio-machines in the processing of organic waste. they are highly active in consuming various easily obtainable organic materials, such as vegetables, fruits, household waste, fishery waste, animal carcasses, and livestock manuscript received: 06 agustus, 2024. revision accepted: 08 october, 2024. published: 30 october, 2024. https://doi.org/10.14421/biomedich.2024.132.511-516 512 biology, medicine, & natural product chemistry 13 (2), 2024: 511-516 manure. this technology enables the decomposition of organic waste quickly, reduces odor, and is sustainable (amandanisa & suryadarma, 2020). waste processing, especially food waste from canteens, is highly needed at uin raden intan lampung. currently, the processing of food waste is not yet optimal, for example, the canteen at the faculty of ushuluddin produces a large amount of food waste daily. based on observations and interviews conducted by researchers with the canteen management, the waste produced consists of vegetables, bones, rice, and fruit. a small portion of this waste is taken home by canteen owners with livestock, while the rest is disposed of in campus-provided facilities (rahayu, 2023). special or environmentally friendly management, such as using maggot larvae (hermetia illucens), can be one solution to food waste. this study aims to determine how maggot larvae (hermetia illucens) can help in processing and decomposing organic waste at the uin raden intan lampung canteen. materials and methods study area the research was conducted in november 2023 at 2 locations. the first location involved collecting organic food waste from 24 canteen units at the faculty of ushuluddin, uin raden intan lampung. the second location was the maggot house (hermetia illucens) at uin raden intan lampung. procedures approach and type of research this research uses a quantitative approach that focuses on variables in human life and analyzes the relationships between these variables using statistical tools and objective theory (hayre and zheng, 2022). this experimental research uses a completely randomized design (crd) method with 3 treatments and 3 replications for each treatment. each medium used weights 0.7 kg or 700 grams, and each therapy involves 300 grams of 12-day-old maggot larvae (hermetia illucens). the treatments applied in this research are as follows: (p1) bone and rice waste medium, (p2) vegetable and fruit waste medium, (p3) mixed vegetable, fruit, rice, and bone waste medium. observation parameters the research parameters observed in this study are the duration of maggot consumption, maggot residue, and maggot weight. tools and materials cthe tools used in this research include maggot bins (hermetia illucens), jars, buckets, digital scales, gloves, masks, trash bags, maggot cages (hermetia illucens), basins, plastic sieves, writing tools for recording observation results, and a mobile phone camera for documenting the research. the materials used in this research include food waste such as vegetables, rice, bones, fruit, and 12-day-old maggot larvae (hermetia illucens). data analysis data analysis is conducted to determine the time required for maggot (hermetia illucens) to consume waste, the residue produced, and the weight of maggot (hermetia illucens) in different media through quantitative data analysis. quantitative data relates to numerical data, whether obtained from measurements, such as measurement or calculation results (notoadmojo, 2010). the data analysis process described in this paragraph includes a homogeneity test using spss 25 software. if the data is found to be homogeneous, a one-way anova test is conducted. if the p-value is less than 0.05, a duncan test is performed at a 5% significance level is performed to determine the differences between treatments. this statistical approach is important for assessing the significance of observed differences in the data, and ensuring the validity and reliability of the research findings (zulias, 2013). results and discussion results duration, residue, and weight of maggots observations on the duration of organic waste processing by maggots (hermetia illucens), the residue produced, and the weight of maggots in each treatment show differences. the treatments consist of p1 (700 grams of rice and bone waste medium), p2 (700 grams of vegetable and fruit waste medium), and p3 (700 grams of a combination of vegetable, fruit, rice, and bone waste), as shown in figure 1 below: figure 1. research data results. listiana et al. – utilization of maggot larvae (hermetia illucens) for processing … 513 durasi of maggots (hermetia illucens) in decomposing organic waste the duration of maggots (hermetia illucens) in decomposing organic waste is the time maggots require to consume the organic waste. observations show the average duration of decomposition varies for each treatment, as seen in figure 2 below: figure 2. the average duration graph of hermetia illucens maggots decomposing organic waste. the duncan test results can be seen in table 1. table 1. duncan test results for maggot duration (hermetia illucens). treatment duration of maggots (hermetia illucens) p1 515b p2 395a p3 496.6b residue produced by maggots (hermetia illucens) in processing food waste residue refers to the remains of waste decomposition by hermetia illucens larvae. observations on the residue produced by maggots in processing food waste from three different treatments show varying average results, as depicted in figure 3 below: figure 3. the average residue graph of maggots (hermetia illucens) the results of the duncan test can be seen in table 2. table 2. duncan test results for maggot residue (hermetia illucens) treatment maggot residue (hermetia illucens) p1 141.3a p2 293a p3 135.6a maggot weight (hermetia illucens) weight is the mass of an organism that has undergone growth (azkaria and herawati, 2021). observation data on the weight of maggots (hermetia illucens) from three different treatments can be seen in figure 4 below: 0 100 200 300 400 500 600 p1 (bone and rice waste medium) p2 (vegetable and fruit waste medium) p3 (mixed vegetable, fruit, rice, and bone waste medium) 515 395 496,6 duration of maggots decomposing organic waste (in minutes) 0 50 100 150 200 250 300 p1 (bone and rice waste medium) p2 (vegetable and fruit waste medium) p3 (mixed vegetable, fruit, rice, and bone waste medium) 141,3 293 135,6 maggot residue in grams 514 biology, medicine, & natural product chemistry 13 (2), 2024: 511-516 figure 4. the average weight graph of maggots (hermetia illucens). the results of the duncan test can be seen in table 3. table 3. duncan test results for maggot weight (hermetia illucens) treatment maggot weight (hermetia illucens) p1 320,67a p2 560,33b p3 430,33b discussion based on figure 2, the average duration of maggots (hermetia illucens) in decomposing organic waste varies. the fastest duration was found in p2 (fruit and vegetable waste medium), with an average of 395 minutes. this is due to the moist characteristics of the food with 60%90% water content, as well as small or mashed pieces that facilitate nutrient absorption by maggots that lack chewing mouths (dewi et al., 2021). next, p3 (mixed waste medium of bones, rice, vegetables, and fruits) had an average duration of 496.6 minutes, slower than p2 because some media in p3 have a harder texture. the slowest duration was found in p1 (bone and rice waste medium) with an average of 515 minutes due to the hard texture of the bones, thus slowing down the decomposition by maggots (hermetia illucens). based on figure 3, maggot (hermetia illucens) residue varies with different treatment media. the research shows that rice and bone waste is reduced by 80%, producing 141.3 grams of residue. fruit and vegetable waste is reduced by 58%, with 293 grams of residue. mixed waste of rice, bones, fruits, and vegetables is reduced by 81%, producing 135.6 grams of residue. the lowest residue is made from mixed waste (135.6 grams), while the highest residue is from fruit and vegetable waste (293 grams) because fruit seeds, fruit peels, and vegetable stems are difficult for maggots to reduce. based on figure 4, p2 (vegetable and fruit waste) showed the highest weight with an average of 560.3 grams, followed by p3 (mixed bones, rice, vegetables, and fruits) with an average of 430.3 grams, and p1 (bones and rice) with the lowest weight of 320.6 grams. the weight in p1 is lower because its nutrients are sufficient to stimulate maggot (hermetia illucens) growth, but the high organic content increases the number of bacteria and organic particles, affecting maggot weight. the results related to the use of maggot larvae (hermetia illucens) to decompose organic waste are simple methods that can be applied by the community, both at the household and industrial scales. maggot larvae cultivation is easy to do at home because the process is simple and requires little capital. with proper management of the larvae life cycle, this method creates environmentally friendly waste processing. maggot larvae do not produce additional waste, and the processing residue, known as frass, can be used as organic fertilizer (raihan, 2022). this research uses waste and maggot larvae in a 2:1 ratio, namely 700 grams of waste (rice, bones, fruits, and vegetables) and 350 grams of 12-day-old maggot larvae per treatment. twelve-day-old maggot larvae are chosen because at 1218 days old, they are effective at consuming and reducing large amounts of organic waste. additionally, maggots are easier to separate from the feed at this age, making it effective for observing residue parameters (yuwita et al, 2022) (bay et al, 2022). regarding the duration of maggots in decomposing organic waste based on analysis of variance (anova), different waste media significantly affect the duration of maggots (hermetia illucens) in decomposing organic waste. maggots (hermetia illucens) will remain in the medium if nutrients and water content are sufficient. optimal water content is key to bioconversion; if water decreases, maggots will stop metabolism, harden, and die. conversely, too much water makes maggots uncomfortable and tend to leave the food source (fahmi, 2018). differences in waste decomposition duration by maggots (hermetia illucens) are due to feed characteristics. effective feed should be moist enough 0 100 200 300 400 500 600 p1 (bone and rice waste medium) p2 (vegetable and fruit waste medium) p3 (mixed vegetable, fruit, rice, and bone waste medium) 320,6 560,3 430,3 maggot weight in grams listiana et al. – utilization of maggot larvae (hermetia illucens) for processing … 515 with 60%-90% water content, and high in protein and carbohydrates for maggot growth. the size of the waste also affects the decomposition speed; waste that is too large takes longer because maggots do not have mouths to chew. therefore, large waste must be ground or chopped to maximize decomposition (yulianingsih and yani, 2023). maggots (hermetia illucens) convert nutrients from organic waste into protein, fat, and other components, while the rest becomes residue from the decomposition process. the research results show that rice and bone waste is reduced by 80% with an average residue of 141.3 grams, fruit and vegetable waste is reduced by 58% with an average residue of 293 grams, and mixed waste of rice, bones, fruits, and vegetables is reduced by 81% with an average residue of 135.6 grams. maggot larvae (hermetia illucens) are most effective in decomposing mixed waste compared to fruit and vegetable waste, which has a residue that is difficult to decompose due to seeds, peels, and stems. the growth of maggot larvae (hermetia illucens) varies in each observation bin, with weight increasing as they grow. observations in figure 4. shows the highest maggot weight in p2 and the lowest in p1, because the feed medium affects maggot growth. nutrients in the feed are crucial for maggot development, if the feed lacks sufficient nutrients, larva growth will be slower (swastoko et al., 2023). p2 medium (fruit and vegetable waste) yields the highest maggot (hermetia illucens) weight compared to other treatments because it contains nutrients and organic matter that support maggot growth. fruit and vegetable waste provides a more complex source of nutrients, increasing the number of bacteria and organic particles from decomposition, which supports maggot growth (faradila et al., 2023), (neneng et al., 2023). conclusions maggots can reduce bone and rice waste by 80%, producing 141.3 grams of residue, with a decomposition duration of 515 minutes and a final weight of 320.67 grams. for fruit and vegetable waste, maggots reduce 58% of the waste, producing 293 grams of residue, with a decomposition duration of 395 minutes, and a final weight of 560.33 grams. for mixed waste, maggots were reduced by 81%, with a decomposition duration of 496.6 minutes and a final weight of 430.3 grams. based on the research results, the author recommends conducting further studies using different media and treatments to make the utilization of maggot larvae (hermetia illucens) in decomposing organic waste more effective. competing interests: all authors declare no competing interests. references amandanisa, a., and suryadarma, p. 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(2022). pendampingan budidaya maggot lalat black soldier fly sebagai pengembangan potensi lokal masyarakat. amalee indones. j. community res. engagem., vol. 3, no. 2, pp. 393–404. doi: https://doi.org/10.37680/amalee.v3i2.1922. zulias, m. z. 2013. mengolah data penelitian menggunakan program sas. jakarta: raja grafindo persada. https://doi.org/10.29080/biotropic.v7i2.1876 https://doi.org/10.29244/jurnal_mpd.v5i2.24626 https://doi.org/10.24252/filogeni.v3i3.35501 https://doi.org/10.37680/amalee.v3i2.1922 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 2, october 2023 | pages: 645-649 | doi: 10.14421/biomedich.2023.122.645-649 issn 2540-9328 (online) potency of biocoagulant from cationic modified starch of balbis banana blossom waste for palm oil wastewater treatment: literature study esa ghanim fadhallah*, alif fikri nur hidayat, arfika julya saffanah, bella amanda iswahyudi, diah shihatin wasalamah, eka cahyani putri, nadila ningtias, yana hijriyatin ummi department of agricultural product technology, faculty of agriculture, universitas lampung, jl. prof. dr. soemantri brojonegoro no. 1, bandar lampung, lampung, 35145, indonesia. corresponding author* esa.ghanim@fp.unila.ac.id manuscript received: 19 september, 2023. revision accepted: 07 december, 2023. published: 31 january, 2024. abstract lampung is one of the provinces producing indonesia's second-largest banana crop. operational activities at the palm oil mill produce a by-product of palm oil mill effluent (pome), which can potentially be the most enormous environmental pollution. communities often use chemicals to treat liquid waste, which causes health problems, cannot be decomposed, and can damage the environment. therefore, treating liquid waste using organic materials that are more environmentally friendly, safe for health and easily degraded is necessary. by modifying cationic starch, natural polymers found in banana blossom waste may be utilized for producing natural biocoagulant that are more effective. this paper aims to review the potential of cationic-modified balbis banana blossom waste starch as a natural biocoagulant for processing palm oil waste. starch was first modified into cationic starch by etherification method with the help of hmmahc cationic reagents. the mechanism of cationic starch as a biocoagulant is the exchange of starch cation ions and waste anions to form bridges between colloidal particles and then form flocs that can precipitate. the implementation technique of this idea is by collaborating with several parties to ensure the successful use of banana hump starch as a biocoagulant material, providing support and facilities for the industry and promoting the use of biocoagulant, conducting literature studies related to the use of banana hump starch as a natural biocoagulant, testing the effectiveness of biocoagulant, implemented in the palm oil processing industry. keywords: banana blossom; biocoagulant; cationic starch; pome. introduction indonesia is home to a predominant presence of banana plants, and one of its prominent regions for banana cultivation is the province of lampung. lampung is the second-largest banana-producing province in indonesia, owing to its highly conducive geographical location, making it suitable for banana cultivation. according to data from the central agency on statistics (2018), the province of lampung has yielded a total banana production of 1,438,559 thousand tons in 2018. bananas are considered one of the tropical fruits that can be processed into food products or consumed fresh. commonly utilized parts of the banana plant include the fruit pulp, the banana heart, and its leaves, while other parts, such as the blossom, often need to be addressed or treated as waste. disposing this waste directly into the environment can lead to pollution, threatening the environmental balance (hanifah et al., 2020). hence, further processing is necessary to mitigate this pollution factor. palm oil mill operations produce primary products such as palm kernel oil (pko), palm kernel (pk), and crude palm oil (cpo), as well as byproducts such as liquid waste, solid waste, and emissions released directly into the atmosphere (ibe et al., 2014). according to ibe et al. (2014), palm oil mill effluent (pome), a sort of liquid waste, is recognized as one of the primary waste streams from the palm oil processing sector with the most significant potential for adverse environmental effects. the potential of liquid waste pollution also arises from the sheer quantity of the generated waste, with the required water around 5-7.5 tons per ton of crude palm oil produced, and more than half of it ended up as pome. bala et al. (2014) reported that pome consists of a viscous brownish liquid with a high-water content (9596%), oil content (0.6-0.7%), and total solids content (45%), primarily derived from fruit debris, and exceptionally high chemical oxygen demand (cod) (cod values frequently exceeding 80,000 mg/l) and biochemical oxygen demand (bod) values. https://doi.org/10.14421/biomedich.2023.122.645-649 646 biology, medicine, & natural product chemistry 12 (2), 2023: 645-649 communities commonly consider this, frequently using chemical substances to treat liquid waste, often requiring a conscious understanding of their potential side effects. for example, using aluminium sulfate in specific quantities and durations can lead to human health disturbances and environmental damage (dwi & gusmawarni, 2021). therefore, there is a need to treat liquid waste using organic materials as coagulants to replace the role of chemical coagulants. this approach is safer for the environment, readily biodegradable naturally, and more cost-effective. banana blossom are considered natural polymers with the potential for development in various industries due to their high starch content. asni (2015) states that the starch content varies across different types of banana blossom. for instance, kepok banana musa acuminata × balbisiana) blossom contain 64.20% starch, ‘mas’ banana (musa acuminata lady finger) blossom contain 67.80% starch, and balbisana banana (musa balbisana) blossom contain 69.13% starch. in order to improve the efficiency of coagulants, the modification of cationic starch modification is needed. in this context, an alternative idea emerges regarding one of the preferred solutions, namely the use of synthetic natural coagulants, including the utilization of biocoagulant derived from natural materials such as banana blossom waste. this waste has often been considered an underutilized resource despite its potential for more cost-effective and environmentally friendly liquid waste treatment. the combination of these materials is intended to enhance the performance of the substances used and address the shortcomings of coagulants or flocculants derived from synthetic sources through an etherification process facilitated by cationic reagents. this paper aims to review the potential of cationicmodified balbis banana blossom waste starch as a natural biocoagulant for processing palm oil waste. methods the method used is an effective literature review following the discussed topic. the method of discussion is based on the research results found by previous study, which are then integrated with other researchers to get solid results and conclusions. results and discussion environmental issue in palm oil industry social issues commonly faced by palm oil companies involve the disposal of liquid and solid waste into rivers, which has garnered serious attention from the indonesian public and government due to its highly detrimental impact on aquatic ecosystems. liquid waste from the palm oil industry is a significant pollutant that can cause adverse environmental effects, particularly within water bodies (chan et al., 2013). the liquid waste generated by the palm oil processing industry is known to be filled with highly concentrated pollutants, resulting in elevated pollution levels in the water, with the lowest recorded total suspended solid (tss) concentration being 5473 mg/l (putra, 2014). the pollution of water bodies stems from the activities of large-scale palm oil plantations operating in the vicinity. land clearing activities lead to the transport of suspended solids, a product of erosion during rainfall events. increased turbidity occurs due to the suspended particles resulting from erosion, which obstructs the penetration of sunlight into the water and reduces the intensity of photosynthesis, consequently impeding phytoplankton growth. considering the high annual production, there is a significant likelihood that the generated waste is substantial, which can impact river water quality, making it cleaner. a coagulant is one of the supplementary substances that can be utilized in clean water treatment. coagulant is a chemical compound added to water to precipitate colloidal particles that are difficult to remove. using coagulants in the coagulation process aims to eliminate charges by compressing or eliminating the diffused layer, leaving only attractive forces among particles (prihatinningtyas, 2013). commonly used coagulants are synthetic, also known as chemical coagulants, such as alum and polyaluminium chloride (pac). some studies have also indicated that alum compounds can trigger alzheimer's disease (putra et al., 2019). monomers present in some synthetic organic polymers, such as pac and alum, are known to possess neurotoxic properties (hendrawati et al., 2013). there is increasing interest in developing natural materials as coagulants due to several advantages, including biodegradability, human safety, and costeffectiveness. according to prihatinningtyas (2013), natural coagulants are readily available as they can be extracted from natural occurring sources, such as plants and animals, including banana plants (musa acuminata). the high banana production in lampung province generates waste in the form of banana peels, stems, leaves, and stalks. therefore, using banana peel starch as a natural biocoagulant could be an alternative for environmentally friendly palm oil waste treatment. banana blossom starch potency as biocoagulant starch is a polysaccharide made up of amylose and amylopectin. amylose is a glucose polymer with a linear structure joined by α-1,4 glycosidic linkages, whereas amylopectin has a branching structure with α-1,6 links (niken & adepristian, 2016). balbis banana is a commodity that contains a significant amount of starch, especially in its unused blossom, with a starch content of 69.13% (nofiandi et al., 2019). starch can be used to treat agro-industrial wastewater, especially palm oil mill effluent, as a coagulant. fadhallah et al. – biocoagulant from cationic modified starch 647 however, before the application, starch needs to undergo physical or chemical modification because, if not modified, it will remain suspended in water, causing turbidity. therefore, a starch modification process is required to enable its use as a natural coagulant. one of the modification methods that can be employed is the use of cationic starch. positive ions have been introduced into cationic starch using functional groups such as amino, imine, ammonium, phosphonium, or sulfonium (din et al., 2015). consequently, quaternary ammonium groups are formed on the starch, making the modified starch capable of adsorbing anionic compounds. researchers have widely employed this method to remove various ions and organic pollutants from wastewater (simanaviciute et al., 2017). furthermore, cationic starch synthesis is relatively cost-effective, exhibits effective coagulation properties, and is readily biodegradable (oladoja, 2015). synthesis of cationic starch cationic starch synthesis is carried out through etherification with the assistance of a cationic reagent. one of the cationic reagents that can be used is hydroxymethyl dimethylamine hydrochloride (hmmahc). the consideration for using hmmahc is based on several advantages, including its costeffectiveness, ease of preparation, and high electrophilic reactivity (jiang et al., 2010). the synthesis of cationic starch is performed using the dry mixing method developed by jiang et al. (2010). the process begins with preparing hmmahc, where a formaldehyde solution is introduced into a flask and stirred. subsequently, dimethylamine is added slowly over 30 minutes. dihydroxymethylation is achieved by gradually adding hydrochloric acid over 30 minutes until hmmahc (ho-ch2-n(ch3)2-hcl) is obtained. hmmahc is then vigorously stirred to ensure a perfect reaction. the temperature is maintained at 5-10°c during the reaction process. next, the synthesis of cationic starch involves mixing banana blossom starch, hmmahc, and water. the water removed by using a rotary vacuum evaporator (60°c) until achieved a constant weight. the mixture is then dried in an oven at high temperature until completely dry. after reaching room temperature, soxhlet extraction with ethanol for 24 hours can be used to extract the product. the subsequent step involves separating chromatography to isolate cationic starch while removing unreacted hmmahc and other byproducts. sen et al. (2017) demonstrating synthesis of cationic starch using base catalyst. cationic starch is produced by adding a positively charged group to the hydrogen atom of the starch hydroxyl group, using naoh as a base catalyst. in a reaction flask, add 5 grams of corn starch, 2.5 grams of gtac (glycidyl trimethyl ammonium chloride) as a cationization reagent, 1.5 ml of 1 mol/l naoh solution, and 1.625 ml of distilled water. then, all reactants in the flask are agitated for 5 hours in a water bath at 60°c. when the reaction is finished, 100 ml of ethanol is added to the flask, allowing the cationic starch to precipitate. to remove unreacted gtac and naoh, the cationic starch is vacuum-filtered and rinsed twice using ethanol. the final product is dried for 6 hours in an oven at 50°c. the resulting white cationic starch is subsequently ground into a fine powder. coagulation mechanism the structure of cationic starch can effectively function as a coagulant through charge neutralization and interparticle bridging. the mechanism of cationic starch absorption of anionic pollutants involves physical adsorption and ion exchange, and it possesses a high adsorption capacity, allowing anionic pollutants to be absorbed into the cationic sites on the surface. longchain polymers of polysaccharide molecules are adsorbed onto the surface of colloidal particles. the unadsorbed "tails" on these colloidal particles can adsorb other colloidal particles, thereby bridging between colloidal particles and forming flocs that can settle (kristianto et al., 2020). tran et al. (2022) reported regarding the application of cationic starch and polyaluminum chloride in wastewater flocculation processes, cationic starch successfully forms bridges to facilitate the aggregation and settling of colloidal particles. additionally, cationic starch can treat a significant amount of nitrogenous and phosphorus compounds in wastewater. the mechanism illustrated in figure 1. figure 1. illustration of interparticle bridging mechanism. further implementation using starch as a biocoagulant material can be implemented by various stakeholders involved in waste management. to realize this concept, the involvement of the government, particularly the ministry of environment and forestry, palm oil processing industries, waste management service providers, banana plantation owners, and banana farmers is essential. industries can leverage this concept to enhance waste treatment 648 biology, medicine, & natural product chemistry 12 (2), 2023: 645-649 efficiency and reduce its adverse environmental impact. the government can provide support and facilities to the industries and promote using starch-based biocoagulant as an environmentally friendly and sustainable waste treatment alternative. banana farmers can also adopt this concept by utilizing banana blossom waste, often considered waste, to produce biocoagulant that can be sold to wastewater treatment companies or other industries. conclusions one of the sources of water pollution is liquid waste from the palm oil industry. this waste is generated due to palm oil plantation activities carried out by several largescale industries around these water bodies. coagulants are chemical substances used in water to precipitate colloidal particles that are difficult to remove. using coagulants in the coagulation process aims to destabilize charges by reducing or eliminating the diffused layer, leaving only attractive forces between particles (prihatinningtyas, 2013). using natural materials as coagulants is being increasingly explored due to several advantages, such as biodegradability, human safety, and improved economic aspects. banana plants, especially banana blossom, can be used as natural coagulants. the province of lampung, which has a high banana production rate, generates waste in the form of banana blossom, stems, leaves, and peels. therefore, using starch from banana blossom as a natural biocoagulant can be an environmentally friendly alternative in palm oil waste treatment. this plan has a high likelihood of success in using banana blossom starch as a biocoagulant material. lampung can serve as a pilot region for this concept's implementation, given that it is the second-largest banana producer in indonesia. parts of banana plants, like banana blossom, are often overlooked or considered waste. advanced treatment is required to prevent environmental pollution resulting from this neglect. one solution is to use organic materials as coagulants in wastewater treatment, which can replace the role of chemical coagulants. this is expected to create a safer and environmentally friendly environment and facilitate natural degradation. banana blossom are natural polymers with high starch content and can be developed for various industrial purposes. this concept is expected to address the pollution issues occurring in indonesia and benefit all stakeholders. authors’ contributions: alif fikri nur hidayat, arfika julya saffanah, bella amanda iswahyudi, diah shihatin, eka cahyani putri, nadila ningtias, and yana hijriyatin ummi wrote the manuscript in bahasa. esa ghanim fadhallah translates to english and proofread the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references alwi, h., idris, j., musa, m., & hamid, k. h. k. 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(2022). study on application of cationic modified starch in combination with poly aluminium chloride for treatment of flocculation of aquatic wastewater. aip conference proceedings, 2610, 0400101 040010-7. https://doi.org/10.1063/5.0099561 https://doi.org/10.22146/jrekpros.57798 https://doi.org/10.1007/s41101-017-0024-4 http://dx.doi.org/10.36434/scientia.v9i1.215 https://doi.org/10.1016/j.jwpe.2015.04.004 https://doi.org/10.22146/teknosains.5999 https://doi.org/10.25077/jfu.3.2.96-101.2014 https://doi.org/10.1016/j.carbpol.2017.04.079 https://doi.org/10.1016/j.ijbiomac.2016.12.006 https://doi.org/10.1063/5.0099561 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1273-1284 | doi: 10.14421/biomedich.2025.142.1273-1284 issn 2540-9328 (online) effect of solvent polarity on extraction yield, phytochemical composition, and antioxidant activity of curcuma xanthorrhiza roxb. and moringa oleifera lam. vina octavia azzahra1*, st mardiana1, suharyadi1, rini juita sianipar1, dwi sapri ramadhan2 1department of chemistry, faculty of mathematics and natural sciences, universitas sumatera utara, medan 20155, indonesia 2department of chemistry, faculty of mathematics and natural sciences, universitas negeri medan, medan 20211, indonesia. corresponding author* vina@usu.ac.id manuscript received: 10 october, 2025. revision accepted: 29 november, 2025. published: 13 december, 2025. abstract the present study investigated the effect of solvent polarity on extraction yield, phytochemical composition, and antioxidant activity of curcuma xanthorrhiza roxb. rhizomes and moringa oleifera lam. leaves. maceration was carried out using ethanol, acetone, ethyl acetate, and n-hexane for 72 hours (1:10 w/v). extraction yield, total phenolic content (tpc), total flavonoid content (tfc), and dpph radical scavenging activity were determined. results demonstrated a clear polarity-dependent trend. ethanol yielded the highest extract recovery for both species (17.55 ± 0.97% and 22.93 ± 0.65%), while n-hexane showed the lowest yield. ethanol extracts exhibited the greatest tpc and tfc values, 47.12 mg gae/g and 6.76 mg qe/g for c. xanthorrhiza, and 25.91 mg gae/g and 4.67 mg qe/g for m. oleifera, respectively. correspondingly, ethanol fractions displayed the strongest antioxidant activity with ic50 values of 22.70 and 29.80 mg/ml, indicating an inverse correlation between phenolic load and radical scavenging capacity. the study confirms that solvent polarity is a critical determinant of phytochemical recovery and antioxidant potency. the novelty of this work lies in the first comparative evaluation of c. xanthorrhiza and m. oleifera extracted under identical solvent systems, providing a rational framework for solvent selection in phytopharmaceutical and nutraceutical applications. further work should isolate and characterize the active antioxidant constituents from the most potent extracts. keywords: antioxidant activity; curcuma xanthorrhiza; extraction yield; moringa oleifera; solvent polarity. abbreviations: tpc = total phenolic content; tfc = total flavonoid content; dpph = 2,2-diphenyl-1-picrylhydrazyl; abts = 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); gae = gallic acid equivalent; qe = quercetin equivalent. introduction oxidative stress, largely stemming from the excessive generation of reactive oxygen species (ros), is widely recognized as a fundamental contributor to cellular damage and a spectrum of chronic diseases, including cancer, diabetes, and cardiovascular disorders. the quest for effective natural antioxidants has thus sparked substantial interest in plant-derived products, which often contain high levels of phytochemicals such as polyphenols and flavonoids capable of neutralizing free radicals and modulate oxidative pathways (el-sherbiny et al., 2024; haroen et al., 2025; lister et al., 2025). within this context, the exploration of medicinal plants as potential antioxidant reservoirs has gained prominence in the search for safer, sustainable alternatives to synthetic antioxidants. curcuma xanthorrhiza roxb. (javanese turmeric) and moringa oleifera lam. are two medicinal plants widely utilized for their diverse bioactive compounds and pronounced antioxidant activities. c. xanthorrhiza, prominent in traditional indonesian medicine, is noted for its curcuminoids, xanthorrhizol, and array polyphenolics that exhibit potent antioxidant, antiinflammatory, and hepatoprotective properties (lister et al., 2025; rosidi et al., 2025; yusnira & ediputra, 2025). beyond its ethnomedicinal role, c. xanthorrhiza is integrated into daily life as a culinary spice and beverage ingredient, and it is extensively exploited in pharmacology, functional foods, and cosmeceutical formulations. aqueous rhizome extracts are rich in primary and secondary metabolites and exhibit a broad spectrum of biological activities, and this attributable to their phytochemical repertoire (abd rashid et al., 2022). likewise, m. oleifera, frequently referred to as the miracle tree, similarly boasts a rich profile of phenolics and flavonoids, particularly in its leaves, which correlate strongly with its robust antioxidant, antibacterial, and cytoprotective properties (el-sherbiny et al., 2024; haroen et al., 2025; liga et al., 2025). the leaves were https://doi.org/10.14421/biomedich.2025.142.1273-1284 mailto:vina@usu.ac.id 1274 biology, medicine, & natural product chemistry 14 (2), 2025: 1273-1284 reported to contain higher levels of minerals, vitamins, and essential phytochemicals than other parts (garofalo et al., 2024). although both species are recognized for their antioxidant potential, comparative evaluation under varying solvent polarity conditions remains limited. efficient extraction of these phytochemicals is crucial for optimizing the therapeutic potential of plant extracts (sun et al., 2025). among various factors influencing extraction yield and composition, solvent polarity is regarded as the most influential. the principle of like dissolves like governs the solubility and yield of different classes of phytochemicals. polar solvents such as methanol, ethanol, water are particularly effective in extracting hydrophilic compounds such as polyphenols and flavonoids, while semi-polar and non-polar solvents such as ethyl acetate or n-hexane favor lipophilic constituents (arya et al., 2025; tourabi et al., 2025). furthermore, the use of intermediate polarity or mixedsolvent systems has been shown to enhance the simultaneous extraction of a broader spectrum of bioactives, leading to increased yield and bioactivity (jaglan et al., 2024). recent studies explicitly support the crucial role of solvent polarity in maximizing extraction efficiency, antioxidant capacity, and phytochemical content of medicinal plant extracts. for instance, ethanol and ethyl acetate have demonstrated superior extraction of phenolic and flavonoid compounds from c. xanthorrhiza, yielding extracts with enhanced free radical scavenging ability (haroen et al., 2025; jonathan & ananingsih, 2025). similarly, ethanolic and methanolic extracts of m. oleifera leaves present higher concentrations of phytochemicals and antioxidant activities in dpph and abts assays compared to less polar extracts (geleta et al., 2025). despite their extensive pharmacological use, direct comparative studies under standardized solvent conditions remain scarce given these considerations, the present study aims to elucidate the influence of solvent polarity on the extraction yield, antioxidant activity, and phytochemical composition of c. xanthorrhiza roxb. and m. oleifera lam. by conducting a parallel evaluation using solvents of differing polarity, this work provides a comparative understanding of solute–solvent interactions in two phytochemically rich medicinal species, offering valuable insights for future optimization of extraction strategies and phytopharmaceutical development. materials and methods materials the rhizomes of c. xanthorrhiza roxb. and the leaves of m. oleifera lam. were collected from deli serdang, north sumatra, indonesia. analytical-grade solvents including ethanol, ethyl acetate, acetone, and n-hexane (merck, germany) were employed for extraction. all other reagents were of analytical purity, including magnesium powder, ferric chloride (fecl3) 5%, hydrochloric acid (hcl) 37%, dragendorff’s, mayer’s, and bouchardat’s reagents, liebermann–burchard and salkowski reagents, ammonia solution (25%), and distilled water. collection and identification of plant materials fresh rhizomes of c. xanthorrhiza and leaves of m. oleifera were harvested in august 2025 from kenangan baru village, percut sei tuan district, deli serdang regency, north sumatra, indonesia (3°36′23.7″n, 98°42′30.2″e). taxonomic identification was performed in the herbarium medanense, universitas sumatera utara, and authenticated by a botanist. voucher specimens were deposited under accession numbers 1152/meda/2025 (c. xanthorrhiza roxb.) and 1153/meda/2025 (m. oleifera lam.). the plant materials were washed with tap water to remove debris, air-dried under shade at ambient temperature (25–28 °c) for seven days, and milled to fine powder using a mechanical grinder. the powdered samples were stored in airtight containers at room temperature until further analysis. preparation of plant extracts extraction was performed separately for each plant using maceration according to the method of harborne, (2008) with minor modifications. twenty five grams (25 g) of powdered sample were soaked in 250 ml of solvent (ethanol, ethyl acetate, acetone, or n-hexane; 1:10 w/v) in sealed erlenmeyer flasks for 72 h at room temperature with intermittent agitation. the mixtures were filtered through whatman no. 1 filter paper, and the filtrates were concentrated under reduced pressure using a rotary evaporator at 40 °c. to ensure complete removal of residual solvent and prevent oxidation of thermolabile constituents, the semisolid extracts were further evaporated under a gentle stream of nitrogen gas (n2). the resulting crude extracts were subsequently air-dried at ambient temperature, weighed, and stored at 4 °c in amber glass vials until further use. determination of extraction yield the extraction yield was determined following the method described by aryanti et al., (2025). the yield was expressed as the percentage ratio between the weight of the dried extract obtained after solvent evaporation and the initial dry weight of the plant material. all yields were calculated on a dry weight basis (w/w) and recorded separately for each solvent type. preliminary phytochemical screening qualitative phytochemical screening was conducted on all solvent extracts of c. xanthorrhiza and m. oleifera to azzahra et al. – solvent polarity and phytochemical composition 1275 identify the major classes of secondary metabolites, including alkaloids, flavonoids, phenolics, terpenoids, steroids, and saponins, following standard procedures with minor modifications (dubale et al., 2023; mohamed et al., 2025; rajkumar et al., 2022). detection of alkaloids (dragendorff’s test) approximately 0.05 g of each extract was dissolved in 1 ml of methanol and filtered. the filtrate was combined with 2 ml of 1% hydrochloric acid and one drop (≈50 µl) of dragendorff’s reagent. the mixture was gently shaken and allowed to stand for 2 minutes. formation of an orange-red or reddish-brown precipitate confirmed the presence of alkaloids, resulting from the complexation of bismuth iodide with the nitrogen atom of alkaloid molecules. a reagent blank without extract served as the negative control. the same procedure was also applied using mayer’s and bouchardat’s reagents to confirm the presence of alkaloids through characteristic whiteyellowish and orange-brown precipitates, respectively. detection of flavonoids (alkaline reagent test) a 0.05 g sample of each extract was dissolved in 1 ml of methanol and treated with two drops of 10% sodium hydroxide (naoh). after 1 minute, several drops of 37% hydrochloric acid (hcl) were added. a transient yellow coloration that disappeared upon acidification indicated the presence of flavonoids, due to the formation and neutralization of flavonoid anions. detection of phenolics (ferric chloride test) two milliliters of 15% ferric chloride (fecl3) solution were added to 1 ml of each extract and mixed thoroughly. the appearance of a dark green color denoted phenolics, whereas a blue-black coloration indicated the presence of tannins. detection of triterpenoid (salkowski reaction) five milligrams (5 mg) of extract were dissolved in 1 ml of chloroform (merck, germany) and sonicated at 40 khz for 5 minutes. the solvent was then evaporated to dryness, and 1 ml of concentrated sulfuric acid (96%) was carefully added along the test tube wall. the solution was heated in a water bath at 75 °c for 2 minutes. the formation of a gray or brownish ring at the interface confirmed the presence of terpenoids. detection of saponins (foam test) an aliquot of 1 ml of extract was dissolved in 8 ml of hot distilled water, filtered, and transferred into a test tube. two milliliters of hot distilled water were added, and the mixture was vigorously shaken for 30 seconds. persistent frothing of 1–10 cm in height that lasted for at least 10 minutes and remained stable upon addition of one drop of concentrated hcl indicated the presence of saponins. determination of total phenolic content (tpc) the total phenolic content of each solvent extract was quantified using the folin–ciocalteu colorimetric assay with slight modifications of (mohamed et al., 2025). approximately 10 mg of the dried extract was dissolved in 10 ml of methanol (99.9 %) and homogenized. the solution was centrifuged at 3000 rpm for 15 min to obtain a clear supernatant. an aliquot of 0.4 ml of this supernatant was mixed with 0.4 ml of folin–ciocalteu reagent (diluted 1:1 v/v with distilled water) and allowed to stand for 5 min in the dark. subsequently, 4.2 ml of sodium carbonate solution (5 % w/v) was added, and the mixture was vortexed for 30 s and incubated for 30 min at room temperature (25 ± 2 °c). the absorbance was measured at 760 nm using a uv–vis spectrophotometer against a reagent blank. a standard calibration curve was constructed using gallic acid solutions in the range of 0–140 mg l-1 (r2 = 0.9667). the total phenolic content of each extract was determined from the calibration curve and expressed as milligrams of gallic acid equivalent per gram of extract (mg gae/g). the concentrations of phenolics were derived from the linear relationship between absorbance and gallic acid concentration using the regression parameters obtained from the standard curve. determination of total flavonoid content (tfc) total flavonoid content was estimated by the aluminum chloride (alcl3) colorimetric method. ten milligrams of each extract were dissolved in 10 ml of ethanol (99.9 %) and homogenized thoroughly. the solution was centrifuged at 3000 rpm for 15 min, and the supernatant was used for color development. an aliquot of 0.5 ml of the supernatant was mixed with 2 ml of distilled water and 0.15 ml of sodium nitrite solution (5 % w/v). after 5 min, 0.3 ml of aluminum chloride solution (10 % w/v) was added, followed by 2 ml of sodium hydroxide (1 % w/v). the total volume was adjusted to 10 ml with distilled water, vortexed, and incubated at room temperature for 30 min. absorbance was read at 415 nm using a uv–vis spectrophotometer against a reagent blank containing all reagents except extract. a standard calibration curve was prepared using quercetin solutions ranging from 0 to 140 mg l-1 (r2 = 0.9825). the total flavonoid content of each extract was calculated from the regression line of the standard curve and expressed as milligrams of quercetin equivalent per gram of extract (mg qe/g). the flavonoid concentrations were derived from the linear relationship between absorbance and quercetin concentration based on the corresponding regression parameters. pearson correlation coefficients (r) were computed to evaluate linear associations between total phenolic content, total flavonoid content, and antioxidant activity (ic50). the strength and direction of the correlations were interpreted based on standard statistical criteria. calculations were performed using microsoft excel 365 2024. 1276 biology, medicine, & natural product chemistry 14 (2), 2025: 1273-1284 results and discussion extraction yield of different solvents extraction yield reflects the overall efficiency of solvent systems in recovering extractable constituents from plant matrices and serves as a preliminary indicator of extraction performance. since the solubility of phytochemicals varies with solvent polarity, solvents of differing polarity often yield distinct quantities of extractable material. in this study, maceration was conducted using four solvents (ethanol, acetone, ethyl acetate, and n-hexane) to evaluate how solvent polarity influences the total extract recovery from c. xanthorrhiza rhizomes and m. oleifera leaves. extraction yield data obtained from maceration (1:10 w/v, 72 h) revealed a consistent solvent-dependent pattern. ethanol produced the highest extract recovery for both c. xanthorrhiza rhizomes and m. oleifera leaves, whereas n-hexane gave the lowest yields (figure 1). for c. xanthorrhiza, mean yields were 17.55 ± 0.97% for ethanol, 15.32 ± 0.77% for ethyl acetate, 15.34 ± 1.14% for acetone, and 9.64 ± 1.63% for n-hexane. while in m. oleifera, ethanol extraction yielded 22.93 ± 0.65%, followed by acetone (11.23 ± 5.11%), ethyl acetate (11.07 ± 2.98%), and n-hexane (8.05 ± 0.35%). when compared to n-hexane, ethanol increased extraction recovery by approximately 81.9% for c. xanthorrhiza and 184.9% for m. oleifera, equivalent to 1.8and 2.9fold higher yields, respectively. these data clearly demonstrate that solvent polarity plays a crucial role in determining extraction efficiency, with ethanol being the most effective solvent under maceration conditions. figure 1. extraction yield (%) of curcuma xanthorrhiza rhizomes and moringa oleifera leaves obtained by maceration (1:10 w/v, 72 h) using four solvents of differing polarity. error bars represent standard deviations of duplicate determinations (n = 2). this pattern aligns with previous reports showing that polar protic solvents, particularly ethanol and aqueous ethanol mixtures, generally yield higher extract recoveries from both leafy and rhizomatous plant matrices. such solvents effectively solubilize a wide range of polar and moderately polar compounds while promoting cell-wall swelling and diffusion. comparative extraction studies on phenolic-rich plants support this observation, confirming that ethanol and other polar solvents often achieve greater total extract weight and phenolic recovery than nonpolar solvents (alara et al., 2021; borges et al., 2020). the observed increase in extraction yield with more polar solvents can be explained by molecular-level interactions between the solvent and plant metabolites. mechanistically, extraction yield is largely governed by the polarity of the solvent and the strength of solute– solvent interactions (chathoth et al., 2025; nawaz et al., 2020). solvents with high polarity and strong hydrogenbonding ability are more efficient at dissolving polar phytochemicals such as phenolics, flavonoid glycosides, and other oxygenated secondary metabolites. conversely, nonpolar solvents tend to extract lipophilic constituents, including fatty acids, terpenes, and hydrocarbons, which are present in smaller quantities in most medicinal plant matrices (bitwell et al., 2023). ethanol, a polar protic solvent, exhibits both hydrogen-bond donor and acceptor properties, enabling it to interact with a wider range of chemical constituents than n-hexane, which is completely nonpolar (shi et al., 2022). consequently, ethanol enhances the solvation of diverse phytochemical classes and facilitates diffusion through plant cell walls. moreover, solvent physical characteristics particularly dielectric constant, viscosity, and surface tension collectively influence the solvent’s ability to penetrate the plant matrix and desorb intracellular compounds, thereby determining overall extraction efficiency (acree & lang, 2023; dai & mumper, 2010; shi et al., 2022). nevertheless, solvent polarity is not the only determinant of yield. several comparative studies report exceptions in which medium-polarity solvents or even nonpolar solvents produce higher yields for particular matrices or target classes (alara et al., 2021; bitwell et al., 2023; tripathi et al., 2025). for example, acetone or hexane may give larger yields when the sample is rich in lipids, waxes or nonpolar terpenoids, conversely, aqueous ethanol mixtures often outperform absolute ethanol for recovering phenolics because a small fraction of water improves solute diffusivity and matrix swelling (quitério et al., 2022; zarrinmehr et al., 2022). these context-dependent outcomes have been observed across crops and medicinal plants and are emphasized in recent comparative reviews. therefore, while the dominant yields in ethanol solvent are expected have rich phenolic compound in matrices such as m. oleifera and c. xanthorrhiza, specific compound classes such as volatile oils or nonpolar terpenes could be better recovered by less polar solvents in targeted extractions. comparative literature on each species reinforces the results. reports on m. oleifera commonly show that azzahra et al. – solvent polarity and phytochemical composition 1277 hydroalcoholic and polar organic solvents produce the highest extract yields and phenolic recovery, with consequent stronger antioxidant activity in dpph and related assays (pop et al., 2022). for curcuma species, polar solvent extracts typically yield higher total extract mass and capture curcuminoids and phenolics effectively, although the optimal solvent strength depends on the matrix and target analytes (budiansyah et al., 2023). these parallels validate the present observations and justify ethanol as an appropriate general-purpose extraction solvent for the dual aims of high yield and recovery of antioxidant phytochemicals. from a practical standpoint, solvent selection should correspond to the intended extraction goal. for broad recovery of phenolic antioxidants, ethanol in aqueous mixtures between 40 and 80% is preferable because it offers an effective balance of polarity, safety, and compatibility with downstream analysis (urías-orona et al., 2017). studies have shown that water–ethanol mixtures often yield higher total phenolic content and antioxidant activity than absolute ethanol due to improved solute diffusion and matrix swelling. in contrast, nonpolar solvents such as n-hexane or dichloromethane are suitable for isolating volatile oils and fatty acids, although their total extract yields are generally lower and less correlated with antioxidant capacity (quitério et al., 2022). extraction efficiency also depends on parameters such as particle size, extraction time, temperature, and solvent-to-solid ratio (jaglan et al., 2024). employing optimization techniques such as response surface methodology can help determine the most effective combination of factors for maximizing yield and antioxidant recovery. it should be noted that extraction yield alone is not a direct indicator of bioactive content, therefore, yield data must be interpreted alongside phytochemical and bioactivity analyses. the present results, obtained through maceration at room temperature using organic solvents, may differ under hydroalcoholic or advanced extraction systems such as sonication, soxhlet, or accelerated solvent extraction. phytochemical composition and solvent polarity effects qualitative phytochemical screening was performed to identify the major classes of secondary metabolites present in the extracts and to examine how solvent polarity influenced their distribution. the colorimetric and precipitation assays followed established phytochemical protocols with slight modifications (alara et al., 2021). these methods remain widely used as preliminary approaches to characterize plant metabolites and to guide further chromatographic and spectrophotometric analysis. the qualitative distribution of secondary metabolites in the extracts of c. xanthorrhiza and m. oleifera is summarized in table 1. while the corresponding heatmap visualization (figure 2) illustrates the solvent-dependent variation in metabolite intensity across phytochemical classes. table 1. preliminary phytochemical screening of c. xanthorrhiza rhizomes and m. oleifera leaves extracted with solvents of varying polarity. phytochemical m. oleifera c. xanthorrhiza ethanol ethyl acetate acetone n-hexane ethanol ethyl acetate acetone n-hexane flavonoids (yellow color) ++ + + – ++ ++ ++ – phenolics (blue color) ++ + + – ++ ++ ++ – alkaloids (mayer, whitish precipitate) ++ + + + + + + + alkaloids (dragendorff, reddishbrown precipitate) ++ ++ ++ +++ + + + – alkaloids (bouchardat, orangebrown precipitate) +++ ++ + + + + + – triterpenoids (reddish-brown precipitate) +++ +++ ++ + ++ + + + saponins (foam test) + – – – + – – – symbols represent relative intensity: + = low, ++ = moderate, +++ = high, and – = not detected. results are based on single determinations and indicate observed distribution trends rather than statistically validated frequencies. the screening revealed a solvent-dependent pattern of metabolite distribution. polar ethanol fractions produced the strongest reactions for flavonoids, phenolics, and saponins, while nonpolar n-hexane extracts were largely inactive. ethanol yielded the most intense coloration for both plants, consistent with the known solubility of phenolic hydroxyl-bearing metabolites in polar protic solvents (pop et al., 2022). the absence of these classes in n-hexane indicates that nonpolar solvents fail to extract hydrophilic compounds. similar findings were reported for ethanol and aqueous-ethanol extracts of dracaena angustifolia, moringa and curcuma species, where polar solvents efficiently solubilized polyphenols and glycosides (karta et al., 2024; nurcholis et al., 2023; pop et al., 2022). 1278 biology, medicine, & natural product chemistry 14 (2), 2025: 1273-1284 figure 2. heatmap visualization of the semi-quantitative phytochemical distribution in m. oleifera (m.o) leaves and c. xanthorrhiza (c.x) rhizomes extracted with solvents of differing polarity. grayscale intensity represents metabolite abundance on a qualitative scale; ee: ethanol extract; eae: ethyl acetate extract; ae: acetone extract; he: n-hexane extract. triterpenoids exhibited moderate to strong reactions across both polar and medium-polarity solvents, consistent with their amphiphilic nature. the mixed solubility of these metabolites explains why ethyl acetate and acetone extracts retained notable triterpenoid presence, while n-hexane yielded only weak reactions. the detection of alkaloids in both polar and mediumpolarity fractions suggests a diverse alkaloid profile containing bases of differing polarity. the strong dragendorff reaction observed in the n-hexane fraction of m. oleifera may result from the partial extraction of lipophilic alkaloid free bases or from cross-reactivity with nitrogen-containing terpenoids (nazar et al., 2020; ngo et al., 2023). the selective detection of saponins exclusively in the ethanol extracts is chemically consistent with their polar glycosidic structure, as these compounds require a polar solvent to disrupt cell membranes and release surfactantactive metabolites. previous work on curcuma longa and m. oleifera leaves also demonstrated that ethanol and methanol extracts contain abundant saponins, phenolics, and flavonoids compared with less polar solvents (fachriyah et al., 2020; grover et al., 2021; royani et al., 2023; wihanto et al., 2023). these findings confirm that solvent polarity plays a decisive role in determining the recovery of functional phytochemical classes. polar solvents facilitate the extraction of hydrophilic antioxidant metabolites such as phenolics, flavonoids, and saponins, whereas nonpolar solvents primarily recover lipophilic constituents like terpenes and hydrocarbons. this solvent-dependent distribution pattern establishes a chemical basis for the quantitative variations observed in total phenolic and flavonoid contents presented in the following section. a comprehensive evaluation of these quantitative parameters will further clarify how solvent polarity not only governs extract composition but also modulates antioxidant potency across both species. total phenolic and flavonoid contents quantitative determination of total phenolic content and total flavonoid content refines the qualitative patterns observed earlier and provides a basis for predicting antioxidant potential. comparative values for both species and solvents are presented in table 2 and show a consistent decline in both phenolic and flavonoid totals as solvent polarity decreases, ethanol producing the highest values and n-hexane the lowest. this solventdependent trend in class totals is commonly reported in extraction studies and reviews. for m. oleifera leaves the measured total phenolic content was 25.91 mg gae/g in the ethanol extract, 18.78 mg gae/g in acetone, 17.03 mg gae/g in ethyl acetate and 16.98 mg gae/g in n-hexane. total flavonoid contents in the same order were 4.67, 4.16, 3.94 and 3.37 mg qe/g. for c. xanthorrhiza rhizomes the corresponding phenolic totals were 47.12, 42.62, 37.12 and 35.62 mg gae/g and flavonoid totals 6.76, 6.14, 5.28 and 4.90 mg qe/g. these profiles confirm that polar ethanol preferentially extracts phenolic and flavonoid constituents under the present maceration conditions (dirar et al., 2019; haroen et al., 2022). to quantify the solvent effect, ethanol versus nhexane relative increases in total phenolic content are 52.6 percent for m. oleifera and 32.3 percent for c. xanthorrhiza. relative increases in total flavonoid content for ethanol versus n-hexane are 38.5 percent for m. oleifera and 38.0 percent for c. xanthorrhiza. these calculated differences indicate that flavonoid extraction responds similarly to solvent polarity in both species while the bulk phenolic response is proportionally larger in m. oleifera. species specific chemistry and matrix factors commonly explain such proportional differences (haroen et al., 2022; yodi et al., 2023). azzahra et al. – solvent polarity and phytochemical composition 1279 table 2. total phenolic and flavonoid contents of m. oleifera leaves and c. xanthorrhiza rhizomes extracted using solvents of varying polarity. plant solvent total phenolic content (mg gae/g extract) total flavonoid content (mg qe/g extract) m. oleifera ethanol 25.91 4.67 acetone 18.78 4.16 ethyl acetate 17.03 3.94 n-hexane 16.98 3.37 c. xanthorrhiza ethanol 47.12 6.76 acetone 42.62 6.14 ethyl acetate 37.12 5.28 n-hexane 35.62 4.90 values are expressed as single determinations. gae: gallic acid equivalent; qe: quercetin equivalent. the data illustrate solventdependent trends rather than statistically validated differences. the markedly higher absolute tpc and tfc measured in c. xanthorrhiza merit specific consideration. rhizomes of curcuma species are known to accumulate curcuminoids and other oxygenated phenolic sesquiterpenes that react strongly in the folin–ciocalteu and aluminum chloride assays, which can drive elevated spectrophotometric totals compared with leafy matrices (segneanu et al., 2022; widyastuti et al., 2020). variation in cultivar, harvest time, drying and sample preparation further modulates absolute concentrations and can produce interstudy differences in reported totals (suryani et al., 2022). consequently elevated spectrophotometric totals in rhizome extracts are chemically plausible but require compound-level confirmation. methodological caveats limit the interpretation of spectrophotometric totals. the folin–ciocalteu assay measures total reducing capacity and therefore responds to nonphenolic reducing agents such as ascorbic acid and certain oxygenated metabolites, which can inflate apparent phenolic values (torres et al., 2024). the aluminum chloride colorimetric method is selective for certain flavonoid structural classes and its response varies with substitution pattern and coextractives (dominguez-lópez et al., 2024). for these reasons tpc and tfc should be regarded as comparative class-level indicators rather than absolute molar inventories. confirmatory chromatographic analysis using hplc with diode array detection and lc–ms is recommended to identify and quantify the individual phenolics and flavonoids responsible for the observed totals. the practical significance of these quantitative findings lies in guiding solvent selection for targeted phytochemical recovery. ethanol and aqueous ethanol systems are recommended for the efficient extraction of antioxidant phenolics and flavonoid glycosides, whereas solvents of intermediate polarity may preferentially concentrate certain triterpenoids, and nonpolar solvents tend to recover volatile constituents that contribute little to overall phenolic yield. prior to formulation or preparative applications, replicate extractions and appropriate statistical validation are essential to confirm the consistency of these patterns and substantiate any comparative claims regarding solvent performance. the following section explores how these quantitative phenolic and flavonoid profiles relate to the antioxidant potency of each extract. effects of solvents on antioxidant activity the antioxidant activity of all solvent extracts was evaluated using the dpph radical scavenging assay, and the results are summarized in table 3. the ic50 value, defined as the concentration required to inhibit 50% of dpph radicals, serves as an inverse indicator of antioxidant potency, where lower ic50 denotes stronger activity (baliyan et al., 2022). ic50 values were obtained from linear regression of inhibition versus concentration plots. under the present maceration conditions, ethanol extracts exhibited the strongest dpph scavenging for both species, with ic50 values of 29.80 mg ml-1 for m. oleifera and 22.70 mg ml-1 for c. xanthorrhiza, while nhexane fractions showed the weakest activity with ic50 values near 100 mg ml-1 for both species. these solvent trends mirror the tpc and tfc patterns reported above and are consistent with many comparative extraction studies reporting ethanol extracts exhibited the highest antioxidant capacity, whereas nonpolar fractions displayed the weakest response, confirming that solvent polarity substantially influences antioxidant performance (haroen et al., 2025; segwatibe et al., 2023). to examine quantitative relationships between phytochemical load and radical scavenging, pearson correlation coefficients (r) were calculated between spectrophotometric totals and ic50 across the four solvents for each species (figure 3). for m. oleifera the correlation between total flavonoid content and ic50 was strong and negative, r = −0.96, indicating that higher flavonoid content is associated with lower ic50 and therefore greater scavenging potency; the corresponding correlation between total phenolics and ic50 was moderately negative, r = −0.68. for c. xanthorrhiza strong negative correlations were observed as well, r = −0.85 for total phenolics versus ic50 and r = −0.89 for total flavonoids versus ic50. when the data for both species were pooled the correlations weakened, r = −0.42 for phenolics and r = −0.63 for flavonoids, this attenuation indicates that the strength of these relationships is species-specific, reflecting distinct 1280 biology, medicine, & natural product chemistry 14 (2), 2025: 1273-1284 phytochemical compositions and antioxidant mechanisms between m. oleifera and c. xanthorrhiza. these results are consistent with previous reports demonstrating inverse correlations between spectrophotometric phenolic metrics and dpph ic50 while emphasizing that the strength of the relationship depends on the chemical identity of the phenolics present (ouamnina et al., 2024; rangani et al., 2023). table 3. antioxidant activity (ic50) of m. oleifera leaf and c. xanthorrhiza rhizome extracts obtained with solvents of varying polarity, and their correlation with total phenolic and flavonoid contents. plant solvent ic50 (mg/ml) antioxidant activity r (tpc vs ic50) r (tfc vs ic50) m. oleifera ethanol 29.80 strong −0.68 −0.96 acetone 44.71 moderate ethyl acetate 52.90 moderate n-hexane 100.00 weak c. xanthorrhiza ethanol 22.70 strong −0.85 −0.89 acetone 30.83 moderate ethyl acetate 48.14 moderate n-hexane 93.94 weak lower ic50 values indicate higher antioxidant potency. “antioxidant activity” is classified as: <50 mg/ml = strong, 50–100 mg/ml = moderate, and >100 mg/ml = weak. pearson correlation coefficients (r) represent linear relationships between total phenolic content (tpc) or total flavonoid content (tfc) and ic50 for each species. all values are based on single determinations; correlations are indicative and not inferential due to limited replication. the inverse correlations depicted in figure 3 reinforce that phenolic and flavonoid constituents are major contributors to the antioxidant potential of both species. these metabolites act primarily through hydrogen atom transfer and single electron donation, mechanisms that underpin the dpph radical scavenging assay. stronger correlations for flavonoids than for total phenolics may reflect the higher reactivity of specific flavonoid subclasses, such as quercetin derivatives in moringa and curcuminoids in curcuma, which possess multiple hydroxyl and methoxy substituents enhancing redox potential (el-sherbiny et al., 2024; pop et al., 2022). although these findings clearly demonstrate a structure–activity association, the correlations are based on single determinations and thus should be considered indicative rather than inferential. future analyses using replicated extractions and compound-specific antioxidant testing would provide more definitive quantitative confirmation. figure 3. scatter plots showing the relationships between (a) total phenolic content (tpc) and antioxidant activity (ic₅₀), and (b) total flavonoid content (tfc) and antioxidant activity (ic₅₀) for moringa oleifera leaves and curcuma xanthorrhiza rhizomes extracted with solvents of differing polarity. beyond simple totals, several factors can explain variance in ic50 across solvents and species. first, the dpph assay measures hydrogen or electron donation in an organic medium and therefore responds differently to structural subclasses of phenolics, flavonoids with orthodihydroxy substitution patterns and conjugated systems typically show stronger activity than simple phenolic acids (gulcin & alwasel, 2023; qi et al., 2025). this structural dependence explains why similar tpc values can have different ic50 depending on compound identity. second, nonphenolic antioxidants such as ascorbic acid and certain terpenoids can contribute to dpph azzahra et al. – solvent polarity and phytochemical composition 1281 scavenging and may inflate apparent activity relative to what spectrophotometric tpc predicts (gulcin, 2025; setiawan et al., 2023). this is particularly relevant for rhizome extracts that may contain highly active curcuminoids and other oxygenated sesquiterpenes. third, assay artefacts including solvent–dpph interactions, differing extract colors, and the fixed dpph concentration used can influence ic50 estimates and complicate cross-study comparisons unless standardized units or the antioxidant activity index are reported (martinez-morales et al., 2020). for correct interstudy comparisons, standardization of dpph concentration and reporting of the antioxidant activity index is recommended. the pattern observed, therefore supports a mechanistic interpretation in which polar solvent fractions, enriched in flavonoids and phenolic glycosides, achieve greater dpph scavenging, while nonpolar fractions dominated by hydrocarbons and nonpolar terpenes exhibit weak radical scavenging. however, to move from correlation to causation it is necessary to isolate and test major constituents, for example by hplc fractionation followed by on-line or off-line dpph assays, and to perform replicate biological testing. such targeted work has frequently revealed that a small number of potent molecules, rather than bulk phenolic mass alone, drive antioxidant potency in complex extracts (al-shebli & al-anbari, 2023; deepali et al., 2025). the overall findings indicate that solvent polarity markedly governs antioxidant performance, with polar ethanol extracts exhibiting superior radical scavenging efficiency compared to less polar fractions. these results collectively demonstrate that the distribution and reactivity of antioxidant constituents are strongly solvent-dependent, providing a mechanistic link between extract composition and bioactivity. conclusions the present study demonstrated that solvent polarity plays a decisive role in determining extraction yield, phytochemical composition, and antioxidant activity in c. xanthorrhiza rhizomes and m. oleifera leaves. among the four solvents tested, ethanol consistently produced the highest extract recovery, total phenolic, and total flavonoid contents, while n-hexane yielded the lowest values.the strong solvent-dependent pattern observed across all analyses reflects the chemical affinity between solvent polarity and metabolite solubility. polar protic solvents such as ethanol effectively extracted hydrophilic antioxidant compounds including phenolics, flavonoids, and saponins, whereas nonpolar solvents preferentially recovered lipophilic terpenes and hydrocarbons. the quantitative data confirmed that higher phenolic and flavonoid concentrations corresponded with stronger antioxidant activity, as indicated by lower ic50 values in the dpph assay, and strong negative correlations between ic50 and both tpc and tfc. these findings highlight ethanol as the most suitable solvent for the broad recovery of antioxidant constituents from both species, offering a balance of extraction efficiency, safety, and bioactive compound selectivity. the results provide a scientific basis for optimizing solvent systems in phytochemical extraction and formulation processes. future research should focus on compound-level identification through chromatographic and spectroscopic techniques, along with biological validation of the major antioxidant constituents responsible for the observed activity. acknowledgements: the authors gratefully acknowledge financial support from the penelitian dosen pemula (pdm) grant scheme, faculty of mathematics and natural sciences, universitas sumatera utara, under contract no. 9/un5.4.10.s/ppm/kptalenta/pdm/2025. the authors also thank the herbarium medanense, universitas sumatera utara, for assistance in plant identification and authentication, and the laboratory of natural product chemistry for providing analytical facilities used in this research. authors’ contributions: vina octavia azzahra conceived and designed the study, supervised the experimental work, performed data interpretation, and prepared the final manuscript. st. mardiana contributed to the methodological framework, validation of analytical procedures, and critical revision of the manuscript. suharyadi assisted in data curation, statistical analysis, and literature review. rini juita sianipar carried out laboratory experiments, sample preparation, and extraction procedures under supervision. dwi sapri ramadhan contributed to manuscript structuring, scientific editing, and discussion refinement for journal submission. all authors have read and approved the final version of this manuscript. competing interests: the authors declare that there are no competing interests related to the content of this manuscript. funding: this work was supported by the penelitian dosen pemula (pdm) grant scheme, faculty of mathematics and natural sciences, universitas sumatera utara (contract no. 9/un5.4.10.s/ppm/kptalenta/pdm/2025). references abd rashid, s. n. a., hasham, r., abd. rashid, z. i., cheng, k. k., aziz, a. a., shafin, n., & kaprawi, a. a. 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(2022). the effect of solvents polarity and extraction conditions on the microalgal lipids yield, fatty acids profile, and biodiesel properties. bioresource technology, 344, 126303. https://doi.org/10.1016/j.biortech.2021.126303 https://doi.org/10.24843/jpsa.2023.v05.i02.p05 https://doi.org/10.1007/s11356-022-23337-6 https://doi.org/10.3389/fphar.2025.1615338 https://doi.org/10.13057/biodiv/d230434 https://doi.org/10.1016/j.algal.2024.103503 https://doi.org/10.1186/s40643-025-00859-8 https://doi.org/10.12944/crnfsj.13.2.27 https://doi.org/https:/ve.scielo.org/pdf/alan/v67n3/2309-5806-alan-67-03-211.pdf https://doi.org/https:/ve.scielo.org/pdf/alan/v67n3/2309-5806-alan-67-03-211.pdf https://doi.org/10.22146/ijcpa.507 https://doi.org/10.20473/ijtid.v11i1.42672 https://doi.org/10.13057/biodiv/d241203 https://doi.org/10.29303/jppipa.v11i8.12108 https://doi.org/10.1016/j.biortech.2021.126303 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 33-44 | doi: 10.14421/biomedich.2023.121.33-44 issn 2540-9328 (online) solanum anomalum leaf extract and fractions attenuate oxidative stress and liver injuries in alloxan-induced diabetic rats idongesit charles etuk1, john akpan udobang2, nwakaego omonigho ebong3, jude efiom okokon1,* 1department of pharmacology and toxicology, faculty of pharmacy, university of uyo, uyo, nigeria. 2department of clinical pharmacology and therapeutics, faculty of basic clinical sciences, university of uyo, uyo, nigeria. 3department of pharmacology and toxicology, faculty of pharmacy, madonna university, elele campus. rivers state, nigeria. corresponding author* judeefiom@yahoo.com manuscript received: 03 september, 2022. revision accepted: 20 september, 2022. published: 04 october, 2022. abstract the leaf of solanum anomalum used in ethnomedicine for the treatment of various ailments such as diabetes was evaluated for antioxidative stress and hepatoprotective potentials against hepatic injuries in alloxan-induced diabetic rats. antioxidative stress and hepatoprotective activities of leaf extract and fractions (70-210 mg/kg) were assessed by determining oxidative stress markers levels, liver function indices and histopathological study of livers of treated rats. the leaf extract and fractions caused significant (p<0.05 – 0.001) increases in the levels of oxidative stress markers (sod, cat, gpx, gsh) in the livers of the treated diabetic rats. the extract/fractions treatment caused reduction in liver enzymes (alt, ast and alp), total and direct bilirubin. histology of the livers revealed absence or significant reductions in pathological features in the treated diabetic rats compared to untreated diabetic rats. the results show that the leaf extract and fractions of s. anomalum has antioxidative stress and hepatoprotective potentials which may be due to the antioxidant activities of their phytochemical constituents. keywords: solanum anomalum; medicinal plant; liver protective; antioxidant; antioxidative stress. abbreviations: gas chromatography mass spectrometry (gcms) introduction diabetes mellitus is a chronic metabolic disorder which is associated with increased generation of free radicals especially reactive oxygen species (ros) (okutana et al., 2005). alloxan biotransformation to dialuric acid in the body is accompanied by generation of h2o2, •oh and superoxide radicals via iron catalyst which attack organs like kidney, liver and pancreas etc (mathews and leiter, 1999) and cause oxidative stress which is implicated in the pathogenesis of diabetes complications in animals and humans (baynes and thorpe,1999). ros produce cellular and tissue injury through covalent binding, dna strand breaking, lipid peroxidation and augment fibrosis which is also implicated in other disease conditions (paradies et al., 2011). medicinal plants which are used traditionally in the management of diabetes, therefore serve as a great repository for antidiabetic remedies having the advantage of being safer and providing many therapeutic effects. solanum anomalum thonn. ex schumach, a plant whose fruits and leaves are used medicinally and nutritionally, is commonly found growing in west and east africa sub-regions. its parts are utilised locally to treat diabetes, gastrointestinal disorders, infections, inflammation and pains (burkill, 2000; bukenya and hall, 1988; offor and ubengama, 2015). hypoglycemic and antidiabetic activities of the fruits and leaves have been reported (offor and ubengama ,2015; okokon et al., 2022). more so, in vivo and in vitro antiplasmodial (okokon et al., 2016; okokon et al., 2017a), antioedema (okokon et al., 2017b), antioxidant and antiulcer (okokon et al., 2019a), anticonvulsant and depressant (okokon et al., 2019b), analgesic (okokon et al., 2020) and antidiarrhoeal (udobang et al., 2022) properties of the leaf extract have also been reported. phytochemical constituents such as alkaloids, flavonoids, saponins, tanins, diosgenin, a diosgenin glycoside (25(r)-diosgenin-3-o-α-l-rhamnopyranosyl(1→4)-β-d-glucopyranoside, uracil, 5-methyluracil, 1octacosanol, and octacosane have been reported on the leaves of the plant (okokon et al., 2016; okokon et al., 2022). we report in this study the effect of the leaf extract and fractions s. anomalum on oxidative stress markers, liver function indices and histology of alloxaninduced diabetic rats. https://doi.org/10.14421/biomedich.2023.121.33-44 34 biology, medicine, & natural product chemistry 12 (1), 2023: 33-44 materials and methods plants collection fresh leaves of solanum anomalum were collected in compounds in uruan area, akwa ibom state, nigeria in august, 2020. the plant was identified and authenticated by a taxonomist in the department of botany and ecological studies, university of uyo, uyo, nigeria. hebarium specimen was deposited at department of pharmacognosy and natural medicine herbarium, university of uyo (uuh.75a). extraction fresh leaves of s. anomalum were washed, cut into smaller pieces and dried under shade for two weeks. the leaves were further pulverized to powder using electric grinder. the powdered leaves material was divided into two parts; one part (1.5 kg) was macerated in 50% ethanol (7.5 l) for 72 hours at room temperature (28 ±2 °c). while the other part, (1.5 kg) was successively and gradiently macerated for 72 hours in each of these solvents (2 x 5l), n-hexane, dichloromethane, ethylacetate and methanol to give corresponding fractions of these solvents. these were thereafter filtered and the liquid filtrates were concentrated and evaporated to dryness in vacuo 40°c using a rotary evaporator (buchilab, switzerland). the extract and fractions were stored in a refrigerator at -4°c, until used for the proposed experiments. animals wistar rats (138-150 g) of either sex were obtained from the university of uyo animal house. they were maintained on standard animal pellets and water ad libitum. permission and approval for animal studies were obtained from the faculty of pharmacy animal ethics committee, university of uyo. induction of experimental diabetes using alloxan monohydrate sixty (60) healthy albino wistar rats (male and female) of known weights were fasted for 24 hours, they were reweighed before the induction by a single intra peritoneal injection of freshly prepared solution of alloxan monohydrate (150 mg/kg) in ice cold 0.9% saline (nacl solution). according to the method of pari and saravanan, (2002), the animals were given 2 ml of 5% dextrose solution using orogastric tube immediately after induction to overcome the drug induced hypoglycaemia. a rest period of 72 hours was allowed during which the rats were allowed access to food and water and the diabetes to be fully developed during these 72 hours. after the rest period, rats with moderate diabetes, having persistent glycosuria, and hyperglycaemia (i.e with blood glucose levels 200 mg/dl and above), (lenzen, 2008) were considered diabetic and selected for the experiments. the diabetic animals were randomised and divided into 9 (nine) treatment groups of 6 rats each. based on the value of previously determined median lethal dose (ld50) (okokon et al., 2022), suitable dose regimens were selected and the rats were treated as follows. group 1 was given 10 ml/kg/day of normal saline orally for 14 days, group 2 was administered with 5 mg/kg/day of glibenclamide orally for 14 days, group 3 was given 70 mg/kg/day of s. anomalum leaf extract orally for 14 days and group 4 rats were given 140 mg/kg/day of s. anomalum leaf extract orally for 14 days, group 5 was administered with 210 mg/kg/day of s. anomalum leaf extract orally for 14 days. groups 6 -9 were respectively administered with 140 mg/kg/day of n-hexane, dichloromethane, ethyl acetate and methanol fractions of s. anomalum leaves orally for 14 days. effect of administration of leaf extract and fractions of s. anomlum on fasting blood glucose of alloxan-induced diabetic rats. the fasting blood glucose (fbg) of all the rats was measured after 14 days of administration of the leaf extract and fractions. the method that was employed was “the tail-tipping method”. the blood obtained from the tail vein of the rats was dropped on the dextrostix reagent pad and the pad inserted into a microprocessor digital blood glucometer and the readings were recorded (who, 1980). all the treatments were administered between 7.008.00 am daily throughout the experimental period and food was withdrawn from the experimental animals 12 hours before measurement of fbg to create the necessary fasting period for measurement of the fasting blood glucose concentrations. determination of the body weights changes of the treated diabetic rats throughout the experimental period the body weights of the experimental animals were monitored and recorded at the following points; just before the fasting in preparation for induction of the diabetes, after induction, on stabilization of diabetes and after the prolonged study collection of blood samples and organs after 14 days of treatment (24 hours after the last administration) the rats were weighed again and sacrificed under light diethyl ether vapour. blood samples were collected by cardiac puncture and used immediately. blood were collected into plain centrifuge tubes and edta bottles. the blood in the centrifuge tubes were centrifuged immediately at 1500 rpm for 15 min to separate of serum at room temperature to avoid haemolysis and used for biochemical assays. blood that was collected into edta bottles were taken for haematological analysis. the livers and kidneys of the diabetic rats were surgically removed, weighed and fixed in 10% formaldehyde for histological process. etuk et al. – biological activities of solanum anomalum 35 hematological study after the animals were sacrificed under diethyl ether anesthesia, blood samples were collected from each rat by cardiac puncture using 21 gauge (21 g) needles mounted on a 5 ml syringe into ethylene diamine tetraacetic acid (edta) – coated sample bottles for analyzed. hematological parameters such as red blood cell count (rbc), hemoglobin, (hb), packed cell volume (pcv), platelet concentration (plc) and total and differential white blood cell count (wbc). these parameters were analyzed using automatic hematological system (sysmex hematology – coagulation system, model mo-1000 i, trans asia, japan). liver function test the following parameters were determined; aspartate transaminase (ast), alanine aminotransferase (alt), total cholesterol, alkaline phosphatase (alp), total and direct bilirubin. the determinations were done spectrophotometrically using randox analytical kits according to standard procedures of manufacturer’s protocols (tietz, 1976) at the chemical pathology department of university of uyo teaching hospital. evaluation of the protective effect of the leaf extract and fractions on biochemical parameters and histology of livers of alloxan-induced diabetic rats serum was separated from the blood of each animal sacrificed and the sera were stored at -20oc until used for biochemical determinations such as total protein, albumin, aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphotase (alp), direct and total bilirubin. the determinations were done spectrophotometrically using randox analytical kits according to standard procedures of manufacturer’s protocols (reitman and frankel, 1957; tietz, 1976)). the livers of the animals were surgically removed, weighed and a part of each fixed in 10% formaldehyde for histological processes, while the other part was washed with ice cold 0.9% nacl and homogenates were made in a ratio of 1 g of wet tissue to 9 ml of 1.25% kcl by using motor driven teflon-pestle. the homogenates were centrifuged at 7000 rpm for 10 min at 4˚c and the supernatants were used for the assays of superoxide dismutase (sod) (marklund and marklund, 1974), catalase (cat) (sinha,1972), glutathione peroxidase (gpx) (lawrence and burk,1976), and reduced glutathione (gsh) (ellman,1959). malondialdehyde (mda) and gutathione-s-transferase (gst) were measured using commercial diagnostic kits (sigmaaldrich, usa) according to standard manufacturer's protocols. gas chromatography-mass spectrometry (gcms) analysis gc-ms analyses of hexane and dichloromethane fractions were performed using an agilent 7890a gas chromatograph connected with an 5975c msd detector (agilent technologies, usa). 1-2 µl of each fraction was injected to a hp5-ms column (5 % phenylmethylpolysiloxane, 30 m × 0.25 mm × 0.25 µm) and eluted with helium gas under a pressure of 10 psi. the oven temperature gradient was: keeping at 150 °c for 3 min, increasing to 300 °c at 10 °c/min, and keeping at 300 °c for 5 min. electron ionization mode of gc-ms at 70 ev was applied (okokon et al., 2022). the compounds in the hexane and dichloromethane fractions were determined by comparison of spectral data in the nist 2011 database. statistical analysis data obtained from this work were analysed statistically using one –way anova followed by tukey-kramer multiple comparison test using instat graphpad software, (san diego, usa). differences between means were considered significant at 5% level of significance i.e. p≤ 0.05. results and discussion effect of leaf extract and fractions on body weights of rats the body weights of the treated and untreated alloxaninduced diabetic rats were observed to have changed considerably within the period of study (table 1). treatment of the diabetic rats with the leaf extract and fractions produced a non – dose dependent increases in the body weight of the diabetic rats with the middle dose (140 mg/kg) having the highest weight increase. these increases which were significant (p<0.05-0.001) when compared to control were highest in the middle dose treated group (10.02%) followed by methanol fractiontreated group (8.77 %). (table 1). effect of extract and fractions on weights of organs treatment of alloxan–induced diabetic rats with leaf extract and fractions of s. anomalum caused prominent decreases in the liver weights of the treated rats though non dose-dependently (table 1). these decreases which were pronounced in groups treated with higher doses of the extract (140 and 210 mg/kg) and n-hexane fraction were only significant (p<0.001) in the group treated with methanol fraction (table 1). antidiabetic activity of the leaf extract and fractions during prolonged treatment the leaf extract produced significant (p<0.05-0.001) reductions in fbg levels of the diabetic rats following repeated treatment. these reductions which were non 36 biology, medicine, & natural product chemistry 12 (1), 2023: 33-44 dose dependent were sustained throughout the duration of the study. these effects were comparable to that the standard drug, glibenclamide. on day 14, the effects were 64.08%, 63.43%, 65.43% and 64.54% for 70, 140, 210 mg/kg and glibenclamide respectively (table 1). the various fractions produced sustained significant (p<0.05-0.001) reductions in fbg of the diabetic rats. the effects of the fractions on day 14 were 67.99, 54.66, 66.08 and 66.77% respectively for n-hexane, dichloromethane, ethyl acetate and methanol fractions. the effects exerted by n-hexane, ethyl acetate and methanol were better than that of standard drug, glibenclamide (table 1). effect of leaf extract and fractions on liver function test parameters of diabetic rats. treatment of the diabetic rats with leaf extract and fractions of s. anomalum caused significant (p<0.050.001) reductions in the levels of total bilirubin, alt, alp and ast when compared to control. direct bilirubin level was not affected by treatment with the extract and fractions. although reductions were observed in ast levels of the treated diabetic rats, it was only significant (p< 0.01-0.001) at the highest dose (210 mg/kg) and methanol fraction treated group (table 2). effect of leaf extract and fraction on liver antioxidant enzymes. treatment of alloxan-induced diabetic rats with leaf extract and fractions of s. anomalum caused significant (p<0.05-0.001) dosedependent elevation in the levels of the antioxidant enzymes (sod, cat, gpx) when compared to control. similarly, gsh level was significantly (p<0.001) elevated following treatment with the extract and fractions when compared to control. n-hexane fraction followed by methanol fraction exerted the highest activity. similarly, there were significant (p<0.05-0.01) reductions in the level of mda of the treated rats with ethyl acetate and dcm fractions having the most significant effects. also, significant (p<0.050.01) increases in enzymes and gsh levels as well as significant (p<0.01) reductions in mda level were observed with the standard drug, glibenclamide (table 3). effect of extract and fractions on the histology of livers of diabetic rats histologic sections of livers of untreated diabetic rats revealed numerous hepatocytes with pyknotic nucleus, cellular and vascular degeneration, vascular congestion, hepatocytes hyperplasia and periportal inflammation. livers of diabetic rats treated with glibenclamide (10 mg/kg) revealed reduced pathological signs with normochromic pyknotic nucleus. (figure 1). livers of rats treated with leaf extract (70 – 210 mg/ kg) revealed hyperchromic cellular profile with vascular congestion and numerous pyknotic nucleus. livers of diabetic rats treated with the middle dose of the extract (140 mg/kg) showed no visible cellular abnormality (figure 1). livers of diabetic rats treated with n-hexane, dichloromethane, ethyl acetate and n-butanol fractions (140 mg/kg) revealed normochromic cellular profile containing periportal inflammation and pyknotic nuclei. there was no obvious cellular abnormality in the livers of diabetic rats treated with ethyl acetate fraction of the leaf. it could be considered slightly affected. (figure 1). effect of leaf extract and fractions on haematological indices of diabetic rats administration of the leaf extract and fractions of s. anomalum caused significant (p< 0.05-0.001) increases in the levels of white blood cells, lymphocytes, and eosinophils, which was significant at the middle dose (140 mg/kg) when compared to control. neutrophils, monocytes, basophils and platelets were reduced, while rbc, hb concentration, platelets and pack cell volume were not affected significantly (p>0.05) by the administration of extract and fraction (table 4). gas chromatography-mass spectroscopy (gcms) analysis the phytochemical analysis of the most active fractions (n-hexane and dichloromethane) of s. anomalum revealed the presence of major compounds. the compounds in n-hexane fraction were bicyclo[3.1.1]heptanes-2,5,6-trimethyl-(1.alpha 2-beta, 5alpha-, squalene and beta.-sitosterol trimethylsilyl ether (table 5). while 3,7,11,15-tetramethyl-2-hexadecen-1ol, squalene and heptacosane were revealed to be present in dcm fraction (table 6). etuk et al. – biological activities of solanum anomalum 37 table 1. effect of leaf extract and fractions of solanum anomalum on body and liver weights of alloxaninduced diabetic rats. treatment dose mg/kg body weight (g) weights of liver (g) fasting blood glucose (mg/dl) day 0 day 15 % increase 0 hr 14th day control normal saline 132.6 ± 18.34 129.3± 20.43 -2.48 6.72±0.98 266.0±17.16 249.0±14.01 glibenclamide 10 132.0 ± 9.45 141.3± 12.33 7.04 6.10±0.15 233.0±12.00 82.6±8.19b extract 70 152.8 ± 15.56 163.0± 9.29 6.67 6.28±0.23 279.3±14.74 100.3±16.69b 140 143.6 ± 12.55 158.0 ± 3.15 10.02 5.80±0.32 260.6±8.32 95.3±11.29 a 210 140.8 ± 13.29 152.6 ± 7.22 8.38 5.82±0.67 257.0±3.71 88.3±8.83b nhexane fraction 140 142.9 ± 8.54 153.6 ± 6.48 7.48 5.82±0.65 264.3±14.50 84.6±40.05c dichloromethane fraction 140 138.4 ± 6.26 144.6± 13.71 4.68 6.14±0.35 236.0±37.60 107.0±14.52b ethyl acetate fraction 140 144.31 ± 8.50 156.6± 9.88 8.51 7.07±0.22 271.3±13.04 92.0±14.29a methanol fraction 140 145.8 ± 7.45 158.6± 10.55 8.77 0.98±0.07c 245.6±12.33 81.6±18.49b data are expressed as mean ± sem, significant at ap<0.05, bp< 0.01, cp< 0.001, when compared to control. (n=6). table 2. effect of s. anomalum leaf extract and fractions on the liver function parameters of alloxan-induced diabetic rats. treatment dose (mg/ kg) alt (iu/l) alp (iu/l) ast (iu/l) total bilirubin (µmol/l ) direct bilirubin (µmol/l) control 10 mg/ml 97.00± 4.35 109.53±8.95 172.63±10.68 2.16±0.14 1.60±0.10 crude extract 70 77.90±9.49 92.11± 2.67 162.42± 1.45 2.06± 0.29 1.16±0.03 140 75.10±12.61 70.73±8.67 149.69±15.94 1.80± 0.05 1.10±0.15 210 53.23±2.25c 61.92±8.67b 128.07±18.57a 1.50± 0.05 1.10±0.00 nhexane fraction 140 63.73±3.91b 100.23±9.68 130.48±8.54 1.60±0.00 1.10±0.10 dichloromethane fraction 140 68.86±3.33a 62.92±6.76b 155.42±18.27 1.43±0.20a 1.10±0.05 ethyl acetate fraction 140 70.40±4.19 67.43±5.44b 145.13±15.21 1.86 ± 0.31 1.10±0.11 methanol fraction 140 58.2±5.51c 53.33±6.09c 106.98±0.75c 1.36± 0.08a 1.10±0.10 glibenclamide 10 61.12±10.94b 60.40±6.54b 122.00±4.00b 1.66± 0.12 1.30±0.10 data is expressed as mean ± sem, significant at ap<0.05, bp< 0.01, cp< 0.001, when compared to control. (n=6). table 3. effect of s. anomalum leaf extract on liver antioxidative stress markers in alloxan-induced diabetic in rats. parameters/ treatment dose mg/kg sod (µg/ml) cat (iu/l) gst (µg/ml) gsh (µg/ml) gpx (µg/ml) mda (µmol/ml) diabetic control 10 ml/kg 0.18±0.03 3.68± 0.28 0.16±0.01 0.71±0.02 0.039±0.001 0.54±0.02 crude extract 70 0.24±0.04a 4.11±0.23 0.21±0.02 0.72±0.03 0.055±0.002 0.52±0.02 140 0.20±0.02 2.95±0.28 0.23±0.01a 0.81±0.02 a 0.056±0.002c 0.48±0.03 210 0.30± 0.02c 3.53±0.17 0.31±0.01c 1.01±0.03c 0.059±0.002c 0.41±0.02c n-hexane fraction 140 0.30±0.02c 5.86± 0.48c 0.23±0.01a 0.98±0.02c 0.057±0.002c 0.44±0.0b dichloromethane fraction 140 0.20 ±0.04 2.87±0.18 0.31±0.01c 0.82±0.01a 0.058±0.002c 0.38±0.01c ethyl acetate fraction 140 0.33 0.02c 4.640.26 0.26±0.01c 0.92± 0.02c 0.048± 0.002a 0.31± 0.02c methanol fraction 140 0.37 0.03c 4.330.56 0.32±0.01 c 0.87± 0.02c 0.049± 0.002a 0.43± 0.02b glibenclamide 10 0.29±0.02c 5.69±0.18 b 0.25±0.02 b 0.85±0.01c 0.051±0.003b 0.40±0.02c data were expressed as mean  sem. significant at ap< 0.05, bp< 0.01, cp< 0.001 when compared to diabetic control. (n = 6). table 4. effect of solanum anomalum leaf extract and fractions on hematological parameters of alloxan-induced diabetic rats. treatment dose mg/kg wbc (l) neut. (%) lym (%) mono (%) eosino (%) baso (%) rbc (l) hgb (g/dl) pcv (%) platelets. (l) control normal saline 10 9.27± 1.95 59.00±1.60 35.66±1.65 4.66± 0.28 0.26± 0.21 1.00± 0.10 6.00± 0.28 12.13± 0.78 43.76±3.22 940.0± 35.76 crude extract 70 11.60±1.56 51.86± 3.16 45.86±2.06 a 2.96± 0.24 a 0.16 ±0.06c 0.90± 0.05 6.72 ± 0.53 12.63± 0.56 43.20±2.36 709.3± 54.14a 140 15.15±3.34 a 50.40±0.64 45.30±0.88 a 2.63± 0.29 a 0.76 ± 0.12c 1.10± 0.20 7.83± 0.78 14.10± 1.25a 53.76±7.61a 684.6± 92.13a 210 9.86±1.66 57.93 ± 0.60 36.53±4.27 3.76± 0.38 0.66± 0.12c 0.50± 0.00 7.17± 0.18 11.10± 0.68 40.80±1.55 925.6± 61.50 nhexane fraction 140 9.90±1.51 59.50 ± 0.60 34.90±1.44 4.96± 0.03 0.43 ± 0.17b 0.60± 0.26 6.08± 0.69 12.46± 0.08 44.50±0.40 768.0± 82.92 dichloromethane fraction 140 9.32±2.31 48.10±14.13 65.30±5.71c 3.40± 0.58 0.36 ± 0.51 0.90 ± 0.05 6.65 ± 0.90 12.56± 1.07 45.90±7.50 723.0± 35.80 ethyl acetate fraction 140 10.31±1.64 50.66±0.82 43.46±0.73 3.60 ± 1.01 0.30± 0.51 0.50± 0.17 7.28± 0.17 12.13± 1.21 43.90±4.43 775.0±59.53 methanolfraction 140 14.55 ± 0.98a 47.60±6.45a 46.93±6.59 a 4.16± 0.72 0.40± 0.15c 0.53 ± 0.03 6.70 ± 0.63 13.33 ± 0.74 48.90±2.82 867.3±74.71 glibenclamide 10 12.03±3.76 33.16±0.79b 61.56±1.66 c 2.23± 0.28 a 0.06± 0.03c 0.55± 0.03 7.56 ± 0.31 12.66±1.18 47.90±2.82 888.0±47.05 data is expressed as mean ± sem, significant at ap<0.05, bp<0.01, cp<0.001, when compared to control. (n=6). 3 8 bio lo g y, m ed ic in e, & n atu ral pro d u c t c h em istry 1 2 (1 ), 2 0 2 3 : 3 3 -4 4 etuk et al. – biological activities of solanum anomalum 39 table 5. gcms analysis of column hexane fraction of s. anomalum. peak rt compound name formula mol. mass 1. 10.044 bicyclo[3.1.1]heptanes-2,5,6-trimethyl-(1.alpha 2-beta, 5alphac19h38o5si4 458.18 2. 18.315 squalene c30h50 410.39 5. 18.326 .beta.-sitosterol trimethylsilyl ether c32h58osi 486.43 table 6. gcms analysis of column dcm fraction of s. anomalum. peak rt (min) compound name formula mol. mass 1. 10.044 cis-pinane c10h18 138.25 2. 10.524 3,7,11,15-tetramethyl-2-hexadecen-1-ol c20h40o 296.1 3. 10.547 1,4-eicosadiene c20h38 278.5 4 18.315 squalene c30h50 410.39 5. 22.836 heptacosane c27h56 380.44 keys: vascular congestion (vc), vascular degeneration (vd), cellular degeneration (cd), periportal inflammation (pi), pyknotic nucleus (pn), red blood cell (rbc), hepatocytes (h), hepatocytes hyperplasia(h). hepatic vein (hv), bile duct (bd), and pyknotic nucleus (pn) figure 1: histological sections of livers of alloxan-induced diabetic rats treated with normal saline 10 ml/kg (1), glibenclamide 10 mg/kg bw (2), leaf extract 70 mg/kg bw (3), leaf extract 140 mg/kg bw (4), leaf extract 210 mg/kg bw (5), n-hexane fraction 140 mg/kg (6) dichloromethane fraction 140 mg/kg (7), ethyl acetate 140 mg/kg (8), methanol fraction 140 m/kg (9), at magnification a(x400), stained with h&e method. discussion the leaf of s. anomalum is use in ethnomedicine for the treatment of various ailments such as malaria, gastrointestinal disorders and diabetes (okokon et al., 2016). this work was designed to evaluate antioxidative and hepatoprotective potentials of leaf extract and fractions of s. anomalum in alloxan-induced diabetic rats. the body weights of diabetic rats were found to increase significantly following treatment with the leaf extract and fractions. diabetes is associated with a severe loss in body weight due to loss or degradation of structural proteins (rajkumar and govindarajulu, 1991). treatment with the leaf extract and fractions remedied this situation perhaps due to the alleviation of hyperglycemic state and stimulation of protein synthesis. 987 6 321 54 40 biology, medicine, & natural product chemistry 12 (1), 2023: 33-44 the leaf extract and fractions treatments were observed in this study to cause significant increases in wbc, lymphocytes and eosinophils, while neutrophils, monocytes and basophils were significantly reduced. other parameters were not affected. the increases maybe due to immunological responses by the body defence mechanism to heal or repair the injuries caused by alloxan (soetan et al., 2013) which can be attributed to the immunostimulatory effect of diosgenin (nimbalkar et al., 2018) and squalene. metabolism of alloxan to dialuric acid causes the production of h2o2, •oh and superoxide radical (mathews and leiter, 1999)., which destroys β-cell necrosis and induces diabetes by partial destruction of pancreatic β-cells of islet of langerhans (cakici et al., 1994; abdel-barry et al., 1997). this reduces insulin levels, raise the blood glucose level thereby leading to type 2 diabetes mellitus, but with residual pancreatic βcells with potentials to secrete insulin (sheela and augusti, 1992; subramoniam et al., 1996). in this study, s. anomalum leaf extract/fractions were observed to demonstrate sustained significant antidiabetic activities during prolonged study with the methanol, n-hexane and ethyl acetate fractions exerting prominent activities. the fasting blood glucose (fbg) levels of the treated diabetic rats were significantly reduced when compared to those of untreated diabetic rats (control). the antidiabetic results observed in this study corroborate that of other species of solanum such as s. nigrum (sengottaiyan et al., 2012; umamageswari et al.,2017), s. trilobatum (doss et al., 2009), s. xanthocarpum (selvi and yogananth, 2016) and solanum villosum (nyaga et al., 2019) thus, confirming strongly the antidiabetic potentials of this plant in ethnomedicine. the leaf extract and fractions were observed in this study to cause significant decrease in weight of liver. generally, internal organs weights are considered as important indicator to injury and toxicities (farah et al., 2013). hypertrophy of organs often indicates toxicity and damaged to organ (ping et al., 2013). this often results from oedema due to inflammation of the organs. free radicals generated during alloxan metabolism cause destruction of hepatic, pancreatic and kidney cells and tissues (mathews and leiter, 1999). the decrease in weights of liver by the extract/fractions especially dcm and methanol fractions-treated group, is as a result of protective effect of the fractions against the effect of free radicals generated by alloxan and diabetic condition perhaps due to its hypoglycemic and antioxidant activities of the phytoconstituents (okokon et al., 2019a) such as diosgenin (kanchan et al., 2016), 1-octacosanol and octacosane (leng et al., 2020; sengupta et al., 2018; rhetso et al., 2018 ), squalene (gunes, 2013; micera et al., 2020), β-sitosterol (gupta et al., 2011; baskar et al., 2012) and phenolic compounds in the leaf extract. metabolism of alloxan leads to the generation of free radicals which attack organs and cause oxidative stress which contributes to diabetes complications in animals or humans (baynes and thorpe, 1999). reactive oxygen species (ros) produce cellular and tissue injury through covalent binding, dna strand breaking, lipid peroxidation (lpo) and augment fibrosis which is also implicated in other disease conditions (sirnivasan et al., 2007; paradies et al., 2011). the results of this study show that oxidative stress was duly induced by alloxan in the diabetic rats as reflected in the marked reductions in the levels of sod, cat, gsh and gpx in hepatic tissues as well as significant increase in mda levels in the liver of the diabetic rats. this finding agrees with earlier findings that the activities of these antioxidants are known to reduce during diabetes (parmar and kar,2008; kostolanska et al., 2009; dixit and kar, 2010). the oxidative stress status of the diabetic rats is further supported by marked elevations in the serum levels of ast, alt, alp, and total bilirubin levels of the diabetic rats. reduction in liver levels of these oxidative markers following treatments with the leaf extract and fractions strongly suggest the great potentials of leaf extract and fractions in attenuating oxidative stress associated with type ii diabetes mellitus which was probably mediated via free radical scavenging activities and improving glutathione status in the tissues by its phytochemical constituents such as diosgenin, 1octacosanol, octacosane and β-sitosterol (gupta et al., 2011; baskar et al., 2012;kanchan et al., 2016;leng et al., 2020; sengupta et al., 2018; rhetso et al., 2018). disease conditions such as diabetes affect the liver and changes the hepatic function as shown by increases in the levels of ast, alt, alp, total and direct bilirubin indicates liver damage which are also detected in human diabetes (takaike et al., 2004). damage to liver cells often results in leakage of enzymes such as aspartate aminotransferase (ast), and alanine aminotransferase (alt) into the blood, while blood levels of alkaline phosphatase (alp) rise when the bile flow is slow or blocked (prat and kaplan, 2000). in this study, treatment of the diabetic rats with the leaf extract and fractions was found to cause significant reductions in the levels of alt, alp and total bilirubin. the integrity of hepatocytes is assessed by levels of the serum liver enzyme markers such as ast, alt and alp (moss and henderson, 1999). serum aminotransferases levels (alt and ast) are two of the most useful measures of liver parenchymal cell injury. ast is raised in acute liver damage, but is also present in rbcs, kidney, testis, cardiac and skeletal muscles, so it is not specific to the liver, while alt is almost exclusively found in the liver (nyeblom et al., 2006). elevated levels of ast and alt indicate liver damage but are not good measures of liver function since they do not reliably reflect the synthetic ability of the liver and may come from tissues other than the liver such as muscles. the leaf extract and fractions produced etuk et al. – biological activities of solanum anomalum 41 significant reduced the elevated levels of these enzymes. this further corroborates histologic findings. the increased activities of transaminases, which are active in the absence of insulin due to the availability of amino acids in the blood of diabetics and are also responsible for the increased gluconeogenesis and ketogenesis (udayakumar et al., 2009). diabetes and hyperlipidaemia also cause cell damage by altering the cell membrane architecture, which results in enhanced activities of alp in diabetic rats (udayakumar et al., 2009). this suggests that alloxan-induced diabetes caused lipid peroxide mediated tissue damage in the liver. the increase in the levels of these enzymes in diabetes may be as a result of the leaking out from the tissues and then migrating into the blood stream. the decrease in ast, alt and alp levels in leaf extract and fractions treated groups indicates the protective effect on the liver. total bilirubin level which was increased in the untreated diabetic rats was observed in this study to be significantly reduced in groups treated with the husk extract and fractions. bilirubin, is a metabolic product of hemoglobin which undergoes conjucation with glucuronic acid in hepatocytes to increase its water solubility. determination of bilirubin represent an index for the assessment of hepatic function, severity of necrosis, conjugation and excretory capacity of hepatocytes and any abnormal increase in the level of bilirubin in the serum indicate hepatobililiary disease and severe disturbance of hepatocellular function (martin and friedman, 1992). decrease in serum bilirubin level of the diabetic rats after treatment with the leaf extract and fractions indicated extract/fractions effectiveness to restore normal functional status of the liver. the above activities of the extract/fraction show that the leaves extract and fractions posses hepatoprotective potential against alloxan-induced hepatic injury. these results corroborate histologic findings which show significant hepatoprotective potentials of the extract/fractions. this effect could be due to the antioxidant /free radical scavenging activities of the extract and fractions (okokon et al., 2019a), squalene, β-sitosterol (ivorra et al., 1988; gupta et al., 2011; baskar et al., 2012; micera et al., 2020) as well as the isolated compounds; diosgenin, 1-octacosanol and octacosane (kanchan et al., 2016; leng et al., 2020; sengupta et al., 2018; rhetso et al., 2018). reports have been indicated that persistent hyperglycemia causes increased production of oxidative stress in alloxan-induced diabetes (bonnefont et al., 2000). hence, excessive ros produced leads to oxidative damage and increased lipid peroxidation (lpo). the results of this study showed a significant increase in lpo levels resulting in high level of mda in alloxan-induced diabetic rats liver with accompanying reduction in the levels of oxidative stress markers (sod, cat, gpx and gsh) in the liver. this reduction in the levels of antioxidant enzymes in this study corroborates those of earlier reports (parmar and kar, 2008; kostolanska et al., 2009; dixit and kar, 2010). in in vivo experimental models, tissue oxidative stress markers such as sod, cat and gsh are useful and reliable markers of antioxidant status while mda is a sensitive and reliable marker for lipid peroxidation (kumar et al., 2010; feillet-coudray et al., 1999). sod plays an important role in oxygen defense metabolism by intercepting and reducing superoxide to hydrogen peroxide, which in mammals is readily reduced to water principally by cat and gpx. gpx plays a pivotal role in minimizing the oxidative stress. gpx and gst work together with gsh and decompose h2o2 and other organic hydroperoxides to non-toxic products. glutathione, reduced form gsh is ubiquitous tripeptide thiol, is a vital intra/extra-cellular protective antioxidant against oxidative/nitrosative stress. glutathione reductase is an enzyme responsible for its conversion back to the reduced state (kayali and tarhan, 2006; okutana et al., 2005). the antioxidant enzymes levels were found to be reduced in untreated alloxan-induced diabetic rats. hence, the changes of these biomarkers is in accordance with the decrease in antioxidant state in the body as other reports (ei-missiry and ei-gindy, 2000; meral et al., 2001). in the present study, administration of the extract and fractions significantly counteract the changes of oxidative stress biomarkers in alloxan-induced rats thus preventing the accumulation of excessive oxidative stress with corresponding increases in the levels of antioxidant enzymes/oxidative stress markers. this activity is due to the antioxidant activities of the phytochemicals such as squalene, β-sitosterol, diosgenin, 1-octacosanol, octacosane and other phenolic compounds found to be present in the leaf extract. similarly, β-sitosterol and diosgenin present in the extract/fractions have been reported to cause reduction in hepatic lipid peroxidation and elevation in the activities of catalase, superoxide dismutase and glutathione (gupta et al., 2011b; baskar et al., 2012; kanchan et al., 2016). the liver of the extract/fractions treated diabetic rats were found to be significantly protected from the effects of free radicals generated by alloxan. this protection was visible as the livers of the treated rats lacked or had reduced pathological signs such as vascular degeneration and congestion as well as periportal inflammation observed in the untreated diabetic rats. these findings corroborated that of chemical pathology and therefore suggest hepatoprotective activity against oxidative stress induced by alloxan. the protection is due to the free radical scavenging potentials of the phytoconstituents of the extract and fractions such as squalene, β-sitosterol, diosgenin, 1-octacosanol, octacosane as well as the antioxidant activity of other phenolic compounds present in the extract (okokon et al., 2019a). the hepatoprotective effect of solanum anomalum in this study corroborate that previously 42 biology, medicine, & natural product chemistry 12 (1), 2023: 33-44 reported on other species of solanum such as s. nigrum, s. macrocapon and s. xanthocarpum (lin et al., 2008; 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(2022). analysis of ethanol extract of solanum anomalum leaves for antidiarhoeal activity. journal of current biomedical research.2(2):145-157. who expert committee on diabetes mellitus. tech. rep. series no.646.world health organisation, geneva, 1980. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1185-1191 | doi: 10.14421/biomedich.2025.142.1185-1191 issn 2540-9328 (online) application of carbon nanodots from cocoa husk waste to improve the mechanical properties of cellulose-carrageenan-based biodegradable films fitri nuraini fadila1, esa ghanim fadhallah1, ahmad sapta zuidar1, teguh setiawan1, gustaf triyoga1*, jihan allya syahar1, hersan pratama ashari1, precillia regitha1, fathan khanifadin2, wulan nur aisyah1, dea meranda1, zaskia rahma andini1 1department of agricultural product technology, faculty of agriculture, university of lampung 2department of physics, faculty of mathematics and natural sciences, university of lampung jl. prof. dr. sumantri brojonegoro no. 1 bandar lampung, 35145, tel +62 721 701609, fax +62 721 702767, indonesia. corresponding author* gustaffftriyogaa@gmail.com abstract biodegradable films made from natural materials such as cellulose and carrageenan are easily degradable but exhibit poor mechanical properties. incorporating carbon nanodots (cds) from cocoa husk waste has the potential to improve their mechanical properties. this study aimed to characterize cds synthesized from cocoa husk waste and evaluate their mechanical properties of cellulose-carrageenanbased biodegradable films. the research stages include the isolation of cocoa husk cellulose, cds synthesis, cds characterization, biodegradable film preparation, and characterization of the mechanical properties of biodegradable films. the experimental design used was a completely randomized design (crd) with the treatment factor of cds addition at six levels (0%, 1%, 3%, 5%, 7%, 9%) with three replicates. the results showed that the average size of cocoa husk cds was 997 nm, and the absorbance peak was detected at 298 nm, validating that the cds had been successfully synthesized with blue light emission. incorporating cds significantly affected the mechanical properties of the biodegradable film. biodegradable cellulose-carrageenan-based films with the addition of cds from cocoa husks had a thickness of 0.35–0.64 mm, tensile strength of 2.94–4.20 mpa, elongation of 36.51–63.28%, and elasticity of 6.00–9.45 mpa. the tensile strength and elongation values meet the jis z 1707 standard. higher concentrations of cds significantly improved tensile strength and elasticity, while reducing the thickness and elongation. keywords: biodegradable film; carbon nanodots; carrageenan; cellulose. introduction plastic has become the most widely used material across all levels of society. according to the latest 2024 report from the ministry of environment and forestry of indonesia (klhk), the country generates approximately 33 million tons of waste annually from daily human activities. within this total, plastic accounts for 19.76% of waste composition, making it the second largest contributor after food waste. more than 40% of this waste is poorly managed, creating a significant risk of environmental pollution. the accumulation of unprocessed plastic waste, in particular, poses serious threats to ecosystems (putra et al., 2025). efforts to mitigate waste accumulation include reducing single-use plastic consumption, promoting recycling initiatives, and substituting conventional plastics with biodegradable materials (sari et al., 2023). biodegradable films are thin layers derived from natural polymers that can be decomposed by microorganisms into simpler products such as co₂, ch₄, h₂o, and energy. the degradation process involves water molecule penetration into the material, cleavage of covalent bonds, and the enzymatic breakdown of cellulose and hemicellulose (safira & purbasari, 2022). these films can serve as edible coatings for products such as sausages, meat, and fruits, or as light protective layers for various applications (rachmawati et al., 2023). the base materials commonly used in biodegradable films are polysaccharides, including starch, cellulose, and lignin, selected depending on the intended application. among these, cellulose-based films are preferred due to their superior film-forming ability. to enhance the performance of biodegradable films, additional materials such as alginate, chitosan, and carrageenan are often incorporated during the thickening process. carrageenan is widely applied as an emulsifier, thickener, stabilizer, and food coating material. when dissolved in water, carrageenan forms gels and exhibits strong thickening properties, thereby facilitating biodegradable film manuscript received: 06 september, 2025. revision accepted: 04 december, 2025. published: 10 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1185-1191 1186 biology, medicine, & natural product chemistry 14 (2), 2025: 1185-1191 production (zanjabila et al., 2023). however, despite these advantages, celluloseand carrageenan-based biodegradable films have limitations such as low elasticity and poor water resistance. reinforcing agents are therefore required to improve their mechanical properties. carbon nanodots (cds) are carbon-based nanomaterials known for their biocompatibility, strong fluorescence, water solubility, low toxicity, and favorable reflective properties. incorporating cds into biodegradable films has been shown to significantly enhance their mechanical performance. for example, millah and dwandaru (2024) reported that the addition of cds synthesized from corn husk waste increased tensile strength by up to 217% and improved the water resistance of corn starch-based edible films. similarly, sari et al. (2023) demonstrated that cds enhance thermal stability and prevent the degradation of organic components in films. cds can be produced through heat treatment of organic feedstocks, including empty fruit bunches of palm oil, peanut shells, and sugar (koshi et al., 2021). a promising source of organic feedstock for cds is agro-industrial waste, particularly cocoa husks. cocoa fruit generally consists of 75% husk, 22% bean shell, and 3% placenta. the large proportion of cocoa husk presents an opportunity for its valorization into sustainable materials such as cds. cocoa husks are rich in lignocellulosic components, containing approximately 35.4% cellulose, 37% hemicellulose, and 14.7% lignin (sena et al., 2021). lignocellulose derived from agricultural residues serves as an abundant carbon source and an effective precursor for cds synthesis (lee & ko, 2025). furthermore, lignocellulosic components are known to enhance the strength of polymer-based materials. thus, the utilization of cellulose from cocoa husks represents a promising pathway for cds production. research on the use of cocoa husks as a precursor for cds remains limited, and their application in improving the mechanical properties of biodegradable films has not been extensively explored (nazibudin, 2022; norman et al., 2022). given the high lignocellulose content of cocoa husks, they are an ideal candidate for cds synthesis. therefore, the objective of this study was to characterize cds synthesized from cocoa husk waste and to investigate the effect of cds incorporation on the mechanical properties of cellulose-carrageenan-based biodegradable films. material and methods materials and equipments cocoa husk waste was obtained from cocoa farmers in sukoharjo 1 village, pringsewu district, lampung. additional materials included distilled water, deionized water, naoh, h₂o₂, carrageenan (indogum), ethanol, acetic acid, and glycerol. the equipment used comprised standard chemical glassware, petri dishes, a grinder, an oven (memmert), an 80-mesh sieve, a 250-mesh nylon filter, a hydrothermal autoclave, an ultrasonic machine, a centrifuge, a particle size analyzer (beckman coulter), an x-ray diffractometer (panalytical), a genesys 10 uvvis spectrophotometer, a digital micrometer (rohs), and a universal testing machine (zwick roel). experimental design the experimental procedure consisted of cellulose isolation from cocoa husk, synthesis and characterization of carbon nanodots, preparation of biodegradable films, and evaluation of their mechanical properties, including thickness, tensile strength, elongation, and elasticity. the study employed a completely randomized design with a single treatment factor, namely the addition of carbon nanodots at six levels (0%, 1%, 3%, 5%, 7%, and 9%), each replicated three times. isolation celluse of cocoa husk cellulose isolation from cocoa husk waste was carried out following the method of sari et al. (2023). one kilogram of cocoa husk was washed with water, sliced into approximately 1 cm pieces, and dried in an oven at 65°c for 3 h. the dried husks were then ground for 5 minutes and sieved through an 80-mesh sieve to obtain fine powder. the powder was subjected to delignification by heating in 1 l of 12% naoh solution for 2 hours. the mixture was filtered, and the residue was washed thoroughly to yield crude cellulose. bleaching was performed by heating the cellulose in 300 ml of 3% h₂o₂ at 100°c for 1 h. the product was then filtered, washed, and dried at 65°c for 3 hours, resulting in cellulose powder from cocoa husk waste. synthesis of carbon nanodots carbon nanodots were synthesized following marphongahtun et al. (2023) with modification of the carbon source. one gram of cocoa husk powder was homogenized with 50 ml of deionized water and transferred into a hydrothermal autoclave, where it was processed for 6 h at 200°c. the resulting product was centrifuged at 3000 rpm for 30 min. the black precipitate was separated, and the supernatant was filtered and stored for further characterization. the supernatant contained the synthesized carbon nanodots, which were characterized using particle size analysis (psa), fourier transform infrared spectroscopy (ftir), x-ray diffraction (xrd), and uv-vis absorbance spectroscopy. characterization of carbon nanodots the synthesized carbon nanodots were analyzed using psa to determine particle size distribution and average diameter at the nanometer scale. ftir spectra were used to identify functional groups based on characteristic absorption peaks. xrd was employed to determine diffraction patterns, phase characteristics, and fadila et al – application of carbon nanodots from cocoa husk waste to … 1187 crystallinity. uv-vis absorbance spectroscopy was conducted in the range of 200–600 nm to evaluate light absorption properties. preparation of biodegradable film biodegradable films were prepared using a solution casting method adapted from koshy et al. (2021). six grams of cellulose, previously isolated from cocoa pod husk, were dissolved in 45 ml of acetic acid at 60°c for 25 minutes. the mixture was then combined with 100 ml of distilled water, 5 ml of glycerol, 4 g of carrageenan (kappa), and carbon nanodots at the designated treatment levels. the solution was stirred at 70°c for 25 minutes, poured into glass plates, and airdried at room temperature for 2 days, followed by oven drying at 60°c for 16 hours. the resulting films were peeled off and subjected to mechanical testing, including thickness, tensile strength, elongation, and elasticity (iso, 2019). characterization of the mechanical properties of the biodegradable film mechanical properties of the films were characterized according to iso (2019). thickness was measured at five different points using a digital micrometer, and the results were expressed in millimeters. tensile strength, elongation, and elasticity were determined using a universal testing machine with 2 × 7 cm film specimens. tensile strength was expressed in mpa, elongation in percentage, and elasticity was calculated as the ratio of tensile strength to elongation, expressed in mpa. data analysis data obtained from this study were analyzed both statistically and descriptively, depending on the parameter. mechanical property data were expressed as mean values with standard deviations and analyzed statistically using one-way anova, followed by duncan’s multiple range test at a significance level of α = 0.05. statistical analysis was performed with spss version 25. characterization data from psa, ftir, xrd, and uv-vis spectroscopy were analyzed descriptively. results and discussion particle size analysis (psa) particle size analysis was conducted to determine the distribution of particle sizes in the carbon nanodots (cds) samples. this technique can be performed using various approaches, including laser diffraction, microscopy, and sedimentation (putri, 2023). in this study, 2 ml of cds suspension was analyzed, and the numerical data were visualized in the form of a particle size distribution graph, as shown in figure 1. the analysis revealed that the average particle size was 997 nm, with a range from 811.1 nm to 3125 nm and a polydispersity index (pi) of 0.401. these values indicate that the product did not fall within the conventional size range of carbon nanodots. according to khan et al. (2021), cds are typically defined as carbon nanoparticles with average sizes below 10 nm and exhibiting either spherical or sheet-like morphology. similar findings were reported by kristian & wahyuni (2022), who observed particle sizes between 38.75 nm and 51.8 nm for cds synthesized from tangerine peel waste, which likewise exceeded the conventional sub-10 nm definition. particle size in cds is strongly influenced by factors such as the synthesis approach (top-down or bottom-up), precursor type, and reaction conditions including temperature, duration, and ph. generally, top-down techniques tend to yield smaller nanoparticles, whereas precursor composition and reaction parameters play a critical role in determining final cds dimensions (mocci et al., 2022). figure 1. particle size analysis test results. fourier transform infrared (ftir) spectroscopy ftir spectroscopy was employed to identify molecular vibrations and predict the chemical structure of the cds samples (sanjiwani et al., 2020). the ftir analysis revealed three major absorption peaks at 3257 cm⁻¹, 2190 cm⁻¹, and 1640 cm⁻¹ (figure 2). the broad peak at 3257 cm⁻¹ corresponds to o–h stretching vibrations, confirming the presence of hydroxyl groups in the molecular structure (adu et al., 2022). the peak at 2190 cm⁻¹ is attributed to triple-bond vibrations (c≡c or c≡n), indicating the presence of alkynes or nitrile groups. the band at 1640 cm⁻¹ is associated with c=c or c=o stretching, suggesting the existence of carbon– carbon or carbon–oxygen double bonds, which are characteristic of carbon-based nanostructures. these results are consistent with those reported by yudhanto & dwandaru (2024), who identified similar functional groups, including o–h, c=c, and c≡n, in cds derived from jackfruit seed waste. the agreement in functional group distribution confirms the successful formation of characteristic cds structures in this study. 1188 biology, medicine, & natural product chemistry 14 (2), 2025: 1185-1191 figure 2. fourier transform infra-red test results. x-ray diffraction (xrd) analysis xrd analysis was conducted to investigate the crystalline structure and lattice parameters of cds synthesized from cocoa husk (millah & dwandaru, 2024). the diffraction pattern obtained is shown in figure 3, which presents the relationship between diffraction angle (2θ) and intensity. the diffraction pattern exhibited several peaks, with a broad main peak appearing around 2θ = 24°, which is typically associated with the interlayer spacing of graphitic structures. additional peaks at approximately 2θ = 43° and 54° were also observed, which may correspond to crystalline phases originating from impurities or secondary structures within the sample. the broad nature of the primary peak indicates the presence of small crystallites with a predominantly amorphous structure. the crystallinity degree of the cds sample was calculated as 11.42%, confirming that the material is primarily amorphous to semi-amorphous in nature. these findings are consistent with those of millah & dwandaru (2024), who reported that cds derived from corn husk waste exhibited an amorphous structure, with broad diffraction peaks between 10° and 35° and no sharp crystalline peaks. thus, the xrd results support the conclusion that cds synthesized from cocoa husk predominantly display amorphous structural characteristics. figure 3. x-ray diffraction test result. uv-vis spectrophotometer testing uv-vis spectroscopy was carried out to determine the absorbance of the synthesized cds in the ultraviolet (uv) and visible (vis) regions at specific wavelengths (dwandaru et al., 2020). the test was performed over a wavelength range of 200–600 nm, and the results are presented in figure 4. the cds synthesized from cocoa husk exhibited absorption peaks at 298, 310, 324, and 328 nm, with the highest absorbance recorded at 298 nm. the absorbance values were primarily observed at uv wavelengths below 380 nm. this finding is consistent with sari et al. (2020), who reported that cds synthesized from betel leaf exhibited absorption peaks between 260–360 nm. the red shift observed in cocoa husk cds, indicated by the absorption peak shifting toward longer wavelengths, suggests an increase in particle size. a higher absorbance intensity also reflects the successful formation of cds (elfino & dwandaru, 2022). dwandaru et al. (2020) reported that cds derived from namnam fruit displayed absorption peaks between 228–268 nm, while sari et al. (2020) observed peaks between 257–320 nm for betel leaf cds. these findings support the conclusion that the optical properties of cds vary depending on precursor materials and synthesis conditions. figure 4. spektrofotometer uv-vis test result. mechanical properties of the biodegradable film the mechanical properties evaluated for the cellulose– carrageenan-based biodegradable film included thickness, tensile strength, elongation at break, and young’s modulus. the results are presented in table 1. fadila et al – application of carbon nanodots from cocoa husk waste to … 1189 table 1. mechanical characteristics of biodegradable film. cds concentration thickness (mm) tensile strength (mpa) elongation (%) young's modulus (mpa) 0% 0.63±0.04 a 3.59±0.06 b 63.28±2.42 a 5.67±0.41 a 1% 0.64±0.03 a 3.68±0.08 b 61.44±1.46 a 6.00±0.19 a 3% 0.35±0.02 d 4.20±0.13 a 44.55±2.01 c 9.45±0.59 b 5% 0.54±0.03 b 2.94±0.35 c 36.51±2.32 c 8.12±1.24 b 7% 0.43±0.04 c 3.51±0.06 b 41.11±1.00 b 8.53±0.12 b 9% 0.44±0.02 c 3.47±0.04 b 39.43±1.83 b 8.80±0.35 b note: different letters following the numbers within the same column indicate statistically significant differences according to duncan’s test at the 5% significance level. film thickness is a critical parameter for determining the structural integrity and quality of biodegradable films in packaging applications (khotimah et al., 2022). table 1 shows that the maximum thickness (0.64 mm) was obtained with 1% cds, while the minimum thickness (0.35 mm) was observed with 3% cds. these results indicate that increasing cds concentrations tends to reduce film thickness. zaky et al. (2021) noted that film thickness can also be influenced by the addition of plasticizers such as carrageenan and glycerol. however, the thickness values obtained in this study exceeded the jis z 1707 standard, which specifies a maximum of 0.25 mm for packaging materials (japanese standards association, 2019). the tensile strength values in table 1 demonstrate a significant effect of cds incorporation. the addition of 3% cds yielded the highest tensile strength (4.20 mpa), significantly higher than the control (3.59 mpa). although the tensile strength decreased slightly at 5% cds (2.94 mpa), an increase was again observed at 7% cds (3.51 mpa), which remained significantly higher than at 5%. this suggests that cd incorporation up to 3% strongly reinforces the polymer matrix, while higher concentrations may cause aggregation before partially stabilizing again. these findings align with millah & dwandaru (2024), who reported increased tensile strength with cds addition. suheti (2022) attributed such improvements to enhanced stress transfer between polymer chains and cds nanofillers. the tensile strength values obtained in this study satisfy the jis requirement for packaging materials, which sets a minimum tensile strength of 0.392 mpa. elongation at break reflects the flexibility of films under tensile stress. the results (table 1) show that the highest elongation (63.28%) occurred in the control film, while the lowest (36.68%) was recorded for 5% cds. this inverse relationship between tensile strength and elongation indicates that cds incorporation increases rigidity while reducing flexibility. similar trends were reported by millah & dwandaru (2024), who observed that cds enhanced tensile strength but reduced elongation. changes in the mechanical properties of the biodegradable film occur due to molecular-level interactions between the surface of nanofillers and the polymer matrix (roy & rhim, 2021). according to jis standards, elongation values above 50% are considered good, while values below 10% are considered poor. based on this criterion, the elongation results in this study can be considered satisfactory. young’s modulus, which represents film stiffness, also varied significantly with cds concentration. the elasticity and strength of a material can be observed through the stress-strain curve or table obtained by performing tensile testing on the material (sakti et al., 2023). the results that showed at the table 1, highest value (9.45 mpa) was recorded at 3% cds, while the control film exhibited the lowest value (5.67 mpa). these results confirm that cds incorporation enhances film stiffness, with higher modulus values corresponding to reduced elasticity. this finding is consistent with suheti (2022), who reported that cds nanofillers improve mechanical rigidity by strengthening interactions between fillers and polymer chains. a similar trend was observed by millah & dwandaru (2024), where the addition of cds from corn husk waste increased the modulus of edible films from 11.40 mpa to 22.53 mpa. conclusions carbon nanodots (cds) were successfully synthesized from cocoa pod husks, as evidenced by their characteristic blue fluorescence, an average particle size of 997 nm, the presence of –oh, c=c, and c≡n functional groups, and a main xrd diffraction peak at 2θ = 24°. the incorporation of these cds into cellulose– carrageenan biodegradable films produced thin layers with thickness values ranging from 0.35 to 0.64 mm, tensile strength of 2.94–4.20 mpa, elongation at break of 36.51–63.28%, and young’s modulus of 6.00–9.45 mpa. increasing cds concentration generally enhanced the tensile strength and young’s modulus of the films, demonstrating their potential as functional nanofillers for improving the mechanical performance of biodegradable packaging materials. authors’ contributions: esa ghanim fadhallah, ahmad sapta zuidar, and teguh setiawan designed the study. 1190 biology, medicine, & natural product chemistry 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2540-9328 (online) the effect of use of local organic microorganism fertilizers on rice media, banana sticks, and tongol fish on the growth of arabika coffee plants (coffea arabica l.) in the gunung karang garden, pandeglang district suyamto*, m. hujaji, hadi susilo, swastika oktavia, mu’jijah biology study program, faculty of pharmaceutical and health sciences, mathla'ul anwar university banten, indonesia. corresponding author* suyamto35@yahoo.co.id manuscript received: 13 march, 2024. revision accepted: 23 july, 2024. published: 04 august, 2024. abstract arabica coffee plants (coffea arabica l.) are plantation commodities with high economic value. efforts that can be made in farming without using chemicals that will damage the environment is by utilizing local microorganisms (mol). for farmers who demand the use of cheap and practical fertilizers, they can be directed to use local microorganism organic fertilizers made from rice, banana stems, and tuna. this study aims to determine the effect of using local microorganism organic fertilizers made from rice, banana stems, and tuna on the growth of c. arabica plants. the type of research to be carried out is experimental research. this research method uses a completely randomized factorial design (crfd) consisting of 2 factors and 3 replications. the first factor is the source material consisting of 3 levels, namely: m1 = stale rice, m2 = banana stem, and m3 = tuna. the second factor is the fermentation period consisting of: h1 = 2 weeks fermentation and h2 = 4 weeks fermentation. the measured parameters of c. arabica plants are height, number of leaves, and stem diameter. the results showed that rice, banana stem, and tuna can be made into mol fertilizers. the use of mol organic fertilizers (stale rice, banana stem, and tuna) affects the growth of c. arabica, which can be seen from the parameters of plant height, number of leaves, and stem diameter. m3h2 mol organic fertilizer (tuna fermented for 4 weeks) provides optimal growth of c. arabica in terms of plant height, number of leaves, and stem diameter. keywords: banana stem; coffea arabica l.; tuna; mol; rice. introduction a plantation commodity with high economic value is the coffee plant (coffea arabica l.), which can provide foreign exchange income for the indonesian state and support national development. the area of coffee plantations in indonesia reached 1,243,441 ha (in 2019), with a coffee production capacity of 716,089 tonnes per year and an export value of 279,961 tonnes (815,933,000 us$) (makmur & karim, 2020). cultivating c. arabica plants to obtain optimal seed production results requires proper fertilization. many c. arabica farmers still do not pay special attention to good fertilization so that the production of c. arabica seeds is still low, which becomes a problem for c. arabica farmers, namely inappropriate fertilization which has an impact on the low production of c. arabica seeds from cultivation. (sapurah et al., 2019). production of c. arabica plants can increase by increasing the planting area and soil fertility with excellent and appropriate fertilization techniques. in farmers in developing countries like indonesia, still use synthetic pesticides and fertilizers to cultivate c. arabica plants. this can lead to dependence on the continuous use of synthetic fertilizers and pesticides, which can cause environmental damage, decrease soil fertility, and ultimately reduce soil productivity (manure, 2014). continuous use of synthetic chemicals (pesticides and chemical fertilizers) over a long period will pollute the environment, kill populations of beneficial microorganisms (playing a role in soil biogeochemical cycles), harm non-target pests, the emergence of resistant pests and nutrients in the soil will decrease. therefore, alternative solutions are needed to replace chemical fertilizers and develop microorganism-based biocontrol that is safer, more effective, has fewer detrimental effects, and is made from natural ingredients (mursalim et al., 2018). the use of local microorganisms or mol for short is an effort made as an alternative to the use of synthetic chemicals (chemical fertilizers) that can damage the environment. mol can utilize local microorganisms which can be used to improve soil structure, fertilize plants which can be a solution for farmers for organic https://doi.org/10.14421/biomedich.2024.131.251-258 252 biology, medicine, & natural product chemistry 13 (1), 2024: 251-258 farming that is environmentally friendly and free from dangerous chemicals. mol can be made by utilizing household waste, agricultural and livestock waste which can be easily obtained and is widely available in the environment so that using mol can save cultivation costs by up to 20-25%. mol is used as a starter in making organic fertilizer in both solid and liquid form (delfiana et al., 2019). mol can improve compost quality and speed up the composting process. mol contains bacterial microorganisms such as azotobacter sp., azospirillum sp., bacillus sp., pseudomonas sp., rhizobium sp., and phosphate solubilizing bacteria which are beneficial for plants and increase soil fertility. mol can be produced independently from surrounding natural materials such as agricultural waste, livestock waste and even household waste (rahayu & tamtomo, 2017). research on household waste-based mol has been conducted using stale rice (arifan et al., 2020); stale rice, banana stems, and tuna fish, which had a significant influence on the growth of mustard greens (brassia juncea) (wet weight, number of leaves, and plant height (mursalim et al., 2018). utilization of household and agricultural waste or waste such as stale rice, tuna and banana stems as organic fertilizer by utilizing mol by farmers so that they obtain fertilizer that is cheap, practical and easy to make but very effective and efficient for farmers in increasing plant fertility and improving plant quality so that crop production will increase. this research aims to determine the effect of using organic fertilizer from local microorganisms on the growth of c. arabica plants rown on rice, banana stems and tuna. research methods this research was conducted in march – october 2022. the research was conducted at gunung karang gardens, pandeglang regency. the tools used in this research were a shovel, a sprayer, a 5 liter bucket, paper (used newspapers), a board container, a glass jar, a plastic bottle, a scale, a 15x20 cm polybag, a handsprayer, a measuring cup, and a caliper. the materials that will be used in this research include arabica coffee seeds (coffea arabica l). water, banana stems, rice washing water, rice, coconut water, brown sugar, naoh, soil and tuna. making mol organic fertilizer mol stale rice put the stale rice in a box-shaped container (20 cm x 15 cm x 7 cm), cover with newspaper and provide an air gap so that air can still enter. the container containing the rice is then placed in a damp and shady place so that mold grows on the entire surface of the rice. rice that has grown mold or microorganisms is selected by discarding rice that contains black mold. the selected rice is then put into the bucket. 500 ml of coconut water and rice washing water and 1 kg of brown sugar were added to the bucket containing the stale rice, then 1,500 ml was added. the bucket was covered with newspaper and stored for 2 weeks (h1) and 4 weeks (h2). mol banana stem media the banana stems are chopped into small sizes then put into a box-shaped container (20 cm x 15 cm x 7 cm), covered with newspaper and given an air gap so that air can still enter. the container containing the chopped banana stems is then placed in a damp and shady place so that the entire surface of the chopped banana stems grows with mold. chopped banana stems that have grown fungi or microorganisms are selected by discarding chopped banana stems that contain black fungi. the chopped banana stems that have been selected are then put into the bucket. add 500 ml of coconut water and rice washing water and 1 kg of brown sugar to the bucket containing chopped banana stems and then add 1,500 ml. the bucket was covered with newspaper and stored for 2 weeks (h1) and 4 weeks (h2). mol tuna fish media 1 kg of tuna is chopped into small pieces and then put into a box-shaped container (20 cm x 15 cm x 7 cm), covered with newspaper and given an air gap so that air can still enter. the container containing chopped tuna is then placed in a damp and shady place so that the entire surface of the tuna is covered with fungus. tuna fish that have grown fungi or microorganisms are selected by removing pieces of tuna that contain black fungi. the tuna that has been selected is then put into the bucket. add 500 ml of coconut water and rice washing water and 1 kg of brown sugar to the bucket containing chopped banana stems and then add 1,500 ml. the bucket was covered with newspaper and stored for 2 weeks (h1) and 4 weeks (h2). mol formulation design this research method uses a factorial completely randomized design (crd) consisting of 2 factors and 3 replications. the first factor is the original ingredients which consist of 3 levels, namely: m1 = stale rice, m2 = banana stems and m3 = tuna. the second factor is the fermentation time which consists of: h1 = 2 weeks fermentation and h2 = 4 weeks fermentation. these two factors were repeated 3 times to obtain 18 repetitions. hujaji et al. – the effect of use of local organic microorganism fertilizers on … 253 table 1. combination of treatments. treatment h1 h2 m1 m1h1 m1h2 m2 m2h1 m2h2 m3 m3h1 m3h2 preparation of research areas and plants preparing the research area is by cleaning the research area from weeds, rocks and rubbish with a hoe. three beds were made of soil and a drainage ditch was made in each bed to avoid waterlogging. a shade is created above the bed to avoid the intensity of sunlight and direct rain exposure to the nursery area. the shelter is made with a bamboo or wood frame measuring 2.5 m x 1.7 m. the growing medium used is topsoil, which is fine fertile soil that has been sifted with a 20 mesh diameter sieve, where the size of the polybag used is 1 kilo with a ratio of soil to sand of 1:1. preparation of planting media preparing the planting media is by filling the planting media into polybags, filling them one by one by mixing 1 part topsoil with 1 part sand into the polybag. selection of c. arabica seeds c. arabica seeds were selected that were the same size, the seeds were not attacked by disease, were not moldy, the color was uniform, the seed leaves had no holes, were healthy and of good quality. planting c. arabica seeds c. arabica seeds are planted into the planting medium by immersing them, namely the back side up and the belly side down. planting c. arabica seeds in polybags c. arabica planting is done by taking the nursery seeds and transferring them into prepared polybags. providing mol treatment mol liquid fertilizer solution (stale rice, banana stems, and tuna) is made by dissolving organic fertilizer according to a predetermined dose in size (50 ml, 100 ml, 150 ml, 200 ml, 250 ml and 300 ml) in each measuring cup by adding one liter of water each then stirring evenly. the mol fertilizer solution (stale rice, banana stems, and tuna) is then put into a handsprayer and then sprayed on c. arabica plants when the stomata are open, namely in the morning between 08.00-10.00. mol is given at 7 weeks after transplanting (mspt). subsequent fertilization every week for 8 weeks. when spraying the c. arabica plants under observation, they were protected using cardboard so that splashes of the solution did not hit other plants with different treatments. maintenance maintenance by watering c. arabica every morning and evening according to the humidity conditions of the planting medium. other maintenance is controlling pests and diseases and removing weeds that grow manually using hands or a sickle. observation parameters plant height (cm) observations were made by measuring the height of the c. arabica plant, measured from the base of the stem at the ground surface to the tip of the stem, using a meter, which was carried out every week for eight observations. number of leaves (strands) observations were made by counting the number of fully formed c. arabica leaves, which was carried out every week for eight observations. stem diameter (cm) observations were made by measuring the middle of the c. arabica stem that appeared on the ground using a caliper, which was carried out every week for eight observations. data analysis data from observations of c. arabica plants were analyzed using analysis of variance to determine the effect of treatment. if there are differences between treatments, duncan's multiple range test is carried out with α=1% and α=5%. results and discussion observation of plant height results of observations of c. arabica plant height at the age of 7 weeks after transplanting (mspt) during 8 weeks of observation (table 2). table 2. observation results of c. arabica plant height. treatment plant height (cm) 0 1 2 3 4 5 6 7 8 m1h1 19,23 21,40 23,87 26,17 28,30 30,63 33,10 35,50 37,70 m1h2 19,23 21,60 24,17 26,40 28,67 31,20 33,47 36,20 38,30 m2h1 19,23 21,80 24,53 27,17 29,70 32,30 34,77 36,50 38,40 m2h2 19,27 22,00 24,80 27,57 30,27 32,90 35,60 37,67 39,50 m3h1 19,27 22,30 25,10 27,93 30,53 33,43 36,20 37,70 39,67 m3h2 19,27 22,40 25,73 28,70 31,77 34,90 38,13 39,30 40,63 control 19,23 19,73 20 20,27 20,70 21,10 21,30 21,80 22,70 254 biology, medicine, & natural product chemistry 13 (1), 2024: 251-258 figure 1. crop high response graph. results of statistical analysis using duncan's test on c. arabica plant height parameters (table 3). table 3. duncan test plant height parameters. treatment plant height (cm) m1h1 28,43ab m1h2 28,80ab m2h1 29,37ab m2h2 29,95ab m3h1 30,23b m3h2 31,20b control 20,75a note: numbers followed by different letters indicate a significant difference based on the duncan test at the 5% level (p≤0.05) plant height is a plant measurement that is often observed as an indicator of growth or as a parameter to measure the influence of the environment or the treatment applied because plant height is the easiest measure of growth to see (ramli et al., 2017). the results of observations of c. arabica plant height after analysis of variance showed that there were variations in plant height. the average height of c. arabica plants in the m1h1 treatment was 28.4333 cm, m1h2 was 28.8037 cm, m2h1 was 29.3778 cm, m2h2 was 29.9519 cm, m3h1 was 30.2370 cm and m3h2 was 31.2037 cm. the highest c. arabica plant height in the 8th week occurred in the m3h2 treatment (mol tuna fermented for 4 weeks) with an average height of 40.3 cm. various types of mol react fully and can be utilized by plants where the administration of various mol begins to show their respective advantages. the results of statistical analysis using the duncan test on c. arabica plant height showed that there were significant differences (significantly different) between the m1h1, m1h2, m2h1, m2h2, m3h1 and m3h2 groups and the control. it is suspected that giving mol contains nutrients that play a role in plant vegetative growth. this is thought to be because the treatment (giving mol rice, banana stems and tuna) can fulfill the need for macro and micro nutrients for plant height growth. a plant will grow and develop well if the nutrients the plant needs are in sufficient quantities and are in a form that is ready to be absorbed. meanwhile, yudiawati & kurniawati. (2019) explained that mol contains a source of nitrogen and phosphorus for plants. where plants need the elements n and p to be able to grow, develop and produce well and stimulate vegetative growth. apart from that, the presence of microorganisms contained in mol is thought to stimulate the media decomposition process, thereby helping to provide nutrients from organic material available in the soil which ultimately can increase nutrient absorption by plants. romero (2014) states that the addition of organic matter to the soil can increase the development of soil microorganisms, because there is a supply of carbon as energy for the development of microorganism activity in the soil, thereby helping the absorption of nutrients from the soil and increasing plant production. the addition of useful microorganisms in mol is thought to have helped supply n for optimal plant growth. mol added with beneficial microorganisms works better in aerobic soil conditions. conditions like this support an increase in the population of soil microorganisms which can help provide nutrients for plant growth. these microorganisms are known to have the ability to provide nutrients and contain growth hormones that plants need. these microorganisms are also able to work well in increasing plant growth. mol contains 7 microorganisms which are very useful for plants, namely: azospirillium, azotobacter, bacillus, aeromonas, aspergillus, phosphate solubilizing microbes and cellulotic microbes. according to suiatna (2010) azospirillium sp is a non-symbiotic n-fixing bacteria in the air. apart from its direct role in increasing plant n content, azospirililum sp. it is also able to produce phytohormones that can stimulate plant growth, including auxin, gibberellins and cytokinins. mol contains aspergillus niger bacteria which can dissolve the p element in the soil. this p element is very important for the growth of plant leaves. aspergillus niger which helps growth and soil fertility. a. niger is a microbe that can dissolve phosphate in the soil. if plants lack this element, plant growth will be hampered. a. niger increased stem growth several times higher than the control treatment. it is hoped that giving high doses of nitrogen will also form high levels of protein so that it will increase the width, length and number of leaves which will expand the surface available for photosynthesis (yuliani, 2015). the results of statistical analysis using the duncan test on c. arabica plant height showed that groups m1h1, m1h2, m2h1, m2h2 and groups m3h1 and m3h2 were significantly different. the m3h1 and m3h2 groups and the control group were also significantly different. this shows that the m3h1 and m3h2 groups have a greater influence on plant height compared to the 0 5 10 15 20 25 30 35 40 45 0 1 2 3 4 5 6 7 8 p la n t h ei g h t (c m ) observation time (week-) m1h1 m1h2 m2h1 m2h2 m3h1 m3h2 kontrol hujaji et al. – the effect of use of local organic microorganism fertilizers on … 255 m1h1, m1h2, m2h1, m2h2 groups. this shows that mol from tuna has the greatest influence on the height growth of c. arabica plants. the results are the same as research from mursalim et al (2018) where the mol treatment of tuna had the most significantly different effect on plant height among the other treatments. according to suaib (2015), tuna nutrition contains the element nitrogen (n) which functions as a fertilizer for the leaves. the protein content of tuna fish is 36.10%, equivalent to nitrogen of 5.78%. pamungkas & supijatno's (2017) research shows that applying nitrogen fertilizer has a significant effect on plant height. it is suspected that the plants have the ability to utilize nitrogen in the soil in the early weeks of observation so that the high yields of the plants are different from other treatments. the availability of nitrogen in the soil is influenced, among other things, by soil organic matter, soil water content, temperature and nitrogen fixation by soil bacteria. observation of the number of leaves results of observations of the number of leaves of c. arabica plants at the age of 7 weeks after transplanting (mspt) during 8 weeks of observation (table 4). table 4. observation results of the number of c. arabica leaves. treatment number of strands 0 1 2 3 4 5 6 7 8 m1h1 5,00 5,33 6,33 6,67 7,33 8,00 8,33 9,33 9,67 m1h2 5,33 5,67 6,67 7,00 8,33 9,00 9,67 10,33 10,67 m2h1 5,00 5,67 7,33 8,00 9,33 9,67 10,33 11,00 11,33 m2h2 5,00 6,00 7,67 8,67 9,67 10,67 11,00 11,67 12,33 m3h1 5,33 6,00 8,33 10,33 12,00 12,33 12,67 13,33 14,00 m3h2 5,33 7,00 9,33 11,33 12,33 13,33 14,33 14,67 15,67 control 5 5 5,33 5,67 5,67 6 6,33 6,67 7 figure 2. leaf count response graph. results of statistical analysis using the duncan test on the parameters of the number of leaves of c. arabica plants (table 5). table 5. duncan test parameters for number of leaves. treatment number of leaves m1h1 7,33ab m1h2 8,07abc m2h1 8,62abc m2h2 9,18abc m3h1 10,48bc m3h2 11,48c control 5,85a note: numbers followed by different letters indicate a significant difference based on the duncan test at the 5% level (p≤0.05) the second vegetative growth parameter observed was the number of leaves. observing the number of leaves is very necessary because apart from being an indicator of growth, leaf number parameters are also needed as supporting data to explain the growth process that occurs (ramli et al., 2017). the results of observations on the number of leaves of c. arabica plants after analysis of variance showed that there was variation in the number of leaves. the average number of c. arabica leaves in the m1h1 treatment was 7.33, m1h2 was 8.07, m2h1 was 8.62, m2h2 was 9.18, m3h1 was 10.48 and m3h2 was 11.48. the highest number of leaves in the 8th week was m3h2 (tongkol fish mol fermented for 4 weeks) with an average of 16 leaves. it is suspected that the increase in the number of leaves is due to leaf formation being influenced by the absorption and availability of nutrients. the nutrients contained in mol from flying fish waste play a major role in plant growth and development. the results of statistical analysis using the duncan test on the number of c. arabica leaves showed that groups m1h1, m1h2, m2h1, m2h2 and groups m3h1 and m3h2 were significantly different. the m3h1 and m3h2 groups and the control group were also significantly different. this is thought to be because there are nutrients in mol. according to the research results of suhastyo (2011), mol contains available n. the benefits of n itself for plants are for the formation or growth of plant parts, such as leaves, stems and roots. plants that lack phosphorus respond to leaf growth. soil that lacks phosphorus also has bad consequences for plants. the visible symptom is that the color of the entire leaf changes to become too 0,00 5,00 10,00 15,00 20,00 0 1 2 3 4 5 6 7 8 p la n t h e ig h t (c m ) observation time (week-) m1h1 m1h2 m2h1 m2h2 m3h1 m3h2 kontrol 256 biology, medicine, & natural product chemistry 13 (1), 2024: 251-258 old, and often appears shiny reddish. on the edges of the leaves of the branches and stems there is a purple red color which gradually turns yellow and will fall off. lack of phosphorus for plants will have an impact on the number of plant leaves. mol contains aspergillus niger bacteria which can dissolve the p element in the soil. this p element is very important for the growth of plant leaves. the taller the plant, the more leaves it has, the taller the plant will be, this is because the formation of carbohydrates resulting from plant assimilation increases, causing an increase in the fresh weight of the plant. this is thought to be because the mol treatment can fulfill the needs of macro and micro nutrients for plant growth (yuliani, 2015). the m3h2 group and the control group were also significantly different. this shows that the m3h2 group has a greater influence on the number of leaves of c. arabica plants compared to the m1h1, m1h2, m2h1, m2h2 and m3h1 groups. this shows that mol from tuna has the greatest influence on the number of leaves of c. arabica plants. the results are the same as research from mursalim et al (2018) where the mol treatment of tuna had the most significantly different effect on plant height among the other treatments. this is due to the optimal nutrient content of tuna mol. observation of the stem diameter results of observations of stem diameter of c. arabica plants at the age of 7 weeks after transplanting (mspt) during 8 weeks of observation (table 6). table 6. observation results of c. arabica stem diameter. treatment rod diameter (mm) 0 1 2 3 4 5 6 7 8 m1h1 4,67 4,67 5,00 5,33 5,33 6,00 6,33 6,67 7,33 m1h2 4,33 4,67 5,33 5,67 6,00 6,33 6,67 7,33 7,67 m2h1 4,33 4,67 5,00 5,33 5,67 6,00 6,33 7,00 7,33 m2h2 4,67 4,67 5,67 6,00 6,00 6,33 7,33 7,67 8,00 m3h1 4,33 5,00 6,00 6,33 6,33 8,00 8,67 9,33 9,67 m3h2 4,33 5,33 6,33 6,67 7,33 9,00 9,67 10,67 11,33 control 4,67 5 5 5,33 5,67 5,67 6 6 6,33 figure 3. bar diameter response chart. results of statistical analysis using duncan's test on c. arabica stem diameter parameters (table 7). table 7. duncan test for bar diameter parameters. treatment bar diameter m1h1 5,70a m1h2 5,74ab m2h1 6.00a m2h2 6,26ab m3h1 7,07ab m3h2 7,85b control 5,51a note: numbers followed by different letters indicate a significant difference based on the duncan test at the 5% level (p≤0.05) the results of observations of the stem diameter of c. arabica after analysis of variance showed that there were variations in stem diameter. the average stem diameter of c. arabica in the m1h1 treatment was 5.70 mm, m1h2 was 5.74 mm, m2h1 was 6.00 mm, m2h2 was 6.26 mm, m3h1 was 7.07 mm and m3h2 was 7.85 mm. according to julita et al (2013), apart from containing bacteria which have the potential to degrade organic matter, mol solution also acts as a growth stimulant to control pests and plant diseases. the results of statistical analysis using the duncan test on c. arabica stem diameter showed that the m1h2, m2h2, m3h1, m3h2 groups and the control group were significantly different. this is thought to be because there are nutrients in mol. it is suspected that the nutrients contained in the various types of mol provided can be fully utilized to increase stem diameter and it is suspected that environmental factors are more dominant in stem diameter, such as the intensity of sunlight. in the relationship between sunlight and plants, there is always a connection between sunlight and the process of photosynthesis. photosynthesis is the process of making food that occurs in green plants with the help of sunlight and enzymes. the results of photosynthesis are used to support plant growth and development. the stem is an area of accumulation of plant growth, especially in younger plants, so the presence of nutrients can 0,00 2,00 4,00 6,00 8,00 10,00 12,00 0 1 2 3 4 5 6 7 8 d ia m a te r b a ta n g ( m m ) waktu pengamatan (minggu ke) m1h1 m1h2 m2h1 m2h2 m3h1 m3h2 kontrol observation time (week-) p la n t h ei g h t (c m ) hujaji et al. – the effect of use of local organic microorganism fertilizers on … 257 encourage plant vegetative growth, including the formation of chlorophyll in the leaves, which will increase the rate of photosynthesis. the higher the rate of photosynthesis, the photosynthate produced will provide a greater increase in stem girth. stem diameter growth is faster in open areas than in shaded areas so that plants planted in open areas tend to be short and stocky, whereas plant diameter growth is closely related to the rate of photosynthesis which will be proportional to the amount of sunlight intensity received and respiration (yudiawati & kurniawati, 2019). the m3h2 group and the control group were also significantly different. this shows that the m3h2 group greatly influences the stem diameter of c. arabica compared to the m1h1, m1h2, m2h1, m2h2 and m3h1 groups. this shows that the mol of tuna has the greatest influence on the stem diameter of c. arabica. the nutrient content in mol is thought to be able to support plant stem diameter. mol from mackerel is usually rich in nutrients such as nitrogen, phosphorus, and potassium, which are important for plant growth and development. apart from that, mol from tuna fish also contains enzymes and amino acids which can help increase soil microbial activity and optimize nutrient availability for plants. therefore, the use of mol organic fertilizer from tuna can help increase plant productivity and quality, including the diameter of plant stems. organic fertilizer from mol mackerel fermented for 4 weeks provides optimal growth of c. arabica in terms of plant height, number of leaves and stem diameter. this is because fish contains many nutrients, namely n (nitrogen), p (phosphorus) and k (potassium), which are components of organic fertilizer. the main role of nitrogen (n) for plants is to stimulate overall growth, especially stems, branches and leaves. apart from that, nitrogen also plays a role in the formation of green leaves which is very useful in the photosynthesis process. another function is to form proteins, fats and various other organic compounds. apart from that, the role of nitrogen in plants can also increase plant weight, leaf diameter and plant height. the work of bacteria in breaking down proteins and amino acids into simpler compounds so they can grow and reproduce produces waste compounds such as nh3. fungus on tuna causes the fish to smell rancid. this odor arises due to the production of ammonia (nh3). the ammonia produced from decomposing mackerel is decomposed by soil microorganisms so that plants can obtain nitrogen properly. fish silage has high levels of n nutrients which are obtained from protein. it is hoped that the use of fish waste will be useful in the plant cultivation process because it has quite good nutritional content and amino acid content. in addition, organic matter in fish can affect growth. conclusion based on the results of the research and discussion, it can be concluded as follows: 1). rice, banana stems and tuna can be made into mol fertilizer. 2.) the use of mol organic fertilizer (stale rice, banana stems, and tuna) influences the growth of c. arabica, namely the parameters of plant height, number of leaves and stem diameter. 3). mol m3h2 organic fertilizer (mol tuna fermented for 4 weeks) which provides optimal growth of c. arabica in terms of plant height, number of leaves and stem diameter. competing interests: the author declares there is no conflict of interest. references arifan, wilis ari setyati, r.td wisnu broto & aprilia larasati dewi. 2020. pemanfaatan nasi basi sebagai mikro organisme lokal (mol) untuk pembuatan pupuk cair organik di desa mendongan kecamatan sumowono kabupaten semarang. jurnal pengabdian vokasi. 01(04): 252-256. batara, noviani lily. 2015. kualitas mikroorganisme lokal (mol) yang digunakan pada penanaman padi (oryza sativa 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jurnal agroscience volume 5 no. 2:-12. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 12, number 1, april 2023 | pages: 133-141 | doi: 10.14421/biomedich.2023.121.133-141 issn 2540-9328 (online) ethnobotanical survey of aromatic and medicinal plants used in traditional medicine and agri-food in the fez-meknes region hamza el finou*, nadia salhi, asma halmoune, lhoussaine el rhaffari department of biology laboratory of bioactives, health and environment, faculty of sciences, moulay ismail university meknes. morocco. corresponding author* h.elfinou@edu.umi.ac.ma manuscript received: 11 november, 2022. revision accepted: 29 december, 2022. published: 14 january, 2023. abstract in order to identify the medicinal plants used in agri-food and traditional medicine by the population of fez-meknes region (morocco), a floristic and ethnobotanical study was carried out in 4 provinces of this region (fez, meknes, azrou, taza). questionnaire forms were used to survey the usual users of the plants and herbalists and to collect as much information as possible on the therapeutic and dietary use of aromatic and medicinal plants in the region. for plants used for food, our survey identified 29 species divided into 26 genera and 16 families, including herbaceous plants (70%), trees (16.60%), shrubs (10%) and bushes (3.30%). the study of the medicinal flora used in traditional medicine also allowed the inventory 81 species belonging to 47 families. leaves are the most commonly used part and the majority of remedies were prepared in the form of infusion (47%) and decoction (26%). among all the diseases treated, digestive diseases are the most cited (25%), followed by dermatological diseases (21%). the present study allowed us to evaluate some traditional practices used by the fez-meknes region population. in this context, it is essential to carry out similar investigations in other regions of the kingdom, in order to safeguard this precious natural heritage by means of a monograph that is as complete as possible and to validate the remedies and preparations identified using rigorous scientific protocols. keywords: agri-food; ethnobotany; medicinal plants; monograph; traditional medicine. introduction according to the who (world health organization), nearly 6377 species of plants are used in africa, of which more than 400 are medicinal plants that constitute 90% of traditional medicine. in 2004, nearly 75% of the african population used plants to treat themselves and do not have access to modern medicines, whose pharmaceutical industry still relies heavily on the diversity of secondary plant metabolites to find new molecules with novel biological properties (mikou et al. 2016) the use of plants in therapy has been known for a long time. due to the richness and original diversity of its flora, morocco constitutes a real phytogenetic reservoir with approximately 4500 species and subspecies of vascular plants. the diversity of the relief and the most varied bio climates associated with them have given rise to a large number of endemic species. the rate of endemism is about 20% of the total number of species (pousset 1989). however, while there is no denying the curative virtues of a large number of plants, a good knowledge of them (toxicity, form of administration, dose and method of preparation) is essential to select in the mass of actions attributed to plants. in this sense, floristic and ethnobotanical studies have been carried out in different regions of morocco and have shown a return of the populations to the traditional use of medicinal plants (hseini and kahouadji (2007), lahsissene and kahouadji (2010) and salhi et al. 2010). the multiplication of these ethnobotanical studies on a national scale will make it possible to gather more information on moroccan medicinal plants, to enhance them and to preserve some of the knowledge acquired by the local population (mikou et al. 2016). these ethnobotanical studies are the most approach for discovering new medical plants or focused on those previously identified for their bioactive ingredients (celestina et al. 2012). in this sense, and in order to highlight the virtues and traditional uses of aromatic and medicinal plants, the present ethnobotanical study was conducted among the population of the fez-meknes region. materials and methods study area the present ethnobotanical study was carried out in the form of a survey using a pre-established questionnaire with various specific questions about the informant, the vernacular identity of the plant, as well as the part used, https://doi.org/10.14421/biomedich.2023.121.134-141 136 biology, medicine, & natural product chemistry 12 (1), 2023: 133-141 the modes of preparation, the therapeutic and traditional uses. study zone located in the north centre of morocco, the fezmeknes region is composed of 2 prefectures (fez and meknes) and 7 provinces (taounate, taza, sefrou, el hajeb, boulemane, my yacoub and ifrane) (figure 1). the region has three types of climates; a continental climate in the northern part, very hot and dry in summer and cold and wet in winter. a cold and humid climate in mountainous zones, very cold and snowy in winter, temperate in summer and a semi-arid climate in the high hills of boulemane, where the average rainfall does not exceed 250 mm. the winter is very cold and snowy. figure 1. the fez-meknes region on the national map. survey procedure in order to ensure a high degree of objectivity of the data obtained from our study, the survey is conducted using a survey form or questionnaire based on four axes:  information about the informant's profile (age, gender, level of education, etc.)  choice between the two medicines (modern and traditional)  information on the nature and pharmaceutical techniques of the plants used (local name, part used, method of preparation, dose)  information on the use of medicinal plants for diseases treatment. data analysis for data analysis, ibm spss statistics-21 software was used. the graph-pad prism 8 software was also used to create graphs. the frequency of citation was determined as follows: 𝐹𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑜𝑓 𝑐𝑖𝑡𝑎𝑡𝑖𝑜𝑛 = ([ 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑖𝑡𝑎𝑡𝑖𝑜𝑛𝑠 𝑥 100 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑖𝑡𝑎𝑡𝑖𝑜𝑛𝑠 ]) results socio-demographic data for the use of aromatic and medicinal plants, the results obtained showed a dominance of women gender with 87.3% against 12.7% for men, and the age group of 40 to 60 years with 51%. for education level, those not attending school come first at 41%. analysis of medicinal plants catalogue inventoried floristic aspect the study of the medicinal flora has allowed the inventory of 81 species belonging to 47 families. among the 47 families, the most used are the lamiaceae with 14 species followed by the asteraceae (7), apiaceae (7), fabaceae (5) and myrtaceae (3). table 1 presents a complete analysis of the plants. plant parts used and preparation modes the plant parts used are ranked in decreasing order of importance: leaves (42%), seeds (27%), fruits (10%), stem (9%), and bark (9%). other parts were presented by 5% (figure 2.a). to facilitate the use of medicinal plants, several preparation methods were used, such as decoction, maceration and infusion. in this sense, we obtained that infusion (47%), decoction (26%), whole plant (16%) and cataplasms (11%) were the most used preparation methods (figure 2.b). leav es ste m s seeds b ark fru it o th er 0 15 30 45 60 42% 9% 27% 9% 10% 5% p e rc e n ta g e % a in fu sio n decoctio n cata pla sm raw p la nt 0 15 30 45 60 47% 26% 11% 16% p er ce n ta g e % b figure 2. percentage of parts used (a) and preparation methods (b). el finou et al. – ethnobotanical survey of aromatic and medicinal plants … 135 why do you use traditional medicine in order to find out the reasons for using traditional medicine, the interviewees were asked this question. the efficacy criteria came first with 48%, followed by the availability of plants and the lower cost with 8% and 7% respectively. while 38% of the respondents stated all three criteria at the same time. the use of medicinal plants constitutes a risk to human health, hence the need to know their toxicity status. for this reason, the question of toxicity was asked to the herbalists and phytotherapists, 89.2% of whom declared the nontoxicity of the plants most often used in the region. this percentage correlates with the good expertise of these professionals. the survey revealed that 53.9% of these professionals had 10 to 20 years of experience in traditional herbal medicine field, and 21% had more than 20 years (figure 3). 0 15 30 45 60 under 10 years between 10 and 20 years more than 20 years 25% 53% 20% pourcentage % figure 3. years of experience of herbalists and practitioners interviewed. symptoms treated with aromatic and medicinal plants of all the diseases treated, digestive diseases are the most cited (25%), followed by dermatological diseases (21%) and cardiovascular diseases (15%) (figure 4). 0 5 10 15 20 25 30 neurological-neueropsychic other osteoarticular respiratory cardiovascular dermatological digestive 5% 7% 9% 13% 15% 21% 25% percentage % figure 4. symptoms treated with aromatic and medicinal plants. table 1. list of plants used for food purposes. common name scienific name family biological form preparation method part used nutritional interest number of citations le bugle ajuga iva lamiaceae herbaceae fresh leaves rich in calcium, potassium, and antioxidants. use for the urinary system diseases. 3 malva, la mouve malva sylvestris malvaceae herbaceae cooked leaves, stem rich in vitamins c and e, unsaturated fatty acids with metabolic and digestive benefits. 5 le palmier nain chamaerops humilis arecaceae shrub fresh fruit, root antioxidants, omega 3, 6 and 9 and vitamins a, b, c, e. interest in blood pressure regulation. 1 l’arbousier arbutus unedo ericaceae shrub fresh fruit rich in vitamin c 2 136 biology, medicine, & natural product chemistry 12 (1), 2023: 133-141 common name scienific name family biological form preparation method part used nutritional interest number of citations -antioxidants regulation of cholesterol synthesis. chardon d’espagne scolymus hispanicus astéraceae herbaceae cooked and fresh leaves, stem composed of inulin (simple sugar) with metabolic interest in passing kidney stones. 6 la ronce rubus fruticosus rosaceae shrub fresh fruit rich in vitamin c and all forms of vitamin b (except b12). 2 cardon cyanara cardunculus astéraceae herbaceae cooked leaves, stem high mineral and vitamin b content. 3 rhus pentaphylla anacardiace ae shrub fresh fruit -1 l’oléastre olea europea oleaceae tree oil fruit rich in fatty acids: omega 3, 6, 9 with digestive and metabolic benefits. 2 romarin rosmarinus officinalis lamiaceae herbaceae cooked leaves metabolic interest and use in the digestive system. 2 fenouil foeniculum vulgare apiaceae herbaceae cooked stem, bulb rich in vitamins b, c, d, e. metabolic interest and use for digestive system. 2 le jujubier zizyphus lotus rhamnaceae tree fresh fruit use for the digestive and genito-urinary system. 5 le thym thymus bleicherianthus lamiaceae herbaceae cooked leaves and whole plants use for the treatment of digestive tract. 4 herbaceae cooked leaves and stem metabolic interest. 4 pourpier potage portulaca oleracea portulacacea e herbaceae cooked leaves and stem rich in omega 3. 4 chêne verte quercus ilex fagaceae tree fresh seeds potassium, calcium, phosphorus with interest in metabolic, regulation of glycemic and digestive tract disorders. 5 menthe à feuille ronde mentha rotundifolia lamiaceae herbaceae cooked leaves and stem digestive tract and respiratory system. 5 le myrte myrtus commuins myrtaceae tree fresh fruit use for fatigue and digestive tract. 4 petite férule elaeoselinum asclepium apiaceae herbaceae fresh and cooked leaves and stem metabolic interest. 1 la férule ferula communise apiaceae shrub cooked flowers anticoagulation. 2 lavande lavandula multifidal lamiaceae shrub cooked leaves treatment of digestive tract and respiratory system. 3 grande oseille rumex acetosa polygonacée ae herbaceae fresh and cooked leaves rich in vitamin c with metabolic interest. 4 ache aquatique apium nodiflorum apiaceae herbaceae cooked leaves high content of calcium, vitamin e, b9, and antioxidants. 4 la bette beta vulgaris amaranthace aeou chenopodiac eae herbaceae cooked leaves vitamin c, potassium and iron. 3 origan compact origanum compactum bentham. lamiaceae herbaceae cooked leaves and whole plants treatment of gastric pains. 4 moutard des champs sinapis arvensis brassicaceae herbaceae cooked leaves metabolic interest. 3 lavande papillon lavandula stoechas lamiaceae herbaceae cooked leaves and whole plants use for the treatment of digestive tract and respiratory system. 4 el finou et al. – ethnobotanical survey of aromatic and medicinal plants … 137 common name scienific name family biological form preparation method part used nutritional interest number of citations caroubier ceratonia siliqua fabaceae tree flour fruit rich in carbohydrates, vitamin a, amino acids and fatty acids. 6 table 2. list of plants for medical use. common name scientific name family therapeutic use frequency of citation percentage of citation globulaire globularia alypum l. plantaginaceae eczema, gout, digestive problems, diabetes, sports perspiration and wound healing. 10 9.25% thym serpolet thymus serpyllum lamiaceae treatment of cough, digestive disorders, colic. 81 80 .14 % la rue d’alep ruta chalepensis.l rutaceae hair care, dermatological, respiratory, oral and digestive diseases. 6 5.55% caroubier ceratonia siliqua l leguminosae (ceasalpiniaceae) regulation of intestinal transit, hypercholesterolemia. 8 7.40% ricin ricinus communis euphorbiaceae hair care. 1 0.92% aubépine crataegus monogyna rosaceae cardiovascular system, stress and sleep disorders. 6 5.70% chénopode chenopodium ambrosioides amaranthaceae treatment of fever, asthma and other ailments. 8 7.25% marrube blanc marrubium vulgare lamiaceae gastrointestinal diseases, cough and bronchitis. 3 2.77% carvi carum carvi l. apiaceae digestion, expectoration, colic, sometimes for rheumatic problems. 14 12.81% laurier rose nerium oleander.l apocynaceae dermatological problems, dental infections, fever. 20 18.51% verveine odorante aloysa citriodora verbenaceae nervousness, sleep disorders. 7 6.48% lavande lavandula angustifolia p.mill lamiaceae genito-urinary problems, digestive tract, hair and skin care. 11 10.18% khella ammi visnaga (l.) lam apiaceae (umbelliferae) oral and dental infections, respiratory disorders. 78 72.22% romarin rosmarinus officinalis l. lamiaceae asthma and colds, digestive tract, hair care. 63 58.33% girofle syzygium aromaticum myrtaceae antiseptic, urinary tract infections. 6 5.55% fenugrec trigonella foenum graecum fabaceae treatment of gastralgia, appetite, digestion, hypercholesterolemia. 32 29.62% harmel peganum harmala zygophyllaceae rheumatism, healing and burns. 4 3.70% souchet long cyperus longus cyperaceae inflammation and pain, anticonvulsant properties. 1 0.92% jujubier ziziphus lotus rhamnaceae soothing; anxiety, regulation of intestinal transit. 6 5.55% roseau à massette typha latifolia l. typhaceae analgesic, carminative. 2 1.85% thuya de barbarie tetraclinis articulata cupressaceae treatment of intestinal infections, fever, hair and skin care. 3 2.77% herniaire herniaria hirsuta caryophyllaceae renal calculi, urinary tract. 1 0.92% achillée des marais achillea odorata l. subsp astéracées rheumatism, tonic, stimulant. 1 0.92% myrte myrtus communis myrtaceae treatment of arthralgia.. 7 6.48% henné lawsonia inermis lythraceae hair colouring, dermatological problems. 12 11.11% cresson alénois lepidium sativum brassicaceae anti-rheumatic, lung diseases, gastric problems, and to boost immunity. 9 8.33% haricots noirs phaseolus vulgaris fabaceae anti-inflammatory properties. 2 1.85% vigne à vin vitis vinifera l. vitaceae against abscesses and constipation. 1 0.92% 138 biology, medicine, & natural product chemistry 12 (1), 2023: 133-141 common name scientific name family therapeutic use frequency of citation percentage of citation le cubèbe piper cubeba l.f. piperaceae tonic, stimulant, culinary, antiashmatic. 1 0.92% menthe odorante mentha suaveolens lamiacées tonic, stomachic and antispasmodic effects. 5 4.62% ortie urtica dioica urticaceae antirheumatic, treatment of inflammation, diuretic, antidiabetic. 1 0.92% souci des champs calendula arvensis asteraceae gastric inflammations. 1 0.92% sauge à feuilles verveine salvia verbenaca lamiaceaa regulation of perspiration; menstrual cycle. 6 5.55% rosier rosa centifolia rosaceae wellness, anxiety. 3 2.77% pin maritime pinus pinaster ait pinaceae treatment of respiratory diseases, pain relief for rheumatic diseases. 1 0.92% laurier laurus nobilis lauraceae antibacterial, antiviral, antiseptic, fungicide, decongestant, nervous system regulator, digestion and anxiety. 8 7.40% inule visqueuse dittrichia viscosa asteraceae constipation, gastrointestinal diseases. 3 2.77% marjolaine origanum majorana lamiacées digestive disorders, flatulence, nausea, intestinal spasms, diarrhea; nervous disorders, migraines, insomnia. 9 8.33% oseille de guinée hibiscus sabdariffa malvaceae treatment of respiratory tract inflammations, high blood pressure, cholesterol, fever and stomach pains. 1 0.92% ail allium sativum amaryllidaceae prevention of cardiovascular disease, cancer. 3 2.77% oursins echinops spinosus asteraceae dermatological diseases. 12 11.11% armoise artemisia vulgaris asteraceae treatment of digestive disorders, joint and muscle pain and insomnia. 11 10.18% magydaris magydaris panacifolia apiaceae hair care and dermatological diseases. 1 0.92% garou daphne gnidium thymelaeaceae hair care and dermatological diseases. 8 7.40% cumin velu ammodaucus leucotrichus apiaceae treatment of respiratory diseases and intestinal disorders. 8 7.40% melissa melissa officinalis lamiaceae calming (nervous disorders, stress, anxiety, anguish), antispasmodic (stomach, intestine), heart problems (tachycardia). 2 1.85% arganier argania spinosa sapotaceae cosmetic application (face and body care). 1 0.92% nerprun alaterne rhamnus alaternus l. rhamnacées anxiety, digestion, constipation. 3 2.77% sauge salvia officinalis lamiacées digestion, stress, anxiety, cough. 5 4.62% eucalyptus eucalyptus gunnii myrtaceae anti-inflammatory and analgesic, treatment of respiratory problems. 9 8.33% jasmin jasminum polyanthum oleaceae anxiety. 1 0.92% lupinus lupinus luteus fabaceae prevention of cardiovascular and skin diseases. 2 1.85% noyer juglans regia juglandaceae natural toothbrush, natural antifungal. 3 2.77% figuier opuntia ficus indica cactaceae diuretic activity. 1 0.92% bergamote citrus bergamia rutaceae digestive disorders, stress, mouth and skin infections. 1 0.92% coriandre coriandrum sativum.l apiaceae digestive disorders and diarrhea, and to induce sleep. 8 7.41% aristoloche aristilochia paucinervis aristolochiaceae digestive disorders, analgesic 1 0.92% origan, thym commun thymus vulgaris lamiaceae tonic, digestive diseases. 29 26.85% https://fr.wikipedia.org/wiki/lauraceae https://www.facebook.com/hashtag/marjolaine?__eep__=6&__cft__%5b0%5d=azxtmc8dyjurivnaleqpm7prq3dusapay0y7x2vfmnx5a-whmrqzjjfkea5tk-zr_gglsyeydi8-bfr8scy9x5ghdvvlbadsvlfzolrmrk_669jegvp2-sxu_lav9yqjojnv2scvukn49aop3pf6tpai&__tn__=*nk-r https://fr.wikipedia.org/wiki/malvaceae https://www.google.fr/search?bih=657&biw=1366&hl=fr&q=amaryllidaceae&stick=h4siaaaaaaaaaongvuluz9u3mcozljdzxmrnmjtyvjmtk5msmjyamaoa1j0dwb4aaaa&sa=x&ved=2ahukewiqypiax_zwahvgzrokhu25bk4qmxmoatalegqijhad https://fr.wikipedia.org/wiki/asteraceae https://fr.wikipedia.org/wiki/asteraceae https://en.wikipedia.org/wiki/apiaceae https://fr.wikipedia.org/wiki/lamiaceae https://fr.wikipedia.org/wiki/lamiaceae https://fr.wikipedia.org/wiki/sapotaceae https://fr.wikipedia.org/wiki/myrtaceae https://fr.wikipedia.org/wiki/fabaceae https://fr.wikipedia.org/wiki/salvadora_persica https://fr.wikipedia.org/wiki/salvadoraceae el finou et al. – ethnobotanical survey of aromatic and medicinal plants … 139 common name scientific name family therapeutic use frequency of citation percentage of citation coquelicot papaver rhoeas papaveraceae insomnia, respiratory diseases. 16 14.81% coloquinte citrurllus colocynthis cucurbitacées diuretic activity. 2 1.85% valériane valeriana officinalis valerianaceae sleep disturbances. 2 1.85% garance rubia tinctorum.l rubiacées stomach diseases, anti diarrhea.. 1 0.92% dauphinelle staphisaigre delphinium staphisagria ranunculaceae hair care. 1 0.92% euphorbe euphorbia resiniphera euphorbiaceae wound healing, ophthalmic diseases. 17 15.74% millepertuis hypericum perforatum hypericaceae anxiety, sleep problems. 14 12.96% colchique d’automne, safran des près conopodium majus koch apiaceae weight gain. 1 0.92% séné senna alexandrina caesalpiniaceae laxative, constipation. 1 0.92% anis étoilé illicium verum illiciaceae anti-inflammatory, intestine. 2 1.85% lin linum usitatissium linaceae inflammation, gastritis, hair care. 3 2.77% fenouil, aneth doux foeniculum vulgare apiaceae anxiety, insomnia, vomiting, gastrointestinal and respiratory disorders. 7 6.48% carline atractylis gummifera asteraceae appetite, stomach. 3 2.77% gingembre zingiber officinale zingibéraceae muscle pain, digestion. 1 0.92% géranium geranium robertianum géraniaceae stress and anxiety. 1 0.92% camommile romaine chamomilla nobilis astéraceae anxiety, insomnia. 2 1.88% discussion at the end of this survey, 29 species of plants for food use were identified, which were divided into 26 genera and 16 families, including herbaceous plants (70%), trees (16.60%), shrubs (10%) and bushes (3.30%). the study of the medicinal flora used in traditional medicine has also allowed the inventory of 81 species belonging to 47 families. for the socio-demographic data, the results obtained showed a dominance of female gender with 87.3% against 12.7% for men, and the age group of 40 to 60 years with 51%. for education level, those not attending school come first at 41%. in this sense, several ethnobotanical studies carried out on a national scale have also confirmed these results (ziyyat et al. 1997), hmamouchi (1999), (jouad et al. 2001), (eddouks et al. 2002), (tahraoui et al. 2007), (mehdioui and kahouadji 2007), (salhi et al. 2010) and (benkhnigue et al. 2012). for the used part, we obtained that the leaves are the most used (42%). then, seeds (27%), fruits (10%), stem (9%), bark (9%), ad other parts were presented by 5%. several studies also confirmed these results (el rhaffari and zaid (2008), bammou et al. 2015) and umartani and nahdi (2021). although the use of leaves is represented by an important percentage, it was noted during the survey, that some users proceed to pull the whole plant instead of being interested only in the desired part. on the other hand, there is a clear relationship between the used part of the plant and the effects of this exploitation on its existence (cunningham 1996), this mode of collection seriously compromises the sustainability of medicinal species, especially bulbous ones. knowing that the leaves are the seat of photosynthesis and sometimes of the storage of secondary metabolites responsible for the biological properties of the plant (bigendako-polygenis and lejoly 1990), the ease and speed of harvesting (bitsindou 1986) may be the cause of the high rate of use of foliage by the region population. the use of medicinal plants is done by several modes of preparation. in this study it was obtained that the majority of the remedies are prepared by infusion (47%) and decoction (26%). as the respondents do not have ideas about the precise quantities and measures in the preparation and dosage of phytomedicines. precision is lacking on several plants such as the quantities of plant organs to be prepared, the solvent or vehicle used, the time needed to prepare the solutions (decoction, infusion, powder, fumigation, poultice, maceration and daubing) and the precise dose to be prescribed. however, the infusion (47%) and decoction (26%) remain the most commonly used methods of preparation. this percentage shows that the local population grows to the decoction mode and finds it adequate to warm the body and disinfect the plant (lahsissene and kahouadji 2010). on the other hand, decoction allows the collection of the most active principles and attenuates or cancels the toxic effect of certain recipes. the results obtained showed that most medicinal plants are widely used in the care of the digestive system 140 biology, medicine, & natural product chemistry 12 (1), 2023: 133-141 (25%). our results are also confirmed by those of hmamouchi and agoumi (1993) who conducted a study in the mechraâ bel ksiri region, hseini and kahouadji (2007) in the region of rabat, mehdioui and kahouadji (2007) in the province of essaouira, salhi et al. (2010) in kenitra city and lahsissene and kahouadji (2010) in the zaër region. other plants are also used to treat dermatological diseases (21%) and cardiovascular diseases (15%). according to respondents and their clients, these herbal recipes have therapeutic action, which suggests that they include many bioactive ingredients that are essential to the living flora's physiological processes. as a result, they are thought to be more compatible with human bodies (agbatutu et al., 2022). conclusions traditional phytotherapy was and still, currently in demand by people who have confidence in trusting in the popular uses of plants and not having resources and access to modern medicine. the present work was carried out with the aim of making as complete an inventory as possible of the medicinal plants used in the fez-meknes region (morocco) and to gather information concerning the practical therapeutic uses in this region. the results obtained showed that of the 47 families inventoried, the lamiaceaes is the most represented with 14 species (29.78%). on the ethnobotanical and pharmacological side, the leaves are the most commonly used part. decoction and infusion are the most commonly used method. similarly, of all the diseases treated, digestive diseases are the most cited. in addition, our results showed an important diversity of wild edible species in the fez-meknes region, which represents a significant asset for food and nutritional diversification. the present study allowed us to evaluate some traditional practices used by the population of fezmeknes region. a wealth of knowledge and expertise has been shown. in this context, it is essential to carry out similar investigations in other regions of the kingdom in order to safeguard this precious natural heritage by means of a monograph that is as complete as possible, and to validate the remedies and preparations identified by means of rigorous scientific protocols. acknowledgements: the authors would like to thank all the herbalists and citizens of the fez-meknes region who agreed to participate in this survey. authors’ contributions: nadia salhi & asma halmoune and hamza el finou designed the study, collection of data. hamza el finou analyzed the data and wrote the manuscript. lhoussaine el rhaffari: validation and supervision. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references agbatutu, a., ogie-odia, e. a., ifedele, m. o., abdulrasaq, f. m., eseigbe, d., & imade, f. n. 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(1997). phytotherapy of hypertension and diabetes in oriental morocco. journal of ethnopharmacology, 58(1). https://doi.org/10.1016/s03788741(97)00077-9 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 977-981 | doi: 10.14421/biomedich.2025.142.977-981 issn 2540-9328 (online) academic stress and blood glucose levels: a correlational study among medical laboratory technology students lisa savitri1*, mochamad hanif hilmi1, elfred rinaldo kasimo1, rochmad krissanjaya1, kharisul ihsan2 1department of medical laboratory technology, faculty of health sciences, kadiri university, jalan selomangleng no. 1, kediri, east java, indonesia 2department of pharmacy, faculty of pharmacy, public health, hospital administration, radiology, universitas strada indonesia, kediri, indonesia. corresponding author* lisasavitri@unik-kediri.ac.id abstract diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose levels, often influenced by various physiological and psychological factors. one such factor is stress, particularly academic stress among university students. this study aims to explore the potential correlation between academic stress levels and random blood glucose levels among medical laboratory technology (d.iv) students at kadiri university. a cross-sectional, quantitative approach was used in this study. a total of 28 eligible students participated. academic stress levels were assessed using the perceived sources of academic stress (psas) questionnaire, while random blood glucose levels were measured using spectrophotometric methods with glucose god fs reagents. data were analyzed using the pearson correlation test to determine the relationship between the two variables. the majority of students experienced moderate academic stress (71.43%), while the rest (28.57%) experienced high stress. no students reported low stress levels. regarding blood glucose, 53.57% of participants had normal levels, and 46.43% had elevated levels. the pearson correlation coefficient was 0.045 with a p-value of 0.819, indicating no statistically significant relationship between academic stress levels and blood glucose levels. regression analysis also confirmed that while there was a slight positive trend (suggesting that higher stress may slightly raise glucose levels), the effect was negligible and not statistically significant. this study found no significant correlation between academic stress and random blood glucose levels among medical laboratory technology students. although stress may contribute to changes in glucose levels through hormonal mechanisms, other factors—such as diet, physical activity, and individual coping strategies—likely play a more substantial role. interventions focusing on stress management, healthy lifestyle habits, and regular health monitoring are recommended to maintain both psychological well-being and metabolic health in students. keywords: academic stress; blood glucose; university students; medical laboratory technology; correlation study. abbreviations: adrenocorticotropic hormone (acth), a hormone involved in stress response and glucose regulation; diabetes mellitus (dm), a chronic metabolic disorder characterized by high blood glucose levels; diploma iv (d.iv), referring to the four-year undergraduate equivalent program; glucose (glu), the parameter code used on the photometer for blood glucose measurement; glucose oxidase (god), an enzyme-based reagent used in glucose testing; international diabetes federation (idf), a global organization focusing on diabetes awareness and data; medical laboratory technology (mlt), the academic program of the student participants; probability value (p-value), used to assess statistical significance in hypothesis testing; perceived sources of academic stress (psas), the questionnaire used to measure academic stress; revolutions per minute (rpm), indicating the speed setting used in the centrifuge during serum preparation; and statistical package for the social sciences (spss), the software used for data analysis. introduction diabetes mellitus (dm) is a chronic condition characterized by elevated blood glucose levels due to impaired insulin function, which normally helps regulate blood sugar to maintain the body’s homeostasis. dm is classified into two main types: type 1, which requires external insulin (e.g., injections) due to the body’s inability to produce insulin, and type 2, which results from the body’s reduced sensitivity to insulin. type 2 is often associated with weight gain and requires lifelong management through healthy eating, regular exercise, and the prevention of hypoglycemia or hyperglycemia (nursucita & handayani, 2022). according to the international diabetes federation (idf, 2021), approximately 537 million adults (ages 20– 79) worldwide are living with diabetes—equivalent to 1 in 10 people—resulting in 6.7 million deaths annually, or one death every 5 seconds. indonesia ranks fifth globally, with 19.47 million people diagnosed out of a population of 179.72 million, indicating a national prevalence rate of 10.6%. the idf also reports that 81% of people with diabetes live in lowand middle-income countries, and 44% of adults with diabetes remain undiagnosed (simarmata & prabawati, 2024). riskesdas (2018) data manuscript received: 07 august, 2025. revision accepted: 27 october, 2025. published: 29 october, 2025. https://doi.org/10.14421/biomedich.2025.142.977-981 mailto:lisasavitri@unik-kediri.ac.id 978 biology, medicine, & natural product chemistry 14 (2), 2025: 977-981 shows that the prevalence of diagnosed diabetes in indonesians aged 15 and older was 2%, up from 1.5% in 2013. based on blood glucose tests, the prevalence increased from 6.9% in 2013 to 8.5% in 2018. only about 25% of people with diabetes are aware of their condition. east java ranks fifth among indonesian provinces with the highest number of diabetes cases (kusuma et al., 2024). blood glucose comes from the carbohydrates we eat, which are stored as glycogen in the liver and skeletal muscles. in people with diabetes, blood sugar levels can rise due to various factors such as diet, physical inactivity, medication, obesity, genetics, and stress. stress can trigger the release of hormones like adrenaline, glucagon, and cortisol, which increase blood sugar levels by reducing insulin sensitivity (fatih, 2023). stress is a physical or emotional response to events or thoughts that cause feelings of sadness, anger, or anxiety. it activates the nervous system, heightens sensory awareness, increases circulation, deepens breathing, and tenses muscles (wilujeng et al., 2023). academic stress refers to stress triggered by academic pressures, such as excessive assignments, tight schedules, unsatisfactory grades, or anxiety about exams (barseli et al., 2017). a study by ramadhani & mastuti (2022) involving 146 students found that 34% experienced moderate stress, 29% high stress, and 6% very high stress. academic load, environmental pressure, and individual personality traits can all contribute to student stress. prolonged stress can increase cortisol levels, which in turn raises blood sugar as the body prepares for increased physical and mental demands (caesaria et al., 2021). meanwhile, a study by natalansyah et al. (2020) involving 39 students showed that among 27 students experiencing moderate stress, 23 had high blood glucose levels. chi-square test results indicated a significant relationship between stress and blood glucose levels (p = 0.006). however, another study by caesaria et al. (2021) on 40 third-year medical laboratory technology (d.iii) students at poltekkes kemenkes palembang showed different findings. average blood glucose levels across different stress levels did not significantly differ (p = 0.98), indicating no correlation between student stress levels and blood glucose. materials and methods research design this study is a quantitative, non-experimental research using a cross-sectional design, which observes the relationship between dependent and independent variables by collecting data at a single point in time (yunitasari et al., 2020). tools and materials to assess academic stress levels, the materials used included the perceived sources of academic stress (psas) questionnaire developed by bedewy & gabriel (2015) and pens. for blood glucose testing, the equipment included a one tech medical komodo 200 photometer, dlab clinical centrifuge, onemed 3 cc syringe, tourniquet, onemed clot activator blood tubes (red), socorex micropipettes, medical gloves, plasters, yellow and blue pipette tips, and a proper medical waste disposal container. the materials used included 70% alcohol swabs, blood serum, and glucose god fs reagent for glucose measurement. procedure academic stress assessment procedure the research began by requesting formal approval from kadiri university, followed by obtaining ethical clearance from the research ethics committee to ensure the study met ethical standards. once permission was granted, the researcher approached potential participants, explained the study’s purpose and procedure, and obtained their informed consent. participants were then given the psas questionnaire to complete by marking checkboxes that best represented their experiences of academic stress. completed questionnaires were collected, coded for confidentiality, and scored to determine each respondent’s academic stress level based on their responses. blood glucose measurement procedure the blood glucose testing began by preparing all necessary tools and materials (e.g., syringe, alcohol swab, tourniquet, plaster, vacuum tube). after handwashing and patient identification, the researcher explained the blood draw procedure to the participant. the participant was seated comfortably, the puncture site was cleaned, and a tourniquet was applied. using a 15– 45° angle, blood was drawn into a syringe. the tourniquet was released, the needle removed, and pressure applied to stop bleeding. a plaster was placed over the puncture site for about 5 minutes. the collected blood was transferred into a labeled clot activator tube and left at room temperature for 15 minutes, then centrifuged at 3000–3500 rpm for 15 minutes to separate the serum. to measure glucose levels, the one tech medical komodo 200 photometer was turned on and set to the "analysis" mode with the “glu” parameter selected. the device was rinsed with distilled water. a standard solution was prepared using 1000 µl glucose reagent + 10 µl standard, and a sample solution using 1000 µl glucose reagent + 10 µl serum. a blank was made using 1000 µl reagent only. all mixtures were incubated at room temperature for 15 minutes. after incubation, readings were taken using the photometer: first zeroing the instrument with distilled water, then measuring the blank, standard, and sample in order. the resulting glucose levels were recorded by the researcher. savitri et al. – academic stress and blood glucose levels 979 data analysis techniques the study employed bivariate analysis to examine the relationship between two variables (sataloff et al., 2018). since the data included ordinal and ratio scales, correlation tests were used. prior to correlation analysis, normality and homogeneity tests were conducted. if the data were not normally distributed, spearman’s rank correlation was applied. if the data met normality assumptions, pearson’s correlation was used to examine the relationship between academic stress and blood glucose levels. all statistical analyses were performed using spss version 24, with a 95% confidence level (α = 0.05). results and discussion results pearson correlation is a statistical method used to evaluate the linear relationship between two variables— typically one dependent and one independent. this analysis produces a correlation coefficient to measure the strength of that relationship. a significance value (pvalue) greater than 0.05 indicates that there is no statistically significant correlation between the two variables. conversely, a p-value less than 0.05 suggests a statistically significant correlation (jabnabillah & margina, 2022). based on the pearson correlation test, the correlation coefficient was 0.045 with a p-value of 0.819, indicating no significant relationship between academic stress levels and blood glucose levels. since the p-value is greater than 0.05, the result is not statistically significant, meaning there's insufficient evidence to confirm a real correlation between the two variables in this population. regression analysis is another statistical method used to examine relationships and predict outcomes between variables. in this context, linear regression helps assess how an independent variable influences a dependent variable and whether the effect is positive or negative (refiantoro, 2022). the regression analysis showed that when the total stress score is zero, the initial blood glucose level (intercept) is 1.400. the regression coefficient for total stress was 0.050, meaning that for every one-unit increase in stress, blood glucose is estimated to increase by 0.050 units. however, the significance value of 0.819 shows that this influence is not statistically significant (as it exceeds 0.05), and the tvalue of 0.231 also confirms that the stress level does not significantly impact blood glucose. in conclusion, the results suggest no meaningful relationship or effect between academic stress and blood glucose levels among the students in this study. discussion out of 32 respondents, 28 students met the inclusion and exclusion criteria. according to the findings, no students experienced mild stress, 20 students (71.43%) experienced moderate stress, and 8 students (28.57%) experienced severe stress. this indicates that most medical laboratory technology (mlt) students at kadiri university experienced moderate academic stress, with a smaller portion reporting high stress. common sources of academic stress include learning demands, pressure to perform, time constraints, numerous assignments, unsatisfactory grades, and exam anxiety (barseli et al., 2017). these results align with a study by mutaqqin et al. (2021) on 75 medical students at fk unpri, where 74.7% of respondents experienced moderate stress, 6.7% high stress, and 18.7% low stress, suggesting that moderate stress levels are common among university students. blood glucose testing (table 5.2) showed that 15 students (53.57%) had normal glucose levels, while 13 students (46.43%) had high glucose levels. this may be because most students are in the 18–25 age range, a period where metabolism tends to function well. physical activity helps regulate blood glucose by improving circulation, enhancing muscle mass, and boosting metabolism (setianto et al., 2023). the finding that nearly half of the students had elevated glucose is consistent with sayekti & yulistari (2022), who found that 10 of their participants had high glucose levels. a hectic academic schedule can lead students to adopt unhealthy habits, including poor eating patterns, irregular sleep, and a lack of exercise. consumption of sugary foods, fast food, and soft drinks is common, often without the balance of physical activity—factors that can lead to higher blood glucose (sattu et al., 2024). normality and homogeneity tests confirmed that the data were normally distributed and homogeneous. the pearson correlation test again showed no significant relationship between academic stress and blood glucose (r = 0.045; p = 0.819). this aligns with research by sayekti & yulistari (2022) on sixth-semester mlt students at itsk insan cendekia medika jombang. of 26 students, 3 had normal stress, 3 low, 5 moderate, 11 high, and 4 very high. meanwhile, 16 students (61.5%) had normal blood glucose, and 10 (38.5%) had high levels. a spearman rank test yielded a p-value of 0.54 (> 0.05), indicating no significant correlation. the regression analysis also supported this, with an intercept of 1.400 and a coefficient of 0.050, suggesting that each unit increase in stress could increase glucose by 0.050 units—but again, the p-value of 0.819 and t-value of 0.231 confirm that this effect is not statistically significant. further breakdown showed that: 12 students with moderate stress had normal glucose levels, likely due to good physical fitness and healthy habits, 8 students with moderate stress had high glucose, possibly due to poor diet and lack of activity, 3 students with high stress had low glucose, likely from healthy behavior and regular exercise, and 5 students with high stress had high glucose, likely caused by elevated acth levels, which stimulate glucocorticoids, leading to increased 980 biology, medicine, & natural product chemistry 14 (2), 2025: 977-981 gluconeogenesis and higher blood glucose (saputra & muflihatin, 2020). academic stress is a common issue among students and can affect both physical and emotional well-being. causes range from lack of guidance and unsatisfactory academic performance to intense exam pressure and unsupportive environments. stress may manifest in various ways, including low self-confidence, poor focus, social withdrawal, irritability, frustration, and in some cases, depression (rahmawati et al., 2021). according to sari et al. (2024), stress is one of several factors that can elevate blood glucose levels, primarily by triggering the sympathetic nervous system and neuroendocrine responses. stress leads to the release of norepinephrine and epinephrine, which increase heart rate and stimulate glycogenolysis in the liver, resulting in rapid glucose release into the bloodstream (jasmine, 2023). based on these findings, the researcher suggests that other factors—such as diet, physical activity, genetics, and individual stress coping ability—may have influenced students’ blood glucose more than stress alone. many students with moderate stress still managed to maintain stable glucose levels through good time management, social support, and effective coping strategies. in conclusion, academic stress does not appear to significantly affect blood glucose levels. however, lifestyle factors like diet and exercise play a protective role in maintaining glucose balance. individuals experiencing moderate to high stress are encouraged to adopt healthy eating habits and regular physical activity to prevent long-term metabolic complications from elevated blood sugar. this study highlights the importance of psychosocial support and stress management education for students—especially those at risk for diabetes. regular blood glucose screening is also recommended for students with high stress levels to allow for early detection and preventive care. conclusions based on the findings of the study, the academic stress levels among medical laboratory technology (tlm) students at kadiri university were as follows: none of the students experienced low stress (0%), 20 students (71.43%) experienced moderate stress, and 8 students (28.57%) experienced high stress. regarding blood glucose levels, 15 students (53.57%) had normal levels, while 13 students (46.43%) had elevated levels. the pearson correlation test showed a correlation coefficient of 0.045 with a p-value of 0.819, indicating no significant relationship between academic stress levels and blood glucose levels. while the positive regression coefficient suggests a slight tendency for blood glucose to increase with higher stress levels, this effect is minimal and statistically insignificant. acknowledgements: thank you to the hematology laboratory of kadiri university and the clinical laboratory of the bhakti wiyata institute of health sciences, kediri, for their support during the completion of this research. authors’ contributions: lisa savitri designed the study, analyzed the data, and wrote the manuscript. all authors wrote the manuscript and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. funding: the authors declare that there are no funding. references barseli, m., ifdil, i., & nikmarijal. (2017). konsep stres akademik siswa. jurnal konseling dan pendidikan, 5(3), 143–148. https://doi.org/10.29210/115700 caesaria, n. d., nurfadilla, h., & puspitasari, d. (2021). hubungan tingkat stres terhadap kadar glukosa darah pada mahasiswa semester 3 prodi d3 analis kesehatan poltekkes kemenkes palembang. jurnal kesehatan dan sains, 6(1), 11– 17. fatih, m. (2023). pengaruh stres terhadap kadar gula darah. jurnal kesehatan bakti husada, 23(1), 45–52. international diabetes federation (idf). (2021). idf diabetes atlas (10th ed.). https://diabetesatlas.org jabnabillah, f., & margina, d. (2022). analisis regresi dan korelasi dalam penelitian kesehatan. jurnal statistika dan kesehatan, 4(2), 27–36. jasmine, p. (2023). mekanisme neuroendokrin stres terhadap kadar glukosa darah. jurnal fisiologi, 12(1), 59–66. kusuma, a. t., prasetya, m., & rahmadi, a. 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(2023). pengaruh aktivitas fisik terhadap kadar gula darah pada usia produktif. jurnal kesehatan olahraga, 11(1), 73–80. simarmata, t., & prabawati, r. (2024). prevalensi dan faktor risiko diabetes mellitus di negara berkembang. jurnal kesehatan global, 3(1), 41–50. wilujeng, s., prasetyo, h., & iskandar, r. (2023). respons fisiologis tubuh terhadap stres dan dampaknya pada kesehatan. jurnal biomedis indonesia, 15(2), 48–55. yunitasari, e., rahmah, m. m., & utami, d. (2020). metodologi penelitian kuantitatif dalam keperawatan. jurnal penelitian kesehatan, 8(1), 1–9. this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1105-1110 | doi: 10.14421/biomedich.2025.142.1105-1110 issn 2540-9328 (online) computational insights into the comparative analysis of insulinlike compounds in bitter melon: targeting gsk-3 protein and insulin receptor fernando nalini prasangika department of chemistry, university of kelaniya, kelaniya 11600, sri lanka. fernando nalini prasangika, university of kelaniya, tel. +94713716698, sri lanka. corresponding author naliniprasangika@gmail.com manuscript received: 06 august, 2025. revision accepted: 21 november, 2025. published: 27 november, 2025. abstract diabetes, a rapidly escalating global health challenge, is defined by chronic hyperglycemia resulting from impaired insulin secretion, impaired insulin action, or both. current treatment options often fall short due to side effects and limited efficacy in addressing long-term complications, underscoring the urgent need for safer, more effective alternatives. this study delves into the potential of insulin-like compounds derived from bitter melon (momordica charantia) to combat diabetes by targeting two pivotal proteins: glycogen synthase kinase-3 (gsk-3) and insulin receptors. these proteins are crucial for glucose regulation and insulin signaling, making them key targets for blood sugar control.through computational molecular docking, we evaluated the binding affinities and inhibition potentials of key bitter melon compounds, including charantin and vicine. molecular structures were sourced from the pubchem database and optimized using density functional theory (b3lyp functional, 6-311g++ (d, p) basis set) with gaussian-09 software. structural data for gsk-3 (pdb id: 1q5k) and insulin receptors (pdb id: 1ir3) were retrieved from the protein data bank, and docking studies were conducted using the lamarckian genetic algorithm in autodock 4.2. protein-ligand interactions, bond lengths, and amino acid residues in binding pockets were analyzed with discovery studio, while admet profiles and toxicity levels were predicted using pkcsm and protoxii.charantin demonstrated the highest binding affinity and inhibition potential against both gsk-3 and insulin receptors. toxicity analysis revealed that charantin, classified under toxicity class 6, is safer than vicine (class 4), with a higher ld50 value indicating lower toxicity. these findings position charantin as a promising multi-target anti-diabetic agent with significant efficacy and minimal side effects. this research paves the way for developing novel, safer anti-diabetic medications derived from natural sources, offering a beacon of hope in the fight against diabetes. keywords: gsk-3 protein; insulin receptor; molecular docking; optimization; toxicity. abbreviations: gsk-3 glycogen synthase kinase-3, irinsulin receptors introduction the medicinal plant bitter melon (momordica charantia) is well known for its possible anti-diabetic properties. according to research, the hypoglycemic qualities of bitter melon are attributed to a number of bioactive substances, mainly charantin, vicine, polypeptide-p, and certain lectins. due in large part to bioactive substances like charantin and vicine that imitate the action of insulin and increase insulin receptor activation, bitter melon (momordica charantia) is a medicinal plant with noteworthy anti-diabetic qualities. bitter melon is a viable natural option for managing diabetes because of these chemicals, which helps control glucose metabolism by boosting glucose uptake, glycogen formation, and enhancing insulin signaling. even though a large number of studies have demonstrated its positive effects, more investigation is required to ensure safe and effective use in conjunction with traditional therapies.(joseph & jini, 2013),(liu et al., 2021) charantin charantin (as shown in figure 1) is a triterpenoid combination of the cucurbitane type that is mostly made up of stigmasteryl glucoside and sitosteryl glucoside. by boosting glucose absorption and encouraging the production of glycogen in the liver, muscles, and adipose tissues, it has shown strong hypoglycemic effects. according to some research, charantin may work better than some oral hypoglycemic medications, such as tolbutamide. it is thought to work similarly to insulin, improving insulin sensitivity and glucose metabolism to assist control blood sugar levels. charantin's individual components, however, had less of an impact, suggesting that the mixture or other unknown elements may be https://doi.org/10.14421/biomedich.2025.142.1105-1110 1106 biology, medicine, & natural product chemistry 14 (2), 2025: 1105-1110 responsible for some of its effectiveness.(desai & tatke, 2015) vicine although it is not as well understood as charantin, another component of bitter melon also exhibits hypoglycemic action. both substances support the general glucose-lowering benefits of bitter melon, which include enhancing insulin receptor sensitivity and encouraging glucose absorption. (shown in figure 1) many of the conventional and experimental applications of bitter melon for the treatment of diabetes are supported by the combined effects of vicine and charantin. their functions in enhancing insulin signaling, reducing blood glucose levels, and possibly providing safer substitutes or supplements to traditional diabetic treatments are all supported by research. though encouraging, more thorough research is required to completely understand their mechanisms and maximize their therapeutic application. (a) (b) figure 1 insulin-like compounds in bitter melon a) vicine, b) charantin. (a) (b) figure 2. a) insulin receptor (pdb id: 1ir3) b) gsk-3 protein (pdb id: 1q5k) (denzel & kästner, 2018; ullah et al., 2023; zahid et al., 2019). insulin receptor two α-subunits that bind insulin and two β-subunits that have tyrosine kinase activity make up the transmembrane protein known as the insulin receptor (show in figure 2). insulin binding causes autophosphorylation of receptors, which starts signaling pathways that improve tissue metabolism, glucose absorption, and glycogen formation. diabetes and insulin resistance are exacerbated by impaired insulin receptor activity. it serves as a crucial molecular switch that transforms the presence of insulin into reactions that control glucose levels in cells.(varma narasimha k, 2022) gsk-3 protein by phosphorylating glycogen synthase, the kinase known as glycogen synthase kinase-3 (gsk-3) prevents the formation of glycogen. insulin promotes the synthesis of glycogen and the absorption of glucose by deactivating gsk-3 through the pi3k/akt pathway. insulin resistance results from overactive gsk-3, which also disrupts insulin signaling. as a result, gsk-3 inhibition improves insulin sensitivity and is a crucial target for the management of diabetes.(hazarika et al., 2012; pandey & degrado, 2016; ullah et al., 2023) molecular docking a popular computational method in drug development, molecular docking simulates the interaction between a target macromolecule, like a protein, and a small molecule (ligand) to determine the optimal binding orientation, or "pose." drug candidate creation and optimization are guided by the estimation of the ligandtarget complex's binding affinity and stability. in molecular docking, the target and ligand structures are prepared, the docking simulation is carried out using algorithms and scoring functions, and the outcomes are assessed for the most advantageous couplings. this approach is essential for both optimizing leads in structure-based drug creation and comprehending molecular recognition.(denzel & kästner, 2018; hase & scuseria, 2003; klein & lukeš, 2006; schlegel, 2011) this study explores the toxicity profiles of vicine and charantin, determines which phytochemical in bitter melon lowers blood glucose levels by which particular pathway, and assesses whether charantin can work through both the gsk-3 and insulin receptor pathways. to do this, the binding affinities of vicine and charantin with gsk-3 and the insulin receptor were ascertained using computational chemistry. the antidiabetic processes of bitter melon's bioactive chemicals, particularly vicine and charantin, are better understood thanks to this study. by evaluating their interactions with two important targets gsk-3 and the insulin receptor using computational chemistry, it becomes clearer which routes these substances use to reduce blood glucose. determining whether charantin can function via both pathways provides a basis for creating treatments that target both pathways. assessing the toxicity profiles of these substances also lends credence to their possible safe application as supplements or natural substitutes for current diabetes therapies. the overall goal of this research is to help find plant-based treatments for diabetes.(prasangika et al., 2025; sneha et al., 2023) materials and methods study area the computational assessment of antidiabetic phytochemicals present in momordica charantia (bitter melon) is the main objective of this work. charantin and prasangika – bitter melon: gsk-3 and ir targets 1107 vicine are molecularly docked with the insulin receptor and gsk-3, two important diabetes-related proteins, to determine their toxicity profiles, possible modes of action, and binding affinities. procedures all the computational calculations, optimizations, and molecular docking studies were done using a workstation with an intel© core i7 5820k cpu (3.3-3.6 ghz) and 32 gb ram, and the processor with intel(r) core (tm) i54570 cpu @ 3.20ghz and 4gb ram. protein preparation the 3d structures of protein molecules were downloaded from the protein data bank (pdb) (https://www.rcsb.org) (pdb id –1q5k and pdb id 1ir3) according to the resolution and method (x-ray crystallography). protein structures were cleaned by using biovia discovery studio 2021. all water molecules, ligands, ions and heteroatoms were removed from the protein molecule.from the cleaned gsk-3 whole protein, two distinct protein chains were separated. gsk-3a and gsk-3b. the ligand was docked with each of these three proteins, gsk-3 whole protein, gsk-3a, and gsk-3b. molecular docking autodock tools 1.5.6 and autodock 4.2 were used to perform molecular docking investigations, utilizing the lamarckian genetic algorithm and the free energy function. while ligands were made with hydrogen atoms and gasteiger charges, protein structures were made with polar hydrogen and kollman charges. blind docking was used to investigate every potential binding site after grid and docking parameter files were created. a population of 300, 2,500,000 energy evaluations, 27,000 generations, 100 genetic algorithm runs, a mutation rate of 0.02, and a crossover rate of 0.80 were all used in the docking process. using identical grid box dimensions, each ligand was docked to the insulin receptor, gsk-3, gsk-3a, and gsk-3b proteins in ten different trials. the ligand was flexible, but the protein stayed rigid. each set's optimal binding energy was chosen for examination. the docking log files (.dlg) were used to obtain inhibition constants and binding energies. plip and discovery studio were utilized to evaluate molecular interactions, bond lengths, and active site residues; pymol was employed for structural analysis and visualization. control inhibitor of the docking approach inhibitors are commonly found in protein structures and are kept in the rcsb protein data library. data from the pubchem database was used to optimize the inhibitors' structures using the same theoretical level. for docking studies, these optimized structures served as ligands. using the same docking procedure that was utilized to bind the protein and phytochemicals in bitter melon, the inhibitor was docked to the gsk-3 protein. autodock tools 1.5.6 was used to examine the docked conformation's binding energies. admet study protox-ii and the pkcsm web server were both used in this investigation to forecast the compounds' admet profiles. while protox-ii delivers a toxicity assessment that includes potential side effects and safety categories, the pkcsm server provides comprehensive pharmacokinetic information. a number of phytochemical toxicity metrics, such as hepatotoxicity, carcinogenicity, mutagenicity, cytotoxicity, ld50 (mg/kg), and toxicity class, were obtained using the protox-ii server. data analysis by choosing the lowest binding energy and matching inhibition constant (ki) from ten trials per protein-ligand pair, the docking data were examined. a comparative analysis revealed that vicine and charantin interacted more strongly with the insulin receptor and gsk-3 (including its a and b variants). plip and discovery studio were utilized to investigate protein-ligand interactions, and pymol was employed to visualize the structure. the protox-ii web server was used to estimate admet and toxicity profiles in order to evaluate the compounds' safety and drug-likeness. results and discussion docking results and optimized results table 1. the results of optimization and docking process. name of the phytochemical optimized energy ( x 104 kcal /mol) lowest binding energy with insulin receptor(kcal/mol) lowest binding energy with gsk-3 protein(kcal/mol) charantin -114.3271 -8.76 -8.49 vicine -71.3229 -6.02 -6.71 according to the results of the molecular docking investigation, charantin has a substantially greater binding affinity than vicine for the gsk-3 protein and the insulin receptor. with the lowest binding energies of https://www.rcsb.org/ 1108 biology, medicine, & natural product chemistry 14 (2), 2025: 1105-1110 -8.76 kcal/mol for the insulin receptor and -8.49 kcal/mol for gsk-3, charantin demonstrated a robust and consistent interaction with these important targets that are involved in insulin signaling and glucose regulation. vicine, on the other hand, had binding energies of -6.02 kcal/mol and -6.71 kcal/mol, respectively, which were significantly lower. charantin's structural stability is further supported by its optimized energy (-114.3271 x 104 kcal/mol), which could be a factor in its enhanced inhibitory capability. these findings demonstrate charantin's potential as a multi-target antidiabetic drug that can alter gsk-3 function and insulin receptor activation, two essential pathways for blood sugar regulation. charantin's ability to improve insulin sensitivity and glucose homeostasis may be supported by its stronger interactions, which also support its potential for development into safer, natural pharmaceutical alternatives for the treatment of diabetes. docking interactions of charantin figure 3. docking analysis of charantin a) interaction surface of insulincharantin complex b) 3d representation of insulin-charantin binding pocket c) interaction surface of gsk-3 protein-charantin complex d) 3d representation of gsk-3 protein-charantin binding pocket. strong polar interactions that support the high binding affinity and stable complex formation are indicated by the interaction analysis of charantin with the gsk-3 protein (as shown in figure 3), which mostly shows conventional hydrogen bonds and carbon hydrogen bonds. these hydrogen bonds are necessary for precisely locating charantin in the active site, which may prevent gsk-3's kinase activity from regulating glucose metabolism. charantin's interaction with the insulin receptor (as shown in figure 3), on the other hand, exhibits a wider variety of non-covalent interactions, such as alkyl, pi-alkyl, and van der waals forces. by enhancing the molecular fit and balancing the polar interactions at the binding site, these hydrophobic contacts improve the binding stability. charantin effectively occupies hydrophobic pockets on the insulin receptor, which may be essential for regulating insulin signaling and receptor activation, according to the existence of alkyl and pi-alkyl interactions. all things considered, charantin's capacity to efficiently bind and modify a variety of protein targets is demonstrated by its potent hydrogen bonding with gsk-3 and its adaptable hydrophobic interactions with the insulin receptor. its potential as an effective anti-diabetic agent that works through complementary mechanisms of gsk-3 inhibition and insulin receptor activation is supported by this complex binding profile. docking interactions of vicine figure 3. docking analysis of vicine a) interaction surface of gsk-3 protein-vicine complex b) 3d representation of gsk-3 protein-vicine binding pocket c) interaction surface of insulin-vicine complex d) 3d representation of insulin-vicine binding pocket. vicine and insulin interact via a variety of noncovalent bonding mechanisms, such as pi-anion interactions, van der waals forces, and traditional hydrogen bonds. by promoting intimate contact and complementarity between vicine and insulin molecules, these interactions aid in the stabilization of the insulin structure. tight binding is ensured by hydrogen bonds and van der waals forces, and the complex's overall stability is enhanced by the possibility of pi-anion interactions between the negatively charged groups on insulin and the aromatic rings in vicine. additionally, vicine attaches to gsk-3 by a combination of pi-alkyl interactions, van der waals forces, conventional hydrogen bonds, and carbon hydrogen bonds. this wide variety of interactions points to a more complex and long-lasting binding with gsk-3. vicine probably fits well into the active or allosteric regions of gsk-3 because carbon hydrogen bonds and pi-alkyl interactions usually improve the specificity and strength of ligandenzyme binding. vicine's binding may alter the activity of gsk-3, an essential enzyme involved in insulin signaling and glycogen metabolism, which could impact cellular functions linked to glucose absorption and prasangika – bitter melon: gsk-3 and ir targets 1109 storage. all things considered, these interactions demonstrate that vicine may influence insulin function in two ways: directly stabilizing insulin molecules and controlling important enzymes such as gsk-3 that are involved in insulin signaling pathways. vicine's pharmacological usefulness in controlling glucose metabolism is highlighted by this multi-targeted interaction pattern, which also points to its potential use in treatment approaches for diabetes and insulin resistance. admet properties of charantin and vicine table 2. comparative analysis of admet properties of two phytochemicals. admet parameter charantin vicine intestinal absorption (%) 79.677 28.87 water solubility(log mol/l) -4.741 -2.689 vdss (human) (logl/kg) -1.163 0.988 total clearance 0.689 0.252 ld50(mg/kg) 8000 1000 toxicity class 6 4 charantin and vicine's drug-likeness and pharmacokinetic profiles differ significantly, according to the admet analysis. when taken orally, charantin exhibits a significantly higher intestinal absorption rate (79.68%) than vicine (28.87%), suggesting superior bioavailability. charantin, on the other hand, is less soluble in aqueous environments than vicine (log mol/l -2.689), which may have an impact on its formulation and absorption dynamics. in terms of distribution, vicine shows a higher potential for distribution (0.988), suggesting that it may spread more widely throughout the body, whereas charantin displays a low volume of distribution (vdss, log l/kg -1.163), suggesting restricted tissue penetration and retention. the fact that charantin's total clearance (0.689) is greater than vicine's (0.252) indicates that charantin gets removed from the body more quickly, which may have an impact on how frequently it is taken. these chemicals are further distinguished by toxicity parameters: vicine's ld50 value is 1000 mg/kg, but charantin's is 8000 mg/kg, suggesting a far lower risk of acute toxicity. (as shown table 2) as a result, charantin may be safer for therapeutic uses because it is a member of a less hazardous class (class 6) than vicine (class 4). (as shown in table 2). despite having a lesser volume of distribution and water solubility than vicine, charantin has more advantageous admet properties overall, including better intestinal absorption and reduced toxicity. although formulation strategies may be required to address its solubility limits, these modifications imply that charantin may have higher oral bioavailability and safety characteristics, making it potentially more suited for therapeutic development. vicine has superior solubility and tissue distribution, but its therapeutic window may be limited by its increased toxicity and poorer absorption. prioritizing these phytochemicals for additional pharmacological and clinical research is made easier by this comparative admet profile. conclusions in conclusion, charantin shows great promise as a multitarget natural antidiabetic drug, outperforming vicine in a number of crucial areas, according to both molecular docking and admet studies. strong hydrogen bonds and a variety of hydrophobic contacts enable charantin to bind to both gsk-3 and the insulin receptor with noticeably increased affinities. these changes result in more stable and efficient adjustments to insulin signaling and glucose regulation systems. even though charantin has certain limits in terms of water solubility and tissue distribution, these molecular characteristics, along with its superior intestinal absorption and significantly reduced toxicity, greatly enhance its drug-likeness and therapeutic prospects. vicine, on the other hand, has a reduced binding potential, limited absorption, and increased toxicity, which may limit its potential as a therapeutic agent even if it has superior water solubility and a wider tissue distribution. overall, charantin's good pharmacokinetic characteristics and potent multi-target protein interactions support its continued development as a safer and more effective natural diabetes medication. these strong arguments support more thorough clinical and pharmacological research to fully understand charantin's potential in the treatment of diabetes. acknowledgements: i want to express my sincere gratitude to all of the university of kelaniya supporters for their crucial advice and help during this project. authors’ contributions: conceptualization, manuscript writing and computational simulations: nalini prasangika. competing interests: the authors declare that there are no competing interests. funding: funding was not requested. references denzel, a., & kästner, j. 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(2019). bitter gourd as the potential source of various bioactive compounds and its use for different diseases: a review. science letters, 7(3), 99– 103. https://doi.org/10.1109/mcise.2003.1208636 https://doi.org/10.6026/97320630008251 https://doi.org/10.1016/s2222-1808(13)60052-3 https://doi.org/10.1021/jp063468i https://doi.org/10.1155/2021/3796265 https://doi.org/10.7150/thno.14334 https://doi.org/10.4038/cjs.v54i3.8728 https://doi.org/10.1002/wcms.34 https://doi.org/10.1590/1519-6984.250179 biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 10, number 2, october 2021 | pages: 117-121 | doi: 10.14421/biomedich.2021.102.117-121 issn 2540-9328 (online) antibacterial activity of mangrove root extracts from ngurah rai mangrove forest, denpasar-bali made dharmesti wijaya1*, anak agung gede indraningrat2 1pharmacology and pharmacy department; 2microbiology and parasitology department, faculty of medicine and health sciences, warmadewa university jl. terompong no 24 denpasar 80235, tel. +62 361 240727, indonesia. corresponding author* dharmestiwijaya@gmail.com manuscript received: 08 october 2021. revision accepted: 15 october, 2021. published: 21 october, 2021. abstract the increasing rate of antimicrobial resistance in the past decades has motivated the search for novel antibacterial compounds to overcome infectious diseases. among diverse natural sources, mangrove ecosystems offer untapped sources of biological active compounds for future antibacterial medicine. this research was aimed to evaluate antibacterial activities of crude extracts of four dominant mangrove plants from the ngurah rai mangrove forest namely rhizophora mucronata, avicennia marina, rhizophora apiculata, and sonneratia alba. roots of these four plants were extracted using methanol, chloroform, and n-hexane. these crude extracts were tested against two gram positive bacteria (staphylococcus aureus and streptococcus mutans) and two gram negative bacteria (escherichia coli dan klebsiella pneumoniae) by disc difussion assay. we found that 3 mg/ml of n-hexane crude extracts from r. apiculata yielded the highest zone of inhibition of 8.64 mm against s. aureus. while, 3 mg/ml of chloroform crude extract of r. apiculata yielded the highest inhibition of 19.83 mm against s. mutans. unfortunately, no zone of inhibition was observed when crude extracts were tested against gram negative indicator strains. our results indicate that the root crude extracts of r. apiculata yielded the highest zone of inhibition against gram positive indicator strains compared to root crude extracts of r. mucronata, s. alba, and a. marina. further research is required to determine the antibacterial activities of the mangrove crude extracts against other bacterial indicator strains to determine their spectrum of activities. keywords: mangrove; roots, antibacterial; rhizopora apiculata. introduction antimicrobial substances play a crucial role during prevention and medication of infectious diseases, postoperation infections, infections after chemotherapy or infections for diabetic patients, late stage of kidney diseases and patients with rheumatoid arthritis (ventola, 2015). however, incidence rate of antimicrobial resistance has increased dramatically in the past decades (ventola, 2015), which cause difficulty to treat infectious diseases, increase cost for medication, and lead to higher mortality rate (world health organization, 2020). world health organization reported approximately 700,000 mortality every year until 2014 due to antimicrobial resistance and it is predicted the casualties of antimicrobial resistance reached 10 million in 2050 (departemen kesehatan republik indonesia, 2016). immediate actions are required to overcome the increasing rate of antimicrobial resistance such as a better diagnose and prescription of antibiotics, optimization of therapy regimen, and prevention of infectious diseases (ventola, 2015). in addition, the search for novel antimicrobial compounds are urgently needed since many antibiotics on the market are no longer effective to eradicate bacterial infections (ventola, 2015). among natural resources, mangrove ecosystems are the ideal habitat to search for novel antimicrobial activities. mangrove refers to ecosystem where seawater and fresh water dissolve, which characterize by plants that can tolerate high salinity and limited oxygen (friess, 2016). a number of studies showed that mangrove plants are rich of various of active compounds such as alkaloid, steroid, terpenoid, saponin, tannin, flavonoid dan poliphenol (dahibhate et al., 2018; gouda et al., 2015; patra & mohanta, 2014). furthermore, diversity of these secondary metabolites led to broad biological activities such as antibacterial, antifungal, antioxidant, antiviral, and anticancer, which overall could potentially be developed as the source of new drugs (patra & thatoi, 2011; saranraj, 2015). a study conducted in kalimantan showed antibacterial activities of root of mangrove plant of rhizophora apiculata (usman, 2018). however, a similar study from mangrove plants found in bali have not been conducted. the ngurah rai mangrove forest is the biggest mangrove ecosystem in bali which has more than 16 different mangrove spesies and the four https://doi.org/10.14421/biomedich.2021.102.117-121 118 biology, medicine, & natural product chemistry 10 (2), 2021: 117-121 dominant species are rhizophora mucronata, avicennia marina, rhizophora apiculata and sonneratia alba (lugina et al., 2017). to date, antibacterial properties of these four dominant mangrove species are rather unexplored. therefore, we aimed to screen for antibacterial activities of crude extract of the root of these four mangrove species by using three different solvents: methanol, chloroform and n-hexane against gram negative and positive indicator strains. it is expected antibacterial activities of these crude extracts will shed light to explore antibacterial active compounds from root of mangrove plants. materials and methods materials roots of the four dominant mangrove plants: rhizophora mucronata, avicennia marina, rhizophora apiculata and sonneratia alba were collected from the ngurah rai mangrove forest (8o43’40.4886” s, 115o11’42.80313” e), bali, indonesia, on august 2019 during the low tide. the species determination was carried out in eka karya botanical garden, bedugul, tabanan, bali, indonesia. the solvents (methanol, chloroform, and n-hexane) were purchased from merck, germany. methods sample extraction for each of mangrove species, 50 g of each dry powder of mangrove root were macerated twice using 250 ml of individual solvent (methanol, chloroform, or n-hexane) at room temperature for 24 hours and filtered using whatmann filter paper no. 1. the extracts were then evaporated using rotary evaporator (ika®, rv8) at 50°c, 90 rpm and stored in closed and air tight vial at 4°c until further used. antibacterial screening from each mangrove root extract, three level of concentrations were prepared: 1 mg/ml, 3 mg/ml and 5 mg/ml. subsequently, each crude extract was exposed against bacterial indicator strains gram positive (staphylococcus aureus dan streptococcus mutans) and gram negative (escherichia coli dan klebsiella pneumoniae) by disc diffusion assay. one hundred microlitre of bacterial indicator strains were spotted on luria bertani agar and spread using a sterile spreader. thirty microlitre of crude extracts were spotted on a 6 mm paper disc and air dried for 30 minutes. paper discs in triplicates containing crude extracts were placed on agar media containing bacterial indicator strains. ampicillin and streptomycin were used as positive controls, while the solvents, methanol, chloroform, and n-hexane were used as negative controls. antibacterial activities were calculated based on average zone of inhibition (zoi) that were formed from triplicate disc. results and discussion the results showed that antibacterial activities were displayed by some extracts against gram positive strains, s. aureus and s. mutans (table 1), but none against gram negative strains, e. coli dan k. pneumoniae. among the four mangrove species, only r. apiculata exhibited antibacterial properties against both gram positive indicator strains. the concentration of 3 mg/ml n-hexane extract showed the highest zoi of 8.64 ± 0.13 mm against s. aureus while 3 mg/ml chloroform extract yielded the highest zoi of 19.83 ± 0.63 mm against s. mutans. a study by seepana and co-workers (2016) also showed that methanol, ethanol, and hexane extract of r. apiculata roots is active against salmonela typhi and s. aureus. their study also showed that methanol, hexane, and ethanol r. apiculata root extracts have no activity against e. coli. however, there are activities against other gram-negative bacteria salmonella typhi, shown by methanol, hexane, and ethanol extracts of r. apiculata root. ethanolic extract of r. apiculata root also displays activity against k. pneumonia (seepana et al. 2016). figure 1. antimicrobial activity of r. apiculata n-hexane extracts in triplicates against s. aureus. the concentration of 3 mg/ml n-hexane extract showed the highest zoi (8.64 ± 0.13 mm). 5 mg/ml 5 mg/ml 5 mg/ml 3 mg/ml 3 mg/ml 1 mg/ml 1 mg/ml 3 mg/ml 1 mg/ml wijaya & indraningrat – antibacterial activity of mangrove root 119 figure 2. antimicrobial activity of r. apiculata chloroform extracts in triplicates against s. mutans. the concentration of 3 mg/ml chloroform extract yielded the highest zoi of 19.83 ± 0.63 mm against s. mutans. table 1. antibacterial activities of mangrove root extracts. bacterial strains samples solvents concentration zoi (mm) staphylococcus aureus rhizopora apiculata methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 8.05 ± 0.64 3 mg/ml 8.64 ± 0.13 5 mg/ml 7.84 ± 0.61 rhizopora mucronata methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 5 mg/ml sonneratia alba methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 5 mg/ml avicennia marina methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 5 mg/ml positive controls ampicillin 10 μg 19.20 ± 0.98 streptomycin 10 μg 15.54 ± 0.22 negative controls methanol chloroform n-hexane 3 mg/ml 3 mg/ml 3 mg/ml 1 mg/ml 1 mg/ml 5 mg/ml 1 mg/ml 5 mg/ml 5 mg/ml 120 biology, medicine, & natural product chemistry 10 (2), 2021: 117-121 bacterial strains samples solvents concentration zoi (mm) streptococcus mutans rhizopora apiculata methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 13.98 ± 1.49 3 mg/ml 19.83 ± 0.63 5 mg/ml 15.92 ± 0.68 n-hexane 1 mg/ml 3 mg/ml 5 mg/ml rhizopora mucronata methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 5 mg/ml 7.32 ± 0.43 sonneratia alba methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 7.39 ± 0.21 5 mg/ml 7.96 ± 0.25 avicennia marina methanol 1 mg/ml 3 mg/ml 5 mg/ml chloroform 1 mg/ml 3 mg/ml 5 mg/ml n-hexane 1 mg/ml 3 mg/ml 5 mg/ml positive controls ampicillin 10 μg 31.72 ± 3.03 streptomycin 10 μg 20.76 ± 1.49 negative controls methanol chloroform n-hexane in addition, other rhizopora species namely r. mucronata also displayed antibacterial activity in which 5 mg/ml n-hexane extract showed zoi of 7.32 ± 0.43 mm against s. mutans. rhizopora species including r. apiculata and r. mucronata contain many medically important secondary metabolites such as essential oils, glycosides, alkaloids, phenolic compounds, and tannins (habib et al., 2018). tannins is known to have antimicrobial properties against both gram-positive and gram-negative bacteria. this compound passes the bacterial cell wall and intrudes the cell metabolism, resulted in bacterial death (kaczmarek, 2020). furthermore, n-hexane extract of s. alba root showed activity against s. mutans in which the concentration of 3 mg/ml and 5 mg/ml yielded zoi of 7.39 ± 0.21 and 7.96 ± 0.25 mm respectively. although study on s. alba root extract is still limited, there are a number of studies focusing on leaves and bark of this plant. a study by saad and co-workers (2012) showed that methanolic extract of s. alba leaves antibacterial activities were observed against gram-negative bacteria e. coli (17,5 mm), as well as gram-positive bacteria namely s. aureus (12.5 mm) and bacillus cereus (12.5 mm) (saad et al., 2012). our result indicate that overall, crude extract of mangrove root displayed much lower antibacterial activities compared to positive control ampicillin (10 μg) dan streptomycin (10 μg). however, zone of inhibition r. apiculata crude extracts using n-hexane (3 mg/l) were comparable to that of streptomycin. therefore, r. apiculata could potentially contain novel antibacterial compounds. however, composition of active compounds in the crude extracts need to be confirmed. it is expected that once this active compound wijaya & indraningrat – antibacterial activity of mangrove root 121 has been identified, it can be isolated and further developed as new antibiotics drugs. conclusion in conclusion, crude extracts of root r. apiculata displayed the highest antibacterial activities compared to other crude extracts of different mangrove plants. we found that extraction using n-hexane yielded the highest inhibition zones compared to methanol and chloroform. further research is required to elucidate the active compounds in n-hexane. acknowledgements: we would like to acknowledged financial support from up2m of faculty of medicines and health sciences under grant no. 417/unwar/fkik/up2m/kp-02/vii/2019. conflicts of interest: the authors declare that there are no conflicts of interest. references dahibhate, n., saddhe, a., & kumar, k. (2018). mangrove plants as a source of bioactive compounds: a review. 08. doi:10.2174/2210315508666180910125328 departemen kesehatan republik indonesia. (2016). mari bersama atasi resistensi antimikroba (amr). friess, d. a. (2016). mangrove forests. current biology magazine, 26, r746-r748. gouda, s., das, g., & patra, j. k. (2015). mangroves: a rich source of natural bioactive compounds. in s. k. panda (ed.), recent advances in natural products. usa: studium press llc. habib, m. a., khatun, f., ruma, m. k., chowdhury, a., silve, a. r., rahman, a., & hossain, m. i. (2018). a review on phytochemical constituents of pharmaceutically important mangrove plants, their medicinal uses and pharmacological activities. kaczmarek, b. (2020). tannic acid with antiviral and antibacterial activity as a promising component of biomaterials—a minireview. materials, 13(14), 3224. retrieved from https://www.mdpi.com/1996-1944/13/14/3224 lugina, m., alviya, i., indartik, & pribadi, m. a. (2017). strategi keberlanjutan pengelolaan hutan mangrove di tahura ngurah rai bali (strategy of mangrove management in ngurah rai grand forest park). jurnal analisis kebijakan kehutanan, 14(1), 61-77. patra, j. k., & mohanta, y. k. (2014). antimicrobial compounds from mangrove plants: a pharmaceutical prospective. the chinese journal of integrated traditional and western medicine, 1-10. doi:10.1007/s11655-014-1747-0 patra, j. k., & thatoi, h. n. (2011). metabolic diversity and bioactivity screening of mangrove plants: a review. acta physiologiae plantarum, 33(4), 1051-1061. doi:10.1007/s11738-010-0667-7 saad, s., taher, m., susanti, d., qaralleh, h., & awang, a. f. i. b. (2012). in vitro antimicrobial activity of mangrove plant sonneratia alba. asian pacific journal of tropical biomedicine, 2(6), 427-429. doi:https://doi.org/10.1016/s2221-1691(12)60069-0 saranraj, p. (2015). mangrove medicinal plants: a review (vol. 7). seepana, r., karthick, p., narayana murthy, k., ramesh, c., mohanraju, r., & vijayakumar, a. (2016). evaluation of antimicrobial properties from the mangrove rhizophora apiculata and bruguiera gymnorrhiza of burmanallah coast, south andaman, india. journal of coastal life medicine, 4(6), 475-478. doi:10.12980/jclm.4.2016j6-52 usman. (2018). uji fitokimia dan uji antibakteri dari akar mangrove rhizopora apiculata terhadap bakteri escherichia coli dan staphylococcus aureus. jurnal kimia dan pendidikan kimia, 2(3), 169-177. doi:10.20961/jkpk.v2i3.11850 ventola, c. l. (2015). the antibiotic resistance crisis: part 1: causes and threats. p & t: a peer-reviewed journal for formulary management, 40(4), 277-283. retrieved from https://www.ncbi.nlm.nih.gov/pubmed/25859123 https://www.ncbi.nlm.nih.gov/pmc/pmc4378521/ ventola, c. l. (2015). the antibiotic resistance crisis: part 2: management strategies and new agents. p t, 40(5), 344-352. world health organization. (2020). global antimicrobial resistance surveillance system (glass) report: early implementation 2020. retrieved from geneva: https://apps.who.int/iris/bitstream/handle/10665/332081/9789 240005587-eng.pdf?ua=1 this page intentionally left blank biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 1, april 2025 | pages: 167-176 | doi: 10.14421/biomedich.2025.141.167-176 issn 2540-9328 (online) identification and screening of enzymatic activity of degrading fungi microplastics in three final processing sites (tpa) in the province lampung sari oktaviyani*, marlina kamelia, aulia ulmillah biology department, faculty of science and technology, uin raden intan lampung, jl. endro suratmin sukarame – bandar lampung 35131, tel. 0721-780887, indonesia. corresponding author* sarioktaviyani50@gmail.com abstract one of the main causes of problems in the final processing site (tpa) of lampung province is the increasing pile of plastic waste produced by human activities. this can be a source of microplastic pollution and is dangerous if it settles in the body of organisms. fungi can be an alternative to help the natural biodegradation process reduce microplastic pollution in the soil ecosystem. this study aims to identify and characterize fungal isolates that can degrade microplastics in the final processing site (tpa) of lampung province. the sampling method was purposive sampling. the growing colonies were identified by observing the clear zone, then characterized morphologically. data analysis using qualitative descriptive methods and measuring the clear zone index. this study successfully identified 13 fungal isolates that showed clear zone areas. four isolates (ba1.2, ba2.5, bu3.4, ka2.3) could degrade three types of microplastics. the highest clear zone: bu3.4 for pet (2,00 mm), ka2.4 for pe (1,50 mm), and ba2.3 and ba2.5 for pp (1,25 mm). identification and morphological characterization showed 8 isolates belonging to the genus aspergillus, 3 isolates of penicillium, 1 isolate of fusarium, and 1 isolate of sclerotium. degradation occurs through hyphal adhesion and secretion of polymer-degrading enzymes. keywords: biodegradation; clear zone index; fungi; landfill; microplastics. introduction lampung province faces significant problems in waste management in several landfills, such as bakung landfill (bandar lampung city), bumiayu landfill (pringsewu regency), and karangrejo landfill (metro city) (umayasari & sandy, 2024). operational facilities are old and unable to accommodate the increasing volume of waste (supratikno et al., 2023). although designed with a sanitary landfill system, the current implementation of the open dumping system hurts the environment and surrounding communities (malihah et al., 2023). plastic is the main contributor to the problem in the lampung province tpa (nisa et al., 2023). plastic waste comes from various human activities, such as packaging, household appliances, and disposable products. types of plastic such as polyethylene terephthalate, polyethylene, polypropylene, polyvinyl chloride, polystyrene, and nylon (shadrina, 2024). this type of plastic is carcinogenic and contains hazardous chemicals, such as dibenzodioxins and polychlorinated dibenzofurans which can cause cancer (fathulloh et al., 2021). the accumulation of potential plastic that exceeds capacity not only hinders management efficiency, but also has the potential to create microplastics that endanger the ecosystem and the health of organisms (shafira et al., 2022). microplastics are plastic fragments with a diameter of <5mm which are divided into two, namely primary microplastics and secondary microplastics (jamika, 2023). primary microplastics are plastic products intentionally made microscopic, such as microbeads found in skin care products (trivantira, 2022). secondary microplastics result from the fragmentation of larger plastics in the form of fibers, fragments, granules, and films (syahadatina et al., 2024). febriyanti et al., (2024), stated that plastic contributes to microplastic pollution in various ecosystems, including soil and water. plastic waste not only remains on the surface of the soil, but is also degraded into microplastics by wind and the activity of living organisms below the surface (suharsono et al., 2021). microplastics that accumulate in landfill soil can migrate through air, water, wind, dust, erosion, and surface water runoff creating a chain impact in the food chain (rezaei et al., 2019). microplastics take hundreds of years to decompose through various physical, chemical and biological processes. manuscript received: 14 may, 2025. revision accepted: 26 may, 2025. published: 27 may, 2025. https://doi.org/10.14421/biomedich.2025.141.167-176 168 biology, medicine, & natural product chemistry 14 (1), 2025: 167-176 based on the results of the vosviewer analysis, it is known that the problem of microplastics is still largely unresolved. one potential solution is to accelerate the degradation of microplastics through biological agents, such as microorganisms. fungi are known to break down microplastics into simple compounds with the help of specific enzymes, thus offering a more sustainable approach to reducing the impact of microplastic pollution. the role of soil microorganisms, especially fungi as agents of microplastic biodegradation still requires further development (arista, 2023). previous studies have shown the potential of indigenous fungal isolates, such as aspergillus sp., penicillium sp., and fusarium sp. in degrading microplastics (karimah, 2023; magnin et al., 2020; najah, 2022; rohmah et al., 2019). therefore, this study aims to identify and characterize fungal isolates that can degrade microplastics at the final processing site (tpa) of lampung province. materials and methods materials the equipment used is spades, clip bags, lion star brand cooler boxes, hirayama brand autoclaves, pyrex brand petri dishes, pyrex brand beakers, pyrex brand measuring cups, onelab brand erlenmeyer flasks, pyrex brand test tubes, test tube racks, ose needles, onelab brand object glass, onelab brand cover glass, biosan brand hot plates, polygon brand magnetic stirrer bars, tweezers, bunsen lamps, pyrex brand glass droppers, eppendorf brand micropipettes, eppendorf brand blue tips, kern brand analytical scales, thermo scientific brand vortex ovens, thermo scientific brand incubators, esco brand laminar air flow and optika italia brand binocular microscopes. the materials used include soil samples from three different landfills, sterile distilled water, alcohol, paseo brand tissue, joyko brand label paper, wrapping paper, cotton pads, whatman brand filter paper, aluminum foil, plastic wrap, gauze, 70% alcohol onemed brand, himedia brand instant potato dextrose agar (pda) media, minimal salt medium (msm) media, hufa brand chloramphenicol, polyethylene terephthalate (pet) plastics, polyethylene (pe), polypropylene (pp) and lactophenol cotton blue (lpcb). methods soil sample preparation sampling used the purposive sampling method (roflin & liberty, 2021). soil samples were taken as much as 1 kg in an area surrounded by decomposed plastic waste with a depth of 11-15 cm (karimah, 2023). samples were stored in a cooler box using labeled plastic (kahula et al., 2024). figure 1 the sampling location is at bakung landfill, bandar lampung city. figure 2. the sampling location is at bumiayu tpa, pringsewu regency. figure 3. the sampling location is at karangrejo tpa, metro city. making pda media and modifying msm the cultivation media used in the study consisted of potato dextrose agar (pda) and mineral salt medium (msm). pda media was made with 39 grams of instant pda in 1000 ml of aquades, added with chloramphenicol as an antibiotic (adiningrum, 2024). msm media is made with a composition of ingredients, oktaviyani et al. – identification and screening of enzymatic activity … 169 such as k2hpo4 (1 g), kh2po4 (0.2 g), nacl (1 g), (nh4)2so4 (1 g), mgso4.7h2o (0.5 g), cacl2·2h2o (0.002 g), feso4.7h2o (0.01 g), mnso4.h2o (0.001 g), cuso4.5h2o (0.001 g), znso4.7h2o (0.001 g), chloramphenicol, agar (15 g) in 1000 ml of distilled water (wardani, 2021). the media is homogenized on a magnetic hot plate stirrer and autoclaved at 121°c and 12 atm for 20 minutes (basarang et al., 2018). msm media was modified by adding microplastic powder, namely polyethylene terephthalate (pet), polyethylene (pe), and polypropylene (pp) which had been sterilized by soaking in alcohol for 30 minutes, rinsing with distilled water for 20 minutes, and airing in laminar air flow for 15 minutes (al ikhsani, 2021). isolation of microplastic degrading fungi a soil sample weighing 1 gram was prepared in a test tube and diluted to 10 ml with sterile distilled water. the suspension was homogenized and serially diluted up to eight (10-8) for dry, non-agglomerated soil samples (karimah, 2023). samples from dilutions 10-5, 10-6, 107, and 10-8 were inoculated on pda media with a volume of 1 ml using the pour plate method. incubate for 3–7 days at room temperature (abna et al., 2024). selected fungal colonies were purified on new pda media. screening of enzymatic activity of microplastic degrading fungi the degradation potential was tested using msm agar media with a microplastic powder content of 1% (w/v). fungal isolates were inoculated at 30-35°c for 8-10 days. the clear area around the colony is characterized by rapid growth and spread over the surface of the medium. this shows the potential of this fungus as a decomposer (wardani, 2021). identification of fungal morphology fungal identification is carried out through macroscopic and microscopic observations. macroscopic observations were carried out directly on fungal colonies grown for 7 days, including the characteristics of the shape, color and texture of the colony (novitasari et al., 2021). the slide culture method with lactophenol cotton blue (lpcb) staining was used for microscopic observations (fitria et al., 2023). pieces of media are placed on microscope slides. the fungal isolate was streaked around the corner of the media with a loop needle, and covered with a sterile cover glass. the slide is placed in a petri dish with filter paper and a slide holder. an incubation period of 35 days will see the growth of fungus around the media. the cover glass of the culture slide was stained with lactophenol cotton blue (lpcb) dye and placed on an object glass with 100x magnification for genus identification (fahmi et al., 2021). characters that can be observed include the shape of the hyphae, branching patterns, spore structure, the presence of septa on the hyphae, exudates and water droplets. data analysis data analysis includes data obtained from field observations and laboratory results. the data was analyzed descriptively qualitatively, then presented as tables or pictures of the results of macroscopic and microscopic morphological characterization, as well as the formation of clear zones for each fungal isolate (rohmah et al., 2019). clear zone index measurements were carried out by measuring the enzymatic index value on the diameter of the clear zone formed around fungal colonies on msm media containing microplastic powder (najah, 2022). ei = diameter koloni + zona bening diameter koloni results and discussion results isolation of microplastic degrading fungi isolation of fungi from three landfills produced 15 isolates with different morphologies, of which 13 isolates showed the potential for microplastic degradation through the formation of clear zones, with the degradation process involving extracellular enzymes such as laccase, peroxidase, and lipase whose effectiveness is influenced by environmental factors and the physicochemical characteristics of microplastics. screening of enzymatic activity of microplastic degrading fungi the results of enzymatic activity screening of 13 fungal isolates from bakung tpa (ba), bumiayu tpa (bu), and karangrejo tpa (ka) showed a high degradation ability. varies against polyethylene terephthalate (pet), polyethylene (pe), and polypropylene (pp) microplastics. this variation in ability is caused by differences in the enzymatic mechanisms possessed by each fungal isolate. the degradation ability is identified by forming a clear zone on msm agar media, indicating the activity of extracellular hydrolytic enzymes (istiqomah, 2020). table 1. clear zone diameter by fungal isolates on msm media. isolate code diameter (mm) clear zone pet pe pp ba1.2 1,33 1,05 1,19 ba2.3 1,27 ba2.4 1,03 ba2.5 1,04 1,40 1,25 ba3.6 1,25 1,17 bu1.1 1,06 bu2.2 1,21 1,17 bu2.3 1,22 bu3.4 2,00 1,21 1,00 ka1.1 1,05 ka2.3 1,25 1,05 1,08 ka2,4 1,50 1,07 ka3.5 1,10 note: pet: polyethylene terephthalate; (pe) polyethylene; (pp) polypropylene. 170 biology, medicine, & natural product chemistry 14 (1), 2025: 167-176 figure 4. fungal isolates showing the highest clear zone according to the content of microplastic types: (a) isolate bu3.4 the highest clear zone for pet reached 2,00 mm (b) isolate ka2.4 the highest clear zone for pe reached 1,50 mm (c) isolate ba2.3 the clear zone for pp reached 1,25 mm (d) isolate ba2.5 the clear zone for pp reached 1,25 mm. discussion isolation of microplastic degrading fungi the morphological characteristics observed in 13 isolates of microplastic-degrading fungi on pda media showed colony diversity and could be grouped into four main suspected genera based on typical macroscopic and microscopic characteristics. morphological identification is an important step in taxonomic characterization and understanding the biodegradation potential of fungal isolates (haya, 2023). based on morphological observations of microplastic degradation mushroom isolates, the result of characterization is obtained as follows: table 2. characterization of microplastic-degrading fungi codes ba1.2 – ba3.6. character morphology isolate code ba1.2 ba2.3 ba2.4 ba2.5 ba3.6 top view bottom view form circular circular circular circular circular margin entire entire entire undulate filaform texture cottony cottony velvety powdery cottony elevation raised raised raised umbonate flat exodus aerial aerial aerial aerial aerial character microscopic suspected genus fusarium aspergillus penicillium penicillium sclerotium the morphological characterization observations in table 2 show that isolate ba1.2 has a purplish pink color on the upper surface and reddish brown on the lower surface. isolate ba2.3 is white on the upper surface and yellowish white on the lower surface. isolate ba2.4 appears transparent with some structure in the center on the upper surface and has a brown color in the center on the lower surface. all three isolates have circular colony a b c d oktaviyani et al. – identification and screening of enzymatic activity … 171 shapes with entire (intact) edges. isolates ba1.2 and ba2.3 have a cottony texture, while ba2.4 has a velvet texture. all isolates show raised elevation with aerial hyphae growth. microscopic observation identified ba1.2 as a suspected genus fusarium with visible septate hyphae and characteristic crescent-shaped (fusiform) macroconidia, ba2.3 as suspected aspergillus with round conidial heads, and ba2.4 as suspected penicillium with brush-like branched conidiophores. isolate ba2.5 has a green colony with yellow edges (top view) and reddish orange (bottom view), a circular shape with undulate (wavy) edges, powdery texture, and umbonate elevation. microscopic observation identified it as the genus penicillium. isolate ba3.6 has a whitish gray color, circular shape with filiform edges, cottony texture, and flat elevation, and is suspected as sclerotium based on its characteristic sclerotia structure and the presence of mycelium with septate hyphae. table 3. characterization of microplastic-degrading fungi codes bu1.1 – ka1.1 . character morphology isolate code bu1.1 bu2.2 bu2.3 bu3.4 ka1.1 top view bottom view form filamentous circular circular circular circular margin filamentous entire entire entire filamentous texture velvety cottony powdery powdery powdery elevation raised raised convex convex raised exodus aerial submerged submerged submerged aerial character microscopic suspected genus aspergillus penicillium aspergillus aspergillus aspergillus table 3 displays the morphological characterization observations of isolates bu1.1, bu2.2, bu2.3, bu3.4, and ka1.1. isolate bu1.1 is blackish white with a spreading pattern, filamentous shape and edge, velvety texture, raised elevation, and suspected as aspergillus with septate hyphae with upright conidiophores bearing conidial heads. isolate bu2.2 has a grayish white colony, circular shape with entire edge, cottony texture, raised elevation, and submerged hyphae growth. microscopic observation identified it as penicillium. isolate bu2.3 is reddish in the center and bu3.4 shows a concentric circle pattern in orange. both are circular with entire edges, powdery texture, convex elevation, submerged hyphae growth, and suspected as aspergillus. isolate ka1.1 is black (top view) and dark gray with a circular pattern (bottom view), circular shape with filamentous edges, powdery texture, raised elevation, aerial hyphae growth, and suspected as aspergillus. 172 biology, medicine, & natural product chemistry 14 (1), 2025: 167-176 table 4. characterization of microplastic-degrading fungi codes ka2.3 – ka3.5. character morphology isolate code ka2.3 ka2.4 ka3.5 top view bottom view form filamentous circular filamentous margin filamenous entire filamentous texture powdery cottony powdery elevation flat raised flat exodus aerial submerged aerial character microscopic suspected genus aspergillus aspergillus aspergillus table 4 shows the morphological characterization observations of isolates ka2.3, ka2.4, and ka3.5. isolate ka1.1 is black (top view) and dark gray with a circular pattern (bottom view), circular shape with filamentous edges, powdery texture, raised elevation, and aerial hyphae growth. isolate ka2.3 is whitish brown to whitish gray, filamentous in shape and edge, powdery texture, flat elevation, and aerial hyphae growth. isolate ka2.4 is brownish green-gray with a dark center and cream brown on the bottom, circular shape with entire edge, cottony texture, raised elevation, and submerged hyphae growth. isolate ka3.5 has a brownish green colony with irregular pattern (top view) and whitish to grayish (bottom view). the colony shape and edge are filamentous, powdery texture, flat elevation, and aerial hyphae growth. this isolate is identified as genus aspergillus on microscopic observation showing unbranched upright conidiophores with round conidial heads. all four isolates are suspected to be genus aspergillus. discussion isolation of microplastic degrading fungi isolation of fungi from soil samples of bakung landfill, bumiayu landfill, and karangrejo landfill was conducted at depths of 11-15 cm, using serial dilution methods (10⁻⁵ to 10⁻⁸) and pour plate technique. the selection of 11-15 cm depth in soil microbiology research is based on previous studies showing that the composition and function of microbial communities are significantly influenced by soil depth, with significant changes in community structure and microbial activity as depth increases (naylor et al., 2022). the isolation process yielded 15 fungal isolates with different morphologies, with 13 isolate codes showing microplastic degradation potential by forming clear zones. indigenous fungi from landfills have resistance to extreme conditions such as temperature fluctuations, high humidity, and the presence of diverse waste. fungal growth is influenced by organic matter content, temperature, humidity, and soil ph (nafiah, 2024). the mechanism of microplastic degradation by fungi involves a gradual process that begins with colonization of the polymer surface, followed by secretion of extracellular enzymes that break down complex chemical bonds. the main enzymes involved in the degradation process include laccase, manganese peroxidase, alkane hydroxylase, lignin peroxidase, versatile peroxidase, and lipase which can break the chemical bonds of plastic oktaviyani et al. – identification and screening of enzymatic activity … 173 polymers (azzahra, 2024). according to rohmah et al., (2019) the isolate aspergillus terreus (lm 1021) showed optimal degradation activity at ph 5 and a temperature of 25°c, capable of degrading phb (44.96%), plastic bags (4.28%), and ldpe (4.9%). another study stated that aspergillus niger can degrade polyethylene up to 16% in 30 days (safdar et al., 2024). studies show that fusarium solani can degrade pet by producing hydrolase enzymes (erlambang et al., 2019). the efficiency of microplastic biodegradation is influenced by the physicochemical characteristics of the polymer, including crystallinity, molecular weight, and functional groups. polymers with high crystalline structures such as pet and hdpe tend to be more resistant to biological degradation compared to amorphous polymers such as phb and pcl. modification of microplastic surfaces through physical or chemical pretreatment can increase the rate of degradation by creating nucleation points for enzyme attachment. optimal conditions for fungal growth are achieved on potato dextrose agar (pda) media with ph 4.5-5.6, temperature 24-29°c, and relative humidity 90100% (basarang et al., 2018). these parameters are important to optimize the cultivation of microplasticdegrading fungi in environmental biotechnology applications. enzymatic activity screening of microplastic degrading fungi table 1 shows that the potential for degradation of polyethylene terephthalate (pet) is the type of microplastic that is most degraded with 11 isolates, followed by polypropylene (pp) with 8 isolates, and polyethylene (pe) with 6 isolates. this degradation pattern is consistent with the molecular characteristics of each related polymer. polyethylene terephthalate (pet) has an ester bond that is relatively easier to hydrolyze by cutinase and enzymes generally produced by soil fungi. polyethylene (pe) and polypropylene (pp) have a more stable and hydrophobic hydrocarbon structure, making them more difficult to degrade (ningrum et al., 2023). isolate bu3.4 was able to degrade polyethylene terephthalate (pet) with the highest clear zone reaching 2,00 mm after 10 days of incubation. this ability can be attributed to the secretion of specific hydrolytic enzymes such as petase and mhetase work synergistically to break the ester bond in the pet structure. the petase enzyme catalyzes the hydrolysis of pet into mhet (mono-2-hydroxyethyl terephthalate), which is then converted into terephthalic acid and ethylene glycol by mhetase. the high degradation ability of the bu3.4 isolate is in line with the research of danso et al. (2019), which reported that several species of fungi from the genus fusarium and humicola can produce the enzyme a potent petase. isolate ka2.4 showed superiority in degrading polyethylene (pe) with a clear zone of 1,50 mm. polyethylene (pe) is known as a polymer that is very resistant to biological degradation because its saturated hydrocarbon chain structure does not have functional groups that are easily attacked by enzymes. the ability of ka2.4 isolates to indicate the production of oxidative enzymes such as laccase or peroxidase that can oxidize the pe surface through the formation of carbonyl groups (asmi, 2020). lysinibacillus xylanilyticus and aspergillus niger can degrade polyethylene plastic (arista, 2023). isolates ba2.3 and ba2.5 showed the ability to degrade polypropylene with a clear zone reaching a size of 1,25 mm. the mechanism of pp degradation by fungi begins with oxidoreductase enzymes such as laccase and peroxidase catalyzing the oxidation of the polymer surface, producing carbonyl and carboxyl groups on the pp chain. monooxygenase and alkane hydroxylase enzymes insert oxygen atoms into the ch bond, forming primary alcohols which are then oxidized to aldehydes by alcohol dehydrogenase. the aldehyde dehydrogenase enzyme converts aldehydes to carboxylic acids, while esterases and lipases facilitate the cleavage of the polymer chain at the ester group formed (nurmalasari & kebumian, 2018). figure 4 shows that there are four isolate codes (ba2.3, ba2.5, bu3.4, and ka2.4) that can potentially degrade the three types of samples tested. this multi-degradation ability is very valuable in biodegradation applications in overcoming microplastic pollution in the environment (fachrul et al., 2021). the fungal isolates that have the highest clear zone according to the content of microplastic types are as follows: isolate code bu3.4 with the highest clear zone for polyethylene terephthalate (pet) reaching 2,00 m, isolate code ka2.4 with the highest clear zone for polypropylene (pe) reaching 1,50 mm and isolate codes ba2.3 and ba2.5 with the highest clear zone for polypropylene (pp) reaching 1,25 mm. msm agar media with 1% microplastic powder content with essential inorganic components such as mineral salts k₂hpo₄, kh₂po₄, (nh₄)₂so₄, cacl₂), dan trace elements (feso₄, znso₄, mnso₄), but deliberately made deficient in organic carbon sources. the composition of this media forces fungi to develop an enzyme system that can degrade microplastics into monomer units that can be used as a source of carbon and energy (wei & zimmermann, 2017). the ability of microplastic biodegradation by fungi is identified by forming a clear zone around the colony on msm media. this clear zone is seen as a transparent area that contrasts with the opaque area around it. msm media adjusted at a ph range of 6.5-7.0 supports optimal activity of degradative enzymes such as laccase, peroxidase, and hydrolase produced by microplastic degrading fungi. the relatively small size of the clear zone (1,00 mm to 2,00 mm) reflects the slow degradation process of microplastics in accordance with the recalcitrant nature of plastic polymers (asmi et al., 2022). the rate of degradation is influenced by various factors including polymer crystallinity, molecular weight, particle size distribution, and the presence of additives such as plasticizers and uv stabilizers that can change the 174 biology, medicine, & natural product chemistry 14 (1), 2025: 167-176 accessibility of the substrate to enzymes. the time and size of the clear zone varied depending on the type of fungal isolate tested, with some isolates starting to form a clear zone from day 6, and the maximum size was reached after 14-21 days of incubation at 30°c (wardani, 2021). the formation of clear zones on nutrient-poor msm agar media indicates that indigenous fungi from the landfill have adapted to use microplastics as an alternative carbon source. this adaptation involves developing an enzyme system that can degrade synthetic polymers into monomer units that can be utilized for mushroom growth. this adaptive ability makes indigenous fungi from landfills in lampung province potentially effective and sustainable biodegradation agents in overcoming microplastic pollution. morphological identification of microplastic degrading fungi this research identified four suspected genera, namely aspergillus, penicillium, fusarium, and sclerotium. the aspergillus genus dominates (isolates ba2.3, bu2.3, bu3.4, ka1.1, ka2.3, ka2.4, and ka3.5) with the advantages of fast mycelial growth and production of antifungal compounds (magnin et al. 2020; wardani, 2021). aspergillus produces laccase, esterase, peroxidase, lipase and urease enzymes which play a role in the degradation of complex polymers (oliveira et al. 2020). penicillium (isolates ba2.4, ba2.5, and bu2.2) is the second most abundant genus, known to have significant synthetic polymer degradation capabilities through the production of enzymes such as cutinase, esterase, and lipase (suresh et al., 2025). the typical morphological structure of penicillium in the form of brush-like branched conidiophores makes it easier to identify microscopically. fusarium (isolate ba1.2) with characteristic crescentshaped (fusiform) macroconidia can degrade various polymers by producing laccase, peroxidase and lipase enzymes. sclerotium (isolate ba3.6) has the potential to degrade complex organic polymers via ligninolytic enzymes. according to sakti et al. (2024), sclerotium rolfsii has the potential to degrade complex organic polymers through the production of ligninolytic enzymes. the distinctive sclerotia structure is the main marker in identifying this genus. although research on sclerotium's ability to degrade microplastics is still limited, the potential of its ligninolytic enzymes opens up new opportunities in the development of plastic biodegradation technology. the dominant powdery texture in many isolates (ba2.5, bu2.3, bu3.4, ka1.1, ka2.3, ka3.5) is related to the production of large amounts of conidia which supports the spread and adaptation of the fungus to various environmental conditions. aerial hyphal growth allows optimal oxygen access for oxidative reactions in polymer degradation, while submerged hyphal growth allows direct contact with the substrate for enzymatic degradation processes. the morphological diversity of microplasticdegrading fungi found in this study provides insight into the different physiological adaptations to microplastic substrates. these isolates have the potential to have different degradation mechanisms and efficiencies. these four indigenous fungal genera have high adaptability and complex enzymatic systems, so they have the potential to be effective microplastic biodegradation agents for applications to restore plastic-polluted environments. conclusions this study successfully isolated 15 fungal isolates with diverse morphologies from three landfills in lampung province, with 13 isolates showing microplastic degradation activity as indicated by the clear zone index value. pet type microplastics were degraded the most (11 isolates), followed by pp (8 isolates) and pe (6 isolates). four isolates (ba2.3, ba2.5, bu3.4, and ka2.4) had the potential to degrade the three types of samples tested. the isolates with the highest clear zones were bu3.4 for pet (2,00 mm), ka2.4 for pe (1,50 mm), and ba2.3 and ba2.5 for pp (1,25 mm). morphological characterization showed 8 isolates suspected of the aspergillus genus, 3 isolates of penicillium, 1 isolate of fusarium, and 1 isolate of sclerotium. microplastic degradation involves hyphal adhesion to the plastic surface and the secretion of extracellular enzymes that break down the chemical bonds of the plastic polymer. indigenous fungi from landfills in lampung province have the potential to be effective and sustainable biodegradation agents in overcoming microplastic pollution in the environment. authors’ contributions: sari oktaviyani & marlina kamelia designed the study. sari oktaviyani performed the laboratory work. sari oktaviyani and marlina kamelia analyzed the data. sari oktaviyani and aulia ulmillah wrote the manuscript. all authors read and approved the final version of the manuscript. competing interests: the authors declare that there are no competing interests. references abna, i. m., nurfitria, s., & mahayasih, p. g. m. w. 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(2017). biocatalysis as a green route for recycling the recalcitrant plastic polyethylene terephthalate. microbial biotechnology, 10(6), 1302–1307. https://doi.org/10.1111/1751-7915.12714. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 14, number 2, october 2025 | pages: 1315-1324 | doi: 10.14421/biomedich.2025.142.1315-1324 issn 2540-9328 (online) ethnobotanical study of plant utilization in the life cycle ceremonies of the kaili tado ethnic group in central sulawesi syech zainal*, supriyatman, aan febriawan, amalia buntu, muh. syarif abd syukur, pahriadi, raya agni department of natural science education, faculty of teacher training and education, tadulako university, jl. soekarno hatta no.km. 9, tondo, kec. mantikulore, kota palu, sulawesi tengah 94148, indonesia. corresponding author* syechzainal97mpd@gmail.com abstract the kaili tado ethnic group of central sulawesi maintains a rich ethnobotanical tradition in which plants play essential roles in life cycle ceremonies that mark the stages of human existence from birth and marriage to death. this study aimed to identify and analyze the species, uses, symbolic meanings, and conservation status of plants employed in the traditional rituals of the kaili tado community. the research was conducted in langko village, lindu district, sigi regency, from july to august 2025, using a qualitative descriptive ethnobotanical approach. data were collected through semi-structured interviews, participatory observation, and field documentation involving twelve key informants, including traditional leaders, ritual practitioners, and elder community members. a total of 11 plant species were recorded, representing diverse functions and cultural symbolism. plants such as oryza sativa (rice), cocos nucifera (coconut), and areca catechu (areca nut) symbolize prosperity, purity, and strength, respectively, while others like kalanchoe pinnata and jatropha curcas serve as protective and purifying agents. most species are easily available around home gardens, but a few such as piper betle and calamus sp. are becoming scarce. the study demonstrates that the kaili tado community’s ritual plant use reflects deep ecological awareness and moral philosophy, emphasizing balance between humans and nature. ethnobotanical documentation of these practices is essential not only for preserving cultural heritage but also for promoting biodiversity conservation and environmental education. keywords: ethnobotany; kaili tado; life cycle ceremonies; traditional knowledge; cultural symbolism; biodiversity conservation. introduction indonesia is known as one of the world’s centers of biodiversity, not only in terms of flora and fauna but also in the diversity of its ethnic groups, cultures, and local wisdom (arifin & nakagoshi 2011; prawiradilaga & soedjito 2013). this rich biodiversity has long been intertwined with cultural traditions and daily life, particularly in rural and indigenous communities that maintain close relationships with nature (sangha et al. 2018). among these, ethnobotany plays a vital role in understanding how local people utilize plants for various purposes, including medicine, food, rituals, and sociocultural practices. ethnobotanical studies provide insights into traditional knowledge systems that have been inherited through generations and reflect the intimate bond between humans and their natural environment (cariño & ferrari 2021; turner et al. 2022; levis et al. 2024). the kaili people are one of the major ethnic groups inhabiting central sulawesi, indonesia, and they are divided into several sub-ethnic groups, including the kaili tado community in lindu district, sigi regency (siradjuddin 2020; siradjuddin 2021). the kaili tado ethnic group maintains a rich cultural heritage characterized by diverse traditional ceremonies that represent different stages of human life, from pregnancy, birth, childhood, and adulthood to marriage and death (septiwiharti et al. 2020). these ceremonies known collectively as upacara siklus hidup or life cycle rituals serve not only as cultural expressions but also as manifestations of gratitude, protection, and continuity of life (wahyuningtyas et al. 2022; hariyanto et al. 2024; manan et al. 2024). each of these traditional ceremonies involves the use of various plant species that possess symbolic, spiritual, and practical meanings (sapkota 2013). for instance, oryza sativa (rice) symbolizes prosperity and sustenance, cocos nucifera (coconut) represents purity and completeness, while kalanchoe pinnata (cocor bebek) and bacopa sp. are used in infant protection rituals symbolizing growth and vitality (liu et al. 2022). the utilization of such plants in rituals reflects the community’s deep understanding of nature, its resources, manuscript received: 07 october, 2025. revision accepted: 29 november, 2025. published: 14 december, 2025. https://doi.org/10.14421/biomedich.2025.142.1315-1324 mailto:syechzainal97mpd@gmail.com 1316 biology, medicine, & natural product chemistry 14 (2), 2025: 1315-1324 and the meanings attached to them. however, these ethnobotanical practices are gradually diminishing due to modernization, acculturation, and the declining transmission of traditional knowledge among younger generations (hanazaki et al. 2013; dean 2024). in recent years, cultural transformation and environmental degradation have posed significant threats to the preservation of indigenous knowledge (nguyen et al. 2023), particularly in small ethnic groups like the kaili tado. the younger generation is increasingly detached from traditional customs, and the documentation of local ethnobotanical knowledge remains scarce (ramavhunga 2025). as a result, valuable information on the identity, symbolic function, and ecological importance of ritual plants risks being lost (geng et al. 2017; prasetyo 2023). such knowledge loss not only threatens cultural identity but also undermines the potential of traditional wisdom as a source for biodiversity conservation, sustainable resource management, and educational development (kakoty 2018; abas et al. 2022; sinthumule 2023). ethnobotanical research offers a scientific approach to preserving and revitalizing traditional ecological knowledge (kumar et al. 2021). by documenting plant species used in rituals and understanding their symbolic, functional, and ecological roles, ethnobotany bridges cultural heritage with scientific inquiry. studies in other regions, such as those conducted by baydoun et al. (2017) and uchôa (2024), have revealed that indigenous rituals often involve the use of medicinal and culturally important plants that play significant roles in maintaining both ecological balance and social harmony. in this context, the kaili tado community provides a valuable example of how traditional beliefs are deeply connected to natural resource use and conservation ethics (malone et al. 2014). furthermore, the ethnobotanical study of ritual plants contributes not only to cultural preservation but also to environmental education (abas et al. 2022). documenting and integrating this knowledge into educational materials can increase local awareness of biodiversity, promote cultural pride, and support character education rooted in local values. for this reason, exploring and analyzing the ethnobotanical aspects of the kaili tado life cycle ceremonies is essential for both scientific and educational purposes (baydoun et al. 2017; uchôa 2024). this study aims to document and analyze the types of plants used in the life cycle ceremonies of the kaili tado ethnic group, their functions, symbolic meanings, and current availability in the local environment. the results are expected to provide comprehensive ethnobotanical data that can serve as a reference for biodiversity conservation, cultural preservation, and future educational material development. in addition, the study contributes to the broader understanding of how traditional societies maintain ecological wisdom through cultural expressions, emphasizing that the relationship between humans and plants goes beyond mere utility it embodies respect, spirituality, and sustainability. materials and methods time, place, and research subjects this research was conducted from july to august 2025 in langko village, lindu district, sigi regency, central sulawesi, indonesia. langko village was chosen purposively because it is one of the main settlements of the kaili tado ethnic group, which still actively preserves traditional cultural practices, particularly the life cycle ceremonies that mark various stages of human life such as birth, marriage, and death. the village is located within the lindu highland ecosystem, an area surrounded by forest vegetation, agricultural land, and traditional settlements, providing rich biodiversity that supports the availability of plant species used in ritual activities. the geographical and cultural characteristics of this location make it an ideal site for studying the relationship between traditional ecological knowledge and plant utilization. the research subjects consisted of members of the kaili tado community who possess deep knowledge and direct experience in performing traditional ceremonies and using ritual plants. informants were selected using a purposive sampling technique, with specific criteria that included: (1) individuals recognized as traditional leaders (to tua adat), (2) elder community members who have participated in and inherited ritual practices, and (3) women practitioners who play key roles in preparing plant-based ritual materials. in total, twelve informants were involved in this study, comprising three traditional leaders, five elder community members, and four women practitioners. these informants were considered credible sources of ethnobotanical knowledge because they represent both the cultural authority and practical experience of the community. their involvement ensured that the collected data reflected the authenticity of local traditions and the continuity of indigenous knowledge transmission within the kaili tado ethnic group. additionally, younger villagers were occasionally included as supporting participants to provide insights into generational perspectives regarding the preservation and transformation of ethnobotanical practices. type of research this research employed a qualitative descriptive approach within the framework of ethnobotanical study. the qualitative method was selected to obtain in-depth understanding of the cultural meanings, symbolic values, and practical functions of plants used in the traditional life cycle ceremonies of the kaili tado ethnic group. the descriptive nature of this study aimed to present a zainal et al. – ethnobotanical study of the kaili tado 1317 comprehensive depiction of how the community identifies, selects, and utilizes various plant species within their ritual context. ethnobotany, as an interdisciplinary science that combines botany, anthropology, and ecology, was used as the main approach to explore the relationship between the kaili tado people and their surrounding plant resources. through this method, the research emphasized naturalistic observation, open-ended interviews, and cultural interpretation rather than experimental manipulation. the aim was to document traditional ecological knowledge that has been transmitted orally through generations and to describe how such knowledge reflects both cultural identity and environmental adaptation. the study also followed the principles of participatory observation, allowing researchers to be directly involved in community activities and ceremonies to gain a holistic understanding of plant usage patterns. furthermore, the research design was aligned with the objectives of ethnobotanical documentation and cultural preservation, ensuring that the findings not only identify the plant species used in rituals but also interpret their symbolic meanings within the socio-cultural system of the kaili tado community. this type of research provides qualitative evidence that connects biodiversity utilization with traditional wisdom, contributing to the broader fields of cultural anthropology, biodiversity conservation, and environmental education. research procedures the research procedures were conducted systematically through several stages to ensure that data collection and analysis were accurate, ethical, and representative of the kaili tado cultural context. preliminary preparation the initial stage involved conducting a comprehensive literature review on ethnobotany, traditional ceremonies, and previous studies related to the kaili ethnic group. this review provided theoretical and contextual foundations for the research. during this phase, the researchers also obtained necessary research permits from the local government and ethical clearance from the faculty of teacher training and education, tadulako university. in addition, coordination meetings were held with community leaders in langko village, lindu district, to introduce the research objectives and secure their consent and support. establishing early communication with the community ensured mutual understanding and ethical collaboration throughout the research process. field observation and informant identification the second stage focused on field reconnaissance and informant identification. researchers visited langko village to directly observe the local environment and social dynamics related to traditional ceremonies. informants were selected using a purposive sampling technique, emphasizing individuals recognized by the community as to tua adat (traditional leaders), ritual practitioners, and women responsible for preparing ritual materials. these individuals were considered the most knowledgeable about plant use in ceremonial practices. interviews during this phase aimed to determine the sequence of life cycle ceremonies, their symbolic components, and the key plant species associated with each ritual. data collection the third stage involved intensive data collection through semi-structured interviews, participant observation, and documentation. semi-structured interviews were used to gather detailed information on local plant names, uses, parts utilized, symbolic meanings, and specific ritual contexts. participant observation was conducted during several ceremonies, including mosaviraka ngana (baby swing ceremony), membua soibou (house-moving ceremony), adam pocembola (marriage ceremony), and motapusi posarara (severing kinship ceremony). researchers observed how plants were selected, prepared, and positioned within rituals, while also recording oral narratives and explanations from community elders. photographs and field notes documented each ritual step, and plant specimens were collected for scientific identification. plant identification and verification all plant specimens collected in the field were analyzed for morphological characteristics and taxonomic classification using references such as flora of sulawesi. to ensure scientific accuracy, the identified specimens were validated by comparison with preserved materials at the herbarium celebense (ceb), tadulako university. this verification ensured that each species listed in the ethnobotanical record corresponded accurately to its scientific identity, supporting the reliability of both cultural and biological data. data organization and interpretation in this stage, data from interviews, field observations, and documentation were compiled, coded, and organized into thematic categories: plant species, local names, plant parts used, symbolic meanings, and conservation status. data were analyzed using the qualitative interactive model proposed by huberman & miles (1994), consisting of data reduction, data display, and conclusion drawing. to enhance analytical depth, the use value (uv) index was calculated following phillips & gentry (1993) to quantify the relative cultural importance of each plant species based on its frequency and diversity of use. validation and reporting the final stage involved data triangulation to confirm validity and reliability. this was achieved by crosschecking information from different informants, 1318 biology, medicine, & natural product chemistry 14 (2), 2025: 1315-1324 comparing interview results with direct observations, and reviewing relevant literature. findings were then presented to community leaders in langko village for verification, ensuring that interpretations accurately reflected cultural realities. finally, the results were compiled into a comprehensive ethnobotanical documentation of plant use in the life cycle ceremonies of the kaili tado ethnic group. the final output contributes to biodiversity conservation, cultural preservation, and local education development providing a scientific foundation for integrating traditional ecological knowledge into modern educational and conservation frameworks. data analysis techniques the data obtained from interviews, observations, and documentation were analyzed using qualitative descriptive techniques following the interactive model of huberman & miles (1994), which consists of three main stages: data reduction, data display, and conclusion drawing or verification. in the data reduction stage, raw data collected from the field were carefully selected, summarized, and categorized according to the main research variables, such as local plant names, scientific names, parts used, ritual types, symbolic meanings, and availability status. this process was intended to simplify the data while retaining essential information relevant to the research objectives. in the data display stage, the organized information was presented in narrative descriptions and tabular formats to show the relationship between plant diversity, ritual function, and cultural significance. the use of tables and descriptive summaries allowed for a clearer interpretation of patterns and relationships between plant species and their ritual applications within the kaili tado community. the conclusion drawing and verification stage involved interpreting the meaning of the data and verifying its accuracy through cross-checking between different sources, including multiple informants, field observations, and supporting literature. triangulation was applied to ensure the validity and reliability of the findings by comparing the consistency of information obtained from various data collection methods. results and discussion types of plant species used in the life cycle ceremonies of the kaili tado ethnic group field observations and interviews with traditional leaders and elder informants revealed a total of 11 plant species commonly used in these rituals. these plants vary in morphology, function, and cultural significance, reflecting the community’s deep ecological knowledge and long-standing relationship with the surrounding natural environment (table 1). table 1. list of plant species used in the life cycle ceremonies of the kaili tado ethnic group. scientific name indonesian name local name oryza sativa l. rice pae cocos nucifera l. coconut kaluku musa paradisiaca l. banana loka dano kalanchae pinnata l. cocor bebek pomata areca catechu l. areca nut kalosu eleusine indica l. goosegrass kavoko hibiscus tiliaceus l. waru kalibau piper betle l. betel boulu sida rhombifolia l. sidaguri sidagori bacopa sp. creeping weed languntale calamus sp. rattan bolago the results identified 11 plant species utilized in various life cycle ceremonies performed by the kaili tado community in langko village, lindu district, central sulawesi. these species span multiple families, predominantly arecaceae, poaceae, malvaceae, and piperaceae. the diversity of plants used reflects the deep ethnobotanical knowledge embedded in kaili tado cultural traditions. each plant fulfills a symbolic and functional purpose within the rituals. for example, oryza sativa (rice) symbolizes sustenance, prosperity, and life continuity values closely tied to agrarian societies. similarly, cocos nucifera (coconut) represents purity, unity, and spiritual strength. the widespread availability of these plants around the community’s home gardens signifies an ecological adaptation and a sustainable relationship between people and their environment. cultural traditions often use plants as mediators between humans and the spiritual realm. in the kaili tado worldview, natural objects such as plants are imbued with spiritual power (pue), which connects the material and immaterial worlds (geng et al. 2017; prasetyo 2023). the repeated use of the same species in multiple rituals indicates their high symbolic rank and perceived potency (kakoty 2018). for instance, areca catechu and piper betle common ritual plants throughout indonesia are also prominent in kaili tado ceremonies, zainal et al. – ethnobotanical study of the kaili tado 1319 reinforcing their significance in fostering social and marital harmony. these findings align with similar ethnobotanical studies among other kaili sub-ethnic groups (hanazaki et al. 2013; nguyen et al. 2023), which documented comparable plant species being employed for healing, cleansing, and symbolic offerings. the consistency across regions underscores a shared cultural framework emphasizing balance between humans and nature (liu et al. 2022). moreover, the predominance of easily cultivated species indicates that the kaili tado community actively conserves culturally significant flora through domestic planting. this ensures long-term availability and supports cultural sustainability (turner et al. 2022; levis et al. 2024). the presence of calamus sp. (rattan), which is ecologically rarer, demonstrates that even forest-dependent species continue to hold strong cultural importance, reflecting traditional ecological ethics that discourage overexploitation (nguyen et al. 2023). status of plant availability in the kaili tado community the availability of ritual plants plays a vital role in determining the continuity of traditional ceremonies among the kaili tado community. plants that are easy to access are more likely to be preserved through ongoing use, while those that have become rare risk being excluded from ritual practices. to assess this aspect, researchers documented the presence and accessibility of each identified plant species within the local ecosystem whether they are cultivated, grow naturally, or require collection from forested areas (table 2). table 2. list of plant species used in the life cycle ceremonies of the kaili tado ethnic group. scientific name indonesian name local name availability status oryza sativa l. rice pae easy cocos nucifera l. coconut kaluku easy musa paradisiaca l. banana loka dano easy kalanchae pinnata l. cocor bebek pomata easy areca catechu l. areca nut kalosu easy eleusine indica l. goosegrass kavoko easy hibiscus tiliaceus l. waru kalibau easy piper betle l. betel boulu difficult sida rhombifolia l. sidaguri sidagori easy bacopa sp. creeping weed languntale rare calamus sp. rattan bolago difficult the majority of plant species used in the kaili tado life cycle rituals are easily accessible within the local environment, particularly in home gardens, village borders, and nearby forests. of the 11 species recorded, eight are categorized as “easy to find,” while three piper betle, bacopa sp., and calamus sp. are classified as rare or difficult to obtain. this availability pattern reflects both ecological and cultural dynamics. plants like rice, banana, coconut, and waru are commonly cultivated due to their daily economic and social importance (arifin & nakagoshi 2011). their abundance facilitates continuous ritual use, ensuring cultural continuity. on the other hand, the decreasing abundance of piper betle and calamus sp. is mainly attributed to habitat alteration, limited replanting, and reduced traditional cultivation practices. the rarity of these plants poses potential risks to the continuity of cultural practices, as certain rituals depend specifically on these species (sangha et al. 2018). the betel plant (piper betle), for instance, plays a crucial role in marriage ceremonies (adam pocembola), symbolizing unity and respect. its scarcity could, therefore, affect ritual performance and meaning transmission. ethnobotanical research in similar contexts (arizona, 2011; kasim et al., 2021) emphasizes that when traditional plants decline, communities often seek substitutes. however, substitution may dilute symbolic meaning, leading to cultural erosion (hanazaki et al. 2013; dean 2024). thus, local awareness of plant conservation is vital. the kaili tado community demonstrates proactive ecological ethics by maintaining small-scale cultivation of sacred plants, illustrating how traditional knowledge contributes to biodiversity conservation (ramavhunga 2025). from an ecological anthropology perspective, the preservation of ritual plants can also be interpreted as an adaptive strategy to environmental changes. the persistence of certain species, despite modernization, reveals a selective conservation pattern driven by cultural necessity rather than mere utility. these practices highlight the reciprocal relationship between cultural resilience and ecological sustainability (geng et al. 2017; abas et al. 2022). utilization of plant parts and functions in ceremonies the kaili tado people do not use entire plants indiscriminately in their rituals; instead, they selectively utilize specific plant parts that hold symbolic and practical significance. this selection is guided by 1320 biology, medicine, & natural product chemistry 14 (2), 2025: 1315-1324 traditional knowledge, passed down orally through generations, regarding the inherent properties and ritual suitability of each plant. the parts used such as leaves, fruits, stems, and seeds serve different roles, including purification, protection, fertility, and unity (table 3). table 3. plant parts utilized and their functions in the life cycle ceremonies of the kaili tado ethnic group. scientific name local name part used function in ceremony oryza sativa l. pae seeds symbol of prosperity and sustenance cocos nucifera l. kaluku fruit, leaves symbol of purity and unity musa paradisiaca l. loka dano fruit, stem symbol of fertility and harmony kalanchae pinnata l. pomata leaves protection against disease and evil spirits areca catechu l. kalosu fruit symbol of strength and endurance eleusine indica l. kavoko leaves coloring and purification hibiscus tiliaceus l. kalibau leaves protection and blessing piper betle l. boulu leaves symbol of unity and respect sida rhombifolia l. sidagori leaves symbol of healing and protection bacopa sp. languntale whole plant protection for infants calamus sp. bolago stem symbol of separation and boundary the use of specific plant parts in kaili tado ceremonies demonstrates a complex integration of symbolic, medicinal, and spiritual functions (sirajuddin 2021). among the 11 plant species identified, leaves are the most frequently utilized plant part, followed by fruits, stems, and seeds. the predominance of leaves highlights their accessibility, flexibility in ritual use, and association with life and renewal. leaves often represent growth and vitality, qualities that align closely with the symbolic essence of life cycle ceremonies. in kaili tado tradition, each plant part is carefully chosen to match the intended symbolic and ritual function. for example, rice (oryza sativa) seeds are used as offerings to symbolize prosperity and continuous sustenance, while coconut (cocos nucifera) fruit and leaves represent purity and wholeness key values in both cleansing and marriage rituals. coconut water is often sprinkled as a purifier, signifying the removal of misfortune. similar ritual use of cocos nucifera has been reported in various indonesian ethnobotanical contexts, including balinese and bugis traditions, where it symbolizes unity between human and divine (giambelli 2021). meanwhile, areca catechu and piper betle are used together in ceremonies involving social bonds such as marriage (adam pocembola). the act of sharing betel quid (a mixture of areca nut, betel leaf, and lime) represents respect, kinship, and alliance values deeply rooted in austronesian cultural symbolism (tusinski 2016). these plants are not chosen arbitrarily; they carry centuries of shared cultural meaning across indonesia and southeast asia. medicinally significant plants, such as kalanchoe pinnata, sida rhombifolia, and bacopa sp., are used to safeguard physical and spiritual well-being. their leaves and sap are believed to prevent disease or repel negative forces. scientific studies have confirmed their pharmacological properties: kalanchoe pinnata contains flavonoids and alkaloids with anti-inflammatory effects, sida rhombifolia possesses antimicrobial compounds, and bacopa sp. is known for its antioxidant and cognitive-enhancing properties. the convergence of symbolic and medicinal use highlights the empirical basis of traditional wisdom, showing how ritual practices also reflect indigenous pharmacology (elendu 2024). furthermore, calamus sp. (rattan) plays a distinctive role in the motapusi posarara ceremony, where it is physically cut to represent the severance of kinship. the rattan’s fibrous strength and flexibility symbolize resilience and the emotional difficulty of separation, linking natural form with cultural metaphor (mchattie & ting 2024). similarly, eleusine indica leaves are crushed to produce red pigment in the nokolontigi ceremony, symbolizing sincerity and the blood bond between individuals a direct reflection of how physical properties of plants inform their symbolic function. the multifunctional use of plants in kaili tado culture thus bridges spiritual belief, ecological awareness, and empirical observation. each plant part carries a layered meaning that embodies the community’s cosmological perspective: humans are part of nature’s cycle, not separate from it. the selection of plants for ritual use demonstrates both cultural continuity and biological adaptation, illustrating how ethnobotanical practices contribute to sustainable ecological relationships and cultural resilience (kumar et al. 2021). ethnobotanical meanings of plants in each life cycle ceremony each traditional ceremony performed by the kaili tado ethnic group involves a distinct combination of plant species, each carrying a unique symbolic meaning aligned with the spiritual and moral purpose of the ritual. these meanings embody the community’s worldview, in which plants are considered mediators between humans and nature. through ethnobotanical interpretation, it becomes evident that every ritual from birth to death represents a continuous cycle of life sustained by plant symbolism (tabel 4). zainal et al. – ethnobotanical study of the kaili tado 1321 table 4. ethnobotanical meanings of plant species used in each life cycle ceremony of the kaili tado ethnic group. ceremony name plant species symbolic meaning mosaviraka ngana (baby swing ceremony) kalanchoe pinnata, bacopa sp. protection and vitality membua soibou (house moving ceremony) oryza sativa, musa paradisiaca prosperity and harmony adat kalabu’ (marriage among relatives) sida rhombifolia purification and protection nokolontigi (pre-marital purification) eleusine indica sincerity and spiritual cleansing adam pocembola (marriage ceremony) cocos nucifera, piper betle, areca catechu, hibiscus tiliaceus unity, respect, strength, and peace motapusi posarara (severing kinship ceremony) calamus sp. separation and independence kamaté (funeral ceremony) jatropha curcas protection and purification the symbolic meanings of plants used in the life cycle ceremonies of the kaili tado ethnic group reveal a sophisticated cultural system where natural elements express moral, spiritual, and social principles (sirajuddin 2020; sirajuddin 2021). each plant is not merely a ritual object but serves as a metaphor of life, linking human existence to nature’s rhythm and divine balance (septiwiharti et al. 2020). in mosaviraka ngana, the baby swing ceremony, kalanchoe pinnata and bacopa sp. embody protection and vitality qualities essential to newborn life. the use of these species underscores the kaili tado belief that plants can act as mediators between the spiritual and physical worlds. this belief reflects animistic cosmology, where every living organism possesses spiritual essence (pue). scientifically, these plants have medicinal functions that align with their symbolic use; kalanchoe pinnata is known to treat inflammation and fever, supporting the concept of protection and health (rahman et al. 2019). in membua soibou, when a family moves into a new house, oryza sativa and musa paradisiaca signify prosperity and harmony core values in domestic life (permanasari 2017). rice, the staple food and life symbol in most indonesian cultures, represents sustenance and gratitude, while banana conveys sweetness and unity (mualimin et al. 2025). the offering of these plants reflects the principle of gotong royong (mutual cooperation), emphasizing balance and community welfare. the adat kalabu’ ritual, involving sida rhombifolia, expresses purification and protection for couples marrying within kinship lines (marasabessy 2025). this aligns with the broader austronesian principle of ritual cleansing before social transitions. similarly, nokolontigi employs eleusine indica to symbolize sincerity through its red pigment interpreted as the essence of life and devotion (kasim et al. 2021). the adam pocembola marriage ceremony is the most elaborate, using cocos nucifera, areca catechu, piper betle, and hibiscus tiliaceus. each species plays a role in symbolizing purity, respect, endurance, and peace. coconut represents divine connection and purity, betel leaf embodies communication and respect, areca nut signifies courage, and waru leaves represent reconciliation (giambelli 2021). the combination of these plants expresses the holistic unity of physical, emotional, and spiritual dimensions in marital life. in contrast, motapusi posarara involves calamus sp., a plant physically cut to symbolize separation of kinship. its durability and elasticity mirror human bonds strong yet severable when required by social ethics (mane 2023). finally, in the kamaté funeral ceremony, jatropha curcas is planted at the grave as protection against malevolent spirits, representing both closure and purification (lautenschläger 2020). these meanings show how kaili tado ethnobotany integrates symbolic thought, ecological knowledge, and moral philosophy. plants serve as media through which humans express gratitude, request blessings, and maintain harmony with the universe (ozdemir 2003). this integration of cultural symbolism with biological function demonstrates that ethnobotany is not simply about plant use but also about the continuity of worldview and identity (dean 2024; luo 2024). preserving these practices thus contributes not only to cultural heritage but also to biocultural conservation, reinforcing the unity of nature and culture that defines indigenous ecological wisdom (asrawijaya 2024). conservation status and cultural continuity understanding the conservation status of ritual plants is essential to evaluating how traditional ecological knowledge supports biodiversity preservation in indigenous communities. the kaili tado people rely on locally available plants, yet several species are now facing scarcity due to habitat loss and declining ritual frequency. assessing the conservation status of each species helps to identify which plants require cultivation efforts or protection initiatives to maintain both ecological and cultural sustainability (table 5). 1322 biology, medicine, & natural product chemistry 14 (2), 2025: 1315-1324 table 5. conservation status and cultural continuity of plant species used in the life cycle ceremonies of the kaili tado ethnic group. scientific name local name availability status conservation notes oryza sativa l. pae easy cultivated widely as staple food; readily available. cocos nucifera l. kaluku easy commonly planted near households; maintained for daily and ritual use. musa paradisiaca l. loka dano easy planted in home gardens; high regeneration rate. kalanchae pinnata l. pomata easy grows naturally in shaded areas; self-propagating species. areca catechu l. kalosu easy cultivated in small plantations; socio-cultural and economic value. eleusine indica l. kavoko easy wild grass species; regenerates naturally in open areas. hibiscus tiliaceus l. kalibau easy common along riverbanks and forest edges; traditionally maintained. piper betle l. boulu difficult cultivation declining due to reduced traditional use. sida rhombifolia l. sidagori easy grows naturally in village surroundings; resilient to disturbance. bacopa sp. languntale rare habitat decreasing; found only near streams. calamus sp. bolago difficult harvested from natural forests; regeneration limited. the conservation status of ritual plants in the kaili tado community reveals the dynamic interplay between cultural practice, ecological adaptation, and modernization pressures (sirajuddin 2021). among the 11 plant species identified, eight are still categorized as easily available, while three piper betle, bacopa sp., and calamus sp. are considered rare or difficult to find. these findings indicate that while traditional rituals continue to sustain certain species, ecological changes and declining ritual frequency threaten the availability of others (tusinski 2016). the persistence of widely available species such as oryza sativa, cocos nucifera, musa paradisiaca, and areca catechu is largely due to their dual role in daily life and ritual use. these plants are cultivated extensively for food, economic value, and cultural functions (rahman et al. 2019; marasabessy 2025). for example, rice cultivation remains central to kaili tado livelihood and spirituality, reinforcing its presence in multiple ceremonial contexts. similarly, coconut and banana trees are integral to both subsistence and symbolic life, ensuring their continuous propagation around homesteads. this indicates that integration of ritual and livelihood functions serves as a natural conservation mechanism within traditional societies (mualimin et al. 2025). in contrast, species that are exclusively used for specific rituals such as piper betle, bacopa sp., and calamus sp. are becoming scarce. informants noted that younger generations rarely cultivate piper betle since its social function (chewing as a symbol of respect) has diminished. the bacopa sp., associated with infant protection rituals (mosaviraka ngana), is rarely used today as modern medical practices replace traditional protection ceremonies. meanwhile, calamus sp., obtained from forest habitats, is vulnerable due to deforestation and limited regeneration. this reflects a broader issue observed in many indigenous communities, where erosion of traditional knowledge leads to loss of culturally significant species (sirajuddin 2020). nevertheless, the kaili tado people exhibit strong awareness of their cultural and ecological heritage. community elders continue to preserve ritual knowledge through oral transmission and direct participation of younger members in ceremonies (manan et al. 2024). this form of intergenerational education acts as a cultural conservation strategy, ensuring that the symbolic meanings and ecological roles of plants are not forgotten. the maintenance of sacred plants near homes such as kalanchoe pinnata, areca catechu, and hibiscus tiliaceus demonstrates how cultural values translate into everyday conservation actions. the interdependence between cultural preservation and biodiversity conservation is evident. ethnobotanical studies, including those by kasim et al. (2021), have shown that traditional ceremonies often promote sustainable plant management because sacred values restrict overexploitation. the kaili tado practice of using minimal plant quantities during rituals reflects ethical use rooted in reverence, not consumption. plants are viewed as living beings possessing spiritual essence (pue), reinforcing attitudes of respect and restraint in their collection. moreover, traditional knowledge provides adaptive strategies for environmental management. the use of easily renewable plant species in most ceremonies indicates that the community consciously avoids overharvesting forest-dependent plants (lautenschläger et al. 2020). this adaptation illustrates the principle of biocultural resilience the ability of cultural systems to adjust to ecological changes while maintaining their symbolic and moral integrity (ozdemir 2003). in the context of cultural continuity, the kaili tado rituals represent more than symbolic acts; they are mechanisms for sustaining ecological wisdom and social cohesion. each ceremony reinforces community identity, moral values, and awareness of environmental balance (septiwiharti et al. 2020). documenting these practices through ethnobotanical research contributes to both cultural revitalization and biodiversity protection, bridging indigenous knowledge with scientific understanding (arifin & nakagoshi 2011). however, modernization and diminishing ritual participation pose challenges for the transmission of this knowledge. therefore, integrating ethnobotanical values into educational materials, such as local wisdom-based zainal et al. – ethnobotanical study of the kaili tado 1323 teaching resources, can ensure continuity of cultural and ecological awareness among younger generations. such integration aligns with the goals of education for sustainable development (esd), emphasizing harmony between humans and nature (sangha et al. 2018; levis et al. 2024). conclusions the ethnobotanical practices of the kaili tado ethnic group reveal that plants play multidimensional roles in sustaining both cultural and ecological systems. each species used in life cycle ceremonies carries symbolic meanings associated with human values such as purity, strength, harmony, and protection. the presence of 11 identified plant species most of which remain accessible in local environments demonstrates the community’s enduring connection to nature. however, several species are becoming less common, indicating a need for community-based conservation initiatives. the findings highlight that ritual plant use among the kaili tado people is not merely ceremonial but represents a form of traditional ecological knowledge that supports sustainable resource management. therefore, preserving this ethnobotanical wisdom is crucial for maintaining cultural identity, ecological balance, and intergenerational transmission of indigenous knowledge. acknowledgements: the authors would like to express their sincere gratitude to the faculty of teacher training and education, tadulako university, for providing research approval and academic guidance throughout the study. special appreciation is extended to the traditional leaders and residents of langko village, lindu district, for their hospitality, cooperation, and invaluable knowledge of kaili tado cultural practices. their openness and participation made it possible to document and interpret the rich ethnobotanical heritage of their community authors’ contributions: conceptualization, syech zainal, supriatman, and amalia buntu; methodology, pahriadi, muh. syarif abd syukur, raya agni, and aan febriawan; analysis, muh. syarif abd syukur and raya agni; writing original draft preparation, syech zainal, supriatman, and amalia buntu; writing, review and editing, all authors. competing interests: the authors declare that there are no competing interests. funding: this research was funded by tadulako university, grant number 4621/un28/hk.02/2025. references abas, a., aziz, a., & awang, a. 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